Design , Synthesis and Pharmacological Evaluation of New Nonsteroidal Anti-Inflammatory Derived from 3-Aminobenzothieno [ 2 , 3-d ] pyrimidines

During the last few years, condensed thienopyrimidine derivatives have received considerable attention. The therapeutic importance of thienopyrimidines prompted us to synthesize some of spiro(benzothieno[2,3-d]pyrimidine-4-one) derivatives. Some of the novel benzothino-pyrimidine derivatives 3a, 9b, 10b, 11a, 11b, and 11c showed considerable potent anti-inflammatory and analgesic activity of superior G.I.T. safety profile in experimental rats in comparing to indomethacin and tramadol as reference drugs.


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used therapeutics, primarily used for the treatment of pain and inflammation in arthritis for significant side effects of gastrointestinal lesions, bleeding, and nephrotoxicity.Therefore, the discovery of new safer anti-inflammatory drugs represents a challenging goal for such a research area, fused pyrimidines continue to attract considerable attention of researchers because of their great practical usefulness, primarily, due to a very wide spectrum of their biological activities.Thienopyrimidines occupy a special position among these compounds.Thienopyrimidine derivatives are characterized by a very broad spectrum of biological activities, such as anticancer [1][2][3][4], antiviral [5,6], antimicrobial [7][8][9], analgesic and anti-inflammatory [10][11][12][13][14] anticonvulsant [15,16], thymidine phosphorylase inhibitors [17], anti-HIV [18], and antihistaminic [19].The present work is an extension of our ongoing efforts towards the synthesis and evaluation of some new substituted thieno [2,3-d]pyrimidine derivatives as analgesic and anti-inflammatory agents.

Anti-Inflammatory Effect
The anti-inflammatory activity of fourteen of the newly synthesized compounds 3a, 3b, 3c, 5, 6, 7, 8a, 8c, 9b, 10a, 10b, 11a, 11b, 11c were evaluated by applying carrageenan-induced paw edema bioassay in rats [20]   indomethacin as a reference standard.Results were expressed as mean ± S.E.Difference between vehicle control and treatment groups were tested using one way ANOVA followed by the least significant difference (L.S.D.).Methods of statistical analysis were done according to Armitage et al. [21].
According to Table 1, administration of many of tested compounds 60 min prior to carrageenan injection at dose of 9 mg/kg b wt caused significant inhibition of paw edema response.Compounds 3a, 3b, 11a and 11c caused significant decreases in paw edema after 2, 3, 4 h after drug administration, while 9b and 10b gave their response after 2 h of administration and continued to the third hour.Compounds 8c and 11b showed the effect only after 2 h but compounds 3c, 5, 7 significantly decreased the paw edema after 4 h post administration.

Analgesic Activity
The analgesic activity of the fourteen derivatives was also evaluated by applying Hot plate test [22] using tramadol as a standard reference.Results were expressed as mean ± S.E.Difference between vehicle control and treatment groups were tested using one way ANOVA followed by the least significant difference (L.S.D.).Methods of statistical analysis were done according to Armitage et al. [21].
According to Table 2, compounds 3a, 8a, 10a, 11a and 11c showed significant analgesic activity higher than that obtained by Tramadol 1 h and 2 h post administration.Compounds 8c, 10b and 11b exhibited significant analgesic activity higher than or slightly equipotent to Tramadol only after 2 h of administration.Compounds 3b and 5 exhibited the analgesic effect after 1 h of administration only.Compounds 3c, 6, 7 and 9b have no analgesic activity Thus, it can be concluded that, compounds 3a, b, 5, 8a, c, 10a, b, 11a-c have significant analgesic activity and compound 11a is the most potent compound.

Ulcerogenic Effect
The ulcerogenic effect of the most active anti-inflammatory and analgesic derivatives 3a, b, 8a, 10a, 11a, c was evaluated [23].According to Table 3, it has been found that, compounds 3a, b, 8a, 11c have very little ulcerogenic effect with in comparison to indomethacin.Interestingly, compound 11a exhibited no ulcerogenic effect in all of the experimental animals.On the other hand compound 10a resulted in ulcer lesions in many of  the experimental rats.Therefore, the potential medicinal value of these compounds as anti-inflammatory and analgesic agents, that they have better safety margin than indomethacin on gastric mucosa.

Conclusion
Different fourteen compounds were evaluated as antiinflammatory and analgesic agents in experimental animals.It has been found that the compounds 3a, 3b, 8a, 10a, 11a, 11c exhibited the dual pharmacological activities with superior gastrointestinal safety profile when compared to indomethacin except 10a which resulted in ulcer lesions in many of the experimental rats.Surprisingly, compound 11a exhibited no ulcerogenic effect in all of the experimental animals.Thus, it can be concluded that spirobenzothienopyrimidine moiety, phenylpyrazolo-thinopyrimidine, morphonyl and piperazinylthinopyrimidine ring systems are important for both anti-inflammatory and analgesic activity of potent safety margin profiles towards gastrointestinal tract.
The second way for preparation.To a warmed ethanolic sodium hydroxide solution (0.40 g in 50 mL ethanol), compound 2 (10 m mol), and carbon disulfide (excess 5 mL) were added.The mixture was heated under reflux for 15 h.The reaction mixture was allowed to cool to 0˚C, the deposited precipitate was filtered off, washed by water (20 mL), dried, and crystallized from ethanol/acetone mixture as brown crystals; in 68% yield, m.p. 180˚C -183˚C.

Materials and Methods
Animals-adult rats of both sexes weighing 150 -200 g and adult mice weighing 20 -25 g were used in the experiments.Animals were housed under standardized conditions for light and temperature and received standard rat chow and tap water and libitum.Animals were randomly assigned to different experimental groups, each kept in a separate cage.All animal procedures were performed after approval from the Ethics committee of the National Research Center and in accordance with the recommendations for the proper care and use of laboratory animals (NIH publication No. 85-23, revised 1985).

Antiinflammatory Testing
The carrageenan rat paw edema model of inflammation was used to evaluate the anti-inflammatory properties of the tested compounds.Rats were randomly assigned to the treatment groups and sterile carrageenan lambda (100 ul of a 1% solution in saline) was injected sub-planter into right hind paw of the rat.Carrageenan caused visible redness and pronounced swelling that was well developed by 4 h and persisted for more than 48 h.Right hind paw was measured with a planimeter [24,25] before, and at 1, 2, 3 and 4 h after carrageenan injection.All the tested compounds were dissolved in DMSO then injected i.p. (9 mg/ kg b wt).The control animals were injected (i.p.) with appropriate volume of DMSO.The standard drug was indomethacin (10 mg/kg b wt).Different compounds or indomethacin were given 1 hr before carrageenan injection.

Analgesic Testing
The hot-plate test was performed on mice by using an electronically controlled hot-plate (Ugo Basile, Italy) heated to 52˚C, for possible centrally mediated analgesic effect of the drugs.Fourteen groups of rats were given vehicle and/or the different compounds and the last group received tramadol (20 mg/kg b wt) 60 min prior to testing.Latency to lick a hind paw or jumping [26] was recorded sequentially before and at 1, 2 h post treatment.

Ulcerogenic Effects
Groups of five male Wistar rats with a weight between 150 and 175 g are used.They are starved 48 h prior to drug administration.The test compounds are administered orally in 10 mL/kg as aqueous suspension.Doses which are highly active in the activity (9 mg/kg) are chosen and used.The animals are sacrificed after 7 h.Stomachs are removed and placed on saline soaked filter paper until inspection.A longitudinal incision along the greater curvature is made with fine scissor.The stomach is inverted over the index finger and the presence or the absence of gastric irritation is determined.The presence of a single or multiple lesions (erosion, ulcer or perforation) is considered to be positive [23].The number of ulcers and the occurrence of hyperemia is noted (determine ulcer index).

Table 2 . Analgesic effect.
aValues represent the mean ± S.E. of six animals for each groups.a P < 0.05: Statistically significant from Control (Dunnett's test).

Table 3 .
Ulcerogenic effect.Values represent the mean ± S.E. of five animals for each group.a P < 0.05: Statistically significant from ethanol treated rats (Kruskal Wallis, followed by Mann Whitney test).