Synthesis and Assessment of the Cytotoxic Effects of Some Novel Biginelli 3,4-Dihydropyrmidine-2-(1H) One Derivatives Containing 2-Mercapto-3-Phenyl-4(3H)-Quinazolinone

Abstract In the present study, a new series of 3,4- dihydropyrmidine2-(1 H) one derivatives(4a-f) which linked to 2-mercapto-3-phenyl-4(3H)-quinazolinone were synthesized by using the nucleophilic substitution reaction of 2-mercapto-3-phenyl-4(3H)-quinazolinone (1) and an appropriate 6-bromomethyl-3,4- dihydropyrmidine2-(1 H) one (3a-f) in the presence of KOH as a base and in CH3CN as solvent. The chemical structures of the synthesized compounds were confirmed by means of IR, 1H-NMR, 13C-NMR, Mass spectroscopy, and Elemental analysis. The cytotoxicity of the newly synthesized compounds was evaluated in vitro against the human breast cancer (MCF-7) cell line and human colon cancer cell line (HT-29) based on the results of the MTT assay. Our results indicated that compound 4b exerted the most cytotoxic effect in the two cell lines and the IC50 values were (4.25 ± 0.16 μM) for MCF-7 cell lines and (12.11 ± 0.76 μM) for HT-29 cell lines. Also, compound 4b induced apoptosis more than other compounds in the two cell lines. Therefore, compound 4b may be a potential anticancer agent.


Introduction
Cancer is a major leading cause of death worldwide.Several approaches are administered to treat cancer such as chemotherapy, immunotherapy, radiotherapy, and surgery. 1 Despite, the existence of approaches to treat cancer, they cannot treat cancer completely.Cancer recurrence and cancer cell resistance to chemotherapy are still confusing problems in cancer chemotherapy.Hence, developing new drugs is vital to effective cancer treatment. 2n recent decades, research interests have been focused on the synthesis of organic compounds which possess medicinal activities.Recently, dihydropyrimidinones (DHPMs) and their derivatives have occupied a distinct and important position in medicine because of their versatile scaffold in organic synthesis and significant template for the development of various therapeutic agents.many proprieties of DHPM's have been reported such as anticancer, [3][4][5][6][7][8] antimicrobial, 9,10 antimalarial 11,12 anti-inflammatory, 13,14 antihypertension 15 anti-HIV-1 activity. 16The synthesis of functionalizing (DHPMs) was reported for the first time by P. Biginelli in 1893.The Biginelli multicomponent reaction, involving a one-pot three-component condensation of aldehyde, b-ketoester, and urea, provides easy access to the preparation of 3,4-dihydropyrmidine2-(1 H) ones. 17Various methods have been developed for the synthesis of these compounds. 18,19Any substitution within the structure of dihydropyrimidinones is associated with changes in medicinal effects.Medicinal chemistry with changes in functional groups of pharmacophore structure seeks the synthesis of compounds with low cytotoxicity.The 4(3H)-quinazolinone derivatives have attracted considerable attention from many scientists because of possessing a broad spectrum of biological properties like antimicrobial, 20 anticancer 21,22 anti-inflammatory, 23 and anticonvulsant. 24According to the heterocyclic structure of the quinazolinone derivatives, they would be capable to bind the DNA structure. 25In tumor cells, the DNA structure is not completely intact and the repair systems are defective, so if an agent can bind the DNA, it can affect the DNA biological function such as replication and transcription which are vital to cell growth. 26he 2-mercapto-3-aryl-4(3H)-quinazolinone derivatives have been reported as monoamine oxidases (MAO) inhibitors. 27,28Apoptosis is a programmed cell death that its occurrence can be induced by chemical agents that can modulate cellular biological functions such as DNA replication, transcriptions, signaling pathways, and mitochondrial membrane.This type of cell death is suitable to eliminate cancer cells because in this type of cell death the body of the dead cells cannot damage peripheral cells.Therefore, apoptosis is an attractive target to treat cancer. 29henever we connect two different and independent pharmacophores with a covalent bond, a new hybrid compound is created.The presence of two or more pharmacophores in a single unit leads to a pharmacological potency greater than the sum of each individual moiety's potencies.The objective of forming these hybrids is an attempt to reach an active antitumor agent with potentiated activity and selectivity toward cancerous cells.In the library search, the binding of 3,4-dihydropyrmidine2-(1 H) ones (DHPMs) to 2-mercapto-3-aryl-4(3H)-quinazolinones has not been reported and since both 2-mercapto-3-aryl-4(3H)-quinazolinones and 3,4-dihydropyrmi-dine2-(1 H) one derivatives have anti-cancer activities, we hope to observe synergistic anti-cancer effects by combining them.Based on the above observations here, we report the synthesis, assessment of the cytotoxic effects and docking study of a new series of Dihydropyrimidine -Quinazolinone hybrids.

Chemistry
Melting points were recorded on a Philip Harris C4954718 apparatus without calibration.FT-IR spectra were recorded on a Thermo Nicolet670 Nexus spectrometer with KBr pellets. 1 H and 13 C NMR spectra were recorded on Bruker 300 or 400 MHz spectrometers using DMSO-d6 as the solvent.Microanalyses were performed on a Leco Analyzer 932.Mass spectra were recorded on Agilent Technologies 5975 C using Electron Impact (EI) 70 eV.Thin-layer chromatography (TLC) analyses were carried out on silica gel plates.All chemicals were purchased from Merck (Tehran, Iran) and used as received in all standard procedures.

Synthesis of 2-mercapto-3-phenylquinazolin-4(3H)-one (1)
A mixture of phenyl isothiocyanate (50 mmol, 6 ml), and anthranilic acid (50 mmol, 6.86 g), was heated under reflux for 6 h in absolute EtOH (70 ml).The obtained solid was washed with EtOH, dissolved in 10% NaOH, precipitated with HCl, washed several times with H 2 O, and dried.It was crystallized from AcOH. 30 Yield ¼ 11.General procedure for the synthesis of ethyl 6-methyl-2-oxo-4-aryl-3,4-dihydropyrimidine-5carboxylate (2a-f) The compounds were prepared according to the procedure described by Dilmaghani et al. 19 In a round-bottom flask (10 mL) equipped with a magnetic stirrer and condenser, freshly prepared H 2 SO 4 /charcoal system (0.4/0.3 g, 133% w/w), n-hexane (2.5 mL) and CH 3 CN (0.5 mL) were added and the mixture was stirred for 1 min at room temperature.After addition of the aldehyde (1 mmol), b-dicarbonyl compound (1 mmol) and urea (1.2 mmol) to the reaction mixture, stirring under reflux conditions was continued for the appropriate time.The progress of the reaction was monitored by TLC.When the reaction was completed, the mixture was cooled to room temperature and a sinter glass funnel removed the residue charcoal.To the filtrate, aqueous NaHCO 3 (10%, 5 mL) was added and the mixture was extracted with EtOAc (3 Â 15 mL).The combined extracts were dried over anhydrous Na 2 SO 4 .Evaporation of the solvent and recrystallization of the crude product in hot MeOH affords DHPMs (2a-f) in 78-87% yield.

Biology reagents
Cell culture and treatment HUVEC, HT-29, and MCF-7 cell lines (Iranian Pasteur's Institute) were maintained in DMEM high glucose-containing penicillin-streptomycin (100 u/ml, Biowest) enriched with FBS 10%(Gibco) and L-glutamine (Sigma) and then incubated in CO 2 -incubator (Memmert) at 37 C.For the cytotoxic assay, 5000 cells were cultured in each well of 96-well plates, then treated with different concentrations of 4a-f compounds (10,20,30, and 40 mM/ml) for 24,48, and 72 h.Next, 10 mL of MTT solution (5 mg/ml, Sigma) was added to the wells and incubated for 4 h.Subsequently, the formazan crystals were dissolved in DMSO (Merck) and the wells absorbency was read in a microplate reader (Biotech) at 570 nm.Finally, based on MTT data, the compounds IC 50 were calculated by COMPUSYN software and presented as a chart designed by excel 2019.

Apoptosis analysis by Hoechst 33342 staining
For Hoechst staining, 2 Â 10 5 cells were cultured in each well of 24-well plates and treated with 20 mM/ml of the (4a-f) compounds for 72 h.After incubation, the cells were centrifuged and washed twice with PBS (phosphate buffered saline).Then, ice-cold methanol was added and the samples were incubated at À20 C for 20 min.Subsequently, the cells were washed twice with PBS and incubated in Hoechst33342 dye solution (1 mg/ml, Sigma) for 30 min at room temperature. 31Ultimately, the cells samples were washed once in PBS, transferred onto slides and observed and then photographed by fluorescent microscopy (Zeiss, Germany).

Docking methods
Crystal structure of DNA dodecamerd(CGCGAATTCGCG) 2 with entry code 1BNA was taken from the Brookhaven Protein Data Bank (RCSB). 32The 3D structures of the compounds were prepared using Gauss view 5.0.Thereafter in order to get the most stable geometry all structures were optimized by using Gaussian 09 software by using the B3LYP hybrid density functional theory at the 6-31 G ÃÃ level. 33Flexible ligand docking calculations were performed with the Auto Dock 4.2 package by using empirical free energy function and Lamarckian genetic algorithm with local search. 34During the preparation of the DNA in Auto Dock Tools environment, water molecules were removed from crystal structure and missing hydrogen atoms and Gasteiger charges were added to the molecule.At first a grid box, containing the whole DNA was used to allow the compounds move freely during docking run.Thereafter, the grid map was set at 40 Å Â 40 Å Â 40 Å with a grid-point spacing of 0.375 Å to cover the binding site.For all docking parameters, default values were used excepting 200 separated docking runs contain of the maximum 25,000,000 energy evaluations.For each compound the docking conformation with the lowest binding energy in the highest populated cluster was selected as the most probable binding mode.

Chemistry
The synthetic pathway to synthesize the new compounds (4a-f) is depicted in the Scheme (1).2-thioxo-3-(phenyl)2,3-dihydroquinazolin-4(1H)-one (1) was obtained by heating anthranilic acid with phenyl isothiocyanate in ethanol.The existence of thiol-thione tautomerism is known for the compounds and generally, the thione form is predominant in the solid state.These constitutional isomers were distinguished by 1 HNMR and 13 CNMR.The thione tautomer was confirmed by the presence of a singlet signal at 13.04 ppm, corresponding to the NH group, and a signal at 175.6 ppm due to C ¼ S. The 3,4-dihydropyrmidine2-(1 H) one(2a-f) were synthesized from the three-component Biginelli reaction, using aromatic aldehydes, ethylacetoactate, urea, and freshly prepared H 2 SO 4 /charcoal as catalyzer according to the procedure reported in the literature. 19 1 NMR spectra of final compounds (2a-f) showed a singlet signal about d ¼ 2.16-2.27ppm due to the Me group at C6 on the pyrimidinone ring system.The proton of the C-H which was attached to C4 appeared as a doublet signal near 5.0-5.The lowest IC50 value is observed in 4b treated cancerous cells.Therefore, the compound has a more cytotoxic effect rather than others.On the other hand, in the HUVEC cell line the 4c showed the most cytotoxic effect.
(4a-f).Compounds (4a-f) were confirmed based on their 1 HNMR spectra which showed the absence of NH group in mercaptoquinazolin (1) at 13.04 ppm and showed new signals as an AB coupling system that involve two doublets with the same coupling constant around 4.24 and 4.55 ppm due to the two protons of the methylene that links the sulfur to the DHPM core.

Biological activity evaluations
In vitro anticancer evaluation of the synthesized compounds In this study, dihydropyrimidine-Quinazolinone hybrid derivatives have been synthesized and their cytotoxic effects on MCF-7 and HT-29 cell lines were evaluated and the IC 50 values were presented in Table 1.On the other hand i Based on Table 1 data, the (4a-f) compounds decreased the viability of the cells and surprisingly the 4b compound exerted the highest cytotoxic effect in both of the two cell lines.Our results were similar with a recent work that reported the quinazolinine and dihydroquonazolinone derivatives exert cytotoxic effects on colorectal and breast cancer cell lines. 35On the other hand, Gupta et al. found that spiroisoquinoline-pyrimidine derivatives decreased MCF-7 cancer cell viability, 36 the results are consistent with our results (Table 1).docking studies showed that the 4c compound has had the most affinity to the analyzed DNA sequence, the docking results are almost similar to its cytotoxic effect on the HT-29 cell line because, in the cells, 4c compound after the 4b compound showed the most cytotoxic effect.As understood from Table 1, the cytotoxic effects of the compounds on the MCF-7 cell line were higher than it in the HT-29 cell line due to lower IC50 values in MCF-7 cells rather than HT-29 cells.As seen in Table 1, the results of the compounds on the HUVEC cell line were almost similar to docking results that suggested 4c has the highest binding affinity to DNA among the compounds.But, due to the significant cytotoxic effect of 4c on the HUVEC cell line,  this compound cannot be considered an anticancer agent among the compounds.Therefore 4b is a better anticancer agent than 4c.Based on differential pathway activity in different cells 37 the two cancerous cell lines and the HUVEC cell line showed different IC 50 .We showed that the compounds induced apoptosis in the two cancer cell lines and interestingly the apoptosis occurrence was correlated to the (4a-f) compounds IC 50 values.Considering Hoechst 33342 staining (Figure 1), the compounds particularly the 4b compound showed the most apoptosis inducing effect (Figure 1).According to the docking study results (Figure 2 and Table 2) the compounds can interact with the minor groove of the applied DNA sequence.Based on its DG and Ki levels, the 4c showed the strongest binding level with the selected DNA sequence.The results of the MTT assay of the HUVEC cells are similar to the docking data that suggest 4c is the strongest compound in binding to DNA.The two most cytotoxic compounds (4b, 4c) contain a hydroxyl group on one ring of their structure.Therefore, it suggested that the presence of the OH group causes the strong hydrogen bonds formation of 4b with DNA (see the supplementary data) in both of the cancerous cells.

Conclusion
In the present study, 3,4-dihydropyrmidine2-(1 H) one-2-mercapto-3-phenyl-4(3H)-quinazolinone hybrids were synthesized in order to produce compounds with greater cytotoxicity.The synthesized compounds were characterized using FT-IR, 1 H-NMR, 13 C-NMR spectral data, ESI Mass, and elemental analysis.The cytotoxic effect of the newly synthesized compounds was evaluated using an MTT assay on human breast cancer (MCF-7) cells and human colon cancer (HT-29) cells.The results showed that compound 4b revealed a higher cytotoxic effect on the cancer cell lines.Therefore, it can be considered a potential agent for cancer therapy.However, additional studies should be done to analyze its accurate anticancer effect.

Figure 1 .
Figure 1.Fluorescence microscopy of cell nuclei in MCF-7 and HT-29 cell line.In the treated cells, the dense and fragmented nuclei are increased that shown by arrows.In 4b treated cells, the dense or fragmented nuclei are more than them in comparison to other treated cells, so, the apoptotic nuclei in 4b treated cells are more than the other compounds treated cells.The lowest apoptotic nuclei are observed in the 4e-treated cells in the two cell lines.

Figure 2 .
Figure 2. The superimposition of the best docking structures of the compounds in the DNA minor groove.All of the compounds can bind to DNA via hydrogen bond formation with the minor groove.

Table 1 .
Effect of the synthesized compounds in IC50 (lM) on the growth of two human tumor cell lines.

Table 2 .
Binding energies and inhibition constants of the compounds in the DNA minor groove.