Facile synthesis and anticancer activity of novel dihydropyrimidinone derivatives


 The enaminone, (2E)-3-(dimethylamino)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one was prepared by refluxing 3,4,5-trimethoxy acetophenone with dimethylformamide dimethylacetal (DMF–DMA) without solvent for 12 h. The dihydropyrimidinone derivatives (1–9) were prepared by reacting enaminone, substituted benzaldehydes and urea in glacial acetic acid. The compounds (1–9) were synthesized in significant yield using one step multicomponent reaction. Structures of all the novel synthesized compounds were characterized and confirmed by various spectroscopic methods. The compounds were evaluated for their anti-cancer activity against HepG2 cancer cell line. Compound 9 displayed significant anti-cancer activity. During the apoptotic assay, it showed a significant increase in necrosis from 1.97% to 12.18% as compared to the control. Mechanism of anti-proliferation was performed by cell cycle distribution assay, which showed a decrease in G2+M from 12.90 to 8.13 as compared to control.


INTRODUCTION
Pyrimidines have played a signifi cant role in medicinal chemistry 1 . Pyrimidines are important scaffolds because of their antihypertensive activity 2 . In 1975, nifedipine (4-aryl-1,4-dihydropyridine) was fi rst introduced as an antihypertensive drug into clinical medicine. The dihydropyridines are reported as the most potent calcium channel modulators 3 . Dihydropyrimidinones have been reported as antihypertensive agents 4-5 . The Biginelli reaction involves one-pot synthesis of 3,4-dihydropyrimidine-2 (1H)-ones using aldehydes, active methylene compounds and (thio) urea in an acidic environment. Dihydropyrimidines are associated with a wide spectrum of pharmacological activities 6−7 . In drug development, dihydropyrimidine derivatives have been tested for antibacterial 8−9 , anti-infl ammatory 10 , anticancer 11 , anti-parkinson 12 , antidiabetic 13 , antihypertensive 14 and antitumor activities 15 . The dihydropyrimidinone derivatives have been reported as potent compounds against HepG2 cancer cell line 16 .
3,4,5-Trimethoxy benzoyl moiety has also played a signifi cant role in medicinal chemistry and has been found in several compounds having anti-cancer activity 17−18 . The compounds containing these two important scaffolds (dihydropyrimidinone and 3,4,5-trimethoxybenzoyl) may have signifi cant therapeutic potential as anticancer agents.

Chemistry
All the solvents were obtained from (Merck, New Jersy USA). Thin-layer chromatography (TLC) was performed on Silica gel 60F 254 (Merck, Millipore, Billerica, MA, USA). Perkin Elmer FT-IR spectrophotometer (Per-kinElmer Inc., Walthum, Ma, USA) was used for IR spectroscopy. Gallenkamp melting point apparatus was used for melting point determination. 1 H and 13 C NMR spectra of the compounds were performed on Bruker NMR 500/700 MHz (Bruker Corporation, Billericia, MA, USA). Mass spectra of compounds were performed on Agilent triple quadrupole 6410 TQ GC/MS equipped with ESI (electrospray ionization). The elemental analysis of the compounds was performed by CHN Elementar (Analysensysteme GmbH, Langenselbold, Germany).

Biological Evaluation
HepG2 hepatocellular carcinoma cells were maintained in RPMI 1640 (Sigma). The HepG2 cells were grown in 96-well plates. Vibrant apoptosis assay kit (Annexin-V, APC conjugate; Molecular Probes™) was applied to study cell viability as per the manufacturer's recommendation. Briefly, the study was performed on both adherent and fl oating cells after collection.

Flow Cytometric analysis of cellular DNA content:
HepG2 cells were fi xed in 1 mL ethanol (70%) for 1 hour at 25 o C. 1 mL Sodium citrate (50 mM) containing 250 μg RNase was used for harvested HepG2 for harvesting and incubated for 1 hour at 50 o C. Further, propidium iodine (PI) in the same buffer was used and cells were incubated for half-hour. Finally, the HepG2 cells were analyzed by fl ow cytometry (Becton Dickinson, San Jose, CA, USA).
In the present study, the effect of compound 9 was investigated using HepG2 cancer cells. After Annexin V and DAPI staining, cells were analyzed by fl ow cytometry (Fig. 4). The results revealed that the treatment did not increase the number of apoptotic cells compared to the control. However, it showed a signifi cant increase in necrosis % from 1.97% to 12.18%.

Cell cycle distribution
Fluorescence-activated cell sorting (FACS) analysis was used to study the effect of drug treatment on cell cycle distribution. HepG2 cells were treated with compound 9 (10 μM) for 48 h. The analysis showed no dramatic change in the accumulation of G1 and S phases. However, there is a decrease in G2+M from 12.90 to 8.13 (Fig. 5).

Apoptosis assay
A hallmark of apoptosis is the exposure of phosphatidylserine on the surface of apoptotic cells, which mediates their recognition and phagocytosis by macrophages. Caspases, a family of cysteine proteases, are specifi cally activated in apoptosis and mediate the series of characteristic morphological changes. Extensive research has proved that the features of apoptotic cells may vary signifi cantly depending on the cell type, the nature of  urea and glacial acetic acid. All the prepared compounds were analyzed and confi rmed by several spectroscopic techniques. All the compounds were screened for anticancer activity against HepG2 cancer cell line. Only compound 9 displayed signifi cant anti-cancer activity. During apoptotic assay, it showed a signifi cant increase in necrosis from 1.97% to 12.18% as compared to control. Mechanism of anti-proliferation by cell cycle distribution assay also confi rmed that there is a decrease in G2+M from 12.90 to 8.13 as compared to control.

ACKNOWLEDGMENTS
Authors are grateful to King Saud University, Riyadh, Saudi Arabia for funding the work through Researchers Supporting Project (No. RSP-2021/359).