AV-412 free base is an EGFR inhibitor (IC50s: 0.75, 0.79, 0.5, 2.3, 19 nM for EGFR, EGFR(T790M), EGFR(L858R), EGFR(L858R/T790M) and ErbB2).

EGFR (L858R/T790M):2.3 nM, EGFR (L858R):0.51 nM, EGFR (T790M):0.79 nM, EGFR:0.75 nM, ErbB2:19 nM

AV-412 inhibits autophosphorylation of EGFR and ErbB2 (IC50: 43 and 282 nM, respectively) and EGF-dependent cell proliferation (IC50: 100 nM). Additionally, AV-412 abrogates EGFR signaling in the gefitinib-resistant H1975 cell line, which harbors a double mutation of L858R and T790M in EGFR.

In cancer xenograft models, AV-412 (30 mg/kg) effectively halts the growth of A431 and BT-474 cell lines, which notably overexpress EGFR and ErbB2, respectively. This compound also inhibits autophosphorylation of EGFR and ErbB2, correlating with its antitumor properties. AV-412 exhibits considerable antitumor activity across different dosing regimens, displaying significant efficacy with daily and every-other-day administrations, but not with once-weekly dosing. Additionally, it significantly combats the gefitinib-resistant, ErbB2-overexpressing breast cancer cell line KPL-4, affirming its potent antitumor effects.

Recombinant intracellular kinase domains of EGFR, EGFRL858R, EGFRT790M, EGFRL858R/T790M, and purified EGFR from A431 cell membranes are used. Kinase reactions are carried out in 8 mM MOPS (pH 7.0), 0.2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM MnCl2, 10 mM Mg acetate, 0.1 mg/mL poly(Glu, Tyr) 4:1, [γ33P-ATP], and 5–10 mU of enzyme, except that 250 μM of the GGMEDIYFEFMGGKKK peptide substrate is used for EGFRT790M. Phosphorylation is initiated by the addition of ATP and is allowed to proceed for 40 min at room temperature. The reaction is stopped by the addition of 3% phosphoric acid, then aliquots of the reaction mixture are spotted onto a filter mat. After rinsing to remove peptides bound non-specifically, the filter is scintillation counted.

To test the effects of AV-412 on growth factor-dependent cell proliferation, A431 and A7r5 cells are cultured for 24 h at 37°C in the presence of 1 ng/mL epidermal growth factor and 50 ng/mL platelet-derived growth factor, respectively. The 3H-thymidine incorporation during this period is measured.

For studies examining the dosing schedule in relation to efficacy against TE-8 tumors, AV-412 is administered either once daily, every other day, or once per week for 2 weeks. Mice are killed 1 day after the final treatment, and the tumors are dissected and weighed. For evaluation of tumor phosphorylation, tumor-bearing mice are given a single administration of AV-412 and tumors are dissected 4 h later.