Evaluation of 4'-substituted bicyclic pyridones as non-steroidal inhibitors of steroid 5alpha-reductase

4'-Substituted bicyclic pyridones were prepared and evaluated as non-steroidal inhibitors of type 1 and 2 steroid 5alpha-reductase (SR). A range of 4'-substituents were incorporated into the bicyclic scaffold to investigate SAR within and across different classes of non-steroidal inhibitors of SR. Bicyclic pyridones containing a 4'-benzoyl or long carbon chain tether showed more potent inhibition against type 1 SR than inhibitors with N-substituted acetamide groups in the 4'-position. SAR derived from 4'-substituted bicyclic pyridones reported here do not correlate with SAR derived from known potent 4'-substituted biaryl acid SR inhibitors. A 4'-benzoyl group is favoured by the active site in both isozymes.     

Tissue distribution and kinetic characteristics of rat steroid 5 alpha-reductase isozymes. Evidence for distinct physiological functions.

The enzyme steroid 5 alpha-reductase (5 alpha-reductase) catalyzes the reduction of delta 4,5 double bonds in a variety of substrates and is thought to play both catabolic and anabolic roles in steroid hormone metabolism. Here, we describe the isolation and characterization of a cDNA encoding the rat type 2 isozyme of 5 alpha-reductase and compare the kinetic properties and tissue-specific expression patterns of this isozyme with those of the type 1 isozyme. The type 2 isozyme has apparent Km values in the nanomolar range for steroid substrates, whereas the type 1 isozyme has micromolar affinities. The isozymes differ in their inhibition by various 4-azasteroids with the type 2 isozyme showing exquisite sensitivity (Ki = 40 pM) to 21,21-pentamethylene-4-aza-5 alpha-pregn-1-ene-3,20-dione. Messenger RNAs encoding the type 2 isozyme are more abundant than type 1 mRNAs in most male reproductive tissues, whereas the type 1 mRNAs predominate in peripheral tissues. Both 5 alpha-reductase mRNAs are more efficiently induced by dihydrotestosterone than by testosterone in the regenerating prostate. The differences in substrate affinities and tissue distributions of the 5 alpha-reductase isozymes suggest that type 2 plays an anabolic role and type 1 a catabolic role in the metabolism of androgens and other steroid hormones.     

Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2

5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure–activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic ω-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2.     

3,3-diphenylpentane skeleton as a steroid skeleton substitute: novel inhibitors of human 5alpha-reductase 1

We designed and synthesized novel type 1 5alpha-reductase inhibitors by using 3,3-diphenylpentane skeleton as a substitute for the usual steroid skeleton. 4-(3-(4-(N-Methylacetamido)phenyl)pentan-3-yl)phenyl dibenzylcarbamate (11k) is a competitive 5alpha-reductase inhibitor with the IC(50) value of 0.84 microM.     

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing.     

Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model

Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.     

Interaction between rat prostatic steroid 5 alpha-reductase and 3-carboxy-17 beta-substituted steroids: novel mechanism of enzyme inhibition

Efforts to identify novel compounds capable of blocking the steroid 5 alpha-reductase (SR) catalyzed conversion of testosterone (T) to 5 alpha-dihydrotestosterone have resulted in the development of 17 beta-substituted-3-androstene-3-carboxylic acids as potent inhibitors of the rat prostatic enzyme. The dead-end inhibition patterns of one of these steroidal acrylates, 17 beta-N-(2-methyl-2-propyl)-carbamoyl-androst-3,5-diene-3-carboxylic acid were best evaluated with a linear uncompetitive kinetic model vs both T (Kii = 11 +/- 1 nM) and NADPH (Kii = 22 +/- 2 nM). To interpret these observations, the kinetic mechanism of the rat prostatic SR was shown to involve the binding of NADPH prior to that of T through a series of dead-end and product inhibition experiments. Within the context of this preferentially ordered kinetic mechanism, it is proposed that the uncompetitive inhibition patterns result from the association of the steroidal acrylate to an enzyme complex containing NADP+ in formation of a dead-end ternary complex of enzyme, NADP+, and inhibitor.     

Synthesis and structure-activity relationship of potent bicyclic lactam thrombin inhibitors

A simple and versatile method for preparation of (D)-Phe-Pro peptidomimetic bicyclic thiazolidine lactams is presented. These bicyclic lactams have chemical diversity alpha to the lactam carbonyl and, when linked to electrophilic arginines, provide potent thrombin inhibitors.     

Studies on the mechanism of steroid 5 alpha-reductase inhibition by 3-carboxy A-ring aryl steroids

The mechanism of interaction between two 3-carboxy A-ring aryl steroids, 17 beta-(N,N-diisopropylcarboxamide)-estra-1,3,5(10)-triene-3-carboxy lic acid (1) and 17 beta-(N-t-butylcarboxamide)-estra-1,3,5(10)-triene-3-carboxylic acid (2), with rat hepatic and human prostatic steroid 5 alpha-reductases has been investigated. Dead-end inhibition plots with 1 and 2 versus both substrates (testosterone and NADPH) were linear-uncompetitive using either enzyme, while double-inhibition analyses indicated cooperative binding to enzyme between NADP+ and 1 or 2. These results were interpreted within the ordered kinetic mechanism of steroid 5 alpha-reductase to result from the preferential association of 3-carboxy A-ring aryl steroids to the enzyme-NADP+ complex. The direct displacement by 2 of a radioligand known to associate to this same enzyme form provides further support for these conclusions.     

Synthesis of 8-chloro-benzo[c]quinolizin-3-ones as potent and selective inhibitors of human steroid 5alpha-reductase 1

The synthesis of a series of differently substituted 8-chloro-benzo[c]quinolizin-3-ones, as potent and selective human steroid 5alpha-reductase type 1 inhibitors, has been accomplished by a four-step procedure based on the TiCl4-promoted tandem Mannich-Michael cyclization of 2-silyloxy-1,3-butadienes with N-t-Boc iminium ions from quinolin-2-ones. The presence on the benzo[c]quinolizinone nucleus of a methyl group and a double bond at positions 6 and 4-4a, respectively, as in compound 1d, gave rise to one of the most potent non-steroidal 5alphaR-1 inhibitors reported so far (IC50 = 14 nM).     

Four-amino acid segment in steroid 5 alpha-reductase 1 confers sensitivity to finasteride, a competitive inhibitor.

The 4-azasteroid 17 beta-(N-t-butyl)carbamoyl-4-aza-5 alpha-androst-1-en-3-one (finasteride) is 100-fold more potent as a competitive inhibitor of the rat NADPH:delta 4-3-oxosteroid-5-alpha- oxidoreductase (steroid 5 alpha-reductase) type 1 enzyme (Ki = 3-5 nM) than of the human type 1 enzyme (Ki greater than or equal to 300 nM). In this study, we exploit this differential sensitivity to map a major determinant of finasteride sensitivity in steroid 5 alpha-reductase. Chimeric steroid 5 alpha-reductase cDNAs composed of different combinations of rat and human exon sequences were created by genetic engineering, expressed in human embryonic kidney 293 cells, and assayed for their sensitivity to finasteride. Hybrid proteins containing sequences encoded by rat exon 1 were found to be as sensitive to finasteride as the parental enzyme. The exchange of progressively smaller protein segments encoded within exon 1 identified a tetrapeptide sequence (Val-Ser-Ile-Val) in the rat enzyme that conferred sensitivity to finasteride. The analogous sequence in the human enzyme (Ala-Val-Phe-Ala) conferred partial resistance to the drug. Finasteride was a competitive inhibitor of the native and all chimeric enzymes tested, suggesting that the tetrapeptide segments form a portion of the substrate-binding domain of steroid 5 alpha-reductase.     

Characterization and chromosomal mapping of a human steroid 5 alpha-reductase gene and pseudogene and mapping of the mouse homologue.

The enzyme steroid 5 alpha-reductase catalyzes the conversion of testosterone into the more powerful androgen, dihydrotestosterone. We previously described the cloning of rat and human cDNAs that encode steroid 5 alpha-reductase and their expression in oocytes and cultured cells. Here, we report the isolation, characterization, and chromosomal mapping of two human steroid 5 alpha-reductase genes. One gene (symbol SRD5A1) is functional, contains five exons separated by four introns, and maps to the distal short arm of chromosome 5. Two informative restriction fragment length polymorphisms are present in exons 1 and 2 of this gene. A second gene (symbol SRD5AP1) has all of the hallmarks of a processed pseudogene and was mapped to the q24-qter region of the X chromosome. In the mouse, a single steroid 5 alpha-reductase gene (Srd5 alpha-1) is linked to Xmv-13 on chromosome 13.     

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.     

Stable expression of human 5alpha-reductase type II in COS1 cells due to chromosomal gene integration: a novel tool for inhibitor identification

Inhibitors of human 5alpha-reductase type II are promising drug candidates for the treatment of benign prostatic hyperplasia which is associated with high prostatic DHT levels. In this study we describe the evaluation of potential inhibitors in a new cell assay. First a plasmid (pRcCMV-5alphaII) for the expression of human 5alpha-reductase type II was constructed by the use of the vector pRcCMV and transfected into the African green monkey fibroblast-like cell line COS1. By selection with G418 sulfate, ten COS1 single cell clones were obtained of which three stably exhibited high 5alpha-reductase activity. One single cell clone (COS1-5alphaIIST) was selected for further investigations. By Southern blot analysis, fluorescence in situ hybridization (FISH) and comparative PCR experiments it turned out that the expression plasmid pRcCMV-5alphaII has been integrated into the chromosome, resulting in a long-term stable expression of the foreign 5alpha-reductase gene. The newly established cell line was used for testing novel compounds on their inhibitory effect on human 5alpha-reductase type II. Using this whole cell assay, inhibitors with IC(50) values in the nanomolar range could be identified.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-substituted 4-(5-indolyl)benzoic acids. Synthesis and evaluation of steroid 5alpha-reductase type I and II inhibitory activity.

The synthesis of N-alkyl and N-arylalkyl substituted 4-(5-indolyl)benzoic acid derivatives as inhibitors of steroid 5alpha-reductases is described. For the human type II isozyme a benzyl substituent (IC50 6.20 microM) and for the human type I isozyme a cyclohexanemethyl substituent (IC50 2.10 microM) on the indole nitrogen proved to be most efficacious, thus providing interesting leads for the development of drugs for the treatment of benign prostatic hyperplasia (BPH).     

Structural/mechanistic insights into the efficacy of nonclassical β‐lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates

Clavulanic acid and avibactam are clinically deployed serine β‐lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo β‐lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine β‐lactamase) but not L1 (metallo β‐lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X‐ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam‐like β‐lactams plus nonclassical β‐lactamase inhibitors, particularly avibactam‐like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.     

Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: elucidation of the kinetic mechanism

A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.     

5-Phenyl substituted 1-methyl-2-pyridones and 4'-substituted biphenyl-4-carboxylic acids. synthesis and evaluation as inhibitors of steroid-5alpha-reductase type 1 and 2.

The synthesis of a series of 5-phenyl substituted 1-methyl-2-pyridones (I) and 4'-substituted biphenyl-4-carboxylic acids (II) as novel A-C ring steroidomimetic inhibitors of 5alpha-reductase (5alphaR) is described. Compounds 1-4 (I) were synthesized by palladium catalyzed cross coupling (Ishikura) reaction between diethyl(3-pyridyl)borane and aryl halides (1b-4b) followed by alpha-oxidation with sodium ferrocyanate of the 1-methyl-pyridinium salt. Inhibitors II (5-18) were obtained either by two successive Friedel-Crafts acylations from biphenyl (5a-10a) followed by saponification to yield the corresponding carboxylic acids (5-10) or by Suzuki cross coupling reaction to give the 4'-substituted biphenyl-4-carbaldehydes 11a-18a. The latter compounds were subjected to a Lindgren oxidation to yield compounds 11-18. The compounds were tested for inhibitory activity toward human and rat 5alphaR1 and 2. The test compounds inhibited 5alphaR, showing a broad range of inhibitory potencies. The best compound in series I was the N-(dicyclohexyl)-4-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl)benzamide 4 exhibiting an IC(50) value for the human type 2 enzyme of 10 microM. In series II, the most active compound toward human type 2 isozyme was the 4'-(dicyclohexyl)acetyl-4-biphenyl carboxylic acid (10; IC(50)=220nM). Both series showed only marginal activity toward the human type 1 isozyme. In conclusion, the biphenyl carboxylic acids (II) are more appropriate for 5alphaR inhibition than the 5-phenyl-1-methyl-2-pyridones (I). Especially the 4'-carbonyl compounds 5-10 represent new lead structures for the development of novel human type 2 inhibitors.     

Properties of a 4-ene-3-ketosteroid-5 alpha-reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144

When Eubacterium sp. 144 was grown in the presence of progesterone, extracts of these cells contained a 4-ene-3-ketosteroid-5 alpha-reductase (5 alpha-reductase). No evidence for the presence of a 5 beta-steroid-reductase or a 5 alpha to 5 beta-steroid-isomerase was found. 5 alpha-Reductase activity was dependent on reduced methyl viologen as the electron donor and this could be generated biologically by adding pyruvate or H2 to cell extracts or chemically by adding sodium dithionite. NADH or NADPH with or without flavin nucleotides were not electron donors for 5 alpha-reductase. Most of the 5 alpha-reductase activity (60-65%) of crude extracts was located in the membrane fraction and the enzyme was solubilized by treatment with 1% Triton X-100. Optimum 5 alpha-reductase activity occurred at pH 7.0-7.5 in potassium phosphate buffer but was stimulated by Tris-HCl buffer (pH 8.0-9.0). 5 alpha-Reductase activity was highest at 10% (v/v) methanol and was progressively inhibited by higher methanol concentrations. Sulfhydryl reagents strongly inhibited 5 alpha-reductase but the enzyme was not affected by other metabolic inhibitors. Extracts prepared from cells induced with 16-dehydroprogesterone and grown without hemin contained 5 alpha-reductase and 16-dehydroprogesterone-reductase activities equivalent to those found in extracts of induced cells grown with hemin. This indicates that hemin is not required for the synthesis of active steroid double bond-reductases in strain 144.