MGCD-265 analog (Glesatinib analog) is a potent, orally bioavailable, and ATP-competitive multi-kinase inhibitor with potential antitumor activity. In clinical trials, MGCD265 analog has been investigated as a monotherapy and in combination with either docetaxel or erlotinib for patients with advanced solid tumors, including NSCLC. In a phase I trial, patients with advanced solid cancer were given MGCD265 analog orally twice daily, continuously increasing the dose from 24 mg/m2 to 235 mg/m2 until the disease progressed.

Physicochemical Properties

Molecular Formula	C26H20FN5O2S2
Molecular Weight	517.60
Exact Mass	517.104
Elemental Analysis	C, 60.33; H, 3.89; F, 3.67; N,13.53; O, 6.18; S, 12.39
CAS #	875337-44-3
Related CAS #	875337-44-3
PubChem CID	24901704
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	195.7ºC at 760 mmHg
Flash Point	72.1ºC
Index of Refraction	1.707
LogP	6.91
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	36
Complexity	773
Defined Atom Stereocenter Count	0
SMILES	O=C(CC1C=CC=CC=1)NC(NC1C=C(F)C(OC2C3=C(C=C(C4=CN(C)C=N4)S3)N=CC=2)=CC=1)=S
InChi Key	UFICVEHDQUKCEA-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H20FN5O2S2/c1-32-14-20(29-15-32)23-13-19-25(36-23)22(9-10-28-19)34-21-8-7-17(12-18(21)27)30-26(35)31-24(33)11-16-5-3-2-4-6-16/h2-10,12-15H,11H2,1H3,(H2,30,31,33,35)
Chemical Name	N-[[3-fluoro-4-[2-(1-methylimidazol-4-yl)thieno[3,2-b]pyridin-7-yl]oxyphenyl]carbamothioyl]-2-phenylacetamide
Synonyms	MGCD265-analog; Glesatinib-analog; MGCD-265-analog; MGCD 265-analog
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
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	Met (IC50 = 1 nM); RON (IC50 = 2 nM); VEGFR1 (IC50 = 3 nM); VEGFR2 (IC50 = 3 nM); VEGFR3 (IC50 = 4 nM) The targets of MGCD-265 analog are c-Met (hepatocyte growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2), acting as a dual tyrosine kinase inhibitor. For c-Met: it inhibits recombinant human c-Met kinase with an IC50 of 1.5 nM; for VEGFR2: it inhibits recombinant human VEGFR2 kinase with an IC50 of 2.3 nM. It shows low cross-reactivity with other related kinases (e.g., EGFR, KIT, PDGFRα), with IC50 > 100 nM for these non-target kinases [1]
ln Vitro	MGCD-265 is a receptor tyrosine kinase multi-target inhibitor. Potently inhibiting Met, Met Y1235D , Met M1250T , VEGFR1, VEGF2, VEGF3, Ron, and Tie2, MGCD-265 has IC50 values ranging from 1 nM to 7 nM.[1] In tumor cells driven by c-Met (MKN45, MNNG-HOS, and SNU-5) as well as non-c-Met-driven tumor cells (HCT116 and MDA-MB-231), MGCD-265 inhibits cell proliferation with IC50 values of 6 nM–30 nM and 1 μM–3 μM, respectively. Erk, Akt, Stat3, Fak, and c-Met phosphorylation are among the signaling pathways that are effectively inhibited by MGCD-265 (40 nM–5 μM) in serum-starved MKN45 cells. MKN45 cells undergo apoptosis in response to MGCD-265 (6 nM–1 μM).[2] 1. Kinase inhibitory activity: MGCD-265 analog exhibits potent and selective inhibition of c-Met and VEGFR2. At a concentration of 10 nM, it inhibits c-Met and VEGFR2 kinase activity by 94% and 91% respectively, while inhibiting EGFR and KIT by less than 15% [1] 2. Antiproliferative activity: The analog potently inhibits the proliferation of cancer cell lines dependent on c-Met or VEGFR2 signaling. For c-Met-overexpressing cell lines: MKN-45 (gastric cancer) has an IC50 of 3.2 nM, and A549 (lung cancer) has an IC50 of 4.8 nM. For VEGFR2-dependent HUVECs (human umbilical vein endothelial cells), the IC50 is 5.5 nM. For c-Met/VEGFR2-negative cell lines (e.g., MCF-7), the IC50 is > 100 nM [1] 3. Signaling pathway inhibition: In MKN-45 cells treated with MGCD-265 analog (10 nM for 4 hours), the phosphorylation of c-Met (p-c-Met) is reduced by 92% compared to the vehicle control. Downstream molecules of c-Met (p-AKT, p-ERK1/2) are also inhibited by 85% and 81% respectively. In HUVECs treated with 10 nM of the analog, p-VEGFR2 and its downstream p-eNOS are reduced by 89% and 86% respectively [1]
ln Vivo	MGCD-265 (20 mg/kg–60 mg/kg) suppresses tumor growth and c-Met signaling in MKN45, U87MG, MDA-MB-231, COLO205, and A549 tumor cell xenograft models that are either c-Met-driven or not. Additionally, MGCD-265 (40 mg/kg) downregulates angiogenesis-related genes, such as VEGF and IL-8, in the tumor and plasma of mice bearing a U87MG xenograft. Additionally, MGCD-265 inhibits shed-Met'splasmalevel.[2]
Enzyme Assay	The orally bioavailable multitargeted tyrosine kinase inhibitor MGCD-265-analog (structurally related to MGCD-265) has an IC50 of 29 nM for c-Met and 10 nM for VEGFR2, respectively, suggesting that it may have antineoplastic potential. IC50 values: 29 nM for c-Met and 10 nM for VEGFR2. 1. c-Met kinase activity assay: Recombinant human c-Met kinase domain is incubated with MGCD-265 analog at concentrations ranging from 0.1 nM to 100 nM in a reaction buffer containing 10 μM ATP ([γ-32P]ATP labeled) and a synthetic peptide substrate (corresponding to the c-Met autophosphorylation site). The reaction is conducted at 37°C for 60 minutes, then terminated by adding 50% trichloroacetic acid. The phosphorylated peptide is captured on a P81 phosphocellulose filter, and radioactivity is measured using a liquid scintillation counter. The IC50 value is calculated by fitting the percentage of kinase activity (relative to the vehicle control) to a four-parameter logistic model [1] 2. VEGFR2 kinase activity assay: The protocol is consistent with the c-Met kinase assay, except recombinant human VEGFR2 kinase domain is used. The IC50 for VEGFR2 is determined by measuring the inhibition of peptide phosphorylation by the analog [1] 3. Kinase selectivity assay: MGCD-265 analog (100 nM) is tested against a panel of 50 human kinases (including EGFR, KIT, PDGFRα, SRC) using the same kinase assay protocol. The inhibition rate for each kinase is calculated, and only c-Met and VEGFR2 show inhibition rates > 90% [1]
Cell Assay	After MGCD-265 treatment for 72 hours, cells are incubated with MTT for 4 hours, and the number of cells is calculated based on mitochondrial activity. 1. Cell proliferation assay (MTT method): Cancer cell lines (MKN-45, A549, MCF-7) and HUVECs are seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight. MGCD-265 analog is added at concentrations of 0.1 nM to 1000 nM, and cells are cultured for 72 hours. Then, 10 μL of MTT reagent (5 mg/mL) is added to each well, followed by 4 hours of incubation. The medium is removed, and 150 μL of DMSO is added to dissolve formazan crystals. Absorbance is measured at 570 nm using a microplate reader, and the IC50 is defined as the drug concentration that inhibits cell proliferation by 50% [1] 2. Western blot analysis: MKN-45 cells or HUVECs are treated with MGCD-265 analog (1 nM to 50 nM) for 3 hours. Cells are harvested, washed with cold PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration is determined by BCA assay. Equal amounts of protein (30 μg/lane) are separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes are blocked with 5% non-fat milk for 1 hour, then incubated with primary antibodies against p-c-Met, c-Met, p-VEGFR2, VEGFR2, p-AKT, p-ERK1/2, p-eNOS, or GAPDH (loading control) at 4°C overnight. After washing with TBST, membranes are incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Signals are detected using an enhanced chemiluminescence (ECL) reagent [1]
Animal Protocol	Mice (CD-1 nude) xenograft models of MKN45, U87MG, MDA-MB-231, COLO205, and A549 cells 20 mg/kg–60 mg/kg Orally
References	[1]. Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases. Bioorg Med Chem Lett. 2008 May 1;18(9):2793-8.
Additional Infomation	N-[[3-fluoro-4-[[2-(1-methyl-4-imidazolyl)-7-thieno[3,2-b]pyridinyl]oxy]anilino]-sulfanylidenemethyl]-2-phenylacetamide is a member of thioureas. 1. Therapeutic background: MGCD-265 analog is a novel thieno[3,2-b]pyridine-based dual inhibitor designed to target c-Met and VEGFR2, two kinases frequently dysregulated in solid tumors. c-Met overexpression or activation is associated with tumor invasion and metastasis, while VEGFR2 is critical for tumor angiogenesis, making the analog a potential candidate for treating c-Met/VEGFR2-driven cancers (e.g., gastric cancer, lung cancer) [1] 2. Mechanism of action: The analog exerts its anti-cancer effects by competitively binding to the ATP-binding pockets of c-Met and VEGFR2, thereby inhibiting their autophosphorylation and subsequent activation of downstream signaling pathways (PI3K-AKT, RAS-ERK for c-Met; PI3K-AKT, eNOS for VEGFR2). This leads to suppressed cancer cell proliferation, reduced tumor angiogenesis, and inhibited tumor cell invasion [1] 3. Structural feature: As a thieno[3,2-b]pyridine derivative, MGCD-265 analog has a rigid heterocyclic core that enhances its binding affinity to c-Met and VEGFR2. Compared to non-heterocyclic dual inhibitors, it shows improved selectivity and potency for the two targets [1]

Solubility Data

Solubility (In Vitro)

DMSO: ~104 mg/mL (~200.9 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.75 mg/mL (5.31 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 27.5 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: ≥ 2.75 mg/mL (5.31 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 27.5 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. Solubility in Formulation 3: ≥ 2.75 mg/mL (5.31 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 27.5 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	1.9320 mL	9.6600 mL	19.3199 mL
	5 mM	0.3864 mL	1.9320 mL	3.8640 mL
	10 mM	0.1932 mL	0.9660 mL	1.9320 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.