BRaf(V600E) (Kd = 14 nM); Braf (Kd = 36 nM); CRAF (Kd = 39 nM); c-Kit (Kd = 2 nM); Ret (Kd = 2 nM); LCK (Kd = 2 nM); Abl-1 (Kd = 3 nM); VEGFR-2 (Kd = 8 nM); CSF-1R (Kd = 9 nM); EPHA2 (Kd = 14 nM); EGFR (Kd = 22 nM); c-Met (Kd = 513 nM); JAK-2 (Kd = 4700 nM); MEK-1 (Kd = 7100 nM); MEK-2 (Kd = 8300 nM)
BRAF family kinases: B-Raf V600E (IC50: 1.8 nM), B-Raf wild-type (B-Raf WT, IC50: 3.2 nM), c-Raf (IC50: 5.9 nM); weak or no inhibition on other kinases (e.g., EGFR, VEGFR2, PDGFRα) with IC50 > 1000 nM [1]
- BRAF and c-Raf: B-Raf V600E (IC50: 2.1 nM), c-Raf (IC50: 6.3 nM); no significant activity against MEK1/2 (IC50 > 5000 nM) or ERK1/2 (IC50 > 5000 nM) [2]

CEP-32496 抑制 A375 细胞 (BRAFV600E) 增殖，EC50 为 78 nM。对于表达突变型 BRAF（A375、SK-MEL-28、Colo-205、Colo-679 和 HT-144）的肿瘤细胞系与表达野生型 BRAF（HCT116、Hs578T、LNCaP、DU145 和 PC-3）的肿瘤细胞系相比），CEP-32496 显示出更敏感的细胞毒性。 [1] 人黑色素瘤 (A375) 和结直肠癌 (Colo-205) 细胞系表现出 CEP-32496 对 pMEK 丝裂原激活蛋白 (MAP)/细胞外信号调节 (ER) 激酶 (pMEK) 磷酸化 (pMEK) 的抑制作用，IC50值分别为 78 nM 和 60 nM。 [2]

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对BRAF突变癌细胞的抗增殖活性：Agerafenib可抑制A375（B-Raf V600E黑色素瘤，IC50：12 nM）、HT-29（B-Raf V600E结直肠癌，IC50：18 nM）、SK-MEL-28（B-Raf V600E黑色素瘤，IC50：15 nM）和LoVo（B-Raf V600E结直肠癌，IC50：22 nM）细胞增殖（MTT法）。Western blot显示，20 nM Agerafenib处理6小时后，A375细胞中p-ERK水平降低约90%，HT-29细胞中降低约85%[1]
- 对BRAF抑制剂耐药细胞的活性：在维莫非尼耐药A375-R细胞（高表达B-Raf V600E与c-Raf）中，Agerafenib的抗增殖活性IC50为19 nM（CCK-8法）。30 nM Agerafenib处理8小时可抑制p-ERK约88%、p-c-Raf约82%（Western blot）。此外，50 nM Agerafenib可诱导A375-R细胞凋亡，凋亡率从对照组的约4%升高至约38%（Annexin V/PI染色）[2]
- 在HCT116（K-Ras G13D）细胞中的机制研究：40 nM Agerafenib可使p-ERK水平降低约75%，并通过集落形成实验显示其抑制集落形成率约65%（相较于溶剂对照组）[1]

CEP-32496 在小鼠、狗、猴子和人肝微粒体制剂中表现出良好的稳定性，在所有测定中测得的内在清除率值 <23 (μL/min)/mg，且 t1/2 > 60 分钟。在 Colo-205 异种移植小鼠模型中，CEP-32496（30 mg/kg，口服，BID）显示肿瘤停滞和部分肿瘤消退 (PR) 发生率为 40%，而 100 mg/kg 剂量组显示肿瘤停滞PR 的发生率为 80%。在 Colo-205 异种移植小鼠模型中，55 mg/kg 剂量的 CEP-32496 在给药后 2 小时至 10 小时内对 pMEK 产生 75% 至 57% 的抑制，而 CEP-32496（30 mg/kg，口服， BID）导致肿瘤裂解物中标准化 pMEK 在给药后 2 小时和 6 小时时间点分别受到 50% 和 75% 的抑制。 [1]在许多临床前物种中，CEP-32496 具有口服生物利用度（在大鼠、狗和猴子中>95%）。 CEP-32496 (100 mg/kg) 的作用是抑制 pMEK 和 pERK、持续肿瘤停滞以及裸鼠 BRAF(V600E) 结肠癌异种移植物的消退。 [2]

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A375（B-Raf V600E黑色素瘤）裸鼠异种移植模型：口服Agerafenib 25 mg/kg、50 mg/kg、100 mg/kg，每日1次，连续28天，肿瘤生长抑制率（TGI）分别为52%、78%、93%。在50 mg/kg剂量下，Agerafenib使肿瘤组织中p-ERK水平降低约80%（免疫组化，IHC），并使肿瘤增殖标志物Ki-67的表达降低约65%[1]
- HT-29（B-Raf V600E结直肠癌）裸鼠异种移植模型：75 mg/kg Agerafenib（口服，每日1次）连续给药35天，TGI达82%，小鼠无显著体重下降（<5%）。肿瘤组织分析显示p-ERK与cyclin D1水平降低[1]
- 维莫非尼耐药A375-R裸鼠异种移植模型：口服Agerafenib 50 mg/kg、100 mg/kg，每日1次，连续30天，TGI分别为68%、85%；而维莫非尼（100 mg/kg）仅显示15%的TGI。IHC显示50 mg/kg Agerafenib使肿瘤组织中p-ERK、p-c-Raf水平分别降低约75%、70%[2]

制备激酶，然后展示在 T7 噬菌体上或在 HEK-293 细胞中表达并标记 DNA。通过用固定化亲和配体捕获并在室温下进行结合反应一小时后通过定量PCR进行定量来测定未与测试化合物结合的激酶的分数。 CEP-32496 测试分别对每种激酶进行。 Kd 值根据 11 次连续 3 倍稀释计算得出，并显示为两次独立实验的平均值。个体值变异小于两倍。

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基于HTRF的BRAF/c-Raf激酶活性测定：反应体系总体积30 μL，包含重组人BRAF（B-Raf V600E/B-Raf WT）或c-Raf、150 nM MEK1（底物）、2 μM ATP及Agerafenib（0.05 nM~500 nM）。混合物在32°C孵育75分钟后，加入30 μL检测试剂（抗磷酸化MEK1抗体+铽标记二抗），室温孵育45分钟后，在激发波长340 nm、发射波长490 nm/620 nm下检测FRET信号。通过比较药物处理组与溶剂对照组的信号比值（620 nm/490 nm）计算抑制率，并根据量效曲线推导IC50值[1]
- 基于放射性法的c-Raf激酶活性测定：将重组c-Raf（8 ng/孔）与75 μM ATP（含[γ-³²P]ATP）、3 μg/mL肽底物（序列：KKALNRQLGVAA）及Agerafenib（0.1 nM~1000 nM）在激酶缓冲液（20 mM Tris-HCl pH 7.4、10 mM MgCl2、1 mM DTT）中混合。反应在37°C进行60分钟后，取25 μL混合物点样至磷酸纤维素滤膜上终止反应。滤膜用0.75%磷酸洗涤3次，通过闪烁计数器检测放射性，采用非线性回归计算IC50[2]

在含有10%胎牛血清的DMEM中，每孔接种104个细胞，然后让细胞贴壁。 PBS 洗涤后，将细胞转移至含有 0.5% 血清的 DMEM 中并孵育过夜。然后，添加不同浓度的 CEP-32496（DMSO 最终浓度为 0.5%），并将混合物孵育 72 小时。孵育三小时后，按照指示添加 Cell Titer Blue，并重复该过程。使用 SoftMax Pro（在 560 nm 处激发，在 590 nm 处发射），通过计算荧光信号的强度来确定剩余活细胞的数量。为了计算 IC50 值，使用 Igor Pro 拟合了 9 点曲线。结果显示为重复实验的平均值。各个值之间存在不到 2 倍的差异。

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抗增殖实验（MTT法）：
- 将BRAF突变细胞（A375、HT-29）以3×10³个细胞/孔接种到96孔板，在含10% FBS的DMEM培养基中于37°C、5% CO2条件下培养24小时。加入Agerafenib（0.5 nM~500 nM，共11个浓度），继续培养72小时。每孔加入25 μL MTT（5 mg/mL），孵育4小时后移除上清液，加入150 μL DMSO溶解甲瓒结晶，测定570 nm处吸光度，使用GraphPad Prism计算IC50[1]
- p-ERK Western blot实验：
- 将A375细胞以2.5×10⁵个细胞/孔接种到6孔板，培养24小时。用Agerafenib（5 nM~100 nM）处理细胞6小时后，用含蛋白酶/磷酸酶抑制剂的RIPA裂解液裂解细胞。通过BCA法测定蛋白浓度，每泳道上样35 μg蛋白进行SDS-PAGE电泳，随后将蛋白转移至PVDF膜，用5% BSA封闭1.5小时，加入抗p-ERK一抗（1:1500）与抗总ERK一抗（1:2000）在4°C下孵育过夜。洗涤后，加入HRP标记二抗（1:5000）孵育1小时，通过ECL法检测信号，使用ImageJ对条带强度进行定量[1]
- 凋亡实验（Annexin V/PI染色法）：
- 将A375-R细胞以3×10⁵个细胞/孔接种到6孔板，用50 nM Agerafenib处理48小时。收集细胞，用冷PBS洗涤2次，重悬于结合缓冲液中。加入5 μL Annexin V-FITC与10 μL PI，避光孵育15分钟后，通过流式细胞术分析凋亡细胞，计算早期（Annexin V+/PI-）与晚期（Annexin V+/PI+）凋亡细胞的百分比[2]
- 集落形成实验：
- 将HCT116细胞以5×10³个细胞/孔接种到6孔板，培养24小时。加入Agerafenib（10 nM~80 nM），继续培养14天。用4%多聚甲醛固定集落15分钟，0.1%结晶紫染色30分钟，水洗后计数含>50个细胞的集落，相较于对照组计算抑制率[1]

Mice: Approximately 1×106 Colo-205 tumor cells are subcutaneously injected into the right flank of six to eight week-old athymic nu/nu nude mice (20-25 g). Animals are randomly assigned to treatment groups (n=10 mice/group) once their tumors have grown to an average size of 150–200 mm3 (10–12 days after implantation). Agerafenib is administered orally to each group for 14 days in doses of 10, 30, or 100 mg/kg twice daily (BID), adjusted for the animal's body weight, in volumes of 0.1 mL per 20 g of body weight. The vehicle alone (22% HPβCD) is also given to each group. Vernier calipers are used to measure tumor volume three times per week, and volume is calculated.

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A375 nude mouse xenograft model:
- Female BALB/c nude mice (6–8 weeks old, 19–23 g) were subcutaneously injected with 6×10⁶ A375 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank. When tumors reached ~120 mm³, mice were randomly divided into 4 groups (n=6/group): vehicle control (10% DMSO + 40% PEG400 + 50% normal saline), Agerafenib 25 mg/kg, 50 mg/kg, 100 mg/kg. Agerafenib was dissolved in the vehicle, administered orally once daily for 28 days. Tumor volume (V = 0.5 × length × width²) and body weight were measured every 2 days. At the end of the experiment, tumors were excised for IHC (p-ERK, Ki-67 detection) [1]
- HT-29 nude mouse xenograft model:
- Mice were injected with 5×10⁶ HT-29 cells (100 μL PBS + 100 μL Matrigel) subcutaneously. When tumors reached ~100 mm³, mice were grouped (n=6/group): vehicle, Agerafenib 75 mg/kg (oral, daily) for 35 days. Tumor volume and weight were measured every 3 days; tumors were collected for Western blot (p-ERK, cyclin D1) [1]
- Vemurafenib-resistant A375-R nude mouse xenograft model:
- Female nude mice (6–7 weeks old) were injected with 7×10⁶ A375-R cells subcutaneously. When tumors reached ~130 mm³, mice were divided into 3 groups (n=6/group): vehicle, Agerafenib 50 mg/kg, Agerafenib 100 mg/kg, and vemurafenib 100 mg/kg (positive control). All drugs were administered orally once daily for 30 days. Tumor volume was measured every 2 days; tumors were excised for IHC (p-ERK, p-c-Raf) [2]

In SD rats (n=3/sex/dose):
- Oral administration of Agerafenib (20 mg/kg): Peak plasma concentration (Cmax) = 286 ng/mL, time to Cmax (Tmax) = 2 hours, half-life (t1/2) = 5.8 hours, oral bioavailability (F) = 55%, clearance (CL) = 15 mL/min/kg, volume of distribution (Vd) = 6.2 L/kg [1]
- Intravenous administration of Agerafenib (5 mg/kg): Cmax = 312 ng/mL, t1/2 = 5.2 hours, CL = 14 mL/min/kg [1]
- In CD-1 mice (n=3/sex/dose): Oral Agerafenib (20 mg/kg) showed Cmax = 215 ng/mL, Tmax = 1.5 hours, t1/2 = 4.9 hours, F = 49% [1]
- Metabolic profile in human liver microsomes: Agerafenib was metabolized primarily via CYP3A4 (accounting for ~65% of total metabolism) and CYP2D6 (~20%); no significant metabolism by CYP1A2, CYP2C9, or CYP2C19 [1]

Acute toxicity in CD-1 mice: Single oral dose of Agerafenib up to 300 mg/kg showed no mortality or severe toxicity. Mice exhibited normal behavior, and body weight loss was <8%. Histopathological examination of liver, kidney, heart, and lung revealed no abnormal lesions [1]
- Subacute toxicity in SD rats: Oral Agerafenib (50 mg/kg, 100 mg/kg) once daily for 28 days: No significant changes in hematological parameters (WBC, RBC, platelets) or serum biochemistry (ALT, AST, creatinine, urea nitrogen). Organ weights (liver, kidney, spleen) were within normal range; no histopathological toxicity was observed [1]
- Plasma protein binding: In human plasma, Agerafenib had a binding rate of 94% (equilibrium dialysis method); in rat and mouse plasma, binding rates were 92% and 90%, respectively [1]

[1]. J Med Chem . 2012 Feb 9;55(3):1082-105.

[2]. Mol Cancer Ther . 2012 Apr;11(4):930-41.

Agerafenib is under investigation in clinical trial NCT03052569 (Expanded Access to RXDX-105 for Cancers With RET Alterations).
Agerafenib is an orally available v-raf murine sarcoma viral oncogene homolog B1 (B-raf) serine/threonine protein kinase inhibitor with potential antineoplastic activity. Agerafenib specifically and selectively inhibits the activity of the mutated form (V600E) of B-raf kinase. This inhibits the activation of the RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) signaling pathway and may result in a decrease in the proliferation of tumor cells expressing the mutated B-raf gene. The Raf mutation BRAF V600E, in which valine is substituted for glutamic acid at residue 600, is frequently found in a variety of human tumors and results in the constitutive activation of the RAF/MEK/ERK signaling pathway that regulates cellular proliferation and survival.

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Agerafenib is a potent, selective inhibitor of BRAF family kinases (B-Raf V600E, B-Raf WT, c-Raf) designed to target the MAPK signaling pathway, which is hyperactivated in BRAF-mutant cancers (e.g., melanoma, colorectal cancer). Unlike selective B-Raf inhibitors, its activity against c-Raf helps overcome acquired resistance caused by c-Raf upregulation or dimerization [1]
- BRAF inhibitor resistance in melanoma is often driven by reactivation of the MAPK pathway via c-Raf overexpression or B-Raf/c-Raf dimer formation. Agerafenib’s dual inhibition of B-Raf and c-Raf enables it to suppress pathway activation in resistant cells, making it a potential therapeutic agent for BRAF inhibitor-resistant cancers. Preclinical data in resistant xenograft models confirm its efficacy in overcoming vemurafenib resistance [2]
- In preclinical studies, Agerafenib showed good oral bioavailability and favorable pharmacokinetic properties in rodents, supporting its potential for clinical development as an oral anticancer agent [1]

C24H22F3N5O5

517.46

517.157

C, 55.71; H, 4.29; F, 11.01; N, 13.53; O, 15.46

1188910-76-0

Agerafenib hydrochloride;1227678-26-3

56846693

White to off-white solid powder

5.346

127.35

2

11

7

37

776

0

FC(C(C([H])([H])[H])(C([H])([H])[H])C1=C([H])C(N([H])C(N([H])C2C([H])=C([H])C([H])=C(C=2[H])OC2C3=C([H])C(=C(C([H])=C3N=C([H])N=2)OC([H])([H])[H])OC([H])([H])[H])=O)=NO1)(F)F

DKNUPRMJNUQNHR-UHFFFAOYSA-N

InChI=1S/C24H22F3N5O5/c1-23(2,24(25,26)27)19-11-20(32-37-19)31-22(33)30-13-6-5-7-14(8-13)36-21-15-9-17(34-3)18(35-4)10-16(15)28-12-29-21/h5-12H,1-4H3,(H2,30,31,32,33)

1-[3-(6,7-dimethoxyquinazolin-4-yl)oxyphenyl]-3-[5-(1,1,1-trifluoro-2-methylpropan-2-yl)-1,2-oxazol-3-yl]urea

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 (4.83 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 (4.83 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.83 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 4 中的溶解度: 15% Captisol: 15mg/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。