Synthesis, Electrochemical Studies, Molecular Docking, and Biological Evaluation as an Antimicrobial Agent of 5-Amino-6-cyano-3-hydroxybenzo[c]coumarin Using Ni-Cu-Al-CO3 Hydrotalcite as a Catalyst.

New insights via a multicomponent cyclocondensation reaction of 2-hydroxy benzaldehyde acetoacetic ester and malononitrile were attained under efficient conditions. A detailed mechanistic study exhibits that the reaction via multicomponent, one-pot, two-step incooperating three-components' reactants under nominal conditions efficiently synthesizes the intermediates in shorter than reported time along with the formation of a novel compound [5-amino-6-cyano-3-hydroxybenzo[c]coumarin] in the presence of Ni-Cu-Al-CO3 hydrotalcite as a heterogeneous catalyst. Hydrotalcite functions as an efficient and versatile catalyst as it is safe, easy to work up, and recyclable many times under ambient conditions, and the reaction time is shorter. The aforementioned conditions make these solid basic heterogeneous catalysts environmentally friendly. The product yield obtained was 89%. The product was characterized using FTIR, LCMS, and NMR. Electrochemical studies were also carried out to check the reduction and oxidation behavior. The synthesized product showed antimicrobial activity. Antimicrobial activity against human pathogens, viz, S. Aureus, P. Aeruginosa, and P. Bulgaria, was studied by using the agar well diffusion method. The molecular docking studies of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin exhibit its suitable attachment at the active center of the type IIA topoisomerases and gyrase enzymes which suggest its potential antibacterial activity when compared using ciprofloxacin drug as the control.

Introduction

Diseases caused by bacteria are potent health hazards internationally due to the growing number of drug-resistant microbes.[1−4] The increasing number of drug-resistant bacteria has focused the scientists to look for the latest drug. Benzo[c]coumarin derivatives show numerous biological actions like antimicrobial, anticancer, anti-inflammatory, antiasthmatic, and antioxidant.[5−9] In recent years, heterocyclic compounds have attracted strong interest due to their pharmacological properties and biological activity.

In molecular studies, type IIA topoisomerase enzymes multiply the eukaryotic topoisomerases II and relax DNA in an ATP-dependent reaction, while the bacterial topoisomerases IV multiply enzymes of decadent DNA and gyrase enzymes of negative supercoils into DNA.[10]

The production of polyfunctionalized pyrans using Mg/La mixed oxides was reported by Seshu et al., which was synthesized using malononitrile, aldehydes, and ethyl acetoacetates as reactants.[11] The literature suggests few organic bases as catalysts,[12,13] viz., tetrabutylammonium bromide, l-proline, rare earth perfluorooctanoates, and hexadecyltrimethylammonium bromide, for the preparation of benzocoumarin and 4H-pyrans using multicomponent synthesis. Also, the multicomponent synthesis of 5-amino-6-cyano-3-hydroxybenzocoumarin has been reported using MgO as catalysts.[14] All these catalysts have an advantage, while some are disadvantaged by restrictions such as strict reaction conditions, low yields, poor recyclability, and tedious workup. Thus, the development of an appropriate solid base catalyst for the efficient synthesis of 4H-pyrans and 5-amino-6-cyano-3-hydroxybenzocoumarin in one pot remains an important target for chemists, as an economically and eco-friendly idea, and the use of environmentally friendly solvents is very desirable. In the past few years, a heterogeneous catalyst derived from alkali earth metallic precursors has found application in catalyzed reactions. Thus, the heterogeneous solid base hydrotalcite catalysts have been recognized as potential alternatives to homogeneous organic basic catalysts.

This paper reports the synthesis and antimicrobial activity of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin. This shows that the compound synthesis follows a different mechanism (Scheme ) from the reported mechanism. In the present work, various solid base catalysts for the preparation of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin have been tried. We have identified that Ni–Cu–Al–CO3 hydrotalcite acts as an efficient catalyst in multicomponent condensation of 2-hydroxy benzaldehyde, acetoacetic ester, and malononitrile (Scheme ). Ni–Cu–Al–CO3 hydrotalcite is a recyclable catalyst, and the presence of nickel, copper, and aluminum increases the basicity of the catalyst. The present catalyst leads to 5-amino-6-cyano-3-hydroxybenzo[c]coumarin in high yields within short reaction times. After that, the molecular docking studies were carried out to evaluate the possible interaction of synthesized compounds with active pockets. After that, the electrochemical studies were carried out to check the response, in which a synthesized compound shows reduction and oxidation behavior.

Scheme 1

Synthesis of 5-Amino-6-cyano-3-hydroxybenzo[c]coumarin by the Ni–Cu–Al–CO3 Hydrotalcite Catalyst

To the best of our knowledge, Ni–Cu–Al–CO3 is being reported as a heterogeneous catalyst for the synthesis of 5-amino-6-cyano-3-hydroxybenzocoumarin under mild reaction conditions. MgO has been studied as a heterogeneous catalyst due to its good activity at low temperature (at room temperature) and low cost. Lowering of catalytic activity due to leaching of MgO has restricted its application. Another advantage of Ni–Cu–Al–CO3 hydrotalcites is high yield (i.e., 89% yield in comparison with MgO which resulted in 80% yield). Hydrotalcites are more basic than MgO due to the presence of more basic metal in their structure. In a previous paper, MgO was used as a catalyst one time, but hydrotalcite was reused three times. The yield, conversion, and purity of the product were determined using various analytical techniques.

Results and Discussion

Synthesis and Characterizations of Ni–Cu–Al–CO3 Hydrotalcite

The synthesis of hydrotalcite is simple, nontoxic, and environmentally friendly. The Ni–Cu–Al–CO3 hydrotalcite was synthesized via the coprecipitation method.[15] The Ni–Cu–Al–CO3 hydrotalcite has been characterized by powder-X-ray diffraction (P-XRD), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), and transmission electron microscopy (TEM).

The hydrotalcite Ni–Cu–Al–CO3 synthesized in ratios of 1:3:1 was dried overnight and after drying exhibited a layered structure of compounds. The XRD patterns confirm the anionic clay-like structures of the crystallized hydrotalcite (Figure a). The 2 theta angles reflected in X-ray were at 12.16°, 23.4°, 35.43°, 48.2°, and 61.5° with corresponding d values of 7.273, 3.79, 2.53, 1.88, and 1.50 Å, respectively, confirming the synthesis of the hydrotalcite. The product exhibited low crystallinity because of the incorporation of the copper in higher molar ratios into the reaction mixtures. These broadenings of the peaks can be suggested due to the small size of the particles of the existence of a strain among crystalline phases. The second reason is that the structures exhibit octahedron coordination, and serious deformations occur via incorporating copper in them. This effect is known as Jahn–Teller distortion. On further increasing the ratio, the copper peak at 12.5 appeared, which corresponds to the hydroxylated structure. The presence of copper oxide was indicated by the black color. Its presence was further confirmed by the presence of the peak at 39.17, which is a typical peak of copper oxide. Therefore, XRD exhibited poor crystallinity. Our findings were supported by other research; i.e., similar results were also reported by Fetter et al., Venugopal et al., and Behamani et al.[16−22]

Figure 1

PXRD of Ni–Cu–Al–CO3 hydrotalcite [a], FTIR of Ni–Cu–Al–CO3 hydrotalcite catalyst [b], TGA of Ni–Cu–Al–CO3 hydrotalcite catalyst [c], and TEM of Ni–Cu–Al–CO3 hydrotalcite (A and B fresh catalyst image and C and D recycle catalyst image) [d].

The bands (Figure b) from the FTIR spectra were similar to the CO3 anion interlamellar-like phases in hydrotalcites. The absorption bands around 3434.88 cm–1 were ascribed to the stretching vibrations of the valence OH groups, which were present in the brucite layer, and the water molecules occupy the spaces in interlamellates which are related to carbonates via hydrogen bonds, as the disordered structure appeared, followed by the unfolding of the νOH band. Another peak appearing at 1629 cm–1 was due to the presence of vibrations of reformatted water molecules which were intercalated in interlamellate spaces. The band located at 1384 cm–1 exhibited the presence of the CO3 anion. All the bands which were recorded in the area of low frequency were ascribed to the vibrations of metallic oxides MO and O–M–O, where M = Cu, Ni, or Al. These bands appear in between 800 and 400 cm–1. These readings which were recorded were supported by several researchers, as reported in the literature.[16,22,23]

The weight loss noticed in the TGA graph was mainly found to be in two places. The TGA curves of Ni–Cu–Al–CO3 HT were carried out up to 800 °C in a N2 gas flow. The first weight loss exhibited (Figure c) at about 230 °C can be attributed to the presence of the water molecules adsorbed on the surface of the material, i.e., because of the presence of the coordinated water molecules with the surface of the catalyst. The second weight loss was at around 390 °C because of the presence of the metal oxide formations. Further, no weight loss was substantially exhibited over the 450 °C temperature. Our readings were similar to the reported readings in the literature.[18,22]

TEM results of the Ni–Cu–Al–CO3 catalyst are shown in Figure d (A and B) as a fresh catalyst or in Figure d (C and D) as a recycled catalyst. The corresponding TEM images of the fresh sample and recycled catalyst sample of the Ni–Cu–Al–CO3 catalyst NiO particles with a diameter around 15–90 nm have been observed in the transmission electron microscope (TEM) analysis of the spent catalyst, and the presence of nickel and copper oxide particles (dark zones) over the mesoporous structure of Al2O3 suggests changes in the surface of the catalyst.

Investigation of Catalytic Activity of Ni–Cu–Al–CO3 Hydrotalcite and Its Best Results Obtained

The reactions were carried out by optimizing various parameters given as follows. The detailed investigation using different hydrotalcite catalysts is shown in Figure A. In the presence of a catalyst (100 mg), the highest yield obtained was 89% with Ni–Cu–Al–CO3 hydrotalcites as catalysts. Because Cu2+ 3d9,4s0 shows strong static John–Teller distortion, CuII has relatively strong electronegativity compared to other transition metals. The basicity is directly proportional to electronegativity, so Ni–Cu–Al–CO3 hydrotalcites are more basic in nature as compared to other hydrotalcites. The lowest yield of 35% was obtained with Ca–Al–CO3 hydrotalcite because Ca2+ and Al3+ have a small size and show higher solvation in protic solvent. So, the basicity of the Ca–Al–CO3 hydrotalcite in protic solvent is reduced. The catalyst could be reused at least four to five times with only a slight reduction in the activity of the catalyst, as shown Figure B. The reaction was highly sensitive to the nature of the solvent, as shown in Figure C below. Our investigations exhibited that the highest yields of the reaction product were obtained by using the polar organic solvents. Salicyaldehyde, ethyl acetoacetate, and malononitrile are soluble in water and miscible in ethanol and become a homogeneous mixture, so that reacting molecules come closer to each other, hence increasing effective collision. Ethanol and water are protic solvents and thus stabilize the transition state in reactant molecules; hence, higher yields are obtained.

Figure 2

Effect of different hydrotalcites as catalysts [A], effect of recycling of Ni–Cu–Al hydrotalcite catalysts [B], and effect of solvents in the presence of a Ni–Cu–Al hydrotalcite catalyst [C].

The effect on the product yield in the presence of different catalyst concentrations of Ni–Cu–Al–CO3 hydrotalcite was also investigated. Initially, with the increase in the concentration of catalysts from 20 to 80 mg, the yield of the products was also enhanced from 60% to 78%, respectively. The highest yield of 89% was obtained with the concentration of 100 mg of catalyst, as shown in Table below. The reaction effected by temperature change was studied in the temperature range (room temp to 120 °C) for the synthesis of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin. According to the collision model, a chemical reaction can occur only when reactant molecules collide with more than a certain amount of kinetic energy and in the proper orientation shown Table .

Table 1

Effect of Concentration of the Ni–Cu–Al Hydrotalcite Catalyst

S. no.	catalyst amount (mg)	yield (%)
1	20	60
2	40	67
3	60	72
4	80	78
5	100	89

Table 2

Effect of Temperature on Product Yield in the Presence of Catalyst

S. no.	temperature (°C)	time (minutes)	yield (%)
1	room temperature	300	60
2	60	250	75
3	80	180	89
4	100	100	85
5	120	90	82

Characterization of Product 5-Amino-6-cyano-3-hydroxybenzo[c]coumarin

In the mass spectra of the final compound, the molecular ion peak is obtained at 252 m/z, matching to the calculated molecular weight of the molecule supporting the proposed structure. In the FTIR spectra of the final compound, a band due to the (CN) group appeared at 2195 cm–1. The bands due to NH2 stretching vibrations were observed at 3351 cm–1. The other characteristic groups (C–H), (C–C), and (C–O) were observed at 3450, 1606, and 1185 cm–1, respectively. These observations support the proposed structure. In the C13 NMR spectrum, the cyclic ester peak is observed at 156.66–152 ppm. Here the carbon is attached to two electronegative oxygen atoms, one of which is sp2 hybridized. Due to this, C13 shows a large chemical shift (deshielding). The nitrile (CN) peak is at 117.60 ppm. The carbon is directly attached to the electronegative nitrogen atom which is SP hybridized and hence shows a large chemical shift value. Aromatic carbon signals are from 129.45 to 129.49 ppm, which show slight deshielding due to the electron-withdrawing group (CN group) which is directly attached to C13. The peak at 125.88 ppm shows a slight shielding due to the electron-donating group attached to C13. The 124.75 ppm peak shows slight shielding due to the electron-donating group (NH2), which is attached directly to C13 (carbon attached to OH shows slight deshielding as compared to a carbon attached to the NH2 group). It is due to the electronegativity of oxygen which is greater than nitrogen, as shown in the spectra.

The purity of the synthesized product was greater than 96%, checked by HPLC techniques. The synthesized product with a flow rate of 0.6 mL/min and mobile phase used was 0.1% formic acid (pump A) and 100% methanol (pump B).

Mechanism of 5-Amino-6-cyano-3-hydroxybenzo[c]coumarin

The possible mechanism for the formation of 5-amino-6-cyano-3-hydroxybenzocoumarin is shown Figure . The first step is the formation of intermediate 3 by the Knoevenagel condensation of 2-hydroxybenzeldehyde 1 and ethyl acetoacetate 2.

Figure 3

Reaction mechanism of 5-amino-6-cyano-3-hydroxybenzocoumarin.

The second pathway involves the initial step that leads to the formation of intermediate 3 by the Knoevenagel condensation of salicylaldehyde (1) and malononitrile (4). This step is followed by Michael addition by the malononitrile anion (4), and subsequent cyclization results in the final product (5), i.e., 5-amino-6-cyano-3-hydroxybenzo[c]coumarin. The synthesized compound of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin is the same. However, our mechanism of this compound is a different mechanism from that found by Valizadeh.[14] They have reported that the intermediate is favored by Knoevenagel condensation of salicylaldehyde and malononitrile, but in our reaction mechanism, the formation of intermediate 3 was a result of Knoevenagel condensation of salicylaldehyde and ethyl acetoacetate. This intermediate (3) is more electrophilic due to an α,β-unsaturated carbonyl group, which decreases the energy of the LUMO intermediate (low in energy of LUMO) as compared to the intermediate found by Valizadeh[14] et al. The intermediate become highly electrophilic and reacted rapidly with a nucleophile (protonated malononitrile).

In our reaction, the Knoevengel condensation reaction is the first step. When the reaction of ethyl acetoacetate (2) with malononitrile (4) was tried, no product was formed, which shows that this reaction is not possible because the methyl active group is present in both components.

Electrochemical Studies

The electrochemical response of the synthesized compound 5-amino-6-cyano-3-hydroxybenzo[c]coumarin was investigated by cyclic voltammetry (CV) using Ag/AgCl, glassy carbon, and silver wire as a reference and working and counter electrodes, respectively. CV was employed to investigate the electrochemical changes of 5 ppb of catalyst in BR buffer (pH 10.5) solution by applying potential ranging from −2.0 to 1.5 V at the scan rate of 50 mV/s, as shown in Figure A. During the scan, an oxidation peak was observed at 0.955 V, which was an indication of the oxidation state in amino-6-cyano-3-hydroxybenzo[c]coumarin (Table ). A cathodic peak was observed at −0.499 V, which corresponds to the reduction of synthesized compounds. The cyclic voltammogram of the title compound was recorded at various scan rates (from 0.02 to 0.1 V/s) in the same potential range as previously shwon in Figure B. On increasing the scan rate, it was observed that the peak current increases. The anodic peak potential was found to shift toward a more positive value, while the cathodic peak potential moves toward a more negative value. The shifting of peak potential and increase in peak current indicate the mode of interaction and the nature of binding of compounds in solution.[31] Based on information of potential separation and linearity at all scan rates, the title compound exhibits reversible behavior in the present electrolyte solution.[32][34]

Figure 4

Cyclic voltammograms were recorded in BR buffer (pH 10.5) containing 0.1 M KCl as electrolyte solution on a glassy carbon electrode at the scan rate of 50 mV/s [A]. Cyclic voltammogram of the 5-amino-6-cyano-3-hydroxybenzo[c]coumarin sample at various scan rates of 0.02 V/s to 0.1 V/s [B]. Represents linearity curves at different scan rates [C].

Table 3

Electrochemical Data for Compound 5-Amino-6-cyano-3-hydroxybenzo[c]coumarin vs Ag/AgClin BR Buffer (pH 10.5) Containing 0.1 M KCl as a Supporting Electrolyte on a Glassy Carbon Electrode at the Scan Rate of 50 mV/s and 25° C

compound	Epa/V	Epc/V	ΔEp/V	E1/2/V
5-amino-6-cyano-3-hydroxybenzo[c]coumarin	0.955	–0.499	1.454	0.228

A plot between the peak current (ip) and the square root of the scan rate (υ1/2) is linear (Figure B and 4C). For the synthesized compound, it is suggested that the reaction that occurs on the electrode surface is reversible, and there is confirmed diffusion-controlled behavior of electrodes.

Molecular Docking Studies

Molecular docking analysis is a crucial tool for analyzing interactions and motions during ligand–protein binding in structural molecular biology and computer-assisted drug discovery.[24,25] This drug was docked with a protein chosen using the Swiss ADME-Target prediction system. Before choosing the protein, the activities of the specific ligand were investigated, and the strong activities were taken into consideration. For the interaction, the protein was received in PDB format from the RCBS protein library,[26,27] as shown in Figure B,D. With the help of AutodockVina[28] and Chimera,[29] the titled molecule is studied further for biological activity. Lowering the value of binding energy more will show biological activity. In order to study the best in silico conformation with compounds under study, the Topoisomerase II alpha as a target receptor was used for docking studies. The titled molecule’s structure was drawn in Chimera by using a canonical smiles structure using Swiss ADME.[30] The structure was reduced to the minimum energy state. The suitable protein was downloaded from the RSB Protein data bank in PDB format, and then protein was prepared for docking by removing water moieties. The docking studies suggest well-established hydrogen bonds with amino acids at active sites. The active sites are locations where Topoisomerase II alpha formed a complex with compounds under evaluation. The active pocket consisted of amino acid residues such as LEU476, HIS475, GLY472, MET340, PHE356, ALA302, and MET295, as shown in Figure c. The energy minimization program was used to create a 3D structure of synthesized compounds; it was then used for protein ligand docking. Figure d exhibits docked images of the standard drug and synthesized compound ciprofloxacin. Figure a and b exhibits the binding energy and inhibition constant of compounds as well as the standard. In silico studies suggest that all the molecules under study showed good binding energy toward the target protein and the value of −0.2 kJ mol–1.

Figure 5

Protein binding active site of control drug ciprofloxacin [A]. Receptor with ligand docking of control drug [B]. Synthesis of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin of the protein binding active site [C]. Receptor with ligand molecule docking autodockvina [D].

Antimicrobial Activity

The MIC of the synthesized compound was evaluated by analyzing the turbidity observed in the culture tubes containing compounds ranging from 0.1 to 1 mg/mL, which suggests bacterial growth, whereas no growth could be seen in culture tubes containing compounds ranging from 0.5 to 10 mg/mL (Table ). A small aliquot of the sample from the culture tube containing compounds ranging from 0.5 to 10 mg/mL were collected and poured in an agar plate, which were allowed to grow for 24 h at ambient temperature conditions. No growth of bacteria was seen, thus indicting the antibacterial property of the compound at this particular concentration. Thus, it can be inferred that the MIC of the compound is observed at a concentration of 0.5 mg/mL.

Table 4

Minimum Inhibitory Concentration of Compoundsa

		bacterial growth at different concentrations
		compound
dilution for same bacterial concentration	sets	10 mg/mL	5 mg/mL	1 mg/mL	0.5 mg/mL	0.1 mg/mL
Staphylococcus aureus	1	-	-	-	-	+
	2	-	-	-	-	+
	3	-	-	-	-	+
Pseudomonas aeroginosa	1	-	-	-	-	+
	2	-	-	-	-	+
	3	-	-	-	-	+
Proteus vulgaris	1	-	-	-	-	-
	2	-	-	-	-	+
	3	-	-	+	-	+

(+) = Turbidity due to microbial growth; (−) = No Turbidity.

Conclusion

The present article reports the synthesis of heterocyclic compound 5-amino-6-cyano-3-hydroxybenzo[c]coumarin using an environmentally friendly procedure via Ni–Cu–Al–CO3 hydrotalcite, which has been used as efficient catalysts. These catalysts are inexpensive and nontoxic powders. Data of spectra and another analytical research supported the formation of 5-amino-6-cyano-3-hydroxybenzo[c]coumarin. The present work offers many merits such as ethanol/water, which were used as solvents, and the reaction conditions were extremely simple. There was operational simplicity; the reaction time was short and easy to work up; and purification was also easy for the products via simple means of recrystallization. The addition of the concentration of catalysts, i.e., hydrotalcites (0.1 g), viz., Ni–Cu–Al–CO3 hydrotalcite, resulted in the formation of product in good yields (89%), and with water the yield obtained was 70%. No yields were obtained when the reaction was carried out in solvents like acetonitrile and acetone. The molecular docking study showed the antimicrobial activity presence in the compounds. This was further confirmed by antimicrobialactivity. The synthesized compounds thus can be considered as promising leads, having biological activity such as antimicrobial. The electrochemical study showed the reduction and oxidation behavior of synthesized compounds.

Experimental Section

Methods and Materials

Nickel sulfate, copper sulfate, and aluminum nitrate were purchased from Rankem Fine Chemicals Ltd. NaOH and Na2CO3 were purchased from Fisher Scientific, respectively. The chemicals and the solvents were procured from laboratory/spectroscopy chemical suppliers and were used without purification. The salicylaldehyde was purchased from Himedia, and malononitrile, ethyl-acetoacetate, and ethanol were purchased from Thermo-Fisher.

All the catalyst patterns were obtained by Rigaku X’Pert diffractometer equipment, R (Modal no Mini Flex 600), using Cu Ka radiation (λ = 1.5406 Å) and a step size of 0.02°, voltage of 40 kV, and tube current of 15 mA. The data collected were in the 2θ range of 3–900. The goniometer used is Mini Flex 300/600, and the scanning speed of the instrument is 10.0000°/min. The incident slit is 1.250°, with a length limiting slit of 10.0 mm. The scanning mode is continuous, and the detector used is D/te X Ultra. The thermal stability of the hydrotalcite was determined using a Shimadzu thermal analyzer, Model No. TGA-50. The weight losses were recorded in the temperature range between 30 and 1000 °C, with a heating rate of 10 °C/min and under a constant flow of nitrogen (20 ± 0.5 mL/min). FTIR is one of the molecular vibrational spectroscopic techniques used to investigate the structural bonding and chemical properties of compounds and hence is helpful in the determination of the quantitative as well as qualitative analysis. The various functional groups have their frequencies, and hence they absorb specific IR radiations which detect the presence or absence of the groups. Infrared spectra were recorded using the PerkinElmer FTIR spectrometer in the range 4000–400 cm–1 using KBr pellets. IR vibration modes of hydroxyl groups and intercalated anions have been used in identifying interlayer species. C13 and 1H NMR spectra of the synthesized compounds were recorded using a Bruker 300 MHz or Bruker Advance III 500 MHz instrument, respectively. Chemical shifts (d) are given in parts per million using TMS as an internal standard. The abbreviations s, d, t, q, and m are used to indicate singlet, doublet, triplet, quadruplet, and multiple signals, respectively. The molecular weight of the compound was determined in an LC-MS instrument manufactured by Shimazdu equipment model number 8030.

HPLC analysis (Shimadzu LC8030 AD) was carried out on a C18 reversed-phase analytical column (150 mm–4.6 mm, particle size 5 mm) at 37 °C using mobile phases A, 0.1% formic acid in distilled water, and B, 100% methanol at a flow rate 0.6 mL min–1 (Table ). The following gradient was applied: linear increase from solution 30% B to 100% B in 15 min. The total flow rate is 0.6000 mL/min, and the pump concentration of B/A is 75:25%. The maximum and minimum pressure limits of both pumps A and B are 300 kgF/cm3 and 0 kgF/cm3, respectively. The detector used is a UV detector along with the wavelength of 254 nm with an auxillary range of 1.04 Au/m, and the temperature of the column oven is 40 °C.

Table 5

Zone of Inhibition Using 5-Amino-6-cyano-3-hydroxybenzo[c]coumarin and Standard Ciprofloxacina

		zone of inhibition of standard drug ciprofloxacin		zone of inhibition of synthesized compound
S. no.	microbes	A	B	A	B
1	Staphylococcus aureus	40 mm	≥40 mm	20 mm	21 mm
2	Pseudomonas aeruginosa	37 mm	40 mm	10 mm	22 mm
3	Proteus bulgaria	39 mm	35 mm	10 mm	21 mm

A is the concentration (100 μg/mL) of the standard drug and synthesized compounds, and B is the concentration (500 μg/mL) of the standard and synthesized compounds.

Preparation of the Ni–Cu–Al–CO3 Hydrotalcite Catalyst

The Ni–Cu–Al–CO3 hydrotalcite was synthesized using a coprecipitation technique at pH 10. In a typical method, 1 M NiSO4 (1.9 g), 3 M CuSO4 (9 g), and 1 M Al2NO3 (2.6 g) corresponding to a Ni/Cu/Al molar ratio of 1:3:1 were dissolved in water (50 mL). Addition of 1 M NaOH and 1 M Na2CO3 results in the precipitation. After the precipitation is completed, the solutions are kept for 6 h. The precipitate was washed several times with deionized water and filtered. The obtained catalyst was dried at 120 °C for 8 h.

General Synthesis of the Product [5-Amino-6-cyano-3-hydroxybenzo[c]coumarin]

A mixture of salicyl-aldehyde (2 mmol) and ethyl-acetoacetate (2 mmol) and 100 mg of Ni–Cu–Al–CO3 hydrotalcite catalyst were mixed in 5 mL of ethanol/water at 80 °C for 30 min. After 30 min, malononitrile (2 mmol) was added to the reaction mixture, keeping the same reaction conditions. After 1 h when the reaction was completed, the reaction mixture was filtered using the Whatman filter paper, and the filtered mass was kept for 24 h for the separation of the catalyst from the product; the solvent was also evaporated from the product. A reported compound named [5-amino-6-cyano-3-hydroxybenzo[c]coumarin] was obtained. The color of the compound obtained was brownish orange which was soluble in ethanol, and the observed melting point was 201 °C. Brownish orange powder, mp 201 °C; 1H NMR (300 MHz, MeOH) (δ ppm) 9.96 (broad OH, 1H), 8.67 (s, 1H), 7.60 (dd, j = 1.42 Hz and j = 8.10 Hz), 7.378 (m, 2H), 7.36 (dd, j = 1.45 Hz, j = 8.12 Hz, 1H), 7.122 (broad, 2H, NH2). 13C NMR (75 MHz CDCl3-d6) δ = 156.66, 152.07, 135.73, 129.49, 129.10, 125.88, 124.75, 118.04, 117.60, 113.73, 103.48, 61.60, 50.33, 14.26, 77.23 (solvent peak). Anal. Calcd (%) for C14H8N2O3; LCMS spectra (ESI) m/z (%) 251 (M–).

1H NMR and 13C NMR Spectra of the 3-Acetylcoumarin [Intermediate]

Yellow powder, mp 120 °C; 1H NMR (CDCl3, 500 MHz) (δ ppm) 2.71 (aliphatic C–H), 7.32–7.65 (Ar–H), 8.45 (benzo-fused-coumarin-H-proton). 13C (CDCl3, 500 MHz) (δ ppm) 134.38 (C7), 130.23 (C5), 124.97 (C6), 124.47 (C8), 118.21 (C9), 116.61 (C3) (all peaks were observed to have a chemical shift), 195.386 (C17-carbonyl carbon), 30.493 (C19-methyl group carbon), 159.146 (C2), 155.251 (C10), 147.395 (C4), 77 (solvent peak); LCMS (ESI) m/z (%) 189 (M+).

Evaluation of Antimicrobial Activities

Working Stock Solution Preparation

The stock solution was prepared by dissolving 0.1, 0.5, 1, 5, and 10 mg of synthesized compound in 1 mL of DMSO. Three prospective pathogenic microbes were evaluated against five different concentrations of synthesized compound.

Microorganisms Used and Their Preparation

Three microbial species were used for the present study which are as follows: Staphylococcus aureus. MTCC 87 it is an example of Gram-positive bacteria, Pseudomonas aeruginosa. MTCC 424 an example for Gram-negative bacteria, Proteus bulgaris (Figure ). MTCC 742 is an example of Gram-negative bacteria. All bacterial samples were cultured into nutrient agar and malt extract broth for incubation overnight at 37 °C.

Figure 6

Antimicrobial activity of the compound 5-amino-6-cyano-3-hydroxybenzo[c]coumarin synthesized using standard drug ciprofloxacin with Staphylococcus aureus [A], Pseudomonas aeruginosa [B], and Proteus bulgaria [C].

Antimicrobial Susceptibility Testing

Well Diffusion Method

Agar well diffusion is a method which is widely used to study the antimicrobial activity of synthesized compounds. Similarly to the procedure used in the disc diffusion method, the agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface. Then, a hole with a diameter of 6–8 mm is punched aseptically with a sterile cork borer or a tip, and a volume (20–100 μL) of the antimicrobial agent or synthesized compound solution at the desired concentration is introduced into the well. Then, agar plates are incubated under suitable conditions depending on the test microorganism. The antimicrobial agents diffuse in the agar medium and inhibit the growth of the microbial strain tested.

Minimum Inhibitory Concentrations (MICs)

The minimum inhibitory concentration of the synthesized compound was then calculated at different concentrations ranging from 0.1, 0.5, 1, 5, and 10 mg/mL against Staphylococcus aureus, Pseudomonas aeroginosa, and Proteus vulgaris by a broth dilution method in nutrient broth. The concentration of culture was adjusted to 0.2 at 568 nm (1 × 108 CFU/mL, 0.5 McFarland’s standard). Positive and negative control was used as a standard. The minimum inhibitory concentration was evaluated by moving the turbidity. Small aliquots of the sample (approximately 50 μL) from the culture tubes showing the least or no turbidity were taken and poured in an agar plate for 24 h, and the bacterial growth was observed. The experiment was performed in triplicates (Krishnan et al., 2015; Cavassin et al., 2015).