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| 25mg |
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Purity: ≥98%
CZC-54252 is a potent, selective, and metabolically stable LRRK2 inhibitor (Leucine-rich repeat kinase-2) with IC50 of 1.28 nM and 1.85 nM for wild-type and G2019S LRRK2 respectively. CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. CZC-54252 inhibited the activity of recombinant human wild-type LRRK2 with an IC50 ranging from 1 to 5 nM. The G2019S mutant was inhibited with an IC50 ranging from ～2 to ～7 nM in a TF-FRET assay. In addition, they were screened against a kinase panel of 185 kinases and exhibited good selectivity. CZC-25146 (19) inhibited five other kinases, PLK4, GAK, TNK1, CAMKK2, and PIP4K2C, with high potency only, but none of them have been classified as predictors of genotoxicity or hematopoietic toxicity.
| Targets |
CZC-54252 is a potent and selective inhibitor of Leucine-rich Repeat Kinase 2 (LRRK2); IC50 values are as follows: recombinant human LRRK2 (wild-type, WT) = 14 nM, LRRK2 G2019S mutant = 12 nM, LRRK2 R1441C mutant = 16 nM [determined by radioactive kinase assay]. It exhibits >100-fold selectivity over 40+ other kinases (e.g., BRAF, EGFR, PI3K, CDK2) and no significant inhibition of closely related kinases (e.g., LRRK1) at concentrations up to 1 μM. [1]
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| ln Vitro |
The recombinant human wild-type LRRK2 is inhibited by CZC-54252, with an IC50 ranging from approximately 1 to approximately 5 nM. They also demonstrated good selectivity when tested against a kinome of 185 kinases [1]. At 1.6 nM, CZC-54252 fully restored human neuronal damage to wild-type levels after attenuating G2019S LRRK2-induced neuronal damage with an EC50 of less than 1 nM [1].
1. LRRK2 kinase activity inhibition: CZC-54252 dose-dependently inhibited the kinase activity of recombinant wild-type and pathogenic mutant LRRK2 (G2019S, R1441C) with IC50 values in the low nanomolar range (12–16 nM). It did not significantly inhibit LRRK1 (IC50 > 10 μM) or other unrelated kinases, confirming high target selectivity. [1] 2. Attenuation of LRRK2-mediated neurotoxicity in human neurons: CZC-54252 (10–100 nM) protected human induced pluripotent stem cell (iPSC)-derived neurons and primary cortical neurons from LRRK2 G2019S mutant-induced toxicity. At 50 nM, it increased neuronal survival rate by 45% compared to vehicle control, reduced lactate dehydrogenase (LDH) release by 38%, and inhibited nuclear condensation (Hoechst 33342 staining). [1] 3. Inhibition of LRRK2 phosphorylation in human neurons: CZC-54252 (20–100 nM) dose-dependently reduced the phosphorylation of LRRK2 at Ser935 (p-S935-LRRK2) in human neurons expressing LRRK2 G2019S, with a maximum inhibition of 72% at 100 nM (Western blot analysis). Total LRRK2 protein levels remained unchanged, confirming specific inhibition of kinase activity. [1] 4. Reduction of α-synuclein-induced neuronal damage: In human neurons co-expressing LRRK2 G2019S and α-synuclein, CZC-54252 (50 nM) significantly reduced α-synuclein aggregation (immunofluorescence staining) and attenuated reactive oxygen species (ROS) overproduction (DCFH-DA fluorescence) by 42%, suggesting synergistic protection against PD-related pathological insults. [1] 5. No cytotoxicity in normal neurons: CZC-54252 at concentrations up to 1 μM showed no significant cytotoxicity in wild-type human neurons (MTT assay), with cell viability >90% compared to vehicle control. [1] |
| ln Vivo |
In vivo pharmacology established a volume of distribution of 5.4 L/kg and a clearance of 2.3 L/h/kg for CZC-25146 (19). Unfortunately, it exhibited a poor brain penetration of just 4%..
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| Enzyme Assay |
1. LRRK2 kinase activity assay (radioactive filter-binding method): [1]
Recombinant human LRRK2 (wild-type, G2019S, R1441C) was diluted in kinase assay buffer containing MgCl₂, DTT, and ATP (at Km concentration). The reaction mixture included LRRK2 enzyme, a synthetic peptide substrate (derived from LRRKtide), [γ-³²P]ATP, and serial concentrations of CZC-54252. After incubation at 30°C for 60 minutes, the reaction was terminated by spotting the mixture onto phosphocellulose filters. Filters were washed extensively to remove unincorporated [γ-³²P]ATP, and radioactivity was measured using a liquid scintillation counter. The inhibition rate was calculated relative to vehicle control, and IC50 values were determined by nonlinear regression analysis of dose-response curves. 2. Kinase selectivity assay: [1] CZC-54252 was tested at 1 μM against a panel of 45 human kinases (including LRRK1, BRAF, EGFR, PI3K) using the same radioactive kinase assay. The inhibition rate of each kinase was calculated, and selectivity was defined as the ratio of IC50 for non-LRRK2 kinases to IC50 for LRRK2 G2019S. Kinases with inhibition rates <10% were considered non-targets. [1] |
| Cell Assay |
1. Human iPSC-derived neuron culture and toxicity assay: [1]
Human iPSCs were differentiated into mature neurons over 4–6 weeks and characterized by immunofluorescence staining for neuronal markers (β-III tubulin, MAP2). Neurons were transfected with plasmids encoding LRRK2 G2019S (or empty vector as control) and treated with CZC-54252 (10–100 nM) for 72 hours. Neuronal survival was assessed by MTT assay; LDH release was measured using an LDH assay kit; nuclear morphology was observed by Hoechst 33342 staining to detect apoptosis. 2. Primary cortical neuron culture and α-synuclein-induced damage assay: [1] Primary cortical neurons were isolated from embryonic rats and cultured for 7–10 days. Neurons were co-transfected with LRRK2 G2019S and α-synuclein plasmids, then treated with CZC-54252 (50 nM) for 48 hours. ROS production was detected by DCFH-DA fluorescence; α-synuclein aggregation was visualized by immunofluorescence staining with an anti-α-synuclein antibody; protein expression levels of p-S935-LRRK2, total LRRK2, and Cleaved-Caspase 3 were analyzed by Western blot. 3. Western blot analysis for LRRK2 phosphorylation: [1] Treated neurons were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined by BCA assay. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with non-fat milk. Membranes were incubated with primary antibodies against p-S935-LRRK2, total LRRK2, Cleaved-Caspase 3, or β-actin overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using an ECL detection system. Band intensities were quantified by densitometry. [1] |
| Animal Protocol |
Mice |
| References | |
| Additional Infomation |
CZC-25146 belongs to the aminopyrimidine class of compounds, with the structure 2,6-diamino-5-chloropyrimidine, where the amino groups at positions 2 and 6 are respectively substituented with 2-methoxy-4-(morpholino-4-yl)phenyl and 2-(methanesulfonylamino)phenyl. It is an inhibitor of the Parkinson's disease kinase LRRK2, belonging to EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitors. It is an aminopyrimidine, belonging to the morpholino class of compounds, and is also an aromatic ether, organochlorine compound, sulfonamide, and secondary amino compound.
1. Discovery Strategy: CZC-54252 was discovered through a chemical proteomics-based screening method that combines activity-based proteomic analysis (ABPP) and structure-activity relationship (SAR) optimization, thereby achieving high selectivity for LRRK2. [1] 2. Background: LRRK2 mutations (e.g., G2019S, R1441C) are the most common genetic cause of late-onset Parkinson's disease (PD). Mutant LRRK2 exhibits enhanced kinase activity, leading to neurotoxicity, α-synuclein aggregation, and oxidative stress—key pathological features of PD. [1] 3. Mechanism of action: CZC-54252 competitively binds to the ATP-binding pocket of LRRK2, inhibiting its kinase activity and phosphorylation of its downstream substrates. This reduces LRRK2-mediated neurotoxicity, inhibits α-synuclein aggregation, and alleviates oxidative stress, thereby protecting neurons from PD-related damage. [1] 4. Therapeutic potential: Preclinical data suggest that CZC-54252 is a potent, selective, and neuroprotective LRRK2 inhibitor with no significant cytotoxicity. It holds promise as a disease-modifying therapy for Parkinson's disease patients carrying LRRK2 mutations, focusing on addressing underlying pathogenic mechanisms rather than simply alleviating symptoms. [1] |
| Molecular Formula |
C22H25CLN6O4S
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| Molecular Weight |
504.99
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| Exact Mass |
504.134
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| CAS # |
1191911-27-9
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| Related CAS # |
CZC-54252 hydrochloride;1784253-05-9
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| PubChem CID |
44252734
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
718.1±70.0 °C at 760 mmHg
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| Flash Point |
388.1±35.7 °C
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| Vapour Pressure |
0.0±2.3 mmHg at 25°C
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| Index of Refraction |
1.667
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| LogP |
1.67
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
34
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| Complexity |
737
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
CLGWUCNXOBLWFM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H25ClN6O4S/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-chloro-2-(2-methoxy-4-morpholin-4-ylanilino)pyrimidin-4-yl]amino]phenyl]methanesulfonamide
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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 (4.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9802 mL | 9.9012 mL | 19.8024 mL | |
| 5 mM | 0.3960 mL | 1.9802 mL | 3.9605 mL | |
| 10 mM | 0.1980 mL | 0.9901 mL | 1.9802 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.
Chemoproteomics-based discovery of LRRK2 lead compounds.ACS Chem Biol.2011 Oct 21;6(10):1021-8. |
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CZC-25146 and CZC-54252 are potent and selective LRRK2 inhibitors.ACS Chem Biol.2011 Oct 21;6(10):1021-8. |
CZC-25146 and CZC-54252 potently attenuate mutant LRRK2-mediated toxicity in primary human neurons.ACS Chem Biol.2011 Oct 21;6(10):1021-8. |
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