Synthesis, Molecular Docking, and Evaluation of Triazole and Chalcone Conjugate As Antitubercular Agent

The aim of this work was to be combine two pharmocophoric nuclei viz, triazole and chalcone and evaluate their antitubercular activity. Propargylated vanillin was condensed with differently substituted acetophenones to produce various chalcones (3a-c). Propargyl chalcones were then made to react with benzyl azides (2a-d) using the technique of Click chemistry and this reaction yielded triazole-chalcone hybrids (4a-l) in good yields, ranged from 34 to 93%. These hybrids were evaluated for their antitubercular activity, from the results it was found that triazole and chalcone on combination exhibited enhanced bioactivity thereby supported the theory of synergistic effect. The conjugate 4a and 4f were found to be most potent with MIC of 1.6 µg/ml. Molecular docking studies of bioactive compounds were in good congruence with in-vitro studies.


Introduction
Mycobacterium tuberculosis is a signi cant health issue and is the world's leading cause of mortality.
The WHO has initiated a global anti-tuberculosis drug resistance project to evaluate the effects of drug resistance. Nevertheless, several new chemical compounds with important anti-tuberculosis activity have recently been identi ed, but only one new drug [] i.e., Bedaquiline has been approved in the last 46 years. It was approved by USA in 2012 and recommended by WHO and US Centers for Disease Control in 2013.
TB is triggered by many types of Mycobacterium tuberculosis (at least nine members), which is nonmotile and rod-shaped and an aerobic disease-causing bacterium. Streptomycin and p-amino salicylic acid were among the rst antibiotics developed with activity against Mycobacterium tuberculosis (Mtb). These were followed in rapid succession by other drugs like isoniazid, pyrazinamide, cycloserine, ethionamide, ethambutol, and rifampicin. However, in spite of discovery of such a large number of antitubercular drugs, it still takes about nine months to cure TB, which is too long, to cure TB in current therapy. Because of emergence of MDR strains, established risks of existing drugs, the treatment for the patients has become di cult. Moreover, there are growing incidence of TB deaths. So because of all these reasons, there is growing need for development of new anti-tuberculosis drugs with novel modes of action so to provide effective treatment and resolve the issue of drug resistance.
In this regard, molecular hybridization (MH) [] has emerged as modern strategy for designing new drugs to ght against drug resistance and to augment the existing therapeutic advances in the treatment of TB.
The technique of MH usually involves combination of more than one pharmacophoric moiety having different bioactivity to form a new hybrid with enhanced potency and a nity, when correlated with the parent molecules. This technique can modify selectivity pro le and decreases the undesired side effects exerted by the compounds. Single multi-functional hybrid drugs can contribute combination therapies.
The research eld of synthesizing hybrids by using approach of MH is expanding at great pace and is attracting more and more researchers throughout the world. New drugs can be developed by combining two bioactive nuclei having enhanced bioactivity [] . The pharmacophoric moieties are chosen on the basis of their known biological activity, and it is presumed that the resultant hybrid molecule may display synergistic or enhanced or additive biological activity [,] Because of immense importance of molecular hybridization approach, we in our research approach, synthesized new novel Triazole-Chalcone hybrids and evaluated them for their antitubercular activity. Chalcone is a simple and versatile molecular scaffold, present in most of the naturally occurring compounds and precursor for avonoids and iso avonoids. In addition, it has been synthesized in laboratory to produce bioactive molecules. Both the natural and synthetic chalcones have displayed several fascinating biological properties with proven potential against different type of diseases.
Chalcones are essential natural products that belong to avonoid family. Due to presence of large number of replaceable hydrogen atoms these can form variety of derivatives. Chalcones are generally present in fruits, tea, vegetables, spices etc. Chalcone as well as their synthetic derivatives shows different types of activities like anticancer [, ] , antitubercular [, ]  Molecular docking studies are now most frequently being used methods in drug design on structural basis as these studies have potential to analyze the binding-conformation of ligand molecules to the appropriate targeted binding sites. [, ] The concept of structural based drug design always promotes the in-silico methods for molecular docking before starting the actual lab screening process. In silico methods predicts the binding sites and predict the possible mechanism of ligand-protein interactions as well as target binding. [, ] Molecular docking is the study of how two or more molecular structures (e.g., drug and enzyme or protein) t together. [, ] Even though triazole and chalcones have been studied for their biological activities but the effect of hybridization on the biological activities needs more extensive speculation. Hence, because of diverse biological activity of both 1,2,3-triazoles and Chalcones, it was envisaged that the bringing together the two biologically active moieties i.e., chalcone and 1,2,3-triazoles may lead to improved biological activities. So, we synthesized triazole based Chalcones and evaluated them for anti-tubercular activity. The docking of the bioactive conjugates has been done so that binding sites and possible mechanism could be predicted. Hence, herein we report the synthesis of a series of 1,2,3-triazole-chalcone hybrids as prospective anti-tubercular agents.

Results And Discussion
Chemistry: The synthesis of chalcone-triazole is being depicted in scheme-I that involves four steps. In the rst step benzyl azide (1a-d) were prepared from benzyl chloride and sodium azide, different substituents on phenyl ring of benzyl chloride are used so that differently substituted triazole could be produced in the later stage. In another step, vanillin was functionalized at O-atom with propargyl group (compound 2) after reacting with propargyl chloride in mildly basic medium. The reaction proceeded very e ciently and gave single product without the formation of side products. The polar solvent is used for the reaction that is DMF but for the formation of pure crystals complete removal of solvent is necessary. Next step involved the formation of chalcone (3a-c), in that propargyl functionalized vanillin (2) was condensed with acetophenones through the aldehydic group via Claisen-Schmidt reaction. The product obtained in this step is not so pure therefore, column chromatography was done to get pure compound. Consequently, the yield of the product was less. The yellow crystals so obtained were recrystallized with ethanol.
The last step is the formation of triazole-chalcone hybrid (4a-l) in that cyclization of propargyl group of functionalized chalcone (3a-c) was done with the aid of Click chemistry tools. In this reaction, azide group of benzyl azides (1a-d) add onto the triple bond of propargyl group of chalcones. The mechanism of the reaction is (3 + 2) cycloaddition. The reaction is catalyzed by CuSO 4 . The reaction mixture was stirred in a sonicator. The rules of green chemistry are followed in this step.

IR analysis:
The presence of a characteristic band at around 2092.83 cm − 1 indicated the presence of Ν = N = N stretching (in case of benzyl azides) in the IR spectra of molecules (1a-d) and it also showed absorption bands at 1647.26cm − 1 attributing to C = C stretching in aromatics. The IR spectra of compound 2 showed absorption band at ν = 3244.38 cm − 1 due to presence of C-H stretching supporting the attachment of propargyl group with vanillin, apart from this the absence of O-H stretching peak supported the replacement of H of hydroxyl group with propargyl group. Apart of these, absorption peaks at 1120.68 cm − 1 due to C-O stretching, 1689.70 cm − 1 (CHO stretching), p-substituted stretching at 810.13 cm − 1 , and o-substituted stretching = 777.34 cm − 1 were also obtained.
The IR spectra of compounds 3(a-c) supported the formation of chalcones with characteristic peaks of C = C stretching at around at 1530.05 cm − 1 , and an intense peak of C = O stretching in the region of 1600-1650 cm − 1 , thereby supporting the presence of α, β-unsaturated carbonyl functional group. Two distinguishing peaks at around 3200 cm − 1 and 2100 cm − 1 due to C-H stretching and CC stretching for alkynes respectively, but these peaks are absent in the spectra of triazoles 4(a-j). In the spectra of triazoles there was a peak around 3150 cm-1 due to = C-H str of triazole ring and another peak at 1010 cm − 1 due to (Ν = N = N) ring.

NMR analysis:
The 1H-NMR of compounds 1(a-d) signals at ∂ 7.24-7.56 ppm for aromatic protons and singlet for methylene proton in the region of 2.50-2.55 ppm. The spectra of compound 2 showed characteristic signal for H-CC for propargyl group at δ 2.58 ppm and at δ 4.86 for 2 protons of -OCH 2 . There was no signal for proton for O-H thereby con rming the attachment of propargyl group. In 1 H-NMR spectra of 3 (a-c), the signal for -CHO was not present rather signals for = C-H proton became visible in aromatic region thereby con rming the formation of chalcones. In spectra of 4 (a-l) peak for H-CC for propargyl group at δ 2.58 ppm disappeared instead a singlet signal for -C-H appeared at δ 7.18 ppm showing the formation of triazole nucleus.
Biological activity: The compound 2-4(h) were evaluated for their Anti-tubercular activity by using H37RV Mycobacterium strain by Alamar blue assay. The Minimum Inhibitor concentration (MIC) is expressed in µg/ml as shown in Table 1. Compound 2 showed activity up to 50µg/ml. Compounds like 3a, 3b, and 3c shows 25 µg/ml, 100 µg/ml and 100 µg/ml. It is worth mentioning that chalcone is unsubstituted it is more potent than the substituted chalcones. Now, when molecular hybrids of these compounds are being formed activity is enhanced. It is seen that unsubstituted triazole exhibit the maximum inhibition at a concentration of 1.6 µg/ml as well when uorine and bromine both electronegative halogens are attached on triazole ring they also exhibit inhibition upto 1.6 µg/ml. In addition to this, when only uorine is attached to triazole ring its activity reduced; however, when di ouro is present on ring its inhibition increased upto 1.6 µg/ml. Moreover, substitution of bromine alone showed promising activity up to 6.2 µg/ml but electronegative atom like cyano if present along with bromine that shows antagonistic effect. [] From the results it could be concluded that activity was enhanced on hybridizing the two pharmacophoric unities. These results support the hypothesis that synergistic effect is observed on molecular hybridization. [] Docking Studies: Molecular docking of biologically active ligands has been performed to study the active binding site in the Mycobacterium protein chain which are available for binding interactions. Moreover, the analysis and interpretation of the binding behavior play a crucial role in potential drug designs and in elucidating fundamentals of biochemical processes. [,] All the ligands show active binding interaction with the reference protein as indicated by -ve value of binding a nity. All the polar contacts of individual ligands are described in the tabular form with amino acid residue with code. From the data, it is quite evident all the ligands show binding interaction with arginine (ARG) amino acid residue except 4c. This might be helpful in explaining its lower IC 50 value as compared to 4a, 4c and 4f. GLN, TYR and SER are other important residue with which ligands binds in similar ways.
Visual presentation of one best binding pose of each ligands is described in gures.

Experimental:
Material: All the chemicals were procured from Sigma Aldrich Ltd. Melting point were recorded in Ko er hot stage apparatus and were uncorrected. 1 H NMR were recorded using BRUKER AVANCE II 400 NMR and TMS was used as an internal standard. Coupling constant values are represented in Hz. Infrared spectra were recorded on FTIR 8400S (Shimadzu) and pellet were prepared in KBr. The absorption values were expressed in cm − 1 .

Methods:
Synthesis of compounds 1. Synthesis of compound 1-(azidomethyl) benzene Reaction starts with benzyl bromide, 2, 5-uorobenzyl bromide, 4-cyanobenzylbromide (5 mmol) and Sodium azide (6 mmol) in DMSO, the reaction mixture was stirred for 4-6 hours at room temperature. Completion of reaction can be con rmed using TLC. After completion of reaction, product was extracted with diethyl ether and the nal extract was washed with distilled water to remove off impurities and DMSO if there. Organic layer was dried over Na 2 SO 4 and ltered the mixture. The ltrate was evaporated under rotatory evaporator to get desired compound 1(a-d).

Characterization of compound 1a
The IR spectra of compound 1(a) was recorded just by putting liquid drop on IR plate in the range of 400-

Synthesis of compound 2-methoxy-4-(prop-2-yn-1-yloxy) benzaldehyde (2):
Reaction starts with Vanillin (1.52 g, 0.009 mol), and propargyl bromide (1.36 ml, 0.018 mol) and potassium carbonate K 2 CO 3 (2.76 g, 0.02 mol) were dissolved in 15 ml of DMF. The reaction mixture was stirred for 3-4 hours at room temperature. Completion of reaction was con rmed using TLC technique. After completion of reaction, extraction was done using (Chloroform) CHCl 3 . Organic layer was separated and washed with excess amount of distilled water to remove off all the impurities and DMF present in the mixture. Final extract was dried over anhydrous Na 2 SO 4 . The solvent was evaporated under rotatory evaporator to get compound 2. And recrystallization can be done in ethanol.
Characterization data of compound 2 The IR of compound 2 was done just by putting crystals in IR plate then examined and the spectrum was shown in Fig. 9.

Characterization data of Compound 3a
The IR analysis of compound 3(a) was done just by putting crystals in IR plate. It Dissolve compound 3(a-c) (0.1g) and azide 1(a-d) (0.1ml) in 20 ml of dry DCM. After that solutions of anhydrous CuSO 4 (0.04 g) and sodium ascorbate (0.25g) in water were added. The solution was stirred at room temperature in sonicator till reaction was completed. Progress of reaction was observed using TLC. After the completion of reaction 50 ml of water was added and extracted with DCM. The organic layer was dried over Na 2 SO 4 and the ltrate was evaporated under rotatory evaporator to get our desired product. And wash the nal product with cold ethanol.

Characterization data of Compound 4a
The Methods to analyze Anti-tubercular activity: The anti-TB activity was done using Alamar blue assay. All the synthesized compounds (Scheme-I) µwere tested against Mycobacterium tuberculosis. 100 µl each of sterile water and Middlebrook 7H9 broth was added in each well of 96 well plates. The concentration of compound was varied from 100 − 0.2 µg/ml. Incubation of plates was done for ve days at 37 0 C. 25 µl of 1:1 mixture of Alamar blue reagent and 10% Tween 80 was added after ve days. Further incubation of 24 h was done. A blue color in well indicated the bacterial cell inhibition whereas pink color indicated cell growth. [xxx] Molecular docking studies: The structures of proteins used in this work were downloaded from the Protein Data Bank (PBD Code: 4Y6U). [] Molecular docking study has been carried out by the free software package Auto-dock Vina. [,] Initially, the protein structure was re-processed before using as receptor for docking. Necessary steps taken in preprocessing of protein were addition of hydrogen atoms, assignment of atomic charges, and elimination of water molecules that are not involved in ligand binding. The Auto-dock tool package was used for grid generation with a maximal size of 2700 Å with 0.6 Å spacing. Pymol was used to visualize the result of docking studies. []

Conclusion
A series of triazole-chalcone conjugates (4a-l) were prepared using tools of Claisen Schimdt condensation and tools of Click chemistry. It could be concluded from the result of antitubercular activity using MABA assay that hybrids combining two pharmacophores were quite active, compound 4a and 4f showed inhibition at a concentration of 1.6 µg/ml. Binding energy as calculated by docking studies coincided with biological activity. Present work gave an insight into synergistic effect of triazole and chalcone moieties and this aspect could be further studied. The hybrid molecules could be considered for more extensive study so that new drugs with less side effects could be introduced.  Tables   Due to technical limitations, table 1 to 3 is only available as a download in the Supplemental Files section. Figure 1 Binding interactions of 4a, 4c, 4e and 4f

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Tables.docx