Starting from 3-methoxyestra-1,3,5(10),16-tetraene-17-carbaldehydes of natural (13β) and epimeric (13α) series, a series of isomeric 3-hydroxy-17-hydroxymethylestra-1,3,5(10)-trienes, including those containing 16α,17α-annulated cyclopropane and cyclohexane ring D', were prepared using the Corey-Chaykovsky and Diels-Alder reactions followed by reduction-demethylation with diisobutylaluminum hydride and hydrogenation. Target compounds showed antiproliferative effects on MCF-7 breast cancer cells to varying degrees superior to that on MCF-10A cells, in low micromolar concentrations. The ERα-mediated luciferase reporter gene assay demonstrated that obtained steroids without an additional carbocycle or with a cyclopropane 16α,17α-annulated carbocycle are effective ERα activators. In this test, steroids of the natural configuration showed high activity at both 10 nM and 100 nM concentrations, whereas 13α-steroids showed a strong dose-dependent effect, surpassing their natural counterparts at a concentration of 100 nM. The 13β-steroid bearing additional 16α,17α-cyclohexane ring had low activity in the test. A simple docking approach using AutoDock Vina was used as a test for a preliminary assessment of the estrogenicity of the compounds. The scope of its applicability and limitations were shown using examples of synthesized molecules.

Data on the influence of solvents, ratios and nature of reagents on the distribution of typical impurities and yields of target products of the reaction of 19-norpregna-1,3,5(10),16-tetraen-20-one derivatives with 4-dimethylamino-, 3,4-dimethoxy-, and 4-bromobenz-aldehydes and 3-pyridinecarbaldehyde were studied and summarized. A mechanism for the formation of 1,3-diketones in the reaction of 17-acetyl-substituted steroids with 4-dimethyl-aminobenzaldehyde in the presence of lithium diisopropylamide in THF was proposed. The Claisen-Schmidt condensation results were shown to be critically affected by the base and the steroid solubility. Optimization possibilities were considered and an improved procedure for the synthesis of new 21-benzylidene-substituted steroids was proposed.

This study aimed to investigate the activities of novel 20(R)-3,20-dihydroxy-19-norpregn-1,3,5(10)-trienes (kuz7 and kuz8b) of natural 13β- and epimeric 13α-series against triple-negative MDA-MB-231 breast cancer cells. High antiproliferative activity of synthesized compounds kuz8b and kuz7 against MDA-MB-231 triple-negative cancer cells was revealed. The steroid kuz7 of natural 13β-configuration was more active against MDA-MB-231 cells than the 13α-steroid kuz8b. Cell cycle analysis revealed common patterns for the action of both tested compounds. The number of cells in the subG1 phase increased in a dose-dependent manner, indicating induction of apoptosis, which was also verified by PARP cleavage. In contrast, the number of cells in the G0/G1 phase decreases with increasing compound concentration. Steroid kuz7 at micromolar concentrations reduced the expression of GLUT1, a glucose transporter. High efficacy of the combination of kuz7 with biguanide metformin was shown, and synergistic effects on MDA-MB-231 cell growth and expression of the anti-apoptotic protein Bcl-2 were revealed. According to the obtained results, including the high activity of kuz7 against triple-negative cancer cells, the detected induction of apoptosis, and the decrease in GLUT1 expression, 13β-steroid kuz7 is of interest for further preclinical studies both alone and in combination with the metabolic drug metformin.

Methods for extending the side chain of 20-oxopregnanes were summarized. The possibilities of alkylation of 3-methoxy-19-norpregna-1,3,5(10)-trien-20-one and its 16α,17α-methano and -butano derivatives with alkyl halides in the presence of lithium diisopropylamide were studied. The Claisen—Schmidt condensation of these steroids with aromatic aldehydes resulted in a series of the corresponding 21-benzylidenesteroids. Carrying out the Claisen—Schmidt condensation in an aprotic solvent in the presence of lithium diisopropylamide yielded, in addition to the target 21-benzylidenesteroids, the corresponding 21-benzoyl derivatives — steroidal 1,3-diketones.

A directed synthesis of two progesterone metabolites, allopregnanolone and 3a-hydroxy-pregn-4-en-20-one, from Δ16-pregnanolone and progesterone, respectively, was carried out by a reduction of the carbonyl groups in positions 3 and subsequent inversion of the configuration of the resulting alcohols by the Mitsunobu reaction. The selectivity of the reduction of the conjugated carbonyl group in position 3 of progesterone with sodium borohydride in the presence of cerium(III) chloride (Luche reduction) was demonstrated. The ef ect of the obtained metabolites on the proliferation of breast cancer cells of the MCF-7 and T47D lines under normal and steroid-free conditions was studied. It is shown that the ef ect of these compounds on the proliferation depends on the presence of additional steroids in the culture medium. Metabolites exerted small cytostatic ef ects on the growth of the MCF-7 cells under standard conditions, while the transfer of the cells to a steroid-free medium weakened these cytotoxic ef ects. In the experiments with the T47D line cells, the cell growth was stimulated under both standard and steroid-free conditions. Allopregnanolone and progesterone stimulate the growth to a greater extent under steroid-free conditions than under standard ones.

Breast cancer is the most common cancer in women. There is an urgent need for new drugs with novel modes of effect on tumors. It is very important to develop compounds with high activity against tumor cells and low toxicity on normal epithelium. The development of new anti-tumor drugs based on steroidal compounds is a promising approach for obtaining agents that combine antiestrogenic, cytostatic and proapoptotic effects. The aim of the work is to study the mechanism of anticancer action of new steroidal compound, 3,20 (R)-dihydroxy-19-norpregnatriene (Kuz7). MCF-7 breast cancer cell line and MCF-10A human mammary epithelial cell line were obtained from the ATCC collection. Antiproliferative activity was measured by MTT. ERα activity was assessed by gene-reporter assay. Protein expression was measured by standard immunoblotting. We have developed a new type of anticancer steroids - a series of 3-hydroxyestra-1,3,5(10)-trienes of natural and epimeric 13α-configuration with 17th-side chain bearing the second hydroxy group and 16,17-fused three- or six-membered carbocycle (or without it). Most of these compounds exhibited cytotoxic and ERα inhibiting activities on MCF-7 cells and have low toxicity in the MCF-10A normal epithelial cells. 3,20(R)-dihydroxy-19-norpregnatriene (Kuz7) was selected as a leader molecule for in-depth study. At low doses, Kuz7 caused significant accumulation of p21 and inhibition of CDK2 and CDK4 in MCF-7 cells. Apoptosis provoked by Kuz7 was determined by fragmented PARP. Compound kuz7 is considered as an effective inhibitor of proliferation and ERα signaling in MCF-7 cells. kuz7 decreased cyclin D1 expression and caused cell arrest in G0 phase. New series of highly active antitumor steroids has been developed. The lead compound, kuz7, blocks the growth of MCF-7 cells through induction of apoptosis and regulation of hormonal pathways and cell cycle. Further studies, in vitro and in vivo, are needed to confirm our findings in relation to luminal type A breast cancer. The work was supported by RFBR (project 19-03-00246).

A convenient preparative synthesis of 3-methoxyestra-1,3,5(10),16-tetraene-17-carb-aldehyde and its epimeric 13α-analog, the key structures in the synthesis of steroid estrogen receptor modulators and potential antitumor agents, was developed starting from estrone and including the generation of intermediate 16-dehydro-17-carbonitriles and their reaction with diisobutylaluminum hydride. The influence of conformational dif erences in the steroid molecules with the natural 13β- and epimeric 13α-configurations on the reactivity of intermediate compounds and the stereoselectivity of the trimethylsilyl cyanide addition to the carbonyl group of methyl ethers of epimeric estrones was considered.