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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.
Physicochemical Properties
| Molecular Formula | C22H25N7O2 | |
| Molecular Weight | 419.48 | |
| Exact Mass | 419.206 | |
| Elemental Analysis | C, 62.99; H, 6.01; N, 23.37; O, 7.63 | |
| CAS # | 952021-60-2 | |
| Related CAS # | 1175132-90-7 (HCl);1071848-28-6 952238-93-6 (?HCl);1247874-19-6 (2HCl);952021-60-2; | |
| PubChem CID | 135565545 | |
| Appearance | Solid powder | |
| Density | 1.6±0.1 g/cm3 | |
| Index of Refraction | 1.790 | |
| LogP | 0.95 | |
| Hydrogen Bond Donor Count | 4 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 4 | |
| Heavy Atom Count | 31 | |
| Complexity | 725 | |
| Defined Atom Stereocenter Count | 1 | |
| 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 | |
| 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 | |
| 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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| HS Tariff Code | 2934.99.9001 | |
| 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) |
Biological Activity
| 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 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. | |
| 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. | |
| Animal Protocol |
Colo205 xenograft mouse model 40 mg/kg intravenous injection |
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| References |
[1]. Mol Cancer Ther . 2008 Aug;7(8):2394-404. [2]. J Biol Chem . 2010 Oct 22;285(43):33104-33112. [3]. Clin Cancer Res . 2009 Jul 15;15(14):4630-40. [4]. Cell Cycle . 2012 Jul 1;11(13):2507-17. |
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| 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. |
Solubility Data
| 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 |