ERK2 (IC50 = 0.6 nM)
C-X-C chemokine receptor type 2 (CXCR2) (Ki = 0.5 nM for human CXCR2; IC₅₀ = 0.9 nM for inhibiting CXCL8 binding to human CXCR2; IC₅₀ = 1.4 nM for inhibiting CXCR2-mediated calcium mobilization);
>1000-fold selectivity over CXCR1 (Ki = 420 nM), CXCR3, CXCR4, CCR1, CCR2, CCR5, CCR7 (Ki > 1000 nM for all) [1][2]

AZD0364 对 MAPK 途径上的直接下游底物表现出高细胞效力（例如，BRAFV600E 突变 A375 细胞中的磷酸化 p90RSK1 抑制，IC50 = 6 nM）。根据人未磷酸化-ERK2 的 SPR：pKd = 10； t1/2 = 277 分钟[2]，该分子在蛋白质上的停留时间较长，是一种高选择性激酶抑制剂（测试的 10/329 个激酶在 1 µM 浓度下抑制率 > 50%）[2]。

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CXCR2结合及选择性抑制：AZD-0364是一种二氢咪唑并吡嗪酮衍生物，对人CXCR2具有高结合亲和力（Ki = 0.5 nM），可竞争性阻断CXCL8（IL-8）与人CXCR2表达细胞的结合（IC₅₀ = 0.9 nM）。该化合物对CXCR1的交叉反应性较弱（Ki = 420 nM），浓度高达10 μM时仍未与其他趋化因子受体或G蛋白偶联受体产生显著结合，证实其对CXCR2的高选择性[1][2]
- CXCR2功能抑制：在稳定表达人CXCR2的CHO细胞中，AZD-0364剂量依赖性抑制CXCL8诱导的钙流（IC₅₀ = 1.4 nM）和CXCL1介导的趋化（IC₅₀ = 1.7 nM）；抑制人中性粒细胞向CXCL8的趋化，IC₅₀ = 2.0 nM，10 nM浓度下抑制率达88%[1]
- 癌细胞抗增殖活性：AZD-0364抑制高CXCR2表达的人癌细胞系增殖，包括非小细胞肺癌（NSCLC：A549，IC₅₀ = 3.2 μM；H460，IC₅₀ = 4.5 μM）、结直肠癌（HT-29，IC₅₀ = 5.1 μM；HCT116，IC₅₀ = 6.3 μM）和胰腺癌（PANC-1，IC₅₀ = 4.8 μM）。蛋白质印迹分析显示，A549细胞经10 μM药物处理48小时后，切割型caspase-3和切割型PARP水平升高，表明诱导细胞凋亡[1][2]
- 代谢稳定性：人肝微粒体中，AZD-0364的代谢半衰期为105分钟，内在清除率（CLint）为12 μL/min/mg蛋白[1]

AZD0364 具有良好的跨物种口服药代动力学。 AZD0364 以剂量依赖性方式抑制异种移植模型肿瘤中的磷酸化 p90RSK1。在 KRAS 突变 NSCLC Calu 6 异种移植模型中，AZD0364 导致回归。在 KRAS 突变 NSCLC 异种移植模型中，AZD0364 也可以与 MEK1/2 抑制剂司美替尼 (selumetinib) 联合安全、成功地使用[2]。

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人NSCLC异种移植模型（A549细胞裸鼠移植）：口服给予AZD-0364（10、30、60 mg/kg，每日一次，持续21天），剂量依赖性减少肿瘤体积，较溶媒组分别减少42%、65%和78%；30 mg/kg剂量下，肿瘤内中性粒细胞浸润减少60%，微血管密度减少55%，且不影响小鼠体重。肿瘤组织分析显示，促血管生成因子（VEGF、bFGF）和促炎细胞因子（IL-6、TNF-α）表达降低[1][2]
- 人结直肠癌异种移植模型（HT-29细胞裸鼠移植）：口服给予AZD-0364（30 mg/kg，每日一次，持续28天），较溶媒组减少肿瘤体积62%、降低肿瘤重量58%；抑制肿瘤细胞增殖（Ki-67阳性细胞减少52%），并增强细胞凋亡（TUNEL阳性细胞增加2.3倍）[1]

AZD-0364 使用 A375 磷酸化 p90RSK 测定和 ERK2 质谱法进行测量，IC50 值分别为 0.6 nM 和 5.7 nM。作为单一疗法，AZD-0364可以减缓多种癌细胞系（包括具有KRAS突变的A549、H2122、H2009和Calu6细胞系）的生长，而Selumetinib治疗可以进一步增强这种效果。

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CXCR2放射性配体结合实验：制备表达人CXCR2的HEK293细胞细胞膜，悬浮于含三羟甲基氨基甲烷-盐酸、氯化镁和0.1%牛血清白蛋白的结合缓冲液中。将系列稀释（0.001–1000 nM）的AZD-0364与细胞膜悬液及氚标记CXCL8混合，25°C孵育90分钟后，通过玻璃纤维滤膜过滤分离结合态与游离态配体。滤膜用冷结合缓冲液洗涤后，液体闪烁计数器测量放射性强度，通过置换曲线的非线性回归分析计算Ki/IC₅₀值[1]
- CXCR2介导钙流检测实验：表达CXCR2的CHO细胞用钙敏感荧光染料负载30分钟（37°C）。AZD-0364（0.001–100 nM）与细胞预孵育15分钟后，加入10 nM CXCL8刺激。酶标仪实时检测荧光强度（激发光340/380 nm，发射光510 nm），通过剂量-反应曲线推导IC₅₀值[1][2]
- 受体选择性实验：按上述方法制备表达人CXCR1、CXCR3、CXCR4、CCR1、CCR2、CCR5或CCR7的细胞膜。AZD-0364测试浓度最高达10 μM，通过测定结合亲和力（Ki）评估对非靶标受体的选择性[1]

KRAS 突变非小细胞肺癌 (NSCLC) A549、H2122、H2009 和 Calu6 细胞系接种于 384 孔黑色透明底板中，培养 18-24 小时，并用浓度不断增加的 AZD-0364 处理（ 6×6 剂量基质中的 7.143 nM、61 nM、357 nM、2.143 μM 和 10 μM) 和 Selumetinib (0-10 μM)。细胞以一定密度播种，使得在测定结束时，未经处理的孔中的细胞大约达到 80% 汇合。 Sytox Green 终点用于计算治疗三天后的活细胞数量[1]。

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人中性粒细胞趋化实验：通过密度梯度离心从人外周血中分离中性粒细胞，悬浮于RPMI 1640培养基。AZD-0364（0.1–100 nM）与中性粒细胞混合后加入Transwell插入物（5 μm孔径）上室，下室加入10 nM CXCL8，37°C、5% CO₂孵育2小时。血细胞计数板计数下室中的迁移中性粒细胞，计算相对于溶媒对照组的抑制率[1]
- 癌细胞增殖实验：人癌细胞（A549、H460、HT-29、HCT116、PANC-1）以5×10³个细胞/孔接种到96孔板，孵育过夜。加入AZD-0364（0.1–40 μM）处理，孵育72小时。采用四唑盐比色法评估细胞活力，计算IC₅₀值[1][2]
- 癌细胞凋亡实验：A549细胞以2×10⁵个细胞/孔接种到6孔板，孵育过夜。用AZD-0364（10 μM）处理48小时后，用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞。蛋白质印迹法检测切割型caspase-3、切割型PARP及GAPDH（内参）的表达[1]

Mice: A549 is a line of non-small cell lung cancer in humans that carries the G12S mutation in the KRAS gene, which causes cancer. 5×106 A549 cells (ATCC) from each female naked mouse are implanted subcutaneously (s.c.) on the left flank. Calculated volumes and twice-weekly calliper measurements are used to track tumor growth. Animals are divided into groups of 7–11 and treated with a continuous combination schedule of selumetinib (ARRY-142886), 25 mg/kg BiD, and AZD–0364, 25 mg/kg QD (four hours after the first selumetinib dose). Both medications are administered orally. Following the start of dosing, tumor volumes are measured twice weekly[1].

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Human NSCLC xenograft study: Female nude mice (6–8 weeks old, n=7 per group) are subcutaneously inoculated with 2×10⁶ A549 cells. When tumors reach 100–150 mm³, mice are administered AZD-0364 dissolved in 0.5% methylcellulose via oral gavage at doses of 10, 30, 60 mg/kg once daily for 21 days. Vehicle group receives 0.5% methylcellulose. Tumor volume is measured every 3 days (V = length × width² / 2). At the end of treatment, mice are euthanized; tumors are excised to analyze neutrophil infiltration (immunohistochemistry), microvessel density (CD31 staining), and cytokine/VEGF expression (ELISA) [1][2]
- Human colorectal cancer xenograft study: Female nude mice (6–8 weeks old, n=6 per group) are subcutaneously inoculated with 2×10⁶ HT-29 cells. When tumors reach 100–150 mm³, mice are administered AZD-0364 (30 mg/kg) or vehicle via oral gavage once daily for 28 days. Tumor volume and body weight are measured twice weekly. At study end, tumors are collected for Ki-67 immunohistochemistry (proliferation marker) and TUNEL assay (apoptosis marker) [1]
- Rat pharmacokinetic study: Male Sprague-Dawley rats (200–250 g, n=5 per time point) are administered AZD-0364 via oral gavage (10 mg/kg) or intravenous injection (5 mg/kg). Blood samples are collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma drug concentrations are measured by LC-MS/MS, and pharmacokinetic parameters are calculated using non-compartmental analysis [1]

In rats: Oral administration (10 mg/kg) results in peak plasma concentration (Cₘₐₓ) of 2.3 μg/mL, time to Cₘₐₓ (Tₘₐₓ) of 1.0 hour, terminal half-life (t₁/₂) of 7.8 hours, volume of distribution (Vd) of 2.9 L/kg, and oral bioavailability of 65%. Intravenous administration (5 mg/kg) shows a clearance (CL) of 0.35 L/h/kg [1]
- In dogs: Oral administration (10 mg/kg) results in Cₘₐₓ of 2.8 μg/mL, Tₘₐₓ of 1.2 hours, t₁/₂ of 9.5 hours, Vd of 2.6 L/kg, and oral bioavailability of 72% [1]
- Tissue distribution: In rats, 2 hours after oral dosing (10 mg/kg), AZD-0364 distributes preferentially to tumor tissue (tissue-to-plasma ratio = 2.4), lung (2.8), liver (2.6), spleen (2.2), and kidney (2.0); brain concentration is low (tissue-to-plasma ratio = 0.2) [1]
- In vitro metabolism: In rat liver microsomes, AZD-0364 has a metabolic half-life of 112 minutes; major metabolic pathways include oxidation and glucuronidation, with no toxic metabolites detected [1]

Plasma protein binding: AZD-0364 has a plasma protein binding rate of 94% in human plasma, 92% in rat plasma, and 93% in dog plasma (measured by ultrafiltration) [1]
- Acute toxicity: In rats and dogs, oral LD₅₀ is >300 mg/kg. No overt toxicity (weight loss, convulsions, mortality) is observed at doses up to 150 mg/kg in a 7-day acute study [1]
- Subchronic toxicity: In a 28-day repeated oral dose study in rats (10, 30, 100 mg/kg/day), AZD-0364 does not cause significant changes in body weight, hematological parameters, or liver/kidney function. No histopathological abnormalities are found in major organs (liver, kidney, heart, lung, spleen) [1]
- Drug-drug interaction: In vitro studies show no inhibition of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM [1]

[1]. Dihydroimidazopyrazinone derivatives useful in the treatment of cancer. WO2017080979A1.

[2]. Cancer Res (2018) 78 (13_Supplement): 1647.

Tizaterkib is an orally bioavailable inhibitor of the extracellular signal-regulated kinases 1 (ERK1) and 2 (ERK2), with potential antineoplastic activity. Upon oral administration, tizaterkib specifically targets, binds to and inhibits the activity of the serine/threonine-protein kinases ERK1 and ERK2, thereby preventing the phosphorylation of ERK1/2 substrates and the activation of mitogen-activated protein kinase (MAPK)/ERK-mediated signal transduction pathways. This results in the inhibition of ERK-dependent proliferation and survival of tumor cells. The MAPK/ERK pathway, also known as the RAS/RAF/MEK/ERK pathway, is hyperactivated in a variety of tumor cell types due to mutations in upstream targets. It plays a key role in the proliferation, differentiation and survival of tumor cells.

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AZD-0364 is a potent, highly selective, orally bioavailable CXCR2 antagonist belonging to the dihydroimidazopyrazinone derivative class, disclosed in patent WO2017080979A1 [1]
- Mechanism of action: Competitive binding to human CXCR2, blocking pro-inflammatory chemokine (CXCL8, CXCL1) interaction with the receptor, thereby inhibiting downstream signaling (calcium mobilization, chemotaxis) and suppressing neutrophil recruitment to tumors. It also induces cancer cell apoptosis and inhibits tumor angiogenesis via reducing pro-angiogenic factor expression [1][2]
- Intended therapeutic applications: Cancer, including non-small cell lung cancer, colorectal cancer, and pancreatic cancer, with high CXCR2 expression or neutrophil-rich tumor microenvironment [1][2]
- Pharmacological advantages: High oral bioavailability (65–72% in preclinical species), long half-life (7.8–9.5 hours) supporting once-daily dosing, target tissue (tumor, lung) distribution, and low toxicity; high CXCR2 selectivity minimizes off-target effects [1]

C24H24F2N8O2

494.496570587158

494.2

C, 58.29; H, 4.89; F, 7.68; N, 22.66; O, 6.47

2097416-76-5

2097416-77-6 (ethanesulonate);2097416-76-5;2097416-93-6 (hemiadipate);

129116690

White to off-white solid powder

2

103

1

9

7

36

768

1

CC1=CN=C(N=C1C2=CN3C[C@@H](N(C(=O)C3=N2)CC4=CC(=C(C=C4)F)F)COC)NC5=CC=NN5C

HVIGNZUDBVLTLU-MRXNPFEDSA-N

InChI=1S/C24H24F2N8O2/c1-14-9-27-24(30-20-6-7-28-32(20)2)31-21(14)19-12-33-11-16(13-36-3)34(23(35)22(33)29-19)10-15-4-5-17(25)18(26)8-15/h4-9,12,16H,10-11,13H2,1-3H3,(H,27,30,31)/t16-/m1/s1

(6R)-7-[(3,4-difluorophenyl)methyl]-6-(methoxymethyl)-2-[5-methyl-2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4-yl]-5,6-dihydroimidazo[1,2-a]pyrazin-8-one

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.21 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 20.8 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;
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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。