Novel D-modified heterocyclic androstane derivatives as potential anticancer agents: Synthesis, characterization, in vitro and in silico studies.

Cancer remains a major health concern worldwide. The most frequently diagnosed types of cancer are caused by abnormal production or action of steroid hormones. In the present study, the synthesis and structural characterization of new heterocyclic androstane derivatives with D-homo lactone, 17α-(pyridine-2''-ylmethyl) or 17(E)-(pyridine-2''-ylmethylidene) moiety are presented. All compounds were evaluated for their anti-proliferative activity against HeLa cervical cancer cell line and non-cancerous kidney MDCK cells, where A-homo lactam compound 9A showed the greatest selectivity. Based on in vitro binding assays, N-formyl lactam compound 18 appeared to be the strong and isoform-selective ligand for ERα, while compound 9A displayed binding affinity for the GR-LBD, but also inhibited aldo-keto reductase 1C4 enzyme. Out of four selected compounds, methylpyrazolo derivative 13 showed potential for aromatase binding, while in silico studies provided insight into experimentally confirmed protein-ligand interactions.

In silico ADMET analysis of the A-, B- and D-modified androstane derivatives with potential anticancer effects.

The major challenge in the fight against cancer is to design new drugs that will be more selective for cancer cells, with fewer side effects. Synthetic steroids such as cyproterone, fulvestrant, exemestane and abiraterone are approved powerful drugs for the treatment of hormone-dependent diseases such as breast and prostate cancers. Therefore, androstane derivatives in 17-substituted, 17a-homo lactone and 16,17-seco series, with potent anticancer activity, were selected for pharmacokinetic and druglike predictions from the absorption, distribution, metabolism and excretion (ADME) models. In silico determination of physico-chemical and ADMET properties was performed using SwissADME and ProTox-II web tools. The possibility of gastrointestinal absorption and brain penetration was analyzed using the BOILED-Egg model, while the in silico evaluation of the similarities between selected steroid derivatives and FDA-approved drugs was carried out using the SwissSimilarity tool. Of all tested, two compounds that showed good in silico ADMET results, in addition to promising cytotoxicity and molecular docking results, could potentially be evaluated in in vivo tests.

Ring-fused 3ß-acetoxyandrost-5-enes as novel neuroprotective agents with cholinesterase inhibitory properties.

Alzheimer´s disease (AD) is an intellectual disorder caused by organic brain damage and cerebral atrophy, characterized by the loss of memory, judgment, and abstract thinking followed by declining cognitive functions, language, and the ability to perform daily living activities. Many efforts have been made to decrease the effects of the disease but also to block the neurodegenerative process. Cholinesterase inhibitors (ChEIs) are a group of medicines that act at the neurotransmission of acetylcholine, preventing its excessive breakdown and helping to improve cognitive functions in patients with AD. In this work, 16 chiral steroids, namely ring-fused 3ß-acetoxyandrost-5-ene derivatives, their precursor and two 16-dehydroprogesterone-derived dioximes, were assessed as cholinesterase inhibitors and neuroprotective agents. The results demonstrated that some of the tested steroids are cholinesterase inhibitors and the majority selective for acetylcholinesterase inhibition. Albeit, one ring-fused 3ß-acetoxyandrost-5-ene containing N-methylpiperidine ring (compound 2g) demonstrated to be a selective and potent inhibitor of the butyrylcholinesterase enzyme. (S)- 4,4a,5,6,7,8-(hexahydronaphthalen-2-one)-fused 3ß-acetoxyandrost-5-ene (compound 6) showed high neuroprotective effect, high ability to restore the mitochondrial membrane potential from glutamate intoxication, and dramatic improvement in cell morphology. The described results provided relevant structure-activity relationship data.

Chemometrics of anisotropic lipophilicity of anticancer androstane derivatives determined by reversed-phase ultra high performance liquid chromatography with polar aprotic and protic modifiers.

The research of the use of steroid compounds in the treatment of various types of cancer has a history of more than 50 years. Numerous steroid derivatives have expressed significant anticancer activity, however the thorough analysis of their physicochemical and toxicological properties is required prior to their clinical application. The present study is focused on the characterization of physicochemical properties of a series of previously synthesized new androstane derivatives (16E-hydroxyimino derivatives, D-homo lactones and D-seco dinitriles) with anticancer activity applying reversed-phase ultra high performance liquid chromatography (RP-UHPLC) system under isocratic conditions and chemometric tools, including in silico determination of their absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The chromatographic analysis was carried out applying two two-component and one ternary mobile phases with aprotic (acetonitrile) and protic (water and methanol) solvents. The conducted quantitative structure-retention relationship modeling resulted in two univariate and nine multivariate linear mathematical models that successfully correlate the retention parameters (logk) with descriptors of molecular polarity/lipophilicity (tPSA, REF, Average LogP, ClogP, ALOGP, LogS-SILICOS-IT, AClogS), descriptors that mainly depend on intermolecular interactions (BP, CP, CT, DE, HL) and ADMET descriptors (SWISSLogKpSP, GPCR, HIA, EI). The quality of the models was proved by the internal and external validation, while the robustness of the models was confirmed by Y-randomization test. Considering the established meaningful relationships between physicochemical/ADMET properties and retention parameters, the determined logk parameters of the analyzed series of steroid derivatives can be considered to be a biopharmaceutical property from the perspective of lipophilicity.

Chemical synthesis, NMR characterization, and anticancer activity of androstene derivatives with a C17-side chain.

Cancer remains one of the leading causes of death, worldwide. In addition, the lack of efficacy and selectivity of chemotherapeutic agents for cancer cells is a challenge that needs to be addressed through the development of new drugs. Since aminosteroids are of interest in fighting cancer, our group previously reported antiproliferative activity on several cancer cell lines of two representatives, RM-133 and RM-581. To extend the structure-activity relationship study of aminosteroids, of which RM-133 (androstane) and RM-581 (estrane) are the main candidates, we performed the chemical synthesis and biological evaluation on lung (SHP-77), breast (T-47D) and prostate (DU-145, PC-3 and LAPC-4) cancer cells of four analogues of RM-581. We moved the functionalized side chain from position 2 of the androstane and estrane derivatives to incorporate it into a new chain located at position 17. Chemical synthesis took place in 2 steps from steroidal side-chain carboxylic acids, allowing to obtain 4 steroid derivatives with acceptable yields, which were fully characterized by nuclear magnetic resonance spectroscopy (1H and 13C NMR). After the evaluation of compounds 12-15, lower antiproliferative activities varying from 12 to 54%, 0-33% and 0-63% were observed for SHP-77, DU-145 and PC-3 cell lines, respectively, while higher activities varying from 33 to 62% and 45-84% were observed for T-47D and LAPC-4 cell lines, respectively, when tested at 10 µM. Overall, it was observed that these aminosteroids have a lower cytotoxic activity than that of RM-581 and, that moving the side chain from steroid position C2 to C17 is clearly detrimental for antiproliferative activity. However, this work has enabled us to expand our knowledge of the structural requirements to maintain the anticancer activity of aminosteroid derivatives.

Cytotoxicity, in silico predictions and molecular studies for androstane heterocycle compounds revealed potential antitumor agent against lung cancer cells.

The IL6/JAK2/STAT3 axis dysregulation and the related downstream pathways are a major contributor to the progression of non-small-cell lung carcinoma (NSCLC) and mainly affect apoptosis. Furthermore, tubulin inhibitors are potential chemotherapeutic agents against NSCLC. In this study, we have provided new molecular insights into the antiproliferative activity of six 3ß-acetoxy-5α-androstane heterocycle compounds against NSCLC. The cell line A549, which represents a good model of NSCLC, was used to evaluate the antitumour activity of tested androstane derivatives, and non-cancerous gingival mesenchymal stem cell line (GMSC) were used to assess the specificity and toxicity of the tested compounds. Further on, molecular docking predictions were used to determine the molecular targets for the most promising cytotoxic compound. To assess apoptosis and cell cycle progression in treated A549 cells, flow cytometry was used. RT-qPCR and ELISA analyses were used to gain deep insights into cellular and molecular mechanisms. Results revealed that compound 4 has potential cytotoxicity on A549 cells, with lower IC50 value (27.36 µM). Moreover, in silico, compound 4 showed a good binding affinity to JAK2 and tubulin-colchicine soblidotin molecular targets. This was further confirmed on the molecular level. Compound 4 has also led to apoptosis and increased fragmentation of DNA, and mitochondrial dysfunction. Our findings have provided good evidence that compound 4 may be a dual inhibitor of IL6/JAK2/STAT3 and tubulin formation in lung cancer. These findings support further molecular exploration of this androstane derivative as promising anti-lung cancer agent.Communicated by Ramaswamy H. Sarma.

Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors.

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines.

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

Modified bile acids and androstanes-Novel promising inhibitors of human cytochrome P450 17A1.

Cytochromes P450 are key enzymes for steroid hormone biosynthesis in human body. They are considered as targets for the screening of novel high efficient drugs. The results of screening of bile acids and androstane derivatives toward human recombinant steroid 17α-hydroxylase/17,20-lyase (CYP17A1) are presented in this paper. A group of steroids, binding with micromolar or submicromolar affinity (in a range from 9 µM - less than 0.1 µM), was identified. Results presented here showed that these steroidal compounds are able to decrease rate of hydroxylation of essential CYP17A1 substrate - progesterone, while some compounds completely inhibited enzyme activity. Structure-activity relationship (SAR) analysis based on in vitro and in silico studies showed that high affinity of the enzyme to bile acids derivatives is correlated with side chain hydrophobicity and presence of hydroxyl or keto group at C3 position. From the other side, bile acid-derived compounds with more polar side chain or substituents at C7 and C12 positions possess higher Kd values. Among androstane-derived steroids couple of Δ5-steroids with hydroxyl group at C3 position, as well as 16,17-secosteroids, were found to be high affinity ligands of this enzyme. The data obtained could be useful for the design of novel highly efficient inhibitors of CYP17A1, since the bile acids-derived compounds are for first time recognized as effective CYP17A1 inhibitors.

Cytotoxic androstane derivatives from Sarcococca ruscifolia.

Sarcorusones A-D (1-4), four new androstane (C19-steroid) derivatives were characterized from Sarcococca ruscifolia along with five known compounds. Their structures were elucidated on the basis of extensive MS and NMR spectroscopic analysis. All the new structures share common 14-hydroxyl and 17-ketone functional groups, and compounds 2-4 feature a seneciamide group connecting to C-3 position. The inhibitory activities of all the isolates against melanoma cell B16F10 and lung cancer cell H1299 were evaluated, and compounds 2, 3, 5, and 6 exhibited moderate cytotoxic activities against B16F10 and H1299 cell lines with IC50 values 2.7-8.0 µM.

New A-homo lactam D-homo lactone androstane derivative: Synthesis and evaluation of cytotoxic and anti-inflammatory activities in vitro.

This paper describes the synthesis of a new A-homo lactam D-homo lactone androstane derivative from dehydroepiandrosterone. To evaluate the impact of the introduction of nitrogen in the parental scaffold on biological activity, a new androstane enamide-type lactam derivative was prepared and characterized. The new compound as well as starting compounds were screened for cytotoxic, anti-angiogenic and anti-inflammatory activities using several human cancer cell lines (MCF-7, MDA-MB-231, PC3, CEM, G-361, HeLa), endothelial (HUVEC) and non-tumour (MRC-5 and BJ) cell lines. Strong cytotoxic and anti-inflammatory activity with a broad therapeutical window was demonstrated by the A-homo lactam D-homo lactone androstane derivative. The induction of apoptosis in treated PC3 cultures was confirmed using apoptotic morphology screening and a fluorescent double-staining method. New A-homo lactam D-homo lactone androstane derivative induced apoptosis more than the tested reference compounds, Formestane and Doxorubicin. An in silico ADME analysis showed that the compounds possess drug-like properties.

Molecular docking, anti-proliferative activity and induction of apoptosis in human liver cancer cells treated with androstane derivatives: Implication of PI3K/AKT/mTOR pathway.

Worldwide, cancer is still an area with high unmet medical need. Lead optimization efforts towards structure-based drug design were employed to discover newly synthesized hetero-steroid derivatives with promising anticancer effects against hepatocellular carcinoma (HCC). The aim of our study is to evaluate the anti-proliferative activity and the mechanism, a dual PI3K/mTOR inhibitor, and mechanism of action of a series of heterocylic androstane derivatives as anti-HCC agent. The cytotoxic effects of different heterocylic androstanes and 5FU as single agents, were assessed against both HepG2 cells and Non-malignant MDCK cell line to assess the toxicity. Then the underlying mechanism of compound 4 as most promising compound was evaluated using molecular docking, MTT assay, cell cycle analysis, DNA fragmentation, and real-time PCR. The results of MTT assay showed potential cytotoxic effect for compound 4 and 5 against liver cancer cell line with IC50 value 39.81 and 57.54 µM, respectively. Inhibition of the PI3K/AKT/mTOR pathway was achieved by compound 4, which was documented by molecular docking and augmented by gene expression analysis. Detailed mechanism revealed that compound 4 induced cell cycle arrest, DNA fragmentation, and induction of apoptosis by inhibition of anti-apoptotic genes, and upregulation of apoptotic genes. Our results shed a light on aminopyrazoloandrostane derivative 4 as an inhibitor of the PI3K/AKT/mTOR pathway, which might be acting as promising anti-liver cancer agent. Our data support further investigation of agents targeting the PI3K/AKT/mTOR.

Design, synthesis, and biological evaluation of novel androst-17ß-amide structurally related compounds as dual 5α-reductase inhibitors and androgen receptor antagonists.

Prostate cancer (PCa) is the second leading cause of death in men. Apart from androgen receptor, 5α-reductase has also been recognized as a potential target. In this study, a series of androst-17ß-amide compounds have been designed and synthesized targeting both AR and 5α-reductase. Their anti-proliferation activities were evaluated in AR + cell line 22RV1 and AR - cell line PC-3. The results indicated that most of the synthesized compounds inhibited the testosterone-stimulated cell proliferation with good selectivity and safety. Among all the compounds, androst[3,2-c]pyrazole derivatives (9a-9d) displayed the best inhibition activity comparable with flutamide. Moreover, most of the synthesized compounds displayed good 5α-reductase inhibitory activities with IC50 lower than 1 µM. The docking result of 9d-AR indicated that AR was forced to expands its binding cavity and maintain an antagonistic conformation since the steric hindrance of 9d impeded H12 transposition. Overall, compound 9d can be identified as a potential dual 5α-reductase inhibitor and AR antagonist, which might be of therapeutic importance for PCa treatment.

The effect of the androstane lung cancer inhibitor content on the cell-selective toxicity of hydroxyapatite-chitosan-PLGA nanocomposites.

An androstane (17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (derivative A)) cancer inhibitor was successfully captured in a carrier made of nano-sized hydroxyapatite (HAp) coated with chitosan-PLGA polymer blends (Ch-PLGA). In our previous studies, we demonstrated that it was convenient to use spherical HAp/Ch-PLGA carriers as vehicles to target the lungs following intravenous administration. In this study, we used emulsification and subsequent freeze-drying to load the spherical HAp/Ch-PLGA carriers with varying contents of the derivative A, in order to examine the selective toxicity towards cancerous/healthy lung cells. The XRD and FT-IR techniques confirmed the drug loading process, and the content of the poorly water soluble derivative A was estimated directly via the DSC technique. The particles were spherical in shape with the d50 distribution varying between 167 and 231 nm, whereas the content of the derivative A ranged from 6.5 to 19.3 wt%. Cell-selective cytotoxicity was examined simultaneously on two cell lines: human lung carcinoma (A549 ATCC CCL 185) and human lung fibroblasts (MRC-5 ATCC CCL 171). All particles exhibited nearly three times larger cytotoxicity towards cancer cells (A549) than towards healthy cells (MRC5), where the particles with the derivative A content of 6.5 wt% allowed for the viability of healthy cells >80%. Ninety-six hours after the treatment of cells with particles with different contents of derivative A (after incubation and recovery), recovery was faster in damaged healthy cells than in cancerous cells.

Multicomponent access to androstano-arylpyrimidines under microwave conditions and evaluation of their anti-cancer activity in vitro.

Novel ring D- and A-fused pyrimidines in the androstane series were efficiently synthesized within 10-15min in polar protic solvents under microwave irradiation via two kinds of multicomponent heterocyclization reactions followed by spontaneous or promoted oxidation. The rates of the one-pot catalyst-free transformations of steroidal ß-ketoaldehydes, ammonium acetate and substituted benzaldehydes in EtOH were found to be affected slightly by the steric and electronic feature of the substituents on the aromatic ring of the arylaldehyde component and the different reactivities of rings D and A of the sterane core. At the same time, the acid-catalyzed Biginelli-type reaction of dihydrotestosterone acetate, urea and arylaldehydes, and subsequent Jones oxidation of the primarily formed dihydropyrimidinones led to the corresponding ring A-fused 1H-pyrimidin-2-ones in moderate yields independently of the substituents on the aromatic moiety. The synthesized compounds were tested in vitro on human cancer cell lines as well as on non-cancerous fibroblast cells by the MTT assay in order to investigate their biological effects. As a result of the pharmacological screen, a remarkable structure-function relationship has been observed as the acetylated Biginelli products exhibited higher toxicity compared to the deacetylated version of each compound. Furthermore, in case of three 2'-arylpyrimidine derivatives a strong prostate cancer cell specific activity has been identified.

Anti-tumor efficacy of new 7α-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cells.

The majority of breast cancer cases are estrogen receptor positive (ER+). Although, third-generation aromatase inhibitors (AIs) are used as first-line treatment in post-menopausal women, they cause endocrine resistance and bone loss, which limits their success. Therefore, there is a demand to discover new potent molecules, with less toxicity that can circumvent these drawbacks. Our group has previously demonstrated that new 7α-substituted steroidal molecules, 7α-(2ξ,3ξ-epoxypropyl)androsta-1,4-diene-3,17-dione (3), 7α-allylandrost-4-ene-3,17-dione (6), 7α-allylandrost-4-en-17-one (9), 7α-allyl-3-oxoandrosta-1,4-dien-17ß-ol (10) and 7α-allylandrosta-1,4-diene-3,17-dione (12) are potent AIs in placental microsomes. In this work, it was investigated their anti-aromatase activity and in vitro effects in sensitive and resistant breast cancer cells. All the steroids efficiently inhibit aromatase in breast cancer cells, allowing to establish new structure-activity relationships for this class of compounds. Moreover, the new AIs can inhibit breast cancer cell growth, by causing cell cycle arrest and apoptosis. The effects of AIs 3 and 12 on sensitive cells were dependent on aromatase inhibition and androgen receptor (AR), while for AI 9 and AI 10 were AR- and ER-dependent, respectively. In addition, it was shown that all the AIs can sensitize resistant cancer cells being their behavior similar to the sensitive cells. In summary, this study contributes to the understanding of the structural modifications in steroidal scaffold that are translated into better aromatase inhibition and anti-tumor properties, providing important information for the rational design/synthesis of more effective AIs. In addition, allowed the discovery of new potent 7α-substituted androstane molecules to inhibit tumor growth and prevent endocrine resistance.

Facile and efficient access to Androsten-17-(1',3',4')-pyrazoles and Androst-17ß-(1',3',4')-pyrazoles via Vilsmeier reagents, and their antiproliferative activity evaluation in vitro.

In this work, twenty-seven novel steroidal pyrazole derivatives were designed and effectively synthesized with two different commercially available staring material, Isopregnanolone 1 and 5,16-Pregnadienolone 7, via the key intermediates, 17ß-(4'-formyl)pyrazolylandrost-3ß-yl formate and 17-(4'-formyl)pyrazolylandrost- 5,16-dienes-3ß-yl formate, which were obtained from the cyclization of steroidal phenylhydrazone with Vilsmeier reagent catalyzed by phosphorous oxychloride followed by hydrolysis, then Borch reduction to afford the target derivatives under mild conditions. Structures of these compounds were identified by 1H NMR, 13C NMR and high resolution mass spectrometry. Based on our previous work, the cytotoxicity of these derivatives were evaluated by the SRB method against 293T cell lines and three cancer cell lines: A549, Hela and MCF-7. The results indicated that compounds 5b-d, and 11a-e exhibited moderate to high cytotoxic activities with IC50 values ranging from 0.62 to 7.51 µM. Among the eight hybrids, compound 11b, with an ethyl amino and a dien-pregn moieties showed the highest potency, with an IC50 values of 0.87 µM and 0.53 µM for 293T cell lines and Hela cell lines, respectively. Some structure-activity relationships among the groups of the twenty-seven derivatives are discussed and identify several determinants important for the activity of these compounds. What's more, further molecular mechanism studies suggested that 11b one of the most potent derivatives caused Hela cell lines apoptosis and arrested the cell cycle at S phase in a concentration dependent manner.

Investigation of the In Vitro and In Vivo efficiency of RM-532-105, a 17ß-hydroxysteroid dehydrogenase type 3 inhibitor, in LAPC-4 prostate cancer cell and tumor models.

In the fight against androgen-sensitive prostate cancer, the enzyme 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is an attractive therapeutic target considering its key role in the formation of androgenic steroids. In this study, we attempted to assess the in vivo efficacy of the compound RM-532-105, an androsterone derivative developed as an inhibitor of 17ß-HSD3, in the prostate cancer model of androgen-sensitive LAPC-4 cells xenografted in nude mice. RM-532-105 did not inhibit the tumor growth induced by 4-androstene-3,17-dione (4-dione); rather, the levels of the androgens testosterone (T) and dihydrotestosterone (DHT) increased within the tumors. In plasma, however, DHT levels increased but T levels did not. In troubleshooting experiments, the non-androgenic potential of RM-532-105 was confirmed by two different assays (LAPC-4 proliferation and androgen receptor transcriptional activity assays). The enzyme 5α-reductase was also revealed to be the predominant enzyme metabolizing 4-dione in LAPC-4 cells, yielding 5α-androstane-3,17-dione and not T. Other 17ß-HSDs than 17ß-HSD3 seem responsible in the androgen synthesis. From experiments with LAPC-4 cells, we fortuitously came across the interesting finding that 17ß-HSD3 inhibitor RM-532-105 is concentrated inside tumors.

A new androstanoid metabolite from a soil fungus Curvularia borreriae strain HS-FG-237.

A new androstanoid metabolite, 4α-methyl-9α-methoxyandrosta-8,15-diene-3,17-dione (1), was isolated from a soil fungus Curvularia borreriae (Pleosporaceae) strain HS-FG-237. Its structure was determined by extensive spectroscopic and X-ray diffraction analyses. Compound 1 exhibited poor cytotoxicity toward HCT-116 cells by CCK-8 assay and weak anti-inflammatory activity in an ANA-1 murine macrophages model.

Selective anticancer activity of hydroxyapatite/chitosan-poly(d,l)-lactide-co-glycolide particles loaded with an androstane-based cancer inhibitor.

In an earlier study we demonstrated that hydroxyapatite nanoparticles coated with chitosan-poly(d,l)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous injection into mice. In this study we utilize an emulsification process and freeze drying to load the composite HAp/Ch-PLGA particles with 17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (A), a chemotherapeutic derivative of androstane and a novel compound with a selective anticancer activity against lung cancer cells. 1H NMR and 13C NMR techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The thermogravimetric and differential thermal analyses coupled with mass spectrometry were used to assess the thermal degradation products and properties of A-loaded HAp/Ch-PLGA. The loading efficiency, as indicated by the comparison of enthalpies of phase transitions in pure A and A-loaded HAp/Ch-PLGA, equaled 7.47wt.%. The release of A from HAp/Ch-PLGA was sustained, neither exhibiting a burst release nor plateauing after three weeks. Atomic force microscopy and particle size distribution analyses were used to confirm that the particles were spherical with a uniform size distribution of d50=168nm. In vitro cytotoxicity testing of A-loaded HAp/Ch-PLGA using MTT and trypan blue dye exclusion assays demonstrated that the particles were cytotoxic to the A549 human lung carcinoma cell line (46±2%), while simultaneously preserving high viability (83±3%) of regular MRC5 human lung fibroblasts and causing no harm to primary mouse lung fibroblasts. In conclusion, composite A-loaded HAp/Ch-PLGA particles could be seen as promising drug delivery platforms for selective cancer therapies, targeting malignant cells for destruction, while having a significantly lesser cytotoxic effect on the healthy cells.