Synthesis and Biological Activity of 1α,2α,25-Trihydroxyvitamin D₃: Active Metabolite of 2α-(3-Hydroxypropoxy)-1α,25-dihydroxyvitamin D₃ by Human CYP3A4

Abstract

Our previous studies revealed that recombinant human CYP3A4 converted 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D 3 (O2C3), which was a more potent binder to vitamin D receptor (VDR) than the natural hormone, 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 , 1), to 1α,2α,25-trihydroxyvitamin D 3 (2). Here, we synthesized 2 using the Trost Pd-mediated coupling reaction between an A-ring precursor and a CD-ring bromoolefin and evaluated its preliminary biological activity. We found that metabolite 2 from O2C3 was still active as a VDR ligand while maintaining human VDR binding affinity (27 1-4) Actually, 1 and several synthetic analogs of 1 have been used clinically in the treatment of bone diseases, secondary hyperparathyroidism, psoriasis, and osteoporosis.1,5) Although 1 is inactivated by CYP24A1-dependent catabolism via C-24 hydroxylation to calcitroic acid for excretion from the body, 1) it was found that some 2α-substituted active vitamin D analogs were highly resistant to CYP24A1, for example, the k cat /K m value of 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D 3 (O2C3), which showed 1.8-times greater binding affinity for vitamin D receptor (VDR) than 1, [6][7][8][9][10] was only 3% of that for 1. 11) CYP24A1 is the specific enzyme induced by the VDR-ligand (1 or its analog) complex in the target tissue and inactivates 1 and its analogs; therefore, CYP24A1-resistant ligands would have long-term biological effects on the target tissues.12) On the other hand, CYP3A4 is a broad-spectrum drug-metabolizing P450 enzyme, 13) but 1 and its analog 2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D 3 (O1C3) are not primary substrates for CYP3A4.12) Recently, however, we demonstrated that O2C3 was metabolized by CYP3A4 and converted to 1α,2α,25-trihydroxyvitamin D 3 12) (2, Fig. 1). Its 2β-isomer (4) is a known compound and shows potent 1α,25(OH) 2 D 3 -like activities, 14) and we report here the synthesis of a new 2α-hydroxylated analog 2 using the Trost Pd-mediated coupling reaction between an A-ring precursor 12 and a CD-ring bromoolefin 13 to evaluate its preliminary biological activity. 15-18)The A-ring precursor 12 for Trost coupling was prepared from the known epoxide 5 19) in 11 steps (Chart 1). Briefly, treatment of 5 with p-methoxybenzyl alkoxide with heating gave methyl 3-O-(p-methoxybenzyl) altropyranoside 6, which had the 2α-hydorxy group (steroidal numbering) required in target molecule 2. After 2-O-silylation, the p-methoxybenzyl (PMB) protecting group was removed by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) to give 7, and the methoxymethyl (MOM) group was introduced instead for the next bromination reaction using N-bromosuccinimide (NBS). NBS treatment for the resulting O-MOM-protected bezylidene acetal gave bromide 8. Activated Zn-reduction in the presence of NaBH 3 CN produced alcohol 9, which was converted to epoxide 10 via tosylation followed by tetrabutylammonium fluoride (TBAF) treatment. Trimethylsilyl (TMS)-ethynylation of 10 afforded enyne 11, and subsequent solvolysis in K 2 CO 3 -MeOH and O-...

The Centennial Collection of VDR Ligands: Metabolites, Analogs, Hybrids and Non-Secosteroidal Ligands

“…Figure 24 (2014-2015) [322][323][324][325][326][327][328][329][330][331][332][333][334][335][336]. 1α,20S,24R-Trihydroxyvitamin D 3 (1410) [332] showed a higher degree of activation, anti-inflammatory activity, and antiproliferative activity than vitamin D 3 receptor.…”