Akt1 (IC50 = 2.7 nM); Akt3 (IC50 = 8.1 nM); Akt2 (IC50 = 14 nM); Leishmania; Akt1 E17K mutant

ARQ 092 阻断非活性 AKT 的膜易位，甚至使膜相关活性形式去磷酸化，从而扰乱 AKT 活性。用 50–500 nM ARQ 092 治疗可显着抑制中性粒细胞 M2 整合素功能，并降低血小板 P-选择素暴露和 Ib/IX/V 介导的凝集。 ARQ 092 抑制多种癌细胞系的增殖，但对白血病、乳腺癌、子宫内膜和结直肠癌细胞系最有效。此外，与具有 wt-PIK3CA/PIK3R1 或 PTEN 突变的癌细胞系相比，ARQ 092 抑制在含有 PIK3CA/PIK3R1 突变的癌细胞系中更为常见。在突变细胞中，ARQ 092 特异性靶向 PI3K/AKT 通路和 AKT，并降低 GSK3 和 GSK3 磷酸化。

短期口服 ARQ 092 或羟基脲（镰状细胞病的主要疗法）可减少接受 TNF-α 攻击的镰状细胞病小鼠小静脉中的异型细胞间相互作用。 ARQ 092 每日连续服用 60 mg 的剂量或每周服用一次 600 mg 的剂量，持续数月，耐受性良好。 ARQ 092 可能会抑制血管内细胞中所有 AKT 同工型的活性，从而减轻 SCD 患者的血栓形成和炎症过程。 ARQ 092 在含有 PI3K 通路基因突变的子宫内膜肿瘤子集中高度活跃。

Miransertib (ARQ-092) 是一种口服生物可利用的、选择性的、有效的变构 Aktin 抑制剂，对 Akt1、Akt2、Akt3 的 IC50 值分别为 2.7 nM、14 nM 和 8.1 nM。

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AKT1（1–480）、AKT2（1–481）和AKT3（1–479）阿尔法筛查分析[1]
使用GSK3衍生的生物素化肽底物、crosstide（生物素GRPRTSSFAEG）和AlphaScreen（扩增发光邻近均匀测定）技术测定AKT活性。在10%DMSO中以所需终浓度的10倍制备试验化合物和对照，并将每种化合物2.5μL加入反应板（Corning 96孔半面积固体白色非结合表面板）的每个孔中。将全长未磷酸化的AKT在测定缓冲液（50 mM Tris，pH 8.0，0.02 mg/mL BSA，10 mM MgCl2，1 mM EGTA，10%甘油，0.2 mM Na3VO4，1 mM DTT，0.1 mMβ-甘油磷酸盐和0.2 mM NaF）中稀释，并以17.5μL的体积加入每个孔中，使25μL反应中的终浓度为8 nM（AKT1）、63 nM（AK T2）或13 nM（AKT3）。在室温下预孵育20分钟后，通过加入5μL在测定缓冲液中稀释的活化混合物来启动激酶反应，该混合物含有生物素化的交叉肽、PDK1、MAPKAPK2、DOPS/DOPC、PtdIns（3,4,5）P3和ATP，最终浓度为60 nM生物素化交叉肽、0.1 nM（AKT1、AKT3）或0.3 nM M（AKT1、AKT2）或18μM（AKT3）ATP。将平板在室温下孵育30分钟，通过加入10μL在测定缓冲液中制备的停止/检测混合物在黑暗中停止反应，该混合物含有EDTA、AlphaScreen链霉抗生物素供体和蛋白A受体珠，以及终浓度为10mM EDTA的磷酸化AKT底物抗体，500 ng/孔的AlphaScreen链霉素供体珠和蛋白A接收器珠，以及最终稀释度为1:350的磷酸化AK T底物抗体。在室温下在黑暗中孵育测定板90分钟，并在PerkinElmer Envision多标记板阅读器上读取板（激发波长，640 nm；发射波长，570 nm）。所有报告的IC50值均为至少n=2次测定的几何平均值。

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CYP450抑制试验[1]
将人肝微粒体（0.25 mg/mL）、探针底物[3μM咪达唑仑（3A4）、5μM丁咯洛尔（2D6）、100μM甲磺丁脲（2C9）、10μM紫杉醇（2C8）、80μM S-美芬妥英（2C19）和50μM非那西丁（1A2）]、3.3 mM MgCl2、1 mM NADPH和溶解在DMSO（0.1%终浓度）中的化合物（0.1-10μM）孵育10分钟，然后使用含利血平（内标）的乙腈停止反应。在35°C的恒定氮气流下蒸发溶剂，将所得化合物溶解在100μL水中进行LC/MS/MS分析。使用由0.5μM酮康唑（3A4）、1μM磺胺苯唑（2C9）、1µM奎尼丁（2D6）、2μM槲皮素（2C8）、1微米诺卡酮（2C19）和0.5μMα-萘黄酮（1A2）组成的抑制剂混合物作为阳性对照。仅含有DMSO（无化合物）的培养基作为100%活性对照。基于100%活性对照的信号计算抑制百分比，并使用XLfit4中的拟合模型205（单部位剂量反应）估算或确定IC50值。

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跨物种NADPH依赖性微粒体稳定性测定[1]
将人、CD-1小鼠和比格犬肝微粒体（0.25 mg/mL）、3.3 mM MgCl2和NADPH再生系统（0.4单位/mL G6PDH、1.3 mM NADP+和3.3 mM G6P）与1μM化合物一起孵育0、3、6、10、15或30分钟。用含华法林的乙腈（内标）停止孵育，并通过LC/MS/MS分析样品。峰面积比用于确定每个时间点与时间零点相比的剩余百分比。使用单指数衰变方程估算半衰期值。咪达唑仑和普萘洛尔（仅雌性小鼠）用作阳性对照。

使用 CellTiter 非放射性细胞增殖测定进行抗增殖细胞测定，该测定利用活细胞从四唑化合物产生甲臜。 ATCC 可以购买 AN3CA 和 A2780 电池。 A2780 细胞在 RPMI 中培养，而 AN3CA 细胞在 DMEM 中培养。培养24小时并处理72小时后，将测试物质以不超过0.5% v/v的最终DMSO浓度施加到96孔板中的细胞上。使用 MTS 库存试剂（DPBS 中 2 mg/mL）将 PMS 库存试剂（DPBS 中 0.92 mg/mL）稀释 20 倍，然后稀释 5 倍并添加到 96 孔板的每个孔中。

Mice：The experiments presented here employ only male offspring, and all mice are kept on outbred C57BL6/J backgrounds that have undergone more than 10 generations of backcrossing. Then, for the next four weeks, either the vehicle or Miransertib (100 mg/kg body weight) is given daily by oral gavage. When established hypertrophy was indicated at 12 weeks of age, administration started and lasted for 4 weeks until the mice were 16 weeks old. SHP2+/+ and SHP2Y279C/+ mice receive only vehicle treatment as controls.

Compounds 9a,b, 21a–c, and 23 did not significantly inhibit CYP450 1A2, 2C8, 2D6, and 3A4. Compounds 9a and 9b inhibited CYP450 2C9 with a submicromolar IC50 value, while 9a also inhibited CYP450 2C19 (IC50 ≤ 1 μM). All compounds were adequately stable in liver microsomes (human, mouse, and dog). In general, we found that compounds from this chemical series possess adequate in vitro ADME properties, and compound ARQ 092 (21a) in particular showed good biochemical inhibition, cellular knockdown of AKT phosphorylation, and ADME properties. In a mouse pharmacokinetic study (po at 100 mg/kg, iv at 5 mg/kg), compound ARQ 092 (21a) showed an oral bioavailability of 23%. An in vivo pharmacodynamic assessment of ARQ 092 (21a) using NCr-M nude mice implanted with AN3CA tumor xenografts was reported in our recent publication. Compound 21a resulted in 99%, 95%, and 58% reductions in p-AKT (S473), p-AKT (T306), and p-PRAS40 (T246), respectively, after tumor-bearing mice were treated with 100 mg/kg po (Table 8). The inhibition of phosphorylation was sustained at 8 h. The plasma concentration of compound 21a at 1 h was 2.1 μM and decreased to 0.26 μM at 8 h, while in the tumor, the concentration was 21.0 μM at 1 h and 9.6 μM at 8 h. The concentrations in the tumor tissues were significantly higher than in the plasma, indicating a marked preference for tissue accumulation compared with the vasculature compartment.[1]

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Definitive single dose pharmacokinetic studies were conducted in rats and monkeys (Table 9). ARQ 092 (21a) showed good absolute oral bioavailability in rats and monkeys with F values of 62% and 49%, respectively. The compound was more slowly absorbed in rats compared to monkeys with Tmax values of 8.0 h for rats versus 4.3 h for monkeys. The half-life was also longer in rats compared to monkeys with t1/2 values of 17 h in rats versus 7 h in monkeys. The Cmax was 198 and 258 ng/mL and the AUCinf was 5496 and 2960 h·ng/mL in rats and monkeys, respectively.

[1]. Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor. J Med Chem. 2016 Jul 14;59(13):6455-69.

[2]. Miransertib (ARQ 092), an orally-available, selective Akt inhibitor is effective against Leishmania. PLoS One. 2018 Nov 6;13(11):e0206920.

Miransertib is under investigation in clinical trial NCT01473095 (Phase 1 Dose Escalation Study of ARQ 092 in Adult Subjects With Advanced Solid Tumors and Recurrent Malignant Lymphoma).
Miransertib is an orally bioavailable inhibitor of the serine/threonine protein kinase AKT (protein kinase B) with potential antineoplastic activity. Miransertib binds to and inhibits the activity of AKT in a non-ATP competitive manner, which may result in the inhibition of the PI3K/AKT signaling pathway. This may lead to the reduction in tumor cell proliferation and the induction of tumor cell apoptosis. The AKT signaling pathway is often deregulated in cancer and is associated with tumor cell proliferation, survival and migration.

C27H24N6

432.5197

432.206

C, 74.98; H, 5.59; N, 19.43

1313881-70-7

Miransertib hydrochloride;1313883-00-9; 1313881-70-7; 1817727-87-9 (3HCl); 1817727-88-0 (mesylate)

53262401

Off-white to yellow solid powder

1.4±0.1 g/cm3

700.8±70.0 °C at 760 mmHg

377.6±35.7 °C

0.0±2.2 mmHg at 25°C

1.742

6.09

95.6

2

5

4

33

653

0

N([H])([H])C1(C2C([H])=C([H])C(=C([H])C=2[H])N2C(C3C([H])=C([H])C([H])=NC=3N([H])[H])=NC3C([H])=C([H])C(C4C([H])=C([H])C([H])=C([H])C=4[H])=NC2=3)C([H])([H])C([H])([H])C1([H])[H]

HNFMVVHMKGFCMB-UHFFFAOYSA-N

InChI=1S/C27H24N6/c28-24-21(8-4-17-30-24)25-32-23-14-13-22(18-6-2-1-3-7-18)31-26(23)33(25)20-11-9-19(10-12-20)27(29)15-5-16-27/h1-4,6-14,17H,5,15-16,29H2,(H2,28,30)

3-[3-[4-(1-aminocyclobutyl)phenyl]-5-phenylimidazo[4,5-b]pyridin-2-yl]pyridin-2-amine

ARQ092; ARQ-092; AKT inhibitor 2; ARQ 092 Free Base; Miransertib [INN]; ARQ 092

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: ~86 mg/mL (~183.4 mM)
Water: <1 mg/mL
Ethanol: ~6 mg/mL (~12.8 mM)

配方 1 中的溶解度: ≥ 1.25 mg/mL (2.89 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 12.5 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。

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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。