Synthesis and evaluation of analogues of 4-androstene-3,17-dione as aromatase inhibitors

Twenty-three synthetic analogues of 4-androstene-3,17-dione (androstenedione) have been evaluated as inhibitors of human placental microsomal aromatase enzyme. Among the most potent of these compounds were the 4-hydroxy, 6 alpha-fluoro, 6 beta-fluoro, and 4-fluoroandrostenediones and 4-fluoro-19-nor-4-androstene-3,17-dione. 4-Hydroxy-4-androstene-3,17-dione (4HAD) is an irreversible inhibitor of aromatase in vitro, whereas the four fluoro analogues are reversible inhibitors. 4HAD and 4-fluoro-4-androstene-3,17-dione caused significant regression of the nitrosomethylurea-induced mammary tumor in rats, but the other fluoro derivatives were inactive.     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.     

An improved radioimmunoassay for 4-androstene-3,17-dione

A radioimmunoassay using an antiserum produced against 6β-hydroxy-4-androstene-3,17-dione-6-succinyl-BSA conjugate is described which permits the rapid determination of 4-androstene-3,17-dione in multiple serum samples that are purified by column chromatography on neutral alumina. Steroids which reacted significantly with the antiserum were found to be 5α-androstane-3,17-dione, 5β-androstane-3,17-dione and 6β-hydroxy-4-androstene-3,17-dione. After column chromatography on alumina, however, the only significantly cross-reacting steroids were the 5α and 5β-androstane-3,17-diones, while cross-reactivity from other steroids was reduced to less than 1%.     

Effect of 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A) on the 5 alpha-reduction of androgens in the rat prostate

The present study reports the effects exerted by 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A), three steroids known to inhibit the aromatization of androgens to estrogens, on the in vitro metabolism of labelled testosterone (T), dihydrotestosterone (DHT) and androstenedione (delta-4-A) in the ventral prostate of adult male rats. It has been found that ATD, in the concentration tested, does not influence the conversion of labelled T into DHT, but decreases the formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol (diols). On the contrary, 4-OH-A and 4-Ac-A simultaneously decrease the formation of DHT and the diols. When T is used as the substrate, the presence in the medium of these three steroids enhances the formation of delta-4-A and of 5 alpha-androstanedione (5 alpha-A). ATD, but not 4-OH-A and 4-Ac-A inhibits the conversion of labelled DHT into the diols. The transformation of labelled delta-4-A into 5 alpha-A is not modified by either ATD or 4-OH-A, while 4-Ac-A exerts only a small inhibition. These results suggest that the three aromatase inhibitors tested are able to profoundly modify the metabolism of T in the ventral prostate of the rat. In particular: 4-OH-A and 4-Ac-A are able to inhibit the conversion of T into DHT; ATD is able to inhibit the conversion of DHT into the diols; ATD and 4-OH-A do not inhibit the process of 5 alpha-reduction of delta-4-A into 5 alpha-A, while 4-Ac-A exerts only a minor effect. It is suggested that in the ventral prostate of the rat there are two different 5 alpha-reductase isoenzymes, one sensitive to the inhibitory effect of the steroid tested and which is responsible for the conversion of T into the 5 alpha-reduced metabolites of the 17-OH series (DHT and the diols), and a second one, insensitive to the effects of the three steroids, which affects the conversion of delta-4-A into 5 alpha-A.     

Ovum transport in cyclic rats rendered hypo-oestrogenic by treatment with 4-hydroxy-4-androstene-3,17-dione

The effects of decreasing oestrogen secretion upon the rate of oocyte transport achieved by the administration of 4-hydroxy-4-androstene-3,17-dione were investigated in cyclic rats. Control animals examined during late pro-oestrus showed that the majority of oocytes had entered the uterus. In contrast, when inhibitor was administered from the afternoon of metoestrus or from late dioestrus through prooestrus, oviducal retention of oocytes was observed. When treatment was delayed until the morning of pro-oestrus, only uterine retention of oocytes was observed. The inhibitor decreased oestradiol concentrations in ovarian vein, while systemic testosterone values were increased. Treatment with exogenous oestradiol counteracted the effect of the inhibitor on ovum transport. The elevation of systemic testosterone concentrations by means of subdermal implants of testosterone failed to alter the normal pattern of ovum transport. These results demonstrate that normal oestrogen secretion during late metoestrus and dioestrus is required for the movement of oocytes from the oviduct to the uterus, whereas the preovulatory oestrogen surge is needed for the expulsion of ova from the uterus.     

Aromatase inhibition by 4-thiosubstituted-4-androstene-3,17-dione derivatives

The synthesis and evaluation of 4-thiosubstituted-4-androstenedione analogs as inhibitors of estrogen synthetase (aromatase) is described. All compounds were prepared by the addition of various thiol reagents to 4 beta,5 beta-epoxyandrostanedione. Inhibitory activity of synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H]4-androstene-3,17-dione as substrate. Synthesized compounds exhibiting high inhibitory activity were further evaluated under initial velocity conditions to determine apparent Ki values. Several compounds were effective competitive inhibitors, and have apparent Ki values ranging from 34 to 52 nM, with the apparent Km for androstenedione being 54 nM. The results of these studies demonstrate a tightly fitted enzyme pocket that can accommodate bulky substituents at the C-4 position of androstenedione not to exceed 4.3 A in width and 5.5 A in length.     

Reduction of 4-androstene-3,17-dione by growing cucumber plants

4-[4-¹⁴C]Androstene-3,17-dione was applied to the leaves of growing cucumber plants, Cucumis sativus, twice a week. Four weeks after the first     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

The influence of 4-hydroxy-4-androstene-3,17-dione on androgen metabolism and action in cultured human foreskin fibroblasts

4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5 alpha-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent Km of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent Ki of 2.7 nM. When 5 alpha-reductase activity was determined in the presence of 200 nM [3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 microM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, androstenedione. The influence of 4-OHA on 5 alpha-reductase activity and androgen receptor binding is minimal.     

Inactivation of aromatase in vitro by 4-hydroxy-4-androstene-3,17-dione and 4-acetoxy-4-androstene-3,17-dione and sustained effects in vivo

4-Hydroxy-4-androstene-3,17-dione (4-OHA) and 4-acetoxy-4-androstene-3,17-dione (4-AcA), in addition to being competitive inhibitors of aromatase, cause time-dependent, irreversible, loss of enzyme activity in both human placental and rat ovarian microsomes. $\text{In vivo}$, treatment of rats with 4-OHA also causes loss of ovarian aromatase activity. To test whether this loss of activity could have $\text{in vivo}$ significance, rats with hormone-dependent, mammary tumors were treated with 4-OHA on alternate weeks. Tumor regression continued to occur during the weeks without treatment. These findings suggest that inactivation of aromatase is important in the mechanism of action of the compounds $\text{in vivo}$.     

Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.     

Photohydration of testosterone and 4-androstene-3,17-dione in aqueous solution.

Irradiation of testosterone, 4-androstene-3,17-dione, or their "half-molecule" 4,4a,5,6,7,8-hexahydro-4a-methyl-2(3H)-naphthalenone in dilute aqueous solutions with ultraviolet light of 254 nm wavelength caused rapid addition of water across the olefinic bond with formation of 5,17 beta-dihydroxy-5 alpha-androstan-3-one, 5-hydroxy-5 alpha-androstane-3-17-dione, and 9-hydroxy-10-methyl-2-decalone, respectively. Time-lapse spectrometry in the ultraviolet region showed that the photohydration of the androgenic steroids was extremely efficient and virtually free of the side reactions. Preparative photolytic reactions carried out in water-methanol solutions allowed isolation and characterization of photoproducts.     

The effect of the aromatase inhibitor, 4-(phenylthio)-4-androstene-3,17-dione, on dimethylbenz(A)anthracene-induced rat mammary tumors

4-(Phenylthio)-4-androstene-3,17-dione (4-PTAD), a known inhibitor of human placental aromatase, was examined as a growth inhibitor of DMBA-induced rat mammary tumors. Subcutaneous administration of 4-PTAD at dose levels of 25 or 50 mg/kg/day caused a significant decrease in hormone-dependent tumor growth. Resumption of tumor growth occurred when either the administration of inhibitor was stopped or when inhibitor was coadministered with estradiol indicating that suppression of tumor growth was due to inhibition of estrogen biosynthesis. Additionally, plasma levels of estradiol were found to be lower in the animals treated with 4-PTAD. The major metabolite of 4-PTAD in vitro was identified as 4-(phenylthio)-4-androstene-17 beta-ol-3-one and was found to have 60% of the aromatase inhibitory activity of 4-PTAD.     

Synthesis of 19-oxygenated derivatives of the competitive inhibitor of aromatase, 5-androstene-4,17-dione.

19-Hydroxy- and 19-oxo-steroids 13 and 15, respectively, which are potential metabolites of the aromatase inhibitor 5-androstene-4,17-dione (3), were synthesized from 19-(tert-butyldimethylsilyloxy)androst-5-en-17-one (5) or 4beta-acetoxyandrost-5-en-17-one (16), respectively, through 5alpha-bromo-4beta-hydroxy-6beta,19-epoxyandrostan+ ++-17-one (10) as a key intermediate in each sequence. Reaction of the 19-siloxy compound 5 with Br2 gave 5alpha-bromo-6beta,19-epoxide 8, which was treated with N,N'-dimethylacetamide followed by reaction with N-bromoacetamide and 0.28 M HCIO4, to yield compound 10. On the other hand, treatment of the 4beta-acetoxy steroid 16 with N-bromoacetamide-HCI04 followed by oxidation with Pb (IV) acetic acid and I2 under irradiation and subsequent hydrolysis with K2CO3 also produced compound 10 and in better yield than that in the above synthesis. Jones oxidation of the 4beta-ol 10 followed by reductive debromination with zinc dust yielded the 19-ol 13 in low yield as well as 6beta,19-epoxy-4-one 12 as the major product. Furthermore, the major product 12 was converted into the 19-ol 13 in moderate yield from compound 12 through acetolysis and subsequent alkaline hydrolysis. The 19-oxo steroid 15 was obtained after treatment of compound 13 with pyridinium dichromate. Compounds 13 and 15 were analyzed as the methoxime-trimethylsilyl and methoxime-dimethylisopropylsilyl derivatives and the methoxime derivative, respectively, using gas chromatography-mass spectrometry.     

Conversion of 4-androstene-3,17-dione to testosterone by Pisum sativum

4-Androstene-3,17-dione-[4-¹⁴C] was applied to the leaves of growing pea plants, Pisum sativum. Within a week, 28% of the administered steroid was specifically reduced to testosterone. Part of the testosterone was present in esterified form, and 5α-androstane-3β,17β-diol was also identified as a metabolite, but neither epitestosterone nor estrogens were detected.     

Identification of N-arachidonylglycine, U18666A, and 4-androstene-3,17-dione as novel insulin Secretagogues

The glucose-induced insulin secretion is fine-tuned by numerous factors. To systematically identify insulinotropic factors, we optimized a primary beta-cell-based functional assay to monitor intracellular Ca2+ flux ([Ca2+]i). By this assay system, we successfully identified several insulinotropic peptides including cholecystokinin, gastrin releasing peptide, vasopressin, and oxytocin from tissue extracts. Screening of an assortment of chemical compounds, we determined three novel insulin secretagogues: N-arachidonylglycine (NAGly), 3beta-(2-diethylamino-ethoxy) androstenone hydrochloride (U18666A), and 4-androstene-3,17-dione. The NAGly increased [Ca2+]i through stimulation of the voltage-dependent Ca2+ channels and it was dependent on extracellular glucose level. On the other hand, U18666A and 4-androstene-3,17-dione increased [Ca2+]i in the presence of K ATP channel opener diazoxide while it was inhibited by the presence of Ca2+ channel blocker nitrendipine, suggesting that their effects are independent of K ATP channel. These unique features will be useful for further development of insulinotropic factors and drugs for treating type 2 diabetes.     

分枝杆菌Mycobacterium ZLP生产雄烯二酮（4-AD）的优化工艺

利用正交实验对分枝杆菌降解植物甾醇生产雄烯二酮（4-AD）的发酵培养基进行优化,同时进行温度和pH的优化。结果表明：培养基的最佳组成为质量分数2．0％葡萄糖、2．0％蛋白胨、0．7％MgSO4、0．8％K2HPO4;最适温度28℃、最适pH7．0,在此基础上发酵96h,4-AD的产率可达到62．15％。