Synthesis and antitubercular evaluation of imidazo[2,1-b][1,3,4]thiadiazole derivatives

1 Introduction

Tuberculosis (TB) is the oldest documented infectious disease. It is a chronic necrotizing bacterial infection with a wide variety of manifestations caused by Mycobacterium tuberculosis, which has plagued humans throughout recorded and archeological history (Dutt and Stead, 1999), as per the recent report it has been estimated that approximately one-third of the world’s population is infected with this microorganism. Today, TB is among the top five causes of global mortality, according to the World Health Organization (WHO) facts sheets, TB kills 2 million people each year. It is estimated that between 2000 and 2020, nearly one billion people will be newly infected if control is not further strengthened (Maher and Floyd, 2002; Dye, 2002). The treatment of mycobacterial infections especially the tuberculosis, has become an important problem due to the emergence of monodrug and multidrug-resistant strains of M. tuberculosis (Rattan et al., 1998). Therefore, there is a need for new drugs of new structural classes and with a novel mechanism of action other than isoniazid (INH), rifampicin (RIF) and pyridazinamide (PZA). In this regard, no new class of antituberculosis agents has been developed since the introduction of rifampin into clinical in 1960s. Therefore, there is an urgent need for the development of innovative anti-TB agents to effectively combat TB, with improved properties such as enhanced activity against MDR strains, reduced toxicity and shortened duration of therapy. From the mid-1990s, this infectious disease was the focus of renewed scientific interest. In the last 10 years, the research on M. tuberculosis has undergone much progress. The thriving accomplishment of the genome of M. tuberculosis has offered a promise of a new generation of potent drugs to combat the emerging epidemic of TB. The emphasis of mycobacterial research now has shifted from gene hunting to interpretation of the biology of the whole organism in an effort to better define which activities are likely to be critical to survival and thus amenable to the development of new drugs (Cole et al., 1998; Barry et al., 2000). In this regard, there have been few additions of some promising new anti-tuberculosis agents, such as the long acting rifamycins, fluoroquinolones, oxazolidinones and nitroimidazopyrans to the existing main-line drugs (Ian, 2001).

Imidazoles certainly belong among the most important, significant and abundant five membered heterocycles, which are constituents of a variety of natural and synthetic products. The advent of sulfur drugs and the later discovery of mesoionic compounds greatly accelerated the rate of progress in the field of thiadiazoles. The thiadiazole and imidazole compounds are extensively studied due to their wide spectrum of bioactivities. Among them the imidazo(2,1-b)-1,3,4-thiadiazole derivatives are pharmacologically important because of their immunostimulant, anti-inflammatory, analgesic, antifungal, antimicrobial, antileishmanial, antitumor, anti-tuberculosis and other activities (Mazzone et al., 1984; Srivastava and Pathak, 1991; Gadad et al., 1999; Terzioglu and Aysel, 2003; Jaquith et al., 2010). In addition, the reports of (Kolavi et al., 2006; Gadad et al., 2004) on the synthesis, antimicrobial and anti-tubercular activity of a series of 2,5,6-trisubstituted imidazo(2,1-b)-1,3,4-thiadiazoles and 2,6-disubstituted imidazo(2,1-b)-1,3,4-thiadiazole derivatives respectively, which have exhibited moderate to excellent anti-tuberculosis activity, are the driving force for selecting imidazo(2,1-b)-1,3,4-thiadiazole nucleus Fig. 1.

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Figure 1 Reported and proposed anti-tubercular imidazo[2,1-b][1,3,4]thiadiazole derivatives (Gadad et al., 2004; Kolavi et al., 2006).

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Therefore, in view of the above facts and in continuation of our search for newer antimycobacterial agents (Gadad et al., 2004), in this paper we report synthesis, spectral studies, antimycobacterial evaluation and cytotoxic activity of various imidazo[2,1-b][1,3,4]thiadiazole derivatives.

2 Chemistry

The synthesis of 2-(1-methyl-1H-imidazol-2-yl)-6-(N-substituted phenyl)-imidazo[2,1-b][1,3,4] thiadiazole derivatives 5(a–j), denominated as imidazo[2,1-b][1,3,4]thiadiazole derivatives was achieved through a versatile and efficient synthetic route outlined in Scheme 1. The starting compound 1-methyl-1H-imidazole-2-carbonitrile 2 was prepared by stirring cyanogen bromide and 4-N,N-dimethylamiopyridine with 1-methyl-1H-imidazole 1 under argon gas at room temperature (Remers et al., 1971). Compound 2 was converted into 5-(1-methyl-1H-imidazo-2-yl)-1,3,4-thiadiazol-2-amine 3 by direct cyclization of thiosemicarbazide (Chauviere et al., 2003). The imidazo[2,1-b][1,3,4]thiadiazole derivatives 5(a–j) were prepared from α-bromoarylketone 4(a–j), by reaction with 5-(1-methyl-1H-imidazo-2-yl)-1,3,4-thiadiazol-2-amine 3 under reflux in dry ethanol.

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Scheme 1 Reagents and conditions: (a) 4-N,N-Dimethylamiopyridine, DMF, Cyanogen bromide, stirred 15 h; (b) thiosemicarbazide, trifluoroacetic acid, reflux 15 h and (c) refluxed in dry ethanol for 18 h.

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3 Biological activity

3.1

3.1 Antimycobacterial activity

All the compounds were evaluated for in vitro antituberculosis activity against M. tuberculosis, as part of the TAACF TB screening program under direction of the US National Institutes of Health, the NIAID division. Rifampicin was used as a reference drug. MIC of compounds 5(a–j) was determined against M. tuberculosis H₃₇Rv (ATTCC 27294) strain by using broth dilution assay method, the Microplate Alamar Blue Assay (MABA) (Suling et al., 2000; Yajko et al., 1995). Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. Compounds effecting <90% inhibition in the primary screening (MIC >6.25 μg/ml) were not generally evaluated further. The active compounds were re-tested by serial dilution beginning at the concentration of 6.25 μg/ml against M. tuberculosis H₃₇Rv to determine the actual minimum inhibitory concentration (MIC) in the BACTEC 12B medium. Rifampicin was used as a reference drug. The MIC is defined as the lowest concentration affecting a reduction in fluorescence of 90% relative to controls (Collins and Franzblau, 1997). The activity data of compounds 5(a–j) are given in Table 1.

Table 1 In vitro antitubercular activity and cytotoxicity of compounds 5 (a–j) .

Compound	R	Inhibition (%)	Activity	MIC (μg/ml)	IC50a	SIb
5a	3-Nitro	91	+	4.34	10.56	2.43
5b	4-Bromo	94	+	5.78	11.4	1.97
5c	4-Chloro	95	+	5.48	12.3	2.24
5d	4-Fluoro	90	+	4.86	8.5	1.74
5e	H	16	−	>6.25	–	–
5f	4-Nitro	98	+	3.14	9.8	3.12
5g	4-Methyl	18	−	>6.25	–	–
5h	3-Methyl	30	−	>6.25	–	–
5i	2,4-Dichloro	92	+	5.66	10.3	1.81
5j	2,4-Dihydroxy	35	−	>6.25	–	–

MIC rifampicin: 0.125–0.25 μg mL⁻¹.

SI (selectivity index) rifampicin >10.

a IC₅₀ inhibition concentration (inhibited 50% of total cells in μM and converted into μg/mL for SI calculation).

b SI selectivity index (ratio between IC₅₀ and M. tuberculosis MIC value).

4 Structure activity relationship (SAR)

In this study a new series of imidazo[2,1-b][1,3,4]thiadiazole derivatives 5(a–j) derivatives were synthesized and evaluated against M. tuberculosis, as part of the TAACF TB screening program under direction of the US National Institutes of Health, the NIAID division. From the ten compounds tested 5(a–j), six (5a, 5b, 5c, 5d, 5f and 5i) displayed significant inhibition effects in the primary screening against M. tuberculosis H37Rv in the BACTEC 12B medium, using the BACTEC 460 radiometric system. Compounds demonstrating at least 90% inhibition in the primary screening were re-tested in order to determine the actual minimum inhibitory concentration (MIC) against M. tuberculosis.

A brief investigation of the structure–activity reveal that the activity is considerably affected by various substituents at the 6th position of imidazo(2,1-b)-1,3,4-thiadiazole nucleus. It has been observed that among the series, the compounds 5a, 5b, 5c, 5d, 5f, and 5i having electron withdrawing group at the 6th position of imidazo(2,1-b)-1,3,4-thiadiazole ring exhibited the significant anti-tubercular activity while other compounds showed poor to moderate activity. Among the electron withdrawing groups, Nitro substituent (5f) at para position shows an enhanced anti-tubercular activity with MIC of 3.14 μg mL⁻¹. On the other hand electron donating substituents (5g and 5h) and unsubstituted (5e) compounds resulted in poor anti-tubercular activity.

5 Result and discussion

6 Conclusion

In the present paper, we reported the synthesis and anti-tubercular activity of a novel series of imidazo-[2,1-b]-1,3,4-thiadiazoles. The preliminary in vitro anti-tubercular activities of these novel series indicate that the presence of phenyl halides at C-6 position of imidazo[2,1-b][1,3,4]thiadiazoles (5a, 5b, 5c, 5d, 5f and 5i) exhibited significant antitubercular activities but not as good as that of the nitro phenyl substituent 5f having MIC of 3.14 μg mL⁻¹. Further, some selected compounds were also assessed for their cytotoxic activity (IC₅₀) against a mammalian Vero cell line using the MTT assay. The results indicated that these compounds exhibit anti-tubercular activity at non-cytotoxic concentrations. The preliminary in vitro antituberculosis screening result of novel imidazo[2,1-b][1,3,4]thiadiazole derivatives reported in the present article evidenced that many of the compounds from the series have emerged as potent antitubercular agents endowed with moderate to good activity. The possible improvements in the activity can be further achieved by a slight modification in the substituent on the basic imidazo[2,1-b][1,3,4]thiadiazole nucleus. Our findings will have an impact on researcher for further investigation in this field in search of potent antitubercular agents.

7 Experimental

All chemicals and solvents were supplied by Merck, S.D. Fine Chemical Limited, Mumbai. All the solvents were distilled and dried before use. The reactions were monitored with the help of thin-layer chromatography using pre-coated aluminum sheets with GF₂₅₄ silica gel, 0.2 mm layer thickness (E. Merck). Melting points of the synthesized compounds were recorded on the Veego (VMP-MP) melting point apparatus. IR spectrum was acquired on a Shimadzu Infra Red Spectrometer, (model FTIR-8400S). Both ¹H NMR (DMSO) and ¹³C NMR (DMSO) spectra of the synthesized compounds were performed with a Bruker Avance-II 400 NMR Spectrometer operating at 400 MHz in SAIF, Punjab University (Chandigarh). Chemical shifts were measured relative to internal standard TMS (δ: 0). Chemical shifts are reported in δ scale (ppm). Mass spectra of the synthesized compounds were recorded at MAT 120 in SAIF, Punjab University.