Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Pharmaceutical Sciences


Medicinal Chemistry

Research Advisor

Wei Li, Ph.D.


Sarka Beranova, Ph.D. Tomoko Fujiwara, Ph.D. Duane D. Miller, Ph.D. Andrzej T. Slominski, Ph.D.


Rheumatoid arthritis (RA) is one of the autoimmune diseases, and is affecting 2.5 million Americans in total. Among the treatment options of RA, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the only steroidal drug used clinically for anti-inflammatory and immune diseases. However, long-term use of 1,25(OH)2D3 (625 µg/day) in human would result in hypercalcemia (toxicity), and 1,25(OH)2D3 has substantial hypercalcemic effects (toxicity) in mice at a dose as low as only 2 µg/kg. Fortunately, during the investigation of novel metabolic pathway of vitamin D3 by cytochrome P450 enzymes, we found 20S-hydroxyvitamin D3 [20S(OH)D3] as a good lead compound. 20S(OH)D3 suppressed disease symptoms at 2 µg/kg in collagen-induced arthritis model, and high doses of 20S(OH)D3 (up to 30 µg/kg) do not cause hypercalcemia in rats or mice. Thus 20S(OH)D3 has the potential to be structurally optimized for providing anti-inflammatory agents without toxicity. In this study, four series of 20S(OH)D3 analogs have been synthesized and studied, they are C20 Gemini analogs, C24-hydroxlated analogs, C23-hydroxlated analogs and C24 modified analogs together with their 1α-hydroxylated derivatives. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20S(OH)D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR. Bioactive vitamin D3 metabolites 20S,24S-dihydroxyvitamin D3 [20S,24S(OH)2D3] and 20S,24R-dihydroxyvitamin D3 [20S,24R(OH)2D3] were chemically synthesized and confirmed to be identical to their enzymatically generated counterparts. The absolute configurations at C24 and its influence on the kinetics of 1α-hydroxylation by CYP27B1 were determined. Their corresponding 1α-hydroxyl derivatives were subsequently produced. Biological comparisons of these products showed different properties with respect to vitamin D3 receptor activation, anti-inflammatory activity, and anti-proliferative activity, with 1α,20S,24R(OH)2D3 being the most potent compound. The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time, and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory and anti-proliferative activities. Four C24 modified analogs of 20S(OH)D3 were chemically synthesized and comprehensively tested against different activities together with their 1α-hydroxyl derivatives. Metabolism of 20S(OH)D3 analogs against cytochrome P450 27B1 (CYP27B1, activation enzyme) and CYP24A1 (catabolism enzyme) suggested that they are better substrates of both enzymes than 20S(OH)D3, and can be activated (1α-hydroxylated) by CYP27B1 except 23-amide which is not a substrate but an inhibitor of CYP27B1. Their 1α-OH derivatives were potent vitamin D receptor (VDR) agonists comparable with 1,25(OH)2D3 although they themselves showed weak or none VDR stimulation activity in three cell lines. To understand the molecular interactions between these analog and VDR, two analogs together with 20S(OH)D3 and 1,25(OH)2D3 were co-crystalized with human VDR. These analogs and 1α-OH derivatives significantly upregulated the mRNA expression of VDR target genes, suggesting their actions via VDR, at least partially. In addition, their anti-inflammatory activities have been investigated in aspect of IFNγ inhibition in splenocytes. This study demonstrates the mechanisms of action of 20S(OH)D3 anlogs, is of great importance for future drug development of anti-inflammatory agents. From the above-mentioned studies, we learned that the introduction of 1α-hydroxy could potentiate the anti-inflammatory activities of 20S(OH)D3 and its anlogs. Thus it would be beneficial to further investigate the 1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3] analogs. 1,20S(OH)2D3 was chemically synthesized for the first time. A semi-reduced intermediate of the Birch reduction for 1α-OH formation was obtained for the first time, and thus was used to propose the reaction mechanism. X-ray crystallography analysis of the key intermediate confirmed the formation of 1α-OH. 1,20S(OH)2D3 binds efficiently in vitamin D receptor (VDR), being similar with its native ligand 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, their co-crystal structures revealed differential molecular interactions of 20S-OH and 25-OH in VDR, which may help understand their biological activities. In addition, 1,20S(OH)2D3 functions as a VDR agonist with stronger/comparable activities than/with 1,25(OH)2D3 in aspects of VDR stimulation and regulating VDR downstream genes, and inhibition of inflammatory markers. This study offers a convenient synthetic route using a novel intermediate 1α,3β-diacetoxypregn-5-en-20-one, and provides molecular basis of design for drug development of 1,20S(OH)2D3 and its analogs. Overall, we have synthesized and biologically evaluated four series of 20S(OH)D3 analogs for their potential applications in anti-inflammatory diseases such as RA. The synthetic scheme of 1,20S(OH)2D3 could pioneer future development of its analogs. These findings will provide important guidance for the development of next generation anti-RA agents using 20S(OH)2D3 scaffold.





One year embargo expires May 2018.

Available for download on Saturday, May 05, 2018