Linker Variation and Structure-Activity Relationship Analyses of Carboxylic Acid-based Small Molecule STAT3 Inhibitors

To guide the design of optimized analogues, the molecular features underlying the activity of three reported STAT3 inhibitors—benzoic acid (SH4-54), salicylic acid (BP-1-102), and benzohydroxamic acid (SH5-07)—were systematically investigated. All three lead compounds share a common N-methylglycinamide scaffold, with its two amine groups derivatized by distinct functional groups. Modifications were made to both the three functional moieties and the methylene (CH₂) group of the glycinamide core. Substituting the pentafluorobenzene or cyclohexylbenzene groups, or replacing the aromatic ring in the carboxylic or hydroxamic acid motifs with heterocycles containing nitrogen or oxygen, resulted in reduced potency.
Notably, analogues featuring an Ala-linker (compounds 1a and 2v) or a Pro-based scaffold (compound 5d), all in the (R)-configuration at the chiral center, exhibited enhanced inhibition of STAT3 DNA-binding activity in vitro, with IC₅₀ values of 3.0 ± 0.9 μM, 1.80 ± 0.94 μM, and 2.4 ± 0.2 μM, respectively. These compounds—along with related analogues—effectively suppressed constitutive STAT3 phosphorylation and activation in human breast cancer and melanoma cell lines. In vitro, they also inhibited tumor cell viability, proliferation, colony formation, and migration.
Among the Pro-based derivatives, compound 5h, which incorporates a more polar tetrahydropyranyl (THP) ring in place of the cyclohexyl group, demonstrated improved cellular permeability. Overall, Pro-based analogues in the (R)-configuration showed the most favorable profiles, including superior physicochemical characteristics such as microsomal metabolic stability and Caco-2 permeability. In particular, compound 5d stood out for its enhanced tumor cell specificity.