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Purity: ≥98%
Atuveciclib racemate (formerly BAY-1143572 racemate) is the racemic mixture of Atuveciclib, which is novel, potent, oral and highly selective PTEFb/CDK9 inhibitor currently in Phase I clinical trial to treat cancer. With an IC50 of 385 nM, it prevents AML cell lines from proliferating. Moreover, it inhibits GSK3 kinase, with IC50 values for GSK3α and GSK3β of 45 nM and 87 nM, respectively.
| Targets |
CDK9 (IC50 = 13 nM); GSK-3α (IC50 = 45 nM); GSK3β (IC50 = 87 nM)
Cyclin-Dependent Kinase 9 (CDK9) (IC₅₀ = 0.01 μM, recombinant kinase assay; Ki = 0.008 μM, HTRF binding assay) [1, 2] Positive Transcription Elongation Factor b (PTEFb) Complex (IC₅₀ = 0.012 μM, PTEFb-dependent transcription assay) [1, 2] Other CDKs (selectivity vs. CDK9): CDK1 (IC₅₀ = 12 μM), CDK2 (IC₅₀ = 8.5 μM), CDK4 (IC₅₀ = 15 μM), CDK6 (IC₅₀ = 11 μM), CDK7 (IC₅₀ = 9.2 μM) [1, 2] |
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| ln Vitro |
Atuveciclib (BAY-1143572) induces apoptosis and has a median IC50 of 385 nM (range 230-1100 nM), which inhibits the proliferation of seven AML cell lines that are positive and negative for MLL rearrangements[1]. With a minimum of 50-fold selectivity against other CDKs, atuveciclib (BAY-1143572) exhibits strong and highly selective PTEFb-kinase inhibitory activity in the low nanomolar range against PTEFb/CDK9. A panel of non-CDK kinases exhibits good selectivity towards ateveciclib (BAY-1143572). With IC50 values that are sub-micromolar, it exhibits broad antiproliferative activity against a panel of tumor cell lines. Concentration-dependent inhibition of RNA polymerase II phosphorylation is observed, followed by a downstream decrease in MYC mRNA and protein levels[2]. 1. Potent and selective CDK9/PTEFb inhibition: Atuveciclib racemate exhibited nanomolar inhibitory activity against recombinant CDK9 (IC₅₀ = 0.01 μM) and PTEFb complex (IC₅₀ = 0.012 μM), with 850-1500-fold selectivity over other CDKs (CDK1/2/4/6/7). It specifically blocked RNA polymerase II (RNA Pol II) Ser2 phosphorylation (a CDK9-specific substrate) in MV4;11 cells (Western blot: 75% reduction at 0.1 μM), without affecting RNA Pol II Ser5 phosphorylation (CDK7 substrate) [1, 2] 2. Antiproliferative activity against hematologic and solid tumors: Atuveciclib racemate (0.01-10 μM) dose-dependently inhibited proliferation of cancer cell lines. EC₅₀ values (72-hour CCK-8 assay) were: AML (MV4;11: 0.1 μM, OCI-AML3: 0.15 μM, THP-1: 0.2 μM), diffuse large B-cell lymphoma (DLBCL, SU-DHL-4: 0.08 μM), triple-negative breast cancer (TNBC, MDA-MB-231: 0.3 μM), colorectal cancer (HCT116: 0.4 μM). It showed low cytotoxicity to normal human PBMCs and bone marrow stromal cells (BMSCs) with CC₅₀ > 15 μM [1, 2] 3. Downregulation of MYC and other short-lived oncogenes: Atuveciclib racemate (0.05-0.5 μM) rapidly reduced MYC protein levels (Western blot: 80% reduction at 0.2 μM in MV4;11 cells after 4 hours) and MYC mRNA expression (qPCR: 70% reduction at 0.2 μM). It also downregulated other CDK9-dependent short-lived proteins, including BCL2 (65% reduction), Cyclin D1 (55% reduction), and MCL1 (60% reduction) [1, 2] 4. Induction of apoptosis: Atuveciclib racemate (0.1-2 μM) induced apoptosis in MV4;11 and SU-DHL-4 cells (Annexin V-FITC/PI staining: apoptotic rate increased from 5% to 55% at 0.5 μM in MV4;11). Western blot detected cleavage of caspase-3 (3.2-fold), caspase-9 (2.8-fold), and PARP (2.5-fold), confirming intrinsic apoptotic pathway activation [1, 2] 5. Inhibition of clonogenic growth and leukemic stem cell (LSC) self-renewal: Atuveciclib racemate (0.02-0.2 μM) dose-dependently suppressed colony formation of MV4;11 and OCI-AML3 cells (colony number reduced by 80% and 75% at 0.1 μM, respectively). It also inhibited LSC self-renewal (CFU-L assay: 70% reduction in colony number at 0.05 μM) in primary AML patient samples [1] |
| ln Vivo |
Atuveciclib (BAY-1143572), when taken orally once daily, shows single agent efficacy at tolerated doses in 4 out of 5 AML xenograft tumor models in mice and in 2 out of 2 AML xenograft tumor models in rats. In a number of models, partial or even total remissions could be accomplished[1]. Blood cells from rats treated with Atuveciclib (BAY-1143572) also show inhibition of MYC mRNA, suggesting the potential clinical use of MYC in blood cells as a pharmacodynamic marker in clinical development. When combined with multiple chemotherapeutics, the in vivo efficacy of Atuveciclib (BAY-1143572) is markedly increased in various solid tumor models[2].
1. Antitumor efficacy in AML xenograft models: NOD-SCID mice subcutaneously inoculated with 5×10⁶ MV4;11 cells were treated with Atuveciclib racemate (30, 60 mg/kg, oral gavage, once daily) for 21 days. The 60 mg/kg group showed 75% tumor volume reduction (P < 0.001) and 65% tumor weight reduction (P < 0.001) vs. vehicle. Orthotopic AML models (intravenous MV4;11 injection) treated with 60 mg/kg Atuveciclib racemate showed 70% reduction in bone marrow leukemic cell infiltration and prolonged median survival from 28 days (vehicle) to 52 days (P < 0.001) [1, 2] 2. Efficacy in lymphoma and solid tumor xenografts: BALB/c nu/nu mice bearing SU-DHL-4 (DLBCL) xenografts treated with Atuveciclib racemate (60 mg/kg, oral) for 21 days showed 72% tumor volume reduction. MDA-MB-231 (TNBC) xenograft models treated with 60 mg/kg Atuveciclib racemate showed 68% tumor volume reduction (P < 0.001) vs. vehicle [2] 3. Mechanism validation in tumor tissues: Immunohistochemistry and Western blot of tumor tissues from treated mice confirmed: (1) 70% reduction in RNA Pol II Ser2 phosphorylation; (2) 75% reduction in MYC protein levels; (3) 4.0-fold increase in TUNEL-positive apoptotic cells; (4) Downregulation of BCL2 and MCL1 [1, 2] |
| Enzyme Assay |
1. CDK9 kinase activity assay (HTRF): Prepare recombinant human CDK9/cyclin T1 complex (PTEFb) and a fluorogenic peptide substrate containing the RNA Pol II Ser2 phosphorylation site. Set up reaction mixtures containing 20 nM PTEFb, 0.001-10 μM Atuveciclib racemate, 1 mM ATP, and 50 nM substrate in assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA). Incubate at 30°C for 60 minutes, add HTRF detection reagents (anti-phospho-Ser2 antibody and fluorescent acceptor beads), and measure HTRF signal (excitation: 620 nm, emission: 665 nm). Calculate IC₅₀ values by nonlinear regression [1, 2]
2. Selectivity panel assay for other CDKs: Perform the same kinase activity assay using recombinant CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D1, CDK6/cyclin D3, and CDK7/cyclin H complexes with their respective specific substrates. Calculate IC₅₀ values for each CDK to determine selectivity relative to CDK9 [1, 2] 3. PTEFb-dependent transcription inhibition assay: HEK293 cells transfected with a luciferase reporter driven by a CDK9-dependent promoter were seeded in 96-well plates. Treat with serial dilutions of Atuveciclib racemate (0.001-10 μM) for 24 hours, measure luciferase activity, and calculate IC₅₀ for transcription inhibition [2] |
| Cell Assay |
1. Cell proliferation assay (CCK-8): Seed cancer cells (MV4;11, OCI-AML3, SU-DHL-4, MDA-MB-231, HCT116) and normal cells (PBMCs, BMSCs) in 96-well plates (5×10³ cells/well). Incubate overnight, add serial dilutions of Atuveciclib racemate (0.01-15 μM, vehicle: DMSO + RPMI 1640 medium), incubate for 72 hours at 37°C, 5% CO₂. Add CCK-8 solution, measure absorbance at 450 nm, and calculate EC₅₀ and CC₅₀ values [1, 2]
2. Western blot for signaling proteins: Seed MV4;11 or SU-DHL-4 cells in 6-well plates (1×10⁶ cells/well), incubate overnight, treat with 0.05-0.5 μM Atuveciclib racemate for 4-24 hours. Lyse cells, extract proteins, separate by SDS-PAGE, transfer to PVDF membranes, and probe with antibodies against MYC, BCL2, MCL1, Cyclin D1, RNA Pol II (total and Ser2-phosphorylated), cleaved caspase-3, cleaved PARP, and GAPDH (loading control) [1, 2] 3. Apoptosis assay: Seed MV4;11 cells in 6-well plates (5×10⁵ cells/well), treat with 0.1-2 μM Atuveciclib racemate for 48 hours. Stain with Annexin V-FITC and PI, analyze apoptotic rate by flow cytometry [1, 2] 4. qPCR for MYC mRNA: Seed MV4;11 cells in 6-well plates (1×10⁶ cells/well), treat with 0.05-0.5 μM Atuveciclib racemate for 4 hours. Extract total RNA, synthesize cDNA, perform qPCR with primers for MYC and GAPDH (internal control) [2] 5. Clonogenic and CFU-L assays: For clonogenic assay: Seed MV4;11 or OCI-AML3 cells (1×10³ cells/well) in 6-well plates, treat with 0.02-0.2 μM Atuveciclib racemate, incubate for 14 days (medium changed every 3 days), fix with methanol, stain with crystal violet, count colonies. For CFU-L assay: Isolate primary AML cells from patient samples, seed in methylcellulose medium with 0.02-0.05 μM Atuveciclib racemate, incubate for 10 days, count CFU-L colonies [1] |
| Animal Protocol |
Immunocompromized NOD/Shi-scid/IL-2Rγ null (NOG) mice xenografted with patient-derived ATL cells and in vivo pharmacokinetic in rats 1. MV4;11 AML subcutaneous xenograft model: Female NOD-SCID mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ MV4;11 cells suspended in 0.2 mL PBS:Matrigel (1:1) into the right flank. When tumors reached 100-150 mm³, Atuveciclib racemate was dissolved in 0.5% methylcellulose to prepare 3 mg/mL and 6 mg/mL solutions. Mice were treated with oral gavage of 30 mg/kg or 60 mg/kg once daily for 21 days; vehicle group received 0.5% methylcellulose. Tumor volume (length × width² / 2) and body weight were measured every 2 days. At study end, tumors were dissected for Western blot and immunohistochemistry [1, 2] 2. MV4;11 AML orthotopic model: Female NOD-SCID mice (6-8 weeks old, n=10 per group) were intravenously injected with 1×10⁶ MV4;11 cells via the tail vein. Seven days post-inoculation, Atuveciclib racemate (60 mg/kg, oral gavage, once daily) or vehicle was administered for 28 days. Body weight was measured every 2 days, and survival was recorded for 60 days. At study end, bone marrow was collected to analyze leukemic cell infiltration by flow cytometry [1] 3. SU-DHL-4 DLBCL and MDA-MB-231 TNBC xenograft models: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ SU-DHL-4 or MDA-MB-231 cells (0.2 mL PBS:Matrigel=1:1). When tumors reached 100-150 mm³, Atuveciclib racemate (60 mg/kg, oral gavage, once daily) or vehicle was given for 21 days. Tumor volume and body weight were monitored every 2 days, and tumors were collected for immunohistochemistry [2] |
| ADME/Pharmacokinetics |
1. Oral absorption and bioavailability: The oral bioavailability of the artevicide racemic mixture was 45% in mice (single oral dose of 60 mg/kg) and 42% in rats (single oral dose of 30 mg/kg). The peak plasma concentration (Cₘₐₓ) was 4.2 μM (mice, 60 mg/kg), reached at 1 hour (Tₘₐₓ) [1, 2]
2. Plasma protein binding: The in vitro human plasma protein binding rate was 93-95% (concentration range: 0.1-10 μM) [1, 2] 3. Half-life and tissue distribution: The terminal elimination half-life (t₁/₂) was 5.8 hours in mice and 6.2 hours in rats. It is widely distributed in tumor tissues (in MV4;11 xenografts, the tumor/plasma ratio was 2.3 at 4 hours) and moderately penetrates the liver, spleen, and bone marrow (bone marrow/plasma ratio was 1.5) [1] 4. Metabolism: The artuvizine racemic is mainly metabolized in the liver via cytochrome P450 3A4 (CYP3A4)-mediated oxidative metabolism. No major active metabolite was detected (IC₅₀ > 10 μM for CDK9) [2] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: The racemic mixture of Atuveciclib showed low cytotoxicity to normal human cells, with CC₅₀ > 15 μM in peripheral blood mononuclear cells (PBMCs) and bone marrow mesenchymal stem cells (BMSCs), and CC₅₀ > 20 μM in normal hepatocytes [1, 2]. 2. In vivo safety: In 21–28 day xenograft studies, the racemic mixture of Atuveciclib (30–60 mg/kg, orally) did not cause significant changes in body weight (mean weight loss < 4%), food intake, or mortality. Serum ALT, AST, BUN, and creatinine levels were all within the normal range. Histopathological examination of the liver, kidneys, heart, lungs and bone marrow revealed no drug-related lesions [1, 2]
3. Acute toxicity: The median lethal dose (LD₅₀) of oral administration of atuveciclib racemate to mice was > 200 mg/kg [2] 4. Hematological safety: After 28 consecutive days of treatment with 60 mg/kg atuveciclib racemate, no significant inhibition of normal hematopoietic function was observed in mice; peripheral blood mononuclear cell (PBMC) count and granulocyte-macrophage colony-forming unit (CFU-GM) formation were not affected [1] |
| References |
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| Additional Infomation |
1. Chemical and structural properties: The artuviceri racemic mixture is a 1:1 mixture of the S- and R-enantiomers of artuviceri (BAY-1143572). The active S-enantiomer (IC₅₀ = 0.007 μM) exhibits approximately 100-fold higher CDK9 inhibitory activity than the R-enantiomer (IC₅₀ = 0.8 μM). The CDK9 inhibitory activity of the racemic mixture (IC₅₀ = 0.01 μM) reflects the contribution of the S-enantiomer [1, 2]. 2. Mechanism of action: The artuviceri racemic mixture selectively binds to the ATP-binding pocket of CDK9, inhibiting the kinase activity of the PTEFb complex (CDK9/cyclin T1). This drug blocks RNA polymerase II Ser2 phosphorylation, inhibiting the transcriptional elongation of short-lived oncogenes (MYC, BCL2, MCL1, Cyclin D1) that are crucial for tumor cell survival and proliferation. Downregulation of these oncogenes induces endogenous apoptosis and inhibits tumor growth [1, 2]. 3. Clinical Status and Therapeutic Potential: Atuveciclib racemic mixture is a first-in-class CDK9/PTEFb inhibitor currently undergoing Phase I clinical trials for the treatment of advanced hematologic malignancies (acute myeloid leukemia, lymphoma) and solid tumors (triple-negative breast cancer, colorectal cancer). In preclinical models of MYC-driven tumors, the drug has shown promising efficacy and holds promise for overcoming resistance to conventional chemotherapy and targeted therapies [1, 2]. 4. Preclinical Advantages: Compared to non-selective CDK inhibitors, the racemic mixture of Atuveciclib exhibits high selectivity for CDK9, minimizing off-target effects on other CDKs (CDK1/2/4/6) that regulate cell cycle progression, thereby reducing hematological and gastrointestinal toxicity. Its favorable oral bioavailability and pharmacokinetic characteristics support its clinical development [1, 2].
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| Molecular Formula |
C18H18FN5O2S
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| Molecular Weight |
387.43
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| Exact Mass |
387.116
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| Elemental Analysis |
C, 55.80; H, 4.68; F, 4.90; N, 18.08; O, 8.26; S, 8.27
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| CAS # |
1414943-88-6
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| Related CAS # |
Atuveciclib;2923012-24-0;Atuveciclib S-Enantiomer;2250279-81-1
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| PubChem CID |
71618220
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
589.9±60.0 °C at 760 mmHg
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| Flash Point |
310.6±32.9 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.639
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| LogP |
1.03
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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 |
27
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| Complexity |
588
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=CC(OC)=C1C1N=C(NC2=CC(=CC=C2)CS(C)(=C=N)=O)N=CN=1)F
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| InChi Key |
ACWKGTGIJRCOOM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H18FN5O2S/c1-26-16-9-13(19)6-7-15(16)17-21-11-22-18(24-17)23-14-5-3-4-12(8-14)10-27(2,20)25/h3-9,11,20H,10H2,1-2H3,(H,21,22,23,24)
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| Chemical Name |
4-(4-fluoro-2-methoxyphenyl)-N-[3-[(methylsulfonimidoyl)methyl]phenyl]-1,3,5-triazin-2-amine
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.45 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5811 mL | 12.9056 mL | 25.8111 mL | |
| 5 mM | 0.5162 mL | 2.5811 mL | 5.1622 mL | |
| 10 mM | 0.2581 mL | 1.2906 mL | 2.5811 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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