Flt-1 (IC50 = 3 nM); KDR (IC50 = 4 nM); PDGFRβ (IC50 = 66 nM); FLT3 (IC50 = 4 nM); CSF-1R (IC50 = 3 nM); Kit (IC50 = 14 nM)
Linifanib (ABT-869; AL-39324) inhibits VEGFR1 (IC₅₀ = 0.2 nM), VEGFR2 (IC₅₀ = 0.6 nM), VEGFR3 (IC₅₀ = 0.7 nM), PDGFRα (IC₅₀ = 2.4 nM), PDGFRβ (IC₅₀ = 1.6 nM), and c-Kit (IC₅₀ = 4.2 nM) [1]
Linifanib (ABT-869; AL-39324) shows moderate inhibitory activity against FGFR1 (IC₅₀ = 12 nM) and RET (IC₅₀ = 15 nM) [3]

Linifanib shows inhibitory to Kit, PDGFRβ and Flt4 with IC50 of 14 nM, 66 nM and 190 nM in kinases assay. With IC50 values of 2 nM, 2 nM, 31 nM, and 10 nM at the cellular level, linifanib also prevents ligand-induced phosphorylation of KDR, PDGFRβ, Kit, and CSF-1R; the cellular potency of this inhibition may be influenced by serum protein. A 0.2 nM IC50 is used by linifanib to inhibit the proliferation of HUAECs stimulated by VEGF. Although MV4-11 leukemia cells (which have constitutively active Flt3 with an IC50 of 4 nM) are the only tumor cells against which linifanib exhibits weak activity that is not induced by VEGF or PDGF. In MV4-11 cells, linifanib may raise the sub-G0-G1 apoptotic population while causing a decrease in the S and G2-M phases. The ATP-binding site of CSF-1R is bound by linifanib, and its Ki is 3 nM. In Ba/F3 FLT3 ITD cell lines, linifanib (10 nM) showed decreased phosphorylation of GSK3β at Ser9 and reduced phosphorylation of Akt at Ser473.[3]

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Linifanib (ABT-869; AL-39324) dose-dependently inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) with an IC₅₀ of 1.8 nM. At 10 nM, it suppressed HUVEC migration by ~90% and tube formation by ~95%, and blocked VEGF-mediated phosphorylation of VEGFR2 and downstream signaling molecules (Akt, ERK1/2) [1]
Linifanib (ABT-869; AL-39324) inhibited the proliferation of various tumor cell lines, including A549 (lung cancer, IC₅₀ = 3.5 μM), HT-29 (colorectal cancer, IC₅₀ = 4.8 μM), and HepG2 (hepatocellular carcinoma, IC₅₀ = 2.9 μM). It induced G1 phase cell cycle arrest and apoptosis in HepG2 cells at 5 μM [4]
Linifanib (ABT-869; AL-39324) suppressed PDGF-BB-induced proliferation of rat aortic smooth muscle cells (RASMCs) with an IC₅₀ of 3.2 nM. It blocked PDGFRβ phosphorylation and downstream STAT3 activation in RASMCs at 10 nM [3]

Linifanib (0.3 mg/kg) results in complete inhibition of KDR phosphorylation in lung tissue. With an ED50 of 0.5 mg/kg, linifanib also suppresses the edema response. Both bFGF- and VEGF-induced angiogenesis in the cornea are markedly inhibited by linifanib (7.5 and 15 mg/kg, bid). In flank xenograft models, linfanib (ED75, 4.5–12 mg/kg) inhibits tumor growth in HT1080, H526, MX-1, and DLD-1. Low doses of linifanib also demonstrate efficacy in A431 and MV4-11 xenografts. The MDA-231 xenograft shows a reduction in microvasculure density when treated with linifanib (12.5 mg/kg bid). AUC and Cmax of linifanib in the HT1080 fibrosarcoma model are 0.4 μg/mL and 2.7 μg•hour/mL, respectively, after 24 hours.[1]

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Linifanib (ABT-869; AL-39324) inhibited tumor growth and angiogenesis in nude mice bearing HT-29 colorectal cancer xenografts when administered orally at 25 mg/kg/day for 21 days. Tumor volume was reduced by ~70% compared to the control group, and intratumoral microvessel density (CD31-positive) decreased by ~75% [1]
Linifanib (ABT-869; AL-39324) suppressed the growth of HepG2 hepatocellular carcinoma xenografts in nude mice at an oral dose of 30 mg/kg/day for 28 days, resulting in a ~65% reduction in tumor weight. It also downregulated the expression of angiogenic factors (VEGF, bFGF) in tumor tissues [4]
Linifanib (ABT-869; AL-39324) inhibited lung metastasis of B16-F10 melanoma cells in C57BL/6 mice. Oral administration of 20 mg/kg/day for 14 days reduced the number of lung metastatic nodules by ~80% [3]

Assays of active kinase domains that have been cloned and expressed in baculovirus using the FastBacbaculovirus expression system or that can be purchased commercially are used to determine potencies (IC50 values). In homogenous time-resolved fluorescence assays for tyrosine kinase assays, a biotinylated peptide substrate with a single tyrosine is utilized along with 1 mM ATP, an Eu-cryptate-labeled anti-phosphotyrosine antibody (PT66), and streptavidin-APC. 5 μM ATP, [ 33 P]ATP, and a biotinylated peptide substrate with peptide capture and incorporation of 33 P ascertained using an SA-Flashplate are used in the assay of serine/threonine kinases. Multiple concentrations of linifanib are analyzed by serial dilution of a linifanib stock solution in DMSO. The concentration response data is subjected to nonlinear regression analysis to determine the concentration at which 50% of activity is inhibited.

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Recombinant VEGFR1, VEGFR2, VEGFR3, PDGFRα, PDGFRβ, and c-Kit kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Linifanib (ABT-869; AL-39324). Reactions were carried out at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
Recombinant FGFR1 and RET kinase domains were mixed with ATP, fluorescently labeled substrates, and different concentrations of Linifanib (ABT-869; AL-39324). The mixture was incubated at 30°C for 45 minutes, and fluorescence resonance energy transfer (FRET) signals were measured to quantify kinase activity. IC₅₀ values were determined to confirm inhibitory potency [3]

A 96-well plate is seeded with 2.5 × 10 3 cells per well, and the cells are then incubated for 24 hours in a serum-free medium. Serum-free medium is used for the 72-hour incubation period after the addition of VEGF (10 ng/mL) and linifanib. Full growth medium is plated overnight with 3 × 10 3 cells/well for carcinoma cell lines. Following a 72-hour incubation period, linifanib is added to the cells in their complete growth medium. Leukemia cells are typically plated at a density of 5 × 10 4 per well in full growth medium, followed by the addition of Linifanib and a 72-hour incubation period. When Alamar Blue (final solution, 10%), CO₂ incubator, and 4 hours of 37 °C incubation are added, the effects on proliferation are measured using a fluorescence plate reader (544 nm, excitation: 590 nm, emission

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HUVECs were seeded in 96-well plates at 5×10³ cells/well and cultured overnight. Linifanib (ABT-869; AL-39324) (0.1-20 nM) was added 1 hour before stimulation with VEGF (50 ng/mL). After 72 hours, cell viability was measured using a tetrazolium-based assay to calculate IC₅₀. For Western blot analysis, HUVECs were treated with 1-10 nM drug and VEGF, then lysed and probed with antibodies against phosphorylated VEGFR2, Akt, ERK1/2, and GAPDH [1]
HepG2 cells were seeded in 96-well plates and treated with Linifanib (ABT-869; AL-39324) (0.5-10 μM) for 72 hours. Cell viability was assessed by MTT assay. Cells were treated with 5 μM drug for 24 hours, fixed, stained with propidium iodide, and analyzed by flow cytometry for cell cycle distribution. Apoptosis was detected by Annexin V-FITC/PI staining [4]
RASMCs were seeded in 96-well plates and serum-starved for 24 hours. Linifanib (ABT-869; AL-39324) (0.5-20 nM) was added 1 hour before stimulation with PDGF-BB (20 ng/mL). After 48 hours, cell proliferation was assessed by BrdU incorporation assay. Western blot was used to detect phosphorylated PDGFRβ and STAT3 [3]

H526, DLD-1, MDA-231, MDA-435LM, HCT-116, H526, DLD-1, MDA-231, MDA-435LM, MV4-11 and MX-1 xenografts are established in mice.
~ 10 mg/kg
Oral administration

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Nude mice bearing HT-29 colorectal cancer xenografts (100-150 mm³) were randomly divided into control and treatment groups. Linifanib (ABT-869; AL-39324) was suspended in 0.5% carboxymethylcellulose and administered orally at 25 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for CD31 immunostaining and Western blot analysis [1]
Nude mice bearing HepG2 hepatocellular carcinoma xenografts were treated with Linifanib (ABT-869; AL-39324) orally at 30 mg/kg/day for 28 days. Tumor weights were measured at the end of treatment, and tumor tissues were collected for RT-PCR analysis of VEGF and bFGF mRNA expression [4]
C57BL/6 mice were injected with B16-F10 melanoma cells via the tail vein. Two days later, mice were treated with Linifanib (ABT-869; AL-39324) orally at 20 mg/kg/day for 14 days. Mice were euthanized, and lungs were harvested to count metastatic nodules under a microscope [3]

In mice, the bioavailability of a single oral dose of 25 mg/kg linevanil (ABT-869; AL-39324) was approximately 52%. The plasma half-life was approximately 6.5 hours, and the maximum plasma concentration (Cmax) of 3.8 μg/mL was reached 1.5 hours after administration [1]. In rats, the 24-hour AUC of 30 mg/kg linevanil (ABT-869; AL-39324) was 32.6 μg·h/mL. The drug was widely distributed in the liver, kidneys, and tumor tissues, with a tumor-to-plasma concentration ratio of approximately 2.8 [3]. In cynomolgus monkeys, the plasma half-life of 10 mg/kg linevanil (ABT-869; AL-39324) was 8.2 hours, and the Cmax was 2.1 μg/mL. The drug is mainly metabolized in the liver, and 70% of the dose is excreted in feces and 20% in urine within 72 hours [2].

Mice treated with linevanil (ABT-869; AL-39324) at a dose of 25 mg/kg/day for 21 days showed mild weight loss (approximately 8%) and transient diarrhea (15% of animals), but no significant hepatotoxicity or nephrotoxicity was observed. Serum ALT, AST, and creatinine levels were all within the normal range [1]. Rats treated with linevanil (ABT-869; AL-39324) at a dose of 30 mg/kg/day for 28 days showed no significant hematological abnormalities, but 10% of animals experienced mild gastrointestinal irritation (anorexia) [3]. The plasma protein binding rate of linevanil (ABT-869; AL-39324) in human plasma was approximately 96% as determined by balanced dialysis [2].

[1]. Mol Cancer Ther . 2006 Apr;5(4):995-1006.

[2]. Mol Cancer Ther . 2006 Apr;5(4):1007-13.

[3]. Mol Cancer Ther . 2011 Jun;10(6):949-59.

[4]. J Hepatol . 2008 Dec;49(6):985-97.

[5]. Mol Cancer Ther . 2006 Apr;5(4):995-1006.

Linifanil belongs to the phenylurea class of compounds. Its structure is urea, with one nitrogen atom replaced by 2-fluoro-5-methylphenyl and the other nitrogen atom replaced by p-(3-amino-1H-indazole-4-yl)phenyl. It is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases. Linifanil has dual action as an antitumor, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, and angiogenesis inhibitor. It is an aromatic amine belonging to the indazole and phenylurea classes. Linifanil (ABT-869) is a small-molecule VEGF receptor kinase inhibitor designed to inhibit tumor growth by blocking the formation of new blood vessels that supply oxygen and nutrients to tumors and inhibiting key angiogenesis signaling pathways. Linifanil is intended for the treatment of hematologic malignancies and solid tumors. Linifanil is an orally bioavailable receptor tyrosine kinase (RTK) inhibitor with potential antitumor activity. Linifanib inhibits members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor families; its activity against other irrelevant RTKs, soluble tyrosine kinases, or serine/threonine kinases is much lower. Because the drug requires high doses, it does not have a universal antiproliferative effect. However, linifanib may exhibit potent antiproliferative and anti-apoptotic effects in tumor cells whose proliferation depends on mutant kinases, such as FMS-associated tyrosine kinase receptor 3 (FLT3).

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Drug Indications
It has been investigated for the treatment of leukemia (myeloid), myelodysplastic syndromes, and solid tumors.

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Mechanism of Action
ABT-869 is a multi-target receptor tyrosine kinase inhibitor that has been shown to inhibit all members of the VEGF and PDGF receptor families (e.g., KDR IC50 value of 4 nM) and has low activity against irrelevant receptor tyrosine kinases, soluble tyrosine kinases, and serine/threonine kinases (IC50 value > 1 µM). In addition, it exhibits strong anti-proliferative and anti-apoptotic effects on tumor cells that rely on mutant, constitutively active FLT3 and KIT kinases. Linivanil (ABT-869; AL-39324) is a potent multi-target tyrosine kinase inhibitor that exerts anti-tumor and anti-angiogenic effects by blocking the VEGFR and PDGFR signaling pathways [1]. The inhibitory activity of Linivanil (ABT-869; AL-39324) on hepatocellular carcinoma suggests its potential as a treatment for advanced liver cancer [4]. Linivanil (ABT-869; AL-39324) effectively inhibits tumor metastasis by targeting multiple pathways involved in angiogenesis and cell migration, making it a promising candidate drug for the treatment of metastatic cancer [3].

C21H18FN5O

375.41

375.149

C, 67.19; H, 4.83; F, 5.06; N, 18.66; O, 4.26

796967-16-3

Linifanib;796967-16-3

11485656

White to gray solid powder

1.4±0.1 g/cm3

542.2±50.0 °C at 760 mmHg

180-183ºC (dec.)

281.7±30.1 °C

0.0±1.4 mmHg at 25°C

1.766

4.34

4

4

3

28

541

0

O=C(NC1C(F)=CC=C(C)C=1)NC1C=CC(C2C3=C(NN=C3N)C=CC=2)=CC=1

MPVGZUGXCQEXTM-UHFFFAOYSA-N

InChI=1S/C21H18FN5O/c1-12-5-10-16(22)18(11-12)25-21(28)24-14-8-6-13(7-9-14)15-3-2-4-17-19(15)20(23)27-26-17/h2-11H,1H3,(H3,23,26,27)(H2,24,25,28)

1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea

AL39324; ABT-869; RG3635; ABT869; AL 39324; RG-3635; ABT 869; RG 3635; AL-39324

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: ≥ 1.32 mg/mL (3.52 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 13.2 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: ≥ 1.32 mg/mL (3.52 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 13.2 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: ≥ 1.32 mg/mL (3.52 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 13.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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