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DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Patrick John Ward, A.B.

*****

The Ohio State University

1996

Dissertation Committee: Approved by

R. W. Brueggemeier L. W. Robertson J. V. Hines Advisor Y.C.Iin College of Pharmacy UMI NUmber: 9630999

UMI Microform 9630999 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 First and last, this work is dedicated to my love and my inspiration, my wife Jacqueline. I am forever grateful to my family, the Ward's and the Ebner's, without whose support and love I never would have finished this work.

When I began this journey, I never dreamt that it would come to an end. As my graduate career draws to a close, I have come to realize that it is never supposed to end - that the point of the journey is not to arrive. It is with humility, and immense pride that for the remainder of my journey, when asked who I am, I may now answer, "I am a scientist." ACKNOWLEDGEMENTS

Most people of my generation do not have heroes. Somehow it isn’t thought proper anymore. My hero is Woody Hayes, and I would like to respectfully acknowledge the inspiration he has provided me in life.

I am fortunate to have had many mentors. I would like to recognize the following people as having been such to me. Dr. Robert Breitenbach, Dr. Dean Metter, and Dr. Roger DeRoos -- gentlemen, your words and their knowledge ring in my ears to this day. Dr. John Neff, Dr. Thomas O’Dorisio, and Dr. Sue ODorisio --1 hope that one day I may follow your example.

I would like to express my deep and sincere gratitude to my advisor, Dr. Robert Brueggemeier. His guidance, encouragement and support were of such fundamental importance that I must state that I would not have been capable of completing my graduate studies without them.

I would like to thank and acknowledge my committee, Dr. Young Lin, Dr. Larry Robertson and Dr. Jennifer Hines. Their guidance and insight were invaluable. I owe a large debt of gratitude to my colleagues in the laboratory, Dr. Carl Lovely, Dr. Jill O’Reilly, Dr. Mike Darby, Muriel Liberto, Anne Quinn, Abijit Bhat, and Damon Sharp. Their assistance, knowledge and insight were invaluable.

I would like to thank and acknowledge Dr. Albert Soloway, Dr. Robert Curley, Dr. Raymond Doskotch, Dr. Duane Miller, Dr. Dennis Feller, Dr. Nigel Preistley, and Dr. Steve Bergmeier for their knowledge and insight. Teaching goes far beyond the simple dissemination of information. These men understand this implicitly.

I would like to acknowledge those who went before me and illuminated the path, Dr. Mustapha Beleh, Dr. Soheila Ebrahemian, Dr. Mike Pcolinsky, Dr. Jeff Cristoff, and Dr. Jill O’Reilly.

Finally, it is with great sadness that I would like to remember two individuals whose stay on this earth ended while I did this work, Dean Thomas Harris and my Mend Herman.

v ...pay no attention to the man behind the curtain. -The Wizard of Oz

Life is too important to be taken too seriously. -Oscar Wilde

We must not be suspicious of the answers nature provides us, for she is always truthful. Rather we should be careful of the questions we ask of her. -A Szent-Georgi VITA

August 9, 1963 ...... Born, Columbus, Ohio

1986 ...... A.B., University of Missouri, Columbia, Missouri

1987-Present ...... Medical Scientist Training Program Fellow, The Ohio State University, Columbus, Ohio

FIELDS OF STUDY

Major Field: Medicinal Chemistry Studies in the biochemistry and endocrinology of steroidal inhibitors of biosynthesis for the purpose of developing effective therapies for the treatment of steroid-dependent tumors of the breast and . TABLE OF CONTENTS

Page DEDICATION...... ii ACKNOWLEDGMENTS...... iv VITA...... vii

LIST OF FIGURES...... xi LIST OF TABLES...... xv LIST OF SCHEMES...... xvi ABSTRACT...... xvii CHAPTER 1 INTRODUCTION...... 1 1.1 ...... 1 1.2 ...... 4 1.2.1 History...... 4 1.2.2 Incidence, Mortality, Prevalence a & Risk ...... 4 1.2.3 Screening ...... 5 1.2.4 Anatomy & Histology ...... 6 1.2.5 Diagnosis & Tumor Grading ...... 8 1.2.6 Clinical Staging & Prognosis ...... 8 1.2.7 Carcinoma of the Prostate vs. Benign Prostatic Hypertrophy ...... 9 1.2.8 Pathophysiology of Disease Progression ...... 12 1.2.8.1 Levels ...... 12 1.2.8.2 Androgen Receptor Mutations ...... 13 1.2.8.3 Intraame/Autocrine/Paracrine Factors ...... 17 1.2.8.3.1 Growth Factors ...... 17

viii 1.2.8.3.2 Other Epithelial-Stromal Interactions ...... 27 1.2.8.3.3 Other Endocrine Factors ...... 28 1.2.8.4 Genetic Alterations: Oncogenes, Tumor Suppressor Genes, & Chromosomal Alterations ...... 32 1.2.8.5 Adaptation vs. Selection ...... 34 1.3 Prostate Cancer Treatment Modalities ...... 34 1.3.1 Surgery, Radiation & Cytotoxic ...... 34 1.3.2 Endocrine Manipulation ...... 35 1.3.2.1 Hypothalamic-Pituitary-Testicular Axis Disruption ...... 36 1.3.2.1.1 Orchiectomy & Hypophysectomy ...... 36 1.3.2.1.2 ...... 37 1.3.2.1.3 Progestins ...... 38 1.3.2.1.4 GnRH ...... 38 1.3.2.2 ...... 41 1.3.2.2.1 Antiandrogens ...... 42 1.3.2.2.2 Steroidal Antiandrogens ...... 43 1.3.2.3 Disruption of Androgen Biosynthesis ...... 47 1.3.2.3.1 5a-Reductase...... 48 1.3.2.3.2 17,20-Lyase...... 54 1.4 Breast Cancer ...... 71 1.4.1 Breast Cancer Treatment ...... 72 1.4.1.1 Hypothalamic-Pituitary-Ovarian Axis Disruption 74 1.4.1.1.1 Oophorectomy, Hypophysectomy & Adrenalectomy ...... 74 1.4.1.1.2 Progestins ...... 75 1.4.1.1.3 GnRH Agonists ...... 75 1.4.1.2 ...... 76 1.4.1.3 17,20-Lyase Inhibitors ...... 79 1.4.1.4 ...... 80 CHAPTER 2 STATEMENT OF PROBLEMS AND OBJECTIVES...... 90 2.1 Objective 1: Evaluation of 7a-Substituted for 17,20-Lyase Inhibition ...... 91

ix 2.2 Objective 2: Evaluation of 7a-Substituted Steroids in vivo...... 93

CHAPTER 3 EXPERIMENTAL METHODS...... 94 3.1 General Procedures - Chemistry ...... 94

3.2 Synthetic Methods ...... 95 3.3 General Procedures - Biochemistry ...... 107 3.4 Biochemical Methods ...... 108 3.5 In Vivo Studies...... 117 CHAPTER 4 RESULTS AND DISCUSSION...... 120 4.1 Chemistry ...... 120 4.2 Biochemistry ...... 128

4.3 In Vivo Studies...... 135 CHAPTER 5 CONCLUSIONS AND SUMMARY...... 146 REFERENCES...... 151 APPENDIX A *H and 13C NMR Spectra of Target Compounds ...... 196

x LIST OF FIGURES

Figure Page

1 Schematic representation of the human prostate, showing the transitional and peripheral zones ...... 7

2 Schematic representation of the functional organization of the human androgen receptor ...... 15

3 Schematic depicting the effects of various growth factors on both normal and malignant prostatic epithelial cells 26

4 Schematic depiction of the possible sites of endocrine manipulation in the treatment of prostatic adenocarcinoma ...... 36

5 Structure of naturally occuring GnRH and several synthetic GnRH agonists ...... 40

6 Structure of nonsteroidal antiandrogens ...... 44

7 Structure of -based steroidal antiandrogens ...... 45

8 Structure of -based steroidal antiandrogens ...... 46

9 Structure of and the 4-azasteroid- based antiandrogens ...... 47

10 Structure of nonsteroidal 5a-reductase inhibitors ...... 49

11 Structure of steroidal 5a-reductase inhibitors ...... 51

xi Figure Eflge

12 Structure of 3-addic substituted and azasteroid 5oc-reductase inhibitors ...... 53

13 Androgen Biosynthesis ...... 56

14 Proposed enzymatic mechanism of 17

15 Proposed enzymatic mechanism of 17,20-lyase ...... 61

16 Structure of nonsteroidal inhibitors of 17,20-lyase ...... 65

17 Structure of steroidal inhibitors of 17,20-lyase ...... 66

18 Structure of steroidal competitive inhibitors of 17,20-lyase...... 69

19 Structure of steroidal irreversible inhibitors of 17,20-lyase...... 70

20 Schematic depiction of the possible sites of endocrine manipulation in the treatment of mammary carcinoma 74

21 Structure of representative antiestrogens ...... 78

22 Estrogen Biosynthesis ...... 81

23 Proposed enzymatic mechanism of aromatase ...... 82

24 Structure of nonsteroidal aromatase inhibitors ...... 84

25 Structure of competitive steroidal aromatase inhibitors ...... 87

26 Structure of steroidal -activated irreversible aromatase inhibitors ...... 89

27 Structure of proposed steroidal 17,20-lyase inhibitors ...... 92

xii Figure Page

28 In vitro experimental values for 7a-APTADD ...... 93

29 Double reciprocal (Lineweaver-Burke) plot of kinetic data evaluating 7p-phenethylandrostenedione (7P-PEA, 98) for 17,20-lyase inhibitory activity ...... 132

30 Log dose-response data and sample calculations evaluating , 48, 7a-APTADD, 87, and 7p-PEA, 98, for 17,20-lyase inhibitory activity ...... 134

31 Inhibition of rat mammary tumor growth by 7a-APTADD.... 137

32 Radioimmunoassay of serum levels ...... 138

33 Radioimmunoassay of serum estradiol levels ...... 142

34 Remaining ovarian aromatase activity ...... 144

35 Relative ovarian aromatase mRNA levels ...... 145

36 1H-NMR of 7a-butylthio-4-pregnen-3,20-dione, 87 ...... 197

37 13C-NMR of 7a-butylthio-4-pregnen-3,20-dione, 87 ...... 198

38 1H-NMR of 7a-(2'-methylpropylthio)-4-pregnen- 3.20-dione, 88 ...... 199

39 13C-NMR of 7a-(2'-methylpropylthio)-4-pregnen- 3.20-dione, 88 ...... 200

40 !H-NMR of 7a-phenylthio-4-pregnen-3,20-dione, 89 ...... 201

41 13C-NMR of 7a-phenylthio-4-pregnen-3,20-dione, 89 ...... 202

42 1H-NMR of 7a-(4'-chlorophenylthio)-4-pregnen- 3.20-dione, 90 ...... 203 xiii £ifiai££ Pace 43 13C-NMR of 7a-(4'-chlorophenyltluo)-4-pregnen- 3.20-dione, 90 ...... 204 44 !H-NMR of 7a-(4'-aminophenylthio)-4-pregnen- 3.20-dione, 91 ...... 205 45 13C-NMR of 7a-(4'-aminophenylthio)-4-pregnen- 3.20-dione, 91 ...... 206 46 ^-NMR of la-phenylthio-4-pregnen-3,20-dione, 92 ...... 207

47 13C-NMR of la-phenylthio-4-pregnen-3,20-dione, 92 ...... 208

xiv LIST OF TABLES

Table Ease 1 Carcinoma of the Prostate: Clinical Staging System ...... 10 2 Growth Factors Implicated in Prostate Cancer ...... 19

3 Structure-activity Relationships of Progestin Analogs 129 4 Structure-activity Relationships of Androgen Analogs 130 5 17,20-Lyase Inhibition ...... 133

xv LIST OF SCHEMES

Scheme Page I Synthesis of pregna-4,6-diene-3,20-dione and pregna-4,6,16-triene-3,20-dione ...... 121 II Synthesis of pregna-l,4-diene-3,20-dione ...... 123 III Synthesis of 7a-thio substituted progestins ...... 124

IV Synthesis of la-phenylthioprogesterone ...... 125

V Synthesis of androsta- l,4,6-triene-3,17-dione (A14>6-ATD)...... 125

VI Synthesis of 7a-APTADD ...... 126

VII Salvage of A1>4>6-ATD from unwanted mono and bis thio addition products ...... 127

xvi DEVELOPMENT OF STEROIDAL INHIBITORS OF CYTOCHROME P450-DEPENDENT ANDROGEN AND ESTROGEN BIOSYNTHESIS

By

Patrick John Ward, Ph.D.

The Ohio State University, 1996

Professor Robert W. Brueggemeier, Advisor

Steroid-dependent tumors of the breast and prostate represent a significant number of cancer diagnoses, morbidity and mortality. and estrogens play a pivotal role in the progression and growth of prostate and breast malignancies. The disruption of their, biosynthesis is a logical approach to depriving steroid-dependent tumor cells of their supply of . Development of steroidal compounds which possess selectivity for these over other steroidogenic enzymes and high affinity for the active site would be desirable both for their potential clinical utility and for possible insights they might provide into the structure of the active site.

17,20-Lyase and aromatase are heme-containing monoxygenases which oxidatively remove small alkyl side-chains. The proposed mechanistic details for these enzymes are similar. It was therefore postulated that similarities may exist between the two enzyme active sites. Specifically, the 17,20-lyase active site may accommodate substitution at the 7a-position as does aromatase. The structure-activity relationships of B-ring substituted progestins and androgens for cytP450i7a inhibition had not been investigated.

la-Phenylthioprogesterone and a series of 7a-thio-substituted progestins were prepared. These and a number of 7a-substituted , were screened for 17,20-lyase inhibition in a rat testis microsomal assay. None of the proposed progestins or androgens displayed significant 17,20-lyase inhibitory activity. This suggests structural differences between the 17,20-lyase and aromatase active sites despite mechanistic similarities. Specifically, the aromatase active site accomodates substitution at the steroid 7a-face, whereas the 17,20-lyase active site does not. Thus, the potent inhibitor, 7a-APTADD, exhibits selectivity for aromatase.

7a-APTADD is a potent enzyme-activated irreversible inhibitor of aromatase which had not been evaluated in vivo. 7a-APTADD showed significant tumor growth inhibition at two doses (25 mg/Kg, and 50 mg/Kg) during a six week treatment period in an estrogen-dependent rat mammary tumor model. When evaluated in normal, cycling adult female rats for its effect on endocrine physiology, it inhibitied serum estradiol levels, ovarian aromatase activity and aromatase mRNA expression. CHAPTER 1

INTRODUCTION

1.1 Cancer

The American Cancer Society (ACS) estimates that 1,252,000 new diagnoses of cancer will be made in 1995. This figure does not include carcinoma in situ, a neoplastic condition in which the tumor has not yet traversed the basement membrane. It also does not include the basal and squamous cell carcinomas of the epidermis. These add an additional 800,000 cases to the previous figure. The ACS also estimates that 547,000 patients will die of cancer in 1995--one in five deaths in the U.S. Over the course of the lifetime of an individual, the aggregate risk of developing a clinically diagnosed cancer is currently estimated to be one in three. Cancer represents the second leading cause of mortality in the United States, exceeded only by heart disease.1,2

Medical science has been comfortable for several decades with the general idea that cancer represents a fundamental derangement in the control of cell growth and differentiation. This disorder arises in rare cells through changes in genes or gene expression which give the altered cells a growth advantage over surrounding cells. These changes are inherited by daughter cells like a stable genetic trait. The net result of these alterations is conversion of the affected cells to the transformed state:

1 2 anaplasia and uncontrolled growth. What has been much less clear until the past 10 to 15 years is the identity of the genes affected in a cancer cell, the nature of the changes in these genes, and how these changes might alter the normal control mechanisms governing the cell cycle, cellular growth, proliferation and differentiation. Many hypotheses have been put forth to account for the origins of cancer. For example, cancer may result from the introduction of viral genes or fragments of viral genes into somatic cells. Cancer may result from the activation of viral genes that preexist in our germ cell lines as a consequence of ancient viral infections. Cancer may arise as a result of inappropriate expression of cellular genes that are beneficial in other cell types, or in the same cell type at another stage of ontogeny. And finally, cancer may result from damage to, or other physical mutations in, an important subset of our genes.

These hypothetical genes which influence carcinogenesis have been proposed to control important cellular processes, such as nuclear events (DNA replication, transcription and repair), the cell cycle, metabolic pathways, or cell surface interactions which regulate growth. When their pattern of expression is normal, these genes are termed proto-oncogenes, and they are expressed at the appropriate time during the cell cycle, at the appropriate time during ontogeny, at appropriate levels, and with appropriate biological activity. When they become altered by carcinogenic mechanisms such that their pattern of expression becomes deranged, or their biologic activity is inappropriate, they are termed oncogenes, and cancer may result.3'5 3

All cancerous processes share in common three interrelated features which characterize the degree of malignancy or aggressiveness of a tumor. These features are growth, invasiveness, and metastasis. Growth, or neoplasia, refers to the size of the tumor and the rate at which mitosis is occurring. Tumor size and doubling time are general indices of malignancy. Tumor neovascularization is intimately related to growth, and as long as expansion of the vascular supply and the rate of tumor cell division are closely matched, tumor necrosis does not occur. Invasiveness refers to the ability of the tumor to breach the extracellular matrix, or basement membrane, surrounding the neoplasm. This involves production and secretion of enzymes, cell motility and tumor/stroma interactions. Metastasis refers to the ability of the primary tumor to colonize distant sites with tumor cells, thus producing a secondary growth. Metastasis is strongly dependent on invasion, cell motility, cell adhesion and the ability of the tumor cell to avoid immune surveillance. The ultimate insulting event (e.g. ischemia, infarction, hemostatic or hemodynamic compromise, hemorrhage, organ system failure) which leads to patient mortality may be the result of any of these features of malignancy, individually or in combination.4

Finally, the lack of basic understanding of the exact pathophysiologic processes which result in the malignant phenotype has resulted in the imprecise and often confusing characterization of neoplasms based on anatomic location, histology and embryology. Thus, carcinoma of a particular anatomic structure may indeed be referring to a multitude of pathologic processes and resultant malignant disease states. Conversely, 4 the same pathologic process may be operative in a multitude of tissues, resulting in a wide variety of malignancies. Fortunately, this situation is changing. As tumor biology and the pathways leading to malignancy become better understood, new therapeutic targets will continue to be identified, and thus, rationally designed therapies and preventive strategies will result.

1.2 Prostate Cancer

1.2.1 History

In 1817, Langstaff6 and Wadd7 independently described the first two reported cases of carcinoma of the prostate. It was not until 1900 that the disease received much attention from the medical community, when Albarran and Halle8 described a number of cases of carcinoma of the prostate from specimens presumed to be benign prostatic hypertrophy. In 1904, Young9 performed the first radical prostatectomy. In 1925, Broder10 published the first prostate tumor grading system as a means of differentiating latent tumors from more aggressive disease.

1.2.2 Incidence, Mortality, Prevalence & Risk

Adenocarcinoma of the prostate represents the leading cause of cancer among men in the United States and the second leading cause of cancer mortality. It leads lung cancer in incidence (2.54 times the lung cancer incidence) and is second to it in mortality (0.42 times the lung cancer mortality). The ACS estimates that 244,000 new diagnoses of prostate cancer will be made in 1995. Presumably because of improved 5 methods of detection, the incidence has grown by 50% in the past 15 years. Further increases in incidence are expected as the widespread use of serum prostate specific antigen (PSA) screening continues to be implemented. The ACS estimates that there will be 40,400 deaths due to prostate cancer in 1995. The incidence of prostate cancer increases with age; with greater than 80% of cases being diagnosed in men age 65 and older. The prevalence of the disease is greatest in North America and western , with black Americans having the highest incidence in the world. The disease is rare in Africa, the near east, central and south America. There is no clear-cut environmental association with the disease explaining its geographic distribution.1,2,11

1.2.3 Screening

Increasingly, focus has turned toward screening and early detection. Despite numerous recent advances in medical and surgical therapy, there has been little change in the prostate cancer mortality rate over the past 30 years.1,2 The ideal screening test employs methodology which is sensitive, inexpensive, has low morbidity and is easy to perform. The goal of screening is to identify disease early enough in its natural history such that therapeutic intervention will increase patient survival and decrease patient morbidity. Traditionally, digital rectal examination (DRE) has been the method used to evaluate the prostate gland. Two newer methods, measurement of serum prostate specific antigen (PSA - a serine protease related to the kallikreins12) levels, and transrectal ultrasonography (TRUS) have begun to be used as screening tests.13 An index termed the 6

PSA density (PSAD), which combines the previous two methods as the ratio of serum PSA to prostate volume, is also being used to distinguish elevated PSA due to malignancy from benign prostatic hypertrophy (BPH). The indications and methods used for prostate cancer screening remain controversial. There is currently a multi-center study in progress, the American Cancer Society National Prostate Cancer Detection Project.14 The goal of this study is to aid in resolving these controversies by providing data on which the justification for screening programs will be based.15

1.2.4 Anatomy & Histology (Figure 1)

The prostate is an accessory gland in the seminal outflow tract. It is a retroperitoneal organ which lies inferior to and encircles the neck of the urinary bladder, as well as the proximal portion of the urethra. It lies anterior to and abuts the rectum. The ejaculatory ducts join with the urethra within the prostate at a prominence referred to as the verumontanum. The ejaculatory ducts are formed by the union of the vas deferens with the ducts of the seminal vesicles along the posterolateral aspect of the gland. The prostate is conical in shape and is comprised of three major lobes; two lateral and one posterior. The region of the prostate which encircles the mouth of the bladder and lies proximal to the verumontanum is referred to as the transitional zone, and the region of the posterior lobe which lies closest to the rectum is called the peripheral zone.

The prostate is a compound tubuloalveolar gland. The parenchyma consists of cuboidal, stratified columnar, or pseudostratified columnar epithelium that forms tubules and alveoli which can at times be irregular 7

Vas Deferens

Seminal Vesicle Bladder

Transitional Ejaculatory Zone — ” Duct Verumontanum

Peripheral Zone

Urethra

Figure 1. Schematic representation of the human prostate, showing the transitional and peripheral zones.

with large cavities containing papillae or folds of parenchyma with a thin core of connective tissue stroma. The epithelial cells of the parenchyma appear to be of two functional types, basal and secretory. The basal lamina is indistinct, and the epithelium rests on a layer of connective tissue with dense networks of elastic fibers and numerous capillaries. The 8 ducts are lined by simple squamous epithelium, and empty into the prostatic urethra on the right and left sides of the verumontanum. The gland is surrounded by a capsule and is invested with a rich fibrous connective tissue stroma consisting of collagen fibers, fibroblasts and smooth muscle cells.16,17

1.2.5 Diagnosis & Tumor Grading

The diagnosis of prostate cancer is made pathologically and is based upon histologic examination of a prostate biopsy specimen and observation of neoplastic changes. Tumor grading is the pathologist’s attempt to prognosticate the behavior of prostate cancer. Briefly, the factors taken into account are the extent of tissue involvement, the degree of nuclear anaplasia (nuclear grade), and the pattern of glandular differentiation (histologic grade).18-25 The problems and controversies associated with grading are numerous and beyond the scope of this text. Irrespective of the grading system used, all systems distinguish the very aggressive tumors with poor prognosis from the non-aggressive tumors with excellent prognosis. Unfortunately, most tumors fall in the intermediate group wherein he the disagreements and controversies.

1.2.6 Clinical Staging & Prognosis

Numerous staging systems for prostatic carcinoma have been devised and because each has shortcomings, there is controversy surrounding them. Although the Tumor-Nodes-Metastasis (TNM) system is gaining wider acceptance,26 the system most commonly used in the U.S. 9 is that of Whitmore.27 The purpose of staging is to evaluate the local extent of disease (vis. tumor grade -- size, invasiveness, anaplasia) and to determine the extent of systemic disease (vis. metastasis -- number, location). Decisions about clinical therapy and prognosis are made based on these findings. The desired goal of staging is to predict the natural history of the disease, response to therapy, and the prognosis for the patient28 (Table 1).

1.2.7 Carcinoma of the Prostate vs. Benign Prostatic Hypertrophy

Although both benign prostatic hypertrophy (BPH) and adenocarcinoma of the prostate exhibit similar epidemiologic characteristics, their coincident temporal occurrence appears to be unrelated.29 Prevalence increases with age. Indeed, it is so high in the seventh and eighth decades of life, that it is argued that BPH is not a disease, but rather a result of the normal aging process.30,31 BPH is characterized by the formation of large, discreet nodules in the periurethral transitional zone of the prostate. Evidence suggests that the cause of BPH is related to androgen and estrogen trophic effects.32"37 Studies in beagle dogs (the only animal that develops BPH with aging) show that in young castrated animals it is possible to induce prostatic hyperplasia by administration of androgens. This effect is enhanced by the simultaneous administration of 17|3-estradiol, thus implicating synergism between the two. 5a- (DHT), which is the product of 5a-reduction of by the NADPH-dependent enzyme 5a-reductase, is the ultimate mediator of prostate growth.35,36,38 10

Table 1 Carcinoma of the Prostate: Clinical Staging System

Description Whitmore27 TNM26 Localized Disease Incidental TURP A Ti • Focal, low grade Ax Tia

* Diffuse, high grade a2 Tib Diagnosed on TRUS- guided biopsy prompted by elevated PSA only lc Clinically Detected B t2

Bj (52cm) T2a (<1 / 2 lobe) • Palpable tumor, 1 lobe B2 (>2 cm) T2b (>1/2 lobe,

but < 1 lobe)

• Palpable tumor, both lobes b3 t2c • Palpable beyond capsule c T3.4

* Extending to lateral sulcus Ci t 3

* Extending to base of seminal vesicle C2 t 3 * Beyond base of seminal vesicle C3 —

• Invades sphincter, bladder neck, or rectum — T4

* Invades levator ani or pelvic side wall — t 4

Metastatic Disease D T1 .4 NJ.3 M0

— • Elevated Acid Phosphatase only D0 • Pelvic lymph nodes only Di Tm NjMo

* Bone, lung, etc. d 2 Tj^Nj^Mjflbone)

• Lung, , brain — T1.4Ni.3M je

• Hormonally refractory d 3 d 3 11

The prostatic epithelium contains large numbers of androgen receptors- the receptor for DHT, and expression of the androgen receptor is enhanced by estrogens. There is no difference in plasma testosterone levels between patients with BPH and those without BPH. In addition, plasma testosterone levels decline with age, particularly after the age of 60, while estradiol levels increase (both absolute and relative to testosterone). Thus, the currently accepted model of BPH etiology holds that estrogens sensitize the prostate to the trophic effects of the androgens.33'36-37

Careful studies by McNeal have demonstrated that BPH originates almost exclusively in the transitional zone of the prostate39 (Figure 1-note the area proximal to the verumontanum corresponding to the periurethral regions of the middle and lateral lobes). This distribution is in striking contrast to that of prostatic carcinoma, which almost exclusively involves the peripheral zone of the posterior lobe40 (Figure 1). In summary, it should be noted that, despite the fact that BPH and prostatic carcinoma share several common clinical features (androgen dependence, age of occurrence), and the earlier assertions that BPH represents a premalignant condition, the evidence does not support any association between the two.41 Rather, it appears that the most likely candidate premalignant lesion for adenocarcinoma of the prostate is prostatic intraepithelial neoplasia (PIN), a condition characterized histologically by cellular proliferation within preexisting ducts, and glands with cytologic changes mimicking cancer, including nuclear and nucleolar enlargement.42,43 12

1.2.8 Pathophysiology of Disease Progression

Because of the earlier work of Huggins and Hodges,44 and due to the clinical observation that androgen ablation therapy yields an initial positive response in almost all cases, it was widely accepted that adenocarcinoma of the prostate is initially androgen-dependent, and then progresses to an androgen-independent state. Subsequent investigations appear to indicate that this model may be an oversimplification.

1.2.8.1 Androgen Receptor Levels

Despite high expectations, measurement of androgen receptor (AR) levels in prostate biopsy specimens has not proved to be a reliable index for predicting androgen dependence, or responsiveness in prostate tumors.45'50 Interestingly, hormone-dependent prostate tumor cells are AR-positive, but not all hormone-independent tumor cells are AR-negative.51 Indeed, the response of prostate tumor cells to androgen varies across a wide range of responses. These include 1) AR-positive, androgen-dependent cells which proliferate upon androgen stimulation, but become apoptotic and die upon androgen withdrawal; 2) AR-positive, androgen-responsive cells which proliferate upon androgen stimulation, but continue to five and proliferate at a markedly slower rate upon androgen withdrawal; 3) AR-(positive or negative), androgen-unresponsive cells which are unaffected by androgen; and 4) AR-positive cells which show a paradoxical decrease in proliferation upon androgen stimulation and increase in proliferation upon androgen withdrawal.52 This relationship is further clouded by two additional observations. The first is that the intracellular distribution of AR differs 13 between primary and metastatic disease implying that the kinetics of receptor activation have changed in metastatic cells. Cytosolic AR content is markedly higher in metastatic cells, whereas nuclear content is only one- fourth that of primary tumor cells.53 The second is that the response to androgen in normal prostatic tissue displays intra-tissue regional variability, even though the androgen receptor is uniformly expressed.54,55

1.2.8.2 Androgen Receptor Mutations

The AR is the product of a member of the superfamily of genes that code for the retinoic acid, vitamin D, thyroid hormone, and steroid hormone receptors. These receptors are -dependent transcription factors which share in common several structural features. These include the carboxyl-terminal hormone-binding domain, the central DNA binding domain, which possesses the familiar finger structural motif that binds to the target hormone response element, and the amino-terminal domain, which is responsible for nuclear membrane translocation and transcriptional activation (Figure 2). The binding of androgen to the AR initiates a conformational change which activates the steroid-AR complex and results in the formation of an AR homodimer. The homodimer interacts with particular sequences of DNA within the promoter region of certain genes, referred to as the androgen response elements (ARE), as well as a number of other nuclear transcription factors. Binding of the steroid- AR homodimer complex to the ARE initiates transcription, thus producing an mRNA coding for the androgen-inducible gene product. The elevated levels of mRNA lead to an increase in the synthesis of the protein product 14 by the endoplasmic reticulum. There is some older evidence which indicates that the steroid-AR complex may also be participating in translation of the protein product. Kibonudeoproteins (RNP) were isolated which were bound to the DHT-AR complex. It was postulated that these steroid-AR-RNP complexes enter the cytosol, participate in protein synthesis, and then are released, thus recycling the AR.56-59

The search for explanations for the androgen independence of prostate tumor cells led, quite logically, to the possibility of mutations in the AR gene. Measurements of AR levels were detected using immunoreactivity and radioligand binding. These experimental techniques are important indicators, but do not offer a complete view of normal AR function. Antibodies are specific for only a small portion of the AR molecule, and thus may not detect anomaly in another part of the molecule. Analogously, ligand binding only measures activity in one of several required functional domains of the AR molecule. Mutant AR’s can be generated that bind steroid but do not activate transcription, or that do not bind steroid but are constitutively active, or whose steroid binding is no longer androgen-specific.60-65

Thus far, a number of mutations in the AR gene have been described. In biopsy specimens taken from Whitmore stage B patients, point mutations were discovered in the highly conserved codon 730. This missense mutation yields a G to A transition, resulting in the substitution of Met for Val in the hormone binding domain.66 In another report, two separate point mutations were observed in the hormone binding domain. 15

1-^ ------Amino Acid Number 910

Hormone Binding Domain

DNA Binding Domain

Nuclear Translocation

Transcriptional Activation

Figure 2. Schematic representation of the functional organization of the human androgen receptor.

The first, detected in primary tumor biopsy specimens, was a T to A transversion in codon 701, resulting in the substitution of His for Leu. The second, found in primary tumor and in tumor metastases, was an A to G transition in codon 877, resulting in the substitution of Ala for Thr.67 Others have reported frequent mutations to the same codon in prostatic tumor specimens derived from transurethral resection of the prostate (TURP) in patients with metastatic disease.68 This mutation has also been observed in the LNCaP tumor cell line, which is derived from a lymph node metastasis of a prostate tumor.69,70 Interestingly, this mutation leads to altered ligand binding characteristics, such that the mutant receptor may be activated not only by the androgens (testosterone and 5a- dihydrotestosterone), but also by estradiol, , and the antiandrogens, , , and .65,71,72 16

There is a recent report describing 8 separate point mutations in the hormone binding domain. These were found in specimens derived from metastases in patients with advanced disease. Four of these findings were single mutations resulting in single amino acid substitutions in the AR molecule, one of which was within the previously mentioned codon 877, yielding substitution of Ser (not Ala as above) for Thr. The remaining isolated mutant AR contained four mutations resulting in four amino acid substitutions in the AR molecule.73 Another recent report describes 18 different mutations found in specimens derived from biopsies of latent primary tumors. These mutations ranged from deletions, resulting in frame shifts in the DNA binding domain, to point mutations, resulting in nonsense or missense substitutions in the hormone binding domain.74 Schoenberg reports 18 base pair deletional mutants in the CAG-repeat microsatellite within exon 1 of the AR gene in samples taken from primary prostate tumor biopsies. This mutation is similar to that associated with Kennedy’s disease, occurring in a poly-Gln sequence within the N-terminal portion of the AR, corresponding to the transcriptional activation portion of the AR molecule.75

Although reports of mutations to the AR in advanced metastatic disease provide compelling evidence for this being at least one of the mechanisms by which prostatic carcinoma progresses to a hormone independent state, there is at least one report which offers a dissenting view. These authors observed no mutations to the AR gene in primary tumor specimens derived from TURP or radical prostatectomy in patients 17 with locally progressive, hormone-refractory disease (Whitmore stage B). They assert that progression is not predicated on mutations in the AR gene, and that such findings represent the minority of cases.50

1.2.8.3 Intracrme/Autocrine/Paracrine Factors

Focus has also turned toward the role of growth factors, , and epithelial/stromal interactions which may contribute to tumorigenesis and the loss of growth and differentiation control in the prostatic epithelium.

1.2.8.3.1 Growth Factors

Growth factors may influence prostatic cancer cells by intracrine, autocrine and paracrine mechanisms. The intracrine scheme involves the production of a growth factor followed by an intracellular interaction between the growth factor and its receptor. The producing cell is also the signaling target, thus no secretion of growth factor is involved. The autocrine pathway also involves an auto-activation scheme. The cell of production is again the target, but this mechanism entails secretion of the growth factor into the extracellular compartment, followed by growth factor interaction with its receptor on the extracellular surface of the cell membrane. The paracrine loop involves the production and secretion of growth factor by one cell, followed by binding of the growth factor to its receptor on a neighboring cell. There are a variety of potential signaling pathways associated with the paracrine mechanism. Some possibilities include stromal cells signaling epithelial tumor cells, tumor cell to stromal 18 cell signaling, tumor cell to tumor cell, stromal cell to stromal cell, and normal epithelial cell to tumor epithelial cell. It must be appreciated that these interactions need not be simple, single-step events, but may involve multiple steps, multiple growth factors and multiple cell types. There also exists the possibility that some paracrine mechanisms may involve more than just a classical dose-response effect. Examples might include: spatial or temporal summation of signaling events; variable signal molecule secretory patterns, such as pulsatile, sinusoidal, or continuous; and resonating circuits.

Many peptide growth factors have been implicated in the growth control of prostate cancer.76'82 Three families of growth factors have been the most extensively studied: the epidermal growth factor (EGF) family, the transforming growth factor-3 (TGF-3) family, and the heparin-binding growth factor (HBGF) family (Table 2).

The EGF family includes epidermal growth factor and transforming growth factor-alpha (Table 2). The members of the EGF family signal through the same molecule, the EGF receptor, which is a 170 kDa transmembrane glycoprotein and protein tyrosine kinase, and is the product of the c-erbBl protooncogene. EGF and TGFa have similar biologic properties, which, in general, include promotion of proliferation, differentiation and angiogenesis. They are also equally capable of binding to and downregulating the EGF receptor.83'85 Although EGF and TGFa signal through the same receptor, they appear to be expressed at different ontogenetic times by the normal prostate. 19

Table 2 Growth Factors Implicated in Prostate Cancer

Epidermal Growth Factor (EGF) Family • Epidermal Growth Factor (EGF) • Heregulin • Heparin-binding EGF • Amphiregulin • Transforming Growth Factor-alpha (TGF-a) • Cripto Transforming Growth Factor-beta (TGF-P) Family • Transforming Growth Factor-pi • Activins • Transforming Growth Factor-p2 • Inhibins • Transforming Growth Factor-P3 Heparin-binding Growth Factor (HBGF) Family • acidic Fibroblast Growth Factor (aFGF) • int-2 protein (HBGF-3) • basic Fibroblast Growth Factor (bFGF) • hst/K S3 protein (HBGF-4) • Fibroblast Growth Factor-5 (FGF-5) • Fibroblast Growth Factor - 6 (FGF-6 ) • Keratinocyte Growth Factor (KGF) Insulin-like Growth Factor (IGF) Family • IGF-I • Relaxin • IGF-II

Platelet Derived Growth Factor (PDGF) Nerve Growth Factor (NGF)______Hepatocyte Growth Factor (HGF)_____

TGFa is expressed embryologically and during neonatal development,86 whereas EGF is expressed at high levels in adult prostatic tissue and is present at high concentration in seminal fluid8788 EGF is also required for prostatic epithelial cells to grow in primary culture 89 The EGF receptor shows a regional pattern of expression within the prostate such that it appears to be preferentially expressed on basal cells rather than secretory cells in normal and hyperplastic prostate.90 Interestingly, the pattern of expression of the EGF receptor protein becomes discontinuous in prostatic intraepithelial neoplasia and is almost lost in frank prostatic malignancy 20 to the extent that tumor cells display only about 25% of the immunohistochemical staining seen in normal prostate.90’93 Attempts to quantify EGF receptor expression and activity in normal, hypertrophic and malignant prostatic tissues by competitive ligand binding assays have yielded conflicting results.42,94’96 This may be explained by the fact that explanted tissue and tissue homogenates contain both normal basal and secretory cells, as well as malignant cells. Thus, these assays are not measuring binding to EGF receptor in any single cell population exclusively, but rather in a heterogeneous mixture of normal and malignant cells. Attempts to measure EGF receptor mRNA levels have encountered the same pitfalls.97 However, assessment of EGF receptor mRNA levels using in situ hybridization methods revealed that all prostatic adenocarcinomas express cytoplasmic EGF receptor mRNA, and that levels of expression may correlate with tumor nuclear grade.98

All well-characterized prostate tumor cell lines (LNCaP, ALVA-101, PC-3, DU-145) synthesize EGF receptor mRNA and protein, in addition to synthesizing and secreting TGFa.99’101 In the androgen-sensitive LNCaP tumor cell line, dihydrotestosterone stimulates cellular proliferation, but does not induce an increase in EGF synthesis. Instead, dihydrotestosterone causes an increase in the expression of TGFa and EGF receptor.100 This suggests that androgens are no longer able to modulate EGF synthesis, and that androgen regulatory control has shifted to expression of TGFa and the EGF receptor in a fashion analogous to that seen in embryogenesis. This switch results in the continuous exploitation of a TGFa autocrine growth stimulatory loop by LNCaP cells, yielding uncontrolled proliferation. A 21 similar observation has been made in androgen-dependent ALVA-101 prostate tumor cells. Again, androgens stimulate TGFa and EGF receptor expression, as well as cellular proliferation. In addition, it appears that TGFa and the androgens have a synergistic stimulatory effect on cellular proliferation which is greater than the effect of either agent alone. Moreover, anti-EGF receptor antibodies were able to block the stimulatory effect of TGFa or androgens.102 Thus, it appears that the mitogenic effects of the androgens are at least in part modulated through a TGFa-EGF receptor autocrine loop. Additional evidence exists which supports the assertion that a constitutive TGFa-EGF receptor autocrine loop is stimulating prostate tumor cell mitogenesis. In the androgen-insensitive tumor cell lines PC-3 and DU-145, either anti-EGF receptor or anti-TGFa antibodies were able to reduce both cellular proliferation and EGF receptor phosphorylation.101-103 In related studies, EGF appears to enhance the invasiveness of PC-3 tumor cells by stimulating the production and secretion of extracellular proteases.104

The heparin-binding growth factor (HBGF) family contains at least 7 members, including acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and keratinocyte growth factor (KGF) (Table 2).105106 Although distinct receptors have been described for aFGF, bFGF and KGF, all members of the HBGF family can signal through common receptors.107 Members of the HBGF family are, in general, mitogenic for epithelial and stromal cells, except for KGF, which is produced by fibroblasts and only stimulates epithelial cells.108 The HBGF family plays an important role in the growth and differentiation of the normal prostate. Both aFGF and 22 bFGF are required for prostatic epithelial cell growth in primary culture.109 In the adult prostate, both epithelial cells and stromal cells synthesize, secrete and respond to bFGF.109'111 As alluded to above, KGF is synthesized and secreted by the adult prostatic stroma, which then binds to and is mitogenic only for prostatic epithelium.108

The role of the heparin-binding growth factors in prostatic carcinoma remains unclear. Only bFGF has been detected in human tumors, but it does not appear to be overexpressed.112 Basic FGF appears to stimulate the proliferation and anchorage-independent growth of the DU-145 and LNCaP tumor cell lines, but does not stimulate the proliferation of the PC- 3 tumor cell line.111,112 Interestingly, in a rat transplanted tumor model, FGF receptors appear to be altered to the extent that they display greatly elevated sensitivity to FGF stimulation.110

The transforming growth factor-beta (TGFp) family contains several members, including TGFpi-TGF(53 (Table 2). TGFp is secreted as an inactive, larger pro-peptide, termed latent TGFP, which must be proteolytically processed to yield the active 25 kDa dimer.113 TGFp has a number of biological properties which vary from tissue to tissue. In general, TGFP inhibits epithelial cell proliferation, and stimulates stromal cell proliferation,114 angiogenesis, and extracellular matrix formation.115 In normal prostatic tissue, TGFp appears to potently inhibit epithelial cell proliferation.116 TGFpl inhibits the growth of normal rat prostatic epithelium in primary tissue culture, while aFGF reverses this effect.109 Also, in primary tissue culture, TGFpi can induce programmed cell death 23

(apoptosis) if the cells are grown in the absence of EGF.117

Transforming growth factors (31-03 have been detected in human prostate tumors.99,118,119 In the Dunning 3327 rat prostate adenocarcinoma model, TGF01 mRNA levels are markedly elevated compared to normal. The more anaplastic and rapidly growing tumor sublines show greater TGF01 mRNA levels than the more differentiated, slower growing sublines. In addition, TGF01 mRNA levels do not appear to be regulated by androgens.120,121 The highly anaplastic, androgen- insensitive Dunning R3327 AT3 tumor subline produces TGF01, but its growth does not appear to be inhibited by TGF01.122 In vivo, prostatic tumor growth does not appear to be inhibited by TGF01, and tumors which overproduce TGF01 are larger, more highly vascularized, less necrotic, more invasive and more extensively metastatic than those which do not.120,121 Human prostate tumors appear to exhibit the same TGF01 overexpression and insensitivity as their animal counterparts.119,123,124 Human prostate tumor cell lines yield conflicting results regarding the expression of TGF01 and TGF0 receptor, depending upon the culture conditions and time of exposure to TGF01.118,125,126 Tumor cells grown in the presence of other growth factors and extracellular matrix are not inhibited by TGFpl.127 Thus, it would seem that TGF01 is not an inhibitor of advanced prostate tumor cell growth. Instead, it is functioning to provide host effects, including suppression of the growth of surrounding epithelial elements, promotion of angiogenesis and tumor neovascularization, and local suppression of immunity for evasion of immune surveillance. Thus, it appears that TGFpl acts in a permissive fashion, by providing an 24 optimized environment for continued tumor growth, invasion and metastasis.

The insulin-like growth factor family (referred to in the older literature as the somatomedins, sulfation factor, or non-suppressible insulin-like activity) includes IGF-I, IGF-II and relaxin. These molecules share a high degree of sequence homology with insulin, and they signal through the IGF-I receptor, a homodimeric transmembrane glycoprotein which possesses an intracellular tyrosine kinase activity. There is a certain degree of cross-talk between the IGF’s and insulin, such that each group is able to signal through the other’s receptors with approximately 100-fold lower affinity. Moreover, it appears that there are heterodimeric insulin/IGF receptor hybrids, in addition to the usual homodimers (IGF/IGF and insulin/insulin). These hybrids appear to have a ligand specificity profile closer to that of IGF receptor than insulin receptor. There is also a recently discovered class of IGF-binding proteins which appear to modulate the effects of IGF. There are a number of hypotheses regarding the function of the IGF-binding proteins, including attenuation of IGF action, maintenance of an IGF storage pool, and the provision of an IGF carrier/transport system for delivery of IGF to its target. The normal cellular functions of the IGF’s include acute anabolic effects on protein and carbohydrate metabolism, and longer term effects on cell division, differentiation and apoptosis.128 Normal prostatic epithelial cells possess IGF-I receptors, whereas normal prostatic stromal cells do not. Conversely, normal prostatic stroma produces and secretes IGF, while normal prostatic epithelium does not. Thus, it appears that an IGF paracrine loop, similar 25 to that seen with KGF, operates within the normal prostate.129,130

The prostate tumor cell lines, PC-3, DU-145 and LNCaP, overproduce the IGF receptor as well as IGF-II, but do not produce IGF- j 131,132 peptide analogs of IGF-I,133 monoclonal antibodies to the IGF receptor,132 and antisense oligonucleotides which block IGF receptor or IGF- II expression132,133 all block the proliferation of these tumor cell lines. Moreover, it appears that the IGF’s are capable of activating androgen receptor-mediated transcription in the absence of androgens.134,135 These findings point toward an IGF autocrine loop in prostate malignancy and implicate the IGF’s in providing prostatic growth stimulation after . Moreover, the IGF autocrine loop may provide a mechanism for continued growth stimulation after the progression of prostatic tumors to androgen-insensitivity.

In summary, it appears that in the normal prostate, EGF, bFGF, KGF and IGF-I induce epithelial growth through paracrine mechanisms. The relative contribution of each to the maintenance of normal growth and differentiation is unclear, but, in the presence of androgens, the stroma and epithelium produce and respond to these growth factors. TGFpl appears to stimulate the growth of fibroblasts and inhibit the growth of normal prostatic epithelium. Both aFGF and bFGF appear to be capable of attenuating the effects of TGF(}1. Thus, the interplay between the stroma and epithelium through stimulatory and inhibitory peptide growth factors, in the presence of androgens, maintains normal prostatic growth (Figure 3). 26

TGFa EGF TGFa IGF’s IGF-II r Immune ty '- ' HBGF's TGFpi \Surveillanc§/ W TGFpi

Prostatic Epithelial Cell Epithelial Cell \JT IGF-R & EGFR/ Normal Malignant

Figure 3. Schematic depicting the effects of various growth factors on both normal and malignant prostatic epithelial cells. Also note increases in EGF and IGF receptor levels in malignant cells, and the effects of hypersecretion of TGFpi on immune surveillance and angiogenesis.

This scenario is contrasted with that of the malignant prostate, where TGFa and IGF-II, in addition to overexpression of the EGF and IGF receptors, as well as possible mutations in FGF receptors, provide aberrant growth stimulation to the prostatic epithelium. TGFpi seems to have no inhibitory effect on the growth of the malignant prostatic epithelium, but does stimulate angiogenesis and neovascularization, in addition to inhibiting immune surveillance.136 Thus, it appears that TGFpi provides a potential growth advantage to the malignant epithelium through permissive host effects (Figure 3). 27

1.2.8.3.2 Other Epithelial-Stromal Interactions

As described previously, the (ER) has been demonstrated in normal prostatic epithelium and the prostatic epithelium of BPH, but reports regarding the presence of ER in the prostatic epithelium of primary tumor biopsies and metastases are conflicting.49’53 137'139 Thus the role of estrogen in prostatic adenocarcinoma is unclear. As previously stated, point mutations in codon 877 of the AR render the AR molecule susceptible to activation by estradiol. In addition to this, Brolin and coworkers have demonstrated the expression of ER in stromal cells of carcinomatous by immunohistochemistry. They were unable to detect ER in the stroma of normal prostate, or BPH. They were also unable to demonstrate epithelial ER in malignant prostate, or in any of 3 tumor cell lines (DU-145, PC-3, and LNCaP).49 These findings suggest the intriguing possibility of estrogen-mediated paracrine stimulation of the prostatic epithelium by the prostatic stroma in malignant prostatic tissue.

Current work appears to indicate that both normal and malignant prostatic epithelium is influenced by the extracellular matrix (ECM). Recent studies demonstrate that type-IV collagen augments the bFGF and TGFa-stimulated proliferation of transplanted LNCaP tumors in athymic nude mice. Moreover, it appears that fibronectin and tenascin are capable of enhancing tumor cell invasiveness. This has been demonstrated by their capacity to induce the soft agar colony forming ability of LNCaP cells in vitro.80 In addition to these ECM proteins, heparan sulfate proteoglycans 28 and the laminins have been implicated in the promotion of prostatic tumorigenesis and growth.140 Interestingly, it appears that the expression of some of the extracellular matrix proteins (e.g. the integnns and type-VII collagen) is down-regulated, or completely repressed, in malignant prostatic tissue.141 The role that extracellular matrix proteins play in prostatic tumor cell proliferation is not entirely dear. Possibilities indude induction of morphologic changes in prostatic tumor epithelial cells, promotion of the invasiveness and metastatic potential of these cells, induction of local growth factor synthesis and secretion, and formation of ECM-growth factor complexes, which could modulate growth factor function at its receptor, or provide a readily accessible, localized storage pool of growth factor.80’140 It also appears that organ-specific fibroblasts are capable of modulating the tumorigenicity of prostatic epithelial cells. Fibroblasts from bone and prostate are able to induce tumor formation, whereas lung and kidney-derived fibroblasts do not appear to have any induction potential.140 Finally, it has been demonstrated that malignant prostatic epithelial cells, which are invasive and possess metastatic potential, synthesize and secrete elevated levels of a number of extracellular proteases. These include the gelatinases, matrilysin, stromelysin, urokinase and cathepsin D.141

1.2.8.3.3 Other Endocrine Factors

Recently, it has been shown that receptors are present on the epithelium of the normal prostate, the epithelium of benign prostatic hypertrophy, and the epithelium of malignant prostate.142-146 It appears 29 that prolactin is trophic in all three of these cases, stimulating epithelial proliferation.147'149 Dopamine, and dopaminergic compounds suppress the synthesis and secretion of prolactin by the posterior pituitary. Although the precise role of prolactin in the development and physiology of the normal prostate, the BPH prostate, and the malignant prostate remains unclear, it does appear that the dopamine receptor agonists bromocriptine and lisuride can suppress tumor growth and reduce bone pain in patients with advanced prostatic malignancy.150,151 Interestingly, dopamine antagonists, such as perphenazine, can stimulate prostatic tumor growth.147

Luteinizing hormone releasing hormone (LHRH) agonists are a mainstay of endocrine ablative therapy in both prostatic carcinoma and estrogen-dependent mammary carcinoma (see detailed discussion below). The of the LHRH agonists is to decrease androgen (and estrogen) biosynthesis through a paradoxical decrease in LH secretion as a result of LHRH receptor tachyphylaxis. There is also evidence which indicates that LHRH agonists have direct inhibitory effects on testosterone secretion at the testicular level.152 Recently, LHRH receptors have been demonstrated on malignant prostatic epithelium in both the human and the rat.146,153,154 Interestingly, LHRH receptors do not appear to be present on the epithelium of the normal or benign hypertrophic prostate.146 The presence of LHRH receptors on malignant prostatic epithelium has prompted workers to investigate their possible role in prostatic malignancy. Animal studies show that LHRH agonists suppress prostatic tumor growth,153,155 but it is apparent that this effect cannot be solely 30

attributed to direct antiproliferative effects at the prostatic epithelial cell level (keep in mind the hypothalamic-pituitary-testicular axis). Subsequent studies in the LNCaP prostatic tumor cell line have demonstrated that LHRH agonists specifically inhibit cellular proliferation in a dose-dependent fashion; that this antiproliferative action is completely reversed by concomitant administration of LHRH antagonists; and that LNCaP cells possess LHRH receptors.156 Based on these findings, it is not unreasonable to assume that the LHRH agonists may be inhibiting prostatic tumor growth not only by inhibiting the production of androgens via hypothalamic-pituitary-testicular axis, but also by exerting a direct antiproliferative effect at the level of the malignant epithelial cell. Related studies have shown that both LNCaP and DU-145 cells express an mRNA coding for LHRH and the protein product.157'159 The precise role of LHRH in the overall regulatory control of tumor cell proliferation is unclear, but it appears that it may be functioning as an autocrine/paracrine mediator of the malignant prostatic epithelial cell response to androgens.158,159

Somatostatin receptors have been demonstrated in the Dunning 3327 rat prostate tumor model.146,153,155,160 In addition, somatostatin analogs have been shown to inhibit tumor growth and induce tumor fibrosis in this model.153,155,161'163 Initial studies attempting to demonstrate somatostatin receptors on human prostatic tumor cells yielded equivocal results,146 but subsequent investigation has established their presence.160,164,165 There is considerable variability in binding affinity among various somatostatin analogs, suggesting the possibility that there 31 are differences in somatostatin receptors between normal and malignant cells, between tumor types and between tissues.160 These findings raise the possibility of mutations in the somatostatin receptor gene, and suggest a potential role for these receptor mutants in tumorigenesis. Somatostatin receptors have been demonstrated in the LNCaP prostate tumor cell line, and somatostatin inhibits LNCaP cell proliferation in vitro.166 A number of studies involving xenografts of both hormone dependent (PC-82, LNCaP) and hormone independent (PC-3, DU-145) prostate tumor cell lines in athymic nude mice have shown that somatostatin analogs inhibit the growth of these tumors. In all of these models, somatostatin receptors were demonstrated on the tumor cells, and it was shown that treatment with somatostatin analogs decreased serum growth hormone, prolactin, and IGF-I levels, in addition to decreasing EGF receptor expression in the tumor cells.167"170 Phase I and II clinical trials of somatostatin analogs for the treatment of hormone refractory prostate adenocarcinoma have shown some promise.171,172 As with LHRH agonists, systemic endocrine effects must be differentiated from local effects. Somatostatin is capable of negative feedback control of prolactin and growth hormone secretion at the level of the pituitary and . In addition, somatostatin can suppress circulating levels of IGF-I. Since prolactin and IGF-I have been demonstrated to be trophic to both normal and malignant prostatic epithelium, it is not unreasonable to conclude that at least part of the prostatic response to treatment with somatostatin analogs in vivo may be attributed to systemic endocrine effects.173 32

There have been investigations into the possible presence of prostatic aromatase activity, leading to the hypothesis that local production of estrogens contributes to prostatic tumorigenesis. These studies have yielded negative results, and thus, it appears that this is not a contributing factor in prostatic malignancy.174 As mentioned in the earlier discussion of BPH, circulating levels of estrogens increase with age, both absolute and relative to testosterone. Thus, although local prostatic aromatization of androgens to estrogens does not appear to occur, peripheral aromatization does occur. The role that the estrogens may be playing in prostatic tumorigenesis is unclear. Other studies exploring the possible role of additional neuroendocrine mechanisms in prostatic tumorigenesis have been undertaken as well.79

1.2.8.4 Genetic Alterations: Oncogenes, Tumor Suppressor Genes, and Chromosomal Aberrations

In an attempt to define the molecular mechanisms that lead to prostatic malignancy, a significant body of work has focused upon investigating the presence and role of genetic alterations associated with this disease. As discussed above, oncogenes are genes which when expressed confer the malignant phenotype upon a cell. Oncogenes are generated by damage to a normal cellular protooncogene, followed by expression of oncogene fimction. Tumor suppressor genes normally function by suppressing tumorigenesis. When these genes become altered by damage or mutation, a permissive or de-repressive state follows, and the cell is converted to malignancy. These genetic alterations may be brought 33 about through a number of mechanisms. Transduction by retroviruses, transfection by DNA viruses, chromosomal translocations or rearrangements, DNA amplification, deletion of regulatory sequences, point mutations, sequence insertions or deletions, and derangement of DNA repair mechanisms are all means by which protooncogenes can be converted to oncogenes, or tumor suppressor genes can become altered such that they lose their normal function. Chromosomal aberrations which have been detected in prostatic carcinoma include alterations in chromosomes 7, 8, 10 and 16.175,176 Other groups report derangements in other chromosomes, but results are conflicting or their significance is unclear.177,178 Tumor suppressor genes which have been studied in prostatic carcinoma include p53, Rb, DCC, APC, hMSH2, and nm23-Hl. It appears that alterations in any of these genes are associated with disease progression.78,179,180 Finally, numerous oncogenes have been investigated in prostatic carcinoma, including ras, myc, jun, fos, sis, int-2, met, bcl-2 and erbB2.181-189 Although it is convenient to categorize these topics separately for the purpose of discussion, it must be appreciated that investigations involving growth factors, growth factor receptors, cell signaling, oncogenes, epithelial/stromal interactions, tumor suppressor genes and apoptosis are not mutually exclusive, but in fact are interrelated and overlap with one another extensively (e.g. a growth factor receptor whose gene, when mutated, is an oncogene). 34

1.2.8.5 Adaptation vs. Selection

As discussed above, all prostatic adenocarcinomas are initially hormone responsive, but eventually progress to a hormone unresponsive state. A critical question which is central to defining the mechanisms by which this progression occurs involves whether the tumor cells adapt to their environment, whether their phenotype is unstable and dedifferentiates to a more primitive state, whether a subset of cells in the tumor population are initially hormone-independent and are simply selected for by the environment, or whether a combination of these is operative. These investigations are being pursued and are covered in depth elsewhere.190-191

1.3 Prostate Cancer Treatment Modalities

1.3.1 Surgery, Radiation and Cytotoxic Chemotherapy

Surgery, radiation therapy and cytotoxic chemotherapy are still largely the primary weapons used to treat cancer. Predictably, these methods are widely utilized in the treatment of prostatic adenocarcinoma as well. There are two possible surgical interventions used to resect the malignant prostate: radical prostatectomy (with or without regional lymph node dissection) and transurethral resection of the prostate (TURP). Historically, radical prostatectomy has been associated with significant morbidity, such as urinary incontinence and . These complications have been reduced by continued refinement of techniques which spare nerve, muscle and vascular tissue. TURP is a much less 35 invasive technique, but has limited utility because it cannot be used to resect the entire gland. Instead this procedure is used to alleviate urethral obstruction due to BPH and for collection of biopsy specimens for diagnostic purposes.192 Radiation therapy for prostatic adenocarcinoma includes a variety of modalities, classified according to the type of emission utilized (gamma-rays, beta-particles, alpha-particles), and the method of delivery (external beam vs. implantable brachytherapy). The goal of this therapy is to deliver a cytotcidal dose of radiation to the malignant tissue without causing collateral tissue damage. There is significant morbidity involved with radiation techniques including chronic enteritis, urinary and intestinal incontinence, and erectile dysfunction.193 Cytotoxic chemotherapy for prostatic carcinoma has largely been a complete failure.194 Factors which have contributed to this are multidrug resistance and the necessity of prohibitively high dosing. Several newer agents in combination, such as etoposide, , vinblastine, and suramin, have shown limited promise.195,196

1.3.2 Endocrine Manipulation

Androgen ablation remains the mainstay of pharmacologic therapy for adenocarcinoma of the prostate. As mentioned previously, most prostate tumors are initially androgen-dependent. Therefore, the goal of endocrine treatment is to deprive these androgen dependent tumor cells of their supply of steroid hormones, thereby inducing death of androgen- dependent tumor cells, and suppressing tumor growth. If one examines the physiology of steroid hormone biosynthesis and action, several logical sites 36 of pharmacologic intervention emerge. These include suppression of androgen secretion through disruption of the normal feedback control of the hypothalamic-pituitary-testicular axis, antagonism of androgen action at its receptor, and ablation of androgen production through inhibition of the androgen biosynthetic enzymes (Figure 4).

Asm s Ligand-Recentor Binding Biosynthesis

C21 Progestins Hypothalamus 17,20-Lyase GnRH AR

Anterior C 19 Androgens Pituitary Androgens 5a-Reductase FSH 7 & LH 5 a-Dihydroandrogens

Testis AR

Figure 4. Schematic depiction of the possible sites of endocrine manipulation in the treatment of prostatic adenocarcinoma.

1.3.2.1 Hypothalamic-Pituitary-Testicular Axis Disruption

1.3.2.1.1 Orchiectomy and Hypophysectomy

Since over 95% of all androgen production occurs in the testes, surgical orchiectomy is a logical method of approaching androgen ablation. Unfortunately, this carries with it significant negative psychosocial 37 implications, since many men see orchiectomy as being emasculating. Thus, other pharmacologic methods of castration are used (see discussion below). Surgical excision of the , or hypophysectomy, would also be an approach to endocrine ablation therapy. This would remove the GnRH-stimulated production and release of the , and thereby remove the stimulus for androgen biosynthesis by the testis. This therapy has obvious, serious drawbacks attributable to the fact that the pituitary is involved in numerous other endocrine functions, and surgical extirpation of the gland would abolish these functions as well.

1.3.2.1.2 Estrogens

The progestins, androgens and estrogens provide negative feedback control of production by the anterior pituitary, and of GnRH secretion by the hypothalamus. Therefore, it has been reasoned that suppression of GnRH and gonadotropin secretion, and by extension, androgen production, could be achieved by treatment with high doses of estrogens. Agents which have been used in this context include ethinyl estradiol, diethylstilbesterol, and the pro-, (TACE). Undesirable side effects include , nausea and vomiting. This type of treatment also carries with it significant risk for cardiovascular complications, including edema and deep vein thrombosis. Because of this, there is an elevated risk of embolus formation and tissue infarction, therefore this therapy has largely been abandoned in favor of other forms of endocrine therapy.197 38

1.3.2.1.3 Progestins

The progestins, as well as the androgens and estrogens, provide negative feedback regulation over the hypothalamus and pituitary. They act by suppressing GnRH and gonadotropin release, thereby removing the endocrine stimulus for testicular androgen production. Progestins have the added benefit of causing fewer adverse effects than estrogens, particularly cardiovascular effects. Synthetic progestins, such as acetate and acetate, have been used as a means of suppressing the hypothalamic-pituitary-testicular axis. However, androgen levels tend to escape suppression within 2-6 months of treatment; thus, progestin treatment has been augmented with mini-dose estrogens. Unfortunately, the adverse cardiovascular and feminizing effects of the estrogens manifest upon supplementation; therefore, this form of therapy has been abandoned.198

1.3.2.1.4 GnRH Agonists

Gonadotropin releasing hormone (GnRH) (also referred to as releasing hormone-LHRH) is a decapeptide, synthesized and secreted by specific neurosecretory cells in the hypothalamus, which acts on the anterior pituitary, causing release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the normal setting, GnRH is secreted by the hypothalamus in a pulsatile fashion into the hypothalamic-hypophyseal portal vein, where it is transported to the anterior pituitary. GnRH then binds to its receptors 39 on the cell surface, thereby stimulating gonadotropin secretion, and inducing coated pit formation. These coated pits form endocytic vesicles. The endosomes then fuse with lysosomes, where the peptide ligands and receptors are subjected to degradation by lysozomal enzymes. Because of the pulsatile nature of normal GnRH secretion, not all GnRH receptors are occupied with hgand-saturation is not achieved. This maintains a "spare reserve" of GnRH receptors, allows the effector cells to replace the degraded receptors, and avoids receptor tachyphylaxis.

On first inspection, it would appear that therapy with GnRH agonists would simply exacerbate tumor growth by further stimulating androgen production. How is it that these agents paradoxically suppress gonadotropin secretion? The answer lies in the fact that GnRH is normally secreted in bursts at 90 minute intervals. Normally, these pulses allow the gonadotropin-secreting cells of the anterior pituitary to recover from GnRH stimulation by synthesizing and replacing the cell membrane-bound GnRH receptors that were endocytosed and degraded. Upon implementation of GnRH treatment, all receptors are initially occupied by a pharmacologic dose of GnRH agonist, thus inducing their endocytic internalization and degradation. GnRH levels are then maintained in high enough concentration such that all newly synthesized receptors are immediately occupied and internalized. Thus, insufficient receptor levels are achieved on the cell membrane to overcome the threshold receptor occupancy needed to effect a response. The net result of this is receptor tachyphylaxis, and suppression of gonadotropin release. 40

1 23456789 10 GnRH pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Leuprolide pyroGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

Tryptorelin pyroGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt pyroGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt

Goserlin pyroGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-AzGly-NH 2

Figure 5. Structure of naturally occuring GnRH and several synthetic GnRH agonists

Synthetic agonists of GnRH are peptide-based analogs of the naturally occurring ligand. Replacement of the glycine at position 6 or L- leucine at position 7 with D-amino acids confers resistance to metabolic degradation. The D-configuration at position 6 is also thought to help stabilize a P-turn in the active conformation of the native molecule.

Introduction of bulky t-butyl ethers of D-serine at position 6 is thought to increase the lipophillicity of the molecule, thereby increasing its volume of distribution, by increasing binding to plasma proteins, in addition to slowing metabolic clearance. Substitution of short chain alkyl amines for the glycine amide at the CC^H-terminus increases the affinity of the compound for the GnRH receptor. Alternatively, substitution of a-aza amino acids, where the a-carbon has been replaced by a nitrogen, for the glycine at position 10, also increase affinity for the GnRH receptor (Figure

5) . 199 41

Ironically, the development of GnKH agonists was originally pursued in order to find agents for the treatment of infertility. It very quickly became apparent from in vivo investigation that these agents decreased fertility by the mechanism described above. Clinical investigation of the effectiveness of GnRH agonists for the treatment of prostatic carcinoma has shown that they can induce castrate androgen levels .200 The objective response rate (partial or complete disease remission) for GnRH agonist treatment varies between 35-70%. Although complete remission is rare, it must be appreciated that studies have included patients with advanced stage disease (stage C and D), and that the possibility of early diagnosis is only a recent occurrence with the advent of the modem screening techniques discussed previously. Thus, these agents may indeed be more effective in the treatment of earlier stage disease than studies indicate .200

1.3.2.2 Antiandrogens

Another site for disruption of androgen action is at the level of the androgen receptor (AR) with agents which can block androgen function by competing for the hormone binding site on the AR. Evaluation of antiandrogens in vivo is dependent upon the biological assay used, and can therefore be problematic. The reason for this variability is likely due to the fact that there are multiple isoforms of the androgen receptor which are the product of a single gene, but are modified post-translationally .201'204 Antiandrogens fall into two general structural classes, steroidal agents and nonsteroidal agents. 42

1.3.2.2.1 Nonsteroidal Antiandrogens

The assertion that there is an absolute requirement for a steroidal molecule in the activation of the AR was invalidated when the potent nonsteroidal flutamide (Figure 6 ; 1) was introduced .205,206 Subsequent receptor binding studies demonstrated that flutamide competed with 5

Nilutamide (RU 23908) (Figure 6 ; 3) has been shown to bind to the androgen receptor 215, as well as inhibit the in vivo trophic effects of testosterone on the prostate and abolish the negative feedback control of

GnRH secretion by the hypothalamus .216 Because of this abolition of the 43 feedback control of GnRH secretion by the hypothalamus, plasma gonadotropin and testosterone levels become greatly elevated after treatment with nilutamide .216 As a result of this, nilutamide has been evaluated as a combination treatment with other agents, such as GnRH agonists or castration, for total androgen ablation. Results have been promising .217’218 Newer analogs of nilutamide (Figure 6 ; 4-6) have been reported which are more active in vitro and in vivo than other antiandrogens, with RU 58841 (4) being the most selective antiandrogen described to date in vivo .219 Casodex (ICI 176,334) (Figure 6 ; 7), a pure antiandrogen, has demonstrated effectiveness as a single agent androgen ablative therapy for prostatic carcinoma, but has significant, adverse effects which preclude its use in BPH or other hyperandrogenic conditions

(e.g. , alopecia, , polycystic ovary syndrome ) .220'223 Other nonsteroidal compounds, including analogs, , coumarin derivatives, cinnamic acid derivatives, seco-steroids, DIMP, bacterial metabolites, and polycyclic aromatic hydrocarbons have been reported as antiandrogens, and are covered thoroughly in recent reviews.224,225

1.3.2.2.2 Steroidal Antiandrogens

Cyproterone acetate (Figure 7; 8 ), an analog of 17a- hydroxyprogesterone, is the original . This compound was first developed in an effort to prepare orally active progestins. It was quickly recognized for its progestational ability to suppress gonadotropin release as well as a surprising ability to compete 44

OH 1: R=H Flutamide 7: Casodex 2: R=OH Hydroxyflutamide

y^~H R 4: Y=0; R=(CH2)4OH RU 58841 3: Nilutamide 5: Y=S; R=(CH2)4OH RU 59063 6:Y=S;R=CH3 RU 56187

Figure 6. Structure of nonsteroidal antiandrogens

with 5a-DHT for the hormone binding site on the AR. The related 16- methylene analog (SCH 12600) (Figure 7; 9) was prepared, and was also shown to be antiandrogenic. Other pregnane-based antiandrogens were reported, including A-norprogestins, B-norprogestins, , and (Figure 8 ; l l ) . 224'226 Recently, a newer pregnane analog, (TZP 4238) (Figure 7; 10), has been reported to be effective at inhibiting tumor growth in the Dunning 3327 rat prostatic carcinoma model .227

A number of androstane-based compounds have been prepared and tested for their antiandrogenic activity. One of the first compounds reported in this series was 17a-methyl-B-nortestosterone (SKF 7690)

(Figure 8 ; 12) 228 Over the next decade, several other androstane-based 45

/ 0 t"OAc

O Cl ci Cl 8: 9: SCH 12600 10:

Figure 7. Structure of pregnane-based steroidal antiandrogens

derivatives were prepared and found to possess antiandrogenic activity, including bromoxymethyltestosterone (BOMT) (Figure 8 ; 13), SC 9420

(Figure 8 ; 14), A-nortestosterone (Figure 8 ; 15), A-nordihydrotestosterone

(Figure 8 ; 16), R 2956 (Figure 8 ; 17), 18,18-dimethyl-19-nortestosterone

(Figure 8 ; 18), and (Figure 8 ; 19).224.229 As might be expected, the mechanism of action of these compounds is to compete with the ligand at the receptor, without the progestational effects on the hypothalamus and pituitary seen with the pregnane-based compounds. In the case of oxendolone and (18), the rationale for alkyl substitution at positions 16 and 18 was that the added steric bulk would interfere with receptor interaction with the 17-hydroxyl, thereby preventing receptor activation .224 Rasmusson and colleagues developed a series of 4-aza-androgens in an effort to find potent inhibitors of 5a-reductase (see below). A number of these compounds also were able to compete with 5a-DHT for the hormone binding site on the AR. 46

SAc 11: Spironolactone 12: SKF 7690 13: BOMT

OH OH

H 16 14: SC9420 15: A-Nortestosterone A-Nordihydro- testosterone OH OH

18 17: R2956 18,18-Dimethyl-19- 19: Oxendolone nortestosterone

Figure 8. Structure of androstane-based steroidal antiandrogens

These compounds displayed affinities which ranged from slightly higher than endogenous androgens to approximately an order of magnitude less (Figure 9; 20-26)230. The recently developed androstane-based antiandrogen, zanoterone (WIN49596) (Figure 9; 27), an A-ring-fused pyrazole derivative similar to the anabolic androgen, stanazol, has been 47 evaluated for the treatment of prostatic carcinoma and as a combination treatment with a 5a-reductase inhibitor .231232

OH OH

CH3 ch 3 ch 3 20 21 22 OH

H3C-S-N.

23: R=OH 27: Zanoterone 24: R=C02CH3 25: R=COCH(CH3)C2H5 26: R=CO(2-pyrrolyl)

Figure 9. Structure of zanoterone and the 4-azasteroid based antiandrogens

1.3.2.3 Disruption of Androgen Biosynthesis

As was illustrated schematically in Figure 4, another target of hormonal ablation is the suppression of androgen biosynthesis. Two sites of pharmacologic intervention have been explored as approaches to the 48 inhibition of androgen biosynthesis. These are the inhibition of 5a- reductase and cytochrome P450 17,20-lyase.

1.3.2.8.1 5a-Reductase

The NADPH-dependent, 4-ene-3-oxosteroid 5a-oxidoreductase (5a- reductase) is a membrane-bound enzyme found in the endoplasmic reticulum which converts the A4-steroids to the corresponding 5a-reduced steroids, such as 5a-dihydrotestosterone. The enzyme has two isoforms, type I and type II, which show distinct tissue distribution, distinct kinetic parameters, and which have amino acid sequences of approximately 50% homology. The type I 5a-reductase is generally found in peripheral tissues with a Km for testosterone in the micromolar range, while the type II isozyme is expressed at higher levels in the male reproductive tissues and displays a Km for testosterone in the nanomolar range. The type I isozyme displays a broad pH optimum (6.0-8.5), while the type II isozyme shows a narrow pH optimum (5.0-5.5). The genes coding for both isozymes (SRD 5A1 and SRD 5A2) have been localized to chromosome 2. The differences in substrate affinity and tissue distribution of the two isozymes suggests that the type I enzyme plays a catabolic role and the type II enzyme plays an anabolic role in the metabolism of the androgens. The chemical mechanism of enzymatic catalysis is not as straight-forward as that seen in the other hydride transfer reactions. Rather, it appears that coenzyme Q of the electron transport system, as well as cytochrome P450 reductase, are also involved with the transfer of electrons to the substrate. Tritium labeling experiments have established that the hydride 49 of NADPH is transferred to the substrate 5a-position, while solvent (H 2O) provides the proton at the 4-position. The kinetic mechanism of 5a- reductase activity is an ordered bi-bi scheme, with the pyridine nucleotide binding first .233 Interestingly, 5a-reductase type I has been shown to have sequence-specific DNA-binding activity in vitro, but no classical

DNA-binding motifs have been identified in the protein sequence .234

29 30

Figure 10. Structure of non-steroidal 5a-reductase inhibitors

A number of nonsteroidal 5a-reductase inhibitors have been reported to be active against the human enzyme (Figure 10; 28-30).225 The benzoquinolinone-based uncompetitive inhibitor, LY 191,704 (Figure 10; 29), displays nanomolar affinity for the type I isozyme, but much weaker 50 affinity for the type II isozyme, thus precluding its use in the treatment of prostatic carcinoma .235 Other nonsteroidal compounds which have been reported to be inhibitors of 5a-reductase include p-substituted benzoic acid derivatives, ZnS 0 4 , phenazine derivatives, and .225-233

The first reported steroidal inhibitor of 5a-reductase was 4- androstene-3-one-17p-carboxylate (Figure 1 1 ; 31). The next group of compounds which were found to have 5a-reductase inhibitory activity were the 16(3-alkyl- 19-nortestosterone analogs, such as oxendolone (Figure 8 ; 19). These compounds were originally developed as androgen receptor antagonists, but were found to have a fortuitous dual mode of androgen ablative action. The allene (Figure 1 1 ; 32) were originally developed as irreversible inhibitors of 3(l- dehydrogenase/A 4 >5 -isomerase, but instead were found to be potent irreversible inhibitors of 5a-reductase. A group of pregnane-based diazoketone-containing compounds, possessing a 4-diazo functionality while maintaining the trans A/B ring junction and the sp2-hybridization at C-3 and C-4, have been reported to be potent mechanism-based inhibitors

(Figure 1 1 ; 33). These compounds are thought to effect irreversible inhibition by alkylating the enzyme active site subsequent to the generation of a reactive diazonium species as the result of C-3 carbonyl protonation in the enolization step of the catalytic mechanism. Another group of mechanism-based inhibitors includes the 6 -exo methylene androstene-based compounds (Figure 1 1 ; 34). These compounds displayed good affinity for the rat type II 5a-reductase. Structure-activity relationship studies in this series revealed that the 4-ene-3-one was 51 essential for inhibitory activity, that the substituents at C-17 influenced inhibitory , that the angular C-19 methyl was not necessary for activity, that the 6 -exo methylene also improved affinity, and that introduction of bulky substituents at C-7 completely abolished inhibitory activity. The pyridyl N-oxide (Figure 11; 35) has recently been prepared and shown high affinity for the type II isozyme (app. K;=31nM). A series of 4-cyano substituted analogs have been reported to be very potent transition state inhibitors with K; values in the nanomolar range (Figure 11; 36). These compounds are thought to be reduced by the enzyme, and then form the corresponding stabilized 3-enols which are tight-binding transition state mimics .225,233

O n 2h 31 32 33 o

o

34 35 36

Figure 11. Structure of steroidal 5a-reductase inhibitors 52

Since the beginning of the 1980's, a number of azasteroids have been reported as inhibitors of 5a-reductase, and this group of compounds is the most extensively studied. The rationale for their design was to mimic the enzyme-bound enolate intermediate by incorporating sp2-hybrid centers at the 3- and 4-positions, as well as maintaining the 5a geometry, while utilizing a lactam functionality which cannot be reduced by the enzyme. One of these compounds, (Figure 12; 40), has shown an approximately 100-fold selectivity for the type II isozyme over type I. This compound is tight-binding, and its off-rate is slow enough that it behaves as an irreversible inhibitor. Finasteride has completed phase III clinical trials for the treatment of BPH. In the Dunning 3327 rat prostatic carcinoma model, finasteride lowered tumor 5a-DHT levels, but was ineffective at slowing tumor progression. Finasteride has been shown to lower serum PSA levels in stage D prostatic carcinoma patients. Finasteride is currently being evaluated in a long-term study to assess its in the prevention of prostatic carcinoma. Other 4-azasteroids (Figure 12; 41-43) have also shown effective inhibition of human 5a- reductase both in vitro and in vivo. Recently, a group of 6 -azasteroids (Figure 12; 44) have been reported which show sub-nanomolar affinities for both the type I and type II isozymes .225’233

A number of transition state analogs have been prepared which possess an acidic functionality at the 3-position (Figure 12; 37-39). The acidic group, as well as the A3-double bond, mimic the presumed enolate intermediate of the native substrate. This was confirmed by the loss of affinity with the saturated 3-carboxylates, or the reduction of the 53 carboxylate to either the aldehyde or the alcohol. Increases in the stenc bulk of the 3-acidic function, for example, replacement of the phosphinic with the phosphonic acid, decreased affinity. The addition of the 5,6-double bond to the 3-ene-3-carboxylate (Figure 12; 38: ) markedly increased affinity. These analogs display uncompetitive kinetics with respect to both NADPH and testosterone, and require NADPH for activity.

-Bu>

37 39 40: Finasteride R = c o 2h , c h 2c o 2h , r = c o 2h , c h 2c o 2h , p h o 2h , p o 3h p h o 2h , p o 3h , s o 3h Z = NHPrj, NHBut, N(Prj)2l N(Bu,)2

38: Epristeride A3'5; R=C02H; Z=NHBu,

44 41: R=N(Et)2 4-MA 42: R=N(Prj)CONHPrj 43: R=NHCH(C6H5)2

Figure 12. Structure of 3-acidic substituted and azasteroid 5a-reductase inhibitors 54

The presumed mechanism of inhibition involves the formation of a ternary complex between the enzyme, the negatively charged inhibitor, and the positively charged NADP+. Replacement of the acidic function in this series with a nitro group generates a potent competitive inhibitor, thus lending further credence to the role of the anion in forming a ternary complex. One of the analogs in this series, epristeride (Figure 1 2 ; 38), is a clinical candidate which shows excellent inhibition of the type II isozyme, and inhibits prostatic tumor growth in several animal models .225,233

1.3.2.3.2 17,20-Lyase

Another opportunity for the pharmacologic disruption of androgen biosynthesis may be found in the conversion of the C -2 1 progestins to the C-19 androgens by cytochrome P450 17a-hydroxylase/l7,20-lyase. In the male, androgens are synthesized and secreted by the Leydig cells of the testis. An indirect contribution to the androgen pool is also made by the adrenal cortex. The (DHEA) and produced by the adrenal cortex indirectly augment the androgen pool because they can be converted to testosterone by peripheral tissues. Other tissues, such as liver and prostate are capable of forming testosterone from precursors, but their contribution to the androgen pool is minimal. The synthesis of the androgens by Leydig cells is regulated by the anterior pituitary through the action of the gonadotrophin, luteinizing hormone (LH), also referred to as interstitial cell-stimulating hormone (ICSH). LH is in turn under the regulation of the hypothalamic peptide GnRH, while both LH and GnRH are under the negative feedback regulation of the 55 androgens, estrogens, and progestins (as discussed previously; see also Figure 4). LH binds to its receptor located on the surface of the Leydig cell, and, through a G-protein-mediated process, activates adenylate cyclase, thereby increasing the intracellular concentration of cyclic AMP (cAMP). The intracellular increase in cAMP induces two sets of temporally distinct responses, an acute set (occurring within seconds to a few minutes), and a chronic set (occurring within several minutes to hours) In the acute limb of the response, cAMP activates protein kinase A, which subsequently phosphorylates, thereby activating, a number of enzymes involved in steroid biosynthesis. One of these, esterase, hydrolyzes cholesterol esters found in intracellular lipid storage sites, thus producing free cholesterol, which is translocated to the mitochondrion. In the chronic limb of the response, cAMP binds to and activates a nuclear transcription factor. This activated protein complex binds to specific DNA elements within certain gene promoter regions, termed cAMP-responsive sequences (CRS). Binding induces an increase in the transcription of a number of genes, including genes which code for steroidogenic enzymes such as cholesterol side chain cleavage enzyme (cytP450scc) and 17a- hydroxylase/17,20-lyase (cytP450i7a). Cholesterol side chain cleavage enzyme, a mixed function oxidase found in the mitochondrion, converts cholesterol to . The conversion of cholesterol to pregnenolone is the rate-limiting step in the biosynthesis of all steroid hormones .236,237

A number of additional, non-mitochondrial enzymes (including cytP450n), which convert pregnenolone and progesterone to testosterone, comprise the remainder of the androgen biosynthetic apparatus. 56

A4-Pathway A5-Pathway

3p-HSDH/A4-5-lsomerase HO' Progesterone Pregnenolone

CytP45017 (17a-Hydroxylase) CytP45017 (17a-Hydroxylase)

=o ••OH

Q., ^ v 3p-HSDH/A4'6-lsomerase 17a-Hydroxyprogesterone 17a-Hydroxypregnenolone

CytP450. (17,20-Lyase) CytP45017 (17,20-Lyase) O

- 3p-HSDH/A4's-lsomerase Androstenedione Dehydroepiandrosterone (DHEA)

17P-HSDH 17P-HSDH OH OH

3p-HSDH/A4S-lsomerase HO Testosterone OH

5a-Reductase

5 a-Di hydrotestosterone

Figure 13. Androgen Biosynthesis 57

This apparatus contains two major pathways, known as the A4- and A5- pathways, which are distinguishable because the A4-pathway converts progesterone to androstenedione, and the A5-pathway converts pregnenolone to DHEA (Figure 13). The first two enzymatic transformations in both pathways are performed by another cytochrome P450 monoxygenase, found in the smooth endoplasmic reticulum, termed cytochrome P450 17a-hydroxylase/17,20-lyase (cytP450i7a). This enzyme oxidatively cleaves the 2-carbon acetyl side-chain attached to carbon-17, corresponding to positions 20 and 21 of the pregnane skeleton. The enzyme also requires NADPH and molecular oxygen. Reducing equivalents are not directly provided by NADPH, rather, electrons are transferred from NADPH to 17,20-lyase by NADPH-cytochrome P450 reductase. The resulting 17-keto androgens, DHEA and androstenedione, are further metabolized to testosterone and 5a-DHT by several hydroxysteroid dehydrogenases and a double bond isomerase (Figure 13).

Early work implicated the progestins, 17a-hydroxyprogesterone or 17a-hydroxypregnenolone, as obligatory intermediates in testosterone biosynthesis which were subsequently metabolized further by a second enzymatic process that cleaved the C 1 7 - C 2 0 bond to yield the C-19 androstane skeleton .238 The accepted belief that two separate enzymes were necessary for the conversion of the C -2 1 progestins to the C-19 androgens was not challenged until 1981 when the proteins containing these enzymatic activities were purified from neonatal pig testis, and were found to be identical .239,240 Instead of simply solving this problem, this finding created yet more controversy, because it questioned how 58 steroidogenic cells control the prevalence of the hydroxylation pathway versus the cleavage pathway. In other words, how do cells in the adrenal cortex stop cytP450i7tt after 17a-hydroxylation, thereby shunting the 17a- hydroxyprogestins into and mineralocorticoid biosynthesis, whereas Leydig cells continue on with C 1 7 - C 2 0 bond cleavage, generating the C-19 androgens? Two explanations of this phenomenon have been put forth. The first involves a mass balance effect where subsequent enzymes in the glucocorticoid and mineralocorticoid biosynthetic pathways compete with cytP450i7a for the 17a-hydroxyprogestins. The second involves the level of expression of cytochrome P450 reductase which transfers electrons from NADPH to cytP450i7a. Since cytP450i7a requires the reductase to transfer electrons in order to activate oxygen and perform oxidative cleavage of the C 1 7 - C 2 0 bond (see discussion of mechanism, and Figures 14 & 15 below), presumably reduced levels of expression of the reductase would impede this reaction, thus effectively stopping catalysis after 17a- hydroxylation. It appears that the second of these scenarios is operative. Briefly, Yanagibashi and Hall found that monoclonal antibodies to 21- hydroxylase effectively blocked this enzymatic activity, but failed to induce a concomitant increase in 17,20-lyase activity. Moreover, they found that addition of purified P450 reductase to microsomes or purified cytP450i7a protein, isolated from either adrenal or testis, increased 17,20-lyase activity in a dose-dependent fashion .241

The 17,20-lyase gene (CYP17) is part of the cytochrome P450 superfamily, and has been localized to the long arm of chromosome 1 0 (10q24.3).242,243 The human, porcine, bovine, murine, and rat full-length 59 cDNAs coding for 17,20-lyase have been isolated from both the gonad

(ovary and testis) and the adrenal gland ,244'246 and several of the parent genes have been isolated and sequenced .247'252 Based on the DNA sequence data obtained from this work, it appears that there are not tissue-specific splicing, or post-transcriptional processing variants, and thus there are not tissue-specific 17,20-lyase isozymes.

7 ( si ibstrate J Enzyme

Enzyme

Figure 14. Proposed enzymatic mechanism of 17a-hydroxylation. Note that electrons are provided by NADPH and are transferred to cytP450n by NADPH-cytochrome P450 reductase. 60

It is beyond the scope of this text to provide an in-depth review of the molecular biology, expression and regulation of the CYP17 gene, but thorough discussions may be found elsewhere.253"259

The proposed enzymatic mechanism of cytP450i7a (Figures 14 and 15) involves two sequential oxidations, and utilizes two equivalents each of NADPH and molecular oxygen. The first step (Figure 14) is a typical cytochrome P450-mediated hydroxylation which stereoselectively introduces a hydroxyl function in the 17a-position. The enzyme first binds the substrate (either progesterone or pregnenolone), which is accompanied by a change from the high to the low spin-state of the heme iron, yielding a type-I difference spectrum. Next, an electron is transferred to the heme iron by NADPH-cytochrome P450 reductase, thus reducing the iron from the Fe111 to the Fe11 oxidation state. The heme iron then binds molecular oxygen, giving up an electron in the process to become re-oxidized to the F e 111 state, and generating a ferric-peroxy radical species. P450 reductase transfers a second electron from NADPH to the ferric-peroxy radical, and the resulting ferric peroxide is protonated by an active-site residue. The ferric hydroperoxide disproportionates to give water and an Fev=0 C'oxene") species. One of the canonical forms of this oxene intermediate is an oxoferrylporphyrin radical cation. This species is proposed to abstract the 17a-hydrogen atom, generating an FeIV-bound hydroxyl radical, and a 17a substrate radical. The two radical species then recombine to form the hydroxylated product.260*263 61

The mechanism of the second oxidation, the C 17-C20 bond cleavage, or 17,20-lyase reaction (Figure 15), appears to proceed through the nucleophilic attack of a ferric peroxide on the C-20 carbonyl, rather than an oxene mechanism. Again, the initial step involves the binding of substrate

02 1e' 1 e ' Enz V "Fem >e" Sub Sub ✓ I O o \ 0«

HB H20 Enzyme o A OH

Figure IS. Proposed enzymatic mechanism of 17,20-lyase. Note that electrons are provided by NADPH and are transferred to cytP450n by NADPH-cytochrome P450 reductase. 62 to the enzyme active site, accompanied by a concomitant shift from the high to the low spin state of the heme iron. The product of the hydroxylation reaction is the substrate for the lyase reaction, and both reactions occur at the same active site. Therefore, the substrate is already bound to the enzyme, though an equilibrium exists between bound and free 17a-hydroxyprogestins. NADPH-cytochrome P450 reductase transfers an electron to the heme iron, converting it to the F e 11 oxidation state. The heme iron then binds molecular oxygen, giving up an electron in the process to become re-oxidized to the F e 111 state, and generating a ferric-peroxy radical species. P450 reductase transfers a second electron from NADPH to the ferric-peroxy radical, generating a ferric-peroxide species. The ferric peroxide performs a nucleophilic attack on the C-20 carbonyl, yielding a ferric peroxy hemiketal which collapses in a heterolytic fashion through a Baeyer-Villiger rearrangement to yield the 17-O-acetoxy hemiketal. General acid-base catalysis by an active site amino acid residue results in elimination of acetate, and generation of the 17-ketone. The remaining Fe111-bound oxide is protonated and then decomposes to give water, thus regenerating the initial form of the active site. An important question regarding the catalysis of C 17-C20 bond cleavage is why this reaction proceeds through the formation of a ferric peroxy hemiketal, rather than proceeding through protonation and disproportionation of the ferric peroxide to form an oxene (as in the 17a-hydroxylation reaction). One explanation involves the disruption of an active site proton shuttle by the substrate 17a-hydroxyl due to hydrogen bonding with one of the active site residues (Arg-346) which participates in this shuttle. This prevents 63 protonation of the ferric peroxide and favors nucieophilic attack on the C-20 carbonyl by the ferric peroxide.260 There is considerably more controversy surrounding the mechanism of the C 17-C20 bond cleavage than 17a- hydroxylation. This is due to the fact that trace amounts of androsta-4,16- diene-3-one and 17a-hydroxyandrost-4-ene-3-one (Figure 17; 56: ) have been isolated from 17,20-lyase incubations. Alternative mechanisms involving homolytic cleavage of the ferric peroxy hemiketal have been put forth to account for these observations.260'263

As with the 5a-reductase inhibitors, 17,20-lyase inhibitors may be divided into two general classes, nonsteroidal and steroidal. The most extensively studied nonsteroidal inhibitors of cytP450i7a are the imidazole antimycotic, , and the antiepileptic agent, (Figure 16; 45, 46).264'269 Objective response rates have been achieved with high doses of these compounds in the treatment of relapsed prostatic carcinoma.270,271 Unfortunately, these compounds display non-specific inhibition of a number of other cytochrome P450's, as well as numerous undesirable adverse effects, thus precluding their widespread use in androgen ablative therapy for prostatic carcinoma. In an attempt to improve both activity and therapeutic index, several thiophenol-based derivatives of aminoglutethimide have been prepared, but were ineffective cytP450i7a inhibitors.272 The only imidazole-based compound which has been extensively developed as a 17,20-lyase inhibitor is the benzimidazole analog, (R 75251) liarozole (Figure 16; 47). In preclinical studies, liarozole has been shown to reduce plasma testosterone and androstenedione levels in vivo,273 as well as inhibit the growth of prostatic tumors in the Dunning 64

R3327 rat model.274 A number of pyridine-containing compounds, such as metyrapone (Figure 16; 48), have been shown to be inhibitors of 17, 20- lyase (Figure 16; 48-51)275'278 These compounds displayed K; values in the low micromolar range, while the Km for the substrate, progesterone is 140nM.279 Bulky esters of pyridine-3- (Figure 16; 52) have been shown to be somewhat better 17, 20-lyase inhibitors, displaying K; values in the sub-micromolar range.280 Pyridine-based compounds yield type-II P450 difference spectra, which indicates that the pyridine nitrogen is coordinating to the heme iron at the active site of cytP450i7tt.281 The nonsteroidal antiandrogens flutamide, hydroxyfhitamide, and nilutamide (Figure 6; 1-3) have been shown to have inhibitory action on 17,20-lyase, although their K; values were in the low micromolar range 282,283 Though the affinities of these antiandrogens for 17, 20-lyase are not high, it would presumably be an added benefit of treatment with these to block biosynthesis as well as androgen receptor binding. Other nonsteroidal compounds which have been reported as inhibitors of 17,20-lyase include: the sedative-hypnotic, etomidate,284 nicotine and cotinine 285 fi-carboline286 and other pineal indoles,287 the anticonvulsant, ,288 ,289 hydroxyfluoroazobenzenes,290 the HMG-CoA reductase inhibitor, mevinolin,291 and suramin.292 None of these exhibit particularly high affinity for 17,20-lyase, and many possess other unnecessary and unwanted pharmacologic activities, thus these compounds have not been further developed for use as 17,20-lyase inhibitors in the treatment of prostate cancer. 45: Ketoconazole 46: Aminoglutethimide 47: Liarozole

och 3 48: Metyrapone 49: n - 0 51: SU 10603 52 50: n = 1

Figure 16. Structure of non-steroidal inhibitors of 17,20-lyase

The earliest reported synthetic steroidal inhibitors of 17,20-lyase were 160-bromo-3P,17a-dihydroxy-5a-pregnane-ll,2O-dione and 170- ureido-androsta-l,4-diene-3-one (Figure 17; 53, 54). These were reported to be irreversible inhibitors which displayed K; values in the sub-micromolar range. These compounds were not rigorously proven to be enzyme-activated irreversible inhibitors because it was not demonstrated that irreversible inactivation was dependent upon catalytic activity. For example, studies demonstrating the dependence of irreversible inactivation upon NADPH and time, the lack of recovery of activity after dialysis, the protection from inactivation by substrate, or the absence of protection by electrophile scavengers were not reported. The authors also did not recognize the 66 possible utility of these compounds in the treatment of prostatic carcinoma, thus they have not been further developed.293,294 A number of progestins, used as oral contraceptives, have been reported to be weak inhibitors of 17,20-lyase 295 All of these compounds possess an acetylenic functionality in the 17a-position (Figure 17; 55: norethindrone). Presumably, cytP450i7O catalytic activity generates a reactive species at this center which alkylates either a heme nitrogen, and/or an amino acid residue at or near the active site. Studies investigating the mechanism of inhibition of these compounds have not been reported. Other 17a- acetylene-containing compounds which have been reported to be 17, 20- lyase inhibitors include, the anabolic androgen, ,296'298 and the antiprogestin , (RU 486).299'302

OH

HO

53 54 55: Norethindrone 56: Epitestosterone

Figure 17. Structure of steroidal inhibitors of 17,20-lyase

17a-Hydroxyandrost-4-ene-3-one (Figure 17; 56: epitestosterone) has been reported to be a potent competitive inhibitor of 17,20-lyase, 67 displaying an apparent K i value in the low nanomolar range.303 This compound effectively represents the 20,21-nor analog of 17a- hydroxyprogesterone. Presumably, it binds to the active site in much the same way as 17a-hydroxyprogesterone, with the 17a-hydroxyl forming a hydrogen bond with the same active site residue(s) as 17a- hydroxyprogesterone, but without possessing the C-20, C-21 side-chain. The enzyme is not able to introduce additional hydroxyl functionality due to the 17a-hydroxyl of epitestosterone disrupting the hypothesized proton shuttle necessary for generation of the oxene intermediate which is proposed to perform hydroxylation,260 and there is no C-20 carbonyl acceptor of ferric peroxide nucleophilic attack (Figures 14 & IS ) .26 0 ' 263 Another competitive inhibitor of cytP450i7a which displays a low nanomolar K; value is 22-amino-23,24-bisnor-5-cholen-3|l-ol (Figure 18; 57: 22-AJBC).304'305 Spectral binding studies demonstrated that 22-ABC produces a type-II difference spectrum, indicating that the amine nitrogen is coordinated to the heme iron at the active site. The rest of the molecule possesses the 5-ene-3(l-ol configuration which is identical to pregnenolone, one of the substrates for cytP450i7tt. Unfortunately, this compound also exhibits a nanomolar K; value for the cholesterol side chain cleavage enzyme. Recently, a series of 17-(3-pyridyl) substituted androstene-based compounds have been described which display sub-nanomolar K; values (Figure 18; 58, 59).306,307 The rationale for the preparation of these compounds was based on the prior observed inhibitory activity of the 3- pyridyl acetate esters (Figure 16; 52). The intent of their design was to prepare analogs which incorporate the steroid skeleton and the pyridine 68 ring so that the lone pair on the pyridine nitrogen could coordinate to the active site heme iron. Interestingly, these compounds exhibit a strict requirement for the 3-pyridyl substitution pattern. 2- and 4-pyridyl substitution results in a dramatic loss of inhibitory activity, as does methylene homologation between the steroid C-17 and the pyridine ring. Although less stringent, these compounds also display a requirement for the C 16-C17 double bond, with reduction of this bond causing an approximately 1 0 -fold reduction in the K; value. In vivo studies have demonstrated the ability of these compounds to reduce prostate and seminal vesicle weights, with no effect on adrenal weight. Additionally, these compounds caused a decrease in plasma testosterone levels, and an increase in plasma LH levels .308 Brodie and coworkers have recently prepared two compounds which they report to be competitive inhibitors of

17,20-lyase (Figure 18; 60: 22-A, 61: 22-oxime ) .309'310 Interestingly, both compounds also appear to exhibit 5a-reductase inhibition, displaying nanomolar K; values. This would be desirable because simultaneous inhibition of both enzymes would presumably provide more effective androgen ablation. The 20p-carboxaldehyde displayed a K; value for 17,20- lyase in the sub-micromolar range, while the 2 2 -oxime exhibited a nanomolar K; value. Although not reported, it is reasonable to hypothesize that the 2 2 -oxime function of 61 is coordinating to the heme iron of the active site. Both compounds were also evaluated in vivo, where they lowered plasma testosterone levels. A group of B-ring substituted progestins have been reported as competitive inhibitors of 17,20-lyase. These are megestrol acetate (Figure 18; 62),311 medroxyprogesterone 69 acetate (Figure 18; 63),311 and 6-methylene-4-pregnene-3,20-dione (Figure 18; 64: LY 207,320).312 Each of these possess small alkyl substituents in the 6-position, implying that B-ring substitution may be accomodated by the 17,20-lyase active site. No other B-ring substituted progestins have been reported as inhibitors of 17,20-lyase, thus the structure-activity relationships pertaining to these types of agents remain to be explored. Other compounds which have been reported to display weak competitive binding to the cytP450i7tt active site include, the progestins promegestrone,313 and 20-hydroxyprogesterone,314 and the anabolic androgen, .315

HO HO 57:22-ABC 58 59 60:22-A

NOH C'OAc OAc

61:22-oxime 62 63 64: LY 207,320 Megestrol Acetate Medroxyprogesterone Acetate

Figure 18. Structure of steroidal competitive inhibitors of 17,20-lyase 70

17p-(Cyclopropylamino)-androst-5-ene-3(3-ol (Figure 19; 65: MDL 27302) has been reported to be an enzyme-activated irreversible inhibitor of 17, 20-lyase.316 The design of this compound was based on the observation that a number of hepatic cytochrome P450 hydroxylases were inactivated by cyclopropylamines through the generation of a P-iminium radical which alkylates the enzyme active site. This compound demonstrated an apparent K; of 90 nM, and it was meticulously demonstrated that irreversible inactivation was dependent upon NADPH and time, that the enzyme was protected from inactivation by substrate, and that irreversible inactivation was unaffected by dialysis or the presence of electrophile scavengers such as dithiothreitol. Another series of mechanism-based irreversible inhibitors have been described (Figure 19; 66, 67). These compounds display micromolar K; values, and do not exhibit specificity for 17,20-lyase, irreversibly inactivating 21-hydroxylase as well.317

? NH R

65 66 67 R = v i n y l A 5 ; R = H , 3 P - O H R = ethinyl A 4 ; R = O

Figure 19. Structure of steroidal irreversible inhibitors of 17,20-lyase 71

An affinity label inhibitor, 17a-bromoacetoxyprogesterone, alkylates a unique cysteine residue which is presumably in the active site of purified cytP450i7«.318

1.4 Breast Cancer

The ACS estimates that 183,400 new diagnoses of breast cancer will be made in 1995. The vast majority of these will be diagnosed in women. Breast cancer is the second leading cause of cancer mortality among women in the U.S., second only to lung cancer. The ACS estimates that there will be 46,000 deaths due to breast cancer in 1995. Depending upon how life span is defined, the estimated risk of a woman developing breast cancer in her lifetime ranges from 1 in 8 to 1 in 10.1

Over the past 60 years, the incidence of breast cancer has steadily increased, while the mortality rate has remained constant. The explanation for this remains unclear, but is likely due to improvements in detection, heightened public awareness of the disease, and increases in life expectancy. Breast cancer incidence increases with age, and exhibits a geographic distribution. The highest rates are observed in Canada, the U.S. and northern Europe, while the lowest rates are found in Mexico, Asia, and developing countries. Explanations which have been put forth to account for this phenomenon rely largely on lifestyle considerations, such as high dietary fat and low fiber intake, increased alcohol consumption, and later age of first full-term pregnancy. Other considered risk factors include early onset of menarche, late onset of menopause, and positive family history. The unifying theme for all of these risk factors is overall 72 exposure to estrogens. Increased exposure is hypothesized to be associated with increased risk. It must be appreciated that fewer than one third of all cases of breast cancer can be linked to any of these risk factors.1,319

1.4.1 Breast Cancer Treatment

Surgery, external beam radiation, and cytotoxic chemotherapy are three of the primary weapons used to treat breast cancer. Currently, there are two surgical interventions which are routinely used to resect malignant breast tissue: modified radical mastectomy (with or without regional lymph node dissection), and tumor extirpation (lumpectomy). Historically, radical mastectomy also involved resection of the pectoralis muscles of the anterior chest wall (the Halsted procedure), which would result in significant post-surgical limitation in the movement of the associated upper extremity. Refinement of surgical technique in the 1970's led to muscle and nerve sparing procedures which have ended these complications. External beam radiation is generally used as an adjuvant to surgery, particularly lumpectomy, and is thus limited to treatment of localized disease. Refinements in the delivery of radiation to the anterior chest wall have lowered the morbidity (e.g. costal osteolysis, pulmonary fibrosis) associated with this form of therapy. Cytotoxic chemotherapy is also used as an adjuvant to surgery. Cytotoxic chemotherapy appears to exhibit a slightly higher success rate in premenopausal patients than in postmenopausal patients. The goal of both radiation and chemotherapy is to deliver a tumoricidal dose of therapeutic agent to malignant tissue, while limiting the collateral damage to normal tissue. The effectiveness of 73 both therapies drops markedly with increased extent of metastatic disease, thus demonstrating that effective treatment and survival are greatly dependent upon early detection. In depth discussions of clinical presentation, diagnostic criteria, tumor grading, clinical staging, treatment regimens, criteria defining choice of a particular treatment regimen, and prognostic indicators of clinical outcome are beyond the scope of this text. The reader is directed elsewhere for thorough recent reviews of these subjects.320,321

A fourth possible therapeutic modality involves endocrine manipulation. Approximately two-thirds of all breast tumors contain estrogen receptors (ER), with one-half of these exhibiting dependence upon estrogen for growth.322,323 Therefore, the goal of endocrine treatment is to deprive these estrogen dependent tumor cells of their supply of steroid hormones, thereby inducing death of estrogen-dependent tumor cells, and suppressing tumor growth. Examination of the physiology of steroid hormone biosynthesis and action suggests several logical sites of pharmacologic intervention. These include suppression of estrogen production through disruption of the normal feedback control of the hypothalamic-pituitary-ovarian axis, antagonism of estrogen action at its receptor, and ablation of estrogen production through inhibition of the estrogen biosynthetic enzymes (Figure 20). There is an added layer of complexity in the manipulation of female reproductive endocrinology attributable to the fact that the postpubertal female population may be divided into two classes, premenopausal and postmenopausal. In the case of premenopausal women, the primary site of estrogen biosynthesis is the 74 ovary, and circulating levels of estrogens (although cyclical) are higher than in postmenopausal women. In postmenopausal women, the primary site of estrogen biosynthesis is peripheral. The adrenals produce androgens, which breast and adipose tissue aromatize to produce estrogens.324'326

Axis Ligapd-Receptpr finding Biosynthesis

C2l Progestins Hypothalamus 17,20-Lyase GnRH ER V Anterior C1B Androgens Pituitary Estrogens Aromatase FSH &LH V C18 Estrogens

Ovary ER

Figure 20. Schematic depiction of the possible sites of endocrine manipulation in the treatment of mammary carcinoma.

1.4.1.1 Hypothalamic-Pituitary-Ovarian Axis Disruption

1.4.1.1.1 Oophorectomy, Hypophysectomy and Adrenalectomy

Until recently, surgical endocrine ablative procedures, such as oophorectomy and hypophysectomy were standard therapies for patients with recurrent breast cancer.322 Comparative trials of aromatase inhibition and antiestrogens with surgery demonstrated similar response 75 rates.327 Because pharmacologic therapies do not require hospitalization or surgery and do not possess drastic endocrine sequelae, they have replaced surgical ablation.

1.4.1.1.2 Progestins

As described previously, the estrogens, androgens and progestins provide negative feedback regulation of the hypothalamus and pituitary; suppressing GnRH and gonadotropin release, thereby removing the stimulus for ovarian estrogen production. Additionally, progestins may also provide negative feedback regulation of pituitary ACTH secretion, thereby indirectly reducing plasma estrogen levels by reducing adrenal production of androgens. In breast tumor cell lines, it has been reported that progestins provide direct antiproliferative effects, and abolish the stimulatory effects of estrogens on growth. Progestins may also exert antiproliferative effects by suppressing ER levels, or by increasing the activity of 17p-hydroxysteroid dehydrogenase, thereby increasing conversion of estradiol to (an estrogen of much lower potency).327 Synthetic progestins such as megestrol acetate and medroxyprogesterone acetate have been utilized in this form of therapy.

1.4.1.1.3 GnRH Agonists

This topic has been covered in depth earlier in this manuscript (please refer to section 1.3.2.1.4). To briefly reiterate, the logic of this form of endocrine ablative therapy is to induce GnRH receptor tachyphylaxis on the cells of the anterior pituitary, thereby effectively removing the signal 76 transduction pathway by which the hypothalamus stimulates the anterior pituitary to secrete the gonadotropins, FSH and LH. Without gonadotrophin-stimulated synthesis of estrogens, plasma estrogen levels are suppressed. GnRH agonists have been used to treat both premenopausal and postmenopausal patients. The response rates in premenopausal women were similar to those seen with oophorectomy (approximately 30-40%). Response rates in postmenopausal women were poor. This may be explained by the lack of functioning ovaries in the postmenopausal woman, and therefore an absent therapeutic target. As described in section 1.2.8.3.3, there is some evidence which indicates that breast and prostate tumor cells possess GnRH receptors and that GnRH agonists have a direct antiproliferative effect on tumor cells.200,328

1.4.1.2 Antiestrogens

Antagonism of estrogen action at its receptor remains the mainstay of endocrine ablative therapy for the treatment of hormone responsive breast cancer. Antiestrogens function by competing with estrogen for the hormone binding site on the estrogen receptor, thereby antagonizing the normal biologic function of the estrogens. The antiestrogens may be divided into two structural classes, nonsteroidal and steroidal.

The first , MER-25 (Figure 21; 68), was developed in the 1950's and was based on the structure of the stilbene-based compounds, diethylstilbesterol and chlorotnanisene. Preclinical studies demonstrated antiestrogenic activity both in vitro and in vivo, but further development of this compound was discontinued due to marked neurotoxic effects.329,330 77

The failure of MER-25 led to the synthesis and development of a second generation of nonsteroidal antiestrogens which possessed the triarylethylene base structure. One of these analogs, clomiphene (Figure 21; 69), was originally developed for use as an antifertility agent. As was the case with the first GnRH agonists, clomiphene, ironically, displayed the opposite activity for which it was developed. It displayed pro-fertility activity. The explanation for this may be found in the fact that in successfully competing with estrogen for its receptor in the hypothalamus and the anterior pituitary, clomiphene removes negative feedback regulatory control and actually induces an elevation in serum gonadotrophin levels, thereby stimulating Graafian follicle development. In clinical trials in patients with advanced stage metastatic disease, both MER-25 and clomiphene exhibited activity by reducing bone pain and tumor size.329,330

Tamoxifen (Figure 21; 70), another of the triarylethylene antiestrogens, was developed in the 1960’s. Preclinical studies both in vitro and in vivo demonstrated that tamoxifen exhibits partial agonist activity, effectively competing with estrogen for the estrogen receptor.330 331 The tamoxifen-occupied estrogen receptor is not entirely biologically inert. It possesses novel signal transduction capability, whose physiologic relevance is currently under investigation.331,332 Structure-activity relationship investigations of tamoxifen have revealed that the Z- configuration is important for antiestrogenic activity 329 78

f r O*^—

,OCH;

OH

68: MER-25 69: Clomiphene 70: Tamoxifen OH

HO

71: R=(CH2)ioCONCH3(CH2)3CH3 IC1164,384 72: R=(CH2)9SO(CH2)3CF2CF3 IC1182,780

Figure 21. Structure of representative antiestrogens

Tamoxifen is widely used clinically, and is the only antiestrogenic agent currently approved for treatment of breast cancer (although and are currently undergoing phase III clinical evaluation). Tamoxifen has been shown to prolong disease-free survival, and appears to also possess serendipitous favorable effects which aid in the prevention of osteoporosis and atherosclerosis.327,333,334

Because tamoxifen is a partial agonist, further development of antiestrogens has focused on the preparation of compounds which exhibit pure antiestrogenic activity. Presumably, agents of this type would provide 79 more complete and longer lasting antitumoral effects. Design of pure antiestrogens has been based on the estradiol carbon skeleton. Introduction of long alkyl chain substituents at the 7a-position (Figure 21; 71, 72) produces analogs which exhibit high affinity for the EE. The steric bulk in the 7a-position is presumably functioning to prevent ligand binding-induced conformational changes in the ER which result in ER activation, thereby maintaining the ER in an inactive conformation. This inactive conformation is hypothesized to be either unable to dimerize and bind to the target estrogen response element found in the promoter region of estrogen-responsive genes or unable to associate with and activate other transcriptional activating factors necessary for initiation of transcription. Preclinical investigation of the effectiveness of ICI 164,384 and ICI 182,780 have demonstrated that these compounds inhibit estrogen-dependent gene expression, decrease cell and tissue ER content in vitro, and cause mammary tumor regression in vivo,332-335 Presently, these compounds are undergoing clinical evaluation. Other compounds which have also been reported to exhibit pure antiestrogenic activity possess other 7a- substituents, or llp-substituents.336

1.4.1.3 17,20-Lyase Inhibitors

This topic was extensively discussed in section 1.3.2.3.2 of this manuscript as an approach to hormonal ablation for the treatment of prostate cancer. Inhibition of 17,20-lyase also has relevance in the treatment of hormone dependent breast cancer. The rationale for utilization of this type of therapy in breast cancer treatment is largely the 80 same as with prostate cancer. If it is possible to disrupt the biosynthesis of the androgens, then by extension disruption of estrogen biosynthesis is achieved because the biochemical precursors of the estrogens are the androgens. Please refer to section 1.3.2.3.2 and Figures 13-18 for in depth discussion of 17,20-lyase.

1.4.1.4 Aromatase

The final and rate-limiting step in the biosynthesis of the estrogens is the conversion of the C-19 androgens to the C-18 estrogens by cytochrome P-450arom aromatase (Figure 21). Because aromatase is the final (rather than an intermediate) enzymatic activity in the estrogen biosynthetic pathway, inhibition of this enzyme would not affect the supply of substrate to other steroid biosynthetic pathways, and therefore would presumably not result in adverse endocrine sequelae. This fact makes it a logical and attractive target for the pharmacologic disruption of estrogen biosynthesis. The purpose of this form of therapy is to deprive estrogen- dependent breast tumors of their supply of steroid hormone. As stated previously, peripheral aromatization, particularly in breast and adipose tissue, is the source of estrogen production in postmenopausal women. Local breast tumor tissue estrogen levels are higher than plasma levels in both pre- and postmenopausal women, with ER+ tumor cells possessing higher levels than ER- tumor cells.319,337 This supports reports of local aromatase activity in malignant breast tissue,324,338'342 and illustrates the necessity of disrupting local estrogen production by tumor cells in order to deprive estrogen-dependent tumor cells of their supply of hormone. Progesterone

CytP45017 (17a-Hydroxylase)

17a-Hydroxyprogesterone

CytP450 (17,20-Lyase)

Aromatase Androstenedione Estrone

17P-HSDH 17p-HSDH

OH

Aromatase Testosterone 17P-Estradiol

Figure 22. Estrogen Biosynthesis 82

Aromatase is found in the smooth endoplasmic reticulum . 343 Aromatase converts the C-19 androgens to the C-18 estrogens by oxidatively removing the angular C-19 methyl group of androstenedione or testosterone (androstenedione is the preferred substrate). This is achieved via three sequential oxidations which utilize three equivalents each of molecular oxygen and NADPH (Figure 23). As is the case with other P450- catalyzed oxidations, the electrons provided by NADPH are transferred to aromatase by NADPH-cytochrome P450 reductase. The first oxidation consists of a typical P450-catalyzed (oxene) hydroxylation (Figure 14) which introduces a hydroxyl group on the angular C-19 methyl group. The second oxidation is again a typical P450-mediated hydroxylation (Figure 14) which stereoselectively removes the pro-R hydrogen of the C-19 methylene hydroxyl, thereby introducing a second hydroxyl function at C- 19.

O OH HO. HO. NADPH NADPH

Androstenedione

NADPH; 02

HO Estrone

Figure 23. Proposed enzymatic mechanism of aromatase 83

The resulting C-19 gem-diol readily dehydrates to yield the 19-oxo androgen. The final oxidation results in the elimination of the l^-hydrogen (which is incorporated into a molecule of water), elimination of C-19 as formic acid, and generation of the A-ring phenol characteristic of the estrogens. The mechanistic details of the oxidative cleavage of the C 10-C19 bond are still the focus of considerable debate .262,344,345

Aromatase inhibitors may be divided into two groups, nonsteroidal and steroidal. Nonsteroidal inhibitors are, in general, competitive, reversible and yield type-II difference spectra. Type-II difference spectra imply that a heteroatom, such as nitrogen or , is coordinating to the heme iron. The original nonsteroidal aromatase inhibitors are (as with 17,20-lyase) the antiepileptic agent, aminoglutethimide, and the imidazole antimycotic, ketoconazole (Figure 16; 45, 46). Lack of specificity, and unacceptable adverse effects provided the impetus for development of other compounds to supplant ketoconazole and aminoglutethimide in the treatment of breast cancer. Imidazole-based compounds, such as and CGS 18320B (Figure 24; 73, 74), have been prepared and have displayed approximately 3 orders of magnitude greater affinity for aromatase than aminoglutethimide. Unfortunately, acceptable selectivity remains an elusive target since these compounds also inhibit the biosynthesis of the progestins, the , and the mineralocorticoids. Several triazole-based compounds have been synthesized and evaluated for aromatase inhibitory activity in vitro and in vivo (Figure 24; 75-77). These compounds exhibit nanomolar K; values, and a very high degree of selectivity for aromatase. These compounds have 84 been reported to possess good oral , and to cause regression of DMBA-induced mammary tumors in a rat model. The structural features which these molecules possess that have been determined to be necessary for activity are a free amine, either as an aniline or within a ring system such as an imidazole or a triazole, which can coordinate to the heme iron at the active site of aromatase, and an aromatic ring which can fill the space corresponding to the A-ring of androstenedione .346’347

CN

NC CN

73: Fadrozole 74: CGS 18320B

NC. CN NC CN 75: Arimidex 76: Vorozole 77:

Figure 24. Structure of nonsteroidal aromatase inhibitors

Preparation of steroidal analogs has led to the development of three categories of compounds: competitive inhibitors, irreversible inhibitors (affinity labels), and enzyme-activated irreversible inhibitors (suicide substrates). The majority of steroidal inhibitors of aromatase compete 85 with substrate (either testosterone or androstenedione) for the enzyme active site. These compounds produce type-I difference spectra, indicating that they are displacing substrate from the active site rather than coordinating a heteroatom to the heme iron. The structural requirements for aromatase inhibition by steroidal compounds were initially determined by the screening of available steroids in a human placenta microsomal assay.348 These requirements include, a C-19 steroid carbon (androstane) skeleton containing a trans A/B ring junction, A 4 unsaturation, a carbonyl at C-3, and either a carbonyl, or a P-hydroxyl at C-17. Subsequent investigation has largely focused on functionalization of the A or B rings, and modification of the C-19 angular methyl group.

A number of competitive inhibitors of aromatase have been reported. The spatial requirements for the A-ring binding to the active site are rather restrictive, therefore only a limited number of effective inhibitors possessing A-ring modifications have been described. One of these, in which the 3-ketone has been replaced with a methylene group, exhibits potent competitive inhibition with a K; value of 4.7 nM (Figure 25; 78). More extensive structural modifications of the B-ring are tolerated. Bulky substituents at the C-7 position have provided a series of very potent aromatase inhibitors. 7a-(4'-amino)phenylthio-androst-4-ene-3,17-dione (Figure 25; 79: 7a-APTA) is a very effective inhibitor, with an apparent K; of 18 nM. This compound has also demonstrated effective aromatase inhibition in mammary tumor cell culture .349 7a-APTA also exhibits effective inhibition of tumor growth in a DMBA-induced, hormone dependent rat mammary tumor model .350 Evaluation of variously 86 substituted aromatic analogs of 7a-APTA provided no correlation between the electronic character of the substituents and inhibitory activity.

Additionally, introduction of A 6 unsaturation reduces inhibitory activity, implying that C-7 substituents must project from the alpha face of the steroid ring system for optimum inhibitory activity. A series of carbon isosteres of 7a-thio substituted androstanes have recently been reported (Figure 25; 80).351 These compounds also exhibit very potent inhibitory activity, with K; values ranging from 15-40 nM. Presumably these compounds would display superior oral bioavailability, since replacement of the sulfur atom with carbon should limit metabolic degradation. Another position on the androstane skeleton which has received considerable investigation is the angular C-19 methyl group-the site of enzymatic oxidative cleavage. In this series, heteroatoms have been introduced in an effort to enhance affinity through coordination of the heteroatom to the active site heme iron. Examples of these C-19 substituted analogs include, thiiranes, oxiranes, thioethers and azides (Figure 25; 81, 82).352,353 A unique group of C-19 substituted analogs, which have recently been reported, contain a C 2-C19 bridge (Figure 25; 83).

These A-ring bridged steroids consist of both 5- and 6 -membered ring analogs which contain carbon, oxygen, nitrogen, or sulfur atoms. Several exhibit mechanism-based irreversible inhibition of aromatase .354'358

A number of irreversible inhibitors of aromatase, both affinity label- type and enzyme-activated, have been reported. Because of the inherent chemical reactivity of affinity labeling agents, this type of has only been examined in vitro. 87

O

79: 7a-APTA O

O O 8281 83 X = O, S R = SCH3, N3 X = (CH2)n=o,i. S, O, NH

Figure 25. Structure of competitive steroidal aromatase inhibitors

Although this class of compound would be of limited, if any, clinical utility, they are quite useful probes of the active site .347,359 Mechanism-based irreversible inhibitors are compounds which mimic the substrate and are converted to a reactive intermediate as a result of enzyme catalytic activity. The reactive intermediate forms a covalent bond with an amino acid residue in the enzyme active site, thereby alkylating and inactivating the enzyme. This type of compound is more useful clinically than the affinity labeling agents since it is inert until activated by the enzyme.

The first compound designed as a mechanism-based inhibitor of aromatase contained a C-19 ethinyl substituent (Figure 26; 84: MDL 18962).360'364 Other reported mechanism-based inhibitors which possess 88 latent chemical groups in the C-19 position include the 19,19-difluoro and 19-thio analogs (Figure 26; 85).365'367 A number of mechanism-based inhibitors have been developed from more detailed biochemical investigation of several inhibitors which were originally thought to be competitive inhibitors. These compounds can be grouped into three general categories: the 4-substituted , 7-substituted androsta- l,4-diene-3,17-diones, and 6 -methylene- or 6 -oxo-androstenediones. The prototype 4-substituted androstenedione is 4-hydroxyandrostenedione

(Figure 26; 8 6 : 4-OHA). 4-OHA and its acetate ester have been evaluated both in vitro and in vivo. Both compounds exhibit enzyme-mediated inactivation of aromatase in vitro, and cause regression of hormone- dependent, DMBA-induced mammary tumors in rats. 4-OHA has undergone clinical evaluation, where it has demonstrated an objective response in the treatment of advanced stage breast cancer in postmenopausal women .368'370 4-Thiol and 4-amine-based analogs of 4- OHA have been prepared, but do not exhibit inhibition as effective as 4-

OHA.371’372

Introduction of a 1,2-double bond into the androstane skeleton results in compounds which display mechanism-based inactivation of aromatase. The simplest of these are androsta-l,4-diene-3,17-dione and androsta-l,4,6-triene-3,17-dione (A1-4- and A 1 4 6 -androstenedione). Introduction of substituents at the 7-position in both A1-4- andA1-4-6- androstenedione has resulted in potent enzyme-activated irreversible inhibitors. 7a-(4'-amino)phenylthio-androsta-l,4-diene-3,17-dione (Figure 26; 87: 7a-APTADD) exhibits high affinity for aromatase, with a K; value of 89

9.9 nM, and possesses the most rapid rate of inactivation reported, with a

^ in act of 8.4 x 10'3 sec1. This compound also displays potent aromatase inhibition in cell culture. Another enzyme-activated irreversible inhibitor which possesses the 1,2-double bond, also contains a C-l methyl group

(Figure 26; 8 8 : ). This analog displays a K; of 6 6 nM and a

&inact of 1 .8 x 1 0 '3 sec.'1. Androstenediones which possess an exocyclic B- ring methylene group also exhibit enzyme-mediated inactivation of aromatase. Bxemestane (Figure 26; 89) possesses an exocyclic double bond at C-6 . This compound exhibits a K£ of 26 nM and a &inact of 4.03 x 10"3 sec.'1, and also induces regression of hormone-dependent mammary tumors in a rat model. The related 6 -oxo analog also displays time-dependent inactivation of aromatase .346,347

OH 84: MDL 18962 85: R = SH, CF2H 86:4-OHA

87: 7a-APTADD 88: Atamestane 89:

Figure 26. Structure of steroidal enzyme-activated irreversible aromatase inhibitors CHAPTER 2

STATEMENT OF PROBLEMS AND OBJECTIVES

As detailed in the previous chapter, androgens play a pivotal role in the progression and growth of prostatic tumors. The disruption of androgen biosynthesis by inhibiting the enzymatic activity of cytochrome P450 17,20-lyase is a logical approach to androgen ablation for the purpose of depriving androgen dependent prostatic tumor cells of their supply of hormone. A number of steroidal and non-steroidal 17,20-lyase inhibitors have been prepared. Until the very recent (ca. 1995) reports of the 17-(3- pyridyl) substituted androstanes (Figure 18; 58, 59 ) 306 and the 22-oxime (Figure 18; 61),310 no potent, selective steroidal inhibitors of 17,20-lyase had been reported. Discovery and development of steroidal compounds which possess both selectivity for 17,20-lyase over the other steroidogenic cytochromes P450, as well as high affinity for the 17,20-lyase active site would be desirable both for their possible clinical utility and for possible insights which they might provide into the structure of the enzyme active site.

As described in the previous discussion of aromatase, a number of 7a-substituted steroids have been reported as quite potent inhibitors of aromatase. Compounds such as 7a-APTA (Figure 25; 79) have been shown to exhibit nanomolar affinity (K i = 18 nM) for aromatase, implying that

90 91 aryl groups in the 7a-position greatly enhance binding to the active site. As detailed in the previous discussions of the proposed 17,20-lyase and aromatase enzymatic mechanisms, both enzymes are heme-containing monoxygenases which perform sequential oxidations to remove small alkyl side-chains. The proposed mechanistic details of these oxidations are quite similar in both enzymatic activities (Figures 14, 15 and 22).260'263 It is therefore reasonable to postulate that similarities may exist in the structures of the two enzyme active sites, and that, by extension, the active site of 17,20-lyase may accommodate substitution in the 7a-position in much the same fashion as does the active site of aromatase. Moreover, as described above, several progestins which possess small alkyl substituents in the 6 -position of the B-ring exhibit competitive inhibition of 17,20-lyase (Figure 18; 62-64), implying that B-ring substitution may be accomodated by the 17,20-lyase active site. To date, these are the only reported B-ring substituted compounds which possess 17,20-lyase inhibitory activity. The structure-activity relationships pertaining to B-ring substituted progestins and androgens for cytP450i7O inhibitory activity have not been investigated.

2.1 Objective 1: Evaluation of 7a-substituted Steroids for 17,20-Lyase Inhibition

A series of 7a-thio substituted progestins will be prepared, and these compounds, as well as la-phenylthioprogesterone and a number of 7a-phenylthio and 7-arylaliphatic substituted androstanes, will be screened for 17,20-lyase inhibitory activity in a rat testis microsomal 92 enzyme assay (Figure 27).

95: PJW-I-85B O 90: PJW-I-77B R = s - O NH; 79: 7a-APTA 91: PJW-I-65B R =

92: PJW-I-61B 96: PJW-I-42A

93: PJW-I-73B » - x Cl 87: 7a-APTADD 94: PJW-I-35B NH2

97,98

Figure 27. Structure of proposed steroidal 17,20-lyase inhibitors

As has been reported, 7a-APTADD (Figures 26 and 27; 87) is a potent enzyme-activated irreversible inhibitor of cytochrome P450 aromatase (Kj = 9.9 nM; feinact = 8.4 x 10 '3 sec/ 1) .373 This compound has not been evaluated in vivo to determine if it displays activity inhibiting mammary tumor growth or inducing mammary tumor regression. It has also not been evaluated in vivo for its effects on normal reproductive endocrinology. 93

2 .2 Objective 2: Evaluation of 7a-substituted Steroids in vivo

Numerous 7a-substituted androgens have been evaluated in vitro using microsomal enzyme assays and human carcinoma cell culture systems.349'373,384 The next step in the development of these compounds is evaluation for efficacy in vivo. In this aspect of the research, 7a-APTADD will be evaluated for tumor growth inhibitory activity in a DMBA-induced, estrogen-dependent Sprague-Dawley rat mammary tumor model. Further, 7a-APTADD will be evaluated for its effects on the estrus cycle, serum estradiol levels, ovarian aromatase activity, and ovarian aromatase mRNA expression in the normal, cycling adult female Sprague-Dawley rat.

A r o m a t a s e

C y t P 4 5 0 19 O

A ndrostenedione E s t r o n e

o

app. Kj=9.9 nM IC50=91.4 nM (M CF-7 cells)

IC50=7.3 nM (JA r cells) app. kinact=8.4x10‘3sec‘1 Q.

7 < x - A P T A D D

Figure 28. In vitro experimental values for 7a-APTADD CHAPTER 3

EXPERIMENTAL METHODS

3.1 General Procedures - Chemistry

Progesterone and androstenedione were purchased from Steraloids (Wilton, N.H.) and other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). The purity of purchased chemicals was checked by thin-layer chromatography, and recrystallization or other purification was performed as needed. All glassware was washed in a sodium isopropoxide base-bath and dried at 1 2 0 °C prior to use. Dioxane was freshly distilled from sodium and benzophenone prior to use. Silica gel column chromatography was performed using Kieselgel 60 (230-400 mesh), which was purchased from E. Merck (Darmstadt, ). Basic aluminum oxide was purchased from Fischer Scientific (Fair Lawn, N.J.). Thin-layer chromatography plates were purchased from Scientific Adsorbents (Atlanta, Ga.). Melting points were determined on a Thomas Hoover capillary melting point apparatus and were not corrected for atmospheric pressure. iH-NMR and 13C-NMR spectra were obtained on a Bruker AF

250 MHz FT-NMR spectrometer using residual CHCI 3 or CH2 CI2 as a reference unless otherwise noted. The NMR data are reported in parts per million (ppm) on the 5 scale as follows: chemical shift [multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet),

94 95 integration, coupling constant (in Hz), interpretation]. Infrared spectra were obtained on a Laser Precision Analytical RFX-40 FT-IR spectrophotometer, utilizing the transept sliding KBr wedge modification of the Michelson interferometer. FT-IR samples were prepared as a KBr pellet. Elemental analyses were performed by Oneida Research Services (Whitesboro, N.Y.) and were within ±0.3% of the theoretical values for the elements indicated. High resolution mass spectra were obtained at the Ohio State University Chemical Instrument Center using either a Kratos MS-30 or VG 70-250S El mass spectrometer.

3.2 Synthetic Methods

Pregna-4,6-diene-3,20-dione (A 4 '6-Progesterone) (99: PJW-I-39B):

O 4-Pregnene-3,20-dione (progesterone) (5 g, 0.016 mol), chloranil (5 g, 0.02 mol, 1.3 equiv.) and tosic acid (1 g, 0.005 mol, cat.) were dissolved in 250 mL of t-butanol. This stirred solution was heated to reflux for 20 hours under an inert atmosphere (Ar). The reaction mixture was cooled to room temperature and filtered. Solvents were removed in vacuo to yield a thick, brown, tar. The tar was dissolved in a minimum volume

(approx. 100 mL) of CHCI 3 and adsorbed to a lOx mass (approx. 50 g) of basic alumina (activity I) by adding the alumina to the CHCI 3 solution and removing solvent in vacuo. The adsorbed alumina was added to the top of a

200 g CHCI3 slurry-packed bed of basic alumina and products eluted with 96

CHCI3 . Solvents were removed in vacuo to yield a pale yellow solid. Crystallization was performed in acetone/hexane and yielded off-white crystals which were dried in vacuo (3 g, 9.6 x 10 '3 mol, 60.0%): m.p. 139-

141°C; Rp 0.31 (15:1 - CH 2Cl2 :ethyl acetate, 20 cm glass-backed silica, 70-

230 mesh, 250pm). Spectral data were as reported .374-375

Pregna-l,4-diene-3>20-dione (A1'4-Progesterone) (100: PJW-I-81B):

O 4-Pregnene-3,20-dione (progesterone)

(1 g, 3.2 x 10' 3 mol), 2,3-dichloro-5,6-

dicyanobenzoquinone (DDQ, 1 g, 4.4 x 1 0 ‘3 mol, 1.3 equiv.) and tosic acid (100 mg, 5 x

10*4 mol, cat.) were dissolved in 100 mL of benzene. This stirred solution was heated to reflux for 8 hours under an inert atmosphere (Ar). The reaction mixture was cooled to room temperature and solvents were removed in vacuo to yield a thick, brown, tar. The tar was dissolved in a minimum volume (approx. 100 mL) of

CHCI3 and adsorbed to a lOx mass (approx. 10 g) of basic alumina (activity

I) by adding the alumina to the CHCI 3 solution and removing solvent in vacuo. The adsorbed alumina was added to the top of a 1 0 0 g CHCI3 slurry-packed bed of basic alumina and products eluted with CHCI 3 . Solvents were removed in vacuo to yield a pale yellow solid. This material was subjected to column chromatography by passing over a 70 g bed of silica gel utilizing 4:1 CH 2 Cl2 :ethyl acetate as the mobile phase. Crystallization was performed in acetone/hexane and yielded white crystals which were dried in vacuo (570 mg, 1.8 x 10 '3 mol, 57.0%): m.p. 97

141-142°C; Rf 0.42 (4:1 - CH2Cl2:ethyl acetate, 20 cm glass-backed silica,

70-230 mesh, 250[im). Spectral data were as reported .374-375

Pregna-4,6,16-triene-3,20-dione (A4 »6 »1 6-Progesterone) (96: PJW-I-42A):

O 4-Pregnene-3,20-dione progesterone) (5 g, 0.016 mol), chloranil (5 g, 0.02 mol, 1.3 equiv.) and tosic acid (1 g, 0.005 mol, cat.) were dissolved in 250 mL of t-butanol. This stirred solution was heated to reflux for 48 hours under an inert atmosphere (Ar). The reaction mixture was cooled to room temperature and filtered. Solvents were removed in vacuo to yield a thick, brown, tar. The tar was dissolved in a minim um volume

(approx. 100 mL) of CHCI 3 and adsorbed to a lOx mass (approx. 50 g) of basic alumina (activity I) by adding the alumina to the CHCI 3 solution and removing solvent in vacuo. The adsorbed alumina was added to the top of a

200 g CHCI3 slurry-packed bed of basic alumina and products eluted with

CHCI3. Solvents were removed in vacuo to yield a pale yellow solid. Crystallization was performed in acetone/hexane and yielded off-white crystals, which were the desired product of this reaction, pregna-4,6-diene- 3,20-dione. The mother liquor from this reaction was concentrated in vacuo and crystallization was performed in acetone/hexane which yielded off- white crystals which were dried in vacuo (1 g, 3.2 x 10'3 mol, 16.0%): m.p. 162-164°C; Rf 0.27 (15:1 - CH^C^ethyl acetate, 20 cm glass-backed silica,

70-230 mesh, 250|im). Spectral data were as reported .374,375 98

7a-Butvlthio-4-pregnene-3.20-dione (90: PJW-I-77BV

Pregna-4,6-diene-3,20-dione (1 g, 3.2

x 1 0 '3 mol) and 30 mL of n-butyl mercaptan (1-butanethiol) were dissolved in 20 mL of freshly distilled (from Na) dioxane. A 300 mg (0.013 mol, 4 equiv.) chip of freshly cut (under hexane & therefore still bright) sodium metal was added to this stirred solution. An inert atmosphere (Ar) was established, and the mixture was heated to 70°C for 3 days. After cooling to room temperature, the reaction mixture was diluted with 100 mL diethyl ether and extracted with two 100 mL portions of saturated NH 4 CI. The organic phase was dried by passing over MgSC >4 and solvents removed in vacuo to yield a pale yellow oil. Crystallization was performed in ethanol and yielded a pale yellow powder which was dried in vacuo (500 mg, 1.24 x 10'3 mol, 38.8%): m.p. 99-101°; Rp .45 (15:2 - CH 2 Cl2 :ethyl acetate, 2 0 cm glass-backed silica, 70-230 mesh, 250 pm); *H NMR (250 MHz, CDCI 3) 8 0.64 (s, 3H,

Cis methyls), 0.88 (t, J- 7.1 Hz, 3H, 7a-side chain methyls), 1.17 (s, 3H, C 19 methyls), 2.09 (s, 3H, C 21 methyls), 3.00 (m, 1H, 7p), 5.75 (d, J= l.l Hz, 1H, vinyl); *3C NMR (CDC13) 8 13.17, 13.49, 17.79, 20.94, 21.92, 22.73, 23.61, 30.69, 31.30, 31.60, 33.83, 35.39, 38.00, 38.35, 38.65, 39.51, 43.65, 46.21, 46.86, 51.90, 63.27 (C7), 126.76 (C4), 167.11 (C5), 198.38 (C3), 208.84 (C20); IR (KBr) 2945, 2929, 2889, 2864, 1701, 1662, 1614, 1414, 1381, 1354, 1331, 1271, 1242, 1219, 1209, 1190, 945 c m 1; MS m /e calc'd for

C25 H38O2S: 402.26, found 402.26; Analysis calc'd for C 25 H38O2S: C, 74.58; H, 9.52; O, 7.95; S, 7.95; found: C, 74.50; H, 9.60. 99

7a-(2'-Methvlpropvlthio>-4-pregnene-3.20-dione (91: PJW-I-65B-):

=0 Pregna-4,6-diene-3,20-dione (1 g, 3.2 x

lO*3 mol) and 30 mL of isobutyl mercaptan (2- methylpropanethiol) were dissolved in 25 mL of freshly distilled (from Na) dioxane. A 300 mg (0.013 mol, 4 equiv.) chip of freshly cut (under hexane & therefore still bright) sodium metal was added to this stirred solution. An inert atmosphere (Ar) was established, and the mixture was heated to 70°C for 3 days. The reaction mixture was diluted with 100 mL diethyl ether and extracted with two 100 mL portions of saturated NH4 CI. The organic phase was dried by passing over MgSC >4 and solvents removed in vacuo to yield a pale yellow oil. Crystallization was performed in acetone/hexane and yielded off-white needles which were dried in vacuo (1.09 g, 2.71 x 10*3 mol, 84.7%): m.p. 153-155°; Rf .45 (15:2 -

CH2Cl2 :ethyl acetate, 20 cm glass-backed silica, 70-230 mesh, 250|im); JH

NMR (250 MHz, CDCI3) 8 0.64 (s, 3H, Ci8 methyls), 0.96 (d, J= 6.7 Hz, 6 H,

7a-side chain methyls), 1.17 (s, 3H, C 19 methyls), 2 .1 0 (s, 3H, C21 methyls),

2.96 (m, 1H, 7p), 5.75 (bs, 1H, vinyl); 13C NMR (62 MHz, CDCI 3) 8 13.27, 17.89, 21.04, 21.97, 22.29, 22.85, 23.76, 28.64, 31.40, 33.93, 35.48, 38.11, 38.45, 38.81, 39.70, 40.60, 43.77, 46.94, 52.02, 63.38 (C7), 126.87 (C4), 167.20 (C5), 198.57 (C3), 208.99 (C20); IR (KBr) 2964, 2941, 2922, 2914, 2895, 2885, 2871, 2848, 1711, 1674, 1622, 1469, 1448, 1437, 1383, 1362,

1232, 1219, 1171 cm*1; MS m /e calc'd for C25 H 38O2S: 402.26, found

402.26; Analysis calc'd for C 25 H3gC>2S: C, 74.58; H, 9.52; found: C, 75.01; H, 9.30. 100

7a-Phenvlthio-4-pregnene-3.20-dione (92; PJW-I-61B):

Pregna-4,6-diene-3,20-dione (1 g, 3.2 x

1 0 ' 3 mol) and 30 mL of thiophenol were dissolved in 25 mL of freshly distilled (from Na) dioxane. A 300 mg (0.013 mol, 4 equiv.) chip of freshly cut (under hexane & therefore still bright) sodium metal was added to this stirred solution. An inert atmosphere (Ar) was established, and the mixture was heated to 70°C for 24 hours. The remaining sodium metal was removed and quenched. The reaction mixture was diluted with 100 mL diethyl ether and extracted with two 100 mL portions of saturated NH 4 CI.

The organic phase was dried by passing over MgSC >4 and solvents removed in vacuo to yield a thick, pale, yellow oil. Crystallization was performed in acetone/hexane to yield off-white needles that were dried in vacuo (720 mg,

1.71 x 10' 3 mol, 53.4%): m.p. 202-203.5°; Rf 0.5 (15:1 - CH 2 Cl2 :ethyl acetate, 20 cm glass-backed silica, 70-230 mesh, 250pm); *H NMR (250

MHz, CD2CI2) 8 0.67 (s, 3H, Cis methyls), 1.18 (s, 3H, C 19 methyls), 2 .1 2 (s,

3H, C21 methyls), 3.43 (m, 1H, 7p), 5.66 (bs, 1H, vinyl), 7.33 (m, 5H, aromatics); 13C NMR (62 MHz, CDCI3) 8 13.31, 17.87, 21.10, 22.91, 23.69, 31.44, 33.98, 35.57, 38.13, 38.20, 38.55, 39.58, 43.67, 47.04, 50.71, 52.23, 63.40, 127.27, 127.56, 129.17, 133.37, 134.23, 166.63, 198.56, 208.89; IR (KBr) 2962, 2943, 2920, 2885, 2873, 2858, 1705, 1670, 1616, 1471, 1437, 1431, 1414, 1387, 1356, 1329, 1269, 1240, 1213, 1188, 1169, 1149, 945,

766, 752, 739, 700 cm'1; MS m /e calc'd for C27H34 O2S: 422.22, found

422.22; Analysis calc'd for C 27H34 O2S: C, 76.74; H, 8.12; found: C, 76.75; H, 101

8.04.

Ta-^-ChlorophepylthioV-d-pregnene-B^O-dione (93: PJW-I-73B):

Pregna-4,6-diene-3,20-dione (1.2 g,

3.84 x 10 ’3 mol) and 20 g (0.1383 mol, 36 eq.) of 4-chlorothiophenol were dissolved in 20 mL of freshly distilled (from Na) dioxane. A 300 mg (0.013 mol, 3.4 equiv.) Cl chip of freshly cut (under hexane & therefore still bright) sodium metal was added to this stirred solution. An inert atmosphere (Ar) was established, and the mixture was heated to 70°C for 3 days. The remaining sodium metal was removed and quenched. The reaction mixture was diluted with 100 mL diethyl ether and extracted with two 100 mL portions of saturated NH 4 CI. The organic phase was dried by passing over MgS 0 4 and solvents removed in vacuo to yield a thick, pale, foul-smelling, yellow oil. Crystallization was performed in acetone/hexane and yielded off-white plates which were dried in vacuo (1.69 g, 3.70 x 10‘3 mol, 96.4%): m.p. 212.5-214°; Rf .49 (15:2 - CH 2Cl2 :ethyl acetate, 20 cm glass-backed silica, 70-230 mesh, 250pm); *11 NMR (250

MHz, CD2CI2) 8 0.67 (s, 3H, Cig methyls), 1.18 (s, 3H, C 19 methyls), 2 .1 2 (s,

3H, C21 methyls), 3.40 (m, 1H, 7p), 5.63 (d, J=1.5 Hz, 1H, vinyl), 7.27 (m,

4H, aromatics); 13C NMR (62 MHz, CDC13) 8 13.28, 17.85, 21.06, 22.88, 23.66, 31.40, 33.95, 35.55, 38.00, 38.14, 38.52, 39.52, 43.84, 47.06, 50.94, 52.20, 63.34 (C7), 127.26 (C4), 129.36 (C2'), 132.73 (Ci'), 133.83 (C4'), 134.54 (C3'), 166.24 (C5), 198.46 (C3), 208.76 (C20); IR (KBr) 2958, 2945, 102

2916, 2879, 1701, 1670, 1616, 1473, 1448, 1429, 1414, 1385, 1363, 1271,

1236, 1219, 1190, 1093, 1007, 833 cm’1; MS m/e calc'd for C2 7 H 3 3 O2 SCI:

456.19, found 456.19; Analysis calc'd for C2 7 H 3 3 O2 SCI: C, 71.02; H, 7.29; found: C, 70.84; H, 7.56.

TnUd'-Aminophenvlthiol^pregnene-a^O-dione (94; PJW-I-35B):

Pregna-4,6-diene-3,20-dione (1.3 g, 4.16 x 10*3 mol) and p-amino-thiophenol (2.6 g, 0.0208 mol, 5 equiv.) were dissolved in 60 mL of freshly distilled (from Na metal) dioxane. A chip (approx.

Nl"*2 300 mg, 0.013 mol, 4 equiv.) of freshly cut (under hexane & therefore still bright) sodium metal was added to this stirred solution. An inert atmosphere (N 2) was established and the mixture was heated to reflux for 5 days. The reaction mixture was then allowed to cool to rt and the remaining sodium metal was removed and quenched. The reaction mixture was then poured into ~350 mL of triple distilled water. The resulting pale yellow precipitate was collected by gravity filtration and recrystallized from acetone/hexane to give yellow crystals which were dried in vacuo (1.0 g, 2.29xl0*3 mol, 55%): m.p. 231-

233° (decomp.); Rf 0.08 (15:1 - CH 2 Cl2:ethyl acetate, 20 cm glass-backed silica, 70-230 mesh, 250pm); NMR (250 MHz, CD 2C12) 8 0..67 (s, 3H,

Ci8 methyls), 1.18 (s, 3H, C 19 methyls), 2 .1 2 (s, 3H, C21 methyls), 3.21 (m,

1H, 7p), 3.76 (bs, 2H, amine), 5.70 (s, 1H, vinyl), 6.90 (m, 4H, aromatics);

!3C NMR (62 MHz, CDC13) 8 13.25, 17.85, 21.05, 22.89, 23.67, 31.42, 103

33.97, 35.52, 37.74, 38.20, 38.52, 39.46, 43.83, 46.73, 51.64, 52.08, 63.42 (C7), 115.63, 121.08, 127.19, 136.45, 146.78, 167.34 (C5), 198.66 (C3), 208.99 (C20); IR (KBr) 3421, 3342, 3244, 3228, 2952, 1703, 1664, 1641,

1618, 1597, 1493, 1169, 829 cm'1; MS m/e calc'd for C27H35 NO2S: 437.24, found 437.24; Analysis calc'd for C 27H35 NO2S: C, 74.10; H, 8.07; N, 3.20; obsv'd. C, 73.96; H, 7.94; N, 3.11. la-Phenylthio-4-pregnene-3.20-dione (95; PJW-I-85B):

Pregna-l,4-diene-3,20-dione (500

mg, 1.6 x 10'3 mol) and 30 mL of thiophenol were dissolved in 25 mL of freshly distilled (from Na) dioxane. A 300 mg (0.013 mol, 4 O equiv.) chip of freshly cut (under hexane & therefore still bright) sodium metal was added to this stirred solution. An inert atmosphere (Ar) was established, and the mixture was heated to 70°C for 24 hours. The remaining sodium metal was removed and quenched. The reaction mixture was diluted with 100 mL diethyl ether and extracted with two 100 mL portions of saturated NH 4 CI. The organic phase wasdried by passing over MgS 0 4 and solvents removed in vacuo to yield athick, pale, yellow oil. Crystallization was performed in acetone/hexane and yielded off-white needles which were dried in vacuo

(500 mg, 1.18 x lO -3 mol, 74.0%): m.p. 212-213.5°; RF 0.45 (15:1 -

CH2Cl2:ethyl acetate, 20 cm glass-backed silica, 70-230 mesh, 250|im); *H

NMR (250 MHz, CDC13) 5 0.67 (s, 3H, Ci8 methyls), 1.34 (s, 3H, C 19 methyls), 2 .1 2 (s, 3H, C21 methyls), 3.54 (m, 1H, lp), 5.80 (bs, 1H, vinyl), 7.33 (m, 5H, aromatics); 13C NMR (62 MHz, CDCI 3) 8 13.34, 19.78, 20.24, 22.86, 24.39, 30.76, 31.49, 32.55, 35.56, 38.36, 39.48, 42.54, 43.89, 47.63, 54.27, 55.90, 63.44 (Ci), 124.43, 127.83, 129.21, 133.58, 134.00

1232, 1185, 1165, 1146, 1 1 2 1 , 1113, 1068, 1026, 896, 854, 705 cm 1; MS m /e calc'd for C27H34 O2S: 422.22, found 422.22; Analysis calc'd for

C27H34 O2S: C, 76.74; H, 8.12; found: C, 76.48; H, 7.88.

Androsta-4,6-diene-3,17-dione (A 4 '6-ADD) (101): O 4-Androstene-3,17-dione (androstenedione) (25 g, 0.087 mol), chloranil (27 g, 0.113 mol, 1.3 equiv.) and tosic acid (1 g, 0.005 mol, cat.) were dissolved in 300 mL of t-butanol. This stirred solution was heated to reflux for 20 hours under an inert atmosphere (Ar). The reaction mixture was cooled to room temperature and filtered. Solvents were removed in vacuo to yield a thick, brown, tar. The tar was dissolved in a minim um volume (approx. 100 mL) of CHC1 3 and adsorbed to approximately 50 g of basic alumina (activity I) by adding the alumina to the CHCI 3 solution and removing solvent in vacuo. The product-adsorbed alumina was added to the top of an approximately 750 g CHC1 3 slurry-packed bed of basic alumina and products eluted with CHCI 3. Solvents were removed in vacuo to yield a pale yellow solid. Crystallization was performed in acetone/hexane and yielded off-white crystals which were dried in vacuo (15 105 g, 0.053 mol, 60.0%): m.p. 172-173°C; Rf 0.31 (15:1 - CH 2Cl2 :ethyl acetate,

2 0 cm glass-backed silica, 70-230 mesh, 250pm). Spectral data were as reported .373

Androsta-l,4,6-triene-3,20-dione (A 1>4 »6-ATD) 102:

O Androsta-4,6-diene-3,17-dione (A4-6- ADD) (15 g, 0.053 mol), 2,3-dichloro-5,6- dicyanobenzoquinone (DDQ, 15 g, 0.066 mol, 1.3 equiv.) and tosic acid (1 g, 0.005 mol, cat.) were dissolved in 250 mL of dioxane. This stirred solution was heated to reflux for 16 hours under an inert atmosphere (Ar). The reaction mixture was cooled to room temperature and solvents were removed in vacuo to yield a thick, brown, tar. The tar was dissolved in a minimum volume (approx. 100 mL) of CHCI 3 and adsorbed to approximately 50 g of basic alumina (activity I) by adding the alumina to the CHCI 3 solution and removing solvent in vacuo. The adsorbed alumina was added to the top of a

500 g CHCI3 slurry-packed bed of basic alumina and products eluted with

CHCI3. Solvents were removed in vacuo to yield 10 g of a pale yellow solid. This material was subjected to column chromatography by passing over a

200 g bed of silica gel utilizing 4:1 CH 2Cl2 :ethyl acetate as the mobile phase. Crystallization was performed in acetone/hexane and yielded white crystals which were dried in vacuo (10 g, 0.035 mol, 65.0%): m.p. 167-

169°C; Rf 0.38 (4:1 - CH 2Cl2:ethyl acetate, 20 cm glass-backed silica, 70-

230 mesh, 250pm). Spectral data were as reported .373 106

7a-(4,-Aminophenylthio)androsta-l,4-diene-3,17-dione (7a-APTADD) 87:

O Androsta-l,4,6-triene-3,17-dione (5 g, 0.0177 mol) and p-aminothiophenol (10 g, 0.08 mol, 4.5 equiv.) were dissolved in 200 mL of freshly distilled NHz (from Na metal) dioxane. Several pieces (approx. 1.5 g, 0.05 mol, 3 equiv.) of freshly cut (under hexane & therefore still bright) sodium metal was added

to this stirred solution. An inert atmosphere (N 2) was established and the

mixture was heated to reflux for 6 hours. The reaction mixture was then allowed to cool to rt and the remaining sodium metal was removed and quenched. The reaction mixture was then poured into -500 mL of triple distilled water and extracted with ethyl acetate. The organic phase was subjected to silica gel column chromatography. The fractions containing the unwanted la- and la-,7a-bis addition products were pooled and

concentrated in vacuo. This material was oxidized with 2 equivalents of MCPBA, and treated with KOH to effect elimination of the resulting sulfoxides to regenerate the androsta-1,4,6-triene-3,17-dione (ATD) starting material, which was reused. The 7a-APTADD-containing fractions were pooled, concentrated in vacuo and the resulting thick, yellow tar was triturated with acetone. The resulting pale yellow precipitate was collected by gravity filtration and subjected to medium pressure liquid chromatography in 1 g portions. Elution was achieved utilizing a step

gradient of 500 mLs each of 9:1, 4:1, and 3:2 CH 2Cl2:ethyl acetate. The 7a- 107

APTADD-containing fractions were pooled, concentrated in vacuo and recrystallized from acetone/hexane to give white crystals which were dried in vacuo (1.0 g, 0.0024 mol, 15%): m.p. 254° C. Spectral data were as previously reported .373

3.3 General Procedures - Biochemistry:

[l,2-3H]-Progesterone was purchased from New England Nuclear (Boston, Ma.) and was purified by thin-layer chromatography. TLC was performed utilizing a 4 x 20 cm plastic-backed, 70-230 mesh, 250 fxm thickness, silica gel plate and 7:3 CHC^ethyl acetate as the mobile phase.

Purified [l,2-3H]-progesterone was visualized using a Berthold model LB 2722-2 radio TLC scanner equipped with a proportional detector. The peak corresponding to an Rf of 0.45 was scraped from the TLC plate, and the desired [l,2-3H]-progesterone was desorbed by stirring in 15 mL ethyl acetate. Silica was removed and rinsed by gravity filtration. Solvents were evaporated with a stream of N 2 . Recovery of radiochemically pure [1,2- 3H]- progesterone was approximately 30%. Purified [l,2- 3H]-progesterone was stored as a 5 pCi/mL ethanolic solution at 4°C which served as a working solution of radiotracer for subsequent use in enzyme assays.

Adult Sprague-Dawley rat testes were purchased from Harlan Industries (Cumberland, Ind.) and were shipped on dry ice on the same day that the animals were sacrificed. Centrifugations were performed on a

Beckman model TJ -6 and J2-21. Ultracentrifugations were performed on a Beckman model L5-50B. UV/VIS absorption and Xmax measurements were performed using a Pharmacia LKB Ultrospec III spectrophotometer. 108

3H-Labelled reagents were detected by liquid scintillation counting employing H# for quench monitoring. Liquid scintillation counting was performed on a Beckman model LS 6800 or LS 8100 utilizing Formula 3a70b cocktail purchased from Research Products International (Mount Prospect, 111.). Tissue was processed using a Tekmar Co. Tissumizer model high torque, variable speed, probe tissue homogenizer. Triple distilled water for the preparation of buffers was purchased from the Ohio State University Reagent Laboratory. Trizma and other buffer salts were purchased from Sigma Chemical Co. (St. Louis, Mo.). Buffer pH determinations were made utilizing a Beckman model $ 43 digital pH meter. Assay incubations were performed in a Forma Scientific model 2564 constant temperature, variable speed shaker water bath. Plastic- backed silica gel TLC plates with indicator (DC Plastikfolien, Kieselgel 60,

F254 ) were purchased from EM Separations Technology (Gibbstown, N.J.). Organic solvents used for extractions and as mobile phase in TLC separations were purchased from Malinkrodt Chemical Co. (Paris, Ky.) Reagents for protein determinations were purchased from BioRad Chemical Co. (Richmond, Ca.).

3.4 Biochemical Methods

Isolation of rat testis microsomes :3 7 6

Testes were obtained from 10-12 week old male Sprague-Dawley rats. Testes were stored at -80°C until processing, at which time they were thawed at 4°C in 0.2 M Tris buffer (pH 7.4) and weighed. Once the tissue had thawed, the seminiferous tubules were dissected away from the tunica 109 albuginea using a #10 scalpel and placed in a 3:1 volume of 0.25 M sucrose solution. Tissue was homogenized in a stainless steel container utilizing a motorized, variable speed, stainless steel, probe tissue homogenizer. Homogenization was done in 10 second, high speed bursts to minimize foaming. The crude homogenate was subjected to further homogenization with a Dounce tissue homogenizer. The final homogenate was centrifuged at 10,000 x g for 30 min. at 4°C using a Beckman JA-14 rotor. The microsome-containing supernatant was collected and the pellet discarded. The supernatant was centrifuged at 11,000 x g for 30 min at 4°C using a Beckman 70-Ti fixed angle ultracentrifuge rotor. The supernatant was collected and the pellet discarded. The resulting supernatant was spun at 120,000 x g for 60 min. at 4°C utilizing a Beckman 50-Ti fixed angle ultracentrifuge rotor. The cytosol-containing supernatant was discarded and the microsomal pellet washed by resuspending in 0.2M Tris buffer (pH 7.4) and spun at 120,000 x g for 60 min. at 4°C. The pellets were collected, divided, placed in 1.5 mL snap cap conical (eppendorf) vials, labeled, and stored at -80°C.

Protein Determination :3 7 7

Protein content in microsomal suspensions was determined by a

BioRad modification of the Bradford procedure .378 Briefly, a standard solution containing 1 |J.g/|iL of bovine serum albumin (BSA) was prepared in 0.1M Tris (pH 7.4) containing 280 (iM Mg CI 2 . From this, a duplicate series of 10-12 dilutions containing from 5 |ig protein to 100 |ng protein, and

Biorad protein reagent were prepared in a total buffer volume of 1 mL. 110

These were used to create a plot on which a linear regression was performed to generate a standard line. Simultaneously, a duplicate series of 2-3 dilutions of the microsomal suspension and biorad reagent were prepared in a 1 mL volume of buffer. All samples were vortexed and left to stand for 15-20 min. Absorbances were taken for each sample at 595 nm. The previously mentioned standard line was generated, and protein content in the microsome-containing samples calculated by linear interpolation.

17.20-Lvase Initial Velocity Kinetics:2 7 9

This assay was performed in 0.1M Tris (pH 7.4) containing 280pM

MgCl2 . Propylene glycol (100 pL) was used as a solubilizing agent. Incubations were performed at 32°C, the physiologic temperature of the rat scrotum .379 Microsomes were thawed at 4°C and resuspended in assay buffer using a Dounce tissue homogenizer. Each assay consisted of triplicate samples of six substrate (progesterone) concentrations ranging from 0.25 x Km to 10 x Km (35 nM, 70 nM, 140nM, 350 nM, 700 nM, 1400 nM), as well as a negative control containing heat-inactivated microsomes

(350 nM progesterone). 0.05 pCi (approx. 100,000 dpm) of [1,2- 3H]- progesterone was used as a radiotracer and was added to each assay sample as a 10 pL ethanolic solution. An NADPH regeneration system was utilized, and consisted of 1.8 mM NADP (Na+ Salt), 3.5 mM glucose- 6 - phosphate (Na +2 salt), and 3 units of glucose- 6 -phosphate dehydrogenase added in a 0.5 mL volume of assay buffer. Incubations were performed in a total volume of 2.6 mL (2.0 mL microsomal suspension, 0.5 mL NADPH I l l regeneration system, 0.1 mL propylene glycol). Assay pre-incubation time was 5 min. and incubation time was 20 min. Assay samples were quenched with 5 mL of 7:3 CHClsiethyl acetate, spiked with a few drops of a 7:3 CHCl3 :ethyl acetate solution of the four possible assay products (progesterone, 17a-hydroxyprogesterone, androstenedione, and testosterone), covered and stored at -20°C until product extractions were performed. Each sample was extracted three times with 5 mL of 7:3

CHCl3 :ethyl acetate, the organic phases were dried by passing over

Na2SC>4 , combined and solvents removed by evaporation under a stream of

N2 . Exactly 1 mL of 7:3 CHCl3 :ethyl acetate was added to each sample, followed by 15-20 min. of ultrasonication. A 50 |llL aliquot of each sample was subjected to thin layer chromatographic resolution. TLC was performed utilizing a 20 x 20 cm plastic-backed, 70-230 mesh, 250 pm thickness, silica gel plate scored into 1 cm lanes and 7:3 CHCI 3: ethyl acetate as the mobile phase. Products were visualized under UV light (230 nM), and the spot corresponding to progesterone (Rf = 0.45) marked. The progesterone spot was cut out and the amount of [l,2- 3H]-progesterone determined by liquid scintillation counting. All enzyme kinetic calculations were performed based on remaining free substrate concentrations. All velocities were normalized to 1 mg/mL of protein as determined by the method of Bradford .378 Data were evaluated by double reciprocal (Lineweaver-Burke) plot and constants (Km and Vma*) were determined by the least squares regression method of Cleland .380 112

17,20-Lyase Inhibitor Screening Assay;

This assay was performed in 0.1M Tris (pH 7.4) containing 280pM

MgCl2 - Propylene glycol (100 pL) was used as a solubilizing agent, and incubations were performed at 37°C. Microsomes were thawed at 4°C and resuspended in assay buffer using a Dounce tissue homogenizer. Each assay consisted of triplicate samples of a single substrate (progesterone) concentration (10 x Km, 1400 nM), and two inhibitor concentrations (3,5 pM and 7.0 pM). Four compounds were screened per assay. The positive control consisted of a triplicate sample of a single substrate (progesterone) concentration (10 x Km, 1400 nM) with no inhibitor, as well as a triplicate negative control containing heat-inactivated microsomes (1400 nM progesterone). 0.05 pCi (approx. 1 0 0 ,0 0 0 dpm) of [l,2- 3H]-progesterone was used as a radiotracer and was added to each assay sample as a 10 pL ethanolic solution. An NADPH regeneration system was utilized, and consisted of 1 .8 mM NADP (Na+ Salt), 3.5 mM glucose- 6 -phosphate (Na +2 salt), and 3 units of glucose- 6 -phosphate dehydrogenase added in a 0.5 mL volume of assay buffer. Incubations were performed in a total volume of 2.6 mL (2.0 mL microsomal suspension, 0.5 mL NADPH regeneration system, 0.1 mL propylene glycol). Assay pre-incubation time was 10 min. and incubation time was 30 min. Assay samples were quenched with 5 mL of

7:3 CHCl3:ethyl acetate, spiked with a few drops of a 7:3 CHCl 3 :ethyl acetate solution of the four possible assay products (progesterone, 17a- hydroxyprogesterone, androstenedione, and testosterone), covered and stored at -20°C until product extractions were performed. Each sample was extracted three times with 5 mL of 7:3 CHCl 3 :ethyl acetate, the 113 organic phases were dried by passing over Na 2SC>4 , combined and solvents removed by evaporation under a stream of N 2 . Exactly 1 mL of 7:3

CHCl3:ethyl acetate was added to each sample, followed by 15-20 min. of ultrasonication. A 50 pL aliquot of each sample was subjected to thin layer chromatographic resolution. TLC was performed utilizing a 20 x 20 cm plastic-backed, 70-230 mesh, 250 pm thickness, silica gel plate scored into 1 cm lanes and 7:3 CHCl 3 :ethyl acetate as the mobile phase. Products were visualized under UV light (230 nM), and the spot corresponding to progesterone (R f = 0.45) marked. The progesterone spot was cut out and the amount of [l,2- 3H]-progesterone determined by liquid scintillation counting. All calculations were performed based on remaining free substrate concentrations. All velocities were normalized to 1 mg/mL of protein as determined by the method of Bradford .378 Data were evaluated by determining percent inhibition.

17.20-Lvase Kj Determination:2 7 9

This assay was performed in 0.1M Tris (pH 7.4) containing 280|iiM

MgCl2 - Propylene glycol (100 pL) was used as a solubilizing agent, and incubations were performed at 37°C. Microsomes were thawed at 4°C and resuspended in assay buffer using a Dounce tissue homogenizer. Each assay consisted of 3 sets of triplicate samples of 5 substrate (progesterone) concentrations ranging from 0.5 x Km to 10 x Km (70 nM, 140nM, 350 nM, 700 nM, 1400 nM) and either no inhibitor, [I] = 700 nM, or [I] = 1.4 pM. The negative control contained heat-inactivated microsomes (350 nM progesterone). 0.05 pCi (approx. 1 0 0 ,0 0 0 dpm) of [l,2- 3H]-progesterone 114 was used as a radiotracer and was added to each assay sample as a 10 pL ethanolic solution. An NADPH regeneration system was utilized, and consisted of 1 .8 mM NADP (Na+ Salt), 3.5 mM glucose- 6 -phosphate (Na +2 salt), and 3 units of glucose- 6 -phosphate dehydrogenase added in a 0.5 mL volume of assay buffer. Incubations were performed in a total volume of 2.6 mL (2.0 mL microsomal suspension, 0.5 mL NADPH regeneration system,

0 .1 mL propylene glycol). Assay pre-incubation time was 15 min. and incubation time was 15 min. Assay samples were quenched with 5 mL of

7:3 CHCla'.ethyl acetate, spiked with a few drops of a 7:3 CHCl 3 :ethyl acetate solution of the four possible assay products (progesterone, 17a- hydroxyprogesterone, androstenedione, and testosterone), covered and stored at -20°C until product extractions were performed. Each sample was extracted three times with 5 mL of 7:3 CHCl 3 :ethyl acetate, the organic phases were dried by passing over Na 2SC>4 , combined and solvents removed by evaporation under a stream of N 2 . Exactly 1 mL of 7:3

CHCl3:ethyl acetate was added to each sample, followed by 15-20 min. of ultrasonication. A 50 pL aliquot of each sample was subjected to thin layer chromatographic resolution. TLC was performed utilizing a 20 x 20 cm plastic-backed, 70-230 mesh, 250 pm thickness, silica gel plate scored into 1 cm lanes and 7:3 CHCl 3:ethyl acetate as the mobile phase. Products were visualized under UV light (230 nM), and the spot corresponding to progesterone (Rf = 0.45) marked. The progesterone spot was cut out and the amount of [l, 2 -3H]-progesterone determined by liquid scintillation counting. All enzyme kinetic calculations were performed based on remaining free substrate concentrations. All velocities were normalized to 1X5

1 mg/mL of protein as determined by the method of Bradford .378 Data were evaluated by double reciprocal (Lineweaver-Burke) plot and constants (Km, Ki, and Vmax) were determined by the least squares regression method of

Cleland .380

17.20-Lvase ICM Determination:

This assay was performed in 0 .1M Tris (pH 7.4) containing 280pM

MgCl2- Propylene glycol (100 |xL) was used as a solubilizing agent, and incubations were performed at 37°C. Microsomes were thawed at 4°C and resuspended in assay buffer using a Dounce tissue homogenizer. Each assay consisted of a log-dose response curve at a single substrate (progesterone) concentration of 0.5 x Km (350 nM) and 5 inhibitor concentrations ranging from [I] = 1 x 10‘7 M, to [I] = 1 x 1 0 3 M. The negative control contained heat-inactivated microsomes (350 nM progesterone), and the positive control contained no inhibitor (350 nM progesterone). 0.05 |iCi (approx. 100,000 dpm) of [l,2- 3H]-progesterone was used as a radiotracer and was added to each assay sample as a 10 jiL ethanolic solution. An NADPH regeneration system was utilized, and consisted of 1.8 mM NADP (Na+ Salt), 3.5 mM glucose- 6 -phosphate (Na +2 salt), and 3 units of glucose- 6 -phosphate dehydrogenase added in a 0.5 mL volume of assay buffer. Incubations were performed in a total volume of 2.6 mL (2.0 mL microsomal suspension, 0.5 mL NADPH regeneration system, 0.1 mL propylene glycol). Assay pre-incubation time was 10 min. and incubation time was 20 min. Assay samples were quenched with 5 mL of

7:3 CHCl3 :ethyl acetate, spiked with a few drops of a 7:3 CHCl 3 :ethyl 116

• acetate solution of the four possible assay products (progesterone, 17a- hydroxyprogesterone, androstenedione, and testosterone), covered and stored at -20°C until product extractions were performed. Each sample

was extracted three times with 5 mL of 7:3 CHCl 3:ethyl acetate, the

organic phases were dried by passing over Na 2SC>4 , combined and solvents

removed by evaporation under a stream of N 2 . Exactly 1 mL of 7:3

CHCl3:ethyl acetate was added to each sample, followed by 15-20 min. of ultrasonication. A 50 pL aliquot of each sample was subjected to thin layer chromatographic resolution. TLC was performed utilizing a 20 x 20 cm plastic-backed, 70-230 mesh, 250 pm thickness, silica gel plate scored

into 1 cm lanes and 7:3 CHCl 3:ethyl acetate as the mobile phase. Products were visualized under UV light (230 nM), and the spot corresponding to

progesterone (R f = 0.45) marked. The progesterone spot was cut out and

the amount of [l,2- 3H]-progesterone determined by liquid scintillation counting. All calculations were performed based on remaining free

substrate concentrations. All velocities were normalized to 1 mg/mL of

protein as determined by the method of Bradford .378 Data were evaluated

graphically by log dose vs response plot and the IC 50 values were

determined by Marquardt regression. IC 50 values represent the concentration of inhibitor which produces half-maximal inhibition of 17,20- lyase activity in rat testis microsomes and were calculated by a non-linear regression analysis using the Marquardt method (SAS Institute, Carey, NC) Ki values were calculated based on the following formula: IC«, = K,(l + ^ l ).381 117

3.5 In Vivo Studies

Tumor Growth Inhibition Studies :3 5 0

Pubertal female Sprague-Dawley rats age 50 days were treated by gastric lavage with a single 15 mg/Kg dose of DMBA (7,12- dimethylbenz(a)anthracene) as a 1 mg/mL solution in com oil .382 Animals were housed in metal cages containing ground corncob, provided Purina laboratory chow and water ad libitum, and maintained in an American Association for Accreditation of Laboratory Animal Care-accredited anim alfacility utilizing a 14 hour diurnal, 1 0 hour nocturnal photoperiod. Animals were checked for the presence of tumors beginning 5 weeks post DMBA treatment. Treatments consisted of either a suspension of 7 a- APTADD in USP sesame oil, USP sesame oil only, or a suspension of 7a- APTADD and estradiol in USP sesame oil. Animals bearing between 1-3 tumors of 0 .8 -1.2 cm diameter were randomly assigned to either: a treated group (25 and 50 mg/Kg/day), an untreated group (sesame oil vehicle only), or a treated + estradiol group (50 mg/Kg/day for 3 weeks then 50 mg/Kg/day

+ 0.3mg/Kg/day E2 for 3 weeks). Animals were treated once per day by hypodermal injection in the flank region alternating sides each day. Tumor measurements were taken 3 times per week and tumor volumes calculated according to the following formula: v = itr\r2. At the conclusion of the study, animals were sacrificed and blood and tissues were harvested. Serum was obtained for radioimmunoassay of estradiol and gonadotrophin levels by cardiac puncture, and collection in a clotting tube followed by centrifugation at 10,000 x g for 15 min. The serum layer was decanted and 118 stored at -80°C until the RIA’s were performed. Tissues were harvested by blunt dissection, rinsed in phosphate-buffered saline, the excess adipose tissue trimmed away, and immediately frozen in liquid nitrogen. The isolated tissues were maintained at -80°C for later use in molecular biology studies. Gross necropsy was performed on carcasses immediately following harvest of serum and tissues.

Endocrine Studies:

Normal, cycling, 10 week old, female, Sprague-Dawley rats were segregated by estrus state into one of several treatment groups: 50 mg/Kg/day treated for 1 , 3, 6 , 7, and 13 days, and untreated (vehicle only) for 1 , 3, 6 , 7, and 13 days. Treatments consisted of either a 25 mg/mL suspension of 7a-APTADD in USP sesame oil, or USP sesame oil only. Animals were treated once daily at the same hour of the morning by hypodermal injection in the flank region alternating sides each day. Animals were housed in metal cages containing ground corncob, provided Purina laboratory chow and water ad libitum, and maintained in an American Association for Accreditation of Laboratory Animal Care- accredited animal facility utilizing a 14 hour diurnal, 10 hour nocturnal photoperiod. At the conclusion of the treatment period, animals were sacrificed and blood and tissues were harvested. Serum was obtained for radioimmunoassay of estradiol and gonadotrophin levels by cardiac puncture, and collection in a clotting tube followed by centrifugation at 10,000 x g for 15 min. The serum layer was decanted and stored at -80°C until the RIA’s were performed. Tissues were harvested by blunt 119 dissection, rinsed in phosphate-buffered saline, the excess adipose tissue trimmed away, and immediately frozen in liquid nitrogen. The isolated tissues were maintained at -80°C for later use in biochemical and molecular biology studies. Carcasses were stored at -80°C for subsequent necropsy examination. CHAPTER 4

RESULTS AND DISCUSSION

4.1 Chemistry:

The first objective was to prepare the different 7a-thio, and la- phenylthio substituted progestin derivatives which were proposed to be potential competitive inhibitors of 17,20-lyase (Figure 27; 90-95). The outlines and discussions pertaining to the synthesis of these compounds are illustrated in Schemes I-IV below.

Synthesis of Pregna-4,6-diene-3,20-dione (A 4 >6-Progesterone; 99):

Preparation of A4,6-progesterone (Scheme I; 99) was acheived utilizing a high potential quinone oxidant under mildly acidic conditions.

According to the earlier work of Turner, selective introduction of the C 6-C7 double bond is most readily acheived using tetrachlorobenzoquinone

(chloranil ) . 374,375 Chloranil was used in slight excess (1.3 equiv.) in the presence of catalytic amounts of tosic acid. Tosic acid aids in the enolization of the 4-ene-3-one to form the proposed 3,5-dienol intermediate necessary for C-7 hydride transfer to chloranil, thereby forming the 4,6- dienone. Workup of this type of oxidation involves passage of the product mixture over a bed of aluminum oxide in order to remove and unreacted quinone. 120 121

Yields were not high, and were on average about 60%. Reaction time was

an important parameter, because short reaction times (e.g. 1 2 hours or less) resulted in inadequate conversion of progesterone to the desired 4,6- dienone, while long reaction times (e.g. 48 hours) resulted in formation of the 4,6,16-triene (Scheme I; 96). Although this material was not a desired product of this reaction, it was purified and saved for later biochemical evaluation as a possible inhibitor of 17,20-lyase.

Chloranil; 1.3 equiv. TsOH; cat. O tBuOH Progesterone A reflux; 16-20 hours 99: A4,6-Progesterone

TsOH; ca Chloranil; 1.3 equiv. tBuOH A reflux; 48 h

96: PJW-I-42A

Scheme I. Synthesis of pregna-4,6-diene-3,20-dione and pregna-4,6,16-triene-3,20-dione 122

Synthesis of Pregna-l,4-diene-3,20-dione (A 1,4 -Progesterone; 1 0 0 ):

Preparation of A1,4-progesterone (Scheme II; 100) was also acheived utilizing a high potential quinone oxidant under mildly acidic conditions.

According to the earlier work of Turner, selective introduction of the C 1-C2 double bond is most readily acheived using 2,3-dichloro-5,6- dicyanobenzoquinone (DDQ) with benzene as the solvent .374,375 DDQ was used in slight excess (1.3 equiv.) in the presence of catalytic amounts of tosic acid. Tosic acid aids in the enolization of the 3-ketone to form the proposed 2-enol intermediate necessary for C-l hydride transfer to DDQ, thereby forming the 1,4-dienone. Workup of this type of oxidation is somewhat more cumbersome than the chloranil oxidation because it involves two column chromatographic purifications. The first is the passage of the product mixture over a bed of aluminum oxide in order to remove hydroquinones and unreacted quinone, while the second involves passage of the product mixture over a bed of silica gel for separation of the desired 1,4-dienone from unwanted starting material, 4,6-dienone and 1,4,6-trienone. Yields were not high, and were often less than 60%. 123

DDQ; 1.3 equiv. TsOH; cat. O Benzene Progesterone A reflux; 8 hours -|oo: A1,4-Progesterone

Scheme II. Synthesis of pregna- l,4-diene-3,20-dione

Synthesis of 7a-Thiosubstituted Proeestins (90-94):

Preparation of the 7a-thiosubstituted progestins (Scheme 111; 90-94) involved conjugate, Michael-type addition of the appropriate thiolate anion to the 4,6-dienone. Generation of the appropriate thiolate anion was acheived in situ by reacting the corresponding thiol with sodium metal in freshly distilled, dry dioxane. This approach to generation of the thiolate is more convenient than the use of another base because it simplifies the synthetic procedure while avoiding base-catalyzed retro-Michael elimination of the thio-substituted product. By using sodium metal, the thiolate is generated in situ, along with hydrogen gas and Na+ , without the need of an initial base-catalyzed thiol deprotonation. Initially, it was reasoned that removal of unreacted thiol starting material could be acheived by basic extraction during workup. This proved to be unwise due to base-catalyzed retro-Michael elimination to give 4,6-dienone starting material. Instead, the product mixture was extracted with saturated

NH4 CI, and directly purified by crystallization from acetone:hexane. Yields 124 ranged from a low of 38% for the n-butyl thiosubstituted analog (90) to a high of 96.4% for the (4'-chloro)phenyl thiosubstituted analog (93).

Dioxane A4'6-Progesterone A 70° c !3 days

Scheme III. Synthesis of 7a-thio substituted progestins

Synthesis of la-Phenvlthio-4-pregnene-3.20-dione (95):

Preparation of la-phenylthioprogesterone (Scheme IV; 95) involved conjugate, Michael-type addition of the phenyl thiolate anion to the 1,4- dienone. Generation of the appropriate thiolate anion was acheived in situ by reacting thiophenol with sodium metal in freshly distilled, dry dioxane. Purification was again acheived by recrystallization from acetone hexane, and the yield was 74%. Spectral data and elemental analysis revealed that recrystallization was effective and yielded quite pure (95). 125

O O

Na° O Dioxane O A1,4-Progesterone A 70° C; 3 days 95 1 a-Phenytthioprogesterone

Scheme IV. Synthesis of la-phenylthioprogesterone

Large-scale S yn th esis o f 7a-APTADD (87):

The second objective was to prepare 7a-APTADD (Figure 27; 87) in multigram quantity and high purity so that it could be evaluated in vivo for tumor growth inhibition and effects on normal endocrine function. The previously published synthesis from androstenedione was a labor-

intensive, low yield process (Schemes V and VI ) .373

O O O

Chloranil; 1.3 equiv. DDQ; 1.3 equiv.

Dk,xane O A reflux 0 Androstenedione 16-20 hours A4,6-ADD 16-20 hours Al4'6-ATD

Scheme V. Synthesis of androsta-1,4,6-triene-3,17-dione (AM> 6-ATD) 126

1 a-APTADD NH; O Na° O Dioxane A reflux; 6 hours 7a-APTADD

Scheme VI. Synthesis of 7a-APTADD

Each oxidation requires column chromatographic purification, while the thiolate addition requires two column chromatographic purifications. This necessitated the formation of a production line of three people, each performing one of the separations. The chloranil and DDQ oxidations were

done on a 25 gram scale, which yielded 10 grams of A 14-6-ATD. The overall

yield of the two oxidation steps which introduce the A 6 and A1 double bonds was only approximately 40%. Since this was such a low yield process, it

was desirable to minimize possible waste of the A 14>6-ATD. Moreover, the yield of highly pure 7a-APTADD was only 15% in the thiolate addition reaction, further pointing out the need to minimize waste of A14>6-ATD starting material. Therefore, a A14-6-ATD salvage scheme was devised 127 where the unwanted la-mono and la,7a-bis-thio addition products were oxidized with MCPBA, and the resulting sulfoxides treated with base in order to effect sulfoxide elimination, thereby regenerating the A1-4>6-ATD starting material (Scheme VII). This procedure resulted in recovery of approximately 60-70% of the lost A 1*4>6-ATD, and therefore greatly reduced the loss afAM.6-ATD starting material.

O 1 a-APTADD 1. MCPBA 2. KOH O

Scheme VII. Salvage of A1,4,6-ATD from unwanted mono and bis thio addition products 128

4.2 Biochemistry:

The initial focus of biochemical studies was to screen the various thio substituted progestins and androgens (Figure 27) for 17,20-lyase inhibitory activity. This was acheived by performing rat testis microsomal incubations at a single concentration of progesterone substrate and two concentrations of inhibitor, typically 2.5 x [S] and 5 x [S]. As with other NADPH-requiring reactions, a typical NADPH regeneration system containing 1.8 mM NADP (Na+ Salt), 3.5 mM glucose- 6-phosphate (Na +2 salt), and 3 units of glucose- 6-phosphate dehydrogenase was utilized, and a preincubation performed. This system possessed an approximately 2500- fold excess capacity for generating the reducing equivalents needed for 17,20-lyase activity, thereby insuring that the supply of NADPH was not a limiting factor in the assay system. An additional caveat must be added with regard to the detection of 17,20-lyase enzymatic activity. Since 17,20- lyase is an intermediate activity in the biosynthesis of most steroid hormones, and a testis microsomal, rather than a purified protein enzyme assay was being used, several fates for the products of 17,20-lyase enzymatic activity are possible. First, catalytic activity may not progress beyond 17a-hydroxylation, and second, additional possible metabolic activities can remove androstenedione from the product pool (e.g. conversion to DHEA, testosterone or 5a-dihydrotestosterone). In order to simplify the assay protocol and to minimize error, it was reasoned that monitoring the disappearance of progesterone substrate, rather than the appearance of androstenedione product was an acceptable method of 129 following 17,20-lyase activity .279 The amount of product formed was calculated simply by subtraction of the final nanomoles of substrate from the initial nanomoles of substrate.

Table 3 Structure-activity Relationships of Progestin Analogs

Compound Concentration % Inhibition 90 3.5 pM 23.4 7cc-n-butylthio 7.0 |iM 36.5 91 3.5 pM 25.8 7a-isobutylthio 7.0 pM 31.2 92 3.5 pM 19.6 7a-phenylthio 7.0 pM 31.5 93 3.5 pM 2 .1 7a-(4'-chloro)phenylthio 7.0 pM 5.3 94 3.5 pM 11.5 7a-(4'-amino)phenylthio 7.0 pM 2 1 .6 95 3.5 pM 26.4 la-phenylthio 7.0 pM 30.2 96 3.5 pM 50.2 A4-6.16- 7.0 pM 54.8 48 3.5 pM 1 0 0 .0 Metyrapone 7.0 pM 1 0 0 .0

As may be seen in Table 3, none of the progestin-based analogs displayed particularly effective 17,20-lyase inhibitory activity. The inhibitor screening assay utilized a progesterone substrate concentration of

10 x Km (1.4 (jlM ) , and two inhibitor concentrations of 25 x Km (3.5 pM) and 130

50 x Km (7.0 pM). The conversion of substrate to products ranged between 18-32%. The least active compound was the (4'-chloro)phenylthio substituted analog, 93, and the most active compound was the A 4 616- pregnatriene, 96. No compound displayed inhibitory activity comparable to metyrapone, 48, under the conditions of the screening assay.

Table 4 Structure-activity Relationships of Androgen Analogs

Compound Concentration % Inhibition 79 3.5 pM 8.7 7a-APTA 7.0 pM 19.3 87 3.5 pM 20.7 7a-APTADD 7.0 pM 77.4 103 3.5 pM 37.6 7a-phenylthio 7.0 pM 46.8 97 3.5 pM 2 0 .8 7|3-BA 7.0 pM 23.4 98 3.5 pM 28.1 7P-PEA 7.0 pM 34.8 104 3.5 pM 15.6 7P-PPA 7.0 pM 20.9 48 3.5 pM 1 0 0 .0 Metyrapone 7.0 pM 1 0 0 .0

As may be seen in Table 4, the androstane-based analogs, as with the pregnane-based analogs, did not exhibit particularly effective 17,20- lyase inhibitory activity. Again, the inhibitor screening assay utilized a 131 progesterone substrate concentration of 10 x Km (1.4 pM), and two inhibitor concentrations of 25 x Km (3.5 |jlM ) and 50 x Km (7.0 pM). The conversion of substrate to products ranged between 11-24%. The least active compound was 7ot-APTA, 79, and the most active compound was 7a-APTADD, 87. No compound displayed inhibitory activity comparable to metyrapone, 48, under the conditions of the screening assay.

Kinetic studies were undertaken in order to further evaluate 70-phenethylandrostenedione (70-PEA), 98. As may be seen in Figure 28, when the results of a typical kinetic experiment are plotted using the familiar double-reciprocal (Lineweaver-Burke) method, Km and Vmo* values obtained from inhibitor-containing samples do not differ from those which do not contain inhibitor. Kiihn-Velten and colleagues have published a detailed kinetic analysis of the 17,20-lyase enzyme, and they report Km and Vmax values of 140 nM and 440 nM/min. respectively .279 A typical experimentally determined Km value in this set of experiments was 181 nM, with no significant departure from this value in the inhibitor- containing samples (ie. K mapp = K m). Since K mapp= Km(l + |I]/K j), K t must be vast in order to make the ratio [I]/K; vanishingly small such that K m aPP = Km. Thus, as determined kinetically, 70-phenethylandrostenedione (70- PEA), 98, is a very weak inhibitor of 17,20-lyase. Vm0x values were normalized to microsomal protein content rather than cytP450 absorbance, and thus cannot be compared to the results of Kiihn-Velten and coworkers. Nevertheless, Vmax were unchanged between inhibitor-containing and non- inhibitor-containing samples. These results suggest that this material does not appear to be a non-competitive inhibitor of 17,20-lyase either. 132

40 -r 1/v

3 0 - - 1A/exp 25-- 1/Vopt 2 0 -- No I 15--

io --

-0.004 0.004 0.008 0.012 0.016

Figure 29. Double reciprocal (Lineweaver-Burke) plot of kinetic data evaluating 7(5-phenethylandrostenedione (7P-PEA, 98) for 17,20-lyase inhibitory activity. Units for the x-axis are nM-1, and for the y-axis are (nmol/min/mg protein)'1. 133

In order to further evaluate 7(3-phenethylandrostenedione (7(3-PEA), 98, as a possible inhibitor of 17,20-lyase, log dose-response experiments were undertaken using a five order of magnitude range in inhibitor concentration. Data were graphically evaluated, and IC 5 0 values determined using Marqhuardt regression analysis. K; values were calculated according to the method described by Cheng and Prusoff .381 As may be seen in Table 5 and Figure 30, when compared with a known inhibitor (metyrapone, 48), 7(3-PEA is a very poor inhibitor, displaying an

IC50 value of 1154.11 |iM, and a K; value of 329.75 |iM. Further, when the enzyme-activated irreversible aromatase inhibitor, 7a-APTADD, 87, is evaluated using this system, it shows poor inhibitory activity as well. Although it is of note that it possesses approximately five times more activity than 7(3-PEA.

Table 5 17,20-Lyase Inhibition

Compound Compound Rat Testis Microsomes Number Name IC 50 Log IC 50 SE app. K; 48 Metyrapone 77.43nM -4.11 .4281 22.12 jiM 87 7a-APTADD 214.15|iM -3.67 .3557 61.19pM 98 7|3-PEA 1154.11(iM -2.94 .3218 329.75pM IC so values represent the concentration of inhibitor which produces half-maximal inhibition of 17,20-lyase activity in rat testis microsomes. The IC5 0 values were derived from log IC f 0 values calculated by a non-linear regression analysis using the Marquardt method (SAS Institute, Carey, NC). SE values were also obtained from this analysis. 134 7cx-APTADD 73-PEA 0.25 n 0.3 n

•s 0.2- 0.25- 0.2-

0.15- 0.1- 0. 1-

0.05- 0.05-

1— I— I— I— I— I— I---- 1— I— I 9876543210 9876543210 ■log[I] -log[I] M etyrapone Calculations

0.35- IC,50 K; = Kmrn = 140 nM 1-JSI [S] = 350tiM K. £ 0 .2 5 -

£ 0 .1 5 -

0.05- n—i—i—i—r 6 5 4 3 2 •logfl]

Figure 30. Log dose-response data, and sample calculations evaluating metyrapone, 48, 7

4.3 In Vivo Studies:

Tiimnr growth inhibition:

The first objective of the in vivo studies was to evaluate the possible tumor growth inhibition effects of 7a-APTADD in a DMBA-induced, hormone-dependent, Sprague-Dawley rat model. Animals were treated with DMBA at age 45-50 days. This corresponds to the pubertal development period in the rat, and thereby maximizes the possibility of exposing the rapidly dividing mammary epithelium to the DNA-alkylating carcinogen derived from metabolism of DMBA. After an induction period of 5 weeks, the animals were examined for the presence of tumors twice per week. It was important to try to insure that each animal entered the study at the same relative point in the natural history of the disease in order to reduce the individual variability in the response to treatment with 7a- APTADD. Therefore, only animals bearing between 1 and 3 tumors of 0.8 to 1 .2 cm diameter were randomly assigned to treatment groups.

The administration of DMBA, a carcinogen, to young female rats results in the formation of estrogen-dependent mammary tumors .382 This well-characterized animal model has been one of the primary methods used in the evaluation of the biology and therapy of hormone-dependent breast tumors. The untreated controls were induced and selected as described above, but were treated with sesame oil vehicle only. Thus, the only difference between these animals and those treated with 7a-APTADD was the presence of 7a-APTADD in the daily injection. Presumably, this group would control for any effects seen from the treatment protocol itself, 136 thereby exposing only the effects of 7a-APTADD in the treated group. Since this study is based on the ability of 7a-APTADD to irreversibly inhibit aromatase, the final enzymatic step in estrogen biosynthesis, it is reasonable to postulate that supplementation of the treatment regimen with a physiologic dose of estradiol would reverse any reduction in tumor growth. Further, it is also reasonable to presume that estrogen supplementation would overcome aromatase inhibition, unless 7a- APTADD had additional effects, outside of aromatase inhibition, upon tumor proliferation. Previous characterization of the DMBA rat model has demonstrated that oophorectomy ablates the estrogen stimulus to mammary tumor growth, and that subsequent supplementation with a physiologic dose of estradiol re-establishes this stimulus .381 Therefore, these controls were not incorporated in these experiments.

As may be seen in Figure 31, when mean tumor volume is plotted versus treatment time, 7a-APTADD at a 50 mg/Kg/day dosage causes an initial slight decrease in mean tumor volume. Tumor volume remained unchanged during the remainder of the study. In the 25 mg/Kg/day treatment group, mean tumor volume remained unchanged for the first three weeks of the study, and then began to increase through the end of the study. By contrast, tumors in untreated animals grew steadily throughout the study. In the case of the animals treated with 50 mg/Kg/day 7a- APTADD supplemented with estradiol, again an initial slight decrease in tumor volume was observed. Upon initiation of estrogen supplementation (0.3 mg/Kg/day) following an initial 3 week 50 mg/Kg/day 7a-APTADD treatment period, tumors began to grow markedly and continued growing 137 through the end of the study. On two occasions, untreated animal tumors had grown to a size where significant necrosis had taken place and the skin overlying the tumor became ulcerated. At this point it became necessary to sacrifice these animals as per protocol. The time point corresponding to untreated animals being sacrificed may be noted by a sudden decrease in the mean tumor volume.

2 . 4 - , Vehicle Only 50 mg/Kg/d 7a-APTADD + 2.2 - 0.3 mg/Kg/d E2 after day 20

^ 1.6 -

1 . 4 - 25 mg/Kg/d 7a-APTADD 1.2 -

0.8-

0.6- 50 mg/Kg/d 7a-APTADD

0 3 6 9 12 15 18 21 24 27 30 33 36 Days Treated

Figure 31. Inhibition of rat mammary tumor growth by 7a-APTADD 138

At the end of the tumor growth inhibition study, animals were sacrificed, serum was collected, and gross necropsy performed. Necropsy revealed no grossly apparent pathology outside of the mammary tumors. Injection sites displayed no evidence of inflammation. Abdominal, pelvic, and thoracic contents appeared normal, displaying no inflammatory changes, fatty changes, grossly evident neoplasia or fibrosis. Serum estradiol levels were measured by radioimmunoassay. As may be seen in the bar graph in Figure 32, serum estradiol levels were markedly elevated above untreated controls in both the treated and treated supplemented with estradiol groups after 6 weeks of treatment.

1 2 0 -i

100 - s ;

! > 8 0 - 3 [o

6 0 - U wCO

I 4 0 ~ ma>

2 0-

0- Treated 50 mg/Kg Untreated Treated + Estradiol

Figure 32. Radioimmunoassay of serum estradiol levels 139

It is of particular note that these animals had been bearing tumor burden for a number of weeks, and in the case of the untreated controls (vehicle only), this tumor burden was quite large. The general state of health of the vehicle only treated animals was quite poor, with the animals appearing pale and cachectic. It is therefore not particularly suprising in light of their generally poor health that the vehicle only treated animals displayed serum estradiol levels which were at the lower limit of detection of the radioimmunoassay system. It is reasonable to postulate that a number of non-essential physiologic systems (e.g. the reproductive system) might suffer under these circumstances.

In collaborative work done with Anne L. Quinn of our research group, upon sacrifice of the animals at the conclusion of the study, ovaries were collected from 50 mg/Kg/day treated, untreated, and 50 mg/Kg/day treated supplemented with estradiol animals. Studies were initiated to examine the effect of the enzyme-activated irreversible inhibitor, 7a-APTADD on the expression of cytochrome P450arom- A kinetic RT-PCR method was used to semi-quantitate ovarian cytochrome P450arom mRNA transcripts in 50 mg/Kg/day treated and vehicle only (untreated) animals.

Six week treated animals showed a consistent increase in cytochrome P-450arom expression over untreated animals. RNA from 4 treated as well as 4 untreated animals was pooled and showed a 3.4-fold increase in cytochrome P -450arom transcripts. The same reverse transcriptase reaction was evaluated for (3-actin expression. The level of (3- actin mRNA was essentially the same in both the treated and untreated 140 groups. This confirms that the difference in cytochrome P-450arom mRNA levels does not correspond to differences in starting total RNA.

The high rate of inactivation of aromatase by 7a-APTADD initially leads to an overall lowering of serum estradiol levels. In intact animals, a decrease in serum estradiol levels would stimulate gonadotrophin secretion through normal feedback regulatory mechanisms. This effect would in turn lead to stimulation of ovarian aromatase synthesis leading to estradiol production and subsequent tumor growth. The radioimmunoassays carried out on serum collected from 6 week treated animals support this hypothesis. Treated animals have over 200% the serum estradiol levels of control animals (Figure 32); an observation consistent with elevated aromatase expression. These observations led to further investigation into the endocrinology of 7a-APTADD treatment in normal, cycling adult female rats.

Endocrine Studies;

As stated above, the interesting observation of elevated aromatase expression, and elevated serum estradiol levels in 6 week treated anim als led to the formulation of a set of experiments which would examine the endocrine effects of treatment with the enzyme-activated irreversible aromatase inhibitor, 7a-APTADD, on the normal, cycling, adult female rat. An unrecognized problem with drawing conclusions regarding the endocrine effects of 7a-APTADD in the tumored animal study was that the estrus state of the animals was unknown. This fact was accounted for in the design of the endocrine study. 141

Ten week old female Spr ague-D awley rats were examined by vaginal lavage with phosphate buffered saline for two weeks in order to establish estrus state. Once this had been established, animals were segregated by estrus state and assigned to one of several treatment groups. Animals were treated with either 50 mg/Kg/day of 7a-APTADD or sesame oil vehicle only for 1 , 3, 6 , 7 and 13 days. Estrus state was monitored daily throughout the treatment period. Both 7a-APTADD treated and vehicle- only treated animals continued to cycle normally, as determined by vaginal lavage, throughout the treatment period. At the end of the treatment period, animals were sacrificed, serum and ovaries were collected, and gross necropsy performed. Necropsy revealed no grossly apparent pathology in animals treated for 1 , 3 or 6 days. Injection sites displayed no evidence of inflammation. Abdominal, pelvic, and thoracic contents appeared normal, displaying no grossly evident inflammatory changes, fatty changes, neoplasia or fibrosis. In 7 and 13 day treated animals, necropsy revealed abundant evidence of pathology. in each anim al displayed numerous poorly circumscribed 1-3 cm areas of firm, rubbery, gray tissue suggestive of fatty, fibrotic or neoplastic changes. In addition, livers displayed numerous narrow white streaks of 1 -2 cm length suggestive of fatty changes. Other abdominal organs including the spleen, omenta, small intestine and large intestine displayed numerous small, approximately 0.5 cm, white nodules suggestive of lymphadenopathy or neoplasia. In one animal, the lungs displayed numerous small, approximately 3-5 mm irregular, dark red, areas suggestive of infarction or hemorrhage. In all cases, pelvic contents appeared normal, and injection 142 sites exhibited no evidence of inflammation.

Serum estradiol levels were measured by radioimmunoassay. As may be seen in the bar graph in Figure 33, serum estradiol levels were suppressed by approximately 33% below vehicle only treated controls in the 50 mg/Kg/d 7a-APTADD treated animals at all time intervals. The suppressive effect of treatment appears to be most pronounced after three days of treatment, and least pronounced after thirteen days of treatment.

50 mg/Kg/d □ Vehicle Only

3 1 2 0 -

3 1 0 0 -

« 8 0 -

to 4 0 -

Day 1 Day 3 Day6 Day 7 Day 13

Figure 33. Radioimmunoassay of serum estradiol levels

At the time of animal sacrifice, ovaries were removed, and one ovary from each animal was assayed for aromatase activity, while the other 143 ovary was flash frozen in liquid nitrogen and stored at -80°C for subsequent measurement of aromatase mRNA levels. As may be seen in

Figure 34, ovarian aromatase activity (expressed as pmol E 2/mg protein/hr) was profoundly reduced as a result of 7a-APTADD treatment. Aromatase activity did not recover to untreated levels for the 13 day duration of the study. Since estrogen biosynthesis, and thus aromatase activity, varies considerably with estrus state, these data were also expressed as remaining aromatase activity vs stage of the estrus cycle at the time of animal sacrifice (Figure 34). As may be seen in Figure 34, aromatase activity was markedly suppressed regardless of the stage of the estrus cycle. Thus, the rat ovary appears not to have been able to overcome irreversible aromatase inhibition by 7a-APTADD at a 50 mg/Kg/d dose. It is not unreasonable to propose that with sufficient stimulation of aromatase production by the gonadotrophins that this effect could be overcome. Analogously, this inhibition may be overcome if 7a-APTADD is administered at lower dose.

When compared with serum estradiol levels (Figure 33), the levels of aromatase activity and mRNA expression (Figures 34 & 35) appear not to correlate. There seems to be an unaccounted for source of estradiol production since the levels of aromatase activity are far lower than would be expected based on the serum estradiol level. This observation may be explained by the fact that the radioimmunoassay only measures one of the estrogens, 17P-estradiol, and not estrone or . Estrone sulfate may be hydrolyzed to estrone by the enzyme sulfatase, and estrone may be converted to estradiol by the action of type I 17p-hydroxysteroid 144 dehydrogenase. In this way, these metabolites may serve as a reservoir of estrogen, and thereby artificially elevate the serum estradiol level after complete inhibition of aromatase.

I 50 mg/Kg/d □ Vehicle Only I h2l 1.2-t

0.8-

B 0 .6 - 0.6-

0.4-

0.2 -

Day1 Day 3 Day6 Day 7 Day 13 Metestrus Estrus Proestrus

Figure 34. Remaining ovarian aromatase activity (pmol E 2/mg protein/hr) (In collaboration with A.L. Quinn, 1995)

As described above, one ovary was collected for subsequent measurement of aromatase mRNA levels. Levels of aromatase mRNA transcript were measured using Northern analysis. As may be seen in Figure 35, aromatase gene transcription was suppressed below control values by 7

1.8 H 50 mg/Kg/d O Vehicle Only 1.6 1.4 1.2

1 0.8 0.6 0.4 0.2

0 Metestrus Estrus Proestrus

Figure 35. Relative ovarian aromatase mRNA levels (In collaboration with A.L. Quinn, 1995) CHAPTER 5

CONCLUSIONS AND SUMMARY

Steroid hormone-dependent tumors of the breast, endometrium and prostate represent a significant number of new cancer diagnoses, as well as significant sources cancer morbidity and mortality . 1 As described previously, androgens play a pivotal role in the progression and growth of prostatic malignancies. The disruption of androgen biosynthesis by inhibiting the enzymatic activity of cytochrome P450 17,20-lyase is a logical approach to androgen ablation for the purpose of depriving androgen dependent prostatic tumor cells of their supply of hormone. A number of steroidal and non-steroidal 17,20-lyase inhibitors have been prepared. Until the very recent (ca. 1995) reports of the 17-(3-pyridyl) substituted androstanes (Figure 18; 58, 59 ) 306 and the 22-oxime (Figure 18; 61),310 no potent, selective steroidal inhibitors of 17,20-lyase had been reported. Discovery and development of steroidal compounds which possess both selectivity for 17,20-lyase over the other steroidogenic cytochromes P450, as well as high affinity for the 17,20-lyase active site would be desirable both for their possible clinical utility and for possible insights which they might provide into the structure of the enzyme active site.

146 147

Estrogens play a pivotal role in the progression and growth of breast malignancies. The disruption of estrogen biosynthesis by inhibiting the enzymatic activity of cytochrome P450 aromatase is a logical approach to estrogen ablation for the purpose of depriving estrogen dependent mammary tumor cells of their supply of hormone. A number of 7a- substituted steroids have been reported as quite potent inhibitors of aromatase. Compounds such as 7a-APTA (Figure 25; 79) have been shown to exhibit nanomolar affinity (Kj = 18 nM) for aromatase, implying that aryl groups in the 7a-position greatly enhance binding to the active site.349,350,359 As detailed in discussions of the proposed 17,20-lyase and aromatase enzymatic mechanisms, both enzymes are heme-containing monoxygenases which perform sequential oxidations to remove small alkyl side-chains. The proposed mechanistic details of these oxidations are quite similar in both enzymatic activities .260"263 It is therefore reasonable to postulate that similarities may exist in the structures of the two enzyme active sites, and that, by extension, the active site of 17,20-lyase may accommodate substitution in the 7a-position in much the same fashion as does the active site of aromatase. Moreover, as described above, several progestins which possess small alkyl substituents in the 6 -position of the B-ring exhibit competitive inhibition of 17,20-lyase (Figure 18; 62-64), implying that B-ring substitution may be accomodated by the 17,20-lyase active site. To date, these are the only reported B-ring substituted compounds which possess 17,20-lyase inhibitory activity. The structure- activity relationships pertaining to B-ring substituted progestins and androgens for cytP450i7tt inhibitory activity have not been investigated. 148

A series of 7a-thio substituted progestins were prepared, and these compounds, as well as la-phenylthioprogesterone and a number of 7a- phenylthio and 7-arylaliphatic substituted androstanes, were evaluated for 17.20-lyase inhibitory activity in a rat testis microsomal enzyme assay system. To briefly summarize, none of the proposed substituted progestins, or androgens displayed significant 17,20-lyase inhibitory activity in a screening assay (Tables 3 & 4), an initial velocity kinetics assay (Figure 28), or a dose-response assay (Figure 29). These results suggest differences between the 17,20-lyase and aromatase active sites despite their mechanistic similarities. More specifically, the aromatase active site accomodates substitution at the steroid 7a-position, whereas the 17,20-lyase active site does not. Although other authors have reported activity in 6-substitued progestins, it appears that only very small substituents in the 6-position (e.g. methyl, methylene) are tolerated by the 17.20-lyase active site (Figure 18; 62-64).311,312 In a recent report by Laughton and colleagues, the authors propose a molecular model for the 17.20-lyase active site based on sequence homology with cytP450cam in which the substrate (progesterone or pregnenolone) contacts the enzyme active site in a sense which is upside-down with respect to aromatase binding of its androgen substrates.383 Specifically, the authors assert that the 17,20-lyase active site possesses an "L-shaped" configuration, with the heme moiety occupying the elbow of this L-shaped pocket. The progestin substrate occupies one limb of the pocket for 17a-hydroxylation catalytic activity, and the other limb of the pocket for 17,20-lyase activity. In either case, they assert that the (3-face of the progestin substrate contacts the 149 pocket of the enzyme active site. It would appear that the results of the experiments involving evaluation of the 7a- and la-thiosubstituted progestins and 7-substituted androgens support their assertions of the steroid P-face contacting the active site since substitution at the steroid a- face in this series of compounds does not confer any added benefit to 17,20- lyase binding affinity. Moreover, 7a-APTADD exhibits high selectivity as evidenced by the 105-fold difference in IC so values between aromatase and 17,20-lyase inhibition.

As has been reported, 7a-APTADD (Figure 27; 87) is a potent enzyme-activated irreversible inhibitor of cytochrome P450 aromatase (Kj = 9.9 nM; kinact = 8.4 x 10'3 sec.'1).373 This compound had not been evaluated in vivo to determine if it displays activity inhibiting mammary tumor growth or inducing mammary tumor regression. It had also not been evaluated in vivo for its effects on normal reproductive endocrinology.

7a-APTADD was evaluated for tumor growth inhibitory activity in a DMBA-induced, estrogen-dependent Sprague-Dawley rat mammary tumor model. Further, 7a-APTADD was evaluated for its effects on the estrus cycle, serum estradiol levels, ovarian aromatase activity, and ovarian aromatase mRNA expression in the normal, cycling adult female Sprague- Dawley rat. Briefly, 7a-APTADD showed significant tumor growth inhibition at two doses (25 mg/Kg, and 50 mg/Kg) during a six week treatment period (Figure 30). When evaluated in normal animals for its effect on normal endocrine physiology, it showed significant inhibition of ovarian aromatase activity and serum estradiol levels (Figures 32 & 33). 150

In addition, 7a-APTADD showed the interesting effect of suppressing aromatase mRNA production in the proestrus and metestrus stages, and stimulating aromatase mRNA production in the estrus stage of the rat reproductive cycle (Figure 34). These results appear to be most logically rationalized based on the normal feedback regulation of the ovarian portion of the rat estrus cycle. Irreversible disruption of ovarian aromatase activity would lead to decreased ovarian production of estrogens. This would, in turn, reduce estrogen negative feedback control on the hypothalamus, thereby inducing an increase in gonadotrophin production. This increase would lead to augmentation of gonadotrophin stimulation of ovarian aromatase gene transcription. Unfortunately, 7a-APTADD also displayed the ability to induce significant gross pathologic changes in treated animals. In 7 and 13 day treated animals, necropsy revealed abundant evidence of pathology. Livers in each animal displayed numerous poorly circumscribed 1-3 cm areas of firm, rubbery, gray tissue suggestive of fatty, fibrotic or neoplastic changes. In addition, livers displayed numerous narrow white streaks of 1-2 cm length suggestive of fatty changes. Other abdominal organs including the spleen, omenta, small intestine and large intestine displayed numerous small, approximately 0.5 cm, white nodules suggestive of lymphadenopathy or neoplasia. In one animal, the lungs displayed numerous small, approximately 3-5 mm irregular, dark red, areas suggestive of infarction or hemorrhage. Presumably, 7a-APTADD and/or one or more of its metabolites is quite toxic, and by an unidentified mechanism is inducing these pathologic changes. REFERENCES

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XH and 13C NMR Spectra of Target Compounds 7-ALPMA-tN-BUTTLTH!01-PROGESTERONE

V325F.4JL DATE 30-3-9* SF 250.133 sr 8B.B 01 *205.770 sr 327GB TO 1633* Stf 230G.977 HZ/PT .17? PM 12.0 RO .320 AO 2.313 RG 1 NS 128 TE 297 FW 3780 02 3200.000 DP 63L PO LB . 200 GB 0.0 CX 37.80 cr 25.00 fi 10.S80P F2 -.099P HZ/CN 71.657 fPH/CN .28G SR 2359.’*

10.0 9.5 7.0 6.0 2.5 PPH 196 Figure 35. 1H-NMR of 7a-butylthio-4-pregnen-3,20-dione, 87 “ L ^ -tfJl'U ^ U P S JU lb itS U N fc UMMli lN|Li)Li-5l *M c(- L iM ri 2 a u « e tt " U ' L* WM *e 3 C

S( r \\W

ORTE 26-5-93 5P 62.896 sr 62.0 01 2170.000 St 327GB TO 3??BB Stf 15151 515 NZ/P1 .925 m 2.0 RD 1.000 aa ! 081 RC 200 MS 731 It 297 rw 19000 02 1000.000 OP *;h At? Le 1 000 ae B.0 c* 37.00 Cr 25.00 FI 220.001P F? -5.000P HZ/CM 502.102 PPM/CM E 081 SR •1 0 1 0 .7 3

10 30 iz 197 Figure 36. 13C-NMR of 7a-butylthio-4-pregnen-3,20-dione, 87 ll'Tfc'CAflL Figure 37. Figure !H-NMR of 7a-(2'-methylpropylthio)-4-pregnen-3,20-dione,88 7~ALPHA-!SOBU?TLTH!OPRQGESTERONE 2BMCIN C0CL3 HS. 338 PH HZ/PT 4232. 2S8.133 01 ST SF 12-4-83 ORTE V3SII FV ie RQ RO sh TO SI S48 32 MS RG 20B1P 270 PPtf/CH 67. 597 2S. HZ/CH F2 FI CT GO 02 297 TE cx 63LPO OP R2859. SR 9B.I 16384 16384 2777. 778 3288. 37. 10. 000P 0 2 . . 320 0 212 0 080 946 69 00 00 200 r.r f - T - F s p v a ie n e .T S C P U T * ! 7H?nn RPr.f 5TF80NE l FBHf. !S CDCl 3i fe

BfiRD ORTE 38-5-93 SF 62.896 ST €2.8 01 2178.888 SI 32/50 TO 3276B 30 15151.SIS H2/PT .925 r* 2.8 RO 4.888 no 1.861 RC 288 NS 1481 IE 29/ FH 19888 02 1888.888 OP 17H BB LB 1.808 GB 8.8 CX 37.80 CT 2s.ee FI 778.804P F2 -4.985P H2/CM 382.*S? PPH/CM B.881 SR -48*5.35

n o :jc u e p.e tee 92 pe*( 199 Figure 38. 13C-NMR of 7a-(2’-methylpropylthio)-4-pregnen-3,20-dione, 88 1NKCIRL 9.0 Figure 39. Figure JH-NMR of 897a-phenylthio-4-pregnen-3,20-dione, N ) 2 1 C 2 0 C IN G H B 2 ! B 1 6 - I - W J P E N O R E T S E G O R p O l H T L T N E H P - l f H P L i f - 7 PPM 5.0 7.0 R3337.86 .273SR PPM/CM 68.210 70.00HI/CM F2 FI cr t c GB 18 3700.000 OP 02 FV Q.820 RO RQ PM .156 H2/PT su 4589.630 S 250.133 01 sr SF DATE 10-10-93 BIET13C.M0 S127 32 MS RC E297 TE ro 32768 t 631PO 60.0 16384 3200 2551.020

37.00 10.090P .0 2 1

0.0 3.211 700 0 .7 .BIBP 200 MK-t-filfi 7-HI PHH-PHFNn IHl'lPRmiFSTFPllNE IN TOC! 5

PJM1BI2 DATE 12-10-93

SF 62.BS6 ST 62.a 01 2170.000 SI 32260 TO 32760 15151.515 sv >T .925

FN 2. 0 RO 4. 000 no 1.081 RC 200 N$ 7606 TE 297 FM 19000 02 3500. 000 r p I7M Bf1 LB 1.000 ue 0. 0 cx 37. 00 CT 22. 00 FI 216. 005P F2 •,. 986P 112/CM 3GB. OG0 PPM/CH 5..065 SP -«B«B, t 3

200 190 H8 P®M 201 Figure 40. 13C-NMR of 7a-phenylthio-4-pregnen-3,20-dione, 89 tH’KUMt Figure 41. Figure ^-NMR of 7a-(4'-chlorophenylthio)-4-pregnen3,20-dione,90 ^-NMR 3 U 0 C K I G K B 2 E H O R E T S E c f O R P O I H t L T N E H P ) O R D L H C - 4 ( - l f H P L l f - 7 i m sr ORTE V5. 355 RO PV H2/PT S120 32 MS RC S0 1630* 4230.061TO SI 01 ST 2BfltP 270 PPM/CH 67. H2/CN F2 FI 3700 02 F0 no X37. cr CX G6 LB 63LPO DP R29S9. SR It ! 9-1-93 .9 0 9 16304 2906. 3200. S.1332SB. 297 25. 0 IP 0 0 10.

1. 0 . 0

00 629 0 977 605 819 000 00 200 <0 202 7-RLPMQ-I1-CHL OROIPHENTLTHI0PROGESTERONE IN CDCL3

((

BIET13C-P10 OflTfc 10-10-93 sr 62.696 sr 62. B 01 217B. 888 SI 32768 TO 32768 sv 151S1. SIS H2/PT . 92S P0 2.8 00 ».080 no I..881 RG 200 rts 6296 TE 297 FU 19808 02 39SB. 088 OP 12H BB LB t. 888 CB 8..8 CX 37. 88 cr 20..88 Fi 2IS. eesp F2 , B89P H l/Ctl J6S..636 PPN/CH S. 813 SR 1016.. 2B

»*PPN 203 Figure 42. 13C-NMR of 7a-(4'-chlorophenylthio)-4-pregnen3,20-dione, 90 7 filPHfi f l H I H O P H E R U T H l D P R Q G E S T E R B N E

OflTE 12*12*81 SF 2 S B .135 S» 9 0 .0 01 # 110.173 51 1659# TO 1B3B# 5V 2 50 0 .0 0 0 HZ/P! .3 8 5 Ptf 5 .0 RO .9 2 0 RO 5 .2 7 7 RC 20 NS #0 TF 7R7 FW 3200 02 3200.000 OP B3L PO LB .2 0 0 CB 0 .0 CX 3 7 .0 0 CT 20.00 FI 10.000P F2 - IM P HZ/CH 67.611 PPH/CN .270 SR 2959.21

1 u _ j AWIL

I ...... 1 1 ■ ’ ■ - 1 »■»* »,mT t | | | i -t I I 1 , . 3. 8 3.5 3.0 7.5 7.0 6.5 6.0 5 .5 5.8 *.5 4.0 3-5 3.1 2 .5 2.2 1 .5 : . 0 .5 PPH 204 Figure 43. XH-NMR of 7a-(4,-aminophenylthio)-4-pregnen-3,20-dione, 91 PM 10 2B 193 168 _ 170 l *0 P8 Tm 90 ' 70 ' 0 3 ^ « 0 5 0 6 ' 0 7 ' 0 9 ' 0 9 m T 8 P ' 2 2 1 1*30 Z M ” T s I V le 0 7 1 _ 8 6 1 3 9 1 20B 0 1 ? Figure 44. Figure 13C-NMR of 7a-(4'-aminophenylthio)-4-pregnen-3,20-dione,91 * 1 0 - 7 8 S 3 - I m - Q t*-OHJMOIPnE*

101 32768 62.0 55.SIS 15151. 44.29 -4046. BE1 12H 19000 7601 2170. 11-161-93 215. 297 6?. 2 00 22. 37.

.0 2. .0 0. 1. 1 1

. . 925 000 996 000 091 00SP 011 00 000 205 'NTtem 9.0 Figure 45. Figure 1H-NMR92 of la-phenylthio-4-pregnen-3,20-dione, N S L C O C !N E M O R E T S E C O R P O t H T L T N E H P - l f H P L B - 1 B 5 9 - I - 8 J P *.55.5 3.5 2.5 2I98P 1B.BBIP 22.11 . 37.IBF2 4IB .211 FI CT CX CB LB OP 3EI9 FM 28 .821 MS 12.flRC AO RO PV .349 H27PT s« 4866.*86 SI 01 5 r SP DATE 12-11-93 Htlll3R.10 Htlll3R.10 R2959.21 .273SR PPH/CM 68.275HI/CH 3201.BIB 02 TE ro 91.1 63L PO 16304 163BI 0 0 2BS7.143

297 259.133 12B

2.067 206 PjW-t-850 50NC/COCL3 1-nLPMfl-PHtNTLTHIOPRlJGESTERONE

TLB2B4BC.001 OfiTE 8-11-03 SF 67. 025 ST 67.9300000 01 2540.000 SI 32769 ro 32769 50 17241.370 H2/Pr 1.0S2 P0 2.0 RO 2.000 no .950 RG 200 N5 2012 Tt 297 F 0 21609 02 4000.000 DP 12H 90 L6 I 000 GB 0.0 CX 37.00 CT 22.00 FI 215.008P F2 -.909P H2/CM 39b.529 PPH7CH 5.039 207 Figure 46. 13C*NMR of la-phenylthio-4-pregnen-3,20-dione, 92