Synthesis of Some Novel Benzotriazole-1-yl-acetic acid substituted aryl hydrazide Derivatives as Anticonvulsant Agents

 

B. Pochaiah*, C.P. Meher, B. Srujana, P. Swarnalatha, A.Muralidhar Rao

Maheshwara Institute of  Pharmacy, Chitkul, Patancheru, Medak, A.P

*Corresponding Author E-mail: balu14886@gmail.com

 

Benzotriazole (I) has been prepared by reacting ortho phenylene diamine with Sodium nitrite in presence of glacial acetic acid at 850 OC. N1-ethylacetate derivative of Benzotriazole (II) was synthesized by reacting the Benzotriazole (I) with ethyl chloroacetate in dry acetone solvent and in presence of anhydrous Potassium carbonate. N-Hydrazine hydrate derivative of Benzotriazole (III) were synthesized by reacting the ethylacetate derivative of Benzotriazole with hydrazine hydrate. Newly synthesized Benzotriazole derivatives of Aromatic Aldehydes (IVc,d) and aromatic ketones (IVa,b ) were prepared by reacting the Hydrazine hydrate derivative of Benzotriazole with 4-OH Benzaldehyde, N, N -dimethyl amino Benzaldehyde, Acetophenone and benzophenone in methanol and these synthesized compounds were confirmed by IR and NMR spectras. Which were purified by recrystallisation and column chromatography. The conformed compounds were evaluated for anticonvulsant activity using phenytoin as standard by MES method.

 

 

INTRODUCTION:

Major efforts to explore novel and useful aspects of benzotriazole chemistry began in 1987 when Katritzky and co-workers carried out systematic studies of the properties and reactions of N-substituent’s in heterocyclic compounds . Since that time, benzotriazole molecule has proven to be a highly valuable synthetic auxiliary and has a wide range of biological activities, e.g., anti-inflammatory^[1]. antimycobacterial activity^[2], anti-nociceptive^[3], anticonvulsant^[4], antimicrobial^[5], protein kinase inhibiting^[6] and immunosuppressive activities. Five key attributes contribute to the success of benzotriazole as a desirable synthetic auxiliary: Introduction and removal from organic compounds easily, Ability to convey multiple activating influences on molecules to which it is attached, intrinsically unreactive and stable, Desirable physical and biological properties, Ready availability^[7].

 

Our main aim is to find out drugs with a higher therapeutic efficiency and a wider spectrum of action, we focused on incorporation of the various aromatic substituent into benzotriazole nucleus, since benzotiazole molecule is containing depressant action we are intrested in finding out anti convulsant effect.

 

Thus, a series of novel Benzotriazol-1-yl-acetic acid (aromatic substituted)-hydrazide derivatives were synthesized and tested for their anti-convulsant effect and found significant results for the activity, especially compound IVc and  IVd  were found to be potent anti convulsant agents.

 

MATERIALS AND METHODS:

All melting points are uncorrected and were taken on electro-thermal capillary melting point apparatus. Infrared spectra were performed (KBr, cm) on JASCO FT/IR-6100.  ¹H-NMR spectra (CDCl₃-d, δppm) was obtained on Jeol 270 MHz and on Jeol sx 500 MHz spectrometer using TMS as an internal standard . All reactions were followed and checked by TLC (aluminum sheets) using hexane and ethyl acetate. These (1:1 V/V) eluent and the plates were sprayed with iodine and also checked under uv light^[8]

 

Step-1: General Procedure for the Preparation of benzotriazole(I):

10.8gms of OPDA dissolved in a mixture of 12gms(11.5ml,0.2mol) of glacial acetic acid and 30ml of water containing 250ml beaker, slight warming may be necessary, cool the clear solution at 15⁰c stir magnetically and then add a solution of 7.5gms(0.11mol) of sodium nitrite in 15ml water in one portion the reaction mixture becomes warm within 2-3mins reaches a temperature of about 85⁰c and begins to cool while the color changes from deep red to pale brown continue the stirring for about 15min by the time the temperature drops to 35-40⁰c and thoroughly chilled in an ice water bath for about 30min. Then collect by vacuum filtration the pale brown solid which separates and wash with three 30ml portions of ice water, add decolorizing agent charcoal, filter and allow the filtrate to cool to about 50⁰c before adding a few crystals of the crude benzotriazole which have been retained for seeding  allow the mixture to attain room temperature slowly and then thoroughly chill in ice and collect the benzotriazole which separates as pale yellow colored needles of  Benzotriazole, M.P was 99-100⁰c^.[9]

 

Step-2: synthesis of 1[2-ethyl acetate]-1, 2, 3-Benzotriazole (II) from I:

1gm benzotriazole was dissolved in 50ml of dry acetone to that add ethyl chloro acetate (1.028ml) and K₂CO₃(1.16mg) and refluxed for 10-12hrs then filtered, filtrate was taken in acetone and was evaporated to form a white color crystallysed product of 1[2-ethyl acetate]-1,2,3-Benzotriazole. Completion of the reaction was confirmed by TLC using ethyl acetate and hexane (1:1 V/V) as solvent system and the product was purified by recrystallisation from acetone.

 

Step-3: synthesis of 1[1-methyl hydrazide]-1, 2,3Benzotriazole (III) from II

Equimolar quantities of 1[2-ethyl acetate]-1, 2, 3-Benzotriazole, hydrazine hydrate were taken and dissolved in methanol, the mixture is allowed to reflex for 2-3hrs to give a resultant product of 1[1-methyl hydrazide]-1,2,3Benzotriazole. TLC was performed by using mobile phase ethyl acetate:hexane (1:1 V/V). Purified by recrystallisation from ethanol.

 

Step 4: general procedure for preparation of final derivatives IVa-d from III:

1[1 - methyl hydrazine] -1, 2, 3 Benzotriazole (III) was treated with various aromatic aldehydes and ketones like 4–OH benzaldehyde, N,N-dimethyl aminobenzaldehyde, acetophenone, Benzophenone in equimolar quantities, by using methanol as solvent and stirred for 1hr. completion of the reaction was confirmed by TLC, RF values were calculated by using hexane and ethyl ecetate(1:1 V/V) as solvent system. Final solid product was recrystillised from methanol, purified by column chromatography and percentage yield was calculated for four compounds and they were named respectively:

·         IVa- Benzotriazole-1-yl-acetic acid (1-phenyl-ethylidene)-hydrazide.

·         IVb- Benzotriazole-1-yl-acetic acid benzhydrylidene-hydrazide.

·         IVc- Benzotriazole-1-yl-acetic acid (4-hydroxy-benzylidene)-hydrazide.

·         IVd- Benzotriazole-1-yl-acetic acid (4-dimethylamino-benzylidene)-hydrazide.

 

 

Table 1. Physical and Analytical data of synthesized compounds

Compound	Mol. formula	Mol. mass	M.P (0C)	Yield (%)
I	C6H5N3	119	98-100	83.12
II	C10H11N3O2	205	45-47	72.36
III	C8H9N5O	191	110-115	66.63
IVa	C16H15N5O	293	175-180	44.83
IVb	C21H17N5O	355	155-158	43.28
IVc	C15H13N5O2	295	194-196	42.13
IVd	C15H18N6O	322	183-186	47.23

 

Table 2. Spectral data of synthesized compounds

Compound	IR KBr (cm–1)	1H NMR (CDCL3-d, δppm)
I	3389.12(N-H), 3012.42(Ar-C-H), 1600.50(C=N), 1450(ArC=C), 1300.13(C-C)	7.0(S, 1H, -NH), 7.45-7.98 (M, 4H, -CH).
II	3400.52(N-H), 3050.54 (Ar-C-H),2900.65(C-H), 1680.80(C=O), 1600.50(C=N)	1.30(S, 3H, -CH3), 4.52 (S, 4H, -CH2), 7.59-7.96(M, 4H, Ar-CH).
III	3423.42(N-H), 3067.74 (Ar-C-H), 2923.65(C-H), 1689.50 (C=O), 1617.59(C=N), 853.94 (C-N).	2(d, 1H, -NH2), 4.62 (S, 4H,     -CH2)7.55-7.92 (M, 4H, Ar-CH), 7.2 (S, 1H, -NH).
Iva	3423.42(N-H), 3067.74 (Ar-C-H), 2923.65(C-H), 1689.50(C=O), 1617.59(C=N),1452 (ArC=C), 1313.13(C-C), 853.94 (C-N)	2.35(S, 3H, -CH 3), 4.62 (S, 2H,-CH2), 7.0(S, 1H, -NH), 7.3-7.98(M, 9H, Ar-H).
IVb	3420.40(N-H), 3045.34(Ar- C-H), 2923.65(C-H), 1680.34(C=O), 1617.59(C=N), 1452.56(Ar C=C), 820.01 (C-N).	4.54(S, 2H, -CH2), 6.89(S, 1H, NH), 7.4-8.4 (M, 14H,Ar-H).
IVc	3611.02(O-H), 3407.04 (N-H), 3107.70(Ar=C-H), 2823.33(=C-H), 1681.70(C=N), 1603.92     (Ar C=O), 1314.02(C-C).	4.62(S, 2H, CH 2), 5(S, 1H, -OH), 6.8-7.98(M, 8H, Ar-H), 8.0(S, 1H, -NH), 8.1(S, 1H, CH).
IVd	3030.33(Ar=C-H), 3487.04(N-H), 3011.18(=C-H), 2110.18(C=N), 1681.70 (Ar C=O), 1600.92(Ar =C-C), 1276.57(C-C).	4.82(S, 2H, CH 2), 6.8-8.1 (M, 8H, Ar-H), 8.7(S, 1H, -NH), 8.2(S, 1H, CH), 2.35(S, 6H, CH 3).

 

 

SCHEME:

 

 

Evaluation of anticonvulsant activity:

Maximal electroshock induced seizures (MES Method):

Albino wister rats of either sex were obtained from the animal house, St.John College of Pharmacy, Warangal, CPCSEA Registration Number: 1278/ac/09/CPCSEA. They were properly housed in separate cages and fed with standard diet and water ad libitum throughout the study period. The Institutional animal ethical committee (IAEC), St.John College of Pharmacy, Warangal, permitted the ethical clearance for the study protocol. The animals were divided into four groups. Each group contains six animals. The Group-1 received vehicle control (1% w/v SCMC,1gm/100ml) whereas Group-2 received standard drug i.e. Phenytoin, 25mg/kg,  intrapertonially. Group- 3, 4, 5 and 6 received IVa, IVb,  IVc and IVd p.o. respectively for 14 days.

 

On the 14^th day, seizures were induced to all groups by using an Electro-convulsometer. Maximal electroshock seizures were elicited by a 60 Hz alternating current of 150Ma intensity for 0.2 sec. A drop of electrolyte solution (0.9% NaCl) with lignocaine was applied to the corneal electrodes prior to application to the rats. This increases the contact and reduces the incidence of fatalities. The duration of various phases of epilepsy were observed. The percentage protection was estimated by observing the number of animals showing abolition of hind leg. Tonic Extension (or) extension not greater than 90 [3].

 

 

Fig. 1 Anti Convulsant activity of different Benzotriazole derivatives

 

Table3. The MES induced epilepsy screening test for the synthesized compounds using Phenytoin as standard and carboxy methyl cellulose as control.

The values are expressed as mean ± SEM of 6 animals. Comparisons were made between: Group I Vs Group II, Group III, Group IV, Group V and Group VI.

Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s –‘t’ test.

**p< 0.01, *p<0.05 and ^nsp- non significant.

 

RESULTS AND DISCUSSION:

Chemistry:

Benzotriazole (I) have been prepared by reacting OPDA with Sodium nitrite in presence of glacial acetic acid at 85⁰C. N₁–ethylacetate derivative of Benzotriazole (II) was synthesized by reacting the Benzotriazole (I) with ethyl acetate in dry acetone solvent and in presence of anhydrous Potassium carbonate. The reaction was carried out for 12 hours and these synthesized compounds were confirmed by melting point and TLC Profile. Which were purified by recrystallization and column chromatography. N-Hydrazine hydrate derivatives of Benzotriazole (III) were synthesized by reacting the ethyl acetate derivative of Benzotriazole with hydrazine hydrate in methanol and these synthesized compounds were confirmed by melting point and TLC Profile which were purified by recrystallization and column chromatography. Newly synthesized Benzotriazole derivative of Aromatic Aldehydes (IVc,d)  were prepared by reacting the  Hydrazine hydrate derivative  of Benzotriazole   with  4 –OH  Benzaldehyde and  N,N –dimethyl amino Benzaldehyde in methanol  and  these  synthesized compounds  were confirmed  by IR and NMR spectras .Which were purified by recrystallization and column chromatography. Newly synthesized Benzotriazole derivative of Aromatic Ketones (IVa,b ) were prepared by reacting the  Hydrazine hydrate derivative of Benzotriazole with Acetophenone and benzophenone in methanol  and  these  synthesized compounds were confirmed by IR and NMR spectras. Which were purified by recrystallization and column chromatography.

 

Characterization of spectral data:

The list of new aromatic aldehydes and ketone derivatives of benzotriazole synthesized in present investigation and their characterization data are presented as follows.

 

IR Specteum (KBr pellet method):

Infrared spectrum (KBr pellet method) of Benzotriazole-1-yl-aceticacid(4-hydroxy-benzylidene)-hydrazide (IVc)  has shown absorption peaks at 1681.70cm^-1 stretching indicating the presence of C=N group of final benzotriazole derivatives two strong bonds were found respectively at 3407.04cm^-1 and 1603.92cm^-1 are due to –NH,-C=O stretching.

 

¹HNMR spectrum:

Benzotriazole-1-yl-aceticacid(4-hydroxy-benzylidene)-hydrazide (IVc) had  shown the absence of two singlet peaks of hydrazine hydrate derivative of benzotriazole this suggests that the hydrogen atoms of amine of hydrazine hydrate derivative of benzotriazole has reacted with 4-hydroxy benzaldehyde to form Benzotriazole-1-yl-aceticacid (4-hydroxy-benzylidene)- hydrazide which is confirmed by characteristic signal at 8.1ppm due to

  having one proton, a singlet at  4.62 ppm was integrated to 2 protons corresponding to –CH_2, a singlet at  5ppm was integrated to one proton corresponding to  –OH, a multiplet at  6.8-7.98ppm integrated to 8 protons indicate the presence of Ar-H and a singlet at 8ppm integrated to 1 proton indicates the presence of –NH group in the compound.

 

Anticonvulsant activity:

Benzotriazoles were tested for anti convulsant activity. Results were shown significant reduction in the hindlimb tonic extensor phase, due to the inhibition of voltage dependent Sodium channel. Our Benzotriazole aldehyde derivatives showed better activity than Benzotriazole ketone derivatives.

 

CONCLUSION:

Benzotriazoles traditionally used for the anti microbial, anti fungal activity. Some newly synthesized benzotriazole derivatives posses CNS activity. So, we synthesized various substituted benzotriazole derivatives which posses CNS depressant activity. These benzotriazole derivatives (IVa-d) had shown significant reduction in the hind limb tonic extensor phase, which is similar to Grand-mal epilepsy in humans; this may be due to the inhibition of voltage dependent Na⁺ channels. By comparing the results of anti convulsant activity it was found that substitution of aromatic aldehydes to N-Hydrazine hydrate derivative of Benzotriazole showed better activity when compared to that of substitution of aromatic ketones. Further investigation must be done to study more about these substituted benzotriazole derivatives.

 

ACKNOWLEDGEMENTS:

We are are thankful to N. Karthik, V. Varun, P. Sandhya Rani, T. Divya and M. Ravali of Rosory College of Pharmacy, Warangal for their help during the entire research work. Also thankful to Indian Institute of Chemical Technology, Hyderabad for the technical support.

 

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Received on 19.12.2012         Modified on 04.01.2013

Accepted on 17.01.2013         © AJRC All right reserved