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Purity: ≥98%
AZD7762 HCl (AZD-7762) is a novel, potent, selective, ATP-competitive, and synthetic small molecule checkpoint kinase (Chk) inhibitor with anticancer activity. It has an IC50 of 5 nM for Chk1. AZD-7762 has potential chemosensitizing activity by binding 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.
| Targets |
Chk1 (IC50 = 5 nM); Chk2 (IC50 = 5 nM)
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| ln Vitro |
In vitro, AZD-7762 hydrochloride corrects Chk1 and Chk2 just as well. AZD-7762 hydrochloride increases the effectiveness of NSC 613327 and SKF 104864A, suppresses DNA damage-induced S and G2 checkpoints, and modifies downstream checkpoint diluting proteins. By using the scintillation taxis assay, the AZD-7762 salt was found to potently block Chk1 phosphorylation of the cdc25C peptide, with an IC50 of 5 nM. It was found that the salt AZD-7762 had a Ki of 3.6 nM. According to kinetic characterization, AZD-7762 salt binds to Chk1's ATP binding site where it is assumed to engage in direct, reversible competition for ATP binding. It was demonstrated that AZD-7762 hydrochloride could completely remove camptothecin-induced G2 blockage, with a maximal elimination range of 100 nM and an average EC50 of 10 nM (n=12) [1].
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| ln Vivo |
AZD-7762 hydrochloride (10 mg/kg and 20 mg/kg) dose-dependently increased the anticancer efficacy of NSC 613327 in H460-DNp53 xenograft experiments. In other words, the inhibition rates (%T /C) drop to 48% and 32%, respectively, as the dose increases. When used in conjunction with CPT-11 in a mouse xenograft research, AZD-7762 hydrochloride dramatically raised CPT-11 activity and %T/C to -66% and -66%, respectively. -67% [1]. When combined with CX-5461, AZD7762 hydrochloride causes both internal and exterior apparent cell death in Tp53-null (Tp53-/-) Eμ-Myc [2].
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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 |
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| Additional Infomation |
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]
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| Molecular Formula |
C17H20CLFN4O2S
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|---|---|
| Molecular Weight |
398.8827
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| Exact Mass |
398.098
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| Elemental Analysis |
C, 51.19; H, 5.05; Cl, 8.89; F, 4.76; N, 14.05; O, 8.02; S, 8.04
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| CAS # |
1246094-78-9
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| Related CAS # |
AZD-7762;860352-01-8
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| PubChem CID |
56972142
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
4.821
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
495
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| Defined Atom Stereocenter Count |
1
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| SMILES |
Cl[H].S1C(C2C([H])=C([H])C([H])=C(C=2[H])F)=C([H])C(=C1C(N([H])[C@]1([H])C([H])([H])N([H])C([H])([H])C([H])([H])C1([H])[H])=O)N([H])C(N([H])[H])=O
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| InChi Key |
WFZBLOIXZRZEDG-YDALLXLXSA-N
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| InChi Code |
InChI=1S/C17H19FN4O2S.ClH/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);1H/t12-;/m0./s1
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| Chemical Name |
(S)-5-(3-Fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide Hydrochloride
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| Synonyms |
AZD-7762 Hydrochloride; AZD-7762 HCl; AZD7762; AZD 7762; AZD-7762.
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5070 mL | 12.5351 mL | 25.0702 mL | |
| 5 mM | 0.5014 mL | 2.5070 mL | 5.0140 mL | |
| 10 mM | 0.2507 mL | 1.2535 mL | 2.5070 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 |
Effect of AZD7762 on cell cycle proteins following treatment of cells with DNA-damaging agents.Mol Cancer Ther.2008 Sep;7(9):2955-66. |
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AZD7762 potentiated gemcitabine and topotecan.Mol Cancer Ther.2008 Sep;7(9):2955-66. |
AZD7762 potentiated gemcitabine in rodent xenograft efficacy models.Mol Cancer Ther.2008 Sep;7(9):2955-66. |
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