Synthesis of 2-substituted indoles and evaluation of their antibacterial activity and inhibitory effects on the efflux pump of methicillin-resistant Staphylococcus aureus

Suttinun Vicharn1, Jitnapa Sirirak1, Weerachai Phutdhawong2, Thongchai Taechowisan3, Waya S. Phutdhawong1* 1Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand. 2Department of Science, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Sean Campus, Nakhon Pathom, Thailand. 3Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand.


INTRODUCTION
The development of new pharmaceutical agents is one of the greatest challenges in overcoming widely spread epidermics and drug resistance. Furthermore, the ineffectiveness of medications such as antimicrobial drugs cause an increase in mortality, morbidity, and public health problems. The major components of resistance to many classes of antimicrobials are efflux mechanisms (Reygaert, 2018). In these mechanisms, efflux pump inhibitors are used to stop bacteria from pumping antibiotics out of cells, and it has been reported that 5-nitro-2-phenylindole '…..(INF55) (1) (Fig. 1) participates in the NorA efflux pump of Gram-positive bacteria and increases the susceptibility of Staphylococcus aureus to antibiotics (Markham et al., 1999). Substituents on the phenyl ring in INF55 (1) are also important for biological activities; that is, the 2,5-dimethoxyl group on the aryl ring (2) increases the inhibitory activity of NorA (Samosorn et al., 2006). This indicated that the substituents on the phenyl ring of the 2-arylindole nucleus affect biological activity. Similarly, the substitutions of the benzene ring of the indole nucleus diverted the biological activity, which were reported in the literature (Lal and Snape, 2012;Naik et al., 2014;Williams et al., 2013). Among these, 5-methoxy-2-phenylindole (3) was reported to be a significant antibacterial activity against Gram-positive Bacillus cereus minimum inhibitory concentrations (MIC) = 3.9 µg/ml. Furthermore, 5-amidine-2-arylindole bearing a 3,5-disubstituted aryl ring (4) remarkably affects the inhibition of acid-sensing ion channels (Kuduk et al., 2009). In addition, the 2-aryl-substituted indole derivative (5) is a bacterial histidine kinase inhibitor (Deschenes et al., 1999).
With the wide-ranging biological activities, the 2-arylindole scaffold is among the privileged frameworks to study the antibacterial activity and its inhibited NorA efflux pumpresistant mechanism. Thus, the goal of this study is to investigate the 2-arylindoles with various substituents on the aryl ring with their antibacterial activities and the ability to potentiate the activity of commercially available drugs and overcome drug resistance.
Indole is synthesized using various synthesis methods, including Fischer indole synthesis (Cho et al., 2009), Bischler synthesis (Cossío et al., 2008), Madelung cyclization (Imanishi et al., 1996), and transition metal catalyzation (Heck and Terpko, 1979;Larock and Yum, 1991). However, in terms of synthesis efficiency, these methods are limited by their requirement of several steps to be completed (Dai et al., 2001;Ezquerra et al., 1996;Rudisill and Stille, 1989;Taylor et al., 1985). Furthermore, only a few methods are available to substitute the indole ring at the C2 position because the C3 position is more reactive than the C2 position for electrophilic aromatic substitution. Nevertheless, an N-protected indole can undergo organometallic substitution at the C2 position.
In this study, Fischer indole synthesis was applied as a simple and effective method to prepare 2-substituted indoles. The antibacterial activities of the synthesized compounds and their synergistic antibacterial potential with tetracyclines against the clinical isolate of methicillin-resistant S. aureus (MRSA) Sp6 were evaluated and described. Their synergistic mechanism was also investigated through molecular docking.

General information
Melting points (m.p.) were determined using a Stuart Scientific SMP 2 m.p. apparatus and uncorrected. 1 H-and 13 C-NMR spectra were obtained with (D) d 6 -DMSO solutions at 300 MHz for 1 H and 75 MHz for 13 C with a Bruker AVANCE 300 spectrometer. Tetramethylsilane was used as an internal standard. Mass spectra were recorded with a Polaris Q or Hewlett Packard 5973 mass spectrometer. Reagents and solvents were supplied by Acros, Aldrich, Fluka, and TCI.

General procedure for the synthesis of 2-substituted indoles (4a-m)
Phenylhydrazine (1) (1.5 eq.) was mixed with ketone (2a-m) (1.0 eq.) in 50% acetic acid (20 ml). The mixture was added to a beaker containing polyphosphoric acid (1.0 eq.), stirred while being heated in an oil bath for 30 minutes. The reaction mixture was cooled to room temperature and added to iced water (50 ml) to yield a black precipitate. The precipitate was crystallized in ethanol to obtain 2-substituted indoles (4a-m).

2-(4-Nitrobenzamidophenyl)-1H-indole (6f)
The title compound was synthesized from 4m (0.11 g, 0.52 mmol), Et 3 N (0.05 ml, 0.36 mmol), and 4-nitrobenzyl chloride (0.05 g, 1 mmol) to obtain 2-(4-nitrobenzamidophenyl)-  8, 110.9, 113.1, 113.8, 120.0, 120.4, 121.0, 122.1, 126.1, 128.9, 136.8, 136.9, 147.1, 152.0, 171.8 Standards, 2000). Kanamycin and chloramphenicol were used as references for antibacterial activity. The test samples were dissolved in DMSO with a range of 512-0.98 μg/ml in nutrient broth supplemented with 10% glucose and 0.05% phenol red (NBGP). Then, 100 μl of each concentration was added to each well (96-well microplate) containing 95 μl of NBGP and 5 μl of inoculum (standardized at 1.5 × 10 6 cfu/ml by adjusting the optical density to 0.1 at 600 nm. The final concentration of DMSO in the well was less than 1% preliminary analyses with 1% (v/v) DMSO/NBGP affected neither the growth of the test organisms nor the change in color because of this growth). The negative control well contained 195 µl of NBGP and 5 µl of the standard inoculum. The plates were covered with a sterile plate sealer, agitated using a plate shaker to mix the content of the wells, and incubated at 37°C for 24 hours. The assay was repeated twice, and microbial growth was determined by observing the change in the color of the content of the wells (i.e., red = without growth, yellow with growth). The lowest concentration showing no color change was considered the MIC.

Synergistic antibacterial assay
MRSA Sp6 was isolated from pus samples of patients in Nakhon Pathom Hospital (Taechowisan et al., 2018). The drug resistance of this strain was detected via a cefoxitin paper method of the American Association of Clinical and Laboratory Standards Institute (CLSI, 2014). Its drug resistance and efflux pump genes were also detected through PCR amplification. The primers of the species-specific S. aureus (femA) gene, the penicillin-binding protein 2 (mecA) gene, and the active efflux pump of the plasmidlocated tetracycline (tetK) gene were utilized for PCR assay as previously described (Taechowisan et al., 2018).
The MICs of methicillin and tetracycline were determined using a twofold dilution method in accordance with the guidelines of the National Committee for Clinical Laboratory Standards (1997). In this procedure, 100 ml of MRSA Sp6 (10 5 cfu/ml) was mixed with different concentrations of methicillin and tetracycline (16, 32, 64, and 128 mg/ml) in a 96-well plate and incubated at 37°C for 24 hours. A control group was prepared by culturing MRSA Sp6 with 0 mg/ml methicillin and tetracycline. Various concentrations (16, 32, 64, and 128 mg/ml) were applied to 100 ml of MRSA Sp6 (10 5 cfu/ml) cells alone or in combination with either methicillin or tetracycline (16, 32, 64, and 128 mg/ ml) in 96-well plates to elucidate the effect of methicillin and tetracycline activities in the presence of 2-arylindole derivatives. The plates were incubated at 37°C for 24 hours. A blank control was also prepared by culturing MRSA Sp6 cells in media only without methicillin and tetracycline or 2-arylindole derivatives.

Molecular modeling
The homology model of NorA from S. aureus was constructed using a Swiss-Model server and the protein EmrD efflux pump from E. coli (PDB ID: 2GFP) because the crystal structure of NorA from S. aureus remained unavailable (Zárate et al., 2019). Molecular docking was performed with iGEMDOCK v2.1 (Hsu et al., 2011) to explore the protein-substrate interactions and binding position of 4g-k and 4 m in the active site of NorA from S. aureus. Ciprofloxacin, which is known as a good inhibitor of NorA efflux pumps, was also docked into the NorA efflux pump, and its binding energy was compared with those of our compounds.

Prediction of ADMET by computational analysis
The computational prediction of the compounds was performed through the online software SwissADME (http:// swissadme.ch) in order to evaluate the pharmacokinetics of the synthesized molecules. The interaction of the synthesized molecules with CYP was used to predict the toxicity properties. Passive human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) were used to predict the pharmacokinetic behaviors of these molecules. Caco-2-permeability was considered as druglikeness property.

RESULTS AND DISCUSSION
Chemistry 2-Arylindoles were prepared in a manner similar to previously reported Fischer indole synthesis (Hughes, 1993)  (Scheme 1). After the reaction parameters were screened, the reaction of 1 (1.5 eq.) and methyl ketone derivatives (2; 1.0 eq.) in 50% acetic acid (20 ml) produced moderate to high yields of indole derivatives (4) via hydrazone derivatives (3). The reaction time and temperature of each product under the optimized conditions are shown in Table 1. The heating duration of most of the aromatic derivatives of 2 at high temperatures should be prolonged. Unidentified products except nitrobenzene substitution (entries 10-11) were obtained at high temperatures (Scheme 2). The structures of all known compounds were confirmed by m.p. and spectroscopic analysis.
The substitutions of amino groups on the aromatic ring were investigated to study the structure-activity relationship (Scheme 2). Amidation using acid halide (5) produced moderated yields of amides (6a-f). Then, the antibacterial activities and synergistic effects of the synthesized 2-arylindole derivatives were further evaluated.

Antibacterial activity
All the synthesized compounds 4a-m and 6a-f were tested against S. aureus ATCC 25932, B. cereus ATCC 7064, B. subtilis ATCC 6633, E. coli ATCC 10536, and S. typhi ATCC 19430 by NCCLS microbroth dilution methods (National Committee for Clinical Laboratory Standards, 1997). Kanamycin and chloramphenicol were used as positive controls, and a solvent was used as a negative control. The results are shown in Table 2 as MIC (μg/ml) unit. Among the compounds tested, 4m was the most active with an MIC of 15.6 μg/ml against B. subtilis and S. typhi. Furthermore, 4c, 4d, and 4f selectively inhibited B. subtilis and had MICs of 31, 125, and 62 μg/ml, respectively. In addition, 4j showed antibacterial activities against S. typhi at 15.6 μg/ml. Thus, the phenyl ring, substituents, and their positions on the phenyl ring are essential for potency. Compounds 4e and 4h with phenyl-bearing electron-donating groups on the metaposition have weak or no activity, while compound 4k bearing electron-withdrawing group on the meta-position has selective activity against S. typhi. On the other hand, the phenyl-bearing hydrophilic substituents at para-position seem to be important for the activity. The protected amino derivatives (6a-f) had weak or no antibacterial activity against all the tested bacteria. This may assume that the amide linkage of 4m affected the hydrophilicity and might obstruct the interaction of these molecules and the bacterial active site.

Synergistic effect
The synthesized compounds were assayed to examine their antibacterial activities against MRSA Sp6. The results revealed that none of them exhibited an inhibitory activity at 128 mg/ml. By comparison, the MIC of tetracycline to MRSA Sp6 was 256 mg/ml. Nevertheless, the MIC of tetracycline decreased notably when the 2-arylindole derivatives were added. This result demonstrated that the sensitivity of MRSA Sp6 to tetracycline improved. Each experiment was repeated three times (Table 3). 4g, 4j, and 4k elicited significant synergistic effects with tetracycline against MRSA Sp6. Thus, the MIC of tetracycline could be reduced from 256 to 32 mg/ml when it was combined with 4g, 4j, and 4k at a concentration of 16 mg/ml.

Molecular docking
Molecular docking was conducted to explore the protein-substrate interactions and binding positions of the active synergistic compounds, namely, 4g-k and 4m, in the active site of NorA from S. aureus. These compounds were docked into the active site of the S. aureus NorA model prepared with the EmrD efflux pump as a template. Their molecular docking results were compared with those of the NorA substrate ciprofloxacin. The findings revealed that 4g-k and 4 m bound to the cavity of the NorA efflux pump in a position similar to    that of ciprofloxacin (Fig. 2). The binding energies of 4g-k and 4m were −92.00 to −85.07 kcal/mol (Table 4), which were slightly higher than that of ciprofloxacin (−99.64 kcal/ mol). Moreover, in the active site of the NorA efflux pump, 4g, 4h, and 4k interacted with key amino acid residues, namely, Ile240, Ala243, and Ala289, respectively, while 4i, 4j, and 4m interacted with Val44, Phe47, and Ile240, respectively (Fig.  3). Therefore, the aromatic moieties of 4g-k and 4m played important roles in the binding of these compounds to the hydrophobic core of the NorA efflux pump.

Prediction of ADMET by computational analysis
The ADMET properties of the synthesized compounds were presented in Table 5. This result suggested that most of the compounds were through to cross BBB permeation. For in vitro Caco-2 cell permeability, most of the compounds except 4h-k and 4m have high permeability and are easy to absorb. All of the compounds showed % HIA in the range of 91.70%-100.00% which were considered to be very well absorbed in the gastrointestinal tract. The toxicity predictions were considered of the cytochrome P450s (CYP), an important enzyme system for drug metabolism that influences clearance rates, toxicity, and interactions with coadministered drugs. All compounds were predicted to be not substrates and not inhibitors for four major CYP isoforms, except for compounds 4a-m, which were predicted to be CYP2C19, CYP2C9, and CYP3A4 inhibitors. This suggested that these compounds may be metabolized in the liver which may have hepatotoxicity.

CONCLUSION
2-Arylindole derivatives were synthesized, and their antibacterial activity and synergistic effects on multidrugresistant S. aureus were evaluated. The results showed that 4j combined with tetracycline showed a potent antibacterial activity against S. typhi and exhibited a synergistic effect against MRSA Sp6 strain. Furthermore, 4 m displayed the most potent antibacterial activity against B. subtilis, whereas 4j was the most potent against S. typhi. All the synthesized compounds improved the sensitivity of MRSA Sp6 to tetracycline. Molecular docking studies on 4g, 4h, and 4k revealed that interaction with key amino acids occurred at the active site of the NorA efflux pump. However, the toxicity study by ADMET was predicted as hepatotoxicity. Therefore, the 2-arylindole derivatives could be considered as an interesting scaffold and further development of the 2-arylindole derivative with no toxicity might be the leading compounds that could potentiate the activity of commercially available drugs against multidrugresistant bacteria.