犁头霉11α-羟基化制备16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯-3,20-二酮

选育到一株对16β-甲基,17α,21-二羟基孕甾-1,4-二烯-3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21-乙酰化(Ⅱb)可明显提高11α-羟基化的能力.在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,11α,21-三羟基孕甾-1,4-二烯-3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认.     

犁头霉11α-羟基化制备16β-甲基-11α，17α，21 三羟基孕甾-1，4 二烯 3，20 二酮

选育到一株对16β-甲基-17α,21-二羟基孕甾-1,4-二烯3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21乙酰化(Ⅱb)可明显提高11α-羟基化的能力。在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认。     

香港坚木中的达玛烷型三萜

从楝科植物香港坚木的树吉中分得了４个新的达玛烷型三萜,基于详细的光谱分析及与同类化合物的光谱数据比较鉴定了其结构,分别命名为：２０Ｒ,２４Ｒ－表－２５－达玛烷烯－３－酮,１６β－羟基达玛烷－２０,２５－二烯－３－酮,２６－羟基达玛烷－２０,２４－二烯－３－酮和３β－乙酰基－７α,２１Ｓ,２５－三羟基－２１Ｓ,２３Ｒ－表－１（１１）－达玛烷烯。     

瘿花香茶菜的微量成分

从唇形科瘿花茶菜（Ｒａｂｄｏｓｉａ ｒｏｓｔｈｏｒｎｉｉ）叶的乙醚抽出物中分出２个新的微量成分,瘿花丙素和丁素。基于详细的光谱分析,包括应用二维核磁共振数据,瘿花丙素和化学结合分别确定为对映－１１α－乙酰氧基－７β,１３β,１９－三羟基贝壳杉－１６烯－１５－酮（１）和对映－１１α－乙酰氧基－７β,１２β,１４α－三羟基贝壳杉－１６烯－１５－酮（２）。     

茜草乔木酮乙和丙的结构

从小红参（Ｒｕｂｉａｙｕｎｎａｎｅｎｓｉｓ）中分离鉴定了２个新的乔木烷型三萜,分别命名为茜草乔木酮乙和丙（ｒｕｂｉａｒｂｏｎｏｎｅＢ,Ｃ）。用光谱和化学方法证明它们的结构分别为：７β,１９α,２８－三羟基乔木－９（１１）－烯－３－酮和２８－乙酰氧基－７β,１９α－二羟基乔木－９（１１）－烯－３－酮。     

毛喉鞘蕊花的微量成分

从唇形科毛喉鞘蕊花全草的氯仿提取物分离到２个新的微量成分,鞘蕊花戊素和已素。基于光谱分析,鞘蕊花戊素和已 素的化学结构分别鉴定为１α,７β－二乙酰氧基－８,１３－环氧－６β羟基勒丹－１４烯－１１－酮（１）和７β－乙酰氧基－８,１３环氧－６β,α－二羟基勒丹－１４烯－１１－酮（２）。     

总状毛霉对4-烯-3-酮甾体的生物转化研究

从土样中筛选到一株能转化甾体的菌株,经形态观察,鉴定为总状毛霉(Mucor racemosus)。首次利用该菌株对4-烯-3-酮类甾体衍生物进行生物转化,目的是合成具有潜在活性的羟基类4-烯-3-酮衍生物。转化条件为27℃,220r/min振荡培养4d。转化产物经乙酸乙酯萃取,用硅胶柱层析法分离,通过红外、质谱和核磁分析确定了甾体转化产物的化学结构。黄体酮生物转化得到的产物是14α-羟基-4-孕甾烯-3,20-二酮和7α,14α-二羟基-4-孕甾烯-3,20-二酮;4-雄烯二酮的转化产物是14α-羟基-雄甾-4-烯-3,17-二酮1、4α,17β-二羟基-雄甾-4-烯-3-酮和6α,17β-二羟基-雄甾-4-烯-3-酮。研究结果表明总状毛霉具有转化甾体的能力,对4-烯-3-酮类甾体进行生物转化的主要产物是14α-羟基甾体衍生物。     

微生物学方法制备16α-甲基-11α，17α，21-三羟基孕甾-1，4-二烯3，20-二酮

本文报道了简单节杆菌A69-2和球孢白僵菌AS69同时存在下对16α-甲基-17α-羟基孕甾-4．烯-3,20-二酮-21-醋酸酯(16MRSA)的协周转化作用。 这种协同转化怍用既能解除16α-甲基-11α,17a,21-三羟基孕甾-4-烯-3,20-二酮(16MllaHC)对球孢白僵菌AS69的11α羟化酶的抑制作用,又可降低高浓度的16M11aHC对节杆菌A69—2脱氢酶活性的影响,同时还能抑制节杆菌脱氢过程的副反应。在底物浓度为0.15％(W／V)时,l6α-甲基-11α,17a,21-三羟基孕甾-1,4-二烯-3,20-二酮(16MDHC)的收率约50%,故是制备1 6MDHC的一种理想的微生物学方法。     

粉红单端孢Trichothecium roseum对雄甾-4-烯-3,17-二酮的生物转化

雄甾-4-烯-3,17-二酮（4AD）是甾体化合物合成过程中的关键中间体,其羟化产物通常具有良好的药理活性或作为工业生产甾体药物的重要中间体。利用粉红单端孢Trichothecium roseum对4AD进行生物转化,从其发酵提取物中共分离鉴定了3个4AD羟基化产物：6β-羟基-雄甾-4-烯-3,17-二酮（6β-OH-4AD,1）,14α-羟基-雄甾-4-烯-3,17-二酮（14α-OH-4AD,2）,6β,14α-双羟基-雄甾-4-烯-3,17-二酮（6β,14α-di-OH-4AD,3）,表明T. roseum对4AD的C-6β位和C-14α位具有较强的羟化能力,其中14α-OH-4AD(2)可作为合成强心甾类化合物毛地黄毒素的重要中间体,6β,14α-di-OH-4AD(3)可作为合成具有抗肿瘤活性的14α-羟基-雄甾-4-烯-3,6,17-三酮的重要中间体。提供了1株能够高效制备活性甾醇中间体14α-OH-4AD和6β,14α-di-OH-4AD的菌株,同时可为研究其他甾醇药物奠定基础。     

云南茯苓皮的化学成分研究

从云南产茯苓表皮中分得６个三萜类化合物,经光谱数据鉴定为：茯苓酸（Ⅰ）,３β－羟基羊毛甾烷－７,９（１１）,２４－三烯－２１－酸（Ⅱ）,茯苓酸Ａ（Ⅲ）,茯苓酸Ｂ（Ⅳ）,去氢齿孔酸（Ⅴ）,齐果酸（Ⅵ     

New steroidal antiinflammatory antedrugs: Methyl 3,20-dioxo-9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-1,4-pregnadiene-16 alpha-carboxylate and its 21-O-acyl derivatives

In a continuing effort to increase local to systemic activity ratios of potent steroidal antiinflammatory antedrugs, a series of 21-O-acyl derivatives of methyl 3,20-dioxo-9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-1,4-pregnadiene-16 alpha-carboxylate, FP16CM, were synthesized. These derivatives were evaluated for antiinflammatory activity and their adverse effects in an acute and semi-chronic croton oil-induced ear edema bioassay. Following a single topical application in the croton oil-induced ear edema bioassay, treatment with all the compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID(50) values (nmol/ear resulting in a 50% reduction of edema) were calculated: prednisolone (Pred); 454, FP16CM; 255, 21-acetate (FP16CM-acetyl); 402, 21-propionate (FP16CM-propionyl); 474, 21-valerate (FP16CM-valeryl); 446 and 21-pivalate (FP16CM-pivalyl); 219 nmol. In a 5-day semi-chronic study at the equipotent doses, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights or plasma corticosterone levels unlike the parent compound Pred. The compounds were assessed for high-affinity glucocorticoid receptor binding and glucocorticoid-mediated inhibition of nitric oxide (NO) generation in an in vitro RAW 264.7 macrophage cell culture system. Binding affinities for cytosolic glucocorticoid receptors were Pred; 85, FP16CM-acetyl; 86, FP16CM-propionyl; 169, FP16CM-valeryl; 149, FP16CM-pivalyl; 126 nM, respectively. Concomitant potencies for inhibition of NO generation by macrophages stimulated with lipopolysaccharide were Pred; 159, FP16CM-acetyl; 377, FP16CM-propionyl; 405, FP16CM-valeryl; 344, FP16CM-pivalyl; 311 nM, respectively. Collectively, results of these investigations suggest that esterification of 21-OH with various anhydrides did not improve receptor binding, inhibition of NO generation and ear edema inhibition, however, serum corticosterone level and local over systemic activities (L/S) were markedly improved.     

New steroidal anti-inflammatory antedrugs: methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-3,20-dioxo-1, 4-pregnadiene-16alpha-carboxylate, methyl 21-desoxy-21-chloro-11beta-hydroxy-3,20-dioxo-1, 4-pregnadiene-16alpha-carboxylate, and their 9alpha-fluoro derivatives*

To a series of 21-desoxy-21-chloro-corticosteroids, a metabolically labile methoxycarbonyl group at C-16 has been incorporated. The approach is to synthesize locally active compounds that are hydrolyzed to inactive and readily excretable acid metabolites upon entry into the systemic circulation. Novel antedrugs were evaluated for anti-inflammatory activity and their adverse effects in an acute and semichronic croton oil-induced ear edema bioassay. Binding affinity to glucocorticoid receptors and induction of L-tyrosine-2-oxoglutarate aminotransferase were studied in hepatoma tissue culture cells. After a single topical application in the croton oil-induced ear edema bioassay, treatment with all the compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID(50) values (nmol/ear resulting in a 50% reduction of edema) were calculated: 540, 618, 454, and 346 nmol for prednisolone (P), methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-3,20-dioxo-1, 4-pregnadien-16alpha-carboxylate (PClCM), methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-9alpha-fl uoro-3, 20-dioxo-1,4-pregnadien-16alpha-carboxylate (FPClCM), and methyl 21-desoxy-21-chloro-9alpha-fluoro-11beta-hydroxy-3,20-dioxo- 1, 4-pregnadien-16alpha-carboxylate (FDPClCM), respectively. Results of the 5-day rat croton oil ear edema bioassay indicated that, in contrast with the parent compound P, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, or plasma corticosterone levels. The binding affinities for cytosolic hepatoma tissue culture glucocorticoid receptors were 33, 201, 471, 5304, and 3765 nM for P, PClCM, FPClCM, methyl 21-desoxy-21-chloro-11beta-hydroxy-3,20-dioxo-1, 4-pregnadien-16alpha-carboxylate (DPClCM), and FDPClCM, respectively. Collectively, results of these investigations suggest that modifications of P, which included replacement of 21-hydroxyl group with chlorine and addition of 16-methoxycarbonyl group with or without 17-hydroxyl moiety, retained the topical anti-inflammatory activity of the parent compound P without significant adverse systemic effects.     

Metabolism of steroidal anti-inflammatory antedrugs in vitro: methyl 3,20-dioxo-11beta,17alpha,21-trihydroxy-1,4-pregnadiene-16alpha-carboxylate; its 9alpha-fluorinated,and their 21-O-acyl derivatives

The in vitro hydrolysis rates of steroidal anti-inflammatory antedrugs, methyl 3,20-dioxo-11beta,17alpha,21-trihydroxy-1,4-pregnadiene-16alpha-carboxylate (P16CM), its 9alpha-fluorinated analogue (FP16CM), and their 21-O-acyl derivatives (P16CM-acetyl, FP16CM-acetyl, FP16CM-propionyl, FP16CM-valeryl, and FP16CM-pivalyl) were investigated in rat plasma. These steroids were synthesized based on the antedrug concept. P16CM and FP16CM were hydrolyzed to inactive steroid-16-carboxylate, with half-lives of 90.0 and 99.4 min, respectively. The metabolite was positively identified by NMR and elemental analysis. To determine the relative hydrolysis rate of the C21-O-acyl versus the C16-methoxycarbonyl group, P16CM- and FP16CM-21-O-acyl derivatives were also studied. The hydrolysis rates of all 21-O-acyl groups were much faster than that of the 16-methoxycarbonyl group. The half-lives of P16CM-acetyl, FP16CM-acetyl, FP16CM-valeryl, and FP16CM-propionyl were 6.3, 16.8, 23.2, and 18.4 min, respectively. On the other hand, FP16CM-pivalyl showed relatively slow hydrolysis rate (T(1/2): 59.7 min). These results clearly indicate that 21-O-acyl group is metabolized first to active compound, P16CM or FP16CM, followed by the hydrolysis of 16-methoxycarbonyl to corresponding inactive steroid-16-carboxylates as the major metabolites. Collectively, the results of the present study support the previous reports where decrease in adverse systemic effects without losing local anti-inflammatory activity was attributed to the hydrolysis of the active agents to inactive acidic metabolites in the systemic circulation. This study thus shows that the incorporation of a 16-methoxycarbonyl coupled with a 21-O-acyl moiety may be a fundamentally sound synthetic strategy in the development of locally active anti-inflammatory steroids having reduced systemic adverse activities.     

Bioconversion of steroids by Cochliobolus lunatus--II. 11 beta-hydroxylation of 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate in dependence of the inducer structure.

The 11 beta-hydroxylase of the filamentous fungus Cochliobolus lunatus m 118 was induced with the substrate 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate (11 beta-deoxyprednisolone 17-acetate) itself, substrate analogues, different pregnane compounds, sterols, intermediates of microbial sterol side-chain degradation or bile acids, together with 24 different steroids in a standardized test system. The resulting 11 beta-hydroxylation rate, leading to prednisolone 17-acetate and prednisolone, respectively, was determined and compared with the hydroxylation rate of non-induced cultures. The transformation yield strongly depended on the inducer structure. The microbial sterol side-chain degradation intermediates (20S)-20-hydroxymethylpregn-4-en-3-one and the corresponding pregna-1,4-diene compound caused the highest induction effects (induction factors 5.1 and 4.9, respectively). The metabolism of (20S)-20-hydroxymethylpregna-1,4-dien-3-one during the cultivation was elucidated. The induction effect decreased with the rising oxidation of the inducer. The significant increase of the 11 beta-hydroxylation rate of 1-dehydro-pregnane substrates by specific induction allows alternative pathways to glucocorticoid partial syntheses.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

Synthesis and pharmacological evaluations of new steroidal anti-inflammatory antedrugs: 9alpha-Fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its derivatives

In continuing efforts to develop potent anti-inflammatory steroids without systemic adverse effects, methyl 9alpha-fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its 16-alkoxycarbonyl derivatives (FP16CE, FP16CP and FP16CB) were synthesized based on the antedrug concept. The steroids were evaluated for their pharmacological activities and adverse systemic effects. All steroidal antedrugs showed both binding affinity to the glucocorticoid receptor in liver cytosol and inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cell. These compounds also inhibited croton-oil-induced ear edema and showed no systemic effects such as thymus atrophy and suppression of corticosterone level after 5-day treatment. Among those compounds tested, FP16CM showed the highest activities in receptor binding, NO inhibition and ear edema, these activities were comparable to those of prednisolone. Hydrolysis study in plasma showed that FP16CB was hydrolyzed rapidly, with the half-live (T1/2) of 3.2 min and the half-lives of other compounds were between 16.9 and 29.4 min. These results support the antedrug concept, of which the decrease in systemic adverse effects is attributed to fast hydrolysis to inactive metabolite in the systemic circulation.     

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues—value precursors in the synthesis of modern glucocorticoids starting from 9-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and ¹H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.     

Characterization of testosterone 16 alpha-hydroxylase (I-P-450(16) alpha) induced by phenobarbital in mice

Cytochrome P-450 (I-P-450(16) alpha), which is associated with phenobarbital-induced testosterone 16 alpha-hydroxylation activity, was purified from livers of phenobarbital-treated female 129/J mice on the basis of the specific hydroxylation activity in fractions eluted from columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified I-P-450(16) alpha fraction was 12.4 nmol/mg of protein, and it had an apparent molecular weight of 54K. The specific activity of reconstituted testosterone 16 alpha-hydroxylation activity with the purified I-P-450(16) alpha fraction was 6-8 nmol min-1 (nmol of cytochrome P-450)-1. Rabbit antibody raised against the purified I-P-450(16) alpha fraction inhibited nearly 100% of the 16 alpha-hydroxylation activity in liver microsomes of phenobarbital-treated female 129/J mice but did not affect hepatic microsomal 16 alpha-hydroxylation activity of untreated male and female 129/J mice at all. In hepatic microsomes of phenobarbital-treated male 129/J mice, 70% of the 16 alpha-hydroxylation activity, at most, was catalyzed by I-P-450(16) alpha, and the residual 30% of the activity was catalyzed by C-P-450(16) alpha. The increase of I-P-450(16) alpha by phenobarbital was due to de novo synthesis of I-P-450(16) alpha, and this induction was not sexually regulated in 129/J mice. Anti-C-P-450(16) alpha [Harada, N., & Negishi, M. (1984) J. Biol. Chem. 259, 12285-12290] did not inhibit the 16 alpha-hydroxylation catalyzed by I-P-450(16) alpha; thus, I-P-450(16) alpha and C-P-450(16) alpha are immunochemically distinct isozymes of testosterone 16 alpha-hydroxylase.     

Mouse liver testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha). Purification, regioselectivity, stereospecificity, and immunochemical characterization

Microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) was purified from the livers of male 129/J mice based on enzyme activity in the eluates from columns of DEAE Bio-Gel A, hydroxylapatite, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified P-450(16) alpha fraction was 9.5 nmol/mg of protein. The specific testosterone 16 alpha-hydroxylation activity of the purified P-450(16) alpha fraction was 80 nmol/min/nmol of cytochrome P-450 or 764 nmol/min/mg of protein, and these values were about 40- and 400-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(16) alpha showed extremely high regioselectivity and stereospecificity for testosterone hydroxylation; more than 90% of the testosterone metabolites formed by the purified P-450(16) alpha fraction was 16 alpha-hydroxytestosterone. The purified anti-P-450(16) alpha antibody exhibited absolute specificity for inhibition of testosterone 16 alpha-hydroxytestosterone was inhibited by the anti-P-450(16) alpha. Anti-P-450(16) alpha inhibited the 16 alpha-hydroxylation activity of intact microsomes prepared from livers of male or female 129/J mice more than 90%, indicating that P-450(16) alpha is the major cytochrome P-450 isozyme catalyzing 16 alpha-hydroxylation activity of testosterone in these microsomal preparations. The purified P-450(16) alpha fraction also possessed high benzphetamine N-demethylation activity relative to the rates found with other xenobiotic substrates tested in this report.