Initially, for optimization the reaction of hydroxylamine hydrochloride (20 mmol), methyl acetoacetate (20 mmol) in 20 mL water: ethanol (19:1, v/v) was stirred at room temperature for 15 min, then 4-Hydroxy-3-methoxybenzaldehyde was added to mixture (Scheme 1).
First, we performed model reaction in presence of Cocos nucifera L. juice at room temperature. We found that when the amount of Cocos nucifera L. juice was only 4.0 mL in reaction mixture then yield of reaction was less (78%) and completion of reaction time was also more (Table 1, Entry 1). Than we increase the amount of Cocos nucifera L. i.e. 6.0 mL, 8.0 mL and 10.0 mL respectively and we observed maximum yield of product (92%) and reaction time was also less (Table1, Entry 4). After having these encouraging results, next we explored the same model reaction in presence of Solanum lycopersicum L. juice and Citrus limetta juice. We observed that maximum yield (90%) (Table 1, Entry 4), and (95%) (Table 1, Entry 4) was obtained in presence of Solanum lycopersicum L. juice (10.0 mL) and Citrus limetta juice (10.0 mL) respectively. With these optimized conditions, further we explored the substrate scope of this methodology by varying substituted benzaldehyde. In this paper, we reported the synthesis of substituted isoxazoles (4a-4h) by one-pot multicomponent reaction between equimolar substituted aldehydes (1a-1h) viz. 4-Chlorobenzaldehyde, 3,4-Dimethoxybenzaldehyde, 4-Methoxybenzaldehyde, 3-Hydroxybenzaldehyde, 2-Hydroxybenzaldehyde, 4-Bromobenzaldehyde, 3,4-Dihydroxybenzaldehyde, 3,4,5-Trimethoxybenzaldehyde and methyl acetoacetate (2a) and hydroxylamine hydrochloride (3a) in presence of green catalyst viz. Cocos nucifera L. juice, Solanum lycopersicum L. juice and Citrus limetta juice at room temperature (Scheme 1). The physical data of this study were presented in Table 2. All the synthesized compounds were fully characterized by FTIR, IHNMR and CHN spectral techniques. All synthesized compounds (4a-4h) were shown in Fig 1. In order to show the beauty of current protocol, the previous protocols and their yields for the synthesis were summarized in Table 3. We observed that Cocos nucifera L. juice, Solanum lycopersicum L. juice and Citrus limetta juice catalyst gives the best catalytic activity in terms of product yield and reaction time as compared to other catalysts in literature. Therefore the present procedure for synthesis of isoxazole derivatives is considered as sustainable and eco-friendly protocol.
The possible mechanism for the formation of substituted isoxazole derivatives is shown in Scheme 2. According to this mechanism first of all there is formation of cyclized adduct (A) by the nucleophilic attack of the amino group and hydroxyl group of hydroxylamine hydrochloride to the carbonyl carbon of methyl acetoacetate in presence of Cocos nucifera L. juice, Solanum lycopersicum L. juice and Citrus limetta juice. The aldehyde was attacked on the cyclized adduct (A) and subsequent Knoevenagel adduct (4a-4h) is formed via removal of the water molecule.
Table 1: Model reaction of 4-Hydroxy-3-methoxybenzaldehyde (20 mmol), methyl acetoacetate (20 mmol) and hydroxylamine hydrochloride (20 mmol) using Cocos nucifera L. juice, Solanum lycopersicum L. juice and Citrus limetta juice as catalyst
Entry
|
Catalyst Concentration (mL)
|
Method A
|
Method B
|
Method C
|
Time (h)
|
Yield (%)
|
Time (h)
|
Yield (%)
|
Time (h)
|
Yield (%)
|
1
|
4.0
|
15
|
78
|
13
|
78
|
11
|
85
|
2
|
6.0
|
13
|
84
|
9
|
85
|
6
|
90
|
3
|
8.0
|
11
|
90
|
6
|
88
|
5
|
92
|
4
|
10.0
|
7
|
92
|
4
|
90
|
4
|
95
|
Table 2: Physical data of substituted isoxazole derivatives (4a-4h)
Entry
|
Products
|
Ar
|
R1
|
Method A
|
Method B
|
Method C
|
M.pt. (ºC)
|
Time (h)
|
Yield (%)
|
Time
(h)
|
Yield (%)
|
Time (h)
|
Yield
(%)
|
1
|
4a
|
Ph-Cl
|
CH3
|
10
|
80
|
7
|
76
|
8
|
89
|
185-187
|
2
|
4b
|
Ph-(OCH3)2
|
CH3
|
8
|
81
|
6
|
89
|
10
|
90
|
210-212, (Lit., 210-211)26
|
3
|
4c
|
Ph-OCH3
|
CH3
|
5
|
92
|
8
|
90
|
8
|
92
|
172-174, (Lit., 173-175)24
|
4
|
4d
|
Ph-OH
|
CH3
|
6
|
90
|
7
|
92
|
8
|
83
|
200-202, (Lit., 200-201)27
|
5
|
4e
|
Ph-OH
|
CH3
|
8
|
85
|
5
|
80
|
4
|
80
|
198-200, (Lit., 200-201)27
|
6
|
4f
|
Ph-Br
|
CH3
|
7
|
80
|
6
|
78
|
8
|
76
|
178-180
|
7
|
4g
|
Ph-(OH)2
|
CH3
|
5
|
78
|
4
|
94
|
10
|
81
|
210-211, (Lit., 212-214)26
|
8
|
4h
|
Ph-(OCH3)3
|
CH3
|
6.5
|
82
|
4
|
91
|
6
|
82
|
194-196
|
Table 3: Comparison for different catalysts used for synthesis of isoxazole derivatives (4a-4h)
S.No.
|
Catalyst
|
Solvent
|
Temperature (oC)
|
Time (min)
|
Yield (%)
|
References
|
1
|
Sodium benzoate (15 mol%)
|
Water
|
RT
|
60
|
85
|
28
|
2
|
Saccharose (20 mol%)
|
Solvent-free
|
100 oC
|
10
|
75
|
29
|
3
|
Cetyltrimethylammonium chloride (30 mol%)
|
Water
|
90 oC
|
240
|
89
|
30
|
4
|
Nano-ZnO (5 mol%)
|
Water
|
70 oC
|
60
|
94
|
31
|
5
|
Nano-CuI (1.2 mol%)
|
Water
|
Reflux
|
40
|
90
|
32
|
6
|
TBABr (10 mol %)
|
Water
|
Reflux
|
15
|
90
|
33
|
7
|
γ-Alumina (30 mol%)
|
Water
|
Reflux
|
50
|
80
|
34
|
8
|
β-Cyclodextrin (10 mol%)
|
Water-ethanol (9:1, v/v)
|
80 oC
|
15
|
92
|
35
|
9
|
Urea (10 mol%)
|
Water-ethanol (1:1, v/v)
|
RT
|
480
|
86
|
36
|
10
|
DABCO (5 mol%)
|
Water
|
Reflux
|
15
|
92
|
37
|
11
|
DCDBTSD (10 mol%)
|
Water
|
80 oC
|
60
|
85
|
38
|
12
|
[Bmim]OH (20 mol%)
|
Solvent-free
|
50-60 oC
|
45
|
90
|
39
|
13
|
Cocos nucifera L. juice
|
Water : ethanol (19:1, v/v)
|
RT
|
4
|
90
|
Present work
|
14
|
Solanum lycopersicum L. juice
|
Water : ethanol (19:1, v/v)
|
RT
|
7
|
92
|
Present work
|
15
|
Citrus limetta juice
|
Water : ethanol (19:1, v/v)
|
RT
|
4
|
95
|
Present work
|
Table 4: Herbicidal activity of substituted isoxazoles (4a-4h)
Compounds
|
Growth Inhibition (%)
|
Root
|
Shoot
|
50 (µg/mL)
|
100 (µg/mL)
|
150 (µg/mL)
|
200 (µg/mL)
|
50 (µg/mL)
|
100 (µg/mL)
|
150 (µg/mL)
|
200 (µg/mL)
|
4a
|
36.66 ± 1.14
|
53.33 ± 1.07
|
63.33 ± 0.87
|
73.33 ± 0.67
|
64.61 ± 1.21
|
69.23 ± 0.93
|
75.38 ± 1.95
|
80.00 ± 0.83
|
4b
|
46.66 ± 0.93
|
66.60 ± 0.90
|
80.00 ± 0.50
|
90.00 ± 1.46
|
67.69 ± 1.11
|
76.92 ± 1.27
|
86.15 ± 0.89
|
93.84 ± 1.18
|
4c
|
34.36 ± 0.72
|
48.36 ± 0.49
|
61.45 ± 0.75
|
78.65 ± 0.55
|
51.39 ± 0.80
|
62.36 ± 0.78
|
71.45 ± 1.48
|
82.36 ± 0.99
|
4d
|
43.33 ± 0.40
|
60.00 ± 1.75
|
73.33 ± 0.40
|
86.66 ± 0.50
|
67.69 ± 1.12
|
76.92 ± 1.27
|
84.61 ± 0.94
|
90.76 ± 1.23
|
4e
|
40.52 ± 1.06
|
58.37 ± 0.99
|
74.50 ± 0.50
|
88.24 ± 1.00
|
49.92 ± 1.05
|
68.28 ± 0.97
|
78.36 ± 0.99
|
90.25 ± 0.94
|
4f
|
35.69 ± 0.43
|
45.48 ± 0.44
|
60.36 ± 1.52
|
72.58 ± 1.32
|
41.30 ± 1.24
|
54.78 ± 1.52
|
71.13 ± 1.01
|
85.48 ± 1.51
|
4g
|
36.66 ± 1.57
|
50.00 ± 0.82
|
63.33 ± 0.68
|
76.66 ± 0.82
|
60.00 ± 0.66
|
66.15 ± 1.00
|
75.38 ± 2.09
|
81.53 ± 1.14
|
4h
|
43.33 ± 0.98
|
63.33 ± 1.47
|
76.66 ± 0.97
|
86.66 ± 0.92
|
75.38 ± 0.95
|
81.53 ± 1.56
|
84.61 ± 1.10
|
89.23 ± 0.94
|
All values are mean ± S.D.
Table 5: Antifungal activity of substituted isoxazoles (4a-4h)
Compounds
|
Growth Inhibition (%)
|
Fungi
|
Rhizoctonia solani (conc.) µg/mL
|
Colletotrichum gloeosporioides (conc.) µg/mL
|
250
|
500
|
1000
|
2000
|
250
|
500
|
1000
|
2000
|
4a
|
12.50 ± 1.07
|
27.50 ± 0.59
|
52.50 ± 0.71
|
75.00 ± 0.72
|
10.23 ± 0.68
|
24.62 ± 1.58
|
48.75 ± 0.69
|
70.23 ± 0.43
|
4b
|
56.00 ± 0.82
|
64.00 ± 1.06
|
76.00 ± 1.17
|
90.00 ± 2.35
|
36.45 ± 0.86
|
58.45 ± 0.95
|
70.23 ± 1.51
|
82.45 ± 1.04
|
4c
|
42.00 ± 1.37
|
56.00 ± 0.87
|
80.00 ± 0.66
|
90.00 ± 1.26
|
30.25 ± 0.73
|
51.36 ± 1.01
|
66.60 ± 0.67
|
79.65 ± 0.95
|
4d
|
a
|
a
|
a
|
a
|
a
|
a
|
36.68 ± 0.62
|
51.78 ± 1.09
|
4e
|
20.75 ± 1.44
|
58.49 ± 1.06
|
66.03 ± 0.56
|
84.90 ± 0.77
|
a
|
a
|
a
|
a
|
4f
|
a
|
a
|
48.12 ± 1.12
|
74.96 ± 2.50
|
a
|
a
|
24.56 ± 1.58
|
42.98 ± 1.30
|
4g
|
23.98 ± 0.93
|
56.42 ± 0.54
|
68.45 ± 0.69
|
87.36 ± 0.75
|
14.25 ± 0.52
|
29.35 ± 1.55
|
48.68 ± 0.35
|
68.45 ± 0.52
|
4h
|
45.23 ± 1.29
|
58.69 ± 1.01
|
81.35 ± 2.09
|
91.23 ± 0.72
|
25.12 ± 0.75
|
47.98 ± 0.87
|
61.89 ± 2.19
|
74.30 ± 1.44
|
All values are mean ± S.D. a: No Growth Inhibition
Table 6: Antibacterial activity of substituted isoxazoles (4a-4h)
Compounds
|
Inhibition Zone (mm)
|
Bacteria
|
Erwinia carotovora (conc.) µg/mL
|
Xanthomonas citri (conc.) µg/mL
|
250
|
500
|
1000
|
2000
|
250
|
500
|
1000
|
2000
|
4a
|
a
|
1.00 ± 0.05
|
2.00 ± 0.25
|
3.00 ± 0.45
|
0.30 ± 0.03
|
0.70 ± 0.05
|
1.00 ± 0.12
|
1.30 ± 0.15
|
4b
|
1.50 ± 0.20
|
2.20 ± 0.05
|
3.00 ± 0.15
|
4.00 ± 0.15
|
a
|
a
|
a
|
a
|
4c
|
a
|
a
|
2.00 ± 0.15
|
5.00 ± 0.45
|
a
|
1.00 ± 0.12
|
2.00 ± 0.13
|
3.00 ± 0.13
|
4d
|
a
|
1.00 ± 0.05
|
2.50 ± 0.25
|
4.00 ± 0.40
|
a
|
a
|
a
|
0.50 ± 0.02
|
4e
|
a
|
a
|
a
|
a
|
a
|
a
|
a
|
a
|
4f
|
0.70 ± 0.07
|
1.00 ± 0.01
|
1.60 ± 0.20
|
2.10 ± 0.25
|
0.10 ± 0.01
|
0.30 ± 0.02
|
0.60 ± 0.04
|
0.70 ± 0.07
|
4g
|
a
|
a
|
0.60 ± 0.07
|
1.20 ± 0.15
|
2.10 ± 0.20
|
2.60 ± 0.26
|
3.10 ± 0.30
|
3.90 ± 0.47
|
4h
|
3.00 ± 0.40
|
5.50 ± 0.05
|
7.00 ± 0.45
|
9.60 ± 0.36
|
1.00 ± 0.12
|
2.20 ± 0.20
|
3.00 ± 0.32
|
5.00 ± 0.55
|
All values are mean ± S.D. ; a: No Inhibition Zone
Characterization data of selected compounds
(Z)-4-(3,4-Dimethoxybenzylidene)-3-methylisoxazol-5(4H)-one (4b): Elemental Analysis found: C, 63.15; H, 5.30; N, 5.67; O, 25.88; Required: C, 62.89; H, 5.32; N, 5.78
(Z)-4-(4-Methoxybenzylidene)-3-methylisoxazol-5(4H)-one (4c): m.p. 172-174ºC; 1H NMR (400 MHz, DMSO-d6): δ 2.25 (s, 3H, CH3); 3.89 (s, 3H, OCH3; 7.74 (s, 1H, =CH); 7.05-8.50 (m, 4H, Ar-H); IR (νmax cm-1) (neat): 1591.5 (C=N), 1619.0 (C=C, aromatic), 1729.9 (C=O); 1431.8 (N-O), 1276.6 (OCH3); Elemental Analysis found: C, 66.35; H, 5.10; N, 6.45; O, 22.10; Required: C, 66.33; H, 5.13; N, 6.62
(Z)-4-(2-Hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (4e): m.p. 198-200ºC; 1H NMR (400 MHz, DMSO-d6): δ 2.25 (s, 3H, CH3); 8.20 (s, 1H, =CH); 6.87-8.77 (m, 4H, Ar-H); 10.85 (s, 1H, OH)
(Z)-4-(4-Bromobenzylidene)-3-methylisoxazol-5(4H)-one (4f): m.p. 178-180ºC; 1H NMR (400 MHz, CDCl3): δ 2.29 (s, 3H, CH3); 7.37 (s, 1H, =CH); 7.59-8.22 (m, 4H, Ar-H)
Herbicidal activity
All synthesized compounds (4a-4h) were screened for herbicidal activity against Raphanus sativus L. at various concentration 200, 150, 100 and 50 µg/mL as shown in Table 4. Results were quoted in form of primary screening. Synthesised compounds were diluted to 1000 µg/mL concentration as a stock solution. Herbicidal activity of synthesized compounds was evaluated against Raphanus sativus L. by inhibitory effect of the compounds on the growth of weed roots and shoots. The percentage of inhibition of growth was calculated from the mean differences between treated and control. From the herbicidal activity data, we identified that compound (Z)-4-(3,4-Dimethoxybenzylidene)-3-methylisoxazol-5(4H)-one (4b) was most active against Raphanus sativus L. seeds. The growth inhibition may be attributed to substitution of methoxy group on phenyl ring. The box plot and graphical representation of herbicidal activity of all synthesized compounds (4a-4h) against Raphanus sativus L. seeds were shown in Figs 2-5.
Antifungal activity
All synthesized compounds (4a-4h) were tested for their in vitro antifungal activity against Rhizoctonia solani and Colletotrichum gloeosporioides. The percentage growth inhibition of compounds against R. Solani and C. Gloeosporioides 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 compound 4b was most likely against both the fungus viz. R. solani and C. gloeosporioides respectively. This result may be due to presence of methoxy group on phenyl ring. The box plot and graphical representation of antifungal activity of all synthesized compounds (4a-4h) against Rhizoctonia solani and Colletotrichum gloeosporioides were shown in Figs 6-9.
Antibacterial activity
All synthesized compounds (4a-4h) were evaluated for their inhibitory effect on the growth of two bacterial species viz. Erwinia carotovora and Xanthomonas citri at various concentrations i.e. 250, 500, 1000 and 2000 µg/mL. Luria- Bertani medium was sterilized by auto-claving at 15 psi pressure at 121oC for 15 to 20 minutes. The plates were prepared by pouring 30-35 mL of the sterilized media into sterilized Petri plates. Media was then allowed to solidify and then suspension of 3-4 days old broth of test organism was then spread on specific medium plates. Sterile filter paper discs moistened with test compounds solution in DMSO were carefully placed on the medium inoculated with the specific bacterial suspension. Sterile filter paper discs dipped in DMSO served as control. These plates were incubated at 25 ± 2oC and the diameter of growth inhibition zone was measured after 24 h. The results of antibacterial screening of synthesized compounds (4a-4h) were presented in Table 6. The results revealed that some of synthesized compounds showed no inhibition against Erwinia carotovora and Xanthomonas citri at all concentrations. Maximum Erwinia carotovora growth was inhibited by compound 4h showing inhibition zone 3.00-9.60 mm. Maximum Xanthomonas citri growth was also inhibited by compound 4h showing inhibition zone 1.00-5.00 mm. This inhibition may be due to presence of methoxy group on phenyl ring. The box plot and graphical representation of antibacterial activity of all synthesized compounds (4a-4h) against Erwinia carotovora and Xanthomonas citri were shown in Figs 10-13.