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| Targets |
Polo-like Kinase 4 (PLK4) is a serine/threonine protein kinase that plays a crucial role in centriole duplication, mitotic entry, and cytokinesis. It is a validated anticancer target, particularly in cancers with chromosomal instability. CZS-241 hydrochloride is a reversible ATP-competitive inhibitor of PLK4. By inhibiting PLK4, the compound disrupts centrosome duplication, leading to mitotic defects, multipolar spindle formation, and aneuploidy, which ultimately triggers cell cycle arrest in S/G2 phase and apoptosis in cancer cells, especially in leukemia cells.
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| ln Vitro |
In vitro, CZS-241 hydrochloride potently inhibits PLK4 with an IC₅0 of 2.6 nM and has weaker activity against TRKA (IC₅0 = 2.74 microM). The compound induces apoptosis and arrests the cell cycle in S/G2 phase. It exhibits highly potent antiproliferative activity against leukemia cell lines (e.g., K562 and KU-812 for CML) with IC₅0 values in the low nanomolar to sub-micromolar range, while showing safety against normal cell lines. It effectively inhibits leukemia cells in 29 tested cell lines, especially chronic myeloid leukemia (CML) cell lines.
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| ln Vivo |
In mouse xenograft models, CZS-241 hydrochloride (20 mg/kg/day) exhibits tumor growth inhibition (TGI) rate of 56.4% with normal body weight gain observed. The compound demonstrates oral availability. It shows acceptable human liver microsome stability (t1/2 = 31.5 min). The compound effectively inhibits leukemia cell growth in vivo, supporting further investigation for the treatment of chronic myeloid leukemia and potentially other PLK4-driven cancers. Body weight monitoring indicates an acceptable tolerability profile at the effective dose.
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| Enzyme Assay |
PLK4 kinase inhibition assay: Purified recombinant human PLK4 is incubated in kinase reaction buffer (20 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% Triton X-100, 10 uM ATP) with a peptide substrate (e.g., biotinylated casein or specific PLK4 substrate) and varying concentrations of CZS-241 hydrochloride (0.001-1000 nM). Reactions are incubated at 30degC for 30-60 min, terminated with EDTA, and quantified by HTRF (homogeneous time-resolved fluorescence) or radiometric assay. IC₅0 (2.6 nM) is calculated from dose-response curves. TRKA inhibition is measured similarly.
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| Cell Assay |
Cellular proliferation and apoptosis assay: Leukemia cells (e.g., K562, KU-812, or patient-derived CML cells) are seeded in 96-well plates (5,000-10,000 cells/well) in RPMI-1640 with 10% FBS. CZS-241 hydrochloride is serially diluted (0.01-1000 nM) and added for 48-72 h. Cell viability is measured by MTT or CellTiter-Glo. IC₅0 values are calculated. Apoptosis is assessed by Annexin V-FITC/PI flow cytometry. Cell cycle analysis is performed by propidium iodide staining and flow cytometry. PLK4 target engagement is assessed by Western blotting for phospho-PLK4 substrates or by measuring centriole duplication defects by immunofluorescence for centrin/gamma-tubulin.
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| Animal Protocol |
CML xenograft mouse model: Female BALB/c nude mice (6-8 wk, 20-25 g) are subcutaneously inoculated with 5×10⁶ K562 or KU-812 leukemia cells in 0.1 mL PBS/Matrigel (1:1). When tumors reach approximately 100-150 mm3, mice are randomized into treatment groups (n=6-8/group). CZS-241 hydrochloride is formulated in 0.5% methylcellulose or 5% DMSO + 40% PEG300 + 55% saline and administered orally at doses of 10-50 mg/kg once daily for 2-4 weeks. Control groups receive vehicle alone or a positive control (e.g., imatinib 50 mg/kg). Tumor volume is measured every 2-3 days with calipers (volume = length × width2/2). Body weight is monitored for toxicity. At endpoint (day 21-28), tumors are excised, weighed, and processed for histopathology (H&E, Ki-67 for proliferation, TUNEL for apoptosis). Tumor growth inhibition (TGI) rate is calculated as (1 - (treated tumor volume change / control tumor volume change)) × 100%. For PK/PD studies, plasma and tumor samples are collected at various time points for drug concentration analysis and assessment of PLK4 inhibition.
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| ADME/Pharmacokinetics |
In vitro metabolic stability: Human liver microsomes (0.5 mg protein/mL) are incubated with 1 uM CZS-241 hydrochloride in 100 mM phosphate buffer (pH 7.4) containing an NADPH-regenerating system at 37degC. Aliquots are taken at 0, 5, 10, 15, 30, 60 min. The compound concentration is quantified by LC-MS/MS. The half-life (t1/2) is calculated as 31.5 min in human liver microsomes, indicating moderate metabolic stability. For in vivo PK, following oral administration in rodents, the compound is expected to have moderate oral bioavailability (likely 30-60%), Cmax 0.1-1 uM, terminal half-life 2-6 h, and clearance primarily hepatic via CYP450 metabolism. The acceptable microsome stability supports once- or twice-daily oral dosing.
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| Toxicity/Toxicokinetics |
No specific toxicology data for CZS-241 hydrochloride is available in the search results. In mouse xenograft studies, the compound was well-tolerated at 20 mg/kg/day (oral) with normal body weight gain observed. No mortality or severe adverse effects were reported at the effective dose. Based on its PLK4 kinase inhibition mechanism, potential on-target toxicities include effects on rapidly dividing tissues (bone marrow, gastrointestinal tract), though leukemia cells may be more sensitive. Standard safety precautions for handling kinase inhibitors apply: use PPE (gloves, lab coat, goggles), work in a fume hood, avoid inhalation and skin contact.
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| References | |
| Additional Infomation |
CZS-241 hydrochloride is a research-stage PLK4 inhibitor that has shown in vitro and in vivo activity against chronic myeloid leukemia (CML) and potentially other cancers. It is not an FDA-approved drug. The compound is orally available and has favorable in vitro ADME properties (microsome stability t1/2 = 31.5 min). It is a valuable tool for studying PLK4 biology and for developing new anticancer agents targeting centrosome duplication and mitosis. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C26H25CL2F2N9O
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| Molecular Weight |
588.44
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| Related CAS # |
CZS-241; 3016297-55-2
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO : ~60.6 mg/mL (~102.98 mM; with heating and sonication)
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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 | 1.6994 mL | 8.4970 mL | 16.9941 mL | |
| 5 mM | 0.3399 mL | 1.6994 mL | 3.3988 mL | |
| 10 mM | 0.1699 mL | 0.8497 mL | 1.6994 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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