Efficient One Pot Synthesis of Xanthene-Triazole-Quinoline/Phenyl Conjugates and Evaluation of their Antimicrobial Activity

compostos apresentou a melhor atividade antibacterial e antifúngica. Posteriormente, os modos de ligação deste composto no sítio ativo da enzima topoisomerase II DNA gyrase B foram investigados. Novel xanthene-triazole-quinoline/phenyl conjugates were synthesized by eco-friendly one pot three-component condensation of 12-aryl-2-hydroxy-tetrahydrobenzo[a]xanthene-11-one, propargyl bromide and 4-azido-7-chloroquinoline/phenyl azide using polyethylene glycol (PEG-400) as a reaction medium with an aim to explore their effect on the in vitro growth of microorganisms causing microbial infection. All newly synthesized xanthene-triazole-quinoline/ phenyl conjugates were fully characterized and were evaluated for in vitro antibacterial and antifungal activity. Antimicrobial activity was evaluated against nine microbial strains. All compounds showed good Gram positive antibacterial and antifungal activity. One of the compounds showed best antibacterial and antifungal activity. Further, binding mode of this compound at the active site of enzyme topoisomerase II DNA gyrase B has also been investigated.


Introduction
During the past few years, the incidence of bacterial and fungal infection has increased to alarming levels because of the resistance to existing drugs and they are collectively a major cause of morbidity and mortality, especially in immunocompromised patients. 1 Therefore, discovery of new classes of antimicrobial agents is crucial to combat multi-drug resistant infections. Benzo [a] xanthene and their derivatives are important heterocyclics with interesting biological activities, such as antibacterial, 2 anti-inflammatory, 3 antiviral, 4 antimalarial, 5 and antitumor. 6 Some classes of xanthenes have also been used as antagonists for paralyzing the action of zoxazolamine and in photodynamic therapy. Nitrogen containing heterocycles such as triazoles and quinolines are common structural motifs in pharmacologically important molecules and alkaloids with activities spanning a diverse range of targets. 1,2,3-triazoles have occupied special place in medicinal chemistry due to their numerous biological activities such as anti-fungal, 7 anti-bacterial, 8 anti-alergic, 9 anti-HIV, 10,11 anti-tubercular. 12,13 The 1,2,3-triazole derivatives can be easily synthesized using click chemistry through copper catalyzed azide alkyne cycloaddition. 14 Quinolines have been of interest as they possesses useful pharmacological activities such as anti-malarial, 15 anti-HIV, 16 anti-tumor, 17 and anti-bacterial. 18 Literature survey reveals that hybridization is a classic strategy in drug design based Vol. 25, No. 7, 2014 on combining two or more different bioactive moieties in a single molecule to get the corresponding conjugate/ hybrid molecules. 19 These conjugates generally show much better activity compared to their precursors and can work by the same or different mechanisms of action compared to the precursors. 20,21 Thus, more effective antimicrobial compounds can be designed by joining two or more biologically active heterocyclic systems together in a single molecular framework. Multicomponent reactions combined with the use of environmental friendly reaction medium are valuable tools for the preparation of structurally diverse conjugates/hybrids of drug-like heterocyclic compounds without additional impact on environment. 22 As a part of our continued research for new antimicrobial agents by multicomponent reactions, 23 and inspired by the biological activity of compounds containing benzo[a]xanthene, 1,2,3-triazoles and quinolines as pharmacophores, we designed the potentially bioactive target molecules by combining these pharmacophores in a single molecule as shown in Scheme 1. We attempted the synthesis of conjugates consisting of benzo[a]xanthene, 1,2,3-triazoles and quinolines moieties by condensation of 12-aryl-2hydroxy-tetrahydrobenzo[a]xanthene-11-one, propargyl bromide and 4-azido-7-chloroquinone/phenyl azide.
The structures of all novel compounds (1a-1o) were confirmed by infrared (IR), 1 H nuclear magnetic resonance (NMR), 13 C NMR, mass spectra and elemental analysis. The 1 H NMR spectra of compound 1a showed two singlets for three protons at d 0.94 and d 1.10, corresponding to two methyl groups. Two methylene protons adjacent to carbonyl group act as AB system and appeared at d 2.24 and d 2.27, while another methylene group of dimedone ring appeared as singlet at d 2.54. The methylene group adjacent to oxygen atom also acts as AB system and appeared at d 5.41 and d 5.30. The one methine proton of benzo[a]xanthene  Figure 1. The crystallographic data collection and structure refinement details for compound 1d and 1p are summarized in Table 3 and Table 4, respectively.
We have also carried out the sequential synthesis of 1a in two steps as depicted in Scheme 4 to demonstrate the advantages of multicomponent reactions in the synthesis of target molecules over sequential process. The reaction of 12-(4-bromophenyl)-2-hydroxy-9,9-dimethyl-8,9,10,12tetrahydrobenzo[a]xanthen-11-one (1.0 mmol) with propargyl bromide (1.2 mmol) in presence of K 2 CO 3 in PEG-400 at 80 °C gave 12-(4-bromophenyl)-9,9-dimethyl-2-(prop-2-ynyloxy)-8,9,10,12-tetrahydrobenzo[a] xanthen-11-one (3) in 82% yield. Structure of the intermediate 3 was confirmed by 1 H NMR, 13 C NMR and mass spectra. The reaction of 3 was then carried out with 4-azido-7-chloroquinoline in PEG-400 in the presence of CuSO 4 .5H 2 O (10 mol%) and sodium ascorbate (20 mol%) and gave the desired product 4 in 89% yield. The overall a logP values were calculated using OSIRIS property explorer software; b reaction was performed using phenyl azide (1 eq.) instead of 4-azido-7-chloroquinoline. sequential reaction required longer reaction time and gave poorer overall sequential reaction yield of 74% compared to the multicomponent process (92%), thereby demonstrating its advantage.  Table 5. All compounds showed MIC values in the range of   1a, 1b, 1i, and 1m) with halogen substituent over phenyl ring of xanthene moiety showed higher activity as compared to other compounds. The replacement of halogen atom with nitro or alkyl group results in a decrease  in the antibacterial activity. The position of substituent over phenyl ring also affects the antibacterial activity of compound. Compound 1c having nitro group at para position over phenyl ring of xanthene moiety showed MIC 64 µg mL -1 against Staphylococcus aureus, while compound 1g with nitro group at meta position showed MIC 128 µg mL -1 against Staphylococcus aureus. The presence of chloroquinoline moiety also enhances the antibacterial activity of these compounds as replacing chloroquinoline moiety with phenyl ring (compound 1p) resulted in decrease in the antibacterial activity ( Figure 2 and Table 5). All compounds were found to be inactive against Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Enterobacter aerogenes). The possible reason for inactivity of these compounds against Gram negative bacteria can be explained by the fact that the susceptibility of microorganisms to a drug depends on the physicochemical characteristics of that drug (hydrophobicity and hydrossolubility) and on the composition of microbial membranes. The outer layer of the outer membrane of Gram negative bacteria is composed of lipopolysaccharide molecules that form a hydrophilic environment providing protection against hydrophobic molecules. 27 The synthesized compounds (1a-1p) are highly hydrophobic as indicated by their high log P values as shown in Table 2 which makes them inactive against Gram negative bacteria.
In order to investigate a plausible mechanism of action of the most active compound 1a against bacteria S. aureus, docking studies of 1a into active site of enzyme topoisomerase II DNA gyrase B were performed. Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. 28 The protein ligand complex was constructed based on the X-ray structure of topoisomerase II DNA gyrase with its bound inhibitor ciprofloxacin that is available through the RCSB Protein Data Bank (PDB entry 2XCT). Docking was performed using auto dock 4 program. 29 The binding mode of inhibitor ciprofloxacin at the active site of topoisomerase II DNA gyrase B is shown in Figure 3. Ciprofloxacin formed two hydrogen bonds with Arg-458 and Ser-1084 residues at the active site of enzyme. 28 Binding mode of 1a at the active site of topoisomerase II DNA gyrase B is shown in Figures 4 and 5. Compound 1a binds in a similar fashion as ciprofloxacin at the active site with binding energy of -5.86 kcal mol -1 . Compound 1a formed hydrogen bonds with Ser1084 residue and  of formation of this bridge, the active site seems poised to cleave the DNA and results in inhibition of bacterial growth. 28 All compounds (1a-1p) were also tested for their in vitro antifungal activity against three fungal strains, namely, A. niger, A. flavus and Penicillum sp. The standard drug fluconazole was used for comparison of the antifungal activity shown by the compounds and results were recorded as a percentage of mycelial growth inhibition and MIC. The results of antifungal activity of all compounds are shown in Table 6. It can be inferred from Table 6 that all the compounds showed good antifungal activity against the three pathogens. From a careful comparison of the results, it is observed that all compounds (1a-1p) showed more than 50% inhibition of mycelial growth against A. niger, A. flavus and Penicillum sp. in comparison with the standard drug. All compounds (1a-1p) showed MIC (µg mL -1 ) values in the range of 16-128 µg mL -1 against A. niger and in the range of 32-128 µg mL -1 against A. flavus and Penicillum sp. species. Compounds (1a, 1b, 1i, and 1m), having a halogen substituent over phenyl ring of xanthene moiety, showed higher antifungal activity as inferred from Table 4. Compound 1a showed excellent antifungal activity against all three strains with mycelial growth inhibition of 72.2% against Aspergillus niger, 65.5% against Aspergillus flavus and 57.7% against Penicillum sp. Compound 1a showed MIC of 16 µg mL -1 against Aspergillus niger as compared to fluconazole with MIC of 12.5 µg mL -1 . Compound 1a also showed a lowest MIC value (32 µg mL -1 ) against Aspergillus flavus and Penicillum sp.

Conclusion
In conclusion, we have reported an efficient synthesis of novel xanthene-triazole-quinoline/phenyl conjugates (1a-1p) by one pot three-component condensation of 12-aryl-2-hydroxy-tetrahydrobenzo[a]xanthene-11ones, propargyl bromide and 4-azido-7-chloroquinoline/ phenyl azide in the presence of K 2 CO 3 as a base and 10 mol% CuSO 4 .5H 2 O and 20 mol% sodium ascorbate in PEG-400 at 80 °C. All newly synthesized compounds were evaluated for antimicrobial activity against nine microbial strains including six bacterial strains and three fungal strains. All compounds exhibited good antibacterial activity and antifungal activity. All compounds showed good Gram positive antibacterial activity and antifungal activity. Compound 1a was found to be most potent antibacterial and antifungal agent with highest activity among all compounds. Binding mode of compound of 1a at the active site of enzyme topoisomerase II DNA gyrase B has also been investigated.

Experimental
All chemicals were purchased from Sigma-Aldrich, Spectrochem and were used as received. F 254 precoated aluminium plates with silica gel 60 from Merck were used to monitor reaction progress. IR (KBr) spectra were recorded on Perkin Elmer FTIR spectrophotometer and the values are expressed as ν max /cm -1 . The NMR ( 1 H and 13 C) spectra were recorded on Jeol JNM ECX-400P at 400 MHz and 100 MHz, respectively. The chemical shift values are recorded on d scale and the coupling constants (J) are in Hertz. The mass spectra were recorded on an Agilent 6520-QTOF LCMS having ESI source in positive mode. Elemental analyses were recorded on VarioEL III elemental analyzer in CHNS mode. Single crystal X-Ray intensity data was collected on Oxford Diffraction Xcalibur CCD diffractometer with graphite monochromatic Mo Kα radiation (λ = 0.71073 Å) at temperature 298 K.
Spectral data for compounds 1a-1p 2-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl) methoxy)-12-(4-bromophenyl)-9, 9-dimethyl-8,9,10,12- The antibacterial activity of all compounds was evaluated by the agar well diffusion method. 30 All the microbial cultures were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately 1.5 × 10 6 cfu mL -1 . 20 mL of Mueller Hinton agar medium was poured into each Petri plate and plates were swabbed with 100 µL inocula of the test microorganisms and kept for 15 min for adsorption. Using sterile cork borer of 8 mm diameter, wells were bored into the seeded agar plates and these were loaded with a 100 µL volume with concentration of 2.0 mg mL -1 of each compound reconstituted in dimethylsulphoxide (DMSO). Vol. 25, No. 7, 2014 All the plates were incubated at 37 °C for 24 h. Antibacterial activity of each compound was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (HiAntibiotic zone scale). DMSO was used as a negative control whereas ciprofloxacin was used as positive control. This procedure was performed in three replicate plates for each organism and the mean values of the diameter of inhibition zones ± standard deviations were calculated.

Determination of minimum inhibitory concentration (MIC) of chemical compounds
MIC of the all compounds against bacterial and yeast strains was determined using macrodilution tube method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). 31 In this method, various test concentrations of newly synthesized compounds were prepared from 128 to 0.25 µg mL -1 in sterile tubes No. 1-10. 100 µL sterile Mueller Hinton Broth (MHB) was poured in each sterile tube followed by the addition of 200 µL test compound in tube 1. Two fold serial dilutions were carried out from tube 1 to tube 12 and excess broth (100 µL) was discarded from the last tube No. 10. To each tube, 100 µL of standard inoculum (1.5 × 10 8 cfu mL -1 ) was added. Turbidity was observed after incubating the inoculated tubes at 37 °C for 24 h. Ciprofloxacin was used as positive control while DMSO was used as negative control.

Procedure for minimum bactericidal concentration (MBC)
MBC is the lowest concentration of antimicrobial compound that will prevent the growth of an organism after subculture on to antibiotic free media. MBCs were determined by spreading the 100 µL compound from one below MIC and MIC itself. All the tubes were incubated for 24 h at 37 °C. The growth was observed on each plate. 32 Experimental procedure for antifungal activity The antifungal activity all compounds was evaluated by poisoned food technique. 32 The molds were grown on Sabouraud dextrose agar (SDA) at 25 °C for 7 days and used as inocula. The 15 mL of molten SDA (45 °C) was poisoned by the addition of 100 µL volume of each compound reconstituted in the DMSO, poured into a sterile Petri plate and allowed it to solidify at room temperature. The solidified poisoned agar plates were inoculated at the center with fungal plugs (8 mm diameter) obtained from the colony margins and incubated at 25 °C for 7 days. DMSO was used as the negative control whereas fluconazole was used as the positive control. The experiments were performed in triplicates. Diameter of fungal colonies was measured and expressed as percent mycelial inhibition.
Percent inhibition of myelial growth = (dc -dt) / dc × 100 where dc = average diameter of fungal colony in negative control sets; dt = average diameter fungal colony in experimental sets.
MIC of all compounds was determined by the macrodilution broth method. A two fold serial dilution of compounds was prepared in Sabouraud dextrose broth to achieve a decreasing concentration range of 512 to 1 µg mL -1 in sterile test tubes. Each dilution was seeded with 100 µL of the standardized fungal inoculums (2 × 10 5 spores mL -1 ).The inoculated culture tubes were incubated at 25 °C for 7 days. A set of tubes containing only broth was kept as control. After incubation, tubes were examined for changes in turbidity as an indicator of growth. The lowest concentration that did not permit any visible growth of a mold was considered as MIC of that compound.

Docking protocol
The automated docking studies were carried out using Auto Dock version 4.0. 29 First, AutoGrid component of the program precalculates a three-dimensional grid of interaction energies based on the macromolecular target using the AMBER force field. Then automated docking studies were carried out to evaluate the binding free energy of the inhibitors within the macromolecules. The threedimensional structures of the aforementioned compounds were constructed using Chem. 3D ultra 11.0 software [Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2009)], then they were energetically minimized by using MOPAC with 100 interations and minimum RMS gradient of 0.10. The Gasteiger-Hückel charges of ligands were assigned. The crystal structures of topoisomerase II DNA gyrase (PDB code: 2XCT) complex were retrieved from the RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do). All bound waters and ligands were eliminated from the protein and the polar hydrogens and the Kollman-united charges were added to the proteins. AutoGrid component of the auto dock program pre-calculates a three-dimensional grid of interaction energies based on the macromolecular target using the AMBER force field. The cubic grid box of size 28 Å, 22 Å, 42 Å along x, y, z directions, respectively with a spacing of 0.375 Å and grid maps were created representing the catalytic active target site region where the native ligand was embedded. Then automated docking studies were carried out to evaluate the binding free energy of the inhibitors within the macromolecules. The GALS search algorithm (genetic algorithm with local search) was chosen to search for the best conformers. The default parameters were set using the software ADT on PC, which is associated with Auto-Dock 4.0. Results differing by less than 0.5 Å in positional root-mean-square deviation (RMSD) were clustered together and the results of the most favorable free energy of binding were selected as the resultant complex structures.

Supplementary material
Copies of 1 H, 13 C NMR spectra of all compounds and crystallographic information file (CIF) of compound 1d and 1p can be found as supplementary information, free of charge at http://jbcs.sbq.org.br as PDF file. Crystallographic data (excluding structure factors) for the structure have been deposited with the Cambridge Crystallographic Data Center with CCDC No. 962611 (compound 1d), and 962663 (compound 1p). These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/ data_request/cif.