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Synthesis and in vitro studies of radiolabeled and fluorescent aromatase inhibitors

Chen, Hsiu-Ho, Ph.D.

The Ohio State University, 1988

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 SYNTHESIS AND IN VITRO STUDIES OF RADIOLABELED AND FLUORESCENT AROMATASE INHIBITORS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By Hsiu-Ho Chen, M.S.

a je s f c

The Ohio State University 1988

Dissertation Committee: Approved by

Robert W. Brueggemeier, Ph.D.

Dennis R. Feller, Ph.D.

Duane D. Miller, Ph.D.

Larry W. Robertson, Ph.D. c / m ~—* Robert W. Brueggemeier College of Pharmacy DEDICATION

To Dad and Mom

- ii - ACKNOWLEDGEMENTS

I am grateful to acknowledge the following individuals and organiza­

tions for their assistance in the completion of my graduate studies:

To professor Robert W. Brueggemeier for all his guidance, concern

and insight throughout my stay at the university.

To Chung-Shan Institute of Science and Technology, R.O.C. for

full fellowship support till dissertation completion date. To all faculty, staff and my fellow graduate students for their friendship, support and valuable suggestions.

To Mr. J. Fowble, Mr. J. Miller, Mr. R. Weisenberger and Mr. D.

Chang for their help in obtaining Mass, NMR and FTIR spectra.

To Dr. Michael Darby and Miss Nancy Katlic for their technical

assistance in preparing radiolabeled compound and MCF-7 cell cultures.

To Mr. Paul Campbell for his great assistance in preparing this document on the College's computer equipment.

Lastly and most importantly, to my parents and husband for their love, understanding and enthusiastic support.

- ill - VITA

May 12, 1953...... Born - Taipei, Taiwan, R.O.C.

June, 1976 ...... B.S. Pharmacy, Taipei Medical College.

August, 1978 ...... M.S. Pharmaceutical Chemistry, National Defense Medical Center(NDMC).

Sept. 1979-Aug. 1984 ...... Instructor, School of Pharmacy, NDMC.

Sept. 1984-present ...... Graduate Student, College of Pharmacy, The Ohio State University.

PUBLICATIONS

Articles:

1. H.H. Chen, "Synthesis and Biological Evaluation of Perimidine Derivatives", M.S. Thesis, NDMC, 1978.

2. K.C. Liu und H.H. Chen, "Synthese und anorektishe wirkung einiger kondensierter Perimidin-Derivate", (1979) Arch. Pharm. (Weinheim), 312, 776-780.

3. K.C. H u and H.H. Chen,"Synthesis of Mannich Bases of 5-Methyl -lH-benzotriazole as Potential Muscle Relaxants", (1981) J.Taiwan Pharm. Ass. 33, 61-66.

4. K.C. Liu and H.H. Chen, "Synthesis of Mannich Bases of 5-Nitro lH-benzotriazole as Potential Muscle Relaxants", (1982) J. Taiwan Pharm. Ass. 34, 108-113.

5. K.C. Liu und H.H. Chen, "Synthese und Anorekitishe Prufung von Thiazole [3,2-a] Perimidin” , (1983) Arch. Pharm. (Weinheim) 316, 728-729.

- iv - 6. K.C. Liu and H.H. Chen, "Reaction of 2-Hydrazinoperimidine with Acetylacetone", (1984) J. Heterocycl. Chem. 211, 911-912.

7. K.C. Liu and H.H. Chen, "Synthesis and Anorectic Evaluation of per- imidone, 2-Azaperimidine and 2,3-Dihydroperimidine- 2-spirocyclhexane", (1984) J. Taiwan Pharm. Ass. 36, 140-143.

8. K.C. Liu and H.H. Chen, "Cyclocondensation of 2-Hydrazino- perimi- dine with Diethyl oxalate and Ethyl pyruvate", (1985) J. Hetero- cycl. Chem. 22, 1363-1364.

9. K.C. H u and H.H. Chen, "Syntheses of 3-Substituted 1H-1,2,4- Tria- zolo [4,3-a] Perimidine", (1985) Arch. Pharm. (Weiheim) 318, 468-470.

Abstracts and Presentations:

1. K.C. Liu and H.H. Chen, "Synthesis and Biological Evaluation of Perimidine Derivatives", 9th International Heterocyclic Chemistry Congress, Tokyo, Japan, August 22-26, 1983.

2. P.K. Li, H.H. Chen, N.E. Katlic, M.V. Darby, and R.tf. Brueggemeier "7a-Substituted Steroidal Aromatase Inhibitors in Hormone-dependent Breast Cancer in Culture and in Vivo", International Conference on Aromatase, Miami, Florida, March 4-7, 1987. Abstract No. 23.

3. R.W. Brueggemeier, N.E. Katlic, P.K. Li, H.H. Chen, and M.V. Darby, "7a-Substituted Steroidal Aromatase Inhibitors in Hormone-dependent Breast Cancer in Culture and In Vivo". 193rd ACS National Meeting, Denver, Colorado, April 5-10, 1987. Abstract No. 50.

FIELD OF STUDY

Major Field: Medicinal Chemistry Steroid Biochemistry

- v - TABLE OF CONTENTS

PAGE

DEDICATION...... ii ACKNOWLEDGMENTS...... iii

VITA...... iv LIST OF FIGURES...... ,. ix

LIST OF TABLES...... xii LIST OF SCHEMES...... xiii

ABBREVIATIONS...... xiv

CHAPTER I. INTRODUCTION...... 1

1.1. The role of aromatase in estrogen biosynthesis ...... 2

1.1.1. Estrogen biosynthesis in the ovary ...... 3

1.1.2. Estrogen biosynthesis in the placenta ...... 6

1.1.3. Extragonadal aromatization ...... 8

1.1.4. Mechanisms of aromatization ...... 10

1.1.5. Purification and characteristics of aromatase 14

1.2. Classification of aromatase inhibitors ...... 17

1.2.1. Steroidal aromatase inhibitors ...... 18

1.2.1.1. A-ring modifications ...... 19 1.2.1.2. Cjg substitutions ...... 23 1.2.1.3. B-ring modifications ...... 32

1.2.1.4. D-ring modifications ...... 37

- vi - 1.2.2. Nonsteroidal aromatase inhibitors ...... 40

1.3. Metabolism of aromatase inhibitors ...... 45

CHAPTER II. STATEMENT OF OBJECTIVES...... 49

2.1. Synthesis of radiolabeled aromatase inhibitors ...... 50

2.2. Synthesis of fluorescent aromatase inhibitors ...... 52

2.3. Synthesis of the mechanism-based inhibitors ...... 53

Summary...... 54 CHAPTER IH. EXPERIMENTAL...... 59

3.1. Synthetic methods ...... 61

3.2. Biochemical methods ...... 75

3.2.1. Studies on placental microsomes ...... 75

3.2.1.1. Competitive inhibition studies for inhibitors 49-57 ...... 76

3.2.1.2. Time-dependent aromatase inactivation studies for inhibitors 54, 55 and 57 ___ 76

3.2.1.3. Aromatase irreversible inactivation by inhibitors 54, 55 and 57 ...... 77

3.2.1.4. Preliminary stability studies for I-IPTA metabolism ...... 78

3.2.1.5. Time course for •^I-IPTA metabolism... 79

3.2.1.6. Various protein concentrations for I-IPTA metabolism ...... 80

3.2.1.7. Various concentrations of IPTA at constant protein concentration metabolism ...... 80 3.2.2. Studies in MCF-7 cell cultures ...... 81

CHAPTER IV. RESULTS AND DISCUSSION...... 82

4.1. Chemistry ...... 82

- vii - 4.1.1. Radiolabeled aromatase inhibitor ...... 82 4.1.2. Fluorescent probes of aromatase inhibitors.. 82 4.1.3. Active-site directed irreversible inhibitors ...... 85 4.2. Biochemistry ...... 85

4.2.1. Kj determinations of synthesized inhibitors ...... 85 4.2.2. Inactivation studies of mechanism-based inhibitors ...... 94

4.2.3. Microsomal metabolism of * 2^I-IPTA...... 114

4.2.4. MCF-7 cell cultures metabolism of * 2 ^I-IPTA 116

CHAPTER V. CONCLUDING REMARKS...... 121

BIBLIOGRAPHY...... 123

APPENDICES

A. PreUminary stability studies for microsomal metabolism of 125I-IPTA ...... 133

B. Time courses for microsomal metabolism * 2 ^I-IPTA ...... 136 19^ C. Microsomal metabolism of I-IPTA at varying protein concentrations ...... 142

D. Microsomal metabolism of -^I-IPTA at varying substrate concentrations ...... 147

E. MCF-7 cell metabolism of 1 2 5I-IPTA...... 151

- viii - LIST OF FIGURES

FIGURES PAGE

1. Steroidogenic pathway of estrogen biosynthesis ...... 2 2. Two-cell theory of follicle estrogen production...... 5

3. Mechanism of aromatization via peroxidative attack at C^g.. 10

4. Mechanisitic alternatives for C^g demethylation via path a

and path b hydroxylated intermediates ...... 12

5. The mechanism of aromatization of C^g-C^g homolytic

cleavage ...... 13

6. Proposed mechanism of PED inactvation of aromatase by

Covey et al ...... 25

7. Proposed mechanism of PED inactvation of aromatase by

Metcalf et al...... 26

8 . Double-reciprocal plots for aromatase inhibition by

inhibitor 50 ...... 86

9. Double-reciprocal plots for aromatase inhibition by

inhibitor 51 ...... 87

10. Double-reciprocal plots for aromatase inhibition by

inhibitor 52 ...... 88

11. Double-reciprocal plots for aromatase inhibition by inhibitor 53 ...... 89

- ix - 12. Double-reciprocal plots for aromatase inhibition by

inhibitor 54 ...... 90

13. Double-reciprocal plots for aromatase inhibition by

inhibitor 55 ...... 91 14. Double-reciprocal plots for aromatase inhibition by inhibitor 57 ...... 92 15. Inactivation of aromatase by inhibitor 54 in

the presence of NADPH ...... 98

16. Inactivation of aromatase by inhibitor 54 in

the absence of NADPH ...... 99

17. Plot of the inactivation half-time (min) vs 1/[I]

nM~* for inhibitor 54 ...... 100

18. Protection of inhibitor 54 inactivation of

aromatase by substrate ...... 101

19. Inactivation of aromatase by inhibitor 54 in

the presence of nucleophilic trapping agent ...... 102 20. Inactivation of aromatase by inhibitor 55 in

the presence of NADPH ...... 103 21. Inactivation of aromatase by inhibitor 55 in

the absence of NADPH ...... 104 22. Plot of the inactivation half-time (min) vs 1/[I]

nM"^ for inhibitor 55 ...... 105

23. Protection of inhibitor 55 inactivation of

aromatase by substrate...... 106

24. Second enzyme pulse inactivation by inhibitor 55 ...... 107

- x - 25. Inactivation of aromatase by inhibitor 57 in the presence of NADPH ...... 108

26. Inactivation of aromatase by inhibitor 57 in the absence of NADPH ...... 109

27. Plot of the inactivation half-time (min) vs 1/[I]

nM ' 1 for inhibitor 57 ...... 110

28. Protection of inhibitor 57 inactivation of

aromatase by substrate ...... I l l

29. Inactivation of aromatase by inhibitor 57 in

the presence of nucleophilic trapping agent ...... 112

30. Double-reciprocal plots for aromatase inhibition by

inhibitor 55 in the presence of mercaptoethanol ...... 113

31. Time course for *^I-7-IPTA metabolism ...... 117

32. Microsomal metabolism of ^^I-IPTA at varying protein concentrations ...... 118 33. Microsomal metabolism of *^I-IPTA at varying substrate concentrations ...... 119

34. Microsomal enzymes involved in the metabolism of

androgens and estrogens ...... 120

- xi - LIST OF TABLES

TABLES PAGE

1. UV absorption and fluorescent emission •wavelength of fluorescent aromatase inhibitors ...... 84

2. The Kjj, for androstenedione and Kj's for the synthesized inhibitors ...... 93 3. Comparison of inactivation kinetics for irreversible

inhibitors of aromatase ...... 113

- xii - LIST OF SCHEMES

SCHEMES PAGE

I. Synthesis of radiolabeled aromatase inhibitor 48 ...... 51

II. Synthesis of fluorescent aromatase inhibitor 49 ...... 55

III. Synthesis of fluorescent aromatase inhibitors 50 and 51 ...... 56

IV. Synthesis of fluorescent aromatase inhibitors 52 and 53 ...... 57

V. Synthesis of mechanism-based aromatase inhibitors 54, 55 and 57...... 58

- xiii - ABBREVIATIONS

1. A = 4-androstene-3,17-dione

2. 1,4-ADD = l,4-androstadiene-3,17-dione 3. 4,6-ADD = 4,6-androstadiene-3,17-dione

4. ATD = 1 ,4 ,6-androstatriene-3,17-dione

5. 7o-APTA = 7o-(4'-amino)phenylthio-4-androstene-3,17-dione

6 . 7a-APTADD = 7a- (4'-amino)phenylthio-l,4-androstadiene-3,17- dione

7. 7a-BrPTADD = 7a-(4'-bromo)phenylthio-l,4-androstadiene-3,17- dione

8 . 7a-HPTADD = 7a-phenylthio-l,4-androstadiene-3,17-dione

9. 7a-IPTA = 7a-(4'-iodo)phenylthio-4-androstene-3,17-dione

10. 7a-IPTADD = 7a-(4l-iodo)phenylthio-l,4-androstadiene-3,17-dione 11. ME = mercaptoethanol

12. PED = 10p-(2-propynyl)estr-4-ene-3,17-dione

- xiv - CHAPTER I

INTRODUCTION

1.1.1. The Role of Aromatase In Estrogen Biosynthesis

As shown in the steroidogenic pathway in Figure 1, the final step of estrogen biosynthesis is catalyzed by aromatase (estrogen synthe­ tase). The enzyme is a unique complex involving a flavoprotein,

NADPH-cytochrome C reductase, that transfers electrons from NADPH to the terminal protein cytochrome P-450 aromatase. This enzyme com­ plex catalyzes the conversion of androgens (male sex hormones) such as androstenedione and testosterone to the respective estrogens (female sex hormones), estrone and estradiol respectively. This weU-known conversion has been termed aromatization.

It is now evident that hormones play an important role not only in the female reproductive system but also in the male. These estrogens are synthesized by the testis and by the extragonadal tissues includ­ ing the brain, breast tumors and adipose tissues in both males and females.

- 1 - cholesterol pregnenolone progesterone

OH

OH

HO'

OH

OH HO.

OH OH

OH HO'

aldosterone •1 .\ 1 OH x ) S ^ , testosterone I cortisol

estradiol estrone

Figure 1. Steroidogenic pathway of estrogen biosynthesis. The enzymes are (a) side chain cleavage,(b) 3p-hydroxystero1d dehydrogenase and A*- , -1sonerase, (c) 17«*-hydroxylase, (d) 21-hydroxy 1 ase, (e) 17,20-lyase, (f) 110-hydroxylase, (gr) 18-hydroxylase, (h) 170-hydroxysterold dehydrogenase, (I) aromatase, and (j) estradiol dehydrogenase. 3 1.1.1. Estrogen Biosynthesis in the ovary

The ovary and placenta are the primary tissues for estrogen bios­ ynthesis. The medium and large foliices in the ovary are the major

sites of aromatization. Many studies have investigated the role of the different cell types within the follicle. McNatty and associates [1]

observed that granulosa cells, thecal tissue, and stromal tissue all

synthesized progesterone, A^-androstenedione, testosterone, dihydro­

testosterone, estrone, and estradiol; the ratio of the steroids varied

with time of the cycle, size and stage of maturation or atresia in folli­

cles from which cells and tissues were isolated.

The two-cell theory of follicle estrogen production [Figure 2] postu­

lated [2-4] that androgen (mainly androstenedione) produced by LH-

stimulated thecal cells is the major substrate for the synthesis of estradiol by FSH-stimulated granulosa cells. When FSII binds to specif­ ic granulosa cell receptors, cAMP production is stimulated, which leads to increase aromatase enzyme and the conversion of theca androgen to

estrogen. However, the exact source of the substrate for granulosa

cells is difficult to identify. In several species, the granulosa cells

are unable to synthesize androstenedione prior to ovulation, and that substrate is provided by the thecal cells. In human follicle, andros­

tenedione is synthesized by the granulosa cells.

Richards and Midgley [5] demonstrated that, although estrogen is mainly produced by the granulosa cells provided the cells are exposed to FSH, the estrogen appears to be derived from de novo synthesis and also by aromatization of thecal-derived androgen. Without FSH, the granulosa cells lose their capacity of synthesizing estrogens or of aro­

matizing thecal androgens; nevertheless, they continue producing androgens. A similar loss in aromatase activity in thecal tissue was found.

Besides gonadotropin stimulation, other factors may be involved in

the conti'ol of estrogen biosynthesis. The addition of testosterone to

rat granulosa cell cultures acts synergistically with the stimulatory

effects of FSH on aromatase due to the mediation of androgen receptors

[6 ]. Armstrong and Dorrington [7] demonstrated that estrogen may play a role in the local regulation of follicular function. Brodie et al.

[8 ] reported substrate inhibition of aromatase in rat ovarian microsomal incubations. Inhibition of estrogen production by rat granulosa cells cultured with concentrations of androstenedione greater than 10 yM was also found by Erickson and Hsueh [9]. Luteinizing Hormone ^ ------LH R eceptor Cholesterol ATPCAMP Theca Cells Androstenedione

Basement Membrane

Androstenedione Estrogen

Aromatase Granulosa Cells ATP CAMP Enzyme

FSH R ecep to r (Follicular Fluid) _ _ -O Follicle Stimulating H o rm o n e

Figure 2. Two-cell theory of follicle estrogen prduction. 6

1.1.2. Estrogen Biosynthesis in the placenta

The main roles of placental cytochrome P-450 are cholesterol side

chain cleavage (CSCC) and the biosynthesis of estrogens from Cjg endogeneous steroids [24,25]. Most of drug substrate biotransforma­ tion reactions occuring in the liver have also been reported in the pla­ centa. In the absence of exposure to inducers, placental cytochrome

P-450 mono-oxygenenases can chiefly catalyze the 19-hydroxylation of

androstenedione, 2 0 , 2 2 -hydroxylation of cholesterol, 2 -hydroxylation of

17 p-estradiol, and N-hydroxylation and N-demethylation [10],

The placenta has shown the ability to form estrogen from andros­ tenedione and testosterone in the perfusion and in v itr o studies [1 1 ].

However, unlike the ovarian 17,20-lyase system, the placenta cannot convert progestrone or cholesterol into estrogens. In addition, the cellular heterogeneity and regularly changing cell populations still remain problems in using ovarian tissue for studying aromatase.

Whereas human term placenta has an unlimited amount of aromatase, the majority of which is bound to the cytoplasmic membrane systems, it serves as a unique model in the aromatization of androgens to estro­ gens.

Various subcellular fractions prepared from the fresh human placen­ ta have been utilized. Ryan was the first to isolated the membrane- bound enzyme complex, termed "aromatase", from the microsomal frac­ tion of the human placental tissue and found the NADPH and molecular oxygen are required for aromatization [12]. Following detection and measurement of the cytochrome P-450 in the human term placenta, they

revealed that a CO-binding pigment with an absorption maximum at 450 my of both mitochondria and microsomes corresponds to cytochrome P-450 of liver microsomes in its reduction by NADPH. The observation that CO did not inhibit the aromatization of androst-4-ene-3,17-dione by microsomes when limiting the concentration of O 2 distinguishes this conversion from other mixed function oxidase reactions involved in steroid hormone biosynthesis. This finding showed cytochrome P-450 does not act in a catalytic role in the rate-limiting, (^-requiring step in the aromatization. It is conceivable that the hydroxylation of C-19 prior to its removal is the controlling step [13]. Although the placen­ tal mitochondrial cytochrome P-450 concentration is several times great­ er than that in the microsomal fraction, both function in steroid hydroxylation s to the same extent [14].

Gibb and Lavoie [15] investigated the substrate specificity of aro­ matase in human placental microsomal preparation by use of mixed substrate experiments. The results were consistent with a single enzyme metabolizing both androstenedione and testosterone. They pro­ posed that human placental microsomes contain a single "high affinity" site for aromatization of both steroids.

Based on the result of [1,2,-^H, 4-^C]-16a-hydroxy androstene­ dione (16a-OHA) incubation with the ovarian microsomes in the pres­ ence of NADPH with or without a monoclonal antibody to human placen­ ta aromatase cytochrome P-450, Osawa and his collaborators reported that the aromatase cytochrome P-450 in the human ovaries is immunochemically similiar to that in the placenta [16]. 8 1.1.3. Extragonadal aromatization

Siiteri, MacDonald and their associates [17] developed an attractive

concept of "extragonadal aromatization". Androgens, particularly androstenedione produced principally in the adrenal glands, are aroma­ tized to estrogens at extraglandular sites, i.e., outside of either the

ovary or the adrenal gland. These sites have not been identified abso­ lutely; however, the aromatization has been shown to occur in fat

[18,171], hairs [19], kidney, muscle, skin [20,21], liver [22], bone marrow [23], and specific nuclei of the hypothalamus [27]. Longcope and coworkers found that adipose and muscle were the main tissue sites of peripheral aromatization [28,172], The extent of extragonadal aromatization is affected by age, sex, and weight. Aging in men and women is associated with a two- to fourfold increase in extraglandular formation of estrone. In men, the production of estrone is mostly extraglandular. In postmenopausal women, extraovarian aromatization of androgens to estrogens increases dramatically and produce nearly all estrogens in these women [29], Moreover, this increase in aromatase enzyme activity in the adipose tissue supports the growth of estrogen- dependent tumors in premenopausal patients with breast cancer [30].

An increased total aromatizing enzyme activity results in feminization in males but not affect females [31], Heavy women have higher conver­ sion rates and higher circulating estrogen concentrations than do slen­ der women [18]. This is a function of increased number of adipose cells rather than an increase in specific activity of aromatase in those cells. Several studies indicated that about 58% of patients with endom­

etrial cancer were obese and had crucially higher estrogen level than

normal postmenopausal women or patients of average weight [32].

Judd et al. [33] and MacDonald et al. [34] reported that higher estro­

gen conversions in obese subjects both with and without endometrial

cancer. The latter pointed out that obesity and increased estrogen lev­

els play an importment role in the increasing the risk of estrogen-

dependent cancer [35].

Excessive peripheral aromatization occurs in men and boys with

familial gynecomastia. Peripheral aromatization increases with age.

Men over 50 years old have about a 50% increase of total plasma estra­

diol levels, with minimal changes (less than 10%) in the free estradiol

levels due to increase in binding of the estradiol by raised

testosterone-estrogen binding globulin levels [36,37].

Abdul-Hajj et al. [38] reported that aromatization occurred in estro­

gen receptor negative tumors to a greater extent than in estrogen

receptor positive tumors. However, others found that there were no correlation between aromatase activity and the presence of progester­

one receptors [39]. Santen et al. [40] suggested that the delivery of

estrogens from systemic sources to the tumor or the local production by conversion of estrone sulfate to estrone may be important. Addi­

tionally, the tumor cells were found to have higher activities of both estrogen sulfatase and estrogen sulfotransferase than normal breast tissue. 10

1.1.4 Mechanism of aromatization

The mechanism of aromatization continues to receive extensive ongo­

ing study by several research groups. There are four reaction

mechanisms being proposed to date.

Mechanism I: Peroxidative attack at C^g.

Three sucessive hydroxylations of the androgen precursor are fol­ lowed by a spontaneous chemical aromatization of the A ring to yield the phenolic estrogens [Figure 3J. Akhtar et al. [42,43] examined the loss of the Cj^g-methyl group as formic acid upon aromatization by use of *®C >2 and intermediates. The pro-R hydrogen of C^g-^H inter­ mediate is eliminated during aromatization and the pro-S hydrogen is incorporated into the extruded formic acid.

O

O O O

o

Figure 3. Mechanism of aromatization via peroxidative attack at C^g. 11

Mechanism II: 10-hydroxylation Two sequential hydroxylations occur at the androgen C-19 position, followed by a third liydroxylation at 13 position [Figure 4, Path a]. Townsley and Brodie [44] suggested that an oxygen is inserted at the 9 Q 13 position, and a A • -enol is then formed with the removal of the

23"H favored stereoselectrically.

Mechanism III: 23-hydroxylation

Fishman and coworkers proposed that two sequential hydroxylations take place at the androgen C-19 position, followed by a third at 23

[Figure 4, Path b]; the products of these hydroxylations are

19-hydroxyandrost-4-ene-3,17-dione, 19-oxoandrost-4-ene-3,17-dione, and estrone. Estrone results from the spontaneous collapse of the intermediate 23-hydroxy-19-oxo-androst-4-ene-3,17-dione with libera­ tion of formic acid. The last step involves a nonenzymatic transforma­ tion. They synthesized the 2a- and 23-19-hydroxy- and the 2a- and

23 ~ 19-oxoandrostenediones, and verified that only the

23"hydroxy-19-oxoandrostenedione was converted to estrone with the elimination of formic acid. Thus, they proposed the 23-position is the major site of rate-determining hydroxylation, and recommended that it may be involved the multiple enzyme sites in the aromatization process: one enzyme for the C^g-hydroxylation, and the other for

23-hydroxylation [45-48],

However, Caspl et al. [49] failed to show incorporation of

2 3 -hydroxyl group into formic acid under enzymatic or nonenzymatic condition with the synthesized [23-*®0, 19-^HJ 23-hydroxy-103"formyl 12 androst-4-ene-3,17-dione. Therefore, Fishman's mechanism is not an

imperative pathway of estrogen biosynthesis.

OH H I HO C = 0 q s f H

path a / 0

path b /OH HC=0 C =0 O— CH HO

Figure 4. Mechanisitic alteratives for Cjg demethylation via path a and path b hydroxylated intermediates. 13

Mechanism IV: CjQ-C^g homolytic cleavage

A new possible mechanism of this radical cleavage as shown in Fig­

ure 5, was reported by Covey in the 193rd ACS National meeting in

1987. He proposed that the third oxygenation of aromatase is required to carry out the hydrogen abstraction at Cj of 19, 19-dihydroxy

androstenedione, homolytic cleavage of Cjq -C^q bond, and then oxygen rebound at C^g [50],

OH OH I I jj * OH 'C-OH vC-0H H-C-OH path 1: I I 'C-OH •OH XX JCQ-XC

L path 2: i « r H H-C-OH J 3 . I HO'

Figure 5. The mechanism of aromatization of Cjq , Cjg homolytic cleavage. 14

1.1.5. Purification and Characteristics of Aromatase

Although the enzyme has been measured in many tissues, traditional

enzymological experiments were very difficult to carry out in the past.

In particular, purification of the cytochrome P-450 monooxygenases

from placenta are difficult because of their membrane-bound nature,

instability and low concentration. In 1981, Pasanen and Pelkonen [51]

showed that phenyl-Sepharose column chromatography proved to be a

rapid and efficient initial purification step for placental P-450s with content of 4-7 nmol/mg protein. In 1982, Osawa and his collaborators

demonstrated that two distinct aromatase-active protein complexes (aro­ matase I and aromatase II) were solubilized by use of deoxycholate and

separated by diethylaminoethyl cellulose chromatography from human term placenta. Aromatase II converted androstenedione to estrone and was found to be the major aromatase, containing about five fold more aromatase activity and reduced NADP-cytochrome C reductase activity than did aromatase I. Using antibodies, they characterized similiar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450 [52]. They also evaluated the distribu­ tion of the total aromatase activity in the homogenate, 900xg pellet and 900xg supernatant of two human term placentas. The ratio of total activity was about 2:1 in fresh placenta, but about 19:1 in the placen­ ta stored at -96°C for 3.5 months. The data showed that aromatase itself is stable for months at -96°C and the 900 x g pellet prepared from the frozen tissue contains almost the total original aromatase 15 activity in the fresh placenta. These results verified the widely accepted idea that fresh tissue and fast processing are not necessarily applicable to placental aromatase [53J. In 1985, Simpson's group [57] isolated the cytochrome P-450arom with a molecular weight of 55K based on immunoaffinity chromatography of monoclonal antibodies and a spe­ cific content of 2 nmol/mg protein. In 1986, Nakajin et al. [58], using an octylamino-agarose column, found aromatase to have a molecular weight of 55,000 and a specific cytochrome P-450 content of 2.7 nmol/ mg protein. Nevertheless, the half-life of this enzyme was only 2.5 days. Hagerman purified the human placenta aromatase by utilization of affinity chromatography [54]. Tan and Muto reported the partial purification of cytochrome P-450arom to a specific content of 4.2 nmol/ mg protein, the enzyme with a turnover number of 4.8 min"^ but elec- trophoretically inhomogeneous [56], In 1987, Kellis and Vickery [55] developed a new isolation procedure for cytochrome P-450arom. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic deter­ gent, Nonident P-40. This procedure yielded a highly purified and active cytochrome P-450arom with the highest specific content of 11.5 nmol of cytochrome P-450 per mg of protein ever reported to date.

Recently, Harada [59] described that a purified aromatase may be a unique and identified form of cytochrome P-450. His result from immu­ nochemical experiments was inconsistent with the conclusion by Osawa et al. [60], who proposed two forms of aromatase (aromatase I and II) with different substrate specificities in human placenta microsomes. 16

Evans et al. [61] have separated a partial complementary DNA clone coding for about 60% of the carboxy-terminal portion of aromatase.

This clone is specific for human cytochrome P-450arom from a phage Xgtll human placental cDNA library, after screening initially with polyclonal antibodies (IgGs) against partially purified human pla­ cental aromatase-system cytochrome P-450. Currently, this clone is being used as a probe to examine regulation of aromatase gene expres­ sion in various tissues [62,63]. Another approach involves restriction mapping and sequencing, and two cDNA inserts complentary to mRNA encoding aromatase cytochrome

P-450 (P-450arom) have been determined [65]. The open reading frame contains at the carboxy terminal end, and is identical to all four cysteine-containing tryptic peptide isolated by Chen et al. from puri­ fied cytochrome P~450arom [64]. It is believed to be heme-binding region of the cytochrome, as reported by Kawajiri et al. [ 6 6 ].

Most recently, Simpson et al. studied the structural analysis of the gene encoding aromatase cytochrome P-450 by use of a 2.5 kb cDNA insert complementary to human cytochrome P-450arom. This cDNA was isolated from a human placenta cDNA library and has a sequence encoding 420 amino acids. Observed changes in the synthesis of cyto­ chrome P-450arom are reflective of changes in the levels of mRNA encoding this protein. Their preliminary results regarding one of genomic fragments of about 9 kb in length showed the presence of sequences corresponding to the 5' end of the cDNA, interrupted by at least two introns [67]. 17

1.2 Classification of Aromatase Inhibitors

One of the methods for treatment of estrogen-dependent breast can­ cers has involved the use of aromatase inhibitors. Due to their ability to decrease the amount of estrogen available to support the growth of tumor cells and the blockade of aromatase in the final step of the estrogen biosynthesis, specific inhibitors of aromatase have the poten­ tial to be effective with a minimum of side effects [41J. Diseases such as endometrial cancer [35], gynecomastia [107], and other conditions such as endometriosis, idiopathic oligospermia, precocious puberty and premature labor have been reported to benefit from treatment with aro­ matase inhibitors [ 6 8 ], Henderson et al. recommended that aromatase inhibitors may be of value for application in non-surgical treatment of benign prostatic hyperplasia [70].

A number of compounds have been investigated in an effort to find an agent which most specifically and effectively inhibits the aromatase enzyme. 18 1.2.1. Steroidal Aromatase Inhibitors

Steroidal compounds act as substrates for the enzyme interfere with

androgen aromatization by binding to the enzyme at the active site and

producing type I high-spin spectra (type I inhibitors) [71]. Many

enzyme inhibitors not only compete with the substrate but are convert­

ed by the catalytic process of the enzyme to intermediates which irre­ versibly bind to the enzyme and produce its inactivation. The equa­

tion of the reaction is E + I E • I -> E • I' -+ El'. This type of inhibitors is called "suicide inhibitors" (or Kcat inhibitors, mechanism-

based inhibitors) and is quite specific because the compounds bind to the active site of the enzyme. Affinity labels, known as active-site directed irreversible aromatase inhibitors, are substrate analogs carry­ ing reactive electrophilic or alkylating groups.

Since 1973, the Brodie’s research group [8,73] evaluated the rela­

tive substrate activity of 100 readily available steroids and some nonst­

eroids for their ability in aromatase inhibitory activity using human

placental microsomes. Subsquently, Siiteri and Thompson confirmed

their results and summarized the structure-activity relationships for aromatase inhibition as follows [74]:

1. Based on the A/B rings, trans structures are better inhibitors than those of the els structures. 2. 3-Keto analogs are most effective. 19

3. Steroids with extended linear conjugation are beneficial, e.g. 4,6-diene-3-one, l,4,6-triene-3-one, and 4-ene-3,6-dione.

4. Cjy substituents are of effectiveness as follows: 17-keto > 173-ol

> 173-formate > 173-acetate > 173-propionate » other derivatives.

5. 19-Norandrostanes are less effective than androstane derivatives.

6 . Estrogen and antiestrogens have poor inhibitory activity.

7. Cjg 4-androsten-3-one compounds are usually better inhibitors than the corresponding ring A reduced (5a or 53) steroids or 4-estren-3-one compounds.

Consequently, the effective aromatase inhibitors are most likely to resemble the natural substrates androstenedione and testosterone.

1.2.1.1. A-ring Modifications

Brodie’s research group focused on the studies of substitution at

C-4 of androstenedione molecule as aromatase inhibitors in v itr o and in v iv o . 4-Hydroxy- and 4-acetoxy-4-androstene-3,17-dione (4-OHA

1, 4-AcetoxyA 2) and 1,4,6-androstatriene-3,17-dione 3 were found to act as competitive and suicide substrates of aromatase inhibitors, giv­ ing rise to aromatase inactivation following pseudo-first-order kinetics.

Further conjugation of the 3-keto-4-ene system to afford

4-hydroxy-4,6-androstene-3,17-dione 4 caused more rapid inactivation of aromatase in rat ovarian microsomes than 4-OHA. However, none of the androstenedione derivatives were more effective than 4-OHA or 4-AcetoxyA [ 75 ]. 20 4-OHA and 4-AcetoxyA act as aromatase inhibitors and inhibit

reproductive process in v iv o, and were effective in causing regression

of DMBA [7,12-dimethylbenz(a)anthracene]-induced hormone-dependent

mammary tumors by reducing estrogen production via aromatase inhib­

ition in the rat [139,140]. 4-OHA is now under investigation in phase

II clinical trial for the treatment of advanced breast cancer in postme­ nopausal women [68,76].

From the initial screening assays for inhibition of aromatase by 4-

(substituted thio)-4-androstene-3,17-dione, Abul-Hajj [103] found that increasing the alkyl side chain resulted in a considerable decrease in the inhibitory activity. Several compounds, such as R=H, COCHg 5,

were effective competitive inhibitors with apparent Kj's of 36-73 nM. Derivatives with a phenyl substituent showed good inhibitory activity,

but the substitution on the phenyl ring decreased or eliminated the

effect. Analogs with an alkyl chain up to three carbons on the 4-thio-

substituted position still functioned as an aromatase inhibitor; how­ ever, longer chain derivatives exhibited no activity. The investigators calculated the enzyme could accomodate the bulky area up to about 5.5 angstroms long. However, the inhibitory activity of the derivatives is lower than the corresponding O isosters (4-OHA and 4-AcetoxyA). A new, orally active irreversible aromatase inhibitor,

4-am inoandrosta-4,6-dione-3,17-dione (FCE 24210) 6 was reported by di

Salle et al.. The in v itr o potency of FCE 24210 (ICgQ= 120 nM) was about 4 times lower than 4-OHA. It was shown to produce NADPH-dependent and time-dependent inactivation of aromatase. The inactivation half-life of FCE 24210 was 4 min, compared to 2 min for

4-OHA. Using pregnant mare serum gonadotropin (PMSG) pretreated rats by the subcutaneous route (s.c.) and measuring the remaining ovarian aromatase activity, the FCE 24210 and 4-OHA were equipotent, with ED^q of 2.7 and 3.0 mg/kg respectively. Orally, the ED^q of

FCE 24210 was 14 mg/kg, whereas that of 4-OHA was 100 mg/kg. More comparative s.c. studies in rats showed that FCE 24210 had no intrinsic estrogenic (uterotrophic) and androgenic effect, but 4-OH did have androgenic effect. Therefore, they claimed that FCE 24210 is a compound worth further investigation [69].

Handerson's research group described aromatase inhibition by l-methyl-l,4-androstadiene-3,17-dione (SH489) 7, which suppressed gonadal and peripheral aromatization in juvenile female rats. SH489 is a potent inhibitor of aromatase in v itr o and in vivo and lacks intrinsic endocrine activity. Therefore, SH489 was thought to be attractive to use for clinical trial in the treatment of estrogen dependent diseases 22

OH OCOCHa

NHg

5: R=H, COCHa 6 23

1.2.1.2. Cj^g Substitutions

10fJ-Propynyl-substituted estr-4-ene-3,17-dione (PED; MDL 18,962) analogs, the first series of mechanism-based enzyme-activated irrevers­ ible inhibitors of human placental aromatase were synthesized and pro­ posed the actions of mechanism by three individual groups.

1. Covey et al. [ 8 6 ] proposed a mechanism for the inactivation of

aromatase by PED [Figure 6 ] in which the unreactive PED 8 was

converted into the reactive conjugated acetylenic ketone, then

covalently modified and inactivated the enzyme. They proved

that PED, propargylic alcohol 8 a and propargylic ketone 8 b inacti­

vate the enzyme in the presence of NADPH. PED showed an

apparent Kj of 23 nM and Kjnacj. of 1.11 x 10"^ sec"^. The inac­

tivation of ketone intermediate 8 b was also observed in the absence of NADPH.

2. Metcalf et al. [77,78] examined the aromatase inhibitory activity of allenic and acetylenic steroids, observed the position of the allenic and propargylic function with specificity toward aromatase,

conferred to be of the active-slte directed irreversible inactiva­

tion. They evaluated PED 8 (MDL 18,962) as a suicide inhibitor

of aromatase and observed an inhibition constant Kj= 4.5 ± 1.3

nM. It was noted that the 2-propynyl group is an important fac­

tor for the time-dependent inactivation [81]. They suggested

that the mechanism of action may involve oxygen insertion into 24 the carbon-carbon triple bond to produce the highly reactive

oxirene species [Figure 7]. Covalent bond formation was

proposed to occur via the oxirene intermediate rather than via a

Michael addition process, because the Kj of propagylic alcohol was about 1,000 times higher than that for PED.

3. Marcotte and Robinson [79] reviewed the design and study on

those irreversible inhibitors of human placental microsomal

aromatase, such as lOP-propargyl ( 8 ), lOp-allenyl (9),

lOp-difluoromethyl (10), and derivatives of estr-4-ene-3,17-dione due to the initial two steps of the mechanism of action involving hydroxylation at the C-19 methyl group. Compound 10 produced

the time-dependent irreversible inactivation of aromatase in the

presence of NADPH, but lOP-fluoromethyl analogs 11 was an

alternate substrate for aromatase and was converted to estrogen

without significant inactivation of the enzyme. Thus, they

postulated that the mechanism of inactivation by 1 0 is through

formation of an acyl fluoride by sequential 19-hydroxylation and

then dehydration. An acyl fluoride analog was also synthesized;

however, it can not prove the proposed mechanism.

Moreover, Kruter et al. [82] studied the effects of PED on androgen action and metabolism in cultured human foreskin fibroblasts. Currently, biological characterization and regression of estrogen-dependent mammary tumors of PED are being evaluated by Johnston [170]. PED could be useful for clinic studies. 25

^ O H

0 0 8

nz _ ,o

0 j x r

8b

Figure 6. Proposed mechanism of PED inactivation of aromatase by Covey et al. [86]. 26

0

8

Figure 7. Proposed mechanism of PED inactivation of aromatase by Metcalf et al. [77]. Shih et al.[83] synthesized novel epoxy steroids 10-(epoxyethyl)estr-4-ene-3,17-diones as active site probes of

aromatase and as inhibitors of estrogen synthesis. The 19R-coumpound

1 2 , proved to be a very powerful competitive inhibitor of human placental microsomal aromatase (Kj= 7 nM) while the 19S-isomer 12 was

less effective (Kj= 75 nM). Childers [84] synthesized novel

108-thiiranyl- 4-estrene-3,17-diones 13 by action of triphenylphosphine

sulphide-picric acid on 1 0 -oxiranyl precursor found potent inhibition of placental aromatase. The 19R-isomers were potent inhibitors and

showed affinity 36-fold (10-oxirane, 12) and 80-fold (10-thiirane, 13) greater than the corresponding 19S-isomers. Further evidences from

spectral titrations of microsomal preparation and purified P-450o___ arom demonstrated that the binding of the 19R-isomers to enzyme shifts the Soret maximum of the ferric enzyme, producing the high spin form of the enzyme. They concluded that the oxygen atom of the 10-oxirane and the sulfur atom of the 1 0 -thiirane are bound to the heme iron in the inhibitor complexes. Additionally, the stereoselective binding of the 19R-isomers indicated C-l and C-2 of the A ring of the Cjg androgen substrates are closely positioned near the heme to permit direct attack by an iron-bound oxidant [85]. 28

9

0

F8HC

10 11

(1 9 R ) 1 2

H8

13 (1 9 S ) 29

Flynn and coworkers (80] synthesized 173-hydroxy-

10-methylthio-l,4-estradlene-3-one 14, and found It had two time-dependent pathways of enzyme inactivation. One pathway is NADPH-dependent pathway, the loss of enzyme activity was induced in the presence of NADPH; the other is a slower inactivation, it was observed in the absence of NADPH. The proposed mechanism of inactivation was that the formation of a sulfenyl ester was major result in the NADPH dependent pathway, whereas the direct active-site alkylation at the methylthiol group occurred in the absence of NADPH.

Nelson's research group [104] replaced the C-19 methyl group in the androgen nucleus with either a thiol 15 or a methylenethiol group

16. Both compounds acted as suicide substrates of aromatase, with

Kj's of 106 and 34 nM respectively. The mechanism of action was further confirmed that aromatase can oxidize a sulfur placed in the

1 0 3 -position, in addition, oxidize 16 to the sulfenyl ester.

OH

0

15: R=SH 14 10: R=CH2SH 30 Akhtar [122] postulated that the sulfur or nitrogen substituents at the Cjg position may coordinate v/ith the heme iron of aromatase resulting in the inhibition. 19-Azido-4-androstene-3,17-dione 17, and

19-methylthio-4-androstene-3,17-dione 18 were found to be potent competitive inhibitors. A Type II P-450 binding spectrum was produced when the 19-methylthio analog was added to aromatase presaturated with androstenedione. The sulfur atom interacts with the heme iron and forms a ligand resulting in the inactivation of arom atase.

Weintraub and his coworkers introduced a trimethylsilyl or a trimethylsilymethyl group to C-l, C-2 or C-19 of androst-4-en-3-one analogs, and reported that only 1 0 -[l-hydroxy- 2 -(trimethylsilyl) ethyl]estr-4-ene-3,17-dione 19, caused inhibition of human aromatase with apparent Kj of 562 ± 12 nM. 19-Hydroxy group of 19 enhanced the affinity for binding to active site [ 8 8 ].

Kinetic evidence indicated by Kellis and Vickery [91] that 19-norandrostenedione (19-nor A) is a competitive inhibitor of androstenedione (A), is 3-fold more sensitive to the heme-iron ligand cyanide. Because A competes with cyanide, which results in steric exclusion of cyanide by the C-19 methyl group of A, whereas 19-nor A promotes cyanide binding to the heme-iron. 31

1 7 1 8

OH

19 32

1.2.1.3. B-ring Modifications

Brueggemeier et al. [94] synthesized la- and 7a-thio substituted

phenyl-androstenediones and found 7a-thio substituted derivatives were very effectitive competitive inhibitors. 7-APTA, 7a-(4'-amino)phenylthio- 4-androstene-3,17-dione 20, has an apparent

Kj of 18 nM, with an apparent substrate of 63 nM. Based on the results of electron-donating and -withdrawing groups on the aromatic ring of 7a-phenylthioandrostenediones, it appeared that there was no

correlation between the electronic character of the substituents and

inhibitory activity [ 95 ].

Further reports from Brueggemeier and Snider [96,99] described the synthesis of 7a-thio substituted derivatives containing alkylating moieties 21, 22, and 23 as active-site directed irreversible inhibitors of aromatase. Those inhibitors produced time-dependent, first-order inactivations of aromatase. Biochemical studies on ^C-radiolabeled inhibitors in v itr o provided the information that the aromatase was covalently labeled with MW of 50-55K.

They also developed an effective mechanism-based inhibitor of aromatase, 7a - (4' -amino) phenylth io -1,4 -andros tadiene -3,17- dione (7a-APTADD) 24, with an apparent K| of 9.9 nM. The half-time of inactivation was 1.4 min [100]. Also, this research group synthesized the 4,6-dienes or 1,4, 6 -trienes derivatives with 7-aryl substitutents 25 and 26 studies in order to combine the extended linear conjugation in the ring A and/or B and with 7a-substituted of androstenedione 33 derivatives. The results showed that 7-benzyl and 7-phenethyl substituents are effective inhibitors with apparent Kj ranges 60.9-174.0

nM, but 7-phenyl derivatives are poor inhibitors. Based on the overlapped minimized-structure of 7cc-APTA and those inhibitors, they

proposed that 7-phenyl group of the inhibitors can only accommodate themselves at a pseudo p position. On the other hand, 7-benzyl and

7-phenethyl groups of those derivatives can orient themselves in a way that the phenyl rings project into the 7a pocket [ 101 ].

In other studies, Solo et al. [102] reported a series of 7a-alkyl testosterone derivatives have the potential as inhibitors of aromatase, especially 7a-(3'-acetoxypropy])-4-androstene-3,17-dione was as effec­ tive as 4,6-androstadiene-3,17-dione. Briefly, the above observations pointed out that the aromatase can tolerate considerable bulk at the

7a- position of 4-androstene-3,17-dione or testosterone. New approaches on xenobiotic steroids, Tan and coworkers synthes­ ized the 6 a- and 6 p-hydro peroxyandrostenediones (27, 28) and found both epimers functioned as substrates and competitive inhibitors of aromatase with Kj's similar to the of androstendione [109], They also Investigated both epimers showed irreversible inactivation of aro­ matase without the presence of NADPH. The inactivation may be decreased by addition of either substrate or dithiothreitol (DTT), an antioxidant; increased by p-hydroxy-mercuribenzoate, a sulfhydryl inhibitor. The data indicated a cysteine residue was present within the active site of enzyme and the inactivation was due to the binding of inhibitor to the active site resulted in the oxidation of the cysteine residue [ 1 1 0 ]. 34

NHR 0 •NR,

20: R=H 21: R=(CH3CH3C1)3 23: R=COCHaBr 22: R=(CH3CH30Ms)3

■NH,

24 25: a=0-2 X=H. N03, NH3

26: a = 0 - 2 X=H, N03, NH3 35

Covey et al. studied 10fl-hydroperoxy-4-estrene-3,17-dione 29, an

aromatase inhibitor like 6 -hydroperoxy compounds, was able to cause a

time-dependent inactivation of aromatase in the absence of NADPH.

Furthermore, the inhibition was to be active-site directed by protection of androstenedione. The addition of DTT was partially reversed by

the inhibitor, indicated the oxidation of sulfhydryl group to sulfenic

acid on the active site of enzyme [111]. The compound in the pres­ ence of the coenzyme prevents the inactivation of enzyme, thus the

application in vivo is not of interest.

Osawa et al. [89] have studied on the stereochemistry of the func­ tional group which determines the mechanism of aromatase inhibition by

6 -bromoandrostenedione (BrA). They found that 6 ot-BrA 30 is one of the most potent competitive inhibitors reported to date, with an appar­ ent Kj of 3.4 nM; while GP-BrA 31 has the characteristics of a mechanism-based inhibitor in v itr o . This is due to the bromo group of 6f5-epimer in the quasi-axial conformation, its 1,3-diaxial interaction to the 19-angular methyl group, the 6 a-epimer has this group in the equatorial position and ideal chair conformation in the ring B. Name­ ly, an active site-directed process at 6 -substituted androgens is involved. This result was also observed by Tan and Rousseau [90],

The substitution at C -6 "front" side of the androgen molecule strongly affected the aromatase activity. It interferes with the binding of the enzyme to that part of the molecule. Those androgens induce a strong type I spectrum as well as competitive inhibition with the natural subs­ trate, 4-androstene-3,17-dione. IB OB

0

0

OOH

0

0 2 LZ

HO

0

9£ 37

1.2.1.4. D-ring modifications

Active-site inactivation of aromatase from human placental micro- somes by 16a-bromoandrogens 32 was studied by Bellino and coworkers [92].

Thompson and Siiteri reported that A^-testololactone 33, a nonan- drogenic augmentor and inhibitor of androgens, inhibited aromatase [105] and may cause aromatase inactivation due to the C-l double bond

[135], Furthermore, it has been used clinically to reduce peripheral aromatization in man with gynecomastia [107] and in postmenopausal woman [108]. It significantly inhibited basal estradiol synthesis in cultured granulosa cells and induced reduction in ovarian-vein estra­ diol levels, as well as those of 4-OHA, was observed by Yoshimura et al. [106]. However, the other pharmacological effects of these com­ pounds may be important to take into account.

Most recently, Covey et al. evaluated the effect of steroid D-ring modifications on suicide inhibition of aromatase by analogs of androsta-l,4-diene-3,17-dione 34, 35 [87]. It appears that inhibitor 34 with D-ring intact are mechanism-based inhibitors, with Kj=

0.49-2.0 yM, limting tj ^/2 *s 5.5-14.3 min, but the D-ring opened 35 are less potent. Therefore, they proposed that, the binding of the steroid D-ring to aromatase has strict geometric requirements. 38

0

•B r

32

33 34 Ri r2 X 35a b: R i =CH3; R2=C0zCH3, C02H a 0 ch 2 35c i: Ri=H; R2=C02CH3>C02H,CHO etc. b OH OH CH c HH ch 2 d 0 ch 2

CO CD 40 1.2.2 Nonsteroidal Aromatase Inhibitors

By virtue of the unique aromatization reaction in steroid biosynthe­

sis, the aromatase inhibitors interfere with steroid hydroxylation by

binding to cytochrome P-450s, which are known as type II inhibitors can produce a different spectral pattern on binding the enzyme [72].

Type II inhibitors are less specific inhibitors since their binding to the cytochrome P-450 moiety will influence the hydroxylating enzymes involved in steroid biosynthesis. The nonsteroidal aromatase inhibitors have a heteroatomic chemical group as a common feature.

Aminoglutethimide [3- (4-aminophenyl)-3-ethyl-piperidine-2, 6 -dione] (AG) 36, an anticonvulsant, is an inhibitor of the first step of steroi­ dogenesis, cholesterol side chain cleavage, and inhibits the production

of all steroid hormones. It was found to act non specifically, but was a

more potent inhibitor of aromatase than of the other steroid hydroxy­ lases [112,113]. AG is a mixture of D- and L- stereoisomers.

D-isomer was 30-fold more potent than the L- form for aromatase

inhibition [114]. Its adrenal dysfunction side effect caused to be withdrawn from use in 1966. Nevertheless, AG was found to be effec­ tive endocrine therapy for metastatic breast cancer in postmenopausal women [115,116]. Treatment with AG and hydrocortisone coadministra­ tion was equally effective as adrenalectomy [117]. The primary amino group on the phenyl ring was thought to be responsible for the steroi­ dogenic inhibitory activity. Kellis and Vickery assumed that the aryiamine in AG caused the 41 inhibition of enzyme, and examined 4-cyclohexylaniline 37, a compound lacking the glutarimide moiety but containing the arylamine, as an aro­ matase inhibitor [118]. Foster et al. employed the pyridyl group to

substitute the aminoplienyl group of AG 38, found it was a good com­

petitive aromatase inhibitor but no inhibition of P-450scc [145]. Other modifications such as pyrrolidinedione replacing the piperidinedione of AG 39, have shown selective inhibition of aromatase, but less effective than AG for their effects on estradiol biosynthesis in the rat [119].

Using molecular modelling techniques, Neidle and Jarman studied the influence of the hydrogen, methyl or ethyl group at C-3 on the con­ formational flexibility of AG. The calculation data showed that AG adopts a small number of energetically non-interconvertible conformer, and is a rather rigid molecule. On the other hand, the hydrogen and methyl substituted analog have much greater flexibility than that of ethyl group. It indicated the former substituents can fit into the dif­ fering receptor sites without changing the energy and conformation.

This also explained why the replacement of the ethyl by hydrogen caused around a 1 0 -fold drop of IC^q value [ 1 2 0 ]. Rowlanda and coworkers synthesized the analogs of AG in which the piperidine- 2 , 6 -dione ring was replaced by substituted or unsubstituted azabicyclohexanedione rings. The substituted analogs 40 and 41 pro­ duced inhibitory activity of aromatase without significant activity toward the CSCC enzyme, with Kj value of 0.015, 0.02 yM, respective­ ly. They are about 100 times more potent than AG [121]. AG and its analogs produced a Type II spectrum by coordination of the amine moiety with the heme iron. 42

In comparison with the potencies of several aromatase inhibitors in vitro, Brodie [134] and Johnston and Metcalf [135] recommend that 4-OHA and lOfi-propargyl-A are much more potent than either testolac- tone or AG in the human placental microsomal preparation. 4-OHA in the rat ovarian system is less effective than it is in the placenta. 4-OHA is about GO times more potent than AG in the human tissue, but only 15 times more potent in the rat tissue [138].

H k 36 30

H R 39 40i R^CiHg 41: R=C5Hu

37 43

Kellis et al. reported that o-naphthoflavone (ANF) 42, an inhibitor

of liver microsomal cytochromes P-450 [124], is a potent competitive

inhibitor of human placental and ovarian aromatase [125]. The metabo­ lites of ANF ■will be discussed in the Section 1.3.

The antifugal agents miconazole 43, ketoconazole, clotrimazole and

other imidazole derivatives have been claimed to be aromatase inhibitors

[128,129]. They produced a Type II binding spectrum involving an interaction between the heme iron and an imidazole nitrogen. Ayub and Levell evaluated the effectiveness of a number of imidazole drugs

compared with 4-OHA and AG as aromatase inhibitors. From the IDqq

values, they found that the order of decreasing inhibitory effect was:

4-OHA > clotrimazole > miconazole > ketoconazole > AG, etc. Those imi­

dazole drugs and AG were reversible inhibitors of aromatase activity,

but 4-OHA was an irreversible inhibitor. The major component of

those imidazoles as inhibitor of aromatase is due to the presence of one

or more aromatic rings on the N-l substituent [130], France et al. [123] described ketoconazole and AG were about 10 times more potent

inhibitors of ovine placental aromatase compared with their effects on human placental aromatase.

Hirsch et al. [131,173] investigated fenarimol [a- (2-chlorophenyl) -ct(4-chlorophenyl) -5-pyrimidine-methanol] (LY56110), 44, a pyrimidine carbinol agricultural fungicide, which caused a dose-dependent decrease in infertility due to inhibition of central nervous system aromatase activity. Lilly research laboratories dem onstrated LY56110 inhibits the aromatization of androstenedione by 44 rat ovarian and term human placental microsomes with ICgQS of 22 and

29 nM respectively. From induction and inhibition studies in the rats, dogs, and monkeys, the compound were found to be more potent than AG or cimetidine. LY56110 induced a reverse Type I binding spectrum with rat ovarian microsomes [ 132 ].

4 3

0 H

4 2

Cl

44 45 1.3. Metabolism of aromatase inhibitors

Alexandre and Balthazart [143] studied the inhibition of testoster­ one metabolism in the brain and cloacal gland of the quail by specific aromatase inhibitors such as 1,4,6-androstatriene-3,17-dione (ATD)

and other antihormones. Surprisingly, they found that ATD not only

inhibited the production of estradiol but also that of

5(5-dihydrotestosterone and small amount to 5a-dihydrotestosterone.

Thus, they suggested when interpreting the results of in vivo studies

with those compounds, one should be concerned with these unexpected

properties.

Knecht, Brodie and Catt [142] presented their studies on aromatase

inhibitors to prevent granulosa cell differentiation for estrogen in

luteinizing hormone receptor expression. They showed the stimulation

of estrogen production by FSH alone or with androstenedione during 48-h culture was prevented by 4-OHA. 4-OHA can initially enhance the FSH-stimulated cAMP production at the first 20-h cell culture, but then reduce from 20-48 h. With concentrations lower than 50 yM,

4-OHA increased the FSH-stimulated cAMP production, and LH receptor formation. Most interestingly, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, impling that

4-OHA or a metabolite may have partial estrogenic or androgenic prop­ erties. However, with higher concentrations of 4-OHA, the inhibitory effect on LH receptor formation was potentiated by keoxifene or flu­ tamide . 46 Abul-Hajj [136] compared the inhibitory activity of AG, 4-OHA,

7a-APTA and cyanoketone on androgen aromatization by human mam­ mary tumors and found all four compounds were equally effective in inhibiting estrogen synthesis from dehydroepiandrosterone, but only three (not cyanoketone) were able to inhibit the aromatization of androstenedione with ranging 81-97%.

Dao [137] investigated the inhibition of aromatase in human breast tumors by A^-testololactone, testololactone, and 6 a- and 6(5-BrA. All four compounds were competitive inhibitors of androstenedione aromati­ zation, and all blocked the estrogen synthesis in five tumors. He recommended a patient with advanced breast cancer be treated with large doses of A^-testololactone before oophorectomy and adrenalecto­ my, the response was presented as a successful treatment. Douglas and Nicholls [144] described that acetylaminoglutethimide (AcAG) is the only major metabolite of AG in humans. Moreover, AcAG is non-inhibitory towards desmolase and aromatase and is an inactivation product [145].

In attempting to develop new antiendocrine drugs, Jarman and coworkers [146] reported on the metabolism-directed design of analogs of AG. They found a new major metabolite in patients’ urine which appears to be an induced metabolite and identified as hydroxylamino- glutethimide, which is also an inactivation product. The percent inhib­ ition of aromatase with concentration of substrate of 20 gg/mL by AG and was 53 and 36% respectively [149]. 47 Kellis and his coworkers [126] studied the inhibitory potency of hydroxylated derivatives of ANF, and found that 9-hydroxy-ANF, a

metabolite of ANF, was the most effective with IgQ of 20 nM. This com­ pound exhibited high binding affinity to enzyme and the same reverse

Type I spectra as the parent compound. Therefore, they proposed a

model for binding of 9-hydroxy-ANF in which the 7,8-benzochromone ring system of the ANF derivatives occupies the steroid ring binding

site of aromatase. In the study of the regiospecific effect of the 19-methyl substituted

steroids on human placenta aromatase, Covey et al. [127] demonstrat- 1 O ed using C>2 that the dihydroxy groups of C-^g facilitate the homolyt-

ic cleavage of the Cjg-C^g bond after the hydrogen abstraction at Cj

initiated the third monooxygenation. In addition, neither the andros­ tenedione analog [10-ethylestr-4-ene-3,17-dione] nor the 19-oxoandrostenedione analog [10-acetylestr-4-ene-3,17-dione] was con­

verted to estrogens or oxygenated metabolites, but both analogs of

19-hydroxyandrostenedione [ 10-[ (lS)/or (lR)-l-hydroxyethylJestr- 4-ene-3,17-dione] were converted to 10-acetylestr-4-ene-3,17-dione in the presence of NADPH or NADH and C> 2 . The enzyme regiospecificity was detected with no change. Metabolism and parmacokinetics of the N- and C-n-octyl analogs of pyrldoglutethimide [3-ethyl-3- (4-pyridyl)piperidine-2, 6 -dione] were

studied in vivo (rabbits and rats) and in v itr o (rat hepatic micro­

somes) by Seago and his collaborators [147]. Based on the in v itr o studies, they demonstrated that both substrates had extensive 48 oxidation. The plasma half-lives were shorter than those of the parent compound, pyridoglutethimide, which may be due to the oxidative metabolism facilitating rapid excretion. The microsomal half-life of

C-n-octyl analogs was of the same order as that observed in vivo in both rat and rabbit. However, the HPLC data of plasma from animals treated with C-n-octyl analogs did not show any peaks corresponding to the metabolites which identified in the extraction of the microsomal incubates.

Lilly researchers [148] patented their studies on a series of aroma­ tase inhibiting N-substituted imidazole and triazole derivatives and their salts which were useful for treatment of estrogen-dependent dis­ eases. For example, l-[bis(4-nitrophenyl)methyl]imidazole reduced the area of established DMBA-induced mammary tumors in rats from 64.5 to o 18.6 mm over 4-week test (in control rats with increasing from 62.8 O to 1087.2 mm ), It also inhibited aromatase in rat ovarian microsomes with an EDgQ of 0.017 yM. CHAPTER II STATEMENT OF OBJECTIVES

In recent years, endocrine therapy has become more important in the treatment of breast cancer due to its lack of toxicity and conse­

quent excellent remissions that it can sometimes furnish. Aromatase

inhibitors block the final step of estrogen biosynthesis. As in afore­

mentioned studies, they would not deplete other steroids, can provide

both selective and effective inhibition of estrogen production, can be

more effective in reducing circulating estrogens than surgical ablative

processes, and are of value in the treatment of endometriosis, idiop­ athic oligospermia, endometrial and breast cancers, and gynecomastia.

Estrogen-responsive human breast cancer cell lines comprise MCF-7, MDA-MB-134, CAMA-1, T47D and ZR-75-1 [154]. MCF-7, a stable epi­ thelioid uncloned human breast cancer cell line, originally derived from the pleural effusion of a female patient with metastatic mammary carci­ noma [150], is the best characterized of these.

MCF-7 cells have numerous enzymes involved in the steroid bio­ chemistry, including steroid dehydrogenases, aromatase and steroid

hydroxylases. In addition, the cells have estrogen, androgen, proges- trone and glucocorticoid receptors has been widely used as a model

- 49 - 50 system for studies on the mechanism of action of estrogen, and the role of estrogen and other hormones in breast cancer [151]. The fac­

tors affecting the responses of MCF-7 cells to steroids have been

reported. Their growth inhibition by antiestrogens, e.g., tamoxifen, can be overcome not only by 17(5-estradiol and diethylstilbesterol but also by androgen and estrogen precursors, such as dehydroepiandros- terone [152,153].

Investigations in our laboratory of 7a-APTA 20 (Kj= 18 nM on human placental microsome) and other aromatase inhibitors in MCF-7 cells revealed that these compounds inhibit estrogen biosynthesis [97],

In particular, 7a-APTA does not interact with the estrogen receptors present in MCF-7 cells, does not increase the levels of progesterone receptors, and does not increase the rate of cell growth.

Due to the major differences in the intact cells or in v iv o , the inhi­ bitory effects may be mediated by metabolites of those inhibitors rather than the native compounds. Studies with radiolabeled inhibitors in cell cultures and human placental microsomes will provide further evidence for enzyme-activated irreversible inhibitors.

2.1. Synthesis of radiolabeled aromatase inhibitors

Recent unpublished data showed that an iodinated 7a-thio analog 48 is an effective inhibitor of aromatase with an apparant Kj of 12 nM

[98], It can be prepared from triazine 47 by use of [*^I]-sodium iodide [95] [Scheme I]. The radiolabeled •^ I analog will be examined for stability, ability to be aromatized, and whether aromatization to 51 Cjg analogs and/or conversion to C^g metabolites occurs, analyzed by reverse-phase HPLC.

HS n h2

NH;

45

NaNOj

HCI

HN(CHj)2

N = N

TFA

N al*

48

Scheme I. Synthesis of radiolabeled inhibitor 48. 52

2.2 Synthesis of fluorescent aromatase inhibitors

Fluorescent probes have been efficiently utilized in quantitative studies on membrane dynamics, such as enzyme activity [155-157],

transport [158], ligand binding [160-163] and receptor mobility [164], However, at present time, no reports on fluorescent probes of aroma­ tase inhibitors have been reported. In general, the dansyl amino acids, sulfonamides which result from the reaction of l-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl

chloride) with amino acids, have been used for end-group determina­

tion in peptide chemistry. The dansyl derivative has an extensive

yellow fluorescence. It is about 100-times more sensitive than the cor­

responding dinitrophenylated derivative [159]. Both can form fluores­

cent complexes with enzyme and possess unique properties as "fluores­

cent probes" of enzyme structure.

The syntheses of fluorescent derivatives 49, 50, 51 can be accom­ plished by alkylation of the free amino group of 7a-APTA with dansyl chloride and dinitrofluorobenzene (DNFB), respectively [Scheme II and III]. The naphthyl derivatives 52 and 53 are prepared from andros­ tenedione and lp- and 2p-naphthalenethiol, respectively [Scheme IV]. It is hoped that those derivatives can be developed for study of spe­ cific interactions of hydrophobic residues of enzyme. To determine whether those compounds retain the properties of aromatase inhibition 53 o£ the parent molecule 7o-APTA, enzyme kinetic studies will be per­ formed .

2.3 Synthesis of the mechanism-based inhibitors

1,4-Androstadiene-3,17-dione (1,4-ADD) is well-known as a subs­ trate and weak irreversible Inhibitor of aromatase. Nevertheless, the mechanism of aromatase inactivation following enzymatic catalysis by various steroids containing the 1,4-ADD structural moiety remained unknown. Moreover, the interaction of 7a-thio substituents on the androstadienedione molecule as inhibitors with the active site of aroma­ tase are not defined. Therefore, it is attractive to design the poten­ tial irreversible inhibitors based on the structure of the competetive inhibitor, 7o-APTA.

Snider and Brueggemeier [100] first synthesized and studied one of the most potent inhibitors reported to date, 7o-APTADD 24 with an apparent Kj of 9.9 nM and inactivation half-time of 1.4 min. In designing inhibitors for metabolism studies, of particular interest is the halogen compounds 55 and 57 [Scheme V] which may confer the electron-withdrawing effect on aromatase as well as may be readily converted to radiolabled probes such as 54

SUMMARY

(I) Chemical objectives:

1. To synthesize the radiolabeled compound 48 for use in metabolism

studies of aromatase inhibitor.

2. To synthesize compounds 49-53 as fluorescent probes of the puri­

fied aromatase and for use in cell culture studies.

3. To synthesize compounds 53-57 as potential enzyme-activated irre­ versible inhibitors of aromatase.

(II) Biochemical objectives:

1. To evaluate the metabolism of *^IPTA 48 in subcellular micro­ somes of human placenta and in the MCF-7 cell cultures.

2. To evaluate inhibitors 49-57 in v itr o using enzyme kinetic studies

with human placental microsomes in order to do further studies on

fluorescent and radiolabeled probes. Dansyl Chloride

NH-S

k b — N(CH3)2

Scheme II. Synthesis of fluorescent aromatase inhibitor 49.

cn DNFB

20 50

DNFB 0,N

NH, NH NO.

24 51

Scheme III. Synthesis of fluorescent aromatase inhibitors 50 and 51.

CT5 o

+

o

SH

+

53

Scheme IV. Synthesis of fluorescent aromatase inhibitors 52 and 53.

^3CP 58 Scheme V. Synthesis of mechanism-based aromatase inhibitors 54, 55 and 57.

NaNO.

HN{CHj)2

N=N*N(CH3)j CHAPTER III

EXPERIMENTAL

All melting points were taken with a Fischer-Johns melting point apparatus and are uncorrected. Proton nuclear magnetic resonance spectra (NMR) were performed on Bruker HX-90E (90 mHz), IBM AF-250 Fourier Transform NMR and multinuclear IBM AF-270 pulse NMR spectrometers and are reported in parts per million on the 6 scale.

Data are reported as follows: chemical shift [multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet), integration coupling con­ stant (in Hz), interpretation]. Infrared spectra (IR) were recorded on a Beckman IR 4230 spectrophotometer. Ultraviolet spectra (UV) were obtained on a Beckman DU-40 spectrophotometer and fluorescent emission spectra were recorded on Farrand spectrofluorometer MK-2 and X-Y recorder. Mass spectra (ms) were taken with Kratos

MS25RFA double focusing or Kratos MS-30 instruments at ionization energy of 70 eV. Samples on which exact masses were measured exhib­ ited no significant peaks of m/e greater than that of the parent. Ele­ mental analyses were examined by Galbraith Laboratories, Inc. (Knox­ ville, TENN.) High performed liquid chromatography (HPLC) were performed on a Beckman model 334 gradient HPLC or Laboratory Data

Control Chromatography Accessory Module HPLC using a reverse-phase

- 59 - column (Altex ultrasphere-ODS, 5 y, 4.6 mm x 15 cm). Solvents and chemical reagents were dried and purified prior to use when deemed necessary: benzene, dioxane (distilled from CaH 2 and sodium metal), hexane. All chemicals were purchased from Aldrich Chemical Co. (Mil­ waukee, WI) and solvents were obtained on campus. Analytical thin- layer chromatography was performed with EM laboratories or Analtech, Inc. (Newark, DE) 0.20 mm thick precoated silica gel 60 F-254 plates.

Column chromatography materials were purchased from EM (Darmstadt,

Germany) and basic aluminum oxide from Fischer Scientific (Fair Lawn,

NJ). HPLC solvents were purchased from Burdick and Jackson Labo­ ratories, Inc. (Muskegon, MI). Biochemicals were purchased from Sig­ ma Chemical Co. (St.Louis, MO). [13-^H] 4-Androstene-3,17-dione

IOC and Na I were purchased from New England Nuclear (Boston, MA). Steroids were obtained from Searle Laboratories (Skokie, IL) or Research Plus (Bayonne, NJ) and checked for purity by melting point,

TLC or NMR. MCF-7 cells were obtained from the Ohio State Universi­ ty Cell Culture Service. A modified Eagle's MEM supplemented with essential amino acids (1.5x), vitamin (1.5x), nonessential amino acids (2x) and L-glutamine (lx) was obtained in powdered form GIBCO

(Long Island, NY). The sterilized liquid media was prepared by the Media Preparation Service of the Ohio State University Comprehensive

Cancer Center by dissolving the powder in water containing sodium chloride (0.487 g/L), pyruvic acid (0.11 g/L), sodium bicarbonate (1.5 g/L) and phenol red (0.01%), and the pH adjusted to 6 . 8 . Fetal calf serum was obtained from K. C. Biological (Lenexa, KS). Tissue 61 culture flasks and supplies were obtained from Corning Glass Works (Corning, NY). Sorvall RC 2-B centrifuge was used for centrifuge samples at 4°C and ultracentrifugation was performed on a Beckman

L5-50B Ultracentrifuge. Radioactive samples were detected with a

Backman Gamma 8000 and Beckman LS 6800 scintillation counter using Formula 963 (New England Nuclear) as the counting solution. The

order of experimental procedures generally follows their orders of appearance in the text.

3.1. SYNTHETIC METHODS

The preparation of compounds 45-24 has been previously described

[93-95,100]. Some conditions were modified to aid in purification or to improve the yield.

4.6-Androstadiene-3.17-dione (4,6-ADD) (45)

45

To a stirred solution of 4-androstene-3,17-dione (3 g, 10.5 mmol) in 50 mL of t-butyl alcohol warmed to 30°C was added 2.84 g (11.54 mmol) of chloranil. The reaction mixture was stirred and refluxed for

6 hr, then cooled and concentrated under reduced pressure. The residue was dissolved in CH 2 CI2 and filtered. The solution was then 62 concentrated in vacuo. The residue was taken up in CH 2 CI2 , and placed on the basic alumina column (70 g). Elution with CH 2 CI2 afford­ ed crude 1.75g. Recrystallization from acetone/haxane gave 1.68 g

(56.4%) of pure ADD of white crystals: mp 168-169°C (lit.

172.5-173°C) XH NME (90 MHz, CDClg) 6 0.93 (s, 3H, C18), 1.05 (s,

3H, C19), 5.78 (s, 1H, C4), 6.26 (s, 2H, Cg & C7) .

7ct-(4' -Amino)phenvlthio-4-androstene-3.17-dione (7a-APTA) (20)

20

4,6-ADD (100 mg, 0.35 mmol) and aminothiophenol (133 g, 1.06 mmol) in a 10-mL round-bottom flask under Argon was heated to

80-85°C and a small piece of sodium metal was added. The reaction proceeded for 30 min at which time no starting material remained as indicated by TLC. The remaining sodium was removed, and the reac­ tion was allowed to cool to room temperature, then washed with ben­ zene. The pale yellow precipitate was filtered. The filtrate was evapo­ rated under reduced pressure. The residue (2.47 g) was crystallized from acetone/hexane to afford 1.98 g analytical pure white crystals APTA (68.7%): mp 2.12-214°C (dec) (lit. 254-256°C). The spectral data were the same as reported [94], 63 7g-4*-(3".3"-Dimethvlazldo)phenylthio-4-androstene-3.17-dione (47)

47

To a solution of 90 mg (0.22 mmol) of 7a-APTA in 5 mL of acetone

and 1.5 mL of 1.0 N HC1 cooled to 0°C was added 18 mg (0.26 mmol)

of NaNC>2 in 0.2 mL of water. The reaction mixture was treated by the similar method as reported by Darby et al. [95]. It was isolated and yielded 98.4 mg (96%) of pure 47: mp 208-209°

7a-(41 -^^ Iodophenvl)thio-4-androstene-3,17-dione (48)

48

To a stirred solution of 47 (2.3 mg, 0.0539 mmol) in 0.1 mL of ben­ zene was added a solution of Nal (75 mg, 0.5 mmol) and trifluoroacetic acid (85 yL, 1.1 mmol) In 1.0 mL of fresh formic acid. To this mix­ ture was added 100 yCi of Na^^^I. The reaction was vortexed for 30 64 min at room temperature, then diluted with 2 mL of benzene. The ben­

zene layer was washed with 2 mL of saturated NaHSOg solution and

dried (Na2 S0 4). After evaporated off the benzene, the residue was

taken up in acetonitrile. It was then purified by reversed-phase

HPLC (acetonitrile-water, 65:35, as mobile phase). The afforded radiolabeled product cochromatographed on tic with authentic 7a-IPTA. The activity of material was 2 mCi and the specific activity was ca. 1.175 yCi/mg.

1.4.6-Androstatriene-3.17-dlone (ATD) (3)

3 To a stirred solution of 4-androstene-3,17-dione (2 g, 6.99 mmol) in

50 mL of dry dioxane was added 3.51 g (15.39 mmol) of 2,3-dichloro-

5,6-dicyano-l,4-benzoquinone (DDQ). The reaction mixture was refluxed under Argon for 24 hr, then cooled and filtered. The filtrate was concentrated in vacuo. The crude residue was chromatographed over 50 g of basic alumina eluted with CH 2 Cl2. Recrystallization from acetone/hexane afforded 0,69 g (35 %) of pure ATD: mp 159-161°C

(lit. 164-165°C). % NMR (250 MHz, CDClg) 6 1.00 (s, 3H, CHg),

1.03 (s, 3H, CH3) 6.02 (dd, 1H, J = 2, 2 Hz, C4 H), 6.09 (dd, 1H, J

- 12, 2 Hz, C2H), 6.26 (dd, 1H, J = 12, 2 Hz, CgH), 6.35 (dd, 1H, J

~ 5, 12 Hz , C?H), 7.05 (d, 1H, J = 12 Hz, CjH). 65

7a-(A* -Amino)phenvlthio-1.4-androstadiene-3.17-dione (7a-APTADD) (24)

NH.

24

To a stirred and siurry solution of 150 mg (0.532 mmol) of ATD in 7 drops of 12 N HC1 was added 70 drops of glacial acetic acid. 75 mg

(0.596 mmol) of 4-aminothiophenol was added. The reaction mixture was

stirred at room temperature for 15 hr. It was then neutralized with

saturated NaHCOg and extracted with ethyl acetate twice. The organic

layers were extracted with distilled HgO and brine twice. The com­

bined organic extracts were concentrated in vacuo. The residue was

chromatographed over 40 g of silica gel (dichloromethane-ethyl acetate, 10:1 and 7:1) to give 86.7 mg (40%) of pure lo-APTADD, 32.5 mg

(15%) of pure desired product 24, and 99.2 mg (35%) of 1,7-diaddition

product. The analytical data were similiar to that reported [100]. 7a-(4'-Dansvlamino)phenylthio-4-androstene-3.17-dione (49)

NH-S

49

To a stirred solution of 20 mg (0.0488 mmol) of 7a-APTA in 5 mL of

the mixture of acetone and 0.1 M of Na 2 COg (1:1), pH 9.5-10 was

added 57 mg (0.211 mmol) of dansyl chloride. The reaction mixture

was stirred at room temperature in the dark place for 10 hr. The solution was filtered and concentrated in vacuo. The residue was

flash-chromatographed over 10 g of silica gel (dichloromethane-ethyl acetate=4:l) to afford a yellow powder 49 (4.5 mg, 14.3%); mp 165°C

(dec.); *H NMR (270 MHz, CDClg) 6 0.90 (s, 3H, C18), 1.20 (s, 3H,

C19), 3.40-3.41 (m, 1H, C?H), 5.58 (d, J = 1.2 Hz, 1H, C4 H),

6.87-6.91 (m, 3H, ArH & NH), 7.16-7.20 (m, 3H, ArH), 7.46 (dd, J =

8.5, 7.5 Hz, 1H, ArH), 7.59 (dd, J = 8.5, 7.5 Hz, 1H, ArH), 8.20

(dd, J = 7.2, 1.2 Hz, 1H, ArH), 8.30 (d, J = 8.5 Hz, 1H, ArH), 8.52

(d, J = 8.5 Hz, 1H, ArH), exact mass calcd for CgyH^gNgO^Sg m/e:

643.2664; found m/e:643.2670. 67 7«-T 4'-(2".4"-Dlnitrophenvl)amino1phenvlthio-4-androstene-3.17-dione (50)

50

20 mg of 7a-APTA (0.0489 mmol) and 40 mg of sodium bicarbonate were dissolved in 0.5 mL of distilled water and to this was added a solution of 40 mg (0.028 mL, 0.223 mmol) of 2 ,4-dinitrofluorobenzene in 1 mL of ethyl alcohol. The mixture was stirred for 15 hrs at room temperature. The solution was concentrated to remove ethyl alcohol and 10 mL of H 2 O was then added. It was extracted with ethyl ace­ tate and dried (NagSO^). After rotary evaporation of solvent, the residue was chromatographed over 10 g of silica gel (dichloromethane- ethyl acetate, 1 0 : 1 ); then, following recrystallization with methyl alco­ hol got 22 mg (78.3%) of pure 50: mp 223-224°C. IR (KBr) 3310,

3080, 2920, 2880, 1735, 1670, 1590, 1490, 1330, 800 cm’ 1; XH NMR (270

MHz, CPCI3 ) 6 0.93 (s, 3H, C18), 1.23 (s, 3H, C1Q), 3.64 (m, 1H,

C?H), 5.68 (d, J = 1.2 Hz, 1H, C4 H), 7.19 (d, J = 9.5 Hz, 1H, ArH coupling), 7.25 (d, J = 8 Hz, 2H, ArH), 7.49 (d, J = 8 Hz, 2H,

ArH), 8.20 (dd, J = 9.5, 2.6 Hz, 1H, ArH), 9.18 (d, J = 2.6 Hz, 1H, ArH), 9.93 (s, 1H, NH). Anal. Calculated for CgjHggNgOgS: C, 64.68; H, 5.78; N, 7.30. Found: C, 64.22; H, 5.78; N, 7.16. 68

7a- f 41 -(2",4"-Dinitrophenyl)amino1phenylthio-1,4-androstadiene-3. 17-dione (51)

51

20 mg of 7a-APTADD (0.049 mmol) and 40 mg of sodium bicarbonate were dissolved in 0.5 mL of distilled water and to this was added a solution of 40 mg (27 yL, 0.223 mmol) of 2,4-dinitrofluorobenzene in 1 mL of ethyl alcohol. The mixture was stirred for 10 hr at room temp­ erature. The solution was concentrated to remove ethyl alcohol and 10 mL of distilled water was then added. It was extracted with ethyl acetate twice. The organic layers were collected and were washed with brine (get pH 6-7). The water layers (pH 8-9) were neutrallized with

IN HC1 and were extracted with ethyl acetate twice. The combined organic layers were dried over Na 2 S0 4. After evaporation of the sol­ vent, the residue was chromatographed over 12 g of silica gel

(dichlorome thane-ethyl acetate, 7:1). Recrystallization with methyl alcohol afforded 22.5 mg (80%) of pure yellow crystals 51: mp 232-234°C. IR (KBr) 3280, 3100, 2940, 2880, 1745, 1660, 1620, 1590,

1335, 800 cm"1; 2H NMR (270 MHz, CDClg) 6 0.95 (s, 3H, C18), 1.27

(s, 3H, Clg ), 3.75 (m, 1H, C?H), 6.01 (s, 1H, C4 H), 6.29 (dd, J =

10, 1.8 Hz, 1H, C 2H), 7.06 (d, J = 10 Hz, 1H, C jH ), 7.19 (d, J = 9.5 Hz, 1H, ArH), 7.25 (d, J = 8.5 Hz, 2H, A of AB q, ArH), 7.49

(d, J = 8.5 Hz, 2H, B of AB q, ArH), 8.19 (dd, J = 9.5, 2.6 Hz,

1H, ArH), 9.17 (d, J = 2.6 Hz, 1H, ArH), 9.94 (s, 1H, NH); exact

mass: calcd for Cg^Hg^NgOgS, m/e: 573.1935; found m/e: 573.1941. Anal. Calculated for CgjHgjNgOgS^I^O: C, 60.87; H, 6.10; N, 6.87. Found: C, 61.17; H, 5.66; N, 6.75.

7n-(l'B-Naphthyl)thlo-4-androstene-3,17-dione (52)

0

To a stirred solution of 4,6-androstadiene-3,17-dione (170 mg, 0.559 mmol) and lf$-naphthalenethiol (0.25 ml, 1.802 mmol) under argon was

added a small piece of sodium metal. The reaction was heated to 75

-80°C in oil bath for 18 hr. The crude solid was obtained when cooled. It was then washed with petroleum ether and was filtered. The residue was chromatographed over 10 g of silica gel (hexane/ethyl

acetate, 2 : 1 ) and was recrystallized with ethyl acetate to afford 202 mg

(76%) nf white crystals 52: mp 228-230°C (dec); IE (KBr) 3010, 2900

(b r), 1740, 1650, 1625 cm '1; XH NMR (270 MHz, CDClg) 6 0.93 (s,

3H, C18), 1.23 (s, 3H, C19), 3.64-3.68 (m, 1H, C?H ), 5.65 (d, J =

Hz, 1H, C4 H), 7.43 (dd, J = 8 , 7 Hz, 1H, ArH), 7.50-7.62 (m, 2H,

ArH), 7.73 (dd, J = 7, 1 Hz, 1H, ArH), 7.86 (dd, J = 10, 8 Hz, 2H, ArH), 8.4 (d, J = 8 Hz, 1H, ArH); ms m/e (relative intensity): 444

(M+, 0.025), 284 (0.354), 160 (0.523), 115 (100). Anal. Calculated for C 2gH3 2 0 2 S: C, 78.34; H, 7.25. Found: C, 78.06; H, 7.13.

7a-(2'p-Naphthyl)thio-4-androstene-3.17-dione (53)

53

To a melted and stirred solution of 2fl-naphthalenethiol (199 mg,

1.244 mmol) warmed at 80°C under Argon was added ADD (170 mg, 0.599 mmol) and a small piece of sodium metal. The reaction was heated at 80°C for 24 hr. The white-powered like crude solid was obtained when cooled slowly. It was then washed with petroleum ether and fil­ tered. The residue was chromatographed over 10 g of silica gel

(hexane/ethyl acetate, 4:1) and recrystallized with ethyl acetate to get

165 mg (62%) of white crystals 53: mp 205-206°C. IR (KBr) 3015, 2900

(b r), 1730, 1660, 1640 cm"1; JH NMR (270 MHz, CDClg) 5 0.95 (s,

3H, C18), 1.25 (s, 3H, C19), 3.69-3.70 (m, 1H, C?H), 5.73 (d, J =

1.5 Hz, 1H, C4 H), 7.44-7.54 (m, 3H, ArH), 7.64-7.89 (m, 4H, ArH); ms m/e (relative intensity): 444 (M+, 0.119), 284 (0.748), 160 (0.951),

136 (1.00), 115 (0.813). Anal. Calculated for C 2 gH3 2 0 2 S: C, 78.34; H, 7.35. Found: C, 78.61; H, 7.23. 71

7«-phenylthlo-1.4-androstadlene-3,17-dione (54)

54

To a stirred solution of ATD (75 mg, 0.266 mmol) and a mixture of

8 drops of 12 N HC1 and 160 drops of glacial acetic acid was added thiophenol (30 yL, 32.23 mg, 0.293 mmol). The reaction was stirred for 24 hr at room temperature. It was then neutralized with saturated

NaOH, and extracted with ethyl acetate. The organic layer was con­

centrated in vacuo. The residue was chromatographed over 5 g of sili­

ca gel (CH2 CL2 ). Recrystallization with ethyl acetate gave 28 mg

(27%) of 54: mp 203-205°C. IR (KBr) 3015, 2900 (b r), 1730, 1660,

795, 750 cm-1 ; % NMR (270 MHz, CDCI 3 ) 6 0.94 (s, 3H, C1Q), 1.25

(s, 3H, Cig ), 3.66-3.67 (m, 1H, C?H), 6.03 (dd, J = 1.5, 1.5 Hz,

1H, C4 H), 6.27 (dd, J = 10, 1.8 Hz, 1H, C 2H), 7.04 (d, J = 10 Hz,

1H, C jH ), 7.26-7.42 (m, 5H, ArH); exact mass calcd for C25^28®2^ m/e: 392.1811, found m/e: 392.1813. Anal. Calculated for

C25H28°2S*1/2H20 : C> 74-78; H» 7.20; S, 7.97. Anal. C, 74.59; H, 7.12; S, 7.85. 72

7g-(4*-bromo)phenvlthio-l.4-androstadiene-3.17-dione (55)

55

To a stirred solution of ATD ( 200 mg, 0.709 mmol) and a mixture of 3 drops of 12 N HC1 and GO drops of glacial acetic acid was added bromothiophenol (110 mg, 0.58 mmol). The reaction mixture was stirred at room temperature for 15 hr. It was concentrated in vacuo and the residue was flash chromatographed twice over 8 g portions of silica gel (ethyl acetate-petroleum ether, 1:4), following recrystalliza­ tion with ethyl acetate to yield white crystals of 55 (50 mg, 15%): mp

215-217°C (dec); IR (KBr) 3020, 2915 (b r), 1730, 1655, 825 cm"1; XH

NMR (270 MHz, CDClg) 6 1.06 (s, 3H, C18), 1.37 (s, 3H, C19),

3.74-3.78 (m, 1H, C?H), 6.12 (dd, J =1.5, 1.5 Hz, 1H, C4 H), 6.39

(dd, J = 10, 2 Hz, 1H, C2 H), 7.15 (d, J = 10 Hz, 1H, CjH ), 7.37 (d,

J = 8.5 Hz, 2H, ArH), 7.55 (d, J = 8.5 Hz, 2H, ArH); ms m/e (rela­ tive intensity): 470 (M+, 52.87), 392 (2.45), 283 (51.36); exact mass: calcd for C2 5 H2 7 0 2 SBr: m/e: 470.0916, found m/e: 470.0938. Anal.

Calculated for C 25H2 7 0 2 SBr: C, 63.69; H, 5.77; S, 6.80; Br, 16.95; Anal. C, 63.32; H, 5.99; S, 6.94; Br, 16.69. 73 7a-4'-(3".3>,-Dimethvlazido)phenylthio-l.4-androstadiene-3.17-dione (56)

56

To a solution of 90 mg (0.22 mmol) of APTADD in 5 mL of acetone

and 1.5 mL of 1.0 N HC1 cooled to 0°C was added 18 mg (0.26 mmol)

of NaNC>2 in 0.2 mL of water and stirred at 0°C for 1 hr. The reac­

tion mixture was treated with 1 mL of 40% aquous dimethylamine solu­

tion, dropwise over a 1 0 -min period, then poured into a mixture of 20 mL of dichloromethane and 20 mL of water. The organic layer was

separated and washed with two 20-mL portions of water, and two

20-mL portions of saturated aqueous sodium chloride. The organic layer was dried (Na2S0 4) and concentrated in vacuo. The residue

(1 2 2 mg) was chromatographed over 10 g of silica gel (dichloromethane -ethylacetate, 25; 1) to give 87 mg (85.2%) of white crystals 56; *H

NMR (300 MHz, CDClg) 6 0.95 (s, 3H, C18), 1.26 (s, 3H, Cig), 3.4

(s, 6H, N(CH3)2), 3.61-3.62 (m, 1H, C?H), 6.07 (br s, 1H, C 4 H),

6.29 (dd, J = 10, 1.8 Hz, 1H, C 2H), 7.05 (d, J - 10 Hz, 1H, CjH ),

7.38 (m, 4H, ArH); exact mass calcd for C 2 7 H3 4 N3 O2 S m/e: 464.2372, found m/e: 464.2361. 74 7a-(4*-Iodophenvl)thi.o-1.4-androstadiene-3.17-dione (57)

57

To a stirred solution of 5G (25 mg, 0.0539 mmol) in 3 mL of benzene was added a solution of Nal (15 mg, 0.1 mmol) and trifluoroacetic acid (10 ]iL, 0.131 mmol) in 0.5 mL of distilled formic acid. The solution was stirred vigorously at room temperature for 1 hr, and then diluted with 5 mL of benzene. The benzene layer was removed, and the form­ ic acid was washed with 5 mL of fresh benzene three times. The com­ bined benzene solutions were dried (K 2 CO3 ) for 30 min, then evapo­ rated to yield 27 mg of crude product as a brown oil. It was chromatographed over 5 g of silica gel (dichloromethane-ethyl acetate,

40:1) to afford 22.5 mg (80.6%) pure product 57 as an off-white solid: mp 193-195°C. IR (KBr) 3040, 2920 (b r), 1740, 1665 cm '1; NMR

(250 MHz, CDCI3 ) 6 0.99 (s, 3H, C18), 1.23 (s, 3H, C19), 3.64-3.71

(m, 1H, C?H), 6.03 (br s, 1H, C 4 H), 6.27 (dd, J = 10.2, 1.8 Hz,

1H, C2H), 7.03 (d, J = 10.2 Hz, 1H, CjH), 7.10 (d, J - 8.3 Hz, 2H,

ArH), 7.62 (d, J = 8.3 Hz, 2H, ArH); exact mass calcd for

C2 5 H2 7 0 2SI m/e: 518.0778, found m/e: 518.0803. 75

3.2 BIOCHEMICAL METHODS

3.2.1. Studies on placental microsomes

Preparation of placental microsomes: Human term placentas were obtained from non-smoking women on delivery at OSU hospital, were transported on ice to the lab and processed immediately at 4°C. All procedures were carried out by a similar method of Brueggemeier et al. [94] to obtain microsomes. The placental tissue was cut free from fetal membrane, large blood vessels and connective tissues, and minced into small pieces, yielding weight about 280-300 g. The tissue was homogenized for two 30-second in a Waring blender with a homogeniza­ tion buffer which containing 0.25 M sucrose, 0.04 M nicotinamide and

0.05 M sodium phosphate pH 7.0 ( 2 parts tissue to 1 part buffer).

The homogenate was poured into twelve chilled Sorvall tubes and cen­ trifuged at 10,000 g for 30 min in a Sorvall RC 2-B centrifuge to remove mitochondria and cell debris. The supernatant was then centri­ fuged at 105,000 g for 1 h in a Beckman L5-50 B ultracentrifuge to obtain the microsomal pellet. The pellet was homogenized and resus­ pended in 0.1 M sodium phosphate buffer pH 7.0 and recentrifuged at

105,000 g for 1 h. This washing procedure was repeated. The result­ ing microsomal pellet was stored at -70°C until needed. The amount of microsomal protein were determined by Lowry protein micro-assay [165,166].

Kinetic studies were carried out under initial velocity conditions and the aromatase activity was measured by the H 2O assay developed by

Thompson and Siiteri [105] as modified by Reed and Ohno [141]. 76

3.2.1.1. Competitive inhibition studies for inhibitors 49-57.

Various concentrations of 4-androstene-3,17-dione (60-500 nM) and

a single concentration of inhibitor were preincubated with propylene

glycol (100 yL), NADP (1.8 mM), glucose- 6 -phosphate (2.85 mM) and

glucose- 6 -phosphate dehydrogenase (5 units) at 37°C for 5 min. Pla­

cental microsomes (0.07-0.12 mg) were homogenized and diluted the

total volume to 3.5 mL with 0.1 M of sodium phosphate buffer solution,

pH 7, then were added to the preincubated mixture. The solution was

incubated at 37°C for 15 min. The reaction was stopped by the addi­

tion of 5 mL of CHClg into the incubate. After vortexing for 20 sec,

the CHClg-quenched sample were centrifuged at 1250g for 10 min.

200 yL of the aqueous layer was mixed with 5 mL of scintillation cock-

q tail, and counted for H radioactivity by liquid scintillation spectrome­

try. Assays were run in duplicate and control samples containing no

inhibitors were run simultaneously. Blank samples were incubated with boiled microsomes. The kinetic data was analysed by Cleland's pro­ grams [167]. Protein concentrations were determined by the Lowry protein assay [165,166],

3.2.1.2. Time-dependent aromatase inactivation studies for inhibitors 54-57.

NADPH (0.2 mM, 0.5 mL) was added into an incubate mixture which contained various concentrations of inhibitor (20-200 nM), placental microsomal protein (0.2-0.3 mg/mL) and propylene glycol (100 yL) in

0.1 M of sodium phosphate buffer solution, pH 7 to a total volume of 10 mL. Aliquots (1.0 mL) were removed at various time peroids (0, 2, 5, 7, 10 min) and immediately diluted 1:10 with ice-cold buffer solu­

tion. The remaining aromatase activity was assayed by addition of the

diluted solution (3 mL) to a mixture of [ip- H] 4-androstene

-3,17-dione (200,000-300,000 dpm), propylene glycol (100 yL), NADP

(1.8 mM), glucose- 6 -phospliate (2.8 mM) and glucose- 6 -phosphate deh­

ydrogenase (5 units) in 0.1 M of sodium phosphate buffer, pH 7, to a

final volume of 3.6 mL, and incubated at 37°C for 30 min. The reac­

tion was terminated by addition of 5 mL of CHClg. The sample was

then vortexed for 20 sec, and centrifuged at 1250g for 10 min. Ali­ quots (1 mL) from the water layer were mixed with 4 mL of scintilla­ tion cocktail to form gels, and counted for radioactivity. Controls were run simultaneously without the inhibitor. The inactivation studies in the absence of NADPH were performed in the same manner as that with NADPH in the initial incubation. Protection studies were carried out analogous to the inactivation studies with unlabeled

4-androstene-3,17-dione (0.1-0.2 yM) and inhibitor (0.1 or 0.2 yM) included in the initial incubation.

3.2.1.3. Aromatase irreversible inactivation by 54, 55, and 57.

The procedure for inactivation studies in the presence of nucleo- philic trapping agents, such as cysteine (0.25 mM, 12.5 mL) or

p-mercaptoethanol (0.5 mM, 18.4 yL), was performed in the same way as above with the trapping agent included in the initial incubation. The enzyme pulse experiment was carried out with two groups (A and B) of samples consisting of a control (without inhibitor) and inhibitor 55 (200 nM), incubated with propylene glycol (100 yL), 78

NADPH (0.1 M) and placental microsomes (1-1.25 mg of protein) in 0.1

M of sodium phosphate buffer, pH 7.0, to make the total volume up to

8 mL at 37°C. Aliquots (1.0 mL) were removed from group A at time

0. 3, 6 , 9 min, respectively, and diluted 1 to 10 with buffer for assay

of the remaining activity. After 12 min of incubation, a second pulse

of fresh microsomes (1-1.25 mg of protein) was added to group B and the inhibitor concentration was readded 200 nM. Aliquots (1.0 mL) were removed at time periods 12, 15, 18 min respectively, diluted 1:10

with buffer immediately, and assayed for the remaining activity. Con­ trol samples contained no inhibitor in the initial incubations.

3.2.1.4. Preliminary stability studies for ^®I-IPTA metabolism.

To each 50-mL Erlenmeyer flask (x 6 ) was added 100 yL of propy­ lene glycol followed by a solution of 13.5 yL (0.217 nmol), 300,000 dpm of •^I-7a-IPTA in 95% ethyl alcohol, and 0.503 nmol (0.262 yg) of cold

7o-IPTA (29.8 yL of a 10 yg/mL solution) preincubated for 10 min at

37°C, with shaking in a Forma Scientific Model 2564 shaker bath.

Duplicate samples were performed under three conditions: 1. with an NADPH-generating system, consisting of NADP (4.7 mg)

and glucose 6 -phosphate (3.1 mg) in 1 mL of buffer, and 2.5

units of glucose- 6 -phosphate dehydrogenase (10 yL). 2. without an NADPH-generating system, 3.0 mL of resuspended microsomes (0.188 mg/mL) in 0.1 M sodium phosphate pH 7.0 was added respectively.

3. with an NADPH-generating system and 3 mL of boiled microsome. 79

The incubation was allowed to continue for 2 h. Ethyl acetate (each 4 mL) was added to stop the incubation, the mixtures were then extract­

ed with ethyl acetate (3x5 mL) three times, and the saved ethyl ace­ tate and water layers separated. The ethyl acetate layers were dried

with Na 2 SC>4 and evaporated under flow at 45-50°C water bath.

After using a Beckman Gamma-8000 spectrometer to count radioactivity,

each sample was dissolved in 200 pL of acetonitrile. HPLC separation

of each was accomplished on a reverse-phase column eluting with 65%

acetonitrile in water. Fractions (0.5 mL/min) were collected for 40

min, and counted in a Beckman Gamma 8000 spectrometer. The amount 1 oc of I-IPTA isolated was determined by Beckman Gamma 8000 count­

ing. Then, each net cpm's vs retention time (min) was plotted using Lotus 1-2-3.

3.2.1.5. Time course for 125I-IPTA metabolism.

To each 50-mL Erlenmeyer flask (x 8 ) was added 100 pL propylene glycol followed by a solution of 13.5 pL (0.217 nmol, 300,000 dpm) of 125i_7a_jpTA in 95% ethyl alcohol, and 0.503 nmol of (0.262 pg) cold

7a-IPTA (29.8 pL of a 10 pg/mL solution) preincubated for 10 min at

37°C, with shaking in a Forma Scientific Model 2564 shaker bath.

Duplicates were done with an NADPH-generating system, consisting of

NADP (4.7 mg) and glucose 6 -phosphate (3.1 mg) in 1 mL of buffer, and 2.5 units of glucose- 6 -phosphate dehydrogenase (10 pL). To each incubate was added 3 mL of microsomal suspension (0.19 mg/mL pro­ tein) . The flasks were allowed to incubate 0, 10, 20, 30, 45, 60, 90,

120 min at 37°C respectively. Ether (each 4 mL) was added to stop 80 the incubation, the mixtures were then extracted with ether (3x5

mL) three times, and saved the ether and water layers separated. The

ether layers were dried with Na 2 SO^ and evaporated under flow at 25°C water bath. After using a Beckman Gamma-8000 spectrometer

to count the residue, each sample was dissolved in 200 yL acetonitrile.

HPLC separation of each sample was accomplished on a reverse-phase

column eluting with 65% acetonitrile in water. Fractions (0.5 mL/min)

were collected for 40 min and counted by Beckman Gamma 8000 (pro­

gram user 2). The amount of * 2 ^I-IPTA isolated was determined by

Beckman Gamma 8000 counting. Then, each net cpm’s vs retention

time (min) was plotted using Lotus 1-2-3.

3.2.1.6. Various protein concentrations for 125I-IPTA metabolism.

Various concentrations of protein (0.3, 0.6, 1, 1.5, 2.0 and 3.0 mg/3 mL solution) were added into six incubate mixtures respectively.

Each incubate (3.6 mL) contained 200 nM (0.72 nmol) of substrate (including 0.217 nmol of radiolabeled IPTA), propylene glycol (100 yL) and an NADPH-generating system. They were incubated for 30 min and treated in the same manner as the above time course studies.

3.2.1.7. Various concentrations of IPTA at constant protein concentra­ tion metabolism.

Various concentrations of unlabeled IPTA (0.18, 0.36, 0.54, 0.72,

1.08 and 1.80 nmol) and radiolabeled IPTA (0.16 nmol) was added to make total concentrations of IPTA of 50, 100, 150, 200, 300, and 500 nM in the 3.6 mL of incubate containing the same amount of the above

NADPH-generating solution and propylene glycol. After preincubation 81 for 3 min, 3 mL of protein (0.5 mg/mL) was added into the mixtures, and incubated for 30 min. The remaining procedures were performed in the same manner as the above time course studies.

3.2.2. Studies in MCF-7 cell cultures. A solution of 225 yL of a 10 yCi/mL of ^^I-IPTA and a 728 yL of

10 yg/mL unlabeled IPTA was prepared. The mixture was then evapo­ rated under N 2 at 35-40°C. The residue was dissolved in 100 yL of 95% ethyl alcohol. Injection of 20 yL of the mixture to each of four O 150-cm flasks of MCF-7 mammary cells containing 20 mL of sterilized liquid medium was added and allowed to incubate 0, 12, 24, 48, 72 hr at 37°C respectively. The removed flasks and the remaining 20 yL of mixture (as a standard) were kept in the freezer. Subsquently, the culture media were thawed and poured into 50-mL plastic centrifuge tubes. The addition of 10 mL of ice-cold 30 % TCA (30% of trichloroa­ cetic acid in water) precipitated the proteins. Tubes were then capped, vortexed and centrifuged in the TJ- 6 R centrifuge at 2500 rpm's for 15 min. The supernatant layer is extracted three times by adding an equal volume of ethyl acetate. Combined ethyl acetate layers are dried over Na 2 SO^ and evaporated under ^ ^ g j to dryness. HPLC separation of each was accomplished on a reverse-phase column eluting with 65% acetonitrile in water. Fractions (0.5 mL/min) were collected for 40 min and counted in a Beckman Gamma 8000 spectrom eter (pro­ gram user 2). The amount of -^I-IPTA isolated was determined by

Beckman Gamma 8000 counting. Each net cpm's vs retention time (min) was plotted using Lotus 1-2-3. CHAPTER IV RESULTS AND DISCUSSION

4.1 Chemistry

The synthetic schemes of the 7o-substituted androst-4-ene and

l,4-diene-3,17-dione derivatives 48-57 are illustrated in schemes I-V (Chapter II).

4.1.1. Radiolabeled aromatase inhibitor

In an attempt to use the radioiodine as a tag in in v itr o and intact •JOC cells for stability and metabolism studies, I-7a-IPTA was prepared

IOC from corresponding triazine using [■lAOI]-sodium iodide. The last step 19^ incorporated the I to the triazine intermediate and was consistent with an ideal radiochemical synthetic method. In the preparation, the purity of formic acid affected the yield of the radiolabeled IPTA. The

125i _70_ip t a was purified by reverse phase HPLC using 65% of aceton- itile in water as mobile phase. The radiochemical purity of 125i_7a_ipTA was about 93.4%. The product was identified by cochro­ matographing on tic with authentic unlabeled IPTA.

4.1.2. Fluorescent aromatase inhibitors

In the syntheses of the dansylamine and dinitrobenzene (DNB) derivatives, pH of the reaction mixtures are critical. The optimal pH

- 82 - for dansylation is 9.5-10.5. If pH is larger than 10.5, the dansyl chloride is hydrolyzed rapidly by base. If pH is less than 9.5, the unreactive protonated form of amino group slows the labeling reaction relative to hydrolysis by water. Therefore, it is necessary to add a several-fold excess of reagent, and allow the unused reagent to hydro­ lyze and form the sulfonic acid (DNS-OH). Increasing the amount of dansyl chloride and increasing pH values in the reaction mixture favor the increased formation of dansyl derivatives. The dansyl derivatives can be detected on tic. Under UV irradiation, the derivatives show an intense yellow fluorescing spots, while DNS-OH has blue-green fluores- ence. However, compound 49 decomposed at room temperature, and should be kept sealed in the dark at 4°C. Dinitrofluorobenzene

(DNFB) is also a reagent of amino groups, and its derivatives are more stable than dansylamine derivatives. Sodium bicarbonate (0.9-1.0 mM) is used in the reaction in order to optimize the pH range of 8-9. The yields of DNB derivatives 50 and 51 were higher than of the dan­ sylamine derivative 49. Naphthylthio derivatives 52 and 53 were syn­ thesized by reaction of 4,6-androstadiene-3,17-dione and 13-/2P-naphthalenethiol under base catalysis. The retro-Michael elimi­ nation forming 4,6-androstadiene-3,17-dione may happen during work­ up procedures. Both afforded yields around 62-76%. Ultraviolet (UV) absorption and fluorescent emission wavelength of synthetic aromatase inhibitors are shown in Table 1. 84

Table 1. UV absorption and fluorescent emission wavelength of fluores­ cent aromatase inhibitors.

Inhibitors UV absorption Fluorescent emission

^ m a x ’ max'

49 229 515

50 242.5 365

51 221 & 234 342

52 224 394

53 224.5 362 85 4.1.3. Active-site directed irreversible inhibitors

Compound 55 and 56 were synthesized from l,4,6-androstatriene~3,17-dione with thioplienol and bromothiophenol, respectively. Their side reactions, such as 1-addition and

1,7-diaddition, resulted in yields of desired products lower than 30%.

Compound 57 was prepared from 7a-APTADD 24 via diazotization in 80% yield. The procedures were modified from those for the preparation of compound 47 [95].

4.2 Biochemistry

4.2.1. K| determinations of synthesized inhibitors

In order to determine the ability of synthesized compounds to inter­ act with aromatase, the potential inhibitors were evaluated in v itr o in enzyme kinetic studies using the human placental microsomes. The ini­ tial velocity studies of inhibitors were performed under the limiting enzyme conditions with varying substrate concentration versus a con­ stant inhibitor concentration. Aromatase activity was assayed by the q radiometric method which measured the amount of released as an index of estrogen formation.

Each compound evaluated exhibited competitive inhibition, as deter­ mined from the Lineweaver-Burk plots. The results were plotted as

1/velocity vs 1/[substrate], Figures 8-14. The apparent Kj's of these inhibitors were calculated by a weighted regression analysis computer program [167]. The kinetic parameters of these inhibitors are listed in Table 2. 86

Aromatase inhibition by 7a- 4' -(2 '\4 ”—dinitrophenyl)amino phenylthio A

60 55 50: h 4 5 £ 34 0 o 351 o 30

^ 20 \ l 5 ^ 10 5 0 ~1— '— I— '— I— 1—r- 0 6 8 10 12 14 16 18 i/M

Figure 8 . Double-reciprocal plots for aromatase inhibition by com­ pound 50. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.23 yM ( + ), or 0.46 yM (n). Velocity is expressed as ymol product/mg protein/min and 1/[S] values have units of yM . Each point represents the aver­ age of two determinations. Competitive inhibition is observed with an apparent K, of 86.7 ± 12.3 nM (K = 68.5 ± 11.1 nM; Vm = 147.9 ± 5.6 nmoi/mg/min). 87

Aromatase inhibition by 7a— 4' -(2 ”,4,,-dinitrophenyl)amino phenylthio ADD

0 2 4 6 8 10 12 14 16 18

Figure 9. Double-reciprocal plots for aromatase inhibition by com­ pound 51. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.23 yM ( + ), or 0.46 yM (□). Velocity is expressed as ymol product/mg protein/min and 1/[S] values have units of yM” . Each point represents the aver­ age of two determinations. The enzyme inhibition is observed with an apparent K, of 67.6 ± 17.6 nM (K = 58.5 ± 17.1 nM; Vm = 233.3 ± 18.8 nmol/mg/min). 88

Aromatase Inhibition by 7a-(1-beta-Naphthyl)thio A

120 110 10 0 : h 90 5 8 0 o 70 S GO fcq 50 £ 40 \ 30 ^ 2 0 10

0 2 4 6 8 10 12 14 16 18

Figure 10. Double-reciprocal plots for aromatase inhibition by com­ pound 52. Varying concentrations of androstenedione were Incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.3 yM ( + ), or 0.6 yM (n). Velocity is expressed as ymol product/mg protein/min and 1/[S] values have units of yM-1. Each point represents the average of two determinations. Competitive inhibition is observed with an apparent Kj of 24.1 ± 7.1 nM (!(,„= 67.9 ± 22.3 nM; Vmax= 230.7 ± 17.8 nmol/mg/min). 89

Aromatase Inhibition by 7a—(2' beta-Naphthyl)Thio A

90 80 70 60

10 A-

0 2 4 6 8 10 12 14 16 18

Figure 11. Double-reciprocal plots for aromatase inhibition by com­ pound 53. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.3 yM ( + ), or 0.6 yM (□). Velocity is expressed as ymol product/mg protein/min and 1/[SJ values have units of yM" . Each point represents the average of two determinations. Competitive inhibition is observed with an apparent K, of 38.9 ± 9.2 nM (K = 53.8 ± 14.5 nM; Vm = 203.6 ± 11.4 nmol/mg/min). 90

Aromatase inhibition by 7a-phenylthio ADD

190 180 170 160 ISO 140 130 h 120 & 110 O 100 o 90 Ed 80 V 70 \ 60 ^ 5 0 40 30 2 0 10 0 i— 1— i— <— i— ■— i— '— i— 1— i— ■— i— ■— i— 1— i— ■— r 0 2 4 6 8 10 12 14 16 18 iM

Figure 12. Double-reciprocal plots for aromatase inhibition by cop- mound 54. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.3 yM ( + ), or 0.6 yM (o). Velocity is expressed as ymol product/mg protein/min and 1/[S] values have units of yM . Each point represents the average of two determinations. The enzyme inhibition is observed with an apparent Kj of 20.0 ± 3.5 nM (1^= 53.3 ± 10.3 nM; Vmax= 217.1 ± 8 .6 nmol/mg/min). 91

Aromatase inhibition by 7 a -(4' - bromo)phenylthio ADD

100 * 90 BO 70

f a 6 0 § s o <43 ^ 40

io -

0 2 4 6 8 10 12 14 16 IB

Figure 13. Double-reciprocal plots for aromatase inhibition by com­ pound 55. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.3 yM (+), or 0.6 yM (n). Velocity is expressed as ymol product/mg protejn/min and 1/[S] values have units of yM . Each point represents the average of two determinations. The enzyme inhibition is observed with an apparent K. of 21.9 ± 5.1 nM (K = 74.8 ± 19.1 nM; Vmax= 236 •4 ± 15.0 nmol/mg/min). 92

Aromatase inhibition by 7a-JPTADD

.3 250-

200 '

100 ' 50'

0 2 4 6 8 10 12 14 16 18

' / [ s ]

Figure 14. Double-reciprocal plots for aromatase inhibition by com­ pound 57. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentrations of 0 yM (A), 0.1 yM ( + ), or 0.3 yM (□). Velocity is expressed as ymol product/mg protein/min and 1/[S] values have units of yM” . Each point represents the average of three determinations. The enzyme inhibition is observed with an apparent K, of 4.4 ± 1.4 nM (K = 63.4 ± 20.0 nM; Vm = 112.8 ±8.1 nmol/mg/min). 93

Table 2. The Km for androstenedione and K/s for the synthesized inhibitors.

Inhibitors Km (nM) Vvmax * Kj(nM)

50 68.5 ±11.1 147.9 ± 5.6 86.7 ±12.3

51 58.5 ±17.1 233.3 ±18.8 67.6 ±17.6

52 67.9 ±22.3 230.7 ±17.8 24.1 ± 7.1

53 53.8 ±14.5 203.6 ±11.4 38.9 ± 9.2

54 53.3 ±10.3 217.1 ± 8 .6 20.0 ± 3.5

55 74.8 ±19.1 236.4 ±15.0 21.9 ±5.1

57 63.4 ±20,0 1 1 2 .8 ± 8 .1 4.4 ± 1.4

* nmol/mg/min 94

4.2.2. Inactivation studies of mechanism-based inhibitors For a compound to be a potential enzyme-activated irreversible aro­ matase inhibitor, it has to meet the following requirements [169]:

1. The inhibitor should produce a pseudo-first order inactivation of the enzyme.

2. The inactivation requires the normal enzyme catalytic reaction.

3. A reversibly binding substrate should protect the enzyme from inactivation. 4. The inhibitor should not diffuse out of the active site while the inactivation takes place. Thus, the nucleophilic trapping agents

should not protect the enzyme from inactivation.

5. The inhibition ought to be irreversible.

Compounds 54, 55, and 57 were examined in inactivation and protection studies.

Protection studies were carried out by including the substrate,

4-androstene-3,17-dione in the incubation mixture containing enzyme, inhibitor and NADPH. The data were plotted as log % control activity against time. Based on the evaluation of the inactivation half-time vs l/[inhibitor] concentration, the first order inactivation rates for inhib­ itors 54, 55 and 57, can be determined by the method of Meloche using the following equation [168]:

1 ‘l/2= ~ ( T x Kinact > * T 95

The initial inactivation rates were measured by determining the time

required for a given inhibitor concentration to cause a 50% loss of

enzyme activity. The apparent Kjnacj. is determined from the slope and represents the amount of inhibitor which will result in a half-

maximal rate of enzyme inactivation. A plot of vs l/[11 should give a straight line. The y-intercept obtained from the plot repre­

sents T, the inactivation half time (T) at infinite inhibitor concentra­ tion. The rate of inactivation (kapp) is calculated as [In 2]/T.

Specifically, an inhibitor in a mechanism-based inactivation process is converted to a highly reactive intermediate at the active site. The inhibitor reacts with enzyme with a quick manner without diffusing out of the active site. Using the nucleophilic trapping agent, mercaptoe- thanol (ME) to test if the activated inhibitors diffused out of the active site.

In the presence of NADPH, varying concentrations of compound 54 showed time-dependent, first-order inactivation of human placental microsomes [Figure 15]. In the absence of NADPH, no inactivation was observed for compound 54 [Figure 16]. Thus, linear plots of compound 54 was observed revealing saturation kinetics [Figure 16].

A plot of tjy 2 vs l/l IJ is shown in Figure 17. Androstenedione at concentrations of 100 nM and 200 nM was incubated with microsomal aromatase, inhibitor (200 nM) and NADPH, and showed protecting the enzyme from inactivation [Figure 18]. Compound 54 in the presence of nucleophilic agent, ME, was shown increasing aromatase inactivation

[Figure 19]. Further verification whether ME stabilized the inhibitor- 96

enzyme complex to synergize the Inactivation was performed by com­

pound 54 which incubated in an identical condition of competitive

inhibition studies in the presence of ME (18.4 yL). The results [Fig­ ure 30] indicated that inhibitor 54 with mercaptoethanol can display

better aromatase inactivation, with an apparent Kj of 3.9 ± 0.9 nM (K]m= 18.4 ± 4.4 nM), which is fivefold lower than that of inhibitor only.

In the absence of NADPH, the inhibitor at concentration of 100 yM failed to produce an Inactivation of aromatase [Figure 21], while in the presence of NADPH, a first order inactivation was observed [Figure

20]. A plot of tj / 2 vs 1/(1) shown in Figure 22. Androstenedione at concentration 100 nM and 200 nM was incubated with microsomal aro­ matase, inhibitor (200 nM) and NADPH, and showed protecting the enzyme from inactivation [Figure 23]. In the presence of mercaptoe­ thanol, compound 55 was unable to show reasonable result due to unstable in the incubate mixture. It was subsequently examined by a second enzyme pulse experiment. If the rate of inactivation of the second pulse is faster than the first one, the activated inhibitor is diffusing from the active site and reentering the incubation solution to inactivate the enzyme. Compound 55 was observed with a half-time of inactivation (tj/ 2 ) 30.0 min [Figure 24], although the control sam­ ples showed degrade the enzyme much more quickly. A second enzyme pulse with equal amount was added into the incubation at time 12 min, the concentration of inhibitor was readjusted to 200 yM. The tjy 2 °f second enzyme pulse was 50.0 min [Figure 24], This indicated the 97

compound was irreversibly bound to the enzyme upon inactivation before diffusing from active site.

Compound 57 in the presence of NADPH exhibited time-dependent, first-order inactivation of human placental microsomes [Figure 25]. The inactivation observed for the incubations containing NADPH were much greater than the observed without NADPH for compound 57 [Fig­ ure 26]. A plot of tjy 2 against 1/[I] is shown in Figure 27. Andros- tenedione at concentration of 100 nM was incubated with microsomal aromatase, inhibitor (100 nM) and NADPH, it showed protecting the enzyme from inactivation [Figure 28]. Compound 57 exhibited no pro­ tection in the presence of ME (0.5 mM) [Figure 29].

Summary;

1. Enzyme inactivations by these compounds are dependent on enzyme catalysis.

2. Androstenedione decreases the inactivations produced by the inhibitors, and the half-life of enzyme activity was lengthened when increasing the concentration of substrate from 0 to 200 nM were used [Figures 18, 23 and 28]. These results suggest that

the inhibitors are interacting at the active site of aromatase. The

comparison of inactivation kinetics for those synthesized com­

pounds with other reported inhibitors was shown in Table 3.

3. To further establish the inactivation kinetics experiments with

differring additional concentration of substrate in the determina­

tion of remaining aromatase activity should be performed. 98

Aromatase Inactivation by HPTADD in the presence of NADPH

100 control — A— .

* * • • • • • 23 nil —A—. —€3— 30 nil

(H > 100nil ■H - A - o 200 nU

Ho £ co CJ

0 2 4 6 B 10 Time (min)

Figure 15. Inactivation of aromatase by compound 54 in the pres­ ence of NADPH. A time-dependent, first order inactivation of aroma­ tase activity is produced at inhibitor concentrations of 25 nM (o), 50 nM (o), 100 nM (A), and 200 nM (•). Control samples contained no inhibitor (*). Each point represents the average of three determina­ tions . 99

Aromatase Inactivation by HPTADD in the absence of NADPH

control

wwiHiini^ nm iiwi 200nU «•— • TT • 200 nU

& >

40 H-o o £c o o

Time (min)

Figure 16. Inactivation of aromatase by compound 54 in the absence of NADPH. In the absence of NADPH, the inhibitor at concentration of 200 nM (□) failed to produce an inactivation of aromatase while in the presence of NADPH, a first-order inactivation was observed (o ). Control samples contained no inhibitor (*). Each point represents the average of three determinations. 100

2 0 y

0 0.01 0.02 0.03 0.04 0.05 1 /[I]

Figure 17. Plot of the inactivation half-time (min) vs 1/[I] nM-* for compound 54. 101

Protection from HPTADD Inactivation of Aromatase by substrate

100 nU

200 nU

•200 nU

Time (m in)

Figure 18. Protection of compound 54 inactivation of aromatase by substrate. Androstenedione at concentration of 0 yM (A), 100 nM (o), 200 nM (□) was incubated with microsomal aromatase, inhibitor (200 nM) and NADPH, and protected the enzyme from inactivation. Control samples were run simultaneously and contained no inhibitor (*). Each point represents the average of three determinations. 102

Protection studies by HPTADD in the presence of nucleophilic trapping agent

control tool 200 nil —e - Cantrol + UC

200 nil + UE s - & - o

o ^4 o b a o o

o 2 4 6 a to Time (m in)

Figure 19. Inactivation of aromatase by compound 54 in the pres- cence of a nucleophilic trapping agent. A first-order inactivation of aromatae by Inhibitor 54 (200 nM) was observed in the presence (A) or absence (o) of mercaptoethanol (ME). Control sample with (□) and without (*) ME contained no inhibitor. Each points represents the average of three determinations. The value for 100% aromatase activity without ME (*) is 0.13 pmol mg min ” 1 and with ME (□) is 0.25 pmol mg min . 103

Aromatase Inactivation by BrPTADD in the presence of NADPH

control 100:

00 lOOnU - A - 200 nU

30

Time (m in)

Figure 20. Inactivation of aromatase by compound 55 in the pres­ ence of NADPH. A time-dependent, first order inactivation of aroma­ tase activity is produced at inhibitor concentrations of 25 nM (o), 50 nM (□), 100 nM (A), and 200 nM (•). Control samples contained no inhibitor (*). Each point represents the average of three determina­ tions . 104

Aromatase Inactivation by BrPTADD in the absence of NADPH

control - o - lOOnM - B - 100 nM

0 2 4 6 0 10 12 Time (min)

Figure 21. Inactivation of aromatase by compound 55 in the absence of NADPH. In the absence of NADPH, the inhibitor at concentration of 100 nM (a) failed to produce an inactivation of aromatase while in the presence of NADPH, a first-order inactivation was observed (o). Control samples contained no inhibitor (*). Each point represents the average of three determinations. ior>

B i

£ 30 • o 5

o 0.01 0.02 0.03 0.04 0.03 i/m

Figure 22. Plot of the inactivation half-time (min) vs 1/[I] nM-1 for compound 55. 10G

Protection of BrPTADD Inactivation of Aromatase by substrate

control —+— 200 nM 100 nM

— A -

4 6 8 Time (min)

Figure 23. Protection of compound 55 inactivation of aromatase by substrate. Androstenedione at concentration of 0 yM (A), 100 nM (a) and 200 nM (o) was incubated with microsomal aromatase, inhibitor (200 nM) and NADPH, and protected the enzyme from inactivation. Control samples were run simultaneously and contained no inhibitor (*). Each point represents the average of three determinations. 107

Aromatase Inactivation by BrPTADD Effect of Addition Enzyme A B 100 Control A

200 nM (A) — ■ © .- Control B

200 nM (B) ~ e —

03 0 0 12 19 10 Time (min)

Figure 24. Second enzyme pulse inactivation by compound 55. A first order inactivation of an initial pulse of enzyme by the inhibitor at 200 nM (o) was observed from 0 to 9 min (A). After 12 min of incu­ bation, a second pulse of enzyme was added to the inactivated samples and a first-order inactivation was produced from 12 to 18 min which paralleled that of the first (B). Control samples contained no inhibitor (*). Each point represents the average of two determinations. The val­ ue for 100% aromatase activity (A) is 0.5 pmol mg’ min" and in (B) is 0 .6 pmol mg mln . 10R

Aromatase Inactivation by IPTADD in the presence of NADPH

control ’"“( t a s - 25 nil

50 nU

75 nil — A - 100 nU

Time (m in)

Figure 25. Inactivation of aromatase by compound 57 in the pres­ ence of NADPH. A time-dependent, first order inactivation of aroma­ tase activity is produced at Inhibitor concentrations of 25 nM (o), 50 nM (o), 75 nM (A), and 100 nM (•). Control samples contained no inhibitor (*). Each point represents the average of three determina­ tions . 109

Aromatase Inactivation by IPTADD in the absence of NADPH

contro

50 nM

100 nM

4 6 8 Time (min)

Figure 26. Inactivation of aromatase by compound 57 in the absence of NADPH. In the absence of NADPII, the inhibitor at con­ centration of 50 nM (o), 100 nM (o) failed to produce an inactivation of aromatase. Control samples contained no inhibitor (*). Each point represents the average of three determinations. 110

£ 2 40 - I .

I-t—t

0 0.01 0.02 0.03 0.04 0.05 i/m

Figure 27. Plot of the inactivation half-time (min) vs 1/[I] nM“* for compound 57. I l l

Protection from IPTADD Inactivation of Aromatase by substrate

100 control

100 nU* ~ 0 ~ 100 nU

0 2 4 6 a 10 Time (min)

Figure 28. Protection of compound 57 inactivation of aromatase by substrate. Androstenedione at concentration of 0 yM (□), 100 nM (o) was Incubated with microsomal aromatase, inhibitor (100 nM) and NADPH, and protected the enzyme from inactivation. Control samples were run simultaneously and contained no inhibitor (*). Each point represents the average of three determinations. 112

Aromatase Inactivation by IPTADD in the presence of nucleophiiic trapping agent

toot [ __ -W ------—. - . g _____ Control E *- Control*

100 nM 60 -...Q- 100 nM* 5 -A-

rHo 30 d o cj K

10 0 2 4 6 8 10 Time (min)

Figure 29. Inactivation of aromatase by compound 57 in the pres- cence of a nucleophiiic trapping agent. A first-order inactivation of aromatae by inhibitor 57 (100 nM) was observed in the presence (A) or absence (□) of mercaptoethanol (ME). Control sample with (o) and without (*) ME contained no inhibitor. Each points represents the average of three determinations. The value for 100% aromatase activity without (*) is 0.25 pmol mg'^min ” 1 and with ME (o) is 0.34 pmol mg min . 113

Aromatase inhibition by 7a-phenylthio ADD

2 7 0 '

2 4 0 '

<2 / 0 ' E*»«

h 1 8 0 '

9 0 '

3 0 '

0 2 4 6 8 10 12 14 16 18 i/\?]

Figure 30. Double-reciprocal plots for aromatase inhibiton by com­ pound 55 in the presence of mercaptoethanol. Varying concentrations of androstenedione were incubated with enzyme at inhibitor concentra­ tions of 0 yM (A), 0.3 yM (+), or 0.6 yM (□). Velocity is expressed as nmol product/mg protein/min and 1/[S] values have units of yM . Each point represents the average of three determinations. The enzyme inhibition is observed with an apparent K« of 3.9 ± 0.9 nM (!*„,= 18.4 ± 4.4 nM; Vmax= .1.12.3 ± 2.9 nmol/mg/mln). 114

Table 3. Comparison of inactivation kinetics for irreversible inhibitors of aromatase.

Inhibitors K^nM) Kinact(^M)

54 20.0 ± 3.5 2.07

55 21.9 ± 5.1 1.26

57 4.4 ± 1.4 1.95

ATD 1 1 0 .0 ND

APTADD 9.9 0.16

4-OH-A 1 0 ,2 ND PED 4.5 ND

ND- not determined.

4.2.3. Microsomal metabolism of 125I-IPTA

7a-[4,-Iodo]phenylthio-4-androstene-3J 17-dione (7a-IPTA) is an effective inhibitor of aromatase with an Kj of 12 nM. The stability of the sulfide linkage and deiod(nation of the radiolabeled probe were

evaluated in the preliminary stability studies. After a 2h-incubation in

the presence of NADPH, O 2 and microsomes, no IPTA was left and

about 60% of net was eluted by reversed-phase column. In the presence of O 2 and microsomes, without NADPH no metabolites were observed, and around 10% deiod(nation was seen. In the presence of

NADPH, C>2 and boiled microsomes, approximately 20% deiodination was observed [Appendix A]. [125ij_70_ipta (200 nM, 300,000 dpm) was incubated with placental microsomes and an NADPH-generating system for various time periods

(0,10,20,30,45,60,90,120 min). After 30 min, deiodination, cleavage of the 7a-thio substituent and the presence of metabolite #1 and #2 were observed, as determined by HPLC [Figure 31, Appendix BJ. The free

1 ^ 1 and -^^I-iodothiophenol were measured at first 10 -min incubation.

The net %-^I formed of -^I-IPTA decreased from 95% to 40% within

30-min period. After 1 h-incubation, the amount of substrate dropped to 16%; and 2 h later, it was lower than 10%. The amount of metabo­ lite #1 was shown increasing from 1.9% to 35.5% within 1 hr, and then present saturation. The amount of metabolite #2 was observed increas­ ing from 0% to 35% within 2-h period and kept rising. It was apparent that 30 min was an appropriate time for studies on the metabolism of the radiolabeled compound. Therefore, the incubation of the various concentrations of protein and [ I]-7o-IPTA1 were also examined at 30 min respectively. Figure 32 and 33 showed the relationships of substrate and l^I-labeled metabolites. The plots of net % ^^1 vs HPLC retention time (min) at varying [protein] and [IPTA] were shown in Appendix C and D respectively. The more protein concentrations were added, the more different metabolites formed. The amount of metabolite #1 was up to 61.28% as the 110 yg/mL of protein was used, but dropped to 32.32% when increasing the concentration of protein up to 540 yg/mL. More­ over, increasing [protein] up to 360 yg/mL produced a particular metabolite #3, and di-^^I-iodotliiophenol. 116

Figure 33 showed the more substrate concentrations were employed, the more free iodides and iodothiophenol produced. Through 30-min

metabolism, the amount of metabolite # 1 showed increases as the con­ centration of substrate increased. However, while the concentration of

IPTA was up to 300 nM, the unmetabolized substrate left in the incu­

bate to be maximum; although further increases in the substrate con­

centration to 500 nM do not cause an increase of its amount.

4.2.4. MCF-7 cell cultures metabolism of * 2 ®I-IPTA.

[ 125I]-7a-IPTA was incubated for 0, 12, 24, 48, 72 hr in MCF-7

cell cultures. Similiar microsomal metabolism was observed in MCF-7

cell cultures after 24 hr incubation; a major metabolite (#1) same as above was analyzed (Appendix F). At 48 and 72 hr incubations, both

showed a significant peak of free *2^I (50-75%) in HPLC analysis, but nearly no metabolite #1 was present. The nromatase inhibitors may undergo retro-Michael elimination to form 4,G-androstadien-3,17-dione 1 oc which proved by the formation of I-iodothiophenol. By coelution the unlabeled compounds as tonatlve identification of the products on HPLC, the metabolite (#1) has the same retention time as

7a-(4,-bromo)phenylthio- 173-hydroxy-4-androsten-3-one. Since micro­ somal enzymes are involved in the metabolism of androgens and estro­ gens, the formation of aromatized estrogenic metabolites and hydroxy- lated metabolites of the steroid is expected [Figure 34]. Thus, IPTA is enzymatically converted to the 173-hydroxyl derivative by 173"hydroxysteroid dehydrogenase present in the crude microsomal preparation. 117

125 Time Course for Microsomal Metabolism of I—IPTA to n -

\ a =» free 1“ •0»- \ b = p—iodothiophenol \ c =» metabolite #1 - \ d d « 7—IPTA u T o n - * A e «■ metabolite #2 M K

9 40*- V c Z _ —------»-■ ...... - i

_ ® A x :

^ i 1 b 1 i 1------1------1------1------1------1------0 20 40 *0 M tOO !20 Incubation Time (min)

Figure 31. Time course for ^**I-7-IPTA metabolism in human pla­ cental microsomes. concentrations.

iue 2 Mcooa mtbls o *®-PA t ayn protein varying at *^®I-IPTA of metabolism Microsomal 32. Figure Net %125 I Form ed rti Dpnet irsml eaoim f I—IPTA of Metabolism Microsomal Dependent Protein 100 20 30% 70% 90% 10 40% 80% 80% 80% 0 % % % % .0 .0 .0 .0 .0 .0 .0 .0 .0 1.80 1.80 1.40 1.20 1.00 0.80 0.80 0.40 0.20 0.00 Poen (mg) [Protein] a = free I” I” free = a b = p-iodothiophenol p-iodothiophenol = b = 7-IPTA - 7 #1 = d etabolite m = c = meaoie #2 etabolite m = e 125 118 rt concentrations. trate iue 3 Mcooa mtbls o 15-PA t ayn subs­ varying at 125I-IPTA of metabolism Microsomal 33. Figure nM Product Formed 100- 200- 120- 100- 140- 2 -b iodothiophenol = b 220- - 240 100 - 100 200- 200 125 40- 00- 00 - O h - - — PA eaoim a vrig ubsrt concentration strate b su varying at I—IPTA metabolism * 1 * 1 = 7—IPTA #1 = d etabolite m = c “ l free = a e e = metabolite #2 #2 metabolite ---- 1 ------IT] (nM) [IPTA] X a ^=*=— 1 ----- > . . — i"' d c e 119 120

aromatase ANDROSTENEDIONE ESTRONE

oxido— oxido- P —450 reductase P —45 0 reductase

aromatase TESTOSTERONE ESTRADIOL

Figure 34. Microsomal enzymes involved in the metabolism of andro­ gens and estrogens. P-450 means cytochrome P-450. Oxidoreductase represents the non P-450 enzyme 17-ketosteroid 17P- oxidoreductase.

Metabolites #2 and #3 were eluted out of reverse-phase column after that of 7a-^^^1-IPTA. Thus, these labeled metabolites are more lipo­ philic than the substrate; and may be of 5a-/5(5-H, or 3a-/3fJ-OH estrogenic metabolites. Interestingly, the retention time of metabolite

#2 was same as one of the side products of IPTA synthesis. However, through mass and FTIR analysis, the side product did not exhibit any significant iodine fragments. The identification of metabolites is still underw ay.

Future studies on the metabolism of lp-^H-BrPTA which made by lp-^H-4-androstene-3,17-dione with bromothiophenol, and 7a-^**I

-IPTADD made by modification of the reported method [95J will provide more evidence of the chemical nature of metabolites. CHAPTER V CONCLUDING REMARKS

7a-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase in v itr o . One of the potent competitive inhibitors in the series, 7o-(4,-iodo)phenylthio-4-androstene-3,17-dione (7a-IPTA) with an apparent Kj of 12 nM, was prepared as a A<:,t'I-radiolabeled1 probe. 1 9S Metabolism studies on human placental microsomes of I-7a-IPTA dem­ onstrated that deiodination, cleavage of the 7ot-thio substituent and metabolites #1, #2 and #3, and di-iodothiophenol are produced. MCF-7 cells metabolism of IPTA showed the metabolite #1 and #2 were the major metabolites. Comparing the retention time and by coelution, metabolite #1 was shown to be identical to 7a-(4'-iodo) phenylthio-17$-hydroxy-4-androsten-3-one. IPTA is enzymatically con­ verted to the 17p-hydroxyl derivative by 17p-hydroxysteroid dehyd­ rogenase present in the crude microsomal preparation.

Synthotic analogs of 7o-substituted 4-ene-3,17-dione or 1,4-diene

-3,17-dione have been evaluated as inhibitors of human placental microsomal aromatase, with apparent Kj ranges of 4.4-86.7 nM at apparent ranges of 53.3-74.8 nM, and Vmax ranges of 112.8-236.4 nmol/mg protein/min. Naphthylthio derivatives are about 3-4 fold more

- 121 - 122 effective than dinitrophenyl derivatives. The 1,4-diene steroids have better aromatase inhibitory activity than 4-ene steroids. The mechanism-based inhibitors such as HPTADD and BrPTADD are as effective as lo-naphthylthio derivative. In particular, IPTADD is the most potent inhibitor in the series. This may be due to the orienta­

tion of the side chain toward the 7o-pocket of the active site of aroma­ tase and the electron-withdrawing effect of the iodine atom.

7o-Substituted l,4-androstadiene-3,17-diones 54, 55 and 57 pro­ duced time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no aromatase inactivation was observed in the absence of NADPH. Nucleophiiic trapping agents such as mercap- toethanol did not protect the enzyme from inactivation by inhibitors 55 and 57. In addition, the inhibitor 55 displayed better aromatase inacti­ vation with mercaptoethanol than without mercaptoethanol. Further­ more, in competitive inhibition studies of 55 in the presence of mercap­ toethanol, an apparent K- of 3.9 nM was observed, which is fivefold lower than that of inhibitor only (apparent of 18.4 nM).

The fluorescent, radiolabeled and mechanism-based inhibitors will be useful for topographical and functional studies, such as the exact bio­ chemical nature of the active sites of purified aromatase. Additionally,

o i n c examination of H- or I- radiolabeled probes in human placental microsomes or in MCF-7 cell cultures will provide further information on the metabolism of these 7a-substituted inhibitors. BIBLIOGRAPHY

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173. Hirsch, K.S., Jones, D., Lindstrom, T.D., Stamm, N.B., Sut­ ton, G.P., Taylor, H.M. and Weaver, D.E. (1987) S te ro id , 50, 201. APPENDIX A

Preliminary stability studies

for microsomal metabolism of ^®I-IPTA

HPLC Conditions

HPLC: Beckman Model 334 gradient Column: Reverse-phase, Altex ultrasphere-ODS (5 y, 4.6 mm x 15 cm) Mobile phase: 65% Acetonitrile in water

Eluting1 flow rate: 1 mL/min

Chart speed: 0.5 cm/min

Fractions: 0.5 mL/min

Detection: UV, 280 nM

- 133 - 1 OR Microsomal Metabolism of I—IPTA (std) to*

s o * - a - 7—IPTA

K M S -

20* -

10* -

OX r r f f Tfr r rTTr r r r r r i nyTWrr 0.0 10.0 2 3 .0 M .03.0 HPLC Retention (min)

Microsomal Metabolism o P I—IPTA (sample 1) 4 0 * - r »*- a a 7—IPTA M X - b = metabolite #1 c = free I — in 29X - CM

K 2 0 * - 40) i z 1 3 * -

1 0 * -

8 * -

OX - PPMfmiol yfin ii rwwrf 10 .0 13.0 3&.0 4 0 .0 HPLC Retention (min) 135

125 Microsomal Metabolism of I—IPTA (sample 2) •ra

90S - a - 7—IPTA 90S - b = free I “

4iV z JOS -

OS &0 29.0 3 0 4 40.0 HPLC Retention (mln)

Microsomal Metabolism of 195 I—IPTA (sample 3) 4 0 S

3 9 S - a = 7—IPTA 9 0 S - b = free I “ in

4ia z 1 9 S -

IC S -

5 S -

OS f»rrfHrTTfnfry>rT»TTrt|l 04 19.0 204 29.0 3 0 4 4 0 4 HPLC Retention (min) APPENDIX B

Time courses for microsomal metabolism of ^ I-IP T A .

HPLC Conditions

HPLC: Beckman Model 334 gradient Column: Reverse-phase, Altex ultrasphere-ODS

(5 y, 4.6 mm x 15 cm)

Mobile phase: 65% Acetonitrile in water

Eluting flow rate: 1 mL/min

Chart speed: 0.5 cm/min

Fractions: 0.5 mL/min

Detection: UV, 280 nM

- 136 - 137

Microsomal Metabolism of ^ 25 j—IPTA (std)

4 1 1

U S -

u s -

t s s - 10* - s* -

OS 0.0 3.0 18.0 30.0 3 3 .0 HPLC Retention (min)

Microsomal Metabolism of 125 I—IPTA (time 0)

sox

40X -

20X -

10X -

OX 03) U 10.0 I S.0 30 .0 3 3 .0 4 0.0 HPLC Retention (min) Net %125! Net %125 ­ u t 1 2 3 14S rn - I M ­ S O n - n SA Microsomal Metabolism I—IPTA of^2^ min) (20 Microsomal Metabolism of125 I—IPTA min) (10 a &0 ce c b o J Bo 10.0 HPLC Retention (min) PC tnton ( n) in (m n tio eten R HPLC a a a a d 0 MLO .0 0 1 10.0 0.0 20 - 7—IPTA - d 1“ free a a p-iodothiophenol b metabolite #2a e « metabolite « #1c d a p—iodothiophenol a b a metabolite a e c T|I1 l» W»i IIT1 l l i n im » lW llT»| IT1W | lT 00 U.0 0 A 3 30.0 7— « M 2 a free metabolite #1 IPTA 1 .0 0 3 “ § 2 "T* .0 0 4 138 139

125 Microsomal Metabolism of I—IPTA (30 min) m

14* - a =* free 1“ b » p—iodothiophenol c metabolite in I OS - d - 7—IPTA CM e «■ metabolite §2 K ■w 9

nw*i i^ wti i wmwl i mOOfi 1M 2 0 .0 2X0 300 U. 0 40.0 HPLC Retention (min)

125 Microsomal Metabolism of I—IPTA (45 min)

10X a *■ free I" b *■ p—iodothiophenol c metabolite #1 d » 7—IPTA in CM e » metabolite #2 t* at

IlllrflltlMMHlMJlllMhM'wMiIWi llr 10.0 iu 2 0 .0 2X0 30.0 U .0 40.0 HPLC Retention (min) 140

Microsomal Metabolism of 12^-IPTA (60 min) 13X

i ox - in CM K z

ox - w I M 103 30 0 300 300 100 400 HPLC Retention (min)

125 Microsomal Metabolism of I—IPTA (90 min)

I3X -

IOX -

ox- m CM T— K ox - 01 *" Z 4X -

3X -

ox-l r'rff'of", ROOw.TOOtfrftffWwi i 100 100 300 300 HPLC Retention (min) Net J5125 I irsml eaoim f I—IPTA min) (120 of Metabolism Microsomal too HPLC Retention (min) PfiOnfWiTTiinU i 3u 125 b =» p-iodothiophenol = free I" = a d « 7— « d I P T metabolite = c A #1 » metabolite » e #2 tt.0 jao mr** U.0 0 0 4 APPENDIX C

Microsomal metabolism of 125I-IPTA at varying protein concentrations.

HPLC Conditions

HPLC: Laboratory Data Control Chromatography

Accessory Module

Column; reverse-phase, Altex ultrasphere-ODS (5 y, 4.6 mm x 15 cm) Mobile phase: 65% Acetonitrile in water

Eluting flow rate: 1 mL/min Chart speed: 0.5 cm/min

Fractions: 0.5 mL/min

Detection: UV, 280 nM

- 142 - 143

125 Microsomal Metabolism of I—IPTA (Protein 0) 4 0

4 0

3 0

3 0 in CM

in - in ‘

n ^ I I H I I I f t | TTII tTI I Ifl rr»nntrini|iiTHfhmn u nifftn tiTii|i r>n»ri| i 04) M 1 M 13.0 SOLO no 1 U 30.1 HPLC Retention (min)

Microsomal Metabolism of ^ ‘’l—IPTA (53 ug protein/mL)

HPLC Retention (min) 144

Microsomal Metabolism of 1 1—IPTA (110 ug proteln/mL) 30S its - a - free 1 in b — p—iodothiophenol - e c *» metabolite #1 MS *■ d - 7—IPTA in” « e — metabolite #2 CM " k ,w : b

n - * 1 • 4S - a d 1 as ll 1 II n i J U I I oo &o too too aoo aoo j o o soo 4 0 0 HPLC Retention (min)

j 2 5 Microsomal Metabolism of I—IPTA (180 ug protein/mL) an in - c a « free 1~ - b - p-lodothlophenol - c - metabolite #1 d - 7-IPTA m las CM - « e » metabolite #2 b :

4S - a 1 d n - rw llll 1 1 1 1 1 4 1 oo so too 1M aoo aoo 300 300 400 HPLC Retention (min) 145

Microsomal Metabolism of^ I—IPTA (270 ug proteln/mL) 90S in - a - free I in - b - p-iodothiophenol m c — metabolite £l MX - d - 7—IPTA m in e — metabolite §2 CM K in - •M e n n 4X H

Wi w fw 111 m l fWwwtfJ ao BM 10L0 1&0 300 9M 300 3*J> 4 U HPLC Retention (min)

Microsomal Metabolism of^5]_jpTA (360 ug protein/mL) t+X

in - a — free I b - p-iodothtophenol in - c *■ metabolite #1 d - 7—IPTA m e - metabolite §2 CM f ® metabolite #3 K g - dl-iodothtophenol za

l i 9 ylllhl.ulllfcwrIII iTWifrw»lfllf»WWPfffMTtWf 1M 1MU> 30 3Ml0 UA 30.0 HPLC Retention (min) 125 Microsomal metabolism of I—IPTA (540 ug protein/mL)

a » free b * p-iodothiophenol c » metabolite #1 d = 7—IPTA e = metabolite #2 f - metabolite #3 g = di—iodothiophenol

200 200 300 300 400 HPLC Retention (min) APPENDIX D

Microsomal metabolism of 125I-IPTA at varying substrate concentrations

HPLC Conditions

HPLC: Laboratory Data Control Chromatography

Accessory Module

Column: reverse-phase, Altex ultrasphere-ODS

(5 y, 4.6 mm x 15 cm)

Mobile phase: 65% Acetonitrile in water

Eluting flow rate: 1 mL/min Chart speed: 0.5 cm/min Fractions: 0.5 mL/min

Detection: UV, 280 nM

- 147 - 148

Microsomal Metabolism of125|-IPTA (50 nM)

17* -

i n ­

is* - a - free I it* - b =» p—iodothiophenol m CM c « metabolite #1 M - d « 7—IPTA M ■*■> e =• metabolite §2 0 7* - z as -

n -

IS - W WWHl>HWMltfnwWi>hipLb, tn IW I I I I II aa u 1M tU 3&0 MS 3 U U S 400 HPLC Retention (min)

Microsomal Metabolism of125I-IPTA (100 nM) 13*

u s - a » free I M b » p—iodothiophenol m cm c a metabolite #1 d « 7—IPTA

4 i e a metabolite #2 • 2 «_

3*

I* dil M M 1U IBS 20.0 20.0 3 0 J uo 4010 HPLC Retention (min) Net %125l Net %125 11 - 13* S 8 1 * 4 1 10 12 2 ax- 0 - * a - * 4 * - * - * - * - * * -W 0.0

Microsomal Metabolism of ^2®I-IPTA nM) (150 i r s m l eaoim f 1—IPTAMicrosomal Metabolism nM) (200 of l u H i i ii i f l w 00 80 00 8 sao a s 284 20.0 18.0 10.0 u HPLC Retention (min) PC eeto (min) Retention HPLC 80 00 80 00 80 40.0 38.0 30.0 28.0 20.0 18.0 *•b p—iodothiophenol =*a free I = re I free = a *»b p—iodothiophenol - 7—IPTA - d = metabolite = c #1 *»c metabolite #1 - 7—IPTA - d = metabolite = §2e =*e metabolite 125 #2 80 40.0 38.0 149 Net %12SI Net %125 30* 10 13* 18* 14* 10 31* 31* 17* - 17* 11* 11* - 13* IS* 18* - 18* 0* a* 3* 4* •x 0* - * 3 8* - 1* 1* 7*- * * ------i 0.0 a.o rl o f l l i W I rrfll

■ a u I ■HJ n w 8.0 b &o L 1 b

I Microsomal Metabolism of12^l-IPTA (500 nM) irsml eaoim f2 —PA 30 nM) I—IPTA of*2^ (300 Metabolism Microsomal wmh c c d 10.0 1 M c metabolite = c #1 d IL I

PC eeto (min) Retention HPLC 1 HPLC Retention (min) e 18.0 80 00 3841 30.0 18.0

30.0

=»a free 1 = p—iodothiophenol = b = 7—IPTA = d =*e metabolite #2 fe 1 free a b a mtblt #1 metabolite a c - 7—IPTA - d e e 38.0 « « «* p-iodothiophenol eaoie #2 metabolite 3041 30.0 38.0 3841 40.0 4041 150 APPENDIX E

MCF-7 ceU metabolism of 125I-IPTA.

HPLC Conditions

HPLC: Laboratory Data Control Chromatography Accessory Module

Column: reverse-phase, Altex ultrasphere-ODS

(5 y, 4.G mm x 15 cm)

Mobile phase: 65% Acetonitrile In water

Eluting flow rate: 1 mL/min

Chart speed: 0.5 cm/min

Fractions: 0.5 mL/min

Detection: UV, 280 nM

- 151 - Net . -125,% I NetM . X>125 im - 70S - S 0 1 - * 0 3 - * 0 4 - 70* - * 0 0 - * 0 0 - s a t 0 * 0 ao ta u j C- Cl Mtbls o lIT (ie 0) (time l-IPTA of Metabolism Cell MCF-7 MCF-7 Cell Metabolism f125 o l-IPTA (12 hr) too 1 a HPLC Retention (min) I HPLC Retention (min) ...... b M 2 M 3 M 4M 3M 3M 3M 2M IM M 3M IM I H U M M il II il M M U I H 125 a metabolite b- #2 - 7—IPTA 7—IPTA 3M uo 400 152 153

MCF-7 Metabolism of 125 |_|PTA (24 hr) 43X - c — 40% - a «■ free 1 39%- b p—Iodothiophenol c — metabolite #1 30%- d - 7—IPTA CMirr 29%- e = metabolite §2 M 20%- 16 19%- am ■ 10%- 1 d 5% - 1 b 1 1 e

OjO so 100 ISO 1U 2S0 wo iu m o HPLC Retention (min)

MCF-7 Metabolism of25 1—IPTA (48 hr) 49% 40* a *= free I 39% b *» p-lodothlophenol c metabolite #1 30X d = 7—IPTA cT e =■ metabolite #2 g <=> dl-lodothlophenol ** 20X % z 15%

10X

5X o 00 SO 100 ISO 200 2S0 MO MO 400 HPLC Retention (min) N in Net % 70S 70S SOX - - sox - sox - sox ' •OX tox - tox X Hfrri OXH ao

L 0 14 00 304 4 0 3 4 0 2 30.0 104 104 U MCF-7 Metabolism of125 l-IPTA hr) (72 HPLC Retention (min) « p-iodothiophenol « b *»a I free 7—IPTA - c TIT* 40.0 154