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

Article abstract of DOI:10.1021/acs.jmedchem.7b01231

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-(2,4,4-trimethylpentan-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.

Full text of DOI:10.1021/acs.jmedchem.7b01231

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Article
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, Kyusik Jang, Inhyub Kim, Xuezhen Xu, Dipesh Harmalkar, Seong-Ah
Shin, Minkoung Kim, Bo-Kyung Kim, Jaehyung Park, Bonsu Ku, Sujin Oh, Misun Won, and Kyeong Lee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01231 • Publication Date (Web): 09 Oct 2017
Downloaded from http://pubs.acs.org on October 9, 2017
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Methyl 3ꢀ(3ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀ
yl)phenoxy)ꢀpropanamido)benzoate as a Novel and
Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor
Targeting Cancer Metabolism
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Ravi Naik , Hyun Seung Ban , Kyusic Jang , Inhyub Kim , Xuezhen Xu , Dipesh
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Harmalkar , SeongꢀAh Shin , Minkyoung Kim , BoꢀKyung Kim , Jaehyung Park , Bonsu ku ,
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Sujin Oh, Misun Won *, and Kyeong Lee *
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College of Pharmacy, Dongguk University, Goyang, 10326, Korea
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Metabolic Regulation Research Center, Korea Research Institute of Bioscience and
Biotechnology, Daejeon, 34141, Korea
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Biomolecular Science, University of Science and Technology, Daejeon, 34113, Korea
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Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience and
Biotechnology, Daejeon, 34141, Korea
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Functional Genomics, University of Science and Technology, Daejeon, 34113, Korea
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Disease Target Structure Research Center, Korea Research Institute of Bioscience and
Biotechnology, Daejeon, 34141, Korea
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New Drug Development Center, Asan Medical Center, Seoul, 05505, Korea
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KEY WORDS: Hypoxia, Malate dehydrogenase 1 and 2, Structureꢀactivity relationship, Cancer
metabolism
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ABSTRACT
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Previously, we reported a hypoxiaꢀinducible factor (HIF)ꢀ1 inhibitor LW6 containing an
aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a
(
chemical biology approach. Structureꢀactivity 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 HIFꢀ1 inhibition.
We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to
target
cancer
metabolism,
and
selected
methylꢀ3ꢀ(3ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀ
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 hypoxiaꢀinduced HIFꢀ1α 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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INTRODUCTION
Metabolic alterations, including aerobic glycolysis, high fatty acid synthesis, and rapid glutamine
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metabolism, are characteristics of cancer cells. Notably, a high glycolysis rate is important for
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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
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during glycolysis into the mitochondria. The malate–aspartate shuttle is operated by two pairs
of enzymes, malate dehydrogenases (MDHs) and glutamate oxaloacetate transaminases (GOTs),
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which are localized to the mitochondria and cytoplasm. Aminoꢀoxyacetic acid (AOA), a specific
inhibitor of the malate–aspartate shuttle, impairs the conversion of glucose into tricarboxylic acid
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(TCA) cycle products and decreases the proliferation of breast adenocarcinoma cells. Moreover,
mitochondrial GOT2 acetylation stimulates the malate–aspartate NADH shuttle activity to
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support ATP production needed for pancreatic tumor growth. Knockdown of the GOT1/2 genes
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inhibits cell proliferation and colony formation of pancreatic cancer cells.
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
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the NAD/NADH cofactor system.
In the cytoplasm, MDH1 reduces OAA to malate while
+
oxidizing NADH to NAD . In the malateꢀaspartate 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.
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(
Insert Figure 1 here)
Although the involvement of MDH in cancer is not currently a major focus in the field of
cancer metabolism, there are evidences for the cancerꢀassociated functions of MDH1 and MDH2.
Glutamineꢀdependent pancreatic ductal adenocarcinoma (PDAC) cells require MDH1 to
maintain their cellular redox state by reprograming glutamine metabolism, and MDH1
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knockdown inhibited the viability of PDAC cells. In addition, knockdown of MDH1 decreased
the viability of BT474, ErbꢀB2 receptor tyrosine kinase 2 (ERBB2)ꢀpositive breast cancer cells,
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by suppressing the fatty acid synthesis pathway. In another study, MDH1 stabilized by glucose
depletion translocates to the nucleus and interacts with p53 to regulate the transcription of the
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p53ꢀdependent metabolic checkpoint. It has also been shown that high expression of MDH2 is
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associated with shorter relapseꢀfree survival and chemoresistance in prostate cancer patients.
MDH2 knockdown suppresses proliferation and enhances docetaxel sensitivity via induced
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metabolic inefficiency of prostate cancer cells.
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 substrateꢀ
associated residues of human MDH1 were wellꢀmatched 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 3ꢀ(2ꢀ(4ꢀ((3r,5r,7r)ꢀadamantanꢀ1ꢀyl)phenoxy)acetamido)ꢀ4ꢀhydroxybenzoate
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(
LW6, 1), a hypoxiaꢀinducible factor (HIF)ꢀ1α inhibitor, using its chemical probe, propꢀ2ꢀynꢀ1ꢀ
yl
3ꢀ(2ꢀ(4ꢀ((3r,5r,7r)ꢀadamantanꢀ1ꢀyl)ꢀ2ꢀ(3ꢀ(trifluoromethyl)ꢀ3Hꢀdiazirinꢀ3ꢀ
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yl)phenoxy)acetamido)ꢀ4ꢀhydroxybenzoate (2, Figure 2). 1 inhibits MDH2 activity by binding
the NADꢀbinding site in a competitive fashion. Cancer cells treated with 1 showed reduced
oxygen consumption and increased local cellular oxygen tension, thereby promoting HIFꢀ1α
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degradation under hypoxic conditions. HIFꢀ1α correlates with the aggressive growth of solid
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tumors, resistance to radiation and chemotherapy, and poor clinical outcomes.
The HIFꢀ1α/β
complex binds to hypoxiaꢀresponsive elements (HREs) in the promoter of hypoxiaꢀresponsive
genes, including vascular endothelial growth factor (VEGF) and glucose transporter 1
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(
GLUT1).
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 structureꢀactivity
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
HIFꢀ1 and cancer cell metabolism. Finally, we identified a methyl 3ꢀ(3ꢀ(4ꢀ(2,4,4ꢀ
trimethylpentanꢀ2ꢀyl)phenoxy)propanamido)benzoate (16c) as a dual inhibitor of MDH1/2 that
showed significant tumor reduction in a colorectal cancer xenograft model.
(Insert Figure 2 here)
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RESULTS AND DISCUSSION
Chemistry. To develop novel MDH inhibitors, copound 1 derivatives were synthesized as
indicated in Schemes 1–4. The carboxylic acid derivatives 4a–l were prepared using a twoꢀstep
synthesis. The hydroxyl groups of phenols 3a–l were subjected to alkylation using ethyl
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chloroacetate in the presence of potassium carbonate (K CO ) in dimethylformamide (DMF) and
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the resulting ethyl ester was hydrolyzed with lithium hydroxide to obtain 4a–l. These resulting
carboxylic acids were subsequently used to prepare amide derivatives (5a–l) using the standard
amide coupling method (Scheme 1).
(Insert scheme 1 here)
Coupling of the commercially available methyl 3ꢀaminoꢀ4ꢀhydroxybenzoate with 4ꢀ
adamantyl phenoxyacetic (4a) or 2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetic acid (4l) in the
.
presence of Nꢀ(3ꢀdimethylaminopropyl)ꢀNʹꢀethylcarbonate hydrochloride (EDC HCl), 1ꢀ
hydroxybenzotriazole (HOBT), and N,Nꢀdiisopropylethylamine (DIPEA) yielded the
hydroxylated analogs, 1 or 6, respectively. Oꢀalkylation of 1 and 6 with alkyl or aryl halide
produced 4ꢀadamantyl 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.
(Insert schemes 2 and 3 here)
Some of the key intermediate (E)ꢀphenoxyacrylic acid derivatives (12a and 12b) were
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synthesized from phenols 3a and 3l using previously described procedures (Scheme 4). The
hydroxyl groups of phenols 3a and 3l were alkylated with methyl propiolate in the presence of
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triphenylphosphine (PPh ) in toluene, and the resulting (E)ꢀphenoxyacrylic methyl esters (9a and
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9b) were hydrolyzed with lithium hydroxide to form 12a and 12b. Other key intermediates, such
as the 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 threeꢀcarbon linker part, phenols 3a and
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l were alkylated with methyl 4ꢀbromobutanoate in the presence of K CO in DMF followed by
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hydrolysis to obtain 11a and 11c. A series of carboxylic acid derivatives were coupled with
methyl 3ꢀamino benzoate or methyl 3ꢀaminoꢀ4ꢀhydroxybenzoate in the presence of EDC, HOBT,
and DIPEA to produce the amide derivatives 14a–f, 15a–b, 16a–c, as shown in Scheme 4.
(Insert Scheme 4 here)
MDH Enzyme Assay. The newly synthesized compounds were evaluated for their ability to
inhibit human recombinant MDH1 and MDH2 enzymatic activity using an oxaloacetateꢀ
dependent 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 4ꢀadamantyl 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 4ꢀadamantyl (aryloxyacetylamino)benzoic ester
analogue 5a, which induced mild inhibitory activity against both MDH1 and MDH2 enzymes
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(
IC = 23.48 ± 1.64 and 15.97 ± 2.02 ꢁM, respectively). Substitution of 4ꢀadamantyl group with
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electron withdrawing groups, such as NO and CF (5b and 5c), did not yield any MDH1/MDH2
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inhibitory activity (>40 ꢁM). Then, the 4ꢀadamantyl group of 5a was modified with a similar
lipophilic group (tꢀbutyl) to yield 5d, which exhibited moderate inhibitory activity (IC = 10.74
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± 1.31) against MDH1, but not against MDH2. The compounds substituted with a simple alkyl
chain (methyl to nꢀpropyl chain) 5e–g failed to inhibit MDH enzymes up to 40 ꢀM. Interestingly,
5h and 5i with nꢀbutyl or nꢀpentyl moiety exhibited medium inhibitory activity against MDH1
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IC = 9.58 ± 0.23, 6.79 ± 0.21 ꢁM, respectively). Compounds 5d, 5h and 5i showed some
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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 2,4,4ꢀtrimethylpentanyl substituent
showed dual inhibition against both MDH1 and MDH2. As listed in table 1, compound 5l
demonstrated considerable inhibition against MDH1 and MDH2 with IC values of 6.65 ± 0.65
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and 8.92 ± 1.39 ꢁM, 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 2,4,4ꢀtrimethylpentanyl substituent or an
equivalent for part A is important for the effective inhibition of MDH enzymes (Table 1).
Insert table 1
Based on 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
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potency against both MDH1 and MDH2. Notably, in this study we found that a known MDH2
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inhibitor 1 showed better activity against MDH1 (IC = 1.10 ± 1.10 µM) than MDH2, whereas
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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
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IC = 1.14 ± 0.09 and 1.89± 0.18 ꢁM, respectively). This finding suggested that OHꢀcontaining
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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 amineꢀsubstituted alkyl
groups or a methoxyꢀsubstituted alkyl group led to analogues 7a–b and 7d–e, which resulted in
the loss of activity. Methoxy compound 7c with 2,4,4ꢀtrimethylpentane scaffold showed 12ꢀfold
selective MDH1 activity over MDH2 (IC = 2.27 ± 0.32 and 27.47± 0.16 ꢁM, respectively). By
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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).
Insert 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
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direct attachment to an aromatic ring, will determine the relative involvement of these two
processes. CF ꢀphenyl compound 8a exhibited less activity than 6 against both MDH1 and MDH
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with IC values of 3.44 ± 0.43 and 1.53 ± 0.08 ꢁM, respectively. Notably, 2ꢀ(4ꢀ(adamantanꢀ1ꢀ
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yl)phenoxy)ꢀ1ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ethanone
(IDFꢀ11774) is a potent HIFꢀ1 inhibitor that is
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approved as a clinical candidate for cancer therapy.
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, piperazineꢀcontaining
compounds 8b and 8c showed moderate activity against both MDH1 and MDH2. Compound 8d
with 1ꢀ(propꢀ2ꢀynyl)piperazine completely lost activity. Interestingly, propargyl ester (8e)
showed potency against MDH1 with an IC value of 0.92 ± 0.28 ꢁM. The hydroxylꢀadamantane
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containing amide 8f showed weak inhibitory activity against both MDH1 and MDH2 (Table 3).
Insert 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 2,4,4ꢀtrimethylpentanyl 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 ꢁM, respectively). Oneꢀ to twoꢀcarbon
homologations (14a–b, 16a, and 16c) to the acetamide 5a and 5l resulted in a significant increase
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in MDH1 and MDH2 enzyme potency, as shown in Table 4. In contrast, oneꢀ to twoꢀcarbon
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 (IC = 1.07 ± 0.07 and 1.06 ± 0.03 ꢁM, respectively). It was found
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that homologation (14a and 16a) for the 4ꢀadamantyl derivatives resulted in lower potency than
that of the trimethylpentane derivatives (14a and 16c). According to previous studies, various
compounds with 4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy pendent groups were reported to be
useful for the treatment of some chronic diseases, including inflammation, diabetes,
30,31
hyperglycemia, and cancers.
For the 2,4,4ꢀtrimethylpentaneꢀbased compounds, the
introduction of a more conformationally constrained oxyacrylic amide linker, as represented by
15b, induced better MDH inhibitory activity. By contrast, the corresponding 4ꢀadamantyl
compound (15a) showed MDH2ꢀselectivity (6ꢀfold) 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 oneꢀcarbon 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 ꢁM,
respectively. Similarly, compound 14c containing two carbon homologated linkers with OH
group was less active against MDH1 and MDH2 enzymes compared to compound 14b (IC =
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1.18 ± 0.08 and 1.11 ± 0.04 ꢁM, 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 IC = 3.00 ± 0.03 and 10.98 ± 0.79
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M, respectively, than the corresponding OHꢀfree compound 14e. This finding suggests that the
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MDH1 and MDH2 inhibitory activities for compounds with or without a OH group at part B was
dependent on the carbon linker.
(Insert Table 4 here)
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To date, several MDH inhibitors have been reported. A thyroid hormone thyroxine (T4) was
8
,32
described to suppresses MDH2 activity with an IC50 value of 43.6 µM.
A staurosporine
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aglycone K252c also inhibits MDH activity with an IC50 value of 8 µM. However, this
compound mainly suppresses PKC and PKA with IC values of 2.45 and 25.7 µM, respectively.
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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 offꢀtarget effects of MDH inhibitors because compounds may
have multiꢀtargeting properties. It may affect the activity of metabolic enzymes involving
NADHꢀNAD conversion. Additionally, it is not easy to increase the selectivity of the compound
towards MHD1 or MDH2 because they share common NADH binding sites of 63% similarity
based on structural superimposition (Fig. S2). Therefore, based on 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
linkerꢀcontaining 15a was selected as a selective MDH2 inhibitor. According to a docking study
using Xꢀray 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).
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CellꢀBased HRE Reporter Assay. Next, we assessed whether the inhibition of MDH1 or
MDH2 enzymes was related to the outputs of the HREꢀluciferase activity, which indicates HIFꢀ
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α inhibition (Table 5). The hypoxiaꢀinduced HIFꢀ1 activities of the representative compounds
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were determined using a cellꢀbased HRE reporter assay in HCT116. We followed a previously
described assay protocol under standard hypoxic assay conditions (1% O , 94% N , and 5%
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CO2). 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
HIFꢀ1 activation (> 40 µM). The MDH1 inhibitors 5i and 7c did not show potent effects in the
HRE assay. However, the MDH2ꢀselective inhibitor 15a and the dual MDH1/2 inhibitors 14b
and 16c strongly suppressed HREꢀluciferase activity. This finding is consistent with the previous
report that inhibition of MDH2 suppresses mitochondrial respiration and increases oxygen
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content, thereby stimulating HIFꢀ1α degradation in cancer cells. Then, to further confirm the
role of MDH1, MDH2, or both MDH1 and MDH2 activity in HIFꢀ1α accumulation, intracellular
oxygen content was determined using a mono azo rhodamine (MAR) fluorescent hypoxia probe,
(
Figure S4, Supporting Information). Based on the MDH enzyme and cellꢀbased HRE assays
results, the MDH1 selective agents (5i and 7c) did not affect the fluorescence of MAR. However,
4b and 16c that showed MDH2 inhibitory activity significantly reduced MAR fluorescence,
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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
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supplies the NADPH required for the synthesis and elongation of fatty acids. It has been
reported that the inhibition of MDH1 induced anticancer activity by suppressing fatty acid
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synthesis. Moreover, MDH2 inhibition reduced the loss of NADH and inhibited mitochondrial
respiration, resulting in increased oxygen contents and stimulation of HIFꢀ1α degradation.
MDH2 knockdown may induce anticancer activity by reducing HIFꢀ1α accumulation and
sensitizing chemoresistant cancer cells.
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In addition to the specific roles of MDH1 and MDH2, both enzymes are associated with the
malateꢀaspartate shuttle. This process involves a reaction between NADH and oxaloacetate in the
cytosol, and the malate is transported into the mitochondria and subsequently reꢀoxidized by the
electron transfer chain. The malateꢀaspartate shuttle facilitates the translocation of electrons
across the mitochondrial membrane, thereby 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 HIFꢀ1. To elucidate the inhibitory mechanism of compound 16c
on MDH1 and MDH2 activity, an enzyme kinetics study was performed using various NADH
concentrations. Doubleꢀreciprocal plots of enzyme kinetics revealed a competitive mode of
MDH1 and MDH2 inhibition by compound 16c with K values of 0.74 and 0.95 µM, respectively
i
(Figure 3A and B). Additionally, we found that the trimethylpentane derivative 16c showed
reasonable water solubility (94.4 ± 0.50 ꢁM at pH 7.4), which was superior to that of another
dual MDH1/2 inhibitor, 14b (45.8 ± 1.45 ꢁM at pH 7.4, Table S1, Supporting Information) and,
therefore, merits further evaluation as a novel anticancer agent for targeting cancer cell
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Effects on HIFꢀ1α Pathway and Oxygen Tension. Following the identification of a novel
MDH1/2 dual inhibitor, we examined the effects of compound 16c on HIFꢀ1α signaling in
HCT116 cells. Under hypoxic conditions, treatment with compound 16c suppressed hypoxiaꢀ
induced HIFꢀ1α accumulation in a concentrationꢀdependent manner (Figure 4A). As expected,
compound 16c inhibited the expression of HIFꢀ1α target genes, including VEGF, GLUT1, and
pyruvate dehydrogenase kinase 1 (PDK1), without affecting the levels of HIFꢀ1α mRNA (Figure
4B).
(
Insert Figure 4 here)
To characterize the inhibitory mechanism of compound 16c against HIFꢀ1α 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 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 acetylꢀCoA carboxylase (ACC), a
downstream target of AMPK (Figure 5B and 5C). Furthermore, staining with the hypoxiaꢀ
detecting probe MAR showed that compound 16c increased intracellular oxygen tension,
presumably because of reduced OCR, resulting in HIFꢀ1α degradation under hypoxic conditions
(Figure 5D).
(Insert Figure 5 here)
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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
complexꢀlinked respiratory process was regulated by the addition of specific substrates and
inhibitors (Figure 6A). Under these experimental conditions, compound 16c significantly
inhibited complex Iꢀdependent respiration (Figure 6B) without affecting complex II/IIIꢀ and IVꢀ
dependent respiration (Figure S5, Supporting Information). These findings suggest that the
inhibition of MDHs by compound 16c reduced complex Iꢀdependent mitochondrial respiration.
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(Insert Figure 6 here)
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 [q.d.]) for 14 days and significant tumor growth
inhibition (63.4%) was observed (Figure 7). Moreover, compound 16c did not cause significant
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 MDH1ꢀselective compound 5i and MDH2ꢀselective 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 MDH1ꢀ and MDH2ꢀselective and MDH1/MDH2 dual
inhibitors. We demonstrated that MDH2, but not MDH1 inhibitors, suppressed HREꢀluciferase
activity and the growth of cancer cells. Considering that dual inhibition of MDH enzymes may
be a potent approach to target cancer cell metabolism, the trimethylpentaneꢀcontaining 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 downꢀregulate
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 hypoxiaꢀinduced HIFꢀ1α accumulation and the expression of HIFꢀ1α 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 tumorꢀsuppressing agent, suggesting that the
simultaneous inhibition of both MDH1 and MDH2 enzymes could be a crucial therapeutic
strategy.
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EXPERIMENTAL SECTION
Chemistry: General
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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
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atmosphere of dried argon in flameꢀdried glassware. The proton nuclear magnetic resonance ( Hꢀ
NMR) spectra were determined on a Varian (300 MHz, 400 MHz or 500 MHz) spectrometer
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(
Varian Medical Systems, Inc., Palo Alto, CA, USA). CꢀNMR 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 highꢀ
resolution mass spectrometry (HRMS) (electron ionization MS) obtained on a JMSꢀ700 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, thinꢀlayer
chromatography on 0.25ꢀmm silica plates (E. Merck; silica gel 60 F254) was used to monitor
reactions. Final product purity was checked by reversed phase highꢀpressure liquid
chromatography (RPꢀHPLC), performed on a Waters Corp. HPLC system equipped with an
ultraviolet (UV) detector set at 254 nm. The mobile phases used were: (A) H O containing 0.05%
2
trifluoroacetic acid; and (B) CH CN. HPLC employed a YMC Hydrosphere C18 (HSꢀ302)
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column (5ꢀµm particle size, 12ꢀnm 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. 3aꢀl) were purchased from Sigma
aldrich, TCI, Ark pharma and Combi blocks. The compound purity was assessed using method A,
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a gradient of 25% B to 100% B in 35 minutes. The purity of all biologically evaluated
compounds was >95% in method A.
General procedure for the synthesis of 5aꢀl: EDCI (1.2 equiv) and HOBt (1.2 equiv) were
added to a solution of appropriate phenoxyacetic acid 4aꢀl (1.0 equiv), methyl 3ꢀaminobenzoate
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(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 MgSO , filtered, and
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concentrated in vacuo. The resulting residue was purified by silica gel column chromatography.
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Methyl 3ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀylꢀphenoxy)acetamido)benzoate (5a)
Methyl 3ꢀ(2ꢀ(4ꢀnitrophenoxy)acetamido)benzoate (5b): Obtained as a white solid (0.21 g, 64.2%
1
yield). HꢀNMR (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),
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.95 (s, 2H), 3.86 (s, 3H); CꢀNMR (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 [C H N O ]:
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29.0852, Found: 329.0676; Purity 97.3% (as determined by RPꢀHPLC, method A, t = 14.62
R
min).
Methyl 3ꢀ(2ꢀ(4ꢀ(trifluoromethyl)phenoxy)acetamido)benzoate (5c): Obtained as a white solid
1
(
0.01g, 40.8% yield). HꢀNMR (CDCl , 400 MHz) δ 8.32 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0
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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 =
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.4 Hz, 2H), 4.68 (s, 2H), 3.93 (s, 3H), 1.62 (s, 2H); CꢀNMR (100 MHz, CDCl )
δ
166.5, 159.1,
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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]
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calcd [C H F NO ]: 354.0875, found: 354.0929; Purity 99.99% (as determined by RPꢀHPLC,
17
15
3
4
method A, t = 17.70 min).
R
2
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Methyl 3ꢀ(2ꢀ(4ꢀtertꢀbutylphenoxy)acetamido)benzoate (5d)
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Methyl 3ꢀ(2ꢀ(pꢀtolyloxy)acetamido)benzoate (5e): Obtained as a white solid (0.10 g, 40.4%
1
yield). HꢀNMR (400 MHz, CDCl )
δ
8.51 (s, 1H), 8.09 (t, J = 8.0 Hz, 1H), 7.99 (m, 1H), 7.80 (d,
3
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),
13
3
.89 (s, 3H), 2.29 (s, 3H); CꢀNMR (100 MHz, CDCl )
δ
166.7, 166.5, 154.9, 137.2, 131.8,
3
130.9, 130.3, 129.2, 125.8, 124.5, 120.9, 114.6, 67.7, 52.3, 20.5; HRMS [M+H] calcd
C H NO ]: 300.1158, Found: 300.1232; Purity 100% (as determined by RPꢀHPLC, method A,
[
1
7
18
4
tR = 16.29 min).
Methyl 3ꢀ(2ꢀ(4ꢀethylphenoxy)acetamido)benzoate (5f): Obtained as a white solid (0.34 g, 97.4%
1
yield). HꢀNMR (400 MHz, CDCl )
δ
8.48 (brs, 1H), 8.00 (m, 1H), 7.81 (m, 1H), 7.42 (t, J = 7.8
3
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,
1
3
2
H), 1.23 (m, 3H); CꢀNMR (100 MHz, CDCl )
δ
166.7, 166.5, 155.0, 138.3, 137.1, 13.9, 129.2,
3
1
29.1, 125.8, 124.5, 120.9, 114.7, 67.7, 52.2, 28.0, 15.8; HRMS [M+H] calcd [C H NO ]:
18 20
4
314.1314, Found: 314.1379; Purity 100% (as determined by RPꢀHPLC, method A, t = 18.06
R
min).
Methyl 3ꢀ(2ꢀ(4ꢀpropylphenoxy)acetamido)benzoate (5g): Obtained as a white solid (0.31 g,
1
9
0.5% yield). HꢀNMR (400 MHz, CDCl )
δ
8.46 (brs, 1H), 8.09 (t, J = 1.8 Hz, 1H), 8.00 (dd, J
3
=
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
1
3
(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); Cꢀ
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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 [C H NO ]: 328.2471, Found:
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328.1546; Purity 100% (as determined by RPꢀHPLC, method A, t = 19.92 min).
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Methyl 3ꢀ(2ꢀ(4ꢀbutylphenoxy)acetamido)benzoate (5h): Obtained as a white solid (0.32 g,
1
98.4% yield). HꢀNMR (400 MHz, CDCl )
δ 8.52 (brs, 1H), 8.10 (s, 1H), 7.99 (dd, J = 8.2, 1.2
3
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 =
.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
8
1
3
(
t, J = 7.4 Hz, 3H); CꢀNMR (100 MHz, CDCl )
δ
166.8, 166.5, 155.0, 137.2, 137.0, 130.9,
29.6, 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
[C H NO ]: 342.1627, Found: 342.1694; Purity 100% (as determined by RPꢀHPLC, method A,
3
1
2
0
24
4
tR = 21.59 min).
Methyl 3ꢀ(2ꢀ(4ꢀpentylphenoxy)acetamido)benzoate (5i): Obtained as a yellow solid (0.13 g,
1
81.2% yield). HꢀNMR (400 MHz, CDCl )
δ 8.40 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H),
3
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), 1.61ꢀ1.57 (m, 2H), 1.34ꢀ1.29 (m, 4H),
13
0
.89 (t, J = 4.0 Hz, 1H); CꢀNMR (100 MHz, CDCl )
δ
166.7, 166.6, 155.0, 137.2, 137.1, 131.1,
3
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 [C H NO ]: 356.1862, Found: 356.1842; Purity 100% (as determined by RPꢀHPLC,
21
26
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method A, t = 23.53 min).
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Methyl 3ꢀ(2ꢀ(4ꢀcyclopentylphenoxy)acetamido)benzoate (5j): Obtained as a yellow solid
1
(
0.10 g, 62.5% yield). HꢀNMR (400 MHz, CDCl )
δ
8.39 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0
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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
1
1
.0 Hz, 2H), 4.61 (s, 2H), 3.93 (s, 3H), 2.98ꢀ2.94 (m, 1H), 2.06ꢀ2.05 (m, 2H), 1.82ꢀ1.77 (m, 2H),
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3
.70ꢀ1.67 (m, 2H), 1.57ꢀ1.52 (m, 4H); CꢀNMR (100 MHz, CDCl )
δ
166.7, 166.6, 155.1, 140.8,
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37.1, 131.1, 129.3, 128.4, 125.9, 124.5, 120.9, 114.7, 67.9, 52.3, 45.2, 34.7, 25.4; HRMS
[M+H] calcd [C H NO ]: 354.1705, Found: 354.1682; Purity 100% (as determined by RPꢀ
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HPLC, method A, t = 22.03 min).
R
Methyl 3ꢀ(2ꢀ(4ꢀcyclohexylphenoxy)acetamido)benzoate (5k): Obtained as a yellow solid (0.11
1
g, 70.5% yield). HꢀNMR (400 MHz, CDCl )
δ 8.39 (s, 1H), 8.07 (s, 1H), 8.00 (d, J = 8.0 Hz,
3
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), 2.50ꢀ2.46 (m, 1H), 1.84ꢀ1.76 (m, 4H), 1.73ꢀ1.58 (m, 2H),
13
1
.44ꢀ1.38 (m, 4H); CꢀNMR (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
[
C H NO ]: 368.1862, Found: 368.1842; Purity 100% (as determined by RPꢀHPLC, method A,
22 26 4
tR = 23.62 min).
Methyl 3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamido)benzoate (5l): Obtained as a
1
white solid (0.10 g, 64.1 % yield). HꢀNMR (400 MHz, CDCl )
δ
8.46 (s, 1H), 8.08 (s, 1H), 7.79
3
(d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.44ꢀ7.32 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 6.90 (d,
1
3
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); CꢀNMR (100
MHz, CDCl3) 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 [C H NO ]: 398.2331,
δ
2
4
32
4
Found: 398.2345; Purity 100% (as determined by RPꢀHPLC, method A, t = 26.11 min).
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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 3ꢀaminoꢀ4ꢀ
hydroxybenzoate (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.
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Methyl 3ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀylꢀphenoxy)acetamido)ꢀ4ꢀhydroxybenzoate (1)
Methyl 4ꢀhydroxyꢀ3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamido)benzoate (6):
1
Obtained as a white solid (1.00 g, 64.1 % yield). HꢀNMR (400 MHz, CDCl )
δ 9.75 (s, 1H),
3
8.60 (s, 1H), 7.85ꢀ7.83 (m, 1H), 7.73 (s, 1H), 7.38ꢀ7.34 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.94ꢀ
1
3
6
.90 (m, 2H), 4.68 (s, 2H), 3.89 (s, 3H), 1.73(s, 2H), 1.36 (s, 6H), 0.72 (s, 9H); CꢀNMR (100
MHz, CDCl3) 168.2, 166.7, 154.5, 147.2, 144.6, 129.2, 128.0, 127.6, 122.3, 121.3, 119.5,
14.2, 67.5, 57.0, 52.3, 38.1, 32.3, 31.8, 31.6; HRMS [M+H] calcd [C H NO ]: 414.2280,
δ
1
2
4
32
5
Found: 414.2249; Purity 100% (as determined by RPꢀHPLC, method A, t = 24.95 min).
R
General procedure for the synthesis of 7aꢀf: 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
MgSO . The solvent was filtered and evaporated under reduced pressure to afford a crude solid
4
which was purified by silica gel column chromatography.
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Methyl 3ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀylꢀphenoxy)acetamido)ꢀ4ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethoxy)benzoate
1
(
7a): Obtained as a white solid (0.10 g, 77.8% yield). HꢀNMR (400 MHz, CDCl ) δ 9.06 (s, 1H),
3
9
.02 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.95ꢀ6.91 (m, 3H), 4.62 (s, 2H),
.22 (t, J = 4.0 Hz, 2H), 3.89 (s, 3H), 2.92 (t, J = 4.0 Hz, 2H), 2.64ꢀ2.62 (m, 4H), 2.09 (brs, 3H),
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19
20
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28
29
30
31
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33
34
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38
39
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47
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57
58
59
60
4
1
3
1.89ꢀ1.88 (m, 6H), 1.77ꢀ1.72 (m, 10H); CꢀNMR (100 MHz, CDCl3)
δ
166.8, 166.5, 155.0,
51.1, 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,
6.7, 35.7, 28.9, 23.6; HRMS [M+H] calcd [C H N O ]: 533.3015, Found: 533.2988; Purity
1
3
1
32 41
2
5
00% (as determined by RPꢀHPLC, method A, t = 13.39 min).
R
Methyl
3ꢀ(2ꢀ(4ꢀadamantanꢀ1ꢀylꢀphenoxy)acetamido)ꢀ4ꢀ(3ꢀmorpholinopropoxy)benzoate
1
(
7b): Obtained as a white solid (0.09 g, 69.7% yield). HꢀNMR (400 MHz, CDCl ) δ 9.05 (s, 1H),
3
9.04 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.94ꢀ6.92 (m, 3H), 4.62 (s, 2H),
4
.16 (t, J = 4.0 Hz, 2H), 3.90 (s, 3H), 3.69ꢀ3.68 (m, 4H), 2.53 (t, J = 4.0 Hz, 2H), 2.42ꢀ2.40 (m,
1
3
4
H), 2.09 (brs, 3H), 2.02 (t, J = 4.0 Hz, 2H), 1.89ꢀ1.88 (m, 6H), 1.77ꢀ1.72 (m, 6H); CꢀNMR
166.8, 166.3, 154.9, 151.1, 145.7, 126.7, 126.5, 126.3, 123.0, 120.9, 114.4,
10.3, 68.0, 66.9, 55.2, 53.7, 52.0, 43.4, 36.7, 35.7, 28.9; HRMS [M+H] calcd [C H N O ]:
(100 MHz, CDCl3) δ
1
33
43
2
6
563.3121, Found: 563.3085; Purity 100% (as determined by RPꢀHPLC, method A, t = 13.38
R
min).
Methyl 4ꢀmethoxyꢀ3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamido)benzoate (7c):
1
Obtained as a white solid (0.08 g, 77.6% yield). HꢀNMR (400 MHz, CDCl )
δ
9.03 (s, 1H), 8.97
3
(
s, 1H), 7.85ꢀ7.83 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 6.93ꢀ6.90 (m, 3H), 4.63 (s, 2H), 3.93 (s, 3H),
1
3
3.89 (s, 3H), 1.71 (s, 2H), 1.35 (s, 6H), 0.71 (s, 9H); CꢀNMR (100 MHz, CDCl ) δ 166.7, 166.5,
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2
2
2
2
2
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1
54.8, 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 [C H NO ]: 428.2437, Found: 428.2416; Purity 100%
25 34
5
(as determined by RPꢀHPLC, method A, t = 27.91 min).
R
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Methyl
4ꢀ(2ꢀmethoxyethoxy)ꢀ3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀ
1
yl)phenoxy)acetamido)benzoate (7d): Obtained as a white solid (0.10 g, 87.7% yield). Hꢀ
NMR (400 MHz, CDCl3)
δ 9.15 (s, 1H), 9.06 (s, 1H), 7.83ꢀ7.80 (m, 1H), 7.32 (d, J = 8.0 Hz,
2H), 6.94ꢀ6.90 (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,
1
3
2H), 3.44 (s, 3H), 1.71 (s, 2H), 1.34 (s, 6H), 0.70 (s, 9H); CꢀNMR (100 MHz, CDCl )
δ
166.7,
66.4, 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 [C H NO ]: 472.2699, Found:
3
1
27
38
6
4
72.2676; Purity 100% (as determined by RPꢀHPLC, method A, t = 27.57 min).
R
Methyl
4ꢀ(2ꢀmorpholinoethoxy)ꢀ3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀ
1
yl)phenoxy)acetamido)benzoate (7e): Obtained as a white solid (0.09 g, 70.8% yield). Hꢀ
NMR (400 MHz, CDCl3)
δ 9.06 (s, 1H), 9.04 (s, 1H), 7.84ꢀ7.81 (m, 1H), 7.32 (d, J = 8.0 Hz,
2H), 6.93ꢀ6.88 (m, 3H), 4.62 (s, 2H), 4.21 (t, J = 4.0 Hz, 2H), 3.90 (s, 3H), 3.69ꢀ3.66 (m, 4H),
1
3
2.83 (t, J = 4.0 Hz, 2H), 2.56ꢀ2.54 (m, 4H), 1.71 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H); CꢀNMR
100 MHz, CDCl3) 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
C H N O ]: 527.3121, Found: 527.3096; Purity 100% (as determined by RPꢀHPLC, method A,
(
δ
[
3
0
43
2
6
tR = 12.90 min).
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Methyl
4ꢀ(propꢀ2ꢀynyloxy)ꢀ3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀ
1
yl)phenoxy)acetamido)benzoate (7f): Obtained as a white solid (0.10 g, 91.7% yield). HꢀNMR
(400 MHz, CDCl3)
δ 9.06 (s, 1H), 9.02 (s, 1H), 7.85ꢀ7.83 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.04
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60
(
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 =
1
3
4
.0 Hz, 2H), 1.71 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H); CꢀNMR (100 MHz, CDCl )
δ
166.6, 166.4,
3
1
54.8, 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 [C H NO ]: 452.2437, Found: 452.2412; Purity
2
7
34
5
1
00% (as determined by RPꢀHPLC, method A, t = 27.33 min).
R
General procedure for the synthesis of 8aꢀf: EDCI (1.2 equiv) and HOBt (1.2 equiv) were
added to a solution of 2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)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 MgSO , filtered, and
4
concentrated in vacuo. The resulting residue was purified by silica gel column chromatography.
Nꢀ(4ꢀ(trifluoromethyl)phenyl)ꢀ2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamide
(8a):
1
Obtained as a white solid (0.11 g, 71.54% yield). H NMR (CDCl , 400 MHz)
δ 8.43 (s, 1H),
3
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,
1
3
2H), 4.62 (s, 2H), 1.72 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H); C NMR (100 MHz, CDCl )
δ
166.8,
54.5, 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 [C H F NO ]: 408.2150, Found: 408.2122; Purity 100% (as determined
3
1
23
29
3
2
by RPꢀHPLC, method A, t = 28.40 min).
R
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Nꢀ(4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)ꢀ2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamide
1
(8b): Obtained as a white solid (0.12 g, 72.7% yield). HꢀNMR (CDCl , 400 MHz)
δ
8.17 (s, 1H),
3
7.45 (d, J = 9.2 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.92ꢀ6.88 (m, 4H), 4.58 (s, 2H), 3.25ꢀ3.23 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
3
4
H), 2.68ꢀ2.67 (m, 4H), 2.42 (s, 3H), 1.71 (s, 2H), 1.31 (s, 6H), 0.71 (s, 9H); CꢀNMR (100
166.2, 154.7, 148.7, 144.1, 129.2, 127.5, 121.6, 116.6, 114.0, 67.7, 57.0, 55.1,
9.4, 46.1, 38.1, 32.3, 31.8, 31.6; HRMS [M+H] calcd [C H N O ]: 438.3121, Found:
MHz, CDCl3)
δ
4
27
40
3
2
438.3100; Purity 100% (as determined by RPꢀHPLC, method A, t = 12.56 min).
R
1ꢀ(4ꢀ(4ꢀ(trifluoromethyl)benzyl)piperazinꢀ1ꢀyl)ꢀ2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀ
1
yl)phenoxy)ethanone (8c): Obtained as a white solid (0.15 g, 81.0% yield). HꢀNMR (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), 3.63ꢀ3.59 (m, 4H), 3.56 (s, 2H), 2.40ꢀ2.39 (m, 4H), 1.70 (s, 2H), 1.38
1
3
(s, 6H), 0.71 (s, 9H); CꢀNMR (100 MHz, CDCl )
δ
166.6, 155.4, 143.2, 142.0, 129.4, 129.1,
27.2, 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 [C H F N O ]: 491.2885, Found: 491.2854; Purity 100% (as determined by RPꢀ
3
1
[
28
38
3
2
2
HPLC, method A, t = 14.31 min).
R
1ꢀ(4ꢀ(propꢀ2ꢀynyl)piperazinꢀ1ꢀyl)ꢀ2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)ethanone (8d):
1
Obtained as a white solid (0.11 g, 78.5% yield). HꢀNMR (400 MHz, CDCl )
δ 7.30 (d, J = 8.0
3
Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 4.67 (s, 2H), 3.68ꢀ3.63 (m, 4H), 3.29 (t, J = 4.0 Hz, 2H), 2.56ꢀ
13
2
.51 (m, 4H), 2.25 (t, J = 4.0 Hz, 1H), 1.69 (s, 2H), 1.33 (s, 6H), 0.70 (s, 9H); CꢀNMR (100
MHz, CDCl3)
1.9, 38.0, 32.3, 31.8, 31.6; HRMS [M+H] calcd [C H N O ]: 371.2699, Found: 371.2680;
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,
4
23
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9
Purity 100% (as determined by RPꢀHPLC, method A, t = 11.38 min).
R
Propꢀ2ꢀynyl
4ꢀhydroxyꢀ3ꢀ(2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamido)benzoate
1
(8e): Obtained as a white solid (0.10 g, 60.6% yield). HꢀNMR (400 MHz, CDCl )
δ = 9.84 (s,
3
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1
H), 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,
13
J = 4.0 Hz, 1H), 1.72 (s, 2H), 1.36 (s, 6H), 0.72 (s, 9H); CꢀNMR (100 MHz, CDCl )
δ 169.0,
3
1
64.9, 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,
2.4, 38.1, 32.3, 31.8, 31.6; HRMS [M+H] calcd [C H NO ]: 438.2280, Found: 438.2247;
5
26
32
5
Purity 100% (as determined by RPꢀHPLC, method A, t = 24.56 min).
R
Nꢀ(3ꢀhydroxyꢀadamantanꢀ1ꢀyl)ꢀ2ꢀ(4ꢀ(2,4,4ꢀtrimethylpentanꢀ2ꢀyl)phenoxy)acetamide (8f):
1
Obtained as a white solid (0.12 g, 76.9% yield). HꢀNMR (400 MHz, CDCl )
δ
8.02 (s, 1H), 7.31
3
(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,
1
3
2H), 1.99 (s, 4H), 1.70 (s, 6H), 1.58 (s, 2H), 1.34 (s, 6H), 0.70 (s, 9H); CꢀNMR (100 MHz,
CDCl3) 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 [C H NO ]: 414.3008, Found: 414.2985; Purity
δ
2
6
40
3
1
00% (as determined by RPꢀHPLC, method A, t = 23.33 min).
R
General procedure for the synthesis of 9a and 9b: The mixture of 3a or 3l (1.0 equiv) and
o
methyl propiolate (2.0 equiv) in toluene was added to Ph P (1.0 equiv) at ꢀ10 C. The mixture was
3
o
then allowed to heat up to 115 C and stirred for 2 hours. The solvent was removed under
reduced pressure and the residue was purified by purified by silica gel column chromatography.
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