H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA).

Erα (Y537S):0.41 nM (Ki), Erα (WT):1 nM (Ki)

H3B-5942以30至300 mg/kg的剂量每日一次口服（q.d.×1），在血浆和肿瘤中的暴露量呈剂量成比例的增加，同时ERαY537S/WT ST941肿瘤模型中的ERα靶基因PGR和NPY1R的表达呈剂量成比例的降低。H3B-5942以200 mg/kg的剂量进行单次或重复给药，可以抑制大量直接的ERα靶基因，其中q.d.×1给药能够在给药后最长维持72小时的靶基因抑制效果，且q.d.×3（连续3天每日一次给药）展现出对PGR和NPY1R的最强抑制。

单次或重复给予H3B-5942（每次200 mg/kg）能够抑制一大组直接ERα靶基因，其中q.d.×1（每天一次）给药可维持靶基因抑制至多达72小时，而q.d.×3（每天三次）给药在PGR和NPY1R上展现出最强的抑制效果。

ERαWT (297–554) and mutant (297–554) proteins were incubated in 50 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 5% glycerol, and 1 mmol/L TCEP with a 2-fold excess of compound (2 μmol/L H3B-5942:1 μmol/L ERα protein solution) at 4°C overnight.?Mass analyses were carried out (ESI source, 4.0 kV ionization voltage, 250°C capillary temperature, 10 arb sheath gas, S-lens RF level 65) coupled with an Accela Open AS 1250.?Samples (10 μL) were desalted on a C4 column ( 2.1 × 150 mm, 2.6 μm) with a gradient from 5% to 95% B over 10 minutes.?Eluent A consisted of 0.1% formic acid in water, and eluent B consisted of 0.1% formic acid in acetonitrile.?The flow was set to 400 nL/minute.?All solvents were LC/MS grade .?The mass spectrometer was run in positive mode collecting full scan at R = 70,000 from m/z 500 to m/z 2,000.?Data were collected with the Xcalibur 3.1 software.

Animals were selected based on TV and randomized into treatment groups of 6 to 8 animals per group.?Single-agent or combination treatments were started on day 0 and continued for the duration of the study.?H3B-5942 was administered orally, tamoxifen was given Q2D, fulvestrant was given?, and palbociclib was administered orally ?Each treatment was administered based on BW (10 mL/kg).?H3B-5942 was formulated daily in 10% 2-Hydroxypropyl-β-CycloDextrin (HPβCD) in 5% dextrose, tamoxifen was formulated in 95% peanut oil/5% ethanol (EtOH), clinical-grade fulvestrant was administered, and palbociclib was formulated in 25 mmol/L sodium bicarbonate, 15 mmol/L lactic acid solution with 2% Cremophor EL.?The BW measurements were performed daily, and tumor measurements were recorded twice a week.