INTRODUCTION:

Although anti-inflammatory drugs are used extensively to treat both acute and chronic inflammatory conditions, none is curative. In general, they suppress rather than abolish inflammatory reactions, thereby providing symptomatic relief. Sometimes limited efficacy reflects the complexity and multi-factorial nature of inflammatory processes. Proteolytic and other tissue-destructive enzymes become involved in chronic inflammation, but much less are known about both these mediators and those substances concerned with tissue repair. Substituted benzoxazole derivatives possessing anti-inflammatory^1,2, analgesic³ , anticancer⁴ and antimicrobial activities⁵ have been reported in some literatures. Apart from the above mentioned activities some interesting efficacy of anthelmintic⁶ , anti – HIV⁷, and protein kinase inhibitors⁸ activities of benzoxazole derivatives were also reported in the literatures.

A large number of heterocyclic compounds containing the benzoxazole rings are associated with diverse pharmacological properties such as anti-inflammatory, analgesic, antibacterial, antifungal, anticonvulsant, antidepressant, antiviral and anticancer activity. In view of this, several articles devoted to the synthesis and biological activity of substituted benzoxazoles.

For this, reason, we have taken up the synthesis of 2-(N¹-Substituted hydrazino)-N-[4-(5-methyl benzoxazol-2-yl)-phenyl]-acetamide to screen them for their possible anti-inflammatory and antimicrobial activity.

4-(5-Methyl-benzoxazol-2-yl)-Phenylamine (I) was obtained by heating 2-amino-4-methyl phenol with 4-amino benzoic acid in polyphosphoric acide between 220-230 ⁰C. Compound (I) on reaction with chloroacetyl chloride in benzene yielded 2-Chloro-N-[4-(5-Methyl-benzoxazol-2-yl)-phenyl]-acetamide(II). Treatment of compound (II) with hydrazine hydrate in ethanol gave 2-Hydrazino-N-[4-(5-methyl-benzoxzol-2-yl)-phenyl]-acetamide(III) Which was converted to Schiff base by the reaction of (III) with various aldehydes in presence of glacial acetic acid to get 2-(N¹-Substituted hydrazino)-N-[4-5-methyl benzoxazol-2-yl)-phenyl]-acetamide (IVa-j) in good yields (Scheme-1). The Characterization of the compounds has been done by spectral data. The physical and analytical datas are represented in Table-1.

EXPERIMENTAL:

The melting points were determined in open capillaries using Veego VMP-1 melting point apparatus and LABINDIA digital melting point apparatus expressed in ^oC and are uncorrected. The reactions were monitored by TLC (precoated-Merck) using Benzene:Acetone and detected by UV Chamber and also using iodine as visualizing agent. The IR spectra of the compounds were recorded on Perkin-Elmer Infrared-283 Spectrophotometer using KBr pellets and are expressed in cm^-1.

Table No:1 Physical and Analytical Data of 2-(N’-Substituted –hydrazino)-N-[4-(5-methyl-benzoxazol-2-yl)-phenyl]-acetamide

Compound No.	Substituents	Molecular Formula	Molecular Weight	Melting Point ⁰C (Range)	Yield (%)	R_f value	TLC Mobile Phase Benzene: Acetone
Compound No.	R	Molecular Formula	Molecular Weight	Melting Point ⁰C (Range)	Yield (%)	R_f value	TLC Mobile Phase Benzene: Acetone
IV_a	C₆H₅	C₂₃H₂₀N₄O₂	384	128-130	76	0.59	4:3
IV_b	C₆H₄NO₂	C₂₃H₁₉N₅O₄	429	226-228	82	0.48	4:1
IV_c	C₆H₅O	C₂₃H₂₀N₄O₃	400	207-209	74	0.64	4:1
IV_d	C₆H₄Cl	C₂₃H₁₉N₄O₂Cl	418	212-214	70	0.60	7:3
IV_e	C₇H₇O₂	C₂₄H₂₂N₄O₄	430	198-200	76	0.55	7:3
IV_f	C₆H₅O	C₂₃H₂₀N₄O₃	400	220-222	79	0.61	4:2
IV_g	C₈H₁₀N	C₂₅H₂₅N₅O₂	427	186-188	72	0.59	4:2
IV_h	C₆H₄F	C₂₃H₁₉N₄O₂F	402	215-217	81	0.58	7:3
IV_i	C₆H₃Cl₂	C₂₃H₁₈N₄O₂Cl₂	452	216-218	70	0.63	4:2
IV_j	C₅H₃O	C₂₁H₁₈N₄O₃	374	200-202	78	0.65	7:3

Solvent for Recrystallization : Acetone

Table No. 2 Effect of some new Benzoxazole derivatives on Carrageenan-induced Paw Edema in Rats

Group	Treatment	Dose (mg/kg, p.o)	Paw volume at the end of 3hr (ml)	% inhibition
1	Control (CMC 0.5% w/v)	0.5	0.43 + 0.03	--
2	Indomethacin	10	0.10 + 0.01*	76.74
3	Compound IV_b	100	0.37 + 0.005**	13.95
4	Compound IV_c	100	0.30 + 0.004*	30.23
5	Compound IV_e	100	0.32 + 0.006**	25.58
6	Compound IV_h	100	0.40 + 0.005**	6.9

Each value represents the mean + S.E.M of six observations. * p < 0.01 ; ** p < 0.05 Vs control Data were analysed one way ANOVA followed by Dunnet Test.

Nuclear Magnetic spectra (¹H-NMR) were obtained from dpx-300 MHx Spectrophotometer using DMSO as solvent with TMS as the internal standard.

Synthesis of 4-(5-Methyl Benzoxazol-2-yl)-Phenylamine (I):¹¹

A mixture of 2-Amino-4-Methyl Phenol (0.02mol) and p-amino benzoic acid (0.02mol) were mixed with Polyphosphoric acid (PPA) to get a stirrable paste. The mixture was heated to reflux for 3 hours using an oil bath at a temperature of 220-230^oC. At the end of the reaction period the mixture was poured on to the crushed eyes with continuous stirring. It was neutralized with 10% Sodium Hydroxide solution. The Precipitated product was then filtered with suction and washed with cold water. The product was dried in hot air oven and recrystallized from ethanol.

IR Spectrum (KBr in cm^-1) exhibited characteristic absorption frequencies at : 3290 (NH₂), 1636 (C=N), 1576 (C=C) and 1320 (C-O-C).

¹H NMR (DMSO, in δ): 2.3 (s, 3H, CH₃), 5.4 (brs, 2H, NH₂), and 7.9-8.3 (m, 7H Ar-H R=Phenyl)

Synthesis of 2-Chloro-N-[-4-(5-Methyl Benzoxazol-2-yl)-Phenyl]-acetamide (II):¹²

To a solution of 4-(5-methyl benzoxazol-2-yl)-Phenylamine (0.005mol) in reflexing benzene (50ml) was added drop wise Chloroacetyl Chloride (0.006mol), and the mixture was further refluxed for 13 minutes. The excess of benzene distilled off and the product was decomposed on to crushed ice and precipitate collected by filtration. The product was washed with cold water, dried and recrystallized from ethanol.

IR Spectrum (KBr in cm^-1) exhibited characteristic absorption frequencies at: 3244 (NH), 1677 (C=O), 16534 (C=N), 1568 (C=C), 1318 (C-O-C) and 803 (C-CI).

¹H NMR (DMSO, in δ): 2.3 (s, 3H, CH₃), 2.6 (s, 2H, CH₂CI), 7.9-8.3 (m, Ar-H R=Phenyl) and 9.6-9.8 (s, 1H, NHCO) MS m/z:300(M+)

Synthesis of 2-Hydrazino-N-[4-(5-methyl-benzoxazol-2-yl)-phenyl]-acetamide (III):¹³

A mixture of 2-Chloro-N-[4-(5-methyl-benxoxazol-2-yl)-phenyl]-acetamide (0.026mol) and hydrazine hydrate (0.13mol) in ethanol (50 ml) were refluxed under stirring for 2 hrs. At the end of reaction period, the mixture was cooled and poured on to crushed ice. The solution was kept aside for 1 hour and the product resulted was filtered and washed with cold water dried with hot air oven and recrystallized from ethanol.

IR Spectrum (KBr in cm^-1) exhibited characteristic absorption frequencies at: 3345 (NH₂), 3274 (NH), 1682 (C=O), 1638 (C=N), 1562 (C=C), 1312 (C-O-C).

¹H NMR (DMSO, in δ): 2.1 (s, 3H, CH₃), 4.2 (brs, 2H, COCH₂), 7.5-7.9 (m, 7H, Ar-H R=Phenyl) and 8.6 (brs, 3H, -NHNH₂) and 9.8 (s, 1H, NHCO) MS m/z: 296 (M+)

Synthesis of 2-(N¹-Substituted-hydrazino)-N-[4-(5-methyl-benzoxazol-2-yl)-phenyl]-acetamide (IVa-j): ¹³

A mixture of 2-Hydrazino-N-[4-(5-methyl-benzoxazol-2-yl)-phenyl]-acetamide (2.0g, 0.007 mol) and appropriate substituted aldehyde (0.007mol) in 15ml glacial acetic acid refluxed for 30minutes. At the end of the reaction period, it was cooled and poured into crushed ice and stirred. It was kept aside for over night and the product obtained was filtered and washed with cold water. It was recrystallized from acetone to get a pure compound.

IR Spectrum (KBr in cm^-1) exhibited characteristic absorption frequencies at 3345 and 3277 (NH and NH -stretching), 1684 (C=O), 1603 (C=N), 1560 (C=C), 1500 (C-N of N-CH-Ar), 1261 (C-O-C) and 794 (C-CI).

¹H NMR (DMSO, in δ): 2.1 (s, 3H, CH₃), 4.3 (brs, 2H, COCH₂), 7.5-7.9 (m, 12H, Ar-H, R=Phenyl), 8.7(s, 1H, N=CH) and 9.8 (s, 1H, NHCO). MS m/z: 384 (M+) (R=Phenyl)

Anti-inflammatory Screening

Carrageenan induced rat hind paw edema⁹.

In LD₅₀ studies it was found that the compounds selected were safe upto a maximum dose of 2000 mg/kg body weight. There were no changes in normal behaviour pattern and no sign and symptoms of toxicity and mortality were observed. Hence the anti-inflammatory studies were carried out at a dose of 100mg/kg body weight. The anti-inflammatory activity of synthesized benzoxazole derivatives were studied using carrageenan induced rat hind paw edema. The effect of the test compounds on carrageenan-induced edema was depicted in Table 2. After 30 minutes, all the test benzoxazole compounds showed statistically significant decrease in the paw volume. Rats treated with compound IV_c showed significant decreased in paw volume (30.23, P < 0.01) when compared to control However, the standard drug Indomethacin showed significant reduction (76.74 %) in paw volume at the end of 3hr.

Antimicrobial activity¹⁰

Antibacterial Screening and Antifungal Screening:

The synthesized compounds were screened for antibacterial activity against three Gram-positive bacteria such as Bacillus subtilis, Bacillus Stearothermophilus, and microccus leuteus, as well as against three Gram-negative bacteria like Klebsiella pneumoniae, Pseudomonas cepacia and Salmonella typhi by the cup plate method. The synthesized compounds were screened for antifungal activity against the fungi Candida albicans, Saccharomyces Cerevisiae and Trichosporon beigelii by the cup plate method.

The samples were taken in the concentration of 1000µg/well in DMSO. Neutrient agar media was used for antibacterial screening and Sabouraud,s dextrose agar media was used for antifungal activity. Streptomycin and Griseofulvin at 50-mcg/ml concentrations in DMSO was used as reference standard for antibacterial and antifungal activity respectively. The plate containing bacteria organisms were incubated at 37 ± 0.5^oC for 48 hrs.The zone of inhibition was calculated by measuring the diameter of the inhibition zone. All the compounds were tested for antibacterial activity against all the bacteria at the concentration of 1000µg/well.

RESULTS AND DISCUSSION:

In case of anti-inflammatory activity, compound IVc showed good anti-inflammatory activity compare to control group and all the compounds were tested for their antimicrobial activities, none of the compounds showed activity against bacteria and fungi.

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