Evaluation of the Antioxidant Activity of some Hydrazone Schiff’s bases bearing Benzotriazole Moiety

 

Salah Eddine Rahmani¹*, Mokhtar Lahrech²

¹Laboratory of Organic Chemistry and Natural Substances, University of Djelfa, 17000, ALGERIA.

²Department of Biology, Faculty of Life and Natural Sciences, University of Djelfa, 17000, ALGERIA.

 

ABSTRACT:

In our work, a series of substituted hydrazone Schiff’s bases of N-benzylideneacetohydrazide 2-(1H-benzo[d] [1,2,3] triazol-1-yl)- (B3-B12) were synthesized from the benzotriazole hydrazide and with the use of various aromatic aldehydes. The structure of the synthesized compounds was confirmed by IR, ¹HNMR and Mass spectra. All the compounds were screened for in vitro antioxidant activity by DPPH scavenging and FRAP assay. The results show that all the compounds have significant antioxidant activity. Compounds substituted with electron donating groups like methoxy and hydroxyl showed higher antioxidant activity.

 

 

 

INTRODUCTION:

In 1864, Hugo Schiff was the first who had ever made a great effort for the synthesis of imine¹. Schiff base compounds have taken much interest in synthetic chemistry.  They are used as substrates in the preparation of a number of industrial and biologically active compounds via different synthetic approaches. In the Hydrazone Schiff bases, the pharmacophore (-CO-NHN=C) is reported to be the cause of biological activity of Schiff bases of hydrazones². Consequently, all the compounds containing this active moiety form an important group of medicinal and pharmaceutical agents³, such as anticancer⁴, antibacterial⁵, antifungal⁶, anti-convalescent⁷, anti-depressant⁸, antioxidant⁹, antimalarial¹⁰, hydrazones are also known for their use in agrochemical industry¹¹.

 

In recent years, a large number of compounds containing benzotriazole system have been investigated through different synthetic approaches.  They are widely used in many important fields such as chemical synthesis¹², materials¹³, water research¹⁴, corrosion inhibitors¹⁵.

 

 

Pharmalogical studies of the 1h-benzotriazol nucleus have been shown to possess a broad spectrum of biological activities¹⁶ which include anticonvulsant¹⁷, anti inflammatory¹⁸, antifungal¹⁹, analgesic²⁰, antitumor agents²¹, anticancer²², antiparasitic²³, antiviral²⁴, antimicrobial²⁵, and Antioxidative²⁶. In addition to these considerable biological applications. They also form an important group of intermediates, protecting groups and final products in organic synthesis²⁷.

 

In the present work, we have synthesized ten hydrazone Schiff bases B3-B12 from the reaction of benzotriazole hydrazide with substituted aromatic benzaldehydes and evaluated there in vitro antioxidant activity by DPPH and FRAP assays.

 

MATERIAL AND METHODS:

All solvents and chemicals were of commercial reagent grade and used as they are received. Melting points are taken in an open capillary tube. The purity of the compound was confirmed by TLC using silica gel precoated plates of 0.25 mm thickness with ethyl acetate and petroleum ether (1:1) as eluent. IR spectra were recorded in KBr on Perkin Elmer FTIR spectrometer. The 1HNMR were recorded on Brucker 300 MHz in DMSO-d6.

 

Experimental:

Preparation of Ethyl Benzotriazole Acetate (B1):

(0.01 mol) of benzotriazole was refluxed with (0.01 mol) of ethylchloroacetate and (1.0 gm) of potassium carbonate in (30 mL) of dry acetone for 10 hr. After completion of the reaction acetone was removed and the residue crystallized from ethanol giving a white solid, Mp: 61-63°C, (yield 80%)²⁸.

 

Preparation of 2-(1H-Benzo[d][1,2,3]Triazol-1-yl)Acetohydrazide (B2):

A mixture of hydrazine hydrate 80% (0.05 mol, 0.25 ml) and   ethyl 2-(1H- benzo[d][1,2,3]triazol-1-yl)acetate B1 ( 0.01 mol) in absolute ethanol (20 ml)  were refluxed for 4 h. The reaction mixture was cooled and poured into water. The crude product was filtered off and recrystallized from ethanol to give the desired hydrazide. Mp: 168-170°C, (yield 65 %)²⁹.

Preparation of Schiff bases 2-(1H-Benzo[d][1,2,3]Triazol-1-yl)-N'-(Substituted Benzylidene)Acetohydrazide (B3-B10):

Hydrazide derivative of benzotriazole B2 (0.01mol) was refluxed with different substituted aromatic aldehydes in equimolar ratio, in 20 ml of methanol, for 6 hrs in the presence of catalytic amount of glacial acetic acid. The reaction mixture was cooled to room temperature, and poured into crushed ice. The resulting mixture was filtered and the solid obtained was washed with cold water dried and recrystallized from ethanol.

 

 

Fig. 1: Scheme of synthesis

 

2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-benzylideneacetohydrazide (B3):

Yield 83 %; Mp: 166°C

IR (KBr, cm-1): 3029(C-H), 1727(C=O), 1658(C=N), 1373(C-N). MS: 279(M+).

1H NMR (DMSO d₆): δ (ppm) 4.8 (s,2H), 7.1-8.0 (m, 9H), 8.1 (s, 1H).

 

N-(4-bromobenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B4): 

Yield 85 %; Mp: 240°C

IR (KBr, cm-1): 2935(C-H), 3367 (NH), 1646(C=O), 1572(C=N). MS: 315(M+).

1H NMR (DMSO d₆): δ (ppm) 4.8 (s, 2H), 7.1-9.5 (m, 8H), 8.0(s, 1H), 8.2(s, 1H)

 

N-(4-methylbenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B5):

Yield 76 %; Mp: 210°C

IR (KBr, cm-1): 2792(C-H), 1715(C=O), 1630(C=N), 1358(C-N). MS: 294(M+).

1H NMR (DMSO d₆): δ (ppm) 2.3 (s,3H), 4.8 (s, 2H), 7.1-8.0 (m, 8H), 8.1 (s, 1H)

 

N-(4-hydroxybenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B6): 

Yield 81 %; Mp: 237°C

IR (KBr, cm-1): 3327 (NH), 2967 (CH), 1651 (C=N), 1690(C=O). MS: 326(M+).

1H NMR (DMSO d₆): δ (ppm) 4.8 (s, 2H), 7.4-9.3 (m, 7H), 8.1(s, 1H), 8.2(s, 1H)

 

N-(2-nitrobenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B7): 

Yield 72 %; Mp: 198°C

IR (KBr, cm-1): 3324 (NH), 2982 (CH), 1729(C=O), 1698 (C=N). MS: 325(M+).

1H NMR (DMSO d₆): δ (ppm) 4.8 (s, 2H), 7.6-9.2 (m, 8H), 8.1(s, 1H), 8.2(s, 1H)

 

N-(4-chlorobenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B8): 

Yield 83 %; Mp: 214°C

IR (KBr, cm-1): 3315 (NH), 2929 (CH), 1675 (C=N), 1715(C=O). MS: 315(M+).

1H NMR (DMSO d₆): δ (ppm) 4.8 (s, 2H), 7.2-9.3 (m, 8H), 8.1(s, 1H), 8.2(s, 1H)

 

N-(4-dimethylaminobenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B9): 

Yield 78 %; Mp: 270°C

IR (KBr, cm-1): 3365 (NH), 2910 (CH), 1587 (C=N), 1705(C=O). MS: 323(M+).

1H NMR (DMSO d₆): δ (ppm) 2.9 (s, 6H), 4.8 (s, 2H), 6.6-9.2 (m, 8H), 8.1(s, 1H), 8.2(s, 1H)

 

N-(2-chlorobenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B10): 

Yield 80 %; Mp: 215°C

IR (KBr, cm-1): 3310 (NH), 2913 (CH), 1663 (C=N), 1712(C=O).MS: 315(M+).

1H NMR (DMSO d₆): δ (ppm) 4.8 (s, 2H), 7.2-9.3 (m, 8H), 8.1(s, 1H), 8.2(s, 1H)

 

N-(2-hydroxybenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B11):

Yield 79 %; Mp: 232°C

IR (KBr): 3042(C-H), 3358 (NH), 1702(C=O), 1641(C=N). MS: 309(M+).

1H NMR (DMSO d₆): δ (ppm) 3.6 (s,3H), 4.7 (s, 2H), 6.7-9.4 (m, 8H), 8.1 (s, 1H), 8.2 (s, 1H)

 

N-(4-methoxybenzylidene)-2-(1H-benzo[d][1,2,3]triazol-1-yl)acetohydrazide (B12): 

Yield 88 %; Mp: 145°C

IR (KBr, cm-1): 2789 (C-H), 1713(C=O), 1651(C=N), 1341(C-N). MS: 309(M+).

1H NMR (DMSO d₆): δ (ppm) 3.7 (s,3H), 4.8 (s, 2H), 6.8-9.2 (m, 8H), 8.06 (s, 1H), 8.1 (s, 1H)

 

Antioxidant Activity:

For screening the antioxidant activity, two in vitro methods, DPPH scavenging and FRAP are used. 

 

DPPH assay:

The free radical scavenging activities of the synthesized compounds S1-S6 were determined by using DPPH method³⁰. 

 

All the Schiff bases were dissolved in DMSO and diluted to prepare the stock solution 5mg/ml. 150µl of freshly prepared DPPH solution (0.004% w/v) was taken in test tubes and stock solutions of the derivatives were added followed by serial dilutions (50 µg to 200 µg) to every test tube and the final volume was made to 3 ml with methanol. After 30 min, the absorbance was read at 517 nm using a spectrophotometer. Ascorbic acid was used as a reference standard (STD) and dissolved in methanol to make the stock solution with the same concentration (5 mg/ml). The control sample was prepared containing the same volume without any derivative and reference (ascorbic acid). Methanol was used as blank. The difference in absorbance between a test sample and a control was considered as activity. The obtained results are presented in fig. 2.

 

FRAP assay:

The FRAP assay was performed using Benzie and Strain method³¹. The principle of this method is based on the reduction of (TPTZ) ferric-tripyridyltriazine complex to its ferrous, coloured form in the presence of antioxidants. 300 mmol/l acetate buffer and 10 mmol/l of 2,4,6-tripyridyl-s-triazine (TPTZ) in a solution of 40 mmol/l (HCl) and 20 mmol/l of ferric chloride (FeCl₃·6H₂O) were mixed in the ratio of 10:1:1 to form the FRAP reagent. 0.5 ml of test sample and 3 ml of FRAP reagent were vortexed.  Absorbance was measured at 593 nm, and readings were calculated after calibration. The obtained results are presented in fig. 3.

 

 

Fig. 2: DPPH scavenging activity

 

 

Fig. 3: FRAP value

 

RESULTS AND DISCUSSION:

In this work, some hydrazone Schiff’s bases were synthesized, in good yield, by refluxing an equimolar mixture of hydrazides and aldehydes by adding catalytic amount of acetic acid over a period of 6 hrs, the reactions were monitored by TLC. The melting points of all synthesized compounds were found in open capillary tubes and readings were uncorrected. The synthesized compounds were confirmed by m/z value of molecular ion peak in mass spectroscopy. 

 

The formation of the title compounds is indicated by the disappearance of peak due to NH₂ of the starting material in IR and ¹HNMR spectrum of all the compounds. The IR spectrum showed the presence of peak absorbed in between at 1580-1700 cm-1, 2780- 3050 cm-1 due to -C=N stretching, =C-H stretching and ¹HNMR spectrum showed the presence of singlet at due to =CH group, and aryl groups at δ 6.6-9.5 of the all synthesized compounds. The mass spectrum of the compound showed molecular ion peaks corresponding to their molecular formula.

 

The synthesized compounds were screened for their in vitro antioxidant activity by DPPH scavenging and FRAP methods show good antioxidant activity, using ascorbic acid as standard. Compounds B3, B5, B6, B8, B11 and B12 gave better scavenging ability in both assays (DPPH and FRAP) as shown in fig.2 and fig. 3.

 

CONCLUSION:

A series of hydrazone schiff bases were prepared by reaction of benzotriazol hydrazide with various aromatic aldehydes. Synthesized compounds were characterized by spectral data and evaluated for in vitro antioxidant activity by DPPH and FRAP assays. Among the synthesized compounds B3, B5, B6, B8, B11 and B12, which are having electron donating groups on the phenyl ring exhibiting good activity with maximum scavenging free radical. Therefore, new ways, for possible pharmacophoric modifications, may help in exploiting the antioxidant activity.

 

ACKNOWLEDGEMENTS:

We are thankful to the Head of the Chemistry and Biology departments at Djelfa University for continued support of our work.

 

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Accepted on 31.05.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 4104-4107.