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

AZD7648 inhibits IR-induced DNA-PK S2056 auto phosphoryalation with an IC50 = 92 nM in A549 non-small cell lung cancer (NSCLC) cells. DNA-PK S2056 autophosphorylation with an IC50 value of 92 nM. A significant increase in the number of cells arrested in the G2/M phase of the cell cycle, a 4-fold increase in the formation of micronuclei, and a 3-fold induction of the formation of H2AX, pATM S1981, and 53BP1 foci are all caused in A549 cells when AZD7648 (1 M) is combined with 2Gy IR for 48 hours[2]. Researchers have developed a highly potent and selective inhibitor of DNA-PK, AZD7648, which inhibits IR-induced DNA-PK S2056 auto phosphoryalation with an IC50 = 92 nM in A549 non-small cell lung cancer (NSCLC) cells. AZD7648 is a potent radiosensitiser where treatment in combination with IR led to a concentration-dependent reduction of the colony survival capacity of A549 and H1299 NSCLC cells (DEF37 at 100 nM = 1.7 and 2.5, respectively). In A549 cells, AZD7648 (≥1 µM) in combination with 2Gy IR for 48 hours led to a significant accumulation of cells arrested in the G2/M of the cell cycle, a 4-fold increase in micronuclei formation, and 3-fold induction of γH2AX, pATM S1981 and 53BP1 foci formation compared with IR alone. AZD7648 was also found to combine synergistically with doxorubicin in a panel of ovarian and triple negative breast cancer (TNBC) cell lines in cell growth inhibition assays when applying the Loewe additivity model (synergy scores 4 - 35).[2]

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This optimisation resulted in AZD7648, which is a potent inhibitor of DNA-PK (0.6 nM in biochemical assay, 89 nM in A549 cells) and >100x selective against 396 other kinases in a Thermofisher selectivity panel. In addition, AZD7648 has good crystalline solubility, metabolic stability and predictable pharmacokinetics in preclinical species. [1]

AZD7648 is a potent and highly selective DNA-PK inhibitor, with good crystalline solubility, permeability and metabolic stability, good bioavailability and predictable pharmacokinetics in preclinical species, and potent knockdown of pRPA and regressions in murine xenograft models when combining with olaparib or radiation[1]. In murine xenograft models, PD was assessed using pRPA (S4/8) and was potently inhibited (in vivo IC50 = 52 nM). Tumour growth inhibition was observed with AZD7648 as monotherapy, and regressions were observed in combination with either olaparib or radiation. The efficacy observed when combining AZD7648 with olaparib was correlated to free cover over the in vivo IC90. AZD7648 is a potent and highly selective DNA-PK inhibitor, with good crystalline solubility, permeability and metabolic stability, good bioavailability and predictable pharmacokinetics in preclinical species, and potent knockdown of pRPA and regressions in murine xenograft models when combining with olaparib or radiation. These features make AZD7648 a suitable clinical candidate, and clinical studies evaluating AZD7648 as a potential cancer treatment are planned in 2019.[1]

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In vivo the combination of AZD7648 with IR (5x 2Gy) induced tumour regression in H1299 and A549 NSCLC xenografts in a dose-dependent manner (84 and 11% regression respectively), while monotherapy treatment only achieved tumour growth inhibition. In these two models the increased activation by IR of three primary DNA-PK pharmacodynamic markers, pDNAPK (S2056), pRPA32 (S4/8) and γH2AX, was inhibited by AZD7648 treatment (70-90% inhibition 2 h after IR + AZD7648). Similarly, liposomal doxorubicin (2.5 mg/kg weekly) in combination with AZD7648 (37.5 mg/kg bid) induced tumour regressions in the BT474c ER+ breast cancer xenograft model and in a TNBC PDX model (63% and 33% regression respectively), while monotherapy treatments only achieved tumour growth inhibition. These data confirm that DNA-PK inhibition with AZD7648 enhances the efficacy of a range of DSB inducing agents in vitro and in vivo, providing a clear rationale for its clinical investigation[2].

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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 [4].

Cellular pharmacology [4]
Primary potency of AZD7648 on DNA-PK activity was evaluated in A549 cells following IR by measuring DNA-PKcs autophosphorylation on Ser2056. The selectivity of AZD7648 was evaluated in other cell assays for its pharmacological activity on ATM (pATM Ser1981) in HT29, ATR (pCHK1 Ser345) in HT29, mTOR (pAKT Ser473) in MDA-MB-468, and PI3K isoforms (pAKT Thr308) in BT474 (PI3Kα), MDA-MB-468 (PI3Kβ), RAW-264 (PI3Kγ) and JEKO-1 (PI3Kδ) cells. IC50 was defined as the concentration required to give a 50% reduction in the phosphorylation of the downstream target for each respective kinase.

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High-content immunofluorescence imaging and analysis [4]
High-content imaging assays were performed in A549 and OAW42 cells to evaluate the cellular response to combination treatment of AZD7648 with IR and doxorubicin, respectively. Cells were fixed and stained for γH2AX, 53BP1 and pATM Ser1981 and imaged on the CV7000 high-content imaging platform.

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

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)

Solubility in Formulation 1: 10 mg/mL (26.29 mM) in 0.5% HPMC 0.1%Tween80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)