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Purity: ≥98%
PF-477736 (also known as PF-736; PF-00477736; PF477736) is a novel, selective, potent and ATP-competitive Chk1 inhibitor with potential antitumor activity. In a cell-free assay, it inhibits Chk1 with a Ki of 0.49 nM as well as VEGFR2, Aurora-A, FGFR3, Flt3, Fms (CSF1R), Ret, and Yes. PF-477736 exhibits selectivity for Chk1 ~100 times greater than Chk2. In tumor cells with intrinsic checkpoint defects, chk1 inhibitor PF-477736 may enhance the antitumor efficacy of different chemotherapeutic agents by bypassing the last checkpoint defense against DNA damaging agent-induced lethal damage.
| Targets |
Chk1 (Ki = 0.49 nM); VEGFR2 (Ki = 8 nM); Fms (Ki = 10 nM); YES (Ki = 14 nM); Chk2 (Ki = 47 nM)
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| ln Vitro |
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| ln Vivo |
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| Enzyme Assay |
The experiment is carried out in a 96-well plate at 30°C for 20 minutes using 0.1 mL of assay buffer that contains 25 mM magnesium chloride, 0.4 M NaCl, 4 mM PEP, 0.15 mM NADH, 28 units of lactate dehydrogenase/mL, 16 units of pyruvate kinase/mL, 3 mM DTT, 0.125 mM Syntide-2, 0.15 mM ATP, and 28 units of lactate dehydrogenase/mL. One nanometer of CHK1 kinase domain is added to start the assay. By measuring initial velocities while PF-477736 is present in different concentrations, the inhibition of CHK1 activity is ascertained. A kinetic model for competitive inhibition is fitted to the data through analysis using Enzyme Kinetic and Excel software, resulting in a Ki value. Examining PF-477736 at 1 μM or 10 μM against a panel 2 of roughly 100 protein kinases allows for the determination of the compound's kinase selectivity.
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| Cell Assay |
The antiproliferative effects of PF-477736 on human cancer cell lines with p53 defects are measured using the IC50 assay. Each line of cells is seeded in a 96-well assay plate with complete medium at an exponentially growing density, and the cells are allowed to attach for 16 hours. After that, PF-477736 is serially diluted, and the proper controls are added to each plate. The drug is incubated in cells for ninety-six hours. Each well is filled with MTT working stock that has been diluted in complete medium, and the cells are incubated for an additional four hours. DMSO is added to each well following centrifugation and supernatant removal, and plates are then read at 540 nm using a SpectraMax plate reader.
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| Animal Protocol |
Colo205 xenograft mouse model
40 mg/kg intravenous injection |
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| References | ||
| Additional Infomation |
PF-00477736 is a diazepinoindole that is 8-amino-4,5-dihydro-6H-[1,2]diazepino[4,5,6-cd]indol-6-one which is substituted at position 2 by a 1-methylpyrazol-4-yl group and in which the amino group at position 8 has undergone condensation with the carboxy group of (2R)-2-cyclohexylglycine to give the corresponding carboxamide. It is an inhibitor of checkpoint kinase 1 (Chk 1). It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an antineoplastic agent. It is an amino acid amide, a member of pyrazoles and a diazepinoindole.
PF-00477736 has been used in trials studying the treatment of Neoplasms. CHK1 Inhibitor PF-477736 is a proprietary compound targeting cell cycle checkpoint kinase 1 (chk1) with potential chemopotentiation activity. Chk1 inhibitor PF-477736 inhibits chk1, an ATP-dependent serine-threonine kinase that is a key component in the DNA replication-monitoring S/G2 checkpoint system. By overriding the last checkpoint defense against DNA damaging agent-induced lethal damage, chk1 inhibitor PF-477736 may potentiate the antitumor efficacy of various chemotherapeutic agents against tumor cells with intrinsic checkpoint defects. |
| Molecular Formula |
C22H25N7O2
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| Molecular Weight |
419.48
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| Exact Mass |
419.206
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| Elemental Analysis |
C, 62.99; H, 6.01; N, 23.37; O, 7.63
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| CAS # |
952021-60-2
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| Related CAS # |
1175132-90-7 (HCl);1071848-28-6 952238-93-6 (?HCl);1247874-19-6 (2HCl);952021-60-2;
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| PubChem CID |
135565545
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| Appearance |
Solid powder
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| Density |
1.6±0.1 g/cm3
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| Index of Refraction |
1.790
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| LogP |
0.95
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
31
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| Complexity |
725
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C1NN=CC2=C(C3=CN(C)N=C3)NC3C2=C1C=C(NC(=O)[C@@H](C1CCCCC1)N)C=3
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| InChi Key |
NDEXUOWTGYUVGA-LJQANCHMSA-N
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| InChi Code |
InChI=1S/C22H25N7O2/c1-29-11-13(9-25-29)20-16-10-24-28-21(30)15-7-14(8-17(27-20)18(15)16)26-22(31)19(23)12-5-3-2-4-6-12/h7-12,19,27H,2-6,23H2,1H3,(H,26,31)(H,28,30)/t19-/m1/s1
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| Chemical Name |
(2R)-2-amino-2-cyclohexyl-N-[2-(1-methylpyrazol-4-yl)-9-oxo-3,10,11-triazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13),11-pentaen-6-yl]acetamide
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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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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3839 mL | 11.9195 mL | 23.8390 mL | |
| 5 mM | 0.4768 mL | 2.3839 mL | 4.7678 mL | |
| 10 mM | 0.2384 mL | 1.1920 mL | 2.3839 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 |
| NCT00437203 | Terminated | Drug: PF-00477736 Drug: gemcitabine |
Neoplasms | Pfizer | December 2006 | Phase 1 |
PF-00477736 abrogates the camptothecin-induced DNA damage checkpoint in a dose-dependent manner.Mol Cancer Ther.2008 Aug;7(8):2394-404. |
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PF-00477736 effectively abrogates the gemcitabine-induced S-phase arrest with a corresponding increase in apoptotic cell populations in the combination treatment compared with the gemcitabine treatment alone.Mol Cancer Ther.2008 Aug;7(8):2394-404. |
A,PF-00477736 enhances gemcitabine-induced cytotoxicity in a time- and dose-dependent manner in HT29 cells as determined by cell survival assay. |
A,PF-00477736 potentiates the antiproliferative effect of gemcitabine.B,in vitrocytotoxicity of PF-00477736 in selected cell lines with different DNA-damaging agents.Mol Cancer Ther.2008 Aug;7(8):2394-404. |
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A,in vitroeffects of gemcitabine ± PF-00477736 on the modulation of proteins involved in the G2DNA damage checkpoint pathway.B,gemcitabine + PF-00477736 combinationin vitroleads to increased DNA damage. |
 A,PF-00477736 potentiation of gemcitabine in human colon Colo205 xenograft model.B,summary of PF-00477736 potentiation of gemcitabine in human colon xenograft models.Mol Cancer Ther.2008 Aug;7(8):2394-404 |
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