AZD3463

IGF-1R; ALK (Ki = 0.75 nM)

体外活性：AZD3463 在 ALK 驱动的临床前模型和各种克唑替尼耐药模型中有效。 AZD3463 能够抑制细胞中的 ALK，其能够降低含有 ALK 融合体的肿瘤细胞系（包括 DEL (ALCL NPM-ALK)、H3122 (NSCLC EML4-ALK) 和 H2228 (NSCLC EML4-ALK)）中 ALK 自磷酸化的能力。 ALK 的抑制与下游信号传导（包括 ERK、AKT 和 STAT3 通路）的扰动相关，导致在体外含有 ALK 融合的细胞系中优先抑制增殖。 AZD3463 对许多临床相关的克唑替尼耐药突变（包括守门突变体 L1196M）保留了良好的活性，其中在含有 EML4-ALK 的 BAF3 细胞系中在体外和体内观察到与野生型 ALK 相当的效力。为了进一步评估 AZD3463 克服其他耐药机制的潜在能力，在体外独立衍生自 H3122 细胞的多种克唑替尼耐药细胞系以及患者衍生的克唑替尼复发模型中评估了抗增殖活性。这些耐药细胞系包含多种耐药机制，包括 L1196M 看门人和 T115Ins 突变、ALK 扩增和/或次要驱动因素（包括 EGFR 和 IGF1R）。对于 12 个体外获得性耐药模型中的 10 个，AZD3463 的抗增殖效力保留在亲代 H3122 细胞的 4 倍以内。激酶测定：ZD-3463 是一种 ALK/IGF1R 抑制剂，可克服克唑替尼获得性耐药的多种机制。

在原位异种移植小鼠模型中，AZD3463（15 mg/kg，腹腔注射）对带有 WT 和 F1174L 致癌突变体 ALK 的神经母细胞瘤肿瘤的生长显示出显着的治疗效果.

---

AZD3463还证明了在体内异种移植物肿瘤中剂量依赖性抑制pALK的能力，导致停滞（H3122）或消退（DEL，H2228）。AZD3463对许多临床相关的克唑替尼耐药突变保持良好的活性，包括看门人突变体L1196M，其中在含有EML4-ALK的BAF3细胞系中观察到与野生型ALK具有同等效力[1]。

---

AZD3463在NB的ALK-WT和F1174L突变正交异性异种移植物小鼠模型中均显示出抗肿瘤疗效[3]
基于AZD3463对NB细胞的体外细胞毒性作用，我们开始评估该药物在NB原位异种移植物小鼠模型中抑制肿瘤生长的作用。在这组体内实验中，将具有稳定萤光素酶基因表达的NGP和SH-SY5Y细胞分别植入裸鼠的左肾。在AZD3463治疗结束时，对对照组和治疗组的异种移植物肿瘤进行解剖和称重。与对照组相比，AZD3463治疗组（SH-SY5Y和NGP）均观察到显著的肿瘤生长抑制。AZD3463对SH-SY5Y异种移植物小鼠的治疗导致肿瘤几乎完全消退，在NGP异种移植物鼠中观察到显著消退（图5A，C）。在小鼠携带SH-SY5Y和NGP肿瘤4周后，通过腹腔注射用AZD3463或DMSO治疗小鼠48小时。然后我们检查了AZD3463对肿瘤组织中PI3K/AKT/mTOR信号传导的影响，发现AZD3463在体内有效地阻断了AKT和RPS6的磷酸化，并诱导了PARP、胱天蛋白酶3和LC3 A/B的切割（图5E）。这些结果表明，AZD3463作为单一药物可有效诱导NB原位异种移植物小鼠模型的凋亡和自噬。

在几种肿瘤类型中观察到间变性淋巴瘤激酶（ALK）的基因组重排，包括60-80%的间变性大细胞淋巴瘤（ALCL）和3-6%的非小细胞肺癌癌症（NSCLC）。尽管ALK抑制剂克唑替尼在选定的ALK阳性NSCLC患者中具有临床疗效，但大多数出现初始反应的患者最终会复发。已经提出了导致耐药性的各种机制，包括ALK扩增和耐药性突变，以及包括EGFR、cKIT和最近的IGF1R在内的替代途径驱动因素。我们发现了一种新型且强效的ALK抑制剂AZD3463，Ki值为0.75nM，它还能以同等效力抑制其他受体酪氨酸激酶，包括胰岛素生长因子受体（IGF1R）。AZD3463抑制细胞中的ALK，这可以通过其减少含有ALK融合物的肿瘤细胞系中ALK自磷酸化的能力来证明，包括DEL（ALCL NPM-ALK）、H3122（NSCLC EML4-ALK）和H2228（NSCLC EML4-ALK）。ALK的抑制与下游信号传导的紊乱有关，包括ERK、AKT和STAT3通路，导致体外含ALK融合细胞系的增殖受到优先抑制。[1]

---

为了进一步评估AZD3463克服其他耐药机制的潜在能力，在体外独立衍生自H3122细胞的多种克唑替尼耐药细胞系以及患者衍生的克唑替尼复发模型中评估了抗增殖活性。这些耐药细胞系包含多种耐药机制，包括L1196M看门人和T115Ins突变、ALK扩增和/或包括EGFR和IGF1R在内的次要驱动因素。AZD3463在体外12种获得性耐药模型中有10种的抗增殖能力保持在亲本H3122细胞的4倍以内。总之，AZD3463是一种强效的ALK抑制剂，可抑制包括IGF1R在内的其他激酶，并在多种耐药机制驱动的许多克唑替尼耐药模型中具有活性。[1]

---

ZD-3463 是一种 ALK/IGF1R 抑制剂，可规避克唑替尼耐药性发展中涉及的多种机制。

细胞增殖和生长试验[2]
将细胞以7000个细胞（H3122、24-H3122和CR-H3122）和4000个细胞（A549）的密度接种在96孔板中，并使其粘附24小时。在测定前，用单独的药物或药物组合处理细胞72小时。如Skehan等人（1990）所述，使用磺基罗丹明B（SRB）测定法评估细胞毒性和增殖率。简而言之，在4°C下用50µL 10%三氯乙酸（TCA）固定细胞30分钟。用50µL SRB对蛋白质进行染色，然后用1%乙酸洗涤孔。然后将板干燥，将SRB溶解在100µL的100μM TRIS缓冲液中。用Spectromax平板读数器在490 nm处读取吸光度，扣除630 nm的背景；细胞生长抑制通过DMSO处理的对照与处理过的细胞的吸光度之比进行评估。所有细胞毒性试验均在三个独立的实验中进行，实验一式三份。在己内酯中测量细胞增殖时间点。

---

细胞周期分析[2]
为了评估克唑替尼对我们先天抗性模型中细胞周期的影响，将H3122细胞以每孔10000个细胞的密度接种在6孔板中，并在治疗前孵育过夜，以使细胞粘附。细胞用克唑替尼（10μM）处理24小时，之后用0.01 M磷酸盐缓冲盐水洗涤细胞，通过离心收集裂解物，并在接下来的12天内用70%乙醇固定。对于细胞周期分析，用0.01 M PBS洗涤细胞，用RNase（20 mg/mL）处理，然后用碘化丙啶染色。用GalliosTM流式细胞仪处理细胞，并用FlowJo LLC版本10进行分析。进行了三个独立的实验，一式三份。

---

免疫印迹[2]
对于蛋白质印迹分析，首先在6孔板中以每孔200000个细胞的密度接种细胞。处理后，收集细胞，在0.01 M PBS中洗涤，并在由50μM Tris-HCl（pH 8）、150 mM NaCl、1μM EDTA、1 mM NaF、1 mM原钒酸钠、1 mM苯甲基磺酰氟（PMSF）、1%TritonX-100和1%SDS组成的缓冲液中裂解。通过离心（4°C下12500 RPM，8分钟）澄清细胞提取物，然后对裂解物进行SDS-PAGE，然后转移到PVDF膜上，用一抗探测感兴趣的蛋白质，用含1%牛血清白蛋白的0.01 mM PBS稀释。

5 to 6-week-old female athymic Ncr nude mice (SH-SY5Y and NGP xenograft tumors bearing mice)[1].
15 mg/kg
Intraperitoneal injection; once daily for 2 days

---

Orthotopic Mouse Model of neuroblastoma (NB) [3]
Five to six-week-old female athymic Ncr nude mice were maintained under barrier conditions (pathogen-free conditions provided by plastic cages with sealed air filters). The preclinical mouse model of NB was established using orthotopic (intrarenal) implantation of the NB cells. Briefly, a transverse incision was created over the left flank of the nude mouse and 1.5 × 106 human luciferase-transduced SH-SY5Y or NGP cells suspended in 0.1 ml of PBS were surgically injected into the left renal capsule and towards the superior pole of the left kidney of the nude mice. After allowing the cells to engraft for 2 to 3 weeks, mice bearing tumors with similar sizes (using bioluminescent imaging to monitor tumor growth) were randomly divided into two groups: a DMSO control group and an AZD3463 treatment group. Both control and experimental groups contained six mice each. Three mice from each group were selected and treated with DMSO or AZD3463 over a 48 hour period via15 mg/kg intraperitoneal (i.p.) injection once daily for 2 days. Tumors were then harvested for WB analysis. The rest were treated for 21 days (15 mg/kg by intraperitoneal (i.p.) injection once daily). At the end of the treatment, all mice were sacrificed. Tumors and the right kidneys (control) were harvested and weighed.

[1]. Cancer Res (2013) 73 (8_Supplement): 919.

[2]. Sci Rep. 2017 Oct 24;7(1):13955.

[3]. Sci Rep. 2016 Jan 20;6:19423. Sci Rep. 2016 Jan 20;6:19423.

AZD3463 is a member of the class of indoles that is 1H-indole substituted by a 2-[4-(4-aminopiperidin-1-yl)-2-methoxyanilino]-5-chloropyrimidin-4-yl group at position 3. It is an orally bioavailable dual inhibitor of ALK and IGF1R with Ki value of 0.75 nM for ALK. It has a role as an apoptosis inducer, an antineoplastic agent, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an autophagy inducer. It is a member of indoles, an aminopiperidine, a monomethoxybenzene, an aminopyrimidine, a secondary amino compound, a tertiary amino compound and an organochlorine compound.

---

ALK positive non-small cell lung cancer is highly responsive to ALK inhibitors such as crizotinib, but drug resistance typically develops within a year of treatment. In this study we investigated whether IGF-1R is an independent druggable target in ALK-positive lung cancer cells. We confirmed that combination ALK and IGF-1R inhibitor treatment is synergistically cytotoxic to ALK-positive lung cancer cells and that this remains the case for at least 12 days after initial exposure to crizotinib. ALK-positive cells with acquired resistance to crizotinib did not acquire cross-resistance to IGF-1R inhibition, though combination treatment in the resistant cells gave additive rather than synergistic cytotoxicity. We concluded that IGF-1R is an independent druggable target in ALK-positive lung cancer and support the trial of combination treatment.[2]

---

ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB.[3]

C24H25CLN6O

448.9479

448.177

C, 64.21; H, 5.61; Cl, 7.90; N, 18.72; O, 3.56

1356962-20-3

1356962-20-3

56599293

Light green to green solid powder

1.3±0.1 g/cm3

723.6±70.0 °C at 760 mmHg

391.4±35.7 °C

0.0±2.3 mmHg at 25°C

1.693

3.11

92.09

3

6

5

32

605

0

ClC(C=NC(NC1=C(OC)C=C(N2CCC(N)CC2)C=C1)=N3)=C3C4=CNC5=C4C=CC=C5

GCYIGMXOIWJGBU-UHFFFAOYSA-N

InChI=1S/C24H25ClN6O/c1-32-22-12-16(31-10-8-15(26)9-11-31)6-7-21(22)29-24-28-14-19(25)23(30-24)18-13-27-20-5-3-2-4-17(18)20/h2-7,12-15,27H,8-11,26H2,1H3,(H,28,29,30)

N-[4-(4-aminopiperidin-1-yl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)pyrimidin-2-amine

AZD 3463; AZD-3463; 1356962-20-3; AZD3463; AZD-3463; ALK/IGF1R inhibitor; N-(4-(4-Aminopiperidin-1-yl)-2-methoxyphenyl)-5-chloro-4-(1H-indol-3-yl)pyrimidin-2-amine; N-[4-(4-aminopiperidin-1-yl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)pyrimidin-2-amine; AZD3463

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: 6~20 mg/mL (13.4~44.6 mM)

配方 1 中的溶解度: ≥ 2 mg/mL (4.45 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.0 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80 +，混匀；然后加入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。

---

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。