Hirshfeld Surface Analysis, DFT Calculations, Development by 3² Factorial Design, and Evaluation for Solubility and Anticancer Activity of Neratinib–3-Hydroxybenzoic Acid Co-Crystals

Objectives Neratinib, a pan-HER tyrosine kinase inhibitor for HER2-positive breast cancer, suffers from extremely poor aqueous solubility (< 0.002 mg/mL), severely limiting oral bioavailability and therapeutic efficacy. This study aimed to develop and optimize neratinib cocrystals using rational co-former selection and factorial design to enhance solubility, dissolution, and anticancer activity. Methods Five potential co-formers were screened at 3 molar ratios (1:1, 1:2, 1:3) using SynthonDock software. Electronic properties and interaction energetics were characterized using density functional theory (DFT) calculations at B3LYP/6-31G(d,p) level. Neratinib–3-hydroxybenzoic acid cocrystals were prepared by solvent evaporation and optimized in 3 2 factorial design using the concentration of co-former (24.8–74.4 mg) and stirring time (30–90 min) as independent variables. The PXRD, FTIR, DSC, SEM and Hirshfeld surface studies were carried out to characterize the compounds. MTT assay was used to evaluate in vitro cytotoxicity against SK-BR-3 cells. Results 3-Hydroxybenzoic acid showed the best binding energy (− 10.05 kcal/mol with a ratio of 1:3) and highest co-crystal score (90.5). Increased reactivity was established using density functional theory (DFT) with the calculation showing diminished HOMO–LUMO gap (0.4324 eV vs 3.3072 eV for pure drug). The 12.4-fold solubility enhancement (0.112 mg/mL vs 0.009 mg/mL) and 4.6 times better dissolution (84.6% vs 18.4% at 60 min) was reached using the third batch PF9 (74.4 mg of 3-hydroxybenzoic acid, 90 minutes.) Cytotoxicity studies showed enhanced anticancer activity (IC 50 = 18.46 µg/mL vs 24.18 µg/mL for pure neratinib) toward HER2-positive breast cancer cells. Conclusion The optimized neratinib–3-hydroxybenzoic acid cocrystal is a clinically relevant pharmaceutical approach to enhance oral bioavailability and therapeutic efficacy in HER2-positive breast cancer for subsequent biological exploration in vivo.