Vitamin D Analogues with a p-Hydroxyphenyl Group at the C25 Position

Sep 27, 2017 - Vitamin D Analogues with a p-Hydroxyphenyl Group at the C25 Position: Crystal Structure of Vitamin D Receptor Ligand-Binding Domain ...
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Article Cite This: J. Med. Chem. 2017, 60, 8394-8406

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Vitamin D Analogues with a p‑Hydroxyphenyl Group at the C25 Position: Crystal Structure of Vitamin D Receptor Ligand-Binding Domain Complexed with the Ligand Explains the Mechanism Underlying Full Antagonistic Action Akira Kato, Makiko Yamao, Yuta Hashihara, Hiroaki Ishida, Toshimasa Itoh, and Keiko Yamamoto* Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan S Supporting Information *

ABSTRACT: Vitamin D receptor (VDR) antagonists can be classified into two categories: the first category of VDR antagonists, which do not stabilize the helix 11−12, and the second category of antagonists, which destabilize the helix 6−7 region. To elucidate the mechanism underlying the first category antagonists by using the crystal structure, we designed and synthesized several VDR ligands with a p-hydroxyphenyl group at the C25-position. Of these, 22S-butyl-25-carbonyl analogue 5b and 25-di-p-hydoroxyphenyl analogues 6a,b showed strong antagonistic activity. We succeeded in cocrystallizing the ligand-binding domain of VDR complexed with 5b and found that the structure showed an alternative conformation of the helix 11−12 that explained the mechanism of the first category antagonists. Taking the present and previous studies together, we could elucidate the mechanisms underlying first and second categories antagonists based on individual crystal structures. This study provides significant insights into antagonism against not only VDR but also nuclear receptors.

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compound 1b13), and lactam compounds 1c, 1d14 (Figure 1). Recently, we reported new VDR antagonists 2a and 2b with bulky substituents at the side chain terminus (Figure 1).15 Only four X-ray crystal structures of the ligand-binding domain (LBD) of VDR complexed with the antagonist had been reported: four antagonists are 1a,12 (23S)-25-dehydro-1αhydroxyvitamin D3-26,23-lactone (TEI9647),16 (25R)-25-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro19,26,27-trinor-20-epi-vitamin D3 (ADTT),17 and (25R)-26adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro19,27-dinor-20-epi-vitamin D3 (ADMI4).17 However, these structures adopt the active conformation observed for VDRLBD complexed with 1,25D3 and synthetic analogs, and thus these crystal structures do not explain the mechanism of antagonism against VDR. It was conjectured that these structures adopt the active conformation by the effects on crystal packing. Therefore, several structural analyses in solution were performed. We analyzed the solution structure of VDR-LBD in the presence of antagonist 1b using a hybrid approach combining small-angle X-ray scattering (SAXS) with molecular dynamics (MD) simulation to show that the helix 11−12 of the 1b/VDR-LBD complex is unfolded and flexible

INTRODUCTION 1α,25-Dihydroxyvitamin D3 (1,25D3) is a hormone that plays significant roles in calcium metabolism, immunomodulation, cellular differentiation, and cellular proliferation.1 1,25D3 exerts these physiological effects by modulating gene transcription through binding to the vitamin D receptor (VDR).2 VDR is a ligand-dependent transcription factor and a member of the nuclear receptor (NR) superfamily. The conformation of VDR changes from the inactive to the active form by binding 1,25D3, and then the complex forms a heterodimer with the retinoid X receptor (RXR).3 The VDR/RXR heterodimer binds to the response element of the target gene and recruits a coactivator, resulting in transcription of the target gene. Coactivator is recruited when the helix 12 of VDR adopts the stable active conformation by binding 1,25D3 or a synthetic agonist. Many vitamin D analogues have been developed, of which more than 10 VDR agonists are used in clinical applications, such as for the treatment of metabolic bone diseases and skin diseases.4,5 VDR antagonists hold promise for treating diseases of VDR hyperfunction, such as Paget’s disease of bone and osteopetrosis,6 but no VDR antagonists are currently available for use as drugs. Several groups have identified various VDR antagonists, such as carboxylic ester compounds (ZK series),7,8 26,23-lactone compounds,9,10 adamantane compounds,11 our laboratory’s 22S-butyl vitamin D analogues (e.g., 1a (JB)12 and © 2017 American Chemical Society

Received: June 5, 2017 Published: September 27, 2017 8394

DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

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Figure 1. Structures of 1α,25-(OH)2D3 (1,25D3) and its analogues.

passively, and this category includes 1b, OU-72, and 2b. The second category of VDR antagonists induces changes in the helix 6−7 conformation but stabilizes the helix 11−12 and comprises 2a and 1c,d. Crystal structures of several second category of antagonists, which change the helix 6−7 conformation, have been reported.15,20 However, no crystal structure of VDR-LBD, which forms an alternative conformation of the helix 11−12, has been achieved to date. Our intent was to ascertain the mechanism of action of the first category of antagonists, using the crystal structure of VDR-LBD. We designed and synthesized new vitamin D analogues with a p-hydroxyphenyl group at the C25-position to capture cocrystals of VDR-LBD showing alternative conformation of the helix 11−12. Here, we report the design, synthesis, and biological evaluation of vitamin D analogues with a 25-p-hydroxyphenyl group (3a,b−6a,b; Figure 1) and X-ray crystallographic analyses of VDR-LBD complexed with these analogues.

compared to the canonical structure of the 1,25D3/VDR-LBD complex.18 In the NMR experiment by Singarapu et al.,19 the complex of VDR-LBD with the antagonist 2-methylene-(22E)(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D3 (OU-72) showed that residues in the helix 11−12 are disordered. In the hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiment which enables analysis of protein dynamics in solution, we analyzed the structure of VDR-LBD in the presence of 2a and 2b.15 This study indicated that 2b does not stabilize the helix 11−12 and actually destabilizes it, thus explaining the antagonistic action of 2b. In contrast, 2a stabilizes the helix 11−12 but destabilizes the helix 6−7 region, resulting in the change of the helix 6−7 conformation, thereby explaining the antagonistic action of 2a. Indeed the change of the helix 6−7 conformation was revealed by the crystal structure of 2a/VDRLBD complex.15 A recent crystallographic study by Asano et al. showed that 1c,d change the helix 6−7 conformation, thereby explaining the antagonistic action of 1c,d.20 Taking these research results together, we believe that VDR antagonists can be reclassified into two categories. The first category of VDR antagonists does not stabilize the helix 11−12 either actively or

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RESULTS Design. All VDR-LBD crystals reported to date show a cognate structure at the helix 11−12, suggesting that the helix

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DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

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Scheme 1. Synthesis of Compounds 3a,b−5a,b

Scheme 2. Synthesis of Compounds 6a,b

designed because the 22S-butyl group is effective for VDR antagonistic activity. Synthesis. As shown in Schemes 1 and 2, analogues 3a,b− 6a,b were synthesized from aldehydes 7a,b or esters 10a,b which were prepared by the procedure reported previously.13,15 (4-Bromophenoxy)-tert-butyldimethylsilane was lithiated with n-BuLi in THF, then reacted with aldehydes 7a,b to give compounds 8a,b, respectively. Compounds 8a,b were the starting structures for several analogues: they were (i) deprotected with HF−pyridine in THF to give the desired compounds 3a,b, respectively; (ii) treated with CSA in methanol to give compounds 4a,b, in which three TBS groups were deprotected and the 25-hydroxy group was methylated; and (iii) oxidized with Dess−Martin periodinane to give 25carbonyl compounds 9a,b, which were then deprotected with CSA to afford compounds 5a,b, respectively. Treatment of

11−12 must adopt the cognate structure for crystal packing and thus cocrystallization. To detect an alternative structure at the helix 11−12 triggered by antagonist binding, we speculated that vitamin D analogues with a bulky substituent to prevent folding of the helix 11−12 and a hydrophilic group for additional interactions (such as a hydrogen bond) might be effective. We therefore designed new analogues 3a−6a with a p-hydroxyphenyl group at the C25-position (Figure 1). Analogue 4a with a 25-methoxy group was designed to evaluate the importance of a hydroxyl group at the C25-position. Analogue 5a with a 25-carbonyl group was expected to induce an alternative helix 11−12 structure due to its rigid side chain structure. Two p-hydroxyphenyl groups were introduced to 6a to increase steric repulsion with the helix 11−12 as compared to 3a. In addition, 3b−6b, each bearing a 22S-butyl group, were 8396

DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

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SMRT222 failed. We then attempted crystallization of these complexes in the presence of the cofactor peptides DRIP205-2 or SRC2-3. DRIP205-2 is often used for the crystallization of rat VDR-LBD, and we recently reported that SRC2-3 is also effective.23 Good quality crystals of the 5b/VDR-LBD complex containing SRC2-3 were obtained, whereas crystallization in the presence of DRIP205-2 failed. X-ray diffraction data of the 5b/ VDR-LBD complex containing SRC2-3 were collected to 2.05 Å resolution. Crystallization of VDR-LBD complexed with the antagonist 6a or 6b also failed under all tested conditions, whereas crystals of the complex with the partial agonist 3a, 4a,b, or 5a were obtained in the presence of DRIP205-2. We solved the crystal structure of all these complexes, and the crystallographic analysis data for the 3a, 4a,b, and 5a,b/VDRLBD complexes are summarized in Table S1. Omit maps of the 3a, 4a,b, and 5a,b/VDR-LBD complexes are shown in Figure S2. The overall protein structure in complex with 3a, 4a,b, or 5a adopted the active conformation observed in the 1,25D3/ VDR-LBD complex (Figure S3). Interestingly, though 3a, 4a, and 4b used in crystallization are a 1:1 mixture of 25R- and 25S-epimers, 3a, 4a, and 4b in crystals are 25R-isomer, 25RSisomers, and 25S-isomer, respectively (Figure S3c−e). These results demonstrate that the 25R-isomer in 3a and the 25Sisomer in 4b form more favorable interactions with VDR-LBD than the 25S-isomer in 3a and the 25R-isomer in 4b, respectively. On the other hand, in compound 4a, the 25Risomer and the 25S-isomer interact with VDR-LBD in almost the same fashion, as the crystal structure shows (Figure S3d). Figure 3a shows VDR-LBD complexed with antagonist 5b superimposed on the 1,25D3/VDR-LBD complex (PDB code 2ZLC24), and significant differences at helix 10/11 are apparent. In the 5b/VDR-LBD complex, there is no electron density for Phe402 and Gln403 on helix 11 (Figure 3b) and helix 11 is unraveled from Arg398 to Gln403 (Figure 3c). Moreover, the main chains of helix 10 and helix 11 are shifted from the canonical position (for example, 4.3 Å at the Cα of Gln396, 6.8 Å at the Cα of Leu400) (Figure 3c,d) due to steric repulsion between the p-hydroxyphenyl group of 5b and His393 on helix 10 (Figure 3e). That is, the p-hydroxyphenyl group of 5b pushes out helix 11 and disorders the helix. On the other hand, helix 12 of the 5b/VDR-LBD complex adopts an almost identical position to that of the active conformation observed in the 1,25D3/VDR-LBD complex (Figure 3a). The 1α- and 3β-hydroxyl groups of 5b form hydrogen bonds with four amino acid residues (Tyr143, Ser274, Ser233, and Arg270), like 1,25D3, but the carbonyl group at the side chain does not form hydrogen bonds with His301 and His393 (Figure 3f). Instead, the phenolic hydroxyl group of 5b forms two water-mediated hydrogen bonds to Asn390, Lys260, Ser261, and Ile264. Furthermore, the phenyl group forms π−π stacking interactions with one of two conformers of His393 (Figure 3f). The Connolly surface of the ligand binding pocket (LBP) in the 4b or 5b/VDR-LBD complexes showed the formation of a butyl pocket to accommodate the 22S-butyl group, but a small channel was generated by only 5b at the top of the butyl pocket (Figure 4a). We observed ligand-specific repositioning of the side chain of Leu305 driven by steric repulsion between Leu305 and the 22S-butyl group (Figure 4b). Moreover, a helix break occurs at the N-terminus of helix 7 due to disappearance of the hydrogen bond between the carbonyl of Leu305 and the NH of Leu309 (Figure 4c). On the basis of these results, the cavity of

esters 10a,b with the appropriate lithium derivative gave bisphenol compounds 11a,b, which were then deprotected with CSA to afford compounds 6a,b, respectively. Compounds 6a,b were not alkylated by CSA due to steric effects unlike 4a,b. Biological Activities. Binding affinities to VDR were evaluated using a previously reported competitive binding assay with [3H]-1,25D3.21 The results are summarized in Table 1. All synthetic compounds showed significant binding to VDR, Table 1. VDR Binding Affinity of Synthetic Analogues 3a,b− 6a,ba relative affinityb and IC50 (nM)c compd

3

4

5

6

a: R = H

0.0044b 36 (0.16)c 0.0094b 17 (0.16)c

0.0049b 102 (0.50)c 0.031b 23 (0.72)c

0.006b 84 (0.50)c 0.028b 20 (0.55)c

0.00096b 520 (0.50)c 0.0042b 130 (0.55)c

b: R = Bu a

Competitive binding of 1,25D3 and synthetic compounds 3a,b−6a,b to the human vitamin D receptor. The experiments were carried out in duplicate. The IC50 values (nM) are derived from dose−response curves and represent the compound concentration required for 50% displacement of radiolabeled 1α,25-dihydroxyvitamin D3 from the receptor protein. bAffinities are presented as relative values, where the reference value of 1,25D3 is defined as 1. cIC50 of synthetic compound. Value in the parentheses is IC50 of 1,25D3 in each experiment.

indicating that they are VDR ligands. Monophenol analogues 3−5 showed stronger affinity than bisphenol analogues 6. All 22S-butyl compounds 3b−6b showed stronger affinity than the corresponding 22-H compounds 3a−6a, indicating that the 22S-butyl group increases VDR affinity. The abilities of synthetic compounds 3a,b−6a,b to induce transcription of vitamin D-responsive genes were examined using the previously reported luciferase reporter gene assay system in Cos7 and HEK293 cells.21 The results are shown in Figure 2. Transcriptional activities in HEK293 cells were stronger than in Cos7 cells, consistent with previous studies. In Cos7 cells, compounds 3b, 5b, and 6a,b did not show transactivation but rather exhibited concentration-dependent inhibition of transactivation induced by 1,25D3, showing that these compounds are VDR antagonists. Compounds 3a, 4b, and 5a showed weak transactivation and inhibited 1,25D3induced transactivation, indicating that they are weak partial agonists. Compound 4a showed full agonistic activity at high concentration. In HEK293 cells, 4a showed full agonistic activity, 3a,b, 4b, and 5a showed partial agonistic activity, and 5b and 6a,b showed antagonistic activity. We concluded that compound 5b is an especially potent VDR antagonist because the IC50 of 5b (20 nM) was 20−40 times lower than the IC50 of 6b (400 nM) and 6a (800 nM) in HEK293 cells. We evaluated the ligand-dependent recruitment of nuclear receptor coactivator SRC-1 and a heterodimer partner, nuclear receptor RXRα, to the VDR using a mammalian two-hybrid assay in HEK293 cells.21 As shown in Figure S1 in Supporting Information, antagonists 5b and 6a,b did not recruit SRC-1, 5b and 6b exhibited only marginal recruitment of RXRα, and 6a weakly recruited RXRα. X-ray Crystal Structure Analysis. To unravel the mechanism underlying the antagonism of compounds 5b, 6a, and 6b, we attempted to crystallize VDR-LBD complex with 5b, 6a, or 6b. Our attempts to crystallize ligand/VDR-LBD complex in the presence or absence of the co-repressor peptide 8397

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Figure 2. Transactivation of synthetic compounds. Transactivation of compounds 3a,b−6a,b was evaluated using Cos7 (a−d) and HEK293 (e−h) cells. Transcriptional activity was evaluated using a dual luciferase assay with 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. In experiments with Cos7 cells, luciferase activity equal to 10−8 M 1,25D3 was defined as 1, and in experiments with HEK293 cells, activity equal to 10−9 M 1,25D3 was defined as 1. Inhibitory effects on transactivation were evaluated in the presence of 1,25D3 (c, d, g, h).

5b/VDR-LBD complex was characterized as belonging to the second type of butyl pocket reported previously.25

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22S-butyl group increased VDR affinity because this group induced formation of a butyl pocket and formed hydrophobic interactions with amino acid residues in the butyl pocket.12,13 Antagonist 5b. In the antagonist 5b/VDR-LBD complex, the 25-carbonyl group of 5b does not interact with any amino acid residue but the phenolic hydroxyl group forms two watermolecule-mediated hydrogen bonds with four residues (Figure 3f). Surprisingly, antagonist 5b induced the alternative structure of the helix 10/11 due to steric repulsion between the rigid side chain of 5b and His393 on helix 10 (Figure 3). In the crystals, amino acid residues at the helix 10/11 stand apart from

DISCUSSION

Synthetic vitamin D analogues 3a,b−6a,b exhibited significant binding affinity for human VDR (hVDR) (Table 1). The monophenol compounds 3a,b−5a,b exhibited more potent affinity than the bisphenol compounds 6a,b, indicating that the bisphenol substituent is too bulky to effectively bind to VDR. 22S-Butyl compounds 3b−6b showed stronger affinity than the corresponding 22-H compounds 3a−6a. Introduction of the 8398

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Figure 3. X-ray crystal structure of the 5b/VDR-LBD complex. (a) Superposition of an overall view of 5b/VDR-LBD (yellow) and 1,25D3/VDRLBD (green). (b) 2Fo − Fc map of the helix 10−12 in 5b/VDR-LBD. The map of ligand 5b is hidden. (c) Conformational change in helix 10/11 of 5b/VDR-LBD. Compared with 1,25D3/VDR-LBD, helix 11 is unraveled from Arg398 to Gln403, and Phe402 and Gln403 were not observed. (d) The main chain of helix 10/11 is shifted from the canonical position. (e) Helix 10/11 is pushed out by the p-hydroxyphenyl group of 5b. (f) Interactions between 5b and VDR-LBD. Red dashed lines show hydrogen bonds between 5b and VDR-LBD; orange dashed lines show the π−π stacking interactions between the p-hydroxyphenyl group of 5b and His393 in VDR-LBD. The phenolic hydroxyl group forms two water-mediated hydrogen bonds with Asn390, Lys260, Ser261, and Ile264.

neighboring VDR-LBD (the closest distance between VDRLBD and neighboring VDR-LBD is 4.8 Å), like the 1,25D3/ VDR-LBD complex (Figure S4), that is, the alternative structure of the helix 10/11 is not influenced by crystal packing. The 25-carbonyl group and 22S-butyl group contribute to the rigidness of the side chain of 5b, and these substituents are important for formation of the alternative conformation of the helix 10/11 resulting in the expression of antagonist activity. Thus, we first analyzed the crystal structure of the antagonist 5b/VDR-LBD complex and clearly showed the alternative conformation at the helix 11 representing the first category of VDR antagonists.

A similar conformational change of helix 11 was previously observed in the crystal structures of LBDs of estrogen receptors α and β (ERα and ERβ). In ERα-LBD complexed with the antagonist 4-hydroxytamoxifen (OHT),26 3-(4-(1,2-diphenylbut-1-enyl)phenyl)acrylic acid (GW5638; tamoxifen analog),27 or (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (AZD9496)27 and in ERβ-LBD complexed with the antagonist N-n-butyl-N-methyl-11-(3,17β-dihydroxyestra-1,3,5(10)-trien-7α-yl)undecanamide (ICI 164,384),28 the bulky structures of these ligands directly prevent helix 12 folding. This ER antagonistic mechanism was classified as “direct antagonism” by Sharma et al.29 In this direct 8399

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Figure 4. Second type of butyl pocket induced by the 22S-butyl group of 5b. (a) Connolly channel surface of the 1,25D3/VDR-LBD (green), 4b/ VDR-LBD (pink), and 5b/VDR-LBD (yellow). The 22S-butyl groups of 4b and 5b are accommodated in a butyl pocket, and a small channel is generated by only 5b. (b) The 22S-butyl group of 5b causes drastic repositioning of the side chain and the main chain of Leu305. (c) The hydrogen bond between the carbonyl of Leu305 and the NH of Leu309, required for α-helix formation, disappears.

Figure 5. Structures of helix 10 to helix 12: (a) crystal structure of 1,25D3/VDR-LBD;24 (b) SAXS-MD structure of antagonist 1b/VDR-LBD;18 (c) crystal structure of antagonist 5b/VDR-LBD. In both the SAXS-MD structure (1b/VDR-LBD) and the crystal structure (5b/VDR-LBD), helix 11 is unraveled from Arg398 onward to form a loop.

the p-hydroxyl phenyl group of the ligand and His393 (Figure 3). Therefore, 5b is classified in the “indirect antagonism”. We recently showed the solution structure of the antagonist 1b/VDR-LBD complex using a SAXS-MD hybrid approach.18 SAXS-MD analysis indicated that helix 11 is unraveled and helix 12 is positioned near the coactivator binding site. These features are similar to those of the OHT/ERα-LBD complex.26 Interestingly, in the 5b/VDR-LBD complex, helix 12 is located in a position similar to its position of the SAXS-MD structure of 1/VDR-LBD. More interestingly, helix 11 is unraveled from Arg398 to Gln403 and formed a loop in both the crystal structure of 5b/VDR-LBD and the SAXS-MD structure of 1b/ VDR-LBD (Figure 5). NMR experiments with the antagonist/

antagonism, the helix 11 is unraveled or disordered by a dramatic conformational change of helix 12. On the other hand, in ERα-LBD complexed with 7-oxabicyclo[2.2.1]hept-5-ene sulfonate (OBHS) derivatives, the bulky substituent of the ligands causes steric repulsion with an amino acid side chain in helix 11.30 These OBHS derivatives are ERα antagonists that displace helix 11 and indirectly destabilize helix 12. This antagonized mechanism was termed “indirect antagonism”.29 Both “direct antagonism” and “indirect antagonism” affect the helix 11−12, and therefore both are classified into the first category of NR antagonists. In the 5b/VDR-LBD complex, helix 11 is unraveled and disordered by steric repulsion between 8400

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Figure 6. Comparison of 5b/VDR-LBD and other compounds/VDR-LBD: (a) superposition of 5b/VDR-LBD (yellow) and 4b/VDR-LBD (pink); (b) superposition of 5b/VDR-LBD and structures of VDR-LBD complexed with 3a (light blue), 4a (orange), 4b and 5a (magenta).

not observed, unlike the 5b/VDR-LBD complex. These results suggest that monophenol compounds require the 25-carbonyl and 22S-butyl groups for antagonist activity. In comparison of 5b/VDR-LBD and 4b/VDR-LBD, phydroxyphenyl group of 5b exists in the region of helix 10/11 in active conformation of 4b/VDR-LBD, whereas p-hydroxyphenyl group of 4b is accommodated between helix 10/11 and helix 3 (Figure 6a). Because both 22S-butyl groups of 5b and 4b are accommodated in the butyl pocket (Figure 4a), conformations of C20−C23 of 5b and 4b are fixed (Figure 6a). Compound 5b has carbonyl group at C25 so that C24, C25, and the following p-hydroxyphenyl group adopt a planar structure (Figure 6a). That is, the side chain terminus (C24-phydroxyphenyl group) of 5b is rigid. We considered that rigid analog 5b is not detained in VDR-LBP, since even flexible 25methoxy analog 4b is jammed into LBP (Figures 4a and 6a). For the above reasons, crystal structure of 5b/VDR-LBD is considered to show an alternative conformation of helix 10/11. Indeed, ligands 3a and 4a, which do not have both 22S-butyl and 25-carbonyl groups, and ligand 5a, which does not have 22S-butyl group, are also jammed into LBP like 4b and do not induce the alternative conformation of helix 11−12 (Figure 6b). Thus, both of 22S-butyl and 25-carbonyl groups are effective to detect alternative conformation of VDR-LBD complexed with an antagonist. Table S2 summarizes structural changes (differences) in VDR-LBD compared with the 1,25D3/VDR-LBD complex. The series of crystal structures obtained in this study indicate that bulky substituents of a ligand newly generate desirable intimate interactions and/or undesirable steric repulsion with the protein, thereby modulating the LBD structure. Therefore, a bulky substituent is highly effective for VDR agonism and antagonism. Moreover, these compounds should change the structure of human VDR-LBD similar to rat VDR-LBD because amino acid residues at human VDR-LBP are almost the same as those at rat VDR-LBP. Taken together, VDR antagonist inducing the alternative conformation of helix 11 and/or helix 12 can be designed and synthesized by introducing substituents with appropriate bulkiness and rigidness into a high-affinity skeleton for VDR. Further, the crystal structure of VDR-LBD/ antagonist complexes can be obtained by devising the crystallization method.

VDR-LBD complex also showed that Lys395−Glu421 is disordered19 and HDX-MS analysis showed that the helix 11−12 is destabilized by antagonist 2b.15 The present study describes the first VDR-LBD crystal structure showing the alternative conformation of the helix 11 and explaining the mechanism underlying the first category of VDR antagonists. Antagonists 6a and 6b. Although crystals of VDR-LBD complexed with 6a or 6b could not be obtained, we deduced that 6a and 6b prevent the helix 11−12 cognate folding (Figure S1). Therefore, 6a and 6b were classified into the first category of VDR antagonists. Compounds 6a,b, with two p-hydroxyphenyl groups, showed antagonistic activity, and 3a,b, with one p-hydroxyphenyl group, were partial agonists in HEK293 cells (Figure 2). These results show that two phenols produce antagonistic activity due to steric repulsion with the protein. We previously reported that 25-diphenyl analogues also showed partial agonistic activity.15 Therefore, phenolic hydroxyl groups are effective substituents for antagonistic activity. Compounds 3a,b, 4a,b, and 5a. Compounds 3a,b showed weak partial agonistic activity in HEK293 cells (Figure 2). The crystal structure of 3a/VDR-LBD complex shows a new hydrogen bond between the phenolic hydroxyl group of 3a and Ala299 on helix 3 (Figure S3c). The helix 10−12 adopts the active conformation, similar to that in the 1,25D3/VDRLBD complex, and no steric repulsion between the helix 11−12 and the ligand was observed. Although no crystal structure with 3b was attained, we assumed that 3b/VDR-LBD complex adopts active conformation similar to that of the 3a/VDR-LBD complex because 3b showed partial agonist activity. The 25-methoxy analogues 4a and 4b showed full agonistic activity at high concentration in HEK293 cells (Figure 2). These compounds do not form hydrogen bonds with His301 and His393, but the phenolic hydroxyl groups form hydrogen bonds with the residues (Gly300, Ala299, Gln396) on helix 10 and/or loop 6−7 (Figure S3d,e). These compounds also interact hydrophobically with His393. Therefore, compounds 4a and 4b induce the active conformation without forming hydrogen bonds with His301 and His393 and show agonist activity. The 25-carbonyl 22-H compound 5a was a weak partial agonist, unlike the 22-Bu compound 5b (Figure 2). In the crystal structure, the 25-carbonyl and phenolic hydroxyl groups of 5a do not interact with any amino acid residue (Figure S3f). However, the helix 10−12 forms the active conformation, and the steric repulsion between the helix 11−12 and the ligand was 8401

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with EtOAc. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (2.5 g) with 30% EtOAc/hexane to afford 4a (3.0 mg, 82%, dr 1:1). 1 H NMR (600 MHz, CDCl3) δ: 0.52, 0.53 (1:1) (3H, s, H-18), 2.58 (1H, m, H-14), 2.83 (2H, m, H-9), 3.14, 3.18 (1:1) (3H, s, OMe), 4.01 (1H, m, H-25), 4.48 (2H, m, H-1,3), 5.09, 5.11 (each 1H, s, C CH2), 5.88 (1H, d, J = 11.1 Hz, H-7), 6.35 (1H, d, J = 11.1 Hz, H-6), 6.81 (2H, d, J = 8.3 Hz, arom-H), 7.15 (2H, d, J = 8.3 Hz, arom-H). 13 C NMR (150 MHz, CDCl3) δ: 11.1, 23.1, 23.9, 24.9, 29.0, 29.5, 33.4, 36.1, 36.2, 38.3, 38.6, 38.9, 40.6, 46.0, 56.5, 58.3, 70.9, 72.0, 83.8, 83.9, 107.9, 115.3, 115.4, 124.5, 128.2, 128.3, 129.0, 131.0, 132.6, 143.7, 152.1, 167.9. HRMS (ESI−) calcd for C32H45O4 (M − H)− 493.3318, found 493.3294. UV (EtOH) λmax 233, 245, 254, 263 nm. 22S-Butyl-25RS-(4′-hydroxyphenyl)-25-methoxy-2-methylidene-19,26,27-trinor-1α-hydroxyvitamin D3 (4b). In a similar manner to that for the synthesis of 4a from 8a, target compound 4b (14.0 mg, 0.0232 mmol) was obtained from 8b (22.5 mg, 0.0287 mmol) in 81% yield (dr 1:1). 1H NMR (300 MHz, CDCl3) δ: 0.52, 0.53 (1:1) (3H, s, H-18), 0.67, 0.75 (1:1) (3H, d, J = 5.8 Hz, H-21), 2.56 (1H, dd, J = 13.4, 3.7 Hz, H-14), 2.86 (1H, m, H-9), 3.17, 3.18 (1:1) (3H, s, OMe), 3.98 (1H, q, J = 6.7 Hz, H-25), 4.48 (2H, m, H1,3), 5.09, 5.12 (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), 6.82 (2H, d, J = 8.3 Hz, arom-H), 7.18 (2H, d, J = 8.3 Hz, arom-H). 13C NMR (75 MHz, CDCl3) δ: 12.2, 14.4, 22.4, 23.4, 23.5, 23.7, 27.8, 28.0, 29.2, 31.0, 36.2, 36.8, 37.3, 38.3, 39.9, 40.0, 40.7, 46.0, 56.5, 56.5, 70.8, 72.0, 84.1, 84.3, 107.9, 115.3 (2 carbons), 115.4, 124.4, 128.2, 128.4, 130.5, 134.8, 143.7, 152.0, 155.3. HRMS (ESI−) calcd for C36H53O4 (M − H)− 549.3949, found 549.3950. IR (neat) 3418, 2924, 2856, 2359, 2341, 1614, 1515, 1261, 1078, 1043, 833, 756, 669 cm−1. UV (DMSO) λmax 245, 253, 263 nm. 25-(4′-Hydroxyphenyl)-2-methylidene-19,26,27-trinor-25oxo-1α-hydroxyvitamin D3 (5a). In a similar manner to that for the synthesis of 4a from 8a, target compound 5a (3.3 mg, 0.00689 mmol) was obtained from 9a (4.6 mg, 0.00776 mmol) in 89% yield. 1H NMR (300 MHz, CDCl3) δ: 0.54 (3H, s, H-18), 0.96 (3H, d, J = 6.4 Hz, H21), 4.50 (2H, m, H-1,3), 5.10, 5.12 (each 1H, s, CCH2), 5.88 (1H, d, J = 11.1 Hz, H-7), 6.36 (1H, d, J = 11.1 Hz, H-6), 6.88 (2H, d, J = 8.9 Hz, arom-H), 7.91 (2H, d, J = 8.9 Hz, arom-H). 13C NMR (75 MHz, CDCl3) δ: 12.2, 18.9, 21.3, 22.4, 23.6, 27.8, 29.1, 35.8, 36.1, 38.3, 38.9, 40.6, 45.9, 46.0, 56.4, 56.5, 70.9, 72.0, 108.0, 115.4 (3 carbons), 124.5, 130.4, 130.5, 130.8 (2 carbons), 143.6, 152.0, 160.0, 199.5. HRMS (ESI−) calcd for C31H41O4 (M − H)− 447.3005, found 477.2996. UV (DMSO) λmax 228, 245, 253, 263 nm. 22S-Butyl-25-(4′-hydroxyphenyl)-2-methylidene-19,26,27trinor-25-oxo-1α-hydroxyvitamin D3 (5b). In a similar manner to that for the synthesis of 4a from 8a, target compound 5b (7.1 mg, 0.0133 mmol) was obtained from 9b (13.2 mg, 0.0150 mmol) in 89% yield. 1H NMR (300 MHz, CDCl3) δ: 0.52 (3H, s, H-18), 0.80 (3H, d, J = 5.8 Hz, H-21), 0.89 (3H, t, J = 7.0 Hz, CH3 of Bu), 2.58 (1H, dd, J = 13.2, 4.0 Hz, H-14), 4.49 (2H, m, H-1,3), 5.11, 5.12 (each 1H, s, CCH2), 5.88 (1 H, d, J = 11.3 Hz, H-7), 6.37 (1 H, d, J = 11.3 Hz, H-6), 6.88 (2H, d, J = 8.7 Hz, arom-H), 7.90 (2H, d, J = 8.7 Hz, aromH). 13C NMR (75 MHz, CDCl3) δ: 12.2, 13.2, 14.4, 22.3, 23.4, 23.7, 27.0, 27.9, 28.7, 29.1, 29.8, 30.9, 36.9, 37.4, 40.0, 40.7, 41.0, 46.0, 53.9, 56.5, 71.0, 71.9, 108.0, 115.4, 115.5 (2 carbons), 124.5, 130.4, 130.5, 130.8 (2 carbons), 143.6, 152.0, 160.2, 199.8. HRMS (ESI+) calcd for C35H50NaO4 (M + Na)+ 557.3601, found 557.3590. UV (DMSO) λmax 257, 266 nm. 25-Di-(4′-hydroxyphenyl)-2-methylidene-19,26,27-trinor1α,25-dihydroxyvitamin D3 (6a). In a similar manner to that for the synthesis of 4a from 8a, target compound 6a (2.1 mg, 0.00367 mmol) was obtained from 11a (6.4 mg, 0.00621 mmol) in 59% yield. 1H NMR (300 MHz, CDCl3) δ: 0.52 (3H, s, H-18), 0.87 (3H, d, J = 6.4 Hz, H-21), 2.58 (1H, dd, J = 13.3, 3.3 Hz, H-14), 2.83 (2H, m, H-9), 4.49 (2H, m, H-1,3), 5.10, 5.12 (each 1H, s, CCH2), 5.88 (1H, d, J = 11.2 Hz, H-7), 6.36 (1H, d, J = 11.2 Hz, H-6), 6.72 (2H, d, J = 8.7 Hz, arom-H), 6.83 (2H, d, J = 8.7 Hz, arom-H), 7.04 (2H, d, J = 8.7 Hz, arom-H), 7.09 (2H, d, J = 8.7 Hz, arom-H). 13C NMR (75 MHz, CDCl3) δ: 12.2, 14.3, 18.9, 22.4, 23.6, 26.7, 27.8, 29.1, 36.0, 36.4, 38.3, 40.2, 40.5, 46.0, 56.5, 56.6, 70.9, 72.0, 77.9, 107.9, 115.0 (2 carbons),

CONCLUSIONS Our previous study showed that the crystal structure of the antagonist/VDR-LBD complex adopts an alternative conformation of helix 6−7, and a similar conformational change of helix 6−7 was observed in the crystal structure reported by Shimizu et al. The present study revealed the crystal structure of the antagonist/VDR-LBD complex, showing an alternative conformation of helix 10/11. Analysis of these crystal structures allowed elucidation of the mechanisms underlying first and second categories of antagonists. These results indicate that antagonists of nuclear receptors, including VDR, variously modulate the protein structure. This study will contribute to the discovery of new antagonists that induce appropriate structural changes in nuclear receptors.

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EXPERIMENTAL SECTION

Synthesis. All reagents were purchased from commercial sources. NMR spectra were recorded at 300 or 600 MHz for 1H NMR and 75 or 150 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-T100LP spectrometer. 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 JASCO V-630BIO 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. 25RS-(4′-Hydroxyphenyl)-2-methylidene-19,26,27-trinor1α,25-dihydroxyvitamin D3 (3a). To a solution of 8a (8.80 mg, 0.0109 mmol) in THF (0.5 mL) was added hydrogen fluoride− pyridine (hydrogen fluoride 65%, 15.1 μL, 0.109 mmol) at 0 °C, and the mixture was stirred for 14 h. The reaction was quenched with saturated NaHCO3 aqueous solution, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (1.5 g) with 40% EtOAc/hexane to afford 3a (4.54 mg, 87%, dr 1:1). 1H NMR (CDCl3) δ 0.52, 0.53 (1:1) (3H, s, H-18), 0.89 (3H, m, H-21), 2.83 (2H, m, H-9), 4.49 (2H, m, H-1, 3), 4.61 (1H, m, H-25), 5.09, 5.11 (each 1H, s, CCH2), 5.88 (1H, d, J = 11.1 Hz, H-7), 6.36 (1H, d, J = 11.1 Hz, H-6), 6.81 (2H, d, J = 8.7 Hz, arom-H), 7.22 (2H, d, J = 8.7 Hz, arom-H). 13C NMR δ 12.2, 18.9, 18.9, 22.4, 22.6, 22.7, 23.6, 27.8, 27.8, 29.1, 35.8, 35.9, 36.1, 38.3, 39.5, 39.6, 40.6, 45.9, 56.5, 56.7, 70.9, 72.0, 74.4, 74.6, 107.8, 115.4 (3 carbons), 124.4, 127.5, 127.5, 130.5, 137.3, 137.4, 143.6, 152.1, 155.2, 155.2. HRMS (ESI−) calcd for C31H43O4 (M − H)− 479.3161, found 479.3157. UV (EtOH) λmax 233, 245, 254, 263 nm. 22S-Butyl-25RS-(4′-hydroxyphenyl)-2-methylidene19,26,27-trinor-1α,25-dihydroxyvitamin D3 (3b). In a similar manner to that for the synthesis of 3a from 8a, target compound 3b (4.40 mg, 0.00817 mmol) was obtained from 8b (11.8 mg, 0.0134 mmol) in 61% yield (dr 1:1). 1H NMR (CDCl3) δ 0.53, 0.54 (1:1) (3H, s, H-18), 0.69, 0.076 (1:1) (3H, d, J = 5.8 Hz, H-21), 2.84 (2H, m, H-9), 4.48 (2H, m, H-1, 3), 4.58 (1H, m, H-25), 5.09, 5.11 (each 1H, s, CCH2), 5.88 (1H, d, J = 11.2 Hz, H-7), 6.36 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ 12.2 (2 carbons), 13.0, 13.2, 14.1 (2 carbons), 22.3, 23.4, 23.7, 27.8, 28.8, 29.2, 31.0, 37.3, 38.3, 40.0, 40.0, 40.6, 45.9, 46.0, 54.0, 56.3, 70.9, 72.0, 74.8, 75.0, 107.9, 115.4 (2 carbons), 124.4, 127.5 (2 carbons), 127.7 (2 carbons), 130.5, 143.6, 152.1, 155.3. HRMS (ESI−) calcd for C35H51O4 (M − H)− 535.3793, found 535.3789. UV (EtOH) λmax 245, 253, 263 nm. 25RS-(4′-Hydroxyphenyl)-25-methoxy-2-methylidene19,26,27-trinor-1α-hydroxyvitamin D3 (4a). To a solution of 8a (6.0 mg, 0.00741 mmol) in MeOH (1.0 mL) was added camphor sulfonic acid (17.2 mg, 0.0741 mmol) at 0 °C, and the mixture was stirred at room temperature for 4 h. The reaction was quenched with saturated NaHCO3 aqueous solution, and the mixture was extracted 8402

DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

Journal of Medicinal Chemistry

Article

115.1 (2 carbons), 115.4, 124.5, 128.6 (2 carbons), 128.8, 130.5, 131.3 (2 carbons), 140.3, 143.6, 152.1, 154.6, 154.7. HRMS (ESI−) calcd for C35H51O4 (M − H)− 571.3429, found 571.3450. UV (EtOH) λmax 245, 253, 263 nm. 22S-Butyl-25-di-(4′-hydroxyphenyl)-2-methylidene19,26,27-trinor-1α,25-dihydroxyvitamin D3 (6b). In a similar manner to that for the synthesis of 4a from 8a, target compound 6b (6.7 mg, 0.0107 mmol) was obtained from 11b (13.5 mg, 0.0124 mmol) in 86% yield. 1H NMR (300 MHz, CDCl3) δ: 0.56 (6H, m, H18, 21), 2.58 (1H, dd, J = 13.3, 3.6 Hz, H-14), 2.85 (2H, m, H-9), 4.58 (2H, m, H-1,3), 5.10, 5.12 (each 1H, s, CCH2), 5.90 (1H, d, J = 11.1 Hz, H-7), 6.36 (1 H, d, J = 11.1 Hz, H-6), 6.73 (1H, d, J = 11.2 Hz, H-6), 6.81 (2H, d, J = 8.7 Hz, arom-H), 7.01 (2H, d, J = 8.7 Hz, arom-H) 7.08 (2H, d, J = 8.7 Hz, arom-H), 7.09 (2H, d, J = 8.7 Hz, arom-H). 13C NMR (75 MHz, CDCl3) δ: 12.2, 13.2, 14.4, 22.4, 23.3, 23.7, 28.0, 28.4, 29.2, 29.8, 30.9, 32.0, 37.5, 38.3, 40.6, 41.3, 46.0, 46.0, 54.0, 56.6, 70.9, 72.0, 78.2, 107.9, 115.1 (4 carbons), 115.4, 124.5, 128.3, 128.6 (2 carbons), 130.5, 131.4 (2 carbons), 141.1, 143.7, 152.0, 154.5, 154.7. HRMS (ESI+) calcd for C41H56KO5 (M + K)+ 667.3765, found 667.3776. IR (neat) 3354, 2926, 2856, 1708, 1512, 1443, 1257, 1041, 833, 756, 686 cm−1. UV (EtOH) λmax 245, 253, 263 nm. 5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2methylidene-9,10-secoestra-5,7-dien-17-yl]-1-{4′-[tert-butyl(dimethyl)silyl]oxy}phenylhexan-1-ol (8a). To a solution of (4bromophenoxy)-tert-butyldimethylsilane (56.8 mg, 0.196 mmol) in dry THF (0.6 mL) at −78 °C was added n-BuLi (1.60 M hexane solution, 61.0 μL, 0.0998 mmol), and the mixture was stirred for 1 h. To this solution was added a solution of aldehyde 7a (22.2 mg, 0.0361 mmol) in dry THF (0.5 mL), and the mixture was stirred for 3 h. The reaction was quenched with saturated NH4Cl aqueous solution and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (10 g) with 5% EtOAc/hexane to afford 8a (19.6 mg, 66%, dr 1:1). 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.19 (6H, s, SiMe), 0.52, 0.53 (1:1) (3H, s, H-18), 0.86, 0.90 (each 9H, s, t-Bu), 0.98 (9H, s, t-Bu), 2.81 (1H, m, H-9), 4.43 (2H, m, H-1, 3), 4.60 (1H, m, H-25), 4.92, 4.97 (each 1H, s, CCH2), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6), 6.81 (2H, m, arom-H), 7.20 (2H, m, arom-H). 13C NMR δ −4.92, −4.76, −4.71 (2 carbons), −4.27 (2 carbons), 12.2, 18.3, 18.3, 18.4, 18.9, 18.9, 22.4, 22.6, 22.7, 23.6, 25.8 (3 carbons), 25.9 (3 carbons), 26.0 (3 carbons), 27.9, 28.9, 35.8, 35.9, 36.2, 38.7, 39.5, 40.7, 45.8, 47.8, 56.4, 56.6, 71.8, 72.7, 74.5, 74.7, 106.4, 116.2, 120.1 (2 carbons), 122.6, 127.2, 127.2, 132.9, 137.8, 137.9, 141.4, 153.1, 155.2, 155.2. HRMS (ESI+) calcd for C49H86NaO4Si3(M + Na)+ 845.5714, found 845.5739. UV (EtOH) λmax 231, 237, 245, 254, 264 nm. (4S)-4-{[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-1-{4′-[tertbutyl(dimethyl)silyl]oxy}phenyloctan-1-ol (8b). In a similar manner to that for the synthesis of 8a from 7a, target compound 8b (11.8 mg, 0.0134 mmol) was obtained from 7b (13.4 mg, 0.0200 mmol) in 67% yield (dr 1:1). 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.19 (6H, s, SiMe), 0.53, 0.54 (1:1) (3H, s, H-18), 0.68, 0.76 (1:1) (3H, d, 5.7 Hz, H-21), 0.87, 0.90 (each 9H, s, t-Bu), 0.98 (9H, s, t-Bu), 2.82 (1H, m, H-9), 4.44 (2H, m, H-1, 3), 4.58 (1H, m, H-25), 4.92, 4.97 (each 1H, s, CCH2), 5.83 (1H, d, J = 11.1 Hz, H7), 6.22 (1H, d, J = 11.1 Hz, H-6), 6.82 (2H, m, arom-H), 7.21 (2H, m, arom-H). 13C NMR δ −4.89, −4.76, −4.71 (2 carbons), −4.27 (2 carbons), 12.2, 13.0, 13.2, 14.4, 18.3 (2 carbons), 18.4, 22.3, 23.4, 23.4, 23.6, 25.8 (3 carbons), 25.9 (3 carbons), 26.0 (3 carbons), 27.9, 28.8, 28.9, 31.0, 37.2, 38.7, 40.0, 40.8, 45.9, 47.8, 54.0, 56.5, 71.7, 72.7, 74.9, 75.1, 106.4, 116.3, 119.6, 122.6, 127.2 (2 carbons), 127.4 (2 carbons), 132.9, 141.4, 153.1, 155.3. HRMS (ESI+) calcd for C53H94NaO4Si3(M + Na)+ 901.63576, found 901.64043. IR (neat) 3421, 2955, 2928, 2856, 1608, 1508, 1256, 1101, 912, 837, 777, 669 cm−1. UV (EtOH) λmax 227, 245, 254, 264 nm. 5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2methylidene-9,10-secoestra-5,7-dien-17-yl]-1,1-{4′-[tert-butyl(dimethyl)silyl]oxy}phenylhexan-1-one (9a). To a solution of 8a (7.6 mg, 0.00923 mmol) in CH2Cl2 (0.4 mL) were added NaHCO3

(14.7 mg, 0.175 mmol) and Dess−Martin periodinane (5.87 mg, 0.0138 mmol) at room temperature, and the mixture was stirred for 1.5 h. The reaction was quenched with saturated NaHCO3 aqueous solution, and the mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (8 g) with 2% EtOAc/ hexane to afford 9a (6.8 mg, 90%). 1H NMR (300 MHz, CDCl3) δ: 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.23 (6H, s, SiMe), 0.54 (3H, s, H-18), 0.86, 0.87 (each 9H, s, t-Bu), 0.97 (3H, d, J = 6.5 Hz, H-21), 0.99 (9H, m, t-Bu), 4.43 (2H, m, H-1,3), 4.92, 4.97 (each 1H, s, C CH2), 5.83 (1 H, d, J = 11.1 Hz, H-7), 6.22 (1 H, d, J = 11.1 Hz, H-6), 6.87 (2H, d, J = 8.7 Hz, arom-H), 7.89 (2H, d, J = 8.7 Hz, arom-H). 13 C NMR (75 MHz, CDCl3) δ: −4.9, −4.8, −4.7 (2 carbons), −4.2 (2 carbons), 12.2, 18.3, 18.4, 18.9, 21.3, 22.4, 23.6, 25.7 (3 carbons), 25.9 (3 carbons), 26.0 (3carbons), 27.8, 28.9, 35.9, 36.2, 38.7, 38.9, 40.7, 45.8 (2 carbons), 47.7, 56.4, 56.4, 71.8, 72.7, 106.4, 116.3, 120.0 (2 carbons), 122.6, 130.4 (2 carbons), 130.9, 132.9, 141.4, 153.1, 160.2, 199.5. UV (EtOH) λmax 246, 254, 263 nm. (4S)-4-{[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-1,1-{4′[tert-butyl(dimethyl)silyl]oxy}phenyloctan-1-one (9b). In a similar manner to that for the synthesis of 9a from 8a, target compound 9b (19.0 mg, 0.0217 mmol) was obtained from 8b (21.7 mg, 0.0247 mmol) in 88% yield. 1H NMR (300 MHz, CDCl3) δ: 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.23 (6H, s, SiMe), 0.56 (3H, s, H18), 0.82 (3H, d, J = 5.4 Hz, H-21), 0.87 (9H, s, t-Bu), 0.90 (12H, m, CH3 of Bu, t-Bu), 0.99 (9H, s, t-Bu), 4.43 (2H, m, H-1,3), 4.93, 4.97 (each 1H, s, CCH2), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.23 (1H, d, J = 11.2 Hz, H-6), 6.88 (2H, d, J = 8.7 Hz, arom-H), 7.92 (2H, d, J = 8.7 Hz, arom-H). 13C NMR (75 MHz, CDCl3) δ: −4.9, −4.8, −4.7, −4.7, −4.2 (2 carbons), 12.2, 13.3, 14.4, 18.3, 18.4, 22.3, 23.4, 23.6, 25.7 (3 carbons), 25.9 (3 carbons), 26.0 (3 carbons), 27.1, 28.0, 28.7, 28.9, 29.9, 31.0, 37.2, 37.6, 38.8, 40.1, 40.8, 45.8, 47.7, 54.0, 56.5, 71.9, 72.6, 106.4, 116.3, 120.0 (2 carbons), 122.6, 130.4 (2 carbons), 130.9, 132.9, 141.3, 153.1, 160.2, 199.9. HRMS (ESI+) calcd for C53H92NaO4Si3 (M + Na)+ 899.6201, found 899.6232. IR (neat) 3431, 2955, 2930, 2858, 1678, 1599, 1258, 1101, 910, 837, 779, 667 cm−1. UV (EtOH) λmax 246, 254, 264 nm. 5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2methylidene-9,10-secoestra-5,7-dien-17-yl]-1,1-{4′-[tert-butyl(dimethyl)silyl]oxy}phenylhexan-1-ol (11a). In a similar manner to that for the synthesis of 8a from 7a, target compound 11a (9.8 mg, 0.00952 mmol) was obtained from 10a (10.0 mg, 0.0155 mmol) in 61% yield. 1H NMR (300 MHz, CDCl3) δ: 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.17 (6H, s, SiMe), 0.18 (6H, s, SiMe), 0.51 (3H, s, H18), 0.84 (12H, m, H-21, t-Bu), 0.89 (9H, s, t-Bu), 0.97 (18H, s, t-Bu), 2.81 (1H, m, H-9), 4.42 (2H, m, H-1,3), 4.92, 4.97 (each 1H, s, C CH2), 5.82 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz, H-6), 6.76 (4H, d, J = 8.7 Hz), 7.22 (4H, d, J = 8.7 Hz). 13C NMR (75 MHz, CDCl3) δ: −4.9, −4.8, −4.7 (2 carbons), −4.3 (4 carbons), 12.2, 14.3, 18.3, 18.4, 18.9, 22.4, 22.8, 23.6, 25.8 (6 carbons), 25.9 (3 carbons), 26.0 (3 carbons), 27.8, 28.9, 29.5, 32.1, 36.2, 36.4, 38.7, 40.7, 45.8, 47.8, 56.4, 56.7, 71.8, 72.7, 78.2, 106.4, 116.2, 119.5 (2 carbons), 119.6 (2 carbons), 122.6, 127.4 (4 carbons), 132.9, 140.3, 140.4, 141.4, 153.1, 154.4, 154.4. UV (EtOH) λmax 230, 245, 254, 264 nm. (4S)-4-{[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-1,1-{4′[tert-butyl(dimethyl)silyl]oxy}phenyloctan-1-ol (11b). In a similar manner to that for the synthesis of 8a from 7a, target compound 11b (26.1 mg, 0.0240 mmol) was obtained from 10b (36.6 mg, 0.0522 mmol) in 46% yield. 1H NMR (300 MHz, CDCl3) δ: 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.18 (12H, s, SiMe), 0.55 (3 H, s, H-18), 0.68 (3 H, d, J = 5.5 Hz, H-21), 0.87, 0.90 (each 9H, m, t-Bu), 0.97 (18H, m, t-Bu), 2.82 (2H, m, H-9), 4.43 (2H, m, H-1,3), 4.93, 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), 6.77 (4H, d, J = 8.7 Hz, arom-H), 7.23 (4H, m, arom-H). 13C NMR (75 MHz, CDCl3) δ: −4.9, −4.8, −4.7, −4.7, −4.3 (4 carbons), 12.2, 13.2, 14.4, 18.3, 18.4, 18.4, 22.3, 23.4, 23.6, 25.8 (6 carbons), 26.0 (3 carbons), 26.0 (3 carbons), 28.0, 28.8, 28.9, 29.8, 31.0, 37.5, 38.8, 40.7, 40.8, 40.9, 45.8 (2 carbons), 47.7, 54.0, 56.5, 71.8, 72.6, 78.4, 8403

DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

Journal of Medicinal Chemistry

Article

106.4, 116.2, 119.6 (4 carbons), 122.6, 127.4 (2 carbons), 127.5 (2 carbons), 132.9, 140.2, 140.4, 141.4, 153.1, 154.4, 154.5. HRMS (ESI+) calcd for C65H112NaO5Si4 (M + Na)+ 1107.7485, found 1107.7470. IR (neat) 3622, 2955, 2928, 2895, 2856, 1720, 1607, 1504, 1256, 1101, 914, 837, 777, 675 cm−1. UV (EtOH) λmax 231, 325, 245, 254, 264 nm. Competitive Binding Assay, Human VDR. The binding affinity for VDR−LBD was evaluated according to the procedure reported previously.13,21 Transfection and Transactivation Assay. Transactivation in Cos7 cells and HEK293 cells was measured by dual luciferase assay according to the procedure reported previously.13,21 Protein Expression and Purification. Expression of rat VDRLBD (residues 116−423, Δ165−211) and the following purification were done by the procedure reported previously.23,31 X-ray Crystallographic Analysis. 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) or coactivator peptide (H2N-KENALLRYLLDKD-CONH2) derived from SRC2 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), 0.05−0.1 M sodium formate, 15−17% (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. Prior to diffraction data collection, crystals were soaked in a LVCO-1 (LV Cryo Oil) (MiTeGen, NY, U.S.). Diffraction data sets of 3a/VDR−LBD complex and 5a/VDR−LBD complex, 4a/VDR−LBD complex, and 4b/VDR− LBD complex and 5b/VDR−LBD complex were collected at 100 K in a stream of nitrogen gas at beamlines BL-5A of KEK-PF, NE3A of KEK-PFAR, and BL-17A of KEK-PF (Tsukuba, Japan), respectively. Reflections were recorded with an oscillation range per image of 1.0°. Diffraction data were indexed, integrated, and scaled using the program iMOSFLM.32,33 The structures of ternary complex were solved by molecular replacement with the software Phaser34 in the CCP4 program35 using rat VDR-LBD coordinates (PDB code 2ZLC), and finalized sets of atomic coordinates were obtained after iterative rounds of model modification with the program Coot36 and refinement with refmac5.37−41

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VDR-LBD complex), 5XPO (5a/VDR-LBD complex), and 5XPL (5b/VDR-LBD complex). Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

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

Keiko Yamamoto: 0000-0001-6642-7961 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Japan Agency for Medical Research and Development (AMED) and a Grant-in-Aid for Scientific Research (Grant 26460155) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to K.Y. This work was also supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2013−2017). Synchrotronradiation experiments were performed at the Photon Factory (Proposal 2015G717), 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; 1,25D3, 1α,25-dihydroxyvitamin D3; NR, nuclear receptor; RXR, retinoid X receptor; SAXS, small-angle X-ray scattering; MD, molecular dynamics; HDX-MS, hydrogen/deuterium exchange coupled with mass spectrometry; ER, estrogen receptor

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REFERENCES

(1) Pike, J. W.; Meyer, M. B.; Bishop, K. A. Regulation of Target Gene Expression by the Vitamin D Receptor - An Update on Mechanisms. Rev. Endocr. Metab. Disord. 2012, 13, 45−55. (2) Jurutka, P. W.; Whitfield, G. K.; Hsieh, J. C.; Thompson, P. D.; Haussler, C. A.; Haussler, M. R. Molecular Nature of the Vitamin D Receptor and Its Role in Regulation of Gene Expression. Rev. Endocr. Metab. Disord. 2001, 2, 203−216. (3) Orlov, I.; Rochel, N.; Moras, D.; Klaholz, B. P. Structure of the Full Human RXR/VDR Nuclear Receptor Heterodimer Complex with Its DR3 Target DNA. EMBO J. 2012, 31, 291−300. (4) Kubodera, N. Pharmaceutical Studies on Vitamin D Derivatives and Practical Syntheses of Six Commercially Available Vitamin D Derivatives That Contribute to Current Clinical Practice. Heterocycles 2010, 80, 83−98. (5) Nagpal, S.; Na, S.; Rathnachalam, R. Noncalcemic Actions of Vitamin D Receptor Ligands. Endocr. Rev. 2005, 26, 662−687. (6) Menaa, C.; Barsony, J.; Reddy, S. V.; Cornish, J.; Cundy, T.; Roodman, G. D. 1,25-Dihydroxyvitamin D3 Hypersensitivity of Osteoclast Precursors from Patients with Paget’s Disease. J. Bone Miner. Res. 2000, 15, 228−236. (7) Bury, Y.; Steinmeyer, A.; Carlberg, C. Structure Activity Relationship of Carboxylic Ester Antagonists of the Vitamin D3 Receptor. Mol. Pharmacol. 2000, 58, 1067−1074. (8) Herdick, M.; Steinmeyer, A.; Carlberg, C. Carboxylic Ester Antagonists of 1α,25-Dihydroxyvitamin D3 Show Cell-Specific Actions. Chem. Biol. 2000, 7, 885−894. (9) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman, A. W.; Ishizuka, S. Antagonistic Action of Novel 1α,25-Dihydrox-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00819. Figure S1 showing SRC-1 and RXRα recruitment to VDR by 1,25D3 and 3a,b−6a,b in HEK293 cells, Figure S2 showing omit maps of the 3a, 4a,b, and 5a,b/VDRLBD complexes, Figure S3 showing X-ray crystal structure of VDR-LBD complexed with 1,25D3, 3a, 4a, 4b, and 5a, Figure S4 showing distances between helix 10/11 of VDR-LBD and neighboring VDR-LBD in crystal structures, Table S1 listing data collection and refinement statistics, and Table S2 listing summary of VDR-LBD structural changes induced by the ligand binding (PDF) Molecular formula strings (CSV) Accession Codes

The coordinate data for the structures were deposited in Protein Data Bank with accession numbers 5XPP (3a/VDRLBD complex), 5XPN (4a/VDR-LBD complex), 5XPM (4b/ 8404

DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.7b00819 J. Med. Chem. 2017, 60, 8394−8406