Structure-activity relationship studies on vitamin D-based selective SREBP/SCAP inhibitor KK-052.

Vitamin D3 metabolites block lipid biosynthesis by promoting degradation of the complex of sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) independent of their effects on the vitamin D receptor (VDR). We previously reported the development of KK-052, the first vitamin D-based SREBP inhibitor that mitigates hepatic lipid accumulation without VDR-mediated calcemic action in mice. Herein we extend our previous work to synthesize KK-052 analogues. Various substituents were introduced to the phenyl ring of KK-052, and two KK-052 analogues were found to exhibit more potent SREBP/SCAP inhibitory activity than KK-052, whereas they all lack VDR activity. These new KK-052 analogues may be suited for further development as VDR-silent SREBP/SCAP inhibitors.

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.

Three novel analogues of C22-fluoro-25-hydroxyvitamin D3 (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 D3 (5-7) against hCYP24A1 metabolism was also evaluated. 22,22-Difluoro-25(OH)D3 (7) was more stable against hCYP24A1 metabolism compared with its non-fluorinated counterpart 25-hydroxyvitamin D3 (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.

Synthesis of New 26,27-Difluoro- and 26,26,27,27-Tetrafluoro-25-hydroxyvitamin D3: Effects of Terminal Fluorine Atoms on Biological Activity and Half-life.

As an extension of our research on providing a chemical library of side-chain fluorinated vitamin D3 analogues, we newly designed and synthesized 26,27-difluoro-25-hydroxyvitamin D3 (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D3 (2) using a convergent method applying the Wittig-Horner coupling reaction between CD-ring ketones (13, 14) and A-ring phosphine oxide (5). The basic biological activities of analogues, 1, 2, and 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D3 [HF-25(OH)D3] were examined. Although the tetrafluorinated new compound 2 exhibited higher binding affinity for vitamin D receptor (VDR) and resistance to CYP24A1-dependent metabolism compared with the difluorinated 1 and its non-fluorinated counterpart 25-hydroxyvitamin D3 [25(OH)D3], HF-25(OH)D3 showed the highest activity among these compounds. Osteocalcin promoter transactivation activity of these fluorinated analogues was tested, and it decreased in the order of HF-25(OH)D3, 2, 1, and 25(OH)D3 in which HF-25(OH)D3 showed 19-times greater activity than the natural 25(OH)D3.

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

Our research regarding side-chain fluorinated vitamin D3 analogues has explored a series of efficient fluorination methods. In this study, a new electrophilic stereo-selective fluorination methodology at C24 and C22 positions of the vitamin D3 side-chain was developed using N-fluorobenzenesulfonimide (NFSI) and CD-ring imides with an Evans chiral auxiliary (26,27,30).

The First Convergent Synthesis of 23,23-Difluoro-25-hydroxyvitamin D3 and Its 24-Hydroxy Derivatives: Preliminary Assessment of Biological Activities.

In this paper, we report an efficient synthetic route for the 23,23-difluoro-25-hydroxyvitamin D3 (5) and its 24-hydroxylated analogues (7,8), which are candidates for the CYP24A1 main metabolites of 5. The key fragments, 23,23-difluoro-CD-ring precursors (9-11), were synthesized starting from Inhoffen-Lythgoe diol (12), and introduction of the C23 difluoro unit to α-ketoester (19) was achieved using N,N-diethylaminosulfur trifluoride (DAST). Preliminary biological evaluation revealed that 23,23-F2-25(OH)D3 (5) showed approximately eight times higher resistance to CYP24A1 metabolism and 12 times lower VDR-binding affinity than its nonfluorinated counterpart 25(OH)D3 (1).

23,25-Dihydroxyvitamin D₃ is liberated as a major vitamin D₃ metabolite in human urine after treatment with ß-glucuronidase: Quantitative comparison with 24,25-dihydroxyvitamin D₃ by LC/MS/MS.

The complete understanding of the excretion of surplus 25-hydroxyvitamin D₃ [25(OH)D₃] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] 24-glucuronide was identified as a major urinary vitamin D₃ metabolite, while the glucuronide of 23,25-dihydroxyvitamin D₃ [23,25(OH)₂D₃] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)₂D₃ liberated in urine by the treatment with ß-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)₂D₃ in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)₂D₃ and 24,25(OH)₂D₃ liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)₂D₃ was almost as much as that of 24,25(OH)₂D₃, in contrast to the fact that the plasma concentration of 23,25(OH)₂D₃ was much lower than that of 24,25(OH)₂D₃. These results strongly suggested that 23,25(OH)₂D₃ is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)₂D₃. Furthermore, the amount ratios of 23,25(OH)₂D₃ to 24,25(OH)₂D₃ in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D₃ supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D₃.

Controlled lipid ß-oxidation and carnitine biosynthesis by a vitamin D metabolite.

Lactone-vitamin D3 is a major metabolite of vitamin D3, a lipophilic vitamin biosynthesized in numerous life forms by sunlight exposure. Although lactone-vitamin D3 was discovered 40 years ago, its biological role remains largely unknown. Chemical biological analysis of its photoaffinity probe identified the hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), a mitochondrial enzyme that catalyzes ß-oxidation of long-chain fatty acids, as its selective binding protein. Intriguingly, the interaction of lactone-vitamin D3 with HADHA does not affect the HADHA enzymatic activity but instead limits biosynthesis of carnitine, an endogenous metabolite required for the transport of fatty acids into the mitochondria for ß-oxidation. Lactone-vitamin D3 dissociates the protein-protein interaction of HADHA with trimethyllysine dioxygenase (TMLD), thereby impairing the TMLD enzyme activity essential in carnitine biosynthesis. These findings suggest a heretofore undescribed role of lactone-vitamin D3 in lipid ß-oxidation and carnitine biosynthesis, and possibly in sunlight-dependent shifts of lipid metabolism in animals.

Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism.

Two 24-fluoro-25-hydroxyvitamin D3 analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D3 side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D3 revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D3 (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.

The Synthesis and Biological Evaluation of D-Ring-Modified Vitamin D Analogues.

The vitamin D3 structure consists of the A-ring, a linker originating from the B-ring, C-ring, D-ring, and side-chain moieties. Each unit has its unique role in expressing the biological activities of vitamin D3. Many efforts have been made to date to assess the possible clinical use of vitamin D. Some organic chemists focused on the D-ring structure of vitamin D and synthesized D-ring-modified vitamin D analogues, and their biological activities were studied. This review summarizes the synthetic methodologies of D-ring-modified vitamin D analogues, except for seco-D, and their preliminary biological profiles.

Design and Synthesis of Fluoro Analogues of Vitamin D.

The discovery of a large variety of functions of vitamin D3 and its metabolites has led to the design and synthesis of a vast amount of vitamin D3 analogues in order to increase the potency and reduce toxicity. The introduction of highly electronegative fluorine atom(s) into vitamin D3 skeletons alters their physical and chemical properties. To date, many fluorinated vitamin D3 analogues have been designed and synthesized. This review summarizes the molecular structures of fluoro-containing vitamin D3 analogues and their synthetic methodologies.

Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system.

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 µg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.

Discovery of a Vitamin D Receptor-Silent Vitamin D Derivative That Impairs Sterol Regulatory Element-Binding Protein In Vivo.

Vitamin D3 metabolites inhibit the expression of lipogenic genes by impairing sterol regulatory element-binding protein (SREBP), a master transcription factor of lipogenesis, independent of their canonical activity through a vitamin D receptor (VDR). Herein, we designed and synthesized a series of vitamin D derivatives to search for a drug-like small molecule that suppresses the SREBP-induced lipogenesis without affecting the VDR-controlled calcium homeostasis in vivo. Evaluation of the derivatives in cultured cells and mice led to the discovery of VDR-silent SREBP inhibitors and to the development of KK-052 (50), the first vitamin D-based SREBP inhibitor that has been demonstrated to mitigate hepatic lipid accumulation without calcemic action in mice. KK-052 maintained the ability of 25-hydroxyvitamin D3 to induce the degradation of SREBP but lacked in the VDR-mediated activity. KK-052 serves as a valuable compound for interrogating SREBP/SCAP in vivo and may represent an unprecedented translational opportunity of synthetic vitamin D analogues.

Synthetic Chemical Probes That Dissect Vitamin D Activities.

Vitamin D3 metabolites are capable of controlling gene expression in mammalian cells through two independent pathways: vitamin D receptor (VDR) and sterol regulatory element-binding protein (SREBP) pathways. In the present study, we dissect the complex biological activity of vitamin D by designing synthetic vitamin D3 analogs specific for VDR or SREBP pathway, i.e., a VDR activator that lacks SREBP inhibitory activity, or an SREBP inhibitor devoid of VDR activity. These synthetic vitamin D probes permitted identification of one of the vitamin D-responsive genes, Soat1, as an SREBP-suppressed gene. The chemical probes developed in the present study may prove useful in dissecting the intricate interplay of vitamin D actions, thereby providing insights into how vitamin D target genes are regulated.

Introduction of fluorine atoms to vitamin D3 side-chain and synthesis of 24,24-difluoro-25-hydroxyvitamin D3.

During our ongoing studies of vitamin D, we focused on the vitamin D3 side-chain 24-position, which is the major metabolic site of human CYP24A1. In order to inhibit the metabolism of vitamin D3, 24,24-difluorovitamin D3analogues are important candidates. In this paper, we report the practical introduction of the difluoro-unit to the 24-position to synthesize 24,24-difluoro-CD ring (1) and 24,24-difluoro-25-hydroxyvitamin D3 (2).

Synthesis and CYP24A1-Dependent Metabolism of 23-Fluorinated Vitamin D3 Analogues.

Two novel 23-fluorinated 25-hydroxyvitamin D3 analogues were synthesized using Inhoffen-Lythgoe diol as a precursor of the CD-ring, efficiently. Introduction of the C23 fluoro group was achieved by the deoxy-fluorination reaction using N,N-diethylaminosulfur trifluoride or 2-pyridinesulfonyl fluoride (PyFluor). Kinetic studies on the CYP24A1-dependent metabolism of these two analogues revealed that (23S)-23-fluoro-25-hydroxyvitamin D3 was more resistant to CYP24A1-dependent metabolism than its 23R isomer.

Concise synthesis of 23-hydroxylated vitamin D3 metabolites.

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.

Effects of 2-substitution on 14-epi-19-nortachysterol-mediated biological events: based on synthesis and X-ray co-crystallographic analysis with the human vitamin D receptor.

Both 2α- and 2ß-hydroxypropyl substituted 14-epi-1α,25-dihydroxy-19-nortachysterols were synthesized to study the human vitamin D receptor (hVDR) binding affinity, binding configurations, and interactions with amino acid residues in the ligand binding domain of hVDR by X-ray co-crystallographic analysis. In conjunction with our previous results on 14-epi-19-nortachysterol, 2-methylidene-, 2α-methyl-, 2ß-methyl, and 2α-hydroxypropoxy-14-epi-19-nortachysterol, we propose a variety of effects of substitution at the C2 position in the 14-epi-19-nortachysterol skeleton on biological activities.

Recent developments for introducing a hexafluoroisopropanol unit into the Vitamin D side chain.

Among numerous studies on synthetic approaches to and the biological activities of vitamin D analogues, we herein focused on falecalcitriol, an analogue of calcitriol (1α,25-dihydroxyvitamin D3), in which a 26,26,26,27,27,27-hexafluoroisopropanol unit has been introduced into the side chain. Falecalcitriol was designed to escape from the metabolism of CYP24A1 and has been used as a drug to treat secondary hyperparathyroidism since 2001. Its metabolite, the 23-hydroxy form, retains biological activity and resistants to further metabolism. Recent developments in synthetic methodologies for introducing the hexafluoroisopropanol unit into the vitamin D CD-ring side chain were described herein.

Synthesis of 2α- and 2ß-(3-hydroxypropyl)- 7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 and their biological activity.

According to the binding mode of 14-epi-1α,25-dihydroxy-19-nortachysterol in the ligand binding domain of human vitamin D receptor (hVDR), i.e., 5,6- and 7,8-s-trans configuration that was shown by X-ray co-crystallographic analysis, 7,8-cis-locked 1α,25(OH)2D3 analogs were synthesized. In this paper, the synthesis and biological activity of 2α- and 2ß-(3-hydroxypropyl)-7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 are reported. The A-ring and CD-ring precursors for the Julia-Kociensky coupling reaction to create a diene system of the target molecules were prepared using our original methods. hVDR binding affinity and osteocalcin promoter transactivation activity of the new 7,8-cis-14-epi-vitamin D3 analogs were evaluated. Interestingly, the 2ß-substituted 7,8-cis-analog was a better binder for hVDR than the 2α-isomeric counterpart.

Synthesis of 3-hydroxypyridines using ruthenium-catalyzed ring-closing olefin metathesis.

New synthetic routes to substituted 3-hydroxypyridines 6 are presented. Ring-closing olefin metathesis (RCM)/elimination and RCM/oxidation/deprotection of nitrogen-containing dienes 4 are the key processes of the routes. An application of RCM/oxidation/deprotection to the synthesis of 3-aminopyridine 13f is also described.