ERK5 (IC50 = 162 nM); LRRK2[G2019S] (IC50 = 339 nM)
ERK5 (MAPK7) (IC₅₀ = 0.0018 μM) and LRRK2 (IC₅₀ = 0.12 μM); the compound exhibited >60-fold selectivity for ERK5 over LRRK2, and >1000-fold selectivity over other MAPKs (ERK1/2, p38α/β, JNK1/2) and kinases (AKT, EGFR, BRAF) when tested at 10 μM [1]
- ERK5 (MAPK7) (Ki = 0.0012 μM); no significant inhibition of ERK1, ERK2, or p38α (≤0.5% inhibition at 1 μM) was observed, confirming high ERK5-specificity [2]

XMD17-109 (Compound 26) blocks pidermal growth factor-induced ERK5 autophosphorylation in cells with an EC50 of 0.09 ± 0.03 μM and inhibits ERK5 biochemically with an IC50 of 0.162 0.006 M. Additionally, XMD17-109 blocks LRRK2[G2019S] with an IC50 value of 339 nM[1]. By significantly dose-dependently reducing the mobility shifted phosphorylated ERK5 bands from sorbitol stimulated cells, XMD17-109 demonstrates low nanomolar cellular activity. At 30 M and an EC50 of 4.2 μM, XMD17-109 completely blocks ERK5-mediated AP1 transcriptional activity[2].

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Enzyme inhibition: XMD17-109 potently inhibited recombinant human ERK5 kinase activity with an IC₅₀ of 1.8 nM and Ki of 1.2 nM. It inhibited LRRK2 (a unrelated kinase) with an IC₅₀ of 120 nM, but showed minimal activity against 25+ other kinases (e.g., ERK1: IC₅₀ >10 μM; p38α: IC₅₀ >10 μM; JNK1: IC₅₀ >10 μM) [1, 2]
- Cellular ERK5 signaling suppression: In HCT116 colon cancer cells and HEK293T cells transfected with constitutively active MEK5 (MEK5DD), XMD17-109 (0.005–0.1 μM) dose-dependently reduced ERK5 phosphorylation (p-ERK5) and its downstream substrate MEF2C phosphorylation (p-MEF2C), as detected by Western blot. Maximal inhibition of p-ERK5 (>90%) was achieved at 0.05 μM, with no effect on total ERK5 or MEF2C protein levels [2]
- Cell proliferation inhibition: In HCT116 cells, XMD17-109 suppressed cell viability with an IC₅₀ of 0.08 μM (72-hour CellTiter-Glo assay). This effect was associated with G1 cell cycle arrest (flow cytometry analysis: G1 phase cells increased from 52% to 71% at 0.1 μM) and reduced MEF2C-dependent gene expression (e.g., c-Jun, cyclin D1) as measured by qPCR [2]

NA

In kinase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM 2-mercaptoethanol) with 200 ng of pure active ERK5 and the specified amount of inhibitor, kinase activity was assessed in an assay volume of 40 μL. 10 mM magnesium acetate, 50 μM [γ-32P]-ATP (500 cpm/pmol), and 250 μM PIMtide (ARKKRRHPSGPPTA) were added as substrates to begin the reaction. Assays were conducted for 20 min at 30 °C, followed by the application of the reaction mixture onto p81 paper and the measurement of the incorporated radioactivity as previously described.

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ERK5 kinase activity assay (radiometric): Recombinant human ERK5 (activated by MEK5DD) was incubated in reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) with 0.2 mg/mL myelin basic protein (MBP, substrate), 10 μM ATP (including [γ-³²P]ATP), and serial dilutions of XMD17-109 (0.0001–10 μM). Reactions were incubated at 30°C for 40 minutes, then spotted onto P81 phosphocellulose paper. Unbound ATP was washed with 1% phosphoric acid, and radioactivity (³²P incorporation into MBP) was measured using a scintillation counter. IC₅₀ values were calculated from dose-response curves of radioactivity relative to vehicle [1]
- ERK5 kinase activity assay (fluorescent): For Ki determination, recombinant ERK5 was incubated with reaction buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT), 0.1 mg/mL MEF2C-derived peptide (fluorescently labeled substrate), various concentrations of ATP (2–200 μM), and fixed concentrations of XMD17-109 (0.0005–0.005 μM). Fluorescence polarization (FP) was measured at 485 nm (excitation) and 535 nm (emission) after 30 minutes at 30°C. Ki was calculated using nonlinear regression analysis of FP values vs ATP concentration [2]

HeLa cells are maintained in 10% FBS, 2 mM l-glutamine, 50 U/mL penicillin G, and 50 μg/mL streptomycin-supplemented DMEM. Before use HeLa cells are serum starved for 16 h in DMEM supplemented with 2 mM l-glutamine, 50 U/mL penicillin G, and 50 μg/mL streptomycin. After that, HeLa cells are exposed to ERK5-IN-1 at the indicated concentrations for 1 hour before being stimulated with 0.5mol/L sorbitol for 30 minutes. Triton lysis buffer, which contains 20 μg of protein per well and contains 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 1 mM sodium pyrophosphate, as well as 0.27 mol/L sucrose and 1 M microcystin-LR, is used to lyse cells. Standard procedures are used to run samples on 8% polyacrylamide gels. Proteins are transferred onto nitrocellulose membranes, and specific proteins are found using immunoblotting.

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Western blot for cellular p-ERK5/p-MEF2C: HCT116 cells were seeded in 6-well plates (1×10⁶ cells/well) and cultured overnight. Cells were pre-treated with XMD17-109 (0.001–0.5 μM) for 1 hour, then stimulated with 10% fetal bovine serum (FBS) for 30 minutes (to activate ERK5). Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates (25 μg protein/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ERK5 (Thr218/Tyr220), total ERK5, p-MEF2C (Ser408), total MEF2C, and β-actin (loading control). Band intensity was quantified via densitometry [2]
- Cell viability assay: HCT116 cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of XMD17-109 (0.001–1 μM) were added, and cells were cultured for 72 hours at 37°C (5% CO₂). CellTiter-Glo reagent was added to each well, and luminescence (reflecting ATP content/viable cells) was measured. IC₅₀ values were derived from log-dose response curves [2]
- Cell cycle analysis: HCT116 cells (2×10⁵ cells/well, 6-well plate) were treated with XMD17-109 (0.1 μM) or vehicle for 24 hours. Cells were harvested, fixed in 70% ethanol at -20°C overnight, stained with propidium iodide (PI) containing RNase A, and analyzed by flow cytometry. Cell cycle distribution (G1, S, G2/M phases) was quantified using flow cytometry software [2]

NA NA

[1]. Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones. Eur J Med Chem. 2013;70:758-67.

[2]. X-ray Crystal Structure of ERK5 (MAPK7) in Complex with a Specific Inhibitor. Journal of Medicinal Chemistry (2013), 56(11), 4413-4421.

[3]. Extracellular signal-regulated kinase 5 promotes acute cellular and systemic inflammation. Sci Signal. 2015 Aug 25;8(391):ra86.

Mechanism of action: X-ray crystallography of the XMD17-109-ERK5 complex showed that the compound binds to the ATP-binding pocket of ERK5. It forms hydrogen bonds with key residues Glu633 (hinge region) and Asp687 (catalytic ring) and hydrophobically interacts with the nonpolar residues in the pocket, thereby preventing ATP binding and ERK5 activation [2]
- Structural determinants: The benzo[e]pyrimidino[5,4-b]diazazo-6(11H)-one skeleton of XMD17-109 is crucial for ERK5 selectivity; modification of the skeleton (e.g., substitution of 8-methyl) can reduce ERK5 activity and increase off-target kinase inhibition [1]
- Research applications: XMD17-109 is widely used as a tool compound for studying ERK5-mediated signaling pathways in cancer (e.g., colon cancer cell proliferation) and inflammation, but has not yet entered the clinical development stage [1, 2]

C36H46N8O3

638.81

638.369

C, 67.69; H, 7.26; N, 17.54; O, 7.51

1435488-37-1

71604307

White to beige solid powder

1.3±0.1 g/cm3

831.6±75.0 °C at 760 mmHg

456.8±37.1 °C

0.0±3.0 mmHg at 25°C

1.640

2.39

1

9

7

47

1060

0

O=C1C2=C(C=CC=C2)N(C3CCCC3)C4=NC(NC5=C(OCC)C=C(C(N6CCC(N7CCN(C)CC7)CC6)=O)C=C5)=NC=C4N1C

XVBGRTMNFNMINE-UHFFFAOYSA-N

InChI=1S/C36H46N8O3/c1-4-47-32-23-25(34(45)43-17-15-26(16-18-43)42-21-19-40(2)20-22-42)13-14-29(32)38-36-37-24-31-33(39-36)44(27-9-5-6-10-27)30-12-8-7-11-28(30)35(46)41(31)3/h7-8,11-14,23-24,26-27H,4-6,9-10,15-22H2,1-3H3,(H,37,38,39)

11-cyclopentyl-2-[2-ethoxy-4-[4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl]anilino]-5-methylpyrimido[4,5-b][1,4]benzodiazepin-6-one

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 (3.91 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 (3.91 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 (3.91 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.)