Facile Synthesis of Some Coumarin Derivatives and Their Cytotoxicity through VEGFR2 and Topoisomerase II Inhibition

Novel semisynthetic coumarin derivatives were synthesized to be developed as chemotherapeutic anticancer agents through topoisomerase II, VEGFR2 inhibition that leads to apoptotic cancer cell death. The coumarin amino acids and dipeptides derivatives were prepared by the reaction of coumarin-3-carboxylic acid with amino acid methyl esters following the N,N-dicyclohexylcarbodiimide (DCC) method and 1-hydroxy-benzotriazole (HOBt), as coupling reagents. The synthesized compounds were screened towards VEGFR2, and topoisomerase IIα proteins to highlight their binding affinities and virtual mechanism of binding. Interestingly, compounds 4k (Tyr) and 6c (β-Ala-L-Met) shared the activity towards the three proteins by forming the same interactions with the key amino acids, such as the co-crystallized ligands. Both compounds 4k and 6c exhibited potent cytotoxic activities against MCF-7 cells with IC50 values of 4.98 and 5.85 µM, respectively causing cell death by 97.82 and 97.35%, respectively. Validating the molecular docking studies, both compounds demonstrated promising VEGFR-2 inhibition with IC50 values of 23.6 and 34.2 µM, compared to Sorafenib (30 µM) and topoisomerase-II inhibition with IC50 values of 4.1 and 8.6 µM compared to Doxorubicin (9.65 µM). Hence, these two promising compounds could be further tested as effective and selective target-oriented active agents against cancer.


Introduction
Cancer is the leading or second leading cause of death from noncommunicable diseases in the world [1]. Cancer is a multifaceted disease characterized by the growth of abnormal cells that are out of control. Tumor formation and development are linked to apoptosis, which is thought to be a highly programmed mechanism of cell death. One of the main reasons for their prominence was their wide range of pharmacological actions. The design

Chemistry
In continuation of our efforts in synthesizing various bioactive molecules [36], it was found desirable to synthesize a series of coumarin-3-amino acid methyl esters and Coumarins are a class of natural compounds commonly found in a variety of plant families [16], as naturally occurring antitumor compounds. Hyaluronic acid, a key component in tumor growth and progression, is inhibited by 4-methylumbelliferone [17]. Cancers of the pancreas, kidney, prostate, ovaries, and breasts are all effectively targeted by this chemopreventive and chemotherapeutic drug [18]. Many different semi-synthetic coumarin compounds, with varying substitution patterns, have been identified as powerful anticancer drugs, as shown in Figure 1B, as examples of some anticancer coumarin-based derivatives (I-VI) [19][20][21][22].
Recently, studies have demonstrated that coumarin-based compounds can inhibit immune regulation, cell growth and differentiation, and cell differentiation through VEGFR2 [23][24][25], and topoisomerase II [26][27][28] inhibition, which have been considered attractive targets for the development of anticancer agents. Almost all cellular responses to VEGF are mediated by VEGFR-2, a type III transmembrane receptor tyrosine kinase (RTK) present in a wide variety of cancer cells. It is considered as the most important transducer of VEGF-dependent apoptosis and angiogenesis [29]. Hence, the VEGFR-2 inhibitory signaling pathway has become a crucial strategy for the identification and development of novel therapeutics for a variety of human malignancies [30].
N.S. Reddy et al. [31] have outlined the growth inhibitory properties of the novel coumarin sulfonamide. These coumarin derivatives activated c-Jun N-terminal kinase 1 (JNK 1) [32]. Reddy et al. have designed and synthesized a series of novel coumarin-3carboxamides and examined the antioxidant, anti-inflammatory and against anti-human immunodeficiency activities [33,34]. For a long time, organic and medicinal chemists have been interested in the synthesis of coumarins and their derivatives. Structure modification on coumarins focused on the attachment of pharmacophoric groups at C-3, C-4 and C-7 positions for biological evaluation, including anti-microbial, anti-HIV, anti-cancer, lipidlowering, anti-oxidant, and anti-coagulation activities [35]. Considering the highly valuable biological and pharmaceutical properties of coumarins, it was aimed to synthesize a series of coumarins containing a variety of pharmacophores, amino acids and peptides at position 3, and to investigate the hit compounds through the molecular docking screening as cytotoxic against HepG2, and MCF-7 cancer cells.

Chemistry
In continuation of our efforts in synthesizing various bioactive molecules [36], it was found desirable to synthesize a series of coumarin-3-amino acid methyl esters and dipeptides. By interacting with the receptor recognition sites, these compounds were designed to enhance the binding affinity of the coumarin moiety.
Condensation of diethyl malonate with salicylaldehyde afforded the coumarin-3carboxylic acid ethyl ester, which upon hydrolysis gave coumarin-3-carboxylic acid (3, Scheme 1) [32]. The acid 3 is an excellent precursor for the structure modification via peptide coupling. Peptide bonds were first introduced in the literature using a variety of coupling reagents, which react carboxylic acid with amino acid methyl ester [37]. A convenient coupling method [38] was employed for the formation of peptides by reaction of the carboxylic acid group with amino acid esters, using N,N-dicyclohexylcarbodiimide (DCC) and 1-hydroxy-benzotriazole (HOBt), as coupling reagents. With its ability to reduce racemization during carbodiimide peptide coupling, HOBt is a common method [39]. The acid 3 is an excellent precursor for the structure modification via peptide coupling. Peptide bonds were first introduced in the literature using a variety of coupling reagents, which react carboxylic acid with amino acid methyl ester [37]. A convenient coupling method [38] was employed for the formation of peptides by reaction of the carboxylic acid group with amino acid esters, using N,N-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt), as coupling reagents. With its ability to reduce racemization during carbodiimide peptide coupling, HOBt is a common method [39].
Thus, the treatment of coumarin-3-carboxylic acid (3) with amino acid methyl esters (glycine, L-alanine, L-leucine, L-serine, L-phenylglycine, β-alanine, L-valine, L-methionine, L-tryptophan, L-tyrosine) in the presence of coupling reagents, DCC and HOBT, afforded the amino ester derivatives 4a-l, respectively, in the 63-87% yield, Scheme 2. The acid 3 is an excellent precursor for the structure modification via peptide coupling. Peptide bonds were first introduced in the literature using a variety of coupling reagents, which react carboxylic acid with amino acid methyl ester [37]. A convenient coupling method [38] was employed for the formation of peptides by reaction of the carboxylic acid group with amino acid esters, using N,N-dicyclohexylcarbodiimide (DCC) and 1-hydroxy-benzotriazole (HOBt), as coupling reagents. With its ability to reduce racemization during carbodiimide peptide coupling, HOBt is a common method [39].
The structure assignment of the amino acid ester 4a-l and dipeptide 6-8(a-c) based on spectral and physicochemical analysis; Figure 2. All the isolated products exhibited a rather interesting, fixed conformation, as represented in Figure 2, and indicated from all 1 H NMR spectra. Thus, the 1 H NMR spectrum of 3-methyl-2-[(2-oxo-2H-chromene-3-carbonyl)amino]-butyric acid methyl ester 4e (L-Val) demonstrated an interesting exchangeable singlet down fielded signal at δ 9.24 ppm corresponding to the NH group participating in an intramolecular hydrogen bond interaction of the type N-H···O=C [40].

Molecular Docking
The synthesized derivatives were screened for their targets using a molecular docking study. They were screened towards VEGFR-2, and topoisomerase IIα, based on the previous studies that highlighted their binding affinities. As seen in Table 1, the compounds/protein interactions were summarized as a result of docking analysis.
Regarding the molecular docking study towards VEGFR2, the co-crystallized ligand of VEGFR2 protein formed three HB interactions with Asp 1064, Cys 919 and Lys 868 as

Molecular Docking
The synthesized derivatives were screened for their targets using a molecular docking study. They were screened towards VEGFR-2, and topoisomerase IIα, based on the previous studies that highlighted their binding affinities. As seen in Table 1, the compounds/protein interactions were summarized as a result of docking analysis. Regarding the molecular docking study towards VEGFR2, the co-crystallized ligand of VEGFR2 protein formed three HB interactions with Asp 1064, Cys 919 and Lys 868 as the key amino acids. Interestingly, most compounds formed one important interaction like the native ligand. Additionally, for the molecular docking study towards topoisomerase IIα, the co-crystallized ligand of Topoisomerase IIα protein formed one HB with Ser 149 as the key amino acid. Thus, both compound 4k and 6c structures represents the important pharmacophoric regions that made them share the activity towards the three proteins by forming the same interactions with the key amino acids, such as the co-crystallized ligands. As observed in Figure 3 with the surface and interactive representations, compound 4k maintained the binding disposition of the co-crystallized ligand of VEGFR2 (A), and Topoisomerase II (B).

ADME Pharmacokinetics
In order to determine the physicochemical and drug-like properties of compounds 4k and 6c towards the examined proteins, bioinformatics study was performed. All of the tested chemicals were readily permeable to and absorbed. As shown in Table 2

ADME Pharmacokinetics
In order to determine the physicochemical and drug-like properties of compounds 4k and 6c towards the examined proteins, bioinformatics study was performed. All of the tested chemicals were readily permeable to and absorbed. As shown in Table 2, typically, compounds contained 2 H-bonding donors and 6 to 7 H-bonding acceptors. In addition, they exhibited log p values (1.81-2.69), so they were well tolerated by cell membranes. The ratable bond number (nrotb) should be less than 10 for effective regulation of conformational changes and oral bioavailability. In addition, the BOILED-Egg model found that the compounds had good absorption in the gastrointestinal tract, as shown in Figure 4. compounds contained 2 H-bonding donors and 6 to 7 H-bonding acceptors. In addition, they exhibited log p values (1.81-2.69), so they were well tolerated by cell membranes. The ratable bond number (nrotb) should be less than 10 for effective regulation of conformational changes and oral bioavailability. In addition, the BOILED-Egg model found that the compounds had good absorption in the gastrointestinal tract, as shown in Figure 4.   . BOILED-Egg model for compounds 4k (A) and 6c (B) using the Swiss ADME. "Points located in the BOILED-Egg's yolk are molecules predicted to passively permeate through the bloodbrain barrier, while points located in the BOILED-Egg's white are molecules predicted to be passively absorbed by gastrointestinal tract".

Cytotoxicity against MCF-7 and HepG2 Cancer Cell Lines
Based on the preliminary of the molecular docking studies of all tested compounds, two hit compounds 4k and 6c with the highest binding affinities towards the tested targets to be screened as cytotoxic against MCF-7 and HepG2 cell lines. As seen in Figure 5, compounds 4k and 6c exhibited promising cytotoxicity against MCF-7 cells with IC50 values of 4.98 and 5.85 µM, respectively, causing cell death by 97.82 and 97.35%, respectively. Additionally, compound 4k exhibited promising cytotoxicity against HepG2 cells with an IC50 value of 9.4 µM, while compound 6c showed moderate cytotoxicity with an IC50 value of 33.88 µM. Hence, these compounds exhibited promise cytotoxic activity. . BOILED-Egg model for compounds 4k (A) and 6c (B) using the Swiss ADME. "Points located in the BOILED-Egg's yolk are molecules predicted to passively permeate through the bloodbrain barrier, while points located in the BOILED-Egg's white are molecules predicted to be passively absorbed by gastrointestinal tract".

Cytotoxicity against MCF-7 and HepG2 Cancer Cell Lines
Based on the preliminary of the molecular docking studies of all tested compounds, two hit compounds 4k and 6c with the highest binding affinities towards the tested targets to be screened as cytotoxic against MCF-7 and HepG2 cell lines. As seen in Figure 5, compounds 4k and 6c exhibited promising cytotoxicity against MCF-7 cells with IC 50 values of 4.98 and 5.85 µM, respectively, causing cell death by 97.82 and 97.35%, respectively. Additionally, compound 4k exhibited promising cytotoxicity against HepG2 cells with an IC 50 value of 9.4 µM, while compound 6c showed moderate cytotoxicity with an IC 50 value of 33.88 µM. Hence, these compounds exhibited promise cytotoxic activity.

Enzyme Target Activity
Based on the primarily molecular docking studies of the highest binding affinities of 4k and 6c, enzymatic target activities of both compounds towards VEGFR2, topoisomerase II proteins were tested. As summarized in Table 3, compounds 4k and 6c demonstrated promising VEGFR-2 inhibition with IC 50 values of 23.6 and 34.2 µM, compared to Sorafenib (30 µM) and topoisomerase-II inhibition with IC 50 values of 4.1 and 8.6 µM compared to Doxorubicin (9.65 µM). Our results of VEGFR2 inhibition agreed with previous studies [23,24] that exhibited promising VEGFR2 inhibition of newly synthesized coumarin derivatives aligned with cytotoxicity in breast cancer cells with promising IC 50 values.
Other studies [26,41,42] designed some novel coumarin derivatives as cytotoxic activities through topoisomerase inhibition in some cancer cell lines including MCF-7 cells. There were previous studies [43,44] linking apoptosis as the mechanistic cell death upon treatment to VEGFR2 and topoisomerase inhibition, that are emerged as a prime approach for discovering new therapies for many apoptosis-dependent anticancer drugs.

Enzyme Target Activity
Based on the primarily molecular docking studies of the highest binding affinities of 4k and 6c, enzymatic target activities of both compounds towards VEGFR2, topoisomerase II proteins were tested. As summarized in Table 3, compounds 4k and 6c demonstrated promising VEGFR-2 inhibition with IC50 values of 23.6 and 34.2 µM, compared to Sorafenib (30 µM) and topoisomerase-II inhibition with IC50 values of 4.1 and 8.6 µM compared to Doxorubicin (9.65 µM). Our results of VEGFR2 inhibition agreed with previous studies [23,24] that exhibited promising VEGFR2 inhibition of newly synthesized coumarin derivatives aligned with cytotoxicity in breast cancer cells with promising IC50 values. Other studies [26,41,42] designed some novel coumarin derivatives as cytotoxic activities through topoisomerase inhibition in some cancer cell lines including MCF-7 cells. There were previous studies [43,44] linking apoptosis as the mechanistic cell death upon treatment to VEGFR2 and topoisomerase inhibition, that are emerged as a prime approach for discovering new therapies for many apoptosis-dependent anticancer drugs.

Synthesis
Solvent was purified and dried in the usual way. The boiling range of the petroleum ether used was 40-60 °C. Thin layer chromatography (TLC): silica gel 60 F254 plastic plates (E. Merck, layer thickness 0.2 mm) detected by UV absorption. Melting points were determined on a Buchi 510 melting-point apparatus and the values are uncorrected. NMR

Synthesis
Solvent was purified and dried in the usual way. The boiling range of the petroleum ether used was 40-60 • C. Thin layer chromatography (TLC): silica gel 60 F 254 plastic plates (E. Merck, layer thickness 0. General procedure for the preparation of coumarin-3-amino acid methyl esters (4a-l): To a cold solution (−5 • C) of the amino acid methyl ester hydrochloride (2.0 mmol) in acetonitrile (20 mL) containing triethyl amine (0.28 mL, 2.0 mmol), coumarin-3-carboxylic acid (3) (0.38 g, 2.0 mmol), dicyclohexylcarbodiimide (DCC) (0.414 g, 2.0 mmol) and 1hydroxybenzotriazole (HOBT) (0.27 g, 2.0 mmol) were added successively. The reaction mixture was stirred at 0 • C for one hour, at 5 • C for one hour, and then at room temperature for 12 h. The precipitated dicyclohexylurea was filtered off and the filtrate was evaporated under reduced pressure. The residue was dissolved in ethyl acetate, filtered and the filtrate was washed with 5% NaHCO 3 , 1N HCl and saturated solution of sodium chloride then dried over anhydrous sodium sulphate. After evaporation of the solvent, the remaining oily residue was triturated with petroleum ether (b.p. 40-60 • C) at 0 • C with scratching. The solid residue was filtered off and crystallized from petroleum ether/ethyl acetate. Charcterization analyses including NMR and MS for the synthesized compounds were supported as supplementry.

Enzyme Target Assays
VEGFR2 (KDR) kinase assay kit "BPS Bioscience Corporation catalog#40325" and Topo isomearse II screening kit "TopoGEN, Inc., Columbus, OH, USA" were utilized to invetstiage the enzyme activity for the most active compounds following the manufacturer instructions. Inhibition of autophosphorylation by tested compounds was determined. The IC 50 values were determined using GraphPad Prism 7 software from curves depicting the percentage of inhibition using eight concentrations of each compound [43,44].

Conclusions
Herein, a novel series of coumarin-based amino acid and dipeptide derivatives synthesized as cytotoxic agents through VEGFR2 and Topoisomerase II inhibition. The coumarin amino acids and dipeptides derivatives were prepared by the reaction of coumarin-3carboxylic acid with amino acid methyl esters following N,N-dicyclohexylcarbodiimide (DCC) and 1-hydroxy-benzotriazole (HOBt), as coupling reagents. Both compounds 4k (Tyr) and 6c (β-Ala-L-Met) exhibited potent cytotoxic activities against MCF-7 cells with IC 50 values of 4.98 and 5.85 µM, respectively, causing cell death by 97.82 and 97.35%, respectively. Validating the molecular docking results, they exhibited promising VEGFR-2 inhibition with IC 50 values of 23.6 and 34.2 µM, compared to Sorafenib (30 µM) and topoisomerase-II inhibition with IC 50 values of 4.1 and 8.6 µM compared to Doxorubicin (9.65 µM). Accordingly, compounds 4k and 6c were singled out as promising therapeutic candidates for the research and development of new anticancer medicines due to their dual inhibitory effect.

Conflicts of Interest:
The authors declare no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.