2024 Volume 72 Issue 4 Pages 399-407
Ryanodine receptor 2 (RyR2) is a large Ca2+-release channel in the sarcoplasmic reticulum (SR) of cardiac muscle cells. It serves to release Ca2+ from the SR into the cytosol to initiate muscle contraction. RyR2 overactivation is associated with arrhythmogenic cardiac disease, but few specific inhibitors have been reported so far. Here, we identified an RyR2-selective inhibitor 1 from the chemical compound library and synthesized it from glycolic acid. Synthesis of various derivatives to investigate the structure–activity relationship of each substructure afforded another two RyR2-selective inhibitors 6 and 7, among which 6 was the most potent. Notably, compound 6 also inhibited Ca2+ release in cells expressing the RyR2 mutants R2474S, R4497C and K4750Q, which are associated with cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT). This inhibitor is expected to be a useful tool for research on the structure and dynamics of RyR2, as well as a lead compound for the development of drug candidates to treat RyR2-related cardiac disease.
Ryanodine receptors (RyRs) are involved in contraction of striated muscles, including skeletal and cardiac muscles, by mediating the release of Ca2+ from the sarcoplasmic reticulum (SR) into the cytosol. The released Ca2+ binds to troponin complexes on actin filaments, leading to the exposure of myosin-binding sites and enabling actin-myosin cross-bridge formation to drive contraction.1–5) RyRs are classified into three types, RyR1, RyR2 and RyR3. RyR1 is mainly expressed in skeletal muscle cells, RyR2 is mainly expressed in cardiac muscle cells, and RyR3 is ubiquitously expressed in the human body.6) The function of RyRs is critical to proper muscle function, and aberrant RyRs can be lethal. For example, malignant hyperthermia (MH) is caused by RyR1 overactivation.7) Dantrolene sodium salt (Dantrolene Na) has been approved to treat MH,8) and other RyR1 inhibitors are still being developed as alternative candidates.9–11) On the other hand, mutations in RyR2 can cause fatal cardiac arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and idiopathic ventricular fibrillation (IVF).12,13) Cohort research found many mutations like R2474S, R4497C and K4750Q were associated with CPVT.14–16) Thus, inhibitors targeting pathologically mutated RyR2 are expected to be drug candidates for treating heart diseases, as well as research tools. However, RyR inhibitor development has been hampered by the difficulty of developing evaluation methods for RyR activity. Our group has recently reported cell-based assays for RyRs,17–19) and identified specific RyR inhibitors,9,10) including RyR2 inhibitors.18,20) In the present work, we screened two chemical libraries for RyR2 inhibitors and identified compound 1 as a hit (Fig. 1). We synthesized this compound from glycolic acid, and investigated the structure–activity relationship (SAR) of its substructures with the aim of increasing the inhibitory potency. Some of the developed compounds also inhibited Ca2+ release from cells expressing pathogenic mutants of RyR2: R2474S, R4497C and K4750Q, as well as wild type (WT) RyR2.
(a) Chemical structure of the known RyR2 inhibitor tetracaine and the hit compound 1. (b) Inhibitory effect of 30 µM 1 on RyR2(WT) and RyR1(R2163C). Fluorescence intensity after treatment (F) is normalized by that before treatment (F0). The dashed line of Tet shows the effect of 1 mM tetracaine on each RyR.
We previously developed an RyR2 inhibitor screening assay and used it to identify candidate inhibitors.18) Briefly, RyR2(WT) was expressed on the endoplasmic reticulum (ER) membrane and the fluorescent Ca2+ indicator R-CEPIA1er was expressed inside the ER in HEK293 cells. The fluorescence intensity of R-CEPIA1er is dependent on the concentration of Ca2+ in the ER, and therefore Ca2+ release from ER into the cytosol can be estimated indirectly from the change in the fluorescence intensity of R-CEPIA1er. Specifically, if RyR2 is inhibited, the Ca2+ concentration in ER is increased, resulting in a higher fluorescence intensity than in the vehicle-treated sample. In the screening, we used this cell-based assay to screen compound libraries from the University of Tokyo and Tokyo Medical and Dental University, and identified a selective RyR2 inhibitor 1 containing ester and amide moieties from the library of the University of Tokyo (Fig. 1a). The inhibitory effect of 30 µM 1 on RyR2 was similar to that of 1 mM tetracaine, which is known as a pan-RyR inhibitor (Fig. 1b, left). We also examined the effects of these compounds on RyR1 according to previously reported methods using RyR1(R2163C) expressing cells.17) Compound 1 had no effect on the function of RyR1 (Fig. 1b, right). Compound 1 contains cyclobutanecarboxylic ester, amide, and diphenyl ether substructures, so we set out to synthesize compound 1 from glycolic acid, which forms the central structure, and started an SAR study of each substructure.
Synthesis of Compound 1 from Glycolic AcidTo examine the roles of the substructures in the RyR2-inhibitory activity, we aimed to synthesize a series of derivatives of 1. Compound 1 itself was synthesized by means of two condensations of the central glycolic acid moiety. First, the reaction between glycolic acid and 4-phenoxyaniline 3 in the presence of condensing agents gave amide intermediate 4 and then further condensation reaction with cyclobutanecarboxylic acid 5 using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N,N-dimethyl-4-aminopyridine (DMAP) afforded the desired compound 1 (Chart 1). The synthesized 1 showed dose-dependent inhibition of RyR2 with an EC50 value to inhibit RyR2 activity of 20.7 µM (100% inhibition was defined as the activity in the presence of 1 mM tetracaine). Compound 1 had no effect on RyR1, even at the concentration of 30 µM (Fig. 2).
The dashed line shows the F/F0 score of 1 mM tetracaine.
To examine the roles of the substructures of 1, we first focused on modifying the cyclobutanecarboxylic ester structure (Table 1). We replaced the cyclobutyl moiety of 1 with other cycloalkanes. Similar ring sizes to 1 were acceptable: the cyclopropyl 6 and cyclopentyl 7 analogs retained the activity, while analog 8 having a larger cyclohexyl ring completely lost the activity. The analog 6 was almost twice as potent as 1 (EC50 = 10.3 µM). Next, since we considered that the cycloalkanecarboxylic ester substructure of 1 might be susceptible to hydrolysis, we introduced a methyl substituent at the cyclopropyl ring to obtain analog 9, which is expected to be resistant to cellular esterases.21) However, introduction of a methyl group next to the carbonyl group resulted in loss of the inhibitory activity towards RyR2, suggesting that the cycloalkyl ring is strictly recognized by RyR2. Since the larger cyclohexyl ring was not acceptable, we designed analogs 10 and 11 with phenyl and 4-pyridine rings, but surprisingly, both analogs showed no inhibitory activity towards RyR2. Interestingly, acetyl analog 12 was also inactive, indicating that the ring structure is necessary for RyR2 inhibition.
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Next, we investigated the diphenyl ether moiety (Table 2). To examine whether a simpler structure retained the activity, we synthesized the methoxy phenyl amide analog 13, in which the terminal phenyl ring was replaced with a methyl group. We also designed a fluorophenyl analog 14 to examine the electron-withdrawing effect on the phenyl ring near the amide bond. However, both analogs were inactive. Thus, we next introduced different aromatic rings instead of the diphenyl ether, synthesizing the anthracene and naphthalene analogs 15 and 16. These analogs also showed no activity. Therefore, we next designed a benzylphenyl analog 17 which had the same structure as 1 except for the replacement of the oxygen of diphenyl ether with carbon. However, 17 also lacked inhibitory activity towards RyR2, indicating that the diphenyl ether is essential for the activity and cannot be modified.
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Finally, we focused on the central glycolic amide moiety (Table 3) and synthesized an amide-amide analog 21 from tert-butoxycarbonyl (Boc)-glycine (Chart 2). We also synthesized the analog 22 methylated at the nitrogen of the diphenyl ether amide, because previous research has shown that an RyR2 inhibitor derivative with secondary amide substructure completely lacked the activity, while a tertiary amide showed significant RyR2 inhibition.20) However, in the case of compound 1, the introduction of methyl group or amide-amide structure had a negative effect. Since the difference between 1 and 21 is the amide of the cyclobutyl moiety, we next introduced a methyl group at the nitrogen of the cyclobutylamide moiety of 21, obtaining analog 25. For this purpose, we used N-methylated 23 as the starting material to introduce the methyl group selectively (Chart 3). However, 25 also lacked RyR2-inhibitory activity.
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Therefore, we fixed the ester and the secondary amide substructures, and introduced various modifications in the glycol amide part by using commercially available modified glycolic acids as starting materials to obtain methyl analog 26, isopropyl analog 27 and phenyl analog 28. However, these three analogs all lacked inhibitory activity towards RyR2. These results indicate that the central glycolic amide substructure is critical for RyR2-inhibitory activity.
Inhibitory Activities towards RyR2 with Pathogenic MutationsOur comprehensive SAR analysis indicated that most of the substructures of 1 are strictly recognized by RyR2, but we identified three active inhibitors of RyR2, 1, 6 and 7. Next, we screened the effects of these compounds on RyR2 bearing three major CPVT-associated mutations (Fig. 3), i.e., R4495C (mouse numbering, corresponding to R4497C in humans), R2474S and K4750Q. Cells expressing these mutant RyR2s were prepared according to our reported method for the preparation of RyR2(WT)-expressing cells.14,18) Compound 6 was the most potent, while 1 and 7 exhibited weaker but equal potency towards RyR2(WT). The same pattern of inhibitory potency was seen towards RyR2(R4495C). All the compounds showed weaker activity towards the R2474S and K4750Q mutants, though 6 still showed the highest potency. Therefore, we concluded that, among our synthesized RyR2 inhibitors, compound 6 showed the most potent inhibition and the broadest range of inhibitory activity towards RyR2 bearing mutations associated with CPVT.
The dashed line shows the F/F0 score of 1 mM tetracaine towards RyR2.
Through screening of two chemical libraries for RyR2-selective inhibitors with our previously developed bioassay, we discovered a novel RyR2 inhibitor 1. Detailed SAR studies revealed that most of the substructures of the hit compound 1 are strictly recognized by RyR2. However, among the synthesized derivatives, we identified another two RyR2-selective inhibitors 6 and 7. Among them, compound 6, having a cyclopropyl ring in place of the cyclobutyl ring of 1, showed the greatest potency. Notably, compound 6 also inhibited RyR2 bearing mutations associated with CPVT. This inhibitor is expected to be useful for research on the structure and dynamics of RyR2, and as a lead compound for the development of drug candidates to treat CPVT.
All reagents were purchased from TCI Chemicals (Tokyo, Japan), FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), or Kanto Kagaku Co., Inc. (Tokyo, Japan), and used without further purification: TLC was performed using silica gel coated with a fluorescent indicator F254 (Merck Millipore, Burlington, MA, U.S.A.; #1.05715.0001). Silica gel column chromatography was performed employing neutral silica gel (60 Å, 40–50 µm) purchased from Kanto Kagaku Co., Inc. NMR spectra were recorded on a Bruker AVANCE 400 (1H: 400 MHz and 13C: 101 MHz) and Bruker AVANCE 500 (1H: 500 MHz and 13C: 125 MHz) spectrometers. Chemical shift values for protons are referenced to the signal of the residual signal chloroform in chloroform-d (δ 7.26), dimethyl sulfoxide-d5 in dimethyl sulfoxide-d6 (δ 2.54) and acetone-d5 in acetone-d6 (δ 2.05), and chemical shift values for carbons are referenced to the signal of the carbon resonance of chloroform-d (δ 77.16) or acetone-d6 (δ 29.84 for CD3). High-resolution mass spectra (HR-MS) were collected on a Bruker Daltonics micrOTOF-2 focus with the electron spray ionization time-of-flight (ESI-TOF) method.
Synthesis2-Hydroxy-N-(4-phenoxyphenyl)acetamide (4)A solution of 4-phenoxyaniline (3, 185.3 mg, 1.0 mmol) and glycolic acid (2, 76.1 mg, 1.0 mmol) in N,N-dimethylformamide (5.0 mL) were added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (230.1 mg, 1.2 mmol) and hydroxy benzotriazole (162.2 mg, 1.2 mmol), and the mixture was stirred overnight at room temperature. The reaction mixture was quenched with water and extracted with ethyl acetate 20 mL and washed with sat.NaHCO3aq. sat.NH4Claq. and brine. The organic layer was dried over by sodium sulfate, concentrated in vacuo gave 195.7 mg of crude as a light brown solid and followed by recrystallization from n-Hexane/ethyl acetate to obtain the target compound 4 (150.2 mg, 62%). 1H-NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H, NH), 7.77 (dd, J = 6.8, 2.0 Hz, 2H), 7.36 (td, J = 6.8, 1.2 Hz, 2H), 7.09 (tt, J = 6.8, 1.2 Hz, 1H), 7.01–6.96 (m, 4H), 4.83 (s, 1H, OH), 4.10 (d, J = 4.4 Hz, 2H); 13C-NMR (100 MHz, Acetone-d6) δ: 170.0, 157.9, 152.8, 134.5, 129.8, 122.9, 121.0, 119.3, 118.1, 62.0; HR-MS (ESI-TOF, m/z) Calcd for C14H14NO3+ [M + H]+, 244.0968. Found 244.0975.
General Procedure A for Syntheses of 1 and 6–12A mixture of the corresponding acid (1.0 equivalent (equiv.)), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv.), 4-dimethylaminopyridine (0.10 equiv.) and compound 4 (1 equiv.) in methylene chloride was stirred at room temperature overnight. Then the reaction mixture was quenched with water and washed with sat.NaHCO3aq., sat.NH4Claq. and brine, then dried over by sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1) to give the corresponding compound.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Cyclobutanecarboxylate (1)Following the general procedure A, compound 1 was obtained as a yellow solid in 37% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.20 (s, 1H, NH), 7.65 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.68 (s, 2H), 3.35–3.26 (m, 1H), 2.37–2.19 (m, 4H), 2.02–1.85 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.0, 165.2, 157.8, 152.9, 134.3, 129.8, 122.9, 121.1, 119.3, 118.1, 62.4, 37.5, 24.8, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C19H20NO4+ [M + H]+, 326.1387. Found 326.1402.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Cyclopropanecarboxylate (6)Following the general procedure A, compound 6 was obtained as a white solid in 24% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.28 (s, 1H, NH), 7.67 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.68 (s, 2H), 1.80–1.74 (m, 1H), 0.95–0.93 (m, 4H); 13C-NMR (125 MHz, Acetone-d6) δ: 173.8, 165.6, 157.8, 152.9, 134.3, 129.8, 123.0, 121.3, 119.3, 118.1, 62.6, 12.2, 8.0; HR-MS (ESI-TOF, m/z) Calcd for C18H17NO4Na+ [M + Na]+, 334.1050. Found 334.1038.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Cyclopentanecarboxylate (7)Following the general procedure A, compound 7 was obtained as a white solid in 32% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.20 (s, 1H, NH), 7.65 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.68 (s, 2H), 2.95–2.87 (m, 1H), 1.97–1.82 (m, 4H), 1.73–1.55 (m, 4H); 13C-NMR (125 MHz, Acetone-d6) δ: 175.3, 165.3, 157.8, 152.9, 134.3, 129.8, 122.9, 121.1, 119.4, 118.1, 62.4, 43.1, 29.6, 25.5; HR-MS (ESI-TOF, m/z) Calcd for C20H22NO4+ [M + H]+, 340.1543. Found 340.1552.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Cyclohexanecarboxylate (8)Following the general procedure A, compound 8 was obtained as a white solid in 53% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.18 (s, 1H, NH), 7.64 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.67 (s, 2H), 2.50–2.42 (m, 1H), 1.97–1.93 (m, 2H), 1.79–1.20 (m, 8H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.4, 165.3, 157.8, 152.9, 134.3, 129.8, 122.9, 121.2, 119.3, 118.1, 62.3, 42.4, 25.6, 25.1; HR-MS (ESI-TOF, m/z) Calcd for C21H24NO4+ [M + H]+, 354.1700. Found 354.1718.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl 1-methylcyclopropane-1-carboxylate (9)Following the general procedure A, compound 9 was obtained as a white solid in 62% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.18 (s, 1H, NH), 7.65 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.67 (s, 2H), 1.33 (s, 3H), 1.27 (dd, J = 6.8, 3.8 Hz, 2H), 0.77 (dd, J = 6.8, 3.8 Hz, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.5, 165.2, 157.8, 152.9, 134.3, 129.8, 122.9, 121.1, 119.4, 118.1, 62.6, 18.5, 18.1, 16.1; HR-MS (ESI-TOF, m/z) Calcd for C19H20NO4+ [M + H]+, 326.1387. Found 326.1403.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Benzoate (10)Following the general procedure A, compound 10 was obtained as a white solid in 69% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.41 (s, 1H, NH), 8.11 (dd, J = 7.6, 1.6 Hz, 2H), 7.70–7.66 (m, 3H), 7.55 (td, J = 7.6, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.0 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.96 (s, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 165.4, 165.1, 157.8, 153.0, 134.3, 133.3, 129.8, 129.7, 129.6, 128.6, 123.0, 121.3, 121.22, 119.3, 118.1, 63.2; HR-MS (ESI-TOF, m/z) Calcd for C21H18NO4+ [M + H]+, 348.1230. Found 348.1245.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Isonicotinate (11)Following the general procedure A, compound 11 was obtained as a white solid in 72% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.39 (s, 1H, NH), 8.74 (dd, J = 4.4, 1.6 Hz, 2H), 8.36 (br s, 1H), 7.83 (dd, J = 4.4, 1.6 Hz, 2H), 7.66 (dd, J = 9.2, 1.2 Hz, 2H), 7.35 (td, J = 7.6, 1.2 Hz, 2H), 7.09 (tt, J = 7.6, 1.2 Hz, 1H), 6.99–6.95 (m, 4H), 4.23 (d, J = 5.6 Hz, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 167.0, 165.4, 157.8, 152.8, 150.4, 141.5, 134.8, 129.8, 122.9, 121.1, 120.9, 119.3, 118.1, 43.5; HR-MS (ESI-TOF, m/z) Calcd for C20H17N3O3Na+ [M + Na]+, 370.1162. Found 370.1172.
2-Oxo-2-((4-phenoxyphenyl)amino)ethyl Acetate (12)Following the general procedure A, compound 12 was obtained as a white solid in 66% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.23 (s, 1H, NH), 7.66 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 7.10 (tt, J = 7.6, 1.2 Hz, 1H), 7.00–6.96 (m, 4H), 4.66 (s, 2H), 2.12 (s, 3H); 13C-NMR (125 MHz, Acetone-d6) δ: 169.7, 165.3, 157.8, 153.0, 134.3, 129.8, 123.0, 121.4, 119.3, 118.1, 62.5, 19.7; HR-MS (ESI-TOF, m/z) Calcd for C16H16NO4+ [M + H]+, 286.1074. Found 286.1085.
General Procedure B for Syntheses of 13–17A solution of corresponding aniline (1.2 equiv.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv.), hydroxy benzotriazole (1.2 equiv.) and glycolic acid (1 equiv.) in N,N-dimethylformamide was stirred at 80 °C overnight. Then the reaction mixture was quenched with water, extracted with ethyl acetate, washed with sat.NaHCO3aq., sat.NH4Claq. and brine, and then dried over by sodium sulfate. After evaporation, the residue was roughly purified by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1) to give the impure intermediates.
A mixture of the intermediate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv.), 4-dimethylaminopyridine (0.10 equiv.) and cyclobutene carboxylic acid (1.2 equiv.) in methylene chloride was stirred at room temperature overnight. Then the reaction mixture quenched with water and washed with sat.NaHCO3aq., sat.NH4Claq. and brine, and then dried over by sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1) to give the corresponding compounds.
2-((4-Methoxyphenyl)amino)-2-oxoethyl Cyclobutanecarboxylate (13)Following the general procedure B, compound 13 was obtained as a pink solid in 65% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.01 (s, 1H, NH), 7.53 (dd, J = 6.8, 2.0 Hz, 2H), 6.87 (dd, J = 6.8, 2.0 Hz, 2H), 4.64 (s, 2H), 3.76 (s, 3H), 3.34–3.25 (m, 1H), 2.36–2.16 (m, 4H), 1.99–1.87 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.1, 165.1, 156.2, 131.6, 121.2, 113.7, 62.4, 54.7, 37.5, 24.8, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C14H17NO4Na+ [M + Na]+, 286.1050. Found 286.1042.
2-((4-Fluorophenyl)amino)-2-oxoethyl Cyclobutanecarboxylate (14)Following the general procedure B, compound 14 was obtained as a white solid in 42% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.23 (s, 1H, NH), 7.66 (ddd, J = 6.8, 2.0 Hz, 2H), 7.09 (td, J = 8.8, 2.4 Hz, 2H), 4.62 (s, 2H), 3.34–3.26 (m, 1H), 2.37–2.19 (m, 4H), 2.02–1.86 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.0, 165.4, 158.9, 134.8, 121.3, 115.1, 62.3, 37.5, 24.8, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C13H14FNO3Na+ [M + Na]+, 274.0850. Found 274.0844.
2-(Anthracen-2-ylamino)-2-oxoethyl Cyclobutanecarboxylate (15)Following the general procedure B, compound 15 was obtained as a brown solid in 62% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.43 (s, 1H, NH), 8.56 (s, 1H), 8.48 (s, 1H), 8.45 (s, 1H), 8.04 (d, J = 8.4 Hz, 3H), 7.60 (dd, J = 8.8, 2.4 Hz, 1H), 7.51–7.43 (m, 2H), 4.77 (s, 2H), 3.37–3.33 (m, 1H), 2.41–2.16 (m, 4H), 2.02–1.88 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.0, 165.8, 132.2, 131.1, 129.1, 128.1, 127.8, 126.0, 125.6, 125.3, 125.0, 120.8, 114.8, 62.5, 37.5, 24.9, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C21H19NO3Na+ [M + Na]+, 356.1257. Found 356.1242.
2-(Naphthalen-1-ylamino)-2-oxoethyl Cyclobutanecarboxylate (16)Following the general procedure B, compound 16 was obtained as a yellow solid in 40% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.26 (s, 1H, NH), 8.09–8.05 (m, 1H), 7.95–7.91 (m, 1H), 7.85 (t, J = 7.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.56–7.48 (m, 3H), 4.87 (s, 2H), 3.41–3.33 (m, 1H), 2.42–2.19 (m, 4H), 2.01–1.88 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.1, 166.1, 134.2, 132.9, 132.8, 128.3, 127.9, 126.0, 125.6, 125.5, 122.0, 121.4, 62.7, 37.6, 24.9, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C17H18NO3+ [M + H]+, 284.1281. Found 284.1295.
2-((4-Benzylphenyl)amino)-2-oxoethyl Cyclobutanecarboxylate (17)Following the general procedure B, compound 17 was obtained as a white solid in 75% yield. 1H-NMR (400 MHz, Acetone-d6) δ: 9.13 (s, 1H, NH), 7.55 (d, J = 8.8 Hz, 2H), 7.27 (td, J = 7.6, 1.6 Hz, 2H), 7.29–7.20 (m, 2H), 7.19–7.15 (m, 3H), 4.66 (s, 2H), 3.93 (s, 2H), 3.33–3.25 (m, 1H), 2.35–2.18 (m, 4H), 2.01–1.84 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.0, 165.2, 141.6, 136.9, 136.6, 129.0, 128.7, 128.3, 125.9, 119.6, 62.4, 40.9, 37.5, 24.8, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C20H22NO3+ [M + H]+, 324.1594. Found 324.1600.
Syntheses of 19–22tert-Butyl(2-oxo-2-((4-phenoxyphenyl)amino)ethyl) Carbamate (19)A solution of N-(tert-Butoxy carbonyl) glycine (100 mg, 0.57 mmol) and 4-phenoxyaniline (126.9 mg, 0.68 mmol) in N,N-dimethylformamide (5.0 mL) were added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (163.9 mg, 0.86 mmol) and hydroxy benzotriazole (115.7 mg, 0.86 mmol), and the mixture was stirred overnight at room temperature. The reaction mixture was quenched with water and extracted with ethyl acetate 30 mL and washed with sat.NaHCO3aq., sat.NH4Claq. and brine. The organic layer was dried over by sodium sulfate. Concentration of the solution in vacuo gave compound 19 as a light brown oil (136 mg, 70%). 1H-NMR (400 MHz, CDCl3) δ: 8.44 (br s, 1H, NH), 7.47 (dd, J = 6.8, 2.0 Hz, 2H), 7.31 (td, J = 7.6, 2.0 Hz, 2H), 7.08 (tt, J = 7.6, 1.2 Hz, 1H), 6.98–6.95 (m, 4H), 5.48 (br s, 1H, NH), 3.94 (d, J = 5.6 Hz, 2H), 1.47 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ: 167.8, 157.4, 156.5, 153.6, 132.9, 129.7, 123.1, 121.7, 119.5, 118.4, 80.7, 45.4, 28.3; HR-MS (ESI-TOF, m/z) Calcd for C19H23N2O4+ [M + H]+, 343.1652. Found 343.1639.
N-(2-Oxo-2-((4-phenoxyphenyl)amino)ethyl) Cyclobutanecarboxamide (21)Compound 19 (135.0 mg, 0.39 mmol) was resolved in 4.0 mL methylene chloride and trifluoroacetic acid (0.50 mL) was added to the solution. Then the mixture was stirred at room temperature overnight and quenched with sat.NaHCO3aq. The mixture was extracted with methylene chloride and the extract was washed with brine, dried over by sodium sulfate and concentrated to provide crude compound 20 as a white solid (56.4 mg), which was used in the next step without further purifications. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (67.1 mg, 0.35 mmol) and hydroxy benzotriazole (47.3 mg, 0.35 mmol) were added to a solution of compound 20 (56.4 mg, 0.23 mmol) and cyclobutanecarboxylic acid (23.4 mg, 0.23 mmol) in N,N-dimethylformamide (5.0 mL), and the mixture was stirred overnight at room temperature. The reaction mixture was quenched with water, extracted with ethyl acetate 20 mL and washed with sat.NaHCO3aq., sat.NH4Claq. and brine. The organic layer was dried over by sodium sulfate, concentrated in vacuo to gave 58.7 mg of crude as a white solid. After recrystallization (n-Hexane/ethyl acetate), the target compound 21 (50.1 mg, 67%) was obtained as a white solid. 1H-NMR (400 MHz, Acetone-d6) δ: 9.25 (s, 1H, NH), 7.64 (dd, J = 8.8, 1.0 Hz, 2H), 7.38 (td, J = 7.6, 1.2 Hz, 2H), 7.24 (br s, 1H, NH), 7.09 (tt, J = 7.6, 1.2 Hz, 1H), 6.99–6.95 (m, 4H), 3.99 (d, J = 5.6 Hz, 2H), 3.24–3.16 (m, 1H), 2.32–2.10 (m, 4H), 2.02–1.78 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.8, 167.6, 157.9, 152.6, 134.8, 129.8, 122.9, 120.8, 119.4, 118.0, 43.1, 39.3, 24.9, 17.9; HR-MS (ESI-TOF, m/z) Calcd for C19H21N2O3+ [M + H]+, 325.1547. Found 325.1539.
N-(2-(Methyl(4-phenoxyphenyl)amino)-2-oxoethyl) Cyclobutanecarboxamide (22)Compound 21 (30.0 mg, 0.09 mmol) was resolved in 3.0 mL N,N-dimethylformamide, then cesium carbonate (44.0 mg, 0.14 mmol) and iodomethane (0.10 mL) step-by-step was added to the solution. The reaction mixture was stirred at room temperature overnight. and the reaction mixture was quenched with water, extracted with ether, and dried over by sodium sulfate. The organic layer was concentrated to yield crude 32.5 mg (a light yellow solid). After purification by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1), the target compound 22 was obtained as a white solid (16.9 mg, 53%). 1H-NMR (400 MHz, CDCl3) δ: 7.39 (td, J = 7.6, 2.0 Hz, 2H), 7.18 (tt, J = 7.6, 0.8 Hz, 1H), 7.12 (dd, J = 6.8, 2.4 Hz, 2H), 7.07 (dd, J = 8.8, 0.8 Hz, 2H), 7.00 (dd, J = 8.8, 2.0 Hz, 2H), 6.36 (br s, 1H, NH), 3.76 (d, J = 4.4 Hz, 2H), 3.28 (s, 3H), 3.09–3.00 (m, 1H), 2.30–2.10 (m, 4H), 2.00–1.81 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ: 174.8, 168.7, 157.9, 156.0, 136.2, 130.0, 128.5, 124.3, 119.8, 119.4, 42.0, 39.7, 37.6, 25.3, 18.2; HR-MS (ESI-TOF, m/z) Calcd for C20H23N2O3+ [M + H]+, 339.1703. Found 339.1719.
Syntheses of 24 and 25Ethyl N-(Cyclobutanecarbonyl)-N-methylglycinate (24)A solution of cyclobutane carboxylic acid (240.3 mg, 1.2 mmol), sarcosine ethyl ester hydrochloride (23, 307.2 mg, 1.0 mmol), hydroxy benzotriazole (324.3 mg, 2.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (460.1 mg, 1.2 mmol) and N,N-diisopropylethylamine (1.0 mL, 3.0 mmol) in N,N-dimethylformamide (4.0 mL) was stirred at 60 °C overnight. The mixture was quenched with water, extracted with ethyl acetate 20 mL, and washed with sat.NaHCO3aq., sat.NH4Claq. and brine. The organic layer was dried over by sodium sulfate and concentrated in vacuo. After purification by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1), the ester intermediate compound 24 was obtained as a yellow oil (40.6 mg, 20%). This compound showed 2 isomer peaks on 1H-NMR and 13C-NMR due to the amide rotamer. 1H-NMR (400 MHz, CDCl3) δ: 4.15–4.09 (m, 2H), 4.02 (s, 0.8 × 2H), 3.88 (s, 0.2 × 2H), 3.33–3.24 (m, 0.8 × 1H), 3.11–3.05 (m, 0.2 × 1H), 2.91 (s, 0.8 × 3H), 2.89 (s, 0.2 × 3H), 2.34–2.24 (m, 2H), 2.16–1.74 (m, 4H), 1.23–1.18 (m, 3H); 13C-NMR (100 MHz, CDCl3) δ: 175.2, 174.8, 169.4, 169.2, 61.4, 61.0, 51.0, 49.4, 37.1, 37.1, 35.7, 34.7, 25.0, 24.7, 17.8, 14.1; HR-MS (ESI-TOF, m/z) Calcd for C10H17NO4Na+ [M + Na]+, 222.1101. Found 222.1107.
N-Methyl-N-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl) Cyclobutanecarboxamide (25)1 N NaOHaq. (2.0 mL) was adde to a solution of the ester compound 24 (40.6 mg, 0.20 mmol) in methanol/water (1 : 1, 2.0 mL) and stirred at room temperature for 12 h. The reaction mixture was acidified with 1 N HCl 20 mL and then extracted with methylene chloride. The organic layer was dried over by sodium sulfate and concentrated to yield the impure carboxylic acid as a yellow oil (34.6 mg), which was used in the next step without further purification. A solution of 4-phenoxyaniline (55.6 mg, 0.3 mmol) and the mixture carboxylic acid (34.6 mg, 0.20 mmol) in N,N-dimethylformamide (3.0 mL) were added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (46.0 mg, 0.24 mmol) and hydroxy benzotriazole (32.4 mg, 0.24 mmol), and the mixture was stirred overnight at room temperature. The reaction mixture was quenched with water, extracted with ethyl acetate and washed with sat.NaHCO3aq., sat.NH4Claq. and brine. The organic layer was dried over by sodium sulfate and concentrated in vacuo to yield 65.4 mg crude as a brown oil. The crude product was purified by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1) and recrystallization from n-Hexane/ethyl acetate, and then title compound 25 was obtained as a white solid (38.6 mg, 57% in two steps). This compound showed 2 isomer peaks on 1H-NMR and 13C-NMR due to the amide rotamer. 1H-NMR (400 MHz, Acetone-d6) δ: 9.30 (s, 0.3 × 1H, NH), 9.18 (s, 0.7 × 1H, NH), 7.67 (dd, J = 8.8, 1.6 Hz, 0.3 × 2H), 7.64 (dd, J = 8.8, 1.6 Hz, 0.7 × 2H), 7.35 (td, J = 7.6, 1.2 Hz, 2H), 7.12–7.07 (m, 1H), 6.99–6.95 (m, 4H), 4.14 (s, 0.7 × 2H), 4.13 (s, 0.3 × 2H), 3.50–3.42 (m, 0.7 × 1H), 3.38–3.30 (m, 0.3 × 1H), 3.05 (s, 0.7 × 3H), 2.92 (s, 0.3 × 3H), 2.35–2.08 (m, 4H), 2.00–1.74 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.5 (major), 174.2 (minor), 167.1 (major), 166.8 (minor), 157.9 (major), 157.8 (minor), 152.8, 134.9 (major), 134.7 (minor), 129.8, 122.9 (minor), 122.8 (major), 121.0 (minor), 120.8 (major), 119.4, 118.1 (minor), 118.0 (major), 52.3 (minor), 51.3 (major), 36.9, 35.4 (major), 33.9 (minor), 24.8 (minor), 24.5 (major), 17.4; HR-MS (ESI-TOF, m/z) Calcd for C20H23N2O3+ [M + H]+, 339.1703. Found 339.1713.
General Procedure C for Syntheses of 26–28A solution of corresponding acid (1.0 equiv.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv.), N,N-diisopropylethylamine (2.0 equiv.), hydroxy benzotriazole (1.2 equiv.) and 4-phenoxyaniline (1 equiv.) in N,N-dimethylformamide was stirred at 70 °C overnight. Then the reaction mixture was quenched with water, extracted with ethyl acetate, washed with sat.NaHCO3aq., sat.NH4Claq. and brine, and dried over by sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1) to give the intermediate products. All intermediates were used in the next step without further purification.
A mixture of the corresponding intermediate (1.0 equiv.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv.), 4-dimethylaminopyridine (0.10 equiv.) and cyclobutene carboxylic acid (1.2 equiv.) in methylene chloride was stirred at room temperature overnight. Then the reaction mixture was quenched with water, extracted with ethyl acetate, washed with sat.NaHCO3aq., sat.NH4Claq. and brine, and then dried over by sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography (n-Hexane/ethyl acetate = 1 : 1) to give the corresponding compound 26–28.
(R)-1-Oxo-1-((4-phenoxyphenyl)amino)propan-2-yl Cyclobutanecarboxylate (26)Following the general procedure C, compound 26 was obtained as a white solid in 3% yield in two steps. [α]D25 = −45.1 (c = 0.10, CHCl3). 1H-NMR (400 MHz, Acetone-d6) δ: 9.18 (s, 1H, NH), 7.68 (dd, J = 7.2, 2.0 Hz, 2H), 7.36 (td, J = 7.6, 1.6 Hz, 2H), 7.10 (tt, J = 7.6, 1.0 Hz, 1H), 7.00–6.96 (m, 4H), 5.15 (q, J = 6.8 Hz, 1H), 3.32–3.23 (m, 1H), 2.36–2.17 (m, 4H), 2.01–1.85 (m, 2H), 1.48 (d, J = 6.8 Hz, 3H); 13C-NMR (125 MHz, Acetone-d6) δ: 173.9, 169.7, 157.9, 152.9, 134.5, 129.8, 122.9, 121.5, 119.3, 118.1, 70.2, 37.6, 24.8, 18.0, 16.9; HR-MS (ESI-TOF, m/z) Calcd for C20H22NO4+ [M + H]+, 340.1543. Found 340.1550.
(R)-1-Oxo-1-((4-phenoxyphenyl)amino)-3-phenylpropan-2-yl Cyclobutanecarboxylate (27)Following the general procedure C, compound 27 was obtained as a white solid in 9% yield in two steps. [α]D25 = −46.7 (c = 0.10, CHCl3). 1H-NMR (400 MHz, Acetone-d6) δ: 9.18 (s, 1H, NH), 7.62 (dd, J = 8.8, 2.0 Hz, 2H), 7.36 (td, J = 7.6, 1.6 Hz, 2H), 7.29 (d, J = 4.4 Hz, 4H), 7.25–7.20 (m, 1H), 7.10 (tt, J = 7.2, 1.0 Hz, 1H), 6.99–6.95 (m, 4H), 5.32 (q, J = 4.8 Hz, 1H), 3.29–3.12 (m, 3H), 2.22–2.09 (m, 4H), 1.98–1.86 (m, 2H); 13C-NMR (125 MHz, Acetone-d6) δ: 173.9, 167.4, 157.8, 153.1, 136.7, 134.2, 129.8, 129.5, 128.2, 126.6, 123.0, 121.5, 119.3, 118.1, 74.3, 37.7, 7.6, 25.0, 24.5, 18.0; HR-MS (ESI-TOF, m/z) Calcd for C26H26NO4+ [M + H]+, 416.1856. Found 416.1843.
(R)-3-Methyl-1-oxo-1-((4-phenoxyphenyl)amino)butan-2-yl Cyclobutanecarboxylate (28)Following the general procedure C, compound 28 was obtained as a white solid in 20% yield in two steps. [α]D25 = −55.2 (c = 0.10, CHCl3). 1H-NMR (400 MHz, Acetone-d6) δ: 9.14 (s, 1H, NH), 7.66 (dd, J = 8.8, 1.6 Hz, 2H), 7.36 (td, J = 7.6, 1.6 Hz, 2H), 7.09 (tt, J = 7.6, 1.6 Hz, 1H), 6.99–6.95 (m, 4H), 4.66 (d, J = 5.2 Hz, 1H), 3.37–3.28 (m, 1H), 2.37–2.16 (m, 5H), 2.02–1.86 (m, 2H), 1.01 (dd, J = 6.8, 2.4 Hz, 6H); 13C-NMR (125 MHz, Acetone-d6) δ: 174.8, 167.9, 157.8, 152.4, 134.8, 130.4, 123.4, 121.6, 119.9, 78.1, 37.5, 30.5, 25.2, 25.0, 19.1, 18.4, 17.8; HR-MS (ESI-TOF, m/z) Calcd for C22H26NO4+ [M + H]+, 368.1856. Found 368.1875.
BiologyAll biology experiments with cells were just followed the reported methods.17,18) As mentioned in these reports, the activity of RyR1(WT) to release Ca2+ is not strong, which is not enough to measure the activity of RyR inhibitors. Thus, we used the mutated RyR1, RyR1(R2163C), for all experiments to investigate the selectivity of RyR2 inhibitors.
This work was partly supported by JSPS KAKENHI (22K15244 to R.I., 22K06652 to N.K. and 22H02805 to T.M.), TMDU priority research areas grant (to R.I.), the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) (JP21am0101086 for screening the chemical library of the University of Tokyo, JP20am0101080 to T.M. and N.K., and JP23ama121043 to R.I. and H.K.), the Practical Research Project for Rare/Intractable Diseases (19ek0109202 to N.K.) from AMED, an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (2–5 to T.M.) and the Vehicle Racing Commemorative Foundation (6303 to T.M.). A part of this research was based on the Cooperative Research Project of the Research Center for Biomedical Engineering.
The authors declare no conflict of interest.
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