Lymphocyte-specific protein tyrosine kinase (Lck): IC₅₀ ≈ 4 nM (recombinant human Lck kinase activity); non-Lck Src family kinases: Src (IC₅₀ > 100 nM), Fyn (IC₅₀ > 100 nM), Yes (IC₅₀ > 200 nM); other kinases: EGFR (IC₅₀ > 1000 nM), Abl (IC₅₀ > 1000 nM), demonstrating high selectivity for Lck [1]

WH-4-023 and the 2-substituted version on Lck show comparable increases in potency [1].

---

In recombinant human Lck kinase activity assays: WH-4-023 (0.1 nM–100 nM) concentration-dependently inhibited Lck activity. At 4 nM, inhibition rate reached ~50% (IC₅₀); at 100 nM, inhibition rate exceeded 95%. It showed minimal cross-inhibition of other Src family kinases (e.g., Src inhibition <10% at 100 nM) and no significant activity against non-Src kinases (e.g., EGFR inhibition <5% at 1000 nM) [1]
- In mouse splenic T cells activated by anti-CD3/CD28: WH-4-023 (1 μM–10 μM) suppressed T cell activation. At 10 μM: (1) IL-2 secretion (measured by ELISA) decreased by ~70% compared to the solvent control; (2) CD69 (early T cell activation marker, detected by flow cytometry) positive cells reduced by ~65%; (3) Western blot revealed reduced phosphorylation of Lck (Tyr394) and its downstream effector ZAP-70 (Tyr493), with no change in total Lck/ZAP-70 protein levels [1]
- In mixed lymphocyte reaction (MLR) assays (mouse splenocytes from C57BL/6 and BALB/c mice co-cultured): WH-4-023 (0.3 μM–10 μM) concentration-dependently inhibited T cell proliferation. At 10 μM, [³H]-thymidine incorporation (proliferation marker) decreased by ~80% vs. control [1]

In mouse delayed-type hypersensitivity (DTH) model (DNFB-induced ear inflammation): Mice were divided into control (solvent) and WH-4-023 groups (3 mg/kg, 10 mg/kg, 30 mg/kg, 100 mg/kg, oral gavage). Mice were sensitized with DNFB on day 0, challenged on day 5, and drugs were administered once daily from day 3 to day 5. Compared to control: (1) Ear swelling (inflammation index) was reduced by ~20% (3 mg/kg), ~32% (10 mg/kg), ~40% (30 mg/kg), and ~65% (100 mg/kg) at 24 hours post-challenge; (2) Histopathology of ear tissues showed decreased inflammatory cell infiltration (neutrophils, lymphocytes) in drug-treated groups [1]
- In rat carrageenan-induced paw edema model: Rats were divided into control (solvent) and WH-4-023 groups (30 mg/kg, 100 mg/kg, oral gavage). Carrageenan was injected into the hind paw on day 0, and drugs were administered 1 hour before injection. Paw volume was measured at 1, 3, 6 hours post-injection. At 3 hours (peak edema): (1) Paw volume increase was inhibited by ~35% (30 mg/kg) and ~55% (100 mg/kg) vs. control; (2) Serum TNF-α and IL-6 levels (pro-inflammatory cytokines, measured by ELISA) were reduced by ~40% (30 mg/kg) and ~60% (100 mg/kg) [1]

Recombinant human Lck kinase activity assay: Recombinant human Lck (residues 22–509, including the kinase domain) was expressed in E. coli and purified via nickel-chelate affinity chromatography. The kinase reaction was performed in a 50 μL volume containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 5 μM ATP (including [γ-³²P]ATP for radioactivity labeling), 20 μM Lck-specific peptide substrate (sequence: EAIYAAPFAKKK), and WH-4-023 at concentrations of 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM (solvent as control). The mixture was incubated at 30°C for 45 minutes, then terminated by adding 25 μL of 0.5 M EDTA. 40 μL of the reaction mixture was spotted onto phosphocellulose filter paper, which was washed three times with 0.75% phosphoric acid (10 minutes each) to remove unincorporated ATP. Filters were dried, placed in scintillation fluid, and radioactivity was measured using a liquid scintillation counter. Inhibition rates were calculated as (1 – radioactivity of drug group / radioactivity of control group) × 100%, and the IC₅₀ value was determined by fitting the inhibition rates to a four-parameter logistic dose-response curve using graphing software [1]
- Non-Lck kinase selectivity assay: The same reaction conditions as above were used, with recombinant Src, Fyn, Yes, EGFR, or Abl kinase domains and their respective specific peptide substrates. WH-4-023 was tested at concentrations up to 1000 nM, and kinase activity was quantified via scintillation counting to assess cross-inhibition [1]

Mouse splenic T cell isolation and activation assay: Spleens were harvested from 6–8 week-old C57BL/6 mice, and single-cell suspensions were prepared by mechanical dissociation and red blood cell lysis. T cells were enriched via nylon wool column separation. Isolated T cells (2×10⁶ cells/mL) were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were pretreated with WH-4-023 (0 μM, 1 μM, 3 μM, 10 μM) for 1 hour at 37°C (5% CO₂), then stimulated with plate-bound anti-mouse CD3 (5 μg/mL) and soluble anti-mouse CD28 (2 μg/mL) for 24 hours [1]
- IL-2 secretion detection (ELISA): After 24 hours of stimulation, cell culture supernatants were collected and centrifuged at 1000×g for 5 minutes to remove cell debris. Microtiter plates were coated with capture anti-mouse IL-2 antibody overnight at 4°C, blocked with 5% non-fat milk in PBS-Tween 20 for 1 hour at room temperature, and then incubated with supernatants (or IL-2 standards) for 2 hours. Plates were washed, incubated with biotin-conjugated detection anti-mouse IL-2 antibody for 1 hour, followed by streptavidin-HRP for 30 minutes. TMB substrate was added, the reaction was stopped with 2 M H₂SO₄, and absorbance was measured at 450 nm. IL-2 concentrations were calculated using a standard curve [1]
- CD69 expression detection (flow cytometry): Stimulated T cells were harvested, washed twice with PBS containing 2% fetal bovine serum, and incubated with fluorochrome-conjugated anti-mouse CD69 antibody for 30 minutes at 4°C in the dark. Cells were washed again, resuspended in PBS, and analyzed using a flow cytometer. The percentage of CD69-positive cells was quantified, with unstimulated T cells as a negative control [1]
- Lck/ZAP-70 phosphorylation detection (Western blot): Stimulated T cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. 30 μg of total protein per lane was separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour at room temperature. Membranes were incubated overnight at 4°C with primary antibodies against phosphorylated Lck (Tyr394), total Lck, phosphorylated ZAP-70 (Tyr493), total ZAP-70, and β-actin (loading control). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, and signals were detected using enhanced chemiluminescence (ECL) reagent. Band intensities were quantified using ImageJ software [1]
- Mixed lymphocyte reaction (MLR) assay: Splenocytes from C57BL/6 mice (responders) and BALB/c mice (stimulators, irradiated with 30 Gy to prevent proliferation) were co-cultured at a 1:1 ratio (2×10⁵ cells/well) in 96-well plates. WH-4-023 (0 μM, 0.3 μM, 1 μM, 3 μM, 10 μM) was added at the start of co-culture. After 72 hours of incubation, 1 μCi of [³H]-thymidine was added to each well, and incubation continued for an additional 18 hours. Cells were harvested onto glass fiber filters, and radioactivity was measured using a beta counter to assess T cell proliferation [1]

Mouse delayed-type hypersensitivity (DTH) model: Female C57BL/6 mice (6–8 weeks old, 18–22 g) were housed in SPF facilities (22–25°C, 12 h light/dark cycle) with free access to food and water. On day 0 (sensitization), the dorsal skin was shaved and painted with 100 μL of 0.5% DNFB in acetone/olive oil (1:1). On day 5 (challenge), 20 μL of 0.2% DNFB was applied to the right ear (left ear as control). WH-4-023 was dissolved in a solvent consisting of 5% DMSO, 10% Cremophor EL, and 85% normal saline. Drug groups received oral gavage of WH-4-023 at doses of 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg (10 μL/g body weight) once daily on days 3, 4, and 5. The control group received the same volume of solvent. At 24 hours post-challenge, mice were euthanized, and ear thickness was measured using a digital caliper (inflammation index = right ear thickness – left ear thickness). Right ear tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE for histopathological analysis of inflammatory cell infiltration [1]
- Rat carrageenan-induced paw edema model: Male Sprague-Dawley rats (200–220 g) were housed in SPF facilities. WH-4-023 was dissolved in the same solvent as the DTH model. Rats were randomly divided into control (solvent) and drug groups (30 mg/kg, 100 mg/kg, oral gavage, n=6/group). One hour after drug administration, 0.1 mL of 1% carrageenan in normal saline was injected subcutaneously into the plantar surface of the right hind paw (left paw as control). Paw volume was measured using a plethysmometer at 1, 3, and 6 hours post-injection (edema index = right paw volume – left paw volume). At 6 hours post-injection, rats were euthanized, and blood was collected via cardiac puncture. Serum was separated by centrifugation (3000×g for 15 minutes), and serum TNF-α and IL-6 levels were measured using rat-specific ELISA kits [1]

In acute toxicity assessment (WH-4-023 in C57BL/6 mice after a single oral dose of 300 mg/kg): no deaths or obvious clinical toxicities (e.g., lethargy, diarrhea, weight loss >10%) were observed within 7 days after administration. Serum biochemical analysis (ALT, AST, creatinine, BUN) at 7 days showed no significant differences between the treatment group and the control group, indicating that no obvious acute hepatotoxicity or nephrotoxicity was observed [1].

[1]. Novel 2-aminopyrimidine carbamates as potent and orally active inhibitors of Lck: synthesis, SAR, and in vivo antiinflammatory activity. J Med Chem. 2006 Aug 10;49(16):4981-91.

N-(2,4-dimethoxyphenyl)-N-[2-[4-(4-methyl-1-piperazinyl)anilino]-4-pyrimidinyl]carbamate (2,6-dimethylphenyl) ester belongs to the piperazine class of compounds. WH-4-023 is a novel, highly effective, and orally available small molecule inhibitor of Lck, belonging to the 2-aminopyrimidine carbamate class of compounds. Its selectivity for Lck is significantly higher than that of other Src family and non-Src kinases, thereby minimizing off-target effects and making it a valuable tool for studying Lck-mediated biological processes [1]. The anti-inflammatory mechanism of WH-4-023 involves inhibiting Lck kinase activity, thereby blocking Lck-dependent T cell activation (reduced IL-2 secretion and CD69 expression) and T cell proliferation (inhibition of mixed lymphocyte responses). This suggests that it has potential therapeutic value in T cell-mediated inflammatory diseases (such as rheumatoid arthritis and psoriasis) and autoimmune diseases [1]
- WH-4-023 showed good oral bioactivity in animal models (delayed-type hypersensitivity and paw edema), with significant anti-inflammatory effects at doses ≥30 mg/kg. This oral activity supports its potential as a lead compound for the development of oral anti-inflammatory drugs targeting Lck [1]

C32H36N6O4

568.67

568.279

837422-57-8

11844351

White to off-white solid powder

1.2±0.1 g/cm3

743.2±70.0 °C at 760 mmHg

403.3±35.7 °C

0.0±2.5 mmHg at 25°C

1.637

3.42

1

9

9

42

826

0

NBTNHSGBRGTFJS-UHFFFAOYSA-N

InChI=1S/C32H36N6O4/c1-22-7-6-8-23(2)30(22)42-32(39)38(27-14-13-26(40-4)21-28(27)41-5)29-15-16-33-31(35-29)34-24-9-11-25(12-10-24)37-19-17-36(3)18-20-37/h6-16,21H,17-20H2,1-5H3,(H,33,34,35)

2,6-dimethylphenyl (2,4-dimethoxyphenyl)(2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)carbamate

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: ≥ 0.77 mg/mL (1.35 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 7.7 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.

---

Solubility in Formulation 2: ≥ 0.77 mg/mL (1.35 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 7.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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