Tetrazole, Schiff’s Base, Pyridine, Anti-tubercular Activity.

All chemicals and solvents were purchased from S D Fine chemicals. All reactions are carried out at laboratory condition. Melting points were determined with open capillary and were uncorrected. FT-IR spectra were recorded on a Jasco FT-IR model 8010 spectrophotometer, 1H NMR spectra were recorded in DMSO on a Varian mercury FT-NMR model YH- 300 instrument using TMS as internal standard.

Step I: General procedure for synthesis of ethyl [5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetate (A)

An equimolar mixture of 2-(1H-tetrazol-5-yl) pyridine (0.02 mol, 5 gm), ethyl chloroacetate (0.02mol, 3.67 ml) and anhydrous potassium carbonate (0.02mol, 3.76 gm) in methanol (40 ml) was refluxed on a water bath for 4 hrs, cooled to room temperature, filtered , dried and recrystalized from ethanol. The compound was separated as white amorphous powder.

Step II: General procedure for synthesis 2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetohydrazide (B)

To a mixture of compound A , (0.03 mol, 9 gm) in methanol (40 ml) , hydrazine hydrate (99% ,0.03 mole, 1.95 ml) was added with continuous stirring to get clear solution.Reflux the reaction mixture on water bath for about 5 hrs. The solution was concentrated and allowed to cool overnight. The resulting solid obtained was filtered, washed with cold water, dried and recrystalized from ethanol. The compound was separated as white powder.

Step III: General procedure the synthesis of N'-[(E)-substituted phenyl methylidene]-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetohydrazide (C1- C10)

Equimolar quantity of the hydrazide compound B, (0.003 mol, 2 gm) and various aromatic aldehydes (0.003 mol) in ethanol and dioxane (50 ml) were heated on a water bath under reflux for 8 hrs. The resulting Schiff’s bases (C1- C10) were cooled and poured into crushed ice. The precipitate thus obtained was filtered, washed with cold water and purified by recrystalisation using ethanol as solvent. The physicochemical and spectral data of the compounds (C1- C10) is described in [Tables 1 and 2].

Figure 1: Synthesis protocol for N'-[(E)-substituted phenyl methylidene]-2-[5-(pyridin-2-yl)-1H-stetrazol-1-yl] acetohydrazide.

Table 1: Physicochemical Characterization of N'-[(E)-substituted phenyl methylidene]-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetohydrazide.

Table 2: Spectral Characterization of N'-[(E)-substituted phenyl methylidene]-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetohydrazide.

In vitro Evaluation of Antitubercular activity of Compounds C1 –C10

The procedure followed for anti-TB activity mainly involves the use of Middlebrook 7H-9 broth and standard strain of M. tb h37Rv. The basal medium is prepared according to manufacture's instructions (Hi-Media) and sterilized by autoclaving. 4.5 ml of broth is poured into each one of the sterile bottles. To this, 0.5ml of ADC supplement is added. This supplement contains catalase, dextrose and bovine serum albumin fraction v. Then a stock solution of the compound is prepared (10 mg / ml). From this appropriate amount of solution is transferred to media bottles to achieve final concentrations of 10, 25, 50, ug / ml. finally 10 ul suspension of M. tubercles strain (100000 organisms / ml, adjusted by McFarland's turbidity standard) is transferred to each of the tubes and incubated at 37?. Along with this one growth control without compound and drug controls are also set up. The bottles are inspected for growth twice a week for a period of three weeks. The appearance of turbidity is considered as growth and indicates resistance to the compound. The growth is confirmed by making a smear from each bottle and performing a ZN stain. Streptomycin was used as standard drug and DMSO as control [28]. The results are presented in Table 3. (-) – Indicated no growth, (+) indicated growth less than 20 colonies, (++) indicated growth more than 20 colonies.

Table 3: Antitubercular activity data of the compounds C1 – C10.

The different Schiff bases of the2-(1H-tetrazol-5-yl) pyridine were synthesized by the reaction of 2-(1H-tetrazol-5-yl) pyridine with ethyl chloroacetate to form ethyl [5-(pyridin-2-yl)-1H-tetrazol-1-yl]acetate (A). it was further treated with hydrazine hydrate to form 2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetohydrazide (B). The compound B was treated with different aromatic aldehyde to form N'-[(E)-substituted phenylmethylidene]-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl] acetohydrazide (Schiff Bases) (C1–C10). The FT-IR spectra of Schiff.s bases (C1-C10) showed the absence of peak of ester groups and the new peaks which appeared at 1610-1650 cm-1 which are attributed to the new azomethine (C=N) group. Some spectral data are listed in Table 2. The FT-IR absorption bands disappearance at (1610-1650) cm-1 is give good evidence for the success step of reaction. These absorption bands due to (NH) amine group stretching frequency are at 3450 cm-1. Also FT-IR spectra of Tetrazole showed clear absorption bands at (1441-1499) cm-1 due to (N=N). The NMR spectra of all the synthesized compounds were determined and the chemical shift values of H NMR shows at 9.60 due to (s, 1H, NH), at 7.90 (s, 1H,N=CH) and 7.91- 6.80 (m, 9H, Ar), 5.55 (s, 2H, -CH2) ppm [29]. This value indicates the presence of all protons which conforms the structure of the synthesized compounds. From the results of antitubercular screening, it is evident that most of the compounds are very weakly active and few are moderately active against Mycobacterium tubercle in comparison with standard. Compounds C4, C10 containing R=Br and –N(CH3)2 showed significant antitubercular activity (25µg amount) against Mycobacterium tubercle in comparison with standard.

In conclusion, our preliminary anti-TB results encourage us to engineer the chemical structure of Tetrazole containing Schiff bases to generate essential pharmacophores features that could lead to the synthesis of a promising candidate to develop anti-TB agents In conclusion, the results of investigation revealed that we have prepared a new series of Schiff bases containing 5- phenyl tetrazole and demonstrated that these compounds possessed good antitubercular activity. Newly synthesized tetrazole-based derivatives exerted also a promising potential in the combined anti-TB therapy are C2, C3 and C8 in which R is 2-Cl, 4-Cl and 4-NO2 respectively and C2, C3 and C5 in which R is 2-Cl,4-Cl and 4-CH3 respectively. The compounds C4 and C10 showed significant antitubercular activity against M. tubercles. Also the comparative evaluation of active compounds is required for further studies; the data reported in this article may be helpful guide for medicinal chemist who is working in this area.

We are highly thankful to University of Pune, Pune for providing the spectral data, Dr. V.K. Deshmukh for providing necessary research facilities to carry out work.

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