J Pharm Pharmaceut Sci (www.cspscanada.org) 10(3):254-262, 2007
Synthesis of some quinoline-2(1H)-one and 1, 2, 4 - triazolo [ 4 , 3 -a ] quinoline derivatives as potent anticonvulsants
Li-Ping Guan,a, b Qing-Hao Jin,b Guan-Rong Tian,d Kyu-Yun Chai,c and Zhe-Shan Quan, a, b,*
a Key Laboratory of Organism Functional Factors of the Changbai Mountain（Yanbian University), Ministry of Education, Yanji, Jilin, 133002, P. R. China. b College of Pharmacy, Yanbian University, Yanji, Jilin, 133000, P. R. China. cDepartment of Chemistry, Wonkwang University, Iksan 570-749, Korea. dDepartment of Chemistry, Yanbian University, Yanji, Jilin, 133000, P. R. China.
Received, December, 12, 2006; Revised March 10, 2007; Accepted April 24, 2007; Published, April 27th, 2007.
Corresponding Author: Zhe-Shan Quan, College of Pharmacy, Yanbian University, No. 121, JuZi Street, Yanji City, Jilin Province, P. R. China Tel.: +86-433-2660606. Fax: +86-433-2660568. E-mail address: zsquan@ybu.edu.cn
ABSTRACT - Purpose. A new series of substituted quinoline-2(1H)-one and 1,2,4-triazolo[4,3-a]-quinoline derivatives were designed and synthesized to meet the structural requirements essential for anticonvulsant properties. Methods. 4-substituted-phenyl-3,4-dihydro-2(1H)-quinolines, 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3a]quinolines and 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo-[4,3-a]quinoline-1-(2H)-ones derivatives were synthesized using 3-substituted-phenyl-N-phenyl-acrylamide as a starting material. Their anticonvulsant activity were evaluated by maximal electroshock (MES) test, subcutaneous pentylenetetrazol (scPTZ) test, and their neurotoxic effects were determined by the rotarod neurotoxicity test. Results. The compounds 4-substitued-phenyl-3,4-dihydro-2(1H)-quinolines (2a-f) had increased anticonvulsant effects compared to the parental compounds. The compounds 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolines (3a-f) had significantly increased anticonvulsant activity compared to 2a-f. However, the compounds 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline-1(2H)-ones(4a-f), exhibited no anticonvulsant effects even under a high dose of 300 mg/kg. Conclusions. The triazole, but not the triazolone, modified series showed stronger anticonvulsant effects than the parent compounds. Among them, compound (3f), 5-(p-fluorophenyl)-4,5–dihydro-1,2,4-triazolo[4,3-a]quinoline, showed the strongest anticonvulsant effect with ED50 of 27.4mg/kg and 22.0mg/kg in the anti-MES and anti-PTZ test, respectively.
Introduction
Epilepsy, a ubiquitous disease characterized by recurrent seizures, inflicts more than sixty million people worldwide according to epidemiological studies (1). The majority of antiepileptic drugs have been in use since 1985. They do not provide satisfactory seizure control in all patients and typically cause notable adverse side effects (2, 3). Research to find more effective and safer antiepileptic drugs, is, therefore, imperative and challenging in medicinal chemistry.
Quinoline derivatives have been known to possess a variety of biological activities such as antitumor (4), antimalarial (5), antiplatelet (6), antidepressant (7), antiulcer (8) and cardiac stimulant (9).
In our previous research on the positive inotropic activity of quinolines (10), 3,4-dihydro-2 (1H)-quinoline (compound I) showed a slight positive anticonvulsant activity with an effective dose of 300mg/kg in the anti-MES test. In order to obtain compounds with better anticonvulsant activity, we synthesized 4-substituted-phenyl-3,4-dihydro-2 (1H)-quinolines (2a-f) using 3,4-dihydro-2 (1H)-quinolione as the lead compound. The hypothesis was that the introduction of a substituted-phenyl into the 4-position of compound I would increase the lipophilic property of the compounds and increase their permeability to the blood-brain barrier which probably enhances their anticonvulsant activity. Subsequently, 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline (3a-f) and 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline-1(2H)-one (4a-f) derivatives were synthesized by incorporating a triazole or triazolone ring into N1-C2 positions of compounds 2a-f, hoping to increase their receptor binding and metabolic stability and, as a result, obtaining compounds with increased anticonvulsant activity. Similar designing approaches have also been reported (11-14).
For instance, when triazole or triazolone was incorporated into 4- and 5-positions of 1-aryl-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one, the anticonvulsant activity of the resulting compounds 11H-triazolo [4,5-c] [2,3] benzodiazepine and 11H-triazolo[4,5-c][2,3]benzodiazepine-3(2H)-ones increased remarkably.
The new compounds were evaluated as anticonvulsant agents in experimental epilepsy models, i.e., maximal electroshock test (MES) and subcutaneous pentylenetetrazol (sc-PTZ) induced seizure in mice.
They were also evaluated for neurotoxicity by the rotarod assay performed in mice.
INSTRUMENTS
Melting points were determined in open capillary tubes and were uncorrected. 1H-NMR and 13C-NMR spectra were measured on a AV-300 (Bruker, Switzerland), and all chemical shifts were given in ppm relative to tetramethysilane. Mass spectra were measured on an HP1100LC (Agilent Technologies , USA). Elemental analyses were performed on a 204Q CHN (Perkin Elmer , USA). Microanalyses of C, N, and H were performed using a Heraeus CHN Rapid Analyzer. The major chemicals were purchased from Alderich chemical corporation. All other chemicals were of analytical grade.
Materials and METHODS
Chemistry
Compounds 2a-f were prepared according to a reported procedure (15). Briefly, they were obtained by the cyclization of 3-substituted-phenyl-N-phenyl-acrylamide using polyphosphoric acid as a catalyst. Compounds 3a-f were prepared as reported (16,17) where the compounds 2a-f were sulfurizated with phosphorous pentasulfide and then cyclized with formhydrazide. Compounds 4a-f were prepared as reported (15,16) where the compounds 2a-f were sulfurizated with phosphorous pentasulfide and then cyclized with methyl hydrazinocarboxylate, as depicted in the following scheme.
The structures of the target compounds were confirmed by 1H-NMR, 13H-NMR, MS and elemental analysis techniques.
Preparation of compounds 2a-f
A mixture of 3-substituted-phenyl-N-phenyl-acrylamide (1.00g) and polyphosphoric acid (20g) was heated to 120oC. After 30 min, the reaction mixture was cooled and hydrolyzed over crushed ice. The reaction product was extracted with three 125 ml portions of dichloromethane. The extracts were combined and dried over anhydrous MgSO4. Evaporation of the solvents gave a crude product that was purified by recrystallization from ethanol. The yield, melting point and spectral data of each compound is given below.
4-Phenyl-3,4-dihydro-2 (1H)-quinolone (2a): Yield 70%, mp 180-18oC. 1H-NMR (CDCl3): 2.97 (m, 2H, J = 7.5 Hz, CH2), 4.35 ( t, H, J = 7.5 Hz, CH), 6.94-7.38 (m, 4H, C6H4-), 7.01-7.34 (m, 5H, C6H5-), 9.41 (s , 1H, NH). 13C-NMR (CDCl3 )δ: 38.40,42.02,115.76,123.30,126.65,127.20,127.81,127.99,128.33,128.89,137.12,141.52,170.89. MS : (M + 1) 224. Anal. Calcd. for C15H13NO: C, 80.69; H, 5.87; N, 6.27. Found: C, 80.41; H, 5.92; N, 6.34.
4-(4-Chlorophenyl)-3,4-dihydro-2 (1H)-quinolone (2b): Yield 93.4%, mp 180-182oC. 1H-NMR (CDCl3): 2.99 (m, 2H, J = 6.9 Hz, CH2), 4.38 (t, H, J = 6.9 Hz, CH), 6.95-7.37 (m, 4H, C6H4-), 7.02-7.35 (m, 4H, C6H4-), 9.50 (s, 1H, NH). 13C - NMR (CDCl3)δ: 38.34, 41.43, 115.86, 123.50, 126.05, 128.28, 128.98, 129.09, 129.14, 133.06, 137.00, 139.99, 170.53. MS: (M+1) 259. Anal. Calcd. for C15H12ClNO: C, 69.91; H, 4.69; N, 5.43. Found: C, 69.64; H, 4.49; N, 5.39.
4-(4-Methoxyphenyl)-3,4-dihydro-2 (1H)-quinolone (2c): Yield 73.5%, mp 138-140oC. 1H-NMR (CDCl3): 2.87 (m, 2H, J = 7.5 Hz, CH2), 4.28 (t, H, J = 7.5 Hz, CH), 3.76 (s , 1H, OCH3), 6.95-7.37 (m , 4H, C6H4-), 6.75-7.08 (m, 4H, C6H4-), 9.48 (s, 1H, NH). 13C-NMR (CDCl3)δ: 38.55, 41.21, 55.27, 114.28, 115.60, 123.34, 127.13, 127.92, 128.33, 128.80, 133.38, 136.93,158.71, 170.83. MS: (M+1) 254. Anal. Calcd. for C16H15NO2: C, 75.87; H, 5.97; N, 5.53. Found: C, 75.71; H, 5.69; N, 5.37.
Chemicals
Midazolam, hydroxymidazolam and bromazepam were a kind gift from Roche Laboratories (Neuilly-sur-Seine. The origin of testosterone, dextromethorphan, bupropion and related metabolites has been detailed in our previous reports (34-36). Ultrapure water was obtained using a Millipore Simplicity 185 apparatus. Dichloromethane, hexane, diethylether, and methanol (SDS, France) were of HPLC grade and used without further purification, and formic acid was from JT Backer (Ville, France). Tissue culture medium TC 199 (10 x concentrated with Earle's salts) and glutamine were purchased from Sigma Aldrich Chimie ( St Quentin Fallavier, France).
Gut sac preparation and incubation
Male Sprague Dawley rats (221-240 g weight, Depré, Saint Doulchard, France) were used in our experiments. Gut sacs were prepared and incubated with the target drugs at the stated concentrations as previously described (34-36). At the appropriate time points, sacs were removed, washed 3 times in saline and blotted dry, and the serosal contents collected. The sacs were digested individually in 25 ml of 1 M NaOH at 37°C for one hour, or in certain cases, homogenised in ultrapure water using an Ultra-turrax blender. The protein content of the digest or homogenate was measured by the method described by Peterson (37) with bovine serum albumin as standard. Samples of the mucosal and serosal fluids and the tissue digest were extracted for LC-MS analysis. From the volume of the sac contents and the quantity of each compound present, the appearance/transport was expressed as nanomoles or picomoles per mg of tissue protein.
Sample preparation
Midazolam (8.2 mg) was dissolved in 0.5 ml of methanol in a 125 ml flask and the volume was then completed with TC199 to give a 100µM solution. The final amount of methanol was 0.7 % (v/v) and 0.16 % (v/v) respectively for testosterone and midazolam in the solutions used for incubation.
The preparation of substrate solutions for incubation and the analytical procedures for testosterone, dextromethorphan and bupropion have been detailed in our earlier reports (34-36) for each compound. The analytical methods were all validated to quantify the substrates and their related metabolites in intestinal tissue after digestion in 1M NaOH. In 1 M NaOH, the extraction recovery was over 80% for midazolam and testosterone but it was only about 55% for dextromethorphan. As bupropion was not stable in 1M NaOH, the amount in the tissue was evaluated from a tissue homogenate. One ml of the homogenate was alkalinized with 20 µl of 1M NaOH and then extracted with 1 ml of diethylether. After manual agitation and centrifugation (5 min, 4000 x g), the organic layer was back extracted with 950 µl of water and 50 µl of formic acid before analysis of the aqueous layer (10µl). Extraction recoveries from homogenates were similar to those obtained in TC 199 (> 75%).
For testosterone, midazolam and dextromethorphan, the extraction procedure from intestine tissues was carried out after complete digestion of the tissue in 1M NaOH. In the experiments with testosterone, 0.5 ml of the resulting solution was neutralized with 43 µl of concentrated HCl, and 5 µl of progesterone (Internal Standard, 16 µM) and 0.375 ml of water were then added before extraction as previously described for serosal and mucosal media. After evaporation of the organic layer, the residue was dissolved in 1 ml of water and analysed by LC-MS, with the final concentration of Internal Standard (IS) at 0.8 µM.
For experiments with dextromethorphan, after complete digestion of the tissue in 1 M NaOH, 50 µl of Internal Standard (codeine, 10 µM) were added to 0.5 ml of the digest, then 28 µl of 1% acetic acid (pH 6) and 0.422 ml of water and the mixture extracted as previously reported for the serosal content (35). The residue was dissolved in 1 ml of water after evaporation of the organic layer and the final codeine concentration was 0.5µM.
For the midazolam experiments, 0.375 ml of TC 199 and 25 µl of bromazepam (6 µM) used as IS were added to 0.1 ml of serosal fluid, and the mixture was then extracted with 0.5 ml of a mixture of hexane/dichloromethane (1/1). After agitation, the organic layer was removed and evaporated to dryness under nitrogen and the residue dissolved in 0.1 ml of water for the quantification of midazolam and hydroxymidazolam. For the mucosal fluid, 50 µl was removed, diluted with 0.2 ml of TC 199 and added to 0.25 ml of the (IS) 6 µM bromazepam). Samples were then extracted as described for serosal fluid and after evaporation the residue was dissolved in 1 ml of water. The final concentration of the IS was 1.5 µM in all samples. When intestinal tissues were completely digested in 1M NaOH, 0.5 ml of the resulting solution was added to 25 µl of IS (6 µM) and 0.375 ml of water and acidified with 0.1 ml of acetic acid before extraction as previously described for serosal and mucosal media. After evaporation, the residue was dissolved in 1 ml of water before LC-MS analysis. The IS concentration was 1.5µM.
LC-MS Analysis
The LC-MS system consisted of a Waters 2690 separation module interfaced to a ZQ mass spectrometer equipped with an electrospray ionisation source (Waters, St Quentin, France). The apparatus was managed with a Masslynx software (Micromass, version 3.5). LC-MS analysis for testosterone, dextromethorphan and bupropion metabolism studies were run in positive mode as previously reported (34-36).
In experiments with midazolam, analyses were run in positive mode (electrospray) with capillary and cone voltages set to 3.5 KV and 35 V, the temperature of the heated capillary at 280° C and the nitrogen nebulizing gas flow at 360 L/h. The mobile phase consisted of a mixture of water/methanol/formic acid 1% (38/42/20) with a flow rate of 0.2 ml/min in isochratic conditions for a run time of 15 min. Quantifications were run in SIR mode by selecting the characteristic (M+H)+ ions: m/z = 326 for midazolam, m/z = 342 for 1-hydroxymidazolam and m/z = 315 for bromazepam used as internal standard at a concentration of 1.5 µM for all the quantifications. The retention times were 3.8 min, 5.8 min and 12.8 min respectively for midazolam, 1-hydroxymidazolam and bromazepam. Any matrix effect was detected by the analysis of TC199 without analytes after extraction.The linearity of the method was verified in the range of 0.025 µM to 3 µM for midazolam and hydroxymidazolam. Intra-day repeatability was determined by analysing, on the same day, a set of midazolam and 1-hydroxymidazolam solutions in TC199 medium at 0; 0.015; 0.025; 0.05; 0.25; 0.4; 1; 2; 3 µM. The solutions were analysed after extraction by the procedure previously described for sample preparation. Quality control (QC) solutions at three concentrations (0.06; 0.75 and 2.5 µM) were analysed three times each. The procedure used for intra-day repeatability was reproduced each day for five days to assess inter-day repeatability (n =12 assays). Intra-day standard deviation (RSD %) for the compounds assayed were between 3.5 % and 17.6 % for midazolam and between 3.1 % and 10.6 % for 1-hydroxymidazolam. Inter-day repeatability (RSD %) was between 5.3 % and 15.9 % for midazolam and between 4.5 % and 15.5 % for 1-hydroxymidazolam. Accuracy (% bias) was 10.56 % (0.06 µM); 4.89 % (0.75 µM) and 1.67 % (2.5µM) for midazolam and 2.50 % (0.06 µM), -2.44 % (0.75 µM) and -2.60 % (2.5 µM) for 1-hydroxymidazolam. Inter day accuracy (% bias ) was 7.64 % (0.06 µM); 7.56 % (0.75 µM) and 4.2 % (2.5 µM) for midazolam and 12.5 % (0.06 µM), -0.56% (0.75 µM) and -0.4 % (2.5 µM) for 1-hydroxymidazolam. The limits of quantification (LOQ, S/N > 10) were 0.0025 µM (4 ng injected) for midazolam and 0.025µM (40 ng injected) for 1-hydroxymidazolam respectively.
Stability
Calibration curves were constructed from stock solutions of substrates and metabolites stored at -20°C. Samples containing the different analytes prepared from the stock solutions were analysed periodically to verify the stability of the compounds. Stock solutions remained stable for all compounds for at least 4 months. Tissues were analysed immediately after complete digestion in 1 M NaOH, however the stability of testosterone, dextromethorphan and midazolam was satisfactory for 24 hours under these conditions.
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Synthesis of compounds 3a-f and 4a-f
4-(4-Methylphenyl)-3,4-dihydro-2 (1H)-quinolone (2d): Yield 74.6%, mp 154-156. 1H-NMR (CDCl3): 2.91 (m, 2H, J = 7.2 Hz, CH2), 4.36 (t, H, J = 7.2 Hz, CH), 2.54 (s,1H, CH3), 6.96-7.38 (m, 4H, C6H4-), 7.01-7.22 (m , 4H, C6H4-), 9.36 (s, 1H, NH). 13C-NMR (CDCl3)δ: 21.03, 38.46, 41.62, 115.64, 123.31, 126.96, 127.69, 127.92, 128.34, 129.58, 136.85, 137.01,138.39, 170.92. MS: (M+1) 238. Anal. Calcd. for C16H15NO2: C, 80.98; H, 6.37; N, 5.90. Found: C, 81.17; H, 6.16; N, 5.73.
4-(4-Fluorophenyl)-3,4-dihydro-2 (1H)-quinolone (2e): Yield 94%, mp 184-186oC. 1H-NMR (CDCl3): 3.01 (m, 2H, J = 6.9 Hz, CH2), 4.39 (t , H, J = 6.9 Hz, CH), 6.97-7.34 (m, 4H, C6H4-), 6.91-7.17 (m, 4H, C6H4-), 9.40 (s, 1H, NH). 13C-NMR (CDCl3)δ: 38.54, 41.30, 115.77 (d, 2JC-F = 21.0 Hz), 115.78, 123.46, 126.42, 128.19, 128.29, 129.30 (d, 3JC-F = 8.3 Hz), 136.97, 137.15, 161.92 (d, 1JC-F = 244.5 Hz) , 170.55. MS: (M+1)228. Anal. Calcd. for C15H12FNO: C, 74.67; H, 5.01; N, 5.81. Found: C, 74.91; H, 4.91; N, 5.97.
4-(3-Fluorophenyl)-3,4-dihydro-2 (1H)-quinolone (2f): Yield 64.5%, mp 177-178oC. 1H-NMR (CDCl3): 3.02 (m, 2H, J = 7.2 Hz, CH2), 4.39 (t , H , J = 7.2 Hz, CH), 6.95-7.34 (m, 4H, C6H4-), 6.75-7.20 (m, 4H, C6H4-), 9.39 (s, 1H, NH ). 13C-NMR (CDCl3)δ: 38.28, 41.76, 114.88, 114.85 (d, 2JC-F = 21.0 Hz), 115.73, 116.48, 123.45, 128.92, 129.67, 129.93, 130.42 (d, 3JC-F = 8.3 Hz), 130.60, 137.02, 163.06 (d, 1JC-F = 244.5 Hz), 169.90. MS: (M+1) 228. Anal. Calcd. for C15H12FNO: C, 74.67; H, 5.01; N, 5.81. Found: C, 74.78; H, 4.89; N, 5.89.
Preparation of compounds 3a-f.
Acetonitrile (60 ml) and triethylamine (40 ml) were placed in a three-necked round-bottomed flask, to which P2S5 (9.99g, 0.06 mol) was added slowly in an ice bath and stirred until dissolved. Then 4-substituted-phenyl-3,4-dihydro-2(1H)-quinolones (0.04 mol) was added while stirring. The mixture was refluxed for 5 h in a nitrogen atmosphere. After removing the solvent under reduced pressure, the residue was dissolved in 120 ml of dichloromethane, washed with water (120 × 3), and dried over anhydrous MgSO4. Evaporation of the solvents gave a crude product, then formhydrazide (2.4g, 0.04 mol), n-butanol (80 ml) and acetic acid (0.5 ml) were added, and the mixture was refluxed for 20 h in a nitrogen atmosphere. Solvents were removed under reduced pressure, and the residue was extracted twice with dichloromethane (60 ml). The dichloromethane layer was washed three times with matured sodium chloride (60 × 3) and dried over anhydrous MgSO4. After removing the solvents, products were purified by silica gel column chromatography (dichloromethane: methanol = 20:1). The yield, melting point and spectral data of each compound is given below.
5-Phenyl-4,5-dihydro-1,2,4- triazolo[4,3-a]quinoline (3a): Yield 82.7%, mp 140-142oC. 1H-NMR (CDCl3): 3.45 (m, 2H, J = 8.0 Hz, CH2), 4.34 (t, H, J = 8.0 Hz, CH), 7.10-7.42 (m, 4H, C6H4-), 7.07-7.25 (m, 4H, C6H5-), 8.69 (s, 1H, H-1). 13C-NMR (CDCl3)δ: 28.54, 42.44, 116.30, 127.42, 127.71, 127.85, 128.71, 129.08, 129.98, 130.49, 131.88, 137.46, 140.70, 149.65. MS: (M+1) 248. Anal. Calcd. for C16H13N3: C, 77.71; H, 5.30; N, 16.99. Found: C, 77.89; H, 5.08; N, 17.28.
5-(4-Chlorophenyl)-4,5-dihydro-1,2,4- triazolo[4,3-a]quinoline (3b): Yield 78.7%, mp 176-178oC. 1H-NMR (CDCl3): 3.48 (m, 2H, J = 8.0 Hz, CH2), 4.37 (t, H, J = 8.0 Hz, CH), 7.11-7.51 (m, 4H, C6H4-), 7.08-7.28 (m, 4H, C6H4-), 8.70 (s, 1H, H-1). 13C-NMR (CDCl3)δ: 28.47, 41.83, 116.47, 127.40, 128.86, 129.16, 129.25, 129.84, 130.14, 131.81, 133.50, 137.50, 139.18, 149.32. MS: (M+1) 284. Anal. Calcd. for C16H12ClN3: C, 68.21; H, 4.29; N, 14.91. Found: C, 68.46; H, 4.64; N, 15.10.
5-(4-Methoxyphenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline (3c): Yield 63.2%, mp 188-190oC. 1H-NMR (CDCl3): 3.46 (m, 2H, J = 6.9 Hz, CH2), 4.33 ( t, H, J = 6.9 Hz, CH ), 3.80 (s, 3H, OCH3), 7.11-7.49 (m, 4H, C6H4-), 6.84-7.28 (m, 4H, C6H4-), 8.68 (s,1H, H-1). 13C-NMR (CDCl3)δ: 28.65, 41.66, 55.28, 114.09, 116.27, 127.22, 128.49, 128.86, 129.91, 130.88, 131.82, 132.62, 137.44, 149.76. 158.95. MS: (M+1) 278. Anal. Calcd. for C17H15N3O: C, 73.63; H, 5.45; N, 15.15. Found: C, 73.39; H, 5.71; N, 14.89.
5-(4-Methylphenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline (3d): Yield 81.6%, mp 158-160oC. 1H-NMR (CDCl3): 3.47 (m , 2H, J = 7.5 Hz, CH2), 4.34 (t, H, J = 7.5 Hz, CH), 2.34 (s, 3H, CH3), 7.13-7.49 (m, 4H, C6H4-), 7.01-7.28 (m, 4H, C6H4-), 8.69 (s, 1H, H-1). 13C-NMR (CDCl3)δ: 21.02, 28.54, 42.03, 116.81, 126.87, 127.71, 128.06, 128.51, 129.27, 129.72, 130.73, 131.82, 137.47, 139.28, 149.76. MS: (M+1) 262. Anal. Calcd. for C17H15N3: C, 78.13; H, 5.79; N, 16.08. Found: C, 77.88; H, 5.95; N, 15.86.
5-(4-Fluorophenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline (3e): Yield 72.8%, mp 168-169oC. 1H-NMR (CDCl3): 3.49 (m, 2H, J = 7.5 Hz, CH2), 4.38 ( t, H, J = 7.5 Hz, CH), 7.10-7.51 (m, 4H, C6H4-), 6.92-7.28 (m, 4H, C6H4-), 8.69 (s, 1H, H-1). 13C-NMR (CDCl3)δ: 28.64, 41.70, 115.28 (d, 2JC-F = 21.0 Hz), 115.83, 116.11, 116.44, 127.37, 129.19, 129.38 (d, 3JC-F = 8.3 Hz), 129.85, 130.22, 131.80, 136.44, 162.07 (d, 1JC-F = 245.3 Hz) . MS: (M+1) 266. Anal. Calcd. for C16H12FN3: C, 72.44; H, 4.56; N, 15.84. Found: C, 72.71; H, 4.32; N, 16.12.
5-(3-Fluorophenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline (3f): Yield 54.5%, mp 158-160oC. 1H-NMR (CDCl3): 3.50 (m, 2H, J = 6.9 Hz, CH2), 4.39 (t, H , J = 6.9 Hz, CH), 7.12-7.52 (m, 4H, C6H4-), 6.81-7.29 (m, 4H, C6H4-), 8.70 (s,1H, H-1) . 13C-NMR (CDCl3)δ: 28.45, 42.10, 114.56 (d, 2JC-F = 18.8 Hz), 114.85 (d, 2JC-F = 18.8 Hz), 116.50, 123.45, 123.48, 127.40, 128.92, 129.91 (d, 3JC-F = 8.3 Hz), 130.58 (d, 3JC-F = 8.3 Hz), 130.69, 131.41, 131.85, 143.31, 163.05 (d, 1JC-F = 245.3 Hz) . MS: (M+1) 266. Anal. Calcd. for C16H12FN3: C, 72.44; H, 4.56; N, 15.84. Found: C, 72.63; H, 4.32; N, 16.06.
Preparation of compounds 4a-f.
To 4-substituted-phenyl-3,4-dihydro-2(1H)-quinolione-thiones (0.04mol),prepared according to the method described by Zappala et al. (13) methyl hydrazinocarboxylate (2.4g, 0.04 mol), n-butanol (80 ml), and acetic acid (0.5ml) was added and the mixture was refluxed for 60 h in a nitrogen atmosphere. Solvents were removed under reduced pressure, and the residue was extracted twice with dichloromethane (60 ml). The dichloromethane layer was washed three times with matured sodium chloride (60 × 3) and dried over anhydrous MgSO4. After removing the solvents, products were purified by silica gel column chromatography (dichloromethane: methanol = 20:1). The yield, melting point and spectral data of each compound are given below.
5-Phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-one (4a): Yield 65.13%. mp 202-204oC. 1H-NMR (CDCl3): 3.20 (m, 2H, J = 6.6 Hz, CH2), 4.35 (t, H, J = 6.6 Hz, CH),7.19-8.46 (m, 4H, C6H4-), 7.01-7.29 (m, 4H, C6H5-), 9.82 (s, 1H, N-H). 13C-NMR (CDCl3)δ: 29.60, 42.38, 114.42, 117.40, 126.09, 127.46, 127.74, 128.49, 128.89, 129.02, 132.71, 140.83, 143.71, 153.13. MS: (M+1) 264. Anal. Calcd. for C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C, 72.76; H, 5.26; N, 15.75.
5-(4-Chlorophenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-one (4b): Yield 52.3%, mp 256-258oC. 1H-NMR (CDCl3): 3.18 (m, 2H, J = 6.5 Hz, CH2), 4.32 (t, H, J = 6.5 Hz, CH),6.92-8.45 (m, 4H, C6H4-), 7.04-7.21 (m, 4H, C6H4-) , 9.67 (s, 1H, N-H). 13C-NMR (CDCl3)δ: 29.57, 41.77, 117.51, 126.24, 128.39, 128.75, 128.88, 129.06, 129.20, 130.10, 132.60, 139.28, 143.38 , 152.81. MS: (M+1) 298. Anal. Calcd. for C16H12ClN3O: C, 64.54; H, 4.06; N, 14.11. Found: C, 64.46; H, 4.29; N, 13.93.
5-(4-Methoxyphenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-one (4c): Yield 57.8%, mp 206-208oC. 1H-NMR (CDCl3): 3.18 (m, 2H, J = 6.9 Hz, CH2), 4.31 (t, H, J = 6.9 Hz, CH),3.80 (s, 3H, OCH3), 6.88-8.45 (m, 4H, C6H4-), 6.85-7.15 (m, 4H, C6H4-), 9.88 (s, 1H, N-H). 13C-NMR (CDCl3)δ: 29.74, 41.59, 55.28, 114.39, 117.37, 126.08, 128.40, 128.75, 128.91, 129.40, 132.61, 132.75, 143.88, 152.99, 158.83. MS: (M+1) 294. Anal. Calcd. for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found: C, 69.49; H, 5.36; N, 14.12.
5-(4-Methylphenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-one (4d): Yield 63.4%, mp 188-190oC. 1H-NMR (CDCl3): 3.20 (m, 2H, J = 6.6 Hz, CH2), 4.32 (t, H, J = 6.6 Hz, CH),2.35 (s, 3H, CH3), 7.00-8.47 (m, 4H, C6H4-), 7.02-7.20 (m, 4H, C6H4-), 9.99 (s, 1H, N-H). 13C-NMR (CDCl3)δ: 21.01, 29.66, 42.00, 117.36, 126.05, 127.60, 128.40, 128.94, 129.27, 129.68, 132.69, 137.13, 137.79, 143.82, 153.09. MS: (M+1) 278. Anal. Calcd. for C17H15N3O: C, 73.63; H, 5.45; N, 15.15. Found: C, 73.83; H, 5.36; N, 15.32.
5-(4-Fluorophenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-one (4e): Yield 51%, mp 255-258oC. 1H-NMR (CDCl3): 3.21 (m, 2H, J = 6.6 Hz, CH2), 4.36 (t, H, J = 6.6 Hz, CH),6.92-8.47 (m, 4H, C6H4-), 6.88-7.17 (m, 4H, C6H4-), 10.10 (s, 1H, N-H). 13C-NMR (CDCl3)δ: 29.73, 41.66, 115.28 (d, 2JC-F = 21.0 Hz), 115.77, 116.06, 117.50, 126.19, 128.75, 129.32 (d, 3JC-F = 8.3 Hz), 132.61, 136.55, 143.48, 152.98, 162.01 (d, 1JC-F = 245.3 Hz). MS: (M+1) 282. Anal. Calcd. for C16H12FN3O: C, 68.32; H, 4.30; N, 14.94. Found: C, 68.06; H, 4.56; N, 14.76.
5-(3-Fluorophenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-one (4f): Yield 50.4%, mp 224-226oC. 1H-NMR (CDCl3 ): 3.21 (m, 2H, J = 6.5 Hz, CH2), 4.37 (t, H, J = 6.5 Hz, CH),6.97-8.49 (m, 4H, C6H4-), 6.81-7.18 (m, 4H, C6H4-), 10.26 (s, 1H, N-H). 13C-NMR (CDCl3) δ: 29.44, 42.07, 114.50 (d, 2JC-F = 21.8 Hz), 114.79 (d, 2JC-F = 21.8 Hz), 117.52, 123.35, 126.22, 128.23, 128.79, 128.92, 130.52 (d, 3JC-F = 8.3 Hz), 130.63 (d, 3JC-F = 8.3 Hz), 132.64, 143.42, 152.12, 163.08 (d, 1JC-F = 246.0 Hz). MS: (M+1) 282. Anal. Calcd. for C16H12FN3O: C, 68.32; H, 4.30; N, 14.94. Found: C, 68.46; H, 4.21; N, 14.86.
Anticonvulsant Tests
The MES test, sc-PTZ test, and rotarod test were carried out by the Antiepileptic Drug Development Program (ADD), Epilepsy Branch, National Institutes of Health, Bethesda, MD, USA (18,19). All compounds were tested for anticonvulsant activity with C57B/6 mice in the 18-25g weight range purchased from the Laboratory of Animal Research, College of Pharmacy, Yanbian University. The tested compounds were dissolved in polyethylene glycol-400.
In Phase I screening (Table 1) each compound was administered at three dose levels (30, 100, and 300 mg/kg i.p., 3 mice for each dose) with anticonvulsant activity and neurotoxicity assessed at 30 min and 4 h intervals after administration. Anticonvulsant efficacy was measured in the MES test and the sc-PTZ test. In the MES test, seizures were elicited with a 60 Hz alternating current of 50 mA intensity in mice. The current was applied via corneal electrodes for 0.2 s. Abolition of the hind-leg tonic-extensor component of the seizure indicated protection against the spread of MES-induced seizures. The sc-PTZ test involved subcutaneous injection of a convulsant dose (CD97) of pentylenetetrazol (85mg/kg in mice). Elevation of the pentylenetrazol-induced seizure threshold was indicated by the absence of clonic spasms for at least 5 s duration over a 30 min period following administration of the test compound. Anticonvulsant drug-induced neurologic deficit was detected in mice using the rotorod ataxia test.
The pharmacologic parameters estimated in phase I screening was quantified for compounds 3a-f in phase II screening (Table 2). Anticonvulsant activity was expressed in terms of the median effective dose (ED50), and neurotoxicity was expressed as the median toxic dose (TD50). For determination of the ED50 and TD50 values, groups of 10 mice were given a range of intraperitoneal doses of the test drug until at least three points were established in the range of 10-90% seizure protection or minimal observed neurotoxicity. From the plot of this data, the respective ED50 and TD50 values, 95% confidence intervals, slope of the regression line, and the standard error of the slope were calculated by means of a computer program written at National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA.
Results of the initial anticonvulsant activity evaluation phase I of the synthesized compounds are presented in Table 1. Based on the result of the preliminary screening, compounds 3a-3f were selected for phase II tests, the results of which are shown in Table 2.
Table 1. Phase I mouse anticonvulsant activity (i.p.)
Compd. |
Dosage |
MESa |
scPTZb |
Rotarodc |
|||
0.5h 4h |
0.5h 4h |
0.5h 4h |
|||||
1 |
300 |
3/3 |
0/3 |
3/3 |
0/3 |
3/3 |
1/3 |
2a |
100 |
2/3 |
0/3 |
2/3 |
0/3 |
0/3 |
0/3 |
2b |
100 |
2/3 |
0/3 |
2/3 |
0/3 |
0/3 |
0/3 |
2c |
100 |
2/3 |
0/3 |
2/3 |
0/3 |
0/3 |
0/3 |
2d |
100 |
1/3 |
0/3 |
1/3 |
0/3 |
0/3 |
0/3 |
2e |
100 |
2/3 |
0/3 |
2/3 |
2/3 |
0/3 |
0/3 |
2f |
100 |
2/3 |
0/3 |
2/3 |
2/3 |
0/3 |
0/3 |
3a |
100 |
3/3 |
1/3 |
3/3 |
1/3 |
1/3 |
0/3 |
3b |
100 |
3/3 |
1/3 |
3/3 |
1/3 |
2/3 |
0/3 |
3c |
100 |
3/3 |
1/3 |
3/3 |
1/3 |
0/3 |
0/3 |
3d |
100 |
2/3 |
0/3 |
2/3 |
0/3 |
0/3 |
0/3 |
3e |
30 |
1/3 |
1/3 |
1/3 |
1/3 |
0/3 |
0/3 |
3f |
30 |
2/3 |
2/3 |
2/3 |
2/3 |
0/3 |
0/3 |
4a |
300 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
4b |
300 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
4c |
300 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
4d |
300 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
4e |
300 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
4f |
300 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
b Subcutaneous pentylenetetrazol test (number of animals protected / number of animals tested).
c Rotorod toxicity (number of animals exhibiting toxicity / number of animals tested).
Table 2. Phase II quantitative anticonvulsant data in mice (Test drug administered i.p.)
Compd. |
ED50a |
Rotarod Toxicity |
PIb |
||
MES |
scPTZ |
TD50c |
MES |
scPTZ |
|
3a |
54.8 (46.3-64.8) e |
42.4 (35.9-50.2) |
141.6 (123.70-161.6) |
2.58 |
3.34 |
3b |
45.6 (39.2-53.1) |
50.9 (42.2-61.5) |
131.2 (109.4-157.5) |
2.88 |
2.58 |
3c |
61.1 (51.3-72.8) |
47.3 (39.7-56.4) |
136.3 (118.2-157.3) |
2.23 |
2.88 |
3d |
84.9 (75.8-95.0) |
84.8 (72.3-99.5) |
203.6 (172.2-240.8) |
2.40 |
2.40 |
3e |
32.9 (27.8-38.9) |
27.4 (22.7-33.1) |
98.2 (85.1-113.3) |
2.99 |
3.58 |
3f |
27.4 (23.4-32.2) |
22.0 (18.5-26.2) |
91.3 (79.1-105.3) |
3.33 |
4.15 |
Phenytoind |
9.5 (8.1-10.4) |
＞300 |
65.5 (52.5-72.9) |
6.9 |
＜0.22 |
Carbamazepind |
8.8 (5.5-14.1) |
＞100 |
71.6 (45.9-135) |
8.1 |
＜0.22 |
phenobarbitald |
21.8 (21.8-25.5) |
13.2 (5.8-15.9) |
69.0 (62.8-72.9) |
3.2 |
5.2 |
Valproated |
272 (247-338) |
149 (123-177) |
426 (369-450) |
1.6 |
2.9 |
a Dose measured in mg/kg. b PI = TD50 / ED50
c Minimal neurotoxicity was determined by the rotarod test 30 min after the tested compounds were administrated.
d Data from Huseyin, U., et al. 1998 18
e The 95% confidence limits
Discussion
The 4-substituted-phenyl-3,4-dihydro-2(1H)-quinolines (2a-f) showed remarkable anticonvulsant activity. Compound I indicated anti-MES effect only under the high dose of 300 mg/kg, whereas compounds 2a-f showed anti-MES and anti-PTZ effect at the medium dose of 100 mg/kg. We reason that the increase in anticonvulsant activity might be due to their easier transport across biological membranes after the introduction of the substituted-phenyl at the fourth position of compound I.
The 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolines (3a-f) were prepared by incorporating a triazole group into 2a-f at the N1-C2 position, which caused stronger anticonvulsant effect. Significantly, compounds 3e and 3f showed anticonvulsant effect at the low dose of 30 mg/kg.
Based on the result of the preliminary screening, compounds 3a-f were selected for phase II tests, where their anticonvulsant activity and neurotoxicity in mice were quantified and expressed in terms of median effective dose (ED50) and median neurotoxic dose (TD50). As shown in Table 2, ED50 of the anti-MES activity of the six compounds were between 27.4－84.9 mg/kg. The structure-activity relationships were concluded as following Introduction of electron donor groups such as methyl or methoxyl to the phenyl ring reduced anticonvulsant activity, whereas introduction of electron-acceptor groups such as chlorine or fluorine to the phenyl ring increased anticonvulsant activity. For example, the activity of compounds 3c, 3d (with donor groups introduced) was lower than that of 3a, while the anti-MES effect of 3b, 3e and 3f (with electron acceptor groups introduced) was higher than that of 3a. The activity was the strongest for the two fluorine introduced compounds where the m-F derivative (3f) had higher activity than the p-F derivative (3e). Compound 3f had an ED50 of 27.4 mg/kg in the anti-MES test, which was higher than the control drug valproate, but was lower than phenytoin, carbamazepin and phenobarbital.
Compounds 3a-f also possessed strong anti-PTZ effect, which was a little higher than their anti-MES effect. The strongest compound was still 3f with ED50 of 22.0 mg/kg, which was stronger than the control drugs phenytoin, carbamazepin and valproate, and only weaker than phenobarbital. scPTZ has been reported to produce seizures by inhibiting gamma-aminobutyic acid (GABA) neurotransmission (20, 21). GABA is the main inhibitory neurotransmitter substance in the brain, and is widely implicated in epilepsy. Inhibition of GABAergic neurotransmission or activity has been shown to promote and facilitate seizures (22-24), while enhancement of GABAergic neurotransmission is known to inhibit or attenuate seizures. The standard anticonvulsant drugs used have also been shown to exert their anticonvulsant action by enhancing GABAergic neurotransmission and activity (23). The findings of the present study tend to suggest that the derivatives in this study might have inhibited or attenuated PTZ-induced seizures in mice by enhancing GABAergic neurotransmission.
The protective index (PI) value of compounds 3a-f was 2.23-3.33 in MES test, among which compound 3f had the best PI value of 3.33, which was better than control the drugs valproate and phenobarbital, but was lower than phenytoin and carbamazepin.
The 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolin-1(2H)-ones (4a-f) were obtained by incorporating a triazolone group into N1-C2 positions of compounds 2a-f, respectively However, none of these compounds showed anticonvulsant effect even at the high dose of 300 mg/kg, which was very much unexpected. It is possible, however, that triazolone incorporation into the substituted quinoline led to dramatic reduction in the lipophilicity of the compounds and made them difficult to pass biological membranes.
CONCLUSION
The 4-substitued-phenyl-3,4-dihydro-2(1H)-quinolines (2a-f), synthesized by introducing substituted-phenyl to 3,4-dihydro-2(1H)-quinoline at the fourth position, had remarkably increased anticonvulsant effects compared to the parent compounds. The 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolines (3a-f), prepared by incorporating a triazole ring into 2a-f at the N1-C2 positions, in turn had significantly increased anticonvulsant activity compared to 2a-f. However, the compounds prepared by incorporating a triazolone into 2a-f at the N1-C2 positions, namely, 5-substituted-phenyl-l-4,5-dihydro- 1,2,4-triazolo[4,3-a] quinoline-1(2H)-ones（4a-f）,exhibited no anticonvulsive effect even under a high dose of 300 mg/kg.
ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation of China (No. 30460151)