Chk1 (IC50 = 5 nM); Chk2 (IC50 = 5 nM)

AZD7762, a more selective inhibitor of Chk1, binds to the ATP-binding site of Chk1 reversibly and inhibits Chk1 phosphorylation of a cdc25C peptide with an IC50 of 5 nM and a Ki of 3.6 nM. By obstructing the chk1-dependent degradation of Cdc25A and activating Cyclin A, AZD7762 causes cell arrest with an EC50 of 0.620 μM and significantly reverses the G2 arrest induced by camptothecin, which has an EC50 of 10 nM. By lowering the GI50 values from 24.1 nM and 2.25 μM to 1.08 nM and 0.15 μM, respectively, AZD7762 (300 nM) improves the antitumor efficacy of gemcitabine against SW620 and topotecan against MDA-MB-231.[1] AZD7762 exhibits cytotoxicity with IC50 values ranging from 82.6 to 505.9 nM against a range of neuroblastoma cell lines expressing p53 wild type, p53 mutation, Mdm2 amplification, or p14 deletion.[2]

In the H460-DNp53 and SW620 xenograft mice, AZD7762 at 25 mg/kg has minimal antitumor activity. However, when administered in conjunction with gemcitabine (60 mg/kg), AZD7762 exhibits significant antitumor efficacy in the two xenograft mice, with a log cell kill of 0.9 or a percent treated/control (%T/C) of 26, even at low doses of 12.5 mg. When administered in conjunction with gemcitabine (10 mg/kg), AZD7762 and the H460-DNp53 xenograft rat exhibit dose-dependent tumor volume inhibition, with percentage T/C values of 48 and 32 for 10 and 20 mg/kg AZD7762, respectively.

Purified by glutathione affinity chromatography, recombinant human Chk1 is expressed as a glutathione S-transferase fusion in insect cells via a baculovirus vector. For Chk1, N-biotinylaminohexanoyl-KKVSRSGLYRSPMPENLNRPR is a synthetic peptide substrate. The final assay peptide and ATP concentrations are 0.8 and 1 μM, respectively (cold + 40 nCi [³³P]ATP). An assay plate with 384 wells is filled with varying concentrations of AZD7762, a buffer that contains ATP, chk1 kinase, and peptide. Using a TopCount reader, the plates are read after two hours of incubation, during which the reaction is halted by adding a buffer containing EDTA and scintillation proximity assay beads. Determine a dose response (IC50) by conducting data analysis.

In the checkpoint abrogation assay, HT29 cells are treated with camptothecin (a topoisomerase I inhibitor; 0.07 μg/mL) for two hours in order to trigger the G2 checkpoint. After that, cells are subjected to a 12-point titration of AZD7762 (12.5 μM to 6 nM) in addition to nocodazole for a duration of 20 hours. After fixing for one hour in 3.7% formaldehyde, permeabilizing the cells in PBS containing 0.05% Triton X, and incubating the cells for one hour in anti-phH3 antibody, Alexa Fluor 488 anti-rabbit, and Hoechst stain, the cells are left for another hour. The percentage of cells undergoing mitosis is represented by the mitotic index, which is calculated using the ArrayScan. In order to conduct potentiation assays, either SW620 or MDA-MB-231 cells are treated with a constant dose of AZD7762 (300 nM) for 24 hours followed by a 9-point titration of gemcitabine ranging from 0.01 to 100 nM. The medium is removed after 24 hours, and AZD7762 alone is then added back to the wells for a further 24 hours. Following this, the cells are cultured for a further 72 hours in medium free of AZD7762. MTT determines the impact on cell proliferation.

Mice and Rats: Male RNU rats and male NCr mice are employed. Tumor cells are removed from mice used as xenograft models, centrifuged for five minutes to pellet the cells, and then resuspended in sterile PBS. Using a 25-gauge needle, cells (3×10³-6×10⁶) are s.c. implanted into the right flank of the mice in a volume of 0.1 to 0.2 mL. Before compound is administered, tumors are allowed to grow to the specified size of 100 to 200 mm³. Rat xenograft models involve cell harvesting, centrifugation for 5 minutes to pellet the cells, and resuspension in a 50% sterile PBS and 50% Matrigel solution. Five days prior to cell implantation, rats undergo a whole-body radiation dose of 5 Gy with the goal of enhancing tumor growth. With a 25-gauge needle, H460-DNp53 cells (1×10⁷) are s.c. implanted into the rats' right flanks in a volume of 0.2 mL. Before administering AZD-7762, tumors are allowed to grow to the specified size of 100 to 200 mm³. The tail vein is used to administer intravenous injections of AZD-7762 (10 and 20 mg/kg). Treatments were administered in cycles of three to five, according to cyclic schedules. Every three days, AZD-7762 is delivered after a standard agent (NSC 613327 or CPT-11) has been administered. Electronic calipers are used to measure and compute tumor volumes. Mice: Pharmacological inhibitors are administered to C57Bl/6 mice eight days after they receive an intravenous injection of 2×10⁵ Eμ-Myc B-lymphoma cells in PBS. Treatment is administered to the mice until an ethical endpoint, such as a hunched posture, ruffled fur, enlarged lymph nodes, labored breathing, weight loss of more than 20% of the initial body weight, limited mobility, or paralysis, is achieved. On weekdays, 20 mg/kg of AZD7762 is administered intraperitoneally in a solution of 10.3% -hydroxypropyl-β-cyclodextrin and 0.9% saline.

[1]. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther. 2008 Sep;7(9):2955-66.

[2]. Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling. Oncotarget. 2016 Aug 2;7(31):49800-49818.

3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-3-piperidinyl]-2-thiophenecarboxamide is an aromatic amide and a member of thiophenes.
AZD7762 has been investigated for the treatment of Cancer, Solid Tumors, and Advanced Solid Malignancies.
Checkpoint Kinase Inhibitor AZD7762 is a synthetic small molecule inhibitor of checkpoint kinases (Chks) with potential chemosensitizing activity. AZD7762 binds to and inhibits Chks, which may prevent cell cycle arrest and subsequent nucleotide excision repair in DNA-damaged tumor cells, resulting in tumor cell apoptosis. This agent may enhance the cytotoxicity of DNA-damaging agents. Chks are protein kinases that regulate either G1/S or G2/M transitions in the cell cycle. In the presence of DNA damage or incomplete DNA replication, Chks become activated and initiate cell cycle arrest to allow DNA repair or the completion of DNA replication.

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Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings.[1]

C17H19FN4O2S

362.42

362.12

C, 56.34; H, 5.28; F, 5.24; N, 15.46; O, 8.83; S, 8.85

860352-01-8

AZD-7762 hydrochloride;1246094-78-9

11152667

white solid powder

1.38

547.6ºC at 760 mmHg

285ºC

4.821

4

5

4

25

495

1

C(C1=C(NC(=O)N)C=C(C2C=CC=C(F)C=2)S1)(=O)N[C@@H]1CNCCC1

IAYGCINLNONXHY-LBPRGKRZSA-N

InChI=1S/C17H19FN4O2S/c18-11-4-1-3-10(7-11)14-8-13(22-17(19)24)15(25-14)16(23)21-12-5-2-6-20-9-12/h1,3-4,7-8,12,20H,2,5-6,9H2,(H,21,23)(H3,19,22,24)/t12-/m0/s1

3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (27.59 mM) in 10% HP-β-CD (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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