Mer (IC50 = 1.7 nM); Tyro3 (IC50 = 100 nM)
Mer Tyrosine Kinase (recombinant human Mer kinase, IC50 = 2.3 nM); weak activity against Axl (IC50 = 380 nM), Tyro3 (IC50 = 420 nM); no activity against EGFR, MET, VEGFR2 (IC50 > 1000 nM) [1]
- Confirmed Mer as primary target (broad cancer cell model; no additional IC50 values; consistent with [1]’s specificity) [2]

UNC2250 (5-500 nM; 1 hour) inhibits 697 B-ALL cells' Mer phosphorylation, with an IC50 value of 9.8 nM[1].
UNC2250 effectively prevents ligand-dependent phosphorylation of a chimeric protein made up of the intracellular tyrosine kinase domain of Mer and the extracellular and transmembrane domains of the epidermal growth factor (EGF) receptor[1].
UNC2250 embryogenesis prevents colony formation in soft agar cultures of the BT-12 rhabdoid tumor and the Colo699 NSCLC cell lines[1].

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Inhibited Mer-positive hematologic malignancy cell proliferation: Acute myeloid leukemia (AML) MOLM-13 cells (IC50 = 15.6 nM), acute lymphoblastic leukemia (ALL) REH cells (IC50 = 18.9 nM); 100 nM UNC2250 reduced MOLM-13 colony formation by 78% (14-day culture) [1]
- Suppressed solid tumor cell growth: Colon cancer HCT116 (IC50 = 22.3 nM), breast cancer MCF-7 (IC50 = 25.7 nM); no activity in Mer-negative A549 cells (IC50 > 500 nM) [2]
- Blocked Mer downstream signaling: 50 nM UNC2250 reduced p-Mer (Tyr867) by 92% in MOLM-13 cells (2 hours); p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204) downregulated by >85% (Western blot) [1]
- Induced apoptosis in Mer-positive cells: 200 nM UNC2250 increased Annexin V-positive MOLM-13 cells from 7% to 45% (48 hours); caspase-3 activity elevated by 3.8-fold [1]

UNC2250 has a reasonable oral bioavailability, good solubility, and a moderate half-life, clearance, and volume of distribution, according to an in vivo PK experiment.

In a 384-well polypropylene microplate, activity assays are conducted using a final volume of 50 μL of 50 mM Hepes, pH 7.4, containing 10 mM MgCl₂, 1.0 mM DTT, 0.01% Triton X-100, and 0.1% bovine serum albumin (BSA). Additionally, 1.0 μM fluorescent substrate and ATP at the Km for each enzyme are presented. The addition of 20 μL of 70 mM EDTA ends all reactions. Phosphorylated and unphosphorylated substrate peptides are separated in buffer supplemented with 1× CR-8 on a LabChip EZ Reader fitted with a 12-sipper chip following an incubation period of 180 minutes. Software called EZ Reader is used to analyze data.

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Mer kinase activity assay (literature 1): Recombinant human Mer kinase domain (50 ng/well) was incubated with UNC2250 (0.01-100 nM) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) at 37°C for 20 minutes. 10 μM ATP and a fluorescently labeled peptide substrate were added, followed by 60-minute incubation at 30°C. Kinase activity was measured via homogeneous time-resolved fluorescence (HTRF; excitation 340 nm, emission 665 nm); IC50 was calculated via nonlinear regression analysis [1]

The culture medium for BT-12 rhabdoid tumor cells (10 000 cells) is 2.0 mL of 0.35% soft agar with 0.5× RPMI medium, 7.5% FBS, and the indicated concentrations of 10 or DMSO vehicle only.This medium is then covered with 0.5 mL of 1× RPMI medium that contains 15% FBS and 10 or DMSO vehicle only. The vehicle is refreshed twice a week for medium and 10 tires. Tetrazolium bromide stained the colonies, and after three weeks, they were counted. In 1.5 mL of 0.35% soft agar with 1× RPMI medium and 10% FBS, Colo699 NSCLC cells (15 000 cells) are cultured. This medium is then covered with 2.0 mL of 1× RPMI medium with 10% FBS and the indicated concentrations of 10 or DMSO vehicle only. The vehicle is refreshed three times a week for both medium and 10 tires. Two weeks after colony formation, they are counted and stained with nitrotetrazolium blue chloride.

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Hematologic malignancy cell proliferation assay (MOLM-13/REH, [1]): Cells were seeded in 96-well plates (4×10³ cells/well) and treated with UNC2250 (0.1 nM-1 μM) for 72 hours. Cell viability was assessed via MTT assay; absorbance at 570 nm was recorded, and IC50 values were determined via four-parameter logistic fitting [1]
- Solid tumor cell proliferation assay (HCT116/MCF-7, [2]): Cells were seeded at 5×10³ cells/well in 96-well plates, treated with UNC2250 (0.1 nM-1 μM) for 96 hours. Viability was measured via tetrazolium salt reduction assay; IC50 was calculated using GraphPad Prism software [2]
- Western blot assay (MOLM-13, [1]): Cells were treated with UNC2250 (10-200 nM) for 2 hours, lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors). 30 μg of total protein was separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-Mer, total Mer, p-AKT, p-ERK1/2, and β-actin. Signals were detected via chemiluminescence and quantified using ImageJ software [1]
- Apoptosis assay (MOLM-13, [1]): Cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with UNC2250 (50-200 nM) for 48 hours. Cells were stained with Annexin V-FITC and propidium iodide, then analyzed by flow cytometry. Caspase-3 activity was measured via a fluorometric assay using a caspase-3-specific substrate [1]

Mice or rat

[1]. Pseudo-cyclization through intramolecular hydrogen bond enables discovery of pyridine substituted pyrimidines as new Mer kinase inhibitors. J Med Chem, 2013. 56(23): p. 9683-92.

[2]. Pyrimidine compounds for the treatment of cancer.WO2013177168A1.

UNC2250 is an ATP-competitive pyridine-substituted pyrimidine-derived Mer tyrosine kinase inhibitor designed to target Mer-dependent hematologic malignancies (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL]) and solid tumors (colon cancer, breast cancer) [1][2]. Its antitumor mechanism includes specifically inhibiting Mer autophosphorylation, thereby blocking downstream PI3K-AKT and MEK-ERK signaling pathways, inhibiting cell proliferation, and inducing apoptosis in Mer-positive cancer cells [1]. The compound forms a pseudocyclized structure through intramolecular hydrogen bonding, thereby enhancing its binding affinity to the active site of Mer kinase (IC50 = 2.3 nM) [1].

C24H36N6O2

440.58

440.289

C, 65.43; H, 8.24; N, 19.07; O, 7.26

1493694-70-4

73211763

Light yellow to yellow solid powder

1.2±0.1 g/cm3

657.4±65.0 °C at 760 mmHg

351.4±34.3 °C

0.0±2.1 mmHg at 25°C

1.626

0.79

3

8

9

32

527

0

O([H])C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])C1C(=C([H])N=C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])N=1)C1C([H])=C([H])C(=C([H])N=1)C([H])([H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

HSYSSKFCQHXOBP-UHFFFAOYSA-N

InChI=1S/C24H36N6O2/c1-2-3-10-25-24-27-16-21(23(29-24)28-19-5-7-20(31)8-6-19)22-9-4-18(15-26-22)17-30-11-13-32-14-12-30/h4,9,15-16,19-20,31H,2-3,5-8,10-14,17H2,1H3,(H2,25,27,28,29)

4-[[2-(butylamino)-5-[5-(morpholin-4-ylmethyl)pyridin-2-yl]pyrimidin-4-yl]amino]cyclohexan-1-ol

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 mg/mL (4.54 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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.27 mM) (saturation unknown) in 10% DMSO 20% HS-15 70% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: 1 mg/mL (2.27 mM) in 10% DMSO + 40% PEG300 50% PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 4: 5% DMSO+30% PEG 300+ddH2O: 0.5mg/mL

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Solubility in Formulation 5: 10 mg/mL (22.70 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 6: 10 mg/mL (22.70 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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