Synthesis of hydrocortisone esters targeting androgen and glucocorticoid receptors in prostate cancer in vitro.

Androgen and glucocorticoid receptors have been recently described as key players in processes related to prostate cancer and mainly androgen receptor's inactivation was shown as an effective way for the prostate cancer treatment. Unfortunately, androgen deprivation therapy usually loses its effectivity and the disease frequently progresses into castration-resistant prostate cancer with poor prognosis. The role of the glucocorticoid receptor is associated with the mechanism of resistance; therefore, pharmacological targeting of glucocorticoid receptor in combination with antiandrogen treatment was shown as an alternative approach in the prostate cancer treatment. We introduce here the synthesis of novel 17α- and/or 21-ester or carbamate derivatives of hydrocortisone and evaluation of their biological activity towards androgen and glucocorticoid receptors in different prostate cancer cell lines. A 17α-butyryloxy-21-(alkyl)carbamoyloxy derivative 14 was found to diminish the transcriptional activity of both receptors (in single-digit micromolar range), with comparable potency to enzalutamide towards the androgen receptor, but weaker potency compared to mifepristone towards the glucocorticoid receptor. Lead compound inhibited proliferation and the formation of cell colonies in both androgen and glucocortiocid receptors-positive prostate cancer cell lines in low micromolar concentrations. Candidate compound 14 showed to interact with both receptors in cells and inhibited the translocation of receptors to nucleus and their activation phoshorylation. Moreover, binding to receptor's ligand binding domains was assessed by molecular modelling. Lead compound also induced the accumulation of cells in G1 phase and its combination with enzalutamide was shown to be more effective than enzalutamide alone.

Novel preparation of substituted oxazolines condensed to d-ring of estrane skeleton and characterization of their antiproliferative properties.

A simple and efficient synthesis of novel estrone 16α,17α-oxazoline derivatives substituted at the D ring (compounds 6a-g) is described. The reduction of 16α-azido-3-methoxyestra-1,3,5-trien-17-one (1) in methanol in the presence of CeCl3 under the condition of the Luche reaction produced two epimeric azido alcohol (16α-azido-17α-hydroxy and 16α-azido-17ß-hydroxy) derivatives of estra-1,3,5(10)-triene-3-methyl ether (compounds 2 and 3) in a yield of 90% and 7.6%. The reaction of the sterically unhindered 16α-azido-17α-hydroxy-estra-1,3,5(10)-triene-3-methyl ether (2) with a range of benzaldehydes under the condition of the Schmidt rearrangement yielded d-ring substituted estrone 16α,17α-oxazoline derivatives 6a-g. The in vitro antiproliferative activities of compounds 1, 2, 3, 6a-g were also determined by means of MTT assays on a panel of human cancer cell lines HeLa, SiHa, C-33 A, A2780, MCF-7, MDA-MB-231 and T47D.

Stereocontrolled synthesis of the four possible 3-methoxy and 3-benzyloxy-16-triazolyl-methyl-estra-17-ol hybrids and their antiproliferative activities.

The four possible isomers of each of 3-methoxy- and 3-benzyloxyestra-1,3,5(10)-trien-17-ols (5-8 and 9-12) were converted through 16-p-tosyloxymethyl- or 16-bromomethyl derivatives into their 3-methoxy- and 3-benzyloxy-16-azidomethylestra(1,3,5(10)-triene derivatives (13-16 and 17-20). The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of these compounds with different terminal alkynes afforded novel 1,4-disubstituted diastereomers (21a-f, 22a-f, 23a-f, 24a-f and 25a-f, 26a-f, 27a-f, 28a-f). The antiproliferative activities of the structurally related triazoles were determined in vitro with the microculture tetrazolium assay on four malignant human cell lines of gynecological origin (Hela, SiHa, MCF-7 and MDA-MB-231).

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.

Synthesis, Biological Evaluation and Docking Studies of 13-Epimeric 10-fluoro- and 10-Chloroestra-1,4-dien-3-ones as Potential Aromatase Inhibitors.

Fluorination of 13-epimeric estrones and their 17-deoxy counterparts was performed with Selectfluor as the reagent. In acetonitrile or trifluoroacetic acid (TFA), 10ß-fluoroestra-1,4-dien-3-ones were formed exclusively. Mechanistic investigations suggest that fluorinations occurred via SET in acetonitrile, but another mechanism was operative in TFA. Simultaneous application of N-chlorosuccinimide (NCS) and Selectfluor in TFA led to a 1.3:1 mixture of 10ß-fluoroestra-1,4-dien-3-one and 10ß-chloroestra-1,4-dien-3-one as the main products. The potential inhibitory action of the 10-fluoro- or 10-chloroestra-1,4-dien-3-one products on human aromatase was investigated via in vitro radiosubstrate incubation. The classical estrane conformation with trans ring anellations and a 13ß-methyl group seems to be crucial for the inhibition of the enzyme, while test compounds bearing the 13ß-methyl group exclusively displayed potent inhibitory action with submicromolar or micromolar IC50 values. Concerning molecular level explanation of biological activity or inactivity, computational simulations were performed. Docking studies reinforced that besides the well-known Met374 H-bond connection, the stereocenter in the 13 position has an important role in the binding affinity. The configuration inversion at C-13 results in weaker binding of 13α-estrone derivatives to the aromatase enzyme.

Stereoselective synthesis of new type of estradiol hybrid molecules and their antiproliferative activities.

To prepare new type of estrane hybrid molecules, we chose 3-methoxy- and 3-benzyloxy-17ß,16ß-epoxymethylene-estra-1,3,5(10)-trienes as starting materials (2 and 5). These steroid oxetanes were transformed with ethylene glycol in the presence of BF3.OEt2 into 3-methoxy- and 3-benzyloxy-16ß-(2'-oxa-4'-hydroxy)butyl-17ß-hydroxy-estra-1,3,5(10)-trien-17ß-ols (3a and 6a). Iodination of the terminal hydroxy group afforded iodo derivatives 3b and 6b, which underwent one-pot 3-O-alkylation with unprotected ascorbic acid to yield 3c and 6c. The same process with salicylic acid led to 2-O-alkylated salicylic acid derivatives 3d and 6d. Iodo derivatives 3b and 6b underwent nucleophilic exchange reaction with NaN3 furnishing the corresponding azido compounds 3e and 6e. These compounds were subjected to azide-alkyne CuAAC reactions with phenylacetylene and their p-substituted derivatives to form 1,4-substituted triazoles 3f-h and 6f-h. The reduction of 3e and 6e with hydrazine hydrate in the presence of Raney Ni provided the corresponding amino derivatives 3i and 6i. These compounds were reacted further with varied substituted benzoic acids to deliver terminal benzamido derivatives 3j-m and 6j-m. We determined the in vitro antiproliferative activities of compounds 2, 5, 3a-m and 6a-m by means of MTT assays on a panel of human adherent cancer cell lines A2780, MCF-7, MB-231 and SiHa.

Synthesis of Artemisinin-Estrogen Hybrids Highly Active against HCMV, P. falciparum, and Cervical and Breast Cancer.

Artemisinin-estrogen hybrids were for the first time both synthesized and investigated for their in vitro biological activity against malaria parasites (Plasmodium falciparum 3D7), human cytomegalovirus (HCMV), and a panel of human malignant cells of gynecological origin containing breast (MCF7, MDA-MB-231, MDA-MB-361, T47D) and cervical tumor cell lines (HeLa, SiHa, C33A). In terms of antimalarial efficacy, hybrid 8 (EC50 = 3.8 nM) was about two times more active than its parent compound artesunic acid (7) (EC50 = 8.9 nM) as well as the standard drug chloroquine (EC50 = 9.8 nM) and was, therefore, comparable to the clinically used dihydroartemisinin (6) (EC50 = 2.4 nM). Furthermore, hybrids 9-12 showed a strong antiviral effect with EC50 values in the submicromolar range (0.22-0.38 µM) and thus possess profoundly stronger anti-HCMV activity (approximately factor 25) than the parent compound artesunic acid (7) (EC50 = 5.41 µM). These compounds also exerted a higher in vitro anti-HCMV efficacy than ganciclovir used as the standard of current antiviral treatment. In addition, hybrids 8-12 elicited substantially more pronounced growth inhibiting action on all cancer cell lines than their parent compounds and the reference drug cisplatin. The most potent agent, hybrid 12, exhibited submicromolar EC50 values (0.15-0.93 µM) against breast cancer and C33A cell lines.

Synthesis and structure-activity relationships of 2- and/or 4-halogenated 13ß- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis.

Ring A halogenated 13α-, 13ß-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure-activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17ß-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.

Antiproliferative Properties of Newly Synthesized 19-Nortestosterone Analogs Without Substantial Androgenic Activity.

19-Nortestosterone C-17 epimers with prominent antiproliferative properties have been previously described. In our present study, five novel 17α-19-nortestosterones (3-7) were synthesized to increase their beneficial biological activities with no associated undesired hormonal effects. The compounds were screened by a viability assay against a panel of human adherent gynecological cancer cell lines. Three of the tested derivatives (3-5) exhibited a remarkable inhibitory effect on the proliferation of HeLa cells with IC50 values lower than that of our reference agent cisplatin (CIS). These three active agents also displayed considerable cancer selectivity as evidenced by their weaker growth inhibitory effect on non-cancerous fibroblast cells compared to CIS. The most potent newly synthesized 17α-chloro derivative (3) was selected for additional experiments in order to characterize its mechanism of action. Since nandrolone (19-nortestosterone, 1) is a structural analog with selective antiproliferative action on cervical carcinoma cells, it was utilized as a positive control in these studies. A lactate dehydrogenase (LDH) assay demonstrated a moderate cytotoxic effect of the test compounds. Cell cycle disturbance and the elevation of the hypodiploid population elicited by the test agents were detected by flow cytometry following propidium staining. The proapoptotic effects of the tested steroids were confirmed by fluorescent microscopy and a caspase-3 activity assay. Treatment-related caspase-9 activation without a substantial change in caspase-8 activity indicates the induction of the intrinsic apoptotic pathway. The selected agents directly influence the rate of tubulin assembly as evidenced by a polymerization assay. Yeast-based reporter gene assay revealed that the androgenic activity of the novel 19-nortestosterone derivative 3 is by multiple orders of magnitude weaker than that of the reference agent 1. Based on the behavior of the examined compounds it can be concluded that a halogen substitution of the 19-nortestosterone scaffold at the 17α position may produce compounds with unique biological activities. The results of the present study support that structurally modified steroids with negligible hormonal activity are a promising basis for the research and development of novel anticancer agents.

The first Pd-catalyzed Buchwald-Hartwig aminations at C-2 or C-4 in the estrone series.

A facile Pd-catalyzed C(sp2)-N coupling to provide a range of 2- or 4-[(subst.)phenyl]amino-13α-estrone derivatives has been achieved under microwave irradiation. The reactions were mediated with the use of Pd(OAc)2 as a catalyst and KOt-Bu as a base in the presence of X-Phos as a ligand. The desired products have been obtained in good to excellent yields. The nature and the position of the aniline substituent at the aromatic ring influenced the outcome of the couplings. 2-Amino-13α-estrone was also synthesized in a two-step protocol including an amination of 2-bromo-13α-estrone 3-benzyl ether with benzophenone imine and subsequent hydrogenolysis.

Antiproliferative and antimetastatic properties of 3-benzyloxy-16-hydroxymethylene-estradiol analogs against breast cancer cell lines.

Despite emerging new therapeutic opportunities, cancer is still a major health problem and a leading cause of death worldwide. Breast tumors are the most frequently diagnosed female malignancies, and the triple-negative subtype is associated with poorer prognosis and lower survival rates than other breast cancer types. The aims of the present study were to determine the anticancer potency of a set of C-3 and C-16 modified estradiol-derivatives against a panel of breast cancer cell lines, and to characterize the mechanism of action of two selected compounds (1 and 5) against the MDA-MB-231 triple-negative breast cancer cell line. Growth-inhibitory properties were investigated by an MTT-assay. Cell cycle analysis by flow cytometry has revealed G1 phase accumulation and indicated the proapoptotic effect of 1 and 5 through the elevation of the apoptotic subG1 phase on MDA-MB-231 cells after 24 h treatment. The antimetastatic activities of these compounds were examined by wound healing and Boyden chamber assays, and both compounds were shown to significantly inhibit the migration and invasion of MDA-MB-231 cells at sub-antiproliferative concentrations. Gelatin zymography assay has indicated that matrix metalloproteinase-2 and -9 are not involved in the antimetastatic action of the molecules. Western blot analysis was performed with 24 h incubation to examine the possible changes in the level of focal adhesion kinase (FAK), and both compounds were found to inhibit the phosphorylation of FAK in a concentration-dependent manner in MDA-MB-231 cells. The results of this study demonstrate that C-3 and C-16 modified estradiol derivatives are potent antiproliferative and antimetastatic compounds against a triple-negative breast cancer cell line with a mechanism of action involving the inhibition of FAK, a novel anticancer therapeutic target. Therefore, these findings can be utilized in the development of promising anticancer agents with steroid skeleton.

Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed azide-alkyne cycloaddition and their inhibitory effect on 17α-hydroxylase/C17,20-lyase.

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 17α- and 17ß-azidoandrost-5-en-3ß-ol epimers (3b and 5b) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a-k and 9a-k). For the preparation of 5'-iodo-1',2',3'-triazoles (8m-n and 9m-n), an improved method was developed, directly from steroidal azides and terminal alkynes, in reaction mediated by CuI and ICl as iodinating agents. Acetolysis and subsequent hydrolysis of 8n and 9n yielded 5'-hydroxy-1',2',3'-triazoles 8o and 9o. The inhibitory effect of 8a-o, 9a-o, 3, and 5 on rat testicular C17,20-lyase was investigated by means of an in vitro radioincubation technique. The results revealed that the C-17 epimers of steroidal triazoles influence the C17,20-lyase effect. Inhibitors were found only in the 17α-triazolyl series (8a-o), whereas in the C-17 azide pair the 17ß compound (5b) was more potent.

Stereoselective synthesis of the four 16-hydroxymethyl-3-methoxy- and 16-hydroxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-ol isomers and their antiproliferative activities.

The reduction of 16-hydroxymethylene-3-methoxy-13α-estra-1,3,5(10)-trien-17-one (14) and 16-hydroxymethylene-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-one (16) yielded a mixture of two diastereomeric diols, the 16α-hydroxymethyl,17ß-hydroxy and 16ß-hydroxymethyl,17α-hydroxy isomers (17a-20a) in a ratio of 6:1. We describe a straightforward synthetic route to transform the isomers with trans functional groups attached to ring D (17a-20a) into isomers with cis functional groups (25a-28a). We determined the in vitro antiproliferative activities of compounds 17a-20a and 25a-28a by means of MTT assays against a panel of human adherent cancer cell lines HeLa, A2780, MCF-7, T47D, MDA-MB-231 and MDA-MB-361.

Investigation of pH and substituent effects on the distribution ratio of novel steroidal ring D- and A-fused arylpyrazole regioisomers and evaluation of their cell-growth inhibitory effects in vitro.

Novel androstanopyrazoles have been efficiently synthesized from steroidal ß-ketoaldehydes with different arylhydrazine hydrochlorides both under acidic and basic conditions. Knorr-type transformations of 16-hydroxymethylene-dehydroepiandrosterone containing its 1,3-dicarbonyl moiety on ring D, proved to be regioselective in pyridine at room temperature, while mixtures of regioisomers were obtained in acidic EtOH under reflux. Contrarily, the cyclocondensation reactions of 2-hydroxymethylene-dihydrotestosterone bearing its reactive functionalities on ring A, led to a mixture of pyrazole regioisomers in varying ratio depending on the applied medium. The regioisomeric distribution was found to depend on the electronic character of the substituent of the phenylhydrazine applied. After separating the related isomers by column chromatography, they were subjected to in vitro pharmacological studies to investigate their antiproliferative activities against three human breast malignant cell lines (MCF7, T47D, MDA-MB-231). Flow cytometry revealed that the most potent agents elicited a cell cycle disturbance on MDA-MB-231 and T47D cells.

Synthesis of novel 13α-estrone derivatives by Sonogashira coupling as potential 17ß-HSD1 inhibitors.

Novel 13α-estrone derivatives were synthesized by Sonogashira coupling. Transformations of 2- or 4-iodo regioisomers of 13α-estrone and its 3-methyl ether were carried out under different conditions in a microwave reactor. The 2-iodo isomers were reacted with para-substituted phenylacetylenes using Pd(PPh3)4 as catalyst and CuI as a cocatalyst. Coupling reactions of 4-iodo derivatives could be achieved by changing the catalyst to Pd(PPh3)2Cl2. The product phenethynyl derivatives were partially or fully saturated. Compounds bearing a phenolic OH group furnished benzofurans under the conditions used for the partial saturation. The inhibitory effects of the compounds on human placental 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated by an in vitro radiosubstrate incubation method. Certain 3-hydroxy-2-phenethynyl or -phenethyl derivatives proved to be potent 17ß-HSD1 inhibitors, displaying submicromolar IC50 values.

Synthesis and in vitro investigation of potential antiproliferative monosaccharide-d-secoestrone bioconjugates.

The syntheses of monosaccharide-d-secoestrone conjugates are reported. They were prepared from 3-(prop-2-inyloxy)-d-secoestrone alcohol or oxime and monosaccharide azides via Cu(I)-catalyzed azide-alkyne cycloaddition reactions (CuAAC). The antiproliferative activities of the conjugates were investigated in vitro against a panel of human adherent cancer cell lines (HeLa, A2780 and MCF-7) by means of MTT assays. The protected d-glucose-containing d-secoestrone oxime bioconjugate (24b) proved to be the most effective with an IC50 value in the low micromolar range against A2780 cell line.

Mechanism of antiproliferative action of a new d-secoestrone-triazole derivative in cervical cancer cells and its effect on cancer cell motility.

Cervical cancer is the fourth most frequently diagnosed tumor and the fourth leading cause of cancer death in females worldwide. Cervical cancer is predominantly related with human papilloma virus (HPV) infection, with the most oncogenic types being HPV-18 and -16. Our previous studies demonstrated that some d-secoestrone derivatives exert pronounced antiproliferative activity. The aim of the current investigation was to characterize the mechanism of action of d-secoestrone-triazole (D-SET) on three cervical cancer cell lines with different pathological backgrounds. The growth-inhibitory effects of D-SET were determined by a standard MTT assay. We have found that D-SET exerts a pronounced growth-inhibitory effect on HPV 18-positive HeLa and HPV-negative C-33 A cells, but it has no substantial inhibitory activity on HPV 16-positive SiHa or on intact fibroblast MRC-5 cell lines. After 24h incubation, cells showed the morphological and biochemical signs of apoptosis determined by fluorescent double staining, flow cytometry and caspase-3 activity assay. Besides the elevation of the ratio of cells in the subG1 phase, flow cytometric analysis revealed a cell cycle arrest at G2/M in both HeLa and C-33 A cell lines. To distinguish the G2/M cell population immunocytochemical flow cytometric analysis was performed on HeLa cells. The results show that D-SET significantly increases the ratio of phosphorylated histone H3, indicating cell accumulation in the M phase. Additionally, D-SET significantly increased the maximum rate of microtube formation measured by an in vitro tubulin polymerization assay. Besides its direct antiproliferative activity, the antimigratory property of D-SET has been investigated. Our results demonstrate that D-SET significantly inhibits the migration and invasion of HeLa cells after 24h incubation. These results suggests that D-SET is a potent antiproliferative agent against HPV 16+ and HPV-negative cervical cancer cell lines, with an efficacious motility-inhibiting activity against HPV 16+ cells. Accordingly D-SET can be regarded as a potential drug candidate with a promising new mechanism of action among the antiproliferative steroids, potentially allowing for the design of novel anticancer agents.

Synthesis, functionalization and biological activity of arylated derivatives of (+)-estrone.

Various novel arylated estrone derivatives, such as 2-aryl-, 4-aryl- and 2,4-diaryl-estrones, by Suzuki-Miyaura reactions. While the synthesis of 4-arylestrones could be carried out under standard conditions, the synthesis of 2-arylestrones and 2,4-diarylestrones required a thorough optimization of the conditions and it proved to be important to use sterically encumbered biaryl ligands. The best results were obtained by the use of RuPhos. Combination of developed Suzuki coupling reactions with subsequent cyclization reactions afforded more complex hybrid structures, containing dibenzofuran, benzocoumarin and steroid moieties. These derivatives were tested as pancreatic lipase inhibitors and it was found that most of the compounds exhibited inhibition of pancreatic lipase but the maximum inhibitory potential was shown by 4-arylestrones. All of the synthesized derivatives showed inhibitory values in the range of 0.82±0.01-59.7±3.12µM. The biological activity was also rationalized on the bases of docking studies.

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).