ERK5-IN-4 (compound 34b) is a selective inhibitor of extracellular signal-related kinase 5 (ERK5). The ERK5-IN-4 inhibitor inhibits the kinase activity of ERK5 full-length kinase and truncated ERK5 (ERK5ΔTAD) in HEK293 cells with IC50s of 77 nM and 300 nM, respectively.

Physicochemical Properties

Molecular Formula	C16H11CL2FN4O2
Molecular Weight	381.188544511795
Exact Mass	380.024
CAS #	1888305-17-6
PubChem CID	118959090
Appearance	Typically exists as solid at room temperature
LogP	2.9
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	526
Defined Atom Stereocenter Count	0
InChi Key	LIEUQFMVFKJKHX-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H11Cl2FN4O2/c1-23-7-9(6-21-23)22-16(25)12-4-8(5-20-12)15(24)13-10(17)2-3-11(18)14(13)19/h2-7,20H,1H3,(H,22,25)
Chemical Name	4-(3,6-dichloro-2-fluorobenzoyl)-N-(1-methylpyrazol-4-yl)-1H-pyrrole-2-carboxamide
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	ERK5-IN-4 (compound 34b) is selective for MAP3K, p38 (IC50>30 μM), and BRD4 (IC50>20 μM), in contrast to several other reported ERK5 inhibitors [1]. ERK5-IN-4 (0-100 μM; 72 h) suppresses HEK293 cells' ERK5 kinase activity, and (0-1 μM; 72 h) causes ERK5 transcriptional activity to be paradoxically activated, producing the C-terminal transcriptional activation domain (TAD), which separates from the nuclear localization sequence (NLS) and results in ERK5 nuclear translocation [1]. After 72 hours of incubation, ERK5-IN-4 suppresses cancer cells with GI50s of 19.6 μM (HEK293), 22.3 μM (A498), 25 μM (SJSA-1), and 26.6 μM (MDA-MB-231). [1]. For ABL1 that is not phosphorylated, AURKA, CSF1R, and DCAMKL1, the kinome selectivity Kd of ERK5-IN-4 is 1.2 μM, 0.29 μM, 0.046 μM, 0.061 μM, 0.18 μM, 0.38 μM, 1.3 μM, 0.42 μM, 0.22 μM, 2.8 μM (DCLK1), ERK5 (MAPK7), FGFR1, JAK3 (JH1 domain catalytic), KIT, LRRK2, MEK5 (MAP2K5).
ln Vivo	ERK5-IN-4 (compound 34b) (oral; 10 mg/kg) shows limited clearance in mice, with an oral bioavailability of 42% [1].
Cell Assay	Western Blot Analysis[1] Cell Types: HeLa cells Tested Concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3 μM Incubation Duration: 1 hour Experimental Results: Upper phosphorylated ERK5 band produced upon EGF stimulation and inhibition.
Animal Protocol	Animal/Disease Models: pharmacokinetic/PK/PK Parameters of ERK5-IN-4[1] Doses: Route of Administration: Experimental Results: Route Dose (mg/kg) Cl (mL/min/kg) Vd (L/kg) t1/2 ( min) BA (%) intravenously (iv) (iv)(iv) or orally10 14 0.6 80 42
References	[1]. Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor. J Med Chem. 2022 May 12. 65(9):6513-6540.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6234 mL	13.1168 mL	26.2336 mL
	5 mM	0.5247 mL	2.6234 mL	5.2467 mL
	10 mM	0.2623 mL	1.3117 mL	2.6234 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.