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.


Introduction
Vitamin D 3 is a fat-soluble vitamin produced from 7-dehydrocholesterol through a two-step pericyclic reaction on the B-ring: a photochemical electrocyclic reaction and a subsequent thermal [1,7]-sigmatropic rearrangement. 25-Hydroxyvitamin D 3 [25(OH)D 3 (1)] and 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 (2)] are important compounds responsible for various physiological functions to maintain homeostasis in the human body. Vitamin D 3 is divided into five constructive units: An A-ring, a linker originating from the B-ring, a C-ring, a D-ring, and a side-chain, each of which plays an essential role in expressing vitamin D-related activities [1,2].
Among these structures, both the A-ring and side-chain, in particular, are vitamin D 3 activation and inactivation sites controlled by vitamin D-metabolizing CYPs [3][4][5], and the introduction of a substituent to and the structural transformation of these sites have been vigorously pursued. On the other hand, the CD-ring does not undergo direct hydroxylation in the activation and inactivation steps of the vitamin D 3 molecule (Scheme 1).
For the synthesis of vitamin D 3 analogues, the CD-ring part is often used directly from the natural vitamin D 3 CD-ring part due to the difficulty of constructing the chiral CD-ring structure. Therefore, it is challenging to introduce functional groups to this part or transform the CD-ring to another skeleton. As a result, the synthesis of vitamin D 3 analogues with a modified CD-ring and evaluation of their biological activities are still less explored than synthesis of vitamin D analogues with a modified A-ring or sidechain. However, organic chemists have attempted the introduction of substituents and/or structural transformation of both C-and D-ring parts and have succeeded in synthesizing a group of analogues and evaluating their biological activities to date [6][7][8][9][10]. The D-ring unit is characterized by the direct attachment of the side chain, and there have been many reports that the introduction of substituents to or structural transformation of the D-ring has significant effects on vitamin D activities.
In this review, we focus on the D-ring, which is one of the essential sites forming the basic framework of the vitamin D 3 molecule. We describe the synthetic approach to vitamin D 3 analogues with substituents or structural transformation on the D-ring, except for seco-D analogues, and their preliminary biological activities.

16-Ene-Vitamin D 3 Analogues
Among D-ring-modified vitamin D 3 analogues, a wide variety of 16-ene-vitamin D 3 analogues have been synthesized and evaluated regarding their biological properties as potential anticancer agents with low calcemic effects.
The first synthetic report on the 16-ene-vitamin D 3 analogue (3) was described by Hoffmann-La Roche's group in 1995 [11], and its biological activities were studied [12][13][14][15]. The synthetic route started from a commercially available steroid, dehydroepiandrosterone (4). The 16-ene unit was constructed utilizing an ene reaction between Z-olefin (5) and aldehyde (6) in the presence of Me 2 AlCl to give a mixture of C22-diastereomeric alcohols (7), and subsequent Barton deoxygenation afforded 16-en-23-yne (8). To construct the triene structure, photochemical conversion and subsequent thermal isomerization were applied on the B-ring diene system (Scheme 2). The final product, 3, was identified as a potential antipsoriatic agent at that time.
In Scheme 3, the synthesis of 1-hydroxymethyl analogues (9-12) is described. Introduction of the key 16-ene unit was accomplished by a Me 2 AlCl-mediated ene reaction between olefin (16) and formaldehyde to afford alcohol (17). Next, 17 was converted to iodide (18), followed by the Zn/Ni-promoted Michael addition of iodide (18) to ethyl acrylate to produce ethyl ester (19). The ethyl ester (19) was treated with methyl magnesium bromide or ethyl magnesium bromide to give alcohol (20,21). After oxidation of 20 and 21, protection of the C25-hydroxy group afforded 8-keto-CD-ring (22) and (23). Each ketone (22,23) and the racemic A-ring moiety (24) were coupled using PhLi, and then protecting groups of the coupling products were removed in the presence of TBAF to afford 16-ene analogues (9-12) (Scheme 3).
For the synthesis of 24-oxo-16-ene analogues (13)(14)(15), alcohol (17) was used as a starting material (Scheme 4). The synthesis began with the oxidation of primary alcohol to give aldehyde (25), followed by the addition of 26 to aldehyde (25) under basic conditions, to afford a mixture of C22-diastereomeric alcohols. Removal of the C22-hydroxy group using the Barton reaction yielded 24-oxo compound 27, which was converted to 8-keto-CDring (28) in four steps. Coupling reactions using 28 and A-rings (24,29) were performed under the same conditions as in Scheme 3. Under the coupling conditions, the 24-oxo unit was inert, i.e., no chemical conversion was observed at the C24 position.   The authors evaluated the antiproliferative activity of the newly synthesized analogues (10,12,14,15) in murine keratinocytes in vitro and found that the 26,27-diethyl-1βhydroxymethyl analogue 12 and the 24-oxo with a natural A-ring analogue 15 showed activity comparable with that of the natural hormone 1α,25(OH) 2 D 3 (2), even at physiologically relevant nanomolar concentrations. The 22-oxo-analogue 15 was an intermediary metabolite of 16-ene-1α,25(OH) 2 D 3 (75 in Figure 1) formed through the (24R)-oxidation pathway (Scheme 1) with a longer half-life than the naturally occurring 24-oxo-1α,25(OH) 2 D 3 and equipotent to parent 75 itself in regulating cell-growth and differentiation [18].
Next, one-carbon-elongated analogues (43,44) and their C24-fluorinated versions (45,46) were prepared in a similar manner. Treatment of iodide (51) with t-butyl methyl sulfone under basic conditions gave a 25-sulfone CD-ring (52). Introduction of the difluoro unit to the C24 position of sulfone (52) was achieved by utilizing a cationic fluorination reagent in the presence of nBuLi to give 53. Desilylation of 52 and 53, followed by oxidation at the C8-OH group, afforded ketones (54,55). Finally, these ketones (54,55) were converted into the desired vitamin D 3 analogues (45-48) using the convergent coupling reaction with a lithium anion of the racemic A-ring moiety (56) (Scheme 7).
Synthesis of 23-ene analogues (47,48) started from aldehyde (36) (Scheme 8). Treatment of 36 with t-butyl methyl sulfone under basic conditions afforded C23-OH adducts (57). Mesylation of C23-OH, followed by elimination gave olefin (58). Although deprotection of the TES protective group was problematic using TBAF because of the Michael addition of fluoride to the C23 position, HF-MeCN instead of TBAF gave 59 in a nearly quantitative yield. The resulting alcohol (59) was subjected to PDC oxidation to afford ketone (60). Finally, 60 was converted to the desired vitamin D 3 analogues (47,48) using the same method as above in Scheme 7.
Next, the vitamin D receptor-mediated transcriptional activity of three analogues (42,45,49) in ROS 17/2.8 cells was evaluated, and 42 and 45 showed strong transcriptional activity only approximately twofold less active than that of 1α,25(OH) 2 D 3 (2). For practical chemotherapeutic uses, the calcemic activity of five analogues (42,43,45,47,49) was tested in vivo, revealing that 42, 45, and 49 did not show calcemic activity at a 10 µg/kg dose for 1 week. In contrast, 47 was moderately calcemic, and 43 showed strong calcemic activity [20]. The synthesis of a C20-epi form of the 16-ene analogue (119) is shown in Scheme 11. The known C17-ketone (123) was used as a starting material, and stereoselective introduction of the 17Z-double bond was accomplished by a Wittig reaction. Treatment of carboxylic acid (124) with methyl lithium twice, followed by protection of the resulting C25-hydroxy group, afforded the desired CD-ring (125). The stereoselective oxidation of 125 with selenium dioxide afforded a C16-hydroxy-CD-ring (126). The C16-hydroxy group was subsequently converted to a leaving group, and the syn-S N 2' type reaction using a higher-order lithium cuprate (Li 2 Cu 3 R 5 , R = Me, nBu, cPr, tBu, Ph) gave varieties of C20-epi-16-ene-CD-rings (128-132). Removal of the TBS protective group of 128 and subsequent PDC oxidation afforded ketone (133). Synthesis of a C20-epi-16-ene analogue (119) with the triene structure was achieved by the convergent method.
The synthesis and biological activities of C20-cyclopropyl-16-ene-1α,25dihydroxyvitamin D 3 analogues (136,137) were reported by Uskoković et al. in 2006 [25]. As shown in Scheme 13, 136 and a 19-nor type of 137 were prepared from ketone 138. After introduction of the cyclopropyl unit by the Wittig reaction, reduction and TBS protection gave 140. The 16-ene unit formation was accomplished by an ene reaction with formaldehyde to afford 141. The alcohol 141 was subsequently oxidized with pyridinium chlorochromate, and the formed aldehyde was converted to alkyne 143 in three steps. Acetylide prepared from alkyne 143 was reacted with acetone, followed by deprotection of the O-TBS group and reduction of the alkynyl moiety to give C25-OH-CD-ring 144. Oxidation of the C8-OH group, followed by trimethylsilylation of the C25-OH group, afforded 8-ketone 145. Finally, synthesis of 16-ene analogues (136,137) was achieved in a convergent manner by connecting with A-ring (29 or 146). Scheme 13. Synthesis of C20-cyclopropyl-16-ene analogues (136,137).
To compare the anti-inflammatory properties of 136, 149, and 2, the authors tested their potency to inhibit the production of cytokines (IFN-γ, TNF-α, IL-12/23p40, and IL-6) in vitro, and concluded that 136 and its 24-oxo metabolite 149 had a similar activity and significantly higher potency compared to the natural hormone (2).
Evaluation of the calcemic activity of 136, 149, and 2 revealed that 136 and 149 were less calcemic than 2, and 149 was the least calcemic among the three analogues.

16-Modified Vitamin D 3 Analogues
In 2003, the C16-substituted analogue 152 and its C20-epi-version 153 were reported by the LEO Pharma group in order to produce specific antibodies to 1α,25(OH) 2 D 3 (2) and its C20-epi form (Scheme 15) [27]. The CD-ring synthons 154 and 155 were constructed from the known ketone 156. Reduction of 156 with NaBH 4 yielded a mixture of (20R)-OH-CD-ring (157) and its 20S-isomer (158) in a ratio of 85:15. After separation of each isomer, 157 and 158 were converted to C20-natural form CD-ring (161) and C20-epi form CDring (162), respectively, in two steps. Tosylation of 161 and 162, followed by nucleophilic substitution to give 163 and C20-epi 164. The key step for introducing the C16-OH group was achieved by hydroboration/oxidation of 163 and C20-epi 164 to afford C16α-OH 165 and C20-epi-16α-OH 166 along with C16β-OH isomers as the minor products. Both C16α-OH-CD-rings (165,166) were converted to 8-keto-CD-ring (154) and C20-epi-8-keto-CD-ring (155), respectively, in four steps. The Wittig-Horner coupling reaction with the lithium salt of the A-ring precursor 29 gave the coupling products. The acetate group of the coupling products was replaced with glutaric acid esters, followed by deprotection with TBAF to afford 152 and 153. Scheme 15. Synthesis of the C16-substituted analogue 152 and its C20-epi-version 153 for the hapten formula. *: an asymmetric carbon atom.
In 2019, Okamoto and coworkers reported the synthesis of 16-oxa-vitamin D 3 analogues (169,170) [29]. They synthesized the 16-oxa-CD-ring moiety (171) starting from 1-chloro-3-methylbut-2-ene (172) in 20 steps (Scheme 16). For the construction of CDring parts, a Ti(II)-mediated enyne cyclization/Cu-catalyzed allylation and a subsequent ring-closing metathesis reaction (RCM) were applied. The synthetic 171 was coupled with the A-ring moieties (173,174) in the presence of PdCl 2 (dppf) and KOH to produce 16-oxa-vitamin D 3 analogues (169,170). The VDR binding affinity of the newly synthesized 16-oxa analogues 169 and 170 was evaluated using the fluorescence polarization vitamin D-receptor competitor assay and time-resolved fluorescence resonance energy transfer VDR co-activator assay in comparison with those of the natural hormone (2) and its 19-nor form. It was demonstrated that both 169 and 170 were potent analogues. However, they were less potent than both the natural hormone (2) and its 19-nor-form.

Decalin-Vitamin D analogues
The natural hormone, 1α,25(OH) 2 D 3 (2), contains a trans-hydrindane CD-ring fragment because of the biological synthetic pathway starting from lanosterol as the original steroidal skeleton. In 1996, Vandewalle et al. expanded the five-membered D-ring to a six-membered ring, and the CD-ring of the new vitamin D consisted of a trans-decalin ring system as shown in Scheme 17 [30]. (S)-Wieland-Miescher ketone 175 [31] was converted to trans-5-oxo-decalin 177a with the natural 20R-configuration at the vitamin D 3 side chain and also 179a with the unnatural 20S-configuration side chain. During the synthesis of 177a and 179a, cis-fused decalins (177b, 179b) were also produced; however, treatment of the mixture of 5-oxo-decalins under basic conditions afforded trans-decalins as the major products in their equilibriums, respectively. These decalin-type CD-rings were connected to the A-ring precursors (29 or 146), respectively, to yield the new class of 1α,25(OH) 2 D 3 analogues 180 and 182 as well as the 19-nor-versions 181 and 183. It is noteworthy that the natural 20R-configuration analogues of 180 and 181 showed higher VDR binding affinity, which was almost at the same level as that of 1α,25(OH) 2 D 3 (2), than their 20-epi-counterparts 182 and 183, and the cell differentiation and proliferation activity of 180 and 181 was one order of magnitude higher than that of 2; however, 20-epi-counterparts 182 and 183 possessed weaker activity than 2. The trans-decalin pair represented one of the rare examples that the 20-epimer exhibited reduced biological activities when compared with the analogues with the natural 20R-configuration [32]. De Clercq's group developed the trans-decalin core structure for pseudo-S 2 -symmetrical vitamin D analogues (188,190) [33]. For the synthesis, bis-triflate 185 was prepared from crystalline diketone 184 [34], and enyne 186 was connected in two-ways using Suzuki-Miyaura coupling and Sonogashira coupling reactions followed by appropriate chemical treatments to afford 188 and 190 in crystalline form, respectively (Scheme 18). However, these analogues did not show VDR-binding and antiproliferative activities.
As shown in Figure 3, the De Clercq group also developed various CD-ring modified analogues 191-195 [35][36][37]. These spiro-analogues showed low calcemic activity. From a natural product diosgenin, a spiro-vitamin D analogue 196 was synthesized by a Japanese group in 2005, and this compound, 196, induced apoptosis in Hep G2 cells through activation of p53 and Bax mRNAs [38].
Verlinden et al. published excellent review articles on CD-ring modified analogues of vitamin D, which included a D-ring-or C-ring lacking vitamin D molecules, and interestingly, these analogues showed moderate vitamin-D-related biological activities [39,40].

Conclusions
Actually, the 16-ene structure of the CD-ring part of vitamin D 3 first appeared in the total synthesis of 1α,25(OH) 2 D 3 (2) reported by the Hoffmann-La Roche group in 1982 [51]. To elongate the side chain at C20 from the (17Z)-ethylidene CD-ring creating the 20R-natural stereochemistry, they utilized the ene reaction with ethyl propionate in the presence of Lewis acid EtAlCl 2 . After the successful ene reaction, the first 16-ene structure was obtained, but the double bond was then reduced stereo-selectively to the saturated CD-ring system that was included in the natural active vitamin D 3 skeleton. A great variety of unique 16-ene vitamin D analogs were synthesized using the ene reaction, and their biological activities were evaluated by Hoffmann-La Roche group and Posner's group as shown in Section 2. It was proved that 16-ene-1α,25(OH) 2 D 3 had higher binding affinity for the VDR, lower affinity for the vitamin D binding protein in circulation, and greater resistance to 24-oxo-mediated catabolism as compared with 1α,25(OH) 2 D 3 . Another strategy for 16-ene construction was developed by Mouriño's group in 1999. They utilized S N 2'-syn-facial displacement toward (17Z)-olefin carbamate from 135 by high-order cuprates (Li 2 Cu 3 Me 5 , etc.) to generate the 20R-natural configuration and the 16-ene double bond with high yield. The leaving group, a carbamate group, for the S N 2' reaction was located at the C16 position, and various types of substituents could be introduced at the C20 position, stereoselectively, by this route. On the other hand, examples of 16-modified vitamin D analogues were few as described in Section 3, and only hydroxylated analogues were synthesized in which Mouriño's synthetic intermediate was included as above. The unique example was Okamoto's 16-oxa analogues of 1α,25(OH) 2 D 3 , even though these analogues exhibited weaker VDR binding affinity. Vandewalle and De Clercq developed a trans-decalin CDring system in the vitamin D structure, and these analogues showed potent biological activities, which is summarized in Section 4. Finally, our synthetic 15-substituted vitamin D 3 analogues that possessed moderate biological activity were described in Section 5, but combination with 16-ene modification brought higher potency in VDR binding and osteocalcin transactivation activity than the natural hormone 2. Although it is known that seco-D-ring system as vitamin D analogues [39], this review covers real ring systems as the vitamin D derivatives with the chemically modified D-ring. Modification on the vitamin D skeleton is worthy of challenging, and only real synthetic molecules are able to tell us benefits for the specific disease treatment.
Author Contributions: Conceptualization, F.K. and A.K.; writing-original draft preparation, F.K. and S.M.; writing-review and editing, A.K.; supervision, A.K.; funding acquisition, A.K. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the grant-in-aid from the Japan Society for the Promotion of Science (no. 18K06556 to AK).

Conflicts of Interest:
The authors declare no conflict of interest.