Design, Synthesis, Biological Activity Evaluation, and Molecular Docking of 2-Aminopyrimidine-Based PKMYT1 Inhibitors.

Introduction

PKMYT1 is a WEE-family G2/M cell cycle checkpoint kinase commonly overexpressed in a broad spectrum of human malignancies. WEE1 exclusively phosphorylates CDK1 at Tyr15, whereas PKMYT1 targets both Thr14 and Tyr15. Unlike WEE1 inhibition, PKMYT1 suppression triggers synthetic lethality with CCNE1. Nearly all disclosed PKMYT1 inhibitors so far fall into structural analogs originating from RP-6306, making the discovery of PKMYT1 inhibitors with chemotypes distinct from RP-6306 crucial.

Methods

The compounds were structurally optimized using CADD, synthesized, and characterized by 1H NMR, 13C NMR, HRMS, and HPLC. They were then assessed for kinase binding affinity via the LanthaScreenTM Eu kinase binding assay, for cellular activity using the CCK-8 assay, and for cell-cycle distribution by flow cytometry, along with investigations into related mechanisms.

Results

This study yielded 24 compounds of 2-aminopyrimidine through substituent derivatization of the pyrimidine scaffold. Among these derivatives, MS13 exhibited potent kinase binding affinity against PKMYT1 (IC50 = 0.86 nM) and demonstrated strong anti-proliferative activity against CCNE1 high-amplification OVCAR3 cells and HCC1569 cells (IC50-OVCAR3 = 1.52 μM, IC50-HCC1569 = 0.66 μM). Additionally, it showed some selectivity towards A549 and HEK293T cells, with SI values of 4.31 (A549/OVCAR3), 9.92 (A549/HCC1569), 2.04 (HEK293T/OVCAR3), and 4.70 (HEK293T/HCC1569). Compound MS13 dose-dependently suppressed clonogenicity and triggered S-phase cell cycle blockade. Pharmacokinetic studies showed moderate hepatic microsomal stability (t1/2 = 32.2 min). Molecular dynamics simulations indicated a favorable binding mode between compound MS13 and PKMYT1 (docking score: -9.322 kcal/mol).

Conclusions

MS13 is a promising highly potent tool compound that provides a clear direction for the future optimization of PKMYT1 inhibitors.