Introduction

Antimicrobial agents are important because they prevent bacteria from multiplying and growing. Antimicrobials are effective against a wide variety of infectious diseases caused by pathogens. In order to combat rising antimicrobial resistance, it is imperative to develop new and potentially beneficial antimicrobials that will be less toxic [1, 2]. The heterocyclic compounds possessing chromene moiety are widely used in many industries, containing pharmaceutics [3], cosmetics [4], biodegradable agrochemicals [5], and pigments [6]. 2-Amino-4H-chromenes as one of the most well-known chromene derivatives have attracted much attention for its medicinal and biological activities, such as antimalarial [7], anti-HIV [8], antifungal [9], antimicrobial [10], antitumor [11], antioxidant [12], antileishmanial [13], anti-inflammatory [14], hypotensive [15], and inhibitors properties [16]. As an example, compounds A [17], B [18], and C [19] exhibited antibacterial activities (Fig. 1). Additionally, they are widely used to treat neurodegenerative diseases by enhancing cognitive function such as, such as Parkinson, Alzheimer, schizophrenia, Down syndrome, myoclonus, and Huntington, as well as amyotrophic lateral sclerosis [20,21,22,23,24].

Fig. 1
figure 1

Some biologically active compounds including 2-amino-4H-chromene moiety

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The importance of 2-amino-4H-chromene derivatives has resulted in the development of a variety of reactions to prepare them. One of the most important reactions in this context is the multicomponent condensation reaction of OH-Acids, aldehyde, and malononitrile. This transformation has been catalyzed with a variety of homogeneous or heterogeneous catalysts, such as, [Cu (bpdo)2.2H2O]2+/montmorillonite [25], Basic ionic liquid [PEMIM][OH] [26], Ruthenia doped fluorapatite (RuO2/FAp) [27], Hyper-crosslinked microporous polyphenanthrene [28], Nano polypropylenimine dendrimer (DAB-PPI-G1) [29], Ca(OH)2 [30], Tungstic acid functionalized mesoporous SBA-15 [31], Na2CO3 [32], CuO/ZnO@N-GQDs@l-proline hexagonal nanocomposite [33], p-Cymene Ru(II) complex [34], 4-(N,N-dimethylamino)pyridine (DMAP) [35], DABCO [36], Tetramethylguanidine [37], Diethylamine [38], Triethylamine [39], as well as Potassium phthalimide- N-oxyl (POPINO) [40]. Despite the fact that these procedures are suitable for synthesizing 4H-chromenes, many of them suffer from one or more drawbacks, such as long reaction times, difficult workups, the use of expensive catalysts, or the need for special equipment.

Multicomponent reactions (MCRs) can provide a cost-effective and time-saving method for constructing a wide range of chemicals including complex organic molecules, pharmaceuticals, and biologically active compounds. They have been extensively used in synthesizing natural products and other biologically active molecules since its discovery. In recent years, MCRs have gained significant popularity due to their various advantages, such as environmental friendliness, simplistic completion, mild conditions, and high efficiency [41].

The results of these findings encourage us to develop catalytically efficient, simple, fast, and green procedures for synthesizing heterocyclic compounds containing 2-amino-4H-chromene systems. Furthermore, as part of our research interest in the synthesis of potentially bioactive heterocyclic compounds [42,43,44,45,46,47,48,49,50], here we present the green protocol for synthesizing novel 2-amino-4H- chromene derivatives through a one-pot three-component condensation reaction of malononitrile, aromatic aldehydes, and 2,4-dihydroxybenzophenone in the presence of the catalytic amount of triethylamine (Scheme 1) and evaluation of their antioxidant and antibacterial activities.

Scheme 1
scheme 1

Preparation of 2-amino-4H-chromenes through a one-pot three-component condensation reaction

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Results and discussion

The optimal reaction conditions were determined by performing a three-component reaction with benzaldehyde (1a), malononitrile (2) with 2,4-dihydroxybenzophenone (3) in 10 mL ethanol with a catalyst. The optimal reaction conditions were investigated by performing a three-component reaction of the benzaldehyde (1a), malononitrile (2) with 2,4-dihydroxybenzophenone (3) in the presence of a catalyst in 10 mL ethanol as a model reaction. As tabulated in Table 1, the reaction was examined in the presence of 0.5 equivalents of several bases such as piperidine, triethylamine, sodium hydroxide, potassium hydroxide, potassium carbonate, as well as a free-catalyst condition (entries 1–6). The results showed that Et3N afforded to the desired product in higher yield than other bases (entry 3 compared to entries 2 and 4–6). Additionally, the reaction was tested with various amounts of Et3N (entries 7–10) and a 25% proportion was determined as optimum (entry 8) and higher amounts of triethylamine did not improve the reaction yield. Following this, the model reaction was tested in various solvents, including MeOH, H2O, EtOH, EtOH:H2O, CH3CN, THF, CHCl3, and Et2O demonstrating that EtOH is the optimal solvent for this reaction (entries 11–17). As a final step, the reaction was investigated at various temperatures (entries 18–20). Compared to entries 18 and 19, entry 20 yielded the highest yield when the reaction was carried out in refluxed ethanol.

Table 1 The optimization of reaction conditions for the synthesis of compound 4a
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Three-component reaction of aromatic aldehydes 1a–m, malononitrile 2, and 2,4-dihydroxybenzophenone 3 in refluxed ethanol and in the presence of Et3N (25%) was carried out to determine the scope and limitations of this reaction. Table 2 shows good to excellent yields of the corresponding products (80–99%). According to the results, the reaction yields increased for aromatic aldehydes containing electron withdrawing substituents at the para position (entries 2–6 compared to entries 1 and 11). These aromatic aldehydes were more effective at obtaining the desired product with higher yield than aromatic aldehydes with electron withdrawing substituents at the ortho position (for example entry 6 compare to entry 12). It can be due to the result of steric hindrance of substituents at their ortho position of aromatic rings of the aldehydes.

Table 2 Synthesis of 2-amino-4H-chromene derivatives 4a–m
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The structure of all the synthesized 2-amino-4H-chromens was confirmed with IR, 1H NMR, 13C NMR, and mass spectrum. The FT-IR spectrum of 4f displays two signals at 3334 cm−1 and 3199 cm−1 for NH2 group, at 3059 cm−1 for the Csp2 –H group, at 2920 cm−1 for the Csp3 –H group, a sharp signal at 2205 cm−1 for the CN group, a strong absorption band at 1659 cm–1 for the carbonyl group, at 1603 cm−1 for the C = C group, and a sharp signal at 1254 cm−1 for the Csp2 –O group. The mass spectrum of this compound exhibits the two molecular ion peaks at m/z = 404 (M+. + 2) and m/z = 402 (M+.), and the base peak at m/z = 291 (M+. -ClC6H4) agrees with the proposed structure.

The 1H NMR spectrum of 4f in DMSO-d6 at 25 °C exhibits a singlet at about 4.76 ppm for the CH group of 4H-pyran moiety, a doublet at about 6.73 ppm (1H, 3JHH = 8.8 Hz) for the aromatic CH proton, a broad signal at about 7.18 ppm for the exchangeable protons of NH2 group, three doublets at about 7.22 (2H, 3JHH = 8.4 Hz), 7.37 (2H, 3JHH = 8.4 Hz), 7.49 (1H, 3JHH = 8.8 Hz), a triplet signal at about 7.55 ppm (2H, 3JHH = 6.8 Hz), a multiple signal at about 7.62–7.64 (3H) for the aromatic CH protons, and a broad signal at about 12.26 ppm for the exchangeable proton of OH group. The 13C NMR spectrum of 4f in DMSO-d6 at 25 °C displays a signal at about 36.1 ppm for the CH group of 4H-pyran moiety, and signals at about 57.1, 160.8, 116.5 and 200.0 ppm for the quaternary carbons (C* = C–N and C = C*–N), CN group, and carbonyl group of ketone, respectively. Also, the 13C NMR spectrum of 4f exhibited 14 signals with relevant chemical shifts for the aromatic carbons.

Scheme 2 illustrates a proposed mechanism for this reaction. Initially, α,β- unsaturated I is formed by the Knoevenagel condensation between aromatic aldehyde 1 and malononitrile 2 in the presence of triethylamine. In the basic condition, 2,4-dihydroxybenzophenone 3 loses its proton and is converted to the enolate ion II that performs Michael addition on Cβ of compound I to obtain intermediate III. In the following, it carried out a cyclization reaction to afford compound V which lead to the corresponding product 4 through a 1,3-proton transfer.

Scheme 2
scheme 2

A proposed mechanism for preparing 2-amino-4H-chromenes 4a–m

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As it can be seen in Scheme 3, there are two possible positions including path A and B for performing Michael’s addition on Cβ of compound I which could be led to products 4 and 5, respectively.

Scheme 3
scheme 3

Two possible positions for performing Michael addition

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Investigation of the 1H NMR spectra of products 4a-m showed that the splitting of HB is appeared as a doublet signal which confirms the reaction has progressed only through path A and only product 4 was obtained. For example, expanded 1H NMR spectrum of compound 4f is shown in Fig. 2. Observing two doublet signals for HA (6.73 ppm) and HB (7.49 ppm) confirms that the reaction mechanism is performed only through path A.

Fig. 2
figure 2

The expanded 1H NMR spectrum of compound 4f

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Antioxidant activity

DPPH radical scavenging was used by Blois's method to assess the in vitro antioxidant activity of 2-amino-4H-chromenes 4a–m and compound 3. Antioxidants containing a high number of heteroatoms, π-electrons, and exchangeable hydrogen atoms are more effective to scavenge free radicals produced by DPPH. An absorption decrease at 517 nm wavelength could indicate the presence of antioxidants by changing the color of the DPPH test solution from dark purple to light yellow. Figure 3 shows that the synthesized compounds inhibited DPPH with potencies ranging from 71 to 95 percent, much better than ascorbic acid as a standard antioxidant (84%). Also, the IC50 values of the antioxidant activities of all tested samples were studied. Among these compounds, 4b exhibited the highest free radical scavenging activity (IC50 = 10.32 ± 0.35 μM), this could be due to the fact that it contains more heteroatoms with lone-pair electrons in its structure than other examined compounds and also includes exchangeable protons in NH2 and OH groups. [51].

Fig. 3
figure 3

Antioxidant activity of the synthesized compounds 3 and 4a–m

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Antibacterial activity

The minimum inhibitory concentration (MIC) method was applied to investigate the antibacterial activity of compounds 3 and 4a–m against Staphylococcus aureus (ATCC2592) as Gram-positive bacteria and Gram-negative bacteria Escherichia coli (ATCC1399), and was compared with Cefixim as a standard antibiotic. According to Table 3, the synthesized compounds 4a–m are generally more effective than starting material (3) against all of the tested microorganisms. In addition, all of the examined samples showed higher antibacterial activity against Gram-positive bacteria than Gram-negative bacteria. Among these compounds, 4b and 4 g exhibited the highest activity against S. aureus as Gram-positive bacteria (MIC = 0.625 mM) and E. coli Gram-negative bacteria (MIC = 1.25 mM).

Table 3 Antibacterial activity of the compounds 4a–m using MIC valuesa
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Conclusion

We have synthesized a novel series of 2-amino-4H-chromene derivatives by the three-component reaction between 2,4-dihydroxybenzophenone, malononitrile and a variety of aromatic aldehydes in the presence of triethylamine as catalyst in ethanol as a green solvent with high to excellent yields of the products. The structure of the synthesized products was characterized by FTIR, 1H, 13C NMR spectroscopy, CHN analyses, and mass spectrometry. In comparison to other reported procedures, this procedure is simple, eco-friendly, requires a short reaction time, and allows for easy separation of the products. The antibacterial and antioxidant activity of all of the products investigated against Staphylococcus aureus (a Gram-positive bacteria) and Escherichia coli (a Gram-negative bacteria) using the MIC method, as well as the radical scavenger DPPH. The results demonstrate that compound 4b showed the highest antioxidant and antibacterial activities. We are investigating novel approaches to synthesize more complex structures that exhibit antimicrobial activities in continuation of our studies in heterocyclic chemistry.

Experimental

The following chemicals were purchased from the Merck Company (Germany): malononitrile, 2,4-dihydroxybenzophenone, triethylamine, aromatic aldehydes, and solvents. The structures of synthesized samples 4a–m were confirmed using the following analyses. The melting point of the compounds 4a–m was determined with Electrothermal IA9100 (Essex, UK). 1HNMR and 13CNMR spectra were recorded using DMSO-d6 as solvent and TMS as an internal reference on a Bruker-400 Avance III spectrometer (Bruker, Germany). The FTIR spectra were recorded using a Bruker vector 22 spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were measured out on Finnigan-MAT 8430 mass spectrometer operating in electron impact mode. The UV/Vis spectrophotometry was achieved by Anthos 2020 Microplate Reader (Anthos, Biochrom, UK).Elemental analyses were accomplished using a Heraeus CHN-O rapid analyzer (Germany). The GC report for compound 4b was performed by a Agilent 7890A (USA).

General procedure for the synthesis of compounds 4a–m

A mixture of aromatic aldehydes 1a–m (1.0 mmol), malononitrile 2 (1.0 mmol), and 2,4-dihydroxybenzophenone 3 (1.0 mmol) in the presence of 25% mole triethylamine was magnetically stirred in 10 ml of ethanol and agitated at the reflux condition for an appropriate time (Table 2). After completion of the reaction (followed by TLC), the mixture was allowed to cool to room temperature. The precipitated product was filtered and crystallized in ethanol to obtain the pure desired product 4a–m.

2-Amino-6-benzoyl-4-phenyl-5-hydroxy-4H-chromene-3-carbonitrile (4a, C 23 H 16 N 2 O 3 )

Cream powder, m.p. 276–278 °C (Reported 280–282 °C [29],), yield: 90%; IR (KBr) (υmax, cm−1): 3501 (OH), 3422 and 3324 (NH2), 3032 (Csp2 –H), 2922 (C sp3–H), 2184 (CN), 1653 (C = O), 1603 (C = C), 1253(C sp2–O). 1H NMR (400.13 MHz, DMSO-d6): δH 4.75 (s, 1H, CH), 6.74 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.13 (s, 2H, NH2), 7.19 (d, 2H, 3JHH = 7.2 Hz, 2CHAr), 7.21 (t, 1H, 3JHH = 8.0 Hz, CHAr), 7.31 (t, 2H, 3JHH = 7.6 Hz, 2CHAr), 7.50 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.55 (t, 2H, 3JHH = 8.4 Hz, 2CHAr), 7.63–7.67 (m, 3H, 3CHAr), 12.41 (s, 1H, OH).13C NMR (100.6 MHz, DMSO-d6): δC 36.6 (CH), 57.6 (C* = C–N), 108.0 and 112.0 (Cq), 116.6 (CN), 120.5 (Cq), 127.2, 127.6, 128.9, 129.3, 132.6 133.9 and 137.6 (CH), 145.2, 154.3 and 160.0 (Cq), 160.7 (C = C*–N), 200.1 (C = O). MS: m/z (%) 368 (M+., 15), 291 (M+ –C6H4, 100), 213 [(M+–(CH3C6 H4 + C6H5)), 57], 185 [(M+–(C6H5 + C6H5CO + H), 5)], 105 (C6H5CO+, 22), 77 (C6H5+, 11); Anal. Calcd for C23H16N2O3 (368.12): C, 74.99; H, 4.38; N, 7.60, Found: C, 75.10; H, 4.37; N, 7.57.

2-Amino-6-benzoyl-4-(2،4-dichlorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4b, C 23 H 14 Cl 2 N 2 O 3 )

Cream powder, m.p. 280–282 °C, yield: > 99%; IR (KBr) (υmax, cm−1): 3402 (OH), 3312 and 3201 (NH2), 3083 (C sp2 –H), 2927 (C sp3–H), 2198 (CN), 1659 (C = O), 1612 (C = C), 1259 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 5.23 (s, 1H, CH), 6.73 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.16 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.19 (s, 2H, NH2), 7.36 (dd, 1H, 3JHH = 8.4 Hz,4JHH = 2.4 Hz, CHAr), 7.52 (dd, 2H, 3JHH = 9.2 Hz, 3JHH = 8.0 Hz, 2CHAr), 7.56–7.57 (m, 2H, 2CHAr), 7.62–7.67(m, 3H, 3CHAr), 12.38 (s, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 33.9 (CH), 55.6 (C* = C–N), 107.9 and 111.0 (Cq), 116.3 (CN), 119.8 and 128.3 (Cq), 128.9, 129.3 (CH), 132.3 (Cq), 132.4, 132.6 and 133.4 (CH), 134.3 (Cq), 137.4 (CH), 141.3 and 141.4 (Cq),154.5 and 159.9 (CH), 160.9 (C = C*–N), 200.0 (C = O). MS: m/z (%) 440 (M+  + 4, 3), 438 (M+  + 2, 7), 436 (M+., 11), 403 ( (M+. + 2)–Cl, 2)), 401 (M+ -Cl, 7), 291 (M+.–Cl2C6H3, 100), 213 [(M+.–(Cl2 C6H3 + C6H5 + H)), 60], 105 (C6H5CO+, 13), 77 (C6H5+, 15); Anal. Calcd for C23H14Cl2N2O3 (436.04): C, 63.17; H, 3.23; N, 6.41, Found: C, 63.06; H, 3.22; N, 6.39.

2-Amino-6-benzoyl-4-(4-formylphenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4c, C 24 H 16 N 2 O 4 )

Cream powder, m.p. 218–220 °C, yield: 98%; IR (KBr) (υmax, cm−1): 3449 (OH), 3348 and 3206 (NH2), 3064 (C sp2 –H), 2924 (C sp3–H), 2850 (CO–H), 2195 (CN), 1692 and 1650 (C = O), 1605 (C = C), 1256 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.86 (s, 1H, CH), 6.77 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.22 (s, 2H, NH2), 7.41 (d, 2H 3JHH = 8.0 Hz, 2CHAr), 7.52 (d, 2H, 3JHH = 8.8 Hz, 2CHAr), 7.55 (d, 2H, 3JHH = 7.6 Hz, 2CHAr), 7.62–7.65 (m, 2H, 2CHAr), 7.87 (d, 2H, 3JHH = 8.4 Hz, 2CHAr), 9.92 (s, 1H, CHO), 12.21 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.8 (CH), 56.7 (C* = C–N), 108.1 and 111.8 (Cq), 116.5 (CN), 120.2 (Cq), 128.5, 128.9, 129.3, 130.4, 132.7, 134.2 and 135.4 (CH), 137.5, 151.7, 154.1 and 159.6 (Cq), 160.7 (C = C*–N), 193.1 and 200.0 (C = O). MS: m/z (%) 396 (M+., 24), 291 (M+.–CHOC6H4, 100), 291 (M+.–CHOC6H4, 100), 213 [(M+.–(CHOC6H4 + C6H5), 46)], 185 [(M+.–(CHOC6H4 + C6H5CO + H), 5)], 105 (C6H5CO+, 10), 77 (C6H5+, 12); Anal. Calcd for C24H16N2O4 (396.11): C, 72.72; H, 4.07; N, 7.07, Found: C, 72.80; H, 4.09; N, 7.05.

2-Amino-6-benzoyl-4-(4-cyanophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4d, C 24 H 15 N 3 O 3 )

Cream powder, m.p. 254–255 °C, yield: 96%; IR (KBr) (υmax, cm−1): 3545 (OH), 3451 and 3351 (NH2), 3105 (C sp2 –H), 2922 (C sp3–H), 2226 and 2197 (CN), 1650 (C = O), 1607 (C = C), 1256 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.87 (s, 1H, CH), 6.75 (d, 1H, 3JHH = 9.2 Hz, CHAr), 7.26 (s, 2H, NH2), 7.39 (d, 2H, 3JHH = 8.4 Hz, 2CHAr), 7.50–7.56 (m, 3H, 3CHAr), 7.63–7.65 (m, 3H, 3CHAr), 7.79 (d, 2H, 3JHH = 7.6 Hz, 2CHAr), 12.30 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 38.8 (CH), 56.5 (C* = C–N), 108.1, 110.1 and 111.5 (Cq), 116.6 and 119.2 (CN), 120.1 (Cq), 128.8, 128.9, 129.3, 132.7 and 133.1 (CH), 134.2 (Cq), 137.5 and 150.5 (CH), 154.1 and 160.0 (CqAr), 160.6 (C = C*–N), 199.9 (C = O). MS: m/z (%) 393 (M+. + 23), 291 (M+.–CNC6H4, 100), 213 [(M+.–(CNC6H4 + C6H4 + H), 60], 185 [(M+.–(CNC6H4 + C6H5CO + H), 105 (C6H5CO+, 15), 77 (C6H5+, 16); Anal. Calcd for C24H15N3O3 (393.11): C, 73.27; H, 3.84; N, 10.68, Found: C, 73.36; H, 3.82; N, 10.70.

2-Amino-6-benzoyl-4-(4-bromorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4e, C 23 H 15 BrN 2 O 3 )

Orange powder, m.p. 254–256 °C (Reported 248–250 °C [29],), yield: 95%; IR (KBr) (υmax, cm−1): 3442 (OH), 3329 and 3251 (NH2), 3054 (C sp2 –H), 2921 (C sp3–H), 2199 (CN), 1662 (C = O), 1607 (C = C), 1256 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.75 (s, 1H, CH), 6.74 (d, 1H, 3JHH = 9.2 Hz, CHAr), 7.15 (d, 2H, 3JHH = 8.0 Hz, 2CHAr), 7.19 (s, 2H, NH2), 7.49–7.53 (m, 3H, 3CHAr), 7.56 (d, 2H 3JHH = 7.2 Hz, 2CHAr), 7.63–7.67 (m, 3H, 3CHAr), 12.40 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.2(CH), 57.0(C* = C–N), 108.1 and 112.2 (Cq), 116.5 (CN), 120.2 and 120.3(Cq), 128.9, 129.3,130.0, 131.8, 132.6, 134.0 and 137.5 (CH), 144.6, 154.1 and 159.9 (Cq), 160.7 (C = C*–N), 200.0 (C = O). MS: m/z (%) 448 (M+. + 2, 14), 446 (M+., 14), 367 (M+. –Br, 5), 291 (M+.–BrC6H4, 100), 213 [(M+.–(BrC6 H4 + C6H5), 64)], 185 [(M+.–(BrC6H4 + C6H5CO + H), 5)], 105 (C6H5CO+, 17), 77 (C6H5+, 19); Anal. Calcd for C23H15BrN2O3 (446.03): C, 61.76; H, 3.38; N, 6.26, Found: C, 61.57; H, 3.40; N, 6.28.

2-Amino-6-benzoyl-4-(4-chlorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4f, C 23 H 15 ClN 2 O 3 )

Yellow powder, m.p. 235–236 °C, yield: 94%; IR (KBr) (υmax, cm−1): 3433 (OH), 3334 and 3199 (NH2), 3059 (Csp2 –H), 2920 (Csp3–H), 2205 (CN), 1659 (C = O), 1603 (C = C), 1254 (Csp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.76 (s, 1H, CH), 6.73 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.18 (s, 2H, Exchangeable with D2O, NH2), 7.22 (d, 2H, 3JHH = 8.4 Hz, 2CHAr), 7.37 (d, 2H, 3JHH = 8.4 Hz, 2CHAr), 7.49 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.55 (t, 2H, 3JHH = 6.8 Hz, 2CHAr), 7.62–7.64 (m, 3H, 3CHAr), 12.26 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.1(CH), 57.1 (C* = C–N), 107.9 and 112.3 (Cq), 116.5 (CN), 120.3 (Cq) 128.9, 129.0, 129.3, 129.6, 131.7 and 132.6 (CH), 134.0 and 137.5 (Cq), 144.2 (CH), 154.1 and 159.9 (Cq), 160.8 (C = C*–N), 200.0 (C = O). MS: m/z (%) 404((M+. + 2), 5), 402 (M+., 17), 291 [(M+. -ClC6H4), 100], 213 [(M+.–(ClC6H4 + C6H5 + H), 57], 185 [(M+.–(ClC6H4 + C6H5CO + H), 5], 105 (C6H5CO+, 13), 77 (C6H5+, 16); Anal. Calcd for C23H15ClN2O3 (402.08): C, 68.58; H, 3.75; N, 6.95, Found: C, 68.67; H, 3.74; N, 6.93.

2-Amino-6-benzoyl-4-(4-fluorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4 g , C 23 H 15 FN 2 O 3 )

Pink powder, m.p. 285–287 °C, yield: 90%; IR (KBr) (υmax, cm−1): 3442 (OH), 3327 and 3411 (NH2), 3211 (C sp2 –H), 3061 (C sp3–H), 2193 (CN), 1658 (C = O), 1608 (C = C), 1261 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.77 (s, 1H, CH), 6.74 (d, 1H, 3JHH = 9.2 Hz, CHAr), 7.13 (t, 2H, 3JHH = 8.8 Hz, 2CHAr), 7.16 (s, 2H, NH2), 7.20–7.24 (m, 2H, 2CHAr), 7.49 (d, 1H 3JHH = 8.8 Hz, CHAr), 7.53–7.57 (m, 2H, 2CHAr), 7.63–7.64 (d, 3H, 3JHH = 7.6 Hz, 3CHAr), 12.42 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.0 (CH), 57.5 (C* = C–N), 108.1 and 112.6 (Cq), 115.6 (d, 2JCF = 21.4 Hz) (CH), 116.5 (CN), 120.4 (Cq), 128.9, 129.3, 129.5 (d, 3JCF = 8.1 Hz), 132.6, 133.9 and 137.6 (CH), 141.4 (d, 4JCF = 2.7 Hz), 154.1 and 159.9 (Cq), 160.7 (C = C*–N), 161.4 (d, 1JCF = 241.2 Hz) (Cq), 200.10 (C = O). MS: m/z (%) 386 (M+., 38), 291 (M+.–FC6H4, 100), 213 [(M+.–(FC6H4 + C6H5), 69)], 185 [(M+.–(FC6H4 + C6H5CO + H), 5)], 105 (C6H5CO+, 15), 77 (C6H5+, 19); Anal. Calcd for C23H15FN2O3 (386.11): C, 71.50; H, 3.91; N, 7.25, Found: C, 71.49; H, 3.93; N, 7.22.

2-Amino-6-benzoyl-4-(3-nitrophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4 h , C 23 H 15 N 3 O 5 )

Brown powder, m.p. 242–244 °C, yield: 90%; IR (KBr) (υmax, cm−1): 3429 (OH), 3338 and 3207 (NH2), 3061 (C sp2 –H), 2922 (C sp3–H), 2195 (CN), 1645 (C = O), 1609 (C = C), 1530 and 1343 (NO2), 1256 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.99 (s, 1H, CH), 6.78 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.29 (s, 2H, NH2), 7.51–7.56 (m, 3H, 3CHAr), 7.63–7.66 (m, 4H, 4CHAr), 7.70 (d, 1H, 3JHH = 7.6 Hz, CHAr), 8.02 (s, 1H, CHAr),), 8.11 (d, 1H, 3JHH = 8.0 Hz, CHAr), 12.34 (s, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.4(CH), 56.6 (C* = C–N), 108.2 and 111.6 (Cq), 116.8 (CN), 120.1 (Cq), 122.1, 122.4, 128.9, 129.4, 130.7 and 132.7 (CH), 134.3 (Cq), 134.6, 137.5 and 147.3 (CH), 148.2, 154.0 and 160.1 (Cq), 160.5 (C = C*–N), 199.9 (C = O). MS: m/z (%) 413 (M+. + 13), 291 (M+.–NO2C6H4,, 100), 213 [(M+.–(NO2C6H4 + C6H5 + H), 37], 185 [(M+.–(NO2C6H4 + C6H5CO + H), 5)], 105 (C6H5CO+, 6), 77 (C6H5 +, 7); Anal. Calcd for C23H15N3O5 (413.10): C, 66.83; H, 3.66; N, 10.16, Found: C, 66.75; H, 3.68; N, 10.18.

2-Amino-6-benzoyl-4-(3-bromorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4i, C 23 H 15 BrN 2 O 3 )

Yellow powder, m.p. 248–250 °C, yield: 90%; IR (KBr) (υmax, cm−1): 3439 (OH), 3340 and 3218 (NH2), 3054 (C sp2 –H), 2918 (C sp3–H), 2193 (CN), 1647 (C = O), 1606 (C = C), 1254 (C sp2–O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.78 (s, 1H, CH), 6.75 (d, 1H, 3JHH = 8.8 Hz, CHAr),7.19 (d, 1H, 3JHH = 8 Hz, CHAr), 7.21 (s, 2H, NH2), 7.29 (t, 1H, 3JHH = 7.6 Hz, CHAr), 7.35 (t, 1H, 4JHH = 1.6 Hz, CHAr), 7.43 (d, 1H, 3JHH = 7.6 Hz, CHAr), 7.51 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.53–7.57 (m, 2H, 2CHAr), 7.63–7.67 (m, 3H, 3CHAr), 12.37 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.3(CH), 57.0(C* = C–N), 108.1, 112.0 (Cq), 116.6 (CN), 120.2 and 122.1 (Cq), 126.9, 128.9, 129.3 130.2 130.3, 131.3, 132.6, 134.1 and 137.5 (CH), 147.8, 154.1 and 160.0 (Cq), 160.6 (C = C*–N), 199.9 (C = O). MS: m/z (%) 448 (M+. + 2, 2), 446 (M+., 2), 367 (M+.–Br, 5), 307 [(M+.–(Br + CN + NH2 + OH + H), 53], 291 (M+.–BrC6H4, 32), 261 [(M+.–(BrC6 H4 + CH + OH), 76)], 189 [(M+. -(BrC6H4 + C6H4 –CN + H, 100)], 105 (C6H4CO+, 6)، 77 (C6H5 +, 7); Anal. Calcd for C23H15BrN2O3 (446.03): C, 61.76; H, 3.38; N, 6.26, Found: C, 61.55; H, 3.35; N, 6.28.

2-Amino-6-benzoyl-4-(3-chlorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4j, C 23 H 15 ClN 2 O 3 )

Orange powder, m.p. 231–233 °C, yield: 90%; IR (KBr) (υmax, cm−1): 3433 (OH), 3334 and 3199 (NH2), 3059 (C sp2 –H), 2920 (C sp2–H), 2205 (CN), 1745 (C = O), 1659 (C = C), 1254 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 4.79 (s, 1H, CH), 6.75 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.15 (d, 1H, 3JHH = 7.6 Hz, CHAr), 7.21 (s, 3H, NH2 + CHAr), 7.28–7.31 (m, 1H), 7.36 (t, 1H, 3JHH = 7.6 Hz, CHAr), 7.51 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.55 (dd, 2H, 3JHH = 8.0 Hz, 3JHH = 8.0 Hz, 2CHAr), 7.64–7.66 (m, 3H, 3CHAr), 12.20 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 36.4 (CH), 57.0 (C* = C–N), 108.1 and 112.0 (Cq), 116.7 (CN), 120.3 and 126.5 (Cq), 127.2, 127.4, 128.9, 129.3, 131.0, 132.6 and 133.4 (CH), 134.1 (Cq), 137.6, and 147.6 (CH), 154.1 and 160.0 (Cq), 160.6 (C = C*–N), 199.9 (C = O). MS: m/z (%) 404 (M+. + 2), 7), 402 (M+., 21), 291 [(M+. -ClC6H4), 100], 213 [(M+.–(ClC6H4 + C6H5 + H), 61], 185 [(M+.–(ClC6H4 + C6H5CO + H), 61)], 105 (C6H5CO+, 12), 77 (C6H5 +, 15)); Anal. Calcd for C23H15ClN2O3 (402.08): C, 68.58; H, 3.75; N, 6.95, Found: C, 68.49; H, 3.73; N, 6.97.

2-Amino-6-benzoyl-4-(4-methoxyphenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4 k , C 24 H 18 N 2 O 4 )

Cream powder, m.p. 238–240 °C (Reported 232–233 °C [29],), yield: 85%; IR (KBr) (υmax, cm−1): 3437 (OH), 3297 and 3331 (NH2), 3065 (C sp2 –H), 2925(C sp3–H), 2199 (CN), 1659 (C = O), 1606 (C = C). 1H NMR (400.1 MHz, DMSO-d6): δH 3.71 (s, 3H, OCH3), 4.68 (s, 1H, CHAr), 6.73 (d, 1H, 3JHH = 8.8 Hz, CHAr), 6.87 (d, 2H 3JHH = 8.8 Hz, CHAr), 7.02 (s, 2H, NH2), 7.10(d, 2H 3JHH = 8.4 Hz, CHAr),7.48 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.55 (t, 2H, 3JHH = 8.0 Hz, CHAr), 7.63–7.67 (m, 3H, CHAr), 12.43(s, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 35.8 (CH), 55.5 (C = C*–CN), 57.9 (OMe), 108.0 (Cq), 113.2 (CN), 116.3 and 120.5 (2Cq), 128.7,128.9, 129.3 and 132.6(4CH), 133.7 (Cq), 137.3 (CH), 137.6, 154.2 and 158.4(3Cq), 159.9 (CH), 160.8 (C = C*–NH2), 200.1 (C = O). MS: m/z (%) 398 (M+. + 23), 291 (M+.–CH3OC6H4, 100), 213 [(M+.–(CH3OC6H4 + C6H4 + H), 60], 185 [(M+.–(CH3OC6H4 + C6H5CO + H), 107 (CH3OC6H4+, 15), 77 (C6H5+, 16)); Anal. Calcd for C24H18N2O4 (398.13): C, 72.35; H, 4.55; N, 7.03, Found: C, 72.26; H, 4.53; N, 7.06.

2-Amino-6-benzoyl-4-(2-chlorophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4 l , C 23 H 15 ClN 2 O 3 )

Yellow powder, m.p. 273–275 °C, yield: 82%; IR (KBr) (υmax, cm−1): 3423 (OH), 3317 and 3204 (NH2), 3050 (C sp2 –H), 2925 (C sp3–H), 2200 (CN), 1659 (C = O), 1612 (C = C), 1262 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6) δH: 5.25 (s, 1H, CH), 6.74 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.12 (d, 1H, 3JHH = 7.6 Hz, CHAr), 7.13 (s, 2H, NH2), 7.22–7.29 (m, 2H, 2CHAr) 7.41 (dd, 1H, 3JHH = 7.6 Hz, 4JHH = 1.4 Hz, CHAr), 7.51 (d, 1H, 3JHH = 9.2 Hz, CHAr), 7.56 (d, 2H, 3JHH = 7.6 Hz, 2CHAr), 7.62 -7.67 (m, 3H, 3CHAr), 12.41 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 34.1 (CH), 56.2 (C* = C–N), 107.9 and 111.5 (Cq), 116.2 (CN), 120.0 (Cq), 128.2, 128.9, 129.0, 129.3 and 129.9 (CH), 130.9 (Cq), 132.5 and 132.6 (CH), 134.2 (Cq), 137.5 and 142.2 (CHAr), 154.6 and 159.9 (Cq), 161.0 (C = C*–N), 200.1 (C = O). MS: m/z (%) 404 ((M+. + 2), 3), 402 (M+., 9), 291 [(M+.–ClC6H4), 100], 213 [(M+.–(ClC6H4 + C6H5 + H), 50], 185 [(M+.–(ClC6H4 + C6H5CO + H), 5)], 105 (C6H5CO+, 54), 77 (C6H5+, 33); Anal. Calcd for C23H15ClN2O3 (402.08): C, 68.58; H, 3.75; N, 6.95, Found: C, 68.69; H, 3.77; N, 6.93.

2-Amino-6-benzoyl-4-(2-methylophenyl)-5-hydroxy-4H-chromene-3-carbonitrile (4 m , C 24 H 18 N 2 O 3 )

Orange powder, m.p. 255–257 °C, yield: 80%; IR (KBr) (υmax, cm−1): 3409 (OH), 3315 and 3194 (NH2), 3057 (Csp2 –H), 2927 (C sp3–H), 2198 (CN), 1660 (C = O), 1613 (C = C), 1264 (C sp2 –O). 1H NMR (400.1 MHz, DMSO-d6): δH 2.52 (s, 3H, CH3), 5.01 (s, 1H, CH), 6.74 (d, 1H, 3JHH = 8.8 Hz, CHAr), 6.86 (d, 1H 3JHH = 8.4 Hz, CHAr), 7.06 (s, 2H, NH2), 7.09 (d, 1H 3JHH = 6.8 Hz, CHAr), 7.15 (d, 1H 3JHH = 6.8 Hz, CHAr), 7.49 (d, 1H, 3JHH = 8.8 Hz, CHAr), 7.52–7.56 (m, 2H, 2CHAr), 7.62–7.65 (m, 3H, 3CHAr), 12.42 (bs, 1H, OH). 13C NMR (100.6 MHz, DMSO-d6): δC 19.5 (CH3), 32.4 (CH), 57.6 (C* = C–N), 107.9 and 113.3 (Cq), 116.1 (CN), 120.5 (Cq), 126.8, 127.0, 128.3, 128.9, 129.3, 130.4, 132.5, 133.8 and 135.1 (CH), 137.5, 144.1, 154.5, and 159.5 (Cq), 160.9 (C = C*–N), 200.1 (C = O). MS: m/z (%) 382 (M+., 30), 291 (M+.–CH3C6H4, 100), 291 (M+ –CH3C6H4, 100), 213 [(M+–(CH3C6 H4 + C6H5)), 57], 185 [(M+.–(CH3C6H4 + C6H5CO + H), 5)], 105(C6H5CO+, 21), 77 (C6H5+, 11); Anal. Calcd for C24H18N2O3 (382.13): C, 75.38; H, 4.74; N, 7.33, Found: C, 75.49; H, 4.73; N, 7.31.

General procedure for evaluation of antioxidant activity

In a spectrophotometric study, the antioxidant activity of compounds 4a–m was examined using the DPPH radical scavenging method [52]. First, triplicate samples of each compound were prepared in methanol solvent at five concentrations (200, 100, 50, 25, and 12.5 µM). Then, 100 μM DPPH methanolic solution was added (1:1 v/v) to each solution and shaken vigorously. The absorbance of solutions was measured at 517 nm after 1 h keeping them in the dark at room temperature. Assays were conducted in triplicate, and the percentage of inhibition was calculated as follows:

%Inhibition \(=\frac{(A\mathrm{c}-\mathrm{As})}{\mathrm{As}}\times 100\)where Ac is the absorbance value of the control sample (DPPH solution), and As is the absorbance value of the tested sample.

General procedure for evaluation of antibacterial activity

The MIC values of compounds 3 and 4a–m were evaluated against S. aureus (ATCC2592) and E. coli (ATCC1399) according to the previously standard protocols documented by Clinical and Laboratory Standards Institute. [39] Firstly, suspensions of samples were prepared in lower concentration ranges from 2 × 10–3−5 mM in DMSO and subsequently, 100 µL of diluted samples were poured into a 96-wells tray. To adjust turbidity, a half McFarland tube was used to prepare a suspension of freshly cultivated bacteria (18–20 h) in normal saline. After dilution with Müller Hinton Broth (1:100), 100 µL of this suspension was added to each well. Each well was tested with 0.5–1 × 106 CFU/mL of bacteria. In each well, the final concentration of test substance was halved by the addition of bacterial suspension (1 × 10–3−2.5 mM). A minimum inhibitory concentration (MIC) was determined after 22 h of incubation at 37 °C.