SYNTHESIS AND BIOLOGICAL EVALUATION OF SCHIFF BASES DERIVED FROM P-DIMEHTYLAMINOBENZALDEHYDE

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1 Journal of Cell and Tissue Research Vol 8(2) (2008) ISS: (Available online at wwwtcrjournalscom) Original Article SYTHESIS AD BIOLOGICAL EVALUATIO OF SCHIFF BASES DERIVED FROM P-DIMEHTYLAMIOBEZALDEHYDE JAI, J, MASAD,, SIHA, R, GARG, V K AD PATIL, V Department of Pharmaceutical Technology, Meerut Institute of Engineering and Technology, Meerut , India E mail: neerajmasand@rediffmailcom Received: April 2, 2008; Accepted: April 25, 2008 Abstract: A series of Schiff bases have been synthesized by condensation of p- Dimehtylaminobenzaldehyde with the corresponding aromatic primary amines Their IR and 1 H MR spectra have been obtained All the synthesized Schiff bases were evaluated for their anticonvulsant activity by maximal electroshock (MES) test and their neurotoxicity effects were determined by the rotorod neurotoxicity test at 30, 100 and 300 mg/kg dose levels at two different time intervals Acute toxicity study and CS depressant activity of these compounds were also screened Compound 1, 2, 3, 6 and 7 were found to be active in MES test Majority of the compounds have shown activity after 05 h Compounds 2 and 6 showed activity after 4 h The compounds 1, 3, 4 and 6 showed neurotoxicity at 300 mg/kg (ip) All tested compounds have shown CS depressant effect of varying degrees at the dose of 1/5 th of LD 50 Key words: Schiff bases, p-dimehtylaminobenzaldehyde? ITRODUCTIO Schiff bases are potentially active compounds and have been reported to possess anti-inflammatory, analgesic [1-4], antifungal, antibacterial [5-9], anticonvulsant [10-12] and antioxidant [13] activities Schiff bases derived from various heterocycles were reported to possess cytotoxic activity [14-16] With this envision in mind, we synthesized a series of Schiff bases derived from p-dimehtylaminobenzaldehyde by condensation with the appropriate aromatic amines The structures of the synthesized compounds were identified using spectroscopic techniques All the compounds were screened for anticonvulsant properties by maximal electroshock (MES) test and neurotoxicity by rotorod test Acute toxicity study and CS depressant activity of these compounds were also screened The CS depressant activity was evaluated by locomotor activity and pentobarbital sleeping time methods All tested compounds have shown CS depressant effect of varying degrees MATERIALS AD METHODS Instruments: The melting point of the compounds was recorded on Perfit Digital Melting Point Apparatus and was uncorrected The purity of the compounds was checked by thin layer chromatography using silica gel G as stationary phase IR spectra were recorded in KBr disc on Shimadzu FTIR-8400S 1 H MR spectra were recorded in CDCl 3 at 400 MHz on BRUKER Spectrometer using tetramethylsilane as internal standard itrogen estimation was determined using Kjeldahl apparatus All chemicals were purchased from Sigma Aldrich Chemical Corporation Preparation of compounds: Compounds 1-7 were prepared according to a reported procedure as shown in Scheme 1 [10] The Schiff bases were prepared by condensation of p-dimehtylaminobenzaldehyde with the corresponding primary aromatic amines using a mixture of glacial acetic acid and hydrobromic acid as catalyst The nitrogen estimation was carried out using Kjeldahl method and the results are comparable 1431

2 J Cell Tissue Research HC O HC Ar + Ar H 2 Gla Ace acid/hbr H 3 C CH 3 H 3 C CH 3 Scheme 1: Compound 1-7 Synthesis of Schiff bases General procedure Compd o Ar Molecular Formula Rf itrogen % Calcd(found) log P Recrystallisation Solvent 1 4-Bromophenyl C 15H 15 2Br 046 a 927 (918) Chloroform 2 4-itrophenyl C 16H 16 2O 062 a ) Methanol 3 4-Methylphenyl C 16H b 1176 (1162) Ether 4 4-Chlorophenyl C 15H 15 2Cl 043 c 1085 (1075) Ether 5 1-aphthalenyl C 19H a 1021 (1014) Ether 6 4-Hydroxyphenyl C 15H 16 2O 020 d 1166 (1159) Ether 7 4-Methoxyphenyl C 16H 18 2O 011 b 1102 (1098) Ether Table 1: Physical properties of PDAB Schiff bases a Chloroform: Ethyl acetate: Benzene (45:02:1), b Chloroform: Benzene: Methanol (1:1:02), c Chloroform: Ethyl acetate: Benzene (35:02:1), d Chloroform: Petroleum ether (1:05) to analytically calculated values The structures of the target compounds were confirmed by IR and 1 H MR techniques Synthesis of Schiff bases: Equimolar quantities of p-dimethylaminobenzaldehyde (001 mol) and primary aromatic amines (001 mol) were dissolved in 10 ml of absolute ethanol in a round bottom flask The ph of the reaction mixture was maintained between 2-3 using a mixture of 4 ml of glacial acetic acid and 2 drops of hydrobromic acid The completion of reaction was monitored with TLC using silica gel G After cooling at room temperature, the reaction mixture was basified with aqueous ammonia solution The precipitate was collected and further washed with dil HCl to remove excess amine It was dried at room temperature and recrystallized from various solvents as specified in table 1 Compound o 1: % Yield= 6623, mp C, IR (KBr): 2904,2821,2794, 1687, 1593, 1548, 1531, 1485, 1313, 825, H MR (CDCl 3 ) (400MHz): δ 304 (6H, s), (2H, d), (2H, d), (2H, d), (2H, d), (1H, s) Compound o 2: % Yield= 7677, mp C, IR (KBr): 3055, 2923, 1668, 1596, 1514, 1442, 1360, H MR (CDCl 3 ) (300MHz): δ 311 (6H, s), (4H, m), (2H, d), (2H, d), 901 (1H, s) Compound o 3: % Yield= 6497, mp C, IR (KBr): 2917, 2871, 1659, 1585, 1544, 1438, 1363, H MR (CDCl 3 ) (300MHz): δ 235 (3H, s), 310 (6H, s), (6H, d), (6H, d), (4H, m), 827 (1H, s) Compound o 4: % Yield= 6876, mp C, IR (KBr): 2926, 2862, 1668, 1600, 1541, 1488, 1315, 829, H MR (CDCl 3 ) (300MHz): δ 314 (6H, s), (6H, d), (6H, d), (4H, m), 817 (1H, s) Compound o 5: % Yield= 5762, mp >330 0 C, IR (KBr): 3055, 3022, 2960, 2867, 1643, 1600, 1510, 1470, 1367, 836 Compound o 6: % Yield= 65, mp C, IR (KBr): 2919, 2824, 1662, 1600, 1553, 1534, 1375, 1315, 1432

3 Table 2: Anticonvulsant and neurotoxicity screening of synthesized compounds 1-7 Doses of 30, 100 and 300 mg/kg were administered and the protection and neurotoxicity were measured after 05 and 4 h The figures in the table indicate the minimum dose at which protection or neurotoxicity was demonstrated in mice The (-) indicates an absence of activity/ neurotoxicity at maximum dose administered (300 mg/kg) Compound o Intraperitoneal Injection in mice MES screen T screen 05 h 4 h % Mortality 05 h 4 h Phenytoin Table 3: Influence of compounds on the spontaneous locomotor activity in mice (n=6) a P<005, b P<002, c P<001, d P<0001; as compared to control, n=6 Jain et al Table 4: Effect of test compounds on Pentobarbital-sleeping time in mice * P<005 compared with control group, # P<0001 compared with control group (Student s t-test) (n= 6) Compound o Control (Pentobarbitone Sodium) Dose mg/kg, ip Mean sleeping time ± SEM (min) ± ± ± 369* ± ± 301* ± ± 055 # ± ± 218* ± ± 431* ± ± 416* ± ± 455 # (6H, d), (6H, d), (4H, m), 812 (1H, s) Group Dose mg/kg (ip) 30 min Mean no of impulses ± SEM 1 h Mean no of impulses ± SEM Control ± ±1183 Diazepam 1 101±617 d 4083±377 d ±436c 34533±336d ±448d 11966±358d ±299d 87±330d ±482 c 35216±506 d ± ±278 d ± ±351 d ±355 d 37366±325 b ±343 d 14616±381 d ±279 d 9883±468 d ±503a 36516±568 a ±384 d 1285±362 d ±555 d 8983±390 d ±283 a 3765±347 b ±348 d 1355± ±292 d 100± ±531 a 3625±432 b ±423 d 9333±206 d ±306 d 7183±300 d ± ±293 b ±453 d 1375±343 d ±384 d 10183±311 d 1168, H MR (CDCl 3 ) (300MHz): δ 308 (6H, s), (4H, m), (4H, m), 973, 1001 (2H, merged with each other) Compound o 7: % Yield= 6433, mp C, IR (KBr): 2918, 2794, 1660, 1600, 1537, 1410, 1373, 1232, 1166, H MR (CDCl 3 ) (300MHz): δ 225 (3H, s), 376 (3H, s), 309 (6H, s), Pharmacological experimental: The experiments were carried out on male and female Wistar-albino mice (body weight of g) in CPCSEA registered animal house (711/02/a/CPCSEA) of the institute The animals were randomly allocated to treatment groups (six animals per group, per treatment) Compounds were administered intraperitoneally (ip) as a suspension in 2%w/v Tween-80 in a volume of 10 ml/kg [17] All the compounds were subjected to: i) Acute toxicity in mice, ii) Anticonvulsant screening and iii) CS depressant activity For later, the compounds were administered in doses equivalent to 1/20, 1/10 and 1/5 th of LD 50, whereas for anticonvulsant screening the test compounds were administered at the doses of 30, 100 and 300 mg/kg Acute toxicity: Acute toxicity was assessed by the Miller and Tainter [18] method and presented as LD 50 The correction factor was applied to 0 and 100 per cent mortality group The per cent mortality values were converted to probit values The probit values were plotted against log doses and LD 50 was calculated as the dose corresponding to probit 5 [18] Anticonvulsant screening: All the compounds were screened for anticonvulsant properties adopting the maximal electroshock (MES) and neurotoxicity screening methods [19] The compounds were administered intraperitoneally to mice, in doses of 30, 100 and 300 mg/kg The anticonvulsant activity was observed at 05 and 40 h duration after the 1433

4 J Cell Tissue Research administration of test compounds Maximal electroshock method: Maximal electroshock was induced by the application of electrical current to the brain via corneal electrodes The stimulus parameter for mice was 50 ma in a pulse of 60 Hz for 02 sec Abolition of the hind limb tonic extensor spasm was recorded as a measure of anticonvulsant activity Rotorod test: The mice were trained to stay on an accelerating rod of diameter 32 cm, rotating at a speed of 10 revolutions per minute Trained animals were selected and administered with test compounds after 05 and 40 h duration eurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one minute in each of the three trials (Table 2) Locomotor activity: Spontaneous locomotor activity in mice was monitored using actophotometer After the intraperitoneal injection of the investigated compounds and standard diazepam (1mg/kg, ip), animals were placed in the actophotometers for a period of 10 min Locomotor activity was recorded and expressed in terms of total photo beam counts for 10 min per animal at 30 and 60 min intervals [20] For details see table 3 Pentobarbital sleeping time method: The experiment was carried out in a quiet room Mice were grouped of six each and basal activity score of all mice was recorded Control and test groups received pentobarbitone sodium (45 mg/kg, ip) 30 min after the intraperitoneal administration of saline and test compounds respectively The effects were recorded as follows: Time elapsed between the administrations of pentobarbital until loss of righting reflex was recorded as the onset of sleep, while the time from the loss of its recovery was considered as the duration of sleep The test compounds were compared against control [21] (Table 4) Statistics: Results obtained were presented as mean ± SEM and evaluated statistically using Student s T- test P<005 was the significant criterion RESULTS The compounds have shown comparable percentage of nitrogen to analytically calculated content All synthesized compounds have shown characteristic - C= stretch in the region of cm -1, Aromatic ring stretch in the region of cm -1, - C-H stretch for CH 3 in the region of cm - 1 Moreover, absence of -H stretch for aromatic H 2 in the region of cm -1 has also indicated product formation All the synthesized compounds have shown characteristic peaks in the region of as singlet integrating for -methyl protons Aromatic protons appeared in the region of and the characteristic singlet for -=C-H protons in the region of The compounds were screened for sedative hypnotic activity by pentobarbital sleeping method and locomotor activity determination method, using pentobarbital and diazepam as standards All tested compounds have shown CS depressant effect of varying degrees DISCUSSIO Based on the reported procedures for the synthesis of Schiff bases in the literature, a scheme of organic reactions was designed and performed Each step was thoroughly investigated and optimized to obtain high yields, by adjusting the reaction conditions For Schiff base formation, anhydrous conditions were required along with glacial acetic acid/hydrobromic acid All the Schiff base formation reactions were carried out in absolute alcohol The quantities of amines and p-dimethylaminobenzaldehyde taken were equimolar to avoid undesired byproduct formation Para-substituted aromatic amines easily reacted with p-dimethylaminobenzaldehyde while ortho and metasubstituted aromatic amines failed to react During recrystallisation, use of organic solvents like chloroform, methanol, petroleum ether, dichloromethane led to recovery of reactants as observed by spots in TLC Hence few of them recrystallised with ether Lipophilicity is important for the interaction of drug with receptors and penetration across the biological membrane For compounds 2, 5 and 7 high partition coefficient values were observed The compounds were screened using 30, 100 and 300 mg/kg dose levels at two different time intervals Among Schiff bases compounds 1, 2, 3, 6 and 7 were found to be active in MES test Majority of the compounds have shown activity after 05 h Compounds 2 and 6 showed activity after 4 h Most 1434

5 of the compounds showed activity at 300 mg/kg dose level eurotoxicity in mice was measured by rotorod method The compounds 1, 3, 4 and 6 showed neurotoxicity at 300 mg/kg (ip) Compound o 7 showed neurotoxicity at 100 mg/kg (ip) Remaining compounds were not found to be neurotoxic at maximum administered dose In locomotor activity determination method, all compounds have displayed dose dependent decrease in motor activity at the time interval of 30 min and 1hr Compounds 1, 2, 4 and 6 were found to be the most potent at the dose level of 100 mg/kg one of the compounds has shown any significant effect at the dose level of 1/10 th and 1/20 th of LD 50 Compounds 1, 2, 3 and 6 showed hypnotic effect at the dose of 1/5 th of LD 50 ACKOWLEDGEMETS Authors are thankful to the Director, Meerut Institute of Engineering and Technology, Meerut for providing facilities to complete the project work Jain et al [12] Sridhar, SK, Pandeya, S, Stables, JP and Ramesh, A: Eur J Pharm Sci, 16: (2002) [13] Jiang, JJ, Tsu-Chang, C, Wen-Lin, H, Hwang, JM and Hsu, LY: Chem Pharm Bull, 51(11): (2003) [14] Holla, BS, Veerndra, B, Shivananda, MK and Poojary, B: Eur J Med Chem, 38: (2003) [15] Maria Loriga, S, Piras, G, Paglietti, MP, Costi, AV: IL Farmaco, 58: (2003) [16] Bekhit, AA, Ola A, El-Sayed, TAK, Al-Allaf, HY, Enein, A and Kunhi, M: Eur J Med Chem, 39: (2004) [17] Mandal, SC, Tapan, K, Maity, J, Das, BP and Pal, SM: J Ethnopharmacol, 72: (2000) [18] Miller and Tainter (1944): In: Handbook of Experimental Pharmacology (Kulkarni, SK), Vallabh Prakashan, Delhi (1999) [19] Aggarwal, and Mishra, P: J Pharm Pharmaceut Sci, 7 (2): (2004) [20] Kurian, R, Arulmozhi, DK, Veeranjaneyulu, A, Bodhankar, SL,: Indian J Pharmacol, 38(5): (2006) [21] Girish, S, Achliya, SD, Wadodkar, A and Dorle K: J Ethnopharmacol, 94: (2004) REFERECES [1] A Geronikaki, D Hadjipavlou-Litina, Maria A: IL Farmaco, 58: (2003) [2] Sondhi, SM, irupma Singh, Ashok Kumar, Lozach, O and Meijer, L: Bioorg Med Chem, 14: (2006) [3] Sridhar, SK and Ramesh, A: Biol Pharm Bull, 24(10): (2001) [4] Iana Vazzana, Emanuela Terranova, Francesca Mattioli, Fabio Sparatore: Arkivoc (v), ISS : (2004) [5] Karthikeyan, MS, Prasad, DJ, Poojary, B, Bhat, KS, Holla, BS and Kumari, S: Bioorg Med Chem, 14: (2006) [6] Parekh, J, Inamdhar P, air, R, Baluja, S and Chanda, S: J Med Chem Soc, 70 (10): (2005) [7] air, R, Shah, A, Baluja, S and Chanda, S: J Serb Chem Soc, 71(7): (2006) [8] Aurea, E, Maria da, G, Vanilde, G, Miller, J and Giesbrecht, A: J Braz Chem Soc, 10(1): (1999) [9] Zhang Li-Xia, Liu, Yi Cia, Li-Hua, Hu Yan-Jun, Yin Jun and Hu Pei-Zhi: Thermochim Acta, 440: (2006) [10] Kucukguzel, I, Kuculguzel, SG, Rollas, S, Otuk- Sanis, G and Ozdemir, O: IL Farmaco, 59: (2004) [11] Verma, M, Pandeya, S, Singh, K and Stables JP: Acta Pharm, 54: (2004) 1435