The objective of this investigation was to synthesize and investigate the anti-inflammatory, antioxidant and antibacterial activities of a three series of novel coumarins derivatives bearing pyrazoles, pyrazolines and pyrans ring system with the hope of discovering new structure leads serving as anti-inflammatory-antioxidant-antibacterial agents.
The biheterocyclic compounds (4a-k) containing both coumarin and pyrazole ring system was synthesized by the condensation process between dibromo chalcone (3a-k) and phenylhydrazine in presence of triethylamine. The coumarinylpyrazolines (5a-k) were obtained by cyclization of chalcones (2a-k) with benzohydrazide in hot pyridine. Finally treatment of chalcone (2a-k) with malanonitrile in presence of piperidine afforded synthesis of coumarinylpyran derivatives (6a-k). The tested compounds did not show toxic effects at doses upto 2000 mg/kg b.w. in acute toxicity experiments.
Among the three series, coumarinylpyrazolines series (5a-k) showed potent anti-inflammatory activity. Amongst the tested compounds, 5c and 5h showed pronounced anti-inflammatory activity (59.77% and 61.71% respectively) against carrageenin induced paw edema that was comparable to diclofenac (71.15%), whereas, compounds 4c, 4d, 4h, 4i, 5d, 6c, 6d, 6h and 6i displayed good anti-inflammatory activity (50.06 to 59.28%). However, none of the newly synthesized compounds was found to be superior over the reference drug. The preliminary in vivo biological activities of these novel compounds evidenced that the presence of chlorine, fluorine and methoxy groups in the aromatic ring of 5th position of the pyrazole and pyrazoline; 4th position of pyran nucleus gave rise to an increased anti-inflammatory activity.
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Compound 6h showed considerable activity (52.13%) in model of chronic inflammation such as adjuvant-induced arthritis and compounds 4d and 5d exhibited moderate chronic anti-inflammatory activity (45.28 and 41.50% repectively) as compared to standard diclofenac (55.67%). Compounds 4d, 5d and 6h causes low ulcerogenicity compared with the standard drug acetyl salicylic acid. Since GI problems due to NSAID continue to be the major impediment to their use in therapeutics, GI protection of the new anti-inflammatory derivatives prove them useful lead molecules for the development of better NSAID with greatly improved therapeutic index (further studies are required to confirm this suggestion).
The binding mode of the tested compounds inside the COX-1 and COX-2 active site was predicted using a docking technique. Molecular docking studies using auto dock of these derivatives with COX-2 and COX-1 receptors are also showed minimum binding energy for all the compounds and may be considered as good inhibitors of COX-2. A Lamarckian genetic algorithm method implemented in the program Auto dock 4.0, was employed. When the results of docking studies considered, it can be suggested that coumarinylpyrazoline (5a-k) and coumarinylpyran (6a-k) derivatives with appropriate substitutions which can fill the adjunct pocket and interact with the relatively polar residues such as His207 and His388 may be useful to propose new molecules with enhanced selectivity towards COX-2. In addition, the substitution of 1H-pyrazole as a suitable central ring template with the coumarin moiety can be suggested as an effective scaffold for further design of selective and potent COX-2 inhibitors.
The 2,2-diphenyl-1-picryl-hydrazyl (DPPH·) radical scavenging activity (RSA) evaluation is a standard assay in antioxidant activity studies and offers a rapid technique for screening the RSA of specific compounds or extracts. The interaction of synthesized compounds 4a-k and 5a-k with stable DPPH free radical indicates their free radical scavenging ability. Majority of the tested compounds in these series showed low to moderate interaction with the DPPH radical at 10-100 µg/mL concentration. Maximum DPPH RSA was observed in compounds 4k and 5k, which have p-OH group as substituent on the phenyl ring. Compounds 4d and 5d which contain -Cl groups on the phenyl ring at position 2 and 4 showed moderate radical scavenging activity but lower than compounds 4k and 5k; whereas the compounds 4h and 5h that contain methoxy groups on the phenyl ring at position 3, did not show very less activity. On the other hand, compounds 6a-k showed scavenging of DPPH radical at varying degrees when compared with the standard antioxidant vitamin C. Antioxidant activity of these compounds is related with their electron or hydrogen radical donating ability to DPPH radical, so that they become stable diamagnetic molecules. This strength is the reason for the higher antioxidant activity of the compounds 4k and 5k.
Compound with 4-methoxy (5b) and 4-hydroxy (5k) substituent in the phenyl ring exhibited highest antioxidant activity from coumarinylpyrazoline series (5a-k) in hydrogen peroxide radical scavenging method. Coumarinylpyrazoles bearing 2,4-dichloro (4d) and 4-hydroxy (4k) substitution on phenyl ring will be responsible for enhanced antioxidant activity.
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Antibacterial activity of synthesized compounds was carried out by using cup-plate agar diffusion method. The result showed that compounds 5g, 6c and 6i exhibited maximum antibacterial activity against all the tested microorganisms at a concentration of 100µg/mL. The activity is considerably affected by substituents present at the para position of phenyl ring. It has been observed that the presence of a strong electron-withdrawing group specially -Cl (6c) and -F (6i) group enhances the electron conjugation throughout the structure. On the other hand, some of the newly synthesized compounds containing-N(Me)2 or -OMe as a substituent (5e and 5h) were able to inhibit the growth of gram-positive as well as gram-negative bacteria.
In comparing the MIC values with the standard, compounds 4h and 6d exhibit the most potent in vitro antibacterial activity against all evaluated organisms; while compounds 4c, 4d, 4i, 5g, 6c and 6i showed respectable antibacterial activity. The MIC values of these novel compounds evidenced that the presence of fluorine, methoxy and chlorine groups at the third position in the aromatic ring of the pyrazole and pyrazoline nucleus and forth position in the aromatic ring of pyran nucleus gave rise to an increased antibacterial potency. Hence, it is accomplished that there is simple scope in developing of tested compounds as a lead compounds.
Finally compounds 4d and 5d might be recognized as the most biologically active component within present study with an interesting anti-inflammatory, antioxidant and antibacterial profile. These results of previous experimental and docking studies strongly suggest that most of molecules synthesized in this study may certainly be capable drug scaffold with interesting pharmacological profile.
Further detailed studies are needed to know the mechanism of action and site of action of these compounds. Consequently coumarins represent a class that needs further investigation with the hope of finding new anti-inflammatory, antioxidant and antibacterial agents.