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1.
The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hydroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (1c)] nor the 19-oxoandrostenedione analogue [10-acetylestr-4-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]estr-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with 18O2 established that 3c generated from 2c retained little 18O (less than 3%), while 3c arising from 2e retained a significant amount of 18O (approximately equal to 70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 microM, 9.9 microM, and 150 nM for 1c, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with 4-hydroxyandrostenedione (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes. 相似文献
3.
Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition. 相似文献
4.
All oxidative functions of aromatase, i.e., estrogen production, 19-oxygenated androgen production and 7-ethoxycoumarin deethylation, were inhibited in parallel in placental microsomes from non-smokers by the mechanism-based, time-dependent inactivators (suicide substrates) 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione. In contrast, the aromatase suicide substrate androst-4-ene-3,6,17-trione had little or no effect on the conversion of androst-4-ene-3,17-dione to 19-hydroxyandrost-4-ene-3,17-dione or on the conversion of the latter to 3,17-dioxoandrost-4-en-19-al while severely limiting the capacity for estrogen production from androst-4-ene-3,17-dione and 19-hydroxyandrost-4-ene-3,17-dione in such microsomal preparations. Androst-4-ene-3,6,17-trione, therefore, appears to uncouple the 19-hydroxylation of androgens from estrogen synthesis. This agent also produced only a minimal inhibition of 7-ethoxycoumarin deethylation, indicating that this major constitutive transformation of a xenobiotic chemical is associated with the steroid 19-hydroxylating function of the aromatase system. 相似文献
5.
Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated. 相似文献
6.
Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities. 相似文献
7.
Treatment of 19-[oxygenated]-androst-4-ene-3,17-dione with Mn(AcO) 3 and ClCH 2COOH in benzene gave epimeric mixtures of the corresponding 2ξ-chloroacetates and 2ξ-acetates. The products were processed to give the title compound. For the synthesis of the 2- 18O analog, ClCH 2C 18OOH was used, which was prepared from ClCH 2COCl. 相似文献
8.
The ester cleavage of R- and S-isomers N-CBZ-leucine p-nitrophenyl ester intermolecularly catalyzed by R- (a) and S-stereoisomers (b) of the Pd(II) metallacycle [Pd(C 6H 4C *HMeNMe 2)Cl(py)] (3) follows the rate expression kobs = ko + kcat [3], where the rate constants kcat equal 25.8 ± 0.4 and 7.6 ± 0.5 dm 3 mol −1 s −1 for the S- and R-ester, respectively, in the case of 3a, but are 5.7 ± 0.6 and 26.7 ± 0.5 dm 3 mol −1 s −1 for the S- and R-ester, respectively, in the case of 3b (pH 6.23 and 25°C). Thus, the best catalysis occurs when the asymmetric carbons of the leucine ester and Pd(II) complex are R and S, or S and R configured, respectively. Molecular modeling suggests that the stereoselection results from the spatial interaction between the CH 2CHMe 2 radical of the ester and the -methyl group of 3. A hydrophobic/stacking contact between the leaving 4-nitrophenolate and the coordinated pyridine also seems to play a role. Less efficient intramolecular enantioselection was observed for the hydrolysis of N-t-BOC- S-metthionine p-nitrophenyl ester with R- and S-enantiomers of [Pd(C 6H 4C*HMeNMe 2)Cl] coordinated to sulfur. 相似文献
9.
A number of inhibitors of estrogen synthesis are now becoming available which could be of value in the treatment of breast cancer. 4-Hydroxyandrostenedione (4-OHA), the first of these compounds to enter the clinic has been found to be effective in postmenopausal patients who have relapsed from tamoxifen. Thus, in studies of 240 patients, 26% patients experienced partial or complete response to treatment. An additional 25% patients had disease stabilization. 4-OHA is a potent selective, steroidal inhibitor which causes inactivation of aromatase in vitro. It is effective in reducing concentrations of ovarian estrogens in rats and of ovarian and peripheral estrogens in non-human primate species. The compound has been shown to lower serum estrogen levels in postmenopausal breast cancer patients. However, not all of these patients experienced disease remission, suggesting that their tumors were hormone insensitive rather than that the dose of 4-OHA was suboptimal. In trials of patients who had not received prior tamoxifen treatment, 4-OHA (250 mg i.m. every 2 weeks) was found to induce complete or partial tumor regression in 33% of patients. The response of patients was not significantly different from that observed in patients treated with tamoxifen (30 mg o.d) of 37%. No significant difference between treatments was observed for disease stabilization, the duration of response or median survival. Several other steroidal aromatase inhibitors have been studied, such as 7-substituted androstenedione derivatives. MDL 18962 [10-(2-propynyl)estr-4-ene-3,17-dione] and FCE 24304 (6-methylen-androsta-1,4-diene-3,17-dione) are currently in clinical trials. Non-steroidal inhibitors of cytochrome P-450 enzymes, such as imidazole and triazole derivatives have been developed which are highly selective for aromatase. Three triazoles which are very potent and selective inhibitors are vorazole (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)-methyl]1-methyl-1H-benzotriazole R 76713, arimidex 2,2′[5-(
-1,2,4-triazol-1-yl methyl)-1,3-phenylene]bis(2-methylpropiononitrile) (ZD1033) and letrozole 4-[1-(cyanophenyl)-1-(1,2,4-triazolyl)methyl]benzonitril (CGS 20267). These compounds reduce serum estradiol concentration to undetectable levels in breast cancer patients. These highly potent inhibitors provide the opportunity to determine whether a further degree of estrogen suppression will be important in producing greater clinical response. With the recent approval of 4-OHA in several countries and the introduction of the potent new compounds, aromatase inhibitors either alone or in combination with the antiestrogen are likely to improve the treatment of breast cancer. 相似文献
10.
The absolute configuration at C-12 of pittosporatobiraside A and B isolated from the leaves of Pittosporum tobira was determined to be S on the basis of the exciton chirality of their dibenzoate derivative. The structures of the two glycosides were thus established to be (1 S,9 S,10 S,11 S,12 S,14 R,16 R)-12-[( Z)-2-methyl-1-oxo-2-butenyl]-6,14-dimethyl-2-methylene-9-(1-methylethyl)-15,17-dioxatricyclo[8.7.0.0 11,16]heptadec-5-en-13-one and (1 S,9 S,10 S,11 S,12 S,14 R,16 R)-12-(3-methyl-1-oxo-2-butenyl)-6,14-dimethyl-2-methylene-9-(1-methylethyl)-15,17-dioxatricyclo [8.7.0.0 11,16]heptadec-5-en-13-one, respectively. 相似文献
11.
19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats. 相似文献
12.
The heterocyclic analogue of ( S)-glutamic acid, ( S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [( S)-AMPA] is a potent and selective AMPA receptor agonist, whereas the enantiomeric compound, ( R)-AMPA, is virtually inactive. We have previously characterized ( RS)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [( RS)-APPA] as a partial AMPA receptor agonist showing about 60% of the efficacy of ( RS)-AMPA. This partial agonism produced by ( RS)-APPA is, however, only apparent, since resolution of ( RS)-APPA has now been shown to provide the full AMPA receptor agonist, ( S)-APPA, whereas ( R)-APPA is a
acid (non-NMDA) receptor antagonist showing preferential AMPA blocking effects. In agreement with classical theories for competitive interaction between agonists and antagonists, the efficacy of depolarizations produced by ( S)-APPA in the rat cortical wedge preparation was shown to be progressively reduced with increasing molar ratios of ( R)-APPA/( S)-APPA. These compounds and the competitive antagonists ( RS)-2-amino-3-(3-carboxymethoxy-5-methyl-4-isoxazolyl)propionic acid [( RS)-AMOA], 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo( f)quinoxalin-2,3-dione (NBQX) were also tested in [ 3H]AMPA and [ 3H]CNQX binding systems, the latter ligand being used in the absence or presence of thiocyanate ions. On the basis of these studies it is suggested that ( RS)-AMPA and the AMPA agonist ( S)-APPA interact with a high-affinity receptor conformation, whereas the competitive antagonists ( RS)-AMOA and ( R)-APPA, derived from these agonists, preferentially bind to a low-affinity AMPA receptor conformation. The competitive antagonists, CNQX and NBQX which are structurally unrelated to ( RS)-AMPA or ( RS)-APPA, do not seem to discriminate between these two AMPA receptor conformations. The modified [ 3H]CNQX binding assay containing thiocyanate ions was shown to provide receptor affinity data for AMPA receptor agonists as well as antagonists, which correlate with the potencies of these compounds in the cortical wedge preparation. Using autoradiographic techniques, ( S)- and ( R)-APPA were shown to exhibit significantly different absolute potencies as inhibitors of [ 3H]AMPA binding in a number of regions of the rat brain. 相似文献
13.
A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. (( S)- tert.-leucine, ( R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and ( S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C 18 solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients ( r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine. 相似文献
14.
Three new crystalline tin selenide salts have been prepared from the reactions of [PPh 4] 2[Sn(Se 43] in supercritical solvents. The starting material pyrolyzes in supercritical acetonitrile to form [PPh 4] 4[Sn 6Se 21] (I), and it also reacts with SnSe in supercritical ammonia leading to a mixture of [PPh 4] 4[Sn 3Se 11] 2 (II). and [PPh 4] 2[Sn(Se 4)(Se 6) 2] (III). All three compounds have been characterized by single crystal X-ray diffraction. Crystallographic data: for I, C 96H 90P 4Se 21Sn 6, space group triclinic, P-1, A = 18.763(3), B = 24.600(4), C = 13.137(1) Å, = 102.63(1), β = 93.66(1), γ = 108.72(1)°, V = 5544(1) Å 3, Z = 2, R = 0.0350, RW = 0.0317: for II, C 96H 80P 4Se 22Sn 6, space group monoclinic P2 1/ c, A = 31.500(4), B = 16.572(3), C = 22.352(3) Å, β = 103.53(1)°, V = 11344(3) Å 3, Z = 4, R = 0.0771, RW = 0.0664: for III, C 48H 40P 2Se 16Sn, space group monoclinic, C2/ c, A = 25.381(2), B = 13.934(4), C = 19.465(3) Å, β = 121.587(8)°, V = 5867(2) Å 3, Z = 4, R = 0.0807, RW = 0.0650. One of the compounds, [PPh 4] 2[Sn(Se 4(Se 62], is a molecular cluster while the other two complexes [PPh 4] 4[Sn 3Se 11] 2 and [PPh 4] 4[Sn 6Se 21], are one dimensional tin selenide chains. The structures of the two chains are related and consits of tetrahedral and distorted trigonal bipyramidal tin(IV) centers bridged by Se 2−, Se 22− and Se 32− chains. 相似文献
15.
The conversion of a molecule of 19-oxoandrost-4-ene-3,17-dione [1a] to estrone [2a] by human placental aromatase requires a molecule of oxygen and of NADPH. An atom of this molecule of oxygen is incorporated into the extruded formic acid derived from C-19 of [1a]. It was proposed that the 0 2 is utilized for the enzymatic 2β-hydroxylation of [1a] and the released intermediate 2β-hydroxy-19-oxoandrost-4-ene-3, 17-dione [5a]aromatizes nonenzymatically. Should [5a] be an obligatory intermediate of estrogen biosynthesis, then all the oxygen of its 2β-hydroxyl must be incorporated into the extruded formic acid. We have previously synthesized [2β- 180;19- 3H][5c] and proved that none of its 2β- 180 was incorporated in the formic acid extruded in the aromatization. On this basis we concluded that [5a] can not be an obligatory precursor of estrogen biosynthesis. The trapping of radioactive androst-4-ene-2β,3β,17β,19-tetrol in a reductively terminated incubation of a mixture of radioactive androst-4-ene-3, 17-dione and [5a] with crude placental aromatase was interpreted as evidence in support of the intermediacy of [5a]. We confirmed that the tetrol can indeed be trapped in the reductively terminated incubations. However, considering that the crude placental enzyme preparation very likely contains numerous activated oxygen species capable of a variety of oxidation reactions, most of which may not be related to estrogen elaboration, and in view of our results quoted above, the origin and the eventual biosynthetic role of the parent compound of the tetrol remains to be determined. 相似文献
16.
Synthesis of lobucavir prodrug, L-valine, [(1 S,2 R,3 R)-3-(2-amino-1,6-dihydro-6-oxo-9 H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1 R,2 R,4 S)-2-(2-amino-6-oxo-1 H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N, N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O, O′-[(1 S,2 R,3 R)-3-(2-amino-6-oxo-1 H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1 R,2 R,4 S)-2-(2-amino-1,6-dihydro-6-oxo-9 H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O, O′-[(1 S,2 R,3 R)-3-(2-amino-6-oxo-1 H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1 S,2 R,4 R)-3-(2-amino-6-oxo-1 H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield). 相似文献
17.
- erythro-5,6,7,8-Tetrahydrobiopterin (BH 4), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH 4 exists as natural (6 R)- and unnatural (6 S)-isomers. In our previous reports, only (6 R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6 R)- and unnatural (6 S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH 4. Thus, TH activity was observed only in the presence of exogenous BH 4. We compared the difference in in situ DOPA formation by TH activity in the presence of (6 R)- or (6 S)-BH 4 in the human TH-transfected cells. The effect of exogenous BH 4 was also compared between (6 R)- and (6 S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 μM endogenous (6 R)-BH 4. The rate of uptake of both BH 4 isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 μM but was nearly saturated at 1 mM BH 4. TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6 R)- or (6 S)-BH 4 dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6 R)-BH 4 was approximately three times as active as (6 S)-BH 4. In contrast, in PC12h cells which contained endogenous (6 R)-BH 4 (approximately 100 μM), exogenous (6 R)-BH 4 activated DOPA formation maximally at 500 μM about 10-fold, while (6 S)-BH 4 activated it only slightly, about 2.5-fold. These results suggest that (6 S)-isomer has lower cofactor activity with TH in the cells than (6 R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell. 相似文献
18.
Androstenedione analogs containing 7-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450 arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7-(4′-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent Kinact for 7-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 × 10 −3 sec −1, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7-(4′-iodo)phenylthio-1,4-androstadienedione, 7-IPTADD, and the reconstituted aromatase system. Incubations with [ 125I]7-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450 arom by gel elctrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450 arom. HPLC radiochromatographic analysis of the isolated cytochrome P50 arom confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P50 arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450 arom complex demonstrates that the radioactive inhibitor is covalently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase activity, and radioiodinated 7-IPTAPP binds covalently to the cytochrome P450 arom. 相似文献
19.
Treatment of the A-ring aromatic steroids estrone 3-methyl ether and β-estradiol 3, 17-dimethyl ether with Mn(CO) 5+BF 4− in CH 2Cl 2 yields the corresponding [(steroid)Mn(CO) 3]BF 4 salts 1 and 2 as mixtures of and β isomers. The X-ray structure of [(estrone 3-methyl ether)Mn(CO) 3]BF 4 · CH 2Cl 2 (1) having the Mn(CO) 3 moiety on the side of the steroid is reported: space group P2 1 with a=10.3958(9), b=10.9020(6), c=12.6848(9) Å, β=111.857(6)°, Z=2, V=1334.3(2) Å 3, calc=.481 cm −3, R=0.0508, and wR=0.0635. The molecule has the traditional ‘piano stool’ structure with a planar arene ring and linear Mn---C---O linkages. The nucleophiles NaBH 4 and LiCH 2C(O)CMe 3 add to [(β-estradiol 3,17-dimethyl ether)Mn(CO) 3]BF 4 (2) in high yield to give the corresponding - and β-cyclohexadienyl manganese tricarbonyl complexes (3). The nucleophiles add meta to the arene -OMe substituent and exo to the metal. The and β isomers of 3 were separated by fractional crystallization and the X-ray structure of the β isomer with an exo-CH 2C(O)CMe 3 substituent is reported (complex 4): space group P2 12 12 1 with a=7.5154(8), b=15.160(2), c=25.230(3) Å, Z=4, V=2874.4(5) Å 3, calc=1.244 g cm −3, R=0.0529 and wR2=0.1176. The molecule 4 has a planar set of dienyl carbon atoms with the saturated C(1) carbon being 0.592 Å out of the plane away from the metal. The results suggest that the manganese-mediated functionalization of aromatic steroids is a viable synthetic procedure with a range of nucleophiles of varying strengths. 相似文献
20.
After intravenous administration of the vitamin D 3 analog, 22-oxacalcitriol (OCT), to normal rats plasma metabolites were investigated by HPLC, GC-MS and LC-MS. Five side-chain oxidation metabolites, 24 R(OH)OCT, 24 S(OH)OCT, (25 R)-26(OH)OCT, (25 S)-26(OH)OCT and 24oxoOCT, were identified by comparison with the corresponding synthetic compounds. These side-chain oxidation metabolites were similar to those of calcitriol [1,25(OH) 2 vitamin D 3] described previously. Besides these five metabolites, two unique side-chain cleavage metabolites, 20 S(OH)-hexanor-OCT and 17,20 S(OH) 2-hexanor-OCT, were identified as main metabolites in plasma by GC-MS and LC-MS using a specific chemical reaction. Our studies suggest that OCT is extensively metabolized and circulates in blood as a number of metabolites as well as unchanged OCT. This metabolism includes both unique pathways of C 23-O 22 cleavage and 17-hydroxylation, in addition to the side-chain oxidation metabolites similar to those of 1,25-(OH) 2D 3. 相似文献
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