Fruit juice mediated multicomponent reaction for the synthesis of substituted isoxazoles and their in vitro bio-evaluation

A simple, efficient and eco-friendly procedure for the synthesis of isoxazole derivatives (4a–4h) using one-pot three-component reaction between substituted aldehydes (1a), methyl acetoacetate (2a) and hydroxylamine hydrochloride (3a) has been achieved in presence of Cocos nucifera L. juice, Solanum lycopersicum L. juice and Citrus limetta juice respectively. The homogeneity of synthesized compounds was confirmed by melting point and thin layer chromatography. The synthesized compounds were characterized by using 1H NMR, FTIR and CHN analyses and evaluated for in vitro herbicidal activity against Raphanus sativus L. (Radish seeds). The compounds (4a–4h) were also screened for their fungicidal activity against Rhizoctonia solani and Colletotrichum gloeosporioides. Antibacterial activity was also tested against Erwinia carotovora and Xanthomonas citri. From bio-evaluation data, it was found that compound 4b was most active against Raphanus sativus L. (root) and Raphanus sativus L. (shoot) respectively. Compound 4b was also found most active against both the fungus viz. R. solani and C. gloeosporioides showing maximum percentage growth inhibition i.e. 90.00 against R. solani and 82.45 against C. gloeosporioides at 2000 µg/mL concentration. Compound 4 h has shown maximum inhibition zone i.e. 3.00–9.60 mm against Erwinia carotovora at 2000 µg/mL concentration. Maximum Xanthomonas citri growth was also inhibited by compound 4 h showing inhibition zone 1.00–5.00 mm at highest concentration.


Entry
Catalyst concentration (mL)   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.

Method A Method B Method C Time (h) Yield (%) Time (h) Yield (%) Time (h) Yield (%)
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. 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 results, we observed that compound (Z)-4-(3,4-Dimethoxybenzylidene)-3-methylisoxazol-5(4H)-one (4b) was exhibited maximum percentage growth inhibition i.e. 90.00 against Raphanus sativus L. (root) and also exhibited maximum percentage growth inhibition i.e. 86.15 against Raphanus sativus L. (shoot) respectively at 200 µg/mL concentration. 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. 3, 4, 5 and 6.
Antifungal activity. All synthesized compounds (4a-4h) were screened for their fungicidal activity against 2 fungal strains viz. Rhizoctonia solani and Colletotrichum gloeosporioides by poisoned food technique method. DMSO was used as negative control against fungal strains. The result of antifungal activity of tested compounds is shown in Table 5. Most of synthesized compounds possess moderate to good activity against R. solani and C. gloeosporioides respectively. Compound 4d showed no antifungal activity at all concentrations against R. solani, may be due to electron releasing nature of -OH group. Compound 4f has shown no growth inhibition upto     Antibacterial activity. The propitious antifungal activity of synthesized compounds (4a-4h) has inspired authors to test further for antibacterial activity. All synthesized compounds (4a-4h) were tested for their in vitro  www.nature.com/scientificreports/ antibacterial activity against two bacterial strains Erwinia carotovora and Xanthomonas citri by inhibition zone method using DMSO as negative control. The results of antibacterial activity of synthesized compounds were shown in Table 6. Compound 4a has shown no inhibition zone at 250 µg/mL concentration. Compound 4a exhibited 1.00 mm, 2.00 mm and 3.00 mm inhibition zone against Erwinia carotovora at 500, 1000 and 2000 µg/ mL concentrations respectively, due to electron withdrawing nature of chlorine group. Compound 4e has shown no inhibition zone at all the concentrations against Erwinia carotovora, may be due to electron releasing nature of -OH group. Compound 4c has shown no inhibition zone at 250 and 500 µg/mL concentrations respectively. Compound 4c exhibited 2.00 mm and 5.00 mm inhibition zone at 1000 and 2000 µg/mL concentrations respectively, may be due to methoxy substitution on phenyl ring. Compound 4d has shown no inhibition zone at 250 µg/mL concentration.  www.nature.com/scientificreports/

Materials and methods
All the required chemicals for experiment were purchased from CDH (Central Drug House), SRL (Sisco Research Laboratory) and Sigma-Aldrich and used without purification. Melting points were determined in open head capillaries and are uncorrected. The reaction was monitored by thin layer chromatography. Infrared spectra (4000-350 cm −1 ) of the synthesized compounds were recorded in KBr pellets on Perkin Elmer FT-IR-R2X  www.nature.com/scientificreports/ spectrophotometer and frequency was expressed in cm −1 . The 1 H NMR spectra were recorded in CDCl 3 or DMSO-d 6 using tetramethyl silane (TMS) as internal reference on "Brucker Ac 400 F" (400 MHz) nuclear magnetic resonance spectrometer. Elemental analysis was performed using ThermoFinnigan CHN elemental analyser. The chemical shifts values were quoted in delta (parts per million, ppm).    www.nature.com/scientificreports/ obtained by holing the fruit with a knife. Then juice was filtered using Whatman filter paper no. 1 for removal of residues to get clear juice, which was used as catalyst 36 .
Preparation of Solanum lycopersicum L. juice. The main constituents per 100 g of Solanum lycopersicum L. juice are water (94.24 g), carbohydrates (3.53 g), protein (0.85 g), fat (0.29 g), ascorbic acid (70.1 mg), sugars (2.58 g) and dietary fibre (0.4 g). Fresh tomatoes were purchased from the local market. Then washed thoroughly under running tap water followed by rinsing thrice with double distilled water. Tomatoes were squeezed and juice were strained initially through a muslin cloth then passed through Whatman filter paper no. 1 37 .
Preparation of Citrus limetta juice. Citrus limetta is a species of citrus. It contains high amount of ascorbic acid due to which it acts as acid catalyst in organic synthesis. First of all wash the sweet limes and pat them dry. Cut them into two halves. Then using a citrus juice squeezer, juice was extracted. Then the juice was filtered through cotton and then through Whatman filter paper no. 1 to remove solid material and to get clear juice which was used as a catalyst.
Screening of herbicidal activity. Solutions of 50 µg/mL, 100 µg/mL, 150 µg/mL and 200 µg/mL of the test compounds in DMSO were prepared. Agar powder (5gm) was put into boiling distilled water (1L) until it dissolved, and then cooled down to 40-50 °C. The solution (2 mL) containing test compounds and melting agar (18 mL) was mixed and this mixture was added to a petridish with 4.5 cm diameter. The agar plate without test compound was used as an untreated control. The 15 seeds of Raphanus sativus L. (Radish) were put on the surface of the agar plate. The Petridishes were covered with glass lids, and the cultivation conditions were kept at 25 ± 1 °C and 12 h in light and 12 h in dark alternating for seven days. Seven days later, the root lengths and shoot lengths of Raphanus sativus L. were measured. The growth inhibitory rate related to untreated control was determined by given formula.
Screening of antifungal activity. Amongst the several methods available, poisoned food technique 38 which is the most common was used for testing antifungal activity. The test fungus was grown on Potato dextrose agar medium. The required amount of synthesized compounds dissolved in 1 mL of DMSO was incorporated aseptically into 99 mL aliquots of sterilized potato dextrose agar cooled at 45 °C after brief shaking. Each lot of medium was poured into Petri dishes and allowed to solidify. 1 mL DMSO in media was taken as control. Each dish was inoculated centrally with a 5 mm mycelial disc cut from the periphery of 2-3 days old fungal colonies. Inoculated Petri plates were incubated in the dark 25 ± 2 °C for 48-72 h and colony diameters were measured periodically till the control dishes were nearly completely covered with fungus growth. Three replicates were used for each concentration of a chemical together with three dishes containing only the solvent and no toxicant.
The degree of inhibition of growth was calculated from the mean differences between treatments and the control as percentage of latter by using the formula.
where Control = mycelial growth in control dish, Treated = mycelial growth in treated dish. www.nature.com/scientificreports/ Screening of antibacterial activity. The inhibition zone method 39 was followed for screening the synthesized compounds for their antibacterial activity. The bacterial suspension was prepared from 48 h old culture. The bacterial growth from five slants was taken and mixed in 100 mL sterilized distilled water aseptically. The medium was melted and cooled at 45 °C, needed medium was poured aseptically in sterilized Petri plates and rotated gently for even distribution of the medium and was allowed to solidify. 250, 500, 1000 and 2000 µg/mL concentrations of synthesized compounds were prepared from the stock solution by taking appropriate amount and diluting with DMSO. The circular paper discs of 10 mm diameter were prepared from Whatman's Filter paper No. 1. The disc were kept in Petri plate and autoclaved at 15 lbs pressure 20 min. Two paper discs were used for each concentration of the synthesized compounds. The excess of solution absorbed by paper discs was removed by holding them vertically by sterile forecep. Such soaked discs were transferred aseptically to Petri plates containing media and bacterial suspension spread over the surface. Each concentration and chemical was replicated 3 times. Such Petri plates were inverted and kept at 5 °C for 2 h for better diffusion of the chemicals in agar medium. Later, on the Petri plates were incubated at 25 ± 2 °C for 48 h. The zone of inhibition for each concentration of the chemicals was recorded in mm after 48 h of incubation.

Statistical analysis.
The experiments were performed in triplicates for each treatment and the mean value were recorded and expressed as mean ± S.D. The descriptive statistics in form of box-and-whisker diagram were also presented in this paper. The spacing between the different parts of the box indicates the degree of dispersion and skewness in the data ( Supplementary Fig. S1).

Conclusions
We have developed a novel route for synthesis of biologically active substituted isoxazole derivatives (4a-4h) via one-pot three-component reaction between substituted aldehydes (1a-1h), methyl acetoacetate (2a) and hydroxylamine hydrochloride (3a) in presence of Cocos nucifera L. juice, Solanum lycopersicum L. juice and Citrus limetta juice. The present protocol offers many advantages such as simple and efficient catalytic system, simple work-up, cost-effective and products were obtained in good to excellent yields. A comparison between current catalysts viz. Cocos nucifera L. juice, Solanum lycopersicum L. juice & Citrus limetta juice and some previous catalysts for synthesis of substituted isoxazole derivatives revealing that these catalysts are superior to other reported catalysts in terms of product yield, reaction time and catalyst loading. All synthesized compounds (4a-4h) were also screened for their bio efficacy in terms of herbicidal activity against Raphanus sativus L.
(Radish) seeds, fungicidal activity against R. solani, C. gloeosporioides and antibacterial activity against Erwinia carotovora and Xanthomonas citri. Based on biological activity data, we concluded that strong electronegative groups at the phenyl ring exhibit a good activity profile compared to electron releasing groups. This research work also encourage organic chemist for the design of novel molecules to identify many more biologically active heterocycles for the benefit of humanity. www.nature.com/scientificreports/