Introduction In-Vitro Antibacterial / Antifungal Screening of 2-Chloroquinoline Scaffold Derivatives

The emergence of multi-drug resistance strains of bacteria and fungi such as Methicilin resistant Staphylococcus aureus (MRSA), Vancomycin resistant enterococcus (VRE) and Fluconazole resistant Candida species have made treatment of infectious diseases difficult and over the time have become a serious medical problem [1-5]. The mechanism of resistant is continuously evolving in pathogenic bacteria to currently used antimicrobials [6-8]. The search for new antimicrobial agents have been an important and challenging task for medicinal chemists [9-11] and discovery of novel and potent antimicrobial agents is still a best way to combat this situation [12-13]. Introduction In-Vitro Antibacterial / Antifungal Screening of 2-Chloroquinoline Scaffold Derivatives Kumar S*1,3, Goel N2, Afzal O1, Ali MR1 and Bawa S*1 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India 2Maharaja Surajmal Institute of Pharmacy, C-4, Janakpuri, New Delhi, India 3Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India *Corresponding author: Bawa S, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India, E-mail: drsbawa@rediffmail.com Citation: Kumar S, Goel N, Afzal O, Ali MR, Bawa S (2015) In-Vitro Antibacterial / Antifungal Screening of 2-Chloroquinoline Scaffold Derivatives. J Antibiot Res 1(1): 101. doi: 10.15744/2574-5980.1.101


In-Vitro Antibacterial / Antifungal Screening of 2-Chloroquinoline Scaffold Derivatives
Some of the recent chemical modifications quinoline include Bedaquiline (R207910) which has shown extraordinary activity against both drug susceptible and drug-resistant strains of M. tuberculosis, exhibiting MIC values of 30-120 ng/ml, [17].Laquinimod is an experimental immunomodulator drug and it is currently under investigation for oral treatment of multiple sclerosis (MS) [18].GSK 299423 is an investigational compound which has shown potent activity against antibiotic-resistant strains of bacteria such as Staphylococus aureus, including methicillin resistance S. aureus (MRSA) and against gram-negative bacteria like E. coli, Pseudomonas, Klebsiella and Acinetobacter [19] (Figure 2).Fascinated by multifarious bioactivity of quinoline various researchers and scientists are still engaged in developing potent molecule based on quinoline such as Saeed et al. [20] have reported the synthesis of conformationally constrained Analogs of N-Substituted Piperazinylquinolones tested for antimicrobial activity.Likewise various 4-pyrazolyl-N-(hetero)arylquinoline were prepared by Nilesh et al. [21] and observed that some of the compounds were more or equipotent against most of the employed strains than commercially available drugs.Impelled by these observations and in continuation of our research for bioactive molecules based on 2-chloroquinoline system [22][23][24], we address here synthesis and in-vitro antimicrobial activity of some newer differentiated 2-chloroquinoline derivatives.
Volume 1 | Issue 1 Melting points were determined by the open capillary method with electrical melting point apparatus and are uncorrected.IR spectra were recorded as KBr (pellet) on Bio Rad FT-IR spectrophotometer and 1 H and 13 C-NMR spectra were recorded on Bruker DPX 300 MHz spectrophotometer using DMSO-d 6 or CDCl 3 as a NMR solvent.Mass spectra (MS-ESI) were recorded on a JEOL-AccuTOF JMS-T100LS mass spectrometer and elemental analysis on Vario-EL III CHNOS-Elemantar analyzer.Thin Layer Chromatography (TLC) was performed to monitor progress of the reaction and purity of the compounds, spot being located under iodine vapors or UV-light.The starting material 2-chloro-3-formyl-quinoline 1 and 2-chloro-3-formyl-6-methylquinoline 2 were prepared according to the literature method [25].

Synthesis of 1,3,4-Oxadiazole Derivatives (7-10)
A mixture hydrazones (3-6) (0.001 mol), chloramines-T (1.14 g, 0.005 mol) and 10 ml of abs.ethanol taken in a round bottom flask and refluxed for 6-8 hr.The progress of the reaction was monitored on TLC.After word the reaction mixture was poured in water and extracted with ether.The combined extract was washed with water and dried over anhydrous sodium sulphate and concentrated under reduced pressure [26].

Synthesis of compounds 11 and 12
To a solution of compound 1 or 2 (0.01 mol) in absolute methanol, solid sodium borohydride (0.45 g, 0.012 mol) was added portion wise over a period of 30 min.with constant stirring at room temperature.After that solvent was evaporated under reduced pressure and the residue was triturated with water and the crystalline product was filtered, washed with water and dried.The product was recrystallized from methanol.

General method for the synthesis of compounds (13-16)
To a solution of 11 or 12 (0.005 mol) in pyridine (10.0 mL) was slowly added benzoyl chloride (0.7 g, 0.005 mol) or p-tuloloyl chloride (0.77 g, 0.005 mol) at room temperature.After stirring for 10 min, the mixture was allowed to warm at room temperature and maintained for 2 h.The mixture was then diluted with cold water (50 mL), the solid product obtained was washed repeatedly to remove pyridine.The dried product was than recrystallized from ethanol.
( Sodium hydroxide (0.132 g, 0.0033 mol) was slowly added over 5 min to a stirred solution of 2-mercaptobenzimidazole (0.21 g, 0.0014 mol) or 6-nitro-2-mercaptobenzimidazole (0.28 g, 0.0014 mol) in ethanol (20 mL).Compound 17 or 18 (0.0016 mol) was slowly added to this solution at 0 º C and stirred for 12-14 hrs at room temperature.The completion of the reaction was monitored on TLC and after that solvent was removed under reduced pressure, the residue was poured into 10% NaHCO 3 solution and extracted with ethyl acetate.The organic layer was dried over Na 2 SO 4 , and concentrated.The residue was crystallized from methanol [27].Potato dextrose agar (PDA) and nutrient agar were used as culture medium for antifungal and antibacterial activity respectively.Normal saline with tween 80 (0.01%) was used to make suspension of fungal and bacterial spore for lawning.Fifty milliliters of PDA medium was poured into each petri dish (15 cm diameter).Five ml of the spore suspension was spread over the solid agar medium and plates were dried in incubator at 37° for 1 hr.Using an agar punch, wells were made on these seeded agar plates and solutions of test compounds in DMSO at conc.range of 6.25, 12.5, 25.0, 50, 100 and 200 µg/ml were added into each well, labeled previously.A control was also prepared using solvent DMSO.The Petri plate were prepared in duplicate and incubated at 30 °C for 72 hr for fungi and 37 °C for 24 hr for bacteria.Antifungal activity was determined by measuring zone of inhibition and the minimum inhibitory concentration (MIC) was noted by seeing the lowest concentration of the test drug at which there was no visible growth.Activity of each compound (3-26) was compared with standard Fluconazole and Ciprofloxacin and results have been summarized as MIC (average zone of inhibition of two reading in millimeter) in Table 1.

Antimicrobial Screening
The crystal structure of bacterial DNA gyrase (PDB code: 3G75, Resolution-2.30Å)was retrieved from Protein Data Bank (PDB) and was utilized for molecular docking studies .Protein was prepared with the Protein Preparation Wizard in Maestro using options: bond orders were assigned, hydrogen atoms were added, formal charges were treated and water molecules were deleted.Hydrogen bonding network was then optimized using the exhaustive sampling option and the protein was minimized to an RMSD limit from the starting structure of 0.3 Å using the Impref module of Impact with the OPLS_2005 force field.Prepared protein structure was used to generate Glide scoring grids for the subsequent docking calculations.Docking grids was generated with the default settings in Glide using the co-crystalized ligand (B48)to define the centre of the grid box (20×20×20 Å).Default parameters were used and no constraints were included during grid generation.The three dimensional coordinates of the most potent compound 21 was generated using Maestro module of Schrodinger.Ligands were prepared using LigPrep 2.6 with Epik 2.4 to expand protonation and tautomeric states at 7.0 ± 2.0 pH units and energy was minimized using the OPLS 2005 force field.The docking calculations were performed by Glide XP docking.

No Antifungal activity Antibacterial activity
A. niger A.flavus C albican P. aurogiosa S. aureus E. coli 100 (7.5) 100 (6.5) 200 (6.0)50 (6.5)50 (7.5)25 ( 6  The physiochemical properties important for ADME (Absorption, Distribution, Metabolism and Excretion) considerations were predicted using QikProp 3.6 (Schrodinger) that calculates properties like molecular weight, molecular volume, no. of H-bond donors, no. of H-bond acceptors, polar surface area, Q Plog Po/w (Predicted octanol/water partition coefficient) and violations related to Lipinski's "Rule of 5" and Jorgensen's "Rule of 3" to filter out compounds with clear-cut undesirable properties.The prerequisite was to neutralize the compounds before being used by QikProp.The neutralization step was carried out using Lig prep after which all the hits from both the approaches were processed for calculation of ADME properties.

Chemistry
The structure of diverse 2-chloroquinoline derivatives was elucidated by combined use of IR, 1 H and 13 C-NMR and mass spectral data.The presence of the 1,3,4-oxadiazole unit in compounds (7-10) was supported by the appearance of two quaternary signals of (C-2, C-5) at δ value 164.5 and 166.7 ppm in 13 C-NMR spectrum of compound 7.This was further supported by mass spectrum of compound 7 (m/z 309.12).The synthesis of compounds (13)(14)(15)(16) was achieved by reacting quinoline carbinol derivatives (11,12) with benzoyl chloride in pyridine.The formation of benzoate derivatives were established by locating characteristics peak of -CH 2 OCO-which was observed in the range at δ value 5.01-5.06ppm integrating for two protons in 1 H-NMR.In 13 C-NMR this particular function was observed at δ value 64.6 ppm for compound 13.In IR spectra the characteristics C=O and C-O band for compounds (13)(14)(15)(16)) were observed at 1724 -1730 and 1117-1123 cm -1 respectively.The synthesis of secondary amines (19)(20)(21)(22) of sulphanilamide/ p-aminophenol was identified by locating -CH 2 NH-function in spectral data.The 1 H-NMR signal due methylene of -CH 2 NH-was observed at δ value 4.59-4.62ppm, while the NH proton was resonated at 4.30-4.38ppm as singlet or broad singlet.The synthesis was further confirmed by mass spectrometry in which molecular ion peak was registered at m/z 347.11 (M + ) and M+2 peak at 349.11 for compound 20.The synthesis of compounds (23)(24)(25)(26) was established by identifying the characteristics -CH 2 S-peak in NMR.In 1 H-NMR spectra of compounds (23)(24)(25)(26) the signal due methylene proton of -CH 2 S-group was resonated at δ value 4.69-4.72integrating for two protons.While in 13 C-NMR, the methylene carbon was identified at δ 38.0 for compound 23.All these observations confirm successful synthesis of compounds.

Antimicrobial activity
Results of antibacterial screening are presented in Table 1 as MIC the conc.at which no visible growth was observed (zone of inhibition in mm).The quinolinyl hydrazones (3)(4)(5)(6) and there corresponding oxadiazoles (7-10) exhibited variable effect on the growth of bacterial strains.The hydrazones and oxadiazoles of INH, compounds (3, 5, 7 and 8) exhibited MIC of 12.5 to 50 µg/ ml against test strains and among these compound 7 and 8 showed MIC of 12.5 µg/ml against the E. coli.While hydrazones and oxadiazoles of benzoic acid hydrazide (4, 6, 9 and 10) showed MIC in the range of 50 to 200 µg/ml.The difference in the MIC within these analogues (3-10) may be attributed to presence of INH residue which itself is a potent antimycobacterial agent.The intermediate compound 11 and 12 showed moderate antibacterial (MIC 50 to 100 µg/ml) activity and their corresponding ester (13)(14)(15)(16) turns from moderately active to weakly active (MIC 100 to 200 µg/ml).The chloromethyl intermediate (17 and 18) were also showed weak activity which was observed at (MIC 200 µg/ml) against the test bacterial strains.While their corresponding secondary amines of sulphanilamide (19,20) and p-aminophenol (21,22) was comparatively more active in inhibiting the growth of the bacteria (MIC 12.5 to 25 µg/ml).Among the 2-mercaptobenzimidazole derivatives (23)(24)(25)(26), the nitro derivatives were more active against the all the bacterial strains and there MIC was observed in the range of 25-50 µg/ml.

Antibacterial activity
The antifungal activity quinolinyl hydrazones (3)(4)(5)(6) and there corresponding oxadiazoles (7-10) derivatives was found to be weak as their MIC were observed in the range of 100 to 200 µg/ml against test strains.The compound 11 and 12 also exhibited weak antifungal activity while there ester analogue (13)(14)(15)(16) were slightly more active than the parent compound and there MIC were observed in between 50 to 100 µg/ml.The antifungal activity of chloromethyl derivatives of 2-chloroquinoline (17 and 18) was found in the range of 50 to 100 µg/ml.The quinolinyl amine derivatives of sulphanilamide and p-amniophenol (19)(20)(21)(22) showed antifungal activity in the range of 25-100 µg/ml.The benzimidazole derivatives (23)(24)(25)(26) showed moderate antifungal activity against the test strain C. albicans, A. niger and A. flavus was and there MIC observed at 25 to 50 µg/ml.To understand the mechanism of action underlying activity of most active compound 21, we proceeded to examine the interaction of compound 21 with bacterial DNA gyrase (PDB code: 3G75) [30].All docking runs were carried out as per Glide XP Docking protocol in Schrodinger 9.4 [31,32].The XP Glide score obtained for compound 21 was found to be -7.62. Figure 4 and Figure 5 shows the binding mode of compound 21 interacting with DNA gyrase and revealed that amino acids ASP57, GLU58, ASH81,ILE51, ILE175, VAL79, ILE102, ILE86 and PRO87 located in the binding pocket played vital roles in the interaction of compound 21 with the enzyme.The hydroxyl substituent at the distal phenyl ring and NH group acts as H-bond donar and formed H-bond network with the amino acid residue ASP57 and GLU58 at 1.57 and 2.01 Å respectively.One nitrogen atom of quinoline nucleus providedadditional H-bond with ASH81 at 1.32 Å.The hydrophobic interactions with ILE51, ILE175, VAL79, ILE102, ILE86 and PRO87 further stabilized the compound in the active site of bacterial DNA gyrase.

Figure 1 :
Figure 1: Chemical tailoring or chemical remodeling of existing antibacterial drugs classes, showing development of Gatifloxacin or Moxifloxacin from Nalidixic acid and investigational molecules PA-824 and OPC-67683 from Metronidazole etc

Figure 2 :
Figure 2: Chemical structures of some investigational quinoline containing antimicrobial molecules

-chloroquinolin-3-yl) methyl benzoate 13:
To a solution of compound 11 or 12 (0.01 mol) in dry benzene, SOCl 2 (1.55 g, 0.013 mol) was added and the mixture refluxed for 4 hr.Solvent was evaporated under reduced pressure and the residue was dissolved in ether, washed with 10% NaHCO 3 and twice with water.Dried over Na 2 SO 4 and concentrated in vacuvo to give a residue which was crystallized from methanol.To a mixture of compound 3 (0.003 mol) and sulphanilamide/ p-aminophenol (0.003 mol) in 20 mL of absolute ethanol, 1 mL of triethylamine (TEA) was added and refluxed for 12-15 h.After completion of the reaction, content of the flask reduced to half and left overnight.The crystalline mass obtained was filtered off, washed with water, dried and recrystallized from ethanol to give19-22.

Table 2 :
QikProp properties of all the compounds (3-26) calculated from QikProp tool of Schrodinger