Synthesis and Pharmacological Evaluation of Fenamate Analogues : 1 , 3 , 4-Oxadiazol-2-ones and 1 , 3 , 4-Oxadiazole-2-thiones

A series of fenamate pyridyl or quinolinyl analogues of 1,3,4-oxadiazol-2-ones 5a-d and 6a-r, and 1,3,4-oxadiazole-2-thiones 5e-g and 6s-v, respectively, have been synthesized and evaluated for their analgesic (hot-plate) , antiinflammatory (carrageenin induced rat's paw edema) and ulcerogenic effects as well as plasma prostaglandin E2 (PGE2) level. The highest analgesic activity was achieved with compound 5a (0.5 ,0.6 ,0.7 mrnolkg b.wt.) in respect with mefenamic acid (0.4 mmollkg b.wt.). Compounds 6h, 6l and 5g showed 93, 88 and 84% inhibition, respectively on the carrageenan-induced rat's paw edema at dose level of 0.1rnrnol/kg b.wt, compared with 58% inhibition of mefenamic acid (0.2mmoll kg b.wt.). Moreover, the highest inhibitory activity on plasma PGE2 level was displayed also with 6h, 6l and 5g (71, 70,68.5% respectively, 0.lmmolkg b.wt.) compared with indomethacin (60%, 0.01 mmolkg b.wt.) as a reference drug. In addition 6i, 6k, 6p, 6r, 6t and 6v were devoid of any ulcerogenicity.


Introduction
Many nonsteroidal antiinflammatories (NSAIDs) are now widely used in the treatment of inflammatory disorders.However , despite their wide spread use , none of the presently available agents are ideal , each has its shortcoming [I].The need for more effective and safer agents for treating inflammatory conditions is reflected in the growing list of new compounds undergoing clinical trials.The fenamates represent a class of NSAIDs that share as their common structural feature an N-arylanthranilic acid [2].Their mechanism of action is inhibtion of cyclooxygenase (COX) activity and thereby the production of prostaglandins [3].The fenamates are differentiated by their aryl substituents as shown by meclofenamic acid (la) [4], mefenamic acid (lb) [5] and flufenamic acid (lc) [6].The most active derivatives have substituents at positions 2 , 3 and 6 of the ring attached to the anthranilic acid nitrogen atom .Also, the pyridyl moiety of clonixin (2a) [7] and niflumic acid (2b) [8] can be considered isosteric to the aryl one of fenamic acid derivatives.Despite the significant potency exhibited by these fenamates, they display various side effects as gastrointestinal disturbances, ulcerogenicity [9] and renal toxicity [lo].Thus, among the attempts to achieve safer agents is the esterification of the carboxylic function.This is well represented by glaphenine (3a) [l 11 and floctaphenine (3b) [l 11, in which the N-aryl ring of fenamic acid is replaced by the substituted heterocylic quinolinyl moiety.3a : X= 7-C1 (Glaphenine); 3b : X= 8-CF3 (Floctaphenine Further development in this area is extended through search for other active heterocylic biological isosteres of N-arylanthranilic acid.Thus, the replacement of the carboxylic acid hnctionality of several fenamates with acidic heterocyles e.g.1,3,4oxadiazole-2-thione 4 provided dual inhibitor of cylooxygenase and 5-lipooxygenase enzymes [12].I Het 5a -g (Table I), 6a-v (Table 2) X = 0 or S RI,R2 = H, Cl, CH3, OCH3 Het = 4-pyridyl (5a-g), 4-quinolinyl(6a-v) All these premises focused the intense interest for the search of novel analgesic and antiinflammatory agents that belong to the class of fenamates having the general framework 5 and 6 in order to modulate the biologic effects and abolish others.

Scheme l a
2-Aminobenzoic acid hydrazides 8a-d [14](Scheme 1).General procedure for the synthesis of 5-(2-Aminoaryl)-1,3,4-oxadiazole-2(3I-3)-thiones 7e-g [15].Method B (Scheme I) To a solution of 1.4 g (0.025 mol) of potassium hydroxide in 5 ml water and 25 ml ethanol was added dropwisely under stirring and cooling (5 OC) a solution of 0.025 mol of the hydrazide 8a or 8c-d in 75 ml of ethanol.Carbon disulfide (0.55 ml, 0.027mol) was added to the previous mixture and the temperature was raised to 50-60°C during lh and heating was continued overnight.To a stirred solution of 0.01 mol of either 7a-d or 7e-g in 30 ml of absolute ethanol was added in portions 1.92 g (0.01 mol) of 4-bromopyridine hydrochloride.The reaction mixture was refluxed for 3h.The organic solvent was concentrated and the residual solid of either 5a-d or 5e-g, respectively, was crystallized from 2-propanol (c.f.Table I).A stirred solution of 0.01 mol of either 7a-d or 7e-g in 30 ml of absolute ethanol containing 1 drop of concentrated hydrochloric acid was heated gradually to 60 OC.The appropriate 4-chloroquinoline derivative (O.Olmo1) was added at once to the previous mixture, then refluxed for 3h.The formed yellow precipitate of either 6a-r or 6s-v was filtered and crystallized from 2-propanol: ethanol (c.f.Table 2).

Pharmacology:
1.Analgesic activity: The analgesic effect of the prepared compounds 5a-g and 6a-v was investigated using the hot-plate method[l7].The method depends on observing the normal response to a pain stimulus in untreated animals and comparing it with the response to the same stimulus after the administration of the tested compound at definite time intervals.The mouse response to heat is a convenient application of this principle.The mice were dropped gently in a dry glass beaker of one litre capacity maintained at 55-55.5"C.The normal reaction time in seconds for all animals was determined.The tested compounds 5a-g and 6a-v in doses of 0.5,0.6 and 0.7 mmollkg b.wt.as well as mefenamic acid in a dose of 0.4 mmolkg b.wt.as reference drug were subcutaneously (s.c.) administered in groups of mice (n=6) as an aqueous suspension in 7% tween-80 and the reaction time was re-determined at 10,20, 30,45 and 60 minutes intervals.Thereafter, the relative potency as well as the duration of action of the test compounds was compared with that of the standard drug.
The inhibitory activity of the tested compounds on carrageenan-induced rat's paw edema were determined according to the method of Winter et a1 [18] .Groups of adult male albino rats (120-160 g) of 6 animals each were fasted for 18 h before beingorally dosed with the tested compounds5a-gand6a-vin a dose of 0.1 mmollkg b.wt.[l9] (as aqueous suspension in 7% tween 80) one hour before carrageenan challenge.Foot paw edema was induced by subplanter injection of 0.05ml of 1% suspension of carrageenan in saline into the plantartissue of one hind paw.An equal volume ofsaline was injected into the other hind paw andserved as control.Four hours after compound administration, the animals were decapitated, blood samples were collected and the paws were rapidly excised.The average weight ofedema was estimated for the treated as well as the control groups and the percentage inhibition of weight of edema was also evaluated [20].Mefenamic acid (0.2 mmollkg b.wt.) [21] was employed as standard against which the tested compounds were compared.

3.Estimation of Plasma Prostaglandin E2 (PGE 2):
Plasma was separated from heparinized blood collected from rats ( n=6) by centrifkgation at 12000g for 2 minutes at 4°C and immediately stored frozen at -20°C until assayed.The Assay Designs'Correlate-EIA Prostaglandin E2 (PGE2) Kit is a competitive immunoassay for the quantitative determination of PGE2 in biological fluids [22].The kit uses a monoclonal antibody to PGE2 to bind, in a competitive manner, the PGE2 in the sample.After simultaneous incubation at room temperature, the excess reagent was washed away and substrate was added.After a short incubation time, the enzyme reaction was stopped and the yellow colour generated was read on a microplate reader (DYNATECH, MR 5000) at 405 nrn.The intensity of the bound yellow colour is inversely proportional to the concentration of PGE2 in either standards or samples.

4-Ulcerogenic effect in rats:
Groups of adult male albino rats of six animals each (120-160 g), were fasted overnight, then orally given the tested compounds (0.1 mmolkg b.wt.).Four hours later, animals were killed, their stomachs were removed, opened along the greater curvature, and the number of ulcers were assessed by adopting the method of Core11 et al [23].The results were compared with that of mefenamic acid (0.2 mmol/kg b.wt.) as reference drug.
Statistical a n d d a t a analysis: Data are expressed as means k s.e.m.Statistical comparison between different groups was done using one way analysis of variance (ANOVA), followed by multiple comparison test (post hoc LSD).Significance was accepted at p <0.05.
Conclusively, analgesic activity was augmented by the pyridyl oxadiazol-2-one and 2thione moieties, in addition to the trifluoromethyl quinolinyl one as achieved with compounds 5a > 5f >6i >6v, respectively at a dose level of 0.6 and 0.7 mmolkg b.wt., 30 to 45 min from compound administration.Their analgesic activities were higher than that of mefenamic acid (0.4 mmoVkg b.wt.) used as reference drug.

Plasma prostaglandin E2 (PGE2) level
The data presented in Table 8, illustrate the effect of compounds 5a-g and 6a-v on plasma PGEz level.In the pyridyl oxadiazol-Zones 5a- level in this group was arranged in the following decreasing order: 6t>6s>6u>6v.In general, the highest inhibitory effect on plasma PGE2 level was achieved with compounds 6h>61>5g (0.1 mmollkg b.wt.) in the previous decreasing order ( Fig. 3).
In conclusion, the highest analgesic activity was achieved with the pyridyl oxadiazol-2-one 5a (0.5, 0.6 and 0.7 mmol /Kg b.wt.).Also, the replacement of both the N-phenyl and carboxylic acid fbnctions of mefenarnic acid, with either substituted quinolinyl and oxadiazol-2-one moieties as represented with 6h and 61 or with pyridyl and oxadiazole-2thione such as 5g, augments the antiinflammatory activity compared with mefenamic acid as reference drug.Regarding the ulcerogenic effect of 5a, 6h and 61, it was much less than that of mefenarnic acid, while the same effect was observed with 5g.Since the mechanism of action of the fenamates is inhibtion of cyclooxygenase (COX) activity and thereby the production of prostaglandins [3] and since carrageenan in the paw edema model, increases COX-2 and PGE2 level [1,24] and inflammation could be blocked by a selective COX-2 inhibitor [25, we can deduce fi-om the present results that the most potent compounds under investigation may act through inhibition of COX-2.

"Table7:
Antiinflammatory activity and ulcerogenic effect of compounds 5a-g and 6a-v .a dose level of 0.1 rnmoY kg b.wt.c Mefenarnic acid at a dose level of 0.2 mmoY kg b.wt.d Each value represents the mean * s.e.m. of the number of animals in each group (n=6).e Each value represents the mean (ulcer number) * s.e.m.of the number of animals in each group (n=6).

Table 6 :
Analgesic activity of compounds 5a-g and 6a-v in adult male albino mice after the respective time from compound administration.

Table 6 (cont.):
At a dose level of 0.5,0.6 and 0.7 mmoY kg b.wt.c Mefenamic acid at a dose level of 0.4 mmoY kg b.wt.d Each value represents the mean reaction time in seconds *s.e.of the number of animals in each group (n=6).