The synthesis of androstane brassinosteroid analogues with α-azido acid ester groups in position 17β

Androstane brassinosteroid analogues with α-azido acid ester groups in position 17β were synthesized from 2α,3α,17β-trihydroxy-5α-androstan-6-one after the protection of the 2α,3α-diols upon treatment with the corresponding α-azido acid and the subsequent deprotection of the diol grouping. Six new castasterone analogues were prepared. The biological activities were evaluated in two bioassays: a rice lamina inclination test and bean second internode bioassays. The activities of the new analogues differ in these two bioassays.     

17(E)-Picolinylidene androstane derivatives as potential inhibitors of prostate cancer cell growth: Antiproliferative activity and molecular docking studies

We report a rapid and efficient synthesis of A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives from dehydroepiandrosterone. Compounds were validated spectroscopically and structurally characterized by X-ray crystallography. Virtual screening by molecular docking against clinical targets of steroidal anticancer drugs (ERα, AR, Aromatase and CYP17A1) suggests that 17(E)-picolinylidene, but not 17α-picolyl androstanes could specifically interact with CYP17A1 (17α-hydroxylase) with similar geometry and affinity as Abiraterone, a 17-pyridinyl androstane drug clinically used in the treatment of prostate cancer. In addition, several 17(E)-picolinylidene androstanes demonstrated selective antiproliferative activity against PC3 prostate cancer cells, which correlates with Abiraterone antiproliferative activity and predicted CYP17A1 binding affinities. Based on these preliminary results, 17(E)-picolinylidene androstane derivatives could be a promising starting point for the development of new compounds for the treatment of prostate cancer.     

Conjugates of abscisic acid, brassinosteroids, ethylene, gibberellins, and jasmonates

Phytohormones, including auxins, abscisic acid, brassinosteroids, cytokinins, ethylene, gibberellins, and jasmonates, are involved in all aspects of plant growth, and developmental processes as well as environmental responses. However, our understanding of hormonal homeostasis is far from complete. Phytohormone conjugation is considered as a part of the mechanism to control cellular levels of these compounds. Active phytohormones are changed into multiple forms by acylation, esterification or glycosylation, for example. It seems that conjugated compounds could serve as pool of inactive phytohormones that can be converted to active forms by de-conjugation reactions. Some conjugates are thought to be temporary storage forms, from which free active hormones can be released after hydrolysis. It is also believed that conjugation serves functions, such as irreversible inactivation, transport, compartmentalization, and protection against degradation. The nature of abscisic acid, brassinosteroid, ethylene, gibberellin, and jasmonate conjugates is discussed.     

Changes in the Mutagenic and Estrogenic Activities of 17β-Estradiol after Treatment with Nitrite

We determined the changes in the mutagenic and estrogenic activities of 17β-estradiol after a nitrite treatment. Nitrite-treated 17β-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17β-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17β-estradiol to have been formed in the reaction mixture. 2-Nitro-17β-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17β-estradiol was found to be lower than that of 17β-estradiol. These data suggest that a daily oral intake of 17β-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

The in vivo metabolism of 7 beta, 17-dimethyltestosterone-6,7-3H.

7 beta, 17-Dimethyltestosterone (17 beta-hydroxy-7 beta, 17-dimethyl-4-androsten-3-one) (I) was given to three subjects in oral doses of 400 mg per day for ten days. The initial dose contained the steroid tritiated in the 6 and 7 positions. Plasma levels and urinary excretion patterns were followed in all three subjects. Isolations were done on the urine, plasma, and stools of one patient. From the urine 7 beta, 17-dimethyl- 5 alpha-androstane-3 beta,17 beta-diol (VI) was isolated from the nonhydrolyzed fractions. Unchanged (I), 7 beta,17-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (III) and 7 beta, 17-dimethyl-5 beta-androstane-3 beta,17 beta-diol (IV) were isolated from the nonhydrolyzed and enzyme-hydrolyzed fractions. 7 beta,17-dimethyl-5 alpha-androstane-3 alpha,17 beta-diol (V) was isolated from the enzymatic fractions. From the stools were isolated unchanged (I), (III), (IV), (V), and (VI). Unchanged (I) and its 5 alpha-dihydro derivative (17 beta-hydroxy-7 beta,17-dimethyl-5 alpha-androstan-3-one) (II) were identified in the plasma. The total recovery of radioactivity in the one patient on whom the isolations were done was 57%; 40% from the urine and 17% from the stools.     

Assessment of candidate reference genes for the expression studies with brassinosteroids in Lolium perenne and Triticum aestivum

Quantitative PCR studies need proper reference genes with expression stability exclusively validated under certain experimental conditions. The expression stability of several genes commonly used as references was tested under 24-epibrassinolide (EBR) and temperature treatment. Different statistical approaches (qBase^PLUS, BestKeeper, NormFinder) were used to prepare rankings of expression stability in two species of an economic importance: common wheat (Triticum aestivum) and perennial ryegrass (Lolium perenne). Candidate reference genes were shown to be regulated differentially in these two plant species. The maximum stability values indicated that the expression stability was higher in T. aestivum. Taking into account of all ranks it seems that TBP-1 and UBI in ryegrass and ACT, ADP and EF1A in wheat should be used as reference genes in the brassinosteroids and temperature involving studies.     

灵芝子实体中两个新的天然三萜类化学成分的分离、纯化和鉴定

本文采用硅胶和MCI柱层析的方法,从灵芝Ganodermalucidum子实体中分离纯化三萜类化合物。从灵芝子实体的氯仿萃取层中,分离纯化到灵芝属中的2个新天然产物,运用现代NMR技术分析确定了它们的结构,分别为methyl7β-hydroxy-3,11,15,23-tetraoxo-5α-lanost-8-en-26-oate(methylganoderateD)(Ⅰ)和methyl12β-acetoxy-3,7,11,15-tetraoxo-5α-lanost-8-en-24-oate(methyllucidenateD)(Ⅱ)。     

Microbial transformation of 17alpha-ethynyl- and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products

The microbial transformation of the 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17alpha-ethyl-17beta-hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17alpha-ethynyl-17beta-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17alpha-ethynyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17alpha-ethyl-17beta-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17alpha-ethyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (6) and 17alpha-ethyl-6alpha,17beta-dihydroxy-5alpha-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC(50)=1.72 microM).     

Inhibitors of type 1 17β-hydroxysteroid dehydrogenase with reduced estrogenic activity: Modifications of the positions 3 and 6 of estradiol

Breast cancer is the second most frequent cancer affecting women. Among all endocrine therapies for the treatment of breast cancer, inhibition of estrogen biosynthesis is becoming an interesting complementary approach to the use of antiestrogens. The enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a critical role in the biosynthesis of estradiol catalyzing preferentially the reduction of estrone into estradiol, the most active estrogen. Consequently, this enzyme is an interesting biological target for designing drugs for the treatment of estrogen-sensitive diseases such as breast cancer. Our group has reported the synthesis and the biological evaluation of N-methyl, N-butyl 6β-(thiaheptamamide)estradiol as a potent reversible inhibitor of type 1 17β-HSD. Unfortunately, this inhibitor has shown an estrogen effect, thus reducing its possible therapeutic interest. Herein three strategies to modify the biological profile (estrogenicity and inhibitory potency) of the initial lead compound were reported. In a first approach, the thioether bond was replaced with a more stable ether bond. Secondly, the hydroxyl group at position 3, which is responsible for a tight binding with the estrogen receptor, was removed. Finally, the amide group of the side-chain was changed to a methyl group. Moreover, the relationship between the inhibitory potency and the configuration of the side-chain at position 6 was investigated. The present study confirmed that the 6β-configuration of the side chain led to a much better inhibition than the 6α-configuration. The replacement of the 3-OH by a hydrogen atom as well as that of the amide group by a methyl was clearly unfavorable for the inhibition of type 1 17β-HSD. Changing the thioether for an ether bond decreased by 10-fold the estrogenic profile of the lead compound while the inhibitory potency on type 1 17β-HSD was only decreased by 5-fold. This study contributes to the knowledge required for the development of compounds with the desired profile, that is, a potent inhibitor of type 1 l7β-HSD without estrogen-like effects.     

YKL-40、CCL27、CXCL10、Th17细胞及其相关细胞因子IL-17与皮肌炎患者疾病活动度和临床指标的相关性分析

摘要 目的：探讨人软骨糖蛋白-39（YKL-40）、趋化因子配体-27（CCL27）、趋化因子配体-10（CXCL10）、辅助性T细胞17（Th17）及其相关细胞因子白细胞介素-17（IL-17）与皮肌炎患者疾病活动度和临床指标的相关性。方法：选择2020年2月至2022年2月河北北方学院附属第一医院皮肤科收治的80例皮肌炎患者为皮肌炎组,另选取同期40名健康体检人员作为对照组,检测对比两组血清YKL-40、CCL27、CXCL10、IL-17水平及外周血Th17细胞比例。皮肌炎患者根据病情分为活动期组和缓解期组,比较两组YKL-40、CCL27、CXCL10、IL-17水平、Th17细胞比例、皮肌炎相关临床指标及肌炎疾病活动性评估视觉模拟量表（MYOACT）评分,采用Pearson相关系数分析YKL-40、CCL27、CXCL10、IL-17水平、Th17细胞比例与MYOACT评分、皮肌炎相关临床指标的相关性。结果：皮肌炎组的YKL-40、CCL27、CXCL10、IL-17水平及Th17细胞比例均高于对照组（P＜0.05）。活动期组的YKL-40、CCL27、CXCL10、IL-17水平及Th17细胞比例均高于缓解期组（P＜0.05）。活动期组的MYOACT评分及红细胞沉降率（ESR）、铁蛋白（Fer）、乳酸脱氢酶（LDH）、肌酸激酶（CK）水平均高于缓解期组（P＜0.05）。Pearson相关性分析显示,皮肌炎患者的YKL-40、CCL27、CXCL10、IL-17水平、Th17细胞比例与MYOACT评分、ESR、Fer、LDH、CK水平均呈正相关（P＜0.05）。结论：血清YKL-40、CCL27、CXCL10、IL-17水平及外周血Th17细胞比例与皮肌炎病情严重程度有一定相关性,可作为皮肌炎病情的辅助评估指标。     

Analysis and characteristics of multiple types of human 17beta-hydroxysteroid dehydrogenase

Androgens and estrogens are not only synthesized in the gonads but also in peripheral target tissues. Accordingly, recent molecular cloning has allowed us to identify multiple types of 17β-hydroxysteroid dehydrogenases (17β-HSD), the key and exclusive enzymes involved in the formation and inactivation of sex steroids. However, only one form, namely, type 3 17β-HSD, is responsible for pseudohermaphroditism in deficient boys. To date, seven human 17β-HSDs have been isolated and characterized. Although they catalyze substrates having a similar structure, 17β-HSDs have very low homology. In intact cells in culture, these enzymes catalyze the reaction in a unidirectional way — types 1, 3, 5 and 7 catalyze the reductive reaction, while types 2, 4 and 8 catalyze the oxidative reaction. It is noteworthy that rat type 6 17β-HSD also catalyzes the reaction in the oxidative direction. In this report, we analyze the different characteristics of the multiple types of human 17β-HSD.     

Systematic investigation on the synthesis of androstane-based 3-, 11- and 17-carboxamides via palladium-catalyzed aminocarbonylation

3,17-Dicarboxamido-androst-3,5,16-triene, 3-carboxamido-androst-3,5-dien-17-one, 17-carboxamido-androst-4,16-dien-3-one and 11-carboxamido-androst-5,9(11)-dien-3,17-dione derivatives were synthesized in homogeneous carbonylation reactions from the corresponding 3,17-diiodo-androst-3,5,16-triene, 3-iodo-androst-3,5-diene-17-ethylene ketal, 17-iodo-androst-5,16-dien-3-ethylene ketal, 11-iodo-androst-5,9(11)-diene-3,17-bis(ethylene ketal) derivatives, respectively. A highly chemoselective palladium-catalyzed aminocarbonylation of the corresponding iodo-alkene, carried out under mild reaction conditions, can be considered as the key-step for the introduction of the carboxamide functionalities. The synthesis of the iodo-alkene substrate is based on the transformation of the corresponding keto derivative to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). The aminocarbonylation reaction is highly tolerant towards the N-nucleophiles, i.e. various primary and secondary amines including amino acid methyl esters can also be used.     

Application of brassinosteroids to rice seeds (Oryza sativa L.) reduced the impact of salt stress on growth, prevented photosynthetic pigment loss and increased nitrate reductase activity

The effect of applying brassinosteroids to seeds on growth, pigment levels and nitrate reductase activity of rice (Oryza sativa L.) plants grown on saline substratum was investigated. Brassinosteroids reduced the impact of salt stress on growth, considerably restored pigment levels and increased of nitrate reductase activity.     

Improved synthesis of 17β-hydroxy-16α-iodo-wortmannin, 17β-hydroxy-16α-iodoPX866, and the [131I] analogue as useful PET tracers for PI3-kinase

An improved method for the synthesis of 17β-hydroxy-16α-iodo-wortmannin along with the first synthesis of 17β-hydroxy-16α-iodoPX866 and [¹³¹I] radiolabeled 17β-hydroxy-16α-[¹³¹I]iodo-wortmannin, as potential PET tracers for PI3K was also described. The differences between wortmannin and its iodo analogue were compared by covalently docking each structure to L833 in PI3K.     

SHY2 as a node in the regulation of root meristem development by auxin,brassinosteroids, and cytokinin

In multicellular organisms, the balance between cell division and differentiation determines organ size, and represents a central unknown in developmental biology. In Arabidopsis roots, this balance is mediated between cytokinin and auxin through a regulatory circuit converging on the IAA3/SHORT HYPOCOTYL 2 (SHY2) gene. Here, we show that crosstalk between brassinosteroids (BRs) and auxin occurs in the vascular transition zone to promote root meristem development. We found that BR increases root meristem size by up‐regulating expression of the PINFORMED 7 (PIN7) gene and down‐regulating expression of the SHY2 gene. In addition, BES1 could directly bind to the promoter regions of both PIN7 and SHY2, indicating that PIN7 and SHY2 mediate the BR‐induced growth of the root meristem by serving as direct targets of BES1. Moreover, the PIN7 overexpression and loss‐of‐function SHY2 mutant were sensitive to the effects of BR and could partially suppress the short‐root phenotypes associated with deficient BR signaling. Interestingly, BRs could inhibit the accumulation of SHY2 protein in response to cytokinin. Taken together, these findings suggest that a complex equilibrium model exists in which regulatory interactions among BRs, auxin, and cytokinin regulate optimal root growth.     

Effect of treatment with different doses of 17-beta-estradiol on the insulin receptor

The mechanism for the development of insulin resistance in normal pregnancy is complex and is associated with serum levels of sex hormones. However, the influence of these hormones on the early steps of insulin action has not been extensively studied, although the potentially beneficial effect of estradiol on glucose homeostasis has been reported. In this paper, we attempted to determine the effect of 17-beta-estradiol on the insulin receptor of ovariectomized rats treated with different doses of hormones. Our results showed a tissue-dependent response to estradiol. We found that low doses of estradiol increased the amount of insulin receptors in liver and muscle on days 6 and 11 of treatment but not in adipose tissue, and after 16 days only the muscle responsed in this way. On the other hand, high doses of estradiol significantly decreased the amount of insulin receptors, at least in muscle and adipose tissue. We believe that the low concentrations of 17-beta-estradiol (similar to early pregnancy) could be responsible for the increase in insulin sensitivity by increasing the amount of insulin receptors in peripheral tissues. When the hormone levels were high (similar to late pregnancy) the amount of insulin receptors decreased in peripheral tissues, and insulin sensitivity is diminished just as in late pregnancy. The specific molecular mechanism for this action is as yet unknown.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4-9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1-3, 5-8, 10 and 12-15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

Ethanol directly increases dihydrotestosterone conversion to 5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 alpha,17 beta-diol in rat Leydig cells

The direct effect of ethanol on dihydrotestosterone (DHT) conversion to 5 alpha-androstan-3 beta,17 beta-diol (3 beta-diol) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) by adult rat Leydig cells was examined. Concentrations of ethanol comparable to blood levels of alcoholic men (2.2 - 65 mM) increased DHT conversion to 3 beta - and 3 alpha-diol, in direct relation to the dose of ethanol added; a 2-fold or greater stimulation was observed. Because this effect was blocked by 4-methylpyrazole or a saturating NADH concentration, these results suggest that this action is mediated by Leydig cell alcohol dehydrogenase activity. These results may have significant impact in the testis and/or other DHT sensitive tissues because ethanol may decrease the availability of the proposed active androgen.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4–9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1–3, 5–8, 10 and 12–15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

Stereoselective synthesis of some 17beta-dihydrooxazinyl steroids, as novel presumed inhibitors of 17alpha-hydroxylase-C17,20-lyase

17beta-Dihydrooxazinyl steroids 5a-l and 6a-l were synthetized. The acid-catalyzed reactions of 21-azidomethyl-20-hydroxy- and 21-hydroxymethyl-20-azidosteroids with substituted aromatic aldehydes led to the formation of androst-5-en-3beta-ols substituted in position 17beta with dihydrooxazine residues. The inhibitory effects of these compounds on rat testicular C(17,20)-lyase were investigated with an in vitro radioincubation technique.