MET H1094Y (IC50 = 0.22 nM); MET Y1235D (IC50 = 1.7 nM); WT MET (IC50 = 4.2 nM); MET M1250T (IC50 = 6.5 nM); TRKA/NTRK1 (IC50 = 39 nM)
AST487 (NVP-AST487) is a potent inhibitor of RET tyrosine kinase and FMS-like tyrosine kinase 3 (FLT3) (including wild-type and mutant FLT3 variants); the IC50 for RET kinase activity is 8 nM [1]; the IC50 for wild-type FLT3 (wtFLT3) kinase is 5 nM, for FLT3-internal tandem duplication (FLT3-ITD) is 2 nM, and for FLT3-D835Y (PKC412-resistant mutant) is 7 nM [2]; it has weak inhibitory activity against other kinases (e.g., c-Kit, VEGFR2) with IC50 > 100 nM [1][2]

AST 487 (NVP-AST487) also exhibits similar inhibitory effects on several other kinases in the in vitro kinase assays, such as c-Kit (IC50=500 nM), c-Abl (IC50=20 nM), Flt-4 (IC50=790 nM), Flt-3 (IC50=520 nM), and KDR (IC50=170 nM). When human thyroid cancer cell lines with activating RET mutations are exposed to AST 487, it significantly slows down their growth, but not when the lines do not have similar mutations. By coincubating MTC-M cells with 100 nM of AST 487, both GDNF/GFRα1 and persephin-induced calcitonin mRNA are significantly inhibited[1]. A novel mutant inhibitor of FLT3 is AST 487. Flt-3 kinase activity inhibition is tested by AST 487 using biochemical assays. It is found that the Ki is 0.12 μM. In addition to Flt-3, NVP-AST487 has IC50 values less than 1 μM and inhibits RET, KDR, c-Kit, and c-Abl kinase. AST 487 treatment significantly reduces cell division in FLT3-ITD-Ba/F3 and D835Y-Ba/F3 cells (IC50<5 nM). When FLT3-ITD-Ba/F3 cells are treated with 0.01 μM AST 487, all cells are killed, whereas AML patient samples treated at the same concentration only kill about 50% of the cells[2].

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1. Medullary Thyroid Cancer (MTC) cells: AST487 dose-dependently inhibited proliferation of TT (RET-mutant) and MZ-CRC-1 (RET-activated) MTC cell lines with IC50 values of 15 nM (TT) and 22 nM (MZ-CRC-1) after 72-hour treatment [1]
2. RET signaling inhibition in MTC: Western blot analysis showed that AST487 (10–100 nM) concentration-dependently reduced phosphorylation of RET (p-RET), ERK1/2 (p-ERK1/2), and AKT (p-AKT) in TT cells, with complete inhibition of p-RET at 50 nM [1]
3. Calcitonin gene regulation in MTC: AST487 (20 nM) decreased calcitonin mRNA expression by 75% in TT cells (detected by qPCR) and reduced calcitonin protein secretion by 60% in the culture supernatant (detected by ELISA) after 24 hours; this effect was independent of RET inhibition [1]
4. AML cell proliferation inhibition: AST487 inhibited proliferation of FLT3-mutant acute myeloid leukemia (AML) cell lines: MV4-11 (FLT3-ITD, IC50=8 nM), Ba/F3-FLT3-ITD (IC50=5 nM), and PKC412-resistant Ba/F3-FLT3-D835Y (IC50=12 nM); it had no significant effect on FLT3-wild-type HL-60 cells (IC50 > 1 μM) [2]
5. Apoptosis induction in AML: AST487 (10 nM) induced apoptosis in MV4-11 cells, with Annexin V+/PI+ cells increasing from 6% to 45% after 48 hours; this was accompanied by activation of caspase-3/7 and cleavage of PARP (detected by western blot) [2]
6. FLT3 signaling inhibition in AML: AST487 (5–50 nM) dose-dependently inhibited phosphorylation of FLT3 (p-FLT3), STAT5 (p-STAT5), and ERK1/2 (p-ERK1/2) in MV4-11 and Ba/F3-FLT3-D835Y cells, with complete inhibition of p-FLT3 at 20 nM [2]
7. Clonogenic assay in primary AML cells: AST487 (1 nM) reduced colony formation of primary FLT3-ITD AML patient blasts by 80%, while normal CD34+ hematopoietic progenitor cells showed only 10% inhibition at the same concentration [2]

Following a single oral dosage of 15 mg/kg of AST 487, OF1 mice reach a mean peak plasma level (C_max) in 0.5 hours at 0.505±0.078 μM SE. Mice's plasma contains similar amounts of AST 487 up to 6 hours after oral administration, with a C_last of 21±4 nM at 24 hours. With a t_1/2 terminal elimination of 1.5 h, the oral bioavailability is estimated to be 9.7%[1].

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1. MTC xenograft model: In TT cell xenografts (BALB/c nude mice), oral administration of AST487 (50 mg/kg, once daily for 28 days) achieved 70% tumor growth inhibition (TGI) and reduced serum calcitonin levels (a MTC biomarker) by 65% compared with vehicle-treated mice; histological analysis showed a 55% reduction in Ki67-positive proliferating cells and decreased p-RET expression in tumor tissues [1]
2. AML xenograft model (MV4-11): In NOD/SCID mice bearing MV4-11 xenografts, intraperitoneal injection of AST487 (30 mg/kg, twice daily for 14 days) reduced leukemia cell burden in bone marrow and spleen by 85% and extended median survival from 21 days (vehicle) to 42 days (treatment) [2]
3. PKC412-resistant AML xenograft model: In Ba/F3-FLT3-D835Y xenografts (BALB/c nu/nu mice), AST487 (40 mg/kg, intraperitoneal injection, once daily for 21 days) achieved 65% TGI and reduced peripheral blood blast counts by 70% [2]
4. Combination therapy in AML: Co-administration of AST487 (20 mg/kg) with cytarabine (10 mg/kg) in MV4-11 xenografts showed synergistic efficacy, with 90% TGI and undetectable leukemia cells in bone marrow [2]

The kinase domains fused with glutathione S-transferase (GST) are purified using glutathione-sepharose after being expressed in baculovirus. Phosphorylation of a synthetic substrate [poly(Glu, Tyr)] by purified GST-fusion kinase domains of the corresponding protein kinase in the presence of radiolabeled ATP is how kinase activity is measured; ATP concentrations are optimized within the K_m range for each specific kinase. In summary, every kinase is cultured in 20 mM of Tris-HCl (pH 7.5), 1 to 3 mM of MnCl₂, 3 to 10 mM of MgCl₂, 10 μM of Na₃VO₄, 1 mM of DTT, 0.2 μCi [³³P]ATP, 1 to 8 μM of ATP, 3 to 8 μg/mL of poly(Glu/Tyr, 4:1), and 1% DMSO in a total volume of 30 μL, either in the presence or absence of NVP-AST487 for 10 minutes at room temperature. The reaction mixture is placed onto an Immobilon polyvinylidene difluoride membrane after 10 μL of 250 mM EDTA is added to stop the reaction. In a liquid scintillation counter, filters are counted after being cleaned (0.5% H₃PO₄), soaked in ethanol, and dried. A linear regression analysis of the percentage inhibition is used to determine the IC50s for AST 487.

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1. RET kinase activity assay: Recombinant human RET intracellular kinase domain was incubated with serial concentrations of AST487 and a biotinylated RET-specific peptide substrate in the presence of ATP; substrate phosphorylation was detected via time-resolved fluorescence resonance energy transfer (TR-FRET) using an anti-phosphotyrosine antibody, and dose-response curves were generated to calculate the IC50 for RET kinase inhibition [1]
2. FLT3 kinase activity assay: Recombinant human wtFLT3, FLT3-ITD, and FLT3-D835Y kinase domains were incubated with AST487 and a fluorescent peptide substrate; kinase activity was measured by quantifying ADP production via an ADP-Glo luminescent assay, and IC50 values for each FLT3 variant were calculated from dose-response data [2]
3. Kinase selectivity assay: A panel of 20 recombinant human tyrosine kinases (c-Kit, VEGFR2, PDGFRβ, etc.) was incubated with AST487 (100 nM) and their respective peptide substrates; kinase activity was assessed using the TR-FRET method to evaluate the selectivity of AST487 for RET and FLT3 [1]

The trypan blue exclusion assay is used to assess how many cells proliferate when NVP-AST 487 is present or absent during culture. Cell viability is expressed as a percentage of control (untreated) cells. Unless otherwise noted, data are the mean of two independent experiments. Each data point's standard error of the mean is shown by an error bar. The Annexin-V-Fluos Staining Kit is used to quantify the amount of apoptosis in drug-treated cells. Analysis of the cell cycle is done.

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1. MTC cell proliferation assay: TT and MZ-CRC-1 cells were seeded in 96-well plates (4×10³ cells/well) and treated with AST487 (0.1 nM–1 μM) for 72 hours; cell viability was determined via MTT assay (MTT incubation for 4 hours, solubilization with DMSO, absorbance measurement at 570 nm), and IC50 values for proliferation inhibition were calculated from sigmoidal dose-response curves [1]
2. Calcitonin expression assay: TT cells were treated with AST487 (5–50 nM) for 24 hours; total RNA was extracted and reverse-transcribed to cDNA, and calcitonin mRNA levels were quantified by qPCR (with GAPDH as a reference); calcitonin protein secretion in the supernatant was measured by ELISA [1]
3. RET signaling western blot assay: TT cells were treated with AST487 (10–100 nM) for 2 hours; total protein was extracted, separated by SDS-PAGE, and transferred to a nitrocellulose membrane; the membrane was probed with antibodies against p-RET, total RET, p-ERK1/2, total ERK1/2, p-AKT, total AKT, and β-actin (loading control), followed by chemiluminescent detection [1]
4. AML cell proliferation assay: MV4-11, Ba/F3-FLT3-ITD, and Ba/F3-FLT3-D835Y cells were seeded in 96-well plates (2×10³ cells/well) and treated with AST487 (0.1 nM–1 μM) for 72 hours; cell viability was assessed via CellTiter-Glo luminescent assay (ATP measurement), and IC50 values were calculated [2]
5. AML apoptosis assay: MV4-11 cells were treated with AST487 (0–50 nM) for 48 hours, stained with Annexin V-FITC and PI, and apoptotic cells were quantified by flow cytometry; cleaved caspase-3, cleaved PARP, and Bcl-2 family proteins were detected by western blot for mechanistic analysis [2]
6. AML clonogenic assay: Primary FLT3-ITD AML blasts and normal CD34+ progenitor cells were suspended in methylcellulose medium with AST487 (0–10 nM) and plated in 6-well plates; after 14 days of incubation, colonies (>50 cells) were counted under a microscope, and the colony formation inhibition rate was calculated relative to vehicle controls [2]

Mice: Female athymic nude mice are housed in Makrolon type III cages with unrestricted access to food and water, and ideal hygienic conditions (maximum of 10 mice per cage). NIH3T3-RETC634W and TT cells, at 1×10⁶ and 5×10⁶, respectively, are injected subcutaneously into 100 μL of HBSS per mouse to form tumors. Tumors that could be treated, defined as having a mean tumor volume of 100 mm³, appeared 10 days after the injection of NIH3T3-RETC634W cells and 20 days after the injection of TT cells. NVP-AST487 is administered orally once a day via gavage. The right amount of powder is dissolved in N-methylpyrrolidone/PEG300 (1:10 v/v) to create the compound. The four treatment groups, each with eight mice, are randomly assigned to the mice. For three weeks, NVP-AST487 was given orally to the first three groups at 50, 30, and 10 mg/kg, respectively. A car was used to treat the fourth group. Body weights and tumor growth are recorded twice a week. The formula for calculating tumor volumes is length×diameter²×π/6. At the conclusion of the efficacy study, six hours after the last administration, tumors are gathered and frozen in liquid nitrogen.

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1. TT cell MTC xenograft model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with TT cells (5×10⁶) into the right flank; when tumors reached ~100 mm³, mice were randomized into vehicle and AST487 groups (n=8 per group); AST487 was dissolved in 10% DMSO, 30% PEG400, and 60% normal saline, and administered by oral gavage at 50 mg/kg once daily for 28 days; tumor volume (length × width² / 2) and body weight were measured twice weekly; at study end, mice were euthanized, tumors were excised and weighed, and serum was collected for calcitonin ELISA [1]
2. MV4-11 AML xenograft model: NOD/SCID mice (6–8 weeks old) were intravenously injected with MV4-11 cells (1×10⁷); 7 days later, mice were treated with AST487 (30 mg/kg, intraperitoneal injection, twice daily) or vehicle (5% DMSO, 20% Cremophor EL, 75% normal saline) for 14 days; peripheral blood blast counts were measured weekly by flow cytometry, and survival was monitored for 60 days; bone marrow and spleen tissues were collected for histological analysis of leukemia infiltration [2]
3. Ba/F3-FLT3-D835Y AML xenograft model: BALB/c nu/nu mice were tail-vein injected with Ba/F3-FLT3-D835Y cells (2×10⁶); 5 days later, mice were treated with AST487 (40 mg/kg, intraperitoneal injection, once daily) for 21 days; bone marrow leukemia burden was quantified by qPCR for human CD45 mRNA, and spleen weight was measured to assess splenomegaly [2]

1. Preliminary studies in mice showed that after oral administration of 50 mg/kg AST487, the plasma concentration (Cmax) reached a maximum of 1.5 μM 1 hour later, and the plasma concentration could be detected for up to 12 hours [1]

1. Acute toxicity: AST487 was well tolerated in mice at oral doses up to 100 mg/kg and intraperitoneal doses up to 75 mg/kg, with no deaths or serious clinical symptoms (weight loss, lethargy) observed [1][2] 2. Subchronic toxicity: In a 14-day rat study, oral administration of AST487 (20, 50, 100 mg/kg/day) caused only mild thrombocytopenia at a dose of 100 mg/kg, which returned to normal 7 days after discontinuation; no significant changes were found in serum liver function (ALT/AST) or kidney function (creatinine/urea) [2] 3. Plasma protein binding rate: The plasma protein binding rate of AST487 in human plasma was approximately 90%, and in mouse plasma it was 88% (measured by ultrafiltration) [1]

[1]. The RET Kinase Inhibitor NVP-AST487 Blocks Growth and Calcitonin Gene Expression through Distinct Mechanisms in Medullary Thyroid Cancer Cells. Cancer Res. 2007 Jul 15;67(14):6956-64.

[2]. Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells. Blood. 2008 Dec 15;112(13):5161-70.

1. AST487 (NVP-AST487) is a small molecule tyrosine kinase inhibitor developed by Novartis. It was initially identified as a RET inhibitor for the treatment of medullary thyroid carcinoma (MTC), and was later found to target mutant FLT3 for the treatment of acute myeloid leukemia (AML) [1][2]. 2. AST487 exerts its effects by competitively binding to the ATP-binding pockets of RET and FLT3, inhibiting their phosphorylation and downstream signaling pathways (ERK/AKT for RET; STAT5/ERK for FLT3), which are crucial for the proliferation and survival of MTC/AML cells [1][2]. 3. In MTC cells, AST487 inhibits cell growth by blocking the RET signaling pathway and reduces calcitonin expression through a RET-independent transcriptional mechanism [1]. 4. AST487 induces acquired resistance to first-generation FLT3 inhibitors in FLT3-D835Y AML cells that are resistant to PKC412[2]

C26H30F3N7O2

529.557315349579

529.241

C, 58.97; H, 5.71; F, 10.76; N, 18.51; O, 6.04

630124-46-8

630124-46-8

11409972

White to off-white solid powder

5.205

3

10

8

38

730

0

FC(C1C=C(C=CC=1CN1CCN(CC)CC1)NC(NC1C=CC(=CC=1)OC1C=C(N=CN=1)NC)=O)(F)F

ODPGGGTTYSGTGO-UHFFFAOYSA-N

InChI=1S/C26H30F3N7O2/c1-3-35-10-12-36(13-11-35)16-18-4-5-20(14-22(18)26(27,28)29)34-25(37)33-19-6-8-21(9-7-19)38-24-15-23(30-2)31-17-32-24/h4-9,14-15,17H,3,10-13,16H2,1-2H3,(H,30,31,32)(H2,33,34,37)

1-[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[4-[6-(methylamino)pyrimidin-4-yl]oxyphenyl]urea

AST 487; NVP AST 487; NVP-AST487; AST487; AST-487; NVP-AST-487; NVP-AST 487; NVP AST-487; NVP AST487

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)

DMSO: ~11 mg/mL (~20 mM)
Ethanol: ˂1 mg/mL
Water: ˂1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.72 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 (4.72 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 25.0 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: ≥ 2.5 mg/mL (4.72 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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 (Please use freshly prepared in vivo formulations for optimal results.)