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| Targets |
Enitociclib (VIP152; BAY-1251152) is a highly potent and selective inhibitor of cyclin-dependent kinase 9 (CDK9) in complex with cyclin T1 (CycT1), a core component of positive transcription elongation factor b (P-TEFb).
- Ki for human CDK9/CycT1: 0.3 nM (surface plasmon resonance, SPR) [3]
- IC₅₀ for human CDK9/CycT1 kinase activity: 0.8 nM (homogeneous time-resolved fluorescence, HTRF assay) [3] - High selectivity over other CDKs/kinases: IC₅₀ > 1000 nM for CDK1/CycB, CDK2/CycE, CDK4/CycD1, CDK6/CycD3, CDK7/CycH; inhibition rate < 10% for 290+ other kinases at 100 nM [3] - In cellular assays, it inhibits P-TEFb-mediated phosphorylation of RNA polymerase II (RNAPII) at Ser2 with IC₅₀ = 1.2 nM [3] |
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| ln Vitro |
1. Antiproliferative activity across cancer types:
- Hematological malignancies: IC₅₀ (72-h CellTiter-Glo) – MV-4-11 (AML, 0.9 nM), Raji (Burkitt lymphoma, 1.2 nM), Jurkat (T-cell leukemia, 1.5 nM), U266 (multiple myeloma, MM, 2.3 nM) [3]
- MYC⁺ lymphoma cells: IC₅₀ – SU-DHL-4 (0.8 nM), OCI-Ly10 (1.1 nM), Farage (1.3 nM); venetoclax-resistant SU-DHL-4 cells (IC₅₀ = 0.9 nM, comparable to parental cells) [5] - Multiple myeloma (MM) cells: IC₅₀ – RPMI-8226 (1.1 nM), U266 (1.5 nM), MM.1S (1.8 nM); CD138⁺ primary MM cells from patients (IC₅₀ range: 1.2–3.5 nM) [6] - Solid tumors: IC₅₀ – HCT116 (colorectal, 3.5 nM), A549 (lung, 4.8 nM), MDA-MB-231 (breast, 5.2 nM) [3] 2. Transcriptional regulation and oncoprotein downregulation: - MYC⁺ lymphoma cells (SU-DHL-4): 1 nM Enitociclib for 4 h reduced p-RNAPII (Ser2) by 70% and MYC protein by 65% (Western blot); 5 nM for 8 h downregulated MCL1 (70%) and BCL2 (60%) [5] - MM cells (RPMI-8226): 2 nM for 6 h decreased p-RNAPII (Ser2) by 65%, MCL1 by 60%, and BCL2 by 55%; qPCR showed 40–50% reduction in short-half-life transcripts (MYC, MCL1) but no effect on housekeeping genes (GAPDH, ACTB) [6] - AML cells (MV-4-11): 5 nM for 8 h reduced MYC (70%) and MCL1 (65%) [3] 3. Apoptosis induction: - SU-DHL-4 cells: 1 nM for 24 h increased Annexin V⁺ cells to 35%, 5 nM to 80% (flow cytometry); caspase-3/7 activity elevated 4.2-fold at 5 nM [5] - RPMI-8226 cells: 2 nM for 24 h induced 55% Annexin V⁺ cells; 5 nM for 48 h increased TUNEL⁺ cells by 70% (immunofluorescence) [6] - MV-4-11 cells: 10 nM for 24 h resulted in 85% Annexin V⁺ cells [3] 4. Early data: - WO2014076091A1: Enitociclib (referred to as "compound X") inhibited CDK9/CycT1 (IC₅₀ < 5 nM) and Raji cell proliferation (IC₅₀ < 5 nM) [1] - Cancer Res 2017/AACR 2017: Potent P-TEFb inhibition (IC₅₀ < 1 nM) and MYC downregulation in AML cells [2][4] |
| ln Vivo |
1. MYC⁺ lymphoma xenografts:
- Model: SU-DHL-4 cells (1×10⁶) subcutaneously implanted in NSG mice; dosing initiated at tumor volume 100–150 mm³.
- Efficacy: IV Enitociclib (10, 20 mg/kg, once weekly × 3 weeks, dissolved in 10% DMSO/40% PEG400/50% saline):
- 10 mg/kg: Tumor growth inhibition (TGI) = 85%, no complete regression (CR);
- 20 mg/kg: TGI = 98%, 5/6 mice achieved CR (tumor < 50 mm³ for > 28 days);
- Mechanism: Tumors from 20 mg/kg group showed 75% reduction in p-RNAPII (Ser2) and 80% reduction in MYC vs. vehicle [5]
2. Multiple myeloma xenografts: - Subcutaneous model (RPMI-8226, NSG mice): IV Enitociclib (15, 30 mg/kg, once weekly × 4 weeks): - 15 mg/kg: TGI = 78%, median survival extended by 30%; - 30 mg/kg: TGI = 92%, median survival extended by 50% (from 35 days to 53 days); - Bone metastasis model (RPMI-8226 via tail vein, NSG mice): 30 mg/kg IV weekly × 4 weeks reduced osteolytic lesions (μCT) by 65% and bone marrow tumor burden (flow cytometry) by 70% [6] 3. Hematological/solid tumor xenografts: - MV-4-11 (AML, SCID mice): 40 mg/kg IV weekly × 3 weeks: TGI = 95%, 3/8 CR; - Raji (lymphoma, nude mice): 20 mg/kg IV weekly × 3 weeks: TGI = 80%, 1/8 CR; - HCT116 (colorectal, nude mice): 40 mg/kg IV weekly × 3 weeks: TGI = 78% [3] 4. Pharmacodynamic (PD)-pharmacokinetic (PK) correlation: - Plasma concentrations > 5 nM (above cellular IC₅₀) for > 24 hours post-dose correlated with maximal TGI; tumor drug concentration was 2.3–2.8-fold higher than plasma at 24 hours [3][5] |
| Enzyme Assay |
1. CDK9/CycT1 kinase activity assay (HTRF):
- Reaction mixture (25 μL): 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 200 μM ATP, 0.5 μg/mL biotinylated RNAPII Ser2 peptide substrate, 0.1 nM recombinant human CDK9/CycT1, and Enitociclib (0.01–100 nM).
- Incubation: 37°C for 60 min; stopped with 25 μL buffer (50 mM HEPES pH 7.5, 0.2 M EDTA, streptavidin-XL665, Eu³⁺-labeled anti-p-RNAPII Ser2 antibody).
- Detection: Time-resolved FRET (TR-FRET) measured at 615 nm (Eu³⁺) and 665 nm (XL665); IC₅₀ calculated via dose-response curves [3]
2. CDK9/CycT1 binding assay (SPR): - Recombinant human CDK9/CycT1 (5 μg/mL) immobilized on a CM5 chip via amine coupling. - Enitociclib (0.1–100 nM) injected at 30 μL/min in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20). - Association (120 s) and dissociation (300 s) recorded; Ki derived from 1:1 binding model [3] 3. Kinase selectivity panel: - Enitociclib (100 nM) tested against 290+ kinases via radiometric/luminescent assays; inhibition < 10% for all except CDK9 [3] |
| Cell Assay |
1. Cell viability assay (CellTiter-Glo):
- Cells seeded in 384-well plates (1×10³ cells/well) and cultured overnight.
- Enitociclib (0.01–100 nM, 10-point dilution) added; incubated at 37°C, 5% CO₂ for 72 h.
- CellTiter-Glo reagent (equal volume) added; luminescence measured. IC₅₀ calculated via four-parameter logistic regression [3][5][6]
2. Western blot for p-RNAPII/oncoprotein: - Cells (5×10⁵ cells/mL) treated with Enitociclib (0.1–10 nM) for 4–8 h. - Lysed in RIPA buffer (with protease/phosphatase inhibitors); 30 μg protein separated by SDS-PAGE, transferred to PVDF membranes. - Probed with antibodies (anti-p-RNAPII Ser2, anti-MYC, anti-MCL1, anti-β-actin) and HRP-conjugated secondary antibodies; bands visualized by ECL, quantified via densitometry [3][5][6] 3. Apoptosis assay (Annexin V/PI): - Cells treated with Enitociclib (1–10 nM) for 24 h. - Washed with PBS, stained with Annexin V-FITC/PI for 15 min at room temperature. - Flow cytometry analysis: Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis) cells quantified [3][5][6] 4. Primary MM cell assay: - CD138⁺ cells isolated from patient bone marrow via magnetic sorting. - Seeded at 5×10⁴ cells/well, treated with Enitociclib (0.1–10 nM) for 72 h. - Viability measured via trypan blue exclusion; IC₅₀ calculated [6] |
| Animal Protocol |
1. MYC⁺ lymphoma xenograft (SU-DHL-4, NSG mice):
- 6–8-week-old female NSG mice (18–22 g) implanted subcutaneously with 1×10⁶ SU-DHL-4 cells (50% Matrigel/PBS).
- Randomized into 3 groups (n=6/group): vehicle (10% DMSO/40% PEG400/50% saline), Enitociclib 10 mg/kg, 20 mg/kg.
- IV injection via tail vein, once weekly for 3 weeks.
- Tumor volume (V = 0.5×length×width²) measured twice weekly; mice euthanized if tumor > 1500 mm³ or weight loss > 20% [5]
2. MM xenografts: - Subcutaneous model (RPMI-8226, NSG mice): 2×10⁶ cells (50% Matrigel/PBS) implanted subcutaneously. Dosed IV (15, 30 mg/kg, weekly × 4 weeks); tumor/weight monitored as above [6] - Bone metastasis model (RPMI-8226, NSG mice): 5×10⁵ cells injected via tail vein. Dosed IV (30 mg/kg, weekly × 4 weeks); bone lesions assessed by μCT, bone marrow tumor burden by flow cytometry [6] 3. AML/lymphoma/solid tumor xenografts: - MV-4-11 (SCID mice): 5×10⁶ cells (50% Matrigel/PBS) implanted subcutaneously; IV Enitociclib (10–40 mg/kg, weekly × 3 weeks) [3] - Raji (nude mice): 1×10⁷ cells (PBS) implanted subcutaneously; IV 20 mg/kg weekly × 3 weeks [3] - HCT116 (nude mice): 5×10⁶ cells (50% Matrigel/PBS) implanted subcutaneously; IV 40 mg/kg weekly × 3 weeks [3] 4. PK study: - Female CD-1 mice (n=3/time point) administered Enitociclib 20 mg/kg IV. - Blood sampled at 0.083–24 h post-dose; plasma separated by centrifugation. - Drug concentration measured by LC-MS/MS; PK parameters: t₁/₂ = 6.8 h, CL = 12.3 mL/min/kg, Vss = 5.2 L/kg [3] |
| ADME/Pharmacokinetics |
- Female CD-1 mice (n=3/time point) administered Enitociclib 20 mg/kg IV.
- Blood sampled at 0.083–24 h post-dose; plasma separated by centrifugation.
- Drug concentration measured by LC-MS/MS; PK parameters: t₁/₂ = 6.8 h, CL = 12.3 mL/min/kg, Vss = 5.2 L/kg [3]
ADME/Pharmacokinetics: 1. Preclinical PK in rodents/dogs: - Mice (IV 20 mg/kg): t₁/₂ = 6.8 h, CL = 12.3 mL/min/kg, Vss = 5.2 L/kg [3] - Rats (IV 10 mg/kg): t₁/₂ = 8.2 h, CL = 9.5 mL/min/kg, Vss = 4.8 L/kg [3] - Dogs (IV 5 mg/kg): t₁/₂ = 12.5 h, CL = 5.1 mL/min/kg, Vss = 3.9 L/kg [3] 2. Oral bioavailability: - Low oral absorption: F < 5% (mice), F < 3% (rats) due to poor solubility and first-pass metabolism; IV administration selected for clinical development [3] 3. Tissue distribution: - Mice (IV 20 mg/kg, 2 h post-dose): Drug concentrations (ng/g) – liver (1200), kidney (850), tumor (SU-DHL-4, 620) vs. plasma (270 ng/mL) [3][5] 4. Metabolism/excretion: - Metabolized primarily by human CYP3A4; major metabolites inactive (no CDK9 inhibition at 100 nM) [3] - Rats (IV dose): 65% excreted in feces (30% unchanged), 20% in urine (5% unchanged) within 72 h [3] |
| Toxicity/Toxicokinetics |
1. Repeat-dose toxicity:
- Rats (IV 2, 5, 10 mg/kg, daily × 14 days): NOAEL = 5 mg/kg; 10 mg/kg caused transient weight loss (≤10%), mild gastric mucosa hyperplasia, and platelet reduction (≤20%) [3]
- Dogs (IV 1, 3, 6 mg/kg, weekly × 4 weeks): NOAEL = 3 mg/kg; 6 mg/kg caused mild anemia (Hb ↓15%), lymphopenia (↓25%), and reversible ALT elevation (×2) [3] 2. Cardiac/genotoxicity: - No hERG inhibition (IC₅₀ > 10 μM, patch-clamp); no QT prolongation in dogs (≤6 mg/kg) [3] - Negative in Ames test, in vitro micronucleus assay, and in vivo comet assay [3] 3. Plasma protein binding: - 98.5% (human plasma), 97.8% (mouse plasma), 98.2% (dog plasma) (equilibrium dialysis) [3] 4. Toxicity in efficacy studies: - MYC⁺ lymphoma model (20 mg/kg IV): No weight loss (>95% of initial weight) or organ histopathology (liver, kidney, heart) [5] - MM model (30 mg/kg IV): No significant changes in ALT, AST, BUN, or creatinine vs. vehicle [6] |
| References |
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| Additional Infomation |
1. Mechanism of action:
- Enitociclib inhibits CDK9/CycT1 (P-TEFb), blocking RNAPII Ser2 phosphorylation and transcription elongation. This selectively downregulates short-half-life oncoproteins (MYC, MCL1, BCL2) critical for cancer survival, without affecting housekeeping genes [3][5][6]
2. Therapeutic focus: - Developed for hematological malignancies (AML, MYC⁺ lymphoma, MM) and solid tumors (colorectal, lung cancer); prioritizes MYC-driven or therapy-resistant (venetoclax-resistant) cancers [2][3][5][6] 3. Formulation rationale: - IV formulation (10% DMSO/40% PEG400/50% saline) selected due to low oral bioavailability. Weekly dosing is supported by PK/PD data (sustained concentrations above IC₅₀ and minimal cumulative toxicity) [3] 4. Patent and early development: - WO2014076091A1: Discloses Enitociclib’s chemical structure (pyrazolopyrimidine derivative) and initial CDK9 inhibitory activity [1] - Cancer Res 2017/AACR 2017: Confirms efficacy in AML xenografts and P-TEFb inhibition [2][4] 5. Clinical relevance: - Efficacy in patient-derived MM cells and bone metastasis models supports potential for treating relapsed/refractory MM [6] - CR in MYC⁺ lymphoma models addresses unmet need for MYC-targeted therapies [5] |
| Molecular Formula |
C19H18F2N4O2S
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|---|---|
| Molecular Weight |
404.43
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| Exact Mass |
404.1119
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| Elemental Analysis |
C, 56.43; H, 4.49; F, 9.40; N, 13.85; O, 7.91; S, 7.93
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| CAS # |
1610408-97-3
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| Related CAS # |
1610408-96-2 (R-isomer); 1610408-97-3 (S-isomer); 1610358-53-6; 1610358-56-9 (+); 1610358-59-2 (-); 1610368-59-6 (R-isomer HCl); 1610368-60-9 (S-isomer HCl);
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| Appearance |
Typically exists as Off-white to yellow solids at room temperature
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| LogP |
4.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
619
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1(=CC(OC)=C(C=C1)C1C=C(N=CC=1F)NC1N=CC=C(C=1)CS(=O)(C)=N)F
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| InChi Key |
YZCUMZWULWOUMD-NDEPHWFRSA-N
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| InChi Code |
InChI=1S/C19H18F2N4O2S/c1-27-17-8-13(20)3-4-14(17)15-9-19(24-10-16(15)21)25-18-7-12(5-6-23-18)11-28(2,22)26/h3-10,22H,11H2,1-2H3,(H,23,24,25)/t28-/m0/s1
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| Chemical Name |
(S)-((2-((5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyridin-2-yl)amino)pyridin-4-yl)methyl)(imino)(methyl)-l6-sulfanone
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| Synonyms |
BAY-1251152; Enitociclib; VIP152; BAY1251152; 1255AT22ZJ; BAY 1251152; UNII-1255AT22ZJ; BAY-1251152; VIP-152; ENITOCICLIB [INN]; ...; 1610408-97-3; VIP 152
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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 | 2.4726 mL | 12.3631 mL | 24.7262 mL | |
| 5 mM | 0.4945 mL | 2.4726 mL | 4.9452 mL | |
| 10 mM | 0.2473 mL | 1.2363 mL | 2.4726 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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