Synthesis and anti-proliferative activity evaluation of novel 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds

Abstract

A series of novel 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds were synthesized in very good yields using one-pot condensation of 2-hydroxy-1,4-naphthoquinone, aldehydes, and 2-substituted 4,6-diaminopyrimidine. The in vitro anti-proliferative activity of these novel compounds was evaluated in SGC7901 and HepG2 cell lines. Almost all the tested compounds showed manifested potent inhibitory activity against the two tested cancer cell lines.

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Introduction

Multicomponent reactions (MCRs) have become a powerful protocol to access pharmaceutically relevant heterocycles in recent years because of their combined prominent features such as high reaction rate and efficiency, atom economy and selectivity, time and energy savings, target product specificity and minimal environmental impact.¹

Natural products and pharmaceuticals embedded with 1,4-naphthoquinone units display potential medicinal properties such as anticancer,² antifungal,³ antibacterial,⁴ antiviral,⁵ anti-inflammatory,⁶ antimalaria,⁷ antiplatelet,⁵ antithrombotic,⁸ and antiallergic, activities.⁹ Furthermore, 1,4-naphthoquinones have also been shown to inhibit human DNA topoisomerase.¹⁰ A number of 1,4-naphthoquinone derivatives having nitrogen atom present in them received a great deal of attention for their anticancer activity.¹¹ Therefore, the development of facile approaches to access these novel targets with structural diversity is highly desirable and valuable for medicinal chemistry and drug discovery.

Nitrogen containing heterocycles constitute an important class of compounds. Among them, pyrido[2,3-d]pyrimidine ring system occurs as a principal core skeleton among the drug scaffolds and also play crucial role as an important component in organic synthesis, and medicinal chemistry. Pyrido[2,3-d]pyrimidine derivatives gained prominence as they exhibit a wide range of biological and medicinal properties such as analgesic,¹² antiviral,¹³ anti-inflammatory,¹⁴ antimicrobial,¹⁵ antifungal,¹⁶ and anticancer activity.¹⁷ Therefore, the synthesis of diverse structures belonging to this class of compounds is very important.

In the design of new drug prototypes, the concept of molecular hybridization is a useful tool and is based on the combination of pharmacophoric moieties of different bioactive substances to produce a new hybrid compound with improved affinity and efficacy. This strategy has resulted in compounds with modified selectivity profile, different and/or dual mode of action and reduced undesired side effects.¹⁸ Based on the versatile bioactivities of the above mentioned structures, it is promising that the integration of pyrido[2,3-d]pyrimidine scaffold with 1,4-naphthoquinone segment might result in the discovery of new drug candidates with unknown or enhanced bioactivities. We herein represent the synthesis of a series of novel 1,4-naphthoquinone and pyrido[2,3-d]pyrimidine hybrids and their anti-proliferative activity against human cancer cell lines in vitro (Scheme 1).

Scheme 1

Results and discussion

Our strategy to synthesize 1,4-naphthoquinones-fused pyrido[2,3-d]pyrimidines involved three-component reaction of 2-hydroxy-1,4-naphthoquinone, aldehydes, and 2-substituted 4,6-diaminopyrimidine. The initial experiments were performed with 2-hydroxy-1,4-naphthoquinone, benzaldehyde, and 2,4,6-triaminopyrimidine in EtOH at 70 °C for 4 h. This set of conditions led to expected 5-phenyl-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione 4a, albeit in a low 15% yield (Table 1, entry 1). Encouraged by this preliminary result, we optimized the reaction conditions further. Solvent effects were first investigated (Table 1, compare entries 1–7). The use of AcOH facilitated the transformation and delivered 4a in a higher yield of 41%, whereas THF, CHCl₃ and H₂O, as reaction media completely suppressed the reaction. Another two solvents, that is, toluene and DMF, were proven ineffective and gave outcomes inferior to that obtained with AcOH. We then attempted to adjust the temperature to improve the reaction efficiency. Elevating the temperature to 118 °C (reflux) proved more efficient, and expected 4a was afforded in 65% yield (Table 1, entry 11).

Table 1 Reaction conditions optimization for the synthesis 5-phenyl-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione

Entry	Solvent	T/°C	Time/h	Yield/%
1	EtOH	70	4	15
2	THF	62	6	6
3	CHCl3	61	6	0
4	H2O	70	8	0
5	Toluene	70	5	10
6	DMF	70	4	39
7	AcOH	70	4	41
8	AcOH	80	4	43
9	AcOH	90	3	49
10	AcOH	100	2	60
11	AcOH	118	2	65

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Under the above optimized conditions, the substrate scope of this three-component cyclocondensation reaction was examined by using readily available starting materials. As revealed in Table 2, 2,4,6-triaminopyrimidine was first subjected to the reaction with 2-hydroxy-1,4-naphthoquinone and different aldehydes in AcOH at reflux without the use of a strong acid or metal catalyst, and expected 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidines scaffolds 4a–p were obtained in good yields. Various aryl aldehydes having substituents at different positions with electron-poor (e.g., fluoro, chloro and nitro), electron-neutral (e.g., H), electron-rich (e.g., methyl and methoxy) groups, aromatic heterocyclic and aliphatic aldehydes were compatible. Next, we selected 2-methylthio-4,6-diaminopyrimidine as representative substrate to expand the synthetic utility of this methodology further. As we expected, these reactions proceeded smoothly to give access to corresponding tetracyclic products 4q–s. Resulting 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds 4 were fully characterized by IR, NMR spectroscopy and HRMS. The IR spectrum of 4a showed absorptions at 1675 and 1633 cm⁻¹ indicating the presence of two C=O bonds. The high resolution mass spectrum of 4a displayed the quasi-molecular ion ([M + Na]⁺) peak at m/z = 392.1114, which was consistent with the 1 : 1 : 1 adduct of 2-hydroxy-1,4-naphthoquinone, benzaldehyde and 2,4,6-diaminopyrimidine with the loss of two water molecule. The ¹H NMR spectrum of 4a showed four singlet was observed (δ = 5.31, 5.82, 6.23 and 8.68 ppm) for the CH group of C-5 position, NH₂ group of C-2, 4 position and NH group of dihydropyridine respectively. The ¹³C NMR spectrum of 4a showed characteristic signals at δ = 34.8 ppm (due to the R¹–CH group), 181.7 and 179.9 ppm (arising from the two nonequivalent carbonyl groups).

Table 2 Preparation of 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds

Entry	R^1	R^2	Time/h	Product	Mp/°C	Yield/%
1	C6H5	NH2	2	4a	168–170	65
2	2-Thienyl	NH2	2	4b	254–255	69
3	2-Furanyl	NH2	3	4c	297–299	59
4	3-NO2-C6H4	NH2	3	4d	>300	53
5	4-Cl-C6H4	NH2	3	4e	165–167	57
6	2,4-Cl-C6H3	NH2	1.5	4f	>300	72
7	4-F-C6H4	NH2	3	4g	157–159	55
8	2-F-C6H4	NH2	1.5	4h	>300	71
9	4-MeO-C6H4	NH2	1	4i	196–198	79
10	2,5-MeO-C6H3	NH2	2	4j	265–266	63
11	3,4,5-MeO-C6H2	NH2	3	4k	276–278	60
12	3-Br-4-MeO-C6H3	NH2	3	4l	234–236	58
13	C4H9	NH2	4	4m	192–194	50
14	C5H11	NH2	4	4n	193–195	51
15	3-OH-C6H4	NH2	2.5	4o	292–293	57
16	2-CH3-C6H4	NH2	3	4p	173–175	66
17	4-Cl-C6H4	SCH3	3	4q	258–260	52
18	C6H5	SCH3	3	4r	237–239	53
19	3-Br-4-OCH3-C6H3	SCH3	3	4s	>300	55

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A reasonable mechanism is proposed for the formation of 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds 4 (Scheme 2). It is conceivable that 2-hydroxy-1,4-naphthoquinone initially reacts with aldehyde 2 to form olefin 5, which underwent a nucleophilic addition of 2-substituted 4,6-diaminopyrimidine to form the corresponding Michael-type type intermediate 6. This step is then followed by an intramolecular dehydration to yield to product 4.

Scheme 2

To evaluate their anti-proliferative potential, the newly synthesized hybrids 4a–s were subjected to in vitro biological assessment against two human cancer cell lines, SGC7901 and HepG2. The results of the cytotoxicity evaluation, as compared to the anticancer reference compound doxorubicin, were summarized in Table 3. As evidenced by these results, the majority of the derivatives exhibited at least moderate cytotoxic activity against the SGC7901 and HepG2 cell lines. Six of the new hybrids (4b, 4d, 4n, 4q, 4r and 4s) even display a considerable activity profile with IC₅₀ values below 10 μM against both cell lines. It is worthwhile to note that the majority compounds have lesser cytotoxicity on non-cancerous L02 cells. These results clearly suggest the relevance of this interesting new class of hybrids in the framework of cancer therapy research and medicinal chemistry. The results in Table 3 showed also some important structure–activity relationships (SARs) for this series of derivatives. First, the nature of substituents at the C-2 position have substantial influence on the anti-proliferative activity, introduction of methylmercapto group into the C-2 position was found to be quite favorable for increasing anti-proliferative activity. Among this series, compound 4s showed the best anti-proliferative activity with IC₅₀ values of 4.39 μM and 5.91 μM against SGC7901 and HepG2 cell lines, respectively. Second, the substituents at the C-2 moiety appeared to have an important effect upon cytotoxicity, introduction of aromatic heterocycle group into the C-5 position was found to be quite favorable for increasing anti-proliferative activity.

Table 3 Anti-proliferative activities of 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds

Compound	IC50 (μM)
	SGC7901	HepG2	L02
4a	38.86 ± 2.38	29.59 ± 1.43	80.26 ± 3.23
4b	5.88 ± 0.15	9.54 ± 0.51	24.54 ± 0.98
4c	10.15 ± 1.01	18.52 ± 1.26	45.23 ± 2.53
4d	9.79 ± 1.12	9.98 ± 1.21	25.31 ± 1.20
4e	31.00 ± 2.78	42.21 ± 1.98	70.21 ± 2.32
4f	>100	79.64 ± 8.42	>100
4g	30.34 ± 1.67	28.16 ± 3.01	63.25 ± 2.99
4h	36.34 ± 1.25	33.88 ± 5.21	>100
4i	10.02 ± 0.89	16.28 ± 1.59	24.29 ± 1.72
4j	33.74 ± 1.26	53.02 ± 2.87	>100
4k	26.45 ± 1.36	21.55 ± 1.53	50.78 ± 2.42
4l	43.63 ± 4.07	67.95 ± 9.01	>100
4m	26.48 ± 1.29	56.81 ± 9.56	>100
4n	9.91 ± 1.01	8.25 ± 0.84	17.53 ± 0.76
4o	25.54 ± 1.04	22.20 ± 1.07	41.20 ± 1.75
4p	51.05 ± 1.32	39.57 ± 1.87	>100
4q	5.61 ± 0.43	9.15 ± 0.85	19.52 ± 0.99
4r	9.46 ± 0.55	9.99 ± 0.98	18.45 ± 1.32
4s	4.39 ± 0.19	5.91 ± 0.73	11.26 ± 1.05
Doxorubicin	3.18 ± 0.54	2.25 ± 0.42	6.22 ± 0.95

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Conclusion

In the present study, a novel hybrids containing 1,4-naphthoquinone and pyrido[2,3-d]pyrimidine has been developed by molecular hybridization strategy. Nineteen hybrids were synthesized and evaluated for their anti-proliferative activities against SGC7901 and HepG2. The results showed that most of the new compounds showed good to potent cytotoxic activities. The most potent derivative 4s displayed significant inhibition of SGC7901 with an IC₅₀ of 4.39 μM and an IC₅₀ value of 5.91 μM. Therefore, these novel 1,4-naphthoquinones fused with bioactive heterocyclic skeletons may find their pharmaceutical applications after further investigations.

Experimental

General

IR spectra were determined on FTS-40 infrared spectrometer. NMR spectra were determined on Bruker AV-400 spectrometer at room temperature using TMS as internal standard. Chemical shifts (d) are given in ppm and coupling constants (J) in Hz. High resolution mass spectra were recorded on a Bruker micrOTOF-QIII mass spectrometer. Elemental analysis were performed by a Vario-III elemental analyzer. Melting points were determined on a XT-4 binocular microscope and were uncorrected. Commercially available reagents were used throughout without further purification unless otherwise stated.

General procedure for the synthesis of compounds 4

To a mixture of 2-hydroxy-1,4-naphthoquinone (1 mmol), aldehyde (1 mmol) and 2-substituted 4,6-diaminopyrimidine (1 mmol), AcOH (10 mL)was added. The mixture was stirred at reflux for an appropriate time (Table 2). After completion of the reaction (TLC), the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then, the crude product was washed sequentially with 20 mL saturated NaHCO₃ and 20 mL brine, purified by silica gel column chromatography using CHCl₃ : ethyl acetate (v/v = 10 : 1) as eluent to afford the pure product 4.

5-Phenyl-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4a). Reddish-brown power, mp 168–170 °C; IR (KBr): ν 3473, 3383, 3302, 3155, 1675, 1633 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.68 (s, 1H), 8.01–7.99 (m, 1H), 7.93–7.91 (m, 1H), 7.84–7.75 (m, 2H), 7.40 (d, 2H, J = 7.2 Hz), 7.21 (t, 2H, J = 7.6 Hz), 7.13 (d, 1H, J = 7.2 Hz), 6.23 (s, 2H), 5.82 (s, 2H), 5.31 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.7, 179.9, 162.6, 162.1, 154.1, 145.9, 139.9, 135.3, 133.5, 132.5, 130.7, 128.5, 128.4, 126.8, 126.3, 126.1, 118.4, 87.4, 34.8; HRMS-ESI (m/z): calc for C₂₁H₁₅N₅NaO₂ [M + Na]⁺: 392.1123, found: 392.1114.

5-(Thien-2-yl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4b). Reddish-brown power, mp 254–255 °C; IR (KBr): ν 3500, 3467, 3387, 3284, 1676, 1623 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.86 (s, 1H), 8.00 (d, 2H, J = 7.6 Hz), 7.87–7.77 (m, 2H), 7.23 (d, 1H, J = 4.0 Hz), 6.94 (d, 1H, J = 0.8 Hz), 6.85–6.83 (m, 1H), 6.40 (s, 2H), 5.85 (m, 2H), 5.70 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.6, 180.0, 162.7, 162.2, 154.0, 149.4, 139.6, 135.4, 133.6, 132.5, 130.7, 126.9, 126.3, 126.2, 125.0, 124.8, 117.4, 87.0, 30.0; HRMS-ESI (m/z): calc for C₁₉H₁₃N₅NaO₂S [M + Na]⁺: 398.0688, found: 398.0684.

5-(Fur-2-yl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4c). Reddish-brown power, mp 297–299 °C; IR (KBr): ν 3458, 3392, 3297, 3139, 1676, 1614 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.78 (s, 1H), 8.03–7.97 (m, 2H), 7.87–7.78 (m, 2H), 7.40 (s, 1H), 6.39 (s, 2H), 6.27–6.23 (m, 2H), 5.84 (m, 2H), 5.51 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.5, 179.8, 162.6, 162.1, 156.0, 154.3, 142.1, 140.8, 135.3, 133.6, 132.5, 130.7, 126.4, 126.2, 114.6, 110.8, 106.2, 84.5, 29.0; HRMS-ESI (m/z): calc for C₁₉H₁₃N₅NaO₃ [M + Na]⁺: 382.0916, found: 382.0909.

5-(3-Nitrophenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4d). Reddish-brown power, mp >300 °C; IR (KBr): ν 3449, 3317, 3189, 3086, 1673, 1672, 1612 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.90 (s, 1H), 8.36 (t, 1H, J = 2.0 Hz), 8.01–7.99 (m, 2H), 7.93–7.91 (m, 1H), 7.83–7.77 (m, 3H), 7.53 (t, 1H, J = 8.0 Hz), 6.37 (s, 2H), 5.90 (s, 2H), 5.52 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.6, 179.7, 162.6, 162.3, 154.2, 147.9, 147.8, 140.4, 135.3, 135.2, 133.6, 132.4, 130.8, 130.1, 126.3, 126.2, 123.1, 122.0, 117.1, 86.6, 34.8; HRMS-ESI (m/z): calc for C₂₁H₁₅N₅NaO₃ [M + Na]⁺: 437.0974, found: 437.0974.

5-(4-Chlorophenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4e). Reddish-brown power, mp 165–167 °C; IR (KBr): ν 3469, 3385, 3309, 3167, 1677, 1617 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.73 (s, 1H), 8.01–7.99 (m, 1H), 7.93–7.91 (m, 1H), 7.84, 7.75 (m, 2H), 7.42 (d, 2H, J = 8.4 Hz), 7.27 (d, 2H, J = 8.4 Hz), 6.28 (s, 2H), 5.85 (s, 2H), 5.34 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.6, 179.8, 162.6, 162.1, 154.1, 144.8, 140.0, 135.3, 133, 132.5, 131.4, 130.7, 130.3, 128.4, 126.3, 126.1, 117.8, 87.0, 34.3; HRMS-ESI (m/z): calc for C₂₁H₁₄N₅O₂ [M + Na]⁺: 426.0734, found: 426.0677.

5-(2,4-Dichlorophenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4f). Reddish-brown power, mp >300 °C; IR (KBr): ν 3520, 3465, 3398, 3338, 1666, 1643 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.89 (s, 1H), 8.02–7.99 (m, 1H), 788–7.75 (m, 3H), 7.63 (d, 1H, J = 8.4 Hz), 7.48 (d, 1H, J = 2.4 Hz), 7.35–7.32 (m, 1H), 5.92 (s, 2H), 5.82 (s, 2H), 5.51 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.4, 179.7, 162.6, 162.1, 154.0, 141.6, 140.7, 135.4, 134.0, 133.6, 132.9, 132.4, 130.6, 130.6, 129.1, 128.0, 126.3, 126.1, 116.2, 86.3, 34.5; HRMS-ESI (m/z): calc for C₂₁H₁₃Cl₂N₅NaO₂ [M + Na]⁺: 460.0344, found: 460.0330.

5-(4-Fluorophenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4g). Reddish-brown power, mp 157–159 °C; IR (KBr): ν 3484, 3377, 3323, 3196, 1674, 1633 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.72 (d, 1H, J = 0.8 Hz), 8.00–7.91 (m, 2H), 7.81–7.76 (m, 2H), 7.45–7.41 (m, 2H), 7.03 (t, 2H, J = 8.8 Hz), 6.29 (s, 2H), 5.87 (s, 2H), 5.34 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.7, 179.8, 162.5, 162.0, 154.0, 142.0, 139.9, 135.3, 133.5, 132.5, 130.7, 130.3, 130.2, 126.3, 126.1, 118.1, 115.3, 115.0, 87.3, 34.1; HRMS-ESI (m/z): calc for C₂₁H₁₄FN₅NaO₂ [M + Na]⁺: 410.1029, found: 410.0994.

5-(2-Fluorophenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4h). Reddish-brown power, mp >300 °C; IR (KBr): ν 3495, 3434, 3392, 3301, 1677, 1638 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.81 (s, 1H), 8.02–8.00 (m, 1H), 7.89–7.75 (m, 3H), 7.83–7.75 (m, 2H), 7.65–7.60 (m, 1H), 7.22–7.16 (m, 1H), 7.10–7.03 (s, 2H), 5.96 (s, 2H), 5.86 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.4, 179.8, 162.5, 162.0, 159.3, 153.9, 140.6, 135.3, 133.5, 132.4, 131.9, 130.6, 129.1, 129.0, 126.3, 126.1, 124.6, 116.2, 86.3, 34.2; HRMS-ESI (m/z): calc for C₂₁H₁₄FN₅NaO₂ [M + Na]⁺: 410.1029, found: 410.1018.

5-(4-Methoxyphenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4i). Reddish-brown power, mp 196–198 °C; IR (KBr): ν 3511, 3397, 3355, 3288, 1626, 1613 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: (8.66 s, 1H), 8.00–7.89 (m, 1H), 7.93–7.91 (m, 1H), 7.83–7.74 (m, 2H), 7.31 (d, 2H, J = 8.8 Hz), 6.77 (d, 2H, J = 8.8 Hz), 6.24 (s, 2H), 5.86 (s, 2H), 5.25 (s, 1H), 3.65 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.7, 179.8, 162.4, 161.8, 158.3, 153.8, 139.5, 138.0, 135.2, 133.5, 132.5, 130.6, 139.4, 126.2, 126.1, 118.7, 113.9, 87.6, 33.9; HRMS-ESI (m/z): calc for C₂₂H₁₇N₅NaO₃ [M + Na]⁺: 422.1229, found: 422.1228.

5-(2,5-Dimethoxyphenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4j). Reddish-brown power, mp 265–266 °C; IR (KBr): ν 3465, 3385, 3310, 3175, 1669, 1646 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.64 (s, 1H), 8.03–8.01 (m, 1H), 7.88–7.76 (m, 3H), 6.94 (d, 1H, J = 8.8 Hz), 6.81 (d, 1H, J = 3.2 Hz), 6.73–6.70 (m, 1H), 6.06 (d, 2H, J = 0.8 Hz), 5.82 (s, 2H), 5.35 (s, 1H), 3.87 (s, 3H), 3.59 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.4, 180.0, 162.4, 161.8, 154.4, 153.6, 149.7, 140.7, 135.8, 135.2, 133.5, 132.5, 130.8, 126.3, 126.0, 117.7, 116.5, 113.8, 112.3, 87.5, 57.6, 55.6, 29.9; HRMS-ESI (m/z): calc for C₂₃H₁₉N₅NaO₄ [M + Na]⁺: 430.1515, found: 430.1508.

5-(3,4,5-Trimethoxyphenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4k). Reddish-brown power, mp 276–278 °C; IR (KBr): ν 3590, 3450, 3351, 3123, 1663, 1633 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.63 (s, 1H), 8.01–7.93 (m, 2H), 7.84–7.75 (m, 2H), 6.73 (s, 2H), 6.26 (s, 2H), 5.82 (s, 2H), 5.22 (s, 1H), 3.68 (s, 6H), 3.57 (s, 3H), ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.8, 179.9, 162.5, 162.0, 154.0, 152.9, 141.6, 139.8, 136.6, 135.2, 133.5132.5, 130.8, 126.3, 126.2, 118.1, 106.0, 87.4, 60.3, 56.3, 35.3; HRMS-ESI (m/z): calc for C₂₄H₂₁N₅NaO₅ [M + Na]⁺: 482.1440, found: 482.1362.

5-(3-Bromo-4-methoxyphenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4l). Reddish-brown power, mp 234–236 °C; IR (KBr): ν 3459, 3360, 3326, 3092, 1666, 1633 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.79 (s, 1H), 7.95–7.88 (m, 2H), 7.78–7.69 (m, 2H), 7.64 (d, 1H, J = 1.2 Hz), 7.31–7.29 (m, 1H), 6.94 (d, 1H, J = 8.8 Hz), 6.34 (s, 2H), 5.92 (s, 2H), 5.26 (1H), 3.73 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.7, 179.8, 162.5, 162.0, 154.3, 154.0, 139.7, 139.6, 135.2, 133.5, 132.7, 130.6, 128.1, 126.2, 126.1, 118.0, 112.9, 110.5, 87.1, 56.5, 33.8; HRMS-ESI (m/z): calc for C₂₂H₁₆N₅NaO₃ [M + Na]⁺: 500.0334, found: 500.0220.

5-Butyl-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4m). Reddish-brown power, mp >300 °C; IR (KBr): ν 3466, 3397, 3327, 3200, 1676, 1621 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.36 (s, 1H), 8.02–7.99 (m, 2H), 7.87–7.79 (m, 2H), 6.34 (s, 2H), 5.75 (s, 2H), 4.29 (t, 1H, J = 4.8 Hz), 1.57–1.43 (m, 2H), 1.14–1.03 (m, 3H), 0.94–0.88 (s, 1H), 0.74 (t, 3H, J = 6.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 182.1, 179.7, 162.6, 161.8, 155.0, 141.3, 135.2, 133.4, 132.7, 130.7, 126.2, 118.0, 85.9, 34.7, 29.1, 23.1, 14.5; HRMS-ESI (m/z): calc for C₁₉H₁₉N₅NaO₂ [M + Na]⁺: 372.1436, found: 372.1426.

5-Amyl-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4n). Reddish-brown power, mp >300 °C; IR (KBr): ν 3468, 3403, 3327, 3201, 1676, 1620 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.37 (s, 1H), 8.00–7.98 (m, 2H), 7.85–7.75 (m, 2H), 6.38 (s, 2H), 5.78 (s, 2H), 4.28 (t, 1H, J = 4.4 Hz), 1.53–1.46 (m, 2H), 1.19–1.09 (m, 5H), 0.89–0.82 (m, 1H), 0.72 (t, 3H, J = 6.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 182.1, 179.6, 162.6, 161.8, 155.0, 141.3, 135.2, 133.4, 132.7, 130.7, 126.2, 118.0, 85.9, 34.8, 29.1, 24.3, 22.5, 14.3; HRMS-ESI (m/z): calc for C₁₉H₁₉N₅NaO₂ [M + Na]⁺: 386.1593, found: 386.1564.

5-(3-Hydroxyphenyl)-5,12-dihydro-2,4-amino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4o). Reddish-brown power, mp 292–293 °C; IR (KBr): ν 3495, 3354, 3215, 3095, 1673, 1607 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.22 (s, 1H), 8.69 (s, 1H), 8.00–7.92 (m, 2H), 7.83–7.74 (m, 2H), 6.99 (t, 1H, J = 8.0 Hz), 6.85 (d, 1H, J = 7.6 Hz), 6.76 (s, 1H), 6.52–6.50 (s, 1H), 6.21 (s, 2H), 5.84 (s, 2H), 5.24 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.7, 179.9, 162.6, 162.0, 157.6, 154.1, 147.1, 139.8, 135.3, 133.5, 132.5, 130.6, 129.3, 126.2, 126.1, 119.3, 118.4, 115.3, 113.9, 87.3, 3.7; HRMS-ESI (m/z): calc for C₂₁H₁₅N₅NaO₃ [M + Na]⁺: 408.1073, found: 408.1066.

5-(2-Methylphenyl)-5,12-dihydro-2,4-diamino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4p). Reddish-brown power, mp 173–175 °C; IR (KBr): ν 3478, 3375, 3326, 3181, 1672, 1635 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.81 (s, 1H), 7.99 (t, 1H, J = 7.6 Hz), 7.87 (t, 1H, J = 6.4 Hz), 7.82–7.74 (m, 2H), 7.29 (t, 1H, J = 7.2 Hz), 7.07–7.01 (m, 3H), 5.87 (s, 2H), 5.62 (s, 2H), 5.31 (s, 1H), 2.63 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 181.8, 179.9, 169.2, 162.8, 161.8, 153.9, 144.7, 139.9, 135.3, 133.5, 132.5, 128.5, 132.2, 132.0, 126.8, 126.3, 126.1, 118.7, 88.5, 33.3, 19.9; HRMS-ESI (m/z): calc for C₂₂H₁₇N₅NaO₂ [M + Na]⁺: 406.1280, found: 406.1262.

2-Methylthio-5-(4-chlorophenyl)-5,12-dihydro-4-amino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4q). Reddish-brown power, mp 157–159 °C; IR (KBr): ν 3390, 3297, 3196, 1688, 1637 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.53 (s, 1H), 8.51 (d, 1H, J = 8.0 Hz), 7.98–7.96 (m, 1H), 7.84–7.79 (m, 1H), 7.66 (t, 1H, J = 7.6 Hz), 7.40 (d, 1H, J = 8.4 Hz), 7.28 (d, 2H, J = 8.2 Hz), 6.97 (s, 2H), 5.31 (s, 1H), 2.46 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.4, 176.0, 168.5, 161.5, 153.7, 146.3, 143.9, 135.1, 131.7, 131.6, 131.1, 130.2, 129.5, 129.0, 128.5, 125.2, 113.7, 93.3, 33.6, 13.6; HRMS-ESI (m/z): calc for C₂₂H₁₅ClN₄NaO₂S [M + Na]⁺: 398.0688, found: 398.0684.

2-Methylthio-5-phenyl-5,12-dihydro-4-amino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4r). Reddish-brown power, mp 157–159 °C; IR (KBr): ν 3455, 3291, 3160, 3082, 1689, 1606 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.51 (s, 1H), 8.51 (d, 1H, J = 7.6 Hz), 7.98–7.96 (m, 1H), 7.83–7.79 (m, 1H), 7.67–7.61 (m, 1H), 7.40 (t, 2H, J = 1.2 Hz), 7.21 (t, 2H, J = 7.2 Hz), 7.14–7.10 (s, 1H), 6.94 (s, 2H), 5.29 (s, 1H), 2.46 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.5, 176.1, 168.3, 161.6, 153.7, 146.3, 145.0, 135.2, 131.6, 131.1, 130.3, 129.0, 128.6, 128.3, 127.0, 125.2, 114.1, 93.8, 34.1, 13.6; HRMS-ESI (m/z): calc for C₂₂H₁₆N₄NaO₂S [M + Na]⁺: 423.0892, found: 423.0887.

2-Methylthio-5-(3-bromo-4-methoxyphenyl)-5,12-dihydro-4-amino-benzo[g]pyrimido[4,5-b]quinoline-6,11-dione (4s). Reddish-brown power, mp 157–159 °C; IR (KBr): ν 3462, 3373, 3304, 3209, 1690, 1600 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.49 (s, 1H), 8.50 (d, 1H, J = 7.6 Hz), 7.97 (d, 1H, J = 7.6 Hz), 7.83–7.79 (m, 1H), 7.67–7.65 (m, 2H), 7.27–7.25 (m, 1H), 6.96 (d, 3H, J = 8.4 Hz), 5.23 (s, 1H), 3.74 (s, 3H), 2.47 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.4, 176.1, 168.5, 161.5, 154.4, 153.6, 146.1, 138.7, 135.2, 132.8, 131.6, 131.1, 130.2, 129.0, 128.6, 125.2, 113.9, 113.0, 110.4, 93.4, 56.6, 33.1, 13.6; HRMS-ESI (m/z): calc for C₂₃H₁₇BrN₄NaO₃S [M + Na]⁺: 531.0102, found: 531.0094.

Anti-proliferative assay

SGC7901, HepG2 and L02 cells were obtained from the Cell Center of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified eagle's media (DMEM, gibco, USA) containing 1% penicillin–streptomycin, supplemented with 10% fetal bovine serum (FBS, gibco, USA) at 37 °C, 5% CO₂. Cells (3 × 10³ cells) were seeded into 96-well plates and incubated at 37 °C in 5% CO₂/95% air condition. Serially two fold diluted test compound solutions of each drug were added 24 h later, and the cells were incubated for the next 48 h. The final concentrations of compounds in the sample wells ranged from 0.103 μM to 50 μM. After 48 h, 20 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 5 mg mL⁻¹) was added to each well and the cells were incubated for an additional 4 h. Then, 100 μL DMSO were added into each well for dissolving the intracellular formazan crystals. Optical density at 570 nm of each plate was measured with a tunable microplate reader. Each group was in triplicate samples and each drug was divided into at least 5 concentrations. The percentage of absorbance from the sample-treated cells compared to that of the vehicle control (treated with DMSO) was calculated. The resulting cytotoxic activities were expressed as IC₅₀ values and IC₅₀ values were determined by analysis software (Graphpad Prism 6).

Acknowledgements

We are pleased to acknowledge the financial support from Xinxiang Medical University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra17032c

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