A Mixed Population of Antagonist and Agonist Binding Conformers in

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A Mixed Population of Antagonist and Agonist Binding Conformers in a Single Crystal Explains Partial Agonism against Vitamin D Receptor: Active Vitamin D Analogues with 22R‑Alkyl Group Yasuaki Anami, Toshimasa Itoh, Daichi Egawa, Nobuko Yoshimoto, and Keiko Yamamoto* Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan ABSTRACT: We are continuing to study the structural basis of vitamin D receptor (VDR) agonism and antagonism by using 22S-alkyl vitamin D analogues. Here we report the synthesis and biological evaluation of 22R-alkyl analogues and the X-ray crystallographic analysis of vitamin D receptor ligand-binding domain (VDR-LBD) complexed with a 22Ranalogue. VDR-LBD complexed with the partial agonist 8a showed that 8a binds to VDR-LBD with two conformations, one of which is the antagonist/VDR-LBD complex structure and the other is the agonist/VDR-LBD complex structure. The results indicate that the partial agonist activity of 8a depends on the sum of antagonistic and agonistic activities caused by the antagonist and agonist binding conformers, respectively. The structural basis observed here must be applicable to the partial agonism of other ligand-dependent nuclear receptors. This is the first report describing the trapping of a conformational subset of the ligand and the nuclear receptor in a single crystal.

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INTRODUCTION 1α,25-Dihydroxyvitamin D3, 1, is an active form of vitamin D and is recognized as a hormone that plays a pivotal role in calcium homeostasis, cell differentiation and proliferation, and immunomodulation.1 It exerts actions through gene transcription by binding to vitamin D receptor (VDR) belonging to the nuclear receptor superfamily.2 VDR changes conformation from an inactive form to an active form by binding 1 as well as various agonists. Upon binding, VDR forms a heterodimer with retinoid X receptor (RXR). The RXR−VDR heterodimer on response element of the target gene recruits coactivator and transcripts the target gene.3 Various vitamin D analogues have been developed by many groups, since hormone 1 is pharmaceutically important. In fact, VDR agonists are clinically used to treat metabolic bone diseases and skin diseases such as psoriasis.4,5 All analogues clinically used are VDR agonists. VDR antagonists are also interesting in terms of the mechanism of VDR antagonism in addition to their potential use for treating VDR hyperfunction. At present, VDR antagonists are classified structurally into three types. The first type is composed of analogues with a bulky side chain, such as carboxylic ester (ZK series) compounds6 (Chart 1) and adamantane compounds.7 The second type is composed of analogues without a bulky side chain, such as (23S)-25-dehydro-1α-hydroxyvitamin D3-26,23lactone (TEI9647)8 (Chart 1) and its derivatives.8−12 The third type is composed of analogues with a 22S-alkyl group, such as 22S-butyl analogue 2 (Figure 1). We identified 22S-butyl analogue 2 as a VDR antagonist13,14 and have been continuing to study the structural basis of VDR © 2014 American Chemical Society

Chart 1

agonism and antagonism by using 22S-alkyl analogues. First, we synthesized 22S-alkylated analogues systematically and evaluated their biological activity. We found that these analogues show strong antagonist, partial agonist, or full agonist activity, depending on the side chain structure.15 Next, we conducted Xray crystallographic analysis of the ligand-binding domain (LBD) of VDR complexed with 22S-analogue and found that the 22S-butyl group induces an extra cavity.13,16 Figure 1 shows the Connolly channel surface of the ligand-binding pocket (LBP) and clearly indicates the generation of an extra cavity (butyl pocket) to accommodate the butyl group (Figure 1b and Figure 1c). These crystal structures provided important evidence for understanding the structural basis of agonism and antagonism. To date, we have confirmed the followReceived: March 13, 2014 Published: April 17, 2014 4351

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Figure 1. Connolly channel surface of the ligand-binding pocket observed in the crystal structure of the VDR-LBD/ligand complex and the ligand structure: (a) hormone 1/VDR-LBD complex,20 (b) antagonist 2/VDR-LBD complex,13 (c) agonist 3/VDR-LBD complex,16 (d) GEMINI 4/VDRLBD complex.23 (b−d) 22S-Butyl group of 2 and 3 or the second side chain of 4 induce butyl pocket formation. Of these, the ligand that poorly interacts with the C-terminus (helix 11/12) of VDR shows antagonistic activity like 2, while the ligand that intimately interacts with C-terminus (helix 11/12) via hydrogen bond with His393 shows agonistic activity like 3 and 4.

Figure 2. Structures of vitamin D analogues.

ing.13−16 (1) Analogues with a 22S-butyl group induce butyl pocket formation to accommodate the butyl group (Figure

1b,c). (2) Among ligands that induce butyl pocket formation, a ligand acts as an antagonist when it interacts poorly with the C4352

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Scheme 1. Synthesis of Compounds 5a,b, 6a,b, and 9a,b

prepared by the procedure reported previously.15,17 Treatment of E-enoate 10 with Bu2CuLi in the presence of HMPA and TMSCl afforded diastereoselective conjugate addition product 11a (81%) with 93% diastereoselectivity.17 Compound 11a was reduced with DIBAL to give alcohol 12a, which was then deprotected with CSA to afford desired compound 5a in 85% yield from 11a. Compound 11a was treated with MeLi to give 24,24-dimethyl compound 13a, which was then deprotected with CSA to afford target compound 6a in 75% yield from 11a. Target compound 9a was obtained by a similar procedure using EtMgBr from 11a in 83% yield. 22R-Ethyl compounds 5b, 6b, and 9b were synthesized by the same synthetic method as shown in Scheme 1. Introduction of the 22R-ethyl group into 10 was performed with 95% diastereoselectivity. 22R-Butyl analogues with the hydroxyl group at the 25position, 7a and 8a, were synthesized as shown in Scheme 2. Tosylate 15a derived from 12a was treated with KCN to give cyano compound 16a, which was then reduced with DIBAL to give aldehyde 17a. The aldehyde 17a was reduced with NaBH4 to alcohol 18a and then deprotected with CSA to afford the target compound 7a. Oxidation of aldehyde 17a to ester 19a was performed by treating with iodine and KOH in methanol. This oxidation reaction was developed by us previously.18 Ester 19a was treated with MeLi and then CSA to afford compound

terminus (helix 11/12) of VDR (Figure 1b), whereas a ligand acts as an agonist when it interacts intimately with the Cterminus via a hydrogen bond with His393 (Figure 1c,d). (3) Ligands that do not induce an extra cavity like a butyl pocket exhibit increased agonistic activity when the ligand increases interactions with the C-terminus of VDR (Figure 1a). Thus, butyl pocket formation in VDR strongly affects the agonistic or antagonistic behavior of ligands. We were interested in determining whether the analogues with a 22R-alkyl group show antagonistic activity and how the side chain of the analogue is accommodated in the LBP. Here we report the synthesis and biological evaluation of 22R-alkyl analogues as a counterpart of 22S-alkyl compounds and the Xray crystallographic analysis of VDR-LBD complexed with a 22R-analogue.

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SYNTHESIS

We designed and synthesized new analogues with a 22R-alkyl substituent, 5a−9a and 5b−9b, to clarify the effects of stereochemistry at the 22-position on biological activity and VDR-LBD conformation (Figure 2). Synthesis was performed using the method reported previously for the synthesis of 22Salkyl analogues.15,16 As shown in Scheme 1, 22R-butyl vitamin D derivatives 5a, 6a, and 9a were synthesized from E-enoate 10, which was 4353

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Scheme 2. Synthesis of Compounds 7a,b and 8a,b

Table 1. VDR Binding Affinity of Synthetic Analogues 5−9, IC50 (nM)a

a

Competitive binding of 1 and synthetic compounds 5−9 to the human vitamin D receptor. The experiments were carried out in duplicate. The IC50 values are derived from dose−response curves and represent the compound concentration required for 50% displacement of radiolabeled 1α,25dihydroxyvitamin D3 from the receptor protein.

analogues (5a−8a) showed stronger affinity than the corresponding ethyl analogues (5b−8b) except for 9a and 9b. Analogues with a dimethyl alcohol at the side chain terminus (6a,b and 8a,b) showed more potent VDR affinity compared to the corresponding analogues with a primary alcohol (5a,b and 7a,b). The transactivation ability of synthetic compounds 5−9 was examined using the mouse osteopontin luciferase reporter gene assay system in Cos7 cells and HEK293 cells. The results are shown in Figure 3. In Cos7 cells, 6a, 7a, and 5b−7b showed little transactivation, while they concentration-dependently inhibited the transactivation induced by the hormone 1.

8a. 22R-Ethyl compounds 7b and 8b were obtained by the same synthetic method (Scheme 2).

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BIOLOGICAL ACTIVITIES

Binding affinity for VDR was evaluated by a competitive binding assay using tritiated hormone 1. Recombinant human VDR-LBD, which was expressed as a C-terminus GST-tagged protein using the pGEX-VDR vector15,19 in E. coli BL21, was used as the VDR. The results are summarized in Table 1. All compounds showed significant and specific affinity for the VDR, indicating that they are VDR ligands. 22R-Butyl 4354

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Figure 3. Transactivation of compounds 5−9 in Cos7 cells (a−d) and in HEK293 cells (e−h). Transcriptional activity was evaluated by the dual luciferase assay using a full-length human VDR expression plasmid (pCMX-hVDR), a reporter plasmid containing three copies of mouse osteopontin VDRE (SPPx3-TK-Luc), and an internal control plasmid containing sea pansy luciferase expression constructs (pRL-CMV) as described previously.13 In Cos7 cells, the luciferase activity of 10−8 M of 1 was defined as 1 (a−d), and in HEK293 cells the activity of 10−9 M of 1 was defined as 1 (e−h). Inhibitory effect on the transactivation induced by 1,25-(OH)2D3 (1) was also evaluated (c, d, g, h).

Table 2. Summary of Data Collection Statistics and Refinement ligand X-ray source wavelength (Å) space group unit cell dimensions bond (Å) angle (deg) resolution range (Å)a total no. of reflections no. of unique reflections % completenessa Rmerge a,b Refinement Statistics resolution range (Å)a R factor (Rfree/Rwork)a,c

8a (crystal A)

8a (crystal B)

9a

KEK-PF BL-5A 1.000 00 C2

KEK-PF BL-5A 1.000 00 C2

KEK-PF BL-5A 1.000 00 C2

a = 127.84, b = 44.81, c = 46.06 α = 90.00, β = 93.57, γ = 90.00 13.52−1.90 (1.94−1.90) 66 487 20 411 98.6 (99.7) 0.061 (0.336)

a = 127.05, b = 44.77, c = 45.11 α = 90.00, β = 94.20, γ = 90.00 44.99−2.00 (2.06−2.00) 63 521 17 061 98.6 (98.9) 0.066 (0.310)

a = 128.21, b = 44.31, c = 45.81 α = 90.00, β = 93.56, γ = 90.00 63.98−2.40 (2.51−2.40) 18 809 8562 84.3 (84.9) 0.058 (0.210)

13.49−1.90 (1.94−1.90) 0.1885/0.1625

45.03−2.00 (2.06−2.00) 0.2240/0.1989

45.76−2.40 (2.51−2.40) 0.1944/0.1762

a Values in parentheses are for the highest-resolution shell. bRmerge = ∑|(Ihkl − ⟨Ihkl⟩)|/(∑Ihkl), where ⟨Ihkl⟩ is the mean intensity of all reflections equivalent to reflection hkl. cRwork (Rfree) = ∑||Fobs| − |Fcalc||/∑|Fobs|, where 5% of randomly selected data were used for Rfree.

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Figure 4. Binding of 22R-butyl vitamin D analogues to VDR-LBD. Electron density maps with refined compound 8a bound to the VDR-LBD and Connolly channel surface with 8a in crystal A (a−d) and in crystal B (e−h) are shown. Electron density map and Connolly channel surface with compound 9a (i, j) are also depicted. (c, g) Superimposition of major and minor conformers.

PEG4000, and 5% ethylene glycol. Distinct crystals of the VDR-LBD/8a complex were also obtained when the reservoir contained 0.1 M MOPS-Na (pH 7.0), 0.075 M sodium formate, 19% (w/v) PEG4000, and 5% ethylene glycol. We termed the former crystals “crystal A” and the latter crystals “crystal B”. Crystals of the VDR-LBD/9a complex were obtained when the reservoir contained 0.1 M MOPS-Na (pH 7.0), 0.125 M sodium formate, 16% (w/v) PEG4000, and 5% ethylene glycol. Each structure of the protein was elucidated by molecular replacement using the structure of the VDR-LBD/1 complex (PDB code 2ZLC).20 Table 2 summarizes the data collection statistics and refinement for the three structures. All three proteins had a space group of C2 with approximate unit cells of a = 128 Å, b = 44 Å, and c = 46 Å. Structures of crystals A and B were determined at 1.9 and 2.0 Å resolution, respectively. The crystal structure of VDR-LBD complexed with 9a was determined at 2.4 Å resolution. All three crystals showed the formation of a butyl pocket to accommodate the 22-butyl group or the original side chain

Therefore, they are VDR antagonists. Compounds 5a, 8a, 8b, and 9b showed weak transactivation and inhibited hormoneinduced activation, indicating that they are partial agonists for VDR. Only 9a showed full agonistic activity. In HEK293 cells, 7a and 5b−7b showed antagonistic activity; 5a, 6a, and 8a showed partial agonistic activity; and 9a, 8b, and 9b showed full agonistic activity.

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X-RAY CRYSTAL STRUCTURE To understand the influence of the 22R-alkyl substituent on biological activity and protein conformation, we attempted cocrystallization of rat VDR-LBD and 22R-alkyl vitamin D derivatives 5−9. In the absence of coactivator peptide, we could not obtain crystals. However, in the presence of coactivator peptide DRIP205 we could obtain crystals of VDR-LBD accommodating partial agonist 8a and crystals accommodating full agonist 9a. Good quality crystals of the VDR-LBD/8a complex were obtained when the reservoir contained 0.1 M MOPS-Na (pH 7.0), 0.1 M sodium formate, 20% (w/v) 4356

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Figure 5. Interactions between a ligand and VDR. (a, d) In the major conformer, the 1α- and 3β-hydroxyl groups of 8a form pincer-type hydrogen bonds with Ser233 and Arg270 and with Tyr143 and Ser274, respectively, while only His301 is used to form the hydrogen bond with the 25hydroxyl group via water. (b, e) In the minor conformer, the 1α- and 3β-hydroxyl groups of 8a form pincer-type hydrogen bonds, as is the case with the major conformer. The 25-hydroxyl group of 8a forms a hydrogen bond with both His301 and His393 via water. (c) The benzene ring of Phe418 in the major conformer is distorted compared with the canonical Phe, but the benzene ring in the minor conformer is not. (f) Compound 9a interacts with VDR-LBD in a fashion similar to the 22S-epimer 3, including hydrogen bond formation and hydrophobic interactions.

From the 2Fo − Fc map, the side chains of His301, His393 and Phe418 lining the LBP of the VDR-LBD were found to adopt two conformations, with the occupancy ratio corresponding to the major conformer (70%) and the minor (30%) (Figure 5a−c). Thus, the protein conformation was coupled to the ligand conformation. The imidazole ring of His301 in the minor conformer was flipped over compared with the canonical His, whereas its orientation in the major conformer was the same as that of the canonical His (Figure 5a,b).20,21 The benzene ring of Phe418 in the major conformer was distorted compared with the canonical Phe because of steric repulsion with the 22R-butyl group of 8a, whereas in the minor conformer, it was not (Figure 5c). Thus, we were successful in trapping a conformational subset of the ligand and the protein in a single crystal. We assigned the minor conformer as the agonist binding form of the VDR-LBD/8a complex because it has a pincer-type hydrogen bond between the 25-hydroxyl group and both His301 and His393 via water and exhibits intimate interactions between the ligand and helix 11/12. In contrast, the major conformer reflects an antagonist binding form of the complex because of the lack of the pincer-type hydrogen bond and the intimate interactions between the ligand and helix 11/12. The fractional populations of the major and minor conformers must therefore contribute to the antagonistic and agonistic activities, respectively.

bearing the hydroxyl group of the ligand (Figure 4). In addition, the main-chain structures of all three crystals were similar to that of the VDR-LBD/2 complex (data not shown). The 2Fo − Fc map of crystal A indicated that partial agonist 8a binds to VDR with two conformations (Figure 4a−c). The ratio was 7:3. In the major conformer (70%), the original side chain bearing the 25-hydroxyl group of 8a occupied the butyl pocket and the 22R-butyl group occupied the canonical pocket, as expected (Figure 4a,d). In contrast, in the minor conformer (30%), the original side chain occupied the canonical pocket of VDR and the 22R-butyl group occupied the butyl pocket (Figure 4b,d). Figure 4c shows a superimposition of Figure 4a and Figure 4b: the 2Fo − Fc map clearly indicates the existence of two conformers. Interestingly, the Connolly surface of the LBP indicated the generation of a small channel opening to the outside at the top of the butyl pocket (Figure 4d). The 1α- and 3β-hydroxyl groups of 8a were found to form pincer-type hydrogen bonds with Ser233 and Arg270 and with Tyr143 and Ser274, respectively (Figure 5a,b), as is the case with most vitamin D analogues.20,21 In the minor conformer, the 25-hydroxyl group at the side chain of 8a forms a hydrogen bond with both His301 and His393 via water (Figure 5b), while in the major conformer, only His301 is used to form a hydrogen bond with the 25-hydroxyl group via water (Figure 5a). 4357

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Figure 6. Ligand-specific repositioning of Leu305 triggers butyl pocket formation. (a) Interactions of VDR with hormone 1 (green), 22S-butyl analogue 2 (cyan), and 22R-butyl analogue 9a (yellow). Ligand-specific repositioning of the side chain of Leu305 was observed. (b) Interactions of VDR with hormone 1 (green), GEMINI 4 (orange), and 22R-butyl analogue 8a (blue). The original side chain of 8a and 4 caused drastic repositioning of not only the side chain of Leu305 but also the main chain of Leu305. (c) Hydrogen bond between the carbonyl of Leu305 and the NH of Leu309 to form the α-helix. The color of each molecule is same as in (a). (d) Disappearance of the hydrogen bond between the carbonyl of Leu305 and the NH of Leu309 for the α-helix formation. The color of each molecule is the same as in (b). (e) New channel generated in the 8a/ VDR-LBD complex. The channel is generated by the repositioning of the main chain of Glu392, which is caused by the repositioning of the side chain of Leu305.

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DISCUSSION Previously, we found that the 22S-alkyl group of vitamin D analogues 2 and 3 was docked into the butyl pocket (Figure 1b and Figure 1c).13,16 Therefore, we assumed that the 22R-alkyl group of the analogues synthesized here would be docked into the canonical pocket of VDR-LBD and, consequently, that the 22R-alkyl analogues would be unable to interact intimately with the C-terminus (helix 11/12) via a hydrogen bond with His393. For this reason, we predicted that the 22R-alkyl analogues would act as a VDR antagonist. The majority of the analogues worked as an antagonist, as expected, but 5a, 8a,b, and 9a,b acted as either a partial agonist or a full agonist. Cocrystallization of VDR-LBD and individual analogues was attempted but was only successful with the partial agonist 8a and the full agonist 9a. Failure to crystallize the VDR-LBD/ antagonist complex is likely due to the lability of the complex. The crystal structures of the 8a/VDR-LBD and 9a/VDR-LBD complexes gave us a structural basis for understanding why 8a and 9a act as a partial and full agonist, respectively. In crystals of the VDR-LBD/8a complex, 8a binds to VDR with two conformations (Figure 4a−c). As mentioned in our previous paper, no crystal structure of the VDR-LBD/ antagonist complex, which would clearly explain the structural basis of the antagonism, has been reported. Indeed, although compound 2 is an antagonist, in the crystal structure of the 2/ VDR-LBD complex, the ligand is bound to the active conformation of VDR.13 Considering the action of compound 2 as a VDR antagonist, the active conformation of the VDR-

The overall structure of crystal B is quite similar to that of crystal A. The 2Fo − Fc map of crystal B also indicated that partial agonist 8a binds to VDR with two conformations, with the ratio 7:3 (Figure 4e−g). The Connolly surface of the LBP indicated that the channel opening to the outside is slightly larger than in crystal A (Figure 4h). Unlike in crystal A, the electron density for His393 was concentrated, indicating that His393 adopts one conformation (Figure 5d,e), whereas electron density for Leu305 was diffuse, indicating that Leu305 adopts two conformations (data not shown). As shown in Figure 4i, the 2Fo − Fc map indicated that the full agonist 9a/VDR-LBD complex was crystallized in only one conformation. In this complex, surprisingly, the 22R-butyl group of 9a occupies the butyl pocket and the side chain with the hydroxyl group occupies the canonical pocket of VDR (Figure 4i,j), similar to the minor conformer of the 8a/VDRLBD complex. This observation was unexpected, as we had observed that the 22S-butyl group of 3, which is the 22-epimer of 9a, occupies the butyl pocket,16 so we had expected that the 22R-butyl group of 9a would occupy the canonical pocket of VDR. Interestingly, 9a interacts with VDR-LBD in a fashion similar to the 22S-epimer 3,16 including hydrogen bond formation and hydrophobic interactions (Figure 5f). The binding mode of 9a is thus surprising but coincides well with the agonistic activity of 9a. 4358

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Figure 7. B-factor of the crystal structures of VDR-LBD complexed with 1, 8a, and 9a. Green and yellow lines show the B-factor of the protein accommodating agonist, 1 and 9a, respectively. Blue and black lines show B-factor of the protein accommodating the partial agonist 8a in crystals A and B, respectively. Relative value of B-factor indicates a value when the B-factor of K236 is defined as 1.00.

group, which was observed in VDR-LBD complexed with 2, 3, and 9a (Figure 1b, Figure 1c, and Figure 4j). This pocket is generated by repulsion between the butyl group and the side chain of Leu305 at the beginning of helix 7 (Figure 6a). The second type of pocket is induced by the original side chain of the ligand, as observed in the complex with GEMINI 4 which is an analogue with two identical side chains23 (Figure 1d) and 8a (Figure 4d and Figure 4h). The second type of pocket is also generated by repulsion of the original side chain of the ligand (Figure 6b). These demonstrated that ligand-specific repositioning of the side chain of Leu305 triggers the formation of the butyl pocket. It is reasonable that the second type of pocket is larger than the first type because the original side chain of GEMINI and 8a is larger than the butyl group. Interestingly, in the VDR-LBD/ligand complex that forms the second type of pocket, a helix break occurs at the beginning of helix 7 because of the disappearance of the hydrogen bond between the carbonyl of Leu305 and the NH of Leu309 to form the α-helix (Figure 6d). Figure 6b clearly indicates that the disappearance of the hydrogen bond causes the obvious shift of the protein main chain. On the other hand, in the complex forming the first type of pocket, no helix break was observed (Figure 6c). By an investigation into crystal structures of other nuclear receptors, we found similar pocket formation at the same position in PPARγ24 and ERα.25 Taken together, it appears that the region surrounded by the N-terminal of helix 7 and helix 11 in nuclear receptors exhibits some flexibility without loss of binding affinity for the ligand. The formation of a new channel at the top of the butyl pocket was observed in the VDR-LBD/8a complex but not in the VDR-LBD/GEMINI complex (Figure 4d, Figure 4h, Figure 1d). The appearance of this new channel was elucidated from the main-chain shift of Glu392, whose shift is caused by the ligand-specific and drastic repositioning of the side chain of Leu305 as shown in Figure 6e. Figure 7 shows the relative values of B-factors for the crystal structure of VDR-LBD accommodating hormone 1, partial agonist 8a, and full agonist 9a. VDR-LBD complexed with full agonist 1 and 9a demonstrated a relatively stable structure, while the VDR-LBD/8a complex showed a rather unstable structure, especially after helix 10. These results indicate that helix 11 of the VDR-LBD/antagonist complex, even in a crystal, is unstable because of the lack of intimate interactions between the ligand and the C-terminus (helix 11/12) of the protein via the hydrogen bond with His393. This observation corroborates a report of the solution structure of VDR-LBD determined using NMR by Markley, in which the residues between Lys395

LBD/2/peptide complex seems to be a temporary and less preferred structure but was selected during the crystallization process. The same interpretation, i.e., a temporary and less preferred structure selected during crystallization, is also applicable to the major conformer of the 8a/VDR-LBD complex, since there is no active conformation network between the side chain of 8a and the C-terminus (helix 11/ 12) (Figure 4d and Figure 5a). Therefore, we are terming the major conformer the antagonist binding conformation. The minor conformer of the VDR-LBD/8a complex is the agonist binding form of VDR, in which intimate interactions between the ligand and the protein terminus are observed via a hydrogen bond with His393 (Figure 4d, Figure 5b). The major and minor conformers must contribute to the antagonistic and agonistic activities, respectively. We concluded that 22R-butyl analogue 8a in living cells shows partial agonist activity reflecting the sum of antagonistic and agonistic activities caused by the major and minor conformers, respectively. Similar structural elucidations regarding partial agonism against nuclear receptor have been reported where distinct crystal structures with different binding modes were observed in separate crystals but not in a single crystal.22 Pochetti et al. reported different binding modes between PPARγ and the partial agonistic ligand observed in separate crystals, which were generated using different crystallization procedures.22a Nettles et al. reported that coupling of receptor conformation and ligand orientation determines the partial activity by investigating estrogen receptor (ER) and a partial agonist.22b They found distinct ER conformations and distinct ligand orientation in different crystals of wild type and mutated ER. We emphasize that the different binding modes reported by Pochetti et al. and Nettles et al. were observed in different (separate) crystals, whereas in our study different binding modes (two conformations) were detected in a single crystal. Thus, this is the first report showing the trapping of a conformational subset of a ligand and the nuclear receptor in a single crystal. The VDR-LBD/9a complex is the agonist binding form of VDR, in which intimate interactions between the protein terminus and the ligand via a hydrogen bond with His393 are observed (Figure 4j, Figure 5f). Interestingly, the whole molecule of 22R-butyl analogue 9a occupies the same spatial region as that of 22S-butyl compound 3; this is accomplished by shifting the C-20, -21, and -22 to the C-terminus of the protein compared to 3. This structure coincides well with the strong agonistic activity of 9a. In our study, two types of butyl pocket were observed. The first type of pocket is formed by an induced fit with the butyl 4359

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Journal of Medicinal Chemistry

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(3R)-3-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}heptan-1-ol (12a). Ester 11a (837.9 mg, 1.19 mmol) in dry THF (15 mL) was treated with DIBAL (1 M in toluene solution, 3.58 mL, 3.58 mmol) at 0 °C for 1.5 h. The reaction was quenched with 1 N HCl and was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (35 g) with 5% AcOEt/hexane to afford 12a (749.1 mg, 95%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.80 (3H, d, J = 6.3 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 3.67 (2H, m, H-24), 4.42 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.86 (2 carbons), 12.0, 13.0, 14.1, 18.2, 18.3, 22.1, 23.1, 23.4, 25.78 (3 carbons), 25.84 (3 carbons), 27.7, 28.8, 30.1, 32.1, 32.5, 36.2, 37.2, 38.6, 40.7, 45.7, 47.6, 53.8, 56.4, 62.4, 71.7, 72.5, 106.2, 116.1, 122.4, 132.8, 141.2, 153.0. MS (ESI) m/z 681.6 [(M + Na)+, 35]. HRMS (ESI) calcd for C40H74NaO3Si2 (M + Na)+ 681.5074, found 681.5118. IR (neat) 3298, 2955, 2928, 2893, 2858, 1472, 1462, 1256, 1101, 1070, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Butyl-2-methylidene-19,25,26,27-tetranor-1α,24-dihydroxyvitamin D3 (5a). To a solution of 12a (2.9 mg, 0.0047 mmol) in MeOH (200 μL) was added camphor sulfonic acid (4.7 mg, 0.0202 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 60% AcOEt/hexane to afford 5a (1.7 mg, 89%). 1H NMR δ 0.57 (3H, s, H-18), 0.79 (3H, d, J = 6.2 Hz, H-21), 0.90 (3H, t, J = 6.6, 6.8 Hz, -(CH2)3-CH3), 3.60 (1H, m, H-24), 3.73 (1H, m, H-24), 4.48 (2H, m, H-1, 3), 5.09, 5.11 (each 1H, s, -CCH2), 5.88 (1H, d, J = 11.3 Hz, H-7), 6.36 (1H, d, J = 11.3 Hz, H-6). MS (EI) m/z 430.4 (M+, 15), 315.3 (11), 297.3 (12), 269.3 (9), 251.2 (10), 185.2 (11), 69.1 (100). HRMS (EI) calcd for C28H46O3 (M+) 430.3447, found 430.3436. IR (neat) 3325, 2949, 2926, 2858, 1659, 1456, 1072, 1049 cm−1. UV (EtOH) λmax 245, 254, 263 nm. (4R)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-2methyloctan-2-ol (13a). To a solution of ester 11a (57.5 mg, 0.082 mmol) in dry THF (1 mL) at −78 °C was added MeLi (452 μL, 0.49 mmol), and the mixture was heated to −50 °C for 1 h. The reaction was quenched with saturated NH4Cl aqueous solution, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 5% AcOEt/hexane to afford 13a (44.1 mg, 78%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.79 (3H, d, J = 6.6 Hz, H-21), 0.87, 0.89 (each 9H, s, t-Bu), 1.23 (6H, s, H-25, 26), 2.82 (1H, m, H-9), 4.43 (H, m, H-1, 3), 4.92, 4.97 (each 1H, s,-CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.9, −4.88, −4.85, 12.1, 12.9, 14.2, 18.2, 18.2, 22.3, 23.1, 23.4, 25.79 (3 carbons), 25.84 (3 carbons), 28.1, 28.8, 30.2, 30.3, 30.5, 33.4, 36.0, 37.6, 38.6, 40.6, 43.0, 45.7, 47.6, 53.5, 56.4, 71.7 (2 carbons), 72.5, 106.2, 116.2, 122.4, 132.8, 141.2, 153.0. MS (EI) m/z 686.5 (M+, 2), 554.4 (18), 536.3 (13), 366.1 (24), 73.0 (100). HRMS (EI) calcd for C42H78O3Si2 (M+) 686.5490, found 686.5483. IR (neat) 2955, 2928, 2856, 1462, 1373, 1256, 1101, 1072, 935, 896, 835, 775 cm−1. UV (hexane) λmax 246, 255, 264 nm. 22R-Butyl-2-methylidene-19,24-dinor-1α,25-dihydroxyvitamin D3 (6a). To a solution of 13a (21.6 mg, 0.0315 mmol) in MeOH (200 μL) was added camphor sulfonic acid (24.6 mg, 0.106 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 40% AcOEt/hexane to afford 6a (13.8 mg, 96%). 1 H NMR δ 0.58 (3H, s, H-18), 0.78 (3H, d, J = 6.6 Hz, H-21), 1.24 (6H, s, H-25, 26), 4.48 (2H, m, H-1, 3), 5.08, 5.10 (each 1H, s, -C

and Glu421 of VDR-LBD complexed with antagonist were demonstrated to be disordered.26

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CONCLUSIONS We observed a mixed population of antagonist and agonist binding conformers in a single crystal. This observation indicates that the partial agonist activity of 22R-butyl analogue 8a depends on the sum of antagonistic and agonistic activities caused by antagonist and agonist binding conformers, respectively. Combined with our previous study, we draw the following conclusions regarding VDR-agonism, partial agonism, and antagonism. Intimate interactions between the ligand and the C-terminus (helix 11/12) of VDR via a hydrogen bond with His393 overcome the structural strain in VDR caused by the formation of butyl pocket, resulting in the ligand acting as a VDR agonist. However, the ligand acts as a VDR antagonist when it interacts weakly with the C-terminus (helix 11/12) of VDR. Partial agonistic activity is dependent on the sum of agonistic behavior and antagonistic behavior of the ligand and VDR. The structural basis of VDR partial agonism observed here must be applicable to the partial agonism of other liganddependent nuclear receptors, such as steroid hormone receptors, retinoid hormone receptors, and so on.

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EXPERIMENTAL SECTION

All reagents were purchased from commercial sources. Unless otherwise stated, NMR spectra were recorded at 300 MHz for 1H NMR and 75 MHz for 13C NMR in CDCl3 solution with TMS as an internal standard and the chemical shifts are given in δ values. High and low resolution mass spectra were obtained with a JEOL JMS D300, JEOL AccuTOF LC-plus JMS-T100LP, and JEOL JMS-HX110A spectrometers. Relative intensities are given in parentheses in low mass. IR spectra were recorded on a Shimadzu FTIR-8400S spectrophotometer, and data are given in cm−1. UV spectra were recorded on a Beckman DU7500 spectrophotometer. All air and moisture sensitive reactions were carried out under argon or nitrogen atmosphere. Purity was determined by HPLC [Pegasil silica SP100, 4.6 mm × 150 mm, hexane/CHCl3/MeOH (100:25:8), flow rate 1.0 mL/ min] and was >95% for all compounds tested. Ethyl (3R)-3-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}heptanoate (11a). A suspension of CuBr/Me2S (2.44 g, 11.86 mmol) in dry THF (10 mL) was cooled to −30 °C, and to this solution was added n-BuLi (1.62 M in hexanes, 14.64 mL, 23.71 mmol), and the mixture was stirred for 15 min. To this solution was added TMSCl (1.90 mL, 14.82 mmol), HMPA (2.58 mL, 14.82 mmol), and a solution of enoate 10 (953 mg, 1.48 mmol) in dry THF (10 mL) in this order. The mixture was stirred at −30 °C for 1.5 h, and the reaction was quenched with 1 N HCl. The mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (200 g, 40−50 μm) with 0.5% AcOEt/hexane to afford 11a (845 mg, 81%) and its 22S-isomer15 (60.0 mg, 6%). 11a: 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.79 (3H, d, J = 6.7 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 1.26 (3H, t, J = 7.1 Hz, -OCH2CH3), 2.83 (1H, m, H-9), 4.13 (2H, q, J = 7.2, -OCH2CH3), 4.42 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.87 (2 carbons), 12.0, 13.1, 14.1, 14.3, 18.2, 18.3, 22.1, 22.9, 23.4, 25.78 (3 carbons), 25.84 (3 carbons), 27.5, 28.7, 29.6, 32.7, 34.9, 37.1, 37.3, 38.6, 40.7, 45.7, 47.6, 54.1, 56.3, 60.1, 71.6, 72.5, 106.3, 116.2, 122.4, 132.9, 141.0, 153.0, 174.5. MS (FAB+) m/z 701.7 [(M + H)+, 8], 569.5 (26). HRMS (FAB+) calcd for C42H77O4Si2 (M + H)+ 701.5360, found 701.5358. IR (neat) 2955, 2928, 2893, 2856, 1738, 1472, 1462, 1377, 1326, 1256, 1166, 1101, 1070, 935, 896, 835, 775 cm−1. UV (hexane) λmax 245, 253, 263 nm. 4360

dx.doi.org/10.1021/jm500392t | J. Med. Chem. 2014, 57, 4351−4367

Journal of Medicinal Chemistry

Article

CH2), 5.88 (1H, d, J = 11.2 Hz, H-7), 6.35 (1H, d, J = 11.2 Hz, H-6). 13 C NMR δ 12.1, 12.9, 14.2, 22.4, 23.1, 23.5, 28.1, 29.0, 30.2, 30.3, 30.4, 33.4, 36.0, 37.5, 38.2, 40.5, 43.0, 45.8, 45.9, 53.5, 56.5, 70.7, 71.7, 71.9, 107.7, 115.3, 124.3, 130.4, 143.4, 152.0. MS (EI) m/z 458.6 (M+, 9), 368.5 (10), 285.3 (15), 161.3 (22), 147.3 (21), 135.3 (39), 69.2 (100). HRMS (EI) calcd for C30H50O3 (M+) 458.3760, found 458.3762. IR (neat) 3356, 2955, 2928, 2868, 1468, 1377, 1074, 1045, 758 cm−1. UV (EtOH) λmax 246, 254, 263 nm. (5R)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-3ethylnonan-3-ol (14a). To a solution of ester 11a (46.9 mg, 0.067 mmol) in dry THF (1.2 mL) was added EtMgBr (1.0 M in THF solution, 201 μL, 0.20 mmol), and the mixture was stirred at room temperature. After 2.5 h, to the mixture was added an additional EtMgBr (1.0 M in THF solution, 134 μL, 0.13 mmol), and the mixture was stirred. In addition, after 2 h, to the mixture was added an additional EtMgBr (1.0 M in THF solution, 134 μL, 0.13 mmol), and the mixture was stirred for 1.5 h. The reaction was quenched with saturated NH4Cl aqueous solution, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 5% AcOEt/hexane to afford 14a (41.4 mg, 86%). 1H NMR δ 0.04, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.53 (3H, s, H-18), 0.79 (3H, d, J = 6.7 Hz, H-21), 0.87, 0.89 (each 9H, s, t-Bu), 2.82 (1H, m, H-9), 4.42 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.89, −4.86, 8.0, 8.1, 12.0, 13.0, 14.2, 18.18, 18.24, 22.3, 23.4, 25.78 (3 carbons), 25.83 (3 carbons), 28.2, 28.8, 30.3, 31.0, 31.5, 33.2, 34.9, 37.7, 38.0, 38.6, 40.6, 45.7, 47.5, 53.6, 56.4, 71.7, 72.4, 75.3, 106.2, 116.1, 122.4, 132.7, 141.2, 153.0. MS (FAB+) m/z 715.6 [(M + H)+, 3)], 583.5 (14). HRMS (FAB+) calcd for C44H83O3Si2 (M + H)+ 715.5881, found 715.5893. IR (neat) 3501, 2955, 2928, 2856, 1732, 1661, 1620, 1462, 1254, 1101, 1072, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Butyl-2-methylidene-26,27-dimethyl-19,24-dinor1α,25-dihydroxyvitamin D3 (9a). A solution of 14a (29 mg, 0.041 mmol) in dry MeOH (0.725 mL) was added camphor sulfonic acid (28.3 mg, 0.122 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 50% AcOEt/ hexane to afford 9a (19.1 mg, 97%). 1H NMR δ 0.58 (3H, s, H-18), 0.79 (3H, d, J = 6.7 Hz, H-21), 0.84−0.92 (9H, m, CH2CH3 × 3), 2.83 (1H, m, H-9), 4.47 (2H, m, H-3, 1), 5.09 (1H, s, CCH2), 5.11 (1H, s, CCH2), 5.89 (1H, d, J = 11.2 Hz, H-7), 6.36 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ 8.0, 8.1, 12.1, 13.0,14.2, 22.4, 23.1, 23.5, 28.1, 29.0, 30.3, 31.1, 31.4, 33.2, 34.9, 37.6, 38.0, 38.1, 40.5, 45.75, 45.85, 53.5, 56.4, 70.6, 71.8, 75.3, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (ESI) m/z 509.4 [(M + Na)+, 100]. HRMS (ESI) calcd for C32H54NaO3 (M + Na)+ 509.3971, found 509.3981. IR (neat) 3346, 2959, 2932, 2874, 1717, 1651, 1614, 1456, 1377, 1078, 1041, 914, 758, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. (3R)-3-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}heptyl 4-Methylbenzenesulfonate (15a). To a solution of alcohol 11a (47.9 mg, 0.073 mmol) in dry pyridine (1 mL) was added ptoluenesulfonyl chloride (20.78 mg, 0.11 mmol) at 0 °C, and the mixture was stirred for 15 h. To this solution was added ice−water and 1 N HCl, and the mixture was extracted with AcOEt. The organic layer was washed with saturated NaHCO3 aqueous solution and brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 5% AcOEt/hexane to afford 15a (55.1 mg, 93%). 1 H NMR δ 0.04, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.53 (3H, s, H18), 0.72 (3H, d, J = 6.7 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.45 (3H, s, Ph-Me), 2.83 (1H, m, H-9), 4.07 (2H, m, H-24), 4.43 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6), 7.35 (2H, d, J = 8.5 Hz, arom-H), 7.80 (2H, d, J = 8.3 Hz, arom-H). 13C NMR δ −5.1, −4.92, −4.88, −4.86, 12.0, 12.8, 14.1, 18.17, 18.24, 21.6, 22.1, 22.9,

23.4, 25.78 (3 carbons), 25.83 (3 carbons), 27.5, 28.6, 28.7, 29.8, 31.5, 35.9, 36.8, 38.6, 40.6, 45.6, 47.6, 53.7, 56.2, 70.0, 71.7, 72.4, 106.3, 116.2, 122.4, 127.9 (2 carbons), 129.8 (2 carbons), 132.9, 133.4, 140.1, 144.6, 153.0. MS (ESI) m/z 835.5 [(M + Na)+, 95]. HRMS (ESI) calcd for C47H80NaO5SSi2 (M + Na)+ 835.5163, found 835.5173. IR (neat) 2955, 2928, 2893, 2856, 1472, 1462, 1362, 1252, 1188, 1178, 1099, 1070, 937, 897, 835, 775, 663 cm−1. UV (hexane) λmax 246, 255, 265 nm. (4R)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}octanenitrile (16a). A solution of tosylate 15a (773 mg, 0.95 mmol) and KCN (173.3 mg, 2.66 mmol) in dry DMSO (24 mL) was stirred at 70 °C for 1 h. The reaction was quenched with ice−water, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (35 g) with 5% AcOEt/hexane to afford 16a (591.2 mg, 93%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.79 (3H, d, J = 6.7 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 4.43 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.87, −4.86, 12.0, 12.9, 14.1, 16.0, 18.2, 18.3, 22.1, 23.0, 23.4, 25.3, 25.78 (3 carbons), 25.84 (3 carbons), 27.7, 28.7, 29.8, 30.8, 36.9, 38.6, 39.2, 40.7, 45.7, 47.6, 53.8, 56.3, 71.7, 72.4, 106.3, 116.3, 120.2, 122.4, 132.9, 140.9, 153.0. MS (ESI) m/z 690.5 [(M + Na)+, 100]. HRMS (ESI) calcd for C41H73NNaO2Si2 (M + Na)+ 690.5078, found 690.5071. IR (neat) 2955, 2929, 2893, 2856, 2245, 1472, 1462, 1252, 1101, 1072, 935, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 264 nm. (4R)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}octanal (17a). Cyanide 16a (36.2 mg, 0.054 mmol) in dry THF (1 mL) was treated with DIBAL (1 M in toluene solution, 70 μL, 0.070 mmol) at −20 °C. After 1.5 h, to the mixture was added an additional DIBAL (1 M in toluene solution, 54 μL, 0.054 mmol), and the mixture was was stirred at 0 °C for 1.5 h. The reaction was quenched with 1 N HCl and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (4 g) with 2% AcOEt/hexane to afford 17a (22.4 mg, 62%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.81 (3H, d, J = 6.1 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 4.42 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6), 9.78 (1H, t, J = 1.9 Hz, −CHO). 13C NMR δ −5.1, −4.92, −4.88, −4.86, 12.0, 12.9, 14.1, 18.17, 18.24, 21.3, 22.1, 23.0, 23.4, 25.78 (3 carbons), 25.83 (3 carbons), 27.7, 28.8, 30.0, 31.2, 37.1, 38.6, 39.5, 40.7, 43.0, 45.7, 47.6, 53.8, 56.3, 71.7, 72.5, 106.2, 116.2, 122.4, 132.8, 141.1, 153.0, 203.2. MS (ESI) m/z 693.5 [(M + Na)+, 55]. HRMS (ESI) calcd for C41H74O3Si2 (M+) 670.5177, found 670.5190. IR (neat) 2955, 2928, 2893, 2856, 2710, 1728, 1472, 1462, 1252, 1101, 1070, 935, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 265 nm. (4R)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}octan1-ol (18a). To a solution of aldehyde 17a (14.3 mg, 0.021 mmol) in dry MeOH/dry CH2Cl2 (1:1, 2 mL) was added NaBH4 (12.89 mg, 0.34 mmol) at 0 °C, and the mixture was stirred at room temperature for 1.5 h. To the solution was added ice−water, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (4 g) with 15% AcOEt/hexane to afford 18a (14.5 mg, quant). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.78 (3H, d, J = 5.9 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 3.64 (2H, t, J = 6.6 Hz, H-25), 4.43 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.88, −4.86, 12.0, 13.0, 14.2, 18.2, 18.3, 22.2, 23.1, 23.5, 25.0, 25.78 (3 carbons), 25.84 (3 carbons), 27.7, 28.8, 30.2, 31.7, 31.9, 37.2, 38.6, 39.8, 40.7, 45.7, 47.6, 53.9, 56.3, 63.6, 71.7, 72.5, 106.2, 116.1, 122.4, 132.8, 141.2, 153.0. MS (FAB+) m/z 673.6 [(M + H)+, 1], 541.6 (4). HRMS (FAB+) calcd for C41H77O3Si2 (M + H)+ 673.5411, 4361

dx.doi.org/10.1021/jm500392t | J. Med. Chem. 2014, 57, 4351−4367

Journal of Medicinal Chemistry

Article

0 °C, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 50% AcOEt/hexane to afford 8a (13.0 mg, 97%). 1 H NMR δ 0.56 (3H, s, H-18), 0.79 (3H, d, J = 6.0 Hz, H-21), 0.90 (3H, m, CH2CH3), 1.22, 1.23 (each 3H, s, H-26, 27), 2.58 (1H, dd, J = 13.2, 3.8 Hz, H-14), 2.84 (2H, m, H-9), 4.48 (2H, m, H-3, 1), 5.09 (1H, s, CCH2), 5.11 (1H, s, CCH2), 5.89 (1H, d, J = 11.3 Hz, H7), 6.37 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ 12.1, 13.2, 14.2, 22.2, 23.1, 23.3, 23.5, 27.7, 29.0, 29.1, 29.5, 30.2, 31.6, 37.1, 38.2, 40.4, 40.5, 43.1, 45.8, 45.9, 54.0, 56.4, 70.7, 71.3, 71.9, 107.7, 115.3, 124.3, 130.4, 143.5, 152.0. MS (ESI) m/z 495.4 [(M + Na)+, 100]. HRMS (ESI) calcd for C31H52NaO3 (M + Na)+ 495.3814, found 495.3820. IR (neat) 3379, 2957, 2926, 2856, 1456, 1379, 1074, 1047, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. Ethyl (3R)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylpentanoate (11b). A suspension of CuBr/Me2S (444.1 mg, 2.12 mmol) in dry THF (4 mL) was cooled to −30 °C, and to this solution was added EtLi (8.50 mL, 4.25 mmol, 0.5 M solution benzene/ cyclohexane = 90/10). The mixture was stirred for 15 min. To this solution were added TMSCl (338 μL, 2.65 mmol), HMPA (463 μL, 2.65 mmol), and a solution of enoate 10 (170.7 mg, 0.27 mmol) in dry THF (2 mL) in this order. The mixture was stirred at −30 °C for 1.5 h, and the reaction was quenched with 1 N HCl. The mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (60 g, 40−50 μm) with 0.3% AcOEt/hexane to afford 11b (130 mg, 73%) and its 22S-isomer15 (7 mg, 4%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.57 (3H, s, H-18), 0.79 (3H, d, J = 6.7 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 1.26 (6H, t, J = 7.1 Hz, -CH2CH3 × 2), 2.83 (1H, m, H-9), 4.13 (2H, m, -OCH2CH3), 4.43 (2H, m, H-3, 1), 4.92 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.9, −4.8 (2 carbons), 12.0, 12.1, 13.0, 14.3, 18.2, 18.3, 22.1, 23.4, 25.7, 25.77 (3 carbons), 25.84 (3 carbons), 27.6, 28.7, 34.7, 36.6, 38.6, 39.2, 40.7, 45.7, 47.6, 54.0, 56.4, 60.1, 71.6, 72.5, 106.3, 116.2, 122.4, 132.9, 141.0, 153.0, 174.5. MS (FAB+) m/z 673.5 [(M + H)+, 4], 541.5 (30). HRMS (FAB+) calcd for C40H73O4Si2 (M + H)+ 673.5047, found 673.5033. IR (neat) 2955, 2928, 2895, 2856, 1738, 1655, 1618, 1462, 1256, 1101, 1072, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. (3R)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylpentan1-ol (12b). Ester 11b (183.6 mg, 0.27 mmol) in dry THF (2 mL) was treated with DIBAL (1 M in toluene solution, 409 μL, 0.41 mmol) at 0 °C. After 1 h, to the mixture was added an additional DIBAL (1 M in toluene solution, 409 μL, 0.41 mmol), and the mixture was stirred at 0 °C for 30 min. The reaction was quenched with 1 N HCl and was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 10% AcOEt/hexane to afford 12b (160.0 mg, 93%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.57 (3H, s, H-18), 0.79 (3H, d, J = 6.5 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 3.67 (2H, m, H-24), 4.42 (2H, m, H-3, 1), 4.92 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.2 Hz, H7), 6.22 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.86 (2 carbons), 12.0, 12.5, 12.9, 18.2, 18.3, 22.1, 23.4, 24.9, 25.78 (3 carbons), 25.84 (3 carbons), 27.8, 28.8, 32.4, 36.4, 38.2, 38.6, 40.7, 45.7, 47.6, 53.7, 56.4, 62.4, 71.6, 72.5, 106.2, 116.2, 122.4, 132.8, 141.1, 153.0. MS (FAB+) m/z 631.5 [(M + H)+, 2], 499.4 (22). HRMS (FAB+) calcd for C38H71O3Si2 (M + H)+ 631.4942, found 631.4931. IR (neat) 3306, 2955, 2930, 2895, 2856, 1732, 1661, 1620, 1472, 1256, 1101, 1070, 1005, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Ethyl-2-methylidene-19,25,26,27-tetranor-1α,24-dihydroxyvitamin D3 (5b). To a solution of 12b (12.9 mg, 0.021 mmol) in dry MeOH (0.37 mL) was added camphor sulfonic acid (14.2 mg, 0.061 mmol) at 0 °C, and the mixture was stirred at room temperature

found 673.5424. IR (neat) 3331, 2953, 2928, 2895, 2856, 1655, 1618, 1472, 1462, 1256, 1101, 1070, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Butyl-2-methylidene-19,26,27-trinor-1α,25-dihydroxyvitamin D3 (7a). To a solution of 18a (29.0 mg, 0.043 mmol) in dry MeOH (0.5 mL) was added camphor sulfonic acid (30.0 mg, 0.13 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 50% AcOEt/hexane to afford 7a (18.3 mg, 96%). 1H NMR δ 0.56 (3H, s, H-18), 0.78 (3H, d, J = 5.9 Hz, H-21), 0.90 (3H, m, CH2CH3), 2.57 (1H, dd, J = 13.3, 3.7 Hz, H-14), 2.84 (2H, m, H-9), 3.64 (2H, t, J = 6.6 Hz, H-25), 4.48 (2H, m, H-3, 1), 5.09 (1H, s, CCH2), 5.11 (1H, s, CCH2), 5.89 (1H, d, J = 11.3 Hz, H-7), 6.36 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ 12.0 13.0, 14.2, 22.2, 23.1, 23.5, 25.0, 27.7, 29.0, 30.2, 31.6, 31.8, 37.1, 38.2, 39.7, 40.5, 45.77, 45.84, 53.9, 56.4, 63.6, 70.7, 71.8, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (ESI) m/z 467.4 [(M + Na)+, 65]. HRMS (ESI) calcd for C29H48NaO3 (M + Na)+ 467.3501, found 467.3510. IR (neat) 3379, 2928, 2866, 1462, 1379, 1074, 1047, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. Methyl (4R)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien17-yl]ethyl}octanoate (19a). To a solution of aldehyde 17a (14.3 mg, 0.021 mmol) in dry MeOH (10 mL) were added KOH (7.2 mg, 0.11 mmol) and then iodine (19.6 mg, 0.055 mmol) at 0 °C, and the mixture was stirred for 1 h. The reaction was quenched with 10% Na2SO3, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 5% AcOEt/ hexane to afford 19a (10.5 mg, 70%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.55 (3H, s, H-18), 0.79 (3H, d, J = 6.0 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 3.68 (3H, s, -OCH3), 4.42 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.89, −4.86, 11.9, 12.9, 14.1, 18.17, 18.24, 22.1, 23.0, 23.4, 24.5, 25.78 (3 carbons), 25.83 (3 carbons), 27.7, 28.8, 29.7, 30.0, 31.2, 33.1, 37.2, 38.6, 39.5, 40.6, 45.7, 47.6, 51.5, 53.7, 56.3, 71.7, 72.5, 106.2, 116.1, 122.4, 132.8, 141.2, 153.0, 174.6. MS (FAB+) m/z 701.6 [(M + H)+, 6], 569.5 (24). HRMS (FAB+) calcd for C42H77O4Si2 (M + H)+ 701.5360, found 701.5377. IR (neat) 2953, 2928, 2895, 2856, 1742, 1466, 1458, 1256, 1101, 1070, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. (5R)-5-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-2methylnonan-2-ol (20a). To a solution of ester 19a (29.3 mg, 0.042 mmol) in dry THF (1 mL) at −78 °C was added MeLi (1.04 M in Et2O solution, 241 μL, 0.25 mmol), and the mixture was stirred. The mixture was heated to −10 °C for 2 h and then stirred for 2 h. The reaction was quenched with saturated NH4Cl aqueous solution, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 10% AcOEt/hexane to afford 20a (25.3 mg, 86%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.55 (3H, s, H-18), 0.79 (3H, d, J = 5.9 Hz, H-21), 0.86, 0.90 (each 9H, s, t-Bu), 1.22, 1.23 (each 3H, s, H-26, 27), 2.83 (1H, m, H9), 4.42 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, C CH2), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13 C NMR δ −5.1, −4.91, −4.88, −4.85, 12.0, 13.2, 14.2, 18.2, 18.3, 22.2, 23.1, 23.3, 23.5, 25.78 (3 carbons), 25.84 (3 carbons), 27.7, 28.8, 29.1, 29.5, 30.2, 31.7, 37.2, 38.6, 40.5, 40.7, 43.1, 45.7, 47.6, 54.0, 56.3, 71.3, 71.7, 72.5, 106.2, 116.1, 122.4, 132.7, 141.3, 153.0. MS (ESI) m/z 701.6 [(M + H)+, 4]. HRMS (ESI) calcd for C43H81O3Si2 (M + H)+ 701.5724, found 701.5725. IR (neat) 3375, 2955, 2928, 2895, 2856, 1472, 1464, 1256, 1101, 1072, 935, 908, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Butyl-2-methylidene-19-nor-1α,25-dihydroxyvitamin D3 (8a). To a solution of 20a (20.0 mg, 0.029 mmol) in dry MeOH (0.5 mL) was added camphor sulfonic acid (19.9 mg, 0.086 mmol) at 4362

dx.doi.org/10.1021/jm500392t | J. Med. Chem. 2014, 57, 4351−4367

Journal of Medicinal Chemistry

Article

0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.79 (3H, d, J = 6.7 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.82 (1H, m, H-9), 4.42 (2H, m, H-3, 1), 4.92 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.22 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.88, −4.86, 12.0, 12.5, 12.9, 18.2, 18.2, 22.3, 23.4, 25.78 (3 carbons), 25.83 (3 carbons), 25.9, 28.2, 28.8, 31.0, 31.5, 36.87, 36.94, 38.0, 38.6, 40.6, 45.7, 47.6, 53.5, 56.4, 71.7, 72.5, 75.2, 106.2, 116.2, 122.4, 132.7, 141.2, 153.0. MS (FAB+) m/z 687.6 [(M + H)+, 4], 555.5 (20). HRMS (FAB+) calcd for C42H79O3Si2 (M + H)+ 687.5568, found 687.5562. IR (neat) 3497, 2955, 2930, 2883, 2856, 1730, 1655, 1618, 1472, 1462, 1256, 1101, 1072, 935, 897, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Ethyl-2-methylidene-26,27-dimethyl-19,24-dinor1α,25-dihydroxyvitamin D3 (9b). To a solution of 14b (33.3 mg, 0.049 mmol) in dry MeOH (1 mL) was added camphor sulfonic acid (33.8 mg, 0.145 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 40% AcOEt/ hexane to afford 9b (22.8 mg, quant). 1H NMR δ 0.59 (3H, s, H-18), 0.78 (3H, d, J = 6.7 Hz, H-21), 0.87 (9H, t, J = 7.6 Hz, CH2CH3 × 3), 2.57 (1H, dd, J = 13.3, 3.8 Hz), 2.83 (2H, m, H-9), 4.48 (2H, m, H-3, 1), 5.09 (1H, s, CCH2), 5.11 (1H, s, CCH2), 5.88 (1H, d, J = 11.3 Hz, H-7), 6.36 (1H, d, J = 11.3 Hz, H-6) .13C NMR δ 8.0, 8.1, 12.0, 12.5, 12.9, 22.4, 23.5, 25.9, 28.2, 29.0, 31.1, 31.4, 36.8, 36.9, 38.0, 38.1, 40.5, 45.8, 45.9, 53.4, 56.5, 70.6, 71.8, 75.3, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (ESI) m/z 481.4 [(M + Na)+, 75]. HRMS (ESI) calcd for C30H50NaO3 (M + Na)+ 481.3658, found 481.3617. IR (neat) 3340, 2962, 2876, 1715, 1651, 1456, 1381, 1074, 1043, 976, 914, 758, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. (3R)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylpentyl 4Methylbenzenesulfonate (15b). To a solution of alcohol 12b (160 mg, 0.25 mmol) in dry pyridine (3 mL) was added p-toluenesulfonyl chloride (72.5 mg, 0.38 mmol) at 0 °C, and the mixture was stirred for 37 h. To this solution was added ice−water and 1 N HCl, and the mixture was extracted with AcOEt. The organic layer was washed with saturated NaHCO3 aqueous solution and brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 5% AcOEt/hexane to afford 15b (164.9 mg, 83%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.53 (3H, s, H-18), 0.72 (3H, d, J = 6.7 Hz, H-21), 0.78 (3H, t, J = 7.3 Hz, CH2CH3), 0.87, 0.90 (each 9H, s, t-Bu), 2.45 (3H, s, Ph-Me), 2.83 (1H, m, H-9), 3.99−4.17 (2H, m, H-24), 4.43 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.83 (1H, d, J = 11.0 Hz, H-7), 6.21 (1H, d, J = 11.0 Hz, H-6), 7.35 (2H, d, J = 8.0 Hz, arom-H), 7.80 (2H, d, J = 8.3 Hz, arom-H). 13C NMR δ −5.1, −4.92, −4.86 (2 carbons), 11.9, 12.2, 12.7, 18.2, 18.3, 21.6, 22.1, 23.4, 24.4, 25.78 (3 carbons), 25.83 (3 carbons), 27.6, 28.6, 28.7, 36.2, 37.8, 38.6, 40.7, 45.6, 47.6, 53.6, 56.3, 70.0, 71.7, 72.5, 106.3, 116.2, 122.4, 127.9 (2 carbons), 129.8 (2 carbons), 132.9, 133.4, 140.1, 144.6, 153.0. MS (APCI) m/z 807.5 [(M + Na)+, 100]. HRMS (APCI) calcd for C45H76NaO5SSi2 (M + Na)+ 807.4850, found 807.4827. IR (neat) 2955, 2928, 2893, 2856, 1472, 1462, 1362, 1252, 1188, 1178, 1099, 1070, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265 nm. (4R)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexanenitrile (16b). A solution of tosylate 15b (160.2 mg, 0.20 mmol) and KCN (37.2 mg, 0.57 mmol) in dry DMSO (5 mL) was stirred at 70 °C for 1.5 h. The reaction was quenched with ice−water, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 2% AcOEt/hexane to afford 16b (116.3 mg, 89%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.57 (3H, s, H-18), 0.79 (3H, d, J = 6.8 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 4.43 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.87 (2 carbons), 12.0, 12.2, 12.8, 16.0, 18.2, 18.3, 22.1, 23.4, 23.6, 25.2, 25.78

for 1.5 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 40% AcOEt/hexane to afford 5b (8.2 mg, quant). 1H NMR δ 0.57 (3H, s, H-18), 0.79 (3H, d, J = 6.5 Hz, H-21), 0.87 (3H, m, CH2CH3), 2.83 (1H, m, H-9), 3.67 (2H, m, H-24), 4.48 (2H, m, H-1,3), 5.09, 5.11 (each 1H, s, -C CH2), 5.89 (1H, d, J = 11.3 Hz, H-7), 6.36 (1H, d, J = 11.3 Hz, H-6). 13 C NMR δ 12.0, 12.4, 12.9, 22.2, 23.5, 24.8, 27.7, 29.0, 32.4, 36.4, 38.1 (2 carbons), 40.5, 45.78, 45.83, 53.7, 56.4, 62.3, 70.7, 71.8, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (ESI) m/z 425.3 [(M + Na)+, 100]. HRMS (ESI) calcd for C28H42NaO3 (M + Na)+ 425.3032, found 425.3069. IR (neat) 3352, 2957, 2872, 1722, 1657, 1616, 1456, 1380, 1072, 1047, 912, 866, 756, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. (4R)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethyl-2-methylhexan-2-ol (13b). To a solution of ester 11b (28.0 mg, 0.042 mmol) in dry THF (0.5 mL) at −78 °C was added MeLi (1.04 M in Et2O solution, 200 μL, 0.21 mmol), and the mixture was stirred for 1 h. The reaction was quenched with saturated NH4Cl aqueous solution, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (20 g) with 2% AcOEt/hexane to afford 13b (21.5 mg, 79%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.78 (3H, J = 6.6 Hz, d, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 1.24 (6H, s, diMe), 2.82 (1H, m, H-9), 4.42 (2H, m, H-1,3), 4.92, 4.97 (each 1H, s, -CCH2), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.22 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.88, −4.86, 12.1, 12.5, 12.9, 18.17, 18.24, 22.3, 23.4, 25.78 (3 carbons), 25.83 (3 carbons), 26.1, 28.2, 28.8, 30.1, 30.5, 36.8, 38.0, 38.6, 40.6, 43.0, 45.7, 47.6, 53.4, 56.4, 71.6, 71.7, 72.5, 106.2, 116.2, 122.4, 132.8, 141.2, 153.0. MS (FAB+) m/z 659.6 [(M + H)+, 2], 527.5 (11). HRMS (FAB+) calcd for C40H75O3Si2 (M + H)+ 659.5255, found 659.5258. IR (neat) 3377, 2955, 2928, 2893, 2856, 1730, 1657, 1618, 1472, 1464, 1381, 1256, 1101, 1072, 935, 897, 835, 777 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Ethyl-2-methylidene-19,24-dinor-1α,25-dihydroxyvitamin D3 (6b). To a solution of 13b (16.1 mg, 0.025 mmol) in dry MeOH (0.45 mL) was added camphor sulfonic acid (17.1 mg, 0.074 mmol) at 0 °C, and the mixture was stirred at room temperature for 1.5 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (15 g) with 40% AcOEt/hexane to afford 6b (10.9 mg, quant). 1H NMR δ 0.59 (3H, s, H-18), 0.78 (3H, J = 6.7 Hz, d, H-21), 0.87 (3H, t, J = 7.3 Hz, CH2CH3), 1.24 (6H, s, diMe), 2.84 (2H, m, H-9), 4.48 (2H, m, H-1,3), 5.09, 5.11 (each 1H, s, -CCH2), 5.89 (1H, d, J = 11.3 Hz, H-7), 6.36 (1H, d, J = 11.3 Hz, H-6). 13C NMR δ 12.1, 12.5, 12.9, 22.4, 23.5, 26.1, 28.1, 29.0, 30.2, 30.4, 36.8, 37.9, 38.2, 40.4, 42.9, 45.8, 45.9, 53.4, 56.5, 70.6, 71.7, 71.8, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (ESI) m/z 453.3 [(M + Na)+, 100]. HRMS (ESI) calcd for C28H46NaO3 (M + Na)+ 453.3345, found 453.3337. IR (neat) 3371, 2961, 2928, 2872, 1718, 1655, 1616, 1464, 1381, 1074, 1045, 903, 756, 665 cm−1. UV (EtOH) λmax 246, 254, 264 nm. (5R,6R)-6-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}3,5-diethylheptan-3-ol (14b). To a solution of ester 11b (56.0 mg, 0.083 mmol) in dry THF (1.5 mL) was added EtMgBr (1.0 M in THF solution, 250 μL, 0.26 mmol), and the mixture was stirred at room temperature. After 1.5 h, to the mixture was added an additional EtMgBr (1.0 M in THF solution, 250 μL, 0.26 mmol), and the mixture was stirred. In addition, after 1.5 h, to the mixture was added an additional EtMgBr (1.0 M in THF solution, 250 μL, 0.26 mmol), and the mixture was stirred for 1.5 h. The reaction was quenched with saturated NH4Cl aqueous solution, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 3% AcOEt/hexane to afford 14b (47.1 mg, 82%). 1H NMR δ 4363

dx.doi.org/10.1021/jm500392t | J. Med. Chem. 2014, 57, 4351−4367

Journal of Medicinal Chemistry

Article

(3 carbons), 25.83 (3 carbons), 27.7, 28.7, 36.1, 38.6, 40.7, 41.1, 45.7, 47.6, 53.7, 56.3, 71.7, 72.5, 106.3, 116.3, 120.2, 122.4, 132.9, 140.9, 153.0. MS (APCI) m/z 662.5 [(M + Na)+, 15]. HRMS (APCI) calcd for C39H69NNaO2Si2 (M + Na)+ 662.4765, found 662.4751. IR (neat) 2955, 2930, 2885, 2856, 2245, 1472, 1462, 1252, 1101, 1070, 935, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 264 nm. (4R)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexanal (17b). Cyanide 16b (10.9 mg, 0.017 mmol) in dry CH2Cl2 (1 mL) was treated with DIBAL (1 M in toluene solution, 34 μL, 0.034 mmol) at −20 °C for 1 h. The reaction was quenched with 1 N HCl and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 2% AcOEt/hexane to afford 17b (9.1 mg, 84%). 1 H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H18), 0.80 (3H, d, J = 6.4 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 4.43 (2H, m, H-3, 1), 4.92 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.22 (1H, d, J = 11.2 Hz, H-6), 9.78 (1H, t, J = 1.9 Hz, -CHO). 13C NMR δ −5.1, −4.92, −4.86 (2 carbons), 11.9, 12.4, 12.8, 18.2, 18.3, 21.2, 22.1, 23.4, 24.0, 25.78 (3 carbons), 25.84 (3 carbons), 27.7, 28.8, 29.7, 36.4, 38.6, 40.7, 41.5, 43.0, 45.7, 47.6, 53.7, 56.3, 71.6, 72.5, 106.2, 116.2, 122.4, 132.8, 141.1, 153.0, 203.2. MS (FAB+) m/z 643.5 [(M + H)+, 1], 511.4 (9). HRMS (FAB+) calcd for C39H71O3Si2 (M + H)+ 643.4942, found 643.4927. IR (neat) 2955, 2928, 2895, 2856, 2709, 1728, 1472, 1463, 1256, 1101, 1070, 935, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 265 nm. (4R)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexan-1ol (18b). To a solution of aldehyde 17b (21.2 mg, 0.033 mmol) in dry MeOH/dry CH2Cl2 (1:1, 4 mL) was added NaBH4 (20.0 mg, 0.53 mmol) at 0 °C, and the mixture was stirred at room temperature for 1.5 h. To the solution was added ice−water, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 10% AcOEt/hexane to afford 18b (20.0 mg, 94%). 1 H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H18), 0.77 (3H, d, J = 6.3 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 3.64 (2H, t, J = 6.5 Hz, H-25), 4.43 (2H, m, H-3, 1), 4.92 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.86 (2 carbons), 12.0, 12.6, 13.0, 18.2, 18.3, 22.2, 23.5, 24.4, 25.0, 25.78 (3 carbons), 25.84 (3 carbons), 27.8, 28.8, 31.9, 36.5, 38.6, 40.7, 41.7, 45.7, 47.6, 53.8, 56.4, 63.6, 71.6, 72.5, 106.2, 116.1, 122.4, 132.8, 141.2, 153.0. MS (APCI) m/z 667.5 [(M + Na)+, 7]. HRMS (APCI) calcd for C39H72NaO3Si2 (M + Na)+ 667.4918, found 667.4952. IR (neat) 3331, 2955, 2928, 2894, 2858, 1655, 1618, 1472, 1462, 1256, 1101, 1070, 935, 908, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Ethyl-2-methylidene-19,26,27-trinor-1α,25-dihydroxyvitamin D3 (7b). A solution of 18b (14.4 mg, 0.022 mmol) in dry MeOH (0.5 mL) was added camphor sulfonic acid (15.5 mg, 0.067 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 40% AcOEt/hexane to afford 7b (8.2 mg, 88%). 1H NMR δ 0.57 (3H, s, H-18), 0.77 (3H, d, J = 6.3 Hz, H-21), 0.87 (3H, m, CH2CH3), 2.58 (1H, dd, J = 13.2, 3.8 Hz, H-14), 2.84 (2H, m, H-9), 3.64 (2H, t, J = 6.6 Hz, H-25), 4.48 (2H, m, H-3, 1), 5.09 (1H, s, CCH2), 5.11 (1H, s, CCH2), 5.89 (1H, d, J = 11.3 Hz, H-7), 6.36 (1H, d, J = 11.3 Hz, H-6). 13C NMR δ 12.0, 12.5, 12.9, 22.2, 23.5, 24.4, 25.0, 27.7, 29.0, 31.8, 36.4, 38.2, 40.5, 41.7, 45.8, 45.9, 53.8, 56.4, 63.6, 70.7, 71.8, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (APCI) m/z 439.3 [(M + Na)+, 17]. HRMS (APCI) calcd for C27H44NaO3 (M + Na)+ 439.3188, found 439.3187. IR (neat) 3352, 2924, 2854, 1379, 1074, 1045, 756, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. Methyl (4R)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexanoate (19b). To a solution of aldehyde 17b (53.1 mg, 0.083

mmol) in dry MeOH (20 mL) was added KOH (24.1 mg, 0.43 mmol) and then iodine (54.5 mg, 0.21 mmol) at 0 °C, and the mixture was stirred for 1.5 h. The reaction was quenched with 10% Na2SO3, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (5 g) with 2% AcOEt/hexane to afford 19b (51.0 mg, 92%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.79 (3H, d, J = 6.2 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 2.83 (1H, m, H-9), 3.68 (3H, s, -OCHH3), 4.43 (2H, m, H-3, 1), 4.93 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.88 (2 carbons), 11.9, 12.4, 12.8, 18.2, 18.3, 22.1, 23.4, 24.0, 24.4, 25.77 (3 carbons), 25.84 (3 carbons), 27.7, 28.8, 33.1, 36.4, 38.6, 40.6, 41.4, 45.7, 47.6, 51.5, 53.6, 56.3, 71.6, 72.5, 106.2, 116.1, 122.4, 132.8, 141.2, 153.0, 174.7. MS (APCI) m/z 695.5 [(M + Na)+, 45]. HRMS (APCI) calcd for C40H72NaO4Si2 (M + Na)+ 695.4867, found 695.4863. IR (neat) 2955, 2930, 2893, 2856, 1742, 1472, 1462, 1256, 1101, 1070, 935, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 265 nm. (5R)-6-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]-5-ethyl-2-methylheptan-2-ol (20b). To a solution of ester 19b (46.7 mg, 0.069 mmol) in dry THF (2 mL) at −78 °C was added MeLi (1.04 M in Et2O solution, 400 μL, 0.42 mmol), and the mixture was stirred. The mixture was heated to −10 °C for 2 h and then stirred for 2 h. The reaction was quenched with saturated NH4Cl aqueous solution, and the mixture was extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 7% AcOEt/hexane to afford 20b (40 mg, 86%). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.79 (3H, d, J = 6.2 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 1.22, 1.23 (each 3H, s, H-26, 27), 2.33 (1H, dd, J = 13.3, 3.2 Hz, H-14), 2.83 (1H, m, H-9), 4.42 (2H, m, H-3, 1), 4.92 (1H, s, CCH2), 4.97 (1H, s, CCH2), 5.84 (1H, d, J = 11.1 Hz, H7), 6.22 (1H, d, J = 11.1 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.88 (2 carbons), 12.0, 12.6, 13.1, 18.2, 18.3, 22.1, 23.2, 23.5, 24.5, 25.77 (3 carbons), 25.84 (3 carbons), 27.8, 28.8, 29.1, 29.5, 36.5, 38.6, 40.7, 42.4, 43.1, 45.7, 47.6, 53.9, 56.3, 71.3, 71.6, 72.5, 106.2, 116.1, 122.4, 132.7, 141.3, 153.0. MS (APCI) m/z 673.6 [(M + H)+, 5], 657.6 (5), 541.5 (45), 523.5 (90), 409.4 (40), 391.4 (100). HRMS (APCI) calcd for C41H77O3Si2 (M + H)+ 673.5411, found 673.5380. IR (neat) 3387, 2957, 2930, 2895, 2856, 1659, 1620, 1472, 1464, 1256, 1101, 1072, 935, 908, 897, 835, 775 cm−1. UV (hexane) λmax 246, 255, 265 nm. 22R-Ethyl-2-methylidene-19-nor-1α,25-dihydroxyvitamin D3 (8b). A solution of 20b (30.1 mg, 0.045 mmol) in dry MeOH (1 mL) was added camphor sulfonic acid (31.15 mg, 0.13 mmol) at 0 °C, and the mixture was stirred at room temperature for 1.5 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 40% AcOEt/hexane to afford 8b (20.5 mg, quant). 1H NMR δ 0.57 (3H, s, H-18), 0.79 (3H, d, J = 6.2 Hz, H-21), 0.86 (3H, m, CH2CH3), 1.217, 1.224 (each 3H, s, H-26, 27), 2.57 (1H, dd, J = 13.4, 3.9 Hz, H-14), 2.84 (2H, m, H-9), 4.47 (2H, m, H-3, 1), 5.09 (1H, s, CCH2), 5.11 (1H, s, CCH2), 5.89 (1H, d, J = 11.2 Hz, H-7), 6.36 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ 12.0, 12.6, 13.1, 22.2, 23.2, 23.5, 24.4, 27.7, 29.0 (2 carbons), 29.5, 36.4, 38.1, 40.5, 42.4, 43.1, 45.8, 45.9, 53.9, 56.4, 70.6, 71.3, 71.8, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS (APCI) m/z 467.4 [(M + Na)+, 15]. HRMS (APCI) calcd for C29H48NaO3 (M + Na)+ 467.3501, found 467.3502. IR (neat) 3364, 2959, 2928, 2870, 1456, 1379, 1151, 1045, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. Competitive Binding Assay, Human VDR. The human recombinant VDR ligand-binding domain (LBD) was expressed as an N-terminal GST-tagged protein in E. coli Rosetta2 (DE3) pLysS.19 The cells were lysed by sonication. The supernatants were diluted approximately 500 times in 50 mM Tris buffer (100 mM KCl, 5 mM DTT, 0.5% CHAPS, pH 7.5) containing bovine serum albumin (100 μg/mL). Binding to GST-hVDR-LBD was evaluated according to the procedure reported.27The receptor solution (570 μL) in an assay tube 4364

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with the software Phaser30 in the CCP4 program31 using rat VDRLBD coordinates (PDB code 2ZLC), and finalized sets of atomic coordinates were obtained after iterative rounds of model modification with the program Coot32 and refinement with CNS and refmac5.33−37 The coordinate data for the structures were deposited in Protein Data Bank with accession numbers 3WT5 (VDR-LBD/8a complex, crystal A), 3WT6 (VDR-LBD/8a complex, crystal B), 3WT7 (VDR-LBD/9a complex).

was incubated with [3H]-1α,25-dihydroxyvitamin D3 (specific activity, 5.85 TBq/mmol, 2000 cpm) together with graded amounts of each vitamin D analogue (0.001−100 nM) or vehicle for 16 h at 4 °C. The bound and free [3H]-1α,25-dihydroxyvitamin D3 molecules were separated by treating with dextran-coated charcoal for 30 min at 4 °C. The assay tubes were centrifuged at 1000g for 10 min. The radioactivity of the supernatant was counted. Nonspecific binding was subtracted. These experiments were done in duplicate. Transfection and Transactivation Assay. COS-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS). Cells were seeded on 24well plates at a density of 2 × 104 per well. After 24 h, the cells were transfected with a reporter plasmid containing three copies of the mouse osteopontin VDRE (5′-GGTTCAcgaGGTTCA, SPPx3-TKLuc), a wild-type or mutant hVDR expression plasmid (pCMXhVDR), and the internal control plasmid containing sea pansy luciferase expression constructs (pRL-CMV) by the lipofection method as described previously.13 After an 8 h incubation, the cells were treated with either the ligand or ethanol vehicle and cultured for 16 h. Cells in each well were harvested with a cell lysis buffer, and the luciferase activity was measured with a luciferase assay kit (Promega, WI, U.S.). Transactivation measured by the luciferase activity was normalized with the internal control. All experiments were done in triplicate. Protein Expression and Purification. Expression of rat VDRLBD (residues 116−423, Δ165−211) and the following cell-lysis and centrifugation were done by the procedure reported previously.16 The supernatant was applied to cOmplete His-tag purification resin (Roche Diagnostics GmbH, Mannheim, Germany), and the resin was thoroughly washed in wash buffer (50 mM Na/K phosphate, pH 7.0, 20 mM imdazole, 500 mM NaCl, 5% glycerol, 1% Tween 20, 1 mM TCEP). The rat VDR-LBD was eluted with elution buffer (250 mM imidazole, 50 mM Na/K phosphate, pH 7.0, 500 mM NaCl, 5% glycerol, 1% Tween 20, 1 mM TCEP). The protein was dialyzed overnight, dialysis with 500 mL of buffer A (20 mM Na/K phosphate, pH 7.0, 5% glycerol, 1 mM EDTA, 0.5 mM DTT), and then loaded onto a HiTrap SP HP (5 mL) column (GE Healthcare) equilibrated with buffer A. The elution was performed by NaCl gradient buffer from 0 to 1.0 M. His-tag of the protein in elution mixture (8 mL) was cleaved by addition of 70 units of thrombin and subsequent incubation at 4 °C for 18 h. Then NaCl (1.0 g) was added to the digested mixture (8 mL), and the resulting mixture was passed through a HiTrap benzamidine FF (1 mL) columun (GE healthcare) with buffer (10 mM Tris-HCl, pH 7.0, 2 M NaCl, 0.1 mM DTT). The flow-through was further purified by Superdex S75 gel filtration (25 mL) column (GE Healthcare) with a buffer (100 mM NaCl, Tris-HCl, pH 7.0). Purified rat VDR-LBD was concentrated in buffer (10 mM Tris-HCl, pH 7.0, 2 mM NaN3, 10 mM DTT) to 7.0 mg/mL, which was estimated by UV absorbance at 280 nm. Crystallization. A mixture of rVDR-LBD and a ligand (5 equiv) was incubated at room temperature for 1 h. Then coactivator peptide (H2N-KNHPMLMNLLKDN-CONH2) derived from DRIP205 in buffer (25 mM Tris-HCl, pH 8.0; 50 mM NaCl; 10 mM DTT; 2 mM NaN3) was added. The mixture of VDR-LBD/ligand/peptide was allowed to crystallize by the vapor diffusion method using a series of precipitant solutions containing 0.1 M MOPS-Na (pH 7.0), 0.075−0.2 M sodium formate, 12−22% (w/v) PEG4000, and 5% ethylene glycol. Droplets for crystallization were prepared by mixing 1 μL of complex solution and 1 μL of precipitant solution, and droplets were equilibrated against 300 μL of precipitant solution at 20 °C. The mixture was stored at 20 °C, and crystals appeared after a few days. X-ray Crystallographic Analysis. Prior to diffraction data collection, crystals were soaked in a cryoprotectant solution containing 0.1 M MOPS-Na, pH 7.0, 0.075−0.2 M sodium formate, 16−20% (w/ v) PEG4000, and 18−24% ethylene glycol. Diffraction data sets were collected at 100 K in a stream of nitrogen gas at beamline BL-5A of KEK-PF (Tsukuba, Japan). Reflections were recorded with an oscillation range per image of 1.0°. Diffraction data were indexed, integrated, and scaled using the program iMOSFLM.28,29 The structures of ternary complex were solved by molecular replacement

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81 42 721 1580. Fax: +81 42 721 1580. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science and by a Grant-in-Aid for Scientific Research (Grant 23590135) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We also thank the Takeda Science Foundation, Japan, for financial support. Synchrotron-radiation experiments were performed at the Photon Factory (Proposal 2011G685), and we are grateful for the assistance provided by the beamline scientists at the Photon Factory.

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ABBREVIATIONS USED VDR, vitamin D receptor; LBD, ligand binding domain; LBP, ligand binding pocket; 1,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; RXR, retinoid X receptor; ER, estrogen receptor

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REFERENCES

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