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Synthesis of Some Bioactive Sulfonamide and Amide Derivatives of Piperazine Incorporating Imidazo[1,2-B]Pyridazine Moiety | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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

Ashish Bhatt*, Ravi Kant and Rajesh K Singh

Department of Chemistry, Mewar University, Chittorgarh, Rajasthan, India

*Corresponding Author:
Ashish Bhatt
Department of Chemistry, Mewar University
Chittorgarh, Rajasthan-312 901, India
Tel: +917742845501
E-mail: [email protected]

Received date: April 11, 2016; Accepted date: April 19, 2016; Published date: April 24, 2016

Citation: Bhatt A, Kant R, Singh RK (2016) Synthesis of Some Bioactive Sulfonamide and Amide Derivatives of Piperazine Incorporating Imidazo[1,2-B] Pyridazine Moiety. Med chem (Los Angeles) 6:257-263. doi:10.4172/2161-0444.1000355

Copyright: © 2016 Bhatt A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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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, 1H 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.

Keywords

Imidazo[1,2-b]pyridazine; Homopiperazine; Antimicrobial and antimalarial activity

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 [7-10].

The piperazine moiety appears in many drugs encompassing a broad range of activities (e.g., Oxatomide, Almitrine, Hydroxyzine, Buclizine, Lomerizine [11]. Nitrogen in piperazine ring plays an important role in exerting biological effects. The basicity of piperazine nitrogen plays an important role in selectivity and potency towards the biological targets. This moiety (monoaryl and diarylpiperazine) also found in drug candidates displaying anti-allergenic [12], antibacterial [13], anti-anxiety [14], anti-emetic [15], antimigraine [16] and platelet antiaggregatory activities [17]. In addition, piperazine moiety is present in many cardiovascular drugs [18] (e.g., Manidipine, Doxazosin, Trimetazidine, Flunarizine, Prazosin) and drug candidates [19,20]. Piperazine and their derivatives also possess antimalarial activity [21], antioxidative activity [22] and antifungal activity [23] and found in many drug molecules such as Meclizine (motion sickness drug), Cyclizine (antiemetic and antihistamine), Clozapine (antipsychotic drug), Imatinib (leukemia drug), Befuraline (stimulant and antidepressant), Antrafenine (analgesic), Trazodone (sedating antidepressant) and Niaprazine (sedating antihistamine) etc.

Piperazine and sulfonamide derivatives represent a category of pharmacologically interesting compounds having diverse biological activities. Intensive research has been carried out on the synthesis and analysis of pharmacological activities of these derivatives. Substituted sulfonamide derivatives are important category of pharmacophores that have a wide spectrum of pharmaceutical accomplishments: as antimalarial [24], anti-microbial [25], anti-bacterial [26,27], anti-cancer [28] anti-fungal [29], anti-oxidant [30], anti-HIV [31], antiplasmodial [32], anti-neoplastic [33], anti-proliferative [34] activities and additionally known to act as 5-HT6, 5-HT7 receptor antagonists [35,36], A2B and CXCR3 antagonists [37,38],11ß-HSD [39], histone deacetylase (HDAC) inhibitors [40], ß-secretase (BACE1) inhibitors [41] and dual PI3K/mTOR inhibitors [42].

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 [43-45], antituberculosis [46-48], 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.

Several imidazo [1,2-b] pyridazine derivative have demonstrated biological activity including inhibitors of the central nervous system [56], antipyritic and hypothermal activity [57], anticonvulsant activity, analgesic and antispasmotic activity [58-60].

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.

Materials and Methods

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 CDCl3 /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

Chemistry

We have prepared some novel sulfonamide or amide derivatives of piperazine ring incorporating imidazo[1,2-b]pyridazine moiety in three steps, using 3-amino-6-chloro pyridazine, 2-bromo-1- substituted aryl(or alkyl)ethanone, homopiperazine and alkyl (or substituted aryl) acid chloride or sulfonyl chloride as the starting materials. 3-amino-6-chloro pyridazine were treated with 2-bromo- 1-substituted aryl(or alkyl)ethanone in ethanol to obtain 6-chloro-2- substituted aryl(or alkyl)imidazo[1,2-b]pyridazine 1(a-d) which on reaction with homopiperazine in NMP results 6-(piperazin-1-yl)-2- substituted aryl(or alkyl)imidazo[1,2-b]pyridazine 2(a-d) and further by reacting with alkyl (or substituted aryl) acid chloride or sulfonyl chloride in presence of triethyl amine in Dichloromethane results the desired sulfonamide or amide derivatives. The clear procedure for the preparation of desired sulfonamide or amide derivatives are given below.

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-6- chloro 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.

Spectral data of intermediate

6-chloro-2-(trifluoromethyl)imidazo[1,2-b]pyridazine 1(a): 1H-NMR (400 MHz, CDCl3): 9.05 (s, 1H, Imidazo-H), 8.36 (d, J=9.6 Hz,1H, pyridazine-H), 7.59 (d, J=9.6 Hz,1H, pyridazine-H). MS: 222.3 (M+). Anal. Calcd for C7H3ClF3N3: C- 37.95%, H- 1.36%, Cl-16.00%, F-25.72%, N-18.96. Found: C- 37.85%, H- 1.33%, Cl-15.96%, F-25.70%, N-18.92.

6-chloro-2-p-tolylimidazo[1,2-b]pyridazine 1(b): 1H-NMR (400 MHz, CDCl3): 8.85 (s, 1H, Imidazo-H), 8.20 (d, J=9.6 Hz,1H, pyridazine-H), 7.93 (d, J=8.4 Hz, 2H, Ar-H), 7.35 (d, J=9.6 Hz, 1H, pyridazine-H), 7.28 (d, J=8.0 Hz,2H, Ar-H), 2.34 (s,3H, Ar-CH3). MS: 244.4 (M+). Anal. Calcd for C13H10ClN3: C- 64.07%, H- 4.14%, Cl- 14.55%, N-17.24. Found: C- 64.04%, H- 4.11%, Cl-14.52%, N-17.21%.

6-chloro-2-(4-(trifluoromethyl)phenyl)imidazo[1,2-b] pyridazine 1(c): 1H-NMR (400 MHz, CDCl3): 9.05 (s, 1H, Imidazo-H), 8.26-8.23 (m, 3H), 7.83 (d, J=8.0 Hz, 2H, Ar-H), 7.41 (d, J=9.6 Hz, 1H, pyridazine-H). MS: 298.2 (M+). Anal. Calcd for C13H7ClF3N3: C- 52.45%, H- 2.37%, Cl-11.91%, F-19.15%, N-14.12%. Found: C- 52.42%, H- 2.33%, Cl-11.89%, F-19.14%, N-14.09%.

6-chloro-2-(2,5-dichlorophenyl)imidazo[1,2-b]pyridazine 1(d): 1H-NMR (400 MHz, CDCl3): 8.92 (s, 1H, Imidazo-H), 8.30 (d, J=9.2 Hz,1H, pyridazine-H), 8.21-8.20 (m, 1H, Ar-H), 7.64 (d, J=8.8 Hz, 1H, Ar-H), 7.51-7.46 (m, 2H). MS: 298.4 (M+). Anal. Calcd for C12H6Cl3N3: C- 48.28%, H- 2.03%, Cl-35.62%, N-14.07%. Found: C- 48.25%, H- 1.99%, Cl-35.60%, N-14.03%.

General procedure for the synthesis of 6-(piperazin-1-yl)-2-substituted 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 Na2SO4. The solvent was evaporated and crude compound was used as such for next step without any purification.

Spectral data of intermediate

6-(piperazin-1-yl)-2-(trifluoromethyl)imidazo[1,2-b] pyridazine 2(a): 1H-NMR (400 MHz, CDCl3): 8.50 (s,1H, Imidazo-H), 7.91 (d, J=10.0 Hz,2H, pyridazine-H), 7.37 (d, J=10.0 Hz,2H, pyridazine-H), 3.43-3.41 (m, 4H, piperazine-H), 3.34 (bs, 1H, NH), 2.80-2.78(m, 4H, piperazine-H), MS:272.6 (M+). Anal. Calcd for C11H12F3N5: C- 48.71%, H- 4.46%, F-21.01%, N-25.82%. Found: C- 48.67%, H- 4.42%, F-20.98%, N-25.79%.

6-(piperazin-1-yl)-2-p-tolylimidazo[1,2-b]pyridazine 2(b): 1H-NMR (400 MHz, CDCl3): 8.40 (s,1H, Imidazo-H), 7.83-7.80 (m,3H), 7.21 (d, J=8.0 Hz,2H, Ar-H), 7.15 (d, J=10.0 Hz,1H, pyridazine-H), 3.39-3.37 (m, 4H, piperazine-H), 3.34 (bs, 1H, NH), 2.82-2.79(m,4H, piperazine-H), 2.31 (s,3H, Ar-H). MS: 294.4 (M+). Anal. Calcd for C17H19N5: C- 69.60%, H- 6.53%, N-23.87%. Found: C- 69.58%, H- 6.51%, N-23.83%.

6-(piperazin-1-yl)-2-(4-(trifluoromethyl)phenyl)imidazo[1,2-b] pyridazine 2(c): 1H-NMR (400 MHz, CDCl3): 8.02 (s,1H, Imidazo-H), 7.97 (d, J=8.4 Hz,2H, Ar-H), 7.80 (d, J=10.0 Hz,1H, pyridazine-H), 7.62 (d, J=8.4 Hz, 2H, Ar-H), 7.30 (d, J=10.0 Hz,1H, pyridazine-H), 3.40-3.37 (m, 4H, piperazine-H), 3.34 (bs, 1H, NH), 2.81-2.78(m,4H, piperazine-H). MS: 348.1 (M+). Anal. Calcd for C17H16F3N5: C- 58.78%, H- 4.64%, F-16.41%, N-20.16%. Found: C- 58.76%, H- 4.61%, F-16.37%, N-20.13%.

2-(2,5-dichlorophenyl)-6-(piperazin-1-yl)imidazo[1,2-b] pyridazine 2(d): 1H-NMR (400 MHz, CDCl3): 8.46 (s,1H, Imidazo-H), 8.20 (d, J=2.4 Hz,1H, Ar-H), 7.89 (d, J=10.0 Hz,1H, pyridazine-H), 7.57 (d, J=8.8 Hz,1H, Ar-H), 7.39 (dd, J1=8.6 Hz, J2=2.6Hz,1H, Ar-H), 7.27 (d, J=10.0 Hz,1H, pyridazine-H), 3.43-3.40 (m, 4H, piperazine-H), 3.33 (bs, 1H, NH), 2.82-2.79(m,4H, piperazine-H). MS: 348.5 (M+). Anal. Calcd for C16H15Cl2N5: C- 55.19%, H- 4.34%, Cl-20.36%, N-20.11%. Found: C- 55.16%, H- 4.31%, Cl-20.33%, N-20.09%.

General procedure for the conversion of 6-(piperazin-1-yl)-2-substituted aryl(or alkyl)imidazo[1,2-b]pyridazine into alkyl (or substituted aryl) sulfonamide or amide 3(a-j)

To a solution of 6-(piperazin-1-yl)-2-substituted aryl(or alkyl) imidazo[1,2-b]pyridazine (0.01 mole) in Dichloromethane was added triethyl amine (0.015 mole) followed by alkyl (or substituted aryl) acid chloride or sulfonyl chloride (0.013 mole) at 0°C and stirred the reaction mixture at room temperature for 2 hrs. The reaction mixture was diluted with water and extracted with Dichloromethne. The organic layer was washed with water, brine and dried over Na2SO4. The solvent was evaporated and crude compound was purified by using column chromatography with 100-200 silica gel to give compound 3(aj) (Scheme 1).

medicinal-chemistry

Scheme 1: Scheme

Spectral data of Desired sulfonamide and amide derivatives

1-(4-(2-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl) piperazin-1-yl)propan-1-one 3(a): 1H-NMR (400 MHz, CDCl3): 8.54 (s,1H, Imidazo-H), 7.97 (d, J=10.0Hz,1H, pyridazine-H), 7.41(d, J=10.0 Hz,1H, pyridazine-H), 3.58-3.51 (m, 8H, piperazine-H), 2.37 (q, J=7.4 Hz, 2H, -COCH2), 1.00 (t, J=7.4 Hz,3H,-CH3). LCMS: 328.4 (M+), Purity-97.5%. Anal. Calcd for C14H16F3N5O: C- 51.37%, H- 4.93%, F-17.41%, N-21.40%, O-4.89%. Found: C- 51.33%, H- 4.91%, F-17.39%, N-21.37%, O-4.87%.

p-tolyl(4-(2-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl) piperazin-1-yl)methanone 3(b): 1H-NMR (400 MHz, CDCl3): 7.93 (s,1H, Imidazo-H), 7.78 (d, J=10.0 Hz,1H, pyridazine-H), 7.35 (d, J=8.0 Hz, 2H, Ar-H), 7.24 (d, J=8.0 Hz,2H, Ar-H), 6.95(d, J=10.0 Hz,1H, pyridazine-H), 3.88-3.56 (m, 8H, piperazine-H), 2.40 (s,3H, Ar-CH3). LCMS: 390.2 (M+), Purity-98.2%. Anal. Calcd for C19H18F3N5O: C- 58.61%, H- 4.66%, F-14.64%, N-17.99%, O-4.11%, Found: C- 58.58%, H- 4.63%, F-14.61%, N-17.96%, O-4.09%.

(4-(2-(trifluoromethyl)imidazo[1,2-b]pyridazin-6-yl)piperazin- 1-yl)(4-(trifluoromethyl) phenyl) methanone (3c): 1H-NMR (400 MHz, CDCl3): 7.94 (s,1H, Imidazo-H), 7.81 (d, J=10.0 Hz,1H, pyridazine-H), 7.72 (d, J=8.0 Hz, 2H, Ar-H), 7.57 (d, J=8.0Hz,2H, Ar-H), 6.94 (d, J=10.0 Hz,1H, pyridazine-H), 3.95-3.56 (m, 8H, piperazine-H). LCMS: 444.3 (M+), Purity-95.7%. Anal. Calcd for C19H15F6N5O: C- 51.47%, H- 3.41%, F-25.71%, N-15.80%, O-3.61%, Found: C- 51.44%, H- 3.39%, F-25.68%, N-15.77%, O-3.57%.

1-(4-(2-p-tolylimidazo[1,2-b]pyridazin-6-yl)piperazin-1- yl)propan-1-one (3d): 1H-NMR (400 MHz, CDCl3): 7.97 (s,1H, Imidazo-H), 7.80 (d, J=8.0 Hz,2H, Ar-H), 7.75 (d, J=10.0 Hz,1H, pyridazine-H), 7.24 (d, J=8.4 Hz, 2H, Ar-H), 6.79 (d, J=9.6 Hz,1H, pyridazine-H), 3.80-3.71 (m,2H, piperazine-H), 3.64-3.62 (m,2H, piperazine-H), 3.55-3.53 (m,2H, piperazine-H), 3.49-3.47 (m,2H, piperazine-H), 2.41 (q, J=7.4 Hz, 2H,-COCH2), 2.38 (s,3H, Ar-CH3), 1.19 (t, J=7.4 Hz, 3H,-CH3). LCMS: 350.3 (M+), Purity-96.4%. Anal. Calcd for C20H23N5O: C- 68.47%, H- 6.63%, N-20.04%, O-4.58%, Found: C-68.45%, H- 6.60%, N-20.01%, O-4.54%.

1-(4-(2-(4-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6- yl)piperazin-1-yl)propan-1-one (3e): 1H-NMR (400 MHz, CDCl3): 8.05 (s,1H, Imidazo-H), 8.01 (d, J=8.4 Hz,2H, Ar-H), 7.77 (d, J=10.0 Hz,1H, pyridazine-H), 7.67 (d, J=8.4 Hz, 2H, Ar-H), 6.85 (d, J=9.6 Hz,1H, pyridazine-H), 3.81-3.78 (m,2H, piperazine-H), 3.64-3.63 (m,2H, piperazine-H), 3.57-3.55 (m,2H, piperazine-H), 3.52-3.49 (m, 2H, piperazine-H), 2.41 (q, J=7.4 Hz, 2H,-COCH2), 1.19 (t, J=7.4 Hz, 3H,-CH3). LCMS: 404.2 (M+), Purity-98.4%, Anal. Calcd for C20H20F3N5O: C- 59.55%, H- 5.00%, F-14.13%, N-17.36%, O-3.97%, Found: C-59.53%, H- 4.97%, F-14.11%, N-17.33%, O-3.94%.

1-(4-(2-(2,5-dichlorophenyl)imidazo[1,2-b]pyridazin-6-yl) piperazin-1-yl)propan-1-one (3f): 1H-NMR (400 MHz, CDCl3): 8.44 (s,1H, Imidazo-H), 8.26 (d, J=2.4 Hz,1H, Ar-H), 7.77 (d, J=10.0 Hz,1H, pyridazine-H), 7.38 (d, J=8.4 Hz, 1H, Ar-H), 7.20 (dd, J1=8.4 Hz, J2=2.4 Hz,1H, Ar-H), 6.87 (d, J=10.0Hz,1H, pyridazine-H),3.81-3.78 (m,2H, piperazine-H), 3.64-3.62 (m,2H, piperazine-H), 3.58-3.56 (m,2H, piperazine-H), 3.52-3.49 (m,2H, piperazine-H), 2.41 (q, J=7.4 Hz, 2H,-COCH2), 1.19 (t, J=7.4 Hz, 3H,-CH3). LCMS: 404.3 (M+), Purity-99.07%, Anal. Calcd for C19H19Cl22N5O: C- 56.45%, H- 4.74%, Cl-17.54%, N-17.32%, O-3.96%, Found: C-56.42%, H- 4.71%, Cl- 17.52%, N-17.29%, O-3.93%

6-(4-(ethylsulfonyl)piperazin-1-yl)-2-(trifluoromethyl) imidazo[1,2-b]pyridazine (3g): 1H-NMR (400 MHz, CDCl3): 7.95 (s,1H, Imidazo-H), 7.79 (d, J=10.0 Hz,1H, pyridazine-H), 6.93(d, J=10.0 Hz,1H, pyridazine-H), 3.65-3.62 (m, 4H, piperazine-H), 3.46- 3.43 (m, 4H, piperazine-H), 3.00 (q, J=7.4 Hz, 2H, -SO2CH2), 1.40 (t, J=7.4 Hz,3H,-CH3). LCMS: 364.2 (M+), Purity-97.39%. Anal. Calcd for C13H16F3N5O2S: C- 42.97%, H- 4.44%, F-15.69%, N-19.27%, O-8.81%, S-8.82%. Found: C-42.93%, H- 4.41%, F-15.66%, N-19.25%, O-8.79%, S-8.78%

6-(4-tosylpiperazin-1-yl)-2-(trifluoromethyl)imidazo[1,2-b] pyridazine (3h): 1H-NMR (400 MHz, CDCl3): 7.90 (s,1H, Imidazo-H), 7.73 (d, J=10.0 Hz,1H, pyridazine-H), 7.66 (d, J=8.0 Hz, 2H, Ar-H), 7.34 (d, J=8.0 Hz,2H, Ar-H), 6.84 (d, J=10.0 Hz,1H, pyridazine-H), 3.64-3.61 (m, 4H, piperazine-H), 3.15-3.12 (m, 4H, piperazine-H), 2.42 (s,3H, Ar-CH3). LCMS: 426.2 (M+), Purity-99.73%. Anal. Calcd for C18H18F3N5O2S: C- 50.82%, H- 4.26%, F-13.40%, N-16.46%, O-7.52%, S-7.54%, Found: C- 50.79%, H- 4.23%, F-13.37%, N-16.43%, O-7.49%, S-7.51%.

2-(trifluoromethyl)-6-(4-(4-(trifluoromethyl)phenylsulfonyl) piperazin-1-yl)imidazo[1,2-b] pyridazine (3i): 1H-NMR (400 MHz, CDCl3): 7.93-7.91 (m,3H), 7.83 (d, J=8.0 Hz, 2H, Ar-H), 7.75 (d, J=10.0 Hz,1H, pyridazine-H), 6.84 (d, J=10.0 Hz,1H, pyridazine-H), 3.66- 3.64 (m, 4H, piperazine-H), 3.21-3.19 (m, 4H, piperazine-H). LCMS: 480.3 (M+), Purity-94.54%. Anal. Calcd for C18H15F6N5O2S: C- 45.10%, H- 3.15%, F-23.78%, N-14.61%, O-6.67%, S-6.69%. Found: C- 45.09%, H- 3.13%, F-23.75%, N-14.59%, O-6.65%, S-6.66%.

6-(4-(ethylsulfonyl)piperazin-1-yl)-2-p-tolylimidazo[1,2-b] pyridazine (3j): 1H-NMR (400 MHz, CDCl3): 7.97 (s,1H, Imidazo-H), 7.80 (d, J=8.0 Hz,2H, Ar-H), 7.75 (d, J=10.0 Hz,1H, pyridazine-H), 7.24 (d, J=8.4 Hz, 2H, Ar-H), 6.78 (d, J=10.0 Hz,1H, pyridazine-H), 3.62-3.60 (m,4H, piperazine-H), 3.46-3.43 (m,4H, piperazine-H), 3.00 (q, J=7.4 Hz, 2H,-SO2CH2), 2.38 (s,3H, Ar-CH3), 1.40 (t, J=7.4 Hz, 3H,- CH3). LCMS: 386.3 (M+), Purity-96.27%. Anal. Calcd for C19H23N5O2S: C- 59.20%, H- 6.01%, N-18.17%, O-8.30%, S-8.32%, Found: C-59.18%, H- 6.00%, N-18.14%, O-8.28%, S-8.29%.

6-(4-(ethylsulfonyl)piperazin-1-yl)-2-(4-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (3k):

1H-NMR (400 MHz, CDCl3): 8.06 (s,1H, Imidazo-H), 8.00 (d, J=8.0 Hz,2H, Ar-H), 7.78 (d, J=10.0 Hz,1H, pyridazine-H), 7.67 (d, J=8.0 Hz, 2H, Ar-H), 6.84 (d, J=10.0 Hz,1H, pyridazine-H), 3.63-3.62 (m,4H, piperazine-H), 3.47-3.45 (m,4H, piperazine-H), 3.00 (q, J=7.2 Hz, 2H,-SO2CH2), 1.41 (t, J=7.4 Hz, 3H,-CH3). LCMS: 440.3 (M+), Purity-92.91% Anal. Calcd for C19H20F3N5O2S: C- 51.93%, H- 4.59%, F-12.97%, N-15.94%, O-7.28%, S-7.30%, Found: C-51.91%, H- 4.55%, F-12.94%, N-15.91%, O-7.25%, S-7.28%.

2-(2,5-dichlorophenyl)-6-(4-(ethylsulfonyl)piperazin-1-yl)imidazo[1,2-b]pyridazine (3l):

1H-NMR (400 MHz, CDCl3): 8.44 (s,1H, Imidazo-H), 8.26 (d, J=2.80 Hz,1H, Ar-H), 7.78 (d, J=10.0 Hz,1H, pyridazine-H), 7.38 (d, J=8.8 Hz, 1H, Ar-H), 7.20 (dd, J1=8.8 Hz, J2=2.4 Hz,1H, Ar-H), 6.85 (d, J=10.0 Hz,1H, pyridazine-H),3.65-3.62 (m,4H, piperazine-H), 3.46- 3.44 (m,4H, piperazine-H), 3.00 (q, J=7.4 Hz, 2H,-SO2CH2), 1.40 (t, J=7.4 Hz, 3H,-CH3). LCMS: 440.5 (M+), Purity-98.9%, Anal. Calcd for C18H19C12N5O2S: C- 49.10%, H- 4.35%, Cl-16.10%, N-15.90%, O-7.27%, S-7.28%, Found: C-49.08%, H- 4.32%, Cl-16.07%, N-15.88%, O-7.24%, S-7.25%.

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 [61-63] 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 108 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 108 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.

Results and Discussion

Chemistry

3-amino-6-chloro pyridazine on reaction with 2-bromo-1- substituted aryl(or alkyl)ethanone in ethanol gives 6-chloro-2- substituted aryl(or alkyl)imidazo[1,2-b]pyridazine 1(a-d). The obtained compound (1) on reaction with homopiperazine in NMP results 6-(piperazin-1-yl)-2-substituted aryl(or alkyl)imidazo[1,2-b] pyridazine 2(a-d) which on further reaction with alkyl (or substituted aryl) acid chloride or sulfonyl chloride gives sulfonamide or amide derivatives of piperazine ring incorporating imidazo[1,2-b]pyridazine moiety 3(a-l). The list of synthesized compound are represented by Table 1.

Compound R1 R2 M.P Yield (%)
3a CF3 C2H5 117 46.2
3b CF3 4CH3- phenyl 156 52.3
3c CF3 4CF3- phenyl 124 49.5
3d 4CH3- phenyl C2H5 172 58.3
3e 4CF3- phenyl C2H5 228 55.7
3f 2,5- Dichloro phenyl C2H5 152 65.3
3g CF3 C2H5 196 63.4
3h CF3 4CH3- phenyl 188 58.4
3i CF3 4CF3- phenyl 207 48.9
3j 4CH3- phenyl C2H5 218 40.2
3k 4CF3- phenyl C2H5 202 61.3
3l 2,5- Dichloro phenyl C2H5 221 39.4

Table 1: List of Synthesized compound.

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.

Compound S. aureus S. pyogenus E. coli P. aeruginosa
3a 100 200 200 200
3b 62.5 125 200 250
3c 100 100 200 200
3d 250 100 62.5 125
3e 125 200 125 100
3f 100 100 100 200
3g 250 200 125 250
3h 250 200 250 500
3i 200 200 250 250
3j 100 250 100 250
3k 250 200 250 100
3l 100 200 250 100
Ampicillin 250 100 100 100
Chloramphenicol 50 50 50 50

Table 2: Antibacterial activity of sulfonamide and amide derivatives (In MIC).

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.

Compound C.albicans A.niger A.clavatus
3a 250 >1000 >1000
3b 1000 1000 1000
3c 500 250 500
3d 1000 >1000 >1000
3e 500 >1000 >1000
3f 1000 500 1000
3g 1000 >1000 >1000
3h 500 500 250
3i 500 500 250
3j 1000 1000 1000
3k >1000 500 500
3l 500 >1000 >1000
Nystatin 100 100 100
Greseofulvin 500 100 100

Table 3: Antifungal activity (in MIC).

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.

Compound Mean IC50 (Micrograme/ml)
3a 0.77
3b 0.97
3c 0.79
3d 0.80
3e 0.89
3f 0.98
3g 1.01
3h 1.12
3i 0.98
3j 1.12
3k 0.79
3l 0.96
Quinine 0.268

Table 4: Antimalarial activity.

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.

Acknowledgements

The authors would like to thank to Dr. Dhanji P. Rajani and Mr. Kalpesh of Microcare Laboratory, and Tuberclosis Research Centre, Surat for conducting the antibacterial, antifungal and antimalarial activity.

References

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Review summary

  1. Jumima
    Posted on Sep 29 2016 at 9:17 pm
    The provided results justify the conclusion that this scaffold is an attractive target for drug discovery. Considering the ease of synthesis, author done larger and diverse library of compounds are screened against a wide panel of microbial targets.

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