Evaluation of anti-bacterial activity of novel 2, 3-diaminoquinoxaline derivatives: design, synthesis, biological screening, and molecular modeling studies

ABSTRACT A series of nonsymmetrical 2,3-diaminoquinoxaline derivatives prepared straight-forwardly by the C-N coupling between chloro-quinoxaline (2,3-DCQ) and various amino substrates. Synthesized products (4a-z) with excellent purity (1HNMR) without performing flash column chromatography purification. Investigated the antibacterial activity of selected nine compounds against Gram-positive, Gram-negative bacterial strains, and additional fungal strains. Overall, the selected screened compounds (4b, 4c, 4 h, and 4 n) presented significant antibacterial activity against bacterial strains. The resulting lead, displaying a novel quinoxaline compound (4c), showed antibacterial activity between 10.5 and 14.89 mm against all strains except A. Niger. Molecular docking studies revealed the binding poses and critical interactions of 2,3-diaminoquinoxaline analogues at the quinolone-binding site of S. aureus DNA gyrase. The docking results are in accordance with the antimicrobial testing data. Binding of these new analogues (as DNA intercalators) at quinolone-binding site involves the contribution from: I) Protein: Ser-1084, II) DNA: DG (G) 9 and DA (H) 13; and III) Mg+2 ion for their binding.


Introduction
Globally, WHO has documented antimicrobial resistance in many first and second-line medications.
[1] Lack of advanced medication, healthcare prescribed most expensive with serve toxicity and even less effective to treat chronic bacterial infection. Because of that, prolonged hospital stays and high cost of treatment with poor outcomes increase the tremendous burden on healthcare and may cause leading cause of death in the future. [2] The most advanced second line, antimicrobial medication (vancomycin and methicillin) has developed resistance against Staphylococcus aureus and a similar trend has been shown in the first line, fluconazole against candida fungal strain. [3] Even though there are many medications existing for antimicrobial treatment, no one causes severe adversative effects [4] such as local inflammation (penicillins), allergic reaction, phototoxicity (tetracyclines), effect on the liver, gray baby disorder, and myelosuppression (chloramphenicol) [5]. The bacterial agent can undergo a mutation that causes resistance to corresponding medication or antibiotics, and finally less effectively binds with target sites like alteration in MOA, chemical alterations of the drug, or by other means. [6] Among that, major threatening is bacteria-produced biofilm to reduced penetration effect or neutralized antibiotics effect. [7,8] Quinoxaline motif is a key structural unit; it exists in various biologically active compounds. In recent research, many small molecules of quinoxaline have a tremendous demand in medicinal chemistry, broadly known as potent antiproliferative and antimicrobial agents. The quinoxaline-bearing antibiotics; including, Triostin A and similar kinds of levomycin and echinomycin to introduce into dsDNA and are advantageously effective toward the bacterial diseases caused by gram-positive bacteria. [9,10] Quinoxaline has also documented to have promising anticancer activity in various research profiles and the U.S. FDA approved the potential anticancer drug for urothelial cancer and bladder cancer under the brand name Balversa (erdafitinib). [11] Quinoxaline scaffold is pharmacology impressive, displaying an array of interesting biological activities such as antibacterial [12][13][14][15][16], anti-tubercular , antimalarial [20-20], anti-viral [23,24], anti-HIV [25], antiinflammatory [26][27][28][29], anti-fungal [30][31][32][33] 53] and also used in anthelmintic drugs. [54] Quinoxaline-based drugs presenting a broad range of diverse classes of biological activities as an outcome of diverse functionality on central structure Figure 1. Quinoxaline also used for crop protection in Agro-industries as a major component of insecticides [55], herbicides [56], and fungicides.
[57] Besides their medicinal and crop protection applications these compounds widely used as dyes for solar cell applications, [58,59] fluorescent materials [60], organic semiconductors, [61][62][63], and corrosion inhibitors for metals. [64] Because of the great call of antimicrobial therapy, continuous innovative research are going on quinoxaline scaffolds to hit novel candidates that may be, save the life for future prospects.

Antimicrobial and antifungal activities
They bought the test microorganisms from the National Collection of Industrial Microorganisms, National Chemical Laboratory (NCL), Pune (India). Nutrient agar, MGYP, and Potato Dextrose agar (25°C, 24 h) were used to cultivate bacteria and fungi, respectively. It measured the zone of inhibition in mm. Disc diffusion method [68,69] using sterile paper disk (diameter 6 mm), against bacterial strain in the concentration of 100 μg/disk screened for antibacterial activity and screened these compounds for antifungal activity against fungi Aspergillus Niger and Candida albicans by the disc diffusion method (

Structure-activity relationship study
The above biological data (Table 2 or Figure 3) of the newly synthesized compounds revealed that all the nine novel derivatives adopted a significant antimicrobial and antifungal profile against the tested bacterial and fungal strains.
The results of antimicrobial screening showed that most of the studied compounds displayed variable growth inhibitory effects on the tested gram-positive, gram-negative bacterial and pathogenic fungal strains. As expected, we distinguish a vibrant dissimilarity in the activity between derivatives within the entire series, directing to the strengthening and diminishing effects of substitution at C-2 and C-3 of the quinoxaline scaffold. The diameter of (Zone of inhibitions) IZs of the antifungal activity has shown almost the same tendency as the antibacterial activities. Concerning the outcome of substitution at the C-3 position on aryl ring is clear that the change of such a substituent may have a remarkable effect on the antimicrobial activity, which may improve or decreased, depending on the electronic nature on the ring. Individually, aryl ring at C-3 having diverse group, fluorinated compound (4c, 4 h) [67] enhanced the activity because of an electron-withdrawing group and the same trend shown in the compound (4b, 4 n) because of the CF 3 group [66] but directing CH 3 and -OCH 3 compound (4 m) decreases activity. Similar steric effect, diminished activities at C-2 position, cyclo-pentane series of compound (4x and 4z) presented inferior activities compare to the cyclo-propyl series of compound (4b and 4 l). The zone of inhibition (mm) shown in Table 3  Among the synthesized derivatives, compound 4c presented moderate activity on C. albicans is 11.33 mm. We do not exactly explain the SAR from few compounds but further, modification in structure can enhance the activity and may identify lead molecules from the same scaffolds.

Molecular modeling study
The synthesized and screened nine 2,3-diamino quinoxaline derivatives were subjected for physicochemical property estimation to understand Table 1. Compounds of the scheme-1 (1-4 series) (N 2 -alkyl-N 3 -phenylquinoxaline-2,3-diamine derivatives): reaction time, yield, and M.P. of each compound. their drug-likeness. Next, molecular docking was employed for predicting the binding modes, interactions and binding affinities of these compounds.

Physicochemical property estimation
The estimated physicochemical properties of top nine compounds are shown in Table-3. Almost all the compounds possess nearly favorable drug-like properties with minor violations (acceptable) for Lipinski's rule of five. These data of screened hit hints for their possibility as potential drug-like candidates.

Binding mode analysis from docking studies
Putative binding modes of both orthosteric or allosteric binders can be accuractly predicted using molecular docking approach [70][71][72]. The molecular docking can also be effectively used for virtual-screening of compounds [73]. Here, we also used molecular docking approach to predict the binding modes of 2,3-diamino quinoxaline derivatives for their antibacterial activity via DNA intercalation. The results of binding mode analysis are summerized in Table-4 . We used chloramphenicol as a reference compound to mimick our earlier experimental design. The binding modes of 2,3-diamino quinoxaline derivatives and reference compound corroborate quite well and is nearly comparable with antimicrobial testing data. The posing of our designs at quinolonebinding site of S. aureus DNA gyrase with DNA is shown in Figure 4 (A-H). The consistency in binding poses of our screened designs at quinolone-binding site is evident from the pose analysis as shown in Figure 4 (4). The interaction with Ser-1084 was seen for all the docked designs. Additionally, the intercalation through pi-pi stacking of quinoxaline moiety between DG (G) 9 and DA (H) 13 DNA base pairs were evident (2D ligand interaction maps in Supplementry Figure 4 figure (4). The important role of metal ion (Mg +2 or Mn +2 ) in mediating coordinated quinolonemetal-bridging is well documented [74]. Similarly in our all docked poses the metalcoordination of pyrazine ring nitrogen with crystal bound Mg +2 ion is observed (indicated in SI 2D ligand interaction maps). The Glide docking scores for nine tested designs were ranged from −5.06 to −7.18 kcal/mol. However the prime MM-GBSA scores of the selected poses ranged from 0.57 to −50.26 kcal/mol showing nearly comparable values to the values from antimicrobial testing data.

Computational modeling
Initially the physicochemical properties of most promising quinoxaline designs were calculated using Schrödinger [75] QikProp module [76]. Then their binding modes were estimated using GLIDE [76,77,] docking module of Schrodinger Suite. The prime MM/GBSA [78,79] G∆ of binding was calculated for evaluating the binding affinities of new designs.

Generation of 3D molecular structures
The quinoxaline derivatives were modeled and minimized using Hyperchem7.5 [80] using MM + force field. The output mol2 files were subjected for ligand preparation at physiological pH of 7.4 with LigPrep module.

Protein structure selection and preparation
The Quinoxaline derivatives binds as DNA intercalators [66] thereby shows antibacterial [12][13][14][15][16], activity. Accordingly, S. aureus DNA gyrase crystal structure (PDB ID: 2XCT) [74] complex with ciprofloxacin and DNA was selected for performing molecular docking studies. Fully automated Protein preparation wizard protocol was used to prepare the protein structure at ionization pH of 7.4. The H-bonding network was optimized and restrained minimization was performed using OPLS-2005 force field with the RMSD convergence criteria of 0.30 Å for all heavy atoms.

Molecular docking study
The fully prepared protein (containing DNA and ciprofloxacin) and ligand structures were considered for the molecular docking study. The GLIDE receptor grid was generated using the centroid of crystal-bound ciprofloxacin molecule within the search space of 14 Å box size for docking ligands 14 Å length at equally spaced 2 Å grids. The standard precision (SP) mode Glide docking was performed with a soft potential function at van der Waals radii scaling of 0.5 with applied core constraints for generating consistent poses of quinoxaline derivatives. The obtained ligand poses were finally selected based on the docking scores and presence of Ser1084 interaction. The images of the ligand poses were generated using Pymol (Schrödinger LLC, 2010) [81][82]. The 2D ligand interaction maps of poses were obtained using ligand-interaction tool from Schrödinger suite.

Instruments and appparatus
General reagents and solvents for the synthesis of compounds were analytical grade (AR) and used without further purification. Air-sensitive reactions were carried out

General procedure for the synthesis of 3-chloro-N-alkylquinoxaline-2-amine (3a-d)
To a stirred solution of 2,3-dichloroquinoxaline (4.0 g, 20 mmol) in DMF (40 mL  CO 3 (40 mmol) were added. The reaction mixture was stirred at room temperature, under nitrogen for 12 h. The completion of the reaction was monitored by TLC and spots were visualized under UV light. The reaction mixture was poured into ice-cold water (400 ml) and stirred for 15 min, then aqueous layer was extracted with ethyl acetate (50 ml X 2). The organic layers were combined, wash with brine solution (50 ml), dried over anhydrous Na 2 SO 4 , filtered and concentrated under-reduced pressure which affords to give crude material that was purified by flash chromatography by using silica gel (230-240 mesh) using 15% ethyl acetate in n-pentane to obtain as white solid product (3a-d)     in ethanol (4 mL) were heated to reflux for 6 h until the reaction completed (monitored by TLC). Then the reaction mixture was cooled, filtered, and the solid was washed with petroleum ether to obtain a solid product (4a-z). -2,3-diamine (4a). Creamish white solid

Conclusions
The aim of the work to design and synthesize novel diamino-quinoxaline scaffolds and products were screened against bacterial and fungal strains. Quinoxaline scaffold presented the preliminary significant antimicrobial activity against a panel of pathogenic microbes including two Grampositive bacteria (S. aureus and B. subtilis), three Gram-negative bacteria (E. coli, K. pneumoniae and Salmonella typhi), and two fungal strains (C. albicans and A. niger). From the quantitative analysis, the compound (4c) presented a broadspectrum (Zone of inhibitions) on all the strains between 10.11 and 14.89 mm. Our docking-based binding mode analysis results are in accordance with the antibacterial testing data. In summary, docking predicts our diamino-quinoxaline analogues as DNA intercalators where they bind at microbial DNA gyrase quinolone-binding site with critical Mg +2 ion metal-coordination, thereby showing their antibacterial activity.