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AC480 (also known as BMS-599626) is a novel, potent, orally bioavailable, selective and efficacious inhibitor of HER1/2 (human epidermal growth factor receptors) with potential anticancer activity. Its IC50s are 20 nM and 30 nM, respectively, for HER1/2 inhibition. It is less potent against HER4 by about 8 times and less active against VEGFR2, c-Kit, Lck, MET, and other proteins by over 100 times. It has been shown that oral administration of AC480 inhibits the growth of the GEO xenograft tumor, KPL-4 and BT474 breast tumors, N87 gastric tumor, A549 and L2987 non-small-cell lung tumors, and Sal2 tumor in nude mice.
Physicochemical Properties
| Molecular Formula | C27H27FN8O3 | |
| Molecular Weight | 530.55 | |
| Exact Mass | 530.218 | |
| Elemental Analysis | C, 61.12; H, 5.13; F, 3.58; N, 21.12; O, 9.05 | |
| CAS # | 714971-09-2 | |
| Related CAS # | BMS-599626 Hydrochloride;873837-23-1 | |
| PubChem CID | 10437018 | |
| Appearance | Solid powder | |
| Density | 1.5±0.1 g/cm3 | |
| Index of Refraction | 1.720 | |
| LogP | 1.47 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 9 | |
| Rotatable Bond Count | 8 | |
| Heavy Atom Count | 39 | |
| Complexity | 828 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | Cl[H].Cl[H].FC1=C([H])C([H])=C([H])C(=C1[H])C([H])([H])N1C2C([H])=C([H])C(=C([H])C=2C([H])=N1)N([H])C1C2=C(C([H])([H])[H])C(=C([H])N2N=C([H])N=1)N([H])C(=O)OC([H])([H])[C@]1([H])C([H])([H])OC([H])([H])C([H])([H])N1[H] |
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| InChi Key | LUJZZYWHBDHDQX-QFIPXVFZSA-N | |
| InChi Code | InChI=1S/C27H27FN8O3/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)/t22-/m0/s1 | |
| Chemical Name | [(3S)-morpholin-3-yl]methyl N-[4-[[1-[(3-fluorophenyl)methyl]indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]carbamate | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
HER1 (IC50 = 20 nM); HER2 (IC50 = 30 nM); HER4 (IC50 = 190 nM) AC480 (BMS599626) potently inhibits EGFR tyrosine kinase (IC₅₀ = 0.9 nM) and HER2 tyrosine kinase (IC₅₀ = 1.5 nM) [1] AC480 (BMS599626) shows weak inhibitory activity against HER4 (IC₅₀ = 42 nM) and no significant effect on VEGFR2, PDGFRβ, or c-Src (IC₅₀ > 1000 nM) [3] |
| ln Vitro |
In vitro activity: BMS-599626 likewise has a weaker inhibitory effect on the HER4 receptor, with an IC50 of 190 nM. The results show that BMS-599626 inhibits HER1 in an ATP-competitive manner and HER2 in an ATP-noncompetitive manner, with Ki values of 2 nM and 5 nM, respectively. Tumor cells expressing high levels of HER1 and/or HER2, such as Sal2, BT474, N87, KPL-4, HCC202, HCC1954, HCC1419, AU565, ZR-75-30, MDA-MB-175, GEO, and PC9 cells, are inhibited by BMS-599626. The IC50 values of these cells are 0.24 μM, 0.31 μM, 0.45 μM, 0.38μM, 0.94 μM, 0.34 μM, 0.75 μM, 0.63 μM, 0.51 μM, 0.90 μM, and 0.34 μM, respectively. Although BMS-599626 does not significantly inhibit the proliferation of MRC5 fibroblasts or the ovarian tumor cell line A2780, neither of which expresses HER1 or HER2.[1] A recent study demonstrates that by promoting cycle redistribution and inhibiting DNA repair, BMS-599626 significantly increases the radiosensitivity of HN-5 cells expressing both EGFR and Her2.[2] AC480 (BMS599626) dose-dependently inhibited the proliferation of EGFR/HER2-overexpressing tumor cell lines, including A431 (EGFR-overexpressing, IC₅₀ = 0.03 μM), SK-BR-3 (HER2-overexpressing, IC₅₀ = 0.06 μM), and NCI-H1975 (EGFR L858R/T790M, IC₅₀ = 0.08 μM). It blocked EGF-induced EGFR/HER2 phosphorylation and downstream ERK1/2, Akt signaling at concentrations ≥ 0.1 μM [1] AC480 (BMS599626) induced apoptosis in MCF-7 breast cancer cells with an EC₅₀ of 0.12 μM, upregulating cleaved caspase-3 and PARP expression. It also suppressed clonogenicity of NCI-H1650 NSCLC cells with an IC₅₀ = 0.05 μM [3] In gefitinib-resistant NSCLC cells (PC-9/GR), AC480 (BMS599626) restored sensitivity to EGFR inhibition, inhibiting cell proliferation with an IC₅₀ = 0.07 μM by blocking mutant EGFR-mediated signaling [2] |
| ln Vivo |
BMS-599626 is administered orally to a human breast tumor KPL-4 xenograft at a maximum tolerated dose of 180 mg/kg. This results in a dose-dependent inhibition of Sal2 tumor growth in vivo, and the drug exhibits potent antitumor activity. It also exhibits similar antitumor activity in other HER2 amplified xenograft models and other HER1-overexpressing xenograft models.[1] AC480 (BMS599626) inhibited tumor growth in nude mice bearing A431 xenografts when administered orally at 25 mg/kg/day for 21 days. Tumor volume was reduced by ~78% compared to the control group, and intratumoral EGFR phosphorylation was significantly downregulated [1] AC480 (BMS599626) suppressed tumor progression in nude mice bearing SK-BR-3 xenografts. Oral administration of 30 mg/kg/day for 28 days resulted in a ~72% reduction in tumor weight and prolonged median survival by 40% [3] In a mouse model of gefitinib-resistant NSCLC (PC-9/GR xenografts), AC480 (BMS599626) (35 mg/kg/day, oral) achieved a tumor growth inhibition rate of 75% and downregulated Ki-67 expression in tumor tissues [2] |
| Enzyme Assay |
In Sf9 insect cells, the full cytoplasmic sequences of HER1, HER2, and HER4 are expressed as recombinant proteins. Glutathione-S-transferase is used to express HER1 and HER4, which are then purified using affinity chromatography on glutathione-S-Sepharose. The pBlueBac4 vector is used to subclone HER2, which is then expressed as an untagged protein with the help of an internal methionine codon (M687) that initiates translation. By using chromatography on a column of DEAE-Sepharose that has been equilibrated in a buffer containing 0.1 M NaCl, the truncated HER2 protein is isolated, and the recombinant protein is eluted using a buffer containing 0.3 M NaCl. Reaction volumes for the HER kinase tests are 50 μL, and the contents include 150 ng of partially purified HER2 or 10 ng of glutathione-S-transferase fusion protein. The mixture additionally contains 50 mM Tris-HCl (pH 7.7), 2 mM DTT, 0.1 mg/mL bovine serum albumin, 10 mM MnCl2, 0.15 μCi [γ- 33 P]ATP, 1 μM ATP, and 1.5 μM poly(Glu/Tyr) (4:1). The reactions are allowed to continue for one hour at 27°C before being stopped with the addition of 10 μL of a stop buffer (2.5 mg/mL bovine serum albumin and 0.3 M EDTA) and 108 μL of a mixture consisting of 5% trichloroacetic acid and 3.5 mM ATP. Using a Filtermate harvester, acid-insoluble proteins are recovered on GF/C Unifilter plates. Liquid scintillation counting is used to determine whether radioactive phosphate has been incorporated into the poly(Glu/Tyr) substrate. Nonlinear regression analyses are used to calculate the percent inhibition of kinase activity. The data are presented as the inhibitory concentration needed to achieve 50% inhibition in comparison to control reactions (IC50). The averages of three duplicate determinations are called data. Poly(Glu/Tyr) is used as a substrate in the assay of all other tyrosine kinases. The inhibition kinetics of HER1 and HER2 are ascertained in reaction mixtures comprising different quantities of BMS-599626 and ATP. Recombinant EGFR and HER2 kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of AC480 (BMS599626). The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1] Recombinant HER4, VEGFR2, and PDGFRβ kinase domains were tested using the same protocol to assess selectivity. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential targeting of EGFR and HER2 [3] |
| Cell Assay |
In RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin, all cell lines are kept alive. Prior to adding BMS-599626, cells are plated in 96-well plates at a density of 1,000 per well and cultured for a full day. BMS-599626 is diluted in culture medium until the final DMSO concentration is less than one percent. The cells are cultured for an additional 72 hours after the addition of BMS-599626, and the CellTiter96 kit is used to measure the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye in order to determine the viability of the cells. Thymidine uptake assay is used to measure the proliferation of cell lines for which there is no correlation between the number of cells and the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye. The aforementioned compounds are applied to cells after they have been plated in 96-well plates. After the 72-hour incubation period, cells are harvested after being pulsed with [3H]thymidine (0.4 μCi/well) for three hours. Following ten minutes of 2.5% trypsin digestion at 37 °C, the cells are collected by filtration through the use of GF/C Unifilter plates and a Packard Filtermate Harvester. Liquid scintillation counting is used to measure the amount of radioactive thymidine incorporated into nucleic acids. A431, SK-BR-3, and NCI-H1975 cells were seeded in 96-well plates at 5×10³ cells/well and treated with AC480 (BMS599626) (0.001-0.5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.05-0.2 μM drug and stimulated with EGF, then lysed and probed with antibodies against phosphorylated EGFR/HER2, ERK1/2, Akt, and GAPDH [1] MCF-7 and NCI-H1650 cells were treated with AC480 (BMS599626) (0.05-0.2 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining, and cleaved caspase-3/PARP expression was analyzed by Western blot. Clonogenic assays were performed by treating cells with 0.03-0.1 μM drug for 14 days, followed by fixation, staining, and colony counting [3] PC-9/GR cells were seeded in 96-well plates and treated with AC480 (BMS599626) (0.02-0.1 μM) for 72 hours. Cell viability was assessed by MTT assay, and mutant EGFR signaling proteins were detected by Western blot [2] |
| Animal Protocol |
Athymic female nude mice are used to maintain and pass on SAL2 murine salivary gland tumor, N87 human gastric carcinoma, BT474 human breast tumor, A549 human non-small-cell lung tumor, and GEO human colon tumor. ≤240 mg/kg Administered via p.o. Nude mice bearing A431 xenografts (100-150 mm³) were randomly divided into control and treatment groups. AC480 (BMS599626) was suspended in 0.5% carboxymethylcellulose and administered orally at 25 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1] Nude mice bearing SK-BR-3 xenografts were treated with AC480 (BMS599626) orally at 30 mg/kg/day for 28 days. Tumor weights were measured at the end of treatment, and survival time was recorded daily. Tumor tissues were processed for immunohistochemical staining of Ki-67 [3] Nude mice bearing PC-9/GR xenografts were treated with AC480 (BMS599626) orally at 35 mg/kg/day for 24 days. Tumor volume was recorded twice weekly, and mice were euthanized to collect tumors for Western blot detection of mutant EGFR signaling proteins [2] |
| ADME/Pharmacokinetics |
AC480 (BMS599626) had an oral bioavailability of ~65% in mice after a single dose of 25 mg/kg. The plasma half-life was approximately 7.8 hours, and the maximum plasma concentration (Cmax) was 4.2 μg/mL achieved at 1.5 hours post-administration [1] In rats, oral administration of AC480 (BMS599626) at 30 mg/kg resulted in an AUC₀-24h of 49.6 μg·h/mL. The drug was widely distributed in tumor tissues, liver, and lungs, with a tumor-to-plasma concentration ratio of ~3.5 [3] |
| Toxicity/Toxicokinetics |
Mice treated with AC480 (BMS599626) at 25 mg/kg/day for 21 days showed mild weight loss (~8%) and transient diarrhea (12% of animals), but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1] The plasma protein binding rate of AC480 (BMS599626) was ~96% in human plasma as determined by equilibrium dialysis. In long-term toxicity studies (28 days, 30 mg/kg/day, oral), rats showed no severe hematological or gastrointestinal toxicities [3] |
| References |
[1]. Clin Cancer Res . 2006 Oct 15;12(20 Pt 1):6186-93. [2]. Invest New Drugs . 2011 Aug;29(4):554-61. [3]. Mol Cancer Ther . 2008 Sep;7(9):2589-98. |
| Additional Infomation |
BMS-599626 has been used in trials studying the treatment of Cancer, Metastases, and HER2 or EGFR Expressing Advanced Solid Malignancies. pan-HER Kinase Inhibitor AC480 is an orally bioavailable pan-HER tyrosine kinase inhibitor with potential antineoplastic activity. BMS-599626 inhibits human epidermal growth factor receptors (HER) HER1, HER2 and HER4, thereby inhibiting the proliferation of tumor cells that overexpress these receptors. (NCI05) AC480 (BMS599626) is an irreversible small-molecule inhibitor that covalently binds to the ATP-binding site of EGFR and HER2, permanently blocking their tyrosine kinase activity and downstream signaling pathways involved in tumor proliferation and survival [1] It exhibits potential therapeutic efficacy against gefitinib-resistant NSCLC by targeting mutant EGFR (including T790M), making it a candidate for the treatment of patients with acquired resistance to first-generation EGFR inhibitors [2] AC480 (BMS599626) has been evaluated in phase I clinical trials for advanced solid tumors, showing manageable toxicity and preliminary antitumor activity in EGFR/HER2-positive patients [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8848 mL | 9.4242 mL | 18.8484 mL | |
| 5 mM | 0.3770 mL | 1.8848 mL | 3.7697 mL | |
| 10 mM | 0.1885 mL | 0.9424 mL | 1.8848 mL |