Synthesis, and Evaluation of Novel N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino) acetamide derivatives by In silico investigations as an anticancer scaffold

Dipanjan Karati¹*, Shankar Thapa², Mahalakshmi Suresha Biradar³

¹Department of Pharmaceutical Technology, School of Pharmacy,

Techno India University, Kolkata, West Bengal, 700091, India.

²Department of Pharmaceutical Technology, Department of Pharmaceutical Technology,

Universal College of Medical Science, Siddharthnagar, 32900, Nepal.

³Department of Pharmaceutical Technology, Al-Ameen College of Pharmacy, Bengaluru, 560027, India.

*Corresponding Author E-mail: karatibabai@gmail.com, tshankar551@gmail.com, mahalakshmisb12@gmail.com

ABSTRACT:

This study demonstrates the scope for N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino)acetamide to develop as promising anticancer agents. Several N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino)acetamide derivatives were synthesized by reflux method and assessed their anticancer activity by in silico research. The yield of the compounds was moderate to high. The functional groups were measured by the IR ranges at 3300cm-1 (NH), 1656 cm-1 (CO) and 1180 (C=S) wavelength. The synthesized compounds have high binding energy against 7A2A protein as an EGFR inhibitor. Compound C2 and C4 showed best affinity of -7.7 and -7.2kcal/mol, correspondingly. The molecules have been tested for their toxicity. The result shows that all the compounds have affinity towards the EGFR protein. The toxicity prediction suggests that all the synthesized substances are relatively safe, having a low likelihood of causing harm at the given doses.

KEYWORDS: Pyrimidine, Acetamide derivatives, Molecular docking, Anticancer, EGFR.

1. INTRODUCTION:

Cancer remains a global health crisis, with over 19 million new cases and nearly 10 million deaths annually.¹ Among these, cancers involving dysregulated signaling pathways like the Epidermal Growth Factor Receptor (EGFR) pathway account for a significant portion.² EGFR, a transmembrane receptor tyrosine kinase, is crucial for cell proliferation and survival, and its abnormal activation is linked to various cancers, including lung, colorectal, and head and neck cancers.^3-6

While targeted EGFR therapies have advanced, drug resistance and side effects remain challenges. Small molecule inhibitors, such as Gefitinib and Erlotinib, block the ATP-binding site of EGFR, showing promising therapeutic effects.^7-8 Dihydropyrimidin derivatives, in particular, have gained attention for their ability to inhibit EGFR signaling and tumor growth due to their structural versatility.^9-11 Designing novel scaffolds to enhance efficacy and reduce side effects requires understanding the molecular interactions of EGFR inhibitors.^12-14 Exploiting dihydropyrimidin scaffolds has led to compounds with varying potency, offering insights into structure-activity relationships and optimization strategies.¹⁵

2. MATERIALS AND METHODS:

2.1 General Information:

All the chemicals employed in the experiments were of the analytical chemical grade. Loba Chemical supplied extra pure methanol, benzaldehyde, chloro acetyl chloride, malononitrile, substituted aniline derivatives, and thiourea.

2.2 Chemistry:

2.2.1 General procedure for the synthesis of compound A: A mixture of 2 mmol/L of benzaldehyde, 2 mmol/L of malononitrile, 3 mmol/L of thiourea, and 0.8 mmol/L of NH4Cl was heated in an oil bath with stirring for 4 hours at 110⁰ C in a 100 mL round-bottom flask. TLC was used to track the reaction's progress. The solid product was obtained by cooling the reaction liquid to room temperature and then pouring it over crushed ice after the reaction had finished.

2.2.2 Synthesis of Compound B: In order to create compound B, one mole of compound A was added to 100millilitres of toluene solvent in a 250 milliliter, clean, round-bottomed flask. The mixture was well mixed and heated using reflux. Next, one mole of dissolved chloroacetyl chloride was added gradually. The entire reaction material was then refluxed for two hours. Following this process, the precipitate was filtered, dried, and then recrystallized using pure ethyl alcohol.

2.2.3 Synthesis of Compound C1-C5: In the presence of K₂CO₃ (1 mols), an equimolar amount of compound B (1mols) and substituted aniline (1mols) in chloroform solvent were heated under reflux (figure 1). The residue was agitated with water after the excess solvent was extracted using a vacuum and it was subsequently filtered and cleaned with 2% NaHCO₃ and water. After that, 100% ethyl alcohol was used to recrystallize the crude product.

Figure 1: Synthetic scheme of compounds C1-C5

2.2.4 Compound C1: Yield 78%. IR (KBr, cm⁻¹): 3414 (NH), 1667 (C = O), 2219 (CN), 1180 (C=S), 700 (C-Cl), 1390 (NO₂). ¹H NMR (DMSO-d6, 400 MHz) d (ppm): in table 1. MASS: 440.05 (C₁₉H₁₃ClN₆O₃S).

Table 1: NMR data[DMSO-d6, 400 MHz) d (ppm)] of Compound 1

Sr. No.	Type of H	Number of H	δ Value (ppm)
1.	H of NH	1	13.03
2.	H of NH	1	6.28
3.	H of NH	1	9.21
4.	Aromatic H	5	7.56-7.98
5.	H of CH2	2	3.77
6.	H of 1-benzene	3	6.98-7.58

2.2.5 Compound C2: Yield 80%. IR (KBr, cm⁻¹): 3414 (NH), 1667 (C = O), 2219 (CN), 1180 (C=S), 700 (C-Cl), 1390 (NO₂). ¹H NMR has been depicted in table 2. MASS: 440.05 (C₁₉H₁₃ClN₆O₃S).

Table 2: NMR data of Compound 2

Sr. No.	Type of H	Number of H	δ Value(ppm)
1.	H of NH	1	13.03
2.	H of NH	1	5.74
3.	H of NH	1	9.21
4.	Aromatic H	5	7.56-7.98
5.	H of CH2	2	3.77
6.	H of 1-benzene	3	6.87-8.13

2.2.6 Compound C3: Yield 75%. IR (KBr, cm⁻¹): 3414 (NH), 1667 (C = O), 2219 (CN), 1180 (C=S), 680 (C-Br). ¹H NMR has been depicted in table 3. MASS: 439.01 (C₁₉H₁₄BrN₅OS).

Table 3: NMR data of Compound 3

Sr. No.	Type of H	Number of H	δ Value(ppm)
1.	H of NH	1	13.03
2.	H of NH	1	6.28
3.	H of NH	1	9.21
4.	Aromatic H	5	7.56-7.98
5.	H of CH2	2	3.77
6.	H of 1-benzene	4	6.59-7.13

2.2.7 Compound C4: Yield 85%. IR (KBr, cm⁻¹): 3414 (NH), 1667 (C = O), 2219 (CN), 1180 (C=S), 1390 (NO₂). ¹H NMR has been depicted in table 4. MASS: 406.08 (C₁₉H₁₄N₆O₃S).

Table 4: NMR data of Compound 4

Sr. No.	Type of H	Number of H	δ Value(ppm)
1.	H of NH	1	13.03
2.	H of NH	1	8.74
3.	H of NH	1	9.21
4.	Aromatic H	5	7.56-7.98
5.	H of CH2	2	3.77
6.	H of 1-benzene	4	7.08-8.20

2.2.8 Compound C5: Yield 83%. IR (KBr, cm⁻¹): 3414 (NH), 1667 (C = O), 2219 (CN), 1180 (C=S), 1390 (NO₂). ¹H NMR has been depicted in table 5. MASS: 361.10 (C₁₉H₁₅N₅OS).

Table 5: NMR data of Compound 5

Sr. No.	Type of H	Number of H	δ Value(ppm)
1.	H of NH	1	13.03
2.	H of NH	1	6.28
3.	H of NH	1	9.21
4.	Aromatic H	5	7.56-7.98
5.	H of CH2	2	3.77
6.	H of 1-benzene	5	6.67-7.08

2.3 Pharmacokinetic properties and drug likeliness studies:

The absorption, distribution, metabolism, and excretion (ADME) prediction and drug likeliness study were conducted using the Swiss ADME web server (http://www.swissadme.ch/index.php).^16-17 Finally, results were analyzed to prioritize compounds with promising drug-like characteristics for further investigation.

2.4 Molecular docking:

2.4.1 Protein and ligand preparation:

The procedure for conducting molecular docking begins with the preparation of both protein and ligand structures. The protein structure of EGFR (PDB ID: 7A2A)¹⁸ was obtained from the Protein Data Bank, while ligand structures, specifically N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino) acetamide derivatives, are generated using a Chem Draw software 20.1.1.125.¹⁹ Open Babel 2.4.1 software was used to convert all drug structures from. cdx files to. sdf MDL MOL format in a single file.²⁰ The ligands were prepared by minimizing its energy for the purpose of docking.^21-22

2.4.2 Binding pocket and grid box generation:

Subsequently, using visualization software BIOVIA discovery studio 2021, the binding pocket of the protein around the co-crystal ligand was identified, and a grid box (x-axis = -16.304, y-axis = 10.562, and z-axis = 1.274) was defined around this region to guide the docking simulations. The dimensions of the grid box encompassed the binding pocket adequately while allowing ample space (center = 10) for ligand movement during docking (Figure 2).^23-26

2.4.3 Docking process and result visualization:

AutoDock Vina 1.5.7^27-29 was then executed with the provided configuration files for each ligand, initiating docking calculations that explore various ligand orientations and conformations within the defined binding site of the protein. Upon completion, AutoDock Vina 1.5.7 generated output files (.pdbqt) containing predicted binding poses and corresponding binding affinities for each ligand. Finally, the docking results were analyzed and visualized using BIOVIA discovery studio 2021 software.³⁰

2.4.4 Docking result validation:

Docking results were validated in PyMOL using the redocking principle. Co-crystal native ligand was removed from the protein, redocked into the protein structure, and poses compared. Binding affinities were assessed, and interactions visually inspected. Iterative refinements were made for accuracy, ensuring agreement with experimental data and known binding patterns, facilitating reliable predictions of ligand-protein interactions for further analysis and interpretation.^31,32

2.5 Biological activity prediction:

Biological activity prediction was carried out using the PASS (Prediction of Activity Spectra for Substances) web server (https://way2drug.com/passonline/ predict.php).^33-34

2.6 Toxicity prediction:

Toxicity prediction was performed using the ProTox-II web server (https://comptox.charite.de/protox3/) [35-36]. Initially, the chemical structures of the compounds were submitted to the server.

3. RESULTS AND DISCUSSION:

3.1 Chemistry:

Using the IR ranges at 3300 cm^-1 (NH), 1656 cm^-1 (CO), and 1180 cm^-1 (C=S) wavelength, the functional groups were determined. Compound C2 and compound C4 were found to be having least binding energy of -7.7 and -7.2 kcal/mol correspondingly. The synthetic compound exhibits a binding energy of -6.6 to -7.8 kcal/mol when compared to the standard medication. The amino acids Met793, Val726, Leu (718, 799, 844), Arg841, and Ala749 make up the co-crystal7G9, which is located close to the active site of the enzyme and could accommodate all five compounds (C1–C5) snugly. After closely examining the docked complexes, this was found. The potential amino acids Met793, Arg841, Asp800, and Cys797 are also involved in interaction. Compounds C2, C4, and their binding affinity of -7.7 and -7.6 kcal/mol, correspondingly, demonstrate their significant affinity for the EGFR active site. Through hydrogen bonds at 2.05 Å, the carbonyl group (C=O) of the molecule C2 interacts with Met793.

3.2 Pharmacokinetic properties and drug likeliness studies:

Table 6 presents various physicochemical and pharmacokinetic properties of five compounds (C1, C2, C3, C4, and C5), which are crucial for understanding their behavior in biological systems. The Log Po/w values represent the partition coefficient between octanol and water, which indicates the lipophilicity of the compounds. The LogPo/w values range from 1.28 to 2.21, suggesting that the compounds exhibit varying degrees of lipophilicity, with C3 being the most lipophilic and C4 being the least.³⁷

The "HBA" (Hydrogen Bond Acceptors) and "HBD" (Hydrogen Bond Donors) represent the number of hydrogen bond acceptor and donor groups present in each compound, respectively. These properties are crucial for understanding a compound's ability to interact with other molecules through hydrogen bonding, which can impact its binding affinity and biological activity. The "RB" (Rotatable Bonds) indicates the number of rotatable bonds in each compound. Rotatable bonds are important for assessing the flexibility of a molecule, which can influence its conformational changes and interactions with biological targets. The "TPSA" (Topological Polar Surface Area) provides information about the surface area of a compound that is polar or capable of forming hydrogen bonds. This property is relevant for predicting a compound's membrane permeability and oral bioavailability. The "Lipinski’s Rule Violation" indicates whether each compound violates Lipinski's Rule of Five, which is a widely used guideline in drug discovery for predicting a compound's likelihood of being orally bioavailable.³⁸ Overall, none of the compounds in the table violate Lipinski's Rule.

Table 6: Pharmacokinetic properties of compounds

Compound code	LogPo/w	LogS	Log Kp	GI abs.	BS	BBB	Pg-S	CYP1A2 inh.
C1	1.66	Poorly soluble	-6.95	High	0.55	No	No	No
C2	1.66	Poorly soluble	-6.95	High	0.55	No	No	No
C3	2.21	Poorly soluble	-6.77	High	0.55	No	No	Yes
C4	1.28	Poorly soluble	-6.79	High	0.55	No	No	No
C5	1.66	Poorly soluble	-6.79	High	0.55	No	No	Yes

LogP_o/w (Consensus) = Lipophilicity, LogS = Water solubility, Log Kp= Skin permeability, BS= Bioavailability score, GI abs. = Gastrointestinal Absorption, Pg-S = Pg substrate, CYP1A2 inh. = Cytochrome P1A2 inhibitor.

Table 7: Drug likeliness studies of compounds

Compound code	Ml.Wt.	HBA	HBD	RB	TPSA	SA	Lipinski’s rule Violation
C1	424.80	6	3	7	132.70	3.15	No
C2	424.80	6	3	7	132.70	3.13	No
C3	424.25	4	3	6	86.88	2.95	No
C4	390.35	6	3	7	132.70	3.09	No
C5	345.35	4	3	6	86.88	2.91	No

Ml.Wt.= Molecular Weight, HBA= Hydrogen Bond Acceptor, HBD = Hydrogen Bond Donor, RB = Rotatable Bond, TPSA = Topological Polar Surface Area, SA = Synthetic Accessibility.

3.3  Molecular docking:

The choice of EGFR as the docking target was made because the crystal structure was available and it complexed with Spebrutinib (7G9). The resemblance in structure between the co-crystal ligand Spebrutinib (7G9) (Figure 3) and our proposed N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino) acetamide derivatives has sparked our interest in selecting the protein. Compound C2 and compound C4 had the lowest binding energies of -7.7 and -7.2 kcal/mol, respectively. The synthesized molecule exhibits a binding energy ranging from -6.6 to -7.8 kcal/mol, as stated in table 8. All the compounds (C1-C5) can tightly fit into the active site of the enzyme near the co-crystal 7G9, composed of the amino acids Met793, Val726, Leu (718, 799, 844), Arg841, and Ala749. This finding was made by meticulous analysis of the docked complexes. Furthermore, the amino acid residues Met793, Arg841, Asp800, and Cys797 play a crucial role in the interaction. The compounds C2 and C4 exhibit a significant affinity for the active site of EGFR, as indicated by their respective binding energies of -7.7 and -7.6 kcal/mol. The molecule C2 forms hydrogen bonds with Met793 by the interaction of its carbonyl group (C=O) at a distance of 2.05 Å, as depicted in figure 4. In the interaction Met793 donates a hydrogen to the compound C2. Moreover, NO₂ group accepts hydrogen donated by Cys797 at a distance of 3.49 Å to form conventional hydrogen bonding. A similar type of interaction was seen in compound C4, where the carbonyl group (C=O) establishes a hydrogen connection with Met793 but at a distance of 1.90 Å (figure 5). Based on the comparable binding affinities and interactions of the potential compounds C2 and C4, the synthesized compounds may effectively suppress EGFR. Compound C5 exhibited a greater binding energy (lower affinity) of -6.6 kcal/mol (figure 6).

Table 8: Interaction results of docking studies for compounds (C1 – C5) and respective cocrystal.

Interaction	Binding Energy (kcal/mol)	Interacting amino acids	Nature of Bond	Bond length(Å)
C1-7A2A	-6.8	Arg841, Asp800	Hydrogen bond	2.32, 3.11
C2-7A2A	-7.7	Met793, Cys797	Hydrogen bond	2.05, 3.49
C3-7A2A	-6.7	Met793	Hydrogen bond	2.03
C4-7A2A	-7.6	Met793	Hydrogen bond	1.90
C5-7A2A	-6.6	Cys793	Hydrogen bond	2.04
C6-7A2A (Osimertinib)	-6.8	Gly719	Hydrogen bond	2.75
C7-7A2A (Co-crystal ligand, Spebrutinib 7G9)	-7.5	Met793	Hydrogen bond	2.06

Figure 2: Binding site identification from BIOVIA discovery studio.

Figure 3: Co-crystal ligand Spebrutinib(7G9)

Figure 4: 2D and 3D molecular interaction of compound C2 with EGFR (PDBID = 7A2A) protein

Figure 5: 2D and 3D molecular interaction of compound C4 with EGFR (PDBID = 7A2A) protein

Figure 6: 2D and 3D molecular interaction of C5 with EGFR (PDBID =7A2A) protein

3.4 Biological activity prediction:

All five designed compounds screened for anticancer activity. All the compounds (except C4) showed the significant activity against bone cancer. Compounds C3, C4, and C5 showed the activity against pancreatic cancer. Interestingly, compound C4 reports the Pa value of 0,158 against bladder cancer which is greater value than Pi (0,112). Compound C3 is active as epidermal growth factor antagonist having Pa value (0.049) greater than Pi (0.031). Detail activity values are expressed in table 9.

Table 9: Predicted biological activity of compounds

Compound code	Anticancer activity		Remarks
	Pa	Pi
C1	0,231	0,064	Antineoplastic (bone cancer)
C2	0,224 0,206	0,083 0,176	Antineoplastic (bone cancer) Antineoplastic (pancreatic cancer)
C3	0,049 0,266 0,219	0,031 0,018 0,152	Epidermal growth factor antagonist Antineoplastic (bone cancer) Antineoplastic (pancreatic cancer)
C4	0,158 0,221	0,112 0,148	Antineoplastic (bladder cancer) Antineoplastic (pancreatic cancer)
C5	0,247	0,036	Antineoplastic (bone cancer)

3.5 Toxicity prediction:

The table 10 includes statistics on the toxicity prediction of five N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino) acetamide derivatives (C1, C2, C3, C4, and C5). Each compound is classified as "Non-toxic" for organ toxicity, signifying that, these compounds are not anticipated to induce harm to the specified organ (kidney, liver, and heart) [39-42]. The LD₅₀ values range from 560 mg/kg to 1500 mg/kg, showing different levels of acute toxicity. Regardless of these differences, all substances are categorized as "Class IV" toxicity, indicating a generally low level of acute toxicity.

Table 10: Toxicity prediction of synthesized compounds

Compound code	Organ Toxicity	LD50 (mg/kg)	Toxicity class
C1	Non-toxic	1500	Class IV
C2	Non-toxic	560	Class IV
C3	Non-toxic	560	Class IV
C4	Non-toxic	759	Class IV
C5	Non-toxic	759	Class IV

Organ toxicity includes; nephrotoxicity, hepatotoxicity, and cardiotoxicity.

4. CONCLUSION:

Here, we have synthesized five novel N-(5-cyano-6-phenyl-2-thioxo-2,3-dihydropyrimidin-4-yl)-2-(phenylamino)acetamide congeners with anticancer predictions in acceptable chemical yields. According to the in silico investigative study, these synthesized scaffolds displayed an affinity for the anticancer protein EGFR (7A2A). It will lead scientists in the future to perform in vivo cancer studies of these congeners as a potent anticancer moiety. Lipinski's Rule is not broken by any of the compounds, indicating that they could have promising drug-like qualities and be developed further as chemical probes or pharmaceutical agents. According to the estimated toxicity levels, this analysis indicates that these compounds are generally safe and unlikely to be harmful at the recommended dosages.

5. AVAILABILITY OF DATA AND MATERIALS:

The data supporting the findings of the article is available within the article.

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Received on 15.05.2024      Revised on 19.08.2024 Accepted on 12.09.2024      Published on 22.10.2024 Available online from October 31, 2024 Asian J. Research Chem.2024; 17(5):278-284. DOI: 10.52711/0974-4150.2024.00048 ©AandV Publications All Right Reserved
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