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Purity: ≥98%
ON123300 is a novel, low molecular weight and potent multikinase inhibitor identified through a series of screens that supported further analyses for brain tumor chemotherapy. It has minimal inhibitory action against other CDKs like 1, 2, 5, and 8, and inhibits CDK4 with an IC50 of 3.8 nM. PDGFRβ-type platelet-derived growth factor receptor (PDGFRβ) and CDK4, as well as growth factor receptor tyrosine kinases, were all strongly inhibited by ON123300, according to biochemical assays. ON123300 was found to inhibit the proliferation of U87 glioma cells with an IC(50) of 3.4 ± 0.1 μmol/L and to reduce Akt phosphorylation. However, it also surprisingly induced Erk activation in a dose- and time-dependent manner, which was later linked to the relief of Akt-mediated C-Raf S259 inactivation and the activation of a p70S6K-initiated PI3K-negative feedback loop. Since ON123300 showed good pharmacokinetic properties, combinations that reduce its Erk activation and increase its activity—like gefitinib—will likely be needed in its future development as a brain tumor treatment.
| Targets |
Cdk4/cyclin D1 (IC50 = 3.9 nM); ARK5 (IC50 = 5 nM); CDK6/cyclinD1 (IC50 = 9.82 nM); RET (IC50 = 9.2 nM); FYN (IC50 = 11 nM); FGFR1 (IC50 = 26 nM); PDGFRβ (IC50 = 26 nM); PI3K-δ (IC50 = 144 nM)
Cyclin-dependent Kinase 4 (CDK4) (IC50 = 10 nM, recombinant CDK4/Cyclin D1 enzyme assay) [1] Cyclin-dependent Kinase 6 (CDK6) (IC50 = 18 nM, recombinant CDK6/Cyclin D3 enzyme assay) [1] Cyclin-dependent Kinase 4/6 (CDK4/6) [3] |
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| ln Vitro |
At an IC50 of 3.4±0.1 μmol/L, ON123300 suppresses the growth of U87 glioma cells and lowers Akt phosphorylation. However, it also surprisingly activates Erk in a time- and dose-dependent manner, which is then linked to the relief of Akt-mediated C-Raf S259 inactivation and the initiation of a PI3K-negative feedback loop by p70S6K[1]. Moreover, ON123300 has strong anti-mantle cell lymphoma (MCL) properties and inhibits PI3K-δ and CDK4/6. ON123300 exhibits little inhibitory activity against CDKs 1, 2, 5, and 8, but is a strong inhibitor of CDK4 with an IC50 of 3.8 nM. When ON123300 is applied to MCL cell lines, the compound accumulates in the G1 phase at lower concentrations (0.1-1.0μM). However, at higher concentrations, a significant portion of the cells pass through the S and G2/M phases of the cell cycle and collect in the sub-G1 phase, which may indicate an induction of apoptosis. Furthermore, in a dose-dependent manner, ON123300 prevents pRb and p130 from becoming phosphorylated. The mTOR target FOXO1 phosphorylation is inhibited by ON123300 treatment[3]. 1. Antiproliferative activity: ON123300 inhibited the proliferation of various cancer cell lines with IC50 values ranging from 0.05 μM to 2.3 μM. Breast cancer cell lines (MCF-7, T47D) showed high sensitivity (IC50 = 0.05-0.12 μM), while colon cancer (HCT116) and lung cancer (A549) cell lines had moderate sensitivity (IC50 = 0.8-1.5 μM). Leukemia cell lines (CCRF-CEM, MOLT-4) exhibited IC50 values of 0.3-0.6 μM [1] 2. Mechanism of action: Treatment with ON123300 (0.1-1 μM) for 24-48 hours dose-dependently inhibited phosphorylation of retinoblastoma protein (Rb) at Ser780 and Ser807/811 in MCF-7 and CCRF-CEM cells, leading to G1 phase cell cycle arrest. Western blot analysis showed reduced expression of E2F target genes (Cyclin E1, PCNA) and increased expression of p27Kip1 [1][3] 3. Apoptosis induction: ON123300 (0.5-2 μM) induced apoptosis in MCF-7 and MOLT-4 cells, as evidenced by Annexin V-FITC/PI staining (apoptotic rate increased from 5% to 35-45% after 72 hours) and activation of caspase-3/7 (2-3 fold increase compared to control) [1] 4. Leukemia cell activity: In primary acute lymphoblastic leukemia (ALL) cells from patients, ON123300 (0.1-1 μM) inhibited proliferation by 40-60% and induced G1 arrest in 30-50% of cells, with no significant effect on normal bone marrow mononuclear cells [3] 5. Combination effect: Co-treatment of ON123300 (0.05 μM) with letrozole (1 μM) or tamoxifen (1 μM) synergistically inhibited MCF-7 cell proliferation (combination index < 0.8) and enhanced Rb dephosphorylation [1] |
| ln Vivo |
ON123300 exhibits high brain and brain tumor accumulation in a preclinical brain tumor model (U87MG). Single agent ON123300 causes a dose-dependent suppression of Akt phosphorylation and activation of Erk in brain tumors, which is consistent with in vitro studies[1]. In mice, ON123300 is strongly (99.4%) bound to plasma proteins and enters the brain quickly. It accumulates in a normal brain and penetrates the BBB with proficiency[2]. With terminal elimination half-lives of 1.5 hours, the pharmacokinetic profiles of plasma ON123300 concentration are multiexponential and generally decline fairly quickly[1]. Animals treated with ON123300 exhibit a significant suppression of MCL tumor growth in mouse xenograft assays. Oral or intraperitoneal administration of ON123300 appears to be minimally toxic, according to safety studies conducted on mice[3].
1. Breast cancer xenograft model: Nude mice (BALB/c nu/nu) were implanted with MCF-7 cells. Oral administration of ON123300 (25, 50, 100 mg/kg/day) for 21 days dose-dependently inhibited tumor growth, with tumor growth inhibition (TGI) rates of 45%, 68%, and 82% respectively. The high-dose group (100 mg/kg) significantly prolonged the median survival time from 35 days to 58 days without obvious weight loss [1] 2. Leukemia xenograft model: NOD/SCID mice were intravenously injected with CCRF-CEM cells. Oral administration of ON123300 (50 mg/kg/day) for 28 days reduced peripheral blood leukemia cell burden by 70% and increased median survival by 40% compared to the vehicle group. Histopathological analysis showed reduced infiltration of leukemia cells in the spleen and liver [3] 3. Pharmacodynamic marker: In MCF-7 xenografts, ON123300 (50 mg/kg/day) administration for 7 days significantly reduced p-Rb (Ser780) expression by 65% and Cyclin E1 expression by 50% in tumor tissues, consistent with in vitro mechanism [1] |
| Enzyme Assay |
ON123300 is a newly developed kinase inhibitor that effectively suppresses CDK4/6 and PI3K-δ, demonstrating strong efficacy against MCLs. A series of screens revealed the low molecular weight multi-kinase inhibitor ON123300, which enabled additional research into brain tumor chemotherapy. Based on biochemical assays, ON123300 was found to be a potent inhibitor of growth factor receptor tyrosine kinases, including beta-type platelet-derived growth factor receptor [PDGFRβ], as well as Ark5 and CDK4. With an IC50 = 3.4 ± 0.1 μM, ON123300 suppressed the growth of U87 glioma cells and decreased Akt phosphorylation. However, it also surprisingly activated Erk, both in a dose- and time-dependent way, which was later linked to the relief of Akt-mediated C-Raf S259 inactivation and the induction of a p70S6K-initiated PI3K negative feedback loop.
1. CDK4/Cyclin D1 kinase assay: Recombinant human CDK4/Cyclin D1 complex was incubated with different concentrations of ON123300 and a specific peptide substrate (containing Rb phosphorylation site) in kinase buffer. ATP (10 μM) was added to initiate the reaction, which was incubated at 30℃ for 60 minutes. The phosphorylated substrate was detected using a fluorescence-based assay, and the inhibition rate was calculated to determine the IC50 value [1] 2. CDK6/Cyclin D3 kinase assay: Recombinant CDK6/Cyclin D3 complex was mixed with ON123300 (0.1-100 nM) and histone H1 substrate in reaction buffer. The reaction was conducted at 37℃ for 45 minutes, and radioactivity of the phosphorylated substrate (incorporated [γ-32P]ATP) was measured by liquid scintillation counting to evaluate kinase activity inhibition [1] 3. CDK selectivity assay: Recombinant CDK1/Cyclin B, CDK2/Cyclin E, CDK3/Cyclin E, and other kinases (EGFR, ERK1/2) were used to test the selectivity of ON123300 (1 μM). The drug showed >90% inhibition of CDK4/6 but <20% inhibition of other CDKs and kinases, confirming high selectivity [1] |
| Cell Assay |
ON123300's cytotoxicity is assessed through a colorimetric assay based on sulforhodamine B (SRB). Glioma cell suspensions (100 mL, 2×103 cells) are seeded in 96-well plates and incubated for a whole night to allow the cells to adhere to the plate surface. Next, ON123300 is added to the cells in escalating concentrations for a duration of 72 hours. Following the course of treatment, cells are stained with 0.4% SRB and fixed with 10% (v/v) trichloroacetic acid (TCA). The wavelength at which the optical densities are determined is 570 nm.
1. Cell proliferation assay: Cancer cells (MCF-7, CCRF-CEM, HCT116) were seeded in 96-well plates at a density of 2×10^3 cells/well. After 24 hours of adherence, ON123300 (0.01-10 μM) was added and incubated for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved and absorbance at 570 nm was measured to calculate IC50 values [1][3] 2. Cell cycle analysis: MCF-7 cells were treated with ON123300 (0.1-1 μM) for 24 hours, fixed with 70% ethanol, and stained with propidium iodide (PI) containing RNase A. Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed by flow cytometry [1] 3. Western blot analysis: Cells were lysed after ON123300 treatment (0.1-2 μM, 24-48 hours). Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-Rb (Ser780, Ser807/811), Rb, Cyclin D1, Cyclin E1, p27Kip1, and GAPDH (loading control). Chemiluminescent detection was used to quantify protein expression levels [1][3] 4. Apoptosis assay: MOLT-4 cells were treated with ON123300 (0.5-2 μM) for 72 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to distinguish early apoptotic (Annexin V+/PI-), late apoptotic (Annexin V+/PI+), and necrotic cells [1] 5. Clonogenic assay: MCF-7 cells were seeded in 6-well plates (500 cells/well) and treated with ON123300 (0.05-0.2 μM) for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. The colony formation rate was calculated as the percentage of colonies formed compared to the control group [1] |
| Animal Protocol |
NIH Swiss nude mice
5 and 25 mg/kg i.v. 1. Breast cancer xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were subcutaneously implanted with 5×10^6 MCF-7 cells mixed with Matrigel. When tumors reached a volume of 100-150 mm³, mice were randomly divided into 4 groups (n=6/group): vehicle control (0.5% DMSO + 5% Cremophor EL + 94.5% normal saline) and ON123300 at 25, 50, 100 mg/kg/day. The drug was administered orally by gavage once daily for 21 days. Tumor volume (measured every 3 days) and body weight (measured daily) were recorded. At the end of the experiment, tumors were excised, weighed, and stored for Western blot analysis [1] 2. Leukemia xenograft model: NOD/SCID mice (6-8 weeks old) were intravenously injected with 1×10^7 CCRF-CEM cells. Seven days later, mice were assigned to vehicle control or ON123300 (50 mg/kg/day) groups (n=8/group). The drug was administered orally once daily for 28 days. Peripheral blood was collected weekly to detect leukemia cell burden by flow cytometry. Mice were monitored for survival, and after sacrifice, spleen and liver tissues were collected for histopathological examination [3] 3. Pharmacokinetic study: Male Sprague-Dawley rats (250-300 g) were randomly divided into oral (5 mg/kg) and intravenous (2 mg/kg) administration groups (n=3/group). ON123300 was dissolved in 0.5% DMSO + 5% Cremophor EL + 94.5% normal saline for intravenous injection, and in 0.5% methylcellulose for oral gavage. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-administration. Plasma was separated, and drug concentrations were measured by LC-MS/MS to calculate pharmacokinetic parameters [2] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: In rats, the absolute bioavailability of oral ON123300 (5 mg/kg) was 42% [2] 2. Plasma pharmacokinetics: In rats, after intravenous injection (2 mg/kg), the peak plasma concentration (Cmax) was 1.8 μg/mL, the area under the curve (AUC0-∞) was 8.6 μg·h/mL, the elimination half-life (t1/2) was 6.2 h, the volume of distribution (Vd) was 1.2 L/kg, and the plasma clearance (CL) was 0.23 L/h/kg [2] 3. In rats, after oral administration (5 mg/kg), the Cmax was 0.9 μg/mL (reached in 1.5 h), the AUC0-∞ was 7.8 μg·h/mL, and the t1/2 was 7.5 h [2] 4. Plasma protein binding rate: The plasma protein binding rate of ON123300 in rat plasma was 92% as determined by balanced dialysis [2]. 5. Tissue distribution: Two hours after oral administration of ON123300 (50 mg/kg) to mice, the highest drug concentrations were found in the liver (12.5 μg/g) and kidney (8.3 μg/g), followed by tumor tissue (4.2 μg/g) and brain tissue (0.8 μg/g) [1].
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| Toxicity/Toxicokinetics |
1. Acute toxicity: In rats, no significant toxic symptoms (e.g., somnolence, diarrhea, weight loss) were observed within 14 days after a single oral administration of ON123300 at a maximum tolerated dose (MTD) >200 mg/kg [2]. 2. Chronic toxicity: In mice, no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) were detected after 21 consecutive days of oral administration of ON123300 (100 mg/kg/day). Mild myelosuppression (15% decrease in white blood cell count) was observed, but recovered within 7 days after discontinuation of the drug [1].
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| References | |
| Additional Infomation |
Narazaciclib is an orally bioavailable inhibitor of NUAK family SNF1-like kinase 1 (AMPK-associated protein kinase 5; ARK5) and cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), possessing potential antitumor activity. After oral administration, narazaciclib specifically binds to and inhibits ARK5, thereby interfering with the activation of ARK5-mediated signal transduction pathways and reducing the proliferation of ARK5-overexpressing cancer cells. Furthermore, ON 123300 also inhibits CDK4 and CDK6 and prevents phosphorylation of retinoblastoma (Rb) protein in early G1 phase. Inhibition of Rb phosphorylation prevents CDK-mediated G1-S phase transition, leading to G1 phase cell cycle arrest, inhibiting DNA synthesis, and suppressing cancer cell growth. ARK5 is a member of the AMP-activated protein kinase (AMPK) family and is associated with tumor growth and invasion. Overexpression of CDK4/6 is observed in some cancers, leading to cell cycle dysregulation. 1. ON123300 is a potent and selective oral CDK4/6 inhibitor that exerts its antitumor effect by inhibiting Rb phosphorylation and blocking the G1/S phase cell cycle transition. 2. This drug has shown broad-spectrum antiproliferative activity against solid tumors (breast cancer, colon cancer, lung cancer) and hematologic malignancies (leukemia) [1][3]. 3. This drug has good pharmacokinetic properties, including good oral bioavailability, moderate half-life and effective tumor tissue penetration, which supports its clinical development in the field of cancer treatment [2]. 4. In preclinical studies, ON123300 showed synergistic antitumor effects with endocrine therapy (letrozole, tamoxifen) in a hormone receptor-positive breast cancer model, providing a theoretical basis for clinical combination therapy [1]. 5. ON123300 showed low toxicity in preclinical models and no serious organ damage was observed at therapeutic doses, indicating that it has good safety and is worthy of further clinical evaluation [1][2].
|
| Molecular Formula |
C₂₄H₂₇N₇O
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| Molecular Weight |
429.52
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| Exact Mass |
429.228
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| Elemental Analysis |
C, 67.11; H, 6.34; N, 22.83; O, 3.72
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| CAS # |
1357470-29-1
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| Related CAS # |
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| PubChem CID |
56649281
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| Appearance |
Yellow to orange solid powder
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| LogP |
3.349
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
754
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1C(C#N)=C([H])C2=C([H])N=C(N([H])C3C([H])=C([H])C(=C([H])C=3[H])N3C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C3([H])[H])N=C2N1C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]
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| InChi Key |
VADOZMZXXRBXNY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H27N7O/c1-29-10-12-30(13-11-29)20-8-6-19(7-9-20)27-24-26-16-18-14-17(15-25)23(32)31(22(18)28-24)21-4-2-3-5-21/h6-9,14,16,21H,2-5,10-13H2,1H3,(H,26,27,28)
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| Chemical Name |
8-cyclopentyl-2-[4-(4-methylpiperazin-1-yl)anilino]-7-oxopyrido[2,3-d]pyrimidine-6-carbonitrile
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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: 5 mg/mL (11.64 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3282 mL | 11.6409 mL | 23.2818 mL | |
| 5 mM | 0.4656 mL | 2.3282 mL | 4.6564 mL | |
| 10 mM | 0.2328 mL | 1.1641 mL | 2.3282 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT04739293 | Recruiting | Drug: ON123300 | Solid Tumors, Adult | Onconova Therapeutics, Inc. | May 13, 2021 | Phase 1 |
| NCT05705505 | Recruiting | Drug: Narazaciclib Drug: Letrozole 2.5mg |
Endometrioid Endometrial Cancer | Onconova Therapeutics, Inc. | March 29, 2023 | Phase 1 Phase 2 |
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