GSK-3β (IC50 = 0.58 nM); GSK-3α (IC50 = 0.65 nM); cdc2 (IC50 = 3700 nM)

CHIR-98014 抑制人 GSK-3β，Ki 为 0.87 nM。 CHIR-98014 对于预防小鼠和大鼠 GSK-3 非常有效。尽管 CHIR-98014 作为 ATP 结合的直接竞争性抑制剂，但与 20 种其他蛋白激酶（例如 Cdc2、ERK2、Tie-2 和 KDR）相比，CHIR-98014 对 GSK-3 的选择性高出 500 倍至 >1000 倍。 CHIR-98014 抑制 Cdc2 的 IC50 为 3.7 M。值得注意的是，CHIR 98014 仅区分 GSK-3 及其最接近的同源物 CDC2 和 ERK2，尽管它对 GSK-3 的高度同源性和亚型表现出相似的效力。当抑制剂 CHIR98014 以不断增加的浓度应用于表达胰岛素受体的 CHO-IR 细胞或原代大鼠肝细胞时，会产生高于基础值 2 至 3 倍的 GS 活性比刺激。对于大鼠肝细胞和 CHO-IR，引起半最大 GS 刺激 (EC50) 的 CHIR-98014 浓度为 107 nM。[1]

GSK-3 抑制剂 CHIR-98014 可激活胰岛素敏感瘦 Zucker 大鼠和胰岛素抵抗 ZDF 大鼠分离的 I 型骨骼肌中的 GS 活性比。从 ZDF 大鼠中分离的比目鱼肌显示出对激活 GS 的胰岛素具有显着抵抗性，但对 500 nM CHIR-98014 的反应程度与来自瘦 Zucker 大鼠的肌肉相同程度（增加 40%）。值得注意的是，胰岛素加 CHIR-98014 的 GS 激活对瘦 Zucker 大鼠的肌肉具有附加作用，并且比 ZDF 大鼠的肌肉具有更大的附加作用。这些细胞和肌肉中的总 GS 活性不会被 CHIR-98014 或胰岛素改变。同时，CHIR-98014 不会影响瘦动物肌肉中的胰岛素剂量反应。高血糖的降低和葡萄糖处理的改善不仅限于db/db小鼠和ZDF大鼠，在ob/ob小鼠、饮食诱导的糖尿病C57BL/6小鼠和用CHIR-治疗的葡萄糖不耐症SHHF大鼠中也观察到类似的结果。 98014。此外，CHIR-98014 还可降低出生后大鼠皮质和海马中 tau 蛋白的磷酸化 (Ser396)。

聚丙烯 96 孔板每孔填充 300 μL 含有激酶的缓冲液（50 mM tris HCl、10 mM MgCl2、1 mM EGTA、1 mM 二硫苏糖醇、25 mM β-甘油磷酸、1 mM NaF、0.01% BSA，pH 7.5） 、肽底物和任何活化剂。在所有无细胞测定中，将 CHIR-98014 或对照添加到 3.5 μL DMSO 中，然后添加 50 μL ATP 库存，以产生 1 μM ATP 的终浓度。孵育后，将一式三份的 100 L 等分试样添加到 Combiplate 8 板中，其中每孔含有 100 µL 浓度的 50 mM ATP 和 20 mM EDTA。将孔用 PBS 冲洗五次，填充 200 L 闪烁液，密封，放置 30 分钟，然后在第一小时后在闪烁计数器中计数。整个过程在室温下进行。

表达人胰岛素受体的 CHO-IR 细胞在含有 10% 胎牛血清且不含次黄嘌呤的 Hamm's F12 培养基中生长至 80% 汇合。将胰蛋白酶处理的细胞以 1 × 106 个细胞/孔的密度接种在 2 mL 不含胎牛血清的培养基中的 6 孔板中。 24 小时后，用 1 mL 含有 GSK-3 抑制剂 CHIR 98014 或对照（最终 DMSO 浓度 0.1%）的无血清培养基替换培养基，在 37 °C 下培养 30 分钟。将细胞在含有 1 mM EDTA、1 mM DTT、100 mM NaF、1 mM 苯甲基磺酰氟和 25 g/mL 亮抑肽素（缓冲液 A）的 50 mM tris (pH 7.8) 中冷冻/解冻，并在 30 ℃ 离心 15 分钟。 4°C/14000 克。使用不存在时的 GS 活性来计算 GS 的活性比。

Drugs and drug administration[2]
SB216763 (30 mg kg−1) and CHIR98014 (30 mg kg−1) were re-suspended in DMSO and injected i.v. AR-A014418 (30 mg kg−1), was dissolved in 100% PEG400 and administered per os (p.o.) Indirubin-3′-monoxime (20 mg kg−1) and Alsterpaullone (20 mg kg−1) were dissolved in 20% DMSO/25% Tween-80 and injected i.p. and s.c., respectively. All drug studies were conducted using P12 rats from the same litter. Control animals were dosed with the respective vehicle and both groups were killed after 1, 2 and 4 h for brain exposure measurements (see the next section), western blotting and GSK-3β activity assays. Experiments measuring the efficacy of each compound were performed at least three times and at a time point determined by brain exposure data. LiCl (100 and 200 mg kg−1) was dissolved in sterile water, and administered p.o. to animals. P12 rats were killed 8 h after injection. Some of the littermates were used as the control group and dosed with NaCl (100 or 200 mg kg−1, p.o.) dissolved in sterile water.[2]

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Brain exposure measurements[2]
Rat brain homogenates were analysed for exposure levels of SB216763, Indirubin-3′-monoxime, Alsterpaullone, CHIR98014 and AR-A014418 using turbulent flow chromatography (HTLC) followed by detection by Tandem mass spectrometry (MS/MS). Four times 70% v w−1acetonitrile was added to the sample and homogenized in an autogizer robot. The brain homogenate was centrifuged at 6000 g for 15 min at 5 °C, and the supernatant was analysed. Calibration curves (1–1000 ng ml−1 brain homogenate) for each compound were prepared using brain homogenate from untreated rats. A total of 25 μl of 10% MeOH with internal standard (citalopram) was added to either 25 μl of brain homogenate or calibration standard, followed by centrifugation at 6000 g for 20 min at 5 °C). Ten microlitres of each sample was injected into the HTLC system using a HTS PAL autosampler. Samples with AR-A014418 were purified using 0.1% HCOOH in water for 15 s (2 ml min−1) using a Cyclone HTLC column (0.5 × 50 mm, 50 μm). The compounds were extracted from the HTLC using 100 μl 0.1% HCOOH/90% acetonitrile placed in the loop and transferred to the analytical column, X-Terra MS C8 (20 × 2.1 mm, 3.5 μm) with 0.1% HCOOH in water over 120 s (0.08 ml min−1) and eluted from the analytical column using a gradient from 0.1% HCOOH/2% MeCN to 0.1% HCOOH/98% acetonitrile for 45 s, followed by elution with 0.1% HCOOH/98% acetonitrile for 120 s flow 0.5 ml min−1). Detection of the compound was performed using Ultima triple-quadropole mass spectrometer (Waters) and positive ionization using multiple reaction monitoring set at optimal conditions. For AR-A014418 the transition 308.9 → 121.7 was used.

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Blood is obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose is measured directly or heparinized plasma is collected for measurement of glucose or insulin. Animals are prebled and randomly assigned to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals are fasted throughout the procedure with food removal early in the morning, 3 h before the first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). Food is taken away 16 hours prior to the test agent being administered when determining the time course of plasma glucose and insulin changes in fasting ZDF rats. The GS activity ratio is calculated as the GS activity in the presence of 5 mM glucose-6-phosphate divided by the activity in the absence of glucose-6-phosphate.

[1]. Diabetes. 2003 Mar;52(3):588-95.

[2]. Br J Pharmacol. 2007 Nov;152(6):959-79.

CHIR-98014 is a member of the class of aminopyrimidines that is pyrimidine substituted by {2-[(6-amino-5-nitropyridin-2-yl)amino]ethyl}amino, 2,4-dichlorophenyl, and 1H-imidazol-1-yl groups at positions 2, 4 and 5, respectively. It is a potent ATP-competitive inhibitor of GSK3alpha and GSK3beta (IC50 values of 0.65 and 0.58 nM, respectively). It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor, an apoptosis inducer, an antineoplastic agent, a hypoglycemic agent, a Wnt signalling activator and a tau aggregation inhibitor. It is a secondary amino compound, a dichlorobenzene, a member of imidazoles, a diaminopyridine, an aminopyrimidine and a C-nitro compound.

C20H17CL2N9O2

486.3141

485.088

C, 49.39; H, 3.52; Cl, 14.58; N, 25.92; O, 6.58

252935-94-7

556813-39-9 (CHIR98024);252935-94-7 (CHIR98014);CHIR98014 HCl;

53396311

Light yellow to yellow solid powder

1.6±0.1 g/cm3

839.0±75.0 °C at 760 mmHg

461.2±37.1 °C

0.0±3.1 mmHg at 25°C

1.753

3.76

158.85

3

9

7

33

645

0

ClC1C([H])=C(C([H])=C([H])C=1C1C(=C([H])N=C(N=1)N([H])C([H])([H])C([H])([H])N([H])C1C([H])=C([H])C(=C(N([H])[H])N=1)[N+](=O)[O-])N1C([H])=NC([H])=C1[H])Cl

MDZCSIDIPDZWKL-UHFFFAOYSA-N

InChI=1S/C20H17Cl2N9O2/c21-12-1-2-13(14(22)9-12)18-16(30-8-7-24-11-30)10-27-20(29-18)26-6-5-25-17-4-3-15(31(32)33)19(23)28-17/h1-4,7-11H,5-6H2,(H3,23,25,28)(H,26,27,29)

6-N-[2-[[4-(2,4-dichlorophenyl)-5-imidazol-1-ylpyrimidin-2-yl]amino]ethyl]-3-nitropyridine-2,6-diamine

CT-98014; CT 98014; CT98014; CHIR 98014; CHIR-98014; 252935-94-7; 6-N-[2-[[4-(2,4-dichlorophenyl)-5-imidazol-1-ylpyrimidin-2-yl]amino]ethyl]-3-nitropyridine-2,6-diamine; CT-98014; 2,6-PYRIDINEDIAMINE, N6-[2-[[4-(2,4-DICHLOROPHENYL)-5-(1H-IMIDAZOL-1-YL)-2-PYRIMIDINYL]AMINO]ETHYL]-3-NITRO-; CHEMBL3185148; N2-(2-((4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-yl)amino)ethyl)-5-nitropyridine-2,6-diamine; N6-[2-[[4-(2,4-DICHLOROPHENYL)-5-(1H-IMIDAZOL-1-YL)-2-PYRIMIDINYL]AMINO]ETHYL]-3-NITRO-2,6-PYRIDINEDIAMINE; CHIR-98014; CHIR98014;CHIR98014 HCl; CHIR-98014 hydrochloride

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: ~8 mg/mL (~16.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL