A Potent Cyclooxygenase-2 Inhibitor for Synthesized Pyrimidine and...

[Full title: A Potent Cyclooxygenase-2 Inhibitor for Synthesized Pyrimidine and Thiazolopyrimidine Derivatives]

OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2016.86.91 Research Article A Potent Cyclooxygenase-2 Inhibitor for Synthesized Pyrimidine and Thiazolopyrimidine Derivatives 1,2 Abd El-Galil E. Amr, 1 Mohamed A. Al-Omar and 3 Mohamed M. Abdalla 1 Department of Pharmaceutical Chemistry, Drug Exploration and Development Chair (DEDC), College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia 2 Department of Applied Chemistry, National Research Center, Dokki, Cairo, Egypt 3 Research Unit, Saco Pharm. Co., 6 th October City 11632, Egypt Abstract Ten pyridine and pyrimidine and thiazolopyrimidine derivatives (1-10) were synthesized and screened as analgesic, anticonvulsant and antiparkinsonian agent before. Herein, all the target compounds showed anti-inflammatory activity. The active compounds showed selective inhibitory activity towards COX-2 enzyme as revealed by the in vitro enzymatic assay. All the tested compounds proved to have superior gastrointestinal (GI) safety profiles as compared to indomethacin, when tested for their ulcerogenic effects Key words: Pyridine derivatives, thiazolopyrimidine, anti-inflammatory activities, COX-2 inhibitors Received: August 13, 2015 Accepted: December 28, 2015 Published: January 15, 2016 Citation: Abd El-Galil E. Amr, Mohamed A. Al-Omar and Mohamed M. Abdalla, 2016. A Potent Cyclooxygenase-2 Inhibitor for Synthesized Pyrimidine and Thiazolopyrimidine Derivatives. Int. J. Pharmacol., 12: 86-91 Corresponding Author: Abd El-Galil E. Amr, Department of Pharmaceutical Chemistry, Drug Exploration and Development Chair (DEDC), College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Copyright: © 2016 Abd El-Galil E. Amr et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 12 (2): 86-91, 2016 Celecoxib F C 3 N N CH 3 NH 2 S O O HO OH HO NH 2 O N N H O HO Br S N N N N Pyrazofurin 3-(5-bromothiophen-2-yl)-1-phenyl-4- (1-phenyl-3-p-tolyl-1 H-pyrazol-5-yl)-1 H-pyrazole INTRODUCTION Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) recognized as the most important class of clinically used agents for the treatment of pain and inflammatory manifestations associated with a number of pathological conditions. However, long term treatments with NSAIDs associated with numerous side effects such as gastrointestinal mucosal damage, bleeding, intolerance and renal toxicity (Sontag, 1986; Flower, 2003; Allison et al ., 1992) Consequently, extensive research work oriented towards improving their pharmacological profile that led to the discovery of multiple isoforms of cyclooxygenase (COX) that are differently regulated (Chandrasekharan et al ., 2002; Song et al ., 1999). The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents free of the undesirable effects associated with old classical NSAIDs. A novel class of selective COX-2 inhibitors has been discovered. Amongest this class, celecoxib (Fig. 1) was shown to be a potent and gastrointestinal (GI) safe anti-inflammatory agent. Chemically it is pyrazole containing, diaryl-heterocyclic template that is known to selectively inhibit COX-2 (Palomer et al ., 2002) Several compounds containing pyrazole moity were also reported to exhibit anti-inflammatory activity with acceptable safty margines (Tsuji et al ., 1998; Beers et al ., 1997). Recently, it was worth to mentioned, benzene sulfonamides (Bekhit et al ., 2008), pyrimidines (Venu et al ., 2008), imidazoles (Salimi et al ., 2007) and thiazolidinones (Hu et al ., 2013) are other important pharmacodynamic heterocyclic nuclei which when incorporated into different heterocyclic templates, have been reported to possess excellent potent anti-inflammatory activity. In view of these observations and in continuation of our previous work in pyridine and pyrimidine chemistry, some heterocyclic compounds containing the pyridine derivatives; thiazolopyrimidine moiety were synthesized and tested their anti-inflammatory activities MATERIALS AND METHODS Chemistry: All the tested compounds were confirmed by physical and spectroscopic evidences according to the previously reported procedures (Amr et al ., 2005) Pharmacological activities Experimental animals: All animals were obtained from National Research Center, Cairo, Egypt, Giza, Egypt and were acclimatized for 10 days under standard housing conditions (24±1 E C; 45-55% RH with 12:12 h light/dark cycle). The animals had free access to rat food and water. The animals were habituated to laboratory conditions for 48 h prior to the experimental protocol to minimize any nonspecific stress. Animals were maintained under standard conditions in the animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) Cotton pellet-induced granuloma bioassay: The experimental method which was used in cotton pelletinduced granuloma bioassay has been adopted from Bekhit et al . (2008) Carrageenan-induced rat paw edema: The experimental method which was used in carrageenan-induced rat paw edema has been adopted from Di Rosa and Willoughby (1971) Human COX-1 and COX-2 enzymatic assay: Human COX-1 and COX-2 activities were determined as described by Wakitani et al (1998) Fig. 1: Structures of celecoxib and reported active pyrazole derivatives 87

Int. J. Pharmacol., 12 (2): 86-91, 2016 O HO HN NH H S OH O H H O O O O O S CN O CI N N O CI CI O N CI CI (1) S CNO ONC S N O O N N O N CI CI O N (2) EtO O O S N N O O S O O OEt (3) O O O H H (4) (5) (6) CI HO HN NH S H OH CI (7) S O H N N H HO OH (8) O N 2 O H N N H S HO OH CI CI (9) O N 2 O H H N N HO OH S (10) Ulcerogenic effects: All target compounds were evaluated for their ulcerogenic potential in rats (Abouzeit-Har et al , 1982) Human, rat and dog microsomal COX assays: The experimental method which was used in human, rat and dog microsomal COX assay bioassay has been adopted from Chan et al . (1999) Acute toxicity: The oral acute toxicity of compounds was investigated using male mice (20 g) according to previously reported methods (Verma et al ., 1994; Litchfield and Wilcoxon, 1949). The animals were divided into groups of six mice each. The compounds were given orally, suspended in 1% gum acacia, in doses of 1, 10, 100, 200, 250 and 300 mg kg G 1 . The mortality percentage in each group was recorded after 24 h. Additionally, the test compounds were investigated for their parenteral acute toxicity in groups of six mice each as reported earlier (Bekhit and Fahmy, 2003). The compounds, or their vehicle propylene glycol (control), were given by intraperitoneal injection in doses of 10, 25, 50, 75 and 100 mg kg G 1 . The percentage survival was followed up to seven days (Bekhit and Fahmy, 2003) Statistical analysis: Results are expressed as Mean±SEM Differences between vehicle control and treatment groups were tested using one-way analysis ANOVA, followed by multiple comparisons by the Dunnettʼs test. A value of p # 0.005 was considered statistically significant. Dose-response curves for percent protection and ulceration were fitted by a four-parameter logistic function using a nonlinear least-squares regression RESULTS AND DISCUSSION Chemistry: Herein a series of spiroalkanones, pyrimidine and thiopyrimidine heterocyclic derivatives 1-10 (Fig. 2) were synthesized and illustrated by physical, chemical and spectroscopic evidences before and screened as analgesic, anticonvulsant and antiparkinsonian agents (Amr et al ., 2005) In this study, we report the activities of these compounds as antiinflammatory agents Pharmacological activities: A series of substituted pyrimidines synthesized and screened for selective COX-2 inhibitors (Tietz et al ., 2013). Many thiazolopyrimidines was Fig. 2: Chemical structure of the tested compounds (1-10) 88

Int. J. Pharmacol., 12 (2): 86-91, 2016 Table 1: Anti-inflammatory activity (ED 50 mmol) and ulcerogenic activity Test compounds ED 50 (µmol) Ulceration (%) 1 1.83±0.0001 0.00 2 1.77±0.0003 5.44 3 1.98±0.0002 0.00 4 1.88±0.0002 0.00 5 1.85±0.0002 0.00 6 1.81±0.0002 0.00 7 1.61±0.0001 7.43 8 1.56±0.0001 8.45 9 1.67±0.0001 6.23 10 1.71±0.0002 6.18 Indomethacin 9.568±0.00078 100 Values were calculated from the mean values of data from three separate experiments and presented as Mean±SEM. All results are significant different from control values at p # 0.005. All results are significant different from reference standard values at p # 0.005 Table 2: Effects of tested compounds on carrageenan-induced rat paw edema (mL), percentage protection and activity relative to indomethcin Test compounds Increase in paw edema (mL)±SEM a,b Protection (%) Control 0.988±0.0006 0 Indomethacin 0.25±0.0005 74.49 1 0.19±0.0007 80.77 2 0.16±0.0002 83.81 3 0.31±0.0001 68.62 4 0.24±0.0003 75.71 5 0.23±0.0004 76.72 6 0.17±0.0004 82.79 7 0.13±0.0003 86.84 8 0.12±0.0001 87.85 9 0.14±0.0002 85.83 10 0.16±0.0004 83.81 Values were calculated from the mean values of data from three separate experiments and presented as Mean±SEM. All results are significant different from control values at p # 0.005. All results are significant different from reference standard values at p # 0.005 synthesised and have potent anti-inflammatory activities and some of them showed potent antipyretic activities (Myakushkene et al ., 1999). On the other hand, some of pyrimidine derivatives were synthesized and have been potent anti-inflammatory activities (Sondhi et al ., 2001, 1999, 2000) Pharmacological activities Anti-inflammatory activities: Determination of the antiiflammatory activities via two animal models the firest was determintining the ED 50 that induce antiinflammatory activities using cotton pellet-induced granuloma bioassay (Table 1), all the tested compounds showed excellent anti-inflammatory activity and the order of activity was 8 (ED 50 = 1.56 µmol), 7 (ED 50 = 1.61 µmol), 9 (ED 50 = 1.67 µmol), 10 (ED 50 = 1.71 µmol), 2 (ED 50 = 1.77 µmol), 6 (ED 50 = 1.81 µmol), 1 (ED 50 = 1.83 µmol), 5 (ED 50 = 1.85 µmol), 4 (ED 50 = 1.88 µmol) and 3 (ED 50 = 1.98 µmol). All tested compounds were more active than indomethacin (ED 50 = 9.568 µmol) The second one involving determination of increasing in paw edema by using carrageenan-induced rat paw edema (Table 2). All the tested compounds showed excellent protection against carrageenan-induced rat paw edema and the decending order of activity was 8 (protection, 87.85%), 7 (protection, 86.84%), 9 (protection, 85.83%), 10 (protection, 83.81%), 2 (protection, 83.81%), 6 (protection, 82.79%), 1 (protection, 80.77%), 5 (protection, 76.72%), 4 (protection, 75.71%) and 3 (protection, 68.62%). All tested compounds were more active than indomethacin (protection, 74.49%). The results obtained by carrageenan-induced rat paw edema considered as a good evidence toll that confirmed the results obtained by the cotton pellet-induced granuloma bioassay Ulcerogenic activities: Compounds 1 (ulceration, 0.00%), 3 (ulceration, 0.00%), 4 (ulceration, 0.00%), 5 (ulceration, 0.00%) and 6 (ulceration, 0.00%) was devoided from ulcerogenic activities, while other showed low ulcerogenic activities Compounds 2 (ulceration, 5.44%), 7 (ulceration, 7.43%), 8 (ulceration, 8.45%), 9 (ulceration, 6.23%) and 10 (ulceration, 6.18%) causing minor ulceration while indomethacin causing 100% ulceration. It was oserved that as the antiinflammatory activities increases the ulcerogenic activities increases (Table 1) In vitro human COX-2 a and COX-1 b enzymes inhibitory activities: In vitro human COX-2 a and COX-1 b enzymes inhibitory activities of compounds revealed that the tested compounds inhibited both COX-2 a and COX-1 b enzymes but greatly on COX-2 a enzyme more than COX-1 b enzyme (Table 3). The selectivity ratio COX-2/COX-1 was determined and was in the following desceniding selectivity order 8 (COX-2/COX-1 selectivity ratio: 1058.333), 7 (COX-2/COX-1 selectivity ratio: 921.429), 9 (COX-2/COX-1 selectivity ratio: 835.294), 10 (COX-2/COX-1 selectivity ratio: 835.000), 2 (COX-2/COX-1 selectivity ratio: 890.476), 6 (COX-2/COX-1 selectivity ratio: 792.000), 1 (COX-2/COX-1 selectivity ratio: 748.148), 5 (COX-2/COX-1 selectivity ratio: 731.034), 4 (COX-2/COX-1 selectivity ratio: 740.000) and 3 (COX-2/COX-1 selectivity ratio: 825.806). Compounds 8, 7, 9, 10, 2, 6, 1 and 5 were more active than celecoxi compound 4 equally active to celecoxib while, compound 3 less active than celecoxib (COX-2/COX-1 selectivity ratio: 333.333) 89

Int. J. Pharmacol., 12 (2): 86-91, 2016 Table 3: In vitro human COX-2 a and COX-1 b enzymes inhibitory activities of compounds Test COX-2 IC 50 c COX-1 IC 50 c Approximate selectivity compounds (µmol) (µmol) ratio COX-2/COX-1 Indomethacin 2.63±0.0005 0.26±0.0001 0.099 Celecoxib 0.30±0.0004 100.00±2.43 333.333 1 0.27±0.0006 202.00±2.58 748.148 2 0.21±0.0004 187.00±2.39 890.476 3 0.31±0.0007 256.00±3.45 825.806 4 0.30±0.0008 222.00±2.36 740.000 5 0.29±0.0009 212.00±3.67 731.034 6 0.25±0.0005 198.00±1.40 792.000 7 0.14±0.0002 129.00±4.63 921.429 8 0.12±0.0001 127.00±2.44 1058.333 9 0.17±0.0003 142.00±2.57 835.294 10 0.20±0.0005 167.00±3.48 835.000 Values were calculated from the mean values of data from three separate experiments and presented as Mean±SEM. All results are significant different from control values at p # 0.005. All results are significant different from reference standard values at p # 0.005 Table 4: Effect of compounds on human, dog and rat microsomal COX activities IC 50 nM ------------------------------------------------------------------ Compounds Human Dog Rat Celecoxib 89 112 132 1 40 65 88 2 29 48 59 3 78 90 156 4 65 87 134 5 51 77 96 6 33 53 71 7 19 25 36 8 10 21 32 9 21 32 39 10 24 37 43 Values were calculated from the mean values of data from three separate experiments and presented as Mean±SEM. All results are significant different from control values at p # 0.005. All results are significant different from reference standard values at p # 0.005 Table 5: Acute toxicity of compounds (1-10) Test compounds LD 50 (mg kg G 1 ) 1 3265.764 2 2531.532 3 2341.211 4 4576.798 5 3421.643 6 2438.543 7 4576.453 8 4323.000 9 2341.456 10 5342.678 Values were calculated from the mean values of data from three separate experiments and presented as Mean±SEM. All results are significant different from control values at p # 0.005 Effect of compounds on human, dog and rat microsomal COX activities: In human, dog and rat kidney microsome preparations (COX-1), celecoxib was substantially less potent than all the tested compounds and the order was as follow 8, 7, 9, 10, 2, 6, 1, 5, 43 and celecoxib (Table 4) Acute toxicity: All tested compounds showed high LD 50 mainly above 2 g kg G 1 . These high LD 50 ensure high sfety and go theraputic windows (Table 5) CONCLUSION Carfull examination of the relation between chemicall structure and apharmacological activities cualminated on the following assumptions Structure activity relationship; C Cyclheptenes fused to heterocyclinc ring (compounds 8 and 7) system essential for higher antiinflammatory activities and greatest COX-2 b selective inhibition C Cyclhexenes fused to heterocyclinc ring (compounds 9 and 10) system were less active than cyclheptene ones C Polycyclic fused ring systems showed moderate antiinflammatory activities with greatest COX-2 b selective inhibition C Chlorine atoms and small size molecules showed lower antiinflammatory ACKNOWLEDGMENT The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGP-0172 REFERENCES Abouzeit-Har, M.S., T. Verimer and J.P. Long, 1982. Effect of long term estrogen and lithium treatment on restraint induced gastric erosion in intact and ovariectomized rats. Die Pharmazie, 37: 593-595 Allison, M.C., A.G. Howatson, C.J. Torrance, F.D. Lee and R.I.N. Russel, 1992. Gastrointestinal damage associated with the use of nonsteroidal antiinflammatory drugs. N. Engl. J. Med., 327: 749-754 Amr, A.E.G.E., H.H. Sayed and M.M. Abdalla, 2005. Synthesis and reactions of some new substituted pyridine and pyrimidine derivatives as analgesic, anticonvulsant and antiparkinsonian agents. Archiv der Pharmazie, 338: 433-440 Beers, S.A., E.A. Malloy, W. Wu, M. Wachter and J. Ansell et al ., 1997 N -(5-substituted) thiophene-2-alkylsulfonamides as potent inhibitors of 5-lipoxygenase. Bioorg. Med. Chem. Lett., 5: 779-786 90

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Statistical analysis, Anti-inflammatory activity, Pharmacological activities, Anti-inflammatory activities, Animal model, Acute toxicity, Anti-inflammatory agent, LD50, Indomethacin, Animal house, Dunnett's test, Carrageenan-induced rat paw edema, Nonsteroidal anti-inflammatory drug, NSAID, Antiinflammatory activity, Paw edema, Cox-2, Renal toxicity, Dose response curve, Analgesic, Structure-activity relationship, Therapeutic window, Intraperitoneal injection, Experimental animal, Side effect, Cyclooxygenase enzyme, Pyridine derivatives, Antiinflammatory activities, Pyrimidine derivatives, Celecoxib, Heterocyclic compound, Thiazolidinones, Pyrimidine, Selective COX-2 inhibitor, Anticonvulsant, Standard housing conditions, Enzymatic assay, Cotton-pellet induced granuloma, CPCSEA, Percentage protection, Pyrazole, Oral acute toxicity, Ulceration, Pyrazole derivative, COX-2 inhibitor, Committee for the Purpose of Control and Supervision on Experiments on Animals, Cyclooxygenase, Ulcerogenic Effect, Gastrointestinal mucosal damage, Cyclooxygenase-2 inhibitor, Chlorine atoms, Deanship of Scientific Research, Benzene sulfonamide, Spectroscopic evidence, COX, Antiinflammatory agent, Research group project, Imidazoles, Chlorine Atom.