Synthesis of a new serie of quinoline-carboxamides based on methylated aminoesters: NMR characterization and antimicrobial activity

Ten new quinoline-carboxamides have been synthesized using the coupling reaction between 2-oxo1,2-dihydroquinoline-4-carboxylic acid as a substrate and five different amino ester at room temperature with basic media (triethylamine). The products were obtained with a good yield ranging from 60 to 80 % and were structurally characterized by H and C NMR spectroscopy and mass spectrometry. The antibacterial activities of the synthesized compounds have been evaluated against 9 strains of bacteria and compared to references (erythromycin, ofloxacin, ticarcillin, oxacillin, ampicillin, norfloxacin, ceftazidim, cefotaxim). The results showed that the majority of carboxamides-quinoline ester groups present a larger inhibition diameters than those of the antibiotics references. The highest antibacterial activity in vitro against the Enterococcus feacalis has been revealed for compound 1a (methyl 2-oxo-1.2-dihydroquinoline-4-yl-L-alaninate).


Introduction
Antibiotics based on heterocycles pharmacophore present a great interest and play a central role in the medicinal sector during this century, especially due to their potential effects against bacterial infections, however bacterial cells have developed some resistance and are able to defend themselves against the antibiotic. The increasing of this resistance phenomenon towards antibiotics has been described as a global public health menace 1 .
Quinolones is among the most synthesized antibiotics that have been widely used by scientists to synthesize new antibacterial agent. In 1962, the first quinolone directly derived from 7-chloroquinoline was discovered to be nalidixic acid ( Figure 1: I), which is indicated for the treatment of urinary tract infection 2 . Other derivatives, such as fluoroquinolones (norfloxacin, levofloxacin, ofloxacine, lomefloxacin, etc), were synthesized by grafting both fluoro group at the position 6 and piperazine at the position 7 of the quinolines substrate. Regarding the norfloxacin (Figure 1: II), it constitutes the second generation of fluoroquinolone presenting an increase in activity against Gram+ 3 . While in the case of 3 rd generation fluoroquinolone, such as, levofloxacin (Figure 1: III), a large spectrum activities was manifested against Gram + and Grambacteria 4 . Concerning the structure-activity of moxifloxacin fluoroquinolone drug of 4 th generation (Figure 1: IV), it was obtained by incorporating methoxyl group at position 8 and bicyclo-1,5-diamine at the position 7. The structural modification induced a change with improving the activity and conferring some efficiency treatment against pneumologic infections 5 .
The development of new synthetic heterocycles quinoline presents a major strategy and challenge for their synthesis and the discovery of new promising drug candidate able to present a potent pharmacological and therapeutic activities.
Amino acids are considered as major constituents of many drugs, such as β-lactams antibiotics 18 and glutamate antagonists 19 . Tyrosine, phenylalanine, and tryptophan are well known for their essential role in the living organism and present a potent and wide range of therapeutic activities 20 and also present antioxidant activity 21 . In order to study the biological and pharmacological activities of amino acids, some researchers have been carried out the modifications on amino acid groups by coupling than to other heterocyclic compounds, such as coumarine 22-23 . Shivaraj et al. 2013 24 have shown that a serie of quinoline-6-carboxamides based on primary amines present an antibacterial activity in vitro against Escherichia coli and Staphyllococcus aureus, converting them with glycosidic ring to surfactants 25 to ligand metal complexes 26 29 have reported that the serie of 2oxo-1,2-dihydroquinoline-4-carboxylate derivatives presents an interesting antioxidant activity. In our case study, we have tried to graft new quinoline heterocyclic compounds to a serie of amino esters groups in order to provide some interesting biological activities.
In this work, we report the synthesis of ten new quinoline-4-carboxamides compounds. Their structural characterizations by both Nuclear Magnetic Resonance 1 H and 13 C and mass-spectrometry, as well as, the evaluation of their antibacterial activities against 9 types of bacteria.

Synthesis of quinoline-carboxamides
Our study concerned the synthesis of two types of serie of quinoline-carboxamides derivatives, 2-oxo-1,2-dihyquinoline-4-carboxamides and 6-bromo-2oxo-1,2-dihyquinoline-4-carboxamides by reacting a quinolinic acid as a substrate with five types of various amino methyl esters groups (L-alanine-OMe, L-phenylalanine-OMe, L-phenylglycine-OMe, L-Serine-OMe and L-tryptophane-OMe) during 12 hours at room temperature with the presence of hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) as a coupling agent in basic medium using triethylamine (TEA) and dimethylformamide (DMF) as a suitable solvent for this type of reaction. The five amino acids (alanine, serine, phenylglycine, phenylalanine and tryptophan) were converted towards their methylated esters groups, The synthesized compounds purified on liquid chromatography column using silica gel as a stationary phase and were obtained with good yields ranging from 60 to 80%. Their chemical structures were elucidated by both techniques 1 H and 13 C NMR and mass-spectrometry.
In order to synthesize the quinoline-carboxamides, the first step requires the preparation of two types of quinoline substrates: 2-oxo-1,2-dihydroquinoline-4carboxylic acid 1 and 6-bromo-2-oxo-1,2dihydroquinoline-4-carboxylic acid 2, which are obtained by reacting the malonic acid on both isatin and its bromo-derivatives with the presence of sodium acetate. The reaction was performed under reflux of acetic acid during 24 hours 30 (Scheme 1).
Before the preparation step of quinolinecarboxamides, the five amino acids were converted to their methylated amino esters by the action of thionyl chloride (SOCl2) on amino acid in methanol under reflux 31 . In the second step, ten new quinolinecarboxamides products (1a1e and 2a2e) were obtained (Scheme 1) by coupling reaction between each substrate (1 and 2) with the five different types of methylated amino esters hydrochloric acid salt (L-alanine-OMe, L-phenylalanine-OMe, L-phenylglycine-OMe, L-serine-OMe and L-tryptophane-OMe). The differents synthesized products are illustrated in both Scheme 1 and Table 1.  Both compounds 1a and 2 showed an antibacterial activity against bacterium Escherichia coli and bacterium Enterococsus feacium, respectively, with the highest diameter of inhibition zones (d=14 mm). However, Listeria monocytogenes and Klebsiella pneumonia strains showed resistance for all the synthesized compounds, no interesting inhibition zones were observed ( Figure 2). In order to test the sensibility of bacterial strains, an antibiotic susceptibility test was performed on eight different types of antibiotics: erythromycin, ofloxacin, ticarcillin, oxacillin, ampicillin, norfloxacin, ceftazidim, cefotaxim. The results showed that all bacterial strains tested present some resistance to the antibiotics, mainly in the case of the bacteria: Acinetobacter baumannii, Escherichia coli and Listeria monocytogenes, as for the following four antibiotics: erythromycin, oxacillin, ticarcillin and ceftazidim. The manifested character was shown at certain bacterial strains without presenting any sensibility for all the strains tested or without matching with those of strains types. The sensibility was very marked for ofloxacin (fluoroquinolone antibiotic family) affecting the following strains Staphylococcus aureus, Klebsiella pneumonia, Haemophilus influenza, and Enterococcus feacalis, as well as, for norfloxacin against bacteria Klebsiella pneumonia, Enterococcus feacalis, Enterobacter sp and Salmonella sp. While in the case of ceftazidim antibiotic, only two strains present some sensitivity towards Klebsiella pneumonia and Salmonella sp, followed by ampicillin towards Salmonella sp. Finally, the most sensitive bacterial strains affected both Klebsiella pneumonia and Salmonella sp followed by Enterococcus feacalis.
The results obtained are encouraging, showed the importance of those quinoline-carboxamides towards bacterial strains, once compared to different antibiotics; and might explain the role of bacterial strains in the acquisition of resistance against antibiotic agents. The nature of amino acid groups could influence the chemical properties of quinolinederivatives, and then modify the action modes of bacterial activities (Table 2).  values of antibacterial activities of tested compounds were determined and illustrated in Table 3. The two marked compounds 1a and 2 showed the lowest antimicrobial MIC at 0.0775 mg/ml against Enterococcus feacalis and 0.155 mg/ml against Acinetobacter baumannii, respectively, and considered as a promising molecules (pharmacophore) for antimicrobial activities test.
The antimicrobial activities were manifested in the case of all compounds against Enterococcus feacalis, Salmonella sp, Staphylococus aureus and Acinetobacter baumannii, respectively, are stronger than those against other bacterial strains. The following compounds 1c, 1d, 2b and 2d presented less microbial activities against tested bacterial strains, while the products 1a, 1b, 2b, 4b and 6b showed moderate effect.

Conclusion
In this work a series of five new compounds of 2-oxo-1,2-dihyquinoline-6-carboxamides (1a, 1b, 1c, 1d and 1e), as well as, another series of 5-bromo-2-oxo- The component 1a presented the highest antibacterial activity against Acinetobacter baumannii strain with the MIC= 77.5 µg/ml, while the most synthesized compounds showed moderate antibacterial activity towards nine different bacteria strains.
Column liquid chromatography was performed on 60 Merck silica gel (230-400 mesh ASTM). Thin layer chromatography (TLC) was performed on Merck aluminum plates coated with 60 F254 Merck silica gel (thickness 0.2 mm), and the synthesized components were revealed by an ultra-violet lamp set at 254 nm. Melting points were determined by an Electrothermal IA 9000 Series digital fusiometer using capillary tubes.
NMR spectra were performed on Bruker DRX-300 AVANCE spectrometer at the "Cité Innovation" Sidi Mohamed Ben Abellah University of Fez . 1 H NMR spectra were recorded at 300 MHz, and 13 C NMR spectra were recorded at 75 MHz. Samples were dissolved in DMSO-d6 or/in CDCl3. The chemical shift of different peaks was expressed in ppm, and the coupling constants n J in Hz. For describing the multiplicity of signals, the following abbreviations have been used: s: singlet, d: doublet, dd: doublet doublet, ddd: doublet doublet doublet, m: multiplet, t: triplet, q: quadruplet.
The high resolution mass spectra (HRMS) were registered in the EI (70 eV) or FAB mode and were reported as m/z (% of relative intensity) at the mass spectrometry service of the University of Valencia, Spain.
A mixture of 5-bromo isatin (0.013 mol), malonic acid (0.016 mol) and sodium acetate (1.9 mmol) dissolved in acetic acid. Medium stirred and heated under reflux for 24 hours. The water was added to the mixture until formation of precipitate and then filtered.
General procedure for the protection of carboxylic acids groups derived from amino acids An amount of SOCl2 (2 mol) was added drop by drop to the methanol (MeOH) at 0 °C. After 15 min, the mixture was added to the amino acid (1 mol), stirred during 2 hours at room temperature and then heated under reflux for 2 hours. The excess of unreacted mixture of MeOH and SOCl2 was removed using rotary evaporator. The remaining residue has been solubilized in MeOH and then an amount of diethyl ether was added until precipitation, followed by filtration.

Disc diffusion method
The disc diffusion method 33 started by seeding the bacterial strains on the surface of Mueller Hinton agar. After 15 min the sterile wattman N°1 disc (diameter of 6mm) is placed on the surface each agar and impregnated 10 µl of compounds 1-2e (10 mg/1ml of DMSO). Then petri dishes were incubated at 37 °C during 24 hours. After incubation, the absence of bacterial growth expressing antimicrobial activity by the presence of a translucent halo around the disc including a diameter measured with a caliper in mm.

Minimum inhibitory concentration (MIC)
The minimum inhibitory concentrations of the synthesized compounds were determined and based on the literature data by the method of Bouhdid et al. 34 with some modification. The product was serially diluted in DMSO and the Brain Heart Infusion (BHI) nutrient agar has been sterilized. 140 µl of the sterilized medium was added to all microplate wells using the microdilution method, containing 20 µl DMSO and a series of test product dilutions ranging from 10mg/ml to 0.0025 mg/ml. Then, 20 μL of bacterial inoculum was added to each well. The 12 th well was considered as growth control. The microplate was incubated at 37 °C during 24 hours and 10µL of triphenyltetrazolium (TTC) chloride was added to each well as a growth indicator. After 2h in incubation at 37°C the MIC is the lowest concentration that does not cause any change in TCC staining and corresponds to the absence of bacterial growth.