Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 Synthesis and Evaluation of Anibamine and Its Analogs as Novel Anti-Prostate Cancer Agents Kendra Haney Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.edu/etd Part of the Chemicals and Drugs Commons © The Author Downloaded from https://scholarscompass.edu/etd/1974 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact libcompass@vcu. SYNTHESIS AND BIOLOGICAL EVALUATION OF ANIBAMINE AND ITS ANALOGS AS NOVEL ANTI-PROSTATE CANCER AGENTS A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University by By Kendra May Haney BS in Biochemistry at Washington and Lee University, 2006 Major Director: YAN ZHANG, Ph.
ASSISTANT PROFESSOR, DEPARTMENT OF MEDICINAL CHEMISTRY Virginia Commonwealth University Richmond, Virginia December, 2009 ii Acknowledgements I would like to take this opportunity to thank my advisor, Dr. Yan Zhang, for his guidance and teaching from the first day I was accepted in to the Department of Medicinal Chemistry until now. I would also like to thank him for providing me with generous financial support. My day to day experiences in the lab could not have been successful without the help from Dr.
Guo Li whose patience is endless. I would also like to thank Dr. Joy Ware in the Department of Pathology for the opportunity to work in her lab and gain valuable experience outside an organic chemistry lab and Amanda Richardson for her infinite help with cell culture as well as for always letting me talk about my mutts. I would like to thank the other members of Dr.
Yan Zhang’s lab, past and present, for helping me learn more about organic chemistry and teamwork. To my friends, I would like to extend my gratitude for giving me friendship, support, and laughter. Thank you Genevieve, for always being willing to take a break. I am especially grateful to Nolan, for sitting through practices of seminars and listening to every detail about my day, and loving me anyway.
I would like to thank my family for tolerating my extended stay in Virginia, for buying many plane tickets, and all their love and support. I would especially like to thank my parents for their constant love and support throughout my life, especially in my higher education years. I would never have come this far in science without the interest and knowledge instilled in me by my high school chemistry teacher, Mr. Bill Cunningham, and my undergraduate chemistry professor, Dr.
And love and thanks to my father and grandfather, without whom I might not have found myself with such a passion for anti-cancer research. iii Table of Contents Acknowledgements….…ii List of Tables…………………………………………………………………….…ix List of Figures………………………………………………….x List of Schemes……………………………………………….……xii List of Abbreviations…………………………………. The Prostate and Prostatic Disorders…………………………………………. Prostate Cancer Cell Lines…………………………………………….
Inflammation and Prostate Cancer…………………………………. CCR5 and CCL5/RANTES in PCa…………………………………………. Prevention and Treatment……………………………………………………. Chemokine and Chemokine Receptor Structure and Signaling………….
Chemokines and the Tumor Microenvironment……………………………. Chemokines and Immunotolerance……………………………. Chemokines and Metastasis…………………………………………. The Chemokine/Chemokine Receptor System in Cancer Therapy……….
CC Chemokine Receptor 5 (CCR5) Structure, Function, and Antagonists…. Natural Products and Drug Discovery…………. Natural Products and Their Target Proteins…………………………………. Structural Attributes of Natural Products…………….
From Traditional Medicine to the NCI Cancer Panel…. From Extract to Drug Candidate……………………. Camptothecin and Taxol as Models of Natural Product Drug Discovery……. Influence of Natural Products on Cancer Biology….
Anibamine, a Natural Product Chemokine Receptor CCR5 Antagonist…. Summary of Impact of Natural Products on Drug Discovery…………. Results and Discussion……………………………………………………………. Chemical Synthesis of Anibamine and Analogs as CCR5 Antagonists……….
Synthesis of key intermediates in each route………………………. Bromination of key intermediates …………………………………………. Hydrogenation of alkyne intermediates……………………………. Exploration of sidechain coupling reactions………………………….
Deprotection and cyclization reactions. Separation of isomers…………………………. Anti-proliferative Activity of Anibamine and its Analogs……………………. Anti-proliferative activity on PC-3 cell line…………………………….
Anti-proliferative activity on DU-145 cell line…. Anti-proliferative activity on M12 cell line…. Anti-proliferative activity of deconstructed analogs…. Anti-proliferative effect over time………….
Dynamics Simulations and Docking of Anibamine Analogs…………………. Modeling of anibamine and its analogs……………………………. Dynamics simulation of prepared homology models………………. GOLD docking of ligands into CCR5 homology models…………….
Analysis of ligand binding to CCR5 model based on 1F88 structure……. Analysis of ligand binding to CCR5 model based on 2RH1 structure………. Comparison of ligand docking in each receptor model. Synthesis of anibamine analogs……………………………….
Intermediates in Anibamine series a………………………………………….86 5-Bromo-2-hydroxy-4,6-dimethylnicotinonitrile (6)…………………. 86 2-Hydroxy-4,6-dimethylpyridine-3,5-dicarbonitrile (2a)……………………….87 2-Bromo-4,6-dimethylpyridine-3,5-dicarbonitrile (8a)…………………………88 1-Methoxy-4-((prop-2-ynyloxy)methyl)benzene (10)………………………….91 Non-1-yl triphenylphosphonium bromide (14)…………………………………. Final products in series a…………………………………………………………. Intermediates in the synthesis of series b………………………………………….
Final products in series b……………………………………………………………105 8-dec-1Z-enyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium chloride (1b)…….105 8-dec-1E-enyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium chloride (17b)….106 8-decyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium (20b)……………………106 5. Intermediates in the synthesis of series c…………………………………………107 3-morpholinobut-2-enenitrile (7)…………………………………………….108 viii 6-bromo-2,4-dimethylnicotinonitrile (8c)………………,……………………108 6-(3-((4-Methoxybenzyloxy)prop-1-ynyl)-2,4- dimethylnicotinonitrile (11c)………………………. Final products in series c……………………………………………………………….115 6-dec-1Z-enyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium chloride (1c)…….115 6-dec-1E-enyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium chloride (17c)….115 6-decyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium (20c)………………. Biological screening of CCR5 antagonists……………………………………………….
Cell culture method …………………………………………………………. Anti-proliferation assay protocol……………………………………………………. Molecular Dynamics simulations and docking of anibamine analogs………………….…120 ix List of Tables page Table 1 Reaction conditions, yields and stereoselectivity of the various coupling reactions….51 Table 2 Half maximal inhibitory concentration (IC50) of 12 compounds in three cell lines at 72 hours…………………………….60 Table 3 Percent inhibition of deconstructed analogs in the M12 cell line at 72 hours….67 Table 4 Percent inhibition of deconstructed analogs in the PC-3 cell line at 72 hours.67 Table 5 Percent inhibition of deconstructed analogs in the DU-145 cell line at 72 hours.67 Table 6 Absorbance values of 20c at three time intervals in three cell lines.……………………69 Table 7 GOLDscores in 1F88 CCR5 model……….74 Table 8 GOLDscores in 2RH1 CCR5 model…………….79 x List of Figures Figure 1. 2-D structure of CCR5 with palmitoylation sites and two disulfide bonds…….
Structures of CCR5 antagonists-Maraviroc, TAK-779 and Anibamine……………. Structures of natural products-nicotine, quinine, and morphine…. Examples of reactive functional groups in natural products…. Structures of taxanes-taxol, 10-deacetylbaccatin III, and taxotere….
Structures of camptothecin and analogs irinotecan and topetecan…. Proposed structural modifications of anibamine……………………………. Structures of anibamine, 1a, and proposed deconstructed analogs 1b and 1c. Key intermediates in each synthetic pathway……………………………….
Geometric isomers of target compounds…. All synthesized compounds…………………………………………………. Metabolic cleavage of WST-1 into soluble formazan dye……………………. Structures of Anibamine and eleven analogs……….
Percent inhibition of PC-3 cell line by 24 at four concentrations…………………. Structures and IC50 of 24, 21, and 1a…………………………………………….62 Figure 16 Percent inhibition of DU-145 cell line by 17a at four concentrations………. Structures of 17a, 22, and 25…………………………………………………….63 Figure 18 Percent inhibition of M12 cell line by 26 at four concentrations…………….64 xi Figure 19 The percent inhibition of both saturated deconstructed analogs in DU145 cell line……………………………………………………………. Structures of anibamine and docked ligands………………………………….
The common indazolinium core of anibamine and all docked ligands with labeled key carbons………………………………………………………………. Binding of anibamine in the CCR5 model based on 1F88……………………. Binding configuration of 18a and anibamine in the 1F88-based model……. Overlay of Anibamine and 1c in 1F88-based model…………………………….
Overlay of 18a and 18c in 1F88-based CCR5 model……………………………. Binding of 1a and 20a in !2-AR based model…………………………………. The binding modes of 17c, 20c and 1c in !2-AR based model.80 xii List of Schemes Scheme 1. Total synthesis of anibamine (1a) from acetyl acetone…………………………….37 Scheme 2 Route 1 to the first intermediate in anibamine synthesis.40 Scheme 3 Route 2 to the first intermediate in anibamine synthesis…………………………….40 Scheme 4: Mechanism of Rosenmund-von Braun reaction…………………………………….41 Scheme 5: Mechanism for domino halide exchange-cyanation of aryl bromides…………….42 Scheme 6 Synthetic route to the first intermediate in the synthesis of 1c …………………….43 Scheme 7 Synthetic route from key intermediate to hydrogenation product………………….44 Scheme 8 Synthesis of PMB protected propargyl alcohol…………………………………….44 Scheme 9: Synthetic route from first hydrogenation product to Wittig product in synthesis of Anibamine…….46 Scheme 10: Synthetic route from first hydrogenation product to Wittg product in synthesis of of 1b and 1c……………………………………………………………………………….47 Scheme 11 Synthesis of non-1-yl triphenyl phosphonium bromide (14).47 Scheme 12: Proposed mechanism for Schlosser modification of the Wittig reaction………….49 Scheme 13: Synthetic route from Wittig product to final product for all routes……………….52 xiii List of Abbreviations Å Angstroms °C degrees Celsius " chemical shift % percent AAH atypical adenomatous hyperplasia 10-CSA 10-camphorsulfonic acid AcOH acetic acid AR androgen receptor br broad peak bFGF basic fibroblast growth factor CCL2 CC chemokine ligand 2 CCL3 CC chemokine ligand 3 CCL4 CC chemokine ligand 4 CCL5 CC chemokine ligand 5 CCL7 CC chemokine ligand 7 CCL8 CC chemokine ligand 8 CCL19 CC chemokine ligand 19 CCL20 CC chemokine ligand 20 CCL21 CC chemokine ligand 21 CCR2 CC chemokine receptor 2 CCR5 CC chemokine receptor 5 CCR7 CC chemokine receptor 7 CDCl3 deuterated chloroform CH2Cl2 dichloromethane CHCl3 chloroform CI chronic inflammation CO2 carbon dioxide CXCL8 CXC chemokine ligand 8 CXCL12 CXC chemokine ligand 12 CXCL16 CXC chemokine ligand 16 CXCR4 CXC chemokine receptor 4 d doublet DC dendritic cells xiv DHT dihydroxytestosterone DIBAL-H di-isobutyl aluminum hydride DMF dimethylformamide DMSO dimethylsulfoxide DU-145 dura mater derived prostate cancer cell line ECM extracellular matrix EGF epidermal growth factor EGF-F epidermal growth factor receptor EL2 extracellular loop 2 EtOAc ethyl acetate Et2O diethyl ether FBS fetal bovine serum FGF-R fibroblast growth factor receptor fs femtoseconds g grams G-CSF granulocyte-colony stimulating factor GOLD genetic optimization for ligand binding GPCR G protein coupled receptor HIV human immunodeficiency disorder HPV human papillomavirus Ic50 half maximal inhibitory concentration IFN interferon IGF insulin-like growth factor IGFB insulin-like growth factor binding protein IGF-I insulin-like growth factor type 1 IGFR insulin-like growth factor receptor IL interleukin IP3 inositol-1,4,5-triphosphate IR infrared ITS insulin, transferrin, selenium kDa kilodalton Kow octanol-water partition coefficient LC50 half maximal lethal concentration LHMDS lithium hexamethyldisalizide LNCaP lymph node derived prostate cancer cell line µM micromolar m multiplet M12 metastatic prostate cancer cell line MeOH methanol xv MHz megahertz mL milliliters mmol millimolar MMP matric metalloproteinase mRNA messenger ribonucleotidic acid MsCl methane sulfonyl chloride NAD: oxidized nicotinamide adenine dinucleotide NADH reduced nicotinamide adenine dinucleotide NH4OH ammonium hydroxide nm nanometers nM nanomolar NMR nuclear magnetic resonance P69 non-neoplastic prostate epithelial cell line P PBS phosphate buffer solution PC-3 bone derived prostate cancer cell line PCa prostate cancer PIA proliferative inflammatory atrophy PIN prostate intraepithelial neoplasia PLC! phospholipase C! PSA prostatic specific antigen RANTES regulated upon activation normal T cell expressed SV40 Simian vacuolating virus 40 T triplet TADC tumor-associated dendritic cells TAM tumor-associated macrophages TBAB tetrabutylammonium bromide TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TIL tumor-infiltrating cells TGF-# transforming growth factor alpha TGF-! transforming growth factor beta TGF-!-R transforming growth factor beta receptor TLC thin layer chromatography TNF tumor necrosis factor xvi Abstract SYNTHESIS AND BIOLOGICAL EVALUATION OF ANIBAMINE AND ITS ANALOGS AS NOVEL ANTI-PROSTATE CANCER AGENTS By Kendra May Haney A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University Virginia Commonwealth University, 2009 Major Director: Yan Zhang, Assistant Professor, Department of Medicinal Chemistry The chemokine receptor CCR5 has been implicated in the pathogenesis of prostate cancer.
A novel natural product, anibamine, was isolated and found to be a micromolar inhibitor of the receptor. Anibamine was used as a new anti-prostate cancer lead compound. To discover the pharmacophore, analogs of anibamine were designed using the “deconstruction- reconstruction-elaboration” approach and synthesized. The establishment of a stereoselective route to only one isomer was explored, to increase yield and eliminate elaborate purification procedures.
Analogs were found to have anti-prostate cancer activity at levels higher than the parent compound. The molecular modeling studies of the deconstructed analogs indicate that due xvii to the psuedo-symmetry of the parent compound, the binding conformation of the deconstructed analogs may not be very different from each other. All this information together may help identify a next generation lead compound for anti-prostate cancer treatment. Introduction Prostate cancer (PCa) is the most common cancer in men after lung cancer.1 The American Cancer Society estimates that nearly 200,000 new cases of PCa will be diagnosed in 2009, with 27,000 deaths attributable to PCa in the United States alone.