Introduction

Quinoline moiety is present in many classes of biologically active compounds (Ganesh et al., 2008; Larghi et al., 2009; Liu et al., 2009; Musiol et al., 2006a, b, 2007; Narender et al., 2006, Ramesh et al., 2009; Righi et al., 2008; Vazquez et al., 2004). The biological activities of quinoline derivatives depend not only on the bicyclic hetero-aromatic pharmacophore but also on the nature of the peripheral substituents and their spatial relationships.

They also exhibit antimalarial (Kaur et al., 2009), antitumor (Behforouz et al., 2007) antioxidant (Abas et al., 2006), antileishmanial (Rocha et al., 2005), and antiplatelet activities (Kuo et al., 2001). In addition, they function as pharmacologically active synthetic compounds (Watson et al., 2001) such as DNA binding capabilities (Atwell et al., 1989) and as DNA-intercalating carrier (Chen et al., 2000). A series of compounds derived from 8-hydroxyquinoline as potential HIV-1 integrate inhibitors were synthesized (Majerz-Maniecka et al., 2005). In addition styrylquinoline derivatives have gained strong attention due to their activities as perspective HIV integrase inhibitors (Jiang et al., 1990; Mekouar et al., 1998; Polanski et al., 2002; Pommier et al., 2005; Thomas and Roy, 2008; Zouhiri et al., 2005) and also for their extensive biological activities (Ganesh et al., 2008; Larghi et al., 2009; Liu et al., 2009; Mekouar et al., 1998; Narender et al., 2006).

In view of the above observations, and in continuation of our program on the chemistry of 4H-pyran derivatives (Abd-El-Aziz et al., 2004, 2007; Bedair et al., 2000, 2001; Eid et al., 2003; El-Agrody, 1994; El-Agrody et al., 1997a, b, 2000, 2001, 2002, 2011; Khafagy et al., 2002; Sayed et al., 2000; Sabry et al., 2011), it seemed interesting to synthesize new 4H-pyrano[3,2-h]quinoline derivatives by means of α-cyano-p-halocinnamonitriles, ethyl α-cyano-p-halocinnamates and evaluatation of their antitumor activities. The chemical structure of the studied compounds and structure–activity relationships (SAR) are discussed in this study.

Chemistry

Condensation of 8-hydroxy-2-methylquinoline (1) with p-chlorobenzaldehyde and p-bromobenzaldehyde in acetic anhydride under reflux afforded (E) 2-(4-chloro/bromo-styryl)-8-hydroxyquinoline (3a,b) via the intermediate (E) 8-acetoxy-2-(4-chloro/bromo-styryl)quinoline (2a,b) (Musiol et al., 2006a, b, 2007), while condensation of 1 with p-chlorobenzaldehyde, p-bromobenzaldehyde, and p-fluorobenzaldehyde under Microwave irradiation furnished (E) 2-(4-chloro/bromo/fluorostyryl)-8-hydroxyquinoline (3a–c) (Chang et al., 2010; Musiol et al., 2006a, b, 2007) (Scheme 1).

Scheme 1
scheme 1

Preparation of (E) 8-acetoxy-2-(4-chloro/bromostyryl)quinoline (2a,b) and (E) 2-(4-chloro/bromo/f luorostyryl)-8-hydroxyquinoline (3a–c)

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The structures of 2 and 3 were established on the basis of spectral data. The IR spectra of 2b showed the presence of a CO stretch at υ 1760 cm−1, while for 3b,c showed the appearance of a OH stretch at υ 3348–3399 cm.−1 The 1H and 13C NMR spectra of 2b revealed the presence of signals at δ 7.65 (d, J = 16 Hz, 1H, =CH), 7.50 (d, J = 16 Hz, 1H, =CH), 2.60 ppm (s, 3H, COCH3) and 134.39 (=CH), 129.40 (=CH), 21.06 ppm (CH3). Characteristic resonances were observed at δ 9.62 (bs, 1H, OH), 8.15–8.13 (d, J = 16 Hz, 1H, =CH), 7.52–7.45 ppm (d, J = 16 Hz, 1H, =CH) and 133.10–133.00 (=CH), 129.14–129.05 ppm (=CH) for 3b,c. The 13C NMR–DEPT spectra at 45°, 90° and 135° of 3b and the MS spectra of 2b and 3b,c provided additional evidences in support of the proposed structures.

The relative (E) configuration of compounds 2 and 3 were established from the coupling constant values (J = 16 Hz).

Treatment of (E) 2-(4-chloro/bromo/fluorostyryl)-8-hydroxyquinoline (3a–c) with α-cyano-p-chloro/bromocinnamonitrile (4a,b) and ethyl α-cyano-p-chloro/bromocinnamate (4c,d) in ethanol and piperidine under reflux afforded (E) 2-amino-4-(4-chloro/bromophenyl)-9-(4-halostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5a–f) and (E) ethyl 2-amino-4-(4-chloro/bromophenyl)-9-(4-halostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6a–f), respectively (Scheme 2).

Scheme 2
scheme 2

Synthesis of 9-(4-halostyryl)-4H-pyrano[3,2-h]quinoline derivatives (5a–f, 6a–f)

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The formation of compounds 5 and 6 indicates that the phenolate anion (C-7) of 3 attacks at the β-carbon of 4 to yield an acyclic Michael adduct, which underwent cyclization (Abd-El-Aziz et al., 2004), as shown in (Scheme 3) to give compounds 5 and 6.

Scheme 3
scheme 3

Mechanism formation of compounds (5,6)

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The structures 5 and 6 were established on the basis of spectral data. The IR spectra of 5a–f showed the appearance of NH2 stretch at υ 3456–3384, 3328–3310, 3200–3168 cm−1 and CN stretch at υ 2198–2187 cm,−1 while NH2 stretch at υ 3412–3380, 3349–3292 cm−1 and CO stretch at υ 1677–1643 cm−1 for 6a–f. The 1H and 13C NMR spectra of 5a–f and 6a–f revealed the presence of 4H signals at δ 5.11–5.00 (s, 1H, H-4) and 40.71–40.09 ppm (C-4). In compound 6a–f the ester group gave 1H signals at 4.13–4.01 (q, J = 7 Hz, 2H, CH2) and 1.22–1.10 (t, J = 7 Hz, 3H, CH3) with the corresponding signals in the 13C spectra at 59.59–58.73 (CH2) and 14.39–14.26 ppm (CH3) respectively. The 13C NMR–DEPT spectra at 45°, 90°, 135°, 13C NMR–APT and the MS spectra of compounds 5 and 6 provided additional evidences in support of the proposed structures.

The relative (E) configuration of compounds 5 and 6 were established from the coupling constant values (J = 16–16.5 Hz).

Antitumor assays

Compounds 5a–f and 6a–f were evaluated for their human tumor cell growth inhibitory activity against three cell lines: breast adenocarcinoma (MCF-7), lung carcinoma (HCT), and hepatocellular carcinoma (HepG-2). The measurement of cell growth and viability were determined as described in the literature (Rahman et al., 2001). In vitro cytotoxicity evaluation using viability assays were performed by the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University using, Vinblastine as standard drug. The inhibitory activity of the synthetic compounds 5a–f and 6a–f against three different human tumor cell lines MCF-7, HCT, and HepG-2 are given in Table 1 and Figs. 1, 2 and 3.

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

Evaluation of cytotoxicity against MCF-7 cell line

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Fig. 2
figure 2

Evaluation of cytotoxicity against HCT cell line

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Fig. 3
figure 3

Evaluation of cytotoxicity against HepG-2 cell line

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Result and discussion

Quinoline derivatives were chosen for this study because it is known that quinoline and fused quinoline derivatives are important families of active compounds with a wide range of pharmacological properties (Ganesh et al., 2008; Larghi et al., 2009; Liu et al., 2009; Musiol et al., 2006a, b, 2007; Narender et al., 2006, Ramesh et al., 2009; Righi et al., 2008; Vazquez et al., 2004). Twelve compounds of 4H-pyrano[3,2-h]quinoline 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 5a–f and 6a–f were tested against three tumor cell lines: MCF-7, HCT and HepG-2. The cytotoxicity evaluation using viability assays, and the inhibitory activities are given in Table 1 and Figs. 1, 2 and 3. Compounds 5a,d and 6a,c,d had activities (IC50 = 2.4–5.6 μg/ml) against MCF-7 more than the standard drug Vinblastine (IC50 = 6.1 μg/ml), while compounds 5b,c and 6b showed activities (IC50 = 10.3–15.9 μg/ml) close to the standard drug Vinblastine (IC50 = 6.1 μg/ml) and compounds 5e,f and 6e,f showed weak activities (IC50 = 37.9–42.5 μg/ml). Compounds 5a and 6c had activities (IC50 = 4.8–6.1 μg/ml) against the HCT close to the standard drug Vinblastine (IC50 = 2.6 μg/ml), while compounds 5b–d and 6a,b,d–f showed moderate activities (IC50 = 10.2–26.2 μg/ml) as compared with the standard drug Vinblastine and compound 5e,f showed weak activities. Finally, compound 6c had activity (IC50 = 3.1 μg/ml) against HepG-2 more than the standard drug Vinblastine (IC50 = 4.6 μg/ml), while compounds 5c,d and 6a,d showed activities (IC50 = 6.1–8.7 μg/ml) close to the standard drug Vinblastine (IC50 = 4.6 μg/ml) and compounds 5b,6b showed moderate activities (IC50 = 23.2–28.6 μg/ml), in addition compounds 5a,e,f and 6e,f showed weak activities (IC50 = 37.1–48.2 μg/ml).

SAR studies

The SAR studies of compound 5a and its analogs revealed that compounds 5d,a have potent antitumor activities against the MCF-7 than the other compounds 5b,c,e,f. These data indicate that the activity of compounds 5d,a are considerably enhanced by the presence of the bromo/bromo or chloro/chloro atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings in combination with the cyano group (electron-withdrawing group) at the 3-position in 4H-pyrano[3,2-h]quinoline moiety, while the presence of the chloro/bromo or bromo/chloro atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings in combination with the cyano group (electron-withdrawing group) at the 3-position in compounds 5b,c slightly decreased their antitumor activities. However the presence of fluoro/chloro and fluoro/bromo atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings resulted in more decreased in the antitumor activities of compounds 5e,f. Replacement of the electron-withdrawing group, cyano group by ester group at the 3-position for compound 6a and its analogs improved the antitumor activities. Compounds 6d,c,a have potent antitumor activities against the MCF-7 than the other compounds 6b,e,f. These data indicate the activities of compounds 6d,c,a are considerably enhanced by the presence of the bromo/bromo, bromo/chloro or chloro/chloro atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings in combination with the ester group (electron-withdrawing group) at the 3-position in 4H-pyrano[3,2-h]quinoline moiety, while the presence of chloro/bromo atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings in combination with the ester group (electron-withdrawing group) at the 3-position for compound 6b slightly decreased its antitumor activity and the presence of fluoro/chloro and fluoro/bromo atoms (electron-withdrawing groups) at the 4-position of the styryl and the phenyl rings resulted in more decreased in the antitumor activities of compounds 6e,f.

In the case of the HCT, an investigation of the SAR for compound 5a and its analogs revealed that compound 5a showed antitumor activities close to the standard durg Vinblastine, this indicated that the activity is considerably affected by the presence of the (electron-withdrawing groups) chloro/chloro atoms at the 4-positions of the styryl and the phenyl rings in combination with the cyano group at the 3-position, while the antitumor activities decreased for compounds 5c,b,d with the presence of the bromo/chloro, chloro/bromo or bromo/bromo atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings in combination with the cyano group at the 3-position. In addition, compounds 5e,f showed weak antitumor activities due to the presence of fluoro/chloro or fluoro/bromo atoms (electron-withdrawing groups). Introduction of ester group at the 3-position for compound 6a and its analogs did not improve the antitumor activities. Compound 6c had activity close to the standard durg Vinblastine, indicating that the activity is considerably affected by the presence of bromo/chloro atoms at the 4-positions of the styryl and the phenyl rings with the ester group (electron-withdrawing groups) at the 3-position as compared with the standard durg Vinblastine, while compounds 6d,a,b showed modearare activities as compared with the standard durg Vinblastine and compounds 6e,f showed weak activities due to the presence of the other halogen atoms and ester group.

Furthermore, an investigation of the SAR for compound 5a and its analogs against the HepG-2 showed that compounds 5c,d have antitumor activities close to the standard durg Vinblastine. This indicates that the activities are considerably affected by the presence of the bromo/chloro or bromo/bromo atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings with the cyano group (electron-withdrawing group) at the 3-position, while the antitumor activity for compound 5b decreased with the presence of the chloro/bromo atoms. In addition, compounds 5a,e,f showed weak antitumor activities due to the presence the other halogen atoms and cyano group. Replacement of the electron-withdrawing group, cyano group by ester group at the 3-position for compound 6a and its analogs improved the antitumor activity. Compound 6c showed more antitumor activity against the HepG-2 than the standard drug Vinblastine, due to the presence of the bromo/chloro atoms (electron-withdrawing groups) at the 4-positions of the styryl and the phenyl rings in combination with the ester group (electron-withdrawing group) at the 3-position in 4H-pyrano[3,2-h]quinoline, while compounds 6d,a showed antitumor activities against the HepG-2 very close to the standard drug Vinblastine. These data indicate that the activities are considerably affected by the presence of bromo/bromo or chloro/chloro atoms (electron-withdrawing groups). Finally, compounds 6b,e,f showed more decreasing in antitumor activity due to the presence the other halogen atoms and ester group.

Conclusion

In this article, we reported the synthesis of some 4H-pyrano[3,2-h]quinoline derivatives and the antitumor evaluation of all the novel compounds. Compounds 5a,d and 6a,c,d had the most potent antitumor activity against the human breast tumor cells (MCF-7), while compounds 5a and 6c had the most potent against the human lung carcinoma (HCT), and compound 6c had the most potent antitumor activity against the human hepatocellular carcinoma cells (HepG2). This potency could be attributed to the presence of the electron-withdrawing groups, bromo/bromo, bromo/chloro or chloro/chloro atoms at the 4-position of the styryl and the phenyl rings in combination with the ester/cyano group at the 3-position in 4H-pyrano[3,2-h]quinoline.

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), with this technique, the signals of CH & CH3 carbon atoms appears normal (up) and the signal of carbon atoms in CH2 environments appears 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 in both the Faculty of Science Cairo University, Cairo and King Saud University, Riyadh.

Reaction of 8-hydroxy-2-methylquinoline (1) with p-halobenzaldehyde

Method (a)

A mixture of 8-hydroxy-2-methylquinoline (1) (0.01 mol), p-chlorobenzaldehyde or p-bromobenzaldehyde (0.08 mol) and acetic anhydride (100 ml) was heated at 150°C for 30 h (TLC monitoring). After cooling, the solvent was removed in vacuum, and the residue was recrystallised from ethanol/benzene to give 2a,b. Compound 2a,b was heated at 100°C for 1 h (TLC monitoring) in pyridine/water (v/v = 4:1) (100 ml). After cooling, the solvent was removed in vacuum to provide the crude product which recrystallised from ethanol to give 3a,b. The physical and spectral data of compounds 2a,b and 3a–c are as follows:

(E) 8-Acetoxy-2-(4-chlorostyryl)quinoline (2a)

Prepared according to the previously reported procedure (El-Agrody et al., 2011).

(E) 8-Acetoxy-2-(4-bromostyryl)quinoline (2b)

Pale yellow crystals from ethanol/benzene; yield 38%; m.p. 130–131°C; IR (KBr) υ (cm−1): 3080, 3045, 3015, 2940 (CH), 1760 (CO); 1H NMR (500 MHz, CDCl3) δ: 8.15–7.29 (m, 9H, aromatic), 7.65 (d, J = 16 Hz, 1H, =CH), 7.50 (d, J = 16.0 Hz, 1H, =CH), 2.60 (s, 3H, COCH3); 13C NMR (125 MHz, CDCl3) δ: 169.81 (CO), 155.44 (C-2), 147.40 (C-8), 140.93 (C-1a), 136.47 (C-4), 134.39 (=CH), 129.40 (=CH), 129.00 (C-4a), 128.63 (C-6), 125.85 (C-5), 121.74 (C-7), 120.22 (C-3), 21.06 (CH3), 134.98, 133.39, 128.85, 125.57 (aromatic) MS m/z (%): 369 (M++2, 1), 367 (M+, 1), 327 (98), 325 (99), 170 (5), 144 (4), 115 (100), 74 (43), 50 (75); Anal. Calcd for C19H14BrNO2: C, 61.97; H, 3.83; N, 3.80. Found: C, 62.01; H, 3.85; N, 3.84%.

(E) 2-(4-Chlorostyryl)-8-hydroxyquinoline (3a)

Prepared according to the previously reported procedure (El-Agrody et al., 2011).

(E) 2-(4-Bromostyryl)-8-hydroxyquinoline (3b)

Yellow needles from ethanol; yield 32%; m.p. 135–136°C; (Musiol et al., 2006a, b, 2007, m.p. 145°C); IR (KBr) υ (cm−1): 3348 (OH), 3070, 3046, 2952, 2800 (CH); 1H NMR (500 MHz, CDCl3) δ: 9.62 (bs, 1H, OH), 8.31–7.10 (m, 9H, aromatic), 8.13 (d, J = 16.0 Hz, 1H, =CH,), 7.52 (d, J = 16.0, Hz 1H, =CH); 13C NMR (125 MHz, CDCl3) δ: 153.08 (C-2), 152.93 (C-8), 138.12 (C-1a), 136.55 (C-4), 133.00 (=CH), 129.05 (=CH), 128.80 (C-4a), 127.17 (C-6), 121.06 (C-3), 117.56 (C-5), 111.21 (C-7), 135.77, 131.82, 127.73, 121.58 (aromatic).; 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.55 (C-4 ↑), 133.00 (=CH ↑), 131.82 (aromatic ↑), 129.05 (=CH ↑), 127.73 (aromatic ↑), 127.17 (C-6 ↑), 121.06 (C-3 ↑), 117.56 (C-5 ↑), 111.21 (C-7 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.55 (C-4 ↑), 133.00 (=CH ↑), 131.82 (aromatic ↑), 129.05 (=CH ↑), 127.73 (aromatic ↑), 127.17 (C-6 ↑), 121.06 (C-3 ↑), 117.56 (C-5 ↑), 111.21 (C-7 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.55 (C-4 ↑), 133.00 (=CH ↑), 131.82 (aromatic ↑), 129.05 (=CH ↑), 127.73 (aromatic ↑), 127.17 (C-6 ↑), 121.06 (C-3 ↑), 117.56 (C-5 ↑), 111.21 (C-7 ↑) .13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 153.08 (C-2 ↓), 152.93 (C-8 ↓), 138.12 (C-1a ↓), 136.55 (C-4 ↑), 135.77 (aromatic ↓), 133.00 (=CH ↑), 131.82 (aromatic ↑), 129.05 (=CH ↑), 128.80 (C-4a ↓), 127.73 (aromatic ↑), 127.17 (C-6 ↑), 121.58 (aromatic ↓), 121.06 (C-3 ↑), 117.56 (C-5 ↑), 111.21 (C-7 ↑); MS m/z (%): 327 (M++2, 98), 325 (M+, 100), 170 (2), 144 (4), 115 (65), 75 (41), 50 (62); C17H12BrNO.

(E) 2-(4-Fluorostyryl)-8-hydroxyquinoline (3c)

Yellow needles from ethanol; yield 26%; m.p. 110–111°C (Chang et al., 2010; Musiol et al., 2006a, b, 2007, m.p. 107°C); IR (KBr) υ (cm−1): 3399 (OH), 3055, 3011, 2830 (CH); 1H NMR (500 MHz, CDCl3) δ: 9.62 (bs, 1H, OH), 8.31–7.12 (m, 9H, aromatic), 8.15 (d, J = 16.0 Hz 1H, =CH), 7.45 (d, J = 16.0 Hz, 1H, =CH); 13C NMR (125 MHz, CDCl3) δ: 153.29 (C-2), 152.88 (C-8), 138.10 (C-1a), 136.49 (C-4), 133.10 (=CH), 129.14 (=CH), 128.76 (C-4a), 127.83 (C-6), 120.92 (C-3), 117.55 (C-5), 111.17 (C-7), 161.21, 133.06, 129.08, 115.89 (aromatic); MS m/z (%): 265 (M+, 37), 264 (100), 170 (3), 145 (6), 116 (34), 74 (20), 50 (32); C17H12FNO.

Method (b)

8-Hydroxy-2-methylquinoline (1) (0.01 mol) and p-chlorobenzaldehyde, p-bromobenzaldehyde or p-flurobenzaldehyde (0.02 mol) were mixed thoroughly using mortar and put in an open vessel. Then the mixture was exposed to Microwave irradiation for 4 min. The oven was operated at 70% power (560 W) in a two-step mode with interval (2 min-30 s-2 min). After the reaction, the mixture was allowed to cool down and Et2O (10 ml) was added. The crude product was filtered, washed with Et2O (15 ml) and purified by recrystallization from ethanol to give 3a–c (m.p. and mixed m.p.) 27–40%; (3a, Musiol et al., 2006a, b, 2007, m.p. 150°C; 3b, Musiol et al., 2006a, b, 2007, m.p. 145°C and 3c, Chang et al., 2010, m.p. 107°C).

Reaction of (E) 8-hydroxy-2-(4-chloro/bromo/fluorostyryl)quinoline (3a–c) with α-cyano-p-chloro/bromocinnamonitrile (4a,b) and ethyl α-cyano-p-chloro/bromocinnamate (4c,d)

General procedure

A solution of (E) 2-(4-chloro/bromo/fluorostyryl)-8-hydroxyquinoline (3a–c) (0.01 mol) in EtOH (30 ml) was treated with α-cyano-p-chloro/bromocinnamonitrile (4a,b) (0.01 mol) or ethyl α-cyano-p-chloro/bromocinnamate (4c,d) (0.01 mol) and piperidine (0.5 ml). The reaction mixture was heated until complete precipitation occurred (reaction times: 30 min. for 4a,b and 45 min. for 4c,d). The solid product which formed was collected by filtration and recrystallised from etanol or benzene to give 5a–f and 6a–f. The physical and spectral data of compounds 5a–f and 6a–f are as follows:

(E) 2-Amino-4-(4-chlorophenyl)-9-(4-chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5a)

Prepared according to the previously reported procedure (El-Agrody et al., 2011).

(E) 2-Amino-4-(4-bromophenyl)-9-(4-chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5b)

Prepared according to the previously reported procedure (El-Agrody et al., 2011).

(E) 2-Amino-4-(4-chlorophenyl)-9-(4-bromostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5c)

Pale yellow needles from benzene; yield 87%; m.p. 240–241°C; IR (KBr) υ (cm−1): 3456, 3323, 3200 (NH2), 3075, 3033, 2940, 2850 (CH), 2193 (CN); 1H NMR (500 MHz, DMSO-d6) δ: 8.33–7.14 (m, 12H, aromatic), 7.98 (d, J = 16.5 Hz, 1H, =CH), 7.54 (d, J = 16.5 Hz, 1H, =CH), 7.24 (bs, 2H, NH2, canceled by D2O), 5.01 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.21 (C-2), 155.12 (C-9), 144.58 (C-10b), 137.44 (C-10a), 136.53 (C-7), 133.50 (=CH), 129.57 (=CH), 126.89 (C-6a), 126.33 (C-5), 121.72 (C-4a), 123.41 (C-6), 121.01 (C-8), 120.31 (CN), 55.70 (C-3), 40.42 (C-4), 142.81, 135.47, 131.86, 131.63, 129.16, 128.94, 128.29, 121.88 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.53 (C-7 ↑), 133.50 (=CH ↑), 131.86 (aromatic ↑), 129.57 (=CH ↑), 129.16 (aromatic ↑), 128.94 (aromatic ↑), 128.29 (aromatic ↑), 126.33 (C-5 ↑), 123.41 (C-6 ↑), 121.01 (C-8 ↑), 40.42 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.53 (C-7 ↑), 133.50 (=CH ↑), 131.86 (aromatic ↑), 129.57 (=CH ↑), 129.16 (aromatic ↑), 128.94 (aromatic ↑), 128.29 (aromatic ↑), 126.33 (C-5 ↑), 123.41 (C-6 ↑), 121.01 (C-8 ↑), 40.42 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.53 (C-7 ↑), 133.50 (=CH ↑), 131.86 (aromatic ↑), 129.57 (=CH ↑), 129.16 (aromatic ↑), 128.94 (aromatic ↑), 128.29 (aromatic ↑), 126.33 (C-5 ↑), 123.41 (C-6 ↑), 121.01 (C-8 ↑), 40.42 (C-4 ↑). 13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 160.21 (C-2 ↓), 155.12 (C-9 ↓), 144.58 (C-10b ↓), 142.81 (aromatic ↓), 137.44, (C-10a ↓), 136.53 (C-7 ↑), 135.47 (aromatic ↓), 133.50 (=CH ↑), 131.86 (aromatic ↑), 131.63 (aromatic ↓), 129.57 (=CH ↑), 129.16 (aromatic ↑), 128.94 (aromatic ↑), 128.29 (aromatic ↑), 126.89 (C-6a ↓), 126.33 (C-5 ↑), 123.41 (C-6 ↑), 121.88 (aromatic ↓), 121.72 (C-4a ↓), 121.01 (C-8 ↑), 120.31 (CN ↓), 55.70 (C-3 ↓), 40.42 (C-4 ↑); MS m/z (%): 517 [M++4] (1), 515 (M++2, 3.19), 513 (M+, 2.55), 406 (90.68), 404 (100), 224 (2), 166 (41), 101 (43), 74 (50), 50 (68); Anal. Calcd for C27H17BrClN3O: C, 62.99; H, 3.33; N, 8.16. Found: C, 63.51; H, 3.17; N, 7.88%.

(E) 2-Amino-4-(4-bromophenyl)-9-(4-bromostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5d)

Pale yellow needles from benzene; yield 88%; m.p. 260–261°C; IR (KBr) υ (cm−1): 3397, 3321, 3195 (NH2), 3085, 3053, 2968, 2871 (CH), 2196 (CN); 1H NMR (500 MHz, DMSO-d6) δ: 8.32–7.10 (m, 12H, aromatic), 8.13 (d, J = 16.0 Hz, 1H, =CH), 7.67 (bs, 2H, NH2, canceled by D2O), 7.52 (d, J = 16.0 Hz, 1H, =CH), 5.00 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.25 (C-2), 153.08 (C-9), 144.59 (C-10b), 138.12, (C-10a), 136.56 (C-7), 132.99 (=CH), 129.95 (=CH), 128.80 (C-5), 127.17 (C-6a), 122.65 (C-4a), 121.58 (C-6), 121.03 (C-8), 117.55 (CN), 56.93 (C-3), 40.46 (C-4), 142.82, 135.77, 133.83, 131.65, 129.17, 129.06, 123.42, 120.15 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.56 (C-7 ↑), 133.83 (aromatic ↑), 131.65 (aromatic ↑), 132.99 (=CH ↑), 129.95 (=CH ↑), 129.17 (aromatic ↑), 129.06, (aromatic ↑), 128. 80 (C-5 ↑), 121.58 (C-6 ↑), 121.03 (C-8 ↑), 40.46 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.56 (C-7 ↑), 133.83 (aromatic ↑), 131.65 (aromatic ↑), 132.99 (=CH ↑), 129.95 (=CH ↑), 129.17 (aromatic ↑), 129.06, (aromatic ↑), 128. 80 (C-5 ↑), 121.58 (C-6 ↑), 121.03 (C-8 ↑), 40.46 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.56 (C-7 ↑), 133.83 (aromatic ↑), 131.65 (aromatic ↑), 132.99 (=CH ↑), 129.95 (=CH ↑), 129.17 (aromatic ↑), 129.06, (aromatic ↑), 128. 80 (C-5 ↑), 121.58 (C-6 ↑), 121.03 (C-8 ↑), 40.46 (C-4 ↑)0.13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 160.25 (C-2 ↓), 153.08 (C-9 ↓), 144.59 (C-10b ↓), 142.82 (aromatic ↓), 138.12, (C-10a ↓), 136.56 (C-7 ↑), 135.77 (aromatic ↓), 133.83 (aromatic ↑), 132.99 (=CH ↑), 131.65 (aromatic ↑), 129.95 (=CH ↑), 129.17 (aromatic ↑), 129.06 (aromatic ↑), 128. 80 (C-5 ↑), 127.17 (C-6a ↓), 123.42 (aromatic ↓), 122.65 (C-4a ↓), 121.58 (C-6 ↑), 121.03 (C-8 ↑), 120.15 (aromatic ↓); 117.55 (CN ↓), 56.93 (C-3 ↓), 40.46 (C-4 ↑); MS m/z (%): 561 [M++4] (7.18), 559 [M++2] (17.31), 557 [M]+ (9.16), 405 (M++2, 21.49), 403 (M+, 25.65), 249 (6), 221 (8), 166 (15), 102 (48), 77 (79), 50 (100); Anal. Calcd for C27H17Br2N3O: C, 57.99; H, 3.06; N, 7.51. Found: C, 57.87; H, 3.03; N, 7.34%.

(E) 2-Amino-4-(4-chlorophenyl)-9-(4-fluorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5e)

Pale yellow needles from ethanol/benzene; yield 84%; m.p. 245–246°C; IR (KBr) υ (cm−1): 3421, 3328, 3197 (NH2), 3085, 3051, 2880 (CH), 2190 (CN); 1H NMR (500 MHz, DMSO-d6) δ: 8.33–7.14 (m, 12H, aromatic), 8.01 (d, J = 16.0 Hz, 1H, =CH), 7.43 (d, J = 16.0 Hz, 1H, =CH), 7.23 (bs, 2H, NH2, canceled by D2O), 5.01 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 163.32 (C-2), 160.25 (C-9), 155.32 (C-10b), 137.41, (C-10a), 136.49 (C-7), 133.62 (=CH), 129.58 (=CH), 128.72 (C-5), 126.83 (C-6a), 121.70 (C-4a), 124.41 (C-6), 120.90 (C-8), 120.32 (CN), 55.96 (C-3), 40.40 (C-4), 161.36, 144.62, 142.79, 132.82, 129.24, 127.97, 126.24, 115.98 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.49 (C-7 ↑), 133.62 (=CH ↑), 129.58 (=CH ↑), 129.24 (aromatic ↑), 128.72 (C-5 ↑), 127.97 (aromatic ↑), 126.24 (aromatic ↑), 124.41 (C-6 ↑), 120.90 (C-8 ↑), 115.98 (aromatic ↑), 40.40 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.49 (C-7 ↑), 133.62 (=CH ↑), 129.58 (=CH ↑), 129.24 (aromatic ↑), 128.72 (C-5 ↑), 127.97 (aromatic ↑), 126.24 (aromatic ↑), 124.41 (C-6 ↑), 120.90 (C-8 ↑), 115.98 (aromatic ↑), 40.40 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.49 (C-7 ↑), 133.62 (=CH ↑), 129.58 (=CH ↑), 129.24 (aromatic ↑), 128.72 (C-5 ↑), 127.97 (aromatic ↑), 126.24 (aromatic ↑), 124.41 (C-6 ↑), 120.90 (C-8 ↑), 115.98 (aromatic ↑), 40.40 (C-4 ↑)0.13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ163.32 (C-2 ↓), 161.36 (aromatic ↓), 160.25 (C-9 ↓), 155.32 (C-10b ↓), 144.62 (aromatic ↓), 142.79 (aromatic ↓), 137.41, (C-10a ↓), 136.49 (C-7 ↑), 133.62 (=CH ↑), 132.82 (aromatic ↓), 129.58 (=CH ↑), 129.24 (aromatic ↑), 128.72 (C-5 ↑), 127.97 (aromatic ↑), 126.83 (C-6a ↓), 126.24 (aromatic ↑), 121.70 (C-4a ↓), 124.41 (C-6 ↑), 120.90 (C-8 ↑), 120.32 (CN ↓), 115.98 (aromatic ↑), 55.96 (C-3 ↓), 40.40 (C-4 ↑); MS m/z (%): 455 (M++2, 12.43), 453 (M+, 29.30), 343 (100), 248 (1), 221 (2), 166 (11), 100 (5), 65 (3); Anal. Calcd for C27H17ClFN3O: C, 71.45; H, 3.78; N, 9.26. Found: C, 71.35; H, 3.57; N, 9.08%.

(E) 2-Amino-4-(4-bromoophenyl)-9-(4-fluorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5f)

Pale yellow needles from ethanol/benzene; yield 81%; m.p. 240–241°C; IR (KBr) υ (cm−1): 3408, 3319, 3168 (NH2), 3050, 2964, 2900, 2860 (CH), 2187 (CN); 1H NMR (500 MHz, DMSO-d6) δ: 8.33–7.14 (m, 12H, aromatic), 8.01 (d, J = 16.0 Hz, 1H, =CH), 7.46 (d, J = 16.0 Hz, 1H, =CH), 7.23 (bs, 2H, NH2, canceled by D2O), 5.00 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 163.32 (C-2), 160.25 (C-9), 155.33 (C-10b), 137.41, (C-10a), 136.49 (C-7), 133.62 (=CH), 129.95 (=CH), 127.99 (C-5), 126.83 (C-6a), 123.41 (C-6), 121.63 (C-4a), 120.90 (C-8), 120.31 (CN), 56.00 (C-3), 40.47 (C-4), 161.36, 145.03, 142.79, 132.82, 129.24, 127.99, 126.24, 115.98 (aromatic); MS m/z (%): 499 (M++2, 39.28), 497 (M+, 40.22), 342 (100), 247 (2), 221 (3), 166 (8), 100 (19), 50 (36); Anal. Calcd for C27H17BrFN3O: C, 65.07; H, 3.44; N, 8.43. Found: C, 65.82; H, 3.33; N, 8.08%.

(E) Ethyl 2-amino-4-(4-chlorophenyl)-9-(4-chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6a)

Prepared according to the previously reported procedure (El-Agrody et al., 2011).

(E) Ethyl 2-amino-4-(4-bromophenyl)-9-(4-chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6b)

Prepared according to the previously reported procedure (El-Agrody et al., 2011).

(E) Ethyl 2-amino-4-(4-chlorophenyl)-9-(4-bromostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6c)

Colorless needles from ethanol; yield 80%; m.p. 191–192°C; IR (KBr) υ (cm−1): 3405, 3292 (NH2), 3034, 2979, 2909, 2850 (CH), 1676 (CO); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.32–7.32 (m, 12H, aromatic), 7.30 (bs, 2H, NH2, canceled by D2O), 7.95 (d, J = 16.0 Hz, 1H, =CH), 7.54 (d, J = 16.0 Hz, 1H, =CH), 5.10 (s, 1H, H-4), 4.01 (q, J = 7.0 Hz, 2H, CH2), 1.10 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ (ppm): 168.15 (CO), 160.82 (C-2), 155.10 (C-9), 146.71 (C-10b), 142.78 (C-10a), 136.49 (C-7), 133.36 (=CH), 129.23 (=CH), 128.20 (C-5), 126.69 (C-6a), 124.65 (C-4a), 123.30 (C-6), 120.59 (C-8), 75.79 (C-3), 58.73 (CH2), 40.10 (C-4), 14.26 (CH3), 137.49, 135.50, 131.86, 130.65, 129.18, 129.14, 126.78, 121.84 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.49 (C-7 ↑), 133.36 (=CH ↑), 131.86 (aromatic ↑), 129.23 (=CH ↑), 129.18 (aromatic ↑), 129.14 (aromatic ↑), 128.20 (C-5 ↑), 126.78 (aromatic ↑), 123.30 (C-6 ↑), 120.59 (C-8 ↑), 58.73 (CH2 ↓), 40.10 (C-4 ↑), 14.26 (CH3↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.49 (C-7 ↑), 133.36 (=CH ↑), 131.86 (aromatic ↑), 129.23 (=CH ↑), 129.18 (aromatic ↑), 129.14 (aromatic ↑), 128.20 (C-5 ↑), 126.78 (aromatic ↑), 123.30 (C-6 ↑), 120.59 (C-8 ↑), 40.10 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.49 (C-7 ↑), 133.36 (=CH ↑), 131.86 (aromatic ↑), 129.23 (=CH ↑), 129.18 (aromatic ↑), 129.14 (aromatic ↑), 128.20 (C-5 ↑), 126.78 (aromatic ↑), 123.30 (C-6 ↑), 120.59 (C-8 ↑), 58.73 (CH2 ↑), 40.10 (C-4 ↑), 14.26 (CH3 ↑). 13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 168.15 (CO ↓), 160.82 (C-2 ↓), 155.10 (C-9 ↓), 146.71 (C-10b ↓), 142.78 (C-10a ↓), 137.49 (aromatic ↓), 136.49 (C-7 ↑), 135.50 (aromatic ↓), 133.36 (=CH ↑), 131.86 (aromatic ↑), 130.65 (aromatic ↓), 129.23 (=CH ↑), 129.18 (aromatic ↑), 129.14 (aromatic ↑), 128.20 (C-5 ↑), 126.78 (aromatic ↑), 126.69 (C-6a ↓), 124.65 (C-4a ↓), 123.30 (C-6 ↑), 121.84 (aromatic ↓), 120.59 (C-8 ↑), 75.79 (C-3 ↓), 58.73 (CH2 ↓), 40.10 (C-4 ↑), 14.26 (CH3 ↑); MS m/z (%): 564 (M++4, 2.87), 562 (M++2, 10.26), 560 (M+, 7.82), 492 (3.31), 490 (10.31), 488 (9.62), 453 (97.59) 451 (100), 296 (21), 228 (34) 150 (20), 111 (84), 75 (93), 50 (85); Anal. Calcd for C29H22BrClN2O3: C, 61.99; H, 3.95; N, 4.99. Found: C, 62.04; H, 4.02; N, 5.03%.

(E) Ethyl 2-amino-4-(4-bromophenyl)-9-(4-bromostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6d)

Colorless needles from ethanol; yield 81%; m.p. 195–196°C; IR (KBr) υ (cm−1): 3412, 3296 (NH2), 3049, 3020, 2979, 2935, 2900, 2873 (CH), 1677 (CO); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.32–7.23 (m, 12H, aromatic), 7.86 (bs, 2H, NH2, canceled by D2O), 7.95 (d, J = 16.5 Hz, 1H, =CH), 7.53 (d, J = 16.5 Hz, 1H, =CH), 5.09 (s, 1H, H-4), 4.00 (q, J = 7.0 Hz, 2H, CH2), 1.10 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ (ppm): 168.13 (CO), 160.82 (C-2), 155.10 (C-9), 147.71 (C-10b), 137.49 (C-10a), 136.51 (C-7), 133.37 (=CH), 129.64 (=CH), 128.82 (C-5), 126.70 (C-6a), 124.60 (C-4a), 123.31 (C-6), 120.62 (C-8), 75.72 (C-3), 58.74 (CH2), 40.10 (C-4), 14.27 (CH3), 142.78, 135.51, 131.88, 131.13, 129.15, 126.79, 121.85, 121.05 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.51 (C-7 ↑), 133.37 (=CH ↑), 131.88 (aromatic ↑), 131.13 (aromatic ↑), 129.64 (=CH ↑), 129.15 (aromatic ↑), 126.79 (aromatic ↑),128.82 (C-5 ↑), 123.31 (C-6 ↑), 120.62 (C-8 ↑), 58.74 (CH2 ↓), 40.10 (C-4 ↑), 14.27 (CH3 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.51 (C-7 ↑), 133.37 (=CH ↑), 131.88 (aromatic ↑), 131.13 (aromatic ↑), 129.64 (=CH ↑), 129.15 (aromatic ↑), 126.79 (aromatic ↑), 128.82 (C-5 ↑), 123.31 (C-6 ↑), 120.62 (C-8 ↑), 40.10 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.51 (C-7 ↑), 133.37 (=CH ↑), 131.88 (aromatic ↑), 131.13 (aromatic ↑), 129.64 (=CH ↑), 129.15 (aromatic ↑), 126.79 (aromatic ↑),128.82 (C-5 ↑), 123.31 (C-6 ↑), 120.62 (C-8 ↑), 58.74 (CH2 ↑), 40.10 (C-4 ↑), 14.27 (CH3 ↑). 13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 168.13 (CO ↓), 160.82 (C-2 ↓), 155.10 (C-9 ↓), 147.71 (C-10b ↓), 142.78 (aromatic ↓), 137.49 (C-10a ↓), 136.51 (C-7 ↑), 135.51 (aromatic ↓), 133.37 (=CH ↑), 131.88 (aromatic ↑), 131.13 (aromatic ↑), 129.64 (=CH ↑), 129.15 (aromatic ↑), 128.82 (C-5 ↑), 126.79 (aromatic ↑), 126.70 (C-6a ↓), 124.60 (C-4a ↓), 123.31 (C-6 ↑), 121.85 (aromatic ↓), 121.05 (aromatic ↓), 120.62 (C-8 ↑), 75.72 (C-3 ↓), 58.74 (CH2 ↓), 40.10 (C-4 ↑), 14.27 (CH3 ↑); MS m/z (%): 608 (M++4, 3.88), 606 (M++2, 7.88), 604 (M+, 3.98), 451 (13.30), 449 (11.72), 293 (5), 249 (6), 157 (20) 116 (21), 76 (74), 50 (100); Anal. Calcd for C29H22Br2N2O3: C, 57.45; H, 3.66; N, 4.82. Found: C, 57.01; H, 3.44; N, 4.63%.

(E) Ethyl 2-amino-4-(4-chlorophenyl)-9-(4-fluorostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6e)

Colorless needles from benzene/ethanol; yield 80%; m.p. 192–193°C; IR (KBr) υ (cm−1): 3410, 3295 (NH2), 3050, 2979, 2900 (CH stretching), 1677 (CO); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.32–7.30 (m, 12H, aromatic), 7.30 (bs, 2H, NH2, canceled by D2O), 7.98 (d, J = 16.5 Hz, 1H, =CH), 7.46 (d, J = 16.5 Hz, 1H, =CH), 5.11 (s, 1H, H-4), 4.01 (q, J = 7.0 Hz, 2H, CH2), 1.10 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ (ppm): 168.15 (CO), 161.35 (C-2), 160.84 (C-9), 155.31 (C-10b), 137.48 (C-10a), 136.45 (C-7), 133.49 (=CH), 129.28 (=CH), 128.12 (C-5), 126.63 (C-6a), 124.63 (C-4a), 123.30 (C-6), 120.48 (C-8), 75.80 (C-3), 58.73 (CH2), 40.11 (C-4), 14.26 (CH3), 163.31, 146.74, 142.78, 130.64, 129.23, 126.67, 115.97 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.45 (C-7 ↑), 133.49 (=CH ↑), 129.28 (=CH ↑), 129.23 (aromatic ↑), 128.12 (C-5 ↑), 126.67 (aromatic ↑), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑), 58.73 (CH2 ↓), 40.11 (C-4 ↑), 14.26 (CH3 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.45 (C-7 ↑), 133.49 (=CH ↑), 129.28 (=CH ↑), 129.23 (aromatic ↑), 128.12 (C-5 ↑), 126.67 (aromatic ↑), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑), 40.11 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.45 (C-7 ↑), 133.49 (=CH ↑), 129.28 (=CH ↑), 129.23 (aromatic ↑), 128.12 (C-5 ↑), 126.67 (aromatic ↑), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑) 58.73 (CH2 ↑), 40.11 (C-4 ↑), 14.26 (CH3 ↑). 13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 168.15 (CO ↓), 163.31 (aromatic ↓), 161.35 (C-2 ↓), 160.84 (C-9 ↓), 155.31 (C-10b ↓), 146.74 (aromatic ↓), 142.78 (aromatic ↓), 137.48 (C-10a ↓), 136.45 (C-7 ↑), 133.49 (=CH ↑), 130.64 (aromatic ↓), 129.28 (=CH ↑), 129.23 (aromatic ↑), 128.12 (C-5 ↑), 126.67 (aromatic ↑), 126.63 (C-6a ↓), 124.63 (C-4a ↓), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑), 75.80 (C-3 ↓), 58.73 (CH2 ↓), 40.11 (C-4 ↑), 14.26 (CH3 ↑); MS m/z (%): 502 (M++2, 3.15), 500 (M+, 10.12). 388 (100), 315 (13), 219 (8), 176 (37), 113 (36), 75 (29); Anal. Calcd for C29H22ClFN2O3: C, 69.53; H, 4.43; N, 5.59. Found: C, 69.63; H, 4.56; N, 5.67%.

(E) Ethyl 2-amino-4-(4-bromophenyl)-9-(4-fluorostyryl)-4H-pyrano[3,2-h]quinoline-3-carboxylate (6f)

Pale yellow needles from benzene/ethanol; yield 79%; m.p. 190–191°C; IR (KBr) υ (cm−1): 3408, 3292 (NH2), 3040, 2977, 2900 (CH stretching), 1675 (CO); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.32–7.23 (m, 12H, aromatic), 7.86 (bs, 2H, NH2, canceled by D2O), 7.98 (d, J = 16.5 Hz, 1H, =CH), 7.32 (d, J = 16.5 Hz, 1H, =CH), 5.09 (s, 1H, H-4), 4.02 (q, J = 7.0 Hz, 2H, CH2), 1.11 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ (ppm): 168.14 (CO), 161.35 (C-2), 160.84 (C-9), 155.31 (C-10b), 137.48 (C-10a), 136.44 (C-7), 133.49 (=CH), 129.63 (=CH), 128.24 (C-5), 126.64 (C-6a), 124.56 (C-4a), 123.30 (C-6), 120.48 (C-8), 75.74 (C-3), 58.74 (CH2), 40.12 (C-4), 14.26 (CH3), 163.31, 147.16, 142.78, 131.13, 129.28, 126.67, 119.12, 115.97 (aromatic); 13C NMR–DEPT spectrum at 135° CH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ 136.44 (C-7 ↑), 133.49 (=CH ↑), 131.13 (aromatic ↑), 129.63 (=CH ↑), 129.28 (aromatic ↑), 128.24 (C-5 ↑), 126.67 (aromatic ↑), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑), 58.74 (CH2 ↓), 40.12 (C-4 ↑), 14.26 (CH3 ↑). In the DEPT spectrum at 90° only CH signals are positive (up) and showed δ 136.44 (C-7 ↑), 133.49 (=CH ↑), 131.13 (aromatic ↑), 129.63 (=CH ↑), 129.28 (aromatic ↑), 128.24 (C-5 ↑), 126.67 (aromatic ↑), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑), 40.12 (C-4 ↑). In the DEPT spectrum at 45° (CH, CH2, and CH3 positive) revealed signals at δ 136.44 (C-7 ↑), 133.49 (=CH ↑), 131.13 (aromatic ↑), 129.63 (=CH ↑), 129.28 (aromatic ↑), 128.24 (C-5 ↑), 126.67 (aromatic ↑), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 115.97 (aromatic ↑), 58.74 (CH2 ↑), 40.12 (C-4 ↑), 14.26 (CH3 ↑). 13C NMR–APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ 168.14 (CO ↓), 161.35 (C-2 ↓), 160.84 (C-9 ↓), 155.31 (C-10b ↓), 137.48 (C-10a ↓), 136.44 (C-7 ↑), 133.49 (=CH ↑), 129.63 (=CH ↑), 128.24 (C-5 ↑), 126.64 (C-6a ↓), 124.56 (C-4a ↓), 123.30 (C-6 ↑), 120.48 (C-8 ↑), 75.74 (C-3 ↓), 58.74 (CH2 ↓), 40.12 (C-4 ↑), 14.26 (CH3 ↑), 163.31 (aromatic ↓), 147.16 (aromatic ↓), 142.78 (aromatic ↓), 131.13 (aromatic ↑), 129.28 (aromatic ↑), 126.67 (aromatic ↑), 119.12 (aromatic ↓), 115.97 (aromatic ↑); MS m/z (%): 546 (M++2, 10.00), 544 (M+, 10.78), 518 (2.52), 516 (3.45), 474 (20.86), 472 (21.51), 435 (100), 361 (6), 266 (2), 221 (3), 176 (24), 114 (6), 75 (6); Anal. Calcd for C29H22BrFN2O3: C, 63.86; H, 4.07; N, 5.14. Found: C, 61.95; H, 3.95; N, 4.74%.

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 cell 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 cytotoxicity activity was studied against three cell lines: breast adenocarcinoma (MCF-7), lung carcinoma (HCT) and hepatocellular carcinoma (HepG-2) using the colorimetric MTT assay as described by Mossman (1983). The cells were seeded in in 96-well microtitre plate 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 microtitre 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, the 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 cell cytotoxic effect of each tested compound was calculated.