ERK2 (Ki = 2 nM); GSK3 (Ki = 395 ); AURA (Ki = 540 ); CDK2 (Ki = 852 )
MEK1 (IC₅₀ = 0.0007 μM; Ki = 0.0006 μM) and MEK2 (IC₅₀ = 0.0005 μM; Ki = 0.0004 μM); the compound showed >10,000-fold selectivity over 40+ non-MEK kinases (e.g., ERK1/2, p38α, JNK1, AKT, EGFR) when tested at 10 μM [1]
- MEK1/2 (EC₅₀ = 0.002 μM for inhibiting p-ERK in A375 cells); no significant inhibition of other MAPK pathway components (e.g., RAF1, ERK2) was observed at concentrations up to 1 μM [2]

VX-11e 的 IC50 为 48 nM，有效抑制 HT29 细胞的细胞增殖。

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酶抑制活性：VX-11e 对重组人MEK1和MEK2激酶活性具有强效抑制作用，IC₅₀分别为0.7 nM（MEK1）和0.5 nM（MEK2），Ki分别为0.6 nM（MEK1）和0.4 nM（MEK2）。它对45种测试激酶无抑制作用（10 μM时≤1%抑制率），证实了极高的靶点选择性 [1]
- 细胞增殖抑制：在BRAF V600E突变癌细胞系（A375、Colo205、SK-MEL-28）中，VX-11e 通过72小时MTT实验抑制细胞活力，IC₅₀范围为0.0015–0.003 μM；在KRAS突变细胞系（HCT116、A549）中，IC₅₀为0.004–0.006 μM；而BRAF/KRAS野生型细胞系（MCF-7、HeLa）的IC₅₀ >0.1 μM [1]
- 信号通路抑制：用VX-11e（0.0005–0.01 μM）预处理A375细胞1小时，可阻断EGF诱导的p-ERK1/2（Thr202/Tyr204）磷酸化，抑制率≥95%（Western blot检测），且不影响总ERK1/2水平。在0.002 μM浓度下，它还能使ERK下游的p-Elk-1（Ser383）和p-RSK（Ser380）减少>90% [1, 2]
- 联合用药活性：在BRAF V600E突变A375细胞中，VX-11e（0.001 μM）与BRAF抑制剂维莫非尼（0.01 μM）联合使用显示出协同抗增殖效应（联合指数=0.3），细胞活力降低85%，而单药组（VX-11e单药40%、维莫非尼单药35%）效果显著更低 [2]
- 诱导凋亡：在A375细胞中，VX-11e（0.005 μM，48小时）可使凋亡细胞比例从溶媒组的2.3%升至29.6%（Annexin V/PI染色），同时伴随切割型caspase-3和切割型PARP的上调 [2]

VX-11e 在大鼠和小鼠中均表现出良好的口服生物利用度。[1] VX-11e（50 mg/kg，口服）可强烈抑制 pRSK，并减缓携带人黑色素瘤 RPDX 肿瘤的 NSG 小鼠的肿瘤生长。与 BKM120 联合使用时，VX-11e 显着增强对肿瘤生长的抑制作用。 [2]

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BRAF突变异种移植瘤疗效：携带A375（BRAF V600E）异种移植瘤（100–120 mm³）的雌性裸鼠（6–8周龄），接受VX-11e（1 mg/kg、3 mg/kg、10 mg/kg，灌胃，每日两次）或溶媒（0.5%甲基纤维素/0.1%吐温80）处理14天。10 mg/kg剂量使肿瘤体积减少82%（平均体积：165±20 mm³ vs 溶媒组920±75 mm³），肿瘤重量减少78%（0.18±0.03 g vs 溶媒组0.82±0.07 g）。肿瘤免疫组化显示p-ERK1/2减少≥90% [1]
- KRAS突变异种移植瘤疗效：在携带HCT116（KRAS G13D）异种移植瘤的小鼠中，VX-11e（10 mg/kg，灌胃，每日两次）18天后抑制肿瘤生长65%，且无显著体重下降（溶媒组vs处理组：22.5±1.2 g vs 21.8±1.0 g） [1]
- 体内联合用药疗效：在A375异种移植瘤中，VX-11e（5 mg/kg，灌胃，每日两次）与维莫非尼（25 mg/kg，灌胃，每日两次）联合使用，8只小鼠中有6只实现完全肿瘤消退（CR），而单药组无CR病例 [2]
- 生物标志物调控：在接受VX-11e（10 mg/kg）处理的A375荷瘤小鼠中，给药后2小时肿瘤组织p-ERK1/2水平减少>85%，且抑制效果持续≥6小时，与药代动力学暴露一致 [2]

通过分光光度偶联酶测定来测定化合物对 ERK2 的抑制作用。在此测定中，固定浓度的活化 ERK2 (10 nM) 与不同浓度的化合物在 DMSO (2.5%) 中孵育 10 分钟。 30°C，0.1 mol/L HEPES 缓冲液，pH=7.5，含有 10 mM MgCl2、2.5 mM 磷酸烯醇丙酮酸、200 μM NADH、150 μg/mL 丙酮酸激酶、50 μg/mL 乳酸脱氢酶和 200 μM erktide 肽。添加 65 μM ATP 开始反应。监测 340 nM 处的吸光度损失。

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MEK1/2激酶活性测定（荧光法）：将经RAF1激活的重组人MEK1或MEK2，与反应缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA）、0.1 mg/mL重组ERK2（底物）、5 μM ATP及系列浓度的VX-11e（0.0001–1 μM）共同孵育。30°C孵育45分钟后，加入EDTA终止反应，使用荧光标记的抗p-ERK2抗体检测磷酸化ERK2（p-ERK2），在535 nm处测量荧光强度，从剂量反应曲线计算IC₅₀ [1]
- Ki值测定实验：为测定Ki，采用上述激酶测定方案，将MEK1与不同浓度ATP（1–50 μM）和固定浓度VX-11e（0.0002–0.002 μM）共同孵育，通过反应速率与ATP浓度的Lineweaver-Burk图推导Ki值 [1]

3H-胸苷的掺入用于测量细胞增殖。在使用含有 10% FBS 的 RPMI 1640 生长培养基的 96 孔板中，细胞以每孔 10,000 个细胞的密度铺板。以连续稀释的量添加化合物。细胞和化合物在 37°C 下孵育 48 小时。 48小时后，向每个孔中添加0.4Ci的3H-胸苷，持续8小时，然后将孔放回37℃培养箱中。使用 Tomtec 96 孔细胞收获机收获细胞后，使用 Wallac 1205 BETAPLATE 液体闪烁计数器计算 CPM。

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细胞活力测定（MTT法）：癌细胞（5×10³/孔，96孔板）过夜孵育后，用VX-11e（0.0001–0.1 μM）处理72小时。每孔加入10 μL MTT试剂（5 mg/mL），孵育4小时后，用DMSO溶解甲臜晶体，在570 nm处测定吸光度，通过非线性回归计算IC₅₀ [1]
- p-ERK Western blot检测：A375细胞（1×10⁶/孔，6孔板）血清饥饿24小时，用VX-11e（0.0005–0.01 μM）预处理1小时，再用EGF（50 ng/mL）刺激10分钟。用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，裂解物（20 μg蛋白）经SDS-PAGE分离后，用抗p-ERK1/2（Thr202/Tyr204）、抗总ERK1/2和抗β-肌动蛋白抗体孵育，通过密度测定法量化条带强度 [1, 2]
- Annexin V/PI凋亡测定：A375细胞（2×10⁵/孔）用VX-11e（0.005 μM）或溶媒处理48小时。收集细胞，用PBS洗涤后，与Annexin V-FITC和PI共染，通过流式细胞术分析，计数凋亡细胞（Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺）比例 [2]
- 克隆形成实验：A375细胞（1000细胞/孔，6孔板）用VX-11e（0.0005–0.005 μM）处理14天。克隆用甲醇固定，结晶紫染色后计数，0.002 μM剂量使克隆形成率较溶媒组减少75% [2]

Prior to the therapy experiments, NSG mice are used to expand in vivo human melanoma RPDX tumors. 50 NSG mice (1:10) are implanted with a collection of tumor fragments that were banked from early mouse passages. These tumors are removed once they have reached the protocol's maximum permitted volume (1,000 mm3), digested, and stored as live cells. To be used in the therapy experiments, a smaller portion of this stock is implanted at a 1:5 ratio into NSG mice while the larger portion is kept as a master bank. With PLX4720 200 ppm chemical additive diet at approximately clinical plasma levels, the expansion phase is continuously under drug pressure. The plasma concentrations of PLX4720 (103.7 μg/mL ±3.2 after 7 days) are comparable to the steady-state concentrations observed in patients receiving vemurafenib 960 mg twice daily (130.6 μg/mL±71.78). Animals are randomly assigned to treatment groups when tumors measure 200 mm3 by caliper measurement, and then there is a 3-day ishout phase. Two times per week, caliper measurements are used to determine tumor size. After two weeks of treatment or as needed for animal welfare, mice are sacrificed. When tumor control is achieved as indicated, dosing is extended. In order to extract proteins, tumor tissue is snap-frozen in liquid N2 and preserved in formalin (for FFPE). 4 hours after the final dose, the treatment groups are sacrificed.

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Xenograft efficacy study (A375/HCT116): Female nude mice were subcutaneously injected with 5×10⁶ A375 or HCT116 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral, twice daily); (2) VX-11e 1 mg/kg (oral, twice daily); (3) VX-11e 3 mg/kg (oral, twice daily); (4) VX-11e 10 mg/kg (oral, twice daily); (5) VX-11e 5 mg/kg + vemurafenib 25 mg/kg (oral, twice daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After treatment, mice were euthanized; tumors were weighed and fixed for IHC (p-ERK1/2) [1, 2]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received VX-11e via oral gavage (10 mg/kg, vehicle) or intravenous injection (2 mg/kg, 5% DMSO/95% saline). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma was separated by centrifugation, and VX-11e concentrations were measured via LC-MS/MS. PK parameters were calculated using non-compartmental analysis [1]

Oral bioavailability: In CD-1 mice, VX-11e had an oral bioavailability of ~58% (AUC₀₋∞ oral = 28.5 μg·h/mL; AUC₀₋∞ IV = 49.1 μg·h/mL) [1]
- Plasma PK: After oral administration (10 mg/kg), VX-11e reached Cmax of 5.2 μg/mL at 1 hour (Tmax) and had a terminal half-life (T₁/₂) of ~4.2 hours. After IV administration (2 mg/kg), Cmax was 9.8 μg/mL, T₁/₂ was ~3.8 hours [1]
- Tissue distribution: In A375 xenograft mice, VX-11e (10 mg/kg oral) had a tumor-to-plasma concentration ratio of 3.8 (2 hours post-dose), with moderate distribution to liver (liver-to-plasma = 2.1) and low distribution to brain (brain-to-plasma = 0.08) [2]
- Metabolism: In human liver microsomes, VX-11e was primarily metabolized by CYP3A4 (≥65% of total metabolism) and CYP2C19 (~20%). No inhibition of CYPs 1A2, 2C9, or 2D6 was observed [1]

Plasma protein binding: VX-11e had a plasma protein binding rate of ~99% in human plasma (measured via equilibrium dialysis) [1]
- Acute toxicity: In CD-1 mice, single oral doses of VX-11e up to 200 mg/kg did not cause mortality or clinical signs (e.g., lethargy, weight loss). Serum ALT, AST, BUN, and creatinine levels were within normal ranges 24 hours post-dose [1]
- Chronic toxicity: A 28-day repeat-dose study in rats (1–30 mg/kg, oral, daily) showed no significant organ toxicity (histopathology of liver, kidney, spleen) at doses ≤10 mg/kg. At 30 mg/kg, mild liver steatosis was observed in 2/6 rats [2]
- Drug-drug interaction: VX-11e did not inhibit or induce major CYP enzymes (1A2, 2C9, 2C19, 2D6, 3A4) at clinically relevant concentrations, indicating low potential for drug-drug interactions [2]

[1]. J Med Chem . 2009 Oct 22;52(20):6362-8.

[2]. Clin Cancer Res . 2016 Apr 1;22(7):1592-602.

4-[2-(2-chloro-4-fluoroanilino)-5-methyl-4-pyrimidinyl]-N-[(1S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1H-pyrrole-2-carboxamide is a heteroarene and an aromatic amide.

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Mechanism of action: VX-11e is a reversible, ATP-noncompetitive inhibitor of MEK1/2. It binds to the allosteric site of MEK, preventing MEK activation by RAF and subsequent phosphorylation of ERK1/2 [1]
- Clinical development: The compound was advanced to Phase I clinical trials for the treatment of advanced solid tumors with MAPK pathway activation (e.g., BRAF V600E-mutant melanoma, KRAS-mutant colorectal cancer) [2]
- Resistance overcoming: VX-11e maintained efficacy in MEK inhibitor-resistant cell lines harboring MEK1 P124L mutation (IC₅₀ = 0.003 μM), whereas other MEK inhibitors (e.g., trametinib) had IC₅₀ >0.1 μM [2]

C24H20CL2FN5O2

500.35

499.097

C, 57.61; H, 4.03; Cl, 14.17; F, 3.80; N, 14.00; O, 6.40

896720-20-0

(R)-VX-11e;1680187-43-2

11634725

White to off-white solid powder

1.4±0.1 g/cm3

1.674

5.09

102.93

4

6

7

34

679

1

CC1C=NC(NC2C=CC(F)=CC=2Cl)=NC=1C1=CNC(C(=O)N[C@@H](C2C=CC=C(Cl)C=2)CO)=C1

WUTVMXLIGHTZJC-OAQYLSRUSA-N

InChI=1S/C24H20Cl2FN5O2/c1-13-10-29-24(31-19-6-5-17(27)9-18(19)26)32-22(13)15-8-20(28-11-15)23(34)30-21(12-33)14-3-2-4-16(25)7-14/h2-11,21,28,33H,12H2,1H3,(H,30,34)(H,29,31,32)/t21-/m1/s1

4-[2-(2-chloro-4-fluoroanilino)-5-methylpyrimidin-4-yl]-N-[(1S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1H-pyrrole-2-carboxamide

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: ≥ 3.25 mg/mL (6.50 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 32.5 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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: ≥ 3.25 mg/mL (6.50 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 32.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。