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| Targets |
CDK12 and CDK13 (cyclin-dependent kinases 12 and 13). BSJ-01-175 is a dual covalent inhibitor targeting CDK12 and CDK13. It binds irreversibly to cysteine residues Cy51039 on CDK12 and Cy51017 on CDK13 via an acrylamide warhead. The IC50 values are 156 nM for CDK12/CycK and 282.6 nM for CDK13/CycK. Inhibition of CDK12/13 reduces phosphorylation of RNA Polymerase II at Serine 2 residues (p-Ser2), which is required for transcription elongation, splicing, and processing of certain genes, particularly DNA damage response genes (e.g., BRCA1, BRCA2, ATR, FANCI, RAD51) and super-enhancer-associated genes. Selective inhibition of CDK12/13 in cancer cells downregulates these genes, leading to genomic instability, DNA damage accumulation, and cell death.
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| 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].
In cell-free biochemical assays, BSJ-01-175 directly inhibits CDK12/CycK and CDK13/CycK activity. IC50 values are 156 nM for CDK12/CycK and 282.6 nM for CDK13/CycK. The inhibition mechanism is covalent and irreversible, as demonstrated by time-dependent inhibition and the presence of a reactive acrylamide warhead. For selectivity profiling, BSJ-01-175 shows excellent selectivity for CDK12/13 over other kinases (including other CDKs) due to the unique placement of the reactive cysteine in the CDK12/13 active site. The compound binds to the ATP-binding site but forms a covalent adduct with the cysteine residue. |
| 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
BSJ-01-175 is significantly selective for cancer cells compared to normal cells. The compound potently inhibits RNA polymerase II phosphorylation (p-Ser2) in cancer cells. It induces downregulation of CDK12-targeted genes, including DNA damage response genes (e.g., BRCA1, BRCA2, ATR, RAD51). In Ewing sarcoma cells, BSJ-01-175 reduces expression of EWS-FLI1 target genes and induces apoptosis. The compound also efficiently reduces CDK6 protein levels via CRBN-mediated E3 ligase activity, thereby inhibiting cell cycle progression and suppressing proliferation in CDK6-dependent cancers. In RH4 and other FP-RMS cells, the compound may have activity similar to other CDK12/13 inhibitors. BSJ-01-175 induces cell cycle arrest at G2/M phase and increases DNA damage markers (gammaH2AX). The compound shows sub-micromolar EC50 in sensitive cancer cell lines. |
| Enzyme Assay |
CDK12/13 kinase activity is measured in a cell-free format using recombinant CDK12/CycK or CDK13/CycK proteins and a suitable peptide substrate (e.g., RNA Pol II CTD peptide, often YSPTSPS with multiple repeats). The kinase reaction is carried out in the presence of ATP (10-100 uM) and varying concentrations of BSJ-01-175 (0.1-1000 nM). After incubation, ADP production is quantitated using a luminescent kinase assay (e.g., Kinase-Glo® or ADP-Glo™). For time-dependent and irreversible inhibition studies, the compound is pre-incubated with the enzyme for various time points (0-120 minutes) before adding ATP and substrate. The IC50 shift over time indicates irreversible binding. Alternatively, mass spectrometry can be used to confirm covalent adduct formation on the target cysteine. For selectivity profiling, BSJ-01-175 is screened against a panel of >100 kinases at 1 uM.
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| 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. Cancer cell lines (e.g., Ewing sarcoma, osteosarcoma, rhabdomyosarcoma) are seeded in 96-well plates and treated with BSJ-01-175 at concentrations ranging from 0.001-10 uM for 48-96 hours. Cell viability is measured using CellTiter-Glo or MTT assays. EC50 values are calculated. For p-RNA Pol II (Ser2) inhibition, cells are treated with BSJ-01-175 (0.1-1 uM) for 2-6 hours, and lysates are analyzed by Western blot using anti-p-Ser2 antibody. For gene expression analysis, cells are treated with BSJ-01-175 (0.1-1 uM) for 6-24 hours, total RNA is extracted, and CDK12-target gene expression (e.g., BRCA1, RAD51, ATR) is measured by qRT-PCR or RNA-seq. For DNA damage assessment, cells are treated for 24-48 hours and stained with anti-gammaH2AX antibody for immunofluorescence or flow cytometry. For cell cycle analysis, cells are stained with propidium iodide and analyzed by flow cytometry. Apoptosis is measured by Annexin V/PI staining and caspase-3/7 activity assays. For CDK6 degradation analysis (if applicable), cells are treated with BSJ-01-175 (0.1-1 uM) for 4-24 hours and CDK6 protein levels are quantified by Western blot. |
| 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. BSJ-01-175 exhibits in vivo efficacy in Ewing sarcoma patient-derived xenograft (PDX) mouse models. In PDX models of Ewing sarcoma, administration of BSJ-01-175 at 10 mg/kg once daily via intraperitoneal injection (IP) inhibits tumor growth. The compound represents the first selective CDK12/13 covalent inhibitor with reported in vivo efficacy. In these studies, tumor volume is measured twice weekly with calipers, and body weight is monitored for tolerability. At study termination, tumors are harvested for biomarker analysis (p-RNA Pol II Ser2 reduction, downregulation of CDK12 target genes, gammaH2AX increase, and apoptosis markers). Additional in vivo efficacy studies may be conducted in other CDK12/13-dependent cancer models, such as triple-negative breast cancer and high-grade serous ovarian cancer. Standard dosing regimens: 10 mg/kg IP daily or every other day for 2-4 weeks. |
| ADME/Pharmacokinetics |
BSJ-01-175 is administered in vivo via intraperitoneal injection at 10 mg/kg once daily. The compound is typically formulated in vehicles such as 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline or similar. The solubility in this formulation is 2-5 mg/mL. The maximum concentration in vivo has not been fully published. Pharmacokinetic parameters (half-life, Cmax, oral bioavailability) for BSJ-01-175 have not been extensively reported. As a covalent inhibitor, the duration of target engagement may exceed the compound's presence in circulation, allowing for less frequent dosing. Absorption, distribution, metabolism, and excretion (ADME) properties typical of covalent inhibitors include moderate-to-high plasma protein binding and distribution to tissues including tumors. Formal PK studies would be required for further development.
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| Toxicity/Toxicokinetics |
Preclinical toxicology data for BSJ-01-175 have not been extensively published. In mouse xenograft studies at the efficacious dose of 10 mg/kg once daily IP, the compound is reported to be well-tolerated with no significant body weight loss or mortality. However, CDK12/13 inhibition is associated with potential on-target toxicities, including effects on normal cell transcription, particularly in highly proliferative tissues (e.g., bone marrow, gastrointestinal tract). In preclinical studies with other CDK12/13 inhibitors (e.g., THZ531), toxicities such as thrombocytopenia, anemia, and gastrointestinal disturbances have been observed at higher doses. For BSJ-01-175, a favorable therapeutic window is suggested by its selectivity for cancer cells over normal cells. Formal toxicology studies (e.g., 28-day repeat-dose in rodents) would be required for clinical development. The compound should be handled as an experimental agent with appropriate safety precautions.
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| References | |
| Additional Infomation |
BSJ-01-175 was discovered through structure-activity relationship (SAR) studies of THZ531 derivatives. It is the first selective CDK12/13 covalent inhibitor with reported in vivo efficacy in Ewing sarcoma PDX models. The compound has a molecular weight of 545.08 and molecular formula C30H33ClN6O2. BSJ-01-175 features an acrylamide warhead that covalently targets cysteine residues in CDK12 (Cys1039) and CDK13 (Cys1017). The compound demonstrates exquisite selectivity over other kinases, including CDK7, CDK8, and CDK9, due to the unique placement of these reactive cysteines. The development of BSJ-01-175 provides a valuable chemical tool for studying CDK12/13 biology and validating these kinases as therapeutic targets in cancers such as Ewing sarcoma, triple-negative breast cancer, high-grade serous ovarian cancer, and other CDK12/13-dependent malignancies. BSJ-01-175 is not approved for human use and is intended for research purposes only. The compound is also known by the catalog numbers T63841 and V41805.
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| Molecular Formula |
C30H33CLN6O2
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| Molecular Weight |
545.075025320053
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| Exact Mass |
544.235
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| CAS # |
2227392-55-2
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| PubChem CID |
134560325
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| Appearance |
White to off-white solid powder
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| LogP |
5.4
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
39
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| Complexity |
806
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| Defined Atom Stereocenter Count |
2
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| 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
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| InChi Key |
DMUSMYYDKCXFKR-MRJIRHQNSA-N
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| 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
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| 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
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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 |
| 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) |
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
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (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.
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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