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Purity: ≥98%
ERK5-IN-2 is a novel, potent, orally bioavailable, sub-micromolar, and selective ERK5 inhibitor with selectivity over p38α and BRD4. For ERK5 and ERK5 MEF2D, it has IC50 values of 0.82 μM, 3 μM, respectively. Both the growth of tumor xenografts and the angiogenesis induced by basic fibroblast growth factor (bFGF) in Matrigel plugs are inhibited by ERK5-IN-2. Endothelial cell angiogenesis and tumor cell motility are two cellular phenotypes that have been linked to extracellular regulated kinase 5 (ERK5) signaling. A series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group were found to exhibit IC50 values in the micromolar range, but had no selectivity against p38 MAP kinase. These novel ERK5 inhibitors, such as ERK5-IN-2, were discovered using high throughput screening. Truncation of the N-substituent attenuated the inhibition of p38α, which was important because it only slightly increased potency (∼3-fold) against ERK5. The selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). This compound's complex crystal structure with ERK5 has been determined (PDB 5O7I). This substance was orally bioavailable and prevented tumor xenograft growth and bFGF-driven angiogenesis in Matrigel plugs. The selective ERK5 inhibitor that is described in this article offers a lead for further development into a tool compound for more in-depth studies looking to understand how ERK5 signaling affects cancer and other diseases.
| Targets |
ERK5 (IC50 = 0.82 μM); ERK5 MEF2D (IC50 = 3 μM)
ERK5 (IC₅₀ = 0.82 ± 0.10 μM), p38α (IC₅₀ > 120 μM) |
|---|---|
| ln Vitro |
46 inhibits ERK5 kinase activity in a biochemical assay with an IC₅₀ of 0.82 μM and shows >146-fold selectivity over p38α.
In a cell-based ERK5-dependent MEF2D reporter gene assay in HEK293 cells, 46 exhibits an IC₅₀ of 3.0 ± 1.2 μM. 46 inhibits the proliferation of PC3 prostate cancer cells with a GI₅₀ of 44 ± 2.8 μM. Kinome-wide screening at 10 μM against 456 kinases revealed that besides ERK5, 46 inhibits only seven other kinases by ≥90% (DCAMKL3, JAK1, SLK, MAP3K15, TYK2, JAK2, MST2, and DCAMKL1). Surface Plasmon Resonance (SPR) analysis confirmed that 46 does not bind to the first bromodomain of BRD4, unlike the control compound XMD8-92. The crystal structure of 46 bound to the ERK5 kinase domain (PDB: 5O7I) was solved at 2.4 Å resolution, revealing its binding mode in the ATP-binding site. |
| ln Vivo |
ERK5-IN-2 (Compound 46) (cartilage; 100 mg/kg; 7 days in CD1 mice and 10 days in CD1 nude (nu/nu) mice) possesses low hemoglobin concentrations and anti-angiogenic properties [1]. In caco-2 cell permeability tests in humans and animals, ERK5-IN-2 (iv or intravenous 10 mg/kg for 0.083-24 hours) shows low internal clearance, high flux, and low efflux ratio (ER)
In a Matrigel plug angiogenesis assay in female CD1 mice, oral administration of 46 (100 mg/kg, twice daily for 7 days) significantly reduced hemoglobin content in plugs (p = 0.0009), indicating inhibition of bFGF-driven angiogenesis. In an A2780 human ovarian carcinoma xenograft model in female CD1 nude mice, oral administration of 46 (100 mg/kg, twice daily for 10 days) significantly reduced tumor volume by 57% compared to the vehicle control (p = 0.002). |
| Enzyme Assay |
ERK5 biochemical activity was measured using a fluorescence polarization (FP) assay. Recombinant ERK5/MEK5 complex (5-10 nM) was incubated with a FAM-labeled peptide substrate, ATP, and serially diluted compound in a reaction buffer for 2 hours at 37°C. Phosphorylated product was detected by adding a binding solution and measuring fluorescence polarization after 2 hours at room temperature.
p38α biochemical activity was measured using a LANCE TR-FRET assay. Full-length p38α (1-3 nM) was incubated with a Ulight-labeled peptide substrate, ATP, and serially diluted compound in a reaction buffer for 1 hour at 37°C. The reaction was quenched with EDTA, and detection was performed by adding a Europium-labeled anti-phospho antibody, incubating for 2 hours at room temperature protected from light, and measuring TR-FRET. |
| Cell Assay |
ERK5-dependent cellular activity was assessed using a dual-luciferase reporter assay in HEK293 cells. Cells were co-transfected with expression vectors for constitutively active MEK5 (MEK5DD), ERK5, and a GAL4-MEF2D fusion protein along with a GAL4-driven luciferase reporter. Four hours post-transfection, cells were treated with compound at various concentrations. After 20 hours, cells were harvested, and firefly and Renilla luciferase activities were measured to determine IC₅₀ values.
Anti-proliferative activity against PC3 cells was determined by measuring cell viability after treatment with compound for a specified duration, and GI₅₀ values were calculated. |
| Animal Protocol |
Animal/Disease Models: Matrigel-vaccinated female CD1 mice (8-10 weeks old) and female CD1 nude (nu/nu) mice (8-10 weeks old) bearing A2780 human ovarian cancer xenografts [1]
Doses: 100 mg/1]. kg Route of Administration: Po; twice (two times) daily; 7 days for CD1 mice, 10 days for CD1 nude mice (nu/nu) Experimental Results: Significant reduction in tumor volume. Animal/Disease Models: 8-10 weeks old female CD1 mice [1] Doses: 10 mg/kg Route of Administration: intravenous (iv) (iv)injection or oral administration; 0.083-24 hrs (hrs (hours)) Experimental Results: The terminal plasma half-life is 38 minutes, and the plasma clearance rate is 27mL/ min/kg, oral bioavailability is 68%. For the Matrigel plug angiogenesis assay, female CD1 mice (8-10 weeks old) were inoculated subcutaneously with 500 μL of Matrigel containing bFGF (500 ng/mL). After 24 hours, mice were randomized to receive vehicle, XMD8-92 (50 mg/kg, i.p., twice daily), or 46 (100 mg/kg, p.o., twice daily) for 7 days. On day 7, plugs were excised, snap-frozen, and hemoglobin content was quantified colorimetrically. For the A2780 xenograft tumor model, female CD1 nude mice (8-10 weeks old) were inoculated subcutaneously with A2780 cells. After 7 days, when tumors reached an average volume of 72 mm³, mice were randomized to receive vehicle, XMD8-92 (50 mg/kg, i.p., twice daily), or 46 (100 mg/kg, p.o., twice daily) for 10 days. Tumor volumes were measured regularly with calipers, and body weight was monitored. For pharmacokinetic studies, female CD1 mice (8-10 weeks old) received a single dose of 46 (10 mg/kg) either intravenously or orally. Blood was collected at multiple time points via cardiac puncture under terminal anesthesia. Plasma was separated and drug concentrations were determined by LC-MS. |
| ADME/Pharmacokinetics |
46 exhibited low intrinsic clearance in both human and mouse liver microsomes (HLM Clᵢₙₜ < 5 μL/min/mg protein; MLM Clᵢₙₜ = 26 μL/min/mg protein). In the Caco-2 cell permeability assay, 46 showed high apparent permeability (Pₐₚₚ > 10 × 10⁻⁶ cm/s) and a low efflux ratio (0.82). The thermodynamic solubility of 46 was estimated to be >100 μM by turbidimetric determination. 46 showed moderate plasma protein binding in mice (fᵤ = 0.06). In mice, following a single intravenous injection of 10 mg/kg, the terminal plasma half-life of 46 was 38 min (T₁/₂), the plasma clearance (CL) was 27 mL/min/kg, and the volume of distribution (Vd) was 1.2 L/kg. Following a single oral administration of 10 mg/kg, 46 reached its maximum plasma concentration (Cₘₐₓ) of 6 μM at 15 minutes (Tₘₐₓ), with an area under the curve (AUC) of 254 μg/mL·min and an oral bioavailability (F) of 68%.
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| Toxicity/Toxicokinetics |
No significant toxicity was observed in the A2780 xenograft study. The lowest body weight in the treatment group was 99% of initial body weight, compared to 96% in the control group and 98% in the XMD8-92 group. The IC₅₀ of 46 against four cytochrome P450 isoenzymes (2C19, 2C9, 2D6, 3A4) was > 5 μM, indicating a low CYP inhibitory potential.
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| References | |
| Additional Infomation |
Compound 46 is a pyrrole-2-carboxamide derivative discovered through optimization of lead compounds in a high-throughput screening program. Structural optimization included truncating the amide substituent, which significantly improved its selectivity for p38α MAP kinase. This compound serves as a selective pharmacology tool for investigating the role of the ERK5 signaling pathway in cancer and angiogenesis, and has demonstrated good oral bioavailability and in vivo efficacy.
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| Molecular Formula |
C17H11BRFN3O2
|
|---|---|
| Molecular Weight |
388.190546274185
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| Exact Mass |
387.001
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| CAS # |
1888305-96-1
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| Related CAS # |
1888305-96-1
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| PubChem CID |
118959080
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| Appearance |
White to off-white solid
|
| LogP |
2.9
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
479
|
| Defined Atom Stereocenter Count |
0
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| InChi Key |
ATKCERYALDNMPL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H11BrFN3O2/c18-12-4-1-5-13(19)15(12)16(23)10-7-14(21-8-10)17(24)22-11-3-2-6-20-9-11/h1-9,21H,(H,22,24)
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| Chemical Name |
4-(2-bromo-6-fluorobenzoyl)-N-pyridin-3-yl-1H-pyrrole-2-carboxamide
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| Synonyms |
ERK5-IN 2; ERK5 IN-2; ERK5-IN-2
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 78~250 mg/mL (200.9~644.0 mM)
Ethanol: ~3 mg/mL (~7.7 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.36 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.36 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5761 mL | 12.8803 mL | 25.7606 mL | |
| 5 mM | 0.5152 mL | 2.5761 mL | 5.1521 mL | |
| 10 mM | 0.2576 mL | 1.2880 mL | 2.5761 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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