Introduction

The chemistry of quinoline derivatives has attracted many researchers due to their applications as biological activities and pharmacological agents. Quinoline moiety is present in many classes of biologically active compounds (El-Agrody et al. 2013; Thomas et al. 2010; Chang et al. 2010; Larghi et al. 2009; Liu et al. 2009; Ramesh et al. 2009; Righi et al. 2008; Ganesh et al. 2008; Musiol et al. 2007). Many compounds that synthesized from 8-hydroxyquinoline and its derivatives have been also shown to have diverse therapeutic activities such as antiprotozoic drugs as well as antineoplastics (Jampilek et al. 2009) and antiproliferative (Musiol et al. 2008; Badawey and Kappe 1997) activities. In addition styrylquinoline derivatives have been explored as perspective HIV integrase inhibitors (Mrozek-Wilczkiewicz et al. 2010; Leonard and Roy 2008; Zouhiri et al. 2005) and biologically active compounds (Chang et al. 2010; Larghi et al. 2009, Ma et al. 2004; Jiang et al. 1990). They also exhibit antimalarial (Kaur et al. 2009), antitumor (Behforouz et al. 2007), antioxidant (Mallesha et al. 2013), antileishmanial (Rocha et al. 2005), and antiplatelet activities (Kuo et al. 2001). In addition several 4H-pyrano[3,2-h]quinoline derivatives has antitumor activity (El-Agrody et al. 2012; Al-Ghamdi et al. 2012) against different human tumor cell lines. Furthermore, they function as pharmacologically active synthetic compounds (Watson et al. 2001) such as DNA binding capabilities (Atwell et al. 1989) and DNA-intercalating carrier (Chen et al. 2000). In view of the above-mentioned findings, I report herein the synthesis of a series of halogenated 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives and their evaluation as antitumor, hoping to add some synergistic biological significance to the target molecules. The structural–activity relationship (SAR) of 4-, 6-, and 9-positions for the prepared compounds and lipophilicity was also discussed.

Results and discussion

Chemistry

Interaction of 8-hydroxyquinoline (1) with α-cyanomono, di or trisubstituted cinnamonitriles (2a–j) in ethanolic piperidine under reflux for 1 h afforded the corresponding 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (3a–j) as depicted in Scheme 1. The 4-position of compounds 3a–j is a chiral center and all the reaction progress was observed by TLC.

Scheme 1
scheme 1

Synthesis of 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (3a–j)

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The assignment of structure 3 was established on the basis of spectral data. The infrared (IR) spectra of 3 showed the appearance of a NH2 stretch at υ 3478–3407, 3371–3322, 3210–3188 cm−1 and a CN stretch at υ 2198–2186 cm−1. The 1H and 13C NMR spectra of 3 revealed the presence of 4H signals at δ 5.56–4.89 (s, 1H, H-4) and 40.80–38.12 ppm (C-4), respectively. The 13C NMR-attached proton test (APT) spectra of compound 3 provided additional evidence in support of the proposed structure. In addition, the mass spectra of compound 3 gave also additional evidences for the proposed structures.

In a similar manner, reaction of 5-chloro-8-hydroxyquinoline (4) with α-cyanomono, di or trisubstituted cinnamonitriles 2a–j in ethanolic piperidine under reflux for 1 h gave 2-amino-4-aryl-6-chloro-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (5a–j) as shown in Scheme 2. The 4-position of compounds 5a–j is a chiral center and all the reaction progress was observed by TLC.

Scheme 2
scheme 2

Synthesis of 2-amino-4-aryl-6-chloro-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (5a–j)

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The assignment of structure 5 was established on the basis of spectral data. The IR spectra of 5 showed the appearance of a NH2 stretch at υ 3479–3432, 3349–3321, 3205–3182 cm−1 and a CN stretch at υ 2201–2192 cm−1. The 1H and 13C NMR spectra of 5 revealed the presence of 4H signals at δ 6.03–5.00 (s, 1H, H-4) and 40.50–36.78 ppm (C-4), respectively. The 13C NMR-APT spectra of compound 5 provided additional evidence in support of the proposed structure. In addition, the mass spectra of compound 5 gave also additional evidences for the proposed structures.

Similarly, reaction of 2-methyl-8-hydroxyquinoline (6) with α-cyanomono, di- or trisubstituted cinnamonitriles 2a–j in ethanolic piperidine under reflux for 1 h gave 2-amino-4-aryl-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (7a–j) as shown in Scheme 3. The 4-position of compounds 7a–j is chiral center and all the reaction progress was observed by TLC.

Scheme 3
scheme 3

Synthesis of 2-amino-4-aryl-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (7a–j)

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The assignment of structure 7 was established on the basis of spectral data. The IR spectra of 7 showed the appearance of a NH2 stretch at υ 3471–3431, 3366–3323, 3205–3182 cm−1 and a CN stretch at υ 2195–2188 cm−1. The 1H and 13C NMR spectra of 7 revealed the presence of 4H signals at δ 6.01–4.97 (s, 1H, H-4) and 40.54–36.68 ppm (C-4), respectively. The 13C NMR-APT spectra of compound 7 provided additional evidence in support of the proposed structure. In addition, the mass spectra of compound 7 gave also additional evidences for the proposed structures.

Antitumor activity

All the synthesized compounds 3a–j, 5a–j, and 7a–j were screened for their in vitro antitumor activity against four human cancer cell lines: breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116), hepatocellular carcinoma (HepG-2), and lung carcinoma (A549) at various concentrations ranging from 0 to 50 μg/ml and the cell viability was measured by the microculture tetrazolium (MTT) assay as described in the literature (Rahman et al. 2001). In vitro cytotoxic evaluation using cell viability assay was performed at the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt using Vinblastine and Colchicine as reference drugs. The inhibitory concentration (IC50, in µg/ml) of target compounds 3a–j, 5a–j, and 7a–j against the four human cancer cell lines MCF-7, HCT-116, HepG-2, and A549 were given in Table 1.

Table 1 SAR of the 4-aryl group and the IC (IC50, in µg/ml) of target compounds against the four human cancer cell lines in comparison with Vinblastine and Colchicine as measured with the MTT method
Full size table

SAR studies

The partition coefficient (Log P), which is well known as an index of lipophilicity, is an important physicochemical parameter was measured by ACD/Labs Log P calculated, ver.14.02 and was cited in Table 1. The SAR studies at the 4-, 6-, and 9-positions and the relationship between lipophilicity and antitumor activity were explored. The SAR studies of compounds 3a–j revealed that compounds 3d, b, h, a has the highest potent antitumor activity (IC50 = 1.17–5.33 μg/ml) against breast adenocarcinoma (MCF-7) as compared to the other compounds 3j, f, e, c, i, g (IC50 = 10.8–38.2 μg/ml) and the reference drug Vinblastine (IC50 = 6.1 μg/ml), while compounds 3d, b, h, a, j, f has the higher potent antitumor activity (IC50 = 1.17–11 μg/ml) against MCF-7 as compared to the compounds 3e, c, i, g (IC50 = 23.8–38.2 μg/ml), and the reference drug Colchicine (IC50 = 17.7 μg/ml). The comparison of IC50 values of the halosubstituted compounds 3a–j at the phenyl ring at 4-position demonstrated that the dichloro atoms at 2,3-position or 3,4-position and the monochloro atom at 3-position or 2-position increased the antitumor activity profile against MCF-7 as compared to Vinblastine and Colchicine, suggesting that the dichloro atoms at 2,3-position or 3,4-position and the monochloro atom at 3-position or 2-position at the phenyl ring (hydrophobic group) at 4-position of the 4H-pyrano[3,2-h]quinoline moiety is preferred over the other substituted groups with decreasing of partition coefficient as shown in Table 1. Replacement of the 6-H for compound 3 with 6-Cl improved of the antitumor activities of compound 5. Compounds 5b, i, e, d (IC50 = 0.9–2.47 µg/ml) have the higher potent antitumor activities against the MCF-7 as compared to Vinblastine (IC50 = 6.1 μg/ml) and the other compounds 5f, h, j, a, c, g (IC50 = 7.76–10.2 and >50 μg/ml), while compounds 5b, i, e, d, f, h, j, a, c displayed good activity (IC50 = 0.9–10.2 µg/ml) against the MCF-7 as compared to Colchicine (IC50 = 17.7 μg/ml) and compound 5g inactive (IC50 = >50 μg/ml). This potency could be attributed to the presence of the monochloro/bromo atom at 3-position or 4-position, the dichloro atoms at 2,4-position or 2,3-position of the phenyl ring (hydrophobic group) at 4-position of the 4H-pyrano[3,2-h]quinoline moiety with chloro atom at 6-position, suggesting that this positions at the phenyl ring at 4-position was superior in inhibiting the growth of MCF-7 than the other positions with increasing of partition coefficient as shown in Table 1. In addition, replacement of the 9-H for compound 3 with 9-Me improved of the antitumor activities of compound 7. Compounds 7h, d, a, j, b, f (IC50 = 1.24–5.92 µg/ml) exhibited very good activities against the MCF-7 as compared to Vinblastine (IC50 = 6.1 μg/ml) and Colchicine (IC50 = 17.7 μg/ml) and the other compounds 7c, e, i, g (IC50 = 27.9–38.9 and >50 μg/ml). These data indicate that the activity of compounds 7h, d, a, j, b, f was considerably attributed to the presence of the dichloro atoms at 3,4-position, 2,3-position, 2,5-position, monochloroatom at 2-position, 3-position and trisubstituted of the phenyl ring (hydrophobic group) at 4-position of the 4H-pyrano[3,2-h]quinoline moiety, suggesting that these positions were superior in inhibiting the growth of MCF-7 with 9-Me and with decreasing of partition coefficient as shown in Table 1.

In the case of human colon carcinoma (HCT-116), investigation of SAR revealed that the 2,3-dichlorosubstituted and the 3-chlorosubstituted phenyl 3d, b exhibited good activity (IC50 = 1.3 and 1.35 μg/ml) as compared to Vinblastine (IC50 = 2.6 μg/ml) and the other compounds 3h, a, j, f, c, e, g, i (IC50 = 3.9–43.7 μg/ml), while compounds 3d, b, h, a, j, f, c, e, g has the higher potent antitumor activity (IC50 = 1.3–31.2 μg/ml) against HCT-116 as compared to Colchicine (IC50 = 42.8 μg/ml) and the compound 3i (IC50 = 43.7 μg/ml). This indicated that the dichloro atoms at 2,3-position and the monochloro atom at 3-position have a variable influence on the cytotoxic activity against HCT-116 with decreasing of partition coefficient as shown in Table 1. Replacement of the 6-H for compound 3 with 6-Cl resulted in reduction of potency for the compound 5. Compounds 5e, b, j exhibited near activities (IC50 = 3.01–3.72 μg/ml) and the other compounds 5i, a, d, c, f, h, g exhibited moderate to lower activities (IC50 = 5.81–34.3 μg/ml) against HCT-116 as compared to Vinblastine (IC50 = 2.6 μg/ml), while compounds 5e, b, j, i, a, d, c, f, h, g exhibited good activity (IC50 = 3.01–34.3 μg/ml) against HCT-116 as compared to Colchicine (IC50 = 42.8 μg/ml) with decreasing of partition coefficient as shown in Table 1, suggesting that the dichloro atoms at 2,4-position and the monochloro atom at 3-position have the more variable influence on the cytotoxic activity against HCT-116 than the another groups. In addition, replacement of the 9-H for compound 3 with 9-Me resulted in more reduction of antitumor activities of compound 7. Compounds 7c, b, i, h, j, a, f, d, e, g exhibited near or moderate to lower activities (IC50 = 15.8–45.5 and >50 μg/ml) against HCT-116 as compared to Vinblastine (IC50 = 2.6 μg/ml), while compounds 7c, b, i, h, j, a, f, d exhibited good activity (IC50 = 15.8–39.5 μg/ml) against HCT-116 as compared to Colchicine (IC50 = 42.8 μg/ml) with increasing of partition coefficient as shown in Table 1, suggesting that monochloro atom at 4-position or 3-position and the monobromo atom at 4-position is preferred over another groups.

Furthermore, compounds 3d, b, h (IC50 = 1.41–2.44 μmol/l) showed good antitumor activities against hepatocellular carcinoma (HepG-2) as compared to Vinblastine (IC50 = 4.6 μg/ml) and the other compounds 3a, j, f, e, g, c (IC50 = 5.57–36.3 and >150 μg/ml) and compound 3i was inactive, while compounds 3d, b, h, a, j (IC50 = 1.41–5.57 μg/ml) exhibited good antitumor activities against HepG-2 as compared to Colchicine (IC50 = 10.6 μg/ml) and the other compounds 3f, e, g, c (IC50 = 11.5–36.3 and >150 μg/ml) and compound 3i was inactive with decreasing of partition coefficient as shown in Table 1, suggesting that the dichloro atoms at 2,3-position, 3,4-position and the monochloro atom at 3-position have the more variable influence on the cytotoxic activity against HepG-2 than the another groups. Replacement of the 6-H for compound 3 with 6-Cl resulted in improvement of potency for the compound 5. Compounds 5b, e, i, d, h, a exhibited good activities (IC50 = 0.7–4.62 μg/ml) and the other compounds 5j, f, c, g exhibited moderate to lower activities (IC50 = 8.97–44.5 μg/ml) against HepG-2 as compared to Vinblastine (IC50 = 4.6 μg/ml), while compounds 5b, e, i, d, h, a, j exhibited good activity (IC50 = 0.7–8.97 μg/ml) against HepG-2 as compared to Colchicine (IC50 = 10.6 μg/ml) with increasing of partition coefficient as shown in Table 1, suggesting that the monochloro/bromo atom at 3-position or 4-position, the dichloro atoms at 2,4-position, 2,3-position have the more variable influence on the cytotoxic activity against HepG-2 than the another groups. In addition, replacement of the 9-H for compound 3 with 9-Me resulted in improvement of antitumor activities of compound 7. Compounds 7c, h, b exhibited good activities (IC50 = 1.5–3.01 μg/ml) against HepG-2 as compared to Vinblastine (IC50 = 4.6 μg/ml), while compounds 7d, i, j, f, a, e exhibited near or moderate to lower activities (IC50 = 5.39–23.9 and >50 μg/ml) and compounds 7c, h, b, d, i, j, f (IC50 = 1.5-10.5 μg/ml) showed good activities against HepG-2 as compared to Colchicine (IC50 = 10.6 μg/ml) and the other compounds 7a, e, g with increasing of partition coefficient as shown in Table 1, suggesting that the monochloro atom at 4-position or 3-position and the dichloro atoms at 3,4-position have the more variable influence on the cytotoxic activity against HepG-2 than the another groups.

Finally, compounds 3b, h (IC50 = 1.23 and 2.05 μg/ml) showed a significant antitumor activities against lung carcinoma (A549) as compared to Vinblastine (IC50 = 3.79 μg/ml) and the other compounds 3a, j, f, e, c, g, d, i (IC50 = 4.07–38 and >50 μg/ml), while compounds 3b, h, a, j, f, e, c (IC50 = 1.23–21.1 μg/ml) exhibited good activities as compared to Colchicine (IC50 = 21.3 μg/ml) and the other compounds 3g, d, i (IC50 = 33.6, 38 and >50 μg/ml) with increasing of partition coefficient as shown in Table 1. This is due to the presence of the monochloro atom at 3-position and the dichloro atoms at 3,4-position which have the more variable influence on the cytotoxic activity against A549 than the another groups. Replacement of the 6-H for compound 3 with 6-Cl resulted in improvement of potency for the compound 5. Compounds 5e, j, b, i exhibited good activities (IC50 = 1.79–3.1 μg/ml) and the other compounds 5a, f, h, d, c, g exhibited moderate to lower activities (IC50 = 5.66–24.3 μg/ml) against A549 as compared to Vinblastine (IC50 = 3.79 μg/ml), while compounds 5e, j, b, i, a, f, h, d (IC50 = 1.79–15.6 μg/ml) showed good activities against A549 as compared to Colchicine (IC50 = 21.3 μg/ml) and the other compounds 5c, g (IC50 = 22.1 and 24.3 μg/ml) with increasing of partition coefficient as shown in Table 1, suggesting that the dichloro atoms at 2,4-position and the trisubstituted have the higher influence on the cytotoxic activity against A549 than the another groups. Furthermore, replacement of the 9-H for compound 3 with 9-Me resulted reduction of antitumor activities of compound 7. All the compound 7 exhibited lower activities (IC50 = 13.7–36.7 and >50 μg/ml) against A549 as compared to Vinblastine (IC50 = 3.79 μg/ml), while compound 7h showed good activity against A549 as compared to Colchicine (IC50 = 21.3 μg/ml) and the other compounds 7j, f, b, a, d, e, i, c, g (IC50 = 22.2–36.7 and >50 μg/ml) with increasing of partition coefficient as shown in Table 1, suggesting that the dichloro atoms at 3,4-position is preferred over than the another groups.

Conclusions

In conclusion, halogenated 4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives 3a–j, 5a–j, and 7a–j was synthesized. Structures of the synthesized compounds were elucidated on the basis of IR, 1H NMR, 13C NMR, and MS data. Among the newly synthesized compounds 3d, b, h, a, 5b, i, e, d, 7h, d, a, j, b, f and 3d, b, h, a, j, f, 5b, i, e, d, f, h, j, a, c, 7h, d, a, j, b, f showed highest inhibition against MCF-7 as compared to Vinblastine and Colchicine respectively, while compounds 3d, b, 5e, b, j and 3d, b, h, a, j, f, c, e, g, 5e, b, j, i, a, d, c, f, h, g, 7c, b, i, h, j, a, f, d exhibited the best growth inhibitory activity against HCT-116 as compared to the standard drugs Vinblastine and Colchicine respectively, and compounds 3d, b, h, 5b, e, i, d, h, a, 7c, h, b and 3d, b, h, a, j, 5b, e, i, d, h, a, j, 7c, h, b, d, i, j, f exhibited the best growth inhibitory activity against HepG-2 as compared to Vinblastine and Colchicine, respectively, while compounds 3b, h, 5e, j, b, i and 3b, h, a, j, f, e, c, 5e, j, b, i, a, f, h, d, 7h showed highest inhibition against A549 as compared to Vinblastine and Colchicine, respectively. On the basis of SAR, lipophilicity and the partition coefficient (Log P) are beneficial for antitumor activity. The highest inhibition activity of the halogenated 4H-pyrano[3,2-h]quinoline derivatives 3a–j, 5a–j and 7a–j is in the following order:

6-Cl analogs 5a–j > unsubstituted analogs 3a–j >9-Me analogs 7a–j

Further investigations are essential to gain deeper insight into structure–activity aspects and to predict the optimal structural parameters, which could be beneficial in development of antitumor therapeutics.

Experimental

All chemicals were purchased from Sigma-Aldrich Chemical Co. Melting points were determined with a Stuart Scientific Co. Ltd apparatus and are uncorrected. 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 500/600 MHz spectrometer. 13C NMR spectra were obtained using 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-QP5 spectrometer. Elemental analyses were performed on a Perkin-Elmer 240 microanalyser. Analytical thin layer chromatography (TLC) on silica gel-precoated F254 Merck plates to check the purity of the compounds.

General procedure for the synthesis of 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3a–j), 2-amino-4-aryl-6-chloro-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5a–j) and 2-amino-4-aryl-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives (7a–j).

A solution of 8-hydroxyquinoline 1, 5-chloro-8-hydroxyquinoline 4, or 8-hydroxy-2-methyl-quinoline 6 (0.01 mol) in EtOH (30 mL) was treated with α-cyanomono, di or trisubstituted cinnamonitriles 2a–j (0.01 mol) and piperidine (0.5 mL). The reaction mixture was heated under reflux with stirring for 1 h. The solid product formed was collected by filtration and recrystallized from a suitable solvent to give 3a–j, 5a–j, and 7a–j. The physical and spectral data of compounds 3a–j, 5a–j, and 7a–j are as follows:

2-amino-4-(2-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3a)

Yellow crystals from ethanol; yield 87 %; m.p. 289–290 °C; IR (KBr, υ max cm−1): 3478, 3323, 3195 (NH2), 2196 (CN), 1658 (C=N) 1H NMR (500 MHz, DMSO-d6) δ: 8.96–7.06 (m, 9H, aromatic), 7.29 (bs, 2H, NH2), 5.44 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.50 (C-2), 150.27 (C-9), 143.27 (C-10b), 137.35 (C-10a), 136.24 (C-7), 128.92 (C-5), 127.93 (C-6a), 124.34 (C-4a), 123.88 (C-8), 122.88 (C-6), 120.51 (CN), 54.63 (C-3), 38.48 (C-4), 142.05, 132.10, 129.83, 128.09, 127.83, 126.09 (aromatic), MS m/z (%): 335 (M++2, 5.51), 333 (M+,17.23) with a base peak at 222 (100); anal. calcd. for C19H12ClN3O: C, 68.37; H, 3.62; N, 12.59. Found: C, 68.41; H, 3.64; N, 12.63 %.

2-amino-4-(3-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3b)

Yellow crystals from ethanol; yield 88 %; m.p. 240-241 °C; IR (KBr, υ max cm−1): 3407, 3322, 3205 (NH2), 2196 (CN), 1656 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.20–7.08 (m, 9H, aromatic), 7.22 (bs, 2H, NH2), 4.89 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.41 (C-2), 150.28 (C-9), 143.04 (C-10b), 136.44 (C-10a), 136.00 (C-7), 128.26 (C-5), 127.80 (C-6a), 124.28 (C-4a), 123.75 (C-8), 122.23 (C-6), 120.25 (CN), 55.41 (C-3), 40.80 (C-4), 148.02, 134.82, 130.84, 130.17, 127.06, 126.71 (aromatic), MS m/z (%): 335 (M++2, 2.95), 333 (M+,8.32) with a base peak at 222 (100); anal. calcd. for C19H12ClN3O: C, 68.37; H, 3.62; N, 12.59. Found: C, 68.40; H, 3.64; N, 12.61 %.

2-amino-4-(4-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3c)

Prepared as previously described (El-Agrody and Al-Ghamdi 2011).

2-amino-4-(2,3-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3d)

Yellow crystals from benzene; yield 87 %; m.p. 281–283 °C; IR (KBr, υ max cm−1): 3466, 3349, 3188 (NH2), 2186 (CN), 1646 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.97–7.08 (m, 8H, aromatic), 7.32 (bs, 2H, NH2), 5.56 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.54 (C-2), 150.35 (C-9), 143.36 (C-10b), 137.33 (C-10a), 136.29 (C-7), 129.17 (C-5), 128.75 (C-6a), 124.42 (C-4a), 123.87 (C-8), 122.90 (C-6), 119.99 (CN), 54.27 (C-3), 39.99 (C-4), 150.09, 133.95, 130.20, 129.51, 127.96, 125.99 (aromatic), MS m/z (%): 371 (M++4, 2.15), 369 (M++2, 12.05), 366 (M+,18.29) with a base peak at 222 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 61.94; H, 2.98; N, 11.38 %.

2-amino-4-(2,4-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3e)

Yellow crystals from ethanol; yield 86 %; m.p. 238–240 °C; IR (KBr, υ max cm−1): 3472, 3322, 3210 (NH2), 2192 (CN), 1657 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.94–7.02 (m, 8H, aromatic), 7.32 (bs, 2H, NH2), 5.42 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.50 (C-2), 150.13 (C-9), 143.32 (C-10b), 137.32 (C-10a), 136.05 (C-7), 128.52 (C-5), 128.11 (C-6a), 125.93 (C-4a), 123.79 (C-8), 122.83 (C-6), 119.93 (CN), 54.23 (C-3), 38.12 (C-4), 149.99, 135.95, 132.97, 131.82, 130.65, 127.75 (aromatic). In 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.50 (C-2 ↓), 150.13 (C-9 ↑), 149.99 (aromatic ↓), 143.32 (C-10b ↓), 137.32 (C-10a ↓), 136.05 (C-7 ↑), 135.95 (aromatic ↓), 132.97 (aromatic ↑), 131.82 (aromatic ↑), 130.65 (aromatic ↑), 128.52 (C-5 ↑), 128.11 (C-6a ↓), 127.75 (aromatic ↓), 125.93 (C-4a ↓), 123.79 (C-8 ↑), 122.83 (C-6 ↑), 119.93 (CN ↓), 54.23 (C-3 ↓), 38.12 (C-4 ↑); MS m/z (%): 371 (M++4, 1.28), 369 (M++2, 7.59), 367 (M+,11.82) with a base peak at 222 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 62.00; H, 3.05; N, 11.45 %.

2-amino-4-(2,5-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3f)

Yellow crystals from benzene/ethanol; yield 84 %; m.p. 293–294 °C; IR (KBr, υ max cm−1): 3475, 3330, 3188 (NH2), 2198 (CN), 1656 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.92–7.05 (m, 8H, aromatic), 7.13 (bs, 2H, NH2), 5.41 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.53 (C-2), 151.15 (C-9), 143.38 (C-10b), 137.39 (C-10a), 136.15 (C-7), 128.72 (C-5), 128.18 (C-6a), 126.03 (C-4a), 123.99 (C-8), 122.91 (C-6), 119.99 (CN), 54.65 (C-3), 38.45 (C-4), 149.78, 131.55, 130.67, 130.12, 129.65, 127.87 (aromatic); MS m/z (%): 371 (M++4, 2.76), 369 (M++2, 14.64), 367 (M+,23.66) with a base peak at 222 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 61.95; H, 3.06; N, 11.46 %.

2-amino-4-(2,6-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3g)

Yellow crystals from benzene; yield 84 %; m.p. 295–296 °C; IR (KBr, υ max cm−1): 3471, 3361, 3188 (NH2), 2196 (CN), 1660 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.96–6.96 (m, 8H, aromatic), 7.26 (bs, 2H, NH2), 6.03 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.88 (C-2), 150.34 (C-9), 143.95 (C-10b), 137.03 (C-10a), 136.04 (C-7), 128.69 (C-5), 128.00 (C-6a), 123.68 (C-4a), 122.26 (C-8), 119.75 (C-6), 118.21 (CN), 51.87 (C-3), 37.20 (C-4), 137.14, 135.63, 134.87, 130.83, 130.65, 130.08, 125.24 (aromatic); MS m/z (%): 371 (M++4, 4.32), 369 (M++2, 23.64), 367 (M+, 34.64) with a base peak at 222 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 61.92; H, 3.10; N, 11.50 %.

2-amino-4-(3,4-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3h)

Yellow crystals from benzene/ethanol; yield 84 %; m.p. 289–290 °C; IR (KBr, υ max cm−1): 3430, 3371, 3196 (NH2), 2187 (CN), 1666 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.97–7.09 (m, 8H, aromatic), 7.32 (bs, 2H, NH2), 5.46 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.57 (C-2), 150.36 (C-9), 143.76 (C-10b), 137.29 (C-10a), 136.03 (C-7), 129.02 (C-5), 127.99 (C-6a), 125.98 (C-4a), 123.85 (C-8), 122.35 (C-6), 119.91 (CN), 53.83 (C-3), 38.88 (C-4), 143.40, 132.29, 131.81, 131.09, 130.80, 130.08 (aromatic); MS m/z (%): 371 (M++4, 2.18), 369 (M++2, 11.75), 367 (M+, 18.14) with a base peak at 255 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 62.07; H, 3.09; N, 11.48 %.

2-amino-4-(4-bromophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3i)

Prepared as previously described (El-Agrody and Al-Ghamdi 2011).

2-amino-4-(3,5-dibromo-2-methoxyphenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (3j)

Pale yellow crystals from ethanol; yield 81 %; m.p. 224–226 °C; IR (KBr, υ max cm−1): 3435, 3337, 3200 (NH2), 2190 (CN), 1657 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.97–7.27 (m, 7H, aromatic), 7.35 (bs, 2H, NH2), 5.13 (s, 1H, H-4), 3.59 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.23 (C-2), 151.01 (C-9), 142.21 (C-10b), 137.99 (C-10a), 133.91 (C-7), 132.06 (C-5), 127.93 (C-6), 125.13 (C-6a), 124.70 (C-4a), 123.34 (C-8), 117.28 (CN), 61.35 (CH3), 54.35 (C-3), 36.88 (C-4), 153.32, 140.99, 132.45, 125.35, 120.11,118.56 (aromatic); MS m/z (%): 489 (M++4, 2.33), 487 (M++2, 4.63), 485 (M+, 2.37) with a base peak at 222 (100); anal. calcd. for C20H13Br2N3O2: C, 49.31; H, 2.69; N, 8.63. Found: C, 49.40; H, 2.71; N, 8.70 %.

2-amino-6-chloro-4-(2-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5a)

Yellow needles from benzene; yield 84 %; m.p. 298–300 °C; IR (KBr, υ max cm−1): 3479, 3326, 3205 (NH2), 2198 (CN), 1659 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.06–7.19 (m, 9H, aromatic), 7.30 (bs, 2H, NH2), 5.44 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.25 (C-2), 151.17 (C-9), 142.88 (C-10b), 138.01 (C-10a), 132.45 (C-7), 130.08 (C-5), 125.22 (C-6), 124.85 (C-6a), 123.41 (C-8), 120.90 (C-4a), 119.75 (CN), 54.45 (C-3), 38.53 (C-4), 141.30, 132.12, 131.40, 129.25, 128.03, 125.66 (aromatic), MS m/z (%): 369 (M++2, 5.1), 367 (M+,15.01) with a base peak at 256 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 61.92; H, 3.06; N, 11.49 %.

2-amino-6-chloro-4-(3-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5b)

Yellow crystals from benzene; yield 85 %; m.p. 273–275 °C; IR (KBr, υ max cm−1): 3441, 3321, 3205 (NH2), 2201 (CN), 1659 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.06-7.12 (m, 9H, aromatic), 7.34 (bs, 2H, NH2), 5.03 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.20 (C-2), 151.18 (C-9), 142.52 (C-10b), 138.14 (C-10a), 132.54 (C-7), 127.33 (C-5), 125.22 (C-6), 124.97 (C-6a), 123.44 (C-8), 121.74 (C-4a), 119.97 (CN), 55.23 (C-3), 40.32 (C-4), 147.47, 133.39, 130.90, 127.44, 126.53, 126.34 (aromatic), MS m/z (%): 369 (M++2, 15.3), 367 (M+, 45.03) with a base peak at 256 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 62.07; H, 3.23; N, 11.61 %.

2-amino-6-chloro-4-(4-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5c)

Yellow crystals from benzene; yield 81 %; m.p. 276–278 °C; IR (KBr, υ max cm−1): 3444, 3349, 3188 (NH2), 2199 (CN), 1657 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.01–7.02 (m, 9H, aromatic), 7.12 (bs, 2H, NH2), 5.01 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.26 (C-2), 151.23 (C-9), 142.55 (C-10b), 138.66 (C-10a), 132.23(C-7), 127.33 (C-5), 125.89 (C-6), 125.05 (C-6a), 123.49 (C-8), 121.84 (C-4a), 120.07 (CN), 55.28 (C-3), 40.11 (C-4), 140.47, 132.32, 130.50, 129.64, 127.93, 127.31 (aromatic), MS m/z (%): 369 (M++2, 6.87), 367 (M+, 20.11) with a base peak at 256 (100); anal. calcd. for C19H11Cl2N3O: C, 61.98; H, 3.01; N, 11.41. Found: C, 61.87; H, 2.94; N, 11.25 %.

2-amino-6-chloro-4-(2,3-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5d)

Pale yellow crystals from benzene; yield 81 %; m.p. 298–300 °C; IR (KBr, υ max cm−1): 3479, 3324, 3182 (NH2), 2195 (CN), 1656 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.55–7.11 (m, 8H, aromatic), 7.38 (bs, 2H, NH2), 5.56 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.30 (C-2), 151.26 (C-9), 143.79 (C-10b), 137.99 (C-10a), 130.34 (C-7), 128.84 (C-5), 125.37 (C-6), 124.99 (C-6a), 123.54 (C-8), 120.39 (C-4a), 119.65 (CN), 54.08 (C-3), 39.98 (C-4), 143.02, 132.53, 130.20, 129.82, 125.64 (aromatic), MS m/z (%): 405 (M++4, 1.55), 403 (M++2, 9.12), 401 (M+, 14.02) with a base peak at 256 (100); anal. calcd. for C19H10Cl3N3O: C, 56.67; H, 2.50; N, 10.44. Found: C, 56.79; H, 2.70; N, 10.60 %.

2-amino-6-chloro-4-(2,4-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5e)

Pale yellow crystals from benzene; yield 80 %; m.p. 277–279 °C; IR (KBr, υ max cm−1): 3471, 3327, 3196 (NH2), 2193 (CN), 1660 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.06–7.20 (m, 8H, aromatic), 7.36 (bs, 2H, NH2), 5.44 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.23 (C-2), 151.21 (C-9), 142.95 (C-10b), 137.97 (C-10a), 132.82 (C-7), 128.21 (C-5), 125.62 (C-6), 124.96 (C-6a), 123.48 (C-8), 120.32 (C-4a), 119.64 (CN), 53.99 (C-3), 38.15 (C-4), 140.34, 133.14, 132.90, 132.47, 129.46, 125.31 (aromatic). In 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.23 (C-2 ↓), 151.21 (C-9 ↑), 142.95 (C-10b ↓), 140.34 (aromatic ↓), 137.97 (C-10a ↓), 133.14 (aromatic ↓), 132.90 (aromatic ↓), 132.82 (C-7 ↑), 132.47 (aromatic ↑), 129.46 (aromatic ↑), 128.21 (C-5 ↑), 125.62 (C-6 ↓), 125,31 (aromatic ↑), 124.96 (C-6a ↓), 123.48 (C-8 ↑), 120.32 (C-4a ↓), 119.64 (CN ↓), 53.99 (C-3 ↓), 38.15 (C-4 ↑). MS m/z (%): 405 (M++4, 3.61), 403 (M++2, 20.48), 401 (M+, 31.19) with a base peak at 256 (100); anal. calcd. for C19H10Cl3N3O: C, 56.67; H, 2.50; N, 10.44. Found: C, 56.47; H, 2.33; N, 10.31 %.

2-amino-6-chloro-4-(2,5-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5f)

Pale yellow crystals from benzene; yield 82 %; m.p. > 300 °C; IR (KBr, υ max cm−1): 3476, 3347, 3196 (NH2), 2196 (CN), 1650 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.58–7.26 (m, 8H, aromatic), 7.38 (bs, 2H, NH2), 5.46 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.32 (C-2), 151.22 (C-9), 143.02 (C-10b), 137.95 (C-10a), 132.48 (C-7), 129.31 (C-5), 125.37 (C-6), 124.95 (C-6a), 123.51 (C-8), 119.83 (C-4a), 119.65 (CN), 53.62 (C-3), 38.89 (C-4), 142.94, 132.34, 132.00, 131.19, 131.04, 125.57 (aromatic); MS m/z (%): 405 (M++4, 2.22), 403 (M++2, 12.26), 401 (M+, 18.11) with a base peak at 256 (100); anal. calcd. for C19H10Cl3N3O: C, 56.67; H, 2.50; N, 10.44. Found: C, 56.70; H, 2.63; N, 10.54 %.

2-amino-6-chloro-4-(2,6-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5g)

Colorless needles from benzene; yield 82 %; m.p. > 300 °C; IR (KBr, υ max cm−1): 3475, 3325, 3188 (NH2), 2199 (CN), 1661 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.07–7.05 (m, 8H, aromatic), 7.42 (bs, 2H, NH2), 6.03 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.59 (C-2), 151.30 (C-9), 143.55 (C-10b), 137.71 (C-10a), 132.53 (C-7), 130.37 (C-5), 125.37 (C-6), 124.73 (C-6a), 123.49 (C-8), 119.38 (C-4a), 118.91 (CN), 51.95 (C-3), 37.04 (C-4), 136.56, 135.54, 134.96, 130.90, 128.85, 124.86 (aromatic); MS m/z (%): 405 (M++4, 2.06), 403 (M++2, 11.77), 401 (M+, 18.14) with a base peak at 256 (100); anal. calcd. for C19H10Cl3N3O: C, 56.67; H, 2.50; N, 10.44. Found: C, 56.80; H, 2.63; N, 10.60 %.

2-amino-6-chloro-4-(3,4-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5h)

Pale yellow needles from benzene; yield 82 %; m.p. 257–259 °C; IR (KBr, υ max cm−1): 3470, 3321, 3199 (NH2), 2199 (CN), 1658 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.04–7.27 (m, 8H, aromatic), 7.39 (bs, 2H, NH2), 5.06 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.18 (C-2), 151.11 (C-9), 145.97 (C-10b), 138.13 (C-10a), 132.44 (C-7), 128.21 (C-5), 125.25 (C-6), 125.04 (C-6a), 123.40 (C-8), 121.22 (C-4a), 119.89 (CN), 54.94 (C-3), 38.97 (C-4), 142.56, 131.34, 131.18, 129.99, 129.65, 126.23 (aromatic); MS m/z (%): 405 (M++4, 1.52), 403 (M++2, 8.89), 401 (M+, 13.37) with a base peak at 256 (100); anal. calcd. for C19H10Cl3N3O: C, 56.67; H, 2.50; N, 10.44. Found: C, 56.57; H, 2.38; N, 10.27 %.

2-amino-6-chloro-4-(4-bromophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5i)

Yellow crystals from ethanol; yield 86 %; m.p. 228-230 °C; IR (KBr, υ max cm−1): 3445, 3330, 3182 (NH2), 2192 (CN), 1658 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.95–7.18 (m, 9H, aromatic), 7.25 (bs, 2H, NH2), 5.00 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.30 (C-2), 150.25 (C-9), 144.95 (C-10b), 137.47 (C-10a), 135.98 (C-7), 127.77 (C-5), 126.67 (C-6), 123.68 (C-6a), 122.20 (C-8), 121.30 (C-4a), 117.68 (CN), 55.59 (C-3), 40.50 (C-4), 143.03, 131.63, 129.94, 127.45, 121.78 (aromatic), MS m/z (%): 380 (M+–Cl+4, 2.08), 378 (M+–Cl+2, 5.02), 376 (M+–Cl, 3.37) with a base peak at 222 (100); anal. calcd. for C19H11BrClN3O: C, 55.30; H, 2.69; N, 10.18. Found: C, 55.37; H, 2.73; N, 10.23 %.

2-amino-6-chloro-4-(3,5-dibromo-2-methoxyphenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (5j)

Pale yellow Crystals from ethanol; yield 81 %; m.p. 223-225 °C; IR (KBr, υ max cm−1): 3432, 3337, 3182 (NH2), 2192 (CN), 1657 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.07–7.29 (m, 7H, aromatic), 7.39 (bs, 2H, NH2), 5.25 (s, 1H, H-4), 3.69 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.43 (C-2), 151.21 (C-9), 142.78 (C-10b), 138.06 (C-10a), 134.91 (C-7), 132.30 (C-5), 125.25 (C-6a), 124.90 (C-4a), 123.43 (C-8), 120.93 (C-6), 117.00 (CN), 61.32 (CH3), 54.19 (C-3), 36.78 (C-4), 154.34, 141.49, 132.50, 125.86, 120.00, 118.29 (aromatic); MS m/z (%): 490 (M+–Cl+6, 0.85), 488 (M+–Cl+4, 3.53), 486 (M+–Cl+2, 4.41), 484 (M+–Cl, 2.19) with a base peak at 222 (100); anal. calcd. for C20H12Br2 ClN3O2: C, 46.05; H, 2.32; 6.80; N, 8.06. Found: C, 46.16; H, 2.41; N, 8.13 %.

2-amino-4-(2-chlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7a)

Yellow crystals from ethanol; yield 88 %; m.p. 238–239 °C; IR (KBr, υ max cm−1): 3470, 3366, 3196 (NH2), 2195 (CN), 1659 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.46–6.82 (m, 8H, aromatic), 7.16 (bs, 2H, NH2), 5.43 (s, 1H, H-4), 2.72 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.54 (C-2), 159.11 (C-9), 142.86 (C-10b), 136.91 (C-10a), 136.30 (C-7), 128.90 (C-5), 125.21 (C-6a), 123.84 (C-8), 122.93 (C-4a), 120.52 (C-6), 120.07 (CN), 54.73 (C-3), 38.56 (C-4), 24.84 (CH3) 142.16, 136.08, 131.97, 130.42, 129.84, 126.20 (aromatic), MS m/z (%): 349 (M++2, 1.56), 347 (M+,4.63) with a base peak at 236 (100); anal. calcd. for C20H14ClN3O: C, 69.07; H, 4.06; N, 12.08. Found: C, 69.11; H, 4.14; N, 12.13 %.

2-amino-4-(3-chlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7b)

Yellow crystals from ethanol; yield 87 %; m.p. 236–238 °C; IR (KBr, υ max cm−1): 3464, 3346, 3200 (NH2), 2188 (CN), 1655 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.19–7.09 (m, 8H, aromatic), 7.17 (bs, 2H, NH2), 4.97 (s, 1H, H-4), 2.69 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.41 (C-2), 159.12 (C-9), 149.51 (C-10b), 137.09 (C-10a), 135.18 (C-7), 128.92 (C-5), 125.79 (C-6a), 122.50 (C-4a), 123.54 (C-8), 121.45 (C-6), 120.38 (CN), 55.84 (C-3), 40.54 (C-4), 144.76, 136.05, 129.58, 131.78, 128.60, 126.17 (aromatic), MS m/z (%): 349 (M++2, 4.35), 347 (M+, 13.27) with a base peak at 236 (100); anal. calcd. for C20H14ClN3O: C, 69.07; H, 4.06; N, 12.08. Found: C, 69.14; H, 4.16; N, 12.15 %.

2-amino-4-(4-chlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7c)

Prepared as previously described (El-Agrody and Al-Ghamdi, 2011).

2-amino-4-(2,3-dichlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7d)

Yellow crystals from benzene; yield 87 %; m.p. 240–241 °C; IR (KBr, υ max cm−1): 3458, 3323, 3196 (NH2), 2193 (CN), 1656 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.22–7.00 (m, 7H, aromatic), 7.23 (bs, 2H, NH2), 5.53 (s, 1H, H-4), 2.71 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.59 (C-2), 159.17 (C-9), 144.69 (C-10b), 136.89 (C-10a), 136.10 (C-7), 128.72 (C-5), 126.32 (C-6a), 123.66 (C-8), 123.01 (C-4a), 119.98 (C-6), 119.95 (CN), 54.38 (C-3), 40.08 (C-4), 24.97 (CH3), 142.94, 136.31, 132.32, 130.16, 129.47, 124.96 (aromatic); MS m/z (%): 385 (M++4, 1.21), 383 (M++2, 8.81), 381 (M+, 12.44) with a base peak at 236 (100); anal. calcd. for C20H13Cl2N3O: C, 62.84; H, 3.43; N, 10.99. Found: C, 62.75; H, 3.56; N, 11.06 %.

2-amino-4-(2,4-dichlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7e)

Yellow crystals from ethanol; yield 86 %; m.p. 249–250 °C; IR (KBr, υ max cm−1): 3475, 3328, 3196 (NH2), 2193 (CN), 1660 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.44–7.02 (m, 7H, aromatic), 7.35 (bs, 2H, NH2), 5.44 (s, 1H, H-4), 2.73 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.52 (C-2), 159.18 (C-9), 144.13 (C-10b), 137.55 (C-10a), 136.34 (C-7), 128.52 (C-5), 126.11 (C-6a), 123.79 (C-8), 123.93 (C-4a), 122.89 (C-6), 120.03 (CN), 54.29 (C-3), 38.19 (C-4), 23.73 (CH3), 149.91, 134.75, 132.97, 131.66, 130.61, 127.45 (aromatic); MS m/z (%): 385 (M++4, 1.27), 383 (M++2, 7.74), 381 (M+, 12.2) with a base peak at 236 (100); anal. calcd. for C20H13Cl2N3O: C, 62.84; H, 3.43; N, 10.99. Found: C, 62.88; H, 3.50; N, 11.05 %.

2-amino-4-(2,5-dichlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7f)

Yellow crystals from benzene/ethanol; yield 84 %; m.p. 266–268 °C; IR (KBr, υ max cm−1): 3471, 3356, 3179 (NH2), 2194 (CN), 1656 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.23–7.01 (m, 7H, aromatic), 7.23 (bs, 2H, NH2), 5.44 (s, 1H, H-4), 2.71 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.63 (C-2), 159.01 (C-9), 143.92 (C-10b), 136.85 (C-10a), 136.08 (C-7), 126.33 (C-5), 124.93 (C-6a), 123.64 (C-8), 123.01 (C-4a), 119.92 (C-6), 119.50 (CN), 55.85 (C-3), 40.14 (C-4), 24.95 (CH3), 142.96, 132.27, 131.77, 131.01, 130.72, 128.95 (aromatic); MS m/z (%): 385 (M++4, 3.61), 383 (M++2, 20.48), 381 (M+, 31.19) with a base peak at 236 (100); anal. calcd. for C20H13Cl2N3O: C, 62.84; H, 3.43; N, 10.99. Found: C, 62.79; H, 3.39; N, 10.71 %.

2-amino-4-(2,6-dichlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7g)

Yellow crystals from benzene; yield 85 %; m.p. 289–290 °C; IR (KBr, υ max cm−1): 3470, 3375, 3187 (NH2), 2189(CN), 1660 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.22–6.87 (m, 7H, aromatic), 7.18 (bs, 2H, NH2), 6.01 (s, 1H, H-4), 2.71 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.93 (C-2), 159.13 (C-9), 143.54 (C-10b), 136.59 (C-10a), 135.64 (C-7), 126.36 (C-6a), 124.21 (C-5), 123.46 (C-8), 122.95 (C-4a), 119.75 (C-6), 118.18 (CN), 52.02 (C-3), 37.24 (C-4), 24.97 (CH3), 137.27, 136.10, 134.83, 130.81, 130.01, 128.65 (aromatic). In 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.93 (C-2 ↓), 159.13 (C-9 ↓), 143.54 (C-10b ↓), 137.27 (aromatic ↓), 136.59 (C-10a ↓), 136.10 (aromatic ↓), 135.64 (C-7 ↑), 134.83 (aromatic ↓), 130.81 (aromatic ↑), 130.01(aromatic ↑), 128.65 (aromatic ↑), 126.36 (C-6a ↓), 124.21 (C-5 ↑), 123.46 (C-8 ↑), 122.95 (C-4a ↓), 119.75 (C-6 ↑), 118.18 (CN ↓), 52.02 (C-3 ↓), 37.24 (C-4 ↑), 24.97 (CH3 ↑). MS m/z (%): 385 (M++4, 2.06), 383 (M++2, 11.77), 381 (M+, 18.14) with a base peak at 236 (100); anal. calcd. for C20H13Cl2N3O: C, 62.84; H, 3.43; N, 10.99. Found: C, 62.88; H, 3.50; N, 11.05 %.

2-amino-4-(3,4-dichlorophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7h)

Yellow crystals from benzene/ethanol; yield 83 %; m.p. 268–270 °C; IR (KBr, υ max cm−1): 3479, 3336, 3196 (NH2), 2190 (CN), 1664 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.96–7.06 (m, 7H, aromatic), 7.30 (bs, 2H, NH2), 5.42 (s, 1H, H-4), 2.73 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.51 (C-2), 151.36 (C-9), 143.45 (C-10b), 137.56 (C-10a), 136.23 (C-7), 129.42 (C-5), 128.09 (C-6a), 126.08 (C-4a), 123.95 (C-8), 122.77 (C-6), 120.11 (CN), 53.88 (C-3), 38.92 (C-4), 24.71 (CH3), 141.40, 132.39, 131.83, 131.19, 130.80, 129.03 (aromatic). MS m/z (%): 385 (M++4, 0.88), 383 (M++2, 4.93), 381 (M+, 7.91) with a base peak at 236 (100); anal. calcd. for C20H13Cl2N3O: C, 62.84; H, 3.43; N, 10.99. Found: C, 62.85; H, 3.47; N, 11.03 %.

2-amino-4-(4-bromophenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7i)

Prepared as previously described (El-Agrody and Al-Ghamdi 2011).

2-amino-4-(3,5-dibromo-2-methoxyphenyl)-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7j)

Pale yellow crystals from ethanol; yield 81 %; m.p. 224–226 °C; IR (KBr, υ max cm−1): 3431, 3333, 3201 (NH2), 2191 (CN), 1659 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.20–7.03 (m, 6H, aromatic), 7.23 (bs, 2H, NH2), 5.25 (s, 1H, H-4), 3.70 (s, 3H, OCH3), 2.70 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.69 (C-2), 159.16 (C-9), 142.75 (C-10b), 142.44 (C-10a), 136.96 (C-7), 132.18 (C-5), 126.22 (C-6a), 123.61 (C-8), 122.95 (C-4a), 120.49 (C-6), 120.27 (CN), 61.36 (Me), 54.59 (C-3), 36.68 (C-4), 24.96 (CH3), 154.14, 136.09, 134.48, 118.26, 117.01 (aromatic); MS m/z (%): 503 (M++4, 1.65), 501 (M++2, 3.05), 499 (M+, 1.65) with a base peak at 236 (100); anal. calcd. for C21H15Br2N3O2: C, 50.33; H, 3.02; N, 8.38. Found: C, 50.40; H, 3.10; N, 8.50 %.

Antitumor screening

Cell culture and cytotoxicity evaluation using viability assay

Compounds 3a–j, 5a–j, and 7a–j were initially evaluated for in vitro antitumor activity against three different human cell lines: MCF-7, HCT-116, HepG-2, and A549 in comparison with Vinblastine and Colchicine. The measurements of cell growth and the viabilities and in vitro cytotoxicity evaluation using viability assay were determined as described in the literature (Rahman et al. 2001) and the result was cited in Table 1.