Identification of the Histidine Residue in Vitamin D Receptor That

Article Cite This: J. Med. Chem. 2018, 61, 6339−6349

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Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands Mami Yoshizawa, Toshimasa Itoh, Tatsuya Hori, Akira Kato, Yasuaki Anami, Nobuko Yoshimoto, and Keiko Yamamoto* Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan

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ABSTRACT: We designed and synthesized vitamin D analogues with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogues 1−4 have an electrophilic enone group at the side chain for conjugate addition to His301 or His393 in the VDR. All compounds showed specific VDR-binding potency and agonistic activity. Covalent bond formations of 1−4 with the ligand-binding domain (LBD) of VDR were evaluated by electrospray ionization mass spectrometry. All compounds were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of 1−4 increased with incubation time. Enone compounds 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compounds. Furthermore, we successfully obtained cocrystals of VDR-LBD with analogues 1−4. X-ray crystallographic analysis showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogues that form a covalent bond with VDR-LBD.

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peroxisome proliferator-activated receptor (PPAR).4,5 Covalent ligands that bind to the VDR are 1,25D3-3β-bromoacetate and (23S)-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone.6,7 These analogues are covalently bound to cysteine in the LBD. Therefore, we aimed to logically create specific ligands that form a covalent bond with residues, other than cysteine, based on the structure of the ligand-binding pocket (LBP), which represents a rational design approach to identifying covalent ligands. 1α,25-Dihydroxyvitamin D3 (1,25D3), the active form of vitamin D3, is anchored by three pincer-type hydrogen bonds with residues in the LBP of VDR. The 25-hydroxyl group of 1,25D3 forms hydrogen bonds with His301 and His393 and is thought to contribute to transactivation.8,9 We focused on the nucleophilicity of histidine in designing covalent modifiers. Few other drugs are known to be covalent modifiers of histidine, such as fumagillin and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone.10,11 While histidine is not a common covalently modified target compared to cysteine and serine, it is one of the available bases under physiological conditions, and a lone pair on the sp2 amine could be expected to be a nucleophile.

INTRODUCTION A number of popular covalent-binding drugs are used in clinical pathology, including penicillin, omeprazole, aspirin, etc. One of the advantages of covalent drugs is that the pharmacological activity after binding persists until degradation of the target protein. Another benefit of covalent bond formation is its large energy, which exceeds van der Waals forces and hydrogen bonds, so that the influence on the stability of the target protein is large. Indeed, the three drugs mentioned above are often used in medical practice because of their strong effects.1 In contrast, covalent modifiers have been excluded from high throughput screening and chemical libraries due to the risks of off-target and false-positive results. Therefore, it is important that covalent drugs show binding selectivity to target molecules as well as high activity at low doses.2 One approach to achieve these characteristics is the appropriate selection of the target residue within the target molecule. The majority of covalent drugs modify cysteine or serine residues.3 In addition, it appears that most covalent drugs have been identified not by design but by serendipity. In regards to nuclear receptor ligands, a few classes are known to have potential as covalent modifiers. Oxo fatty acids, reported by us, and 2-chloro-5-nitrobenzanilide are irreversible ligands of © 2018 American Chemical Society

Received: May 15, 2018 Published: June 23, 2018 6339

DOI: 10.1021/acs.jmedchem.8b00774 J. Med. Chem. 2018, 61, 6339−6349

Journal of Medicinal Chemistry

Article

Figure 1. Structures of target compounds 1−4.

Scheme 1. Synthesis of Compounds 1 and 2

Hydrogen bond formation with His301 and His393 is a specific interaction to the VDR, and a nuclear receptor ligand that forms a covalent bond with histidine has not yet been reported. Therefore, we designed VDR ligands that have an electrophile at the side chain for conjugate addition with histidine. 1,25D3 regulates many biological functions, including calcium metabolism, cell differentiation, and immune responses, by gene transcription following binding to VDR.12,13 A covalent modifier for the VDR is expected to exhibit high activity and long-term effects, and to be applicable to the development of drugs for the treatment of diseases such as osteoporosis, psoriasis, and cancer.14,15

In this report, we describe the design, synthesis, and biological activities of vitamin D analogues with an electrophilic enone group for conjugate addition to the VDR, and also X-ray crystallographic analysis of the VDR ligand-binding domain (LBD) complexed with enone analogues.

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RESULTS Design and Synthesis. We designed vitamin D analogues with electrophilic covalent modifiers for the VDR (Figure 1). Novel vitamin D analogues 1−4 have an electrophilic enone group at the side chain for conjugate addition to the functionally important residues His301 and His393 in the VDR. The β-

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Scheme 2. Synthesis of Compounds 3 and 4

Table 1. VDR Binding Affinity of Synthetic Analogues 1−4a

a

Competitive binding of 1,25D3 and synthetic compounds 1−4 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 1,25D3 from the receptor protein. bUpper row: Affinities are presented as relative values, the reference value of 1,25D3 being defined as 1. cLower row: IC50 of synthetic compound. Value in the parentheses is IC50 of 1,25D3 in each experiment.

synthesized from the protected Weinreb amide 7, which was coupled with the corresponding Grignard reagent to afford the enone 9 (92%). The deprotection of 9 with CSA afforded compound 2. Ene-ynone compound 3 and dienone compound 4 were synthesized from the previously reported aldehyde 10 (Scheme 2).16 The conversion of 10 to alkyne via Corey−Fuchs alkyne synthesis afforded the diastereomeric-mixture of 11 (dr = 3:2) in 80% yield.18 Alkyne 11 was oxidized by Dess−Martin periodinane to provide ene-ynone 12 (95%) and then deprotected with CSA to afford the target compound 3 in 86% yield. For the synthesis of compound 4, alkyne 11 was selectively reduced from a triple bond to diene alcohol 13 using Vitride (sodium bis (2-methoxyethoxy) aluminumhydride) in 89% yield as a diastereomeric mixture (dr = 3:2). Dess−Martin oxidation of alcohol 13 afforded ketone 14, which was deprotected to provide the target compound 4. Biological Activities. Binding affinity for VDR was evaluated by a competitive binding assay using [3H]-1,25D3 according to a previously reported method.16,19 Recombinant human VDR-LBD was expressed as a C-terminus GST-tagged

carbon of enones was introduced to the position C26 corresponding to the oxygen atom of the 25-hydroxyl group of 1,25D3, which forms hydrogen bonds with His301 and His393 in the VDR-LBP. It was designed such that the β-carbon is subject to nucleophilic attack by the nitrogen atom of histidine. Analogues 1 and 2 were designed for investigation of the difference in reactivity depending on the presence or absence of a substituent of β-carbon. Analogues 3 and 4 have two reaction points expected for 1,4-addition and 1,6-addition. Enone compounds 1 and 2 were synthesized from the previously reported methyl ester 5 (Scheme 1).16 Compound 5 was hydrolyzed to carboxylic acid 6 at 85% yield. The carboxyl group in 6 transformed to the Weinreb amide in 7 in 95% yield.17 After the deprotection of 7 with CSA, Grignard reagent reacted with the deprotected Weinreb amide 8 to produce compound 1 in 72% yield. For the synthesis of the target compound 1, 1,4-conjugate addition of MeOH as a solvent occurred when deprotection was performed after introducing an enone group, so that compound 1 could not be obtained. For this reason, a Grignard reaction with the deprotected Weinreb amide was carried out. However, the target compound 2 was 6341



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Figure 2. Transactivation of compounds 1−4 was evaluated using Cos7 and HEK293 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 construct (pRL-CMV), as described previously.16 In experiments in Cos7 cells, luciferase activity equal to 10−8 M 1,25D3 was defined as 1, and in experiments in HEK293 cells, activity equal to 10−9 M 1,25D3 was defined as 1.

protein using the pGEX-VDR vector in E. coli BL21. The results are summarized in Table 1. All compounds showed specific VDR-binding potency and high affinity with IC50 values in the nanomolar range. The results indicated that compounds 1−4 are potent VDR ligands. The transcriptional activity of compounds 1−4 was tested using the rat osteopontin luciferase reporter gene assay system in Cos7 cells and HEK293 cells, according to a previously reported method.16 As shown in Figure 2, all compounds showed full agonistic activity in both cells, but they seem to be less potent. Enone compound 2 was the most potent compound. ESI-MS Analysis. Covalent bond formations of 1−4 with the rat VDR-LBD were evaluated by electrospray ionization mass spectrometry (ESI-MS).20 Figure 3 shows the ESI-MS spectra of the VDR-LBD in the absence and presence of ligands. We obtained a peak of m/z = 30,553 for apo VDR-LBD as shown in Figure 3a. In the presence of 5 equiv of 1,25D3, a single peak for the unmodified protein calculated mass 30,553 was observed (Figure 3b). When 5 equiv of compounds 1−3 were incubated with VDR-LBD for 24 h, the mass spectra showed peaks for the VDR-LBD increased according to the molecular weight of each ligand (Figure 3c−e). These results indicate that ligands 1−3 are covalently bound to the VDR-LBD. In contrast, dienone 4 showed only a peak for the unmodified protein at room temperature. However, after incubation for 30 h at 37 °C, the mass spectrum showed a peak for the unmodified protein and a second peak for the protein covalently modified with ligand 4 (Figure 3f). The peak intensity of the modified VDR was half that of the unmodified VDR, indicating that the covalent bond formation of 4 was not complete under this condition. These results indicate that compounds 1−3 are more potent VDR ligands than compound 4 with respect to covalent modification.

Figure 3. ESI-MS analysis of rat VDR-LBD chemically modified by 1− 4. (a) Mass spectrum of VDR-LBD in absence of ligands. (b) Mass spectrum of VDR-LBD with 5 equiv of 1,25D3. (c−f) Mass spectra of VDR-LBD with 5 equiv of 1−4.

Next, we verified whether the reaction was reversible or irreversible. After 2 equiv of compounds 1−3 were incubated 6342



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Figure 4. Time-dependent ESI-MS of VDR-LBD chemically modified by 1−3.

Figure 5. Crystal structures of rat VDR-LBD bound to enone ligands 1−4. Electron density maps of covalent targets His301 and His393 with (a) 1 (5ZWE), (b) 2 (5ZWF), (c) 3 (5ZWH), and (d) 4 (5ZWI). Fo−Fc maps (unclear electron density) displayed in green mesh at 2.5σ level, together with 2Fo−Fc map displayed in blue mesh at 1σ.

with the VDR-LBD for 24 h, we confirmed covalent bond formation with the VDR using ESI-MS. Subsequently, we

exchanged the sample buffer for ligand-free buffer, and 10 equiv of 1,25D3 was added. After incubation for 24 h, the mass spectra 6343



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Figure 6. Conformational preferences of compound 2. (a) In VDR-LBD/2 complexes, compound 2 (orange) had the 26S configuration. The s-cis model of 2 (light green) based on the positive density map is displayed in green mesh at 2.5σ level. The s-cis model interacts with Leu223 with too close contact. (b) Steric repulsion between Leu223 and the s-cis isomer made it easier to convert the conformation to s-trans isomer. If the histidine nucleophilic attack of the β-carbon of the s-trans isomer is from the si face, the stereochemistry of the product has a 26S configuration.

Arg270 and Tyr143/Ser274, respectively, as previously described for the VDR-LBD/1,25D3 complex (Figure S1b). The lack of polar groups of the side chain corresponding to the 25-hydroxyl group of 1,25D3 resulted in ligand 1 not forming hydrogen bonds with His301 and His393. The same was true for the VDR-LBD/2−4 complexes (Figure S1c−f). However, as shown in Figure 5a, the electron density map (2Fo−Fc map) showed a continuous electron density between the side chain of 1 and His301. This shows that enone compound 1 was covalently bound to His301. Similarly, enone compound 2 and ene-ynone compound 3 formed covalent bond with His301 (Figure 5b,c). The new C−N bond formation took place between the β-position of the enone (1,4-adduct) and the nitrogen of the imidazole ring of His301. In contrast, dienone compound 4 did not form a covalent bond with histidine (Figure 5d). Interestingly, His393 formed a hydrogen bond with a water molecule (Figure S1c−f). The covalent bond formation of 1 and His301 was found in the major conformer (occupancy 0.7). The difference density map, Fo−Fc map, showed unclear localized electron density near the side chain of 1, but we could not identify other minor conformers (Figure 5a). In the VDR-LBD/2 complex, the major conformer (occupancy 0.7) forms the covalent bond, and there is also an unclear electron density near the side chain of 2. The positive density in the Fo−Fc maps (green mesh) of 1 and 2 was localized at the far side to His301. The VDR-LBD/3 complex indicated that the ligand adopts two conformations, covalently and noncovalently binding to VDR (Figure 5c). The ratio of covalently and noncovalently binding ligand is 1 to 1. The noncovalently binding conformation of 3 is similar to that of the VDR-LBD/4 complex (Figure 5d).

showed the only peaks for the covalently modified VDR without exchange of the ligand. Since 1,25D3 possess stronger affinity than ligands 1−3, we considered that the covalent bond between ligands 1−3 and VDR is irreversible. Lastly, we investigated the reactivity of the conjugate addition between ligands 1−3 and the VDR-LBD. As shown in Figure 4, the peaks for covalently modified protein increased in an incubation time-dependent manner. The formation of covalent bonds of 1−3 with the VDR-LBD was time-dependent. The time for enone 1 and 2 to completely form covalent bonds with the VDR was 90 and 180 min, respectively. However, covalent bond formation of ene-ynone 3 remained incomplete after 6 h. The mass spectrum at 6 h showed two peaks for the covalently modified and intact VDR, and the intensity ratio of the peaks was about 1 to 1. From the above results, enone 2 is the most reactive of the ligands. Ene-ynone 3 showed a reduced rate of covalent binding to the VDR compared to the enone compounds. We considered the differences in observed reactivity of the ligands to the VDR. Enones 1 and 2 have a single bond at C22, which allows free rotation of the side chain in the VDR-LBP. In contrast, ene-ynone 3 and dienone 4 have double bonds at C22, making the side chain rigid. These structural features restrict the binding conformation in the VDR-LBP. Enones have a clear advantage over dienones in the formation of covalent bonds. It is assumed that the flexibility of C22 participates in the speed of the conjugate addition reaction. X-ray Crystal Structure. To investigate the binding site and the interactions of compounds 1−4 with the VDR, we performed cocrystallization of these analogues and the rat VDR-LBD. We successfully obtained cocrystals of the VDRLBD with 1−4 and the coactivator peptide DRIP205 and subsequently carried out X-ray crystallographic analysis. The space group of these crystals was identical to that of known rat VDR-LBD crystals. The data collection and statistics of structure refinement are summarized in Table S1. The overall crystal structure of the VDR-LBD complexed with 1 was similar to the VDR-LBD/1,25D3 complex (PDB code 2ZLC, Figure S1a).21 The A-ring and CD-ring of ligand 1 were positioned almost the same as those of 1,25D3. The 1α- and 3βhydroxyl groups of 1 made hydrogen bonds with Ser233/

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DISCUSSION We synthesized four vitamin D ligands 1−4 for conjugate addition with His301 and His393 of VDR. All synthetic compounds showed binding affinity in the nanomolar range for VDR. Despite the lack of a hydroxyl group of the end of the side chain, these ligands were full agonists of the VDR. Covalent bond formation of all ligands and VDR-LBD was observed using 6344



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covalent bond formation with 2, His301 seems to stabilize Gln396. For this reason, we suggest that covalent bond formation with His301 stabilizes helix 12 at the canonical position; thus 2, can activate VDR.

ESI-MS. The covalent bond formation of ligands 1 and 2 was significantly faster than that of ene-ynone 3 and dienone 4. Enone compounds 1 and 2 have an sp3-carbon at C22, allowing free rotation of the side chain in the ligand-binding site of VDR. In contrast, ene-ynone compound 3 and dienone compound 4 have a carbonyl group at C22 conjugated to the sp or sp2 carbon at the side chain, making the side chain rigid. These structural features would restrict the binding conformation in the ligandbinding site of VDR. Of our synthesized compounds, enone ligands 1 and 2 have a clear advantage over 3 and 4 to form covalent bonds. It is suggested that the flexibility of the side chain participates in determining the rate of the conjugate addition reaction. X-ray crystallographic analysis showed compounds 1−3 formed covalent bonds with His301 of VDR. However, dienone compound 4 did not form an adduct with histidine under the crystallization condition. In the VDR-LBD/1,2 complexes, the covalent binding conformer of compounds 1 and 2 was a major conformer (occupancy 0.7). We were unable to identify other minor conformers; however, the unclear localized electron density that suggests the minor conformations was confirmed. In compound 2, s-cis conformation should be preferred to the s-trans conformation because of an allylic strain in α,βunsaturated ketone in solution.22 Based on the VDR-LBD/2 complex, it is able to dock the s-cis form of 2 so that the conformer fits to the positive density map (Figure 6a). However, the distance between the methyl group of 2 and Leu223 at helix3 is only 2.5 Å. This indicates that the methyl group can cause steric repulsion with Leu223 by perturbation of the protein. Thus, the s-cis conformation can flip to s-trans conformation with ease. Thus, we expect that covalent bond formation proceeds from the s-trans conformation in this mechanism. In addition, compound 2 had the 26S configuration in the cocrystal structure, suggesting that covalent bond formation was achieved by si face attack of His301 to s-trans conformation of 2 (Figure 6a,b). The reactivity of the conjugate addition reaction with His301 is thought to be associated with the predominance of the s-trans conformation. In general, increasing substituents at the β-carbon is disadvantageous to conjugate reactions. Inexplicably, the covalent bond formation of 2 as evaluated by ESI-MS was faster than 1 (Figure 4). In the model of the s-cis form of 2, the terminal methyl group of 2 interacts with Leu223 at a relatively close distance of 2.5 Å. Steric repulsion with Leu223 tends to occur, and therefore, compound 2 would heighten the formation of the s-trans conformation compared to compound 1 (Figure 6a,b). At the Aring of 2, the mode of hydrogen bond formation with VDR-LBD is identical to that of 1,25D3. Although the hydrogen bonds with His301 and His393 are important for activity, agonist 2 lacked hydrogen bonds with these histidines and instead formed a covalent bond with His301. Among previously reported ligands, ligands lacking hydrogen bonding with His301 or His393 tended to show low affinity and efficacy.22−26 In the VDR/2 complex, His301 formed hydrogen bond with Gln396 on helix11, as observed for the 1,25D3/VDR-LBD complex (Figure S2). Helix11 is a highly mobile helix as indicated by small-angle X-ray scattering and molecular dynamics (SAXS-MD) experiments and the hydrogen/ deuterium exchange coupled with mass spectrometry (HDXMS) experiment by us.25,27 Gln396 is an originating point of the helix break, and helix 12, which creates the activation function 2 (AF-2) surface, moves in cooperation with helix 11.27,28 By

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CONCLUSION We designed and synthesized vitamin D analogues with electrophilic covalent modifiers for VDR. ESI-MS analysis revealed the covalent bond formation of ligands and VDR. From the comparison of the side chain structure of 1−4, the flexibility of the side chain appeared to be important for the high conjugation reactivity with VDR. X-ray crystallographic analysis showed the covalent bonds with compounds 1−3 and His301 in the VDR-LBD. Since there are few reports of compounds that covalently bind to histidine in proteins, this study is an example showing the usefulness of histidine as a covalently modified target. Additionally, in enone compounds 1 and 2, covalent bond formation occurred by s-trans conformation, which is more unstable than s-cis conformation. The steric repulsion of the terminal side chain and Leu223 may accelerate the change from s-cis conformation to s-trans conformation. In general, covalent modifiers are inhibitors of target enzymes or antagonists of receptors. In contrast, our ligands show full agonistic activity. Thus, our results encourage the design of covalent modifiers as full agonists.

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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. 6-[(1R,3R,7E,17β)-1,3-Dihydroxy-2-methylidene-9,10-secoestra5,7-dien-17-yl]-hept-1-en-3-one (1). To a solution of 8 (12.6 mg, 0.029 mmol) in dry THF (1 mL) was added vinyl magnesium bromide (1.0 M solution in THF, 292 μL, 0.29 mmol), and the mixture was stirred at 0 °C. After 1 h, to the mixture was added an additional vinyl magnesium bromide (1.0 M solution in THF, 146 μL, 0.15 mmol), and the mixture was stirred for 1 h. The reaction was quenched with saturated NH4Cl and 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 45% AcOEt/hexane to afford 1 (8.4 mg, 72%). 1 H NMR δ: 0.55 (3H, s, H-18), 0.95 (3H, d, J = 6.2 Hz, H-21), 4.48 (2H, m, H-1, 3), 5.09, 5.11 (each 1H, s, CCH2), 5.82 (1H, dd, J = 10.2, 1.5 Hz, H-26), 5.88 (1H, d, J = 10.9 Hz, H-7) 6.22 (1H, dd, J = 17.5, 1.5 Hz, H-26), 6.36 (2H, m, H-6, H-25). 13C NMR δ: 12.1, 18.6, 22.3, 23.5, 27.6, 28.9, 30.0, 35.8, 36.7, 38.2, 40.4, 45.8 (2 carbons), 56.3 (2 carbons), 70.7, 71.8, 107.7, 115.4, 124.2, 127.8, 130.5, 136.6, 143.2, 152.0, 201.4. LRMS (ESI+) m/z 421.3 [(M + Na)+, 100]. HRMS (ESI+) calcd. for C26H38Na1O3 (M + Na)+ 421.2719, found 421.2729. IR (neat) 3391, 3306, 2945, 2924, 2891, 2872, 2839, 1732, 1651, 1634, 1464, 1454, 1416, 1373, 1333, 1292, 1204, 1076, 1059, 995, 966, 904, 867, 847, 831, 758, 673 cm−1. UV (MeOH) λmax 244.5, 252.5, 262.0 nm. 7-[(1R,3R,7E,17β)-1,3-Dihydroxy-2-methylidene-9,10-secoestra5,7-dien-17-yl]-oct-2-en-4-one (2). To a solution of 9 (17.0 mg, 0.027 mmol) in MeOH (1 mL) was added camphor sulfonic acid (51.1 mg, 0.22 mmol) at 0 °C, and the mixture was stirred at room temperature 6345



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4-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-N-methoxy-N-methylpentanamide (7). To a solution of 6 (129.7 mg, 0.210 mmol) in dry CH2Cl2 (3 mL) at 0 °C were added N,O-dimethylhydroxylamine hydrochloride (28.6 mg, 0.29 mmol), 1-hydroxy-1H-benzotriazol hydrate (43.7 mg, 0.32 mmol), N-methylmorpholine (65 μL, 0.59 mmol), and 1-ethyl-3(3-(dimethylamino)propyl)-carbodiimide hydrochloride (61.8 mg, 0.32 mmol). The mixture was stirred at room temperature for 2 h. The reaction was quenched with water and 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 5% AcOEt/hexane to afford 7 (131.4 mg, 95%). 1 H NMR δ: 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.55 (3H, s, H18), 0.86, 0.90 (each 9H, s, tert-Bu), 0.96 (3H, d, J = 6.3 Hz, H-21), 2.83 (1H, m, H-9), 3.18 (3H, s, NMe), 3.70 (3H, s, OMe), 4.42 (2H, m, H-1, 3), 4.92, 4.97 (each 1H, s, CCH2), 5.84 (1H, d, J = 11.5 Hz, H-7), 6.22 (1H, d, J = 11.4 Hz, H-6). 13C NMR (75 MHz, CDCl3) δ: −5.1, −4.94, −4.89 (2 carbons), 12.1, 18.13, 18.22, 18.6, 22.2, 23.4, 25.75 (3 carbons), 25.81 (3 carbons), 27.6, 28.7, 29.7, 30.7, 32.2, 35.9, 38.5, 40.6, 45.6, 47.6, 56.2, 56.3, 61.2, 71.6, 72.5, 106.2, 116.2, 122.4, 132.8, 141.0, 152.9, 175.2; LRMS (ESI+) m/z 682.6 [(M + Na)+, 100]. HRMS (ESI+) calcd. for C38H69N1Na1O4Si2 (M + Na)+ 682.4663, found 682.4626. IR (neat) 2955, 2926, 2854, 1678, 1470, 1256, 1101, 1072, 1005, 935, 897, 835, 775, 711, 673 cm−1. UV (MeOH) λmax 245.0, 253.5, 263.0 nm. 4-[(1R,3R,7E,17β)-1,3-Dihydroxy-2-methylidene-9,10-secoestra5,7-dien-17-yl]-N-methoxy-N-methylpentanamide (8). Compound 7 (31.8 mg, 0.048 mmol) in MeOH (1 mL) was treated with camphor sulfonic acid (89.4 mg, 0.378 mmol) by the procedure described in preparation of compound 2 to give 8 (15.1 mg, 73%). 1 H NMR δ: 0.56 (3H, s, H-18), 0.96 (3H, d, J = 6.2 Hz, H-21), 3.18 (3H, s, NMe), 3.70 (3H, s, OMe), 4.47 (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, 18.6, 22.3, 23.5, 27.5, 28.9 (2 carbons), 30.7, 32.2, 35.9, 38.2, 40.4, 45.8 (2 carbons), 56.28, 56.32, 61.2, 70.7, 71.8, 107.7, 115.4, 124.2, 130.5, 143.2, 152.0, 175.2. LRMS (ESI+) m/z 454.3[(M + Na)+, 100]. HRMS (ESI+) calcd. for C26H41N1Na1O4 (M + Na)+ 454.2933, found 454.2934. IR (neat) 3418, 2926, 2854, 1728, 1643, 1461, 1456, 1381, 1277, 1248, 1188, 1148, 1122, 1076, 1045, 1009, 910, 866, 831, 746, 673 cm−1. UV (MeOH) λmax 244.5, 252.5, 262.0 nm. 7-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-oct-2-en-4-one (9). To a suspension of Mg turnings (51.2 mg, 2.10 mmol) in dry THF (1 mL) were added a small piece of iodine and trans-1-bromo-1-propen (114 μL, 1.33 mmol) in dry THF (250 μL) . The mixture was heated by a heat gun and stirred for 0.5 h. To the resulting Grignard reagent was added 7 (58.3 mg, 0.088 mmol) in THF (1 mL) at room temperature, and the mixture was stirred for 0.5 h. The reaction was quenched with saturated NH4Cl and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (50 g) with 2% AcOEt/hexane to afford 8 (52.3 mg, 92%). 1 H NMR δ: 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.54 (3H, s, H18), 0.86, 0.90 (each 9H, s, tert-Bu), 0.94 (3H, d, J = 6.2 Hz, H-21), 1.90 (3H, dd, J = 6.8, 1.7 Hz, H-27), 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 = 11.1 Hz, H-7), 6.13 (1H, dq, J = 15.9, 1.7 Hz, H-25), 6.22 (1H, d, J = 11.1 Hz, H-6), 6.85 (1H, dq, J = 15.7, 6.8 Hz, H-26). 13C NMR δ: −5.1, −4.92, −4.87 (2 carbons), 12.1, 18.15, 18.20, 18.24, 18.7, 22.2, 23.4, 25.77 (3 carbons), 25.83 (3 carbons), 27.6, 28.7, 30.3, 35.8, 37.0, 38.5, 40.6, 45.7, 47.6, 56.2, 56.3, 71.6, 72.5, 106.2, 116.2, 122.4, 131.9, 132.8, 141.0, 142.2, 153.0, 201.1. LRMS (ESI+): m/z 663.5 [(M + Na)+, 100]. HRMS (ESI+) calcd. for C39H68Na1O3Si2 (M + Na)+ 663.4605, found 663.4626. IR (neat) 2955, 2928, 2854, 1732, 1703, 1682, 1634, 1472, 1377, 1362, 1259, 1100, 1072, 1024, 935, 897, 835, 800, 777, 709, 667 cm−1. UV (MeOH) λmax 245.0, 253.5, 263.0 nm. 2-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-oct-6-en-4-yn-3-ol (11). To a solution of 1,1-dibromo penta-1,3-diene (73.8 mg, 0.33 mmol) in dry

for 1 h. The reaction was quenched with saturated NaHCO3 and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (25 g) with 40% AcOEt/hexane to afford 2 (7.1 mg, 65%). 1 H NMR δ: 0.55 (3H, s, H-18), 0.94 (3H, d, J = 6.2 Hz, H-21), 1.91 (3H, dd, J = 6.8, 1.7 Hz, H-27), 4.49 (2H, m, H-1, 3), 5.09, 5.11 (each 1H, s, CCH2), 5.89 (1H, d, J = 11.3 Hz, H-7), 6.13 (1H, dq, J = 15.7, 1.6 Hz, H-25), 6.36 (1H, d, J = 11.4 Hz, H-6), 6.82 (1H, dq, J = 15.7, 6.8 Hz, H-26). 13C NMR δ: 12.1, 18.2, 18.6, 22.2, 23.5, 27.5, 28.9, 30.3, 35.8, 37.0, 38.2, 40.4, 45.8 (2 carbons), 56.28, 56.30, 70.7, 71.8, 107.7, 115.4, 124.2, 130.5, 131.9, 142.3, 143.2, 152.0, 201.1. LRMS (ESI+): m/ z 435.3 [(M + Na)+, 100]. HRMS (ESI+) calcd. for C27H40Na1O3 (M + Na)+ 435.2875, found 435.2880. IR (neat) 3389, 2918, 2872, 2849, 1722, 1693, 1661, 1634, 1464, 1441, 1377, 1321, 1292, 1259, 1215, 1194, 1148, 1124, 1076, 1047, 972, 912, 868, 833, 754, 667 cm−1. UV (MeOH) λmax 244.5, 252.5, 262.0 nm. 2-[(1R,3R,7E,17β)-1,3-Dihydroxy-2-methylidene-9,10-secoestra5,7-dien-17-yl] -oct-6-en-4-yn-3-one (3). Compound 12 (23.9 mg, 0.038 mmol) in MeOH (1 mL) was treated with camphor sulfonic acid (87.0 mg, 0.3750 mmol) by the procedure described in preparation of compound 2 to give desired product 3 (13.2 mg, 86%). 1 H NMR δ: 0.58 (3H, s, H-18), 1.23 (3H, d, J = 6.8 Hz, H-21), 1.89 (3H, dd, J = 6.9, 1.8 Hz, H-27), 4.48 (2H, m, H-1,3), 5.09, 5.11 (each 1H, s, CCH2), 5.66 (1H, dq, J = 15.8, 1.8 Hz, H-25), 5.89 (1H, d, J = 11.2 Hz, H-7), 6.35 (1H, d, J = 11.2 Hz, H-6), 6.53 (1H, dq, J = 15.8, 6.9 Hz, H-26). 13C NMR δ: 12.4, 16.5, 19.2, 22.5, 23.4, 26.7, 28.9, 38.1, 40.3, 45.8, 46.0, 52.1, 52.3, 55.8, 70.6, 71.8, 86.0, 90.9, 107.8, 109.0, 115.7, 124.0, 140.3, 142.4, 147.5, 151.9, 191.9; MS (ESI+) m/z 431.3 [(M + Na)+, 100]. HRMS (ESI) calcd. for C27H36 Na1O3 (M + Na)+ 431.2562, found 431.2580. IR (neat) 3387, 2926, 2872, 2179, 1659, 1443, 1373, 1290, 1256, 1229, 1184, 1072, 1045, 982, 953, 899, 752, 667 cm−1. UV (EtOH) λmax 245, 253, 262 nm. 2-[(1R,3R,7E,17β)-1,3-Dihydroxy-2-methylidene-9,10-secoestra5,7-dien-17-yl]-octa-4,6-dien-3-one (4). Compound 14 (26.9 mg, 0.0421 mmol) in MeOH (1 mL) was treated with camphor sulfonic acid (97.8 mg, 0.4210 mmol) by the procedure described in preparation of compound 2 to give desired product 4 (15.9 mg, 92%). 1 H NMR δ: 0.60 (3H, s, H-18), 1.14 (3H, d, J = 7.0 Hz, H-21), 1.87 (3H, d, J = 4.7 Hz, H-27), 2.83 (1H, m, H-9), 4.47 (2H, m, H-1,3), 5.09, 5.11 (each 1H, s, CCH2), 5.88 (1H, d, J = 11.1 Hz, H-7), 6.13 (1H, d, J = 15.0 Hz, H-23), 6.22 (2H, m, H-25,26), 6.35 (1H, d, J = 11.3 Hz, H6), 7.19 (1H, ddd, J = 15.4, 6.5, 3.4 Hz, H-24). 13C NMR δ: 12.4, 16.9, 18.8, 22.5, 23.4, 27.0, 28.9, 38.1, 40.3, 45.8, 45.9, 47.6, 52.6, 55.7, 70.6, 71.7, 107.7, 115.6, 124.0, 126.3, 130.3, 130.8, 140.4, 142.6, 143.1, 151.9, 204.1. MS (ESI+) m/z 411.3 [(M + H)+, 100]. HRMS (ESI) calcd. for C27H39O3 (M + Na)+ 411.2899, found 411.2934. IR (neat) 3420, 2932, 2872, 1632, 1589, 1443, 1381, 1333, 1074, 1047, 1001, 752, 667 cm−1. UV (EtOH) λmax 245, 253, 263, 277 nm. 4-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-pentanoate (6). Ester 5 (150.5 mg, 0.239 mmol) was solved in 5% KOH/MeOH:H2O (19:1) (3 mL) at 60 °C, and the mixture was stirred at room temperature for 3 h. The reaction was quenched with aqueous citric acid and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was purified by the medium-pressure liquid chromatography (MPLC) using a column (KP-C18-HS) with 100% MeOH to afford 6 (127.7 mg, 87%). 1 H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.55 (3H, s, H18), 0.86, 0.90 (each 9H, s, t-Bu), 0.95 (3H, d, J = 6.3 Hz, H-21), 2.83 (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 = 11.0 Hz, H-7), 6.23 (1H, d, J = 10.9 Hz, H-6). 13C NMR δ −5.1, −4.92, −4.87 (2 carbons), 12.1, 18.16, 18.24, 18.4, 22.2, 23.4, 25.77 (3 carbons), 25.83 (3 carbons), 27.6, 28.7, 30.7, 31.0, 35.7, 38.6, 40.6, 45.7, 47.6, 56.18, 56.21, 71.6, 72.5, 106.3, 116.2, 122.4, 132.9, 140.9, 152.9, 180.1. MS (ESI+) m/z 639.5 [(M + Na)+, 100]. HRMS (ESI+) calcd. for C36H64Na1O4Si2 (M + Na)+ 639.4241, found 639.4223. IR (neat) 3431, 2953, 2928, 2895, 2854, 1709, 1650, 1620, 1470, 1462, 1252, 1101, 1070, 1003, 935, 897, 835, 775, 711, 673 cm−1. UV (MeOH) λmax 245.0, 253.5, 263.0 nm. 6346



Article

THF (1.2 mL) was added n-BuLi (1.60 M in hexane, 408 μL, 0.65 mmol) at −78 °C, and the mixture was allowed to warm to 0 °C for 1 h. To a solution of 1,1-dibromo penta-1,3-diene (73.8 mg, 0.33 mmol) in dry THF (1.2 mL) was added n-BuLi (1.60 M in hexane, 408 μL, 0.65 mmol) at −78 °C, and the mixture was stirred. After 1 h, the mixture was stirred at 0 °C for 1 h. The resulting lithium acetylide reagent was added to a solution of 10 (62.4 mg, 0.11 mmol) in dry THF (1.1 mL) at −78 °C, and the mixture was stirred for 10 min at 0 °C. The reaction was quenched with saturated NH4Cl and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (75 g) with 6% AcOEt/hexane to afford 11 (55.7 mg, 80%, 3:2 mixture of diastereomers). The diastereomeric mixture 11 was used without further purification. 1 H NMR (11-major*/11 minor**) δ: 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18)*, 0.57 (3H, s, H-18)**, 0.86, 0.90 (each 9H, s, tert-Bu), 1.07 (3H, d, J = 6.4 Hz, H-21)**, 1.13 (3H, d, J = 6.4 Hz, H-21)*, 1.79 (3H, m, H-27), 4.42 (2H, m, H-1,3), 4.57 (1H, s, br, H-22)**, 4.60 (1H, s, br, H-22)*, 4.92, 4.97 (each 1H, s, CCH2), 5.52 (1H, m, H-25), 5.85 (1H, d, J = 11.1 Hz, H-7), 6.15 (1H, dq, J = 15.5, 6.8 Hz, H-26)*, 6.16 (1H, dq, J = 15.8, 6.8 Hz, H-26)**, 6.21 (1H, d, J = 11.2 Hz, H-6). 13C NMR δ: −5.11 (2 carbons), −4.91 (2 carbons), −4.86 (4 carbons), 12.0, 12.3, 13.0, 13.4, 18.15 (2 carbons), 18.24 (2 carbons), 18.56 (2 carbons), 22.2, 22.3, 23.4 (2 carbons), 25.78 (6 carbons), 25.83 (6 carbons), 26.96, 27.09, 28.7, 28.8, 38.6 (2 carbons), 40.36, 40.46, 42.63, 42.67, 45.54, 45.83, 47.60 (2 carbons), 52.2, 53.3, 55.8, 56.1, 65.9, 66.0, 71.6, 71.7, 72.50 (2 carbons), 83.9, 84.9, 86.0, 88.4, 106.3 (2 carbons), 110.2, 110.3, 116.3 (2 carbons), 122.3 (2 carbons), 132.9, 133.0, 140.0, 140.1, 140.79, 140.84, 153.0 (2 carbons). MS (ESI+) m/z 661.4 [(M + Na)+, 100]. HRMS (ESI) calcd. for C39H66Na1O3Si2 (M + Na)+ 661.4448, found 661.4448. IR (neat) 3406, 2953, 2928, 2893, 2856, 1659, 1620, 1472, 1461, 1444, 1377, 1360, 1256, 1101, 1070, 1024, 1003, 947, 935, 908, 897, 835, 775, 735, 673 cm−1; UV (EtOH) λmax 245, 254, 263 nm. 2-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-oct-6-en-4-yn-3-one (12). To a solution of 11 (35.1 mg, 0.055 mmol) and NaHCO3 (23.1 mg, 0.2745 mmol) in dry CH2Cl2 (550 μL) was added Dess−Martin periodinane (15% in CH2Cl2, 228 μL, 0.1098 mmol) at 0 °C. The mixture was stirred for 10 min at room temperature. The reaction was quenched with saturated NaHCO3 at 0 °C 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 12 (33.1 mg, 95%). 1 H NMR δ: 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.58 (3H, s, H18), 0.86, 0.90 (each 9H, s, tert-Bu), 1.24 (3H, d, J = 6.8 Hz, H-21), 1.88 (3H, dd, J = 6.9, 1.8 Hz, H-27), 4.42 (2H, m, H-1,3), 4.92, 4.98 (each 1H, s, CCH2), 5.66 (1H, dq, J = 15.8, 1.8 Hz, H-25), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6), 7.20 (1H, dq, J = 15.8, 6.9 Hz, H-26). 13C NMR δ: −5.11, −4.91, −4.86 (2 carbons), 12.4, 16.5, 18.1, 18.2, 19.2, 22.5, 23.3, 25.7 (3 carbons), 25.8 (3 carbons), 26.7, 28.7, 38.5, 40.5, 45.9, 47.6, 52.2, 52.3, 55.8, 71.6, 72.5, 86.1, 90.8, 106.3, 109.0, 116.5, 122.2, 133.3, 140.3, 147.4, 152.9, 192.0. MS (ESI+) m/z 659.4 [(M + Na)+, 100]. HRMS (ESI) calcd. for C39H64Na1O3Si2 (M + Na)+ 659.4292, found 659.4297. IR (neat) 2955, 2930, 2893, 2856, 2183, 1666, 1630, 1462, 1253, 1101, 1070, 935, 897, 835, 775, 673 cm−1. UV (EtOH) λmax 245, 253, 263 nm. 2-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-octa-4,6-dien-3ol (13). To a solution of 11 (165 mg, 0.26 mmol) in dry THF (3.2 mL) was added sodium bis(2-methoxyethoxy)aluminum hydride solution (70% in toluene, 590 μL, 2.07 mmol) at 0 °C. The mixture was stirred for 2.5 h at room temperature. The reaction was quenched with saturated potassium sodium tartrate at 0 °C and extracted with Et2O. The organic layer was washed with brine, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel (17 g) with 6% AcOEt/ hexane to afford 13 (146.8 mg, 89%, 3:2 mixture of diastereomers). The diastereomeric mixture 13 was used without further purification. 1 H NMR (13-major*/13 minor**) δ: 0.03, 0.05, 0.07, 0.08 (each 3H, s, SiMe), 0.55 (3H, s, H-18)*, 0.57 (3H, s, H-18)**, 0.86, 0.90

(each 9H, s, tert-Bu), 0.91 (3H, d, J = 6.8 Hz, H-21)*, 0.97 (3H, d, J = 6.7 Hz, H-21)**, 1.76 (3H, m, H-27), 2.83 (1H, m, H-9), 4.22 (1H, m, H-22)**, 4.31 (1H, m, H-22)*, 4.43 (2H, m, H-1,3), 4.92, 4.97 (each 1H, s, CCH2), 5.68 (2H, m, H-23,26), 5.84 (1H, d, J = 10.8 Hz, H7)**, 5.85 (1H, d, J = 11.1 Hz, H-7)*, 6.13 (2H, m, H-24, 25), 6.22 (1H, d, J = 10.8 Hz, H-6). 13C NMR δ: −5.10 (2 carbons), −4.91 (2 carbons), −4.86 (4 carbons), 12.0, 12.2, 12.3, 12.6, 18.08, 18.10, 18.16 (2 carbons), 18.23 (2 carbons), 22.1, 22.3, 23.4 (2 carbons), 25.78 (6 carbons), 25.83 (6 carbons), 27.1, 27.3, 28.73, 28.76, 38.6 (2 carbons), 40.47, 40.49, 41.9, 42.5, 45.6, 45.9, 47.6 (2 carbons), 52.9, 53.4, 55.8, 56.1, 71.6 (2 carbons), 72.5 (2 carbons), 74.0, 74.5, 106.3 (2 carbons), 116.2 (2 carbons), 122.4 (2 carbons), 129.0, 129.1, 129.82, 129.89, 130.9, 131.0, 132.4, 132.88, 132.92, 133.4, 140.9, 141.0, 153.0 (2 carbons). MS (ESI+) m/z 663.5 [(M + Na)+, 100]. HRMS (ESI) calcd. for C39H68Na1O3Si2 (M + Na)+ 663.46047, found 663.46392. IR (neat) 3404, 2953, 2930, 2887, 2856, 1659, 1620, 1472, 1461, 1447, 1400, 1377, 1361, 1328, 1256, 1190, 1101, 1070, 1024, 989, 935, 908, 897, 835, 775, 735, 673 cm−1. UV (EtOH) λmax 236, 244, 253, 263 nm. 2-[(1R,3R,7E,17β)-1,3-Bis{[(tert-butyl(dimethyl)silyl]-oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-octa-4,6-dien-3-one (14). To a solution of 13 (31.2 mg, 0.049 mmol) and NaHCO3 (20.5 mg, 0.2435 mmol) in dry CH2Cl2 (490 μL) was added Dess−Martin periodinane (15% in CH2Cl2, 202 μL, 0.0974 mmol) at 0 °C. The mixture was stirred for 10 min at room temperature. The reaction was quenched with saturated NaHCO3 at 0 °C 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 14 (22.6 mg, 73%). 1 H NMR δ: 0.02, 0.05, 0.06, 0.08 (each 3H, s, SiMe), 0.60 (3H, s, H18), 0.86, 0.90 (each 9H, s, tert-Bu), 1.15 (3H, d, J = 6.8 Hz, H-21), 1.87 (3H, d, J = 4.7 Hz, H-27), 2.84 (1H, m, H-9), 4.42 (2H, m, H-1,3), 4.92, 4.97 (each 1H, s, CCH2), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.14 (1H, d, J = 14.9 Hz, H-23), 6.21 (3H, m, H-7,25,26), 7.20 (1H, ddd, J = 15.3, 6.5, 3.6 Hz, H-24). 13C NMR δ: −5.11, − 4.92, − 4.87 (2 carbons), 12.4, 16.9, 18.1, 18.2, 18.8, 22.4, 23.3, 25.76 (3 carbons), 25.82 (3 carbons), 27.0, 28.7, 38.6, 40.5, 45.8, 47.6, 47.8, 52.6, 55.6, 71.6, 72.5, 106.3, 116.4, 122.3, 126.3, 130.4, 133.1, 140.3, 140.5, 143.0, 152.9, 204.1. MS (ESI+) m/z 661.4 [(M + Na)+, 100]. HRMS (ESI) calcd. for C39H66Na1O3Si2 (M + Na)+ 661.4448, found 661.4482. IR (neat) 2955, 2930, 2887, 2856, 1686, 1659, 1637, 1593, 1472, 1462, 1445, 1400, 1377, 1333, 1254, 1190, 1101, 1070, 1003, 935, 908, 897, 835, 777, 735, 673 cm−1. UV (EtOH) λmax 246, 254, 263, 277 nm. Binding Affinity. The binding affinity for human VDR-LBD was evaluated according to a procedure reported previously.16 Transactivation. Transactivation in Cos7 cells or HEK293 cells was measured using a dual luciferase assay according to the procedure reported previously.16 Protein Expression and Purification. Expression of rat VDRLBD (residues 116−423, Δ165−211) and the following purification were conducted using a procedure reported previously.25,29,30 ESI-MS Analysis. The rat VDR-LBD was concentrated in 50 mM ammonium acetate, pH 6.5. The ligands (5 equiv) were added to rat VDR-LBD solution (10 μM, 250 μL), and mixtures were incubated at room temperature. ESI-MS measurements were performed on a JEOL JMS-T100LP spectrometer. The samples were introduced into the ion source at a flow rate of 30 μL/min using a syringe pump (Harvard Apparatus) and a 1 mL Hamilton syringe. Data collection was carried out with the following interface parameters: positive ion mode, capillary voltage (VCap) 2000 V, orifice 1 voltage 130 V, orifice 2 voltage 3 V, ring lens voltage 15 V, ion guide voltage 2500 V, detector voltage 2600 V, orifice 1 temperature 80 °C, desolvation temperature 350 °C, nebulizer gas 0.5 L min−1, drying gas 1.5 L min−1. ESI spectra were recorded in the mass range m/z 100−4000. Data analysis was performed using the ESI deconvolution Ver. 2.01 of JEOL. Crystallographic Analysis. A mixture of rat VDR-LBD in buffer (10 mM Tris-HCl, pH 7.0; 2 mM TCEP; 2 mM NaN3) and a ligand (1 equiv for 1 and 3, 0.5 equiv for 2, and 1.2 equiv for 4) was incubated at room temperature for 30 min. The following purification was performed according to a procedure reported previously.25,30 Diffraction data sets of 1/VDR-LBD, 2/VDR-LBD, 3/VDR-LBD, 6347



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and 4/VDR-LBD were collected at 100 K in a stream of nitrogen gas at beamlines BL-5A of KEK-PF, NE3A of KEK-PFAR, and NW12A of KEK-PFAR (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.31,32 The ternary complex structures were solved by molecular replacement with the software Phaser33 in the CCP4 program34 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 Coot35 and refinement with refmac5.36−40

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00774. X-ray crystal structure of the VDR-LBD complexed with 1,25D3, 1, 2, 3 and 4; comparison of the VDR-LBD/2 complex with the VDR-LBD/1,25D3 complex; data collection and refinement statistics; 1H NMR, 13C NMR, and HPLC spectra data (PDF) Molecular formula strings (CSV) Accession Codes

The coordinate data for the structures were deposited in Protein Data Bank with accession numbers 5ZWE (1/VDR-LBD complex), 5ZWF (2/VDR-LBD complex), 5ZWH (3/VDRLBD complex), and 5ZWI (4/VDR-LBD complex).

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Article

AUTHOR INFORMATION

Corresponding Author

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

Yasuaki Anami: 0000-0001-5136-7708 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 (No. 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). Synchrotron radiation experiments were performed at the Photon Factory (Proposal No. 2013G656, 2015G717), and we are grateful for the assistance provided by the beamline scientists at the Photon Factory.

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ABBREVIATIONS USED 1,25D3, 1α,25-dihydroxyvitamin D3; HDX-MS, hydrogen/ deuterium exchange coupled with mass spectrometry; LBD, ligand binding domain; LBP, ligand binding pocket; MD, molecular dynamics; NR, nuclear receptor; PPAR, peroxisome proliferator-activated receptor; SAXS, small-angle X-ray scattering; VDR, vitamin D receptor 6348



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Identification of the Histidine Residue in Vitamin D Receptor That

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