Synthesis and in vitro antibacterial and antifungal evaluation of quinoline analogue azetidin and thiazolidin derivatives

A library of quinoline analog two novel series of azetidin (SH 1-5 ) and thiazolidin (SH a-e ) derivatives were designed and synthesized with simple and eco-friendly methodologies. The structures of the compounds were elucidated with the aid of elemental analysis, IR, 1 H-NMR and mass spectral data. These novel synthesized compounds were evaluated for antibacterial activity against two gram-positive bacteria ( Staphylococcus aureus , Staphylococcus pyogenus ) and two gram-negative bacteria ( Pseudomonas aeruginosa , Escherichia coli ). The title compounds were also studied for their antifungal activity with Candida albicans , Aspergillus niger , Aspergillus clavatus using the broth dilution technique. Most of the compounds were the best bio-active desired antibacterial analog with less MIC value against different tested strains.


INTRODUCTION
Quinoline derivatives in general are known to have a broad range of applications in medicinal, bio-organic, industrial as well as in the fields of synthetic organic chemistry.Their derivatives have been found to possess various biological activities like anti-malarial, antibacterial, anti-fungal, anti-asthmatic, antihypertensive, anti-inflammatory and anti-platelet activity [1].They also exhibit anti-tubercular and immune depressant activities [2].Few promising compounds with the quinoline ring system like, pamaquine (1), chloroquine (2), tafenoquine (3), bulaquine (4), quinine (5) and mefloquine (6) as an antimalarial agent and amodiaquine (7) as an antimalarial as well as anti-inflammatory agent (Figure 1) [3][4][5].Quinoline or 1-aza-napthalene or benzo pyridine is one of the most nitrogen containing heterocyclic aromatic compound having a variety of applications in medicinal, bioorganic, industrial as well as in the fields of synthetic organic chemistry, becomes reason to pick out quinoline as N-heterocyclic compound for our study (Figure 2).Moreover, in recent years there are greater societal expectations that chemists should produce greener and more sustainable chemical processes.So we have tried to follow green methods for the synthesis, as far as possible.During our literature for the synthesis of quinoline derivatives, Nafees Ahmed et al. studied structural activity relationship of quinoline-2,4-diols for anti-HIV activity and they also conclude that the activity is due to unsubstituted ring B and two free -OH groups [6].Therefore, we found quinoline-2,4-diol having hydroxyl group at 2 nd and 4 th positions will became quite interesting for the study.We also tried to incorporate the azetidin-2-one moiety and thiazolidin-4-one moiety in quinoline-2,4-diol scaffold to enhance its activity.
Besides the above applications, thiazolidinones moiety is also associated with a broad spectrum of biological activities including antibacterial [23,24], antifungal [25], antiinflammatory [26,28], hypnotic, anticonvulsant, antitubercular [29], antiviral [30,31], antihistamines, anthelmintic, cardiovascular and anticancer [32].Quinolines could be synthesized using various methodologies and recently we have reported one review article on it [33].Due to the environmental pollution, we have tried to carry out all the transformations using green chemistry principle.We have used microwave irradiation for this multi-step synthesis.In view of the need to discover potent novel antimicrobial agents, we have synthesized novel quinoline 2,4-diol analogue 2-azitidinone/ thiazolidin-4 one derivatives (Figure 5) and evaluated their antimicrobial and antifungal activity against different strains of bacteria and fungal using the broth micro dilution method and compared with some reference drugs.

MATERIALS AND METHODS
All Starting materials and other reagents were purchased from commercial suppliers and were used without any further purification unless otherwise indicated.The reactions were assayed by thin layer chromatography (TLC) and terminated as judged by the consumption of starting material.Analytical thin-layer chromatography (TLC) was performed on silica gel G 60 F254 (Merck) plates and eluted with the appropriate solvent ratios (v/v).The melting points were recorded in optimelt automated melting point system and were uncorrected.IR spectra were recorded on a Perkin-Elmer 377 spectrophotometer, 1 H NMR spectra was measured in Bruker AV 400 MHz using CDCl3 as a solvent and TMS as an internal standard.Mass spectra was recorded on Advion Expression CMS, USA, using Methanol: Water: Formic acid (80: 20: 0.1) as mobile phase.Elemental analysis was performed on the Vario MICRO cube, elementary CHN analyzer serial no.: 15084053.Here we have used the CEM Discover microwave system for synthesis.Its model no.: 908010 and made CEM Matthews.Inc, USA.

1. General procedure for the synthesis of 4-Hydroxyquinolin-2(1H)-one (S A ).
Aniline (7 mL, 5 mmol) and malonic acid (5.2 g, 5 mmol) was thoroughly mixed with 20 g poly phosphoric acid (PPA) {P 2 O 5 (287.9g) was added to 85 % phosphoric acid (200 g, 118.4 mL) with stirring and microwave heating.The mixture was then heated for next 15 min.} in 250 mL flask and heated with stirring in a microwave reactor (CEM-S-discover) at 400 W during 2×20 min with 5 min interval.The temperature reached 210 °C.Then the mixture was poured into crushed ice and a cream colored solid was filtered.The crude product was purified by extraction with EtOH and a white crystalline analytical sample was obtained [21].

General Procedure for the Synthesis of 4-Hydroxy-3-formyl-quinolin-2(1H)-one (S B )
Sodium hydroxide (80 ml of 15 %) was charged with 0.0124 mole of 4hydroxyquinolin-2(1H)-one and was cooled to 4 ºC and stirred.Subsequently the temperature inside the flask was maintained at 80 ºC on a water bath.At 80 ºC, 40 mL (0.5mole) of chloroform was introduced in three portions at intervals of fifteen minutes down the condenser on hot water bath.Stirring was continued for 12 hours, and then the reactionmixture was cooled to RT.The orange colored liquid was acidified with dilute sulfuric acid.It was extracted with ethyl acetate and dried with anhydrous sodium sulfate.Ethyl acetate was removed under vacuum and the solid obtained was purified by column chromatography (SiO 2 ) in hexane, ethyl acetate (7:3) mixture.

3. General Procedure for the Synthesis of 4-Hydroxy-3-formylideneamino-quinolin-2(1H)-one (S 1 -S 5 )
To a solution quinoline aldehyde (1 mmol) in dichloromethane and various aromatic anilines (1 mmol) added into the reaction mixture.It was stirred for 8 hours at room temperature.The reaction mixture was poured into ice-water to afforded crude products which was purified by either crystallization in ethanol or purified by column chromatography using pet ether and ethyl acetate (9:1) as eluent.They are conformed by spectral as well as analytical analysis.

4. General Procedure for the Synthesis of 3-chloro-4-(2,4-dihydroxyquinolin-3-yl)-1azetidin-2-one (SH 1 -SH 5 )
To the solution of Schiff base (1 mmol) in dimethylformamide (DMF), triethylamine (TEA) (1.2 mmol) and chloroacetyl chloride (1 mmol) were added, the reaction mixture was stirred properly and then place it at 300 W under microwave irradiation.Progress of the reaction was checked by TLC (Ethyl acetate/Toluene, 2:1).After completion of the reaction mixture was poured into ice-water.The crude product was obtained by filtration and crystallized in methanol.They are confirmed by spectral as well as analytical analysis.
Here, Gentamycin, Ampicillin and Chloramphenicol were used as a standard control drug for antibacterial activity, whereas Nystatin and Greseofulvin were used as a standard control drug for antifungal activity.To determine the minimum inhibitory concentration, a stock solution of the final synthesized compounds (2000 μg/mL) was prepared in dimethyl sulfoxide and then test compounds were incorporated in a specified quantity of molten sterile agar, i.e., nutrient agar and dextrose agar for antibacterial and for antifungal screening respectively.

1. 1. Determination of minimal inhibitory concentration (MIC)
The sterile test tubes containing 1ml of sterile media were added to 1 ml of different serially diluted test samples.To these tubes, 0.1 ml of suspension of respective microorganism was added in normal saline and incubated at 37 ±2 ºC for 24 hr.After 24 hr a loop full of samples was streaked in zigzag fashion over the agar medium in a petri plate from the culture and this was incubated at 37 ±2 ºC for 24 hours.Then the lowest concentration of the sample that inhibited the microbial growth in the petri dish was determined and this is considered as MIC.This procedure was performed to confirm the MIC.The results are summarized in Table 1.The inoculums were prepared by taking a loopful of stock culture to about 100 mL of nutrient broth, in 250 mL clean and sterilized conical flasks.The flasks were incubated at 27 °C for 24 hr before use.The plates were kept undisturbed for at least two hours at room temperature toallow diffusion of the solution properly, into potato-dextrose-agar medium.Then theplates were incubated at 25 °C for 48 hr.The highest dilution showing at least 99 % inhibition zone is taken as MIC.The result of this is much affected by the size of the Inoculum.The experiments were performed in triplicate in order to minimize the errors.The results of antifungal activity are given in the Table 2.

1. Chemistry
The title compounds, quinoline analoge azetidin and thiazolidin derivatives (Table 1) were synthesized via four step reactions starting from the 4-Hydroxyquinolin-2(1H)-one (S A ). Here, 4-Hydroxyquinolin-2(1H)-one firstly undergoes Reimer-Tiemann formaylation reaction in the presence of sodium hydroxide and chloroform to afforded 4-Hydroxy-3 formylquinolin-2(1H)-one (S B ) in good yield.This formaylated compound condensed with various aromatic amines in the presence of acetic acid to afforded various schiff-base in excellent yields (S 1-5 ).(Scheme1).Finally these Schiff-base were incorporated in the synthesis of azetidin (SH 1-5 ) and thiazolidin (SH a-e ) derivatives by using chloroacetyl chloride and thioglycolic acid respectively.The structures of products (SH 1-5 ) and (SH a-e ) were confirmed by different spectroscopic techniques like IR, 1 H NMR, Mass and elemental analysis.Both an electron withdrawing and electron donating substituents on the aromatic ring afforded excellent yield of the products.Both the azetidin (SH 1-5 ) and thiazolidin (SH a-e ) derivatives were prepared under microwave irradiation, which facile the conversion of the reaction within short period of time compared to conventional procedure.

2. Antibacterial activity
The antibacterial activity of the synthesized compounds SH 1-5 and SH a-e was determined in-vitro using MIC (Broth Dilution Method) against four pathogenic micro-organisms viz.E. coli, P. aeruginosa (Gm -ve) and S. aureus, S. pyogenus (Gm +ve) at various concentrations.Table 2 shows that all the newly synthesized quinoline scaffolds were found to exhibit good to moderate activity against the specific microbial strain, among which, SH 4 and SH c exhibited high potent activity against E. coli at MIC of 62.5 µg/ml.SH d and SH 3 shows activity against E. Coli equivalent to Ampicillin and Chloramphenicol respectively.SH 2 , shows equipotent activity against P. aeruginosa while SH 1 , SH 5 , SH b and SH e showed activity against P. Aeruginosa at MIC of 100 µg/mL, which is equivalent to standard drug ampicillin.
All synthesized compounds showed good activity against S. aureus, however SH 4 and SH d exhibited best potent activity against S. aureus.SH 2 shows activity against S. pyogenus at MIC of 50 µg/mL which is equivalent to standard drug Chloramphenicol.While SH SH a shows potent activity against S. Pyogenus at MIC of 100 µg/mL, which is equivalent to standard drug ampicillin.All the remaining final quninoline derivatives were found to demonstrate good-to-poor activity profiles at minimum inhibitory concentration levels.

3. Antifungal activity
The antifungal activity of the synthesized compounds SH 1-5 and SH a-e was determined in-vitro using an Agar plate method against three strains viz.C. albicans, A.niger and A.clavatus at different concentration ranging between 100 µg/ml to 1250 µg/ml.Out of the tested compounds SH 4 exhibited a significant activity against C.albicans at concentration of -209-250 µg/ml reference standard Griseofulvin at concentration 500 µg/mL.SH 1 , SH 5 , SH d and SH e shows equipotent antifungal activity at MIC 500 µg/ml.

CONCLUSION
In this article, we have elaborated the initial efforts made toward the sighting of novel, potentially active quinoline analog two novel series of azetidin (SH 1-5 ) and thiazolidin (SH a-e ) derivatives which were synthesized by simple and efficient method.From the bioassay it is clear that the introduction of appropriate halo, nitro and the methoxy substituent on the phenyl ring would lead to the more active antimicrobial derivatives.Compounds were screened against wide range of pathogenic bacteria and fungal strains shows better activity.Overall, from the bioassay results, we just conclude that findings of the present study will have a good impact on medicinal chemists to synthesize similar analogous which will show enhanced bioactivity.

Figure 1 .
Figure 1.Few promising compounds with quinoline ring system.

Table 1 .
Result of antibacterial by MIC method of the synthesized compounds.

Table 2 .
Result of antifungal activity for the synthesized compounds.