AZD-7648

DNA-PKcs (IC50 = 91.3 nM); PI3Kγ (IC50 = 1.37 μM); ATM (IC50 = 117.93 μM)

AZD7648 在 A549 非小细胞肺癌 (NSCLC) 细胞中抑制 IR 诱导的 DNA-PK S2056 自动磷酸化，IC50 = 92 nM。 DNA-PK S2056 自磷酸化，IC50 值为 92 nM。停滞在细胞周期 G2/M 期的细胞数量显着增加，微核形成增加 4 倍，H2AX、pATM S1981 和 53BP1 焦点形成诱导 3 倍当 AZD7648 (1 M) 与 2Gy IR 结合 48 小时时，所有这些都是在 A549 细胞中引起的[2]。 研究人员开发了一种高效和选择性的DNA-PK抑制剂AZD7648，它在A549非小细胞肺癌（NSCLC）细胞中以IC50=92nM抑制IR-诱导的DNA-PKS2056自动磷酸化。AZD7648是一种强效的放射增敏剂，其中与IR联合治疗导致A549和H1299 NSCLC细胞集落存活能力的浓度依赖性降低（DEF37在100 nM时分别为1.7和2.5）。在A549细胞中，与单独使用IR相比，AZD7648（≥1µM）与2Gy IR联合使用48小时，导致细胞明显积聚在细胞周期的G2/M期，微核形成增加4倍，γH2AX、pATM S1981和53BP1病灶形成诱导增加3倍。在应用Loewe加性模型时，在一组卵巢和三阴性乳腺癌症（TNBC）细胞系的细胞生长抑制试验中，AZD7648也被发现与阿霉素协同结合（协同得分4-35）。[2]

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这种优化产生了AZD7648，它是DNA-PK的强效抑制剂（生化测定中为0.6 nM，A549细胞中为89 nM），在Thermofisher选择性面板中对396种其他激酶的选择性大于100倍。此外，AZD7648在临床前物种中具有良好的晶体溶解性、代谢稳定性和可预测的药代动力学。

AZD7648 是一种有效的、高选择性的 DNA-PK 抑制剂，具有良好的结晶溶解度、渗透性和代谢稳定性，在临床前物种中具有良好的生物利用度和可预测的药代动力学，与奥拉帕尼或放疗联合使用时，可有效敲低 pRPA 并在小鼠异种移植模型中实现回归[1 ]。 在小鼠异种移植物模型中，使用pRPA（S4/8）评估PD，并有效抑制PD（体内IC50=52 nM）。AZD7648作为单一疗法观察到肿瘤生长抑制，与奥拉帕尼或放疗联合使用观察到消退。AZD7648与奥拉帕尼联合使用时观察到的疗效与体内IC90的自由覆盖有关。AZD7648是一种强效、高选择性的DNA-PK抑制剂，具有良好的结晶溶解性、渗透性和代谢稳定性，在临床前物种中具有良好的生物利用度和可预测的药代动力学，与奥拉帕尼或辐射联合使用时，在小鼠异种移植物模型中具有强效的pRPA敲除和回归作用。这些特征使AZD7648成为一种合适的临床候选药物，并计划在2019年进行临床研究，评估其作为潜在的癌症治疗方法。[1]

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在体内，AZD7648与IR（5x 2Gy）的组合以剂量依赖的方式诱导H1299和A549 NSCLC异种移植物的肿瘤消退（分别为84%和11%的消退），而单药治疗仅实现了肿瘤生长抑制。在这两种模型中，AZD7648治疗抑制了IR对三种主要DNA-PK药效标志物pDNAPK（S2056）、pRPA32（S4/8）和γH2AX的激活增加（IR+AZD7648治疗2小时后抑制率为70-90%）。类似地，在BT474c ER+乳腺癌症异种移植物模型和TNBC PDX模型中，脂质体阿霉素（每周2.5 mg/kg）与AZD7648（37.5 mg/kg bid）联合诱导肿瘤消退（分别为63%和33%消退），而单药治疗仅实现肿瘤生长抑制。这些数据证实，AZD7648对DNA-PK的抑制增强了一系列体外和体内DSB诱导剂的疗效，为其临床研究提供了明确的理论基础[2]。

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AZD7648在异种移植物和PDX模型中的一系列剂量和方案中增强了奥拉帕尼的疗效，实现了持续的肿瘤消退，并为其临床研究提供了明确的理论基础[4]。

细胞药理学[4]
通过测量Ser2056上的DNA-PKcs自磷酸化，在IR后在A549细胞中评估AZD7648对DNA-PK活性的主要效力。在其他细胞试验中评估了AZD7648对HT29中ATM（pATM Ser1981）、HT29中ATR（pCHK1 Ser345）、MDA-MB-468中mTOR（pAKT Ser473）和BT474（PI3Kα）、MDAM-B-468（PI3Kβ）、RAW-264（PI3Kγ）和JEKO-1（PI3Kδ）细胞中PI3K亚型（pAKT Thr308）的选择性。IC50定义为使每种相应激酶的下游靶标磷酸化减少50%所需的浓度。

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高含量免疫荧光成像和分析[4]
在A549和OAW42细胞中进行了高含量成像分析，以分别评估AZD7648与IR和阿霉素联合治疗的细胞反应。将细胞固定并进行γH2AX、53BP1和pATM Ser1981染色，并在CV7000高含量成像平台上成像。

Immunocompromised SCID (C.B-17/IcrHan®Hsd-Prkdcscid) or Hsd:Athymic Nude-Foxn1nu female mic were used for tumour implantation. AZD7648 was formulated in 0.5% hydroxypropyl methylcellulose/0.1% Tween80 (HPMC/T) and orally dosed (4–100 mg kg−1). When dosed twice daily, the time between the morning and evening doses was 8 h. Targeted irradiation of 2 Gy was delivered over 2 min daily over the first 5 days of treatment. Liposomal doxorubicin was diluted in physiological saline and intravenously dosed at 2.5 or 5 mg kg−1 once per week. Olaparib was formulated in 10% DMSO/30% Kleptose and orally dosed at 100 mg kg−1 once daily. All three combinations were dosed 1 h after the morning dose of AZD7648 or its vehicle HPMC/T. Tumour growth inhibition from start of treatment was assessed by comparison of the mean change in tumour volume for the control and treated groups, using the Mousetrap application and represented as TGI. Statistical significance was evaluated using a one-tailed t-test. All in vivo studies complied with all relevant ethical regulations for animal testing and research, followed AstraZeneca’s global bioethics policy and received ethical approval from the AstraZeneca ethical committee. HBCx-17 PDX study was carried out at XenTech, France in accordance with French regulatory legislation concerning the protection of laboratory animals. [4]

[1]. Cancer Res (2019) 79 (13_Supplement): DDT01-02.

[2]. Cancer Res (2019) 79 (13_Supplement): 3512.

[3]. Nat Commun. 2022 Jul 22;13(1):4240.

[3]. Nat Commun. 2019 Nov 7;10(1):5065.

DNA-PK inhibitor AZD7648 is an orally bioavailable ATP-competitive inhibitor of DNA-dependent protein kinase (DNA-PK), with potential chemo/radiosensitizing and antineoplastic activites. Upon oral administration, DNA-PK inhibitor AZD7648 selectively targets, binds to and inhibits the activity of DNA-PK, thereby interfering with the non-homologous end joining (NHEJ) process and preventing repair of DNA double strand breaks (DSBs) caused by ionizing radiation or chemotherapeutic treatment. This increases chemo- and radiotherapy cytotoxicity leading to enhanced tumor cell death. AZD7648 may also increase the effect of poly(ADP-ribose) polymerase (PARP) inhibitors and may work as a monotherapy in tumors with high endogenous levels of DNA damage resulting from defects in other DNA repair pathways. The enhanced ability of tumor cells to repair DSBs plays a major role in the resistance of tumor cells to chemo- and radiotherapy. DNA-PK plays a key role in the NHEJ pathway and DSB repair.

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DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.[4]

C18H20N8O2

380.4038

380.17

C, 56.83; H, 5.30; N, 29.46; O, 8.41

2230820-11-6

2230820-11-6

135151360

Off-white to light yellow solid powder

1

101

1

7

3

28

588

0

CC1=CC2=NC=NN2C=C1NC3=NC=C(N(C)C(N4C5CCOCC5)=O)C4=N3

XISVSTPEXYIKJL-UHFFFAOYSA-N

InChI=1S/C18H20N8O2/c1-11-7-15-20-10-21-25(15)9-13(11)22-17-19-8-14-16(23-17)26(18(27)24(14)2)12-3-5-28-6-4-12/h7-10,12H,3-6H2,1-2H3,(H,19,22,23)

7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(oxan-4-yl)purin-8-one

AZD7648; AZD 7648; 2230820-11-6; AZD7648; 7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(oxan-4-yl)purin-8-one; 7-Methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-9-(tetrahydro-2H-pyran-4-yl)-7H-purin-8(9H)-one; 97A09L5JCK; 7,9-Dihydro-7-methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-8H-purin-8-one; compound 16 [PMID: 31851518]; AZD-7648

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: 8.3~9 mg/mL (21.9~23.7 mM)
Ethanol: ~3 mg/mL (~7.9 mM)

配方 1 中的溶解度: 10 mg/mL (26.29 mM) in 0.5% HPMC 0.1%Tween80 (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。

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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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