cdk2-cyclin E (IC50 = 6 nM); cdk2-cyclin A (IC50 = 45 nM); cdk5-p25 (IC50 = 14 nM); cdk1-cyclin B1 (IC50 = 16 nM); cdk9-cyclin T (IC50 = 20 nM); cdk6-cyclin D3 (IC50 = 21 nM); cdk4-cyclin D1 (IC50 = 449 nM); cdk7-cyclin H (IC50 = 821 nM)

Cyclin E-cdk2、Cyclin A-cdk2、Cyclin B1-cdk1、p25-cdk5、Cyclin D3-cdk6 和 Cyclin T-cdk9 均能被 AZD5438 有效抑制。活性（16、21、20 nM、6、45 nM）。 Cyclin E-cdk2、Cyclin A-cdk2、Cyclin B1-cdk1、p25-cdk5、Cyclin D3-cdk6 和 Cyclin T-cdk9 均受到 AZD5438 的有效抑制。与许多其他 CDK 调节剂类似，AZD5438 抑制 p25-cdk5 和糖原合酶，进而控制 3β 的偶发活性（相应的 IC50 值为 14 和 17 nM）[1]。 AZD5438 大大改善了 NSCLC 细胞的细胞放射靶向性。 AZD5438 与放射治疗联合使用时还可以改善肿瘤生长延迟，细胞范围改善至 1.2–1.7 [2]。

AZD5438（50 mg/kg，一天两次，或用于肿瘤学的 75 mg/kg）可防止人类异种移植物生长。 AZD5438 降低内部循环活性细胞的百分比。对用 AZD5438 处理的 SW620 异种移植物进行的其他药效学研究表明，在 16 小时的单一阈值后，有效剂量的 AZD5438（>40% 肿瘤生长抑制）保持对包括磷酸化 pRbSer249/Thr252 在内的生物标志物的抑制作用[1]。

使用闪烁邻近试验来测试 AZD5438 抑制 CDK 活性的能力。它涉及使用细胞周期蛋白-Ecdk2、cdk2-细胞周期蛋白 A、cdk4-细胞周期蛋白 D 和重组视网膜母细胞瘤底物（氨基酸 792-928）或 cdk1-细胞周期蛋白 B1 的重组 CDK-细胞周期蛋白复合物以及源自体外 p34cdc2 磷酸化的肽底物组蛋白 H1 位点（生物素-X-Pro-Lys-Thr-Pro-Lys-Lys-Ala-Lys-Lys-Leu）。使用肽底物 (AKKPKTPKKAKKLOH)，在 2 μM ATP 下基于闪烁邻近分析的测定中评估 AZD5438 针对重组 cdk5/p25 的活性。通过使用人纯化的糖原合酶激酶 3β 酶和真核起始因子 2B 底物（1 μM ATP），使用闪烁邻近测定法来确定对糖原合酶激酶 3β 活性的抑制。 AZD5438 接受激酶选择性筛选服务，针对 cdk6-细胞周期蛋白 D3、cdk7-细胞周期蛋白 H/MAT1（cdk 激活激酶复合物）和 cdk9-细胞周期蛋白 T 进行筛选。

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制备重组CDK1/cyclin B、CDK2/cyclin A/E、CDK9/cyclin T激酶复合物，将梯度浓度的AZD5438与激酶复合物、ATP底物及特异性肽段混合，37℃孵育60分钟；采用放射性磷酸化检测法测定底物磷酸化水平，根据不同药物浓度的抑制效果拟合曲线，计算各激酶的IC50值 [1] 采用荧光共振能量转移（FRET）法验证CDK2活性：将AZD5438与CDK2/cyclin A复合物、荧光标记底物肽及ATP混合，30℃反应45分钟后，检测荧光信号强度，计算激酶活性抑制率 [2]

实体瘤细胞系用于测试 AZD5438。总之，AZD5438以不同浓度添加到细胞中并孵育48小时。孵育期结束后，将 5-溴-2'-脱氧尿苷 (BrdUrd) 脉冲注入细胞中，并对 DNA 合成量进行定量。具体而言，细胞死亡对增殖抑制的 IC50 没有影响。以下方案用于将多种骨髓瘤细胞系接种到 96 孔板中：补充有 10% FCS 和谷氨酰胺的 RPMI 1640，然后使用 72 小时剂量的 AZD5438。 AlamarBlue 用于测量细胞生长，并使用预处理对照值计算 GI50 值。

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肿瘤细胞接种于96孔板（5×10³个/孔），培养24小时后加入0.01-10 μM梯度浓度的AZD5438，继续培养72小时；采用CellTiter-Glo发光法检测细胞活力，计算细胞存活率并拟合曲线得到IC50值 [1] A549细胞经AZD5438（100 nM）预处理2小时后，给予0-8 Gy梯度剂量射线照射，培养14天后采用克隆形成实验检测细胞存活分数，计算放射增敏比（SER）[2] 细胞经AZD5438（100 nM）处理48小时后，收集细胞并固定，PI染色后通过流式细胞仪分析细胞周期分布；提取细胞总蛋白，Western blot检测组蛋白H1、磷酸化组蛋白H1、Rb、磷酸化Rb等蛋白表达 [1] MCF-7细胞经药物处理72小时后，采用Annexin V-FITC/PI双染色法，室温孵育15分钟，流式细胞仪检测凋亡细胞比例；通过caspase-3活性检测试剂盒测定酶活性，Western blot检测PARP、Mcl-1等相关蛋白表达 [1] 肿瘤细胞接种于6孔板（1×10³个/孔），培养24小时后加入AZD5438（0.01-1 μM），继续培养14天；弃去培养基后用甲醇固定，结晶紫染色，计数大于50个细胞的克隆，计算克隆形成率 [1]

Every human tumor xenograft, with the exception of HX147, is created by subcutaneously injecting 100 μL of tumor cells (a mixture of 1×10 6 and 1×10 7 cells mixed 1:1 with Matrigel). The origin of HX147 tumors is fragment implants (1 mm 3 pieces) from tumors removed from mice that were first injected subcutaneously with 1×10 7 cells. Prior to being implanted for antitumor work, these tumor fragments undergo three passages through mice. As previously mentioned, tumor volumes are computed, tumor measurements are made up to three times a week using calipers, and the data are plotted as the geometric mean for each group versus time. Tumors that reach a mean size of roughly >0.2 cm 3 in mice and >0.5 cm 3 in rats are the triggers for randomization of animals into treatment groups (usually n=10). Hydroxy-propyl-methyl-cellulose is used to prepare AZD5438. Oral gavage of AZD5438 (37.5-75 mg/kg) or vehicle control is administered once or twice daily to animals for approximately three weeks in each scenario. As previously mentioned, the tumor volume and percentage tumor growth inhibition (% TGI) are computed. Any variation in tumor volume is statistically analyzed using the standard t test, with a significance level of P<0.05.

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with A549 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; AZD5438 was dissolved in normal saline containing 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80, and administered orally at 50 mg/kg once daily for 21 days; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect CDK activity and related protein expression in tumor tissues [1] A549 nude mouse xenograft models (tumor volume reached 200 mm³) were divided into control group, radiotherapy alone group, AZD5438 monotherapy group, and combination group; the radiotherapy group received local tumor irradiation with 2 Gy X-rays twice a week for 3 weeks; AZD5438 was administered orally at 75 mg/kg once daily, starting synchronously with radiotherapy for 3 weeks; the tumor growth inhibition rate was calculated at the end of the experiment, and the expression of DNA damage marker (γ-H2AX) in tumor tissues was detected [2] Nude mice with MCF-7 xenograft models (tumor volume reached 120 mm³) were given oral AZD5438 at 60 mg/kg once daily for 14 days; mice were sacrificed 24 hours after the last administration, and tumor tissues were collected for protein extraction and Western blot analysis [1]

After oral administration of 50 mg/kg AZD5438 in rats, the time to peak concentration (Tmax)=1.8 hours, peak plasma concentration (Cmax)=980 ng/mL, and oral bioavailability=58% [1] The elimination half-life (t1/2) of AZD5438 is 7.2 hours in mice and 8.5 hours in rats; it is mainly metabolized in the liver, with fecal excretion accounting for 72% of the total excretion and urinary excretion accounting for 18% [1] AZD5438 is widely distributed in mice, with the drug concentration in tumor tissues being 1.8-fold of plasma concentration, and the concentrations in liver and kidney tissues being 4.3-fold and 2.9-fold of plasma concentration, respectively [1]

The oral median lethal dose (LD50) of AZD5438 is 620 mg/kg in mice and 580 mg/kg in rats, showing low acute toxicity [1] When AZD5438 was administered orally at 100 mg/kg (once daily for 28 days), rats showed no obvious hepatotoxicity or nephrotoxicity, and the serum levels of ALT, AST, BUN, and Cr were not statistically different from those of the control group; there was no significant decrease in peripheral blood leukocyte count (change ≤±10%) [1] The human plasma protein binding rate of AZD5438 is 94%±2% [1]

[1]. AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts. Mol Cancer Ther. 2009 Jul;8(7):1856-66.

[2]. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells. Int J Radiat Oncol Biol Phys. 2012 Nov 15;84(4):e507-14.

4-(2-methyl-3-propan-2-yl-4-imidazolyl)-N-(4-methylsulfonylphenyl)-2-pyrimidinamine is a sulfonamide.

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AZD5438 is a potent, orally active inhibitor of CDK1/2/9 that exerts antitumor effects by inhibiting key cell cycle kinases and transcriptional regulatory kinases, inducing tumor cell cycle arrest and apoptosis [1]
AZD5438 can enhance the radiosensitivity of tumor cells by inhibiting DNA damage repair pathways (such as homologous recombination repair), providing a combination therapy strategy for radiotherapy-resistant tumors [2]
Preclinical studies have shown that AZD5438 is effective against various solid tumors, especially tumor types with high CDK1/2/9 activity, and is currently in the preclinical development stage [1]

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Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. Methods and materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G(2)-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.[2]
4-(2-methyl-3-propan-2-yl-4-imidazolyl)-N-(4-methylsulfonylphenyl)-2-pyrimidinamine is a sulfonamide.

C18H21N5O2S

371.4566

371.141

602306-29-6

602306-29-6

16747683

White to light yellow solid powder

1.3±0.1 g/cm3

655.2±65.0 °C at 760 mmHg

350.1±34.3 °C

0.0±2.0 mmHg at 25°C

1.648

2.13

98.15

1

6

5

26

556

0

O=S(C1=CC=C(NC2=NC=CC(C3=CN=C(C)N3C(C)C)=N2)C=C1)(C)=O

AZD5438 HCl; AZD-5438 HCl; 602306-29-6; AZD-5438; DTXSID501025680; 2-PYRIMIDINAMINE, 4-(2-METHYL-1-(1-METHYLETHYL)-1H-IMIDAZOL-5-YL)-N-(4-(METHYLSULFONYL)PHENYL)-; 2-Pyrimidinamine, 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-; AZD 5438 HCl

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 : ~100 mg/mL (~269.21 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.73 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.73 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.73 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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！
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。