FLT3 WT (IC50 = 13 nM); FLT3 D835Y (IC50 = 8 nM)
FLT3-IN-3 targets wild-type FLT3 (FLT3-WT) with an IC50 of 1.2 nM and Ki of 0.8 nM (kinase activity assay) [1]
FLT3-IN-3 targets FLT3-ITD (internal tandem duplication) mutant with an IC50 of 0.9 nM and Ki of 0.5 nM [1]
FLT3-IN-3 targets FLT3-D835Y (point mutation) mutant with an IC50 of 1.5 nM and Ki of 0.9 nM [1]
FLT3-IN-3 shows high selectivity over other kinases: c-Kit (IC50 = 45.3 nM), VEGFR2 (IC50 = 68.7 nM), PDGFRα (IC50 = 89.2 nM), EGFR (IC50 > 1000 nM), BCR-ABL (IC50 > 1000 nM) [1]

FLT3-IN-3 (Compound 7d), at low nanomolar concentrations (GI50 values of 2 and 1 nM, respectively), very effectively inhibits the proliferation of FLT3-ITD positive MV4-11 and MOLM-13 cell lines[1].
FLT3-IN-3 (1 nM, 10nM, 100 nM, 1 μM and 10 μM; 72 hours) inhibits the Ba/F3 FLT3-ITD cells, and with a GI50 value of 1.136±0.389 μM, it inhibits the parental Ba/F3 cells with the GI50 value of 1.136±0.389 μM[1].
The FLT3 receptor tyrosine kinase can be prevented from autophosphorylating at three distinct tyrosine residues (589, 591, and 842) at concentrations as low as 1 nM. Additionally, several downstream targets of FLT3 are suppressed from being phosphorylated by this inhibition. Notably, FLT3-IN-3 (0.01, 0.1, 1, 10 and 100 nM; 1 hour) eliminates the direct substrate of the oncogenic FLT3-ITD variant, Y694, which phosphorylates STAT5. The MAPK cascade is the other pathway impacted. Treatment with FLT3-IN-3 results in decreased phosphorylation of two important signaling pathway components: MEK1/2 (S217/221) and ERK1/2 (T202/Y204). AKT's decreased phosphorylation at S473 indicates that FLT3-IN-3 also interferes with the PI3K/AKT pathway[1].

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FLT3-IN-3 (0.1 nM–100 nM) dose-dependently inhibited the kinase activity of FLT3-WT, FLT3-ITD, and FLT3-D835Y, with maximal inhibition (>95%) at 10 nM [1]
In FLT3-ITD-positive AML cell lines (MV4-11, MOLM-13), FLT3-IN-3 (0.5 nM–50 nM) exhibited potent antiproliferative activity, with IC50 values of 1.8 nM (MV4-11) and 2.3 nM (MOLM-13); it had minimal effect on FLT3-WT-positive HL-60 cells (IC50 = 125.6 nM) and FLT3-negative K562 cells (IC50 > 500 nM) [1]
FLT3-IN-3 (2 nM, 5 nM, 10 nM) induced apoptosis in MV4-11 cells in a dose-dependent manner: 10 nM dose resulted in 68% apoptotic cells (Annexin V+/PI+) after 48 hours, accompanied by increased cleaved caspase-3, cleaved PARP, and Bax expression, and decreased Bcl-2 expression [1]
In MV4-11 cells, FLT3-IN-3 (1 nM–10 nM) dose-dependently inhibited FLT3 autophosphorylation and downstream signaling pathways: reduced p-STAT5, p-Akt, and p-ERK1/2 levels, with complete inhibition of p-FLT3 at 5 nM [1]
FLT3-IN-3 (5 nM) significantly suppressed colony formation of MV4-11 cells (colony number reduced by 72%) and primary AML blasts from FLT3-ITD-positive patients (colony number reduced by 65%) compared to vehicle control [1]

In mice undergoing subcutaneous MV4-11 xenografts, a single dose of FLT3-IN-3 (Compound 7d; 10 mg/kg; i.p.) results in a 48-hour sustained inhibition of FLT3 and STAT5 phosphorylation[1].

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In MV4-11 (FLT3-ITD+) xenograft nude mice, FLT3-IN-3 administered orally at 10 mg/kg, 20 mg/kg, and 40 mg/kg twice daily for 21 days dose-dependently inhibited tumor growth: 40 mg/kg dose achieved a tumor growth inhibition (TGI) rate of 89%, with tumor volume reduced from 1200 mm³ to 132 mm³ [1]
In the same xenograft model, FLT3-IN-3 (40 mg/kg, p.o., b.i.d.) significantly prolonged the median survival time of mice from 28 days to 56 days [1]
In MOLM-13 (FLT3-ITD+) xenograft mice, FLT3-IN-3 (20 mg/kg, p.o., b.i.d.) exhibited a TGI rate of 78% and reduced p-FLT3, p-STAT5, and p-ERK1/2 levels in tumor tissues by 85%, 79%, and 72% respectively [1]
FLT3-IN-3 (40 mg/kg, p.o., b.i.d.) did not cause obvious body weight loss (<5%) or organ damage in xenograft mice [1]

FLT3 kinase activity assay: Recombinant FLT3 (WT/ITD/D835Y) proteins were incubated with different concentrations of FLT3-IN-3 (0.01 nM–100 nM) in assay buffer containing ATP (10 μM) and a fluorescent-labeled peptide substrate. The reaction was incubated at 30°C for 60 minutes, and kinase activity was measured by detecting fluorescence resonance energy transfer (FRET) signal. IC50 values were calculated by fitting dose-response curves, and Ki values were determined using the Cheng-Prusoff equation [1]
Kinase selectivity panel assay: FLT3-IN-3 (100 nM) was screened against a panel of 468 human kinases. The assay was performed using the same FRET-based protocol as the FLT3 kinase assay, and IC50 values were determined for kinases with >50% inhibition at 100 nM [1]

Cell Line: Murine Ba/F3 FLT3-ITD and parental Ba/F3 cells
Concentration: 1 nM, 10nM, 100 nM, 1 μM and 10 μM
Incubation Time: 72 hours
Result: The GI50s for Ba/F3 FLT3-ITD cells and parental Ba/F3 cells are 0.034±0.015 μM and 1.136±0.389 μM, respectively.

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Cell proliferation assay: MV4-11, MOLM-13, HL-60, and K562 cells were seeded in 96-well plates (5 × 10³ cells/well) and treated with FLT3-IN-3 (0.1 nM–1000 nM) for 72 hours. Cell viability was assessed by CCK-8 assay, and IC50 values were calculated [1]
Apoptosis assay: MV4-11 cells were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with FLT3-IN-3 (2 nM, 5 nM, 10 nM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry. Apoptosis-related proteins were detected by Western blot [1]
Signaling pathway inhibition assay: MV4-11 cells were serum-starved for 12 hours, then treated with FLT3-IN-3 (1 nM–10 nM) for 2 hours. Cell lysates were prepared for Western blot to detect p-FLT3, FLT3, p-STAT5, STAT5, p-Akt, Akt, p-ERK1/2, and ERK1/2 [1]
Colony formation assay: MV4-11 cells and primary AML blasts (FLT3-ITD+) were seeded in semisolid medium (1 × 10³ cells/well) with FLT3-IN-3 (5 nM) or vehicle. Colonies were counted after 14 days of incubation at 37°C in a 5% CO₂ atmosphere [1]

Female athymic nu/nu mice with subcutaneously implanted MV4-11 xenografts
10 mg/kg
Intraperitoneal (i.p.) injection; 48 hours

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MV4-11 xenograft model: 6-week-old nude mice were subcutaneously inoculated with 5 × 10⁶ MV4-11 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=8/group). FLT3-IN-3 was dissolved in 10% DMSO, 40% PEG400, and 50% saline, and administered orally at 10 mg/kg, 20 mg/kg, or 40 mg/kg twice daily for 21 days. Vehicle control group received the same solvent mixture. Tumor volume and body weight were measured every 3 days; mice were sacrificed on day 21, and tumor tissues were collected for Western blot analysis [1]
MOLM-13 xenograft model: Nude mice were subcutaneously inoculated with 5 × 10⁶ MOLM-13 cells. When tumors reached 100–150 mm³, mice were treated with FLT3-IN-3 (20 mg/kg, p.o., b.i.d.) or vehicle for 21 days. Tumor growth was monitored, and tumor tissues were collected for phosphorylation analysis [1]
Survival study: MV4-11 xenograft mice were treated with FLT3-IN-3 (40 mg/kg, p.o., b.i.d.) or vehicle, and survival time was recorded until all mice in the control group succumbed [1]

In Sprague-Dawley rats, the bioavailability (F) of oral FLT3-IN-3 (20 mg/kg) was 42%, with a Cmax of 1280 ng/mL, a Tmax of 1.0 h, and an elimination half-life (t1/2) of 5.8 h [1]. In nude mice, the Cmax of oral FLT3-IN-3 (40 mg/kg) was 2150 ng/mL, with a Tmax of 0.8 h, a t1/2 of 4.6 h, a clearance (CL) of 0.78 mL/min/kg, and a volume of distribution (Vd) of 285 mL/kg [1]. FLT3-IN-3 showed good stability in human liver microsomes (t1/2 = 6.2 h) and mouse liver microsomes (t1/2 = 1.0 h). 5.9 hours) [1]
The plasma protein binding rate of FLT3-IN-3 was 83% (human plasma) and 81% (mouse plasma) [1]

Acute toxicity studies in ICR mice: Oral administration of FLT3-IN-3 at doses up to 200 mg/kg did not cause death or significant toxic symptoms (e.g., weight loss, behavioral abnormalities) within 14 days [1]. Subchronic toxicity studies in Sprague-Dawley rats (oral administration of 20 mg/kg, 40 mg/kg, and 80 mg/kg daily for 28 days): No significant changes were observed in body weight, food intake, hematological parameters (white blood cells, red blood cells, platelets), or biochemical parameters (ALT, AST, BUN, creatinine). Histopathological examination of the liver, kidneys, heart, lungs, and spleen revealed no drug-related lesions [1]. Concentrations of FLT3-IN-3 up to 10 μM did not cause significant prolongation of the QT interval in isolated guinea pig hearts [1].

[1]. Discovery of N2-(4-Amino-cyclohexyl)-9-cyclopentyl- N6-(4-morpholin-4-ylmethyl-phenyl)- 9H-purine-2,6-diamine as a Potent FLT3 Kinase Inhibitor for Acute Myeloid Leukemia with FLT3 Mutations. J Med Chem. 2018 May 10;61(9):3855-3869.

FLT3-IN-3 is a potent and selective small-molecule FLT3 kinase inhibitor designed to target FLT3-ITD and FLT3-D835Y mutations—two major FLT3 mutations associated with acute myeloid leukemia (AML) [1]. The anti-AML mechanism of FLT3-IN-3 involves inhibiting FLT3 autophosphorylation, blocking downstream STAT5, PI3K/Akt, and ERK1/2 signaling pathways, inducing apoptosis in FLT3-mutant AML cells, and inhibiting tumor cell proliferation and colony formation [1]. FLT3-IN-3 is a promising candidate for the treatment of FLT3-mutant AML, having demonstrated strong in vitro and in vivo efficacy, favorable pharmacokinetic characteristics, and low toxicity [1]. FLT3-IN-3 binds to FLT3-IN-3. The ATP-binding pocket of FLT3 kinase forms hydrogen bonds with key residues (Asp835, Glu836) and forms hydrophobic interactions with the gating residue Phe830, thereby conferring high selectivity and potency [1].

C27H38N8O

490.6436

490.32

C, 66.09; H, 7.81; N, 22.84; O, 3.26

2229050-90-0

2229050-90-0

133081975

White to off-white solid powder

3.3

3

8

7

36

671

0

SAEGVASGMTZGFI-UHFFFAOYSA-N

InChI=1S/C27H38N8O/c28-20-7-11-22(12-8-20)31-27-32-25(24-26(33-27)35(18-29-24)23-3-1-2-4-23)30-21-9-5-19(6-10-21)17-34-13-15-36-16-14-34/h5-6,9-10,18,20,22-23H,1-4,7-8,11-17,28H2,(H2,30,31,32,33)

2-N-(4-aminocyclohexyl)-9-cyclopentyl-6-N-[4-(morpholin-4-ylmethyl)phenyl]purine-2,6-diamine

SAN50900; SAN 50900; SAN-50900; FLT3-IN3; FLT3-IN 3; FLT3-IN-3

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: ~250 mg/mL (~509.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.24 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 20.8 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: ≥ 2.08 mg/mL (4.24 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 20.8 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: ≥ 2.08 mg/mL (4.24 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 20.8 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.)