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| 靶点 |
In vitro activity: AT9283 leads to a clear polyploid phenotype by inhibiting the activity of Aurora B kinase in HCT116 cells with IC50 of 30 nM. Furthermore, AT9283 also produces the potent inhibition on HCT116 colony formation.
Kinase Assay: Assays for Aurora A and B are performed in a DELFIA format. Aurora A enzyme is incubated with AT9283 and 3 μM cross-tide substrate (biotin-CGPKGPGRRGRRRTSSFAEG) in 10 mM MOPS, pH 7, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP, and 2.5% DMSO. Aurora B enzyme is incubated with AT9283, 3 μM of the above substrate in 25 mM Tris, pH 8.5, 5 mM MgCl2, 0.1 mg/mL BSA, 0.025% Tween-20, 1 mM DTT, 15 μM ATP, and 2.5% DMSO. Reactions are allowed to proceed for 60 minutes and 45-90 minutes for Aurora A and Aurora B, respectively, before quenching with EDTA. The reaction mixtures are then transferred to a neutravidin-coated plate, and phosphorylated peptide is quantified by means of a phospho-specific antibody and a europium labeled secondary antibody using time-resolved fluorescence (excitation, 337 nm; emission, 620 nm). IC50 values for the control compounds are 92 nM (Aurora A assay) and 17 nM (Aurora B). Cell Assay: HCT 116 cells are cultured in DMEM + 10% FBS + GLUTAMAX I. Black 96-well flat-bottomed (clear) tissue culture treated plates are seeded in 200 μL of medium and incubated for approximately 16 hours at 37°C in a humidified atmosphere of 5% CO2 in air. Cells are treated with test compound at nine different concentrations (spanning 1 nM to 10 μM, plus DMSO vehicle control) and then incubated for 72 hours. Polyploidy morphological observations of the cells are then noted. The concentration of AT9283 required to produce a distinct polyploid phenotype is reported. Cells are seeded at a concentration of 75−100 cells/mL relevant culture media onto 6- or 24-well tissue culture plates and allowed to recover for 16 hours. Test compound (11 concentrations spanning 0.1 nM to 10 μM) or vehicle control (DMSO) is added to duplicate wells to give a final DMSO concentration of 0.1%. Following compound addition, colonies are allowed to grow between 10 and 14 days for optimum discrete colony counting. Colonies are fixed in 2 mL of Carnoys fixative (25% acetic acid, 75% MeOH) and stained in 2 mL of 0.4% w/v crystal violet. The numbers of colonies in each well is counted. IC50 values are calculated by sigmoidal dose-response (variable slope) IC50 curves using Prism Graphpad software. |
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| 体外研究 (In Vitro) |
体外活性：AT9283 通过抑制 HCT116 细胞中 Aurora B 激酶的活性，产生明显的多倍体表型，IC50 为 30 nM。此外，AT9283 还对 HCT116 集落形成产生有效抑制。激酶测定：Aurora A 和 B 的测定以 DELFIA 格式进行。将 Aurora A 酶与 AT9283 和 3 μM 跨潮底物 (生物素-CGPKGPGRRGRRRTSSFAEG) 在 10 mM MOPS、pH 7、0.1 mg/mL BSA、0.001% Brij-35、0.5% 甘油、0.2 mM EDTA、10 mM MgCl2 中孵育、0.01% β-巯基乙醇、15 μM ATP 和 2.5% DMSO。 Aurora B 酶与 AT9283、3 μM 上述底物在 25 mM Tris（pH 8.5）、5 mM MgCl2、0.1 mg/mL BSA、0.025% Tween-20、1 mM DTT、15 μM ATP 和 2.5% DMSO 中一起孵育。 Aurora A 和 Aurora B 的反应分别进行 60 分钟和 45-90 分钟，然后用 EDTA 猝灭。然后将反应混合物转移至中性抗生物素蛋白包被的板中，并使用时间分辨荧光（激发，337 nm；发射，620 nm）通过磷酸特异性抗体和铕标记二抗对磷酸化肽进行定量。对照化合物的 IC50 值为 92 nM（Aurora A 测定）和 17 nM（Aurora B）。细胞测定：HCT 116 细胞在 DMEM + 10% FBS + GLUTAMAX I 中培养。将黑色 96 孔平底（透明）组织培养处理板接种到 200 μL 培养基中，并在 37°C 下孵育约 16 小时。空气中含有 5% CO2 的潮湿气氛。用九种不同浓度的测试化合物（1 nM 至 10 μM，加上 DMSO 载体对照）处理细胞，然后孵育 72 小时。然后记录细胞的多倍体形态学观察结果。报告了产生独特的多倍体表型所需的 AT9283 浓度。将细胞以 75−100 个细胞/mL 相关培养基的浓度接种到 6 或 24 孔组织培养板上，并允许恢复 16 小时。将测试化合物（0.1 nM 至 10 μM 的 11 种浓度）或载体对照 (DMSO) 添加到重复孔中，得到 0.1% 的最终 DMSO 浓度。添加化合物后，菌落可生长 10 至 14 天，以实现最佳的离散菌落计数。将集落固定在 2 mL Carnoys 固定剂（25% 乙酸、75% MeOH）中，并在 2 mL 0.4% w/v 结晶紫中染色。计算每个孔中的菌落数。使用 Prism Graphpad 软件通过 S 形剂量反应（可变斜率）IC50 曲线计算 IC50 值。
AT-9283 (化合物 16) 是一种强效的双重 Aurora A/Aurora B 抑制剂，IC₅₀ 值在低纳摩尔范围（约 3 nM）。它能抑制 HCT116 结肠癌细胞的生长和存活，在 0.03 µM 浓度下诱导出典型的 Aurora B 激酶抑制相关的多倍体细胞表型。 在 HCT116 集落形成二次实验中，AT-9283 表现出强效的抗增殖活性，IC₅₀ 为 12 nM。 该化合物对 CYP450 酶系的抑制谱很干净，对 CYP3A4、2D6、1A2、2C9 和 2C19 的 IC₅₀ 值均大于 10 µM。 [1] |
| 体内研究 (In Vivo) |
在 HCT116 人类结肠癌异种移植小鼠中，AT9283 治疗（15 mg/kg 和 20 mg/kg）16 天可分别显着抑制 67% 和 76% 的肿瘤生长。此外，与血浆（0.5 小时）相比，AT9283 在肿瘤中的半衰期（2.5 小时）也显着延长，并且在小鼠中的口服生物利用度适中（Fp.o. = 24%）。
AT-9283 在免疫功能低下小鼠的早期 HCT116 人结肠癌异种移植模型中显示出显著的体内抗肿瘤功效。 当以间歇性方案腹腔注射（15 和 20 mg/kg，每天两次，持续 2 天，然后停药 2 天，重复 5 个周期）时，与载体对照组相比，在治疗第 16 天分别产生了 67%（%T/C = 33%）和 76%（%T/C = 24%）的显著肿瘤生长抑制。剂量耐受性良好，平均体重维持在起始体重的 90% 以上。 [1] |
| 酶活实验 |
Aurora A 和 Aurora B 激酶活性实验采用 DELFIA 形式进行。
对于 Aurora A，将酶与测试化合物和生物素化的肽底物在含有 MOPS、BSA、MgCl₂、ATP 和 DMSO 的缓冲液中孵育。反应进行 60 分钟后用 EDTA 淬灭。使用磷酸化特异性一抗和铕标记的二抗，通过时间分辨荧光法量化磷酸化肽。 对于 Aurora B，采用类似的程序，使用 Tris 基缓冲液，反应进行 45-90 分钟。IC₅₀ 值从剂量反应曲线中确定。 [1] |
| 细胞实验 |
HCT116 初代细胞实验：将 HCT116 细胞接种在 96 孔板中过夜贴壁。用一系列浓度的 AT-9283（1 nM 至 10 µM）或 DMSO 载体对照处理细胞 72 小时。孵育后，在显微镜下检查细胞的多倍体形态特征。报告产生明显多倍体表型所需的最低浓度。
HCT116 集落形成实验（二次实验）：将细胞以低密度接种在多孔板中。恢复后，用 11 个浓度（0.1 nM 至 10 µM）的 AT-9283 或载体对照处理细胞。让集落生长 10-14 天，然后固定、染色并计数。使用 S 形剂量反应曲线拟合计算抑制集落形成的 IC₅₀ 值。 [1] |
| 动物实验 |
Dissolved in 10% DMSO, 20% water, 70% hydroxypropyl- β-cyclodextrin (25% w/v aq).; 15 and 20 mg/kg; administrated i.p.HCT116 cells are injected s.c. Into the hind flank of male BALB/c mice.
Efficacy Study: The hydrochloride salt of AT-9283 was formulated in 10% DMSO, 20% water, and 70% hydroxypropyl-β-cyclodextrin (25% w/v aqueous solution). Mice bearing HCT116 xenograft tumors (~100 mm³) were dosed intraperitoneally (ip) with 15 or 20 mg/kg of AT-9283, twice daily for 2 days, followed by 2 days without treatment. This cycle was repeated five times. Tumor dimensions were measured periodically, and volumes were calculated. Control animals received the vehicle only on the same schedule. [1] Pharmacokinetic/Tumor Distribution Study: For iv dosing, AT-9283 hydrochloride was administered in 100% saline at 5 or 10 mg/kg. For oral (po) dosing, it was given at 10 mg/kg in 10% saline and 90% water. For ip dosing, it was administered at 20 mg/kg in a vehicle of 10% DMSO, 20% water, and 70% hydroxypropyl-β-cyclodextrin (25% w/v aqueous). Plasma and tumor samples were collected at timed intervals for analysis. [1] |
| 药代性质 (ADME/PK) |
Following intravenous (iv) administration at 5 mg/kg in mice, AT-9283 exhibited a plasma clearance of 114 mL/min/kg, a volume of distribution at steady state (Vss) of 3.9 L/kg, and a plasma half-life (T₁/₂) of 0.5 hours.
The half-life in tumors was significantly longer at 2.5 hours following a 10 mg/kg iv dose. Following oral (po) administration at 10 mg/kg, the maximum concentration (Cmax) was 0.45 µM, AUC₀–t was 0.91 µM·h, and oral bioavailability (F) was 24%. Following intraperitoneal (ip) administration at 20 mg/kg, absorption was essentially complete (F ≈ 100%), with a Cmax of 8.4 µM and an AUC₀–t of 7.7 µM·h. Mouse plasma protein binding was 81.5%. The compound showed good thermodynamic solubility (2.0 mg/mL at pH 7.0 and 13 mg/mL at pH 5.5). [1] |
| 参考文献 |
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| 其他信息 |
AT-9283 (Compound 16) is a multitargeted kinase inhibitor discovered via fragment-based optimization of a pyrazole-benzimidazole core. It potently inhibits Aurora A and Aurora B kinases, leading to cell cycle defects (polyploidy) and apoptosis.
Its kinase inhibition profile extends to other targets implicated in cancer, such as JAK2 and Abl (T315I), potentially offering advantages in treating malignancies driven by multiple kinases. Based on its balanced in vitro potency, physicochemical properties, and in vivo efficacy, AT-9283 was selected for preclinical development and has advanced into phase I clinical trials for the treatment of cancer. [1] |
| 分子式 |
C22H29N7O5
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|---|---|---|
| 分子量 |
471.52
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| 精确质量 |
381.191
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| CAS号 |
896466-76-5
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| 相关CAS号 |
AT9283;896466-04-9
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| PubChem CID |
135566103
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| 外观&性状 |
Typically exists as solid at room temperature
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| 密度 |
1.5±0.1 g/cm3
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| 折射率 |
1.715
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| LogP |
0.92
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| tPSA |
169
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| 氢键供体(HBD)数目 |
6
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| 氢键受体(HBA)数目 |
8
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| 可旋转键数目(RBC) |
6
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| 重原子数目 |
34
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| 分子复杂度/Complexity |
614
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| 定义原子立体中心数目 |
1
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| SMILES |
O=C(NC1CC1)NC2=CNN=C2C=3NC4=C(N3)C=C(C=C4)CN5CCOCC5.C[C@H](O)C(O)=O
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| InChi Key |
RWYNKTMXFIMBFE-WNQIDUERSA-N
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| InChi Code |
InChI=1S/C19H23N7O2.C3H6O3/c27-19(21-13-2-3-13)24-16-10-20-25-17(16)18-22-14-4-1-12(9-15(14)23-18)11-26-5-7-28-8-6-26;1-2(4)3(5)6/h1,4,9-10,13H,2-3,5-8,11H2,(H,20,25)(H,22,23)(H2,21,24,27);2,4H,1H3,(H,5,6)/t;2-/m.0/s1
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| 化学名 |
1-cyclopropyl-3-[5-[6-(morpholin-4-ylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea;(2S)-2-hydroxypropanoic acid
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| 别名 |
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| HS Tariff Code |
2934.99.9001
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| 存储方式 |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| 运输条件 |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| 溶解度 (体外实验) |
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| 制备储备液 | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1208 mL | 10.6040 mL | 21.2080 mL | |
| 5 mM | 0.4242 mL | 2.1208 mL | 4.2416 mL | |
| 10 mM | 0.2121 mL | 1.0604 mL | 2.1208 mL |
1、根据实验需要选择合适的溶剂配制储备液 (母液)：对于大多数产品，InvivoChem推荐用DMSO配置母液 (比如：5、10、20mM或者10、20、50 mg/mL浓度），个别水溶性高的产品可直接溶于水。产品在DMSO 、水或其他溶剂中的具体溶解度详见上”溶解度 (体外)”部分;
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3、建议使用下列计算器进行相关计算（摩尔浓度计算器、稀释计算器、分子量计算器、重组计算器等）;
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计算结果:
工作液浓度： mg/mL；
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体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL ddH2O，混匀澄清。
(1) 请确保溶液澄清之后，再加入下一种溶剂 (助溶剂) 。可利用涡旋、超声或水浴加热等方法助溶；
(2) 一定要按顺序加入溶剂 (助溶剂) 。
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