Synthesis and Biological Evaluation of Some Novel Pyrazoline incorporated 2-Quinolones

 

Abhishek Kumar*, Jennifer Fernandes, Pankaj Kumar

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Nitte University, Paneer, Deralakatte-575018, Mangalore, Karnataka.

 

ABSTRACT:

A series of novel substituted 1-amino-3-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)quinolin-2(1H)-one (AJP1-AJP8) have been synthesized upon reaction with 1-amino-3-cinnamoyl-quinolin-2(1H)-one using hydrazine hydrate as cyclising agent in alcohol medium. 1-amino-3-cinnamoyl-quinolin-2(1H)-one were synthesized by condensing 3-acetyl-1-amino-quinolin-2-one with different substituted benzaldehyde in presence of ethanolic KOH. The structures of the final synthesized compounds were characterized by IR, ¹H NMR and mass spectra. The final synthesized compounds were screened for their antioxidant activity by DPPH radical scavenging method and in vitro cytotoxic activity against Ehrlich Ascites Carcinoma cells (EAC) by Trypan blue exclusion method.

 

 

 

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 antitumor, anti-inflammatory, antiplatelet, antitubercular, antioxidant and antidepressant activity. Many substituted quinolin-2-one derivatives have recently shown great interest in chemotherapy as antitumor drugs². Pyrazolines are dihydro derivatives of pyrazoles and are well known five membered nitrogen containing compounds. Formation of pyrazolines have been reported by the action of nucleophile like hydrazine hydrate or phenyl hydrazine using alcohol as solvent.

 

Pyrazolines are known to exhibit different biological and pharmacological activities like antimicrobial³, antitubercular, anti-inflammatory and anti-depressant⁴. Some other activities are also exhibited by them such as anticonvulsant, antitumor, analgesic and antiandrogenic. The incorporation of two moieties increases the biological activity of both and thus it was of value to synthesize some new heterocyclic derivatives having two moiety in the same molecule. Hence an attempt was made towards the incorporation of pyrazolines with 2-quinolone moiety and to probe how this combination could influence the biological activity. All the synthesized compounds were evaluated for their antioxidant and cytotoxic activity and compared with standard drugs.

 

MATERIALS AND METHODS:

All the chemicals were of analytical grade: Substituted salicylaldehyde, Ethylacetoacetate, Absolute ethanol, Piperidine, Glacial acetic acid, 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 vapors. The homogeneity of the compounds were checked on silica gel-G coated plate by using n-Hexane: Ethylacetate (7:3) as solvent. 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 Substituted 1-amino-3-cinnamoyl-quinolin-2(1H)-one⁵

A mixture of substituted 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 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.

 

Synthesis of Substituted Pyrazolines⁶ (AJP1-AJP8) :

A mixture of substituted 1-amino-3-cinnamoyl-quinolin-2(1H)-one (0.1 mol) and hydrazine hydrate (0.01 mol) in 25 ml ethyl alcohol containing 1-2 drops of glacial acetic acid was refluxed for 4-5 hours. The reaction mixture was monitored by TLC. It was then cooled and added to ice cold water. The precipitated solid obtained was filtered, washed with water and recrystallised from ethanol.

 

Figure 1: Reaction scheme for Pyrazoline derivatives

 

 

Spectral data:

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

IR (cm^-1):

1519 (Ar C=C str), 2948 (C-H aliphatic str), 1690 (C=O str), 3397 (NH₂ str), 1341 (Ar-NO₂ str).

 

¹H NMR (400 MHz, DMSO-d₆):

δ 7.46-8.96 (m, 9H, Ar-H), 4.81 (d, 2H of CH=CH), 3.35 (s, 2H, NH₂).

MS (M⁺): m/z 335.

 

1-amino-3-(5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)quinolin-2(1H)-one (AJP1) IR (cm^-1):

1502 (Ar C=C str), 854 (Ar C-H bend), 2961 (C-H aliphatic str), 1727 (C=O str), 1622 (C=N str), 3422 (NH₂ str), 1428 (Ar-NO₂ str), 1272 (N-H str), 960 (N-N str).

 

¹H NMR (400 MHz, DMSO-d₆):

δ 7.04-8.14 (m, 9H, Ar-H), 4.30 (s, 2H, NH₂), 3.38 (d, 2H, CH₂), 9.60 (s, IH, NH).  

 

Mass (m/z):

349 (M⁺)

 

1-amino-6-nitro-3-(5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)quinolin-2(1H)-one (AJP7) IR (cm^-1):

1504 (Ar C=C str), 853 (Ar C-H bend), 2902 (C-H aliphatic str), 3396 (NH₂ str), 1427 (Ar-NO₂ str), 1273 (N-H str), 960 (N-N str).

 

¹H NMR (400 MHz, DMSO-d₆):

δ 7.60-8.34 (m, 8H, Ar-H), 3.92(s, 2H, NH₂), 2.50 (d, 2H, CH₂), 7.62 (s, IH, NH).  

Mass (m/z): 394 (M⁺) 

 

Antioxidant activity:

Antioxidant activity is a prerequisite for performing many related biological activities, including anticancer, antiallergic, anti inflammatory, antidiabetic etc. The antioxidant activity of the synthesised test compounds was done using DPPH (1, 1-diphenyl-2-picryl hydrazyl) radical scavenging method⁷. The sample size that can lower the initial absorbance of DPPH solution by 50% has been chosen as the endpoint for measuring the antioxidant activity. The assay was carried out in a 96 well microtitre plate. To 100µl of methanol solution, 100µl of each of the test sample or the standard ascorbic acid solution was added separately in wells of the microtitre plate in triplicate. Control was prepared by adding 100µl methanol in 100µl DPPH solution. The plates were incubated at 37˚C for 20 minutes and the absorbance of each solution was measured at 540nm using ELISA reader against the corresponding test and standard blanks and the remaining DPPH was calculated.IC₅₀ is the concentration of the sample required to scavenge 50 % of DPPH free radicals.

                                      Control – Test

% inhibition =                                            ×100    

                                      Control

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 Trypan blue exclusion method⁸. 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 trypan blue exclusion method.

 

 

 

RESULTS AND DISCUSSION:

Table 1: Physicochemical data of Substituted Pyrazolines

Comp. code	R	R1	Mol. formula	Mol. wt	m.p oC	Rf Value	% Yield
AJP1	H	3-NO2	C18H15N5O3	349	212-214	0.68	77
AJP2	H	3,4,5-OCH3	C21H22N4O4	394	148-150	0.52	72
AJP3	H	4-CH3	C19H18N4O	318	170-172	0.58	78
AJP4	H	4-OH	C18H16N4O2	320	182-184	0.64	80
AJP5	H	2-Cl	C18H15ClN4O	338	156-158	0.56	74
AJP6	H	2-NO2	C18H15N5O3	349	192-194	0.72	77
AJP7	6-NO2	3-NO2	C18H14N6O5	394	224-226	0.6	54
AJP8	6-NO2	3,4,5-OCH3	C21H21N5O6	439	160-162	0.56	50

 

Table 2: Evaluation of antioxidant activity of Substituted Pyrazolines (AJP1-AJP8) by DPPH radical scavenging.

Conc. µg/ml	% DPPH RADICAL SCAVENGING
	AJP1	AJP2	AJP3	AJP4	AJP5	AJP6	AJP7	AJP8
3.9	25.9	26.8	12.28	42.3	2.56	23.58	30.28	23.41
7.8	28.52	27.61	36.32	53.83	3.26	23.8	38.23	26.58
15.6	30.54	27.5	37.43	76.93	15.45	29.34	49.26	30.55
31.25	32.88	30	35.47	93.32	17.81	37.19	87.62	39.14
62.5	40.3	35.2	30.91	90.26	18.29	50.89	88.58	50.12
125	52.46	43.5	29.64	88.56	20.73	70.22	87.23	60.85
250	63.43	47.29	34.86	87.19	20.88	84.52	65.18	72.82
500	65.64	71.14	20.32	85.81	22.59	86.57	85.35	79.12
IC50	104	255.2	-	213	-	40.6	60.8	49.85

 

 

 

Figure 2: Antioxidant activity of Substituted Pyrazolines (AJP1-AJP8)

Table 3: Cytotoxic activity of Substituted Pyrazolines (AJP1-AJP8) by Trypan Blue exclusion method

Compounds	No. of dead cells (%) at different concentrations (µg/ml)
	50	100	200	250
Control	-
AJP1	22	35	57	68
AJP2	25	30	44	51
AJP3	23	32	42	53
AJP4	20	31	40	49
AJP5	20	36	52	62
AJP6	24	35	49	65
AJP7	28	37	55	69
AJP8	23	35	46	68
5-Fluorouracil	36	51	88	96

 

Antioxidant activity:

The antioxidant activity of the synthesized test compounds was done using DPPH (1, 1-diphenyl-2-picryl hydrazyl) radical scavenging method. Compounds such as, AJP6, AJP7 and AJP8 showed inhibitory concentration for 50% inhibition below 100 µg/ml when compared to that of Ascorbic acid at IC₅₀ value of 3.69 µg/ml. The presence of 2-quinolone moiety with electron withdrawing group like nitro resulted in increased antioxidant activity.

 

Cytotoxicity Activity:

The test compounds were subjected to in vitro cytotoxicity against Ehrlich Ascites Carcinoma (EAC) cells using Trypan Blue exclusion method. The damaged cells are stained blue by Trypan blue stain and can be distinguished from viable cells. Compounds such as, AJP1, AJP5, AJP6, AJP7 and AJP8 induced the greatest effect on EAC cells with an activity more than 60% at a concentration of 250µg/ml. The presence of 2-quinolone moiety with nitro, chloro and methoxy resulted in increased cytotoxic activity.

 

CONCLUSION:

The study reports the successful synthesis of substituted pyrazoline incorporated 2-quinolones from quinolinyl chalcones with moderate yields and most of the synthesized compounds showed good antioxidant and cytotoxic 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 spectral data and Amala Cancer Research Institute, Thrissur for cytotoxic studies.

 

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Accepted on 06.11.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(12):2257-2260.