Antimicrobial and docking studies of (−)-catechin derivatives

Abstract

Antimicrobial activities of (−)-catechin derivatives were assayed for their antibacterial and antifungal activities against gram positive, gram negative bacteria, and fungi. Most of the compounds significantly active among which Compounds 1a, 1b, and 1c showed excellent antibacterial for both gram negative and gram positive bacteria, these compounds also exhibited excellent antifungal activity more than the standard drug. Molecular docking studies of Compounds 1a and 1b established good binding affinity with ATP-binding pocket of DNA gyrase and are in favor of the observed biological activity. These data collectively suggest that Compounds 1a and 1b could serve as a novel antimicrobial agent.

Introduction

Increasing resistance of microorganisms and multidrug-resistance (MDR) against the available microbial drug is one of the major concerns among researchers and clinicians across the world which led to an increasing search for new antibiotics (Paul et al. 2010). In short periods of time, new and multidrug resistance against the microbes is occurring rapidly (Alanis 2005). New drugs development with increased antimicrobial activity with no adverse effects against MDR bacteria is urgently needed for the human kind worldwide (Fernandes et al. 2013). It is also expected to develop drugs that can reverse the resistance observed overturning the actual bacterial profile, and search for new drug with potent activity is always needed.

Green tea, a widely consumed beverage next to water worldwide is obtained from the leaves of Camellia sinensis (Graham 1992). Catechins and its derivatives showed various pharmacological activities viz antimutagenic (Paschka et al. 1998), antioxidant (Gadow et al. 1997), antibacterial (Hu et al. 2001; Stapleton et al. 2004; Park and Cho 2010), anticancer activities (Uesato et al. 2000; Kumar et al. 2015), and prevention of atherosclerosis (Miura et al. 2001; Chyu et al. 2004). Catechins face one of the major limitations. They have low bioavailability, and low stability in neutral or slightly alkaline solutions and inability to cross cellular membranes (Hong et al. 2002). Thus, derivatives of catechins are of great interest, and have become targets for synthetic chemists and biologists. 3-O-alkyl and acyl analogs of (−)-epicatechin with moderate alkyl chains, exhibited greater improvements in their activity and also prevented from enzymatic or non-enzymatic cleavage (Park et al. 2004). Introduction of moderate-sized aliphatic chain at the C-3 hydroxyl position (lipophilic substituent) significantly improved the inhibitory effect of microbial growth (Park and Cho 2010). In view of this, we synthesized the derivatives of (−)-catechins and studied their activities of particular interest and, in continuation of our studies, on biologically active chemical entities (Husain et al. 2012; Xie et al. 2014; Kumar et al. 2015). Herein we report the antimicrobial activities and docking studies of (−)-catechin analogs (1a–l) with an optimal alkyl chain length for their enhanced activities.

Materials and methods

Chemistry

Series of (−)-catechin derivatives (Fig. 1) were synthesized and characterized according to our reported procedure (Kumar et al. 2015).

Fig. 1

Synthesized (−)-catechin derivatives

Antimicrobial activity

The antimicrobial activity of the synthesized catechin derivatives was determined using well diffusion method (Amsterdam 1996) against different pathogenic bacterial and Candida reference strains procured from the Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India. The pathogenic bacterial and Candida reference strains were seeded on the surface of the media petri plates, containing Muller-Hinton agar with 0.1 mL of previously prepared microbial suspensions individually containing 1.5 × 10⁸ cfu mL⁻¹ (equal to 0.5 McFarland). Wells of 6.0 mm diameter were prepared in the media petri plates using a cork borer and the synthesized compounds at a dose range of 300–1.17 µg well⁻¹ were added in each well under sterile conditions in a laminar air flow chamber. Standard antibiotic solution of Neomycin and Miconazole at a dose range of 300–1.17 µg well⁻¹ and the well containing methanol served as positive and negative controls, respectively. The plates were incubated for 24 h at 37 and 30 °C for bacterial and Candida strains respectively, and the well containing the least concentration showing the inhibition zone was considered as the minimum inhibitory concentration. All experiments were carried out in duplicates and mean values are represented.

Docking studies

DNA gyrase (PDB code: 1AJ6) was selected as the receptor for docking simulation in ATP-binding pocket (Holdgate et al. 1997; Gradisar et al. 2007). After removing the ligand and solvent molecules, hydrogen atoms and Kollman charges were added to each protein atom. Coordinates of each compound were generated using Chemdraw11 followed by MM2 energy minimization. Docking was carried out by AutoDock4 in ATP-binding pocket (Morris et al. 2009). Grid map in Autodock that defines the interaction of protein and ligands in binding pocket was defined. The grid map was used with 60 points in each x, y, and z direction, equally spaced at 0.375 Å. Docking was performed using the Lamarckian genetic algorithm. Each docking experiment was performed 100 times, yielding 100 docked conformations. Parameters used for the docking were as follows: population size of 150; random starting position and conformation; maximal mutation of 2 Å in translation and 50^o in rotations; elitism of 1; mutation rate of 0.02 and crossover rate of 0.8; and local search rate of 0.06. Simulations were performed with a maximum of 1.5 million energy evaluations and a maximum of 50,000 generations. Final docked conformations were clustered using a tolerance of 1.0 Å root mean square deviation. The best model was picked based on the best stabilization energy.

Results and discussion

Twelve derivatives of (−)-catechin (Fig. 1) were synthesized and characterized according to our reported procedure from the starting material 2,4,6-trihydroxyacetophenone and 3,4-bis(benzyloxy)benzaldehyde in 10 steps with an yields of 75–86 % (Kumar et al. 2015). The antimicrobial activity of the synthesized (−)-catechin derivatives was determined using well diffusion method. All the synthesized compounds were screened for their antibacterial activity against Staphylococcus aureus (S. aureus MTCC 96, MLS16, MTCC 2940), Micrococcus luteus (M. luteus MTCC 2470), Bacillus subtilis (B. subtilis MTCC 121), Klebsiella planticola (K. planticola MTCC 530), Escherichia coli (E. coli MTCC 739), and Pseudomonas aeruginosa (P. aeruginosa MTCC 2453) bacterial species. Standard antibiotic solution of Neomycin at a dose range of 300–1.17 µg well⁻¹ and the well containing methanol served as positive and negative controls, respectively. The plates were incubated for 24 h at 37 and 30 °C for bacterial and Candida strains respectively, the well containing least concentration showing the inhibition zone which was considered as the minimum inhibitory concentration.

The antimicrobial screening data showed that Compounds 1a, 1b, and 1c exhibited excellent activity against Bacillus subtilis (B. subtilis MTCC 121), with MIC 2.34 µg mL⁻¹ and moderate activity against S. aureus MTCC 96 2940 with MIC-75 μg mL⁻¹ and it showed a significant activity toward other gram positive bacteria. Other compounds showed weak activity against Micrococcus luteus MTCC 2470 (Fig. 2). Among all tested Compounds 1a, 1b, and 1c showed good antibacterial activity. The cLog P values of all catechin analogs were calculated using ACD/ChemSketch software version 12.01 (listed in Table 1) and were used for structure–activity relationship (SAR) studies. SAR analyses indicate that the Compounds 1a, 1b, and 1c showed excellent activity when compared to other analogs. The increase in the activity may be attributed due to ester-linked analogs. However, other derivatives did not show good activity, which means that replacement of other substituent is not necessary. This shows that ether or ester link to hydroxyl group or simply hydroxyl group is necessary for the activity.

Fig. 2

Antibacterial activities of Compounds 1a–l

Table 1 cLog P values of (−)-catechin derivatives

The antifungal activity of all Compounds 1a–1l was evaluated against Candida albicans (C. albicans MTCC 1962) in which Compounds 1a, 1b, and 1c exhibited excellent activity as shown in Fig. 3. Compounds 1a, 1b, and 1c were evaluated for other strains of fungi such as C. albicans MTCC 183 (C. A1), C. albicans MTCC 854 (C. A2), C. albicans MTCC 1637 (C. A3), C. albicans MTCC 3018 (C. A4), C. albicans MTCC 4748 (C. A5), C. albicans MTCC 7315 (C. A6), C. albicans MTCC 3019 (C. A7), C. albicans MTCC (C.A8), Candida aaseri (C. aaseri MTCC 1962), Issatchenkia hanoiensis (I. hanoiensis MTCC 4755), Candida glabrata (C. glabrata MTCC 3019), Issatchenkia orientalis (I. orientalis MTCC 3020), Candida parapsilosis (C. parapsilosis MTCC 1744), and they showed excellent activity against all type of fungi, which was found to be more than the standard drug. Compounds 1a, 1b, and 1c showed very high inhibitory activity (MIC = 2.4 µg mL⁻¹) when compared with the standard drug, which is illustrated in Fig. 4. Introduction of lipophilic group at C-3 position influences the pharmacokinetic properties.

Fig. 3

Antifungal activity of the (−)-catechin derivatives against candida strain

Fig. 4

Antifungal activities of 1a, 1b, and 1c against different strains of fungus

Docking of catechin derivatives in ATP-binding pocket of DNA gyrase B was performed by Autodock4 (Holdgate et al. 1997; Gradisar et al. 2007; Morris et al. 2009). Two hydroxyl groups of chroman-5,7-diol participate in hydrogen bond with main chains of Val97 and Gly117, and the side chain of Ser121. The 9-alkoxynonan-1-ol group of Compound 1a makes hydrogen bond with Asp73, while methoxynonyloxy of 1b not involving in hydrogen but it can accommodate in ATP-binding pocket (Fig. 5A, B). Compound 1c has nonyl acetate group which is difficult to accommodate in ATP-binding pocket. Compounds 1d, 1e, 1f, 1g, 1h, and 1i have alkyl chain containing bulky group that is difficult to fit in ATP-binding pocket of DNA gyrase. The presence of small hydroxy and methoxy groups in Compounds 1a and 1b makes it easy to be accommodated in ATP-binding pocket of DNA gyrase, which might be a reason for good activity against microbial strain.

Fig. 5

A Docking pose of 1a and 1b on ATP-binding pocket of DNA gyrase B (PDB code: 1AJ6). Panel A represents overall binding of compounds. B Docking pose of 1a and 1b on ATP-binding pocket of DNA gyrase B (PDB code: 1AJ6). Panel B, Stick representation of selected amino acids of DNA gyrase B interacting with Compounds 1a and 1b. Red dot line represents hydrogen bonds

In summary, antimicrobial and docking studies of (−)-catechin derivatives have been studied. Compounds 1a, 1b, and 1c showed excellent antibacterial activity for gram negative and gram positive bacteria. These compounds also exhibit excellent antifungal activity more than the standard drug “neomycin, miconazole,” respectively. This indicates that (−)-catechin derivatives can be considered as potential antimicrobial agents. Molecular docking studies also exhibited good binding affinity of Compounds 1a and 1b which have hydroxyl and methoxy group respectively. Both groups are small and easy to accommodate in ATP-binding pocket of DNA gyrase. Finally, it is conceived that further derivation of such compounds could serve as new templates for antibacterial drugs.