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Purity: ≥98%
AZD7762 (AZD-7762; AZD 7762) is a selective and ATP-competitive inhibitor of Chk1 (checkpoint kinases) with potential anticancer activity. In a cell-free assay, it inhibits CHK1 with an IC50 of 5 nM. A serine/threonine kinase, CHK1 controls the course of mitosis by reacting to replication stress and DNA damage.
Targets |
Chk1 (IC50 = 5 nM); Chk2 (IC50 = 5 nM)
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ln Vitro |
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]
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ln Vivo |
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.
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Enzyme Assay |
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.
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Cell Assay |
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.
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Animal Protocol |
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.
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References |
Molecular Formula |
C17H19FN4O2S
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Molecular Weight |
362.42
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Exact Mass |
362.12
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Elemental Analysis |
C, 56.34; H, 5.28; F, 5.24; N, 15.46; O, 8.83; S, 8.85
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CAS # |
860352-01-8
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Appearance |
white solid powder
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SMILES |
C1C[C@@H](CNC1)NC(=O)C2=C(C=C(S2)C3=CC(=CC=C3)F)NC(=O)N
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InChi Key |
IAYGCINLNONXHY-LBPRGKRZSA-N
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InChi Code |
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
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Chemical Name |
3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide
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Synonyms |
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.7592 mL | 13.7961 mL | 27.5923 mL | |
5 mM | 0.5518 mL | 2.7592 mL | 5.5185 mL | |
10 mM | 0.2759 mL | 1.3796 mL | 2.7592 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT00413686 | Completed | Drug: AZD7762 Drug: Gemcitabine |
Solid Tumors | AstraZeneca | December 2006 | Phase 1 |
NCT00473616 | Terminated | Drug: AZD7762 Drug: Irinotecan |
Advanced Solid Tumors Cancer |
AstraZeneca | May 2007 | Phase 1 |
NCT00937664 | Terminated | Drug: AZD7762 Drug: gemcitabine |
Cancer Solid Tumors |
AstraZeneca | July 2009 | Phase 1 |
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Effect of AZD7762 on cell cycle proteins following treatment of cells with DNA-damaging agents.Mol Cancer Ther.2008 Sep;7(9):2955-66. th> |
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AZD7762 potentiated gemcitabine and topotecan.Mol Cancer Ther.2008 Sep;7(9):2955-66. td> |
AZD7762 potentiated gemcitabine in rodent xenograft efficacy models.Mol Cancer Ther.2008 Sep;7(9):2955-66. td> |