Synthesis and antitumor activity evaluation of novel substituted 5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-diones

Abstract

A series of novel substituted 5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-diones, designed via a molecular hybridization approach, were synthesized in very good yields using one-pot condensation of 2-hydroxy-1,4-naphthoquinone, aldehydes, and 5-substituted-2-amino-1,3,4-thiadiazole. The antitumor activities of all the synthesized compounds were assessed on two different human cancer cell lines (HCT116 and HepG2), and the results showed that most of the new compounds showed good to potent cytotoxic activities.

---

Introduction

Cancer is one of the major causes of death worldwide.¹ Currently there is no cure for the large majority of cancers, and as such, development of effective anticancer drugs is of urgent clinical importance.

Quinones are widely distributed in nature as a constituent of biologically active molecules in living organisms.² These compounds show pronounced biological activities associated with, inter alia, antitumor,^3,4 anti-inflammatory,⁵ antifungal,⁶ antiparasitic,⁷ antioxidant,⁸ trypanocidal,⁹ and antiviral¹⁰ properties. Recently, 1,2-naphthoquinones include β-lapachone,¹¹ tanshinone IIA,¹² dunnione,¹³ mansonone F¹⁴ and salvicine¹⁵ (Fig. 1) have been reported to show remarkable antitumor activities through different mechanisms. One of these quinones was β-lapachone, a natural tetrahydropyran-fused ortho-naphthoquinone, which possesses potent in vitro and in vivo activities against malignant tumor cells, especially in some human solid tumor models, and has now entered phase II clinical trials.¹⁶ It has been reported to kill many human cancers selectively through rapid reactive oxygen species (ROS) generation mediated by NQO1 bioreduction.¹⁷ In view of the limited structure of natural ortho-quinones, it is urgent to develop novel, efficient and diversified ortho-quinones as antitumor agents.

Fig. 1 Structures of potent anticancer 1,2-naphthoquinone analogues.

1,3,4-Thiadiazolo[3,2-a]pyrimidines are one of the most important classes of heterocyclic compounds that occur widely in natural products and drug-like molecules. They have a wide range of biological activities such as antitumor,¹⁸ antibacterial,¹⁹ fungicidal,²⁰ and herbicidal.²¹ Thus functionalized 1,3,4-thiadiazolo[3,2-a]pyrimidines have been used as key building blocks for the preparation of a variety of novel bioactive agents.²²

Over the last few years, molecular hybridization strategy has emerged as a novel approach in modern medicinal chemistry for the exploration of novel and highly active compounds. The molecular hybridization is a strategy of rational design of such ligands or prototypes based on the recognition of pharmacophoric subunits in the molecular structure of two or more known bioactive derivatives which, through the adequate fusion of these sub-units, lead to the design of new hybrid architectures that maintain preselected characteristics of the original templates. Hybrid formation is a classic strategy in drug design based in combining different bioactive fragments or molecules to get the corresponding “hybrids” or “conjugates”, which present different and/or dual modes of action, modify selectivity profile and reduce undesired side effects.²³

As a part of our ongoing research aiming on the development of novel bioactive hybrid molecules,^24–26 we report herein one-pot synthesis and antitumor activity evaluation of novel substituted 5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-diones (Scheme 1). Compared with the previous studies for 1,3,4-thiadiazolo[3,2-a]pyrimidines, one of the biggest innovations in the paper was that a series of novel 1,2-naphthoquinone derivatives possessing 1,3,4-thiadiazolo[3,2-a] pyrimidine scaffolds was designed via molecular hybridization approach, and synthesized in very good yields through one-pot multicomponent reactions, and investigated as antitumor agents which was ground-breaking.

Scheme 1

Results and discussion

The choice of an appropriate reaction medium is of crucial importance for successful synthesis. Initially, the three-component reaction of 2-hydroxy-1,4-naphthoquinone, benzaldehyde, and 5-methyl-2-amino-1,3,4-thiadiazole as a simple model substrate was investigated to establish the feasibility of the strategy and to optimize the reaction conditions (Table 1). The exploration of different solvents, such as EtOH, CH₃CN, CH₃COOH, DMSO, toluene, and DMF showed that the solvent had an obvious influence on the reaction yield. The best result was obtained in DMF. Then, the optimal reaction temperature was further investigated (entries 7–12), and 130 °C with the highest yield of 26% (entry 11) was chosen as the best suitable condition for all further reactions.

Table 1 Optimization of the reaction conditions for the synthesis of 4d

Entry	Reaction conditions	Time/h	Yield^a/%
a Isolated yield.
1	Solvent-free, 130 °C	10	18
2	EtOH, reflux	24	0
3	CH3CN, reflux	24	0
4	CH3COOH, reflux	10	Trace
5	DMSO, 130 °C	8	20
6	Toluene, reflux	10	22
7	DMF, 25 °C	24	0
8	DMF, 100 °C	8	16
9	DMF, 110 °C	8	23
10	DMF, 120 °C	6	25
11	DMF, 130 °C	6	26
12	DMF, 140 °C	6	26

---

Diversity-oriented synthesis is a strategy used by chemical biologists to create a huge diversity of small molecules with potentially useful properties. With optimal conditions in hand, we then carried out the design and diversity-oriented synthesis of novel substituted 5H-benzo[i][1,3,4]thiadiazolo [3,2-a]quinazoline-6,7-diones. Different 2-amino-1,3,4-thiadiazoles and aldehydes were applied to this reaction for the aim to access target molecules with structural diversity. As shown in Table 2, the protocol is amenable to a wide scope of aldehydes and 2-amino-1,3,4-thiadiazoles. In all cases this three-component reaction led regioselectively to 1,2-naphthoquinone derivatives 4 and their structures were characterized by spectroscopic and analytical methods. For example, the IR spectrum of 4g showed absorptions at 1690 and 1624 cm⁻¹ indicating the presence of two C=O bonds, the high resolution mass spectrum of 4g displayed the quasi-molecular ion ([M + Na]⁺) peak at m/z = 396.0777, which was consistent with the 1 : 1 : 1 adduct of 2-hydroxy-1,4-naphthoquinone, 2-methylbenzaldehyde and 5-methyl-2-amino-1,3,4-thiadiazoles with the loss of two water molecule. The ¹H NMR spectrum of 4g exhibited one singlet for the CH group of C-5 position at δ = 6.60 ppm, two singlets due to the methyl protons at δ = 2.53 ppm and δ = 2.30 ppm. ¹³C NMR spectrum of 4g showed 21 distinct resonances. Among them, three characteristic signals at δ = 60.2 ppm (due to the Ar–CH group), 179.6 and 175.9 ppm (arising from the two nonequivalent carbonyl groups) were shown. The HMBC experiments can observe typical two-bond and three-bond proton to carbon couplings. It could be observe that the long-range correlations of carbonyl carbons C-6 to the protons of dihydropyrimidine-fused ring and C-7 to the protons of the benzene-fused ring in HMBC spectrum of 4g, which was able to prove that the formation of the ortho-quinone units in the reaction (Fig. 2).

Table 2 Preparation of substituted 5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-diones

Entry	R^1	R^2	Time/h	Product	Mp/°C	Yield^a/%
a Isolated yield.
1	CH3(CH2)5CH2	H	5	4a	164–166	15
2	3,5-(MeO)2–C6H3	H	7	4b	178–180	18
3	C6H5	Et	6	4c	177–179	24
4	C6H5	Me	6	4d	216–218	26
5	3,5-(MeO)2–C6H3	Me	5	4e	200–201	30
6	3-MeO–C6H4	Me	5	4f	192–194	42
7	2-Me–C6H4	Me	5	4g	269–270	34
8	3,5-(MeO)2–C6H3	Et	7	4h	168–170	32
9	3-MeO–C6H4	Et	7	4i	196–197	37
10	3-OH–C6H4	Me	8	4j	255–257	22
11	3-OH–C6H4	Et	8	4k	207–208	20
12	2-Br-5-OH–C6H3	Me	8	4l	230–231	16
13	3-Me–C6H4	Me	6	4m	207–208	23
14	3,4-(MeO)3–C6H3	Et	6	4n	207–208	19
15	3,4,5-(MeO)3–C6H2	Me	8	4o	187–188	14
16	3-OH-4-MeO–C6H3	Me	6	4p	230–231	28

---

Fig. 2 (a) HMBC of 4g. (b) Locally amplified HMBC of 4g.

The formation of isomeric systems (ortho- and para-quinone units) is possible in the reaction. So, we considered it desirable to obtain independent chemical evidence for the presence of ortho- or para-quinone units in 4. To this end, we reacted 4g with o-phenylenediamine for 30 min under solvent-free conditions, affording compound 5 in 90% yield, confirming the ortho-quinone structure (Scheme 2). The structure of 5 was fully characterized by spectroscopic data and analytical methods, the H-12 and H-15 occur as a multiplet at 8.83–9.34 ppm, more downfield than expected of aromatic protons. This is explicable by the close proximity of these protons to the lone pairs of the neighbouring nitrogens and the consequent anisotropic and van der Waals deshielding (the local steric van der Waals potential leads to a marked deshielding effect). The lack of any carbonyl signal and the presence of two imine carbon signals at 150.3 and 141.6 ppm in ¹³C NMR spectrum of 5, and the fact that 5 is formed by the reaction of one molecule of 4g with one molecule of o-phenylenediamine clearly support the structure of 5, which, in turn, further corroborates the structure of 4 and the regiochemistry of its formation.

Scheme 2

A suggested pathway for the formation of the hybrid is shown in Scheme 3. 2-Hydroxy-1,4-naphthoquinone (1) undergoes Knoevenagel condensation with aldehyde (2) to form olefin (6). The olefin have high reactivity, which can react with varied amines by Michael-type addition to produce the corresponding β-amino derivatives. As a result, subsequent Michael-type addition of 5-substituted-2-amino-1,3,4-thiadiazole 3 to the olefin followed by intramolecular nucleophilic cyclization and dehydration affords the desired product 4.

Scheme 3

To evaluate their antitumor potential, the newly synthesized hybrids 4a–p were subjected to in vitro biological assessment against two human cancer cell lines, HCT116 and HepG2. The results of the cytotoxicity evaluation, as compared to the anticancer reference compound Taxol, were summarized in Table 3. As evidenced by these results, the majority of the derivatives exhibited at least moderate cytotoxic activity against the HCT116 and HepG2 cell lines. Nine of the new hybrids (4a, 4b, 4g, 4j, 4k, 4m, 4n, 4o and 4p) even display a considerable activity profile with IC₅₀ values below 5.0 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-5 position have substantial influence on the antitumor activity, introduction of 3-methylphenyl, 3-methoxyphenyl or 3-hydroxyphenyl group into the C-5 position was found to be quite favorable for increasing antitumor activity. Among this series, compound 4m showed the best antitumor activity with IC₅₀ values of 3.57 μM and 3.57 μM against HCT116 and HepG2 cell lines, respectively, which was less than Taxol. Second, the ortho-quinone moiety appeared to have an important effect upon cytotoxicity, compounds 5 was less potent cytotoxicity, than the corresponding analogues with an ortho-quinone moiety.

Table 3 Antitumor activities of substituted 5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-diones

Compounds	IC50^a (μM)
	HCT116	HepG2	L02
a The means of triplicates ± SD.
4a	5.05 ± 1.39	4.59 ± 0.52	9.56 ± 0.72
4b	4.82 ± 0.46	4.01 ± 0.56	8.05 ± 0.62
4c	8.65 ± 1.96	5.87 ± 1.47	13.25 ± 1.95
4d	8.48 ± 2.83	8.65 ± 1.22	20.10 ± 2.30
4e	5.83 ± 0.27	6.8 ± 0.62	12.02 ± 0.54
4f	9.25 ± 1.96	11.35 ± 4.66	21.20 ± 3.46
4g	3.88 ± 0.54	3.6 ± 0.65	5.98 ± 0.65
4h	7.12 ± 0.65	4.00 ± 0.18	9.56 ± 1.45
4i	6.97 ± 0.59	4.19 ± 0.53	16.23 ± 0.88
4j	3.84 ± 0.5	4.33 ± 0.63	7.56 ± 0.85
4k	4.44 ± 0.25	4.02 ± 0.29	9.66 ± 0.68
4l	5.07 ± 0.68	5.42 ± 1.09	11.26 ± 0.98
4m	3.57 ± 0.17	3.39 ± 0.43	5.36 ± 0.42
4n	4.40 ± 0.34	4.53 ± 0.95	10.23 ± 1.68
4o	3.65 ± 0.42	3.61 ± 0.61	5.78 ± 0.74
4p	3.48 ± 0.18	3.43 ± 0.38	6.01 ± 0.29
5	>200	>200	>200
Taxol	3.76 ± 0.45	19.26 ± 2.53	25.62 ± 2.12

---

Conclusion

In conclusion, the efficient and straightforward preparation of a series of 1,2-naphthoquinone derivatives possessing 1,3,4-thiadiazolo[3,2-a]pyrimidine scaffolds was described, using a one-pot multicomponent approach employing 2-hydroxy-1,4-naphthoquinone, aldehydes, and 5-substituted-2-amino-1,3,4-thiadiazole. Subsequent biological assessment pointed out the relevance of a number of these novel scaffolds in terms of their cytotoxic activity, in general all the tested compounds 4a–4p possessed good deal of antitumor activity against HCT116 and HepG2 cell lines as compared to the standard drugs. On the whole among all the compounds tested, compound 4m exhibited showed the best antitumor activity against the two human cell lines. Therefore, these novel 1,2-naphthoquinones fused with bioactive heterocyclic skeletons may find their pharmaceutical applications after further investigations.

Experimental

General

UV-vis were determined on TU-1950 UV/Vis spectrophotometer. 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-Q III 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 DMF 10 mL, 5-substituted-2-amino-1,3,4-thiadiazole (0.1 mmol) was added. The mixture was stirred at 130 °C for an appropriate time (Table 2). After completion of the reaction (TLC), the reaction mixture was cooled to room temperature and added to the ice water. The precipitate was collected by filtration and the crude product purified by silica gel column chromatography using petroleum ether : ethyl acetate (v/v = 2 : 1) as eluent to afford the pure product 4.

5-Heptyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4a). Red brown powder, mp 164–166 °C; UV-vis (MeOH) λ_max: 552, 498, 397, 381, 376, 362, 356, 317, 267 nm (ε_max: 91 752, 76 903, 89 161, 86 860, 80 564, 81 713, 93 290, 141 435, 80 200 L mol⁻¹ cm⁻¹); IR (KBr): v 3073, 2922, 2852, 1688, 1609, 1535, 1459, 1426, 1401, 1377, 1285, 1247, 1138, 1094, 9858, 896, 851, 778, 729, 563 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.41 (d, 1H, J = 2.0 Hz), 8.27 (d, 1H, J = 7.6 Hz), 8.11 (d, 1H, J = 7.6 Hz), 7.70–7.66 (m, 1H), 7.55 (t, 1H, J = 7.6 Hz), 5.88 (t, 1H, J = 4.0 Hz), 1.98–1.92 (m, 2H), 1.23–1.18 (m, 10H), 0.83 (t, 3H, J = 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 179.9, 176.2, 167.3, 154.2, 141.5, 134.6, 134.4, 130.9, 130.8, 128.7, 126.7, 110.5, 57.7, 34.6, 31.7, 29.2, 29.1, 23.8, 22.5, 14.1; HRMS-ESI (m/z): calcd for C₂₀H₂₁N₃O₂S [M + Na]⁺: 390.1252, found: 390.1229.

5-(3,5-Dimethoxyphenyl)-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4b). Red brown powder, mp 178–180 °C; UV-vis (MeOH) λ_max: 490, 381, 374, 371, 365, 362, 346, 335, 316, 292, 284, 270, 267, 264, 247, 245, 232 nm (ε_max: 27 694, 61 959, 65 510, 70 632, 69 530, 74 449, 95 326, 115 551, 155 857, 90 490, 90 429, 88 469, 88 388, 88 245, 89 592, 91 041, 90 307 L mol⁻¹ cm⁻¹); IR (KBr): v 3062, 2927, 2837, 1685, 1609, 1537, 1460, 1428, 1375, 1156, 1091, 723 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.32 (d, 1H, J = 6.0 Hz), 8.08 (d, 1H, J = 7.2 Hz), 7.70 (t, 1H, J = 7.6 Hz), 7.55 (d, 1H, J = 7.2 Hz), 6.60 (d, 4H, J = 26 Hz), 6.36 (s, 1H), 3.75 (s, 6H); ¹³C NMR (100 MH, CDCl₃) δ: 179.6, 176.1, 166.3, 161.1, 152.4, 142.3, 142.1, 134.7, 134.4, 131.1, 130.9, 128.9, 126.9, 111.4, 107.2, 106.1, 105.8, 100.4, 60.5, 55.4; HRMS-ESI (m/z): calcd for C₂₁H₁₅N₃O₄S [M + Na]⁺: 428.0681, found: 428.0669.

5-Phenyl-2-ethyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4c). Red brown powder, mp 177–179 °C; UV-vis (MeOH) λ_max: 557, 547, 405, 396, 362, 357, 346, 343, 337, 316, 247, 242 nm (ε_max: 75 166, 85 740, 76 443, 87 017, 77 740, 93 277, 105 036, 105 018, 104 980, 141 425, 81 277, 79 836 L mol⁻¹ cm⁻¹); IR (KBr): v 2923, 1688, 1623, 1544, 1465, 1439, 1376, 1234, 1158, 1089, 961, 779, 729, 700 cm⁻¹; ¹N NMR (400 MHz, CDCl₃) δ: 8.32 (d, 1H, J = 7.6 Hz), 8.09 (dd, 2H, J = 7.6, 7.6 Hz), 7.67 (t, 1H, J = 7.2 Hz), 7.54–7.47 (m, 1H), 7.41 (d, 2H, J = 7.2 Hz), 7.29 (t, 2H, J = 7.6 Hz), 6.60 (s, 1H), 2.83 (dd, 2H, J = 13.6, 14.0 Hz), 1.32 (t, 3H, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 179.7, 175.8, 167.5, 160.5, 152.5, 140.2, 134.6, 134.5, 133.6, 131.1, 130.9, 130.2, 128.8, 128.7, 128.5, 127.4, 126.8, 111.6, 60.3, 24.8, 12.7; HRMS-ESI (m/z): calcd for C₂₁H₁₅N₃O₂S [M + H]⁺: 374.0963, found: 374.0962.

5-Phenyl-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4d). Red brown powder, mp 216–218 °C; UV-vis (MeOH) λ_max: 527, 376, 316, 309, 265, 258, 247 nm (ε_max: 21 392, 31 977, 35 298, 35 391, 74 497, 77 765, 78 354 L mol⁻¹ cm⁻¹); IR (KBr): v 2924, 2836, 1688, 1624, 1545, 1489, 1462, 1374, 1234, 1190, 1080, 773, 729 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.36 (d, 1H, J = 7.6 Hz), 8.12 (dd, 2H, J = 12.4, 12.8 Hz), 7.72 (t, 1H, J = 7.2 Hz), 7.62 (d, 1H, J = 7.2), 7.58–7.42 (m, 2H), 7.32 (dd, 2H, J = 14.0, 14.8 Hz), 6.64 (s, 1H), 2.53 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 179.7, 176.0, 171.2, 167.8, 154.2, 152.4, 140.3, 134.6, 133.7, 131.2, 130.9, 130.2, 129.3, 128.8, 128.5, 127.5, 126.8, 111.7, 60.4, 17.0; HRMS-ESI (m/z): calcd for C₂₀H₁₃N₃O₂S [M + Na]⁺: 382.0626, found: 382.0614.

5-(3,5-Dimethoxyphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4e). Red brown powder, mp 200–201 °C; UV-vis (MeOH) λ_max: 533, 394, 389, 381, 376, 362, 323, 295 nm (ε_max: 78 708, 114 332, 116 666, 111 479, 106 582, 100 333, 134 312, 92 395 L mol⁻¹ cm⁻¹); IR (KBr): v 2929, 2831, 1690, 1612, 1597, 1541, 1490, 1462, 1437, 1372, 1296, 1230, 1201, 1157, 1090, 1052, 966, 837, 721, 696, 534 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.29 (d, 1H, J = 7.2 Hz), 8.06 (d, 1H. J = 6.8 Hz), 7.66 (d, 1H, J = 6.4 Hz), 7.52 (d, 1H. J = 6.4 Hz), 6.53 (d, 3H, J = 15.6 Hz), 6.35 (s, 1H), 3.75 (s, 6H), 2.53 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 179.7, 175.9, 167.7, 161.0, 154.2, 152.4, 142.5, 134.6, 131.1, 128.7, 126.8, 111.5, 105.8, 100.3, 60.2, 55.4, 17.0; HRMS-ESI (m/z): calcd for C₂₂H₁₇N₃O₄S [M + Na]⁺: 442.0837, found: 442.0830.

5-(3-Methoxyphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4f). Red brown powder, mp 192–194 °C; UV-vis (MeOH) λ_max: 531, 498, 400, 397, 381, 376, 362, 353, 334, 331, 329, 286 nm (ε_max: 100 058, 84 862, 85 921, 113 960, 118 414, 92 666, 93 646, 98 764, 111 018, 116 999, 117 000, 86 881 L mol⁻¹ cm⁻¹); IR (KBr): v 2929, 2820, 1691, 1617, 1548, 1459, 1441, 1373, 1274, 1234, 1159, 1090, 1041, 964, 774, 732, 532 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.34 (d, 1H, J = 7.6 Hz), 8.09 (t, 1H, J = 6.8 Hz), 7.73–7.68 (m, 1H), 7.55 (dd, 1H, J = 7.6, 7.6 Hz), 7.24 (dd, 1H, J = 16.0, 20.4 Hz), 6.99 (t, 2H, J = 4.8 Hz), 6.82 (dd, 1H, J = 8.0, 8.4 Hz), 6.60 (s, 1H), 3.80 (s, 3H), 2.54 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 179.7, 176.0, 167.7, 159.8, 154.2, 152.3, 141.7, 134.6, 134.5, 131.1, 130.8, 129.8, 128.8, 126.8, 119.6, 114.1, 113.5, 111.6, 60.2, 55.3, 17.0; HRMS-ESI (m/z): calcd for C₂₁H₁₅N₃O₃S [M + Na]⁺: 412.0732, found: 412.0732.

5-(3-Methylphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4g). Red brown powder, mp 269–270 °C; UV-vis (MeOH) λ_max: 529, 376, 318, 274, 265, 247, 231 nm (ε_max: 26 240, 39 054, 44 146, 69 553, 77 275, 81 294, 81 998 L mol⁻¹ cm⁻¹); IR (KBr): v 2971, 2923, 1690, 1624, 1540, 1463, 1441, 1374, 1236, 1158, 813, 769, 727 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.36 (d, 1H, J = 7.6 Hz), 8.09 (d, 1H, J = 7.6 Hz), 7.71 (t, 1H, J = 7.6 Hz), 7.55 (t, 1H, J = 7.2 Hz), 7.30 (t, 2H, J = 8.0 Hz), 7.12 (d, 2H, J = 8.0 Hz), 6.60 (s, 1H), 2.53 (s, 3H), 2.30 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 179.6, 175.9, 167.5, 154.3, 152.1, 138.8, 137.4, 134.6, 134.4, 131.1, 130.9, 130.2, 129.5, 129.2, 128.8, 127.4, 126.7, 111.9, 60.2, 21.2, 17.0; HRMS-ESI (m/z): calcd for C₂₁H₁₅N₃O₂S [M + Na]⁺: 396.0783, found: 396.0777.

5-(3,5-Dimethoxyphenyl)-2-ethyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4h). Red brown powder, mp 168–170 °C; UV-vis (MeOH) λ_max: 548, 454, 425, 422, 412, 397, 376, 362, 319, 299, 275, 255 nm (ε_max: 107 695, 92 412, 71 521, 77 716, 94 650, 102 178, 89 786, 90 829, 166 050, 96 481, 94 260, 95 416 L mol⁻¹ cm⁻¹); IR (KBr): v 2935, 2836, 1689, 1608, 1542, 1461, 1439, 1373, 1227, 1156, 723 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.33 (d, 1H, J = 7.6 Hz), 8.09 (d, 1H, J = 7.6 Hz), 7.71 (dd, 1H, J = 7.6, 14.0 Hz), 7.54 (t, 1H, J = 7.6 Hz), 6.56 (d, 3H, J = 6.0 Hz), 6.37 (s, 1H), 3.77 (s, 6H), 2.87 (dd, 2H, J = 7.2, 7.6 Hz), 1.35 (t, 3H, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 179.8, 176.0, 167.4, 161.0, 160.3, 152.4, 142.4, 134.6, 131.1, 130.9, 128.7, 126.8, 111.4, 105.8, 100.3, 60.2, 55.4, 24.8, 12.7; HRMS-ESI (m/z): calcd for C₂₃H₁₉N₃O₄S [M + Na]⁺: 456.0994, found: 456.0986.

5-(3-Methoxyphenyl)-2-ethyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4i). Red brown powder, mp 196–197 °C; UV-vis (MeOH) λ_max: 493, 381, 379, 374, 371, 366, 361, 350, 337, 328, 325, 322, 299, 263 nm (ε_max: 25 820, 67 360, 65 760, 71 060, 76 680, 75 360, 82 263, 88 380, 100 620, 119 340, 119 300, 128 940, 88 740, 87 260 L mol⁻¹ cm⁻¹); IR (KBr): v 3061, 2976, 2935, 1688, 1624, 1544, 1469, 1412, 1376, 1233, 1160, 1056, 962, 773, 729 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.32 (d, 1H, J = 7.6 Hz), 8.06 (d, 1H, J = 7.6 Hz), 7.67 (t, 1H, J = 7.2 Hz), 7.52 (t, 1H, J = 7.2 Hz), 7.22 (dd, 2H, J = 15.6, 27.2 Hz), 6.97 (t, 2H, J = 5.2 Hz), 6.58 (s, 1H), 3.78 (s, 3H), 2.84 (dd, 2H, J = 7.2, 15.2 Hz), 1.31 (t, 3H, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 179.7, 175.9, 167.5, 160.4, 159.8, 152.5, 141.7, 134.6, 131.1, 130.9, 129.8, 128.7, 127.2, 126.8, 119.6, 114.0, 113.5, 111.5, 60.2, 55.3, 24.8, 12.7; HRMS-ESI (m/z): calcd for C₂₂H₁₇N₃O₃S [M + Na]⁺: 426.0888, found: 426.0883.

5-(3-Hydroxyphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4j). Red brown powder, mp 255–257 °C; UV-vis (MeOH) λ_max: 562, 539, 508, 407, 397, 381, 376, 362, 326, 232 nm (ε_max: 99 113, 103 415, 93 696, 103 641, 188 672, 188 670, 105 603, 111 207, 121 641, 83 490 L mol⁻¹ cm⁻¹); IR (KBr): v 3264, 2972, 2933, 1687, 1595, 1528, 1460, 1429, 1378, 1278, 1230, 1163, 1089, 776 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.50 (s, 1H), 8.32 (d, 1H, J = 7.6 Hz), 7.95 (d, 1H, J = 7.6 Hz), 7.83–7.64 (m, 2H), 6.80–6.77 (m, 1H), 6.68–6.62 (m, 4H), 6.38 (m, 1H), 2.54 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.7, 175.4, 167.8, 158.0, 156.4, 152.1, 142.5, 135.3, 134.6, 131.6, 131.3, 130.2, 128.5, 126.9, 118.6, 116.0, 114.6, 111.5, 60.1, 17.1; HRMS-ESI (m/z): calcd for C₂₀H₁₃N₃O₃S [M + Na]⁺: 398.0575, found: 398.0575.

5-(3-Hydroxyphenyl)-2-ethyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4k). Red brown powder, mp 207–208 °C; UV-vis (MeOH) λ_max: 490, 392, 389, 387, 381, 376, 362, 348, 316, 299, 275 nm (ε_max: 34 823, 72 490, 79 568, 79 562, 95 490, 92 687, 94 931, 98 726, 149 764, 87 000, 85 058 L mol⁻¹ cm⁻¹); IR (KBr): v 3252, 2972, 2935, 1690, 1608, 1586, 1539, 1488, 1460, 1437, 1380, 1277, 1239, 1162, 1090, 782, 729 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.50 (s, 1H), 8.28 (d, 1H, J = 7.6 Hz), 7.94–7.77 (m, 3H), 7.12 (t, 1H, J = 8.0 Hz), 6.79–6.65 (m, 3H), 6.35 (s, 1H), 2.90 (dd, 2H, J = 7.2, 14.4 Hz), 1.21 (t, 3H, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.7, 175.3, 167.5, 161.8, 158.0, 152.0, 142.3, 135.2, 134.5, 131.5, 131.3, 130.2, 128.4, 126.8, 118.5, 116.3, 114.6, 111.4, 60.1, 24.5, 12.9; HRMS-ESI (m/z): calcd for C₂₀H₁₅N₃O₃S [M + Na]⁺: 412.0733, found: 412.0734.

5-(2-Bromo-5-hydroxyphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4l). Red brown powder, mp 230–231 °C; UV-vis (MeOH) λ_max: 498, 371, 366, 316, 299, 292, 286, 275, 232 nm (ε_max: 29 977, 58 681, 59 250, 173 568, 100 818, 99 818, 100 727, 98 568, 100 568 L mol⁻¹ cm⁻¹); IR (KBr): v 3405, 2970, 2926, 1649, 1606, 1536, 1461, 1437, 1390, 1369, 1279, 1235, 1160, 967, 726, 591 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.74 (s, 1H), 8.34 (d, 1H, J = 7.6 Hz), 7.96 (d, 1H, J = 7.2 Hz), 7.83 (t, 1H, J = 7.2 Hz), 7.69–7.65 (m, 1H), 7.36 (d, 1H, J = 8.8 Hz), 6.86 (s, 1H), 6.73 (s, 1H), 6.66–6.63 (m, 1H), 2.53 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.6, 175.2, 168.0, 1666.6, 157.8, 156.5, 156.3, 152.5, 141.0, 135.3, 134.5, 131.7, 131.3, 128.4, 127.0, 118.4, 113.1, 109.4, 60.4, 17.1; HRMS-ESI (m/z): calcd for C₂₀H₁₂BrN₃O₃S [M + Na]⁺: 475.9680, found: 475.9673.

5-(3-Methylphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4m). Red brown powder, mp 207–209 °C; UV-vis (MeOH) λ_max: 527, 375, 264, 254 nm (ε_max: 21 367, 35 037, 91 112, 93 259 L mol⁻¹ cm⁻¹); IR (KBr): v 3060, 2970, 2921, 1686, 1624, 1543, 1490, 1463, 1443, 1374, 1297, 1226, 1160, 1089, 729, 535 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.37 (d, 1H, J = 8.0 Hz), 8.10 (d, 1H, J = 7.6 Hz), 7.72 (t, 1H, J = 7.6 Hz), 7.56 (t, 1H, J = 7.6 Hz), 7.29–7.10 (m, 4H), 6.59 (s, 1H), 2.54 (s, 3H), 2.32 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 179.1, 175.9, 167.7, 154.2, 152.2, 140.2, 138.5, 134.6, 134.5, 131.1, 130.9, 129.7, 128.8, 128.7, 128.2, 126.7, 124.5, 111.8, 60.4, 21.5, 17.0; HRMS-ESI (m/z): calcd for C₂₁H₁₅N₃O₂S [M + Na]⁺: 396.0783, found: 396.0773.

5-(3,4-Dimethoxyphenyl)-2-ethyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4n). Red brown powder, mp 207–208 °C; UV-vis (MeOH) λ_max: 532, 371, 312, 253, 251, 248 nm (ε_max: 22 195, 30 001, 43 413, 102 608, 105 869, 105 899 L mol⁻¹ cm⁻¹); IR (KBr): v 2936, 2836, 1687, 1623, 1545, 1463, 1442, 1376, 1265, 1141, 1022, 779, 719 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.35 (d, 1H, J = 7.6 Hz), 8.10 (d, 1H, J = 7.6 Hz), 7.73–7.54 (m, 2H), 7.04 (d, 1H, J = 0.8 Hz), 6.88 (d, 1H, J = 8.4 Hz), 6.78 (d, 1H, J = 8.4 Hz), 6.59 (s, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 2.88 (dd, 2H, J = 10.8, 13.2 Hz), 1.35 (t, 3H, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 179.8, 176.0, 167.3, 160.3, 152.4, 149.3, 149.0, 134.6, 134.5, 133.1, 131.1, 130.9, 128.8, 126.7, 119.5, 111.7, 111.2, 111.1, 60.0, 56.0, 55.9, 24.8, 12.7; HRMS-ESI (m/z): calcd for C₂₃H₁₉N₃O₄S [M + Na]⁺: 456.0994, found: 456.0993.

5-(3,4,5-Trimethoxyphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-6,7-dione (4o). Red brown powder, mp 187–188 °C; UV-vis (MeOH) λ_max: 534, 424, 408, 397, 375, 362, 354, 317, 314, 300, 286, 262 nm (ε_max: 75 560, 55 700, 97 940, 196 071, 102 321, 104 726, 100 763, 152 763, 152 751, 88 742, 88 646, 87 245 L mol⁻¹ cm⁻¹); IR (KBr): v 2969, 2929, 1686, 1625, 1590, 1505, 1490, 1439, 1374, 1328, 1231, 1098, 1034, 764, 731 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 8.36 (d, 1H, J = 7.6 Hz), 8.11 (d, 1H, J = 7.2 Hz), 7.74–7.55 (m, 2H), 6.63 (s, 2H), 6.56 (s, 1H), 3.81 (s, 6H), 3.80 (s, 3H), 2.58 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 179.6, 176.1, 167.7, 154.2, 153.5, 152.2, 138.4, 135.9, 134.7, 134.4, 131.1, 131.0, 128.9, 128.8, 126.9, 126.8, 111.6, 104.7, 103.8, 60.8, 56.3, 29.7, 17.1; HRMS-ESI (m/z): calcd for C₂₃H₁₉N₃O₅S [M + H]⁺: 450.1124, found: 450.1127.

5-(3-Hydroxy-4-methoxyphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-7,8-dione (4p). Mp 230–231 °C; UV-vis (MeOH) λ_max: 527, 371, 366, 317, 314, 297, 284, 271 nm (ε_max: 40 821, 64 271, 63 943, 89 298, 92 923, 96 163, 108 621, 106 643 L mol⁻¹ cm⁻¹); IR (KBr): v 3443, 2971, 2926, 1687, 1621, 1542, 1461, 1421, 1377, 1280, 1210, 1160, 1090, 946, 782, 761 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.10 (s, 1H), 8.30 (d, 1H, J = 8.0 Hz), 7.94 (d, 1H, J = 7.6 Hz), 7.81 (d, 1H, J = 7.6 Hz), 7.65 (d, 1H, J = 7.6 Hz), 6.86–6.76 (m, 3H), 6.31 (m, 1H), 3.71 (s, 3H), 2.54 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.7, 175.4, 167.6, 156.2, 151.9, 148.4, 147.0, 135.2, 134.6, 1333.8, 131.5, 131.3, 128.4, 126.8, 119.0, 114.9, 112.5, 111.6, 59.8, 56.0, 17.1; HRMS-ESI (m/z): calcd for C₂₁H₁₅N₃O₄S [M + Na]⁺: 428.0681, found: 428.0686.

Typical procedure for the synthesis of compounds 5

A mixture of 5-(3-methylphenyl)-2-methyl-5H-benzo[i][1,3,4]thiadiazolo[3,2-a]quinazoline-7,8-dione (1 mmol) and o-phenylenediamine (1.5 mmol) was heated at 110 °C for 30 min. The reaction mixture was then cooled to room temperature and diluted with cold water (40 mL). The solid product was collected by filtration and was purified by recrystallization from 95% EtOH to afford the desired pure products 5 as an orange red powder, mp 222–223 °C; UV-vis (MeOH) λ_max: 453, 334, 312, 309, 270, 266, 259, 247, 195 nm (ε_max: 428 777, 50 533, 47 778, 48 244, 85 955, 83 778, 86 111, 89 511 L mol⁻¹ cm⁻¹); IR (KBr): v 3051, 2921, 2954, 1598, 1539, 1512, 1489, 1412, 1350, 1145, 1058, 736, 678, 602 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ: 9.34 (d, 1H, J = 7.6 Hz), 8.83 (d, 1H, J = 7.6 Hz), 8.23 (t, 1H, J = 7.2 Hz), 8.13 (t, 1H, J = 1.6 Hz), 7.89–7.71 (m, 4H), 7.53 (t, 3H, J = 11.2 Hz), 7.05 (d, 2H, J = 8.0 Hz), 2.48 (s, 3H), 2.23 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 164.2, 150.4, 150.3, 142.4, 141.6, 141.5, 140.4, 139.5, 137.8, 135.8, 131.3, 129.9, 129.5, 129.4, 129.0, 128.9, 128.5, 128.1, 127.8, 125.1, 124.8, 111.3, 60.7, 21.2, 17.1; HRMS-ESI (m/z): calcd for C₂₇H₁₉N₅S [M + H]⁺: 446.1439, found: 446.1427.

Antitumor assay

HCT116 cells and HepG2 cells were maintained in DMEM (Gibco, Invitrogen Corporation, NY, USA) medium supplemented with 10% FBS, streptomycin (100 μg mL⁻¹) and penicillin (100 units per mL) and incubated at 37 °C, 5% CO₂. HCT116 cells (5000 cells per well) and HepG2 cells (10 000 cells per well) were seeded into 96-well plates and incubated at 37 °C in 5%CO₂/95% air condition. Serially twofold 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 Scientific Research Fund of Xinxiang Medical University (No. 2013QN130) and Key Scientific Research Projects in Universities of Henan Province (No. 15A350011).

References

1. M. Gallorini, A. Cataldi and V. di Giacomo, BioDrugs, 2012, 26, 377.
2. R. H. Thompson, Naturally Occurring Quinones IV: Recents Advances, Champman & Hall, London, 1997.
3. C. Asche, Mini-Rev. Med. Chem., 2005, 5, 449.
4. E. B. Skibo, C. Xing and R. T. Dorr, J. Med. Chem., 2001, 44, 3545.
5. E. R. de Almeida, A. A. S. Filho, E. R. dos Santos and C. A. C. Lopes, J. Ethnopharmacol., 1990, 29, 239.
6. S. Gafner, J. L. Wolfender, M. Nianga, H. Stoeckli-Evans and K. Hostettmann, Phytochemistry, 1996, 42, 1315.
7. E. N. Silva Jr, R. F. S. Menna-Barreto, M. C. F. R. Pinto, R. S. F. Silva, D. V. Teixeira, M. C. B. V. de Souza, C. A. Simone, S. L. de Castro, V. F. Ferreira and A. V. Pinto, Eur. J. Med. Chem., 2008, 43, 1774.
8. J. P. Kim, W. G. Kim, H. Koshino, J. Jung and I. Yoo, Phytochemistry, 1996, 43, 425.
9. C. O. Salas, M. Faúndez, A. Morello, J. D. Maya and R. A. Tapia, Curr. Med. Chem., 2011, 18, 144.
10. V. K. Tandon, R. V. Singh and D. B. Yadav, Bioorg. Med. Chem. Lett., 2004, 14, 2901.
11. M. Dubin, S. H. Fernandez Villamil and A. O. Stoppani, Medicina, 2001, 61, 343.
12. L. Zhou, W. K. Chan, N. Xu, K. Xiao, H. Luo, K. Q. Luo and D. C. Chang, Life Sci., 2008, 83, 394.
13. M. E. Dolan, B. Frydman, C. B. Thompson, A. M. Dlamond, B. J. Garblras, A. R. Safe, W. T. Beck and L. J. Marton, Anticancer Drugs, 1998, 9, 437.
14. D. Wang, M. Y. Xia, Z. Cui, S. I. Tashiro, S. Onodera and T. Ikejima, Biol. Pharm. Bull., 2004, 27, 1025.
15. J. S. Zhang, J. Ding, Q. M. Tang, M. Li, M. Zhao, L. J. Lu, L. J. Chen and S. T. Yuan, Bioorg. Med. Chem. Lett., 1999, 9, 2731.
16. J. Bian, B. Deng, L. Xu, X. Xu, N. Wang, T. Hu, Z. Yao, J. Du, L. Yang, H. Sun, X. Zhang and Q. You, Eur. J. Med. Chem., 2014, 82, 56.
17. L. S. Li, E. A. Bey, Y. Dong, J. Meng, B. Patra, J. Yan, X. J. Xie, R. A. Brekken, C. C. Barnett, W. G. Bornmann, J. Gao and D. A. Boothman, Clin. Cancer Res., 2011, 17, 275.
18. N. S. El-Sayed, E. R. El-Bendary, S. M. El-Ashry and M. M. El-Kerdawy, Eur. J. Med. Chem., 2011, 46, 3714.
19. A. K. Gadad, C. S. Mahajanshetti, S. Nimbalkar and A. Raichurkar, Eur. J. Med. Chem., 2000, 35, 853.
20. S. S. Shukurov, M. A. Kukaniev, I. M. Nasyrov, K. S. Zakharov and R. A. Karakhanov, Russ. Chem. Bull., 1993, 11, 1871.
21. J. A. Montgomery, Med. Res. Rev., 1982, 2, 271.
22. S. J. Xue, L. P. Duan, S. Y. Ke, J. Z. Li and Y. L. Guo, Chin. J. Struct. Chem., 2005, 24, 703.
23. L. Q. Wu, C. Zhang and W. L. Li, Bioorg. Med. Chem. Lett., 2013, 23, 5002.
24. L. Q. Wu, C. Zhang and W. L. Li, Bioorg. Med. Chem. Lett., 2014, 14, 1462.
25. X. J. Yang, C. Zhang and L. Q. Wu, RSC Adv., 2015, 5, 18945.
26. X. J. Yang, C. Zhang and L. Q. Wu, RSC Adv., 2015, 5, 25115.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03323g

---