Original article Design, synthesis and antimicrobial activity of novel benzothiazole analogs Manavendra K. Singh a , Ragini Tilak b , Gopal Nath b , Satish K. Awasthi c, * , Alka Agarwal a, * a Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India b Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India c Chemical Biology Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India article info Article history: Received 30 November 2012 Received in revised form 19 February 2013 Accepted 21 February 2013 Available online 1 March 2013 Keywords: Benzothiazole Click chemistry 1,2,3-Triazole Antibacterial activity Antifungal activity abstract In an attempt to design and synthesize a new class of antimicrobials, dialkyne substituted 2-aminobenzothiazole was reacted with various substituted aryl azides to generate a small library of 20 compounds (3aet) by click chemistry. Structures of the newly synthesized compounds were established on the basis of spectral data. These compounds were screened for their antibacterial activity against Gramþ bacteria (Staphylococcus aureus and Enterococcus faecalis), Grambacteria (Salmonella typhi, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella boydii) and antifungal activity against Candida tropicalis, Candida albicans, Candida krusei, Cryptococcus neoformans) as well as molds (Aspergillus niger, Aspergillus fumigatus). The compound 3e showed maximum potency against all Gramþ/grambacterial strains with MIC value 3.12 mg/ml, which is two fold more active as compared to standard drug ciprofloxacin (MIC 6.25 mg/ml). However, all compounds were found ineffective against S. boydii (clinical isolate). Further, only one compound 3n was found to be the most active against all fungal strains with MIC value in the range of 1.56 mg/mle12.5 mg/ml while the remaining compounds showed moderate to weak antifungal activity. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Benzothiazole analogs are one of the most versatile class of com- pounds which most commonly occur as various marine and terres- trial natural products [1,2]. It is a unique scaffold for further molecular exploration to synthesize novel compounds. It is well documented that benzothiazole derivatives find wide application in non-invasive diagnosis of Alzheimer’s disease [3,4], antitubercular [5], antima- larial [6], antihelmintic [7] and also antidiabetic [8] etc. In addition, several benzothiazole analogs have also been screened as potential amyloid-binding diagnostic agents in neurodegenerative disease [9,10] and as selective fatty acid amide hydrolase inhibitors [11]. 2-Aminobenzothiazole moiety is present in various bioactive molecules such as imaging agents for b-amyloid plaques [9], pho- tosensitizers [12], inhibitors of stearoyl-coenzyme A d-9 desaturase [13], antitumor I [14], antimicrobial [5], orexin receptor antagonist II [15] and the Gramþ selective antibacterial III [16]. Antifungal flu- conazole IV and other marketed drugs, voriconazole V and albaco- nazole VI contain 1,2,4-triazole (Fig. 1). However, 1,2,3-triazole ring is not found in any marketed drugs. The click chemistry developed by Sharpless et al. is an excellent approach for regioselective syn- thesis of 1,2,3-triazole ring system in presence of various functional groups. Sincere efforts have been made to incorporate 1,2,3-triazole in existing drugs, still more research is needed to find lead molecule [17]. Although, 1,2,3-triazole structural moiety does not occur in nature, it is present in several compounds showing various bio- logical activities including anti-HIV [18], anti-bacterial [19], anti- allergic [20], anticonvulsant [21], b-lactamase inhibitory [22], and anti-tuberculosis activities [23]. 1,2,3-triazole has been extensively studied owing to its importance in industrially interesting materials, such as dyes, anticorrosive agents, photo stabilizers, photographic materials, and agrochemicals [17]. We therefore found it interesting to design new molecules within the scope of synthetic procedure using benzothiazole scaf- fold followed by suitable modification to generate diversified compounds for antimicrobial activity. In this study, we exploited click chemistry for synthesis of diversified benzothiazole com- pounds mainly for the two reasons; first, it can tolerate wide range of functional groups and easy to do eco-friendly reactions at room temperature either in water or mixture of water and organic sol- vents, secondly; this approach will generate compounds having 1,2,3-triazole functionality rather than 1,2,4-triazole. These * Corresponding authors. E-mail addresses: [email protected], [email protected], skawasthi@ chemistry.du.ac.in, [email protected](A. Agarwal). Contents lists available at SciVerse ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.02.027 European Journal of Medicinal Chemistry 63 (2013) 635e644
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at SciVerse ScienceDirect
European Journal of Medicinal Chemistry 63 (2013) 635e644
Design, synthesis and antimicrobial activity of novel benzothiazoleanalogs
Manavendra K. Singh a, Ragini Tilak b, Gopal Nath b, Satish K. Awasthi c,*, Alka Agarwal a,*aDepartment of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, IndiabDepartment of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, IndiacChemical Biology Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
Article history:Received 30 November 2012Received in revised form19 February 2013Accepted 21 February 2013Available online 1 March 2013
0223-5234/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2013.02.027
a b s t r a c t
In an attempt to design and synthesize a new class of antimicrobials, dialkyne substituted2-aminobenzothiazole was reacted with various substituted aryl azides to generate a small library of 20compounds (3aet) by click chemistry. Structures of the newly synthesized compounds were establishedon the basis of spectral data. These compounds were screened for their antibacterial activity againstGramþ bacteria (Staphylococcus aureus and Enterococcus faecalis), Gram� bacteria (Salmonella typhi,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella boydii) and antifungal activityagainst Candida tropicalis, Candida albicans, Candida krusei, Cryptococcus neoformans) as well as molds(Aspergillus niger, Aspergillus fumigatus). The compound 3e showed maximum potency against allGramþ/gram� bacterial strains with MIC value 3.12 mg/ml, which is two fold more active as compared tostandard drug ciprofloxacin (MIC 6.25 mg/ml). However, all compounds were found ineffective againstS. boydii (clinical isolate). Further, only one compound 3n was found to be the most active against allfungal strains with MIC value in the range of 1.56 mg/mle12.5 mg/ml while the remaining compoundsshowed moderate to weak antifungal activity.
� 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Benzothiazole analogs are one of the most versatile class of com-pounds which most commonly occur as various marine and terres-trial natural products [1,2]. It is a unique scaffold for furthermolecularexploration to synthesize novel compounds. It is well documentedthat benzothiazole derivatives find wide application in non-invasivediagnosis of Alzheimer’s disease [3,4], antitubercular [5], antima-larial [6], antihelmintic [7] and also antidiabetic [8] etc. In addition,several benzothiazole analogs have also been screened as potentialamyloid-binding diagnostic agents in neurodegenerative disease[9,10] and as selective fatty acid amide hydrolase inhibitors [11].
2-Aminobenzothiazole moiety is present in various bioactivemolecules such as imaging agents for b-amyloid plaques [9], pho-tosensitizers [12], inhibitors of stearoyl-coenzyme A d-9 desaturase[13], antitumor I [14], antimicrobial [5], orexin receptor antagonist II[15] and the Gramþ selective antibacterial III [16]. Antifungal flu-conazole IV and other marketed drugs, voriconazole V and albaco-nazole VI contain 1,2,4-triazole (Fig. 1). However, 1,2,3-triazole ring
is not found in any marketed drugs. The click chemistry developedby Sharpless et al. is an excellent approach for regioselective syn-thesis of 1,2,3-triazole ring system in presence of various functionalgroups. Sincere efforts have been made to incorporate 1,2,3-triazolein existing drugs, still more research is needed to find lead molecule[17]. Although, 1,2,3-triazole structural moiety does not occur innature, it is present in several compounds showing various bio-logical activities including anti-HIV [18], anti-bacterial [19], anti-allergic [20], anticonvulsant [21], b-lactamase inhibitory [22], andanti-tuberculosis activities [23]. 1,2,3-triazole has been extensivelystudied owing to its importance in industrially interestingmaterials,such as dyes, anticorrosive agents, photo stabilizers, photographicmaterials, and agrochemicals [17].
We therefore found it interesting to design new moleculeswithin the scope of synthetic procedure using benzothiazole scaf-fold followed by suitable modification to generate diversifiedcompounds for antimicrobial activity. In this study, we exploitedclick chemistry for synthesis of diversified benzothiazole com-pounds mainly for the two reasons; first, it can tolerate wide rangeof functional groups and easy to do eco-friendly reactions at roomtemperature either in water or mixture of water and organic sol-vents, secondly; this approach will generate compounds having1,2,3-triazole functionality rather than 1,2,4-triazole. These
Fig. 1. Chemical structure of pharmacologically important benzothiazoles and fluconazoles.
Scheme 1. Schematic representation of synthesis of benzothiazole 1,2,3-triazoleanalogs.
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644636
compounds can be screened for antibacterial and antifungal ac-tivities. Structureeactivity relationship (SAR) can be done on leadmolecule to find the most potent molecule as a clinical candidate.Moreover, synthesis of these compounds will also provide an op-portunity to compare antimicrobial activity with compounds con-taining 1,2,4-triazole, a common moiety found in several clinicalantimicrobial drugs.
In view of the above mentioned facts, and in continuation toongoing research work in our laboratory on design and synthesis ofnew antimicrobial agents [24e26] and X-rays analysis of smallmolecules [27e30], we further designed and synthesized small li-brary of 20 compounds of benzothiazole-1,2,3-triazole analogs andscreened them for antimicrobial activity.
2. Chemistry
The benzothiazol-2-yl-di-prop-2-ynyl-amine (1a) was synthe-sized in good yield by simple alkylation involving reaction of2-aminobenzothiazole and propargyl bromide as outlined inScheme 1. The compound 1a was further reacted with varioussubstituted aromatic azides (2aet) to give 20 different compounds(3aet). The detailed general synthesis procedure of the compoundsis mentioned in the Experimental section. All synthesized com-pounds (3aet) were characterized by ESI-MS, 1H and 13C NMR. Thestructure of the compound 3e was also confirmed by X-ray crys-tallographic analysis and reported elsewhere (CCDC no 896668).The crystal structure of compound 1b was published earlier [29].
3. Results and discussion
Various compounds were generated by reacting 2-amino-benzothiazole with propargyl bromide in presence of base K2CO3
in dry acetone which yielded 1a (major) and 1b (minor). Thecompound 1a containing propargyl group at 2-positionwas used assubstrate to further generate small 1,4-disubstituted 1,2,3-triazolelibrary of 20-compounds (3aet) by reacting various substitutedaromatic azides using click chemistry. All compounds were purified
by column chromatography or by recrystallization, well character-ized by 1H and 13C NMR, ESI-MS etc. Various substituted1,2,3-triazoles 3aet were screened for their antibacterial activity(MIC) against various Gramþ and gram� bacterial strains and ac-tivity data are summarized in Table 1. We chose two strains ofGramþ bacteria i.e. Staphylococcus aureus (ATCC 25323) andEnterococcus faecalis (clinical isolate) and five gram� bacterialstrains i.e., Escherichia coli (ATCC 35218), Salmonella typhi (MTCC3216), Klebsiella pneumoniae (clinical isolated), Pseudomonas aeru-ginosa (ATCC 27893) and Shigella boydii (clinical isolate) for study.
It is evident from Table 1 that six compounds viz. 3b, 3c, 3e, 3g,3h and 3r were found more potent with either less or equal MIC as
Table 1Antibacterial activity (MIC mg/ml) and % hemolysis of compounds 3aet.
Compoundno.
R Gram þve strain Gram �ve strain % hemolysis
S. aureus(ATCC 25323)
E. faecalis(clinicalisolate)
S. typhi(MTCC 3216)
E. coli(ATCC 35218)
K. pneumoniae(clinical isolate)
P. aeruginosa(ATCC 27893)
S. boydii(clinicalisolate)
3a
Cl
Cl
50 50 >100 50 >100 >100 >100 4.54
3b
F
Cl
6.25 3.12 6.25 25 50 3.12 100 5.32
3c
Cl
6.25 3.12 6.25 12.5 50 6.25 >100 4.35
3dF
>100 >100 >100 >100 >100 >100 >100 26.52
3e
F
F
3.12 3.12 3.12 3.12 3.12 3.12 100 2.47
3f
OCH3
100 >100 >100 50 >100 >100 >100 7.54
3g
F
Cl
50 6.25 6.25 3.12 100 6.25 100 12.31
3hCl
25 6.25 6.25 12.5 50 12.5 >100 19.48
3i
F
>100 >100 >100 >100 >100 >100 >100 29.79
(continued on next page)
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644 637
Table 1 (continued )
Compoundno.
R Gram þve strain Gram �ve strain % hemolysis
S. aureus(ATCC 25323)
E. faecalis(clinicalisolate)
S. typhi(MTCC 3216)
E. coli(ATCC 35218)
K. pneumoniae(clinical isolate)
P. aeruginosa(ATCC 27893)
S. boydii(clinicalisolate)
3jCl
Cl
50 >100 100 >100 >100 >100 >100 37.66
3k
F
CH3
>100 >100 >100 50 >100 >100 >100 29.79
3l
H3C
>100 >100 >100 50 >100 >100 >100 48.27
3m
CH3
CH3
>100 >100 >100 >100 >100 >100 >100 43.05
3n
Br
>100 >100 >100 >100 >100 >100 >100 12.37
3o
OCH3
OCH3H3CO
>100 >100 >100 >100 >100 >100 >100 36.60
3p >100 >100 >100 >100 >100 >100 >100 34.04
3q
NO2
>100 >100 >100 >100 >100 >100 >100 27.85
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644638
Table 1 (continued )
Compoundno.
R Gram þve strain Gram �ve strain % hemolysis
S. aureus(ATCC 25323)
E. faecalis(clinicalisolate)
S. typhi(MTCC 3216)
E. coli(ATCC 35218)
K. pneumoniae(clinical isolate)
P. aeruginosa(ATCC 27893)
S. boydii(clinicalisolate)
3r
OCF3
12.5 12.5 >100 25 12.5 25 100 9.12
3s
C5H11
>100 >100 >100 >100 >100 >100 >100 3.28
3t
CH3H3C
100 >100 100 100 >100 >100 100 7.42
Ciprofloxacin e 6.25 6.25 6.25 6.25 6.25 3.12 6.25 e
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644 639
compared to control drug ciprofloxacin. DMSO was also taken in acontrol experiment which showed no effect in the experiment.
The compound 3a containing 2,4-dichlorosubstituted benzenering showed moderate to weak activity in the range of 50 mg/ml to100 mg/ml while ciprofloxacin showed MIC 6.25 mg/ml in all strainsexcept P. aeruginosa where MIC is 3.12 mg/ml. The compounds 3band 3c having 3-chloro-4-fluoro and 4-chlorosubstituted benzenering, respectively were found to be the most potent with MIC3.12 mg/ml in E. faecalis. Further, the same compounds also showedMIC 6.25 mg/ml in S. aureus and S. typhi. Again, the compound3b was found to be the most potent with MIC 3.12 mg/ml whilethe compound 3c exhibited good activity with MIC 6.25 mg/mlagainst strain P. aeruginosa. Further, the compound 3b showed MIC25 mg/ml while compound 3c showedMIC 12.5 mg/ml against E. coli.In other strains, the compounds 3b and 3c showed moderate toweak activity in the range of 50 mg/ml to100 mg/ml. The compound3e containing 2,4-difluorosubstituted benzene showed excellentpotency against six strains having MIC 3.12 mg/ml except S. boydii,clinical isolate strain (MIC 100 mg/ml). Further, the compound 3ghaving 2-chloro-4-fluorosubstituted benzene ring showed potentinhibitory activity against E. faecalis, S. typhi and P. aeruginosa withMIC 6.25 mg/ml. It also showed two folds strong antibacterialactivity against E. coli with MIC 3.12 mg/ml while standard drughas MIC 6.25 mg/ml and showed moderate to weak activity in therange of 50 mg/ml to 100 mg/ml against remaining strains. Thecompound 3h showed MIC 6.25 mg/ml against E. faecalis, S. typhiand E. coli while against P. aeruginosa showed MIC 12.5 mg/mland against remaining strains showed moderate to weak activitywith MIC 25 mg/ml to 100 mg/ml. Again, the compounds 3i to 3qshowed moderate to weak activity against all strains in the range of50 mg/ml to 100 mg/ml or>100 mg/ml. The compound 3r containing4-OCF3 substituted benzene ring was found active against threebacterial strains viz. S. aureus, E. faecalis and K. pneumoniae with
MIC 12.5 mg/ml while against remaining strains showed moderatetoweak susceptibility withMIC in the range of 25 mg/mle100 mg/mlor >100 mg/ml. Further, the compounds 3s and 3t were foundinactive with MIC 100 mg/ml or >100 mg/ml against all strains. Ingeneral, control drug ciprofloxacin showed MIC 3.12 mg/ml againstP. aeruginosa while against the remaining strains showed MIC6.25 mg/ml.
It is obvious from the analysis of activity results that electronwithdrawing groups such as fluoro, chloro or combination of thesehave strong effects in determining the antibacterial activity. Thisobservation is supported by the highest activity shown by thecompound 3e against six bacterial strains (both Gramþ and gram�strains) except S. boydii. Further, the position of functional groups inbenzene ring also play critical role toward activity. For example,compound 3g containing chloro and fluoro groups at position-2 & 4in benzene ring, showed potency while the compound 3i withfluoro group at position-3 in benzene ring lost activity. The pres-ence of chloro at position-2 & 3 in benzene ring (compound 3j) alsolost activity. However, the presence of Br group at 4-position inbenzene ring of compound also lost activity. Thus, we hypothesizethat the position-2 is critical for antibacterial activity and fluorogroup has significant effect on antibacterial activity as compared tochloro group.
Further, the presence of electron releasing groups such asmethyl, methoxy and pentyl groups irrespective of position in thebenzene rings have shown strong adverse effect on antibacterialactivity and thus the compounds containing these functionalgroups showed weak or moderate activity as compared to electronwithdrawing groups. However, nitro group at position-4 alsoresulted in decreased activity. In order to ascertain this hypothesis,we need to do more systematic design and synthesis of diversifiedcompounds using multifunctional substituted benzene ring. Oncelead compound is identified from in vitro screening, more
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644640
systematic structureeactivity relationship (SAR) is needed to getmolecule for in vivo studies which may eventually clinical candi-date for future.
Further, these compounds 3aet were also screened against fivefungal strains viz. Candida tropicalis (ATCC 750), Candida albicans(clinical), C. albicans (ATCC 90028), Candida krusei (ATCC 6258),Cryptococcus neoformans (clinical) and two molds Aspergillus niger(clinical) and Aspergillus fumigatus (clinical) for their antifungalactivity. These compounds showed weak to moderate antifungalactivity as shown in Table 2. Fluconazole was taken as a standarddrug in the experiment.
Compound 3a having 2,4-dichlorosubstituted benzene ring wasfound to be the most potent. It showed antifungal activity with MIC1.56 mg/ml against clinical strains A. niger and A. fumigatus which iscomparable to standard drug fluconazole with MIC 1.56 mg/ml.Again, the compound 3a also showed potent inhibitory activityagainst strains C. neoformans with MIC 3.12 mg/ml while against theremaining strains showed MIC in the range between 6.25 mg/ml and12.5 mg/ml. The compound 3b containing 3-chloro-4-fluorobenzenering showed moderate activity against C. krusei and C. neoformanswith MIC 3.12 mg/ml and 1.56 mg/ml, respectively while against theremaining strains showed MIC in between 6.25 mg/ml and 25 mg/ml.
The compound 3c containing 4-chlorobenzene ring showedgood activity against C. neoformans with MIC 3.12 mg/ml while themoderate to weak activity in the range of 3.12 mg/mle25 mg/mlwas observed with other strains. The compound 3f having3-methoxysubstituted benzene ring showed good activity againstthree strains viz. C. neoformanswithMIC 1.56 mg/ml and A. niger andA. fumigatus with MIC 3.12 mg/ml while fluconazole showed MIC0.78 mg/ml and 1.56 mg/m respectively. Again, compound 3g con-taining 2-chloro-4-flurosubstituted benzene ring showed goodactivity against C. neoformans with MIC 1.56 mg/ml and showedmoderate to weak activity against remaining six strains in between6.25 mg/ml and 50 mg/ml. Further, the compound 3j having 2,3-dichlorosubstituted benzene ring showed reasonable good activ-ity against C. neoformans with MIC 3.12 mg/ml and weak or no ac-tivity against remaining strains with MIC 25 mg/ml to >100 mg/ml.Further, the compound 3n having 4-bromosubstituted benzenering had shown good activity against C. neoformans with MIC
Table 2Antifungal activity (MIC mg/ml) of compounds 3aet.
1.56 mg/ml while against A. niger & A. fumigatus showed MIC3.12 mg/ml. Further, the same compound also showed moderateactivity in the range of 6.25 mg/mle12.5 mg/ml against remainingstrains.
The compound 3r containing 4-OCF3 group in benzene ring alsoshowed reasonable good activity with MIC 3.12 mg/ml against twostrains C. albicans and C. neoformans. Further, the moderate activitywas seen with the other remaining strains in between 12.5 mg/mland 25 mg/ml.
Again, the compound 3s having 4-pentyl group was found inac-tive against most of strains except C. tropicalis with MIC 12.5 mg/ml.The compound 3t containing 3,5-dimethyl groups in benzeneshowed potent activity with MIC 3.12 mg/ml against two strainsC. albicans & C. neoformans while the remaining strains showedmoderate activity with MIC in between 12.5 mg/ml or >100 mg/ml.The fluconazole was taken as a control drug which showed MICvalue in between 0.78 mg/ml and 3.12 mg/ml against various strains.Thus, it is obvious from the above antifungal screening data that bothpresence of electron withdrawing groups such as bromo or chlorogroup and electron releasing groups such as methoxy group onposition-2, 3 or 4 showed potent antifungal activity. Further, pres-ence of dihalogen in benzene ring has better antifungal activity thanmonohalogen. Interestingly, 4-bromosubstituted benzene showedstrong antifungal activity while it showed weak antibacterialactivity.
We hypothesize that groups such asmethoxy, alkyls and halogenspresent on suitable position in benzene ring enhanced antifungalactivity. Dichlorocompounds showed better antifungal activity thandifluorocompounds. Moreover, we need to use multifunctionalsubstituted benzene ring in click chemistry to generate large numberof diverse compounds for in vitro antifungal screening. Further, thescreening of these compounds will also help to predict positionaleffect of functional groups on antimicrobial activity. Structureeactivity relationship (SAR) needs to be done on most potentmolecule to find lead molecule which can be evaluated for in vivoactivity. Thus, more systematic design and synthesis of secondgeneration compounds with various functional groups are neededto established meaningful structureeactivity relation (SAR). We areworking in this direction.
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644 641
3.1. Hemolytic activity
To test toxicity of synthesized compounds 3aet, the hemolyticactivities were carried out according to the procedure developed byNielson et al. [31] on human hRBC at a fixed concentration of100 mM. The analysis results showed that these compounds caused2e48% hemolysis. Most of the compounds (3ae3c, 3ee3h, 3n, and3re3t) showed less than 20% hemolysis suggesting that most of thecompounds are less toxic. In general, the compounds with betterantimicrobial activity showed negligible or very low toxicity profile.The compound 3e showed only 2.47% hemolysis at very high con-centration i.e. 100 mM. Thus, these results further support the sig-nificance of the study. The antibacterial as well hemolysis resultsare depicted in Table 1.
4. Conclusion
The objective of the present work was to design, synthesize andscreen the antibacterial and antifungal activities of novel benzo-thiazole analogs with the hope of discovering new structure leadsas the most potent antimicrobial agents. Our aim has been achievedby the synthesis of small benzothiazole library of 20 compoundswith diver functionalities by exploiting click chemistry andscreened them against various Gramþ and gram� bacterial as wellas fungal strains. The compounds 3b, 3c and 3e were found to bethe most potent than standard drug ciprofloxacin with MIC3.12 mg/ml against E. faecalis (clinical isolate) and E. coli (ATCC35318) bacterial strains respectively and the compounds viz. 3b, 3c,3g and 3h were also active with MIC 6.25 mg/ml, which is same asciprofloxacin against S. aureus (ATCC 25323), E. faecalis (clinicalisolate), S. typhi (MTCC 3216) and P. aeruginosa (ATCC 27893). Mostof the compounds showed very little toxicity as revealed by he-molysis data.
Further, the compound 3ewas found to be themost active agentagainst all bacterial strains except S. boydii (clinical isolate). Theelectron withdrawing groups at position-2, 4 in benzene ringenhanced the inhibitory activity while electron releasing groupsdecrease the inhibitory activity in bacteria. Furthermore, thecompound 3a was found to be the most potent with MIC at1.56 mg/ml which shows similar activity as fluconazole againstA. niger (clinical) and A. fumigatus (clinical). The only compound 3nshowed good activity against all fungal strains while remainingcompounds showed moderate to weak activity. The obtained re-sults clearly demonstrate that the compounds derived from ben-zothiazole exhibited better antimicrobial activity. This ispreliminary results and to reach more appropriate conclusion, 2ndand 3rd generation of compounds should be synthesized in order toestablish meaningful structureeactivity relationship (SAR).
5. Experimental
Chemicals and solvents used in this study were purchased fromE. Merck (India) and SigmaeAldrich chemicals. Melting points weredetermined by using open capillary method and are uncorrected.1H NMR spectral data were recorded on Brucker Advance spec-trometer at 300 MHz and Jeol JNM ECX spectrometer at 300 MHzusing TMS as an internal standard. The chemical shifts values wererecorded on d scale and the coupling constants (J) in Hertz. Thefollowing abbreviations were used in reporting spectra: s ¼ singlet,d ¼ doublet, dd ¼ double doublet, t ¼ triplet, td ¼ triple doublet,q ¼ quartet, m ¼ multiple. ESI-MS spectra were obtained on aWaters Micromass LCT Mass spectrometer. Elemental analysis wasdone on Elementar GmbH VarioEl analyzer.
5.1. General procedure for the synthesis of benzothiazol-2-yl-di-prop-2-ynyl-amine (1a)
The synthesis of alkyne was carried out according to the liter-ature procedure. Briefly, to a solution of amine (6 mmol) in dryacetone was added anhydrous K2CO3 (32 mmol) and reactionmixture was further refluxed for 15e30 min. Subsequently, KI(3 mmol) and propargyl bromide (7.2 mmol) were added and thereaction mixture was further refluxed for 18 h. The completion ofreaction was monitored by TLC. After completion of the reaction,mixture was cooled, filtered, and the filtrate was evaporated invacuo to give the two products (1a & 1b), which were purified bycolumn chromatography using hexane and dichloromethane(65:35) as eluent. The major product was compound 1awhich wasused for synthesis of next step compounds (3aet) by click chem-istry while the compound 1bwas crystallized and the structurewasestablished by X-rays analysis. All 20 compounds were screened forantibacterial and antifungal activities.
5.2. General procedure for the synthesis of azide 2 (aet)
The synthesis of various azides was carried out according to theliterature procedure [32]. Briefly, aniline (1 eq, 5 mmol) was dis-solved in 6 N HCl solution (20 ml) at room temperature and cooledup to 0 �C, followed by addition of a solution of NaNO2 (1 eq,5 mmol). The reaction mixture was stirred for 10 min at 0e5 �C.Sodium azide (1.2 eq, 6 mmol) was added and mixture wasfurther stirred at room temperature for 2 h. The reaction wasworked up by dilution with ethyl acetate. The organic layer waswashed with brine solution and dried over sodium sulfate. Afterevaporation of the solvent, the crude product 2 (aet) was pureenough for further reactions. All the synthesized azides were storedat �20 �C.
5.3. General procedure for the synthesis of compounds (3aet)
The synthesis of compounds (3aet) was carried out according tothe literature procedure [33]. Briefly, benzothiazol-2-yl-di-prop-2-ynyl-amine (1a) (1 mmol) and various aromatic azide (2 mmol)were suspended in N,N0-dimethylformamide (10 ml). Sodiumascorbate (0.3 mmol, in water) was added, followed by copper (II)sulfatepentahydrate (0.03 mmol, in water). The heterogenousmixture was stirred vigorously overnight, and the completion ofreaction was monitored by TLC. After completion of the reaction,the reaction mixture was diluted with water, cooled in ice, and theprecipitate was collected by filtration.
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644642
A total of seven bacterial strains viz. S. aureus (ATCC 25323),S. typhi (MTCC 3216), E. coli (ATCC 35318), E. faecalis (clinicalisolate), P. aeruginosa (ATCC 27893), S. boydii (clinical isolate) andK. pneumoniae (clinical isolate) and seven fungal strains viz.C. tropicalis (ATCC 750), C. albicans (clinical), C. albicans (ATCC90028), C. krusei (ATCC 6258), A. niger (clinical), A. fumigatus(clinical), C. neoformans (clinical) were used in the investigation forantimicrobial assay. All cultures were preserved at Department ofMicrobiology, Institute of Medical Sciences, Banaras Hindu Uni-versity, Varanasi, India which were obtained from American TypeCulture Collection (ATCC), MTCC and clinical strain. The fresh mi-crobial broth cultures were prepared in normal saline before thescreening procedure. Ciprofloxacin was used as standard drug forantibacterial activity and fluconazole was used as standard drug forantifungal activity.
Minimum inhibitory concentration (MIC) of all compounds wasdetermined by micro-dilution method [34] using serially diluted(8 folds) compounds. MIC of the compounds was determined byseries of dilution at various concentrations. Different concentrationof the compounds (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml,6.25 mg/ml, 3.12 mg/ml, 1.56 mg/ml, 0.78 mg/ml) were serially diluted
M.K. Singh et al. / European Journal of Medicinal Chemistry 63 (2013) 635e644644
in microtiter plate. Specifically, 0.1 ml of standardized inoculums(1e2 � 107 cfu/ml) was added in each tube of microtiter plate. Theplates were incubated aerobically at 37 �C for 18e24 h. The lowestconcentration (highest dilution) of the compounds showed novisible bacterial growth no turbidity in the solution when it wascompared with the control was regarded as the MIC. Mueller-Hinton agar and Luria broth (Hi-media, Mumbai, India), was usedfor antibacterial activity.
Similar protocol was followed for determination of MIC ofantifungal compounds except Sabouraud dextrose agar pH 7.3� 0.2(Hi-media) was used.
5.5. Determination of hemolytic activity of compounds on humanred blood cells (hRBC)
The hemolysis assay was carried out according to the proceduredeveloped by Nielson et al. [31]. Briefly, the fresh human blood wascollected from the hospital and washed three times in sterilephosphate buffered saline (PBS) solution. After each washing step,the cells were centrifuged at 3000 rpm for 7 min at room tem-perature and supernatant was discarded after each washing. ThehRBC were re-suspended in PBS and adjusted the final concentra-tion of 5�108 cells/ml. An aliquot (10 ml) of the cell suspensionwasadded in 100 mL buffer solution containing 100 mM test compoundsin 1% v/v DMSO in PBS. Further, controls were also taken as 1% v/vDMSO in PBS and sterilewater. The cell suspensionswere incubatedat 37 �C for 1 h with constant shaking. After 1 h, the solution wascentrifuged at 1300 rpm for 5 min at room temperature andabsorbance was recorded at 540 nm. The UV absorbance values ofthe test compounds were expressed as a % of the absorbance ofsterile water (equivalent to 100% hemolysis) to give % hemolysisresults.
Acknowledgment
AA is thankful to (UGC), New Delhi, India (scheme no. 34-311/2008) and Banaras Hindu University, Varanasi, India for financialsupport. This work was partly supported by GN and RT Departmentof Microbiology, Institute of Medical Sciences, Banaras Hindu Uni-versity. SKA is thankful to the University of Delhi, Delhi, India forfinancial support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2013.02.027.
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