Synthesis of Novel 1,2,4-Triazolyl Coumarin Derivatives as Potential Anticancer Agents

A series of novel coumarin derivatives carrying 1,2,4-triazole or 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole moieties were prepared and evaluated in vitro as anticancer in the human colon cancer (HCT116) cell line. -e derivatives 4c and 8c exhibited marked anticancer activity with IC50 values 4.363 and 2.656 μM, respectively.-emolecular docking studies suggested possible interaction with tyrosine kinases (CDK2).


Introduction
Coumarin (2H-1-benzopyran-2-one; 2H-chromen-2-one) derivatives are a large class of important naturally occurring and synthetic oxygen-containing heterocycles.is type of benzopyrone structure enables such derivatives to interact with diversity of enzymes and receptors in organisms through weak bond interactions, thereby exhibiting wide potentiality as bioactive drugs [1].More than 1300 coumarin derivatives were identified as secondary metabolites from plants, bacteria, and fungi [2].Coumarin derivatives were early recognized as important agents for the prevention and treatment of diseases [3,4].Several coumarin-based derivatives are currently used as anticoagulant drugs and as rodenticides [5][6][7].e naturally occurring coumarins such as novobiocin, chlorobiocin, and coumermycin have been found to be an unprecedented class of antibiotics, specifically against Gram-positive bacteria [8].e natural coumarin derivatives possessing long-chain hydrocarbon substitutions such as ammoresinol and ostruthin [9], grandivittin, agasyllin, and osthole displayed significant antibacterial activity against clinically isolated Gram-positive and Gram-negative bacterial strains [10,11].In addition, the coumarin glycoside fraxin displayed free-radical scavenging effect and cell protective effect against hydrogen peroxide-mediated oxidative stress [12,13].Meanwhile, the coumarin derivatives ensaculin [14] and AP2243 [15] are known acetylcholine esterase inhibitors which have been used clinically in treating Alzheimer's disease for a long time.
Coumarin compounds as potential anticancer agents have become a rapidly developing, extremely active, and attractive topic.e coumarin derivative STX 64, a potent estrogen antagonist, is currently under clinical trials as anticancer agents against breast carcinoma [16].Moreover, auraptene has been reported as anticancer agent against the liver, skin, tongue, esophagus, and colon cancers [17] (Figure 1).

In Vitro Anticancer Activity.
e antitumor activity of compounds 4a-c, 5a-c, and 8a-c was investigated against the human colorectal cancer cell line (HCT116) following the previously reported colorimetric cytotoxicity assay of Skehan et al. [31].e results of the preliminary cytotoxic activity of compounds 4a-c, 5a-c, and 8a-c and the anticancer drug doxorubicin [32] are shown in Table 2.

Molecular Docking Studies.
To understand the mechanism of action of the anticancer activities of the newly synthesized compounds, we carried out molecular docking, which is used to predict the binding mode of ligands within the binding site of target proteins [33].Taking into consideration the previously reported tyrosine kinases (CDK2) inhibitory activity of the structurally related chromene anticancer agents genistein [34] and quercetin [35], we docked the synthesized compounds in CDK2 active site.To validate and specify the target protein for the antitumor activity of newly synthesized triazolyl coumarin derivatives, CDK2 protein was selected and downloaded from the protein data bank (PDB ID: 1KE8) [36].Docking studies of compound 4c into the active site of CDK2 enzyme showed two hydrogen bonds with THR160 and GLU81 and a good electrostatic interaction with the active site (Figure 2).
Similarly, docking conformation of the active compound 8c showed good interactions with the active site residues of this protein.Compound 8c formed two hydrogen bond interactions between amino groups moiety, as it acts as a hydrogen bond donor with the side chain of HIE84 and ASP86 residues with strength of 45%.Furthermore, it showed Van der Waals interaction with PHE80 and PHE82 (Figure 3).Finally, there was a good correlation between the docking studies and the biological pro les.

Materials and Methods
Melting points ( °C) were measured in open-glass capillaries using a Gallenkamp melting point apparatus and are uncorrected.IR spectra were recorded in potassium bromide discs on a Shimadzu FTIR 8101 PC infrared spectrophotometer.e NMR spectra were recorded on a BRUKER VX-500 NMR spectrometer. 1 H spectra were run at 500 MHz, and 13 C spectra were run at 125 MHz in deuterated dimethylsulphoxide (DMSO-d 6 ) using TMS as an internal standard.Electron impact mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 eV.Elemental analyses were performed using a Vario LIII CHNS analyzer, and the analytical data (C, H & N) were in agreement with the proposed structures within ±0.4% of the theoretical values.e biological evaluation of the products was carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt.e excess phosphoryl chloride was distilled off under reduced pressure.e resulted residue was triturated with dry pyridine (2 mL), and ice-cold water (250 mL) was added and the mixture was kept for 10 minutes.e precipitated crude product was filtered, washed with water, and crystallized from ethanol to yield 2.2 g (80%) of 3 as light brown powder.IR (cm −1 ): 3280 (NH), 3169, 3155 (NH 2 ), 2895 (Aliphatic CH), 1715 (C�O). 1

Method A.
To a suspension of the arylideneamino analogues 4a-c (50 mmol), in dry acetonitrile (70 mL), iodine (1.27 g, 50 mmol) was added, the reaction mixture was heated under reflux, and the reaction progression was monitored by TLC using ethyl acetate : hexane (9 : 1) as mobile phase.Upon completion of the reaction, the reaction mixture was poured onto ice-cold water (200 mL), and a solution of sodium thiosulfate was added to destroy excess iodine.e greenish precipitate thus formed was filtered, washed with ethyl acetate, and recrystallized from DMF-EtOH.
e separated solid precipitate was filtered, washed with water, and crystallized from DMF-Ethanol to yield compounds 8a-c as reddish brown crystals.

Conclusions
In the current study, a novel series of triazolyl coumarin derivatives were synthesized and evaluated as anticancer agents against human colorectal cancer cell line (HCT116).
e compounds 4c and 8b exhibited marked cytotoxic activity with 59.62 and 97.93% relative potency, respectively, compared to the potent anticancer drug doxorubicin.e molecular docking studies of the active compounds revealed that these compounds might act via inhibition of tyrosine kinases (CDK2).e active compounds are considered to be good candidates as newer anticancer agents, and further studies including preparation of newer analogues and toxicity testing are required for optimization of the activity which are being undertaken.

Figure 2 :Figure 3 :
Figure 2: (a) 2D and (b) 3D interactions of compound 4c with ATP active site of CDK2, which showed hydrogen bond and electrostatic interactions.

Table 1 :
Crystallization solvents, melting points, yield percentages, molecular formulae, and molecular weights of compounds 3