Chemical Synthesis and Biological Evaluation of 3-Substituted Estrone/Estradiol Derivatives as 17ß-Hydroxysteroid Dehydrogenase Type 1 Inhibitors Acting via a Reverse Orientation of the Natural Substrate Estrone.

Estradiol (E2) plays an important role in the progression of diseases such as breast cancer and endometriosis. Inhibition of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), the enzyme that catalyzes the last step in the biosynthesis of the estrogenic hormone E2, therefore constitutes an interesting approach for the treatment of these two estrogen-dependent diseases. In order to obtain new inhibitors of 17ß-HSD1, the impact of a m-carbamoylphenyloxy group at position three of an estrane nucleus was evaluated by preparing three derivatives of estrone (E1) and E2 using a microwave-assisted synthesis of diaryl ethers. Their inhibitory activity was addressed on two cell lines (T-47D and Z-12) representative of breast cancer and endometriosis, respectively, but unlike T-47D cells, Z-12 cells were not found suitable for testing potential 17ß-HSD1 inhibitors. Thus, the addition of the m-carbamoylphenyl group at C3 of E1 (compound 5) did not increase the inhibition of E1 to E2 transformation by 17ß-HSD1 present in T-47D cells (IC50 = 0.31 and 0.21 µM for 5 and E1, respectively), and this negative effect was more obvious for E2 derivatives 6 and 10 (IC50 = 1.2 and 1.3 µM, respectively). Molecular docking allowed us to identify key interactions with 17ß-HSD1 and to highlight these new inhibitors' actions through an opposite orientation than natural enzyme substrate E1's classical one. Furthermore, molecular modeling experiments explain the better inhibitory activity of E1-ether derivative 5, as opposed to the E2-ether derivatives 6 and 10. Finally, when tested on T-47D and Z-12 cells, compounds 5, 6 and 10 did not stimulate the proliferation of these two estrogen-dependent cell lines. In fact, they reduced it.

Transcriptional coactivator with PDZ-binding motif suppresses the expression of steroidogenic enzymes by nuclear receptor 4 A1 in Leydig cells.

Transcriptional coactivator with PDZ-binding motif (TAZ) plays crucial role in maintaining testicular structure and function via regulation of senescence of spermatogenic cells. However, it remains unclear whether TAZ is involved in testosterone biosynthesis in testicular Leydig cells. We found that TAZ deficiency caused aberrant Leydig cell expansion and increased lipid droplet formation, which was significantly associated with increased lipogenic enzyme expression. Additionally, the expression of key steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 (CYP) 11A1, CYP17A1, and 3ß-hydroxysteroid dehydrogenase, was greatly increased in TAZ-deficient testes and primary Leydig cells. Interestingly, the transcriptional activity of nuclear receptor 4 A1 (NR4A1) was dramatically suppressed by TAZ; however, the protein expression and the subcellular localization of NR4A1 were not affected by TAZ. TAZ directly associated with the N-terminal region of NR4A1 and substantially suppressed its DNA-binding and transcriptional activities. Stable expression of TAZ in the mouse Leydig TM3 cell line decreased the expression of key steroidogenic enzymes, whereas knockdown of endogenous TAZ in TM3 cells increased transcripts of steroidogenic genes induced by NR4A1. Consistently, testosterone production was enhanced within TAZ-deficient Leydig cells. However, TAZ deficiency resulted in decreased testosterone secretion caused by dysfunctional mitochondria and lysosomes. Therefore, TAZ plays essential role in NR4A1-induced steroidogenic enzyme expression and testosterone production in Leydig cells.

Cloning and identification of a new repressor of 3,17ß-Hydroxysteroid dehydrogenase of Comamonas testosteroni.

BACKGROUND: 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17ß-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17ß-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17ß-HSD are still unknown. METHODS AND RESULTS: In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17ß-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17ß-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17ß-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17ß-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. CONCLUSIONS: The second tetR gene acts as a negative regulator in 3,17ß-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.

Preclinical Pharmacokinetics, Tissue Distribution and Primary Safety Evaluation of a Novel Curcumin Analogue H10 Suspension, a Potential 17ß Hydroxysteroid Dehydrogenase Type 3 Inhibitor.

17ß Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is the key enzyme in the biosynthesis of testosterone, which is an attractive therapeutic target for prostate cancer (PCa). H10, a novel curcumin analogue, was identified as a potential 17ß-HSD3 inhibitor. The pharmacokinetic study of H10 in rats were performed by intraperitoneal (i.p.), intravenous (i.v.) and oral (p.o.) administration. In addition, the inhibitory effects of H10 against liver CYP3A4 were investigated in vitro using human liver microsomes (HLMs). The acute and chronic toxicological characteristics were characterized using single-dose and 30 d administration. All the mice were alive after i.p. H10 with dose of no more than 100 mg/kg which are nearly the maximum solubility in acute toxicity test. The pharmacokinetic characteristics of H10 fitted with linear dynamics model after single dose. Furthermore, H10 could bioaccumulate in testis, which was the target organ of 17ß-HSD3 inhibitor. H10 distributed highest in spleen, and then in liver both after single and multiple i.p. administration. Moreover, H10 showed weak inhibition towards liver CYP3A4, and did not cause significant changes in aspartate transaminase (AST) and alanine transaminase (ALT) levels after treated with H10 for continuously 30 d. Taken together, these preclinical characteristics laid the foundation for further clinical studies of H10.

Substituted Aryl Benzylamines as Potent and Selective Inhibitors of 17ß-Hydroxysteroid Dehydrogenase Type 3.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed at high levels in testes and seminal vesicles; it is also present in prostate tissue and involved in gonadal and non-gonadal testosterone biosynthesis. The enzyme is membrane-bound, and a crystal structure is not yet available. Selective aryl benzylamine-based inhibitors were designed and synthesised as potential agents for prostate cancer therapeutics through structure-based design, using a previously built homology model with docking studies. Potent, selective, low nanomolar IC50 17ß-HSD3 inhibitors were discovered using N-(2-([2-(4-chlorophenoxy)phenylamino]methyl)phenyl)acetamide (1). The most potent compounds have IC50 values of approximately 75 nM. Compound 29, N-[2-(1-Acetylpiperidin-4-ylamino)benzyl]-N-[2-(4-chlorophenoxy)phenyl]acetamide, has an IC50 of 76 nM, while compound 30, N-(2-(1-[2-(4-chlorophenoxy)-phenylamino]ethyl)phenyl)acetamide, has an IC50 of 74 nM. Racemic C-allyl derivative 26 (IC50 of 520 nM) was easily formed from 1 in good yield and, to determine binding directionality, its enantiomers were separated by chiral chromatography. Absolute configuration was determined using single crystal X-ray crystallography. Only the S-(+)-enantiomer (32) was active with an IC50 of 370 nM. Binding directionality was predictable through our in silico docking studies, giving confidence to our model. Importantly, all novel inhibitors are selective over the type 2 isozyme of 17ß-HSD2 and show <20% inhibition when tested at 10 µM. Lead compounds from this series are worthy of further optimisation and development as inhibitors of testosterone production by 17ß-HSD3 and as inhibitors of prostate cancer cell growth.

Characterization of essential domains in HSD17B13 for cellular localization and enzymatic activity.

Human genetic studies recently identified an association of SNPs in the 17-ß hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/ß-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.

A novel hybrid method named electron conformational genetic algorithm as a 4D QSAR investigation to calculate the biological activity of the tetrahydrodibenzazosines.

To understand the structure-activity correlation of a group of tetrahydrodibenzazocines as inhibitors of 17ß-hydroxysteroid dehydrogenase type 3, we have performed a combined genetic algorithm (GA) and four-dimensional quantitative structure-activity relationship (4D-QSAR) modeling study. The computed electronic and geometry structure descriptors were regulated as a matrix and named as electron-conformational matrix of contiguity (ECMC). A chemical property-based pharmacophore model was developed for series of tetrahydrodibenzazocines by EMRE software package. GA was employed to choose an optimal combination of parameters. A model has been developed for estimating anticancer activity quantitatively. All QSAR models were established with 40 compounds (training set), then they were considered for selective capability with additional nine compounds (test set). A statistically valid 4D-QSAR ( Rtraining2=0.856 , Rtest2=0.851 and q2 = 0.650) with good external set prediction was obtained.

Synthesis of substituted 15ß-alkoxy estrone derivatives and their cofactor-dependent inhibitory effect on 17ß-HSD1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a key enzyme in the biosynthesis of 17ß-estradiol. Novel estrone-based compounds bearing various 15ß-oxa-linked substituents and hydroxy, methoxy, benzyloxy, and sulfamate groups in position C3 as potential 17ß-HSD1 inhibitors have been synthesized. In addition, in vitro inhibitory potentials measured in the presence of excess amount of NADPH or NADH were investigated. We observed substantial inhibitory potentials for several derivatives (IC50 < 1 µM) and increased binding affinities compared to unsubstituted core molecules. Binding and inhibition were found to be cofactor-dependent for some of the compounds and we propose structural explanations for this phenomenon. Our results may contribute to the development of new 17ß-HSD1 inhibitors, potential drug candidates for antiestrogen therapy of hormone-dependent gynecological cancers.

Novel Benzothiazole-based Ureas as 17ß-HSD10 Inhibitors, A Potential Alzheimer's Disease Treatment.

: It has long been established that mitochondrial dysfunction in Alzheimer's disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10), also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17ß-HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17ß-HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.

Identification of steroidal derivatives inhibiting the transformations of allopregnanolone and estradiol by 17ß-hydroxysteroid dehydrogenase type 10.

17ß-Hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is a mitochondrial enzyme known for its potential role in Alzheimer's Disease (AD). 17ß-HSD10, by its oxidative activity, could decrease the concentration of two important neurosteroids, allopregnanolone (ALLOP) and 17ß-estradiol (E2), respectively preventing their neurogenesis and neuroprotective effects. Since the inhibition of 17ß-HSD10 could lead to a new treatment for AD, we developed two biological assays using labeled ALLOP or E2 as substrates to measure the inhibitory activity of compounds against pure 17ß-HSD10 protein. After the optimization of different parameters (time, concentration of enzyme, substrate and cofactor), analogs of the first reported steroidal inhibitor of 17ß-HSD10 in intact cells were screened to determine their inhibitory potency for the ALLOP or the E2 oxidation. One compound, androstane derivative 5, possesses the best dual inhibition against both transformations (ALLOP, IC50 = 235 µM and E2, IC50 = 610 µM). Some compounds are dual inhibitors to a lesser extent, and others seem selective for one of the transformations in particular. By developing two reliable assays and by identifying a first generation of steroidal inhibitors of pure 17ß-HSD10, this preliminary study opens the door to new and more potent inhibitors.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives.

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-ß-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5 µM and, furthermore, three of them produced ≥68% inhibition at 1 µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-ß-hydroxysteroid dehydrogenase 2 - a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.

Impact of androstane A- and D-ring inversion on 17ß-hydroxysteroid dehydrogenase type 3 inhibitory activity, androgenic effect and metabolic stability.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a major player in human endocrinology, being one of the most important enzymes involved in testosterone production. To capitalize on the discovery of RM-532-105, a steroidal 17ß-HSD3 inhibitor, we explored the effect of its backbone configuration on inhibitory activity, androgenic profile, and metabolic stability. Two modifications that greatly alter the natural shape of steroids, i.e. inversion of the methyl on carbon 13 (13α-CH3 instead of 13ß-CH3) and inversion of the hydrogen on carbon 5 (5ß-H instead of 5α-H), were tested after the syntheses in 6 steps of 2 isomeric forms (5α/13α-RM-532-105 (6a) and 5ß/13ß-RM-532-105 (6b), respectively) of the 17ß-HSD3 inhibitor RM-532-105 (5α/13ß-configurations). For compound 6b, a cis/trans junction of the A/B rings did not significantly alter the inhibitory activity on 17ß-HSD3 (IC50=0.15µM) as well as the liver microsomal stability (16.6% of 6b remaining after 1h incubation) compared to RM-532-105 (IC50=0.11µM and 14.1% remaining). In contrast, a trans/cis junction of C/D rings reduced the inhibitory activity on 17ß-HSD3 (IC50=1.09µM) but increased the metabolic stability with 29.4% of compound 6a remaining after incubation. The structural modifications represented by compounds 6a and 6b did not change the non-androgenicity profile of an androsterone derivative such as RM-532-105, but slightly increased its cytotoxic activity.

Potential Antiosteoporotic Natural Product Lead Compounds That Inhibit 17ß-Hydroxysteroid Dehydrogenase Type 2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) converts the active steroid hormones estradiol, testosterone, and 5α-dihydrotestosterone into their weakly active forms estrone, Δ4-androstene-3,17-dione, and 5α-androstane-3,17-dione, respectively, thereby regulating cell- and tissue-specific steroid action. As reduced levels of active steroids are associated with compromised bone health and onset of osteoporosis, 17ß-HSD2 is considered a target for antiosteoporotic treatment. In this study, a pharmacophore model based on 17ß-HSD2 inhibitors was applied to a virtual screening of various databases containing natural products in order to discover new lead structures from nature. In total, 36 hit molecules were selected for biological evaluation. Of these compounds, 12 inhibited 17ß-HSD2 with nanomolar to low micromolar IC50 values. The most potent compounds, nordihydroguaiaretic acid (1), IC50 0.38 ± 0.04 µM, (-)-dihydroguaiaretic acid (4), IC50 0.94 ± 0.02 µM, isoliquiritigenin (6), IC50 0.36 ± 0.08 µM, and ethyl vanillate (12), IC50 1.28 ± 0.26 µM, showed 8-fold or higher selectivity over 17ß-HSD1. As some of the identified compounds belong to the same structural class, structure-activity relationships were derived for these molecules. Thus, this study describes new 17ß-HSD2 inhibitors from nature and provides insights into the binding pocket of 17ß-HSD2, offering a promising starting point for further research in this area.

The protective effects of zinc in lead-induced testicular and epididymal toxicity in Wistar rats.

The aim of this study was to investigate the beneficial effects of zinc (Zn) in preventing lead (Pb)-induced reproductive toxicity in Wistar rats. The rats were divided into four groups, namely, control group, Pb group, Zn group, and Pb + Zn group. Animals were exposed to Pb (819 mg of Pb/L) or Zn (71 mg of Zn/L) or both through drinking water for 65 days. Rats exposed to Pb showed decreased weights of testes and accessory sex organs. Significant decrease in the testicular daily sperm production, epididymal sperm count, motility, viability, and number of hypoosmotic tail coiled sperm was observed in Pb-exposed rats. Testicular 3ß- and 17ß-hydroxysteroid dehydrogenase activity levels and circulatory testosterone levels were also decreased significantly in Pb-exposed rats. A significant increase in the lipid peroxidation products with a significant decrease in the activities of catalase and superoxide dismutase were observed in the testes and epididymis of Pb-exposed rats. Moreover, the testicular architecture showed lumens devoid of sperm in Pb-exposed rats. Supplementation of Zn mitigated Pb-induced oxidative stress and restored the spermatogenesis and steroidogenesis in Pb-exposed rats. In conclusion, cotreatment of Zn is effective for recovering suppressed spermatogenesis, steroidogenesis, elevated oxidative status, and histological damage in the testis of rats treated with Pb.

Interference of Paraben Compounds with Estrogen Metabolism by Inhibition of 17ß-Hydroxysteroid Dehydrogenases.

Parabens are effective preservatives widely used in cosmetic products and processed food, with high human exposure. Recent evidence suggests that parabens exert estrogenic effects. This work investigated the potential interference of parabens with the estrogen-activating enzyme 17ß-hydroxysteroid dehydrogenase (17ß-HSD) 1 and the estrogen-inactivating 17ß-HSD2. A ligand-based 17ß-HSD2 pharmacophore model was applied to screen a cosmetic chemicals database, followed by in vitro testing of selected paraben compounds for inhibition of 17ß-HSD1 and 17ß-HSD2 activities. All tested parabens and paraben-like compounds, except their common metabolite p-hydroxybenzoic acid, inhibited 17ß-HSD2. Ethylparaben and ethyl vanillate inhibited 17ß-HSD2 with IC50 values of 4.6 ± 0.8 and 1.3 ± 0.3 µM, respectively. Additionally, parabens size-dependently inhibited 17ß-HSD1, whereby hexyl- and heptylparaben were most active with IC50 values of 2.6 ± 0.6 and 1.8 ± 0.3 µM. Low micromolar concentrations of hexyl- and heptylparaben decreased 17ß-HSD1 activity, and ethylparaben and ethyl vanillate decreased 17ß-HSD2 activity. However, regarding the very rapid metabolism of these compounds to the inactive p-hydroxybenzoic acid by esterases, it needs to be determined under which conditions low micromolar concentrations of these parabens or their mixtures can occur in target cells to effectively disturb estrogen effects in vivo.

Sirtuin 4 Regulates Lipopolysaccharide Mediated Leydig Cell Dysfunction.

Bacterial lipopolysaccharide (LPS) is the most important contributing factor in pathogenesis of bacterial infection in male accessory glands; and it has shown to inhibit testicular steroidogenesis and induce apoptosis. The present study demonstrates that LPS causes mitochondrial dysfunction via suppression of sirtuin 4 (SIRT4); which in turn affects Leydig cell function by modulating steroidogenesis and apoptosis. LC-540 Leydig cells treated with LPS (10 µg/ml) showed impaired steroidogenesis and increased cellular apoptosis. The mRNA and protein expression of SIRT4 were decreased in LPS treated cells when compared to controls. The obtained data suggest that the c-Jun N-terminal kinase (JNK) activation suppresses SIRT4 expression in LPS treated Leydig cells. Furthermore, the overexpression of SIRT4 prevented LPS induced impaired steroidogenesis and cellular apoptosis by improving mitochondrial function. These findings provide valuable information that SIRT4 regulates LPS mediated Leydig cell dysfunction.

Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17ß-hydroxysteroid dehydrogenase.

We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17ß-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3ß-hydroxysteroid dehydrogenase activity toward 5ß-androstanes, 5ß-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17ß-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.

Synthesis of a dansyl-labeled inhibitor of 17ß-hydroxysteroid dehydrogenase type 3 for optical imaging.

The steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a therapeutic target in the management of androgen-sensitive diseases such as prostate cancer and benign prostate hyperplasia. In this Letter, we designed and synthesized the first fluorescent inhibitor of this enzyme by combining a fluorogenic dansyl moiety to the chemical structure of a known inhibitor of 17ß-HSD3. The synthesized compound 3 is a potent fluorogenic compound (λex=348 nm and λ em=498 nm). It crosses the cell membrane, keeps its fluorescent properties and is distributed inside the LNCaP cells overexpressing 17ß-HSD3, where it inhibits the transformation of 4-androstene-3,17-dione into the androgen testosterone (IC50=262 nM).

Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17ß-hydroxysteroid dehydrogenase.

The enzyme type 5 17ß-hydroxysteroid dehydrogenase 5 (17ß-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17ß-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ(4)-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17ß-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17ß-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia.

Synthesis and Biological Evaluation of Triazolyl 13α-Estrone-Nucleoside Bioconjugates.

2'-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne-azide click reaction (CuAAC). For the introduction of the azido group the 5'-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3'-hydroxy groups of the nucleosides were protected by acetyl groups and the 5'-hydroxy groups were modified by the tosyl-azide exchange method. The commonly used conditions for click reaction between the protected-5'-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17ß-hydroxysteroid dehydrogenase 1 (17ß-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC50 value of 9 µM). The thymidine conjugate displayed considerable 17ß-HSD1 inhibitory activity (IC50 = 19 µM).