Synthesis, Characterization and biological evaluation of Novel Carboxamides, Oxadiazoles and Isoindoline-1,3-diones derived from 2-substituted phenylquinoline-4-carbohydrazides.

The new 2-arylquinoline-4-carboxylic acid derivatives 4(a-f) and 5(a-i) , were tested by qualitative and quantitative methods on various bacterial and fungal strains and proved to be active at low concentrations against Gram-positive and Gram-negative bacteria as well as fungi. The MIC values were determined for test compounds as well as for reference standards. Compounds 4b and 5d showed better antibacterial and antifungal activity than clinically prevalent drugs (Gentamicin, Ampicillin and Fluconazole) against Staphylococcus aureus and Candida albicans . The structures of newly synthesized compounds have been characterized on the basis of their spectroscopic data. The study revealed the potential of newly synthesized compounds as a novel group of antimicrobials. and Antimicrobial activity. The hydrazides were characterized by their physical, analytical and spectral data. IR spectra of the hydrazides showed NH and C=O stretching bands at 3264-3356cm -1 and 1640-1662cm -1 , respectively. The absorption bands associated with other functional groups appeared in the expected region. In the 1 HNMR spectra of the hydrazides, the amine(NH 2 ) proton appeared at 4.7-5.1 ppm as a sharp D 2 O exchangeable singlet, whereas a broad more downfield D 2 O exchangeable singlet at 9.8-10.1 ppm was characteristic of the NH proton (CONH group), the other protons appeared at the expected chemical shifts and integral values. Reaction of 1(a-c) with salicylaldehyde or 2-hydroxynapthaldehyde in ethanol in presence of catalytic amount of hydrochloric acid furnished N'-(2-hydroxybenzylidene)-2-(4-substituted phenyl)quinoline-4-carbohydrazides or N'-((3-hydroxynaphthalen-2-yl)methylene)-2-(4-substituted phenyl) quinoline-4-carbohydrazides 2(a-f) . Then the reduction of 2(a-f) using sodium borohydride in methanol gave N'-(2-hydroxybenzyl)-2-(4-substitutedphenyl)quinoline-4-carbohydrazides or N'-((3-hydroxy naphthalene-2-yl)methyl-2-(4-substituted phenyl) quinoline-4-carbohydrazides 3(a-f) . The internal Mannich reaction of 3(a-f) with formaldehyde in ethanol afforded N-(2H-benzo[e][1,3] oxazin-3(4H)-yl)-2-(4-substitutedphenyl)quinoline-4-carboxamides or


Experimental
Melting points were determined with an Electro thermal melting point apparatus and are uncorrected. Reactions were monitored by TLC, performed on silica gel glass plates, visualization on TLC were achieved by iodine indicator. I.R. spectra (potassium bromide) were recorded on Perkin-Elmer FTIR spectrophotometer (ν max in cm -1 ); 1 H and 13 C-NMR spectra were recorded on Bruker 200/300 MHz instruments using CDCl3 and DMSO-d6 as solvents. Chemical shifts (δ) are reported in ppm downfield from internal TMS standard. ESI MS mass spectra were recorded on a Va 70-70H mass spectrometer (Manchester, UK) at 70 eV, with a trap current of 200 μA and 4 kV of acceleration voltage and ESI mode positive ion trap detector. Elemental Analysis was performed on a Perkin-Elmer 2400 series II elemental CHNS analyzer. All chemicals and reagents were obtained from Aldrich, Lancaster, Merck, Sdfine or Spectrochem Pvt. Ltd and were used without further purification.

General Method for the preparation of 5(a-i).
A mixture of 2-(4-substituted phenyl)-quinoline-4-carbohydrazide 1 (0.01 mol), quinoline-carboxylic acid, furoic acid or phthalimido acetic acid (0.01 mol) and phosphorus oxychloride (10 ml) was refluxed ~ 12h. The reaction mixture was cooled and allowed to stand at room temperature for 2 h. It was then poured on to crushed ice. The solid thus obtained were collected and treated with sodium bicarbonate solution (5%), then with water, filtered and recrystalised from mixture of ethanol and dimethyl formamide (2:1) to get compounds 5(a-i).

2-((5-(2-p-tolylquinolin-4-yl)-1,3,4-oxadiazol-2-yl)methyl)isoindoline1,3-dione(5i):
Light brown crystals [Ethanol and Dimethyl Formamide (2:1)] ( To synthesized 5i mixture of 2-(4-methyl phenyl)-quinoline-4-carbohydrazide 1c (0.01 mol), phthalimido acetic acid (0.01 mol) and phosphorus oxychloride (10 ml) was refluxed ~ 12h and then followed the above general procedure. It was obtained as a light brown crystals.); yield ~79 %; Rf value: 0.29 ( [46] . The MIC was determined by using two fold serial dilution method [47,48] . Gentamicin, Ampicillin and Fluconazole were used as reference standards to compare the antibacterial and antifungal activities, respectively. For determining both antibacterial and antifungal activities, the synthesized compounds were dissolved in chloroform (stock solution 5mg/mL). In order to ensure that the solvent had no effect on bacterial growth, a control test was also performed containing broth supplemented with only chloroform at the same dilution used as in our experiment. The solvent used for evaluation of compounds exhibited no antimicrobial activity. This property represented a practical advantage for the antimicrobial evaluation of these water insoluble compounds. Further dilution was prepared at the required quantities of 100, 50, 25, 12.5, 6.25 and 3.125 μg /mL concentration. The MIC values were obtained from the lowest concentration of the test compound where the tubes remain clear, indicating that the bacterial growth was completely inhibited at this concentration. The Diameter of zone of inhibition is expressed in mm and, MIC values in μg/mL. The results are shown in Table 1

In vitro antibacterial Assay
The cultures obtained in Muller-Hinton broth for all the bacteria after 24 hr of incubation at 37 0 C. Testing was carried out on Muller-Hinton broth at pH 7.4 using two fold serial dilution techniques. The final inoculum size was 106 CFU/mL. A set of tubes containing only inoculated broth was kept as control. After incubation for 24h at 37 0 C, the last tube with no growth of microorganism was recorded to represent MIC expressed in μg/mL. Every experiment in the antibacterial assay was replicated twice in order to define the MIC values. Comparison of antibacterial activity of 4(a-f) and 5(a-i) with that of antibacterial drugs, Gentamicin and Ampicillin showed that compounds 4b and 5d had better activity while compounds 4c and 5i exhibited milder activity and compound 4f showed poor activity against Staphylococcus aureus (ATCC-9144). Compound 5d also exhibited milder activity against Escherichia coli (MTCC-739). Compound 4b (MIC 3.12μg/mL) and 5d (MIC 3.12μg/mL) had shown promising antibacterial profiles on comparison with antibacterial drugs, Gentamicin (MIC 6.25μg/mL) and Ampicillin (MIC 6.25μg/mL), against Staphylococcus aureus (ATCC-9144) ( Table 1) as exhibited in Fig  1. Compounds 4(a-f) and 5(a-i) were also screened against Bacillus subtilis (ATCC-6633), Pseudomonas aeruginosa (ATCC-25615), Klebsiella pneumoniae (MTCC-2405), and Escherichia coli (MTCC-739) but did not exhibit significant antibacterial activity except 5d, which exhibited milder activity against the mentioned strains.

In vitro antifungal Assay
The cultures were obtained in sabouraud dextrose broth after incubation for 24 hr at 35 0 C. Testing was performed in sabouraud dextrose broth at pH 7.4 using two fold serial dilution techniques. The final inoculum size was 105 CFU/mL. A set of tubes containing only inoculated broth was kept as control. After incubation for 48hr at 35 0 C, the last tube with no growth of microorganism was recorded to represent MIC expressed in μg/mL. Every experiment in the antifungal assay was replicated twice in order to define the MIC values. Comparison of antifungal activity of compounds 4(a-f) and 5(a-i) with that of antifungal drug, Fluconazole, showed that compound 4b (MIC 3.12μg /mL)and 5d (MIC 3.12μg/mL) had better antifungal activity against Candida albicans (ATCC-24433). 4c, 5c, 5i and 5a exhibited milder antifungal activity against Candida albicans (ATCC-24433) and Aspergillus niger (MTCC-1344) respectively. Compound 4b and 5d (MIC 3.12μg/mL) had shown promising antifungal profiles against Candida albicans (ATCC-24433) as exhibited in Fig 2. Compounds 4(a-f) and 5(a-i) were also screened against Aspergillus niger (MTCC-872), Aspergillus fumigatus (MTCC-343) and Penicillium chrysogenum (MTCC-2725) but did not exhibit significant antifungal activity except 5a, which showed some activity.
The compounds tested, exhibited specific antimicrobial activity against different bacterial and fungal strains with MIC values in a range of 3.12-100 μg/mL. Many of these compounds showed significant activity comparable to the standard drugs at the tested concentrations. The attachment of N-benzoxazine group to 1(a-c) leading to 4(a-c), improved the antimicrobial activity, since 4b and 4c possess superior activity than other derivatives 4(d-f) of quinazoline -4carboxamide. The electronic property of para substituent of 2-phenyl ring of quinazoline seems to have slight effect on the antimicrobial activity. Both electron withdrawing (Cl) and electron donating (CH3) groups afforded good antimicrobial activity. The result suggests that the volume of the substituents may play an important role for the activity as compounds with N-benzoxazine structural motif have a good activity than the compounds with bulky N-naphthoxazine group. The better activity of 5d can be explained on the basis, that the presence of two quinazoline pharmacophores in a molecule reinforces its antimicrobial action. Consistent with these results, compounds 4b and 5d were found to be most potent among the tested compounds and exhibited better antimicrobial activity than the clinically prevalent antimicrobial drugs N o v e m b e r 0 1 , 2 0 1 4 such as Gentamicin, Ampicillin and Fluconazole. Interestingly, all the target compounds were found to be devoid of antimicrobial activity against P.aeruginosa, K.pneumoniae, A.fumigatus and P.chrysogenum.
In general, compounds 4(b, c& e), 5(a, c, d, g &i), showed significant to moderate activity, whereas rest of the compounds are inactive against all tested bacterial as well as fungal strains.