6-Alkyl- and 6-arylandrost-4-ene-3,17-diones as aromatase inhibitors. Synthesis and structure-activity relationships.

Two series of 6 beta- and 6 alpha-substituted androst-4-ene-3,17-diones (5 and 6) were synthesized as aromatase inhibitors to gain insights of structure-activity relationships of varying substituents (methyl, ethyl, n-propyl, isopropyl, n-butyl, phenyl, benzyl, vinyl, and ethynyl) to the inhibitory activity. All of the inhibitors synthesized prevented human placental aromatase in a competitive manner. The inhibition activities of all the 6-n-alkylated steroids 5a-d and 6a-d (Ki = 1.4-12 nM) as well as the 6 beta-vinyl (5h), 6 alpha-benzyl (6g), and 6-methylene (10) compounds (Ki = 5.1, 10, and 4.9 nM, respectively) were very powerful whereas those of the 6-isopropyl (5e and 6e), 6-phenyl (5f and 6f), 6 beta-benzyl (5g), and 6 beta-ethynyl (5i) steroids, having a bulky or polar substituent, were relatively weak. The 6 beta-ethyl derivative 5b was the most potent inhibitor among those synthesized. Inhibitors 5a, 5f, 5h, 5i, 6b, and 10 did not cause a time-dependent inactivation of aromatase. The 6 beta-alkyl steroids essentially had higher affinity for the enzyme than the corresponding 6 alpha-isomers, whereas the opposite relation was observed in a series of the aryl steroids. These results along with molecular modeling with the PM3 method clearly indicate that aromatase has a hydrophobic binding pocket with a limited accessible volume in the active site in the region corresponding to the beta-side rather than the alpha-side of the C-6 position of the substrate.

Time-dependent inactivation of aromatase by 6-alkylandrosta-1,4-diene-3,17-diones. Effects of length and configuration of 6-alkyl group.

Series of 6alpha- and 6beta-alkylandrosta-1,4-diene-3,17-diones (3 and 4) were synthesized and evaluated as time-dependent inactivators of aromatase in human placental microsomes to gain insights to the structure-activity relationship of varying the 6-n-alkyl substituents (C-1--C-7) to the time-dependent inactivation activity. All of the inhibitors synthesized were powerful to good competitive inhibitors of aromatase, with apparent Ki's ranging from 4.7 to 54 nM. The 6beta-ethyl (4b) and 6beta-n-pentyl (4e) compounds were the most potent among them (Ki = 4.7 and 5.0 nM for 4b and 4e, respectively). In a series of the 6alpha-alkyl steroids, the inhibitors 3a-d having C-1--C-4 at the 6-position as well as the 6 alpha-n-heptyl (3g) compounds did not. In contrast, in the 6beta-alkyl steroid series, only the methyl analog 4a inactivated aromatase in a time-dependent manner, and the other alkyl steroids having more than two carbons at C-6beta did not. The inactivations were prevented by the substrate androstenedione, and no significant effects of L-cysteine on the inactivation were observed in each case. These results along with molecular modeling with the PM3 method indicate that both length and stereochemistry of a straight alkyl substituent at the C-6 position of androsta-1.4-diene-3,17-dione (3h) play an important role in the cause of a time-dependent inactivation of aromatase. No significant correlation between affinity for the enzyme and the inactivation ability in the 6-alkylandrosta-1,4-diene-3,17-diones is observed.

Synthesis of androst-5-en-7-ones and androsta-3,5-dien-7-ones and their related 7-deoxy analogs as conformational and catalytic probes for the active site of aromatase.

A series of androst-5-en-7-ones and androsta-3,5-dien-7-ones and their 7-deoxy derivatives, respectively, were synthesized and tested for their abilities to inhibit aromatase in human placental microsomes. All the steroids inhibited the enzyme in a competitive manner with Ki's ranging from 0.058 to 45 microM. The inhibitory activities of 17-oxo compounds were much more potent than those of the corresponding 17 beta-alcohols in each series. Steroids having an oxygen function (hydroxy or carbonyl) at C-19 were less potent inhibitors than the corresponding parent compounds having a 19-methyl group. 3,5-Dien-7-one 24 and its 19-hydroxy and 19-oxo derivatives (12 and 13) as well as 19-oxo-5-en-7-one 3 caused a time-dependent inactivation of aromatase only in the presence of NADPH in which the kinact values of 19-als 3 and 13 (0.143 and 0.189 min-1, respectively) were larger than those of the corresponding 19-methyl (23 and 24) and 19-hydroxy (1 and 12) steroids, respectively. 19-Nor-5-en-7-one 4 but not its 3,5-diene derivative 14 also inactivated the enzyme in a time-dependent manner. In contrast, 7-deoxy steroids 21 and 27, having a 19-methyl group, did not cause it. The inactivations were prevented by the substrate androstenedione, and no significant effects of L-cysteine on the inactivations were observed in each case. The results suggest that oxygenation at C-19 would be at least in part involved in the inactivations caused by the inhibitors 23 and 24. The conjugated enone structures should play a critical role in the inactivation sequences.

Synthesis and structure-activity relationships of 6-substituted androst-4-ene analogs as aromatase inhibitors.

Series of 6 alpha- and 6 beta-alkyl-substituted androst-4-en-17-ones (18 and 19) and their 17 beta-reduced derivatives (14 and 15)(alkyl: methyl, ethyl, n-propyl, n-pentyl, n-octyl) were synthesized and evaluated as aromatase inhibitors. Androst-4-en-17-ones having an oxygen function (hydroxy, acetoxy, or methoxy group) at C-6 alpha and C-6 beta (4 and 5) were also tested for their abilities to inhibit aromatase. All of the steroids studied inhibited human placental aromatase in a competitive manner. The inhibitory activities of the 6 alpha- and 6 beta-methyl-17-keto steroids 18a and 19a (Ki = 3.1 and 5.3 nM, respectively) as well as the 6 beta- alcohol 5a (Ki = 6.0 nM) were high, and their apparent Ki values were lower than that of the parent 6-unsubstituted 3-deoxy steroid 1 (Ki = 6.8 nM). Elongation of the methyl group decreased affinity for aromatase in relation to carbon number of the alkyl chain in each series, in which the 6 alpha- alkyl steroids 18 essentially had higher affinity for the enzyme than the corresponding 6 beta- isomers 19. The inhibitory activities of the 17 beta-hydroxy analogs 14 and 15 were less potent than those of the corresponding 17-keto steroids. The 6 alpha-ethyl compound 18b, the 6 alpha-oxygenated derivatives 4, and the 6 beta-acetoxy and 6 beta-methoxy analogs 5b and 5c were powerful inhibitors (Ki = 12-24 nM). The methyl steroids (18a and 19a) produced "type I" difference spectra upon interaction with aromatase. These results along with molecular modeling with the PM3 method suggest that compounds 18a and 19a may produce a thermodynamically stable enzyme-inhibitor complex in the hydrophobic binding pocket with a limited accessible volume. A carbonyl group at C-17 of the 6-alkylandrost-4-enes is essential for the tight binding. Moreover, the binding pocket also tolerates a polar hydroxy group at the 6 beta-position rather than at the 6 alpha-position.

Role of hydrophilic interaction in binding of hydroxylated 3-deoxy C(19) steroids to the active site of aromatase.

As part of our investigation into the structure-activity relationship of a novel class of aromatase inhibitors, C(19) steroids having no oxygen function at C-3, we tested aromatase inhibition activity of polar diol compounds 4,19-dihydroxyandrost-5-en-17-ones (25 and 27) and 6,19-dihydroxyandrost-4-en-17-ones (36 and 37). 4alpha,19-Diol 25 was synthesized from tert-butyldimethylsilyoxyandrost-4-ene steroid (9) through its OsO(4) oxidation, giving the 4alpha,5alpha-dihydroxy derivative 12, as a key reaction. Acetylation of 5beta,6alpha-dihydroxy-19-acetate 30 and its 5alpha,6beta-analogue 31 followed by dehydration with SOCl(2) and alkaline hydroxysis gave 6alpha,19-diol 36 and its 6beta-isomer 37, respectively. The stereochemistry of a hydroxy group at C-4 of compound 25 and that at C-6 of compounds 36 and 37 were determined on the basis of (1)H NMR spectroscopy in each case. 4beta,19-Diol 27, previously synthesized, was identified as an extremely powerful competitive inhibitor of aromatase (K(i) = 3.4 nM). In contrast, its 4alpha,19-dihydroxy isomer 25 and other series of diol compounds, 6,19-dihydroxy-4-en-17-one steroids, were moderate to poor competitive inhibitors (K(i) = 110-800 nM). Through this series of analyses, it was concluded that hydrophilic interaction of a 4beta,19-diol function with the active site of aromatase plays a critical role in the tight binding of 3-deoxy-5-ene steroids.

Synthesis and biochemical studies of 16- or 19-substituted androst-4-enes as aromatase inhibitors.

Androst-4-en-17-one derivatives [19-acetoxide 4, 16-bromides 14 and 15, 19,19-difluoride 18, and (19R,S)-19-acetylenic alcohol 25] and androst-4-en-17 beta-ol derivatives 3, 5, 10, 12, and 19 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All the 17-oxo steroids, except compound 25 and 17,19-diol 3 of this series, were effective competitive inhibitors with apparent Ki's ranging from 170 to 455 nM. 19,19-Difluoro steroid 18 and 19-acetylenic alcohol 25, a weak competitive inhibitor (Ki = 7.75 microM), caused a time-dependent, pseudo-first-order inactivation of aromatase activity with kinact's of 0.0213 and 0.1053 min-1 for compounds 18 and 25, respectively. NADPH and oxygen were required for the time-dependent inactivation, and the substrate, androst-4-ene-3,17-dione, prevented it, but a nucleophile, L-cysteine, did not in each case. The results strongly suggest that aromatase would attack the 19-carbon of steroids 18 and 25.

Metabolic aspects of the 1 beta-proton and the 19-methyl group of androst-4-ene-3,6,17-trione during aromatization by placental microsomes and inactivation of aromatase.

Aromatase catalyzes the conversion of androst-4-ene-3,17-dione to estrogen through sequential oxygenations at the 19-methyl group. Androst-4-ene-3,6,17-trione (AT) is a suicide substrate of aromatase, and the mechanism of inactivation of aromatase has been postulated to involve enzymatic oxygenation at the 19-position. [1 beta-3H,4-14C]-, [19-3H3,4-14C]-, and [1 beta-3H,19-14C]ATs, with high specific activities, were synthesized to study metabolic aspects and the inactivation mechanism. Incubation of the labeled AT with human placental microsomes yielded the 19-oxygenated derivatives, 19-hydroxy-AT and 19-oxo-AT, as well as the aromatization products, 6-oxoestrone and 6-oxoestradiol. A stereospecific 1 beta-proton elimination occurred during the aromatization of [1 beta-3H,4-14C]AT, and a marked tritium isotope effect was observed in the first hydroxylation at C-19 of [19-3H3,4-14C]AT. After incubation of the three double-labeled ATs, the solubilized proteins were subjected to SDS-PAGE and the 3H/14C ratio of the aromatase-bound metabolite in a 46-69 kDa fraction was analyzed. A marked decrease of the 3H/14C ratio of the metabolite was observed in the experiment using [19-3H3,4-14C]AT, compared with that of the labeled AT used, but there were no significant changes in the other experiments, indicating that the adduct retains the 1 beta-proton, the 19-carbon, and one of the three 19-methyl protons of AT. Thus, we conclude that further oxygenation of 19-oxo-AT produced by the two initial hydroxylations of AT at C-19 yields not only 6-oxoestrogen (by a mechanism similar to that involved in the aromatization of the natural substrate) but also a reactive electrophile that immediately binds to the active site in an irreversible manner, resulting in inactivation of aromatase.

Mechanism for aromatase inactivation by a suicide substrate, androst-4-ene-3,6,17-trione. The 4 beta, 5 beta-epoxy-19-oxo derivative as a reactive electrophile irreversibly binding to the active site.

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione to estrone through three sequential oxygenations of the 19-methyl group. Androst-4-ene-3,6,17-trione (1) is a suicide substrate of aromatase. The inactivation mechanism for steroid 1 has been studied to show that the inactivation reaction proceeds through the 19-oxo intermediate 3. To further clarify the mechanism, 4 beta, 5 beta-epoxyandrosta-3,6,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase, upon treatment of compound 3 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 inhibited human placental aromatase in a competitive manner (Ki = 30 microM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K1 = 88 microM, kinact = 0.071 min-1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K1 in either case (kinact: 0.133 or 0.091 min-1, in the presence of NADPH or BSA, respectively). The substrate androst-4-ene-3,17-dione protected the inactivation, but a nucleophile, L-cysteine, did not. When both the epoxide 6 and its 19-methyl analog 4 were subjected separately to reaction with N-acetyl-L-cysteine in the presence of NaHCO3, the 19-oxo steroid 6 disappeared from the reaction mixture more rapidly (T1/2 = 40 sec) than the 19-methyl analog 4 (T1/2 = 3.0 min). The results clearly indicate that the 4 beta, 5 beta-epoxy-19-oxo compound 6, which is possibly produced from 19-oxo-4-ene steroid 3 through the 19-hydroxy-19-hydroperoxide intermediate, is a reactive electrophile that irreversibly binds to the active site of aromatase.

6-Phenylaliphatic-substituted androst-4-ene-3,17-diones as aromatase inhibitors: structure-activity relationships.

Two series of 6alpha- and 6beta-phenylaliphatic-substituted androst-4-ene-3,17-diones (3 and 5) were synthesized as aromatase inhibitors to gain insights of structure-activity relationships of varying the n-alkyl moiety (C2 to C5) of the 6-phenylaliphatic substituents to the inhibitory activity. All of the inhibitors synthesized inhibited human placental aromatase in a competitive manner with apparent Ki values ranging from 16 to 115 nM. The 6alpha-phenethyl analog 3a and the 6beta-phenbutyl analog 5c (Ki=16 nM for the two inhibitors, respectively) were the most potent inhibitors in each series. The inhibitory activities of the 6beta-substituted steroids 5 except for the phenethyl compound 5a were more powerful than those of the corresponding 6alpha-isomers 3. Elongation of the alkyl moiety of the 6-substituent of the 6alpha-phenethyl steroid 3a up to five methylene units decreased affinity to aromatase in all cases, whereas the addition of two more methylene units to the 6-side chain of the 6beta-phenethyl analog 5a increased the affinity in relation to carbon number of the 6-substituent. These results along with molecular modelling with the PM3 method, would give a new information about the formation of thermodynamically stable enzyme-inhibitor complex in a hydrophobic binding pocket in the active site of aromatase.

Biological aromatization of delta4,6- and delta1,4,6-androgens and their 6-alkyl analogs, potent inhibitors of aromatase.

Enzymic aromatization of delta6- and delta1,6-derivatives of the natural substrate androstenedione with human placental aromatase was first studied using gas-chromatography-mass spectrometry. The two steroids were aromatized with apparent Km and Vmax values of 62 nM and 32 pmol/min/mg protein for the delta6-steroid and 167 nM and 10 pmol/min/mg protein for the delta1,6-steroid, respectively. We next explored the aromatization of a series of 6-alkyl (methyl, ethyl, n-propyl, and n-pentyl)-substituted delta6-androstenediones and their delta1,6-analogs, potent competitive inhibitors of aromatase, to gain insight into the relationships between the inhibitory activity of the 6-alkyl-C19 steroids and their ability to serve as a substrate of aromatase. In a series of the delta1,6-androstenediones, all the 6-alkyl steroids were more efficient substrates than the parent delta1,6-steroid in which the aromatization rates of the alkyl steroids were about 2-fold that of the parent steroid, in contrast, all of the 6-alkyl-substituted delta6-androstenediones were converted into the corresponding 6-alkyl-delta6-estrogens with the rates of less than about a half that of the parent steroid. These results indicate that the 6-alkyl function decreases the aromatization rate of the delta6-steroid but enhances that of the delta1,6-steroid. The relative apparent Km values for the C19 steroids obtained in this study are different from the relative Ki values obtained previously, indicating that a good inhibitor is not essentially a good substrate in the 6-alkyl-substituted delta6- and delta1,6-androstenedione series.

Inhibition study of human placental aromatase with 3-deoxy- and 6-oxo-steroids using [1 beta-3H]16 alpha-hydroxyandrostenedione as a substrate: a comparison to that using [1 beta-3H]androstenedione.

Inhibition of aromatase activity in human placental microsomes with 3-deoxy- (1, 2, 4, 6 and 8), 6-oxo- (3, 5 and 9), and 16 alpha-bromo- (7) steroids was studied using [1 beta-3H]16 alpha-hydroxyandrostenedione (16 alpha-OHAD) as a substrate and compared to that with the conventional substrate, [1 beta-3H]androstenedione. All the steroids inhibited both 16 alpha-OHAD and [1 beta-3H]androstenedione aromatization in a competitive manner. Based on Ki/Km ratios obtained in both series of experiments, their relative inhibitory activities in the series with 16 alpha-OHAD were not necessarily identical with those in the other series. The results would be important to understand the relationship of structure to function of aromatase in human placental microsomes.

4-Oxygenated androst-5-en-17-ones and their 7-oxo derivatives as aromatase inhibitors.

A series of androst-5-ene-4,7-diones and 4-oxygenated androst-5-enes were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids examined inhibited the enzyme in a competitive manner. The inhibitory activity of 4beta-hydroxy-5-ene steroid 7 (Ki = 25 nM) was much more powerful than that of the parent 5-ene steroid 11 (Ki = 78 nM), whereas 4beta-acetate 8 and 4-oxo analog 5 (Ki = 90 and 120 nM, respectively) were less potent than compound 11. This indicates that a hydrogen bonding between a hydroxy group of the 4beta-ol 7 and a residue of the active site of aromatase plays an important role in its binding. The 5-en-4-one steroid 5 did not cause a time-dependent inactivation of aromatase. In contrast, 5-ene-4,7-dione 13 as well as its 19-hydroxy and 19-oxo analogs 19 and 20 caused the time-dependent inactivation only in the presence of NADPH in air with the k(inact) values ranging from 0.057 to 0.192 min(-1), although their affinities for the enzyme were not high (Ki = 430-6300 nM). The inactivation was prevented by androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results suggest that oxygenation at C-19 would be at least in part involved in the inactivation caused by the inhibitor 13.

19-oxygenations of 3-deoxy androgens, potent competitive inhibitors of estrogen biosynthesis, with human placental aromatase.

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of 3-deoxy derivative of AD, compound 1, and its 5-ene isomer 4, which are potent competitive inhibitors of aromatase, to serve as a substrate, we studied their 19-oxygenation by human placental aromatase and the metabolites isolated were analyzed by gas chromatography-mass spectrometry. Inhibitors 1 and 4 were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 2 and 5 and 19-oxo derivatives 3 and 6 as well as the 17beta-reduced 19-hydroxy compounds 7 and 8. Kinetic studies indicated that the 5-ene steroid 4 was surprisingly a good substrate for the aromatase-catalyzing 19-oxygenation with the V(max) value of 45 pmol/min per mg prot which was approx. four times higher than that of the other. The relative K(m) value for steroids 1 and 4 obtained in this study is opposite from the relative K(i) value obtained previously in the inhibition study. The results reveal that there is a difference between a binding suitable for serving as an inhibitor of aromatase and a binding suitable for serving as a substrate of the enzyme in the 3-deoxy steroid series and the C-3 carbonyl group of AD is essential for a proper binding as a substrate to the active site of aromatase.

A time-dependent inactivation of aromatase by 19-oxygenated androst-4-ene-3,6,17-triones.

19-Hydroxyandrost-4-ene-3,6,17-trione (19-OHAT), its 19-oxo derivative (19-oxo AT) and 4 beta, 5 beta-epoxyandrostane-3,6,17-trione (5) were synthesized as possible intermediates involved in a mechanism-based inactivation of aromatase caused by androst-4-ene-3,6,17-trione (AT). These compounds, inhibited the enzyme in a competitive manner with Ki's of 0.61, 7.5 and 5.1 microM for 19-OHAT, 19-oxo AT, and compound 5. The two 19-oxygenated steroids showed a time-dependent, pseudo-first order rate of inactivation of aromatase with kinact's of 0.222 and 0.076 min-1 for 19-OHAT and 19-oxo AT, respectively, while compound 5 did not. NADPH and oxygen were required for the inactivation. Androstenedione blocked the inactivation, while L-cysteine partially prevented that of 19-OHAT and almost completely that of 19-oxo AT. When the 19-oxygenated steroids were separately subjected to reaction with N-acetyl-L-cysteine, these rapidly disappeared from the reaction mixture with t1/2 of 25 min (19-OHAT) and 20 s (19-oxo AT). This finding indicates that L-cysteine prevents inactivation by a chemical dependent elimination of the inhibitors from the incubate. These results suggest that the 19-oxygenation rather than the 4,5-epoxidation may be involved in the time-dependent inactivation by AT.

Determination of estrogen ring D glucuronides in pregnancy plasma by direct radioimmunoassay without hydrolysis.

A direct radioimmunoassay method using highly specific antisera without prior deconjugation has been developed for determination of estradiol 17-glucuronide and estriol 16-glucuronide in human plasma. Antisera were elicited in the rabbit by immunization with antigens in which the steroid haptens are linked to a carrier protein through the C-2 or C-4 position. After treatment with Rivanol the protein of albumin-free antiserum was covalently bound to a p-arylamine glass bead support through the cross-linkage with glutaraldehyde. A simple and reliable assay method employing the antibody-glass preparation was established and applied to measurement of estrogen ring D glucuronide concentration in peripheral plasma throughout normal pregnancy.

Studies directed toward a mechanistic evaluation of aromatase inhibition by androst-5-ene-7,17-dione. Time-dependent inactivation by the 19-nor and 5 beta, 6 beta-epoxy derivatives.

To gain further insight into the mechanism for inactivation of aromatase by androst-5-ene-7,17-dione (1) and its 19-nor analog 4, 10 beta-oxygenated steroids 5 and 6, delta 1(10)-steroid 7, and 19-oxo-5 beta,6 beta-epoxy compound 8 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids studied inhibited the enzyme in a competitive manner with apparent Ki values ranging from 1.1 to 35 microM. The delta 1(10)-compound 7 was the most potent inhibitor among them. All of the inhibitors caused a time-dependent inactivation of aromatase in the presence of NADPH in air with the kinact values ranging from 0.036 to 0.190 min-1. The substrate androstenedione protected the inactivation, but a nucleophile, L-cysteine, did not, in each case. In contrast, each inhibitor did not cause the time-dependent inactivation in the absence of NADPH. These results show that the 5 beta,6 beta-epoxide 8 and/or the dienone 7 are not a reactive electrophile involved in the irreversible binding to the active site of aromatase during the mechanism-based inactivation caused by the suicide substrates 1 and/or 4.

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.

Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.

Synthesis of 16 alpha-bromoacetoxy androgens and 17 beta-bromoacetylamino-4-androsten-3-one: potential affinity labels of human placental aromatase.

A novel synthesis of 16 alpha-hydroxy-4-androstene-3,17-dione (3), 16 alpha-hydroxy-4-androstene-3,6,17-trione (4), 17 beta-amino-5-androsten-3 beta-o1 (10) and 17 beta-amino-4-androsten-3-one (14) is described. 16 alpha-Bromoacetoxy-4-androstene-3,17-dione (5), 16 alpha-bromoacetoxy-4-androstene-3,6,17-trione (6) and 17 beta-bromoacetylamino-4-androsten-3-one (15) were synthesized as potentially selective irreversible inhibitors of androgen aromatases. 16 alpha-Bromo-4-androstene-3,17-dione (1) and 16 alpha-bromo-4-androstene-3,6,17-trione (2) were converted to compounds 3 and 4 in 80-90% yield by controlled stereospecific hydrolysis using sodium hydroxide in aqueous pyridine. Reductive amination of 3 beta-hydroxy-5-androsten-17-one and 3-methoxy-3,5-androstadien-17-one (11) using ammonium acetate and sodium cyanohydridoborate (NaBH3CN) and a subsequent treatment with acid gave the amines 10 and 14 respectively, as a salt. The corresponding 17-imino compounds 9 and 13 were also isolated from the reaction mixtures when methanol was used as a solvent for the reaction. The 16 alpha-hydroxyl compounds 3 and 4 and the 17 beta-amino compound 14 were converted to the corresponding bromoacetyl derivatives, 5, 6, and 15, with bromoacetic acid and N,N'-dicyclohexylcarbodiimide.

Controlled alkaline hydrolysis of steroidal alpha-bromoketones: new conditions and synthesis of 2 alpha-hydroxy-3-ones.

Controlled alkaline hydrolysis of 16 alpha-bromo-17-keto steroids 1, 5 and 7 with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu4NOH) and synthesis of 2 alpha-hydroxy-3-ones 11, 13 and 16 by the controlled hydrolysis of the corresponding 2 alpha-bromo-3-ones 9, 12 and 15 are described. Treatment of the bromoketones 1,5 and 7 with potassium carbonate in aqueous acetone or with n-Bu4NOH in aqueous dimethylformamide (DMF) gave 16 alpha-hydroxy-17-ones 3m 6 and 8 in 85-90% yield, respectively. 2 alpha-Hydroxy-3-ones 11, 13 and 16 were obtained by hydrolysis of the corresponding bromoketones 9, 12 and 15 in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2 alpha-bromoketone 9 and the 2 beta-bromo isomer 10 precedes the formation of the ketol 11 in which the true intermediate might be the 2 beta-isomer 10. However, rearranged androstane derivatives, 3 beta-hydroxy-2-one 18 and 20, were stereoselectively obtained by treatment of the bromoketones 12 and 15 with an excess amount of sodium hydroxide.