After completion of the reaction, the solid products were collected by simple filtration and then recrystallized with ethylacetate to afford pure pyrazoles derivatives (4a-4i) as shown in Fig. 1. The physical data of substituted pyrazoles derivatives (4a-4i) was shown in Table 1. The structure of synthesized compounds was confirmed by 1HNMR, FTIR spectral analysis as well as comparison of their melting points with those of reported compounds. The optimization of reaction conditions has been presented in Table 2. The comparison of activity of different catalysts with respect to time and yield of reaction as shown in Table 3. It was found that Guar-gum gives the best catalytic activity in terms of products yield, reaction conditions and reaction time as compared to other catalyst in literature viz. I2/K2CO3, ZnCl2, Al2O3, NaH, Chloranil and Glycine. The catalyst used in present study is nature derived, easily available and inexpensive which makes this procedure eco-friendly. The plausible mechanism for the formation of substituted pyrazoles derivatives (4a-4i) is shown in Scheme 2. First, nitrile ion is activated by removal of acidic hydrogen from malononitrile catalyzed by acidic catalyst. Finally, the arylidene nitrile intermediate formed through the Knoevenagel condensation reaction between the intermediate nitrile anion and substituted aldehydes. On the other hand, the reaction of 2,4-dinitrophenyl hydrazine with arylidene nitrile afforded an intramolecular cyclized product.
Table 5
Antifungal activity of substituted pyrazoles (4a-4i)
Products
|
Growth inhibition (%)
|
Fungi
|
Rhizoctonia solani
|
Aspergillus niger
|
250 (µg/mL)
|
500 (µg/mL)
|
1000 (µg/mL)
|
2000 (µg/mL)
|
250 (µg/mL)
|
500 (µg/mL)
|
1000 (µg/mL)
|
2000 (µg/mL)
|
4a
|
37.78
|
47.12
|
58.68
|
69.55
|
40.13
|
49.18
|
58.24
|
66.68
|
4b
|
40.65
|
52.02
|
61.72
|
70.03
|
39.81
|
48.34
|
58.13
|
69.12
|
4c
|
38.75
|
49.52
|
60.00
|
70.72
|
41.23
|
51.00
|
60.78
|
69.85
|
4d
|
a
|
a
|
A
|
a
|
37.83
|
48.12
|
58
|
68.75
|
4e
|
41.52
|
51.22
|
62.00
|
70.88
|
40.85
|
49.52
|
57.26
|
68.81
|
4f
|
39.89
|
49.53
|
58.64
|
68.01
|
38.71
|
48.00
|
56.71
|
67.00
|
4g
|
40.72
|
49.53
|
58.65
|
69.71
|
a
|
A
|
a
|
a
|
4h
|
35.17
|
47.00
|
56.12
|
66.89
|
42.12
|
52.11
|
62.00
|
71.09
|
4i
|
39.67
|
48.14
|
58.00
|
69.12
|
40.05
|
49.54
|
58.13
|
69.00
|
Table 6: Antioxidant activity of substituted pyrazoles (4a-4i)
Products
|
DPPH free radical scavenging activity (%)
|
IC50 (µg/mL)
|
25 (µg/mL)
|
50 (µg/mL)
|
75 (µg/mL)
|
100 (µg/mL)
|
4a
|
7.27
|
11.01
|
23.11
|
32.12
|
122.30
|
4b
|
a
|
a
|
a
|
a
|
a
|
4c
|
a
|
a
|
a
|
a
|
a
|
4d
|
12.21
|
32.67
|
47.27
|
52.67
|
92.34
|
4e
|
16.36
|
24.72
|
48.10
|
61.47
|
87.14
|
4f
|
7.51
|
19.57
|
31.45
|
58.41
|
96.36
|
4g
|
a
|
a
|
a
|
a
|
a
|
4h
|
6.21
|
19.41
|
21.42
|
34.36
|
150.94
|
4i
|
a
|
a
|
a
|
a
|
a
|
a: no growth inhibition
Characterization data of synthesized compounds
5-amino-1-(2,4-dinitrophenyl)-3-(4-methoxyphenyl)-1H-pyrazole-4-carbonitrile (4a): m.p. 228-229ºC [ Lit. 229-230ºC, Weber (2002)]; 1H NMR (400 MHz, CDCl3): δ 2.54 (s, 3H, OCH3); 6.09-7.09 (m, 3H, Ar-H); 7.11-7.59 (m, 4H, Ar-H); 9.67 (s, 2H, NH2); IR (νmax cm-1) (neat): 3237.0 (NH), 3031.0 (C=CH, aromatic), 2296.0 (CN), 1616.0 (C=C, aromatic), 1335.0 (NO2)
5-amino-3-(4-chlorophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazole-4-carbonitrile (4b): m.p. 177-179ºC; 1H NMR (400 MHz, CDCl3): δ 7.39-7.67 (m, 3H, Ar-H); 7.21-7.57 (m, 4H, Ar-H); 8.72 (s, 2H, NH2); IR (νmax cm-1) (neat): 3259.0 (NH), 3107.0 (C=CH, aromatic), 2223.0 (CN), 1616.0 (C=C, aromatic), 1335.0 (NO2), 744.0 (C-Cl)
5-amino-1-(2,4-dinitrophenyl)-3-(4-methylphenyl)-1H-pyrazole-4-carbonitrile (4c): m.p. 183-184ºC; 1H NMR (400 MHz, CDCl3): δ 2.42 (s, 3H, CH3); 7.32-7.44 (m, 3H, Ar-H); 7.66-7.83 (m, 4H, Ar-H); 8.13 (s, 2H, NH2); IR (νmax cm-1) (neat): 3262.0 (NH), 3110.0 (C=CH, aromatic), 2221.0 (CN), 1614.0 (C=C, aromatic), 1331.0 (NO2)
5-amino-3-(2-chlorophenyl)-1-(2,4-dinitrophenyl) -1H-pyrazole-4-carbonitrile (4d): m.p. 167-168ºC [ Lit. 168ºC, Hawang et al., (2011)]; IR (νmax cm-1) (neat): 3288.0 (NH), 3052.0 (C=CH, aromatic), 2229.0 (CN), 1614.0 (C=C, aromatic), 1336.0 (NO2), 758 (C-Cl)
5-amino-1-(2,4-dinitrophenyl)-3-(3-nitrophenyl) -1H-pyrazole-4-carbonitrile (4e): m.p. 179-180ºC; IR (νmax cm-1) (neat): 3267.0 (NH), 3110.0 (C=CH, aromatic), 2228.0 (CN), 1614.0 (C=C, aromatic), 1335.0 (NO2)
5-amino-1-(2,4-dinitrophenyl)-3-(2-hydroxyphenyl)-1H-pyrazole-4-carbonitrile (4f): m.p. 233-235ºC; 1H NMR (400 MHz, CDCl3): δ 7.41-7.55 (m, 3H, Ar-H); 7.69-7.80 (m, 4H, Ar-H); 8.15 (s, 2H, NH2); IR (νmax cm-1) (neat): 3483.0 (OH), 3293.0 (NH), 3112.0 (C=CH, aromatic), 2241.0 (CN), 1614.0 (C=C, aromatic), 1332.0 (NO2)
5-amino-1-(2,4-dinitrophenyl)-3-(4-hydroxyphenyl) -1H-pyrazole-4-carbonitrile (4g): m.p. 187-188ºC; IR (νmax cm-1) (neat): 3498.0 (OH), 3310.0 (NH), 3108.0 (C=CH, aromatic), 2221.0 (CN), 1616.0 (C=C, aromatic), 1316.0 (NO2)
5-amino-1-(3,4-dihydroxyphenyl)-1-(2,4-dinitrophenyl)-1H-pyrazole-4-carbonitrile (4h): m.p. 214-215ºC; 1H NMR (400 MHz, CDCl3): δ 7.14-7.19 (m, 3H, Ar-H); 7.13-8.03 (m, 3H, Ar-H); 8.12 (s, 2H, NH2); IR (νmax cm-1) (neat): 3816.0 (OH), 3292.0 (NH), 2941.0 (C=CH, aromatic), 2226.0 (CN), 1623.0 (C=C, aromatic), 1332.0 (NO2)
5-amino-1-(2,4-dinitrophenyl)-3-(3-hydroxyphenyl) -1H-pyrazole-4-carbonitrile (4i): m.p. 243-245ºC; 1H NMR (400 MHz, CDCl3): δ 7.10-7.18 (m, 3H, Ar-H); 7.12-8.07 (m, 4H, Ar-H); 8.16 (s, 2H, NH2); IR (νmax cm-1) (neat): 3481.0 (OH), 3292.0 (NH), 3112.0 (C=CH, aromatic), 2241.0 (CN), 1614.0 (C=C, aromatic), 1332.0 (NO2)
Herbicidal activity
All synthesized compounds (4a-4i) were tested for herbicidal activity against Raphanus sativus L. at 200, 150, 100 and 50 µg/mL concentrations (Table 4). Results were shown in the form of primary screening. All compounds were diluted to 1000 µg/mL concentration as a stock solution. Herbicidal activity of compounds was evaluated against Raphanus sativus L. by inhibitory effect of compounds on the growth of weed roots and shoots. The percentage of inhibition growth was calculated from mean differences between treated and control. From the herbicidal activity data, we observed that compound 4f was exhibited maximum percentage growth inhibition i.e. 91.00 against Raphanus sativus L. (root) and also compound 4f was exhibited maximum percentage growth inhibition i.e. 89.07 against Raphanus sativus L. (shoot) respectively at 200 µg/mL concentration. This growth inhibition may be attributed to substitution of hydroxy group on phenyl ring. The box plot and graphical representation of herbicidal activity of all synthesized compounds (4a-4i) against Raphanus sativus L. were shown in Figs 2-5.
Antifungal activity
All synthesized compounds (4a-4i) were screened for their in vitro antifungal activity against Rhizoctonia solani and Aspergillus niger. The percentage growth inhibition of compounds against R. Solani and A. niger is presented in Table 5. DMSO was used as control against both the test fungi. A culture of test fungi was grown on Potato Dextrose Agar (PDA) medium at ambient temperature (25 ± 2oC). The stock solution (2000 µg/mL) of test compounds were prepared in DMSO and further dilutions were made to 1000, 500 and 250 µg/mL concentrations and stored at 4oC for further use. Potato Dextrose Agar Media, containing specific concentration of test compounds was poured on Petri plates. After solidification, small disc (0.5 cm diameter) of the fungus culture was cut with a sterile cork borer and transferred aseptically upside down in centre of Petri plate. Petri plates were incubated in BOD incubator at 25 ± 2oC. Growth of fungal colony was measured after every 24 h till the fungus in control plates (containing DMSO) completely occupied it. From the fungicidal activity results, we concluded that compounds 4e and 4h were found to be most likely against R. solani and A. niger respectively. This result may be due to presence of nitro and hydroxy groups on phenyl ring. The box plot and graphical representation of antifungal activity of all synthesized compounds (4a-4i) against Rhizoctonia solani and Aspergillus niger were shown in Figs 6-9.
Antioxidant activity
The antioxidant activity data (Table 6) revealed that compounds 4a, 4d, 4e, 4f and 4h have exhibited DPPH free radical scavenging activity at 25, 50, 75 and 100 mg/mL concentrations. Compound 4a exhibited DPPH free radical scavenging activity 1.e. 7.27, 11.01, 23.11 and 32.12% at 25, 50, 75 and 100 mg/mL concentrations respectively. Compounds 4b and 4c have shown no DPPH free radical scavenging activity at all concentrations. Compounds 4d, 4e and 4f exhibited DPPH free radical scavenging activity 1.e. 12.21, 32.67, 47.27, 52.67%, 16.36, 24.72 48.10, 61.47% and 7.51, 19.57, 31.45, 58.41% respectively. Compounds 4g and 4i have shown no DPPH free radical scavenging capacity at 25, 50, 75 and 100 mg/mL concentrations. Compound 4h exhibited DPPH free radical scavenging activity 1.e. 6.21, 19.41, 21.42 and 34.36% at 25, 50, 75 and 100 mg/mL concentrations. From the antioxidant activity results, we conclude that compound 4e displayed potent free radical scavenging activity 1.e. 61.47% at 100 µg/mL with least IC50 value 87.14 mg/mL. This result may be due to presence of nitro group on phenyl ring. The box plot and graphical representation of antioxidant activity of all synthesized compounds (4a-4i) were shown in Figs 10-11.