WH-4-023

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 和 Lck 上的 2 取代版本显示出可比的效力增加 [1]。

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在重组人Lck激酶活性实验中：WH-4-023（0.1 nM–100 nM）浓度依赖性抑制Lck活性。4 nM时抑制率达50%（IC₅₀），100 nM时抑制率超过95%；对其他Src家族激酶交叉抑制作用微弱（如100 nM时对Src抑制率<10%），对非Src激酶无显著活性（如1000 nM时对EGFR抑制率<5%）[1]
- 在抗CD3/CD28激活的小鼠脾脏T细胞中：WH-4-023（1 μM–10 μM）抑制T细胞活化。10 μM时：（1）IL-2分泌（ELISA检测）较溶剂对照组减少约70%；（2）CD69（T细胞早期活化标志物，流式细胞术检测）阳性细胞比例降低约65%；（3）Western blot显示Lck（Tyr394）及其下游效应分子ZAP-70（Tyr493）的磷酸化水平降低，而总Lck/ZAP-70蛋白水平无变化[1]
- 在混合淋巴细胞反应（MLR）实验中（C57BL/6小鼠脾细胞与BALB/c小鼠脾细胞共培养）：WH-4-023（0.3 μM–10 μM）浓度依赖性抑制T细胞增殖。10 μM时，[³H]-胸腺嘧啶掺入量（增殖标志物）较对照组减少约80%[1]

在小鼠迟发型超敏反应（DTH，DNFB诱导耳炎症）模型中：小鼠分为对照组（溶剂）和WH-4-023处理组（3 mg/kg、10 mg/kg、30 mg/kg、100 mg/kg，口服灌胃）。第0天用DNFB致敏，第5天激发，药物从第3天至第5天每日给药1次。与对照组相比：（1）激发后24小时耳肿胀度（炎症指数）在3 mg/kg、10 mg/kg、30 mg/kg、100 mg/kg剂量下分别降低约20%、32%、40%、65%；（2）耳组织病理显示药物处理组炎症细胞（中性粒细胞、淋巴细胞）浸润减少[1]
- 在大鼠角叉菜胶诱导足肿胀模型中：大鼠分为对照组（溶剂）和WH-4-023处理组（30 mg/kg、100 mg/kg，口服灌胃）。第0天向 hind paw 注射角叉菜胶，给药在注射前1小时进行。分别在注射后1、3、6小时测量足体积。3小时（肿胀峰值）时：（1）足体积增加量在30 mg/kg、100 mg/kg剂量下分别较对照组抑制约35%、55%；（2）血清促炎细胞因子（TNF-α、IL-6，ELISA检测）水平在30 mg/kg、100 mg/kg剂量下分别降低约40%、60%[1]

重组人Lck激酶活性测定实验：在大肠杆菌中表达重组人Lck（残基22–509，含激酶结构域），通过镍螯合亲和层析纯化。激酶反应在50 μL体系中进行，含50 mM Tris-HCl（pH7.5）、10 mM MgCl₂、1 mM DTT、5 μM ATP（含[γ-³²P]ATP用于放射性标记）、20 μM Lck特异性肽底物（序列：EAIYAAPFAKKK），以及浓度为0.1 nM、0.3 nM、1 nM、3 nM、10 nM、30 nM、100 nM的WH-4-023（溶剂为对照）。混合物30℃孵育45分钟后，加入25 μL 0.5 M EDTA终止反应。取40 μL反应液点样至磷酸纤维素滤纸上，用0.75%磷酸洗涤3次（每次10分钟）以去除未掺入的ATP。滤纸干燥后加入闪烁液，通过液体闪烁计数器测定放射性强度。抑制率按（1–药物组放射性/对照组放射性）×100%计算，使用绘图软件将抑制率拟合至四参数逻辑斯蒂剂量-反应曲线，确定IC₅₀值[1]
- 非Lck激酶选择性实验：采用与上述相同的反应条件，使用重组Src、Fyn、Yes、EGFR或Abl激酶结构域及其各自的特异性肽底物。WH-4-023测试浓度最高达1000 nM，通过闪烁计数量化激酶活性，评估交叉抑制情况[1]

小鼠脾脏T细胞分离与活化实验：从6–8周龄C57BL/6小鼠中获取脾脏，机械解离并裂解红细胞制备单细胞悬液，通过尼龙毛柱分离富集T细胞。纯化的T细胞（2×10⁶个细胞/mL）重悬于含10%胎牛血清、100 U/mL青霉素、100 μg/mL链霉素的RPMI 1640培养基中，在37℃（5% CO₂）下用WH-4-023（0 μM、1 μM、3 μM、10 μM）预处理1小时，随后用包被于平板的抗小鼠CD3（5 μg/mL）和可溶性抗小鼠CD28（2 μg/mL）刺激24小时[1]
- IL-2分泌检测（ELISA）：刺激24小时后收集细胞培养上清，1000×g离心5分钟去除细胞碎片。微孔板用捕获抗小鼠IL-2抗体4℃包被过夜，室温下用含5%脱脂牛奶的PBS-Tween 20封闭1小时，加入上清液（或IL-2标准品）孵育2小时。洗涤后加入生物素偶联的检测抗小鼠IL-2抗体孵育1小时，再加入链霉亲和素-HRP孵育30分钟。加入TMB底物，用2 M H₂SO₄终止反应，测定450 nm处吸光度，通过标准曲线计算IL-2浓度[1]
- CD69表达检测（流式细胞术）：收集刺激后的T细胞，用含2%胎牛血清的PBS洗涤2次，4℃避光条件下用荧光素偶联的抗小鼠CD69抗体孵育30分钟。再次洗涤后重悬于PBS，用流式细胞仪分析，量化CD69阳性细胞百分比，以未刺激T细胞作为阴性对照[1]
- Lck/ZAP-70磷酸化检测（Western blot）：用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解刺激后的T细胞，BCA法测定蛋白浓度。每泳道上样30 μg总蛋白，经10% SDS-PAGE分离后转印至PVDF膜，室温下用5%脱脂牛奶封闭1小时。膜用抗磷酸化Lck（Tyr394）、总Lck、抗磷酸化ZAP-70（Tyr493）、总ZAP-70及抗β-actin（内参）一抗4℃孵育过夜。洗涤后用HRP偶联二抗室温孵育1小时，增强化学发光（ECL）显影，ImageJ定量条带灰度值[1]
- 混合淋巴细胞反应（MLR）实验：将C57BL/6小鼠脾细胞（反应细胞）与BALB/c小鼠脾细胞（刺激细胞，30 Gy照射以抑制增殖）按1:1比例（2×10⁵个细胞/孔）在96孔板中共培养，培养开始时加入WH-4-023（0 μM、0.3 μM、1 μM、3 μM、10 μM）。孵育72小时后，每孔加入1 μCi [³H]-胸腺嘧啶，继续孵育18小时。将细胞收集至玻璃纤维滤纸上，通过β计数器测定放射性强度，评估T细胞增殖[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 (single oral dose of WH-4-023 at 300 mg/kg in C57BL/6 mice): No mortality or significant clinical signs of toxicity (e.g., lethargy, diarrhea, weight loss >10%) were observed within 7 days post-administration. Serum biochemical analysis (ALT, AST, creatinine, BUN) at 7 days showed no significant differences between drug-treated and control mice, indicating no obvious acute hepatotoxicity or nephrotoxicity [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]carbamic acid (2,6-dimethylphenyl) ester is a member of piperazines.

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WH-4-023 is a novel, potent, and orally active small-molecule inhibitor of Lck, belonging to the 2-aminopyrimidine carbamate chemical class. Its high selectivity for Lck over other Src family and non-Src kinases minimizes off-target effects, making it a valuable tool for studying Lck-mediated biological processes [1]
- The anti-inflammatory mechanism of WH-4-023 involves inhibition of Lck kinase activity, which blocks Lck-dependent T cell activation (reduced IL-2 secretion, CD69 expression) and T cell proliferation (suppressed MLR). This suggests potential therapeutic applications in T cell-mediated inflammatory diseases (e.g., rheumatoid arthritis, psoriasis) and autoimmune disorders [1]
- WH-4-023 exhibits good oral bioactivity in animal models (DTH and paw edema), with significant anti-inflammatory effects at doses ≥30 mg/kg. This oral activity supports its potential as a lead compound for developing 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

92.29

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)

配方 1 中的溶解度: ≥ 0.77 mg/mL (1.35 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 7.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.77 mg/mL (1.35 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 7.7 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。