凤眼草的化学成分研究

目的 对苦木科臭椿属植物臭椿(Ailanthus altissima(Mill．)Swingle)的果实，即凤眼草的乙醇提取物进行分离并鉴定。方法 经反复纯化制备化合物，并根据化合物的理化性质分析、波谱解析、对照品比较及与文献报道对比鉴定化合物的化学结构。结果 从质量分数为95％的乙醇室温浸提物中得到6个甾类化合物和3个萜类化合物，根据理化性质和谱学解析分别鉴定为stignmst-4-ene-3，6-dione(Ⅰ)、β-sitosterol(Ⅱ)、6β-hydroxystigmast-4-en-3-one(Ⅲ)、5α，8α-epidioxyergesta-6，22-dien-3β-ol(Ⅳ)、stigmast-4-ene-3β，6β-diol(V)、9，19-cyclolanost-23(Z)-ene-3β，25-diol(Ⅵ)、3-epi-ursoloic acid(Ⅶ)、12β，20(S)-dihydroxydammar-24-ell-3-one(Ⅶ)、daucosteml(Ⅸ)。结论 化合物Ⅰ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ、Ⅶ、Ⅸ均为从本属首次分离得到。     

芫花花蕾中甾醇类化合物的分离与鉴定

目的对瑞香科瑞香属植物芫花(Daphne genkwa Sieb.et Zucc.)花蕾中的甾醇类化学成分进行分离与结构鉴定。方法运用硅胶﹑Sephadex LH-20柱色谱﹑重结晶等分离手段进行甾醇类化合物的分离纯化,根据理化性质及波谱数据鉴定其化学结构。结果从芫花花蕾的体积分数95%乙醇提取物中分离得到6个化合物,分别鉴定为:豆甾-5-烯-3β,7α-二醇(stigmasta-5-ene-3β,7α-diol,1)、豆甾-5-烯-3β,7β-二醇(stigmasta-5-ene-3β,7β-diol,2)、豆甾-4-烯-3β,6β-二醇(stigmasta-4-ene-3β,6β-diol,3)、7-ketositosterol(3β-hydroxysitost-5-ene-7-one,4)、过氧化麦角固醇(ergosterol peroxide,5)和β谷甾醇-3β-吡喃葡萄糖苷-6'-O-棕榈酸酯(β-sitosteryl-3β-glucopyranoside-6'-O-palmitate,6)。结论化合物2⁶为首次从瑞香属植物中分离得到,化合物1为首次从芫花植物中分离得到。     

内生真菌Sporormiella irregularis(No.71-11-4-1)中一个新的二裂甾体

目的研究一株地衣内生真菌Sporormiella irregularis(No.71-11-4-1)的次生代谢产物。方法采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、ODS柱色谱以及HPLC等多种色谱手段对其发酵提取物进行分离纯化,并借助光谱学数据对单体化合物进行结构鉴定。结果分离得到9个甾类化合物,分别鉴定为22E-3,7-epoxy-5,10∶8,9-disecoergosta-9(10),22-diene-5,8-dione(1)、22E-5α,6α-ep-oxyergosta-8(14),22-diene-3β,7α-diol(2)、22E-5α,6α-epoxyergosta-8(9),22-diene-3β,7α-diol(3)、22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol(4)、22E-3β-hydroxy-5α,6α-epoxyergosta-22-en-7-one(5)、22E-ergosta-7,22-diene-3β,5α,6β-triol(6)、22E-6β-methoxyergosta-7,22-diene-3β,5α-diol(7)、22E-5α,8α-epidioxyergosta-6,22-dien-3β-ol(8)、22E-ergosta-4,6,8(14),22-tetraen-3-one(9)。结论其中化合物1是新化合物,属于5,10∶8,9二裂甾体;化合物2～9均为首次从Sporormiella属真菌中分离得到。     

海绵共附生真菌Penicillium chrysogenum次级代谢产物的化学成分及生物活性研究

目的 对分离自西沙隋氏蒂壳海绵共附生真菌Penicilliumchrysogenum的次级代谢产物进行化学成分及其生物活性研究,以期发现结构特异并且活性良好的次级代谢产物。方法 对隋氏蒂壳海绵共附生真菌Penicilliumchrysogenum用真菌2号培养基发酵,发酵后的菌丝体采用溶剂提取、萃取和现代色谱分离纯化手段,再运用现代核磁波谱技术并结合高分辨质谱鉴定化合物结构。基于微阵列技术的表面等离子体共振成像（SPRi）系统,检测化合物与肿瘤相关蛋白的相互作用,并提供化合物与肿瘤相关蛋白的结合动力学数据。结果 通过分离隋氏蒂壳海绵共附生真Penicilliumchrysogenum的菌丝体提取物,从中分离鉴定了7个单体化合物,鉴定结果为conidiogenone（1）、2-acetylquinazolin-4(3H)-one（2）、15β-hydroxyl-(22E,24R)-ergosta-3,5,8,22-tetraen-on（3）、ergosta-4,6,8(14),22-tetraen-3-one（4）、 2-((2E,4E)-hexa-2,4-dienoyl)-5,6-dihydroxy-4,6-dimethylcyclohex-4-ene-1,3-dione（5）、(22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol（6）、2-(1-hydroxyethyl)quinazolin-4(3H)-one（7）。结论 化合物3和6是从Penicillium属内第一次分离得到,化合物4是从真菌Penicilliumchrysogenum中第一次分离得到,化合物5与肿瘤蛋白VEGFR-1、FGFR有亲和作用,KD的数值分别为7.78×10-3和1.44×10-1 μmol/L。     

中国南海海绵Spheciospongia sp.中的甾体化合物

目的研究中国南海海绵Spheciospongia sp.的化学成分。方法采用多种色谱方法分离纯化,依据理化性质、波谱数据和文献对照的方法鉴定结构。结果从中国南海海绵Spheciospongia sp.石油醚提取物中分离鉴定了6个甾体类化合物:胆甾醇(cholesterol,1),胆甾-4-稀-3-酮(cho-lest-4-en-3-one,2),胆甾-3,6-二酮(cholest-3,6-dione,3),3β-羟基-胆甾-5-稀-7-酮(3β-hydroxy-cholest-5-en-7-one,4),6β-羟基-胆甾-4-稀-3-酮(6β-hydroxy-cholest-4-en-3-one,5),胆甾-3β,5α,6β-三醇(cholest-3β,5α,6β-triol,6)。结论化合物2-6为首次从该属海绵中分离得到。     

板栗种皮化学成分的分离与鉴定

目的更好地开发利用板栗(Castanea mollissimaBlume)的药用资源。方法采用硅胶柱色谱、凝胶柱色谱、氧化铝柱色谱和重结晶等方法对板栗种皮的体积分数为75%的乙醇提取物进行分离;通过NMR谱和薄层色谱等方法对分离得到的化合物进行结构鉴定。结果分离得到8个化合物,分别鉴定为对羟基苯甲酸(p-hydroxy benzoic acid,1)、原儿茶酸(protocatechuic acid,2)、没食子酸(gallic acid,3)、香草酸(vanillic acid,4)、东莨菪内酯(scopoletin,5)、豆甾-4-烯-6β-羟基-3-酮(stigmast-4-en-6β-ol-3-one,6)、豆甾-4-烯-3,6-二酮(stigmast-4-en-3,6-dione,7)、豆甾烷-3β,6α-二醇(stigmastane-3β,6α-diol,8)。结论化合物7、8为首次从栗属植物中分离得到。     

姜黄化学成分的分离与鉴定

目的研究姜黄的化学成分。方法采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱法和HPLC法等方法对姜黄体积分数为95%的乙醇提取物的乙酸乙酯层的化学成分进行分离与纯化,并根据理化性质、NMR、MS等波谱数据鉴定化合物的结构。结果分离得到6个化合物,分别鉴定为:芳姜黄酮(ar-tumerone,1)、14-羟基芳姜黄酮(6-(4-hydroxy methyl phenyl)-2-methyl-hept-2-ene-4-one,2)、isobisabolone A(3)、姜黄酮(turmeronol,4)、2-methyl-6-(4-hydroxyphenyl)-2-hepten-4-one(5)、2-methyl-6-(2-cyclohexen-4-one)-2-hepten-4-one(6)。结论化合物2为新的天然产物,化合物6为首次从姜黄属中分离得到。     

氢溴酸樟柳碱

氢溴酸樟柳碱是从茄科植物唐古特山莨菪 Scopolia tangutica Maxim [Anisodus tanguticus (Maxim) Pascher] 中提出的一种新生物碱的氢溴酸盐。【化学名】3α-(2′-苯基-2′,3′-二羟基丙酰氧基)-6,7β-环氧莨菪烷。     

红球菌g26中C17位脱氢酶的克隆表达及生物转化

目的对红球菌g26中的短链脱氢酶基因进行克隆表达并考察其能否将甾体化合物雄甾-4-烯-3.17-二酮(androst-4-ene-3,17-dione,AD) C17位羰基还原为羟基。方法通过检索数据库获得红球菌g26中的两个短链脱氢酶基因,应用PCR、重组表达载体的构建等生物技术在大肠杆菌中进行外源表达,破碎细胞后应用SDS-PAGE对可溶性蛋白进行分析,筛选出合适的目的蛋白诱导条件,进而进行酶的体外转化反应,应用薄层色谱分析、高效液相分析和质谱法鉴定产物结构。结果与结论红球菌g26中的短链脱氢酶可以转化甾体化合物AD为睾丸酮(TS)。红球菌g26内的短链脱氢酶参与了甾体化合物C17位羰基到羟基的还原反应。     

延龄草化学成分的分离与鉴定

目的研究延龄草(Trillium tschonoskii Maxim.)根及根茎的化学成分。方法利用硅胶柱色谱、凝胶柱色谱、ODS(反相C18键合硅胶)柱色谱、高效液相色谱等色谱技术对延龄草的化学成分进行分离和纯化,通过理化性质和NMR等波谱数据分析确定化合物的结构。结果从延龄草根及根茎70%(φ)乙醇提取物中分离得到11个已知成分,分别确定为重楼皂苷Ⅵ(chonglouosideⅥ,1)、偏诺皂苷元-3β-O-α-L-吡喃鼠李糖基-(1→4)-[O-α-L-吡喃鼠李糖基-(1→2)]-O-β-D-吡喃葡萄糖苷(penogenin-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glu-copyranoside,2)、重楼皂苷Ⅶ(chonglouosideⅦ,3)、偏诺皂苷元-3β-O-β-D-吡喃葡萄糖基-(1→6)-[O-α-L-吡喃鼠李糖基-(1→2)]-O-β-D-吡喃葡萄糖苷(trikamsteroside B,4)、(25S)-27-羟基偏诺皂苷元-3β-O-α-L-吡喃鼠李糖基-(1→2)-O-β-D-吡喃葡萄糖苷((25S)-27-hydroxypenogenin-3β-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranoside,5)、(25S)-27-羟基偏诺皂苷元-3β-O-α-L-吡喃鼠李糖基-(1→4)-[O-α-L-吡喃鼠李糖基-(1→2)]-O-β-D-吡喃葡萄糖苷((25S)-27-hydroxypenogenin-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside,6)、(25S)-27-羟基偏诺皂苷元-3β-O-α-L-吡喃鼠李糖基-(1→4)-O-α-L-吡喃鼠李糖基-(1→4)-[O-α-L-吡喃鼠李糖基-(1→2)]-O-β-D-吡喃葡萄糖苷(polyphyllosideⅢ,7)、(23S,24S,25S)-螺甾-5-烯-1β,3β,21,23,24-五羟基-1β-O-β-D-呋喃芹糖基-(1→3)-O-α-L-吡喃鼠李糖基-(1→2)-[O-β-D-吡喃木糖基-(1→3)]-O-α-L-吡喃阿拉伯糖苷(trikamsteroside E,8)、3β-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosylhomo-aro-cholest-5-ene-26-O-β-D-glucopyranoside(parispseudoside B,9)、3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranosylhomo-aro-cholest-5-ene-26-O-β-D-glucopyranoside(aethioside A,10)、3β-O-α-L-rhamnopyranosyl-(1→4)-O-α-L-rham-nopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranosylhomo-aro-cholest-5-ene-26-O-β-D-glucopyranoside(parispseudoside A,11)。结论化合物9-11为首次从百合科植物中分离得到,7为首次从延龄草属植物中分离得到,4-6、8为首次从延龄草中分离得到。     

7α,17α-二甲基-17β-羟基雄甾-4-烯-3酮的若干A环骈杂环衍生物的合成

将7α-甲基引进甲基睾丸素后,经甲酰化反应制得2-羟次甲基衍生物(Ⅳ),再先后转变为单肟(Ⅴ)、双肟(Ⅵ),环合得70,17α-二甲基-17β-羟基-雄甾-4-烯骈[2,3-c]呋咱(Ⅶ)。也可使(Ⅳ)与水合肼或盐酸羟胺作用,分别制得相应的雄甾-4-烯骈[3,2-c]吡唑(Ⅷ)及雄甾2,4-二烯骈[2,3-d]异(口恶)唑(Ⅸ)。在酸性反应条件下合成(Ⅸ)时,还分离出它的重排脱水产物7α,17α,17β-三甲基雄甾-2,4,13-三烯骈[2,3-d]异(口恶)唑(Ⅹ)。     

Degradation of urine samples and its influence on the ¹³C/¹²C ratios of excreted steroids

The degradation processes in deficiently stored urine samples are well investigated regarding steroid concentrations and diagnostic ratios, such as the quotient of testosterone divided by epitestosterone. In contrast, nothing is known about the influence on carbon isotope ratios (CIR) by inappropriate storage conditions. In general, it is assumed that degradation, i.e. deconjugation or dehydrogenation, does not change CIR and thus CIR can be used in cases where the steroid profile turns out to be invalid. Therefore, the CIR of urinary steroids was investigated in different urine samples during the course of degradation over a time period of six months. Several steroids excreted as glucuronides (androsterone (A), etiocholanolone (E), testosterone, pregnanediol (PD) and 5α- and 5β-androstane-3α,17β-diol) or sulfo-conjugated (A, E and androst-5-ene-3β,17β-diol (5EN17b)) were investigated together with their unconjugated correspondents (A, E, PD and 5EN17b) and the main dehydrogenation products (5α- and 5β-androstane-3,17-dion and androst-4-ene-3,17-dion). For this purpose, the exiting methods for CIR determination were extended and validated. In addition, the urinary concentrations of all investigated steroids were monitored. Particular attention was paid to dehydroepiandrosterone conjugated and unconjugated together with its degradation product 3α,5-cyclo-5α-androstan-6β-ol-17-one as here the strongest influence on CIR was expected.     

Induction of CYP3A expression by dehydroepiandrosterone: involvement of the pregnane X receptor.

Dehydroepiandrosterone (DHEA) is a steroid produced by the human adrenal gland. Administration of pharmacological doses of DHEA to rats changes expression of many genes, including the cytochrome P450 family members CYP4A1 and CYP3A23. It is known that induction of CYP4A expression by DHEA requires the peroxisome proliferator-activated receptor alpha (PPAR(alpha)). In the current study, PPAR(alpha)-null mice were used to examine the role of PPAR(alpha) in expression of CYP3A. In wild-type mice, 150 mg/kg DHEA-sulfate induced Cyp4a and Cyp3a11 mRNAs by 5- and 2-fold, respectively. Induction of Cyp4a expression by DHEA-sulfate was not observed in PPAR(alpha)-null mice, whereas induction of Cyp3a11 expression by DHEA-sulfate was similar between genotypes. This suggests that PPAR(alpha) is not involved in induction of Cyp3a11 expression by DHEA. Because expression of CYP3A family members can be induced by activation of another member of the nuclear receptor superfamily, the pregnane X receptor (PXR), we examined the ability of DHEA to activate PXR. In transient transfection assays, DHEA and its metabolites androst-5-ene-3beta,17beta-diol (ADIOL), androst-5-ene-3,17-dione, and androst-4-ene-3,17-dione were activators of PXR. Maximal induction of a PXR-responsive reporter gene of approximately 3-fold was observed at concentrations of 50 to 100 microM, indicating that these steroids are relatively weak activators of PXR. Human and murine PXR exhibited different specificities for DHEA and its metabolites. ADIOL activated reporter gene expression in the presence of murine but not human PXR. Results of these studies suggest that the induction of rodent CYP3A expression upon treatment with high doses of DHEA occurs through activation of PXR.     

Patent Evaluation: 2β,19-Methyleneamino Bridged Steroids as Aromatase Inhibitors

Summary

Novelty: The compounds 2β-19(methyleneamino)androst-4-ene-3,17-dione and its 17β-ol are disclosed. They are said to be aromatase inhibitors and are potentially useful for the treatment of hyperoestrogenaemia. They may also be useful as antifertility agents, and could be used to treat breast cancer and other oestrogen-induced or oestrogen-stimulated tumours or hyperplastic tissue disorders.

Biology: The activity of the 3,17-dione in the inhibition of aromatase was demonstrated using the method of Johnston et al. (Endocrinology (1984) 115:776) and Burkhart et al. (Steroids (1985) 45:357). The inhibitor is pre-incubated with enzyme prior to assaying for activity in the presence of high substrate levels. The compound showed K_i = 259 nM, Λ₅₀ = 2.66 min, k^cat/K_i = 16 760.

Chemistry: Detailed synthesis of two end products and three intermediates is described. These are prepared by cyclization of 19(N-protected ((2-methoxyethoxy)methylamino)steroid using titanium tetrachloride followed by selective reduction of the 17-ketone 2β,19-(methylene amino) androst-4-ene, to give the 3,17-dione and its 17β-ol.     

Steroid 9alpha-hydroxylation during testosterone degradation by resting rhodococcus equi cells

The conversion pathway of testosterone to androst-4-ene-3,17-dione and 9alpha-hydroxy androstane metabolites, 9alpha-hydroxyandrost-4-ene-3,17-dione and 9alpha,17beta-dihydroxyandrost-4-en-3-one was proposed for the ring degradation in steroids by a minimal liquid medium (NMMP)-dispersed Rhodococcus equi ATCC 14887. The microorganism produced 9alpha-hydroxy androstane metabolites from testosterone at high conversion ratio without the addition of ring degradation inhibitory agents. Several NMMP-based media showed the similar effect on the microbial transformation, in which the respective molar yields of 9alpha-hydroxyandrost-4-ene-3,17-dione and 9alpha,17beta-dihydroxyandrost-4-en-3-one were approx. 3 to 47% and approx. 3 to 11%, respectively, whereas nutrient broth, a rich medium, basically showed no accumulation. On the basis of this evidence, magnesium sulfate and casamino acids among the components of NMMP were found to compromise the determinant for the production of the 9alpha-hydroxy androstane metabolites without appreciable decomposition of the steroid ring system.     

国产甲基炔诺酮的杂质分离和鉴定

本文报道了甲基炔诺酮(norgestrel)中两个杂质的分离和鉴定。用薄层层析和高效液相色谱对甲基炔诺酮进行分析,发现除主成分甲基炔诺酮外尚含有六个杂质。用柱层析和制备型薄层层析预分,经径向加压柱对其中两个杂质进行高效液相色谱制备。经紫外、红外和质谱分析并与对照品比较,确认这两个杂质分别为(±)13β-乙基-17β-羟基-甾烷-4-烯-3-酮及(±)13β-乙基-甾烷-4-烯-3,17-二酮。     

A new isopimarane-type diterpene and a new natural atisane-type diterpene from Excoecaria agallocha

From the woods of Excoecaria agallocha, a new isopimarane-type diterpene, 3α,11β-dihydroxy-ent-isopimara-8(14),15-dien-2-one (1) and a new natural atisane-type diterpene, 16β-hydroxy-ent-atisan-3-one (2) were isolated together with three known compounds, ribenone (3), ent-labda-8(17),13E-diene-3β,15-diol (4), and ent-3β-hydroxybeyer-15-ene-2,12-dione (5). Their structures were determined by spectral data and x-ray crystallography evidence.     

Thiol-containing androgens as suicide substrates of aromatase

The thiol-containing androgens 17 beta-hydroxy-10 beta-mercaptoestr-4-en-3-one and 19-mercaptoandrost-4-ene-3,17-dione were synthesized and tested in human placental microsomes for their ability to suicide inhibit aromatase. Both compounds showed time-dependent, pseudo-first-order rates of inactivation of aromatase with Ki's of 106 and 34 nM and kcat's of 3.2 X 10(-3) and 1.2 X 10(-3) s-1 respectively for 1 and 2 at 30 degrees C. Diffusion dialysis failed to reactivate aromatase previously inactivated by either compound, and both compounds required that NADPH and O2 be present for the time-dependent inactivation of the enzyme. The presence of the substrate, androst-4-ene-3,17-dione (5.0 microM), protected the enzyme from inactivation while cysteine (1.0 mM) failed to protect aromatase from inactivation by either compound. The above evidence demonstrates that both compounds are potent suicide inhibitors of aromatase.     

Detection of dihydrotestosterone gel, oral dehydroepiandrosterone, and testosterone gel misuse through the quantification of testosterone metabolites released after alkaline treatment

The natural occurrence of endogenous anabolic steroids together with their availability in different administration forms makes the detection of their misuse a great challenge for doping control laboratories. Nowadays, the detection of endogenous steroids abuse is performed by the analysis of the steroid profile. Recently, androst-1,4-dien-3,17-dione (1,4-AD), androst-4,6-dien-3,17-dione (4,6-AD), 17β-hydroxy-androst-4,6-dien-3-one (6-T), and androst-15-en-3,17-dione (15-AD) have been described as testosterone (T) metabolites released after basic treatment of the urine. In the present work, the usefulness of these metabolites has been evaluated detecting the use of three different forms of endogenous steroids in a single dose: dihydrotestosterone gel (DHT), oral dehydroepiandrosterone (DHEA), and T gel. After the independent administration of these endogenous steroids, a rise in the value of several of the ratios calculated between the tested metabolites was noticed. For DHT, a small increase was observed for the ratios 1,4-AD/15-AD, 6-T/15-AD and 4,6-AD/15-AD although only for one volunteer. Better results were obtained for oral DHEA and T gel where an increase was observed in all volunteers for several of the tested ratios. The detection time in which the misuse can be detected (DT) has been evaluated using two different approaches: (1) comparison with population based reference limits, and (2) comparison with individual threshold levels. The obtained DTs were compared with the results of previously published markers for the misuse of such substances. When using basic released metabolites, shorter DTs were obtained for DHT, similar DTs for DHEA, and the detectability was substantially improved for T gel.     

Interactions of thiol-containing androgens with human placental aromatase

A series of thiol androgens were synthesized and investigated to characterize structural features important for the inhibition of aromatase. Analogues of androstenedione with thiol groups in either the 2 alpha-, 10 beta-, or 19-positions caused time-dependent inhibition of human placental aromatase. When their KI and kcat values were compared with those of 4-hydroxyandrost-4-ene-3,17-dione (4-OHa) and 10 beta-propargylestr-4-ene-3,17-dione (PED), the thiol androgen 10 beta-mercaptoestr-4-ene-3,17-dione (10 beta-SHnorA) proved to be the most potent suicide substrate. However, 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) was the best all-around inhibitor. All compounds except 19-SHA exhibited normal type I P-450 difference spectra with partially purified/solubilized, human placental aromatase. The Ks values for the series of compounds compared qualitatively to the KI values determined from the time and concentration-dependent inhibition experiments. 19-SHA induced split Soret peaks at 380 and 474 nm, which suggested binding of the 19-thiolate directly to the ferric iron of aromatase. This binding could be displaced by aminoglutethimide but not by androstenedione. The inhibitory activity of 19-SHA may be explained by two independent mechanisms: (1) suicide inactivation of aromatase in the ferrous state; and (2) a direct "hyper-type II" binding to the remaining portion of the cytochrome in the ferric state. A free thiol group was necessary for the suicide inhibitory activity of 19-SHA; time-dependent inactivation of aromatase by 19-(acetylthio)androst-4-ene-3,17-dione (19-SAcA) and 19-xanthogenylandrost-4-ene-3,17-dione (19-XanA) could be prevented if the microsomes were preincubated with a carboxyesterase inhibitor. Aromatase previously inactivated by either thiol androgens,4-OHA, or PED could not be reactivated after incubation with the disulfide reducing agent dithiothreitol, which suggests that a disulfide bond may not be involved in aromatase inactivation by these inhibitors.