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Estriol-16α-glucosiduronate in the urine and serum of pregnant women was measured by a direct radioimmunoassay which was highly specific for this compound and did not require hydrolysis or chromatography. Urinary and serum estriol-16α-glucosiduronate increased as pregnancy progressed. Pregnant women with presumptive renal failure (toxemia of pregnancy, malignant hypertension, post renal transplantation, etc) showed low urinary levels of estriol-16α-glucosiduronate but normal serum levels. Their renal clearance of estriol-16α-glucosiduronate was calculated provisionally and compared to that of creatinine. The results suggest that some serum estriol is conjugated by the kidney and that the measurement of estriol-16α-glucosiduronate can be used as a clinical index of renal failure during pregnancy.

A series of 2-phenylaliphatic-substituted androsta-1,4-diene-3,17-diones (6) as well as their androstenedione derivatives (5) were synthesized as aromatase inhibitors to gain insights of structure–activity relationships of varying the alkyl moiety (C1 to C4) of the 2-phenylaliphatic substituents as well as introducing a methyl- or trifluoromethyl function to p-position of a phenethyl moiety to the inhibitory activity. The inhibitors examined showed a competitive type inhibition. The 2-phenpropylandrosta-1,4-diene 6c was the most powerful inhibitor (Ki: 16.1 nM) among them. Compounds 6c along with the phenethyl derivative 6b caused a time-dependent inactivation of aromatase (kinact: 0.0293 and 0.0454 min−1 for 6b and 6c, respectively). The inactivation was prevented by the substrate androstenedione, and no significant effect of l -cysteine on the inactivation was observed in each case. Molecular docking of the phenpropyl compound 6c to aromatase was conducted to demonstrate that the phenpropyl group orients to a hydrophobic binding pocket in the active site to result in the formation of thermodynamically stable enzyme–inhibitor complex.

We developed highly sensitive detection of testosterone (T) and 5α-dihydrotestosterone (DHT) by liquid chromatography–electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17β-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN–MeOH–H2O–formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3 mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New 6beta,19-bridged steroid analogs of androstenedione, 6beta,19-epithio- and 6beta,19-methano compounds 11 and 17, were synthesized starting from 19-hydroxyandrostenedione (6) and 19-formylandrost-5-ene-3beta,17beta-yl diacetate (12), respectively, as aromatase inhibitors. All of the compounds including known steroids 6beta,19-epoxyandrostenedione (4) and 6beta,19-cycloandrostenedione (5) tested were weak to poor competitive inhibitors of aromatase and, among them, 6beta,19-epoxy steroid 4 provided only moderate inhibition (K(i): 2.2 microM). These results show that the 6beta,19-bridged groups of the inhibitors interfere with binding in active site of aromatase.

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 microl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 microl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 +/- 1.1 pmol, which was in complete agreement with the observed X(0) = 15.0 +/- 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.

To gain the structure-activity relationship of Delta(1)-androstenediones (Delta(1)-ADs) as mechanism-based inactivator of aromatase, series of 2-alkyl- and 2-alkoxy-substituted Delta(1)-ADs (6 and 9) as well as 2-bromo-Delta(1)-AD (14) were synthesized and tested. All of the inhibitors examined blocked aromatase in human placental microsomes in a competitive manner. In a series of 2-alkyl-Delta(1)-ADs (6), n-hexyl compound 6f was the most powerful inhibitor with an apparent K(i) value of 31 nM. The inhibitory activities of 2-alkoxy steroids 9 decreased in relation to length of the alkyl chain up to n-hexyloxy group (K(i): 95 nM for methoxy 9a). All of the alkyl steroids 6 along with the alkoxy steroid 9, except for the ethyl and n-propyl compounds 6b and 6c, caused a time-dependent inactivation of aromatase. The inactivation rates (k(inact): 0.020-0.084 min(-1)) were comparable to that of the parent compound Delta(1)-AD. The inactivation was prevented by the substrate AD, and no significant effect of l-cysteine on the inactivation was observed in each case. The results indicate that the 2-hexyl compound 6f act as the most powerful mechanism-based inactivator of aromatase among Delta(1)-AD analogs and may be submitted to the preclinical study in estrogen-dependent breast cancer.

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.

A simultaneous method for quantification of four catechol estrogens, namely, 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, and 4-hydroxyestradiol by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed in a positive mode for analyzing a simple picolinyl derivatization. Reaction of these estrogens with picolinyl derivatization reagent resulted in the formation of di- or tri-picolinyl derivatives, and the derivatives exhibited single charged protonated molecules as base peaks in a positive mode in electrospray ionization/mass spectrometry (ESI-MS). Picolinyl derivatives of these catechol estrogens provided approximately 10 times greater ESI responses in LC-ESI-MS/MS (selected reaction monitoring) compared with those of underivatized molecules in a negative mode. The use of picolinyl derivatizatives, solid-phase extraction, and a 13C-labeled internal standard helped us in determining these catechol estrogens whose quantification limit was 20 pg for every sample. The present method was applied for quantifying these catechol estrogens in a biological sample.

Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

Simultaneous quantification method of three major metabolites of cortisone and cortisol, tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using a recently developed picolinyl derivatization. Conversion of each steroid into the corresponding picolinyl derivatives (1b, 2b or 3b) was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 3, 21-dipicolinyl derivatives. Positive ion-ESI mass spectra of the picolinyl derivatives were dominated by an appearance of [M+H](+) as base peaks in all cases. The picolinyl derivatives provided 15 to 80-fold higher ESI response in the LC-ESI-MS/MS (selected reaction monitoring: SRM) when compared to those of underivatized molecules in a positive LC-ESI mode. The use of the picolinyl ester, solid-phase extraction, and deuterium labeled internal standards enabled the concentrations of these metabolites in human urine to be determined simultaneously by LC-ESI-MS/MS (SRM) with a small sample volume of less than 1microl urine.

Treatment of aldosterone with 35% HCl in EtOH or in MeOH followed by the picolinyl derivatization gave the picolinyl derivative of aldosterone-ethyl ether, 8, or methyl ether, 9, as a single and well-shaped liquid chromatographic peak. Picolinyl derivatization of aldosterone produced 21-picolinyl derivative of 18,20-anhydro-hemiacetal derivatives, 6, with poor chromatographic peak with wide half-width. Further conversion of 6 to 8 required long reaction time (>4 h). Structure of each picolinyl or alkyl ether-picolinyl derivative, was carefully elucidated by nuclear magnetic resonance spectroscopy, electron ionization mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Enhancement of sensitivity (approximately 10-fold) in positive-LC-ESI-MS/MS of aldosterone was confirmed by the use of the alkyl ether-picolinyl derivatization when compared to the underivatized molecule.

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering estrogen level in patients with estrogen-dependent breast cancer. We prepared and tested series of the pyridine- and other heterocyclic ring-containing derivatives of 2- and 4-aminoestrones, estrone, and estradiol, compounds 5, 10, 12 and 15. The isonicotinyl derivatives of 2- and 4-aminoestrone, compounds 5c and 10c, were fairly potent competitive inhibitors of aromatase (K(i), 2.1+/-0.14 and 1.53+/-0.08 microM for 5c and 10c, respectively) and other compounds did not show, to a significant extent, the aromatase inhibitory activity. This result suggests that the isonicotinyl-substituted derivatives 5c and 10c would be accessible to the active site of aromatase.

To gain insight into the mechanistic features for aromatase inactivation by the typical suicide substrates, androsta-1,4-diene-3,17-dione (ADD, 1) and its 6-ene derivative 2, we synthesized 19-substituted (methyl and halogeno) ADD and 1,4,6-triene derivatives 8 and 10 along with 4,6-diene derivatives 9 and tested for their ability to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. 19-Methyl-substituted steroids were the most powerful competitive inhibitors of aromatase (K(i): 8.2-40 nM) in each series. Among the 19-substituted inhibitors examined, 19-chloro-ADD and its 6-ene derivatives (7b and 9b) inactivated aromatase in a time-dependent manner in the presence of NADPH in air while the other ones did not. The time-dependent inactivation was blocked by the substrate AD and required NADPH. Only the time-dependent inactivators 7b and 9b in series of 1,4-diene and 1,4,6-triene steroids as well as all of 4,6-diene steroids 9, except for the methyl compound 9a, served as a substrate for aromatase to yield estradiol and/or its 6-ene estradiol with lower conversion rates compared to the corresponding parent steroids 1,4-diene, 1,4,6-triene and 4,6-diene derivatives. The present findings strongly suggest that the aromatase reaction, 19-oxygenation, at least in part, would be involved in the time-dependent inactivation of aromatase by the suicide substrates 1 and 2, where the 19-substitutent would play a critical role in the aromatase reaction probably though steric and electronic reasons.

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.

Our rationale is based on the finding that estrone 3-sulfamate (EMATE, 2d), a typical estrone sulfatase (ES) inhibitor, can be hydrolyzed and the pharmacological effect of the free estrogen contributes to the bioactivity of the sulfamate. A number of 3-sulfamoylated derivatives of the good aromatase inhibitors, 2- and 4-halogeno (F, Cl, and Br) estrones and their estradiol analogs as well as 6beta-methyl and phenyl estrones, were synthesized and evaluated as inhibitors of ES in human placental microsomes in comparison with the lead compound EMATE. Among them, 2-chloro- and 2-bromoestrone 3-sulfamates (2b and 2c), along with their estradiol analogs 3b and 3c, were powerful competitive inhibitors with K(i)'s ranging between 4.0 and 11.3 nM (K(i) for EMATE, 73 nM). These four sulfamates as well as the 2-fluoro analogs 2a and 3a inactivated ES in a time-dependent manner more efficiently than EMATE, and 2-halogeno estrone sulfamates 2 also caused a concentration-dependent loss of ES activity. The results may be useful for developing a new class of drugs having a dual function, ES inhibition and aromatase inhibition, for the treatment of breast cancer.

Aromatase catalyzes the conversion of androstenedione (1a, AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the spatial nature of the AD binding (active) site of aromatase in relation to the catalytic function of the enzyme, we tested for the ability of 2alpha-substituted (halogeno, alkyl, hydroxy, and alkoxy) ADs (1b-1i) to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. All of the steroids inhibited the enzyme in a competitive manner with the apparent K(i)'s ranging from 45 to 1150 nM. 2alpha-Halogeno (F, Cl, and Br) and 2alpha-alkyl (CH3 and CH2CH3) steroids 1b-1f were powerful to good inhibitors (Ki=45-171 nM) whereas steroids 1g-1i, having an oxygen function (hydroxy or alkoxy) at C-2alpha, were poor inhibitors (Ki=670-1150 nM). Aromatization of some of the steroids with placental microsomes was analyzed by gas chromatography-mass spectrometry, indicating that the aromatization rate of the bromide 1d was about two-fold that of the natural substrate AD and that of 2alpha-methoxide 1h was similar to that of AD. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.

Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxidations of the 19-methyl group. 3-DeoxyAD ( 1 ) and its 5-ene isomer 4 are potent and good competitive aromatase inhibitors, which are converted by aromatase to the aldehyde derivatives 3 and 6 , respectively, through 19-hydroxy intermediates 2 and 5 , respectively. To study the deuterium isotope effect on the conversions of 19-ols 2 and 5 into the corresponding 19-als 3 and 6 , we initially synthesized [19,19- 2 H 2 ]19-ols 2 and 5 starting from the corresponding non-labeled 19-als 3 and 6 through NaB 2 H 4 reduction of the 19-aldehyde group, followed by oxidation with pyridinium dichromate, and a subsequent NaB 2 H 4 reduction. Approximately 1:1 mixtures of non-labeled ( d 0 ) and deuterated ( d 2 ) 19-ols 2 and 5 were separately incubated with human placental microsomes in the presence of NADPH under an air atmosphere, and deuterium contents of the recovered substrates and the 19-aldehyde products were determined by gas chromatography–mass spectrometry. In each experiment, the ratio of d 0 to d 2 of the recovered substrate along with that of d 0 to d 1 of the product were identical to the d 0 to d 2 ratio of the employed substrate irrespective of the incubation time, indicating that the 19-oxygenations of the 3-deoxy steroids 2 and 5 proceeded without a detectable isotope effect, as seen in the aromatization sequence of the natural substrate AD.

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.

To study the stereochemical aspects of the aromatase reaction of androst-4-en-17-one (1) and its 5-ene isomer 4, competitive inhibitors of aromatase, the [19S-(3)H]- and [19R-(3)H]-labeled 19-hydroxy derivatives 2 and 5, were synthesized through NaB(3)H(4) reduction of the corresponding 19-aldehydes 3 and 6 as a key reaction. The hitherto unknown stereochemistry of the NaB(3)H(4) reduction was established based on the deuterium-labeling experiments with NaB(2)H(4). A comparison of (1)H-NMR spectra of the NaB(2)H(4) reduction products of 19-als 3 and 6 with those of the respective authentic steroids revealed that the ratios of 19S-(2)H to 19R-(2)H were 90 : 10 for the 4-ene steroid 2 and 70 : 30 for the 5-ene isomer 5, respectively. Jones oxidation of the [19S-(2)H]19-ols, followed by the non-labeled NaBH(4) reduction, gave the corresponding [19R-(2)H]19-ols 2 and 5 (R-(2)H : S-(2)H=90 : 10 for steroid 2 and 70 : 30 for steroid 5). The stereoselectively (3)H-labeled compounds 2 and 5 were similarly obtained in these sequences.

To gain insight into the mechanism for irreversible inactivation of aromatase by 6beta-bromoandrostenedione (1), one of the earliest discovered suicide substrates, in relation to the catalytic function of the enzyme, the 2,2-dimethyl derivative of compound 1, steroid 4, and its 6alpha-isomer 5, as well as 2-methyl-1,4-diene steroid 8 and its 6alpha-bromide 10, were synthesized. All of the steroids inhibited aromatase activity in human placental microsomes with apparent K(i)'s ranging between 10 and 81 nM. The 2,2-dimethyl-6beta- and 6alpha-bromo steroids 4 and 5 were extremely powerful inhibitors (K(i): 14 and 10 nM, respectively), but these two did not cause a time-dependent inactivation of aromatase in the presence of NADPH; in contrast, the 2-methyl-1,4-diene steroids 8 and 10 caused time-dependent inactivation with apparent k(inact) of 0.035 and 0.071 min(-1), respectively, in a suicide manner. These results indicate that the 2,2-dimethyl function of the 6beta-bromide 4 would prevent the inactivation of aromatase caused by inhibitor 1 in a suicide manner, probably through steric activity, whereas the 2-methyl group of steroid 8 did not significantly affect the suicidal inactivation by the parent 1,4-diene steroid, a typical suicide substrate.

As part of our investigation into the structure–activity relationship of a novel class of aromatase inhibitors, two series of 3-deoxy androgens, androst-5-en-17-ones with a non-polar alkoxy ( 5 and 6 ), alkyl ( 20 − 22 ), or phenylalkyl ( 23 and 24 ) group at C-4β and 4-acyloxyandrost-4-en-17-ones ( 29 – 32 , and 34 ) were synthesized and evaluated. The 4β-alkyl and 4β-phenylalkyl compounds were obtained through reaction of 4α,5α-epoxy steroid ( 8 ) with RMgBr (R: alkyl and phenylalkyl) followed by dehydration of the 4β-substituted 5α-hydroxy products ( 15 − 19 ) with SOCl 2 as key reactions. Acylation of 4α,5α-diol ( 25 ) with (RCO) 2 O in pyridine and subsequent dehydration with SOCl 2 gave the 4-acyloxy steroids. All of the steroids studied, except for 4-acetoxy-19-ol ( 34 ) that was a non-competitive inhibitor of human placental aromatase, blocked aromatase activity in a competitive manner. 4-Benzoyloxy- and 4-acetoxy steroids ( 31 ) and ( 32 ) were the most powerful inhibitors of aromatase ( K i =70 and 60 nM, respectively). Elongation of an acetoxy group in a series of 4-acyloxy steroids or a methyl group in a series of 4β-alkyl steroids decreased affinity for aromatase principally in relation to carbon number of the acyl or alkyl function. The present findings are potentially useful for understanding the spatial and electronic nature of the binding site of aromatase as well as for developing effective aromatase inhibitors.

To gain insight into the catalytic function of aromatase, we studied aromatization of a series of 6α- and 6β-ether-substituted (methoxy, ethoxy, and n -butoxy) androst-4-ene-3,17-dione (AD) steroids ( 1 and 2 ) and their androsta-1,4-diene-3,17-dione (ADD) derivatives ( 3 and 4 ) with human placental aromatase by gas chromatography–mass spectrometry (GC–MS). Among the steroids examined, 6β-methoxy and 6β-ethoxyADDs ( 4a and 4b ) are suicide substrates of aromatase. All of the steroids were found to be converted into the corresponding 6-alkoxy estrogens. Introduction of the alkoxy groups at C-6 of AD or ADD decreased the ability of these to serve as a substrate of aromatase. In 6α-alkoxy steroid series, compounds 1 and 3 , the aromatization rate increased by elongating the 6-methoxy group up to the n -butoxy group whereas, in the 6β-isomers series, 2 and 4 , the rate decreased due to this structural modification. 6β-Alkoxy steroids, 2 and 4 , including the suicide substrates, were extremely poor substrates for the aromatization reaction. Apparent K m values obtained for 6α-alkoxy compounds 1 and 3 were similar to each other, ranging from 92 to 111 nM, as shown by their previously-obtained K i values. The findings indicate that the stereochemistry as well as the bulkiness of the 6-ether-substituent play an important role in the ability to serve as a substrate. It is also predicted that the aromatization reaction and the mechanism-based inactivation reaction would be related and have a definite partition number which is characteristic to the compound in a series of suicide substrates.

To gain insight into the nature of the substrate binding site and the catalytic function of aromatase, we studied the inhibition of androstenedione aromatization by 4beta,5beta-epoxy-16alpha-hydroxyandrostenedione (4) and its 19-hydroxy and 19-oxo derivatives, 5 and 6, as well as the biochemical aromatization of these steroids in human placental microsomes. The 19-methyl and 19-oxo compounds, 4 and 6, were weak competitive inhibitors of aromatase, with apparent K(i) values of 246 microM and 270 microM, respectively, whereas the 19-hydroxy compound 5 inhibited aromatase in a non-competitive manner with the K(i) of 135 microM. The 19-methyl compound 4 inactivated aromatase in a time-dependent manner with k(inact) of 0.213 min(-1) in the presence of NADPH in air, but the other two did not cause it. The conversion of the three epoxides into estrogen, as well as 19-oxygenation of 19-methyl steroid 4 with human placental microsomes in the presence of NADPH in air, were not detected by gas chromatography-mass spectrometry. The present results are consistent with the two binding sites theory in the active site of aromatase.

Treatment of enol acetates of 3β-acetoxyandrost-5-en-17-one and its 5α-reduced analog, 5α-androstan-17-one, and estrone acetate, 1–4 , with Pb(OCOCH 3 ) 4 in acetic acid and acetic anhydride gave the previously unreported products, 16β-(acetoxy)acetoxy-17-ketones 8–10 and 12 , in 9–15% yields along with the known major products, 16β-acetoxy-17-ketones 5–7 and 11 . Similar treatment of the 16β-acetoxy-17-ketones with the lead reagent did not yield the corresponding (acetoxy)acetates. Reaction of the enol acetate 3 with Pb(OCOCD 3 ) 4 in CD 3 COOD yielded principally the labeled (acetoxy)acetate 10 - d 3 , which had a CD 3 COOCH 2 COO moiety at C-16β. In contrast, when the deuterated enol acetate 3 - d 3 , which was obtained by treatment of the 17-ketone 14 with (CD 3 CO) 2 O in the presence of LDA and which had a CD 3 COO moiety at C-17, was reacted with Pb(OCOCH 3 ) 4 , the resulting product was the labeled compound 10 - d 2 . This product had a CH 3 COOCD 2 COO function at C-16β. Based on these results, along with further isotope-labeling experiments, it seems likely that the (acetoxy)acetate is produced through a lead (IV) acetate-catalyzed migration of the 17-acetyl function of the enol acetate to the C-16β-position followed by attack of an acetoxy anion of the lead reagent.

To gain insight into the binding aspects of 6alpha- and 6beta-methylandrostenediones (1 and 2), potent competitive inhibitors and effective substrates of aromatase, at the active site of the enzyme, we synthesized their 19-hydroxy and 19-oxo derivatives to determine their inhibition of aromatase activity as well as their aromatization rates in human placental microsomes. The 6beta- and 6alpha-methyl-19-ols 12 and 13 were produced from 19-(tert-butyldimethylsiloxy)androstenedione (6) in 6 steps in which the Grignard reaction of 5alpha,6alpha-epoxy steroid 8 with CH3MgBr was employed as a key reaction. Oxidation of the 19-ols 12 and 13 yielded the corresponding 19-als 14 and 15. The 6alpha-methyl steroids 13 and 15 were good competitive inhibitors of aromatase (Ki< or =100 nM), and their aromatization rates obtained by gas chromatography-mass spectrometric analysis were 110 and 205 pmol/min/mg protein, respectively. In contrast, the 6beta-methyl isomers 12 and 14 were non-competitive inhibitors, with Ki values of more than 500 nM, and they were aromatized at rates of 16 and 20 pmol/min/mg protein, respectively. These results reveal that there is a marked difference in binding to the active site between the 19-oxygenated 6alpha-methyl and 6beta-methyl inhibitors where the binding manner of the 6alpha-steroids, rather than the 6beta-isomers, is suitable as a substrate for aromatase.

4Beta,5beta-epoxy derivatives of 16alpha-hydroxyandrostenedione (2), one of the natural substrates for aromatase, and its 19-oxygenated compounds 4 and 5 were synthesized as mechanistic and catalytic probes for the enzyme reaction. Treatment of 16alpha-bromoandrostenedione (13) or its 19-hydroxy analog 19 which was prepared from 3beta-hydroxy-19-(tert-butyldimethylsiloxy)androst-5-en-17-one (16) in three steps, with H2O2 and NaOH followed by controlled alkaline hydrolysis with NaOH in aqueous pyridine stereospecifically yielded 4beta,5beta-epoxy-16alpha-ol 15 or 4beta,5beta-epoxy-16alpha,19-diol 22, respectively. Oxidation of 16beta-bromo-4beta,5beta-epoxy-19-ol 21 with pyridinium dichromate followed by controlled alkaline hydrolysis produced 4beta,5beta-epoxy-16alpha-hydroxy-19-al 24.

16Alpha-hydroxy-17-keto steroids, 1, 3, and 8, and their 17beta-hydroxy-16-keto isomers, 4, 5, and 9, were transformed into the corresponding 17beta-alkoxy-16-keto derivatives on treatment with trimethylsilyl iodide (TMSI) in the presence of alkyl alcohol in CHCl3 in poor to high yields.

Deoxygenation reaction of steroidal 16,17-ketols 1, 2 and 6 as well as their silyl ethers 3 and 7 and 16- and 17-iodoketone analogs 11, 12, and 14 with trimethylsilyl iodide (TMSI) or HI under various conditions was examined. The results indicate that the deoxygenation producing 16- and 17-ketones 9 and 8 proceeds through multiple reaction pathways; a direct iodination of a siloxy group of the ketol silyl ethers by iodide ion to give the iodoketones (path b), addition of TMSI to a carbonyl group of the ketol silyl ethers to yield diiodo derivatives 22 and 23 through iodo-bis-TMS compounds 20 and 21 (path a), and cleavage of ether bond of dimers 15-18 initially produced are, at least, involved. In these sequences, rearrangement of the 16-ketols 1 and 2 to the 17β-ketol 6 also plays a significant role. The yields of the ketones 9 and 8 and their relative amounts would be dependent on the relative importance of each pathway in the reaction.

[3β, 7, 7, 17α-2H4]Androst-5-ene-17β, 19-diol (6) and [3, 3, 7, 7, -2H4]androst-4-ene-17β, 19-diol (15-d4) were synthesized as internal standards for gas chromatographic-mass spectrometric analysis of the 19-hydroxylation of androst-5-en-17-one (1) and its 4-ene isomer 2, inhibitors of estrogen biosynthesis, using human placental aromatase. Treatment of [7, 7-2H2]3β-tosylate 3 with Zn-NaI-D2O, followed by reduction with NaBD4, gave compound 6 (d4, 79.8 atom%). Compound 15-d4 was synthesized via 3β, 17β-dihydroxy-5-en-7-one 10 as a key intermediate. Deoxygenation of the 5-en-7-one 10 and [7, 7-2H2]17β-hydroxy-4-en-3-one 13-d2, produced from compound 10 in two steps, with AlCl2D2 was used for the deuterium labeling reaction, producing compound 15-d4 (d4, 93.5 atom%). The 19-oxygenation products of aromatase inhibitors 1 and 2 could be analyzed, after NaBH4 reduction, as the corresponding 17β, 19-diol bis-trimethylsilyl ethers using the internal standards 6 and 15-d4.

Reaction of various steroidal 16, 17-ketols, 16α-hydroxy-17-ketones 1-3, and 15, 16β-hydroxy-17-ketone 4, and 17β-hydroxy-16-ketones 5-7, and 17, along with methyl ethers of 16α- and 17β-ketols, 1 and 5, with an excess of trimethylsilyl iodide (TMSI) or with HI in CHCl3, produced the deoxygenated products, a mixture of the corresponding 17-and 16-ketones, in low to quantitative yields, in which the 17-ketone was the major product in each case. When the 16β-deuterated 16α-ketol 3 and the 17α-deuterated 17β-ketol 7 were reached with TMSI for a brief period (15 min), the deuterium content at C-16β and C-17α of the recovered steroids 3 and 7 was reduced by 17 and 35%, respectively. The present results indicate that the deoxygenation proceeds not only through a direct iodination pathway producing α-iodoketone but also through other reaction pathways.

Two series of 6-alkylandrosta-4,6-diene-3,17-diones (5) and their 1,4,6-triene analogs 6 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between varying the 6-n-alkyl substituents (C1–C7) and inhibitory activity. All of the steroids synthesized were extremely powerful competitive inhibitors of aromatase in human placental microsomes, with apparent Ki values for the 6-alkyl-4,6-diene steroids 5 ranging from 17 to 36 nM and with those for the 1,4,6-triene steroids 6 ranging from 2.5 to 58 nM. The 6-ethyl-1,4,6-triene compound 6b (Ki = 2.5 nM) was the most potent inhibitor among them. The 6-alkyl-1,4,6-triene steroids 6, except for the 6-methyl analog 6a, had higher affinity for aromatase than the natural substrate androstenedione (Km = 24 nM), and their inhibitory activities were more potent than the corresponding 4,6-diene steroids 5. In a series of the 4,6-diene steroids 5, compounds 5c–f with the n-alkyl chain substituents (C3 to C6) also had slightly higher affinity than androstenedione for aromatase. All of the 1,4,6-triene steroids 6 inactivated aromatase in a time-dependent manner, with kinact values ranging from 0.021 to 0.074 min−1; in contrast, the 4,6-diene analogs 5 did not. The inactivation was prevented by androstenedione, and no significant effect of l -cysteine on the inactivation was observed in each case. These results indicate that the length of the n-alkyl substituent at C-6 of androsta-1,4,6-triene-3,17-dione (6h), rather than its 4,6-diene analog 5h, plays a critical role in tight binding to the active site of aromatase. No significant correlation was observed between affinity for the enzyme and the inactivation ability of the 6-alkyl-1,4,6-trienes.

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, 4β,5β-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 μM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (KI = 88 μM, fkinact = 0.071 min−1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K, 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 ( T 1 2 = 40 sec ) than the 19-methyl analog 4 ( T 1 2 = 3.0 min ). The results clearly indicate that the 4β,5β-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. BIOCHEM PHARMACOL 52;8:1253–1259, 1996.

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.

A series of 6α- and 6β-alkylestradiols (5 and 6) (alkyl : methyl, ethyl, n-propyl, n-pentyl, and n-heptyl) were synthesized as possible aromatase reaction products of 6-alkylandrost-4-ene-3, 17-diones and their Δ1-derivatives, potent competitive and mechanism-based inhibitors of aromatase. Treatment of 6-oxoestradiol with Grignard reagents followed by acid-catalyzed dehydration and subsequent catalytic hydrogenation over Pd-C gave the 6-alkylestradiols (5 and 6). GC-MS (electron impact mode) of trimethylsilyl derivatives of bis-trimethylsilyl derivatives of compounds 5 and 6 revealed that the 6α-alkyl compounds, 5, emerged with a longer retention time compared to the corresponding 6β-alkyl isomers, 6, where the retention time was in proportion to the length of the 6-alkyl chain in each series. In the MS, a molecular ion (M+) peak was the base peak for all the 6-alkylestrogens, with strong and characteristic fragment ion peaks corresponding to M+-131 and at m/z 325. A selected ion monitoring method using a molecular ion will be sensitive enough for analysis of the aromatization reaction of the 6-alkylandrogens.

To gain further insight on the relationship between 6-alkylandrost-4-ene-3,17-diones and their aromatase inhibition activity, a series of alkyl steroids with long alkyl chains (n-pentyl, n-hexyl, or n-octyl) at C-6 alpha and 6 beta were synthesized. All of the steroids studied inhibited human placental aromatase in a competitive manner with apparent Ki values ranging from 2.8 to 80 nM. The 6 beta-pentyl analog 4a (Ki = 2.8 nM) was the most potent inhibitor. The inhibitory activities of the 6 beta-alkyl steroids 4 were more powerful than those of the corresponding 6 alpha-isomers 5. The addition of one methylene unit to the 6 alpha- and 6 beta-n-butyl moieties of androst-4-ene-3,17-dione markedly increased the affinity to aromatase, whereas further elongation of the n-pentyl group decreased affinity in relation to the carbon number of the alkyl chain. These results, along with molecular modeling with the PM3 method, suggest that the increased affinities of the pentyl steroids 4a and 5a may essentially depend on the formation of thermodynamically stable enzyme-inhibitor complex in the hydrophobic binding pocket.

To gain insight into the catalytic function of aromatase and its substrate specificity, we studied reversible inhibition of 16alpha-hydroxyandrostenedione (16alpha-OHAD) aromatization in human placental microsomes by several suicide substrates of androstenedione (AD) aromatization, including 4-hydroxyAD (1), 6-oxoAD (2) and its 19-hydroxy analogue 3, androst-5-ene-4,7,17-trione (4), and 10beta-acetoxyandrost-5-en-7,17-dione (5) that, in contrast, do not cause a suicide inactivation of 16alpha-OHAD aromatization. All inhibitors examined blocked 16alpha-OHAD aromatization in a competitive manner with apparent K(i) values ranging from 0.50 to 980 nM. The relative K(i) values between inhibitors 1-5 obtained in the 16alpha-OHAD aromatization experiments were markedly different from those obtained in the AD aromatization experiments. The results predict that all inhibitors examined bind to the 16alpha-OHAD binding site in a manner that does not cause suicide inactivation of 16alpha-OHAD aromatization. These findings would be useful for understanding the active (binding) site structure as well as the catalytic function of aromatase.

2,2-Dimethylandrost-4-ene-3,6,17-trione ( 5 ) and its 4-methoxy- ( 7 ) and 4-hydroxy- ( 8 ) derivatives were synthesized. 7α-Acetoxy-4-ene-3,6-dione steroid 2 was also prepared by the improved method involving the lead tetraacetate oxidation of androst-4-ene-3,6,17-trione ( 1 ). These steroids along with the 2-acetoxy- ( 11 and 12 ), 2-substituted 1-ene- ( 9 and 10 ), and 4-substituted ( 13–15 ) derivatives of compound 1 were evaluated as inhibitors of human placental aromatase. All the steroids, except the 2-acetoxy-1-ene 10 and the 2β-acetate 11 of which K i values were not determined because of their poor inhibitory activities, blocked aromatase in a competitive manner. Compounds 5 and 8 as well as the 4-hydroxy steroid 15 were potent inhibitors (K i : 25–42 nM) whereas the inhibitory activities of steroids 2, 7, 9, 13 , and 14 were good to fair, respectively (K i : 160–810 nM). Inhibitors 2 and 15 inactivated the enzyme in a time-dependent manner in the presence of NADPH but the 2,3-dimethyl derivatives 5 and 8 did not. Androstenedione blocked the inactivation by l -cysteine did not. The results suggest that the 2β-methyl group would prevent the aromatase-catalyzed oxygenation at C-19 of the dimethyl steroids 5 and 8 most likely through the steric reasons.

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.

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.

4β,5β-Epoxyandrostane-3,6,17-trione 2 undergoes cleavage of the epoxy ring as a result of nucleophilic attack by water to give both 2-hydroxy-1,4-diene-3,6-dione 3 and 7α-hydroxy-4-ene-3,6-dione 9 upon treatment with sulfuric acid in aqueous acetone. In the similar reaction in acetic acid, 2α-acetoxy-4-ene-3,6-dione 6 and its 2β-isomer 7 are produced along with the diosphenol 3. Reaction of the epoxide 2 with sodium hydroxide in methanol affords 4-methoxy-4-ene-3,6-dione 12 the dealkylation of which with hydrochloric acid yields the 4-hydroxy derivative 14.

Aromatization of 16alpha-hydroxyandrostenedione (16alpha-OH AD) with aromatase in human placental microsomes was studied by gas chromatography-mass spectrometry (GC-MS) using 12,4,6,6,9alpha,16beta,17alpha-2H7]estriol as an internal standard. 16alpha-OH AD was incubated with the microsomes in the presence of NADPH in air. The metabolite was extracted with ethyl acetate and treated with NaBH4. The reduced product, estriol, was isolated by Sep-Pak C18 cartridge and then analyzed as the tris(trimethylsilyl)ether by a GC-MS (EI mode). The production of estriol was dependent upon protein concentration and incubation time. Apparent Km and Vmax values of the microsomal aromatase for 16alpha-OH AD were 568 nM and 25.5 pmol/min/mg protein, respectively. In this assay, aromatase activity, estriol formation, could be determined at a level as low as 1 pmol/min/mg protein. Aromatase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estriol formation in a competitive manner with 25 and 30 nM of apparent Ki values, respectively.

Report an efficient deoxygenation of the 21-alkoxy group and the conversion of the 17α-hydroxy 20-ketone to a D-homosteroid upon treatment with TMSI in the presence of MeOH

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.

4β,5β-Epoxyandrostane-3,6,17-trione 2 undergoes cleavage of the epoxy ring as a result of nucleophilic attack by water to give both 2-hydroxy-1,4-diene-3,6-dione 3 and 7α-hydroxy-4-ene-3,6-dione 9 upon treatment with sulfuric acid in aqueous acetone. In the similar reaction in acetic acid, 2α-acetoxy-4-ene-3,6-dione 6 and its 2β-isomer 7 are produced along with the diosphenol 3. Reaction of the epoxide 2 with sodium hydroxide in methanol affords 4-methoxy-4-ene-3,6-dione 12 the dealkylation of which with hydrochloric acid yields the 4-hydroxy derivative 14.