Design, Synthesis, and Biological Evaluation of Triazolyl- and Triazinyl-Quinazolinediones as Potential Antitumor Agents

Al-Romaizan, Abeer N.; Ahmed, Nesreen S.; Elfeky, Sherin M.

doi:https://doi.org/10.1155/2019/9104653

Journal of Chemistry

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Abstract Introduction Experimental Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2019 | Article ID 9104653 | https://doi.org/10.1155/2019/9104653

Design, Synthesis, and Biological Evaluation of Triazolyl- and Triazinyl-Quinazolinediones as Potential Antitumor Agents

Abeer N. Al-Romaizan,¹Nesreen S. Ahmed,^1,2and Sherin M. Elfeky^1,3

Academic Editor: Zhong-Wen Liu

Received22 Oct 2018

Revised12 Dec 2018

Accepted25 Dec 2018

Published03 Feb 2019

Abstract

Novel 6(3-1H-1,2,4-triazol-1-yl)-3-phenylquinazoline-2,4(1H,3H)-diones (7a–e) were synthesized from different enaminones (6a–e) with 6-hydrazinyl-3-phenylquinazoline-2,4(1H,3H)-dione. 2,6(4-2-Substituted-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-diones (8a–k) were synthesized from the reaction of 1-(2,4-dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)thiourea, urea, or guanidine (3a–c) with enaminones (6a–e), and a series from 3-substituted-2-imino-1,3,5-triazin-1(2H)-yl-sulfonyl-phenyl-1-methylquinazoline-2,4(1H,3H)-dione (12a–j) were obtained from the reaction of N-(diaminomethylene)-4-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)benzenesulfonamide (11) with the enaminone (6a–j). The antitumor activity of the synthesized compounds was evaluated against two human cell lines: human colon carcinoma HCT116 and human hepatocellular carcinoma HEP-G2. Some of the tested compounds showed significant potency compared to the reference drug staurosporin.

1. Introduction

Quinazolines are such interesting scaffolds that cover a wide range of biological activity depending on the nature and position of substituents and are reported to act as an antidepressant, antipsychotic, sedative, analgesic, antibacterial, anti-inflammatory, and others [1]. There are different classes of quinazolinones depending on the position of substituents: 2-substituted-4(3H)-quinazolinones, 3-substituted-4(3H)-quinazolinones, 4-substituted quinazolinones, 2,3-disubstituted-4(3H)-quinazolinones, and 2,4-disubstituted-4(3H)-quinazolinones. Depending on the position of the oxo group, these quinazolinones can be further classified into three main groups: 2(1H)-quinazolinones, 4(3H)-quinazolinones, and 2,4(1H,3H)-quinazolinones. They differ in their synthetic approaches and in their starting materials [2]. Quinazolinones were reported to act as antitumor agents through different mechanisms. Methoxylated-2-benzyl thioquinazoline-4(3H) ones were reported to act as antitumor agents through targeting dihydrofolate reductase enzyme (DHFR) [3]. A series of 2[(3-substituted-4(3H)-quinazoline-2-yl-thio]N-(3,4,5)trimethoxy phenyl)acetamides were reported to act as antitumor agents against a number of cancer cell lines [4]. Also a series of 6-methyl-2-thioxoquinazoline-4(1H)-ones were reported to act as antitumor candidates through targeting and inhibiting the epidermal growth factor receptor (EGFR) [5]. EGFR is a transmembrane protein that is closely related to the receptor tyrosine kinase. Both play a significant role in cell proliferation differentiation, survival, and metabolism. Overexpression of EGFR is associated with the development of various tumors where 30% of breast cancer was reported to have a correlation with overexpression of the growth factor receptor ErbB2. Blocking EGFR binding site would thus prevent EGFR expressing tumors [6, 7]. Erlotinib is an antitumor agent that is used for nonsmall cell lung cancer and pancreatic cancer, and it is reported that it acts through the inhibition of EGFR [8]. Gefitinib is a marketed antitumor agent that acts through inhibiting EGFR as well [9]. Both erlotinib and gefitinib (Figure 1) belong to a class of EGFR inhibitors that are 4-anilinoquinazolines [10]. Another class of reported EGFR inhibitors is 2-styryl-4aminoquinazolines (SQ-I) (Figure 1). This class was reported to show potent in vitro antiproliferative activity against different cell lines compared to gefitinib [11]. In the present work, new compounds of quinazoline-2,4(1H,3H)-diones were designed through introducing substituted N-containing ring systems: triazole or 2-oxo-, 2-thia-, 2-imino-triazine moieties at C6, or introduction of 2- imino-triazine through a phenyl sulfonyl linker at N₃ of the quinazolinone ring (Figure 2). The antitumor activity of the new derivatives was evaluated via the MTT assay.

(a)

(b)

(c)

2. Experimental

2.1. Chemistry

Melting points were recorded using Stuart melting apparatus. IR spectra (KBr) were recorded on an FT-IR spectrometer (ν_max/cm⁻¹). Nuclear magnetic resonance (¹H NMR and ¹³C NMR) spectra were recorded on Bruker 400 MHz and 300 MHz spectrometer using DMSO-d6 or CDCl₃ as solvents; the chemical shifts are expressed in δ ppm using TMS as an internal standard. Mass spectra were recorded on Shimadzu QP-GC/MS mass spectrometers at the microanalytical unit of Faculty of Science of Cairo University. Elemental microanalyses were carried out, and the results were within ±0.3 from the theoretical values. Solvent evaporation was performed under reduced pressure using Buchi R-3000 Rotacool Rotary Evaporator, and thin layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates (E. Merck, Germany); compounds were detected with 254 nm UV lamp. All chemicals and starting materials were commercial chemicals obtained from Sigma-Aldrich.

2.1.1. Synthesis of 6(3-1H-1,2,4-Triazol-1-yl)-3-phenylquinazoline-2,4(1H,3H)-diones and 6(4-2-Substituted-1,3,5-Triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-diones

(1) Synthesis of 6-Bromo-3-phenylquinazoline-2,4(1H,3H)-dione (1). 5-Bromoisatoic anhydride (1 mmol) reacted with aniline (1 mmol) in ethanol (20 ml) in presence of few drops of acetic acid under ultrasonic irradiation at 80°C for 25 min to afford only one isolable product examined by TLC. The solution was concentrated under suction poured on crushed ice, filtered off, and recrystallized from aqueous ethanol to obtain the desired product in yields of 95% (254–255°C [12], m.p. 249–252°C).

(2) Synthesis of 6-Hydrazinyl-3-phenylquinazoline-2,4(1H,3H)-dione (2). 6-Bromo-3-phenylquinazoline-2,4(1H,3H)-dione (1) (1 mmol) was allowed to reflux in neat hydrazine hydrate (10 ml) for 12 hours, and the reaction was monitored with TLC. The solution was poured on ice and allowed to stir till the hydrazide accumulated and then was filtered off and recrystallized from aqueous ethanol. Yield: 88%, m.p.: 150–152°C, and IR (ν_max/cm⁻¹) 1659 and 1639 (2C=O), 3470, 3389, and 3373 (NH, NH₂); ¹H NMR (DMSO-d₆): δ 8.53 (s, 1H, C₅-H), 7.80 (d, 1H, C₈-H), 7.72 (d, 1H, C₇-H), 7.27 (m, 4H, Ar-H), 7.21 (m, 1H, Ar-H), 6.51 (s, 1H, -NH), and 3.48 (m, 3H, -NHNH₂).

(3) General Procedure for Synthesis of 3a–c. 6-Bromo-3-phenylquinazoline-2,4(1H,3H)-dione (1) (1 mmol) was allowed to reflux with urea, thiourea, or guanidine (1 mmol) in ethanol (20 ml) for 12 hours. The reaction was monitored by TLC until no starting materials were present. The precipitate formed on cooling was filtered off and recrystallized from aqueous ethanol in yields of 90–92%. The following compounds were prepared: 1-(2,4-Dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)urea ( 3a ). Yield 92% and m.p.:130–132°C; ¹H NMR (DMSO-d₆): δ 9.24 (s, 1H, NH), 8.10 (s, 1H, C₅-H), 7.82 (m, 4H, Ar-H), 7.74 (d, 1H, C₈-H), 7.24 (d, 1H, C₇-H), 6.65 (m, 1H, Ar-H), 5.52 (s, 1H, N₁-H), and 3.67 (s, 2H, NH₂) 1-(2,4-Dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)thiourea ( 3b ). Yield 91% and m.p.: 173–175°C; ¹H NMR (DMSO-d₆): δ 8.75 (s, 1H, NH), 8.53 (s, 1H, C₅-H), 7.81 (d, 1H, C₈-H), 7.73 (d, 1H, C₇-H), 7.22 (m, 2H, Ar-H), 7.14 (m, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 6.96 (s, 1H, N₁-H), and 4.06 (s, 2H, NH₂) 1-(2,4-Dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)guanidine ( 3c ). Yield 90%, m.p.: 160–162°C, and IR (ν_max/cm⁻¹) 1718 and 1679 (C=O), 3423, 3393 and 3383 (NH, NH₂); ¹H NMR (DMSO-d₆): δ 8.61 (s, 1H, NH), 8.40 (s, 1H, C₅-H), 8.04 (d, 1H, C₈-H), 7.86 (d, 1H, C₇-H), 7.52 (m, 3H, Ar-H), 7.45 (m, 1H, Ar-H), 7.00 (s, 1H, N₁-H), 5.35 (s, 2H, NH₂), and 4.04 (s, 1H, NH)

(4) General Procedure for Synthesis of 6a–e. Substituted acetophenones or 2-acetyl thiophene (furan) (1 mmol) were refluxed with DMF-DMA (1.2 mmol) for 8–12 hours in ethanol (20 ml). The reaction was monitored with TLC till no starting materials were detectable. The corresponding enaminones derivatives were afforded in yields of 92–97%. The solutions were evaporated under suction. The precipitates were collected and recrystallized from aqueous ethanol. The following compounds were prepared: (E)-3-(Dimethylamino)-1-(furan-2-yl)prop-2-en-1-one ( 6a ). 92°C [13], yield: 93%, and m.p.: 95–97°C (E)-3-(Dimethylamino)-1-(thiophen-2-yl)prop-2-en-1-one ( 6b ). 147–149°C [14], yield 92%, and m.p.: 140–141°C (E)-3-(Dimethylamino)-1-phenylprop-2-en-1-one ( 6c ). 92°C [15], yield 95%, and m.p.: 93–95°C (E)-3-(Dimethylamino)-1-(4-nitrophenyl)prop-2-en-1-one ( 6d ). 139–141°C [16], yield: 96%, and m.p.: 151–153°C (E)-3-(Dimethylamino)-1-(4-fluorophenyl)prop-2-en-1-one ( 6e ). 94–96°C [16], yield: 97%, and m.p.: 100–101°C

(5) General Procedure for Synthesis of 7a–e. Enaminones (6a–e) (1 mmol) and 6-hydrazinyl-3-phenylquinazoline-2,4(1H,3H)-dione (2) (1 mmol) were refluxed together in ethanol (20 ml) for 24 hours. The reaction was monitored using TLC till termination. The solutions were dried under suction, and the obtained precipitates were recrystallized from petroleum ether in yields of 93–96%. The following compounds were prepared: 6-(3-(Furan-2-yl)-1H-1,2,4-triazol-1-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 7a ). Yield: 95%, m.p.: 154–156°C, and IR (ν_max/cm⁻¹) 1707 and 1638 (2C=O), 3383 (NH); ¹H NMR (DMSO-d₆): δ 10.07 (s, 1H, C-H trizole), 7.70 (m, 3H, C-H quinazoline), 7.30–7.11 (m, 6H, Ar-H), 6.79 (m, 2H, Ar-H), and 6.43 (s, 1H, N-H quinazoline). Ms: [M⁺ 371] consistent with the molecular formula C₂₀H₁₃N₅O₃. 3-Phenyl-6-(3-(thiophen-2-yl)-1H-1,2,4-triazol-1-yl)quinazoline-2,4(1H,3H)-dione ( 7b ). Yield: 94%, m.p.: 160–162°C, and IR (ν_max/cm⁻¹) 1702 and 1637 (2C=O), 3375 (NH); ¹H NMR (DMSO-d₆): δ 10.00 (s, 1H, C-H trizole), 7.70 (d, 3H, C-H quinazoline), 7.01–7.33 (m, 6H, Ar-H), 6.79 (m, 2H, Ar-H), and 6.43 (s, 1H, N-H quinazoline). 3-Phenyl-6-(3-phenyl-1H-1,2,4-triazol-1-yl)quinazoline-2,4(1H,3H)-dione ( 7c ). Yield: 93%, m.p.: 80–82°C, and IR (ν_max/cm⁻¹) 1712 and 1660 (2C=O), 3492 (NH); ¹H NMR (CDCl₃): δ 9.88 (s, 1H, C-H trizole), 8.40 (s, 1H, C₅-H), 7.85 (d, 1H, J = 8.0 Hz, C₈-H), 7.64 (d, 1H, J = 8.1 Hz, C₇-H), 7.62–7.26 (m, 10 H, Ar-H), and 6.38 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 129.5, 129.7, 129.9, 130.1, 133.1, 133.3, 133.5, 134.1, 136.4, 138.2, 146.2, 146.4, 150.0, 152.7, 161.0, and 161.2. 6-(3-(4-Nitrophenyl)-1H-1,2,4-triazol-1-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 7d ). Yield: 96%, m.p.: 177–179°C, and IR (ν_max/cm⁻¹) 1706 and 1677 (2C=O), 3430 (NH); ¹H NMR (CDCl₃): δ 9.80 (s, 1H, C-H triazole), 8.33–8.10 (m, 3H, C-H quinazoline), 8.12 (d, 2H, Ar-H), 7.75 (d, 3H, Ar-H), 7.41 (m, 4H, Ar-H), and 6.38 (s, 1H, N-H quinaozline);¹³C NMR (CDCl₃): δ 115.7, 122.6, 123.4, 124.8, 126.5, 126.9, 127.8, 129.28, 129.3, 129.4, 129.7, 132.3, 133.6, 135.0, 137.2, 140.2, 146.2, 146.4,149.5, 159.7, 161.1, and 162.1. 6-(3-(4-Fluorophenyl)-1H-1,2,4-triazol-1-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 7e ). Yield: 96%, m.p.: 110–112°C, and IR (ν_max/cm⁻¹) 1701 and 1675 (2C=O), 3433 (NH); ¹H NMR (CDCl₃): δ 9.96 (s,1H, C-H triazole), 8.12 (s, 1H, C₅-H), 7.74 (d, 1H, J = 8.3 Hz, C₈-H), 7.56 (d, 1H, J = 8.3 Hz, C₇-H), 7.52–7.42 (m, 6H, Ar-H), 7.41–7.14 (m, 3H, Ar-H), and 6.40 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 115.2, 117.9, 123.4, 124.4, 126.5, 126.9, 128.5, 129.3, 129.7, 131.3, 132.1, 133.9, 133.9, 134.7, 137.1, 138.2, 139.0, 140.0, 146.8, 159.7, 161.2, 162.6, and 171.2.

(6) General Procedure for Synthesis of 8a–k. Enaminones (6a–e) (1 mmol) and 1-(2,4-dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)thiourea, urea, or guanidine (3a–c) (1 mmol) were refluxed in ethanol (20 ml) for 24 hours. The reaction was monitored using TLC till termination. The solutions were dried under suction. The obtained precipitates were recrystallized from petroleum ether to obtain products in yields of 92–97%. The following compounds were prepared: 6-(4-(Furan-2-yl)-2-oxo-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8a ). Yield: 92%, m.p.: 83–85°C, and IR (ν_max/cm⁻¹) 1707, 1688 and 1631 (3C=O), 3432 (NH); ¹H NMR (CDCl₃): δ 9.40 (s, 1H, C-H triazine), 8.09 (s, 1H, C₅-H), 7.82 (d, 1H, J = 8.3 Hz, C₈-H), 7.72 (d, 1H, J = 8.3 Hz, C₇-H), 7.70–7.34 (m, 5H, Ar-H), 6.99 (s, 1H, N-H quinazoline), and 6.64–6.59 (m, 3H, Ar-H); ¹³C NMR (CDCl₃): δ 108.7, 111.2, 112.6, 117.7, 120.7, 122.0, 124.8, 126.7, 129.5, 130.4, 131.9, 132.8, 136.4, 137.7, 144.6, 147.1, 151.2, 153.3, 157.9, 162.8, and 164.5. 6-(2-Oxo-4-(thiophen-2-yl)-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8b ). Yield: 93%, m.p.: 96–98°C, and IR (ν_max/cm⁻¹) 1711, 1682 and 1642 (3C=O), 3454 (NH); ¹H NMR (CDCl₃): δ 9.06 (s, 1H, C-H triazine), 8.53 (s, 1H, C₅-H), 8.20 (d, 1H, J = 8, C₈-H), 7.79 (d, 1H, J = 8.2 Hz., C₇-H), 7.77–7.50 (m, 8H, Ar-H), and 6.77 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 118.3, 126.4, 128.2, 128.4, 129.6, 131.5, 132.5, 134.7, 134.8, 135.3, 135.4, 137.5, 138.9, 142.6, 143.2, 143.7, 150.3, 154.1, 155.4, 167.0, and 169.8. 6-(2-Oxo-4-phenyl-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8c ). Yield: 92%, m.p.: 80–82°C, and IR (ν_max/cm⁻¹)1723, 1656 and 1630 (3C=O), 3431 (NH); ¹H NMR (CDCl₃): δ 9.08 (s, 1H, C-H triazine), 8.53 (s, 1H, C₅-H),7.87 (d, 1H, J = 8.4 Hz, C₈-H), 7.72 (d, 1H, 8.4 Hz., C₇-H),7.71 (m, 2H, Ar-H), 7.65–7.61 (m, 3H, Ar-H), 7.60–7.20 (m, 5H, Ar-H), and 6.19 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 119.9, 124.3,127.3, 127.7, 128.6, 128.9, 129.5, 129.9, 131.3, 132.4, 132.9, 134.0, 134.3, 135.3, 137.0, 138.1, 142.0, 142.8, 151.2, 157.2, 157.4, 160.5, and 162.4. 6-(4-(4-Nitrophenyl)-2-oxo-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8d ). Yield: 95%, m.p.: 168–170°C, and IR (ν_max/cm⁻¹) 1723, 1657 and 1637, (3C=O), 3431 (NH); ¹H NMR (CDCl₃): δ 9.11 (s, 1H, C-H triazine), 8.47–8.00 (m, 7H, Ar-H), 7.83–7.76 (m, 4H, Ar-H), 7.17 (m, 1H, Ar-H), and 6.99 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 118.8, 120.3, 120.9, 123.5, 123.9, 124.0, 124.2, 128.3, 130.2, 130.6, 130.7, 134.0, 135.9, 137.4, 138.6, 139.8, 140.8, 150.5, 151.3, 153.9, 156.4, 158.4, and 164.7. 6-(4-(4-Fluorophenyl)-2-oxo-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8e ). Yield: 96%, m.p.: 115–117°C, and IR (ν_max/cm⁻¹) 1722, 1682 and 1642 (3C=O), 3441 (NH); ¹H NMR (CDCl₃): δ 9.02 (s, 1H, C-H triazine), 8.33–7.90 (m, 4H, Ar-H), 7.89 (d, 1H, J = 8.5 Hz., C₈-H), 7.83 (d, 1H, J = 8.5 Hz., C₇-H), 7.80–7.23 (m,6H, Ar-H), and 6.85 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 115.7, 115.9, 115.9, 116.1, 119.6, 129.2, 129.3, 130.0, 130.3, 130.8, 131.1, 132.5, 132.6, 132.8, 133.3, 133.7, 134.3, 138.2, 138.3, 151.0, 159.3, 164.4, 165.5, and 171.2. 6-(4-(Furan-2-yl)-2-thioxo-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8f ). Yield: 94%, m.p.: 146–148°C, and IR (ν_max/cm⁻¹) 1732 and 1629 (2C=O), 3436 (NH); ¹H NMR (CDCl₃): δ 8.81 (s, 1H, C-H triazine), 8.09 (s, 1H, C₅-H), 7.92–7.42 (m, 6H, Ar-H), 7.23 (m, 1H, Ar-H), 6.95–6.89 (m, 3H, Ar-H), and 6.66 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 111.7, 113.0, 116.0, 119.0, 121.1, 123.3, 124.6, 126.1, 127.1, 128.2, 129.5, 134.2, 138.1, 141.1, 142.6, 146.7, 149.5, 150.3, 164.3, 165.6, and 180.8. Ms: [M⁺ 415]. Consistent with the molecular formula C₂₁H₁₃N₅O₃S. 3-Phenyl-6-(4-(thiophen-2-yl)-2-thioxo-1,3,5-triazin-1(2H)-yl)quinazoline-2,4(1H,3H)-dione ( 8g ). Yield: 93%, m.p.:111–113°C, and IR (ν_max/cm⁻¹) 1721 and 1641 (2C=O), 3431 (NH); ¹H NMR (CDCl₃): δ 9.02 (s, 1H, C-H triazine), 8.53–7.72 (m, 7H, Ar-H), 7.21–7.09 (m, 4H, Ar-H), and 6.76 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 125.3, 128.1, 128.2, 128.3, 128.4, 128.5, 128.7, 129.4, 130.9, 131.1, 132.5, 132.6, 132.7, 135.4, 135.4, 135.5, 138.8, 142.5, 157.9, 159.5, and 186.2. Ms: [M⁺ 431]. Consistent with the molecular formula C₂₁H₁₃N₅O₂S₂. 3-Phenyl-6-(4-phenyl-2-thioxo-1,3,5-triazin-1(2H)-yl)quinazoline-2,4(1H,3H)-dione ( 8h ). Yield: 93%, m.p.:110–112°C, and IR (ν_max/cm⁻¹) 1653 and 1717 (2C=O), 3431 (NH); ¹H NMR (CDCl₃): δ 9.07 (s, 1H, C-H triazine), 8.71 (s, 1H, C₅-H), 8.03 (d, 1H, J = 7.6 Hz, C₈-H), 7.53 (d, 1H, J = 7.6 Hz., C₇-H) 7.53–7.47 (m, 4H, Ar-H), 7.47–7.07 (m,6H, Ar-H), and 6.99 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 120.1, 122.8, 124.6, 125.4, 127.2, 128.70, 128.9, 130.1, 131.4, 133.7, 134.5, 135.7, 138.3, 139.7, 141.1, 142.4, 143.1, 143.8, 145.5, 146.5, 158.3, 167.4, and 178.5. Ms: [M⁺ 425]. Consistent with the molecular formula C₂₃H₁₅N₅O₂S. 6-(4-(4-Nitrophenyl)-2-thioxo-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8i ). Yield: 95%, m.p.: 198–200°C, and IR (ν_max/cm⁻¹) 1708 and 1625 (2C=O), 3430 (NH); ¹H NMR (CDCl₃): δ 9.11 (s, 1H, C-H triazine), 8.82 (s, 1H, C₅-H), 8.80 (d, 1H, J = 8.2 Hz, C₈-H), 8.72 (d, 1H, J = 8.2 Hz, C₇-H), 8.31 (d, 2H, J = 6.8 Hz, Ar-H),8.03 (d, 2H, J = 6.8 Hz, Ar-H) 7.55–7.20 (m, 5H, Ar-H), and 6.99 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 120.3, 122.1, 123.6, 124.1, 124.8, 127.4, 128.2, 130.3, 131.2, 133.8, 135.7, 137.4, 138.8, 141.4, 143.2, 147.4, 148.8, 149.9, 153.3, 159.1, 162.2, 164.8, and 177.8. Ms: [M⁺ 470]. Consistent with the molecular formula C₂₃H₁₄N₆O₄S. 6-(4-(4-Fluorophenyl)-2-thioxo-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8j ). Yield: 97%, m.p.:118–120°C, and IR (ν_max/cm⁻¹) 1704 and 1657 (C=O), 3393 (NH); ¹H NMR (CDCl₃): δ 9.00 (s, 1H, C-H triazine), 8.60 (d, 2H, J = 5.2 Hz., Ar-H), 8.53 (s, 1H, C₅-H), 8.06 (d, 2H, J = 5.2 Hz., Ar-H), 7.89 (d, 1H, J = 8.3 Hz, C₈-H),7.78 (d, 1H, J = 8.3 Hz., C₇-H) 7.42–7.23 (m, 1H, Ar-H), 7.21–7.09 (m, 5H, Ar-H), and 6.99 (s, 1H, N-H quinazoline); ¹³C NMR (CDCl₃): δ 111.2, 113.0, 116.2, 117.7, 122.0, 127.1, 129.3, 131.6, 133.8, 136.2, 138.5, 139.4, 143.9, 145.6, 148.4, 151.4, 154.6, 157.0, 158.3, 161.7, 164.1, 165.2, 171.2, and 178.9. 6-(2-Imino-4-(thiophen-2-yl)-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-dione ( 8k ). Yield: 94%, m.p.:172–174°C, and IR (ν_max/cm⁻¹) 1708 and 1638 (2C=O), 3240 and 3434 (2NH); ¹H NMR (CDCl₃): δ 9.26 (s, 1H, C-H triazine), 8.53 (s, 1H, C₅-H), 7.85 (d, 1H, J = 8.4 Hz., C₈-H), 7.80 (d, 1H, J = 8.4 Hz., C₇-H), 7.73–7.45 (m, 3H, Ar-H), 7.22–7.13 (m, 5H, Ar-H), 6.99 (s, 1H, N-H quinazoline), and 6.74 (s, 1H, NH); ¹³C NMR (CDCl₃): δ 111.7, 116.4, 119.2, 124.1, 128.4, 129.7, 132.5, 133.4, 135.4, 135.4, 137.2, 138.9, 140.2, 142.6, 144.5, 146.2, 149.9, 152.7, 155.9, 164.9, and 166.4. Ms: [M⁺ 414]. Consistent with the molecular formula C₂₁H₁₄N₆O₂S.

2.1.2. Synthesis of 3-Substituted-2-Imino-1,3,5-triazin-1(2H)-yl-sulfonyl-phenyl-1-methylquinazoline-2,4(1H,3H)-dione (12a–j)

(1) Synthesis of N-(Diaminomethylene)-4-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)benzenesulfonamide ( 11 ). N-methyl isatoic anhydride (9) (2 mmol) was refluxed with sulfaguanidine (10) (2 mmol) in glacial acetic acid (30 ml) under ultrasound irradiation at 80°C for 90 min. The reaction was monitored with TLC till no starting materials were detectable. The solution was poured on crushed ice. The precipitate was collected by filtration and recrystallized from aqueous ethanol. Yield (97%), m.p.: 172–174°C, and IR (ν_max/cm⁻¹) 1705 and 1632 (2C=O), 3431, 3345 and 3240 (NH, NH₂); ¹H NMR (DMSO-d₆): δ 10.33 (s, 1H, NH), 7.84 (d, 2H, Ar- H), 7.40 (m, 4H, Ar-H), 6.75 (m, 2H, Ar-H), 5.71 (s, 2H, NH₂), 2.81 (s, 3H, CH₃), and 1.92 (s, 1H, NH). Ms: [M⁺ 373]. Consistent with the molecular formula C₁₆H₁₅N₅O₄S.

(2) General Procedure for Synthesis of 6f–j. Different substituted acetophenones (1 mmol) were refluxed with DMF-DMA (1.2 mmol) for 8–12 hours. In ethanol (20 ml), reaction was monitored with TLC till no starting materials were detectable. The corresponding enaminones derivatives were afforded in yields of 92–97%. The solutions were evaporated under suction. The precipitates were collected and recrystallized from aqueous ethanol. The following compounds were prepared: (E)-1-(2-Bromophenyl)-3-(dimethylamino)prop-2-en-1-one ( 6f ). Liq. [17]; yield: 92% and liq. (E)-3-(Dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one ( 6g ). Liq. [18]; yield 92% and liq. (E)-3-(Dimethylamino)-1-(p-tolyl)prop-2-en-1-one ( 6h ). m.p.: 112–114°C [19]; yield 94% and m.p.: 115–117°C (E)-3-(Dimethylamino)-1-(pyridin-2-yl)prop-2-en-1-one ( 6i ). m.p.: 135–136°C [20]; yield 97% and m.p.: 136–138°C (E)-3-(Dimethylamino)-1-(4-methoxyphenyl)prop-2-en-1-one ( 6j ). m.p.:98–100°C [21]; yield 96% and m.p.: 104–106°C

(3) General Procedure for Synthesis of 12a–j. N-(Diaminomethylene)-4-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)benzenesulfonamide (11) (1 mmol) and (E)-3-(dimethylamino)-1-arylprop-2-en-1-ones (6a–j) (1 mmol) in DMF/ethanol were refluxed for 8–12 hours. The reaction was monitored with TLC till no starting materials were detectable. The solutions were concentrated under suction. Poured on ice, the precipitates formed were collected by filtration and recrystallized from aqueous ethanol, and the products were obtained in yields of 93–98%. The following compounds were prepared: 3-(4-((4-(Furan-2-yl)-2-imino-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12a ). Yield 95%, m.p.: 230–232°C, and IR (ν_max/cm⁻¹) 1707 and 1660 (C=O), and 3345 (NH); ¹H NMR (CDCl₃): δ 10.30 (s, 1H, NH), 8.09 (d, 2H, Ar-H), 8.02 (d, 2H, Ar-H), 7.87 (m, 3H, Ar-H), 7.76 (s, 1H, C-H triazine), 7.74–7.38 (m, 4H, C-H quinazoline), and 3.64 (s, 3H, CH₃); ¹³C NMR (CDCl₃): δ 29.7, 108.8, 111.3, 115.9, 117.8, 118.9, 123.8, 125.1, 127.7, 128.6, 129.1, 131.2, 133.9, 135.8, 136.7, 140.8, 145.7, 147.9, 152.0, 153.9, 160.1, and 162.5. Ms: [M⁺ 476]. Consistent with the molecular formula C₂₂H₁₆N₆O₅S. 3-(4-((2-Imino-4-(thiophen-2-yl)-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12b ). Yield 94%, m.p.: 257–259°C, and IR (ν_max/cm⁻¹) 1701 and 1631 (2C=O), and 3347 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 7.84–7.71 (m, 5H, Ar-H), 7.69 (s, 1H, C-H triazine), 7.41–7.36 (m, 6H, Ar-H), and 3.35 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 29.3, 110.7, 114.0, 115.1, 119.7, 120.3, 121.4, 122.5, 123.8, 126.2, 127.2, 129.0, 129.8, 133.0, 134.8, 136.4, 138.9, 141.6, 150.1, 158.0, 166.7, and 168.25. Ms: [M⁺ 492]. Consistent with the molecular formula C₂₂H₁₆N₆O₄S₂. 3-(4-((2-Imino-4-phenyl-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12c ). Yield 94%, m.p.: 255–257°C, and IR (ν_max/cm⁻¹) 1704 and 1631 (C=O) and 3347 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 7.85–7.70 (m, 4H, Ar-H),7.69 (s, 1H, C-H triazine), 7.40 (m, 3H, Ar-H), 6.72–6.64 (m, 6H, Ar-H), and 3.37 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 29.3, 110.7, 114.0, 115.1, 117.2, 119.7,120.3, 122.4, 123.6, 126.2, 126.2, 127.1, 129.0, 130.1, 133.0, 133.2, 134.7, 138.9, 141.6, 150.1, 158.0, 160.3, 166.0, and 168.2. Ms: [M⁺ 486]. Consistent with the molecular formula C₂₄H₁₈N₆O₄S. 3-(4-((2-Imino-4-(4-nitrophenyl)-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12d ). Yield 93%, m.p.: 262–264°C, and IR (ν_max/cm⁻¹) 1705 and 1632 (2C=O) and 3350 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 8.36 (m, 4H, Ar-H), 7.96–7.74 (m, 4H, Ar-H),7.72 (s, 1H, C-H triazine), 7.39 (m, 1H, C₈-H), 7.31 (m, 1H, C₅-H), 6.69 (m, 2H, C-H quinazoline), and 3.40 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 29.3, 110.7, 114.0, 115.1, 119.8, 124.0, 124.3, 126.2, 126.8, 128.9, 129.1, 130.2, 133.1, 137.9, 138.9, 141.6, 144.5, 145.9, 150.1, 152.04, 154.4, 155.8, 158.0, and 168.2. Ms: [M⁺ 531]. Consistent with the molecular formula C₂₄H₁₇N₇O₆S. 3-(4-((4-(4-Fluorophenyl)-2-imino-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12e ). Yield 96%, m.p.: 248–250°C, and IR (ν_max/cm⁻¹) 1717 and 1631 (2C=O) and 3350 (NH); ¹H NMR (CDCl₃): δ 10.03 (s, 1H, NH), 8.09 (d, 2H, J = 8.3 Hz., Ar-H), 7.92 (d, 2H, J = 8.3 Hz, Ar-H), 7.89–7.38 (m, 8H, Ar-H), 7.07 (s, 1H, C-H triazine), and 3.55 (s, 3H, CH3); ¹³C NMR (CDCl₃): δ 30.9, 115.6, 117.4, 119.3, 123.8, 124.9, 126.2, 127.2, 128.0, 128.6, 129.0, 130.0, 131.3, 133.1, 137.2, 139.8, 143.6, 147.5, 150.0, 153.9, 157.4, 159.1, 165.7, 167.9, and 171.2. Ms: [M⁺ 504]. Consistent with the molecular formula C₂₄H₁₇FN₆O₄S. 3-(4-((4-(2-Bromophenyl)-2-imino-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12f ). Yield 93%, m.p.:220–222°C, and IR (ν_max/cm⁻¹) 1702 and 1632 (2C=O) and 3342 (NH); ¹H NMR (CDCl₃): δ 10.17 (s, 1H, NH), 8.11 (d, 2H, J = 8.8 Hz, Ar-H), 8.01 (d, 2H, J = 8.8 Hz, Ar-H), 7.84–7.77 (m, 4H, Ar-H), 7.61 (s, 1H, C-H triazine), 7.60–7.30 (m, 4H, Ar-H), and 3.52 (s, 3H,CH₃); ¹³C NMR (CDCl₃): δ 29.7, 117.2, 118.8, 121.5, 122.3, 123.2, 125.2, 126.9, 128.1, 128.6, 130.1, 131.9, 132.8, 133.3, 135.5, 137.6, 143.4, 148.4, 149.3, 149.4, 150.9, 152.0, 156.3, and 160.9. Ms: [M⁺ 564]. Consistent with the molecular formula C₂₄H₁₇BrN₆O₄S. 3-(4-((4-(2-Hydroxyphenyl)-2-imino-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12g ). Yield 93%, m.p.: 252–254°C, and IR (ν_max/cm⁻¹) 1705 and 1632 (2C=O) and 3349 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 7.85–7.65 (m, 4H, Ar-H),7.65 (s, 1H, C-H triazine), 7.61–7.30 (m, 4H, C-H quinazoline), 6.72–6.64 (m, 6H, Ar-H), 5.67 (br, 1H, OH), and 3.34 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 29.4, 110.7, 114.0, 114.2, 115.2, 118.8, 119.8, 121.9, 122.3, 124.1, 126.3, 127,5, 129.0, 130.5, 133.1, 134.5, 138.9, 141.7, 150.2, 152.2, 155.6, 158.0, 162.4, and 168.2. Ms: [M⁺ 502]. Consistent with the molecular formula C₂₄H₁₈N₆O₅S. 3-(4-((2-Imino-4-(p-tolyl)-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12h ). Yield 94%, m.p.: 277–279°C, and IR (ν_max/cm⁻¹) 1702 and 1631 (C=O) and 3348 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 7.84 (d, 2H, Ar-H), 7.68 (m, 2H, Ar-H), 7.67 (s, 1H, C-H triazine),7.66–7.30 (m, 4H, C-H quinazoline), 6.72–6.64 (m, 4H, Ar-H), 3.36 (s, 3H, CH₃), and 2.90 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 20.9, 29.4, 110.7, 114.1, 115.1, 119.8, 121.2, 123.3, 124,4, 126.3, 127.2, 128.1, 129.0, 130.1, 131.4, 133.1, 134.3, 138.9, 140.5, 141.7, 146.2, 150.2, 152.2, 158.0, and 168.2. Ms: [M⁺ 500]. Consistent with the molecular formula C₂₅H₂₀N₆O₄S. 3-(4-((2-Imino-4-(pyridin-2-yl)-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12i ). Yield 98%, m.p.: 253–255°C, and IR (ν_max/cm⁻¹) 1631 and 1705 (2C=O), 3347 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 7.85 (d, 2H, Ar-H), 7.79–7.70 (m, 2H, Ar-H),7.68 (s, 1H, C-H triazine) 7.65–7.30 (m, 4H, Ar-H), 6.72–6.64 (m, 4H, Ar-H), and 3.36 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 29.4, 110.7, 114.1, 115.1, 118.2, 119.8, 121.2, 123.1, 123.2, 124.2, 126.3, 129.0, 130.1, 131.3, 133.1, 135.2, 137.5, 138.9, 141.7, 146.3, 150.2, 158.1, and 168.2. Ms: [M⁺ 487]. Consistent with the molecular formula C₂₃H₁₇N₇O₄S. 3-(4-((2-Imino-4-(4-methoxyphenyl)-1,3,5-triazin-1(2H)-yl)sulfonyl)phenyl)-1-methylquinazoline-2,4(1H,3H)-dione ( 12j ). Yield 97%, m.p.: 271–273°C, and IR (ν_max/cm⁻¹) 1631 and 1705 (2C=O) and 3345 (NH); ¹H NMR (DMSO-d₆): δ 10.30 (s, 1H, NH), 7.85 (d, 2H, J = 8.2 Hz. Ar-H), 7.74 (d, 2H, J = 8.2 Hz., Ar-H),7.69 (s, 1H, C-H triazine), 7.61–7.31 (m, 4H, C-H quinazoline), 6.72–6.64 (m, 4H, Ar-H), 3.84 (s, 3H, CH₃), and 3.34 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆): δ 29.3, 54.8, 110.7, 112.8, 113.1, 114.0, 115.1, 115.7, 119.7, 122.4, 126.2, 129.0, 132.3, 133.0, 134.2, 137.3, 138.4, 138.9, 141.6, 147.4, 150.1, 157.5, 158.0, 167.2, and 168.2. Ms: [M⁺ 516]. Consistent with the molecular formula C₂₅H₂₀N₆O₅S.

2.2. Antitumor Activity

Standard MTT method was used to evaluate the antitumor activity of the synthesized compounds against HepG2 and HCT116 human tumor cell lines. The quantitative assay depends on the ability of the mitochondrial dehydrogenase of viable cells to cleave the tetrazolium ring of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The produced purple color is measured spectrophotometrically, and thus the increase or decrease in the cell number can indicate the antitumor activity of tested compounds. The antitumor activity was conveyed as the concentration of the compound that caused 50% growth inhibition (IC₅₀, mean ± SEM) in comparison to the growth of untreated cells. Cells for cell line were obtained from American Type Culture Collection and cultured using DMEM (Invitrogen) supplemented with 10% FBS (Hyclone,), 10 μg/ml of insulin (Sigma), and 1% penicillin-streptomycin. 96-well plate was used for the test. Cells were treated with serial concentrations of test compounds and incubated for 48 hours at 37°c, and then the plate was examined under inverted microscope before the MTT assay. The cultures were removed from the incubator to laminar flow hood, and MTT was added as 10% of the culture medium volume and then incubated for 2–4 hours. After removal from the incubator, the formed formazan crystals were dissolved; using MTT solubilizing solution, the absorbance was measured at a wavelength of 570 nm [22, 23].

3. Results and Discussion

3.1. Chemistry

3.1.1. Synthesis of 6(3-1H-1,2,4-Triazol-1-yl)-3-phenylquinazoline-2,4(1H,3H)-diones and 6(4-2-Substituted-1,3,5-Triazin-1(2H)-yl)-3-phenylquinazoline-2,4(1H,3H)-diones

Synthesis of quinazolinones involves different starting materials: one of which is isatoic anhydride derivatives. In the present work, 6-bromo-3-phenylquinazoline-2,4(1H,3H)-dione (1) was prepared. Adapting the procedure of Niranjan et al. [24] where 5-bromoisatoic anhydride reacted with aniline in ethanol in presence of few drops of acetic acid under ultrasonic irradiation, the intermediate was taken to the next step, where it reacted with hydrazine hydrate to afford 6-hydrazinyl-3-phenylquinazoline-2,4(1H,3H)-dione (2). The intermediate was characterized by IR spectrum that showed two carbonyl bands at 1639 cm⁻¹ and 1659 cm⁻¹; two NH broad bands appeared at 3373 cm⁻¹ and 3389 cm⁻¹ and a spike characteristic for NH₂ at 3470 cm⁻¹. The hydrazinyl derivative was also characterized by ¹H NMR where 4 protons exchangeable with D₂O appeared at 6.5 ppm and 3.4 ppm corresponding to the proton at NH of the quinazoline ring and the three hydrazine protons, respectively. 6-Bromo-3-phenylquinazoline-2,4(1H,3H)-dione (1) also reacted with urea, thiourea, or guanidine in ethanol to afford 1-(2,4-dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)thiourea, urea, or guanidine (3a–c). Urea derivative (3a) was characterized by ¹H NMR that showed three peaks exchangeable with D₂O: one peak was upfield at 3.6 ppm corresponding to two protons of the urea’s NH₂ group, and the other was downfield at 9.2 ppm corresponding to one proton of the urea’s NH group. The third peak was at 5.5 ppm corresponding to one proton of NH of the quinazoline ring. Similarly, thiourea derivative (3b) was characterized by three peaks in ¹H NMR at 4.0 ppm corresponding to two protons of the thiourea’s NH₂ group, at 8.7 ppm corresponding to one proton of thiourea’s NH proton, and at 6.9 ppm corresponding to the NH proton of the quinazoline ring. Guanidine derivative (3c), on the other hand, was characterized by IR spectrum that showed two carbonyl stretching bands at 1679 cm⁻¹ and 1718 cm⁻¹, two NH stretching bands at 3383 and 3393, and NH₂ stretching band at 3423 cm⁻¹. It was also characterized by ¹H NMR that showed four peaks that were exchangeable with the D₂O one peak downfield at 8.6 ppm and another at 7.0 ppm. Each corresponds to one proton: one of the guanidine NH group and the other for the NH proton of the quinazoline ring. A peak appeared downfield at 5.3 ppm corresponding to two protons of the terminal amino group of the guanidine, while a peak appeared upfield at 4.0 ppm corresponding to one proton of the guanidine NH group. Dimethylformamide dimethyl acetal (DMF-DMA) is a very valuable reagent for organic synthesis. It has an electrophilic site represented by the carbon atom carrying two methoxy groups and nucleophilic site represented by dimethyl amino carrying a lone pair of electrons. Thus, DMF-DMA is involved in two types of reactions: methylation and formylation. Methylation involves the synthesis of methyl esters from acids. Formylation involves the formation of enaminones from active methylenes. It can be further cyclized into heterocyclic ring systems, where enaminones can react with hydrazides to afford the corresponding azoles. It can react with urea, thiourea, and guanidine to afford the corresponding triazines [25, 26]. In the current work, differently substituted acetophenones or 2-acetyl thiophene (furan) reacted with DMF-DMA to prepare the corresponding enaminone derivatives. This was taken to the next step, where enaminones (6a–e) in a cyclization reaction with 6-hydrazinyl-3-phenylquinazoline-2,4(1H,3H)-dione (2) afforded the corresponding 6(3-1H-1,2,4-triazol-1-yl)-3-phenylquinazoline-2,4-(1H,3H)-diones (7a–e), as shown in Scheme 1.

The series was characterized by ¹³C NMR where two carbonyl peaks appeared downfield at above 160 ppm. In ¹H NMR, the characteristic peaks of NH and NH₂ of the hydrazine moiety disappeared indicating the cyclization to triazole. The compounds were also characterized by mass spectrometry, IR spectroscopy showed characteristic carbonyl bands at above 1600 cm⁻¹, and the stretching bands of NH and NH₂ disappeared. Enaminones (6a–e) also reacted with 1-(2,4-dioxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl)thiourea, urea, or guanidine (3a–c) to afford the corresponding 6(4-2-substituted-1,3,5-triazin-1(2H)-yl)-3-phenylquinazoline-2,4-(1H,3H)-diones (8a–k) through a cyclization reaction as shown in Scheme 1. The series were characterized by mass spectrometry, IR spectroscopy, ¹H NMR, and ¹³C NMR. The 2-oxo-1,3,5-triazinyl derivatives (8a–e) showed three characteristic carbonyl stretching bands above 1600 cm⁻¹ in the IR spectrum with only one secondary amine stretching band above 3200 cm⁻¹ corresponding to NH of the quinazoline ring, and the stretching bands of the terminal urea moiety disappeared indicting cyclization. In ¹³C NMR, the compounds showed three peaks downfield at above 160 ppm corresponding to carbonyl carbons. The 2-thioxo-1,3,5-triazinyl derivatives (8f–8j), on the other hand, showed only two carbonyl stretching bands above 1600 cm⁻¹, where the thioxo group shows no characteristic stretching bands. And only one stretching band of the NH secondary amine is above 3200 cm⁻¹. In ¹³C NMR, the compounds showed two downfield peaks at above 160 ppm corresponding to carbonyl carbons and one thioxo carbon at above 170 ppm. The 2-imino-1,3,5-triazinyl derivative (8k) showed two characteristic carbonyl bands in the IR spectrum above 1600 cm⁻¹ and two characteristic secondary amine bands above 3200 cm⁻¹ one for NH of the quinazoline ring and the other for the 2-imino group of the substituting triazine ring at C6. The imino group appeared as a singlet downfield at 9.2 ppm in ¹H NMR.

3.1.2. Synthesis of 3-Substituted-2-Imino-1,3,5-triazin-1(2H)-yl-sulfonyl-phenyl-1-methylquinazoline-2,4(1H,3H)-dione (12a–j)

N-methyl isatoic anhydride (9) reacted with sulfaguanidine (10) in glacial acetic acid under ultrasound irradiation to afford synthesis of N-(diaminomethylene)-4-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)benzenesulfonamide (11). The intermediate was characterized by mass spectrometry, IR, and ¹H NMR spectroscopy. The IR spectrum showed two carbonyl stretching bands at above 1600 cm⁻¹ and two secondary amines stretching bands at 3240 cm⁻¹ and 3345 cm⁻¹ and one primary amine stretching band at 3431 cm⁻¹. In ¹H NMR spectrum, the compound showed a singlet upfield at 2.8 ppm corresponding to three protons of the N-methyl. It also showed three peaks exchangeable with D₂O: one downfield at 10.3 ppm corresponding to the imino group proton, at 5.7 ppm corresponding to two NH₂ protons, and one upfield at 1.9 ppm corresponding to the NH proton. That intermediate reacted in the cyclization reaction with different enaminones to afford 3-substituted-2-imino-1,3,5-triazin-1(2H)-yl-sulfonyl-phenyl-1-methylquinazoline-2,4(1H,3H)-dione (12a–j), as shown in Scheme 2. The compounds were characterized by mass spectrometry, IR, ¹H NMR, and ¹³C NMR spectroscopy. The primary amine stretching bands and one of the secondary amine stretching bands disappeared in the IR spectrum, indicating the cyclization. And two of the D₂O exchangeable peaks disappeared in ¹H NMR: the one at 1.9 ppm, and the peak of NH₂ at 5.7 ppm as additional evidence that the cyclization reaction took place to yield the desired products.

3.2. Antitumor Screening against HEP-G2 and HCT116 Cell Lines

The antitumor activity of compounds 7a, 7b, 8a, 8b, 8c, 8f, 8j, 12d, 12e, and 12j was investigated compared to staurosporin as a reference drug against human hepatocellular carcinoma cell line (HEP-G2) and human colon carcinoma cell line (HCT116) using the standard MTT assay method [22]. Tumor cells were incubated either alone (negative control) or with different concentrations of the test compounds (100, 25, 6.25, 1.56, and 0.39 μg). According to Table 1, the tested compounds exhibited cytotoxic activity of variant degrees depending upon the substituted heterocyclic ring hybridized with the quinazoline scaffold. With respect to HepG-2 cancer cell lines, the derivatives 2-oxo-4-phenyl-1,3,5-triazinylquinazoline 8c and 2-thioxo-4-p-fluorophenyl-1,3,5-triazinylquinazoline 8j represented significant potency of about 2.6 folds higher than that of the reference drug (IC₅₀; 2.68, 2.52 µM, respectively vs IC₅₀ staurosporin; 7.18 µM). Slight reduction in the activity was detected by the 4-furan-2-oxo-1,3,5-triazine analogue 8a but still was 1.5 fold more potent than staurosporin (IC₅₀, 4.53 µM). An observable decrease in the potency was detected upon replacement of the furan heterocyclic ring with thiophene core as compound 8b (IC₅₀; 23.72 µM). Unfortunately, drastic drop in the sensitivity of the cancer cells was observed by the 2-thioxo-4-furan analogue 8f (IC₅₀; 118.1 µM). It could be noted that the conjugation of aromatic homocyclic substituents at triazine-C4 produces greater cytotoxic activity against liver cancer cell (HepG-2) than that obtained by the heterocyclic moieties. Similarly, the five-membered heterocyclic triazole derivative 7c bearing a phenyl ring at triazole-C3 represented about 2.1 folds more potent cytotoxic activity against HepG-2 cancer cell lines than that of the standard drug of IC₅₀; 3.20 µM, while the activity slightly decreased upon the attachment of a furan moiety at the triazole-C3 instead of the aromatic homocyclic ring as compound 7a (IC₅₀; 9.24 µM). It could be noted that the attachment of aromatic homocyclic moieties to the triazine/triazole rings enhances the cytotoxic activity of the compounds more than that of the heterocyclic moieties, which can be explained due to the increase in the hydrophobicity of the derivatives which enables them to cause a rapid membrane disruption mechanism to kill varying cancer cells [27]. On the other hand, the attachment of the 2-imino-triazine ring via sulfonylphenyl linker to the quinazoline ring-N₃ as compounds 12d, 12e, and 12f led to 2-3-fold reduction in the cytotoxic potency of IC₅₀ values of 13.53, 24.54, and 14.39 µM, respectively, in comparison to staurosporin.

With respect to HCT116 cancer cell lines, interestingly, the compound 8c produced the most potent activity against the tested cancer cells of the IC₅₀ value: 1.57 µM vs IC₅₀ staurosporin; 11.26 µM, followed by 8a and 8b of IC₅₀ 2.21, 4.75 µM. The activity is about 11-fold greater than the reference drug. The sensitivity of colon cancer cells observably decreased against 8j (IC₅₀; 30.93 µM), while dramatic drop in the sensitivity was detected against compound 8f of IC₅₀ (206.6 µM). The triazole derivative 7c produced equipotent activity to that of the reference drug, while 7a exhibited a great decrease in the potency of IC₅₀ (99.43 µM). Despite the activity of the sulphonyl derivatives 12d and 12e being 2–3 folds less than staurosporin in case of HepG-2 cancer cells, their potency increased to about 1.5 folds higher than that of the reference drug in case of HCT116 cancer cells (IC₅₀: 6.59 and 7.70 µM), while 12f produced dramatic drop in the activity (IC₅₀: 109.3 µM). It could be concluded the new compounds are promising cytotoxic agents; the six-membered triazine derivatives 8 produced potent broad cytotoxic activity against the two tested cancer cell lines followed by the triazole analogues 7a and 7c and then the sulphonyl derivatives 12d and 12e. Further derivatization is required for the previous compounds to optimize the anticancer activity.

4. Conclusion

In this study, novel quinazoline-2,4(1H,3H)-diones (7a–e), (8a–k), and (12a–j) were developed for cytotoxic evaluation against liver Hep-G2 and colon HCT116 cell lines. The new compounds are promising cytotoxic agents. Among the tested compounds, the six-membered triazine derivatives 8 produced potent broad cytotoxic activity against Hep-G2 and HCT116 cancer cell lines compared to staurosporin followed by the triazole analogues 7a and 7c and then the sulphonyl derivatives 12d and 12e.

Data Availability

All data are available as data file in the website of the journal.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. G-130-247-39. The authors, therefore, acknowledge with thanks DSR for technical and financial support.

Supplementary Materials

The supplementary materials are the PDF files for the original spectroscopic date in the following order (¹³C NMR, ¹H NMR, Mass, and IR). (Supplementary Materials)

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Copyright © 2019 Abeer N. Al-Romaizan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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