Synthesis of New Steroidal Inhibitors of P-Glycoprotein-Mediated Multidrug Resistance and Biological Evaluation on K562/R7 Erythroleukemia Cells

Abstract

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/β-pregnane-3,20-dione or 5β-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/β-pregnane-3,20-dione, among which the 5β-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 μM versus 1.2 and 10.6 μM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5β-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 μM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

Asclepiasterol, a novel C21 steroidal glycoside derived fromAsclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression

“…The natural steroids can be classified into C 18 , C 19 , C 21 , C 23 (mainly cardenolides with a five-membered lactone ring), C 24 (mainly bufadienolides with a six-membered lactone ring) and C 27 structural types which are different in the ring structures and side chain at position C-17 [ 41 ]. The P-gp inhibitors identified by de Ravel and Zeino et al [ 39 , 40 ] belong to cardiotonic steroid derivatives. In contrast, asclepiasterol is a natural C 21 steroid, which has been known to possess less toxicity than the cardenolides and bufadienolids [ 42 ].…”

In vitromodulation of multidrug resistance by pregnane steroids andin vivoinhibition of tumour development by 7α-OBz-11α(R)-OTHP-5β-pregnanedione in K562/R7 and H295R cell xenografts

“…Doxorubicin, colchicine, vincristine, vinblastine, vinorelbine, paclitaxel, mitoxantrone, and cyclosporin A were purchased from Sigma. Syntheses of steroid modulators were previously described 15 . For in vitro experiments, dilutions in culture medium were extemporaneously made from 10 mM solutions in DMSO (or in water for cyclosporin A).…”

“…The NCI-H295R human adrenocortical carcinoma cells (donated by M. Bégeot, INSERM-U864) previously characterised as expressing pathways of steroid biosynthesis 16 were grown at 37 °C in a 5% CO 2 atmosphere using a 1:1 mixture of DMEM and Ham’s F-12 medium, supplemented with L-glutamine (2 mM), antibiotics (50 µg/mL streptomycin, 50 U/mL penicillin), ITS + 1 (mixture of insulin, transferrin, and sodium selenite, from Sigma), 2% Ultroser G and SF (Life Science Technologies). The K562 human erythroleukemia cell line and the doxorubicin-resistant K562/R7 cell line (provided by C. Dumontet) were cultured as previously described 15 . The human promyelocytic HL60-MRP1 cell line overexpressing the Multidrug Resistance Protein 1 (MRP1/ABCC1) and resistant to doxorubicin and the human breast adenocarcinoma MCF7-MTX cell line expressing the Breast Cancer Resistance Protein (BCRP/ABCG2) and resistant to mitoxantrone (donated by A. di Pietro, IBCP-CNRS) were both cultured at 37 °C in a 5% CO 2 atmosphere using either (HL60-MRP1 cells) an RPMI 1640 medium supplemented with 10% FCS, 58 nM of doxorubicin and 70 nM of vincristine or (MCF7-MTX cells) a 1:1 mixture of DMEM and Ham’s F-12 medium supplemented with 10% FCS, L-glutamine (2 mM), antibiotics (50 µg/mL streptomycin and 50 U/mL penicillin).…”

“…Total RNA extraction from NCI-H295R cells, first-strand cDNAs synthesis and real-time PCR were performed as previously described for K562/R7 cells 15 . Results were expressed as relative levels after normalisation by G3PDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA.…”

“…Moreover, steroids are multifunctional molecules facilitating structural modifications for the optimisation of pharmacological activities and for the control of metabolic degradation in vivo . Recently, our laboratory has proposed a simple methodology based on the conversion of hydroxylated precursors of 5α/β-pregnane and 5β-cholane series to ether and/or ester derivatives which provided efficient modulators of P-gp mediated MDR when tested in vitro on the resistant non-steroidogenic K562/R7 human erythroleukemia cell line 15 using doxorubicin as cytotoxic drug (Figure 1).…”