Ultrasound-assisted synthesis and biological significance of substituted 4H-chromene-3- carbonitrile using greenery approaches

Article history: Received June 1, 2020 Received in revised form June 26, 2020 Accepted July 28, 2020 Available online July 28, 2020 A synthesis of 2-amino-4-(R)-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3carbonitrile derivatives (4a-l) in single flasks containing aromatic aldehydes (1a-l), malononitrile (2), and dimedone (3) by using green catalyst, extract of orange without solvent under ultrasound irradiations and reflux condition. Synthesis of 2-amino-4-(R)-7,7-dimethyl5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile derivatives (4a-l) show potent in-vitro anti-inflammatory activity. All synthesized compounds were characterized by FTIR, 1H NMR, 13C NMR and mass spectroscopic data & elemental analysis. © 2021 Growing Science Ltd. All rights reserved.


Introduction
Recently, twelve principles of green chemistry have been considered as important factors in the field of organic synthesis due to environment pollution issues. Organic synthetic field is one of the environment pollution contributors. A number of chemical and pharmaceutical industries release hazardous gases and water by using hazardous materials, during their step reactions while manufacturing processes. However, researchers adopt green synthetic routes for synthesis purposes in the view of principles of green chemistry. 1 One pot multi-components (MCRs) condensation is one of the major routes to reduce the environmental pollutions, energy, reaction time and quantity of initial materials at the time of organic synthesis. 2 Also, green solvents and catalysts reduce pollutions and they are easily available and inexpensive. In organic synthesis, excellent yields occur in water due to great polarity of water. 3 What have attracted us in synthesis of 4H-chromene-3-carbonitrile derivatives is their medicinal properties that includes antibacterial, anticancer, anticoagulant, diuretic, antianaphylactin, spasmolytic, and anti HIV agent. [4][5][6] Also, their use to neuro degeration diseases such as Parkinson's, down's syndrome, schizophrenia, alzheimer's and AIDS are associated with dementia diseases. 7 Furthermore, these derivatives are broadly employed as cosmetic agents, food additives and potential biodegradable agrochemicals. 8 From the details of literature, the derivatives of 4H-chromene-3-carbonitrile have been synthesized by using catalysts such as TBABr, 9 I2 and K2CO3, 10 RE(PFO)3, 11 DBU[Ac], 12 HTMAB, 13 TEBA, 14 DBSA, 15 KF/Al2O3, 16 K4PO4, 17 TBAF, 18 MCM-41-NH2, 19 MgO, 18 20 triethylamine, 21 piperidine, 22,23 (NH4)HPO4, 24 etc. in different solvents. Also, different methods have been used such as microwave and ultrasonication, [25][26][27] for its synthesis.
Sonochemistry is one the best applications of ultrasound for chemical reaction and synthesis. Huge number of studies have been done on fundamental of sonochemistry by Luche & co-workers. [28][29] Recently, ultrasonic method has been widely used in the field of organic synthesis [30][31][32][33] as it improves the product of yield and selectivity, shorter reaction time and milder reaction condition. [34][35][36] Herein, we synthesized substituted 4H-chromene-3-carbonitrile in one pot by using extract of orange juice as a catalyst without solvent under ultrasonication and reflux technique.

Result and Discussion
Although malononitrile has been extensively used in knoevenagel condensation reaction, but there are limited examples of 4H-chromene-3-carbonitrile derivatives synthesized from such reaction. Present work, describes a mild and efficient approach for a synthesis of 4H-benzo[b]pyren derivatives via knoevenagel condensation using orange extract as a catalyst as well as solvent with moderate to excellent yield. This reaction was firstly examined by stirring a mixture of benzaldehyde (1e), Malononitrile (2), dimedone (3) with orange extract (3ml) at room temperature in ethanol for 2 hrs ( Table 1). α-cyanocinnamonitrile (5) was generated, in situ by the interaction of malononitrile and aromatic aldehydes, but final product was not observed. Then, the same reaction was performed in refluxing C2H5OH for 30 minutes at 110 o C and 65 % product was observed. The same reaction was stirred, but without solvent, for 2 hrs at 80 o C, yielded 50 % final product. Then, once again the same reaction was refluxed without solvent for 30 minutes at 110 o C and 79 % final product was observed. The results have shown the significant effect of temperature on the improvement of reaction product; hence, we examined all different aldehydes through entry 4 ( Table 1) due to suitable condition for method-A. Further, we performed similar experiment under ultrasonic irradiation technique with C2H5OH at room temperature for 2 h, and 60 % was final product yield. At, changed temperature (70 o C), this reaction completed in 30 minute and yield was 90 % final product. Similar reaction examined without solvent for 30 minutes, yielded 92 % final product. Thus, we observe the effect of C2H5OH, without solvent and mainly ultrasonic irradiations on product of yields ( Table 1). From the results above, it has been shown that benzaldehyde (1a); Malononitrile (2) and dimedone (3) with orange extract (3ml) at 70 o C under ultrasonic irradiations presented an efficient procedure in the terms of excellent yield in shorter reaction time (method-B) ( Table 1). Scheme 1. Synthesis of 4H-chromene-3-carbonitrile derivatives via method-A and method-B We examined different varieties of aromatic aldehydes (1a-l) conventional as well as ultrasonic irradiation techniques ( Table 2), promoted catalytic Knoevenagel condensation reaction and Michael addition reaction. In every case ultrasonic irradiation improved the yields and the reactions time, but in refluxing case, long time was required for complete reactions. In method -B, products of yields were excellent compared to method-A due to ultrasonic effect and orange juice containing polar water molecules ( Table 2). In continue, the effect of electron withdrawing, electron releasing, and halogen groups in different positions on ring of aromatic aldehydes in the synthesis of 4H-chromene-3-carbonitrile derivatives (4al) were studied and illustrated in Table 2. Aldehyde bearing electron withdrawing groups decreased the reaction time with slightly increased the percentage of yields and electron releasing groups increased the reaction times no outstanding effect on the percentage of yields due to ultrasonic effect with aldehyde groups containing electrophilic nature. Consequently, this protocol gives the desired products in good yields and in relatively short reaction times ( Table 2).
In these works, ultrasonic effect is a major factor observed on each reactions of ultrasonic method, method-B (ultrasonic) gave excellent yields of each products within 30 minutes compared to method-A (refluxing) ( Table 2).

General Methods
All A. R. grade chemicals were purchased from Sigma Aldrich and Avra chemical lab. All reactions were examined in Ultrasonication (frequency of 50 Hz and power of 250 V AC, 5.5 L) at 70 o C temperature. Melting points were recorded on open capillary tube. 1 H NMR spectra were recorded on Bruker (400 MHz) and 13 C NMR spectra were recorded on Brucker (100 MHz) in DMSO solvent using Me4Si as an internal standard. Mass spectra were recorded on Micromass -QUATTRO-II of WATER mass spectrometer and IR spectra were recorded on Perkin-Elmer spectrometer.

General procedure for synthesis of 4H-chromene-3-carbonitrilederivatives
We have taken freshly one orange, crushed, compressed in cotton cloth in centrifuge tube and centrifuged. Collect supernent clear liquid and measured its pH by pH-meter. This was used for catalytic purpose. A mixture of aromatic aldehydes (1a-l) (1mmol), Malononitrile (2) (1mmol), dimedone (3) (1mmol) and orange juice (3 ml) was taken in one pot (Scheme 1). Reaction pot was irradiated at 70 o C in ultrasonic bath as well as same reaction mixture was refluxed at 110 o C. Ultrasonic bath temperature was regulated by adding cold water time to time. After changing some physical properties of mixture, reaction condition was checked by TLC using 7:3 solvent ratio (n-hexane:ethyl acetoacetate). After completion of reaction, mixture was poured in crushed ice containing beaker while stirring, and filtered through a small sintered glass funnel. Product was washed with 10 ml cold water and dry and finally, recrystallized using pure ethanol.

Biological study: Anti-inflammatory activity
All synthesized 4H-chromene-3-carbonitrile compounds (4a-l) were tested for In vitro antiinflammatory activity by 'HRBC membrane stabilization method'. [37][38][39][40] Protocol -The fresh blood was collected from a healthy human volunteer who had not taken any NSAIDS for 2 weeks prior to the experiment, mixed with an equal volume of Alsever's solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid, and 0.42% NaCl), and centrifuged at 3,000 rpm. The packed cells were washed with isosaline, and a 10% suspension was made. Various concentrations of samples were prepared (05, 25, 50, 100 and 250 ppm) using distilled water, and to each concentration 1 mL of phosphate buffer, 2 mL hyposaline, and 0.5 mL of HRBC suspension were added. It was incubated at 37 o C for 30 min and centrifuged at 3,000 rpm for 20 minutes. The hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560 nm. Dichlorofenac sodium was used as standard and distilled water as control in this study. Where the blood control represents 100% hemolysis or zero percent stability, the percentage of HRBC hemolysis calculated by formula: The percentage of hemolysis of HRBC membrane as calculated as follows,