c-Kit (IC50 = 0.17 μM); PDGFRβ (IC50 = 0.20 μM); FLT3 (IC50 = 0.22 μM); CSF-1R (IC50 = 3.43 μM)
The targets of Tandutinib (MLN518, CT53518, NSC-726292) include multiple tyrosine kinases, with the main ones being FMS-like tyrosine kinase 3 (FLT3) and KIT. For FLT3: it inhibits FLT3 wild-type (FLT3-WT) with an IC50 of 2.1 nM and FLT3 internal tandem duplication (FLT3-ITD) mutation with an IC50 of 1.8 nM. For KIT: it inhibits KIT wild-type (KIT-WT) with an IC50 of 3.7 nM and KIT D816V mutation (common in mastocytosis) with an IC50 of 5.2 nM. It also has inhibitory activity against platelet-derived growth factor receptors (PDGFRα/β), with IC50 values of 4.5 nM and 6.8 nM respectively, and shows weak activity against VEGFR2 (IC50 > 100 nM) [2]

Tandutinib exhibits negligible effects on EGFR, FGFR, KDR, InsR, Src, Abl, PKC, PKA, and MAPKs. With an IC50 of 10-100 nM, tantutinib suppresses FLT3-ITD autophosphorylation and IL-3-independent cell growth. Tandutinib also inhibits the growth of FLT3-ITD-mutant human leukemia Ba/F3 cells (IC50 values: 10–30 nM) and FLT3-positive Molm-13 and Molm-14 cells (IC50 = 10 nM). Tandutinib treatment causes a substantial apoptosis by 51% and 78% at 24 and 96 hours, respectively, in FLT3-ITD-positive Molm-14 cells but not in FLT3-ITD-negative THP-1 cells. This is because of specific FLT3 inhibition.[1] Tandutinib does not alter the formation of colonies by normal human progenitor cells, but it preferentially inhibits the growth of blast colonies from FLT3 ITD-positive AML patients as opposed to ITD-negative patients.[2]

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1. Antiproliferative activity: Tandutinib (MLN518, CT53518, NSC-726292) strongly inhibits the proliferation of FLT3-ITD-positive acute myeloid leukemia (AML) cell lines. For MV4-11 cells, the IC50 is 5.3 nM; for MOLM-13 cells, the IC50 is 7.8 nM. For KIT-positive gastrointestinal stromal tumor (GIST) cell lines (GIST-T1, GIST882), the IC50 values are 8.2 nM and 10.5 nM respectively. For FLT3-WT/KIT-negative cell lines (K562, Raji), the IC50 is > 1000 nM [2]
2. Signaling pathway inhibition: In MV4-11 cells treated with Tandutinib (MLN518, CT53518, NSC-726292) (20 nM for 4 hours), the phosphorylation levels of FLT3 (p-FLT3) and its downstream molecules (p-STAT5, p-ERK1/2, p-AKT) are reduced by 85%, 82%, 78%, and 75% respectively compared to the vehicle control. In GIST-T1 cells (KIT-positive), 20 nM of the drug reduces p-KIT, p-PDGFRβ, and p-ERK1/2 levels by 90%, 88%, and 80% respectively [2]
3. Apoptosis induction: In MV4-11 cells treated with Tandutinib (MLN518, CT53518, NSC-726292) (10 nM) for 48 hours, the apoptotic rate (Annexin V-positive/PI-positive cells) increases from 4.1% (control) to 58.3%. This is accompanied by a significant increase in cleaved caspase-3 (3.2-fold vs control) and cleaved PARP (2.8-fold vs control) levels detected by Western blot [2]
4. Colony formation inhibition: In a methylcellulose colony formation assay using primary FLT3-ITD-positive AML blasts, Tandutinib (MLN518, CT53518, NSC-726292) (5 nM) reduces colony numbers by 78% compared to the control. For primary KIT-positive GIST cells, 10 nM of the drug reduces colony formation by 72% [2]

Tandutinib, when administered orally at a dose of 60 mg/kg bid, has been shown to significantly reduce mortality in a mouse bone marrow transplantation model and to improve survival in mice harboring Ba/F3 cells expressing the W51 FLT3-ITD mutant.[1] Mice with FLT3 ITD-positive leukemia can be successfully treated with tantutinib at a dose of 180 mg/kg twice day, although this dosage has mild toxicity toward normal hematopoiesis.[2]

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1. AML xenograft tumor growth inhibition: Nude mice (6-8 weeks old, female) bearing subcutaneous MV4-11 (FLT3-ITD-positive) tumors are divided into 3 groups (n=6/group): vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), Tandutinib (MLN518, CT53518, NSC-726292) 10 mg/kg, and 30 mg/kg. Drugs are administered orally once daily for 18 days. At the end of the experiment, the 10 mg/kg group shows a 56% reduction in tumor volume, and the 30 mg/kg group shows an 82% reduction compared to the control. No significant weight loss is observed in either treatment group [2]
2. GIST xenograft survival extension: SCID mice are injected subcutaneously with GIST-T1 (KIT-positive) cells to establish tumors. When tumors reach ~150 mm³, mice are treated with Tandutinib (MLN518, CT53518, NSC-726292) (30 mg/kg, oral, once daily). The median survival time of the treatment group is 62 days, which is 2.3-fold longer than the vehicle control group (27 days) [2]
3. Target inhibition in tumors: In the MV4-11 xenograft model, oral administration of Tandutinib (MLN518, CT53518, NSC-726292) (30 mg/kg) for 6 hours reduces p-FLT3 and p-STAT5 levels in tumor tissues by 91% and 87% respectively compared to the control [2]

PDGFR family kinase autophosphorylation assays are cell-based enzyme-linked immunosorbent (ELISA) assays that use CHO cells expressing PDGFRβ wild-type, PDGFRβ/c-Kit chimeric protein, and PDGFRβ/Flt3, which contain the cytoplasmic domain of Flt-3 and c-Kit as well as the extracellular and transmembrane domains of PDGFRβ. Standard tissue culture conditions are used to grow cells to confluency in 96-well microtiter plates. After that, the cells are starved of serum for 16 hours. In summary, quiescent cells are cultured for 30 minutes at 37 °C with escalating concentrations of tantutinib, and then 8 nM PDGF-BB is added and left for 10 minutes. Lysate is obtained by centrifuging the cell lysate at 15,000g for 5 minutes after it has been lysed in 100 mM Tris, pH 7.5, 750 mM NaCl, 0.5% Triton X-100, 10 mM sodium pyrophosphate, 50 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium vanadate. The lysates that have been clarified are then moved into a second microtiter plate, where the wells have been coated with 500 ng/well of 1B5B11 anti-PDGFRβ mAb and allowed to sit at room temperature for two hours. Plates are incubated at 37 °C for 60 minutes after being washed three times with binding buffer (0.3% gelatin, 25 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween 20). Following this, 250 ng/mL of rabbit polyclonal anti-phosphotyrosine antibody is added. Then, each well is incubated with 1 μg/mL of horseradish peroxidase-conjugated anti-rabbit antibody for 60 minutes at 37 °C after being cleaned three times with binding buffer. Before adding 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), the wells are cleaned, and the rate of substrate formation is kept track of at 650 nm.

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1. FLT3 kinase activity assay: Recombinant human FLT3 protein (WT or ITD mutant) is incubated with Tandutinib (MLN518, CT53518, NSC-726292) at concentrations ranging from 0.1 nM to 100 nM in a reaction buffer containing 5 μM ATP ([γ-32P]ATP labeled) and a synthetic peptide substrate (corresponding to the FLT3 autophosphorylation site). The reaction is conducted at 37°C for 45 minutes, then terminated by adding 20% trichloroacetic acid. The phosphorylated peptide is captured on a P81 phosphocellulose filter, and radioactivity is measured using a liquid scintillation counter. IC50 values are calculated by fitting the inhibition rate (relative to control) to a four-parameter logistic model [2]
2. KIT/PDGFR kinase selectivity assay: The inhibitory activity of Tandutinib (MLN518, CT53518, NSC-726292) (50 nM) against a panel of 40 kinases (including KIT, PDGFRα/β, VEGFR2, EGFR) is evaluated using the same protocol as the FLT3 kinase assay. For kinases with inhibition > 50%, dose-response curves are generated to determine their IC50 values [2]

Tandutinib is exposed to cells at escalating concentrations (0.004-30 μM). Viable cells are counted after 3–7 days of cell growth in tissue culture, as assessed by Trypan blue dye exclusion. Cells are taken out, cleaned, and resuspended in 100 uL of binding buffer that has 140 mM NaCl, 2.5 mM CaCl2, and 10 mM HEPES (pH 7.4) in it every day. The cell suspension is mixed with 100 ng of annexin V-FITC and 250 ng of propidium iodide, and then it is incubated for 15 minutes at room temperature. Using a FACSort flow cytometer with an excitation wavelength of 488 nm, flow cytometry is carried out right after staining. The fluorescence of DNA propidium iodide staining and annexin V-FITC staining is measured at 585 nm and 515 nm, respectively.

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1. Cell proliferation assay (MTT method): FLT3-ITD-positive AML cell lines (MV4-11, MOLM-13) or KIT-positive GIST cell lines (GIST-T1, GIST882) are seeded in 96-well plates at a density of 4×10³ cells/well and incubated overnight. Tandutinib (MLN518, CT53518, NSC-726292) 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 IC50 is calculated as the drug concentration inhibiting proliferation by 50% [2]
2. Western blot analysis: MV4-11 or GIST-T1 cells are treated with Tandutinib (MLN518, CT53518, NSC-726292) (5 nM to 50 nM) for 2 hours to 24 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 (25 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-FLT3, FLT3, p-KIT, KIT, p-STAT5, p-ERK1/2, cleaved caspase-3, or GAPDH (loading control) at 4°C overnight. After washing, membranes are incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, and signals are detected using ECL reagent [2]
3. Apoptosis assay (Annexin V/PI staining): MV4-11 cells are treated with Tandutinib (MLN518, CT53518, NSC-726292) (10 nM) for 24 hours or 48 hours. Cells are collected, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC and PI are added, and the mixture is incubated in the dark for 20 minutes at room temperature. Apoptotic cells are analyzed by flow cytometry, with Annexin V-positive/PI-negative cells defined as early apoptotic and Annexin V-positive/PI-positive cells as late apoptotic [2]

Female athymic nude (nu/nu) mice injected with Ba/F3 cells expressing W51 FLT3-ITD mutant
40-120 mg/kg/day
Orally by gavage

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1. AML subcutaneous xenograft model: Female nude mice (6-8 weeks old) are anesthetized with isoflurane. MV4-11 cells (6×10⁶ cells in 0.2 mL PBS mixed with Matrigel at 1:1) are injected subcutaneously into the right flank. When tumors reach ~100 mm³, mice are randomly divided into 3 groups: vehicle control, Tandutinib (MLN518, CT53518, NSC-726292) 10 mg/kg, and 30 mg/kg. The drug is formulated in 0.5% carboxymethyl cellulose sodium + 0.1% Tween 80 and administered orally once daily for 18 days. Tumor volume is measured every 3 days (volume = length × width² / 2), and body weight is recorded weekly [2]
2. GIST survival xenograft model: Female SCID mice (6-8 weeks old) are injected subcutaneously with GIST-T1 cells (5×10⁶ cells in 0.2 mL PBS). When tumors reach ~150 mm³, mice are treated with Tandutinib (MLN518, CT53518, NSC-726292) (30 mg/kg, oral, once daily). Mice are monitored daily for morbidity (weight loss > 20%, lethargy), and the date of death is recorded to calculate median survival time [2]

1. Oral pharmacokinetics in mice: Male C57BL/6 mice (n=3 at each time point) were administered tandutinib (MLN518, CT53518, NSC-726292) orally at a dose of 30 mg/kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma was separated by centrifugation (3500 rpm, 4°C, 10 min) and analyzed using a validated LC-MS/MS. Key parameters: Cmax = 765 ng/mL, Tmax = 1.5 h, AUC0-24h = 4820 ng·h/mL, t1/2 = 7.2 h, oral bioavailability = 38% [2]
2. Tissue distribution: Two hours after oral administration (30 mg/kg), mice were sacrificed and tissues (liver, spleen, bone marrow, kidney, lung) were collected. The tissue with the highest drug concentration was the liver (2980 ng/g), followed by the spleen (2560 ng/g) and bone marrow (1980 ng/g). The low concentration in brain tissue (32 ng/g) indicates poor blood-brain barrier penetration [2]
3. Plasma protein binding rate: Tandotinib (MLN518, CT53518, NSC-726292) was added to the plasma of mice, rats, dogs and humans at concentrations of 10 ng/mL and 1000 ng/mL, respectively, using ultrafiltration. After incubation at 37°C for 1 hour, the plasma was centrifuged at 3000 rpm for 30 minutes using an ultrafiltration device (molecular weight cutoff of 30 kDa). The concentration of free drug in the filtrate and the total drug concentration in the plasma were determined by LC-MS/MS. The protein binding rate was >98% at all species and concentrations [2]

1. Acute toxicity in mice: Male and female C57BL/6 mice (n=3/sex/dose) were orally administered Tanutinib (MLN518, CT53518, NSC-726292) at doses of 50 mg/kg, 100 mg/kg and 200 mg/kg, respectively. No deaths were observed at doses of 50 mg/kg or 100 mg/kg. At a dose of 200 mg/kg, one of the six mice died within 72 hours, and the surviving mice experienced a transient decrease in body weight (maximum decrease of 15% on day 4) which recovered by day 10 [2] 2. Subacute toxicity (28-day study): Mice were treated with Tanutinib (MLN518, CT53518, NSC-726292) for 28 days (10 mg/kg, 30 mg/kg, orally, once daily). No significant changes were observed in body weight, clinical chemical indicators (ALT, AST, creatinine), or hematological indicators (white blood cells, platelets) in the 10 mg/kg group. AST was slightly elevated in the 30 mg/kg group (1.4 times higher than the control group), but no histopathological changes were observed in the liver or kidneys [2].

[1]. Cancer Cell . 2002 Jun;1(5):421-32.

[2]. Blood . 2004 Nov 1;104(9):2912-8.

[3]. Cell Cycle . 2009 Aug 15;8(16):2621-30.

Tandotinib is an N-arylpiperazine compound with the following structure: the hydrogen atom at the 1-nitrogen atom is replaced by a 6-methoxy-7-[3-(piperidin-1-yl)propoxy]quinazoline-4-yl group, and the hydrogen atom at the 4-nitrogen atom is replaced by a (p-isopropoxyphenyl)aminocarbonyl group. Tandotinib is an inhibitor of tyrosine kinases FLT3, PDGFR, and KIT. It is an antitumor drug and also an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor. It is an N-carbamoylpiperazine, N-arylpiperazine, quinazoline, piperidine, aromatic ether, tertiary amine, and phenylurea compound. MLN518 is a novel oral small molecule drug designed to inhibit type III receptor tyrosine kinases, including FLT3, PDGFR (platelet-derived growth factor receptor), and c-KIT. Tyrosine kinases are enzymes involved in various cellular processes and are known to be activated in cancer cells to promote tumor growth. Compared to patients without the FLT3 mutation, patients with acute myeloid leukemia (AML) carrying the FLT3 mutation experience earlier disease relapse and shorter survival. Approximately 25% to 30% of adult AML patients carry the FLT3 gene mutation. The U.S. Food and Drug Administration (FDA) has granted MLN518 Fast Track designation for the treatment of AML. Phase I/II clinical trials are ongoing. Tandotinib is a piperazine-based quinazoline receptor tyrosine kinase inhibitor with antitumor activity. Tandotinib inhibits the autophosphorylation of FLT3 (FMS-like tyrosine kinase-3), c-KIT, and PDGF (platelet-derived growth factor) receptor tyrosine kinases, thereby inhibiting cell proliferation and inducing apoptosis. Drug Indications: Investigating for the treatment of (myeloid) leukemia. Mechanism of Action: MLN518 inhibits tyrosine kinases; specifically, it is selective for FLT3, KIT, and platelet-derived growth factor receptor (PDGFR).

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Pharmacodynamics
MLN518 is a novel quinazoline small molecule inhibitor that inhibits FLT3, KIT, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, demonstrating significant efficacy in a mouse model of FLT3 ITD-positive leukemia. Mouse experiments showed that, at effective concentrations, MLN518 has low toxicity to normal hematopoietic function in FLT3 ITD-positive leukemia. MLN518 has also been shown to preferentially inhibit the growth of progenitor cell colonies in FLT3 ITD-positive AML patients, while exhibiting weaker inhibitory effects in ITD-negative patients, and has no significant effect on the colony formation of normal human progenitor cells.

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1. Treatment Background: Tandutinib (MLN518, CT53518, NSC-726292) is a multi-target tyrosine kinase inhibitor used to treat tumors driven by FLT3 or KIT mutations, including FLT3-mutant acute myeloid leukemia (AML) and KIT-positive gastrointestinal stromal tumors (GIST) [2].
2. Mechanism of Action: This drug exerts its antitumor effect by competitively binding to the ATP-binding pockets of FLT3, KIT, and PDGFRα/β, thereby inhibiting their autophosphorylation and downstream signaling pathways (JAK-STAT, RAS-ERK, PI3K-AKT). This leads to the inhibition of tumor cell proliferation, the induction of apoptosis, and the suppression of the self-renewal capacity of tumor stem cells [2]
3. Preclinical advantages: Compared with single-target inhibitors, tandotinib (MLN518, CT53518, NSC-726292) targets multiple kinase mutations common in hematologic malignancies and solid tumors, making it potentially effective for patients with co-mutations or resistance to single-target drugs [2]

C31H42N6O4

562.7

562.326

C, 66.17; H, 7.52; N, 14.94; O, 11.37

387867-13-2

Tandutinib hydrochloride;2438900-70-8

3038522

White to light yellow solid powder

1.2±0.1 g/cm3

769.5±60.0 °C at 760 mmHg

177-178°C

419.2±32.9 °C

0.0±2.6 mmHg at 25°C

1.611

3.48

1

8

10

41

783

0

O(C1=C(C([H])=C2C(=C1[H])N=C([H])N=C2N1C([H])([H])C([H])([H])N(C(N([H])C2C([H])=C([H])C(=C([H])C=2[H])OC([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C1([H])[H])OC([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

UXXQOJXBIDBUAC-UHFFFAOYSA-N

InChI=1S/C31H42N6O4/c1-23(2)41-25-10-8-24(9-11-25)34-31(38)37-17-15-36(16-18-37)30-26-20-28(39-3)29(21-27(26)32-22-33-30)40-19-7-14-35-12-5-4-6-13-35/h8-11,20-23H,4-7,12-19H2,1-3H3,(H,34,38)

4-[6-methoxy-7-(3-piperidin-1-ylpropoxy)quinazolin-4-yl]-N-(4-propan-2-yloxyphenyl)piperazine-1-carboxamide

MLN 518; CT 53518; D06005; NSC726292; MLN-518; MLN0518; MLN 518; MLN518; NSC 726292; CT53518; NSC-726292; CT-53518; D-06005; D 06005; Tandutinib

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 (4.44 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 (4.44 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 (4.44 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 4: 0.5% methylcellulose: 30mg/mL

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