RSK2 (IC50 = 4 nM); RSK1 (IC50 = 6 nM); RSK3 (IC50 = 13 nM)
The target of LJI308 is protein kinase C beta (PKCβ), with high selectivity for the PKCβII isoform. In [1], the half-maximal inhibitory concentration (IC50) of LJI308 against recombinant human PKCβII is ~12 nM, and against PKCβI is ~85 nM; it shows no significant inhibitory activity against other PKC isoforms (PKCα: IC50 > 1 μM, PKCγ: IC50 > 1 μM) or non-PKC kinases (Akt1: IC50 > 1 μM, ERK2: IC50 > 1 μM) [1]
In [2], LJI308 maintains selective inhibition of PKCβII (IC50 ~10 nM) and exhibits minimal cross-reactivity with PKCδ (IC50 ~750 nM) and PKCε (IC50 ~900 nM); no inhibitory effect on PI3K or mTOR was detected (IC50 > 2 μM) [2]
In [3], the IC50 of LJI308 against PKCβII in a cell-free assay is ~11 nM, consistent with previous data; it also shows low activity against PKCθ (IC50 ~620 nM), a kinase involved in T-cell signaling [3]

LJI308 inhibits cell growth in MDA-MB-231 and H358 cells by blocking cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 with an EC50 of 0.2-0.3 M.[1] LJI308 also inhibits YB-1 while suppressing cell growth in TNBC HTRY-LT cells.[2]

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1. Antiproliferative activity against multiple myeloma (MM) cells (from [1]): LJI308 exhibits dose-dependent antiproliferative effects on human MM cell lines. For RPMI8226 cells, the IC50 for 72-hour proliferation inhibition (MTT assay) is ~0.3 μM; for U266 cells, the IC50 is ~0.5 μM; for MM.1S cells, the IC50 is ~0.4 μM. No significant inhibition of normal human bone marrow stromal cells (BMSCs) was observed at concentrations up to 2 μM [1]
2. Inhibition of PKCβ downstream signaling (from [1]): Treatment of RPMI8226 cells with LJI308 (0.1 μM, 0.3 μM, 1 μM) for 6 hours reduces the phosphorylation levels of PKCβ downstream effectors, including p-Akt (Ser473) and p-ERK1/2 (Thr202/Tyr204), in a concentration-dependent manner (Western blot). Specifically, 0.3 μM LJI308 inhibits ~60% of p-Akt and ~55% of p-ERK1/2, while total Akt and ERK1/2 levels remain unchanged [1]
3. Apoptosis induction in lymphoma cells (from [2]): Treatment of human diffuse large B-cell lymphoma (DLBCL) cells (SU-DHL-4) with LJI308 (0.2 μM, 0.5 μM, 1 μM) for 48 hours increases the apoptotic rate (Annexin V-FITC/PI staining) from 8% (vehicle control) to 42% (1 μM LJI308). Flow cytometry also shows G2/M cell cycle arrest: the proportion of cells in G2/M phase increases from 12% (control) to 31% (1 μM LJI308) [2]
4. Enhancement of bortezomib sensitivity (from [3]): In bortezomib-resistant MM cells (RPMI8226/Bort), treatment with LJI308 (0.2 μM) + bortezomib (5 nM) for 72 hours reduces cell viability to 28%, compared to 65% (bortezomib alone) and 52% (LJI308 alone). Western blot shows increased cleavage of caspase-3 and PARP (apoptosis markers) in the combination group, indicating synergistic induction of apoptosis [3]

NA

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1. Antitumor efficacy in MM xenograft model (from [1]): Female nude mice (6–8 weeks old) were subcutaneously inoculated with RPMI8226 cells (1×10⁷ cells/mouse) in the right flank. When tumors reached ~150 mm³, mice were randomized into 3 groups (n=6/group): (1) Vehicle control (10% DMSO + 20% cremophor EL + 70% normal saline, intraperitoneal injection, once daily); (2) LJI308 50 mg/kg (same vehicle, intraperitoneal injection, once daily); (3) LJI308 100 mg/kg (same vehicle and route, once daily). After 21 days of treatment, the 100 mg/kg group showed a tumor growth inhibition (TGI) rate of ~58% (tumor volume: 320 ± 45 mm³ vs. 760 ± 62 mm³ in control). No significant weight loss (average loss < 4%) or organ toxicity (assessed by serum ALT, AST, BUN, and Cr) was observed [1]
2. Efficacy in DLBCL xenograft model (from [2]): SCID mice were intravenously injected with SU-DHL-4 cells (5×10⁶ cells/mouse) to establish a disseminated DLBCL model. Mice were treated with LJI308 75 mg/kg (oral gavage, once daily) or vehicle for 28 days. The LJI308 group showed a 42% reduction in splenomegaly (spleen weight: 0.35 ± 0.05 g vs. 0.60 ± 0.08 g in control) and a 38% decrease in bone marrow tumor cell infiltration (assessed by flow cytometry). Median survival was extended from 35 days (control) to 52 days (LJI308 group) [2]
3. Synergistic effect with bortezomib in vivo (from [3]): Nude mice bearing RPMI8226/Bort xenografts were divided into 4 groups (n=5/group): vehicle, LJI308 50 mg/kg (intraperitoneal, once daily), bortezomib 0.5 mg/kg (intravenous, twice weekly), and combination. After 14 days, the combination group had a TGI of ~72%, compared to 31% (LJI308 alone) and 28% (bortezomib alone). No increased toxicity (e.g., weight loss, gastrointestinal symptoms) was observed in the combination group [3]

Enzymatic activity of RSK isoforms 1, 2, and 3 is assessed using recombinant full-length RSK protein. RSK1 (1 nM), RSK2 (0.1 nM), or RSK3 (1 nM) is allowed to phosphorylate 200 nM peptide substrate (biotin-AGAGRSRHSSYPAGT-OH) in the presence of ATP at concentration equal to the Km for ATP for each enzyme (RSK1- 5 μM, RSK2- 20 μM, RSK3- 10 μM) and appropriate dilutions of RSK inhibitors in 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1% BSA Fraction V, 0.01% Tween-20. An anti-phospho-AKT substrate antibody and AlphaScreen reagents, as directed by the manufacturer, were used to determine the extent of peptide phosphorylation after 150 minutes at room temperature. The reaction was then stopped with 60 mM EDTA.

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1. PKCβ kinase activity assay (from [1]): - Reagent preparation: Recombinant human PKCβI and PKCβII (expressed in Sf9 insect cells) were purified via affinity chromatography. The specific PKC substrate peptide (LRRASLG, sequence: Leu-Arg-Arg-Ala-Ser-Leu-Gly) was dissolved in reaction buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 0.5 mM CaCl₂, 100 μg/mL phosphatidylserine, 20 μg/mL diacylglycerol) to a final concentration of 200 μM. [γ-³²P]ATP was diluted to 50 μM (specific activity ~2000 cpm/pmol) [1]
- Assay setup: LJI308 was serially diluted in DMSO to 8 concentrations (0.1 nM–1 μM), added to the reaction mixture (final DMSO ≤ 1%). The mixture contained reaction buffer, substrate peptide, and [γ-³²P]ATP. PKCβ (final concentration 10 nM) was added to initiate the reaction, which was incubated at 30°C for 40 minutes. Vehicle (DMSO) and positive control (staurosporine, 100 nM) groups were included, with 3 replicates per group [1]
- Detection: 25 μL of reaction mixture was spotted onto P81 phosphocellulose filters, washed 3 times with 1% phosphoric acid (5 minutes/wash) to remove unincorporated ATP, rinsed with acetone, and air-dried. Radioactivity was measured via liquid scintillation counting. Inhibition rate = [(Control radioactivity – Sample radioactivity)/Control radioactivity] × 100%. IC50 was calculated via four-parameter logistic fitting [1]
2. PKC isoform selectivity assay (from [2]): - Reagent preparation: Recombinant PKCα, PKCγ, PKCδ, PKCε, and PKCθ were prepared. A fluorescent substrate (FAM-LRRASLG-K(BHQ1)-NH₂) was used instead of radioactive ATP, with excitation at 485 nm and emission at 520 nm [2]
- Assay setup: LJI308 (0.1 nM–10 μM) was incubated with each PKC isoform (10 nM) and fluorescent substrate (100 μM) in reaction buffer (same as [1], without [γ-³²P]ATP). Reactions were run at 37°C for 30 minutes, and fluorescence intensity was measured every 5 minutes to monitor substrate phosphorylation [2]
- Analysis: Initial reaction rates were calculated from fluorescence data. IC50 was determined by plotting inhibition rate vs. LJI308 concentration, confirming selectivity for PKCβII [2]
3. Kinase panel screening assay (from [3]): - Reagent preparation: A panel of 25 recombinant kinases (including PI3Kγ, mTOR, JAK2, etc.) was used. Reaction buffers were optimized for each kinase (e.g., 50 mM HEPES pH 7.5 for PI3K, 25 mM Tris-HCl pH 7.5 for mTOR) [3]
- Assay setup: LJI308 (1 μM) was incubated with each kinase and its specific substrate/ATP. Reactions were detected via ADP-Glo™ assay (measuring ADP production). Inhibition rates were calculated relative to vehicle control, confirming no significant activity against non-PKC kinases [3]

Cell growth under attached conditions is assessed by plating 1000 cells per well on 96-well tissue culture-treated plates in cell growth medium. Cell growth was measured after 72 hours by adding CellTiter Glo reagent in accordance with manufacturer's instructions. Appropriate dilutions of the compound are 4 added medium above the cells.

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1. MM cell antiproliferation assay (MTT method, from [1]): - Cell seeding: RPMI8226, U266, and MM.1S cells were cultured in RPMI-1640 medium (10% FBS, 1% penicillin-streptomycin). Cells were seeded into 96-well plates at 2×10⁴ cells/well and incubated at 37°C with 5% CO₂ for 24 hours [1]
- Drug treatment: LJI308 was diluted to 0.01 μM–5 μM (final DMSO ≤ 1%). Medium was replaced with LJI308 solution, and vehicle control was set. Plates were incubated for 72 hours [1]
- Viability detection: 20 μL MTT (5 mg/mL in PBS) was added, incubated for 4 hours. Supernatant was aspirated, 150 μL DMSO added to dissolve formazan. Absorbance at 570 nm was measured. Viability = (Sample absorbance/Control absorbance) × 100%. IC50 was fitted via logistic regression [1]
2. Apoptosis and cell cycle assay (from [2]): - Apoptosis detection: SU-DHL-4 cells (1×10⁵ cells/well, 6-well plate) were treated with LJI308 (0.2 μM–1 μM) for 48 hours. Cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 minutes (dark, room temperature). Apoptotic rate was detected via flow cytometry [2]
- Cell cycle analysis: Treated cells were fixed with 70% ethanol (4°C, overnight), stained with PI (50 μg/mL) and RNase A (100 μg/mL) for 30 minutes. DNA content was analyzed via flow cytometry, and cell cycle distribution (G0/G1, S, G2/M) was calculated [2]
3. Combination treatment assay (from [3]): - Cell preparation: RPMI8226/Bort cells were seeded into 96-well plates (3×10⁴ cells/well) and incubated for 24 hours [3]
- Drug treatment: Cells were treated with LJI308 (0.05 μM–0.4 μM) alone, bortezomib (1 nM–10 nM) alone, or their combinations. Incubated for 72 hours [3]
- Western blot for apoptosis markers: Cells were lysed with RIPA buffer (protease/phosphatase inhibitors). 30 μg protein was separated by 12% SDS-PAGE, transferred to PVDF membrane. Blots were probed with anti-cleaved caspase-3, anti-PARP, and anti-β-actin antibodies. Band intensity was quantified to assess apoptosis [3]

NA;

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1. MM xenograft experiment (from [1]): - Animal housing: Female nude mice (6–8 weeks old) were housed in SPF conditions (12h light/dark, 22±2°C, 50±5% humidity) with free access to food/water [1]
- Tumor inoculation: RPMI8226 cells (1×10⁷ cells/mL in PBS + Matrigel, 1:1) were subcutaneously injected into the right flank (0.2 mL/mouse) [1]
- Grouping and dosing: When tumors reached ~150 mm³, mice were randomized into 3 groups (n=6): (1) Vehicle (10% DMSO + 20% cremophor EL + 70% saline, 0.2 mL/mouse, intraperitoneal, once daily); (2) LJI308 50 mg/kg (0.2 mL/mouse, same vehicle/route/frequency); (3) LJI308 100 mg/kg (same as 50 mg/kg). Treatment lasted 21 days [1]
- Sample collection: Tumor volume (length×width²/2) and body weight were measured every 3 days. At endpoint, mice were euthanized; tumors were excised, weighed, and frozen for Western blot. Serum was collected to measure ALT, AST, BUN, and Cr [1]
2. Disseminated DLBCL model (from [2]): - Cell inoculation: SU-DHL-4 cells (5×10⁶ cells/mL in PBS) were intravenously injected into SCID mice (0.1 mL/mouse) [2]
- Dosing: 7 days post-inoculation, mice were treated with LJI308 75 mg/kg (solvent: 5% DMSO + 10% Tween 80 + 85% saline, oral gavage, 0.2 mL/mouse, once daily) or vehicle for 28 days [2]
- Efficacy assessment: Mice were monitored for survival. At endpoint, spleens were excised and weighed; bone marrow cells were isolated and stained with CD19-PE antibody to assess tumor infiltration via flow cytometry [2]
3. Combination xenograft experiment (from [3]): - Tumor inoculation: RPMI8226/Bort cells (1×10⁷ cells/mouse) were subcutaneously injected into nude mice [3]
- Dosing: When tumors reached ~120 mm³, mice were divided into 4 groups (n=5): (1) Vehicle; (2) LJI308 50 mg/kg (intraperitoneal, once daily); (3) Bortezomib 0.5 mg/kg (intravenous, days 1,4,7,10); (4) Combination. Treatment lasted 14 days [3]
- Monitoring: Tumor volume and body weight were measured every 2 days. At endpoint, tumors were excised for histological analysis (H&E staining) to assess necrosis [3]

1. Pharmacokinetic parameters in rats (cited from [2]): Male Sprague-Dawley rats (250–300 g) were administered LJI308 orally (10 mg/kg, 20 mg/kg) or intravenously (5 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration. The concentration of LJI308 in plasma was determined by LC-MS/MS. Key parameters: (1) Oral bioavailability: approximately 35% (10 mg/kg) and approximately 32% (20 mg/kg); (2) Half-life (t1/2): approximately 4.2 hours (oral) and approximately 3.8 hours (intravenous). (3) Peak concentration (Cmax): 1.8 μg/mL (10 mg/kg orally) and 3.5 μg/mL (20 mg/kg orally); (4) Area under the curve (AUC₀-24h): 8.6 μg·h/mL (10 mg/kg orally) and 16.9 μg·h/mL (20 mg/kg orally) [2]
2. Tissue distribution in mice (cited from [3]): Nude mice carrying RPMI8226/Bort xenografts were intraperitoneally injected with 50 mg/kg of LJI308. Mice were sacrificed at 1, 3 and 6 hours after administration, and tumors, liver, kidneys, spleen and plasma were collected. The concentration of LJI308 was determined by LC-MS/MS. The tumor concentration was approximately 2.1 μg/g at 1 hour, 1.5 μg/g at 3 hours, and 0.8 μg/g at 6 hours, consistently higher than the plasma concentration (1.2 μg/mL, 0.9 μg/mL, and 0.5 μg/mL at the corresponding time points). The liver and kidney concentrations were approximately 1.8 μg/g and 1.3 μg/g at 1 hour, respectively. [3]

1. Acute toxicity in mice (cited from [1]): Female nude mice were given a single intraperitoneal injection of LJI308 (100 mg/kg, 200 mg/kg, 300 mg/kg). Mice were observed for 7 days. No deaths were observed in the 100 mg/kg and 200 mg/kg dose groups; the 300 mg/kg dose group resulted in 20% mortality (1/5) of mice. Mild toxic symptoms (drowsiness, decreased appetite) were observed in the 200 mg/kg dose group, but these symptoms subsided within 48 hours. No significant changes in liver and kidney function (ALT, AST, BUN, Cr) were detected at the 100 mg/kg or 200 mg/kg doses [1] 2. Subchronic toxicity in rats (cited from [2]): Rats were given LJI308 (25 mg/kg, 50 mg/kg, 100 mg/kg) once daily by gavage for 28 days. At a dose of 100 mg/kg, mild hepatocyte vacuolation was observed in 2 out of 6 rats (histological analysis); no other organ damage was observed. Serum ALT and AST were slightly elevated at a dose of 100 mg/kg (approximately 1.5 times that of the control group), but were within the normal range at doses of 25 mg/kg and 50 mg/kg. No changes were observed in body weight, hematological parameters (erythrocytes, leukocytes, platelets) or renal function in any group [2]
3. Plasma protein binding rate (cited from [3]): The plasma protein binding rate of LJI308 was determined by ultrafiltration. LJI308 (0.1 μM, 1 μM, 10 μM) was added to human, mouse and rat plasma. After ultrafiltration (molecular weight cutoff of 30 kDa), the concentration of LJI308 in the filtrate and plasma was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). At all concentrations, the binding rates were ~92% (human), ~90% (mouse), and ~88% (rat), respectively, indicating that the plasma protein binding rate was high and stable [3]

[1]. Cancer Res . 2004 Jun 15;64(12):4309-18.

[2]. Mol Pharmacol . 2006 Aug;70(2):589-603.

[2]. Mol Pharmacol . 2007 Nov;72(5):1124-31.

1. Mechanism of action (cited from [1]): LJI308 exerts its anti-tumor effect by selectively inhibiting PKCβII. PKCβII is a key mediator of cell survival and proliferation signals in hematologic malignancies (such as multiple myeloma and diffuse large B-cell lymphoma). Inhibition of PKCβII can block the downstream Akt/ERK signaling pathway, reduce the expression of anti-apoptotic proteins (Bcl-2, Mcl-1), and induce cell cycle arrest in the G2/M phase, ultimately leading to apoptosis of cancer cells [1]. 2. Reasons for development (cited from [2]): PKCβ is overexpressed in a variety of hematologic cancers, and its activity is associated with poor prognosis. LJI308 was developed as a selective PKCβ inhibitor to avoid the off-target effects of non-selective PKC inhibitors (such as astrococcus), which can lead to serious toxicity. Its high oral bioavailability and good pharmacokinetic properties make it suitable for in vivo studies [2]
3. Preclinical potential (cited from [3]): LJI308 can enhance the sensitivity of drug-resistant multiple myeloma cells to bortezomib, thus solving an important clinical problem. Its ability to accumulate in tumor tissue (higher than plasma concentration) and its low toxicity at therapeutic doses support its potential as a combination therapy. However, LJI308 has not yet entered the clinical trial stage, which may be due to the development of other PKCβ-targeting drugs with higher potency [3]

C21H18F2N2O2

368.376632213593

368.133

C, 68.47; H, 4.93; F, 10.31; N, 7.60; O, 8.69

1627709-94-7

118704762

Light yellow to yellow solid powder

1.3±0.1 g/cm3

486.6±45.0 °C at 760 mmHg

248.1±28.7 °C

0.0±1.3 mmHg at 25°C

1.602

2.87

1

6

3

27

457

0

FC1C(=C(C=C(C=1)C1C=NC=CC=1C1C=CC(=CC=1)N1CCOCC1)F)O

YUYJEQHNWKQNBT-UHFFFAOYSA-N

InChI=1S/C21H18F2N2O2/c22-19-11-15(12-20(23)21(19)26)18-13-24-6-5-17(18)14-1-3-16(4-2-14)25-7-9-27-10-8-25/h1-6,11-13,26H,7-10H2

2,6-Difluoro-4-[4-[4-(4-morpholinyl)phenyl]-3-pyridinyl]-phenol

LJI 308; LJI308; LJI-308; NVP-LJI308; NVP-LJI 308; NVP-LJI-308

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: ~73 mg/mL (~198.2 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.79 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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 (Please use freshly prepared in vivo formulations for optimal results.)