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

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-phenylquinazoline2,4(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-phenyl1-methylquinazoline-2,4(1H,3H)-dione (12a–j) were obtained from the reaction of N-(diaminomethylene)-4-(1-methyl-2,4-dioxo1,2-dihydroquinazolin-3(4H)-yl)benzenesulfonamide (11) with the enaminone (6a–j). -e 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.


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].ere are different classes of quinazolinones depending on the position of substituents: 2-substituted-4(3H)-quinazolinones, 3-substituted-4(3H)quinazolinones, 4-substituted quinazolinones, 2,3disubstituted-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. ey 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-2thioxoquinazoline-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). is 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 2oxo-, 2-thia-, 2-imino-triazine moieties at C6, or introduction of 2-imino-triazine through a phenyl sulfonyl linker at N 3 of the quinazolinone ring (Figure 2).e antitumor activity of the new derivatives was evaluated via the MTT assay.

Experimental
2.1.Chemistry.Melting points were recorded using Stuart melting apparatus.IR spectra (KBr) were recorded on an FT-IR spectrometer (] max /cm −1 ).Nuclear magnetic resonance ( 1 H NMR and 13 C NMR) spectra were recorded on Bruker 400 MHz and 300 MHz spectrometer using DMSO-d6 or CDCl 3 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.
e 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).

Synthesis of 3-Substituted
e solutions were evaporated under suction.e precipitates were collected and recrystallized from aqueous ethanol.
e 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%.e following compounds were prepared:

Antitumor Activity.
Standard MTT method was used to evaluate the antitumor activity of the synthesized compounds against HepG2 and HCT116 human tumor cell lines.
e produced purple color is measured spectrophotometrically, and thus the increase or decrease in the cell number can indicate the antitumor activity of tested compounds.e antitumor activity was conveyed as the concentration of the compound that caused 50% growth inhibition (IC 50 , 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% penicillinstreptomycin. 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 MTTassay.e 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].

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 6hydrazinyl-3-phenylquinazoline-2,4(1H,3H)-dione (2).e 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 NH 2 at 3470 cm −1 .e hydrazinyl derivative was also characterized by 1 H NMR where 4 protons exchangeable with D 2 O 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 1 H NMR that showed three peaks exchangeable with D 2 O: one peak was upfield at 3.6 ppm corresponding to two protons of the urea's NH 2 group, and the other was downfield at 9.2 ppm corresponding to one proton of the urea's NH group.e 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 1 H NMR at 4.0 ppm corresponding to two protons of the thiourea's NH 2 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 NH 2 stretching band at 3423 cm −1 .It was also characterized by 1 H NMR that showed four peaks that were exchangeable with the D 2 O 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.us, 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 2acetyl thiophene (furan) reacted with DMF-DMA to prepare the corresponding enaminone derivatives.is 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.
e series was characterized by 13 C NMR where two carbonyl peaks appeared downfield at above 160 ppm.In 1 H NMR, the characteristic peaks of NH and NH 2 of the hydrazine moiety disappeared indicating the cyclization to triazole.
e 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 NH 2 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)-3phenylquinazoline-2,4-(1H,3H)-diones (8a-k) through a cyclization reaction as shown in Scheme 1. e series were characterized by mass spectrometry, IR spectroscopy, 1 H NMR, and 13 C NMR. e 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 13 C NMR, the compounds showed three peaks downfield at above 160 ppm corresponding to carbonyl Journal of Chemistry carbons.e 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 13 C NMR, the compounds showed two down eld peaks at above 160 ppm corresponding to carbonyl carbons and one thioxo carbon at above 170 ppm.e 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. e imino group appeared as a singlet down eld at 9.2 ppm in 1 H NMR. e 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 1 H NMR spectrum, the compound showed a singlet up eld at 2.8 ppm corresponding to three protons of the N-methyl.It also showed three peaks exchangeable with D 2 O: one down eld at 10.3 ppm corresponding to the imino group proton, at Journal of Chemistry 5.7 ppm corresponding to two NH 2 protons, and one upfield at 1.9 ppm corresponding to the NH proton.at 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. e compounds were characterized by mass spectrometry, IR, 1 H NMR, and 13 C NMR spectroscopy.e primary amine stretching bands and one of the secondary amine stretching bands disappeared in the IR spectrum, indicating the cyclization.And two of the D 2 O exchangeable peaks disappeared in 1 H NMR: the one at 1.9 ppm, and the peak of NH 2 at 5.7 ppm as additional evidence that the cyclization reaction took place to yield the desired products.

Antitumor Screening against HEP-G2 and HCT116 Cell
Lines.
e 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 2thioxo-4-p-fluorophenyl-1,3,5-triazinylquinazoline 8j represented significant potency of about 2.6 folds higher than that of the reference drug (IC 50 ; 2.68, 2.52 µM, respectively vs IC 50 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 (IC 50 , 4.53 µM).An observable decrease in the potency was detected upon replacement of the furan heterocyclic ring with thiophene core as compound 8b (IC 50 ; 23.72 µM).Unfortunately, drastic drop in the sensitivity of the cancer cells was observed by the 2-thioxo-4-furan analogue 8f (IC 50 ; 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 IC 50 ; 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 (IC 50 ; 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-N 3 as compounds 12d, 12e, and 12f led to 2-3-fold reduction in the cytotoxic potency of IC 50 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 IC 50 value: 1.57 µM vs IC 50 staurosporin; 11.26 µM, followed by 8a and 8b of IC 50 2.21, 4.75 µM. e activity is about 11-fold greater than the reference drug.e sensitivity of colon cancer cells observably decreased against 8j (IC 50 ; 30.93 µM), while dramatic drop in the sensitivity was detected against compound 8f of IC 50 (206.6µM).e triazole derivative 7c produced equipotent activity to that of the reference drug, while 7a exhibited a great decrease in the potency of IC 50 (99.43µM).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 (IC 50 : 6.59 and 7.70 µM), while 12f produced dramatic drop in the activity (IC 50 : 109.3 µM).It could be concluded the new compounds are promising cytotoxic agents; the sixmembered 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.

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.e 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.

Figure 2 :
Figure 2: Design of new quinazolinones as antitumor agents.