MNK1 (IC50 = 64 nM); MNK2 (IC50 = 86 nM)
ETC-206 (compound 48) is a potent and selective inhibitor of mitogen-activated protein kinase interacting kinases 1 and 2 (MNK1/2). It inhibits MNK1 and MNK2 enzymatic activity with IC50 values of 0.064 μM and 0.086 μM, respectively. [1]

In the HeLa cell line, tinodasertib (ETC-206) inhibits eIF4E phosphorylation with an IC50 of 321 nM. The CellTiter-Glo viability assay was used in vitro to assess the anti-stress effects of ETC-206 against 25 blood cell lines, including the K562 cell line that overexpresses cardiovascular eIF4E (K562 o/ e eIF4E). for Ramos.2G6.4C10, AHH-1, MC 116, P3HR-1, DOHH2, MPC-11, SU-DHL-6, GK-5, and K562 o/e eIF4E cells [1].

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In HeLa cell-based assays, compound 48 inhibits the phosphorylation of eIF4E (Ser209), a direct substrate of MNK1/2, with an IC50 of 0.321 μM. [1]
Compound 48 was screened against a panel of 104 kinases at a concentration of 1 μM. It demonstrated excellent selectivity, inhibiting only two kinases (MEK and MNK2) at greater than 65% inhibition, and only MNK2 at greater than 90% inhibition. [1]
The antiproliferative activity of compound 48 was assessed using CellTiter-Glo viability assays against 25 hematological cancer cell lines. The IC50 values were generally in the micromolar range, consistent with published data for MNK inhibitors. [1]
When combined with dasatinib, compound 48 suppressed the ability of leukemic stem cells from blast crisis chronic myeloid leukemia patients to form colonies (data to be published elsewhere). [1]
The permeability of compound 48 was assessed in Caco-2 assays, showing an A-B permeability (Papp) of 30.50 × 10⁻⁶ cm/s and an efflux ratio of 1.42, indicating good permeability and low efflux. [1]

Next, using the K562 e/o eIF4E mouse xenograft model, the antitumor effects of ETC-206 were assessed. The drug was administered lateralized at 25, 50, or 100 mg/kg either by itself or in combination with a fixed dose of dasatinib at 2.5 mg/kg for the duration of the study. following the union. A dose of 2.5 mg/kg of dasatinib resulted in 88% tumor growth inhibition (TGI). Conversely, at the highest dose of 100 mg/kg, ETC-206 produced only 23 % of the maximum TGI, which was comparable to untreated animals and did not inhibit tumor growth. More significantly, 2, 5 of the 8 animals at 25, 50, and 100 mg/kg and 8 were tumor-free when ETC-206 and dasatinib at 2.5 mg/kg were combined. This increased tumor growth inhibition in a dose-dependent manner. kilograms, in that order. At all tested doses, the combination of dasatinib and ETC-206 inhibited tumor growth; no weight loss was observed. In syngeneic mouse xenograft models, the combination of dasatinib and ETC-206, as well as dual MNK1/2 and BCR-ABL1, inhibited tumor growth. The terminal elimination half-life of ETC-206 is regulated and is 1.77 hours for mice given 5 mg/kg po and t1/2=1.7 hours for mice given 1 mg/kg iv[1].

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In a K562/eIF4E mouse xenograft model of chronic myeloid leukemia, compound 48 administered orally once daily for 14 days at doses of 25, 50, and 100 mg/kg as a single agent showed minimal antitumor activity, with a maximum tumor growth inhibition of 23% at the highest dose. [1]
When combined with dasatinib (2.5 mg/kg, 5 days on/2 days off schedule for two cycles), compound 48 enhanced dasatinib's antitumor activity in a dose-dependent manner. The combination resulted in tumor-free animals: 2 out of 8 at 25 mg/kg, 5 out of 8 at 50 mg/kg, and 8 out of 8 at 100 mg/kg of compound 48. [1]
Biomarker analysis by Western blot on tumor samples from mice treated with a single dose of 100 mg/kg compound 48 and 2.5 mg/kg dasatinib showed a maximum reduction in phosphorylated eIF4E from 1 to 8 hours post-dose, and a reduction in phosphorylated CRKL (a BCR-ABL1 substrate) from 4 to 8 hours post-dose. This confirms in vivo inhibition of both MNK1/2 and BCR-ABL1 kinases. [1]
In a maximum tolerated dose study, oral administration of compound 48 to healthy mice at doses up to 200 mg/kg daily for 7 consecutive days showed no weight loss and no clinical adverse effects during a 14-day observation period. [1]

MNK1/2 enzymatic inhibition assay: Compounds were tested in a biochemical assay to determine their inhibitory activity against MNK1 and MNK2. The assays were performed with ATP at Km concentrations (1.2 mM for MNK1 and 250 μM for MNK2). IC50 values were determined by nonlinear regression using GraphPad Prism, and the reported values are the average of at least two experiments. [1]

ETC-206's ability to inhibit cell proliferation in 25 hematological cancer cell lines, including the K562 cell line that overexpresses eIF4E (K562 o/e eIF4E), is tested in vitro using the CellTiter-Glo viability assay. Generally speaking, the IC50 values fall into the micromolar range[1].

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eIF4E phosphorylation inhibition assay in HeLa cells: The ability of compounds to inhibit MNK1/2-mediated phosphorylation of eIF4E on Ser209 was assessed using a sandwich immunoassay in HeLa cells. Compounds were tested in a dose-dependent manner in duplicate using eight points from a 3-fold serial dilution. IC50 values were determined by nonlinear regression using GraphPad Prism, and the values reported are the average of at least two experiments. [1]
Antiproliferative activity assay: The antiproliferative effects of compound 48 were assessed in vitro using the CellTiter-Glo viability assay against a panel of 25 hematological cancer cell lines, including the K562 cell line overexpressing eIF4E. [1]
Caco-2 permeability assay: Caco-2 cells were seeded in 24-well plates at 1 × 10⁵ cells/well in culture medium and allowed to grow for 21 days. The permeability of compound 48 was measured in the apical-to-basolateral direction at a concentration of 10 μM in the presence and absence of a P-gp inhibitor. Samples were analyzed by LC-MS/MS, and the apparent permeability coefficient and efflux ratio were calculated. [1]

Mice: CD-1 female mice (6-8 weeks old) are weighed, and those selected for dosing are 24±2 g. Each group of three mice is chosen at random. ETC-206 is given to mice as a single injection into a tail vein at a dose of 1 mg/kg or as a single oral gavage at a dose of 5 mg/kg. 4 mL/kg and 8 mL/kg, respectively, are the injection volumes for intravenous (i.v.) and oral (p.o.) administration[1].

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Maximum tolerated dose study: Female NOD-SCID mice (7-9 weeks old) were randomly assigned to groups of three. They received either vehicle or a single dose of 10, 50, 100, or 200 mg/kg of compound 48 via oral gavage daily for 7 consecutive days. After treatment, mice were observed for delayed clinical abnormalities for an additional 7 days. Mice were sacrificed using CO₂ gas at the end of the 14-day study. [1]
In vivo antitumor efficacy study: K562-eIF4E cells (10⁷ cells/mouse) were subcutaneously implanted into the right flank of female SCID mice. When tumors reached a volume between 75 and 196 mm³, mice were randomly distributed into groups of 8. Compound 48 was dissolved in 0.5% MC/0.1% TW80 solution and administered orally once daily for 14 days at doses of 25, 50, or 100 mg/kg. Dasatinib (2.5 mg/kg) was administered orally on a 5 days on/2 days off schedule for two cycles. For combination treatment, mice received dasatinib followed by compound 48 1 hour later, following the same schedules. Tumor volume was monitored twice weekly using a digital caliper and calculated as (width² × length)/2. Tumor growth inhibition and T/C ratio were calculated at day 14. [1]
Formulation for in vivo studies: For oral administration, compound 48 was formulated in 0.5% methylcellulose with 0.1% Tween 80 in sterile Milli-Q water. For intravenous administration, it was formulated in 10% NMP, 40% PEG300, and 50% sterile MQ water or 10% Cremophor, 10% DMA, and 80% sterile MQ water. [1]
Pharmacokinetic study: Male CD-1 mice (n=15 per compound, 3 per time point) received compound 48 intravenously at 1 mg/kg or orally at 5 mg/kg. Blood samples were collected at various time points via cardiac puncture, and plasma was obtained by centrifugation. Compound concentrations were analyzed by LC-MS/MS, and pharmacokinetic parameters were calculated using WinNonlin. [1]
Western blot biomarker study: K562-eIF4E tumor-bearing CB17 SCID mice received a single oral dose of 100 mg/kg compound 48 and 2.5 mg/kg dasatinib. Mice were sacrificed at pre-dose, 1, 4, and 8 hours post-dose. Tumors were excised, flash frozen, and homogenized. Lysates were analyzed by Western blot using antibodies against eIF4E, phospho-eIF4E (S209), CRKL, and phospho-CRKL. [1]

Caco-2 permeability: Compound 48 showed high permeability in Caco-2 assays with Papp A-B of 30.50 × 10⁻⁶ cm/s and an efflux ratio of 1.42, indicating good absorption potential and low efflux. [1]
LogD: The distribution coefficient at pH 7.4 for compound 48 was 2.61. [1]
Solubility: The aqueous solubility of compound 48 was 55.4 μg/mL. [1]
Microsomal stability: Compound 48 showed good metabolic stability in liver microsomes from multiple species. Half-life values were: human (48 min), mouse (56 min), rat (>60 min), and dog (>60 min). [1]
CYP inhibition: Compound 48 showed no significant inhibition of CYP3A4 or CYP2D6 (IC50 > 20 μM for both). [1]
Plasma protein binding: Compound 48 exhibited high plasma protein binding in both human (99.7%) and mouse (84.2%). [1]
Mouse pharmacokinetics: After intravenous administration at 1 mg/kg, compound 48 had a C0 of 2379 ng/mL, clearance of 0.56 L/h/kg, and half-life of 1.77 h. After oral administration at 5 mg/kg, it reached a Cmax of 2481 ng/mL at Tmax of 0.5 h, with an AUC0-t of 15484 ng·h/mL and oral bioavailability of 64%. [1]

In a maximum tolerated dose study in healthy mice, oral administration of compound 48 at doses up to 200 mg/kg daily for 7 consecutive days showed no weight loss and no clinical adverse effects during a 14-day observation period, indicating a favorable safety profile. [1]
Compound 48 showed no significant inhibition of CYP3A4 or CYP2D6 (IC50 > 20 μM), suggesting low potential for drug-drug interactions via these major CYP enzymes. [1]

[1]. Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia. J Med Chem. 2018 May 24;61(10):4348-4369.

Tinodasertib is a selective mitogen-activated protein kinase (MAPK)-interacting protein kinase (MNK) type 1/2 inhibitor with potential antitumor activity. After administration, Tinodasertib inhibits MNK1/2-dependent phosphorylation of eukaryotic initiation factor 4E (eIF4E), thereby interfering with its role in mRNA translation. eIF4E is an oncoprotein that must be phosphorylated to promote tumor cell proliferation and progression. MNK is a serine/threonine kinase closely related to oncogenic transformation and tumor progression.

C25H20N4O2

408.45

408.16

C, 73.51; H, 4.94; N, 13.72; O, 7.83

1464151-33-4

1464151-33-4

71766360

Light yellow to yellow solid powder

3.9

0

4

3

31

668

0

C1(C2=CC=C(C(=O)N3CCOCC3)C=C2)=CN2C(C3C=CC(C#N)=CC=3)=CN=C2C=C1

FWRFPHJSGLYXTD-UHFFFAOYSA-N

InChI=1S/C25H20N4O2/c26-15-18-1-3-20(4-2-18)23-16-27-24-10-9-22(17-29(23)24)19-5-7-21(8-6-19)25(30)28-11-13-31-14-12-28/h1-10,16-17H,11-14H2

4-[6-[4-(morpholine-4-carbonyl)phenyl]imidazo[1,2-a]pyridin-3-yl]benzonitrile

Tinodasertib; ETC-206; 4-(6-(4-(morpholine-4-carbonyl)phenyl)imidazo[1,2-a]pyridin-3-yl)benzonitrile; AUM001; ETC 206; ETC206

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: ~82 mg/mL (~200.8 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (12.24 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 50.0 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: ≥ 5 mg/mL (12.24 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 50.0 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: ≥ 5 mg/mL (12.24 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 50.0 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.)