sample thesis title description

Enjoy a completely custom, expertly-written dissertation. Choose from hundreds of writers, all of whom are career specialists in your subject.

Guide for Writing a Thesis Title

A thesis title refers to a paper’s short header comprising of two parts. The first section comprises the information regarding the work’s topic while the second part covers the research methods. The primary objective of a title is to capture the reader’s attention while briefly describing the paper. Consequently, students should know how to compose a good title when writing a dissertation.

Ideally, thesis titles express the arguments and subjects of the papers. Therefore, researchers should write titles after writing their theses. That’s because they know the course of their arguments after completing their theses. Remember that this title is the first thing that readers see upon receiving the paper. Therefore, this section should provide a concise topic view that the paper addresses.

To ensure your thesis title captures the reader’s attention and effectively describes your paper, consider seeking the assistance of a professional dissertation writer . Our experts will help you craft a compelling and informative title that accurately reflects the content of your dissertation. With the guidance of a professional dissertation writer, you can enhance the impact of your research and make a strong impression on your readers.

Why a Thesis Title Matters

As hinted, a dissertation title is the text’s hallmark. It reveals the essence of your paper while framing the central argument in an academic paper. While it’s a short phrase, it tells your audience more about the content. This section of the text should give readers a glimpse of your study. That’s why you should invest your time in creating a brilliant title of your paper. Ideally, you should think about this part for your paper as its packaging.

The title should be sufficiently pretty to capture the right audience’s attention. What’s more, the topic should meet certain requirements, depending on the academic writing format of your paper. Thus, whether you’re writing an APA, MLA, or PPA paper will determine aspects like quotation, abbreviation, and capitalization.

Since a title enables you to make your first contact with your readers, make it sufficiently compelling while using it to set the pace for your content. It can also entice your audience to read the entire paper.

Primary Components of a Dissertation Title

The topic of your thesis paper should be as distinct as the text it describes. However, a good title exhibits certain fundamental factors. Whether it is political science, economics, or social sciences, these elements apply to this part of a paper. And they should guide you when writing titles for the theses that the audiences find worth reading.

Formatting: Students should never submit their thesis without checking to ensure that their titles meet the formatting standards of their academic writing styles. While not all academic papers require formatting, styles differ, depending on institutions and disciplines. Formatting requirements are essential because they influence how learners write citations and quotations. What’s more, your writing style dictates how you organize the piece. Your educator might also specify the instructions to follow regarding your thesis’ tone. Therefore, consider such elements carefully to write a brilliant title. Also, remember capitalization rules when writing your topic.
Interest areas: Your study’s objectives are a significant part of the title. What you want to accomplish with the study should set a tone for your paper. Therefore, make sure that your title reflects those objectives. Your interest areas should give your paper its broad scope. However, factor in your specifics. For instance, if writing a thesis about social media marketing’s impacts on the purchasing process provides a broad scope to work with. Nevertheless, you can focus on specific networks like Instagram and Twitter. Therefore, your title should mention specific social media websites. Thus, your interest area should provide a rough guide regarding your title.
Internal Consistency: Effective thesis titles are not just attractive and precise. They are also internally consistent. Your title should accurately reflect your study. When a reader sees your title, they should get a glue of the content of your paper. If your title is about a case study approach, readers expect to find an introduction, abstract, and methodology section in the paper. Lacking consistency can create a disconnect that may push some readers away. Therefore, pay attention to the style and language of your writing to avoid misleading or losing your audience along the way.

The best dissertation titles are precise, concise, and relevant. They are also brief because many words discourage some audiences. However, a good title is not too short. Instead, it comprises over four words while thriving on specificity.

How to Title a Thesis

The title of your thesis paper should summarize your study’s main idea. It should also comprise as few words as possible, while adequately describing the purpose and/or content of the research paper. Most people read the title first and the most. If it’s too long, it will have unnecessary words. And if it’s too short, it uses too general words. Therefore, focus on creating a title that provides information regarding the focus of your work.

If your goal is to learn how to write a thesis title, these parameters should help you formulate a suitable topic.

Your research objectives or purpose
Your paper’s narrative tone, typically defined by your research type
Your research methods

Always remember to focus your title on capturing your audience’s attention while drawing their interest to the research problem that you intend to investigate.

Write the final title after completing your research to ensure that it accurately captures what you did. That means you can have a working title that you develop early during the research process. That’s because your working title can anchor the focus of your study the way a research problem does. Essentially, you should consistently refer to your working title to avoid forgetting the main purpose of your study. That way, you can avoid drifting off on the tangent when writing. Final thesis titles have several characteristics that make them effective.

These include:

Accurate indication of the study subject and scope
Wording that stimulates the reader’s interest while creating a positive impression
They do not use abbreviations
They use the current study field’s nomenclature
A revelation of the paper’s organization
Identification of independent and dependent variables
A suggestion of a relationship between the variables that support the primary hypothesis
A limit to substantive words
Can be in a question or phrase form
Correct capitalization and grammar with capital last and first words

The title of a thesis is the only aspect that readers will find when searching indexing databases or search engines. Therefore, it should be persuasive and clear to tell leaders what your research is about.

Sample Dissertation Titles

Using samples is a great way to master the art of writing brilliant titles. And the internet is awash with dissertation title examples. An ideal title should summarize your manuscript’s main idea while informing the readers about your dissertation’s nature and main topic. It can also mention your research’s subjects, location, and methodology. It may also specify theoretical issues or variables you investigated and their relationship. Often, a title should indicate your discovery.

Effective titles have eloquent and interesting wording that provides precise and necessary details. Their vocabulary can also bear relevant allusions and nuances. However, they are short and informative. Universities, departments, and style guides set strict character or word limits for titles. For instance, the APA’s publication manual limits a title to 12 words.

Since search engines use titles, words that lack a specific relationship with research become extra baggage. Thus, such titles might not work in bringing the right audience. As such, there are reasons to avoid unnecessary adjectives and adverbs. Essentially, use them sparingly to maximize your title’s effect. Words like methods, study, and methods are extraneous. However, some titles identifying the study type and dissertation methodologies can include such words.

Reading and analyzing quality samples can help you learn how to make a dissertation title. Nevertheless, check samples that fit in your study field to understand what educators in your area look for in titles.

Sample Dissertation Titles Law Students can Use

Educators require law students in the US and UK universities to write dissertations or theses at some point. In most cases, this task is the last hurdle for learners before graduating from law graduate schools. The requirement evokes horror and excitement in equal measures. But, this task provides a chance for learners to interrogate their interest area academically. Nevertheless, completing this task is a monumental responsibility. Here are dissertation titles samples that law students can use as their guide when writing this paper.

A comprehensive evaluation of female and male rape legislations: How do they differ?
Analysis of lie detectors usage in criminal justice: Are they effective?
Challenges that parties face in Vienna Convention on Contracts application for international sales
A comparison of human right law gaps in different countries
How family law has changed over the years
What are the repercussions for females vs. males involved in domestic violence?
A literature review of religion and employment laws convergence in the US
Evaluating sexual harassment at the workplace
Assessing corporate social responsibility and its mediating role in companies performance
How do medical law and ethics coexist?

Dissertations are long papers. Therefore, their topics are crucial because they determine the difficulty or simplicity of completing them. Use these samples to guide you when creating a topic for your thesis if you’re a law student.

Sample PR Dissertation Titles

When writing dissertations, public relations students should make reasonable arguments and answer research questions. Their hypotheses should provide evidence to serve as their basis. And educators expect learners to time collecting and documenting the evidence. An ideal title can make this task simple and interesting. Therefore, students should select titles that align with their developing practice area. Here are sample topics that PR students can consider exploring in their studies and writing about.

How fake and truth news change the operations of public relations offers
How essential is storytelling versus truth?
How should public relations practitioners ensure that their messages resonate well in the current fake news era?
How transparency looks like in public relations
Analyzing effective reputation and crisis management in the mobile and social media’s world
How public relations has changed- The shifting skillset for modern public relations practitioners
How mobile has affected public relations
Inbound marketing and public relations- Can PR be inbound?
How public relation practitioners are adapting to social media
Public relations monitoring and measurement- How to determine PR ROI

Public relations students can use these topic samples as their guide for creating value-adding and industry-relevant topics. However, learners should develop topics they are passionate about to enjoy their writing process.

Sample Dissertation Titles Sociology Students will Love

Several issues in social science can be a good foundation for a sociology dissertation topic. If looking for the best title for your sociology thesis, here are sample topics to consider.

Analyzing the differences in gender and sexual issues between males and females
How religious beliefs vary according to the practices and customs of a country
How modern social science studies link education and religion
How social change is taking over the world- The link between religion and social change
What are the effects of education’s sociological policies after World War II?
How immigrants’ foreign culture affects the practices and values of the indigenous people
Examining counterculture’s shifting fundamentals
How Japan’s culture compares to that of the UK
Examining the dimensions and trends of gender voting in British and American political systems
Examining the influence and power of minority interests in a society

These ideas can help you come up with a title for your thesis. However, create a title you will find interesting to research and write about. That’s the only way you will enjoy working on your thesis.

Sample Med Dissertation Titles

If pursuing medical studies, you’ll need a good topic for your dissertation at some point. Medical studies present a broad field. However, your topic should capture specific objectives and goals of your research. Here are sample topics that medical students can explore.

How to manage and take care of patients suffering from acute pain
Medical management and psychological treatment of prisoners with drug dependence problems
How midwives can improve the pregnancy outcomes
How midwives can help in high-risk pregnancies improvement
Occupational health psychology in stress management
How to prevent work-related injuries and illnesses
How to prevent the side effects of mineral fertilizers on plant workers and the environment
How emergency doctors’ mental health and their life quality relate
How to ensure personnel mental health in a security company
Occupational safety- Why is it essential for factory workers?

Whether you need an undergraduate or a Ph.D. thesis title, each of these ideas can provide a basis for formulating your topic. Nevertheless, make sure that you will be comfortable working with your title.

Sample Dissertation Titles for Business Management

A business management dissertation can cover different areas in business studies. When writing this paper, a student should focus on answering specific questions. Here are sample topics that students majoring in business management can explore in their papers.

How remote workers affect business management
How businesses can manage collaborations and communications with remote workers
Effect of wages changes on business costs
How investing in artificial intelligence enables business managers to satisfy their customers
Risk management by companies and focusing performance on the competitive advantage mediating role
Effective management models for the tourism sector
An empirical investigation of cost-leadership, business performance, and market orientation
Why intellectual capital management matters in business
Hyper-competitiveness in modern business environments- What is it about?
How banks can enhance their international connectivity with enterprise customers

This category has brilliant undergraduate thesis title samples. However, learners should take their time to identify topics they can confidently and comfortably work on. That way, they can enjoy their dissertation writing process.

Sample Interior Design Dissertation Titles

When pursuing interior design studies, your educator might ask you to write a dissertation. If allowed to select your title, consider exploring these ideas.

Why interior design is not for the wealthy people only
The interior design concept for people with tight budgets
How long interior design should take when working on a standard house
Benefits of terracotta tiles combined with woven rugs
Effects of modern trends on interior design
How to rework a retirement home from an interior designer’s perspective
The link between fashion and interior design- How each borrows ideas from the other
Why you should use your kitchen floor mats for your home’s design
How a building’s design affects the owner’s mental health
How a good design can help in managing workplace distractions

This category has some of the best titles that interior design students can explore in their papers. But like with the other categories, learners should settle on topics they can comfortably research and write about.

Sample Primary Education Dissertation Titles

Education is among the broadest study fields. The purpose of dissertation assignments in this field is to help learners explore and understand different learning approaches and education types. Here are sample topics to explore in this study field.

How the COVID-19 pandemic has affected primary education
How to maintain social distance in primary schools
How the COVID-19 pandemic has increased online primary education
The practice and theory of primary education games as tools for enhancing learning
How the learning ability of children affect their performance
How to create efficient learning settings for enhancing early childhood education
Factors enhancing and inhibiting creativity in primary schools
How primary education can develop life skills among pupils
Effective ways teachers can evaluate and monitor students in primary schools
How computer-based programs can enhance learning in primary schools

Primary education is compulsory in most developed and developing countries. This education helps in establishing foundations in mathematics, geography, history, social sciences, and science. Students that want to become primary teachers can explore these ideas when writing dissertations.

Sample Art History Dissertation Titles

Art history entails studying the objects that humans have made for aesthetic pleasure purposes. And this study field is varied and wide. If looking for a thesis title example in this field, here are brilliant ideas to consider.

How humans have exemplified their desire to touch and see God in art
How Gothic architecture is more than pointed arch
Describe the change in Egyptian art over time
How does the Gertrude Stein picture by Picasso marks his development as an artist?
Examining Picasso from the perspectives of social and political movements of his time
Describe Miro’s contribution to a surrealist movement
Discuss biomorphic in 20 th -century painting
How humans have appropriated sculpture for political display
Did the British architectural style provide a basis for the Delhi center?
How necessary is aesthetic and art appreciation?

If pursuing art history, consider any of these ideas for your dissertation, but make sure that it’s a topic you will be happy to research and write about.

Sample Globalization Dissertation Titles

When writing globalization dissertations, learners have a wide range of topic ideas they can use as the basis of their work. Here are sample topics to consider for your globalization thesis.

How globalization can affect your identity
Effects of globalization in sports
How trade relates to globalization
How globalization affects economic growth
Analysis of workers’ interests from a globalization perspective
The Cold War globalization
Is globalization bad or good for mankind?
How water scarcity affects globalization
How globalization affects the poor
Globalization and feminism

These are brilliant ideas to explore when writing a globalization thesis paper. Nevertheless, students must research their topics to come up with excellent papers about these topics.

Sample LLM Dissertation Titles

LLM dissertations topics cover the subject areas that students pursue during LLM program modules. This paper can tackle doctrinal, theoretical, policy, and jurisprudential issues that are relevant in modern legal and policy affairs. Here are sample titles for LLM dissertations.

Speech freedom and privacy right in the media and press- Should governments restrict it?
What are the weak and strong points of the judicial review process?
How to justify civil liberties restriction for public safety’s sake
How effective are anti-corruption laws in a country?
Precautions for preventing mistakes and abuse of assisted suicides legalization
National and international law- Which one should prevail?
Migrating with a minor- What legal gaps do people face when relocating?
Dividing assets after divorce- Is the law fair for the involved parties?
Effective legal mechanisms for preventing child labor
How to ease conflict when protecting trade secrets within the business law

If pursuing legal studies, you can find a title of thesis your educator will find interesting to read. But pay attention to select an interesting topic you’ll be glad to research and work with.

Sample Ph.D. Thesis Titles

A title for a Ph.D. thesis should tell the readers what you examined during your research. Thus, it should summarize your work and indicate the topic. Here are examples of attention-grabbing and catchy titles for Ph.D. theses.

Small business strategies and how to adjust them to globalization
Human resource management and strategies in non-profit organizations
Risks and benefits of international joint revenue
Outsourcing as a practice in business
Gender equality in business- Effective management approaches
Working remotely versus modern workplaces
How mentoring influences individual success
How business size impacts financial decisions
Financial risks for modern businesses
How to reduce risks at the workplace

These are brilliant thesis titles to explore when writing a Ph.D. dissertation. However, you can tweak your preferred title to make it unique and suitable for your study field.

Tips for Creating Thesis Titles

Even with the above samples, some learners can have difficulties creating titles for their thesis. These tips will make creating the best thesis title for high school students, undergraduates, masters, and Ph.D. learners easier.

Select the words to use in your title carefully
Seek advice from the professor, a friend, or classmate
Follow the format specified by your department or school
Write the final title after writing the paper
Make your title informative, brief, and catchy
Avoid abbreviations, initials, and acronyms

To ensure the creation of an exceptional thesis title, consider seeking the assistance of a professional dissertation writers . Experts have the experience and expertise to guide you in selecting the most appropriate words and crafting an informative, brief, and catchy title. Additionally, they can help you follow the format specified by your department or school while avoiding the use of abbreviations, initials, and acronyms.

Final Thoughts

The title of your thesis should indicate the subject and scope of your research. It should be engaging, concise, explanatory, and descriptive. Also, avoid abbreviations, jargon, acronyms, initials, and redundant words. Additionally, follow the requirements of your academic formatting styles and use examples to create a good title for your thesis.

Frequently Asked Questions

Richard Ginger is a dissertation writer and freelance columnist with a wealth of knowledge and expertise in the writing industry. He handles every project he works on with precision while keeping attention to details and ensuring that every work he does is unique.

Succeed With A Perfect Dissertation

Business/Finance

Should Personalization Be Optional in Paid Streaming Platforms?: Investigating User Data as an Indirect Compensation for Paid Streaming Platforms (2022)
The Influence of Live Streaming Ecommerce on Customer Engagement on the Social Media Platforms (2022)
An overview of the COVID-19 Pandemic Impact on Small Businesses in the U.S (2022)
Exploring Key Predictors of Subsequent IPO Performance in the United States between 2016 -2021 (2022)
The relationship between executive incentives and corporate performance under the background of mixed reform—Based on the empirical analysis of A-share listed companies from 2016 to 2018 (2022)
How Sovereign Credit Rating Changes Impact Private Investment (2022)
Chinese Mutual Fund Manager Style Analysis Based on Natural Language Processing (2022)
The Influence of COVID-19 on Cryptocurrency Price (2022)
Does Weather matter on E-commerce? Weather and E-commerce consumer behavior of Americans in four U.S. cities (2021)
ModellingCFPB Consumer Complaint Topics Using Unsupervised Learning (2021)
Vote For The Environment: Quantitative characteristics of shareholder resolution votes on environmental issues (2021)
Social Capital’s Role in Accessing PPP Funds & the Evolving Nature of Online Lenders in the Small Business Ecosystem (2021)
Predicting stock returns with Twitter: A test of semi-strong form EMH (2017)
Who Receives Climate Finance and Why? A Quantitative Analysis of Climate Adaptation and Mitigation Funds Allocation during 2003-2013 (2014)
The American Dream—Deferred (2013)
Job Satisfaction and Employee Turnover Intention: What does Organizational Culture Have To Do With It? (2013)
What Factors Are Associated With Poor Households Engaging in Entrepreneurship? (2013)
Uncertainty in measuring Sustainable Development: An application for the Sustainability-adjusted HDI (2012)
Homeownership and Child Welfare in Unstable Times (2012)
On the Evaluation of Conditional Cash Transfer Programs (2012)
Financial Crisis and Bank Failure Prediction: Learning Lessons from the Great Recession (2011)
Starbucks and its Peers: Corporate Social Responsibility and Corporate Financial Performance (2011)
Statistical Arbitrage Strategies and Profit Potential in Commodity Futures Markets (2011)
An Approach to Lending with Heterogeneous Borrowers (2010)
Changes in Perceived Risk and Liquidity Shocks and Its Impact on Risk Premiums (2010)
Equity Risk Premium Puzzle and Investors' Behavioral Analysis: A Theoretical and Empirical Explanation from the Stock Markets in the U.S. & China (2010)
Investing in Microfinance: A Portfolio Optimization Approach (2010)
Empirical Analysis of Value Investing Strategy in Times of Subprime Mortgage Crisis 2007-08 (2009)
Two Engines of Monetary Policy: The Federal Reserve and the European Central Bank: Different Approaches. Different Results? (2008)
Searching for the "Sweet Spot": The Optimal Mix of Executive Compensation to Maximize Firm Performance (2005)
Differentials in Firm-Level Productivity and Corporate Governance: Evidence from Japanese Firm Data in 1998-2001 (2004)
Where's the Brand Equity?: Further Investigations Into the Role of Brand Equity in Experiential, Luxury, and Other Products (2003)
An Account of Worth through Corporate Communication (2002)
Deciphering Federal Reserve Bank Statements Using Natural Language Processing (2022)
Gender Wage Gaps (2022)
The Relationship between the Overall Sentiment on Twitter and Stock Market Performance during COVID-19 Pandemic in 2020 (2022)
The U.S. Stock Market’s Influence on China Stock Market between 2014 and the first half of 2019 (2022)
Social Protection and the SDGs: A Data-Driven Bayesian Network Analysis (2022)
Overeducation: The Effects of the Great Recession on the Labor Market (2021)
Investor Sentiment and Stock Returns: Evidence from China's A-Share Market (2021)
Difference-in-Differences Analysis (2017)
Rapid Transition: A Comparison of Subway Usage and Rent Data to Predict Gentrification in New York City (2017)
Female Labor Force Participation Rate and Economic Development: Time-Series Evidence in China (2016)
Linkage Between Stock and Commodity Markets' Volitility in Both the U.S. and China (2016)
Will Urbanization be the Next Economic Growth Engine for China? (2014)
Solar Electricity's Impact on Germany's Wholesale Electricity Market (2014)
How Does Quantitative Easing Policy Impact Emerging Markets: Evidence from the Effects on Long-Term Yields Structure of Hong Kong and Singapore (2014)
The Effect of Income Taxes in Mexico: Evidence and Implications for Permanent Taxpayers (2014)
Jumping on the Bandwagon: Conformity and Herd Behavior (2014)
Effects of War After War: A Quantitative Comparison of the Economic Performance of Jewish World War II Veterans to Non-Jewish World War II Veterans (2013)
Basel III Agreement: Will Higher & More Strictly Defined Capital Standards Impede on the Growth of Small and Medium-Sized Enterprises? (2013)
Unemployment and Economic Growth in Peru: 2001-2012 (2013)
The Informal Market for Foreign Direct Investment: The Attractive Power of Country-Specific Characteristics (2012)
Evaluating the impact of the Workfare Income Supplement Scheme on Singapore's Labour Market (2012)
Innovation and Fiscal Decentralization in Transitional Economies (2012)
International Trade and Economic Growth: Evidence from Singapore (2012)
Economic Openness and Welfare Spending in Latin America (2012)
Assessing the Costs of Fractional Reserve Banking: A Theoretical Exposition and Examination of Post-Meiji Japan (2012)
Pricing Emerging Market Corporate Bonds: An Approach Using the CDS-Bond Basis Spread (2012)
The Geographical Distribution of Mixed-Income Housing in Low-Income Housing Tax Credit Developments (2012)
An Economic Theory of Voting: Can we Explain, through Digital Inequalities, Why People Vote Less? (2011)
Super-Pornstar Economics: Investigating the Wage Premium for Pornstar-Escorts (2011)
The Dynamic Linkages among International Stock Markets: The Case of BRICs and the U.S. (2011)
Revisiting the Financing Gap: An Empirical Test from 1965 to 2007 (2010)
Antitrust Law and the Promotion of Democracy and Economic Growth (2010)
An Analysis of Keynesian Economics (2010)
Who Will Pay to Reduce Global Warming? A Multivariate Analysis of Concern, Efficacy, and Action (2010)
Wage Difference Between White, Non-White, Local, and International Professional Players in the NBA (2010)
Is Microlending Sustainable? Discerning the Relationship Between Microfinancial Participation, Measures of Acute Morbidity, and Expectations of the Characteristics of Village Organizations (2009)
Application of Multi-Attribute Utility Theory to Consumers' Choices about Environmentally Responsible Decisions (2009)
Trade Openness and Poverty Reduction: What is the Evidence? (2009)
Crude Oil Prices: Mean Reversion in the Spot? Futures Know the Future? (2008)
Evaluating the Impact of Supply-side Factors on Conditional Cash Transfer Programs: The Case of Nicaragua (2008)
Females: Less Likely to Be Entrepreneurs? A Multi-level Analysis of the Effect of Gender on Entrepreneurial Activity (2008)
Banking the Mexican Immigrant Population: Analysis of Profiling Variables (2008)
A Comparison of Microfranchising to Independent Microenterprises in Ghana (2008)
From Autarky to Free Trade: Will China Overtake the U.S. as the Major Trading Power in the Global Economy? (2006)
Cluster Patterns of Age and Racial/Ethnic Groups Within Privately Developed Section 8 HUD Rent Subsidy Properties in New York City (2004)
The Impact of Decimalization on Market Volatility and Liquidity (2004)
Strategic Delegation with Unobservable Incentive Contracts: An Experiment (2002)
Exchange Rate Market Pressure and The Quality of Governance (2001)

Public Health

Analysing the Performance of Supervised ML models in Breast Cancer Diagnosis (2022)
Portability of Polygenic Scores for QuantitativeTraits using Continuous Genetic Distance in the UK Biobank (2021)
A Report on the Correlation between COVID-19 pandemic and Unemployment Rate through Visualization (2021)
Spatial Summary of Outdoor Dining and COVID-19 Rates in NYC (2021)
The COVID-19 Infodemic: Narratives from the US & India (2021)
Exploring the Experiences of People Living with HIV in the United States: Modelling Muscle Ache/Pain and Medicaid Expansion (2017)
An Ounce of Prevention is Worth a Pound of Cure: An Algorithm Using Non-Health Indicators to Predict Health Risks of an Individual (2017)
Does Racial Concordance in Clinical Encounters improve Providers’ Accessibility and Patients’ Satisfaction with Providers? (2016)
Proportionality of Death Sentences in Alabama (2014)
Zombies, Brains, and Tweets: The Neural and Emotional Correlates of Social Media (2013)
Asexuality as a Spectrum: A National Probability Sample Comparison to the Sexual Community in the UK (2013)
Parent-reported and Child Self-reported Symptoms of Psychiatric Disorder and their Relationships to Independent Living Skills in a Clinical Sample of Perinatally HIV-infected and Perinatally HIV-exposed but Uninfected Adolescents: An Exploratory Analysis (2013)
The Sperm Shopper: How Consumer Segments and Evolutionary Pyschology Shape Choice of Sperm Donor (2012)
Social Context and Impoverished Youths' General Health Outcomes: Community Disorder and Violence Predicting Self-Rated Health and Body Mass Index (2012)
Location Theory and the Supply of Primary Care Physicians in Rural America (2012)
Perception of Neighborhood Safety and Overweight/Obesity Status among Non-Metropolitan Adolescents in the U.S. (2011)
Factors Affecting the Extent of Depression Treatment (2011)
Beyond Gender Binary in Survey Design (2010)
Junk Food and BMI: A Look at Schools Banning Candy, Snacks, and Soft Drinks and the Effect on Fifth Graders' BMI (2009)
Delivering Maternal Health: An Examination of Maternal Mortality on a National Scale (2008)
Public Health and the Conrad Visa Waiver Program (2007)
Alzheimer's Disease, Migration, and Social Environment: A Study of Caribbean Hispanics (2005)
The Influence of Physician Attributes on Cesarean Likelihood (2004)
Natural or Human-Made Disaster: Dimensions of Impact Measurement (2003)
Healthy Life Choices Project: Efficacy of Nutritional Intervention with Normal Foods and Cognitive/Behavioral Skill Building on HIV/AIDS Associated Diarrhea and Quality of Life (2002)

Political Science

Encouraging Voter Registration Among Minority Voters: A Field Experiment Using Radio Advertisements (2022)
Public Opinion Transition in China: Evidence from Weibo (2022)
Gender and Co-sponsorship in U.S. Congress (2017)
Accessing Social Influences of Congressmen with Keyword Network (2016)
How presidential election in 2016 affects the stock market – A Twitter sentiment analysis perspective (2016)
Assessing Assessors: A Study on Anti-Corruption Strategies in New York City’s Property Tax System (2016)
Demographic Trends in Virginia 2013
The determinants of Party and Coalition Identification in Chile: The effect of long and short-term factors (2013)
Radical Moderation: Factors Affecting Support for Islamic Extremism (2012)
Accommodationists versus Hardliners in Slovakia: Correlates of Public Opinion on Selected Foreign Policy Topics 2004 - 2010 (2012)
Measurement and Belief: Determinants of Federal Funding for Public Diplomacy Programs (2010)
Consumerism and Political Connectedness in Socialist Czechoslovakia (2010) - History
Civilizations and Social Tolerance: A Multi-Level Analysis of 58 Countries (2008)
How Does the 1965 Immigration Act Matter? (2006)
7200 Revolutions per Minute: An Economic Analysis of the Struggle between the Recording Industry and Peer-to-Peer File Sharing Networks (2005)
Classifying Myers-Briggs Personality Type based on Text (2021)
Hiding Behind the Computer Screen: Imposter Phenomenon in the Tech Industry (2022)
Relation between dark tourism on-site experience and visitors’ satisfaction (2022)
Evaluating the Impact of Self-perceptions of Creativity and DemographicFactors on Arts Participation: Evidence from the United States (2021)
Running head: QUEER HAPPINESS AND SUPPORTExamining Happiness in LGBTQ+ People and its Relationshipwith Worsened Parental Relationships After Coming Out (2021)
The Impact of Donating Behavior on the Level of Happiness (2021)
Birds of a Feather, or Do Opposites Attract? THE IMPACT OF PERSONALITY TRAITS ON CONSTRAINT AND HOMOPHILY WITHIN SOCIAL NETWORKS (2017)
Predicting Social Value Orientation from Personal Information and Survey Metadata (2017)
All the Feels: Sentiment Analysis Between Emoji and Text (2017)
Social Media Interface and the Next Generation Cognitive Mapping in New York City (2016)
Is Prospective Memory Ability Flexible? Manipulating Value to Increase Goal Significance (2011)
Will a Nation Be Happier with a More Even Income Distribution? (2007)
Behavioral Extensions to the Topology of Fear: A Gedankenexperimen (2007)
Psychological Control and Preschoolers' Externalizing and Internalizing Behaviors in China (2003)
Prevalence and success of diversity-and-inclusion projects on education crowdfunding platform (2022)
Does gentrification cause the displacement of urban black populations? (2022)
Feedback and Gender in the Workplace: Should You Expect Equal Evaluation from Men and Women? (2021)
What are the determinants for art practitioners to choose self-employment? (2022)
An empirical research for studying the influence of star popularity on the box office of movies (2022)
Couple Dissolution Between Couples Who Meet Offline Versus Couples Who Meet Offline (2021)
Masculine Men Who Wear Makeup: Exploring the Evolving Masculinity (2021)
Do Individual Or Environmental Factors Play a Greater Role in Shaping the Intentions of Female High School Students to Enrol in STEM (2021) Programmes in University?:Evidence from the High School Longitudinal Study of 2009 (2021)
COVID-19 Information Narrative Beliefs Across Social Media Platforms (2021)
Spatial Wage Penalty for Young Mothers: Exploring the Discrepancy of Education Return between Metro and Non-metro Areas (2016)
Inequality Matters: A new Empirical Framework for Studying the Impact of Rising Socioeconomic Inequality on the Poor (2016)
Immigration, Income, and Occupation: Peruvian Immigrants in the Chilean Labor Market (2014)
Preferring France's 35-Hour Workweek: The Effects of Media on Work-Life Balance Preference Formation (2014)
The Effect of College Education on Individual Social Trust in the United States– An Examination of the Causal Mechanisms (2013)
Socio-economic Inequality and Socio-emotional Relationship Quality: Cause and effect? (2013)
Examination of the Relationship between mother's employment status and one's family gender role attitudes (2012)
A Study of Materialism Level among Mid-Atlantic residents (2012)
Relation Recombination - A Sociological Patent Analysis (2012)
The Relationship between Religious Attitudes and Concern for the Environment (2012)
Marrying Down: The Gender Gap in Post-Secondary Completion & Education Hypogamy between 1960 and 2010 (2012)
2.0 Social Networks Have an Impact on our Real Lives (2011)
Evidence of Ethnic Solidarity in Marriage Patterns of Hmong and Sino-Vietnamese in United States (2011)
What Explains the Racial Disparity in Employment Discrimination Case Outcomes? (2010)
Reading Race: The Changing Views of Human Difference in American History Textbooks, 1870-1930 (2010)
Satisfaction with Life (2010)
Entering the "Real World": An Empirical Investigation of College Graduates' Satisfaction with Life (2010)
The Relationship between the Establishment of Marine Protected Areas and Biomass Productivity of Municipal Fisheries in the Philippines (2010)
Performance Surveys, Citizen Respondents, and Satisfaction of Public Services: An Analysis of NYC Feedback Citywide Customer Survey (2009)
Analysis of Job Retention Programs of the Center for Employment Opportunities of the Formerly Incarcerated (2009)
The Intergenerational Transfer of Human Capital: The Role of Grandparents' Education in Grandchildren's Cognitive Abilities (2009)
Are Homicide Trends Fads? Diffusion Analysis of the Urban-rural Spillover Effects on Homicide Incidents from 1960-1990 in the South Atlantic States (2008)
Rejection Sensitivity and the Contagious Effect of Mood Regulation in Romantic Couples (2008)
Women and the Homeostasis of the Inmate Population
An Examination of the Relationship between Government Funding Allocation and Services Provided by Nonprofit Organizations in Brooklyn and the Bronx, 1997-2000 (2007)
The Concurrent Validity of Maternal Self-report: The Impact of Social Desirability on Substance Use and Prenatal Care (2006)
The Effect of Housing Programs on the Economic Outcomes: Utilizing Observation Study Results from Minnesota Family Investment Program (2005)
The Influences of Physician Attributes on Cesarean Likelihood (2004)
Effects of Unemployment, Female Labor Force Participation, and Divorce on Suicide in Turkey: A Durkheimian Evaluation in a non-Western Milieu (2004)
An Experimental Study of the Small World Problem (2002)
The Relationship between Welfare Participation and Social Support (2002)
Sound and Silence: A Structural Analysis of Conversation Topics (2002)
A Reexamination of the Police and Crime Relationship: The New Role Community Policing Plays in Crime Prevention (2001)
DNA Evidence in Court: Jurors, Statistical Training, and Pre-instruction in the Procedural Law (2001)
The Role of Race in Education: An Analysis of Children in Brazil (2001)

Statistics/Computer Science

Predicting Spotify's songs' popularity (2022)
Hiding Behind the Computer Screen: Imposter Phenomenon in the Tech Industry (2021)
An Unsupervised Learning Approach to Address Crime in Mexico, 2012 – 2016 (2017)
Imputation of a variable completely unobserved in one wave of a panel: father’s earnings in the Fragile Families and Child Wellbeing Study (2016)

An Analysis of Pairwise Preference (2016)

Measuring Political Risk and Market Returns (2014)
Which Yelp Reviews will be Voted Useful?- Predicting the Number of Useful Votes Yelp Reviews will get using Machine Learning Algorithms (2014)
Polities and Size: Legitimizing or Limiting? (2013)
The Role of Domain Knowledge in Environmental Concern and Willingness-to-Pay for Environmental Protection: Results from a U.S. Survey of Public Opinion (2013)
The Power to Judge: Social Power Influences Moral Judgments of Simple and Complex Transgressions (2013)
A Time Series Analysis of Crime Rates and Concern for Crime in the United States: 1973-2010 (2012)
TV Gets Social: Evaluating Social Media Data to Explain Variability among Nielsen TV Ratings (2012)
Unit Root or Mean Reversion in Stock Index: Evidence from Nigeria (2010)
Homogeneity in Political Discussion Networks and its Factors (2007)
Why Shift Policy? (2006)
Point Detection for Poisson Disorder - Application in Earthquake Occurrence in Northern California, 1910 - 1999 (2004)
Stock Volatility and Economic Activity: A Causal Analysis (2004)
Strategic Information Transmission in Lobbying (2003)
Economic Theory and Happiness in Mexico: An Extension (2001)
Sales Forecasting Methods: A Consumer Products Company's Perspective (2001)
Soccer Teams Need to Win at Home: The Fans that Increase those Chances (2001)
The impact of school management on student performance (2022)
An investigation of the relationship between educational attainment and COVID-19 vaccination hesitancy in the US (2022)
Does Accountability Help or HinderSchools?: The Mississippi School Accountability Model and its Effect on School Performance (2021)
The Relationship between Education and Health (2021)
Quantifying Variation in American School Safety with Explainable Machine Learning:An Application of Machine Learning Feature Importances for the Social Sciences (2021)
Age, Gender, and Comorbidities Affect Prevalence of Dyscalculia and Dyslexia, A Large-Scale Study of Specific Learning Disabilities Among Chinese Children (2021)
Validation of Fitbit for use in Objective Measurement of Physical Activity and Sleep in Children and Adults (2014)
Do Experienced Principals Fare Better? Estimates of Principal Value-Added (2014)
Beyond the Test Score Gap: Non-Cognitive Skills, High School Graduation, and Post-Secondary Employment (2012)
The Impact of the Level of Native Language Proficiency on the Literacy Achievement of English Language Leisures (2012)
The Effect of School Building Design on Student Achievement (2011)
Measuring Universal Primary Education Using Household Survey Data: The Case of the Millennium Villages Project (2011)
An Additional Burden for Urban Schools: Teacher Transfer Policies and School Performance (2011)
Evaluating Dual Enrollment Programs: Do Location and Instructor Matter? (2010)
A Multi-level Growth-curve Analysis of the Association between Student Body Composition and English Literacy Development among Language Minority Students in New York City Public Schools (2010)
Methods Supporting Policies in Education Reform (2010)
Have Inclusionary Policies in Higher Education Really Helped?: Looking at College Accessibility and the College-wage Premium, 1962-2007 (2010)
NCLB and Curriculum Standards: What Really Impacts Teachers' Decisions to Leave the Profession? (2010)
Exploring the Relationship between Video Games and Academic Achievement via Cross-sectional and Longitudinal Analyses (2009)
Racial Disparities in Collegiate Cognitive Gains: A Multi-level Analysis of Institutional Influences on Learning and its Equitable Distribution (2009)
Hoping for Higher Ed: The Differential Effects of Parental Expectations of Education Attainment (2009)
The Impact of Family Communication on Risk Behavior among Boston Public High School Students (2009)
Path Towards an Attainable Future: The Effect of College Access Programs on High School Dropout (2009)
Traditional vs. Non-traditional College Students and Future Job Satisfaction: A Statistical Approach (2008)
A Multi-level Analysis of Student Assignment to Out-of-field and Uncertified High School Math Teachers: Implications for Educational Equity and Access (2008)
The Impact of Obesity on Education (2005)
The Gender Gap in Standardized Math Tests: Do the Gender Gaps in Math Self-concept and Other Affective Variables Contribute to the Gender Gap in Scores? (2004)
An Alternative Approach to Selection Bias in School Choice: Using Propensity Score Matching to Examine School Sector and Teacher Quality Impact on Educational Outcomes (2003)

Join the QMSS Prospective Mailing List!

Latest tweet.

@QMSS at Columbia 4/4 Gold medalists (1st %ile or >): Kechengjie Zhu, Computer Science Nicholas Milinkovich, QMSS Vijay S Kalmath, Computer Science Apr. 29, 2022
@QMSS at Columbia 3/4 Silver medalists (5th %ile or >): Anh-Vu NGUYEN, DSI Pruthvi Reddy Panati, QMSS Alan Luo, Computer Science Apr. 29, 2022
Address: 420 W 118th St, Suite 807 New York, NY 10027
Email: [email protected]
Phone: +1 212-853-3910
Monday - Friday: 9:00 am - 5:00 pm

When you choose to publish with PLOS, your research makes an impact. Make your work accessible to all, without restrictions, and accelerate scientific discovery with options like preprints and published peer review that make your work more Open.

PLOS Biology
PLOS Climate
PLOS Computational Biology
PLOS Digital Health
PLOS Genetics
PLOS Global Public Health
PLOS Medicine
PLOS Neglected Tropical Diseases
PLOS Pathogens
PLOS Sustainability and Transformation
PLOS Collections
How to Write a Great Title

Maximize search-ability and engage your readers from the very beginning

Your title is the first thing anyone who reads your article is going to see, and for many it will be where they stop reading. Learn how to write a title that helps readers find your article, draws your audience in and sets the stage for your research!

How your title impacts the success of your article

Researchers are busy and there will always be more articles to read than time to read them. Good titles help readers find your research, and decide whether to keep reading. Search engines use titles to retrieve relevant articles based on users’ keyword searches. Once readers find your article, they’ll use the title as the first filter to decide whether your research is what they’re looking for. A strong and specific title is the first step toward citations, inclusion in meta-analyses, and influencing your field.

What to include in a title

Include the most important information that will signal to your target audience that they should keep reading.

Key information about the study design

Important keywords

What you discovered

Writing tips

Getting the title right can be more difficult than it seems, and researchers refine their writing skills throughout their career. Some journals even help editors to re-write their titles during the publication process!

Keep it concise and informative What’s appropriate for titles varies greatly across disciplines. Take a look at some articles published in your field, and check the journal guidelines for character limits. Aim for fewer than 12 words, and check for journal specific word limits.
Write for your audience Consider who your primary audience is: are they specialists in your specific field, are they cross-disciplinary, are they non-specialists?
Entice the reader Find a way to pique your readers’ interest, give them enough information to keep them reading.
Incorporate important keywords Consider what about your article will be most interesting to your audience: Most readers come to an article from a search engine, so take some time and include the important ones in your title!
Write in sentence case In scientific writing, titles are given in sentence case. Capitalize only the first word of the text, proper nouns, and genus names. See our examples below.

Don’t

Write your title as a question In most cases, you shouldn’t need to frame your title as a question. You have the answers, you know what you found. Writing your title as a question might draw your readers in, but it’s more likely to put them off.
Sensationalize your research Be honest with yourself about what you truly discovered. A sensationalized or dramatic title might make a few extra people read a bit further into your article, but you don’t want them disappointed when they get to the results.

Examples…

Format: Prevalence of [disease] in [population] in [location]

Example: Prevalence of tuberculosis in homeless women in San Francisco

Format: Risk factors for [condition] among [population] in [location]

Example: Risk factors for preterm births among low-income women in Mexico City

Format (systematic review/meta-analysis): Effectiveness of [treatment] for [disease] in [population] for [outcome] : A systematic review and meta-analysis

Example: Effectiveness of Hepatitis B treatment in HIV-infected adolescents in the prevention of liver disease: A systematic review and meta-analysis

Format (clinical trial): [Intervention] improved [symptoms] of [disease] in [population] : A randomized controlled clinical trial

Example: Using a sleep app lessened insomnia in post-menopausal women in southwest United States: A randomized controlled clinical trial

Format (general molecular studies): Characterization/identification/evaluation of [molecule name] in/from [organism/tissue] (b y [specific biological methods] )

Example: Identification of putative Type-I sex pheromone biosynthesis-related genes expressed in the female pheromone gland of Streltzoviella insularis

Format (general molecular studies): [specific methods/analysis] of organism/tissue reveal insights into [function/role] of [molecule name] in [biological process]

Example: Transcriptome landscape of Rafflesia cantleyi floral buds reveals insights into the roles of transcription factors and phytohormones in flower development

Format (software/method papers): [tool/method/software] for [what purpose] in [what research area]

Example: CRISPR-based tools for targeted transcriptional and epigenetic regulation in plants

Tip: How to edit your work

Editing is challenging, especially if you are acting as both a writer and an editor. Read our guidelines for advice on how to refine your work, including useful tips for setting your intentions, re-review, and consultation with colleagues.

How to Write an Abstract
How to Write Your Methods
How to Report Statistics
How to Write Discussions and Conclusions
How to Edit Your Work

There is no excerpt because this is a protected post.

There’s a lot to consider when deciding where to submit your work. Learn how to choose a journal that will help your study reach its audience, while reflecting your values as a researcher…

Thesis and Dissertation Guide

« Thesis & Dissertation Resources
The Graduate School Home

Introduction

Copyright Page

Dedication, acknowledgements, preface (optional), table of contents.

List of Tables, Figures, and Illustrations

List of Abbreviations

List of symbols.

Non-Traditional Formats
Font Type and Size
Spacing and Indentation
Tables, Figures, and Illustrations
Formatting Previously Published Work
Internet Distribution
Open Access
Registering Copyright
Using Copyrighted Materials
Use of Your Own Previously Published Materials
Submission Steps
Submission Checklist
Sample Pages

I. Order and Components

Please see the sample thesis or dissertation pages throughout and at the end of this document for illustrations. The following order is required for components of your thesis or dissertation:

Dedication, Acknowledgements, and Preface (each optional)
Table of Contents, with page numbers
List of Tables, List of Figures, or List of Illustrations, with titles and page numbers (if applicable)
List of Abbreviations (if applicable)
List of Symbols (if applicable)
Introduction, if any
Main body, with consistent subheadings as appropriate
Appendices (if applicable)
Endnotes (if applicable)
References (see section on References for options)

Many of the components following the title and copyright pages have required headings and formatting guidelines, which are described in the following sections.

Please consult the Sample Pages to compare your document to the requirements. A Checklist is provided to assist you in ensuring your thesis or dissertation meets all formatting guidelines.

The title page of a thesis or dissertation must include the following information:

Title Page with mesaurements described in surrounding text

The title of the thesis or dissertation in all capital letters and centered 2″ below the top of the page.
Your name, centered 1″ below the title. Do not include titles, degrees, or identifiers. The name you use here does not need to exactly match the name on your university records, but we recommend considering how you will want your name to appear in professional publications in the future.

Notes on this statement:

When indicating your degree in the second bracketed space, use the full degree name (i.e., Doctor of Philosophy, not Ph.D. or PHD; Master of Public Health, not M.P.H. or MPH; Master of Social Work, not M.S.W. or MSW).
List your department, school, or curriculum rather than your subject area or specialty discipline in the third bracketed space. You may include your subject area or specialty discipline in parentheses (i.e., Department of Romance Languages (French); School of Pharmacy (Molecular Pharmaceutics); School of Education (School Psychology); or similar official area).
If you wish to include both your department and school names, list the school at the end of the statement (i.e., Department of Pharmacology in the School of Medicine).
A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Public Policy.
A thesis submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the School of Dentistry (Endodontics).
A thesis submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department of Nutrition in the Gillings School of Global Public Health.
A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Education (Cultural Studies and Literacies).
The words “Chapel Hill” must be centered 1″ below the statement.
One single-spaced line below that, center the year in which your committee approves the completed thesis or dissertation. This need not be the year you graduate.
Approximately 2/3 of the way across the page on the right-hand side of the page, 1″ below the year, include the phrase “Approved by:” (with colon) followed by each faculty member's name on subsequent double-spaced lines. Do not include titles such as Professor, Doctor, Dr., PhD, or any identifiers such as “chair” or “advisor” before or after any names. Line up the first letter of each name on the left under the “A” in the “Approved by:” line. If a name is too long to fit on one line, move this entire section of text slightly to the left so that formatting can be maintained.
No signatures, signature lines, or page numbers should be included on the title page.

Include a copyright page with the following information single-spaced and centered 2″ above the bottom of the page:

Copyright Page with mesaurements described in surrounding text

This page immediately follows the title page. It should be numbered with the lower case Roman numeral ii centered with a 1/2″ margin from the bottom edge.

Inclusion of this page offers you, as the author, additional protection against copyright infringement as it eliminates any question of authorship and copyright ownership. You do not need to file for copyright in order to include this statement in your thesis or dissertation. However, filing for copyright can offer other protections.

See Section IV for more information on copyrighting your thesis or dissertation.

Include an abstract page following these guidelines:

Abstract page with mesaurements described in surrounding text

Include the heading “ABSTRACT” in all capital letters, and center it 2″ below the top of the page.
One double-spaced line below “ABSTRACT”, center your name, followed by a colon and the title of the thesis or dissertation. Use as many lines as necessary. Be sure that your name and the title exactly match the name and title used on the Title page.
One single-spaced line below the title, center the phrase “(Under the direction of [advisor's name])”. Include the phrase in parentheses. Include the first and last name(s) of your advisor or formal co-advisors. Do not include the name of other committee members. Use the advisor's name only; do not include any professional titles such as PhD, Professor, or Dr. or any identifiers such as “chair” or “advisor”.
Skip one double-spaced line and begin the abstract. The text of your abstract must be double-spaced and aligned with the document's left margin with the exception of indenting new paragraphs. Do not center or right-justify the abstract.
Abstracts cannot exceed 150 words for a thesis or 350 words for a dissertation.
Number the abstract page with the lower case Roman numeral iii (and iv, if more than one page) centered with a 1/2″ margin from the bottom edge.

Please write and proofread your abstract carefully. When possible, avoid including symbols or foreign words in your abstract, as they cannot be indexed or searched. Avoid mathematical formulas, diagrams, and other illustrative materials in the abstract. Offer a brief description of your thesis or dissertation and a concise summary of its conclusions. Be sure to describe the subject and focus of your work with clear details and avoid including lengthy explanations or opinions.

Your title and abstract will be used by search engines to help potential audiences locate your work, so clarity will help to draw the attention of your targeted readers.

You have an option to include a dedication, acknowledgements, or preface. If you choose to include any or all of these elements, give each its own page(s).

Dedication page with mesaurements described in surrounding text

A dedication is a message from the author prefixed to a work in tribute to a person, group, or cause. Most dedications are short statements of tribute beginning with “To…” such as “To my family”.

Acknowledgements are the author's statement of gratitude to and recognition of the people and institutions that helped the author's research and writing.

A preface is a statement of the author's reasons for undertaking the work and other personal comments that are not directly germane to the materials presented in other sections of the thesis or dissertation. These reasons tend to be of a personal nature.

Any of the pages must be prepared following these guidelines:

Do not place a heading on the dedication page.
The text of short dedications must be centered and begin 2″ from the top of the page.
Headings are required for the “ACKNOWLEDGEMENTS” and “PREFACE” pages. Headings must be in all capital letters and centered 2″ below the top of the page.
The text of the acknowledgements and preface pages must begin one double-spaced line below the heading, be double-spaced, and be aligned with the document's left margin with the exception of indenting new paragraphs.
Subsequent pages of text return to the 1″ top margin.
The page(s) must be numbered with consecutive lower case Roman numerals (starting with the page number after the abstract) centered with a 1/2″ margin from the bottom edge.

Include a table of contents following these guidelines:

Table of Contents page with mesaurements described in surrounding text

Include the heading “TABLE OF CONTENTS” in all capital letters, and center it 2″ below the top of the page.
Include one double-spaced line between the heading and the first entry.
The table of contents should not contain listings for the pages that precede it, but it must list all parts of the thesis or dissertation that follow it.
If relevant, be sure to list all appendices and a references section in your table of contents. Include page numbers for these items but do not assign separate chapter numbers.
Entries must align with the document's left margin or be indented to the right of the left page margin using consistent tabs.
Major subheadings within chapters must be included in the table of contents. The subheading(s) should be indented to the right of the left page margin using consistent tabs.
If an entry takes up more than one line, break up the entry about three-fourths of the way across the page and place the rest of the text on a second line, single-spacing the two lines.
Include one double-spaced line between each entry.
Page numbers listed in the table of contents must be located just inside the right page margin with leaders (lines of periods) filling out the space between the end of the entry and the page number. The last digit of each number must line up on the right margin.
Information included in the table of contents must match the headings, major subheadings, and numbering used in the body of the thesis or dissertation.
The Table of Contents page(s) must be numbered with consecutive lower case Roman numerals centered with a 1/2″ margin from the bottom edge.

Lists of Tables, Figures, and Illustrations

If applicable, include a list of tables, list of figures, and/or list of illustrations following these guidelines:

Lists of Figures page with mesaurements described in surrounding text

Include the heading(s) in all capital letters, centered 1″ below the top of the page.
Each entry must include a number, title, and page number.
Assign each table, figure, or illustration in your thesis or dissertation an Arabic numeral. You may number consecutively throughout the entire work (e.g., Figure 1, Figure 2, etc.), or you may assign a two-part Arabic numeral with the first number designating the chapter in which it appears, separated by a period, followed by a second number to indicate its consecutive placement in the chapter (e.g., Table 3.2 is the second table in Chapter Three).
Numerals and titles must align with the document's left margin or be indented to the right of the left page margin using consistent tabs.
Page numbers must be located just inside the right page margin with leaders (lines of periods) filling out the space between the end of the entry and the page number. The last digit of each number must line up on the right margin.
Numbers, titles, and page numbers must each match the corresponding numbers, titles, and page numbers appearing in the thesis or dissertation.
All Lists of Tables, Figures, and Illustrations page(s) must be numbered with consecutive lower case Roman numerals centered with a 1/2″ margin from the bottom edge.

If you use abbreviations extensively in your thesis or dissertation, you must include a list of abbreviations and their corresponding definitions following these guidelines:

List of Abbreviations with mesaurements described in surrounding text

Include the heading “LIST OF ABBREVIATIONS” in all capital letters, and center it 1″ below the top of the page.
Arrange your abbreviations alphabetically.
Abbreviations must align with the document's left margin or be indented to the right of the left page margin using consistent tabs.
If an entry takes up more than one line, single-space between the two lines.
The List of Abbreviations page(s) must be numbered with consecutive lower case Roman numerals centered with a 1/2″ margin from the bottom edge.

If you use symbols in your thesis or dissertation, you may combine them with your abbreviations, titling the section “LIST OF ABBREVIATIONS AND SYMBOLS”, or you may set up a separate list of symbols and their definitions by following the formatting instructions above for abbreviations. The heading you choose must be in all capital letters and centered 1″ below the top of the page.

Previous: Introduction

Next: Format

Affiliate Program

UNITED STATES
台灣 (TAIWAN)
TÜRKIYE (TURKEY)
Academic Editing Services
- Research Paper
- Journal Manuscript
- Dissertation
- College & University Assignments
Admissions Editing Services
- Application Essay
- Personal Statement
- Recommendation Letter
- Cover Letter
- CV/Resume
Business Editing Services
- Business Documents
- Report & Brochure
- Website & Blog
Writer Editing Services
- Script & Screenplay
Our Editors
Client Reviews
Editing & Proofreading Prices
Wordvice Points
Partner Discount
Plagiarism Checker
APA Citation Generator
MLA Citation Generator
Chicago Citation Generator
Vancouver Citation Generator
- APA Style
- MLA Style
- Chicago Style
- Vancouver Style
Writing & Editing Guide
Academic Resources
Admissions Resources

How to Make a Research Paper Title with Examples

What is a research paper title and why does it matter?

A research paper title summarizes the aim and purpose of your research study. Making a title for your research is one of the most important decisions when writing an article to publish in journals. The research title is the first thing that journal editors and reviewers see when they look at your paper and the only piece of information that fellow researchers will see in a database or search engine query. Good titles that are concise and contain all the relevant terms have been shown to increase citation counts and Altmetric scores .

Therefore, when you title research work, make sure it captures all of the relevant aspects of your study, including the specific topic and problem being investigated. It also should present these elements in a way that is accessible and will captivate readers. Follow these steps to learn how to make a good research title for your work.

How to Make a Research Paper Title in 5 Steps

You might wonder how you are supposed to pick a title from all the content that your manuscript contains—how are you supposed to choose? What will make your research paper title come up in search engines and what will make the people in your field read it?

In a nutshell, your research title should accurately capture what you have done, it should sound interesting to the people who work on the same or a similar topic, and it should contain the important title keywords that other researchers use when looking for literature in databases. To make the title writing process as simple as possible, we have broken it down into 5 simple steps.

Step 1: Answer some key questions about your research paper

What does your paper seek to answer and what does it accomplish? Try to answer these questions as briefly as possible. You can create these questions by going through each section of your paper and finding the MOST relevant information to make a research title.

Step 2: Identify research study keywords

Now that you have answers to your research questions, find the most important parts of these responses and make these your study keywords. Note that you should only choose the most important terms for your keywords–journals usually request anywhere from 3 to 8 keywords maximum.

Step 3: Research title writing: use these keywords

“We employed a case study of 60 liver transplant patients around the US aged 20-50 years to assess how waiting list volume affects the outcomes of liver transplantation in patients; results indicate a positive correlation between increased waiting list volume and negative prognosis after the transplant procedure.”

The sentence above is clearly much too long for a research paper title. This is why you will trim and polish your title in the next two steps.

Step 4: Create a working research paper title

To create a working title, remove elements that make it a complete “sentence” but keep everything that is important to what the study is about. Delete all unnecessary and redundant words that are not central to the study or that researchers would most likely not use in a database search.

“ We employed a case study of 60 liver transplant patients around the US aged 20-50 years to assess how the waiting list volume affects the outcome of liver transplantation in patients ; results indicate a positive correlation between increased waiting list volume and a negative prognosis after transplant procedure ”

Now shift some words around for proper syntax and rephrase it a bit to shorten the length and make it leaner and more natural. What you are left with is:

“A case study of 60 liver transplant patients around the US aged 20-50 years assessing the impact of waiting list volume on outcome of transplantation and showing a positive correlation between increased waiting list volume and a negative prognosis” (Word Count: 38)

This text is getting closer to what we want in a research title, which is just the most important information. But note that the word count for this working title is still 38 words, whereas the average length of published journal article titles is 16 words or fewer. Therefore, we should eliminate some words and phrases that are not essential to this title.

Step 5: Remove any nonessential words and phrases from your title

Because the number of patients studied and the exact outcome are not the most essential parts of this paper, remove these elements first:

“A case study of 60 liver transplant patients around the US aged 20-50 years assessing the impact of waiting list volume on outcomes of transplantation and showing a positive correlation between increased waiting list volume and a negative prognosis” (Word Count: 19)

In addition, the methods used in a study are not usually the most searched-for keywords in databases and represent additional details that you may want to remove to make your title leaner. So what is left is:

“Assessing the impact of waiting list volume on outcome and prognosis in liver transplantation patients” (Word Count: 15)

In this final version of the title, one can immediately recognize the subject and what objectives the study aims to achieve. Note that the most important terms appear at the beginning and end of the title: “Assessing,” which is the main action of the study, is placed at the beginning; and “liver transplantation patients,” the specific subject of the study, is placed at the end.

This will aid significantly in your research paper title being found in search engines and database queries, which means that a lot more researchers will be able to locate your article once it is published. In fact, a 2014 review of more than 150,000 papers submitted to the UK’s Research Excellence Framework (REF) database found the style of a paper’s title impacted the number of citations it would typically receive. In most disciplines, articles with shorter, more concise titles yielded more citations.

Adding a Research Paper Subtitle

If your title might require a subtitle to provide more immediate details about your methodology or sample, you can do this by adding this information after a colon:

“ : a case study of US adult patients ages 20-25”

If we abide strictly by our word count rule this may not be necessary or recommended. But every journal has its own standard formatting and style guidelines for research paper titles, so it is a good idea to be aware of the specific journal author instructions , not just when you write the manuscript but also to decide how to create a good title for it.

Research Paper Title Examples

The title examples in the following table illustrate how a title can be interesting but incomplete, complete by uninteresting, complete and interesting but too informal in tone, or some other combination of these. A good research paper title should meet all the requirements in the four columns below.

Tips on Formulating a Good Research Paper Title

In addition to the steps given above, there are a few other important things you want to keep in mind when it comes to how to write a research paper title, regarding formatting, word count, and content:

Write the title after you’ve written your paper and abstract
Include all of the essential terms in your paper
Keep it short and to the point (~16 words or fewer)
Avoid unnecessary jargon and abbreviations
Use keywords that capture the content of your paper
Never include a period at the end—your title is NOT a sentence

Research Paper Writing Resources

We hope this article has been helpful in teaching you how to craft your research paper title. But you might still want to dig deeper into different journal title formats and categories that might be more suitable for specific article types or need help with writing a cover letter for your manuscript submission.

In addition to getting English proofreading services , including paper editing services , before submission to journals, be sure to visit our academic resources papers. Here you can find dozens of articles on manuscript writing, from drafting an outline to finding a target journal to submit to.

Academia.edu no longer supports Internet Explorer.

To browse Academia.edu and the wider internet faster and more securely, please take a few seconds to upgrade your browser .

Enter the email address you signed up with and we'll email you a reset link.

We're Hiring!
Help Center

SAMPLE THESIS TITLE WITH A CONCISE AND ACCURATE DESCRIPTION THAT INCLUDES KEY WORDS AND AVOIDS USING SCIENTIFIC FORMULAS

Related Papers

JOSE EDUARDO BARRERA LOPEZ

Phuc.CX4074 2004074

Wan Abdullah

Dazelle Anne Lizada

Journal of Computational Biology

Rohit Singh

Dhansy Naidu

Lian Tze Lim

Credits to the owner

An Amazing Collection Of 20 Thesis Paper Title Examples

Sometimes the most difficult part of writing a research paper is picking your thesis. Are you looking for a thesis paper title that will grab your reader’s attention? You definitely want your professor’s first impression to be a positive one. Hot topics and headlines are always a great place to look for your inspiration. Your thesis should always attempt to solve a real problem and contain existing research on the topic. You want to aim for a conclusion at which no one has arrived at prior to your report.

We have compiled an amazing collection of 20 thesis paper title examples that are sure to make people stop and read.

Should the United States implement a uniform National Healthcare Plan?
Are children being unnecessarily overmedicated? What is the reason behind this trend?
Should there be mandatory drug testing for all welfare recipients? Should recipients automatically lose benefits for failed tests?
Should all law enforcement be required to wear body cams?
Should juveniles be charged as adults for certain crimes?
Should abortion be allowed for unborn children of violence/rape?
What is the parent responsibility vs. school responsibility for on-campus violence?
Are today’s children smarter due to the internet?
Should immoral advertisements be banned from TV?
Should the government have control over what types of food are served to children in public schools?
How has the legalization of marijuana impacted Colorado’s economy?
Should cigarette smoking be banned in all public places?
Should it be illegal to use animals for medical studies?
Should college athletes be paid?
Is censorship needed on the internet?
Should public schools do away with standardized testing?
What is the correlation between fracking and earthquakes?
Should women who kill abusive husbands/partners be punished for their murder?
Do gays, lesbians, bisexuals, or transgender people need special rights for protection?
Has there been a rise in sexual assaults on college campuses in recent years?

This is a project that you will be spending quite a bit of time working on; make sure you pick a topic that will keep you interested throughout. If you are really passionate about an issue, this would be a good way to present your argument. Picking a topic that relates to you will make this task go smoothly.

Indian psychology
HR management
Expert essay writers for hire

Get More Ideas

Plan. Structure. Write. Edit.

Organize Your Process

Format your paper, improve your skills, revise your paper.

1 Shields Ave, Davis,

United States

+1 444-348-5800

[email protected]

+49 621 123 321 124

Attention! Your ePaper is waiting for publication!

By publishing your document, the content will be optimally indexed by Google via AI and sorted into the right category for over 500 million ePaper readers on YUMPU.

This will ensure high visibility and many readers!

Your ePaper is now published and live on YUMPU!

You can find your publication here:

Share your interactive ePaper on all platforms and on your website with our embed function

sample thesis title with a concise and accurate description

description
circle.ubc.ca

Create successful ePaper yourself

Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.

FUNCTION ORIENTED SYNTHESIS OF BIOACTIVE MARINE NATURAL

PRODUCTS AND THEIR PHARMACOPHORE ANALOGUES

by

Labros G. Meimetis

B.Sc., Simon Fraser University, 2006

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

Doctor of Philosophy

in

THE FACULTY OF GRADUATE STUDIES

(Chemistry)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

July 2012

Abstract

Natural products play a central role in drug discovery. The Andersen lab focuses its

efforts on the isolation and structure elucidation of compounds from the marine environment.

Many of these compounds possess biological activity, and often their total synthesis is

undertaken, to provide structure-activity relationship (SAR) studies for new pharmacophores,

and to provide material to probe in vivo biological effects. When possible, small molecule

probes are designed based on the structure of the natural product, to provide insight into the

mechanism of interaction between the active compound and its biological target. Several

projects probing the biological activities of natural products and their analogues by synthesis

are detailed in this thesis

The second chapter describes the construction of water-soluble activators of SHIP1, a

phosphatase that is a negative regulator of the PI3K signal transduction pathway in

hematopoietic cells. The structure of a known SHIP1 activator (2.18) was used to develop the

water-soluble analogues 2.20 and 2.42 in order to enhance the drug-like properties of 2.18.

The third chapter describes the total synthesis of two novel marine natural products,

(S)-niphatenone A (3.23) and B (3.35). Both compounds inhibit transcriptional activity in

prostate cancer cells. Their total synthesis was completed to verify their proposed structures

and to supply material for biological testing. Several analogues of (R)-niphatenone B (3.31)

were constructed providing a clear SAR for the natural product.

The fourth chapter describes a terpenoid natural product 4.1, which was found to be

an antagonist of the androgen receptor. Using lead compound 4.1, a semisynthetic SAR study

was completed and it was determined that analogue 4.4 has enhanced potency relative to 4.1.

Synthetic attempts to construct 4.4 analogues by an epoxide-initiated cascade are described.

The fifth chapter describes synthetic efforts towards the novel peptide-aldehyde

natural product lichostatinal (5.4). Lichostatinal (5.4) is a potent cathepsin K inhibitor. Its

total synthesis would provide material to support structure elucidation efforts and biological

testing.

Preface

Chapter 2 is based on work done at UBC, the BC Cancer Agency, and Aquinox

Pharmaceuticals Inc. The author is responsible for the synthesis and characterization of all

2.18 analogues. In addition to the author, Dr. Matt Nodwell and Dr. Chelsea Wang

constructed 2.20/2.34/2.42/2.43 for biological testing. Single crystal X-ray diffraction

analysis was carried out by Dr. Brian O. Patrick. STDD NOE NMR experiments were run by

Dr. David Williams. The SHIP1 phosphatase assay screening was done by Andrew Ming-

Lum under the guidance of Dr. Alice-F. Mui at the BC Cancer Agency. The solubility of 2.18

analogues, the AKT phosphorylation inhibition assay, some measurements of SHIP1

enzymatic assay, and the in vivo PCA model was completed by Aquinox Pharmaceuticals

Inc.

The work in chapter 3 has been published: Meimetis, Labros G.; Williams David E.;

Mawji, Nasrin R.; Banuelos, Adriana.; Lal, Aaron A.; Park, Jacob J.; de Voogdt, Nicole J.;

Fernandez J.C.; Sadar, Marianne D.; and Andersen, Raymond J. Niphatenones A and B,

glycerol ethers from the sponge Niphates Digitalis block androgen receptor transcriptional

activity in prostate cancer cells: structure elucidation, synthesis, and biological activity. J.

http://pubs.acs.org/doi/pdf/10.1021/jm2014056. The author was responsible for writing the

experimental section as it relates to all synthetic compounds. The author is responsible for

the construction and characterization of all synthetic compounds. Dr. David Williams is

responsible for the isolation and structure determination of niphatenones A (3.8) and B (3.9).

Nasrin R. Mawji, Adriana Banuelos, Aaron A. Lal, and Jacob J. Park under the leadership of

Dr. Marianne D. Sadar at the BC Cancer Agency were responsible for generating all of the

iological data and “click” chemistry experimental data, in addition to Dr. Javier Garcia who

provided insight for the “click” chemistry experiments. The potential fluorescent probe 3.78

was completed as a collaboration between Dr. Javier Garcia and the author.

Chapter 4 is work done at UBC, and the BC Cancer Agency. The lead compound

(4.1) along with its subsequent hydrogenation product (4.2) was isolated and characterized by

Dr. Gavin Carr. All other semisynthetic analogues, in addition to the synthetic study towards

analogues of the natural product 4.4 was completed by the author. All biological data was

acquired by Kevin Yang under the leadership of Dr. Marianne Sadar at the BC Cancer

Agency.

Chapter 5 is work done at UBC. The isolation of lichostatinal (5.4) was completed by

Vincent Paul Lavalee under the supervision of Dr. Dieter Bromme, using an actinomycete

culture extract provided by the laboratory of Dr. Julian Davies. All synthetic compounds

were constructed and characterized by the author.

Table of Contents

Abstract .................................................................................................................................... ii

Preface ..................................................................................................................................... iv

Table of Contents ................................................................................................................... vi

List of Tables .......................................................................................................................... ix

List of Figures .......................................................................................................................... x

List of Schemes ...................................................................................................................... xx

List of Symbols and Abbreviations .................................................................................. xxiii

Acknowledgements ........................................................................................................... xxvii

Dedication ......................................................................................................................... xxviii

Chapter 1: Marine Natural Products From Benchtop to Bedside .................................... 1

1.1 Nature as a Source of Medicine ................................................................................ 1

1.2 Synthesis as an Aid to Structure Elucidation ............................................................ 6

1.3 Translating a Drug-Lead Into a Drug by Structure Activity Relationship (SAR)

Studies ……………………………………………………………………………………..10

1.4 Bioactive Marine Natural Products Reveal Novel Mechanism of Action .............. 13

1.5 Scope of Thesis ....................................................................................................... 14

Chapter 2: Synthesis and Biological Evaluation of SHIP1 Activators ........................... 16

2.1 Inhibition of PI3K Signaling by Activation of SHIP1 ............................................ 16

2.2 First Generation SHIP1 Agonist Pelorol and Analogues ........................................ 18

2.3 Synthesis of SHIP1 Activators Using a Polyene-Initiated Cationic Cascade ......... 21

2.4 Synthesis of Water Soluble Analogues to Facilitate Drug Administration ............ 25

2.5 Saturation Transfer Difference (STD) Spectroscopy NMR.................................... 38

2.6 Synthesis of a Benzophenone Photoaffinity Probe ................................................. 43

2.7 Biological Results ................................................................................................... 51

2.8 Conclusion .............................................................................................................. 57

2.9 Experimental ........................................................................................................... 60

Chapter 3: Glycerol Ethers from the Sponge Niphates digitalis that Block Androgen

Receptor Transcriptional Activity in Prostate Cancer Cells .......................................... 101

3.1 Castration Recurrent Prostate Cancer (CRPC) ..................................................... 101

3.2 The AR NTD as a Novel Therapeutic Target for Treating CRPC ........................ 103

3.3 Synthetic Analogues of (R)-Niphatenone B (3.31) ............................................... 111

3.4 Synthesis of a Click Chemistry Probe and Fluorescent Probe .............................. 117

3.5 Biological Results ................................................................................................. 122

3.6 Conclusion ............................................................................................................ 126

3.7 Experimental ......................................................................................................... 128

Chapter 4: Synthetic Efforts Towards a Novel AR Antagonist (4.1) ............................ 203

4.1 Terpene AR Antagonist (4.1) ................................................................................ 203

4.2 Epoxide-Initiated Cationic Cascade Towards AR Antagonist 4.6 ........................ 207

4.3 Furan Blocking Group as a Strategy to Construct Desired Regioisomer 4.24 ..... 211

4.4 Biological Results ................................................................................................. 217

4.5 Conclusion ............................................................................................................ 220

4.6 Experimental ......................................................................................................... 222

Chapter 5: Synthetic Efforts Towards Lichostatinal (5.4): A Potent Cathepsin K

Inhibitor ............................................................................................................................... 249

5.1 Cysteine Protease Inhibitors ................................................................................. 249

5.2 Lichostatinal (5.4) a Novel Peptide-Aldehyde Inhibitor of Cathepsin K ............. 252

5.3 Conclusion ............................................................................................................ 262

5.4 Experimental ......................................................................................................... 264

Chapter 6: Conclusion ....................................................................................................... 301

6.1 Biological and Pharmacokinetic Evaluation for Water Soluble SHIP1 Activators

……………………………………………………………………………………301

6.2 Additional Biological Evaluations of the Niphatenones and their Analogues ..... 302

6.3 Future Synthetic Strategies Towards Terpene 4.4: An LBD AR Antagonist ....... 303

6.4 Alternative Synthetic Strategy Towards Lichostatinal (5.4) ................................. 307

References ............................................................................................................................ 309

Appendix A: X-Ray Structure Reports............................................................................. 326

A.1 Compound 2.30 ..................................................................................................... 326

A.2 Compound 2.32 ..................................................................................................... 330

A.3 Compound 2.42 ..................................................................................................... 334

List of Tables

Table 1.1 The properties of natural products compared with drugs, and synthetics. ............... 3

Table 2.1 Solubilities of pelorol (2.1) analogues. ................................................................... 53

Table 2.2 AKT phosphorylation inhibition of racemic amine mixture 2.20/2.42. ................. 53

Table 2.3 Activation of SHIP1 enzyme. ................................................................................. 54

Table 4.1 EC50 values for tested compounds in an AR competitor in vitro assay. ............... 219

Table 5.1 Results of arginol (5.24) oxidation model study................................................... 258

Table 5.2 Results of an O-benzyl deprotection model study of intermediate 5.15. .............. 260

List of Figures

Figure 1.1 Bioactive compounds of plants commonly used throughout human history. ......... 1

Figure 1.2 Inhibitors of the 20S proteasome salinosporamide A (1.5) and omuralide (1.6). ... 4

Figure 1.3 Salinosporamide A (1.5) mechanism of action. ...................................................... 4

Figure 1.4 Marine natural product identification at the nM scale............................................. 7

Figure 1.5 Determination of the absolute configuration of muironolide A (1.11) using

chemical degradation. ............................................................................................................... 8

Figure 1.6 Family of imidazole marine natural products. ......................................................... 9

Figure 1.7 Barans retrosynthesis of (±)-palau’amine (1.22). .................................................. 10

Figure 1.8 The antimitotic marine natural product (+)-spongistatin 1 (1.27). ........................ 11

Figure 1.9 A pharmacophore analogue (1.28) of (+)-spongistatin 1 (1.27). ........................... 12

Figure 1.10 A marine peptide with novel biological activity. ................................................ 13

Figure 2.1 The PI3K pathway. ................................................................................................ 16

Figure 2.2 The first selective SHIP1 activator marine natural product pelorol (2.1). ............ 18

Figure 2.3 Pelorol (2.1) analogue 2.2. .................................................................................... 19

Figure 2.4 Chemical and enzymatic oxidation of catechol and subsequent 1,6-addition. ...... 20

Figure 2.5 Analogue 2.3 without the catechol functionality. .................................................. 20

Figure 2.6 Natural products made by biomimetically-inspired syntheses. ............................. 22

Figure 2.7 Carbocation generation .......................................................................................... 22

Figure 2.8 Typical propagation and termination of a polyene cascade. ................................. 23

Figure 2.9 Water soluble prodrug analogues of 2.3. ............................................................... 26

Figure 2.10 Analogue 2.18...................................................................................................... 27

Figure 2.11 Water soluble analogue design. ........................................................................... 27

Figure 2.12 CLogP values of 2.18 and related analogues. ...................................................... 28

Figure 2.13 ORTEP diagram of Mosher ester 2.32. ............................................................... 33

Figure 2.14 ORTEP diagram of alcohol 2.30. ........................................................................ 33

Figure 2.15 Analogues 2.42 and 2.43 constructed using a D-fructose derived Shi catalyst. .. 35

Figure 2.16 ORTEP diagram of racemic amine mixture 2.20/2.42. ....................................... 36

Figure 2.17 STD NMR. .......................................................................................................... 39

Figure 2.18 STDD NMR sample preparation. ........................................................................ 41

Figure 2.19 STDD NOE NMR of 2.42. .................................................................................. 42

Figure 2.20 Components of a photoaffinity probe. ................................................................. 43

Figure 2.21 Examples of photoreactive groups. ..................................................................... 44

Figure 2.22 Commonly used tags. .......................................................................................... 45

Figure 2.23 Examples of photoaffinity probes. ...................................................................... 46

Figure 2.24 Principle of 2.3 based photoaffinity probe (2.53). ............................................... 47

Figure 2.25 SHIP1 phosphatase assay. ................................................................................... 52

Figure 2.26 Efficacy of 2.20/2.42 versus cyproheptadine (Cyp, 1 mg/kg) in a mouse (PCA)

model....................................................................................................................................... 56

Figure 2.27 1 H and 13 C NMR spectra of 2.12 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 62

Figure 2.28 1 H and 13 C NMR spectra of 2.28 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 65

Figure 2.29 1 H and 13 C NMR spectra of 2.26 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 68

Figure 2.30 1 H and 13 C NMR spectra of 2.30 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 70

Figure 2.31 1 H and 13 C NMR spectra of 2.31 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 73

Figure 2.32 1 H and 13 C NMR spectra of 2.32 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 75

Figure 2.33 1 H and 13 C NMR spectra of 2.33 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 77

Figure 2.34 1 H and 13 C NMR spectra of 2.19 recorded in (CD3)2CO at 400 MHz and 100

MHz respectively. ................................................................................................................... 79

Figure 2.35 1 H and 13 C NMR spectra of 2.20 recorded in CD3OD at 600 MHz and 150 MHz

respectively. ............................................................................................................................ 82

Figure 2.36 1 H and 13 C NMR spectra of 2.34 recorded in CD3OD at 600 MHz and 150 MHz

respectively. ............................................................................................................................ 83

Figure 2.37 1 H and 13 C NMR spectra of 2.40 recorded in (CD3)2CO at 400 MHz and 100

MHz respectively. ................................................................................................................... 86

Figure 2.38 1 H and 13 C NMR spectra of 2.44 recorded in CD3OD at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 88

Figure 2.39 1 H and 13 C NMR spectra of 2.45 recorded in CD3OD at 600 MHz and 150 MHz

respectively. ............................................................................................................................ 90

Figure 2.40 1 H and 13 C NMR spectra of 2.61 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 94

Figure 2.41 1 H and 13 C NMR spectra of 2.53 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. ............................................................................................................................ 96

Figure 2.42 NOESY spectrum of 2.53 in CDCl3 at 400 MHz. ............................................... 97

Figure 3.1 Endogenous androgens found in humans. ........................................................... 101

Figure 3.2 Examples of antiandrogens.................................................................................. 103

Figure 3.3 Androgen receptor structure. ............................................................................... 104

Figure 3.4 Small molecule antagonists of the AR NTD. ...................................................... 105

Figure 3.5 Niphatenones A (3.8) and B (3.9). ...................................................................... 105

Figure 3.6 Ceratodictyol A (3.36) and B (3.37). ................................................................... 111

Figure 3.7 Proposed SAR of (R)-niphatenone B (3.31). ....................................................... 111

Figure 3.8 Fluorescent probe mode of action. ...................................................................... 118

Figure 3.9 Non-targeting and targeting fluorescent probes. ................................................. 119

Figure 3.10 Cellular imaging agent of glutathione. .............................................................. 121

Figure 3.11 AR transcriptional activity assay of the niphatenones and their analogues. ..... 123

Figure 3.12 Androgen induced proliferation assay. .............................................................. 125

Figure 3.13 Binding between alkyne probe 3.67 and the NTD AF1 region of the AR. ....... 126

Figure 3.14 1 H and 13 C NMR spectra of 3.16 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 130

Figure 3.15 1 H and 13 C NMR spectra of 3.17 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 132

Figure 3.16 1 H and 13 C NMR spectra of 3.18 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 134

Figure 3.17 1 H and 13 C NMR spectra of 3.20 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 137

Figure 3.18 1 H and 13 C NMR spectra of 3.21 recorded in C6D6 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 139

Figure 3.19 1 H and 13 C NMR spectra of 3.27 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 142

Figure 3.20 1 H and 13 C NMR spectra of 3.28 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 144

Figure 3.21 1 H and 13 C NMR spectra of 3.29 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 146

Figure 3.22 1 H and 13 C NMR spectra of 3.30 recorded in C6D6 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 148

Figure 3.23 1 H and 13 C NMR spectra of 3.31 recorded in C6D6 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 150

Figure 3.24 1 H and 13 C NMR spectra of 3.33 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 152

Figure 3.25 1 H and 13 C NMR spectra of 3.34 recorded in CDCl3 at 300 MHz and 75 MHz

respectively. .......................................................................................................................... 154

Figure 3.26 1 H and 13 C NMR spectra of 3.25 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 156

Figure 3.27 1 H and 13 C NMR spectra of 3.62 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 158

Figure 3.28 1 H and 13 C NMR spectra of 3.63 recorded in C6D6 at 400 MHz and 75 MHz

respectively. .......................................................................................................................... 160

Figure 3.29 1 H and 13 C NMR spectra of 3.41 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 162

Figure 3.30 1 H and 13 C NMR spectra of 3.42 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 164

Figure 3.31 1 H and 13 C NMR spectra of 3.43 recorded in CD2Cl2 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 166

Figure 3.32 1 H and 13 C NMR spectra of 3.45 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 168

Figure 3.33 1 H and 13 C NMR spectra of 3.46 recorded in C6D6 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 170

Figure 3.34 1 H and 13 C NMR spectra of 3.48 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 172

Figure 3.35 1 H and 13 C NMR spectra of 3.49 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 174

Figure 3.36 1 H and 13 C NMR spectra of 3.50 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 177

Figure 3.37 1 H and 13 C NMR spectra of 3.52 recorded in C6D6 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 179

Figure 3.38 1 H and 13 C NMR spectra of 3.53 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 181

Figure 3.39 1 H and 13 C NMR spectra of 3.54 recorded in CD2Cl2 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 183

Figure 3.40 1 H and 13 C NMR spectra of 3.57 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 186

Figure 3.41 1 H and 13 C NMR spectra of 3.58 recorded in CDCl3 at 400 MHz and 150 MHz

respectively. .......................................................................................................................... 188

Figure 3.42 1 H and 13 C NMR spectra of 3.59 recorded in CDCl3 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 190

Figure 3.43 1 H and 13 C NMR spectra of 3.60 recorded in CDCl3 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 192

Figure 3.44 1 H and 13 C NMR spectra of 3.61 recorded in CDCl3 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 194

Figure 3.45 1 H and 13 C NMR spectra of 3.67 recorded in CDCl3 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 196

Figure 3.46 1 H and 13 C NMR spectra of 3.77 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 198

Figure 3.47 1 H and 13 C NMR spectra of 3.78 recorded in CD2Cl2 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 200

Figure 4.1 AR antagonist lead compound 4.1....................................................................... 203

Figure 4.2 Structural similarity between 4.1 and a steroid carbon skeleton. ........................ 204

Figure 4.3 Delocalization of charge in a furan ring system. ................................................. 207

Figure 4.4 Favored and disfavored regioisomers. ................................................................. 211

Figure 4.5 Blocking group to construct A-ring analogues of 4.4. ........................................ 211

Figure 4.6 AR competitor in vitro assay. .............................................................................. 218

Figure 4.7 1 H and 13 C NMR spectra of 4.16 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 224

Figure 4.8 1 H and 13 C NMR spectra of 4.18 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 226

Figure 4.9 1 H and 13 C NMR spectra of 4.19 recorded in CDCl3 at 300 MHz and 75 MHz

respectively. .......................................................................................................................... 228

Figure 4.10 1 H and 13 C NMR spectra of 4.20 recorded in CDCl3 at 300 MHz and 75 MHz

respectively. .......................................................................................................................... 230

Figure 4.11 1 H and 13 C NMR spectra of 4.21 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 232

Figure 4.12 1 H and 13 C NMR spectra of 4.22 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 234

Figure 4.13 1 H and 13 C NMR spectra of 4.23 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 236

Figure 4.14 1 H spectrum of 4.24 recorded in CDCl3 at 400 MHz. ....................................... 238

Figure 4.15 1 H and 13 C NMR spectra of 4.40 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 240

Figure 4.16 1 H and 13 C NMR spectra of 4.42 recorded in CD2Cl2 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 243

Figure 4.17 1 H and 13 C NMR spectra of 4.43 recorded in CDCl3 at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 245

Figure 4.18 1 H and 13 C NMR spectra of 4.44 recorded in CDCl3 at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 247

Figure 5.1 Hydrolysis of a peptide by a cysteine protease. .................................................. 249

Figure 5.2 Peptide-aldehyde inhibitors of cysteine proteases. .............................................. 251

Figure 5.3 Covalent binding mechanism between a peptide-aldehyde natural product and a

cysteine protease. .................................................................................................................. 252

Figure 5.4 Novel peptide-aldehyde lichostatinal (5.4). ......................................................... 253

Figure 5.5 1 H and 13 C NMR spectra of 5.12 recorded in (CD3)2SO at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 266

Figure 5.6 1 H and 13 C NMR spectra of 5.13 recorded in D2O at 400 MHz and 100 MHz

respectively. .......................................................................................................................... 268

Figure 5.7 1 H and 13 C NMR spectra of 5.15 recorded in (CD3)2SO at 600 MHz and CD2Cl2 at

150 MHz respectively. .......................................................................................................... 270

Figure 5.8 1 H and 13 C NMR spectra of 5.16 recorded in (CD3)2SO at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 272

Figure 5.9 1 H and 13 C NMR spectra of 5.18 recorded in (CD3)2SO at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 275

Figure 5.10 1 H and 13 C NMR spectra of 5.20 recorded in (CD3)2SO at 600 MHz and 150

MHz respectively. ................................................................................................................. 278

Figure 5.11 1 H and 13 C NMR spectra of 5.24 recorded in (CD3)2SO at 600 MHz and 150

MHz respectively. ................................................................................................................. 280

Figure 5.12 1 H and 13 C NMR spectra of 5.29 recorded in CD2Cl2 at 600 MHz and 100 MHz

respectively. .......................................................................................................................... 282

Figure 5.13 1 H and 13 C NMR spectra of 5.30 recorded in (CD3)2SO at 600 MHz and 150

MHz respectively. ................................................................................................................. 284

Figure 5.14 1 H and 13 C NMR spectra of 5.32 recorded in (CD3)2SO at 600 MHz and 150

MHz respectively. ................................................................................................................. 286

Figure 5.15 1 H and 13 C NMR spectra of 5.33 recorded in D2O and (CD3)2SO at 400 MHz and

100 MHz respectively. .......................................................................................................... 288

Figure 5.16 1 H and 13 C NMR spectra of 5.35 recorded in CD3OD at 400 MHz and (CD3)2SO

at 150 MHz respectively. ...................................................................................................... 290

Figure 5.17 1 H and 13 C NMR spectra of 5.36 recorded in CD3OD at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 292

Figure 5.18 1 H and 13 C NMR spectra of 5.37 recorded in CD3OD at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 294

Figure 5.19 1 H and 13 C NMR spectra of 5.38 recorded in (CD3)2SO at 600 MHz and 150

MHz respectively. ................................................................................................................. 296

Figure 5.20 1 H and 13 C NMR spectra of 5.39 recorded in CD3OD at 600 MHz and 150 MHz

respectively. .......................................................................................................................... 298

Figure 5.21 1 H and 13 C NMR spectra of 5.40 recorded in (CD3)2SO at 600 MHz and 150

MHz respectively. ................................................................................................................. 300

Figure 6.1 Alternative route to lichostatinal (5.4). ................................................................ 308

List of Schemes

Scheme 2.1 Synthetic route to drug lead 2.3. ......................................................................... 21

Scheme 2.2 Retrosynthetic analysis of 2.3. ............................................................................ 24

Scheme 2.3 Synthesis of (±)-2.3. ............................................................................................ 24

Scheme 2.4 Elimination side products of chlorosulfonic acid cyclization. ............................ 25

Scheme 2.5 Inspiration for biomimetic synthesis: steroid biosynthesis from squalene (2.21).

................................................................................................................................................. 29

Scheme 2.6 Retrosynthetic analysis of 2.20. .......................................................................... 30

Scheme 2.7 Synthesis of amine analogues 2.20 and 2.34. ...................................................... 31

Scheme 2.8 Examples of indium tribromide promoted annulation methodology .................. 32

Scheme 2.9 Alternative amination strategies towards 2.20 and 2.34. .................................... 34

Scheme 2.10 Racemic synthesis of (±)-2.20 using mCPBA. .................................................. 36

Scheme 2.11 Polar analogues of 2.18. .................................................................................... 37

Scheme 2.12 Synthesis of the 2.18 enantiomer (2.48). ........................................................... 38

Scheme 2.13 Retrosynthetic analysis of 2.53. ........................................................................ 48

Scheme 2.14 Benzophenone photoaffinity probe (2.53) synthesis. ........................................ 49

Scheme 2.15 Fries rearrangement and NOE observance in 2.53. ........................................... 50

Scheme 2.16 Rational drug design from a marine natural product (2.1) to drug lead

(2.20/2.42). .............................................................................................................................. 60

Scheme 3.1 Retrosynthetic analysis of niphatenone A (3.8). ............................................... 106

Scheme 3.2 Synthesis of (R)-niphatenone A (3.21). ............................................................. 107

Scheme 3.3 Synthesis of (S)-niphatenone A (3.23). ............................................................. 108

Scheme 3.4 Retrosynthesis of niphatenone B (3.9). ............................................................. 108

Scheme 3.5 Synthesis of (R)-niphatenone B (3.31). ............................................................. 109

Scheme 3.6 Synthesis of (S)-niphatenone B (3.35). ............................................................. 110

Scheme 3.7 Synthesis of a long chain (R)-niphatenone B (3.31) analogue 3.43. ................. 112

Scheme 3.8 Synthesis of a short chain (R)-niphatenone B (3.31) analogue 3.46. ................ 113

Scheme 3.9 (R)-Niphatenone B (3.31) glycerol analogues. .................................................. 114

Scheme 3.10 All carbon backbone (R)-niphatenone B (3.31) analogue 3.61. ...................... 115

Scheme 3.11 Dihydro (R)-niphatenone B (3.31) analogue 3.63. .......................................... 116

Scheme 3.12 Propargyl ether analogue 3.67. ........................................................................ 119

Scheme 3.13 Click chemistry of probe 3.67 and fluorophore 3.69. ..................................... 120

Scheme 3.14 Synthesis of a potential AR NTD fluorescent probe (3.78). ........................... 122

Scheme 4.1 Semisynthesis of 4.1 analogues. ........................................................................ 205

Scheme 4.2 Zoretic’s synthesis of an A-ring 4.4 analogue (4.6). ......................................... 206

Scheme 4.3 Retrosynthesis of a 4.4 A-ring analogue 4.6. .................................................... 206

Scheme 4.4 Synthesis of furanyl isoprene 4.13. ................................................................... 208

Scheme 4.5 Synthesis of regioisomer 4.24. .......................................................................... 209

Scheme 4.6 Goldsmiths furan C-2 silylation strategy. ......................................................... 212

Scheme 4.7 Failed selective TMS deprotection. ................................................................... 212

Scheme 4.8 Synthetic attempt towards C-2 silylated intermediate 4.35. .............................. 213

Scheme 4.9 Attempt at constructing thiolated analogue 4.38. .............................................. 214

Scheme 4.10 Mono-thiolated analogues of 4.12. .................................................................. 215

Scheme 4.11 Di-thiolated analogues of 4.12. ....................................................................... 215

Scheme 4.12 Epoxide-initiated cationic cascade using a thiol blocking group. ................... 216

Scheme 5.1 Retrosynthetic analysis of lichostatinal (5.4). ................................................... 254

Scheme 5.2 Synthesis toward lichostatinal (5.4). ................................................................. 255

Scheme 5.3 Ito’s reduction of Cbz protected arginine 5.22. ................................................. 257

Scheme 5.4 Oxidation of Arginol (5.24). ............................................................................. 258

Scheme 5.5 Synthesis towards lichostatinal (5.4). ................................................................ 259

Scheme 5.6 Alternative route to lichostatinal (5.4). ............................................................. 261

Scheme 6.1 Syntheses towards polar analogues of 4.4. ........................................................ 304

Scheme 6.2 Lithiation strategy to construct A-ring analogues of 4.4. .................................. 305

Scheme 6.3 Deprotection of regiosisomer 4.24. ................................................................... 306

Scheme 6.4 Alternative synthesis of regioisomer (±)-6.9. .................................................... 307

List of Symbols and Abbreviations

��

- degree(s)

% - percent

(�) - racemic

AcOH - acetic acid

AF1 - activation function 1

AIBN - azobisisobutyronitrile

AKT - protein kinase B

Ala - alanine

AR - androgen receptor

Asp - aspartic acid

br - broad

Bn - benzyl

Boc - t-butoxycarbonyl

BRSM - based on recovered starting material

Bu - butyl

n-BuLi - n-butyllithium

t-BuLi - t-butyllithium

�C - degrees Celsius

15C-5 - 1,4,7,10,13-pentaoxacyclopentadecane

18C-6 - 1,4,7,10,13,16-hexaoxacyclooctadecane

CA - California

calcd - calculated

Cbz - carboxybenzyl

CDI - carbonyldiimidazole

CLogP - calculated octanol/water partition coefficient

COSY - two-dimensional correlation spectroscopy

CRPC - castrate recurrent prostate cancer

Cyp - cyproheptadine

Cys - cysteine

� - chemical shift in parts per million

d - doublet

DBU - 1,8-diazabicyclo[5.4.0]undec-7-ene

DCM - dichloromethane

dd - doublet of doublets

DHEA - dehydroepiandrosterone

DIBAL-H - diisobutylaluminium hydride

DIEA - N,N-diisopropylethylamine

DIPT - diisopropyl tartrate

DMAP - 4-dimethylaminopyridine

DMF - N,N-dimethylformamide

DMP - Dess–Martin periodinane

DMS - dimethyl sulfide

DMSO - dimethyl sulfoxide

DNA - deoxyribonucleic acid

DNP - dinitrophenyl

DOM - directed ortho metalation

DPBS - Dulbecco’s phosphate buffered saline

dppp - 1,3-bis(diphenylphosphino)propane

dt - doublet of triplets

DTT - dithiothreitol

ED50

- effective dose

eqiv. - equivalent(s)

EIMS - electron impact mass spectrometry

ESIMS - electrospray ionization mass spectrometry

Et - ethyl

EtOAc - ethyl acetate

EtOH - ethanol

FBS - fetal bovine serum

FMOC - fluorenylmethyloxycarbonyl

g - gram(s)

Gly - glycine

GSH - glutathione

h - hour(s)

HATU - (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate)

HCT-116 - human carcinoma cell line

HPLC - high-performance liquid chromatography

HRESIMS - high resolution electrospray mass spectrometry

HRP - horseradish peroxidase

HSA - human serum albumin

HTS - high throughput screening

HWE - Horner–Wadsworth–Emmons

Hz - hertz

IBX - 2-iodoxybenzoic acid

IC50

- median inhibitory concentration

IgE - immunoglobulin E

IgG - immunoglobulin G

IL-1 - interleukin-1

IL-6 - interleukin-6

J - coupling constant in hertz

Kb - kilo-base pair

Kg - kilogram

KHMDS - potassium bis(trimethylsilyl)amide

L - levorotatory

lb. - pound

LBD - ligand binding domain

LCMS - liquid-chromatography mass spectrometry

LDA - lithium diisopropyl amine

Leu - leucine

LPS - lipopolysaccharide

Lys - lysine

m - multiplet

M - molar concentration

mCPBA - meta-chloroperbenzoic acid

Me - methyl

MeCN - acetonitrile

MeOH - methanol

Met - methionine

mg - milligram(s)

MHz - megahertz

min - minute

mL - milliliter(s)

mm - millimeter(s)

mM - millimolar

MM - multiple myeloma

mmol - millimol(s)

µl - microliter

�M - micromolar

mRNA - messenger ribonucleic acid

MS - mass spectrometry

MTPA - α-methoxy-α-trifluoromethylphenylacetic acid

nM - nanomolar

NCS - N-chlorosuccinimide

ng - nanogram

nm - nanometers

NMR - nuclear magnetic resonance

NOE - nuclear overhauser enhancement

NOESY - nuclear overhauser effect spectroscopy

NTD - n-terminal domain

OSC - oxidosqualene cyclase

PCC - pyridinium chlorochromate

PCR - polymerase chain reaction

PBS - phosphate buffered saline

PEG - poly(ethylene)glycol

pH - -log10[H + ]

Ph - phenyl

PIP3

- phosphatidylinositol-3,4,5-triphosphate

PIP2

- phosphatidylinositol-4,5-biphosphate

PI3K - phosphatidylinositol-3-kinase

ppm - parts per million

i-Pr - isopropyl group

PSA - prostate specific antigen

PtdIns(4,5)P2 - phosphatidylinositol-4,5-biphosphate

PtdIns(3,4,5)P3 - phosphatidylinositol-3,4,5-triphosphate

PtdIns(3,4)P2 - phosphatidylinositol-3,4-biphosphate

PTEN - phosphatase and tensin homologue

Pyr - pyridine

q - quartet

RF - radio frequency

rpm - revolutions per minute

rt - room temperature

s - singlet

SAR - structure-activity relationship

SCUBA - self-contained underwater breathing apparatus

SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE - squalene epoxidase

Ser - serine

SHC - squalene hopene cyclase

SHIP - src homology 2-containing Inositol 5’-phosphatase

sSHIP - stem cell src homology 2-containing Inositol 5’-phosphatase

SNAr - addition-elimination

sp. - species

STD - saturation transfer difference

STDD - saturation transfer double difference

t - triplet

TBA - tetrabutylammonium

TBDPSCl - tert-butylchlorodiphenylsilane

TDPQ - 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]

quinoline

TEA - triethylamine

TEMPO - (2,2,6,6-tetramethylpiperidin-1-yl)oxyl

TFA - trifluoroacetic acid

TFAA - trifluoroacetic anhydride

THF - tetrahydrofuran

Thr - threonine

TIPS - triisopropylsilyl

TLC - thin-layer chromatography

TMS - trimethylsilyl

TNF - tumor necrosis factor

TOCSY - total correlation spectroscopy

tol - toluene

Tyr - tyrosine

ug - microgram

U.S.A. - United States of America

VCAM - vascular cell adhesion molecule

v/v - volume to volume

Acknowledgements

What a trip. First, I must thank the ones closest to me, for without them none of this

would be possible. I thank my beautiful wife Eszter for supporting my crazy and for being

the 50-foot tall woman in a 5’8” package. You Rock! Tentacles! Keep building that death

star! My parents for seeing me at my worst and still deciding to keep me instead of putting

me up for adoption. Thank you. I also thank all of my family in Greece, and the country

itself. Haters gonna hate!

I thank my supervisor Dr. Raymond Andersen for his guidance and support over the

years. The diversity of chemistry that I had the privilege to work on was amazing, and the

discoveries I made were thrilling. There is no challenge I cannot conquer and I have

Raymond to thank for that.

I thank Dr. David Williams who has played a role in all my projects, and to whom I

owe countless bags of chips. I thank Dr. Javier Garcia Fernandez for helping me make sense

of it all and for all the laughs. Chamon MoFo! I thank all of my biological collaborators at

UBC and the BC Cancer Agency. Dr. Glenn Sammis and Dr. Gregory Dake get thanks for

their helpful discussions over the years. A thank you goes to everybody that gathered data for

me in the NMR, MS, and X-ray labs, especially Maria Ezhova in the NMR lab. To all current

and past Andersen group members we laughed, we cried, we had a good time. Mike I

promise to take my silica over to the Chem department this time…let me just run this

column.

I have many people to thank at SFU for many reasons. Thanks goes to Dr. Robert

Britton for continuously supporting me from the time of broken glassware and chemical spill

cover-ups. To Matt, Jeff, and Pat. As I am writing this, I want to grab a few pints with you

guys. The sign of true friendship. You are all kings amongst men. Finally, in random order

(not really I hate you Bart) the pets…Bubbles, Hammy, Fermi, Kosta, Jammy, Julia, Puppy,

Smokey…Bart.

Lastly, I thank myself for all of my efforts.

‘Buy the ticket, take the ride…’

-Hunter S. Thompson

Dedication

To my parents George and Martha,

for all the car rides and sandwiches.

It’s Friday.

Chapter 1: Marine Natural Products From Benchtop to Bedside

1.1 Nature as a Source of Medicine

Mankind’s ability to harness nature as a source of medicine was a key step in the

evolutionary success of our species. In the early stages of human history, plants were the

main source of medicine. Phyta of different varieties were used for many purposes including

seduction, probing the boundaries of consciousness, pain relief, and even suicide. 1,2,3,4

The 20 th century brought about a new era of scientific enlightenment, allowing us to

probe the structure of these once esoteric biologically active molecules. No longer would

myth and folklore surround plants such as mint, peyote, willow, and hemlock, instead the

active components could now be isolated and identified (Figure 1.1). Furthermore, new

chemical entities could be classified as terpenes, 5 alkaloids, 6 polyketides, 7 and nonribosomal

peptides 8 based on their common structural motifs and putative biogenetic origins.

Figure 1.1 Bioactive compounds of plants commonly used throughout human history.

With the commercialization of SCUBA in the 1950’s, the shallow marine

environment could be routinely explored. This catalyzed a renaissance in the field of natural

products. It was realized that if bioactive compounds could be discovered from terrestrial

sources, then the marine environment should provide a plethora of novel compounds as well.

While this has held true over the years, translating bioactive marine drug leads from

benchtop to bedside has been an uphill battle. Over the past 25 years, the advent of high-

throughput screening 9 (HTS) facilitated rapid large-scale analysis of synthetic compounds in

a number of different assays. The most efficient automation systems currently employed test

up to 100,000 compounds a day, 10 with the most current research in HTS suggesting the

screening of 100 million reaction products in ten hours. 11 This development of HTS in the

last couple of decades resulted in compound supply lagging behind testing capabilities. This

stimulated the birth of combinatorial chemistry. 12,13 Combinatorial chemistry allows for the

rapid syntheses of different analogues with structural similarity. HTS proved too alluring for

many pharmaceutical companies in the 90’s, leading giants such as GlaxoSmithKline and

Pfizer to phase out screening of their natural product libraries. However, at the height of HTS

in 2001, there was a twenty year low 14 in the number of new chemical entities reaching the

market. As of 2012, the promise of HTS and combinatorial chemistry has yet to be realized.

The Achilles heel for combinatorial chemistry is its dependence on functional group

interconversion. This results in libraries of oligomers and peptides that, compared to natural

products, have limited chemical diversity. Roche 15 conducted a study in which trade drugs

(compounds with a trade name associated with them), were compared to a natural product

library and a purely synthetic compound library with no biological lineage (Table 1.1).

Approximately 10 % of drugs violated two or more of the Lipinski 16 “drug-like” guidelines

(Chapter 2, Section 2.4) compared to 12 % for natural products; in essence little difference.

Additionally, synthetics relied more on nitrogen, sulfur, and halogens, whereas natural

products contained more oxygen functional groups.

Table 1.1 The properties of natural products compared with drugs, and synthetics.

Natural Products Drugs Synthetics

Molecular weight 360-414 340-356 393

LogP 2.4-2.9 2.1-2.2 4.3

Number of chiral centers 3.2-6.2 1.2-2.3 0.1-0.4

Number of N atoms 0.84 1.64 2.69

Number of O atoms 5.9 4.03 2.77

% of rings that are aromatic 31 % 55 % 80 %

A significant difference 17 between natural products and synthetic compounds is that

natural products have more steric complexity and rigidity due to a large number of rings,

chiral centers, and bridgeheads. The origin of this spatial complexity is that natural products

have evolved to bind biological targets, which have a defined spatial orientation.

Furthermore, natural product building blocks are limited in variety and for an organism to

compete in the environment nature has utilized space effectively resulting in complex three-

dimensional shapes.

A great example illustrating how nature’s palette has created a bioactive compound

well-suited for human physiology is salinosporamide A (1.5). Salinosporamide A (1.5) is a

hybrid polyketide/nonribosomal peptide isolated from the deep-sea actinomycete Salinispora

tropica. 18 Initial testing showed in vitro cytotoxic activity with an IC50 of 11 ng/mL towards

HCT-116, a human colon carcinoma cell line. Structural similarity of 1.5 to omuralide (1.6)

prompted its testing as a 20S proteasome inhibitor. 19 20S proteasome is a multicatalytic,

proteolytic complex involved in intracellular processes such as cell cycle regulation and

cytokine stimulated signal transduction cascades. It plays an important role in

neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. Salinosporamide A

(1.5) was found to be 35 times more potent than omuralide (1.6).

Figure 1.2 Inhibitors of the 20S proteasome salinosporamide A (1.5) and omuralide (1.6).

Investigation of the substantial difference in activity between these two compounds

revealed the mode of action between salinosporamide A (1.5) and 20S proteasome. 20 By

obtaining a crystal structure of the ligand-enzyme complex, it was shown that the interaction

between the ligand and the peptide was covalent (Figure 1.3).

Figure 1.3 Salinosporamide A (1.5) mechanism of action.

The proposed mechanism involves attack of the lactone carbonyl by a threonine

residue forming a covalent bond in ester 1.7 (Figure 1.3). The amine moiety of threonine then

deprotonates the secondary alcohol of intermediate 1.7, followed by an intramolecular

annulation to form a tetrahydrofuran ring in 1.8 (Figure 1.3). The covalent nature of this

interaction is necessary for the activity of salinosporamide A (1.5), however, it is the

presence of the chloro-ethyl functionality which gives 1.5 enhanced potency relative to

omuralide (1.6). The tetrahydrofuran ring in 1.8 was found to provide a barrier to water

molecules, which would otherwise hydrolyze the ligand-enzyme complex 1.8. Additionally,

the protonated threonine amine in 1.8 is now deactivated as the protonated amine, which

under normal circumstances would catalyze hydrolysis (as in the case with omuralide (1.6)).

The hydrogen bonding interaction between the protonated amine and the tetrahydrofuran ring

tighten the ligand-enzyme complex providing an additional barrier to water.

An understanding of chemical complexity as it relates to drug-enzyme interactions

has provided natural products with a second wind. Considering that the majority of current

natural product based therapeutics are terrestrial in origin, the question that needs to be asked

is: “why look to the marine environment for drug development?”

There is promising news regarding the molecular complexity of marine natural

products as shown in a recent review. 21 Approximately 71 % of marine molecular

frameworks are used exclusively by marine organisms, with approximately 50 % having a

one-time occurrence.

Marine natural products also show a higher occurrence of bioactivity when compared

with terrestrial natural products. 22 In a preclinical cytotoxicity screen completed by the

National Cancer Institute, it was shown that approximately 1 % of crude marine natural

product extracts showed anti-tumor activity compared to 0.1 % of crude terrestrial natural

product extracts. The last couple years has seen an increase to approximately 1000 new

marine natural products with varying biological profiles being isolated per annum. 22

This diversity in marine natural products may provide future drug candidates.

However, for any compound to achieve drug status 23 several major hurdles have to be

overcome. It can be shown that synthetic organic chemistry plays a major role in each stage

of development, from benchtop to bedside.

1.2 Synthesis as an Aid to Structure Elucidation

The compounds first identified by marine natural product chemists were “low

hanging fruit” that were present in large quantities in the source organisms, and were easily

separable from other constituents in the extracts. This was a direct result of limited separation

and spectroscopic technologies available in the early days of marine natural product

discovery. As techniques for chemical identification became more sophisticated, smaller

amounts of material could provide the necessary data needed to elucidate a chemical

structure.

A modern example is the macrolide phorboxazole A (1.9) that was isolated 24 from the

marine sponge Phorbas sp. collected in Western Australia (Figure 1.4). Phorboxazole A (1.9)

(95.1 mg) was isolated in the mid 1990’s at masses sufficient for characterization. A number

of smaller peaks in the HPLC trace (0.78-3.0 mg) of the major metabolite (1.9) alluded to

additional compounds. However, the NMR technology at the time was too insensitive for

characterization of these minor components. Fifteen years would pass before phorbaside A 25

(1.10) and muironolide A 26 (1.11) were characterized (Figure 1.4).

Figure 1.4 Marine natural product identification at the nM scale

Muironolide A (1.11) is an example of how synthesis can help in the structure

determination of novel marine natural products. Only 90.0 µg of pure muironolide A (1.11)

was obtained and a 1.7 mm NMR CryoProbe TM (600 MHz) was used for its structure

elucidation. Unfortunately, the relative configuration of the chloro-cyclopropane ring moiety

could not be assigned, since NOESY correlations could not be made to any of the macrolide

ring stereocenters. However, synthesis was used to determine the absolute configuration of

the chloro-cyclopropane functionality. Muironolide A (1.11) was degraded and its chemical

degradation product was compared with known compounds in the literature in order to assign

the absolute configuration of the chloro-cyclopropane ring moiety (Figure 1.5).

chemical degradation.

Muironolide A (1.11) was saponified and then esterified with α-bromo-ketone 1.12 to

yield degradation fragment 1.13 (Figure 1.5). A set of standards with known configuration

was prepared starting from menthol-methyl methacrylate 1.14. These standards were

compared with the muironolide degradation product 1.13. Chiral LCMS was used to show

that the muironolide A (1.11) degradation product and synthetic standard 1.13 were identical,

assigning the (3S,4S,5R) configuration to the natural product (Figure 1.5).

Another example of how synthesis can assist structural elucidation efforts is the

imidazole alkaloid (–)-palau’amine (1.22). (–)-Palau’amine (1.22) is an alkaloid produced by

the marine sponge Stylotella agminata. It was isolated in 1993 27 and represents the pinnacle

of structural complexity in a family of imidazole containing natural products including (–)-

sceptrin 28 (1.20), and (–)-ageliferin 29 (1.21) (Figure 1.6). The key structural features of (–)-

palau’amine (1.22) are eight contiguous stereocenters, six rings, and nine nitrogen’s. The

originally assigned relative configuration of (–)-palau’amine had a cis relationship between

the protons in the pyrrolidine core, but this assignment was revised 30 to a trans relationship

almost fifteen years later (Figure 1.6).

Figure 1.6 Family of imidazole marine natural products.

The bioactivity of (–)-palau’amine (1.22) suggested, antifungal, antitumor, and

immunosuppressive properties. Having an intricate structure that had been debated, in

addition to its potential therapeutic effects made it an appealing target among synthetic

organic chemists. Several manuscripts reporting studies towards the synthesis of (–)-

palau’amine (1.22) were published subsequent to its isolation and structural revision. In

2010, Baran 31 and co-workers published the first synthesis of (±)-palau’amine (1.22) (Figure

1.7).

Figure 1.7 Barans retrosynthesis of (±)-palau’amine (1.22).

The synthesis, which proceeded in 25 steps from commercially available starting

material, was a six-year endeavor. The total synthesis of (±)-palau’amine (1.22) confirmed

the revised structure. Furthermore, it was a testament to how the structural complexity that is

offered by marine natural products forces synthetic organic chemistry to evolve. 32

Studies

Constructing bioactive marine natural products can be a synthetic challenge. Often,

the complexity of the target compound is such that producing the quantities necessary for

clinical trials, let alone clinical use, is not feasible. SAR studies are used in an attempt to

simplify the structure, yet maintain the biological activity of the lead compound. The

polyketide (+)-spongistatin 1 (1.27) isolated 33 from the marine sponge Hyrtius erecta is a

good example of how SAR can produce a simplified analogue of the natural product, while

still maintaining much of the activity (Figure 1.8).

Figure 1.8 The antimitotic marine natural product (+)-spongistatin 1 (1.27).

The macrolactone (+)-spongistatin 1 (1.27) is one of the most cytotoxic agents

known, with an average IC50 of 0.12 nM tested against sixty human cancer cell lines. 34

Preliminary in vivo data showed an inhibition of mitosis and microtubule assembly. Despite

the alluring biological data, all known syntheses of (+)-spongistatin 1 (1.27) are in excess of

100 steps, 35,36 which is too lengthy for clinical appeal.

To address this issue, SAR studies were undertaken to identify the structural

components that are responsible for the sub nM activity. Inspired by the marine natural

product derived drug Halaven ® (an analogue of the polyketide halichondrin B 37 ) the potential

pharmacophore of the compound was identified. Through computational calculations 38 the

four lowest energy solution conformations of (+)-spongistatin 1 (1.27) were determined. The

results suggest that the western hemisphere including the ABEF rings have a common

conformation that does not change between the four lowest energy states (Figure 1.9). The

eastern hemisphere including the DC rings have structurally significant differences in

conformations between the four lowest energy states. This data suggested that the western

hemisphere is rigid, while the eastern hemisphere is more flexible. It was hypothesized that

the binding domain was located within the conformationally rigid portion of the molecule.

Subsequently, analogues of (+)-spongistatin 1 (1.27) were constructed, which maintained the

western hemisphere and ABEF rings, but had a simplified eastern hemisphere (Figure 1.9).

Figure 1.9 A pharmacophore analogue (1.28) of (+)-spongistatin 1 (1.27).

Compound 1.28 represents a thirty-step decrease in its construction when compared

to the natural product (1.27), and maintained nM activity with an IC50 of 60.5 nM in a U937

lymphoma cell line (1.27 displayed an IC50 of 0.06 nM). SAR compliments total synthesis

allowing for construction of simplified pharmacophore analogues derived from complex

marine natural products. 39

1.4 Bioactive Marine Natural Products Reveal Novel Mechanism of Action

The large chemical diversity associated with marine natural products often results in

the identification of novel therapeutic targets. Ziconotide 40 (1.29) (PRIALT ® ) is an analgesic,

and the first marine natural product peptide to be approved for use in the clinic. Its potency is

1000 times greater than morphine, and it does not elicit tolerance as with opiate-based

therapies.

Figure 1.10 A marine peptide with novel biological activity.

This twenty-five amino acid peptide is the synthetic counterpart of ω-conotoxin, a

secondary metabolite found in the venom of the marine snail Conus magus. Ziconotide (1.29)

has an unprecedented mechanism of action. By blocking N-type voltage sensitive calcium

channels, a subset of neurons including primary nociceptors is inhibited, which is responsible

for sending signals to the spinal cord and brain in response to pain.

This unique mode of action 41 for ziconotide (1.29) provides a new avenue for

treatment of severe chronic pain. In effect, the discovery of bioactive marine natural products

such as ziconotide (1.29) broadens our understanding of human biology and allows drug

development to move forward.

1.5 Scope of Thesis

The body of work presented in this thesis details the use of synthetic organic

chemistry to facilitate drug discovery through structure verification, SAR, and probe

development.

Chapter 2 describes the construction of water-soluble SHIP1 activators in an attempt

to develop compounds with enhanced drug-like properties. Small molecule activators of

SHIP1 may be used as novel therapies for hematopoietic malignancies as well as

inflammatory disorders and they could be an alternative to PI3K inhibition. Biological

studies on the constructed SHIP1 activators were conducted and include enzymatic, in vitro,

and in vivo assays.

Chapter 3 describes the total syntheses of two novel glycerol ether marine natural

products to aid in structural elucidation efforts and to provide synthetic material for

biological testing. The natural products are AR-NTD antagonists and represent a novel

pharmacophore. SAR of the natural products was conducted to observe the biological effects

that structural modification may have. A probe based on one of the natural products was used

to show that the binding mechanism between the ligand and the drug target was covalent in

nature. In vitro biological studies were conducted on the natural products and their SAR

analogues to probe the biological effects of these novel AR-NTD pharmacophores.

Chapter 4 describes a known marine natural product that was shown to be an AR-

LBD antagonist. Semisynthesis using the lead compound produced an analogue of enhanced

potency, and the synthetic efforts towards constructing analogues of this novel AR-LBD

antagonist are detailed.

Chapter 5 describes the synthetic efforts toward a novel peptide-aldehyde terrestrial

natural product, which was found to be a potent inhibitor of cathepsin K. The purpose of the

synthesis was to aid in structure elucidation efforts, and to provide additional material for

biological testing.

Chapter 2: Synthesis and Biological Evaluation of SHIP1 Activators

2.1 Inhibition of PI3K Signaling by Activation of SHIP1

Figure 2.1 The PI3K pathway.

The Phosphoinositide 3-kinase (PI3K) signal transduction pathway (Figure 2.1)

regulates many cellular processes such as cell proliferation, activation, and growth. 42,43,44,45

Specific binding between extracellular ligands and receptors on the cell surface activate the

PI3K pathway, which leads to phosphorylation of phosphatidylinositol-4,5-bisphosphate (PI-

4,5-P2, or PIP2). This generates an important second messenger phosphatidylinositol-3,4,5-

trisphosphate (PI-3,4,5-P3, or PIP3) in the plasma membrane.

The levels of PIP3 in unstimulated cells are very low. However, PIP3 is rapidly

synthesized from PIP2 in response to extracellular stimuli. PIP3 concentrations are controlled

by the tumor suppressor phosphatase and tensin homolog (PTEN). PTEN hydrolyzes PIP3

back to PIP2, and the Src homology 2-containing inositol 5-phosphatases 46 (SHIP1, sSHIP,

and SHIP2), which hydrolyze PIP3 to phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2).

Both of these events dampen the signal. Elevated PIP3 concentrations result in amplification

of the signal transduction cascade leading to human diseases such as cancer and

inflammation. 42,43,44,45 Consequently, a number of drugs targeting the PI3K signaling

pathway are being developed. 47 Most of these therapeutics are designed to prevent formation

of the second messenger PIP3 by inhibiting PI3K, or to inhibit downstream targets in the

signal transduction cascade. An alternative method of down-regulating PIP3 production is to

activate PTEN. However, PTEN mutation or silencing occurs in many human malignancies 48

therefore suitable treatment utilizing this pathway remains elusive.

Unlike PTEN, SHIP1 has not been found to mutate during human malignancies.

Activation of the phosphatase SHIP1 49 is an attractive alternative to current therapies since

its expression is restricted to hematopoietic cells, which should minimize off-target tissue

effects.

2.2 First Generation SHIP1 Agonist Pelorol and Analogues

The discovery of the SHIP enzyme by Dr. Gerald Krystal, 50 led to a collaboration in

which the Andersen natural product library of extracts was screened by Dr. Alice Mui in an

effort to discover small molecule activators of SHIP1. A methanol extract of the sponge

Dactylospongia elegans collected in Papua New Guinea exhibited promising activity in the

screening assay. Bioassay-guided fractionation of the extract led to the identification of the

meroterpenoid pelorol (2.1) as a selective SHIP1 activator (Figure 2.2). 51

Figure 2.2 The first selective SHIP1 activator marine natural product pelorol (2.1).

While biological evaluation of pelorol (2.1) was underway in the Mui and Andersen

labs, pelorol (2.1) was isolated by the Konig 52 and Schmitz 53 groups from the sponges

Dactylospongia elegans, and Petrosaspongia metachromia collected at the Great Barrier

Reef and Yap in the Federated States of Micronesia, respectively. In order to fully explore

the potential biological impact of this drug lead, the total synthesis of pelorol (2.1) was

completed by Lu Yang in the Andersen group, 51 along with a preliminary SAR study which

generated a small number of analogues.

Figure 2.3 Pelorol (2.1) analogue 2.2.

It was revealed that analogue 2.2 (Figure 2.3), was more potent than the natural

product and more readily synthesized. With a scalable synthesis to the potent SHIP1 agonist

2.2 in hand, enough material was produced to allow for both in vitro and in vivo evaluation.

In vitro data suggested that 2.2 and pelorol (2.1) suppressed mast cell degranulation and

subsequent TNF release in cells expressing SHIP1. The same effect was not observed in cells

lacking SHIP1 providing evidence for the selective targeting of SHIP1. This suggested that

pelorol (2.1) and 2.2 were promising lead structures for developing a drug candidate.

Pelorol (2.1) and analogue 2.2 provided preliminary proof of principle for the

biological activity resulting from small molecule activation of SHIP1, however, an inherent

structural lability present in the structures needed to be addressed. Catechol functionalities

can be oxidized to ortho-quinones (Figure 2.4), making the molecule susceptible to 1,6-

addition resulting in potential off-target effects, which may have carcinogenic

consequences. 54

Figure 2.4 Chemical and enzymatic oxidation of catechol and subsequent 1,6-addition.

A more in-depth SAR study based on analogue 2.2 was completed to overcome these

unwanted effects. The majority of these new analogues proved to be active in the SHIP1

assay. Compound 2.3 (Figure 2.5), prepared by Matt Nodwell in the Andersen lab, lacked the

labile catechol functionality and had enhanced potency relative to pelorol (2.1).

Figure 2.5 Analogue 2.3 without the catechol functionality.

2.3 Synthesis of SHIP1 Activators Using a Polyene-Initiated Cationic Cascade

With the promising new drug lead 2.3 identified, it was necessary to provide enough

material to fully explore its biological activities. The synthesis of 2.3 used (+)-sclareolide

(Scheme 2.1) as the starting material and followed a reaction sequence similar to that used

for the chiral synthesis of pelorol (2.1).

Scheme 2.1 Synthetic route to drug lead 2.3.

Since larger amounts of 2.3 were needed for further testing, a more concise synthesis

was required. A biomimetically-inspired synthesis utilizing a polyene-initiated cationic

cascade was devised. The concept of polyene cyclizations is not a recent one. 55 It has been

used in many biomimetically-inspired syntheses of terpenes and steroids such as germanicol

(2.10) and progesterone (2.11) shown in Figure 2.6. 56,57

Figure 2.6 Natural products made by biomimetically-inspired syntheses.

Many variations of polyene cyclizations exist. However, a few themes occur in the

course of all these annulations. Cyclization initiation begins with the generation of a

carbocation. Various functional groups are used to generate a carbocation, such as sulfonate

esters, 58 acetals, 59 allylic alcohols, 60 epoxides, 56 and N-acyl iminiums. 61

Figure 2.7 Carbocation generation.

The methods of carbocation generation include protonation, solvolysis, or the

presence of a Lewis acid (Figure 2.7). Once the cation is generated, it undergoes an

intramolecular attack by a nucleophile. Different nucleophiles such as vinylic, 62 acetylenic, 63

aromatic, 64 and heterocyclic 65 functionalities exist (Figure 2.8). Finally, termination occurs

by the elimination of a proton.

Figure 2.8 Typical propagation and termination of a polyene cascade.

A retrosynthetic analysis of 2.3 (Scheme 2.2) reveals the polyene needed to undergo

the cascade. With this in mind, construction of polyene 2.12 began. Aryl bromide 2.5

underwent halogen-metal exchange followed by cuprate formation in situ. The cuprate was

coupled to trans,trans-farnesyl bromide (2.13) to give polyene 2.12 in 45 % yield (Scheme

2.3).

Scheme 2.2 Retrosynthetic analysis of 2.3.

With the polyene in hand, both tin(IV) chloride 64 and chlorosulfonic acid 66 were

chosen as possible cyclization initiators. It was found that cyclization with chlorosulfonic

acid only resulted in partial cyclization (Scheme 2.4). Cyclization with tin(IV) chloride gave

a complex mixture not easily purified by flash column chromatography so the crude mixture

was deprotected with BBr3 followed by reversed phase HPLC purification to give 2.3/2.14 in

29 % yield (Scheme 2.3).

Scheme 2.3 Synthesis of (±)-2.3.

Scheme 2.4 Elimination side products of chlorosulfonic acid cyclization.

This method allows the synthesis of racemic 2.3 in three linear steps as opposed to

nine from (+)-sclareolide in the chiral synthesis of 2.3 (Scheme 2.1). Furthermore, the

racemic synthesis produced the antipodal configuration of the lead compound pelorol (2.1) in

analogue 2.14, which had not been constructed up to that point.

2.4 Synthesis of Water Soluble Analogues to Facilitate Drug Administration

CLogP is the partition coefficient of a compound between water and octanol. It is a

good predictor of oral bioavailability, 67 which is an important pharmacokinetic property.

Having a CLogP of less than five is in agreement with the Lipinski rules of five. 16 These

guidelines were devised by Christopher A. Lipinski, a chemist at Pfizer, after the

observations he made on the pharmacokinetic properties of the current drugs on the market.

The guidelines are meant to predict the drug-likeness of a molecule but not pharmacological

activity. In addition to having a CLogP of less than five, an orally active drug should have no

more than five hydrogen bond donors, ten hydrogen bond acceptors, and a molecular mass of

less than 500 daltons.

Pelorol (2.1) has a CLogP of 5.74, well outside of the Lipinski range. Similarly,

synthetic analogues 2.2 (CLogP = 5.56), and 2.3 (CLogP = 6.16) are too lipophilic to be

considered drug-like. To address the issue of water solubility, prodrugs based on 2.3 were

constructed by Matt Nodwell in the Andersen group (Figure 2.9).

Figure 2.9 Water soluble prodrug analogues of 2.3.

The purpose of a prodrug is to aid in the delivery of an active compound to the

cellular environment. Upon entering the cell, the prodrug is metabolically transformed into

the active compound. 68 In the case of prodrugs 2.15, 2.16, and 2.17, the ester functionality

would be cleaved in vitro to release a biological promoiety and the parent compound (2.3).

An enzymatic SHIP1 assay was used to evaluate prodrugs 2.15, 2.16, and 2.17, and it

was shown that none successfully activated SHIP1. This was not surprising since cleavage of

the prodrug was not expected in the enzymatic assay. The data was consistent with the

hypothesis that the aryl moiety is an important part of the pharmacophore of 2.3. Prodrug

2.15 was tested in vitro and found to inhibit the release of TNF to the same extent as 2.3.

Prodrug 2.15 was further evaluated by dissolving it in a pH 7.4 buffer solution to mimic

cellular conditions. It was determined that cleavage of the prodrug did occur. However, the

parent compound (2.3) precipitated in the aqueous medium, not solving the problem of

solubility of the active form (2.3) in plasma.

As this issue of water solubility in 2.3 was being investigated, a new analogue (2.18)

was constructed and determined to be a more effective SHIP1 activator than 2.3 (Figure

2.10). Analogue 2.18 has a CLogP of 4.99 just under the Lipinski rule of five. However, this

CLogP value is not ideal and an alternative approach was needed to develop a water-soluble

analogue.

Figure 2.10 Analogue 2.18.

We decided to construct compounds with polar functionality incorporated into the

A-ring far from the putative pharmacophore (Figure 2.11) to develop analogues of 2.18 with

acceptable CLogP values and at the same time robust enough to construct the quantities

required for animal testing. By comparing CLogP values of potential A-ring analogues, it

became clear they might have potential for enhanced water solubility (Figure 2.12).

Figure 2.11 Water soluble analogue design.

The C-3 ketone analogue 2.19 has a CLogP of 3.23, and the corresponding C-3

neutral amino derivative 2.20 has a CLogP of 3.58 (Figure 2.12). The amino derivative 2.20

was of particular interest because the amino group should be protonated at physiological pH.

The resulting ammonium ion salt would be expected to have increased water solubility.

Figure 2.12 CLogP values of 2.18 and related analogues.

To access these compounds an epoxide-initiated cationic cascade was proposed.

Epoxide-initiated cascades, similar to the aforementioned polyene-initiated cascade, are

utilized by nature. A well-known example from nature is the biosynthesis of steroids from

squalene (2.21) (Scheme 2.5). 69

In this biosynthetic pathway, squalene (2.21) is converted to hopene (2.22) by

squalene hopene cyclase (SHC) via a polyene-initiated cascade in an enantioselective and

diastereoselective fashion. Similarly, (3S)-2,3-oxidosqualene (2.23) undergoes an

epoxide-initiated cationic cascade in the presence of oxidosqualene cyclase (OSC) to give

lanosterol (2.24), or cycloartenol (2.25), depending on which OSC is used. It is biosynthetic

pathways such as these that have inspired biomimetic syntheses. Many examples exist, 70,71

which utilize epoxide-initiated cascades. Having specific analogues in mind (Figure 2.12),

the retrosynthetic analysis shown in Scheme 2.6 reveals the terminal epoxide required (2.26)

to construct polar analogues of 2.18 using an epoxide-initiated cascade.

Scheme 2.6 Retrosynthetic analysis of 2.20.

The synthetic route to amine 2.20 begins with the construction of polyene 2.26, which

was accomplished by lithiation of aryl bromide 2.27 followed by alkylation with trans,trans-

farnesyl bromide 2.13 (Scheme 2.7). 72 With the original, as well as the antipodal

configuration of pelorol (2.1) in mind, we designed a chiral synthesis. For this, we needed to

construct the terminal epoxide enantioselectively. Several asymmetric epoxidation methods

have been developed 73 for alkyl-substituted olefins and we felt the Shi 74 epoxidation would

suit our needs. Using Shi catalyst 2.29, which is derived from L-fructose, we were able to

obtain (S) terminal epoxide 2.26 in 28 % yield.

Scheme 2.7 Synthesis of amine analogues 2.20 and 2.34.

The key step of the synthesis was next, which is the epoxide-initiated cyclization

cascade. A number of Lewis acids are effective at opening epoxides to initiate a cascade,

such as tin(IV) chloride, 75 methylaluminum dichloride, 76 and scandium triflate. 77 However,

during this project new methodology utilizing indium tribromide to promote arene-

terminated epoxide opening cyclizations was published by Zhao et al. which used similar

substrates as 2.26 (Scheme 2.8). 78

Scheme 2.8 Examples of indium tribromide promoted annulation methodology

Enantio-enriched epoxide 2.26 was exposed to two equivalents of indium tribromide

in methylene chloride to give a bright orange solution. After the workup step, secondary

alcohol 2.30 was found to be present as part of a complex product mixture. A small quantity

of secondary alcohol 2.30, which was devoid of any side products that was obtained from

flash column chromatography, was reacted with (R)-MTPA-Cl to give Mosher esters 79 2.31

and 2.32 in 95 % and 2.0 %, yield, respectively. Analysis of the Mosher’s esters 2.31 and

2.32 via NMR allowed us to determine the combined enantiomeric excess of the Shi

epoxidation and the epoxide-initiated cascade, which was 94 % ee. In addition, single crystal

X-ray diffraction analysis of Mosher ester 2.32 confirmed its absolute and relative

configuration (Figure 2.13).

Figure 2.13 ORTEP diagram of Mosher ester 2.32.

Since larger quantities were required for in vivo testing, another purification method

for 2.30 was necessary. Single recrystallization from boiling hexanes:ethyl acetate of the

crude product mixture after cyclization gave > 99.5 % of the single enantiomer in alcohol

2.30 (Figure 2.14).

Figure 2.14 ORTEP diagram of alcohol 2.30.

The enantiomeric excess of 2.30 after recrystallization was determined by esterifying

the crystalline material with (R)-MTPA-Cl and observing only the single diastereomer 2.31

by NMR. Next, Dess–Martin periodinane (DMP) oxidation of the secondary alcohol 2.30

gave the ketone 2.33 (Scheme 2.7). Removal of the methyl ether protecting groups of 2.33

with BBr3 gave the resorcinol intermediate 2.19.

Scheme 2.9 Alternative amination strategies towards 2.20 and 2.34.

With the deprotected ketone 2.19 in hand, the remaining step was a reductive

amination. Several routes were explored 80,81 to address the overall yield for the amination,

and to discover which method gave the highest diastereoselectivity (Scheme 2.9). Reductive

amination of the bis-benzyl protected ketone 2.39, followed by deprotection of the benzyl

groups gave unsatisfactory yields. Platinum oxide catalyzed hydrogenation of oxime 2.40,

was promising, but the yields were too irreproducible.

Reductive amination of oxime 2.41 gave unsatisfactory yields. It was found that

reductive amination 82 with sodium cyanoborohydride and ammonium acetate, followed by

acidification, extraction, and subsequent purification using a C18 solid phase cartridge gave

the amine analogues 2.20 and 2.34 as the hydrochloride salts in a 20:3 ratio and 46 % yield

(Scheme 2.7). Repeating the synthesis with the Shi catalyst that is the optical antipode of

2.29 prepared from D-fructose using literature methods 83 gave the amine analogues 2.42 and

2.43 (Figure 2.15). This was to determine whether the absolute configuration would play a

role in the activity of these analogues. The �-amines 2.20/2.42 were chosen as the lead

compounds since they were formed in much higher yields compared to their �-amine

epimers 2.34/2.43.

Figure 2.15 Analogues 2.42 and 2.43 constructed using a D-fructose derived Shi catalyst.

The synthesis was also completed using mCPBA 84 as an oxidant (Scheme 2.10). This

yielded racemic epoxide (�) 2.26 which was carried through the already established synthetic

route (Scheme 2.7) to give 2.20 and 2.42 as a racemic mixture (Scheme 2.10). Single crystal

X-ray diffraction analysis of the racemic mixture 2.20/2.42 confirmed the relative

configuration (Figure 2.16).

Scheme 2.10 Racemic synthesis of (±)-2.20 using mCPBA.

Figure 2.16 ORTEP diagram of racemic amine mixture 2.20/2.42.

While primary amines 2.20 and 2.42 were chosen as the lead compounds for

biological testing, a variety of polar analogues were constructed to further probe the SAR

(Scheme 2.11). Intermediate 2.19, was exposed to hydroxylamine hydrochloride under basic

conditions to give oxime 2.40 85 (Scheme 2.11).

Scheme 2.11 Polar analogues of 2.18.

Oxime 2.40 underwent a Beckmann rearrangement in the presence of trifluoroacetic

anhydride to give lactam 2.44 in quantitative yield. 86 Lactam 2.44 was reduced with LAH in

refluxing tetrahydrofuran, followed by an acidic workup and C18 solid phase purification to

give secondary amine hydrochloride salt 2.45. 87 These analogues utilize various nitrogen

functional groups for enhanced polarity, and have CLogP values of 3.48, 2.68, and 3.84 for

the oxime (2.40), lactam (2.44), and secondary amine (2.45), respectively.

Scheme 2.12 Synthesis of the 2.18 enantiomer (2.48).

Along with these new polar analogues, we were able to access the antipodal

configuration of 2.18, which had not been constructed up to this point (Scheme 2.12). This

would provide insight as to whether or not configuration played a role in the activity of 2.18.

The optical antipode of Shi catalyst 2.29 was used to construct secondary alcohol

intermediate 2.46, which was deprotonated with NaH followed by quenching with CS2 and

MeI. The crude mixture was subjected to Barton-McCombie deoxygenation conditions 88

followed by deprotection of the aryl methyl ethers by boron tribromide to give resorcinol

analogue 2.48. This three step reaction sequence gave a 54 % overall yield.

2.5 Saturation Transfer Difference (STD) Spectroscopy NMR

Having constructed polar analogues of 2.18, we wanted to show that these

compounds interact with the SHIP1 enzyme. Furthermore, we wanted to prove that the aryl

moiety is an essential part of the pharmacophore of not only 2.18, but of all analogues of

pelorol (2.1) that had only been alluded to through synthesis thus far. A classical method of

observing ligand-enzyme interactions is X-ray analysis of co-crystallized ligand-enzyme

complexes. 89,90,91 However, growing a crystal of this type is often challenging.

Therefore, we turned our attention to saturation transfer difference NMR

spectroscopy (STD NMR) to answer the key questions about which moiety of the ligand

interacts with SHIP1 and if there is a preference for a specific configuration. STD NMR was

developed by Mayer and Meyer 92 to observe binding interactions between an enzyme and

potential ligands. Various NMR experiments had previously been developed to study binding

processes such as competitive binding spectroscopy, 93 SAR by NMR 94 transferred nuclear

Overhauser effect, 95 and NOE pumping. 96 However, these methods do not have the high

sensitivity that STD NMR has, or the ability to observe enzyme-ligand interactions in vitro. 97

STD NMR measures the transfer of magnetization from an enzyme to a ligand. Only

ligands that are bound to the enzyme show an STD effect. Thus, STD NMR lends itself to a

number of experiments, providing structural insight to the binding regions of the ligand, the

peptide, or both. STD versions of TOCSY, COSY, NOESY, and 1D NOE experiments all

exist. 92

Figure 2.17 STD NMR.

The focus of our efforts was a STD 1D NOE experiment. In order to achieve this

effect, the experiment is performed by first magnetizing the peptide (Figure 2.17). This is

accomplished by irradiating the peptide, or “saturating” the peptide with magnetization, at a

radio frequency (RF) that only contains the protein envelope resonances (on-resonance

irradiation). 98 Under these irradiation conditions, the protein transfers some magnetization to

the surrounding locality as it begins to relax. The protein also transfers some of its

magnetization to any bound ligand through spin diffusion. A series of RF irradiations at a

resonance that does not contain the protein envelope or ligand resonances (off-resonance

irradiation) is then carried out. Subtraction of the spectrum without peptide magnetization

(off-resonance irradiation) from the spectrum with peptide magnetization (on-resonance

irradiation) gives the final STD NOE NMR. This only shows resonances for hydrogen atoms

of the ligand that have NOE’s due to their close contact with the peptide in the bound state.

A further refinement of this technique by Meyers and co-workers 92 is known as

saturated transfer double difference (STDD). This technique was developed to overcome the

poor signal to noise ratios due to biological components typically found in these experiments,

such as buffers, glucose, and host cells. These undesired components produce regions of

extreme overlap making detection of ligand resonances difficult. The technique of STDD is

complementary to that of STD. Along with a sample containing the biological component

and ligand there is a second sample that contains the biological components and no ligand.

The former provides an STD NOE spectrum of the signals arising from ligand-peptide

interaction along with background noise. The latter provides a STD NOE NMR of just

background noise. A subtraction of these two spectra yields the STDD NOE spectrum, which

shows the resonances of the ligand that have received NOE enhancement from binding to the

peptide (Figure 2.18). The main advantage of the STDD experiment is a better signal to noise

ratio.

Figure 2.18 STDD NMR sample preparation.

Having constructed a water-soluble analogue in 2.20 along with its enantiomer 2.42,

we wished to use the method of STDD NOE NMR to observe binding interactions with a

fragment of SHIP1, that still contains phosphatase activity and is subject to allosteric

regulation. This fragment was previously found to be activated by 2.3, 99 and we hoped that

amine analogues 2.20 and 2.42 would have a similar binding interaction. Due to the small

quantities of the SHIP1 fragment available for the experiment, we decided to test both

analogues sequentially in the same NMR tube. The author was responsible for sample

preparation and David Williams ran the STDD experiments.

First, we combined the peptide and 2.42 at a 200:1 ligand to peptide ratio 100 and

subtracted an STD NOE NMR spectrum of the peptide by itself from the resultant STD NOE

NMR spectrum of the peptide and 2.42 to give an STDD NOE spectrum of 2.42. Next, to the

2.42/peptide sample was added 2.20 giving a 100:1 ligand to peptide ratio 97 and an STD

NOE NMR was generated. From this was subtracted an STD NOE NMR spectrum of the

peptide by itself and the STDD NOE NMR spectrum of 2.42, in order to observe STD NOE

effects for 2.20 alone. This generated an STDD NOE spectrum of 2.20. The STDD NOE

NMR spectrum of 2.20 showed the absence of any ligand resonances, suggesting that there

was little to no binding to the peptide. The STDD NOE NMR spectrum of 2.42, however,

showed ligand resonances suggesting binding to the peptide (Figure 2.19).

Figure 2.19 STDD NOE NMR of 2.42.

This data provided evidence supporting the hypothesis that the pharmacophore of

pelorol (2.1) and all subsequent analogues includes the aryl moiety, due to STD NOE signals

for H19 and H17. Furthermore, the data indicates that the methyl groups Me21 and Me14 also

play a role in SHIP1 binding. Since Me21 and Me14 have a cis relationship, and no other

STDD effects are observed for proton resonances on the ABC ring system, this suggests that

there is a potential facial selectivity in the binding of 2.42 to SHIP1.

A surprising outcome of this experiment is that the antipodal configuration of pelorol

(2.1) in analogue 2.42 was found to bind. However, the analogue 2.20 having the absolute

configuration of the natural product pelorol (2.1) was found not to bind. This suggests that a

positive charge on the A-ring moiety of analogue 2.42 may play a role in binding to SHIP1.

Additional experiments are necessary for further evidence to support this hypothesis.

2.6 Synthesis of a Benzophenone Photoaffinity Probe

Photoaffinity probes are a powerful tool for obtaining structural information about the

binding site of a ligand, to facilitate crystallization of an enzyme-ligand complex, and in the

identification of previously unknown biological targets. 101 With the STDD NOE NMR

evidence for binding to SHIP1 of 2.42 in hand, we decided to construct a photoaffinity probe

(photoprobe). The goal was to provide proof of interaction between a ligand and SHIP1 in

cells and structural information about the protein-binding site, which had not been

characterized thus far. To construct a successful photoaffinity probe several structural motifs

are typically present (Figure 2.20).

Figure 2.20 Components of a photoaffinity probe.

The first is a reactive group, which is an electrophilic or a photoreactive moiety, that

covalently binds to the enzyme. Common electrophilic groups are electrophilic

phosphonates 102,103 and �-halomethyl ketones. 104 Often fine-tuning of the electrophilic group

incorporated into the probe is necessary for successful reactivity towards a residue in the

enzyme-binding pocket. Photoreactive substructures are another type of reactive group that

bind covalently to a residue in an enzyme active site only after UV irradiation. Various

photoreactive groups have been utilized, including diazirine 105 and benzophenone 106

photophores (Figure 2.21).

Figure 2.21 Examples of photoreactive groups.

The second required element in a photoaffinity probe is the tag or reporter group. It is

responsible for target identification and its identity dictates the type of analysis used later on.

Examples include fluorophores 107 and biotin, 108 along with chemical handles such as

azides, 109 or alkynes, 110 which can be modified by copper catalyzed Huisgen cycloaddition 111

(click chemistry) to allow for visualization of targets post labeling (Figure 2.22).

Figure 2.22 Commonly used tags.

The third and last required component is a binding group, which is the component

that has an affinity for a biological target. The most important characteristic of a

photoaffinity probe is that it must maintain biological activity. Compound 2.51 is a

photoaffinity probe analogue of HUN-7293, a fungal cyclodepsipeptide that was first

identified as an inhibitor of vascular cell adhesion molecule (VCAM) expression 107 (Figure

2.23). In this case, the photoprobe incorporates a diazirine photophore and an alkyne tag.

Another example is 2.52, the photoprobe version of SecinPP, the first example of a

cytoplasmic regulatory protein inhibitor. Its structure incorporates a benzophenone

photoprobe along with a biotin tag (Figure 2.23). 112

Figure 2.23 Examples of photoaffinity probes.

In our own efforts to design a photoaffinity probe of pelorol (2.1), we took all

previous considerations into account. There were several analogues of pelorol (2.1) to choose

from, with pelorol (2.1) not being a satisfactory candidate, since it contained a labile catechol

functionality that could potentially bind off-target (Figure 2.4). Amine 2.42 had proved

successful in generating STDD NOE data showing binding to SHIP1, however, the lack of

biological data (at the time) did not make it an appealing candidate. Analogue 2.3 was

designed at an earlier stage and had biological data showing it activates SHIP1. 99 Also, from

a practical standpoint our laboratory possessed 1 kg of material from a previous contract

synthesis. Therefore, 2.3 was chosen as the starting material for construction of a

photoaffinity probe.

A benzophenone moiety was chosen as the photophore for several reasons. One is

that this photophore is known to preferentially label to hydrophobic binding regions. 113 When

considering the lipophilicity of pelorol (2.1) (CLogP = 5.74) it is likely that the region of

SHIP1 responsible for binding is hydrophobic. Secondly, it was thought that one-half of the

enzophenone fragment can come from the aryl group already incorporated in 2.3. This

works well with the STDD NOE data suggesting the aryl moiety plays a role in binding to

SHIP1. In order to incorporate the tag into the molecule an alkyne moiety was selected. This

would provide a conservative modification in order to preserve the biological potency of 2.3.

One potential probe (2.53) along with its subsequent retrieval of the ligand-enzyme complex

from the cellular environment is shown in Figure 2.24.

Figure 2.24 Principle of 2.3 based photoaffinity probe (2.53).

For target identification, we desired to use the aforementioned click chemistry

between the alkyne and an azide bound to biotin after covalent binding between the ligand

and peptide. The purpose of biotin is to retrieve the ligand-enzyme complex (Figure 2.24),

and this can be accomplished by utilizing Streptavidin chromatography. 114 Streptavidin

chromatography is used to separate the biotin-ligand-enzyme complex from all other cellular

components due to the high affinity Streptavidin has for biotin. 115 Finally, polyacrylamide gel

electrophoresis (PAGE) can be done on the purified complex and compared with the SHIP1

enzyme alone resulting in bands with similar retention 116 (Figure 2.24). Typically, the gel is

exposed separately to a SHIP1 antibody and a biotin antibody with fluorescent markers. If

the band of interest fluoresces with both antibodies, then the ligand-enzyme complex is

present. This along with mass spectral data of the complex may provide concrete evidence of

binding by the ligand to SHIP1.

Retrosynthetic analysis of the desired photoprobe 2.53 reveals that it can come from

ester 2.56 by a Fries rearrangement. 117 Ester 2.56 can come from acid chloride 2.57 and 2.3

(Scheme 2.13).

Scheme 2.13 Retrosynthetic analysis of 2.53.

The synthesis started with commercially available acid 2.58. Etherification of 2.58

with propargyl bromide (2.59) gave 2.60 in 65 % yield (Scheme 2.14). Chlorination of 2.60

with thionyl chloride gave acid chloride 2.57, which under basic conditions and 2.3 gave the

key ester intermediate 2.61. This set up the key step of the synthesis, which is the Fries

rearrangement.

Scheme 2.14 Benzophenone photoaffinity probe (2.53) synthesis.

Common acids used in the Fries rearrangement include HF, 118 AlCl3, 119 BF3, 120

TiCl4 121 and SnCl4. 122 Aluminum trichloride gave no observable product. However, aryl ester

2.61 in the presence of titanium tetrachloride at 140 � C, gave a complex reaction mixture

after a short reaction time. The crude mixture was purified and the major compound isolated.

To understand the potential product distribution for the final step of this synthesis, we

had to look at the mechanism of the Fries rearrangement. The Fries rearrangement generates

an acylium carbocation 2.63 (Scheme 2.15). This electrophilic intermediate can be attacked

y two possible aryl positions in a Friedel-Crafts type mechanism, resulting in two potential

products 2.53 and 2.62 (Scheme 2.15).

Scheme 2.15 Fries rearrangement and NOE observance in 2.53.

These two regioisomers could not be definitively distinguished with 1D 1 H NMR.

Therefore, the major product of the reaction was analyzed by a NOESY experiment to

determine the site of benzoyl attachment. An NOE was observed between the aryl proton H19

and Me20, which is only possible in compound 2.53, thus identifying the correct regioisomer.

The ability of photoprobe 2.53 to bind covalently to SHIP1 upon irradiation is currently

being investigated in the laboratory of Dr. Alice Mui at the BC Cancer Agency.

2.7 Biological Results

The biological activity of selected compounds was investigated by our collaborator

Dr. Alice Mui at the BC Cancer Agency, Aquinox pharmaceuticals, and SignalChem. The

first set of compounds that was analyzed included 2.48 and the corresponding racemate

2.48/2.18. The goal was to gain insight about the role of the absolute configuration of these

analogues in activating SHIP1. In addition, the benzophenone photoprobe 2.53 was tested for

SHIP1 activity.

The SHIP1 activating properties of these compounds were evaluated using a

chromogenic kinetic assay for SHIP1 phosphatase activity. In this assay, SHIP1 was

incubated with compounds 2.3, 2.53, 2.48, and 2.48/2.18 for 15 minutes before the addition

of inositol-1,3,4,5-tetrakisphosphate. At this point SHIP1 dephosphorylates inositol-1,3,4,5-

tetrakisphosphate, and a phosphate group is released into solution. At a particular time

period, Malachite green along with molybdate are added, and together with free

orthophosphate, they form a green complex that can be measured on a spectrophotometer at

650 nm. The magnitude of the reading on the spectrophotometer is directly related to the

amount of green complex formed, and correlates to the degree to which SHIP1 is activated.

The results are shown in Figure 2.25.

Figure 2.25 SHIP1 phosphatase assay.

Interestingly, 2.48, and 2.48/2.18 have almost the same activity, which indicates that

the absolute configuration does not seem to play a major role in SHIP1 activation. Another

important finding of this study is that photoprobe 2.53 activates SHIP1. This result is a

requirement for it to be a successful photoaffinity probe.

Our attention then turned to the A-ring analogues of 2.18. The first piece of

information was to determine the solubility of amine 2.20 versus its less polar counterparts

2.3 and 2.18. The method to determine solubility in water is as follows. A compound

(2.3, 2.18, or 2.20) of known mass was dissolved in water to a predetermined concentration.

This sample was stirred at room temperature for a specific time period after which the sample

was filtered, and then centrifuged. The supernatant was then analyzed by HPLC using a

standard of known concentration.

Table 2.1 Solubilities of pelorol (2.1) analogues.

Compound Solubility in Water (�g/mL) Solubility in Tris Buffer (�g/mL)

2.3 - 0.003

2.18 - 0.92

2.20 1,400 -

Data are summarized in table 2.1. There is an approximate 500,000 fold increase in

solubility going from 2.3 to 2.20 and an approximate 1500 fold increase going from 2.18 to

2.20. This demonstrated that we had achieved our goal of constructing an analogue with

enhanced solubility in water. The next goal was to ensure that the racemic mixture 2.20/2.42

activated SHIP1 in cells. In vitro evidence for inhibition of AKT phosphorylation was

obtained by incubating 2.20/2.42 with MOLT-4 (SHIP1+) cells and Jurkat (SHIP1–) cells

stimulated with LPS. It was found that AKT phosphorylation was inhibited only in the

MOLT-4 cells that express the SHIP1 enzyme, whereas there was no AKT phosphorylation

inhibition in the Jurkat cells missing SHIP1 (Table 2.2). This is evidence that the inhibition

of AKT phosphorylation by 2.20/2.42 is SHIP1 dependent.

Table 2.2 AKT phosphorylation inhibition of racemic amine mixture 2.20/2.42.

Compound MOLT-4 (SHIP1+) JURKAT (SHIP1–)

2.20/2.42 Inhibition No Effect

Next, a SHIP1 chromogenic kinetic assay was carried out to test the less polar

analogues 2.3 and 2.18 alongside polar analogues 2.19, 2.20/2.42, 2.34/2.43, 2.20, and 2.42

and the results are presented in Table 2.3. Percent (%) activation of SHIP1 is expressed as a

percentage increase relative to background. Scoring is expressed as follows: + (< 25 %); ++

(≥ 25 % but < 50 %); +++ (≥ 50 %).

Table 2.3 Activation of SHIP1 enzyme.

Compound Scoring

2.3 ++

2.18 +++

2.19 +++

2.20/2.42 ++

2.34/2.43 ++

2.20 +

2.42 ++

Several conclusions can be made from these enzymatic assay results. Analogue 2.18,

which is missing A-ring derivatization, along with its A-ring ketone counterpart 2.19, both

activate SHIP1 the most effectively. None of the amines activated SHIP1 as much as 2.18 or

2.19. Interestingly, the antipodal configuration to pelorol (2.1) in amine 2.42 was more active

than amine 2.20, which retained the natural product configuration and yet was almost

inactive. This is in contrast to the difference in activity between 2.48 and 2.48/2.18 (Figure

2.25), which was negligible. This is encouraging evidence as it was shown in the STDD NOE

NMR of 2.20, and 2.42, that only 2.42 had observable binding to SHIP1, an outcome also

verified by this enzymatic study. This leads us to believe that having a positive charge on the

A-ring must affect the binding in a manner yet to be determined. Another outcome is that the

minor �-amine epimers 2.34/2.43 showed no difference in activity compared to the �-amine

epimers 2.20/2.42.

Racemic amine mixture 2.20/2.42 was then evaluated in a standard mouse passive

cutaneous anaphylaxis (PCA) ear model of inflammation. 123 The right ear of the mouse is

inoculated with anti-DNP-IgE, which is an antigen that binds to mast cells in the local

environment. After 24 hours, the compound (2.20/2.42) is administered by oral gavage,

followed by an injection of Evans blue and DNP-HSA. DNP-HSA is an antigen that causes

cross-linking of anti-DNP-Ige molecules bound on the mast cells. This cross-linking triggers

the release of histamine and other small molecules from mast cell granules. Evans blue is

injected to allow for visualization of increased vascular permeability and will stain the tissue

blue where excess extravasation has occurred.

Ideally, the compound administered to the mouse before the DNP-HAS/Evans blue

injection is taken up by the cells, and will prevent the anaphylactic response, thus the ear will

stain minimally blue. Afterwards, the mice are sacrificed and a portion of the inoculated ear

is taken, extracted into solution, and analyzed using a spectrophotometer at 620 nm. A

smaller reading suggests that less Evans blue was retained in the tissue indicating the

effectiveness of the compound at preventing anaphylaxis.

model.

When administered by oral gavage, racemate 2.20/2.42 showed effective anti-

inflammatory activity with a clear dose response and an ED50 of approximately 0.1 mg/kg

(Figure 2.26). This is a promising result since the desired biological effect is concentration

dependent, a step in the right direction for constructing a compound with drug-like

properties.

2.8 Conclusion

The activation of SHIP1 is a novel and selective method to modulate the PI3K cell-

signaling pathway. It is an alternative to classic kinase therapeutic targets and may be useful

for the development of drugs to treat hematopoietic diseases that involve PI3K signaling. The

lead compound pelorol (2.1) was identified as the first SHIP1 activator, and provided a proof

of principle test for our target hypothesis. Pelorol (2.1) was synthesized to confirm its

absolute configuration, and a number of analogues were constructed by Lu Yang to further

probe the SAR. Analogue 2.2 was found to have increased potency relative to pelorol (2.1)

and was easier to construct. However, the labile catechol functionality prevented it from

being a viable drug lead.

An SAR study was done by Matt Nodwell to construct analogues, which maintained

the biological activity of 2.2 but without the labile catechol functionality. This culminated in

analogue 2.3. However, its low solubility (CLogP = 6.16) in water posed a problem for drug

administration. Analogue 2.18 with resorcinol functionality was constructed in order to

enhance water solubility and retain its SHIP1 activation property. Resorcinol 2.18 was found

to have enhanced activity relative to 2.3 and while it’s CLogP (4.99) did benefit from an

extra aryl alcohol, it still fell short of ideal.

The focus of this chapter is a third generation of SAR based on analogue 2.18 in order

to construct water-soluble compounds that activate SHIP1. All of the SAR completed by Lu

Yang and Matt Nodwell was devoted to the aryl moiety of pelorol (2.1) because this was

considered as the putative pharmacophore. With this in mind, the author constructed

analogues of 2.18 that had synthetic modifications to the A-ring, far from the putative

pharmacophore. Amines 2.20 and 2.42 were chosen as the main synthetic targets with a

CLogP value of 3.58, below the Lipinski rule of five.

We used an epoxide-initiated cationic cascade to construct these analogues, and we

utilized an asymmetric Shi epoxidation to affect a chiral synthesis. The syntheses of amines

2.20 and 2.42 are more concise than any of the previous syntheses used to construct SHIP1

activators, such as pelorol (2.1) and 2.3, which require ten and nine steps respectively. In

contrast, the syntheses of 2.20 and 2.42 proceed in six steps starting from achiral

commercially available starting material. This allows rapidly access to enantiopure SHIP1

activators that are highly soluble in water, and active in vitro and in vivo. The solubility of

the racemic amine mixture 2.20/2.42 in water was determined and compared to 2.3 and 2.18.

There was an approximate 500,000, and 1500 fold increase in water solubility respectively.

This result validated our hypothesis of enhanced water solubility of amine analogues 2.20

and 2.42.

Next, the racemic amine mixture 2.20/2.42 was tested in an in vitro AKT

phosphorylation inhibition assay to determine if the SHIP1 enzyme was activated by these

analogues. It was found that SHIP1 was being selectively activated, making 2.20/2.42

suitable candidates for additional biological testing.

The amine analogues 2.20 and 2.42 were tested in a chromogenic kinetic assay

against their less polar counterpart 2.18 to determine their effectiveness as SHIP1 activators.

It was found that neither amine activated SHIP1 to the extent of 2.18. However, these polar

analogues did activate SHIP1 to the same degree as 2.3 which has precedent in SHIP1

activation. 99 Further validation of the amine analogues 2.20 and 2.42 as potential drug

candidates included in vivo testing of the corresponding racemic mixture 2.20/2.42 in a PCA

mouse model. The compounds were effective in diminishing the anaphylactic response

caused by administered antigens. There was a clear dose response, with an ED50 of

approximately 0.1 mg/kg. Making these analogues viable drug candidates for further testing.

One of the interesting outcomes of the biological testing was that amine 2.20, which

has the same configuration as pelorol (2.1), was less active than 2.42, which has the opposite

configuration. We used the method of STDD NOE NMR to determine which moieties in 2.42

and 2.20 were binding to SHIP1 in order to gain information about to the pharmacophore of

these analogues. It was shown that the aryl group had binding interactions with the enzyme,

validating the putative pharmacophore of pelorol (2.1) and all subsequent analogues.

Furthermore, both cis methyl groups on the B/C ring junctions showed binding interactions,

suggesting facial selectivity in binding to SHIP1. The work done in this chapter represents

the continuation of a rational marine product based drug design starting from an initial lead

in pelorol (2.1) and evolving to an analogue with enhanced drug-like properties in 2.42 and

2.20 with promising in vitro and in vivo activity (Scheme 2.16).

(2.20/2.42).

2.9 Experimental

General Methods: All non-aqueous reactions were carried out in flame-dried

glassware and under an Ar or N2 atmosphere unless otherwise noted. Air and moisture

sensitive liquid reagents were manipulated via a dry syringe. All solvents and reagents were

used as obtained from commercial sources without further purification. 1 H and 13 C NMR

spectra were obtained on Bruker Avance 400 direct, 300 direct, or Bruker Avance 600

CryoProbe spectrometers at room temperature. Flash column chromatography was performed

using Silicycle Ultra-Pure silica gel (230-400 mesh). Analytical thin-layer chromatography

(TLC) plates were aluminum-backed ultrapure silica gel 250 μm. Electrospray ionization

mass spectrometry (ESI-MS) spectra were recorded on a Micromass LCT instrument. Optical

otations were measured with a JASCO P-1010 polarimeter at 24 �C and 589 nm (sodium D

line) in methanol (g/100 mL).

Preparation of 2.12:

To a mixture of benzene (7 mL) and ether (3.5 mL) was added bromine 2.5 (180 mg,

0.895 mmol) followed by the dropwise addition of n-BuLi (0.69 mL, 0.98 mmol) at room

temperature. Halogen metal exchange was monitored using thin layer chromatography, after

which CuBr · DMS (92 mg, 0.45 mmol) was added and the reaction mixture was allowed to

stir for 1 hr. Trans,trans-farnesyl bromide (2.13) was then added neat and allowed to stir

overnight. The reaction mixture was quenched with saturated NH4Cl (aq) (25 mL), and the

aqueous layer was extracted with ethyl acetate (100 mL). The combined organic extracts

were washed with 10 % NH4OH followed by brine, dried with MgSO4 and concentrated

using a rotary evaporator. Purification by flash column chromatography (hexane:methylene

chloride 9:2) yielded polyene 2.12 as a light yellow oil (131.5 mg, 0.40 mmol, 45 %). 1 H

NMR (400 MHz, CDCl3) � 6.64 (s, 1H), 6.58 (s, 2H), 5.36 (t, J = 7.3 Hz, 1H), 5.14 (m, 2H),

3.81 (s, 3H), 3.34 (d, J = 7.3 Hz, 2H), 2.33 (s, 3H), 2.15-1.95 (m, 8H), 1.75 (s, 3H), 1.71 (s,

3H). 1.63 (s, 6H); 13 C NMR (100 MHz, CDCl3) � 159.7, 143.2, 139.2, 136.1, 135.0, 131.2,

124.4, 124.1, 122.9, 121.6, 111.8, 111.1, 55.1, 39.7, 39.7, 34.2, 26.7, 26.6, 25.7, 21.5, 17.7,

16.2, 16.0. HRESIMS [M+Na] + calcd for C23H34ONa 349.2507, found 349.2502.

respectively.

Preparation of 2.3/2.14:

To a solution of 2.12 (40 mg, 0.12 mmol) in methylene chloride at –�� C was added

SnCl4 (127.7 mg, 0.49 mmol) dropwise and allowed to stir for 2 hours. The mixture was

quenched with 1 mL of methanol, and then saturated NH4Cl (aq) (25 mL) was added. The

aqueous layer was extracted with ethyl acetate (100 mL) and the combined organic extracts

were washed with brine, dried with MgSO4, and concentrated using a rotary evaporator. The

crude was then deprotected in a similar fashion as 2.47, and purified using flash column

chromatography (hexane:ethyl acetate 7:1) to yield a mixture of diastereomers and

regioisomers. This mixture was further purified with reversed phase HPLC to give racemic

2.3/2.14 (11.1 mg, 0.035 mmol, 29 % over two steps). 1 H and 13 C NMR spectra are identical

to previously reported (Matt Nodwell). HRESIMS [M + H] + calcd for C22H33O 313.2531,

found 313.2526.

Preparation of 2.28:

Bromide 2.27 (1.0 g, 4.60 mmol) was dissolved in 23 mL of tetrahydrofuran and

brought to –78 � C, to which 3.45 mL of 1.6 M n-BuLi in hexanes (5.52 mmol) was added

dropwise, and allowed to stir for 15 minutes. To this solution was added 1.38 mL of 0.1 M

Li2CuCl4 in tetrahydrofuran (0.13 mmol) and allowed to stir for 10 minutes at -�� C, finally

trans,trans-farnesyl bromide 2.13 (1.64 g, 5.75 mmol) dissolved in 20 mL of tetrahydrofuran

was added dropwise and allowed to warm with stirring from -�� C to room temperature

overnight. The reaction was quenched with saturated NH4Cl (aq) (50 mL), and the aqueous

phase was extracted three times with methylene chloride (200 mL). The organic extracts

were dried with MgSO4, and concentrated using a rotary evaporator. The crude mixture was

purified using flash column chromatography (hexanes:ethyl acetate 30:1), to give 2.28 as a

clear oil (1.02 g, 2.99 mmol, 65 %). 1 H NMR (400 MHz, CDCl3) ��6.36 (d, J = 2.3 Hz, 2H),

6.30 (t, J = 2.7 Hz, 1H), 5.34 (t, J = 7.3 Hz, 1H), 5.15-5.08 (m, 2H), 3.78 (s, 6H), 3.31 (d,

2H), 2.16-1.96 (m, 8H), 1.71 (s, 3H), 1.69 (s, 3H), 1.60 (s, 6H); 13 C NMR (100 MHz, CDCl3)

� 160.8, 160.8, 144.2, 136.5, 135.1, 131.2, 124.4, 124.1, 122.6, 106.4, 106.4, 97.6, 55.2, 55.2,

39.7, 39.7, 34.4, 26.7, 26.6, 25.7, 17.6, 16.2, 16.0. HRESIMS [M+Na] + calcd for C23H35O2

343.2637, found 343.2628.

Preparation of 2.26:

Epoxide 2.26 was prepared according to literature procedures 74 using the ent-Shi

catalyst 2.29. Polyene 2.28 (1 g, 2.90 mmol) was oxidized to 2.26 (113.0 mg, 0.315 mmol, 11

%), which was purified by flash column chromatography (hexanes:ethyl acetate 12:1). Based

on recovered starting material 2.28 (622.5 mg, 1.82 mmol), the yield of 2.26 was 28 %. 1 H

NMR (400 MHz, CDCl3) � 6.34 (d, J = 2.2 Hz, 2H), 6.29 (t, J = 2.2 Hz, 1H), 5.33 (t, J = 6.1

Hz, 1H), 5.18 (t, J = 5.7 Hz, 1H), 3.76 (s, 6H), 3.29 (d, J = 7.2 Hz, 2H), 2.68 (t, J = 6.2 Hz,

1H), 2.11-2.14 (m, 4H), 2.04-2.08 (m, 4H), 1.71 (s, 3H), 1.62 (s, 3H), 1.29 (s, 3H), 1.25 (s,

3H); 13 C NMR (100 MHz, CDCl3) � 160.6, 143.9, 136.1, 134.0, 124.6, 122.7, 106.3, 97.4,

63.9, 58.0, 55.0, 39.5, 36.1, 34.3, 27.3, 26.4, 24.7, 18.6, 16.0, 15.9. HRESIMS [M + Na] +

calcd for C23H34O3Na 381.2406, found 381.2415.

Preparation of (±)-2.26:

To polyene 2.28 (9.62 g, 27.9 mmol) dissolved in 100 mL of methylene chloride was

added a solution of mCPBA (5.06 g, 29.3 mmol) in 100 mL of methylene chloride. The

reaction was allowed to stir at room temperature for 3.5 h, after which it was quenched with

saturated NaHCO3, and extracted three times with methylene chloride. The organic extracts

were combined, dried with MgSO4, and concentrated using a rotary evaporator. The crude

mixture was purified using flash column chromatography to give (±)-2.26 (2.24 g, 6.24

mmol, 22 %). 1 H, 13 C, and mass spectrometry data matched that of 2.26 (page 66).

Preparation of 2.30:

To epoxide 2.26 (3.53 g, 9.86 mmol) dissolved in 105 mL of methylene chloride was

added InBr3 (6.99 g, 19.7 mmol), and allowed to stir for 1 hour at room temperature. The

reaction mixture was then quenched with saturated NaHCO3 (100 mL) and the aqueous layer

was extracted three times with methylene chloride (500 mL). The organic extracts were

combined and dried with MgSO4, then concentrated using a rotary evaporator. The crude

reaction mixture was purified using flash column chromatography (hexanes:ethyl acetate

3:1), to give a mixture of 2.30 and various uncyclized products. The product mixture was

then crystallized using boiling solvent (hexanes:ethyl acetate 15:1), to give 2.30 (860.3 mg,

2.39 mmol, 24 %). 1 H NMR (400 MHz, CDCl3) �� 6.40 (d, J = 1.8 Hz, 1H), 6.26 (d, J = 2 Hz,

1H), 3.77 (s, 3H), 3.75 (s, 3H), 3.22 (m, 1H), 2.62 (m, 1H), 2.50 (dd, J = 14.4, 6.2 Hz, 1H),

2.45 (dd, J = 9.5, 3.2 Hz, 1H), 1.74-1.55 (m, 7H), 1.18-1.11 (m, 1H), 1.08 (s, 3H), 1.03 (s,

3H), 0.99 (s, 3H). 0.91 (m, 1H), 0.84 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 159.3, 155.8,

144.8, 133.4, 101.9, 96.8, 79.1, 64.4, 56.1, 55.4, 55.0, 46.0, 38.8, 38.5, 38.2, 36.6, 29.5, 27.9,

27.1, 20.3, 19.2, 16.2, 15.1. HRESIMS [M+Na] + calcd for C23H34O3Na 381.2406, found

381.2415; [α] 24 D = +18.86��(c 0.16).

Preparation of 2.31:

Alcohol 2.30 (219.4 mg, 0.61 mmol, after recrystallization), dissolved in 4 mL of

methylene chloride and to this mixture was added pyridine (0.074 mL, 0.92 mmol), and

DMAP (7.4 mg, 0.061 mmol) then cooled to 0 � C. To the reaction was added (R)-(–)-MTPA-

Cl (169.5 mg, 0.671 mmol) and allowed to warm to room temperature overnight. The

reaction was quenched with saturated NH4Cl (aq) (25 mL) and the aqueous layer was

extracted three times with methylene chloride (100 mL). The organic extracts were combined

and dried with MgSO4 and concentrated using a rotary evaporator. A 1 H spectrum of the

crude mixture showed the absence of diastereomers, meaning that alcohol 2.30 was

enantiopure (at least > 99.5 %) after crystallization. The product mixture was then purified

using flash column chromatography (hexanes:ethyl acetate 12:1), to give 2.31 quantitatively.

1 H NMR (400 MHz, CDCl3) ��7.54 (m, 2H), 7.41 (m, 3H), 6.40 (d, J = 1.9 Hz, 1H), 6.26 (d,

J = 1.9 Hz, 1H), 4.72 (dd, J = 11.5, 4.9 Hz, 1H), 3.77 (s, 3H), 3.75 (s, 3H), 3.54 (s, 3H), 2.62

(m, 1H), 2.51 (dd, J = 14.5, 6.1 Hz, 1H), 2.46 (dd, J = 8.4, 2.9 Hz, 1H), 1.85-1.76 (m, 1H),

1.74-1.67 (m, 3H), 1.65-1.57 (m, 3H), 1.23 (td, J = 13.5, 4.2 Hz, 1H), 1.07 (s, 3H), 1.04 (s,

3H), 1.04 (m, 1H), 0.92 (s, 3H), 0.87 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 166.3, 159.5,

155.8, 144.7, 133.2, 132.3, 129.5, 128.3, 128.3, 127.6, 127.6, 124.9, 122.0 101.9, 96.9, 84.5,

64.2, 56.2, 55.4, 55.3, 55.1, 46.1, 38.1, 38.0, 37.9, 36.5, 29.5, 28.1, 23.0, 20.3, 19.0, 16.2,

16.1. HRESIMS [M+Na] + calcd for C33H41O5F3Na 597.2804, found 597.2814; [α] 24 D = -

31.76��(c 0.65).

Preparation of 2.32

The preparation is identical to 2.31, with the exception that alcohol 2.30 was used

without crystallization. Integration of diastereomers in the 1 H crude gave a diastereomeric

ratio of 97:3 (2.31:2.32) meaning that the epoxidation proceeded in 94 % ee.

Recrystallization from boiling solvents (hexanes:ethyl acetate 30:1) gave crystals from which

a crystal structure was obtained. 1 H NMR (400 MHz, CDCl3)��7.57 (m, 2H), 7.41 (t, 3H),

6.41 (s, 1H), 6.26 (d, J = 1.5 Hz, 1H), 4.75 (dd, J = 10.4, 5.9 Hz, 1H), 3.77 (s. 3H), 3.75 (s,

3H), 3.58 (s, 3H), 2.64 (t, J = 13.8 Hz, 1H), 2.52 (dd, J = 14.3, 6.1 Hz, 1H), 2.46 (m, 1H),

1.91-1.79 (m, 2H), 1.75-1.70 (m, 2H), 1.66-1.62 (m, 4H), 1.25 (td, J = 12.5, 4.9 Hz, 1H),

1.08 (s, 3H), 1.07 (s, 3H), 0.86 (s, 3H), 0.83 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 166.1,

159.5, 155.8, 144.7, 133.2, 132.7, 129.5, 128.3, 127.3, 124.9, 122.1, 101.9, 96.9, 84.3, 64.2,

56.2, 55.42, 55.39, 55.1, 46.1, 38.1, 38.0, 37.9, 36.5, 29.5, 27.7, 23.4, 20.3, 19.0, 16.2, 15.9.

HRESIMS [M+Na] + calcd for C33H41O5F3Na 597.2804, found 597.2814; [α] 24 D = -22.87� (c

1.20).

Preparation of 2.33:

To alcohol 2.30 (600.0 mg, 1.67 mmol) dissolved in 85 mL of methylene chloride

was added Dess–Martin periodinane (1.41 g, 3.34 mmol), and the mixture was allowed to stir

at room temperature for 1.5 hours. Upon completion, saturated NaHCO3 (100 mL) was added

and the aqueous phase was extracted three times with methylene chloride (250 mL). The

organic extracts were combined, then dried with MgSO4, and concentrated using a rotary

evaporator. The crude mixture was purified using flash column chromatography

(hexanes:ethyl acetate 7:1), to give 2.33 (476 mg, 1.34 mmol, 80 %) as a white crystalline

solid. 1 H NMR (400 MHz, CDCl3) ��6.41 (s, 1H), 6.27 (s, 1H), 3.77 (s, 3H), 3.75 (s, 3H),

2.71-2.65 (m, 1H), 2.64-2.56 (m, 2H), 2.54-2.41 (m, 2H), 1.87-1.81 (m, 1H), 1.78-1.73 (m,

2H), 1.71-1.48 (m, 4H), 1.13 (s, 3H), 1.11 (s, 6H), 1.09 (s, 3H); 13 C NMR (100 MHz, CDCl3)

� 217.2, 159.5, 155.8, 144.4, 132.9, 101.9, 96.8, 63.4, 55.4, 55.3, 54.9, 47.5, 45.8, 38.8, 37.4,

36.2, 33.9, 29.5, 26.5, 20.7, 20.5, 19.8, 15.5. HRESIMS [M+Na] + calcd for C23H32O3Na

379.2249, found 379.2243; [α] 24 D = +26.78��(c 0.083).

Preparation of 2.19:

To ketone 2.33 (240 mg, 0.67 mmol), dissolved in 20 mL of methylene chloride

stirring at �� C was added 2.7 mL of BBr3 (1.0 M in methylene chloride, 2.69 mmol). After

stirring for one hour at 0 � C, 2.33 was still present, and the reaction was allowed to reach

room temperature over the next two hours. The reaction was quenched with 1 mL of

methanol and 50 mL of water was added to the mixture. The aqueous phase was extracted

three times with methylene chloride (150 mL). The combined organic extracts were dried

with MgSO4 and concentrated using a rotary evaporator. The crude mixture was purified

using flash column chromatography to give 2.19 (190 mg, 0.58 mmol, 86 %) as a white

crystalline solid. 1 H NMR (400 MHz, (CD3)2CO) �� 7.76 (s, 1H), 7.73 (s, 1H), 6.24 (t, J = 1.0

Hz, 1H), 6.13 (d, J = 1.6 Hz), 2.66-2.61 (m, 1H), 2.59-2.52 (m, 2H), 2.49-2.46 (m, 1H), 2.44-

2.37 (m, 1H), 1.86-1.82 (m, 1H), 1.81-1.77 (m, 1H), 1.76-1.71 (m, 1H), 1.67-1.55 (m, 4H),

1.15 (s, 3H), 1.13 (s, 3H), 1.07 (s, 3H), 1.09 (s, 3H); 13 C NMR (100 MHz, (CD3)2CO) �

216.9, 158.5, 154.8, 146.7, 131.9, 105.8, 102.8, 65.4, 57.2, 48.9, 47.3, 40.5, 39.6, 38.0, 35.4,

30.9, 27.9, 22.2, 22.1, 21.3, 16.9. HRESIMS [M+Na] + calcd for C21H28O3Na 351.1936,

found 351.1929; [α] 24 D = +24.07��(c 0.81).

MHz respectively.

Preparation of 2.20 and 2.34

To a suspension of ketone 2.19 (250 mg, 0.76 mmol) in 50 mL of methanol was

added NaBH3CN (71.7 mg, 1.14 mmol) and NH4OAc (586.5 mg, 7.61 mmol). The reaction

was heated to 70 � C overnight. Upon the disappearance of staring material, the reaction was

cooled to rt, then concentrated using a rotary evaporator. The crude material was partitioned

between water (200 mL) and methylene chloride (100 mL) and acidified to pH 5 with 6 M

HCl. The aqueous phase was then extracted five times with methylene chloride (400 mL).

The resultant aqueous phase was frozen and lyophilized overnight, which gave a white

amorphous solid. To this solid was added 10 mL of water and sonicated to give a

heterogeneous mixture, which was loaded on to a 10 g reversed phase sep-pak (which was

washed with 100 mL of methanol followed by 100 mL of water). Once loaded the column

was flushed with water (100 mL), 60:40 water:methanol (100 mL, 3x), and methanol. The

fractions of interest were the first 60:40 water:methanol fraction containing 78.2 mg of 2.20

pure, and the second and third fraction contained 50.7 mg of a 2:1 mixture of 2.20 and 2.34

as the HCl salt and the freebase, which was acidified and subsequently purified using the

same method. When fully purified the result was 2.20 (112 mg, 0.31 mmol, 40 %), and 2.34

(16.9 mg, 0.046 mmol, 6 %), with a diastereomeric ratio between the two epimers of 20:3.

Compound 2.20: 1 H NMR (600 MHz, CD3OD) � 6.16 (s, 1H), 6.02, (d, J = 1.5 Hz,

1H), 2.93 (dd, J = 12.5, 4.3 Hz, 1H), 2.57 (t, J = 13.6 Hz, 1H), 2.51 (dt, J = 12.5, 3.0 Hz, 1H),

2.42 (dd, J = 14.2, 6.0 Hz, 1H), 1.85 (qd, J = 13.1 , 3.4 Hz, 1H), 1.79-1.73 (m, 2H), 1.71-1.67

(m, 3H), 1.63 (td, J = 12.2, 3.4 Hz, 1H), 1.27 (td, J = 13.3, 3.3 Hz, 1H), 1.11 (dd, J = 11.3,

2.3 Hz, 1H), 1.085 (s, 3H), 1.081 (s, 3H), 1.076 (s, 3H), 0.94 (s, 3H); 13 C NMR (150 MHz,

CD3OD) � 157.5, 154.2, 145.9, 131.7, 104.7, 101.9, 65.6, 61.7, 57.6, 46.8, 39.6, 39.2, 37.8,

37.7, 30.1, 28.2, 24.3, 20.8, 20.2, 16.4, 15.9. HRESIMS [M+H] + calcd for C21H32NO2

330.2433, found 330.2440; [α] 24 D = -8.27��(c 3.68).

Compound 2.34: 1 H NMR (600 MHz, CD3OD) � 6.16 (s, 1H), 6.02 (d, J = 1.2 Hz,

1H), 3.09 (m, 1H), 2.58 (t, J = 13.7 Hz, 1H), 2.51 (m, 1H), 2.44 (dd, J = 14.2, 6.0 Hz, 1H),

2.29 (tt, J = 14.8, 3.3 Hz, 1H), 1.83 (dd, J = 12.9, 6.1 Hz, 1H), 1.71-1.69 (m, 1H), 1.68-1.66

(m, 1H), 1.64-1.59 (m, 1H), 1.49 (dt, J = 11.4, 2.1 Hz, 1H) 1.39 (d, J = 8.5 Hz, 1H), 1.34-

1.27 (m, 2H), 1.11 (s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 1.01 (s, 3H); 13 C NMR (150 MHz,

CD3OD) � 157.5, 154.2, 146.0, 131.8, 104.7, 101.9, 65.1, 59.5, 51.4, 47.1, 39.6, 37.9, 36.6,

34.1, 30.1, 28.4, 22.9, 22.7, 20.9, 20.2, 16.5. HRESIMS [M+H] + calcd for C21H32NO2

330.2433, found 330.2441; [α] 24 D = +6.98��(c 1.80).

Preparation of 2.42 and 2.43:

Identical to 2.20 and 2.34.

For 2.42 HRESIMS [M+H] + calcd for C21H32NO2 330.2433, found 330.2440; [α] 24 D = +

8.3��(c 3.21).

For 2.43 HRESIMS [M+H] + calcd for C21H32NO2 330.2433, found 330.2440; [α] 24 D = -

5.7��(c 1.47).

Preparation of 2.40:

To 2.19 (20 mg, 0.060 mmol) dissolved in 1 mL of pyridine was added

hydroxylamine hydrochloride (33.8 mg, 0.49 mmol), and heated at 50 � C. After three and a

half hours the reaction mixture was cooled to room temperature, saturated NH4Cl (aq) solution

was added (20 mL), and the aqueous phase was extracted three times with methylene

chloride (100 mL). The organic extracts were combined and dried with MgSO4, and

concentrated using a rotary evaporator. The crude was purified using flash column

chromatography (hexanes:ethyl acetate 1:1) to give 2.40 as a white solid (15.5 mg, 0.045

mmol, 75 %). 1 H NMR (400 MHz, (CD3)2CO) �� 6.22 (s, 1H), 6.12 (s, 1H), 3.06 (ddd, J =

15.9, 3.2, 2.7 Hz, 1H), 2.59 (t, J = 14.1 Hz, 1H), 2.53 (dt, J = 12.1, 3.1 Hz, 1H), 2.44 (dd, J =

14.4, 6.1 Hz, 1H), 2.29 (ddd, J = 18.5, 6.4, 5.8 Hz, 1H), 1.75-1.72 (m, 1H), 1.71-1.69 (m,

2H), 1.68-1.66 (m, 1H), 1.65-1.61 (m, 1H), 1.29-1.23 (m ,2H), 1.15 (s, 3H), 1.14 (s, 3H),

1.11 (s, 3H), 1.08 (s, 3H); 13 C NMR (100 MHz, (CD3)2CO) � 171.9, 165.8, 158.5, 154.8,

146.8, 132.1, 105.8, 102.8, 65.9, 61.5, 58.3, 47.4, 41.7, 40.0, 38.4, 29.2, 24.3, 21.7, 18.1,

16.8, 15.5. HRESIMS [M+H] + calcd for C21H30O3N 344.2226, found 344.2230; [α] 24 D = -

14.94��(c 0.02).

Preparation of 2.44:

To 2.40 (10.3 mg, 0.029 mmol) dissolved in 1 mL of methylene chloride at 0 � C was

added trfifluoroacetic anhydride (0.11 mL, 0.79 mmol) and allowed to stir for 1 hour. The

reaction was quenched with 0.1 mL of water and the reaction mixture was concentrated

under a stream of nitrogen. The crude was purified using flash column chromatography

(methylene chloride:methanol 12:1) to give 2.44 (10.0 mg, 0.029 mmol, 100 %) as a white

solid. 1 H NMR (400 MHz, CD3OD) �� 6.17 (d, J = 1.7 Hz, 1H), 6.02 (d, J = 1.9 Hz, 1H), 3.45

(s, 1H), 2.65-2.56 (m, 2H), 2.51-2.43 (m, 3H), 1.83-1.70 (m, 4H), 1.69-1.61 (m, 2H), 1.59-

1.52 (m, 1H), 1.33 (s, 3H), 1.32 (s, 3H), 1.22 (s, 3H), 1.11 (s, 3H); 13 C NMR (100 MHz,

CD3OD) � 179.3, 157.5, 154.3, 145.8, 131.6, 104.6, 101.9, 65.5, 57.8, 56.9, 46.6, 41.2, 39.3,

39.0, 33.7, 32.6, 30.6, 26.4, 24.3, 20.3, 17.5. HRESIMS [M+Na] + calcd for C21H29NO3Na

366.2045, found 366.2035; [α] 24 D = +122.1��(c 0.07).

Preparation of 2.45:

To 2.44 (8.0 mg, 0.023 mmol) dissolved in 4 mL of tetrahydrofuran was added

LiAlH4 (0.072 mL. 2.0 M in tetrahydrofuran, 0.14 mmol) and heated to reflux overnight.

Upon completion, the reaction was quenched with 0.1 mL methanol, and 0.1 mL of 6 M HCl

added. The mixture was concentrated using a rotary evaporator, then lyophilized and purified

using a 2 g reversed phase sep-pak (which was washed with 10 mL of methanol followed by

10 mL of water). Once loaded the column was flushed with water (20 mL), 60:40

water:methanol (50 mL), and methanol (50 mL). The water:methanol fraction after

concentration and lyophilization contained 2.45 (4 mg, 0.011 mmol, 47 %) as a white solid.

1 H NMR (600 MHz, CD3OD) � 6.16 (s, 1H), 6.03 (d, J = 1.5 Hz, 1H), 3.23-3.15 (m, 2H),

2.62 (t, J = 13.9 Hz, 1H), 2.57 (dd, J = 8.2 Hz, 1H), 2.52 (dt, J = 12.9, 2.9 Hz, 1H), 2.00-1.96

(m, 1H), 1.94-1.87 (m, 3H), 1.75-1.72 (m, 2H), 1.70-1.66 (m, 1H), 1.64-1.58 (m, 1H), 1.48

(s, 3H), 1.42 (s, 3H), 1.39 (m, 1H), 1.22 (s, 3H), 1.13 (s, 3H); 13 C NMR (150 MHz, CD3OD)

� 157.7, 154.3, 145.2, 131.2, 104.4, 101.9, 64.6, 63.6, 54.7, 46.2, 43.9, 42.6, 42.2, 38.3, 31.2,

27.9, 24.9, 24.3, 24.0, 19.5, 14.9. HREIMS [M] + calcd for C21H31NO2 329.23548, found

329.23569; [α] 24 D = +20.50��(c 0.02).

Preparation of 2.47:

To sodium hydride (8.0 mg, 60 % in mineral oil, 0.20 mmol) washed two times with

hexanes was added 0.5 mL of tetrahydrofuran. To this heterogeneous mixture was added 2.46

(24.3 mg, 0.067 mmol) dissolved in 2.0 mL of tetrahydrofuran and allowed to stir for 10

minutes. To this mixture was added carbon disulfide (0.024 mL, 0.402 mmol), and allowed

to stir for thirty minutes. Then methyl iodide (0.037 mL, 0.60 mmol) was added neat and the

mixture allowed to stir overnight at room temperature. The mixture was quenched with 0.1

mL of methanol, concentrated under a stream of nitrogen and filter through a plug of silica

with 20 mL of hexanes:ethyl acetate (3:1), and concentrated using a rotary evaporator. The

crude mixture was used in the following step without further purification. The mixture was

dissolved in 3 mL of toluene to which was added tributyltin hydride (87.5 mg, 0.30 mmol)

and AIBN (0.06 mL, 0.012 mmol), and the reaction mixture heated to 120 � C for thirty

minutes. The reaction was then allowed to cool down to room temperature, and concentrated

under a stream of nitrogen. The crude mixture was purified using flash column

chromatography (hexanes:ethyl acetate 15:1), to give 2.47 (18.8 mg, 0.054 mmol, 82 % over

two steps). The 1 H and 13 C NMR spectra were identical to previously reported data (Matt

Nodwell). HRESIMS [M+H] + calcd for C23H35O2 343.2637, found 343.2640; [α] 24 D = -

4.25��(c 0.07).

Preparation of 2.48:

To 2.47 (20 mg, 0.058 mmol) dissolved in 20 mL methylene chloride was added

boron tribromide (0.17 mL, 0.175 mmol, 1.0 M solution in methylene chloride). After

stirring for ninety minutes the reaction was quenched with the addition of 0.1 mL of

methanol, and concentrated under a stream of nitrogen. The crude mixture was then purified

using flash column chromatography to give 2.48 (12.1 mg, 0.038 mmol, 66 %). The 1 H and

13 C NMR spectra were identical to 2.18 (Matt Nodwell). HRESIMS [M+H] + calcd for

C21H29O2 313.2176, found 313.2168; [α] 24 D = -18.1� (c 0.05).

Preparation of 2.61:

To acid chloride 2.57 (28.7 mg, 0.163 mmol) dissolved in 2 mL of tetrahydrofuran

was added 2.3 (56.0 mg, 0.179 mmol) and triethylamine (0.22 mL, 1.57 mmol) at 0 � C, and

the reaction mixture allowed to warm to room temperature overnight. The crude reaction

mixture was concentrated under a stream of nitrogen, and purified using flash column

chromatography (hexanes:ethyl acetate 12:1) to give 2.61 (30.0 mg, 0.063 mmol, 38.6 %). 1 H

NMR (400 MHz, CDCl3) ��8.17 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.91 (s, 1H),

6.73, (2, 1H), 4.77 (s, 2H), 2.7 (m, 1H), 2.60-2.55 (m, 2H), 2.40 (dt, J = 12.0, 3.2 Hz, 1H),

2.35 (s, 3H), 1.85-1.44 (m, 8H), 1.24 (td, J = 14.4, 4.8 Hz, 1H), 1.14 (s, 3H), 1.08 (s, 3H),

1.04 (m, 1H), 1.01 (m, 1H), 0.92 (s, 6H); 13 C NMR (100 MHz, CDCl3) � 165.2, 161.7, 149.3,

148.9, 144.5, 133.2, 132.3, 123.2, 121.6, 116.2, 114.8, 78.0, 76.4, 64.5, 57.2, 56.0, 47.6, 42.7,

40.2, 38.8, 37.2, 33.5, 33.2, 29.1, 21.3, 20.4, 19.7, 19.1, 18.5, 16.3. HRESIMS [M+H] + calcd

for C32H39O3 471.2899, found 471.2829; [α] 24 D = +12.63��(c 2.2).

Preparation of 2.53:

To 2.61 (754 mg, 1.60 mmol) was added titanium tetrachloride (0.35 mL, 3.20 mmol)

and the mixture exposed to an oil bath at 140 � C for fifteen minutes. The black mixture was

then quenched with 50 mL of 0.1 N HCl and extracted three times with methylene chloride

(250 mL). The organic extracts were combined, concentrated using a rotary evaporator, and

purified using flash column chromatography (hexanes:ethyl acetate 7:1) to give 2.53 (69.3

mg, 0.147 mmol, 9.1 %). 1 H NMR (400 MHz, CDCl3) ��9.97 (s, 1H), 7.63 (d, J = 9.2 Hz,

2H), 7.03 (d, J = 8.8 Hz, 2H), 6.60 (s, 1H), 4.79 (s, 2H), 2.56 (t, J = 2.4 Hz, 1H), 2.33 (s, 3H),

2.26-2.19 (m, 1H), 1.98 (dd, J = 15.2, 6.0 Hz, 1H), 1.71-1.16 (m, 9H), 1.13 (s, 3H), 1.07-0.98

(m, 1H), 0.90 (s, 3H), 0.87 (m, 1H), 0.84 (s, 3H), 0.82 (s, 3H), 0.77 (td, J = 12, 4 Hz, 1H);

13 C NMR (100 MHz, CDCl3) � 200.0, 161.0, 158.2, 144.5, 144.5, 140.1, 133.7, 131.2, 117.9,

117.5, 114.7, 77.4, 76.2, 64.3, 57.0, 56.0, 46.7, 42.5, 39.8, 38.9, 36.9, 33.4, 33.1, 31.4, 21.2,

20.4, 19.7, 19.6, 18.2, 16.0. HRESIMS [M+Na] + calcd for C32H38O3Na 493.2719, found

493.2726; [α] 24 D = +59.49� (c 3.46).

Figure 2.42 NOESY spectrum of 2.53 in CDCl3 at 400 MHz.

His-hSHIP1 Enzyme Assay: His-hSHIP1 enzyme assay was performed in 96-well

microtiter plates with 2.5 to 10 ng enzyme/well and 50 ng/well, respectively, in a total

volume of 25 �L of SHIP1 assay buffer (20 mM Tris HCl (pH 7.5), 10 mM MgCl2 and 0.02

% Tween-20). Recombinant His-hSHIP1 enzyme was incubated with test articles or vehicle

(2 % ethanol) and 50 �M inositol-1,3,4,5-tetrakisphosphate (IP4) for 15 min at 37 �C in a

shaking incubator. After 15 min at 37 �C, the amount of inorganic phosphate released was

assessed by the addition of BIOMOL GREEN reagent and incubation for 20 min at room

temperature before measuring the absorbance at 650 nm.

Akt Activation Assay: MOLT-4 and Jurkat T-ALL cells were cultured in RPMI

1640 containing 10 % FBS and 1 % penicillin/streptomycin at 37 � C in a water-jacketed CO2

(5 %) incubator. Cells were seeded at 0.2-0.3 x 10 6 cells/mL and grown for 2 to 3 days before

passaging. Cells that exceeded 25 passages were not used for studies and were discarded.

Akt Phosphorylation Assay: Cells were cultured in serum free RPMI at 1 x 10 6

cells/mL. After overnight culture, 2-3 x 10 6 cells were treated in a 15 mL conical tube with

test article for 30 min followed by IGF-1 stimulation at 0.1 μg/ml for 60 min. The final

concentration of the drug vehicle (dimethyl sulfoxide) was 0.1 %. After treatment, cells were

washed once with ice cold DPBS and lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 140

mM NaCl, 1 % NP-40, Complete Mini Protease Inhibitor Cocktail, 10 mM NaF, 1 mM

Na3VO4, and 1 mM β-glycerol phosphate) on ice for 30 min with vortexing every 10 min.

Samples were then centrifuged at 14,000 rpm for 20 min, and supernatants were collected as

total cell lysates. Akt phosphorylation at S473 in each sample was determined by western

blotting.

Western Blotting: Protein concentration in each sample was determined

colorimetrically using bicinchoninic acid (BCA) assay. Approximately 15-20 μg of total

protein from each sample was mixed with 6 x sample loading buffer (250 mM Tris-HCl, pH

6.8, 30 % glycerol, 10 % SDS, 0.012 % bromophenol blue and 0.6 M DTT) and boiled for 5

min before loading onto a polyacrylamide gel for SDS-PAGE. Proteins from each sample

were separated on a 4-12 % Tris-Glycine gel for 1.5 h with a constant voltage of 125 Volts.

After electrophoresis, proteins were transferred to a nitrocellulose membrane using the iBlot

Dry Transfer system (Life Technologies, Carlsbad, CA, USA). The membrane was then

blocked in 5 % BSA in PBS containing 0.1 % Tween-20 (PBS-T) for 1 h at room

temperature before probing with primary antibodies overnight at 4 � C. The following

antibodies were used: mouse anti-SHIP1 (1:500; v/v), rabbit anti-pAkt (S473) (1:1000; v/v),

rabbit anti-Akt (1:2000), and rabbit anti-β-actin (1:2000; v/v). The membrane was then

incubated with goat anti-rabbit IgG HRP-linked or goat anti-mouse IgG HRP-linked

secondary antibodies (1:3000; v/v) for 1 h at room temperature. Target proteins on the

membrane were detected with ECL solution and exposed on a film.

Mouse Passive Cutaneous Anaphylaxis Model: The in vivo animal study protocols

were approved by the local ethics committee. Forty BALB/c male mice (8 weeks old) were

obtained from Charles River Laboratories (Hollister, CA, USA). Animals were acclimated

for a minimum of five days prior to the start of the study. They were housed five animals per

cage in polypropylene cages, and were allowed free access to food and water. A 12 h

light/dark cycle was maintained. Each animal was injected intradermally in the right ear with

25 ng of anti-DNP-IgE in 20 μL PBS. The left ears were not injected and served as a negative

control. 24 h post-injection, 2.20/2.42 was administered once by oral gavage in saline (0.01,

0.1, or 10 mg/kg; 10.0 ml/kg dose volume) in a model of IgE-mediated passive cutaneous

anaphylaxis. Sixty minutes after dosing, each animal was given a tail vein injection of 2 %

Evans’ blue (0.22 μm filtered, in 200 μL saline) followed by a second tail intravenous

injection of 100 μg DNP-HSA (in 200 μl PBS) (Sigma). Sixty minutes following the DNP-

HSA injection, mice were euthanized using CO2 inhalation. Ear biopsies were performed

with four-millimeter punches from both ears placed into 100 μL formamide in 96 well PCR

plates to elute the Evans’ Blue dye. To minimize evaporation, plates were then sealed during

incubation in a 70 � C water bath overnight. Eighty μL of eluents were transferred to flat-

bottom 96-well plates and read using a SpectraMax M5 spectrophotometer (Molecular

Devices, Sunnyvale, CA, USA) at 620 nm. Background readings from all samples were taken

at 740 nm and subtracted from the 620 nm readings. A blank reading was made on a sample

of formamide and subtracted from all readings. Data are reported as optical density.

Water Solubility of Pelorol Analogues: Samples were weighed accurately in

duplicate (~3 mg each) in 4 mL glass vials. The appropriate amount of deionized water was

added to obtain a final concentration of 6 mg/mL. A stir bar was placed in the vial and the

two mixtures were stirred for 24 h, after which the samples were filtered using a glass filter

membrane. The resulting filtrate was centrifuged in a glass conical tube for 10 min at 10,000

rpm to sediment any precipitate that may have passed through the glass membrane. The

supernatant was sampled for HPLC analysis. The concentration of the test compound was

determined by HPLC using a six-point standard curve of the compound prepared in

methanol.

Chapter 3: Glycerol Ethers from the Sponge Niphates digitalis that Block

Androgen Receptor Transcriptional Activity in Prostate Cancer Cells

3.1 Castration Recurrent Prostate Cancer (CRPC)

Prostate cancer ranks second among most often diagnosed cancers in Canadian men.

It ranks third as a cause of cancer death in Canadian men aged 65 and over. A key nuclear

receptor associated with the prostate is the androgen receptor (AR). Endogenous androgens

such as testosterone (3.1) and dihydrotestosterone (3.2) mediate their biological effects

through the AR (Figure 3.1). 124

Figure 3.1 Endogenous androgens found in humans.

The rate of prostate tissue growth is dependent on the levels of endogenous androgens

in the body. 125 This is typified during puberty when increased androgen production causes

growth of the prostate. Conversely, there is a decrease in prostate volume when androgen

levels diminish. 126 The dependence on androgens by the prostate for mitogenic stimulus is

currently used as a means to treat prostate cancer.

The first line treatment for low grade tumors 127 is prostatectomy (prostate removal)

and localized radiation therapy. Unfortunately, tumour recurrence occurs in approximately

20-40 % of patients treated with first line therapies, at which point androgen ablation therapy

is used. Androgen ablation therapy is achieved either by surgical castration (testes removal),

or by chemical castration. The goal of these therapeutic treatments is to diminish the levels of

androgens in the body in order to decrease prostate tumour size. There are two ways of

achieving chemical castration. The first is the use of small molecules, which inhibit the

production of androgens. Abiraterone (3.3) is a good example (Figure 3.2). 128 Abiraterone

(3.3) inhibits 17 α-hydroxylase/C17,20-lyase 129 (CYP17A1), an enzyme that is expressed in

testicular, adrenal, and prostatic tumor tissues. The enzyme CYP17A1 is responsible for the

production of dehydroepiandrosterone and androstenedione, both of which are endogenous

androgens and precursors to testosterone (3.1). By decreasing the level of

dehydroepiandrosterone in the body, abiraterone (3.3) decreases the level of testosterone

(3.1) resulting in a decrease in prostate tissue volume.

The second method of achieving chemical castration is by using androgen receptor

antagonists. 130 Antagonists are ligands which have binding affinity to a biological target but

do not trigger the biological response of the target upon binding. Instead they block androgen

binding and, therefore, prevent the agonist (androgens) mediated response. Examples include

bicalutamide (3.3) 131 and MDV3100 (3.5) 132 (phase III clinical trials) (Figure 3.2).

Figure 3.2 Examples of antiandrogens.

Chemical methods of decreasing the level of androgens are only effective for a short

period of time. Eventually, the malignancy will begin to grow in the absence of androgens to

form castration recurrent prostate cancer 133 (CRPC). Once CRPC has been established,

alternative treatments are employed which do not specifically target the AR (docetaxel or

sipuleucel-T), however, they only result in an increased life expectancy of two to four

months. There is currently no successful treatment of CRPC.

3.2 The AR NTD as a Novel Therapeutic Target for Treating CRPC

The AR comprises a C-terminal ligand binding domain (LBD), a DNA binding

domain (DBD), a hinge region, and an N-terminus domain (NTD), containing the activation

function-1 (AF1) region (Figure 3.3). 127 Current therapies for treating prostate cancer involve

targeting the LBD of the AR. Although these therapies are effective initially, these

approaches eventually fail. 134 Evidence supporting several mechanisms for the failure of

antiandrogens exist and include overexpression of the AR, 135 mutations allowing for the

activation of the AR by androgens or antiandrogens, 136 AR activation by non-ligands, 137 and

expression of splice variants of the AR which lack the LBD altogether. 138 These factors result

in the failure of treating CRPC by small molecule antagonists of the LBD of the AR.

Figure 3.3 Androgen receptor structure.

Targeting the AR through the DBD is an alternative method, since it has been

crystallized, 139 and so could potentially allow for rational drug design. However, the high

degree of homology found in the DBD to other steroid receptors sites prevents it from being

a promising biological target in the development of a therapeutic candidate.

An important component of the AR is the NTD, a disordered region of the protein.

The NTD plays a critical role in transcriptional activity of the AR in the absence of

androgens, resulting in androgen-independent proliferation of prostate cancer cells. No AR

transcriptional activity is possible without a functioning NTD AF1 region. This has made

targeting of the NTD by small molecules an appealing avenue of exploration for treating

CRPC. A novel cell-based assay developed by Sadar 140 et al. has provided a method to

screen extract libraries for compounds that are NTD antagonists. Use of this assay led to the

isolation and structural elucidation of the AR NTD antagonists sintokamide A (3.6) 140 and

EPI-001 (3.7) 141 (Figure 3.4).

Figure 3.4 Small molecule antagonists of the AR NTD.

Continued efforts to discover new AR NTD antagonists led to further screening of the

Andersen natural product library. The crude methanol extracts of the marine sponge Niphates

digitalis collected in Dominica showed strong activity in the screening assay. Bioactivity-

guided fractionation of the extracts led to the isolation of the glycerol ethers niphatenone A

(3.8) and B (3.9) as the active compounds by David Williams (Figure 3.5).

Figure 3.5 Niphatenones A (3.8) and B (3.9).

The small quantity of niphatenone A (3.8) (0.1 mg) and B (3.9) (0.1 mg) available

from the Niphates digitalis extract made it impossible to determine the absolute configuration

of the natural products, or carry out further evaluation of their biological activities. Hence, a

total synthesis of (S) and (R)-niphatenone A (3.8) and (S) and (R)-niphatenone B (3.9) was

undertaken. Niphatenone A (3.8) was the first of the two natural products to be constructed.

Retrosynthetic analysis indicated that the E-enone of the natural product may come from

phosphorane 3.10 and aldehyde 3.11. Phosphorane 3.10 can be constructed from glycerol

3.12, alkyl bromide 3.13, and phosphorane 3.14 (Scheme 3.1).

Scheme 3.1 Retrosynthetic analysis of niphatenone A (3.8).

The configuration of the natural product was unknown, therefore both stereoisomers

needed to be constructed and compared with the natural material. The synthesis of (R)-

niphatenone A (3.21) began with commercially available (S)-dioxolane (3.15). This would

provide the only stereocenter of the natural product (Scheme 3.2). Deprotonation of 3.15 with

sodium hydride in the presence of 15C-5 followed by treatment with alkyl bromide 3.13 gave

the benzyl protected glycerol ether 3.16. 142 Hydrogenolysis of 3.16 with 10 %

palladium/charcoal under hydrogen (1 atm) gave the primary alcohol 3.17. Intermediate 3.17

was converted to bromide 3.18 by the Appel reaction. 143 Deprotonation of commercially

available phosphorane 144 3.14 with n-BuLi, followed by alkylation with bromide 3.18,

provided the Wittig reagent 3.19 (Scheme 3.2).

Scheme 3.2 Synthesis of (R)-niphatenone A (3.21).

Without any further purification, intermediate 3.19 was used in the subsequent

olefination with commercially available aldehyde 3.11 to give enone 3.20 in a 63 % yield

over two steps. Attempts to remove the acetonide protecting group of 3.20 with p-

toluenesulfonic acid in methanol gave poor yields (17 %). However, HCl in a mixture of

tetrahydrofuran and water 145 gave (R)-niphatenone (3.21) in high yield (Scheme 3.2).

Repeating the synthesis using (R)-dioxolane (3.22) as the starting material gave (S)-

niphatenone A (3.23) (Scheme 3.3).

Scheme 3.3 Synthesis of (S)-niphatenone A (3.23).

Next, we wanted to construct niphatenone B (3.9). Using retrosynthetic analysis, we

see that the E-enone in 3.9 can come from a Horner–Wadsworth–Emmons 146 (HWE)

olefination between aldehyde 3.24 and phosphonate 3.25. Aldehyde 3.24 can be furnished

from coupling glycerol 3.12 and bromide 3.26 (Scheme 3.4).

Scheme 3.4 Retrosynthesis of niphatenone B (3.9).

The synthesis of (R)-niphatenone B (3.31) started with the deprotonation of (S)-

dioxolane (3.15) with sodium hydride followed by alkylation with bromide 3.26 to give ether

3.27 (Scheme 3.5). Deprotection of the benzyl protecting group of 3.27, followed by Dess–

Martin periodinane oxidation 147 under basic conditions gave aldehyde 3.29.

Scheme 3.5 Synthesis of (R)-niphatenone B (3.31).

For the HWE olefination, phosphonate 3.25 was required (Scheme 3.5). Treatment of

commercially available epoxide 3.32 with cerous chloride 148 and sodium bromide gave

bromohydrin 3.33. Intermediate 3.33 was then oxidized with DMP to afford α-bromo-ketone

3.34. Refluxing 3.34 in the presence of triethylphosphite gave phosphonate 3.25 149 in high

yield. Sodium hydride was initially used as the base for the HWE step. However, it provided

poor yields (< 40 %). It was found that barium hydroxide 150 in a mixture of tetrahydrofuran

and water gave 3.30 in a good yield. Finally, deprotection of acetonide 3.30 provided (R)-

niphatenone B (3.31) in an 86 % yield (Scheme 3.5). Repeating the synthesis using (R)-

dioxolane (3.22) as the starting material gave (S)-niphatenone B (3.35) (Scheme 3.6).

Scheme 3.6 Synthesis of (S)-niphatenone B (3.35).

Comparison of the 1 H NMR, 13 C NMR, and MS data of the natural product with the

synthetic material confirmed the proposed structures of niphatenone A (3.8) and B (3.9).

Furthermore, having constructed all four stereoisomers of niphatenone A (3.8) and B (3.9), it

was possible to determine the absolute configuration of the natural product. The natural

product was re-isolated from the sponge by the author, and chiral HPLC analysis completed

by David Williams suggested that the natural product configuration for both niphatenones A

(3.8) and B (3.9) was the (S) configuration.

This configuration was in agreement with ceratodictyol A (3.36) and B (3.37), that

are homologs of (S)-niphatenone A (3.23) and B (3.35) (Figure 3.6). Ceratodictyol A (3.36)

and B (3.37) were isolated by Matsunaga 151 and coworkers from the red algae/sponge

assembly Ceratodictyon spongiosum/Haliclona cymaeformis. The ceratodictyols were

reported to have modest in vitro cytotoxicity against a human cervical cancer cell line (IC50 =

67 �M).

Figure 3.6 Ceratodictyol A (3.36) and B (3.37).

3.3 Synthetic Analogues of (R)-Niphatenone B (3.31)

Having confirmed the structure and absolute configuration of both niphatenone A

(3.8) and B (3.9), we wanted to probe the SAR of these novel AR antagonists. (R)-

Niphatenone B (3.31) was chosen as the lead structure since preliminary biological data

suggested it was the most potent of the four natural and synthetic compounds constructed

(Section 3.5). We were interested in how modifications to the glycerol ether, double bond,

and alkyl chain moieties would affect the activity of these compounds in vitro (Figure 3.7).

Figure 3.7 Proposed SAR of (R)-niphatenone B (3.31).

The first goal was to examine the effect of chain length modification. An analogue of

(R)-niphatenone B (3.31) was constructed with a chain length exceeding the natural product

by six carbon units (3.43) (Scheme 3.7). The synthesis was analogous to that of (R)-

niphatenone B (3.31) except for the structure of the phosphonate (3.41) required for the HWE

olefination step.

Scheme 3.7 Synthesis of a long chain (R)-niphatenone B (3.31) analogue 3.43.

Similarly, an analogue with ten carbon units less was constructed (Scheme 3.8). This

synthesis utilized commercially available phosphonate 3.44 to provide the methyl ketone

moiety in analogue 3.46.

Scheme 3.8 Synthesis of a short chain (R)-niphatenone B (3.31) analogue 3.46.

Next, modifications to the glycerol ether portion of (R)-niphatenone B (3.31) were

undertaken. An epoxide intermediate was designed to provide access to several different

glycerol-modified analogues. The synthesis began with deprotonation of glycidol 3.47 (98 %

ee) in the presence of 15C-5 and bromide 3.26 to yield benzyl-protected alcohol 3.48.

Deprotection of the benzyl group followed by DMP oxidation and HWE olefination with

phosphonate 3.25 yielded the key epoxide intermediate 3.50 (Scheme 3.9).

Scheme 3.9 (R)-Niphatenone B (3.31) glycerol analogues.

In an effort to enhance the solubility of (R)-niphatenone B (3.31) (CLogP = 4.59) in

water, a pegylated 152 version was constructed. Epoxide 3.50 in triethylene glycol (3.51) as

the solvent in the presence of catalytic amounts of bismuth triflate gave the PEG analogue

3.52 (CLogP = 4.39). The glycerol fragment of the niphatenones is also found in EPI-001 141

(3.6) (Figure 3.4), a highly effective antagonist of the AR NTD. It was found that the

chlorohydrin functionality of EPI-001 (3.6) is required for its AR NTD antagonist properties.

Therefore, it was of interest to construct the chlorohydrin analogue of (R)-niphatenone B

(3.31). This was accomplished by opening the epoxide of 3.50 with cerous chloride to give

chlorohydrin 3.53 (Scheme 3.9).

It has been shown that fluorine substitution in the context of small molecule drug

development allows for enhanced physical chemical properties and metabolic stabilities. 153

With this in mind, fluorohydrin 3.54 was prepared from 3.31 using Xtal-FluorE ®154 (Scheme

3.9).

Scheme 3.10 All carbon backbone (R)-niphatenone B (3.31) analogue 3.61.

Next we constructed the all carbon backbone version of (R)-niphatenone B (3.31)

(Scheme 3.10). The synthesis began by brominating primary alcohol 3.55 using the Appel

reaction to provide bromide 3.56. Next, bromide 3.26 was dissolved in tetrahydrofuran and in

the presence of magnesium metal formed the Grignard reagent in situ, followed by alkylation

with bromide 3.56 to give benzyl protected acetonide 3.57 (Scheme 3.10). Deprotection of

3.57 with 10 % palladium/charcoal under H2 (1 atm) followed by DMP oxidation afforded

aldehyde 3.59. Subsequent HWE olefination between aldehyde 3.59 and phosphonate 3.25

gave intermediate 3.60. Acetonide deprotection of enone 3.60 gave the all carbon backbone

analogue 3.61 (Scheme 3.10). In addition to the alkyl and glycerol moieties, the niphatenones

also contain an enone functionality, which may be a potential Michael acceptor to the

biological target. In order to determine if there is a covalent binding mechanism between the

niphatenones, and the AR by a Michael addition, a reduced version of (R)-niphatenone B

(3.31) was constructed in analogue 3.63 (Scheme 3.11).

Scheme 3.11 Dihydro (R)-niphatenone B (3.31) analogue 3.63.

Acetonide 3.30 was hydrogenated in the presence of 10 % palladium/charcoal and H2

(1 atm) to give 3.62. Compound 3.62 was deprotected with HCl in a mixture of

tetrahydrofuran and water to give analogue 3.63 (Scheme 3.11). Biological testing of 3.63

(Section 3.5) showed attenuated activity relative to (R)-niphatenone B (3.31), which

indicated a potential covalent interaction between the natural products and the NTD of the

AR.

3.4 Synthesis of a Click Chemistry Probe and Fluorescent Probe

The decreased biological activity that was obtained from the reduced enone analogue

3.63 suggested covalent binding between the natural product and the NTD of the AR. To test

our hypothesis, a chemical probe (Chapter 2, Section 2.5) based on (R)-niphatenone B (3.31)

was constructed. The probe needed to incorporate functionality that would allow for the

attachment of a fluorophore for visualization of the probe-ligand complex after covalent

binding had occurred (Figure 3.8).

Figure 3.8 Fluorescent probe mode of action.

Fluorescent probes have been developed for in vitro and in vivo imaging of different

biological targets. 155 There are two main types of fluorescent imaging probes (Figure 3.9).

Non-targeting probes do not have affinity for a specific biological target, and are typically

used to observe a biological process. An example is indocyanine green (3.64), 156 which is in

current clinical use for evaluating blood flow and clearance (Figure 3.9).

The second type of fluorescent imaging probe is a targeting probe in which a

fluorophore is bound to a ligand that has a high affinity for a biological target. Accumulation

of the fluorophore at the target site allows for visualization of the desired tissue. An example

of an active probe is the quinoline derivative TDPQ (3.65), 157 which is a selective non-

steroidal AR antagonist, with fluorescent properties (Figure 3.9).

Figure 3.9 Non-targeting and targeting fluorescent probes.

We chose click chemistry 111 to attach the fluorophore to the probe-ligand complex.

This led to the construction of the propargyl ether probe 3.67. The synthesis started with

epoxide analogue 3.50 and ring opening with propargyl alcohol (3.66) in the presence of

catalytic amounts of bismuth triflate gave the propargyl ether analogue 3.67 (Scheme 3.12).

Scheme 3.12 Propargyl ether analogue 3.67.

The fluorescent tag chosen was a fluorescein derivative (3.69) carrying an azide 158

moiety, which allows for attachment using click chemistry. Scheme 3.13 illustrates a

potential mechanism for the covalent binding between probe 3.67 and the NTD of the AR.

The results are described in Section 3.5.

Scheme 3.13 Click chemistry of probe 3.67 and fluorophore 3.69.

An alternative fluorescent probe for the NTD of the AR was also designed based on

(S)-niphatenone B (3.35). Instead of using a tagged approach as in probe 3.67, the enone

functionality was incorporated into the fluorophore, similar to a fluorescent imaging agent

developed to detect cellular glutathione by Kim et al. (Figure 3.10). 159 The basis of the

design is that the unreacted form in 3.71 emits a green fluorescence, however, when the

conjugation is disrupted by the nucleophilic attack of GSH to give 3.72 a blue shift in

fluorescence emission occurs. This allowed for cellular imaging and detection of GSH in

vivo. Since the coumarin-based fluorescent probe in 3.71 contained an enone functionality

that was also found in the niphatenones, 3.71 was incorporated into the natural product,

similar to some reported for fluorescent estrogen receptor ligands. 160

Figure 3.10 Cellular imaging agent of glutathione.

The synthesis of a potential fluorescent probe for the NTD of the AR started with

commercially available aldehyde 3.73 which was cyclized with diethyl malonate under basic

conditions, followed by refluxing in acid to give coumarin 3.75 (Scheme 3.14). Intermediate

3.75 underwent the subsequent Vilsmeier-Haack reaction to give aldehyde 3.76 following

known protocol. 161 Aldehyde 3.76 was then reacted with Wittig reagent 3.19 used in the

synthesis of (R) (3.31) and (S)-niphatenone B (3.35) (Scheme 3.1), to give acetonide 3.77.

Deprotection of 3.77 yielded coumarin analogue 3.78, a potential fluorescent probe of the

NTD AR (Scheme 3.14). Dr. Marianne Sadar at the BC Cancer Agency is currently

investigating compound 3.78.

Scheme 3.14 Synthesis of a potential AR NTD fluorescent probe (3.78).

3.5 Biological Results

The biological activities of the natural product (S)-niphatenones A (3.23), and B

(3.35), along with selected synthetic analogues were tested in an assay that measures AR

transcriptional activity 140 (Figure 3.11). This assay consists of transfecting LNCaP human

prostate cancer cells that express functional AR, with a luciferase reporter that is regulated by

the AR in response to androgen. If a compound is an AR antagonist, the amount of light

emission produced by luciferase when cells are stimulated with a synthetic androgen or

forskolin is diminished.

All the compounds were tested at a concentration of 7 �M with the exception of 3.43,

which was limited to 3.5 �M due to poor solubility. (S)-Niphatenone B (3.35) shows

approximately 50 % inhibition at the test concentration, while (S)-niphatenone A (3.23)

shows weaker activity.

Figure 3.11 AR transcriptional activity assay of the niphatenones and their analogues.

The synthetic unnatural enantiomers (R)-niphatenone A (3.21) and (R)-niphatenone B

(3.31) are both more active than the corresponding natural (S) isomers 3.23 and 3.35. (R)-

niphatenone B (3.31) is the most active compound tested in this series. The reduced enone

analogue (3.63) is less active than (R)-niphatenone B (3.31), but only slightly less active than

(S)-niphatenone B (3.35). This indicates that the enone moiety plays a role in the activity of

(R)-niphatenone B (3.31), and most likely plays a role for the other analogues. However,

covalent binding is not required for activity in the assay. Lengthened alkyl chain analogue

3.43 is about half as active as (R)-niphatenone B (3.31) at half the concentration. Analogue

3.46 with a one-carbon alkyl substituent has essentially lost all activity. This indicates that

the alkyl substituent is necessary for activity and that there is most likely an ideal chain

length.

Removing the glycerol ether oxygen atom from the linear skeleton of (R)-niphatenone

B (3.31) to give the all carbon chain analogue 3.61 produced a compound with roughly half

the activity of 3.31. This indicates that the glycerol ether moiety plays a role in the activity,

most likely through hydrogen bonding. Similarly, acetonide 3.30 and epoxide 3.50 are less

active than 3.31. PEG ether (3.52), chlorohydrin (3.53), and alkyne ether (3.67) analogues are

all less active than (R)-niphatenone B (3.31) and comparable in activity to the natural product

(S)-niphatenone B (3.35). The diminished activity observed for analogues 3.52, 3.53, and

3.67 can perhaps be attributed to the loss of the glycerol primary alcohol as it was also the

case for acetonide 3.30, and the epoxide 3.50. While these modifications were successful for

EPI-001 (3.7), they were unfruitful for the niphatenones and suggest a different binding

mechanism.

Next, an in vitro assay was completed to observe whether the effects of the

niphatenones are due to non-specific toxicity, or due to a specific interaction with the AR.

This was accomplished by comparing the proliferation of LNCaP (AR–) cells versus PC3

(AR+) cells (Figure 3.12). The assay works by incubating LNCaP cells in the presence of a

known androgen R1881 (metribolone), causing mitogenic effects, in addition to the test

compound. If the compound inhibits the AR there should be little to no R1881 stimulated

proliferation of LNCaP cells. The reference experiment is done with bicalutamide (3.4), a

known AR antagonist resulting in no R1881 stimulated proliferation. When (S)-niphatenone

B (3.35) and (R)-niphatenone B (3.31) were tested, they inhibited R1881-induced

proliferation of LNCaP cells at comparable concentrations to bicalutamide (3.4). The same

concentration of either (S) or (R) niphatenone B (3.35) or (3.31) had no effect on the

proliferation of PC3 cells that do not express functional AR. This suggests that the

niphatenones are AR antagonists and that their activity is not due to non-specific toxicity

(Figure 3.12).

Figure 3.12 Androgen induced proliferation assay.

To determine if (R)-niphatenone B (3.31) is an AR NTD antagonist, the click

chemistry probe 3.67 was utilized. Since 3.67 was found to be active in the AR

transcriptional activity assay (Figure 3.11) it was a viable click chemistry probe. The click

chemistry experiment began by exposing analogue 3.67 to recombinant NTD AF1 protein.

After 50 minutes of exposure at 0 � C, click chemistry was used to attach a fluorescein tag

(3.69, Scheme 3.13) and the protein was analyzed using a SDS PAGE gel. Figure 3.13 shows

that the band corresponding to the AF1 protein is labeled with the fluorescent tag,

demonstrating that the probe 3.67 binds covalently to the NTD AF1 protein. The

niphatenones represent the first AR NTD antagonists that were shown to bind covalently.

Figure 3.13 Binding between alkyne probe 3.67 and the NTD AF1 region of the AR.

3.6 Conclusion

The NTD of the AR has been identified as a novel therapeutic target in the

development of small molecules for combating CRPC, which is an advanced stage of

prostate cancer. Current therapies are ineffective for the treatment of CRPC. In a continued

effort to identify small molecule AR NTD antagonists isolated from the marine environment,

the glycerol ethers niphatenone A (3.8) and B (3.9) were identified. These marine natural

products represent a novel class of AR NTD antagonist pharmacophores. However, the small

quantities isolated from the sponge prevented the assignment of the absolute configuration

and any further biological testing.

Each natural product contains one stereocenter, and the synthesis of all four possible

stereoisomers of the two natural products was completed. The synthesis of (R) (3.21) and (S)-

niphatenone A (3.23) was completed in an 18 % overall yield. The synthesis of (R) (3.31)

and (S)-niphatenone B (3.35) was completed in a 32 % overall yield. Chiral HPLC analysis

using synthetic and natural material revealed that both natural products have the (S)

configuration.

Having designed a robust synthesis for both natural products, further biological

testing was possible. Both (R) (3.31) and (S)-niphatenone B (3.35) were found to inhibit

androgen induced proliferation in an in vitro assay using LNCaP cells expressing the AR.

Comparison of in vitro data with PC3 cells lacking the AR showed no inhibition. This

suggested that these compounds are antagonists of the AR and that their activity was not due

to cell toxicity. This result suggests target specificity, which is a key step towards developing

therapeutic compounds.

To gain more insight into this novel pharmacophore, a number of analogues based on

(R)-niphatenone B (3.31) were constructed and assayed. Several conclusions were drawn

from this data: 1) the unnatural (R) configuration is more potent than the natural (S)

configuration, 2) any modification to the glycerol moiety attenuates the activity, 3) there is an

ideal chain length for enhanced activity, 4) the enone moiety plays a role in the activity but is

not required. These results suggest a clear SAR and the potential to construct analogues that

retain much of the activity without the undesirable enone functionality.

Finally, a propargyl ether analogue (3.67) was constructed for use as a click

chemistry probe that was used to demonstrate that (R)-niphatenone B (3.31), binds covalently

to the AF1 region of the NTD AR. (R)-niphatenone B (3.31) represents the first known NTD

AR antagonist with a binding mechanism elucidated using click chemistry. Having

completed an SAR study along with determining the mode of interaction to the NTD AR, the

niphatenones and their corresponding analogues represent potential lead compounds for the

development of therapeutics for the treatment of CRPC.

3.7 Experimental

rotations were measured with a JASCO P-1010 polarimeter at 24 �C and 589 nm (sodium D

line) in ethyl acetate (g/mL).

Preparation of 3.16:

Tetrahydrofuran (45 mL) was added to sodium hydride (436 mg, 10.9 mmol, 60 %

suspension in oil) that was washed twice with hexanes (20.0 mL total) and the suspension

was cooled to 0 � C. To this was added alcohol 3.15 (720 mg, 5.45 mmol) neat, along with

15C-5 (240 mg, 1.09 mmol). The mixture was then allowed to stir at room temperature for 1

h, after which bromide 3.13 (2.5 g, 10.9 mmol) was added dropwise. After 4.5 h the reaction

mixture was cooled to 0 � C and quenched with the addition of 50 mL of water, and the

aqueous phase was extracted three times with methylene chloride (250 mL). The organic

extracts were dried with MgSO4, and concentrated using a rotary evaporator. The crude

mixture was purified using flash column chromatography (hexanes:ethyl acetate 9:1) to give

3.16 as a clear oil (623 mg, 2.22 mmol, 40 %). 1 H NMR (400 MHz, CDCl3) � 7.33 (m, 4H),

7.28 (m, 1H), 4.50 (s, 2H), 4.25 (quin, J = 5.8 Hz, 1H), 4.04 (dd, J = 6.5, 1.7 Hz, 1H), 3.71

(dd, J = 6.1, 2.1 Hz, 1H), 3.56 (m, 5H), 3.42 (dd, J = 5.5, 4.4 Hz, 1H), 1.90 (quin, J = 6.1 Hz,

2H), 1.42 (s, 3H), 1.37 (s. 3H); 13 C NMR (100 MHz, CDCl3) � 138.7, 128.5, 127.8, 127.7,

109.5, 74.9, 73.1, 72.1, 68.8, 67.3, 66.9, 30.2, 26.9, 25.6. HRESIMS [M + H] + calcd for

C16H25O4 281.1753, found 281.1747; [α] 24 D = +13.6� (c 0.12).

Preparation of 3.17:

To compound 3.16 (575 mg, 2.05 mmol) dissolved in 2.6 mL of ethanol was added

10 % Pd/C (133 mg), in a round bottom and the system flushed with H2. The reaction

mixture was then exposed to 1 atm of H2 (balloon) overnight. Upon completion, the

heterogeneous mixture was filtered and the filtrate washed three times with ethanol (60 mL).

The organic extracts were concentrated using a rotary evaporator, and the crude mixture was

purified using flash column chromatography (hexanes:ethyl acetate 1:1), to give 3.17 as a

clear oil (375 mg, 1.97 mmol, 96 %). 1 H NMR (400 MHz, CDCl3) � 4.16 (quin, J = 5.8 Hz,

1H), 3.94 (dd, J = 8.2, 6.5 Hz, 1H), 3.62 (m, 3H), 3.54 (t, J = 6.0 Hz, 2H), 3.39 (m, 2H), 3.02

(s, 1H), 1.72 (quin, J = 6.0 Hz, 2H), 1.31 (s, 3H), 1.25 (s, 3H); 13 C NMR (100 MHz, CDCl3)

� 109.5, 74.7, 71.9, 69.9, 66.6, 60.6, 32.2, 26.7, 25.4. HRESIMS [M + Na] + calcd for

C9H18O4Na 213.1103, found 213.1105; [α] 24 D = +16.3��(c 0.12).

Preparation of 3.18:

To alcohol 3.17 (150 mg, 0.788 mmol) dissolved in 2 mL of methylene chloride was

added PPh3 (248 mg, 0.946 mmol) and the solution was cooled to 0 � C. CBr4 was added to

this solution and it was stirred for 1 h after which the mixture was concentrated under a

stream of nitrogen. The crude mixture was purified using flash column chromatography

(hexanes:ethyl acetate 12:1), to give 3.18 as a clear oil (166 mg, 0.657 mmol, 83 %). 1 H NMR

(400 MHz, CDCl3) � 4.19 (quin, J = 6.0 Hz, 1H), 3.98 (dd, J = 6.5, 1.7 Hz, 1H), 3.65 (dd, J =

6.5, 1.7 Hz, 1H), 3.54 (td, J = 5.9, 2.6 Hz, 2H), 3.43 (m, 4H), 2.04 (td, J = 6.2, 2.9 Hz, 2H),

1.35 (s, 3H), 1.29 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 109.5, 74.7, 72.2, 69.0, 66.7, 32.8,

30.6, 26.9, 25.5. HRESIMS [M + H] + calcd for C9H18O3 79 Br 253.0439, found 253.0439.

Preparation of 3.20:

Phosphorane 3.14 (248 mg, 0.78 mmol) was dissolved in 5 mL of tetrahydrofuran and

the solution was cooled to –78 � C. To this solution was added 1.6 M n-BuLi (0.53 mL, 0.86

mmol) and it was stirred for 20 min. To this solution, bromide 3.18 (166 mg, 0.66 mmol)

dissolved in 0.5 mL methylene chloride was added dropwise, and the mixture was allowed to

warm from 0 � C to room temperature overnight. The reaction mixture was then diluted with

50 mL of water and extracted three times with methylene chloride (250 mL). The organic

extracts were dried with MgSO4 and concentrated using a rotary evaporator. The crude

phosphorane (3.19) was then used immediately in the follow up Wittig reaction by dissolving

it in 1 mL of methylene chloride and adding aldehyde 3.11 (121 mg, 0.657 mmol) before

stirring at room temperature overnight. The reaction mixture was concentrated under a

stream of N2. The crude mixture was purified using flash column chromatography

(hexanes:ethyl acetate 7:1), to give 3.20 as a clear oil (165 mg, 0.415 mmol, 63 % over 2

steps). 1 H NMR (400 MHz, CDCl3) � 4.19 (dt, J = 16.0, 7.2 Hz, 1H), 6.01 (d, J = 16.0 Hz,

1H), 4.19 (quin, J = 6.1 Hz, 1H), 3.99 (dd, J = 8.2, 6.5 Hz, 1H), 3.66 (dd, J = 6.5, 1.7 Hz,

1H), 3.42 (m, 4H), 2.50 (t, J = 7.2 Hz, 2H), 2.15 (q, J = 6.8 Hz, 2H), 1.57 (m, 4H), 1.40 (m,

1H), 1.35 (s, 3H), 1.30 (s, 3H), 1.21 (m, 17H), 0.82 (t, J = 6.8 Hz, 3H); 13 C NMR (100 MHz,

CDCl3) � 200.4, 147.5, 130.3, 109.4, 74.8, 71.9, 71.5, 66.9, 39.7, 32.5, 32.0, 29.7, 29.7, 29.6,

29.5, 29.4, 29.3, 29.2, 28.2, 26.9, 25.5, 22.8, 20.9, 14.2. HRESIMS [M + H] + calcd for

C24H45O4 397.3318, found 397.3310; [α] 24 D = +11.4��(c 0.09).

Preparation of 3.21:

To acetonide 3.20 (45 mg, 0.11 mmol), dissolved in 1.65 mL of a

tetrahydrofuran:water mixture (5:1), was added 12.4 M HCl (0.045 mL, 0.56 mmol), and the

solution was stirred at room temperature for 30 min. The reaction mixture was then quenched

with the addition of saturated NaHCO3 (50 mL) and the aqueous phase was extracted three

times with methylene chloride (100 mL). The organic extracts were dried with MgSO4 and

concentrated using a rotary evaporator. The crude mixture was purified using flash column

chromatography (hexanes:ethyl acetate 1:4) to give 3.21 as a white solid (35 mg, 0.099

mmol, 90 %). 1 H NMR (600MHz, C6D6) � 6.70 (dt, J = 15.6, 7.2 Hz, 1H), 6.03 (d, J = 15.6

Hz, 1H), 3.91 (bs, 1H), 3.81 (bs, 1H), 3.73 (m, 1H), 3.66 (m, 1H), 3.41 (m, 2H), 3.26 (m,

2H), 3.28 (bs, 1H), 2.29 (t, J = 7.2 Hz, 2H), 1.90 (q, J = 6.6 Hz, 2H), 1.67 (quin, J = 7.2 Hz,

2H), 1.48 (quin, J = 6.6 Hz, 2H), 1.28 (m, 18H), 0.91 (t, J = 4.8 Hz, 3H); 13 C NMR (150

MHz, C6D6) � 199.2, 146.6, 130.6, 72.6, 71.3, 71.3, 64.4, 39.8, 32.5, 32.3, 30.0, 30.0, 29.9,

29.8, 29.8, 29.6, 29.4, 28.4, 23.1, 21.0, 14.3. HRESIMS [M + Na] + calcd for C21H40O4Na

379.2824, found 379.2814; [α] 24 D = +2.8��(c 0.07).

Preparation of 3.23:

Procedures identical to those used to prepare 3.21, only using (R)-4-hydroxymethyl-

2,2-dimethyl-1,3-dioxolane (3.22) as the starting material. HRESIMS [M + Na] + calcd for

C21H40O4Na 379.2824, found 379.2813; [α] 24 D = -2.5��(c 0.08).

Preparation of 3.27:

NaH (330 mg, 8.2 mmol, 60 % suspension in oil) was washed twice with hexanes (20

mL total) and added to 4 mL of tetrahydrofuran to give a suspension that was cooled to 0 � C.

To this suspension was added alcohol 3.15 (271 mg, 2.1 mmol) dissolved in 1 mL of

tetrahydrofuran along with 15C-5 (90 mg, 0.41 mmol). The mixture was then allowed to stir

at room temperature for 30 min. after which bromide 3.26 (1.0 g, 4.1 mmol) was added

dropwise. After 4.5 h the reaction mixture was cooled to 0 � C and quenched with the addition

of 100 mL of water, and the aqueous phase was extracted three times with methylene

chloride (250 mL). The organic extracts were dried with MgSO4 and concentrated using a

rotary evaporator. The crude mixture was purified using flash column chromatography

(hexanes:ethyl acetate 9:1), to give 3.27 as a clear oil (350 mg, 1.18 mmol, 58 %). 1 H NMR

(400 MHz, CDCl3) � 7.32 (m, 2H), 7.31 (m, 2H), 7.25 (m, 1H), 4.47 (s, 2H), 4.23 (quin, J =

6.0 Hz, 1H), 4.01 (dd, J = 8.2, 6.5 Hz, 1H), 3.70 (dd, J = 8.2, 6.5 Hz 1H), 3.45 (m, 5H), 3.39

(m, 1H), 1.66 (m, 4H), 1.41 (s, 3H), 1.34 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 138.7,

128.5, 127.7, 127.6, 109.4, 74.9, 72.9, 71.9, 71.6, 70.2, 67.0, 26.9, 26.9, 26.5, 26.5, 25.5.

HRESIMS [M + Na] + calcd for C17H26O4Na 317.1729, found 317.1733; [α] 24 D = +9.9��(c

0.35).

Preparation of 3.28:

To compound 3.27 (760 mg, 2.6 mmol) dissolved in 4 mL of ethanol in a round

bottom flask was added 10 % Pd/C (200 mg) and the system was flushed with H2. The

reaction mixture was then exposed to 1 atm of H2 (balloon) overnight. Upon completion, the

The organic extracts were concentrated using a rotary evaporator and the crude mixture was

purified using flash column chromatography (hexanes:ethyl acetate 2:1), to give 3.28 as a

clear oil (515 mg, 2.52 mmol, 98 %). 1 H NMR (400 MHz, CDCl3) � 4.20 (quin, J = 5.8 Hz,

1H), 3.99 (dd, J = 8.2, 6.5 Hz, 1H), 3.66 (dd, J = 8.4, 6.3 Hz, 1H), 3.57 (t, J = 5.8 Hz, 2H),

3.46 (m, 3H), 3.39 (m, 1H), 2.59 (bs, 1H), 1.60 (m, 4H), 1.36 (s, 3H), 1.31 (m, 3H); 13 C

NMR (100 MHz, CDCl3) � 109.5, 74.7, 71.9, 71.7, 66.9, 62.5, 29.9, 26.8, 26.4, 25.5.

HRESIMS [M + Na] + calcd for C10H20O4Na 227.1259, found 227.1265; [α] 24 D = +12��(c

0.09).

Preparation of 3.29:

To Dess–Martin periodinane (622 mg, 1.46 mmol) was added 9 mL of methylene

chloride, followed by pyridine (387 mg, 4.9 mmol). To this mixture was added alcohol 3.28

(190 mg, 0.97 mmol) dissolved in 0.5 mL of methylene chloride, and the solution was stirred

at room temperature for 30 min. The crude mixture was then concentrated using a rotary

evaporator and purified using flash column chromatography (hexanes:ethyl acetate 2:1), to

give 3.29 as a clear oil (150 mg, 0.74 mmol, 80 %). 1 H NMR (400 MHz, CDCl3) ��9.75 (s,

1H), 4.21 (quin, J = 6.0 Hz, 1H), 4.02 (dd, J = 8.4, 6.7 Hz, 1H), 3.66 (dd, J = 8.2, 6.5 Hz,

1H), 3.48 (m, 3H), 3.39 (m, 1H), 2.49 (td, J = 7.2, 1.2 Hz, 2H), 1.89 (quin, J = 6.8 Hz, 2H),

1.39 (s, 3H), 1.33 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 202.3, 109.5, 74.8, 71.9, 70.6, 66.8,

40.9, 26.8, 25.5, 22.5. HRESIMS [M + Na] + calcd for C10H18O4Na 225.1103, found

225.1098; [α] 24 D = +13��(c 0.03).

Preparation of 3.30:

To phosphonate 3.25 (353 mg, 1.0 mmol) dissolved in 3.0 mL tetrahydrofuran was

added Ba(OH)2 (254 mg, 0.80 mmol) and the suspension was stirred at rt for 30 min. To the

suspension was added aldehyde 3.29 (205 mg, 1.0 mmol), dissolved in 5.2 mL of a

tetrahydrofuran:water mixture (40:1) and was stirred at room temperature for 1 h. The

reaction mixture was then diluted with 100 mL of water and the aqueous phase was extracted

three times with methylene chloride (250 mL). The organic extracts were dried with MgSO4

and concentrated using a rotary evaporator. The crude mixture was purified using flash

column chromatography (hexanes:ethyl acetate 6:1) to give 3.30 as a clear oil (326 mg, 0.82

mmol, 81 %). 1 H NMR (400 MHz, C6D6) � 6.65 (dt, J = 15.6, 7.2 Hz, 1H), 6.02 (d, J = 15.6

Hz, 1H), 4.12 (quin, J = 5.2 Hz, 1H), 3.83 (dd, J = 6.4, 1.6 Hz, 1H), 3.67 (dd, J = 6.4, 2.0 Hz,

1H), 3.32 (dd, J = 6.4, 4.8 Hz, 1H), 3.19 (dd, J = 6.0, 3.6 Hz, 1H), 3.11 (t, J = 6.8 Hz, 2H),

2.27 (t, J = 7.3 Hz, 2H), 1.96 (q, J = 7.6 Hz, 2H), 1.66 (m, 2H), 1.42 (s, 3H), 1.38 (m, 2H),

1.30 (s, 3H), 1.28 (m, 18H), 0.90 (t, J = 6.8 Hz, 3H); 13 C NMR (100 MHz, C6D6) � 198.5,

145.2, 130.8, 109.3, 75.1, 72.1, 70.5, 67.1, 40.4, 32.3, 30.0, 30.0, 29.9, 29.9, 29.7, 29.7, 29.7,

29.1, 28.5, 27.1, 25.6, 24.4, 23.0, 14.3. HRESIMS [M + Na] + calcd for C24H44O4Na

419.3124, found 419.3130; [α] 24 D = +10��(c 0.15).

Preparation of 3.31:

To acetonide 3.30 (218 mg, 0.55 mmol) dissolved in 8 mL of a tetrahydrofuran:water

mixture (5:1) was added 12.4 M HCl (0.22 mL, 2.7 mmol) and the solution was stirred at

room temperature for 30 min. The reaction mixture was then quenched with the addition of

saturated NaHCO3 (50 mL) and the aqueous phase was extracted three times with methylene

chloride (150 mL). The organic extracts were dried with MgSO4 and concentrated using a

rotary evaporator. The crude mixture was purified using flash column chromatography (ethyl

acetate) to give 3.31 as a white solid (168 mg, 0.47 mmol, 86 %). 1 H NMR (600 MHz, C6D6)

� 6.69 (dt, J = 14.4, 7.2 Hz, 1H), 6.02 (d, J = 15.6 Hz, 1H), 3.87 (m, 1H), 3.70 (m, 1H), 3.63

(m, 2H), 3.33 (m, 3H), 3.16 (m, 2H), 2.31 (t, J = 6.6 Hz, 2H), 1.99 (q, J = 6.6 Hz, 2H), 1.65

(m, 2H), 1.45 (quin, J = 7.2 Hz, 2H), 1.28 (s, 18H), 0.91 (t, J = 7.2 Hz, 3H); 13 C NMR (150

MHz, C6D6) � 199.3, 145.8, 130.7, 72.5, 71.2, 70.5, 64.3, 40.4, 32.3, 30.1, 30.1, 30.0, 30.0,

29.9, 29.8, 29.7, 29.2, 28.3, 24.4, 23.0, 14.3. HRESIMS [M + Na] + calcd for C21H40O4Na

379.2835, found 379.2830; [α] 24 D = +2.9��(c 0.16).

Preparation of 3.35:

Procedures identical to those used to prepare 3.31, only using (R)-4-hydroxymethyl-

C21H40O4Na 379.2824, found 379.2816; [α] 24 D = -2.7��(c 0.04).

Preparation of 3.33:

To epoxide 3.32 (2.55 g, 11.9 mmol) dissolved in 150 mL of acetonitrile was added

NaBr (1.45 g, 14.1 mmol) followed by cerium(III) chloride heptahydrate (5.26 g, 14.1 mmol)

and the solution was stirred at room temperature for 24 h. The reaction mixture was then

concentrated using a rotary evaporator and extracted three times with ethyl acetate (150 mL).

The ethyl acetate fractions wee combined, concentrated in vacuo, and purified using flash

column chromatography (hexanes:ethyl acetate gradient 15:1, 10:1, 7:1) to give bromohydrin

3.33 as a white solid (3.19 g, 10.9 mmol, 91 %). 1 H NMR (400 MHz, CDCl3) � 3.78 (bs,

1H), 3.53 (dd, J = 6.8, 3.2 Hz, 1H), 3.38 (dd, J = 6.8, 3.2 Hz, 1H), 2.26 (m, 1H), 1.53 (m,

2H), 1.26 (m, 20H), 0.88 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl3) � 71.2, 40.8, 35.3,

32.1, 29.83/29.80/29.72/29.67/29.52 (all four signals account for a total of 7C), 25.7, 22.8,

14.3; Elemental analysis calcd. (found) for C14H29BrO: Theoretical to be C, 57.33 (57.51);

H, 9.97 (10.04).

Preparation of 3.34:

To bromohydrin 3.33 (158 mg, 0.54 mmol) dissolved in 6 mL of methylene chloride

at room temperature was added Dess–Martin periodinane (455 mg, 1.07 mmol) and the

mixture was stirred for 1 h. The reaction mixture was then quenched with the addition of

chloride (150 mL). The organic extracts were dried with MgSO4 and concentrated in vacuo.

The crude mixture was purified using flash column chromatography (hexanes:ethyl acetate

30:1), to give 3.34 as a white solid (126 mg, 0.43 mmol, 100 %). 1 H NMR (300 MHz,

CDCl3) � 3.89 (s, 2H), 2.64 (t, J = 7.4 Hz, 2H), 1.60 (m, 2H), 1.26 (m, 18H), 0.88 (t, J = 6.7

Hz, 3H); 13 C NMR (75 MHz, CDCl3) � 202.2, 39.9, 34.4, 32.0, 31.7, 29.8, 29.7, 29.6, 29.6,

29.5, 29.2, 24.0, 22.9, 14.2. HRESIMS [M + Na] + calcd for C14H27ONa 79 Br 313.1143, found

313.1136.

Preparation of 3.25:

To bromide 3.34 (80 mg, 0.27 mmol) dissolved in 1 mL of toluene was added

triethylphosphite (228 mg, 1.4 mmol). The reaction mixture was heated to reflux for 24 h,

after which it was cooled to room temperature and concentrated under a stream of N2. The

crude mixture was purified using flash column chromatography (hexanes:ethyl acetate 1:1),

to give 3.25 as a clear oil (89 mg, 0.25 mmol, 95 %). 1 H NMR (400 MHz, CDCl3) ��4.09 (t, J

= 7.2 Hz, 4H), 3.02 (m, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.53 (m, 2H), 1.28 (t, J = 7.0 Hz, 6H),

1.19 (m, 18H), 0.82 (t, J = 6.0 Hz, 3H); 13 C NMR (100 MHz, CDCl3) � 202.3, 62.6, 44.2,

43.1, 41.8, 32.0, 29.7, 29.6(2C), 29.5, 29.48, 29.43, 29.1, 23.5, 22.8, 16.4, 16.3, 14.2.

HRESIMS [M + Na] + calcd for C18H38O4P 349.2508, found 349.2517.

Preparation of 3.62:

To acetonide 3.30 (20 mg, 0.050 mmol) dissolved in 1 mL of

tetrahydrofuran:methanol (1:1), was added 5 mg of 10 % Pd/C, and the system subjected to 1

atm of H2 for 12 h. The reaction was then filtered using 20 mL of methanol, and evaporated

chromatography (hexanes:ethyl acetate 6:1) to give 3.62 as a white solid (19.9 mg, 0.05

mmol, 100 %). 1 H NMR (400 MHz, CDCl3) δ 4.25 (quin, J = 5.5 Hz, 1H), 4.05 (dd, J = 6.1,

2.0 Hz, 1H), 3.71 (dd, J = 6.5, 1.7 Hz, 1H), 3.47 (m, 4H), 2.38 (q, J = 7.5 Hz, 4H), 1.58 (m,

7H), 1.42 (s, 3H), 1.36 (s, 3H), 1.31 (m, 1H), 1.25 (m, 19H), 0.88 (t, J = 6.8 Hz, 3H); 13 C

NMR (100 MHz, CDCl3) δ 211.5, 109.5, 100.1, 74.9, 72.0, 71.7, 67.0, 43.0, 42.8, 32.0, 29.8,

29.7, 29.6, 29.5, 29.5, 29.4, 29.4, 26.9, 25.9, 25.6, 24.0, 23.7, 22.8, 14.2. HRESIMS [M+Na] +

calcd for C24H46O4Na 421.3294, found 421.3283; [α] 24 D = +18.10°�(c 0.04).

Preparation of 3.63:

To acetonide 3.62 (12 mg, 0.030 mmol), dissolved in 0.5 mL of a

tetrahydrofuran:water mixture (5:1) was added 12.4 M HCl (0.012 ml, 0.14 mmol), and

allowed to stir at room temperature for 30 min. The reaction mixture was then quenched with

the addition of saturated NaHCO3 (15 mL) and the aqueous phase was extracted three times

with methylene chloride (50 mL). The organic extracts were dried with MgSO4, and

chromatography (hexanes:ethyl acetate 1:4) to give 3.63 as a white solid (10 mg, 0.027

mmol, 92.7 %). 1 H NMR (400 MHz, C6D6) δ 3.69 (quin, J = 4.8 Hz, 1H), 3.54 (dd, J = 10.9,

4.3 Hz, 1H), 3.46 (dd, J = 10.9, 5.2 Hz, 1H), 3.24 (m, 2H), 3.14 (m, 2H), 2.46 (bs, 1H), 2.00

(m, 4H), 1.85 (bs, 1H), 1.51 (m, 5H), 1.38 (m, 4H), 1.29 (m, 14H), 1.20 (m, 3H), 0.91 (t, J =

6.7 Hz, 3H); 13 C NMR (75 MHz, C6D6) δ 208.9, 72.7, 71.2, 70.7, 64.2, 42.7, 42.4, 32.2, 30.1,

30.1, 30.1, 30.0, 29.9, 29.9, 29.7, 29.6, 26.0, 24.1, 23.6, 23.0, 14.3. ESIMS [M+Na] + calcd

for C21H42O4Na 381.2981, found 381.2968; [α] 24 D = +6.90°�(c 0.01).

Preparation of 3.41:

To ketone 3.40 (546.5 mg, 1.45 mmol) was added 4 mL was added triethylphosphite

(2.53 mL, 14.55 mmol). The reaction mixture was heated to reflux for 48 h, after which it

was cooled to room temperature, and then concentrated under a stream of nitrogen. The crude

mixture was then purified using flash column chromatography (hexanes:ethyl acetate 1:1), to

give 3.41 as a clear oil (358.4 mg, 0.82 mmol, 57.1 %). 1 H NMR (400 MHz, CDCl3) � 4.06

(m, 4H), 3.0 (m, 2H), 2.53 (t, J = 7.31 Hz, 2H), 1.49 (m, 2H), 1.26 (t, J = 7.0 Hz, 8H), 1.18

(m, 28H), 0.80 (t, J = 5.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) � 202.1(doublet), 62.6

(doublet), 44.1, 43.0, 47.7, 32.0, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.7, 29.6,

29.5, 29.4, 29.1, 23.5, 27.8, 16.4, 16.3, 14.2. HRESIMS [M+H] + calcd for C24H50O4P

433.3447, found 433.3438.

Preparation of 3.42:

To phosphonate 3.41 (57 mg, 0.131 mmol) dissolved in 1 mL tetrahydrofuran, was

added Ba(OH)2 (33.2 mg, 0.104 mmol), and allowed to stir at room temperature for 30 min.

To the suspension was added aldehyde 3.29 (26.6 mg, 0.131 mmol), dissolved in 1.6 mL of a

tetrahydrofuran:water mixture (40:1), and allowed to stir at room temperature for 1 h. The

reaction mixture was then diluted with 50 mL of water and the aqueous phase was extracted

three times with methylene chloride (100 mL). The organic extracts were dried with MgSO4,

column chromatography (hexanes:ethyl acetate 8:1) to give 3.42 as a white solid (44.4 mg,

0.092 mmol, 70.4 %). 1 H NMR (400 MHz, CDCl3) � 6.84 (dt, J = 16.0, 6.8 Hz, 1H), 6.10 (d,

J = 15.7 Hz, 1H), 4.25 (quin, J = 5.8 Hz, 1H), 4.05 (dd, J = 6.5, 1.7 Hz, 1H), 3.72 (dd, J =

6.5, 1.7 Hz, 1H), 3.47 (m, 4H), 2.51 (t, J = 7.5 Hz, 2H), 2.29 (q, J = 6.8 Hz, 2H), 1.75 (quin, J

= 6.8 Hz, 2H), 1.59 (m, 2H), 1.42 (s, 3H), 1.36 (s, 3H), 1.25 (m, 30 H), 0.87 (t, J = 6.8 Hz,

3H); 13 C NMR (100 MHz, CDCl3) � 200.9, 146.4, 130.7, 109.6, 100.1, 74.9, 72.1, 70.8, 66.9,

40.4, 32.1, 29.8, 29.8, 29.8, 29.7, 29.6, 29.6, 29.5, 29.2, 28.2, 26.9, 25.6, 24.4, 22.8, 14.3.

HRESIMS [M+Na] + calcd for C30H56O4Na 503.4076, found 503.4089; [α] 24 D = +7.02��(c

0.08).

�

Preparation of 3.43:

To acetonide 3.42 (30 mg, 0.062 mmol), dissolved in 0.6 mL of a

tetrahydrofuran:water mixture (5:1) was added 12.4 M HCl (0.025 mL, 0.31 mmol), and

the addition of saturated NaHCO3 (25 mL) and the aqueous phase was extracted three times

with methylene chloride (100 mL). The organic extracts were dried with MgSO4, and

chromatography (ethyl acetate) to give 3.43 as a white solid (25.3 mg, 0.057 mmol, 92.6 %).

1 H NMR (600 MHz, CD2Cl2) � 6.82 (dt, J = 15.9, 7.1 Hz, 1H), 6.11 (d, J = 15.9 Hz, 1H),

3.81 (m, 1H), 3.66 (dd, J = 11.3, 3.5 Hz, 1H), 3.58 (dd, J = 11.3, 5.6 Hz, 1H), 3.48 (m, 4H),

2.51 (t, J = 7.2 Hz, 2H), 2.42 (bs, 2H), 2.29 (q, J = 7.7 Hz, 2H), 1.75 (quin, J = 7.2 Hz, 2H),

1.57 (m, 2H), 1.27 (m, 30H), 0.89 (t, J = 7.2 Hz, 3H); 13 C NMR (150 MHz, CD2Cl2) � 200.0,

145.6, 130.0, 71.9, 70.2, 70.1, 63.6, 39.7, 31.5, 29.2, 29.2, 29.2, 29.09, 29.03, 28.9, 28.8,

28.6, 27.7, 23.8, 22.2, 13.4. HRESIMS [M+Na] + calcd for C27H52O4Na 463.3763, found

463.3756; [α] 24 D = +2.43��(c 0.08).

Preparation of 3.45:

To phosphonate 3.44 (57.4 mg, 0.296 mmol) dissolved in 1 mL tetrahydrofuran, was

added Ba(OH)2 (75 mg, 0.237 mmol), and allowed to stir at room temperature for 30 min. To

the suspension was added aldehyde 3.29 (60 mg, 0.296 mmol), dissolved in 1.6 mL of a

three times with methylene chloride (150 mL). The organic extracts were dried with MgSO4,

column chromatography (hexanes:ethyl acetate 2:1 ) to give 3.45 as a clear liquid (63.1 mg,

0.26 mmol, 87.9 %). 1 H NMR (400 MHz, CDCl3) � 6.79 (dt, J = 16.0, 6.8 Hz, 1H), 6.06 (d, J

= 16.0 Hz, 1H), 4.24 (quin, J = 5.8 Hz, 1H), 4.03 (dd, J = 6.5, 1.7 Hz, 1H), 3.69 (dd, J = 6.5,

1.7 Hz, 1H), 3.46 (m, 4H), 2.30 (q, J = 8 Hz, 2H), 2.22 (s, 3H), 1.74 (q, J = 6.8 Hz, 2H), 1.39

(s, 3H), 1.34 (s, 3H); 13 C NMR (100 MHz, CDCl3) � 198.7, 147.8, 131.6, 109.6, 74.9, 72.1,

70.8, 66.9, 29.3, 28.2, 27.0, 26.9, 25.5. HRESIMS [M+Na] + calcd for C13H22O4Na 265.1416,

found 265.1413; [α] 24 D = +19.25��(c 0.12).

Preparation of 3.46:

To acetonide 3.45 (30 mg, 0.123 mmol), dissolved in 1.2 mL of a

tetrahydrofuran:water mixture (5:1) was added 12.4 M HCl (0.05 mL, 0.62 mmol), and

concentrated using a rotary evaporator. The crude mixture was purified using a reversed

phase sepak (water:methanol 6:4) to give 3.46 as a clear liquid (7.46 mg, 0.037 mmol, 30 %).

1 H NMR (600 MHz, C6D6) � 6.43 (dt, J = 15.9, 6.6 Hz, 1H), 5.94 (d, J = 15.9 Hz, 1H), 3.75

(quin, J = 5.1 Hz, 1H), 3.59 (dd, J = 7.2, 4.1 Hz, 1H), 3.52 (dd, J = 11.3, 5.6 Hz, 1H), 3.24

(m, 2H), 3.05 (m, 2H), 2.93 (bs, 1H), 2.51 (bs, 1H), 1.87 (s, 3H), 1.85 (m, 2H), 1.33 (quin, J

= 6.6 Hz, 2H); 13 C NMR (150 MHz, C6D6) � 196.9, 146.6, 131.5, 72.5, 71.0, 70.4, 64.2, 29.1,

28.1, 26.7. HRESIMS [M+Na] + calcd for C10H18O4Na 225.1103, found 225.1100; [α] 24 D =

+5.35��(c 0.01).

Preparation of 3.48:

Sodium hydride (2.46 g, 61.7 mmol, 60 % suspension in oil) was washed twice with

hexanes (50.0 mL total), to which was added 260 mL of tetrahydrofuran, was cooled to 0 � C.

To this suspension was added alcohol 3.47 (3.04 g, 41.1 mmol) neat, along with 15C-5 (1.81

g, 8.2 mmol). The mixture was then allowed to stir at room temperature for 1 h. after which

bromine 3.26 (5.0 g, 20.5 mmol) was added dropwise, and the reaction mixture was allowed

to warm to room temperature overnight. The reaction mixture was quenched with the

addition of 100 mL of water, and the aqueous phase was extracted three times with

methylene chloride (500 mL). The organic extracts were dried with MgSO4, and concentrated

using a rotary evaporator. The crude mixture was purified using flash column

chromatography (hexanes:ethyl acetate 4:1), to give 3.48 as a clear oil (996.0 mg, 4.21 mmol,

20.5 %). 1 H NMR (400 MHz, CDCl3) � 7.34 (m, 3H), 7.29 (m, 1H), 4.51 (s, 2H), 3.70 (dd, J

= 11.6, 3.2 Hz, 1H), 3.50 (m, 4H), 3.38 (dd, J = 11.6, 6 Hz, 1H), 3.14 (m, 1H), 2.79 (t, J = 4

Hz, 1H), 2.60 (dd, J = 5.2, 2.8 Hz, 1H), 1.69 (m, 4H); 13 C NMR (100 MHz, CDCl3) � 138.8,

128.5, 128.5, 127.8, 127.8, 127.6, 73.0, 71.6, 71.5, 70.2, 51.0, 44.5, 26.6, 26.5. HRESIMS

[M+H] + calcd for C14H21O3 237.1491, found 237.1494.

Preparation of 3.49:

To compound 3.48 (900 mg, 3.80 mmol) dissolved in 5 mL of ethanol was added

10 % Pd/C dry (200 mg), in a round bottom and the system flushed with H2. The reaction

mixture was then exposed to 1 atm of H2 (balloon) overnight. Upon completion the

purified using flash column chromatography (hexanes:ethyl acetate 3:2), to give 3.49 as a

volatile clear oil (360.5 mg, 2.46 mmol, 64.7 %). 1 H NMR (400 MHz, CDCl3) � 3.70 (dd, J =

11.6, 2.8 Hz, 1H), 3.58 (m, 2H), 3.48 (m, 2H), 3.33 (dd, J = 11.6, 6.0 Hz, 1H), 3.1 (m, 1H),

2.75 (t, J = 4.0 Hz, 1H), 2.6 (bs, 1H), 2.56 (m, 1H), 1.62 (m, 4H); 13 C NMR (100 MHz,

CDCl3) � 71.6, 71.6, 62.6, 50.9, 44.4, 29.9, 26.5. HRESIMS [M+H] + calcd for C7H15O3

147.1021, found 147.1018.

Preparation of 3.50:

To Dess–Martin periodinane (638.2 mg, 1.50 mmol) was added 12 mL of methylene

chloride, followed by triethylamine (553.7 mg, 5.47 mmol). To this reaction mixture was

added alcohol 3.49 (200 mg, 1.36 mmol) dissolved on 1.0 mL of methylene chloride, and

was stirred at room temperature for 1.5 h. The crude mixture was then concentrated using a

rotary evaporator and filtered through a pad of silica (50 mL of 2:3 hexanes:ethyl acetate),

and again concentrated using a rotary evaporator. This crude mixture dissolved in 6.0 mL of

a tetrahydrofuran:water mixture (40:1) was then added to a solution of phosphonate 3.25

(300 mg, 0.86 mmol) in tetrahydrofuran (3 mL) and Ba(OH)2 (215 mg) which had been

stirring at room temperature for 30 min. After stirring for 2 h the reaction mixture was

diluted with 100 mL of water and the aqueous phase was extracted three times with

methylene chloride (350 mL). The organic extracts were dried with MgSO4 and concentrated

chromatography (hexanes:ethyl acetate 5:1), to give 3.50 as a clear oil (211.7 mg, 0.62 mmol,

45.6 %). 1 H NMR (400 MHz, CDCl3) � 6.84 (dt, J = 14.0, 6.8 Hz, 1H), 6.10 (d, J = 16.0 Hz,

1H), 3.73 (dd, J = 11.6, 2.8 Hz, 1H), 3.5 (m, 2H), 3.35 (dd, J = 11.6, 6.0 Hz, 1H), 3.14 (m,

1H), 2.80 (t, J = 4.4 Hz, 1H), 2.60 (dd, J = 5.2, 2.8 Hz, 1H), 2.51 (t, J = 7.2 Hz, 2H), 2.30 (q,

J = 7.2 Hz, 2H), 1.77 (quin, J = 7.6 Hz, 2H), 1.59 (m, 2H), 1.25 (m, 18H), 0.87 (t, J = 6.8 Hz,

3H); 13 C NMR (100 MHz, CDCl3) � 201.0, 146.4, 130.7, 71.7, 70.6, 50.9, 44.3, 40.4, 32.1,

29.8, 29.7, 29.7, 29.65, 29.60, 29.5, 29.5, 29.2, 28.3, 24.5, 22.8, 14.3. HRESIMS [M+H] +

calcd for C21H39O3 339.2899, found 339.2896; [α] 24 D = -6.49��(c 0.04).

Preparation of 3.52:

To epoxide 3.50 (20.0 mg, 0.059 mmol) dissolved in 1 mL of triethylene glycol

(3.51) (7.32 mmol) was added bismuth(III) trifluoromethanesulfonate (3.8 mg, 0.0059

mmol), and the mixture allowed to stir at room temperature. After stirring for 1.5 h the

reaction mixture was diluted with 25 mL of water and the aqueous phase was extracted three

times with methylene chloride (100 mL). The organic extracts were dried with MgSO4, and

chromatography (methylene chloride:methanol 95:5), to give 3.52 as a clear oil (24.7 mg,

0.050 mmol, 84.7 %). 1 H NMR (400 MHz, C6D6) � 6.67 (dt, J = 14.0, 7.2 Hz, 1H), 6.04 (d, J

= 16.0 Hz, 1H), 4.09 (m, 1H), 3.62 (m, 2H), 3.56-3.43 (m, 6H), 3.34 (m, 2H), 3.29 (m, 2H),

3.34 (m, 2H), 3.19 (m, 6H), 2.28 (t, J = 7.2 Hz, 2H), 1.97 (q, J = 7.2 Hz, 2H), 1.66 (m, 2H),

1.43 (quin, J = 7.6 Hz, 2H), 1.28 (m, 18H), 0.91 (t, J = 6.8 Hz, 3H); 13 C NMR (100 MHz,

C6D6) � 198.7, 145.6, 130.8, 74.0, 73.3, 72.1, 70.7, 70.6, 70.6, 70.6, 70.5, 70.0, 69.8, 61.7,

40.3, 32.3, 30.1, 30.1, 29.9, 29.9, 29.7, 29.7, 29.2, 28.5, 24.4, 23.0, 14.3. HRESIMS [M+Na] +

calcd for C27H52O7Na 511.3611, found 511.3616; [α] 24 D = -11.9��(c 0.007).

Preparation of 3.53:

To epoxide 3.50 (23.4 mg, 0.069 mmol) dissolved in 1.5 mL of acetonitrile was

added cerium(III) chloride heptahydrate (12.87 mg, 0.034 mmol) and heated at reflux for 4 h.

The reaction mixture was then concentrated using a rotary evaporator and extracted three

times with ethyl acetate (60 mL). The crude mixture was then concentrated using a rotary

evaporator and purified using flash column chromatography (hexanes: ethylacetate gradient

6:1 - 4:1) to give chlorohydrin 3.53 as a clear oil (22.1 mg, 0.058 mmol, 85.3 %). 1 H NMR

(400 MHz, CDCl3) � 6.76 (dt, J = 14.0, 6.8 Hz, 1H), 6.10 (d, J = 16.0 Hz, 1H), 3.78 (bs, 1H),

3.42 (m, 2H), 3.29 (m, 1H), 3.24 (m, 1H), 3.10 (t, J = 6.4 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H),

2.23 (bs, 1H), 2.00 (q, J = 7.2 Hz, 2H), 1.78 (m, 2H), 1.39 (m, 20H), 1.02 (t, J = 6.4 Hz, 3H);

13 C NMR (100 MHz, CDCl3) � 198.6, 145.1, 130.7, 71.5, 70.39, 70.33, 46.2, 40.5, 32.2, 30.0,

30.0, 30.0, 29.9, 29.9, 29.7, 29.6, 29.0, 28.2, 24.4, 23.0, 14.3. HRESIMS [M+H] + calcd for

C21H40O3 35 Cl 375.2666, found 375.2676; [α] 24 D = +7.61��(c 0.009).

Preparation of 3.54:

To diol 3.31 (9.8 mg, 0.027 mmol) dissolved in 1 mL of methylene chloride at –78 � C

was added DBU (8.2 mg, 0.054 mmol), followed by XtalFluor-E (12.58 mg, 0.054 mmol),

and the reaction allowed to stir for one hour. After stirring for one hour at –78 � C the reaction

mixture was allowed to warm to room temperature over 12 hours, after which it was

concentrated under a stream of nitrogen and the crude reaction mixture was purified using

flash column chromatography (hexanes:ethyl acetate 3:1 to ethyl acetate) to give

fluorohydrin 3.54 (3.5 mg, 0.0097 mmol, 35.9 %). 1 H NMR (600 MHz, CD2Cl2) � 6.81 (dt, J

= 13.8, 6.6 Hz, 1H), 6.10 (d, J = 15.6 Hz, 1H), 4.51-4.35 (m ,2H), 3.99 (m, 1H), 3.51-3.46

(m, 3H), 2.51 (t, J = 7.2 Hz, 2 H), 2.30 (m, 2 H), 1.75 (quin, J = 6.6 Hz, 2H), 1.56 (m, 8H),

1.27 (m, 14H), 0.89 (t, J = 6.6 Hz, 3H); 13 C NMR (150 MHz, CD2Cl2) � 199.9, 145.4, 130.1,

81.0, 70.48, 70.2, 68.5, 39.6, 31.5, 29.2, 29.1, 29.1 29.0, 29.0, 28.9, 28.8, 28.4, 27.6, 23.7,

22.2, 13.4. HRESIMS [M+Na] + calcd for C21H39O3FNa 381.2781, found 381.2783.

Preparation of 3.56:

To acetonide 3.55 (1.0 g, 6.25 mmol), dissolved in 15 mL of methylene chloride, was

added triphenyl phosphine (1.80 g, 6.87 mmol), followed by carbon tetrabromide (2.28 g,

6.87 mmol) and allowed to stir at room temperature. After stirring for 5 h the reaction

mixture was loaded on to a pad of silica and washed with 150 mL of hexanes:ethyl acetate

(2:1). The organic extract was the concentrated using a rotary evaporator and the crude

mixture was purified using flash column chromatography (hexanes:ethyl acetate 15:1) to give

known bromine 3.56 (259 mg, 1.23 mmol, 19.6 %).

Preparation of 3.57:

To bromine 3.26 (562 mg, 2.31 mmol), dissolved in 5 mL of tetrahydrofuran, was

added sanded magnesium turnings (84.3 g, 6.87 mmol). After the reaction mixture cooled to

room temperature (~ 1 h), it was transferred to an empty N2 filled round bottom with a

syringe. This was cooled to 0 � C and Li2CuCl4 (0.1 M in tetrahydrofuran, 0.115 mmol, 1.115

mL) added, followed by bromine 3.56 (259 mg, 1.23 mmol) dissolved in 2.5 mL of

tetrahydrofuran, and the reaction mixture was allowed to warm to room temperature. After 4

h, the reaction mixture was quenched with 0.5 mL of methanol, and loaded on to a pad of

silica and washed with 200 mL of hexanes:ethyl acetate (4:1). The crude mixture was

concentrated using a rotary evaporator and purified using flash column chromatography

(hexanes:ethyl acetate 12:1) to give acetonide 3.57 (177.7 mg, 0.607 mmol, 49.3 %). 1 H

NMR (400 MHz, CDCl3) � 7.33 (m, 4H), 7.27 (m, 1H), 4.49 (s, 2H), 4.06 (quin, J = 6.0 Hz,

1H), 4.00 (m, 1H), 3.46 (m, 3H), 1.62 (m, 2H), 1.52-1.29 (m, 8H), 1.41 (s, 3H), 1.35 (s, 3H);

13 C NMR (100 MHz, CDCl3) � 138.9, 128.5, 127.7, 127.6, 108.7, 76.2, 73.0, 70.5, 69.6, 33.7,

29.8, 29.6, 27.1, 27.1, 26.3, 25.9. HRESIMS [M+Na] + calcd for C18H28O3Na 315.1936,

found 315.1938; [α] 24 D = +5.36��(c 0.04).

respectively.�

Preparation of 3.58:�

To 3.57 (2.5 g, 8.54 mmol) dissolved in 10 mL of ethanol was added 200 mg 10 %

Pd/C, and exposed to 1 atm of H2 and allowed to stir at room temperature overnight. The

mixture was then filtered through filter paper and concentrated using a rotary evaporator then

purified using flash column chromatography (hexanes:ethyl acetate 2:1) to give alcohol 3.58

(315.1 mg, 1.55 mmol, 18.1 %). 1 H NMR (400 MHz, CDCl3) � 3.98 (quin, J = 6.4 Hz, 1H),

3.93 (m, 1H), 3.50 (t, J = 6.4 Hz, 2H), 3.40 (t, J = 7.2 Hz, 1H), 2.71 (bs, 1H), 1.59-1.17 (m,

10H), 1.31 (s, 3H), 1.26 (s, 3H); 13 C NMR (150 MHz, CDCl3) � 108.1, 75.6, 68.9, 62.1, 33.0,

33.0, 32.1, 28.9, 26.4, 25.2, 25.1. HRESIMS [M+Na] + calcd for C11H22O3Na 225.1467,

found 225.1459; [α] 24 D = +9.02��(c 0.04).

Preparation of 3.59:�

To alcohol 3.58 (150 mg, 0.741 mmol) dissolved in 7 mL of methylene chloride, was

added pyridine (0.45 mL, 5.55 mmol), and Dess–Martin periodinane (471.0 mg, 1.11 mmol),

and allowed to stir at room temperature for 30 min. The crude reaction mixture was

(hexanes:ethyl acetate 5:1) to give aldehyde 3.59 (144.2 mg, 0.72 mmol, 97.1 %). 1 H NMR

(600 MHz, CDCl3) � 9.78 (s, 1H), 4.18 (m, 1H), 4.08 (t, J = 6.0 Hz, 1H), 4.04 (m, 1H), 3.5 (t,

J = 6.6 Hz, 1H), 2.44 (t, J = 7.2 Hz, 2H), 1.66 (quin, J = 7.2 Hz, 4H), 1.54-1.37 (m, 3H), 1.41

(s, 3H), 1.36 (s, 3H); 13 C NMR (150 MHz, CDCl3) � 202.2, 108.2, 75.5, 69.0, 43.3, 32.9,

28.7, 26.5, 25.3, 25.1, 21.5. HRESIMS [M+Na] + calcd for C11H20O3Na 223.1310, found

223.1315; [α] 24 D = +7.94��(c 0.01).

Preparation of 3.60:

To phosphonate 3.25 (191 mg, 0.55 mmol) in 2.5 mL of tetrahydrofuran, was treated

with barium hydroxide (185.7 mg, 1.08 mmol) and allowed to stir at room temperature for 30

min. To this heterogeneous mixture was added aldehyde 3.59 (110.0 mg, 0.55 mmol)

dissolved in 4.1 mL of a tetrahydrofuran:water mixture (40:1), and the reaction mixture was

allowed to stir at room temperature overnight. The reaction mixture was then filtered through

a pad of silica and washed with 100 mL of hexanes:ethyl acetate 3:1, and concentrated using

a rotary evaporator. The crude mixture was purified using flash column chromatography

(hexanes:ethyl acetate 9:1) to give acetonide 3.60 (129.9 mg, 0.329 mmol, 59.8 %). 1 H NMR

(600 MHz, CDCl3) � 6.83 (dt, J = 6.6, 16.2 Hz, 1H), 6.10 (d, J = 16.2 Hz, 1H), 4.08 (sep, J =

6.6 Hz, 1H), 4.04 (dd, J = 6.0, 1.8 Hz, 1H), 3.51 (t, J = 6.6 Hz, 1H), 2.52 (t, J = 7.8 Hz, 2H),

2.22 (m, 2H), 1.00 (m, 4H), 1.49 (m, 6H), 1.41 (s, 3H), 1.36 (s, 3H), 1.26 (m, 18H), 0.87 (t, J

= 7.2 Hz, 3H); 13 C NMR (150 MHz, CDCl3) � 200.5, 146.5, 129.9, 108.2, 75.6, 69.0, 39.7,

33.0, 31.9, 31.5, 29.2, 29.2, 29.0, 29.0, 28.9, 28.9, 28.7, 28.7, 27.5, 26.5, 25.3, 25.1, 23.9,

22.2, 13.7. HRESIMS [M+Na] + calcd for C25H46O3Na 417.3345, found 417.3351; [α] 24 D =

+4.56��(c 0.03).

Preparation of 3.61:

To acetonide 3.60 (90.0 mg, 0.228 mmol) dissolved in 5 mL of a

tetrahydrofuran:water mixture (5:1) was added 12.4 M HCl (0.91 mL, 11.28 mmol), and

allowed to stir at room temperature for thirty minutes. The reaction mixture was then

concentrated under a stream of nitrogen and purified using flash column chromatography

(hexanes:ethyl acetate 2:3) to give diol 3.61 (73.8 mg, 0.208 mmol, 91.2 %). 1 H NMR (600

MHz, CDCl3) � 6.83 (dt, J = 15.6, 7.2 Hz, 1H), 6.10 (d, J = 15.6 Hz, 1H), 3.7 (m, 1H), 3.66

(m, 1H), 3.45 (dd, J = 7.2, 3.6 Hz, 1H), 2.53 (t, J = 7.2 Hz, 2H), 2.48 (bs, 2H), 2.24 (q, J =

7.8 Hz, 2H), 1.60 (m, 2H), 1.51-1.44 (m, 4H), 1.37 (m, 4H), 1.26 (m, 18H), 0.89 (t, J = 6.6

Hz, 3H); 13 C NMR (150 MHz, CDCl3) � 200.8, 146.7, 129.9, 71.7, 66.3, 39.7, 32.5, 31.9,

31.5, 29.2, 29.2, 29.2, 29.0, 29.0, 28.9, 28.9, 28.7, 27.5, 24.9, 23.9, 22.2, 13.7. HRESIMS

[M+Na] + calcd for C22H42O3Na 377.3032, found 377.3030; [α] 24 D = +3.69��(c 0.01).

Preparation of 3.67:

To epoxide 3.50 (80.0 mg, 0.236 mmol) along with propargyl alcohol (13.2 mg, 0.236

mmol) was added bismuth(III) trifluoromethanesulfonate (14.77 mg, 0.0236 mmol), and the

mixture allowed to stir at room temperature. After stirring for 30 min the reaction mixture

was purified using flash column chromatography (hexanes:ethyl acetate gradient 3:1-2:1), to

give 3.67 as a clear oil (25.4 mg, 0.064 mmol, 27.1 %). 1 H NMR (600 MHz, CDCl3) � 6.76

(dt, J = 9.6, 4.8 Hz, 1H), 6.09 (d, J = 10.4 Hz, 1H), 4.00 (q, J = 4.0 Hz, 1H), 3.92 (m, 2H),

3.57 (m, 2H), 3.39 (d, J = 3.6 Hz, 2H), 3.18 (m, 2H), 2.38 (t, J = 5.2 Hz, 2H), 2.11 (t, J = 1.6

Hz, 1H), 2.02 (q, J = 4.8 Hz, 2H), 1.76 (m, 2H), 1.47 (q, J = 4.8 Hz, 3H), 1.38 (m, 18H), 1.00

(t, J = 4.4 Hz, 3H); 13 C NMR (150 MHz, CDCl3) � 198.0, 144.7, 130.1, 79.3, 74.1, 71.6,

70.8, 69.8, 68.9, 57.9, 31.7, 29.5, 29.5, 29.4, 29.4, 29.33, 29.30, 29.15, 29.09, 28.5, 27.8,

23.8, 22.5, 13.7. HRESIMS [M+H] + calcd for C24H43O4 395.3161, found 395.3169; [α] 24 D = -

3.39��(c 0.01).

Preparation of 3.77:

To phosphonate 3.19 (~0.79 mmol, crude) in 3 mL of methylene chloride was added

aldehyde 161 3.76 (106.4 mg, 0.43 mmol), and allowed to stir at room temperature overnight.

The solvent was evaporated and the crude mixture was purified using flash column

chromatography (hexanes:ethyl acetate 2:1-3:2) to give 3.77 (28.9 mg, 0.063 mmol, 14.6 %).

1 H NMR (400 MHz, CDCl3) � 7.74 (s, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.28 (t, J = 8.8 Hz,

1H), 7.20 (d, J = 15.6 Hz, 1H), 6.60 (dd, J = 8.8, 2.4 Hz, 1H), 6.47 (s, 1H), 4.25 (q, J = 6.4

Hz, 1H), 4.04 (t, J = 6.4 Hz, 1H), 3.72 (t, J = 6.4 Hz, 1H), 3.54-3.40 (m, 8H), 2.65 (t, J = 7.2

Hz, 2H), 1.72 (m, 2H), 1.64 (m, 2H), 1.41 (s, 3H), 1.35 (m, 3H), 1.33 (t, J = 7.2 Hz, 6H); 13 C

NMR (100 MHz, CDCl3) � 200.8, 160.6, 157.0, 151.9, 144.8, 137.2, 130.0, 126.6, 114.7,

109.6, 109.5, 108.9, 97.1, 74.9, 72.0, 71.5, 67.0, 45.1, 45.1, 41.5, 29.2, 26.9, 25.5, 21.0, 12.6,

12.6. HRESIMS [M+H] + calcd for C26H36NO6 458.2543, found 458.2536.

Preparation of 3.78:

To 3.77 (20.0 mg, 0.043 mmol) in 1 mL of a tetrahydrofuran:water mixture (5:1) was

added 12.4 M HCl (17.6 �l, 0.22 mmol), and allowed to stir at room temperature for 2 hours.

The crude reaction mixture was concentrated under a stream of nitrogen and purified using

flash column chromatography (ethyl acetate) to give 3.78 (4.0 mg, 0.0095 mmol, 22.1 %). 1 H

NMR (600 MHz, CD2Cl2) � 7.82 (s, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H),

7.18 (d, J = 15.6 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.57 (s, 1H), 3.82 (m, 1H), 3.67 (dd, J =

11.4, 4.2 Hz, 1H), 3.61 (dd, J = 11.4, 6.0 Hz, 1H), 3.52-3.44 (m, 8H), 2.67 (t, J = 6 Hz, 2H),

1.72 (m, 2H), 1.63 (m, 2H), 1.23 (t, J = 7.2 Hz, 6H); 13 C NMR (150 MHz, CD2Cl2) � 199.8,

144.0, 136.4, 129.5, 129.5, 125.9, 109.6, 109.5, 96.9, 71.9, 70.7, 70.7, 70.0, 63.6, 44.9, 44.9,

40.5, 28.6, 20.4, 11.6, 11.6. HRESIMS [M+Na] + calcd for C21H31NO6Na 440.2049, found

440.2055.

AR Transcriptional Activity Assay: AR transcriptional activity was measured using

the PSA (6.1 kb)-luciferase reporter gene construct transiently transfected into LNCaP

human prostate cancer cells which express functional AR. This reporter contains several

well-characterized androgen response elements to which the AR specifically binds to

increase luciferase activity in response to androgen such as R1881. LNCaP (2.5 x 10 4

cell/well) cells were seeded on 24-well plates overnight before transfection with PSA (6.1

kb)-luc, (0.5 �g/well) in serum-free, phenol red-free media using lipofectin (Invitrogen)

according to published methods. For SAR analysis, LNCaP cells in 12-well plates were pre-

treated 1 hr with (S)-niphatenone B (3.35), (R)-niphatenone B (3.31) or their SAR analogues

(all added at 7 �M, with the exception of 3.43, which was tested at 3.5 �M due to solubility

limitations) prior to addition of 1 nM R1881 (Metribolone). After 48 h of exposure, cells

were harvested, luciferase activity measured and normalized to protein concentration

determined by the Bradford assay.

Proliferation Assay: Experiments using LNCaP human prostate cancer cells were

done in phenol red-free RPMI 1640 medium with 0.5 % (v/v) fetal bovine serum, while for

PC3 human prostate cancer cells, they were done in phenol red DMEM medium with 5 %

(v/v) fetal bovine serum. Both media were supplemented with, 100 units/mL penicillin, and

100 mg/mL streptomycin. Cells were seeded in 96-well plates for 24 hrs before pre-treatment

for 1 h with bicalutamide (10 µM), (S)-niphatenone B (3.35) and (R)-niphatenone B (3.31)

(~14 �M) before treatment with 0.1 nM R1881 for LNCaP cells. LNCaP cells were incubated

for 72 h with R1881, while the duration of the experiment was 24 h for PC3 cells. BrdU was

added to the cells for an additional 2 h. Cells were fixed prior to incubation for 1.5 h with the

anti-BrdU-POD antibody (Roche). BrdU incorporation was measured at 570 nm via

VersaMax ELISA Microplate Reader (Molecular Devices).

Expression and Purification of Recombinant Protein: AR AF1 recombinant

protein was expressed and purified as described previously. The Ni 2+ -agarose affinity

chromatography purified recombinant AR AF1 protein was further purified by size exclusion

chromatography.

In vitro Binding Assay: The binding reaction was carried out by incubating 10 mM

AR AF1 protein with 20 mM of compound 3.67 containing an alkyne group or dimethyl

sulfoxide on ice for 50 min. The binding reaction was diluted in half (i.e. 5 mM AR AF1

protein and 10 mM compound 3.67) for fluorescein labeling on compound. Labeling was

done by a Click chemistry reaction with 10 mM fluorescein azide, 0.1 mM ascorbic acid and

0.1 mM Copper(II)-TBTA complex (Lumiporbe) and incubated at room temperature for 30 –

40 min. Samples were separated on 12.5 % SDS-PAGE. Fluorescein was detected by an

image analyzer (Fujifilm FLA-7000, GE Healthcare). The same gel was stained with

Coomassie blue.

Chapter 4: Synthetic Efforts Towards a Novel AR Antagonist (4.1)

4.1 Terpene AR Antagonist (4.1)

Small molecule antagonists 130 of the AR are used as a means to treat prostate cancer

in patients suffering from the disease (Chapter 3, Section 3.1). In an ongoing effort to

identify marine natural products that are AR antagonists, the Andersen natural product

extract library was screened using Dr. Marianne Sadar’s cell based transcriptional activity

assay 140 at the BC Cancer Agency. The assay uses LNCaP prostate cancer cells containing an

engineered PSA gene with a luciferase reporter. Stimulation of the cells with forskolin to

stimulate protein kinase A (PKA) or interleukin 6 (IL6), activates the AR NTD leading to

AR-dependent production of PSA-luciferase that generates light upon addition of luciferin. 140

Antagonists of the AR inhibit light production, providing a positive hit in the assay.

Bioassay-guided fractionation of a marine sponge extract carried out by Gavin Carr in the

Andersen group identified the terpene 4.1 as the active compound (Figure 4.1).

Figure 4.1 AR antagonist lead compound 4.1.

Prior to its discovery in the Andersen and Sadar laboratories terpene 4.1 162,163 had no

previously known biological activity. Lead compound 4.1 was found to inhibit the

transcriptional activity of the AR in vitro. The carbon skeleton structure of 4.1 resembles that

of a steroid (Figure 4.2) and it was proposed that the mode of binding is through the LBD of

the AR, which is the same as current antiandrogens (Chapter 3, Figure 3.2).

Figure 4.2 Structural similarity between 4.1 and a steroid carbon skeleton.

The two functional groups of this natural product are a D-ring substituted furan and a

neopentyl carboxylic acid moiety. Analogues of 4.1 were constructed by semisynthesis in a

preliminary SAR study to address the role of these functional groups in the AR antagonist

activity (Scheme 4.1).

First, we desired to construct an analogue that would have a modified furan functional

group. This was accomplished by hydrogenating 164 compound 4.1 (previously synthesized by

Gavin Carr) to give the tetrahydrofuran analogue 4.2 (Scheme 4.1). Next, we attempted to

reduce the carboxylic acid moiety in 4.1 directly to the primary alcohol with LiAlH4,

however, this gave poor yields (15 %). Alternatively, 4.1 was converted to the methyl ester 165

(4.3) with sodium hydroxide and methyl iodide. Reduction of 4.3 with DIBAL at –78 � C

gave alcohol 4.4 in a quantitative yield.

Scheme 4.1 Semisynthesis of 4.1 analogues.

Analogue 4.4 is a known 166 marine natural product found to inhibit the lyase activity

of DNA polymerase β. However, no AR antagonist properties have been previously reported.

Analogues 4.4 and 4.2 in addition to the lead compound (4.1) were tested in Sadar’s cell

based transcriptional activity assay to determine how well they inhibit the transcriptional

activity of the AR receptor. The biological data revealed that by reducing the natural product

(4.1) to the tetrahydrofuran analogue (4.2), there was a drastic decrease in the activity. In

addition, primary alcohol 4.4 was shown to be more active than the lead compound (4.1), and

had in vitro activity similar to that of the known antiandrogen bicalutamide (3.4) (Chapter 3,

Figure 3.2). With this promising biological activity, efforts towards constructing A-ring

analogues of 4.4 were undertaken to probe the SAR. Furthermore, analogue 4.4 has limited

solubility in water (CLogP = 5.88), therefore, water soluble analogues may potentially

enhance the drug-like properties (Chapter 2, Section 2.4) of this novel AR antagonist

pharmacophore. A-ring analogues of 4.4 have been previously synthesized 167 by Zoretic et

al. However, the synthesis is lengthy at fifteen linear steps starting from commercially

available methacrolein 168,169 (4.5) (Scheme 4.2).

Scheme 4.2 Zoretic’s synthesis of an A-ring 4.4 analogue (4.6).

We envisioned an epoxide-initiated cationic cascade (Chapter 2, Section 2.4) to

construct A-ring analogues of 4.4. Retrosynthetic analysis of A-ring analogue 4.6 revealed

the precursors needed. Analogue 4.6 could come from the epoxide-initiated cascade of 4.7.

Terminal epoxide 4.7 could come from geranyl bromide 4.8, furan 4.9, and epoxide 4.10

(Scheme 4.3).

Scheme 4.3 Retrosynthesis of a 4.4 A-ring analogue 4.6.

This synthetic route was devised with full recognition of the inherent reactivity of the

furan ring system toward an electrophile. Furan ring systems are known to undergo

preferential attack by the C-2 position (Figure 4.3) due to enhanced stabilization, through

greater charge delocalization when compared with nucleophilic attack by the C-3 position.

This results in a lower transition state energy for C-2 attack versus C-3 attack.

Figure 4.3 Delocalization of charge in a furan ring system.

This preference in reactivity towards the C-2 position differs from what we desire in

order to construct analogue 4.6, which is reactivity at the C-3 position. It was hypothesized

that the cyclization would yield at least a small amount of the disfavored C-3 regioisomer to

provide us with the desired analogue 4.6, while the C-2 regioisomer analogue would help

broaden the SAR.

4.2 Epoxide-Initiated Cationic Cascade Towards AR Antagonist 4.6

With the retrosynthesis in mind, construction of 4.6 began. The furanyl isoprene

fragment (4.13) was synthesized first (Scheme 4.4). Compound 4.13 is known and was

constructed using the literature protocol. 170 Commercially available 3-furan methanol 4.9 was

converted to chloride 4.11, which was then exposed to sanded magnesium turnings in

tetrahydrofuran to form the Grignard reagent in situ (Scheme 4.4). To the Grignard reagent

was added copper iodide followed by vinyl epoxide 4.10 to give alcohol 4.12. The published

procedure to brominate 4.12 used PBr3, however, it was found that this methodology gave

low yields (< 30 %). Changing the bromination strategy to the Appel 143 reaction resulted in a

higher yield for 4.13 (Scheme 4.4).

Scheme 4.4 Synthesis of furanyl isoprene 4.13.

The geranyl building block intermediate 4.21 (Scheme 4.5) was constructed next.

Geranyl bromide 4.8 was converted to the allylic sulfone 171 4.14 using p-toluenesulfinate and

tetrabutylammonium iodide. Sulfone 4.14 was oxidized using mCPBA to give epoxide 4.15,

which had been previously synthesized by Matt Nodwell in the Andersen group.

Scheme 4.5 Synthesis of regioisomer 4.24.

Epoxide 4.15 was cleaved using periodic acid 172 to give aldehyde 4.16. Phosphonate

4.17 was prepared following literature protocol 173 and was used in a Still–Gennari modified

Horner–Wadsworth–Emmons olefination 174 with aldehyde 4.16 to give the Z-ethyl ester 4.18

in a moderate yield (Scheme 4.5). Attempts to reduce ester 4.18 with LiAlH4 were

unsuccessful. However, using DIBAL to reduce ester 4.18 gave allylic alcohol 4.19 in a high

yield. Alcohol 4.19 was then oxidized to chiral epoxide 4.20 using the Sharpless 175

asymmetric epoxidation method with (–)-diisopropyl tartrate (DIPT) serving as the chiral

ligand. Changing the chiral ligand from (–) to (+)-DIPT, would allow us to access the

antipodal configuration of the natural product (4.1) for further SAR studies. At this stage of

the synthesis, the primary alcohol of 4.20 was protected, in order to prevent any coordination

to it by a Lewis acid in a future annulation step. A tert-butyldiphenylsilyl protecting group

was chosen for its steric bulk, shielding the alcohol during the epoxide-initiated cascade step.

Alcohol 4.20 was protected as the TBDPS-ether (4.21) in a quantitative yield (Scheme 4.5).

Sulfone 4.21 was deprotonated with potassium tert-butoxide 176 at –78 � C and coupled to

bromide 4.13 to give sulfone intermediate 4.22. Palladium catalyzed reductive elimination 177

of the tosyl sulfone moiety in 4.22 gave the terminal epoxide intermediate (4.23).

With the terminal epoxide in hand, the epoxide-initiated cascade step was next.

Indium tribromide served as the Lewis acid to initiate the cascade since it worked well for the

construction of SHIP1 activators (Chapter 2, Section 2.4). The resultant crude annulation

mixture contained a number of compounds. Using semisynthetic material 4.4 as a reference

for comparing 1 H NMR spectra, there was no evidence of the desired regioisomer in the

synthetic annulation reaction mixture. The two proton signals at the C-2 and C-5 positions

(Figure 4.4) in semisynthetic analogue 4.4 are found at 7.11 and 7.05 ppm respectively in the

1 H NMR spectrum. However, all isolated synthetic compounds that showed annulation to the

furan moiety had furan resonances at 7.17 and 6.10 ppm. The resonance at 6.10 ppm is

indicative of the furan C-4 proton, suggesting the exclusive formation of regioisomer 4.24

(Figure 4.4).

Figure 4.4 Favored and disfavored regioisomers.

4.3 Furan Blocking Group as a Strategy to Construct Desired Regioisomer 4.24

Having failed to construct the desired A-ring analogue of AR antagonist 4.4, we

decided to modify the furan functionality by installing a blocking group at the C-2 position

(Figure 4.5). Silyl functional groups were chosen as the best candidate to succeed for a

number of reasons. Silyl groups come in a range of sizes from the large triisoproply silyl

(TIPS) to the small trimethyl silyl (TMS) group, and this allows for potential tuning of the

steric component.

Figure 4.5 Blocking group to construct A-ring analogues of 4.4.

In addition, silicon is electropositive relative to carbon with an electronegativity of

1.90 versus 2.55 respectively (Pauling scale). This should result in an inductive effect, which

should increase the electron density of the furan ring, thus enhancing the nucleophilicity at

the C-4 position. Goldsmith 178 et al. reported a directed ortho metalation (DOM) of 3-furan

ethanol (4.27) and subsequent quenching with TMSCl to give the di-silylated intermediate

4.28. Subsequent deprotection with HCl resulted in TMS removal from the O-silyl group, but

left the C-silyl group intact (Scheme 4.6).

Scheme 4.6 Goldsmiths furan C-2 silylation strategy.

This was used as an inspiration to construct a 4.13 C-2 silyl analogue (Scheme 4.7).

3-Furan methanol 4.9 was protected as the di-silyl intermediate 4.30. Unfortunately, attempts

to selectively deprotect the O-silyl ether as was shown by Goldsmith et al. proved

unsuccessful and resulted in complete deprotection to yield starting material 4.9 (Scheme

4.7). Silylation of the furan ring by this route was abandoned.

Scheme 4.7 Failed selective TMS deprotection.

An alternative strategy using 3-furoic acid (4.31) was employed (Scheme 4.8). 179

Deprotonation of 3-furoic acid (4.31) by LDA followed by quenching with TMSCl gave C-2

silyl intermediate 4.32.

Scheme 4.8 Synthetic attempt towards C-2 silylated intermediate 4.35.

Reduction of the acid moiety in 4.32 to the primary alcohol by LiAlH4 gave

intermediate 4.33. Chlorination of 4.33 with thionyl chloride in the presence of triethylamine

gave a complex mixture. The reactive nature of chlorinated furan analogues such as 4.34

warranted its immediate use without further purification. 178 Intermediate 4.34 was exposed to

sanded magnesium turnings in tetrahydrofuran, after which epoxide 4.10 was added.

Purification of the reaction mixture failed to provide any evidence of silylated compound

4.35.

With the failed attempts of constructing C-2 silyl analogues of 4.12, thiol based

blocking groups were considered as an alternative to silyl groups. Sulfur with an

electronegativity of 2.58 versus 2.55 (Pauling scale) of carbon is slightly more

electronegative and, therefore, through-bond inductive effects to increase electron density in

the ring as with silicon is unlikely. However, thiol groups were chosen to provide steric

hindrance at the C-2 position. Furthermore, literature precedent in constructing thiol based

analogues of furan 178 existed allowing for their rapid development.

The construction of thiolated furan analogues began in a similar fashion as to the

silylated analogues. 3-Furan methanol (4.9) was treated with n-BuLi followed by quenching

with phenyl disulfide to give thiol 4.36 178 (Scheme 4.9). A reaction sequence analogous to

the one shown in Scheme 4.8 was attempted to access the thiolated analogue 4.38 without

success (Scheme 4.9).

Scheme 4.9 Attempt at constructing thiolated analogue 4.38.

Without successfully constructing substituted furan analogues by DOM of 3-furan

methanol (4.9) at an early stage of the synthesis, we turned our attention towards furan

substitution at a later stage of the synthesis. Lithiation 180 of intermediate 4.12 gave a mixture

of mono-thiolated compounds 4.39 and 4.38 at a 2:1 ratio respectively (Scheme 4.10).

Scheme 4.10 Mono-thiolated analogues of 4.12.

While this strategy was successful in generating thiol substituted furan analogues, the

regioselectivity of the reaction was poor due to the absence of a directing group for lithiation.

Furthermore, compounds 4.39 and 4.38 are inseparable by flash column chromatography,

which complicates characterization and future steps. A variation of this route was completed

in which successive lithiations gave the di-thiol substituted furan intermediate 4.41 (Scheme

4.11).

Scheme 4.11 Di-thiolated analogues of 4.12.

This provided a better overall yield along with no purification issues when compared

to the mono-thiolated synthesis. Bromination of 4.40 with carbon tetrabromide and triphenyl

phosphine gave 4.41 (Scheme 4.11). Having constructed a furan analogue that was

substituted at both the C-2 and C-5 position with phenyl sulfide, we continued the synthesis.

Sulfone 4.21 was coupled with bromine 4.41 to give 4.42, and subsequent de-sulfonylation

provided key epoxide intermediate 4.43 (Scheme 4.12).

Scheme 4.12 Epoxide-initiated cationic cascade using a thiol blocking group.

Cyclization with indium tribromide gave a complex reaction mixture, which was

purified using flash column chromatography. None of the desired product was observed and

the major annulation compound in the mixture was 4.44, which had the undesired

regiochemistry. The exclusive formation of 4.44 provided evidence that using a blocking

group as the only means to prevent nucleophilic attack by the more reactive C-2 position in

this furan ring system is not sufficient.

4.4 Biological Results

The semisynthetic analogues 4.2 and 4.4 were tested alongside the lead compound 4.1

in an androgen receptor competitive binding assay. Synthetic androgen R1881 (metribolone)

and clinically approved antiandrogen bicalutamide (3.4) were used as positive controls. This

assay utilizes the property of fluorescence polarization 181 to determine the affinity of a ligand

to the AR. When fluorescent molecules in solution are excited with plane-polarized light, the

light they emit is also in a fixed plane (polarized light). However, this only occurs if the

fluorophore remains stationary during the excitation.

The assay consists of AR-LBD and an androgen ligand with a fluorescent tag

(Fluormone TM AL Green) in the presence of a competitor ligand. The formation of the AL

Green/AR-LBD complex results in the fluorophore being in a bound state. When excited

with polarized light, the slow rotation of the peptide-bound fluorophore results in the

emission of polarized light. A ligand with affinity to the AR-LBD causes the displacement of

AL Green from its bound stationary state. This will result in a decrease in polarization of the

emitted light due to the enhanced tumbling rate of the small molecule fluorophore in

solution. The difference in net polarization in the presence of a test compound is used to

determine the affinity for the AR-LBD. The binding curves of the cell-based assay are shown

in Figure 4.7.

Figure 4.6 AR competitor in vitro assay.

Half-maximal effective concentration (EC50) values were calculated for each

compound from the binding curves. The values from a representative experiment are shown

in Table 4.1. The biological results of the in vitro assay provide a clear SAR for the lead

compound 4.1 and semisynthetic analogues 4.2 and 4.4. There is an approximate fivefold

increase in the EC50 value between 4.1 and reduced analogue 4.2. This suggests that the

aromaticity of the furan moiety in 4.1 is necessary in order to maintain activity.

Table 4.1 EC50 values for tested compounds in an AR competitor in vitro assay.

Compound EC50 (nM)

R1881 2.09

3.4 2014

4.1 14718

4.2 75569

4.4 4249

There is an approximate 3.5 fold decrease in the EC50 value when comparing the lead

compound 4.1 to its semisynthetic analogue in alcohol 4.4. This result indicates that there is

enhanced potency in converting the neopentyl carboxylic acid to a neopentyl alcohol in the

natural product (4.1). In fact, the affinity towards the AR of analogue 4.4 is comparable to

the clinically approved antiandrogen drug bicalutamide (3.4) (Chapter 3, Figure 3.2), which

is currently used as a treatment for prostate cancer.

Lead compound 4.1 in addition to analogues 4.2 and 4.4 were also tested in an in

vitro assay to determine their effect on AR transcriptional activity. This assay is similar to the

assay completed for the niphatenones (Chapter 3, Figure 3.11). It was found that

tetrahydrofuran analogue 4.2 had no effect on inhibiting the transcriptional activity of the

AR. This is further evidence that the furan moiety plays an important role in the biological

activity. The data also suggested that both 4.1 and 4.4 inhibited AR transcriptional activity,

however, analogue 4.4 was almost three times as potent. These results reinforce those of the

in vitro AR affinity assay (Table 4.1). The detailed results of this experiment will be

described elsewhere. The biological results of both in vitro assays are promising, and suggest

a clear SAR for the natural product (4.1), and semisynthetic analogues 4.2 and 4.4.

4.5 Conclusion

Small molecule antagonists 130 of the AR are currently used as a therapeutic treatment

for prostate cancer in patients suffering from the disease. In an ongoing effort to identify

marine natural products that are AR antagonists, the Andersen natural product extract library

was screened using Dr. Marianne Sadar’s cell based transcriptional assay at the BC Cancer

Bioassay-guided fractionation of the crude methanol extract of a marine sponge led to

the identification of terpene 4.1 as an antagonist of the AR. Using compound 4.1, a

semisynthesis was completed to probe this novel AR pharmacophore. Two semisynthetic

analogues were constructed providing reduced analogue 4.2 and alcohol 4.4. An in vitro

assay was completed to determine the binding affinity to the AR of lead compound 4.1 and

analogues 4.2 and 4.4. There were several conclusions that were made from this data: 1) the

furan moiety is necessary in order to have affinity for the AR, 2) the carboxylic acid moiety

found in the natural product is responsible for attenuated activity when compared to its

primary alcohol analogue in 4.4, and 3) analogue 4.4 is almost as active as the clinical drug

bicalutamide (3.4) in its ability to displace a fluorescently-tagged androgen ligand bound to

the AR.

Furthermore, an in vitro assay was completed to determine the ability of the natural

product (4.1) and its analogues 4.2 and 4.4 at inhibiting AR transcriptional activity. The

results of this assay mirror those of the in vitro AR affinity assay. The tetrahydrofuran

analogue 4.2 had lost all activity while analogue 4.4 was more active than the lead compound

(4.1). The results of both in vitro assays suggest a clear SAR for these novel AR

pharmacophores.

The focus of this chapter is dedicated towards the synthesis of 4.4 analogues to

broaden the SAR, and to enhance the solubility of these compounds in water for enhanced

drug-like effects. An epoxide-initiated cationic cascade was devised as a method to construct

A-ring analogues of 4.4 (Scheme 4.3). Annulation of the key terminal epoxide intermediate

4.23 provided the undesired regioisomer 4.24 exclusively (Scheme 4.5). This was caused by

the inherent reactivity of the furan C-2 center versus the C-4 center (Figure 4.3). To address

the issue of furan reactivity, analogues of terminal epoxide 4.23 with a silyl group at the C-2

position was proposed (Figure 4.5). The purpose of this silylated analogue was to block

attack by the reactive C-2 position, and increase the electron density of the ring, all in the

hopes that the C-4 position would react. Unfortunately, our attempts to construct silylated

analogues failed.

Alternatively, thiol groups were installed in the C-2 position of 4.23, in order to

attenuate the reactivity of the C-2 position by blocking nucleophilic attack. Epoxide-initiated

cationic cascade of thiol-substituted furan 4.43, gave exclusively the C-2 substituted

analogue 4.44 with the undesired regiochemistry (Scheme 4.12). This outcome was a clear

indication that steric factors alone are not sufficient to form the desired regioisomer. The

synthesis of 4.4 analogues using an epoxide-initiated cascade is a continuing project in the

Andersen lab. Lead compound 4.1 and semisynthetic analogue 4.4 represent promising drug

leads in the development of small molecule antagonists of the AR.

4.6 Experimental

mass spectrometry (ESI-MS) spectra were recorded on a Micromass LCT instrument.

Preparation of 4.16:

To a solution of metaperiodic acid (918.5 mg, 4.78 mmol, 3.60 mL water) was added

epoxide 4.15 (1.12 g, 3.63 mmol) dissolved in 7 mL of tetrahydrofuran at 0 � C and allowed

to stir for half an hour. To this mixture was added brine (100 mL) and the aqueous layer was

extracted three times with methylene chloride (350 mL). The combined organic extracts were

dried with MgSO4 and concentrated using a rotary evaporator. The crude mixture was

purified using flash column chromatography (hexanes:ethyl acetate 2:1) to give 4.16 (869.8

mg, 3.26 mmol, 89.8 %). 1 H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 7.65 (d, J = 8.0 Hz,

2H), 7.28 (d, J = 8.4 Hz, 2H), 5.15 (t, J = 8.0 Hz, 1H), 3.72 (d, J = 8.0 Hz, 2H), 2.45 (t, J =

7.6 Hz, 2H), 2.39 (s, 3H), 2.27 (t, J = 7.6 Hz, 2H), 1.31 (s, 3H); 13 C NMR (100 MHz, CDCl3)

δ 201.5, 144.8, 144.3, 135.8, 129.8, 128.5, 111.6, 56.0, 41.6, 31.7, 21.7, 16.4. HRESIMS [M

+ H] + calcd for C14H19O3 32 S 267.1055, found 267.1059.

Preparation of 4.18:

To phosphonate 4.17 (440 mg, 1.27 mmol) dissolved in 1.88 mL tetrahydrofuran was

added 18C-6 (1.00 g, 3.81 mmol) and the solution was cooled to –78 � C. To this solution was

added KHMDS (278.9 mg, 1.39 mmol) dissolved in 1.5 mL of toluene, and allowed to stir

for thirty minutes, after which aldehyde 4.16 (320.4 mg, 1.27 mmol) dissolved in 2 mL of

tetrahydrofuran was added dropwise using a syringe. After stirring at –78 � C for one hour,

the reaction mixture was quenched with saturated NH4Cl (aq) (100 mL) and allowed to warm

to room temperature. The aqueous layer was extracted three times with methylene chloride

(350 mL). The combined organic extracts were dried with MgSO4 and concentrated using a

(hexanes:ethyl acetate 5:1) to give 4.18 (259.6 mg, 0.74 mmol, 58.2 %). 1 H NMR (400 MHz,

CDCl3) δ 7.62 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 5.76 (t, J = 7.6 Hz, 1H), 5.11 (t, J

= 8.0 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.70 (d, J = 8.0 Hz, 2H), 2.43 (q, J = 7.6 Hz, 2H),

2.33 (s, 3H), 2.00 (t, J = 7.6 Hz, 2H), 1.80 (s, 3H), 1.24 (s, 3H), 1.20 (t, J = 7.2 Hz, 3H); 13 C

NMR (100 MHz, CDCl3) δ 167.8, 145.5, 144.5, 141.6, 135.9, 129.7, 128.5, 127.8, 111.2,

60.1, 56.1, 39.2, 27.6, 21.6, 20.7, 16.1, 14.4. HRESIMS [M + Na] + calcd for C19H26O4Na 32 S

373.1450, found 373.1457.

Preparation of 4.19:

To 4.18 (333 mg, 0.95 mmol) dissolved in 8 mL of tetrahydrofuran cooled to –78 � C

was added a 1.0 M solution of DIBAL (2.85 mL, 2.85 mmol) dropwise using a syringe. After

stirring at –78 � C for two hours, the reaction mixture was quenched with saturated NH4Cl (aq)

(100 mL) and allowed to warm to room temperature. The aqueous layer was extracted three

times with methylene chloride (250 mL). The combined organic extracts were dried with

MgSO4 and concentrated using a rotary evaporator. The crude mixture was purified using

flash column chromatography (hexanes:ethyl acetate 3:2) to give 4.19 (277.5 mg, 0.89 mmol,

94.6 %). 1 H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H),

5.18 (m, 2H), 4.12 (s, 2H), 3.80 (d, J = 7.8 Hz, 2H), 2.45 (s, 3H), 2.13 (quin, J = 6.9 Hz, 2H),

2.04 (m, 2H), 1.91 (bs, 1H), 1.81 (s, 3H), 1.40 (s, 3H); 13 C NMR (75 MHz, CDCl3) δ 145.9,

144.8, 135.9, 135.4, 129.8, 128.6, 127.2, 110.8, 61.6, 56.2, 39.8, 25.7, 21.8, 21.5, 16.5.

HRESIMS [M + Na] + calcd for C17H24O3Na 32 S 331.1344, found 331.1349.

Preparation of 4.20:

To (–)-DIPT (0.115 mL, 0.549 mmol) and Ti(O-i-Pr)4 (0.115 mL, 0.329 mmol) in 10

mL of methylene chloride was added crushed 4 Å molecular sieves (1.07 g) and allowed to

stir for twenty minutes at -20 � C, after which was added a 5.5 M solution of t-BuOOH (1.44

mL, 7.9 mmol) in decane and the mixture allowed to stir for an additional twenty minutes. To

this mixture was added alcohol 4.19 (1.21 g, 3.92 mmol) dissolved in 6.5 mL of methylene

chloride. After stirring for 2.5 hours TLC indicated the presence of starting materials, and so

(+)-DIPT (0.115 mL, 0.549 mmol), Ti(O-i-Pr)4 (0.115 mL, 0.329 mmol), and t-BuOOH (0.75

mL, 4.1 mmol) was added to the reaction mixture and it was allowed to warm to room

temperature overnight. The crude reaction mixture was then filtered through filter paper and

to it added 100 mL of 2 M NaOH, and allowed to stir until two distinct layers were visible (5

hours). The aqueous layer was extracted three times with methylene chloride (400 mL). The

combined organic extracts were dried with MgSO4 and concentrated using a rotary

(hexanes:ethyl acetate 3:2) to give 4.20 (935.5 mg, 2.88 mmol, 73.5 %). 1 H NMR (300 MHz,

CDCl3) δ 7.66 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 5.17 (t, J = 6.6 Hz, 1H), 3.76 (d, J

= 7.8 Hz, 2H), 3.59 (s, 2H), 2.73 (dd, J = 6.9, 5.4 Hz, 1H), 2.61 (bs, 1H), 2.39 (s, 3H), 2.10

(oct, J = 7.8 Hz, 2H), 1.69-1.49 (m, 2H), 1.34 (s, 3H), 1.33 (s, 3H); 13 C NMR (75 MHz,

CDCl3) δ 145.4, 144.8, 135.8, 129.8, 128.5, 111.2, 64.1, 63.9, 61.2, 56.1, 36.6, 26.4, 21.7,

20.2, 16.4. HRESIMS [M + Na] + calcd for C17H24O4Na 32 S 347.1293, found 347.1299.

Preparation of 4.21:

To alcohol 4.20 (65 mg, 0.20 mmol) dissolved in 0.5 mL of methylene chloride was

added DMAP (2.44 mg, 0.019 mmol) and triethylamine (0.033 mL, 0.24 mmol) and the

reaction mixture was cooled to 0 � C, after which TBDPSCl (0.056 mL, 0.22 mmol) was

added and allowed to warm to room temperature overnight. The crude reaction mixture was

concentrated under a stream of nitrogen and was purified using flash column chromatography

(hexanes:ethyl acetate 4:1) to give 4.21 (113 mg, 0.20 mmol, 100 %). 1 H NMR (400 MHz,

CDCl3) δ 7.72-7.66 (m, 6H), 7.46-7.37 (m, 6H), 7.28 (d, J = 8.4 Hz, 2H), 5.17 (t, J = 6.8 Hz,

1H), 3.78 (d, J = 8.0 Hz, 2H), 3.70 (d, J = 10.8 Hz, 1H), 3.63 (d, J = 11.2 Hz, 1H), 2.39 (s,

3H), 2.14 (m, 2H), 1.56-1.49 (m, 1H), 1.42-1.38 (m, 1H), 1.44 (s, 3H), 1.32 (s, 3H), 1.26 (t, J

= 7.2 Hz, 1H), 1.09 (s, 9H); 13 C NMR (100 MHz, CDCl3) δ 145.3, 144.7, 135.8, 135.7,

133.5, 133.1, 130.0, 128.6, 127.9, 111.3, 65.5, 63.6, 60.9, 56.1, 36.6, 26.9, 26.5, 20.5, 19.4,

16.3, 14.4. HRESIMS [M + Na] + calcd for C33H42O4Na 28 Si 32 S 585.2498, found 585.2485.

Preparation of 4.22:

To bromide 4.13 (55.0 mg, 0.238 mmol) and sulfone 4.21 (96 mg, 0.17 mmol),

dissolved in 2 mL of tetrahydrofuran cooled to –78 � C was added KO t Bu (28.6 mg, 0.25

mmol), and the reaction mixture allowed to stir for three hours. The reaction was then

quenched with the addition of 0.1 mL of methanol, and concentrated under a stream of

nitrogen, and purified using flash column chromatography (hexanes:ethyl acetate 6:1) to give

4.22 (8.3 mg, 0.011 mmol, 9.5 % brsm). 1 H NMR (400 MHz, CDCl3) δ 7.68 (m, 6H), 7.46-

7.37 (m, 6H), 7.30-7.21 (m, 3H), 7.12 (s, 1H), 6.20 (s, 1H), 5.13 (t, J = 6.8 Hz, 1H), 4.89 (d, J

= 10.4 Hz, 1H), 3.89-3.83 (m, 1H), 3.69-3.60 (m, 2H), 2.87 (d, J = 13.2 Hz, 1H), 2.72 (m,

1H), 2.38 (s, 3H), 2.35 (m, 2H), 2.24 (m, 2H), 2.17 (m, 2H), 2.06 (m, 2H), 1.49 (s, 3H), 1.42

(s, 3H), 1.20 (m, 4H), 1.08 (s, 9H); 13 C NMR (100 MHz, CDCl3) δ 142.7, 138.9, 135.8,

135.7, 133.5, 133.2, 130.0, 129.5, 129.4, 129.3, 127.9, 127.9, 118.3, 118.3, 111.1, 65.5, 63.5,

63.5, 61.0, 36.6, 36.6, 28.5, 26.9, 26.5, 24.8, 21.7, 20.5, 19.5, 16.5, 16.2. HRESIMS [M +

Na] + calcd for C43H54O5Na 28 Si 32 S 733.3359, found 733.3342.

Preparation of 4.23:

To sulfone 4.22 (46.6 mg, 0.065 mmol) dissolved in 1 mL of tetrahydrofuran was

added PdCl2(dppp) (7.7 mg, 0.013 mmol) and cooled to 0 � C, to which a 1.0 M solution of

LiBEt3H (0.13 mL, 0.13 mmol) was added dropwise using a syringe. After stirring for one

hour, the reaction was quenched with 0.1 mL of methanol and was concentrated under a

(hexanes:ethyl acetate 10:1), to give epoxide 4.23 (32.4 mg, 0.058 mmol, 88.5 %, yield

includes small impurity). 1 H NMR (400 MHz, CDCl3) δ 7.70-7.66 (m, 5H), 7.46-7.34 (m,

5H), 7.34 (t, J = 1.6 Hz, 1H), 7.21 (s, 1H), 6.28 (s, 1H), 5.17 (t, J = 6.8 Hz, 1H), 5.11 (t, J =

6.8 Hz, 1H), 3.70 (d, J = 10.8 Hz, 1H), 3.64 (d, J = 10.8 Hz, 1H), 2.78 (t, J = 6.4 Hz, 1H),

2.45 (t, J = 7.2 Hz, 2H), 2.25 (m, 2H), 2.12-1.98 (m, 6H), 1.59 (s, 3H), 1.55 (s, 3H), 1.51 (m,

2H), 1.43 (s, 3H), 1.08 (s, 9H); 13 C NMR (100 MHz, CDCl3) δ 142.5, 138.8, 135.7, 135.5,

133.9, 133.4, 133.1, 129.7, 127.7, 125.0, 124.8, 123.8, 111.1, 65.4, 64.0, 60.7, 39.6, 36.4,

28.5, 26.8, 26.7, 26.6, 25.0, 20.3, 19.3, 16.1. HRESIMS [M + Na] + calcd for C36H48O3Na 28 Si

579.3270, found 579.3265.

Preparation of 4.24:

To epoxide 4.23 (32 mg, 0.057 mmol) dissolved in 1 mL of methylene chloride was

added InBr3 (40.7 mg, 0.11 mmol), and allowed to stir at room temperature for forty minutes.

The reaction was quenched with 4 drops of saturated NaHCO3 and concetrated under a

stream of nitrogen. The mixture was purified using flash column chromatography

(hexanes:ethyl acetate 12:1) to give a crude mixture of 4.24 and side products. A 1 H NMR

spectrum of a mixed fraction believed to contain 4.24 shows resonances at 7.17 and 6.10

indicative of protons at the C-5 and C-4 positions respectively. HRESIMS [M + H] + calcd for

C36H49O3 28 Si 557.3451, found 557.3446.

Figure 4.14 1 H spectrum of 4.24 recorded in CDCl3 at 400 MHz.

Preparation of 4.40:

To furan 4.12 (100 mg, 0.60 mmol) dissolved in 3 mL of tetrahydrofuran and cooled

to –20 � C was added a 1.6 M solution of n-BuLi (0.8 mL, 1.28 mmol) and cooled to –10 � C.

The mixture was allowed to stir for 30 minutes after which diphenyl disulfide (151 mg, 0.69

mmol) was added and the mixture stirred at –10 � C for 30 minutes then room temperature for

one hour. The mixture was then cooled to –20 � C and to it was added a 1.6 M solution of

n-BuLi (0.8 mL, 1.28 mmol) and cooled to –10 � C. The mixture was allowed to stir for 30

minutes after which diphenyl disulfide (151 mg, 0.69 mmol) was added and the mixture was

allowed to warm to room temperature overnight. The mixture was then quenched with the

addition of 0.5 mL of methanol, concentrated using a rotary evaporator, and purified using

flash column chromatography (hexanes:ethyl acetate 6:1 – 4:1) to give 4.40 (183.6 mg, 0.48

mmol, 80 %). 1 H NMR (400 MHz, CDCl3) δ 7.29-7.13 (m, 10H), 6.71 (s, 1H), 5.37 (t, J =

8.4 Hz, 1H), 3.92 (s, 2H), 2.61 (t, J = 7.2 Hz, 2H), 2.29 (q, J = 7.2 Hz, 2H), 1.58 (s, 3H), 1.38

(s, 1H); 13 C NMR (100 MHz, CDCl3) δ 146.8, 143.4, 136.4, 136.2, 135.5, 135.2, 129.3,

129.2, 128.5, 127.5, 126.9, 126.5, 124.5, 121.3, 68.8, 28.1, 25.7, 13.8. HRESIMS [M + Na] +

calcd for C22H22O2Na 32 S2 405.0959, found 405.0956.

Preparation of 4.41:

To alcohol 4.40 (64.3 mg, 0.17 mmol) dissolved in 2 mL of methylene chloride was

added triphenylphosphine (57.2 mg, 0.22 mmol) and cooled to 0 � C, after which carbon

tetrabromide (72.3 mg, 0.22 mmol) was added and the reaction allowed to stir for 45 minutes

at 0 � C. The crude reaction mixture was concentrated using a rotary evaporator and filter

through a plug of silica (hexanes:ethyl acetate 4:1) to give 4.41 (59.0 mg, 0.13 mmol, 78 %

estimated from crude material) and immediately used in the next step.

Preparation of 4.42:

To sulfone 4.21 (900 mg, 1.74 mmol) and bromine 4.41 (895.3 mg, 2.0 mmol)

dissolved in 18 mL of tetrahydrofuran and cooled to –78 � C was added KO t Bu (234.3 mg,

2.08 mmol) and the reaction allowed to warm to room temperature overnight. The crude

reaction mixture was quenched with 1 mL of methanol, concentrated under a stream of

nitrogen and purified using flash column chromatography (hexanes:ethyl acetate 6:1-4:1) to

give 4.42 (597.9 mg, 0.64 mmol, 58.2 %, brsm, 272.2 mg of 4.21). 1 H NMR (400 MHz,

CD2Cl2) δ 7.22-7.63 (m, 6H), 7.47-7.40 (m, 6H), 7.31-7.10 (m, 12H), 6.88 (s, 1H), 5.11 (t, J

= 7.2 Hz, 1H), 4.88 (d, J = 10.8 Hz, 1H), 3.84 (m, 1H), 3.67 (d, J = 1.6 Hz, 2H), 2.80 (d, J =

12.8 Hz, 1H), 2.68 (q, J = 7.6 Hz, 1H), 2.48 (t, J = 7.6 Hz, 2H), 2.38 (s, 3H), 2.22-2.11 (m,

3H), 2.04 (t, J = 7.2 Hz, 2H), 1.56 (s, 1H), 1.45 (s, 3H), 1.41 (s, 3H), 1.36-1.30 (m, 1H), 1.17

(dd, J = 8, 1.2 Hz, 3H), 1.09 (s, 9H); 13 C NMR (100 MHz, CD2Cl2) δ 146.5, 144.62, 144.1,

143.8, 136.2, 135.7, 135.6, 135.4, 135.3, 133.6, 133.3, 133.3, 131.5, 129.9, 129.5, 129.3,

129.2, 129.2, 128.3, 127.9, 127.8, 127.4, 127.0, 127.0, 126.9, 126.5, 121.3, 118.1, 65.6, 63.3,

63.3, 60.7, 37.7, 37.7, 36.5, 36.5, 28.4, 26.7, 26.5, 25.6, 21.4, 20.2, 19.3, 16.1, 15.8.

HRESIMS [M + Na] + calcd for C55H62O5Na 28 Si 32 S3 949.3426, found 949.3448.

Preparation of 4.43:

To sulfone 4.42 (600 mg, 0.65 mmol) and PdCl2(dppp) (76.5 mg, 0.13 mmol) in 16

mL of tetrahydrofuran cooled to 0 � C was added a 1 M solution of LiBEt3H (1.3 mL, 1.3

mmol) dropwise using a syringe. After stirring for four hours, the mixture was quenched with

saturated NH4Cl (aq) (150 mL) and allowed to warm to room temperature. The aqueous layer

was extracted three times with methylene chloride (400 mL). The combined organic extracts

were dried with MgSO4 and concentrated using a rotary evaporator. The crude mixture was

purified using flash column chromatography (hexanes:ethyl acetate 15:1) to give 4.43 (376.4

mg, 0.48 mmol, 73.8 %). 1 H NMR (400 MHz, CDCl3) δ 7.67 (t, J = 8.0 Hz, 6H), 7.45-7.36

(m, 7H), 7.25-7.11 (m, 7H), 6.69 (s, 1H), 5.11 (q, J = 6.4 Hz, 2H), 3.69 (d, J = 11.2 Hz, 1H),

3.63 (d, J = 10.8 Hz, 1H), 2.76 (t, J = 6.4 Hz, 1H), 2.55 (t, J = 7.2 Hz, 2H), 2.22 (q, J = 7.2

Hz, 2H), 2.09 (q, J = 6.4 Hz, 2H), 2.04-1.91 (m, 4H), 1.53 (m, 8H), 1.42 (s, 3H), 1.08 (s, 9H);

13 C NMR (100 MHz, CDCl3) δ 146.5, 136.5, 135.8, 135.7, 135.6, 133.3, 129.9, 129.2, 129.1,

128.4, 127.9, 127.8, 127.5, 126.8, 126.3, 124.9, 123.1, 121.5, 118.6, 111.8, 65.5, 64.1, 39.7,

36.5, 28.5, 27.0, 26.9, 26.8, 26.7, 26.0, 22.1, 20.5, 19.4, 16.2, 16.1. HRESIMS [M + Na] +

calcd for C48H56O3Na 28 Si 32 S2 795.3338, found 795.3355.

Preparation of 4.44:

To polyene 4.43 (300 mg, 0.38 mmol) dissolved in 10 mL of methylene chloride was

added InBr3 (275 mg, 0.77 mmol) and allowed to stir at room temperature for one hour. The

mixture was quenched with saturated NaHCO3 (100 mL) and the aqueous layer was extracted

three times with methylene chloride (250 mL). The combined organic extracts were dried

using flash column chromatography (hexanes:ethyl acetate 15:1) to give 4.44 (71.3 mg, 0.10

mmol, 28.4 %, yield includes a small impurity). 1 H NMR (600 MHz, CDCl3) δ 7.72 (dd, J =

16.2, 7.2 Hz, 4H), 7.49-7.45 (m, 5H), 7.24 (t, J = 4.8 Hz, 2H), 7.13 (t, J = 5.2 Hz, 2H), 7.09

(m, 2H), 6.47 (s, 1H), 4.20 (d, J = 10.2 Hz, 1H), 3.47 (d, J = 10.2 Hz, 1H), 3.32 (m, 1H), 2.49

(dd, J = 15.6, 5.4 Hz, 1H), 2.32 (m, 1H), 2.17 (dt, J = 13.2, 3.0 Hz, 1H), 1.90 (m, 1H), 1.78

(m, 2H), 1.70-1.61 (m, 3H), 1.47 (m, 1H), 1.39 (m, 2H), 1.34 (s, 6H), 1.30-1.18 (m, 2H),

1.08 (s, 9H), 0.99 (m, 1H), 0.62 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 163.6, 135.3, 135.2,

132.1, 131.7, 129.5, 128.5, 127.4, 126.0, 125.3, 120.3, 115.9, 80.0, 65.5, 55.9, 55.8, 47.5,

42.4, 37.7, 36.7, 35.3, 35.2, 27.5, 26.4, 22.5, 22.3, 21.8, 18.0, 17.6, 16.6. HRESIMS [M +

H] + calcd for C42H53O3 28 Si 32 S 665.3485, found 665.3474 .

Ligand-Binding Affinities of Terpenes in AR: Androgen Receptor Competitor

Assay Kit (Invitrogen TM ) was employed for the in vitro binding assay. Recombinant AR-

LBD and fluorescently labeled androgen ligand were used to test the binding affinities of

semisynthetic analogues 4.1, 4.2, and 4.4 using synthetic androgen R1881 (metribolone) and

clinically approved antiandrogen bicalutamide (3.4) as positive controls. Each testing

compound underwent serial dilutions and was added in triplicates to a Greiner 384-well black

clear bottom plate. At excitation wavelength of 470 nm and emission of 535 nm,

fluorescence polarization was measured by Infinite M1000 (TECAN®). The graph was

generated by plotting polarization value (mP) against concentration (Log(nM)) using Prism

software (GraphPad Software) as nonlinear regression fit for One-site Competition curves.

Representative data of a single experiment is plotted. Error bars represent the mean ± SEM of

technical triplicates for each data point measured.

Chapter 5: Synthetic Efforts Towards Lichostatinal (5.4): A Potent

Cathepsin K Inhibitor

5.1 Cysteine Protease Inhibitors

Proteases are ubiquitous in human physiology where they play important roles in

apoptosis, 182 breakdown of intracellular proteins, 183 and clearance of organic particulates and

microorganisms. Although a variety of proteases exists, many have a similar mechanism of

action. A nucleophilic amino acid residue in the protease is responsible for amide bond

cleavage, resulting in the hydrolysis of a peptide into smaller units (Figure 5.1). 183

Figure 5.1 Hydrolysis of a peptide by a cysteine protease.

Proteases are classified as serine, 184 threonine, cysteine, 185 aspartate 186 and

metalloproteases. The mechanism of proteolysis by cysteine proteases 187,188 is exemplified in

Figure 5.1. The first step entails deprotonation of the cysteine residue in the active site of the

protease by a nearby basic moiety, typically a histidine residue (Figure 5.1). The next step is

attack of the carbonyl carbon of the peptide substrate by the sulfide anion of cysteine,

liberating a new N-terminus subunit. The resulting thioester intermediate is then hydrolyzed

to regenerate the cysteine and histidine residues along with a carboxylic acid subunit (Figure

5.1).

Unlike many other reversible post-translational modifications that peptides typically

undergo, such as phosphorylation, 189 proteolysis is irreversible. Once hydrolyzed, the only

means available to regain the peptide is for mRNA translation to occur. Because of this

irreversible effect on peptides, proteases are compartmentalized in the lysosome 190 and/or in

the endosome 191 within the cell. However, low concentrations of proteases are found

throughout the body. The activity of cysteine proteases is kept in check by endogenous

cysteine protease inhibitors such as cystatins 192 and serpins. 193 These inhibitors are found in

excess concentrations in the cytoplasm and extracellular space. An imbalance between

cysteine proteases and their inhibitors can lead to human diseases such as muscular

dystrophy, 194 arthritis, 195 bone resorption, 196 and Alzheimer’s disease. 197

Caspase 198 and cathepsin 199 proteases make up the two groups of cysteine proteases.

Cathepsin K is expressed in osteoclasts 200 and has been implicated in bone resorption. The

biological role of cathepsin K has led to the development of small molecule inhibitors as

potential therapeutics to combat osteoporosis, 201 with natural products playing an important

role in lead compound discovery. The first cysteine protease inhibitor to be discovered was

leupeptin 202 (5.1), a peptide-aldehyde produced in culture by various actinomycetes bacteria.

Shortly afterwards, additional peptide-aldehyde natural product inhibitors of cathepsin such

as chymostatin 203 (5.2) and antipain 204 (5.3) were also isolated (Figure 5.2).

Figure 5.2 Peptide-aldehyde inhibitors of cysteine proteases.

While these peptide-aldehyde natural products are potent inhibitors of cathepsin K,

they also show activity towards other cathepsins and serine proteases. In addition, these

inhibitors bind cysteine proteases covalently. Typically, the thiol or alcohol side chain in a

cysteine and/or serine will attack the electrophilic aldehyde moiety in the natural product as

shown in Figure 5.3.

cysteine protease.

This covalent binding mechanism along with the chronic nature of osteoporosis

treatment leads to these peptide-aldehyde inhibitors as being unlikely drug candidates due to

potential off-target effects. 205 However, these inhibitors often find use as chemical tools

when an enzyme is being studied in an in vitro setting. In these studies the cell is lysed,

which releases the once compartmentalized proteases. The addition of a small molecule

protease inhibitor stops proteolysis of potential target enzymes allowing them to be studied.

5.2 Lichostatinal (5.4) a Novel Peptide-Aldehyde Inhibitor of Cathepsin K

Several cathepsin K inhibitors are currently in development and include balicatib 206

(Novartis), relacatib 207 (GlaxoSmithKline), and odanacatib 208 (Merck & Co.). However, there

is currently no available therapeutic cathepsin K inhibitor for the treatment of osteoporosis.

The lack of available therapeutics led to a collaboration between the laboratories of

Dr. Julian Davies and Dr. Dieter Bromme at the University of British Columbia. The

laboratory of Dr. Davies has a collection of more than 2000 strains of streptomyces, and of

those, 384 were screened in a cathepsin K inhibition assay by Vincent Paul Lavallée in Dr.

Bromme’s laboratory. Attempts to purify the most active extract by reversed phase HPLC

purification failed. However, samples of enriched purity were isolated that showed activity in

the nM range in a cathepsin K enzyme inhibition assay. Co-crystallization of the ligand-

substrate complex was successful by soaking the active fraction with cathepsin K. The crystal

was analyzed by a beamline, 209 which is a light source of high-energy (X-rays) emitted from

a synchrotron and often used for protein crystallography. The structure of the active

metabolite was elucidated to be the novel peptide-aldehyde lichostatinal (5.4) (Figure 5.4).

Lichostatinal (5.4) is the first example of a natural product to be isolated from a complex

mixture by co-crystallization with its biological target.

Figure 5.4 Novel peptide-aldehyde lichostatinal (5.4).

Lichostatinal (5.4) is a peptide tetramer comprised of arginine, valine, serine, and

agmatine residues, with a urea linkage between serine and arginine. Peptide 5.4 is structurally

similar to leupeptin (5.1) and antipain (5.3), in that the arginine residue bears the reactive

aldehyde functionality that presumably covalently binds to cathepsin K. While the beamline

data was successful in providing a structure for the active metabolite, additional

spectroscopic data was required to further verify the structure of lichostatinal (5.4) and

compare it with biological data gathered on the crude sample.

The role of the author was to construct synthetic lichostatinal (5.4) to verify the

proposed structure through biological testing and co-crystallization with cathepsin K. The

retrosynthetic analysis of lichostatinal (5.4) shows that it can be derived from linking

agmatine (5.6) and tripeptide 5.5 by a urea bond. Tripeptide 5.5 may be constructed from

coupling the amino acids L-arginine (5.7), L-valine (5.8), and L-serine (5.9) (Scheme 5.1).

Scheme 5.1 Retrosynthetic analysis of lichostatinal (5.4).

The first step in the synthesis was the coupling of 5.10 to L-valine-N-Fmoc (5.11),

using standard coupling methodology (Scheme 5.2). Various peptide coupling reagents 210

exist, and are typically used in a 1:1 ratio to the corresponding amino acid. HATU 211 was

used to couple nitro-protected arginine 5.10 with Fmoc protected L-valine (5.11) to construct

5.12. Intermediate 5.12 was then deprotected 212 with diethylamine in acetonitrile to give

amine 5.13. HATU was again used to couple 5.13 to O-benzyl protected L-serine-N-Boc

(5.14) to give intermediate 5.15. The Boc-protecting group was removed with concentrated

HCl to give amine 5.16.

Scheme 5.2 Synthesis toward lichostatinal (5.4).

At this point, coupling commercially available agmatine sulfate (5.6) to 5.16 with

CDI 213 as the coupling reagent was thought to be the most expeditious route. Unfortunately,

agmatine sulfate (5.6) is sparingly soluble in N,N-dimethylformamide, dimethyl sulfoxide, or

tetrahydrofuran, which are classic CDI coupling solvents. Agmatine sulfate (5.6) and its

corresponding freebase are only freely soluble in water, which decomposes CDI.

Nevertheless, the coupling was attempted using the aforementioned solvents, to no avail

(Scheme 5.2). Alternatively, mono-Boc protected putrescine 214 5.17 was coupled with 5.16.

Varying the reaction conditions was done to optimize the yields for this step. This involved

changing the order of amine addition to CDI. It was found that exposing CDI to tripeptide

5.16 for several hours, followed by addition of 5.17, failed to yield product. However,

exposing CDI to 5.17, followed by addition of 5.16 resulted in product formation (5.18)

(Scheme 5.2). Dimethyl sulfoxide and N,N-dimethylformamide were both tested as solvents

for the reaction and provided similar yields. N,N-dimethylformamide was favored due to ease

of handling when compared with dimethyl sulfoxide. An alternative method in forming the

urea linkage was attempted using triphosgene, 215 however, this was unsuccessful in the hands

of the author.

Next, methods of forming the aldehyde moiety were considered. One way to access

an amino aldehyde is to reduce the ester with DIBAL at –78 � C as shown by Ito 216 et al.

(Scheme 5.3), however, their published results of a 21 % yield in reducing N-Cbz protected

nitro arginol was not promising. Nevertheless, the DIBAL reduction of intermediate 5.15 was

attempted and gave a complex reaction mixture. The crude 1 H NMR spectra showed trace

amounts of reduction products (< 5 %). Alternatively, reducing the ester moiety to the

primary alcohol and oxidizing it back to the aldehyde at a later step was considered (Scheme

5.2).

Scheme 5.3 Ito’s reduction of Cbz protected arginine 5.22.

The synthesis carried on with intermediate 5.18 being reduced with LiBH4, 217

followed by deprotection with HCl, and guanylation 218 with 5.19 to give intermediate 5.20

(Scheme 5.2). Amino alcohol oxidation of 5.20 with IBX 219 in dimethyl sulfoxide under

acidic conditions was used to prepare the corresponding aldehyde. However, only trace

amounts of benzyl-protected lichostatinal (5.21) were observed (< 5 %), and modifying the

reaction conditions had no effect. To address this apparent inertness of our amino alcohol

(5.20) towards oxidative conditions, the literature was examined for precedent in amino

alcohol oxidation. In the original synthesis of leupeptin (5.1) by Shimizu 220 et al. it was

shown that arginol oxidation to the corresponding aldehyde resulted in cyclization to give a

mixture of a hemiaminal in addition to the expected aldehyde. Arginol oxidation was

investigated in a model study by oxidizing 5.24 using a variety of conditions (Scheme

5.4). 221,222,223,224,225

Scheme 5.4 Oxidation of Arginol (5.24).

Table 5.1 Results of arginol (5.24) oxidation model study.

Reagents Solvent Temperature Yield a

IBX dimethyl sulfoxide rt < 5 %

IBX acetone reflux no rxn

DMP, pyridine methylene chloride rt ~ 10 %

SO3 pyr, dmso, i-PrNEt methylene chloride rt ~ 20 %

oxalyl chloride, dmso, NEt3 methylene chloride –78 � C ~ 40 %

TEMPO, NaBr,

toluene, ethyl acetate

rt no reaction

NaOCl, NaHCO3

water

TEMPO, TBACl,

methylene chloride rt no reaction

NaHCO3, K2CO3, NCS

PCC methylene chloride rt no reaction

a Based on the crude 1 H NMR spectra of 5.25/5.26 mixture.

The results of the model study are summarized in Table 5.1. Oxidation of 5.24 with

IBX is low yielding. Oxidations using TEMPO and PCC as oxidants did not yield any

product. Dess–Martin and Parikh–Doering oxidation conditions were more successful. Swern

oxidation of 5.24 gave the highest yields, so it was selected as the favored oxidation method.

The synthesis continued by deprotecting 5.18 followed by guanylation 226 with di-Boc

protected reagent 5.28 to give 5.29 (Scheme 5.5). Guanylation using di-Cbz protected 5.27

was attempted in order to shorten future deprotection steps. The nitro functional group on the

arginine moiety and the Cbz group on the guanidine can be removed by hydrogenation

simultaneously. Unfortunately, even under reflux the guanylation of 5.18 with 5.27 was

unsuccessful (Scheme 5.5).

Scheme 5.5 Synthesis towards lichostatinal (5.4).

The synthesis continued with intermediate 5.29 being reduced with LiBH4 to give

5.30, followed by Swern oxidation to afford the expected aldehyde/hemiaminal mixture.

Attempted deprotection of the O-benzyl serine residue on this intermediate with 10 %

palladium/charcoal under H2 (1 atm) was unsuccessful. This was a surprising outcome, and a

model study was undertaken in which intermediate 5.15 was exposed to different O-benzyl

deprotection conditions (Table 5.2). 227,228,229,230

Table 5.2 Results of an O-benzyl deprotection model study of intermediate 5.15.

Reagent Solvent Temperature/H2 Product Observed

10 % Pd/C ethanol rt/1 atm none

10 % Pd/C ethanol reflux/1 atm none

10 % Pd/C/TFA ethanol rt/1 atm none

10 % Pd/C ethanol rt/15 atm none

20 % Pd(OH)2 ethanol rt/1 atm none

20 % Pd(OH)2 ethanol reflux/1 atm none

NH4HCO2/Zn methanol rt none

NH4HCO2, 10 % Pd/C methanol/tetrahydrofuran rt none

Et3SiH, 10 % Pd/C methanol rt none

Unfortunately, none of the conditions was successful in benzyl deprotection and this

synthetic route was abandoned. However, a similar route in which the serine residue could be

left unprotected was devised (Scheme 5.6). This synthesis does not have the problems

associated with the benzyl deprotection, and may provide a regioisomer of lichostatinal (5.4)

to broaden the SAR. The synthesis began with amine 5.13 being coupled to N-Fmoc-Serine

5.31 with HATU to give intermediate 5.32 in high yield. Intermediate 5.32 was deprotected

with diethylamine to yield 5.33, and subsequently coupled with CDI and mono Fmoc 231

protected putrescine 5.34 to provide 5.35 in a modest yield (Scheme 5.6).

Scheme 5.6 Alternative route to lichostatinal (5.4).

Intermediate 5.35 was guanylated with 5.28 to give 5.36, which was reduced with

LiBH4 to give 5.37. Intermediate 5.37 was oxidized by a Swern oxidation. The crude material

was carried through to the next step, which was deprotection of the nitro group with 10 %

palladium/charcoal and hydrogen (1 atm). The Boc protecting groups were then removed

with a 1:1 mixture of trifluoroacetic acid:methylene chloride to give a complex mixture

(Scheme 5.6). A 1 H NMR resonance at approximately 5.5 ppm suggested that the aldehyde

had cyclized to produce the hemiaminal as shown in Shimizu’s synthesis of leupeptin 220

(5.1). However, absolute confirmation of the presence of lichostatinal (5.4) was not verified.

Nevertheless, this complex mixture was given to Vincent Paul Lavallée in Dr. Bromme's lab

for biological testing in a cathepsin K enzymatic assay. Unfortunately, the crude mixture

showed activity in the µM range in contrast to the nM range found for the natural product

(5.4). This suggested that we were unsuccessful in constructing lichostatinal (5.4). The

synthesis of lichostatinal (5.4) is an on-going project in the Andersen lab.

5.3 Conclusion

Cathepsin K is a cysteine protease that has been implicated in bone resorption, 196

which has led to the development of small molecule inhibitors of cathepsin K. In a continued

effort to identify natural product inhibitors of cathepsin K, the Davies’ natural product extract

library was screened using a cathepsin K enzymatic inhibition assay in the laboratory of Dr.

Bromme. This led to the isolation and identification of lichostatinal (5.4), which is a novel

peptide-aldehyde inhibitor of cathepsin K.

Lichostatinal (5.4) is the first natural product known to be isolated by co-

crystallization with its biological target from a complex mixture. The crystal of the enzyme-

ligand complex was analyzed by a beamline, and the structure of lichostatinal (5.4) was

elucidated. This chapter describes the synthetic effort towards constructing lichostatinal (5.4)

in order to verify its structure by NMR.

Construction of the peptide utilized solution phase peptide chemistry to assemble the

necessary amino acids to obtain intermediate 5.16 (Scheme 5.1). Initial attempts to couple

5.16 to agmatine sulfate (5.6) were unsuccessful. However, by modification of the synthesis

by using amine building block 5.17, it was possible to construct intermediate 5.18. Oxidation

of alcohol 5.20 with IBX gave low yields of the desired aldehyde. A corresponding model

study was undertaken using compound 5.24 in order to examine the yields for oxidation

based on known protocol.

Compound 5.24 was exposed to various oxidative conditions (Table 5.1). A number

of known amino alcohol oxidation methods provided no product or gave only poor yields.

However, Swern oxidation was found to give the highest and most reproducible yield.

Having chosen an appropriate oxidative method, the synthesis moved forward (Scheme 5.5).

Attempts to deprotect the benzyl group on the serine residue with known conditions were

fruitless and a model study to examine benzyl deprotection was completed using intermediate

5.15 as a substrate (Scheme 5.2). None of the attempted literature protocols were successful

in removing the benzyl functionality from 5.15.

A benzyl protecting group free synthetic route to construct lichostatinal (5.4) and its

regioisomer was pursued (Scheme 5.6). From a medicinal chemistry perspective, this is

advantageous since it would broaden the SAR and potentially yield an analogue with

enhanced or comparable potency. Unfortunately, biological testing of a complex reaction

mixture thought to contain lichostatinal (5.4) and its regioisomer was less active than the

natural product (5.4) suggesting that the synthesis had failed to construct the natural product.

Completion of the synthesis of the novel peptide-aldehyde lichostatinal (5.4) is currently

ongoing in the Andersen lab.

5.4 Experimental

Preparation of 5.12:

To Fmoc-L-valine 5.11 (1.25 g, 3.70 mmol) dissolved in 5 mL of N,N-

dimethylformamide was added HATU (1.48 g, 3.89 mmol) and N,N-diisopropylethylamine

(1.29 mL, 7.41 mmol) at 0 � C. To this mixture was added ester 5.10 (1.0 g, 3.70 mmol)

dissolved in 5 mL of N,N-dimethylformamide, and allowed to warm to room temperature

overnight. To the crude mixture was added saturated NaHCO3 (150 mL) and the aqueous

layer extracted three times with methylene chloride (300 mL). The organic extracts were

combined and washed three times with brine, then concentrated using a rotary evaporator,

filtered through a pad of silica (methylene chloride:methanol 4:1), and concentrated again

using a rotary evaporator. Upon the addition of methanol a precipitate formed. This was

triturated with additional methanol (500 mL) and the solid was determined to be 5.12 (1.97 g,

3.55 mmol, 95.9 %). Alternatively 5.12 may by purified using flash column chromatography

(methylene chloride:methanol 95:5). 1 H NMR (400 MHz, (CD3)2SO) δ 8.49 (bs, 1H), 8.34

(d, J = 7.2 Hz, 1H), 7.87 (d, J = 7.2 Hz, 2H), 7.72 (t, J = 6.8 Hz, 2H), 7.39 (t, J = 7.6, Hz,

2H), 7.30 (t, J = 7.2 Hz, 2H), 4.31-4.19 (m, 4H), 3.88 (t, J = 7.6 Hz, 1H), 3.59 (s, 3H), 3.14

(m, 2H), 1.96 (quin, J = 6.8 Hz, 1H), 1.73 (m, 1H), 1.62 (m, 1H), 1.5 (m, 2H), 0.89 (d, J = 6.8

Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); 13 C NMR (100 MHz, (CD3)2SO) δ 172.8, 172.2, 162.9,

156.7, 144.5, 144.3, 141.3, 128.3, 127.7, 125.9, 120.7, 66.3, 60.5, 52.4, 47.3, 40.7, 31.0, 28.3,

19.6, 18.9. HRESIMS [M + Na] + calcd for C27H34N6O7Na 577.2387, found 577.2373.

Preparation of 5.13:

To 5.12 (7.0 g, 12.6 mmol) dissolved in 400 mL of acetonitrile was added

diethylamine (26.1 mL, 252.4 mmol) dropwise, and the mixture allowed to stir at room

temperature for ninety minutes, after which the reaction mixture was filtered through a pad of

silica and the acetonitrile eluent discarded, the pad of silica was then washed with 200 mL of

methylene chloride:methanol (4:1), then concentrated using a rotary evaporator to give 5.13

(4.05 g, 12.2 mmol, 96.7 %). 1 H NMR (400 MHz, D2O) δ 4.36 (dd, J = 8.0 Hz, 1H), 3.8 (d, J

= 6.0 Hz, 1H), 3.63 (s, 3H), 3.17 (m, 2H), 2.15 (quin, J = 6.4 Hz, 1H), 1.80 (m, 1H), 1.71 (m,

1H), 1.58 (m, 2H), 0.94 (d, J = 6.8 Hz, 6H); 13 C NMR (100 MHz, D2O) δ 173.5, 169.5,

158.7, 58.4, 53.1, 52.8, 40.6, 30.2, 27.6, 24.0, 17.6, 16.9. HRESIMS [M + H] + calcd for

C12H25N6O5 333.1886, found 333.1884.

Preparation of 5.15:

To O-benzyl-Boc-L-serine 5.14 (888.6 mg, 3.0 mmol) and N,N-diisopropylethylamine

(1.04 mL, 6.0 mmol) dissolved in 30 mL of N,N-dimethylformamide and cooled to 0 � C was

added HATU (1.2 g, 3.15 mmol) followed by amine 5.13 (1.0 g, 3.0 mmol) and the reaction

allowed to warm to room temperature overnight. The reaction mixture was diluted with

methylene chloride (250 mL) and washed with saturated NaHCO3 (150 mL) followed by

brine (200 mL) and dried with MgSO4 and concentrated using a rotary evaporator. The crude

mixture was purified using flash column chromatography (methylene chloride:methanol

96:4) to give 5.15 (1.54 g, 2.53 mmol, 84.3 %). 1 H NMR (600 MHz, (CD3)2SO) δ 8.47 (bs,

1H), 8.39 (d, J = 7.2 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.28 (m, 5H), 7.24 (m, 1H), 7.09 (d, J

= 9.0 Hz, 1H), 4.4 (s, 2H), 4.23 (t, J = 6.6 Hz, 2H), 4.17 (q, J = 7.8 Hz, 1H), 3.58 (s, 3H),

3.54 (m, 1H), 3.31 (s, 2H), 3.09 (m, 2H), 1.92 (quin, J = 7.2 Hz, 1H), 1.69 (m, 1H), 1.58 (m,

1H), 1.47 (m, 2H), 1.36 (s, 9H), 0.84 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 6.6 Hz, 3H); 13 C NMR

(150 MHz, CD2Cl2) δ 171.6, 171.4, 170.5, 158.9, 155.8, 137.2, 127.9, 127.3, 127.2, 79.9,

72.9, 69.3, 58.2, 54.5, 51.8, 50.9, 40.1, 30.4, 28.6, 27.6, 24.0, 18.5, 17.3. HRESIMS [M +

Na] + calcd for C27H43N7O9Na 632.3020, found 632.3016.

Figure 5.7 1 H and 13 C NMR spectra of 5.15 recorded in (CD3)2SO at 600 MHz and CD2Cl2

at 150 MHz respectively.

Preparation of 5.16:

To 5.15 (232.5 mg. 0.38 mmol) dissolved in 4 mL of methanol was added 12.4 M

HCl (0.5 mL, 6.2 mmol) and the mixture allowed to stir at room temperature overnight. The

reaction was then diluted with water (100 mL) and extracted three times with methylene

chloride (200 mL), and the organic layers were discarded. To the acidic aqueous extract was

added enough NaHCO3 to basify and was extracted three times with methylene chloride (250

mL), dried with MgSO4 and concentrated using a rotary evaporator to give 5.16 (193.4, 0.38

mmol, 100 %, yield includes small impurity). 1 H NMR (600 MHz, (CD3)2SO) δ 8.48 (bs,

1H), 8.44 (d, J = 6.6 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.29 (m, 5H), 7.25 (m, 1H), 4.45 (s,

2H), 4.23 (t, J = 7.2 Hz, 1H), 4.19 (q, J = 6.0 Hz, 1H), 3.58 (s, 3H), 3.52 (m, 2H), 3.41 (m,

1H), 3.1 (m, 2H), 2.86 (s, 1H), 2.70 (s, 1H), 2.2 (bs, 1H), 1.92 (quin, J = 6.6 Hz, 1H), 1.69

(m, 1H), 1.58 (m, 1H), 1.48 (m, 2H), 0.84 (d, J = 6.6 Hz, 3H), 0.78 (d, J = 6.6 Hz, 3H); 13 C

NMR (150 MHz, (CD3)2SO) δ 172.2, 171.0, 171.0, 162.3, 138.3, 128.2, 127.4, 127.3, 72.5,

72.1, 56.5, 54.6, 51.8, 51.7, 40.1, 31.4, 27.8, 24.7, 19.0, 17.7. HRESIMS [M + H] + calcd for

C22H36N7O7 510.2676, found 510.2675.

Preparation of 5.18:

Mono Boc protected putrescine freebase 5.17 (340.9 mg, 1.81 mmol) and CDI (293.4

mg, 1.81 mmol) dissolved in 8 mL of N,N-dimethylformamide were allowed to stir at room

temperature for ten hours, after which amine 5.16 (307.4 mg, 0.60 mmol) dissolved in 3.0 L

of N,N-dimethylformamide was added and the mixture allowed to stir overnight at room

temperature. TLC indicated the presence of starting material so the mixture was heated at 80

� C overnight, after which the reaction was allowed to cool and the mixture evaporated using

a lyophilizer. The crude mixture was purified using flash column chromatography

(methylene chloride:methanol 96:4) to give 5.18 (416.2 mg, 0.57 mmol, 95.8 %). 1 H NMR

(600 MHz, (CD3)2SO) δ 8.49 (bs, 1H), 8.38 (d, J = 7.2 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.29

(m, 5H), 6.77 (t, J = 4.8 Hz, 1H), 6.24 (t, J = 4.8 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H), 4.45 (m,

2H), 4.39 (m, 1H), 4.25 (t, J = 9.0 Hz, 1H), 4.19 (q, J = 7.8 Hz, 1H), 3.65 (dd, J = 9.6, 4.8 Hz,

1H), 3.59 (s, 3H), 3.51 (dd, J = 9.6, 4.8 Hz, 1H), 3.33 (s, 3H), 3.11 (m, 2H), 2.96 (m, 2H),

2.89 (m, 2H), 1.96 (quin, J = 6.6 Hz, 1H), 1.70 (m, 1H), 1.59 (m, 1H), 1.49 (m, 2H), 1.36 (s,

9H), 1.32 (bs, 3H), 0.87 (dd, J = 6.6 Hz, 3H), 0.82 (dd, J = 6.6 Hz, 3H); 13 C NMR (150 MHz,

(CD3)2SO) δ 172.9, 171.8, 171.3, 160.1, 158.3, 156.4, 139.0, 128.9, 128.2, 128.1, 78.1, 72.9,

71.6, 57.8, 53.9, 52.6, 52.6, 40.9, 39.8, 31.9, 29.1, 28.6, 28.2, 28.2, 27.8, 25.4, 19.8, 18.6.

HRESIMS [M + Na] + calcd for C32H53N9O10Na 746.3812, found 746.3822.

Preparation of 5.20:

To 5.18 (102.6 mg, 0.14 mmol) reduced in a similar fashion to 5.36, was filtered

through a plug of silica (methylene chloride:methanol 9:1) and used in the subsequent

reaction without further purification, by dissolving it in 5 mL of methanol and addition of a

12.4 M solution of HCl (0.35 mL, 4.3 mmol) and allowing the mixture to stir at room

temperature overnight. The mixture was concentrated under a stream of nitrogen and

evaporated using a lyophilizer overnight. To this crude mixture dissolved in 1.5 mL of N,N-

dimethylformamide was added triethylamine (0.041 mL, 0.30 mmol) and guanylating reagent

5.19 (22.0 mg, 0.15 mmol) and heated to at 60 � C overnight, acidified with 1 M HCl, then

concentrated using a lyophilizer. The crude mixture was purified using a 5 g C18 sep pak

(water:methanol, 100 %, 9:1, 3:1, 3:2, 100% methanol) to give 5.20 (80.9 mg, 0.12 mmol,

84.6 %). 1 H NMR (600 MHz, (CD3)2SO) δ 8.45 (bs, 1H), 7.79 (m, 1H), 7.74 (d, J = 9.0 Hz,

1H), 7.42 (bs, 1H), 7.27 (m, 5H), 6.98 (bs, 1H), 5.22 (bs, 5H), 4.42 (m, 2H), 4.34 (t, J = 4.2

Hz, 1H), 4.10 (t, J = 7.8 Hz, 1H), 3.62 (m, 2H), 3.49 (dd, J = 9.0, 3.6, Hz, 1H), 3.29 (dd, J =

10.8, 4.8 Hz, 1H), 3.18 (dd, J = 9.6, 6.0 Hz, 1H), 3.06 (m, 4H), 2.96 (m, 2H), 1.92 (quin, J =

6.6 Hz, 1H), 1.52 (m, 2H), 1.41 (m, 3H), 1.36 (m, 4H), 1.21 (bs, 1H), 0.80 (dd, J = 6.6 Hz,

3H), 0.77 (dd, J = 6.6 Hz, 3H); 13 C NMR (150 MHz, (CD3)2SO) δ 170.6, 170.5, 138.1, 128.1,

127.5, 127.4, 72.2, 70.6, 63.1, 57.8, 53.3, 50.3, 40.6, 40.4, 38.6, 30.7, 27.8, 27.8, 27.1, 27.1,

25.9, 25.9, 24.7, 19.1, 18.0. HRESIMS [M + H] + calcd for C27H48N11O7 638.3738, found

638.3736.

Preparation of 5.24:

To 5.15 (356.5 mg, 0.58 mmol) dissolved in 5 mL of tetrahydrofuran and 0.22 mL of

methanol was added LiBH4 (44.5 mg, 2.0 mmol) dissolved in 10 mL of tetrahydrofuran and

the reaction allowed to stir at room temperature for two hours after which the reaction was

diluted with water (100 mL) and extracted three times with methylene chloride (250 mL),

purified using flash column chromatography (methylene chloride:methanol 10:0.5) to give

5.24 (313.6 mg, 0.54 mmol, 92.5 %). 1 H NMR (600 MHz, (CD3)2SO) δ 8.47 (bs, 1H), 7.71

(m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.29 (m, 5H), 7.24 (m, 1H), 7.10 (d, J = 7.2 Hz, 1H), 4.64,

(m, 1H), 4.44 (s, 2H), 4.23 (m, 1H), 4.13 (t, J = 6.6 Hz, 1H), 3.67 (m, 1H), 3.56 (m, 2H), 3.32

(m, 2H), 3.19 (t, J = 4.8 Hz, 1H), 3.11 (m, 1H), 3.06 (t, J = 5.4 Hz, 1H), 1.90 (quin, J = 6.0

Hz, 1H), 1.54 (m, 1H), 1.44 (m, 1H), 1.36 (s, 9H), 1.30 (bs, 1H), 1.24 (m, 1H), 0.81 (d, J =

6.0 Hz, 3H), 0.79 (d, J = 6.0 Hz, 3H); 13 C NMR (150 MHz, (CD3)2SO) δ 170.4, 169.6, 159.3,

155.3, 138.2, 128.1, 127.5, 127.4, 78.4, 71.9, 69.8, 63.2, 57.5, 54.5, 50.4, 40.6, 30.9, 28.1,

27.9, 24.8, 19.1, 17.9. HRESIMS [M + Na] + calcd for C26H43N7O8Na 604.3071, found

604.3063.

Preparation of 5.29:

To 5.18 (45.7 mg, 0.064mmol) deprotected in a similar fashion to 5.15 was dissolved

in 2 mL of N,N-dimethylformamide was added triethylamine (0.01 mL, 0.074 mmol)

followed by Boc protected guanylating reagent 5.28 (20.9 mg, 0.067 mmol). The reaction

mixture was allowed to stir at room temperature for 48 hours, after which the solvent was

evaporated on a lyophilizer and the crude mixture purified using flash column

chromatography (methylene chloride:methanol 5:1) to give 5.29 (37.3 mg, 0.048 mmol, 76

%). 1 H NMR (600 MHz, CD2Cl2) δ 11.42 (s, 1H), 8.45 (s, 1H), 7.29 (m, 5H), 7.14 (s, 2H),

6.37 (s, 1H), 6.00 (s, 1H), 4.5 (m, 3H), 4.43 (s, 1H), 4.27 (t, J = 7.8 Hz, 1H), 3.91 (s, 1H),

3.71 (s, 3H), 3.69 (dd, J = 9.6, 4.2 Hz, 1H), 3.35 (m, 3H), 3.22 (m, 3H), 2.07 (s, 1H), 1.90

(m, 1H), 1.68 (s, 2H), 1.61 (m, 3H), 1.51 (m, 7H), 1.51 (s, 9H), 1.46 (s, 9H), 0.93 (d, J = 6.6

Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H); 13 C NMR (100 MHz, CD2Cl2) δ 172.2, 172.0, 172.0,

163.4, 162.5, 159.7, 158.8, 156.3, 153.2, 149.5, 137.9, 128.4, 127.8, 83.2, 79.2, 73.4, 70.4,

59.0, 55.0, 52.3, 51.4, 40.6, 40.2, 36.3, 31.2, 30.4, 29.1, 28.1, 27.9, 27.8, 26.8, 19.1, 18.0.

HRESIMS [M + H] + calcd for C38H63N11O12 866.4736, found 866.4734.

Preparation of 5.30:

To 5.29 (42.8 mg, 0.049 mmol) dissolved in 1 mL of tetrahydrofuran was added 18

µL methanol and the mixture cooled to 0 � C. To this was added LiBH4 (3.7 mg, 0.017 mmol)

dissolved in 3 mL of tetrahydrofuran, and the reaction mixture allowed to stir for two hours.

The reaction mixture was quenched with the addition of water (20 mL), and concentrated

using a lyophilizer. The crude material was purified using flash column chromatography

(methylene chloride:methanol 20:1) to give 5.30 (37.3 mg, 0.044 mmol, 89.7 %). 1 H NMR

(600 MHz, (CD3)2CO) δ 11.6 (s, 1H), 8.32 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.32 (m, 5H),

7.25 (t, J = 6.6 Hz, 1H), 6.30 (t, J = 4.8 Hz, 2H), 4.52 (m, 2H), 4.40 (m, 1H), 4.21 (t, J = 6.6

Hz, 1H), 4.01 (s, 1H), 3.97 (s, 1H), 3.88 (dd, J = 9.0, 4.2 Hz, 1H), 3.70 (dd, J = 9.6, 3.6 Hz,

1H), 3.53 (s, 2H), 3.39 (quin, J = 6.0 Hz, 2H), 3.22 (m, 3H), 2.85 (m, 2H), 2.14 (s, 1H), 1.61

(m, 6H). 1.52 (m, 4H), 1.49 (s, 9H), 1.42 (s, 9H). 0.92 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6

Hz, 3H); 13 C NMR (150 MHz, (CD3)2CO) δ 171.2, 170.9, 163.2, 159.5, 158.1, 155.6, 152.4,

137.7, 127.7, 127.1, 127.0, 82.3, 77.7, 72.3, 69.7, 63.9, 58.5, 54.5, 50.0, 40.3, 39.8, 39.1,

28.9, 28.8, 28.6, 28.5, 28.4, 28.2, 27.9, 27.1, 26.8, 26.7, 26.0, 18.5, 17.0. HRESIMS [M +

H] + calcd for C37H64N11O11 838.4787, found 838.4791.

Preparation of 5.32:

To Fmoc-L-serine 5.31 (318.6 mg, 0.97 mmol) dissolved in 2 mL of N,N-

dimethylformamide cooled to 0 � C was added HATU (388 mg, 1.02 mmol) and N,N-

diisopropylethylamine (0.34 mL, 1.95 mmol) followed by 5.13 (323.4 mg, 0.97 mmol)

dissolved in 2 mL of N,N-dimethylformamide and the mixture was allowed to warm to room

temperature overnight. To the reaction was added saturated NaHCO3 (100 mL) and the

aqueous layer was washed three times with methylene chloride (250 mL). The organic

extracts were combined and washed three times with brine and dried with MgSO4 and

chromatography (methylene chloride:methanol 95:5) to give 5.32 (591.3 mg, 0.92 mmol,

94.8 %). 1 H NMR (600 MHz, (CD3)2SO) δ 8.49 (bs, 1H), 8.35 (d, J = 6.6 Hz, 1H), 7.87 (d, J

= 7.2 Hz, 2H), 7.72 (t, J = 7.8 Hz, 3H), 7.41 (d, J = 4.2 Hz, 1H), 7.39 (d, J = 7.8 Hz, 2H),

7.30 (t, J = 7.2 Hz, 2H), 4.91 (t, J = 6.0 Hz, 1H), 4.27-4.13 (m, 7H), 3.58 (s, 3H), 3.56-3.51

(m, 2H), 3.12 (m, 2H), 2.0 (s, 1H), 1.97 (quin, J = 6.6 Hz, 1H), 1.70 (m, 1H), 1.60 (m, 1H),

1.50 (m, 2H), 0.86 (d, J = 7.2 Hz, 3H), 0.81 (d, J = 7.2 Hz, 3H); 13 C NMR (150 MHz,

(CD3)2SO) δ 172.1, 171.1, 170.0, 159.3, 155.9, 143.8, 143.7, 140.7, 127.6, 127.1, 125.3,

125.2, 120.1, 65.7, 61.9, 57.1, 57.0, 54.9, 51.8, 51.7, 46.6, 40.1, 30.8, 27.8, 24.6, 19.0, 17.7.

HRESIMS [M + H] + calcd for C30H40N7O9 642.2888, found 642.2829.

Preparation of 5.33:

5.32 (591.3 mg, 0.92 mmol) was deprotected in a similar fashion to 5.12. The crude

freebase was purified using a 5 g C18 Sep-Pak (water:methanol 9:1) to give 5.33 (539.0 mg,

0.84 mmol, 91.3 %). 1 H NMR (400 MHz, D2O) δ 4.37 (dd, J = 8.4, 4.4 Hz, 1H), 4.05 (m,

1H), 3.77-3.69 (m, 1H), 3.66 (m, 1H), 3.64 (s, 3H), 3.60 (q, J = 4.8 Hz, 1H), 3.19 (t, J = 6.4

Hz, 2H), 1.99 (sep, J = 6.8 Hz, 1H), 1.83 (m, 1H), 1.70 (m, 1H), 1.58 (m, 2H), 0.86 (t, J = 6.8

Hz, 6H); 13 C NMR (100 MHz, (CD3)2SO) δ 172.7, 172.2, 171.1, 159.4, 64.1, 59.7, 56.4, 51.8,

51.7, 40.0, 31.3, 30.6, 27.8, 19.0, 17.7. HRESIMS [M + H] + calcd for C15H30N7O7 420.2207,

found 420.2212.

Figure 5.15 1 H and 13 C NMR spectra of 5.33 recorded in D2O and (CD3)2SO at 400 MHz

and 100 MHz respectively.

Preparation of 5.35:

To the Fmoc protected putrescine HBr salt 5.34 (58.1 mg, 0.15 mmol) and CDI (28.1

mg, 0.18 mmol) dissolved in 1.25 mL of N,N-dimethylformamide was allowed to stir for

three hours at room temperature, after which amine 5.33 (58.1 mg, 0.15 mmol) dissolved in

0.6 mL of N,N-dimethylformamide was added and the mixture was allowed to stir at room

temperature overnight. The crude mixture was evaporated by a lyophilizer and purified using

flash column chromatography (methylene chloride:methanol 10:1) to give 5.35 (58.9 mg,

0.078 mmol, 52.4 %). 1 H NMR (400 MHz, CD3OD) δ 7.80 (d, J = 7.6 Hz, 2H), 7.65 (d, J =

7.6 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 4.50 (m, 1H), 4.33 (t, J = 6.8

Hz, 3H), 4.20 (t, J = 6.0 Hz, 2H), 3.83 (dd, J = 11.2, 5.2 Hz, 1H), 3.70-3.66 (m, 2H), 3.69 (s,

3H), 3.22 (t, J = 7.2 Hz, 2H), 3.12 (m, 4H), 2.11 (sep, J = 6.8 Hz, 1H), 1.9 (m, 1H), 1.65 (m,

3H), 1.5 (m, 3H), 0.98 (t, J = 6.8 Hz, 6H); 13 C NMR (150 MHz, (CD3)2SO) δ 172.1, 171.1,

171.0, 159.3, 157.6, 156.1, 143.9, 140.7, 127.6, 127.1, 125.1, 120.1, 65.2, 62.4, 59.7, 56.9,

54.9, 51.8, 46.7, 40.0, 39.9, 38.9, 30.7, 27.8, 27.3, 26.9, 19.1, 17.6, 14.1. HRESIMS [M +

Na] + calcd for C35H49N9O10Na 778.3500, found 778.3502.

Preparation of 5.36:

Compound 5.35 (67.8 mg, 0.11 mmol) was deprotected in a similar fashion to 5.12

and purified in a similar manner. It was used in the following guanylation by dissolving it in

1 mL of N,N-dimethylformamide and adding guanylating agent 5.28 (40 mg, 0.13 mmol) and

triethylamine (0.1 mL, 0.71 mmol) and the reaction allowed to stir at room temperature for

48 hours. The crude mixture was then concentrated using a lyophilizer and purified using

flash column chromatography (methylene chloride:methanol 97:3) to give 5.36 (85.6 mg,

0.11 mmol, 100 %). 1 H NMR (600 MHz, CD3OD) δ 4.47 (s, 1H), 4.32 (t, J = 4.8 Hz, 1H),

4.18 (s, 1H), 3.81 (dd, J = 10.8, 4.8 Hz, 1H), 3.70 (s, 3H), 3.68 (m, 1H), 3.58 (t, J = 6.6 Hz,

2H), 3.22 (t, J = 6.6 Hz, 2H), 3.16 (t, J = 6.6 Hz, 2H), 2.10 (quin, J = 6.6 Hz, 1H), 1.93 (s,

1H), 1.67 (m, 3H), 1.61 (m, 2H), 1.53 (m, 2H), 1.51 (s, 9H), 1.46 (s, 9H), 0.99 (d, J = 6.6 Hz,

3H), 0.97 (d, J = 6.6 Hz, 3H); 13 C NMR (150 MHz, CD3OD) δ 172.1, 171.9, 171.6, 162.7,

159.1, 158.6, 155.7, 152.3, 82.5, 78.5, 61.9, 58.5, 55.0, 50.9, 39.7, 39.7, 38.8, 29.8, 27.6,

26.7, 26.6, 26.4, 25.8, 25.8, 23.8, 17.8, 16.8. HRESIMS [M + H] + calcd for C31H58N11O12

776.4263, found 776.4266.

Preparation of 5.37:

To 5.36 (13.7 mg, 0.017 mmol) dissolved in 0.5 mL of tetrahydrofuran was added

methanol 5.8 µl and the mixture cooled to 0 � C. To the cooled solution was added LiBH4 (1.3

mg, 0.061 mmol) dissolved in 1 mL of tetrahydrofuran, and allowed to stir for 1 hour, after

which 0.1 mL of water was added and mixture concentrated on a lyophilizer. The crude was

purified using flash column chromatography (methylene chloride:methanol 9:1) to give 5.37

(10.4 mg, 0.013 mmol, 81.8 %). 1 H NMR (600 MHz, CD3OD) δ 4.31 (t, J = 5.4 Hz, 1H), 4.10

(s, 1H), 3.92 (s, 1H), 3.82 (dd, J = 10.8, 4.8 Hz, 1H), 3.67 (dd, J = 10.8, 5.4 Hz, 1H), 3.59

(dd, J = 13.2, 5.4 Hz, 1H), 3.48 (dd, J = 9.0, 5.4 Hz, 2H), 3.35 (t, J = 7.2 Hz, 3H), 3.31 (m,

1H), 3.16 (t, J = 6.6 Hz, 3H), 2.12 (quin, J = 6.6 Hz, 1H), 1.66 (s, 2H), 1.59 (m, 3H), 1.53 (m,

1H), 1.51 (s, 9H), 1.46 (s, 9H), 0.97 (t, J = 7.2 Hz, 6H); 13 C NMR (150 MHz, CD3OD) δ

172.3, 171.9, 162.7, 159.1, 158.6, 155.7, 152.3, 82.6, 78.5, 63.2, 61.9, 60.6, 59.1, 55.1, 52.5,

40.1, 39.7, 38.8, 29.4, 27.3, 26.6, 26.3, 25.7, 23.9, 17.9, 16.8. HRESIMS [M + H] + calcd for

C30H58N11O11 748.4325, found 748.4317.

Preparation of 5.38:

To 5.18 (100 mg, 0.14 mmol) dissolved in 2 mL of tetrahydrofuran and 0.06 mL

methanol was added LiBH4 (14 mg, 0.64 mmol) dissolved in 3 mL of tetrahydrofuran and the

reaction allowed to stir at room temperature for thirty minutes, after which the reaction was

diluted with water (50 mL) and extracted three times with methylene chloride (150 mL),

dried with MgSO4 and concentrated using a rotary evaporator. The crude was purified using

flash column chromatography (methylene chloride:methanol 9:1) to give 5.38 (82.8 mg, 0.12

mmol, 85.5 %). 1 H NMR (600 MHz, (CD3)2SO) δ 8.45 (bs, 1H), 7.67 (m, 2H), 7.28 (m, 5H),

6.75 (m, 1H), 6.24 (m, 1H), 6.19 (m, 1H), 4.63 (m, 1H), 4.43 (m, 2H), 4.36 (dd, J = 7.8, 4.8

Hz, 1H), 4.12 (m, 1H), 4.08 (t, J = 4.8 Hz, 1H), 3.67 (bs, 1H), 3.61 (dd, J = 8.4, 3.6 Hz, 1H),

3.49 (dd, J = 9.6, 4.8 Hz, 1H), 3.29 (m, 3H), 3.18 (m, 1H), 3.12 (t, J = 4.8 Hz, 2H), 3.04 (m,

1H), 2.94 (m, 2H), 2.86 (m, 2H), 1.91 (m, 1H), 1.52 (m, 1H), 1.42 (m, 2H), 1.34 (s, 9H), 1.31

(m, 2H), 1.21 (m, 1H), 0.81 (d, J = 6.0 Hz, 3H), 0.80 (d, J = 6.0 Hz, 3H); 13 C NMR (150

MHz, (CD3)2SO) δ 170.5, 170.5, 159.3, 157.5, 155.6, 138.1, 128.1, 127.4, 127.3, 77.3, 72.1,

70.7, 63.2, 57.7, 53.2, 50.3, 40.5, 38.9, 30.8, 28.3, 27.9, 27.3, 27.3, 27.0, 24.7, 19.1, 17.9.

HRESIMS [M + Na] + calcd for C31H53N9O9Na 718.3864, found 718.3870.

Preparation of 5.39:

To Boc protected putrescine 5.17 (57.0 mg, 0.30 mmol) dissolved in 3 mL of N,N-

dimethylformamide was added CDI (49.1 mg, 0.30 mmol), and the reaction mixture allowed

to stir at room temperature for 8 hours. To this was added peptide 5.33 (84.8 mg, 0.20 mmol)

dissolved 4 mL of N,N-dimethylformamide and the reaction was heated to 80 � C overnight.

The crude was concentrated using a lyophilizer and purified using flash column

chromatography (methylene chloride:methanol 23:2) to give 5.39 (46.6 mg, 0.071 mmol,

23.6 %). 1 H NMR (600 MHz, CD3OD) δ 4.59 (s, 1H), 4.48 (s, 1H), 4.31 (s, 1H), 4.16 (m,

1H), 3.81 (dd, J = 10.2, 4.8 Hz, 1H), 3.70 (s, 3H), 3.67 (m, 1H), 3.22 (t, J = 6.6 Hz, 2H), 3.12

(s, 2H), 3.03 (s, 2H), 2.09 (quin, J = 6.6 Hz, 1H), 1.92 (s, 1H), 1.67 (m, 3H), 1.47 (m, 5H),

1,41 (s, 9H), 0.98 (t, J = 7.8 Hz, 6H); 13 C NMR (150 MHz, CD3OD) δ 172.1, 172.0, 171.6,

159.1, 158.6, 156.7, 77.9, 61.9, 58.6, 55.0, 50.8, 39.6, 39.2, 38.8, 29.8, 29.8, 27.6, 26.9, 26.9,

26.6, 26.4, 23.7, 17.7, 16.8. HRESIMS [M + Na] + calcd for C25H47N9O10Na 656.3344, found

656.3351.

Preparation of 5.40:

To 5.18 (300 mg, 0.41 mmol) dissolved in 10 mL of methanol was added 12.4 M HCl

(1.0 mL, 12.0 mmol) and the mixture allowed to stir at room temperature overnight. The

mixture was concentrated under a stream of nitrogen and the crude evaporated overnight

using a lyophilizer, and used in the subsequent guanylation without further purification. This

primary amine HCl salt was dissolved in 3 mL of N,N-dimethylformamide followed by the

addition of triethylamine (0.12 mL, 0.87 mmol) and guanylating reagent 5.19 (63.7 mg, 0.43

mmol). The mixture was then heated at 80 � C overnight after which it was concentrated using

a lyophilizer, and purified using a 5 g C18 Sep-Pak (water:methanol 100 % water, 9:1, 3:2,

100 % methanol) to give 5.40 (269.6 mg, 0.38 mmol, 92.7 % 2 steps). 1 H NMR (600 MHz,

(CD3)2SO) δ 8.47 (bs, 1H), 8.40 (d, J = 6.0 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.66 (m, 1H),

7.28 (m, 5H), 6.85 (bs, 1H), 6.35 (bs, 1H), 4.85 (bs, 5H), 4.43 (m, 2H), 4.37 (m, 1H), 4.24 (t,

J = 6.6 Hz, 1H), 4.14 (m, 1H), 3.63 (m, 1H), 3.57 (s, 3H), 3.50 (m, 1H), 3.07 (m, 4H), 2.97

(m, 2H), 1.95 (quin, J = 6.0 Hz, 1H), 1.68 (m, 1H), 1.57 (m, 1H), 1.47 (m, 2H), 1.42 (m, 2H),

1.35 (m, 2H), 0.84 (d, J = 6.0 Hz, 3H), 0.80 (d, J = 6.0 Hz, 3H); 13 C NMR (150 MHz,

(CD3)2SO) δ 172.1, 170.9, 170.4, 159.3, 157.6, 156.8, 138.2, 128.1, 127.4, 127.3, 72.2, 70.8,

56.9, 53.2, 51.8, 51.7, 40.4, 40.1, 38.6, 31.1, 27.8, 27.2, 25.9, 24.4, 19.0, 17.9. HRESIMS [M

+ H] + calcd for C28H48N11O8 666.3687, found 666.3695.

Chapter 6: Conclusion

It has been proposed that small molecule activators of SHIP1 49 may be used as a

novel therapy for hematopoietic malignancies as well as inflammatory disorders.

Furthermore, activation of the SHIP1 enzymatic pathway may also provide an alternative to

PI3K inhibition. 47 Chapter 2 describes the continuation of a comprehensive SAR study,

which began with the lead compound pelorol 51 (2.1) (Figure 2.2), a marine natural product.

The goal of the SAR study was to construct water-soluble analogues of compound 2.18

(Figure 2.10) for the purpose of enhancing its drug-like properties. 16 Compound 2.18 is an

analogue of pelorol (2.1) and a SHIP1 activator.

Several objectives need to be completed to provide additional biological insight. Lead

compounds 2.20 and 2.42 should be evaluated for their pharmacokinetic properties 232 in

order to understand their absorption and distribution properties in vivo. This would further

validate the concept of enhanced water solubility in providing enhanced drug-like effects in

analogues 2.20 and 2.42. Additional biological testing in animal models other than

inflammation such as multiple myeloma, which has been implicated with SHIP1, 233 should

be completed. Furthermore, analogues 2.40, 2.44, and 2.45 (Scheme 2.11) and their

enantiomers should be evaluated in a SHIP1 enzymatic assay. 51 This would provide

additional information as to the role of configuration in the activity of these analogues.

Additional STDD NOE NMR 92 experiments of analogues 2.20 and 2.42 (Figure 2.19)

should be completed. The future experiment should be performed so that each analogue is

evaluated in a separate sample, rather than testing each analogue sequentially in the same

NMR tube. This may provide more accurate evidence for binding of these analogues to

SHIP1, since potential intermolecular interactions between 2.20 and 2.42, which may affect

the experiment, would be eliminated. Finally, the benzophenone photoaffinity probe 101 2.53

(Scheme 2.14), found to be active in a SHIP1 enzymatic assay, should be used to identify the

SHIP1 amino acid residue(s) responsible for binding, and for additional proof of interaction

in vivo.

6.2 Additional Biological Evaluations of the Niphatenones and their Analogues

It has been proposed 140 that small molecule antagonists of the NTD of the AR are an

appealing avenue of exploration for treating CRPC, an advanced form of prostate cancer

resistant to current therapies. Chapter 3 describes the total syntheses of the marine natural

products niphatenone A (3.8) and B (3.9) (Figure 3.5). These marine metabolites represent a

novel NTD-AR antagonist pharmacophore. The purpose of the total syntheses was to aid in

structure determination. In consequence, the (S) configuration was assigned to the natural

products. An SAR study was completed, and it was shown that the unnatural (R)

configuration of niphatenone B in 3.31 (Figure 3.11) was the most active compound.

A few areas in the project remain to be completed. Fluorinated analogue 3.54

(Scheme 3.9) should be tested in an in vitro AR transcriptional activity assay. 140 Potential

fluorescent 155 probe 3.78 (Scheme 3.14) should be tested in vitro to determine if it is an

NTD-AR antagonist. If found to be active, it may be determined through fluorescence

imaging if the photochemical properties of the ligand (3.78) change due to covalent binding

to the NTD-AR, for use as a fluorescent imaging agent. 160 Furthermore, in vivo studies using

either niphatenone A (3.8) and/or B (3.9), or a suitable analogue, should be completed to

properly evaluate if these AR antagonists have any effect on prostate tissue growth.

6.3 Future Synthetic Strategies Towards Terpene 4.4: An LBD AR Antagonist

Small molecule AR antagonists are currently used as a therapeutic treatment for

prostate cancer. 130 Chapter 4 describes the semisynthesis of analogues based on the marine

natural product lead compound 4.1, which was discovered to be an AR antagonist (Figure

4.1). The semisynthetic analogue 4.4 has enhanced activity compared to the lead compound

4.1 in both an in vitro AR transcriptional activity assay as well as an in vitro AR affinity

assay (Figure 4.7).

A number of questions remain in order to elucidate the biological effect of this novel

LBD-AR antagonist (4.4). Analogue 4.4 should be tested in vivo to observe its effects on

prostate tumor tissue. Furthermore, prodrugs 68 of 4.4 could be constructed using

semisynthetic material (4.4) in order to decrease the CLogP of analogue 4.4. 16

Scheme 6.1 Syntheses towards polar analogues of 4.4.

One potential prodrug of analogue 4.4 would be a PEG analogue 152 (Scheme 6.1).

Compound 4.4 could be esterified using bromoacetylbromide (6.1) to give intermediate 6.2.

Bromide 6.2 may be reacted with PEG thiol 6.3 to afford the highly water soluble pegylated

analogue 6.4. Alternatively, the amine-derivatized prodrug 6.6 could be constructed.

Esterification of analogue 4.4 with Boc-protected glycine 6.5 followed by a deprotection and

ion exchange should yield prodrug 6.6 as the hydrochloride salt (Scheme 6.1).

The method of accessing A-ring analogues of 4.4, by an epoxide-initiated cationic

cascade 69 should be pursued. One avenue of exploration would be to lithiate the terminal

epoxide intermediate 4.23 (Scheme 6.2). Lithiation of 4.23 may be accomplished by using a

variety of bases such as n-BuLi, sec-BuLi, and tert-BuLi. By quenching the resultant alkyl

lithium with D2O, 234 one should be able to observe deuterium incorporation in the furan ring

system by 1 H NMR and establish ideal deprotonation conditions.

Scheme 6.2 Lithiation strategy to construct A-ring analogues of 4.4.

Once the conditions have been identified, quenching with various electrophiles

should be attempted. Initial focus should be on the aforementioned silyl electrophiles

(Section 4.3). This would allow for rapid construction of several functionalized

intermediates, which could then be cyclized. The resulting reaction mixtures can then be

analyzed for the desired product regioselectivity.

Furthermore, previously synthesized intermediate 4.24 (Scheme 4.5) should be

deprotected to yield analogue 6.10 for biological testing (Scheme 6.3). The regioisomer 6.9

should be readily accessible due to the inherent reactivity of the furan ring system (Section

4.1). Compound 6.9 could be synthesized by a Barton-McCombie deoxygenation 88 followed

by a TBDPS deprotection (Scheme 6.3).

Scheme 6.3 Deprotection of regiosisomer 4.24.

Another interesting compound would be analogue (±)-6.9. A synthetic route for

constructing regiosisomer (±)-6.9 is shown in Scheme 6.4. By using previously synthesized

intermediates (4.19) and a similar route (Scheme 4.5) the construction of (±)-6.9 should be

readily accessible. Polyene 55 6.13 could be cyclized with indium tribromide, 78 then

deprotected to give (±)-6.9. This synthetic route also has the advantage of providing the

antipodal configuration to the lead compound (4.1) which would help broaden the SAR.

Scheme 6.4 Alternative synthesis of regioisomer (±)-6.9.

6.4 Alternative Synthetic Strategy Towards Lichostatinal (5.4)

The biological role of cathepsin K in bone resorption 196 has led to the development of

inhibitors of cathepsin K as potential therapeutics to combat osteoporosis. 201 However, there

are currently no available cathepsin K inhibitors for the treatment of osteoporosis. The lack

of available cathepsin K inhibitors led to a collaboration between the laboratories of Dr.

Julian Davies and Dr. Dieter Bromme at the University of British Columbia. This culminated

in the discovery of lichostatinal (5.4), a peptide-aldehyde natural product isolated from

cultures of a terrestrial actinomycete. Lichostatinal (5.4) is the first natural product to be

isolated by co-crystallization with its biological target from a complex mixture.

Chapter 5 details the synthetic efforts towards lichostatinal (5.4), in order to verify its

structure and to provide additional material for biological testing. A reasonable alternative to

the syntheses presented in Chapter 5 is to utilize the chemistry developed for the construction

of leupeptin 220 (5.1), the first natural product cathepsin K inhibitor. In Shimizu’s synthesis of

leupeptin (5.1), the aldehyde/hemiaminal mixture that results from arginol oxidation was

converted entirely to the semicarbazone with semicarbazide. This simplifies the mixture to

give only a single compound (6.14), which should be easily purified by flash column

chromatography (Figure 6.1). This alternative may prove useful if a benzyl-protected serine

is utilized (Figure 6.1). Since the aldehyde/hemiaminal mixture is protected as the

semicarbazone, it would allow for exploration of alternative benzyl deprotection strategies

without having a labile aldehyde moiety present in the molecule.

Figure 6.1 Alternative route to lichostatinal (5.4).

References

(1) McGovern, P. E.; Mirzoian, A.; Hall, G. R. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,

7361–7366.

(2) Móró, L.; Simon, K.; Bárd, I.; Rácz, J. J. Psychoact. Drugs 2011, 43, 188–198.

(3) Mahdi, J. G.; Mahdi, a J.; Bowen, I. D. Cell prolif. 2006, 39, 147–155.

(4) Mahdi, J. G.; Mahdi, a J.; Bowen, I. D. Phytochem. 2005, 66, 1399–1406.

(5) Eisenreich, W.; Schwarz, M.; Zenk, M. H.; Bacherl, A.; Cartayrade, A.; Arigoni, D.

Chem. Biol. 1998, 5, 221–233.

(6) Facchini, P. J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 29–66.

(7) Staunton, J.; Weissman, K. J. Nat. Prod. Rep. 2001, 18, 380–416.

(8) Kleinkauf, H.; Von Doehren, H. Eur. J. Biochem. 1990, 192, 1–15.

(9) Zhu, C.; Jiang, L.; Chen, T. Eur. J. Med. Chem. 2002, 37, 399–407.

(10) Hann, M. M.; Oprea, T. I. Curr. Opin. Chem. Biol. 2004, 8, 255–263.

(11) Agresti, J. J.; Antipov, E.; Abate, A. R.; Ahn, K.; Rowat, A. C.; Baret, J.-C.; Marquez,

M.; Klibanov, A. M.; Griffiths, A. D.; Weitz, D. Proc. Natl. Acad. Sci. U.S.A 2010,

107, 4004–4009.

(12) Schwarz, H.; Karreman, M.; Agronskaia, A. Curr. Biol. 1994, 4, 564–567.

(13) Ortholand, J.-Y.; Ganesan, A. Curr. Opin. Chem. Biol. 2004, 8, 271–280.

(14) Montaser, R.; Luesch, H. Future Med. Chem. 2011, 3, 1475–1489.

(15) Lee, M. L.; Schneider, G. J. Comb. Chem. 2001, 3, 284–289.

(16) Lipinski, C.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.

2001, 46, 3–26.

(17) Oprea, T. I. Curr. Opin. Chem. Biol. 2002, 6, 384–389.

(18) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.; Jensen, P. R.; Fenical,

W.; John, D. Angew. Chem. Int. Ed. 2003, 42, 355–357.

(19) Groll, M.; Ditzel, L.; Lowe, J.; Stock, D.; Bochtler, M.; Bartunik, H. Nature 1997,

386, 463–471.

(20) Groll, M.; Huber, R.; Potts, B. C. M. J. Am. Chem. Soc. 2006, 128, 5136–5141.

(21) Kong, D.-X.; Jiang, Y.-Y.; Zhang, H.-Y. Drug Discov. Today 2010, 15, 884–886.

(22) Munro, M. H.; Blunt, J. W.; Dumdei, E. J.; Hickford, S. J.; Lill, R. E.; Li, S.;

Battershill, C. N.; Duckworth, R. J. Biotechnol. 1999, 70, 15–25.

(23) Mayer, A. M. S.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem, W.; Little, R. D.;

McIntosh, J. M.; Newman, D. J.; Potts, B. C.; Shuster, D. E. Trends Pharmacol. Sci.

2010, 31, 255–265.

(24) Searle, P.; Molinski, T. F. J. Am. Chem. Soc. 1995, 117, 8126–8131.

(25) Macmillan, J. B.; Xiong-zhou, G.; Skepper, C. K.; Molinski, T. F. J. Org. Chem. 2008,

73, 3699–3706.

(26) Dalisay, D. S.; Morinaka, B. I.; Skepper, C. K.; Molinski, T. F. J. Am. Chem. Soc.

2009, 131, 7552–7553.

(27) Kinnel, R. B.; Gehrken, H.-peter; Scheuer, P. J. J. Am. Chem. Soc. 1993, 115, 3376–

3377.

(28) Cipres, A.; Li, K.; Finlay, D.; Baran, P. S.; Vuori, K. ACS. Chem. Biol. 2010, 5, 195–

202.

(29) Holt, T. O. M. G.; Fregeau, N. L.; Jr, T. J.; Sam, R. J. Nat. Prod. 1990, 53, 771–792.

(30) Buchanan, M. S.; Carroll. A. R.; Addepalli. R.; Avery. V. M.; Hooper. J. N. A.;

Quinn. R. J. J. Org. Chem. 2007, 72, 2309–2317.

(31) Seiple, I. B.; Su, S.; Young, I. S.; Lewis, C.; Yamaguchi, J.; Baran, P. S. Angew.

Chem. Int. Ed. 2010, 49, 1095–1098.

(32) Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6699–6702.

(33) Bai, R.; Cichacz, Z.; Herald, C. L.; Pettit, G. R.; Hamel, E. Mol. Pharmacol. 1993, 44,

757–766.

(34) Schyschka, L.; Rudy, A.; Jeremias, I.; Barth, N.; Pettit, G. R.; Vollmar, a M. Leukemia

2008, 22, 1737–1745.

(35) Smith, A. B.; Tomioka, T.; Risatti, C.; Sperry, J. B.; Sfouggatakis, C. Org. Lett. 2008,

10, 4359–4362.

(36) Ball, M.; Gaunt, M. J.; Hook, D. F.; Jessiman, A. S.; Kawahara, S.; Orsini, P.; Scolaro,

A.; Talbot, A. C.; Tanner, H. R.; Yamanoi, S.; Ley, S. V. Angew. Chem. Int. Ed. 2005,

44, 5433–5438.

(37) Towle, M. J.; Salvato, K. A.; Budrow, J.; Wels, B. F.; Kuznetsov, G.; Aalfs, K. K.;

Welsh, S.; Zheng, W.; Seletsky, B. M.; Palme, M. H.; Habgood, G. J.; Singer, L. A.;

Dipietro, L. V.; Wang, Y.; Chen, J. J.; Quincy, D. A.; Davis, A.; Yoshimatsu, K.;

Kishi, Y.; Yu, M. J.; Littlefield, B. A. Cancer Res. 2001, 61, 1013–1021.

(38) Smith, A. B.; Risatti, C.; Atasoylu, O.; Bennett, C. S.; Liu, J.; Cheng, H.; TenDyke,

K.; Xu, Q. J. Am. Chem. Soc. 2011, 133, 14042–14053.

(39) Schumacher, M.; Kelkel, M.; Dicato, M.; Diederich, M. Molecules 2011, 16, 5629–

5646.

(40) Miljanich, G. P. Curr. Med. Chem. 2004, 11, 3029–3040.

(41) Mcgivern, J. G. Acute Pain. 2006, 11, 245–253.

(42) Sasaki, T.; Takasuga, S.; Sasaki, J.; Kofuji, S.; Eguchi, S.; Yamazaki, M.; Suzuki, A.

Prog. Lipid Res. 2009, 48, 307–343.

(43) Bunney, T. D.; Katan, M. Nat. Rev. Cancer 2010, 10, 342–352.

(44) Martelli, A. M.; Evangelisti, C.; Chiarini, F.; Grimaldi, C.; Manzoli, L.; McCubrey, J.

Expert Opin. Invest. Drugs 2009, 18, 1333–1349.

(45) Harris, S. J.; Parry, R. V.; Westwick, J.; Ward, S. G. J. Biol. Chem. 2008, 283, 2465–

2469.

(46) Ware, M. D.; Rosten, P.; Damen, J. E.; Liu, L.; Humphries, R. K.; Krystal, G. Blood

1996, 88, 2833–2840.

(47) Jänne, P.; Gray, N.; Settleman, J. Nat. Rev. Drug Discovery 2009, 8, 709–723.

(48) Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489–501.

(49) Ong, C. J.; Ming-Lum, A.; Nodwell, M.; Ghanipour, A.; Yang, L.; Williams, D. E.;

Kim, J.; Demirjian, L.; Qasimi, P.; Ruschmann, J.; Cao, L.-P.; Ma, K.; Chung, S. W.;

Duronio, V.; Andersen, R. J.; Krystal, G.; Mui, A. L.-F. Blood 2007, 110, 1942–1949.

(50) Damen, J. E.; Liu, L.; Rosten, P.; Humphries, R. K.; Jefferson, A. B.; Majerus, P. W.;

Krystal, G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1689–1693.

(51) Yang, L.; Williams, D. E.; Mui, A.; Ong, C.; Krystal, G.; van Soest, R.; Andersen, R.

J. Org. Lett. 2005, 7, 1073–1076.

(52) Goclik, E.; König, G. M.; Wright, a D.; Kaminsky, R. J. Nat. Prod. 2000, 63, 1150–

1152.

(53) Kwak, J. H.; Schmitz, F. J.; Kelly, M. J. Nat. Prod. 2000, 63, 1153–1156.

(54) Cavalieri, E. L.; Li, K.-ming; Balu, N.; Saeed, M.; Devanesan, P.; Higginbotham, S.;

Zhao, J.; Gross, M. L.; Rogan, E. G. Carcinogenesis 2002, 23, 1071–1077.

(55) Woodward, R.; Bloch, K. J. Am. Chem. Soc. 1953, 75, 2023–2024.

(56) Surendra, K.; Corey, E. J. J. Am. Chem. Soc. 2008, 130, 8865–8869.

(57) Johnson, W. S.; Gravestock, M. B.; McCarry, B. E. J. Am. Chem. Soc. 1971, 93,

4332–4334.

(58) Johnson, W. S.; Crandall, J. K. J. Org. Chem. 1965, 30, 1785–1790.

(59) Johnson, W. S.; Kinnel, R. B. J. Am. Chem. Soc. 1966, 88, 3861–3862.

(60) Johnson, W. S.; Semmelhack, M. F.; Sultanbawa, M.; Dolak, L. A. J. Am. Chem. Soc.

1968, 90, 2994–2996.

(61) Dijkink, J.; Speckamp, W. Tetrahedron Lett. 1977, 18, 935–938.

(62) Johnson, W. S.; Daub, G. W.; Lyle, T. A.; Niwa, M. J. Am. Chem. Soc. 1980, 102,

7800–7802.

(63) Johnson, W. S.; Hughes, L. R.; Carlson, J. L. J. Am. Chem. Soc. 1979, 101, 1281–

1282.

(64) Bartlett, P. A.; Johnson, W. S. J. Am. Chem. Soc. 1973, 95, 7501–7502.

(65) Tanis, S. P.; Herrinton, P. M. J. Org. Chem. 1983, 48, 4572–4580.

(66) Linares-Palomino, P. J.; Salido, S.; Altarejos, J.; Sánchez, A. Tetrahedron Lett. 2003,

44, 6651–6655.

(67) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525–616.

(68) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen, T.;

Savolainen, J. Nat. Rev. Drug Discovery 2008, 7, 255–270.

(69) Yoder, R.; Johnston, J. N. Chem. Rev. 2005, 105, 4730–4756.

(70) Vilotijevic, I.; Jamison, T. F. Angew. Chem. Int. Ed. 2009, 48, 5250–5281.

(71) Xiong, Z.; Busch, R.; Corey, E. Org. Lett. 2010, 12, 1512–1514.

(72) Nunomoto, S.; Kawakami, Y.; Yamashita, Y. J. Org. Chem. 1983, 48, 1912–1914.

(73) Zhang, W.; Loebach, J. L.; Wilson, S. R.; Jacobsen, E. N. J. Am. Chem. Soc. 1990,

112, 2801–2803.

(74) Wang, Z. X.; Tu, Y.; Frohn, M.; Zhang, J. R.; Shi, Y. J. Am. Chem. Soc. 1997, 119,

11224–11235.

(75) Van Tamelen, E.; Seiler, M.; Wierenga, W. J. Am. Chem. Soc. 1972, 94, 8229–8231.

(76) Corey, E.; Wood Jr, H. B. J. Am. Chem. Soc. 1996, 118, 11982–11983.

(77) Bogenstätter, M.; Limberg, A.; Overman, L. E.; Tomasi, A. L. J. Am. Chem. Soc.

1999, 121, 12206–12207.

(78) Zhao, J.-F.; Zhao, Y.-J.; Loh, T.-P. Chem. Commun. 2008, 1, 1353–1355.

(79) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543–2549.

(80) Mukherjee, R.; Jaggi, M.; Rajendran, P.; Srivastava, S. K.; Siddiqui, M. J.; Vardhan,

A.; Burman, A. C. Bioorg. Med. Chem. Lett. 2004, 14, 3169–3172.

(81) Scribner, A.; Moore, J.; Ouvry, G.; Fisher, M.; Wyvratt, M.; Leavitt, P.; Liberator, P.;

Gurnett, A.; Brown, C.; Mathew, J.; Thompson, D.; Schmatz, D.; Biftu, T. Bioorg.

Med. Chem. Lett. 2009, 19, 1517–1521.

(82) Borsch, R.-F.; Bernstein, M.-D.; Durst, H.-D. J. Am. Chem. Soc. 1971, 93, 2897–2904.

(83) Zhao, M.-X.; Shi, Y. J. Org. Chem. 2006, 71, 5377–5379.

(84) Mori, K. Tetrahedron Asymm. 2011, 22, 1006–1010.

(85) Qian, K.; Yu, D.; Chen, C.-H.; Huang, L.; Morris-Natschke, S. L.; Nitz, T. J.;

Salzwedel, K.; Reddick, M.; Allaway, G. P.; Lee, K.-H. J. Med. Chem. 2009, 52,

3248–3258.

(86) Parra-Delgado, H.; Compadre, C. M.; Ramírez-Apan, T.; Muñoz-Fambuena, M. J.;

Compadre, R. L.; Ostrosky-Wegman, P.; Martínez-Vázquez, M. Bioorg. Med. Chem.

2006, 14, 1889–1901.

(87) Holland, H. Tetrahedron Lett. 1979, 3395–3396.

(88) Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin Trans. 1. 1975, 1574–1585.

(89) Witschel, M. C.; Höffken, H. W.; Seet, M.; Parra, L.; Mietzner, T.; Thater, F.;

Niggeweg, R.; Röhl, F.; Illarionov, B.; Rohdich, F.; Kaiser, J.; Fischer, M.; Bacher,

A.; Diederich, F. Angew. Chem. Int. Ed. 2011, 50, 7931–7935.

(90) Takatsuka, Y.; Chen, C.; Nikaido, H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 6559–

6565.

(91) Dror, R. O.; Arlow, D. H.; Borhani, D. W.; Jensen, M. Ø.; Piana, S.; Shaw, D. E.

Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 4689–4694.

(92) Mayer, M.; Meyer, B. Angew. Chem. Int. Ed. 1999, 38, 1784–1788.

(93) Siriwardena, A. H.; Tian, F.; Noble, S.; Prestegard, J. H. Angew. Chem. Int. Ed. 2002,

41, 3454–3457.

(94) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531–

1534.

(95) Meyer, B.; Weimar, T.; Peters, T. Eur. J. Biochem. 1997, 246, 705–709.

(96) Chen, A.; Shapiro, M. J. J. Am. Chem. Soc. 1998, 120, 10258–10259.

(97) Mayer, M.; Meyer, B. J. Am. Chem. Soc. 2001, 123, 6108–6117.

(98) Claasen, B.; Axmann, M.; Meinecke, R.; Meyer, B. J. Am. Chem. Soc. 2005, 127,

916–919.

(99) Ong, C. J.; Ming-Lum, A.; Nodwell, M.; Ghanipour, A.; Yang, L.; Williams, D. E.;

(100) Hernández Daranas, A.; Koteich Khatib, S.; Lysek, R.; Vogel, P.; Gavín, J. Chemistry

Open 2012, 1, 13–16.

(101) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev. Biochem. 2008, 77, 383–414.

(102) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694–

14699.

(103) Pan, Z.; Jeffery, D.; Chehade, K.; Beltman, J.; Clark, J. M.; Grothaus, P.; Bogyo, M.;

Baruch, A. Bioorg. Med. Chem. Lett. 2006, 16, 2882–2885.

(104) Faleiro, L.; Kobayashi, R.; Fearnhead, H.; Lazebnik, Y. EMBO. J. 1997, 16, 2271–

2281.

(105) Das, J. Chem. Rev. 2011, 111, 4405–4417.

(106) Saghatelian, A.; Jessani, N.; Joseph, A.; Humphrey, M.; Cravatt, B. F. Proc. Natl.

Acad. Sci. U.S.A. 2004, 101, 10000–10005.

(107) MacKinnon, A. L.; Garrison, J. L.; Hegde, R. S.; Taunton, J. J. Am. Chem. Soc. 2007,

129, 14560–14561.

(108) Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Chem. Biol. 2000,

7, 569–581.

(109) Vocadlo, D. J.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2004, 43, 5338–5342.

(110) Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179–1187.

(111) Kolb, H. C.; Sharpless, K. B. Drug Discov. Today 2003, 8, 1128–1137.

(112) Bi, X.; Schmitz, A.; Hayallah, A. M.; Song, J.-N.; Famulok, M. Angew. Chem. Int. Ed.

2008, 47, 9565–9568.

(113) Desantis, G.; Jones, J. B. Bioorg. Med. Chem. 2000, 8, 563–570.

(114) Okerberg, E. S.; Wu, J.; Zhang, B.; Samii, B.; Blackford, K.; Winn, D. T.; Shreder, K.

R.; Burbaum, J. J.; Patricelli, M. P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4996–

5001.

(115) Finn, F. M.; Titus, G.; Horstman, D.; Hofmann, K. Proc. Natl. Acad. Sci. U.S.A. 1984,

81, 7328–7332.

(116) Greenbaum, D. Mol. Cell. Proteomics 2001, 1, 60–68.

(117) Tisserand, S.; Baati, R.; Nicolas, M.; Mioskowski, C. J. Org. Chem. 2004, 69, 8982–

8983.

(118) Norell, J. R. J. Org. Chem. 1973, 38, 1924–1928.

(119) Chen, X.; Tordeux, M.; Desmurs, J.-R.; Wakselman, C. J. Fluorine Chem. 2003, 123,

51–56.

(120) Boyer, J.; Krum, J.; Myers, M.; Fazal, A.; Wigal, C. J. Org. Chem. 2000, 65, 4712–

4714.

(121) Rozenberg, V.; Danilova, T.; Sergeeva, E.; Vorontsov, E.; Starikova, Z.; Lysenko, K.;

Belokon, Y. Eur. J. Org. Chem. 2000, 2000, 3295–3303.

(122) Cullinane, N.; Woolhouse, R.; Edwards, B. J. Chem. Soc. 1961, 3842–3845.

(123) Halpern, B. N.; Neveu, T.; Spector, S. Br. J. Pharmacol. 1963, 20, 389–398.

(124) Burnstein, K. L. J. Cell. Biochem. 2005, 95, 657–669.

(125) Joseph, I. B.; Nelson, J. B.; Denmeade, S. R.; Isaacs, J. T. Clin. Cancer Res. 1997, 3,

2507–2511.

(126) Tang, D. G.; Porter, a T. Prostate 1997, 32, 284–293.

(127) Sadar, M. D. Cancer Res. 2011, 71, 1208–1213.

(128) O’Donnell, A.; Judson, I.; Dowsett, M.; Raynaud, F.; Dearnaley, D.; Mason, M.;

Harland, S.; Robbins, A.; Halbert, G.; Nutley, B.; Jarman, M. Br. J. Cancer 2004, 90,

2317–2325.

(129) Attard, G.; Belldegrun, A. S.; de Bono, J. S. BJU. Int. 2005, 96, 1241–1246.

(130) Singh, S. M.; Gauthier, S.; Labrie, F. Curr. Med. Chem. 2000, 7, 211–247.

(131) Fradet, Y. Expert Rev. Anticancer Ther. 2004, 4, 37–48.

(132) Jung, M. E.; Ouk, S.; Yoo, D.; Sawyers, C. L.; Chen, C.; Tran, C.; Wongvipat, J. J.

Med. Chem. 2010, 53, 2779–2796.

(133) Scher, H. I.; Sawyers, C. L. J. Clin. Oncol. 2005, 23, 8253–8261.

(134) Montgomery, R. B.; Mostaghel, E.; Vessella, R.; Hess, D. L.; Kalhorn, T. F.; Higano,

C. S.; True, L. D.; Nelson, P. S. Cancer Res. 2008, 68, 4447–4454.

(135) Chen, Y.; Clegg, N. J.; Scher, H. I. Lancet. Oncol. 2009, 10, 981-991.

(136) Taplin, M. E.; Bubley, G. J.; Shuster, T. D.; Frantz, M. E.; Spooner, A. E.; Ogata, G.

K.; Keer, H. N.; Balk, S. P. N. Engl. J. Med. 1995, 332, 1393–1398.

(137) Jenster, G. J. Pathol. 2000, 191, 227–228.

(138) Guo, Z.; Yang, X.; Sun, F.; Jiang, R.; Linn, D. E.; Chen, H.; Chen, H.; Kong, X.;

Melamed, J.; Tepper, C. G.; Kung, H.-J.; Brodie, A. M. H.; Edwards, J.; Qiu, Y.

Cancer Res. 2009, 69, 2305–2313.

(139) Shaffer, P. L.; Jivan, A.; Dollins, D. E.; Claessens, F.; Gewirth, D. T. Proc. Natl.

Acad. Sci. U.S.A. 2004, 101, 4758–4763.

(140) Sadar, M. D.; Williams, D. E.; Mawji, N. R.; Patrick, B. O.; Wikanta, T.; Chasanah,

E.; Irianto, H. E.; Soest, R. V.; Andersen, R. J. Org. Lett. 2008, 10, 4947–4950.

(141) Andersen, R. J.; Mawji, N. R.; Wang, J.; Wang, G.; Haile, S.; Myung, J.-K.; Watt, K.;

Tam, T.; Yang, Y. C.; Bañuelos, C.; Williams, D. E.; McEwan, I. J.; Wang, Y.; Sadar,

M. D. Cancer Cell 2010, 17, 535–546.

(142) Versteegen, R. M.; van Beek, D. J. M.; Sijbesma, R. P.; Vlassopoulos, D.; Fytas, G.;

Meijer, E. W. J. Am. Chem. Soc. 2005, 127, 13862–13868.

(143) Appel, R. Angew. Chem. Int. Ed. 1975, 14, 801–811.

(144) Shearman, G. C.; Yahioglu, G.; Kirstein, J.; Milgrom, L. R.; Seddon, J. M. J. Mater.

Chem. 2009, 19, 598–604.

(145) Mehta, G.; Mohanrao, R.; Katukojvala, S.; Landais, Y.; Sen, S. Tetrahedron Lett.

2011, 52, 2893–2897.

(146) Wadsworth, W. S.; Emmons, W. D. J. Am. Chem. Soc. 1961, 83, 1733–1738.

(147) Garg, N. K.; Hiebert, S.; Overman, L. E. Angew. Chem. Int. Ed. 2006, 45, 2912–1215.

(148) Sabitha, G.; Satheesh Babu, R.; Rajkumar, M.; Reddy, C. S.; Yadav, J. Tetrahedron

Lett. 2001, 42, 3955–3958.

(149) Bulger, P. G.; Moloney, M. G.; Trippier, P. C. Org. Biomol. Chem. 2003, 1, 3726–

3737.

(150) Zhu, W.; Jime nez, M.; Jung, W. H.; Camarco, D. P.; Balachandran, R.; Vogt, A.;

Day, B. W.; Curran, D. P. J. Am. Chem. Soc. 2010, 132, 9175–9187.

(151) Akiyama, T.; Ueoka, R.; van Soest, R. W. M.; Matsunaga, S. J. Nat. Prod. 2009, 72,

1552–1554.

(152) Veronese, F. M.; Pasut, G. Drug Discov. Today 2005, 10, 1451–1458.

(153) Filler, R.; Saha, R. Future Med. Chem. 2009, 1, 777–791.

(154) Beaulieu, F.; Beauregard, L.-P.; Courchesne, G.; Couturier, M.; LaFlamme, F.;

L’Heureux, A. Org. Lett. 2009, 11, 5050–5053.

(155) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Curr. Opin. Biotechnol. 2007, 18, 17–25.

(156) Ntziachristos, V.; Yodh, a G.; Schnall, M.; Chance, B. Proc. Natl. Acad. Sci. U.S.A

2000, 97, 2767–2772.

(157) Bilski, P. J.; Risek, B.; Chignell, C. F.; Schrader, W. T. Photochem. Photobiol. 2009,

85, 1225–1232.

(158) Gierlich, J.; Burley, G.; Gramlich, P. M. E.; Hammond, D. M.; Carell, T. Org. Lett.

2006, 8, 3639–3642.

(159) Kim, G.-J.; Lee, K.; Kwon, H.; Kim, H.-J. Org. Lett. 2011, 13, 2799–2801.

(160) Sakai, H.; Hirano, T.; Mori, S.; Fujii, S.; Masuno, H.; Kinoshita, M.; Kagechika, H.;

Tanatani, A. J. Med. Chem. 2011, 54, 7055–7065.

(161) Wu, J.-S.; Liu, W.-M.; Zhuang, X.-Q.; Wang, F.; Wang, P.-F.; Tao, S.-L.; Zhang, X.-

H.; Wu, S.-K.; Lee, S.-T. Org. Lett. 2007, 9, 33–36.

(162) Li, C. J.; Schmitz, F. J.; Kelly-Borges, M. J. Nat. Prod. 1999, 62, 287–290.

(163) Hyosu, M.; Kimura, J. J. Nat. Prod. 2000, 63, 422–423.

(164) Takadoi, M.; Katoh, T.; Ishiwata, A.; Terashima, S. Tetrahedron 2002, 58, 9903–

9923.

(165) San-miguelc, N. S. A. M. A.; Taranc, M.; Delmond, B. Tetrahedron 1991, 47, 9187–

9194.

(166) Chaturvedula, V. S. P.; Gao, Z.; Thomas, S. H.; Hecht, S. M.; Kingston, D. G. I.

Tetrahedron 2004, 60, 9991–9995.

(167) Zoretic, P.; Wang, M.; Zhang, Y.; Shen, Z.; Ribeiro, A. J. Org. Chem. 1996, 61,

1806–1813.

(168) McGrath, N.; Lee, C.; Araki, H.; Brichacek, M.; Njardarson, J. T. Angew. Chem. Int.

Ed. 2008, 47, 9450–9453.

(169) Zoretic, P. A.; Weng, X.; Caspar, M. L.; Davis, D. G. Tetrahedron Lett. 1991, 32,

4819–4822.

(170) Snider, B. Synth. Commun. 2001, 31, 3753–3758.

(171) Tong, R.; Valentine, J. C.; McDonald, F. E.; Cao, R.; Fang, X.; Hardcastle, K. I. J.

Am. Chem. Soc. 2007, 129, 1050–1051.

(172) Mori, K. Tetrahed. Asym. 2011, 22, 1006–1010.

(173) Seifert, A.; Vomund, S.; Grohmann, K.; Kriening, S.; Urlacher, V. B.; Laschat, S.;

Pleiss, J. Chem. Bio. Chem. 2009, 10, 853–861.

(174) Malerich, J. P.; Trauner, D. J. Am. Chem. Soc. 2003, 125, 9554–9555.

(175) Rossiter, B. E.; Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1981, 103, 464–465.

(176) Kuk, J.; Kim, B. S.; Jung, H.; Choi, S.; Park, J.-Y.; Koo, S. J. Org. Chem. 2008, 73,

1991–1994.

(177) Hutchins, R. O.; Learn, K. J. Org. Chem. 1982, 47, 4380–4382.

(178) Goldsmith, D.; Liotta, D.; Saindane, M.; Waykole, L.; Bowen, P. Tetrahed Asym.

1983, 24, 5835–5838.

(179) Dong, J.-Q.; Wong, H. N. C. Angew. Chem. Int. Ed. 2009, 48, 2351–2354.

(180) Tofi, M.; Georgiou, T.; Montagnon, T.; Vassilikogiannakis, G. Org. Lett. 2005, 7,

3347–3350.

(181) Moerke, N. Curr. Protoc. Chem Biol. 2009, 1, 1–15.

(182) Patel, T.; Gores, G. J.; Kaufmann, S. H. FASEB. J. 1996, 10, 587–597.

(183) Ciechanover, A. Biochim. Biophys. Acta. 2005, 44, 5944–5967.

(184) Hedstrom, L. Chem. Rev. 2002, 102, 4501–4524.

(185) Chapman, H.; Riese, R. J.; Shi, G. P. Annu. Rev. Physiol. 1997, 59, 63–88.

(186) Seelmeier, S.; Schmidt, H.; Turk, V.; von der Helm, K. Proc. Natl. Acad. Sci. U.S.A.

1988, 85, 6612–6616.

(187) Turk, B.; Turk, D.; Turk, V. Biochim. Biophys. Acta. 2000, 1477, 98–111.

(188) McGrath, M. E. Annu. Rev. Bioph. Biom. 1999, 28, 181–204.

(189) Barford, D.; Das, K.; Egloff, M. P. Annu. Rev. Bioph. Biom. 1998, 27, 133–164.

(190) Turk, V.; Turk, B. EMBO. J. 2001, 20, 4629–4633.

(191) Fineschi, B.; Jim, M. Trends Biochem. Sci. 1997, 22, 377–382.

(192) Barrett, A. J. Trends Biochem. Sci 1987, 12, 193-196.

(193) Gettins, P. Chem. Rev. 2002, 102, 4751–4804.

(194) Katunuma, N.; Kominami, E. Rev. Physiol. Bioch. P. 1987, 108, 1–20.

(195) Jørgensen, I.; Kos, J.; Krašovec, M.; Troelsen, L.; Klarlund, M.; Jensen, T. W.;

Hansen, M. S.; Jacobsen, S. Clin. Rheumatol. 2011, 30, 633–638.

(196) Bone, L.; Everts, V.; Korper, W.; Hoeben, K. A.; Jansen, I. D. C.; Bromme, D.;

Cleutjens, K. B. J. M.; Heeneman, S.; Peters, C.; Reinheckel, T.; Saftig, P.; Beertsen,

W. J. Bone Miner. Res. 2006, 21, 1399–1408.

(197) Lemere, C. A.; Munger, J. S.; Shi, G. P.; Natkin, L.; Haass, C.; Chapman, H. A.;

Selkoe, D. J. Am. J. Pathol. 1995, 146, 848–860.

(198) Lamkanfi, M.; Festjens, N.; Declercq, W.; Vanden Berghe, T.; Vandenabeele, P. Cell

Death Differ. 2007, 14, 44–55.

(199) Mohamed, M. M.; Sloane, B. F. Nat. Rev. Cancer 2006, 6, 764–775.

(200) Identification, S.; Bossard, M. J.; Tomaszek, T. A.; Thompson, S. K.; Amegadzie, B.

Y.; Hanning, C. R.; Jones, C.; Kurdyla, J. T.; Mcnulty, D. E.; Drake, F. H.; Gowen,

M.; Levy, M. A.; Chem, M. J. B. Biochemistry 1996, 271, 12517–12524.

(201) Stoch, S.; Wagner, J. a Pharmacol. Ther. Part C. 2008, 83, 172–176.

(202) Hozumi, M.; Ogawa, M.; Sugimura, T.; Takeuchi, T.; Umezawa, H. Cancer Res.

1972, 32, 1725–1728.

(203) Umezawa, H.; Aoyagi, T.; Morishima, H.; Kunimoto, S.; Matsuzaki, M. J. Antibiot.

1970, 23, 425–427.

(204) Umezawa, S.; Tatsuta, K.; Fujimoto, K.; Tsuchiya, T.; Umezawa, H. J. Antibiot. 1972,

25, 267–270.

(205) Brömme, D.; Lecaille, F. Expet. Opin. Investig. Drugs 2009, 18, 585–600.

(206) Jerome, C.; Missbach, M.; Gamse, R. Osteoporosis Int. 2012, 23, 339–349.

(207) Kumar, S.; Dare, L.; Vasko-Moser, J.; James, I. E.; Blake, S. M.; Rickard, D. J.;

Hwang, S.-M.; Tomaszek, T.; Yamashita, D. S.; Marquis, R. W.; Oh, H.; Jeong, J. U.;

Veber, D. F.; Gowen, M.; Lark, M. W.; Stroup, G. Bone 2007, 40, 122–131.

(208) Eisman, J.; Bone, H. G.; Hosking, D. J.; McClung, M. R.; Reid, I. R.; Rizzoli, R.;

Resch, H.; Verbruggen, N.; Hustad, C. M.; DaSilva, C.; Petrovic, R.; Santora, A. C.;

Ince, B. A.; Lombardi, A. J. Bone Miner. Res. 2011, 26, 242–251.

(209) Arzt, S.; Beteva, A.; Cipriani, F.; Delageniere, S.; Felisaz, F.; Förstner, G.; Gordon,

E.; Launer, L.; Lavault, B.; Leonard, G.; Mairs, T.; McCarthy, A.; McCarthy, J.;

McSweeney, S.; Meyer, J.; Mitchell, E.; Monaco, S.; Nurizzo, D.; Ravelli, R.; Rey,

V.; Shepard, W.; Spruce, D.; Svensson, O.; Theveneau, P. Prog. Biophys. Mol. Biol.

2005, 89, 124–152.

(210) Valeur, E.; Bradley, M. Chem. Soc. Rev. 2009, 38, 606–631.

(211) Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem. 1995, 60, 3561–3564.

(212) Lee, J. T.; Chen, D. Y.; Yang, Z.; Ramos, A. D.; Hsieh, J. J.-D.; Bogyo, M. Bioorg.

Med. Chem. Lett. 2009, 19, 5086–5090.

(213) Zheng, C.; Combs, A. P. J. Comb. Chem. 2002, 4, 38–43.

(214) Singh, P.; Samor, C.; Toma, F. M.; Bussy, C.; Nunes, A.; Al-Jamal, K. T.; Ménard-

Moyon, C.; Prato, M.; Kostarelos, K.; Bianco, A. J. Mater. Chem. 2011, 21, 4850–

4860.

(215) Majer, P.; Randad, R. S. J. Org. Chem. 1994, 59, 1937–1938.

(216) Ito, A.; Takahashi, R.; Baba, Y. Chem. Pharm. Bull. 1975, 23, 3081–3087.

(217) Brown, H.; Narasimhan, S.; Choi, Y. M. J. Org. Chem. 1982, 47, 4702–4708.

(218) Coffey, D. S.; Overman, L. E.; Stappenbeck, F. J. Am. Chem. Soc. 2000, 122, 4904–

4914.

(219) Frigerio, M.; Santagostino, M.; Sputore, S.; Palmisano, G. J. Org. Chem. 1995, 60,

7272–7276.

(220) Shimizu, B.; Saito, A.; Ito, A.; Tokawa, K. J. Antibiot. 1972, 25, 515–523.

(221) Leanna, M. R.; Sowin, T.; Morton, H. E. Tetrahedron Lett. 1992, 33, 5029–5032.

(222) Einhorn, J.; Einhorn, C.; Ratajczak, F.; Pierre, J.-L. J. Org. Chem. 1996, 61, 7452–

7454.

(223) Yasuma, T.; Oi, S.; Choh, N.; Nomura, T.; Furuyama, N.; Nishimura, A.; Fujisawa,

Y.; Sohda, T. J. Med. Chem. 1998, 41, 4301–4308.

(224) Shimokawa, J.; Ishiwata, T.; Shirai, K.; Koshino, H.; Tanatani, A.; Nakata, T.;

Hashimoto, Y.; Nagasawa, K. Chem. Eur. J. 2005, 11, 6878–6888.

(225) Myers, A. G.; Zhong, B.; Movassaghi, M.; Kung, D. W.; Lanman, B. A.; Kwon, S.

Tetrahedron Lett. 2000, 41, 1359–1362.

(226) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett. 1993, 34, 3389–3392.

(227) Mandal, P. K.; McMurray, J. S. J. Org. Chem. 2007, 72, 6599–6601.

(228) Srinivasa, G. R.; Narendra Babu, S. N.; Lakshmi, C.; Gowda, D. C. Synth. Commun.

2004, 34, 1831–1837.

(229) Lemke, A.; Büschleb, M.; Ducho, C. Tetrahedron 2010, 66, 208–214.

(230) Yu, X.; Dai, Y.; Yang, T.; Gagné, M. R.; Gong, H. Tetrahedron 2011, 67, 144–151.

(231) Adamo, I.; Ballico, M.; Campaner, P.; Drioli, S.; Bonora, G. M. Eur. J. Org. Chem.

2004, 2603–2609.

(232) Undevia, S. D.; Gomez-Abuin, G.; Ratain, M. J. Nat. Rev. Cancer 2005, 5, 447–548.

(233) Fuhler, G. M.; Brooks, R.; Toms, B.; Iyer, S.; Gengo, E.; Park, M.-Y.; Gumbleton, M.;

Viernes, D. R.; Chisholm, J. D.; Kerr, W. G. Mol. Med. 2012, 18, 65–75.

(234) Cho, I.; Meimetis, L.; Britton, R. Org. Lett. 2009, 11, 1903–1906.

Appendix A : X-Ray Structure Reports

A.1 Compound 2.30

Data Collection:

A colorless plate crystal of C23H34O3 having approximate dimensions of 0.15 x 0.40 x

0.40 mm was mounted on a glass fiber. All measurements were made on a Bruker APEX II

diffractometer with graphite monochromated Mo-Kα radiation. The data were collected at a

temperature of -170.0 + 0.1 o C to a maximum 2� value of 56.0 o . Data were collected in a

series of ��and � scans in 0.50 o oscillations with 10.0-second exposures. The crystal-to-

detector distance was 40.00 mm.

Data Reduction:

Of the 25982 reflections that were collected, 4780 were unique (Rint = 0.033);

equivalent reflections were merged. Data were collected and integrated using the Bruker

SAINT software package. The linear absorption coefficient, �, for Mo-K� radiation is 0.78

cm -1 . Data were corrected for absorption effects using the multi-scan technique (SADABS),

with minimum and maximum transmission coefficients of 0.913 and 0.988, respectively. The

data were corrected for Lorentz and polarization effects.

Structure Solution and Refinement:

The structure was solved by direct methods. All non-hydrogen atoms were refined

anisotropically. Hydrogen H3O was located in a difference map and refined isotropically. All

other hydrogen atoms were placed in calculated positions. The absolute configuration was

determined on the basis of Bijvoet-pair intensity differences. The results suggest that if the

sample is enantiopure there is a 93 % probability the configuration has been properly

assigned.

Crystal Data:

Empirical Formula C23H34O3n

Formula Weight 358.50

Crystal Color, Habit colorless, plate

Crystal Dimensions 0.15 X 0.40 X 0.40 mm

Crystal System orthorhombic

Lattice Type primitive

Lattice Parameters a = 7.0872(3) Å

b = 10.9264(6) Å

c = 25.447(2) Å

� = 90 o

V = 10269.2(6) Å 3

Space Group P 212121 (#19)

Z value 4

Dcalc

F000

1.208 g/cm 3

784.00

��(MoK�) 0.78 cm -1

Intensity Measurements:

Diffractometer Bruker X8 APEX II

Radiation MoK� (� = 0.71073 Å)

graphite monochromated

Data Images 1535 exposures @ 10.0 seconds

Detector Position 40.00 mm

2�max

56.0 o

No. of Reflections Measured Total: 25982

Unique: 4780 (Rint = 0.033)

Corrections Absorption (Tmin = 0.913, Tmax= 0.988)

Lorentz-polarization

Structure Solution Direct Methods (SIR97)

Refinement Full-matrix least-squares on F 2

Function Minimized � w (Fo 2 - Fc 2 ) 2

Least Squares Weights w=1/(� 2 (Fo 2 )+(0.0513P) 2 + 0.3340P)

Anomalous Dispersion All non-hydrogen atoms

No. Observations (I>0.00�(I)) 4780

No. Variables 245

Reflection/Parameter Ratio 19.51

Residuals (refined on F 2 , all data): R1; wR2 0.041; 0.090

Goodness of Fit Indicator 1.03

No. Observations (I>2.00�(I)) 4366

Residuals (refined on F): R1; wR2 0.035; 0.086

Max Shift/Error in Final Cycle 0.00

Maximum peak in Final Diff. Map 0.26 e - /Å 3

Minimum peak in Final Diff. Map -0.21 e - /Å 3

A.2 Compound 2.32

A colorless plate crystal of C33H41F3O5 having approximate dimensions of 0.02 x 0.12

x 0.41 mm was mounted on a glass fiber. All measurements were made on a Bruker APEX II

diffractometer with graphite monochromator and a CuKα IμS MX microsource (λ = 1.54178

Å). The data were collected at a temperature of -173.1 + 0.1 � C to a maximum 2� value of

136.2 � . Data were collected in a series of ��and � scans in 0.50 � oscillations with 10.0-

second exposures. The crystal-to-detector distance was 49.88 mm.

Of the 10010 reflections that were collected, 4408 were unique (Rint = 0.025;

Friedels not merged); equivalent reflections were merged. Data were collected and integrated

using the Bruker SAINT software package. The linear absorption coefficient, �, for Cu-K�

radiation is 8.49 cm -1 . Data were corrected for absorption effects using the multi-scan

technique (SADABS), with minimum and maximum transmission coefficients of 0.847 and

0.983, respectively. The data were corrected for Lorentz and polarization effects.

anisotropically. All C-H hydrogen atoms were placed in calculated positions with only

isotropic displacement parameters refined. The absolute configuration was determined based

on the refined Flack parameter.

Empirical Formula C33H41F3O5

Formula Weight 574.66

Crystal Dimensions 0.02 X 0.12 X 0.41 mm

Crystal System monoclinic

Lattice Type C-centered

Lattice Parameters a = 13.3572(6) Å

b = 6.8023(3) Å

c = 31.3214(14) Å

� = 95.788(2) o

V = 2831.3(2) Å 3

Space Group C 2 (#5)

1.348 g/cm 3

1224.00

�(CuK�) 8.49 cm -1

Data Images 6821 exposures @ 10.0 seconds

Detector Position 49.88 mm

136.2 o

No. of Reflections Measured Total: 10010

Unique: 4408 (Rint = 0.025; Friedels not

merged)

Corrections Absorption (Tmin = 0.847, Tmax= 0.983)

Least Squares Weights w=1/(� 2 (Fo 2 )+(0.0289P) 2 + 1.3029P)

No. Observations (I>0.00�(I)) 4408

No. Variables 419

Reflection/Parameter Ratio 10.52

Residuals (refined on F 2 , all data): R1; wR2 0.029; 0.068

No. Observations (I>2.00�(I)) 4218

Residuals (refined on F): R1; wR2 0.028; 0.067

Maximum peak in Final Diff. Map 0.20 e - /Å 3

Minimum peak in Final Diff. Map -0.15 e - /Å 3

A.3 Compound 2.42

A colorless plate crystal of C23H39NO4.HCl having approximate dimensions of 0.10 x

0.12 x 0.29 mm was mounted on a glass fiber. All measurements were made on a Bruker

APEX DUO diffractometer with graphite monochromated Mo-Kα radiation. The data were

collected at a temperature of -183.0 + 0.1 o C to a maximum 2� value of 60.2 o . Data were

collected in a series of ��and � scans in 0.5 o oscillations using 3.0-second exposures. The

crystal-to-detector distance was 59.84 mm.

The material crystallizes as a two-component twin with components one and two

related by a 179.3º rotation about the (1 0 0) real axis. Data were integrated for both twin

components, including both overlapped and non-overlapped reflections. In total 49944

reflections were integrated (13488 from component one only, 12668 from component two

only, 23788 overlapped). Data were collected and integrated using the Bruker SAINT

software packages. The linear absorption coefficient, �, for Mo-K� radiation is 1.97 cm -1 .

Data were corrected for absorption effects using the multi-scan technique (TWINABS 2 ), with

minimum and maximum transmission coefficients of 0.740 and 0.980, respectively. The data

were corrected for Lorentz and polarization effects.

The structure was solved by direct methods using non-overlapped data from the major

twin component. The material crystallizes with two molecules of methanol in the asymmetric

unit Subsequent refinements were carried out using an HKLF 5 format data set containing

complete data from component one and overlapped reflections from component two. All

non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in

calculated positions.

Empirical Formula C23H40NO4Cl

Formula Weight 430.01

Crystal Dimensions 0.10 x 0.12 x 0.29 mm

Lattice Parameters a = 29.741(3) Å

b = 7.3000(7) Å

c = 21.452(2) Å

� = 102.638(2) o

V = 4544.7(7) Å 3

Space Group C 2/c (#15)

Z value 8

1.257 g/cm 3

1872.00

�(Mo-K�) 1.97 cm -1

Diffractometer Bruker APEX DUO

Radiation Mo-K� (� = 0.71073 Å)

Data Images 2243 exposures @ 3.0 seconds

Detector Position 59.84 mm

60.2 o

No. of Reflections Measured Total: 49944

Unique: 6679 (Rint = 0.045)

Corrections Absorption (Tmin = 0.740, Tmax= 0.980)

Least Squares Weights w=1/(� 2 (Fo 2 )+(0.0580P) 2 + 9.3161P)

No. Observations (I>0.00�(I)) 6679

No. Variables 297

Reflection/Parameter Ratio 22.49

Residuals (refined on F 2 , all data): R1; wR2 0.064; 0.146

Goodness of Fit Indicator 1.12

No. Observations (I>2.00�(I)) 5968

Residuals (refined on F): R1; wR2 0.055; 0.142

Maximum peak in Final Diff. Map 0.43 e - /Å 3

Minimum peak in Final Diff. Map -0.52 e - /Å 3

More documents
Recommendations

UBC_1963_A5 O7 A4.pdf - cIRcle - University of British Columbia

FUNCTION ORIENTED SYNTHESIS OF BIOACTIVE MARINE NATURAL PRODUCTS AND THEIR PHARMACOPHORE ANALOGUES by Labros G. Meimetis B.Sc., Simon Fraser University, 2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2012 © Labros G. Meimetis, 2012

Page 2 and 3: Abstract Natural products play a ce
Page 4 and 5: Preface Chapter 2 is based on work
Page 6 and 7: Table of Contents Abstract ........
Page 8 and 9: 5.2 Lichostatinal (5.4) a Novel Pep
Page 10 and 11: List of Figures Figure 1.1 Bioactiv
Page 12 and 13: Figure 2.30 1 H and 13 C NMR spectr
Page 14 and 15: Figure 3.17 1 H and 13 C NMR spectr
Page 16 and 17: Figure 3.39 1 H and 13 C NMR spectr
Page 18 and 19: Figure 4.18 1 H and 13 C NMR spectr
Page 20 and 21: List of Schemes Scheme 2.1 Syntheti
Page 22 and 23: Scheme 5.2 Synthesis toward lichost
Page 24 and 25: DNP - dinitrophenyl DOM - directed
Page 26 and 27: Pyr - pyridine q - quartet RF - rad
Page 28 and 29: Dedication To my parents George and
Page 30 and 31: While this has held true over the y
Page 32 and 33: neurodegenerative diseases such as
Page 34 and 35: product extracts. The last couple y
Page 36 and 37: Figure 1.5 Determination of the abs
Page 38 and 39: Figure 1.7 Barans retrosynthesis of
Page 40 and 41: four lowest energy solution conform
Page 42 and 43: such as ziconotide (1.29) broadens
Page 44 and 45: Chapter 2: Synthesis and Biological
Page 46 and 47: 2.2 First Generation SHIP1 Agonist
Page 48 and 49: Figure 2.4 Chemical and enzymatic o
Page 50 and 51: used in many biomimetically-inspire

Scheme 2.2 Retrosynthetic analysis

considered drug-like. To address th

The C-3 ketone analogue 2.19 has a

Scheme 2.6 Retrosynthetic analysis

Scheme 2.8 Examples of indium tribr

The enantiomeric excess of 2.30 aft

Scheme 2.10 Racemic synthesis of (

Scheme 2.12 Synthesis of the 2.18 e

the surrounding locality as it begi

peptide by itself and the STDD NOE

The first is a reactive group, whic

Figure 2.23 Examples of photoaffini

and peptide. The purpose of biotin

y two possible aryl positions in a

amount of green complex formed, and

Next, a SHIP1 chromogenic kinetic a

Figure 2.26 Efficacy of 2.20/2.42 v

pharmacophore. Amines 2.20 and 2.42

Scheme 2.16 Rational drug design fr

Figure 2.27 1 H and 13 C NMR spectr

Preparation of 2.28: Bromide 2.27 (

Preparation of 2.26: Epoxide 2.26 w

Figure 2.29 1 H and 13 C NMR spectr

Figure 2.30 1 H and 13 C NMR spectr

16.1. HRESIMS [M+Na] + calcd for C3

Preparation of 2.32 The preparation

Preparation of 2.33: To alcohol 2.3

Preparation of 2.19: To ketone 2.33

Preparation of 2.20 and 2.34 To a s

Figure 2.35 1 H and 13 C NMR spectr

Preparation of 2.42 and 2.43: Ident

Figure 2.37 1 H and 13 C NMR spectr

Figure 2.38 1 H and 13 C NMR spectr

Figure 2.39 1 H and 13 C NMR spectr

Preparation of 2.48: To 2.47 (20 mg

Figure 2.40 1 H and 13 C NMR spectr

Figure 2.41 1 H and 13 C NMR spectr

Akt Activation Assay: MOLT-4 and Ju

plates to elute the Evans’ Blue d

20-40 % of patients treated with fi

expression of splice variants of th

of the natural products, or carry o

tetrahydrofuran and water 145 gave

For the HWE olefination, phosphonat

The first goal was to examine the e

Scheme 3.9 (R)-Niphatenone B (3.31)

the presence of magnesium metal for

Figure 3.8 Fluorescent probe mode o

potential mechanism for the covalen

Scheme 3.14 Synthesis of a potentia

the alkyl substituent is necessary

demonstrating that the probe 3.67 b

AR antagonist with a binding mechan

Figure 3.14 1 H and 13 C NMR spectr

Figure 3.15 1 H and 13 C NMR spectr

Figure 3.16 1 H and 13 C NMR spectr

29.5, 29.4, 29.3, 29.2, 28.2, 26.9,

Preparation of 3.21: To acetonide 3

Preparation of 3.23: Procedures ide

Figure 3.19 1 H and 13 C NMR spectr

Figure 3.20 1 H and 13 C NMR spectr

Figure 3.21 1 H and 13 C NMR spectr

Figure 3.22 1 H and 13 C NMR spectr

Figure 3.23 1 H and 13 C NMR spectr

Figure 3.24 1 H and 13 C NMR spectr

Figure 3.25 1 H and 13 C NMR spectr

Figure 3.26 1 H and 13 C NMR spectr

Figure 3.27 1 H and 13 C NMR spectr

Figure 3.28 1 H and 13 C NMR spectr

Figure 3.29 1 H and 13 C NMR spectr

Figure 3.30 1 H and 13 C NMR spectr

Figure 3.31 1 H and 13 C NMR spectr

Figure 3.32 1 H and 13 C NMR spectr

Figure 3.33 1 H and 13 C NMR spectr

Figure 3.34 1 H and 13 C NMR spectr

Figure 3.35 1 H and 13 C NMR spectr

29.8, 29.7, 29.7, 29.65, 29.60, 29.

Preparation of 3.52: To epoxide 3.5

Preparation of 3.53: To epoxide 3.5

Preparation of 3.54: To diol 3.31 (

Preparation of 3.56: To acetonide 3

Figure 3.40 1 H and 13 C NMR spectr

Figure 3.41 1 H and 13 C NMR spectr

Figure 3.42 1 H and 13 C NMR spectr

Figure 3.43 1 H and 13 C NMR spectr

Figure 3.44 1 H and 13 C NMR spectr

Figure 3.45 1 H and 13 C NMR spectr

Figure 3.46 1 H and 13 C NMR spectr

Figure 3.47 1 H and 13 C NMR spectr

anti-BrdU-POD antibody (Roche). Brd

of a steroid (Figure 4.2) and it wa

al. However, the synthesis is lengt

converted to chloride 4.11, which w

ligand. Changing the chiral ligand

should increase the electron densit

electronegative and, therefore, thr

Sulfone 4.21 was coupled with bromi

Figure 4.6 AR competitor in vitro a

4.5 Conclusion Small molecule antag

4.6 Experimental General Methods: A

Figure 4.7 1 H and 13 C NMR spectra

Figure 4.8 1 H and 13 C NMR spectra

Figure 4.9 1 H and 13 C NMR spectra

Figure 4.10 1 H and 13 C NMR spectr

Figure 4.11 1 H and 13 C NMR spectr

Figure 4.12 1 H and 13 C NMR spectr

Figure 4.13 1 H and 13 C NMR spectr

Figure 4.14 1 H spectrum of 4.24 re

Figure 4.15 1 H and 13 C NMR spectr

Preparation of 4.42: To sulfone 4.2

Preparation of 4.43: To sulfone 4.4

Preparation of 4.44: To polyene 4.4

Ligand-Binding Affinities of Terpen

Figure 5.1. The first step entails

Figure 5.3 Covalent binding mechani

agmatine (5.6) and tripeptide 5.5 b

At this point, coupling commerciall

Scheme 5.4 Oxidation of Arginol (5.

The synthesis continued with interm

Intermediate 5.35 was guanylated wi

natural product (5.4) suggesting th

Figure 5.5 1 H and 13 C NMR spectra

Figure 5.6 1 H and 13 C NMR spectra

Figure 5.7 1 H and 13 C NMR spectra

Figure 5.8 1 H and 13 C NMR spectra

71.6, 57.8, 53.9, 52.6, 52.6, 40.9,

Preparation of 5.20: To 5.18 (102.6

Figure 5.10 1 H and 13 C NMR spectr

Figure 5.11 1 H and 13 C NMR spectr

Figure 5.12 1 H and 13 C NMR spectr

Figure 5.13 1 H and 13 C NMR spectr

Figure 5.14 1 H and 13 C NMR spectr

Figure 5.15 1 H and 13 C NMR spectr

Figure 5.16 1 H and 13 C NMR spectr

Figure 5.17 1 H and 13 C NMR spectr

Figure 5.18 1 H and 13 C NMR spectr

Figure 5.19 1 H and 13 C NMR spectr

Figure 5.20 1 H and 13 C NMR spectr

Figure 5.21 1 H and 13 C NMR spectr

NMR tube. This may provide more acc

Scheme 6.1 Syntheses towards polar

Scheme 6.3 Deprotection of regiosis

converted entirely to the semicarba

(18) Feling, R. H.; Buchanan, G. O.

(42) Sasaki, T.; Takasuga, S.; Sasa

(72) Nunomoto, S.; Kawakami, Y.; Ya

(99) Ong, C. J.; Ming-Lum, A.; Nodw

(126) Tang, D. G.; Porter, a T. Pro

(153) Filler, R.; Saha, R. Future M

(182) Patel, T.; Gores, G. J.; Kauf

V.; Shepard, W.; Spruce, D.; Svenss

Appendix A : X-Ray Structure Report

Intensity Measurements: Diffractome

A.2 Compound 2.32 Data Collection:

A.3 Compound 2.42 Data Collection:

Extended embed settings

Inappropriate

You have already flagged this document. Thank you, for helping us keep this platform clean. The editors will have a look at it as soon as possible.

Mail this publication

Delete template.

Are you sure you want to delete your template?

For this magazine there is no download available

Magazine: sample thesis title with a concise and accurate description

Save as template?

Help & Support
tuxbrain.com
ooomacros.org
nubuntu.org
Terms of service
Privacy policy
Cookie policy
Cookie settings

Choose your language

Main languages

Further languages

Bahasa Indonesia

Performing this action will revert the following features to their default settings:

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!

Have a language expert improve your writing

Run a free plagiarism check in 10 minutes, generate accurate citations for free.

Knowledge Base
How to write a descriptive essay | Example & tips

How to Write a Descriptive Essay | Example & Tips

Published on July 30, 2020 by Jack Caulfield . Revised on August 14, 2023.

A descriptive essay gives a vivid, detailed description of something—generally a place or object, but possibly something more abstract like an emotion. This type of essay , like the narrative essay , is more creative than most academic writing .

Descriptive essay topics, tips for writing descriptively, descriptive essay example, other interesting articles, frequently asked questions about descriptive essays.

When you are assigned a descriptive essay, you’ll normally be given a specific prompt or choice of prompts. They will often ask you to describe something from your own experience.

Describe a place you love to spend time in.
Describe an object that has sentimental value for you.

You might also be asked to describe something outside your own experience, in which case you’ll have to use your imagination.

Describe the experience of a soldier in the trenches of World War I.
Describe what it might be like to live on another planet.

Sometimes you’ll be asked to describe something more abstract, like an emotion.

If you’re not given a specific prompt, try to think of something you feel confident describing in detail. Think of objects and places you know well, that provoke specific feelings or sensations, and that you can describe in an interesting way.

Prevent plagiarism. Run a free check.

The key to writing an effective descriptive essay is to find ways of bringing your subject to life for the reader. You’re not limited to providing a literal description as you would be in more formal essay types.

Make use of figurative language, sensory details, and strong word choices to create a memorable description.

Use figurative language

Figurative language consists of devices like metaphor and simile that use words in non-literal ways to create a memorable effect. This is essential in a descriptive essay; it’s what gives your writing its creative edge and makes your description unique.

Take the following description of a park.

This tells us something about the place, but it’s a bit too literal and not likely to be memorable.

If we want to make the description more likely to stick in the reader’s mind, we can use some figurative language.

Here we have used a simile to compare the park to a face and the trees to facial hair. This is memorable because it’s not what the reader expects; it makes them look at the park from a different angle.

You don’t have to fill every sentence with figurative language, but using these devices in an original way at various points throughout your essay will keep the reader engaged and convey your unique perspective on your subject.

Use your senses

Another key aspect of descriptive writing is the use of sensory details. This means referring not only to what something looks like, but also to smell, sound, touch, and taste.

Obviously not all senses will apply to every subject, but it’s always a good idea to explore what’s interesting about your subject beyond just what it looks like.

Even when your subject is more abstract, you might find a way to incorporate the senses more metaphorically, as in this descriptive essay about fear.

Choose the right words

Writing descriptively involves choosing your words carefully. The use of effective adjectives is important, but so is your choice of adverbs , verbs , and even nouns.

It’s easy to end up using clichéd phrases—“cold as ice,” “free as a bird”—but try to reflect further and make more precise, original word choices. Clichés provide conventional ways of describing things, but they don’t tell the reader anything about your unique perspective on what you’re describing.

Try looking over your sentences to find places where a different word would convey your impression more precisely or vividly. Using a thesaurus can help you find alternative word choices.

My cat runs across the garden quickly and jumps onto the fence to watch it from above.
My cat crosses the garden nimbly and leaps onto the fence to survey it from above.

However, exercise care in your choices; don’t just look for the most impressive-looking synonym you can find for every word. Overuse of a thesaurus can result in ridiculous sentences like this one:

My feline perambulates the allotment proficiently and capers atop the palisade to regard it from aloft.

An example of a short descriptive essay, written in response to the prompt “Describe a place you love to spend time in,” is shown below.

Hover over different parts of the text to see how a descriptive essay works.

On Sunday afternoons I like to spend my time in the garden behind my house. The garden is narrow but long, a corridor of green extending from the back of the house, and I sit on a lawn chair at the far end to read and relax. I am in my small peaceful paradise: the shade of the tree, the feel of the grass on my feet, the gentle activity of the fish in the pond beside me.

My cat crosses the garden nimbly and leaps onto the fence to survey it from above. From his perch he can watch over his little kingdom and keep an eye on the neighbours. He does this until the barking of next door’s dog scares him from his post and he bolts for the cat flap to govern from the safety of the kitchen.

With that, I am left alone with the fish, whose whole world is the pond by my feet. The fish explore the pond every day as if for the first time, prodding and inspecting every stone. I sometimes feel the same about sitting here in the garden; I know the place better than anyone, but whenever I return I still feel compelled to pay attention to all its details and novelties—a new bird perched in the tree, the growth of the grass, and the movement of the insects it shelters…

Sitting out in the garden, I feel serene. I feel at home. And yet I always feel there is more to discover. The bounds of my garden may be small, but there is a whole world contained within it, and it is one I will never get tired of inhabiting.

If you want to know more about AI tools , college essays , or fallacies make sure to check out some of our other articles with explanations and examples or go directly to our tools!

Ad hominem fallacy
Post hoc fallacy
Appeal to authority fallacy
False cause fallacy
Sunk cost fallacy

College essays

Choosing Essay Topic
Write a College Essay
Write a Diversity Essay
College Essay Format & Structure
Comparing and Contrasting in an Essay

(AI) Tools

Grammar Checker
Paraphrasing Tool
Text Summarizer
AI Detector
Plagiarism Checker
Citation Generator

Here's why students love Scribbr's proofreading services

Discover proofreading & editing

The key difference is that a narrative essay is designed to tell a complete story, while a descriptive essay is meant to convey an intense description of a particular place, object, or concept.

Narrative and descriptive essays both allow you to write more personally and creatively than other kinds of essays , and similar writing skills can apply to both.

If you’re not given a specific prompt for your descriptive essay , think about places and objects you know well, that you can think of interesting ways to describe, or that have strong personal significance for you.

The best kind of object for a descriptive essay is one specific enough that you can describe its particular features in detail—don’t choose something too vague or general.

Cite this Scribbr article

If you want to cite this source, you can copy and paste the citation or click the “Cite this Scribbr article” button to automatically add the citation to our free Citation Generator.

Caulfield, J. (2023, August 14). How to Write a Descriptive Essay | Example & Tips. Scribbr. Retrieved September 5, 2023, from https://www.scribbr.com/academic-essay/descriptive-essay/

Is this article helpful?

Jack Caulfield

Other students also liked, how to write a narrative essay | example & tips, how to write a literary analysis essay | a step-by-step guide, how to write an expository essay.

Jack Caulfield (Scribbr Team)

Thanks for reading! Hope you found this article helpful. If anything is still unclear, or if you didn’t find what you were looking for here, leave a comment and we’ll see if we can help.

Still have questions?

What is your plagiarism score.

IMAGES

Sample thesis template34504
Sample Title Page and Spine Sept2017
Sample thesis
Thesis Examples for Android
免费 Sample Thesis Title Page
(TITLE PAGE TEMPLATE) (Thesis Title) Master’s Thesis

VIDEO

How to Write a Thesis? #thesis #shorts
Writing a Thesis Statement
What to include in your dissertation title
PhD Thesis Defense. Akshay Vishwanathan
Fixed Point Theory. Ph.D. Thesis Defense
How to Write Thesis? Thesis Writing Tips #thesis #shorts #assignment

COMMENTS

DOCX Sample Thesis Title With a Concise and Accurate Description
SAMPLE THESIS TITLE WITH A CONCISE AND ACCURATE DESCRIPTIONTHAT INCLUDES KEY WORDS AND AVOIDS USING SCIENTIFIC FORMULAS by John Doe B.A., The University of British Columbia, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES
Guide for Writing a Thesis Title
A thesis title refers to a paper's short header comprising of two parts. The first section comprises the information regarding the work's topic while the second part covers the research methods. The primary objective of a title is to capture the reader's attention while briefly describing the paper.
What Is a Thesis?
A thesis statement is a very common component of an essay, particularly in the humanities. It usually comprises 1 or 2 sentences in the introduction of your essay, and should clearly and concisely summarize the central points of your academic essay. A thesis is a long-form piece of academic writing, often taking more than a full semester to ...
PDF Sample Thesis Title Page
Sample Thesis Title Page . SELF-REFERENCE AND ENCODING SPECIFICITY . EFFECTS ON THE RECALL OF EXPOSITORY TEXT . by . Gail M. Sikking . A THESIS . Presented to the Faculty of . The Graduate College at the University of Nebraska . In Partial Fulfillment of Requirements . For the Degree of Master of Arts (or appropriate degree) Major: Educational ...
Thesis & Dissertation Title Page
Published on May 19, 2022 by Tegan George . Revised on July 18, 2023. The title page (or cover page) of your thesis, dissertation, or research paper should contain all the key information about your document. It usually includes: Dissertation or thesis title Your name The type of document (e.g., dissertation, research paper)
Sample Thesis Titles
For Students Sample Thesis Titles Completing a thesis is the capstone experience of the QMSS program. Students take this opportunity to apply the tools and methodologies developed through their coursework to questions of particular interest to them.
Choosing a Title
The title is the part of a paper that is read the most, and it is usually read first. It is, therefore, the most important element that defines the research study. With this in mind, avoid the following when creating a title: ... With these examples in mind, think about what type of subtitle reflects the overall approach to your study. This ...
PDF Sample Thesis Title With a Concise and Accurate Description
sample thesis title with a concise and accurate description that includes key words and avoids using scientific formulas by john doe b.a., the university of british columbia, 2008 a thesis submitted in partial fulfillment of the requirements for the degree of master of arts in the college of graduate studies (education)
PDF FORMAT GUIDELINES for THESES AND DISSERTATIONS
The majority of students submit an electronic version of their thesis or dissertation to the Graduate School. Electronic versions, once approved for format by the Graduate School, are ... including the dissertation title. A sample is provided on page 13. Majors are listed on page 29-30. Title Pages Title pages must be printed on white, 8 ½ x ...
DOCX Sample Thesis Title With a Concise and Accurate Description
sample thesis title with a concise and accurate description that includes key words and avoids using scientific formulas. by. jane doe. b.sc., dalhousie university, 2002. a thesis submitted in partial fulfillment of. the requirements for the degree of. master of science. in. the college of graduate studies (biology) the university of british ...
How to configure an appropriate title for your PhD thesis
A good title contains the fewest possible words needed to adequately describe the content and/or purpose of your research paper. The working title should be developed early in the research process ...
Format of theThesis
Format of the Thesis. In form, the thesis is a lengthy experimental, design, or theoretical report, ... Include the title, author, thesis supervisor, place, and date. Abstract. Briefly state the (1) research problem, (2) methodology, (3) key results, and (4) conclusion. Generally, abstracts are between 100 and 150 words--roughly 5-10 sentences.
How to Write a Thesis Statement
Placement of the thesis statement. Step 1: Start with a question. Step 2: Write your initial answer. Step 3: Develop your answer. Step 4: Refine your thesis statement. Types of thesis statements. Other interesting articles. Frequently asked questions about thesis statements.
How to Write a Great Title
Entice the reader. Find a way to pique your readers' interest, give them enough information to keep them reading. Incorporate important keywords. Consider what about your article will be most interesting to your audience: Most readers come to an article from a search engine, so take some time and include the important ones in your title ...
Dissertation & Thesis Outline
Dissertation & Thesis Outline | Example & Free Templates. Published on June 7, 2022 by Tegan George.Revised on July 18, 2023. A thesis or dissertation outline is one of the most critical early steps in your writing process.It helps you to lay out and organize your ideas and can provide you with a roadmap for deciding the specifics of your dissertation topic and showcasing its relevance to your ...
Order and Components
The title page of a thesis or dissertation must include the following information: The title of the thesis or dissertation in all capital letters and centered 2″ below the top of the page. Your name, centered 1″ below the title. Do not include titles, degrees, or identifiers. The name you use here does not need to exactly match the name on ...
How to Make a Research Paper Title with Examples
How-to-Write-a-Title-for-Your-Research-Paper How to Write a Research Paper Title Watch on What is a research paper title and why does it matter? A research paper title summarizes the aim and purpose of your research study. Making a title for your research is one of the most important decisions when writing an article to publish in journals.
PDF A Sample Thesis
Sample Thesis With a Subtitle by Michael McNeil Forbes Sc., The University of British Columbia, 1999 M.Sc., The University of British Columbia, 2001 SUBMITTED TO THE DEPARTMENT OF PHYSICS AND ASTRONOMY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Sample Thesis Title With a Concise and Accurate Description That
SAMPLE THESIS TITLE WITH A CONCISE AND ACCURATE DESCRIPTION THAT INCLUDES KEY WORDS AND AVOIDS USING SCIENTIFIC FORMULAS Nia Jennica Timoteo See Full PDF Download PDF Related Papers Dilip Datta in 24 Hours A Practical Guide for Scientific Writing JOSE EDUARDO BARRERA LOPEZ Download Free PDF View PDF Thesis review Phuc.CX4074 2004074
A List Of Twenty Interesting Thesis Paper Title Examples
Hot topics and headlines are always a great place to look for your inspiration. Your thesis should always attempt to solve a real problem and contain existing research on the topic. You want to aim for a conclusion at which no one has arrived at prior to your report. We have compiled an amazing collection of 20 thesis paper title examples that ...
sample thesis title with a concise and accurate description
sample thesis title with a concise and accurate description. EN. English Deutsch Français Español Português Italiano Român Nederlands Latina Dansk Svenska Norsk Magyar Bahasa Indonesia Türkçe Suomi Latvian Lithuanian česk ... sample thesis title with a concise and accurate description . sample thesis title with a concise and accurate ...
What Is a Thesis Statement? (Tips and Examples)
A thesis statement is a sentence written for a specific audience that conveys the purpose and topic of your paper. The thesis statement appears in the introductory paragraph and is commonly the final sentence of your introduction. A good thesis statement generates interest in the topic and makes the reader want to continue reading.
How to Write a Descriptive Essay
Published on July 30, 2020 by Jack Caulfield . Revised on August 14, 2023. A descriptive essay gives a vivid, detailed description of something—generally a place or object, but possibly something more abstract like an emotion. This type of essay, like the narrative essay, is more creative than most academic writing.