Synthesis of Some Bioactive Sulfonamide and Amide Derivatives of Piperazine Incorporating Imidazo[1,2-B]Pyridazine Moiety

Sulfonamide and amide derivatives comprise an important class of drugs with diverse biological applications. Sulfonamides are widely used as antimicrobial [1,2], anticancer [3,4], anti-inflammatory [5] and antiviral agents as well as HIV protease inhibitors [6]. Sulfonamides were the first effective chemotherapeutic agents to be utilized efficiently to prevent and cure the bacterial infection in human beings [7-10].


Introduction
Sulfonamide and amide derivatives comprise an important class of drugs with diverse biological applications. Sulfonamides are widely used as antimicrobial [1,2], anticancer [3,4], anti-inflammatory [5] and antiviral agents as well as HIV protease inhibitors [6]. Sulfonamides were the first effective chemotherapeutic agents to be utilized efficiently to prevent and cure the bacterial infection in human beings [7][8][9][10].
The chemistry of pyridazines and their fused heterocyclic derivatives has received considerable attention owing to their synthetic and effective biological importance. Pyridazines have been reported to possess antimicrobial [43][44][45], antituberculosis [46][47][48], antifungal [49], anticancer [50], anti-hypertensive [51], herbicidal [52], antiinflammatory [53] activities and protein tyrosine phosphatise 1B(PTP1B) inhibitors [54]. They also have an immense potential in agricultural science as plant growth regulators and crop protection agents [55]. The incorporation of two moieties increases biological activity of both and thus it was of value to synthesize some new heterocyclic derivatives having two moieties in the same molecules.
Looking at the importance of these heterocyclic nuclei, it is thought of interest to accommodate sulphonamide and amide of piperazine with imidazo [1,2-b] pyridazine moieties in single molecular framework and screen them for their various biological activities.

General procedures
Reagent grade chemicals were used without further purification. All the melting points were taken in open capillaries and are uncorrected. The purity and mass of the synthesized compounds was checked by LCMS. 1H NMR spectral was recorded in CDCl 3 /DMSO with tetramethylsilane (TMS) as the internal standard at 400 MHz on a Bruker DRTX-400 spectrophotometer. The chemical shifts are reported as parts per million (ppm). Elemental analysis was performed using a (EURO EA 3000 instrument). Acme silica gel-G and Merck silica gel (100 to 200, 60 to 120 meshes) were used for analytical TLC and Column chromatography respectively Abstract Some new sulfonamide and amide derivatives containing piperazine ring and imidazo [1,2-b]pyridazine moiety have been synthesized by the reaction of 6-chloro-2-substituted aryl(or alkyl)imidazo [1,2-b]pyridazine derivatives [obtained by the reaction of 3-amino-6-chloro pyridazine with 2-bromo-1-substituted aryl(or alkyl)ethanone] with homopiperazine in NMP and followed by reaction with alkyl (or substituted aryl) acid chloride or sulfonyl chloride in presence of triethyl amine and dichloromethane. All the synthesized compounds were characterized by elemental analysis, 1 H NMR and LCMS. These were screened for in vitro antimicrobial activity against two gram positive (Bacillus subtilis and Staphylococcus aureus) and two gram negative bacteria (Pseudomonas fluorescens and Escherichia coli), as well as for antifungal and antimalarial activity.

Preparation of novel sulfonamide and amide derivatives of piperazine incorporating imidazo[1,2-b]pyridazine moiety
General procedure for the synthesis of 6-chloro-2-substituted aryl(or alkyl)imidazo[1,2-b]pyridazine: To a solution of 3-amino-6chloro pyridazine (0.01 mole) in ethanol (10 mL) was added 2-bromo-1-substituted aryl (or alkyl) ethanone at room temperature. Then the reaction mixture was refluxed at 80°C for 4 hrs. The reaction mixture was then cooled and poured into ice-cold water. The resulting precipitate was filtered, washed several times with water, dried and recrystallized from ethanol.

General procedure for the synthesis of 6-(piperazin-1-yl)-2substituted aryl(or alkyl)imidazo[1,2-b]pyridazine
The mixture of 6-chloro-2-substituted aryl(or alkyl)imidazo [1,2-b] pyridazine (0.01 mole) and homopiperazine (0.05 mole) in NMP (5 mL) was heated at 150°C for 1 hr. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over Na 2 SO 4 . The solvent was evaporated and crude compound was used as such for next step without any purification.

Antimicrobial activity
All the synthesized compounds were tested against two gram positive bacteria (Staphylococcus aureus, Streptococcus pyogenes) and two gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa) using micro broth dilution method [61][62][63] for the determination of minimal inhibition concentration. For the antifungal activity the common standard strains that were used, are C. albicans, A. niger and A. clavatus. Muller Hinton broth (Microcare laboratory and Tuberculosis Research Centre, Surat-3, India) was used as nutrient medium to grow and dilute the drug suspension for the test bacteria. Inoculum Size for Test Strain was adjust to 10 8 Cfu [Colony Forming Unit] per milliliter by comparing the turbidity. DMSO was used as diluents / vehicle to get desired concentration of drugs to test upon Standard bacterial strains. Serial dilutions were prepared in primary and secondary screening. In primary screening 1000 micro/ml, 500 micro/ml, and 250 micro/ ml concentrations of the synthesized compounds were taken. The active synthesized compounds found in this primary screening were further tested in a second set of dilution against all microorganisms. The highest dilution showing at least 99% inhibition zone is taken as MIC. The test mixture should contain 10 8 organism/ml. Standard drugs Ampicillin and Chloramphenicol were used as antibacterial for comparison. Standard drugs Nystatin and Greseofulvin were used as antifungal for comparison.

Antimalarial activity
The in vitro antimalarial assay was carried out in 96 well microtitre plates according to the microassay protocol reference. The cultures of P. falciparum strain were maintained in medium RPMI 1640 supplemented with 25 mM HEPES, 1% D-glucose, 0.23% sodium bicarbonate and 10% heat inactivated human serum. The asynchronous parasites of P. falciparum were synchronized after 5% D-sorbitol treatment to obtain only the ring stage parasitized cells. For carrying out the assay, an initial ring stage parasitaemia of 0.8 to 1.5% at 3% haematocrit in a total volume of 200 µl of medium RPMI-1640 was determined by Jaswant Singh Bhattacharya (JSB) staining to assess the percent parasitaemia (rings) and uniformally maintained with 50% RBCs (O + ). A stock solution of 5 mg/ml of each of the test samples was prepared in DMSO and subsequent dilutions were prepared with culture medium. The diluted samples in 20 µl volume were added to the test wells so as to obtain final concentrations (at five fold dilutions) ranging between 0.4 µg/ml to 100 µg/ml in duplicate well containing parasitized cell preparation. The culture plates were incubated at 37°C in a candle jar. After 36 to 40 h incubation, thin blood smears from each well were prepared and stained with JSB stain. The slides were microscopically observed to record maturation of ring stage parasites into trophozoites and schizonts in presence of different concentrations of the test agents. The test concentration which inhibited the complete maturation into schizonts was recorded as the minimum inhibitory concentrations (MIC). Quinine was taken as the reference drug.

Antibacterial activity
The antibacterial activity of all the synthesized compounds were tested in-vitro against pathogenic E. coli, P. aeruginosa, S. aureus and S. pyogenus and the results were compared with standard drugs (Ampicillin and Chloramphenicol). In case of S. aureus, Compound 3b shows higher activity and compounds 3a, 3c, 3e, 3f, 3j, and 3l exhibit good activity while 3d, 3g, 3h, 3i and 3k show moderate activity. In case of S. pyogenus compounds 3c, 3d and 3f exhibit good activity and compound 3b show moderate activity while 3a, 3b, 3e, 3g, 3h, 3i, 3j, 3k and 3l possess less activity. In case of E. coli Compound 3d exhibit higher activity and compounds 3f, 3j show moderate activity while rest of the compounds possess less activity. In case of P. aeruginosa compounds 3e, 3k and 3l exhibit good activity than the rest of the compounds. The results are given in Table 2.

Antifungal activity
The antifungal activity of all the synthesized compounds were tested in-vitro against fungi C. albicans, A. niger and A. clavatus and the results were compared with standard drugs (Nystatin and Greseofulvin). In case of C. albicans compound 3a exhibit higher activity and 3c, 3e, 3h, 3i, and 3l, show good activity while compounds rest of the compouns possess less activity. In case of A. niger and A. clavatus all the compounds possess less activity. The results are given in Table 3.

Antimalarial activity
For antimalarial activity, Compounds 3a, 3c and 3k exhibit good activity closer to reference compound Quinine while rest of the compounds possess less activity. The results are given in Table 4.

Conclusion
All the newly synthesized compounds were screened for antibacterial, antifungal and antimalarial activity. The data in the Tables 2 and 3 indicate that among the synthesized compound 3a, 3b and 3c possesses good antimicrobial activity. However, the activities of the tested compounds are much less than those of standard agents used. These compounds also show potent antimalarial activity. From the results of various biological activities it is clear that these compounds would be of better use in drug development to combat bacterial infections and as antimalarial agents in the future.