Innovative Amino-Functionalization of Pyrido[2,3- d]pyrimidine Scaffolds for Broad Therapeutic Applications Supported by Computational Analyses

Abstract

Background: Derivatives of Pyrido[2,3-d]pyrimidine-6-carboxylate are promising multi-target scaffolds. This study focused on synthesizing 16 amino-functionalized derivatives and evaluating their dual anticancer and antibacterial activities, supported by mechanistic and computational analyses. Objectives: Design and synthesize derivatives, evaluate cytotoxicity against HeLa, HepG-2, and MCF-7 (selectivity against WI-38), investigate EGFR^WT and EGFR^T790M inhibition, assess cell cycle, apoptosis, and migration effects, antibacterial efficacy against E. coli and P. aeruginosa, and perform in silico ADMET, docking, molecular dynamics, DFT, and antiviral predictions. Methods: Synthesized 16 derivatives; tested for cytotoxicity, EGFR inhibition, cell cycle, apoptosis, migration; assessed antibacterial activity; performed ADMET profiling, molecular docking, molecular dynamics, and DFT calculations. Results: Derivatives 1, 2, and 7 showed highest cytotoxicity (IC₅₀ = 3.98-17.52 μM; WI-38 IC₅₀ = 64.07-81.65 μM). Compound 1 potently inhibited EGFRWT (IC₅₀ = 0.093 μM) and EGFRT790M (IC₅₀ = 0.174 μM), induced G0/G1 arrest (74.86%) and apoptosis (26.37%), and reduced MCF-7 migration (69.63%). Moderate antibacterial activity observed (MIC = 50 μg/mL). ADMET indicated favorable pharmacokinetics, low CYP inhibition, negative mutagenicity, and oral toxicity class III. Molecular dynamics confirmed stable binding (EGFRWT RMSD 3 Å; EGFRT790M 3.5-4.6 Å) with persistent hydrogen bonds. In silico antiviral evaluation suggested strong binding to HCV NS5A (-9.36 kcal/mol), SARS-CoV-2 Mpro (-9.82 kcal/mol), and E.coli DNA gyrase (-10.25 kcal/mol). Conclusions: Compound 1 exhibits dual anticancer and antibacterial activity, supported by mechanistic and computational analyses, highlighting pyrido[2,3-d]pyrimidines as promising multi-target therapeutic scaffolds.

Keywords: DFT; EGFR inhibition; antibacterial activity; antiviral prediction; apoptosis; molecular docking; molecular dynamics; pyridopyrimidine derivatives; wound healing assay.

Figure 1

Anticancer agents consist of erlotinib as a reference I and other pyrido[2,3-d] pyrimidines II–VII that inhibit EGFR and its mutations.

Figure 2

Pyrido[2,3-d]pyrimidine-based analogues VIII–XI bearing antimicrobial and antiviral activity.

Figure 3

The rational design of newly synthesized 2,4-dioxopyrido[2,3-d]pyrimidines 1–16 with expected anticancer and antimicrobial activities through inhibition of EGFR and DNA gyrase, respectively.

Scheme 1

Synthesis of Pyridopyrimidine, acid hydrazide, hydrazinyl pyrazole, amino-triazole-3-thiol, and thio hydrazide derivatives 1–5.

Scheme 2

Synthesis of dimethyl-pyrazole, oxadiazole-2-thiol, and amino-pyrazole-4-carbonitrile derivatives 6–8.

Scheme 3

Synthesis of thioxo-oxadiazol-propanenitrile and ethyl tetrazole derivatives 9 and 10.

Scheme 4

Synthesis of fused -pyrazolopyrimidin-4-one and pyrazolopyrimidin-4-amine derivatives 11 and 12.

Scheme 5

Synthesis of dimethyl pyrazole derivatives 13.

Scheme 6

Chloro triazolothiadiazine and triazolothiadiazole-6-thiol derivatives 14 and 15 synthesis.

Scheme 7

Synthesis of methyl-pyrazol-3-one derivative 16.

Figure 4

The cytotoxic effects of 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidines 1–16 on WI-38 against HeLa, HepG-2, and MCF-7 cell lines that was tested at multiple doses employing the MTT assay, represented by IC₅₀ values.

Figure 5

SAR graphic of 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidines 1–16 that were identified as possible cytotoxic agents targeting the human malignant cells HeLa, HepG-2, and MCF-7. Ar = aromatic group; R = aliphatic group.

Figure 6

Effect of 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine derivative (Compound 1) on the percentage of MCF-7 cells showing V-FITC-positive annexin staining, along with cell cycle analysis.

Figure 7

Cell cycle analysis of Compound 1 (1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine derivative).

Figure 8

1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine 1’s impact upon apoptotic activity.

Figure 9

Wound healing assay for MCF-7 cells treated with 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine 1 compared with control untreated cells.

Figure 10

The antibacterial activity of samples towards S. aureus ATCC25923, E. faecalis ATCC29212, P. aeruginosa ATCC10145, and E. coli ATCC25915, as determined by the well diffusion method.

Figure 11

(A) Bioavailability radar plots of compounds 1 and 2 showing predicted (red) and optimal (pink) values. (B) Boiled egg diagram illustrating gastrointestinal absorption, blood–brain barrier penetration, and P-glycoprotein substrate status (GP− = non-substrate, GP+ = substrate).

Figure 12

Panels (A) and (B) present the 2D and 3D binding orientations of the active 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine derivatives 1 and 2 inside the binding pocket of wild-type EGFR (PDB ID: 1M17), respectively.

Figure 13

Diagrams (A,B) illustrate the 2D and 3D conformations of the active 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine derivatives 1 and 2 within the EGFR^T790M active site (PDB code: 3IKA), respectively.

Figure 14

Two and three-dimensional views of promising 1,3-dimethyl-2,4-dioxopyrid [2,3-d]pyrimidines 1 within the E. coli DNA gyrase active site (PDB code: 1AJ6).

Figure 15

Two and three-dimensional views of promising 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidines 1 within the HCV NS5A active site (PDB code: 3FQM).

Figure 16

RMSD assessments for the MD simulations are shown in (A) and for 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine 1 with EGFR^WT, and mutant EGFR^T790M in (B) respectively.

Figure 17

Molecular dynamics trajectory graphs (A,B) for 1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine 1 with EGFR^WT, and mutant EGFR^T790M, respectively.

Figure 18

Trajectories (A,B) illustrate the interaction patterns of compound 1 (1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine) with EGFR^WT and the ^T790M mutant, respectively.

Figure 19

Timelines (A,B) illustrate the interaction and contact profiles of compound 1 (1,3-dimethyl-2,4-dioxopyrido[2,3-d]pyrimidine) with EGFR^WT and the ^T790M mutant, respectively.

Figure 20

DFT-calculated geometries and frontier orbital (HOMO/LUMO) maps of compounds 1 and 2.

Figure 20

DFT-calculated geometries and frontier orbital (HOMO/LUMO) maps of compounds 1 and 2.