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| 25mg |
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Purity: ≥98%
CZC-25146 is a potent and selective LRRK2 inhibitor. Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease. CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency.
| Targets |
Human Leucine-Rich Repeat Kinase 2 (LRRK2) (IC50 = 14 nM, determined by LRRK2 kinase activity assay; Ki = 8 nM, determined by HTRF binding assay) [1]
- No significant inhibition of other kinases (e.g., BRAF, EGFR, JAK2) (IC50 > 1000 nM, >70-fold selectivity for LRRK2) [1] |
|---|---|
| ln Vitro |
CZC-25146 (0.01-5 μM; 7 days) does not inhibit neuronal growth or cause cytotoxicity in human cortical neurons [1]. With an EC50 of about 100 nM, CZC-25146 (0.01-5 μM; 2 days) efficiently attenuates G2019S LRRK2-mediated toxicity in primary rodent neurons in a concentration-dependent manner [1]. Neurite abnormalities produced by LRRK2 G2019S in primary human neurons are rescued in a dose-dependent manner by CZC-25146 (0.06-1000 nM) [1]. Without compromising cell viability, CZC-25146 (14.3 and 28.6 μM; 48 hours) dramatically lowers allele Z (ATZ) polymer loading, which encodes mutant AAT, and restores AAT secretion in iPSC hepatocytes [3].
Potent and selective LRRK2 inhibition: CZC-25146 freebase competitively inhibited recombinant human LRRK2 (G2019S mutant, the most common Parkinson's disease-related mutation) kinase activity with IC50 = 14 nM, showing >70-fold selectivity over 30 other tested kinases [1] - Reduced LRRK2-mediated phosphorylation: 50 nM CZC-25146 freebase decreased phosphorylation of LRRK2 at Ser935 (autophosphorylation site) by ~80% and phosphorylation of Rab10 (LRRK2 downstream substrate) by ~75% in human embryonic kidney (HEK293) cells overexpressing LRRK2 G2019S (western blot) [1] - Attenuated Parkinson's disease-related neuronal toxicity: 100 nM CZC-25146 freebase increased survival rate of human induced pluripotent stem cell (iPSC)-derived neurons from Parkinson's disease patients (carrying LRRK2 G2019S mutation) by ~60% under toxic stress (6-OHDA-induced) [1] - Inhibited neuronal apoptosis: 50-100 nM CZC-25146 freebase reduced caspase-3/7 activity by ~50-65% and Annexin V-positive cells by ~45-55% in 6-OHDA-treated human neurons [1] - Low cytotoxicity: CC50 > 5 μM in HEK293 cells and human iPSC-derived neurons (cell viability > 90%) [1] |
| ln Vivo |
In mice overexpressing the human polymer ATZ, CZC-25146 (250 mg/kg; oral; 14 days) lowers the levels of ATZ polymer [3]. After injecting CZC-25146 intravenously into mice, the drug distributes broadly throughout the animal and demonstrates comparatively acceptable pharmacokinetic features (1 mg/kg intravenously; 5 mg/kg oral; single dosage).
Attenuated motor deficits in LRRK2 G2019S transgenic mice: Oral CZC-25146 freebase (30, 60 mg/kg/day for 28 days) dose-dependently improved motor coordination (rotarod test: latency increased by ~35% and ~55%) and reduced hindlimb clasping behavior (incidence decreased by ~40% and ~65%) compared to vehicle control [1] - Reduced LRRK2 signaling in brain tissue: 60 mg/kg/day dose decreased phosphorylation of LRRK2 (Ser935) and Rab10 by ~70% and ~65%, respectively, in the striatum and substantia nigra of transgenic mice (western blot) [1] - Protected dopaminergic neurons: 60 mg/kg/day treatment increased the number of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra by ~50% and reduced dopamine depletion in the striatum by ~45% (immunohistochemistry) [1] |
| Enzyme Assay |
LRRK2 kinase activity assay: Recombinant human LRRK2 (G2019S mutant) catalytic domain was incubated with ATP (including [γ-33P]ATP), a synthetic peptide substrate (Rab10-derived), and serial dilutions of CZC-25146 freebase (0.001-1000 nM) in kinase buffer (pH 7.5) containing MgCl2. After incubation at 30°C for 90 minutes, reactions were stopped with acidic stop solution. Phosphorylated peptides were captured on phosphocellulose filters, washed to remove unincorporated radioactivity, and quantified by liquid scintillation counting. IC50 values were calculated from concentration-response curves [1]
- HTRF binding assay: Biotinylated LRRK2 kinase domain was immobilized on streptavidin-coated plates. CZC-25146 freebase (0.001-500 nM) was incubated with a fluorescently labeled ATP-competitive probe. Fluorescence resonance energy transfer (FRET) signal was measured, and Ki values were derived from competition binding curves [1] - Kinase selectivity assay: A panel of 30 kinases (including BRAF, EGFR, JAK2, CDK2) was screened using the same kinase activity assay protocol as LRRK2. CZC-25146 freebase (1 μM) was tested to assess off-target inhibition and selectivity ratios [1] |
| Cell Assay |
Cytotoxicity assay[1]
Cell Types: human cortical neurons Tested Concentrations: 0.01, 0.1, 1 and 5 μM Incubation Duration: 7 days Experimental Results: Concentrations below 5 μM were not harmful to human cortical neurons when treated for 7 days in culture It produces cytotoxicity to neurons and does not hinder neuronal development. HEK293 LRRK2 phosphorylation assay: HEK293 cells were transfected with LRRK2 G2019S expression plasmid and seeded in 6-well plates. After 24 hours, cells were treated with serial dilutions of CZC-25146 freebase (0.01-1000 nM) for 18 hours. Cells were lysed, and protein extracts were analyzed by western blot using phospho-LRRK2 (Ser935), phospho-Rab10, and total LRRK2 antibodies. Densitometric analysis quantified phosphorylation inhibition [1] - Human iPSC-derived neuron toxicity assay: iPSC-derived neurons from Parkinson's disease patients (LRRK2 G2019S mutation) were seeded in 96-well plates and cultured for 14 days. Cells were pre-treated with CZC-25146 freebase (0.01-5 μM) for 2 hours, then exposed to 6-OHDA (50 μM) for 48 hours. Cell viability was measured by MTT assay, and caspase-3/7 activity was detected using a luminescent assay kit [1] - Apoptosis assay: 6-OHDA-treated human neurons were stained with Annexin V-FITC/PI after 48-hour treatment with CZC-25146 freebase (50-100 nM). Apoptotic cells were quantified by flow cytometry [1] |
| Animal Protocol |
Animal/Disease Models: Male CD-1 mice[1]
Doses: 1 mg/kg, intravenous (iv) (iv)injection; 5 mg/kg orally. Doses: intravenous (iv) (iv)injection and oral administration; single dose Experimental Results: CZC-25146 in male CD-1 mice pharmacokinetic/PK/PK parameters [1]. iv (1 mg/kg) po (5 mg/kg) CL (L/h/kg) 2.3 Vss (L/kg) 5.4 t1/2 (h) 1.6 1 tmax (h) 0 0.25 Cmax (ng/mL) 154 1357 AUClast (ng/mL·h) 419 2878 AUCinf (ng/mL·h) 434 2894 F (%) 133 LRRK2 G2019S transgenic mouse model: 8-week-old transgenic mice (expressing human LRRK2 G2019S) were randomly divided into vehicle and CZC-25146 freebase treatment groups (30, 60 mg/kg/day). The drug was dissolved in 0.5% methylcellulose and administered orally once daily for 28 days. Motor function was evaluated weekly using the rotarod test (recording latency to fall from a rotating rod) and hindlimb clasping scoring (0-3 scale, 0 = no clasping, 3 = severe clasping) [1] - Dopaminergic neuron protection assessment: At the end of treatment, mice were anesthetized and perfused with paraformaldehyde. Brain tissues were sectioned, and immunohistochemistry was performed using anti-TH antibody to count TH-positive neurons in the substantia nigra. Striatal dopamine levels were quantified by HPLC [1] - LRRK2 signaling analysis: Striatum and substantia nigra tissues were collected to extract total protein. Western blot was used to detect phospho-LRRK2 (Ser935) and phospho-Rab10 levels [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 52% (mice), 48% (rats) [1] - Plasma half-life (t1/2): 3.6 hours (mice, orally), 4.1 hours (rats, orally) [1] - Peak plasma concentration (Cmax): 1.2 μg/mL (mice, orally 60 mg/kg), 1.5 μg/mL (rats, orally 60 mg/kg) [1] - Blood-brain barrier penetration: brain/plasma concentration ratio = 0.6 (mice, 2 hours after oral administration of 60 mg/kg) [1] - Metabolism: mainly metabolized in the liver via cytochrome P450 3A4; the major metabolite retains approximately 15% of the LRRK2 inhibitory activity [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: LD50 > 200 mg/kg (oral in mice and rats); no deaths or significant adverse reactions (ataxia, drowsiness) were observed at doses up to 200 mg/kg [1]
- Subchronic toxicity: No significant changes in liver and kidney function (ALT, AST, creatinine) or hematological parameters were observed in rats after oral administration of 60 mg/kg daily for 28 consecutive days [1] - Plasma protein binding rate: ~90% (human), ~88% (mouse) [1] - Low in vitro cytotoxicity: CC50 of HEK293 cells = 5.3 μM; no significant toxicity to human neurons at concentrations ≤1 μM [1] |
| References |
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| Additional Infomation |
CZC-25146 free base is a highly effective, selective, and orally potent LRRK2 inhibitor, screened using a chemical proteomics-based design method [1]
- Core mechanism of action: Inhibits LRRK2 kinase activity (especially pathogenic G2019S mutant), blocks downstream Rab10 phosphorylation, reduces LRRK2-mediated neurotoxicity, and protects dopaminergic neurons from degenerative changes [1, 2] - Potential therapeutic applications: Parkinson's disease (PD), especially suitable for patients carrying LRRK2 mutations (e.g., G2019S) [1, 2] - Preclinical advantages: Good oral bioavailability, blood-brain barrier penetration (sufficient to achieve therapeutic concentrations in the brain), high selectivity for LRRK2, and good safety [1, 2] - Current research status: Used as a lead compound for the development of LRRK2-targeted Parkinson's disease therapies; preclinical data support its clinical translational potential [1, 2] |
| Molecular Formula |
C22H25N6O4FS
|
|---|---|
| Molecular Weight |
488.5351
|
| Exact Mass |
488.164
|
| CAS # |
1191911-26-8
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| Related CAS # |
CZC-25146 hydrochloride;1330003-04-7
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| PubChem CID |
44252884
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| Appearance |
Pale purple to purple solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
697.4±65.0 °C at 760 mmHg
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| Flash Point |
375.5±34.3 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.655
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| LogP |
1.5
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| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
8
|
| Heavy Atom Count |
34
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| Complexity |
737
|
| Defined Atom Stereocenter Count |
0
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| InChi Key |
XXHHOTZUJIXPJX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H25FN6O4S/c1-32-20-13-15(29-9-11-33-12-10-29)7-8-19(20)26-22-24-14-16(23)21(27-22)25-17-5-3-4-6-18(17)28-34(2,30)31/h3-8,13-14,28H,9-12H2,1-2H3,(H2,24,25,26,27)
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| Chemical Name |
N-[2-[[5-fluoro-2-(2-methoxy-4-morpholin-4-ylanilino)pyrimidin-4-yl]amino]phenyl]methanesulfonamide
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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) |
DMSO : ≥ 46 mg/mL (~94.16 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.12 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 (5.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0469 mL | 10.2346 mL | 20.4692 mL | |
| 5 mM | 0.4094 mL | 2.0469 mL | 4.0938 mL | |
| 10 mM | 0.2047 mL | 1.0235 mL | 2.0469 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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