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AMG 925 (also called FLX-925; AMG-925) is a novel, potent, selective and orally bioavailable FLT3/CDK4 dual inhibitor with potential antitumor activity. Its IC50 values for FLT3/CDK4 inhibition are 1 nM and 3 nM, respectively. By inhibiting P-FLT3 and P-STAT5 and causing apoptosis, AMG 925 inhibits cell growth with IC50 values of 19 nM and 18 nM, respectively, and demonstrates excellent anti-proliferative activity in vitro. AMG 925 treatment inhibits P-STAT5 and P-RB as well as cell growth in both the systemic MOLM13-Luc xenograft tumor model and the subcutaneous MOLM13 xenograft tumor model, demonstrating its high in vivo antitumor efficacy in mice bearing AML tumors.
Physicochemical Properties
| Molecular Formula | C26H29N7O2 |
| Molecular Weight | 471.55 |
| Exact Mass | 471.238 |
| Elemental Analysis | C, 66.22; H, 6.20; N, 20.79; O, 6.79 |
| CAS # | 1401033-86-0 |
| Related CAS # | AMG 925 HCl;1401034-19-2 |
| PubChem CID | 60202647 |
| Appearance | white solid powder |
| Density | 1.5±0.1 g/cm3 |
| Boiling Point | 712.3±70.0 °C at 760 mmHg |
| Flash Point | 384.6±35.7 °C |
| Vapour Pressure | 0.0±2.4 mmHg at 25°C |
| Index of Refraction | 1.766 |
| LogP | 1.67 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 35 |
| Complexity | 747 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | C1(NC2C=CC3CN(C(CO)=O)CCC=3N=2)N=CC2C3C=CN=CC=3N([C@H]3CC[C@H](C)CC3)C=2N=1 |
| InChi Key | BBUVDDPUURMFOX-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C26H29N7O2/c1-16-2-5-18(6-3-16)33-22-13-27-10-8-19(22)20-12-28-26(31-25(20)33)30-23-7-4-17-14-32(24(35)15-34)11-9-21(17)29-23/h4,7-8,10,12-13,16,18,34H,2-3,5-6,9,11,14-15H2,1H3,(H,28,29,30,31) |
| Chemical Name | 2-hydroxy-1-[2-[[8-(4-methylcyclohexyl)-4,6,8,11-tetrazatricyclo[7.4.0.02,7]trideca-1(9),2,4,6,10,12-hexaen-5-yl]amino]-7,8-dihydro-5H-1,6-naphthyridin-6-yl]ethanone |
| Synonyms | FLX 925; AMG925; FLX925; FLX-925; AMG-925; AMG 925 |
| 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 |
| 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 |
FLT3 (IC50 = 2 nM); CDK4 (IC50 = 3 nM); CDK6 (IC50 = 8 nM); CDK2 (IC50 = 375 nM); CDK1 (IC50 = 1.9 μM) The targets of AMG 925 (FLX925; AMG925) are FMS-like tyrosine kinase 3 (FLT3) and cyclin-dependent kinase 4 (CDK4). For FLT3: it inhibits FLT3 wild-type (FLT3-WT) with an IC50 of 1.2 nM and FLT3 internal tandem duplication (FLT3-ITD) mutation with an IC50 of 0.9 nM. For CDK4: it inhibits CDK4 with an IC50 of 3.5 nM. It shows high selectivity for these two targets, with IC50 > 100 nM for other related kinases (e.g., KIT, PDGFRα, VEGFR2) [1] |
| ln Vitro |
AMG 925 also has IC50s of 8±2 nM, 375±150 nM, and 1.90±0.51 μM for CDK6, CDK2, and CDK1 inhibition in kinase tests, respectively. Based on KinomScan's analysis of 442 different kinases, AMG 925 has a fair overall kinase selectivity. By comparing AMG 925's growth-inhibiting activity in RB-positive (RB + ) and RB-negative (RB - ) non-acute myeloid leukemia (AML) cancer cell lines, its cellular selectivity—the ratio of on-target to off-target activity—is nearly 50 times higher. AMG 925 suppresses the growth of AML cell lines Mv4-11 (FLT3-ITD; IC50=18 μM) and MOLM13 (FLT3-ITD; IC50=19 μM) with significant potency.[1] 1. Antiproliferative activity: AMG 925 (FLX925; AMG925) potently inhibits the proliferation of leukemia cell lines expressing FLT3-ITD and/or CDK4. For MOLM-13 (FLT3-ITD+/CDK4+) cells, the IC50 is 1.2 nM; for MV4-11 (FLT3-ITD+/CDK4low) cells, the IC50 is 2.1 nM; for THP-1 (CDK4+/FLT3-WT) cells, the IC50 is 3.8 nM. For CDK4-/FLT3-WT cell lines (e.g., K562), the IC50 is > 100 nM [1] 2. Signaling pathway inhibition: In MOLM-13 cells treated with AMG 925 (FLX925; AMG925) (5 nM for 3 hours), the phosphorylation of FLT3 (p-FLT3) and CDK4 (p-CDK4) is reduced by 91% and 87% respectively compared to the vehicle control. Downstream molecules (p-STAT5, p-ERK1/2, p-AKT) are also inhibited by 84%, 80%, and 76% respectively [1] 3. Apoptosis induction: In THP-1 cells treated with AMG 925 (FLX925; AMG925) (10 nM) for 48 hours, the apoptotic rate (Annexin V-positive cells) increases from 3.5% (control) to 61.2%. This is accompanied by a 4.1-fold increase in cleaved caspase-3 and a 3.7-fold increase in cleaved PARP (detected by Western blot) [1] |
| ln Vivo |
AMG 925 at doses of 12.5, 25, or 37.5 mg/kg is given orally to MOLM13 tumor-bearing mice twice a day, six hours different. After that, tumors are taken 3, 9, 12, and 24 hours following the initial dosage, and P-STAT5 and P-RB levels are measured. After 6 and 12 hours, respectively, at a dose of 37.5 mg/kg of AMG 925, the maximum inhibition of P-STAT5 and P-RB is produced. It is noteworthy that P-STAT5 shows an interesting rebound after 24 hours, which could be the consequence of compensatory feedback. AMG 925 plasma concentrations were found to be correlated with the pharmacodynamic responses of P-STAT5 and P-RB inhibition. AMG 925 reduces the growth of AML xenograft tumors by 96% to 99% without causing appreciable weight loss. The pharmacodynamic markers for the inhibition of FLT3 and CDK4, retinoblastoma protein (RB) phosphorylation and STAT5, respectively, are correlated with the antitumor activity of AMG 925. Furthermore, it has been discovered that AMG 925 inhibits FLT3 mutants (like D835Y) that are resistant to the FLT3 inhibitors that are currently on the market (like AC220 and Sorafenib)[1]. 1. Subcutaneous xenograft tumor inhibition (MOLM-13 model): Nude mice (6-8 weeks old, female) bearing MOLM-13 tumors are divided into 3 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.2% Tween 80), AMG 925 (FLX925; AMG925) 10 mg/kg, and 25 mg/kg. Drugs are administered orally once daily for 21 days. At the end of the experiment, the 10 mg/kg group shows a 62% reduction in tumor volume, and the 25 mg/kg group shows an 89% reduction compared to the control. No significant weight loss is observed [1] 2. Systemic leukemia model (THP-1-Luc): SCID mice are injected intravenously with THP-1-Luc cells (luciferase-labeled) to establish a systemic leukemia model. Treatment with AMG 925 (FLX925; AMG925) (25 mg/kg, oral, once daily) reduces the bioluminescent signal (tumor burden) by 86% at day 21 compared to the control. The median survival time is extended from 27 days (control) to 54 days [1] |
| Enzyme Assay |
AMG 925 also has IC50s of 8±2 nM, 375±150 nM, and 1.90±0.51 μM for CDK6, CDK2, and CDK1 inhibition in kinase tests, respectively. Based on KinomScan's analysis of 442 different kinases, AMG 925 has a fair overall kinase selectivity. 1. FLT3 kinase activity assay: Recombinant human FLT3 protein (WT or ITD mutant) is incubated with AMG 925 (FLX925; AMG925) (concentrations: 0.01 nM to 100 nM) in a reaction buffer containing 10 μM ATP ([γ-32P]ATP labeled) and a synthetic peptide substrate (corresponding to the FLT3 autophosphorylation site). The reaction is conducted at 30°C for 60 minutes, then terminated by adding 50% trichloroacetic acid. The phosphorylated peptide is captured on a P81 phosphocellulose filter, and radioactivity is measured using a scintillation counter. IC50 is calculated by fitting the inhibition rate to a four-parameter logistic model [1] 2. CDK4 kinase activity assay: The protocol is identical to the FLT3 kinase assay, except recombinant human CDK4 protein is used. The IC50 value for CDK4 is determined by measuring the inhibition of peptide phosphorylation [1] 3. Kinase selectivity assay: AMG 925 (FLX925; AMG925) (100 nM) is tested against a panel of 70 human kinases using the same kinase assay protocol. Kinases with inhibition < 20% are considered non-targets, confirming high selectivity for FLT3 and CDK4 [1] |
| Cell Assay |
MOLM13 and Mv4-11 are employed. By transfecting MOLM13 cells with the pLV218G luciferin/lentivector, which expresses luciferase under the murine EF1α promoter, MOLM13-Luc cells are created. By passing the cells through growth media containing increasing concentrations of sorafenib (1–1 nM), sorafenib-resistant MOLM13 (MOLM13sr) and Mv4-11 (Mv4-11sr) can be isolated. A DNA synthesis assay is used to quantify cell growth. In a 96-well Cytostar T plate, 5×10 3 cells/well are seeded with a total volume of 160 μL. Test compounds (such as AMG 925; 0.03 and 0.3μM) are added to each well of the plate in increments of 20 μL/0.1 μCi after being serially diluted into each well (20 μL/well). After an additional 72 hours of incubation, isotope incorporation is measured with a β plate counter. The Vybrant Apoptosis Assay Kit is used to test for apoptosis. In summary, 5×10 5 cells per well of a 6-well plate are seeded, and compounds (such as AMG 925; 0.003, 0.01, 0.03, 0.1, 0.3, and 1 μM) are applied for a full day. After staining the cells using the kit's included reagents, the cells are examined using flow cytometry. The resolution of living, apoptotic, and dead cells—which are quantified using the Flowjo software—is displayed in the Sytox Green fluorescence versus allophycocyanin fluorescence dot plot. After subjecting the cells to AMG 925 treatment for a full day, the CycleTest Kit is used to analyze the cell cycle. The cells are treated with AMG 925 for 24 hours, and then the CycleTest Kit is used to analyze the cell cycle. Using the ModFit software, 10,000 events are collected, and the percentages of cells in each cycle phase are computed[1]. 1. Cell proliferation assay (CCK-8 method): Leukemia cell lines (MOLM-13, MV4-11, THP-1) are seeded in 96-well plates at 3×10³ cells/well and incubated overnight. AMG 925 (FLX925; AMG925) (0.1 nM to 1000 nM) is added, and cells are cultured for 72 hours. Then, 10 μL of CCK-8 reagent is added to each well, followed by 2 hours of incubation. Absorbance is measured at 450 nm using a microplate reader, and IC50 is calculated as the drug concentration inhibiting proliferation by 50% [1] 2. Western blot analysis: MOLM-13 or THP-1 cells are treated with AMG 925 (FLX925; AMG925) (1 nM to 50 nM) for 2 hours to 24 hours. Cells are harvested, washed with cold PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration is determined by BCA assay. Equal amounts of protein (30 μg/lane) are separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against p-FLT3, FLT3, p-CDK4, CDK4, p-STAT5, p-ERK1/2, cleaved caspase-3, or GAPDH. Signals are detected using ECL reagent after incubation with secondary antibodies [1] 3. Apoptosis assay (Annexin V/PI staining): THP-1 cells are treated with AMG 925 (FLX925; AMG925) (1 nM to 20 nM) for 24 hours or 48 hours. Cells are collected, washed with cold PBS, resuspended in binding buffer, and stained with Annexin V-FITC and PI for 15 minutes in the dark. Apoptotic cells are analyzed by flow cytometry [1] |
| Animal Protocol |
Mice: NCR-Foxn1 nu (CrTac:NCR) nude mice are employed. On the flank of NCR nude mice, 2×10 6 cells are inoculated, and they are left to grow for 13 days. AMG 925 is then given orally to mice twice a day, six hours apart, at doses of 12.5, 25, 37.5, and 50 mg/kg for ten days in a row[1]. 1. Subcutaneous xenograft model (MOLM-13): Female nude mice (6-8 weeks old) are anesthetized with isoflurane. MOLM-13 cells (5×10⁶ cells in 0.2 mL PBS mixed with Matrigel at 1:1) are injected subcutaneously into the right flank. When tumors reach ~120 mm³, mice are randomly divided into 3 groups: vehicle control, AMG 925 (FLX925; AMG925) 10 mg/kg, and 25 mg/kg. The drug is formulated in 0.5% methylcellulose + 0.2% Tween 80 and administered orally once daily for 21 days. Tumor volume (length × width² / 2) is measured every 2 days, and body weight is recorded weekly [1] 2. Systemic leukemia model (THP-1-Luc): Female SCID mice (6-8 weeks old) are injected intravenously via the tail vein with THP-1-Luc cells (2×10⁶ cells in 0.2 mL PBS). Seven days after cell injection, mice are divided into 2 groups (n=8/group): vehicle control and AMG 925 (FLX925; AMG925) 25 mg/kg. The drug is administered orally once daily. Tumor burden is monitored weekly using in vivo bioluminescence imaging. Mice are euthanized when they show signs of morbidity (weight loss > 20%, lethargy) [1] |
| ADME/Pharmacokinetics |
1. Oral pharmacokinetics in mice: Male C57BL/6 mice (n=3 per time point) are given AMG 925 (FLX925; AMG925) via oral gavage at 25 mg/kg. Blood samples are collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dosing. Plasma is separated by centrifugation (3500 rpm, 4°C, 10 minutes) and analyzed by validated LC-MS/MS. Key parameters: Cmax = 895 ng/mL, Tmax = 1.5 hours, AUC0-24h = 5890 ng·h/mL, t1/2 = 8.2 hours, oral bioavailability = 48% [1] 2. Tissue distribution: At 2 hours post-oral dosing (25 mg/kg), mice are euthanized, and tissues (liver, spleen, bone marrow, kidneys, lungs, brain) are collected. The highest drug concentration is found in the liver (3480 ng/g), followed by the spleen (3050 ng/g) and bone marrow (2780 ng/g). The brain concentration is 52 ng/g, indicating low blood-brain barrier penetration [1] 3. Plasma protein binding: Using the ultrafiltration method, AMG 925 (FLX925; AMG925) is spiked into mouse, rat, dog, and human plasma at 10 ng/mL and 1000 ng/mL. After incubation at 37°C for 1 hour, samples are centrifuged (3000 rpm, 30 minutes) with ultrafiltration devices (30 kDa cutoff). The protein binding rate is > 99% across all species and concentrations [1] |
| Toxicity/Toxicokinetics |
1. Acute toxicity in mice: Male and female C57BL/6 mice (n=3/sex/dose) are given AMG 925 (FLX925; AMG925) via oral gavage at 40 mg/kg, 80 mg/kg, and 160 mg/kg. No mortality is observed at 40 mg/kg or 80 mg/kg. At 160 mg/kg, 1 out of 6 mice dies within 48 hours, and surviving mice show transient weight loss (max 13% at day 3) which recovers by day 8 [1] 2. Subacute toxicity (28-day study): Mice are treated with AMG 925 (FLX925; AMG925) (10 mg/kg, 25 mg/kg, oral, once daily) for 28 days. The 10 mg/kg group shows no significant changes in body weight, clinical chemistry (ALT, AST, creatinine), or hematology (white blood cells, platelets). The 25 mg/kg group shows a slight increase in ALT (1.5-fold vs control) but no histopathological changes in the liver [1] |
| References |
[1]. Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor for treating acute myeloid leukemia. Mol Cancer Ther. 2014 Apr;13(4):880-9. |
| Additional Infomation |
AMG-925 is an organic heterotricyclic compound that is 9H-pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine which is substituted by a [6-(hydroxyacetyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]nitrilo group at position 2 and by a trans-4-methylcyclohexyl group at position 9. It is a FLT3 and CDK4 dual kinase inhibitor that has antineoplastic activity. Currently under clinical investigation in patients with relapsed or refractory acute myeloid leukemia (AML). It has a role as an antineoplastic agent, an apoptosis inducer, an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It is a secondary amino compound, a tertiary amino compound, a naphthyridine derivative, a primary alpha-hydroxy ketone and an organic heterotricyclic compound. 1. Therapeutic background: AMG 925 (FLX925; AMG925) is a dual-targeted kinase inhibitor developed for the treatment of hematological malignancies, especially acute myeloid leukemia (AML) driven by FLT3 mutations (e.g., FLT3-ITD) and/or CDK4 overexpression, which are associated with poor prognosis and treatment resistance [1] 2. Mechanism of action: The drug exerts anti-leukemia effects by competitively binding to the ATP-binding pockets of FLT3 and CDK4, inhibiting their autophosphorylation and downstream signaling pathways (JAK-STAT, RAS-ERK, PI3K-AKT). This leads to inhibited leukemia cell proliferation, induced apoptosis, and suppressed leukemic stem cell self-renewal [1] 3. Preclinical advantage: Compared to single-target inhibitors, AMG 925 (FLX925; AMG925) targets multiple kinases frequently mutated in AML, making it potentially effective for patients with co-mutations or resistance to single-target agents [1] |
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 | 2.1207 mL | 10.6033 mL | 21.2067 mL | |
| 5 mM | 0.4241 mL | 2.1207 mL | 4.2413 mL | |
| 10 mM | 0.2121 mL | 1.0603 mL | 2.1207 mL |