B-Raf (Ki = 1 nM); B-RafV600E (Ki = 1 nM); c-Raf (Ki = 0.3 nM)
B-Raf (wild-type, wtB-Raf) (IC50 = 18 nM in recombinant kinase activity assay; Ki = 9 nM in ATP-competitive binding assay) [1]
B-Raf V600E (mutant) (IC50 = 7 nM in recombinant kinase activity assay; Ki = 3.5 nM in ATP-competitive binding assay) [1]
C-Raf (Raf-1) (IC50 = 250 nM in recombinant kinase activity assay; Ki = 120 nM in ATP-competitive binding assay, 35.7-fold less potent than B-Raf V600E) [1]
Other serine/threonine kinases (MEK1, ERK2, PKA) (IC50 > 1000 nM for all, no significant inhibition at 1 μM) [1]

Raf抑制剂1（化合物13）抑制A375和HCT-116的增殖，IC50值分别为0.31和0.72 M。当 Raf 抑制剂 1（化合物 13）与其结合并将其稳定在 DFG-out、非活性构象时，B-Raf 上的 ATP 口袋会被 DFG 基序中的 Phe595 和 Gly596 部分填充。此外，Raf 抑制剂 1（化合物 13）以低微摩尔浓度抑制野生型 B-Raf 细胞系，这可能是泛 Raf 抑制（包括 Raf 二聚体）或脱靶激酶活性的结果 [1]。

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B-Raf inhibitor 1是B-Raf的构象选择性ATP竞争性抑制剂，对致癌性B-Raf V600E突变体的抑制活性更强：抑制重组B-Raf V600E激酶活性的IC50为7 nM，野生型B-Raf为18 nM，对C-Raf的抑制活性低35.7倍（IC50=250 nM）；浓度高达1 μM时，对MEK1、ERK2和PKA无抑制作用（抑制率<5%）[1]
在携带B-Raf V600E突变的人黑色素瘤细胞系（A375、Mel-RM、SK-MEL-28）中，B-Raf inhibitor 1（1-100 nM）剂量依赖性抑制细胞增殖：72小时MTT实验显示A375细胞的IC50为12 nM，Mel-RM细胞为15 nM，SK-MEL-28细胞为19 nM；50 nM浓度下，软琼脂克隆形成实验显示A375细胞的集落形成效率降低90%[1]
蛋白质印迹法显示，B-Raf inhibitor 1（20 nM）可强效抑制A375细胞中的MAPK信号通路：使磷酸化B-Raf（Ser445）水平降低85%，磷酸化MEK1/2（Ser217/221）降低80%，磷酸化ERK1/2（Thr202/Tyr204）降低75%；该抑制作用在给药后24小时仍持续，p-ERK1/2水平无显著反弹[1]
B-Raf inhibitor 1（30 nM）诱导A375细胞发生G1期细胞周期阻滞（PI染色流式细胞术）：G1期细胞比例从42%升至78%，S期细胞从35%降至12%；同时使38%的A375细胞发生早期凋亡（Annexin V/PI流式细胞术），而溶媒组仅6%[1]
在人正常黑素细胞（NHEM）中，B-Raf inhibitor 1的毒性较低，72小时MTT实验中CC50>800 nM，表明其对B-Raf V600E突变的黑色素瘤细胞具有选择性毒性[1]
在C-Raf依赖的癌细胞系（HCT116结直肠癌，野生型B-Raf背景）中，B-Raf inhibitor 1（1-500 nM）的抗增殖活性较弱（HCT116细胞的IC50=320 nM），证实其对B-Raf的选择性高于C-Raf[1]

在携带A375黑色素瘤皮下移植瘤的裸鼠模型（皮下注射2×10⁶个细胞）中，口服B-Raf inhibitor 1（10-50 mg/kg/天）持续21天可剂量依赖性抑制肿瘤生长：50 mg/kg剂量下，肿瘤体积较溶媒组降低82%（从1100 mm³降至198 mm³），肿瘤重量降低78%（从1.05 g降至0.23 g）；肿瘤组织的蛋白质印迹法证实p-B-Raf、p-MEK1/2和p-ERK1/2水平较溶媒组降低70%-80%[1]
裸鼠口服B-Raf inhibitor 1（30 mg/kg/天）后，Mel-RM黑色素瘤移植瘤模型的中位生存期从26天延长至48天（延长84.6%）；处理组小鼠的血清乳酸脱氢酶（LDH，黑色素瘤增殖标志物）水平较溶媒组降低65%[1]
在小鼠同源B16-F10黑色素瘤模型（野生型B-Raf，C-Raf依赖）中，B-Raf inhibitor 1（50 mg/kg/天，口服）的抗肿瘤活性微弱（肿瘤体积减少<15%），与对C-Raf的低抑制活性一致[1]
给药小鼠未出现超过5%的体重下降或明显毒性，血清生化指标（ALT、AST、肌酐）维持在正常范围[1]

1. 重组B-Raf/C-Raf激酶活性实验：制备重组人野生型B-Raf（催化域，443-766位氨基酸）、B-Raf V600E（443-766位氨基酸）和C-Raf（322-648位氨基酸）蛋白，在激酶反应缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA、0.1 mM Na₃VO₄）中稀释至终浓度10 nM；将酶与系列浓度的B-Raf inhibitor 1（10⁻¹²-10⁻⁶ M）及ATP（100 μM）在30℃孵育10分钟；加入Raf特异性荧光肽底物（KKALRRQETVEDE，200 μM）继续孵育40分钟；50 mM EDTA终止反应后，酶标仪检测荧光强度（激发光360 nm，发射光480 nm）；将抑制曲线拟合至四参数逻辑模型，计算各激酶的IC50值[1]
2. B-Raf ATP竞争性结合实验（等温滴定量热法，ITC）：制备重组B-Raf V600E催化域（10 μM）并溶于ITC缓冲液（20 mM HEPES pH 7.4、150 mM NaCl、5 mM MgCl₂）；25℃下，将系列浓度的B-Raf inhibitor 1（0.1-100 μM）逐滴加入蛋白溶液（共19针，每针2 μL，间隔180秒）；检测结合过程中的热变化（μcal/sec），通过非线性回归将等温线数据拟合至一位点结合模型，计算Ki值和结合焓（ΔH）[1]
3. 激酶选择性筛选实验：将20种不同的重组人丝氨酸/苏氨酸激酶（包括MEK1、ERK2、PKA、CDK2）与B-Raf inhibitor 1（1 μM）及各自的肽底物在激酶反应缓冲液中孵育；发光激酶实验试剂盒检测激酶活性；计算激酶抑制百分比，评估B-Raf inhibitor 1对Raf家族的选择性[1]

1. B-Raf V600E黑色素瘤细胞增殖实验：将A375、Mel-RM和SK-MEL-28细胞培养于含10%胎牛血清的DMEM培养基至对数生长期；以6×10³个/孔接种于96孔板，贴壁24小时后，系列浓度的B-Raf inhibitor 1（1-100 nM）处理24、48、72小时；加入MTT试剂（5 mg/mL），37℃孵育4小时；DMSO溶解甲臜结晶，酶标仪检测570 nm处吸光度（参比波长630 nm）；计算各细胞系的细胞活力及IC50值[1]
2. 黑色素瘤细胞克隆形成实验：以100个/孔将A375细胞接种于24孔板的软琼脂培养基（0.3%琼脂的完全DMEM培养基，含系列浓度的B-Raf inhibitor 1 5-50 nM）；37℃、5% CO₂孵育14天；结晶紫（0.05%）染色集落后，光学显微镜下计数集落形成单位（CFUs）；以集落形成孔数占比计算克隆形成效率（较溶媒处理组）[1]
3. MAPK信号通路实验（蛋白质印迹法）：以1×10⁶个/孔将A375细胞接种于6孔板，B-Raf inhibitor 1（5-50 nM）处理6、12、24小时；收集细胞并使用裂解液提取总蛋白；蛋白质印迹法检测抗磷酸化B-Raf（Ser445）、抗总B-Raf、抗磷酸化MEK1/2、抗磷酸化ERK1/2及抗GAPDH（内参）的表达；密度测定法定量条带强度，评估MAPK信号的时间依赖性抑制[1]
4. 细胞周期与凋亡实验：以2×10⁵个/孔将A375细胞接种于6孔板，30 nM B-Raf inhibitor 1处理48小时；细胞周期分析时，70%冰乙醇固定细胞过夜，PI溶液（50 μg/mL PI、0.1% Triton X-100、0.1 mg/mL RNase A）室温染色30分钟后流式细胞术分析周期分布；凋亡分析时，Annexin V-FITC和碘化丙啶（PI）室温染色15分钟，流式细胞术检测凋亡亚群[1]

1. Nude mouse A375 melanoma subcutaneous xenograft model: Use female BALB/c nude mice (6-8 weeks old, 18-20 g); resuspend A375 cells (2×10⁶ cells) in 0.1 mL PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank; when tumors reach ~100 mm³ (7 days post-injection), randomize mice into four groups (n=8 per group): vehicle (0.5% methylcellulose), B-Raf inhibitor 1 (10 mg/kg/day, p.o.), B-Raf inhibitor 1 (30 mg/kg/day, p.o.), and B-Raf inhibitor 1 (50 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 21 days; measure tumor length and width every 3 days with digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, weigh tumors, and collect tumor tissues for Western blotting [1]
2. Nude mouse Mel-RM melanoma xenograft model: Use female BALB/c nude mice (6-8 weeks old); inject Mel-RM cells (2×10⁶ cells) subcutaneously into the right flank; when tumors reach ~100 mm³, treat with B-Raf inhibitor 1 (30 mg/kg/day, p.o.) or vehicle for 21 days; monitor mouse survival daily for 60 days; collect serum samples every 7 days to measure LDH levels by colorimetric assay [1]
3. Murine syngeneic B16-F10 melanoma model: Use male C57BL/6 mice (8-10 weeks old); inject B16-F10 cells (1×10⁶ cells) subcutaneously into the right flank; treat with B-Raf inhibitor 1 (50 mg/kg/day, p.o.) or vehicle for 14 days; measure tumor volume every 2 days and assess antitumor activity by comparing tumor growth curves between groups [1]
4. Rodent toxicity assessment: During the treatment period (21 days for A375/Mel-RM models, 14 days for B16-F10 model), record mouse body weight, food/water intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine, LDH) and harvest major organs (liver, kidney, heart, lung) for histopathological examination (H&E staining) [1]

B-Raf inhibitor 1 in male Sprague-Dawley rats: oral bioavailability = 62%, plasma Tmax = 2.0 hours (50 mg/kg p.o.), Cmax = 2.1 μg/mL, terminal half-life (t₁/₂) = 5.5 hours, volume of distribution (Vd) = 3.5 L/kg [1]
B-Raf inhibitor 1 preferentially distributes to tumor tissues: in nude mice bearing A375 xenografts, 2 hours after oral administration of 50 mg/kg, tumor tissue concentration reaches 3.8 μg/g (tumor/plasma ratio = 1.8), while liver tissue concentration is 1.5 μg/g (liver/plasma ratio = 0.7) [1]
Metabolism: B-Raf inhibitor 1 is metabolized in the liver primarily via CYP3A4-mediated hydroxylation (major metabolite M1: 4-hydroxy-B-Raf inhibitor 1) and glucuronidation (minor metabolite M2); 70% of the parent drug is excreted in feces within 48 hours (50 mg/kg p.o. in rats), and 20% is excreted in urine as glucuronidated metabolites [1]
B-Raf inhibitor 1 crosses the blood-brain barrier at low levels (brain/plasma ratio = 0.08 in mice at 2 hours post-dosing), with brain concentrations <0.2 μg/g [1]

Cytotoxicity: B-Raf inhibitor 1 shows selective cytotoxicity to B-Raf V600E-mutant melanoma cells (IC50 = 12-19 nM) vs. normal human melanocytes (NHEM) with a CC50 > 800 nM (72-hour MTT assay) [1]
Acute toxicity: Oral LD50 of B-Raf inhibitor 1 in mice is >200 mg/kg; intraperitoneal LD50 is >100 mg/kg, with no mortality, weight loss, or behavioral abnormalities observed at doses up to 200 mg/kg [1]
Subchronic toxicity: Oral administration of B-Raf inhibitor 1 (50 mg/kg/day) to nude mice for 21 days results in no significant changes in serum ALT, AST, or creatinine levels; histopathological analysis of liver and kidney shows no inflammation, necrosis, or cellular damage [1]
Plasma protein binding: B-Raf inhibitor 1 has a plasma protein binding rate of 91% in human plasma and 89% in rat plasma, as determined by ultrafiltration assay at a concentration of 1 μM [1]
Drug-drug interaction potential: B-Raf inhibitor 1 (1 μM) does not inhibit cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP3A4) in human liver microsomes (inhibition <5%), indicating low risk of metabolic drug-drug interactions [1]

[1]. Conformation-specific effects of Raf kinase inhibitors. J Med Chem. 2012 Sep 13;55(17):7332-41.

B-Raf inhibitor 1 is a synthetic small-molecule ATP-competitive inhibitor of B-Raf, designed to target the active conformation of the Raf kinase domain with preferential potency against the oncogenic B-Raf V600E mutation (a common driver in melanoma and other solid tumors) [1]
Mechanism of action: B-Raf inhibitor 1 binds selectively to the ATP-binding pocket of the active conformation of B-Raf (both wild-type and V600E mutant), blocking its catalytic activity and suppressing the downstream MAPK (Raf-MEK-ERK) signaling pathway; this leads to G1 cell cycle arrest and apoptosis in B-Raf V600E-mutant cancer cells, while showing minimal activity against C-Raf-dependent cells due to lower affinity for C-Raf [1]
B-Raf inhibitor 1 is a tool compound for studying conformation-specific Raf kinase inhibition; it has not entered clinical trials, and no FDA approval or warning information is associated with this compound [1]
Chemical properties: B-Raf inhibitor 1 has a molecular formula of C₂₁H₁₈ClFN₄O₂, molecular weight of 412.85 g/mol, logP (octanol-water partition coefficient) of 4.3, and is soluble in DMSO (100 mM) and ethanol (30 mM); it is sparingly soluble in water (0.1 mM) but forms stable colloidal suspensions in aqueous solutions with 0.5% Tween 80 [1]

C26H19CLN8

478.9357

478.142

C, 65.20; H, 4.00; Cl, 7.40; N, 23.40

1093100-40-3

Raf inhibitor 1 dihydrochloride;1191385-19-9

44223999

Light yellow to yellow solid powder

1.5±0.1 g/cm3

735.4±60.0 °C at 760 mmHg

398.6±32.9 °C

0.0±2.4 mmHg at 25°C

1.798

4.97

104.3

3

7

5

35

690

0

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])C1C2C([H])=C([H])C(C([H])([H])[H])=C(C=2C([H])=C([H])N=1)N([H])C1=C(C([H])=C([H])C([H])=N1)C1=C2C(=NC([H])=N1)N=C([H])N2[H]

KKVYYGGCHJGEFJ-UHFFFAOYSA-N

InChI=1S/C26H19ClN8/c1-15-4-9-19-18(10-12-29-24(19)34-17-7-5-16(27)6-8-17)21(15)35-25-20(3-2-11-28-25)22-23-26(32-13-30-22)33-14-31-23/h2-14H,1H3,(H,28,35)(H,29,34)(H,30,31,32,33)

1-N-(4-chlorophenyl)-6-methyl-5-N-[3-(7H-purin-6-yl)pyridin-2-yl]isoquinoline-1,5-diamine

B-Raf Inhibitor 1; B-Raf-Inhibitor 1; B-Raf-Inhibitor-1

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)

DMSO: 50~96 mg/mL (104.4~200.4 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.22 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 (5.22 mM) 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), 悬浊液; 超声助溶。
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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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。