A protectin DX (PDX) analog with in vitro activity against influenza A(H1N1) viruses.

Antiviral therapy based on neuraminidase (oseltamivir) or polymerase (baloxavir marboxil) inhibitors plays an important role in the management of influenza infections. However, the emergence of drug resistance and the uncontrolled inflammatory response are major limitations in the treatment of severe influenza disease. Protectins D1 (PD1) and DX (PDX), part of a family of pro-resolving mediators, have previously demonstrated anti-influenza activity as well as anti-inflammatory properties in various clinical contexts. Herein, we synthetized a series of simplified PDX analogs and assessed their in vitro antiviral activity against influenza A(H1N1) viruses, including oseltamivir- and baloxavir-resistant variants. In ST6GalI-MDCK cells, the PDX analog AN-137B reduced viral replication in a dose-dependent manner with IC50 values of 23.8 for A/Puerto Rico/8/1934 (H1N1) and between 32.6 and 36.7 µM for susceptible and resistant A(H1N1)pdm09 viruses. In MTS-based cell viability experiments, AN-137B showed a 50% cellular cytotoxicity (CC50 ) of 638.7 µM with a resulting selectivity index of 26.8. Of greater importance, the combination of AN-137B with oseltamivir or baloxavir resulted in synergistic and additive in vitro effects, respectively. Treatment of lipopolysaccharide (LPS)-stimulated macrophages with AN-137B resulted in a decrease of iNOS activity as shown by the reduction of nitrite production, suggesting an anti-inflammatory effect. In conclusion, our results indicate that the protectin analog AN-137B constitutes an interesting therapeutic modality against influenza A virus, warranting further evaluation in animal models.

17ß-hydroxysteroid dehydrogenase type 2 activity, expression and cellular localization in abdominal adipose tissues from women.

CONTEXT: 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2) may be involved in the local modulation of estradiol (E2) availability in adipose tissues. OBJECTIVE: To assess the conversion of E2 into estrone (E1) as well as the expression of 17ß-HSD2 and its localization in omental (OM) and subcutaneous (SC) adipose tissues obtained from women. METHODS: Rates of 14 C-E1 formation from 14 C-E2 were measured in OM and SC adipose tissue homogenates from 29 women. Specific 17ß-HSD2 inhibitor EM-919 was tested in OM and SC adipose tissue homogenates (n = 6). 17ß-HSD2 mRNA expression was measured in whole OM and SC adipose tissues (n = 14). Cellular localization of the enzyme was examined using immunohistochemistry. Anthropometric measurements were obtained and body composition as well as body fat distribution were measured. RESULTS: Significant 14 C-E1 formation from 14 C-E2 in OM and SC tissue homogenates was detected. The rate of 14 C-E1 formation was significantly higher in OM than SC adipose tissue (p < .0001). The conversion of 14 C-E2 to 14 C-E1 was significantly inhibited by EM-919 in OM (p < .05) and SC (p < .05) adipose tissues. Significantly higher expression of 17ß-HSD2 mRNA in OM versus SC fat was found (p = .03). 17ß-HSD2 was localized in the vasculature of OM and SC tissues. Significant negative associations were detected between OM 17ß-HSD2 activity and body mass index, WC, lean body mass as well as SC adipose tissue areas. CONCLUSION: 17ß-HSD2 converts E2 to E1 in OM and SC adipose tissues of women. The activity of this enzyme decreases with increasing adiposity.

A Concise, Gram-Scale Total Synthesis of Protectin DX and Related Labeled Versions via a Key Stereoselective Reduction of Enediyne.

We report a gram-scale total synthesis of protectin DX (PDX) following a convergent synthetic route (24 steps) from l-malic acid. This novel synthetic strategy is based on the assembly of three main building blocks using a Sonogashira coupling reaction (blocks A and B) and Wittig olefination (block C) to provide the 22-carbon backbone of PDX. A key stereoselective reduction of enediyne leads to a central E,Z,E-trienic system of PDX and also gives access to its labeled versions (D and T).

Description of Chemical Synthesis, Nuclear Magnetic Resonance Characterization and Biological Activity of Estrane-Based Inhibitors/Activators of Steroidogenesis.

Steroid hormones play a crucial role in several aspects of human life, and steroidogenesis is the process by which hormones are produced from cholesterol using several enzymes that work in concert to obtain the appropriate levels of each hormone at the right time. Unfortunately, many diseases, such as cancer, endometriosis, and osteoporosis as examples, are caused by an increase in the production of certain hormones. For these diseases, the use of an inhibitor to block the activity of an enzyme and, in doing so, the production of a key hormone is a proven therapeutic strategy whose development continues. This account-type article focuses on seven inhibitors (compounds 1-7) and an activator (compound 8) of six enzymes involved in steroidogenesis, namely steroid sulfatase, aldo-keto reductase 1C3, types 1, 2, 3, and 12 of the 17ß-hydroxysteroid dehydrogenases. For these steroid derivatives, three topics will be addressed: (1) Their chemical synthesis from the same starting material, estrone, (2) their structural characterization using nuclear magnetic resonance, and (3) their in vitro or in vivo biological activities. These bioactive molecules constitute potential therapeutic or mechanistic tools that could be used to better understand the role of certain hormones in steroidogenesis.

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.

Chemical synthesis of fluorinated and iodinated 17ß-HSD3 inhibitors and evaluation for imaging prostate cancer tumors and tissue biodistribution.

Prostate cancer is the most common cancer among men and the development of new therapeutic agents is needed for its treatment and/or diagnosis. 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is involved in the production of androgens, which stimulates the proliferation of prostate cancer cells. Piperazinomethyl-androsterone sulfonamide derivatives were developed as 17ß-HSD3 inhibitors and the concentration of a representative sulfonamide derivative (compound 1) was found to accumulate in prostate tumor tissues relatively to plasma in a mouse xenograft experiment. This finding gives us the opportunity to specifically target the prostate cancer tumors through the development of a radiolabelled version of compound 1 toward targeted molecular radiotherapy or radioimaging diagnosis. The chemical synthesis of fluorinated and iodinated analogs of compound 1 was achieved, leading to a series of compounds with similar levels of inhibition as the initial candidate. From 17ß-HSD3 inhibition activity, molecular modeling and mouse plasma-concentration studies, the most promising compound of this series was selected, its 18F-radiolabelled version (18F-3) synthesized, and imaging/biodistribution studies engaged. When injected in mice, however, 18F-3 uptake in the target tissues (LNCaP[17ß-HSD3] tumors and testicles) was not sufficient to allow their visualization by positron emission tomography. Plasma concentration values of compounds 3-8 administered orally, however, showed that the para-iodo compound 7 is the most metabolically stable and could therefore be an interesting alternative for radiolabelling and radiotreatment.

Induction of Endoplasmic Reticulum Stress-Mediated Apoptosis by Aminosteroid RM-581 Efficiently Blocks the Growth of PC-3 Cancer Cells and Tumors Resistant or Not to Docetaxel.

Aminosteroid derivative RM-581 was previously identified as an endoplasmic-reticulum (ER) stress inducer with potent in vitro and in vivo anticancer activities. We report its evaluation in androgen-independent prostate cancer (PC-3) cells. RM-581 efficiently blocks PC-3 cell proliferation with stronger activity than that of a selection of known antineoplastic agents. This later also showed a synergistic effect with docetaxel, able to block the proliferation of docetaxel-resistant PC-3 cells and, contrary to docetaxel, did not induce cell resistance. RM-581 induced an increase in the expression level of ER stress-related markers of apoptosis, potentially triggered by the presence of RM-581 in the ER of PC-3 cells. These in vitro results were then successfully translated in vivo in a PC-3 xenograft tumor model in nude mice, showing superior blockade than that of docetaxel. RM-581 was also able to stop the progression of PC-3 cells when they had become resistant to docetaxel treatment. Concomitantly, we observed a decrease in gene markers of mevalonate and fatty acid pathways, and intratumoral levels of cholesterol by 19% and fatty acids by 22%. Overall, this work demonstrates the potential of an ER stress inducer as an anticancer agent for the treatment of prostate cancers that are refractory to commonly used chemotherapy treatments.

Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17ß-diol in prostate cancer LAPC-4 cells - Identifying inhibitors of non-classical pathways producing the most potent androgen.

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17ß-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-14C]-3α-diol from [4-14C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC50 = 1.2-14.1 µM). The most potent inhibitor 25 (IC50 = 1.2 µM) possesses a 4-(4-CF3-3-CH3O-benzyl)piperazinyl methyl side chain at C3ß and 17ß-OH/17α-CCH functionalities at C17 of a 5α-androstane core.

Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for optical imaging.

Steroid sulfatase (STS) is an important enzyme regulating the conversion of sulfated steroids into their active hydroxylated forms. Notably, the inhibition of STS has been shown to decrease the levels of active estrogens and was translated into clinical trials for the treatment of breast cancer. Based on quantitative structure-activity relationship (QSAR) and molecular modeling studies, we herein report the design of fluorescent inhibitors of STS by adding a dansyl group on an estrane scaffold. Synthesis of 17α-dansylaminomethyl-estradiol (7) and its sulfamoylated analog 8 were achieved from estrone in 5 and 6 steps, respectively. Inhibition assays on HEK-293 cells expressing exogenous STS revealed a high level of inhibition for compound 7 (IC50 = 69 nM), a value close to the QSAR model prediction (IC50 = 46 nM). As an irreversible inhibitor, sulfamate 8 led to an even more potent inhibition in the low nanomolar value (IC50 = 2.1 nM). In addition, we show that the potent STS inhibitor 8 can be employed as an optical imaging tool to investigate intracellular enzyme sub-localization as well as inhibitory behavior. As a result, confocal microscopy analysis confirmed good penetration of the STS fluorescent inhibitor 8 in cells and its localization in the endoplasmic reticulum where STS is localized.

Induction of endoplasmic reticulum stress by aminosteroid derivative RM-581 leads to tumor regression in PANC-1 xenograft model.

The high fatality and morbidity of pancreatic cancer have remained almost unchanged over the last decades and new clinical therapeutic tools are urgently needed. We determined the cytotoxic activity of aminosteroid derivatives RM-133 (androstane) and RM-581 (estrane) in three human pancreatic cancer cell lines (BxPC3, Hs766T and PANC-1). In PANC-1, a similar level of antiproliferative activity was observed for RM-581 and RM-133 (IC50 = 3.9 and 4.3 µM, respectively), but RM-581 provided a higher selectivity index (SI = 12.8) for cancer cells over normal pancreatic cells than RM-133 (SI = 2.8). We also confirmed that RM-581 induces the same ER stress-apoptosis markers (BIP, CHOP and HERP) than RM-133 in PANC-1 cells, pointing out to a similar mechanism of action. Finally, these relevant in vitro results have been successfully translated in vivo by testing RM-581 using different doses (10-60 mg/kg/day) and modes of administration in PANC-1 xenograft models, which have led to tumor regression without any sign of toxicity in mice (animal weight, behavior and histology). Interestingly, RM-581 fully reduced the pancreatic tumor growth when administered orally in mice.

Total Synthesis of the Antidiabetic (Type 2) Lipid Mediator Protectin DX/PDX.

The first total synthesis of a lipid mediator derived from natural ω-3-fatty acid docosahexaenoic acid (DHA), 10 S,17 S-diHDHA (also referred to as protectin DX/PDX), was achieved in a convergent route (29 steps). The two chiral hydroxyl groups at C-10 and C-17 were derived from readily available ( S)-1,2,4-butanetriol and ( R)-glycidol, respectively. The two stereodefined E-double bonds were generated by a Takai olefination, and the skipped diene side chain was introduced with a stereocontrolled Wittig olefination. Importantly, the sensitive conjugated E, Z, E-triene intermediate was generated by a Boland reduction of the central triple bond of a E, E-dienyne. Overall, this synthetic strategy should allow the preparation of a larger quantity of PDX, which is inaccessible via previously reported biosynthetic approaches.

Implication of STARD5 and cholesterol homeostasis disturbance in the endoplasmic reticulum stress-related response induced by pro-apoptotic aminosteroid RM-133.

The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use.

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.

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.

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).

Targeting cytochrome P450 (CYP) 1B1 with steroid derivatives.

Inhibition of cytochrome P450 1B1 (CYP1B1) represents a promising therapeutic strategy, because it would enable action at three different levels: (1) by inhibiting the formation of mutagenic 4-hydroxy-estradiol, (2) by inhibiting the bioactivation of procarcinogens, and (3) by reducing drug-resistance. Surprisingly, few steroids were reported as inhibitors of CYP1B1. From a screening performed with 90 steroid derivatives, we identified thioestrone (B19) as an inhibitor (IC50=3.4µM) of CYP1B1. Molecular modeling studies showed that the 3-SH group of B19 is closer (3.36Å) to the iron atom of the heme system than the 3-OH group of enzyme substrates estrone and estradiol (4.26Å and 3.58Å, respectively). B19 also produced a better docking GOLD score that correlated with the inhibitory results obtained. The estrane derivative B19 represents an interesting lead compound that can be easily modified to extend the structure-activity relationship study and to provide a next generation of more powerful CYP1B1 inhibitors.

Synthesis and Characterization of Novel Cu(II), Pd(II) and Pt(II) Complexes with 8-Ethyl-2-hydroxytricyclo(7.3.1.0(2,7))tridecan-13-one-thiosemicarbazone: Antimicrobial and in Vitro Antiproliferative Activity.

New Cu(II), Pd(II) and Pt(II) complexes, (Cu(L)(H2O)2(OAc)) (1), (Cu(HL)(H2O)2(SO4)) (2), (Cu(L)(H2O)2(NO3)) (3), (Cu(L)(H2O)2(ClO4)) (4), (Cu(L)2(H2O)2) (5), (Pd(L)(OAc))H2O (6), and (Pt(L)2) (7) were synthesized from 8-ethyl-2-hydroxytricyclo(7.3.1.0(2,7))tridecan-13-one thiosemicarbazone (HL). The ligand and its metal complexes were characterized by IR, ¹H-NMR, (13)C-NMR, UV-Vis, FAB, EPR, mass spectroscopy, elemental and thermal analysis, magnetic susceptibility measurements and molar electric conductivity. The free ligand and the metal complexes have been tested for their antimicrobial activity against E. coli, S. enteritidis, S. aureus, E. faecalis, C. albicans and cytotoxicity against the NCI-H1573 lung adenocarcinoma, SKBR-3 human breast, MCF-7 human breast, A375 human melanoma and HL-60 human promyelocytic leukemia cell lines. Copper complex 2 exhibited the best antiproliferative activities against MCF-7 human breast cancer cells. A significant inhibition of malignant HL-60 cell growth was observed for copper complex 2, palladium complex 6 and platinum complex 7, with IC50 values of 1.6 µM, 6.5 µM and 6.4 µM, respectively.

In vitro evaluation of a tetrahydroisoquinoline derivative as a steroid sulfatase inhibitor and a selective estrogen receptor modulator.

Selective estrogen receptor modulators (SERMs) are currently in use in the hormonal therapy of breast cancer. In that respect, a new hormone-related approach is the therapeutical inhibition of steroid sulfatase (STS), which converts inactive, sulfated steroids into active hormones. We investigated the potential of 6-EO-14, a non-steroidal STS inhibitor with SERM potential. The latter compound, which exhibits a sulfamate moiety, releases the phenol derivative 8-EO-14 after the irreversible inhibition of STS. STS was inhibited by 6-EO-14 (IC50 = 0.3 µM), but not 8-EO-14, in HEK-293 cells transfected with an STS expression vector. The SERM potential of 8-EO-14 was assessed in osteoblast-like Saos-2 cells by investigating its effect on cell proliferation and on the activity of alkaline phosphatase (ALP), a specific differentiation marker. Saos-2 cell proliferation was increased by 21 % following 8-EO-14 addition (1 µM), and 8-EO-14 induced ALP activity (31 % increase at 0.1 nM) via estrogen receptor alpha (ERα) similarly to the SERM raloxifene. As compared to estradiol (E2) (100 %), the relative binding affinity of 6-EO-14 and 8-EO-14) for ERα was found to be weak (0.09 and 0.01 %, respectively). When assessed in two estrogen-dependent human breast cancer cell lines (MCF-7 and T-47D), 8-EO-14 did not support MCF-7 cell proliferation, whereas both 8-EO-14 and 6-EO-14 exhibited estrogen-like growth stimulation in T-47D cells. These two compounds were also unable to block E2-induced cell proliferation, suggesting their lack of antiestrogenic activity. Despite the known potency of 6-EO-14 as an STS inhibitor, the observed trophic activity of this new scaffold towards ERα-positive cells needs to be carefully considered prior to its potential utilization as a therapeutic agent.

Design, chemical synthesis and biological evaluation of 3-spiromorpholinone/3-spirocarbamate androsterone derivatives as inhibitors of 17ß-hydroxysteroid dehydrogenase type 3.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a key enzyme involved in the biosynthesis of testosterone and dihydrotestosterone. These hormones are known to stimulate androgen-dependent prostate cancer. In order to generate effective inhibitors of androgen biosynthesis without androgenic effect, we synthesized a new family of 3-spiromorpholinone and 3-spirocarbamate androsterone derivatives bearing diversified hydrophobic groups. We also tested their inhibitory activity in a microsomal fraction of 17ß-HSD3-containing rat testes, and their androgenic effect on androgen-sensitive LAPC-4 cells. From our first structure-activity relationship (SAR) study, we noted that compound 7e inhibited 17ß-HSD3 (77% at 0.1 µM) compared to our reference compound RM-532-105 (76% at 0.1 µM), but exhibited a residual androgenic effect. A library of 7e analogue compounds was next synthesized in order to generate compounds with reduced androgenic activity. In this new SAR study, the sulfonamide compound 7e21 and the carboxamide compound 7e22 inhibited 17ß-HSD3 (IC50 = 28 and 88 nM, respectively). These two compounds were not androgenic and not cytotoxic even at the highest concentration tested, but their inhibitory activity decreased in intact LNCaP cells overexpressing 17ß-HSD3 (LNCaP[17ß-HSD3]). Structural modifications of these two lead compounds could however be tested to produce a second generation of 17ß-HSD3 inhibitors.

Chemical synthesis, cytotoxicity, selectivity and bioavailability of 5α-androstane-3α,17ß-diol derivatives.

Aminosteroid derivatives represent a new family of compounds with promising antiproliferative activity over different cancer cell lines. Among all the aminosteroid derivatives synthesised in our laboratory, we have identified E-37P as one of the more potent when tested in vitro. Unfortunately, the pharmacokinetic properties of E-37P decrease its effectiveness when tested in vivo. To improve the bioavailability and increase the efficiency of aminosteroid E-37P, two series of analog compounds were synthesised by classic chemical synthesis, they were then characterized, and the concentration that inhibits 50% of cell proliferation (IC50) was determined on different cell lines. RM-133, a 5α-androstane-3α,17ß-diol derivative with a quinoline nucleus at the end of the piperazine-proline side-chain at position 2ß and an ethinyl at position 17α, showed very good antiproliferative activity among the five cancer cell lines studied (IC50=0.1, 0.1, 0.1, 2.0 and 1.1 µM for HL-60, MCF-7, T-47D, LNCaP and WEHI-3, respectively). Moreover, the plasmatic concentration of RM-133 at 3h, when injected subcutaneously in rats, was 2.3-fold higher than that of E-37P (151 vs 64.8 ng/mL). Furthermore, RM-133 weakly inhibited the two representative liver enzymes, CYP3A4 and CYP2D6, indicating a very low risk of drug-drug interactions. The cytotoxicity of RM-133 against normal cells was tested on peripheral blood lymphocytes (PBL) obtained from different donors and previously activated with phytohemagglutinin-L. PBL responded differently to treatment with RM-133, we observed a stimulation of cell proliferation and/or cytotoxicity in a dose-dependent manner. Based on these results, additional studies are currently underway to evaluate the selectivity of our lead compound against normal cell lines in a more detailed fashion.