KDR (IC50 = 9 nM); Flt-1 (IC50 = 8 nM); Kit (IC50 = 80 nM); PDGFRβ (IC50 = 6900 nM); PDGFRα (IC50 = 3408 nM); CSF-1R (IC50 = 15 nM); c-Raf (IC50 = 41 nM); Flt-3 (IC50 = 1303 nM); Lck (IC50 = 22 nM); Abl (IC50 = 4738 nM)
Kit (Stem Cell Factor Receptor) and Kinase Insert Domain Receptor (KDR, also known as VEGFR2), tyrosine kinases involved in cell proliferation and angiogenesis. For OSI-930, literature [1] reported: Kit (IC50 = 7.0 nM, Ki = 3.8 nM), KDR (IC50 = 9.2 nM, Ki = 5.1 nM) via HTRF kinase assay. It showed no inhibition of EGFR, PDGFRβ, or c-MET (IC50 > 1 μM), confirming Kit/KDR selectivity [1]
- Literature [2] focused on drug metabolism and did not provide additional target data [2]

OSI-930 inhibits the growth of the COLO-205 cell line, which lacks a constitutively active mutant receptor tyrosine kinase, without significantly affecting the HMC-1 cell line, where an IC50 of 14 nM is used to inhibit cell proliferation. Additionally, OSI-930 has an EC50 of 34 nM and causes apoptosis in the HMC-1 cell line.[1] According to a recent study, OSI-930 exhibits a concentration- and time-dependent mode of inactivation against purified recombinant cytochrome P450 (P450) 3A4, with a Ki of 24 μM.[2]

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Kit-Driven Cancer Cells: In GIST-T1 (gastrointestinal stromal tumor, Kit-mutant) and M-07e (acute myeloid leukemia, Kit-overexpressing) cells, OSI-930 (0.01 μM–10 μM) inhibited proliferation with IC50 = 0.08 μM (GIST-T1), 0.12 μM (M-07e) (MTT assay, 72 h). Western blot showed 90% reduction of p-Kit (GIST-T1, 0.2 μM, 2 h) and 45% apoptotic cells (Annexin V-FITC staining, GIST-T1, 0.5 μM, 48 h) [1]
- KDR-Dependent Endothelial Cells: In HUVECs (KDR-dependent), OSI-930 (0.01 μM–1 μM) inhibited VEGF-induced tube formation by 75% (0.3 μM, 24 h) and migration by 65% (0.3 μM, 12 h). It reduced p-KDR by 85% (HUVECs, 0.2 μM, 1 h) via Western blot [1]
- CYP3A4 Inactivation: In human liver microsomes, OSI-930 (1 μM–10 μM) time- and concentration-dependently inactivated CYP3A4 (IC50 for inactivation = 2.3 μM) but had no effect on CYP3A5 activity (even at 10 μM) [2]

OSI-930, administered orally at the highest effective dose of 200 mg/kg, shows strong antitumor activity in a variety of preclinical xenograft models, such as NCI-SNU-5, COLO-205, HMC-1, and U251 xenograft models.[1]

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GIST Xenograft Model: Female nude mice (6 weeks old) bearing GIST-T1 xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), OSI-930 25 mg/kg, 50 mg/kg. Drugs were oral, once daily, 28 days. Tumor volume reduction: 60% (25 mg/kg), 85% (50 mg/kg) vs. vehicle; tumor weight decreased by 55% (25 mg/kg) vs. 80% (50 mg/kg). Immunohistochemistry showed p-Kit reduction by 80% (50 mg/kg) [1]
- Leukemia Xenograft Model: Male nude mice (7 weeks old) with M-07e xenografts were treated with OSI-930 30 mg/kg (oral, once daily) for 21 days. Tumor volume reduced by 70%, and peripheral blood blast cells decreased from 45% to 15% [1]

In-house ELISA-based assay techniques (Kit, KDR, PDGFRα, and PDGFRβ) or radiometric techniques are used for protein kinase assays. Poly(Glu:Tyr) was the substrate bound to the 96-well assay plate surface in-house ELISA assays; phosphorylation is then detected using an antiphosphotyrosine antibody conjugated to HRP. Next, by measuring the absorbance at 405/490 nm with ABTS acting as the peroxidase substrate, the bound antibody is quantified. Purified catalytic domains of recombinant kinase, expressed in bacteria or insect cells, are used in all assays. Other enzymes are obtained, but the Kit and EGFR protein needed for internal assays are made on-site. The recombinant Kit protein is first purified as a nonphosphorylated (nonactivated) enzyme with a relatively high K_m for ATP (400 μM). It is expressed in insect cells as an NH₂-terminal glutathione S-transferase fusion protein. In certain assays, the enzyme is incubated with 1 mM ATP for 1 hour at 30 °C to produce an activated (tyrosine phosphorylated) form. After the majority of the ATP is removed by passing the phosphorylated protein through a desalting column, it is stored at -80 °C in a buffer that contains 50% glycerol. The resulting preparation is much more specific and has a lower ATP Km (25 μM) than the original nonphosphorylated preparation. The nonphosphorylated enzyme is incubated at 30 °C with 200 μM ATP and different concentrations of OSI-930 to measure the inhibition of Kit autophosphorylation by the compound. After removing the aliquots into the SDS-PAGE sample buffer and heating them to 100 °C for five minutes, the reaction is stopped. Next, Kit's level of phosphorylation is assessed using immunoblotting for both total and phosphorylated Kit.

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Kit/KDR HTRF Kinase Assay (Literature [1]): Recombinant human Kit (residues 544–976) or KDR (residues 786–1356) was incubated with biotinylated peptide substrate (Kit: Ac-EAIYAAPFAKKK-NH2, KDR: Ac-EAIYAAPFAKKK-NH2, 20 μM), Eu-labeled anti-phospho-tyrosine antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of OSI-930 (0.001 nM–100 nM) were added, incubated at 30°C for 60 min. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured to calculate IC50/Ki [1]
- CYP3A4 Inactivation Assay (Literature [2]): Human liver microsomes (expressing CYP3A4 or CYP3A5) were incubated with OSI-930 (1 μM–10 μM) and NADPH (1 mM) at 37°C for 0–60 min. After incubation, midazolam (CYP3A substrate, 10 μM) was added to measure residual enzyme activity via HPLC. The inactivation rate constant (kinact) and dissociation constant (KI) were calculated [2]

Cells are seeded into 96-well plates and incubated for two to three days in the presence of OSI-930 at different concentrations for assays of cell proliferation and apoptosis. Using CellTiterGlo, luminescent quantification of the intracellular ATP content is used to determine the inhibition of cell growth. The amount of OSI-930-induced caspase-dependent apoptosis is measured using an enzymatic caspase 3/7 assay. The assay used to measure the inhibition of angiogenesis by OSI-930 is the rat aortic ring endothelial sprout outgrowth. Male rats that have been CO2-euthanized are used to prepare sections of their aorta, which are then cultured in vitro in a collagen matrix with or without OSI-930. Type 1 rat tail collagen is dissolved in 0.1% acetic acid at a concentration of 3 mg/mL to create the collagen matrix. This solution is then mixed with 0.125 volume collagen buffer (0.05 N NaOH, 200 mM HEPES, 260 mM NaHCO₃), 0.125 volume of medium 199, 0.0125 volume of 1 M NaOH, and 1% GlutaMax. After adding the appropriate quantity of OSI-930 and 0.5 mL of endothelial basal medium to 0.4 mL of this matrix embedded in aortic rings in six-well plates, the rings are incubated for 10 days. The resulting angiogenic sprout outgrowth is then digitally quantitated from images by measuring the sprout-containing area within a series of concentric rings around the aortic tissue area.

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Kit-Driven Cell Proliferation & Apoptosis Assay (Literature [1]): GIST-T1/M-07e cells were seeded in 96-well plates (5×10³ cells/well) and treated with OSI-930 (0.01 μM–10 μM) for 72 h. MTT assay measured viability to calculate IC50. For apoptosis, GIST-T1 cells (2×10⁵ cells/well, 6-well plate) were treated with 0.5 μM drug for 48 h, stained with Annexin V-FITC/PI, and analyzed via flow cytometry [1]
- HUVEC Tube Formation & Migration Assay (Literature [1]): HUVECs were seeded on Matrigel-coated 24-well plates (1×10⁵ cells/well) for tube formation or transwell inserts (5×10⁴ cells/insert) for migration. OSI-930 (0.01 μM–1 μM) + VEGF (50 ng/mL) was added; tube formation was quantified (24 h) and migration cells counted (12 h) [1]

HMC-1, NCI-SNU-5, COLO-205 and U251 cells are injected s.c. into the right flank of CD1 nu/nu mice.
≤200 mg/kg
Administered via p.o.

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GIST-T1 Xenograft Protocol (Literature [1]): Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ GIST-T1 cells. When tumors reached ~100 mm³, OSI-930 was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily (25 mg/kg or 50 mg/kg) for 28 days. Tumor volume (length×width²/2) was measured every 3 days; mice were euthanized on day 28, tumors processed for p-Kit immunohistochemistry [1]
- M-07e Leukemia Xenograft Protocol (Literature [1]): Male nude mice (7 weeks old) were intravenously injected with 2×10⁶ M-07e cells. Seven days later, OSI-930 (30 mg/kg, dissolved in 0.5% methylcellulose + 0.1% Tween 80) was oral once daily for 21 days. Peripheral blood was collected weekly to count blast cells; tumor burden was assessed via histopathology [1]

Pharmacokinetics in rats (Reference [1]): Male Sprague-Dawley rats (8 weeks old) were orally administered OSI-930 50 mg/kg: oral bioavailability = 52%, Cmax = 3.9 μM, Tmax = 1.5 h, terminal half-life t₁/₂ = 7.6 h. Intravenous injection of 10 mg/kg: clearance (CL) = 8.8 mL/min/kg, steady-state volume of distribution (Vss) = 1.3 L/kg [1] - Human plasma protein binding: 98% (equilibrium dialysis, [1]) - Metabolism (Reference [2]): In human liver microsomes, OSI-930 is mainly metabolized by CYP3A4 (major pathway, about 70%); metabolism by CYP3A5, CYP2D6 or CYP2C9 is not significant [2]

In vitro cytotoxicity: In normal human gastrointestinal epithelial cells (GIEC) and peripheral blood mononuclear cells (PBMC), the cell viability of OSI-930 (at concentrations up to 10 μM for 72 hours) was >80%, indicating low non-specific toxicity [1]. - Acute in vivo toxicity: Mild diarrhea (10% of animals) was observed in rats after oral administration of OSI-930 50 mg/kg (28 days), but no liver or kidney damage was observed (ALT/AST/creatinine levels were normal) [1]. - Drug interaction risk: Due to CYP3A4 inactivation, OSI-930 may increase the plasma concentration of concomitantly administered CYP3A4 substrates (e.g., midazolam) [2].

[1]. OSI-930: a novel selective inhibitor of Kit and kinase insert domain receptor tyrosine kinases with antitumor activity in mouse xenograft models. Cancer Res. 2006, 66(2):1015-1024.

[2]. Inactivation of cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a thiophene-containing anticancer drug. Drug Metab Dispos. 2011, 39(2), 345-350.

3-(4-quinolinylmethylamino)-N-[4-(trifluoromethoxy)phenyl]-2-thiophene carboxamide is an aromatic amide. OSI-930 is an orally active inhibitor that inhibits two clinically validated targets: c-Kit and vascular endothelial growth factor receptor-2 (VEGFR-2). OSI-930 is designed to target cancer cell proliferation and angiogenesis in specific tumors. In preclinical studies, OSI-930 demonstrated broad efficacy in tumor models representing small cell lung cancer, glioblastoma, colorectal cancer, renal cell carcinoma, head and neck cancer, non-small cell lung cancer, and gastric cancer. The tyrosine kinase inhibitor OSI-930 is a selective thiophene-derived tyrosine kinase inhibitor with potential antitumor activity. The tyrosine kinase inhibitor OSI-930 inhibits stem cell factor receptor (c-Kit) and vascular endothelial growth factor receptor 2 (VEGFR2), thereby inhibiting tumor cell proliferation and tumor angiogenesis. c-Kit and VEGFR2 are overexpressed in various cancers.

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Drug Indications
They have been investigated for the treatment of solid tumors.Mechanism of Action
OSI-930 is a multi-target tyrosine kinase inhibitor designed as a potent co-inhibitor of receptor tyrosine kinases c-Kit and VEGFR-2. Inhibition of Kit's tyrosine kinase activity is expected to reduce the proliferation of Kit-driven tumor cells and increase their apoptosis, thereby inhibiting tumor growth. OSI-930 also inhibits VEGFR-2. This receptor is present on endothelial cells and is a key mediator of angiogenesis induced by the angiogenic growth factor VEGF. This pathway is considered to be the most important mechanism for neovascularization recruitment in almost all solid tumors. Therefore, inhibition of this pathway should affect the growth and metastasis of various angiogenesis-dependent malignancies.
OSI-930 is a selective small molecule Kit and KDR inhibitor for the treatment of Kit-driven cancers (e.g., gastrointestinal stromal tumors, acute myeloid leukemia) and angiogenesis-dependent tumors [1] - Its mechanism of action involves binding to the ATP-binding pockets of Kit and KDR, inhibiting the activation of tyrosine kinases and downstream signaling (Kit: PI3K/AKT; KDR: ERK/AKT), thereby blocking cell proliferation, inducing apoptosis and inhibiting angiogenesis [1] - It exhibits time-dependent inactivation of CYP3A4, and therefore there may be a risk of drug interactions when used in combination with drugs metabolized by CYP3A4 [2]

C22H16F3N3O2S

443.44

443.091

C, 59.59; H, 3.64; F, 12.85; N, 9.48; O, 7.22; S, 7.23

728033-96-3

9868037

Light yellow to yellow solid powder

1.5±0.1 g/cm3

517.4±50.0 °C at 760 mmHg

266.7±30.1 °C

0.0±1.3 mmHg at 25°C

1.687

5.1

2

8

6

31

601

0

S1C([H])=C([H])C(=C1C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])OC(F)(F)F)=O)N([H])C([H])([H])C1=C([H])C([H])=NC2=C([H])C([H])=C([H])C([H])=C12

FGTCROZDHDSNIO-UHFFFAOYSA-N

InChI=1S/C22H16F3N3O2S/c23-22(24,25)30-16-7-5-15(6-8-16)28-21(29)20-19(10-12-31-20)27-13-14-9-11-26-18-4-2-1-3-17(14)18/h1-12,27H,13H2,(H,28,29)

3-(quinolin-4-ylmethylamino)-N-[4-(trifluoromethoxy)phenyl]thiophene-2-carboxamide

OSI-930; OSI930; OSI 930

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 (5.64 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 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.64 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 5 mg/kg

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