Synthesis of Some N-(4-(Aryl)-2-Thioxo-1,3-Thiazol-3(2H)-yl)Pyridine-4-Carboxamide as Antimicrobial and Anti-inflammatory Agents

There has been a constant battle between humans and the multitude of microorganisms that cause infections and diseases; the treatment of bacterial infections remains a challenging job because of the increasing number of multidrug-resistant microbial pathogens. Despite the many chemotherapeutics available, the emergence of old and new antibiotic-resistant bacterial strains, mutations in microbial genomes, the incorrect use of antibiotics has been thoroughly demonstrated to greatly increase the development of resistant genotypes has generated a substantial need for new classes of anti-bacterial agents [1-2]. Various 2-thioxo-1,3-thiazol have been extensively investigated due to their application in different areas of biological activity such as antimicrobial [2-3], hypoglycemic [4], anti-inflammatory and analgesic agents [5], antidiabetic [6], antihyperglycemic [7]. Above mentioned facts prompted us to synthesis a series of N-(4-(aryl)-2-thioxo-1,3-thiazol3(2H)-yl)pyridine-4-carboxamide compounds having antimicrobial and anti-inflammatory activity. The structures of the compounds were confirmed by FT-IR, 1H-NMR, GC-mass spectroscopy and elemental analysis data studies; their antibacterial, antifungal, and antiinflammatory activities were performed by MIC (Minimum Inhibitory Concentration) method.


Introduction
There has been a constant battle between humans and the multitude of microorganisms that cause infections and diseases; the treatment of bacterial infections remains a challenging job because of the increasing number of multidrug-resistant microbial pathogens. Despite the many chemotherapeutics available, the emergence of old and new antibiotic-resistant bacterial strains, mutations in microbial genomes, the incorrect use of antibiotics has been thoroughly demonstrated to greatly increase the development of resistant genotypes has generated a substantial need for new classes of anti-bacterial agents [1][2]. Various 2-thioxo-1,3-thiazol have been extensively investigated due to their application in different areas of biological activity such as antimicrobial [2][3], hypoglycemic [4], anti-inflammatory and analgesic agents [5], antidiabetic [6], antihyperglycemic [7]. Above mentioned facts prompted us to synthesis a series of N-(4-(aryl)-2-thioxo-1,3-thiazol-3(2H)-yl)pyridine-4-carboxamide compounds having antimicrobial and anti-inflammatory activity. The structures of the compounds were confirmed by FT-IR, 1 H-NMR, GC-mass spectroscopy and elemental analysis data studies; their antibacterial, antifungal, and antiinflammatory activities were performed by MIC (Minimum Inhibitory Concentration) method.

Experimental Material and methods
All reagents and solvents used in the present study were of analytical grade and procured from Loba Chemie (India). The progress of the reactions were monitored by TLC using Merck silica gel precoated plate, with appropriate mobile phase, visualization by iodine vapour and UV chamber and product are purified by recrystallization technique. All the melting points recorded on a Veego apparatus (Mumbai, India) and were uncorrected. All the synthesized compounds were characterized by their FT-IR, 1 H-NMR, GC Mass spectroscopy. FT-IR spectra were recorded in KBr on Bruker FT-IR instrument (Germany), 1 H-NMR spectra were recorded on Bruker Avance 1 H-NMR spectrometer (Germany), at 400 MHz in DMSO-d6, by using varian instrument using TMS as internal standard and chemical shift values are given in ppm downfield to TMS (tetramethylsilane), GC Mass were recorded on GCMS-QP-5050 Schimadzu (Japan), and Perkin Elmer 2400 Series II CHN Elemental Analyzer. The standard drugs norfloxacin, ketoconazole, and ibuprofen were obtained as gift sample from Wockhardt Ltd., Aurangabad, India.

Synthesis of potassium-pyridine-dithiocarbazate (I)
In a 250 ml round bottom flask, isoniazide (0.075 mol, 10.28 g) was dissolved in a solution of potassium hydroxide (0.075 mol, 4.2 g) in 100 ml of absolute ethanol and carbon disulphide (0.075 mol). The reaction mixture was agitated overnight and diluted with 200 ml of dry ether. The solid obtained was filtered and washed with dry ether, yield 15.05 g ( 80%) [8].

Biological evaluation
Antimicrobial activity [11]: All the synthesized derivatives were screened for in vitro antimicrobial activity against two gram positive strains S .aureus (S. aureus, NCIM 2079), B. subtilis (B. subtilis, NCIM 2711) and two gram negative strains E. coli (E. coli, NCIM 2685), K. pneumonia (K. pneumoniae, NCIM 2957) and two fungal strains A. nigar (A. nigar, NCIM 596), S. cerevisiae (S. cerevisiae, NCIM 3102), using the broth micro dilution method. Minimum inhibitory concentration (MIC) was determined and compared with standard drugs norfloxacin for antibacterial and ketoconazole for antifungal activity and statistical analysis was performed using ANOVA to find the significance of the test, dimethyl sulfoxide used as inert solvent (Table 2).

In vitro anti-inflammatory activity by inhibition of protein denaturation [12]:
The standard drug and synthesized derivative were dissolved in minimum quantity of dimethyl formamide (DMF) and diluted with phosphate buffer ((0.2M, pH 7.4). Final concentration of DMF in all solution was less than 2.5%. Test solution (1ml) containing different concentrations of drug was mixed with 1 ml of 1mM albumin solution in phosphate buffer and incubated at 27° ± 1°C in BOD incubator for 15 min. Denaturation was induced by keeping the reaction mixture at 60° ± 1°C in water bath for 10 min. After cooling, the turbidity was measured at 660 nm (UV-Visible Spectrophotometer). Percentage inhibition of denaturation was calculated from control where no drug was added and the ibuprofen was used as standard drug.
The percentage inhibition of denaturation was calculated by using following formula and statistical analysis was performed using ANOVA to find the significance of the test ( Table 3).

Structure Activity Relationship
The general structural formula of basic compound can be written as follows: The relationship between chemical structure and antimicrobial, anti-inflammatory activity is summarized as follows.
The aryl ring should contain one substituent. Some substituent's that seem to enhance antimicrobial, anti-inflammatory activity are chloro, methyl, methoxy, hydroxyl, amino and nitro groups. Compounds containing the p-Cl or -OH substituent are orders of broad spectrum than the original (first generation) compounds. It is believed that the high activity of these compounds is a function of the electron withdrawing group's substitution on aryl ring at position no. 4 on thiazol ring. Among these compounds, it is thought that the spatial relationship between the electron donating groups contain compounds are less or inactive.

Antimicrobial and anti-inflammatory activity
From in vitro antibacterial activity, In case of E. coli, K. pneumonia, S. aureus and B. subtilis compounds IIc, IIg, IIj and IIm (p-Cl-Ph, p-OCH 3 -Ph, p-OH, and m-NO 2 -Ph ) were found to have significant activity which is 1 folds less than the standard drug norfloxacin, while in vitro antifungal activity, In case of A. nigar and S. cerevisiae compounds IIc, IIf, and IIj (p-Cl-Ph, p-CH 3 -Ph, and p-OH) were found to have significant activity which is 1 folds less than the standard drug ketoconazole. In anti-inflammatory activity, compounds IIc, IIf, IIh, and IIj at 100 mg/mL (p-Cl-Ph, p-CH 3 -Ph, p-NH 2 -Ph, and p-OH) were found to have significant activity which is 1/10 th less than the standard drug ibuprofen and at 200 mg/mL compound IIl (p-NO 2 -Ph) found significant active which is 1/10 th less than standard drug ibuprofen (Tables 2 and 3). Thus from the obtained antibacterial, antifungal and anti-inflammatory activity data we could conclude that the electronwithdrawing groups substituted at specific position on phenyl ring i.e., (p-Cl-Ph, p-OCH 3 -Ph, p-OH, m-NO 2 -Ph, p-CH 3 -Ph, p-NH 2 -Ph and p-NO 2 -Ph) are contributing positively for antibacterial and antiinflammatory activity.

Conclusion
A series of N-(4-(aryl)-2-thioxo-1,3-thiazol-3(2H)-yl)pyridine-4carboxamide had been synthesized in quantitative yields with the use of conventional method and evaluated for their in vitro antimicrobial and anti-inflammatory activity result are shown in Tables 2 and 3. On  The results are expressed as mean ± SDM (n=6). Significance was calculated by using one-way ANOVA with Dunnet's t-test. the basis of observed results, we concluded that additions of different functional groups have varying effects. In addition, the greater biological profiles were observed when the electron-withdrawing groups were incorporated at o-, m-and p-position of the phenyl ring.