FGFR1 (IC50 = 9.3 nM); FGFR2 (IC50 = 7.6 nM); FGFR3 (IC50 = 22 nM); FGFR4 (IC50 = 290 nM)

体外活性：在基于细胞的检测中，CH5183284 在 DMS114（FGFR1 扩增）、SNU-16（FGFR2 扩增）和 KMS11 中以 100 至 300 nM 浓度阻止 FGFR1、FGFR2 和 FGFR3 的自身磷酸化 [t(4;14)易位和 FGFR3 Y373C 突变]细胞系。因此，CH5183284 对具有 FGFR 基因改变的癌细胞系产生选择性抗增殖活性。 CH5183284 还抑制含有一种类型的看门突变 (V564F) 的 FGFR2，这种突变会导致对其他 FGFR 抑制剂产生耐药性。激酶测定：CH5183284/Debio 1347 对 FGFR1 的抑制活性使用辐射滤光测定通过微板闪烁计数器测量 33Pi 的掺入情况进行评估。 LCK、EGFR、KIT、MET、SRC、BRK、FGFR2、Flt3、LTK、INSR、YES、ABL、EPHA2、ZAP70、Fyn、IGF1R、KDR 和 PDGFR 对底物肽的磷酸化活性通过均相时间分辨测定根据标准方法，使用 LANCE Eu-W1024 标记的抗磷酸酪氨酸 PT66 抗体进行荧光测定。使用 EnVision HTS 酶标仪测量时间分辨荧光。 Aurora A、Akt1/PKBα、PKA、Cdk1/cyclin B、Cdk2/cyclin A、PKCα、PKCβ1 和 PKCβ2 对底物肽的活性通过 IMAP FP Screening Express Progressive Binding System 测定。使用 EnVision HTS 酶标仪测量荧光偏振。细胞测定：将细胞系（327 人类肿瘤细胞系）添加到含有 0.076 至 10,000 nM CH5183284/Debio 1347 的 96 孔板的孔中，并在 37°C 下孵育。孵育 4 天后，添加 Cell Counting Kit-8 溶液，再孵育几个小时后，使用 iMark 酶标仪测量 450 nm 处的吸光度。使用公式（1 - T/C）×100（%）计算抗增殖活性，其中T和C代表药物处理的细胞（T）和未处理的对照细胞（C）在450 nm处的吸光度。 IC50 值使用 Microsoft Excel 2007 计算。

CH5183284（100 mg/kg/天，口服）对具有 FGFR 基因改变的异种移植物显示出选择性和显着的抗肿瘤活性，例如 KG1（白血病、FGFR1OP-FGFR1 融合）、SNU-16（胃癌、FGFR2 扩增）、MFE-280（子宫内膜癌，FGFR2 S252W 突变）、UM-UC-14（膀胱癌，FGFR3 S249C 突变）和 RT112/84（膀胱癌，FGFR3-TACC3 融合）。

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CH5183284/Debio 1347在体内的FGFR选择性抗肿瘤活性[1]
为了确认CH5183284/Debio 1347在体内和体外对携带FGFR基因改变的癌症的选择性抗肿瘤活性，我们评估了其在异种移植物小鼠模型中的体内疗效。CH5183284/Debio 1347对具有FGFR基因改变的异种移植物显示出显著的抗肿瘤活性，如KG1[白血病，FGFR1OP-FGFR1融合；最大肿瘤生长抑制（TGI），134%]、SNU-16（癌症，FGFR2扩增；最大TGI，147%）、MFE-280（癌症，FGFR2-S252W突变；最大TGI100%）、UM-UC-14（膀胱癌症，FGFR3-S249C突变；最大TG，116%）和RT112/84（膀胱癌症，FGFR3-7ACC3融合；最大TGI/125%）。在这种情况下。相反，MKN-45（癌症、WT FGFR、MET扩增）对CH5183284/Debio 1347不敏感（MTD时最大TGI 8%；图4A）。这些数据与体外观察结果一致。然后，我们通过在单次给药后进行蛋白质印迹和免疫组织化学研究了肿瘤组织中FGFR信号传导的抑制情况。CH5183284/Debio 1347在SNU-16异种移植物组织中抑制磷酸化FGFR至少7小时（图4B），以及下游信号传导，如磷酸化FRS、磷酸化ERK和磷酸化-S6的减少所示（图4C）。这些结果表明，CH5183284/Debio 1347通过抑制FGFR信号通路，在体外和体内对携带FGFR基因改变的癌症具有选择性抗肿瘤活性。

使用微孔板闪烁计数器进行辐射过滤测定来测量 ³³Pi 的掺入情况，以评估 CH5183284/Debio 1347 对 FGFR1 的抑制活性。标准技术用于均相时间分辨荧光测定，以测量 LCK、EGFR、KIT、MET、SRC、BRK、FGFR2、Flt3、LTK、INSR、YES、ABL、EPHA2、ZAP70、Fyn、IGF1R、底物肽上的 KDR 和 PDGFR。使用 EnVision HTS 酶标仪对时间分辨荧光进行定量。 IMAP FP Screening Express Progressive Binding System 可测量底物肽上所有蛋白质的活性，包括 PKA、Akt1/PKBα、PKA、Cdk1/cyclin B、Cdk2/cyclin A、PKCα、PKCβ1 和 PKCβ2。它使用 EnVision HTS 酶标仪来测量荧光偏振。

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蛋白激酶测定和结合模式的确定[1]
通过用微孔板闪烁计数器测量33Pi的掺入，使用放射性滤光片测定法评估了CH5183284/Debio 1347对FGFR1的抑制活性。在抑制剂CH5183284/Debio 1347存在或不存在的情况下，以与FGFR1的IC10至IC75相对应的七种浓度进行了ATP含量增加（1-200μM）的剂量反应分析，每种ATP浓度都进行了两次测量
在没有化合物的情况下，确定激酶自磷酸化、底物和ATP背景作为对照。基于非线性回归分析计算了不同浓度CH5183284/Debio 1347影响下FGFR1的酶参数Km[ATP]和Vmax值，并采用Lineweaver和Burk描述的方法绘制结果。线性图与x轴在-1/Km处相交，与y轴在1/Vmax处相交。

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试管形成试验[1]
血管生成试剂盒中装有最终浓度为0.1或1µM的CH5183284/Debio 1347或0.01或0.1µM的cediranib的测试化合物，并在含10ng/ml VEGF的培养基中的CO2培养箱（37℃，5%）中孵育。孵育11天后，形成的毛细管状管用70%乙醇固定，并用CD31染色试剂盒进行可视化。在显微镜下，拍摄孔的染色图像（x4物镜）并将其存储为图像文件，并使用Kurabo血管生成图像分析软件定量测量毛细血管样管形成面积。

将细胞系填充到含有 0.076−10,000 nM 测试化合物 (CH5183284) 的 96 孔板孔中，然后将板在 37°C 下孵育。孵育 4 天后添加 Cell Counting Kit-8 溶液，数小时后测量 450 nm 处的吸光度。抗增殖活性计算公式为1-T/C)×100(%)，其中T和C分别代表药物处理细胞(T)和未处理对照细胞(C)在450 nm处的吸光度[1]。

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细胞增殖试验[1]
所有细胞系均由细胞库通过细胞遗传学分析、DNA分析或生长特性进行鉴定，并在复苏后繁殖不到6个月。此外，所有细胞系均按照供应商的说明进行培养。将细胞系加入含有0.076至10000 nmol/LCH5183284/Debio 1347的96孔板孔中，并在37°C下孵育。孵育4天后，加入细胞计数试剂盒-8溶液，再孵育几个小时后，用iMark微孔板阅读器测量450nm处的吸光度。使用公式（1-T/C）×100（%）计算抗增殖活性，其中T和C表示用药物处理的细胞（T）和未处理的对照细胞（C）在450 nm处的吸光度。IC50值使用Microsoft Excel 2007计算。

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蛋白质印迹分析[1]
用0.1%DMSO或CH5183284/Debio 1347处理细胞2小时，并用含有蛋白酶和磷酸酶抑制剂的细胞裂解缓冲液裂解。在裂解之前，使用BioMasher将移植的肿瘤均质化。裂解物用含有还原剂的样品缓冲溶液变性进行SDS-PAGE，然后进行SDS-PAGE。电印迹后，如前所述进行蛋白质印迹分析。本研究中使用的抗体可在补充材料和方法中获得。

Rats: To evaluate the effects on blood pressure (BP), male Wistar rats weighing between 340 and 390 grams are implanted with a telemetry transmitter. Oral gavage of either the vehicle (0.5% carmellose sodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol in purified water) or CH5183284/Debio 1347 (10 and 30 mg/kg) is performed once daily for four days in a row. Five-minute intervals of continuously recorded, automatically analyzed blood pressure data are provided[2].
Mice: SNU-16 xenograft-bearing mice are used to assess the in vivo efficacy. The mice are given CH5183284 orally once a day for 11 days, and the tumor volume and body weight are recorded twice a week[1].

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Telemetry study in rats [1]
Male Wistar rats (340–390 g) implanted with a telemetry transmitter were used for the assessment of effects on blood pressure (BP; ref. 24). Vehicle (0.5% carmellose sodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol in purified water) or CH5183284/Debio 1347 (10 and 30 mg/kg) were administered by oral gavage once a day for 4 consecutive days. Data for blood pressure were automatically analyzed and continuously recorded at 5-minute intervals. Baseline blood pressure was determined by the 24-hour mean of blood pressure before administration, and change in blood pressure from the baseline value (ΔBP) is represented as mean ± SD. The statistical significance between the vehicle group and each dose of the CH5183284/Debio 1347 group was evaluated using the Dunnett test following confirmation of the homogeneity of variance.

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Mouse xenograft study [1]
Female BALB-nu/nu mice (CAnN.Cg-Foxn1/CrlCrlj nu/nu) were kept under specified pathogen-free conditions. Cells (4 × 106 to 1.1 × 107) were suspended in 100 to 200 μL serum-free culture medium and injected subcutaneously into the right flank of the mice. Tumor size was measured using a gauge twice per week, and tumor volume (TV) was calculated using the following formula: TV = ab2/2, where a is the length of the tumor and b is the width. Once the tumors reached a volume of approximately 200 to 300 mm3, animals were randomized into groups (n = 3, 4, or 5 in each group), and treatment was initiated. CH5183284/Debio 1347 or AZD4547 were orally administered once a day.

[1]. The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor. Mol Cancer Ther. 2014 Nov;13(11):2547-58.

[2]. Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1. Mol Cancer Ther. 2015 Mar;14(3):704-12.

[3]. ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor. Mol Cancer Ther. 2015 Dec;14(12):2831-9.

Ch5183284 has been used in trials studying the treatment of Solid Tumours.
Zoligratinib is an orally bioavailable inhibitor of the fibroblast growth factor receptor subtypes 1 (FGFR-1), 2 (FGFR-2) and 3 (FGFR-3), with potential antineoplastic activity. Zoligratinib binds to and inhibits FGFR-1, -2, and -3, which result in the inhibition of FGFR-mediated signal transduction pathways. This leads to the inhibition of both tumor cell proliferation and angiogenesis, and causes cell death in FGFR-overexpressing tumor cells. FGFR, a family of receptor tyrosine kinases upregulated in many tumor cell types, is essential for tumor cellular proliferation, differentiation and survival.

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The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations.[1]

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Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3-BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3-BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3-BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3-BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-ΔBAR cells expressing an FGFR3-BAIAP2L1 variant lacking the Bin-Amphiphysin-Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-ΔBAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity.[2]

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Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. We performed a transcriptome analysis to characterize the response of various cancer cell lines to a selective fibroblast growth factor receptor (FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase (MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the extracellular signal-regulated kinase (ERK) gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor-sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor-sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers.[3]

C20H16N6O

356.38

356.139

C, 67.40; H, 4.53; N, 23.58; O, 4.49

1265229-25-1

1265229-25-1;1265231-80-8;

66555680

Off-white to yellow solid powder

3.932

105.38

3

4

3

27

573

0

O=C(C1=C([H])C2=C([H])C([H])=C([H])C([H])=C2N1[H])C1C([H])=NN(C=1N([H])[H])C1C([H])=C([H])C2=C(C=1[H])N([H])C(C([H])([H])[H])=N2

BEMNJULZEQTDJY-UHFFFAOYSA-N

InChI=1S/C20H16N6O/c1-11-23-16-7-6-13(9-17(16)24-11)26-20(21)14(10-22-26)19(27)18-8-12-4-2-3-5-15(12)25-18/h2-10,25H,21H2,1H3,(H,23,24)

[5-amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone

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: ≥ 2.08 mg/mL (5.84 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (5.84 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 20.8 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: ≥ 2.08 mg/mL (5.84 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 20.8 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。