JS25 is a selective covalent inhibitor of BTK (IC50=5.8 nM), which inactivates BTK by chelating Tyr551. JS25 inhibits cancer cell proliferation/growth, induces cell death, and has an ameliorative effect on the Burkitt lymphoma xenograft model in mice. JS25 is able to cross the BBB (blood-brain barrier).

Physicochemical Properties

Molecular Formula	C29H24N4O4S
Molecular Weight	524.590265274048
Exact Mass	524.151
CAS #	2411771-95-2
PubChem CID	155368276
Appearance	Light yellow to light brown solid powder
LogP	4.1
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	6
Heavy Atom Count	38
Complexity	1030
Defined Atom Stereocenter Count	0
SMILES	C(NC1=CC(N2C3=C(C=NC4=CC(C5=CC=C(NS(C)(=O)=O)C=C5)=CC=C43)C=CC2=O)=CC(C)=C1)(=O)C=C
InChi Key	WPVJPKXENBARHX-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H24N4O4S/c1-4-27(34)31-23-13-18(2)14-24(16-23)33-28(35)12-8-21-17-30-26-15-20(7-11-25(26)29(21)33)19-5-9-22(10-6-19)32-38(3,36)37/h4-17,32H,1H2,2-3H3,(H,31,34)
Chemical Name	N-[3-[8-[4-(methanesulfonamido)phenyl]-2-oxobenzo[h][1,6]naphthyridin-1-yl]-5-methylphenyl]prop-2-enamide
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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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

Targets	IC50: 5.8 nM (BTK), 49.0 nM (BMX), 440 nM (ITK), 190 nM (TXK), 220 nM (TEC), 2.60 μM (BLK)[1]
ln Vitro	JS25 (0-50 μM; 72 h) prevents myeloid and lymphoid B-cell cancer cell lines from proliferating. With IC50s of 28.5 nM, 49.0 nM, 0.44 μM, 0.19 μM, 0.22 μM, and 2.60 μM, respectively, JS25 demonstrates its inhibitory effectiveness against BTK, BMX, ITK, TXK, TEC, and BLK; however, it exhibits minimal inhibition against additional BTK pathway-related proteins (EGFR, ERBB2, and JAK3). Comparing JS25 to Ibrutinib and Acalabrutinib, the selectivity profile of JS25 is preferable[1]. BTK is degraded by JS25 (10 μM; 0, 4, 15 h), which also prevents BTK from being expressed in tumor cells and from having catalytic activity[1]. In primary diffuse large B-cell lymphoma (DLBCL) samples, JS25 (10 μM; 72 h) causes selective ex vivo cytotoxicity and suppresses the growth of Burkitt's lymphoma tumors[1].
ln Vivo	In a mouse xenograft model of Burkitt's lymphoma, JS25 (10 mg/kg and 20 mg/kg; ip; every 2 days, for 14 d) suppresses tumor growth and significantly reduces the creation of secondary tumors[1]. JS25 (1, 2.5, and 5 μM; injection; once daily for two days) is more effective than isbrutinib at reducing tumor burden in xenografts of chronic lymphocytic leukemia generated from zebrafish patients[1].
Animal Protocol	Animal/Disease Models: Female adult BALB/c/NSG mice with Raji cells (sc)[1] Doses: 10 mg/kg and 20 mg/kg Route of Administration: intraperitoneal (ip) injection; every 2 days for 14 days Experimental Results: Caused a 30-40% reduction of the subcutaneous (sc) tumor and an overall reduction in the percentage of metastasis and secondary tumor formation.
References	[1]. Selective Inhibition of Bruton’s Tyrosine Kinase by a Designed Covalent Ligand Leads to Potent Therapeutic Efficacy in Blood Cancers Relative to Clinically Used Inhibitors. ACS Pharmacology & Translational Science, 2022.

Solubility Data

Solubility (In Vitro)

DMSO: 20 mg/mL (38.13 mM)

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	1.9063 mL	9.5313 mL	19.0625 mL
	5 mM	0.3813 mL	1.9063 mL	3.8125 mL
	10 mM	0.1906 mL	0.9531 mL	1.9063 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.