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Full Paper Synthesis of Carbamide Derivatives Bearing Tetrahydroisoquinoline Moieties and Biological Evaluation as Analgesia Drugs in Mice Qianqian Qiu1, Jingjie Wang1,2y, Xin Deng1, Hai Qian1, Haiyan Lin3y, and Wenlong Huang1 1
2 3
State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, Nanjing, China WuXiAppTec (Wuhan) Co., Ltd., Wuhan, China Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, China
Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated non-selective cation channel that is considered to be an important pain integrator. Tetrahydroisoquinoline, the prototypical antagonist of TRPV1, has a clear therapeutic potential. Here, a series of carbamide derivatives of tetrahydroisoquinoline were designed and synthesized. Preliminary biological tests suggested that the compounds I 1, I 2, and I 9 had favorable TRPV1 antagonism activity. In further studies, I 1 exhibited better antinociceptive activity than the positive control BCTC in diverse pain models. All of these results suggested that I 1 can be considered as the lead candidate for the further development of antinociceptive drugs. Keywords: Analgesia drugs / Tetrahydroisoquinoline derivatives / Transient receptor potential vanilloid 1 antagonist Received: December 18, 2014; Revised: February 9, 2015; Accepted: February 17, 2015 DOI 10.1002/ardp.201400455
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Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Introduction Transient receptor potential vanilloid 1 (TRPV1) is the best characterized member of the TRP superfamily of ion channels, which are activated by a range of stimuli, including capsaicin as well as protons (pH 43°C) stimuli among others [1, 2]. In the last decade, significant efforts have been devoted to the design and synthesis of TRPV1 antagonists as antinociceptive agents [3]. A review of the literature on the development of the first TRPV1 antagonist, capsazepine 1, revealed that the tetrahydroisoquinoline moiety is a core pharmacophore of TRPV1 antagonists [4–6]. While less potent than capsazepine 1, compound 2 was a weak and partial antagonist of the TRPV1 receptor. In a previous research, we got two derivatives of tetrahydroisoquinoline (3 and 4) as shown in Fig. 1. However, TRPV1 antagonistic potency made a big difference between
the two compounds, that compound 4 was much superior to compound 3. Based on the preliminary result, we hypothesized that the nitro group at 5-position of tetrahydroisoquinoline moiety should possess the necessary for activity at TRPV1. In this letter, according to the structure of 4, intended for searching derivatives of tetrahydroisoquinoline with better TRPV1 antagonistic potency and antinociceptive activity, the thiourea group in 4 was replaced with urea group, to obtain a series of TRPV1 antagonists with slight structure difference but similar impressive potency. As a result, 11 carbamide derivatives of tetrahydroisoquinoline were synthesized and their activities were evaluated.
Results and discussion Chemistry The synthetic route of target compounds I 1–11 is depicted in Scheme 1. Compound 6 was synthesized by nitration of
Correspondence: Prof. Wenlong Huang, Centre of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China. E-mail: [email protected] Fax: þ86 25 83271480
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y
Contributed equally to the first author. Additional correspondence: Dr. Haiyan Lin, E-mail: [email protected]
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Figure 1. The TRPV1 antagonists with tetrahydroisoquinoline moiety.
Scheme 1. Synthesis of target compounds I 1–11. Reagents and conditions: (a) HNO3, H2SO4; (b) NaBH4, acetic acid; (c) substituted anilines, triphosgene/Et3N, one pot.
isoquinoline with nitric acid in concentrated sulfuric acid at 0°C for 2 h, followed by adjusting the solution pH to 9–10. The formation of compound 7 was conducted by sodium borohydride and compound 6 in acetic acid (10 mL) and THF (20 mL) at 0–25°C. After completion of reaction, the pH of the solution was adjusted to 8 with NaOH. Followed by extraction with ethyl acetate, compound 7 was obtained by concentration in vacuo. The desirable target compounds I 1–11 were produced by one-pot synthesis with substituted phenylamines, compound 7, triphosgene, and Et3N in CH2Cl2 at 10°C. The structures of all target compounds are presented in Table 1.
Table 1. The structures and TRPV1 antagonistic activity of target compounds I 1–11. NO2
O
Compounds I1 I2 I3 I4 I5 I6 I7 I8 I9 I 10 I 11 4 BCTCb)
Biological studies Evaluation of TRPV1 antagonism activity Firstly, all of the new compounds were tested for TRPV1 antagonistic activity. In the TRPV1 antagonistic activity highthroughput screening model, all the compounds inhibited Ca2þ inflow. The data in Table 1 show that compounds I 1, I 2, and I 9 emerged as preferable inhibition of TRPV1. Especially, I 1 exerted potency similarity with the positive control BCTC and compound 4. The results indicated that the varying TRPV1 antagonistic potencies of the derivatives were attributed to the diverse substitution groups of the benzene ring. Furthermore, we determined the IC50s of the optimal compounds I 1 and BCTC. Their values were 157.3 0.85 and 27.9 0.12 nM, respectively. To develop antinociceptive candidates in vivo, we chose compounds I 1, I 2, and I 9 to be assayed in antinociceptive tests.
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H N
N
R 4-tBu 4-CH3 2-OCH3 H 2-C2H5 4-F 2-Cl 3-Cl 2-NO2 2-CH3 2-CH3, 5-Cl
R
Inhibition ratio (%)a) 89.02 0.04 59.20 0.10 20.55 0.06 8.58 0.11 8.58 0.43 6.94 0.56 1.63 1.16 7.26 1.23 48.14 0.32 4.03 0.85 8.93 0.73 99.73 0.03 99.98 0.05
The concentration of the compound is 10 mM. The results (percentage inhibition, %) are the mean SEM of three independent experiments. b) BCTC means N-(4-tert-butyl-phenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide, the positive control as the TRPV1 antagonist. a)
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Arch. Pharm. Chem. Life Sci. 2015, 348, 347–352 Tetrahydroisoquinoline Derivatives as Analgesia Drugs
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Figure 2. Antinociceptive activities of synthesized compounds (30 mg/kg, i.g.) and positive controls 4 and BCTC (30 mg/kg, i.g.). 0.5% sodium carboxymethyl cellulose was given to vehicle animals as blank control. The antinociceptive effects of synthesized compounds in the capsaicin test (a); suppression of acetic acid-induced writhing response (b); inhibition of thermal nociception by synthesized compounds (c). Each bar represents the mean SEM (n ¼ 8). Statistical analysis was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. p < 0.05; p < 0.01; compared with the vehicle group.
Evaluation of antinociceptive activity in vivo To attest the preferable compounds, three different models of pain were selected for antinociceptive activity in vivo (Fig. 2). In the capsaicin test, I 1 and I 9 seemed to be as effective as compound 4, while I 2 showed weak effects. Compounds I 1 and I 9 significantly reduced the number of writhes in protoninduced pain models, especially I 1 was superior to positive control BCTC and compound 4, while I 2 showed nearly no effects. In treatment of heat-induced pain I 1, I 2, and I 9 showed similar %MPE as BCTC and compound 4 in the tail-flick test. All the test compounds had antinociceptive activity to a certain extend. Compounds I 1 and I 9 showed effective treatment in proton- and heat-induced pain, and I 1 was better than I 9 in its impressive TRPV1 antagonistic activity. Compound I 2 could weakly inhibit mouse’s reaction to pain and nociception in the gross models. We assume that the different effects of test compounds in the three models of pain may be attributed to the different stimuli to TRPV1. Compound I 1 exhibited good antinociceptive potency in proton-induced and heat-induced pain models. Focusing in particular on better determining the efficacy of I 1, dose– response experiments had been carried out (Fig. 3). In the
capsaicin test, I 1 dose-dependently reduced the total time spent licking the paw. For comparison, BCTC were found to also significantly reduce the paw-licking nocifensive response to capsaicin, and seemed to be more effective than I 1. Compound I 1 dose-dependently reduced the number of writhes in the abdominal constriction test, the effect was significant and much stronger than BCTC did. Antinociception produced by I 1 in the tail-flick test was also dose-dependent, I 1 (30 mg/kg) had a similar %MPE to BCTC (30 mg/kg). All the antinociceptive activity tests indicated that I 1 had good antinociceptive potency in treatment of proton-induced and heat-induced pain and triggers the antinociception effect in a dose-dependent manner. All the results of biological assays in vitro and in vivo proved that I 1 may be considered as a promising candidate for development of potent agents for treatment of pain.
Conclusion To sum up, we have designed and synthesized 11 carbamide derivatives of tetrahydroisoquinoline. Preliminary biological
Figure 3. Antinociceptive activities of compound I 1 at different doses (2, 10, and 30 mg/kg, i.g.) and BCTC (30 mg/kg, i.g.). 0.5% sodium carboxymethyl cellulose was given to vehicle animals as blank control. The antinociceptive effects of synthesized compounds in the capsaicin test (a); suppression of acetic acid-induced writhing response (b); inhibition of thermal nociception (c). Each bar represents the mean SEM (n ¼ 8). Statistical analysis was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. p < 0.05; p < 0.01; compared with the vehicle group.
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evaluation revealed that several compounds presented TRPV1 antagonistic activity and antinociceptive activity. Especially, I 1 showed superior potency in proton-induced and heat-induced pain models and held the most promising potential for further development.
Experimental Chemistry All reagents and solvents were reagent grade and were used without further purification unless otherwise stated. All of the target compounds were analyzed by 1H NMR and 13C NMR (Bruker ACF-300Q, 300 MHz; Bruker Instruments, Inc., Billerica, MA, USA), MS (1100 LC/MSD spectrometer; Hewlett–Packard, Palo Alto, CA, USA), and elemental analyses (CHN-O-Rapid Instrument; Elementar, Hanau, Germany); melting points were measured using a Mel-TEMP II melting point apparatus, which was uncorrected. Thin-layer chromatography (TLC) was performed on GF/UV 254 plates and the chromatograms were visualized under UV light at 254 and 365 nm.
General procedure for the preparation of 6 Nitric acid (1.7 mL, 38.7 mmol) was added to a stirred solution of isoquinoline (5 g, 38.7 mmol) in concentrated sulfuric acid (20 mL) at 5°C. The reaction mixture was stirred at 0°C for 2 h. The mixture was added to 200 g ice water. The pH was adjusted by the solution of 2 N NaOH to 9–10. The mixture was cooled overnight and filtered. The residue was recrystallized by the mixture of petroleum and ethyl acetate (250 mL/50 mL). Compound 6 (3.5 g) was obtained and the yield was 52.0%.
General procedure for the preparation of 7 Sodium borohydride (0.44 g, 11.5 mmol) was added to a stirred solution of 6 (0.5 g, 2.87 mmol) in acetic acid (10 mL) and THF (20 mL) at 0°C. The reaction mixture was stirred at 25°C monitored by TLC. The pH was adjusted by the saturation solution of NaOH to 8. The mixture was extracted with ethyl acetate (30 mL 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. Saturated HCl ethyl acetate solution (15 mL) was added to the residue and the solution was stirred at 25°C for 1 h and filtered. Compound 7 (0.50 g) was obtained and the yield was 80.64%.
General procedure for the preparation of I 1–11
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N-(4-(tert-Butyl)phenyl)-5-nitro-3,4-dihydroisoquinoline2(1H)-carboxamide (I 1)
Yield 14.6%, white solid, m.p. 190–192°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.61 (s, 1H, NH), 7.86 (d, J ¼ 7.89 Hz, 1H, Ar–H), 7.58 (d, J ¼ 7.53 Hz, 2H, Ar–H), 7.48 (d, J ¼ 7.89 Hz, 1H, Ar–H), 7.40 (d, J ¼ 8.64 Hz, 2H, Ar–H), 7.26 (d, J ¼ 8.64 Hz, 2H, Ar–H), 4.73 (s, 2H, CCH2N), 3.70 (t, J ¼ 5.75 Hz, 2H, CH2CH2N), 3.05 (t, J ¼ 5.51 Hz, 2H, CH2CH2N), 1.25 (s, 9H, Ar-C(CH3)3); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.95, 149.04, 144.16, 137.64, 137.19, 131.67, 129.63, 126.87, 124.87, 122.46, 119.59, 45.61, 40.70, 33.80, 31.22, 25.73; MS (ESI, m/z): 376.4 [MþNa]þ; IR (KBr, cm1): 3268 (nN–H), 1633 (nC –– O), 1597, 1527 (aromatic); Anal. calcd. for C20H23N3O3: C, 67.97; H, 6.56; N, 11.89%. Found: C, 68.11; H, 6.39; N, 11.75%.
5-Nitro-N-(p-tolyl)-3,4-dihydroisoquinoline-2(1H)carboxamide (I 2)
Yield 18.8%, white solid, m.p. 138–140°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.56 (s, 1H, NH), 7.86 (d, J ¼ 7.80 Hz, 1H, Ar–H), 7.57 (d, J ¼ 7.44 Hz, 2H, Ar–H), 7.47 (d, J ¼ 7.81 Hz, 1H, Ar–H), 7.36 (d, J ¼ 8.22 Hz, 2H, Ar–H), 7.05 (d, J ¼ 8.10 Hz, 2H, Ar–H), 4.73 (s, 2H, CCH2N), 3.70 (t, J ¼ 5.62 Hz, 2H, CH2CH2N), 3.04 (t, J ¼ 5.30 Hz, 2H, CH2CH2N), 2.23 (s, 3H, Ar–CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.91, 149.05, 137.67, 137.19, 131.67, 130.69, 129.63, 128.69, 126.88, 122.47, 119.91, 45.61, 40.66, 25.70, 20.29; MS (ESI, m/z): 334.4 [MþNa]þ; IR (KBr, cm1): 3291 (nN–H), 1635 (nC –– O), 1596, 1536 (aromatic); Anal. calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50%. Found: C, 65.72; H, 5.39; N, 13.62%.
N-(2-Methoxyphenyl)-5-nitro-3,4-dihydroisoquinoline2(1H)-carboxamide (I 3)
Yield 23.4%, white solid, m.p. 126–128°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 7.88–7.83 (m, 2H, NH and Ar–H), 7.61 (t, J ¼ 7.61 Hz, 2H, Ar–H), 7.46 (d, J ¼ 7.63 Hz, 1H, Ar–H), 7.08– 6.97 (m, 2H, Ar–H), 6.90–6.87 (m, 1H, Ar–H), 4.73 (s, 2H, CCH2N), 3.81 (s, 3H, Ar–OCH3), 3.69 (s, 2H, CH2CH2N), 3.06 (s, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.81, 150.45, 148.98, 137.13, 131.71, 129.72, 128.28, 126.88, 123.70, 122.87, 122.48, 120.13, 110.95, 55.63, 45.40, 40.66, 25.53; MS (ESI, m/z): 350.3 [MþNa]þ; IR (KBr, cm1): 3434 (nN–H), 1661 (nC –– O), 1600, 1523 (aromatic); Anal. calcd. for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84%. Found: C, 62.47; H, 5.11; N, 12.91%.
5-Nitro-N-phenyl-3,4-dihydroisoquinoline-2(1H)carboxamide (I 4)
The solution of substituted phenylamines (2.35 mmol) and Et3N (0.24 g, 6.99 mmol) in CH2Cl2 (20 mL) was added to a stirred solution of triphosgene (0.23 g, 0.78 mmol) in CH2Cl2 (20 mL) at 10°C. The reaction mixture was stirred for 0.5 h and solution of 7 (0.50 g, 2.55 mmol) was added in CH2Cl2. The reaction mixture was monitored by TLC and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using ethyl acetate/hexane (1:6) as eluent to obtain I 1–11.
Yield 27.5%, white solid, m.p. 124–126°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.78 (s, 1H, NH), 7.87 (d, J ¼ 7.77 Hz, 1H, Ar–H), 7.58 (d, J ¼ 7.50 Hz, 2H, Ar–H), 7.49–7.42 (m, 3H, Ar–H), 7.24 (t, J ¼ 7.86 Hz, 2H, Ar–H), 6.95 (t, J ¼ 7.32 Hz, 2H, Ar–H), 4.75 (s, 2H, CCH2N), 3.71 (t, J ¼ 5.84 Hz, 2H, CH2CH2N), 3.05 (t, J ¼ 5.70 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.84, 149.05, 140.29, 137.13, 131.69, 129.62, 128.27, 126.90, 122.49, 121.86, 119.72, 45.62, 40.70, 25.72; MS (ESI, m/z): 320.4 [MþNa]þ; IR (KBr, cm1): 3291 (nN–H), 1728, 1637 (nC –– O),
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1593, 1538 (aromatic); Anal. calcd. for C16H15N3O3: C, 64.64; H, 5.09; N, 14.13%. Found: C, 64.81; H, 5.21; N, 14.01%.
N-(2-Ethylphenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 5)
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(nN–H), 1659 (nC –– O), 1596, 1531 (aromatic); Anal. calcd. for C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67%. Found: C, 57.64; H, 4.49; N, 12.61%.
N-(2-Nitrophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 9)
Yield 26.7%, white solid, m.p. 143–145°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.20 (s, 1H, NH), 7.86 (d, J ¼ 7.74 Hz, 1H, Ar–H), 7.57 (t, J ¼ 7.29 Hz, 1H, Ar–H), 7.46 (t, J ¼ 7.74 Hz, 1H, Ar–H), 7.19–7.12 (m, 4H, Ar–H), 4.74 (s, 2H, CCH2N), 3.71 (s, 2H, Ar–OCH3), 3.05 (s, 2H, CH2CH2N), 2.59–2.50 (m, 2H, Ar–CH2CH3), 1.08 (t, J ¼ 7.43 Hz, 1H, Ar–CH2CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 155.80, 149.11, 139.37, 137.36, 136.98, 131.65, 129.60, 128.11, 127.18, 126.88, 125.71, 125.30, 122.45, 45.70, 40.88, 25.53, 23.73, 13.93; MS (ESI, m/z): 348.4 [MþNa]þ; IR (KBr, cm1): 3035 (nN–H), 1620 (nC –– O), 1603, 1521 (aromatic); Anal. calcd. for C18H19N3O3: C, 66.45; H, 5.89; N, 12.91%. Found: C, 66.22; H, 5.82; N, 12.75%.
Yield 35.4%, white solid, m.p. 153–155°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 9.42 (s, 1H, NH), 7.91 (q, 2H, Ar–H), 7.71–7.60 (m, 3H, Ar–H), 7.48 (t, J ¼ 7.84 Hz, 1H, Ar–H), 7.26–7.10 (m, 1H, Ar–H), 4.76 (s, 2H, CCH2N), 3.72 (t, J ¼ 5.80 Hz, 2H, CH2CH2N), 3.09 (t, J ¼ 5.61 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.06, 149.01, 140.78, 136.72, 134.28, 134.00, 131.69, 129.59, 127.00, 124.98, 123.98, 123.10, 122.61, 45.37, 40.79, 25.60; MS (ESI, m/z): 365.3 [MþNa]þ; IR (KBr, cm1): 3362 (nN–H), 1672 (nC –– O), 1607, 1525 (aromatic); Anal. calcd. for C16H14N4O5: C, 56.14; H, 4.12; N, 16.37%. Found: C, 56.35; H, 4.21; N, 16.09%.
N-(4-Fluorophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 6)
N-(o-Tolyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 10)
Yield 41.1%, white solid, m.p. 164–166°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.71 (s, 1H, NH), 7.87 (d, J ¼ 7.83 Hz, 1H, Ar–H), 7.51–7.44 (m, 3H, Ar–H), 7.08 (t, J ¼ 8.86 Hz, 1H, Ar–H), 4.74 (s, 2H, CCH2N), 3.70 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.05 (t, J ¼ 5.55 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 159.02, 154.86, 149.04, 137.09, 136.60, 131.67, 129.61, 126.90, 122.50, 121.54, 121.44, 114.90, 114.61, 45.56, 40.64, 25.69; MS (ESI, m/z): 338.3 [MþNa]þ; IR (KBr, cm1): 3038 (nN–H), 1635 (nC –– O), 1607, 1526 (aromatic); Anal. calcd. for C16H14FN3O3: C, 60.95; H, 4.48; N, 13.33%. Found: C, 60.77; H, 4.63; N, 13.17%.
N-(2-Chlorophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 7)
Yield 25.9%, white solid, m.p. 156–158°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.38 (s, 1H, NH), 7.88 (d, J ¼ 7.86 Hz, 1H, Ar–H), 7.59 (d, J ¼ 7.53 Hz, 1H, Ar–H), 7.52–7.40 (m, 3H, Ar–H), 7.32– 7.27 (m, 1H, Ar–H), 7.18–7.06 (m, 1H, Ar–H), 4.75 (s, 2H, CCH2N), 3.73 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.07 (t, J ¼ 5.59 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.83, 149.06, 137.08, 136.38, 131.68, 129.61, 129.21, 128.30, 127.20, 126.92, 125.76, 122.52, 45.53, 40.79, 25.53; MS (ESI, m/z): 354.3 [MþNa]þ; IR (KBr, cm1): 3203 (nN–H), 1625 (nC –– O), 1583, 1520 (aromatic); Anal. calcd. for C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67%. Found: C, 58.11; H, 4.34; N, 12.51%.
N-(3-Chlorophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 8)
Yield 61.1%, white solid, m.p. 154–156°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.23 (s, 1H, NH), 7.86 (d, J ¼ 7.86 Hz, 1H, Ar–H), 7.57 (d, J ¼ 7.44 Hz, 1H, Ar–H), 7.46 (d, J ¼ 7.63 Hz, 1H, Ar–H), 7.22–7.05 (m, 4H, Ar–H), 4.74 (s, 2H, CCH2N), 3.71 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.06 (t, J ¼ 5.64 Hz, 2H, CH2CH2N), 2.16 (s, Ar–CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 155.35, 149.08, 137.69, 137.29, 133.82, 133.14, 131.67, 129.63, 126.88, 126.47, 126.06, 124.69, 122.47, 45.64, 40.75, 25.59, 17.86; MS (ESI, m/z): 334.3 [MþNa]þ; IR (KBr, cm1): 3204 (nN–H), 1718, 1621 (nC –– O), 1574, 1521 (aromatic); Anal. calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50%. Found: C, 65.49; H, 5.56; N, 13.41%.
N-(2-Methyl-5-chlorophenyl)-5-nitro-3,4dihydroisoquinoline-2(1H)-carboxamide (I 11)
Yield 28.7%, white solid, m.p. 181–183°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.28 (s, 1H, NH), 7.87 (d, J ¼ 7.14 Hz, 1H, Ar–H), 7.59–7.46 (m, 1H, Ar–H), 7.35 (s, 1H, Ar–H), 7.21–7.09 (m, 2H, Ar–H), 4.74 (s, 2H, CCH2N), 3.71 (s, 2H, CH2CH2N), 3.06 (s, 2H, CH2CH2N), 2.15 (s, Ar–CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 155.04, 148.99, 139.09, 137.00, 131.68, 131.49, 129.58, 126.94, 124.94, 124.12, 122.53, 45.55, 40.73, 25.52, 17.28; MS (ESI, m/z): 368.0 [MþNa]þ; IR (KBr, cm1): 3200 (nN–H), 1701, 1623 (nC –– O), 1560, 1522 (aromatic); Anal. calcd. for C17H16ClN3O3: C, 59.05; H, 4.66; N, 12.15%. Found: C, 59.24; H, 4.51; N, 11.02%.
Biological studies
Yield 44.2%, white solid, m.p. 148–150°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.86 (s, 1H, NH), 7.87 (d, J ¼ 7.83 Hz, 1H, Ar–H), 7.67 (s, 1H, Ar–H), 7.59 (d, J ¼ 7.50 Hz, 1H, Ar–H), 7.50–7.42 (m, 2H, Ar–H), 7.27 (t, J ¼ 8.08 Hz, 1H, Ar–H), 7.00–6.98 (m, 1H, Ar–H), 4.75 (s, 2H, CCH2N), 3.71 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.06 (t, J ¼ 5.58 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.45, 149.01, 141.96, 136.94, 132.70, 131.69, 129.92, 129.59, 126.92, 122.54, 121.37, 118.85, 117.74, 45.54, 40.69, 25.69; MS (ESI, m/z): 354.2 [MþNa]þ; IR (KBr, cm1): 3405
The TRPV1 aequorin cells (PerkinElmer, USA) were collected from culture plates with Ca2þ and Mg2þ-free phosphatebuffered saline supplemented with 5 mM ethylenediaminetetra-acetic acid; pelleted for 2 min at 1000g; resuspended in Dulbecco’s minimum essential medium–F12 medium with 15 mM HEPES (pH 7.0) and 0.1% BSA (assay buffer) at a density of 3 105 cells/mL; incubated for 4 h in the dark in the presence of 5 mM coelenterazine h (Promega, USA). After loading, cells
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were diluted with assay buffer to a concentration of 5 106 cells/mL. Twenty microliters of cells were injected over 20 mL of the sample solution plated on 384-well plates, respectively, unless otherwise indicated. The digitonin, ATP (Sigma–Aldrich, USA) assay buffer was added in the blank control wells for reference, and final concentrations of digitonin and ATP were 100 mM and 50 mM. The sample solution and the cells were incubated for 2.5 min before adding agonist capsaicin (Tocris, England), then immediately detected. The light emission was recorded during variable times using EnVision 2014 Multilabel Reader (PerkinElmer, USA) [7, 8].
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Statistical analysis of the data Statistical analyses were performed using specific software (GraphPadInStat version 5.00; GraphPad Software, San Diego, CA, USA). Unpaired comparisons were analyzed using the two-tailed Student t-test, unless otherwise stated. This study was supported by the Natural Science Foundation of Jiangsu Province of China (No. BK2012759) and the Fundamental Research Funds for the Central Universities of China (No. JKQ2009003). The authors have declared no conflict of interest.
Evaluation of antinociceptive activity in vivo Capsaicin test: The capsaicin test was assessed as previously described [9]. Following an acclimation period of 30 min, 20 mL of solution of capsaicin (0.8 mg/mL) was injected subcutaneously into the dorsal aspect of the right hind paw. The mouse was then placed in an individual cage. Mice were observed for a continuous period of 5 min. The amount of time spent licking the injected paw was measured and expressed as the cumulative licking time during the 5 min observation period.
References
Tail-flick test: Tail-flick test was performed as described previously [11]. In the experiment evaluating the time course of antinociception, we elected to use the warm water tail withdrawal test in order to minimize damage to the tail because of the repeated testing. In brief, the distal one-third of the tail was immersed into 52.0°C water and latency to withdrawal the tail was recorded before and after drug treatment. The antinociception response was presented as percent maximal possible effect (%MPE) as defined by percent maximal possible effect ¼ 100% (drug response time basal response time)/(cut-off time basal response time). To avoid tissue damage, the cut-off time was suitable for 12 s.
[1] A. Patapoutian, S. Tate, C. J. Woolf, Nat. Rev. Drug Discov. 2009, 8 (1), 55–68. [2] R. Brito, S. Sheth, D. Mukherjea, L. P. Rybak, V. Ramkumar, Cells 2014, 3 (2), 517–545. [3] A. Gomtsyan, C. R. Faltynek, Vanilloid Receptor TRPV1 in Drug Discovery: Targeting Pain and Other Pathological Disorders, John Wiley & Sons, Hoboken 2010. [4] C. S. Walpole, S. Bevan, G. Bovermann, J. J. Boelsterli, R. Breckenridge, J. W. Davies, G. A. Hughes, I. James, L. Oberer, J. Med. Chem. 1994, 37 (13), 1942–1954. [5] D. K. O’Dell, N. Rimmerman, S. R. Pickens, J. M. Walker, Bioorg. Med. Chem. 2007, 15 (18), 6164–6169. [6] R. G. Schmidt, E. K. Bayburt, S. P. Latshaw, J. R. Koenig, J. F. Daanen, H. A. McDonald, B. R. Bianchi, C. Zhong, S. Joshi, P. Honore, Bioorg. Med. Chem. Lett. 2011, 21 (5), 1338–1341. [7] E. Le Poul, S. Hisada, Y. Mizuguchi, V. J. Dupriez, E. Burgeon, M. Detheux, J. Biomol. Screen. 2002, 7 (1), 57–65. [8] S. G. Lehto, R. Tamir, H. Deng, L. Klionsky, R. Kuang, A. Le, D. Lee, J.-C. Louis, E. Magal, B. H. Manning, J. Pharmacol. Exp. Ther. 2008, 326 (1), 218–229. [9] J. Wang, D. Dai, Q. Qiu, X. Deng, H. Lin, H. Qian, W. Huang, Chem. Biol. Drug Des. 2014, in press. DOI: 10.1111/cbdd.12316 [10] H. Qian, Z. Fu, W. Huang, H. Zhang, J. Zhou, L. Ge, R. Lin, H. Lin, X. Hu, Med. Chem. 2010, 6 (4), 205–210. [11] L. Ge, X. Zhang, H. Zhang, H. Qian, W. Huang, J. Zhou, Lett. Drug Des. Discov. 2011, 8 (1), 76–81.
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Abdominal constriction test: Abdominal constriction test was performed as described previously [10]. Mice were placed in individual glass cylinders for a 30 min acclimatization period. The writhes were induced by injection with 0.6% acetic acid (0.1 mL/10 g/mouse i.p.), and immediately placed inside transparent glass cylinders. The number of muscular contractions was counted for 15 min after acetic acid injection.
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Full Paper Synthesis of Carbamide Derivatives Bearing Tetrahydroisoquinoline Moieties and Biological Evaluation as Analgesia Drugs in Mice Qianqian Qiu1, Jingjie Wang1,2y, Xin Deng1, Hai Qian1, Haiyan Lin3y, and Wenlong Huang1 1
2 3
State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, Nanjing, China WuXiAppTec (Wuhan) Co., Ltd., Wuhan, China Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, China
Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated non-selective cation channel that is considered to be an important pain integrator. Tetrahydroisoquinoline, the prototypical antagonist of TRPV1, has a clear therapeutic potential. Here, a series of carbamide derivatives of tetrahydroisoquinoline were designed and synthesized. Preliminary biological tests suggested that the compounds I 1, I 2, and I 9 had favorable TRPV1 antagonism activity. In further studies, I 1 exhibited better antinociceptive activity than the positive control BCTC in diverse pain models. All of these results suggested that I 1 can be considered as the lead candidate for the further development of antinociceptive drugs. Keywords: Analgesia drugs / Tetrahydroisoquinoline derivatives / Transient receptor potential vanilloid 1 antagonist Received: December 18, 2014; Revised: February 9, 2015; Accepted: February 17, 2015 DOI 10.1002/ardp.201400455
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Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Introduction Transient receptor potential vanilloid 1 (TRPV1) is the best characterized member of the TRP superfamily of ion channels, which are activated by a range of stimuli, including capsaicin as well as protons (pH 43°C) stimuli among others [1, 2]. In the last decade, significant efforts have been devoted to the design and synthesis of TRPV1 antagonists as antinociceptive agents [3]. A review of the literature on the development of the first TRPV1 antagonist, capsazepine 1, revealed that the tetrahydroisoquinoline moiety is a core pharmacophore of TRPV1 antagonists [4–6]. While less potent than capsazepine 1, compound 2 was a weak and partial antagonist of the TRPV1 receptor. In a previous research, we got two derivatives of tetrahydroisoquinoline (3 and 4) as shown in Fig. 1. However, TRPV1 antagonistic potency made a big difference between
the two compounds, that compound 4 was much superior to compound 3. Based on the preliminary result, we hypothesized that the nitro group at 5-position of tetrahydroisoquinoline moiety should possess the necessary for activity at TRPV1. In this letter, according to the structure of 4, intended for searching derivatives of tetrahydroisoquinoline with better TRPV1 antagonistic potency and antinociceptive activity, the thiourea group in 4 was replaced with urea group, to obtain a series of TRPV1 antagonists with slight structure difference but similar impressive potency. As a result, 11 carbamide derivatives of tetrahydroisoquinoline were synthesized and their activities were evaluated.
Results and discussion Chemistry The synthetic route of target compounds I 1–11 is depicted in Scheme 1. Compound 6 was synthesized by nitration of
Correspondence: Prof. Wenlong Huang, Centre of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China. E-mail: [email protected] Fax: þ86 25 83271480
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y
Contributed equally to the first author. Additional correspondence: Dr. Haiyan Lin, E-mail: [email protected]
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Figure 1. The TRPV1 antagonists with tetrahydroisoquinoline moiety.
Scheme 1. Synthesis of target compounds I 1–11. Reagents and conditions: (a) HNO3, H2SO4; (b) NaBH4, acetic acid; (c) substituted anilines, triphosgene/Et3N, one pot.
isoquinoline with nitric acid in concentrated sulfuric acid at 0°C for 2 h, followed by adjusting the solution pH to 9–10. The formation of compound 7 was conducted by sodium borohydride and compound 6 in acetic acid (10 mL) and THF (20 mL) at 0–25°C. After completion of reaction, the pH of the solution was adjusted to 8 with NaOH. Followed by extraction with ethyl acetate, compound 7 was obtained by concentration in vacuo. The desirable target compounds I 1–11 were produced by one-pot synthesis with substituted phenylamines, compound 7, triphosgene, and Et3N in CH2Cl2 at 10°C. The structures of all target compounds are presented in Table 1.
Table 1. The structures and TRPV1 antagonistic activity of target compounds I 1–11. NO2
O
Compounds I1 I2 I3 I4 I5 I6 I7 I8 I9 I 10 I 11 4 BCTCb)
Biological studies Evaluation of TRPV1 antagonism activity Firstly, all of the new compounds were tested for TRPV1 antagonistic activity. In the TRPV1 antagonistic activity highthroughput screening model, all the compounds inhibited Ca2þ inflow. The data in Table 1 show that compounds I 1, I 2, and I 9 emerged as preferable inhibition of TRPV1. Especially, I 1 exerted potency similarity with the positive control BCTC and compound 4. The results indicated that the varying TRPV1 antagonistic potencies of the derivatives were attributed to the diverse substitution groups of the benzene ring. Furthermore, we determined the IC50s of the optimal compounds I 1 and BCTC. Their values were 157.3 0.85 and 27.9 0.12 nM, respectively. To develop antinociceptive candidates in vivo, we chose compounds I 1, I 2, and I 9 to be assayed in antinociceptive tests.
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H N
N
R 4-tBu 4-CH3 2-OCH3 H 2-C2H5 4-F 2-Cl 3-Cl 2-NO2 2-CH3 2-CH3, 5-Cl
R
Inhibition ratio (%)a) 89.02 0.04 59.20 0.10 20.55 0.06 8.58 0.11 8.58 0.43 6.94 0.56 1.63 1.16 7.26 1.23 48.14 0.32 4.03 0.85 8.93 0.73 99.73 0.03 99.98 0.05
The concentration of the compound is 10 mM. The results (percentage inhibition, %) are the mean SEM of three independent experiments. b) BCTC means N-(4-tert-butyl-phenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide, the positive control as the TRPV1 antagonist. a)
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Figure 2. Antinociceptive activities of synthesized compounds (30 mg/kg, i.g.) and positive controls 4 and BCTC (30 mg/kg, i.g.). 0.5% sodium carboxymethyl cellulose was given to vehicle animals as blank control. The antinociceptive effects of synthesized compounds in the capsaicin test (a); suppression of acetic acid-induced writhing response (b); inhibition of thermal nociception by synthesized compounds (c). Each bar represents the mean SEM (n ¼ 8). Statistical analysis was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. p < 0.05; p < 0.01; compared with the vehicle group.
Evaluation of antinociceptive activity in vivo To attest the preferable compounds, three different models of pain were selected for antinociceptive activity in vivo (Fig. 2). In the capsaicin test, I 1 and I 9 seemed to be as effective as compound 4, while I 2 showed weak effects. Compounds I 1 and I 9 significantly reduced the number of writhes in protoninduced pain models, especially I 1 was superior to positive control BCTC and compound 4, while I 2 showed nearly no effects. In treatment of heat-induced pain I 1, I 2, and I 9 showed similar %MPE as BCTC and compound 4 in the tail-flick test. All the test compounds had antinociceptive activity to a certain extend. Compounds I 1 and I 9 showed effective treatment in proton- and heat-induced pain, and I 1 was better than I 9 in its impressive TRPV1 antagonistic activity. Compound I 2 could weakly inhibit mouse’s reaction to pain and nociception in the gross models. We assume that the different effects of test compounds in the three models of pain may be attributed to the different stimuli to TRPV1. Compound I 1 exhibited good antinociceptive potency in proton-induced and heat-induced pain models. Focusing in particular on better determining the efficacy of I 1, dose– response experiments had been carried out (Fig. 3). In the
capsaicin test, I 1 dose-dependently reduced the total time spent licking the paw. For comparison, BCTC were found to also significantly reduce the paw-licking nocifensive response to capsaicin, and seemed to be more effective than I 1. Compound I 1 dose-dependently reduced the number of writhes in the abdominal constriction test, the effect was significant and much stronger than BCTC did. Antinociception produced by I 1 in the tail-flick test was also dose-dependent, I 1 (30 mg/kg) had a similar %MPE to BCTC (30 mg/kg). All the antinociceptive activity tests indicated that I 1 had good antinociceptive potency in treatment of proton-induced and heat-induced pain and triggers the antinociception effect in a dose-dependent manner. All the results of biological assays in vitro and in vivo proved that I 1 may be considered as a promising candidate for development of potent agents for treatment of pain.
Conclusion To sum up, we have designed and synthesized 11 carbamide derivatives of tetrahydroisoquinoline. Preliminary biological
Figure 3. Antinociceptive activities of compound I 1 at different doses (2, 10, and 30 mg/kg, i.g.) and BCTC (30 mg/kg, i.g.). 0.5% sodium carboxymethyl cellulose was given to vehicle animals as blank control. The antinociceptive effects of synthesized compounds in the capsaicin test (a); suppression of acetic acid-induced writhing response (b); inhibition of thermal nociception (c). Each bar represents the mean SEM (n ¼ 8). Statistical analysis was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. p < 0.05; p < 0.01; compared with the vehicle group.
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evaluation revealed that several compounds presented TRPV1 antagonistic activity and antinociceptive activity. Especially, I 1 showed superior potency in proton-induced and heat-induced pain models and held the most promising potential for further development.
Experimental Chemistry All reagents and solvents were reagent grade and were used without further purification unless otherwise stated. All of the target compounds were analyzed by 1H NMR and 13C NMR (Bruker ACF-300Q, 300 MHz; Bruker Instruments, Inc., Billerica, MA, USA), MS (1100 LC/MSD spectrometer; Hewlett–Packard, Palo Alto, CA, USA), and elemental analyses (CHN-O-Rapid Instrument; Elementar, Hanau, Germany); melting points were measured using a Mel-TEMP II melting point apparatus, which was uncorrected. Thin-layer chromatography (TLC) was performed on GF/UV 254 plates and the chromatograms were visualized under UV light at 254 and 365 nm.
General procedure for the preparation of 6 Nitric acid (1.7 mL, 38.7 mmol) was added to a stirred solution of isoquinoline (5 g, 38.7 mmol) in concentrated sulfuric acid (20 mL) at 5°C. The reaction mixture was stirred at 0°C for 2 h. The mixture was added to 200 g ice water. The pH was adjusted by the solution of 2 N NaOH to 9–10. The mixture was cooled overnight and filtered. The residue was recrystallized by the mixture of petroleum and ethyl acetate (250 mL/50 mL). Compound 6 (3.5 g) was obtained and the yield was 52.0%.
General procedure for the preparation of 7 Sodium borohydride (0.44 g, 11.5 mmol) was added to a stirred solution of 6 (0.5 g, 2.87 mmol) in acetic acid (10 mL) and THF (20 mL) at 0°C. The reaction mixture was stirred at 25°C monitored by TLC. The pH was adjusted by the saturation solution of NaOH to 8. The mixture was extracted with ethyl acetate (30 mL 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. Saturated HCl ethyl acetate solution (15 mL) was added to the residue and the solution was stirred at 25°C for 1 h and filtered. Compound 7 (0.50 g) was obtained and the yield was 80.64%.
General procedure for the preparation of I 1–11
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N-(4-(tert-Butyl)phenyl)-5-nitro-3,4-dihydroisoquinoline2(1H)-carboxamide (I 1)
Yield 14.6%, white solid, m.p. 190–192°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.61 (s, 1H, NH), 7.86 (d, J ¼ 7.89 Hz, 1H, Ar–H), 7.58 (d, J ¼ 7.53 Hz, 2H, Ar–H), 7.48 (d, J ¼ 7.89 Hz, 1H, Ar–H), 7.40 (d, J ¼ 8.64 Hz, 2H, Ar–H), 7.26 (d, J ¼ 8.64 Hz, 2H, Ar–H), 4.73 (s, 2H, CCH2N), 3.70 (t, J ¼ 5.75 Hz, 2H, CH2CH2N), 3.05 (t, J ¼ 5.51 Hz, 2H, CH2CH2N), 1.25 (s, 9H, Ar-C(CH3)3); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.95, 149.04, 144.16, 137.64, 137.19, 131.67, 129.63, 126.87, 124.87, 122.46, 119.59, 45.61, 40.70, 33.80, 31.22, 25.73; MS (ESI, m/z): 376.4 [MþNa]þ; IR (KBr, cm1): 3268 (nN–H), 1633 (nC –– O), 1597, 1527 (aromatic); Anal. calcd. for C20H23N3O3: C, 67.97; H, 6.56; N, 11.89%. Found: C, 68.11; H, 6.39; N, 11.75%.
5-Nitro-N-(p-tolyl)-3,4-dihydroisoquinoline-2(1H)carboxamide (I 2)
Yield 18.8%, white solid, m.p. 138–140°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.56 (s, 1H, NH), 7.86 (d, J ¼ 7.80 Hz, 1H, Ar–H), 7.57 (d, J ¼ 7.44 Hz, 2H, Ar–H), 7.47 (d, J ¼ 7.81 Hz, 1H, Ar–H), 7.36 (d, J ¼ 8.22 Hz, 2H, Ar–H), 7.05 (d, J ¼ 8.10 Hz, 2H, Ar–H), 4.73 (s, 2H, CCH2N), 3.70 (t, J ¼ 5.62 Hz, 2H, CH2CH2N), 3.04 (t, J ¼ 5.30 Hz, 2H, CH2CH2N), 2.23 (s, 3H, Ar–CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.91, 149.05, 137.67, 137.19, 131.67, 130.69, 129.63, 128.69, 126.88, 122.47, 119.91, 45.61, 40.66, 25.70, 20.29; MS (ESI, m/z): 334.4 [MþNa]þ; IR (KBr, cm1): 3291 (nN–H), 1635 (nC –– O), 1596, 1536 (aromatic); Anal. calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50%. Found: C, 65.72; H, 5.39; N, 13.62%.
N-(2-Methoxyphenyl)-5-nitro-3,4-dihydroisoquinoline2(1H)-carboxamide (I 3)
Yield 23.4%, white solid, m.p. 126–128°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 7.88–7.83 (m, 2H, NH and Ar–H), 7.61 (t, J ¼ 7.61 Hz, 2H, Ar–H), 7.46 (d, J ¼ 7.63 Hz, 1H, Ar–H), 7.08– 6.97 (m, 2H, Ar–H), 6.90–6.87 (m, 1H, Ar–H), 4.73 (s, 2H, CCH2N), 3.81 (s, 3H, Ar–OCH3), 3.69 (s, 2H, CH2CH2N), 3.06 (s, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.81, 150.45, 148.98, 137.13, 131.71, 129.72, 128.28, 126.88, 123.70, 122.87, 122.48, 120.13, 110.95, 55.63, 45.40, 40.66, 25.53; MS (ESI, m/z): 350.3 [MþNa]þ; IR (KBr, cm1): 3434 (nN–H), 1661 (nC –– O), 1600, 1523 (aromatic); Anal. calcd. for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84%. Found: C, 62.47; H, 5.11; N, 12.91%.
5-Nitro-N-phenyl-3,4-dihydroisoquinoline-2(1H)carboxamide (I 4)
The solution of substituted phenylamines (2.35 mmol) and Et3N (0.24 g, 6.99 mmol) in CH2Cl2 (20 mL) was added to a stirred solution of triphosgene (0.23 g, 0.78 mmol) in CH2Cl2 (20 mL) at 10°C. The reaction mixture was stirred for 0.5 h and solution of 7 (0.50 g, 2.55 mmol) was added in CH2Cl2. The reaction mixture was monitored by TLC and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using ethyl acetate/hexane (1:6) as eluent to obtain I 1–11.
Yield 27.5%, white solid, m.p. 124–126°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.78 (s, 1H, NH), 7.87 (d, J ¼ 7.77 Hz, 1H, Ar–H), 7.58 (d, J ¼ 7.50 Hz, 2H, Ar–H), 7.49–7.42 (m, 3H, Ar–H), 7.24 (t, J ¼ 7.86 Hz, 2H, Ar–H), 6.95 (t, J ¼ 7.32 Hz, 2H, Ar–H), 4.75 (s, 2H, CCH2N), 3.71 (t, J ¼ 5.84 Hz, 2H, CH2CH2N), 3.05 (t, J ¼ 5.70 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.84, 149.05, 140.29, 137.13, 131.69, 129.62, 128.27, 126.90, 122.49, 121.86, 119.72, 45.62, 40.70, 25.72; MS (ESI, m/z): 320.4 [MþNa]þ; IR (KBr, cm1): 3291 (nN–H), 1728, 1637 (nC –– O),
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1593, 1538 (aromatic); Anal. calcd. for C16H15N3O3: C, 64.64; H, 5.09; N, 14.13%. Found: C, 64.81; H, 5.21; N, 14.01%.
N-(2-Ethylphenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 5)
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(nN–H), 1659 (nC –– O), 1596, 1531 (aromatic); Anal. calcd. for C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67%. Found: C, 57.64; H, 4.49; N, 12.61%.
N-(2-Nitrophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 9)
Yield 26.7%, white solid, m.p. 143–145°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.20 (s, 1H, NH), 7.86 (d, J ¼ 7.74 Hz, 1H, Ar–H), 7.57 (t, J ¼ 7.29 Hz, 1H, Ar–H), 7.46 (t, J ¼ 7.74 Hz, 1H, Ar–H), 7.19–7.12 (m, 4H, Ar–H), 4.74 (s, 2H, CCH2N), 3.71 (s, 2H, Ar–OCH3), 3.05 (s, 2H, CH2CH2N), 2.59–2.50 (m, 2H, Ar–CH2CH3), 1.08 (t, J ¼ 7.43 Hz, 1H, Ar–CH2CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 155.80, 149.11, 139.37, 137.36, 136.98, 131.65, 129.60, 128.11, 127.18, 126.88, 125.71, 125.30, 122.45, 45.70, 40.88, 25.53, 23.73, 13.93; MS (ESI, m/z): 348.4 [MþNa]þ; IR (KBr, cm1): 3035 (nN–H), 1620 (nC –– O), 1603, 1521 (aromatic); Anal. calcd. for C18H19N3O3: C, 66.45; H, 5.89; N, 12.91%. Found: C, 66.22; H, 5.82; N, 12.75%.
Yield 35.4%, white solid, m.p. 153–155°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 9.42 (s, 1H, NH), 7.91 (q, 2H, Ar–H), 7.71–7.60 (m, 3H, Ar–H), 7.48 (t, J ¼ 7.84 Hz, 1H, Ar–H), 7.26–7.10 (m, 1H, Ar–H), 4.76 (s, 2H, CCH2N), 3.72 (t, J ¼ 5.80 Hz, 2H, CH2CH2N), 3.09 (t, J ¼ 5.61 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.06, 149.01, 140.78, 136.72, 134.28, 134.00, 131.69, 129.59, 127.00, 124.98, 123.98, 123.10, 122.61, 45.37, 40.79, 25.60; MS (ESI, m/z): 365.3 [MþNa]þ; IR (KBr, cm1): 3362 (nN–H), 1672 (nC –– O), 1607, 1525 (aromatic); Anal. calcd. for C16H14N4O5: C, 56.14; H, 4.12; N, 16.37%. Found: C, 56.35; H, 4.21; N, 16.09%.
N-(4-Fluorophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 6)
N-(o-Tolyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 10)
Yield 41.1%, white solid, m.p. 164–166°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.71 (s, 1H, NH), 7.87 (d, J ¼ 7.83 Hz, 1H, Ar–H), 7.51–7.44 (m, 3H, Ar–H), 7.08 (t, J ¼ 8.86 Hz, 1H, Ar–H), 4.74 (s, 2H, CCH2N), 3.70 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.05 (t, J ¼ 5.55 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 159.02, 154.86, 149.04, 137.09, 136.60, 131.67, 129.61, 126.90, 122.50, 121.54, 121.44, 114.90, 114.61, 45.56, 40.64, 25.69; MS (ESI, m/z): 338.3 [MþNa]þ; IR (KBr, cm1): 3038 (nN–H), 1635 (nC –– O), 1607, 1526 (aromatic); Anal. calcd. for C16H14FN3O3: C, 60.95; H, 4.48; N, 13.33%. Found: C, 60.77; H, 4.63; N, 13.17%.
N-(2-Chlorophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 7)
Yield 25.9%, white solid, m.p. 156–158°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.38 (s, 1H, NH), 7.88 (d, J ¼ 7.86 Hz, 1H, Ar–H), 7.59 (d, J ¼ 7.53 Hz, 1H, Ar–H), 7.52–7.40 (m, 3H, Ar–H), 7.32– 7.27 (m, 1H, Ar–H), 7.18–7.06 (m, 1H, Ar–H), 4.75 (s, 2H, CCH2N), 3.73 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.07 (t, J ¼ 5.59 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.83, 149.06, 137.08, 136.38, 131.68, 129.61, 129.21, 128.30, 127.20, 126.92, 125.76, 122.52, 45.53, 40.79, 25.53; MS (ESI, m/z): 354.3 [MþNa]þ; IR (KBr, cm1): 3203 (nN–H), 1625 (nC –– O), 1583, 1520 (aromatic); Anal. calcd. for C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67%. Found: C, 58.11; H, 4.34; N, 12.51%.
N-(3-Chlorophenyl)-5-nitro-3,4-dihydroisoquinoline-2(1H)carboxamide (I 8)
Yield 61.1%, white solid, m.p. 154–156°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.23 (s, 1H, NH), 7.86 (d, J ¼ 7.86 Hz, 1H, Ar–H), 7.57 (d, J ¼ 7.44 Hz, 1H, Ar–H), 7.46 (d, J ¼ 7.63 Hz, 1H, Ar–H), 7.22–7.05 (m, 4H, Ar–H), 4.74 (s, 2H, CCH2N), 3.71 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.06 (t, J ¼ 5.64 Hz, 2H, CH2CH2N), 2.16 (s, Ar–CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 155.35, 149.08, 137.69, 137.29, 133.82, 133.14, 131.67, 129.63, 126.88, 126.47, 126.06, 124.69, 122.47, 45.64, 40.75, 25.59, 17.86; MS (ESI, m/z): 334.3 [MþNa]þ; IR (KBr, cm1): 3204 (nN–H), 1718, 1621 (nC –– O), 1574, 1521 (aromatic); Anal. calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50%. Found: C, 65.49; H, 5.56; N, 13.41%.
N-(2-Methyl-5-chlorophenyl)-5-nitro-3,4dihydroisoquinoline-2(1H)-carboxamide (I 11)
Yield 28.7%, white solid, m.p. 181–183°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.28 (s, 1H, NH), 7.87 (d, J ¼ 7.14 Hz, 1H, Ar–H), 7.59–7.46 (m, 1H, Ar–H), 7.35 (s, 1H, Ar–H), 7.21–7.09 (m, 2H, Ar–H), 4.74 (s, 2H, CCH2N), 3.71 (s, 2H, CH2CH2N), 3.06 (s, 2H, CH2CH2N), 2.15 (s, Ar–CH3); 13C NMR (DMSO-d6, 75 MHz): d ppm 155.04, 148.99, 139.09, 137.00, 131.68, 131.49, 129.58, 126.94, 124.94, 124.12, 122.53, 45.55, 40.73, 25.52, 17.28; MS (ESI, m/z): 368.0 [MþNa]þ; IR (KBr, cm1): 3200 (nN–H), 1701, 1623 (nC –– O), 1560, 1522 (aromatic); Anal. calcd. for C17H16ClN3O3: C, 59.05; H, 4.66; N, 12.15%. Found: C, 59.24; H, 4.51; N, 11.02%.
Biological studies
Yield 44.2%, white solid, m.p. 148–150°C; 1H NMR (DMSO-d6, 300 MHz): d ppm 8.86 (s, 1H, NH), 7.87 (d, J ¼ 7.83 Hz, 1H, Ar–H), 7.67 (s, 1H, Ar–H), 7.59 (d, J ¼ 7.50 Hz, 1H, Ar–H), 7.50–7.42 (m, 2H, Ar–H), 7.27 (t, J ¼ 8.08 Hz, 1H, Ar–H), 7.00–6.98 (m, 1H, Ar–H), 4.75 (s, 2H, CCH2N), 3.71 (t, J ¼ 5.79 Hz, 2H, CH2CH2N), 3.06 (t, J ¼ 5.58 Hz, 2H, CH2CH2N); 13C NMR (DMSO-d6, 75 MHz): d ppm 154.45, 149.01, 141.96, 136.94, 132.70, 131.69, 129.92, 129.59, 126.92, 122.54, 121.37, 118.85, 117.74, 45.54, 40.69, 25.69; MS (ESI, m/z): 354.2 [MþNa]þ; IR (KBr, cm1): 3405
The TRPV1 aequorin cells (PerkinElmer, USA) were collected from culture plates with Ca2þ and Mg2þ-free phosphatebuffered saline supplemented with 5 mM ethylenediaminetetra-acetic acid; pelleted for 2 min at 1000g; resuspended in Dulbecco’s minimum essential medium–F12 medium with 15 mM HEPES (pH 7.0) and 0.1% BSA (assay buffer) at a density of 3 105 cells/mL; incubated for 4 h in the dark in the presence of 5 mM coelenterazine h (Promega, USA). After loading, cells
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Arch. Pharm. Chem. Life Sci. 2015, 348, 347–352 Q. Qiu et al.
were diluted with assay buffer to a concentration of 5 106 cells/mL. Twenty microliters of cells were injected over 20 mL of the sample solution plated on 384-well plates, respectively, unless otherwise indicated. The digitonin, ATP (Sigma–Aldrich, USA) assay buffer was added in the blank control wells for reference, and final concentrations of digitonin and ATP were 100 mM and 50 mM. The sample solution and the cells were incubated for 2.5 min before adding agonist capsaicin (Tocris, England), then immediately detected. The light emission was recorded during variable times using EnVision 2014 Multilabel Reader (PerkinElmer, USA) [7, 8].
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Statistical analysis of the data Statistical analyses were performed using specific software (GraphPadInStat version 5.00; GraphPad Software, San Diego, CA, USA). Unpaired comparisons were analyzed using the two-tailed Student t-test, unless otherwise stated. This study was supported by the Natural Science Foundation of Jiangsu Province of China (No. BK2012759) and the Fundamental Research Funds for the Central Universities of China (No. JKQ2009003). The authors have declared no conflict of interest.
Evaluation of antinociceptive activity in vivo Capsaicin test: The capsaicin test was assessed as previously described [9]. Following an acclimation period of 30 min, 20 mL of solution of capsaicin (0.8 mg/mL) was injected subcutaneously into the dorsal aspect of the right hind paw. The mouse was then placed in an individual cage. Mice were observed for a continuous period of 5 min. The amount of time spent licking the injected paw was measured and expressed as the cumulative licking time during the 5 min observation period.
References
Tail-flick test: Tail-flick test was performed as described previously [11]. In the experiment evaluating the time course of antinociception, we elected to use the warm water tail withdrawal test in order to minimize damage to the tail because of the repeated testing. In brief, the distal one-third of the tail was immersed into 52.0°C water and latency to withdrawal the tail was recorded before and after drug treatment. The antinociception response was presented as percent maximal possible effect (%MPE) as defined by percent maximal possible effect ¼ 100% (drug response time basal response time)/(cut-off time basal response time). To avoid tissue damage, the cut-off time was suitable for 12 s.
[1] A. Patapoutian, S. Tate, C. J. Woolf, Nat. Rev. Drug Discov. 2009, 8 (1), 55–68. [2] R. Brito, S. Sheth, D. Mukherjea, L. P. Rybak, V. Ramkumar, Cells 2014, 3 (2), 517–545. [3] A. Gomtsyan, C. R. Faltynek, Vanilloid Receptor TRPV1 in Drug Discovery: Targeting Pain and Other Pathological Disorders, John Wiley & Sons, Hoboken 2010. [4] C. S. Walpole, S. Bevan, G. Bovermann, J. J. Boelsterli, R. Breckenridge, J. W. Davies, G. A. Hughes, I. James, L. Oberer, J. Med. Chem. 1994, 37 (13), 1942–1954. [5] D. K. O’Dell, N. Rimmerman, S. R. Pickens, J. M. Walker, Bioorg. Med. Chem. 2007, 15 (18), 6164–6169. [6] R. G. Schmidt, E. K. Bayburt, S. P. Latshaw, J. R. Koenig, J. F. Daanen, H. A. McDonald, B. R. Bianchi, C. Zhong, S. Joshi, P. Honore, Bioorg. Med. Chem. Lett. 2011, 21 (5), 1338–1341. [7] E. Le Poul, S. Hisada, Y. Mizuguchi, V. J. Dupriez, E. Burgeon, M. Detheux, J. Biomol. Screen. 2002, 7 (1), 57–65. [8] S. G. Lehto, R. Tamir, H. Deng, L. Klionsky, R. Kuang, A. Le, D. Lee, J.-C. Louis, E. Magal, B. H. Manning, J. Pharmacol. Exp. Ther. 2008, 326 (1), 218–229. [9] J. Wang, D. Dai, Q. Qiu, X. Deng, H. Lin, H. Qian, W. Huang, Chem. Biol. Drug Des. 2014, in press. DOI: 10.1111/cbdd.12316 [10] H. Qian, Z. Fu, W. Huang, H. Zhang, J. Zhou, L. Ge, R. Lin, H. Lin, X. Hu, Med. Chem. 2010, 6 (4), 205–210. [11] L. Ge, X. Zhang, H. Zhang, H. Qian, W. Huang, J. Zhou, Lett. Drug Des. Discov. 2011, 8 (1), 76–81.
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Abdominal constriction test: Abdominal constriction test was performed as described previously [10]. Mice were placed in individual glass cylinders for a 30 min acclimatization period. The writhes were induced by injection with 0.6% acetic acid (0.1 mL/10 g/mouse i.p.), and immediately placed inside transparent glass cylinders. The number of muscular contractions was counted for 15 min after acetic acid injection.
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