Synthesis of carboxyphosphonates and bisphosphonates as potential GGTase II and GGDPS inhibitors

Transcription

1 University of Iowa Iowa Research Online Theses and Dissertations Spring 2018 Synthesis of carboxyphosphonates and bisphosphonates as potential GGTase II and GGDPS inhibitors Robert Armin Matthiesen University of Iowa Copyright 2018 Robert Armin Matthiesen This dissertation is available at Iowa Research Online: Recommended Citation Matthiesen, Robert Armin. "Synthesis of carboxyphosphonates and bisphosphonates as potential GGTase II and GGDPS inhibitors." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Chemistry Commons

2 SYTHESIS OF CARBOXYPHOSPHOATES AD BISPHOSPHOATES AS POTETIAL GGTASE II AD GGDPS IHIBITORS by Robert Armin Matthiesen A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Chemistry in the Graduate College of The University of Iowa May 2018 Thesis Supervisor: Professor David F. Wiemer

3 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL This is to certify that the Ph.D. thesis of PH.D. THESIS Robert Armin Matthiesen has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Chemistry at the May 2018 graduation. Thesis Committee: David F. Wiemer, Thesis Supervisor James B. Gloer Sarah A. Holstein Elizabeth A. Stone Alexei V. Tivanski

4 To my Family and Friends ii

5 The greatest joy: Giving/Helping others. ever pass up an opportunity to share what you have. Dedicated leaders understand they have tremendous power to help by setting a solid example and demonstrating the highest standards. Edward A. Matthiesen Dad Advice #2 iii

6 ACKOWLEDGEMETS The journey began with my first class in chemistry with Mrs. Karen Hohenstein at Delano High School followed by receiving my chemistry major at Luther College, which led to my studies as a chemistry graduate student at the University of Iowa under the direction of Dr. David F. Wiemer. This journey started with little to no interest in chemistry, but over the years I realized that I both enjoyed it, through some dangerous backyard experiments as a teenager, and was pretty good at much of the subject matter. I am grateful for the people and educational opportunities I have found through my pursuit of chemistry, I would like to thank those who have assisted in my studies as a chemist and progression as a person. I would like to thank my Ph.D. mentor, Dr. David F. Wiemer. Dr. Wiemer accepted me into his research group when I was very unsure of what I wanted to study as a new graduate student. Over the years, I learned so much on how to conduct myself in lab and in the classroom through following his practices and listening to how he conducted himself. These traits helped me win the prestigious university teaching award along with several other teaching awards. His direction and suggestions in many of my research projects both helped me to refocus and complete much of my research. I have the upmost respect and gratitude for his patience, understanding, kindness, and his suggestions, some he would consider crazy. The result of many of our conversations through either group meetings or personal talks would always leave me feeling confident and encouraged to do science. Dr. Wiemer is what a leader should strive to be, and out of all of the bosses that I have had over the years, which were all amazing, he has been, bar none, the best. iv

7 I also must thank all of the Wiemer group members, both past and present. Deciding to conduct research under the direction of Dr. Wiemer was made easy due to the open and friendly nature of the group members. They all offered valuable chemistry knowledge, support, friendship, and time in helping with preparation for my group presentations, seminar, comprehensive exam, and final defense. I am especially thankful for Dr. Rebekah Shippy, who taught me lab techniques, corrected slides, and provided valuable knowledge about dealing with phosphorous containing compounds, and being a lab mate I dearly miss. Thanks to Dr. Veronica Wills and Dr. Xiang Zhou for guidance, providing some much needed starting material, and suggestions on many of my carboxyphosphonate and bisphosphonate compounds, with their prior work and knowledge I made many great scientific discoveries to further advance the GGDPS project. A very special thanks to Alex Rier as he provided some late stage key intermediates for synthesizing final targets, as well as all the hard work and other contributions he has made over the year. Alex has been the ideal undergraduate student and one of the hardest workers and brightest students I have worked with. I am unbelievably grateful to have been friends with Chloe Schroeder and Benjamin Foust, the times we spent playing board games or talking are always highlights to my day. I would like to thank my committee members, Dr. James B. Gloer, Dr. Sarah A. Holstein, Dr. Elizabeth A. Stone, and Dr. Alexei V. Tivanski. Dr. Gloer provided much needed guidance on several key MRs and specific MR experiments to help determine a compounds structure, along with HPLC and chromatography suggestions. Dr. Stone was an excellent resource for helping get the Chemistry Safety and Responsibility Stewards (CSaRS) up and running and assisting in the push for a greater safety culture in the chemistry v

8 department led by students. I would also like to thank Dr. Sarah A. Holstein and her research group for providing all of the biological assay data on the carboxyphosphonate and bisphosphonate compounds describe in this thesis and being able to explain all of the data in terms that a chemist could understand. Thanks to the University of Iowa Department of Chemistry Staff. These are the people that make this place such a great and easy place to work in. Thanks to Sharon Robertson and Lindsay Elliott for making sure I was ready for each step in the graduate program. Janet Kugley was the most welcoming and friendly person one could meet in the department and made me feel immediately comfortable when I first arrived. Thanks to Dr. Santhana Velupillai and his staff, they provided great suggestions for resolving difficult to interpret MR. Dr. Lynn Teesch and Vic Parcell are greatly appreciated for the work they accomplished in the mass spectrometry facility when I needed to obtain HRMS data for compounds described in this thesis. Financial support provided by IH, Roy J. Carver Charitable Trust, a Heltibride Summer Fellowship, a Graduate College Post Comprehensive Summer Fellowship, and a Strategic Initiative Funding Fellowship is greatly appreciated. Finally, I would like to thank my family and friends. To my mom and dad that have always told me, you re smart, you will succeed. Your ever loving and encouragement has never gone unappreciated, even if I don t sound like I m thankful for it. Thanks to my science friends from high school that play video games every other night, you have offered a place for me to unwind and get outside advice, especially my brother John. Finally thanks to my girlfriend Rebecca Ritter, your encouragement and willingness to just listen has made many of my bad days better and my good days great. I would not be here without all of your support and advice, thank you for helping me make it through graduate school! vi

9 ABSTRACT Inhibition of enzymes in the isoprenoid biosynthetic pathway (IBP) plays an important role in the treatment of bone diseases and hyperlipidemia. The IBP begins with the enzyme HMG-CoA reductase catalyzing the conversion of 3-hydroxy-3- methylglutaryl-coa (HMG-CoA) to mevalonic acid. Mevalonic acid is then converted to isopentenyl pyrophosphate (IPP) via the intermediate mevalonate-5-diphosphate. Three molecules of IPP are joined by the enzyme farnesyl diphosphate synthase (FDPS), which yields the intermediate farnesyl pyrophosphate (FPP). FPP is an important substrate and represents the branch point in the pathway. Further downstream from FDPS the IBP includes the key enzyme geranylgeranyl diphosphate synthase (GGDPS), which is responsible for the production of geranylgeranyl pyrophosphate (GGPP) and necessary for protein prenylation of the proteins Ras, Rho, and Rab. Compounds which disrupt this pathway at FDPS include risedronate and zoledronate. It is believed that these compounds express their pharmacological effects by impacting geranylgeranylated proteins, particularly Rab proteins in osteoclasts. Geranylgeranyl transferase II (GGTase II) is responsible for the transfer of GGPP to Rab proteins. The Wiemer and Holstein research groups have been interested in targeting Rab proteins as a novel therapeutic strategy for treatment of multiple myeloma. Rab proteins regulate many aspects of intracellular trafficking processes. In myeloma cells, disrupting Rab protein function by inhibiting Rab geranylgeranylation results in the disruption of monoclonal protein secretion, which leads to myeloma cell death. vii

10 Rab geranylgeranylation can be targeted through two different strategies. The first would be an indirect route, which would aim to inhibit the enzymes upstream from GGTase II. This could include the enzymes HMG-CoA reductase, FDPS, and GGDPS. Drugs that inhibit these earlier steps in the IBP indirectly prevent protein geranylgeranylation by depleting cells of GGPP, but they also limit formation of other key intermediates so it would be preferable to disrupt the pathway as far downstream as possible (i.e. GGDPS). The second approach would be direct inhibition of the enzyme responsible for protein prenylation, GGTase II. There are very few known inhibitors of GGTase II. One such inhibitor is the carboxyphosphonate 3-PEHPC, a mimic of the bisphosphonate risedronate. Unfortunately, 3-PEHPC is not sufficiently potent to allow for its use clinically. Our efforts to develop a more potent inhibitor of the enzyme GGTase II began with a focus on focus on preparation of a family of carboxyphosphonates containing a triazole core, and these compounds were prepared via click chemistry. Their activity has been studied, but the salts that were successfully made were ultimately inactive in comparison to 3-PEHPC. Focus then was switched to alteration of isoprenoid bisphosphonate triazoles as GGDPS inhibitors in attempts to increase activity against this enzyme. The biological activity of these bisphosphonates was found to be selective and potent inhibitors of GGDPS, with little to no activity towards the GGTase II or FDPS enzymes. A family of isoprenoid bisphosphonate compounds was synthesized in an effort to increase the biological activity against GGDPS. These bisphosphonates were prepared through click chemistry and tested for their activity against GGTase II and GGDPS. Of the new compounds synthesized it was found that a bisphosphonate head group is an important feature for potent inhibitors of GGDPS. Modifications of the viii

11 bisphosphonate head group yielded promising results, through the installation of a prodrug, which resulted in the most potent triazole bisphosphonate to date with an IC50 of ~5 nm. Methylation of the α-carbon also increased activity of the bisphosphonate triazoles when the alpha methylated compounds were compared to their non-methylated counterparts. Additionally, while a mixture of olefin isomers is more potent than the individual olefin isomers in the non-methylated series, reflecting a synergistic effect, in the methylated series the individual olefin isomers are more potent than the mixture. This is very attractive from the standpoint of drug design. ix

12 PUBLIC ABSTRACT Inhibitors of enzymes in the isoprenoid biosynthetic pathway (IBP), such as the prescription drugs Actonel (risedronate) and Zometa (zoledronate), are clinically used for treatment of osteoporosis and bone diseases, such as Paget s disease. They also are commonly used for patients with multiple myeloma. These drugs inhibit early steps in the IBP, but are believed to express their pharmacological effects further downstream by preventing protein geranylgeranylation via the enzyme GGTase II. Inhibition of the enzyme GGTase II is an attractive therapeutic target as it would stop the trafficking and secretion of monoclonal proteins, especially for multiple myeloma, which is characterized by the over-production of antibodies. However, if these compounds do ultimately prevent protein geranylgeranylation they also inhibit other functions in the IBP, which may give unwanted side effects. An alternative approach would be to directly target the enzyme responsible for protein geranylgeranylation, GGTase II. My research aimed at the synthesis of novel carboxyphosphonates and bisphosphonates, and their biological activity was tested by our collaborators. Previous efforts to synthesize both carboxyphosphonates and bisphosphonates have been successful to a certain degree, and some have been effective inhibitors of the key enzyme geranylgeranyl diphosphate synthase. My goal has been to make structural modifications of these compounds in three different regions, and then provide them to our collaborators so that the biological activity of these compounds could be studied. Modification of one of the three sites, the bisphosphonate head group, resulted in the most potent triazole bisphosphonate to date by installation of a prodrug. A methylated series of bisphosphonates x

13 were found to be more potent inhibitors of the enzyme geranylgeranyl diphosphate synthase, in comparison to their non-methylated counterparts. Additionally, while a mixture of olefin isomers is more potent than the individual olefin isomers in the nonmethylated series, reflecting a synergistic effect, in the methylated series the individual olefin isomers are more potent than the mixture. These discoveries offer greater insight into the sensitivity of the general molecule to minor modifications and might have potential value in drug development. The details of the synthesis of several novel carboxyphosphonates and bisphosphonates, and their biological activities, will be discussed. xi

14 TABLE OF COTETS LIST OF TABLES... xiii LIST OF FIGURES... xiv LIST OF ABBREVIATIOS... xxii CHAPTERS 1. A BRIEF ITRODUCTIO OF IHIBITORS OF THE ISOPREOID BIOSYTHETIC PATHWAY SYTHESIS AD BIOLOGICAL ACTIVITY OF CARBOXYPHOSPHOATES SYTHESIS AD BIOLOGICAL ACTIVITY OF HOMOISOPREOID TRIAZOLE BISPHOSPHOATES SYTHETIC STRATEGIES TOWARDS A ISOPREE TRIAZOLE TETRA POM BISPHOSPHOATE SYTHESIS AD BIOLOGICAL ACTIVITY OF METHYLATED BISPHOSPHOATES SUMMARY AD FUTURE DIRECTIOS EXPERIMETAL PROCEDURES APPEDIX: SELECTED MR SPECTRA REFERECES xii

15 LIST OF TABLES Table 1. Conditions examined to accomplish hydrolysis to the carboxyphosphonate salt or acid Table 2. Impact of isoprene length on bisphosphonate activity Table 3. Inhibition of GGDPS and FDPS with homoisoprene triazole bisphosphonates Table 4. Base-mediated POM conditions for farnesyl triazole bisphosphonate Table 5. Base-mediated POM conditions for geranyl triazole bisphosphonate Table 6. Conditions to obtain a tetra POM species from compound Table 7. Inhibitory effects of novel triazole bisphosphonates on prenyl synthase xiii

16 LIST OF FIGURES Figure 1. Depiction of multiple myeloma in bone marrow... 4 Figure 2. Geminal bisphosphonate and pyrophosphate core structure... 6 Figure 3. List of clinically used bisphosphonates... 7 Figure 4. Isoprenoid biosynthetic pathway in mammals... 8 Figure 5. HMG-CoA reductase inhibitors Figure 6. on-nitrogenous bisphosphonates, nitrogenous bisphosphonates and carboxyphosphonates Figure 7. General triazole bisphosphonate/ carboxyphosphonate structure Figure 8. Carboxyphosphonate GGTase II inhibitors and bisphosphonate parent compounds Figure 9. Derivatives of 3-PEHPC Figure 10. Retrosynthesis of isoprene triazole carboxyphosphonate sodium salts Figure 11. Initial carboxyphosphonate triazoles as phenyl and naphthyl Figure 12. Attempted hydrolysis methods towards active acid or salt Figure 13. Synthesis of click reaction precursors Figure 14. Formation of carboxyphosphonate triazoles as the sodium salt Figure 15. Allylic azide rearrangement xiv

17 Figure 16. Click reaction of isoprene epoxides Figure 17. Formation of single olefin isomer triazoles Figure 18. Different isoprene chain lengths on a bisphosphonate Figure 19. Reverse click precursors and general product Figure 20. Formation of isoprene alkynes Figure 21. Reverse triazole click reactions through the use of sonication Figure 22. Assayed isoprenoid triazole carboxyphosphonates Figure 23. Effects of carboxyphosphonate disruption in protein geranylgeranylation of Rap1a Figure 24. Effects of carboxyphosphonates inducing accumulation of intracellular light chain Figure 25. Synthesis of Compound 8 (VSW-1198) Figure 26. Comparison of geranyl and neryl isoprene chains Figure 27. Preparation of homonerol Figure 28. Preparation of homogeraniol Figure 29. Formation of homoallylic azides Figure 30. Click reaction and hydrolysis to homoisoprene triazoles Figure 31. Comparison of mix 96 and homoneryl Figure 32. Illustration of the prodrug concept xv

18 Figure 33. Phosphoantigen prodrugs Figure 34. Activation of POM prodrug on phosphoantigen Figure 35. General route for synthesis of a tetra POM bisphosphonate Figure 36. Base mediated method to access POM phosphonate prodrug Figure 37. Attempted conversion of a tetraethyl bisphosphonate to a tetra-pom bisphosphonate triazole Figure 38. Attempted conversion of a tetramethyl bisphosphonate to a tetra POM bisphosphonate with geranyl chain Figure 39. Attempted conversion to tetra POM bisphosphonate from compound Figure 40. Attempted preparation of a triazole bisphosphonate prodrug Figure 41. Formation of the triazole bisphosphonate prodrug Figure 42. Tetra POM triazole Figure 43. Previously synthesized triazole bisphosphonates as the sodium salts Figure 44. Preparation of methylated triazole bisphosphonates Figure 45. Preparation of homoisoprene methylated triazole bisphosphonates Figure 46. Comparison of the effects of the -methylated triazole bisphosphonates 155 and 161 to the non-methylated analogues 96 and 95 on protein geranylgeranylation Figure 47. Effects of olefin stereochemistry on activity of the -methylated triazole bisphosphonates Figure 48. Synthesis of ethylated bisphosphonate derivative xvi

19 Figure 49. Cellular activity of an α-ethylated triazole bisphosphonate Figure 50. Compound list of tested triazole bisphosphonates Figure 52. Alternative routes to hetero linked bisphosphonates Figure 53. The 1,4-substitued product and the 1,5-substitued product Figure 54. Attempts towards 1,5-substutued triazole bisphosphonates Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound xvii

20 Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound xviii

21 Figure A H MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound xix

22 Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound xx

23 Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound Figure A H MR spectrum of compound Figure A C MR spectrum of compound xxi

24 LIST OF ABBREVIATIOS Å Ac AcOH ATP AC br brsm C calcd d DBU DCM DGBP DMAPP DMF dt EC50 EI ELISA ESI Et Angstrom acetate acetic acid adenosine triphosphate acetonitrile broad (MR) based on recovered starting material celsius calculated doublet (MR) 1,8-Diazabicyclo[5.4.0]undec-7-ene dichloromethane digeranyl bisphosphonate dimethylallyl pyrophosphate dimethylformamide doublet of triplets (MR) half maximal effective concentration electron impact enzyme-linked immunosorbent assay electrospray ionization ethyl xxii

25 Et2O EtOAc FDPS FPP FPPS FTase g GGDPS GGPP diethyl ether ethyl acetate farnesyl disphosphate synthase farnesyl pyrophosphate farnesyl pyrophosphate synthase farnesyl transferase gram geranylgeranyl diphosphate synthase geranylgeranyl pyrophosphate GGTase I geranylgeranyl transferase type 1 GGTase II geranylgeranyl transferase type 2 HMG-CoA HG HMPA H HPLC HRMS Hz IBP IC50 IPP J KHMDS 3-hydroxy-3-methylglutaryl coenzyme A homogeranyl hexamethylphosphoramide homoneryl high-performance liquid chromatography high resolution mass spectrometry hertz isoprenoid biosynthetic pathway concentration for 50% enzyme inhibition isoprenoid biosynthetic pathway coupling constant (MR) potassium hexamthyldisilazane xxiii

26 LCMS LDA M m Me mg min MHz MK ml liquid chromatography mass spectrometry lithium diisopropylamide molar multiplet (MR) methyl milligram minute megahertz mevalonate kinase milliliter µm micromolar mm mmol MM Ms m/z BS n BuLi nm MP MR PMK millimolar millimole multiple myeloma methanesulfonyl mass/charge ratio normal bromosuccinimide n butyllithium nanomolar -methyl-2-pyrrolidone nuclear magnetic resonance phosphomevalonate kinase xxiv

27 POM ppm q Rf RPMI-8226 rt s SQS t TBAF TBHP t BuOH TEA TFA TFAA THF TLC TMS TsOH tt UV δ pivaloyloxymethyl parts per million quartet (MR) retardation factor human-derived myeloma cell line room temperature singlet (MR) squalene synthase triplet (MR) tetrabutylammonium fluoride tert butyl hydroperoxide tert butanol triethylamine trifluoroacetic acid trifluoroacetic anhydride tetrahydrofuran thin layer chromatography trimethylsilyl p toluene sulfonic acid triplet of triplets (MR) ultraviolet chemical shift (MR) xxv

28 CHAPTER 1 A BRIEF ITRODUCTIO OF IHIBITORS OF THE ISOPREOID BIOSYTHETIC PATHWAY Bone diseases are conditions and disorders that make bones weaker, increase the likelihood of fractures, cause abnormal development, and/or inhibit/impair normal bone development. 1,2 Many of these problems occur before birth, resulting from genetic abnormalities and other defects, but more well-known bone disease also occur later in life. 3 There are many disorders of the bone including osteoporosis, Paget s disease of bone, and multiple myeloma. This only scratches the surface of the vast number and different types of bone diseases. After about the age of 20, a typical human will naturally lose bone density due to aging, but this can also occur at a great rate or be more prominent in those that have nutrient deficiencies, hormonal imbalance, or cell abnormalities. 1,3 Osteoporosis is characterized by low bone mass, due to the body losing too much bone, making too little bone, or both, resulting in weak bone, which can ultimately result in breaks from falls or even minor bump. 4 The World Health Organization classifies osteoporosis as having a standardized score of bone mineral density 2.5 standard deviation units below the mean for young healthy women, and severe osteoporosis at the same rate but with a history of a fracture. 3,5 Osteoporosis is a fairly common bone disease as it affects roughly 200 million women worldwide, and it is typically found in both men and women over the age of 50. 4,6 Though common in both sexes, osteoporosis affects approximately one in every two women compared to about one in every 12 men. 7 But why are women so much more at risk than men? As stated earlier hormones play a role in bone development, 1

29 and estrogen will decrease rapidly when a women reaches menopause along with women having smaller bones in comparison to men. 7 ot only that but women will start with a lower bone density and will lose bone mass much more quickly than their male counterpart. In fact women will on average, after the age of 18, lose roughly one third of their hip bone mass in comparison to men losing, on average, one fourth of their bone mass. 6 This does not mean men are unlikely to suffer from osteoporosis, only that the incidence is higher in women. Another bone disease is a branch of osteoporosis, termed as osteitis deformans, known as Paget s disease of the bone and named after Sir James Paget. 8 Paget s disease is localized bone-remodeling that will affect noncontiguous areas of the skeleton due to a disorder of osteoblast cells, which are in charge of breaking down, rebuilding and remolding bone tissue thus causing weakened, misshapen, and/or fractured bones. 1,9 Unlike osteoporosis, which can affect all of the bones in the skeleton, Paget s disease is most commonly found in the pelvis (70% of cases) but also is found in the femur (55%), Lumbar spine (53%), skull (42%), and tibia (32%). 10,11 Though there is not a cure for Paget s disease, bisphosphonate medication is known to lessen the pain and control the disorder, but the medications will be discussed later. Unlike osteoporosis, Paget s disease affects a much smaller percent of the populations, about 8%, and is found more commonly in males than females. 11 However, similar to osteoporosis, Paget s disease is found to be rare in the younger population, and is more commonly found in patients who are over the age of What is quite interesting is that Paget s disease is more commonly found in patients of European descent and is rare in those from the Indian subcontinent, Asia, and Africa. 12,13 This suggests that the disease 2

30 has a higher propensity to affect descendants of patients, but it is also known that this disease is associated with a viral infection, paramyxoviridae. Finally evidence also shows that air pollution can play a vital role in the development of Paget s disease. 11,14 Fortunately, over the years, Paget s disease has been on a decline due to improved nutrition, western medicine, and an epidemiological understanding of the importance of reducing infection. 15 Cancer is the uncontrolled growth of abnormal cells that can spread to nearly any part of the body and crowd normal cells. If not controlled, it can result in death. 16 The American Cancer Society has stated that more than 15.5 million Americans were alive with a history of cancer on January 1, 2016 and an estimated 16.9 million new cancer cases are expected to be diagnosed in An even more sobering fact is that about 600,920 American are expected to pass away in 2017 from cancer. 17 Multiple myeloma is a cancer formed in plasma cells in the bone marrow. These plasma cells produce abnormal clonal antibodies, or monoclonal protein. (Figure 1). 18 Because monoclonal proteins offer no significant benefit to the body, the vast increase in them ultimately crowds out normal functioning immunoglobulins and can lead to blood thickening and kidney problems. 19 3

31 Figure 1. Depiction of multiple myeloma in bone marrow Several features are characteristic of multiple myeloma. Low blood counts, due to plasma cells overcrowding the bone marrow can lead to increased risks of bleeding and infection.. 16 Bone problems are common in the form of fractures and breaks, due to the osteoclasts reacting to a substance myeloma cells make, which will signal breaking down bones faster than osteoblasts can make new bone. This can also be seen in an increased calcium level in the blood. 16,20 Finally, the antibodies, or monoclonal proteins, made by the malignant plasma cells can cause kidney damage and even kidney failure. 21 Multiple myeloma globally affects roughly 500,000 people and was the cause of over 100,000 deaths in 2015, up from 50,000 in ,22,23 Men have a higher risk of being affected by multiple myeloma than women, by an approximate ratio of 1.54 to

32 Similarly to previous mentioned diseases, multiple myeloma is more common in individuals of the African American and ative Pacific Islander race, and is typically more prominent in patients at older ages, roughly above the age of Those diagnosed with multiple myeloma who undergo treatment typically live for another 10 years, but those who don t seek treatment typically live for about seven months. 25 Treatment for these bone diseases is commonly done through a three tier system. The base, or first tier, relies on maintaining bone health and severely reducing the likelihood of a bone fracture. This can be done in a plethora of ways. Healthy bones can be maintained by meeting the recommend daily intake of calcium and vitamin D, which is responsible for mineralization of bone (bone hardening). 26 Physical activity can help maintain and increase bone mass. 3 Finally, fall prevention will greatly reduce the risk of a bone fracture through recognizing environmental problems that lead to a fall, which can be done through actions such as installation of night lights or a grab bar in the shower. 3 The second tier is simply just assessing the secondary factors that may cause a bone disease like multiple myeloma or osteoporosis. Finally, the third tier is pharmacotherapy through the use of antiresorptives and/or anabolic agents. Bisphosphonates exhibit their ability to inhibit bone resorption through their influence on osteoclasts either directly, through intracellular uptake or cell binding, or indirectly through interactions with other cells. 27 Geminal bisphosphonates consist of a P C P structure and are direct analogs of pyrophosphates, which contain an oxygen instead of a carbon as the core (Figure 2). Because geminal bisphosphonates in almost all cases project strong activity towards bone diseases, they will simply be referred to as bisphosphonates. 5

33 O O P O R 1 C R 2 O P O O Geminal bisphosphonate (bisphosphonate) O O O P O P O O O Pyrophosphate Figure 2. Geminal bisphosphonate and pyrophosphate core structure Bisphosphonates mechanism of action is through their ability to mimic the pyrophosphate structure, thus inhibiting enzymes that employ pyrophosphates. 27 Because bisphosphates have two phosphonate groups, they have a high affinity for metal ions such as magnesium and calcium, and because calcium is abundant in bone, they typically accumulate in high concentration in bones when the drug is administered. 28 That being said, there are two types of bisphosphonates utilized for bone disease treatment: non-nitrogenous bisphosphonates and nitrogenous bisphosphonates (Figure 3). on-nitrogenous bisphosphonates like etidronate, clodronate, and tiludronate are direct mimics of pyrophosphate and as such can replace the terminal pyrophosphate of adenosine triphosphate (ATP), thus creating a molecule that is ultimately non-functional, but that competes with ATP as a non-hydrolyzable analogue. 27 This new ATP analog inhibits cellular energy metabolism in osteoclasts initiating apoptosis and leading to programed cell death. Unfortunately, non-nitrogenous bisphosphonates have more negative effects in comparison to their nitrogenous bisphosphonate counterparts, along with overall having a lower potency to inhibit bone resorption. Thus, they are much less likely to be prescribed. 29 6

34 on-nitrogenous bisphosphonates H 2 itrogenous bisphosphonates HO HO P O OH OH P OH O Etidronate (Didronel ) HO HO P O OH OH P OH O H 2 HO HO P O OH OH P OH O HO HO P O OH OH P OH O Cl HO P HO O Cl P O Clodronate (Bonefos ) OH OH eridronate (erixia ) H 2 Pamidronate (Aredia ) Ibandronate (Boniva ) HO HO P O S P O OH OH Cl HO HO P O OH OH P OH O Aledronate (Fosamax ) HO HO P O OH OH P OH O Olpadronate HO HO P O OH OH P OH O Risedronate (Actonel ) Tiludronate (Skelid ) HO HO P O OH OH P OH O HO HO P O OH OH P OH O Zoledronate (Zometa ) Minodronate Figure 3. List of clinically used bisphosphonates In the other vein are the nitrogenous bisphosphonates. These compounds are inhibitors of the mevalonate pathway due to their ability to dock into the pyrophosphate binding site of the enzyme farnesyl pyrophosphate synthase (FDPS). 30 Binding inhibits the prenylation of geranylgeranyl proteins, (Rab, RhoA, and Rap1a) in osteoclasts and disrupts them. But to understand the overall effect of the nitrogenous bisphosphonates, it is 7

35 important to understand the mevalonate pathway or isoprenoid biosynthetic pathway (IBP, Figure 4). HMG-CoA HMG-CoA Reductase Mevalonic Acid 5 C isoprene unit Statins Enzyme IPP Inhibitor BP FDPS Cholesterol Squalene SQS FPP FTase F proteins (Ras, RhoB) Farnesyl BP GGDPS FTI DGBP GGPP GGTase I GGTI GG proteins (RhoA, Rap1a) GGTase II GG proteins (Rab) Figure 4. Isoprenoid biosynthetic pathway in mammals The IBP begins with production of mevalonic acid from 3-hydoxy-3- methylglutaryl-coa (HMG-CoA) through the enzyme HMG-CoA reductase, which is the rate limiting enzyme of this pathway. A phosphorylation reaction of mevalonic acid to mevalonate-5-phosphate is catalyzed by mevalonate kinase (MK) and ATP is used to transfer a phosphate group. Mevalonate-5-phosphate undergoes a reaction catalyzed by phosphomevalonate kinase (PMK) that requires ATP to yield mevalonate-5-diphosphate. Mevalonate-5-diphosphate undergoes a decarboxylation reaction to yield the five carbon compound 3-isopentenyl pyrophosphate (IPP). The next reaction is catalyzed by the 8

36 enzyme farnesyl diphosphate synthase (FDPS) where the addition of two molecules of IPP to a molecule of dimethylallyl pyrophosphate (DMAPP) results in formation of the 15- carbon compound farnesyl pyrophosphate (FPP). This represents a key point of divergence in the pathway. The enzyme squalene synthase (SQS) can catalyze dimerization of FPP to begin the production of squalene, which ultimately can be converted to cholesterol through a multitude of steps. 31 Another use of FPP is the production of the farnesylated-proteins, which include Ras and RhoB, through the action of the enzyme farnesyl transferase (FTase). Further downstream, IPP can be added to FPP in a process catalyzed by the enzyme geranylgeranyl diphosphate synthase (GGDPS) to yield the 20-carbon intermediate geranylgeranyl diphosphate (GGPP). 31,32 GGPP can be added to a protein either by the enzyme geranylgeranyl transferase I (GGTase I), which modifies proteins such as RhoA and Rap1a, or serve as a substrate for the enzyme geranylgeranyl transferase II (GGTase II) to give another set of G-proteins, including the Rabs. 33 These G-proteins are important in intracellular signaling once they become isoprenylated, which has a great impact on cytoskeletal formation which is responsible for maintaining the contact between osteoclasts and the bone surface and cell growth regulation. 31,34,35 Because there are several key enzymes along the IBP and they ultimately play an important role in metabolism, they are several attractive therapeutic targets. Statins are HMG-CoA reductase inhibitors that are ultimately used to lower cholesterol levels. Examples include lovastatin, atorvastatin, and mevastatin (Figure 5). However, because they are so far upstream in the IBP, they also reduce the synthesis of FPP and GGPP and thus express an effect on limiting protein prenylation. 36 However, studies have shown that statins increase bone mineral density, reducing a user s rate of fracture. 9

37 HO O O OH OH O HO O O O H OH O O O H O H Lovastatin (Mevcor ) Atorvastatin (Lipitor ) F Mevastatin (Compactin ) Figure 5. HMG-CoA reductase inhibitors The nitrogenous bisphosphonates mentioned earlier, including risedronate and zoledronate, are drugs that function as FDPS inhibitors. 37,38 By inhibiting the enzyme FDPS these drugs will express activity further downstream in the IBP by depleting cells of both FPP and GGPP. However, these compounds may be exhibiting their true effects due to depletion of GGPP as it has been shown that farnesylation is not critical to bone resorption but has a strong correlation with geranylgeranylation. 39 Therefore, to avoid unnecessary cellular effects, it would be advantageous to develop an enzyme inhibitor that targets downstream of FDPS to ultimately inhibit protein geranylgeranylation. The Wiemer group has prepared a number of non-nitrogenous bisphosphonates, 40,41 nitrogenous bisphosphonates, 42 and carboxyphosphonates 43 as shown in Figure 6. The non-nitrogenous bisphosphonates were found to be inhibitors of GGDPS, where the compound digeranyl bisphosphonate (1, DGBP) garnered a GGDPS IC50 value of 0.2 µm. 41 What might be just as interesting, most non-nitrogenous bisphosphonates, including etidronate, clodronate, and tiludronate, work by creating non-functional ATP analogues that compete with ATP making the molecule nonhydrolyzable and initiating apoptosis. Interestingly, these aren t 10

38 inhibitors of enzymes in the IBP, while DGBP, a non-nitrogenous bisphosphonate is selective towards a IBP enzyme GGDPS. 44 H (ao) 2 (O)P P(O)(Oa) (ao) 2 (O)P P(O)(Oa) 2 (HO) 2 (O)P HOOC OH COOH P(O)(OH) PEHPC OH COOH P(O)(OH) 2 4 (+)-(S)-3-IPEHPC 5 (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P Figure 6. on-nitrogenous bisphosphonates, nitrogenous bisphosphonates and carboxyphosphonates As mentioned previously, protein prenylation is in part facilitated by the enzyme GGTase II, which plays a critical role in intracellular membrane trafficking by geranylgeranylation of Rab proteins. 45 This could be an attractive strategy for treatment of diseases like multiple myeloma that are highly secretory. This direct approach is more attractive than the previous designed drugs that operate through an indirect approach to express their pharmacological effects by inhibiting further upstream from GGTase II. Currently there are few known inhibitors of the enzyme GGTase II. Known inhibitors include 3-PEHPC (3) 46 and (+)-(S)-3-IPEHPC (4) 47 (Figure 6), which are mimics of the bisphosphonates risedronate and minodronate, respectively. The main difference is the polar head group, one being a carboxyphosphonate and the other a bisphosphonate. The bisphosphonate is almost always a selective inhibitor of FDPS while their carboxyphosphonate counterparts are selective inhibitors of GGTase II. Unfortunately, the 11

39 carboxyphosphonate compounds are not very potent inhibitors and a millimolar concentration is required in enzyme assays for inhibition of Rab geranylgeranylation. 46 The Wiemer group has prepared a library of triazole bisphosphonates and a few carboxyphosphonates such as compounds ,49,50 Their synthesis and biological activity will be discussed in later chapters. The Wiemer group also has attempted to synthesize new GGTase II inhibitors ranging from 3-PEHPC mimics to triazole carboxyphosphonates and bisphosphonate compounds with a hydrophobic tail as isoprene mimics. Triazole carboxyphosphonates and bisphosphonates are attractive due to ease of compound modification, and are based on analysis of the active site of GGTase II bound with a GGPP molecule. 51 Three sites were found for binding: 1) the polar head group; 2) a Zn binding site; and 3) an isoprene binding pocket. The triazole compounds can be broken into three sections (Figure 7). Modification can begin at the pyrophosphate mimic as the bisphosphonate head group (pink). The head group can also be a carboxyphosphonate, an α hydroxy phosphonate, or any other α substituted group. The work in the following chapters describes the different effects certain modifications at this point generate. Another point of modification is the first tether group (red), which arises from a click reaction between an acetylene and an azide, and the length of this tether could be modified. In previous research, the nitrogen-containing heterocycle, a triazole group, proved to be a promising Zn-binding group 52 that, as previously stated, could arise from a click reaction. The final modification that can be made would be the hydrophobic isoprene tail (blue), exploring the best possible length and shape to occupy the enzyme pocket. Work exploring the neryl and geranyl analogues along with modification at the internal olefin will be discussed here. The synthesis and biological 12

40 activity of these triazole carboxyphosphonate and bisphosphonate compounds as potential GGDPS and GGTase II inhibitors will be described in Chapters 2 5. (HO) 2 (O)P (HO) 2 (O)P zinc binding pyrophosphate group mimic tether 1 R m n tether 2 terminal isoprenoid (or mimic) pyrophosphate mimics Reactive acetylene and tether one Origin of isoprenoid (or mimicing) tail (including tether two) (HO) 2 (O)P (HO) 2 (O)P R (HO) 2 (O)P HOOC R Br (or R) HO (HO) 2 (O)P R R = H, OH, alkyl O-alkyl Br Br Br (CH 2 ) 4 Br Br Br Figure 7. General triazole bisphosphonate/ carboxyphosphonate structure 13

41 CHAPTER 2 SYTHESIS AD BIOLOGICAL ACTIVITY OF CARBOXYPHOSPHOATES As discussed in the previous chapter, compounds that inhibit various enzymes along the isoprenoid biosynthetic pathway (IBP) include the statins, nitrogenous bisphosphonates, digeranyl bisphosphonate (DGBP, and its analogues), and carboxyphosphonates, and some are used clinically for the treatment of bone diseases. 53,54 Inhibition of enzymes in the IBP will lead to the disruption in protein post-translational processing further downstream in the pathways. Therefore compounds like lovastatin, risedronate, and DGBP will ultimately lead to the indirect inhibition of the enzyme geranylgeranyl transferase II (GGTase II), which is responsible for the transfer of geranylgeranyl chains from geranylgeranyl pyrophosphate (GGPP) to the Rab proteins. Because the Rab proteins are important for intracellular membrane trafficking and protein secretion, they are a good target for treatment of diseases like multiple myeloma, which are characterized by over-secretion of monoclonal proteins. 55 By inhibiting Rab geranylgeranylation, it is possible to diminish protein trafficking and secretion, causing a buildup of monoclonal proteins and ultimately leading to apoptosis of the myeloma cells. 56 This is an indirect approach and might result in disruption of other cellular processes in the IBP upstream of GGTase II, such as the production of cholesterol. Therefore, a direct approach to inhibiting GGTase II would be most desirable. However, GGTase II inhibitors are both rare and suffer from low enzymatic activity. Two inhibitors of GGTase II previously described are 3-PEHPC and (+)-(S)-3-IPEHPC (Figure 8), where 3-PEHPC shows an IC50 of 600 µm and (+)-(S)-3-IPEHPC was roughly 25 times more potent. 46,47 14

42 Both are carboxyphosphonate mimics of nitrogenous bisphosphonates, risedronate and minodronate, respectively. OH COOH OH P(O)(OH) 2 OH COOH OH P(O)(OH) 2 P(O)(OH) PEHPC P(O)(OH) 2 risedronate P(O)(OH) 2 4 (+)-(S)-3-IPEHPC minodronate P(O)(OH) 2 Figure 8. Carboxyphosphonate GGTase II inhibitors and bisphosphonate parent compounds Previously, the Wiemer group has synthesized analogues of 3-PEHPC such as the -oxide derivatives, and several carboxyphosphonate and bisphosphonate triazoles as potential direct inhibitors of GGTase II. The derivatives of 3-PEHPC, including the - oxides, were originally synthesized by Dr. Xiang Zhou 57 (Figure 9). These compounds were tested for biological activity by Dr. Sarah A. Holstein in an enzyme assay with GGTase II. 43 However, these -oxide carboxyphosphonate derivatives of 3-PEHPC showed no improvement in potency when compared to 3-PEHPC. In fact, compounds 9 12 did not potently inhibit GGTase II or the enzyme more commonly inhibited by bisphosphonates, FDPS. This is both good and bad for future development of inhibitors of GGTase II. It is unfortunate that close mimics of 3-PEHPC could not more potently inhibit the enzyme GGTase II, but as a carboxyphosphonate it helps confirm the theory that with this head group it is possible to avoid the inhibition of further upstream enzymes. 15

43 OH COOH OH COOH COOH COOH COOa P(O)(OH) PEHPC O 9 P(O)(OH) PEPC P(O)(OH) 2 P(O)(OH) 2 P(O)(Oa) 2 O 11 O 12 Figure 9. Derivatives of 3-PEHPC Again, utilizing known information for development of a compound as a potential inhibitor of GGTase II, our approach was to analyze the crystal structure of GGPP bound to the enzyme GGTase II. Development of an inhibitor would include different sites to satisfy several different important requirements, a pyrophosphate mimic to bind the polar head group of GGPP, an isoprene chain to occupy the isoprene pocket, and a Zn binding site. More modifications could be made to the general compound to afford more potent inhibitors. This directed the Wiemer group to the general triazole structures shown in Figure 7. These compounds would be 3-PEHPC mimics with the carboxyphosphonate head group, an isoprene chain to occupy the pocket that holds the distal olefin of GGPP s C-20 chain, and a triazole core as the potential Zn binding site. Retrosynthetically, the isoprene triazole carboxyphosphonate sodium salt 13, would be obtained via hydrolysis of the corresponding carboxyphosphonate ester 14 (Figure 10). 57,58 The triazole would arise in turn from a click reaction of an isoprenoid azide 16 and an acetylene carboxyphosphonate 15. The azides can be prepared from their corresponding bromides, produced in turn by reaction of the alcohol 18. Acetylene 15 can be obtained from carboxyphosphonate 17 via condensation with formaldehyde, followed by dehydration and conjugate addition of an acetylide. 59,60 16

44 (ao) 2 (O)P aooc R (EtO) 2 (O)P EtOOC R R = Isoprene Chain (EtO) 2 (O)P EtOOC 15 R 3 16 (EtO) 2 (O)P EtOOC 17 ROH 18 Figure 10. Retrosynthesis of isoprene triazole carboxyphosphonate sodium salts Originally, Dr. Zhou had synthesized a pair of carboxyphosphonate esters compounds 21 and 22 as shown in Figure 11, which were hydrolyzed using concentrated HCl. However, these compounds showed little to no activity as inhibitors of the GGTase II enzyme or with FDPS. This might be due in part to the possibility that the phenyl and naphthyl groups were unable to successfully occupy the pocket that holds the distal olefin of GGPP s C-20 chain. To synthesize a compound that falls closer in line with the general triazole structure, an isoprene tail would have to be incorporated. Dr. Zhou was successful in synthesizing a few isoprene carboxyphosphonate triazoles as the esters, but when subjected to a plethora of hydrolysis conditions, the desired compound as the fully hydrolyzed carboxyphosphonate could not be isolated. 17

45 (EtO) 2 (O)P (EtO) 2 (O)P EtOOC EtOOC HCl, reflux HCl, reflux (HO) 2 (O)P HOOC 21 (HO) 2 (O)P HOOC 22 Figure 11. Initial carboxyphosphonate triazoles as phenyl and naphthyl The multitude of hydrolysis methods that Dr. Zhou examined can be seen in Figure 12, starting with attempted hydrolysis through similar conditions that were used to convert esters 19 and 20 to their corresponding acids. However, when compound 23 was subjected to these harsh conditions, only decomposition was observed. This can be rationalized by the sensitivity of the olefins in the isoprene chain R group. In an attempt to reduce the likelihood of reactions occurring at other sites, a milder set of conditions was used for the hydrolysis in the form of TMSBr and collidine to generate the collidinium salt intermediate, which was then converted to the desired acid under conditions similar to those of McKenna. 61 However, this only resulted in a mixture of partially hydrolyzed products 25, 26, and 27. Several other attempts at converting the esters to the free acid again proved challenging when trying to avoid conditions that would afford reaction at the isoprene olefin sites. A basic hydrolysis followed by an acid work-up yielded a partially hydrolyzed product 28. A stepwise set of conditions, where the phosphonate ester was converted to the 18

46 acid followed by a base hydrolysis utilizing aoh, also failed to give the desired compound 30. R = Isoprene Chain (HO) 2 (O)P HOOC 23 R 12M HCl, reflux (EtO) 2 (O)P HOOC 24 R (HO) 2 (O)P EtOOC R TMSBr MeOH (EtO) 2 (O)P EtOOC R TMSBr, 2,4,6-collidine MeOH (HO)(EtO)(O)P EtOOC R (ao) 2 (O)P aooc 1M aoh, reflux R (HO)(EtO)(O)P EtOOC 1) 1M aoh, reflux 2) 1M HCl R (HO) 2 (O)P EtOOC 26 R Figure 12. Attempted hydrolysis methods towards active acid or salt In an effort to explore different hydrolysis conditions that would yield a fully hydrolyzed product, a pair of carboxyphosphonate esters was synthesized. The synthesis begins with the formation of the click precursors, alkyne 15 and the isoprenoid azides 34 and 37. Construction of acetylene 15 begins with triethyl phosphonacetate and a condensation with formaldehyde followed by a dehydration through the use of tosic acid to yield the vinyl carboxyphosphonate 31. With compound 31 in hand, a conjugate addition of sodium acetylide afforded the desired acetylene 15 in good yield. 62 Azides 34 and 37 were formed from their respective alcohols prenol (32) and geraniol (35), which were converted to the bromides 33 and 36 through the use of PBr3, and a final reaction with sodium azide to afford the desired azides. 19

47 1) Paraformaldehyde, piperidine, MeOH, reflux, 3 h 2) Phosphonate, reflux, 18 h 3) TsOH H 2 O, Dean Stark toluene, reflux, 16 h 32 OH acch THF, -7 C 16 h (EtO) 2 (O)P COOEt (EtO) 2 (O)P COOEt (EtO) 2 (O)P COOEt 80% 61% PBr 3 a 3 Et 2 O, 0 C 2 h DMF, 16 h Br 70% 65% OH PBr 3 Et 2 O, 0 C 2 h 73% 36 Br a 3 DMF, 16 h 75% 37 3 Figure 13. Synthesis of click reaction precursors With the desired click starting materials in hand, a copper-catalyzed click reaction utilizing CuSO4 as the catalyst to form in situ the Cu (I) species through the reducing agent sodium ascorbate in 4:1 t-buoh:h2o as the solvent gave the desired triazoles 22 and 23 (Figure 14). 62 Both compounds were subjected to a myriad of different hydrolysis conditions in efforts to obtain the desired fully hydrolyzed carboxyphosphonate triazole (Table 1). Attempts at utilizing similar methods to the McKenna hydrolysis, a reaction that the Wiemer group uses for hydrolysis of bisphosphonates, were proven to be unsuccessful in part due to two factors: it was difficult to precipitate any product and the reaction ultimately yielded only partial hydrolysis. Attempts at a reaction very similar to the previously stated one, intended to recover the tri-acid species instead of the sodium salt, also proved unsuccessful as the desired product could not be recovered. The use of TMSI over TMSBr was hypothesized as a way to increase the conversion to a silyl intermediate that would be easier to convert to the desired trisodium salt. Unfortunately, this afforded only multiple byproducts. 63 Another effort employed conditions similar to those that Dr. 20

48 Zhou had first used for hydrolysis of a carboxyphosphonate system with concentrated 12M HCl and heat, but with a shorter reaction time to reduce the chance of addition to the olefins. Again, this only afforded a decomposed mixture. Finally, a set of conditions yielded the desired hydrolyzed product by first converting the carboxylate ester to a carboxylic acid and then employing McKenna hydrolysis conditions over the course of 15 days. 64 Although these reaction conditions were successful, it was a low yielding reaction and required almost two weeks. These compounds were sent to Dr. Sarah A. Holstein for biological assays. Those results will be discussed later in this chapter. (EtO) 2 (O)P 15 COOEt sodium ascorbate CuSO 4 H 2 O (sat.) t-buoh/h 2 O (4:1) 67% (EtO) 2 (O)P EtOOC 38 1) 10% aoh/etoh, reflux 3.5 h 2) 1M HCl 3) TMSBr, Collidine DCM, 0 C to rt 16 h 4) 1M aoh, 15 d 63% (ao) 2 (O)P aooc (EtO) 2 (O)P COOEt 3 37 sodium ascorbate CuSO 4 H 2 O (sat.) t-buoh/h 2 O (4:1) 63% (EtO) 2 (O)P EtOOC 39 1) 10% aoh/etoh, reflux 3.5 h 2) 1M HCl 3) TMSBr, Collidine DCM, 0 C to rt 16 h 4) 1M aoh, 15 d (ao) 2 (O)P aooc 41 24% Figure 14. Formation of carboxyphosphonate triazoles as the sodium salt 21

49 Hydrolysis Method 1) TMSBr, Collidine, DCM 0 C to rt 16h 2) 1M aoh 1) TMSBr, DCM 0 C to rt 16h 2) MeOH/H2O (3:1) 3) 10% aoh/etoh 4) 1M HCl 1) TMSI, 100 C h 2) 2M aoh 1) 12M HCl, reflux 5 h 2) aoh 1) TMSBr, Collidine, DCM 0 C to rt 16 h 2) 1M aoh 3) 10% aoh/etoh, reflux 3. 5 h 4) 1M HCl 1) 10% aoh/etoh, reflux 3.5 h 2) 1M HCl 3) TMSBr, Collidine, DCM 0 C to rt 16 h 4) 1M aoh, 15 d Yield Partial hydrolysis Multiple byproducts Multiple byproducts Decomposition Multiple byproducts 24 63% Table 1. Conditions examined to accomplish hydrolysis to the carboxyphosphonate salt or acid Synthesis of single olefin isomers of isoprenoid triazole carboxyphosphonates With successful discovery of a method to hydrolyze the carboxyphosphonate esters to their corresponding sodium salts, the next step was to synthesis both the pure neryl and geranyl triazoles. This strategy must avoid isomerization due to an allylic azide rearrangement (Figure 15). 65 An allylic azide 42 (as the trans isomer), goes through a [3,3]- sigmatropic rearrangement via the transition state 43, which then yields the secondary azide 44. The secondary azide 44 is then able to form the cis olefin isomer 45. Additional examples of this rearrangement also are shown in Figure 15, one between primary (46 and 49) and tertiary (47 and 50) azides. The two important takeaways from this are: 1) only the 22

50 primary azide will yield the desired triazole product and, 2) when the primary azide ultimately reacts it will yield a greater amount of the E-olefin isomer from the allylic azide rearrangement independent of the original olefin stereochemistry. To determine the ratio of E and Z isomers, a 1 H MR spectrum was used to observe the integrated ratio of the internal olefins methyl group. Generally, this rearrangement would give a 2:1 ratio of E to Z olefin isomers, confirming the results discovered by Sharpless. 65 In fact, Dr. Zhou also confirmed that changing the initial bromide from a geranyl bromide to a neryl bromide and converting each to the corresponding azide, left the ~2:1 ratio of E to Z olefin isomers unaffected. 57 R 43 fast slow R 3 R 42 R Primary vs. Tertiary Azides 3 3 phenylacetylene CuSO 4 5H 2 O sodium ascorbate tbuoh/h 2 O (1:1), rt 12 h Ph HO 46 70% % (47% trans, 8% cis) HO 50 45% 47 30% 3 85% phenylacetylene CuSO 4 5H 2 O sodium ascorbate tbuoh/h 2 O (1:1), rt 12 h 83% HO 48 Ph 51 (91% trans, 9% cis) Figure 15. Allylic azide rearrangement 23

51 With this knowledge, the synthesis of a single olefin isomer of both the neryl and geranyl triazoles began with a regioselective epoxidation of both farnesol and geraniol through the use of tert-butyl hydroperoxide and vanadyl acetylacetonate to separately afford epoxides 53 and 54 (Figure 16). 58,66,67 The epoxides were formed as racemic mixtures, but because the epoxide is acting as an olefin protecting group, the absolute stereochemistry was not important. The alcohols 53 and 54 were converted to the intermediate mesylate through the use of triethyl-amine and methanesulfonyl chloride. Each intermediate was then converted to the bromide with lithium bromide to afford bromides 55 and 56 in high yields. 68 Once the bromides were in hand, conversion to the azides 58, 69, and 60 proceeded smoothly through the use of sodium azide. A click reaction, with previously prepared acetylene 15 and each isoprene azide protected as the epoxide, proceeded under previously described reaction conditions to afford the desired triazole carboxyphosphonates 61, 62, and 63 in good to low yield

52 VO(acac) 2, TBHP OH 1,2-dichloroethane C 2 h 53 O OH 35 OH F = 79% G = 44% 54 O OH F = 82% G = 99% 1) MsCl, Et 3 CH 2 Cl 2, 0 C 2 h 2) LiBr acetone, reflux 1.5 h (EtO) 2 (O)P EtOOC O O 3 a 3 DMF, 16 h 59 3 F = 92% G = 99% 56 = 86% 55 O O Br Br 60 O 3 57 O provided by VSW Br F = 75% G = 48% = 34% sodium ascorbate CuSO 4 H 2 O tbuoh/h 2 O (4:1) (EtO) 2 (O)P EtOOC (EtO) 2 (O)P EtOOC O O (EtO) 2 (O)P EtOOC 63 O Figure 16. Click reaction of isoprene epoxides With the newly formed triazoles in hand, the next step was reduction of the epoxides to regenerate the olefin. Based on the precedents from Dr. Zhou and Dr. Wills work, when epoxides 61, 62, and 63 were treated with sodium iodide and trifluoroacetic anhydride (TFAA) 69 they were successfully converted to the olefins and retained their original olefin stereochemistry (Figure 17). During analysis of the 13 C MR spectra of these compounds, 25

53 two key resonances at ~32 ppm and ~39 ppm are particularly important as they correspond to the CH2 group adjacent to the more substituted carbon of the regenerated olefin, with ~32 ppm corresponding to the Z-olefin isomer and ~39 ppm corresponding to the E-olefin isomer. For compound 65, a resonance was found at 39.6 ppm and for compound 66 a resonance was found at 32.3, confirming the preservation of the olefin stereochemistry. For the final step, the carboxyphosphonate esters were treated under the previously mentioned conditions. However, the neryl compound, 69, was the only compound completely hydrolyzed. Both compounds 67 and 68 were subjected to additional attempts at hydrolyzing the esters, but only the partially hydrolyzed products were obtained in each case. The partial hydrolysis might be explained by the hydrophobic isoprene tail and hydrophilic head of the carboxyphosphonate esters, which make them function as a soap and ultimately complicate the hydrolysis. Unfortunately, the desired products were not able to be recovered and the partially hydrolyzed compounds 67 and 68 along with the fully hydrolyzed compound 69 were sent to Dr. Sarah A. Holstein for biological assays. Those assay results will be discussed later in the chapter. 26

54 (EtO) 2 (O)P O EtOOC 61 (EtO) 2 (O)P EtOOC 62 O ai, TFAA AC/THF (1:1) 0 C to rt 16 h F = 99% G = 99% = 99% (EtO) 2 (O)P EtOOC 64 (EtO) 2 (O)P EtOOC 65 (EtO) 2 (O)P EtOOC 63 O (EtO) 2 (O)P EtOOC 66 1) 10% aoh/etoh reflux 3.5 h 2) 1 HCl 3) TMSBr, Collidine DCM, 0 C to rt 16 h 4) 1M aoh (ao)(eto)(o)p aooc 47% 67 (ao)(eto)(o)p aooc 68 70% (ao) 2 (O)P aooc 69 44% Figure 17. Formation of single olefin isomer triazoles Synthesis of the farnesyl linked triazole was pursued due to previous results from Dr. Zhou s studies of several bisphosphonates (Figure 18). 50 What should be noted in Table 2 is that, the longer the isoprene chain, the greater the bisphosphonate s activity and selectivity towards inhibition of GGTase II in comparison to GGDPS. The hypothesis was that there was a high probability that the carboxyphosphonate series would follow the same trends as the bisphosphonate series in terms of chain length. Unfortunately, studies of the farnesyl linked triazole did not give the tri-sodium salt. Ideally, a geranylgeranyl linked carboxyphosphonate might be preferred, but limitations to the hydrolysis method might make it ever more challenging. 27

55 (ao) 2 (O)P (ao) 2 (O)P A: n = 1 B: n = 2 C: n = 3 H n Figure 18. Different isoprene chain lengths on a bisphosphonate Compound GGDPS IC50 (µm) GGTase II IC50 (µm) A 2.2 >1000 B C Table 2. Impact of isoprene length on bisphosphonate activity 50 Synthesis of an isomeric triazole carboxyphosphonate In an attempt to synthesize an isomeric triazole carboxyphosphonate, it was envisioned that the triazole core might be constructed from a carboxyphosphonate azide and might give new and potentially potent inhibitors of GGTase II or GGDPS. One way to reformulate how the triazole core is constructed is to switch the click precursors, that is, use the azide 70 and the alkyne 71, as shown in Figure 19. After the newly formed triazole 72 is prepared, hydrolysis to the analogous acids or salts would be necessary. 28

56 3 + (EtO) 2 (O)P COOEt n H (EtO) 2 (O)P EtOOC 72 H n Figure 19. Reverse click precursors and general product Formation of the alkyne click precursors proved to be more challenging than initially anticipated. Originally it was thought that a simple Sn2 alkylation of sodium acetylide with geranyl bromide 36, would afford the desired alkyne 73. Unfortunately, this was not the case as it was believed both Sn2 and Sn2 products were formed along with several other products that were unable to be successfully separated under typical column chromatography conditions. Even after several reactions changing variables such as reaction time, temperature, and concentration, the desired product was unable to be recovered. A different approach using conditions presented by Mori 70 also was explored, alkylating the anion of ethynyltrimethylsilane with geranyl bromide followed by deprotection of the silyl group to afford the desired alkyne 73 in poor yield. To synthesize a homogeranyl alkyne 74, a similar reaction was employed. In this case, the anion of 1- (trimethylsilyl)propyne was added to geranyl bromide 36 and the silyl group was removed by treatment with tetrabutylammonium fluoride (TBAF) to afford the desired alkyne 74 in poor yield. Despite the low yield, this strategy was far better than the previous reaction. 71,72 29

57 In a parallel fashion, nerol was converted to neryl bromide 76, which was allowed to react in the presence of the anion of ethynyltrimethylsilane followed by a deprotection with TBAF to afford compound 77 in very low yield. It is believed that the low yield can be attributed to the difficulty in separating such hydrophobic compounds from one another using common silica gel column chromatography. Along with the geranyl and neryl based alkynes, attempts were made at preparation of a prenyl based alkyne. Again, a similar reaction using sodium acetylide as the nucleophile was explored with prenyl bromide 33. Unsurprisingly, this approach did not yield the desired alkyne 78. Using a more successful reaction, 1-(trimethylsilyl)propyne was deprotonated with n-buli and the resulting anion was allowed to react in the presence of prenyl bromide. A final attempted deprotection using TBAF did not afford the desired compound

58 OH 75% 35 1) ethynyltrimethylsilane n-buli, HMPA, THF -78 C 2) geranyl bromide -70 C to rt 3) K 2 CO 3, MeOH/H 2 O 15% PBr 3, Et 2 O 0 C 2 h 36 Br acch THF, 0 C 16 h 73 1) 1-(trimethylsilyl)propyne n-buli, THF -78 C 2) geranyl bromide 3) TBAF, -78 C to rt 39% PBr 3, Et 2 O 0 C 2 h 1) ethynyltrimethylsilane n-buli, HMPA THF -40 C to -10 C 2) neryl bromide -40 C to rt 3) TBAF 75 OH 75% 76 Br 3% 32 OH PBr 3, Et 2 O 0 C 2 h 44% 33 Br acch THF, 0 C 16 h 1) 1-(trimethylsilyl)propyne n-buli, THF -78 C 2) preynl bromide 3) TBAF, -78 C to rt Figure 20. Formation of isoprene alkynes With some alkynes in hand, the next step was to use them in click reactions with appropriate azides to secure the triazoles. As shown in Figure 21, the click reactions attempted with the various alkynes and the carboxyphosphonate were not successful. The carboxyphosphonate 31 was allowed to react in the presence of sodium azide and acetic acid to form the intermediate azide 70. To the intermediate 70 was added the corresponding alkyne 80 or 74, CuSO4 H2O (sat.) and sodium ascorbate to form Cu (I) in situ, and the 31

59 reaction was allowed to proceed for 9 hours under sonication. 73 Unfortunately, neither of the desired triazoles was successfully formed. The reaction was run again, but this time the reaction was allowed to proceed under sonication for roughly 16 hours. Again, no triazole product was recovered. Both Dr. Wills and I attempted several more reactions on the same click precursors increasing both the sonication power and the reaction time. However, these efforts to obtain the desired carboxyphosphonate triazole were not rewarded. During these reactions, it was clear that the intermediate azide 70 was formed by analysis of the 31 P MR spectrum of the reaction mixture, which gave a resonance for compound 70 at 21 ppm. A slightly different approach to the reaction was to replace the alkyne with a commercial alkyne that was verified by 1 H MR (Figure 21). Again, using a procedure described by Duan, 73 and the commercial alkyne 1-decyne, along with a carboxyphosphonate as the trimethyl ester (84), the reaction did not afford a recoverable amount of desired triazole 85. The crude material was analyzed in the Mass Spectrometry Facility by LCMS to determine if compound 85 was in the sample. The results suggested that the triazole 85 was present, but the quantity of material in was very low comparison to other products. To secure the reverse triazoles would require a new set of reaction conditions or different ways to make Cu (I) in situ, or a different solvent system. 32

60 (EtO) 2 (O)P COOEt 31 (EtO) 2 (O)P COOEt provided by VSW 74 1) a 3, AcOH/H 2 O 15 min 2) alkyne, CuSO 4 H 2 O (sat.) sodium ascorbate sonication 9 h 1) a 3, AcOH/H 2 O 15 min 2) alkyne, CuSO 4 H 2 O (sat.) sodium ascorbate sonication 9 h (EtO) 2 (O)P EtOOC (EtO) 2 (O)P EtOOC ) paraformaldehyde piperidine, MeOH, reflux 2) TsOH H 2 O toluene, reflux (MeO) 2 (O)P COOMe (MeO) 2 (O)P COOMe 83 63% 84 1) a 3, AcOH/H 2 O 2) 1-decyne, CuSO 4 H 2 O (sat.) sodium ascorbate sonicate, 9h (MeO) 2 (O)P MeOOC 85 Figure 21. Reverse triazole click reactions through the use of sonication Bioassay results Several of the synthesized carboxyphosphonate triazoles discussed in this chapter were assayed by our collaborators in Dr. Sarah A. Holstein s research group, in both cellular and enzyme assays to establish their ability to inhibit IBP enzymes (Figure 22). Again, the hypothesis was that the carboxyphosphonate head group should be more selective to GGTase II than the corresponding bisphosphonate compounds. A compelling argument for this view can be made by comparison of 3-PEHPC, a carboxyphosphonate, and risedronate, its bisphosphonate analogue. To understand if the compounds summarized in Figure 22 resulted in inhibition of GGTase II, they were tested for their impact on inhibition of prenylation of Rap1a and Rab6. The proteins Rap1a and Rab6 are substrates of GGTase I and GGTase II, respectively. For recognition of Rap1a, the assay utilizes an antibody that only detects unmodified Rap1a. 49 Therefore, in a western blot experiment, a dark spot will signify the presence of the unmodified protein due to direct inhibition of 33

61 GGTase I or lack of GGPP via inhibition of GGPPS inhibition. For Rab6, the assays are run using a Triton X-114 lysis to generate two fractions, one aqueous and one detergent. 49 In typical control conditions, Rab6 is found in the detergent fraction, but when a compound prevents Rab geranylgeranylation, the unmodified protein will be found in the aqueous fraction. 49 Therefore when looking at the Rab6 western blots, a dark spot for the Rab6 aqueous western blot will signify disruption of Rab6 geranylgeranylation. If a tested compound is a GGTase II inhibitor, the compound should only show disruption of Rab6 geranylgeranylation and not Rap1a disruption or any other disruption further upstream in the IBP. If a compound were to disrupt geranylgeranylation from inhibition further upstream, it would be viewed as an indirect inhibitor and not the preferred direct inhibitor. (ao) 2 (O)P aooc 40 (ao) 2 (O)P aooc 41 (ao) 2 (O)P aooc 69 Figure 22. Assayed isoprenoid triazole carboxyphosphonates The compounds listed in Figure 22 were tested for their ability to inhibit GGTase II. Unfortunately, none of these newly synthesized compounds showed significant ability 34

62 to inhibit GGTase II (data not shown). However, these compounds showed a weak propensity to inhibit the enzyme GGDPS. Inhibition of GGDPS results in disruption of proteins modified by both GGTase I and GGTase II, which indicates that use of just the Rap1a western blot is sufficient. Upon consideration of the assay results in Figure 23 for compounds 40, 41, and 69, it is clear that none of them potently inhibit the geranylgeranylation of the protein and thus are at best weak inhibitors. Lovastatin, an HMG-CoA reductase inhibitor, was used as a control along with tubulin as a loading control. Disruption of protein geranylgeranylation as analyzed in a western blot of Rap1a showed that compound 40, bearing the prenyl chain, did not potently induce the accumulation of unmodified Rap1a. This is not surprising, but even at a 1 mm concentration, no disruption was seen. The two more promising compounds, 41 and 69, showed very weak activity towards inducing accumulation of unmodified Rap1a. Compound 41, as the mixture of isomers, was active at a concentration of about 100 µm whereas compound 69, the neryl isomer, was active between 100 and 250 µm. One other conclusion that can be drawn from these assay results is that the mixture is slightly more potent than the neryl isomer, which might suggest that the geranyl isomer is even more active than both the mixture and the neryl isomer alone. 35

63 Figure 23. Effects of carboxyphosphonate disruption in protein geranylgeranylation of Rap1a 36

64 Along with the western blot assays, enzyme-linked immunosorbent assay (ELISA) analysis of lambda light chain levels were determined after treatment with compounds 40, 41, and 69 in human myeloma cells RPMI RPMI-8226 cells were incubated for 48 hours in the presence of compound 40, 41, or 69 at varying concentrations in three independent experiments. Figure 24 shows that compounds 40, 41, and 69 poorly induce accumulation of intracellular light chain. Compound 40, shows little to no activity in inducing accumulation of light chains, while both 41 and 69 almost equally induce accumulation of intracellular light chains, but only at a concentration of approximately 100 µm. Figure 24. Effects of carboxyphosphonates inducing accumulation of intracellular light chain In conclusion, a small family of isoprenoid triazole carboxyphosphonates was synthesized. This was accomplished by developing a hydrolysis method to successfully hydrolyze the carboxyphosphonate esters in the presence of an isoprene chain and avoiding 37

65 conditions that rely upon the use of refluxing concentrated HCl. However, these conditions need more study to improve the low yields and would also benefit from a better purification method. Attempts at making the single olefin isomers for the farnesyl, geranyl, and neryl chain were successful in some cases. The farnesyl and geranyl linked isoprene chains were unable to be successfully recovered as the fully hydrolyzed products, but were isolated as partially hydrolyzed carboxyphosphonate esters. The neryl isoprene triazole was successfully isolated and was assayed along with the mixture of E and Z isomers and the prenyl analogue. All three of these compounds were not specific inhibitors of GGTase II, but they were found to be weak inhibitors of GGDPS. Most notably, compound 41 exhibited GGDPS inhibitor activity at a concentration of approximately 100 µm in cell culture studies and was similarly as active compared to compound 69. Along with these triazoles, attempts were made at preparation of a reverse triazole system, but the desired product was unable to be recovered due to very low yields. 38

66 CHAPTER 3 SYTHESIS AD BIOLOGICAL ACTIVITY OF HOMOISOPREOID TRIAZOLE BISPHOSPHOATES As discussed in previous chapters, nitrogenous bisphosphonates such as risedronate and zoledronate are clinically used as a treatment method for osteoporosis, multiple myeloma, and other diseases of the bone like Paget s disease. 74 These drugs function through inhibition of the IBP enzyme farnesyl diphosphate synthase (FDPS). 75 These compounds ultimately deplete cellular levels of farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP), which leads to an indirect disruption of GGTase II activity. Therefore, inhibition of the downstream enzyme GGDPS would lead to a desired inhibition of GGTase II, but would also avoid potential disruption of the production of cholesterol and other products further upstream in the IBP. The Wiemer group has synthesized several potent GGDPS inhibitors as bisphosphonates, and the most iconic was digeranyl bisphosphonate which showed an IC50 of about 260 nm. Some recent efforts have aimed to develop compounds that improve the importance of the V-shape motif, 76,49 which would improve upon how the isoprene chains occupy the FPP and GGPP sites. However, as explained earlier, some of the most potent inhibitors the Wiemer group has made are nitrogenous bisphosphonates assembled through click chemistry which bear a single isoprenoid chain. The most potent triazole inhibitor at the time was compound 8, more commonly known as VSW-1198 (Figure 6, page 12). 48 The synthesis of this compound was repeated to obtain material that could be used to generate new analogues, and to provide Dr. Sarah Holstein s group with a supply to use 39

67 for animal studies. The synthesis begins with ketone 87 undergoing a Grignard reaction with cyclopropyl magnesium bromide to form the tertiary alcohol 88 in an 89% yield. Opening of the cylcopropyl ring was accomplished by treatment with magnesium bromide, to afford the homogeranyl bromide 89 as a mixture of isomers. Conversion to the azide click precursor 90 was accomplished via a reaction between bromide 89 and sodium azide. To form the bisphosphonate alkyne 93, tetraethyl methylenediphosphate was converted to the vinyl bisphosphonate through condensation with formaldehyde followed by dehydration to yield compound 92, conditions similar to those used for formation of the vinyl carboxyphosphonates (Chapter 2). The vinyl bisphosphonate was then allowed to react in the presence of sodium acetylide to afford, compound 93, in almost quantitative yield. A click reaction using azide 90 and alkyne 93, in the presence of copper sulfate (CuSO4) and sodium ascorbate, yielded the desired triazole 94. A final McKenna hydrolysis 61 afforded the tetra-sodium salt 95 as a 3:1 mixture of E/Z olefin isomers in near quantitative yield. 40

68 O 87 MgBr OH 88 (EtO) 2 (O)P P(O)(OEt) 2 91 (ao) 2 (O)P (ao) 2 (O)P 1) THF, 0 C 2) H 2 O 89% 95 MgBr 2 Et 2 O, 3 h 87% 1) paraformaldehyde Et 2 H, MeOH reflux 16 h 2) TsOH H 2 O, toluene reflux 16 h 69% Br 89 (EtO) 2 (O)P P(O)(OEt) 2 99% (EtO) 2 (O)P P(O)(OEt) ) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 1M aoh 98% a 3 DMF, 16 h 93% acch, THF -15 C to rt 16 h (EtO) 2 (O)P (EtO) 2 (O)P sodium ascorbate CuSO 4 H 2 O (sat.) tbuoh/h 2 O (4:1) 86% 94 (3:1 mix E/Z) Figure 25. Synthesis of Compound 8 (VSW-1198) Although this synthesis was accomplished before, 48 I was able to prepare several grams of the final product 95 through development of very high yielding steps all along the sequence. Compound 95 has an IC50 value of ~45 nm as a 3:1 mixture of E:Z olefin isomers, making it the most potent triazole bisphosphonate GGDPS inhibitor. However, while some variation in the reaction conditions did improve the ratio in favor of the E-isomer, the ring opening rearrangement to afford compound 89 could not be controlled to highly favor either isomer. 77 Synthesis of the individual olefin isomers is of importance to allow comparison of compounds 96, 6, and 7 (Figure 26) in terms of their GGDPS IC50 values. Originally, compound 96 was synthesized by Dr. Zhou and garnered a GGDPS IC50 value 41

69 of 2.2 µm as a 2:1 mixture of E- and Z-olefin isomers, which were not readily separable. 50 This led to the synthesis of the individual isomers 6 and 7, where it was believed initially that the geranyl-linked chain would be more active than the neryl isomer and more active than the mixture. It was found that compound 6 had a GGDPS IC50 of 17 µm and was slightly more selective towards GGDPS than FDPS. 49 On the other hand, compound 7 had an IC50 of 380 nm towards GGDPS and was much more selective towards GGDPS over FDPS. 49 This knowledge made the synthesis of the homonerol isomer of special interest. (ao) 2 (O)P (ao) 2 (O)P 96 (ao) 2 (O)P (ao) 2 (O)P 6 (ao) 2 (O)P (ao) 2 (O)P 7 Figure 26. Comparison of geranyl and neryl isoprene chains Initial efforts by Dr. Wills 77 to synthesize homonerol used the THP derivative of 3- bromopropanol 78 as a Wittig reagent that was shown to have Z-isomer selectivity in reactions with aldehydes. 79 Unfortunately, a condensation with this reagent and 6-methyl- 5-hepten-2-one gave homonerol as only a 1.4:1 mixture in favor of the Z-olefin isomer, and 42

70 the two isomers were not readily separable. 77 There have been several reports on the synthesis of homonerol and homogeraniol, 79,80,81 but to obtain the pure individual olefin isomers a new sequence was derived from research described by Wessjohann. 82 The synthesis begins with conversion of tert-butyl acetoacetate 100 to the alpha brominated compound 101 through a radical reaction with -bromosuccinimide in acetone that proceeds in near quantitative yield (Figure 27). 83 A substitution reaction with sodium acetate in DMF provided the desired acetate Deprotonation of the alpha hydrogen of compound 102 followed by an alkylation with neryl bromide provided the isoprenoid It should be noted that the stereochemistry of the sp 3 carbon does not matter as it will be removed later in the sequence. A decarboxylation reaction catalyzed with TsOH afforded racemic keto acetate 104 with a yield of 99%. 84 Reduction of acetate 104 through the use of lithium aluminum hydride afforded the diol 105 as a mixture of stereoisomers. An oxidative cleavage reaction of diol 105 by use of sodium periodate on silica gel afforded the desired aldehyde 106 in quantitative yield. The final step to yield the desired homonerol 107 proceeded smoothly through a reduction of the aldehyde to the primary alcohol in high yield. 43

71 O O BS O O aoac O O acetone 4.5 h DMF 2 h Ot-Bu Ot-Bu Ot-Bu 97% Br 93% 100 OAc ah, neryl bromide THF, 0 C 16 h 95% OH OH LiAlH 4, Et 2 O 0 C 2 h 89% OAc O p-tsoh, C 6 H 6 78 C 2 h to rt 16 h 99% O AcO CO 2 t-bu 103 aio 4 on SiO 2 DCM, 30 min H O % LiAlH 4, Et 2 O 0 C to rt 16 h 95% HO 107 Figure 27. Preparation of homonerol A similar sequence was conducted to obtain homogeraniol (112), where the β-keto acetate 108 was prepared using geranyl bromide and the branch point compound 102 (Figure 28). With both the desired homonerol (107) and homogeraniol (112) in hand, conversion to the azide click precursor was the next step. O OAc 109 LiAlH 4, Et 2 O 0 C 2 h p-tsoh, C 6 H 6 78 C 2 h to rt 16 h 81% O AcO CO 2 t-bu 108 ah, geranyl bromide THF, 0 C 16 h 87% O O Ot-Bu OAc % OH OH 110 aio 4 on SiO 2 DCM, 30 min 100% H O LiAlH 4, Et 2 O 0 C to rt 16 h HO 96% Figure 28. Preparation of homogeraniol 44

72 Alcohols 108 and 112 were converted to their respective mesylates 113 and 114, which were carried onto the next step without further purification (Figure 29). Typically, the mesylate would be converted to a bromide, which would then be converted to the azide. However, by heating the reaction, the mesylates 113 and 114 were allowed to react in the presence of sodium azide to afford the alkyl azide click precursors 115 and 116 directly, and they were used immediately for the following click reaction. HO HO c-5 MsCl, Et 3 DCM, 0 C 95% MsO 107 MsCl, Et DCM, 0 C 95% MsO c a 3 DMF, 40 C 84% a 3 DMF, 40 C 79% Figure 29. Formation of homoallylic azides Similar to previously mentioned copper catalyzed click reactions, azides 115 and 116 were allowed to react in the presence of the bisphosphonate alkyne 93, copper sulfate, and sodium ascorbate as the reductant, in separate reactions to afford the desired triazoles 117 and 118, respectively (Figure 30). Hydrolysis of the tetraethyl esters 117 and 118 under standard McKenna conditions 61 afforded the desired sodium salts 119 and 120 as their individual olefin isomers. The final products 119 and 120, along with the tetraethyl ester intermediates 117 and 118, were identified as single olefin isomers based on specific shifts in their 13 C MR spectra. The methylene group at the C-5 position in both nerol and geraniol gives different shifts in their 13 C MR spectra (32.2 and 39.7 ppm, respectively), 85 45

73 which compare very well to the corresponding C-5 methylene resonances for homonerol and homogeraniol (32.2 and 40.0 ppm, respectively) (Figure 29). In triazole 117, which is soluble in CDCl3, the C-5 resonance was observed at 32.2 ppm; in the sodium salt 119, which is soluble in D2O, the C-5 resonance was observed at 31.5 ppm. However, the alpha carbon in both 13 C MR spectra is also found in this range, but can easily be identified due to its enormous coupling to phosphorus (~120 Hz). For the geranyl linked triazole 118, the C-5 resonance was observed at 39.8 ppm in CDCl3, and for the sodium salt 120 the C-5 resonance was observed at 39.3 ppm in D2O. Thus, it was clear that both isomers had been prepared cleanly. (ao) 2 (O)P (ao) 2 (O)P (EtO) 2 (O)P P(O)(OEt) 2 93 sodium ascorbate CuSO 4 H 2 O (sat.) tbuoh/h 2 O (4:1) 80% (EtO) 2 (O)P (EtO) 2 (O)P 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 1M aoh % C (EtO) 2 (O)P P(O)(OEt) 2 93 sodium ascorbate CuSO 4 H 2 O (sat.) tbuoh/h 2 O (4:1) 65% (EtO) 2 (O)P (EtO) 2 (O)P 118 C-5 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 1M aoh (ao) 2 (O)P (ao) 2 (O)P % Figure 30. Click reaction and hydrolysis to homoisoprene triazoles 46

74 Both cis and trans isomers of the homo isoprene compounds were individually synthesized through this classic acetoacetate chemistry. The final tetra-sodium salts were produced on a gram scale, which were then made available for biological investigation. The biological activity of both triazole salts 119 and 120 will be discussed in detail and the mixture of olefin isomers 95 will be used as a comparison point. Bioassay Results As previously mentioned, our past work with the geranyl and neryl triazole bisphosphonates, compounds 6 and 7 respectively, demonstrated that the neryl isomer was more potent than either the geranyl isomer or the originally reported mixture of the two isomers. 49 On this basis, it was hypothesized that the homoneryl isomer would not only be more potent than the homogeranyl isomer, but would also be more potent than the original mixture 96. The in vitro enzyme assay results (Table 3) confirmed that homoneryl is more potent than the homogeranyl isomer, but surprisingly it is not more potent than the mixture (96). Furthermore, all of the homoisoprene isomers show great selectivity towards GGDPS over FDPS. GGDPS IC 50 µm FDPS IC 50 µm Homogeranyl ± ± 8.2 Homoneryl ± ± 5.6 Mixture ± ± 5.0 Table 3. Inhibition of GGDPS and FDPS with homoisoprene triazole bisphosphonates

75 Additionally, cell culture studies were performed with homogeranyl 120 and homoneryl 119 isomers to determine their relative potencies in disrupting geranylgeranylation. 86 The homoneryl isomer more potently induces accumulation of intracellular light chain and unmodified Rap1a as compared to the homogeranyl isomer. In cellular studies, the homoneryl bisphosphonate is roughly 2-3 times more potent than homogeranyl. Studies were therefore performed to compare more directly the cellular activity of the mixture to that of the pure homoneryl isomer. The activity of the mixture 96 and homoneryl isomer 119 was very similar across a concentration range of nm (Figure 31). Because the original mixture is a 3:1 ratio in favor of the less potent isomer (3:1 homogeranyl:homoneryl) 48 these results suggested that the two isomers might affect the target enzyme in a synergistic manner. 48

76 Figure 31. Comparison of mix 96 and homoneryl With this information, one would imagine that if an assay were done with a ratio in favor of the homoneryl isomer it would ultimately be a more active mixture. However, no enhancement in activity was observed when cells were treated with a 1:3 ratio of HG:H. 86 Even combinations involving a 1:1 ratio showed some enhancement in activity relative to the single isomer, but less so than with the 3:1 mixture. To determine whether the results of the cell culture combination studies are a consequence of interaction of the two isomers at the level of the enzyme, in vitro enzyme studies were performed. The combination of the two isomers (3:1 HG:H) inhibited 49

77 GGDPS activity more potently than either isomer alone. It was revealed a synergistic interaction was taking place. These assays demonstrated that the homoneryl isomer is more active than its homogeranyl counterpart, which is consistent with the previous data on compounds 6 and However, it was determined that the homoneryl and homogeranyl isomers were less active than the 3:1 mixture of homogeranyl and homoneryl isomers, respectively. In fact, even switching the ratio to favor the more potent of the two individual isomers did not increase activity in comparison to the original 3:1 mixture. Because the combination of isomers more potently inhibits GGDPS than the two individual isomers, it is suggested that there is a synergistic interaction at play. Thus, surprisingly, compound 96, originally synthesized by Dr. Wills, remained the most active individual triazole bisphosphonate at this point in time. 50

78 CHAPTER 4 SYTHETIC STRATEGIES TOWARDS A ISOPREE TRIAZOLE TETRA POM BISPHOSPHOATE Of the compounds reported above and ones synthesized by other researchers in our group, including Dr. Zhou and Dr. Wills, all of the final bisphosphonates are tested as either the sodium salt or the phosphonic acid. This is ideal for both stability s sake and because this is the form that would appear in the cell as the active compound. However, if these drugs are to be used in a clinical setting, administration of a bisphosphonate tetra salt or acid would be feasible due to other bisphosphonates clinically used as the salt, but the fact that highly ionizable compounds are not transported readily across the cell membrane by passive diffusion would not make it ideal. 87 To avoid this issue of a highly charged and polar species, medicinal chemists mask the charged sites by developing moieties which would act as a biolabile protecting group, one that would liberate the active compound once inside the cell through the action of non-specific esterases or another enzymatic process (Figure 32). Pharmaceuticals that include such moieties are classified as prodrugs. 88 One of the most commonly used prodrugs is aspirin, first synthesized by Felix Hoffman in 1897, which releases salicylic acid and acetic acid upon metabolic cleavage. Historically, phosphonate-containing drug salts have used prodrugs moieties to mask their charge

79 Figure 32. Illustration of the prodrug concept 90 There are a number of potential advantages for use of prodrug design in the synthesis of active compounds, including improved solubility, chemical stability, absorption, and prolonged duration of action. From this perspective, prodrug moieties are increasingly being used to deliver compounds for antiviral and antitumor therapy. 91 Such an example is the pivaloyloxymethyl (POM) derivative Adefovir Dipivoxil, a bis-pom phosphonate prodrug which will ultimately release Adefovir as the active agent through the action of a non-specific esterase. Adefovir is used clinically for the treatment of hepatitis B and herpes simplex virus. 92,93,94 Recently the Wiemer group has utilized POM prodrug moieties on a system other than the bisphosphonates presented earlier. Specifically, POM protected phosphoantigens have been synthesized and their biological activity explored to study mechanistic aspects 52

80 of γδ T-cell stimulation. 95 Compounds 121, 122, and 123 as the dimethyl ester, disodium salt, and the dipom ester, respectively (Figure 33), were evaluated for their biological activity. The dimethyl compound 121 had an effective concentration (EC50) >10 µm, while the disodium salt and dipom compounds, 122 and 123, had EC50 values of 4.0 µm and µm, respectively. 95 The dipom compound was almost 1000 times more active than the same compound as the disodium salt in these cell base bioassays. CH 3 O CH 3 O O P O O ao POMO OH P OH P ao POMO OH O O = POM Figure 33. Phosphoantigen prodrugs Although the above results are very promising and yield synthetic inspiration for modification of sodium salts of phosphonate drugs that have not had a prodrug moiety prepared yet, such as the bisphosphonate triazole series, it should be noted that these are EC50 values for a much different biological assay compared to the enzymes of interest here (GGDPS and GGTase II). evertheless, developing a synthetic route to a tetra POM bisphosphonate triazole should yield a compound with better bioavailability and would have a high likelihood of improved cellular activity. When the POM prodrug moiety is cleaved via a non-specific esterase it will release three compounds, formaldehyde, pivalic acid, and the desired drug as the active compound (Figure 34). However, release of formaldehyde and pivalic acid as the metabolic byproducts of POM cleavage 96 brings up concerns of formaldehydes toxicity 97,98 and the potentially more dangerous pivalic acid as a carnitine analogue. 53

81 O O O O O O O P 123 OH esterase O O O O O O O P 124 OH chemical rearrangement H O H OH O O O O P 126 OH 2 nd cycle O O O O P 125 OH O O OH H H Figure 34. Activation of POM prodrug on phosphoantigen Even though the bisphosphonates that have been synthesized show sub micromolar activity as inhibitors of GGDPS in enzyme assays, the goal is to further improve potency even more. Despite the potential toxicity of the byproducts, the potential gain in cellular activity and increase in bioavailability are especially attractive. A general synthesis of the tetra POM bisphosphonate triazole of a typical homoisoprenoid is shown in Figure 35, where the tetramethyl bisphosphonate would be allowed to react with sodium iodide and chloromethyl pivalate to yield the desired final product as the tetra POM bisphosphonate. (MeO) 2 (O)P (MeO) 2 (O)P + O O ai, CH 3 C 80 C (POMO) 2 (O)P (POMO) 2 (O)P Figure 35. General route for synthesis of a tetra POM bisphosphonate 54

82 The synthetic route to obtain a tetramethyl bisphosphonate triazole would require tetramethyl methylenediphosphonate as a starting material, which is prohibitively expensive. Therefore, several different strategies were explored to install the POM moiety through different base-mediated couplings (Figure 36). Presumably the phosphonate ester 129 could be hydrolyzed to its corresponding phosphonic acid 130, most likely through some form of McKenna hydrolysis conditions. 61 The phosphonic acid 130 could then undergo a base-mediated reaction with chloromethyl pivalate to yield the tetra POM species 131. (EtO) 2 (O)P (EtO) 2 (O)P Phosphonate deprotection (HO) 2 (O)P (HO) 2 (O)P POM-Cl, base coupling (POMO) 2 (O)P (POMO) 2 (O)P Figure 36. Base mediated method to access POM phosphonate prodrug 99 Many attempts were made to convert a late stage tetraethyl bisphosphonate ester to the desired tetra POM prodrug. In Table 4 is displayed a list of different methods explored to convert the ester of the POM species 133 (Figure 37). In the first reaction, the tetraethyl ester was hydrolyzed through reaction with TMSBr, and then it was converted to the acid intermediate through a work-up with water. The free acid intermediate was allowed to react in the presence of silver carbonate and chloromethyl pivalate in acetonitrile at reflux. 100 However, these conditions did not afford the desired tetra POM product, but rather a difficult to separate mixture that was believed to contain partially converted POM phosphonate esters. ext a similar hydrolysis was used, but once the free acid was isolated, 55

83 similar base-mediated reaction with POMCl was attempted over a significantly longer reaction time. Unfortunately, only partially converted product was observed once again. Based on the assumption that more mild conditions for the hydrolysis might improve access to the desired product, collidine was used to help convert the free phosphonic acid to a salt. In both reactions 3 and 4, the intermediates were subjected to similar based-mediated POM reaction conditions with silver carbonate and chloromethyl pivalate over 16 hours and three days, respectively. either reaction yielded the desired product. At this point, the hydrolysis conditions were changed to include a work-up with toluene and water instead of acetonitrile and water. Analysis of the intermediate 133 by 31 P MR suggested that the phosphonic acid was formed. With the phosphonic acid intermediate in hand again, another base-mediated coupling reaction with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and chloromethyl pivalate in 1,4-dioxane at 80 C over 16 hours or 3 days was attempted, but these conditions did not afford the desired tetra POM product. In this case, the farnesyl chain was used due to abundant supply of this bisphosphonate. It was believed that the long isoprene chain made for a non-polar soap-like molecule when the intermediate acid was isolated and that formation of the tetra POM prodrug was unlikely to be completed due to such a large non-polar chain. With this in mind, the starting material was changed to a shorter length isoprene chain for further studies. (EtO) 2 (O)P (EtO) 2 (O)P 132 conditions (POMO) 2 (O)P (POMO) 2 (O)P 133 Figure 37. Attempted conversion of a tetraethyl bisphosphonate to a tetra-pom bisphosphonate triazole 56

84 Reaction Hydrolysis Condition Base Mediated POM Addition Yield ) TMSBr, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min 1) TMSBr, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min 1) TMSBr, collidine, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min 1) TMSBr, collidine, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min 1) TMSBr, DCM 0 C to rt 16 h 2) Toluene/H2O (6:1) 20 min 1) TMSBr, DCM 0 C to rt 16 h 2) Toluene/H2O (6:1) 20 min POMCl, Ag2CO3 AC, 80 C 16 h POMCl, Ag2CO3 AC, 80 C (dark) 2 d to rt then 8 d POMCl, Ag2CO3 AC, 80 C 16 h POMCl, Ag2CO3 AC, 80 C 3 d POMCl, DBU 1,4-dioxane, 80 C 16 h POMCl, DBU 1,4-dioxane, 80 C (dark) 3 d R R R R R R Table 4. Base-mediated POM conditions for farnesyl triazole bisphosphonate 132. The next attempt was performed on the monoterpenoid chain 134 under many of the same conditions used earlier for the sesquiterpenoid analogue. The tetraethyl bisphosphonate 134 (Figure 38) was again subjected to hydrolysis conditions followed by a base-mediated POM addition 100 with triethylamine (TEA) in -methyl-2-pyrrolidone (MP) over the course of 16 hours at either 60 C or 80 C. Both reactions failed to yield a recoverable amount of desired tetra POM product. With this information, it was hypothesized that the triazole ring system may disrupt the installation of the POM prodrug, given that two of the triazole nitrogens are nucleophilic. 57

85 (EtO) 2 (O)P (EtO) 2 (O)P conditions (POMO) 2 (O)P (POMO) 2 (O)P Figure 38. Attempted conversion of a tetramethyl bisphosphonate to a tetra POM bisphosphonate with geranyl chain Reaction Hydrolysis Condition Base Mediated POM Addition Yield 1 2 1) TMSBr, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min 1) TMSBr, collidine, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min POMCl, TEA MP, 60 C 16 h POMCl, TEA MP, 80 C 16 h R R Table 5. Base-mediated POM conditions for geranyl triazole bisphosphonate In an effort to use an ethyl phosphonate ester, tetraethyl methylenediphosphonate 91, was converted to the phosphonic acid intermediate and then allowed to react in the presence of a base-mediated coupling with silver carbonate and chloromethyl pivalate in acetone over three days (Figure 39 and Table 5). o change was observed consistent with formation of the final product from the intermediate. After a number of base-mediated hydrolysis conditions were explored, it was determined that more research would need to be done with these conditions to achieve the desired tetra POM product. At this point, all attempts to form a tetra POM compound from a tetraethyl phosphonate ester in a triazole had gone unrewarded. Therefore, compound 91 was allowed to react under similar reaction conditions to introduce the POM moiety from the phosphonate methyl ester. When bisphosphonate 91 was allowed to react in the presence of chloromethyl pivalate and sodium iodide in acetonitrile at reflux, the initial results 58

86 appeared to be more promising. After four days the reaction was checked by 31 P MR and a single peak was observed. However, after workup and purification it was found that only starting material was obtained. The reaction was attempted again, but after eight days the reaction progress was checked by phosphorus MR and only starting material was observed. (EtO) 2 (O)P P(O)(OEt) 2 conditions 91 (POMO) 2 (O)P P(O)(OPOM) Figure 39. Attempted conversion to tetra POM bisphosphonate from compound 91 Reaction Hydrolysis Condition POM Addition Yield ) TMSBr, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min 1) TMSBr, collidine, DCM 0 C to rt 16 h 2) AC/H2O (4:1) 20 min POMCl, TEA MP, 60 C 16 h POMCl, TEA MP, 80 C 16 h POMCl, ai AC, reflux 4 d POMCl, ai AC, reflux, 8 d R R R R Table 6. Conditions to obtain a tetra POM species from compound 91. At this point, all efforts at conversion of a tetraethyl bisphosphonate to the desired POM moiety were unsuccessful. Focus was then shifted to the more expensive route, which would utilize a tetramethyl bisphosphonate that would be converted to the tetra POM 59

87 moiety through the use of sodium iodide and chloromethyl pivalate. 101 It was envisioned that the POM groups might be installed at different point in the synthetic sequence. As shown in Figure 40, tetramethyl methylenediphosphonate 137 was converted to the olefin 138 under standard conditions by condensation with formaldehyde followed by dehydration with tosic acid and azeotropic removal of water through the use of a Dean Stark trap. 102,103 A subsequent reaction with chloromethyl pivalate and sodium iodide at reflux over the course of a day afforded the desired tetra POM moiety 139 in good yield. 101 Unfortunately, when this alkene was subjected to the conjugate addition conditions previously used to obtain the alkyne from the tetraethyl bisphosphonate, 59 the desired terminal acetylene 140 was unable to be recovered. It was believed that there was a competing reaction with the POM groups, which ultimately resulted in a partially hydrolyzed product, or potential decomposition of the POM groups. If it had been formed, compound 140 would have been isolated and then used in a click reaction to form the desired triazole 144. Fortunately, there are several other options to install the POM moiety. Conversion of alkene 138 to the alkyne 141 was accomplished by conjugate addition of sodium acetylide to the olefin 138. However, in this process, the desired alkyne click precursor 141 was accompanied by a significant amount of the α-methylated product 142, and the separation of these two compounds, 141 and 142, was not readily accomplished. This was due in part to the minor change in polarity upon methylation and the resulting Rf values were virtually identical which made for a very difficult separation. The two alkyne compounds formed in a 4:1 ratio in favor of the non-methylated product 141. The methylation was believed to have occurred due to the methyl esters of the bisphosphonate 60

88 acting as methylating agents causing the alpha methylation to occur in the presence of the sodium acetylide functioning as a base. To confirm that the methylation was taking place at the alpha position to the bisphosphonate, two experiments were conducted. The first was a 1 H MR spectrum revealing a triplet with an integration of 3 at ~1.4 ppm, which suggested a methyl group that displayed strong coupling to the two phosphorus atoms. The next experiment was to prepare the methylated product 142 from compound 141 by deprotonating the alpha position through reaction with sodium hydride, then allowing the resulting anion to react with methyl iodide to yield the methylated species 142. Because the methylated and non-methylated compounds were not readably separable, compound 141 was carried on to only compound 142 by this methylation. With one alkyne click precursor in hand, compound 142 was allowed to react under standard click conditions in the presence of geranyl azide as a mixture of olefin isomers, to yield triazole 143 in low yield. Unfortunately, efforts to convert the tetramethyl ester 143 to the desired tetra POM compound 144 were unsuccessful. It was believed that the triazole ring system was reacting with the chloromethyl pivalate yielding a mixture of POM products. 61

89 (MeO) 2 (O)P P(O)(OMe) ) paraformaldehyde Et 2 H, MeOH reflux, 2 h 2) p-tsoh, toluene reflux, 16 h 71% (MeO) 2 (O)P P(O)(OMe) ai, POMCl AC, 80 C 16 h 60% acch, THF -15 C to rt 16 h 54% (4:1, 141:142) (MeO) 2 (O)P P(O)(OMe) (MeO) 2 (O)P P(O)(OMe) ah, CH 3 I THF 0 C to rt 5 h (POMO) 2 (O)P P(O)(OPOM) acch, THF -15 C to rt 16 h 28% 3 sodium ascorbate CuSO 4 H 2 O tbuoh/h 2 O (4:1) (POMO) 2 (O)P P(O)(OPOM) (MeO) 2 (O)P (MeO) 2 (O)P 143 steps ai, POMCl AC, refulx (POMO) 2 (O)P (POMO) 2 (O)P 144 Figure 40. Attempted preparation of a triazole bisphosphonate prodrug Because the synthetic strategy has two other places in the sequence to install the POM moiety, a second route was explored. The first place the POM moiety could be installed is the first step of the synthetic sequence shown above in Figure 40. The tetramethyl methylenediphosphonate 137 could be converted to a tetra POM methylenediphosphonate 139. However, if this synthetic route were pursued, problems 62

90 later in the sequence would occur when trying to convert the extended alkene product to the alkyne through a conjugate addition with sodium acetylide. Another problem with this early installation of the POM group is that formation of the vinyl bisphosphonate required a dehydration reaction, which uses a catalytic amount of para-toluenesulfonic acid. The advent of any amount of acid in the reaction flask would most likely remove the previously installed POM moiety and ultimately release pivalic acid and formaldehyde. This then leads to consideration of the possibility of installation of the POM moiety after the conjugate addition with sodium acetylide and before the click reaction. If this fails, a very different route would have to be used. The next option, shown in Figure 41, would be to install the POM moiety immediately after the formation of compound 138. However, this strategy would require a new synthetic plan to synthesize the alpha methyl acetylene 142. Again, the reason to form compound 142 is that formation of compound 141 is accompanied by generation of 142 in significant quantities, and the two are not readily separable. Therefore an α-methylated POM derivative like compound 144 became the target. In the revised sequence, compound 137 was converted to compound 138 through a condensation with formaldehyde followed by a dehydration with p-toluenesulfonic acid. 102,103 The vinyl bisphosphonate was reduced via catalytic hydrogenation with the use of a Parr shaker to afford the methylated bisphosphonate 145. This reduction avoids a dialkylation problem if compound 137 were allowed to react in the presence of a strong base followed by alkylation with methyl iodide. With compound 145 in hand, the next step was to convert it to the alkyne 142. Originally, bisphosphonate 145 was allowed to react in the presence sodium hydride at 0 C to form the alpha anion of the bisphosphonate, followed by alkylation of propargyl bromide over 63

91 the course of 16 hours while warming to room temperature. Under these conditions, two products were formed in about equal amounts, the alkyne 142 and the allene 146. It was discovered by Delain-Bioton 104 that temperature control was the key to forming the alkyne over the allene in a similar system. That is, if the reaction temperature rose above -10 C the allene would form in an equal amount. However, if the reaction were kept cold (approximately -78 C) preferential formation of the alkyne would occur. In a similar reaction to the one described earlier, bisphosphonate 145 was deprotonated by reaction with sodium hydride at -78 C followed by addition of propargyl bromide. The reaction was kept at -78 C for 12 hours, and then allowed to warm to room temperature to afford the desired alkyne 142 in moderate yield. Treatment of compound 142 with sodium iodide and chloromethyl pivalate yielded the tetra POM bisphosphonate in good yield with minimal purification. However, due to the limited stability of this material, the tetra POM alkyne 147 was immediately carried onto the next step. The alkyne was subjected to a click reaction using CuSO4 and sodium ascorbate in water and tbuoh to afford triazole 148, as the homoneryl isomer. Only a small amount of the crude material was purified through HPLC, but this was sufficient to allow bioassays. The biological results will be discussed in the next chapter. 64

92 1) paraformaldehyde diethylamine MeOH, reflux 2 h 2) p-tsoh H 2 O toluene, reflux 16 h (MeO) 2 (O)P P(O)(OMe) 2 (MeO) 2 (O)P P(O)(OMe) % 138 H 2, Pd/C 10% EtOAc, 3 atm 4 h 95% 1) ah,15-c-5, THF, 0 C 30 min 2) propargyl bromide 0 C to rt 16 h (MeO) 2 (O)P P(O)(OMe) 2 (MeO) 2 (O)P P(O)(OMe) 2 (MeO) 2 (O)P P(O)(OMe) % (1:1 146:142) 145 1) ah, THF, -78 C 1 h 2) propargyl bromide -78 C 12 h to rt 6 h 41% POMCl, ai AC, reflux (POMO) 2 (O)P P(O)(OPOM) % (MeO) 2 (O)P P(O)(OMe) provided by Alex Rier (POMO) 2 (O)P (POMO) 2 (O)P sodium ascorbate CuSO 4 H 2 O tbuoh/h 2 O (4:1) 148 Figure 41. Formation of the triazole bisphosphonate prodrug 148 Purification of the tetra POM 148 triazole was particularly challenging due to the acid sensitive nature of the POM moiety. Originally compound 144 (Figure 42), as the mixture of geranyl and neryl isomers, was subjected to a myriad of attempted purifications. The first approach was a normal phase silica gel column with purification conditions similar to those used for many triazole bisphosphonates. However, the silica gel has an acidic nature, which caused the degradation of the tetra POM compound. Deactivation of 65

93 the silica gel through the use of triethylamine again yielded a decomposed product. Even with altered solvent conditions and a small percent of triethylamine to deactivate the silica gel, separation of compound 144 from azide 37 and the mixture of POMCl/POMI was unsuccessful. Using basic alumina as the stationary phase did not yield the desired degree of separation. Instead the crude material that was used was ultimately recovered with little to no separation. It was then determined that a reverse phase C-8 column on an HPLC would have to be used to afford the pure desired compound. After exploring different solvent conditions with compound 148, the use of a water and methanol gradient was able to yield pure triazole 148. However, the UV/vis trace at a wavelength of 210 nm showed multiple peaks that correlated to a partially hydrolyzed POM compound. Future purifications of this tetra POM compound would need to be explored to avoid loss of product and to decrease the time to collect large samples. sodium ascorbate CuSO 4 H 2 O (POMO) tbuoh/h 2 O (4:1) 2 (O)P + (POMO) 2 (O)P P(O)(OPOM) 2 3 (POMO) 2 (O)P Figure 42. Tetra POM triazole 144 With the tetra POM triazole 148 in hand, biological assays comparing it to the corresponding non-methylated salts would not be a fair comparison. In the next chapter, the methylated bisphosphonate sodium salts are described and the results of their biological assays are compared to the POM compounds. 66

94 In conclusion, the tetra POM prodrug moiety was successfully isolated and synthesized after various points in the synthetic scheme were explored to install this prodrug form. Installation had to occur after the formation of the alkyne click precursor, due to reaction conditions yielding a decomposed product. Installation after the formation of the triazole ring system also would be problematic because the basic nitrogens would interfere with the chloromethyl pivalate addition. It was also found that conversion of the tetraethyl ester bisphosphonates to the phosphonic acid and then an alkylation to chloromethyl pivalate was unsuccessful. Ultimately, through correct timing in the sequence to install the POM group and careful purification, compound 148 was obtained in sufficient quantity that it could be sent for bioassay. 67

95 CHAPTER 5 SYTHESIS AD BIOLOGICAL ACTIVITY OF METHYLATED BISPHOSPHOATES Previously discussed was the synthesis of compounds 95, 119, and 120, shown in Figure 43, and their biological activities were very encouraging as inhibitors of the target enzyme GGDPS. To enhance cell uptake, preparation of a prodrug form of the bisphosphonate as the tetra POM prodrug moiety was attractive. 101 However, previously described efforts to synthesize the prodrug of compound 95 were met with great challenge in part due to the acidity of the bisphosphonate s alpha position. To circumvent this issue, a methyl group was installed at the alpha position to replace the acidic proton. However, installation of the methyl group resulted in a slightly different class of triazole bisphosphonates. Because preparation of the methylated tetra POM prodrug 148 was successful, methylated analogs of the sodium salt were needed for direct comparison. If alpha methylation could maintain, or even improve, the activity of the corresponding bisphosphonates, it would eliminate concern over the acidity at the alpha position in the parent compounds. Below is reported the synthesis and biological activity of a new group of α-methylated isoprenoid triazole bisphosphonates. 68

96 (ao) 2 (O)P (ao) 2 (O)P 95 (ao) 2 (O)P (ao) 2 (O)P 119 (ao) 2 (O)P (ao) 2 (O)P 120 Figure 43. Previously synthesized triazole bisphosphonates as the sodium salts The methylation of these geminal bisphosphonates is straightforward. The methyl group could be installed at various points in the synthesis, but would ideally be installed after the formation of the triazole core, which would offer a more divergent synthesis. Methylation starts by treatment of the bisphosphonate tetraester with sodium hydride in the presence of 15-crown-5 to form the anion, which is then allowed to react with methyl iodide. The E/Z-mixture 149, along with the individual Z-isomer 150 and E-isomer 151, all undergo methylation smoothly under these conditions to afford the methylated products 152, 153, and 154 in moderate to good yield (Figure 44). Hydrolysis of the methylated bisphosphonates under the standard McKenna conditions provided the desired sodium salts 155, 156, and 157 in modest to good yield. However, sufficient material was obtained in all three cases to conduct the desired bioassays, so each reaction was conducted only once. 69

97 (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P ) ah, 15-C-5 THF, 0 C 30 min 2) MeI, 0 C 5 h 88% 1) ah, 15-C-5 THF, 0 C 30 min 2) MeI, 0 C 5 h 66% 1) ah, 15-C-5 THF, 0 C 30 min 2) MeI, 0 C 5 h 41% (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P (EtO) 2 (O)P ) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 2M aoh, rt 16 h 67% 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 2M aoh, rt 16 h 29% 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 2M aoh, rt 16 h 43% (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P Figure 44. Preparation of methylated triazole bisphosphonates In a parallel fashion, the homologated compounds also were converted to their methylated counterparts. The E/Z-mixture 94, along with the individual Z-isomer 117 and the E-isomer 118 all undergo methylation as previously described to afford the desired products 158, 159, and 160 in excellent yield, as shown in Figure 45. These methylated bisphosphonates also were converted to their corresponding sodium salts, compounds , in moderate to good yield under previously described McKenna hydrolysis conditions. 61 All six of these new methylated compounds were tested for biological activity by Dr. Sarah A. Holstein, and the results of those bioassays are described below. 70

98 (EtO) 2 (O)P (EtO) 2 (O)P 94 (EtO) 2 (O)P (EtO) 2 (O)P 117 (EtO) 2 (O)P (EtO) 2 (O)P 118 1) ah, 15-C-5 THF, 0 C 30 min 2) MeI, 0 C 5 h 81% 1) ah, 15-C-5 THF, 0 C 30 min 2) MeI, 0 C 5 h 96% 1) ah, 15-C-5 THF, 0 C 30 min 2) MeI, 0 C 5 h 96% (EtO) 2 (O)P (EtO) 2 (O)P 158 (EtO) 2 (O)P (EtO) 2 (O)P 159 (EtO) 2 (O)P (EtO) 2 (O)P 160 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 2M aoh, rt 16 h 35% 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 2M aoh, rt 16 h 56% 1) TMSBr, Collidine DCM, 0 C to rt 16 h 2) 2M aoh, rt 16 h 78% (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P Figure 45. Preparation of homoisoprene methylated triazole bisphosphonates The impact of substituents at the -carbon of the isoprenoid triazole bisphosphonates on cellular activity was assessed in myeloma cells. Impairment of cellular protein geranylgeranylation was determined via two methods: 1) ELISA for intracellular lambda light chain which is a marker for disruption of Rab geranylgeranylation 105 (Figure 46A); and, 2) immunoblot analysis for unmodified Rap1a (a substrate of GGTase I) (Figure 46B). Lovastatin, an HMG-CoA reductase inhibitor, was included in these experiments as a positive control

99 (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P Figure 46. Comparison of the effects of the -methylated triazole bisphosphonates 155 and 161 to the non-methylated analogues 96 and 95 on protein geranylgeranylation In both the geranyl/neryl/mixture series and the homogeranyl/homoneryl/mixture series, the addition of a methyl group at the -carbon resulted in enhanced cellular activity, with a larger magnitude of change observed in the geranyl/neryl length compounds. Furthermore, the homologated series again proved to be more potent than the parent 72

100 compounds. To extend the conclusion that addition of a methyl group at the -carbon resulted in enhanced cellular activity, parallel assays were run with both individual neryl and individual geranyl chains in the non-homo series and the homo series. The additional bioassays also showed that a methyl group improves activity. ext, the impact of the olefin stereochemistry on biological activity was determined. As shown in Figure 47, the Z-configuration of the geranyl-length compound 156 was approximately 50-fold more potent than the E-isomer 157. Interestingly, however, the activity of the two C 11 compounds (163 and 162) was very similar, with the E-isomer (163) only slightly more potent than the Z-isomer (162). These bioassay results reiterate that the Z-isomer is more potent than the E-isomer, now in the methylated series as well as with the non-methylated compounds, although in the methylated series the difference is small (a difference of less than 10 nm). 73

101 (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P (ao) 2 (O)P Figure 47. Effects of olefin stereochemistry on activity of the -methylated triazole bisphosphonates Finally, to investigate the effect of a larger substituent at the α-position compound 96 was converted to its ethyl derivative. This synthesis was straightforward given preparation of the α-methyl compounds above. Treatment of bisphosphonate 96 with sodium hydride in the presence of 15-crown-5 to form the anion followed by addition of ethyl iodide gave the alkylated derivative 164 in modest yield, and hydrolysis of the ethyl 74