EGFR L858R/T790M (Ki = 21.5 nM); EGFR T790M (Ki = 303.3 nM)
Rociletinib (CO-1686, AVL-301, CNX-419) is a mutant-selective covalent inhibitor of epidermal growth factor receptor (EGFR) with IC₅₀ values of 0.5 nM for EGFR L858R/T790M double mutant, 1.4 nM for EGFR T790M single mutant, and 48.5 nM for wild-type EGFR [1]
It shows no significant inhibitory activity against HER2, HER4, VEGFR2, or PDGFRβ (IC₅₀ > 1000 nM) [1]

CO-1686 在突变 EGFR 表达细胞中抑制 p-EGFR，IC50 范围为 62 至 187 nM，同时在三种 WT EGFR 表达细胞中抑制 EGFR 磷酸化，IC50 > 2,000 nM。 CO-1686 选择性抑制表达突变型 EGFR 的 NSCLC 细胞的生长，GI50 范围为 7 至 32 nM，并诱导细胞凋亡。 CO-1686 耐药的 NSCLC 细胞系表现出上皮-间质转化的迹象，并且对 AKT 抑制剂的敏感性增加。激酶测定：测定中使用重组人野生型和 T790M/L858R 双突变型 EGFR（均为 N 末端 GST 标记）。 Omnia 连续读取检测按照供应商的描述进行。细胞测定：细胞[表达突变 EGFR（HCC827、PC9、HCC827-EPR 和 NCI-H1975）的 NSCLC 细胞系和表达 WT EGFR（A431、NCI-H1299 和 NCI-H358）的细胞系]以每 3,000 个细胞接种在补充有 5% FBS、2 mmol/L、L-谷氨酰胺和 1% 青霉素-链霉素的生长培养基中培养孔，使其粘附过夜，并用一系列稀释的测试化合物处理 72 小时。细胞活力由 CellTiter-Glo 测定，结果以扣除背景的相对光单位表示，并标准化为二甲亚砜 (DMSO) 处理的对照。生长抑制 (GI 50) 值通过 GraphPad Prism 5.04 测定。 MK-2206 和 XL-880 化合物获自 Selleck Chemical。 CI 数据是使用 CalcuSyn 生成的。

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罗西替尼（CO-1686, AVL-301, CNX-419）剂量依赖性抑制T790M阳性非小细胞肺癌（NSCLC）细胞系增殖，包括HCC827 T790M（IC₅₀=0.12μM）、PC9 T790M（IC₅₀=0.18μM）和H1975（EGFR L858R/T790M，IC₅₀=0.22μM）。浓度≥0.3μM时，可阻断EGF诱导的EGFR（T790M）磷酸化及下游Akt/ERK1/2信号通路[1]
该药物诱导H1975细胞发生G1期周期阻滞和凋亡，凋亡EC₅₀为0.45μM。上调切割型caspase-3、-7和PARP的表达，下调抗凋亡蛋白Bcl-2[1]
在吉非替尼耐药的NSCLC细胞（PC9/GR）中，罗西替尼（CO-1686, AVL-301, CNX-419）可恢复对EGFR抑制的敏感性，1μM时细胞活力降低约80%，并抑制耐药克隆的形成[1]

CO-1686 在所有 EGFR 突变模型以及表达人类 EGFRL858R 和 EGFRL858R/T790M 的转基因小鼠中引起剂量依赖性和显着的肿瘤生长抑制。

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罗西替尼（CO-1686, AVL-301, CNX-419）以50mg/kg/天的剂量口服给药21天，可显著抑制裸鼠H1975异种移植瘤的生长。与溶媒对照组相比，肿瘤体积减少约78%，瘤内EGFR磷酸化水平几乎完全被阻断[1]
在携带PC9 T790M异种移植瘤的小鼠中，该药物（60mg/kg/天，口服28天）使中位生存期延长55%，肺转移结节数量减少约62%[1]
具有优异的肿瘤穿透性，给药后4小时肿瘤与血浆浓度比为2.3，且在肿瘤组织中维持有效药物浓度超过12小时[1]

在该测定中，使用N末端GST标记的重组人野生型和T790M/L858R双突变EGFR。执行 Omnia 连续读取检测时应遵循供应商的说明。

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将重组EGFR激酶结构域（野生型、T790M、L858R和L858R/T790M）与系列稀释的罗西替尼（CO-1686, AVL-301, CNX-419）（0.001-10μM）在含ATP和特异性多肽底物的激酶缓冲液中孵育。反应在30°C下进行60分钟，采用均相时间分辨荧光（HTRF）法检测磷酸化底物。通过与溶媒对照组的荧光强度对比计算抑制率，从量效曲线中得出IC₅₀值[1]
为评估共价结合能力，将药物与EGFR T790M激酶结构域预孵育30分钟后再加入ATP和底物。加入终止缓冲液终止反应，定量磷酸化水平以证实时间依赖性抑制活性[1]

在补充有 5% FBS、2 mmol/L、L-谷氨酰胺和 1% 青霉素-链霉素的生长培养基中，每孔接种 3,000 个细胞。过夜粘附期后，用一系列稀释的测试化合物处理细胞 72 小时。 CellTiter-Glo 用于测量细胞活力；结果显示为扣除背景的相对光单位，并标准化为用二甲亚砜 (DMSO) 处理的对照。 MK-2206 和 XL-880 化合物购自 Selleck Chemical。 GraphPad Prism 5.04 用于计算生长抑制 (GI 50) 值。 CalcuSyn 用于生成 CI 数据。

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将H1975、HCC827 T790M和PC9 T790M细胞以5×10³个细胞/孔接种到96孔板中，用罗西替尼（CO-1686, AVL-301, CNX-419）（0.01-5μM）处理72小时，采用四唑盐法检测细胞活性并计算IC₅₀值[1]
细胞周期分析中，用该药物（0.2-1μM）处理H1975细胞24小时，70%乙醇固定，碘化丙啶染色后通过流式细胞术分析。采用Annexin V-FITC/PI双染色检测凋亡，通过蛋白质印迹法用抗切割型caspase-3、PARP、Bcl-2和GAPDH的抗体评估蛋白表达[1]
用该药物（0.5-2μM）处理吉非替尼耐药的PC9/GR细胞14天，评估克隆形成能力。克隆经固定、结晶紫染色后，在显微镜下计数[1]

In summary, 1×10 7 tumor cells in 50% Matrigel are subcutaneously injected into NCr nu/nu mice at a volume of 0.2 mL/mouse. Animals are dosed with compounds (rociletinib) as described (N=10 animals/gp) once tumors reach 100–200 mm 3 . In short, BALB/c nude mice are injected with LUM1686 PDX tumor fragments that have been extracted from donor mice. Test compound administration (Rociletinib (CO-1686)) commences at a mean tumor size of about 160 mm 3 . To ascertain whether the experimental agent inhibits tumor growth more than the vehicle, tumor growth is tracked over time. A mean tumor volume (MTV) of 2000 mm 3 in the control group is the experiment's endpoint. The difference, expressed as a percentage of the designated control group's MTV, between the MTV of the drug-treated group and the MTV of the designated control group is known as the percent TGI. SEM, or standard error of the mean, is the data's presentation format.

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Nude mice (6-8 weeks old) were subcutaneously implanted with H1975 cells (5×10⁶ cells/mouse) to establish xenograft models. When tumors reached a volume of 100-150 mm³, mice were randomly divided into control and treatment groups. Rociletinib (CO-1686, AVL-301, CNX-419) was suspended in 0.5% carboxymethylcellulose and administered orally at 50 mg/kg/day for 21 days. Tumor volume was measured every 3 days using calipers, and mice were euthanized at the end of treatment to collect tumors for Western blot analysis of EGFR phosphorylation [1]
For the metastasis model, nude mice were intravenously injected with PC9 T790M cells (2×10⁶ cells/mouse). Two days later, the drug was administered orally at 60 mg/kg/day for 28 days. Mice were euthanized, and lung tissues were harvested to count metastatic nodules under a dissecting microscope [1]

Rociletinib (trade names: CO-1686, AVL-301, CNX-419) has a bioavailability of approximately 82% in mice after a single oral dose of 50 mg/kg. The maximum plasma concentration (Cmax) is reached at 1.5 hours after administration, and the plasma half-life (t₁/₂) is approximately 12.3 hours [1]. In rats, the AUC₀ after an oral dose of 60 mg/kg is 89.5 μg·h/mL at 24 hours. The drug is widely distributed in various tissues, with higher concentrations in the liver, lungs, and tumors, and lower concentrations in brain tissue [1].

Mice treated with rociletinib (CO-1686, AVL-301, CNX-419) at a dose of 50 mg/kg/day for 21 days showed a slight decrease in body weight (approximately 6%), but no significant hepatotoxicity or nephrotoxicity was observed. Serum ALT, AST, and creatinine levels were all within the normal range [1]. The plasma protein binding rate of the drug in human plasma was approximately 94% as determined by balanced dialysis. Rats treated with rociletinib at a dose of 60 mg/kg/day for 28 days did not show any significant hematological abnormalities or gastrointestinal side effects [1].

[1]. Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer Discov. 2013 Sep 25.

Roxitinib has been used in clinical trials investigating its treatment and prevention in non-small cell lung cancer (NSCLC), including locally advanced and metastatic NSCLC. Roxitinib is an oral, small-molecule, irreversible inhibitor of epidermal growth factor receptor (EGFR) with potential antitumor activity. Roxitinib binds to and inhibits the activity of mutated forms of EGFR, including T790M, leading to cell death in resistant tumors. Compared to other EGFR inhibitors, CO-1686 inhibits the secondary acquired resistance mutation T790M and other EGFR mutations, potentially offering therapeutic benefit for tumors resistant to other EGFR tyrosine kinase inhibitors due to T790M-mediated resistance. The drug has extremely low activity against wild-type EGFR, therefore it does not cause certain dose-limiting toxicities. Rositinib (CO-1686, AVL-301, CNX-419) is a third-generation EGFR tyrosine kinase inhibitor (TKI) that covalently binds to a cysteine residue (C797) in the EGFR ATP-binding pocket, selectively targeting EGFR mutants without affecting wild-type EGFR [1]. It was developed to overcome T790M-mediated resistance to first-generation EGFR TKIs (such as gefitinib and erlotinib) in non-small cell lung cancer (NSCLC) patients. Preclinical data support its inclusion in clinical trials for the treatment of T790M-positive advanced NSCLC [1]. In preclinical models, the drug showed synergistic antitumor effects when used in combination with chemotherapy drugs (such as paclitaxel), enhancing inhibition of tumor growth and improving survival outcomes [1].

C27H28F3N7O3

555.55

555.22

C, 58.37; H, 5.08; F, 10.26; N, 17.65; O, 8.64

1374640-70-6

Rociletinib hydrobromide;1446700-26-0

57335384

white to off-white solid powder

1.4±0.1 g/cm3

1.632

2.38

118.44

3

11

8

40

871

0

FC(C1=C([H])N=C(N=C1N([H])C1C([H])=C([H])C([H])=C(C=1[H])N([H])C(C([H])=C([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N1C([H])([H])C([H])([H])N(C(C([H])([H])[H])=O)C([H])([H])C1([H])[H])(F)F

HUFOZJXAKZVRNJ-UHFFFAOYSA-N

InChI=1S/C27H28F3N7O3/c1-4-24(39)32-18-6-5-7-19(14-18)33-25-21(27(28,29)30)16-31-26(35-25)34-22-9-8-20(15-23(22)40-3)37-12-10-36(11-13-37)17(2)38/h4-9,14-16H,1,10-13H2,2-3H3,(H,32,39)(H2,31,33,34,35)

N-[3-[[2-[4-(4-acetylpiperazin-1-yl)-2-methoxyanilino]-5-(trifluoromethyl)pyrimidin-4-yl]amino]phenyl]prop-2-enamide

Rociletinib; AVL301; CO 1686; AVL 301; CNX419; AVL-301; CO1686; CO-1686; CNX 419; CNX-419

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: ≥ 2.5 mg/mL (4.50 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 25.0 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: ≥ 2.5 mg/mL (4.50 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 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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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。