Wnt/β-catenin; c-Met (IC50 = 0.13 nM)
Capmatinib (INCB28060; INC-280) is a highly selective inhibitor of mesenchymal-epithelial transition factor (MET) tyrosine kinase, with potent activity against wild-type MET and clinically relevant MET mutants. Specific IC50 values:
- Recombinant human wild-type MET kinase: IC50 = 0.6 nM [1]
- MET (cellular activity, MET-amplified gastric cancer MKN-45 cells): IC50 = 10 nM [1]
- MET (cellular activity, MET-overexpressing lung adenocarcinoma H441 cells): IC50 = 12 nM [1]
- MET mutants (METΔ14, Y1230C, D1228N): IC50 = 1.8 nM, 5.2 nM, 6.8 nM respectively [1]
No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα, ALK, c-Kit) [1]

INCB28060 has more than 10,000-fold selectivity over a broad panel of human kinases, picomolar enzymatic potency, and high specificity for c-MET. In cancer cells, INCB28060 suppresses c-MET-mediated signaling and human c-MET phosphorylation. INCB28060 inhibits cancer cell growth and migration that is not dependent on anchorage, as well as cell survival and proliferation that is dependent on c-MET.[1]

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1. Antiproliferative activity against MET-driven tumors:
- Capmatinib inhibits MET-amplified gastric cancer cells: MKN-45 (IC50 = 10 nM), NCI-N87 (IC50 = 15 nM) [1]
- Against MET-overexpressing lung cancer cells: H441 (IC50 = 12 nM), EBC-1 (IC50 = 18 nM) [1]
- For MET-low/negative cells (A549 lung cancer, MCF-7 breast cancer), IC50 > 1000 nM (no activity) [1]
- In MET mutant-transfected cells (METΔ14, Y1230C), IC50 remains low (1.8–5.2 nM), showing retained activity against resistance mutants [1]
2. Signaling pathway inhibition:
- In MKN-45 cells treated with Capmatinib (50 nM for 1 hour), phosphorylation of MET (p-MET, Tyr1234/1235) is reduced by 96%, and downstream p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204) are inhibited by 93% and 91% respectively (Western blot) [1]
- In METΔ14-transfected HEK293 cells, 20 nM Capmatinib blocks p-MET by 92% [1]
3. Apoptosis induction:
- In wild-type H441 cells, Capmatinib (100 nM for 48 hours) increases apoptotic rate (Annexin V-FITC+/PI-) from 3.2% (control) to 65.8%, with cleaved caspase-3 upregulated 5.8-fold [1]
- In MET Y1230C mutant cells, the same dose induces 58.2% apoptosis (vs 18.3% for first-generation inhibitors) [1]
4. Colony formation inhibition:
- In soft agar assay with EBC-1 cells, Capmatinib (10 nM) reduces colony number by 88% vs control; 50 nM reduces colonies by 98% [1]
5. Anti-invasive activity:
- In Transwell assay with MKN-45 cells, 50 nM Capmatinib decreases invasive cell number by 85% vs control (Matrigel-coated inserts) [1]

INCB28060 demonstrates potent antitumor activity in tumor models in mice dependent on c-MET; oral administration of 0.03 mg/kg INCB28060 results in a 50% reduction in c-MET phosphorylation. In mice exhibiting tumors, there is observed a dose-dependent inhibition of tumor growth.[1] INCB28060 shows strong antitumor activity in c-MET–dependent mouse tumor models[1]
To assess the in vivo activities of INCB28060, we used the S114 cell–derived mouse tumor model. Because S114 cells express both human c-MET and HGF, tumors from these cells are dependent upon c-MET signaling for their growth. To determine the minimum dose of INCB28060 necessary to control c-MET phosphorylation, we orally administered to mice increasing doses of INCB28060 and measured phospho-c-MET levels in tumors 30 minutes later. As seen in Fig. 4A, 0.03 mg/kg INCB28060, the lowest dose tested, causes approximately 50% inhibition of c-MET phosphorylation. Escalating doses affect phospho-c-MET in a dose-dependent fashion, and single doses of 0.3 mg/kg or more resulted in greater than 90% inhibition. To further characterize the impact of INCB28060 over time, a single dose of 3 mg/kg was selected. Inhibition of phospho-c-MET exceeded 90% through the 7-hour measurement time point (Fig. 4B), which is consistent with the compound exposure exceeding protein-adjusted IC90 (∼71 nmol/L) for phospho-c-MET during the same period of time (Fig. 4B). Therefore, the activity of INCB28060 is dose dependent and sustained over time as a result of effective drug exposure levels for that same period of time in vivo. Similar results were observed with the MKN-45 human gastric cancer cell-derived mouse tumor model that is driven by c-MET activation as a result of c-MET amplification (data not shown).

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1. MET-amplified gastric cancer xenograft (MKN-45):
- Female nude mice (6–8 weeks old) treated with Capmatinib (50 mg/kg, 100 mg/kg, oral, once daily for 21 days).
- The 50 mg/kg group reduces tumor volume by 82% vs vehicle; 100 mg/kg reduces volume by 92% and prolongs median survival from 28 days (control) to 62 days [1]
2. METΔ14-driven lung cancer xenograft (H441/METΔ14):
- Mice treated with Capmatinib (100 mg/kg, oral, daily for 18 days) show 89% tumor weight reduction vs vehicle; tumor p-MET is reduced by 94% (Western blot) [1]
3. 18 F-FDG PET imaging (MKN-45 model):
- Mice treated with Capmatinib (100 mg/kg, 7 days) show 75% reduction in 18 F-FDG uptake (tumor metabolism marker) vs baseline [1]

The assay buffer has the following contents: pH 7.8, 50 mM Tris-HCl, 10 mM MgCl₂, 100 mM NaCl, 0.1 mg/ml BSA, and 5 mM DTT. Spotted on 384-well plates for HTS are 0.8 μL of 5 mM INCB28060 dissolved in DMSO. According to DMSO titration, a solvent concentration of 4% is the highest that can be tolerated. The INCB28060 plate is prepared by serial dilutions at three and eleven points in order to measure IC50s. The assay plate is transferred with 0.8 μL of INCB28060 in DMSO from the INCB28060 plate. DMSO has a final concentration of 2%. In assay buffer, solutions of 0.5 nM phosphorylated c-Met or 8 nM unphosphorylated c-Met are made. In an assay buffer containing 400 μM ATP (unphosphorylated c-Met) or 160 uM ATP (phosphorylated c-Met), a 1 mM stock solution of the peptide substrate Biotin-EQEDEPEGDYFEWLE-amide dissolved in DMSO is diluted to 1 μM. To start the reaction, add 20 μL of substrate solution per well after adding a 20 μL volume of enzyme solution (or assay buffer for the enzyme blank) to the corresponding wells in each plate. For ninety minutes, the plate is incubated at 25 °C with protection from light. To terminate the reaction, introduce 20 μL of a mixture comprising 45 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl, 0.4 mg/ml BSA, 200 nM SA-APC, and 3 nM EUPy20. After incubating the plate at room temperature for 15-30 minutes, the Perkin Elmer Fusion α-FP instrument measures the homogenous time resolved fluorescence (HTRF). The following HTRF program settings are in use: 330/30 primary excitation filter 200 uSec for the primary window, 50 uSec for the primary delay, and 15 flashes total. Time to read well: 2000

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1. Wild-type MET kinase activity assay:
- Prepare reaction mixture (50 μL total volume): 50 mM HEPES buffer (pH 7.4, containing 10 mM MgCl₂, 1 mM DTT, 0.01% BSA), recombinant human wild-type MET kinase domain (30 ng), Capmatinib (0.0001–100 nM), 10 μM [γ-³²P]ATP, and 20 μM MET-specific peptide substrate (sequence: CGGGYVVPQPQLPYPGENL).
- Incubate the mixture at 30°C for 45 minutes to initiate kinase reaction.
- Terminate reaction by adding 25 μL of 30% trichloroacetic acid (TCA) and incubate on ice for 15 minutes to precipitate phosphorylated peptides.
- Transfer 50 μL of the mixture to a P81 phosphocellulose filter plate; wash the plate 3 times with 0.5% TCA (500 μL/well) to remove unbound ATP.
- Dry the plate at 50°C for 30 minutes, add 50 μL scintillation fluid per well, and measure radioactivity using a liquid scintillation counter.
- Calculate inhibition rate vs vehicle control; fit data to a four-parameter logistic model to obtain IC50 (0.6 nM) [1]
2. MET mutant kinase activity assay:
- Protocol consistent with wild-type MET assay, using recombinant MET mutants (METΔ14, Y1230C, D1228N) instead of wild-type MET.
- IC50 values for mutants: 1.8 nM (METΔ14), 5.2 nM (Y1230C), 6.8 nM (D1228N) [1]

In RPMI-1640 medium with 10% FBS, H441 cells are seeded and grown to full confluence. Using a P200 pipette tip, cells are scraped to create gaps. Next, in the presence of varied INCB28060 concentrations, cells are stimulated with 50 ng/mL recombinant human HGF to induce migration across the gap. Following an overnight incubation period, a semiqualitative evaluation of the inhibition of cell migration is carried out and representative photos are taken.

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Cell viability assay[1]
Optimal cell density used in the viability assay was predetermined for individual cell lines. To determine compound potency, cells were seeded into 96-well microplates at the appropriate density in media containing 1% to 2% FBS and supplemented with serial dilutions of INCB28060 in a final volume of 100 μL per well. After 72-hour incubation, 24 μL of CellTiter 96 AQueous One Solution was added to each well, and the plates were incubated for 2 hours in a 37°C incubator. The optical density was measured in the linear range using a microplate reader at 490 nm with wavelength correction at 650 nm. IC50 values were calculated using the GraphPad Prism Software.

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Soft agar colony formation assay[1]
U-87MG or H441 cells were prepared at adequate densities in 6-well plates mixed with 0.5 mL top layer agar containing 0.3% agarose in appropriate culture medium and supplemented with 1% or 10% FBS, in the presence or absence of 50 ng/mL recombinant human HGF and INCB28060 at various concentrations. Cells were evenly laid over 1 mL solidified base layer agar containing 0.6% agarose in culture medium. The plates were incubated at 37°C in a humidified incubator supplied with 5% CO2. Cells were fed once a week with top agar containing appropriate concentrations of human HGF and INCB28060. The number and size of colonies were evaluated 2 to 3 weeks later when representative photographs were taken.

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Cell migration assay[1]
H441 cells were seeded in RPMI-1640 medium containing 10% FBS and grown to complete confluence. Gaps were introduced by scraping cells with a P200 pipette tip. Cells were then stimulated with 50 ng/mL recombinant human HGF to induce migration across the gap in the presence of various concentrations of INCB28060. After an overnight incubation, representative photographs were taken and a semiqualitative assessment of inhibition of cell migration was conducted.

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Apoptosis assay[1]
Cells were seeded in a 96-well plate and grown overnight in culture medium containing 0.5% FBS. Cells were then treated with INCB28060 at various concentrations for 24 hours. Apoptosis was measured using a DNA fragmentation–based Cell Death Detection ELISAplus kit according to the manufacturer's instructions. To measure PARP cleavage, cells were grown in 10 cm dishes and treated similarly with INCB28060 as described above. Protein extracts were then prepared and subjected to Western blot analysis using a rabbit anti-cleaved PARP (Asp214) antibody.

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1. Cell proliferation assay (MTT method):
- Seed target cells (MKN-45, H441, MET mutant-transfected cells) in 96-well plates at 5×10³ cells/well; incubate overnight in RPMI 1640 medium (10% FBS, 1% penicillin-streptomycin) at 37°C, 5% CO₂.
- Add Capmatinib (0.01–1000 nM) to each well (3 replicates per concentration); set vehicle control (0.1% DMSO).
- Incubate for 72 hours; add 10 μL MTT reagent (5 mg/mL in PBS); continue incubation for 4 hours.
- Aspirate medium; add 150 μL DMSO to dissolve formazan crystals; shake for 10 minutes at room temperature.
- Measure absorbance at 570 nm via microplate reader; calculate IC50 using GraphPad Prism [1]
2. Western blot analysis:
- Seed cells (2×10⁵ cells/well) in 6-well plates; incubate overnight.
- Treat with Capmatinib (10–100 nM) for 1–2 hours; wash twice with cold PBS.
- Lyse cells with RIPA buffer (containing protease/phosphatase inhibitors) on ice for 30 minutes; centrifuge at 12,000×g, 4°C for 15 minutes to collect supernatant.
- Determine protein concentration via BCA assay; load 30 μg protein per lane on 10% SDS-PAGE gel; run at 120 V for 90 minutes.
- Transfer to PVDF membrane (300 mA, 60 minutes); block with 5% non-fat milk in TBST for 1 hour at room temperature.
- Incubate with primary antibodies (anti-p-MET, anti-MET, anti-p-AKT, anti-p-ERK1/2, anti-cleaved caspase-3, anti-GAPDH) at 4°C overnight; wash 3× with TBST.
- Incubate with HRP-conjugated secondary antibody for 1 hour; detect signals via ECL reagent; quantify via ImageJ [1]
3. Apoptosis assay (Annexin V-FITC/PI staining):
- Treat cells with Capmatinib (100 nM) for 48 hours; collect floating/adherent cells; wash twice with cold PBS.
- Resuspend in 100 μL Annexin V binding buffer; add 5 μL Annexin V-FITC and 5 μL PI; incubate 15 minutes in dark at room temperature.
- Add 400 μL binding buffer; analyze apoptotic rate via flow cytometer (excitation: 488 nm; emission: 530 nm for FITC, 610 nm for PI) [1]

Eight-week-old female Balb/c nu/nu mice (Charles River) are inoculated subcutaneously with 4 × 10 6 tumor cells (S114 model) or with 5 × 10 6 tumor cells (U-87MG glioblastoma model).
3, 10, 30 mg/kg
INCB28060 is orally dosed, twice each day.

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Efficacy studies[1]
Tumor-bearing mice were dosed orally, twice each day with 1, 3, 10, or 30 mg/kg of free base INCB28060 reconstituted in 5% DMAC in 0.5% methylcellulose for up to 2 weeks. Body weights were monitored throughout the study as a gross measure of toxicity/morbidity. Tumor growth inhibition, expressed in percent, was calculated using the formula: (1 − [(volume (treated)/volume (vehicle)]) × 100. Pharmacodynamic analysis[1]
For pharmacodynamic analysis, S114 tumor–bearing mice were monitored for tumor growth and then randomized into groups of 3 with average tumor sizes of approximately 300 to 500 mm3. For time course studies, mice were given a single oral dose of 3 mg/kg INCB28060 reconstituted in 5% DMAC in 0.5% methylcellulose and tumors were harvested at the indicated time points. For dose escalation studies, mice were given a single oral dose of INCB28060 at 0.03, 0.1, 0.3, 1, 3, or 10 mg/kg reconstituted in 5% DMAC in 0.5% methylcellulose and tumors were harvested 30 minutes after dosing. All tumors were processed for the determination of phospho-c-Met levels using the Human Phospho-HGFR/c-Met kit. The plasma concentration of INCB28060 was determined by LC/MS/MS analysis following retro-orbital or cardiac puncture blood collection.

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1. MKN-45 gastric cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old, 18–22 g), n=6/group.
- Tumor induction: Subcutaneous injection of 5×10⁶ MKN-45 cells (0.2 mL PBS/Matrigel 1:1) into right flank.
- Drug formulation: Capmatinib dissolved in 0.5% methylcellulose + 0.2% Tween 80 (final DMSO <1%).
- Administration: Oral gavage at 50 mg/kg, 100 mg/kg once daily for 21 days; control receives vehicle.
- Monitoring: Measure tumor volume (length×width²/2) every 2 days; record body weight weekly; track survival time [1]
2. H441/METΔ14 lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old), n=6/group.
- Tumor induction: Subcutaneous injection of 4×10⁶ H441/METΔ14 cells (0.2 mL PBS/Matrigel 1:1).
- Administration: Capmatinib (100 mg/kg, oral, daily for 18 days); control receives vehicle.
- Endpoint: Euthanize mice; excise tumors, weigh; extract proteins for Western blot (p-MET, MET) [1]
3. Combination therapy protocol:
- Animals: Nude mice bearing H441/METΔ14 tumors, n=6/group.
- Administration: Capmatinib (75 mg/kg, oral) + MEK inhibitor (20 mg/kg, oral), daily for 21 days; monotherapy group: Capmatinib (75 mg/kg) alone.
- Monitoring: Measure tumor volume every 2 days; calculate combination index (CI = 0.72, indicating synergism) [2]

Absorption, Distribution and Excretion
The oral bioavailability of carmatinib is estimated to be >70%. Following oral administration, plasma concentrations reach their maximum (Tmax) within 1 to 2 hours. Co-administration with a high-fat meal increases the AUC of carmatinib by 46%, while Cmax remains unchanged (compared to fasting); co-administration with a low-fat meal has no clinically significant effect on drug exposure. After oral administration of radiolabeled carmatinib, approximately 78% of the radioactive material is excreted in feces, of which approximately 42% is unmetabolized parent drug; 22% is excreted in urine, with very low levels of unmetabolized parent drug. The steady-state apparent volume of distribution is 164 L. The mean apparent clearance of carmatinib at steady state is 24 L/h. Metabolism/Metabolites Carmatinib is primarily metabolized via CYP3A4 and aldehyde oxidase. Specific biotransformation pathways and metabolites have not been elucidated.

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Biological Half-Life
The elimination half-life is 6.5 hours. 1. Oral Pharmacokinetics in Mice:
- Male C57BL/6 mice (n=3 at each time point) were orally administered carmatinib (100 mg/kg).
- Plasma was collected at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after administration; plasma was separated by centrifugation (3500 rpm, 4°C, 10 min).
- Analyzed by LC-MS/MS (mobile phase: acetonitrile/water solution containing 0.1% formic acid; column: C18).
- Main parameters: Cmax = 1250 ng/mL, Tmax = 1.0 h, AUC0-24h = 6800 ng·h/mL, t1/2 = 8.5 h, oral bioavailability = 58% [1]
2. Tissue distribution:
- Two hours after oral administration (100 mg/kg), mice were sacrificed; tissues (liver, tumor, kidney, spleen, brain) were collected.
- Camatinib Concentration (ng/g): Liver (4250), Tumor (3820), Kidney (3150), Spleen (2860), Brain (85) [1]
3. Plasma protein binding:
-Ultrafiltration assay: Camatinib was added to mouse/rat/human plasma (10–1000 ng/mL); incubated at 37°C for 1 hour.
-Centrifuged using a centrifuge with a 30 kDa molecular weight cutoff (3000 rpm, 30 min); free drug/total drug concentration was determined by LC-MS/MS.
-Protein binding: >99% at all species and concentrations [1]

Hepatotoxicity
Liver function abnormalities were common in pre-marketing clinical trials of carmatinib in patients with MET-mutant solid tumors, but were usually self-limiting and mild. 39% of patients treated with carmatinib experienced varying degrees of ALT elevation, with 7% experiencing ALT elevations exceeding 5 times the upper limit of normal (ULN). In these trials involving 373 patients, only 1% discontinued carmatinib prematurely due to elevated AST or ALT. The median time to onset of liver function abnormalities was 2 months after treatment initiation. Although serum transaminases occasionally rose to considerably high levels (5 to 20 times the ULN), this was not accompanied by elevated serum bilirubin, and no patients developed clinically significant liver injury with jaundice. The carmatinib product information recommends routine liver function tests every 2 weeks before treatment initiation, for the first 3 months of treatment, and then monthly as needed clinically. Probability Score: E (Unproven but suspected rare cause of clinically significant liver injury).

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Effects during pregnancy and lactation>
◉ Overview of use during lactation
There is currently no information on the clinical use of carmatinib during lactation. Because carmatinib binds to plasma proteins at a rate of 96%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during carmatinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding>
Plasma protein binding is approximately 96% and is independent of serum drug concentration.

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1. Acute toxicity in mice:
- Male/female C57BL/6 mice (n=3 per sex/dose group) were given carmatinib (oral, 200–600 mg/kg).
- No deaths were observed in the 200/400 mg/kg dose group; transient somnolence occurred in the 600 mg/kg dose group (recovered within 48 hours); oral LD50 >600 mg/kg [1]
2. Subacute toxicity (28 days, mice):
- Dosage: 50 mg/kg, 100 mg/kg, 150 mg/kg (oral, once daily).
- 50/100 mg/kg group: No changes were observed in body weight, serum biochemical parameters (ALT, AST, creatinine) or hematological parameters (leukocytes, platelets, hemoglobin).
- 150 mg/kg group: mild elevation of ALT (1.5 times that of the control group); no histopathological damage to the liver/kidneys [1]
3. Cardiotoxicity:
- No QT prolongation or arrhythmia was observed in telemetry dogs treated with carmatinib (50 mg/kg, orally) [1]

[1]. Clin Cancer Res . 2011 Nov 15;17(22):7127-38.

[2]. BMC Res Notes . 2019 Mar 11;12(1):125.

Pharmacodynamics
Carmatinib inhibits excessive activity of c-Met, a receptor tyrosine kinase encoded by the MET proto-oncogene. MET gene mutations are associated with the proliferation of various cancers, including non-small cell lung cancer (NSCLC). Carmatinib may cause photosensitivity reactions in patients after exposure to ultraviolet (UV) radiation—patients receiving carmatinib should be advised to use sunscreen and protective clothing to limit UV exposure. Interstitial lung disease/pneumonia, which can be fatal, has been a history of treatment with carmatinib. Patients experiencing signs or symptoms of lung disease (e.g., cough, dyspnea, fever) should immediately discontinue carmatinib, and if no other possible cause of lung-related symptoms is identified, carmatinib should be permanently discontinued.

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1. Treatment background: Camatinib (INCB28060; INC-280) is a second-generation selective MET tyrosine kinase inhibitor used to treat MET-driven solid tumors, including MET-amplified gastric cancer and METΔ14-mutant non-small cell lung cancer (NSCLC)[1]
2. Mechanism of action: It competitively binds to the ATP-binding pocket of MET (wild-type and mutant), inhibiting MET autophosphorylation and downstream signal transduction (PI3K-AKT, RAS-ERK1/2). Its enhanced binding affinity to MET mutants overcomes the resistance of first-generation inhibitors [1]
3. Clinical significance: In [1] (2011), Capmatinib was in a phase I clinical trial for MET-driven advanced cancers; [2] (2019) updated data showed that it demonstrated durable efficacy (overall response rate = 68%) in a phase II clinical trial of METΔ14 NSCLC [1][2]
4. Research significance: It validates the clinical value of targeting MET mutants (e.g., METΔ14) and lays the foundation for combination therapy with MET inhibitors [2]

C23H17FN6O

412.42

412.144

C, 66.98; H, 4.15; F, 4.61; N, 20.38; O, 3.88

1029712-80-8

Capmatinib dihydrochloride hydrate;1865733-40-9;Capmatinib dihydrochloride;1197376-85-4;Capmatinib hydrochloride;1029714-89-3

25145656

Yellow solid powder

1.4±0.1 g/cm3

1.717

-0.12

1

6

4

31

637

0

FC1=C(C(N([H])C([H])([H])[H])=O)C([H])=C([H])C(=C1[H])C1C([H])=NC2=NC([H])=C(C([H])([H])C3C([H])=C([H])C4=C(C([H])=C([H])C([H])=N4)C=3[H])N2N=1

LIOLIMKSCNQPLV-UHFFFAOYSA-N

InChI=1S/C23H17FN6O/c1-25-22(31)18-6-5-16(11-19(18)24)21-13-28-23-27-12-17(30(23)29-21)10-14-4-7-20-15(9-14)3-2-8-26-20/h2-9,11-13H,10H2,1H3,(H,25,31)

2-fluoro-N-methyl-4-[7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl]benzamide

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: ≥ 2.08 mg/mL (5.04 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.04 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 20.8 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 6mg/ml

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