Insights of a Lead Optimization Study and Biological Evaluation of

Nov 2, 2016 - Insights of a Lead Optimization Study and Biological Evaluation of Novel 4-Hydroxytamoxifen Analogs as Estrogen-Related Receptor γ (ERR...
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Insights of a Lead Optimization Study and Biological Evaluation of Novel 4‑Hydroxytamoxifen Analogs as Estrogen-Related Receptor γ (ERRγ) Inverse Agonists Jina Kim,†,∇ Seo Yeon Woo,†,∇ Chun Young Im,† Eun Kyung Yoo,‡ Seungmi Lee,‡ Hyo-Ji Kim,† Hee-Jong Hwang,† Joong-heui Cho,† Won Seok Lee,† Heeseok Yoon,† Shinae Kim,† Oh-bin Kwon,† Hayoung Hwang,† Kyung-Hee Kim,† Jae-Han Jeon,‡,§ Thoudam Debraj Singh,∥ Sang Wook Kim,⊥ Sung Yeoun Hwang,⊥ Hueng-Sik Choi,# In-Kyu Lee,‡,§ Seong Heon Kim,†,○ Yong Hyun Jeon,*,‡,∥ Jungwook Chin,*,† and Sung Jin Cho*,†,‡ †

New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Korea Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Korea § Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Korea ∥ Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Korea ⊥ Korea Bio-Medical Science Institute, Seoul 06106, Republic of Korea # National Creative Research Initiatives Center for Nuclear Receptor Signals and Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea ‡

S Supporting Information *

ABSTRACT: We evaluated the in vitro pharmacology as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of chemical entities that not only were shown to be highly selective agonists for ERRγ but also exhibited enhanced pharmacokinetic profile compared with 3 (GSK5182). 6g and 10b had comparable potency to 3 and were far more selective for ERRγ over the ERRα, -β, and ERα. The in vivo pharmacokinetic profiles of 6g and 10b were further evaluated, as they possessed superior in vitro ADMET profiles compared to the other compounds. Additionally, we observed a significant increase of fully glycosylated NIS protein, key protein for radioiodine therapy in anaplastic thyroid cancer (ATC), in 6g- or 10btreated CAL62 cells, which indicated that these compounds could be promising enhancers for restoring NIS protein function in ATC cells. Thus, 6g and 10b possess advantageous druglike properties and can be used to potentially treat various ERRγ-related disorders.

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INTRODUCTION

human metabolic diseases including obesity, diabetes, heart failure, and cancer.5 In particular, the ERRγ subtype identified as the last member of the ERR superfamily6 is expressed in various tissues associated with high metabolic needs including the heart, skeletal muscle, and brown adipose tissue;7 thus, they are involved in the transcriptional control of mitochondrial biogenesis and cellular energy.8

The estrogen-related receptors (ERRs; ERRα, -β, and -γ), which are members of the NR3B subfamily of orphan nuclear receptors closely related to the estrogen receptors (ERs; ERα and -β), are constitutively active nuclear receptors that do not bind to endogenous small ligands such as estrogen.1−3 ERRs are primarily expressed in tissues associated with high metabolic demands such as the heart, kidney, and central nervous system, and they are mainly involved in the transcriptional regulation of cellular energy metabolism and homeostasis.4 Therefore, they are regarded as leading therapeutic targets for the treatment of © 2016 American Chemical Society

Received: August 10, 2016 Published: November 2, 2016 10209

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fibrosis in mouse heart.25 The inhibition of endogenous ERRγ expression using 3 attenuated vascular calcification and osteogenic gene expression in vitro and in vivo.26 Intravitreal injection of 3 into the oxygen-induced retinopathy (OIR) model inhibited retinal Vegfa mRNA expression by suppressing hypoxia-induced VEGF expression via ERRγ.27 Compound 3 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice.28 On the basis of the structural motifs of 3, we have successfully demonstrated the synthesis of compound libraries that are more selective against ERRγ inverse agonists with improved absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles.29,30 Moreover, we have broadened the therapeutic scope of these compounds based on our findings that 3 facilitates the responsiveness to radioiodine therapy by modulating sodium iodine symporter (NIS) function in anaplastic thyroid cancer (ATC) cells via ERRγ and MAP kinase signaling pathway.31 On the basis of these encouraging results, we have validated the most promising compound of 3 analog from our previous studies for its ability to enhance NIS protein function, which is a key protein for radioiodine therapy, in ATC cells.30 However, we remain attentive to the development of small ERRγ ligands with much more enhanced pharmacological and ADMET profiles. As part of our continued efforts and achievements, we focused on synthetic strategies in this report for the optimization of B/C-rings of the phenyl group with various substituents to improve the in vitro ADMET profile for subsequent in vivo assessments. Therefore, our objective is to further optimize the new ERRγ inverse agonists that possess a tetrasubstituted scaffold similar to that of 3, with increased in vitro potency and selectivity, including in vitro ADMET and in vivo pharmacokinetic (PK) profiles better than those of currently available agents. From the previous efforts, our focus has shifted to lead optimization efforts by modifying Cring phenyl group with various substituted aryl, heterocyclic, fused cyclic, or bicyclic substituents. We also made minor modifications to the B-ring for adequately improving the in vitro ADMET and in vivo PK profiles for subsequent in vivo efficacy assessments. Herein, we report the chemical synthesis and structure−activity relationship (SAR) results obtained for this campaign, as well as the validation study on NIS function in ATC cells and in vitro ADMET/in vivo PK profiles.

Accumulating evidence suggests that ERRγ is involved in metabolic diseases such as diabetes mellitus type 2, liver injury, alcohol-induced oxidative stress, microbial infections caused by impaired hepatic gluconeogenesis,9,10 damaged hepatic insulin signaling,11 and impaired iron metabolism.12 ERRγ has been shown to promote tamoxifen (TAM, 1) resistance in ERpositive (ER+) ductal and lobular carcinoma cell culture models via extracellular signal-regulated kinase (ERK) mediated activation (phosphorylation).13,14 ERRγ is clinically upregulated in hepatocellular carcinoma (HCC), and its inhibition by either small interfering RNA (siRNA) mediated knockdown or an inverse agonist suppresses liver cancer cell proliferation through G1 arrest.15 Furthermore, ERRγ is thought to play an important role in the metabolic transcriptional pathway depending on the cellular environment of cancer cells.16 It also helps in regulating metabolic reprogramming in primarily hormone-dependent, breast, and prostate cancers by modulating a Warburg-like effect.17 Despite its wide range of biological application, especially for treating certain metabolic disorders18 and cancers,19 only a limited number of studies have investigated ERRγ modulators over the past decades. Consequently, at present, the synthetic compounds for targeting ERRγ with useful pharmacological activity are scarce (Figure 1).

Figure 1. Synthetic selective estrogen receptor modulator (SERM) and estrogen-related receptor γ (ERRγ) ligands: tamoxifen (TAM, 1), a selective estrogen receptor modulator; 4-hydroxytamoxifen (4-OHT, 2), an estrogen receptor α (ERα) antagonist/ERRγ inverse agonist; 3 (GSK5182), a selective ERRγ inverse agonist.

1 is the first clinically useful selective estrogen receptor modulator (SERM) and effective FDA-approved drug for ER+ breast cancer and has been used for more than 30 years.20 4Hydroxytamoxifen (4-OHT, 2) is the active metabolite of 1. It originally served as an ER antagonist, which is a nonsteroidal agent with potent in vitro antiestrogenic properties and competes with estrogen for binding sites in breast and other tissues.21 It was also identified as a nonselective ERRγ inverse agonist.22 A research group reported acyl hydrazine compounds as more selective ERRγ agonists that exhibited mixed ERRγ/β functional activities, but these compounds have not been able to attain reasonable selectivity against the subgroups.23 However, their substantial efforts rendered a more selective inverse agonist for ERRγ, 3 (GSK5182), which is a 2 analog that showed better selectivity for ERRγ than the structurally related nuclear ERα.24 The selective ERRγ inverse agonist 3 mitigates diabetes by inhibiting hepatic gluconeogenesis through peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α).9,10 In addition, it exhibits antimicrobial effects by reducing the expression of ERRγ-mediated hepcidin mRNA.12 Compound 3 completely blocked cardiac hypertrophy in cardiomyocytes and prevented aortic banding-induced cardiac hypertrophy and

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RESULTS AND DISCUSSION Chemistry. A short and efficient synthesis of compounds 6a−m and 7a is described in Scheme 1. Compounds 5a−m were derived using Mitsunobu reaction between 4 and the corresponding alcohol. Phenols 6a−m were derived by the simultaneous reduction of the methyl ester group and the deprotection of pivaloyl (Piv) group by LiAlH4.24 The HCl salts of 7a were formed by treatment of 6a with 1 M aqueous HCl mixed with dichloromethane (CH2Cl2, DCM)/methanol (MeOH) solution. Compounds 10a−d were synthesized as shown in Scheme 2. Mitsunobu reaction of compound 8 with various commercially available alcohols yielded compounds 9a−d. Compounds 9a−d were reduced with DIBAL-H and then treated with 1 M HCl to give salts of compounds 10a−d. Palladium-catalyzed three-component reaction was applied to synthesize various tetrasubstituted olefins shown in Scheme 3. The reaction of iodobenzene (A-ring), internal alkynes (B-ring, 10210

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Scheme 1. Synthesis of Compounds 6 and 7a

Reagents and conditions: (a) R-OH, DIAD, PPh3, CH2Cl2, 15−99%; (b) LiAlH4, THF, 0 °C, 3−99%; (c) TFA, CH2Cl2, rt, 17%; (d) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

a

Scheme 2. Synthesis of Compounds 10a−da

Reagents and conditions: (a) R-OH, DIAD, PPh3, CH2Cl2, 27−66%; (b) DIBAL-H, THF, 0 °C, 0.7−32%; (c) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C. a

-R1) with different substituents on the phenyl ring and either commercially available or internally prepared arylborates (Cring, -R2) in DMF/water with cesium carbonate (base) furnished tetrasubstituted olefins shown as 15a−p.32 The treatment of compounds 15e and 15f with trifluoroacetic acid (TFA) released the Boc protecting group. The resulting indoline and indole were alkylated with the corresponding alkyl halides to afford compounds 18 and 21, respectively. The pivalate was cleaved and the methyl ester was reduced in one-pot reaction with LiAlH4, and the resulting compounds were treated with 1 M HCl to provide compounds 19 and 22, respectively, as described in Scheme 4. Various chemical entities bearing different substituents in the B- and C-ring were constructed as shown in Scheme 5. Compounds 15g−i were deprotected by TFA, reduced using LiAlH4 or LiBH4, and then treated with 1 M HCl at 0 °C to yield compounds 23−26, respectively. In the preparation of compound 27, compound 15k was first reduced with DIBAL-

H, and the above-mentioned procedure for the formation of salts was applied. As shown in Scheme 6, compounds 28a−f were formed by the alkylation of compound 15l with various secondary cyclic amines. The treatment of compounds 28a−f with LiAlH4 simultaneously reduced the ester and deprotected the pivaloyl functionalities. The resulting compounds were treated with 1 M HCl solution to obtain their corresponding HCl salts (29a−f), and those compounds containing a Boc group, such as compounds 29e and 29f, were deblocked in acidic media to yield compounds 30 and 31, respectively. In Scheme 7, compound 15m was alkylated in a similar fashion as mentioned in Scheme 6 to yield compounds 32a−e. Treatment with LiAlH4 simultaneously affected the ester and pivaloyl group, and the treatment of 1 M HCl solution produced the corresponding HCl salts. The compounds with Boc functionality were deprotected in this condition to produce compounds 33a−e. 10211

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Scheme 3. Synthesis of Compounds 15 and 16a

Reagents and conditions: (a) PdCl2(PhCN)2, Cs2CO3, DMF/H2O, 45 °C, 4−95%; (b) LiAlH4, THF, 0 °C, 21−60%; (c) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

a

Compounds 15o and -p were subjected to reductive amination using 1-methylpiperazine to generate the corresponding amines, which were then treated with LiAlH4 or LiBH4 to afford primary alcohols. Compounds 41a and -b were produced by the treatment of 1 M HCl in a mixture of MeOH/ DCM at 0 °C (Scheme 9). Structure−Activity Relationship (SAR) Study. A lead optimization campaign of novel 2 (4-OHT) analog was performed to increase its ERRγ inverse agonist potency and

Compounds 34 and 37 were produced by the alkylation of compound 15n using the same protocol as seen in Schemes 6 and 7. The reduction and deprotection of the ethyl and pivaloyl groups, respectively, with LiAlH4 and/or TFA, followed by treatment with 1 M HCl furnished the desired HCl salts of compounds 35 and 39, respectively (Scheme 8). To construct compound 36, compound 35 was treated with LiAlH4 in a harsher condition (60 °C), and the resulting compound was treated with 1 M HCl solution to generate compound 36. 10212

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Scheme 4. Synthesis of Compounds 19 and 22a

a Reagents and conditions: (a) TFA, CH2Cl2, rt, quant; (b) alkyl halide, K2CO3, NaI, DMF, rt, 11−31%; (c) LiAlH4, THF, 0 °C, 18−55%; (d) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

Scheme 5. Synthesis of Compounds 23−27a

Reagents and conditions: (a) TFA, CH2Cl2, rt; (b) LiAlH4 or LiBH4, THF, 0 °C, 2−38% for 2 steps; (c) DIBAL-H, THF, 0 °C, 44%; (d) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

a

The results of previous molecular docking analyses suggested that the developed inverse agonists bound to the active site via hydrogen bonding with Asp 273, Glu 275, and Asn 346 residues of ERRγ.29 Therefore, the focus of this study was to examine the SAR of new compounds with a tetrasubstituted backbone similar to that of 3 with increased in vitro potency and selectivity. First, we explored the role of the C-ring of compound 3 by

selectivity. We report the rational design, synthesis, and development of novel ERRγ inverse agonists exhibiting druglike profiles comparable to those of compound 2 based on in vitro evaluations and ADMET properties. To assist rational drug design, we investigated well-defined structural modifications designed to enhance the therapeutic window based on molecular modeling and docking studies as well as our fruitful results,29,30 while adhering to accessible synthetic strategies. 10213

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Scheme 6. Synthesis of Compounds 29−31a

Reagents and conditions: (a) R-H (amines), TEA, NaI, DMF, 80 °C, 22−80%; (b) LiAlH4, THF, 0 °C, 31−62%; (c) TFA, CH2Cl2, rt, 44−72%; (d) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

a

Scheme 7. Synthesis of Compound 33a

Reagents and conditions: (a) R-H, TEA, NaI, DMF, 80 °C, 11−99%; (b) LiAlH4, THF, 0 °C, 25−50%; (c) TFA, CH2Cl2, rt, 39−55% in 2 steps; (d) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

a

Scheme 8. Synthesis of Compounds 35, 36 and 39a

Reagents and conditions: (a) R-H, TEA, NaI, DMF, 80 °C, 80−99%; (b) LiAlH4, THF, 0 °C, 28−39%; (c) LiAlH4, THF, 60 °C, 6%; (d) TFA, CH2Cl2, rt, 38%; (e) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C. a

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Scheme 9. Synthesis of Compound 41a

Reagents and conditions: (a) 1-methylpiperazine, NaBH(OAc)3, DCE, 99%; (b) LiAlH4 or LiBH4, THF, 0 °C, 18−73%; (c) 1 M HCl in H2O, MeOH/CH2Cl2 (1:1), 0 °C.

a

Table 1. Structure and ERRγ Binding and Functional Screening of 24 Compounds

a

Binding assays of ERRγ were conducted in duplicates using TR-FRET assay as shown in the Experimental Section. The binding activity in the presence of compounds was represented as the percentage of control binding activity, and the IC50 value was calculated with nonlinear regression fit with four parameters using the Prism 6 software. All IC50 values were statistically evaluated by R2 value of goodness-of-fit, which was over 0.9 (see raw data in molecular formula strings file in Supporting Information for more details). IC50, half-maximal inhibitory concentration. bNormalized luciferase activity of ERRγ at 10 μM of the tested compounds. Data are expressed as percentage activity relative to the absence of compound. Measurements reported are the mean of three determinations. The standard deviation is not shown, as all three assays were found to be reproducible on repeated testing (see raw data including SD in molecular formula strings file for more details). cThe reported ERRγ binding IC50 of 3 is 0.079 μM.24 dND, not determined. 10215

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Table 2. Structure and ERRγ Binding and Functional Screening of 15 Compounds

a

Binding assays of ERRγ were conducted in duplicates using TR-FRET assay as shown in the Experimental Section. The binding activity in the presence of compounds was represented as the percentage of control binding activity, and the IC50 value was calculated with nonlinear regression fit with four parameters using the Prism 6 software. All IC50 values were statistically evaluated by R2 value of goodness-of-fit, which was over 0.9 (see raw data in molecular formula strings file in Supporting Information for more details). IC50, half-maximal inhibitory concentration. bNormalized luciferase activity of ERRγ at 10 μM of the tested compounds. Data are expressed as percentage activity relative to the absence of compound. Measurements reported are the mean of three determinations. The standard deviation is not shown, as all three assays were found to be reproducible on repeated testing (see raw data including SD in molecular formula strings file for more details). cThe reported ERRγ binding IC50 of 3 is 0.079 μM.24 dND, not determined.

incorporating various dimethyl-, cyclic-, bicyclic-, spiro amines at the para position of the phenyl ring as the R-ether linkage.

These initial modifications produced several valuable compounds 6f, -g, 6i−k, 10b, and 29c, which displayed a promising 10216

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Table 3. Structure and ERRγ Binding and Functional Screening of 8 Compounds

a Binding assays of ERRγ were conducted in duplicates using TR-FRET assay as shown in the Experimental Section. The binding activity in the presence of compounds was represented as the percentage of control binding activity, and the IC50 value was calculated with nonlinear regression fit with four parameters using the Prism 6 software. All IC50 values were statistically evaluated by R2 value of goodness-of-fit, which was over 0.9 (see raw data in molecular formula strings file in Supporting Information for more details). IC50, half-maximal inhibitory concentration. bNormalized luciferase activity of ERRγ at 10 μM of the tested compounds. Data are expressed as percentage activity relative to the absence of compound. Measurements reported are the mean of three determinations. The standard deviation is not shown, as all three assays were found to be reproducible on repeated testing (see raw data including SD in molecular formula strings file for more details). cThe reported ERRγ binding IC50 of 3 is 0.079 μM.24 dND, not determined.

binding potency against hERRγ (IC50 ≤ 0.070 μM; Table 1). In addition, these compounds showed an effective inhibition of constitutively active hERRγ activity at the 10 μM functional screening as well. Piperazine-containing compounds, 6b and 7a, did not exhibit sufficient binding potency that met the screening criteria (IC50 < 1 μM), while compound 6c bearing a morpholine moiety exhibited moderate binding potency. On the basis of the comparison of the size of substituted cyclic amines on the Cring (6d−f), it was observed that compounds with six- to threemembered amine (piperidine to aziridine) moiety showed similar activities that were acceptable for the screening purpose. Early SAR studies indicated that the presence of a hydroxyl substituent at the para position (6f, -g, -i, and -l) of the B-ring produced compounds with better activities than those of the corresponding bromo-substituted compounds (10a−d). A comparison of the compounds 6e and 29d revealed that an additional hydroxymethyl group on the pyrrolidine did not

have much effect on the screening campaign. Subsequently, these results also provided informative data concerning the stereospecificity of the substituents on the N-methylpyrrolidine analog. (S)-Isomer (6g) seemed to be slightly more active than the corresponding (R)-isomer (6h) in terms of binding potency. However, in the case of one-carbon extended compounds (6i−k), (S)- and (R)-isomers including the racemic mixture (6i) produced the similar assay results. Longer-chain attached N-methylaziridine compound (6l) and pure isoforms (6m and 10d) were unexpectedly synthesized, which are of no avail. The bicyclic amine compounds (29a, -b and 30; IC50 = 0.079, 0.083, and 0.165, respectively) expressed comparable activities. Additionally, data showed that the presence of a spirocyclic amine system (29c and 31) is not pivotal for the ERRγ inverse agonistic activity. We prepared a series of cyclic amine containing derivatives with different linkers on the C-ring by replacing the oxygen 10217

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Table 4. In Vitro Binding Selectivity of the Screened 20 Compounds

a

Binding assays of ERRγ and other nuclear receptors were conducted in duplicates using TR-FRET assay as shown in the Experimental Section. The binding activity in the presence of compounds was represented as the percentage of control binding activity, and IC50 value was calculated with nonlinear regression fit with four parameters using the Prism 6 software. All IC50 values were statistically evaluated by R2 value of goodness-of-fit, which was over 0.9. (see raw data in molecular formula strings file in Supporting Information for examples). IC50, half-maximal inhibitory concentration.

atom on the ether linkage with other atoms such as nitrogen, carbon, and silicon or applying amide linkage (Table 2). None of these linkers, including directly connected morpholine (16a), amide-linked cyclic amines (24, 35, and 39), siliconeconjugated cyclic piperazines and fused amine (33a−c), piperidine attached as a secondary amine linker (36), and methylene linked methylpiperazine (41a) got through the first screening campaign except for two silicone-conjugated cyclic amine compounds (33d and -e). The previous modeling and experimental results suggested that the para-hydroxyl group on the B-ring of 3 is pivotal for ERRγ binding by hydrogen bonds. To check the importance of the 4-OH, we synthesized several compounds by replacing the B-ring hydroxyl group with bromine or nitrate (26, 27, and 41b). A direct comparison could only be done between -OH compound (41a) and -NO2 compound (41b) because incorporating a bromine or nitrate group in the para position of B-ring seemed to show relatively poor pharmacological activities. Therefore, we did not produce more derivatives

lacking the essential -OH functional group on the B-ring further in this regard. We planned to continue SAR study focused on the hetero and aromatic functionality of the C-ring with seven more compounds, which were fairly inactive overall (Table 3). Notably, one of those compounds, 16c, that comprises a benzofuran connected with ethylpiperidine instead of a plain phenyl ring on the R-group, showed more potent binding and functional activity than other heteroaryl compounds despite the relatively minor conformational changes. Through a funnel-like process, 20 hit compounds in Table 4 were screened for driving additional binding assays of subtype selectivities. Among them, 10 compounds were selected based on the criteria that subtype selectivity for each isoform should be more than 5-fold and ERRγ binding IC50 should be less than that of 3 (0.110 μM). The selected 10 compounds were further confirmed by CYP inhibition assay and metabolic stability testing (see Table S1 in Supporting Information for more details). 10218

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Table 5. In Vitro Binding and Functional Analysis of in Vitro Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Profiles of the Representative 5 Compounds

a Cytochrome P450 (CYP450) enzyme activity was measured by the incubation of CYP450 enzyme isotype-specific substrates and liver microsomes in the presence of the test compound for 15 min. Data are expressed as percentage inhibitory activity relative to the absence of compound. The measurements reported are the mean of duplications. The standard deviation is not shown, as the two assays were found to be reproducible on repeated testing. bMS, microsomal stability. The test compound was incubated with microsomes for 30 min, and the percentage of the remaining compound was measured by LC−MS/MS. The measurements reported are the mean of duplications. The standard deviation is not shown, as the two assays were found to be reproducible on repeated testing. cThe effective permeability after 5 h was calculated as described in Supporting Information. The measurements reported are the mean of four determinations (see raw data including SD in molecular formular strings file in Supporting Information for more details). dhERG channel binding assay was performed using predictor hERG fluorescence polarization assay. IC50 value was calculated with nonlinear regression fit with four parameters. The measurements reported are the mean of duplications. The standard deviation is not shown, as the two assays were found to be reproducible on repeated testing.

Figure 2. Upregulation of NIS protein in CAL62 cells by 6g. (A) CAL62 cells were treated with 6g for 24 h, and immunoblotting was done with ERRγ, phosphorylated-p44/42, and NIS specific antibody. (B−D) Quantitative analysis of ERRγ, phosphorylated-ERK1/2, and NIS protein levels using ImageJ software. Data are the mean ± SD of three samples per group expressed in arbitrary units: (∗) P < 0.05, when compared with untreated cells.

The properties of compounds that exhibited promising activities in in vitro screening tests were subsequently determined using a binding selectivity test, cytochrome P450

(CYP) screening, liver microsomal metabolic stability test, and parallel artificial membrane permeability assay (PAMPA) (Table 5). The binding selectivity profile of 6g and 10b to 10219

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Figure 3. Upregulation of NIS protein in CAL62 cells by 10b. (A) Immunoblotting was performed with ERRγ, phosphorylated-p44/42, and NIS specific antibody following collection of total cell lysates of 10b-treated CAL62 cells. (B−D) Quantitative analysis of ERRγ, phosphorylated-ERK1/2, and NIS protein levels using ImageJ software. Data are the mean ± SD of three samples per group expressed in arbitrary units: (∗) P < 0.05, when compared with untreated cells. NS = not significant.

good PK properties in rats, compound 10b was selected for further in vivo animal studies. Increase of NIS Protein in ATC Cells by 6g or 10b. It has been documented that ATC cells exhibit the absence or reduced expression of fully glycosylated NIS protein leading to radioiodine treatment resistance.33 Previously, our reports have shown that inverse agonists of ERRγ are able to restore the ability of radioiodine uptake in ATC cells by an increase of NIS protein that results from the downregulation of endogenous ERRγ protein and activation of MAP kinase.31,30 Thus, we examined the effects of 6g and 10b on NIS, ERRγ, and phosphorylated ERK1/2 levels in ATC cells, CAL62 cells. 6g and 10b induced significant inhibition of endogenous ERRγ protein levels in CAL62 cells in a dose-dependent manner (Figure 2A,B and Figure 3A,B). The downregulation of ERRγ protein levels was higher in 6g-treated CAL62 cells than 10btreated cells. A marked increase of phosphorylated MAP kinases such as p44 and p42 extracellular signal-regulated kinase (ERK) was determined in 6g- and 10b-treated CAL62 cells (Figure 2A,C and Figure 3A,C). Importantly, we can find a significant increase of fully glycosylated NIS protein in 6g- and 10b-treated CAL62 cells (Figure 2A,D and Figure 3A,D). 6g effectively induced the increase of fully glycosylated NIS protein in a dose-dependent manner, showing 1.5- and 2.5-fold increase at concentrations of 10 and 20 μM, respectively. However, the upregulation of NIS protein in 10b-treated CAL62 cells was only seen at a concentration of 10 μM. The mechanism underlying the failure of NIS protein upregulation in CAL62 cells at a concentration of 20 μM 10b could not be addressed. However, we suggest that it might be due to the

ERRγ was comparable to that of 3 with no observable affinity for ERRα, ERRβ, and ERα at the maximum concentration tested (10 μM). 10 μM 10b exhibited 55% moderate inhibition only for CYP3A4 but did not measurably inhibit the other CYP subtypes, while the control compound 3 inhibited the CYP enzymes by 15−27%. We also used PAMPA to compare the permeability of the newly synthesized compounds and compound 3; compounds 6g and 10b had a much greater permeability (2.11 × 10−6 and 0.93 × 10−6 cm/s, respectively) than the other compounds tested in Table 5. Compound 10b inverse agonists showed improved metabolic stability in both human and rat liver microsomes, while compounds 3 and 6g showed relatively poor metabolic stability in rat studies. In addition to the binding assay, assessing the functional activity is a key step in the screening and selection of potent therapeutic candidates for subsequent in vivo testing. Compound 6g exhibited the best functional IC50 activity, 0.014 μM. However, the result with 10b (IC50 = 0.268 μM) fell short of our expectation, considering its great binding potency. After selecting the two compounds 6g and 10b based on careful consideration of all the profiles mentioned in Table 5, we investigated the in vivo PK properties, and the results were compared with those of the reference compound 3 (see Table S2 in Supporting Information for more details). Although compound 10b showed a slightly higher clearance (CL = 74.4 ± 7.7 mL min−1 kg−1) than 3 (CL = 45.6 ± 9.7 mL min−1 kg−1) and a moderate volume of distribution (Vss =16.1 ± 4.9 L/kg) in the rat PK study, it showed appropriate PK profiles in general, which includes extended half-life (t1/2 = 3.4 ± 0.5 h) and much enhanced bioavailability (F = 38.2%). Owing to its 10220

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Figure 4. Effects of 6g on radioiodine uptake in CAL62 cells. (A) Increase of radioiodine uptake (125I) in 6g-treated CAL-62 cells in a dosedependent manner. CAL62 cell was treated with various doses of 6g for 24 h, followed by 125I uptake assay. (B) Inhibition of 125I uptake in 6gtreated CAL62 cells after treatment of 300 μM of KClO4. (∗) P < 0.05, when compared with 6g-treated cells. NS = not significant.

Figure 5. Enhanced killing effects of 131I by 6g against CAL62 cells. (A) CAL62 cells were pre-exposed with or without 6g prior to incubation with 131 I. One day later cells were washed and further co-incubated with 50 μCi 131I for 6 h. Shown are representative photographic images of plates after clonogenic assay. (B) Quantification of percentage of colony numbers. (∗) P < 0.05, when compared with untreated cells. NS = not significant.

concentration of 10 μM, no significant differences were shown between the vehicle-treated and 6g-treated CAL62 cells. To examine whether the enhanced radioiodine uptake is associated with the modulation of NIS function by 6g, inhibition studies were performed on 6g-treated CAL62 cells by the use of KClO4, a specific inhibitor of NIS. From the inhibition assay, it was observed that the treatment of KClO4 resulted in complete inhibition of enhanced radioiodine uptake in 6g-treated CAL62 cells to the basal level (Figure 4B). These results indicate that the enhanced radioiodine uptake is closely related to the improvement of NIS function induced by 6g in CAL62 cells. Increase of 131I-Mediated Cytotoxicity in CAL62 Cells by 6g. The effective accumulation of radioiodine in thyroid cancer is an important factor for successful radioiodine therapy. We presume that the increase of radioiodine uptake by 6g may lead to more enhanced cytotoxic effects of 131I, which is a clinically used therapeutic radioiodine emitting β radionuclide, against CAL62 cells. A clonogenic assay with 131I demonstrated minimal cytotoxic effects in the CAL62 cells treated with either 6g or 131I alone

induction of apoptosis. Recently, we showed that 3 as an ERRγ inverse agonist inhibits the proliferation of human hepatoma cancer cell via induction of cell cycle arrest.15 We also observed that the treatment with 20 μM 10b reduced the proliferation of CAL62 cells, whereas 10 μM 10b did not. Thus, we suggest that apoptosis induced by 20 μM 10b may lead to the turbulence of signaling cascade associated with the effective restoration of NIS protein level. Enhanced Radioiodine Uptake in CAL62 Cells by 6g. Next, we investigated whether an increase in NIS protein level can enhance the radioiodine uptake in CAL62 cells. Since 6g shows effective upregulation of fully glycosylated NIS protein in CAL62 cells, we tried to focus on the effects of 6g on radioiodine uptake in CAL62 cells. Treatment with 6g led to a significant increase of radioiodine uptake in CAL62 cells (Figure 4A). The highest radioiodine uptake in CAL62 cells was observed at a 6g concentration of 20 μM. The relative increase of radioiodine uptake was 2.0-fold in 6g-treated cells compared with the vehicle-treated cells at a concentration of 20 μM. Even though the radioiodine uptake was slightly escalated in 6g-treated CAL62 cells at a 10221

DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227

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(Figure 5). The relative colony-forming ability in the 131I- and 6g-treated cells was 97.5 ± 2% and 93 ± 6%, respectively (Figure 5B). However, the combination of 6g with 131I resulted in the drastic reduction of colony-forming ability to approximately 67.4 ± 9% in CAL62 cells (P < 0.05).

7.05−7.02 (m, 2H), 6.85−6.78 (m, 4H), 6.66−6.63 (m, 2H), 4.19 (t, J = 4.6 Hz, 2H), 3.50−3.37 (m, 12H), 2.94 (s, 3H), 2.53 (t, J = 2.8 Hz, 2H), 1.53 (m, 2H). HRMS (EI+) m/z calcd for C30H36N2O3 472.2726; found 472.2795. (Z)-4-(5-Hydroxy-2-phenyl-1-(4-(2-(piperidin-1-yl)ethoxy)phenyl)pent-1-en-1-yl)phenol Hydrochloride Salt (6c). The title compound was synthesized from 5c (0.11 g, 0.20 mmol), 1 M LiAlH4 in THF (0.3 mL, 0.3 mmol) in THF (10 mL) according to synthetic procedure for 6b (20 mg, 22% yield). 1H NMR (DMSO-d6, 400 MHz) δ 7.17 (m, 2H), 7.10 (m, 3H), 6.97 (d, J = 8.5 Hz, 2H), 6.75 (m, 4H), 6.66 (d, J = 8.8 Hz, 2H), 4.24 (t, J = 4.3 Hz, 2H), 3.93 (m, 2H), 3.74 (m, 2H), 3.24 (t, J = 6.7 Hz, 2H), 3.15 (s, 2H), 2.54 (s, 4H), 2.39 (m, 2H), 1.38 (m, 2H). HRMS (EI+) m/z calcd for C29H33NO4 459.2410; found 459.2479. (Z)-4-(5-Hydroxy-1-(4-(2-morpholinoethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6d). The title compound was synthesized from 5d (0.1 g, 0.18 mmol), 1 M LiAlH4 in THF (0.27 mL, 0.27 mmol) in THF (2 mL) according to synthetic procedure for 6b (13 mg, 15% yield). 1H NMR (CD3OD, 400 MHz) δ 7.18−7.09 (m, 5H), 7.04 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 4.25 (t, J = 4.8 Hz, 2H), 3.58 (m, 2H), 3.50 (t, J = 4.9 Hz, 2H), 3.45 (t, J = 6.8 Hz, 2H), 3.04 (m, 2H), 2.54 (m, 2H), 1.96 (m, 2H), 1.82 (m, 3H), 1.55 (m, 3H). HRMS (EI+) m/z calcd for C29H33NO4 457.2617; found 457.2686. (Z)-4-(5-Hydroxy-2-phenyl-1-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)pent-1-en-1-yl)phenol Hydrochloride Salt (6e). The title compound was synthesized from 5e (45 mg, 0.08 mmol), 1 M LiAlH4 in THF (0.12 mL, 0.12 mmol) in THF (2 mL) according to synthetic procedure for 6b (6 mg, 17% yield). 1H NMR (CD3OD, 400 MHz) δ 7.18−7.09 (m, 5H), 7.04 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 4.21 (t, J = 4.8 Hz, 2H), 3.69 (m, 2H), 3.59 (t, J = 4.9 Hz, 2H), 3.43 (t, J = 6.8 Hz, 2H), 3.18 (m, 2H), 2.54 (m, 2H), 2.18 (m, 2H), 2.04 (m, 2H), 1.56 (m, 2H). HRMS (EI+) m/z calcd for C29H33NO3 433.2460; found 443.2529. (Z)-4-(1-(4-(2-(Aziridin-1-yl)ethoxy)phenyl)-5-hydroxy-2phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6f). The title compound was synthesized from 5f (0.25 g, 0.47 mmol), 1 M LiAlH4 in THF (0.71 mL, 0.71 mmol) in THF (10 mL) according to synthetic procedure for 6b (6 mg, 3% yield). 1H NMR (CD3OD, 400 MHz) δ 7.17−1.08 (m, 5H), 7.04 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.7 Hz, 2H), 4.20 (t, J = 4.5 Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 3.50 (m, 4H), 3.44 (t, J = 6.8 Hz, 2H), 2.53 (m, 2H), 1.55 (m, 2H). (S,Z)-4-(5-Hydroxy-1-(4-((1-methylpyrrolidin-2-yl)methoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6g). The title compound was synthesized from 5g (50 mg, 0.11 mmol), 1 M LiAlH4 in THF (0.17 mL, 0.17 mmol) in THF (2 mL) according to synthetic procedure for 6b (2 mg, 4% yield). 1H NMR (CD3OD, 400 MHz) δ 7.17−7.08 (m, 5H), 7.05 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.7 Hz, 2H), 4.30 (m, 1H), 4.09 (m, 1H), 3.82 (m, 1H), 3.68 (m, 1H), 3.43 (t, J = 6.7 Hz, 2H), 3.21 (m, 1H), 3.02 (s, 3H), 2.54 (m, 2H), 2.35 (m, 1H), 2.20 (m, 1H), 2.02 (m, 2H), 1.56 (m, 2H). 13C NMR (CD3OD, 100 MHz) δ 155.85, 142.73, 139.38, 138.75, 137.31, 134.53, 131.78, 130.10, 129.43, 127.62, 125.69, 114.60, 113.06, 67.89, 65.19, 61.61, 56.98, 40.33, 32.01, 31.66, 26.10, 22.25. HRMS (EI+) m/z calcd for C29H33NO3 443.2460; found 443.2530. (R,Z)-4-(5-Hydroxy-1-(4-((1-methylpyrrolidin-2-yl)methoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6h). The title compound was synthesized from 5h (50 mg, 0.11 mmol), 1 M LiAlH4 in THF (0.17 mL, 0.17 mmol) in THF (2 mL) according to synthetic procedure for 6b (8 mg, 15% yield). 1H NMR (CD3OD, 400 MHz) δ 7.17−7.08 (m, 5H), 7.05 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 4.28 (m,. 1H), 4.09 (m, 1H), 3.83 (m, 1H), 3.69 (m, 1H), 3.43 (t, J = 6.7 Hz, 2H), 3.21 (m, 1H), 3.02 (s, 3H), 2.54 (m, 2H), 2.35 (m, 1H), 2.22 (m, 1H), 1.99 (m, 2H), 1.56 (m, 2H). HRMS (EI+) m/z calcd for C29H33NO3 443.2460; found 443.2531.

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CONCLUSION A structure-guided strategy of a new series of 2 (4-OHT) analog was performed to enhance its ERRγ inverse agonist potency and selectivity. In this study, we report the rational design, synthesis, and development of novel ERRγ inverse agonists exhibiting druglike profiles comparable to those of compound 3, based on the replacement of B/C-ring, in vitro evaluations, and ADMET properties. For this approach, those analogs that possess much more enhanced in vitro ADMET profiles and PK parameters compared to the reference compound 3 were selected. These compounds are subtypeselective inverse agonists of ERRγ. In vitro binding and functional analysis and in vitro ADMET profiling are critical steps in the selection of potent candidates for subsequent in vivo therapeutic testing. Compounds 6g and 10b have promising pharmacological and ADMET profiles to be considered as lead compounds for treating various ERRγrelated disorders. With this notion, further studies toward radioiodine therapy have been conducted. 6g and 10b increased NIS protein expression in ATC cells, and we found that 6g increased 131I-mediated cytotoxicity in CAL62 cells in clonogenic assay, while 10b produced an advanced PK profile. Additional studies analyzing compounds 6g and 10b in relevant animal disease models targeting ERRγ are currently underway.

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EXPERIMENTAL SECTION

General. All NMR experiments were carried out using an Avance III 400 MHz NMR spectrometer equipped with a 5 mm broadbandobserved probe head (Bruker, Billerica, MA, USA). The NMR spectrum optimization was conducted using the Bruker Topspin 3.1 software, and all parameters were set in it. The compounds were dissolved in CDCl3 or CD3OD, and the spectra were acquired at 25 °C. Mass spectra were measured in positive electrospray ionization (ESI) mode on LCMS-2020 system (Shimadzu, Tokyo, Japan). Column chromatography was performed using a CombiFlash Rf system with RediSep Rf (Teledyne Isco, Lincoln, NE, USA). For the final compounds, further purification was performed by preparative HPLC on Kinetex 5 μm biphenyl, 100 Å (GX-281 HPLC system, Gilson, Middleton, WI, USA; column tube 250 mm × 21.2 mm i.d.), with ACN/H2O as eluent. The purity of the target compounds was determined to be >95% by analytical HPLC using dual different wavelength UV detector. Starting materials were obtained from Aldrich (St. Louis, MO, USA) or Alfa Aesar (Ward Hill, MA, USA). Solvents were obtained from Fisher Scientific (Hampton, NH, USA) or Aldrich and were used without further purification unless noted otherwise. Chemistry. (Z)-4-(5-Hydroxy-1-(4-(2-(4-methylpiperazin-1yl)ethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Dihydrochloride Salt (6b). A solution of 5b (0.13 g, 0.22 mmol) was dissolved in THF (10 mL), and to the mixture was added 1 M LiAlH4 in THF (0.33 mL, 0.33 mmol) dropwise at 0 °C. The solution was stirred at rt for 1 h and was quenched by adding water. The resulting solution was extracted with EtOAc, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30). To a solution of this compound in MeOH/CH2Cl2 (1:1) was added a 1 M solution of hydrochloric acid in water at 0 °C. After stirring for 10 min, the volatiles were removed in vacuum to yield the desired compound (37 mg, 36%). 1H NMR (CD3OD, 400 MHz) δ 7.19−7.08 (m, 5H), 10222

DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227

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2H). HRMS (EI+) m/z calcd for C29H34N2O3 458.2569; found 458.2639. (E)-5-(4-(2-(Aziridin-1-yl)ethoxy)phenyl)-5-(4-bromophenyl)4-phenylpent-4-en-1-ol Hydrochloride Salt (10a). The title compound was synthesized from 9a (44 mg, 0.09 mmol), 1 M DIBAL-H in THF (0.26 mL, 0.26 mmol) in THF (2 mL) according to synthetic procedure for 6b (0.3 mg, 0.7% yield). 1H NMR (CD3OD, 400 MHz) δ 7.51 (d, J = 8.4 Hz, 2H), 7.16−7.09 (m, 7H), 6.83 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 4.18 (t, J = 4.7 Hz, 2H), 3.89 (t, J = 5.6 Hz, 2H), 3.49 (m, 4H), 3.41 (t, J = 6.6 Hz, 2H), 2.49 (m, 2H), 1.53 (m, 2H). (S,E)-5-(4-Bromophenyl)-5-(4-((1-methylpyrrolidin-2-yl)methoxy)phenyl)-4-phenylpent-4-en-1-ol Hydrochloride Salt (10b). The title compound was synthesized from 9b (23 mg, 0.04 mmol), 1 M DIBAL-H in THF (0.35 mL, 0.35 mmol) in THF (2 mL) according to synthetic procedure for 6b (7 mg, 32% yield). 1H NMR (CD3OD, 400 MHz) δ 7.51 (d, J = 8.4 Hz, 2H), 7.16−7.09 (m, 7H), 6.84 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 4.27 (m, 1H), 4.08 (m, 1H), 3.80 (m, 1H), 3.66 (m, 1H), 3.41 (t, J = 6.5 Hz, 2H), 3.19 (m, 1H), 3.00 (s, 3H), 2.50 (m, 2H), 2.33 (m, 1H), 2.20 (m, 1H), 1.98 (m, 2H), 1.52 (m, 2H). 13C NMR (CD3OD, 100 MHz) δ 155.94, 142.42, 142.03, 140.75, 137.66, 136.26, 132.42, 131.74, 131.70, 131.64, 131.06, 131.02, 129.33, 128.66, 128.54, 127.65, 126.05, 120.27, 113.32, 67.88, 65.28, 61.41, 57.05, 40.40, 31.99, 31.45, 26.15, 22.25. HRMS (EI+) m/z calcd for C29H32BrNO2 505.1616; found 505.1688. (E)-5-(4-Bromophenyl)-5-(4-(2-(1-methylpyrrolidin-2-yl)ethoxy)phenyl)-4-phenylpent-4-en-1-ol Hydrochloride Salt (10c). The title compound was synthesized from 9c (58 mg, 0.11 mmol), 1 M DIBAL-H in THF (0.21 mL, 0.21 mmol) in THF (5 mL) according to synthetic procedure for 6b (15 mg, 28% yield). 1H NMR (CD3OD, 400 MHz) δ 7.52 (d, J = 8.4 Hz, 2H), 7.15 (m, 7H), 6.80 (m, 2H), 6.64 (m, 2H), 4.05 (m, 1H), 3.50 (m, 2H), 3.43 (m, 2H), 3.15 (m, 2H), 2.92 (m, 3H), 2.50 (m, 2H), 2.09 (m, 6H), 1.55 (m, 2H). 13C NMR (CD3OD, 100 MHz) δ 142.55, 142.07, 140.46, 137.74, 135.61, 131.72, 131.63, 131.04, 129.33, 127.64, 126.03, 120.22, 114.77, 113.16, 72.77, 65.80, 61.42, 56.94, 50.66, 41.23, 31.73, 29.73, 28.21, 20.16. HRMS (EI+) m/z calcd for C30H34BrNO2 519.1773; found 519.1845. (S,E)-5-(4-Bromophenyl)-5-(4-(3-(1-methylaziridin-2-yl)propoxy)phenyl)-4-phenylpent-4-en-1-ol Hydrochloride Salt (10d). The title compound was synthesized from 9d (23 mg, 0.04 mmol), 1 M DIBAL-H in THF (0.35 mL, 0.35 mmol) in THF (2 mL) according to synthetic procedure for 6b (1 mg, 6% yield). 1H NMR (CD3OD, 400 MHz) δ 7.52 (d, J = 8.4 Hz, 2H), 7.17−7.12 (m, 7H), 6.84 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 3.61 (m, 1H), 3.41 (m, 4H), 3.23 (m, 1H), 3.02 (m, 1H), 2.85 (s, 3H), 2.49 (m, 2H), 2.02 (m, 2H), 1.77 (m, 1H), 1.65 (m, 2H), 1.53 (m, 2H). HRMS (EI+) m/ z calcd for C29H32BrNO2 505.1616; found 505.1689. (E)-4-(5-Hydroxy-1-(4-morpholinophenyl)-2-phenylpent-1en-1-yl)phenol Hydrochloride Salt (16a). The title compound was synthesized from 15a (59 mg, 0.11 mmol) and 1 M LiAlH4 in THF (0.17 mL, 0.17 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (11 mg, 21% yield). 1H NMR (CD3OD, 400 MHz) δ 7.42 (d, J = 8.7 Hz, 2H), 7.20−7.12 (m, 7H), 7.07 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H), 4.07 (m, 4H), 3.62 (m, 4H), 3.44 (t, J = 6.6 Hz, 2H), 2.56 (m, 2H), 1.57 (m, 2H). HRMS (EI+) m/z calcd for C27H29NO3 415.2147; found 415.2218. (Z)-tert-Butyl 4-(4-(5-Hydroxy-1-(4-hydroxyphenyl)-2-phenylpent-1-en-1-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate Hydrochloride Salt (16b). The title compound was synthesized from 15b (7 mg, 0.01 mmol) and 1 M LiAlH4 in THF (12 μL, 0.01 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (2 mg, 31% yield). 1H NMR (CD3OD, 400 MHz) δ 7.38 (m, 2H), 7.31 (m, 1H), 7.22 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.51 (s, 1H), 6.43 (s, 1H), 4.03 (m, 3H), 3.35 (t, J = 6.9 Hz, 2H), 2.32 (m, 2H), 1.81 (m, 2H), 1.59 (m, 6H), 1.44 (s, 9H). HRMS (EI+) m/z calcd for C30H37N3O4 503.2784; found 503.2855. (Z)-4-(5-Hydroxy-2-phenyl-1-(2-(2-(piperidin-1-yl)ethyl)benzofuran-5-yl)pent-1-en-1-yl)phenol Hydrochloride Salt (16c). The title compound was synthesized from 15c (11 mg, 0.02 mmol) and 1 M LiAlH4 in THF (28 μL, 0.03 mmol) in THF (1 mL)

(Z)-4-(5-Hydroxy-1-(4-(2-(1-methylpyrrolidin-2-yl)ethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6i). The title compound was synthesized from 5i (0.1 g, 0.18 mmol), 1 M LiAlH4 in THF (0.26 mL, 0.26 mmol) in THF (2 mL) according to synthetic procedure for 6b (7 mg, 9% yield). 1H NMR (CD3OD, 400 MHz) δ 7.18−7.09 (m, 5H), 7.04 (d, J = 8.5 Hz, 2H), 6.80 (m, 4H), 6.62 (d, J = 8.6 Hz, 2H), 4.09−3.97 (m, 2H), 3.67 (m, 1H), 3.52 (m, 1H), 3.43 (t, J = 6.7 Hz, 2H), 3.16 (m, 1H), 2.94 (s, 3H), 2.53 (t, J = 7.8 Hz, 2H), 2.38 (m, 2H), 2.19−2.01 (m, 3H), 1.85 (m, 1H), 1.54 (m, 2H). HRMS (EI+) m/z calcd for C30H35NO3 457.2617; found 457.2685. (S,Z)-4-(5-Hydroxy-1-(4-(2-(1-methylpyrrolidin-2-yl)ethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6j). The title compound was synthesized from 5j (40 mg, 0.07 mmol), 1 M LiAlH4 in THF (0.1 mL, 0.1 mmol) in THF (2 mL) according to synthetic procedure for 6b (8 mg, 27% yield). 1H NMR (CD3OD, 400 MHz) δ 7.16−7.07 (m, 5H), 7.02 (d, J = 8.6 Hz, 2H), 6.78 (m, 4H), 6.60 (d, J = 8.8 Hz, 2H), 4.03 (m, 2H), 3.67 (m, 1H), 3.51 (m, 1H), 3.41 (t, J = 6.8 Hz, 2H), 3.15 (m, 1H), 2.92 (s, 3H), 2.51 (m, 2H), 2.38 (m, 1H), 2.08 (m, 4H), 1.84 (m, 1H), 1.55 (m, 2H). HRMS (EI+) m/z calcd for C30H35NO3 457.2617; found 457.2686. (R,Z)-4-(5-Hydroxy-1-(4-(2-(1-methylpyrrolidin-2-yl)ethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6k). The title compound was synthesized from 5k (10 mg, 0.02 mmol), 1 M LiAlH4 in THF (0.1 mL, 0.1 mmol) in THF (2 mL) according to synthetic procedure for 6b (3 mg, 39% yield). 1H NMR (CD3OD, 400 MHz) δ 7.16−7.07 (m, 5H), 7.02 (d, J = 6.6 Hz, 2H), 6.78 (m, 4H), 6.60 (d, J = 8.8 Hz, 2H), 4.01 (m, 2H), 3.67 (m, 1H), 3.50 (m, 1H), 3.41 (t, J = 6.7 Hz, 2H), 3.15 (m, 1H), 2.92 (s, 3H), 2.51 (m, 2H), 2.37 (m, 1H), 2.07 (m, 4H), 1.84 (m, 1H), 1.54 (m, 2H). HRMS (EI+) m/z calcd for C30H35NO3 457.2617; found 457.2685. (S,Z)-4-(5-Hydroxy-1-(4-(3-(1-methylaziridin-2-yl)propoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6l). The title compound was synthesized from 5l (50 mg, 0.11 mmol), 1 M LiAlH4 in THF (0.17 mL, 0.17 mmol) in THF (2 mL) according to synthetic procedure for 6b (2 mg, 4% yield). 1H NMR (CD3OD, 400 MHz) δ 7.18−7.09(m, 5H), 7.05 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 3.63 (m, 1H), 3.49 (m, 2H), 3.43 (t, J = 6.8 Hz, 2H), 3.25 (m, 1H), 3.04 (m, 1H), 2.87 (s, 3H), 2.54 (m, 2H), 2.04 (m, 2H), 2.04 (m, 2H), 1.79 (m, 1H), 1.66 (m, 1H), 1.55 (m, 2H). HRMS (EI+) m/z calcd for C29H33NO3 443.2460; found 443.2531. (R,Z)-4-(5-Hydroxy-1-(4-(3-(1-methylaziridin-2-yl)propoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (6m). The title compound was synthesized from 5m (50 mg, 0.11 mmol), 1 M LiAlH4 in THF (0.17 mL, 0.17 mmol) in THF (2 mL) according to synthetic procedure for 6b (8 mg, 16% yield). 1H NMR (CD3OD, 400 MHz) δ 7.17−7.09 (m, 5H), 7.04 (d, J = 2H), 6.84 (m, 2H), 6.78 (m, 2H), 6.71 (m, 2H), 3.62 (m, 1H), 3.43 (m, 4H), 3.26 (m, 1H), 3.04 (m, 1H), 2.87 (s, 3H), 2.53 (m, 2H), 2.05 (m, 2H), 1.77 (m, 1H), 1.69 (m, 1H), 1.54 (m, 2H). HRMS (EI+) m/z calcd for C29H33NO3 443.2460; found 443.2530. (Z)-4-(5-Hydroxy-2-phenyl-1-(4-(2-(piperazin-1-yl)ethoxy)phenyl)pent-1-en-1-yl)phenol Dihydrochloride Salt (7a). A solution of 6a (28 mg, 0.05 mmol) was dissolved in CH2Cl2 (5 mL), and to the mixture was added TFA (77 μL, 1.00 mmol) dropwise at 0 °C. The solution was stirred at rt for 1 h and was quenched by adding water. The resulting solution was extracted with EtOAc, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30). To a solution of this compound in MeOH/CH2Cl2 (1:1) was added a 1 M solution of hydrochloric acid in water at 0 °C. After stirring for 10 min, the volatiles were removed in vacuum to yield the desired compound (4 mg, 17%). 1H NMR (CD3OD, 400 MHz) δ 7.16−7.07 (m, 5H), 7.01 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.6 Hz, 2H), 4.31 (t, J = 4.2 Hz, 2H), 3.66 (m, 10H), 3.41 (t, J = 6.8 Hz, 2H), 2.51 (m, 2H), 1.54 (m, 10223

DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227

Journal of Medicinal Chemistry

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according to synthetic procedure for 16a−d (5 mg, 60% yield). 1H NMR (CD3OD, 400 MHz) δ 7.93 (s, 1H), 7.05−7.12 (m, 9H), 6.85 (d, J = 10.0 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.47(s, 1H), 3.61 (m, 2H), 3.48 (m, 4H), 3.25 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 10.4 Hz, 2H), 2.59 (m, 2H), 1.99 (m, 2H), 1.77−1.87 (m, 3H), 1.51−1.61 (m, 3H). HRMS (EI+) m/z calcd for C32H35NO3 481.2617; found 481.2688. (E)-4-(1-(4-(1-(2-(Dimethylamino)ethyl)-1H-1,2,3-triazol-4yl)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (16d). The title compound was synthesized from 15d (11 mg, 0.02 mmol) and 1 M LiAlH4 in THF (28 μL, 0.03 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (5 mg, 60% yield). 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.34 (d, J = 8.2 Hz, 2H), 6.99 (m, 5H), 6.93 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.2 Hz, 2H), 6.65 (d, J = 8.5 Hz, 2H), 4.78 (m, 2H), 3.66 (t, J = 5.8 Hz, 2H), 3.29 (t, J = 6.7 Hz, 2H), 2.84 (s, 6H), 2.42 (m, 2H), 1.43 (m, 2H). HRMS (EI+) m/z calcd for C29H32N4O2 468.2525; found 468.2597. (Z)-4-(5-hydroxy-2-phenyl-1-(1-(2-(pyrrolidin-1-yl)ethyl)indolin-5-yl)pent-1-en-1-yl)phenol Dihydrochloride Salt (19). The title compound was synthesized from 18 (3 mg, 4.7 μmol) and 1 M LiAlH4 in THF (14 μL, 0.01 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (1 mg, 55% yield). 1H NMR (CD3OD, 400 MHz) δ 7.19−6.97 (m, 7H), 6.77 (d, J = 8.5 Hz, 2H), 6.64 (m, 2H), 6.41 (d, J = 8.7 Hz, 2H), 3.67 (m, 2H), 3.41 (m, 2H), 3.51 (m, 2H), 2.06 (m, 6H), 1.55 (m, 2H), 0.89 (m, 4H). HRMS (EI+) m/z calcd for C31H36N2O2 468.2777; found 468.2751. (Z)-4-(5-Hydroxy-2-phenyl-1-(1-(2-(piperidin-1-yl)ethyl)-1Hindol-5-yl)pent-1-en-1-yl)phenol Dihydrochloride Salt (22). The title compound was synthesized from 21 (9 mg, 0.02 mmol) and 1 M LiAlH4 in THF (46 μL, 0.05 mmol) in THF (1 mL) according to synthetic procedure for 19 (2 mg, 18%). 1H NMR (CD3OD, 400 MHz) δ 7.30−6.95 (m, 14H), 4.41 (m, 2H), 3.61 (m, 4H), 3.44 (m, 2H), 3.11 (m, 2H), 2.56 (m, 2H), 1.93 (m, 6H), 1.57 (m, 2H). HRMS (EI+) m/z calcd for C32H36N2O2 480.2777; found 480.2852. (Z)-4-(5-Hydroxy-2-phenyl-1-(2-(2-(piperazin-1-yl)ethyl)benzofuran-5-yl)pent-1-en-1-yl)phenol Dihydrochloride Salt (23). The title compound was synthesized from 15g (20 mg, 0.03 mmol), TFA (35 μL, 0.46 mmol), and 1 M LiAlH4 in THF (29 μL, 0.03 mmol) in CH2Cl2 (1 mL), THF (1 mL) according to synthetic procedure for 23−26 (5 mg, 38%). 1H NMR (CD3OD, 400 MHz) δ 7.02−6.93 (m, 9H), 6.72 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H), 6.37 (s, 1H), 3.45 (m, 10H), 3.33 (t, J = 6.8 Hz, 2H), 3.15 (m, 2H), 2.45 (m, 2H), 1.46 (m, 2H). HRMS (EI+) m/z calcd for C31H34N2O3 482.2569; found 482.2639. (E)-N-(4-(5-Hydroxy-1-(4-hydroxyphenyl)-2-phenylpent-1en-1-yl)phenyl)pyrrolidine-2-carboxamide Dihydrochloride Salt (24). The title compound was synthesized from 15h (80 mg, 0.12 mmol), TFA (43 μL, 0.55 mmol), and 1 M LiAlH4 in THF (0.18 mL, 0.18 mmol) in CH2Cl2 (1 mL), THF (1 mL) according to synthetic procedure for 23−26 (6 mg, 35%). 1H NMR (CD3OD, 400 MHz) δ 7.25 (d, J = 8.7 Hz, 2H), 7.17−7.12 (m, 5H), 7.05 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 4.31 (m, 1H), 3.44 (m, 4H), 2.55 (m, 2H), 2.47 (m, 1H), 2.09 (m, 3H), 1.56 (m, 2H). HRMS (EI+) m/z calcd for C28H30N2O3 442.2256; found 442.2328. (Z)-4-Phenyl-5-(2-(2-(piperazin-1-yl)ethyl)benzofuran-5-yl)5-(p-tolyl)pent-4-en-1-ol Dihydrochloride Salt (25). The title compound was synthesized from 15i (14 mg, 0.02 mmol), TFA (28 μL, 0.37 mmol), and 1 M LiAlH4 in THF (23 μL, 0.02 mmol) in CH2Cl2 (1 mL), THF (1 mL) according to synthetic procedure for 23−26 (3 mg, 28%). 1H NMR (CD3OD, 400 MHz) δ 7.20−7.06 (m, 11H), 6.84 (d, J = 8.5 Hz, 1H), 6.49 (s, 1H), 3.54 (m, 10H), 3.43 (t, J = 6.8 Hz, 2H), 3.27 (t, J = 7.7 Hz, 2H), 2.55 (m, 2H), 2.36 (s, 3H), 1.58 (m, 2H). HRMS (EI+) m/z calcd for C32H36N2O2 480.2777; found 480.2848. (E)-5-(4-(2,7-Diazaspiro[4.4]nonan-2-yl)phenyl)-5-(4-nitrophenyl)-4-phenylpent-4-en-1-ol Dihydrochloride Salt (26). The title compound was synthesized from 15j (4 mg, 5.72 μmol), TFA (8 μL, 0.10 mmol), and 2 M LiBH4 in THF (11 μL, 0.02 mmol) in CH2Cl2 (1 mL), THF (1 mL) according to synthetic procedure for 23−26 (0.4 mg, 2%). 1H NMR (CD3OD, 400 MHz) δ 8.26 (d, J = 8.6

Hz, 2H) 7.47 (d, J = 8.6 Hz, 2H), 7.21−7.09 (m, 5H), 6.64 (d, J = 8.4 Hz, 2H), 6.30 (d, J = 8.7 Hz, 2H), 3.48 (m, 2H), 3.25 (m, 2H), 3.15 (m, 2H), 2.51 (m, 2H), 2.12 (m, 4H), 1.59 (m, 4H), 1.15 (m, 2H). HRMS (EI+) m/z calcd for C30H33N3O3 483.2522; found 483.2593. (E)-5-(4-Bromophenyl)-4-phenyl-5-(4-(piperidin-1-ylmethyl)phenyl)pent-4-en-1-ol Hydrochloride Salt (27). A solution of 15k (29 mg, 0.06 mmol) was dissolved in THF (1 mL), and to the mixture was added 1 M DIBAL-H in THF (0.3 mL, 0.30 mmol) dropwise at 0 °C. The solution was stirred at rt for 1 h and was quenched by adding water. The resulting solution was extracted with EtOAc, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30). To a solution of this compound in MeOH/CH2Cl2 (1:1) was added a 1 M solution of hydrochloric acid in water at 0 °C. After stirring for 10 min, the volatiles were removed in vacuum to yield the desired compound (12 mg, 44%). 1H NMR (CD3OD, 400 MHz) δ 7.52 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.17−7.10 (m, 5H), 7.03 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 8.1 Hz, 2H), 3.54 (s, 2H), 3.42 (t, J = 6.6 Hz, 2H), 2.52 (m, 6H), 1.62−1.48 (m, 8H). HRMS (EI+) m/z calcd for C29H32BrNO 489.1667; found 489.1739. (Z)-4-(1-(4-(2-(3-Azabicyclo[3.1.0]hexan-3-yl)ethoxy)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (29a). The title compound was synthesized from 28a (6 mg, 0.01 mmol) and 1 M LiAlH4 in THF (30 μL, 0.03 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (3 mg, 62% yield). 1H NMR (CD3OD, 400 MHz) δ 7.16−7.07 (m, 5H), 7.02 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 6.65 (m, 2H), 4.17 (t, J = 4.4 Hz, 2H), 3.70 (m, 2H), 3.57 (t, J = 4.6 Hz, 2H), 3.46 (m, 2H), 3.41 (t, J = 6.7 Hz, 2H), 2.52 (m, 2H), 1.86 (m, 2H), 1.54 (m, 2H), 0.84 (m, 1H), 0.63 (m, 1H). HRMS (EI+) m/z calcd for C30H33NO3 455.2460; found 455.2531. 4-((Z)-5-Hydroxy-1-(4-(2-((1R,2S,5S)-2-(hydroxymethyl)-3azabicyclo[3.1.0]hexan-3-yl)ethoxy)phenyl)-2-phenylpent-1en-1-yl)phenol Hydrochloride Salt (29b). The title compound was synthesized from 28b (5 mg, 0.01 mmol) and 1 M LiAlH4 in THF (13 μL, 0.01 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (2 mg, 57% yield). 1H NMR (CD3OD, 400 MHz) δ 7.93 (s, 1H), 7.13−7.22 (m, 5H), 7.04 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 4.23 (m, 1H), 4.08−4.16 (m, 3H), 3.93 (m, 1H), 3.81 (m, 1H), 3.63 (m, 1H), 3.44 (t, J = 6.8 Hz, 3H), 2.55 (m, 2H), 1.92 (m, 1H), 1.79 (m, 1H), 1.59 (m, 2H), 0.93 (m, 2H). HRMS (EI+) m/z calcd for C31H35NO4 485.2566; found 485.2639. (Z)-4-(1-(4-(2-(2-Azaspiro[4.4]nonan-2-yl)ethoxy)phenyl)-5hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (29c). The title compound was synthesized from 28c (6 mg, 0.01 mmol) and 1 M LiAlH4 in THF (30 μL, 0.03 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (2 mg, 31% yield). 1H NMR (CD3OD, 400 MHz) δ 7.16−7.07 (m, 5H), 7.02 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.8 Hz, 2H), 4.19 (t, J = 4.7 Hz, 2H), 3.72 (m, 1H), 3.60 (m, 2H), 3.50 (m, 1H), 3.41 (t, J = 6.8 Hz, 2H), 3.15 (m, 1H), 2.52 (m, 2H), 2.06 (m, 1H), 1.92 (m, 1H), 1.69 (m, 9H), 1.54 (m, 2H). HRMS (EI+) m/ z calcd for C33H39NO3 497.2930; found 497.2999. (R,Z)-4-(5-Hydroxy-1-(4-(2-(2-(hydroxymethyl)pyrrolidin-1yl)ethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (29d). The title compound was synthesized from 28d (15 mg, 0.03 mmol) and 1 M LiAlH4 in THF (38 μL, 0.04 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (5 mg, 45% yield). 1H NMR (CD3OD, 400 MHz) δ 7.93 (s, 1H), 7.13−7.15 (m, 5H), 7.05 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 4.28 (t, J = 10.0 Hz, 2H), 3.90 (m, 2H), 3.75 (m, 4H), 3.50 (m, 1H), 3.44 (t, J = 6.8 Hz, 2H), 2.55 (m, 2H), 1.90−2.22 (m, 4H), 1.57 (m, 2H). HRMS (EI+) m/z calcd for C30H35NO4 473.2566; found 473.2639. 4-((Z)-1-(4-(2-((3aR,6aS)-Hexahydropyrrolo[3,4-c]pyrrol2(1H)-yl)ethoxy)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (30). A solution of 29e (12 mg, 0.02 mmol) was dissolved in CH2Cl2 (1 mL), and to the mixture was added TFA (25 μL, 0.33 mmol) dropwise at 0 °C. The solution was stirred at 10224

DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227

Journal of Medicinal Chemistry

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rt for 1 h and was quenched by adding water. The resulting solution was extracted with EtOAc, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30). To a solution of this compound in MeOH/CH2Cl2 (1:1) was added a 1 M solution of hydrochloric acid (1.2−2.2 equiv) in water at 0 °C. After stirring for 10 min, the volatiles were removed in vacuum to yield the desired compound (2 mg, 19% yield). 1H NMR (CD3OD, 400 MHz) δ 7.08− 7.16 (m, 5H), 7.04 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 4.31 (m, 2H), 4.04 (m, 2H), 3.83(m, 1H), 3.68 (m, 3H), 3.36−3.49 (m, 8H), 2.55 (m, 2H), 1.57 (m, 2H). HRMS (EI+) m/z calcd for C31H36N2O3 484.2726; found 484.2796. (Z)-4-(1-(4-(2-(2,7-Diazaspiro[4.4]nonan-2-yl)ethoxy)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (31). The title compound was synthesized from 29f (4 mg, 6.7 μmol) and TFA (25 μL, 0.33 mmol) in CH2Cl2 (1 mL) according to synthetic procedure for 30 (0.8 mg, 24% yield). 1H NMR (CD3OD, 400 MHz) δ 7.18−7.07 (m, 5H), 7.00 (d, J = 7.0 Hz, 2H), 6.83 (d, J = 7.2 Hz, 2H), 6.76 (d, J = 7.0 Hz, 2H), 6.68 (d, J = 7.6 Hz, 2H), 4.23 (s, 2H), 3.81 (m, 2H), 3.66 (m, 2H), 3.41 (m, 8H), 2.51 (m, 2H), 2.21 (m, 4H), 1.54 (m, 2H). HRMS (EI+) m/z calcd for C32H38N2O3 498.2882; found 498.2952. (Z)-4-(1-(4-(Dimethyl(piperazin-1-ylmethyl)silyl)phenyl)-5hydroxy-2-phenylpent-1-en-1-yl)phenol Dihydrochloride Salt (33a). The title compound was synthesized from 32a (12 mg, 0.02 mmol), TFA (21 μL, 0.27 mmol), and 1 M LiAlH4 in THF (50 μL, 0.05 mmol) in CH2Cl2 (1 mL), THF (1 mL) according to synthetic procedure for 23−26 (3 mg, 39% yield). 1H NMR (CD3OD, 400 MHz) δ 7.34 (d, J = 8.0 Hz, 2H), 7.17−7.12 (m, 5H), 7.05 (m, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 3.56 (s, 8H), 3.43 (t, J = 6.7 Hz, 2H), 3.02 (s, 2H), 2.55 (m, 2H), 1.56 (m, 2H), 0.52 (s, 6H). HRMS (EI+) m/z calcd for C30H38N2O2Si 486.2703; found 486.2773. (Z)-4-(1-(4-(Dimethyl((4-methylpiperazin-1-yl)methyl)silyl)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol Dihydrochloride Salt (33b). The title compound was synthesized from 32b (8 mg, 0.01 mmol) and 1 M LiAlH4 in THF (38 μL, 0.04 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (3 mg, 44% yield). 1H NMR (CD3OD, 400 MHz) δ 7.34 (d, J = 7.6 Hz, 2H), 7.20−7.13 (m, 5H), 7.05 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 7.6 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 3.67 (m, 8H), 3.43 (t, J = 6.7 Hz, 2H), 3.01 (m, 5H), 2.55 (m, 2H), 1.56 (m, 2H), 0.52 (s, 6H). HRMS (EI+) m/z calcd for C31H40N2O2Si 500.2859; found 500.2929. 4-((Z)-1-(4-((((3aR,6aS)-Hexahydropyrrolo[3,4-c]pyrrol-2(1H)yl)methyl)dimethylsilyl)phenyl)-5-hydroxy-2-phenylpent-1en-1-yl)phenol Dihydrochloride salt (33c). The title compound was synthesized from 32c (3 mg, 4.1 μmol), TFA (5 μL, 0.07 mmol), and 1 M LiAlH4 in THF (12 μL, 0.01 mmol) in CH2Cl2 (1 mL), THF (1 mL) according to synthetic procedure for 23−26 (1 mg, 55% yield). 1H NMR (CD3OD, 400 MHz) δ 7.30 (m, 2H), 7.13 (m, 5H), 7.01 (m, 4H), 6.77 (m, 2H), 4.26 (t, J = 6.2 Hz, 2H), 3.74 (m, 2H), 3.52 (m, 2H), 3.40 (m, 2H), 3.25 (m, 2H), 3.12 (m, 2H), 2.99 (s, 2H), 2.60 (m, 2H), 1.75 (m, 2H), 1.54 (m, 2H), 0.44 (s, 6H). HRMS (EI+) m/z calcd for C32H40N2O2Si 512.2859; found 512.2930. (Z)-4-(1-(4-(Dimethyl(piperidin-1-ylmethyl)silyl)phenyl)-5hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (33d). The title compound was synthesized from 32d (54 mg, 0.09 mmol) and 1 M LiAlH4 in THF (0.18 mL, 0.18 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (12 mg, 25% yield). 1 H NMR (CD3OD, 400 MHz) δ 7.29 (m, 2H), 7.12 (m, 5H), 7.00 (m, 4H), 6.77 (m, 2H), 3.42 (m, 2H), 3.25 (m, 2H), 2.83 (m, 4H), 2.54 (m, 2H), 1.77 (m, 4H), 1.55 (m, 2H). (Z)-4-(1-(4-(Dimethyl(pyrrolidin-1-ylmethyl)silyl)phenyl)-5hydroxy-2-phenylpent-1-en-1-yl)phenol Hydrochloride Salt (33e). The title compound was synthesized from 32e (21 mg, 0.04 mmol) and 1 M LiAlH4 in THF (0.11 mL, 0.11 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (9 mg, 50% yield). 1 H NMR (CD3OD, 400 MHz) δ 7.31 (m, 2H), 7.15−6.99 (m, 9H), 6.79 (d, J = 8.6 Hz, 2H), 3.44 (m, 4H), 2.93 (s, 2H), 2.83 (m, 2H),

2.56 (m, 2H), 2.06 (m, 2H), 1.93 (m, 2H), 1.57 (m, 2H), 0.44 (s, 6H). HRMS (EI+) m/z calcd for C30H37NO2Si 471.2594; found 471.2664. (E)-N-(4-(5-Hydroxy-1-(4-hydroxyphenyl)-2-phenylpent-1en-1-yl)phenyl)-2-(piperidin-1-yl)acetamide Hydrochloride Salt (35). The title compound was synthesized from 34 (7 mg, 0.01 mmol) and 1 M LiAlH4 in THF (18 μL, 0.02 mmol) in THF (1 mL) according to synthetic procedure for 16a−d (2 mg, 28% yield). 1H NMR (CD3OD, 400 MHz) δ 7.25 (d, J = 7.8 Hz, 2H), 7.17−7.10 (m, 5H), 7.05 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 4.01 (s, 2H), 3.57 (m, 2H), 3.44 (t, J = 6.7 Hz, 2H), 3.05 (m, 2H), 2.55 (m, 2H), 1.90 (m, 6H), 1.56 (m, 2H). (E)-4-(5-Hydroxy-2-phenyl-1-(4-((2-(piperidin-1-yl)ethyl)amino)phenyl)pent-1-en-1-yl)phenol Dihydrochloride Salt (36). A solution of 35 (10 mg, 0.02 mmol) was dissolved in THF (1 mL), and to the mixture was added 1 M LiAlH4 in THF (51 μL, 0.05 mmol) dropwise at 0 °C. The solution was heated at 60 °C for 1 h and was quenched by adding water. The resulting solution was extracted with EtOAc, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30). To a solution of this compound in MeOH/CH2Cl2 (1:1) was added a 1 M solution of hydrochloric acid (1.2 equiv) in water at 0 °C. After stirring for 10 min, the volatiles were removed in vacuum to yield the desired compound (0.5 mg, 6% yield). 1H NMR (CD3OD, 400 MHz) δ 7.15− 7.07 (m, 5H), 7.01 (d, J = 8.4 Hz, 2H), 6.74 (m, 4H), 6.50 (d, J = 8.4 Hz, 2H), 3.61 (m, 2H), 3.48 (m, 2H), 3.40 (m, 6H), 2.51 (m, 2H), 1.81 (m, 4H), 1.55 (m, 4H). HRMS (EI+) m/z calcd for C30H36N2O2 456.2777; found 456.2847. (E)-4-(5-Hydroxy-1-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-2-phenylpent-1-en-1-yl)phenol Dihydrochloride Salt (41a). The title compound was synthesized from 40a (12 mg, 0.02 mmol) and 1 M LiAlH4 in THF (43 μL, 0.04 mmol) in THF (2 mL) according to synthetic procedure for 16a−d (2 mg, 18% yield). 1H NMR (CD3OD, 400 MHz) δ 7.19 (m, 2H), 7.16−7.10 (m, 5H), 7.07 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 7.8 Hz, 2H), 6.80 (d, J = 8.3 Hz, 2H), 4.17 (s, 2H), 3.55 (m, 8H), 3.43 (t, J = 6.7 Hz, 2H), 2.96 (s, 3H), 2.55 (m, 2H), 1.56 (m, 2H). HRMS (EI+) m/z calcd for C29H34N2O2 442.2620; found 442.2690. (E)-5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-5-(4-nitrophenyl)-4-phenylpent-4-en-1-ol Dihydrochloride Salt (41b). The title compound was synthesized from 40b (12 mg, 0.02 mmol) and 2 M LiBH4 in THF (47 μL, 0.09 mmol) in THF (2 mL) according to synthetic procedure for 16a−d (8 mg, 73% yield). 1H NMR (CD3OD, 400 MHz) δ 8.26 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.31 (m, 2H), 7.17 (m, 5H), 7.06 (d, J = 7.2 Hz, 2H), 4.32 (s, 2H), 3.61 (m, 8H), 3.43 (t, J = 6.2 Hz, 2H), 2.98 (s, 3H), 2.53 (m, 2H), 1.56 (m, 2H). HRMS (EI+) m/z calcd for C29H33N3O3 471.2522; found 471.2593. ERRγ Binding Assay. ERRγ binding assay was conducted with Lantascreen assay system (Life Technologies, Grand Island, NY, USA), which was based on TR-FRET, according to the manufacturer’s instructions. Briefly, compounds were serially diluted with 2-fold starting from 10 μM and 10 μL of each diluent was added to a 384well plate. Then, GST-conjugated ERRγ LBD (ligand-binding domain) was added to give 5 nM. Next, the mixture of fluoresceinconjugated coactivator PGC-1α (final concentration to be 500 nM) and Tb-a-GST antibody (final concentration to be 5 nM) was added. The reaction mixture was incubated at rt in dark state for 1 h. TRFRET activity was measured at 340 nm excitation and 495 nm/520 nm dual emission using microplate reader (Biotek, SynergyNeo, Winooski, VT, USA), and IC50 value was calculated with the Prism 6 software.34 ERRα, ERRβ, ERα Binding Assay. ERRs and ERα binding assay used the same assay system as ERRγ’s Lanthascreen assay system. Therefore, the procedure was the same as the ERRγ binding assay except for tested enzymes (ERRα, ERRβ, ERα). Used reference compounds were 4-OHT (2) for ERα and ERRβ binding assay, and XCT790 for ERRα binding assay. 10225

DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227

Journal of Medicinal Chemistry

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ERRγ Functional Assay. AD293 cells cultured in Dulbecco’ modified Eagle medium (DMEM) high glucose were supplemented with 0.5% FBS (Hyclone, Logan, UT, USA). After 24 h, the culture medium was changed with DMEM high glucose containing 10% FBS. DNA constructs, which include pCMX-Gal4-ERRγ, pFR-luciferase reporter plasmid, pCMV-β-gal, were transiently transfected into AD293 cells using TransIT-LT1 transfection reagent (Mirus, Madison, WI, USA) for 24 h. Then, transfected cells were treated with compounds for 24 h and harvested for detection of luciferase activity and β-gal activity. All cells were incubated at 37 °C under 5% CO2 in a humidified incubator.10

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anaplastic thyroid cancer; IC50, half-maximal inhibitory concentration; MS, microsomal stability; PAMPA, parallel artificial membrane permeability assay; SAR, structure−activity relationship

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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.6b01204. ADMET assay methods including metabolic stability assay, CYP inhibition assay, PAMPA, hERG assays, and PK study; biological methods for in vitro validation; chemistry of intermediates; NMR spectra and HPLC chromatograms of the title compounds (PDF) Molecular formula strings and some data (CSV) Molecular formual strings, chemical structures, and some data (XLSX)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Y.H.J.: phone, +82-53-200-3149; e-mail, jeon9014@gmail. com. *J.C.: phone, +82-53-790-5257; fax, +82-53-790-5219; e-mail, [email protected]. *S.J.C.: phone, +82-53-790-5226; fax, +82-53-790-5219; e-mail, [email protected]. Present Address ○

S.H.K.: Boryung R&D Center, Chemistry Research Unit, Gyeonggi-do 15425, Korea.

Author Contributions ∇

J.K. and S.Y.W. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI16C1501). Y.H.J was supported by National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIP) (Grant 2014R1A1A1003323). H.-S.C was supported by National Creative Research Initiatives (Grant 20110018305) through the National Research Foundation of Korea (NRF) funded by the Korean government (Ministry of Science, ICT & Future Planning). S.J.C was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (Grant 2016M3A9D9905123).

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ABBREVIATIONS USED 4-OHT, 4-hydroxytamoxifen; ADMET, absorption, distribution, metabolism, excretion, and toxicity; CYP, cytochrome P450; DCM, dichloromethane; ER, estrogen receptor; ERR, estrogen-related receptor; NIS, sodium iodide symporter; ATC, 10226

DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227

Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.6b01204 J. Med. Chem. 2016, 59, 10209−10227