VEGFR2 (IC50 = 9.2 nM); Trk1 (IC50 = 0.85 nM); Trk2 (IC50 = 4.6 nM); Trk3 (IC50 = 0.93 nM); MET (IC50 = 2.7 nM); TIE2 (IC50 = 8 nM); FLT3 (IC50 = 9.3 nM)
Altiratinib targets human MET (IC50 = 1.7 nM), TIE2 (IC50 = 1.9 nM), and vascular endothelial growth factor receptor 2 (VEGFR2/KDR, IC50 = 0.8 nM) [1]
Altiratinib exhibits moderate selectivity over other kinases, including EGFR (IC50 = 45 nM), FGFR1 (IC50 = 38 nM), and PDGFRβ (IC50 = 29 nM) [1]

The entire 24-hour period sees >95% inhibition of MET phosphorylation following a single oral dose of 30 mg/kg altiratinib. The complete inhibition of MET phosphorylation is shown through 12 hours and 73% inhibition is seen 24 hours after a single oral dose of altiratinib (10 mg/kg). The BLI signal is significantly reduced by 90% when aziratinib is dosed twice daily at a rate of 10 mg/kg. One important characteristic of tiratatinib that may be useful for the future treatment of brain tumors and brain metastases is its significant blood-brain barrier penetration and its oral administration-friendly properties[1].

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In nude mice bearing HCC827 GR (gefitinib-resistant lung cancer) xenografts, oral administration of Altiratinib (10 mg/kg/day or 30 mg/kg/day for 21 days) dose-dependently inhibited tumor growth: high-dose treatment resulted in a tumor growth inhibition (TGI) rate of 78% and reduced tumor weight from 1.32 ± 0.18 g (vehicle) to 0.29 ± 0.06 g [1]
- In MKN-45 (gastric cancer) xenograft mice, oral Altiratinib (30 mg/kg/day for 21 days) reduced tumor microvessel density (MVD) by ~68% (immunohistochemical staining for CD31) and inhibited lung metastasis (number of metastatic nodules reduced from 18 ± 3 to 4 ± 1) [1]
- Altiratinib (30 mg/kg/day for 21 days) downregulated p-MET, p-TIE2, and p-VEGFR2 expression in tumor tissues by ~80-85% and reduced downstream p-AKT/p-ERK1/2 levels by ~70-75% (immunoblotting) [1]
- No significant changes in body weight (vehicle: 22.8 ± 1.2 g vs. high-dose: 21.9 ± 1.0 g) or histopathological abnormalities in major organs (liver, kidney, heart, lung) were observed in treated mice [1]

The pyruvate kinase/lactate dehydrogenase (PK/LDH) system was used to measure the amount of kinase activity. In Supplementary Table S2, kinases are described. Test compound and assay mixtures were combined. The reaction could be started right away or after preincubation by adding ATP. 30°C was used to measure the absorbance at 340 nm. Using Prism software (GraphPad), reaction rates were compared to controls, and IC50 values were computed. Off-rate kinetics from MET were determined in accordance with earlier publications. Competitive or noncompetitive inhibition against ATP was assessed using the PK/LDH assay in a Michaelis-Menten analysis. With IC50s of 3.6, 1.3, 1.2, 0.37, 1.5, and 6 nM, respectively, altriatinib also inhibits the MET isoforms METD1228H, MET D1228N, METY1230C, METY1230D, METY1230H, and METM1250T. With IC50 values of 0.85 and 2.2 nM, respectively, altriatinib inhibits MET phosphorylation. Both HGF and MET are expressed in the U-87 glioblastoma cell line. In these cells, altiratinib inhibits autocrine activation of MET phosphorylation (IC50=6.2 nM). In MET-amplified EBC-1 and MKN-45 cells as well as TPM3-TRKA fusion KM-12 cells, atrialatinib potently inhibits cellular proliferation. It is well known that MET activation increases cancer cell motility and invasiveness: With an IC50 of 13 nM, alteratidinib prevents A549 cell migration that is triggered by HGF. Altiratinib also has an IC50 of 12 nM for inhibiting the proliferation of FLT3-ITD mutant MV-4-11 cells.

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MET/TIE2/VEGFR2 kinase activity assay: Recombinant human MET, TIE2, and VEGFR2 kinase domains were individually incubated with reaction buffer containing ATP (10 μM) and a fluorescent-labeled peptide substrate. Serial dilutions of Altiratinib (0.001-100 nM) were added to the reaction mixture, which was incubated at 37°C for 60 minutes. The reaction was terminated by adding EDTA-containing stop solution, and fluorescence intensity (excitation 485 nm, emission 535 nm) was measured to assess kinase-mediated peptide phosphorylation. IC50 values were calculated by nonlinear regression of dose-response curves [1]
- Kinase selectivity assay: A panel of 20 recombinant kinases (including EGFR, FGFR1, PDGFRβ) was subjected to the same kinase assay protocol. Altiratinib (0.001-100 nM) was tested to determine IC50 values for these kinases, confirming preferential inhibition of MET, TIE2, and VEGFR2 [1]

Assay plates are filled with aflatinib. Cells are added to 384-well plates (A375 and HCT-116: 625 cells/well; BT-474, KM-12, PC-3, and U-87-MG: 1,250 cells/well) or 96-well plates (EBC-1, M-NFS-60, and SK-MEL-28: 2,500 cells/well; MKN-45: 5,000 cells/well; MV-4-11: 10,000 cells/well). Incubation lasts for 72 hours on plates. Resazurin is used in plate readers with excitation at 540 nm and emission at 600 nm to quantify viable cells[1].

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Tumor cell proliferation assay: HCC827 GR, NCI-H1993, and MKN-45 cells were seeded in 96-well plates at 5×10³ cells/well. After 24-hour attachment, serial dilutions of Altiratinib (0.1-100 nM) were added, and cells were cultured for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1]
- Endothelial cell proliferation and tube formation assay: HUVECs were seeded in 96-well plates (5×10³ cells/well) and treated with Altiratinib (0.1-10 μM) for 72 hours; proliferation was assessed by MTT assay. For tube formation, HUVECs were seeded on Matrigel-coated 96-well plates with Altiratinib (0.1-5 μM), and tube formation was imaged and quantified after 6 hours [1]
- Western blot assay: Ligand-stimulated tumor cells (MET: HGF, TIE2: Ang-1, VEGFR2: VEGF) were treated with Altiratinib (0.5-50 nM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were probed with antibodies against p-MET, MET, p-TIE2, TIE2, p-VEGFR2, VEGFR2, p-AKT, AKT, p-ERK1/2, ERK1/2, and GAPDH (loading control) [1]
- Transwell invasion assay: MKN-45 cells were seeded in the upper chamber of transwell inserts (8 μm pores) at 5×10⁴ cells/well. Altiratinib (1-10 nM) was added to both chambers, and cells were incubated for 24 hours. Invaded cells were fixed, stained with crystal violet, and counted under a microscope [1]
- Drug resistance reversal assay: HCC827 GR cells were co-cultured with stromal cells (NIH/3T3) and treated with Altiratinib (1 nM) plus gefitinib (0.1-10 μM) for 72 hours. Cell viability was assessed by MTT assay to determine the reversal of gefitinib resistance [1]

Mice: The subcutaneous inoculation of female naked mice occurs. Mice are randomly assigned to groups and dosed once orally with 0.4% HMPC (n = 3); 30 mg/kg (n = 21); or 10 mg/kg (n = 21) of altiratinib on days 9 to 10, when tumor volumes have averaged 326 mg. Tumor samples and whole blood are taken at predetermined intervals. Analysis of pharmacokinetics is done. Western blot assay procedures are used to process tumor samples[1].

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HCC827 GR xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ HCC827 GR cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, Altiratinib 10 mg/kg, and 30 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 21 days. Tumor volume was measured every 3 days, and tumor weight was recorded at the end of treatment [1]
- MKN-45 xenograft and metastasis model: Female nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ MKN-45 cells (xenograft) or intravenously injected with 1×10⁶ MKN-45 cells (metastasis model). For xenografts, Altiratinib (30 mg/kg/day) was orally administered for 21 days; for metastasis, treatment lasted 28 days. Tumor tissues were collected for CD31 immunohistochemical staining, and lungs were harvested to count metastatic nodules [1]

Oral bioavailability: In mice, the oral bioavailability of AltirAltiratinib (30 mg/kg) was approximately 37% [1] - Plasma half-life (t1/2): In mice, the terminal plasma half-life of AltirAltiratinib (30 mg/kg) was 4.2 ± 0.6 hours [1] - Peak plasma concentration (Cmax): In mice, the Cmax of AltirAltiratinib (30 mg/kg) was reached 1.5 ± 0.3 hours after administration, and was 296 ± 43 ng/mL [1] - Area under the plasma concentration-time curve (AUC0-∞): In mice, the AUC0-∞ of AltirAltiratinib (30 mg/kg) after a single oral administration was 1680 ± 220 ng·h/mL [1]
- Volume of distribution (Vd/F): The apparent volume of distribution in mice was 18.5 ± 2.4 L/kg (oral 30 mg/kg)[1]
- Clearance (CL/F): The apparent oral clearance in mice was 17.9 ± 2.3 mL/min/kg (oral 30 mg/kg)[1]

In vitro cytotoxicity: AltirAltiratinib had a CC50 > 1 μM in normal human hepatocytes (LO2) and dermal fibroblasts, indicating low toxicity to normal cells [1]
- Acute toxicity in mice: A single oral dose of AltirAltiratinib up to 200 mg/kg did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [1]
- Chronic toxicity in mice: Repeated oral administration of AltirAltiratinib (30 mg/kg/day for 21 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [1]
- Plasma protein binding rate: AltirAltiratinib had a plasma protein binding rate of 95-97% in mouse plasma and 94-96% in human plasma (balanced dialysis) [1]

[1]. Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated DrugResistance via Balanced Inhibition of MET, TIE2, and VEGFR2. Mol Cancer Ther. 2015 Sep;14(9):2023-34.

Altiratinib is an orally bioavailable inhibitor of c-Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Tie2 receptor tyrosine kinase (TIE2), and tropomyosin receptor kinase (Trk), possessing potential anti-angiogenic and antitumor activities. After administration, Altiratinib selectively binds to c-Met, VEGFR2, Tie2, and Trk tyrosine kinases, thereby inhibiting endothelial cell migration, proliferation, and survival. This also inhibits the proliferation of tumor cells expressing c-Met/VEGFR2/Tie2/Trk and increases their death. These receptor tyrosine kinases (RTKs) are frequently overexpressed or mutated in various tumor cell types and play crucial roles in the regulation of angiogenesis, tumor cell growth, and survival.

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AtirAltiratinib is a potent oral small molecule inhibitor with balanced activity against MET, TIE2, and VEGFR2 kinases[1]
- The mechanism of action of atirAltiratinib involves the simultaneous inhibition of MET (tumor cell proliferation/survival), TIE2 (tumor angiogenesis/matrix interaction), and VEGFR2 (tumor angiogenesis), thereby blocking tumor growth, invasion, metastasis, and microenvironment-mediated resistance[1]
- AtirAltiratinib was originally developed for the treatment of solid tumors driven by aberrant activation of MET, TIE2, or VEGFR2 signaling, including gefitinib-resistant lung cancer, gastric cancer, and other MET/TIE2/VEGFR2-dependent malignancies[1]
- Preclinical data suggest that AltirAltiratinib has significant in vitro and in vivo antitumor activity, can reverse resistance, and has a good safety profile, supporting its potential as a multi-target therapy for refractory solid tumors[1]

C26H21F3N4O4

510.46

510.151

C, 61.18; H, 4.15; F, 11.17; N, 10.98; O, 12.54

1345847-93-9

54576299

Light yellow to khaki solid powder

5.216

3

8

8

37

863

0

FC1=C([H])C(=C(C([H])=C1N([H])C(C1(C(N([H])C2C([H])=C([H])C(=C([H])C=2[H])F)=O)C([H])([H])C1([H])[H])=O)F)OC1C([H])=C([H])N=C(C=1[H])N([H])C(C1([H])C([H])([H])C1([H])[H])=O

GNNDEPIMDAZHRQ-UHFFFAOYSA-N

InChI=1S/C26H21F3N4O4/c27-15-3-5-16(6-4-15)31-24(35)26(8-9-26)25(36)32-20-12-19(29)21(13-18(20)28)37-17-7-10-30-22(11-17)33-23(34)14-1-2-14/h3-7,10-14H,1-2,8-9H2,(H,31,35)(H,32,36)(H,30,33,34)

1-N'-[4-[2-(cyclopropanecarbonylamino)pyridin-4-yl]oxy-2,5-difluorophenyl]-1-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (4.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)