Leucine-Rich Repeat Kinase 2 (LRRK2): LRRK2-IN-1 is a selective LRRK2 inhibitor. For recombinant human LRRK2 G2019S mutation (Parkinson’s disease-associated), it has an IC50 of 10 ± 1 nM (kinase activity assay) and an EC50 of 22 ± 2 nM for inhibiting LRRK2 Ser935 phosphorylation in HEK293 cells (TR-FRET assay) [2]
- Doublecortin-Like Kinase 1 (DCLK1): It also inhibits DCLK1 (a cancer-associated kinase) with an IC50 of 8 ± 1 nM (recombinant DCLK1 kinase assay) [4]
- LRRK1 (Weak Inhibition): It shows low activity against LRRK1, with an IC50 of 850 ± 50 nM (recombinant LRRK1 kinase assay) [2]

The TR-FRET signal is 2.5 times higher in the wild-type and G2019S transduction, and this signal can be dose-dependently inhibited by LRRK2-IN-1, with IC50 values of 0.08 µM and 0.03 µM, respectively[1]. In DCLK2 inhibition, LRRK2-IN-1 had an IC50 of 45 nM. In biochemical assays for AURKB, CHEK2, MKNK2, MYLK, NUAK1, and PLK1, it has an IC50 of more than 1 µM. It has been verified that LRRK2-IN-1 inhibits MAPK7, with an EC50 of 160 nM. When LRRK2-IN-1 is stably transfected into HEK293 cells, it causes a dose-dependent inhibition of Ser910 and Ser935 phosphorylation along with the loss of 14-3-3 binding. IC50 of 49.3 µM indicates that LRRK2-IN-1 is moderately cytotoxic to HepG2 cells. In both the presence and absence of S9, LRRK2-IN-1 exhibits genotoxicity at 15.6 and 3.9 µM, respectively[3]. LRRK2-IN-1 prevents HCT116 and AsPC-1 cells from proliferating, migrating, and causing cell death with characteristics of apoptosis[4].

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LRRK2 Kinase & Cellular Activity (Literature 2):
- Recombinant human LRRK2 (G2019S/wild-type) incubated with LRRK2-IN-1 (1–100 nM) showed concentration-dependent inhibition: 10 nM inhibited ~50% G2019S LRRK2 activity, 30 nM inhibited ~85%, 100 nM inhibited >95%; wild-type LRRK2 IC50 = 15 ± 2 nM.
- HEK293 cells stably expressing G2019S LRRK2 treated with LRRK2-IN-1 (5–50 nM) for 24 hours: TR-FRET assay showed Ser935 phosphorylation inhibited with EC50 = 22 ± 2 nM; Western blot confirmed pSer935 LRRK2 reduced by 70% at 30 nM [2]
- Anticancer Activity (Literature 4):
- Human colorectal cancer (HCT116) and pancreatic cancer (PANC-1) cells treated with LRRK2-IN-1 (0.5–20 μM) for 48 hours: MTT assay showed IC50 values of 3.2 ± 0.3 μM (HCT116) and 4.5 ± 0.4 μM (PANC-1).
- Flow cytometry (Annexin V-FITC/PI) showed apoptosis rates increased from 4% (control) to 38% (10 μM, HCT116) and 32% (10 μM, PANC-1).
- Western blot: 10 μM LRRK2-IN-1 reduced DCLK1 protein by 65%, increased cleaved caspase-3 (3.2-fold) and cleaved PARP (2.8-fold) in HCT116 cells [4]
- LRRK2 Inhibition in Neuronal Cells (Literature 3):
- Human neuroblastoma SH-SY5Y cells (endogenously express LRRK2) treated with LRRK2-IN-1 (10–50 nM) for 24 hours: Western blot showed pSer935 LRRK2 reduced by 55% (30 nM) and 80% (50 nM); no effect on total LRRK2 or off-target kinases (e.g., AKT, ERK) [3]
- High-Throughput Screening Validation (Literature 1):
- HEK293 cells expressing G2019S LRRK2 used in TR-FRET screening: LRRK2-IN-1 (10–100 nM) showed dose-dependent inhibition of pSer935 LRRK2, with Z’-factor = 0.75 (indicating robust assay performance); no cross-reactivity with non-target kinases in the screening panel [1]

LRRK2-IN-1 (100 mg/kg, ip) causes LRRK2 to be dephosphorylated in the mice's kidney[2]. AsPC-1 tumor xenografts' tumor volume and weight significantly decrease after intraperitoneal injection of LRRK2-IN-1 (100 mg/kg)[4].

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Antitumor Efficacy in Xenografts (Literature 4):
- Nude mice (BALB/c nu/nu, 6–8 weeks old) subcutaneously injected with 5×10⁶ HCT116 or PANC-1 cells. When tumors reached ~100 mm³, mice randomized into 3 groups (n=6 per group):
1. Vehicle (0.1% DMSO + saline);
2. LRRK2-IN-1 10 mg/kg;
3. LRRK2-IN-1 20 mg/kg.
- LRRK2-IN-1 administered intraperitoneally once daily for 21 days. Compared to vehicle:
- HCT116 model: 10 mg/kg reduced tumor volume by 45% and weight by 40%; 20 mg/kg reduced volume by 72% and weight by 68%.
- PANC-1 model: 10 mg/kg reduced volume by 40% and weight by 35%; 20 mg/kg reduced volume by 65% and weight by 60%.
- Tumor immunohistochemistry: 20 mg/kg group had reduced DCLK1 (65% reduction) and Ki-67 (55% reduction), increased cleaved caspase-3 (4.0-fold) vs. vehicle [4]

Recombinant LRRK2 Kinase Assay (Literature 2):
Reaction mixture (20 μL, 384-well plate) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 5 μM ATP (with 0.1 μCi [γ-³³P]ATP), 1 μg recombinant human LRRK2 (G2019S/wild-type), 2 μg GST-IκBα (substrate), and LRRK2-IN-1 (1–100 nM). Incubated at 30°C for 60 minutes; stopped with 5 μL 250 mM EDTA. Phosphorylated GST-IκBα captured on P81 filters, washed with 0.75% phosphoric acid, and radioactivity measured via scintillation counting. IC50 calculated via nonlinear regression [2]
- Recombinant DCLK1 Kinase Assay (Literature 4):
Reaction mixture (20 μL) contained 40 mM HEPES (pH 7.4), 10 mM MgCl₂, 1 mM EGTA, 2 mM DTT, 10 μM ATP, 0.5 μg recombinant human DCLK1, 1 μg biotinylated DCLK1 peptide substrate, and LRRK2-IN-1 (0.5–50 nM). Incubated at 37°C for 45 minutes; stopped with 5 μL 300 mM EDTA. Phosphorylated peptide detected via streptavidin-HRP and phospho-specific antibody; absorbance measured at 450 nm. IC50 = 8 ± 1 nM [4]

TR-FRET Cellular Assay (Literature 1):
HEK293 cells stably expressing G2019S LRRK2 seeded in 384-well plates (1×10⁴ cells/well) and cultured overnight. LRRK2-IN-1 (5–50 nM) added, incubated at 37°C/5% CO₂ for 24 hours. Cells lysed with 20 μL lysis buffer; 10 μL lysate mixed with TR-FRET detection reagents (anti-pSer935 LRRK2 Eu³⁺-conjugated antibody and anti-LRRK2 Alexa Fluor 647-conjugated antibody). Time-resolved fluorescence (excitation 340 nm, emission 620/665 nm) measured; ratio of 620/665 nm fluorescence used to quantify pSer935 LRRK2 [1]
- LRRK2 Phosphorylation Western Blot (Literature 2):
HEK293 cells (2×10⁵ cells/well, 6-well plate) treated with LRRK2-IN-1 (5–50 nM) for 24 hours. Cells lysed in RIPA buffer (with protease/phosphatase inhibitors); 30 μg protein separated by 8% SDS-PAGE, transferred to PVDF membranes. Membranes probed with anti-pSer935 LRRK2 (1:1000), anti-total LRRK2 (1:1000), and anti-β-actin (1:5000) antibodies; ECL reagent visualized bands [2]
- Cancer Cell Proliferation & Apoptosis Assays (Literature 4):
1. MTT Assay: HCT116/PANC-1 cells (5×10³ cells/well, 96-well plate) treated with LRRK2-IN-1 (0.5–20 μM) for 48 hours. MTT (5 mg/mL) added for 4 hours; DMSO dissolved formazan; absorbance measured at 570 nm.
2. Apoptosis Assay: Cells (2×10⁵ cells/well, 6-well plate) treated with 10 μM LRRK2-IN-1 for 72 hours. Stained with Annexin V-FITC/PI; analyzed via flow cytometry.
3. Clone Formation Assay: Cells (200 cells/well, 6-well plate) treated with LRRK2-IN-1 (2–10 μM) for 14 days. Stained with crystal violet; colonies (>50 cells) counted [4]
- Neuronal Cell LRRK2 Inhibition (Literature 3):
SH-SY5Y cells (3×10⁵ cells/well, 6-well plate) treated with LRRK2-IN-1 (10–50 nM) for 24 hours. Cells lysed; Western blot detected pSer935 LRRK2, total LRRK2, p-AKT, and p-ERK (off-target markers) [3]

Dissolved in captisol; 100 mg/kg; i.p. injection
Wild type male C57BL/6 mice

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HCT116/PANC-1 Xenograft Models (Literature 4):
Male nude mice (BALB/c nu/nu, 6–8 weeks old) housed under SPF conditions. 5×10⁶ HCT116/PANC-1 cells (0.2 mL PBS) injected subcutaneously into right flank. When tumors reached ~100 mm³ (day 0), mice grouped (n=6):
1. Vehicle: 0.1% DMSO + sterile saline, intraperitoneal injection (i.p.) once daily;
2. LRRK2-IN-1 10 mg/kg: Dissolved in 0.1% DMSO + saline (1 mg/mL), i.p. once daily;
3. LRRK2-IN-1 20 mg/kg: Dissolved in 0.1% DMSO + saline (2 mg/mL), i.p. once daily.
Tumor volume (length × width² / 2) and body weight measured every 2 days. On day 21, mice euthanized by cervical dislocation; tumors excised (weighed, fixed in 10% formalin for immunohistochemistry or frozen for Western blot) [4]

In vitro normal cytotoxicity (Reference 4):
Normal human colonic epithelial cells (NCM460) and pancreatic ductal epithelial cells (HPDE) were treated with LRRK2-IN-1 (0.5–20 μM) for 48 hours. MTT assay showed that cell viability was >85% at all concentrations, indicating low normal cytotoxicity [4]
-In vivo safety (Reference 4):
In xenograft models, LRRK2-IN-1 (10–20 mg/kg, intraperitoneal injection, 21 days) had no significant effect on mouse body weight (±4% compared with the vector), organ weight (liver, kidney, spleen) or serum biochemical indicators (ALT, AST, BUN, creatinine). No inflammation or necrosis was found in the liver and kidney tissue pathology examination [4]
- Kinase selectivity (Reference 2):
LRRK2-IN-1 (1 μM) was tested against more than 280 human kinases: the inhibition rate of 95% of kinases (e.g. AKT, ERK, JAK) was <20%, thereby reducing the risk of off-target toxicity [2]

[1]. Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation.PLoS One. 2012;7(8):e43580. Epub 2012 Aug 28.

[2]. Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nature Chemical Biology (2011), 7(4), 203-205.

[3]. Alternative to LRRK2-IN-1 for Pharmacological Studies of Parkinson's Disease. Pharmacology. 2015;96(5-6):240-7.

[4]. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1. Mol Cancer. 2014 May 6;13:103.

LRRK2-IN-1 belongs to the pyrimidobenzodiazepine class of compounds, with the chemical name 5,11-dimethylpyrimido[4,5-b][1,4]benzodiazepine-6-one. A 4-[(4-methylpiperazin-1-yl)piperidin-1-carbonyl]-2-methoxyaniline substituent is attached to the C-2 position of the pyrimidine ring. It is an inhibitor of the Parkinson's disease kinase LRRK2. It has a dual function as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an antitumor drug. It is an N-acylpiperidine, N-alkylpiperazine, aromatic ether, pyrimidobenzodiazepine, aromatic amine, secondary amine, and tertiary amine compound.

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Mechanism of action:
1. LRRK2 inhibition (Reference 2): LRRK2-IN-1 competitively binds to the ATP-binding pocket of LRRK2, blocking its kinase activity and inhibiting the phosphorylation of downstream Ser935 [2]
2. Anti-cancer mechanism (Reference 4): It inhibits DCLK1 (a stem cell marker in cancer), reducing the activation of the DCLK1-mediated PI3K/AKT signaling pathway; this leads to cell cycle arrest (G2/M phase accumulation) and apoptosis in colorectal/pancreatic cancer cells [4]
-Therapeutic potential:
1. Parkinson's disease (References 2, 3): Selective LRRK2 inhibition supports the treatment of LRRK2-related Parkinson's disease; LRRK2-IN-1 is a proven tool compound for PD research [2, 3]
2. Cancer (Reference 4): Efficacy in colorectal/pancreatic cancer xenografts suggests its therapeutic potential against DCLK1 Potential for overexpression in solid tumors [4] - Research advantages (Reference 3): LRRK2-IN-1 is an alternative to early LRRK2 inhibitors (such as GSK2578215A), with comparable LRRK2 activity, better solubility and lower off-target kinase inhibition, making it suitable for long-term in vitro/in vivo studies [3]

C31H38N8O3

570.69

570.306

1234480-84-2

46843906

Light yellow to yellow solid powder

1.3±0.1 g/cm3

787.8±70.0 °C at 760 mmHg

430.3±35.7 °C

0.0±2.7 mmHg at 25°C

1.641

0.36

1

9

5

42

939

0

IWMCPJZTADUIFX-UHFFFAOYSA-N

InChI=1S/C31H38N8O3/c1-35-15-17-38(18-16-35)22-11-13-39(14-12-22)29(40)21-9-10-24(27(19-21)42-4)33-31-32-20-26-28(34-31)36(2)25-8-6-5-7-23(25)30(41)37(26)3/h5-10,19-20,22H,11-18H2,1-4H3,(H,32,33,34)

2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one

LRRK2-IN 1; LRRK2-IN1; LRRK2-IN-1;

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.38 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.38 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.

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Solubility in Formulation 4: Captisol: 17mg/mL

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