ERα antagonism (IC50 = 0.28 nM); ERα downregulation (IC50 = 0.14 nM); ERα binding (IC50 = 0.82 nM)

AZD9496 表现出 ERα 结合、下调和拮抗作用的效力，IC50 值分别为 0.82 nM、0.14 nM 和 0.28 nM。 AZD9496 的 EC50 为 0.04 nM，可显着抑制 MCF-7 细胞生长[1]。研究发现AZD9496对以下测试的核激素受体表现出高度选择性：黄体酮受体（PR），IC50=0.54 μM；糖皮质激素受体（GR），IC50=9.2 μM；雄激素受体（AR），IC50=30 μM[2]。

---

AZD9496是一种选择性ERα拮抗剂、下调剂和ER+肿瘤细胞生长抑制剂。 AZD9496在体外直接靶向ERα进行下调。AZD9496在体外拮抗和下调突变ER。[1]

---

化合物30b（AZD9496）显示出与前体27b大致相当的总体性质特征（表2），同时保持了优异的跨物种PK（表3）。以化合物2（氟维司群）和雌二醇（E2）为对照化合物的蛋白质印迹分析证实了化合物30b对ERα的显著降解（图7）。化合物30b对其他测试的核激素受体的选择性很高：雄激素受体（AR），IC50=30μM；糖皮质激素受体（GR），IC50=9.2μM；孕酮受体（PR），IC50=0.54μM（参见雌激素受体（ERα），IC50=0.0008μM）。 [2]

在雌激素依赖性 MCF-7 异种移植模型中，低至 0.5 mg/kg 的剂量即可显着抑制肿瘤生长。这种作用伴随着 PR 蛋白水平的剂量依赖性降低，表明是有效的拮抗剂。与单独治疗相比，AZD9496联合CDK4/6抑制剂和PI3K通路具有额外的生长抑制作用。当每天口服一次 AZD9496，剂量为 5 和 25 mg/kg 时，与 ICI 182780 对照相比，子宫重量显着增加（P<0.001），但不如 ICI 47699 显着（P=0.001）。 1]。 AZD9496 还在 HCC-1428 LTED 细胞系的长期雌激素剥夺模型 (LTED) 中进行了测试，该细胞系被认为是最准确的芳香酶抑制模型，因为它能够在缺乏雌激素的情况下生长。 AZD9496有实质性影响；在此模型中，5 mg/kg 剂量下可观察到肿瘤消退[2]。

---

AZD9496是体内有效的口服乳腺肿瘤生长抑制剂[1]
在MCF-7人乳腺异种移植物中，作为代表性的ER+/PR+/HER2+乳腺癌症模型，探讨了慢性口服给药AZD9496的效果。良好的生物利用度和高清除率使小鼠口服给药后的终末t1/2为5-6小时，并导致显著的剂量依赖性肿瘤生长抑制，在50mg/kg时抑制率为96%，与赋形剂对照组相比没有毒性或体重减轻（图4A）。为了确认AZD9496靶向ER通路，在研究结束时采集的肿瘤样本中测量了PR蛋白水平，发现PR显著降低，这与肿瘤生长抑制有关。10和50 mg/kg剂量的PR均降低了90%以上，即使0.5 mg/kg剂量也降低了75%，表明AZD9496可以明显拮抗ER途径（图4B）。与每周3次给予5mg/小鼠氟维司群和每天口服10mg/kg他莫昔芬相比，给予5mg/kg AZD9496（显著抑制肿瘤所需的最小剂量）可产生更大的肿瘤生长抑制作用（图4C）。进行了一系列体内药效学研究，以测量达到PR水平最大抑制所需的时间和恢复到基础水平所需的时光。给予5mg/kg AZD9496三天，PR蛋白减少98%，48小时后继续抑制蛋白水平（图4D），72小时后完全恢复（数据未显示），这表明体内药物半衰期较长。在一周内以3×5mg/只小鼠的剂量给予Fulvestrant，在很长一段时间内PR蛋白减少了60%，测得的血浆水平比临床上使用500mg Fulvestrant在稳态下达到的水平高出约8倍（图4E）。由于已知雌激素本身会下调ER蛋白，与MCF-7模型中使用AZD9496或氟维司群的对照动物相比，我们无法检测到ERα蛋白的进一步降低，这可能是由于在采集肿瘤样本时植入颗粒的雌激素循环血浆水平很高（补充图S5）。在循环血浆中检测到AZD9496的小鼠特异性代谢产物，其水平与AZD9469相似，并显示出相似的药代动力学特征。在体外MCF-7试验中测试这种代谢产物导致ERα拮抗活性比AZD9496低约5倍，ERα下调活性低7倍（数据未显示）。使用基于PR抑制数据的药代动力学/药效学模型，在5mg/kg的体内剂量下，PR的抑制率为98%，当代谢产物的活性被折现时，仅归因于母体化合物的抑制活性为85%。

AZD9496 是一种有效的、口服生物可利用的、选择性的雌激素受体拮抗剂和下调剂 (Ki=0.7 nM)。

---

SILAC检测[1]
MCF-7细胞在含有13C615N4精氨酸的无氨基酸（SILAC）酚红RPMI培养基（重培养基）的稳定同位素标记中生长至少三代，以完全标记肽。然后，细胞在添加了5%透析CSS的重培养基中生长24小时，然后用PBS洗涤，并切换到含有未标记精氨酸和AZD9496、氟维司群、三苯氧胺、雌二醇或DMSO的标准无酚红RPMI培养基。在裂解缓冲液中制备蛋白质裂解物之前，将化合物孵育48小时。使用抗ERα单克隆抗体（SP1）在4℃下对掺有内标物（仅用13C6赖氨酸标记的MCF-7细胞的裂解物）的等浓度样品蛋白质进行免疫沉淀过夜，然后在37℃下用0.4μg胰蛋白酶在50 mmol/L碳酸氢铵中消化过夜，然后通过选择反应监测（SRM）使用相对肽定量进行质谱分析。使用GraphPad PRISM中的单相指数衰减方程（Y=Span.e-K.X+Plateau）测量降解半衰期，其中X是时间，Y是响应，从Span+Plateau开始，以速率常数K减少到Plateau。

---

双核亲和度测量[1]
对于BIAcore亲和力测量，将四His抗体固定在20 mM HEPES、pH 7.4、150 mM NaCl、0.005%T20（HBS-T）运行缓冲液和在AZD9496存在下捕获的6His ERα蛋白中的双核CM5生物传感器芯片上。使用BIA评估软件计算缔合（kass）和解离（kdiss）速率常数和（KD），并将ERαLBD:AZD9496相互作用拟合为1:1 Langmuir结合相互作用模型。

---

生化和体外细胞测定[1]
如前所述，进行了结合、ER激动、拮抗、下调和细胞增殖试验（Biomol Screen 2015；20:748–59). 补充方法中描述了化合物处理细胞的BIAcore亲和力测量和免疫印迹。

---

蛋白质生产、结晶和结构测定[1]
ERα配体结合结构域的蛋白质表达、纯化和结晶如前所述进行。在ADSC探测器上的光束线ID23-1上的ESRF处收集X射线衍射数据。数据使用EDNA中实现的XDS进行处理，并使用SCALA进行缩放和合并。使用AmoRE和内部ERα结构作为搜索模型，通过分子置换求解了与AZD9496复合物中ERα的结构。使用Coot中的验证工具对蛋白质结构进行了质量检查。最终结构已存入蛋白质数据库，ID代码见补充表S1。

AZD9496、ICI 182780 和 ICI 47699 对 MCF-7 细胞 ERα 肽周转的影响。维持无类固醇条件指定的持续时间，并且细胞在含有 13C615N4 L-精氨酸的 SILAC 培养基中生长，以将 ERα 肽标记为“重”（蓝线）。然后，将培养物切换到未标记的 L-精氨酸，用 0.1% DMSO、300 nM Tamoxife、100 nM AZD9496 或 100 nM ICI 182780 将新合成的蛋白质标记为“正常”（红线）。显示的数据是平均值两个独立的实验[1]。

---

免疫印迹[1]
细胞在25 mmol/L Tris/HCL pH6.8、3 mmol/L EDTA、3 mmol/L EGTA、50 mmol/L NaF、2 mmol/L原钒酸钠、270 mmol/L蔗糖、10 mmol/Lγ-甘油磷酸、5 mmol/L焦磷酸钠和0.5%Triton X-100中裂解，补充蛋白酶抑制剂和磷酸酶抑制剂，蛋白质在4%至12%Tris-HCL预制凝胶上运行。用一抗探测膜过夜，然后用HRP标记的二抗孵育，并在Syngene ChemiGenius上用Super Signal West Dura化学发光底物进行可视化。

Mice: The effectiveness of AZD9496 in an MCF-7 xenograft model in vivo. PEG/captisol (vehicle) or AZD9496 (0.02, 0.1, 0.5, 10, and 50 mg/kg, p.o., q.d.) are the daily doses given to MCF-7 xenografts cultivated in male SCID mice. Every few months, the growth of the tumor is measured with a caliper, and the mean tumor volumes for every dose group are plotted.

---

Rat uterine and xenograft studies [1]
MCF-7 cells (5 x 106 ) were implanted subcutaneously in the hind flank of immuno-compromised (SCID) male mice the day after each mouse was surgically implanted with a 0.5 mg/21 day estrogen pellet. HCC1428 LTED (10 x 106 cells) were implanted transdermally into the fourth mammary fat pad of immuno-compromised (NSG) female mice 7 days after they were surgically ovariectomized. CTC-714 PDX model was derived from patient CTC cultures. EpCAM+CD44+ cells were suspended in phosphate buffered saline (PBS) mixed with high concentration matrigel (BD Biosciences) at 10 mg/ml and ~ 650 cells were injected into the third mammary fat pad of a NOD/SCID (Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse. In tumour transplantation study, 2 × 2 mm pieces of tumour tissue from CTC-derived tumour xenografts were implanted in the mammary fad pad of Beige Nude XID mice. Tumour growth was calculated weekly by bilateral caliper measurement (length x width) and mice randomised into vehicle or treatment groups with approximate mean start size of 0.2 to 0.4 cm3 for efficacy studies or 0.5 to 0.8 cm3 for PD studies. Mice were dosed once daily by oral gavage or subcutaneous (s.c.) injection for fulvestrant at the times and doses indicated for the duration of the treatment period. Tumour growth inhibition from start of treatment was assessed by comparison of the mean change in tumour volume for the control and treated groups. Statistical significance was evaluated using a one-tailed Student t test. Tumours were excised at specific time points and fragments either fixed in 10% buffered formalin or snap-frozen in liquid nitrogen and stored at -80 oC and terminal bleeds plasma PK samples collected. Measurement of estradiol levels in mouse plasma from the MCF-7 xenograft model with implanted estrogen pellets was done using a custom-made immunoassay kit from Meso scale Discovery (MSD).

---

Rat Uterine Model [1]
Sexually immature female Han Wistar rats were randomised and dosed either with vehicle, AZD9496 or tamoxifen once daily for 3 days by oral gavage or a single s.c dose of fulvestrant. At 24 hours after the final dose of each agent was given, rats were euthanised, terminal plasma samples collected and uterine tissue removed with both horns intact, blotted dry and weighed. Protein extracts were prepared for immunoblot analysis as described for xenograft studies.

---

Pharmacokinetic studies [1]
The pharmacokinetics of AZD9496 and its active mouse metabolite was investigated using combined data from multiple studies and analysed via population PK modelling. Specific PK studies consisted of IV bolus and PO doses of the parent, and IV bolus dose of the metabolite with multiple time points per animal, 2 animals per time point and were designed to establish a parent-metabolite pharmacokinetic model. The concentration of AZD9496 and its active metabolite in plasma samples was determined within AstraZeneca Oncology DMPK. Samples were analysed for parent and metabolite using LC-MS/MS detection using analytical standards over a final concentration range of 1 nM – 10,000 nM before being analysed using Masslynx and processed using Quanlynx.

Oral bioavailability (F) = 63%, 128%, 79% in rat, mouse and dog, respectively. [2]

---

The effect of chronic, oral dosing of AZD9496 was explored in MCF-7 human breast xenografts, as a representative ER+/PR+/HER2+ breast cancer model. Good bioavailability and high clearance gave a terminal t1/2 of 5–6 hours after oral dosing in the mouse and resulted in significant dose-dependent tumor growth inhibition with 96% inhibition at 50 mg/kg and no toxicity or weight loss relative to the vehicle control group. [1]

[1]. AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models. Cancer Res. 2016 Jun 1;76(11):3307-18.

[2]. Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a Potent and Orally Bioavailable Selective Estrogen.

AZD-9496 is under investigation in clinical trial NCT02248090 (AZD9496 First Time in Patients Ascending Dose Study).
Selective Estrogen Receptor Degrader AZD9496 is an orally available selective estrogen receptor degrader (SERD), with potential antineoplastic activity. Upon administration, SERD AZD9496 binds to the estrogen receptor (ER) and induces a conformational change that results in the degradation of the receptor. This prevents ER-mediated signaling and inhibits the growth and survival of ER-expressing cancer cells.

---

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. [1]

---

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.[2]

C29H29F3N2O6

558.5456

442.19

C, 67.86; H, 5.70; F, 12.88; N, 6.33; O, 7.23

1639042-28-6

AZD9496;1639042-08-2

92044376

Light yellow to brown solid powder

131

4

10

7

40

824

2

C[C@@H]1CC2=C([C@H](N1CC(C)(C)F)C3=C(C=C(C=C3F)/C=C/C(=O)O)F)NC4=CC=CC=C24.C(=C\C(=O)O)\C(=O)O

GYRUZXFUFGWTAG-DMSKNVGDSA-N

InChI=1S/C25H25F3N2O2.C4H4O4/c1-14-10-17-16-6-4-5-7-20(16)29-23(17)24(30(14)13-25(2,3)28)22-18(26)11-15(12-19(22)27)8-9-21(31)32;5-3(6)1-2-4(7)8/h4-9,11-12,14,24,29H,10,13H2,1-3H3,(H,31,32);1-2H,(H,5,6)(H,7,8)/b9-8+;2-1-/t14-,24-;/m1./s1

(Z)-but-2-enedioic acid;(E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： (1). 本产品在运输和储存过程中需避光。  (2). 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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: ≥ 2.75 mg/mL (4.92 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 27.5 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。