INTRODUCTION:

Chalcones represent an important class of natural as well as synthetic products and some of them are having wide range of pharmaceutical activity such as antibacterial^1,2, antitumour^1,21, anticancer^1,3,5, antitubercular², antiinflammatory¹, antioxidant¹, antimalarial¹, antileishmanial¹ etc. The presence of unsaturated ketogroup in the chalcones is found to be amenable for their biological activity⁴.

From the previous studies it was found that N containing heterocyclic compounds from chalcones possesses wide variety of activities such as potential activities like chalcone with efficiency⁶. Pyrazolines are the five member heterocyclic compounds containing two nitrogen atoms⁷.

As all heterocycles are well known for their biological utilizations, pyrazolines also shows variety of biological applications⁸ same as of chalcones. Taking in to consideration the broad spectrum of utilities one cannot ignore the existence of pyrazolines in the field of synthesis⁹.

Therefore in the present work, chalcones have been prepared by condensing various ketones¹⁰, the synthesis of some novel pyrazolines was also carried out for some beneficial use. Led by these retainers, it appeared the interest in synthesis of novel pyrazoline derivatives and screened for their antimicrobial activities.

MATERIAL AND METHODS:

The chalcones have been prepared according to the standard protocol of claisen-schimidt¹¹ condensation by condensing various ketones. The synthesis of novel pyrazolines using chalcones under basic condition in presence of ethanol as a solvent media¹². The reaction is carried out in refluxing condition.

Synthesis of substituted 3-Cinnamoyl-4-Hydroxy-6- Methyl-2-Pyrones (MBCI-MBCV):

A solution of (10 mmol) of dehydroacetic acid of the aromatic aldehyde of (10mmol) and 8-10 drop of piperedine as a catalyst was dissolved in 30 ml of ethanol solvent, the reaction mixture was refluxed for a reaction time 12-15 hrs. After reaction time check the TLC then filter the reaction mixture dried and recrystallized with suitable solvent as shown in (Fig1 Scheme I).

IR spectra were recorded in KBr spectrometer. ¹HNMR spectra were developed on Advance 300 MHz spectrometer with chloroform as a solvent and TMS internal standards chemical shift, the chemical shift values are expressed in part per million (ppm) downfield from the internal standards and signals are quoted as s (singlet), d (doublet), t (triplet) and m (multiplet) purity of the compound is checked by TLC plate using n-hexane and ethyl acetate as an eluent in the ratio of (7:3 v/v).

Spectroscopic data of synthesized chalcone (MBCI-MBCV):

MBCI: 1-(4-hydroxy-6-methyl-2-oxa-2H-Pyran-3-yl)-3-(3-Nitrophenyl)-2-Propenone

IR (cm^-1, KBr): 3114 (OH), 3062 (CH aromatic), 2900 (CH₃), 1729 (Lactone C=O), 1648 (C=O), 1614 (CH=CH).

¹HNMR (CDCl_3, δ/ ppm): 2.30 (3H, s, CH₃), 6.0 (1H, s, C⁵DHA), 7.3 (1H, dd, -C=OCH), 8.2 (1H, dd, =CH-Ar), 6.5-8.4 (4H, m, Ar-H), 14.4 (1H, s, OH).

MBCII: 1-(4-hydroxy-6-methyl-2-oxa-2H-Pyran-3-yl)-3-(3-3,4,5-Trimethoxy)-2- Propenone

IR (cm^-1, KBr): 3121 (OH), 2954 (CH₃), 1726 (Lactone C=O), 1651 (C=O), 1598 (CH=CH)

¹HNMR (CDCl_3, δ/ ppm), 2.4 (3H, s, CH₃), 3.93 (9H, s, 3xOCH₃), 6.0 (1H, s, C⁵ DHA), 7.9 (1H, dd, -C=OCH), 8.2 (1H, dd, =CH-Ar) 6.6-7.2 (2H, m, Ar-H), 13.2 (1H, s, OH).

MBCIII: 1-(4-hydroxy-6-methyl-2-oxa-2H-Pyran-3-yl)-3-(3-methoxyphenyl)-2, Propenone

IR (cm^-1, KBr): 3117 (OH), 2969 (CH₃), 1722 (Lactone C=O), 1655 (C=O), 1597 (CH=CH), 1512 (CH=CH-Ar), 1487 (CH=CH).

¹HNMR (CDCl_3, δ/ ppm): 2.1 (3H, s, CH₃); 3.9 (3H, s, OCH₃), 6.0 (1H, s, C⁵ DHA), 7.9 (1H, dd, C=OCH), 8.2, (1H, dd, =CH-Ar), 6.2 -7.4 (4H, m, Ar-H), 14.6 (1H, s, OH).

MBCIV:1-(4-hydroxy-6-methyl-2-oxa-2H-Pyran-3-yl)-3-(3,4-Dimethoxyphenyl)-2-Propenone

IR (cm^-1, KBr): 3104 (OH), 2981 (CH₃), 1715 (Lactone C=O), 1658 (C=O), 1589 (CH=CH).

¹HNMR (CDCl_3, δ/ ppm): 2.4 (3H, s, CH₃), 3.9-4.0 (6H, s, 2xOCH3), 6.0 (1H, s, C⁵ DHA), 7.9 (1H, dd, C=OCH), 8.3 (1H, dd, =CH-Ar), 6.8-7.3 (3H, m, Ar-H), 16.4 (1H, s, OH).

MBCV: 1-(4-hydroxy-6-methyl-2-oxa-2H-Pyran-3-yl)-3-(2-Fluorophenyl)- 2-Propenone

IR (cm^-1, KBr): 3103 (OH), 2973 (CH₃), 1719 (Lactone C=O), 1646 (C=O), 1608 (CH=CH),

¹HNMR (CDCl_3, δ/ ppm): 2.3 (3H, s, CH₃), 6.0 (1H, s, C⁵ DHA), 7.8 (1H, dd, C=OCH), 8.4 (1H, dd, =CH-Ar), 7.1-8.2 (4H, m, Ar-H), 15.8 (1H, s, OH).

General procedure for synthesis of phenyl pyrazoline:

A mixture of chalcone(0.001 mol)and phenyl hydrazine hydrated (0.002 mol) in 10 ml ethanol was refluxed for 3 hours. After completing of reaction (monitored by TLC), the reaction mixture was distilled off to remove the excess solvent and then it was poured into crushed ice¹³as shown in (Fig1 Scheme II). The solid obtained was washed with water and recrystallized with the help of ethanol. The compounds were stored at room temperature in vials.

Spectroscopic data of synthesized pyrazoline derivatives (MBPI-MBPV)

MBPI: 3-(4-hydroxy-6-methyl-2oxa-2H pyran -3-yl) - 5- (3-Nitrophenyl) -1-phenyl -4, 5- dihydro-2-Pyrazoline

IR (KBr, cm^-1); 3327 (OH str.), 2970 (C-H str. Of –CH₃), 1722 (C=O Lactone), 1612 (C=N str. of pyrazoline ring), 1575, 1532, 1447 (C=C str. of aromatic ring), 1230 (C-N str. of pyrazoline ring), 751 (C-H bending)

¹HNMR (CDCl₃, δ/ ppm): 2.2 (3H, s, CH₃), 3.1 (1H, dd, -CH₂pyraz), 3.9 (1H, dd, -CH₂pyraz), 5.2 (1H, t, -CH pyraz), 6.0 (1H, C⁵ DHA), 6.1-8.2 (9H, m, Ar-H), 13.4 (1H, s, OH)

Mass (m/z): (M+1) 392

MBPII:3-(4-hydroxy-6-methyl-2-oxa-2H-pyran-3-yl)-5-(3,4,5-trimethoxyphenyl)-1-phenyl-4,5-dihydro-2-pyrazoline

IR (KBr, cm^-1): 3376 (OH str.), 2980 (C-H str. of CH₃), 1720 (C=O lactone), 1614 (C=N str. of pyrazoline ring), 1577, 1531, 1497 (C=C str. of aromatic ring), 1232 (C-N str. of pyrazoline ring), 750 (OH bending).

¹HNMR (CDCl₃, δ/ ppm): 2.2 (3H, s, CH₃), 3.8-3.9 (9H, s, 3xOCH₃), 3.2 (1H, dd, -CH₂pyraz), 3.6 (1H, dd, -CH₂pyrazoline), 5.6 (1H, t, -CH pyraz), 5.9 (1H, s, C⁵ DHA), 6.0-7.9 (7H, m, Ar-H), 13.5 (1H, s, OH)

Mass (m/z): (M+1) 437

MBPIII: 3-(4-hydroxy-6-methyl-2-oxa-2H-pyran-3-yl)-5-(3-Methoxyphenyl)--1-phenyl-4, 5-dihydro-2- pyrazoline

IR (KBr, cm^-1): 3330 (OH Str.), 2970 (C-H str. of CH₃), 1730 (C=O lactone), 1610 (C=N str. of pyrazoline ring), 1585, 1530, 1448 (C=C str. of aromatic ring), 1228 (C-N str. of pyrazoline ring), 749 (C- H bending).

¹HNMR (CDCl₃, δ/ ppm): 2.3 (3H, s, CH₃), 3.8 (3H, S, OCH3), 3.3 (1H, dd, CH₂pyraz), 3.8 (1H, dd, CH₂pyraz), 5.5 (1H, t, -CH pyraz),6.0 (1H, s, C⁵ DHA), 6.2-8.0 (9H, m, Ar-H), 14.4 (1H, s, OH)

Mass (m/z): (M+1) 374

MBPIV:3-(-hydroxy-6-methyl-2-oxa-2H-pyran-3-Yl)-5-(3,4,-dimethoxy phenyl)-1-phenyl-4, 5-dihydro-2-pyrazoline

IR (KBr, cm^-1): 3391 (OH str.), 2975(C-H str. of CH₃), 1725 (C=O lactone), 1608 (C=N str. of pyrazoline ring), 1595, 1528, 1485 (C=C str. of aromatic ring), 1230 (C-N str. of pyrazoline ring), 752 (C-H bending)

¹HNMR (CDCl₃, δ/ ppm): 2.5 (3H, s, CH₃), 3.8-3.9 (6H, s, 2xOCH3), 3.4 (1H, dd, CH₂

pyraz), 3.5 (1H, dd, CH₂pyraz), 5.7 (1H, t, CH pyraz), 6.0 (1H, s, C⁵ DHA), 6.2-8.2 (8H, m, Ar-H), 14.2 (1H, s, OH)

Mass (m/z): (M+1) 407

MBPV:3-(4-hydroxy-6-methyl-2.oxa-2H-pyran-3-yl)-5-(2-florophenyl)1-phenyl-4, 5-dihydro-2-pyrazoline.

IR (KBr, cm^-1): 3405 (OH str.), 2985 (C-H str. of CH₃), 1730 (C=O lactone), 1596 (C=N str. of pyrazoline ring), 1575, 1520, 491 (C=C str. of aromatics ring), 1228 (C-N str. of pyrazoline ring), 748 (CH bending)

¹HNMR (CDCl₃, δ/ ppm): 2.3 (3H, s, CH₃), 3.6 (1H, dd, CH₂, puraz), 3.9 (1H, dd, CH₂pyraz), 5.8 (1H, t, CH pyraz), 6.0 (H, s, C⁵DHA), 6.4-8.4 (9H, m, Ar-H), 14.4 (1H, s, OH)

Mass (m/z): (M+1) 365

Biological Activity:

Antibacterial activity:

The synthesized compounds were tested in in vitro for antimicrobial activity against bacterial species like S. aureus, E. coli, S. Typhi and fungi species like Fusariumoxysporum, Candida albicans and Aspergillusflavus at the concentrations 100μg/mL each, by agar plate diffusion method. The concentrations used for screening were confirmed after estimating the MICs of each compound. The solvent used for assay was dimethyl sulfoxide (DMSO) which further diluted with water. Nutrient agar and PDA (Potato Dextrose Agar) was used as the growth medium for the bacterial and fungal species respectively. DMSO was used as a control. The results were compared with standard drug penicillin for antimicrobial activity by measuring the zone of inhibition in mm at 100 μg/mL. Antimicrobial activity was measured as a function of diameter of zone of inhibition (mm)¹⁴.

Fig. 1 Schematic representation of synthesized schemes I and II

RESULTS AND DISCUSSIONS:

The chalcones of DHA were synthesized by claisenschmith condensation in good to excellent yield (scheme I) as shown in Table 1. The structures of all the compounds were established from IR and ¹HNMR spectral analysis is mentioned above. The IR spectrum of compound MBCI to MBCV shows a broad band for OH group at (3000-3125 cm^-1) sharp and strong bands were observed at 1700-1750 cm^-1for lactone carbonyl group. Another sharp band observed at 1598-1650 cm^-1due to the presence of carbonyl group and carbon-carbon band of α, β unsaturated chalcone system. The ¹HNMR spectra of MBCI to MBCV showed a characteristic singlet due to C⁵-H proton δ 5.9-6.0 ppm for lactone unit. We also noted that the olefinic protons of reactive α, β unsaturated keto function occur as doublet around 7.9-8.4 respectively and broad singlet around at δ13.5-16.5 OH group of Lactone unit¹⁵.

A series of some novel phenyl pyrazoline derivatives were synthesized by refluxing chalcone for the synthesis of phenyl pyrazoline have been investigated¹⁶. The presence of nitro methoxy, bromo, fluoro, chloro and methyl group in different position of benzene ring of the chalcones and the use of phenyl hydrazine hydrate resulted in synthesis of new phenyl pyrazoline derivatives with significantly high yield as mentioned in Table 2.

The structure of synthesized compound (MBP I-V) were confirmed on the basis of spectral analysis The IR spectrum of (MBP I–V) exhibited a band due to 2970-2980 (CH₃), 1720-1720 (lactone C=O), 1612-1614 (C=N), 3327-3376 (-OH), 1422 (CH₂ of pyrazoline ring). Further in their ¹HNMR (CHCl₃) spectra the appearance of a signal at δ 3.1-3.2 (1H, dd, H_Apyrazoline), δ 3.6-3.9 (1H, dd, H_B, pyrazoline), δ 5.2-5.6 (1H, t, H_X pyrazoline) δ 13.4-13.5 (1H , s, -OH) confirms the presence of the phenyl pyrazoline ring¹⁷.

All the synthesized compounds screened for antibacterial activity and it was found that there is significant effect on the inhibition zone of microbial growth. The control showed no activity against the strains of microorganisms used. Both the organism’s i.e bacteria and fungi showed maximum zone of inhibition that were 16 mm and 16 mm, respectively. Other strains were also tested against the standard drug^{18, 19,20}. The results are given in Table 3.

Table 1.Physical properties of chalcones

Compound	R’	Molecular formula	M. P (ºC)	Yield %
MBCI	3-NO₂	C₁₅H₁₁O₆N	190	70
MBCII	3,4,5-OCH₃	C₁₈H₁₈O₇	198	80
MBCIII	3- OCH₃	C₁₆H₁₄O₅	195	82
MBCIV	3, 4-OCH₃	C₁₇H₁₆O₆	176	80
MBCV	2-F	C₁₅H₁₁O₄F	160	84

Table 2. Physical properties of pyrazoline derivatives (MBPI-V)

Compound	R’	Molecular formula	M. P (ºC)	Yield %
MBPI	3-NO₂	C₂₁H₁₇O₅N₃	215	52
MBPII	3,4,5-OCH₃	C₂₄H₂₄O₆N₂	235	58
MBPIII	3- OCH₃	C₂₂H₁₇O₄N₂	211	57
MBPIV	3, 4-OCH₃	C₂₃H₂₂O₅N₂	200	56
MBPV	2-F	C₂₁H₁₇O₃N₂F	240	52

Table 3. Antimicrobial activity of pyrazoline derivatives (MBPI-MBPV)

Compound	Bacteria (Zone of Inhibition in mm)			Fungi (Zone of Inhibition in mm)
Compound	A	B	C	D	E	F
MBPI	15	16	12	8	10	13
MBPII	12	10	14	6	16	15
MBPIII	13	14	16	9	12	14
MBPIV	14	11	10	11	14	12
MBPV	11	10	15	9	11	11
Penicillin	11	9	9	9	12	9

CONCLUSION:

In conclusion, we have reported some novel pyrazoline compound shows considerable antimicrobial activity against tested bacteria and fungi species comparatively with standard used in study. In this study the synthesized pyrazoline derivatives will be having importance in the pharmacophoric possession and it’s may provide us the fruitful results in biological and medicinal purposes. So these compounds will be having importance in the medical and agriculture field for management of diseases and pest.

ACKNOWLEDGEMENT:

The authors are thankful to the Director IICT, Hyderabad and Dr. Makarand Kulkarni, Solapur University, Solapur for making availability of spectral analysis facilities for the research work.

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Received on 11.02.2017 Modified on 15.02.2017

Asian J. Research Chem. 2017; 10(2):149-153.

DOI: 10.5958/0974-4150.2017.00025.6