Schiff bases of N-methyl and N-acetyl isatin derivatives with different aryl amines have been synthesized and screened for anticonvulsant activities against maximal electroshock (MES) and subcutaneous metrazole (ScMet). N-methyl-5-bromo-3-(p-chlorophenylimino) isatin 2 exhibited anticonvulsant activity in MES and ScMet with LD50 > 600 mg kg-1, showing better activity than the standard drugs phenytoin, carbamazepine and valproic acid. Thus, compound 2 may be chosen as a prototype for development of new anticonvulsants.Schiffove baze N-metil i N-acetil derivata izatina s različitim aromatskim aminima sintetizirane su i ispitane na sposobnost suzbijanja konvulzija uzrokovanih elektrošokom (MES) i subkutanom primjenom metrazola (ScMet). N-metil-5-bromo-3-(p-klorofenilimino) izatin 2 pokazao je nisku neurotoksičnost i jače antikonvulzivno djelovanje nego standardni antikonvulzivi fenitoin, karbamazepin i valproična kiselina. Zbog toga se spoj 2 može smatrati prototipom za razvoj novih antikonvulziva

JAMES P. STABLES

KRISHNA NAND SINGH

MANJUSHA VERMA

SURENDRA NATH PANDEYA

English

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Epilepsy is a disease of complex nature and of different etiology (1). A large numberof populations of different age groups and sex are affected by this disease. Mainly, twokinds of epilepsy have been identified, one with grand mal and the other with petit mal.Anticonvulsant drugs with MES (maximal electroshock) activity are generally useful ingrand mal, while ScMet (subcutaneous metrazole) antagonists are effective in petit mal.Many drugs have been marketed recently for the treatment of epilepsy (2–4). These in-clude milecemide, zonisamide, lamotrigine, felbamate and tiagabine. Recently, semicar-bazones are emerging as novel anticonvulsant drugs. 4-Bromobenzaldeyde semicarba-zone and 4-bromophenyl semicarbazones have shown promising activities (5–9). Theproposed pharmacophoric requirements in the semicarbazone molecules are:(i) aryl binding site with a hydrophobic group;(ii) hydrogen bonding domain exemplified by the presence of the -NHCO- grouping;(iii) two electron donor system;(iv) hydrophobic binding site whose size determines the type of activity.49Acta Pharm. 54 (2004) 49–56 Original research paperAnticonvulsant activity of Schiff basesof isatin derivativesMANJUSHA VERMA1SURENDRA NATH PANDEYA1KRISHNA NAND SINGH1*JAMES P. STABLES21 Department of Applied ChemistryInstitute of TechnologyBanaras Hindu UniversityVaranasi-221005, India2 Department of Health andHuman ServiceNational Institute of HealthBethesda, Maryland-20892, USAReceived June 26, 2003Accepted December 15, 2003Schiff bases of N-methyl and N-acetyl isatin derivativeswith different aryl amines have been synthesized andscreened for anticonvulsant activities against maximalelectroshock (MES) and subcutaneous metrazole (ScMet).N-methyl-5-bromo-3-(p-chlorophenylimino) isatin (2) ex-hibited anticonvulsant activity in MES and ScMet withLD50 > 600 mg kg–1, showing better activity than the stan-dard drugs phenytoin, carbamazepine and valproic acid.Thus, compound 2 may be chosen as a prototype for de-velopment of new anticonvulsants.Keywords: Schiff base, isatin, anticonvulsant* Correspondence, e-mail: knsinghbhu@yahoo.co.inThe objective of the present work was to test this hypothesis and as a result, a newseries of compounds have been prepared by reacting N-methyl or N-acetyl isatins withdifferent aryl amines (Scheme 1):(i) Isatin has been selected because during initial screening it has shown activity in theMES test (10–14). In the isatin molecule, bromo substituent has been selected becauseof its high potency. Further, an electron withdrawing group, such as NO2, has alsobeen included for SAR studies (15).(ii) Schiff bases of aromatic amines have been prepared because they contain a two elec-tron donor system.(iii) The N-methyl and N-acetyl groups have been incorporated to increase the lipophili-city of the molecules.EXPERIMENTALThe melting point of the compounds was measured in an open capillary and wasuncorrected. The purity of the compounds was confirmed by thin layer chromatographyusing silica gel glass plates as the stationary phase and benzene and ethanol (9:1) as themobile phase. IR spectra were recorded on a JASCO FT/IR-5300 spectrophotometer, Ja-pan. NMR spectra were run on a JEOL FT-NMR spectrometer FX-90Q (Japan) with TMSas internal reference. Isatin derivatives were prepared by the literature methods (16).They were characterized by melting point, elemental analysis and spectral data.The aryl amines were procured from Aldrich (USA) and were used as received.50M. Verma et al.: Anticonvulsant activity of Schiff bases of isatin derivatives, Acta Pharm. 54 (2004) 49–56.NOR 1OR1+ H2N R2N R2glacial CH COOH3RRONR: Br, NOR : CH , COCHR : NO , COOH, OCH , Cl, F21 3 32 2 3Scheme 1Synthesis of Schiff bases. General procedureEquimolar quantities of 5-substituted N-methyl/N-acetyl isatin (0.003 mol) and aro-matic amine (0.003 mol) were added into 20 mL of absolute ethanol containing a fewdrops of glacial acetic acid in a 250-mL round bottom flask. The reaction mixture wasrefluxed for half an hour and then checked for completion by TLC. The solvent wasstripped off and the product was recrystallized from ethanol (99.5%) and characterizedby elemental analysis, IR and 1H NMR.The physical and spectral data of the synthesized compounds are listed in Tables Iand II, respectively.51M. Verma et al.: Anticonvulsant activity of Schiff bases of isatin derivatives, Acta Pharm. 54 (2004) 49–56.Table I. Physical properties of isatin Schiff basesCompd.No.R R1 R2M.p.(°C)Yield(%)Molecularformula(Mr)RfaElementalanalysis (%)(Calcd./found)C H N1 Br CH3 OCH3 210 77C16H13BrN2O2(345.19)0.36155.67 3.80 8.1155.59 4.00 7.952 Br CH3 Cl 243 79C15H10ClBrN2O(349.61)0.40051.53 2.88 8.0151.24 2.73 8.103 Br CH3 NO2 104 89C15H10BrN3O3(360.16)0.54550.02 2.80 11.6750.11 2.52 11.584 Br CH3 COOH 153 91C16H11BrN2O3(359.17)0.21953.50 3.08 7.7953.39 2.89 7.625 Br CH3 F 231 70C15H10FBrN2O(333.15)0.25054.08 3.02 8.4154.84 3.33 8.806 Br COCH3 OCH3 86 90C17H13BrN2O3(373.20)0.47354.71 3.51 7.5154.68 3.31 7.747 Br COCH3 Cl 211 44C16H10ClBrN2O2(377.62)0.31850.89 2.67 7.4250.69 2.32 7.248 Br COCH3 NO2 115 44C16H10BrN3O4(388.17)0.46149.51 2.59 10.8349.31 2.33 10.529 Br COCH3 COOH 230 56C17H11BrN2O4(387.19)0.31552.73 2.86 7.2353.42 2.54 7.4510 Br COCH3 F 117 50C16H10FBrN2O2(361.17)0.55053.21 2.79 7.7653.01 2.52 7.6011 NO2 COCH3 OCH3 98 91C17H13N3O5(339.31)0.27760.18 3.86 12.3860.41 4.26 12.60NRNOR1R252M. Verma et al.: Anticonvulsant activity of Schiff bases of isatin derivatives, Acta Pharm. 54 (2004) 49–56.12 NO2 COCH3 Cl 111 49C16H10ClN3O4(343.66)0.42155.92 2.91 12.2255.75 2.58 12.0013 NO2 COCH3 NO2 55 57C16H10N4O6(354.21)0.27054.22 2.82 15.8156.61 2.43 15.5614 NO2 COCH3 COOH 78 49C17H11N3O6(353.29)0.37557.79 3.13 11.8955.49 3.36 11.8015 NO2 COCH3 F 178 64C16H10FN3O4(327.28)0.27558.72 3.05 12.8458.50 3.00 12.67a Mobile phase: benzene/ethanol 9:1Table I. ContinuedTable II. Spectral data of isatin Schiff basesCompd.No.IR (KBr), (cm–1) 1H-NMR (, ppm)1 3349, 1700, 1702, 1268, 1050, 846, 6002.8 (s, 3H, N-CH3), 3.73 (s, 3H, OCH3),6.3–7.7 (m, 7H, Ar-H), 8.4 (s, 1H, CH=N)2 3450, 1672, 1603, 821, 708, 5042.8 (s, 3H, N-CH3), 6.3–7.0 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)3 3364, 1750, 1613, 1470, 1294, 845, 5772.8 (s, 3H, N-CH3), 6.3–7.9 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)4 3383, 3000, 1749, 1701, 1610, 844, 6092.8 (s, 3H, N-CH3), 6.3–7.9 (m, 7H, Ar-H),11.0 (s, 1H, COOH), 8.4 (s, 1H, CH=N)5 3451, 1673, 1605, 1388, 823, 5062.8 (s, 3H, N-CH3), 6.3–6.7 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)6 3349, 1700, 1702, 1268, 1050, 846, 6002.4 (s, 3H, COCH3), 3.7 (s, 3H, OCH3),6.3–8.0 (m, 7H, Ar-H), 8.4 (s, 1H, CH=N)7 3252, 1687, 1672, 1603, 821, 708, 5032.4 (s, 3H, COCH3), 3.7 (s, 3H, OCH3),6.3–8.0 (m, 7H, Ar-H), 8.4 (s, 1H, CH=N)8 3362, 1746, 1631, 1601, 1539, 1302, 841, 5342.4 (s, 3H, COCH3), 6.3–8.0 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)9 3000, 1739, 1682, 1604, 1535, 760, 5192.4 (s, 3H, COCH3), 6.3–7.9 (m, 7H, Ar-H),8.4 (s, 1H, CH=N), 11.0 (s, 1H, COOH)10 3451, 1710, 1673, 1605, 1388, 823, 5062.4 (s, 3H, COCH3), 6.3–8.0 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)11 3348, 1700, 1702, 1570, 1340, 1268, 1050, 8462.4 (s, 3H, COCH3), 3.7 (s, 3H, OCH3),6.7–8.7 (m, 7H, Ar-H), 8.4 (s, 1H, CH=N)12 3253, 1689, 1670, 1605, 1573, 1333, 820, 7102.4 (s, 3H, COCH3), 6.4–8.7 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)13 3364, 1747, 1633, 1601, 1539, 1302, 8452.4 (s, 3H, COCH3), 6.7–8.7 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)14 3000, 1710, 1684, 1607, 1564, 1332, 7622.4 (s, 3H, COCH3), 6.7–8.7 (m, 7H, Ar-H),8.4 (s, 1H, CH=N), 11.0 (s, 1H, COOH)15 3453, 1711, 1672, 1608, 1568, 1390, 1351, 8252.4 (s, 3H, COCH3), 6.4–8.7 (m, 7H, Ar-H),8.4 (s, 1H, CH=N)Anticonvulsant screeningAll the compounds were screened for anticonvulsant properties adopting the anti-convulsant drug development (ADD) program protocol (17, 18). The mice used wereCarworth Farms No. 1, weighing from 19 to 25.5 g, either sex and 22–33 days old. Ac-commodation conditions were maintained at 20 °C and the number of animals used was1, 3, 5 and 8 in different experiments. Methyl cellulose was used for dissolving the rsetcompounds in ScMet and Rotorod Test, while polyethylene glycol was used for MES.The control experiments were performed with solvents alone. Three animals were usedin the control test. The compounds were administered intraperitoneally (0.01 mL g–1 bodymass) to mice, at doses of 30, 100 and 300 mg kg–1 to 1 to 4 mice. The activities of thecompounds in maximum electroshock (MES) and subcutaneous metrazole (ScMet) testalong with their neurotoxicity are presented in Table III.Electroshock method. – Maximal seizures were induced by the application of electricalcurrent to the brain via corneal electrodes. The stimulus parameters for mice were 50mA in a pulse of 60 Hz for 200 ms. The mice were given the test drug dissolved in poly-ethylene glycol. Abolition of the hind limb tonic extensor spasm was recorded as a mea-sure of anticonvulsant activity.Subcutaneous metrazole seizure pattern test. – A metrazole dose of 85 mg kg–1 adminis-tered subcutaneously to mice causes seizures in more than 97% of the animals. This iscalled the convulsive dose 97 (CD97). The test was carried out by giving the metrazoleinjection approximately 10 minutes before the anticipated time of the peak anticonvul-sant drug action. The animals were observed during the following 4 hours for the occur-rence of seizures. A threshold convulsion is defined as one episode of clonic spasms whichpersists for at least 5 seconds. Absence of even a threshold convulsion during the periodof observation is taken as the endpoint in this test.Rotorod test. – The mice were trained to stand on an accelerating rotorod rotating at10 rev min–1. The rod diameter was 3.2 cm. Trained animals were given the test com-pounds intraperitoneally at doses of 30, 100 and 300 mg kg–1 in methyl cellulose after 30minutes and up to 24 hours. The mice were placed on the rotorod to measure the effectof the drug on their motor performance. The dose at which animals fell off the rotorodwas determined.The compounds were also screened at an oral dose of 30 mg kg–1 and the MES ac-tivity was noted at different time intervals up to 4 hours.Some compounds with OCH3/Cl substituent in phenyl, N-COCH3 and a substituentBr/NO2 at the 5th position in the isatin moiety (compounds 6, 7, 11 and 12) and the com-pound with fluoro substituent in the phenyl ring, N-COCH3 and a substituent Br at the5th position in the isatin structure (compound 10) were screened at 6 Hz shock in mice ata dose of 100 mg kg–1 up to 4 hours. The compounds were dissolved in methyl cellulose,introduced via the i.p. route and the mice were given a current shock of 32 mA.53M. Verma et al.: Anticonvulsant activity of Schiff bases of isatin derivatives, Acta Pharm. 54 (2004) 49–56.RESULTS AND DISCUSSIONThe results of anticonvulsant screening are given in Table III. The two primaryscreens MES and ScMet were performed in mice at doses of 30, 100, 300 mg kg–1 (intra-peritoneally). As compared to isatin, the 5-Br, N-CH3 isatin derivatives 2, 3, 4 with Cl,NO2, COOH groups, respectively, in the phenyl ring, require a lower dose in MES whilethe 5-Br, N-COCH3 isatin derivatives 9, 10 with F, COOH groups, respectively, in thephenyl ring and 5-NO2, N-COCH3 isatin derivative 12 with NO2 group in the phenylring showed activity in ScMet. In general, compounds with N-CH3 were active in theMES test whereas those having N-COCH3 group showed activity in the ScMet test. TheN-methyl-5-bromo-3-(p-nitrophenylimino) isatin 3 and N-acetyl-5-bromo-3-(p-chloro-phenylimino) isatin 10 have shown activity at a dose of 100 mg kg–1 in MES and ScMet,54M. Verma et al.: Anticonvulsant activity of Schiff bases of isatin derivatives, Acta Pharm. 54 (2004) 49–56.Table III. Anticonvulsant and neurotoxicity screening of isatin Schiff basesCompd.No.Concentration (mg kg–1 body mass)aMESa,b ScMeta,b Neurotoxicitya,b LD50 (mg kg–1)1 – – 30 (25 %) 272 300 (100%) 300 (20%) – > 6003 100 (33.3%) – 100 (50%) 1004 300 (100%) – 300 (50%) > 3005 – – 300 (50%) > 3006 – – – > 3007 – – 30 (25%) 278 – – 300 (25%) > 3009 – 300 (60%) 100 (12.5%) 20010 – 100 (20%) – > 30011 – – – > 30012 – 300 (80%) – 2713 – – 100 (25%) 15014 – – 30 (50%) 4215 – – 30 (50%) 28Isatin 400 (100%) – – > 400Phenytoin 30 (100%) – 100 170  13Carbamazepine 30 (100%) 100 (100%) 100 350Valproic acid – 300 (100%) – > 1000MES – maximal electroshock seizure, ScMet – subcutaneous metrazole seizure– No activity.a The two primary screens of MES, ScMet and toxicity were performed by intraperitoneal injection in mice at do-ses of 30, 100, 300 mg kg–1.b Values in parentheses in the MES and ScMet tests indicate the number of animals protected against the number ofanimals tested and in the neurotoxicity test they indicate the number of animals exhibiting toxicity against thenumber of animals tested. No protection of the solvent in control experiments was observed.respectively. Compound 2 with bromo substituent in the isatin ring and chloro substi-tuent in the phenyl ring showed activity both in MES and ScMet tests and no toxicity atthe stipulated doses. In 6 Hz excitation, the N-acetyl-5-nitro-3-(p-chlorophenylimino)isatin 12 protected mice (25%) at a dose of 100 mg kg–1 after 15 minutes of drug adminis-tration. In the oral test, N-methyl-5-bromo-3-(p-carboxyphenyl imino) isatin 4 showed25% activity after half an hour at the dose of 30 mg kg–1 and N-acetyl-5-nitro-3-(p-ni-trophenylimino) isatin 13 showed 25% activity after 2 hours at the dose of 50 mg kg–1.N-methyl-5-bromo-3-(p-nitrophenylimino) isatin (3) showed activity in MES (100 mg kg–1with 33.3% protection) as well as N-methyl-5-bromo-3-(p-aminophenylimino) isatin (4)(300 mg kg–1 with 100% protection). N-methyl-5-bromo-3-(p-bromophenylimino) isatin(2) showed activity both in MES (300 mg kg–1 with 100% protection) and ScMet (300 mgkg–1 with 20% protection). The reference compound, valproic acid, was active only inScMet (300 mg kg–1 with 100% protection) whereas phenytoin was active in MES only.Another reference compound, carbamazepine, was however active both in MES (30 mgkg–1 with 100% protection) and in ScMet (100 mg kg–1 with 100% protection). It is worth-wile to mention that the compound 2, being active both MES and ScMet, had LD50 > 600mg kg–1 as compared to carbamazepine with the LD50 of 350 mg kg–1. Thus, compound2 could be chosen as a lead compound for further modification aimed at improving theanticonvulsant activity.CONCLUSIONSCompound 2 with bromo substituent in the isatin ring and chloro substituent in thephenyl ring exhibited a broad-spectrum activity with no neurotoxicity at the stipulateddose and can hence be chosen as a prototype for the development of new anticonvulsants.Acknowledgement. – The authors are thankful to the All India Council for Technical Education,New Delhi, for financial support.REFERENCES1. J. O. McNamara, Drugs Effective in the Therapy of the Epilepsies, in The Pharmacological Basis ofTherapeutics (Eds. J. G. Hardman, P. B. Molinoff, R. W. Ruddon and A. G. Gilman), 9th ed., Mc Graw-Hill, New York 1990, pp. 461–486.2. A. Sabers and L. Gram, Newer anticonvulsants comparative review of drug interactions andadverse effects, Drugs 60 (2000) 23–33.3. J. W. Britton and E. L. So, New antiepileptic drugs: prospects for the future, J. Epilepsy 8 (1995)267–281.4. 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Proc. 44 (1985) 2645–2649.S A @ E T A KAntikonvulzivno djelovanje Schiffovih baza – derivata isatinaMANJUSHA VERMA, SURENDRA NATH PANDEYA, KRISHNA NAND SINGH i JAMES P. STABLESSchiffove baze N-metil i N-acetil derivata izatina s razli~itim aromatskim aminimasintetizirane su i ispitane na sposobnost suzbijanja konvulzija uzrokovanih elektro{okom(MES) i subkutanom primjenom metrazola (ScMet). N-metil-5-bromo-3-(p-klorofenilimi-no) izatin 2 pokazao je nisku neurotoksi~nost i ja~e antikonvulzivno djelovanje negostandardni antikonvulzivi fenitoin, karbamazepin i valproi~na kiselina. Zbog toga sespoj 2 mo`e smatrati prototipom za razvoj novih antikonvulziva.Klju~ne rije~i: Schiffova baza, izatin, antikonvulzivDepartment of Applied Chemistry, Institute of Technology, Banaras Hindu UniversityVaranasi-221005, IndiaDepartment of Health and Human Service, National Institute of HealthBethesda, Maryland-20892, USA56M. Verma et al.: Anticonvulsant activity of Schiff bases of isatin derivatives, Acta Pharm. 54 (2004) 49–56.

Antikonvulzivno djelovanje Schiffovih baza- derivata isatina

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