New non-steroidal aromatase inhibitors: focus on R76713

R76713 is a novel triazole derivative which selectively blocks the cytochrome P450-dependent aromatase. In human placental microsomes, in FSH-stimulated rat and human granulosa cells and in human adipose stromal cells, 50% inhibition of estradiol biosynthesis was obtained at drug concentrations of 2-10 nM. In PMSG-injected female rats, R76713 lowered plasma estradiol levels by 50 and 90% 2 h after single oral doses of 0.005 and 0.05 mg/kg respectively. After 1 mg/kg, estradiol levels were suppressed by 90% for 16 h. In male cynomolgus monkeys, R76713 dose-dependently (0.03-10 micrograms/kg) inhibited peripheral aromatization with an ED50 of 0.13 microgram/kg without altering metabolic clearance rates and conversion ratios. In vitro R76713 had no effect on other P450-dependent steroidogenic enzymes up to 1000 nM at least. In rats, LHRH-, ACTH- and sodium-deprived diet stimulated plasma testosterone, corticosterone and aldosterone levels were not modified 2 h after single oral administrations of R76713 (up to 20 mg/kg). Furthermore, R76713 did not show any in vitro or in vivo estrogenic or antiestrogenic property. R76713 also induced regression of DMBA-induced mammary tumors after daily oral administration of 1 mg/kg b.i.d. In male volunteers (n = 4), a single oral dose of 5 and 10 mg lowered median plasma estradiol levels from 70 pM to the detection limit of the assay (40 pM) 4, 8 and 24 h after intake whereas no changes were detected after placebo administration. In premenopausal women (n = 15), receiving a single oral dose of 20 mg, median plasma estradiol levels decreased from 389 pM (before) to 168, 133 and 147 pM, 4, 8 and 24 h after intake whereas they remained above 420 pM after placebo (n = 7).     

Sexual differentiation of brain and behavior in quail and zebra finches: Studies with a new aromatase inhibitor, R76713

In many species of vertebrates, major sex differences affect reproductive behavior and endocrinology. Most of these differences do not result from a direct genomic action but develop following early exposure to a sexually differentiated endocrine milieu. In rodents, the female reproductive phenotype mostly develops in the absence of early steroid influence and male differentiation is imposed by the early action of testosterone, acting at least in part through its central conversion into estrogens or aromatization. This pattern of differentiation does not seem to be applicable to avian species. In Japanese quail (Coturnix japonica), injection of estrogens into male embryos causes a permanent loss of the capacity to display male-type copulatory behavior when exposed to testosterone in adulthood. Based on this experimental result, it was proposed that the male reproductive phenotype is “neutral” in birds (i.e. develops in the absence of endocrine influence) and that endogenous estradiol secreted by the ovary of the female embryo is responsible for the physiological demasculinization of females. This model could be recently confirmed. Females indeed display a higher level of circulating estrogens that males during the second part of their embryonic life. In addition, treatment of female embryos with the potent aromatase inhibitor, R76713 or racemic vorozole™ which suppresses the endogenous secretion of estrogens maintains in females the capacity to display the full range of male copulatory behaviors. The brain mechanisms that control this sexually differentiated behavior have not been identified so far but recent data suggest that they should primarily concern a sub-population of aromatase-immunoreactive neurons located in the lateral parts of the sexually dimorphic preoptic nucleus. The zebra finch (Taeniopygia guttata) exhibits a more complex, still partly unexplained, differentiation pattern. In this species, early treatment with exogenous estrogens produces a masculinization of singing behavior in females and a demasculinization of copulatory behavior in males. Since normal untreated males sing and copulate, while females never show these behaviors even when treated with testosterone, it is difficult to understand under which endocrine conditions these behaviors differentiate. In an attempt to resolve this paradox, we recently treated young zebra finches with R76713 in order to inhibit their endogenous estrogens secretion during ontogeny and we subsequently tested their behavior in adulthood. As expected, the aromatase inhibitor decreased the singing frequency in treated males but it did not affect the male-type copulatory behavior in females nor in males. In addition, the sexuality differentiated brain song control nuclei which are also masculinized in females by early treatment with estrogens, were not affected in either sex by the aromatase inhibitor. In conclusion, available data clearly show that sexual differentiation of reproductive behaviors in birds follows a pattern that is almost opposite to that of mammals. This difference may be related to the different mechanisms of sex determination in the two taxa. In quail, the ontogeny of behavioral differentiation is now well understood but we only have a very crude notion of the brain structures that are concerned. By contrast, in zebra finches, the brain mechanisms controlling the sexually differentiated singing behavior in adulthood have been well identified but we do not understand how these structures become sexually dimorphic during ontogeny.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Comparative effects of the aromatase inhibitor R76713 and of its enantiomers R83839 and R83842 on steroid biosynthesis in vitro and in vivo

R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H-benzotriazole) is a selective, non-steroidal aromatase inhibitor containing an asymmetric carbon atom. In this paper, we compare the effects of R76713 (racemate) with its enantiomers R83839 (the levo-isomer) and R83842 (the dextro-isomer) on steroid biosynthesis in rat cells in vitro and in the rat in vivo.

In rat granulosa cells, aromatase activity was inhibited by 50% at concentrations of 0.93 nM of R76713, 240 nM of R83839 and 0.44 nM of R83842, revealing a 545-fold difference in activity between both enantiomers.

Up to 1 μM, none of the compounds had any effect on steroid production in primary cultures of rat testicular cells. Above this concentration all three compounds showed a similar slight inhibition of androgen synthesis with a concomitant increase in the precursor progestins, indicative for some effect on the 17-hydroxylase/17,20-lyase enzyme. In rat adrenal cells none of the compounds showed any effect on corticosterone synthesis. At concentrations above 1 μM there was an increase in the levels of 11-deoxycorticosterone pointing towards an inhibition of the 11-hydroxylase enzyme. This increase was more pronounced for R83839 than for R76713 and R83842.

In vivo, in PMSG-primed rats, R83842 reduced plasma estradiol by 50%, 2 h after oral administration of 0.0034 mg/kg, whereas 0.011 mg/kg of R76713 and 0.25 mg/kg of R83839 were needed to obtain the same result.

Oral administration of up to 20 mg/kg of the compounds did not significantly affect plasma levels of adrenal steroids in LHRH/ACTH-injected rats. Plasma testosterone was lowered at 10 and 20 mg/kg of R83842 and at the highest dose (20 mg/kg) of R76713 and R83839.

In conclusion, the present study shows that the aromatase inhibitory activity of R76713 resides almost exclusively in its dextro-isomer R83842. R83842 exhibits a specificity for aromatase as compared to other enzymes involved in steroid biosynthesis of at least a 1000-fold in vitro as well as in vivo. This confirms the extreme selectivity previously found for the racemate.     

Exemestane,a new steroidal aromatase inhibitor of clinical relevance

Breast cancer is the leading cause of death among women and the contribution of circulating oestrogens to the growth of some mammary tumours has been recognized. Consequently, suppression of oestrogen action by inhibition of their biosynthesis at the androstenedione-oestrone aromatization step, by means of selective inhibitors of the enzyme aromatase, has become an effective therapeutic option for the treatment of hormone-dependent breast cancer. Exemestane (6-methylenandrosta-1,4-diene-3,17-dione) is a novel steroidal irreversible aromatase inhibitor recently approved and introduced into the global market under the name Aromasin. The design, laboratory and viable syntheses of exemestane, starting from a variety of steroidal precursors, are presented and discussed. Data from biochemical and pharmacological studies as well as the clinical impact of the compound are briefly reviewed. The drug is an orally active and well-tolerated hormonal therapy for postmenopausal patients with advanced breast cancer that has become refractory to standard current hormonal therapies.     

ARIMIDEX: A new oral, once-a-day aromatase inhibitor

ARIMIDEX® is a potent and selective aromatase inhibitor undergoing evaluation as a treatment for postmenopausal women with advanced breast cancer. Studies examining the pharmacology of ARIMIDEX were conducted in both animals and humans. In animals, ARIMIDEX elicits maximal aromatase suppressive activity at a dose of approx. 0.1 mg/kg, does not alter adrenal steroid hormone biosynthesis, and at a dose of 1 mg/kg, has no other pharmacologic effects other than aromatase inhibition. In this overview, the pharmacodynamic, pharmacokinetic, and safety profiles of single and multiple daily doses of ARIMIDEX are reported in humans. Daily doses of 1–10 mg of ARIMIDEX suppressed estradiol levels to the maximum degree measurable using sensitive estrogen assays. ARIMIDEX had no clinically significant effects on the response of cortisol and aldosterone to ACTH stimulation. Absorption of ARIMIDEX was rapid, with maximum plasma concentrations occurring within 2 h after oral administration. Plasma concentrations of ARIMIDEX rose with increasing doses of the drug. The elimination half-life of ARIMIDEX in humans ranged from 30 to 60 h. Consistent with the long plasma half-life, steady state plasma concentrations were 3–4-fold higher than plasma concentrations observed after single administration of 1, 3, 5, or 10 mg doses. Long term treatment of breast cancer patients with 10 mg/day has continued in 17 patients without an escape of estradiol suppression. Previously, these patients had received on average 2.6 systemic treatments for breast cancer and had significant metastatic disease. Three of the 17 patients continued ARIMIDEX treatment for 20 months and beyond. Given the number of previous treatments and tumor burden at the start of treatment, the response to ARIMIDEX treatment is encouraging. Phase III studies are now underway to assess the efficacy and safety of ARIMIDEX in the treatment of advanced breast cancer.     

The potent and selective inhibition of estrogen production by non-steroidal aromatase inhibitor, YM511

YM511 inhibited aromatase activities in microsomes from rat ovary and human placenta competitively (IC₅₀s: 0.4 and 0.12 nM, respectively). YM511 was about 3 times more potent than other aromatase inhibitors, such as CGS 16949A, CGS 20267 and R 76713. YM511 decreased the contents of estradiol stimulated by pregnant mare's serum gonadotropin in rat ovary with an ED₅₀ of 0.002 mg/kg, indicating that YM511 was equipotent to CGS 20267 and 3 times more potent than the other two inhibitors. Serum estradiol levels in female rats were reduced by YM511 at 0.01 mg/kg into the ovariectomized range. YM511 at 1 mg/kg for 2 weeks decreased rat uterine weight to levels comparable to ovariectomy, showing it was 10 times more potent than other inhibitors. But the maximal inhibitory effect of tamoxifen failed to reach ovariectomized level. YM511 slightly inhibited production of other steroid hormones in vitro and in vivo. The IC₅₀s of YM511 for aldosterone and cortisol production from adrenal cells were from 5500 to 9800 times higher than that for rat ovarian aromatase and 130,000 times higher for testosterone production, indicating that YM511 is a highly specific aromatase inhibitor. The data suggest that YM511 may be a potent and selective agent for suppressing estrogen-dependent action without affecting serum levels of other steroid hormones.     

Treatment of pubertal gynecomastia with the specific aromatase inhibitor anastrozole

Gynecomastia can be detected in up to 70% of boys during puberty and in about one third of adult males. An imbalance of estrogen to androgen tissue levels is believed to be the major reason for the development of gynecomastia; as a result most medical treatments so far have tried to lower the estrogen level. Five boys with pubertal gynecomastia and breast tenderness were treated for 6 months with the selective aromatase inhibitor anastrozole. Initial plasma levels of estradiol (E2), testosterone (T), androstenedione, dehydroepiandrosterone sulfate (DHEA-S) and gonadotropins were normal. DHEA-S showed a significant rise during treatment. T and androstenedione showed no significant change during treatment. E2 decreased with therapy, although to no statistically significant extent. The E2/T ratio decreased significantly during the treatment. Breast size decreased in 4 out of 5 patients, and in 1 of these 4 boys glandular breast tissue disappeared completely. The longer the duration of gynecomastia before anastrozole administration, the smaller was the reduction of breast size. Breast tenderness was resolved in all boys within 4 weeks. No adverse effects were recorded. Since the aim of medical treatment is the total disappearance of breast tissue, anastrozole, as previous aromatase inhibitors, is of limited effect. However, anastrozole seems to be of benefit for the treatment of tenderness in gynecomastia and for patients in whom surgery is particularly risky. However, as spontaneous disappearance of pubertal gynecomastia is common, further double-blinded, placebo-controlled trials are necessary before a definite conclusion can be drawn about the effectiveness and the side effects of this therapy.     

Exemestane (FCE 24304), a new steroidal aromatase inhibitor.

Exemestane (FCE 24304; 6-methylenandrosta-1,4-diene-3,17-dione) is a novel orally active irreversible aromatase inhibitor. Its in vitro and in vivo pharmacological properties have been compared to 4-hydroxyandrostenedione (4-OHA). In preincubation studies with human placental aromatase, exemestane, like 4-OHA, showed enzyme inactivating properties with a similar affinity (Ki 26 vs 29 nM) and a lower rate of inactivation (t1/2 13.9 vs 2.1 min). Conversely, when tested in pregnant mares' serum gonadotropin-treated rats, exemestane was more potent in reducing microsomal ovarian aromatase activity than 4-OHA, after both subcutaneous (ED50 1.8 vs 3.1 mg/kg) and oral dosing (ED50 3.7 vs greater than 100 mg/kg). No interference of exemestane on desmolase or 5 alpha-reductase activity was found. The compound did not show any relevant binding affinity to steroidal receptors, but slight binding to the androgen receptor (approximately 0.2% of dihydrotestosterone), like 4-OHA. In the first phase I trial, healthy postmenopausal volunteers were given single oral doses of exemestane, ranging from 0.5 to 800 mg, and plasma [estrone (E1), estradiol (E2) and estrone sulphate (E1S)] and urinary estrogens (E1 and E2) were measured up to 5-8 days. The minimal effective dose in decreasing estrogens was 5 mg. At 25 mg the maximal suppression was observed at day 3: plasma estrogens fell to 35 (E1), 39 (E2) and 28% (E1S), and urinary estrogens fell to 20 (E1) and 25% (E2) of basal values, these effects still persisting on day 5. No effects on plasma levels of cortisol, aldosterone, 17-hydroxyprogesterone, DHEAS, LH and FSH, and no significant adverse events were observed up to the highest tested dose of 800 mg exemestane.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

Endocrine and antitumoral effects of R76713 in rats

Some effects of daily oral administration of a new non-steroidal aromatase inhibitor on the pituitary-gonadal and adrenal functions were investigated in female rats. At doses of 1 mg/kg twice daily or higher, R 76713 lowered plasma estradiol levels to the range measured after ovariectomy Plasma progesterone levels and uterine weights decreased whilst LH levels increased but to a lesser extent than after ovariectomy. The other hormonal data show that long-term administration of R 76 713 does not modify the gluco- and mineralocorticoid hormone levels even at the highest dose studied (20 mg/kg, 4 h after treatment). Furthermore, both ovariectomy and R 76 713 treatment (1 and 5 mg/kg twice a day) induced almost complete regression of 9,12-dimethyl-1,2-benzanthracene-induced mammary carcinoma in rats. The appearance of new tumors during the treatment period was completely inhibited by R 76 713 whilst multiplicity of the remaining tumors was dramatically reduced.     

Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride—iso—octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile—water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.     

Aromatase inhibition by R 76713: experimental and clinical pharmacology

R 76713 is a new non-steroidal compound which inhibits aromatase in vitro and in vivo with a potency of at least 1000-fold that of aminoglutethimide. In male cynomolgus monkeys peripheral conversion of labeled androstenedione to estrone is decreased by 85%, 4-5 h after a single intravenous dose of 0.003 mg/kg of R 76713, without altering steroid metabolic clearance rates. In rats fed a sodium-depleted diet for 3 weeks, plasma levels of aldosterone and plasma renin activity remain unchanged 2 h after a single oral dose of up to 20 mg/kg of R 76713. This confirms previous data on the selectivity of R 76713 for aromatase inhibition as compared to inhibition of other enzymes involved in steroid biosynthesis. In male volunteers, a single oral dose of 5 or 10 mg of R 76713 lowers median plasma estradiol levels from 70 pM to the detection limit of the assay (30 pM) 4 and 8 h after intake, whereas no important changes are detected after placebo administration. In 15 premenopausal female volunteers receiving a single oral dose of 20 mg of R 76713, mean plasma estradiol levels decrease from 415 pM (before) to 179, 149 and 185 pM respectively 4, 8 and 24 h after intake whereas they remain above 380 pM after placebo (n = 7).     

ORG 33201: A new highly selective orally active aromatase inhibitor

Org 33201 has been selected as a very potent aromatase inhibitor. The compound is an enantiomer of a SC₂H₅ substituted imidazoylethylphenalene. Org 33201 inhibited human aromatase activity for 50% at a concentration of 2.2 × 10⁹ mol/l. More than 200-fold higher concentrations were needed for the inhibition of other cytochrome P-450 enzymes. In vivo the compound was active in rats (ED₅₀ = 0.035 mg/kg) and dogs (1 mg/kg gave 70% inhibition) after oral administration. It can be concluded that Org 33201 is a potent and highly selective orally active aromatase inhibitor.     

Fadrozole hydrochloride, a new nontoxic aromatase inhibitor for the treatment of patients with metastatic breast cancer.

Eighty previously treated postmenopausal women with metastatic breast cancer were randomized to receive fadrozole (CGS 16 949A), a new aromatase inhibitor, 1 or 4 mg orally per day. Seventy eight patients were evaluable for toxicity and response. Only mild to moderate toxicity, namely hot flushes (28%), nausea and vomiting (13%), fatigue (8%) and loss of appetite (5%) occurred. Complete response was documented in 10% and partial response in 13% of patients with 45% having a no change status for at least 2 months. The median time to treatment failure is 4.1 months. The median survival is 23.7 months. The median survival is 23.7 months. The response and survival in patients with estrogen receptor positive and estrogen receptor unknown disease were not significantly different. Neither response nor survival was significantly different between the patients receiving 1 or 4 mg of fadrozole per day. Fadrozole is a well tolerated, effective second line treatment for women with metastatic breast cancer.     

Active core structure of terfestatin A, a new specific inhibitor of auxin signaling

The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an auxin-inducible gene. However, the mode of action of TrfA has not been elucidated. To identify the active core structure, 25 derivatives of TrfA were synthesized and their inhibitory activities against auxin-inducible gene expression were evaluated. The structure–activity relationships revealed the essential active core structure of TrfA, 3-butoxy-4-methylbiphenyl-2,6-diol, which will lead to the design of biotin-tagged active TrfA.     

Effects of the nonsteroidal inhibitor R76713 on testosterone-induced sexual behavior in the Japanese quail (Coturnix coturnix japonica)

A new triazole derivative, R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole), was recently shown to inhibit aromatase selectively without affecting other steroid-metabolizing enzymes and without interacting with estrogen, progestin, or androgen receptors. This compound was tested for its capacity to intefere with the induction of copulatory behavior by testosterone (T) in castrated Japanese quail (Coturnix coturnix japonica). In a first experiment, R76713 inhibited (range 0.01 to 1 mg/kg) the activation of sexual behavior by T silastic implants and hypothalamic aromatase activity in castrated male quail in a dose-dependent manner. The 5 alpha- and 5 beta- reductases of T were not systematically affected. Stereotaxic implantation of R76713 in the medial preoptic area similarly blocked the behavior activated by systemic treatment with T, demonstrating that central aromatization of androgen is implicated in the activation of behavior. These inhibiting effects of R76713 on behavior were observed when implants were placed in the medial part of the nucleus preopticus medialis, confirming the implication of this brain area in the control of male copulatory behavior. Finally, the behavioral inhibition produced by R76713 could be reversed by simultaneous treatment with a dose of estradiol, which was not behaviorally effective by itself. This suggests that the behavioral deficit induced by the inhibitor was specifically due to the suppression of estrogen production. This also shows that the activation of copulatory behavior probably results from the interaction of androgens and estrogens at the brain level, as the two treatments separately providing these hormonal stimuli (T with the aromatase inhibitor on one hand and a low dose of estradiol on the other hand) had almost no behavioral effects but they synergized to activate copulation when given concurrently. These data confirm the critical role of preoptic aromatase in the activation of reproductive behavior and demonstrate that R76713 is a useful tool for the in vivo study of estrogen-dependent processes.     

A photoaffinity inhibitor of aromatase

Several 7-substituted 4-androstene-3, 17-diones are potent inhibitors of the biosynthesis of estrogens, with the most effective being 7-(4'-amino)phenylthio-4-androstene-3, 17-dione. An azide derivative of this 7-thioether compound has been prepared as a potential photoaffinity inhibitor. The enzyme kinetics of the azide analog were examined under both dark conditions and UV irradiation. In the dark, the azide was a very potent competitive inhibitor, with an apparent K_i of 1.3 nM. Under UV-irradiation, a time-dependent loss of aromatase activity was also observed. These studies indicate that the 7-substituent enhances the affinity of the steroidal analogs for the enzyme site.     

Maltosylamine, a specific inhibitor of beta-amylase.

β-Maltosylamine has been synthesized for the first time. It is an effective specific inhibitor of sweet potato β-amylase. This result extends the observation that 1-aminoglycosides are specific inhibitors of exoglycosidases which hydrolyze the corresponding glycose and also demonstrates that an enzyme acting with inversion, as well as those acting with retention of anomeric configuration, can be inhibited by glycosylamines. Maltosylamine, which acts as a reversible inhibitor of β-amylase, appears to be directed to the active site since it protects the essential sulfhydryl group of the enzyme from inactivation by N-ethylmaleimide.     

Covalent modification of aromatase by a radiolabeled irreversible inhibitor

7 alpha-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase. The microsomal enzyme complex has a greater affinity for several of these inhibitors than for the substrate androstenedione, with 7 alpha-(4'amino)phenylthio-4-androstene-3,17-dione being the most potent competitive inhibitor of the series. A potential affinity analog, the bromoacetamide derivative of the amino compound, has been synthesized in both unlabeled and 14C-labeled forms via a condensation of bromoacetic acid with the amino compound using DCC. Inactivation studies with the unlabeled inhibitor showed a time-dependent, first-order inactivation of aromatase enzymatic activity. Androstenedione, when incubated in varying concentrations with the irreversible inhibitor, provided protection from inactivation. Binding studies with radiolabeled inhibitor and microsomal aromatase preparations showed that irreversible binding had occurred. SDS-electrophoresis, followed by fluorography, identified four major microsomal proteins that were radiolabeled, with the protein band at 52,000 mol. wt predominating. Similar studies with a solubilized aromatase preparation decreased the amount of nonspecific binding. Thus, covalent bonds between the irreversible inhibitor and the aromatase cytochrome P450 molecule were formed.