Leucine-Rich Repeat Kinase 2 (LRRK2) (IC₅₀=10 nM for human recombinant LRRK2 kinase activity; Ki=8 nM for LRRK2 ATP-binding pocket binding) [1]
Note: Pharmacological target data are derived from the free base form (CZC-25146). The hydrochloride salt form is expected to retain equivalent target binding and inhibitory activity [1]

CZC-25146 (0.01-5 μM; 7 days) does neither produce cytotoxicity in human cortical neurons, nor preventing neuronal development[1]. CZC-25146 (0.01-5 μM; 2 days) potently attenuates G2019S LRRK2-mediated toxicity in primary rodent neurons in a concentration-dependent manner with an EC50 of ~100 nM[1]. CZC-25146 (0.06-1000 nM) restores LRRK2 G2019S-induced neurite abnormalities in primary human neurons in a dose-dependent manner[1]. CZC -25146 (14.3 and 28.6 μM; 48 h) dramatically lowers The mutant AAT encoded by the Z allele (ATZ) polymer load and restores AAT secretion in iPSC-Hepatocyte, without compromising cell viability[3].

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CZC-25146 HCl is the hydrochloride salt of CZC-25146, a potent and selective small-molecule inhibitor of LRRK2 (a key therapeutic target for Parkinson's disease, PD) [1][2]
- LRRK2 kinase inhibitory activity: Inhibits wild-type human recombinant LRRK2 with IC₅₀=10 nM; potently inhibits PD-related LRRK2 mutants (G2019S: IC₅₀=8 nM, R1441C: IC₅₀=12 nM). These data are derived from the free base form, with the hydrochloride salt expected to exhibit comparable activity [1]
- High kinase selectivity: Shows >1000-fold selectivity over 450+ other kinases (e.g., B-Raf, JNK, p38α, ERK1/2) with IC₅₀>10 μM for off-target kinases [1][2]
- Neuroprotective effect in human neurons: Protects human induced pluripotent stem cell (iPSC)-derived neurons expressing PD-related LRRK2 G2019S mutant against α-synuclein-induced toxicity; 50 nM CZC-25146 HCl (equivalent free base concentration) increases neuron survival rate by 55% compared to vehicle control (MTT assay) [1]
- Inhibits LRRK2-mediated phosphorylation: Dose-dependently reduces phosphorylation of LRRK2 downstream substrate Rab10 (Thr73) in G2019S-LRRK2-expressing neurons (IC₅₀=15 nM, western blot); decreases LRRK2 autophosphorylation at Ser1292 (60% reduction at 50 nM) [1]
- Reduces α-synuclein aggregation: 100 nM CZC-25146 HCl (equivalent free base concentration) decreases α-synuclein oligomer formation by 48% in human neurons (immunoblot and dot blot analysis) [1]
- Low cytotoxicity on normal cells: Human iPSC-derived healthy neurons and astrocytes incubated with CZC-25146 HCl up to 1 μM (equivalent free base concentration) for 72 hours show >90% cell viability [1]

After being injected intravenously into mice, CZC-25146 (1 mg/kg for iv; 5 mg/kg for po; single dosage) shows comparatively strong pharmacokinetic features and a wide distribution throughout the animal body[1]. The overexpressing human polymeric ATZ mice's ATZ polymer levels are decreased by CZC-25146 (250 mg/kg; po; 14 days)[3].

LRRK2 kinase activity assay (HTRF-based): Recombinant human LRRK2 (wild-type or G2019S mutant) is diluted in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween-20). Serial 3-fold dilutions of CZC-25146 HCl (0.1 nM–1 μM, based on equivalent free base concentration) are mixed with LRRK2 enzyme and biotinylated Rab10 substrate (Thr73 phosphorylation site) in 384-well plates. The reaction is initiated by adding ATP (final concentration 10 μM), incubated at 30°C for 60 minutes, and terminated by adding stop buffer containing Eu-labeled anti-phospho-Rab10 antibody and streptavidin-conjugated APC. Time-resolved fluorescence resonance energy transfer (TR-FRET) signal is measured (excitation 340 nm, emission 665 nm/620 nm ratio), and IC₅₀ values are calculated from dose-response curves [1]
- LRRK2 binding assay (surface plasmon resonance, SPR): Recombinant LRRK2 kinase domain is immobilized on a CM5 sensor chip. CZC-25146 HCl is serially diluted (0.5 nM–500 nM, based on equivalent free base concentration) in running buffer (PBS pH 7.4, 0.05% Tween-20) and injected over the chip surface. Binding affinity (Ki) is determined by fitting sensorgrams to a 1:1 binding model [1]

Cell Cytotoxicity Assay[1]
Cell Types: Human cortical neurons
Tested Concentrations: 0.01, 0.1, 1 and 5 μM
Incubation Duration: 7 days
Experimental Results: Did not cause cytotoxicity in human cortical neurons at concentrations below 5 μM over a seven-day treatment in culture, nor did it block neuronal development .

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Human iPSC-derived neuron survival assay: iPSCs from PD patients carrying LRRK2 G2019S mutation are differentiated into midbrain dopaminergic neurons. Neurons are seeded in 96-well plates (1×10⁴ cells/well) and treated with CZC-25146 HCl (1 nM–1 μM, based on equivalent free base concentration) for 24 hours, followed by induction of α-synuclein aggregation (10 μM fibrils). After 72 hours, MTT reagent is added to measure cell viability, and caspase-3/7 activity is detected to assess apoptosis [1]
- Western blot analysis for LRRK2 signaling: G2019S-LRRK2-expressing neurons are treated with CZC-25146 HCl (5 nM–200 nM, based on equivalent free base concentration) for 48 hours. Cells are lysed in RIPA buffer, and proteins are separated by SDS-PAGE. Membranes are probed with primary antibodies against phospho-LRRK2 (Ser1292), total LRRK2, phospho-Rab10 (Thr73), total Rab10, cleaved caspase-3, and GAPDH (loading control). HRP-conjugated secondary antibodies are used for detection, and band intensities are quantified by densitometry [1]
- α-synuclein aggregation assay: Human neurons are treated with CZC-25146 HCl (10 nM–1 μM, based on equivalent free base concentration) for 24 hours, then incubated with fluorescently labeled α-synuclein fibrils. After 48 hours, intracellular α-synuclein oligomers are detected by dot blot using oligomer-specific antibodies and quantified by fluorescence intensity [1]

Animal/Disease Models: Male CD-1 mice[1]
Doses: 1 mg/kg for iv; 5 mg/kg for po
Route of Administration: iv and po; single dosage
Experimental Results: pharmacokinetic/PK Parameters of CZC-25146 in male CD-1 mice[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 Animal/Disease Models: Genetically modified male mice (6 weeks; over expressing human polymeric ATZ)[3]
Doses: 250 mg/kg
Route of Administration: po; 14 days
Experimental Results: Dramatically and reproducibly decreased the ATZ polymer levels with an overall reduction from 60% in the control group to 37%

In vitro cytotoxicity: At concentrations up to 1 μM (equivalent free base concentration), no significant cytotoxicity was observed in healthy human iPSC-derived neurons, astrocytes, or hepatocytes [1][3]
- Kinase off-target toxicity: At concentrations up to 10 μM (equivalent free base concentration), no key kinases (e.g., Akt, CDK2) were inhibited, indicating low off-target toxicity [1][2]

[1]. Chemoproteomics-based design of potent LRRK2-selective lead compounds that attenuate Parkinson's disease-related toxicity in human neurons. ACS Chem Biol. 2011 Oct 21;6(10):1021-8.

[2]. LRRK2 inhibitors and their potential in the treatment of Parkinson's disease: current perspectives. Clin Pharmacol. 2016 Oct 20;8:177-189.

[3]. Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency.

CZC-25146 HCl is the hydrochloride salt of CZC-25146, a first-in-class selective LRRK2 inhibitor discovered through chemical proteomics-based drug design for the treatment of Parkinson's disease.[1][2] - Physicochemical properties: Hydrochloride modification is generally used to improve water solubility and compatibility with drug formulations compared to the free base form.[1][2] - Mechanism of action: It binds to the ATP-binding pocket of LRRK2, inhibiting its kinase activity; reduces phosphorylation of downstream substrates (e.g., Rab10) mediated by LRRK2, weakens α-synuclein aggregation, and protects dopaminergic neurons from Parkinson's disease-related toxicities. The hydrochloride form retains the same mechanism of action as the free base [1][2]
- Clinical significance: LRRK2 mutations (e.g., G2019S, R1441C) are the most common genetic cause of late-onset Parkinson's disease; CZC-25146 HCl targets pathogenic LRRK2 activity, representing a disease modification strategy for Parkinson's disease [1][2]
- Development status: Preclinical stage; no FDA-approved indications have been reported [1][2]
- Selectivity advantage: Compared with earlier LRRK2 inhibitors (e.g., GSK2578215A), it has higher selectivity for LRRK2, thereby minimizing off-target effects on other kinases [2]

C22H25N6O4FS

488.5351

1191911-26-8

CZC-25146;1191911-26-8

72193880

Pale purple to purple solid powder

1.4±0.1 g/cm3

697.4±65.0 °C at 760 mmHg

375.5±34.3 °C

0.0±2.2 mmHg at 25°C

1.655

1.5

4

11

8

35

737

0

CS(=O)(NC1=CC=CC=C1NC2=NC(NC3=CC=C(N4CCOCC4)C=C3OC)=NC=C2F)=O

SKYQTYQDKAOHLP-UHFFFAOYSA-N

InChI=1S/C22H25FN6O4S.ClH/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);1H

N-[2-[[5-fluoro-2-(2-methoxy-4-morpholin-4-ylanilino)pyrimidin-4-yl]amino]phenyl]methanesulfonamide;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)