Antimicrobial studies of unsymmetrical bis-1,2,3-triazoles

Aryl azides were treated with allenylmagnesium bromide to generate 1,5-disubstituted butynyl 1,2,3-triazoles in a domino fashion, which upon Cu(I) catalyzed 1,3-dipolar cycloaddition with aryl azides afforded novel bis-1,2,3-triazoles in quantitative yields. The final products were analyzed for their antimicrobial activities against a panel of bacterial and fungal strains which revealed the products to be potent antimicrobials.


Introduction
Most of the nitrogen-containing molecules are pharmacologically very active which can be attributed to the fact that nitrogenous compounds are part and parcel of the biomolecular diversity [1][2][3][4][5][6][7]. Amongst the pharmacologically active nitrogenous compounds, a large number of 1,2,3-triazoles and their derivatives attracted considerable attention for the past few decades due to their chemotherapeutical value. Many 1,2,3-triazoles, including bis-triazoles, are found to be potent antimicrobial, analgesic, anti-inflammatory, local anesthetic, anti-convulsant, anti-neoplastic, anti-malarial, and antiviral agents [8][9][10]. Some of them exhibited anti-proliferative, anticancer activity, and several are used as DNA cleaving agents and potassium channel activators. Such type of diverse biological functions is also reported with a variety of bis-triazoles. The 'click chemistry' approach has been the most widely used method for the synthesis of libraries of a large number of biologically active molecular frameworks particularly for the regioselective synthesis of 1,2,3-triazoles, which involves the copper(I)catalyzed cycloaddition reaction between azides and terminal alkynes (CuAAC). This reaction has been termed as the 'cream of the crop' of 'click reactions' and has found application in various facets of drug discovery as it enables a modular approach to generate novel pharmacophores utilizing a collection of reliable chemical reactions [11,12]. Thus, the development of the copper(I)-catalyzed 'triazole click chemistry' has led to many interesting applications including the synthesis, medicinal chemistry, molecular biology, and material science. The bioorthogonality of azide and alkynes [13] has allowed the use of their [3 + 2] cycloaddition in various biological applications including target guided synthesis [14] and activity-based protein profiling [15]. Of particular interest would be the dimeric heterocyclebased ligands which are designed for specific target interactions. Various approaches reported for the synthesis of biologically relevant bis-triazoles include Cu(I)catalyzed 1,3-dipolar cycloaddition of monoazides with diacetylenes or that of monoacetylenes with diazides. For example, the synthesis of bis-triazoles is reported by the reactions of bis(azidomethyl)benzenes with several substituted acetylenes [16]. Recently, much attention has been paid toward the synthesis and pharmacological evaluation of triazoles and bis-triazoles as potent HIV-1 protease inhibitors [17,18] and size-specific ligands for mRNA Hairpin loops [19], respectively. Keeping into consideration the tremendous biological potence of triazoles and bis-triazoles in general and the antimicrobial activity in particular, we, in our continuous endeavor toward the synthesis of pharmacologically active molecules, designed the synthesis of novel unsymmetrical bis-1,2,3-triazoles and then evaluated them for antimicrobial activities. The biological results obtained were very interesting and revealed most of the synthesized molecules to be potent antimicrobials.

General methods
Melting points were recorded on Buchi Melting point apparatus D-545; IR spectra (KBr disks) were recorded on Bruker Vector 22 instrument. NMR spectra were recorded on Bruker DPX200 instrument in CDCl 3 with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in δ (ppm) and coupling constants are given in Hz. Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. The progress of all reactions was monitored by TLC on 2 × 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromatograms were visualized under UV 254-366 nm and iodine.
The analytical data of all the isolated bis-triazoles is given as under.    The test organisms were cultured on agar slants, incubated 24 h at 37 ± 0.5°C and 24-48 h at 27 ± 0.2°C for bacteria and fungi, respectively, to get the freshly prepared cultures. The steroidal derivatives were evaluated for antimicrobial activity against these freshly prepared strains of test organisms by agar diffusion method [20,21]. Muller Hinton Agar (MHA) and Potato Dextrose Agar (PDA) were used as nutrient media for bacterial and fungal strains, respectively. The media (MHA &PDA) were prepared using distilled water and 20 mL of it was transferred into 50-mL test tubes, the test tubes were tightly plugged with cotton and sterilized in autoclave at 15 lb/in 2 for 15 min as directed by the manufacturer. After sterilization, the medium was inoculated with freshly cultured bacterial strains under sterile condition, i.e., under Laminar Flow. The inoculation was done when the temperature of the medium reached 50-40°C, so that test organism may not die at higher temperature. The medium inoculated with test microorganisms was transferred into the plates of 90mm size under sterile conditions. The medium was allowed to solidify and the wells (4/plate) of 6-mm-diameter and 50 μL volume were bored on it using sterile cork borer. The solution of test compound 1000 μg/mL was prepared in DMSO and the wells bored on the medium were each filled (50 μg) with test compound using micropipette (20-200 μL). Four wells were bored on the plates and each filled with same compound and two plates for each test compound were taken and the experiment was repeated twice. The disks of Kenamycin and Flucanazole were also incorporated into the medium for comparison (10-30 μg). The plates containing test organism and test material in contact were incubated at 37 ± 0.5°C for 24 h. Same procedure was employed for antifungal activity; however, the culture strains of fungi were maintained on PDA and spores were transferred into the PDA medium and the plates were incubated at 27 ± 0.2°C for 24-48 h. Inhibition of growth of test organisms (bacterial & fungal) in presence of test material and standard was measured with the help of standard scale and the mean values of inhibition zones are reported in Table 2. Table 2 gives the antimicrobial screening data obtained after treating different microbial strains with test doses of the different bis-triazolyl derivatives and the values are reported in terms of zone of inhibition in "mm".
It is clear from the above data that all the compounds 3a-l showed significant antimicrobial activity against all microbial strains used for testing. It is evident from the data that even the position of substituent on the aromatic ring influences the relative activity which can be attributed to their differences in either the bioavailability or the protein-binding properties.

Results and discussions
The wide range of pharmacological activities especially the antimicrobial potential [14,[22][23][24][25][26][27] of triazole and bis-triazole systems prompted us to design the synthesis of a library of unsymmetrical bis-1,2,3-triazoles based on a stepwise synthetic route involving domino addition of allenylmagnesium bromide to aryl azides resulting in a serendipitous formation of 5-butynylated triazoles in good yields (> 70%) instead of 4-butynylated triazoles. 5-Butynylated triazoles upon Cu(I) catalyzed 1,3-dipolar cycloaddition with aryl azides generated bis-1,2,3-triazoles in quantitative yields. The products together with the approach for their synthesis being novel, the intermediate 5-butynylated triazoles and the final product, the bistriazole, were characterized by IR, 1 H/ 13 C NMR, and mass spectral analysis. The intermediate 3 undergoes a high yielding regioselective Cu(I) catalyzed 1,3dipolar cycloaddition with aryl azides (click reaction) to afford quantitative yields of the product, i.e., bis-1,2,3triazoles, which were isolated in pure form after precipitation. The isolated products were evaluated for their antimicrobial activities against a panel of bacterial and fungal cell lines. The biological results were highly encouraging paving a way for the futuristic medicinal chemistry work based on these scaffolds.

Conclusion
In conclusion, we have developed an unprecedented, convenient strategy for the synthesis of novel, biologically important bis-1,2,3-triazoles employing a domino reaction followed by the copper catalyzed 'click' protocol. The products thus obtained were found to be potent antimicrobial agents.