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Oct 11, 2017 - Sciences, Kunming 650201, China. ‡. State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, RuiJin Hospital, ...
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Article Cite This: J. Med. Chem. 2017, 60, 9053-9066

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Design, Synthesis, and Biological Activities of Vibsanin B Derivatives: A New Class of HSP90 C‑Terminal Inhibitors Li-Dong Shao,†,∥ Jia Su,†,∥ Baixin Ye,‡,∥ Jiang-Xin Liu,†,∥ Zhi-Li Zuo,† Yan Li,† Yue-Ying Wang,*,‡ Chengfeng Xia,*,† and Qin-Shi Zhao*,†,§ †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China ‡ State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, RuiJin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China § University of Chinese Academy of Science, Beijing 100049, China S Supporting Information *

ABSTRACT: Previously, vibsanin B (ViB) was found to preferentially target HSP90β compared to HSP90α. In this study, multiple experiments, including pull-down assays of biotin-ViB with recombinant HSP90β-NTD, MD, CTD, and full-length HSP90β, molecular docking of ViB and its derivatives to the HSP90 CTD, and a inhibition assay of interaction of the HSP90β CTD with GST-tagged cyclophilin 40 (Cyp40) by ViB derivatives, suggest that ViB can directly bind to the HSP90 C-terminus. On the basis of the docking predictions and primary structure−activity relationships (SARs), a series of ViB analogues devised with focus on the C18 position, along with compounds derivatized at the C4, C7, and C8 positions, were designed and chemically synthesized. Compound 12f (IC50 = 1.12 μM against SK-BR-3) exhibits great potency with drug-like properties. Overall, our findings demonstrate that compounds with the vibsanin B scaffold are a new class of HSP90 C-terminal inhibitors with considerable potential as anticancer agents.

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INTRODUCTION As an ATP-dependent molecular chaperone, heat shock protein 90 (HSP90) is responsible for the conformational maturation, activation, and stability of more than 200 client proteins involved in signal transduction which is essential for oncogenic development, cancer cell survival, and drug resistance development. There are two major cytoplasmic isoforms of HSP90, namely HSP90α (inducible type) and HSP90β (constitutive type). Although these two HSP90 isoforms share ∼86% sequence identity and exhibit significant functional redundancy, they also display distinct cellular functions. The other HSP90 isoforms are glucose-regulated protein 94 kDa (Grp94), which is located in the endoplasmic reticulum, and tumor necrosis receptor-associated protein 1 (Trap1), which is located in the mitochondria. All these HSP90s comprise three domains: an Nterminal domain (NTD), which contains an ATP-binding pocket, a middle domain (MD), which is involved in client protein recognition and binding, and a C-terminal domain (CTD), which is a dimerization domain for cochaperone binding. Upon HSP90 inhibition, more than 30 oncoproteins involved in multiple oncogenic pathways are simultaneously disrupted, resulting in a combinatorial attack on cancer.1 Moreover, in eukaryotic cells, HSP90 is upregulated from its © 2017 American Chemical Society

native content of 1−2% to 4−6% only under cellular stress and is continuously overexpressed in cancerous cells but not in normal cells. Targeting HSP90 thus may be effective in killing cancer cells while inducing fewer side effects.2 Therefore, HSP90 inhibitors may have promising applications in the treatment of cancers. Classical small-molecule HSP90 inhibitors such as geldanamycin (1, Figure 1),3 radicicol (2),4 and purine analogues,5 bind to the ATP pocket at HSP90 NTD, thereby impairing the intrinsic ATPase activity and blocking the formation of the mature complex. Consequently, the client proteins (e.g., p53 and ErbB2) degrade through the ubiquitin−proteasome pathway.1a To date, 17 HSP90 NTD inhibitors shown to exert potent antitumor activity in preclinical models have been in clinical trials. However, these have exhibited disappointing results because of their inadequate physicochemical properties, their poor safety profiles, and, most likely, their multiple targets in addition to HSP90 in cancer cells.6 Moreover, HSP90 inhibition by NTD inhibitors leads to displacement of the HSP90-bound transcription factor, heat shock factor-1 (HSFReceived: September 20, 2017 Published: October 11, 2017 9053

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

Journal of Medicinal Chemistry

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Figure 1. Structures of natural HSP90 inhibitors.

Figure 2. Design of ViB C18 analogues. (A) Structures of ViB, its primarily designed C18 analogues 7−10, and ViB-4-OH and their cytotoxicity to cancer cells (only MCF-7 is shown, for more details, see Supporting Information (SI), Table S1). (B) Primary SARs of ViB derivatives. (C) Binding mode of 7. Red-dotted lines denote real interactions in the docking prediction, blue-dotted line is a probable interaction, and the purple circle is a promising modified region near the residues Glu333 and Glu431. (D) Incorporation of a positive charge-containing motif (e.g., tertiary amines and oximes) at C18 likely stabilizes the binding.

inhibitors, such as the coumarin-containing natural product novobiocin (3, Figure 1),9 the natural cyclic pentapeptide sansalvamide A (5),10 and their derivatives, have been identified as potential anticancer drug modalities.11 HSP90 CTD inhibitors, which are highlighted by recent studies as a promising class of HSP90 modulators, exhibit unique effects

1), and induction of the pro-survival heat shock response, which considerably impairs the anticancer effect of these compounds and results in drug resistance in cancer cells.7 More recently, gambogic acid (4) was identified as a selective inhibitor that binds to the MD of HSP90β.8 With the recent discovery of the HSP90 CTD ATP-binding site, specific CTD 9054

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

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Scheme 1a

(a) CCl3CN, PPh3, DCM, RT, 30 min; (b) corresponding secondary amine, NaI, THF, RT, 8−16 h; (c) DAST, DCM, −78 °C, 2 h; (d) TESCl, TEA, DCM, RT, 6 h for13a, TBSCl or TBDPSCl, imidazole, DMAP, DCM, RT, 6−8 h for 13b or 13c; (e) Ac2O, TEA, DMAP, DCM, RT, 4−20 h for 8 and 8a, MeI, Ag2O, DCM, RT, 6−24 h for 8b and 8c; (f) Ac2O, TEA, DMAP, DCM, RT, 2 days for 8d, MeI, Ag2O, sealed, RT, 2 days for 8e; (g) silica gel, DCM, 35 °C, 15 min for 8d and 8e; (h) Pd/C, H2, DCM/hexane, RT, 30 min; (i) MOMCl, DIPEA, DCM, RT, 2 h; (j) SOCl2, pyridine, 0 °C, 5 min; (k) TBAF, THF, 0 °C to RT, 2 h. a

binding model, a series of ViB derivatives were synthesized, and their structure−activity relationships (SARs) for antiproliferative activity were then elucidated. Therefore, we herein reported the design and subsequent biological evaluation of ViB derivatives as a new class of HSP90 C-terminal inhibitors.

on HSP90s conformation, activity, and interactions with cochaperones and clients without eliciting the heat shock response.2c,7 However, the limited quantity of HSP90 CTD inhibitor scaffolds cannot satisfy the requirements for molecular biological interrogation of HSP90 functions and pharmacologic evaluation of anticancer drugs, especially in the context of the intractable development of HSP90 NTD inhibitors. Therefore, there is still a need to investigate novel HSP90 C-terminal inhibitor scaffolds with higher selectivity and affinity. Vibsanin B (ViB; 6, Figure 2A) is a natural diterpene isolated from Viburnum odoratissimum Ker-Gawl.12 In our recent study, ViB was found to show a high selectivity for binding HSP90β compared to HSP90α, inhibit leukocyte chemotactic migration in human monocytic cell lines and zebrafish model, and ameliorate experimental autoimmune encephalomyelitis (EAE) in mice.13 Given that HSP90 is closely associated with the pathogenesis of cancers and that ViB has shown cytotoxicity against KB cells,14 it is interesting to determine which domain of HSP90 (NTD, MD, or CTD?) ViB binds to when acting as an HSP90 inhibitor and whether its antiproliferative activity is retained in other cancer cell lines. In fact, some observations from our most recent research have already implied that the CTD is likely the binding domain of ViB in HSP90.13 In the present study, to validate this hypothesis, pull-down assays of four HSP90 mutants (flag-tagged full-length HSP90β and HSP90β-NTD, MD, and CTD) were performed using biotinViB. For further confirmation, molecular docking analysis and a competitive inhibition assay of novobiocin with ViB were conducted. Subsequently, the antiproliferative activity of ViB against five cancer cell lines (MCF7, SMMC7721, A549, HL60, and SW480) was tested. Furthermore, on the basis of ViB’s

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RESULTS AND DISCUSSION Vibsanin B Binds to the HSP90 C-Terminus. Our previous work showed that ViB can bind to HSP90β with a high selectivity compared to HSP90α.13 To reveal the specific binding site, the plasmids of HSP90 recombinant proteins including flag-tagged full-length HSP90β and HSP90β-NTD, MD, and CTD, were transfected and expressed in the 293T cell line, and then pull-down assays were performed to evaluate their binding with ViB. In these assays, lysates of 293T cells were incubated with 30 μM biotin-ViB13 and purified by streptavidin-agarose beads. As shown in Figure S1A, Supporting Information (SI)), Western blot analysis revealed that the biotin-ViB-bound agarose beads could pull-down the flagtagged HSP90β-CTD (strong binding) and MD (weak binding) but not the flag-tagged HSP90β-NTD, suggesting the direct binding of ViB to the HSP90β-CTD and some interactions involving the overlapped domain of CTD and MD. To further validate this observation, we also purified the recombinant protein His-tagged HSP90β-CTD and performed an in vitro binding assay as previously described (Figure S1B, SI).13 Our data showed that biotin-ViB can directly bind to HSP90β-CTD and that this interaction could be largely disrupted by free 6 or 7 (Figure 2A) but not by the inactive derivative ViB-4-OH,13 further supporting that ViB can directly target the HSP90β-CTD. Moreover, to confirm these results, 9055

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

Journal of Medicinal Chemistry

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Scheme 2a

(a) NaN3, PPh3, DMF/CCl4, 50 °C, 1 h; (b) propargyl alcohol, sodium ascorbate, CuSO4·5H2O, DMF/H2O, sonic, RT, 30 min; (c) NaBH4, CeCl3·7H2O, MeOH, −78 °C, 2 h; (d) MnO2, DCM, RT, 8 h; (e) methoxyamine hydrochloride, MeOH, RT, 4 h; (f) TEMPO, PIDA, DCM, RT, 2 h; (g) p-nitrobenzoyl chloride, TEA, DCM, RT, 30 min; (h) NaH, CS2, THF, −20 °C, 2 h, then MeI, RT, overnight; (i) IBX, DMSO/DCM, RT, 2 h; (j) NaBH(OAc)3, corresponding secondary amine, DCM, RT, 12−18 h; (k) NaClO2, NaH2PO4, 2-methyl-2-butene, H2O, RT, 10 min; (l) corresponding secondary amine, EDCI, DMAP, DCM, 0 °C to RT, 12−24 h. a

allosteric pocket in the literature.11i,15 Specifically, the hydrogen bonding interactions with Phe332 (Figure S2A,D, SI), Glu333 (Figure S2B, SI), and Lys423 (Figure S2A−C, SI) of chain B and Gln596 (Figure S2A,B, SI) of chain A, and the hydrophobic interactions with Pro504, Ile505, and Tyr508, account for the inhibitory activities of these compounds and further support the results of pull-down assays that biotin-ViB can pull down both flag-tagged HSP90 MD and CTD. Although Figure 2C shows that two Glu residues (Glu333 and Glu 431) are located near the C18 position of 7, we found in the docking analysis that the C18 fluoride interacts with only Glu333 (red dotted line). These two Glu residues feature a negative charge and together could likely chelate ligands that contain a positive charge (“privileged” N-containing fragments that can be protonated in the physiological environment, including tertiary amines16 and oximes,17 Figure 2D) to further stabilize the binding complex. Thus, our design of ViB analogues was focused on changes at C18 that improve the interactions with Glu333 and Glu431. To obtain a systematic SAR, positions C8, C4, and C7 were also modified. Preparation of C18-Modified Analogues. Consequently, derivatives with C18-halides (7, 11), tertiary amines (12a− 12f), amides (24a−24f), esters (8, 16, 19), ethers (13a−13c, 8c, 15), aldehyde (9), acid (10), 1,2,3-trizole (18), and oxime (22) (Schemes 1,2) were designed and subsequently synthesized. The C18 chloride (11) analogue was prepared in excellent yield by reacting of 6 with trichloroacetonitrile and triphenylphosphine. C18 fluoride (7) was obtained by treating

an HSP90 competitive binding assay against FITC-geldanamycin (a classical HSP90 NTD inhibitor) was conducted using both HSP90α and HSP90β. As expected, ViB did not compete with FITC-geldanamycin, thereby further demonstrating that ViB is a C-terminal, not an N-terminal, HSP90 inhibitor (data not shown). All these evidence suggest that ViB binds to the HSP90 C-terminus and that ViB derivatives may represent a new class of HSP90 C-terminal inhibitors. Design of Vibsanin B Derivatives. In addition, we found that designed ViB C18-analogues (7−10, Figure 2A) primarily showed dramatic antiproliferative activity against five cancer cell lines (Table S1, SI). The C18 fluoride (7) exhibited remarkable potency against all tested cancer cell lines with IC50 values of 0.14−0.62 μM and was more potent than ViB (IC50 values ranging from 2.64−3.46 μM). The C18 aldehyde analogue (9) was less potent than ViB, whereas the carboxyl analogue (10) was almost inactive (IC50 > 10 μM was defined as inactive in this study). When the C18 hydroxyl group was acetylated, the resulting analogue (8) retained its antiproliferative activity with IC50 values of 2.95−4.63 μM. On the basis of this primary SAR, as shown in Figure 2B, we speculated that the C18 position of ViB was probably the key interacting fragment upon binding to HSP90. Next, molecular docking experiments on the whole HSP90 CTD (PDB 2cg9)11i were performed with compounds 6, 7, 9, and 10 (Figure S2A−D, SI). The docking results demonstrated that the binding site is located at the dimerization site surface (residues 331−333 and 588−595, chain B; 592−596, chain A), which is described as a major 9056

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

Journal of Medicinal Chemistry

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Scheme 3a

(a) NaBH4, CeCl3·7H2O, MeOH, −78 °C, 2 h; (b) TESCl, DIPEA, DCM, RT, 6 h; (c) LiBH4, THF/Et2O, 0 °C to RT, overnight; (d) 26b, corresponding acid, DCC, DMAP, DCM, 0 °C to RT, 12−24 h for 27b−27e, 26c, MOMCl, DIPEA, DCM, RT, 2 h for 27f; (e) AcOH, THF, H2O, RT, 2−4 h; (f) IBX, DMSO/DCM, RT, 2−6 h; (g) TBAF, AcOH, THF, RT, 1−4 h. a

direct saponification of the α,β-unsaturated ester failed because of the high reactivity of the primary hydroxyl group and carbonyl group of ViB. To avoid possible side reaction during the subsequent steps, a protecting group strategy was developed (Scheme 3). Reduction of 13b with NaBH4/ CeCl3·7H2O afforded alcohol 25b, and protection of newly generated hydroxyl group at C4 of 25b with TESCl followed by reduction of C8 unsaturated ester with LiBH4 gave the common intermediate 26b in 74% yield over three steps. Subsequent condensation of the C8 hydroxyl group of 26b with various acids in the presence of DCC and DMAP generated the corresponding esters, which underwent deprotection of C4OTES and then IBX oxidation of the C4 hydroxyl group to afford 27b−27e. Subsequent deprotection of C18-OTBS gave C8 ester analogues 28b−28e. Furthermore, to prepare C8 methoxyl methyl ether 28f, another intermediate, 25c, was used because of the ability to deprotect C18-OTBS using AcOH/ THF/H2O when the C8 hydroxyl group was protected with methoxymethyl ether. As shown in Scheme 2, reduction of the C4 carbonyl group of ViB under standard Luche conditions provided C4 hydroxyl analogues 20a (ViB-4-OH) and 20b in a ratio of 4:1. C4 methoxyl analogues 29a and 29b were synthesized from 25b in two steps (Scheme 3). Further attempts to convert the C4 carbonyl group to a methylene functionality failed. We then examined catalytic hydrogenation conditions for selective removing the double bonds of ViB. However, standard Pd/C, PtO2, Raney Ni, and Wilkinson catalyst hydrogenations afforded a complex mixture of products with hydrogenated double bonds as well as hydrogenation of the C4 carbonyl group. Conversely, reduction in a DCM/hexane solvent system (5/1, v/v) with Pd/C provided 14 as the major product in 50% yield. To our delight, the yield was increased to 70% using Zn/ AcOH. Moreover, intermediate 13a was utilized to synthesize C7 analogues 8d and 8e in two steps (Scheme 1). Derivative 8f

ViB with diethylamino sulfur trifluoride (DAST) at −78 °C.13 To enhance the interaction with the carboxyl groups of Glu333 and Glu431 and further improve the aqueous solubility, tertiary amine analogues 12a−12f were prepared by reacting chloride 11 with the corresponding N-containing fragments16 in the presence of catalytic NaI. Silyl ethers 13a−13c, methyl ether 8c, and methoxymethyl ether 15 were also synthesized by the reaction of 6 with the corresponding alkylsilyl chlorides, MeI, and methoxymethyl chloride (MOMCl), respectively. Additionally, silyl ethers 13a−13c were used as intermediates in the synthesis of other analogues. The ester analogues 8 and 16 were prepared by the condensation of 6 with acetic anhydride and p-nitrobenzoyl chloride, respectively, in the presence of dimethylaminopyridine (DMAP, Scheme 2). Xanthate 19 was synthesized by the addition reaction of 6 to carbon disulfide and subsequent Smethylation with MeI. Moreover, to introduce more hetero atoms at C18, azide 17 and 1,2,3-trizole 18 were synthesized successively from 6 via well-established methods.18 Methyl oxime 22 was generated in a four-step sequence from 6 through Luche reduction, chemoselective oxidation with MnO2, condensation of aldehyde 21 with methoxyamine hydrochloride, and TEMPO/phenyliodine(III) diacetate (PIDA) oxidation.19 Considering the diversity of molecular functionalities, aldehyde 9, acid 10, and amides 24a−24f were prepared consecutively by IBX oxidation, Pinnick oxidation, and condensation with the corresponding N-containing fragments in the presence of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDCI), respectively. Initially, the attempted reductive amination of 9 with the corresponding seco-amines to synthesize compounds 12a−12f was unsuccessful; instead, ring-opening products 23a−23c were obtained.20 Preparation of C8-Modified Analogues. To investigate the necessity of the C8 side chain, we designed C8 analogues 28b−28f. The attempt to modify the C8 side chain through 9057

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

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Figure 3. Crystal structures of 13c (A) and 8d (B).

Table 1. Antiproliferative Activity of C18-Modified Vibsanin B Derivatives (IC50, μM) compd 11 12a 12b 12c 12d 12e 12f 13a 13b 13c 8c 15 16 17 18 19 22 23a 23b 23c 24a 24b 24c 24d 24e 24f ViB DDPa a

MCF-7 0.61 0.69 0.13 0.78 0.22 0.52 0.55 3.15 6.04 15.77 4.13 3.12 0.67 4.89 2.93 0.90 2.51 12.20 12.33 8.84 3.67 12.05 17.15 17.18 3.36 6.30 2.85 16.80

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.08 0.03 0.05 0.04 0.09 0.07 0.09 0.81 0.77 0.30 0.13 0.13 0.12 0.05 0.10 0.19 0.32 0.43 0.22 0.15 0.64 0.91 2.67 0.08 0.62 0.03 0.46

SMMC-7721 0.84 0.82 0.57 0.34 0.14 1.14 2.94 3.46 4.00 38.02 10.97 3.25 2.56 3.08 3.56 0.72 3.47 10.25 7.44 3.09 3.67 13.78 17.17 14.86 3.68 11.72 3.25 9.58

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

A-549

0.14 0.03 0.05 0.04 0.01 0.04 0.09 0.14 0.33 0.52 0.59 0.08 0.06 0.30 0.02 0.13 0.08 0.55 0.52 0.83 0.29 0.70 0.95 0.75 0.21 1.28 0.14 0.24

0.15 0.59 0.13 0.65 0.18 0.66 0.94 3.10 4.20 12.96 3.95 2.98 1.31 1.72 3.47 0.38 3.47 11.16 8.73 2.66 2.70 11.27 13.46 13.03 1.67 8.44 3.46 14.09

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02 0.01 0.03 0.01 0.01 0.01 0.08 0.01 0.48 0.17 0.05 0.06 0.36 0.21 0.02 0.05 0.46 0.28 0.34 0.34 0.98 1.13 1.49 0.05 0.45 0.21 0.76

HL-60 0.16 0.14 0.14 0.74 0.16 1.11 0.84 2.81 2.77 14.43 3.07 2.84 0.83 0.91 3.20 0.18 4.76 4.69 4.06 1.06 3.34 9.08 12.72 12.27 3.27 5.47 2.64 1.69

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.02 0.03 0.01 0.08 0.07 0.30 0.15 0.44 0.11 0.26 0.03 0.04 0.23 0.01 0.12 0.20 0.06 0.13 0.24 0.89 0.86 0.96 0.38 0.73 0.06 0.03

SW480 0.39 0.48 0.27 0.75 0.31 0.77 0.80 2.69 3.35 20.68 3.85 3.71 0.57 4.23 2.87 0.39 2.63 10.30 9.88 12.36 3.30 11.73 16.03 15.14 2.80 4.52 2.65 25.46

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.07 0.06 0.01 0.07 0.13 0.13 0.19 0.21 0.09 0.08 0.15 0.06 0.06 0.20 0.09 0.27 0.24 0.19 0.53 0.17 0.57 1.60 0.26 0.26 0.33 0.17 0.72

Cisplatin was used as a positive control.

potent than ViB. C18 ether derivatives (13a, 13b, 8c, 15) exhibit the same level of antiproliferative activities as ViB, except 13c (C18-OTBDPS, IC50 > 10 μM), suggesting that the steric bulk of the group at C18 should be smaller than that of −OTBS. Similar to fluoride 7, C18-chloride 11 shows excellent antiproliferative activity (IC50 range of 0.15−0.61 μM). To our delight, introducing of secondary amines at C18 resulted in highly active tertiary amines 12a−12f (IC50 range of 0.13−1.14 μM). However, the C18 tertiary amides are exceptions. Amides 24a (IC50 range of 2.70−3.67 μM) and 24e (IC50 range of 1.67−3.68 μM) show comparable activities to ViB, yet 24b− 24d and 24f (IC50 range of 9.08−17.18 μM) are almost inactive because of C18 tertiary amines are more easily protonated than C18 tertiary amides and thus are facilely chelated by Glu333 and Glu431. Moreover, C18 azide 17 (IC50 range of 0.91−4.89 μM), triazole 18 (IC50 range of 2.87−3.56 μM), and oxime 22

was prepared from 13b through elimination using SOCl2/ pyridine and subsequent removal of the TBS protecting group. In view of molecular lipophilicity, C7,18-diester 8a and diether 8b were designed and synthesized from 6 in one step (Scheme 1). C18 Has an Important Effect on Antiproliferative Activity. To investigate the essential functions of each fragment required for HSP90 inhibition, the antiproliferative activities of the ViB derivatives were first evaluated against the human cancer cell lines MCF7, SMMC7721, A549, HL60, and SW480 (Table 1−3). Our results strongly suggest that the types of substitutions at C18 play an important role in the antiproliferative activity (Table 1). C18 ester 8 (IC50 range of 2.95−4.63 μM, Table S1, SI) is less potent than the parent compound ViB, whereas C18 ester 16 (IC50 range of 0.57−2.56 μM) and xanthate 19 (IC50 range of 0.18−0.90 μM) are more 9058

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

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Table 2. Antiproliferative Activity of C4, C4/C5, C7, and Combined Modified Vibsanin B Derivatives (IC50, μM)

a

compd

MCF-7

SMMC-7721

A-549

HL-60

SW480

20a 20b 29a 29b 14 8a 8b 8d 8e 8f 21 ViB DDPa

17.38 ± 2.38 32.98 33.30 ± 4.53 18.53 ± 0.46 >40 3.99 ± 0.08 16.49 ± 0.52 2.32 ± 0.17 14.27 ± 0.74 2.75 ± 0.09 2.32 ± 0.06 2.85 ± 0.03 16.80 ± 0.46

16.37 ± 0.59 >40 38.83 ± 1.12 38.13 ± 2.66 >40 4.13 ± 0.27 9.38 ± 1.31 3.08 ± 0.12 15.83 ± 0.53 4.30 ± 0.12 0.73 ± 0.03 3.25 ± 0.14 9.58 ± 0.24

18.65 ± 0.34 >40 11.23 ± 2.60 19.17 ± 2.11 >40 15.14 ± 1.24 4.18 ± 0.76 3.15 ± 0.17 14.35 ± 0.42 3.09 ± 0.03 0.57 ± 0.06 3.46 ± 0.21 14.09 ± 0.76

15.90 ± 0.61 >40 13.81 ± 1.82 14.85 ± 1.67 >40 3.48 ± 0.20 4.05 ± 0.28 2.37 ± 0.38 14.31 ± 1.20 2.81 ± 0.21 0.24 ± 0.03 2.64 ± 0.06 1.69 ± 0.03

19.67 ± 1.37 >40 12.77 ± 0.20 16.62 ± 0.36 >40 3.52 ± 0.06 14.55 ± 1.63 2.01 ± 0.10 15.16 ± 0.61 2.97 ± 0.29 1.43 ± 0.05 2.65 ± 0.17 25.46 ± 0.72

Cisplatin was used as a positive control.

Table 3. Antiproliferative Activity of C8-Modified Vibsanin B Derivatives (IC50, μM)

a

compd

MCF-7

SMMC-7721

A-549

HL-60

SW480

28b 28c 28d 28e 28f ViB DDPa

7.89 ± 0.19 11.67 ± 0.34 6.04 ± 0.45 27.03 ± 1.26 >40 2.85 ± 0.03 16.80 ± 0.46

9.87 ± 0.69 10.40 ± 0.21 7.81 ± 0.54 20.26 ± 0.65 >40 3.25 ± 0.14 9.58 ± 0.24

8.67 ± 0.53 10.77 ± 0.74 4.10 ± 0.17 15.79 ± 1.11 >40 3.46 ± 0.21 14.09 ± 0.76

5.05 ± 0.20 4.00 ± 0.09 4.18 ± 0.19 22.62 ± 0.20 >40 2.64 ± 0.06 1.69 ± 0.03

4.90 ± 0.72 8.51 ± 0.68 2.51 ± 0.30 10.29 ± 0.81 >40 2.65 ± 0.17 25.46 ± 0.72

Cisplatin was used as a positive control.

Figure 4. Summary of the SARs of the ViB derivatives.

(IC50 range of 2.51−4.76 μM) retain the antiproliferative activity. The Michael Receptor within the Skeleton Is a Structural Requirement. In our previous study, we demonstrated the significance of the Michael acceptor in the anti-inflammatiory activity of ViB.13 Similarly, reduction of the C4 carbonyl group of ViB resulted in two inactive derivatives, 20a (IC50 > 10 μM) and 20b (IC50 > 10 μM), and their corresponding methyl ethers 29a (IC50 > 10 μM) and 29b (IC50 > 10 μM) are also inactive (Table 2). As expected, hydrogenated C5/C6 double bond analogue 14 (IC50 > 10 μM) totally lose its antiproliferative activity, indicating that the C4−C6 Michael acceptor is a structural requirement of ViB. As mentioned above, because the C4−C6 Michael acceptor of ViB is responsible for its biological activity, any modification is intolerable. However, compound 21 (IC50 range of 0.24− 2.32 μM) bearing a C4 hydroxyl group (destroying the C4−C6 Michael acceptor) and a newly formed Michael receptor (C2−

C18) is as potent as ViB. Conversely, compound 9, which contains two Michael receptors (C4−C6 and C2−C18) within the 11-membered ring skeleton, shows poor antiproliferative activity against all tested cell lines. These results suggest that the C4−C6 Michael receptor of ViB could be replaced by a new one elsewhere in the skeleton and that one Michael acceptor in the skeleton is favored. C7 Has Some Effects on Antiproliferative Activity. C7 acetate 8d (IC50 range of 2.01−3.15 μM) is as potent as ViB and more potent than its C7 methyl ether 8e (IC50 > 10 μM). The C7, C18 diacetyl ester 8a and dimethyl ether 8b are less active in some cell lines; for example, 8a is inactive against A549 (IC50 > 10 μM), and 8b is inactive against MCF-7 (IC50 > 10 μM) and SW480 (IC50 > 10 μM). By contrast, C7 hydroxyleliminated analogue 8f retains activity against all tested cell lines (IC50 range of 2.75−4.30 μM). Additionally, the crystal structures of 13c21 and 8d for the first time confirm the two stable conformations21 of ViB proposed by Fukuyama et al.22 9059

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using molecular modeling (Figure 3). The solid-state conformation of 13c exists in BC form, whereas that of 8d exists in CT form (details see Figure S2, SI). C8 Has Some Effects on Antiproliferative Activity. All C8-ester derivatives (28b−28e) exhibit lower antiproliferative activities compared with parent compound 6 (Table 3). The antiproliferative activity of isobutyryl ester derivative 28c (IC50 range of 4.00−11.67 μM) decreases slightly compared to ViB, and benzoyl ester derivative 28e (IC50 > 10 μM) is clearly inactive. Additionally, acetyl ester 28b (IC50 range of 4.90−9.87 μM) and n-butyl ester 28d (IC50 range of 2.51−7.81 μM) are more potent than 28c and 28e, indicating that the steric hindrance effect may be critical at the C8 position. Furthermore, C8-ether derivative 28f (IC50 > 10 μM) is inactive, implying that C8 position does not tolerate ether substitution. Summary of the SARs. As shown in Figure 4, a Michael acceptor within the skeleton (e.g., the C4−C6 Michael acceptor) is necessary for antiproliferative activity. Consequently, any modification of C4 or the C5/C6 double bond can lead to a loss of activity. However, when this Michael acceptor is destroyed, forming a new Michael acceptor in ViB skeleton is favored. The C18 position can be substituted with various functionalities. Among these functionalities, tertiary amines provide the most potent analogues, and fluoride, chloride, pnitrobenzoyl, and xanthate groups are also favored. Azide, triazole, acetate, methyl ether, and methoxymethyl ether are tolerable, whereas amides, aldehyde, and carboxyl are unfavorable. Moreover, the steric bulk of C18 should be less than that of −OTBS. Although methylation of the C7 hydroxyl group is unfavorable, acetylation or elimination of the hydroxyl group is tolerated. Small ester groups are favored at the C8 position, whereas introducing ether group at that position has a negative effect on the activity. Inhibition of HSP90-Dependent Luciferase Refolding. To investigate the HSP90 inhibitory activity of the ViB derivatives, a denatured luciferase refolding assay of selected ViB analogues (6, 7, 8d, 8f, 11, 12a, 12d, and 12f) was performed. The assay identifies inhibitors that obstruct the chaperone activity of HSP90, either by directly binding to its Nterminal or C-terminal nucleotide binding sites or by interfering with the ability of the chaperone to switch conformations. Figure 5 shows that compounds 6, 7, 8f, 11, 12a, and 12f were identified as potent HSP90 inhibitors, whereas the inhibition of HSP90 activity by derivatives 8d and 12d are less than 50%. Moreover, our results of the inhibition of HSP90-dependent luciferase refolding assays of compounds 6, 7, 8f, 11, 12a, and 12f are consistent with the results of those in antiproliferative activity assays (Table S6, SI) Antitumor Spectrum and Primary Toxicity Evaluation of Vibsanin B Derivatives. Among the selected analogues (6, 7, 8d, 8f, 11, 12a, 12d, and 12f), 12f is more active and has more potential as a candidate because of its privileged Nmethylpiperazine motif, which could provide better aqueous solubility.16d Although fluoride 7 is more active, it is difficult to dissolve in water.13 To comprehensively evaluate their antiproliferative effects, 7 and 12f were tested for their activities against human cancer cell lines, namely SK-BR-3 (breast cancer), MDA-MB-468 (breast cancer), HepG2 (liver cancer), AGS (gastric cancer), Huh-7 (liver cancer), HeLa (cervical cancer), BT474 (breast cancer), and BEAS-2B (lung epithelial cell). As shown in Table 4, both compounds 7 and 12f show a broad antiproliferative spectrum, and compound 7 (IC50 = 0.75

Figure 5. Inhibition of firefly luciferase renaturation by selected compounds. Thermally denatured luciferase was incubated in rabbit reticulocyte in the presence of DMSO, indicated as the vehicle control (DMSO), the positive control (novobiocin at 500 μM), or selected compounds (10 μM). After 30 min incubation, luciferase activity was measured as described in the Experimental Section. All tested compounds ViB, 7, 8d, 8f, 11, 12a, 12d, and 12f did not inhibit native luciferase activity. The results are representative of three independent experiments. ***p < 0.001 vs control.

μM to BEAS-2B) is more toxic than 12f (IC50 = 2.84 μM to BEAS-2B), a result that is consistent with in vivo trials in our previous work.13 Moreover, 12f in parallel with 7 was further investigated for primary toxicity in zebrafish embryo model with ViB as a reference (Table S2, SI). Similar to cell assay, 12f is less toxic than 7 and, more importantly, is less toxic than the parent compound ViB (Figure S3, SI). In addition, the ADMET properties of 7 and 12f including solubility, absorption, polar surface area (PSA) (an indicator of oral bioavailability), hepatotoxicity, and rat oral LD50, were predicted using the ADMET and Toxicity Prediction modules of Discovery Studio 4.0 (Table S3, SI). The results demonstrate that compared to 7, 12f is more soluble and less hepatotoxic and has a lower rat oral LD50 value, which further supports the findings of our toxicity tests in BEAS-2B cells and the zebrafish embryo model. HSP90 C-Terminal Inhibition and Binding Mode. On the basis of the above results, the specific binding of the HSP90 C-terminus by 12f was investigated via its interference with the interaction between the HSP90β C-terminus and CyP40, a tetratricopeptide repeat (TPR)-containing immunophilin and a cochaperone to HSP90. CyP40 specifically binds to the MEEVD sequence of the HSP90 C-terminus via the TPR domain.23 In this study, the novobiocin binding site of the HSP90β C-terminus (amino acid 538−728, including the MEEVD domain) was incubated with GST-tagged CyP40. As shown in Figure 6, 12f, as well as novobiocin, significantly disrupts the interaction between the HSP90β C-terminus and CyP40. Moreover, to confirm this result, an HSP90α/β competitive binding assay against FITC-geldanamycin was also conducted. The results revealed that 12f does not compete with FITC-geldanamycin, further demonstrating that 12f is an HSP90 C-terminal inhibitor not an N-terminal inhibitor (data not shown). To further investigate the HSP90 C-terminal binding, we performed molecular docking analysis to obtain the putative binding mode of 12f with the HSP90 C-terminus (PDB 2cg9).11i,15 As predicted, the interactions between the C18 functionality and residues Glu333 and Glu431 play a critical 9060

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Table 4. Antiproliferative Activity Spectrum of Vibsanin B Derivatives (IC50, μM)

a

compd

SK-BR-3

MDA-MB-468

7 12f ViB DDPb

0.33 ± 0.17 1.12 ± 0.15 9.19 ± 0.32 nda

0.31 ± 0.08 0.76 ± 0.04 nda 2.66 ± 0.20

HepG2 0.86 2.26 19.13 4.97

± ± ± ±

0.06 0.10 0.16 0.53

AGS 0.74 0.86 9.68 18.77

± ± ± ±

Huh-7 0.03 0.06 0.29 2.28

0.54 ± 0.07 0.86 ± 0.06 nda 4.83 ± 0.05

Hela 0.88 0.59 2.48 3.32

± ± ± ±

0.02 0.10 0.23 0.19

BT474

BEAS-2B

± ± ± ±

0.77 ± 0.04 2.84 ± 0.13 nda 7.14 ± 1.97

0.94 2.64 4.55 21.92

0.21 0.72 0.12 4.03

nd: not determined. bCisplatin was used as a positive control.

Figure 6. Inhibition of the interaction of the HSP90β C-terminus with Cyp40 by 12f. Inhibition of the HSP90β C-terminus of compounds was tested in an Alphascreen assay. (A) The inhibition rates of novobiocin (500 μM), 12f (100 μM), and ViB (100 μM). (B) Inhibition of the HSP90β C-terminus after treatment with 12f or novobiocin at concentrations of 1, 3.3, 10, 33, and 100 μM. The fluorescence of each well was measured as described in the Experimental Section. The results are representative of three independent experiments. **p < 0.01, 12f vs novobiocin; ***p < 0.001 vs control.

Figure 7. Molecular docking model of 12f bound to the HSP90 C-terminus (PDB 2cg9). (A) Ligand is colored by element type (C, yellow; O, red; N, blue; polar H, white), whereas key residues are shown as sticks (C, green; O, red; N, blue; polar H, white), and key interactions are denoted by a red-dotted line. (B) Solid surface map of the interaction pocket with compound 12f. Red, blue, and white colored regions correspond to negatively charged, positively charged, and neutral areas, respectively.

the mechanism of action is ongoing in our laboratory and will be reported in due course. Evaluation of 12f as an HSP90 Inhibitor in a Cellular System. To clarify the correlation between the antiproliferative activity and HSP90 inhibitory activity of 12f, we examined the ability of 12f to inhibit HSP90 in the human cancer cell line BT474. A decrease in HSP90 client proteins and a compensatory increase in HSPs are the two most common biomarkers used for the inhibition of HSP90 in cells. As expected, 12f is an effective HSP90 inhibitor, as shown by decreases in HSP90 client proteins, including c-Raf, Her2, pAkt, and CDK4, and the compensatory induction of HSP70 and 27 (Figure 8A). Moreover, the cell cycle distribution in the BT474 cells was evaluated. As shown in Figure 8B, treatment with 12f increased the number of G2/M phase cells from

role in binding and activity; thus, modifications that can improve these interactions may have positive effects. For example, C18-fluoride 7 and chloride 11 show excellent antiproliferative activity against all tested cancer cell lines because of the formation of halogen bonds between the C18 fluoride or chloride and Glu333. Regarding 12f, in addition to the interactions of the carbonyl group in the side chain with Lys423 and of the C7 hydroxyl group with Gln596, two stronger interactions, so-called salt bridges24 between N2 of the piperazine ring and Glu333 and Glu431, are observed in the docking mode (Figure 7). These stronger interactions could explain the better potency of 12f compared to novobiocin in the HSP90 C-terminal inhibition assay. A much more detailed molecular biological study elucidating this chelated binding and 9061

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Figure 8. Effects of 12f on HSP90 in cellular systems. (A) BT474 cells were treated with 12f for 24 h. Cells were harvested and subjected to Western blot analysis with the antibodies indicated. β-Actin was used as a loading control. The experiment was carried out in triplicate. (B) Compound 12f induces G2/M phase arrest and apoptosis in BT474 cells. BT474 cells were treated with 12f for 16 h. Cells were harvested and subjected to cell cycle analysis. The percentages of the cells distributed in different phases of cell cycle were determined using CellQuest software (Becton Dickinson). Experiments were performed independently in triplicate. Data shown are from one of three independent experiments. *p < 0.05.

designed and synthesized ViB derivative 12f by linking the privileged N-methylpiperazine moiety to the C18 position of ViB and subsequently proved its potential as a new type of HSP90 C-terminal inhibitor and as an anticancer agent. Compound 12f showed a broad spectrum of antiproliferative activity (IC50 range of 0.55−2.94 μM) and was more potent than the classical HSP90 C-terminal inhibitor novobiocin (IC50 = 700 μM against SK-BR-3) and parent compound 6 (IC50 range of 2.64−3.46 μM). Moreover, compound 12f is primarily a maturation HSP90 C-terminal inhibitor with improved ADMET properties. In this study, ViB and 12f are thermally stable in both PBS (pH = 7.4) at 37 °C and deionized water at 100 °C. Furthermore, our present efforts highlight the importance of target-based drug design, natural product scaffold-based drug discovery, and privileged structures in medicinal chemistry.

15.79% to 25.60%. Collectively, our results show a good correlation between HSP90 binding affinity and cellular activities, suggesting that the antiproliferative effect of 12f is a consequence of its HSP90 chaperone inhibitory properties. In Vitro Stability of Vibsanin B Derivatives. As the highly functionalized groups in ViB especially the C5−C10 unit that forms a very reactive [3,3]-σ system can be quickly transformed to seven-membered ring vibsanins when heated in toluene,20,22 the in vitro stability, including thermostability and solution stability, of selected ViB derivatives (6, 8d, 8f, and 12f) was tested according to the standard method using HPLC.25 As shown in Figure S9A, SI, both ViB and 12f decomposed quickly in toluene at 110 °C [half-life (t1/2 ∼ 5 min)]; otherwise, 12f decomposed more slowly than ViB in toluene at 60 °C with estimated t1/2 values of ∼20 and ∼9 min, respectively. Interestingly, both ViB and 12f are very stable in deionized water at 100 °C for 10 min with 2.8% and 2.3% decomposition, respectively (Figure S10C, SI). In fact, ViB is stable in boiling ethyl alcohol (data not shown). These results demonstrate that ViB derivatives are likely thermostable in protic solvents such as water but not in nonprotic solvents such as toluene. Moreover, ViB derivatives 8d, 8f, and 12f show fairly good solution stability in PBS (pH = 7.4) at 37 °C, whereas 4.92% of ViB is degraded after incubating for 12 h (Figure S9B, SI). In addition, we found that 12f is stable in DMSO stock solution (0.1 M) at 4 °C for 4 weeks (Figure S10A, SI). To our delight, the thermostability test of 8f in boiling toluene indicated that the thermal instability of ViB derivatives in nonprotic solvent can be improved by eliminating the C7 hydroxyl group (Figure S10B, SI).

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EXPERIMENTAL SECTION

Chemistry. All reactions were carried out under an atmosphere of argon in dry flask and were monitored by analytical thin-layer chromatography (TLC), which was visualized by ultraviolet light (254 nm). The reagents were obtained commercially and used without further purification. THF, Et2O, and toluene were distilled from Na, DCM, and DMF were distilled from CaH2 prior to use. ViB was isolated from Viburnum odoratissimum Ker-Gawl. var. sessilif lorum according to the literature method.22a Purification of products was accomplished by flash column chromatography using silica gel (200− 300 mesh). All NMR spectra were recorded with a Bruker Avance III 400 MHz or Avance III 600 MHz (1H NMR) spectrometer and 100 or 150 MHz (13C NMR) in CDCl3: chemical shifts (δ) are given in ppm, coupling constants (J) in Hz, and the solvent signals were used as references (CDCl3: δC = 77.16 ppm; residual CHCl3 in CDCl3: δH = 7.26 ppm). High-resolution mass spectra were recorded on a Waters AutoSpec Premier P776 spectrometer. Optical rotations were measured with a Horiba SEPA-300 polarimeter. The purity of all tested compounds was determined by HPLC (Agilent Technologies 1200 series) equipped with a C-18 bounded-phase column (Zorbax, SB-C18, 4.6 mm × 250 mm); all tested compounds were >95% pure. Melting points were obtained on a WRX-4 apparatus and are uncorrected.

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CONCLUSION Vibsanins are a class of biologically important natural diterpenes. For example, vibsanin A was found to target PKC and thus induce the differentiation of myeloid leukemia cells,26 and ViB (6) was found to inhibit interstitial leukocyte migration-related inflammation which led to the discovery of its binding to HSP90β. In the present study, we successfully 9062

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18-Fluoro Vibsanin B (7).13 To a stirred solution of ViB (50 mg) in DCM (10 mL) at −78 °C under argon was added DAST (38 μL). The resulting solution was stirred for 2 h, then quenched by addition of NaHCO3 saturated aqueous solution (10 mL). The mixture was warmed to room temperature, and water (15 mL) along wtih ethyl acetate (25 mL) were added. The aqueous layer was extracted with ethyl acetate (25 mL × 2). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude material was purified by chromatography on silica gel (petroleum ether/ethyl acetate 40:1−6:1 v/v) to afford compound 7 (15.1 mg, 30% yield) as a white foam. The NMR data were identical with those reported in the literature.13 18-Chloro Vibsanin B (11). To a solution of ViB (1 g) and trichloroacetonitrile (0.48 mL) in DCM (20 mL) at 0 °C was added PPh3 (1.26 g) in portions. The reaction was allowed to warm to RT, and the mixture was stirred for 30 min. Then 2.5 g of silica gel (80− 100 mesh) was added to the mixture, and the solvent was evaporated under vacuum and the residue was purified by chromatography on silica gel (petroleum ether/ethyl acetate 20:1−10:1 v/v) to afford 11 (940 mg, 90%) as a slightly yellow foam; [α]17D +97 (c 2.0, CH2Cl2). 1 H NMR (400 MHz, CDCl3) δ 6.59 (d, J = 16.4 Hz, 1H), 6.16−6.01 (m, 2H), 5.78 (d, J = 5.6 Hz, 1H), 5.65 (d, J = 16.1 Hz, 1H), 5.40 (d, J = 9.1 Hz, 1H), 5.21 (dd, J = 16.1, 9.2 Hz, 1H), 5.09 (t, J = 7.7 Hz, 1H), 4.46 (d, J = 11.6 Hz, 1H), 4.05 (d, J = 11.2 Hz, 1H), 2.19 (s, 3H), 1.93 (s, 3H), 1.68 (s, 3H), 1.58 (s, 3H), 1.38 (s, 3H), 1.04 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 199.9, 167.3, 159.6, 155.1, 145.2, 139.7, 132.2, 132.0, 128.6, 124.2, 123.7, 115.4, 81.5, 74.4, 53.6, 46.1, 44.8, 40.9, 38.9, 27.7, 25.8, 23.3, 20.6, 18.6, 17.8. HRMS (ESI): [M + Na]+ calcd for C25H35ClO4Na 457.2116, found 457.2118. 18-N,N-Dimethyl Vibsanin B (12a). To a solution of dimethylamine hydrochloride (8 mg) in dry THF (1 mL) was added AcONa (8 mg), the resulting mixture was stirred for 30 min at RT, and then the resulting suspension was added to a solution of 11 (20 mg) and NaI (14 mg) in THF (1 mL) via syringe at 0 °C. The reaction was allowed to warm to RT and stirred for 12 h. The solvent was evaporated under vacuum, and the residue was purified by chromatography on silica gel (petroleum ether/ethyl acetate 1:1−1:2 v/v) to afford compound 12a (9 mg, 45%) as a yellow oil; [α]23D +30 (c 15.0, CH2Cl2). 1H NMR (600 MHz, CDCl3) δ 6.51 (d, J = 16.4 Hz, 1H), 6.07 (d, J = 16.4 Hz, 1H), 6.02 (d, J = 8.6 Hz, 1H), 5.78 (d, J = 6.2 Hz, 1H), 5.68 (d, J = 16.1 Hz, 1H), 5.38 (d, J = 9.1 Hz, 1H), 5.20 (dd, J = 16.2, 9.1 Hz, 1H), 5.11 (t, J = 7.0 Hz, 1H), 5.05 (s, 1H), 3.44 (d, J = 13.5 Hz, 1H), 2.37 (s, 6H), 2.20 (s, 3H), 1.94 (s, 3H), 1.68 (s, 3H), 1.59 (s, 3H), 1.38 (s, 3H), 1.03 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 201.8, 167.3, 159.6, 154.0, 145.7, 131.9, 131.1, 129.0, 128.7, 124.4, 123.6, 115.4, 81.8, 74.3, 63.1, 45.3, 44.9, 40.8, 39.0, 29.8, 27.8, 25.8, 23.3, 23.0, 20.6, 18.6, 17.9. HRMS (ESI): [M + C3H5]+ calcd for C30H46NO4 484.3421, found 484.3423. General Procedure for Synthesis of 18-N,N-Dialkyl Vibsanin B (12b−12f). To a solution of compound 11 (20 mg) and NaI (14 mg) in dry THF (2 mL) at 0 °C was added corresponding amine (0.092 mmol), and the resulting mixture was allowed to warm to RT and stirred for 8−16 h. The solvent was evaporated under vacuum, and the residue was purified by chromatography on silica gel. 18-N,N-Diethyl Vibsanin B (12b). Yield 56%; yellow oil; [α]23D +39 (c 8.0, CH2Cl2). 1H NMR (600 MHz, CDCl3) δ 6.42 (d, J = 16.4 Hz, 1H), 6.04 (d, J = 16.4 Hz, 1H), 5.83 (dd, J = 12.8, 3.6 Hz, 1H), 5.79 (s, 2H), 5.64 (d, J = 16.2 Hz, 1H), 5.61 (s, 1H), 5.34 (d, J = 9.1 Hz, 1H), 5.19 (dd, J = 16.2, 9.1 Hz, 1H), 5.12 (t, J = 6.9 Hz, 1H), 3.46 (d, J = 14.3 Hz, 1H), 2.92 (d, J = 14.7 Hz, 1H), 2.62 (dq, J = 14.3, 7.2 Hz, 3H), 2.44 (td, J = 13.8, 6.9 Hz, 4H), 2.20 (s, 3H), 2.03 (d, J = 14.5 Hz, 1H), 1.94 (s, 3H), 1.69 (s, 3H), 1.59 (s, 3H), 1.39 (s, 6H), 1.05−0.98 (m, 12H). 13C NMR (150 MHz, CDCl3) δ 203.0, 167.3, 159.3, 153.8, 145.9, 142.3, 131.9, 129.0, 128.4, 124.4, 123.3, 115.5, 81.8, 74.3, 63.3, 57.7, 53.0, 46.9, 44.8, 40.6, 39.0, 27.7, 23.2, 23.0, 20.6, 18.6, 17.8, 11.9, 8.2. HRMS (ESI): [M + H]+ calcd for C29H46NO4 472.3421, found 472.3412. 18-N,N-Diisopropyl Vibsanin B (12c). Yield 32%; yellow oil; [α]17D +4 (c 2.0, CH2Cl2). 1H NMR (600 MHz, CDCl3) δ 6.41 (d, J = 16.3 Hz, 1H), 6.05 (t, J = 13.6 Hz, 1H), 5.86 (t, J = 15.1 Hz, 1H), 5.80 (dd,

J = 10.0, 8.8 Hz, 2H), 5.61 (d, J = 16.0 Hz, 1H), 5.37−5.29 (m, 1H), 5.21−5.15 (m, 1H), 5.15−5.10 (m, 1H), 3.43 (t, J = 29.5 Hz, 3H), 3.14−2.98 (m, overlapped, 6H), 2.20 (s, 3H), 1.94 (s, 6H), 1.69 (s, 3H), 1.38 (s, 3H), 1.02 (s, 3H), 1.01 (s, 3H), 1.00 (s, 6H), 0.99 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 203.8, 167.5, 159.6, 153.8, 146.1, 143.9, 131.9, 129.1, 126.9, 124.5, 123.0, 115.4, 82.2, 74.4, 49.4, 49.1, 47.8 (×2), 44.6, 40.6, 39.0, 27.8, 25.8, 23.3, 23.0, 21.9, 20.6, 20.5, 19.7, 18.7, 17.8. HRMS (ESI): [M + H]+ calcd for C31H50NO4 500.3734, found 500.3746. 18-Pyrrolidinyl Vibsanin B (12d). Yield 23%; yellow oil; [α]17D +13 (c 1.0, CH2Cl2). 1H NMR (600 MHz, CDCl3) δ 6.46 (d, J = 16.4 Hz, 1H), 6.03 (d, J = 16.4 Hz, 1H), 5.88 (m, 1H), 5.78 (s, 1H), 5.65 (d, J = 16.2 Hz, 1H), 5.34 (d, J = 9.1 Hz, 1H), 5.17 (dd, J = 16.2, 9.1 Hz, 2H), 5.09 (t, J = 7.0 Hz, 1H), 3.44 (d, J = 13.9 Hz, 1H), 3.09 (d, J = 13.9 Hz, 1H), 2.54 (m, 4H), 2.19 (s, 3H), 2.04 (s, 3H), 1.67 (s, 3H), 1.57 (s, 3H), 1.36 (s, 3H), 1.25 (t, J = 7.1 Hz, 4H), 1.01 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 202.6, 167.4, 159.6, 153.8, 145.9, 131.9, 131.1, 128.9, 128.8, 124.4, 123.3, 115.4, 82.0, 74.4, 60.0, 54.6, 44.9, 40.7, 39.1, 27.8, 25.8, 23.7, 23.3, 23.0, 20.6, 18.6, 17.8. HRMS (ESI): [M + H]+ calcd for C29H44NO4 470.3265, found 470.3268. 18-Morpholinyl Vibsanin B (12e). Yield 91%; yellow oil; [α]17D +106 (c 1.0, CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 6.37 (d, J = 16.4 Hz, 1H), 6.04 (d, J = 16.4 Hz, 1H), 5.82 (dd, J = 12.6, 3.3 Hz, 1H), 5.78 (s, 1H), 5.60 (d, J = 16.0 Hz, 2H), 5.31 (d, J = 9.1 Hz, 1H), 5.18 (dd, J = 16.1, 9.1 Hz, 1H), 5.10 (t, J = 6.7 Hz, 1H), 3.69 (brs, 4H), 3.33 (d, J = 13.7 Hz, 1H), 2.93 (d, J = 13.9 Hz, 1H), 2.49 (brd, J = 25.7 Hz, 4H), 2.20 (s, 3H), 1.94 (s, 3H), 1.68 (s, 3H), 1.59 (s, 3H), 1.37 (s, 3H), 1.02 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 202.3, 167.6, 159.8, 153.9, 145.8, 140.6, 140.3, 132.0, 129.1, 124.3, 123.5, 115.4, 82.3, 74.5, 67.2, 67.0, 62.7, 53.9, 44.9, 40.6, 39.1, 29.8, 27.8, 25.8, 23.3, 23.1, 20.7, 18.8, 17.8. HRMS (ESI): [M + H]+ calcd for C29H44NO5 486.3214, found 486.3218. 18-(N-Methylpiperazinyl) Vibsanin B (12f). Yield 50%; yellow oil; [α]20D +33 (c 7.0, CH2Cl2). 1H NMR (800 MHz, CDCl3) δ 6.40 (d, J = 16.4 Hz, 1H), 6.03 (d, J = 16.4 Hz, 1H), 5.82 (dd, J = 12.8, 3.7 Hz, 1H), 5.79 (s, 1H), 5.61 (d, J = 16.1 Hz, 1H), 5.32 (d, J = 9.1 Hz, 1H), 5.19 (dd, J = 16.2, 9.3 Hz, 1H), 5.11 (t, J = 6.9 Hz, 1H), 3.51−3.45 (m, 1H), 3.03 (d, J = 13.6 Hz, 1H), 2.76 (brd, J = 46.9 Hz, 8H), 2.57 (d, J = 13.8 Hz, 1H), 2.51 (s, 3H), 2.20 (s, 3H), 1.94 (s, 3H), 1.69 (s, 3H), 1.59 (s, 3H), 1.37 (s, 3H), 1.03 (s, 3H). 13C NMR (200 MHz, CDCl3) δ 201.9, 167.6, 159.9, 154.1, 145.7, 140.3, 137.3, 132.0, 130.3, 128.6, 124.3, 115.4, 82.5, 74.4, 62.1, 54.6, 51.2, 44.9, 40.6, 39.2, 36.4, 30.5, 27.8, 25.8, 23.3, 23.1, 20.7, 18.7, 17.9, 17.8. HRMS (ESI): [M + H]+ calcd for C30H46N2O4 499.3530, found 499.3541. Biology. Cytotoxicity Assay and IC50 Determination. Cell viability was evaluated by MTT assay. Cells were treated with the indicated compounds at concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in 96well plates. After 48 h, MTT solution was added to each well, for a final concentration of 20%. Cells were then incubated at 37 °C for 4 h, and the absorbance was measured at 570 nm by spectrophotometry. The IC50 values were determined by nonlinear regression analysis using GraphPad Prism software (GraphPad, Inc., San Diego, CA). HSP90-Dependent Luciferase Refolding Assay. The refolding of denatured firefly luciferase evaluated using the rabbit reticulocyte lysate system, as it contains HSP90 and all the requisite cochaperones and partner proteins necessary to facilitate HSP90 client protein folding, maturation, and activation. Firefly luciferase was prewarmed (41 °C) for 10 min to denature the activity. Compounds (10 μM) were added to a black 96-well plate, and a renaturation mixture containing denatured firefly luciferase and rabbit reticulocyte lysate was dispensed in each well. The renaturation system was incubated at room temperature for 3 h. HSP90-mediated luciferase refolding was determined by the measurement of luciferase activity by adding 40 μL of luciferin substrate solution and then reading the luminescence on a SynergyTMHT multidetection reader. As a control, the inhibition of native luciferase by active compounds, such as 12f, was measured as previously described.27 The IC50 value was calculated as the concentration required to inhibit the recovery of luciferase activity by 50% relative to the DMSO control. The effect of experimental compounds on the activity of native luciferase was also assayed as a 9063

DOI: 10.1021/acs.jmedchem.7b01395 J. Med. Chem. 2017, 60, 9053−9066

Journal of Medicinal Chemistry

Article

control. Curve fitting was performed using GraphPad Prism software program (GraphPad, Inc., San Diego, U.S.)27 Alphascreen-Based Screening of HSP90 C-Terminal Inhibitors. The interaction between the HSP90 C-terminus and the tested compounds was tested by a modified Alphascreen assay system (PerkinElmer, Waltham, MA), which consisted of biotin-labeled HSP90β C-terminus (amino acids 527−724, BPS Bioscience), specific substrate GST-tagged cyclophilin 40 (Cyp40), and reaction buffer.28 Compounds were diluted to concentrations of 1, 3.3, 10, and 100 μM. All the mixed components were incubated at room temperature for 30 min. Glutathione acceptor beads were added to the mixture, which was then incubated for another 30 min. Streptavidin-coated donor beads were added and mixed gently. The system was incubated for 10 min, and the fluorescence of each well was measured in a 2390 EnSpire Alpha Reader with excitation at 680 nm and emission in the range of 520−620 nm. Percent activity = {(Acompound − Abackground)/(Aenzyme − Abackground)} × 100%. Western Blotting. Total cell lysates were prepared by direct lysis in 2× Laemmli buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.004% bromophenol blue). Samples were then fractionated in 12% acrylamide gel and transferred to a PVDF membrane (Bio-Rad). The immunoblot was incubated overnight at 4 °C with blocking solution (5% skim milk) and incubated with anti-HER2/ErbB2, anti-pAkt, anti-CDK4, anti-HSP90, anti-HSP70, and anti-HSP27 antibodies (Cell Signaling Technologies, Boston, MA) for 1 h at room temperature followed by the corresponding peroxidase-conjugated secondary antibodies. Proteins of interest were visualized by chemiluminescent detection. Cell Cycle Analysis. BT474 cells (5 × 105 cells/well in 12-well plates) were incubated with 12f (1.25, 2.5, and 5 μM) for 6 h. The cells were collected and washed twice with PBS and were fixed with 70% ethanol overnight. Fixed cells were washed with PBS and then incubated with 20 μL of RNase A (1 mg/mL) for 30 min and stained with propidium iodide (PI) solution (50 mg/mL final concentration) in the dark for 1 h. Fluorescence intensity was analyzed by a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Statistical Analysis. The results are expressed as the means ± SD. One-Way ANOVA was applied for comparison between two groups, and values of P < 0.05 were considered significant. Statistical analyses were performed using the SAS software package (SAS Institute, Cary, NC). Molecular Modeling. Docking of Vibsanin B Derivatives and ADMET Prediction in Silico. DiscoveryStudio 4.0 software was used for the preparation of the ligand and receptor. Autodock Tools v1.5629 was used to perform grid and docking according to the literature.15a,29 Docking parameters were set as the default values, except “The Number of GA Runs,” which was set to 50 on AutoGrid v4.01 and AutoDock v4.01. Docking conformations were classified into different clusters by binding energy, and the cluster with the lowest binding energy was selected. In the selected cluster, conformations with the lowest binding energy and RMSD (