Synthesis of 2,2-dimethyl-4-hydroxy-4-androstene-3,17-dione as an inhibitor of aromatase

2,2-Dimethyl-4-hydroxy-4-androstene-3,17-dione (4) has been synthesized and has been shown to be a powerful competitive inhibitor of aromatase (Ki = 11.4 nM). However, compound 4 does not cause time-dependent loss of enzyme activity, in contrast to the unmethylated parent compound, 4-OHA.     

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.     

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.     

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.     

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.     

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.     

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.     

Selection of 19-(ethyldithio)-androst-4-ene-3,17-dione (ORG 30958): a potent aromatase inhibitor in vivo.

19-Mercaptoandrost-4-ene-3,17-dione (ORG 30365) has been reported to be both a competitive and irreversible inhibitor of aromatase. In comparison to the known aromatase inhibitors 4-hydroxy-androst-4-ene-3,17-dione (4OH-AD) and 1-methyl-1,4-androstadiene-3,17-dione (SH 489), ORG 30365 was found to be, respectively, about 16 and 8 times more active in vitro using human placental microsomes. Although the activity profile of ORG 30365 is very attractive, this compound was not selected for further development because it has limited pharmaceutical stability, which is probably due to its free--SH group and therefore a number of more stable dithio-derivatives of ORG 30365 have been synthesized. These derivatives are considered to be converted to ORG 30365 before they become active. The in vivo aromatase inhibiting activity of these derivatives was determined in hypophysectomized rats treated with the estrogen precursor dehydroepiandrosterone sulphate (DHEAS) using inhibition of cornification of vaginal epithelium as parameter. The 19-(ethyldithio)-derivative (ORG 30958) appeared to be the most active inhibitor in this series being twice as active as ORG 30365 and about 8 times as active as inhibitors like 4OH-AD and SH 489. Besides inhibition of cornification of vaginal epithelium ORG 30958 decreased ovarian aromatase and plasma E2 levels in DHEAS-treated hypophysectomized rats. Plasma estradiol levels were also lowered by ORG 30958 in dogs which were treated with pregnant mare serum gonadotrophin in order to induce pro-estrus. ORG 30958 displayed much less than 1/400th of the androgenic activity of testosterone propionate in immature castrated rats and appeared to be devoid of estrogenic and anti-estrogenic activity in ovariectomized mature rats. A twice daily dose of 1.5 mg ORG 30958/kg postponed ovulation in mature female rats. In conclusion: ORG 30958 is a potent aromatase inhibitor in vivo. It probably becomes active after cleavage of the -S-S- bond yielding ORG 30365 a potent irreversible aromatase inhibitor. ORG 30958 does not display other hormonal activities making it an attractive candidate for the treatment of estrogen-dependent diseases.     

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.     

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3α,11β,17α,21-tetrahydroxy-5β-pregnane-20-one), allo-tetrahydrocortisol (allo-THF, 3α,11β,17α,21-tetrahydroxy-5α-pregnane-20-one) and tetrahydrocortisone (THE, 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione) in human plasma and urine is described. [1,2,3,4,5-²H₅]THF (THF-d₅), allo-[1,2,3,4,5-²H₅]THF (allo-THF-d₅) and [1,2,3,4,5-²H₅]THE (THE-d₅) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M−30]⁺) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.     

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 $\text{in vivo}$.     

Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400 ng/L, and the limit of detection was 14 ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.     

Chenodeoxycholic acid pool size determination from children using isotope dilution mass spectrometry

An isotope dilution mass spectrometry method is described for determining chenodeoxycholic acid pool size in children. The stable isotopically labeled tracer, (11,12-2H2) chenodeoxycholic acid, was administered orally to children, and the enrichment of bile was measured by selected ion monitoring gas chromatography mass spectrometry. The level of (11,12-2H2)chenodeoxycholic acid enrichment found in the patient samples was in the range of 0.5 to 5%. Data are presented illustrating the duplication of this method in two independent laboratories using standard quadrupole mass spectrometers. This procedure provides the clinician with a non-radioactive method for determining chenodeoxycholic acid pool size which is especially beneficial in studies involving children and pregnant women.     

Microbial conversion of solasodine to 1-androstene-3,17-dione (AD), a key intermediate for androgen synthesis

The spirosolane side chain of solasodine has been cleaved by cholesterol preinduced Mycobacterium sp. NRRL B-3805 to yield 1-androstene-3,17-dione (AD), a key intermediate for the synthesis of androgenic drugs. Conversion up to 34% has been recorded in shake flask culture after 192 h incubation period using dimethyl-formamide as carrier for solasodine addition.     

Antitumor effect of a specific aromatase inhibitor, 1-methyl-androsta-1,4-diene-3,17-dione (atamestane), in female rats bearing DMBA-induced mammary tumors

Atamestane is a potent competitive inhibitor of estrogen biosynthesis (aromatase) in several species, in vitro and in vivo, and has no endocrine side effects. In this study, the efficacy of atamestane in suppressing tumor growth was evaluated in comparing with that of a non-steroidal aromatase inhibitor (CGS 16949A, Ciba-Geigy) and ovariectomy. Female Sprague-Dawley rats bearing DMBA-tumors were treated s.c. once daily either with 30 or 150 mg/kg atamestane or with 0.1 or 0.5 mg/kg CGS 16949A for 4 weeks. At these biologically equivalent doses both aromatase inhibitors effectively inhibited tumor growth: at the end of treatment they caused a marked reduction in tumor size (up to 70%), while ovariectomy led to a complete remission of tumor growth. The histo-morphological pictures of the mammary tumors from treated animals were qualitatively almost similar to those of the control. In hosts, neither compound exerted any influence on the weight of genital organs (ovary, uterus and vagina), although the peripheral LH levels were significantly elevated by the higher dose of the aromatase inhibitors. This effect on LH levels is probably due to the elimination of the negative feed-back effect of estrogens on gonadotropin secretion (counter regulation). The serum prolactin levels were decreased by the aromatase inhibitors, indicating a diminution of estrogen levels in the treated animals. The present results clearly demonstrate that, in spite of the counter regulation, a pure aromatase inhibitor such as atamestane in sufficiently high doses is able to inhibit the growth of DMBA-induced mammary tumors in intact female rats.     

6-Methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversible aromatase inhibitor

FCE 24304 (6-methylenandrosta-1,4-diene-3,17-dione), a new irreversible aromatase inhibitor, has been identified and characterized in vitro and in vivo. The compound caused time-dependent inactivation of human placental aromatase with a t1/2 of 13.9 min and ki of 26 nM. When tested in PMSG-treated rats, ovarian aromatase activity was reduced 24 h after dosing by both the s.c. (ED50 1.8 mg/kg) and the oral (ED50 3.7 mg/kg) routes. No interference with 5 alpha-reductase activity nor any significant binding affinity for estrogen receptor was found. Slight binding affinity for the androgen receptor (RBA 0.2% of DHT) was observed.     

Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.