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Flumatinib (HHGV-678; HHGV678; Hansoh Xinfu), the first approved 2nd generation TKI in China and an imatinib derivative, is a potent multi-kinase inhibitor with anticancer activity. It has IC50 values of 1.2 nM, 307.6 nM, and 2662 nM for c-Abl, PDGFRβ, and c-Kit inhibition, respectively. It functions as a BCR-ABL/PDGFR/KIT selective inhibitor, basically. Some KIT mutants with activation loop mutations (D820G, N822K, Y823D, and A829P) were successfully treated with flumatinib to overcome their drug resistance. Studies conducted in vivo have consistently indicated that flumatinib is more effective than imatinib or sunitinib in treating 32D cells that have the secondary mutation Y823D. Clinical trials for flumatinib are presently taking place in Phase I/II settings to treat chronic myelogenous leukemia (CML).
Physicochemical Properties
| Molecular Formula | C29H29F3N8O | |
| Molecular Weight | 562.59 | |
| Exact Mass | 562.242 | |
| Elemental Analysis | C, 61.91; H, 5.20; F, 10.13; N, 19.92; O, 2.84 | |
| CAS # | 895519-90-1 | |
| Related CAS # | Flumatinib mesylate;895519-91-2;Flumatinib-d3 | |
| PubChem CID | 46848036 | |
| Appearance | Off-white to yellow solid powder | |
| LogP | 5.336 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 11 | |
| Rotatable Bond Count | 7 | |
| Heavy Atom Count | 41 | |
| Complexity | 841 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(C1C=C(C(F)(F)F)C(CN2CCN(C)CC2)=CC=1)NC1C=C(NC2N=C(C3C=CC=NC=3)C=CN=2)C(C)=NC=1 |
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| InChi Key | BJCJYEYYYGBROF-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C29H29F3N8O/c1-19-26(38-28-34-9-7-25(37-28)21-4-3-8-33-16-21)15-23(17-35-19)36-27(41)20-5-6-22(24(14-20)29(30,31)32)18-40-12-10-39(2)11-13-40/h3-9,14-17H,10-13,18H2,1-2H3,(H,36,41)(H,34,37,38) | |
| Chemical Name | 4-[(4-methylpiperazin-1-yl)methyl]-N-[6-methyl-5-[(4-pyridin-3-ylpyrimidin-2-yl)amino]pyridin-3-yl]-3-(trifluoromethyl)benzamide | |
| 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 |
c-Abl (IC50 = 1.2 nM); PDGFRβ (IC50 = 307.6 nM); c-Kit (IC50 = 2662 nM)
Bcr-Abl [1] BCR-ABL/PDGFR (Platelet-Derived Growth Factor Receptor)/KIT (Proto-Oncogene Proteins c-kit) [2] |
| ln Vitro |
Flumatinib (HH-GV-678) can primarily inhibit Bcr-Abl'sautophosphorylation in K562 cell. At greater concentrations, flumatinib can prevent the phosphorylation of PDGFR in Swiss3T3 cells and c-Kit in Mo7e cells; however, it has little to no effect on EGFR, KDR, c-Src, and HER2 tyrosine kinases [1]. Drug resistance in some KIT mutants with activation loop mutations (D820G, N822K, Y823D, and A829P) was successfully overcome by flumatinib (HHGV678)[2]. Flumatinib (HH-GV-678) acts as a novel selective inhibitor of Bcr-Abl, which exhibits superior efficacy to imatinib and can effectively override imatinib resistance in Bcr-Abl-positive leukemic cells; it inhibits the proliferation of Bcr-Abl-positive tumor cells, induces cell apoptosis, and reduces the phosphorylation level of Bcr-Abl protein as detected by Western blotting [1] Flumatinib (HH-GV-678) effectively overcomes the drug resistance of certain KIT mutants with activation loop mutations (D820G, N822K, Y823D, and A829P) in transformed 32D cells; it inhibits the phosphorylation of KIT and its downstream signaling effectors ERK1/2 and STAT3, and shows a more potent inhibitory effect on these phosphorylated proteins in 32D-V559D+Y823D cells compared with imatinib and sunitinib [2] |
| ln Vivo |
Female Balb/cA-nu/nu mice, six weeks old and weighing 17–19 g each, were acquired from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and raised in a particular environment free of pathogens. One million KIT mutant transformed 32D cells were injected subcutaneously (s.c.) into the right flank of each mouse. For the next 14 days, mice were divided into groups based on randomization (n = 8–10 per group) and given oral gavage treatments with vehicle, imatinib, flumatinib, or sunitinib. In nude mouse xenograft models bearing Bcr-Abl-positive leukemia cells, Flumatinib (HH-GV-678) demonstrates better efficacy than imatinib, significantly inhibiting tumor growth and improving the survival rate of mice [1] In BALB/c nude mice subcutaneously injected with 32D-V559D+Y823D cells, Flumatinib (HH-GV-678) has superior efficacy to imatinib or sunitinib in improving the survival of mice; a single dose of 75 mg/kg of this drug can be detected in mouse plasma and tumor tissues, and it continuously inhibits the phosphorylation of KIT, ERK1/2, and STAT3 in tumor tissues at different time points after administration [2] |
| Enzyme Assay | Retroviral constructs based on murine stem cell viruses that carried either activating mutant D816V (816 Asp→Val) KIT cDNA or murine–human hybrid WT KIT cDNA were kindly provided by Michael H. Tomasson (Washington University School of Medicine, St. Louis, MO, USA). The intracellular region of human KIT was fused in-frame with the extracellular and transmembrane regions of murine KIT to create hybrid KIT alleles. It has been demonstrated that substituting homologous murine sequences for the human extracellular and transmembrane domains of KIT can increase the expression efficiency and preserve the capacity to transform some KIT mutants in murine cells. The enhanced GFP cassette from the downstream internal ribosomal entry site causes KIT alleles to coexpress with enhanced GFP. In accordance with Molecular Cloning's third edition of Protocol 3, mutagenesis, the KIT point mutations were produced. Mutagenic primers were created to avoid the deleted sequence in insertion mutagenesis and to harbor the deleted sequence in deletion mutagenesis, respectively. Primestar Hot Start DNA polymerase (Takara, Dalian, China) with high fidelity was used in all of the PCRs mentioned above. Takara was also the source of additional enzymes used in the aforementioned experiments. By using direct sequencing, the sequences of every mutant in this study were confirmed. |
| Cell Assay |
In triplicate, cells (5 × 103) were incubated with different concentrations of imatinib, flumatinib, or sunitinib in 96-well plates for 72 hours, using 200 μL medium containing or lacking IL-3. The cells were incubated for 4 hours after we added MTT. The insoluble purple formazan product was dissolved into a colored solution by adding a solubilization solution, which is a solution of the detergent SDS in diluted hydrochloric acid. A spectrophotometer was used to measure the absorbance of this colored solution at 570 nm using a 650 nm reference filter. The ratio of average absorbance in drug-treated wells to no-drug controls was used to plot growth inhibition. GraphPad Prism version 5, a program for curve-fitting, was used to determine the IC50 values. Cultivate Bcr-Abl-positive leukemic cells in vitro, treat the cells with different concentrations of Flumatinib (HH-GV-678) and imatinib for a specific period, detect cell proliferation and apoptosis through cell viability assays and flow cytometry; extract total cell lysates and use Western blotting to determine the phosphorylation level of Bcr-Abl and the expression of downstream signaling pathway proteins, and apply reverse transcriptase polymerase chain reaction to detect the mRNA expression of related genes [1] Cultivate 32D cells transformed by various KIT mutants in vitro, treat the cells with Flumatinib (HH-GV-678), imatinib, and sunitinib at the indicated concentrations for 4 hours, extract total cell lysates, and analyze the levels of phosphorylated and total KIT, ERK1/2, and STAT3 proteins by Western blotting [2] |
| Animal Protocol |
75mg/kg; Oral gavage Six-week-old female Balb/cA-nu/nu mice weighing 17–19 g bearing 32D-V559D or 32D-V559D+Y823D tumors Establish xenograft models by subcutaneously inoculating Bcr-Abl-positive leukemic cells into nude mice, randomly divide the mice into groups, and administer Flumatinib (HH-GV-678) and imatinib to the mice by oral gavage according to a certain dosage regimen; continuously observe and record the tumor growth volume and the survival status of the mice [1] Construct tumor models by subcutaneously injecting 32D-V559D or 32D-V559D+Y823D cells into BALB/c nude mice, randomly allocate the animals into groups, and treat them by oral gavage with vehicle, imatinib (150 mg/kg), Flumatinib (HH-GV-678) (75 mg/kg), or sunitinib (50 mg/kg) according to the indicated dosage regimen and dosing period; for the single-dose pharmacokinetic experiment, mice bearing 32D-V559D+Y823D tumors are given a single dose of 75 mg/kg of this drug, and the mice are sacrificed at different times post-dosing to collect plasma and tumor tissue samples [2] |
| ADME/Pharmacokinetics |
The parent drug Flumatinib (HH-GV-678) is the main form recovered in human plasma, urine, and feces of chronic myelogenous leukemia (CML) patients [3] Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry reveals 34 metabolites of Flumatinib (HH-GV-678) in CML patients, with 7 primary metabolites confirmed by comparison with synthetic reference standards [3] The main metabolic pathways of Flumatinib (HH-GV-678) in humans include N-demethylation, N-oxidation, hydroxylation, and amide hydrolysis; several phase II glucuronidation and acetylation products are also detected in plasma, urine, and feces [3] Flumatinib (HH-GV-678) is predominantly metabolized by amide bond cleavage to yield two corresponding hydrolytic products, and the electron-withdrawing groups of trifluoromethyl and pyridine facilitate the amide bond cleavage [3] |
| References |
[1]. HH-GV-678, a novel selective inhibitor of Bcr-Abl, outperforms imatinib and effectively overrides imatinib resistance. Leukemia. 2010 Oct;24(10):1807-9. [2]. Flumatinib, a selective inhibitor of BCR-ABL/PDGFR/KIT, effectively overcomes drug resistance of certain KIT mutants. Cancer Sci. 2013 Nov 10. [3]. Metabolism of flumatinib, a novel antineoplastic tyrosine kinase inhibitor, in chronic myelogenous leukemia patients. Drug Metab Dispos. 2010 Aug;38(8):1328-40. |
| Additional Infomation |
Flumatinib has been used in trials studying the treatment of Myelogenous Leukemia, Chronic. Flumbatinib is an orally bioavailable tyrosine kinase inhibitor, with potential antineoplastic activity. Upon administration, flumbatinib inhibits the wild-type forms of Bcr-Abl, platelet-derived growth factor receptor (PDGFR) and mast/stem cell growth factor receptor (SCFR; c-Kit) and forms of these proteins with certain point mutations. This results in the inhibition of both Bcr-Abl-, PDGFR- and c-Kit-mediated signal transduction pathways, and the proliferation of tumor cells in which these kinases are overexpressed. Bcr-Abl fusion protein is an abnormal, constitutively active enzyme expressed in Philadelphia chromosome positive chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML). PDGFR, upregulated in many tumor cell types, is a receptor tyrosine kinase essential to cell migration and the development of the microvasculature. c-kit, a receptor tyrosine kinase mutated and constitutively activated in certain tumors, plays a key role in tumor cell survival, proliferation, and differentiation. Flumatinib (HH-GV-678) is a novel antineoplastic tyrosine kinase inhibitor, with the chemical name of 4-(4-Methyl-piperazin-1-ylmethyl)-N-[6-methyl-5-(4-pyridin-3-yl-pyrimidin-2-ylamino)-pyridin-3-yl]-3-trifluoromethyl-benzamide [3] Flumatinib (HH-GV-678) is currently in Phase I clinical trials in China for the treatment of chronic myelogenous leukemia (CML) [3] Molecular modeling of Flumatinib (HH-GV-678) docked to the KIT kinase domain suggests a special mechanism underlying its capability to overcome the drug resistance conferred by KIT activation loop mutations [2] Flumatinib (HH-GV-678) could be a promising therapeutic agent against gastrointestinal stromal tumors (GISTs) resistant to both imatinib and sunitinib due to secondary mutations in the KIT activation loop [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.70 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (3.70 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7775 mL | 8.8875 mL | 17.7749 mL | |
| 5 mM | 0.3555 mL | 1.7775 mL | 3.5550 mL | |
| 10 mM | 0.1777 mL | 0.8887 mL | 1.7775 mL |