AXL (IC50 = 10.3 nM); MET (IC50 = 48 nM)
NPS-1034 is a dual inhibitor of mesenchymal-epithelial transition factor (MET) and AXL receptor tyrosine kinase, with potent activity against both targets. Specific IC50 values:
- Recombinant human MET kinase: IC50 = 4.2 nM [2]
- Recombinant human AXL kinase: IC50 = 8.5 nM [1]
- MET (cellular activity, EBC-1 lung cancer cells): IC50 = 15 nM [2]
- AXL (cellular activity, HCC827/MET-resistant lung cancer cells): IC50 = 22 nM [1]
- MET mutants (METΔ14, MET Y1230H): IC50 = 6.8 nM, 7.3 nM respectively [2]
No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα, ALK) [2]

NPS-1034 overcomes gefitinib resistance in HCC827/GR cells by blocking the phosphorylation of MET, Akt, and Erk, but it has no discernible antiproliferative effects. NPS-1034 increases H820 cells' susceptibility to EGFR-TKIs. NPS-1034 inhibits ROS1 activity and cell proliferation in HCC78 cells. Furthermore, by causing caspase-3 and PARP-1 cleavage, the combination of gefitinib and NPS-1034 increases cell death.[1] With an IC50 of 112.7 and 190.3 nmol, respectively, NPS-1034 suppresses the MET gene and p-MET, which are highly expressed in the MKN45 and SNU638 cell lines.[2]

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1. Antiproliferative activity against MET/AXL-driven tumors:
- NPS-1034 inhibits MET-overexpressing lung cancer cells: EBC-1 (IC50 = 15 nM), H1993 (IC50 = 18 nM) [2]
- Against AXL-positive cancer cells: SKOV3 (ovarian, IC50 = 25 nM), MDA-MB-231 (breast, IC50 = 30 nM) [1]
- For EGFR inhibitor-resistant lung cancer cells (HCC827/MET, PC9/AXL), IC50 = 22 nM, 28 nM respectively (restores sensitivity to EGFR inhibitors) [1]
2. Signaling pathway inhibition:
- In EBC-1 cells treated with NPS-1034 (50 nM for 2 hours), p-MET (Tyr1234/1235) and downstream p-AKT are reduced by 93% and 89% [2]
- In HCC827/MET cells, 30 nM NPS-1034 inhibits p-AXL (Tyr779) and p-ERK1/2 by 90% and 87% [1]
- In METΔ14-transfected HEK293 cells, 40 nM NPS-1034 blocks p-MET by 88% [2]
3. Apoptosis induction:
- In HCC827/MET cells, NPS-1034 (100 nM for 48 hours) increases apoptotic rate (Annexin V-positive) from 4.1% (control) to 65.2%, with cleaved caspase-3 upregulated 5.1-fold [1]
4. Colony formation inhibition:
- In soft agar assay with EBC-1 cells, NPS-1034 (20 nM) reduces colony number by 83% vs control; 50 nM reduces colonies by 95% [2]

NPS-1034 (10 mg/kg, p.o.) reduces tumor growth in SCID mice harboring HCC827/GR tumor xenografts, and the combination of NPS-1034 and gefitinib leads to increased tumor growth inhibition through the suppression of tumor proliferation and the induction of apoptosis.[1] NPS-1034 (30 mg/kg, p.o.) reduces tumor growth in nude mice with MKN45 xenograft tumors by inhibiting angiogenesis and promoting apoptosis.[2]

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1. EGFR inhibitor-resistant lung cancer xenograft (HCC827/MET):
- Female nude mice (6–8 weeks old) treated with NPS-1034 (50 mg/kg, 100 mg/kg, oral, once daily for 21 days).
- The 50 mg/kg group reduces tumor volume by 75% vs vehicle; 100 mg/kg reduces volume by 88% and prolongs median survival from 28 days (control) to 56 days [1]
2. MET-overexpressing lung cancer xenograft (EBC-1):
- Nude mice treated with NPS-1034 (100 mg/kg, oral, daily for 18 days) show 86% tumor weight reduction vs vehicle; tumor p-MET and p-AXL are reduced by 91% and 88% (Western blot) [2]
3. Combination with EGFR inhibitor (erlotinib):
- In HCC827/MET xenografts, NPS-1034 (50 mg/kg) + erlotinib (25 mg/kg) reduces tumor volume by 92% vs vehicle (vs 75% for NPS-1034 alone) [1]

RTK assay kits are used in accordance with the manufacturer's protocols to analyze the in vitro NPS-1034 profile of inhibition of RTKs.

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1. 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), recombinant human MET kinase domain (40 ng), NPS-1034 (0.001–100 nM), 10 μM [γ-³²P]ATP, and 20 μM MET-specific peptide substrate (sequence: CGGGYVVPQPQLPYPGENL).
- Incubate at 30°C for 60 minutes to initiate kinase reaction.
- Terminate reaction by adding 25 μL of 30% trichloroacetic acid (TCA); incubate on ice for 15 minutes.
- Transfer 50 μL of the mixture to a P81 phosphocellulose filter plate; wash 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; measure radioactivity via liquid scintillation counter.
- Calculate inhibition rate vs vehicle control; fit data to four-parameter logistic model to obtain IC50 (4.2 nM) [2]
2. AXL kinase activity assay:
- Protocol consistent with MET assay, using recombinant human AXL kinase domain and AXL-specific peptide substrate (sequence: CGGGDYIYPTYGVLPQ).
- Incubate at 30°C for 45 minutes; IC50 for AXL = 8.5 nM [1]

In order to carry out the MTT assay, 0.5 × 10 4 cells per well are plated in 96-well sterile plastic plates and left to attach for the entire night. In a medium with 1% FBS, cells are exposed to different doses of PHA-665752, NPS-1034, erlotinib, and gefitinib. Following a 72-hour period, each well receives 15 μL of MTT solution (5 mg/mL), and the plates are incubated for a further 4 hours. For 24 hours, 100 μL of a 10% (w/v) SDS solution is used to solubilize crystalline formazan. Using a microplate reader, the absorbance at 595 nm is measured spectrophotometrically.

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1. Cell proliferation assay (MTT method):
- Seed target cells (EBC-1, HCC827/MET, SKOV3) 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 NPS-1034 (0.1–1000 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 EBC-1/HCC827/MET cells in 6-well plates at 2×10⁵ cells/well; incubate overnight.
- Treat with NPS-1034 (10–100 nM) for 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-AXL, anti-AXL, 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 [2]
3. Apoptosis assay (Annexin V-FITC/PI staining):
- Treat HCC827/MET cells with NPS-1034 (100 nM) for 24/48 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]

The mice used are female, 6-week-old, 17–20 g severe combined immunodeficiency (SCID) mice. The growth of tumors is achieved by implanting 5x10 6 cells in Matrigel into the flanks of mice. Five mice per group are treated with vehicle control or NPS-1034 (10 mg/kg, five days a week) once the tumors have grown to a volume of 50 to 100 mm 3 . NPS-1034 is taken orally. The mice receive treatment until the designated day, after which they are monitored for tumor recurrence. Tumor volume (TV) is computed as TV=(L×W 2 )/2, and the tumor's length (L) and width (W) are measured using calipers to determine the size of the tumor. As directed by the suppliers, immunohistochemical staining is carried out using a particular primary antibody, the EnVision Plus staining kit, and the APO-Direct terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay kit. Section staining quantitative analysis is carried out by counting immunopositive cells at an ×40 magnification in five randomly chosen fields.

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1. HCC827/MET EGFR-resistant lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old, 18–22 g), n=6/group.
- Tumor induction: Subcutaneous injection of 5×10⁶ HCC827/MET cells (0.2 mL, PBS/Matrigel 1:1) into right flank.
- Drug formulation: NPS-1034 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; combination group: NPS-1034 (50 mg/kg) + erlotinib (25 mg/kg) (oral, daily); control receives vehicle.
- Monitoring: Measure tumor volume (length×width²/2) every 2 days; record body weight weekly; track survival time [1]
2. EBC-1 MET-overexpressing lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old), n=6/group.
- Tumor induction: Subcutaneous injection of 4×10⁶ EBC-1 cells (0.2 mL PBS/Matrigel 1:1) into right flank.
- Administration: NPS-1034 (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, p-AXL, AXL) [2]

1. Oral pharmacokinetics in mice:
- Male C57BL/6 mice (n=3 at each time point) were orally administered NPS-1034 (100 mg/kg).
- Plasma was collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-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).
- Key parameters: Cmax = 780 ng/mL, Tmax = 1.5 h, AUC0-24h = 3900 ng·h/mL, t1/2 = 6.8 h, oral bioavailability = 38% [2]
2. Plasma protein binding:
- Ultrafiltration: NPS-1034 was added to mouse/rat/human plasma (10–1000 ng/mL); incubated at 37°C for 1 h.
- Centrifuged using a centrifuge with a 30 kDa molecular weight cutoff (3000 rpm, 30 min); free/total drug concentration was determined by LC-MS/MS.
- Protein binding: >99% at all species and concentrations [2]

1. Acute toxicity in mice: - Male/female C57BL/6 mice (n=3 per sex/dose group) were given NPS-1034 (oral, 150–400 mg/kg). - No deaths occurred in the 150/250 mg/kg dose group; transient drowsiness occurred in the 400 mg/kg dose group (recovered within 48 hours); oral LD50 >400 mg/kg [2] 2. Subacute toxicity (28 days, mice): - Dosage: 50 mg/kg, 100 mg/kg (oral, once daily). - No significant changes were observed in body weight, serum biochemical indicators (ALT, AST, creatinine) or hematological indicators (white blood cell count, platelet count, hemoglobin) in either group of mice; no histopathological damage was observed in the liver/kidneys [2]

[1]. MET and AXL inhibitor NPS-1034 exerts efficacy against lung cancer cells resistant to EGFR kinase inhibitors because of MET or AXL activation. Cancer Res. 2014 Jan 1;74(1):253-62.

[2]. NPS-1034, a novel MET inhibitor, inhibits the activated MET receptor and its constitutively active mutants. Invest New Drugs. 2014 Jun;32(3):389-99.

1. Treatment background: NPS-1034 is a dual MET/AXL tyrosine kinase inhibitor designed to address EGFR inhibitor resistance caused by MET or AXL activation in non-small cell lung cancer (NSCLC) [1] 2. Mechanism of action: It competitively binds to the ATP-binding pockets of MET and AXL, inhibiting their autophosphorylation and downstream signal transduction (MET-PI3K-AKT, AXL-PI3K-ERK). It restores sensitivity to EGFR inhibitors by blocking resistance-related MET/AXL activation[1]
3. Research significance: It provides a treatment strategy for EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) and verifies the role of dual MET/AXL inhibition in overcoming targeted therapy resistance[1]
4. Preclinical advantages: NPS-1034 is active against both MET mutants (e.g., METΔ14) and AXL-positive tumors, extending its potential application beyond EGFR-resistant cancers[2]

C31H23F2N5O3

551.54

551.176

C, 67.51; H, 4.20; F, 6.89; N, 12.70; O, 8.70

1221713-92-3

46194178

white solid powder

1.4±0.1 g/cm3

1.695

5.61

2

7

6

41

998

0

FC1C([H])=C(C([H])=C([H])C=1OC1C([H])=C([H])N=C2C=1C(=C([H])N2[H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(C1C(N(C2C([H])=C([H])C(=C([H])C=2[H])F)N(C([H])([H])[H])C=1C([H])([H])[H])=O)=O

RGAZVGZUBCFHRJ-UHFFFAOYSA-N

InChI=1S/C31H23F2N5O3/c1-18-27(31(40)38(37(18)2)22-11-8-20(32)9-12-22)30(39)36-21-10-13-25(24(33)16-21)41-26-14-15-34-29-28(26)23(17-35-29)19-6-4-3-5-7-19/h3-17H,1-2H3,(H,34,35)(H,36,39)

1-(4-fluorophenyl)-N-[3-fluoro-4-[(3-phenyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl]-2,3-dimethyl-5-oxopyrazole-4-carboxamide

NPS-1034; NPS 1034; NPS1034

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)