INTRODUCTION:

2-Quinolones (carbostyrils or 1-aza coumarins) are isosteric with coumarins and isomeric to 4-quinolones could become the probable potential candidate for antibacterial activity¹. 2-Quinolone derivatives were found to be associated with various biological activities such as antitumor², antimalarial³, antiplatelet⁴, antiulcer⁵, antioxidant activity⁶ and antidepressant⁷. Many substituted quinolin-2-one derivatives have recently craned great interest in chemotherapy as antitumor drugs⁸. Chalcones show impressive physiological properties and some of them possess wide range of activities such as antibacterial⁹, antitubercular¹⁰, anticancer¹¹, antimalarial¹², anti-inflammatory¹³ and antiplatelet¹⁴. Due to the presence of reactive α, β carbonyl functional chalcones are the versatile reagents of prime importance in both synthetic and pharmaceutical field.

By considering the above facts and their increasing importance in pharmaceutical and biological field, it was considered of interest to synthesize some new chemical entities incorporating the two active pharmacophores in a single molecular frame work and to evaluate their biological activities. This inculcated the need to synthesize some novel quinolinyl chalcones. The synthesized compounds were screened for their antimicrobial and cytotoxicity activities.

MATERIALS AND METHODS:

All the chemicals were of analytical grade: substituted salicylaldehyde, ethylacetoacetate, absolute ethanol, Piperidine, Hydrazine hydrate and substituted benzaldehyde.

Melting points were determined by open capillary method and are uncorrected. The purity of the compounds was monitored by thin layer chromatography (TLC) using silica gel G plates. The spots were visualized under UV light and by the exposure to iodine vapours. The homogeneity of the compounds were checked on silica gel-G coated plate by using n-hexane: ethylacetate (7:3) as solvent. All IR spectra were recorded in Alpha Bruker using ATR method. ¹H NMR spectra were recorded on Bruker spectrophotometer (400 MHz) in DMSO-d₆ solvent using tetra methyl silane (TMS) as an internal standard. Mass spectra was recorded by LCMS method.

General Procedure:

Synthesis of 3-acetyl-1-amino-quinolin-2-one¹⁵

3-acetyl coumarin (0.01 mol) with excess hydrazine hydrate 99% (0.1 mol) in 25 ml ethanol was refluxed for 12 hours. It was then cooled and poured into crushed ice with stirring. The solid product formed was filtered and recrystallised from ethanol.

Synthesis of Substituted 1-amino-3-cinnamoyl-quinolin-2(1H)-one¹⁶ (AJC1-AJC12):

A mixture of 3-acetyl-1-amino-quinolin-2-one (0.01 mol) and different substituted benzaldehyde (0.01 mol) in 20 ml absolute ethanol was stirred together at room temperature for 24 hours in the presence of 40% KOH. The completion of the reaction was monitored by TLC. The reaction mixture was then poured into crushed ice and acidified with 2N HCl with stirring. The product obtained was filtered, washed with water and recrystallised from ethanol.

Antimicrobial Activity:

All the synthesized compounds were evaluated for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and antifungal activity against Candida albicans and Aspergillus niger using cup plate method. The synthesized test compounds were tested at a concentration of 100 µg/50µl and the standard compound i.e. Amoxicillin and Fluconazole were tested at 25 µg/50µl. Dimethyl formamide (DMF) was used as control. In this technique, melted agar inoculated with microorganisms is poured into petridishes. Wells are made in the agar plate and a specific volume of the antimicrobial substances are placed in them, plates were incubated at a temperature of 37⁰C for 24 hrs and 25⁰C for 48 hrs, in case antibacterial and antifungal activity. The antimicrobial substance diffuses through agar around its well and produces a clear zone of inhibition. The diameter of this zone (mm) gives an estimation of the degree of activity of the antimicrobial substance.

Cytotoxicity Activity:

All the test compounds were studied for short term in vitro cytotoxicity against Ehrlich Ascites Carcinoma cells (EAC) cells. The tumor cells aspirated from peritoneal cavity of tumor bearing mice was washed thrice with normal saline and checked for viability using Tryphan blue exclusion method. The cell suspension (1 million cells in 0.1 ml) was added to tubes containing various concentrations of the test compounds and volume was made upto 1 ml using phosphate buffered saline. Control tubes contained only cell suspension. The assay mixtures were incubated for 3 h, at 37^οC and then percent of dead cells were evaluated by tryphan blue exclusion method.

Spectral data:

1-amino-3-(3-nitrophenyl)acryloyl)quinolin-2(1H)-one (AJC1)

IR KBr (cm^-1): 1506(Ar C=C str), 829 (Ar C-H bend), 2950(C-H aliphatic str), 1701 (C=O str), 3398 (NH₂str), 1350 (Ar-NO₂ str).

¹H NMR (400 MHz, DMSO-d₆): δ 7.12-8.28 (m, 9H, Ar-H), 4.81 (d, 2H of CH=CH), 3.73(s, 2H, NH₂).

MS (M⁺): m/z 235.

1-amino-6-chloro-3-(3-p-tolylacryloyl)quinolin-2(1H)-one (AJC9)

IR KBr (cm^-1): 1502(Ar C=C str), 826 (Ar C-H bend), 2948(C-H aliphatic str), 1698 (C=O str), 3356 (NH₂str), 780 (C-Cl str), 1377 (C-H str Ar-CH₃).

¹H NMR (400 MHz, DMSO-d₆): δ 7.13-8.26 (m, 8H, Ar-H), 4.86 (d, 2H of CH=CH), 3.78(s, 2H, NH₂).

MS (M+1): m/z 339.

Table 1: Physicochemical data of the compounds (AJC1-AJC12);

Comp. code	R	R₁	Mol. Formula	Mol. wt	M.P ^oC	R_f Value	% Yield
AJC-1	H	3-NO₂	C₁₈H₁₃N₃O₄	335	186-188	0.68	82
AJC-2	H	3,4,5-OCH₃	C₂₁H₂₀N₂O₅	380	138-140	0.58	76
AJC-3	H	4-CH₃	C₁₉H₁₆N₂O₂	304	156-158	0.60	72
AJC-4	H	4-OH	C₁₈H₁₄N₂O₃	306	162-163	0.52	78
AJC-5	H	2-Cl	C₁₈H₁₃ClN₂O₂	324	146-148	0.56	80
AJC-6	H	2-NO₂	C₁₈H₁₃N₃O₄	335	174-176	0.78	75
AJC-7	6-NO₂	3-NO₂	C₁₈H₁₂N₄O₆	380	196-198	0.70	62
AJC-8	6-NO₂	3,4,5-OCH₃	C₂₁H₁₉N₃O₇	425	152-154	0.62	65
AJC-9	6-Cl	4-CH₃	C₁₉H₁₅ClN₂O₂	338	170-172	0.66	60
AJC-10	6-Cl	3,4,5-OCH₃	C₂₁H₁₉ClN₂O₅	414	146-148	0.60	70
AJC-11	6-Cl	4-OH	C₁₈H₁₃ClN₂O₃	340	176-178	0.56	64
AJC-12	6-Cl	2-NO₂	C₁₈H₁₂ClN₃O₄	369	190-192	0.82	69

Antimicrobial Activity of the synthesized compounds (AJC1-AJC-12) by cup plate method.

SL. No.	Compd	Diameter of zone of inhibition(mm)
SL. No.	Compd	*S.aureus*	*B.subtilis*	*E.coli*	*P.aureginosa*	*C.albicans*	*A.niger*
1	AJC-1	12	10	13	14	11	15
2	AJC-2	18	15	16	22	07	08
3	AJC-3	09	11	13	10	13	16
4	AJC-4	07	10	11	13	-	07
5	AJC-5	-	-	11	16	08	10
6	AJC-6	20	16	17	23	14	17
7	AJC-7	10	09	12	13	-	-
8	AJC-8	19	17	18	21	06	09
9	AJC-9	10	08	-	-	10	14
10	AJC-10	18	16	15	20	-	-
11	AJC-11	11	13	10	15	08	07
12	AJC-12	21	17	15	21	12	15
13	Amoxicillin	26	23	25	30	-	-
14	Fluconazole	-	-	-	-	23	26
15	Control	-	-	-	-	-	-

Table 3: Cytotoxicity activity of compounds (AJC-1 to AJC-8) by Trypan Blue exclusion method .

Compounds	No. of dead cells (%) at different concentrations(µg/ml)
Compounds	50	100	200	250
Control	-
AJC-1	13	25	45	68
AJC-2	12	27	36	48
AJC-3	18	33	49	65
AJC-4	10	24	35	50
AJC-5	20	38	51	70
AJC-6	14	30	42	54
AJC-7	10	22	31	45
AJC-8	28	42	56	74
5-Fluorouracil	36	51	88	96

DISCUSSION:

3-acetyl-1-amino-quinolin-2-one was synthesized from 3-acetyl coumarin in presence of hydrazine hydrate and ethanol. The proposed mechanism for this reaction is nucleophilic substitution through attacking the carbonyl carbon atom by the lone pair of electrons of nitrogen atom in hydrazine hydrate. This step is accompanied by ring opening and then recyclisation of the intermediate gives N-amino-2-quinolone compounds. A series of N-substituted Quinolinyl chalcones (AJC1-AJC12) were synthesized by condensing substituted 3-acetyl-1-amino-quinolin-2-one with various substituted benzaldehyde in presence of ethanolic KOH. The percentage yield of the synthesized

quinolinyl chalcones were obtained in the range of 60-82%. The physicochemical data is presented in Table 1.

Antimicrobial activity:

The in vitro antibacterial and antifungal activity of the synthesized compounds were determined by using cup-plate method¹⁷. The results of antibacterial and antifungal activity of newly synthesized compounds are reported against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and two fungi Candida albicans and Aspergillus niger. Compounds AJC2, AJC6, AJC8, AJC10 and AJC12 showed good antibacterial activity compared to the standard drug amoxicillin. Compounds AJC1, AJC3, AJC6, AJC9 and AJC12 showed moderate antifungal activity compared to the standard drug fluconazole. The results of the antimicrobial activity is presented in Table 2.

Cytotoxicity Activity:

The test compounds were subjected to in vitro cytotoxicity against Ehrlich Ascites Carcinoma (EAC) cells using Tryphan Blue exclusion method¹⁸. The damaged cells are stained blue by Tryphan blue stain and can be distinguished from viable cells. Compounds AJC₁, AJC₃, AJC₅ and AJC₈ induced the greatest effect on EAC cells with an activity more than 60% at a concentration of 250µg/ml. The results of the cytotoxicity activity is presented in Table 3.

CONCLUSION:

The above results proved that quinolinyl chalcones are found to be interesting lead molecules as antimicrobial and cytotoxicity agents. The study reports the successful synthesis of substituted quinolinyl chalcones with moderate yields and most of the synthesized compounds showed good antimicrobial and cytotoxicity activity.

ACKNOWLEDGEMENTS:

The authors are thankful to Nitte University for providing the necessary facilities to carry out this research. The authors are grateful to Sequent Research Ltd, Mangalore and Central Instrumentation Facility, MIT Manipal for providing spectroscopic data, Amala Cancer Research Centre, Thrissur.

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Received on 17.09.2013 Modified on 25.10.2013

Research J. Pharm. and Tech. 6(12): Dec. 2013; Page 1336-1339