BSJ-01-175 is a potent and specific covalent/irreversible inhibitor of CDK12/13. BSJ-01-175 displays excellent selectivity against cancer/tumor cells, potent inhibition of phosphorylated RNA polymerase II, and significant downregulation of CDK12-targeted genes.

Physicochemical Properties

Molecular Formula	C30H33CLN6O2
Molecular Weight	545.075025320053
Exact Mass	544.235
CAS #	2227392-55-2
PubChem CID	134560325
Appearance	White to off-white solid powder
LogP	5.4
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	9
Heavy Atom Count	39
Complexity	806
Defined Atom Stereocenter Count	2
SMILES	ClC1C=NC(=NC=1C1=CNC2C=CC=CC1=2)N[C@@H]1CCC[C@H](C1)OC1C=CC(=CC=1)NC(/C=C/CN(C)C)=O
InChi Key	DMUSMYYDKCXFKR-MRJIRHQNSA-N
InChi Code	InChI=1S/C30H33ClN6O2/c1-37(2)16-6-11-28(38)34-20-12-14-22(15-13-20)39-23-8-5-7-21(17-23)35-30-33-19-26(31)29(36-30)25-18-32-27-10-4-3-9-24(25)27/h3-4,6,9-15,18-19,21,23,32H,5,7-8,16-17H2,1-2H3,(H,34,38)(H,33,35,36)/b11-6+/t21-,23-/m1/s1
Chemical Name	(E)-N-[4-[(1R,3R)-3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]cyclohexyl]oxyphenyl]-4-(dimethylamino)but-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
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	When compared to wild type (WT), BSJ-01-175 (0–10 μM; 72 hours) boosts cell survival five times, showing a substantial dependence on Cys1039 covalent bond formation [1]. When compared to THZ531, BSJ-01-175 (0–10 μM; 72 hours) marginally decreased the activity of TC71 Ewing sarcoma cells [1]. Specifically targeting CDK12/13, BSJ-01–175 (0–5 μM) suppresses BRAC1 and BRAC2 transcription [1].
ln Vivo	BSJ-01–175 (10 mg/kg; intraperitoneal injection; once daily for 3 weeks) effectively reduced tumor growth during the 3-week medication treatment period [1]. Evaluation of the pharmacokinetic (PK) properties of BSJ-01-175 in mice [1]. Route dose (mg/kg) Tmax (h) Cmax (ng/mL) AUClast (h·ng/mL) T1/2 (h) CL (mL/min/kg) VSS (L/kg) F (%) IV 3 1511 1832 2.2 24.9 3.9 PO 10 2 272 1043 17
Cell Assay	Cell Viability Assay[1] Cell Types: Kelly wild type or CDK12C1039F Cell Tested Concentrations: 0-10 μM Incubation Duration: 72 hrs (hours) Experimental Results: A 5-fold increase in cell viability was observed compared to wild type (WT), indicating an increase in cell viability. Strongly dependent on covalent bond formation with Cys1039. Cell proliferation assay[1] Cell Types: TC71 Ewing Sarcoma Cells Tested Concentrations: 0-10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Slight decrease in activity compared to THZ531.
Animal Protocol	Animal/Disease Models: Female nude mice (BALB/c, 7-8 weeks) carrying TC71 Ewing sarcoma cells [1] Doses: 10 mg/kg Route of Administration: intraperitoneal (ip) injection; one time/day for 3 weeks Experimental Results: Tumor growth was Dramatically inhibited throughout the 3 weeks of drug treatment.
References	[1]. Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma. Eur J Med Chem. 2021;221:113481.

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	1.8346 mL	9.1730 mL	18.3459 mL
	5 mM	0.3669 mL	1.8346 mL	3.6692 mL
	10 mM	0.1835 mL	0.9173 mL	1.8346 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.