Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases.In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.     

7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties

7alpha,11beta-Dimethyl-19-nortestosterone, made by 1,6-methyl addition to 17beta-acetoxy-11beta-methylestra-4,6-dien-3-one, was a highly potent and selective androgen response modulator, with enhanced androgen receptor binding, androgenic activity and anabolic:androgenic ratio over its two monomethyl homologs.     

N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone synthase (CYP11B2)

A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit human steroid-11β-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most potent and selective CYP11B2 inhibitors (IC₅₀ values 53-166 nM) were further evaluated for their potential to inhibit human CYP17 and CYP19, and no inhibition was observed. Clear evidence was shown for N-(Pyridin-3-yl)benzamides to be a highly selective class of CYP11B2 inhibitors in vitro.     

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.     

Atg11 links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway

Proteins are selectively packaged into vesicles at specific sites and then delivered correctly to the various organelles where they function, which is critical to the proper physiology of each organelle. The precursor form of the vacuolar hydrolase aminopeptidase I is a selective cargo molecule of the cytoplasm to vacuole targeting (Cvt) pathway and autophagy. Precursor Ape1 along with its receptor Atg19 forms the Cvt complex, which is transported to the pre-autophagosomal structure (PAS), the putative site of Cvt vesicle formation, in a process dependent on Atg11. Here, we show that this interaction occurs through the Atg11 C terminus; subsequent recruitment of the Cvt complex to the PAS depends on central regions within Atg11. Atg11 was shown to physically link several proteins, although the timing of these interactions and their importance are unknown. Our mapping shows that the Atg11 coiled-coil domains are involved in self-assembly and the interaction with other proteins, including two previously unidentified partners, Atg17 and Atg20. Atg11 mutants defective in the transport of the Cvt complex to the PAS affect the localization of other Atg components, supporting the idea that the cargo facilitates the organization of the PAS in selective autophagy. These findings suggest that Atg11 plays an integral role in connecting cargo molecules with components of the vesicle-forming machinery.     

Atg11的功能研究进展

Atg11利用自身众多螺旋结构域作为支架蛋白,主要介导选择性自噬过程中自噬体的形成.选择性自噬可特异性清除损坏的生物大分子和细胞器,在真核生物的胞内物质周转及细胞器质量控制中起重要作用.本文首先介绍了Atg11的结构特点,其次重点介绍了Atg11在3种选择性自噬(细胞质到液泡靶向(Cvt)途径、过氧化物酶体自噬和线粒体自噬)中的作用,最后概括了Atg11的其他功能.本文系统总结了近几年关于Atg11的研究进展,以期为自噬体形成机制研究及Atg11在自噬体形成过程中的功能研究提供参考.     

The measurement of 4-androstene-3, 11, 17-trione (11-oxo-androstenedione) by radioimmunoassay in human plasma

A radioimmunoassay (RIA) method is described for the determination of 4-androstene-3, 11, 17-trione (11-oxo-androstenedione) in human plasma. 4-androstene-3, 11, 17-trione 3-(0-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate highly specific antiserum in rabbits. Cross reactivities of several other steroids with the antiserum were less than 4%. [1,2-3H] 4-androstene-3, 11, 17-trione was synthesized from [1,2-3H] 17 alpha, 21-dihydroxy-4-pregnene-3, 11, 20-trione. The intra- and interassay variation was 7.3% and 9.8%, respectively. The mean serum 4-androstene-3, 11, 17-trione level for healthy young subjects was 2.37 +/- 0.56 nM (X +/- SD) in males and 3.16 +/- 0.43 nM in females at 8 a.m. During the night, there was a marked decrease in serum level, giving at 11 p.m. 0.87 +/- 0.33 and 1.15 +/- 0.52 nM, respectively. During ACTH stimulation tests, 4-androstene-3, 11, 17-trione increased from 1.81 +/- 0.58 to 2.32 +/- 0.69 nM, while in dexamethasone suppression tests a decrease from 3.20 +/- 0.03 nM was seen. In contrast, HCG administration on 3 consecutive days did not influence plasma concentrations of 4-androstene-3, 11, 17-trione.     

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.     

Synthesis and identification of novel 11beta-aryl-4',5'-dihydrospiro[estra-4,9-diene-17beta,4'-oxazole] analogs with dissociated antiprogesterone activities

A series of novel 11beta-aryl-4',5'-dihydrospiro[estra-4,9-diene-17beta,4'-oxazole] analogs have been evaluated for their antagonist hormonal properties using the T47D cell-based alkaline phosphatase assay and the A549 cell-based functional assay. Some of the compounds showed highly potent, and more selective antiprogestational activity against antiglucocorticoid activity than mifepristone (RU 486).     

Discovery and initial SAR of arylsulfonylpiperazine inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)

High-throughput screening of a small-molecule compound library resulted in the identification of a series of arylsulfonylpiperazines that are potent and selective inhibitors of human 11beta-Hydroxysteroid Dehydrogenase Type 1 (11beta-HSD1). Optimization of the initial lead resulted in the discovery of compound (R)-45 (11beta-HSD1 IC(50)=3nM).     

Structure-activity relationships of estrogens: effects of esterification of the 11 beta-hydroxyl group

Fourteen esters (formate, acetate, propionate, butyrate, hexanoate, heptanoate, and benzoate) located at C-11 of 11 beta-hydroxyesterone and 11 beta-hydroxyestradiol-17 beta were synthesized and evaluated for uterotropic and gonadotropin release inhibition in rats, as well as their ability to displace (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor. The most potent uterotropic agent was 11 beta-formoxyestrone which was 1,625 or 2,500 times as active as 11 beta-hydroxyesterone in the uterotropic or gonadotropin release inhibition assay, respectively. 11 beta-Formoxyestrone was 7.5 times as uterotropic as estradiol-17 beta and equal to estradiol-17 beta in inhibiting gonadotropin release. However, the most potent inhibitor of gonadotropin release was 11 beta-acetoxy-estradiol-17 beta which had 133% of the activity of estradiol-17 beta, although it had only 38% of the activity of estradiol-17 beta in the uterotropic assay. Esters larger than the acetoxy group showed sharply decreased activities in either assay. Despite the high estrogenic potency of the 11-formates or 11-acetates, they were rather weak (6% to 35% as active as estradiol-17 beta) in displacing (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor.     

Adamantane 11-beta-HSD-1 inhibitors: Application of an isocyanide multicomponent reaction

A series of potent and selective adamantane aminoamide 11-beta-HSD-1 inhibitors has been optimized. Chemically these studies were expedited by utilizing readily obtained amino acids as starting materials or an isocyanide multicomponent reaction. Structure-activity relationship studies resulted in the discovery of dual human and mouse 11-beta-HSD-1 potent and selective inhibitors like adamantane 11 and related compounds with high metabolic stability and robust pharmacokinetic profiles.     

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii)

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17β-estradiol (E2). The results showed cyp11a was significantly increased at 10⁻⁶ mol ml^–1 of 17α-MT and 10⁻⁸ mol ml^–1 of E2 in ovary and 10⁻¹⁰ mol ml^–1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.     

11-Oxa and 17alpha-hydroxymethyl analogues of steroid hormones and their derivatives.

The syntheses of 11-oxa and 17alpha-hydroxymethyl analogues of steroid hormones and their derivatives are reported and some of their biological activities are discussed. Generally, the replacement of the 11-methylene group by oxygen results in a diminution of the progestational, androgenic-anabolic, and estrogenic activities. This effect is least pronounced in the case of the progestational activity of 11-oxa-ethisterone and particularly strong in the case of the uterotropic activity of 11-oxa-estradiol. 17alpha-acetoxymethylprogesterone and 17alpha-hydroxymethylprogesterone were synthesized by 2 pathways, 1 of which can be advantageously applied also to the synthesis of 17alpha-acyloxymethyl and 17alpha-hydroxymethyl glucocorticoids. 17alpha-acetoxymethylprogesterone was inactive in the Clauberg test even at high doses.     

Steroids and related products. XLI. (1) the synthesis of 11-oxa steroids. III. (2) 17-acetoxy-11-oxaprogesterone (3).

The synthesis of 17-acetoxy-11-oxaprogesterone, the 11-oxa analogue of the orally active progestational and anti-fertility agent 17-acetoxyprogesterone, is described. An intermediate in the synthesis of 11-oxaprogesterone, 11-oxa-5alpha-pregnane-3,20-dione, available from hecogenin, was used as starting material and the 17-hydroxy function was introduced by a modified Barton oxidation. The new hormone analogue shows only extremely weak progestational activity in the oral Clauberg assay.     

Steroids and related products. LIII. The synthesis of 11-oxa steroids. V. The synthesis of 17-ethynyl-11-oxatestosterone

The synthesis of 17-ethynyl-11-oxatestosterone, both from 11-oxa-5 alpha-pregnane-3,20-dione and, via a 3,17-dioxygenated 9-oxo 9,12-seco 11-nor 5 alpha-androstane-12-oic ester, from 3 beta-acetoxy-17-hydroxy-5 alpha-pregnan-12-one--two products available from hecogenin--is reported. The new hormone analogue shows significant progestational activity in the Clauberg test and relatively weak activity in a post-coital antifertility assay.     

A radioimmunoassay for serum 11-deoxy-17-ketosteroids

A simplified method for evaluating serum 11-deoxy-17-ketosteroids (11-deoxy-17-KS) equivalent to dehydroepiandrosterone sulfate (DHEAS) has been developed without solvolysis and chromatography. 5μl of serum or plasma was added to 1 ml of ethanol, mixed, and centrifuged. 10 or 20 μ1 of the supernatant was evaporated to dryness and incubated with anti-11-deoxy-17-KS antiserum obtained by immunizing a rabbit with DHEA-3·O·CO-BSA which was prepared from DHEA-3·O·COC1 and containing DHEAS-7α³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEAS-7α³H. The accuracy, precision and sensitivity were satisfactory. The blank values could not be differentiated from zero. As the antiserum reacted not only on DHEAS but also on androsterone sulfate and etiocholanolone sulfate, serum 11-deoxy-17KS obtained by the radioimmunoassay expressed nearly the sum of 100% of DHEAS, 45% of androsterone sulfate and 35% of etiocholanolone sulfate in the serum. A good correlation was found between serum 11-deoxy-17-KS and DHEAS obtained by the radioimmunoassay described in a preveous paper (1). The present radioimmunoassay is the simplest method for the evaluation of the concentrations of C₁₉ steroids in the serum.     

Discovery and biological evaluation of adamantyl amide 11beta-HSD1 inhibitors

A series of adamantyl amide 11beta-HSD1 inhibitors has been discovered and chemically modified. Selected compounds are selective for 11beta-HSD1 over 11beta-HSD2 and possess excellent cellular potency in human and murine 11beta-HSD1 assays. Good pharmacodynamic characteristics are observed in ex vivo assays.     

Novel 18beta-glycyrrhetinic acid analogues as potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenases

Extensive structural modifications to the 18beta-glycyrrhetinic acid template are described and their effects on the SAR of the 11beta-hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. Isoform selective inhibitors have been discovered and compound 7 N-(2-hydroxyethyl)-3beta-hydroxy-11-oxo-18beta-olean-12-en-30-oic acid amide is highlighted as a very potent selective inhibitor of 11beta-hydroxysteroid dehydrogenase 2 with an IC(50) = 4pM.     

Hydrolytic behavior of 5alpha-hydroxy-11beta- and 5beta-hydroxy-11alpha-substituted 19-norsteroids

Teutsch G. and Bélanger A. treated 5alpha,10alpha epoxides with Grignard-reagents catalyzed by copper(I) ions. The reaction with steroidal epoxides proceeded with complete regio- and stereospecificity, leading exclusively to the 11beta-substituted compounds. According to our synthetic strategy, the 5,10 epoxide isomers were not separated; instead, the pure 11beta, and in some cases, 11alpha-substituted molecules were isolated after the conjugate addition of the Grignard-reagents, followed by deketalization and dehydration. Surprisingly, appearance of a third compound was generally observed beside the expected deprotected products, and this compound turned out to have a 3-keto-5(10),9(11) structural unit. Starting from pure 3-ethylenedioxy-5alpha,10alpha-epoxy-estr-9(11)-ene-17-one and 3-ethylenedioxy-5beta,10beta-epoxy-estr-9(11)-ene-17-one, four model compounds were synthesized (11alpha- and 11beta-[4-[1,1-(ethylenedioxy)-ethyl]phenyl]-estra-, as well as 11alpha- and 11beta-cyclohexyl-estra-derivatives) to study the process of deprotection and dehydration. 3-keto-5(10),9(11)-derivatives were found to form after deketalization and dehydration only from 11alpha-substituted derivatives, while 11beta-derivatives resulted in only the expected 3-keto-5,9-diene structure. After observing this remarkable difference between the behavior of 11alpha-, 11beta-substituted isomers we decided to take a closer look at the processes of deketalization and dehydration. In order to carry out the hydrolysis under mild conditions, pyridinium paratoluenesulfonate, a weakly acidic salt, was applied. All the intermediate products observed by TLC were isolated. The outcome of the deprotection and elimination reactions can be rationalized by two factors: conjugation of olefins (with the 3-oxo-group or the 11-phenyl group) and orientation of groups to be eliminated.