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.

For slowing or preventing the biological degradation of the VD 3 side chain, replacing C-H with C-F bond(s) at appropriate positions should prolong their half-life in vivo, since a C-F bond is stronger than a C-H bond chemically. The introduction of fluorine atoms into VD 3 analogues can also alter electron distribution, which can confer lower pKa at the hydroxy group(s), change the dipole moment, and influence the conformation because of their marked electron-withdrawing properties. Because of these unique properties, both academic institutions and industries have designed and synthesized numerous fluorinated VD 3 analogues, similarly to other bioactive compounds with fluorine atoms. Most of them show fluorination at the main metabolic site(s) and/or neighboring hydroxy group of the VD 3 molecule, namely either an A-ring or a side-chain moiety or both. This review introduces fluorinated VD 3 analogues and their synthetic methodologies, including some basic biological activities. For slowing or preventing the biological degradation of the VD3 side chain, replacing C-H with C-F bond(s) at appropriate positions should prolong their half-life in vivo, since a C-F bond is stronger than a C-H bond chemically. The introduction of fluorine atoms into VD3 analogues can also alter electron distribution, which can confer lower pKa at the hydroxy group(s), change the dipole moment, and influence the conformation because of their marked electron-withdrawing properties. Because of these unique properties, both academic institutions and industries have designed and synthesized numerous fluorinated VD3 analogues, similarly to other bioactive compounds with fluorine atoms. Most of them show fluorination at the main metabolic site(s) and/or neighboring hydroxy group of the VD3 molecule, namely either an A-ring or a side-chain moiety or both. This review introduces fluorinated VD3 analogues and their synthetic methodologies, including some basic biological activities.

A-Ring Fluorinated VD3 Analogues
On the VD3 A-ring, 1α-hydroxylation is the final and essential step to produce the hormonal form of VD3 from 25(OH)D3 (2), and 1α,25(OH)2D3 (1) exerts a range of physiological activities by binding to the vitamin D receptor (VDR). The A-ring moiety is anchored in the VDR ligand-binding pocket through four hydrogen-bonding interactions, i.e., the 1α-hydroxy group to Ser233 and Arg270, and the 3β-hydroxy group to Tyr143 and Ser274 [21]. As expected, based on the importance of the A-ring moiety, many A-ring fluorinated VD3 analogues have been synthesized and evaluated regarding their biological activities.

1-Fluorinated VD3 Analogues
DeLuca and coworkers described the first synthesis of 1-fluoro-VD3 for the purpose of studying the possibility of using it as a kind of VD3 antagonist against 1α-hydroxylase in 1979 [22]. They prepared 1α-hydroxyvitamin D3-3-acetate by the selective acetylation of 1α-hydroxyvitamin D3 [1α(OH)D3 (5)] and used it as a starting material. The fluorination step was achieved by N,N-(diethylamino)sulfur trifluoride (DAST) to afford 1-fluorovitamin D3 (6) (Scheme 2). The authors did not assign its C1 configuration in the report. The biological evaluation revealed that 6 demonstrated a relative preference for stimulating bone calcium mobilization with respect to intestinal calcium transport after metabolism in vivo, and 6 was a weak agonist that could not be used as an anti-vitamin D agent. Scheme 1. Deactivation metabolic pathways of 25(OH)D 3 (2) and 1α,25(OH) 2 D 3 (1).

A-Ring Fluorinated VD 3 Analogues
On the VD 3 A-ring, 1α-hydroxylation is the final and essential step to produce the hormonal form of VD 3 from 25(OH)D 3 (2), and 1α,25(OH) 2 D 3 (1) exerts a range of physiological activities by binding to the vitamin D receptor (VDR). The A-ring moiety is anchored in the VDR ligand-binding pocket through four hydrogen-bonding interactions, i.e., the 1αhydroxy group to Ser233 and Arg270, and the 3β-hydroxy group to Tyr143 and Ser274 [21]. As expected, based on the importance of the A-ring moiety, many A-ring fluorinated VD 3 analogues have been synthesized and evaluated regarding their biological activities.

1-Fluorinated VD 3 Analogues
DeLuca and coworkers described the first synthesis of 1-fluoro-VD 3 for the purpose of studying the possibility of using it as a kind of VD 3 antagonist against 1α-hydroxylase in 1979 [22]. They prepared 1α-hydroxyvitamin D 3 -3-acetate by the selective acetylation of 1αhydroxyvitamin D 3 [1α(OH)D 3 (5)] and used it as a starting material. The fluorination step was achieved by N,N-(diethylamino)sulfur trifluoride (DAST) to afford 1-fluorovitamin D 3 (6) (Scheme 2). The authors did not assign its C1 configuration in the report. The biological evaluation revealed that 6 demonstrated a relative preference for stimulating bone calcium mobilization with respect to intestinal calcium transport after metabolism in vivo, and 6 was a weak agonist that could not be used as an anti-vitamin D agent.
On the other hand, although 1α-fluoro-25(OH)D3 (11) showed no stimulation of intestinal calcium transport or bone calcium mobilization activities at a dosage level of 1.3 μg, its binding affinity to chick intestine VDR was 30 times greater than that of 25(OH)D3.
On the other hand, although 1α-fluoro-25(OH)D 3 (11) showed no stimulation of intestinal calcium transport or bone calcium mobilization activities at a dosage level of 1.3 µg, its binding affinity to chick intestine VDR was 30 times greater than that of 25(OH)D 3 .
Next, 2α-fluorovitamin D 3 (27) was also synthesized, and its biological activity was tested in 1986 [32]. Electrophilic 2α-fluorination of 3,6β-diacetoxycholest-2-ene (28) using CsSO 4 F gave 2α-fluoroketone (29). To construct the B-secosteroidal structure, conventional photochemical conversion and subsequent thermal isomerization were applied (Scheme 10) [32]. The biological effects of 27 on intestinal calcium transport and bone calcium mobilization as well as the serum calcium concentration at a dosage level of 500 ng for rats were measured, and the activities were found to be essentially equivalent to those of VD 3 itself. Next, 2α-fluorovitamin D3 (27) was also synthesized, and its biological activity was tested in 1986 [32]. Electrophilic 2α-fluorination of 3,6β-diacetoxycholest-2-ene (28) using CsSO4F gave 2α-fluoroketone (29). To construct the B-secosteroidal structure, conventional photochemical conversion and subsequent thermal isomerization were applied (Scheme 10) [32]. The biological effects of 27 on intestinal calcium transport and bone calcium mobilization as well as the serum calcium concentration at a dosage level of 500 ng for rats were measured, and the activities were found to be essentially equivalent to those of VD3 itself. The synthesis and biological activities of 2β-fluoro-1α,25-dihydroxyvitamin D3 (30) were reported by Scheddin et al. in 1998 [33]. The synthetic route was similar to Ikekawa's [31], as shown in Scheme 11, and the biological evaluation revealed that the synthetic 2βfluoro-1α,25-(OH)2D3 (30) exhibited greater potency in vitro, for example, six-times higher affinity for VDR, nearly identical affinity for the vitamin D-binding protein (DBP), and 90times higher antiproliferative activity toward C3H10T1/2 cells under serum-containing conditions, as well as five-times greater adipogenesis inhibitory activity than the natural hormone 1α,25(OH)2D3 (1).

Scheme 10.
Introduction of the 2α-fluoro group to VD 3 using an electrophilic fluorination reaction.
The catalytic asymmetric stereoselective synthesis of the A-ring precursor of the 19nor type 2α-fluorovitamin D3 analogue (31) and its synthesis were reported by Mikami et al. [34,35]. The regio-and stereo-selective 2α-fluorination was achieved via a ring opening reaction of chiral epoxide (32) mediated by HfF4/Bu4NH2F3, the asymmetric catalytic carbonyl-ene cyclization was used to construct the 6-membered A-ring precursor, and the subsequent coupling reaction with the CD ring afforded 2α-fluoro-19-normaxacalcitol (31) (Scheme 12). This 2α-fluoro-22-oxa-19-nor analogue (31) had very low DBP-binding affinity but four-times stronger VDR-binding potency than its 22-oxa-19-nor counterpart and also showed significant transactivation activity [34]. It was also shown that 31 was The catalytic asymmetric stereoselective synthesis of the A-ring precursor of the 19-nor type 2α-fluorovitamin D 3 analogue (31) and its synthesis were reported by Mikami et al. [34,35]. The regio-and stereo-selective 2α-fluorination was achieved via a ring opening reaction of chiral epoxide (32) mediated by HfF 4 /Bu 4 NH 2 F 3 , the asymmetric catalytic carbonyl-ene cyclization was used to construct the 6-membered A-ring precursor, and the subsequent coupling reaction with the CD ring afforded 2α-fluoro-19-normaxacalcitol (31) (Scheme 12). This 2α-fluoro-22-oxa-19-nor analogue (31) had very low DBP-binding affinity but four-times stronger VDR-binding potency than its 22-oxa-19-nor counterpart and also showed significant transactivation activity [34]. It was also shown that 31 was highly effective in inhibiting metastatic tumor growth in vivo without toxicity in terms of hypercalcemia and weight loss [35].

3-Fluorinated VD 3 Analogues
The C3 position of VD 3 has a β-hydroxy group, and substitution of the hydroxy with a fluorine atom is of fundamental interest. There are several reports of replacing the C3-hydroxy group with a fluorine atom in order to demonstrate the expected positive effects on biological activity.

3-Fluorinated VD3 Analogues
The C3 position of VD3 has a β-hydroxy group, and substitution of the hydroxy with a fluorine atom is of fundamental interest. There are several reports of replacing the C3hydroxy group with a fluorine atom in order to demonstrate the expected positive effects on biological activity.

CD-Ring Fluorinated VD 3 Analogues: 11-Fluorinated VD 3 Analogues
To our knowledge, only one report has been published on the synthesis of CD-ring fluoro-VD 3 analogues. In 1994, De Clercq and coworkers designed and synthesized 11αand 11β-fluorovitamin D 3 analogues (61-64) with the aim of inducing a conformational change from s-trans to s-cis at the C6-C7 single bond via expected hydrogen bond formation between the C11-F and C1α-OH groups [48]. Enone (65), readily available from Grundmann's ketone, was used as a starting material. Epoxidation of 65, followed by reductive opening with lithium dimethylcuprate, gave the C11α-OH functional group (66). After coupling with A-ring phosphine oxides, the C11α-OH group was treated with N-(2-chloro-1,1,2-trifluoroethyl)diethylamine (FAR) to give C11α-F and C11β-F isomers as well as an elimination product at a ratio of 3:1:1 (Scheme 21). NMR spectra analyses of the 11-fluoro-1α(OH)D 3 showed that all analogues had a large J 6-7 coupling constant, reflecting an almost exclusive s-trans extended geometry without intramolecular hydrogen bonding between C11-F and 1α-OH.

CD-Ring Fluorinated VD3 Analogues: 11-Fluorinated VD3 Analogues
To our knowledge, only one report has been published on the synthesis of CD-ring fluoro-VD3 analogues. In 1994, De Clercq and coworkers designed and synthesized 11αand 11β-fluorovitamin D3 analogues (61-64) with the aim of inducing a conformational change from s-trans to s-cis at the C6-C7 single bond via expected hydrogen bond formation between the C11-F and C1α-OH groups [48]. Enone (65), readily available from Grundmann's ketone, was used as a starting material. Epoxidation of 65, followed by reductive opening with lithium dimethylcuprate, gave the C11α-OH functional group (66). After coupling with A-ring phosphine oxides, the C11α-OH group was treated with N-(2chloro-1,1,2-trifluoroethyl)diethylamine (FAR) to give C11α-F and C11β-F isomers as well as an elimination product at a ratio of 3:1:1 (Scheme 21). NMR spectra analyses of the 11fluoro-1α(OH)D3 showed that all analogues had a large J6-7 coupling constant, reflecting an almost exclusive s-trans extended geometry without intramolecular hydrogen bonding between C11-F and 1α-OH.

Side-Chain Fluorinated VD3 Analogues
The CYP24A1 pathway is well-known as the deactivation pathway of both 25(OH)D3 (2) and 1α,25(OH)2D3 (1) [19]. Varieties of side-chain-fluorinated VD3 analogues have been actively synthesized because of the expected slower catabolism resulting from the presence of fluorine atoms at the oxidation site or adjacent area.

22-Fluorinated VD3 Analogues
In 1986, Kumar and coworkers described the synthesis of 22-fluorovitamin D3 (67) starting from (22S)-cholest-5-ene-3β,22-diol (68) [49]. Selective protection of the C3 hydroxy group as an acetate, followed by fluorination at the C22 position by DAST, gave 22fluorocholest-5-en-3β-acetate (69). After forming the C5-7 diene unit in the B ring, photolysis and thermal isomerization yielded the target 22-fluorovitamin D3 (67) without assigning C22 stereochemistry (Scheme 22). They tested the biological activities of the analogue in vitro and in vivo, referring to the potency of intestinal calcium transport, serum calcium level, calcium-binding protein induction, plasma vitamin D-binding protein (DBP), and VDR affinities, and concluded that the introduction of a fluorine atom to C22 resulted in the compound, with weak biological activities and poor binding to DBP compared with VD3 itself.

Side-Chain Fluorinated VD 3 Analogues
The CYP24A1 pathway is well-known as the deactivation pathway of both 25(OH)D 3 (2) and 1α,25(OH) 2 D 3 (1) [19]. Varieties of side-chain-fluorinated VD 3 analogues have been actively synthesized because of the expected slower catabolism resulting from the presence of fluorine atoms at the oxidation site or adjacent area.

22-Fluorinated VD 3 Analogues
In 1986, Kumar and coworkers described the synthesis of 22-fluorovitamin D 3 (67) starting from (22S)-cholest-5-ene-3β,22-diol (68) [49]. Selective protection of the C3 hydroxy group as an acetate, followed by fluorination at the C22 position by DAST, gave 22-fluorocholest-5-en-3β-acetate (69). After forming the C5-7 diene unit in the B ring, photolysis and thermal isomerization yielded the target 22-fluorovitamin D 3 (67) without assigning C22 stereochemistry (Scheme 22). They tested the biological activities of the analogue in vitro and in vivo, referring to the potency of intestinal calcium transport, serum calcium level, calcium-binding protein induction, plasma vitamin D-binding protein (DBP), and VDR affinities, and concluded that the introduction of a fluorine atom to C22 resulted in the compound, with weak biological activities and poor binding to DBP compared with VD 3 itself.

CD-Ring Fluorinated VD3 Analogues: 11-Fluorinated VD3 Analogues
To our knowledge, only one report has been published on the synthesis of CD-ring fluoro-VD3 analogues. In 1994, De Clercq and coworkers designed and synthesized 11αand 11β-fluorovitamin D3 analogues (61)(62)(63)(64) with the aim of inducing a conformational change from s-trans to s-cis at the C6-C7 single bond via expected hydrogen bond formation between the C11-F and C1α-OH groups [48]. Enone (65), readily available from Grundmann's ketone, was used as a starting material. Epoxidation of 65, followed by reductive opening with lithium dimethylcuprate, gave the C11α-OH functional group (66). After coupling with A-ring phosphine oxides, the C11α-OH group was treated with N-(2chloro-1,1,2-trifluoroethyl)diethylamine (FAR) to give C11α-F and C11β-F isomers as well as an elimination product at a ratio of 3:1:1 (Scheme 21). NMR spectra analyses of the 11fluoro-1α(OH)D3 showed that all analogues had a large J6-7 coupling constant, reflecting an almost exclusive s-trans extended geometry without intramolecular hydrogen bonding between C11-F and 1α-OH.

Side-Chain Fluorinated VD3 Analogues
The CYP24A1 pathway is well-known as the deactivation pathway of both 25(OH)D3 (2) and 1α,25(OH)2D3 (1) [19]. Varieties of side-chain-fluorinated VD3 analogues have been actively synthesized because of the expected slower catabolism resulting from the presence of fluorine atoms at the oxidation site or adjacent area.

22-Fluorinated VD3 Analogues
In 1986, Kumar and coworkers described the synthesis of 22-fluorovitamin D3 (67) starting from (22S)-cholest-5-ene-3β,22-diol (68) [49]. Selective protection of the C3 hydroxy group as an acetate, followed by fluorination at the C22 position by DAST, gave 22fluorocholest-5-en-3β-acetate (69). After forming the C5-7 diene unit in the B ring, photolysis and thermal isomerization yielded the target 22-fluorovitamin D3 (67) without assigning C22 stereochemistry (Scheme 22). They tested the biological activities of the analogue in vitro and in vivo, referring to the potency of intestinal calcium transport, serum calcium level, calcium-binding protein induction, plasma vitamin D-binding protein (DBP), and VDR affinities, and concluded that the introduction of a fluorine atom to C22 resulted in the compound, with weak biological activities and poor binding to DBP compared with VD3 itself.

23-Fluorinated VD 3 Analogues
As mentioned in the Introduction, the C23 position of VD 3 is one of the essential metabolic sites of CYP24A1; therefore, C23-fluorinated VD 3 analogues have been designed and synthesized based on the idea of blocking the oxidative position. Ikekawa and colleagues achieved the first synthesis of a C23-fluoro-VD 3 analogue, 23,23-difluoro-25(OH)D 3 (70), in 1984 [50]. For the synthesis of 70, the triene structure was constructed by applying the well-established route through 5,7-diene steroids, and the difluoro unit was introduced using DAST into a reactive α-ketoester (71) (Scheme 23).

23-Fluorinated VD3 Analogues
As mentioned in the Introduction, the C23 position of VD3 is one of the essential metabolic sites of CYP24A1; therefore, C23-fluorinated VD3 analogues have been designed and synthesized based on the idea of blocking the oxidative position. Ikekawa and colleagues achieved the first synthesis of a C23-fluoro-VD3 analogue, 23,23-difluoro-25(OH)D3 (70), in 1984 [50]. For the synthesis of 70, the triene structure was constructed by applying the well-established route through 5,7-diene steroids, and the difluoro unit was introduced using DAST into a reactive α-ketoester (71) (Scheme 23).  [52]. The starting methyl ketone was available from VD2, and a subsequent hexafluoroacetone (HFA) aldol reaction gave a 23-oxo derivative, which was reduced to 23R-and 23S-secondary alcohols that could be separated and subjected to deoxyfluorination using DAST to afford 73 and 74 (Scheme 24). Both analogues showed higher VDR-binding affinity and HL-60 cell differentiation activity than falecalcitriol.  [53]. The preliminary biological evaluation revealed that the 23S-isomer (76) showed higher resistance to CYP24A1 metabolism than its 23R-isomer (75).  (73) and its 23S isomer (74) in 2000 [52]. The starting methyl ketone was available from VD 2 , and a subsequent hexafluoroacetone (HFA) aldol reaction gave a 23-oxo derivative, which was reduced to 23R-and 23S-secondary alcohols that could be separated and subjected to deoxyfluorination using DAST to afford 73 and 74 (Scheme 24). Both analogues showed higher VDR-binding affinity and HL-60 cell differentiation activity than falecalcitriol.

24-Fluorinated VD 3 Analogues
Since oxidation of the hydroxy function at the C24 position catalyzed by CYP24A1 is one of the important pathways to deactivate both 25(OH)D 3 and 1α,25(OH) 2 D 3 , developing practical methods to construct the C24-fluoro unit on the VD 3 skeleton has been pursued since 1979. The first synthesis of the 24,24-difluorovitamin D 3 analogue was reported independently by Takayama's group [54] and Kobayashi-Ikekawa's group [55]. Both synthetic routes involved the key intermediate (79), and the two groups synthesized the same analogue, 24,24-difluoro-25(OH)D 3 (80). For the construction of the 24,24-difluoro unit, Takayama and coworkers utilized the reaction of α-ketoester (81)

25-Fluorinated VD 3 Analogues
The 25-hydroxylation is the initial metabolic conversion of VD 3 by CYP2R1 or CYP27A1 [18], and 25(OH)D 3 (2) is known as the major circulating metabolite in the human body. As described in Section 2.1, DeLuca's group also synthesized 1,25-difluorovitamin D3 in 1981, and this compound was devoid of any biological activity [21].
Using the reaction of acetylides with excess HFA gas as the key step, syntheses of the hexafluoroalkynyl-VD3 analogue (104), C-seco-hexafluoroalkynyl-VD3 analogue (105), and the previously mentioned two-side-chain analogues 17-22 including alkene side chains (Section 2.1.) were reported by Ohira et al. in 1992 [78], by Wu et al. in 2002 [79], and by Maehr et al. in 2009 [28], respectively (Scheme 36). Compound 104 had a potent inducing effect on the differentiation of cancer cells, with little calcium mobilization activity. Compound 105 showed comparable VDR-binding affinity to the natural hormone 1 and had strong antiproliferative activity against four cancer cell lines in vitro, with 1% calcemic activity compared with 1 in vivo. More recently, Sigüeiro et al. synthesized C22-diyne analogues with a C17-methyl group, including the hexafluoropropanol unit at the terminal (106), which showed potent VDR-binding affinity [80]. Using the reaction of acetylides with excess HFA gas as the key step, syntheses of the hexafluoroalkynyl-VD 3 analogue (104), C-seco-hexafluoroalkynyl-VD 3 analogue (105), and the previously mentioned two-side-chain analogues 17-22 including alkene side chains (Section 2.1) were reported by Ohira et al. in 1992 [78], by Wu et al. in 2002 [79], and by Maehr et al. in 2009 [28], respectively (Scheme 36). Compound 104 had a potent inducing effect on the differentiation of cancer cells, with little calcium mobilization activity. Compound 105 showed comparable VDR-binding affinity to the natural hormone 1 and had strong antiproliferative activity against four cancer cell lines in vitro, with 1% calcemic activity compared with 1 in vivo. More recently, Sigüeiro et al. synthesized C22-diyne analogues with a C17-methyl group, including the hexafluoropropanol unit at the terminal (106), which showed potent VDR-binding affinity [80].

Scheme 36.
Combination of the hexafluoropropanol unit derived from hexafluoroacetone (HFA) and triple bond(s), which brought conformational rigidity to the side chain.
Hayashi and colleagues described the introduction of the 26,26,26,27,27,27-hexafluoro unit utilizing an aldol reaction [52]. The C23 ketone was treated with HFA in the presence of LiHMDS to afford the HFA adduct (see Scheme 24 in Section 5.2).

Summary
This review summarized the historical fluorinated VD3 analogues with modification from the A-ring to the end of the side-chain, including their synthetic methods. With the aim of preventing or slowing their activation or degradation by CYPs, the A-ring and sidechain have been mainly focused on, and numerous VD3 analogues containing the fluorine atom(s) have been synthesized. Hydroxylation at the C25 and C1α positions of VD3 is Scheme 36. Combination of the hexafluoropropanol unit derived from hexafluoroacetone (HFA) and triple bond(s), which brought conformational rigidity to the side chain.
Hayashi and colleagues described the introduction of the 26,26,26,27,27,27-hexafluoro unit utilizing an aldol reaction [52]. The C23 ketone was treated with HFA in the presence of LiHMDS to afford the HFA adduct (see Scheme 24 in Section 5.2).

Summary
This review summarized the historical fluorinated VD3 analogues with modification from the A-ring to the end of the side-chain, including their synthetic methods. With the aim of preventing or slowing their activation or degradation by CYPs, the A-ring and sidechain have been mainly focused on, and numerous VD3 analogues containing the fluorine atom(s) have been synthesized. Hydroxylation at the C25 and C1α positions of VD3 is

Summary
This review summarized the historical fluorinated VD 3 analogues with modification from the A-ring to the end of the side-chain, including their synthetic methods. With the aim of preventing or slowing their activation or degradation by CYPs, the A-ring and side-chain have been mainly focused on, and numerous VD 3 analogues containing the fluorine atom(s) have been synthesized. Hydroxylation at the C25 and C1α positions of VD 3 is necessary for the activation process of the molecule by CYP2R1/CYP27A1 and CYP27B1, respectively; therefore, in general, the introduction of fluorine to these positions decreases the biological activity through VDR if compared to non-fluorinated 25(OH)D 3 or 1α,25(OH) 2 D 3 as each parent VDR ligand. On the other hand, fluorination at the side chain C23, C24, and C26 (27) of VD 3 , where deactivating hydroxylation occurs based on CYP24A1 metabolism, produces strong VDR agonists that have a long half-life in vivo. Among them, falecalcitriol was successfully approved for the treatment of secondary hyperparathyroidism in Japan. Discovery of the new functions of VD 3 continues; for example, the potent SREBP-inhibitory activity of 25(OH)D 3 [82] and the new fluorinated analogues with their efficient synthetic methods may contribute to the treatment of patients with VD 3 -function-related disease in the future.