INTRODUCTION:

Non-steroidal anti-inflammatory drug (NSAIDs) are most preferred category of drugs used in the treatment of fever, pain, inflammatory diseases and rheumatoid arthritis¹. Chronic use of NSAIDs gives different side effect like GI irritation, bleeding and ulceration. The GI damage from NSAIDs is generally attributed to two factors, local irritation by the carboxylic acid moiety, which are common to most NSAIDs (topical effect) and decreased tissue prostaglandin production, which undermines the physiological role of cytoprotective prostaglandins in maintaining the GI health and homeostasis².

It has been reported that the derivatization of the carboxyl function of representative NSAIDs, resulted in an increased anti-inflammatory activity with reduced ulcerogenic effect³.

The replacement of the carboxylic acid functionality of some NSAIDs, eg, flufenamic and meclofenamic acids, with a tetrazole group not only retained COX inhibitory activity of the parent drug, but also introduced 5-lipooxygenase (5-LOX) inhibition⁴. Several other heterocyclic compounds, including di-tert-butylphenyloxadiazoles, thiazoles or imidazoles and substituted oxadiazole and thiadiazole derivatives^5-7 have been studies and proved to be potent COX/5-LOX inhibitors.

Synthetic approaches based upon NSAIDs chemical modification have been taken with the aim of improving NSAID safety profile. It has been reported in literature that certain compounds bearing 1,3,4-oxadiazole nucleus possess significant anti-inflammatory activity with reduced ulcerogenicity^8-9. In continuation¹⁰of our attempt to discover new and useful agents for treatment of inflammatory diseases, we have synthesized 1,3,4-oxadiazole derivatives, which have been found to possess an interesting profile of anti-inflammatory activity with significant reduction in their ulcerogenic effect.

MATERIAL AND METHODS:

Chemicals used in this synthetic work were purchased from S.D. Fine Chem. Ltd. Mumbai, and Sigma Aldrich India (Merck). Solvents except laboratory reagent grade were dried and purified according to the literature when necessary. Purity of the compounds was checked on TLC plates using silica gel G as stationary phase and iodine vapors as visualizing agent. Melting points of synthesized compounds were determined using Thermonik melting point apparatus and are uncorrected, IR spectra were recorded on Thermo Nicolet Spectrophotometer by using KBr pellets. The ¹HNMR was recorded on Bruker Avance II NMR 500 MHz instruments using appropriated solvent and TMS as internal standard, chemical shifts are expressed as δ values (ppm).

Synthesis and spectral studies:

The title compounds were synthesized as given in the scheme 1.

Synthesis of methyl ester of Asprin (methyl 2-(acetyloxy) benzoate) (I):

The methyl ester was prepared as per procedure reported in the literature¹¹. M.P. 152-154°C, yield: 76.43%. IR Spectra showed bands at 3082(C-H), 1730(C=O), 1238(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm): 7.93-7.12(m, 4H, Ar), 2.45 (s, 3H of CH₃), 2.15 (s,3H of COOCH₃),

Synthesis of 2-(hydrazinylcarbonyl)phenyl acetate (II):

Compound I (0.01mol) and hydrazine hydrate (99%) (0.02mol) were refluxed in absolute methanol (50ml) for 20 hours (monitored by TLC). The mixture was concentrated, cooled and poured in ice cold water. The solid thus separated out was filtered, dried and recrystallized from ethanol: water (4:1). M.P. 164-166°C, yield: 75%. IR Spectra showed bands at 3325(N-H), 3022(C-H), 1637(C=O). ¹H NMR chemical shift at (CDCl₃, δ ppm): 7.45 (s,1H of CONH), 7.45-7.02(m, 4H, Ar), 4.15 (s,2H of NH₂ ), 2.35 (s, 3H of CH₃).

Synthesis of 2-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl) phenyl acetate (III):

A mixture of II (0.005mol) KOH (0.005mol) and carbon disulphide (5ml) in methanol (50ml) was refluxed on a steam bath for 12 hours (monitored by TLC). The solution was then concentrated, cooled and acidified with dil. HCl. The solid mass that separated out was filtered, washed with ethanol, dried and recrystallized from ethanol: water (4:1). M.P. 182-184°C, yield: 72.68%. IR Spectra showed bands at 3373(N-H), 3086(C-H), 1577(C-N), 1162(C=S). ¹H NMR chemical shift at (CDCl₃, δ ppm): 10.85(s, 1H, N-H of 1,3,4-oxadiazole), 8.15-7.82(m, 4H, Ar), 2.73 (s, 3H of CH₃).

Scheme 1

General procedure for the synthesis of derivatives (IVa-h):

To a solution of III (0.01mol) in ethanol, a mixture of formaldehyde (0.015mol) and a secondary amine (0.01 mol) in ethanol was added with stirring. After complete addition, the stirring was continued overnight at room temperature. The precipitated solids were filtered, washed with water and recrystallized from methanol.

2-[4-(morpholin-4-ylmethyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl]phenyl acetate (IVa): This was obtained by reacting III, (0.01mol) and morpholine (0.015mol) as described in general procedure M.P.208-210°C, yield: 73.78%. IR Spectra showed bands at 3325(N-H), 3068(C-H), 1576(C=N), 1511(C=C), 1312(C=S), 1249(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm):8.10-7.82(m, 4H, Ar), 2.85 (s, 3H of CH₃), 4.83(s, 2H, N-CH₂-N), 3.58-3.65(t, 4H, morpholine), 2.69-2.76(t, 4H, morpholine).

2-[4-(piperidin-1-ylmethyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl]phenyl acetate (IVb): This was obtained by reacting (III, 0.01mol) and piperidine (0.015mol) as described in general procedure. M.P.178-180°C, yield: 72.68%. IR Spectra showed bands at 3377(N-H), 2926(C-H), 1575(C=N), 1505(C=C), 1352(C=S), 1245(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm): 7.95-7.72(m, 4H, Ar), 2.45 (s, 3H of CH₃), 4.85(s, 2H, N-CH₂-N), 2.82-2.72(t, 4H, piperidine), 1.64(m, 2H, piperidine), 1.51-1.24(m, 4H, piperidine).

2-{4-[(2-methylpiperidin-1-yl)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}phenyl acetate (IVc): This was obtained by reacting (III, 0.01mol) and 2-methyl piperidine (0.015mol) as described in general procedure. M.P.221-223°C, yield: 77.85%. IR Spectra showed bands at 3307(N-H), 2942(C-H), 1578(C=N), 1507(C=C), 1299(C=S), 1237(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm):8.55-7.72(m, 4H, Ar), 2.55 (s, 3H of CH₃), 4.33(s, 2H, N-CH₂-N),), 2.80-2.72(m, 1H, piperidine), 1.74-1.55(m, 6H, piperidine), 1.37-1.35(d, 3H, 2-methyl piperidine), 1.25(m, 2H, piperidine).

2-[4-(piperazin-1-ylmethyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl]phenyl acetate (IVd): This was obtained by reacting (III, 0.01mol) and piprazine (0.015mol) as described in general procedure. M.P.218-220 °C, yield: 73.80%. IR Spectra showed bands at 3317(N-H), 2952(C-H), 1588(C=N), 1515(C=C), 1310(C=S), 1247(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm): 7.90-7.82(m, 4H, Ar), 2.75 (s, 3H of CH₃), 4.33(s, 2H, N-CH₂-N), 3.95 (s, 1H, piperazine-NH), 3.88-3.65(t, 4H, piprazine), 2.59-2.77(t, 4H, piprazine).

2-{4-[(4-methylpiperazin-1-yl)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}phenyl acetate (IVe): This was obtained by reacting (III, 0.01mol) and N-methyl piprazine (0.015mol) as described in general procedure. M.P.188-190°C, yield: 74.78%. IR Spectra showed bands at 3324(N-H), 2938(C-H), 1578(C=N), 1504(C=C), 1288(C=S). ¹H NMR chemical shift at (CDCl₃, δ ppm): 8.18-7.62(m, 4H, Ar), 2.65 (s, 3H of CH₃), 4.63(s, 2H, N-CH₂-N), 2.43(s, 3H, CH₃), 2.32-2.77(t, 4H, piperazine), 2.59-2.32(t, 4H, piperazine).

2-{4-[(diethylamino)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}phenyl acetate (IVf): This was obtained by reacting 1 (III, 0.01mol) and diethyl amine(0.015mol) as described in general procedure. M.P.198-200°C, yield: 76.78%. IR Spectra showed bands at 3347(N-H), 2940(C-H), 1580(C=N), 1520(C=C), 1280(C=S), 1257(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm): 8.28-7.62(m, 4H, Ar), 2.45 (s, 3H of CH₃), 4.43(s, 2H, N-CH₂-N), 2.80-2.70 (m, 4H, CH₂, CH₂), 1.59-1.48(t, 6H, CH₃, CH₃).

2-{4-[(diphenylamino)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}phenyl acetate (IVg): This was obtained by reacting (III, 0.01mol) and diphenyl amine (0.015mol) as described in general procedure. M.P.228-230°C, yield: 70.50%. IR Spectra showed bands at 3327(N-H), 2962(C-H), 1548(C=N), 1527(C=C), 1279(C=S), 1267(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm) 7.76-7.55 (m,4H, Aromatic), 7.12-6.95 (m,10H of diphenyl amine, 2.55 (s, 3H of CH₃), 4.23(s, 2H, N-CH₂-N).

2-{4-[(cyclohexylamino)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}phenyl acetate (IVh): This was obtained by reacting (III, 0.01mol) and cyclohexyl amine (0.015mol) as described in general procedure. M.P.192-194 °C, yield: 75.50%. IR Spectra showed bands at 3347(N-H), 2952(C-H), 1576(C=N), 1512(C=C), 1299(C=S), 1277(C-O-C). ¹H NMR chemical shift at (CDCl₃, δ ppm) 7.96-7.55 (m,4H, Aromatic), 2.65 (s, 3H of CH₃), 4.73(s, 2H, N-CH₂-N), 2.57-2.25 (m,11H cyclohexyl amine), 2.22 (s,1H NH).

Anti-inflammatory activity¹²

Table No- 2 reveals acute anti-inflammatory activity of title compounds IVa-h at a dose of 10mg/kg by carrageenan induced rat paw edema method. As shown in table 2, the entire investigated compound exhibited moderate to good anti-inflammatory activity. The anti-inflammatory activity of those compounds was comparable to that of the standard drug aspirin. Compounds that showed good anti-inflammatory activity profile were further evaluated for analgesic, antipyretic and ulcerogenic activity.

Analgesic activity¹³

Table No- 3 reveals the analgesic activity of title compounds at the dose of 10mg/kg by acetic acid induced writhing test in mice. From the result, it was noticed that all compounds possess significant analgesic activity.

Antipyretic activity:¹⁴

Furthermore, the encouraging results from anti-inflammatory and analgesic activity prompted us to carry out the antipyretic activity in yeast-induced pyrexia for 10mg/kg in rats. Results of antipyretic activity are given in table no 4.

Ulcerogenic activity:¹⁵

The major drawback of NSAIDs is their gastric ulcer formation due to gastric irritation. The extent of ulcerogenic effect was evaluated for compounds IVa and IVb in rat stress model at the therapeutic dose (i.e. 10 mg/kg). The gastric ulcerogenic potential was evaluated by calculating the ulcer index in treated and control animals. Result are given in Table no-5 which indicate that the compounds IVa induces less ulcer compared to standard drug aspirin. Hence gastric tolerance to these compounds was better than that of standard drug.

RESULT AND DISCUSSION:

The Aspirin was converted to its methyl esters (I) by esterification. This methyl ester was reacted with hydrazine hydrate, gave carbohydrazide 2-(hydrazinylcarbonyl) phenyl acetate (II). This carbohydrazide was treated with CS₂/ KOH in methanol gave 1,3,4-oxadiazole with aspirin moiety 2-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl) phenyl acetate (III). The purity of the compounds was confirmed by melting point, TLC and structure was confirmed by IR and ¹HNMR spectral data.

Treatment of 2-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl) phenyl acetate (III) with various amines in presence of formaldehyde gave the title compounds IVa-h. The purity of these compounds were assessed by melting point, TLC and structure were confirmed by IR, and ¹HNMR. Physicochemical data of the different synthesized title compounds (IVa-h) are given in Table 1.

All these newly synthesized compounds (IVa-h) were screened for acute anti-inflammatory activity using carrageenan induced paw edema method and analgesic activity by acetic acid induced writhing method. Results of acute anti-inflammatory studies showed compounds IVa, IVb, IVd, and IVe showed promising activity and in analgesic studies compounds IVa and IVb showed better activity as compared to standard drug Aspirin. The potent compounds were further screened for antipyretic and ulcerogenic activities.

Compound IVa showed good antipyretic activity and less ulcerogenic activity as compared to standard drug ibuprofen.

Further detailed studies are needed to know the mechanism of action and site of action of these compounds.

Table 1: Physicochemical data of the different synthesized title compounds (IVa-h)

Compound	R	M.P. (°C)	Yield (%)	Rf ^*	Molecular formula	Molecular Weight
IVa		208-210	73.78	0.45	C₁₅H₁₇N₃O₄S	335
IVb		178-180	72.68	0.48	C₁₆H₁₉N₃O₃S	333
IVc		221-223	77.85	0.36	C₁₇H₂₁N₃O₃S	347
IVd		218-220	73.80	0.58	C₁₅H₁₈N₄O₃S	334
IVe		1188-190	74.78	0.56	C₁₆H₂₀N₄O₃S	348
IVf		198-200	76.78	0.38	C₁₅H₁₉N₃O₃S	321
IVg		228-230	70.50	0.43	C₂₃H₁₉N₃O₃S	417
IVh		192-194	75.50	0.53	C₁₇H₂₁N₃O₃S	347

All compounds were recrystallized by methanol.

Stationary phase^*- silica gel G.

Mobile phase- ethyl acetate: chloroform (4:1).

Visualizing agent- iodine vapours.

Table No-2 Anti-inflammatory activity of title compounds (IVa-h) by carrageenan induced paw edema.

Compound	Paw volume		% Inhibition of edema
Compound	3 hr	5 hr	3 hr	5 hr
Control	2.49±0.13	2.90±0.07	-	-
IVa	0.66±0.01	0.83±0.01	73.1^*	71.0^*
IVb	0.87±0.07	1.03±0.06	65.2^*	64.5^*
IVc	1.05±0.24	1.57±0.10	57.8^*	45.9^*
IVd	0.92±0.07	1.02±0.07	63.1^*	64.8^*
IVe	0.92±0.07	1.02±0.07	62.1^*	64.8^*
IVf	1.17±0.05	1.57±0.05	53.0^*	45.9^*
IVg	1.57±0.10	1.92±0.05	36.9^*	33.8^*
IVh	1.45±0.08	1.71±0.09	42.8^*	41.0^*
Aspirin	0.85±0.02	0.94±0.07	65.9^*	67.5^*

Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6), *P < 0.001 significant from control.

Table No-3 Analgesic activity compounds IVa, IVb, IVd and IVe by acetic acid induced writhing method.

Compound	No. of writhes in 15 min.	% Protection
Control	29	-
IVa	5.50	81.0^*
IVb	6.00	79.3^*
IVd	7.00	75.9^*
IVe	7.33	74.7^*
Aspirin	6.33	78.2^*

Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6), *P < 0.001 significant from control.

Table No-4 Antipyretic activity of IVa and IVb, by yeast induced pyrexia

Compound	Mean temperature in °C at intervals^b
Compound	0^a	1	2	3	4	5	TI^c
Control	38.30	38.26	38.20	38.11	38.00	37.89	- 1.04
IVa	38.10	37.70^***	37.55^***	37.13^***	37.06^***	37.15^***	- 3.91
IVb	38.01	37.74^*	37.77^*	37.36^***	37.06^***	37.11^**	- 3.06
Paracetamol	38.37	38.10^*	37.90^***	37.76^***	37.51^***	37.36^***	- 3.22

Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6), *P< 0.05, ** P< 0.01, ***P<0.001 significant from control. a Eighteenth hour after yeast injection was considered as 0 h. b Temperature was recorded hourly from 0 to 5 h after dosing. c Temperature index (TI) is the sum of mean temperature changes from the 0 h.

Table No-5 Ulcer index of IVa and IVb

Compound	Ulcer index(±SEM)
Control	0.75 (±0.17)
IVa	1.93 (±0.36)^*
IVb	2.08 (±0.24)^*
Aspirin	2.15 (±0.25)^a

Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6). ^aP < 0.001, as compared to control. ^*P < 0.001, as compared to standard.

CONCLUSION:

Various oxadiazole derivatives derived from Aspirin was prepared with the objective of developing better anti-inflammatory molecules with minimum ulcerogenic activity and also to evaluate their analgesic and antipyretic activity. All these newly synthesized compounds IVa-h were screened for acute anti-inflammatory activity using carrageenan induced paw edema method. Results of acute anti-inflammatory studies showed compounds IVa, IVb, IVd and IVe showed promising activity. The title compounds were also found to have significant analgesic activity in the acetic acid induced writhing model and antipyretic activity in yeast-induced pyrexia model. In addition, the tested compounds were also found to possess less degree of ulcerogenic potential as compared to aspirin. Thus these compounds constitute an interesting template for the evaluation of new inflammatory inhibitors and may be helpful for the design of new therapeutic tools against inflammation.

ACKNOWLEDGEMENT:

We express our thanks to Indian Institute of science education and research, Bhopal and Indian institute of science, Bangalore for providing spectral analysis of synthesized compounds. We are grateful to Deshpande Laboratories Pvt. Ltd., Bhopal, for permission to screen the pharmacological activities of synthesized compounds.

CONFLICT OF INTEREST:

None

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Received on 17.11.2019 Modified on 05.02.2020

Research J. Pharm. and Tech. 2020; 13(12):5898-5902.

DOI: 10.5958/0974-360X.2020.01029.X