SYNTHESIS AND PHARMACOLOGICAL EVALUATION : ANTIMICROBIAL , ANTI-INFLAMMATORY , ANALGESIC , ULCEROGENIC PROPERTIES OF SEVERAL BIS-HETEROCYCLIC DERIVATIVES

Email: dr_h.panwar@rediffmail.com ABSTRACT In Medicinal chemistry, heterocyclic derivatives contributed the largest classical division of organic chemistry. Thiadiazole compounds have been known for many biological activities such as hypoglycemic, anticancer, anti-inflammatory, anti-asthmatic, antihypertensive etc. On this research, several 1,1-bis [2-{2-(5(arylidene)imino-1,3,4-thiadiazol-2-yl) methyl amino}1,3,4thiadiazo-5-yl] cyclopropane 5(a-h) have been synthesized from cyclopropane dicarboxylic acid comprising thiadiazole moieties. All the synthesized compounds have been characterized by elemental (C,H,N) and spectral (I.R., HNMR, Mass) analysis. Furthermore, all synthesized compounds were screened for their antifungal, antimicrobial, anti-inflammatory, analgesic, ulcerogenic and toxic activities. The Compound 5f, 3-chlorophenyl substituted was found the most potent in antimicrobial spectrum against all the used microbes. On the other hand, compound 5g, 4-hydroxy3-methoxy phenyl substituted was found the most potent in antiinflammatory, analgesic, and ulcerogenic activities which further evaluated for lesser toxicity test.


INTRODUCTION
Currently using non-steroidal antiinflammatory drugs (NSAIDs) viz.ibuprofen, diclofenac, indomethacin and flurbiprofen cause gastric toxicity.Their prolong use resulted into gastro-intestinal (GI) ulceration, bleeding and nephrotoxicity (Kimmey 1992).On the other hand, there is no chemotherapeutic agent which is found to show anti-inflammatory as well as known antibacterial activities.In medicinal chemistry, heterocyclic derivatives contributed the largest classical division of organic chemistry.Organic chemistry is providing facile and efficient methodology for the synthesis of different heterocyclic moieties due to their versatile biological spectrum.Microbial infections cause pain and inflammation.In the current study, we have focused on thiadiazoles due to its versatile biological profile such as hypoglycemic (Avetisyan et al., 1981), anticancer (Parkanyi et al. 1992), anti-inflammatory (Boschelli et al., 1993;Labanauskas et al., 2001;Schenone et al., 2001), anti-asthmatic (Bhattacharya et al., 2005), antihypertensive (Vio et al., 1989), antidepressant (Clerici et al., 2001), anxiolytic (Clerici et al., 2001), fungicidal (Chen and Hen, 2000;Zou et al., 2002;Zou et al., 2002) and antituberculosis (Foroumadi et al., 2001).On the other hand, esters of 1,1-cyclopropane dicarboxylic acid were found to possess important insecticidal activity (Ronald and Leland 1998).Furthermore bulk of literature is available to explore the biological importance of Schiff bases (Ibrahim et al., 2011;Sah et al., 2011;Sanjeeva et al., 2010) especially antimicrobial and thus aroused our interest in the synthesis of some novel bis Schiff bases.Based on the wide spectrum of biological profile of 1,1-propane dicarboxylic acid, thiadiazole, Schiff base and their importance in pharmaceutical, biological field and in continuation of our ongoing research on biologically active heterocycles (Panwar et al., 2006;Goel et al., 2004;Kumar et al., 2003;Panwar et al., 2011;Panwar et al., 2011), it was thought of interest to accommodate 1,1propane dicarboxylic acid, thiadiazole and aryl aldehydes in a single molecular frame work of designing to synthesize some novel bis heterocycles for enhancing biological activity with lesser toxicity.

MATERIAL AND METHODS
All the chemicals used for the preparation of desired derivatives, were obtained from Sisco Research Laboratories (SRL), Mumbai, India; Qualigen Fine Chemicals, Mumbai, India; E. Merck Ltd., New Delhi, India.Reference drugs ampicillin trihydrate and fluconazole were procured from Ind-Swift Pharmaceutical, Panjab, India and Macleods Pharmaceutical, Mumbai, India respectively.The melting points of the compounds were determined in open glass capillaries with the help of thermonic melting points apparatus (Campbell Electronics, Mumbai, India) and are uncorrected.The homogenity of all the newly synthesized compounds were routinely checked by TLC on silica gel G plates and spots were located by using iodine chamber.Elemental analysis was performed in Heraeus C H N rapid analyser.The results were found within the ±0.4% of theoretical values.Infrared spectra were recorded on KBr pellets on a Perkin Elmer system 2000 FTIR spectrometer and 1 H-NMR spectra on Bruker DPX 200 using TMS as internal standard.

Anti-inflammatory activity
Anti-inflammatory activity was determined against carrageenan induced rat's paw edema (Winter et al., 1962).The rats were divided into three groups (control, drug treated, and standard, each groups contain 6 animals).A freshly prepared suspension of carrageenan (1% in 0.9% saline).0.05mL was injected under the planter aponeurosis of the right hind paw of each rat.Test compounds and standard drug were administered orally to the animals of drug treated groups and the standard drug group, respectively, 1 h before the carrageenan injection.The paw volume of each rat was measured before 1 and after 3h of carrageenan treatment with the help of a plethymometer.The percent anti-inflammatory activity was calculated according to the formula given below: Percentage of inhibition of edema : 1 -Vt X 100 Vc Where, Vt and Vc are the mean increase in paw volume of rats of the treated and the control group, respectively.Results obtained were statistically analyzed.

Analgesic activity
Analgesic activity was performed following the method of Berkowitz et al., 1977.This method is based on the property of the test compound to antagonize the phenylquinone-induced pain syndrome in mice.Groups of five mice were injected intraperitonealy with 0.25mL of a 0.02% solution of phenylquinone in ethanol (5%) 1h after oral administration of the test compound.The number of writhes induced in each mouse was counted for 5min (between 5 and 10min) after injection of an irritant.The analgesic effect was expressed as percent protection in comparison to control.% protection = (1 -mean no. of writhes in mice of test groups/ mean number of writhes in mice of control group) X 100

Ulcerogenic activity
Ulcerogenic labilities of newly synthesized compounds were checked (Verma et al., 1981) in albino mices.Albino rats were fasted for 24 h prior to drug administration.All animals were sacrificed 8 h after drug treatment, and their stomachs and small intestines were microscopically examined to assess the incidence of hyperemia, shedding of epithelium, petechial and frank hemorrhages and erosion or discrete ulceration with or without perforation.The presence of any one of these criteria was considered to be an evidence of ulcerogenic activity.

COX-1 and COX-2 activities
The compounds prepared were tested for cyclooxygenase-1 and cyclooxygenase-2 inhibitory activities (Copeland et al., 1994) followed to determine the IC50 values.The enzyme activity is measured using chromogenic assay based on oxidation of N, N, N ' , N 'tetramethyl-p-phenylenediamine (TMPD) during the reduction of prostaglandin G2 to prostaglandin H2 by COX-1 and COX-2 enzymes.COX-1 and COX-2 enzymes used in the assay were purified from microsomal fraction.The compounds were dissolved in DMSO and stock solution was diluted to required assay concentration.The assay mixture consists of Tris-HCl.Tris-HCl buffer (pH 8.0, 100μM).hematin (15μM), EDTA (3mm), enzyme (COX-1 or COX-2, 100μM) and test compound.The mixture was pre-incubated at 25°C for 15min and then the reaction was initiated by the addition of arachidonic acid (100μM) and TMPD (120μM) in total volume of 1.0mL.The enzyme activity was measured by estimating the initial velocity of TMPD oxidation for the first 25s of the reaction following the increase in absorbance at 603nm.IC50 values are calculated from four parameter least squares non-linear regression analysis of the log dose vs percentage inhibition plot.However, none of the compound studied here exhibited significant inhibitory activity when compared to standard inhibitors indomethacin (for COX-1) and celecoxib (for COX-2).

Antimicrobial screening
All the newly synthesized compounds were screened for their antibacterial and antifungal activity.All the bacterial and fungal strains were clinical isolates, identified with conventional morphological and biochemical methods.Bacterial inhibition diameter was measured in mm.Microorganisms employed antibacterial studies were Staphylococcus aureus, Escherichia coli, Klabsiella pneumoniae and Proteus vulgaris.Disk diffusion method (Cruickshank et al., 1975;Collins 1976) was used for determination of the preliminary antibacterial activity.Sterile 5mm Whatman no. 1 filter paper discs were used in the disc diffusion method.Batches of 100 disks were dispensed to each screw-capped bottle and sterilized by dry heat at 140°C for 1h.The test compounds were prepared with different concentrations using DMF.One hundred milliliters of the microbial suspension was spread onto nutrient agar plates.Disks of each concentration were for placed in triplicate in nutrient agar medium seeded with fresh bacteria separately.The incubation was carried out at 37°C for 24h.Ampicillin trihydrate was used as a standard drug.Solvent and growth controls were kept and zones of inhibition were noted (Table I).
On the other hand, the newly prepared compounds were screened for their in vitro antifungal activity against Aspergillus fumigatus (plant isolate), Candida glabrata, Candida albacans and Candida krusei in DMSO by the serial plate dilution method (Khan, 1997;Varma, 1998).Fluconazole (antifungal) was used as reference drug.Sabouraud's agar media were prepared by dissolving peptone (1g), D-glucose (4g), and agar (2g) in distilled water (100mL) and adjusting the pH to 5.7.Normal saline was used to make a suspension of the spore of fungal strain for lawning.A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of the corresponding species.Agar media (20mL) was poured into each petri dish.Excess suspension was decanted and the plates were dried by placing in an incubator at 37°C for 1h.Using an agar punch wells were made into each well labeled.A control was also prepared in triplicate and maintained at 37°C for 3-4 days.Antifungal activity was determined by measuring the diameter of the inhibition zone in mm (Table I).

Acute toxicity test
Lethal dose (LD50) (Carrol 1952) of test compounds were determined in albino mice.After 24h of drug administration, percent mortality in each group was observed from the data obtained LD50.Data revealed that compound 5f does not show any toxicity up to dose of 9.95mg/mL body weight in mice.

Pharmacology
Compounds 4, 5a-f wrere evaluated for antimicrobial, anti-inflammatory, analgesic and ulcerogenic activities.The biological evaluation data were summarized in table I and II.
Synthetically derived molecules 4, 5a-f were assayed by using the disk diffusion method and serial plate method for antimicrobial activity against selected pathogenic clinical strains.The screening results were compared with standard ampicillin trihydrate and fluconazole respectively for antibacterial and antifungal testing respectively.From the antimicrobial screening data of compounds 4 and 5a-h, it was found that conversion of thiadiazole derivative i.e. 4 into Schiff bases i.e. 5a-h, brought enhancing antibacterial and antifungal activity.Compound 5f claimed the most remarkable antimicrobial spectrum against all the used microbes.It showed more potency against K. pneumoniae, P. vulgaris and comparable against S. aureus with standard drug ampicillin trihydrate while on the other hand, remaining tested compounds exhibited mild to moderate activity.Among the derivatives 5(a-h), derivative 5b, 5c and 5d showed moderate potency while compounds 5a, 5e, 5g and 5h showed poor antimicrobial activity against the used pathogenic strains.

CONCLUSION
Antimicrobial evaluation of compound 4 and 1,1-bis (2-phenyl-5-arylidine-1,3thiadiazolidin-4-one) cyclopropanes (5a-h) revealed that conversion of compound 4 into Schiff bases (5a-h) enhances biological potency viz.antimicrobial, anti-inflammatory, analgesic and ulcerogenic activities.Compound 5f possessing 3-chlorophenyl substitution; displayed the most potent antimicrobial spectrum as well as anti-inflammatory and analgesic activity.On the other hand, compound 5g, 4-hydroxy-3-methoxy phenyl substituted was found the most potent in antiinflammatory, analgesic, and ulcerogenic activities and deserves further investigation in order to clarify the mode of action at molecular level, responsible for the activity observed.
. Moreover, rest of the compounds showed moderate degree of COX-1 and COX-2 inhibitory actions suggesting that these compounds showed anti-inhibitory activity by inhibition of both COX-1 and COX-2 enzymes.ALD50 of all these compounds were >800 mg/kg/ i.o.(Table-II).

Table IIa .
Antimicrobial data for the synthesized compounds 4 and 5(a-h).