benzoate as a Novel and Dual Malate ... - ACS Publications

Article Cite This: J. Med. Chem. 2017, 60, 8631-8646

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Methyl 3‑(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)propanamido)benzoate as a Novel and Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor Targeting Cancer Metabolism Ravi Naik,† Hyun Seung Ban,‡,§ Kyusic Jang,† Inhyub Kim,∥,⊥ Xuezhen Xu,† Dipesh Harmalkar,† Seong-Ah Shin,∥ Minkyoung Kim,† Bo-Kyung Kim,∥ Jaehyung Park,† Bonsu Ku,# Sujin Oh,∇ Misun Won,*,∥,⊥ and Kyeong Lee*,† †

College of Pharmacy, Dongguk University, Goyang 10326, Korea Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea § Biomolecular Science, University of Science and Technology, Daejeon 34113, Korea ∥ Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea ⊥ Functional Genomics, University of Science and Technology, Daejeon 34113, Korea # Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea ∇ New Drug Development Center, Asan Medical Center, Seoul 05505, Korea ‡

S Supporting Information *

ABSTRACT: Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure−activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1α accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth.

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tumor growth.6 Knockdown of the GOT1/2 genes inhibits cell proliferation and colony formation of pancreatic cancer cells.7 The two essential MDH isoenzymes, MDH1 and MDH2, are encoded by distinct MDH genes in humans and are localized in the cytosol and mitochondrial matrix, respectively. MDH1 and MDH2 catalyze the reversible conversion of malate and oxaloacetate (OAA) using the NAD/NADH cofactor system.7,8 In the cytoplasm, MDH1 reduces OAA to malate while oxidizing NADH to NAD+. In the malate−aspartate shuttle, malate is transported to the interior of the mitochondrion and oxidized once again to OAA by mitochondrial MDH2, thereby generating NADH, which enters the ETC to produce ATP. Notably, MDH2 is also involved in the TCA cycle which is needed for energy production through respiration. Although the involvement of MDH in cancer is not currently a major focus in the field of cancer metabolism, there are

INTRODUCTION

Metabolic alterations, including aerobic glycolysis, high fatty acid synthesis, and rapid glutamine metabolism, are characteristics of cancer cells.1 Notably, a high glycolysis rate is important for ATP production, biosynthesis, and redox control in cancer cells. In addition, the malate−aspartate shuttle has been demonstrated to be crucial for the net transfer of cytosolic NADH produced during glycolysis into the mitochondria.2,3 The malate−aspartate shuttle is operated by two pairs of enzymes, malate dehydrogenases (MDHs) and glutamate oxaloacetate transaminases (GOTs), which are localized to the mitochondria and cytoplasm (Figure 1).4 Amino-oxyacetic acid (AOA), a specific inhibitor of the malate−aspartate shuttle, impairs the conversion of glucose into tricarboxylic acid (TCA) cycle products and decreases the proliferation of breast adenocarcinoma cells.5 Moreover, mitochondrial GOT2 acetylation stimulates the malate−aspartate NADH shuttle activity to support ATP production needed for pancreatic © 2017 American Chemical Society

Received: September 1, 2017 Published: October 9, 2017 8631

DOI: 10.1021/acs.jmedchem.7b01231 J. Med. Chem. 2017, 60, 8631−8646

Journal of Medicinal Chemistry

Article

Figure 1. Schematic overview of the malate−aspartate shuttle. The malate−aspartate shuttle is operated by malate dehydrogenases (MDHs) and glutamate oxaloacetate transaminases (GOTs). MDH1 in the cytoplasm reduces oxaloacetate to malate while oxidizing NADH to NAD+. Then, malate is transported to the interior of the mitochondrion and oxidized once again to oxaloacetate by mitochondrial MDH2, thereby generating NADH to produce ATP in the electron transport chain (ETC).

Figure 2. Malate dehydrogenase (MDH) inhibitor design.

endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1).27 Although the functional importance of MDH1 and MDH2 has been elucidated, the rationale for the development of MDH inhibitors as anticancer agents has not yet been reported. Therefore, in this study, we attempted to develop potent MDH inhibitors based on the structure−activity relationship (SAR) of compound 1. We then determined the inhibitory activity of these derivatives against MDH and evaluated the relationship between their activity and inhibition of HIF-1 and cancer cell metabolism. Finally, we identified a methyl 3-(3-(4-(2,4,4trimethylpentan-2-yl)phenoxy)propanamido)benzoate (16c) as a dual inhibitor of MDH1/2 that showed significant tumor reduction in a colorectal cancer xenograft model.

evidence for the cancer-associated functions of MDH1 and MDH2. Glutamine-dependent pancreatic ductal adenocarcinoma (PDAC) cells require MDH1 to maintain their cellular redox state by reprograming glutamine metabolism, and MDH1 knockdown inhibited the viability of PDAC cells.7,9 In addition, knockdown of MDH1 decreased the viability of BT474, Erb-B2 receptor tyrosine kinase 2 (ERBB2)-positive breast cancer cells, by suppressing the fatty acid synthesis pathway.10 In another study, MDH1 stabilized by glucose depletion translocates to the nucleus and interacts with p53 to regulate the transcription of the p53-dependent metabolic checkpoint.11 It has also been shown that high expression of MDH2 is associated with shorter relapse-free survival and chemoresistance in prostate cancer patients.6,8 MDH2 knockdown suppresses proliferation and enhances docetaxel sensitivity via induced metabolic inefficiency of prostate cancer cells.12 Homology analysis of MDH1 and MDH2 reveals 26% identity and 43% similarity as measured by Protein−Protein Blast (Figure S1, Supporting Information). Of note, the substrate-associated residues of human MDH1 were wellmatched to those of MDH2 in a sequence alignment as they showed 63% similarity (Figure S2, Supporting Information), which is greater than the homology between the entire regions of the two proteins. Considering that the structures of the substrate binding and active sites of MDH1 and MDH2 are very similar, we hypothesized that the development of an MDH1/2 dual inhibitor might be an achievable and attractive approach to target cancer metabolism. Recently, we identified mitochondrial MDH2 as a target molecule of methyl 3-(2-(4((3r,5r,7r)-adamantan-1-yl)phenoxy)acetamido)-4-hydroxybenzoate (LW6, 1),13 a hypoxia-inducible factor (HIF)-1α inhibitor, using its chemical probe, prop-2-yn-1-yl 3-(2-(4((3r,5r,7r)-adamantan-1-yl)-2-(3-(trifluoromethyl)-3H-diazirin3-yl)phenoxy)acetamido)-4-hydroxybenzoate (2, Figure 2).8 1 inhibits MDH2 activity by binding the NAD-binding site in a competitive fashion. Cancer cells treated with 1 showed reduced oxygen consumption and increased local cellular oxygen tension, thereby promoting HIF-1α degradation under hypoxic conditions.8 HIF-1α correlates with the aggressive growth of solid tumors, resistance to radiation and chemotherapy, and poor clinical outcomes.14−26 The HIF-1α/β complex binds to hypoxia-responsive elements (HREs) in the promoter of hypoxia-responsive genes, including vascular

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RESULTS AND DISCUSSION Chemistry. To develop novel MDH inhibitors, compound 1 derivatives were synthesized as indicated in Schemes 1−4. The carboxylic acid derivatives 4a−l were prepared using a twostep synthesis. The hydroxyl groups of phenols 3a−l were subjected to alkylation using ethyl chloroacetate in the presence of potassium carbonate (K2CO3) in dimethylformamide (DMF), and the resulting ethyl ester was hydrolyzed with lithium hydroxide to obtain 4a−l.21 These resulting carboxylic acids were subsequently used to prepare amide derivatives (5a−l) using the standard amide coupling method (Scheme 1). Coupling of the commercially available methyl 3-amino-4hydroxybenzoate with 4-adamantyl phenoxyacetic (4a) or 2-(4(2,4,4-trimethylpentan-2-yl)phenoxy)acetic acid (4l) in the presence of N-(3-(dimethylamino)propyl)-N′-ethylcarbonate hydrochloride (EDC·HCl), 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIPEA) yielded the hydroxylated analogues, 1 or 6, respectively. O-Alkylation of 1 and 6 with alkyl or aryl halide produced 4-adamantyl and trimethylpentanyl derivatives (7a−f, Scheme 2). Additionally, 4l was coupled with various amines to produce the respective amide derivatives (8a−f), as shown in Scheme 3. Some of the key intermediate (E)-phenoxyacrylic acid derivatives (12a and 12b) were synthesized from phenols 3a and 3l using previously described procedures25 (Scheme 4). The hydroxyl groups of phenols 3a and 3l were alkylated with methyl propiolate in the presence of triphenylphosphine (PPh3) in toluene, and the resulting (E)-phenoxyacrylic methyl esters (9a and 9b) were hydrolyzed with lithium hydroxide to form 12a and 12b. Other key intermediates, such as the 8632



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Scheme 1. Synthesis of Amide Derivatives 5a−la

a

Reagents and conditions: (a) EDC·HCl, N-(3-(dimethylamino)propyl)-N′-ethylcarbonate hydrochloride; HOBT, 1-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide.

Scheme 2. Synthesis of 4-Adamantyl and Trimethylpentane Derivatives 7a−fa

a

Reagents and conditions: (a) methyl 3-amino-4-hydroxybenzoate EDC·HCl, N-(3-(dimethylamino)propyl)-N′-ethylcarbonate hydrochloride; HOBT, 1-hydroxybenzotriazole; DIPEA, N,N-di-isopropylethylamine; DMF, dimethylformamide; (b) alkyl or aryl halide, potassium carbonate (K2CO3), DMF.

propionic acids (13a and 13b) and butanoic acids (11a and 11c), and the branched propanoic acids (11b and 11d) were also synthesized from 3a or 3l, respectively (Scheme 4). The phenoxyacrylic portions of 9a and 9b were saturated using palladium on carbon (Pd/C) catalyzed hydrogenation to generate 10a and 10b. To prepare the three-carbon linker part, phenols 3a and 3l were alkylated with methyl 4bromobutanoate in the presence of K2CO3 in DMF followed by hydrolysis to obtain 11a and 11c. A series of carboxylic acid derivatives were coupled with methyl 3-amino benzoate or methyl 3-amino-4-hydroxybenzoate in the presence of EDC, HOBT, and DIPEA to produce the amide derivatives 14a−f, 15a−b, and 16a−c, as shown in Scheme 4. MDH Enzyme Assay. The newly synthesized compounds were evaluated for their ability to inhibit human recombinant

MDH1 and MDH2 enzymatic activity using an oxaloacetatedependent NADH oxidation assay (Tables 1−4). Compound 1 showed good inhibitory activities toward both enzymes. To define the key structural requirements for biological activity of the aryloxyacetylamino benzoic acid derivatives, our optimization strategy focused on three discrete areas: (A) the 4adamantyl phenyl ring, which could be replaced by a phenyl ring containing various substituents, (B) the aminophenyl ring, and (C) modifications that included replacing the oxyacetylamide linker portion (Figure 1). For SAR for part A, we started with a 4-adamantyl (aryloxyacetylamino)benzoic ester analogue 5a, which induced mild inhibitory activity against both MDH1 and MDH2 enzymes (IC50 = 23.48 ± 1.64 and 15.97 ± 2.02 μM, respectively). Substitution of 4-adamantyl group with electron 8633



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Scheme 3. Synthesis of Trimethylpentane Derivatives 8a−fa

a

Reagents and conditions: (a) amines, EDC·HCl, N-(3-(dimethylamino)propyl)-N′-ethylcarbonate hydrochloride; HOBT, 1-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide.

found that a known MDH2 inhibitor 113 showed better activity against MDH1 (IC50 = 1.10 ± 1.10 μM) than MDH2, whereas the corresponding trimethylpentanyl compound 6 exhibited more potency and dual inhibitory activity against MDH1 and MDH2 (Table 2). Interestingly, compound 5 (with phenolic OH at part B) exhibited greater inhibitory activity than 5l against both MDH1 and MDH2 enzymes (IC50 = 1.14 ± 0.09 and 1.89 ± 0.18 μM, respectively). This finding suggested that OH-containing compounds (i.e., 1 and 6) with an oxyacetamide linker shows better feasibility for MDH enzyme inhibition. The alkylation of the hydroxyl group (1 or 6) with cyclic amine-substituted alkyl groups or a methoxy-substituted alkyl group led to analogues 7a,b and 7d,e, which resulted in the loss of activity. Methoxy compound 7c with 2,4,4trimethylpentane scaffold showed 12-fold selective MDH1 activity over MDH2 (IC50 = 2.27 ± 0.32 and 27.47 ± 0.16 μM, respectively). By contrast, the propargylated compound 7f maintained its relative inhibitory activity against both MDH1 and MDH2. This finding suggests that the free OH group in 1 or 6 is important for its interaction with the protein (Table 2). Using the best analogue, 6, among the compounds shown in Tables 1−2, we explored part B with further modifications using a variety of amines (Figure 2, Table 3). The trifluoromethyl group will pull or withdraw electrons through either resonance or induction to the aromatic group, as shown for compound 8a. Adjacent functional groups, as well as the presence or absence of direct attachment to an aromatic ring, will determine the relative involvement of these two processes. CF3-phenyl compound 8a exhibited less activity than 6 against both MDH1 and MDH 2 with IC50 values of 3.44 ± 0.43 and

withdrawing groups, such as NO2 and CF3 (5b and 5c), did not yield any MDH1/MDH2 inhibitory activity (>40 μM). Then, the 4-adamantyl group of 5a was modified with a similar lipophilic group (t-butyl) to yield 5d, which exhibited moderate inhibitory activity (IC50 = 10.74 ± 1.31) against MDH1 but not against MDH2. The compounds substituted with a simple alkyl chain (methyl to n-propyl chain) 5e−g failed to inhibit MDH enzymes up to 40 μM. Interestingly, 5h and 5i with n-butyl or n-pentyl moiety exhibited medium inhibitory activity against MDH1 (IC50 = 9.58 ± 0.23, 6.79 ± 0.21 μM, respectively). Compounds 5d, 5h, and 5i showed some selectivity to MDH1 over MDH2. For cyclopentyl 5j and cyclohexyl 5k, potent effects was not observed in the MDH enzyme assay. Similar to 5a, 5l with 2,4,4-trimethylpentanyl substituent showed dual inhibition against both MDH1 and MDH2. As listed in Table 1, compound 5l demonstrated considerable inhibition against MDH1 and MDH2 with IC50 values of 6.65 ± 0.65 and 8.92 ± 1.39 μM, respectively. The presence of a trimethylpentanyl moiety may be more beneficial for MDH inhibition than an adamantyl group for the phenoxyacetamido platform (5a vs 5l). These findings suggested that the presence of a 2,4,4trimethylpentanyl substituent or an equivalent for part A is important for the effective inhibition of MDH enzymes (Table 1). On the basis of the encouraging results for compound 5l, a further optimization study focused on analogues with an adamantly or trimethylpentanyl moiety. In addition, we observed that the introduction of a hydroxyl group into the phenyl ring at part B (5a vs 1) resulted in increased potency against both MDH1 and MDH2. Notably, in this study, we 8634



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Scheme 4. Synthesis of 4-Adamantyl and Trimethylpentane Derivatives 14a−f, 15a−b, and 16a−ca

a

Reagents and conditions: (a) (i) K2CO3, methyl 4-bromobutanoate for 11a and 11c and methyl 2-bromo-2-methylpropanoate for 11b and 11d, DMF, (ii) LiOH, THF/H2O; (b) PPh3, methyl propiolate, toluene; (c) Pd/C, H2, MeOH; (d,e) LiOH, THF/H2O; (f,g,h) EDC·HCl, HOBT, DIPEA, DMF, methyl 3-amino-4-hydroxybenzoate for 14c, 14f, and 16b and methyl 3-aminobenzoate for 14a,b, 14d,e, 15a,b, and 16a,c.

1.53 ± 0.08 μM, respectively. Notably, 2-(4-(adamantan-1yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (IDF-11774) is a potent HIF-1 inhibitor that is approved as a clinical candidate for cancer therapy.28,29 A number of piperazine compounds showed various biological activities with favorable properties. Herewith, we prepared amide derivatives with various piperazine groups in different positions. However, piperazine-containing compounds 8b and 8c showed moderate activity against both MDH1 and MDH2. Compound 8d, with 1-(prop-2-ynyl)piperazine, completely lost activity. Interestingly, propargyl ester (8e) showed potency against MDH1 with an IC50 value of 0.92 ± 0.28 μM. The hydroxyl-adamantane containing amide 8f showed weak inhibitory activity against both MDH1 and MDH2 (Table 3). As summarized in Table 4, our SAR study showed that altering the carbon linker (part C) between the alkyl substituted phenyl ring (part A) and amino phenyl (part B) of the phenoxyacetamides was a useful strategy for inducing MDH inhibition. Notably, the addition of branched carbons (14d and 14e) to the corresponding acetamides apparently increased potency against both MDH1 and MDH2. Interestingly, the 2,4,4-trimethylpentanyl derivative with branched carbon and hydroxyl groups in the phenyl ring (14f) was more selective to MDH1 than MDH2 (IC50 = 3.00 ± 0.03 and 10.98 ± 0.79 μM, respectively). One- to two-carbon homologations (14a,b, 16a, and 16c) to the acetamide 5a and 5l resulted in a significant increase in MDH1 and MDH2 enzyme potency, as shown in Table 4. In contrast, one- to two-carbon homologations (14c and 16b) to the OH containing acetamide 6 was not helpful for improvement of activity. Among the

series, compound 16c showed the most potent and dual inhibitory activity against both MDH enzymes (IC50 = 1.07 ± 0.07 and 1.06 ± 0.03 μM, respectively). It was found that homologation (14a and 16a) for the 4-adamantyl derivatives resulted in lower potency than that of the trimethylpentane derivatives (14a and 16c). According to previous studies, various compounds with 4-(2,4,4-trimethylpentan-2-yl)phenoxy pendent groups were reported to be useful for the treatment of some chronic diseases, including inflammation, diabetes, hyperglycemia, and cancers.30,31 For the 2,4,4trimethylpentane-based compounds, the introduction of a more conformationally constrained oxyacrylic amide linker, as represented by 15b, induced better MDH inhibitory activity. By contrast, the corresponding 4-adamantyl compound (15a) showed MDH2-selectivity (6-fold) over MDH1. For compounds that contained OH groups with a modified linker (14c, 14f, and 16b), the MDH1 and MDH2 enzymes inhibitory activity profiles were different for acetamide scaffold with OH groups (i.e., 1 and 6). The one-carbon homologated compound 16b (with OH group) was less potent than 16c (without OH) against MDH1 and MDH2 enzymes with IC50 = 1.54 ± 0.02 and 4.60 ± 0.32 μM, respectively. Similarly, compound 14c containing two carbon homologated linkers with OH group was less active against MDH1 and MDH2 enzymes compared to compound 14b (IC50 = 1.18 ± 0.08 and 1.11 ± 0.04 μM, respectively). This pattern did not change for branched carbon linker compounds with or without a OH group (14e vs 14f). Compound 14f (with −OH group) lost some potency against MDH1 and MDH2 enzymes with IC50 = 3.00 ± 0.03 and 10.98 ± 0.79 μM, respectively, than the corresponding OH-free 8635



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Table 1. In Vitro MDH1 and MDH2 Inhibitory Activity of Amide Derivatives 5a−la

Table 2. In Vitro MDH1 and MDH2 Inhibitory Activity of Amide Derivatives 1, 6, and 7a−fa

a

a

Values are the means of three experiments.

compound toward MHD1 or MDH2 because they share common NADH binding sites of 63% similarity based on structural superimposition (Figure S2, Supporting Information). Therefore, on the basis of these considerations, compounds such as 5i,7, 16a, 14b, and 16c may have advantages in both activity and selectivity as MDH inhibitors. As listed in Table 5, compounds 5i and 7c were categorized as selective MDH1 inhibitors, while most compounds that inhibited MDH2 activity also inhibited MDH1, as demonstrated by 14b and 16c, which were termed dual MDH1/2 inhibitors. The phenoxyacrylic linker-containing 15a was selected as a selective MDH2 inhibitor. According to a docking study using X-ray crystal data for MDH1 (5MDH) and MDH2 (2DFD), the flexible nature of the homologated linker domain of 16c may be helpful for binding both NAD sites of the enzymes (Figure S3, Supporting Information). Cell-Based HRE Reporter Assay. Next, we assessed whether the inhibition of MDH1 or MDH2 enzymes was related to the outputs of the HRE-luciferase activity, which indicates HIF-1α inhibition (Table 5). The hypoxia-induced HIF-1 activities of the representative compounds were determined using a cell-based HRE reporter assay in HCT116. We followed a previously described assay protocol under standard hypoxic assay conditions (1% O2, 94% N2, and 5% CO2).13 Cell viability, as measured using the methylene blue assay, showed that all of the compounds had no significant cytotoxicity at concentrations at which they effectively inhibited HIF-1 activation (>40 μM). The MDH1 inhibitors 5i and 7c


compound 14e. This finding suggests that the MDH1 and MDH2 inhibitory activities for compounds with or without a OH group at part B was dependent on the carbon linker. To date, several MDH inhibitors have been reported. A thyroid hormone thyroxine (T4) was described to suppresses MDH2 activity with an IC50 value of 43.6 μM.8,32 A staurosporine aglycone K252c also inhibits MDH activity with an IC50 value of 8 μM.33 However, this compound mainly suppresses PKC and PKA with IC50 values of 2.45 and 25.7 μM, respectively. Considering the importance of MDH enzymes in cancer metabolism, more potent and selective inhibitors are required for in-depth mechanistic studies and drug development. However, we cannot exclude the possibility of off-target effects of MDH inhibitors because compounds may have multitargeting properties. It may affect the activity of metabolic enzymes involving NADH−NAD conversion. Additionally, it is not easy to increase the selectivity of the 8636



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Table 3. In Vitro MDH1 and MDH2 Inhibitory Activity of Amide Derivatives 8a−fa

a

Table 4. In Vitro MDH1 and MDH2 Inhibitory Activity of Linker Modified Derivatives 14a−f, 15a−c, and 16a−ca


did not show potent effects in the HRE assay. However, the MDH2-selective inhibitor 15a and the dual MDH1/2 inhibitors 14b and 16c strongly suppressed HRE-luciferase activity. This finding is consistent with the previous report that inhibition of MDH2 suppresses mitochondrial respiration and increases oxygen content, thereby stimulating HIF-1α degradation in cancer cells.11 Then, to further confirm the role of MDH1, MDH2, or both MDH1 and MDH2 activity in HIF-1α accumulation, intracellular oxygen content was determined using a mono azo rhodamine (MAR) fluorescent hypoxia probe, (Figure S4, Supporting Information). On the basis of the MDH enzyme and cell-based HRE assays results, the MDH1 selective agents (5i and 7c) did not affect the fluorescence of MAR. However, 14b and 16c that showed MDH2 inhibitory activity significantly reduced MAR fluorescence, indicating increased intracellular oxygen contents. As previously reported, the roles of MDH1 and MDH2 are related to cancer cell metabolism. MDH1 and ME1 convert malate to pyruvate for palmitate synthesis to generate NADH, which supplies the NADPH required for the synthesis and elongation of fatty acids.34 It has been reported that the inhibition of MDH1 induced anticancer activity by suppressing fatty acid synthesis. Moreover, MDH2 inhibition reduced the loss of NADH and inhibited mitochondrial respiration, resulting in increased oxygen contents and stimulation of HIF-1α degradation. MDH2 knockdown may induce anti-

a


Table 5. Cell-Based HRE-Luc Inhibitory Activity by the Representative Compoundsa HRE

a

compd

IC50 (μM)

MDH selectivity

5i 7c

>40 >40

selective MDH1 inhibitor

15a

0.74 ± 0.06

selective MDH2 inhibitor

14b 16c

1.40 ± 0.38 1.08 ± 0.06

dual MDH1/2 inhibitor


cancer activity by reducing HIF-1α accumulation and sensitizing chemoresistant cancer cells. In addition to the specific roles of MDH1 and MDH2, both enzymes are associated with the malate−aspartate shuttle. This process involves a reaction between NADH and oxaloacetate in the cytosol, and the malate is transported into the mitochondria and subsequently reoxidized by the electron transfer chain. The malate−aspartate shuttle facilitates the translocation of electrons across the mitochondrial membrane, thereby 8637



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Figure 3. Mode of malate dehydrogenase (MDH) inhibition by compound 16c. Enzyme kinetics of (A) MDH1 and (B) MDH2 were performed at various concentrations of NADH. Concentrations of compound 16c: 0 μM (●), 0.625 μM (○), 1.25 μM (▼closed triangle), and 2.5 μM (▽).

regulating metabolic coordination between the cytosol and mitochondria. Indeed, the malate−aspartate shuttle is crucial for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis to support rapid tumor cell growth. Therefore, compounds that inhibit both MDH1 and MDH2, such as 14b and 16c, are expected to have considerable anticancer activity. Among the series described in this study, we discovered that compound 16c exhibited the strongest inhibition of both MDH1 and MDH2 and showed potent inhibition against HIF-1. To elucidate the inhibitory mechanism of compound 16c on MDH1 and MDH2 activity, an enzyme kinetics study was performed using various NADH concentrations. Double-reciprocal plots of enzyme kinetics revealed a competitive mode of MDH1 and MDH2 inhibition by compound 16c with Ki values of 0.74 and 0.95 μM, respectively (Figure 3A,B). Additionally, we found that the trimethylpentane derivative 16c showed reasonable water solubility (94.4 ± 0.50 μM at pH 7.4), which was superior to that of another dual MDH1/2 inhibitor, 14b (45.8 ± 1.45 μM at pH 7.4, Table S1, Supporting Information) and, therefore, merits further evaluation as a novel anticancer agent for targeting cancer cell metabolism. Effects on HIF-1α Pathway and Oxygen Tension. Following the identification of a novel MDH1/2 dual inhibitor, we examined the effects of compound 16c on HIF-1α signaling in HCT116 cells. Under hypoxic conditions, treatment with compound 16c suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner (Figure 4A). As expected, compound 16c inhibited the expression of HIF-1α target genes, including VEGF, GLUT1, and pyruvate dehydrogenase kinase 1 (PDK1), without affecting the levels of HIF-1α mRNA (Figure 4B). To characterize the inhibitory mechanism of compound 16c against HIF-1α accumulation, we further examined its effects on mitochondrial respiration using an XF24 extracellular flux analyzer. Compound 16c reduced the oxygen consumption rate (OCR), indicating the inhibition of mitochondrial respiration

Figure 4. Inhibition of hypoxia-induced hypoxia-inducible factor-1 (HIF-1)-α activation by compound 16c. (A) Effects of compound 16c on hypoxia-induced HIF-1α accumulation. After treatment of HCT116 cells with compound 16c under hypoxic conditions, proteins were detected using immunoblot analysis in HCT116 cells. (B) Effects of compound 16c on mRNA expression levels of HIF-1α target genes were detected using reverse transcription−polymerase chain reaction (RT-PCR) analysis.

(Figure 5A). In the presence of compound 16c, the reduction of intracellular ATP levels resulted in the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which inactivated acetyl-CoA carboxylase (ACC), a downstream target of AMPK (Figure 5B,C). Furthermore, staining with the hypoxia-detecting probe MAR showed that compound 16c increased intracellular oxygen tension, presumably because of reduced OCR, resulting in HIF-1α degradation under hypoxic conditions (Figure 5D). We next examined sequential electron flow of the electron transport chain (ETC) to further understand the inhibitory mechanism of mitochondrial respiration of compound 16c. Each complex-linked respiratory process was regulated by the addition of specific substrates and inhibitors (Figure 6A). Under these experimental conditions, compound 16c significantly inhibited complex I-dependent respiration (Figure 6B) without affecting complex II/III- and IV-dependent respiration (Figure S5, Supporting Information). These findings suggest 8638



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Figure 5. Inhibition of mitochondrial respiration by compound 16c. (A) Oxygen consumption rate (OCR) was measured using XF24 extracellular flux analyzer by adding oligomycin (1 μM), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 0.5 μM), and rotenone (1 μM)/ antimycin A (1 μM) to compound 16c-treated HCT116 cells. (B) Adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation was determined using immunoblot analysis with each specific antibody. (C) The intracellular adenosine triphosphate (ATP) content of compound 16c-treated HCT116 cells was measured using a luciferase-based assay system. (D) Intracellular oxygen tension was determined using a hypoxiasensitive probe mono azo rhodamine (MAR, 0.5 μM). Scale bar, 100 μm. Fluorescence intensity of the MAR probe is shown by vertical bars; *P < 0.05 and **P < 0.01, compared with hypoxic control.

Figure 6. Inhibition of mitochondria complex I activity by compound 16c. (A) The exogenous substrate-dependent mitochondrial respiration rate was measured in digitonin-permeabilized HCT116 cells by adding a substrate and inhibitor of each complex. Substrates and inhibitors used were as follows: pyruvate (5 mM), malate (5 mM), rotenone (1 μM), succinate (5 mM), antimycin A (1 μM), ascorbate (5 mM), and tetramethyl-pphenylenediamine (TMPD, 2 mM). (B) Inhibitory activity of compound 16c against mitochondria complex I was determined by adding compound 16c (30 μM) after treatment with complex I substrates.

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metabolism, the trimethylpentane-containing ester compound 16c was identified as a potent and dual inhibitor of MDH1/ MDH2. Measurements of OCR revealed that compound 16c inhibited mitochondrial respiration, thereby reducing APT production. Therefore, the phosphorylation of AMPK was activated to down-regulate mechanistic target of rapamycin (mTOR) signaling. In addition, decreased respiration increased cellular oxygen tension, as shown by the hypoxia probe MAR. Expectedly, compound 16c suppressed hypoxia-induced HIF1α accumulation and the expression of HIF-1α target genes. We also discovered that compound 16c significantly suppressed the activity of complex I in the ETC. Moreover, in the mouse xenograft assay, compound 16c showed strong in vivo antitumor efficacy in an HCT116 cell model. Therefore, the dual MDH1 and MDH2 inhibitor compound 16c was demonstrated to be a potential tumor-suppressing agent, suggesting that the simultaneous inhibition of both MDH1 and MDH2 enzymes could be a crucial therapeutic strategy.

that the inhibition of MDHs by compound 16c reduced complex I-dependent mitochondrial respiration. In Vivo Growth Inhibition. The in vivo antitumor efficacy of compound 16c was analyzed using a xenograft assay of an HCT116 cell mouse model. Compound 16c was administered intraperitoneally (20 mg/kg, once daily [qd]) for 14 days, and significant tumor growth inhibition (63.4%) was observed (Figure 7). Moreover, compound 16c did not cause significant

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EXPERIMENTAL SECTION

Chemistry: General. All the commercial chemicals were of reagent grade and were used without further purification. Solvents were dried with standard procedures. All the reactions were carried out under an atmosphere of dried argon in flame-dried glassware. The proton nuclear magnetic resonance (1H NMR) spectra were determined on a Varian (300, 400, or 500 MHz) spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA). 13C NMR spectra were recorded on a Varian (100 MHz) spectrometer. The chemical shifts are provided in parts per million (ppm) downfield with coupling constants in hertz (Hz). The mass spectra were recorded using high-resolution mass spectrometry (HRMS) (electron ionization MS) obtained on a JMS-700 mass spectrometer (Jeol, Japan) or using HRMS (electrospray ionization MS) obtained on a G2 QTOF mass spectrometer. The products from all the reactions were purified by flash column chromatography using silica gel 60 (230−400 mesh Kieselgel 60). Additionally, thin-layer chromatography on 0.25 mm silica plates (E. Merck; silica gel 60 F254) was used to monitor reactions. Final product purity was checked by reversed phase high-pressure liquid chromatography (RP-HPLC), performed on a Waters Corp. HPLC system equipped with an ultraviolet (UV) detector set at 254 nm. The mobile phases used were: (A) H2O containing 0.05% trifluoroacetic acid, and (B) CH3CN. HPLC employed a YMC Hydrosphere C18 (HS-302) column (5 μm particle size, 12 nm pore size) that was 4.6 mm in diameter × 150 mm in size with a flow rate of 1.0 mL/min. Some of the starting chemicals (i.e., 3a−l) were purchased from Sigma aldrich, TCI, Ark Pharma, and Combi Blocks. The compound purity was assessed using method A, a gradient of 25% B to 100% B in 35 min. The purity of all biologically evaluated compounds was >95% in method A. General Procedure for the Synthesis of 5a−l. EDCI (1.2 equiv) and HOBt (1.2 equiv) were added to a solution of appropriate phenoxyacetic acid 4a−l (1.0 equiv), methyl 3-aminobenzoate (1.0 equiv), and DIPEA (2.5 equiv) in DMF, and the reaction mixture was stirred at room temperature until completion as monitored by TLC. The reaction mixture was diluted with EtOAc brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography. Methyl 3-(2-(4-Adamantan-1-yl-phenoxy)acetamido)benzoate (5a).21 See ref 21. Methyl 3-(2-(4-Nitrophenoxy)acetamido)benzoate (5b). Obtained as a white solid (0.21 g, 64.2% yield). 1H NMR (400 MHz, DMSO) δ 10.5 (brs, 1H), 8.33 (t, J = 1.8 Hz, 1H), 8.25 (d, J = 9.4 Hz, 2H), 7.89 (m, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 9.0 Hz, 2H), 4.95 (s, 2H), 3.86 (s, 3H). 13C NMR (100 MHz, DMSO) δ 166.4, 163.5, 141.7, 139.1, 130.6, 129.7, 126.3, 124.8, 124.5, 120.5, 115.7, 67.6, 52.7. HRMS [M + H] calcd [C16H15N2O6]

Figure 7. In vivo antitumor effects of compound 16c in a HCT116 cell xenograft model. (A) HCT116 cells (5 × 106) were subcutaneously inoculated into the right flank of female nude mice. Drugs were administered intraperitoneally once a day for 14 days after the tumor reached a volume of 80−100 mm3. (B) Image and weight of tumors at the end of the experiment; *P < 0.05 and **P < 0.01, compared with vehicle control.

adverse effects in mice, including changes in body weight and abnormalities of internal organs and behavior (Figure S6, Supporting Information). In addition, we examined the combined effect of MDH1-selective compound 5i and MDH2-selective compound 15a on the growth of HCT116 cell xenografts (Figure S7, Supporting Information). Treatment with 5i (10 mg/kg) and 15a (10 mg/kg) showed 34.9% and 26.2% antitumor efficacy, respectively. However, combined treatment with 6i (10 mg/kg) and 15a (10 mg/kg) demonstrated 79.7% inhibition of tumor growth, indicating a combination of 5i and 15a was more efficient than single compounds. This finding suggests that the inhibition of both MDH1 and MDH2 may induce greater efficacy of anticancer therapeutics.

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CONCLUSION This study reported a trimethylpentane derivative as a novel scaffold that exhibited MDH1 and MDH2 dual inhibitory activity. A series of trimethylpentane derivatives were newly synthesized and evaluated as potential MDH1 or MDH2 inhibitors or dual inhibitors. The SAR study of this series identified both MDH1- and MDH2-selective and MDH1/ MDH2 dual inhibitors. We demonstrated that MDH2, but not MDH1 inhibitors, suppressed HRE-luciferase activity and the growth of cancer cells. Considering that dual inhibition of MDH enzymes may be a potent approach to target cancer cell 8640



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67.9, 52.3, 45.2, 34.7, 25.4. HRMS [M + H] calcd [C21H24NO4] 354.1705, found 354.1682. Purity 100% (as determined by RP-HPLC, method A, tR = 22.03 min). Methyl 3-(2-(4-Cyclohexylphenoxy)acetamido)benzoate (5k). Obtained as a yellow solid (0.11 g, 70.5% yield). 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 8.07 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 12.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 4.60 (s, 2H), 3.92 (s, 3H), 2.50−2.46 (m, 1H), 1.84−1.76 (m, 4H), 1.73−1.58 (m, 2H), 1.44−1.38 (m, 4H). 13 C NMR (100 MHz, CDCl3) δ 166.7, 166.6, 155.1, 142.4, 137.1, 131.1, 129.3, 128.1, 125.9, 124.5, 120.9, 114.7, 67.8, 52.3, 43.3, 34.6, 26.9, 26.1. HRMS [M + H] calcd [C22H26NO4] 368.1862, found 368.1842. Purity 100% (as determined by RP-HPLC, method A, tR = 23.62 min). Methyl 3-(2-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)acetamido)benzoate (5l). Obtained as a white solid (0.10 g, 64.1% yield). 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.08 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.44−7.32 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H), 4.60 (s, 2H), 3.91 (s, 3H) 1.71 (s, 2H), 1.35 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.6, 154.6, 144.3, 137.1, 131.0, 129.3, 127.5, 125.8, 124.5, 120.9, 114.0, 67.6, 56.9, 52.3, 30.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C24H32NO4] 398.2331, found 398.2345. Purity 100% (as determined by RP-HPLC, method A, tR = 26.11 min). General Procedure for the Synthesis of 1 and 6. EDCI (1.2 equiv) and HOBt (1.2 equiv) were added to a solution of appropriate phenoxyacetic acid 4a or 4l (1.0 equiv), methyl 3-amino-4hydroxybenzoate (1.0 equiv), and DIPEA (2.5 equiv) in DMF, and the reaction mixture was stirred at room temperature until completion as monitored by TLC. The reaction mixture was diluted with EtOAc brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography. Methyl 3-(2-(4-Adamantan-1-yl-phenoxy)acetamido)-4-hydroxybenzoate (1).8 See ref 8. Methyl 4-Hydroxy-3-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acetamido)benzoate (6). Obtained as a white solid (1.00 g, 64.1% yield). 1H NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 8.60 (s, 1H), 7.85−7.83 (m, 1H), 7.73 (s, 1H), 7.38−7.34 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.94−6.90 (m, 2H), 4.68 (s, 2H), 3.89 (s, 3H), 1.73 (s, 2H), 1.36 (s, 6H), 0.72 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 168.2, 166.7, 154.5, 147.2, 144.6, 129.2, 128.0, 127.6, 122.3, 121.3, 119.5, 114.2, 67.5, 57.0, 52.3, 38.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C24H32NO5] 414.2280, found 414.2249. Purity 100% (as determined by RP-HPLC, method A, tR = 24.95 min). General Procedure for the Synthesis of 7a−f. The mixture of 1 or 6 (1.0 equiv) and anhydrous potassium carbonate (3.0 equiv), alkyl or aryl halide (1.0 equiv) in DMF was stirred overnight at room temperature. The reaction mixture was diluted with EtOAc and subsequently washed with aqueous sodium bicarbonate, brine, and water. The organic layer was dried over anhydrous MgSO4. The solvent was filtered and evaporated under reduced pressure to afford a crude solid which was purified by silica gel column chromatography. Methyl 3-(2-(4-Adamantan-1-yl-phenoxy)acetamido)-4-(2-(pyrrolidin-1-yl)ethoxy)benzoate (7a). Obtained as a white solid (0.10 g, 77.8% yield). 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 9.02 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.95−6.91 (m, 3H), 4.62 (s, 2H), 4.22 (t, J = 4.0 Hz, 2H), 3.89 (s, 3H), 2.92 (t, J = 4.0 Hz, 2H), 2.64−2.62 (m, 4H), 2.09 (brs, 3H), 1.89−1.88 (m, 6H), 1.77−1.72 (m, 10H). 13C NMR (100 MHz, CDCl3) δ 166.8, 166.5, 155.0, 151.1, 145.5, 126.8, 126.7, 126.2, 123.2, 121.1, 114.4, 110.6, 68.3, 68.0, 54.8, 54.7, 52.0, 43.4, 36.7, 35.7, 28.9, 23.6. HRMS [M + H] calcd [C32H41N2O5] 533.3015, found 533.2988. Purity 100% (as determined by RP-HPLC, method A, tR = 13.39 min). Methyl 3-(2-(4-Adamantan-1-yl-phenoxy)acetamido)-4-(3morpholinopropoxy)benzoate (7b). Obtained as a white solid (0.09 g, 69.7% yield). 1H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 9.04 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.94−6.92 (m, 3H), 4.62 (s, 2H), 4.16 (t, J = 4.0 Hz, 2H), 3.90 (s, 3H), 3.69−3.68 (m, 4H), 2.53 (t, J = 4.0 Hz, 2H), 2.42−2.40 (m, 4H), 2.09 (brs, 3H),

329.0852, found 329.0676. Purity 97.3% (as determined by RP-HPLC, method A, tR = 14.62 min). Methyl 3-(2-(4-(Trifluoromethyl)phenoxy)acetamido)benzoate (5c). Obtained as a white solid (0.01g, 40.8% yield). 1H NMR (CDCl3, 400 MHz) δ 8.32 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.46 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 4.68 (s, 2H), 3.93 (s, 3H), 1.62 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 166.5, 159.1, 136.8, 131.0, 129.4, 127.5, 127.4, 127.4, 126.1, 124.6, 120.9, 114.9, 67.4, 52.3. HRMS [M + H] calcd [C17H15F3NO4] 354.0875, found 354.0929. Purity 99.99% (as determined by RP-HPLC, method A, tR = 17.70 min). Methyl 3-(2-(4-tert-Butylphenoxy)acetamido)benzoate (5d).21 See ref 21. Methyl 3-(2-(p-Tolyloxy)acetamido)benzoate (5e). Obtained as a white solid (0.10 g, 40.4% yield). 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.09 (t, J = 8.0 Hz, 1H), 7.99 (m, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.2 Hz, 2H), 6.87 (m, 2H), 4.56 (s, 2H), 3.89 (s, 3H), 2.29 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.5, 154.9, 137.2, 131.8, 130.9, 130.3, 129.2, 125.8, 124.5, 120.9, 114.6, 67.7, 52.3, 20.5. HRMS [M + H] calcd [C17H18NO4] 300.1158, found 300.1232. Purity 100% (as determined by RP-HPLC, method A, tR = 16.29 min). Methyl 3-(2-(4-Ethylphenoxy)acetamido)benzoate (5f). Obtained as a white solid (0.34 g, 97.4% yield). 1H NMR (400 MHz, CDCl3) δ 8.48 (brs, 1H), 8.00 (m, 1H), 7.81 (m, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 8.0 Hz, 2H), 6.91 (m, 2H), 4.59 (s, 2H), 3.90 (s, 3H), 2.61 (q, J = 7.4 Hz, 2H), 1.23 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.5, 155.0, 138.3, 137.1, 13.9, 129.2, 129.1, 125.8, 124.5, 120.9, 114.7, 67.7, 52.2, 28.0, 15.8. HRMS [M + H] calcd [C18H20NO4] 314.1314, found 314.1379. Purity 100% (as determined by RP-HPLC, method A, tR = 18.06 min). Methyl 3-(2-(4-Propylphenoxy)acetamido)benzoate (5g). Obtained as a white solid (0.31 g, 90.5% yield). 1H NMR (400 MHz, CDCl3) δ 8.46 (brs, 1H), 8.09 (t, J = 1.8 Hz, 1H), 8.00 (dd, J = 8.2 Hz, 1H), 7.82 (dd, J = 7.8 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.90 (m, 2H), 4.59 (s, 2H), 3.91 (s, 3H), 2.54 (m, 2H), 1.61 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.6, 155.0, 137.1, 136.8, 131.0, 129.7, 129.2, 125.8, 124.5, 120.9, 114.6, 67.7, 52.3, 37.1, 24.7, 13.7. HRMS [M + H] calcd [C19H22NO4] 328.2471, found 328.1546. Purity 100% (as determined by RP-HPLC, method A, tR = 19.92 min). Methyl 3-(2-(4-Butylphenoxy)acetamido)benzoate (5h). Obtained as a white solid (0.32 g, 98.4% yield). 1H NMR (400 MHz, CDCl3) δ 8.52 (brs, 1H), 8.10 (s, 1H), 7.99 (dd, J = 8.2, 1.2 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H), 4.57 (s, 2H), 3.89 (s, 3H), 2.56 (t, J = 7.6 Hz, 2H), 1.56 (m, 2H), 1.34 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.8, 166.5, 155.0, 137.2, 137.0, 130.9, 129.6, 129.2, 125.8, 124.5, 120.9, 114.6, 67.7, 52.2, 34.7, 33.8, 22.3, 14.0. HRMS [M + H] calcd [C20H24NO4] 342.1627, found 342.1694. Purity 100% (as determined by RP-HPLC, method A, tR = 21.59 min). Methyl 3-(2-(4-Pentylphenoxy)acetamido)benzoate (5i). Obtained as a yellow solid (0.13 g, 81.2% yield). 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 12.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 4.60 (s, 2H), 3.92 (s, 3H), 2.56 (t, J = 8.0 Hz, 2H), 1.61−1.57 (m, 2H), 1.34−1.29 (m, 4H), 0.89 (t, J = 4.0 Hz, 1H). 13 C NMR (100 MHz, CDCl3) δ 166.7, 166.6, 155.0, 137.2, 137.1, 131.1, 129.7, 129.3, 125.9, 124.5, 120.9, 114.7, 67.8, 52.3, 35.0, 31.4, 31.3, 22.5, 14.0. HRMS [M + H] calcd [C21H26NO4] 356.1862, found 356.1842. Purity 100% (as determined by RP-HPLC, method A, tR = 23.53 min). Methyl 3-(2-(4-Cyclopentylphenoxy)acetamido)benzoate (5j). Obtained as a yellow solid (0.10 g, 62.5% yield). 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 12.0 Hz, 1H), 7.22 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 4.61 (s, 2H), 3.93 (s, 3H), 2.98−2.94 (m, 1H), 2.06−2.05 (m, 2H), 1.82−1.77 (m, 2H), 1.70−1.67 (m, 2H), 1.57−1.52 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.6, 155.1, 140.8, 137.1, 131.1, 129.3, 128.4, 125.9, 124.5, 120.9, 114.7, 8641



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2.02 (t, J = 4.0 Hz, 2H), 1.89−1.88 (m, 6H), 1.77−1.72 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 166.8, 166.3, 154.9, 151.1, 145.7, 126.7, 126.5, 126.3, 123.0, 120.9, 114.4, 110.3, 68.0, 66.9, 55.2, 53.7, 52.0, 43.4, 36.7, 35.7, 28.9. HRMS [M + H] calcd [C33H43N2O6] 563.3121, found 563.3085. Purity 100% (as determined by RP-HPLC, method A, tR = 13.38 min). Methyl 4-Methoxy-3-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acetamido)benzoate (7c). Obtained as a white solid (0.08 g, 77.6% yield). 1H NMR (400 MHz, CDCl3) δ 9.03 (s, 1H), 8.97 (s, 1H), 7.85−7.83 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 6.93−6.90 (m, 3H), 4.63 (s, 2H), 3.93 (s, 3H), 3.89 (s, 3H), 1.71 (s, 2H), 1.35 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.5, 154.8, 151.9, 144.1, 127.5, 126.8, 126.5, 123.1, 121.1, 114.1, 109.6, 67.9, 56.9, 56.1, 52.0, 38.1, 32.3, 31.8, 31.7. HRMS [M + H] calcd [C25H34NO5] 428.2437, found 428.2416. Purity 100% (as determined by RP-HPLC, method A, tR = 27.91 min). Methyl 4-(2-Methoxyethoxy)-3-(2-(4-(2,4,4-trimethylpentan-2yl)phenoxy)acetamido)benzoate (7d). Obtained as a white solid (0.10 g, 87.7% yield). 1H NMR (400 MHz, CDCl3) δ 9.15 (s, 1H), 9.06 (s, 1H), 7.83−7.80 (m, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.94−6.90 (m, 3H), 4.62 (s, 2H), 4.23 (t, J = 4.0 Hz, 2H), 3.89 (s, 3H), 3.78 (t, J = 4.0 Hz, 2H), 3.44 (s, 3H), 1.71 (s, 2H), 1.34 (s, 6H), 0.70 (s, 9H). 13 C NMR (100 MHz, CDCl3) δ 166.7, 166.4, 154.8, 151.0, 144.0, 127.4, 126.9, 126.6, 123.4, 121.0, 114.0, 110.7, 70.7, 68.4, 67.0, 59.2, 57.0, 52.0, 38.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C27H38NO6] 472.2699, found 472.2676. Purity 100% (as determined by RP-HPLC, method A, tR = 27.57 min). Methyl 4-(2-Morpholinoethoxy)-3-(2-(4-(2,4,4-trimethylpentan2-yl)phenoxy)acetamido)benzoate (7e). Obtained as a white solid (0.09 g, 70.8% yield). 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 9.04 (s, 1H), 7.84−7.81 (m, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.93−6.88 (m, 3H), 4.62 (s, 2H), 4.21 (t, J = 4.0 Hz, 2H), 3.90 (s, 3H), 3.69− 3.66 (m, 4H), 2.83 (t, J = 4.0 Hz, 2H), 2.56−2.54 (m, 4H), 1.71 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.7, 166.4, 154.8, 151.0, 144.3, 127.5, 126.7, 123.3, 121.1, 114.1, 110.6, 68.1, 66.9, 66.8, 57.5, 56.9, 54.0, 52.1, 38.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C30H43N2O6] 527.3121, found 527.3096. Purity 100% (as determined by RP-HPLC, method A, tR = 12.90 min). Methyl 4-(Prop-2-ynyloxy)-3-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acetamido)benzoate (7f). Obtained as a white solid (0.10 g, 91.7% yield). 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 9.02 (s, 1H), 7.85−7.83 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 1H), 6.92(d, J = 8.0 Hz, 2H), 4.81 (s, 2H), 4.63 (s, 2H), 3.90 (s, 3H), 2.60 (t, J = 4.0 Hz, 2H), 1.71 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.6, 166.4, 154.8, 149.8, 144.1, 127.4, 127.0, 126.5, 124.1, 121.4, 114.1, 111.1, 67.8, 57.0, 56.8, 52.1, 38.1, 32.3, 31.9, 31.8, 31.6. HRMS [M + H] calcd [C27H34NO5] 452.2437, found 452.2412. Purity 100% (as determined by RP-HPLC, method A, tR = 27.33 min). General Procedure for the Synthesis of 8a−f. EDCI (1.2 equiv) and HOBt (1.2 equiv) were added to a solution of 2-(4-(2,4,4trimethylpentan-2-yl)phenoxy)acetic acid 4l (1.0 equiv), amine (1.0 equiv), and DIPEA (2.5 equiv) in DMF, and the reaction mixture was stirred at room temperature until completion as monitored by TLC. The reaction mixture was diluted with EtOAc brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography. N-(4-(Trifluoromethyl)phenyl)-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acetamide (8a). Obtained as a white solid (0.11 g, 71.54% yield). 1H NMR (CDCl3, 400 MHz) δ 8.43 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 6.4 Hz, 2H), 6.91 (d, J = 6.8 Hz, 2H), 4.62 (s, 2H), 1.72 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H). 13 C NMR (100 MHz, CDCl3) δ 166.8, 154.5, 144.5, 139.9, 127.6, 126.8, 126.4, 126.3, 120.0, 114.1, 67.6, 56.9, 38.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C23H29F3NO2] 408.2150, found 408.2122. Purity 100% (as determined by RP-HPLC, method A, tR = 28.40 min). N-(4-(4-Methylpiperazin-1-yl)phenyl)-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acetamide (8b). Obtained as a white solid (0.12 g, 72.7% yield). 1H NMR (CDCl3, 400 MHz) δ 8.17 (s, 1H), 7.45 (d, J =

9.2 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.92−6.88 (m, 4H), 4.58 (s, 2H), 3.25−3.23 (m, 4H), 2.68−2.67 (m, 4H), 2.42 (s, 3H), 1.71 (s, 2H), 1.31 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.2, 154.7, 148.7, 144.1, 129.2, 127.5, 121.6, 116.6, 114.0, 67.7, 57.0, 55.1, 49.4, 46.1, 38.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C27H40N3O2] 438.3121, found 438.3100. Purity 100% (as determined by RP-HPLC, method A, tR = 12.56 min). 1-(4-(4-(Trifluoromethyl)benzyl)piperazin-1-yl)-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethanone (8c). Obtained as a white solid (0.15 g, 81.0% yield). 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 4.66 (s, 2H), 3.63−3.59 (m, 4H), 3.56 (s, 2H), 2.40− 2.39 (m, 4H), 1.70 (s, 2H), 1.38 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.6, 155.4, 143.2, 142.0, 129.4, 129.1, 127.2, 125.3, 125.2, 113.8, 67.9, 62.2, 57.0, 53.2, 52.7, 45.4, 42.1, 38.0, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C28H38F3N2O2] 491.2885, found 491.2854. Purity 100% (as determined by RP-HPLC, method A, tR = 14.31 min). 1-(4-(Prop-2-ynyl)piperazin-1-yl)-2-(4-(2,4,4-trimethylpentan-2yl)phenoxy)ethanone (8d). Obtained as a white solid (0.11 g, 78.5% yield). 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 4.67 (s, 2H), 3.68−3.63 (m, 4H), 3.29 (t, J = 4.0 Hz, 2H), 2.56−2.51 (m, 4H), 2.25 (t, J = 4.0 Hz, 1H), 1.69 (s, 2H), 1.33 (s, 6H), 0.70 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 166.6, 155.4, 143.3, 127.2, 113.8, 78.1, 73.6, 67.9, 57.0, 51.9, 51.6, 46.8, 45.2, 41.9, 38.0, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C23H35N2O2] 371.2699, found 371.2680. Purity 100% (as determined by RP-HPLC, method A, tR = 11.38 min). Prop-2-ynyl 4-Hydroxy-3-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acetamido)benzoate (8e). Obtained as a white solid (0.10 g, 60.6% yield). 1H NMR (400 MHz, CDCl3) δ = 9.84 (s, 1H), 8.63 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 4.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 2H), 4.91 (d, J = 4.0 Hz, 2H), 4.69 (s, 2H), 2.51 (t, J = 4.0 Hz, 1H), 1.72 (s, 2H), 1.36 (s, 6H), 0.72 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 169.0, 164.9, 154.2, 153.7, 144.8, 129.6, 127.7, 124.6, 124.5, 121.6, 120.2, 114.1, 77.7, 75.1, 67.1, 56.9, 52.4, 38.1, 32.3, 31.8, 31.6. HRMS [M + H] calcd [C26H32NO5] 438.2280, found 438.2247. Purity 100% (as determined by RP-HPLC, method A, tR = 24.56 min). N-(3-Hydroxy-adamantan-1-yl)-2-(4-(2,4,4-trimethylpentan-2yl)phenoxy)acetamide (8f). Obtained as a white solid (0.12 g, 76.9% yield). 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.31 (d, J = 4.0 Hz, 2H), 6.81 (d, J = 12.0 Hz, 2H), 6.31 (s, 1H), 4.36 (s, 2H), 2.28 (s, 2H), 2.02 (s, 2H), 1.99 (s, 4H), 1.70 (s, 6H), 1.58 (s, 2H), 1.34 (s, 6H), 0.70 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 167.4, 154.7, 143.7, 127.4, 113.9, 69.0, 67.6, 57.0, 54.2, 48.9, 44.0, 40.2, 38.0, 34.8, 32.3, 31.7, 31.6, 30.6. HRMS [M + H] calcd [C26H40NO3] 414.3008, found 414.2985. Purity 100% (as determined by RP-HPLC, method A, tR = 23.33 min). General Procedure for the Synthesis of 9a and 9b. The mixture of 3a or 3l (1.0 equiv) and methyl propiolate (2.0 equiv) in toluene was added to Ph3P (1.0 equiv) at −10 °C. The mixture was then allowed to heat up to 115 °C and stirred for 2 h. The solvent was removed under reduced pressure, and the residue was purified by purified by silica gel column chromatography. (E)-3-(4-Adamantan-1-yl-phenoxy)-acrylic Acid Methyl Ester (9a). Obtained as a white solid (2.48 g, 90.6% yield). 1H NMR (300 MHz, CDCl3) δ 7.81 (d, J = 12.3 Hz, 1H), 7.35 (d, J = 9.3 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 5.53 (d, J = 12.3 Hz, 1H), 3.73 (s, 3H), 2.10 (brs, 3H), 1.89−1.88 (m, 6H), 1.77−1.71 (m, 6H). MS (EI) m/z 312 (M+). (E)-3-[4-(2,4,4-Trimethylpentan-2-yl)phenoxy]acrylic Acid Methyl Ester (9b). Purified by silica gel column chromatography, white solid (2.00 g, 71.4% yield). 1H NMR (300 MHz, CDCl3) δ 7.8 (d, J = 12.3 Hz, 1H), 7.36 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 5.53 (d, J = 12.0 Hz, 1H), 3.73 (s, 3H), 1.73 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H). MS (EI) m/z 290 (M+). General Procedure for the Synthesis of 11a−d. The mixture of 3a or 3l (1.0 equiv), anhydrous potassium carbonate (2.0 equiv), and methyl 4-bromobutanoate or methyl α-bromoisobutyrate (2.0 8642



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3-(4-Adamantan-1-yl-phenoxy)-propanoic Acid (13a). Obtained as a white solid (0.45 g, 94.3% yield). 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 7.6 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 4.21 (t, J = 5.6 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.07 (brs, 3H), 1.88−1.86 (m, 6H), 1.77−1.71 (m, 6H). MS (EI) m/z 300 (M+). 3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)propanoic Acid (13b). Obtained as a white solid (0.44 g, 92.4% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 8.0 Hz, 2H), 4.11 (t, J = 4.0 Hz, 2H), 2.65 (t, J = 4.0 Hz, 2H), 1.68 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H). MS (EI) m/z 278 (M+). General Procedure for the Synthesis of 14a−f, 15a,b, and 16a−c. EDCI (1.2 equiv) and HOBt (1.2 equiv) were added to a solution of acid 11a−d or 12a,b or 13a,b (1.0 equiv), amine (1.0 equiv), and DIPEA (2.5 equiv) in DMF, and the reaction mixture was stirred at room temperature until completion as monitored by TLC. The reaction mixture was diluted with EtOAc brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography. Methyl 3-[4-(4-Adamantan-1-yl-phenoxy)butanamido]benzoate (14a).15 See ref 15. Methyl 3-(4-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)butanamido)benzoate (14b). Obtained as a white solid (0.12 g, 82.7% yield). 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.49 (s, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 4.04 (t, J = 6.6 Hz, 4H), 3.91 (s, 3H), 2.61 (t, J = 7.2 Hz, 2H), 2.26−2.19 (m, 2H), 1.70 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.5, 166.9, 156.3, 142.4, 138.4, 130.6, 129.0, 127.0, 125.1, 124.5, 120.8, 113.6, 66.8, 56.9, 52.2, 37.9, 34.0, 32.7, 31.7, 31.6, 25.1. HRMS (EI) m/z calcd for C26H36NO4 [M + H] 426.2644, found 426.2610. Purity 100% (as determined by RP-HPLC, method A, tR = 26.93 min). Methyl 4-Hydroxy-3-(4-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)butanamido)benzoate (14c). Obtained as a white solid (0.10 g, 66.2% yield). 1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.80 (dd, J = 2.0 Hz, 6.4 Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.8 Hz, 2H), 4.06 (t, J = 5.6 Hz, 2H), 3.86 (s, 3H), 2.72 (t, J = 7.2 Hz, 6.8 Hz, 2H), 2.24−2.21 (m, 2H), 1.69 (s, 2H), 1.33 (s, 6H), 0.70 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 173.8, 166.8, 156.1, 153.4, 142.8, 128.7, 127.1, 125.8, 124.3, 121.9, 119.6, 113.6 66.7, 56.9, 52.2, 38.0, 33.6, 32.3, 31.8, 31.6, 25.2. MS (EI) m/z 448 (M + H). HRMS (EI) m/z calcd for C26H36NO5 [M + H] 442.2593, found 442.2570. Purity 100% (as determined by RPHPLC, method A, tR = 25.63 min). Methyl 3-(2-(4-Adamantan-1-yl-phenoxy)-2methylpropanamido)benzoate (14d). Obtained as a white solid (0.12 g, 84.5% yield). 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H) 8.14 (t, J = 2.0 Hz, 1H), 7.95−7.92 (m, 1H), 7.82−7.79 (m, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 9.2 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 3.91 (s, 3H), 2.09 (brs, 3H), 1.88−1.86 (m, 6H), 1.76−1.74 (m, 6H), 1.57 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 173.5, 166.7, 151.3, 147.2, 137.8, 131.0, 129.2, 125.8, 125.4, 124.1, 121.5, 120.6, 81.8, 52.2, 43.3, 36.7, 35.8, 28.9, 25.0. HRMS (EI) m/z calcd for C28H34NO4 [M + H] 448.2488, found 448.2463. Purity 100% (as determined by RPHPLC, method A, tR = 29.60 min). Methyl 3-(2-Methyl-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)benzoate (14e). Obtained as a white solid (0.11 g, 75.8% yield). 1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.14 (t, J = 2.0 Hz, 1H), 7.96−7.93 (m, 1H), 7.82−7.80 (m, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 9.2 Hz, 2H), 3.91 (s, 3H), 1.71 (s, 2H), 1.55 (s, 6H), 1.35 (s, 6H), 0.70 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 173.4, 166.6, 151.1, 145.8, 137.8, 130.9, 129.1, 127.1, 125.4, 124.1, 121.4, 120.7, 81.8, 57.1, 52.1, 38.1, 32.3, 31.7, 31.6, 24.9. HRMS (EI) m/z calcd for C26H36NO4 [M + H] 426.2644, found 426.2623. Purity 100% (as determined by RP-HPLC, method A, tR = 28.88 min). Methyl 4-Hydroxy-3-(2-methyl-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)benzoate (14f). Obtained as a white solid (0.25 g, 83.2% yield). 1H NMR (400 MHz, CDCl3) δ 10.1 (s, 1H), 9.09 (s, 1H), 7.82 (dd, J = 8.6 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.32

equiv) in DMF was stirred overnight at room temperature. The reaction mixture was diluted with EtOAc and subsequentially washed with aqueous sodium bicarbonate, brine, and water. The organic layer was dried over anhydrous MgSO4. The solvent was filtered and evaporated under reduced pressure to afford a crude solid which was purified by silica gel column chromatography to give ester compound. A suspension of ester compound (1.0 equiv) in THF/H2O (1:1) was added to lithium hydroxide monohydrate (3.0 equiv) and stirred overnight at room temperature. The reaction mixture was neutralized with 10% HCl, diluted with EtOAc, and subsequentially washed with aqueous sodium bicarbonate, brine, and water. The organic layer was dried over anhydrous MgSO4. The solvent was filtered and evaporated under reduced pressure to afford a crude solid which was purified by silica gel column chromatography. 4-(4-Adamantan-1-yl-phenoxy)-butanoic Acid (11a). Obtained as a white solid (0.48 g, 88.2% yield). 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 7.6 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 4.00 (t, J = 5.6 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.08−2.12 (m, 2H), 2.07 (brs, 3H), 1.88−1.87 (m, 6H), 1.77−1.76 (m, 6H). MS (EI) [M + H] m/z 315. 2-(4-Adamantan-1-yl-phenoxy)-2-methylpropanoic Acid (11b). Obtained as a white solid (0.92 g, 96.13% yield). 1H NMR (500 MHz, DMSO-d6) δ 12.94 (brs, 1H), 7.23 (d, J = 8.0 Hz, 2H), 6.74 (d, J = 7.2 Hz, 2H), 2.03 (brs, 3H), 1.81−1.80 (m, 6H), 1.75−1.71 (m, 6H), 1.47 (s, 6H). MS (EI) m/z 314 (M+). 4-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)butanoic Acid (11c). Obtained as a white solid (0.47 g, 86.9% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.04 (s, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.82 (d, J = 8.0 Hz, 2H), 3.90 (t, J = 4.0 Hz, 2H), 2.35 (t, J = 4.0 Hz, 2H), 1.98 (t, J = 4.0 Hz, 2H), 1.71 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H). MS (EI) m/z 292 (M+). 2-Methyl-2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic Acid (11d). Obtained as a white solid (0.50 g, 96.1% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.46 (brs, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.74 (d, J = 7.2 Hz, 2H), 1.66 (s, 2H), 1.45 (s, 6H), 1.29 (s, 6H), 0.66 (s, 9H). MS (EI) m/z 292 (M+). General Procedure for the Synthesis of 12a and 12b. A suspension of ester 9a or 9b (1.0 equiv) in THF/H2O (1:1) was added to lithium hydroxide monohydrate (3.0 equiv) and stirred overnight at room temperature. The reaction mixture was neutralized with 10% HCl, diluted with EtOAc, and subsequentially washed with aqueous sodium bicarbonate, brine, and water. The organic layer was dried over anhydrous MgSO4. The solvent was filtered and evaporated under reduced pressure to afford a crude solid which was purified by silica gel column chromatography. (E)-3-(4-Adamantan-1-yl-phenoxy)-acrylic Acid (12a). Obtained as a white solid (0.91 g, 95.8% yield). 1H NMR (300 MHz, DMSD-d6) δ 12.07 (s, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 5.4 Hz, 2H), 7.11 (d, J = 5.1 Hz, 2H), 5.44 (d, J = 7.2 Hz, 1H), 2.06 (brs, 3H), 1.85− 1.83 (m, 6H), 1.75−1.72 (m, 6H). MS (EI) m/z 298 (M+). (E)-3-[4-(2,4,4-Trimethylpentan-2-yl)phenoxy]acrylic Acid (12b). Obtained as a white solid (0.90 g, 94.7% yield). 1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 11.7 Hz, 1H), 7.37 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 5.50 (d, J = 12.3 Hz, 1H), 1.73 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H). MS (EI) m/z 276 (M+). General Procedure for the Synthesis of 13a and 13b. A solution of acrylic acid methyl ester 9a or 9b (1.0 equiv) in methanol was treated with 10% Pd/C (10% w/w). The reaction was subjected to hydrogenation under 1 atm hydrogen gas pressure at room temperature, and the reaction mixture was stirred overnight. After completion of the reaction, the mixture was filtered through a Celite pad and concentrated. The resulting residue was purified by silica gel column chromatography. A suspension of methyl ester 10a or 10b (1.0 equiv) in THF/H2O (1:1) was added to lithium hydroxide monohydrate (4.0 equiv) and stirred overnight at room temperature. The reaction mixture was neutralized with 10% HCl, diluted with EtOAc, and subsequently washed with aqueous sodium bicarbonate, brine, and water. The organic layer was dried over anhydrous MgSO4. The solvent was filtered and evaporated under reduced pressure to afford a crude solid which was purified by silica gel column chromatography. 8643



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(d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 3.87 (s, 3H), 1.72 (s, 2H), 1.57 (s, 6H), 1.37 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 175.7, 166.3, 153.2, 150.5, 146.5, 128.9, 127.2, 124.9, 124.2, 122.2, 122.0, 119.7, 81.7, 57.1, 52.0, 38.2, 32.3, 31.7, 31.5, 24.9. HRMS [M + H] calcd [C26H36NO5] 442.2515, found 442.2576. Purity 100% (as determined by RP-HPLC, method A, tR = 16.29 min). (E)-3-[3-(4-Adamantan-1-ylphenoxy)acryloylamino]benzoic Acid Methyl Ester (15a).25 See ref 25. (E)-3-{3-[4-(2,4,4-Trimethylpentan-2-yl)phenoxy]acrylamido}benzoic Acid Methyl Ester (15b).25 See ref 25. Methyl 3-[3-(4-Adamantan-1-y-phenoxy)propanamido]benzoate (16a).25 See ref 25. Methyl 4-Hydroxy-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)benzoate (16b). Obtained as a white solid (0.01 g, 75.0% yield). 1H NMR (CDCl3, 400 MHz) δ 9.74 (s, 1H), 8.36 (s, 1H), 7.81 (dd, J1 = 2.0 Hz, J 2 = 2.0 Hz, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 2H), 4.35 (t, J = 5.4 Hz, 2H), 3.88 (s, 3H), 2.94 (t, J = 5.6 Hz, 2H), 1.71 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.7, 166.4, 155.3, 153.2, 143.7, 128.9, 127.3, 125.5, 124.2, 122.1, 119.7, 113.9, 63.8, 56.9, 52.1, 38.0, 36.8, 32.3, 31.7, 31.6. HRMS (EI) m/z calcd for C25H34NO5 [M + H] 428.2359, found 428.2431. Purity 99.99% (as determined by RP-HPLC, method A, tR = 24.09 min). Methyl 3-(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)propanamido)benzoate (16c). Obtained as a white solid (0.13 g, 88.4% yield). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.07 (t, J = 1.6 Hz, 1H), 7.88−7.86 (m, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 9.2 Hz, 2H), 4.32 (t, J = 4.0 Hz, 2H), 3.89 (s, 3H), 2.85 (t, J = 4.0 Hz, 2H), 1.70 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 169.2, 166.7, 155.6, 143.4, 138.0, 130.9, 129.2, 127.3, 125.4, 124.4, 120.8, 113.8, 64.1, 56.9, 52.2, 38.0, 37.8, 32.3, 31.8, 31.6, 29.7. HRMS (EI) m/z calcd for C25H34NO4 [M + H] 412.2488, found 412.2463. Purity 100% (as determined by RP-HPLC, method A, tR = 26.00 min). Cell Culture. Human colorectal carcinoma HCT116 cells were cultured in a 5% CO2 atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (Gibco, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). A hypoxic condition was achieved by replacing the cells with 1% O2, 94% N2, and 5% CO2 in a multigas incubator (Sanyo, Osaka, Japan). MDH Activity Assay. The enzyme activity of MDH1 and MDH2 was determined by oxaloacetate-dependent NADH oxidation assay. The reaction was performed in 100 mM potassium phosphate buffer (pH 7.4) with 0.25 nM human recombinant MDH1 (BioVision) or MDH2 (ref. JEM 2014), 200 μM oxaloacetic acid, and 200 μM NADH. In the case of the kinetic assay, the reaction was performed with 0.25 nM rhMDH2, 600 μM oxaloacetic acid, and various concentrations of NADH (60, 75, 100, 150, and 300 μM). After 30 min, the NADH concentration in the mixture was determined by measuring absorbance at 340 nm. The Vmax and Km were determined from double-reciprocal Lineweaver−Burk plots using Sigmaplot 13.0 (Systat Software), and the velocity was plotted against the NADH concentration. HRE-Luciferase Reporter Assay. HCT116 cells stably expressing a hypoxia response element (HRE)-dependent firefly luciferase reporter construct (HRE-Luc) and a CMV-Renilla luciferase reporter construct were established with Cignal Lenti Reporter assay system (SABiosciences, Frederick, MD, USA) according to the manufacturer’s instructions. Cells were incubated with drugs for 12 h under normoxic or hypoxic conditions. After removing the supernatant, luciferase activity was measured with a Dual-Luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions with a Victor X Light luminescence reader (PerkinElmer, Boston, MA, USA). Immunoblot Analysis. Cells were lysed with a RIPA buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 5 mM sodium fluoride, 10 μg/mL phenylmethylsulfonylfluoride (PMSF), and 1 mM sodium vanadate) for 15 min at 4 °C and

centrifuged at 13000 rpm for 15 min. Lysates were then boiled for 5 min in 5× sample buffer (50 mM Tris, pH 7.4, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 4% 2-thioethanol, and 50 μg/mL Bromophenol blue) at a ratio of 4:1. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred to PVDF membranes (Millipore, Billerica, MA), and immunoblotted with the following antibodies: HIF-1α (BD Transduction Laboratories, San Diego, CA, USA), P-ACC (Cell Signaling, Danvers, MA, USA), ACC (Cell Signaling), P-AMPK (Cell Signaling), AMPK (Cell Signaling), P-mTOR (Cell Signaling), mTOR (Cell Signaling), and β-actin (Abcam, Cambridge, UK). Protein expression was visualized on Kodak Biomax X-ray film (Kodak, Rochester, NY, USA). RT-PCR. Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First, 1 μg of total RNA was reverse transcribed using TOPscript RT DryMIX kit (Enzynomics, Seoul, Korea). The PCR primers used were 5′-GCAGCCAGATCTCGGCGAAG-3′ (forward) and 5′-CTGTGTCCAGTTAGTTCAAA-3′ (reverse) for HIF-1α, 5′-GGTGGACATCTTCCAGGAGT-3′ (forward) and 5′-GGCTTGTCACATCTGCAAGT-3′ (reverse) for VEGF, 5′-ATGATTGGCTCCTTCTCTGT-3′ (forward) and 5′-TCAGCATCTCAAAGGACTTG3′ (reverse) for GLUT1, 5′-GGCCAGGTGGACTTCTA-3′ (forward) and 5′-TCATTGTGTCGGTTTCTGAT-3′ (reverse) for PDK1, and 5′-CATCGTGGCTAAACAGGTAC-3′ (forward) and 5′-GCACGACCTTGAGGGCAGC-3′ (reverse) for RPL13A. Then the amplified products were subjected to electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. Measurement of Oxygen Consumption. The oxygen consumption rate (OCR) in cells was measured using the XF24 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA). After determining basal OCR of HCT116 cells, oligomycin (1 μM), carbonylcyanide p-trifluoromethoxyphenylhydrazone (0.5 μM), and rotenone (1 μM)/antimycin A (1 μM) were added sequentially, and OCR was determined following each addition. The oxygen consumption of each respiratory chain was measured using an Oxygraph (Hansatech Instruments, Norfolk, UK). After permeabilization of HCT116 cells (1 × 107 cells) with digitonin (30 μM), substrates and inhibitors for each complex were added. The substrates and inhibitors used were: pyruvate (5 mM), malate (5 mM), rotenone (1 μM), succinate (5 mM), antimycin A (1 μM), ascorbate (5 mM), tetramethyl-p-phenylenediamine (TMPD) (2 mM), and KCN (1 mM). Detection of Oxygen Tension. The intracellular oxygen tension was detected using a hypoxia-detecting probe mono azo rhodamine (MAR, Goryo Chemical, Japan). HCT116 cells were incubated for 6 h with drug and 500 nM MAR under hypoxia and then analyzed with an IncuCyte live cell imaging system (Essen BioScience, Ann Arbor, MI, USA). Xenograft Model. The in vivo antitumor activity of compounds 5i, 7c, 15a, and 16c was determined in a HCT116 xenograft mouse model. All animal experimental protocols were approved by the bioethics committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Five-week-old female nude mice were subcutaneously inoculated with 5 × 106 HCT116 cells in the right flank. When the tumor volume reached 80−100 mm3, drugs were administered intraperitoneally once a day for 14 days. The drug was formulated in a mixture of 10% DMSO, 10% Cremophor EL, and 80% distilled water. Tumor volume was measured every other day with calipers and calculated using following equation: V (mm3) = (length × width × height) × 0.5. Statistical Analysis. The statistical significance of the results was analyzed using the Student’s t test for unpaired observations and Dunnett’s test for multiple comparisons.

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Protein−protein Blast of MDH1 and MDH2, structural and sequence alignment of the active site regions from MDH1 and MDH2, molecular docking study of compounds, effects of compounds on intracellular oxygen tension, effects on mitochondria complex II, III, and IV activity, body weight change in HCT116 xenograft model, in vivo antitumor effect of compound 5i and 15a in HCT116 xenograft model, and in vitro water solubility of 14b and 16c (PDF) Molecular formula strings (CSV)

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Corresponding Authors

*For K.L.: phone, 82-31-961-5214; fax, 82-31-961-5206; Email, [email protected]. *For M.W.: phone, 82-42-860-4174; E-mail, [email protected]. kr. ORCID

Hyun Seung Ban: 0000-0002-2698-6037 Kyeong Lee: 0000-0002-5455-9956 Author Contributions

This manuscript was written with contributions from all the coauthors. All coauthors have approved the final version of the manuscript. R.N and H.S.B. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by National Research Foundation (NRF) grants (2012M3A9C1053532 and 2015M3A6A4065734), Health Technology R&D (HI13C2162), and the KRIBB Initiative of the Korea Research Council of Fundamental Science and Technology.

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ABBREVIATIONS USED AOA, amino-oxyacetic acid; ERBB2, Erb-B2 receptor tyrosine kinase 2; ETC, electron transport chain; GLUT1, glucose transporter 1; GOT, glutamate oxaloacetate transaminase; HIF1, hypoxia-inducible factor-1; HRE, hypoxia-response element; MDH, malate dehydrogenase; mTOR, mechanistic target of rapamycin; OCR, oxygen consumption rate; PDAC, pancreatic ductal adenocarcinoma; PDK1, pyruvate dehydrogenase kinase 1; TCA, tricarboxylic acid

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REFERENCES

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