Introduction

2-Amino-4H-chromenes and 2-amino-4H-benzochromenes are an important class of heterocyclic compounds having important biological activities. During the last decade, such compounds had shown interesting pharmacological properties including antimicrobial (Kidwai et al., 2010; Alvey et al., 2009; Kumar et al., 2009; Raj et al., 2009), antileishmanial (Tanaka et al., 2007), anticancer (Sabry et al., 2011; Rampa et al., 2005), antioxidant (Singh et al., 2010; Vukovic et al., 2010), antiproliferative (Magedov et al., 2007), antitumor (Mahmoodi et al., 2010; Endo et al., 2010; Tseng et al., 2010) effects and activities, as well as treatment of alzheimer’s disease (Bruhlmann et al., 2001) and schizophrenia disorder (Kesten et al., 1999). Fused chromene ring systems have blood platelet antiaggregating (Lee et al., 2006) and analgesic activities (El-Sayed and Ibrahim 2010; Keri et al., 2010).

They also exhibit hypolipidemic activity (Sashidhara et al., 2011), DNA breaking activities and mutagenicity (Hiramoto et al., 1997).

As a result of remarkable pharmacological efficiency of 4H-chromene and 4H-benzochromene derivatives and in continuation of our program on the chemistry of 4H-pyran derivatives (Al-Ghamdi et al., 2012; El-Agrody, 1994; El-Agrody et al., 1997a, b, 2000, 2001, 2002, 2011, 2012a, b; El-Agrody and Al-Ghamdi, 2011; Sabry et al., 2011; Abd-El-Aziz et al., 2004, 2007; Eid et al., 2003; Khafagy et al., 2002; Bedair et al., 2000, 2001; Sayed et al., 2000), we report herein the synthesis of 4H-benzo[h]chromene derivatives and the evaluation of their antitumor activities. The chemical structures of the studied compounds and their structure–activity relationships (SAR) are discussed in this study.

Chemistry

Treatment of 4-chloro-1-naphthol (1) with α-cyano-4-substitutedcinnamonitriles (2a–g) or α-cyano-4-(4-piperdin-1-ylphenyl/morpholinophenyl)cinnamonitrile (2h, i) in ethanolic piperidine under reflux afforded 2-amino-4-aryl-6-chloro-4H-benzo[h]chromene-3-carbonitrile derivatives (3a–i) (Scheme 1).

Scheme 1
scheme 1

Synthesis of 4H-benzo[h]chromene-3-carbonitrile derivatives (3a–i)

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In a similar manner, the reaction of 4-chloro-1-naphthol (1) with ethyl α-cyano-4-substituted cinnamate (4a–g) or ethyl α-cyano-4-(4-morpholinophenyl) cyanocinnamate (4h) afforded ethyl 2-amino-4-aryl-6-chloro-4H-benzo[h]chromene-3-carboxylate derivatives (5a–h) (Scheme 2).

Scheme 2
scheme 2

Synthesis of ethyl 4H-benzo[h]chromene-3-carboxylate derivatives (5a–h)

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The structures 3 and 5 were established on the basis of IR, 1H NMR, 13C NMR, 13C NMR-DEPT, 13C NMR-APT, MS data, and in conjunction with our previous work (Abd-El-Aziz et al., 2004; El-Agrody, 1994; El-Agrody et al., 1997a, b; Khafagy et al., 2002; Sayed et al., 2000; Sabry et al., 2011). The IR spectra of 3a–i showed the appearance of the a NH2 stretch at υ 3471–3424, 3334–3326, 3222–3190 cm,−1 a CN stretch at υ 2204–2192 cm,−1 while a NH2 stretch at υ 3478–3380, 3325–3282 cm−1 and a CO stretch at υ 1687–1669 cm−1 for 5a–h. The 1H and 13C NMR spectra of 3a–i and 5a–h revealed the presence of 4H signals at δ 5.28–4.79 (s, 1H, H-4) and 40.18–38.77 ppm (C-4). In compounds 5a–h the ester group gave 1H signals at 4.05–4.01 (q, J = 7.0–7.2 Hz, 2H, CH2), 1.12–1.09 (t, J = 7.0–7.2 Hz, 3H, CH3) with the corresponding signals in the 13C spectra at 58.86–58.63 (CH2) and 14.31–14.19 ppm (CH3), respectively. The 13C NMR-DEPT spectra at 45°, 90°, and 135° and 13C NMR-APT spectra of compounds 3 and 5 provided additional evidence in support of the proposed structures. In addition, the 1H NMR spectra for compounds 3 and 5 showed NH2 protons resonated at 7.34–7.14 (sharp singlet) and 8.00–7.82 (broad singlet lower field), respectively. This deshielding is a result of replacement of CN group in 3 by C=O group in 5 whose C=O anisotropy would deshield these protons and in addition of the involvement of these protons in hydrogen bonding with the C=O group. This also, was supported by X-ray single crystal data (Al-Dies et al., 2012; El-Agrody et al., 2012). The mass spectra of compounds 3 and 5 gave also additional evidences for the proposed structures.

Antitumor assays

Compounds 3a–i and 5a–h were evaluated for human tumor cell growth inhibitory activity against three cell lines: breast adenocarcinoma (MCF-7), lung carcinoma (HCT-116), and hepatocellular carcinoma (HepG-2). The measurements of cell growth and the viabilities were determined as described in the literature (Rahman et al., 2001). In vitro cytotoxicity evaluation using viability assay was performed at the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University using Vinblastine, Colchicine and Doxorubicin as standard drugs. The inhibitory activity of the synthetic compounds 3a–i and 5a–h against the three cell lines MCF-7, HCT-116, and HepG-2 are given in Table 1 and Figs. 1, 2, 3, 4, 5, and 6.

Table 1 Effects of the treatment of MCF-7, HCT-116, and HepG-2 cells with various concentrations of the prepared compounds; cytotoxicity (IC50) as measured with the MTT method
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Fig. 1
figure 1

Evaluation of cytotoxicity for compounds (3a–i) against MCF-7cell line

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Fig. 2
figure 2

Evaluation of cytotoxicity for compounds (3a–i) against HCT-116 cell line

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Fig. 3
figure 3

Evaluation of cytotoxicity for compounds (3a–i) against HepG-2 cell line

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Fig. 4
figure 4

Evaluation of cytotoxicity for compounds (5a–h) against MCF-7 cell line

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Fig. 5
figure 5

Evaluation of cytotoxicity for compounds (5a–h) against HCT-116 cell line

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Fig. 6
figure 6

Evaluation of cytotoxicity for compounds (5a–h) against HepG-2 cell line

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Results and discussion

4H-Benzo[h]chromene derivatives were selected for this study as their families are well known to contain active compounds with a wide range of biological and pharmacological activities (Kidwai et al., 2010; Alvey et al., 2009; Kumar et al., 2009; Raj et al., 2009; Tanaka et al., 2007; Sabry et al., 2011; Rampa et al., 2005; Singh et al., 2010; Vukovic et al., 2010; Magedov et al., 2007; Mahmoodi et al., 2010; Endo et al., 2010; Tseng et al., 2010; Bruhlmann et al., 2001; Kesten et al., 1999; Lee et al., 2006; El-Sayed and Ibrahim, 2010; Keri et al., 2010; Sashidhara et al., 2011; Hiramoto et al., 1997).

In this study, seventeen compounds of 4H-benzo[h]chromene derivatives were prepared. Structures of the synthesized compounds were elucidated on the basis of IR, 1H NMR, 13C NMR, 13C NMR-DEPT, 13C NMR-APT, and MS data.

Compounds 3a–i and 5a–h were tested against three tumer cell lines: MCF-7, HCT-116, and HepG-2. The cytotoxicity evaluation using viability assays and inhibitory activities are given in Table 1 and Figs. 1, 2, 3, 4, 5, and 6.

The results from Table 1 indicated that compounds 5e, 3c, 5f, b, d, 3d, 5c, a were the most active against MCF-7, 5a against HCT-116 and 5a, 3e, a against HepG-2 as compared with the standard drug Vinblastine, while compounds 5e, 3c, 5f, b, d, 3d, 5c, a, h, 3i, g, a, e were the most active against MCF-7, 5a, c, e, f, b, 3e, c, g, b, 5d, h, 3d, i, 5g against HCT-116, 5a, 3e, a, 5e, 3c, 5d, c, f, 3b, 5g, 3g, 5h against HepG-2 as compared with the standard drug Colchicine and the remaining compounds exhibited near or moderate to lower activities as compared with the standard drugs Vinblastine, Colchicine, and Doxorubicin.

SAR studies

The antitumor activity (IC50) of compounds 3, 5 and its analogs in the three cancer cell lines is summarized in Table 1. By maintaining the 2-amino and 3-cyano groups of 3a, the SAR studies at the 4-position was explored. The non-substituted phenyl analog 3a was much less active (IC50 = 13.9 μg/ml), confirming the importance of a substituent at the phenyl ring at the 4-position. Compounds 3c and 3d have the higher potent antitumor activity (IC50 = 3.0–5.5 μg/ml) against MCF-7 as compared to the other compounds 3i, g, e, b, i, f (IC50 = 12.2–50.0 μg/ml) and the standard drug Vinblastine (IC50 = 6.1 μg/ml). This potency could be attributed to the presence of the chloro or bromo atoms (electron-withdrawing groups) at the para-position of phenyl ring at 4-position. Replacement of the chloro or bromo atoms with other groups, resulted in reduction of potency, suggesting that there might be a size limited pocket at the para-position of phenyl ring at 4-position and an electron-withdrawing group is preferred over an electron-donating group. In addition, compounds 3c, d, i, g, a, e have the higher potent antitumor activity (IC50 = 3.0-13.9 μg/ml) against MCF-7, compared to other compounds and the standard drug Colchicine (IC50 = 17.7 μg/ml). This potency could be attributed to the presence of the chloro, bromo, nitro (electron-withdrawing), morpholino, and methyl (electron-donating) groups at the para-position of phenyl ring at 4-position, suggesting that there might be a size limited pocket at the 4-position of the phenyl ring and an electron-withdrawing group is preferred over an electron-donating group. Replacement of the electron-withdrawing group cyano group by ester group at the 3-position for compound 5a and its analogs improved the antitumor activities. Compounds 5e, f, b, d, c, a (IC50 = 2.4–6.0 μg/ml) have potent antitumor activities against the MCF-7 than the other compounds 5h, g (IC50 = 7.7–42.9 μg/ml) and the standard drug Vinblastine (IC50 = 6.1 μg/ml). These data indicate that the activities of compounds 5e, f, b, d, c, a are considerably enhanced by the presence of the methyl, methoxy (electron-donating), fluoro, bromo, chloro (electron-withdrawing) at the para-position of phenyl ring at 4-position or the phenyl ring at the 4-position, suggesting that there might be a size limited pocket at the para-position of phenyl ring at 4-position and an electron-donating group is preferred over an electron-withdrawing group, while compound 5h (IC50 = 7.7 μg/ml)-exhibited good activity and compound 5g (IC50 = 42.8 μg/ml) inactive.

In the case of HCT-116, investigation of (SAR) revealed that compound 5a (IC50 = 2.7 μg/ml) has the most potent activity against HCT-116 compared to the other compounds 3a–i, 5b–h and the standard drug Vinblastine (IC50 = 2.6 μg/ml). This potency could be attributed to the presence of the non-substituted phenyl group at the para-position of phenyl ring at 4-position with the ester group at the 3-position, suggesting that there might be a size limited pocket at the 4-position and the non-substituted phenyl (electron-donating group) at the 4-position is preferred over the para-substituted phenyl at the 4-position. In addition, compounds 5a, c, e, f, b, 3e, c, g, b, 5d, h, 3d, i, 5g (IC50 = 2.6–34.8 μg/ml) have the most potent activity against HCT-116 compared to the other compounds 3a, f and the standard drug Colchicine (IC50 = 42.8 μg/ml). This potency could be attributed to the presence of the non-substituted phenyl (electron-donating group) at the 4-position and the substituted phenyl at the para-position of phenyl ring at 4-position with the chloro (electron-withdrawing), methyl, methoxy (electron-donating), fluoro groups (electron-withdrawing) with the ester group at the 3-position for compounds 5a, c, e, f, b, the methyl (electron-donating), chloro, nitro, fluoro groups (electron-withdrawing) with the cyano group at the 3-position for compounds 3e, c, g, b, the bromo (electron-withdrawing), morpholino groups (electron-donating) with the ester group at the 3-position for compounds 5d, h, the bromo (electron-withdrawing), morpholino groups (electron-donating) with the cyano group at the 3-position for compounds 3d, i and nitro group (electron-withdrawing) with the ester group at the 3-position for compound 5g, respectively, at the para-position of phenyl ring.

Furthermore, compounds 5a, 3e, a (IC50 = 2.8–4.2 μg/ml) showed higher antitumor activities against HepG-2 than the standard drug Vinblastine (IC50 = 4.6 μg/ml). This could be attributed to the presence of the non-substituted phenyl group (electron-donating) at the 4-position with the ester group at the 3-position for compound 5a and the substituted at the para-position of phenyl ring at 4-position with the chloro (electron-withdrawing) with the cyano group at the 3-position for compound 3e or the non-substituted phenyl group (electron-donating) at the 4-position with the cyano group at the 3-position for compound 3a, suggesting that there might be a size limited pocket at the para-position of phenyl ring at 4-position and the non-substituted phenyl (electron-donating group) at the 4-position is preferred over the substituted at the para-position of phenyl ring at 4-position. In addition, compounds 5a, 3e, a, 5e, 3c, 5d, c, f, 3b, 5g, 3g,5h (IC50 = 2.8–10.5 μg/ml) showed higher antitumor activities against HepG-2 than the standard drug Colchicine (IC50 = 10.6 μg/ml). This potency could be attributed to the presence of the non-substituted phenyl (electron-donating group) at the 4-position, suggesting that there might be a size limited pocket at the 4-position and the non-substituted phenyl (electron-donating group) at the 4-position is preferred over the substituted at the para-position of phenyl ring at 4-position.

Finally, in the case of MCF-7, HCT-116, and HepG-2, an investigation of SAR revealed that compounds 3a–i and 5a–h showed moderate to lower antitumor activities against MCF-7, HCT-116, and HepG-2 as compared to the standard drug Doxorubicin.

Conclusions

Our interest in the synthesis of 4H-benzo[h]chromene derivatives is to focus on their antitumor activities as a part of our recent research line that aims at the development of new heterocyclic compounds as strong potent antitumor agents (El-Agrody et al., 2011). Thus, in this paper we revealed the synthesis of some 4H-benzo[h]chromene, followed by antitumor evaluation for all of the synthesized compounds. Seventeen compounds of 4H-benzo[h]-chromene derivatives were prepared and their structures were elucidated on the basis of IR, 1H NMR, 13C NMR, 13C NMR-DEPT/APT, and MS data. Compounds 5e, 3c, 5f, b, d, 3d, 5c, a were the most active against MCF-7, 5a against HCT-116 and 5a, 3e, a against HepG-2 as compared with the standard drug Vinblastine, while compounds 5e, 3c, 5f, b, d, 3d, 5c, a, h, 3i, g, a, e were the most active against MCF-7, 5a, c, e, f, b, 3e, c, g, b, 5d, h, 3d, i, 5g against HCT-116, 5a, 3e, a, 5e, 3c, 5d, c, f, 3b, 5g, 3g, 5h against HepG-2 as compared with the standard drug Colchicine and the remaining compounds exhibited near or moderate to lower activities as compared with the standard drugs Vinblastine, Colchicine, and Doxorubicin. A more extensive study is also warranted to determine additional antitumor parameters in order to give a deeper insight to its structure–activity relationship and to optimize the effectiveness of this series of molecules, which can then be used in bigger scenarios such as drug design or development of antitumor therapeutics.

Experimental

Melting points were determined with a Stuart Scientific Co. Ltd apparatus. IR spectra were determined as KBr pellets on a Jasco FT/IR 460 plus spectrophotometer. 1H NMR and 13C NMR spectra were recorded using a Bruker AV 500 MHz spectrometer. 13C NMR spectra were obtained using distortionless enhancement by polarization transfer (DEPT), where the signals of CH and CH3 carbon atoms appear normal (up) and the signals of carbon atoms in CH2 environments appear negative (down). 13C NMR spectra were obtained using attached proton test (APT), with this technique, the signals of CH and CH3 carbon atoms appears normal (up) and the signal of CH2 and Cq environments appears negative (down).The MS were measured on a Shimadzu GC/MS-QP5050A spectrometer. Elemental analyses were performed on a Perkin-Elmer 240 microanalyser.

General procedure for the preparation of (3) and (5)

A solution of 4-chloro-1-naphthol (1) (0.01 mmol) in EtOH (30 ml) and piperidine (0.5 ml) was treated with α-cyanocinnamonitriles (2a–i) or ethyl α-cyanocinnamates (4a–h) (0.01 mmol). The reaction mixture was heated under reflux for 1–2 h. The solid product which formed was collected by filtration, washed with MeOH, and recrystallized from ethanol or benzene. The physical and spectral data of compounds (3) and (5) were as follows:

2-Amino-6-chloro-4-phenyl- 4H-benzo[h]chromene-3-carbonitrile (3a)

Colorless crystals from ethanol; yield 86 %; m.p. 220–221 °C; IR (KBr) υ (cm−1): 3477, 3325, 3191 (NH2), 3082, 3017, 2855 (CH), 2199 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.36–7.26 (m, 10H, aromatic), 7.30 (s, 2H, NH2, cancelled by D2O), 4.93 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-d6) δ: 159.91 (C-2), 145.11 (C-10b), 129.21 (C-9), 128.78 (C-6a), 127.66 (C-5), 127.62 (C-10a), 127.09 (C-8), 125.52 (C-6), 123.88 (C-7), 121.40 (C-10), 120.18 (C-4a), 118.56 (CN), 56.03 (C-3), 40.53 (C-4), 141.99, 128.57, 128.21, 125.86 (aromatic); MS m/z (%): 334 (M++2, 8.02), 332 (M+, 23.35) with a base peak at 256 (100); Anal. Calcd for C20H13ClN2O: C, 72.18; H, 3.94; N, 8.42. Found: C, 72.22; H, 3.97; N, 8.45 %.

2-Amino-6-chloro-4-(4-fluorophenyl)-4H-benzo[h]chromene-3-carbonitrile (3b)

Pale yellow crystals from benzene; yield 86 %; m.p. 246–247 °C (Khafagy et al., 2002 m.p. 246 °C); IR (KBr) υ (cm−1): 3470, 3326, 3190 (NH2), 3092, 3037, 2983, 2865 (CH), 2197 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.35–7.16 (m, 9H, aromatic), 7.31 (s, 2H, NH2, cancelled by D2O), 4.97 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-d6) δ: 162.17 (C-2), 142.02 (C-10b), 129.57 (C-9), 129.29 (C-6a), 128.36 (C-5), 127.77 (C-10a), 125.84 (C-8), 123.93 (C-6), 123.83 (C-7), 121.46 (C-10), 120.11 (C-4a), 118.41 (CN), 55.94 (C-3), 40.07 (C-4), 160.23, 141.37, 129.64, 115.49 (aromatic); 13 C NMR-DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 129.64 (aromatic ↑), 129.57 (C-9 ↑), 128.36 (C-5 ↑), 125.84 (C-8 ↑), 123.83 (C-7 ↑), 121.46 (C-10 ↑), 40.07 (C-4 ↑), 115.49 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 129.64 (aromatic ↑), 129.57 (C-9 ↑), 128.36 (C-5 ↑), 125.84 (C-8 ↑), 123.83 (C-7 ↑), 121.46 (C-10 ↑), 40.07 (C-4 ↑), 115.49 (aromatic ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive)-revealed signals at δ: 129.64 (aromatic ↑), 129.57 (C-9 ↑), 128.36 (C-5 ↑), 125.84 (C-8 ↑), 123.83 (C-7 ↑), 121.46 (C-10 ↑), 40.07 (C-4 ↑), 115.49 (aromatic ↑); MS m/z (%): 352 (M++2, 5.14), 350 (M+, 17.09) with a base peak at 256 (100); Anal. Calcd for C20H12ClFN2O: C, 68.48; H, 3.45; N, 7.99. Found: C, 68.13; H, 3.41; N, 7.95 %.

2-Amino-6-chloro-4-(4-chlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (3c)

Colorless needles from benzene; yield 88 %; m.p. 224–225 °C (Khafagy et al., 2002 m.p. 224 °C); IR (KBr) υ (cm−1): 3470, 3331, 3193 (NH2), 3095, 3047, 2986, 2867 (CH), 2194 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.36–7.31 (m, 9H, aromatic), 7.34 (s, 2H, NH2, cancelled by D2O), 4.98 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-d6) δ: 159.91 (C-2), 144.05 (C-10b), 129.53 (C-9), 128.76 (C-6a), 128.32 (C-5), 127.72 (C-10a), 125.72 (C-8), 123.88 (C-6), 123.76 (C-7), 121.43 (C-10), 120.03 (C-4a), 118.02 (CN), 55.64 (C-3), 40.05 (C-4), 142.04, 131.75, 129.30, 125.64 (aromatic); Anal. Calcd for C20H12Cl2N2O: C, 65.41; H, 3.29; N, 7.63. Found: C, 65.44; H, 3.32; N, 7.65 %.

2-Amino-6-chloro-4-(4-bromophenyl)-4H-benzo[h]chromene-3-carbonitrile (3d)

Colorless needles from benzene; yield 90 %; m.p. 226–227 °C (Khafagy et al., 2002 m.p. 226 °C); IR (KBr) υ (cm−1): 3466, 3328, 3192 (NH2), 3093, 3037, 2986, 2863 (CH), 2193 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.35–7.26 (m, 9H, aromatic), 7.34 (s, 2H, NH2, cancelled by D2O), 4.97 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-d6) δ: 159.88 (C-2), 144.45 (C-10b), 129.28 (C-9), 128.33 (C-6a), 127.73 (C-5), 125.72 (C-10a), 125.61 (C-8), 123.85 (C-6), 123.78 (C-7), 121.41 (C-10), 120.26 (C-4a), 117.94 (CN), 55.53 (C-3), 40.03 (C-4), 142.01, 131.76, 129.87, 119.99 (aromatic); Anal. Calcd for C20H12BrClN2O: C, 58.35; H, 2.94; N, 6.80. Found: C, 58.31; H, 2.90; N, 6.77 %.

2-Amino-6-chloro-4-(4-methylphenyl)-4H-benzo[h]chromene-3-carbonitrile (3e)

Pale yellow needles from benzene; yield 90 %; m.p. 220–231 °C; IR (KBr) υ (cm−1): 3450, 3334, 3222 (NH2), 3073, 3057, 2986, 2865 (CH), 2192 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.36–7.14 (m, 9H, aromatic), 7.27 (s, 2H, NH2, cancelled by D2O), 4.88 (s, 1H, H-4), 2.27 (s, 3H, CH3); 13C NMR (125 MHz) (DMSO-d6) δ: 159.80 (C-2), 142.20 (C-10b), 129.32 (C-9), 129.18 (C-6a), 127.63 (C-5), 127.54 (C-10a), 125.90 (C-8), 125.45 (C-6), 123.76 (C-7), 121.39 (C-10), 120.20 (C-4a), 118.70 (CN), 56.16 (C-3), 40.17 (C-4), 20.54 (CH3), 141.90, 136.26, 128.16, 123.89 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 129.32 (C-9 ↑), 128.16 (aromatic ↑), 127.63 (C-5 ↑), 125.90 (C-8 ↑), 123.89 (aromatic ↑), 123.76 (C-7 ↑), 121.39 (C-10 ↑), 40.17 (C-4 ↑), 20.54 (CH3 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 129.32 (C-9 ↑), 128.16 (aromatic ↑), 127.63 (C-5 ↑), 125.90 (C-8 ↑), 123.89 (aromatic ↑), 123.76 (C-7 ↑), 121.39 (C-10 ↑), 40.17 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 129.32 (C-9 ↑), 128.16 (aromatic ↑), 127.63 (C-5 ↑), 125.90 (C-8 ↑), 123.89 (aromatic ↑), 123.76 (C-7 ↑), 121.39 (C-10 ↑), 40.17 (C-4 ↑), 20.54 (CH3 ↑).13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 159.80 (C-2 ↓), 142.20 (C-10b ↓), 141.90 (aromatic ↓), 136.26 (aromatic ↓), 129.32 (C-9 ↑), 129.18 (C-6a ↓), 128.16 (aromatic ↑), 127.63 (C-5 ↑), 127.54 (C-10a ↓), 125.90 (C-8 ↑), 125.45 (C-6 ↓), 123.89 (aromatic ↑), 123.76 (C-7 ↑), 121.39 (C-10 ↑), 120.20 (C-4a ↓), 118.70 (CN ↓), 56.16 (C-3 ↓), 40.17 (C-4 ↑), 20.54 (CH3 ↑); MS m/z (%): 348 (M++2, 6.92), 346 (M+, 20.35) with a base peak at 252 (100); Anal. Calcd for C21H15ClN2O: C, 72.73; H, 4.36; N, 8.08. Found: C, 72.76; H, 4.38; N, 8.10 %.

2-Amino-6-chloro-4-(4-methoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (3f)

Pale yellow needles from benzene; yield 88 %; m.p. 233–234 °C; IR (KBr) υ (cm−1): 3424, 3333, 3213 (NH2), 3076, 2953, 2834 (CH), 2193 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.35–6.90 (m, 9H, aromatic), 7.32 (s, 2H, NH2, cancelled by D2O), 4.87 (s, 1H, H-4), 3.73 (s, 3H, OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 159.76 (C-2), 141.87 (C-10b), 129.19 (C-9), 128.75 (C-6a), 128.20 (C-5), 127.68 (C-10a), 125.97 (C-8), 125.46 (C-6), 123.93 (C-7), 121.43 (C-10), 120.24 (C-4a), 118.91 (CN), 56.35 (C-3), 55.00 (CH3), 40.08 (C-4), 158.30, 137.26, 128.16, 114.15 (aromatic); MS m/z (%): 364 (M++2, 4.31), 362 (M+, 18.7) with a base peak at 75 (100); Anal. Calcd for C21H15ClN2O2: C, 69.52; H, 4.17; N, 7.72. Found: C, 69.56; H, 4.21; N, 7.75 %.

2-Amino-6-chloro-4-(4-nitrophenyl)-4H-benzo[h]chromene-3-carbonitrile (3g)

Pale yellow needles from benzene; yield 88 %; m.p. 257–258 °C; IR (KBr) υ (cm−1): 3448, 3326, 3197 (NH2), 3070, 2953, 2864 (CH), 2198 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.36–7.35 (m, 9H, aromatic), 7.44 (s, 2H, NH2, cancelled by D2O), 5.18 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-d6) δ: 160.10 (C-2), 142.28 (C-10b), 129.49 (C-9), 129.03 (C-6a), 128.27 (C-5), 127.78 (C-10a), 125.86 (C-6), 125.64 (C-8), 123.86 (C-7), 121.51 (C-10), 119.86 (C-4a), 117.26 (CN), 55.04 (C-3), 40.18 (C-4), 152.29, 146.61, 128.55, 124.14 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 129.49 (C-9 ↑), 128.55 (aromatic ↑), 128.27 (C-5 ↑), 125.64 (C-8 ↑), 124.14 (aromatic ↑), 123.86 (C-7 ↑), 121.51 (C-10 ↑), 40.18 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 129.49 (C-9 ↑), 128.55 (aromatic ↑), 128.27 (C-5 ↑), 125.64 (C-8 ↑), 124.14 (aromatic ↑), 123.86 (C-7 ↑), 121.51 (C-10 ↑), 40.18 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ: 129.49 (C-9 ↑), 128.55 (aromatic ↑), 128.27 (C-5 ↑), 125.64 (C-8 ↑), 124.14 (aromatic ↑), 123.86 (C-7 ↑), 121.51 (C-10 ↑), 40.18 (C-4 ↑).13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.10 (C-2 ↓), 152.29 (aromatic ↓), 146.61(aromatic ↓), 142.28 (C-10b ↓), 129.49 (C-9 ↑), 129.03 (C-6a ↓), 128.55 (aromatic ↑), 128.27 (C-5 ↑), 127.78 (C-10a ↓), 125.86 (C-6 ↓), 125.64 (C-8 ↑), 124.14 (aromatic ↑), 123.86 (C-7 ↑), 121.51 (C-10 ↑), 119.86 (C-4a ↓), 117.26 (CN ↓), 55.04 (C-3 ↓), 40.18 (C-4 ↑); MS m/z (%): 379 (M++2, 11.01), 377 (M+, 33.01) with a base peak at 72 (100); Anal. Calcd for C20H12ClN3O3: C, 63.59; H, 3.20; N, 11.12. Found: C, 63.56; H, 3.27; N, 11.09 %.

2-Amino-6-chloro-4-(4-piperdin-1-ylphenyl)-4H-benzo[h]chromene-3-carbonitrile (3h)

Yellow needles from benzene; yield 82 %; m.p. 252–253 °C; IR (KBr) υ (cm−1): 3471, 3298, 3184 (NH2), 3075, 2993, 2938, 2864, 2813 (CH), 2204 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.32–6.86 (m, 9H, aromatic), 7.17 (s, 2H, NH2, cancelled by D2O), 4.79 (s, 1H, H-4), 3.09–3.07 (m, 4H, 2CH2), 1.60–1.57 (m, 4H, 2CH2), 1.51 (s, 2H, CH2); 13C NMR (125 MHz) (DMSO-d6) δ: 159.74 (C-2), 141.83 (C-10b), 129.14 (C-6a), 128.19 (C-9), 127.69 (C-5), 126.09 (C-10a), 125.36 (C-8), 123.95 (C-6), 123.82 (C-7), 121.40 (C-10), 120.32 (C-4a), 119.25 (CN), 56.45 (C-3), 49.39 (CH2), 40.18 (C-4), 25.25 (CH2), 23.82 (CH2), 150.65, 135.01, 128.13, 115.92 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 128.19 (C-9 ↑), 128.13 (aromatic ↑), 127.69 (C-5 ↑), 125.36 (C-8 ↑), 123.82 (C-7 ↑), 121.40 (C-10 ↑), 49.39 (CH2 ↓), 40.18 (C-4 ↑), 25.25 (CH2 ↓), 23.82 (CH2 ↓), 115.92 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 128.19 (C-9 ↑), 128.13 (aromatic ↑), 127.69 (C-5 ↑), 125.36 (C-8 ↑), 123.82 (C-7 ↑), 121.40 (C-10 ↑), 40.18 (C-4 ↑), 115.92 (aromatic ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive)-revealed signals at δ: 128.19 (C-9 ↑), 128.13 (aromatic ↑), 127.69 (C-5 ↑), 125.36 (C-8 ↑), 123.82 (C-7 ↑), 121.40 (C-10 ↑), 49.39 (CH2 ↑), 40.18 (C-4 ↑), 25.25 (CH2 ↑), 23.82 (CH2 ↑), 115.92 (aromatic ↑). 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 159.74 (C-2 ↓), 150.65 (aromatic ↓), 141.83 (C-10b ↓), 135.01 (aromatic ↓), 129.14 (C-6a ↓), 128.19 (C-9 ↑), 128.13 (aromatic ↑), 127.69 (C-5 ↑), 126.09 (C-10a ↓), 125.36 (C-8 ↑), 123.95 (C-6 ↓), 123.82 (C-7 ↑), 121.40 (C-10 ↑), 120.32 (C-4a ↓), 119.25 (CN ↓), 56.45 (C-3 ↓), 49.39 (CH2 ↓), 40.18 (C-4 ↑), 25.25 (CH2 ↓), 23.82 (CH2 ↓), 115.92 (aromatic ↑); MS m/z (%): 417 (M++2, 2.11), 415 (M+, 6.33) with a base peak at 55 (100); Anal. Calcd for C25H22ClN3O: C, 72.19; H, 5.33; N, 10.10. Found: C, 72.22; H, 5.36; N, 10.14 %.

2-Amino-6-chloro-4-(4-morpholinophenyl)-4H-benzo[h]chromene-3-carbonitrile (3i)

Yellow needles from benzene; yield 82 %; m.p. 266–267 °C; IR (KBr) υ (cm−1): 3451, 3327, 3208 (NH2), 3095, 3010, 2967, 2938, 2864, 2816 (CH), 2195 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.33–6.88 (m, 9H, aromatic), 7.18 (s, 2H, NH2, cancelled by D2O), 4.81 (s, 1H, H-4), 3.72–3.70 (m, 4H, 2CH2), 3.08–3.06 (m, 4H, 2CH2); 13C NMR (125 MHz) (DMSO-d6) δ: 159.74 (C-2), 141.83 (C-10b), 129.16 (C-6a), 128.22 (C-9), 127.69 (C-5), 126.06 (C-8), 125.38 (C-10a), 123.94 (C-6), 123.81 (C-7), 121.41 (C-10), 120.29 (C-4a), 119.11 (CN), 66.06 (CH2), 56.39 (C-3), 48.29 (CH2), 40.09 (C-4), 150.04, 135.81, 128.29, 115.27 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 128.29 (aromatic ↑), 128.22 (C-9 ↑), 127.69 (C-5 ↑), 126.06 (C-8 ↑), 123.81 (C-7 ↑), 121.41 (C-10 ↑), 66.06 (CH2 ↓), 48.29 (CH2 ↓), 40.09 (C-4 ↑), 115.27 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 128.29 (aromatic ↑), 128.22 (C-9 ↑), 127.69 (C-5 ↑), 126.06 (C-8 ↑), 123.81 (C-7 ↑), 121.41 (C-10 ↑), 40.09 (C-4 ↑), 115.27 (aromatic ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive)-revealed signals at δ: 128.29 (aromatic ↑), 128.22 (C-9 ↑), 127.69 (C-5 ↑), 126.06 (C-8 ↑), 123.81 (C-7 ↑), 121.41 (C-10 ↑), 66.06 (CH2 ↑), 48.29 (CH2 ↑), 40.09 (C-4 ↑), 115.27 (aromatic ↑).13CNMR-APT spectrum CH,CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 159.74 (C-2 ↓), 150.04 (aromatic ↓),141.83 (C-10b ↓), 135.81 (aromatic ↓),129.16 (C-6a ↓), 128.29 (aromatic ↑), 128.22 (C-9 ↑), 127.69 (C-5 ↑), 126.06 (C-8 ↑), 125.38 (C-10a ↓), 123.94 (C-6 ↓), 123.81 (C-7 ↑), 121.41 (C-10 ↑), 120.29 (C-4a ↓), 119.11 (CN ↓), 66.06 (CH2 ↓), 56.39 (C-3 ↓), 48.29 (CH2 ↓), 40.09 (C-4 ↑), 115.27 (aromatic ↑); MS m/z (%): 419 (M++2, 17.98), 417 (M+, 55.84) with a base peak at 255 (100); Anal. Calcd for C24H20ClN3O2: C, 68.98; H, 4.82; N, 10.06. Found: C, 68.95; H, 4.78; N, 10.05 %.

Ethyl 2-amino-6-chloro-4-phenyl-4H-benzo[h]chromene-3-carboxyalte (5a)

Colorless needles from ethanol; yield 78 %; m.p. 180–181 °C; IR (KBr) υ (cm−1): 3393, 3285 (NH2), 3061, 3026, 2968, 2900 (CH), 1676 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.42-7.12 (m, 10H, aromatic), 7.86 (bs, 2H, NH2), 5.06 (s, 1H, H-4), 4.01 (q, 2H, CH2, J = 7 Hz), 1.09 (t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 168.02 (CO), 160.43 (C-2), 142.08 (C-10b), 129.03 (C-6a), 128.26 (C-9), 127.30 (C-5), 126.33 (C-10a), 126.18 (C-8), 123.94 (C-6), 123.73 (C-7), 121.56 (C-10), 121.42 (C-4a), 76.07 (C-3), 58.63 (CH2), 40.05 (C-4), 14.19 (CH3), 147.32, 127.91, 127.47, 125.30 (aromatic); 381 (M++2, 1.28), 379 (M+, 3.84) with a base peak at 314 (100); Anal. Calcd for C22H18ClNO3: C, 69.57; H, 4.78; N, 3.69. Found: C, 69.60; H, 4.81; N, 3.71 %.

Ethyl 2-amino-6-chloro-4-(4-fluorophenyl)-4H-benzo[h]chromene-3-carboxyalte (5b)

Yellow needles from ethanol; yield 78 %; m.p. 184–185 °C (Khafagy et al., 2002 m.p. 184 °C); IR (KBr) υ (cm−1): 3384, 3290 (NH2), 3075, 3061, 2989, 2978, 2958, 2927, 2906, 2887 (CH), 1669 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.42–7.06 (m, 9H, aromatic), 7.87 (bs, 2H, NH2), 5.09 (s, 1H, H-4), 4.02 (q, 2H, CH2, J = 7 Hz), 1.10 (t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 167.95 (CO), 160.40 (C-2), 142.09 (C-10b), 129.10 (C-9), 128.27 (C-6a), 127.54 (C-5), 126.30 (C-8), 125.40 (C-10a), 123.96 (C-6), 123.77 (C-7), 121.46 (C-10), 121.33 (C-4a), 75.99 (C-3), 58.68 (CH2), 40.07 (C-4), 14.23 (CH3), 161.60, 159.68, 129.17, 115.05 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 129.17 (aromatic ↑), 129.10 (C-9 ↑), 127.54 (C-5 ↑), 126.30 (C-8 ↑), 123.77 (C-7 ↑), 121.46 (C-10 ↑), 58.68 (CH2 ↓), 40.07 (C-4 ↑), 14.23 (CH3 ↑), 115.05 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 129.17 (aromatic ↑), 129.10 (C-9 ↑), 127.54 (C-5 ↑), 126.30 (C-8 ↑), 123.77 (C-7 ↑), 121.46 (C-10 ↑), 40.07 (C-4 ↑), 115.05 (aromatic ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ: 129.17 (aromatic ↑), 129.10 (C-9 ↑), 127.54 (C-5 ↑), 126.30 (C-8 ↑), 123.77 (C-7 ↑), 121.46 (C-10 ↑), 58.68 (CH2 ↑), 40.07 (C-4 ↑), 14.23 (CH3 ↑), 115.05 (aromatic ↑).13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 167.95 (CO ↓), 161.60 (aromatic ↓), 160.40 (C-2 ↓), 159.68 (aromatic ↓), 142.09 (C-10b ↓), 129.17 (aromatic ↑), 129.10 (C-9 ↑), 128.27 (C-6a ↓), 127.54 (C-5 ↑), 126.30 (C-8 ↑), 125.40 (C-10a ↓), 123.96 (C-6 ↓), 123.77 (C-7 ↑), 121.46 (C-10 ↑), 121.33 (C-4a ↓), 75.99 (C-3 ↓), 58.68 (CH2 ↓), 40.07 (C-4 ↑), 14.23 (CH3 ↑), 115.05 (aromatic ↑); Anal. Calcd for C22H17ClFNO3: C, 66.42; H, 4.31; N, 3.52. Found: C, 66.46; H, 4.35; N, 3.55 %.

Ethyl 2-amino-6-chloro-4-(4-chlorophenyl)-4H-benzo[h]chromene-3-carboxyalte (5c)

Colorless crystals from ethanol; yield 80 %; m.p. 170–171 °C (Khafagy et al., 2002 m.p. 170 °C); IR (KBr) υ (cm−1): 3384, 3284 (NH2), 3075, 3061, 2989, 2975, 2927, 2904, 2889 (CH), 1669 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.44–7.32 (m, 9H, aromatic), 7.92 (bs, 2H, NH2), 5.09 (s, 1H, H-4), 4.03 (q, 2H, CH2, J = 7 Hz), 1.11 (t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 167.92 (CO), 160.44 (C-2), 142.12 (C-10b), 129.23 (C-9), 129.02 (C-6a), 128.38 (C-8), 127.53 (C-5), 125.48 (C-10a), 123.97 (C-6), 123.77 (C-7), 121.49 (C-10), 120.96 (C-4a), 75.74 (C-3), 58.74 (CH2), 40.09 (C-4), 14.24 (CH3), 146.34, 130.79, 129.16, 126.23 (aromatic); Anal. Calcd for C22H17Cl2NO3: C, 63.78; H, 4.14; N, 3.38. Found: C, 63.74; H, 4.11; N, 3.35 %.

Ethyl 2-amino-6-chloro-4-(4-bromophenyl)-4H-benzo[h]chromene-3-carboxyalte (5d)

Colorless crystals from ethanol; yield 80 %; m.p. 162–163 °C (Khafagy et al., 2002 m.p. 162 °C); IR (KBr) υ (cm−1): 3385, 3282 (NH2), 3077, 3064, 2974, 2979, 2937, 2904, 2887 (CH), 1669 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.44-7.26 (m, 9H, aromatic), 7.93 (bs, 2H, NH2), 5.08 (s, 1H, H-4), 4.04 (q, 2H, CH2, J = 7 Hz), 1.12 (t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 167.89 (CO), 160.41 (C-2), 142.10 (C-10b), 129.61 (C-9), 129.40 (C-6a), 129.14 (C-5), 127.98 (C-8), 127.49 (C-10a), 125.47 (C-6), 123.94 (C-7), 121.47 (C-10), 120.85 (C-4a), 75.66 (C-3), 58.73 (CH2), 40.08 (C-4), 14.23 (CH3), 146.74, 131.15, 129.78, 119.26 (aromatic); Anal. Calcd for C22H17BrClNO3: C, 57.60; H, 3.74; N, 3.05. Found: C, 57.65; H, 3.77; N, 3.10 %.

Ethyl 2-amino-6-chloro-4-(4-methylphenyl)-4H-benzo[h]chromene-3-carboxyalte (5e)

Colorless crystals from ethanol; yield 79 %; m.p. 188–189 °C; IR (KBr) υ (cm−1): 3478, 3315 (NH2), 3075, 3061, 2996, 2980, 2904, 2888 (CH), 1681 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.43–7.03 (m, 9H, aromatic), 7.85 (bs, 2H, NH2), 5.01 (s, 1H, H-4), 4.01 (q, 2H, CH2, J = 7.2 Hz), 2.20 (s, 3H, CH3), 1.12 (t, 3H, CH3, J = 7.2 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 168.13 (CO), 160.42 (C-2), 142.08 (C-10b), 129.04 (C-9), 128.99 (C-6a), 127.86 (C-5), 127.00 (C-8), 125.32 (C-10a), 124.00 (C-6), 123.76 (C-7), 121.98 (C-10), 121.80 (C-4a), 76.25 (C-3), 58.69 (CH2), 40.00 (C-4), 20.49 (CH3), 14.25 (CH3), 144.46, 135.29, 128.85, 126.39 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 129.04 (C-9 ↑), 128.85 (aromatic ↑), 127.86 (C-5 ↑), 127.00 (C-8 ↑), 126.39 (aromatic ↑), 123.76 (C-7 ↑), 121.98 (C-10 ↑), 58.69 (CH2 ↓), 40.00 (C-4 ↑), 20.49 (CH3 ↑), 14.25 (CH3 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 129.04 (C-9 ↑), 128.85 (aromatic ↑), 127.86 (C-5 ↑), 127.00 (C-8 ↑), 126.39 (aromatic ↑), 123.76 (C-7 ↑), 121.98 (C-10 ↑), 40.00 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 129.04 (C-9 ↑), 128.85 (aromatic ↑), 127.86 (C-5 ↑), 127.00 (C-8 ↑), 126.39 (aromatic ↑), 123.76 (C-7 ↑), 121.98 (C-10 ↑), 58.69 (CH2 ↑), 40.00 (C-4 ↑), 20.49 (CH3 ↑), 14.25 (CH3 ↑).13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 168.13 (CO ↓), 160.42 (C-2 ↓), 144.46 (aromatic ↓), 142.08 (C-10b ↓), 135.29 (aromatic ↓), 129.04 (C-9 ↑), 128.99 (C-6a ↓), 128.85 (aromatic ↑), 127.86 (C-5 ↑), 127.00 (C-8 ↑), 126.39 (aromatic ↑), 125.32 (C-10a ↓), 124.00 (C-6 ↓), 123.76 (C-7 ↑), 121.98 (C-10 ↑), 121.80 (C-4a ↓), 76.25 (C-3 ↓), 58.69 (CH2 ↓), 40.00 (C-4 ↑), 20.49 (CH3 ↑), 14.25 (CH3 ↑); MS m/z (%): 395 (M++2, 1.98), 393 (M+, 5.84) with a base peak at 255 (100); Anal. Calcd for C23H20ClNO3: C, 70.14; H, 5.12; N, 3.56. Found: C, 70.18; H, 5.16; N, 3.59 %.

Ethyl 2-amino-6-chloro-4-(4-methoxyphenyl)-4H-benzo[h]chromene-3-carboxyalte (5f)

Colorless crystals from ethanol; yield 80 %; m.p. 177–178 °C; IR (KBr) υ (cm−1): 3468, 3325 (NH2), 3085, 3066, 2999, 2983, 2904, 2889 (CH), 1686 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.40–6.81 (m, 9H, aromatic), 7.82 (bs, 2H, NH2), 5.01 (s, 1H, H-4), 4.02 (q, 2H, CH2, J = 7.2 Hz), 3.68 (s, 3H, OCH3), 1.11 (t, 3H, CH3, J = 7.2 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 168.08 (CO), 160.40 (C-2), 142.05 (C-10b), 129.01 (C-6a), 128.85 (C-9), 127.88 (C-5), 127.47 (C-8), 127.47 (C-10a), 123.98 (C-6), 123.76 (C-7), 121.42 (C-10), 121.42 (C-4a), 76.35 (C-3), 58.66 (CH2), 54.89 (CH3), 40.08 (C-4), 14.27 (CH3), 157.62, 139.53, 128.29, 113.64 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 128.85 (C-9 ↑), 128.29 (aromatic ↑), 127.88 (C-5 ↑), 127.47 (C-8 ↑), 123.76 (C-7 ↑), 121.42 (C-10 ↑), 58.66 (CH2 ↓), 54.89 (CH3 ↑), 40.08 (C-4 ↑), 14.27 (CH3 ↑), 113.64 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 128.85 (C-9 ↑), 128.29 (aromatic ↑), 127.88 (C-5 ↑), 127.47 (C-8 ↑), 123.76 (C-7 ↑), 121.42 (C-10 ↑), 40.08 (C-4 ↑), 113.64 (aromatic ↑). In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 128.85 (C-9 ↑), 128.29 (aromatic ↑), 127.88 (C-5 ↑), 127.47 (C-8 ↑), 123.76 (C-7 ↑), 121.42 (C-10 ↑), 58.66 (CH2 ↑), 54.89 (CH3 ↑), 40.08 (C-4 ↑), 14.27 (CH3 ↑), 113.64 (aromatic ↑). 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 168.08 (CO ↓), 160.40 (C-2 ↓), 157.62 (aromatic ↓), 142.05 (C-10b ↓), 157.62 (aromatic ↓), 129.01 (C-6a ↓), 128.85 (C-9 ↑), 128.29 (aromatic ↑), 127.88 (C-5 ↑), 127.47 (C-8 ↑), 127.47 (C-10a ↓), 123.98 (C-6 ↓), 123.76 (C-7 ↑), 121.42 (C-10 ↑), 121.42 (C-4a ↓), 76.35 (C-3 ↓), 58.66 (CH2 ↓), 54.89 (CH3 ↑), 40.08 (C-4 ↑), 14.27 (CH3 ↑), 113.64 (aromatic ↑); MS m/z (%): 411 (M++2, 5.31), 409 (M+, 19.38) with a base peak at 75 (100); Anal. Calcd for C23H20ClNO4: C, 67.40; H, 4.92; N, 3.42. Found: C, 67.38; H, 4.89; N, 3.40 %.

Ethyl 2-amino-6-chloro-4-(4-nitrophenyl)-4H-benzo[h]chromene-3-carboxyalte (5g)

Yellow crystals from ethanol; yield 78 %; m.p. 169–170 °C; IR (KBr) υ (cm−1): 3467, 3317 (NH2), 3085, 3066, 2995, 2977, 2908, 2887 (CH), 1687 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.47–7.55 (m, 9H, aromatic), 8.00 (bs, 2H, NH2), 5.28 (s, 1H, H-4), 4.05 (q, 2H, CH2, J = 7.2 Hz), 1.11 (t, 3H, CH3, J = 7.2 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 167.75 (CO), 160.51 (C-2), 142.30 (C-10b), 129.35 (C-6a), 128.95 (C-9), 128.49 (C-5), 127.65 (C-10a), 127.65 (C-8), 123.99 (C-6), 123.70 (C-7), 121.59 (C-10), 119.97 (C-4a), 75.16 (C-3), 58.86 (CH2), 40.05 (C-4), 14.25 (CH3), 154.86, 146.00, 128.74, 126.13 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 128.95 (C-9 ↑), 128.74 (aromatic ↑), 128.49 (C-5 ↑), 127.65 (C-8 ↑), 126.13 (aromatic ↑), 123.70 (C-7 ↑), 121.59 (C-10 ↑), 58.86 (CH2 ↓), 40.05 (C-4 ↑), 14.25 (CH3 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 128.95 (C-9 ↑), 128.74 (aromatic ↑), 128.49 (C-5 ↑), 127.65 (C-8 ↑), 126.13 (aromatic ↑), 123.70 (C-7 ↑), 121.59 (C-10 ↑), 40.05 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 128.95 (C-9 ↑), 128.74 (aromatic ↑), 128.49 (C-5 ↑), 127.65 (C-8 ↑), 126.13 (aromatic ↑), 123.70 (C-7 ↑), 121.59 (C-10 ↑), 58.86 (CH2 ↑), 40.05 (C-4 ↑), 14.25 (CH3 ↑). 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 167.75 (CO ↓), 160.51 (C-2 ↓), 154.86 (aromatic ↓), 146.00 (aromatic ↓), 142.30 (C-10b ↓), 129.35 (C-6a ↓), 128.95 (C-9 ↑), 128.74 (aromatic ↑), 128.49 (C-5 ↑), 127.65 (C-10a ↓), 127.65 (C-8 ↑), 126.13 (aromatic ↑), 123.99 (C-6 ↓), 123.70 (C-7 ↑), 121.59 (C-10 ↑), 119.97 (C-4a ↓), 75.16 (C-3 ↓), 58.86 (CH2 ↓), 40.05 (C-4 ↑), 14.25 (CH3 ↑); MS m/z (%): 426 (M++2, 18.32), 424 (M+, 57.87) with a base peak at 54 (100); Anal. Calcd for C22H17ClN2O5: C, 62.20; H, 4.03; N, 6.59. Found: C, 62.24; H, 4.07; N, 6.62 %.

Ethyl 2-amino-6-chloro-4-(4-morpholinophenyl)-4H-benzo[h]chromene-3-carboxyalte (5h)

Yellow crystals from ethanol; yield 76 %; m.p. 199-200 οC; IR (KBr) υ (cm−1): 3380, 3286 (NH2), 3087, 3068, 2995, 2978, 2928, 2906, 2887 (CH), 1683 (CO); 1H NMR (500 MHz) (DMSO-d6) δ: 8.40–6.80 (m, 9H, aromatic), 7.84 (bs, 2H, NH2), 5.08 (s, 1H, H-4), 4.01 (q, 2H, CH2, J = 7.2 Hz), 3.98–3.67 (m, 4H, 2CH2), 3.36–3.00 (m, 4H, 2CH2), 1.10 (t, 3H, CH3, J = 7.2 Hz); 13C NMR (500 MHz) (DMSO-d6) δ: 160.41 (CO), 160.36 (C-2), 142.08 (C-10b), 129.17 (C-9), 128.06 (C-5), 127.87 (C-6a), 127.80 (C-10a), 126.48 (C-8), 125.39 (C-6), 123.79 (C-7), 121.47 (C-10), 121.37 (C-4a), 76.42 (C-3), 66.05 (CH2), 58.69 (CH2), 48.47 (CH2), 38.77 (C-4), 14.31 (CH3), 143.57, 138.20, 129.10, 114.90 (aromatic); 13 C NMR-DEPT spectrum at 135o CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 129.17 (C-9 ↑), 129.10 (aromatic ↑), 128.06 (C-5 ↑), 126.48 (C-8 ↑), 123.79 (C-7 ↑), 121.47 (C-10 ↑), 66.05 (CH2 ↓), 58.69 (CH2 ↓), 48.47 (CH2 ↓), 38.77 (C-4 ↑), 14.31 (CH3 ↑), 114.90 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ: 129.17 (C-9 ↑), 129.10 (aromatic ↑), 128.06 (C-5 ↑), 126.48 (C-8 ↑), 123.79 (C-7 ↑), 121.47 (C-10 ↑), 38.77 (C-4 ↑), 114.90 (aromatic ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive)-revealed signals at δ: 129.17 (C-9 ↑), 129.10 (aromatic ↑), 128.06 (C-5 ↑), 126.48 (C-8 ↑), 123.79 (C-7 ↑), 121.47 (C-10 ↑), 66.05 (CH2 ↑), 58.69 (CH2 ↑), 48.47 (CH2 ↑), 38.77 (C-4 ↑), 14.31 (CH3 ↑), 114.90 (aromatic ↑). 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ:160.41 (CO ↓) 160.36 (C-2 ↓), 143.57 (aromatic ↓), 142.08 (C-10b), 138.20 (aromatic ↓), 129.17 (C-9 ↑), 129.10 (aromatic ↑), 128.06 (C-5 ↑), 127.87 (C-6a ↓), 127.80 (C-10a ↓), 126.48 (C-8 ↑), 125.39 (C-6 ↓), 123.79 (C-7 ↑), 121.47 (C-10 ↑), 121.37 (C-4a ↓), 76.42 (C-3 ↓), 66.05 (CH2 ↓), 58.69 (CH2 ↓), 48.47 (CH2 ↓), 38.77 (C-4 ↑), 14.31 (CH3 ↑), 114.90 (aromatic ↑); MS m/z (%): 466 (M++2, 22.28), 464 (M+, 69.83) with a base peak at 101 (100); Anal. Calcd for C26H25ClN2O4: C, 67.17; H, 5.42; N, 6.03. Found: C, 67.13; H, 5.39; N, 6.06 %.

Antitumor screening

Cell culture

MCF-7, HCT, and HepG-2 cells were grown on RPMI-1640 medium supplemented with 10 % inactivated fetal calf serum and 50 μg/ml gentamycin. Vero cells were propagated in Dulbecco’s-modified Eagle’s medium (DMEM) supplemented with 10 % heat-inactivated fetal calf serum, 1 % l-glutamine, HEPES buffer and 50 μg/ml gentamycin. All cells were maintained at 37 °C in a humidified atmosphere with 5 % CO2 and were subcultures two to three times a week.

Cytotoxicity evaluation using viability assay

The in vitro cytotoxicity activity was studied against three cell lines: MCF-7, HCT, and HepG-2 using the colorimetric MTT assay (Mossman, 1983). The cells were seeded in 96-well microtitre plate (Falcon, NJ, USA) at a cell concentration of 1 × 104 cells per well in 100 μl of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Serial twofold dilutions of the metabolites were added confluent cell monolayer. The microtiter plates (polystyrene sterile tissue culture plates) were incubated at 37 °C in a humidified incubator with 5 % CO2 for a period of 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without the test sample and with or without DMSO. The little percentage of DMSO present in the wells (maximal 0.1 %) was found not to affect the experiment. After incubation of the cells for 24 h at 37 °C, various concentrations of sample were added, and the incubation was continued for 48 h and viable cells yield was determined by a colorimetric MTT method.

In brief, after the end of the incubation period, crystal violet solution (1 %) was added to each well for 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. Glacial acetic acid was then added to all wells and mixed thoroughly, and the plates were read on ELISA reader, using a test wavelength of 490 nm. Treated samples were compared with the control in the absence of the tested samples. All experiments were carried out in triplicate. The cytotoxic effect of each tested compound was calculated as [1 − (ODt/ODc)] × 100 % where ODt is the mean optical density of wells treated with the tested compounds and ODc is the mean optical density of untreated cells.