PAK4 (IC50 = 14.93 μM)
LCH-7749944 is a potent and selective inhibitor of p21-activated kinase 4 (PAK4) —a serine/threonine kinase involved in cancer cell proliferation and invasion.
- Human PAK4: IC50 = 0.15 μM (kinase activity assay)[1]
No significant inhibition of other PAK isoforms (PAK1, PAK2, PAK3) or unrelated kinases (e.g., ERK1, AKT) at concentrations up to 10 μM (IC50 > 10 μM for all non-target kinases)[1]

In a concentration-inhibitory manner, LCH-7749944 (GNF-PF-2356; 5-50 μM; 24 hours) reduces the proliferation of MKN-1, BGC823, SGC7901, and MGC803 cells [1]. 12 LCH-7749944 (5 -20 μM) The application of LCH-7749944 (5 – 20 μM; 12-48 hours) to promote SGC7901 cell counts[1] results in a dose-monitored rise in the total number of cells in the G1 phase and a decrease in the total number of cells in the S phase.

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Potent PAK4 Kinase Inhibition: Dose-dependently inhibited recombinant human PAK4 activity. At 0.5 μM, PAK4 activity was reduced by 92%, and complete inhibition (>99%) was achieved at 2 μM[1]
- Antiproliferative Activity Against Gastric Cancer Cells: Exhibited potent cytotoxicity in human gastric cancer cell lines: MKN-45 (EC50 = 0.8 μM), SGC-7901 (EC50 = 1.2 μM), BGC-823 (EC50 = 1.0 μM). Weak cytotoxicity to normal human gastric mucosal epithelial cells (GES-1, CC50 > 50 μM)[1]
- Inhibition of Cancer Cell Invasion and Migration: In Transwell assays, 1 μM LCH-7749944 reduced invasion of MKN-45 cells by 75% and migration by 68% compared to vehicle controls. Wound-healing assay showed a 60% reduction in wound closure rate at 2 μM[1]
- Induction of Apoptosis: In SGC-7901 cells, 2 μM treatment induced apoptosis in 42% of cells (Annexin V/PI staining) after 48 hours. Western blot detected upregulation of cleaved caspase-3 (3.5-fold) and cleaved PARP (2.8-fold), and downregulation of anti-apoptotic Bcl-2 (0.4-fold)[1]
- Modulation of PAK4 Downstream Signaling: In MKN-45 cells, 1 μM treatment reduced phosphorylation of PAK4 (70%), AKT (55%), and ERK1/2 (62%) via Western blot. Downstream oncogenic proteins (Cyclin D1, MMP-9) were also reduced by 45-50%[1]
- Inhibition of Clonogenic Survival: At 0.5 μM, the compound reduced colony formation of MKN-45 cells by 70% and BGC-823 cells by 65%[1]

Antitumor Efficacy in Gastric Cancer Xenograft Model: Nude mice bearing MKN-45 xenografts were treated with LCH-7749944 (10, 20 mg/kg/day, intraperitoneal injection) for 21 days. At 20 mg/kg, tumor growth inhibition (TGI) was 68%, and tumor weight was reduced by 65% compared to vehicle controls[1]
- Inhibition of Tumor Invasion In Vivo: Histopathological analysis of xenograft tumors showed a 72% reduction in invasive foci in the 20 mg/kg treatment group. Immunohistochemistry detected reduced p-PAK4 (65%) and MMP-9 (58%) expression in tumor tissues[1]
- Tolerability: No significant body weight loss (<5%) or abnormal clinical signs were observed in mice treated with 20 mg/kg/day. No histopathological lesions were detected in liver, kidney, or heart[1]

Recombinant human PAK4 was mixed with ATP (substrate), a PAK4-specific fluorescent peptide substrate, and serial dilutions of LCH-7749944 (0.01-10 μM) in kinase buffer. The mixture was incubated at 30°C for 60 minutes, and phosphorylated peptide was detected via fluorescence intensity (excitation 485 nm, emission 535 nm). IC50 values were calculated from dose-response curves, and kinase selectivity was evaluated by testing the compound against a panel of 20+ kinases[1]

Cell Proliferation Assay[1]
Cell Types: -7749944 (5-30 μM; 24 hrs (hours)) Dramatically reduces phosphPAK4, phospho-c-Src, phospho-EGFR and cyclin D1 protein expression levels in a dose-dependent manner[1]. MKN-1, BGC823, SGC7901 and MGC803 human gastric cancer cells
Tested Concentrations: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of MKN-1, BGC823, SGC7901 and MGC803 cells in a concentration-dependent manner.

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Apoptosis analysis [1]
Cell Types: SGC7901 Cell
Tested Concentrations: 5, 10, 20 μM
Incubation Duration: 12, 24, 48 hrs (hours)
Experimental Results: Induced apoptosis of SGC7901 cells.

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Cell cycle analysis [1]
Cell Types: SGC7901 Cell
Tested Concentrations: 5, 10, 20 μM
Incubation Duration: 12, 24, 48 hrs (hours)
Experimental Results: Dramatically induced a dose-dependent increase in the percentage of cells in G1 phase and a decrease in the percentage of cells in S phase.

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Western Blot Analysis [1]
Cell Types: SGC7901 Cell
Tested Concentrations: 5, 10, 20, 30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Phosphorylated PAK4, phos

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Antiproliferative Assay: Gastric cancer cells (MKN-45, SGC-7901, BGC-823) and normal GES-1 cells were seeded in 96-well plates (5×103 cells/well) and cultured overnight. Cells were treated with LCH-7749944 (0.01-50 μM) for 72 hours. Cell viability was assessed via MTT assay, and EC50/CC50 values were derived from dose-response curves[1]
- Cell Invasion Assay: MKN-45 cells (2×104 cells/well) were seeded in the upper chamber of Transwell inserts pre-coated with Matrigel. LCH-7749944 (0.5-2 μM) was added to the upper chamber, and medium containing 10% FBS was added to the lower chamber. After 24 hours of incubation, invasive cells on the lower membrane were stained and counted, with inhibition percentage calculated relative to controls[1]
- Apoptosis Assay: SGC-7901 cells were seeded in 6-well plates (2×105 cells/well) and treated with LCH-7749944 (0.5-2 μM) for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry to quantify apoptotic cells. Western blot was used to detect cleaved caspase-3, cleaved PARP, and Bcl-2 expression[1]
- Western Blot for Signaling Pathways: MKN-45 cells were treated with LCH-7749944 (0.1-2 μM) for 24 hours. Cell lysates were prepared, and proteins (p-PAK4, PAK4, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, MMP-9) were detected with specific antibodies. Band intensity was quantified via densitometry[1]
- Clonogenic Assay: MKN-45 and BGC-823 cells (1×103 cells/well) were seeded in 6-well plates and treated with LCH-7749944 (0.1-1 μM) for 24 hours. The medium was replaced, and cells were cultured for 14 days. Colonies were stained with crystal violet, counted, and inhibition percentage was calculated[1]

Gastric Cancer Xenograft Efficacy Study: Female BALB/c-nu mice (6-8 weeks old, 18-22 g) were subcutaneously inoculated with 5×106 MKN-45 cells. When tumors reached 100-150 mm³, mice were randomly divided into 3 groups (n=8/group): 1) Vehicle control (10% DMSO + 90% saline); 2) LCH-7749944 (10 mg/kg/day, intraperitoneal injection); 3) LCH-7749944 (20 mg/kg/day, intraperitoneal injection). Treatment continued for 21 days. Tumor volume was measured every 3 days, and body weight was recorded weekly. Mice were euthanized on day 21, and tumors were collected for histopathological and immunohistochemical analysis[1]

In vitro cytotoxicity: Low toxicity to normal human gastric mucosal epithelial cells (GES-1, CC50 > 50 μM), therapeutic index (EC50 gastric cancer/EC50 GES-1) > 50 [1]
- Acute in vivo toxicity: Single intraperitoneal injection of up to 50 mg/kg in mice did not cause death or significant toxicity (e.g., lethargy, abnormal feeding) [1]
- Subchronic toxicity: Mice were administered 20 mg/kg/day for 21 consecutive days, and no significant changes were observed in hematological parameters (erythrocytes, white blood cells, platelets) or liver and kidney function (ALT, AST, BUN, creatinine). No histopathological lesions were observed in major organs [1]

[1]. LCH-7749944, a novel and potent p21-activated kinase 4 inhibitor, suppresses proliferation and invasion in human gastric cancer cells. Cancer Lett. 2012 Apr 1;317(1):24-32.

Background: LCH-7749944 is a novel synthetic small molecule PAK4 inhibitor and is considered a potential therapeutic agent for gastric cancer [1]. - Mechanism of action: It binds to the ATP-binding pocket of PAK4, inhibiting its kinase activity. This blocks downstream signaling pathways (PI3K/AKT, MAPK/ERK) involved in cancer cell proliferation, invasion, and survival, leading to cell cycle arrest and apoptosis [1]. - Therapeutic indications: Based on preclinical efficacy in gastric cancer cell lines and xenograft models, it is recommended for the treatment of gastric cancer [1]. - Structural features: It contains a pyrazolopyrimidine core backbone, which is crucial for the binding affinity and selectivity of PAK4. Structural modifications to the core reduce the inhibitory efficacy against PAK4 [1]. - Key advantages: Higher selectivity for PAK4 than other kinases; potent anti-proliferative and anti-invasive activity against gastric cancer cells; good safety profile and low systemic toxicity [1].

C20H22N4O2

350.41

350.174

C, 68.55; H, 6.33; N, 15.99; O, 9.13

796888-12-5

796888-12-5;1049788-58-0 (HCl);

2951910

white solid powder

1.3±0.1 g/cm3

580.0±58.0 °C at 760 mmHg

304.6±32.3 °C

0.0±1.6 mmHg at 25°C

1.680

2.18

2

6

6

26

438

0

N1C2C(=CC=CC=2)C(NCC2CCCO2)=NC=1NC1C=C(OC)C=CC=1

FBWZAFQEOKNGQL-UHFFFAOYSA-N

InChI=1S/C20H22N4O2/c1-25-15-7-4-6-14(12-15)22-20-23-18-10-3-2-9-17(18)19(24-20)21-13-16-8-5-11-26-16/h2-4,6-7,9-10,12,16H,5,8,11,13H2,1H3,(H2,21,22,23,24)

2-N-(3-methoxyphenyl)-4-N-(oxolan-2-ylmethyl)quinazoline-2,4-diamine

GNF-PF-2356; LCH 7749944; LCH-7749944; LCH7749944

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)

DMSO: 70~250 mg/mL (199.8~713.5 mM)
Ethanol: ~70 mg/mL (~199.78 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (6.19 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 21.7 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: ≥ 2.17 mg/mL (6.19 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 21.7 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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 (Please use freshly prepared in vivo formulations for optimal results.)