Design, Synthesis and Biological Evaluation of Novel MDH Inhibitors Targeting Tumor Microenvironment

Abstract

MDH1 and MDH2 enzymes play an important role in the survival of lung cancer. In this study, a novel series of dual MDH1/2 inhibitors for lung cancer was rationally designed and synthesized, and their SAR was carefully investigated. Among the tested compounds, compound 50 containing a piperidine ring displayed an improved growth inhibition of A549 and H460 lung cancer cell lines compared with LW1497. Compound 50 reduced the total ATP content in A549 cells in a dose-dependent manner; it also significantly suppressed the accumulation of hypoxia-inducible factor 1-alpha (HIF-1α) and the expression of HIF-1α target genes such as GLUT1 and pyruvate dehydrogenase kinase 1 (PDK1) in a dose-dependent manner. Furthermore, compound 50 inhibited HIF-1α-regulated CD73 expression under hypoxia in A549 lung cancer cells. Collectively, these results indicate that compound 50 may pave the way for the development of next-generation dual MDH1/2 inhibitors to target lung cancer.

1. Introduction

The rapid growth of cancer cells requires an efficient ATP supply. Cancer cells show alterations in the metabolic pathways that are related to energy production and biosynthetic processes, such as increased uptake of glucose and amino acids and increased breakdown of glutamine and fatty acids. Mutations in EGFR, KRAS, MYC, PI3K, AKT, LKB1, and p53 are known to be involved in cancer metabolism. Targeting energy metabolism has been suggested as an effective strategy for inhibiting cancer cells [1,2,3,4].

To obtain ATP in the electron transport pathway in cells, the cytosolic NADH must be transported into the mitochondria. Because NADH itself cannot be transported into the mitochondria, cells transport reducing equivalents across the mitochondrial membrane using a malate–aspartate shuttle (MAS). The MAS is operated by two pairs of enzymes, malate dehydrogenases (MDH1 and MDH2) and glutamate oxaloacetate transaminases (GOT1 and GOT2), which are localized in the mitochondria and cytoplasm. MDH1 and MDH2 catalyze the reversible conversion of malate to oxaloacetate (OAA) using the NAD/NADH cofactor system [5,6,7,8,9]. Aminooxyacetic acid (AOA), a specific MAS inhibitor, decreases the proliferation of breast adenocarcinoma cells by suppressing GOT1 and GOT2 [10].

The association of MDH1 and MDH2 have been reported in several cancers such as pancreatic and lung cancers. The expression levels of MDH1 and MDH2 are high in patients with lung cancer [4,10]. High expression of MDH1 is significantly correlated with patient survival, and its knockdown affects cell viability compared with that of MDH2 [10,11]. A previous report presented LW1497, a dual inhibitor of MDH1 and MDH2, which showed significant in vivo antitumor effects against colon cancer through the inhibition of mitochondrial respiration and hypoxia-inducible factor-1 alpha (HIF-1α) accumulation [12,13]. Another report showed that LW1497 downregulates Slug expression to impede the progression of A549 lung cancer cells by inhibiting epithelial-mesenchymal transition [14].

In this study, to develop a potent dual inhibitor of MDH1/2 for lung cancer, LW1497 derivatives were synthesized based on their structure–activity relationships (SARs). Finally, we identified compound 50 as a dual inhibitor of MDH1/2 in lung cancer cells (Figure 1).

2. Results

2.1. Chemistry

The synthesis of the amide compounds 5a–f was completed in four steps, as shown in Scheme 1. The hydroxyl group of phenol 1 was alkylated with methyl propiolate in the presence of triphenylphosphine (PPh₃) in toluene, and the resulting (E)-phenoxyacrylic methyl ester (2) was saturated with palladium on carbon (Pd/C) under H₂ balloon pressure followed by hydrolysis with lithium hydroxide to furnish carboxylic acid 4 at 81% yield [12]. Finally, the T3P-mediated coupling of the resultant carboxylic acid 4 with corresponding amines yielded a series of amide derivatives 5a–f.

Compound 14 was synthesized in seven steps, as illustrated in Scheme 2. Friedel–Crafts acylation of anisole with in situ-generated acid chloride yielded 8 [15], which was then demethylated with aluminum chloride (AlCl₃) to yield phenol 9. The reduction of 9 with lithium aluminum hydride (LiAlH₄) followed by the alkylation of phenol 10 with commercially available benzyl propiolate yielded phenoxy acrylate 12. Saturation of the phenoxy acrylate portion and debenzylation of 12 were completed using palladium carbon (Pd/C) under H₂ balloon pressure, yielding 13. Interestingly, under the palladium hydrogenation conditions, along with double bond reduction and debenzylation, dehydroxylation of 12 also occurred. The T3P-mediated coupling of the resultant carboxylic acid 13 with the corresponding amine yielded 14.

Trimethylpentane-modified derivatives 21 and 22 were synthesized in seven steps, as shown in Scheme 3. 4-Aminophenol (15) was treated with Boc anhydride to yield 16, which was then alkylated with benzyl propiolate to yield 17. The reduction of the double bond and removal of the benzyl group in 17 using palladium carbon (Pd/C) under H₂ balloon pressure yielded 18. The T3P-mediated coupling of the resultant carboxylic acid 18 with 3-(trifluoromethoxy)aniline yielded 19. The removal of the Boc group in 19 with TFA followed by the reductive amination of 20 with pivalaldehyde yielded 21. The reductive amination of 21 with paraformaldehyde provided 22.

Compound 30 was synthesized as shown in Scheme 4. Friedel–Crafts alkylation of anisole with 3-methylbut-2-enoic acid (23) yielded 24 [16]. The resulting acid 24 was converted to methyl ester 25 and followed by demethylation; then, the Grignard reaction of ester 26 with methyl magnesium bromide yielded the tertiary alcohol 27. The hydroxy group of phenol 27 was alkylated with benzyl propiolate, followed by the double bond reduction and benzyl ester cleavage of 28 by treatment with palladium carbon (Pd/C) under H₂ balloon pressure to yield 29. The T3P-mediated coupling of the resultant carboxylic acid 29 with 3-(trifluoromethoxy)aniline yielded 30.

Biphenyl and naphthalene derivatives 35a–b and 39a–d were synthesized as shown in Scheme 5 and Scheme 6. The hydroxy group of phenols 31 and 36a–d was treated with an excess of ethyl and/or methyl acrylate in the presence of DMAP under microwave irradiation to produce methyl and/or ethyl esters 32 and 37a–b. The esters were hydrolyzed with lithium hydroxide, and the resulting acids were coupled with 3-(trifluoromethoxy)aniline using T3P-mediated amide formation to produce 34 and 39a–d. A Suzuki coupling reaction of 34 with corresponding boronic acids in the presence of Pd(PPh₃)₄ yielded 35a–b.

Compounds 44a–b were synthesized in four steps, as illustrated in Scheme 7. The precursor for the synthesis of 42 was obtained by Boc protection of 2-bromoethanamine HBr with Boc anhydride [17]; then, the hydroxy group of phenol 1 was alkylated to yield 42. Boc was then deprotected using TFA to yield 43. Subsequently, 43 was treated with acid and/or sulfonyl chloride to obtain compounds 44a–b.

Compounds 50 and 51 were synthesized, as illustrated in Scheme 8. The hydroxyl group of phenol 1 was reacted with trifluoromethanesulfonic anhydride to yield 45 [18]. The Buchwald–Hartwig coupling of 45 with methyl piperidine-4-carboxylate and/or tert-butyl piperazine-1-carboxylate yielded 46 and 48, respectively. The base-mediated hydrolysis of 46 yielded the acid 47. Amide coupling of 47 with 3-(trifluoromethoxy)aniline yielded compound 50. The trifluoroacetic-acid-mediated deprotection of the Boc from 48 provided 49, which was then treated with 3-(trifluoromethoxy)benzoyl chloride in the presence of triethylamine to yield 51.

Compounds 56 and 57a–d were synthesized as shown in Scheme 9. The Buchwald–Hartwig coupling reaction of 45 with benzophenone imine followed by the deprotection of benzophenone yielded 53. Alkylation of 53 with methyl 3-bromopropionate in acetonitrile yielded 54. The methyl ester was hydrolyzed using lithium hydroxide to yield 55, and the resultant acid was coupled with 3-(trifluoromethoxy)aniline to give 56. The reductive amination of 56 with ketones or aldehydes in the presence of sodium cyanoborohydride (NaBH₃CN) yielded 57a–c, respectively. Acetyl chloride was reacted with 56 in the presence of triethylamine to yield 57d.

2.2. Biological Evaluation

2.2.1. MDH Enzyme Activity

To develop the dual inhibitors of MDH1 and MDH2, we screened the newly synthesized LW1497 derivatives using MDH and MDH2 enzyme assays. LW1497 was used as the positive control for the MDH1/2 dual inhibitor. We evaluated the ability of the synthesized compounds to inhibit human recombinant MDH1 and MDH2 enzymatic activity using an oxaloacetate-dependent NADH oxidation assay (Table 1, Table 2, Table 3, Table 4, Table 5 and Table 6). Our SAR optimization strategy focused on four different parts of LW1497: (A) the introduction of various groups at different positions on the amide phenyl ring; (B) the replacement of the 3-(4-(2,4,4-trimethylpentan-2-yl) alkyl chain; (C) the alteration of the amide portion; and (D) the modification of the oxypropanamide linker portion (Figure 1).

As a starting point for the SAR studies, we first examined the substitution of various groups on the amide phenyl ring (Part A). We started with N-(3-methoxyphenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide 5a having an electron-donating group (OMe) at the meta position, which showed loss of activity in MDH1 and moderate inhibitory activity in MDH2 (IC₅₀ = 6.18 ± 0.74 and 1.5 ± 0.01 μM, respectively) (Table 1). The replacement of the -OMe group at the meta position of the phenyl ring with electron-withdrawing groups such as -CN and -CF₃ (5b and 5c) resulted in a loss of activity. Interestingly, the -OCF₃ group (5d) regained inhibitory activity against both MDH1 and MDH2 enzymes (IC₅₀ = 0.94 ± 0.05 and 1.24 ± 0.13 μM, respectively) and showed equivalent potency compared with LW1497. However, when the OCF₃ group was shifted from the meta to the ortho and para positions of the phenyl ring (5e and 5f), both led to an approximately three-fold activity loss against both MDH1 and MDH2 compared with 5d. These results implied that the -OCF₃ group at the meta position of the amide phenyl ring was well tolerated (Table 1).

Based on the results for compound 5d, we focused our SAR exploration on the replacement of the 3-(4-(2,4,4-trimethylpentan-2-yl) side chain (portion B) by fixing the -OCF₃ group at the meta position on the amide phenyl ring. The 3-(4-(2,4,4-trimethylpentan-2-yl) moiety was replaced with the 4-(3,3-dimethylbutyl) moiety (14), which maintained moderate MDH1 and MDH2 inhibition (IC₅₀ = 3.38 ± 0.36 and 1.53 ± 0.08 μM, respectively). Further replacement with the amine-linked aliphatic chains (21 and 22) resulted in the loss of activity against both MDH1 and MDH2. In addition, one of the methyl groups from terminal trimethyl was replaced with a hydroxy group (30), resulting in a decrease in the MDH1 and MDH2 inhibitory activities (IC₅₀ = >5 μM). Replacing the portion B fragment from the aliphatic chain to an aromatic ring (35a) retained dual activity against both MDH1 and MDH2 (IC₅₀ = 0.78 ± 0.08 and 1.38 ± 0.59 μM, respectively) compared with the active compound 5d, whereas the 4-F substituted phenyl ring (35b) led to significant activity loss compared with 35a (Table 2). However, when we modified the 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy) moiety with 3-(naphthalen-2-yloxy) moiety (39a), loss of activity against both MDH enzymes was noted (IC₅₀ = >5 μM). Interestingly, substitutions on the naphthalene ring with electron withdrawing groups, such as 7-bromo (39b), 6-bromo (39c), and 6-fluoro (39d), demonstrated selective inhibitory activity against MDH1 than MDH2 (IC₅₀ = 1.61 ± 0.25, 1.61 ± 0.21, 2.25 ± 0.77 and >5 μM, respectively; Table 3). These results suggest that the 4-(3,3-dimethylbutyl)-like alkyl chain moiety is required for the dual inhibitory activity. Replacing the amide linkage of 5d with the reverse amide linkage (44a) maintained moderate inhibitory activity against MDH1 and MDH2 enzymes (IC₅₀ = 3.15 ± 0.11, 2.22 ± 0.28 μM, respectively). Switching the amide group from (44a) to the sulfonamide group (44b) showed moderate activity against MDH1 (IC₅₀ = 2.85 ± 0.45 μM), while it lost activity against MDH2 (Table 4).

For the SAR exploration of portion D, we chose to start with compound 5d by keeping the -OCF₃ group intact at the meta position on the amide phenyl ring in portion A and (4-(3,3-dimethylbutyl) alkyl chain in portion B. Replacement of the flexible ether-linked aliphatic chain from 5d with an amine-linked piperidine ring (50) exhibited dual inhibitory activity against MDH1 and MDH2 (IC₅₀ = 3.33 ± 0.18 and 2.24 ± 0.09 μM, respectively). Compound 51, which contains a piperazine group, showed decreased dual inhibitory activity (Table 5). Replacing the amine-linked piperidine with a more flexible amine-linked aliphatic chain (56) led to an increase in the activity against MDH1 (IC₅₀ = 1.79 ± 0.04 μM) but also maintained similar inhibitory activity against MDH2. When the hydrogen atom in 56 was substituted with aliphatic groups, such as methyl (57a), isopropyl (57b), and cyclobutyl (57c), it retained similar potency against both MDH1 and resulted in a two- to three-fold improvement in the inhibitory activity against MDH2. Interestingly, the N-acylated derivative (57d) was more selective for MDH1 than for MDH2 (IC₅₀ = 1.41 ± 0.47 and >5 μM, respectively) (Table 6).

The combined structure optimization and SAR studies resulted in the identification of novel compounds, such as 5d, 5f, 14, 35a, 44a, 50, 56, 57a, and 57b, which showed strong inhibition on the activities of both MDH1 and MDH2. However, compounds 39b, 39c, 39d, and 57d showed MDH1 activity, whereas 5b and 5c only showed MDH2 activity.

2.2.2. Molecular Docking Study for Compound 50

Molecular docking is a vital component of computer-aided drug design as it offers a range of valuable applications, including the prediction of ligand–target binding modes, the ranking of compounds based on their docking scores, and the correlation of these scores with potential activity [19,20]. Visualizations of the interactions generated through docking studies serves as a guide for optimizing the affinity characteristics of the studied ligands. In this study, compound 50 was subjected to an in-depth molecular examination against both MDH1 and MDH2 to determine its binding patterns. The results showed that compound 50 displayed a docking score of −4.9 Kcal/mol against MDH1, which can be attributed to the number of hydrogen bonds formed by compound 50 against MDH1, as depicted in Figure 2. Compound 50 formed two hydrogen bonds with Gln14 and Asn130, highlighting the importance of forming a hydrogen bond with this amino acid residue for activity.

The molecular docking study of compound 50 against MDH2 revealed a docking score of approximately −5.9 Kcal/mol. Compound 50 formed a hydrogen bond with the Gly35 residue within the binding site and two additional hydrogen bonds with the Ile36 and Lys105 residues, as shown in Figure 3.

2.2.3. Growth Inhibition Assay of Lung Cancer Cells

At the same time, we investigated the effect of the compounds on the growth inhibition of A549 and H460 lung cancer cells bearing KRAS and LKB1 mutations. Compounds 5f, 57, and 57a showed moderate growth inhibition of lung cancer cells A549 (IC₅₀ = 5.97 ± 0.71, 6.06 ± 0.01, 4.41 ± 0.06 μM, respectively) and H460 (IC₅₀ = 3.77 ± 0.02, 5.3 ± 0.01, 4.34 ± 0.03 μM, respectively). Especially, compound 50 demonstrated a two-fold improved growth inhibition of lung cancer cells A549 (IC₅₀ = 3.94 ± 0.04 μM) and H460 (IC₅₀ = 3.67 ± 0.06 μM) compared with LW1497 in A549 (IC₅₀ = 7.98 ± 0.53 μM) and H460 (IC₅₀ = 6.14 ± 0.29 μM) cells. Compound 50 containing a piperidine linker was selected for further analysis.

2.2.4. ATP Production in the Presence of MDH Inhibitor

Malate transported to the interior of the mitochondria is oxidized to OAA by mitochondrial MDH2, thereby generating NADH, which then enters the ETC to produce ATP. Because inhibition of MAS is expected to reduce ATP production in cancer cells, the total amount of ATP was measured after treatment with compound 50. Cells treated with compound 50 showed reduced ATP level in A549 cells (Figure 4A). In addition, an increase in ADP/ATP was observed, indicating the inhibition of ATP synthesis by oxidative phosphorylation (Figure 4B).

We then examined whether the ATP production pattern was altered by drug treatment using an XF analyzer for real-time metabolic analysis (Figure 5). As expected, the total ATP decreased by 14.6% and 28.6% in cells treated with 5 µM and 10 µM of compound 50, respectively (Figure 5).

2.2.5. Compound 50 Inhibits Hypoxia-Induced Accumulation of HIF-1α

Previously, it has been reported that the inhibition of MDH1 or MDH2 enzymes is related to HIF-1a expression levels [12]. We investigated whether compound 50 decreases HIF-1α accumulation in A549 cells (Figure 6). Compound 50 decreased HIF-1α accumulation in a dose-dependent manner (Figure 6). Additionally, we confirmed that compound 50 suppressed the protein expression of HIF-1α target genes, such as GLUT1 and PDK1 (Figure 6), as clearly shown by the Western blot assay.

2.2.6. Compound 50 Suppresses the Expression of CD73 Regulated by HIF-1α

It has been reported that CD73 expression is induced by HIF-1α under hypoxic conditions [21]. Therefore, we examined whether the expression of CD73 is inhibited by compound 50 under hypoxia in A549 lung cancer cells. As shown, CD73 expression increased by HIF-1α was significantly decreased by compound 50 in a concentration-dependent manner. To confirm the effect of compound 50 on CD73 expression, we performed ELISA for CD73. Compound 50 reduced CD73 levels in A549 cells in a dose-dependent manner (Figure 7). These results confirmed that compound 50 showed a significant inhibition of hypoxia-induced transcription and CD73 activity.

3. Discussion

The reprogramming of the core metabolism in tumors confers selective growth advantages, such as the ability to enhance cell proliferation and promote tumor growth and progression [22,23,24,25,26,27]. One of the mechanisms to suppress tumor growth is the impairment of the cancer cell’s metabolism. MAS plays a crucial role in the net transfer of the NADH produced in the cytoplasm into the mitochondria. The transportation of cytosolic NADH as an electron donor into the mitochondria is a major purpose of MAS in cancer cells. Therefore, MDH1 and MDH2, which are components of MAS, are the key molecules involved in ATP production via the process of mitochondrial oxidative phosphorylation [7,28,29]. In this study, we synthesized compound 50, a potent MDH1/MDH2 dual inhibitor that suppressed MAS function in A549 lung cancer cells.

First, we measured ATP production to elucidate the inhibitory effects of compound 50 on mitochondrial respiration. Total ATP production was significantly reduced in A549 cells treated with compound 50, which increased the ADP/ATP ratio. These results suggest that compound 50 inhibits the growth of A549 cells by inhibiting energy metabolism. In all cancer types, ATP supply is solely dependent on glycolysis and OXPHOS. Therefore, the selective inhibition of prevalent tumor energy metabolism may have a significant impact on cancer treatment. However, it can be argued that such a treatment may also severely affect the host’s energy metabolism.

Cancer cells significantly depend on cytosolic NADH production from glucose, fatty acids, and glutamine for ATP production [30]. Elevated glycolysis in cancer cells has been proposed as a mechanism that accelerates oxidative phosphorylation. MAS exerts control over NADH/NAD⁺ homeostasis to maintain the activity of mitochondrial lactate dehydrogenase and enables the aerobic oxidation of glycolytic l-lactate in the mitochondria.

In contrast, high aerobic glycolysis distinguishes cancer cells from normal cells and has been exploited to detect tumors in vivo. Lactate consumption in cells treated with compound 50 did not change significantly. Cancer cells consume the secreted lactate to produce ATP through the TCA cycle and oxidative phosphorylation processes [31,32]. Some cancer cells use lactate as a substrate for TCA intermediates via monocarboxylate transporters (MCT1/4) as well as for ATP production [32]. Lactate can be converted to pyruvate using lactate dehydrogenase (LDH) and can be further converted to acetyl-CoA by ATP-citrate lyase for fatty acid synthesis.

Under hypoxia, HIF-1α increases the transcriptional expression of various genes that are involved in cancer progression, metastasis, angiogenesis, and resistance to therapy [33]. We found that compound 50 decreased the expression of HIF-1α and its target genes, such as glucose transporters (GLUT1 and GLUT3) and PDK1, preventing pyruvate entry into the TCA cycle [5,6]. We also observed a decrease in CD73 expression in the HIF-dependent adenosinergic pathway, which impairs NK cell function in tumors. Recently, the development of agents that can either block CD73 and/or target HIFs concurrently with NK cell-based therapies has emerged as an immunotherapeutic strategy with significant potential for the treatment of solid tumors.

4. Materials and Methods

4.1. Chemistry

4.1.1. General Procedures

The commercial chemicals and solvents used were of reagent grade. All reactions were performed under a nitrogen atmosphere in oven-dried glassware. Reactions were monitored using thin-layer chromatography on 0.25-milimeter silica plates (E. Merck; silica gel 60 F254). The products were purified using flash column chromatography (Biotage) using silica gel 60 (230–400-mesh Kieselgel 60). Proton nuclear magnetic resonance (NMR) spectra were recorded on a Varian 400 MHz spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA). The chemical shifts are provided in δ values (ppm), and the coupling constants are in hertz (Hz). ¹³C NMR spectra were recorded on the Varian 100 MHz spectrometer. The melting points were measured using the Thermo Scientific 9200 melting point apparatus. The mass spectra were recorded using high-resolution mass spectrometry (HRMS) (electron ionization MS) on a Waters G2 QTOF mass spectrometer. The purity of the final products was determined using reversed-phase high-performance liquid chromatography (RP-HPLC). The Waters Corp. HPLC system employed a YMC C18 (HS12S05-1564WT) column (5 μM, 12 nm) that had the dimensions of 4.6 mm × 150 mm and was equipped with an ultraviolet (UV) detector set at 254 nm. The RP-HPLC mobile phases used were (A) water with 0.05% trifluoroacetic acid (TFA) and (B) acetonitrile. The purity of the compounds was assessed using a gradient of 25% B to 100% B in 35 min. The purity of all biologically evaluated compounds was >95%.

4.1.2. General Procedure for Acid–Amine Coupling

To a solution of acid (1.0 equiv), amine (1.1 equiv), and N, N-diisopropylethylamine (DIPEA; 3.0 equiv) in tetrahydrofuran (THF; 25 mL), we added 50% propylphosphonic anhydride (T3P) in ethyl acetate (EtOAc; 2.0 equiv). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with water (20 mL), and the aqueous phase was extracted using EtOAc (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (15–45% EtOAc in hexane).

4.1.3. General Procedure for the Michael Addition of Phenol with Acrylates

To a solution of phenol (1.0 equiv) in methyl or ethyl acrylate (20 mL), we added 4-dimethylaminopyridine (DMAP; 1.0 equiv) at room temperature. The resulting mixture was stirred at 100 °C in a microwave for 2 h. The reaction was quenched using saturated sodium bicarbonate solution (20 mL), and the aqueous phase was extracted using EtOAc (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–15% EtOAc in hexane).

4.1.4. General Procedure for Ester Hydrolysis

To a solution of methyl or ethyl ester (1.0 equiv) in THF:H₂O (10:1), we added lithium hydroxide (LiOH; 2.0 equiv). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure, and the crude residue was redissolved in water (20 mL) and acidified with 3 N hydrochloric acid (HCl; pH 6). The resulting precipitate was collected via suction filtration and dried under vacuum.

4.1.5. General Procedure for Suzuki Coupling

To a solution of halogen compound (1.0 equiv) and corresponding boronic acid (1.2 equiv) in 1,4-dioxane: H₂O (9:1), we added cesium carbonate (Cs₂CO₃; 3.0 equiv). The resulting mixture was degassed with nitrogen for 5 min. Tetrakis(triphenylphosphine)palladium (Pd(PPh₃)₄; 0.05 equiv) was added to the degassed solution, and the mixture was heated at 100 °C for 12 h in a sealed tube. The reaction mixture was cooled to room temperature and diluted with water (20 mL), and the aqueous phase was extracted using ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude residue was purified using silica gel column chromatography (10–15% EtOAc in hexane).

4.1.6. General Procedure for Reductive Amination

To a solution of amine (1 equiv) in dichloromethane (DCM; 30 mL), we added aldehyde and/or ketone (1.2 equiv) and acetic acid (0.1 mL). The mixture was stirred at room temperature for 2 h, and sodium cyanoborohydride (NaCNBH₃; 1.0 equiv.) was then added to the stirring solution. The resulting mixture was stirred at room temperature for 12 h. The reaction was quenched with aqueous sodium bicarbonate solution (30 mL), and the aqueous phase was extracted using DCM (3 × 40 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–25% EtOAc in hexane). The spectral data for the final compounds is available in the supplementary information (Figures S5–S98).

(E)-Methyl 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acrylate (2)

To a solution of 4-(2,4,4-trimethylpentan-2-yl)phenol (1) (2.00 g, 9.69 mmol) and methyl propiolate (1.60 g, 19.3 mmol) in toluene (30 mL), we added triphenylphosphine (2.54 g, 9.69 mmol) at −10 °C. The resulting mixture was heated at 115 °C for 2 h. The mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (15–20% EtOAc in hexane) to yield 2 as a white solid (2.00 g, 71.4% yield). ¹H NMR (400 MHz, deuterated chloroform [CDCl₃]): δ 7.80 (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); HRMS (electrospray ionization [ESI]): [M + H]⁺ calculated for C₁₈H₂₇O₃ 291.1960, found 291.1950.

3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)propanoic acid (4)

To a solution of (E)-methyl 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)acrylate (2) (1.00 g, 3.44 mmol) in methanol (20 mL), we added 10% Pd/C (73.0 mg, 0.68 mmol). The resulting mixture was stirred at room temperature for 12 h under a hydrogen gas atmosphere (1 atm) using a balloon. After the completion of the reaction, the mixture was filtered through a celite pad, and the crude filtrate was evaporated under reduced pressure. To a suspension of methyl ester (1.0 equiv) in THF:H2O (1:1), we added lithium hydroxide monohydrate (0.57 g, 13.7 mmol), and the mixture was stirred at room temperature for 12 h. The reaction mixture was evaporated and acidified with 3N HCl (pH 6), and the resulting precipitate was isolated via suction filtration and dried under vacuum to obtain 4 as a white solid (0.78 g, 81.5% yield). ¹H NMR (400 MHz, dimethyl sulfoxide [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); HRMS (ESI): [M + H]⁺ calculated for C₁₇H₂₇O₃ 279.1960, found 279.1950.

N-(3-Methoxyphenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide (5a)

The compound was prepared from 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic acid (4) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (15–20% EtOAc in hexane) yielded 5a as an off-white solid (65.0 mg, 47.4% yield). Mp: 97−99 °C. ¹H NMR (400 MHz, CDCl₃): δ 8.01 (s, 1H), 7.30 (t, J = 8.4 Hz, 3H), 7.19 (t, J = 8.2 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 11.2 Hz, 2H), 6.65 (d, J = 8.0 Hz, 1H), 4.30 (t, J = 6.0 Hz, 2H), 3.78 (s, 3H), 2.87 (t, J = 6.0 Hz, 2H), 1.70 (s, 2H), 1.33 (s, 6H), 0.71 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.11, 160.11, 155.62, 143.23, 139.07, 129.63, 127.26, 113.79, 112.0, 110.14, 115.14, 105.61, 64.09, 56.93, 55.29, 55.26, 37.99, 37.79, 32.32, 31.77, 31.65; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₄NO₃ 384.2539, found 384.2530; RP-HPLC purity ≥ 99.7%, t_R = 25.9 min.

N-(3-Cyanophenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide (5b)

The compound was prepared from 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic acid (4) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (15–20% EtOAc in hexane) yielded 5b as an off-white solid (50.0 mg, 37.0% yield). Mp: 112−114 °C. ¹H NMR (400 MHz, CDCl₃): δ 8.10 (br s, 1H), 7.93 (s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.41 (q, J = 8.3 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 4.33 (t, J = 5.6 Hz, 2H), 2.87 (t, J = 5.4 Hz, 2H), 1.71 (s, 2H), 1.35 (s, 6H), 0.71 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.45, 155.39, 146.63, 138.66, 129.85, 127.69, 127.39, 123.88, 122.90, 118.49, 113.76, 112.99, 63.96, 56.91, 38.03, 37.71, 32.32, 31.77, 31.64; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₁N₂O₂ 379.2386, found 379.2377; RP-HPLC purity ≥99.8%, t_R = 25.7 min.

N-(3-(Trifluoromethyl)phenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide (5c)

The compound was prepared from 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic acid (4) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (15–20% EtOAc in hexane) yielded 5c as an off-white solid (55.0 mg, 36.4% yield). Mp: 88−90 °C. ¹H NMR (400 MHz, CDCl₃): δ 8.12 (s, 1H), 7.83 (s, 1H), 7.7 (d, J = 8.4 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 6.87 (d, J = 8.8 Hz, 2H), 4.33 (t, J = 5.8 Hz, 2H), 2.86 (t, J = 5.6 Hz, 2H), 1.70 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.34, 155.49, 143.51, 138.51, 131.54, 131.23, 129.52, 127.35, 125.17, 122.88, 122.47, 120.83, 116.55, 113.80, 64.02, 56.93, 38.03, 37.76, 32.32, 31.76, 31.65; ¹⁹F NMR (376 MHz, CDCl₃): δ -62.80; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₁F₃NO₂ 422.2307, found 422.2304; RP-HPLC purity ≥97.6%, t_R = 28.2 min.

N-(3-(Trifluoromethoxy)phenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide (5d)

The compound was prepared from 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic acid (4) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (15–20% EtOAc in hexane) yielded 5d as an off-white semisolid (60.0 mg, 62.7% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.02 (s, 1H), 7.58 (s, 1H), 7.33 (q, J = 8.0 Hz, 4H), 6.96(d, J = 7.6 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 4.32 (t, J = 5.6 Hz, 2H), 2.84(t, J = 5.8 Hz, 2H), 1.71(s, 2H), 1.34(s, 6H), 0.71(s, 9H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.73, 156.39, 148.95, 148.93, 141.96, 141.19, 130.89, 127.34, 121.79, 119.23, 117.99, 115.60, 114.00, 111.54, 63.89, 56.73, 37.98, 36.91, 32.42, 31.97; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₁F₃NO₃ 438.2256, found 438.2250; RP-HPLC purity ≥99.1%, t_R = 28.6 min.

N-(2-(Trifluoromethoxy)phenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide (5e)

The compound was prepared from 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic acid (4) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (15−20% EtOAc in hexane) yielded 5e as a colorless liquid (45.0 mg, 11.9% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.34 (s, 1H), 7.32–7.25 (m, 4H), 7.10 (dd, J = 11.3, 4.3 Hz, 1H), 6.87 (d, J = 8.8 Hz, 2H), 5.30 (s, 1H), 4.32 (t, J = 5.6 Hz, 2H), 2.89 (t, J = 5.6 Hz, 2H), 2.04 (s, 3H), 1.71 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.09, 155.86, 149.57, 139.14, 134.41, 130.34, 129.53, 129.25, 126.95, 126.27, 124.43, 123.70, 121.70, 119.14, 118.56, 118.18, 117.47, 116.51, 113.02, 107.53, 63.93, 37.55; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.58; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₁F₃NO₃ 438.2256, found 438.2247; RP-HPLC purity ≥ 100%, t_R = 28.5 min.

N-(4-(Trifluoromethoxy)phenyl)-3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamide (5f)

The compound was prepared from 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanoic acid (4) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (15–20% EtOAc in hexane) yielded 5f as a colorless liquid (37.0 mg, 35.0% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.01 (s, 1H), 7.54 (d, J = 8.8 Hz, 2H),7.31 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 4.32 (t, J = 5.6 Hz, 2H), 2.85 (t, J = 5.4 Hz, 2H), 1.71 (s, 2H), 1.35 (s, 6H), 0.71 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.14, 155.46, 145.23, 143.50, 136.47, 127.36, 121.78, 121.74, 120.98, 119.18, 113.76, 64.08, 56.92, 38.02, 37.73, 32.33, 31. 77, 31.65; ¹⁹F NMR (376 MHz, CDCl₃): δ -58.18; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₁F₃NO₃ 438.2256, found 438.2261; RP-HPLC purity ≥ 99.9%, t_R = 28.8 min.

1-(4-Methoxyphenyl)-3,3-dimethylbutan-1-one (8)

3,3-Dimethylbutanoic acid (6) (6.00 g, 51.6 mmol) was dissolved in thionyl chloride (10 mL), and the mixture was stirred for 12 h at room temperature under an inert condition. The excess thionyl chloride was removed via evaporation. The residue was redissolved in dichloromethane (DCM; 10 mL) and then added portion-wise to a solution of AlCl₃ (10.3 g, 77.4 mmol) and anisole (5.58 g, 51.6 mmol) in DCM (50 mL) at room temperature. The resulting solution was stirred at room temperature for 2 h. The reaction was quenched with a mixture of HCl (40 mL) in ice-cold water (200 mL), and the aqueous phase was extracted using DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–15% EtOAc in hexane) to yield 8 as an off-white liquid (5.20 g, 48.8% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.92–7.88 (m, 2H), 6.98–6.93 (m, 2H), 3.86 (s, 3H), 2.82 (s, 2H), 1.05 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₃H₁₉O₂ 207.1385, found 207.1376.

1-(4-Hydroxyphenyl)-3,3-dimethylbutan-1-one (9)

To a solution of 1-(4-methoxyphenyl)-3,3-dimethylbutan-1-one (8) (4.00 g, 19.3 mmol) in DCE (25 mL), we added aluminum chloride (AlCl₃; 5.17 g, 38.6 mmol) at 0 °C. The resulting mixture was stirred at 75 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with a mixture of HCl (20 mL) in ice-cold water (100 mL), and the aqueous phase was extracted using DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–12% EtOAc in hexane) to yield 9 as an off-white oil (3.10 g, 83.7% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.92–7.88 (m, 2H), 6.98–6.93 (m, 2H), 2.82 (s, 2H), 1.05 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₂H₁₇O₂ 193.1229, found 193.1221.

4-(1-Hydroxy-3,3-dimethylbutyl)phenol (10)

To a solution of 1-(4-hydroxyphenyl)-3,3-dimethylbutan-1-one (9) (1.30 g, 6.76 mmol) in THF (20 mL) at 0 °C, we added lithium aluminum hydride (LAH; 0.38 g, 10.1 mmol) portion-wise. The resulting mixture was stirred at the same temperature for 2 h. The reaction was quenched with saturated ammonium chloride solution (20 mL), and the aqueous phase was extracted using EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–20% EtOAc in hexane) to yield 10 as an off-white solid (0.91 g, 69.2% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 9.18 (s, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.70–6.63 (m, 2H), 4.72 (d, J = 4.5 Hz, 1H), 4.53 (dt, J = 8.3, 4.1 Hz, 1H), 1.57 (dd, J = 14.0, 8.4 Hz, 1H), 1.37 (dd, J = 14.0, 3.8 Hz, 1H), 0.91 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₂H₁₉O₂ 195.1385; the mass was not observed.

(E)-Benzyl-3-(4-(1-hydroxy-3,3-dimethylbutyl)phenoxy)acrylate (12)

To a solution of 4-(1-hydroxy-3,3-dimethylbutyl)phenol (10) (0.90 g, 4.63 mmol) and N-methyl morpholine (0.05 mL, 0.463 mmol) in acetonitrile (25 mL), we added benzyl propiolate (0.89 g, 5.55 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–10% EtOAc in hexane) to yield 12 as a colorless oil (1.20 g, 73.1% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.87 (d, J = 12.2 Hz, 1H), 7.41–7.29 (m, 6H), 7.14 (d, J = 8.7 Hz, 2H), 5.59 (d, J = 12.2 Hz, 1H), 5.15 (s, 2H), 5.00 (d, J = 4.8 Hz, 1H), 4.70–4.62 (m, 1H), 1.58 (dd, J = 14.1, 8.7 Hz, 1H), 1.39 (dd, J = 14.1, 3.1 Hz, 1H), 0.94 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₂₂H₂₇O₄ 355.1909, found 355.1909.

3-(4-(3,3-Dimethylbutyl)phenoxy)propanoic acid (13)

To a solution of ((E)-benzyl 3-(4-(1-hydroxy-3,3-dimethylbutyl)phenoxy)acrylate (12) (1.20 g, 3.38 mmol) in THF (25 mL), we added 10% Pd/C (0.35 g, 0.33 mmol). The resulting mixture was stirred at room temperature for 12 h under a hydrogen gas atmosphere (1 atm) using a balloon. After completion of the reaction, the reaction mixture was filtered through a celite pad, and the crude filtrate was evaporated under reduced pressure to yield 13 as an off-white powder (0.70 g, 82.7% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.08 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz,2H), 4.11 (t, J = 6.1 Hz, 2H), 2.64 (t, J = 6.1 Hz, 2H), 2.48–2.42 (m, 2H), 1.45–1.32 (m, 2H), 0.92 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₅H₂₃O 251.1647, found 251.1656.

Methyl-3-(3-(4-(4-hydroxy-2,4-dimethylpentan-2-yl)phenoxy)propanamido)benzoate (14)

The compound was prepared from 3-(4-(3,3-dimethylbutyl)phenoxy)propanoic acid (13) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (20% EtOAc in hexane) yielded 14 as an off-white oil (0.09 g, 55.2% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.00 (s, 1H), 7.59 (s, 1H), 7.36–7.28 (m, 2H), 7.13 (d, J = 8.1 Hz, 2H), 6.96 (d, J = 7.0 Hz, 1H), 6.87 (d, J = 7.7 Hz, 2H), 4.32 (t, J = 5.6 Hz, 2H), 2.85 (t, J = 5.6 Hz, 2H), 2.57–2.47 (m, 2H), 1.46 (dd, J = 10.6, 6.5 Hz, 2H), 0.95 (s, 9H); ¹³C NMR (100 MHz, CD₃OD): δ 170.68, 156.62, 149.23, 140.14, 135.78, 129.42, 128.90, 121.75, 119.20, 117.38, 117.08, 116.35, 115.76, 114.87, 114.12, 113.57, 63.73, 36.67, 29.95, 28.33; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.81; HRMS (ESI): [M + H]⁺ calculated for C₂₂H₂₇F₃NO₃ 410.1943, found 410.1932; RP-HPLC purity ≥ 98.3%, t_R = 27.1 min.

tert-Butyl (4-hydroxyphenyl)carbamate (16)

To a solution of 4-aminophenol (15) (3.00 g, 27.4 mmol) in THF (25 mL), we added triethylamine (Et₃N; 11.5 mL, 82.4 mmol) followed by Boc anhydride (7.47 mL, 32.8 mmol). The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with ice-cold water (100 mL), and the aqueous phase was extracted using EtOAc (3 × 70 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–30% EtOAc in hexane) to yield 16 (4.50 g, 79.6% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.19 (d, J = 8.4 Hz, 2H), 6.77–6.73 (m, 2H), 6.33 (s, 1H), 4.99 (s, 1H), 1.51 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₁H₁₆NO₃ 210.1130; the mass was not observed.

(E)-Benzyl-3-(4-((tert-butoxycarbonyl)amino)phenoxy)acrylate (17)

To a solution of tert-butyl(4-hydroxyphenyl)carbamate (16) (4.00 g, 19.1 mmol) and N-methyl morpholine (0.84 mL, 7.64 mmol) in acetonitrile (25 mL), was added benzyl propiolate (3.67 g, 22.9 mmol). The reaction mixture was stirred at ambient temperature for 2 h. The reaction mixture was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–20% EtOAc in hexane) to yield 17 (0.80 g, 11.3% yield) as a colorless semisolid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.39 (s, 1H), 7.82 (d, J = 12.2 Hz, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.37 (t, J = 5.6 Hz, 4H), 7.33 (dd, J = 8.5, 3.9 Hz, 1H), 7.10 (d, J = 8.9 Hz, 2H), 5.54 (d, J = 12.2 Hz, 1H), 5.15 (s, 2H), 1.47 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₂₁H₂₄NO₅ 370.1654, found 370.1650.

3-(4-((tert-Butoxycarbonyl)amino)phenoxy)propanoic acid (18)

To a solution of (E)-benzyl 3-(4-((tert-butoxycarbonyl)amino)phenoxy)acrylate (17) (0.80 g, 2.16 mmol) in THF (25 mL), was added 10% Pd/C (0.46 g, 0.43 mmol). The resulting mixture was stirred at room temperature for 12 h under a hydrogen gas atmosphere (1 atm) using a balloon. After completion of the reaction, the reaction mixture was filtered through a celite pad, and the crude filtrate was evaporated under reduced pressure to yield 18 as an off-white solid (0.35 g, 69.8% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 9.11 (s, 1H), 7.33 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.9 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 2.65 (t, J = 6.0 Hz, 2H), 1.46 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₄H₂₀NO₅ 282.1341, found 282.1333.

tert-Butyl(4-(3-oxo-3-((3-(trifluoromethoxy)phenyl)amino)propoxy)phenyl)carbamate (19)

The compound was prepared from 3-(4-((tert-butoxycarbonyl)amino)phenoxy)propanoic acid (18) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (20% EtOAc in hexane) yielded 19 as an off-white solid (1.35 g, 51.0% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.92 (s, 1H), 7.59 (s, 1H), 7.32 (d, J = 6.5 Hz, 2H), 7.30 (d, J = 8.7 Hz, 2H), 6.97 (s, 1H), 6.89 (d, J = 9.0 Hz, 2H), 6.37 (s, 1H), 4.31 (t, J = 5.8 Hz, 2H), 2.83 (t, J = 5.7 Hz, 2H), 1.51 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₂₁H₂₄F₃N₂O₅ 441.1637, found 441.1635.

3-(4-Aminophenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (20)

To a solution of tert-butyl(4-(3-oxo-3-((3-(trifluoromethoxy)phenyl)amino)propoxy)phenyl)carbamate (19) (1.30 g, 2.95 mmol) in DCM (25 mL), we added trifluoroacetic acid (TFA; 5 mL). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was basified with sodium bicarbonate solution (50 mL), and the aqueous phase was extracted using DCM (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–60% EtOAc in hexane) to yield 20 as an off-brown solid (1.00 g, 99.0% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 10.38 (s, 1H), 7.80 (s, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 7.2 Hz, 1H), 6.99 (d, J = 8.9 Hz, 2H), 4.24 (t, J = 5.9 Hz, 2H), 2.80 (t, J = 5.8 Hz, 2H), NH₂ protons were not observed; HRMS (ESI): [M + H]⁺ calculated for C₁₆H₁₆F₃N₂O₃ 341.1113, found 341.1119.

3-(4-(Neopentylamino)phenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (21)

To a solution of 3-(4-aminophenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (20) (1.0 g, 2.93 mmol) in DCM:MeOH (30 mL, 9:1), we added pivalaldehyde (0.5 mL, 4.40 mmol) followed by three drops of acetic acid; the mixture was stirred at room temperature for 1 h. Then, NaCNBH₃ (0.37 g, 5.86 mmol) was added to the stirring solution. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was basified with 50 mL of a sodium bicarbonate solution, and the aqueous phase was extracted using DCM (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–30% EtOAc in hexane) to yield 21 as a colorless liquid (0.60 g, 56.4% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.20 (s, 1H), 7.58 (s, 1H), 7.36–7.27 (m, 2H), 6.95 (d, J = 7.4 Hz, 1H), 6.82 (d, J = 8.8 Hz, 2H), 6.60 (d, J = 8.8 Hz, 2H), 4.25 (t, J = 5.7 Hz, 2H), 2.85 (s, 2H), 2.81 (t, J = 5.6 Hz, 2H), 0.99 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.58, 149.70, 144.49, 139.32, 130.27, 121.69, 119.16, 118.15, 116.91, 116.14, 114.00, 112.96, 65.29, 56.75, 37.78, 31.78, 27.73; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.77; HRMS (ESI): [M + H]⁺ calculated for C₂₁H₂₆F₃N₂O₃ 411.1896, found 411.1901; RP-HPLC purity ≥ 97.2%, t_R = 12.1 min.

3-(4-(Methyl(neopentyl)amino)phenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (22)

To a solution of 3-(4-(neopentylamino)phenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (21) (0.40 g, 0.97 mmol) in DCM:MeOH (30 mL, 9:1), we added para formaldehyde (0.06 g, 1.94 mmol) followed by three drops of acetic acid; the mixture was stirred at room temperature for 1 h. NaCNBH₃ (0.122 g, 1.94 mmol) was added to the stirring solution. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was basified with sodium bicarbonate solution (50 mL), and the aqueous phase was extracted using DCM (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (0–30% EtOAc in hexane) to yield 22 as a colorless liquid (0.26 g, 63.2% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.23 (s, 1H), 7.59 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 6.86 (d, J = 9.2 Hz, 2H), 6.71 (d, J = 9.2 Hz, 2H), 4.26 (t, J = 5.6 Hz, 2H), 3.07 (s, 2H), 2.94 (s, 3H), 2.81 (t, J = 5.6 Hz, 2H), 0.98 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.86, 149.70, 148.95, 148.93, 145.88, 141.20, 130.85, 121.79, 119.24, 117.99, 115.72, 115.57, 113.25, 111.53, 64.68, 64.36, 41.81, 37.14, 35.12, 28.67; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.76; HRMS (ESI): [M + H]⁺ calculated for C₂₂H₂₈F₃N₂O₃ 425.2052, found 425.2059; RP-HPLC purity ≥ 97.1%, t_R = 12.8 min.

3-(4-Methoxyphenyl)-3-methylbutanoic acid (24)

To a mixture of 3-bromo-3-methylbutanoic acid (23) (12.8 g, 71.1 mmol) and anisole (7.69 g, 71.1 mmol) in DCM (100 mL), we added AlCl₃ (28.4 g, 213.3 mmol) portion-wise at 0 °C. The resulting mixture was stirred at 65 °C for 3 h. The reaction was quenched with a mixture of HCl (40 mL) in 200 mL of ice-cold water, and the aqueous phase was extracted using DCM (3 × 100 mL). The combined organic layers were washed with 10% sodium hydroxide (NaOH) solution. The aqueous phase was acidified to pH 1 using 2 M of HCl and extracted using DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–40% EtOAc in hexane) to yield 24 as an off-white solid (3.00 g, 20.2% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 11.83 (s, 1H), 7.31–7.26 (m, 2H), 6.85–6.82 (m, 2H), 3.72 (s, 3H), 2.52 (s, 2H), 1.35 (s, 6H); HRMS (ESI): [M + H]⁺ calculated for C₁₂H₁₇O₃ 209.2650; the mass was not observed.

Methyl 3-(4-methoxyphenyl)-3-methylbutanoate (25)

To a solution of 3-(4-methoxyphenyl)-3-methylbutanoic acid (24) (2.93 g, 14.0 mmol) in MeOH (20.0 mL), we added concentrated sulfuric acid (H₂SO₄; 1.00 mL). The resulting mixture was stirred at room temperature for 12 h. The reaction was quenched with saturated sodium bicarbonate solution (40 mL), and the aqueous phase was extracted using DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–5% EtOAc in hexane) to yield 25 as an off-colorless liquid (2.12 g, 67.9% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.27 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.3 Hz, 2H), 3.72 (s, 3H), 3.44 (s, 3H), 2.60 (s, 2H), 1.34 (s, 6H); HRMS (ESI): [M + H]⁺ calculated for C₁₃H₁₉O₃ 223.1334, found 223.1335.

Methyl 3-(4-hydroxyphenyl)-3-methylbutanoate (26)

To a solution of methyl 3-(4-methoxyphenyl)-3-methylbutanoate (25) (2.04 g, 9.16 mmol) in DCM (25 mL), we added boron tribromide (1.0 M BBr₃ in DCM; 1.37 mL, 13.7 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with methanol (MeOH; 5 mL) and diluted with water (30 mL). The aqueous phase was extracted using DCM (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–5% EtOAc in hexane) to yield 26 as an off-brown solid (1.27 g, 66.5% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.27 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.3 Hz, 2H), 3.72 (s, 3H), 3.44 (s, 3H), 2.60 (s, 2H), 1.34 (s, 6H); HRMS (ESI): [M + H]⁺ calculated for C₁₂H₁₇O₃ 209.1178, found 209.1171.

4-(4-Hydroxy-2,4-dimethylpentan-2-yl)phenol (27)

To a solution of methyl 3-(4-hydroxyphenyl)-3-methylbutanoate (26) (1.21 g, 5.83 mmol) in THF (25 mL), we added 3.0 M of methyl magnesium bromide in ether (2.09 g, 17.5 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with water (40 mL), and the aqueous phase was extracted using EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (5−15% EtOAc in hexane) to yield 27 as an off-white solid (0.86 g, 71.1% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 9.05 (s, 1H), 7.13 (d, J = 8.7 Hz, 2H), 6.65 (d, J = 8.7 Hz, 2H), 3.85 (s, 1H), 1.81 (s, 2H), 1.28 (s, 6H), 0.79 (s, 6H). HRMS (ESI): [M + H]⁺ calculated for C₁₃H₂₁O₃ 209.3090; the mass was not observed.

Benzyl (E)-3-(4-(4-hydroxy-2,4-dimethylpentan-2-yl)phenoxy)acrylate (28)

To a mixture of 4-(4-hydroxy-2,4-dimethylpentan-2-yl)phenol (27) (0.77 g, 3.70 mmol) and benzyl propiolate (0.65 g, 4.07 mmol) in acetonitrile (25 mL), we added N-methyl morpholine (0.05 g, 0.52 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–10% EtOAc in hexane) to yield 28 as an off-white solid (1.3 g, 95.6% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.87 (d, J = 12.2 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 4.4 Hz, 4H), 7.35–7.31 (m, 1H), 7.10 (d, J = 8.8 Hz, 2H), 5.57 (d, J = 12.2 Hz, 1H), 5.15 (s, 2H), 3.94 (s, 1H), 1.87 (s, 2H), 1.34 (s, 6H), 0.79 (s, 6H).

3-(4-(4-Hydroxy-2,4-dimethylpentan-2-yl)phenoxy)propanoic acid (29)

To a solution of benzyl (E)-3-(4-(4-hydroxy-2,4-dimethylpentan-2-yl)phenoxy)acrylate (28) (1.40 g, 3.80 mmol) in THF (100 mL), we added 10% Pd/C (0.80 g, 0.75 mmol). The resulting mixture was stirred at room temperature for 18 h under a hydrogen gas atmosphere (1 atm) using a balloon. After completion of the reaction, the reaction mixture was filtered through a celite pad, and the crude filtrate was evaporated under reduced pressure to yield 29 as an off-brown liquid (0.89 g, 83.5% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.26 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.89 (s, 1H), 2.66 (t, J = 6.0 Hz, 2H), 1.84 (s, 2H), 1.30 (s, 6H), 0.79 (s, 6H); HRMS (ESI): [M + H]⁺ calculated for C₁₆H₂₅O₄ 281.1753, found 281.1758.

3-(4-(4-Hydroxy-2,4-dimethylpentan-2-yl)phenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (30)

The compound was prepared from 3-(4-(4-hydroxy-2,4-dimethylpentan-2-yl)phenoxy)propanoic acid (29) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–25% EtOAc in hexane) yielded 30 as an off-yellow oil (0.97 g, 76.4% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.26 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.89 (s, 1H), 2.66 (t, J = 6.0 Hz, 2H), 1.84 (s, 2H), 1.30 (s, 6H), 0.79 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆): δ 169.76 (s), 156.31 (s), 148.95 (s), 142.61 (s), 141.16 (s), 130.90 (s), 127.27 (s), 124.33 (s), 121.79 (s), 119.24 (s), 118.02 (s), 116.69 (s), 115.63 (s), 114.06 (s), 111.53 (s), 70.66 (s), 63.92 (s), 56.22 (s), 37.20 (s), 36.92 (s), 31.71 (s), 31.51 (s); ¹⁹F NMR (376 MHz, CDCl₃): δ -57.77; HRMS (ESI): [M + H]⁺ calculated for C₂₃H₂₉F₃NO₄ 440.2048, found 440.2038; RP-HPLC purity ≥ 97.4%, t_R = 19.6 min.

Ethyl 3-(4-bromophenoxy)propanoate (32)

The compound was prepared from 4-bromophenol (31) according to the general procedure for the Michael addition of phenol with acrylates. Purification via silica gel column chromatography (0–5% EtOAc in hexane) yielded 32 as an off-white solid (2.30 g, 38.9% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.37 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.7 Hz, 2H), 4.24–4.15 (m, 4H), 2.77 (t, J = 6.4 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); HRMS (ESI): [M + H]⁺ calculated for C₁₀H₁₂BrO₃ 258.9970; the mass was not observed.

3-(4-Bromophenoxy)propanoic acid (33)

The compound was prepared from ethyl 3-(4-bromophenoxy)propanoate (32) according to the general procedure for ester hydrolysis, yielding 33 as an off-white solid (1.50 g, 69.0% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.38 (d, J = 9.2 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 4.22 (t, J = 6.2 Hz, 2H), 2.82 (t, J = 6.2 Hz, 2H); HRMS (ESI): [M + H]⁺ calculated for C₉H₁₀BrO₃ 248.9813; the mass was not observed.

3-(4-Bromophenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (34)

The compound was prepared from 3-(4-bromophenoxy)propanoic acid (33) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 34 as an off-white solid (0.31 g, 62.7% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.25 (s, 1H), 7.57 (s, 1H), 7.35 (dd, J = 6.7, 2.3 Hz, 3H), 7.27 (dd, J = 10.8, 5.1 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 4.25 (t, J = 5.9 Hz, 2H), 2.80 (t, J = 5.9 Hz, 2H); HRMS (ESI): [M + H]⁺ calculated for C₁₆H₁₄BrF₃NO₃ 404.0109, found 404.0096.

3-([1,1′-Biphenyl]-4-yloxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (35a)

The compound was prepared from 3-(4-bromophenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (34) according to the general Suzuki coupling procedure. Purification via silica gel column chromatography (0–25% EtOAc in hexane) yielded (35a) as a white solid (0.05 g, 60.4% yield). Mp: 132–134 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.87–7.84 (s, 1H), 7.60 (s, 1H), 7.55 (d, J = 8.7 Hz, 4H), 7.42 (t, J = 7.4 Hz, 2H), 7.35 (s, 3H), 7.03 (d, J = 8.6 Hz, 2H), 6.99–6.96 (m, 1H), 4.39 (d, J = 5.5 Hz, 2H), 2.88 (s, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 168.97, 157.46, 149.55, 140.50, 139.11, 134.80, 130.01, 128.76, 128.37, 126.87, 126.75, 121.26, 119.56, 117.77, 116.43, 114.88, 112.67, 64.13, 37.69; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78; HRMS (ESI): [M + H]⁺ calculated for C₂₂H₁₉F₃NO₃ 402.1317, found 402.1312; RP-HPLC purity ≥ 99.8%, t_R = 22.8 min.

3-((4′-Fluoro-[1,1′-biphenyl]-4-yl)oxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (35b)

The compound was prepared from 3-(4-bromophenoxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (34) according to the general Suzuki coupling procedure. Purification via silica gel column chromatography (0–25% EtOAc in hexane) yielded (35b) as a white solid (0.04 g, 41.5% yield). Mp: 101–103 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.84 (s, 1H), 7.60 (s, 1H), 7.49 (dt, J = 8.9, 2.7 Hz, 4H), 7.34 (dt, J = 16.0, 8.2 Hz, 2H), 7.11 (t, J = 8.7 Hz, 2H), 7.00 (dd, J = 15.4, 8.0 Hz, 3H), 4.39 (t, J = 5.8 Hz, 2H), 2.88 (t, J = 5.7 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 169.03, 163.36, 160.96, 157.50, 149.61, 139.14, 136.64, 133.75, 130.39, 128.56, 127.88, 121.69, 119.13, 117.49, 116.51, 115.68, 115.01, 114.20, 113.00, 112.38, 64.10, 37.62; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78, -116.36; HRMS (ESI): [M + H]⁺ calculated for C₂₂H₁₈F₄NO₃ 420.1223, found 420.1216; RP-HPLC purity ≥ 97.6%, t_R = 23.0 min.

Methyl 3-(naphthalen-2-yloxy)propanoate (37a)

The compound was prepared from naphthalen-2-ol (36a) according to the general procedure for the Michael addition of phenol with acrylates. Purification via silica gel column chromatography (0–5% EtOAc in hexane) yielded 37a as an off-colorless liquid (0.50 g, 31.4% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.76 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 6.1 Hz, 2H), 7.46–7.41 (m, 1H), 7.36–7.31 (m, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.13 (dd, J = 8.8, 2.5 Hz,1H), 4.38 (t, J = 6.5 Hz, 2H), 3.75 (s, 3H), 2.88 (t, J = 6.5 Hz, 2H); HRMS (ESI): [M + H]⁺ calculated for C₁₄H₁₅O₃ 231.1021, found 231.1015.

Methyl 3-((7-bromonaphthalen-2-yl)oxy)propanoate (37b)

The compound was prepared from 7-bromonaphthalen-2-ol (36b) according to the general procedure for the Michael addition of phenol with acrylates. Purification via silica gel column chromatography (0–5% EtOAc in hexane) yielded 37b as an off-colorless liquid (0.43 g, 31.1% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.89 (s, 1H), 7.70 (d, J = 9.6 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 8.6 Hz, 1H), 7.14 (d, J = 6.7 Hz, 1H), 7.06 (s, 1H), 4.36 (t, J = 5.1 Hz, 2H), 3.76 (s, 3H), 2.88 (t, J = 6.1 Hz, 2H); HRMS (ESI): [M + H]⁺ calculated for C₁₄H₁₄BrO₃ 309.0126; the mass was not observed.

Methyl 3-((6-bromonaphthalen-2-yl)oxy)propanoate (37c)

The compound was prepared from 6-bromonaphthalen-2-ol (36c) according to the general procedure for the Michael addition of phenol with acrylates. Purification via silica gel column chromatography (0–5% EtOAc in hexane) yielded 37c as an off-colorless liquid (0.53 g, 38.9% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.92 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 15.2 Hz, 2H), 4.36 (t, J = 6.4 Hz, 2H), 3.75 (s, 3H), 2.88 (t, J = 6.4 Hz, 2H); HRMS (ESI): [M + H]⁺ calculated for C₁₄H₁₄BrO₃ 309.0126; the mass was not observed.

Ethyl 3-((6-fluoronaphthalen-2-yl)oxy)propanoate (37d)

The compound was prepared from 6-fluoronaphthalen-2-ol (36d) according to the general procedure for the Michael addition of phenol with acrylates. Purification via silica gel column chromatography (0–5% EtOAc in hexane) yielded 37d as an off-white solid (0.27 g, 16.8% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.72–7.69 (m, 1H), 7.67 (d, J = 9.5 Hz, 1H), 7.38 (dd, J = 9.8, 2.6 Hz, 1H), 7.22 (td, J = 8.8, 2.6 Hz, 1H), 7.18–7.15 (m, 2H), 4.36 (t, J = 6.5 Hz, 2H), 4.21 (q, J = 7.1 Hz, 2H), 2.86 (t, J = 6.4 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H); HRMS (ESI): [M + H]⁺ calculated for C₁₅H₁₆FO₃ 263.1083, found 263.1090.

3-(Naphthalen-2-yloxy)propanoic acid (38a)

The compound was prepared from methyl 3-(naphthalen-2-yloxy)propanoate (37a) according to the general procedure for ester hydrolysis, yielding 38a as an off-white solid (0.35 g, 74.6% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 7.73 (dd, J = 8.4, 5.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.38–7.30 (m, 3H), 7.23 (t, J = 7.0 Hz, 1H), 7.12 (dd, J = 8.9, 2.5 Hz, 1H), 4.27 (t, J = 6.1 Hz, 2H), 2.75 (t, J = 6.1 Hz, 2H); the acid proton was not observed; HRMS (ESI): [M + H]⁺ calculated for C₁₃H₁₃O₃ 217.0865, found 217.0859.

3-((7-Bromonaphthalen-2-yl)oxy)propanoic acid (38b)

The compound was prepared from methyl 3-((7-bromonaphthalen-2-yl)oxy)propanoate (37b) according to the general procedure for ester hydrolysis, yielding 38b as an off-white solid (0.43 g, 31.1% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.18 (d, J = 10.8 Hz, 1H), 4.28 (t, J = 5.9 Hz, 2H), 2.78 (t, J = 5.9 Hz, 2H); the acid proton was not observed; HRMS (ESI): [M + H]⁺ calculated for C₁₃H₁₂BrO₃ 294.9970; the mass was not observed.

3-((6-Bromonaphthalen-2-yl)oxy)propanoic acid (38c)

The compound was prepared from methyl 3-((6-bromonaphthalen-2-yl)oxy)propanoate (37c) according to the general procedure for ester hydrolysis, yielding 38c as an off-white solid (0.23 g, 53.6% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.01 (s, 1H), 7.79 (d, J = 9.9 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.37 (s,1H), 7.10 (d, J = 7.7 Hz, 1H), 4.25 (t, J = 6.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H); the acid proton was not observed; HRMS (ESI): [M + H]⁺ calculated for C₁₃H₁₂BrO₃ 294.9970; the mass was not observed.

3-((6-Fluoronaphthalen-2-yl)oxy)propanoic acid (38d)

The compound was prepared from ethyl 3-((6-fluoronaphthalen-2-yl)oxy)propanoate (37d) according to the general procedure for ester hydrolysis, yielding 38d as an off-white solid (0.20 g, 86.2% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 9.72 (s, 1H), 7.77(d, 1H), 7.72 (d, 1H), 7.41 (d, J = 5.7 Hz, 1H), 7.20 (d, J = 8.9 Hz, 1H), 7.17–7.10 (m, 2H), 4.28 (t, J = 6.1 Hz, 2H), 2.77 (t, J = 6.1 Hz, 2H); HRMS (ESI): [M + H]⁺ calculated for C₁₃H₁₂FO₃ 235.0770; the mass was not observed.

3-(Naphthalen-2-yloxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (39a)

The compound was prepared from 3-(naphthalen-2-yloxy)propanoic acid (38a) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 39a as an off-ivory solid (0.15 g, 27.8% yield). Mp: 82–85 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.87 (s, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.60 (s, 1H), 7.46 (t, J = 6.9 Hz, 1H), 7.37 (s, 1H), 7.35 (s, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.21 (s, 1H), 7.17 (dd, J = 8.9, 2.5 Hz, 1H), 6.97 (d, J = 7.1 Hz, 1H), 4.48 (t, J = 5.8 Hz, 2H), 2.93 (t, J = 5.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 169.09, 155.86, 149.57, 139.14, 134.41, 130.34, 129.53, 129.25, 126.95, 126.27, 124.43, 123.70, 121.70, 119.14, 118.56, 118.18, 117.47, 116.51, 113.02, 107.53, 63.93, 37.55; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78; HRMS (ESI): [M + H]⁺ calculated for C₂₀H₁₇F₃NO₃ 376.1161, found 376.1163; RP-HPLC purity ≥ 97.9%, t_R = 21.5 min.

3-((7-Bromonaphthalen-2-yl)oxy)-N-(3-trifluoromethoxy)phenyl)propanamide (39b)

The compound was prepared from 3-((7-bromonaphthalen-2-yl)oxy)propanoic acid (38b) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 39b as an off-ivory solid (0.02 g, 17.5% yield). Mp: 113–115 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.90 (s, 1H), 7.74 (d, J = 8.9 Hz, 2H), 7.66–7.59 (m, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.6 Hz, 1H), 7.11 (s, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.46 (t, J = 5.7 Hz, 2H), 2.93 (t, J = 5.8 Hz, 2H); ¹³C NMR (150 MHz, CDCl₃): δ 169.02, 156.68, 149.53, 139.01, 135.61, 130.03, 129.65, 129.26, 128.79, 127.58, 127.33, 121.25, 120.78, 118.74, 117.89, 116.54, 112.74, 106.23, 63.90, 31.58; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78; HRMS (ESI): [M + H]⁺ calculated for C₂₀H₁₆BrF₃NO₃ 454.0266, found 454.0254; RP-HPLC purity ≥ 98.8%, t_R = 24.3 min.

3-((6-Bromonaphthalen-2-yl)oxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (39c)

The compound was prepared from 3-((6-bromonaphthalen-2-yl)oxy)propanoic acid (38c) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 39c as an off-white solid (0.06 g, 24.7% yield). Mp: 128–130 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.93 (s, 1H), 7.76 (s, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.7 Hz, 1H), 7.34 (d, J = 7.9 Hz, 2H), 7.17 (s, 2H), 6.98 (d, J = 8.4 Hz, 1H), 4.46 (t, J = 5.7 Hz, 2H), 2.92 (t, J = 5.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 168.73, 156.19, 149.54, 139.00, 132.85, 130.27, 130.01, 129.76, 129.55, 128.93, 128.67, 128.62, 128.30, 119.45, 119.10, 117.54, 116.50, 107.20, 63.95, 37.56; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.79; HRMS (ESI): [M + H]⁺ calculated for C₂₀H₁₆BrF₃NO₃ 454.0266, found 454.0251; RP-HPLC purity ≥ 98.2%, t_R = 24.4 min.

3-((6-Fluoronaphthalen-2-yl)oxy)-N-(3-(trifluoromethoxy)phenyl)propanamide (39d)

The compound was prepared from 3-((6-fluoronaphthalen-2-yl)oxy)propanoic acid (38d) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 39d as an off-white solid (0.04 g, 26.0% yield). Mp: 102–104 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.78 (s, 1H), 7.73 (d, J = 4.6 Hz, 1H), 7.71 (d, J = 4.3 Hz, 1H), 7.60 (s, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.35 (s, 1H), 7.32 (d, J = 7.9 Hz, 1H), 7.21 (s, 2H), 7.19 (s, 1H), 6.98 (d, J = 8.1 Hz, 1H), 4.46 (t, J = 6.0 Hz, 2H), 2.92 (t, J = 5.7 Hz, 2H); ¹³C NMR (100 MHz, cdcl3): δ 168.81, 160.73, 158.31, 155.36–155.35, 149.58, 139.02, 131.22, 129.05, 128.82, 119.44, 119.32, 117.84, 117.01, 116.48, 112.74, 112.53, 110.85, 107.46, 64.01, 37.62; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78, -117.6; HRMS (ESI): [M + H]⁺ calculated for C₂₀H₁₆F₄NO₃ 394.1066, found 394.1060; RP-HPLC purity ≥ 97.0%, t_R = 22.1 min.

tert-Butyl(2-bromoethyl)carbamate (41)

To a solution of 2-bromoethanamine HBr salt (40) (3.00 g, 14.7 mmol) in DCM (20 mL), we added Et₃N (4.12 mL, 29.5 mmol) followed by Boc anhydride (4.07 mL, 17.7 mmol). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with ice-cold water (100 mL), and the aqueous phase was extracted using DCM (3 × 70 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–10% EtOAc in hexane) to yield 41 as an off-colorless liquid (2.5 g, 78.0% yield). ¹H NMR (400 MHz, CDCl₃): δ 4.94 (br s, 1H), 3.67 (t, J = 8.2 Hz, 2H), 3.45 (t, J = 7.2 Hz, 2H), 1.45 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₇H₁₅BrNO₂ 224.0286, found 224.0288.

tert-Butyl(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethyl)carbamate (42)

To a solution of 4-(2,4,4-trimethylpentan-2-yl)phenol (1) (0.25 g, 12.1 mmol) in acetone 50 mL, we added potassium carbonate (K₂CO₃; 5.02 g, 36.3 mmol) followed by tert-butyl(2-bromoethyl)carbamate (41) (3.25 g, 14.5 mmol). The resulting mixture was stirred at 80 °C for 12 h. The reaction mixture was cooled to room temperature and evaporated under reduced pressure to remove acetone. The residue was diluted with water (50 mL), and the aqueous phase was extracted using EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–15% EtOAc in hexane) to yield 42 as an off-white solid (2.00 g, 47.2% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.22 (d, J = 7.2 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 4.03 (t, J = 19.4 Hz, 2H), 3.52 (d, J = 5.2 Hz, 2H), 1.70 (s, 2H), 1.45 (s, 9H), 1.33 (s, 6H), 0.71 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₂₁H₃₅NO₃ 350.2695, found 350.2685.

2-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)ethanamine (43)

To a solution of tert-butyl (2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethyl)carbamate (42) (2.0 g, 5.72 mmol) in DCM (25 mL), we added TFA (10 mL) at room temperature. The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–80% EtOAc in hexane) to yield 43 as an off-white solid (1.1 g, 77.1% yield). ¹H NMR (400 MHz, DMSO): δ 7.97 (d, J = 808 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 4.13 (t, J = 2.6 Hz, 2H), 3.22 (d, J = 2.4 Hz, 2H), 1.69 (s, 2H), 1.30 (s, 6H), 0.67 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₆H₂₈NO 250.2171, found 250.2162.

3-(Trifluoromethoxy)-N-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethyl)benzamide (44a)

To a solution of 2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethanamine (43) (0.2 g, 0.80 mmol), and Et₃N (0.33 mL, 2.40 mmol) in DCM (20 mL), we added 3-(trifluoromethoxy)benzoyl chloride (0.21 g, 0.96 mmol). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with ice-cold water (20 mL), and the aqueous phase was extracted using DCM (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–20% EtOAc in hexane) to yield 44a as an off-white solid (0.085 g, 24.2% yield). Mp: 65–67 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.68 (d, J = 7.2 Hz, 2H), 7.47 (t, J = 8.2 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.59 (s, 1H), 4.15 (t, J = 4.8 Hz, 2H), 3.88 (dd, J = 10.3, 5.1 Hz, 2H), 1.70 (s, 2H), 1.34 (s, 5H), 0.71 (s, 8H); ¹³C NMR (100 MHz, CDCl₃): δ 166.10, 155.98, 149.36, 142.97, 136.47, 130.03, 127.19, 125.07, 123.82, 121.64, 120.02, 119.04, 113.61, 66.47, 56.89, 39.75, 37.95, 32.28, 31.74, 31.64; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.84; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₁F₃NO₃ 438.2256, found 438.2250; RP-HPLC purity ≥ 98.0%, t_R = 28.1 min.

3-(Trifluoromethoxy)-N-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethyl)benzenesulfonamide (44b)

To a solution of 2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethanamine (43) (0.2 g, 0.80 mmol), and Et₃N (0.33 mL, 2.40 mmol) in DCM (20 mL), we added 3-(trifluoromethoxy)benzenesulfonyl chloride (0.25 g, 0.96 mmol). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with ice-cold water (20 mL), and the aqueous phase was extracted using DCM (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–20% EtOAc in hexane) to yield 44b as an off-colorless oil (0.03 g, 7.90% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.82 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.24 (s, 1H), 6.71 (d, J = 8.4 Hz, 2H), 5.01 (s, 1H), 3.99 (t, J = 4.7 Hz, 2H), 3.40 (dd, J = 10.6, 5.4 Hz, 2H), 1.69 (s, 2H), 1.33 (s, 6H), 0.70 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 155.44, 149.35, 143.21, 142.14, 130.85, 127.18, 125.21, 125.03, 119.68, 113.49, 65.95, 56.89, 42.74, 37.95, 32.29, 31.73, 31.63; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.95; HRMS (ESI): [M + H]⁺ calculated for C₂₃H₃₁F₃NO₄S 474.1926, found 474.1931; RP-HPLC purity ≥ 99.9%, t_R = 28.1 min.

4-(2,4,4-Trimethylpentan-2-yl)phenyl trifluoromethanesulfonate (45)

To a solution of 4-(2,4,4-trimethylpentan-2-yl)phenol (1) (15.0 g, 72.7 mmol) in DCM (20 mL), we added Et₃N (3.4 mL, 87.2 mmol). The reaction mixture was cooled to 0 °C, and trifluoromethanesulfonic anhydride (14.6 mL, 87.2 mmol) was added dropwise to the stirring solution. The resulting mixture was stirred at the same temperature for 1 h. The reaction mixture was diluted with ice-cold water (100 mL), and the aqueous phase was extracted using DCM (3 × 70 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–1% EtOAc in hexane) to yield 45 as an off-colorless oil (20.5 g, 83.3% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.43 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 9.2 Hz, 2H), 1.74 (s, 2H), 1.37 (s, 6H), 0.70 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C1₅H₂₂F₃O₃S 339.1242; the mass was not observed.

Methyl 1-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperidine-4-carboxylate (46)

To a solution of 4-(2,4,4-trimethylpentan-2-yl)phenyl trifluoromethanesulfonate (45) (3.0 g, 8.86 mmol), methyl piperidine-4-carboxylate (1.9 g, 10.6 mmol) in toluene (30 mL), we added Cs₂CO₃ (8.67 g, 26.5 mmol). The mixture was degassed with argon for 10 min, and 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropylbiphenyl (Xphos; 0.84 g, 1.77 mmol) and tris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃; 1.62 g, 1.77 mmol) were added. The resulting mixture was stirred at 110 °C for 12 h in a sealed tube. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), and filtered through a celite bed. The filtrate was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–7% EtOAc in hexane) to yield 46 as an off-white solid (1.74 g, 59.3% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.25 (d, J = 4.6 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 3.70 (s, 3H), 3.61 (d, J = 12.3 Hz, 2H), 2.74 (td, J = 11.9, 2.5 Hz, 2H), 2.06–1.99 (m, 2H), 1.89 (dt, J = 21.0, 7.5 Hz, 2H), 1.69 (s, 2H), 1.33 (s, 6H), 0.71 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₂₁H₃₄NO₂ 332.2590, found 332.2577.

1-(4-(2,4,4-Trimethylpentan-2-yl)phenyl)piperidine-4-carboxylic acid (47)

The compound was prepared from methyl 1-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperidine-4-carboxylate (46) according to the general procedure for ester hydrolysis, yielding 47 as an off-white solid (1.58 g, 99.1% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.19 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.56 (d, J = 9.1 Hz, 2H), 2.67 (t, J = 13.0 Hz, 2H), 1.88 (d, J = 10.8 Hz, 2H), 1.66 (s, 2H), 1.60 (dd, J = 17.8, 6.6 Hz, 2H), 1.28 (s, 6H), 0.68 (s 9H); the acid proton was not observed; HRMS (ESI): [M + H]⁺ calculated for C₂₀H₃₃NO₂ 318.2433, found 318.2417.

tert-Butyl 4-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperazine-1-carboxylate (48)

To a solution of 4-(2,4,4-trimethylpentan-2-yl)phenyl trifluoromethanesulfonate (45) (1.0 g, 2.95 mmol), 1-boc-piperazine (0.66 g, 3.54 mmol) in 30 mL of toluene, we added Cs₂CO₃ (2.88 g, 8.86 mmol). The mixture was degassed with argon for 10 min, and Xphos (0.28 g, 0.59 mmol) and Pd₂(dba)₃ (0.54 g, 0.59 mmol) were added. The resulting mixture was stirred at 110 °C for 12 h in a sealed tube. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), and filtered through a celite bed. The filtrate was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–15% EtOAc in hexane) to yield 48 as an off-white solid (1.03 g, 93.2% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.26 (d, 2H), 6.85 (d, J = 8.7 Hz, 2H), 3.60–3.54 (m, 4H), 3.13–3.06 (m, 4H), 1.69 (s, 2H), 1.48 (s, 9H), 1.33 (s, 6H), 0.71 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₂₃H₃₉N₂O₂ 375.3012; the mass was not observed.

1-(4-(2,4,4-Trimethylpentan-2-yl)phenyl)piperazine (49)

To a solution of tert-butyl 4-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperazine-1-carboxylate (48) (1.0 g) in DCM (30 mL), we added TFA (5 mL) at room temperature. The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–10% EtOAc in hexane) to yield 49 as an off-white solid (0.61 g, 83.3% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.86 (s, 1H), 7.25 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 3.27 (dd, J = 13.2 Hz, 4H), 3.24 (s, J = 13.2 Hz, 4H), 1.68 (s, 2H), 1.29 (s, 6H), 0.68 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₈H₃₁N₂ 275.2487, found 275.2475.

N-(3-(Trifluoromethoxy)phenyl)-1-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperidine-4-carboxamide (50)

The compound was prepared from 1-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperidine-4-carboxylic acid (47) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 50 as an off-white solid (0.11 g, 59.0% yield). Mp: 139–140 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 10.26 (s, 1H), 7.84 (s, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.21 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 9.2 Hz, 1H), 6.87 (d, J = 8.8 Hz, 2H), 3.72 (d, J = 12.5 Hz, 2H), 2.64 (t, J = 11.8 Hz, 2H), 1.88 (d, J = 14.9 Hz, 2H), 1.75 (t, J = 10.4 Hz, 2H), 1.67 (s, 2H), 1.28 (s, 6H), 0.69 (s, 9H); ¹³C NMR (100 MHz, DMSO): δ 174.34, 149.11, 148.90, 141.42, 140.09, 130.82, 126.80, 121.78, 119.23, 118.02, 116.68, 115.80, 115.48, 111.50, 56.71, 48.95, 43.24, 37.82, 32.45, 32.02, 31.93, 28.48; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.78; HRMS (ESI): [M + H]⁺ calculated for C₂₇H₃₇F₃N₂O₂ 477.2729, found 477.2726; RP-HPLC purity ≥ 98.3%, t_R = 16.0 min.

(3-(Trifluoromethoxy)phenyl)(4-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperazin-1-yl)methanone (51)

To a solution of 1-(4-(2,4,4-trimethylpentan-2-yl)phenyl)piperazine (49) (0.1 g, 0.36 mmol) and Et₃N (0.15 mL, 1.09 mmol) in DCM (20 mL), we added 3-(trifluoromethoxy)benzoyl chloride (0.1 mL, 0.54 mmol). The resulting mixture was stirred at room temperature for 12 h. After the completion of the reaction, the solvent was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–15% EtOAc in hexane) to yield 51 as an off-white solid (0.06 g, 36.0% yield). Mp: 102–104 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.50–7.45 (m, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.30 (d, J = 6.2 Hz, 3H), 7.27 (s, 1H), 6.86 (d, J = 8.8 Hz, 2H), 3.94 (s, 2H), 3.57 (s, 2H), 3.18 (d, J = 59.8 Hz, 4H), 1.70 (s, 2H), 1.34 (s, 6H), 0.71 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 168.53, 149.14, 148.18, 142.52, 137.52, 126.96, 126.80, 125.54, 125.35, 122.24, 122.05, 121.63, 119.89, 119.72, 119.07, 116.14, 116.10, 77.31, 76.99, 76.68, 56.87, 37.88, 32.29, 31.80, 31.69, 31.55, 31.49.; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.85; HRMS (ESI): [M + H]⁺ calculated for C₂₆H₃₄F₃N₂O₂ 463.2572, found 463.2564; RP-HPLC purity ≥ 99.8%, t_R = 28.2 min.

4-(2,4,4-Trimethylpentan-2-yl)aniline (53)

To a solution of 4-(2,4,4-trimethylpentan-2-yl)phenyl trifluoromethanesulfonate (45) (5.0 g, 14.7 mmol) and diphenylmethanimine (2.42 mL, 17.7 mmol) in toluene (30 mL), we added Cs₂CO₃ (14.4 g, 44.3 mmol). The mixture was degassed with argon for 10 min, and 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (BINAP; 1.8 g, 2.95 mmol) and bis(dibenzylideneacetone)palladium (Pd(dba)₂; 1.7 g, 2.95 mmol) were added. The resulting mixture was stirred at 100 °C for 12 h in a sealed tube. The reaction mixture was cooled to room temperature, diluted with EtOAc (20 mL), and filtered through a celite bed. The filtrate was evaporated under reduced pressure to yield crude (52). The crude product was dissolved in methanol (40 mL) followed by the addition of 35% HCl (10 mL). The resulting mixture was stirred at 70 °C for 2 h. The reaction mixture was cooled to room temperature, and excess methanol was evaporated under pressure. The obtained crude extract was basified with 100 mL of saturated sodium bicarbonate solution, and the aqueous phase was extracted using EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–20% EtOAc in hexane) to yield 52 as a yellow oil (1.19 g, 39.2% yield). ¹H NMR (400 MHz, DMSO): δ 7.00 (d, J = 8.3 Hz, 2H), 6.50 (dd, J = 26.5, 8.2 Hz, 2H), 4.76 (s, 2H), 1.61 (s, 2H), 1.24 (s, 6H), 0.67 (s, 9H); HRMS (ESI): [M + H]⁺ calculated for C₁₄H₂₅N 206.1909, found 206.1903.

Methyl 3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanoate (54)

Methyl 3-bromopropionate (5.00 mL, excess) was added to a stirring solution of 4-(2,4,4-trimethylpentan-2-yl)aniline (53) (1.20 g, 5.84 mmol), Cs₂CO₃ (5.71 g, 17.5 mmol) and sodium iodide (0.43 g, 2.92 mmol) in acetonitrile (30 mL). The resulting mixture was stirred at 70 °C for 12 h in a sealed tube. The reaction mixture was cooled to room temperature, diluted with EtOAc (20 mL), and filtered through a celite bed. The filtrate was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–7% EtOAc in hexane) to yield 54 as a yellow solid (0.61 g, 34.7% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.17 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 8.3 Hz, 2H), 3.70 (s, 3H), 3.44 (t, J = 6.4 Hz, 2H), 2.62 (t, J = 6.4 Hz, 2H), 1.67 (s, 2H), 1.32 (s, 6H), 0.72 (s, 9H); the NH proton was not observed; HRMS (ESI): [M + H]⁺ calculated for C₁₈H₃₀NO₂ 292.2277, found 292.2290.

3-((4-(2,4,4-Trimethylpentan-2-yl)phenyl)amino)propanoic acid (55)

The compound was prepared from methyl 3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanoate (54) according to the general procedure for ester hydrolysis, yielding 55 as a white solid (0.56 g, 98.0% yield). ¹H NMR (400 MHz, DMSO): δ 7.53 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 3.43 (t, J = 7.1 Hz, 2H), 2.71 (t, J = 7.1 Hz, 2H), 1.74 (s, 2H), 1.33 (s, 6H), 0.68 (s, 9H); NH, the acid proton was not observed; HRMS (ESI): [M + H]⁺ calculated for C₁₇H₂₈NO₂ 278.2120, found 278.2122.

N-(3-(Trifluoromethoxy)phenyl)-3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanamide (56)

The compound was prepared from 3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanoic acid (55) according to the general acid–amine coupling procedure. Purification via silica gel column chromatography (0–25% EtOAc in Hexane) yielded 56 as an off-white oil (0.12 g, 27.7% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.09 (s, 1H), 7.53 (s, 1H), 7.38–7.31 (m, 1H), 7.28 (s, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H), 6.99–6.90 (m, 1H), 6.66 (d, J = 8.6 Hz, 1H), 6.37 (d, J = 7.9 Hz, 1H), 5.30 (s, 1H), 3.55 (t, J = 5.5 Hz, 2H), 2.72–2.52 (m, 2H), 1.68 (s, 2H), 1.33 (s, 6H), 0.72 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.44, 149.49, 144.57, 140.67, 139.17, 130.28, 129.54, 127.40, 126.76, 124.24, 121.68, 119.12, 118.19, 117.46, 117.06, 116.31, 115.55, 113.66, 113.28, 113.00, 112.32, 56.95, 40.61, 37.82, 36.75, 32.31, 31.68; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.79; HRMS (ESI): [M + H]⁺ calculated for C₂₄H₃₂F₃N₂O₂ 437.2416, found 437.2401; RP-HPLC purity ≥ 99.9%, t_R = 19.6 min.

3-(Methyl(4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)-N-(3-(trifluoromethoxy)phenyl)propanamide (57a)

The compound was prepared from N-(3-(trifluoromethoxy)phenyl)-3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanamide (56) according to the general reductive amination procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 57a as an off-white solid (0.05 g, 55.3% yield). Mp: 101–103 °C. ¹H NMR (400 MHz, CDCl₃): δ 8.53 (s, 1H), 7.50 (s, 1H), 7.29 (d, J = 8.9 Hz, 2H), 7.23 (d, J = 8.1 Hz, 1H), 6.93 (d, J = 7.1 Hz, 1H), 6.83 (d, J = 8.6 Hz, 2H), 3.61 (t, J = 6.1 Hz, 2H), 2.92 (s, 3H), 2.62 (t, J = 6.1 Hz, 2H), 1.70 (s, 2H), 1.35 (s, 6H), 0.72 (s,9H); ¹³C NMR (100 MHz, CD₃OD): δ 171.88, 149.25, 146.53, 140.21, 138.05, 129.94, 129.36, 126.70, 126.23, 121.75, 119.21, 118.06, 117.43, 116.21, 115.59, 114.74, 113.16, 112.58, 112.35, 111.86, 56.54, 49.17, 37.11, 33.89, 31.68, 30.75; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.77; HRMS (ESI): [M + H]⁺ calculated for C₂₅H₃₄F₃N₂O₂ 451.2572, found 451.2571; RP-HPLC purity ≥ 99.7%, t_R = 19.4 min.

3-(Isopropyl(4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)-N-(3-(trifluoromethoxy)phenyl)propanamide (57b)

The compound was prepared from N-(3-(trifluoromethoxy)phenyl)-3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanamide (56) according to the general reductive amination procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 57b as an off-green liquid (0.01 g, 9.10% yield). ¹H NMR (400 MHz, CDCl₃): δ 10.10 (s, 1H), 7.59 (s, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.5 Hz, 3H), 6.98 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.3 Hz, 1H), 3.63 (dt, J = 13.0, 6.5 Hz, 1H), 3.44–3.38 (t, 2H), 2.46–2.41 (t, 2H), 1.70 (s, 2H), 1.36 (s, 6H), 1.13 (d, J = 6.6 Hz, 6H), 0.70 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.00, 148.66, 144.39, 142.55, 138.93, 129.24, 126.26, 121.34, 120.76, 120.57, 118.20, 116.61, 114.70, 111.50, 56.12, 54.10, 41.55, 37.21, 33.31, 31.38, 30.81, 30.32, 18.70, 0.09; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.74; HRMS (ESI): [M + H]⁺ calculated for C₂₇H₃₉F₃N₂O₂ 479.2885, found 479.2873; RP-HPLC purity ≥ 96.1%, t_R = 15.3 min.

3-(Cyclobutyl(4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)-N-(3-(trifluoromethoxy)phenyl)propanamide (57c)

The compound was prepared from N-(3-(trifluoromethoxy)phenyl)-3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanamide (56) according to the general reductive amination procedure. Purification via silica gel column chromatography (0–20% EtOAc in hexane) yielded 57c as an off-colorless liquid (0.04 g, 38.0% yield). ¹H NMR (400 MHz, CDCl₃): δ 10.10 (s, 1H), 7.62 (s, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.31 (d, J = 8.8 Hz, 3H), 6.96 (t, J = 7.9 Hz, 3H), 3.71 (dt, J = 15.9, 8.1 Hz, 1H), 3.35 (t, J = 6.0 Hz, 2H), 2.38 (t, J = 6.0 Hz, 2H), 2.11 (dd, J = 15.9, 8.0 Hz, 2H), 2.03–1.89 (m, 2H), 1.70 (s, 2H), 1.64 (m, 2H), 1.35 (s, 6H), 0.69 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.89, 149.61, 145.84, 143.38, 139.91, 127.38, 126.85, 121.83, 121.68, 121.04, 115.83, 112.43, 111.88, 58.29, 58.09, 57.08, 46.02, 38.19, 34.19, 32.32, 31.74, 31.54, 31.27, 28.56, 14.34; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.74; HRMS (ESI): [M + H]⁺ calculated for C₂₈H₃₉F₃N₂O₂ 491.2885, found 491.2888; RP-HPLC purity ≥ 96.3%, t_R = 16.8 min.

N-(3-(Trifluoromethoxy)phenyl)-3-(N-(4-(2,4,4-trimethylpentan-2-yl)phenyl)acetamido)propanamide (57d)

To a solution of N-(3-(trifluoromethoxy)phenyl)-3-((4-(2,4,4-trimethylpentan-2-yl)phenyl)amino)propanamide (56) (0.06 g, 0.13 mmol) in DCM (20 mL), we added triethyl amine (0.04 mL, 0.41 mmol) followed by acetyl chloride (0.03 mL, 0.41 mmol). The resulting mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated sodium bicarbonate solution (40 mL), and the aqueous phase was extracted using DCM (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0–15% EtOAc in Hexane) to yield 57d as an off-white solid (0.04 g, 60.8% yield). Mp: 164–166 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.72 (d, J = 13.1 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H), 7.07 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.2 Hz, 1H), 4.09 (t, J = 6.3 Hz, 2H), 2.70 (t, J = 6.3 Hz, 2H), 1.87 (s, 2H), 1.74 (s, 3H) 1.38 (s, 6H), 0.71 (s, 9H); amide proton was not observed; ¹³C NMR (100 MHz, CDCl₃): δ 172.34, 168.90, 151.01, 149.48, 139.84, 139.02, 130.10, 128.18, 127.73, 127.52, 127.14, 126.79, 126.22, 121.72, 119.17, 57.04, 45.18, 38.61, 36.82, 32.37, 31.75, 31.58, 31.44, 31.23; ¹⁹F NMR (376 MHz, CDCl₃): δ -57.74; HRMS (ESI): [M + H]⁺ calculated for C₂₆H₃₃F₃N₂O₃ 479.2522, found 479.2508; RP-HPLC purity ≥ 99.1%, t_R = 26.5 min.

4.2. Biology

4.2.1. Cell Culture

Human lung carcinoma A549 cells were cultured in Dulbecco’s modified Eagle’s medium and H460 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with a 5% Co₂ hypoxia experimental condition. The cells were grown in the hypoxia chamber (1% O₂, 94% N₂, and 5% Co₂ in a multigas incubator)

4.2.2. Cell Growth Inhibition Assay

Cell growth inhibition was assessed using sulforhodamine B (SRB) as described in [34]. The cells were fixed with 10% formalin (Sigma-Aldrich) and stained with 0.4% SRB. Protein-bound dye was dissolved in 10 mM of Tris, and its optical density was measured at 540 nm.

4.2.3. MDH1 and MDH2 Enzyme Activity Assay

The activities of MDH1 and MDH2 were confirmed using an oxaloacetate-dependent NADH oxidation assay. Experiments were conducted using 100 mM of potassium phosphate buffer (pH 7.4) and human recombinant MDH1 (BioVision) and MDH2 (BioVision), and the results were obtained by measuring the absorbance at 340 nm. Enzyme activity was measured using a plate reader at a wavelength of 340 nm.

4.2.4. Western Blot Analysis

The cells were lysed with RIPA buffer (Millipore, Billerica, MA, USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) for 15 min at 4 °C and centrifuged at 12,000 rpm for 10 min. The lysates were quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates were subjected to an SDS-PAGE analysis and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The proteins were identified using the appropriate antibodies: HIF-1α (BD Transduction Laboratories, San Diego, CA, USA), pyruvate dehydrogenase kinase 1 (PDK1) (Cell Signaling, Danvers, MA, USA), GLUT1 and β-action (Santa Cruz Biotechnology, Danvers, MA, USA), and CD73 (Proteintech, Rosemont, IL, USA).

4.2.5. APT Contents

A549 cells (1 × 10⁴ cells/96-well plate) were incubated for 6 h with or without drugs. After removing the supernatant, an ATP assay was performed using ATPlite (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. ATP content was measured using a luminometer (Synergy HTX multimode reader, Winoski, VT, USA).

4.2.6. ADP and ATP Measurement

The ADP/ATP ratio assay kit (Sigma-Aldrich, USA) was used for ADP/ATP determination according to the manufacturer’s instructions. Samples for analysis were prepared using the ADP/ATP ratio assay kit. A549 cells were cultured directly in an assay microplate. The culture medium was removed, and then 90 µL of ATP reagent was added to each well and incubated for 1 min at room temperature. Luminescence was measured on a luminometer for ATP (RLU_A), and the plate was incubated for an additional 10 min. Next, the luminescence was read on a luminometer for ATP (RLU_B). Five microliters of ADP reagent were added to each well. After 1 min, the luminescence was read (RLU_C).

4.2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

The prepared samples were incubated with the same amount of antibody cocktail for 1 h at room temperature and then washed three times with PT buffer. After washing, the plates were developed by adding 100 µL of TMB development solution, and the reaction was stopped by adding 100 µL of stop solution. The amount of CD73 was quantitatively measured according to the instructions on the Human CD73 Simplestep ELISA kit (Abcam, Cambridge, UK).

4.3. Docking Methodology

4.3.1. Ligand Setting

The chemical structure preparation procedure was performed using ChemDraw software. Ligands were prepared by sketching the 2D structure of each ligand, and 3D structures of all ligands were produced using a molecular mechanical (MM) geometry optimization approach. The Epik function in the LigPrep module of Maestro was used in accordance with the Hammett and Taft methods to return the pKa values and 3D structure files for multiple tautomers and ionization states that are likely to exist under the specified conditions. This was accomplished by setting the physiological pH (pH 7.4) at the protonation state step and the geometry by repeating the optimization step. Accordingly, Epik can be automatically used by LigPrep to enumerate the tautomers and protonation states.

4.3.2. Receptor Preparation

The crystal structures of MDH1 (PDB:5MDH, resolution 2.4 Å) and MDH2 (PDB:4WLO, resolution 2.5 Å) were retrieved from the Protein Data Bank server [35,36]. The protein structures were subjected to the protein preparation procedure using Schrodinger software 2020, which included: (i) removing all water molecules from the crystal structure (implicit solvation was used); (ii) assignment of bond orders; (iii) adding hydrogen atoms; (iv) setting the physiological pH (pH 7) via the protonation states of the corresponding amino acid residues using PROPKA software 20 in the Schrodinger molecular modeling package; and (v) restraining the minimization of added hydrogen atoms. The active site used in the docking study was constructed from chain A of the crystal structure.

5. Conclusions

In this study, various structural modifications were employed for the hit compound LW1497 to design new scaffolds. Accordingly, a series of novel (2,4,4-trimethylpentan-2-yl)benzene derivatives were synthesized and screened for their inhibitory effect against MDH1 and MDH2 enzymes. Optimization of the SAR study resulted in the identification of a series of novel dual or selective MDH1 and MDH2 inhibitors. Compounds 5d and 35a showed significant inhibitory activity against both MDH1 and MDH2, whereas compounds 5f, 14, 44a, 50, 56, 57a, and 57b showed similar activity against both enzymes, compared with the positive control LW1497. Compounds 39b, 39c, 39d, and 57d displayed selectivity towards MDH1, whereas 5b and 5c showed selective MDH2 activity. In addition, the growth inhibition results of all tested compounds revealed that compound 50, which contains a piperidine linker, showed a two-fold improved potency in A549 (IC₅₀ = 3.94 ± 0.04 μM) and H460 (IC₅₀ = 3.67 ± 0.06 μM) cell lines compared with the hit compound LW1497. The most potent compound 50 decreased ATP production by inhibiting the MAS shuttle. As expected, compound 50 suppressed the expression of HIF-1α target genes as well as hypoxia-induced HIF-1α accumulation. Therefore, compound 50, now LW2393, may serve as a promising lead for the development of novel MDH1/2 inhibitors for the treatment of lung cancer.

Author Contributions

Conceptualization, M.W. and K.L.; methodology, S.G., D.-I.K., J.L., H.N. and J.-K.K.; validation, S.G. and J.-R.H.; formal analysis, S.G., J.-R.H., J.-K.K. and H.-A.Y.; investigation, J.-Y.I., C.H.L., H.S.B. and Y.C.; resources, Y.C., M.W. and K.L.; data curation, S.G., M.K. and J.-R.H.; visualization, S.G., H.N., J.-R.H., J.-K.K. and H.S.B.; writing—original draft preparation, all authors; writing—review and editing, all authors; supervision, K.L.; project administration K.L.; funding acquisition, K.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Acknowledgments

This work was inspired by the interdisciplinary environments of the JSPS Core-to-Core Program (“Asian Chemical Biology Initiative”).

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures, Schemes and Tables

Figure 1. Chemical structure of a known MDH inhibitor and schematic design for SAR exploration.

Scheme 1. Synthesis of trimethylpentane derivatives 5a–f a. a Reagents and conditions: (a) PPh3, methyl propiolate, toluene, rt, 12 h, 71%; (b) 10% Pd/C, H2 (balloon), EtOAc, rt, 12 h; (c) LiOH, THF, H2O, rt, 12 h, 81% over two steps; (d) amines, T3P, DIPEA, THF, rt, 12 h, 11–47%.

Scheme 2. Synthesis of trimethylpentane-modified derivative 14 a. a Reagents and conditions: (a) SOCl2, rt, 12 h; (b) anisole, AlCl3, DCM, 0 °C to rt, 1 h, 48% over two steps; (c) AlCl3, DCE, 75 °C, 16 h, 84%; (d) LiAlH4, THF, rt, 12 h, 69%; (e) N-methyl morpholine, acetonitrile, rt, 12 h, 73%; (f) 10% Pd/C, H2 (balloon), EtOAc, rt, 12 h, 82%; (g) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h, 55%.

Scheme 3. Synthesis of trimethylpentane-modified derivatives 21 and 22 a. a Reagents and conditions: (a) Boc2O, Et3N, THF, rt, 12 h, 79%; (b) N-methyl morpholine, acetonitrile, rt, 12 h, 11%; (c) 10% Pd/C, H2 (balloon), EtOAc, rt, 12 h, 69%; (d) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h, 51%; (e) TFA, DCM, rt, 2 h, 99%; (f) pivalaldehyde, NaCNBH3, AcOH, DCM, MeOH, rt, 12 h, 56%; (g) paraformaldehyde, NaCNBH3, AcOH, DCM, MeOH, rt, 12 h, 63%.

Scheme 4. Synthesis of 3-(4-(4-hydroxy-2,4-dimethylpentan-2-yl)phenoxy)propanamide derivative 30 a. a Reagents and conditions: (a) anisole, AlCl3, DCM, 65 °C, 2 h, 20%; (b) H2SO4, MeOH, rt, 12 h, 68%; (c) 1.0 M BBr3 in DCM, DCM, rt, 2 h, 66%; (d) 3.0 M CH3MgBr in ether, THF, rt, 16 h, 71%; (e) N-methyl morpholine, acetonitrile, rt, 12 h, 95%; (f) 10% Pd/C, H2 (balloon), EtOAc, rt, 12 h, 83%; (g) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h, 76%.

Scheme 5. Synthesis of biphenyloxypropanamide derivatives 35a–35b a. a Reagents and conditions: (a) DMAP, ethyl acrylate, 100 °C, 3 h, 39%; (b) LiOH, THF, H2O, rt, 12 h, 69%; (c) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h, 62%; (d) boronic acids, Pd(PPh3)4, Cs2CO3, 1,4-dioxnae, H2O, 100 °C, 12 h, 60% for 35a and 41% for 35b.

Scheme 6. Synthesis of naphthalene derivatives 39a–d a. a Reagents and conditions: (a) DMAP, 100 °C, 2 h, 16–38%; (b) LiOH, THF, H2O, rt, 12 h, 31–86%; (c) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h 17–27%.

Scheme 7. Synthesis of intermediate 43 and its amide and sulfonamide derivatives 44a–b a. a Reagents and conditions: (a) Boc2O, Et3N, DCM, rt, 1 h, 78%; (b) K2CO3, acetone, 70 °C, 12 h, 47%; (c) TFA, DCM, rt, 12 h, 77%; (d) acid chloride (44a) or sulfonyl chloride (44b) Et3N, DCM, rt, 1 h, 24% for 44a and 8% for 44b.

Scheme 8. Synthesis of piperidine, piperazine derivatives 50 and 51 a. a Reagents and conditions: (a) trifluoromethanesulfonic anhydride, Et3N, DCM, 0 °C, 1 h, 83%; (b) methyl piperidine-4-carboxylate for 46 and tert-butyl piperazine-1-carboxylate for 48, XPhos, Pd2(dba)3, Cs2CO3, toluene, 110 °C, 12 h, 59% for 46 and 93% for 48; (c) LiOH, THF, H2O, rt, 6 h, 99%; (d) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h, 59%; (e) TFA, DCM, rt, 12 h, 83%; (f) 3-(trifluoromethoxy)benzoyl chloride, Et3N, DCM, rt, 1 h, 36%.

Scheme 9. Synthesis of linker-modified derivatives 56 and 57a–d a. a Reagents and conditions: (a) diphenylmethanimine, BINAP, Pd(dba)2, Cs2CO3, toluene, 100 °C, 12 h; (b) HCl, MeOH, 80 °C, 6 h, 39% over two steps; (c) NaI, methyl 3-bromopropionate, Cs2CO3, acetonitrile, 70 °C, 12 h, 35%; (d) LiOH, THF, H2O, rt, 12 h, 98%; (e) 3-(trifluoromethoxy)aniline, T3P, DIPEA, THF, rt, 12 h, 28%; (f) 73a–c: aldehyde or ketone, NaCNBH3, AcOH, DCM, MeOH, rt, 12 h, 9–55%; 57d: acetyl chloride, Et3N, DCM, rt, 1 h, 61%.

Figure 2. Docked complex of compound 50 with MDH1. (A) a 3D model of MDH1 complexed with compound 50; (B) a 3D depiction of the binding cavity of compound 50 complexed with MDH1 (PDB ID: 5MDH); (C) a 2D interaction pattern of compound 50 with the MDH1 active site. Favorable interactions are color-coded as follows: green—hydrogen bonds, light blue—halogen interaction, orange—π–charge, dark pink—π–π stacking interactions, purple—π–sigma interactions, light pink—hydrophobic interactions. Residues shown in light green form weak van der Waals interactions.

Figure 3. Docked complex of compound 50 with MDH2. (A) A 3D model of MDH2 complexed with compound 50; (B) a 3D depiction of the binding cavity of compound 50 complexed with MDH2 (PDB ID: 2DFD); (C) a 2D interaction pattern of compound 50 with the MDH2 active site. Favorable interactions are color coded as follows: green—hydrogen bonds, light blue—halogen interaction, orange—π–charge, dark pink—π–π stacking interactions, purple—π–sigma interactions, light pink—hydrophobic interactions. Residues shown in light green form weak van der Waals interactions.

Figure 4. (A) The intracellular ATP content of compound 50 treated with A549 cells. (B) Determination of ADP/ATP ratio; * p ≤ 0.05 and ** p ≤ 0.01, compared with the control.

Figure 5. A measurement of the intracellular ATP production rates in A549 cells in a dose-dependent manner.

Figure 6. Dose-dependent inhibition of hypoxia-induced hypoxia-inducible factor-1 expression and its target genes by compound 50 in A549 cells.

Figure 7. Dose-dependent inhibition of CD73 expression under hypoxic conditions by compound 50 in A549 cells.

Table 1

In vitro MDH inhibitory activity and cell viability of amide derivatives 5a−f.

[Image omitted. Please see PDF.]
Compd.	Ar	MDH1IC50 (µM) a	MDH2IC50 (µM) a		Cell ViabilityIC50 (µM) a
					A549	H460
5a	[Image omitted. Please see PDF.]	6.18 ± 0.74		1.5 ± 0.01	11.1 ± 0.77	8.83 ± 0.34
5b	[Image omitted. Please see PDF.]	6.96 ± 0.12		2 ± 0.05	5.25 ± 0.21	5.84 ± 0.74
5c	[Image omitted. Please see PDF.]	8.35 ± 2.86		1.04 ± 0	6.47 ± 0.06	5.68 ± 0.23
5d	[Image omitted. Please see PDF.]	0.94 ± 0.05		1.24 ± 0.13	6.42 ± 0.06	6.81 ± 0.04
5e	[Image omitted. Please see PDF.]	3.14 ± 0.14		3.09 ± 0.2	>10	>10
5f	[Image omitted. Please see PDF.]	3.45 ± 0.07		2.12 ± 0.21	5.97 ± 0.71	3.77 ± 0.02
LW1497		2.24 ± 0.11		1.59 ± 0.04	7.98 ± 0.53	6.14 ± 0.29

^a Values are the means of three experiments.

Table 2

In vitro MDH inhibitory activity and cell viability of trimethylpentane-modified derivatives 14, 21, 22, 30, and 35a–b.

[Image omitted. Please see PDF.]
Compd.	R	MDH1IC50 (µM) a	MDH2IC50 (µM) a	Cell ViabilityIC50 (µM) a
				A549	H460
14	[Image omitted. Please see PDF.]	3.05 ± 0.29	1.53 ± 0.08	14.44 ± 0.1	12.77 ± 1.36
21	[Image omitted. Please see PDF.]	>5	>5	>10	>10
22	[Image omitted. Please see PDF.]	>5	3.13 ± 0.29	>10	>10
30	[Image omitted. Please see PDF.]	>5	>5	8.28 ± 0.76	ND
35a	[Image omitted. Please see PDF.]	0.78 ± 0.08	1.38 ± 0.59	>10	>10
35b	[Image omitted. Please see PDF.]	>5	>5	>10	>10
LW1497		2.24 ± 0.11	1.59 ± 0.04	7.98 ± 0.53	6.14 ± 0.29

^a Values are the means of three experiments.

Table 3

In vitro MDH inhibitory activity and cell viability of naphthalene derivatives 39a–d.

[Image omitted. Please see PDF.]
Compd.	R	R1	MDH1IC50 (µM) a	MDH2IC50 (µM) a	Cell ViabilityIC50 (µM) a
					A549	H460
39a	H	H	>5	>5	>20	>20
39b	Br	H	1.61 ± 0.25	>5	14.19 ± 0.2	14.86 ± 0.07
39c	H	Br	1.61 ± 0.21	>5	17.66 ± 0.18	14.75 ± 0.18
39d	H	F	2.25 ± 0.77	18.29 ± 1.8	>20	ND
LW1497			2.24 ± 0.11	1.59 ± 0.04	7.98 ± 0.53	6.14 ± 0.29

^a Values are the means of three experiments.

Table 4

In vitro MDH inhibitory activity and cell viability of amide and sulfonamide derivatives 44a–b.

[Image omitted. Please see PDF.]
Compd.	R	MDH1IC50 (µM) a	MDH2IC50 (µM) a	Cell ViabilityIC50 (µM) a
				A549	H460
44a	[Image omitted. Please see PDF.]	3.15 ± 0.11	2.22 ± 0.28	13.15 ± 1.3	12.41 ± 0.21
44b	[Image omitted. Please see PDF.]	2.85 ± 0.45	>5	7.32 ± 0.02	7.61 ± 0.24
LW1497		2.24 ± 0.11	1.59 ± 0.04	7.98 ± 0.53	6.14 ± 0.29

^a Values are the means of three experiments.

Table 5

In vitro MDH inhibitory activity and cell viability of linker-modified derivatives 50 and 51.

[Image omitted. Please see PDF.]
Compd.	X	MDH1IC50 (µM) a	MDH2IC50 (µM) a	Cell ViabilityIC50 (µM) a
				A549	H460
50	[Image omitted. Please see PDF.]	3.33 ± 0.18	2.24 ± 0.09	3.94 ± 0.04	3.67 ± 0.06
	Y
51	[Image omitted. Please see PDF.]	5 ± 2.49	>5	20>	18.4 ± 1.55
LW1497		2.24 ± 0.11	1.59 ± 0.04	7.98 ± 0.53	6.14 ± 0.29

^a Values are the means of three experiments.

Table 6

In vitro MDH inhibitory activity and cell viability of linker-modified derivatives 56, 57a–d.

[Image omitted. Please see PDF.]
Compd.	R	MDH1IC50 (µM) a	MDH2IC50 (µM) a	Cell ViabilityIC50 (µM) a
				A549	H460
56	H	1.79 ± 0.04	3.18 ± 0.18	6.06 ± 0.01	5.3 ± 0.01
57a	Me	2.42 ± 0.04	1.54 ± 0.07	4.41 ± 0.06	4.34 ± 0.03
57b	[Image omitted. Please see PDF.]	1.61 ± 0.02	1.09 ± 0.06	6.47 ± 0.06	6.06 ± 0.06
57c	[Image omitted. Please see PDF.]	2.16 ± 0.01	1.18 ± 0.01	7.24 ± 0.11	7.11 ± 0.03
57d	[Image omitted. Please see PDF.]	1.41 ± 0.47	>5	>20	>20
LW1497		2.24 ± 0.11	1.59 ± 0.04	7.98 ± 0.53	6.14 ± 0.29

^a Values are the means of three experiments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph16050683/s1, Figures S1–S4: ATP contents, production and CD73 expression; Figures S5–S98: 1H NMR, ¹³C NMR and ¹⁹F NMR spectra of the final compounds, and HPLC and HR-ESIMS data.

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