HRO-761

A simple and sensitive liquid chromatography tandem mass spectrometry method was established and validated for determination of HRO761 in rat plasma. After prepared by protein precipitation with acetonitrile, HRO761 and internal standard were separated on a Waters BEH C18 column using acetonitrile containing 0.1% formic acid and 0.1% formic acid in water as mobile phase by gradient elution. The method showed excellent linearity over the range of 5–5000 ng/mL with acceptable intraday and interday precision, accuracy, matrix effect, and recovery. The stability assay indicated that HRO761 was stable during the sample acquisition, preparation, and storage. The method was applied to pharmacokinetic study of HRO761 in rats. The result suggested that after intravenous administration at dose of 1 mg/kg, HRO761 was quickly eliminated from the plasma with the elimination half‐life of 1.9 h. After oral administration at doses of 5, 10, and 20 mg/kg, HRO761 was quickly absorbed into plasma and reach the peak concentration (Cmax) of 2598.1–9379.2 ng/mL at 1.0–4.0 h. The exposure increased proportionally with the dose. The oral bioavailability was 79.0%–99.1% over the range of 5–20 mg/kg. This study provides useful information for its further development in clinic.

Werner syndrome RecQ helicase (WRN), a member of the RecQ helicase family, has recently been identified as a synthetic lethal target in microsatellite instability (MSI) tumors. The triazolo-pyrimidine compound HRO761 is the first WRN inhibitor to enter clinical trials, but research on this scaffold remains limited. Here, we designed a series of derivatives to systematically study the structure-activity relationship (SAR) of triazolo-pyrimidine scaffolds, leading to the discovery of compound S35. S35 exhibited excellent WRN helicase inhibitory activity (ADP-Glo kinase assay IC₅₀ = 16.1 nM, fluorometric helicase assay IC₅₀ = 23.5 nM). Additionally, S35 exhibited excellent cellular selectivity, with antiproliferative activity against multiple MSI cell lines (GI₅₀ = 36.4-306 nM), while the GI₅₀ values for multiple microsatellite stability (MSS) cell lines were greater than 20,000 nM. Furthermore, we observed that compound S35 induced DNA damage and caused G2/M cell cycle arrest in MSI cells, which did not occur in MSS cells. S35 demonstrated favorable oral pharmacokinetic properties, with oral administration resulting in dose-dependent tumor growth inhibition in the SW48 xenograft model. These findings provide a promising outlook for the development of WRN inhibitors for the treatment of MSI tumors.