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.

Adamantyl carboxamides and acetamides as potent human 11ß-hydroxysteroid dehydrogenase type 1 inhibitors.

The modulation of 11ß-HSD1 activity with selective inhibitors has beneficial effects on various metabolic disorders including insulin resistance, dyslipidemia and obesity. Here we report the discovery of a series of novel adamantyl carboxamide and acetamide derivatives as selective inhibitors of human 11ß-HSD1 in HEK-293 cells transfected with the HSD11B1 gene. Optimization based on an initially identified 11ß-HSD1 inhibitor (3) led to the discovery of potent inhibitors with IC(50) values in the 100 nM range. These compounds are also highly selective 11ß-HSD1 inhibitors with no activity against 11ß-HSD2 and 17ß-HSD1. Compound 15 (IC(50)=114 nM) with weak inhibitory activity against the key human cytochrome P450 enzymes and moderate stability in incubation with human liver microsomes is worthy of further development. Importantly, compound 41 (IC(50)=280 nM) provides a new lead that incorporates an adamantyl group surrogate and should enable further series diversification.

Rapid and Efficient Microwave-Assisted Friedländer Quinoline Synthesis.

A microwave-based methodology facilitates reaction of 2-aminophenylketones with cyclic ketones to form a quinoline scaffold. Syntheses of amido- and amino-linked 17ß-hydroxysteroid dehydrogenase type 3 inhibitors with a benzophenone-linked motif were pursued using 2-aminobenzophenone as building block. Two amido-linked targets were achieved in modest yield, but when using microwave-assisted reductive amination for the amino-linked counterparts an unexpected product was observed. X-ray crystallography revealed it as a quinoline derivative, leading to optimisation of a simple and efficient modification of Friedländer methodology. Using reagents and acetic acid catalyst in organic solvent the unassisted reaction proceeds only over several days and in very poor yield. However, by employing neat acetic acid as both solvent and acid catalyst with microwave irradiation at 160 °C quinoline synthesis is achieved in 5 minutes in excellent yield. This has advantages over the previously reported high temperatures or strong acids required, not least given the green credentials of acetic acid, and examples using diverse ketones illustrate applicability. Additionally, he unassisted reaction proceeds effectively at room temperature, albeit much more slowly.

Discovery of novel inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenases (11beta-HSDs) are key enzymes regulating the pre-receptor metabolism of glucocorticoid hormones, which play essential roles in various vital physiological processes. The modulation of 11beta-HSD type 1 activity with selective inhibitors has beneficial effects on various conditions including insulin resistance, dyslipidemia and obesity. Therefore, inhibition of tissue-specific glucocorticoid action by regulating 11beta-HSD1 constitutes a promising treatment for metabolic and cardiovascular diseases. Here we report the discovery of a series of novel adamantyl carboxamides as selective inhibitors of human 11beta-HSD1 in HEK-293 cells transfected with the HSD11B1 gene. Compounds 9 and 14 show inhibitory activity against 11beta-HSD1 with IC(50) values in 100nM range. Docking studies with the potent compound 8 into the crystal structure of human 11beta-HSD1 (1XU9) reveals how the molecule may interact with the enzyme and cofactor.

Development of hormone-dependent prostate cancer models for the evaluation of inhibitors of 17beta-hydroxysteroid dehydrogenase type 3.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

The design of novel 17beta-hydroxysteroid dehydrogenase type 3 inhibitors.

17beta-Hydroxysteroid dehydrogenase type 3 (17beta-HSD3) is expressed at high levels in the testes and seminal vesicles but has also been shown to be present in prostate tissue, suggesting its potential involvement in both gonadal and non-gonadal testosterone biosynthesis. The role of 17beta-HSD3 in testosterone biosynthesis makes this enzyme an attractive molecular target for small molecule inhibitors for the treatment of prostate cancer. Here we report the design of selective inhibitors of 17beta-HSD3 as potential anti-cancer agents. Due to 17beta-HSD3 being a membrane-bound protein a crystal structure is not yet available. A homology model of 17beta-HSD3 has been built to aid structure-based drug design. This model has been used with docking studies to identify a series of lead compounds that may give an insight as to how inhibitors interact with the active site. Compound 1 was identified as a potent selective inhibitor of 17beta-HSD3 with an IC(50)=700nM resulting in the discovery of a novel lead series for further optimisation. Using our homology model as a tool for inhibitor design compound 5 was discovered as a novel potent and selective inhibitor of 17beta-HSD3 with an IC(50) approximately 200nM.

17beta-hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for endocrine therapy of hormone-dependent breast cancer.

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

Novel inhibitors of 17beta-hydroxysteroid dehydrogenase type 1: templates for design.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone (E1) to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Syntheses and biological evaluation of novel non-steroidal inhibitors designed to mimic the E1 template are reported using information from potent steroidal inhibitors. Of the templates investigated biphenyl ethanone was promising and led to inhibitors with IC(50) values in the low micromolar range.

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11beta-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11beta-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18beta-glycyrrhetinic acid (18beta-GA) derivatives (2-5) and their inhibitory activities against rat hepatic11beta-HSD1 and rat renal 11beta-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds' ability to inhibit human 11beta-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18beta-GA derivatives 2 and 3 with apparent selectivity for rat 11beta-HSD1 showed a high percentage inhibition for human microsomal 11beta-HSD1 at 10 microM and exhibited IC50 values of 400 and 1100 nM, respectively. The side chain modified 18beta-GA derivatives 4 and 5, although showing selectivity for rat 11beta-HSD1 inhibited human microsomal 11beta-HSD1 with IC50 values in the low micromolar range.

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11beta-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11beta-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11beta-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11beta-HSD1 inhibitors that inhibit human 11beta-HSD1 in the low micromolar range. Docking studies with 1-3 into the crystal structure of human 11beta-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.

Modification of estrone at the 6, 16, and 17 positions: novel potent inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Here we report the syntheses and biological evaluation of novel inhibitors based on the estrone or estradiol template. These have been investigated by modification at the 6, 16 or 17 positions or combinations of these in order to gain information about structure-activity relationships by probing different areas in the enzyme active site. Activity data have been incorporated into a QSAR with predictive power, and the X-ray crystal structures of compounds 15 and 16c have been determined. Compound 15 has an IC50 of 320 nM for 17beta-HSD1 and is selective for 17beta-HSD1 over 17beta-HSD2. Three libraries of amides are also reported that led to the identification of inhibitors 19e and 20a, which have IC50 values of 510 and 380 nM respectively, and 20 h which, having an IC50 value of 37 nM, is the most potent inhibitor of 17beta-HSD1 reported to date. These amides are also selective for 17beta-HSD1 over 17beta-HSD2.

Benzothiazole derivatives as novel inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1.

Selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) have considerable potential as treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here, we report the discovery and synthesis of a series of novel benzothiazole derivatives and their inhibitory activities against 11beta-HSD1 from human hepatic microsomes measured using a radioimmunoassay (RIA) method. The benzothiazole derivatives 1 and 2 showed greater than 80% inhibition for 11beta-HSD1 at 10 microM and exhibited IC50 values in the low micromolar range. The preliminary SAR study suggested the introduction of a chlorine substituent at the 4 position of the benzothiazole ring greatly enhanced the inhibitory activities. Docking studies with the benzothiazole derivative 1 into the crystal structure of human 11beta-HSD1 revealed how the molecule may interact with the enzyme and cofactor.

Novel, potent inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

Many breast tumours are hormone-responsive and rely on estrogens for their sustained growth and development. The enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is primarily responsible for the conversion of estrone (E1) into the most potent of the human estrogens 17beta-estradiol (E2). Here we report the syntheses, inhibitory activities and docking studies for a novel series of pyrazole amides which have been discovered with the aim of probing the structure activity relationships (SAR) for such a template and of using this template to mimic the potent inhibitor 1 (Fig. 1). Amides containing an aromatic pyridyl moiety have been found to give the best inhibition, indicating that the pyridyl group interacts beneficially in the active site. This work has shown that extension from this position on the pyrazole template is well tolerated and the optimization of such systems is under investigation.

17Beta-hydroxysteroid dehydrogenase Type 1 and Type 2: association between mRNA expression and activity in cell lines.

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) are a family of enzymes that regulate steroid availability within a tissue by catalysing the interconversion of active and inactive forms. Type 1 is up-regulated in many breast tumours, and is responsible for the reduction of oestrone to active oestradiol which stimulates cell proliferation within the tumour. Type 2 oxidises many active steroids to their inactive forms, including oestradiol to oestrone. In this study, we have compared the mRNA expression and enzyme activities of Type 1 and Type 2 in MCF-7, MDA-MB-231, T47D, JEG3 and 293-EBNA cell lines. Also studied were two cell lines stably expressing transfected Type 1 cDNA. RT-PCR indicated that little Type 1 mRNA is expressed in two of the breast cancer cell lines, MCF-7 and MDA-MB-231, and in 293-EBNA cells, but that expression is much higher in the T47D breast cancer cell line, and in the choriocarcinoma cell line, JEG3. However, a higher level of expression of Type 1 is seen in the transfected cell lines MCF-7.8H and 293-EBNA[His617beta-HSD1]. Activity assays show that there is high association between mRNA expression and enzyme activity. Assays indicate that, with the exception of MDA-MB-231 cells, Type 2 activity is low in these lines. The study of the basal activities of these enzymes will be used in future studies investigating the regulation of the enzymes by endogenous and exogenous factors. An understanding of their regulation in both healthy and malignant tissues may lead to future therapeutic intervention at the regulatory level.

Novel and potent 17beta-hydroxysteroid dehydrogenase type 1 inhibitors.

Structure-based drug design using the crystal structure of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) led to the discovery of novel, selective, and the most potent inhibitors of 17beta-HSD1 reported to date. Compounds 1 and 2 contain a side chain with an m-pyridylmethyl-amide functionality extended from the 16beta position of a steroid scaffold. A mode of binding is proposed for these inhibitors, and 2 is a steroid-based 17beta-HSD1 inhibitor with the potential for further development.

E-ring modified steroids as novel potent inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are an important class of steroidogenic enzymes that regulate the bioavailability of active estrogens and androgens and are as yet a relatively unexploited therapeutic target. Based on our investigations and those of others, E-ring modified steroids were identified as a useful template for the design of inhibitors of 17beta-HSD type 1, an enzyme involved in the conversion of estrone into estradiol. The synthesis and biological evaluation of a new series of N- and C-substituted 1,3,5(10)-estratrien-[17,16-c]-pyrazoles and the corresponding SAR are discussed. Among the N-alkylated analogues, the most potent inhibitor was the 1'-methoxyethyl derivative, 41, with an IC(50) of 530 nM in T47-D human breast cancer cells. The X-ray crystal structure of the 1'-isobutyl derivative, was determined. Further optimization of the template using parallel synthesis resulted in a library of C5'-linked amides from which 73 emerged. This pyridylethyl amide had an IC(50) of 300 nM and its activity, with that of 41, suggests the importance of hydrogen bond acceptor groups in the pyrazole side chain. Both 41 and 73 displayed selectivity over 17beta-HSD type 2, and preliminary investigations showed 41 to be nonestrogenic in vitro in a luciferase reporter gene assay in contrast to the parent pyrazole 25. Molecular modeling studies, which support these findings, and a QSAR, the predictive power of which was demonstrated, are also presented.

Structure-activity relationships for pyrido-, imidazo-, pyrazolo-, pyrazino-, and pyrrolophenazinecarboxamides as topoisomerase-targeted anticancer agents.

Heterocyclic phenazinecarboxamides were prepared by condensation of aminoheterocycles and 2-halo-3-nitrobenzoic acids, followed by reductive ring closure and amidation. They showed similar inhibition of paired cell lines that underexpressed topo II or overexpressed P-glycoprotein, indicating a non topo II mechanism of cytotoxicity and indifference to P-glycoprotein mediated multidrug resistance. Compounds with a fused five-membered heterocyclic ring were generally less potent than the pyrido[4,3-a]phenazines. A 4-methoxypyrido[4,3-a]phenazine (IC(50)s 2.5-26 nM) gave modest (ca. 5 day) growth delays in H69/P xenografts with oral dosing.

Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance.

Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.

Novel angular benzophenazines: dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents.

A series of substituted angular benzophenazines were prepared using a new synthetic route via a novel regiocontrolled condensation of 1,2-naphthoquinones and 2,3-diaminobenzoic acids. The synthesis and biological activity of this new series of substituted 8,9-benzo[a]phenazine carboxamide systems are described. The analogues were evaluated against the H69 parental human small cell lung carcinoma cell line and H69/LX4 resistant cell line which overexpresses P-glycoprotein. Selected analogues were evaluated against the COR-L23 parental human non small cell lung carcinoma cell line and the COR-L23/R resistant cell line which overexpresses multidrug resistance protein. This series of novel angular benzophenazines were potent cytotoxic agents in these cell lines and may be able to circumvent multidrug resistance mechanisms which result in the lack of efficacy of many drugs in cancer chemotherapy. These compounds show dual inhibition of topoisomerase I and topoisomerase II and thus target two key enzymes responsible for the topology of DNA that are active at different points in the cell cycle. The introduction of chirality into the carboxamide side chain of these novel benzophenazine carboxamides has resulted in the discovery of a potent enantiospecific series of cytotoxic agents, exemplified by 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576 ((R)-4j' '). In vivo activity has been demonstrated for 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576, after intravenous administration to female mice, and this compound has been selected as a development candidate for further evaluation.