Sesquiterpene Lactones and Flavonoids from Psephellus pyrrhoblepharus with Antiproliferative Activity on Human Gynecological Cancer Cell Lines.

Multistep chromatographic separations of the chloroform extract of the Turkish endemic plant Psephellus pyrrhoblepharus (Boiss.) Wagenitz (syn. Centaurea pyrrhoblephara Boiss.) resulted in the isolation of six guaianolid-type sesquiterpenes, chlorojanerin (1), 19-deoxychlorojanerin (2), 15-hydroxyjanerin (3), aguerin B (4), cynaropicrin (5), eleganin (6); three flavonoids, apigenin, 6-methoxyluteolin and jaceosidine; two glycosides, benzyl-1-O-ß-d-glucoside and 3(Z)-hexenyl-1-O-ß-d-glucoside; and the coumarin scopoletin. The structures were established by the interpretation of their ESI-MS and 1D and 2D NMR data including 1H-NMR, JMOD, 1H,1H-COSY, HSQC, HMBC, and NOESY experiments. All compounds were isolated for the first time from P. pyrrhoblepharus. Compounds 1-6, the isolated flavonoids and scopoletin were evaluated for their antiproliferative activities on human gynecological cancer cell lines (SiHa, HeLa, and MDA-MB-231 cells) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Chlorojanerin (1), 19-deoxychlorojanerin (2), aguerin B (4), cynaropicrin (5), eleganin (6) were shown to have noteworthy effects on all of the tested cell lines, while apigenin, jaceosidine, and 6-methoxyluteolin were moderately active on HeLa cells. The highest activities were demonstrated by the chlorine-containing derivatives chlorojanerin (1) and 19-deoxychlorojanerin (2) with IC50 values of 2.21 and 2.88 µM, respectively, against the triple negative breast cancer model MDA-MB-231 cells.

Microwave-assisted synthesis of biologically relevant steroidal 17-exo-pyrazol-5'-ones from a norpregnene precursor by a side-chain elongation/heterocyclization sequence.

Multistep syntheses of novel 17ß-pyrazol-5'-ones in the Δ5-androstane series were efficiently carried out from pregnenolone acetate. A steroidal 17-carboxylic acid was first synthesized as a norpregnene precursor by the bromoform reaction and subsequent acetylation. Its CDI-activated acylimidazole derivative was then converted to a ß-ketoester containing a two carbon atom-elongated side chain than that of the starting material. A Knorr cyclization of the bifunctional 1,3-dicarbonyl compound with hydrazine and its monosubstituted derivatives in AcOH under microwave heating conditions led to the regioselective formation of 17-exo-heterocycles in good to excellent yields. The suppression of an acid-catalyzed thermal decarboxylation of the ß-ketoester and thus a significant improvement in the yield of the desired heterocyclic products could be achieved by the preliminary liberation of the arylhydrazines from their hydrochloride salts in EtOH in the presence of NaOAc. The reaction rates were found to depend on the electronic character of the substituent present in the phenylhydrazine applied. The antiproliferative activities of the structurally related steroidal pyrazol-5'-ones and their deacetylated analogs were screened on three human adherent breast cancer cell lines (MCF7, T47D and MDA-MB-231): the microculture tetrazolium assay revealed that some of the presented derivatives exerted cell growth inhibitory effects on some of these cell lines comparable to those of the reference compound, cisplatin.

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.

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 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).

Synthesis of trans-16-triazolyl-13α-methyl-17-estradiol diastereomers and the effects of structural modifications on their in vitro antiproliferative activities.

Novel 16-triazoles in the 13α-estrone series were synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition of the two diastereomeric (on C-16 and on C-17) 16-azido-13α-estra-1,3,5(10)-trien-17-ol 3-benzyl ethers with substituted phenylacetylenes. The new heterocyclic derivatives were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A431, A2780, T47D, MDA-MB-231 and MDA-MB-361). The inversion of the configurations at C-16 and C-17 selectively affected the growth-inhibitory properties of the tested compounds. The 16ß,17α isomers generally proved to be potent on all cell lines, with IC50 values comparable to those of the reference agent cisplatin. Change of the substitution pattern of the phenyl group of the acetylene led to great differences in antiproliferative properties. Exclusively the p-phenyl-substituted triazoles exerted high cytostatic effects. One of the most potent compounds activated caspase-3 and caspase-9 without influencing caspase-8, confirming the induction of apoptosis via the intrinsic pathway.