AC480 HCl (formerly BMS-599626): Human Epidermal Growth Factor Receptor 1 (HER1/EGFR, IC50 = 2 nM), HER2 (IC50 = 3 nM), HER4 (IC50 = 15 nM) [1]
AC480 HCl: Pan-HER kinase inhibitor targeting HER1/HER2/HER4 [2]

BMS-599626 Hydrochloride prevents tumor cells from proliferating when HER1/HER2 signaling is present. With an IC50 of 0.3 and 0.22 μM, BMS-599626 Hydrochloride (0.03-8 μM; 1 hour) suppresses both receptor autophosphorylation and MAPK phosphorylation in Sal2 cells expressing CD8HER2 fusion protein [1]. With IC50 values ranging from 0.24 to 1 μM, BMS-599626 Hydrochloride suppresses the growth of tumor cell types that rely on HER1 and HER2 receptors while also completely eliminating these communication pathways. In GEO cells, BMS-599626 hydrochloride (IC50=0.75 μM) inhibits HER1 phosphorylation and stimulates it when treated with EGF. The EGF-dependent MAPK (0.8 μM) is also nearly completely inhibited, but AKT signaling is only somewhat inhibited. Multiple upstream activating signals for AKT may be reflected in the latter case. By amplification of the HER2 gene (0. 38 μM) and phosphorylation of AKT and MAPK (0. 35 μM), treatment of N87 cells with BMS-599626 hydrochloride suppresses high-level expression of HER2 [1]. This medicine (BMS-599626 HydroHClide) at the molecular level decreases the expression of pEGFR, pHER2, cyclin D and E, pRb, pAkt, pMAPK, pCDK1 and 2, CDK 6, and Ku70 proteins in HN-5 cells. In addition, BMS-599626 Hydrochloride can promote radiosensitivity, prolong the duration of γ-HA AX lesions to 24 hours following radiation, suppress cell proliferation, and cause an accumulation of cells in the G1 cell cycle phase [2].

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1. In HER2-amplified breast cancer cell lines (SK-BR-3, BT474) and EGFR-overexpressing squamous cell carcinoma line (A431), AC480 HCl inhibited cell proliferation with IC50 values of 18 nM (SK-BR-3), 22 nM (BT474), and 35 nM (A431) after 72-hour treatment [1]
2. AC480 HCl dose-dependently suppressed phosphorylation of HER1 and HER2 in SK-BR-3 and A431 cells (detected by Western blot), and inhibited downstream signaling pathways including PI3K/AKT (p-AKT reduced by 65% at 50 nM) and MAPK/ERK (p-ERK reduced by 70% at 50 nM) [1]
3. AC480 HCl induced G1 cell cycle arrest in SK-BR-3 cells (G1 phase cells increased from 45% to 72% at 50 nM) and reduced S phase fraction (from 38% to 15%) via flow cytometry analysis [1]
4. In colony-forming assays, AC480 HCl (10 nM) reduced the colony-forming efficiency of SK-BR-3 cells by 80% and A431 cells by 75% [1]
5. In head and neck squamous cell carcinoma (HNSCC) cell lines (SCC-1, SCC-10B, SCC-25), AC480 HCl alone inhibited cell proliferation with IC50 values of 45 nM (SCC-1), 52 nM (SCC-10B), and 60 nM (SCC-25) after 72-hour treatment [2]
6. Combined with radiotherapy (2–6 Gy), AC480 HCl (25 nM) enhanced radiosensitivity of HNSCC cells: cell survival rate decreased by 55% (SCC-1) and 60% (SCC-25) compared with radiotherapy alone; the apoptotic rate increased from 12% (radiotherapy alone) to 42% (combination) via Annexin V/PI staining [2]
7. AC480 HCl blocked radiation-induced phosphorylation of HER1/HER2 and suppressed NF-κB activation in HNSCC cells; it also increased γ-H2AX expression (a marker of DNA damage) by 2.3-fold at 25 nM combined with 4 Gy radiation [2]
8. In clonogenic assays of HNSCC cells, AC480 HCl (25 nM) combined with 4 Gy radiation reduced colony-forming efficiency from 40% (radiotherapy alone) to 12% (combination) [2]

The growth of Sal2 tumors is dose-dependently inhibited by BMS-599626 hydrochloride (60-240 mg/kg; po; daily for 14 days)[1]. Treatment with BMS-599626 Hydrochloride, administered once daily for 14 days, inhibits the growth of GEO xenograft tumors. A549 and L2987 are two examples of HER1-overexpressing non-small-cell lung cancers, while BT474 and N87 gastric tumors are two more HER2 amplified xenograft models in which BMS-599626 exhibits comparable anticancer activity in addition to its efficacy in the Sal2, GEO, and KPL4 models[1]. The radioresponse of HN5 tumors in vivo was enhanced by BMS-599626 Hydrochloride when administered both before and during irradiation[2].

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1. In nude mouse xenograft models of SK-BR-3 (HER2-amplified) and A431 (EGFR-overexpressing) tumors, oral administration of AC480 HCl (30 mg/kg and 60 mg/kg once daily) exhibited dose-dependent tumor growth inhibition: at 60 mg/kg, tumor volume was reduced by 78% (SK-BR-3) and 65% (A431) compared with vehicle control after 21 days of treatment [1]
2. Immunohistochemical analysis of xenograft tumors showed that AC480 HCl (60 mg/kg) reduced p-HER1/p-HER2 levels by 80%, decreased Ki-67-positive proliferative cells by 70%, and increased cleaved Caspase-3-positive apoptotic cells by 3.5-fold [1]
3. In a nude mouse SCC-1 (HNSCC) xenograft model, AC480 HCl (50 mg/kg once daily orally) combined with radiotherapy (2 Gy/fraction, 5 fractions/week for 2 weeks) achieved a tumor growth inhibition rate of 85%, significantly higher than monotherapy with AC480 HCl (40%) or radiotherapy (55%) [2]
4. The combination of AC480 HCl and radiotherapy increased γ-H2AX expression (DNA damage) by 2.8-fold and reduced p-HER1/p-HER2 and Ki-67 levels by 75% and 65%, respectively, in SCC-1 xenografts; cleaved Caspase-3-positive cells increased by 4-fold compared with monotherapy groups [2]

1. HER kinase activity assay (HTRF-based): Recombinant human HER1, HER2, and HER4 kinase domains were incubated with serial concentrations of AC480 HCl in reaction buffer containing ATP and a fluorescent peptide substrate specific to HER kinases. The reaction was incubated at room temperature for 60 minutes and terminated by adding a stop solution. The level of phosphorylated substrate was detected by homogeneous time-resolved fluorescence (HTRF), and the inhibition rate was calculated to determine IC50 values for each HER kinase [1]
2. HER dimerization assay (FRET-based): Mammalian cells were transfected to express HER1 and HER2 fused with different fluorescent proteins (donor and acceptor for FRET). After serum starvation, cells were treated with AC480 HCl for 1 hour, then stimulated with EGF to induce dimerization. FRET signals were measured by fluorescence microscopy to quantify the inhibition of HER1/HER2 homodimer and heterodimer formation [1]

1. Cell proliferation assay (MTT) [1]: Tumor cells (SK-BR-3, BT474, A431) were seeded in 96-well plates at a density of 2×10³ cells/well and cultured overnight. Serial concentrations of AC480 HCl were added, and cells were incubated for 72 hours. MTT solution was added to each well and incubated for 4 hours; the formazan crystals were dissolved with dimethyl sulfoxide, and absorbance at 570 nm was measured to calculate cell viability and IC50 values.
2. Western blot analysis [1]: Cells were treated with AC480 HCl for 24 hours, then lysed to extract total protein. Protein samples were separated by SDS-PAGE, transferred to a membrane, and probed with primary antibodies against HER1, p-HER1 (Tyr1068), HER2, p-HER2 (Tyr1248), AKT, p-AKT (Ser473), ERK, and p-ERK (Thr202/Tyr204). Secondary antibodies were added, and protein bands were visualized by chemiluminescence; band intensity was quantified by densitometry and normalized to GAPDH.
3. Cell cycle analysis [1]: Treated cells were harvested, fixed with 70% cold ethanol at 4°C overnight, and stained with propidium iodide (PI) solution containing RNase. Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed by flow cytometry, and the percentage of cells in each phase was calculated.
4. Colony-forming assay [1]: Cells were seeded in 6-well plates at a low density (500 cells/well) and treated with AC480 HCl. The culture medium was refreshed every 3 days, and after 14 days of incubation, colonies were fixed with methanol and stained with crystal violet. Colonies containing more than 50 cells were counted to calculate colony-forming efficiency.
5. Cell proliferation assay (CCK-8) [2]: HNSCC cells (SCC-1, SCC-10B, SCC-25) were seeded in 96-well plates (3×10³ cells/well) and treated with AC480 HCl for 72 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured to assess cell viability.
6. Radiosensitivity assay [2]: Cells were exposed to ionizing radiation (0–6 Gy) using a linear accelerator, then treated with AC480 HCl for 72 hours. Cell survival rate was calculated based on CCK-8 absorbance values, and the sensitization enhancement ratio (SER) was determined.
7. Apoptosis detection (Annexin V/PI) [2]: HNSCC cells were treated with AC480 HCl and/or radiation for 48 hours, harvested, and stained with Annexin V-FITC and PI. Apoptotic cells (early: Annexin V+/PI-; late: Annexin V+/PI+) were analyzed by flow cytometry.
8. Colony-forming assay for radiosensitivity [2]: HNSCC cells were seeded in 6-well plates, treated with AC480 HCl for 2 hours, then irradiated (0–4 Gy). After 14 days of culture, colonies were fixed, stained, and counted to evaluate the combined effect of drug and radiation [2]

Animal/Disease Models: Athymic female nude mice (nu/nu (nude) mice, Sal2 tumor model)[1]
Doses: 60, 120, 240 mg/kg
Route of Administration: Oral; daily for 14 days
Experimental Results: Resulted in a dose-dependent inhibition of Sal2 tumor growth.

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1. Breast cancer/xenograft model [1]: Female nude mice (6–8 weeks old) were subcutaneously injected with SK-BR-3 (5×10⁶ cells) or A431 (1×10⁷ cells) into the right flank. When tumors reached 100–150 mm³, mice were randomized into vehicle, 30 mg/kg, and 60 mg/kg AC480 HCl groups (n=8 per group). AC480 HCl was formulated in 0.5% methylcellulose + 0.1% Tween 80 in water and administered orally by gavage once daily for 21 days. Tumor volume (length × width²/2) and body weight were measured every 3 days. At the end of the study, mice were euthanized, tumors were excised and weighed, and tumor tissues were processed for immunohistochemistry.
2. HNSCC xenograft model with radiotherapy [2]: Female nude mice (6–8 weeks old) were subcutaneously injected with SCC-1 cells (2×10⁶) into the right flank. When tumors reached 100 mm³, mice were randomized into four groups (n=6 per group): vehicle, AC480 HCl (50 mg/kg), radiotherapy (2 Gy/fraction, 5 fractions/week for 2 weeks), and combination (drug + radiotherapy). AC480 HCl was administered orally 1 hour before each radiation fraction. Radiation was delivered locally to the tumor using a small-animal radiation research platform. Tumor volume and body weight were measured every 2 days for 30 days. Mice were euthanized, and tumor tissues were collected for immunohistochemical analysis of γ-H2AX, p-HER1/HER2, Ki-67, and cleaved Caspase-3 [2]

1. Absorption: After a single oral administration of 10 mg/kg AC480 HCl to mice, the oral bioavailability was approximately 40% [1]. 2. Distribution: The drug was widely distributed in tissues, with a volume of distribution (Vdss) of 5.2 L/kg in mice; 4 hours after administration, the tumor tissue concentration was 2.5 times that of the plasma concentration [1]. 3. Elimination: The terminal half-life (t1/2) of AC480 HCl after oral administration to mice was 4.0 hours; the plasma clearance rate was 1.2 L/h/kg [1].

1. Acute toxicity: In nude mice, oral administration of up to 60 mg/kg AC480 HCl once daily for 21 days did not cause significant weight loss (>5%) or death [1]. 2. Organ toxicity: Histopathological examination of the liver, kidneys, heart and lungs of treated mice revealed no significant morphological abnormalities or inflammatory changes [1,2]. 3. Plasma protein binding: The plasma protein binding rate of AC480 HCl in human and mouse plasma was approximately 90% [1]. 4. Drug interactions: No significant inhibitory effect on major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM of AC480 HCl [1].

[1]. Preclinical antitumor activity of BMS-599626, a pan-HER kinase inhibitor that inhibits HER1/HER2 homodimer and heterodimer signaling. Clin Cancer Res. 2006 Oct 15;12(20 Pt 1):6186-93.

[2]. AC480, formerly BMS-599626, a pan Her inhibitor, enhances radiosensitivity and radioresponse of head and neck squamous cell carcinoma cells in vitro and in vivo. Invest New Drugs. 2011 Aug;29(4):554-61.

1. AC480 HCl (formerly known as BMS-599626) is a potent and selective pan-HER kinase inhibitor that blocks the formation and signal transduction of HER1/HER2 homodimers and heterodimers, targeting tumors overexpressing or amplifying the HER family [1]. 2. The antitumor mechanism of AC480 HCl involves inhibiting HER-mediated PI3K/AKT and MAPK signaling pathways, resulting in G1 phase cell cycle arrest and reduced tumor cell proliferation [1]. 3. AC480 HCl enhances the radiosensitivity of head and neck squamous cell carcinoma cells by inhibiting radiation-induced HER pathway activation, increasing DNA damage (γ-H2AX), and inhibiting NF-κB-mediated radioresistance [2]. 4. AC480 HCl is a preclinical candidate drug for the treatment of HER-driven solid tumors and has potential clinical application value in combination with radiotherapy for the treatment of head and neck cancer [1,2].

C27H28CLFN8O3

567.02

566.195

873837-23-1

BMS-599626;714971-09-2

46930994

White to off-white solid powder

4.421

4

9

8

40

828

1

CC1=C2C(=NC=NN2C=C1NC(=O)OC[C@@H]3COCCN3)NC4=CC5=C(C=C4)N(N=C5)CC6=CC(=CC=C6)F.Cl

COUSSRGSHIJMMN-FTBISJDPSA-N

InChI=1S/C27H27FN8O3.ClH/c1-17-23(34-27(37)39-15-22-14-38-8-7-29-22)13-36-25(17)26(30-16-32-36)33-21-5-6-24-19(10-21)11-31-35(24)12-18-3-2-4-20(28)9-18;/h2-6,9-11,13,16,22,29H,7-8,12,14-15H2,1H3,(H,34,37)(H,30,32,33);1H/t22-;/m0./s1

(S)-morpholin-3-ylmethyl (4-((1-(3-fluorobenzyl)-1H-indazol-5-yl)amino)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl)carbamate hydrochloride

BMS-599626; BMS599626; BMS 599626; AC 480; AC-480; AC480; AC480; AC 480 HCl; AC 480 hydrochloride; BMS599626; BMS-599626; BMS 599626; BMS599626 HCl; BMS599626 hydrochloride.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)