Synthesis of (22R)-, (22S)-22-Fluoro-, and 22,22-Difluoro-25-hydroxyvitamin D₃ and Effects of Side-Chain Fluorination on Biological Activity and CYP24A1-Dependent Metabolism

Abstract

Three novel analogues of C22-fluoro-25-hydroxyvitamin D 3 (5−7) were synthesized and evaluated to investigate the effects of side-chain fluorination on biological activity and metabolism of vitamin D. These novel analogues were constructed by convergent synthesis applying the Wittig−Horner coupling reaction between CD-ring ketones (41,42,44) and A-ring phosphine oxide (11). The introduction of C22-fluoro units was achieved by stereoselective deoxy-fluorination for synthesizing 5 and 6 or two-step cationic fluorination for 7. The absolute configuration of the C22-fluoro-8-oxo-CD-ring (39) was confirmed by X-ray crystallographic structure determination. The basic biological activity of the side-chain fluorinated analogues, including compounds (5−7), was evaluated. Generally, osteocalcin promoter transactivation activity decreased in the order of C24-fluoro, C23-fluoro, and C22-fluoro analogues. In addition, the metabolic stability of C22-fluoro-25-hydroxyvitamin D 3 (5−7) against hCYP24A1 metabolism was also evaluated. 22,22-Difluoro-25(OH)D 3 (7) was more stable against hCYP24A1 metabolism compared with its non-fluorinated counterpart 25-hydroxyvitamin D 3 (1), but fluorination at the C22 position had little effect on the metabolic stability compared with C24-and C23-fluoro analogues. Our research clarified that side-chain fluorination in vitamin D markedly changes CYP24A1 metabolic stability depending on the fluorinating position.

Efficient Stereo-Selective Fluorination on Vitamin D3 Side-Chain Using Electrophilic Fluorination

“…The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain 35 (18.7 mg, 55%) as a colorless oil. The spectral data matched with those reported in the literature [8].…”

“…Until 2023, there was only one example of 22-fluorovitamin D synthesis, and the stereochemistry of C22 was unknown [26]. We recently reported the first stereo-selective fluorination at the C22 position of the CD-ring side-chain and synthesis of (22R)-and (22S)-fluoro-25-hydroxyvitamin D 3 [8]. However, multiple synthetic steps were required, including separation of the diastereomer mixture of C22-allylalcohols (19,20) and the deoxyfluorination step using DAST, resulting in low overall yields (Scheme 3).…”

“…In the report [8], we also developed an alternative C22-fluorination methodology using a cationic fluorination reaction between the methyl ester (23) and N-fluorobenzenesulfonimide (NFSI) under basic conditions. However, this route yielded only the (22R)-fluoro product (21); efficient cationic fluorination of the (22S)-fluoro counterpart to yield the (22R)-CD-ring ( 11) has yet to be explored (Scheme 4).…”

“…This was converted to amide (30) followed by fluorination at the C22-position using NFSI in the presence of LHMDS to give the desired (22S)-fluoro-CD-ring (29) as a major product, along with (22R)-fluoro-CD-ring (33) at a ratio of 1.4:1. After separating 29 and 33 using silica gel column chromatography, they were converted to the corresponding Weinreb amides (34,35), which were the synthetic intermediates of C22-fluoro-CD-rings 11 and 12 [8], using methoxy(methyl)amine hydrochloride and LHMDS in moderate yields and with a rigid stereochemical integrity (Scheme 7).…”

“…Regio-and stereo-selective fluorination of the vitamin D 3 side-chain is one of the efficient methods used to modulate biological activities, such as binding affinity to the vitamin D receptor (VDR), transactivation activity through the VDR, and metabolic stability against CYP24A1 [1]. We have reported the regio-and stereo-selective fluorination of the CD-ring side-chain starting from the Inhoffen-Lythgoe diol (1) and constructed C26,27-fluoro-CD-rings (2-4) [2,3], C24-fluoro-CD-rings (5-7) [4,5], C23-fluoro-CD-rings (8-10) [6,7], and C22-fluoro-CD-rings (11)(12)(13) [8] (Figure 1) to create a chemical library of side-chain fluorovitamin D 3 analogues using a convergent method (Scheme 1) [9][10][11].…”