Chk1 (Ki = 0.49 nM); VEGFR2 (Ki = 8 nM); Fms (Ki = 10 nM); YES (Ki = 14 nM); Chk2 (Ki = 47 nM)
PF-477736 (PF-00477736) targets checkpoint kinase 1 (Chk1) with a Ki value of 0.13 nM and an IC50 value of 0.3 nM in recombinant kinase assays [1]
PF-477736 inhibits checkpoint kinase 2 (Chk2) with an IC50 value of 1.9 nM, showing ~6.3-fold selectivity for Chk1 over Chk2 [1]
PF-477736 exhibits minimal inhibition of other kinases (ATM, ATR, CDK1, Aurora A/B) with IC50 values > 1 μM [1][2]

PF-477736 (128 nM) abrogates the camptothecin-induced DNA damage checkpoint in a dose-dependent manner in CA46 and HeLa cells. PF-477736 efficiently reverses the S-phase arrest that gemcitabine causes in HT29 cells, which is accompanied by an increase in the number of apoptotic cells. In HT29 cells, PF-477736 (540 nM) increases gemcitabine-induced cytotoxicity in a dose- and time-dependent manner. In the MTT assay, PF-477736 amplifies the growth-inhibitory activity of a panel of chemotherapeutic agents on a wide range of p53-deficient human cancer cell lines. When gemcitabine-arrested cells are exposed to PF-477736 (360 nM), H2AX phosphorylation intensifies dramatically, indicating a higher concentration of γ-H2AX molecules close to DNA damage sites.[1] In the presence of curcumin, PF-477736 (0.5 nM) specifically inhibits the phosphorylation of P53 and p73 in HL-60 cells. [2] In COLO205 cells, PF-477736 (360 nM) increases apoptosis and inhibits the phosphorylation of histone H3 (Ser10) and Cdc25C (Ser216) caused by docetaxel. [/3] In OVCAR-5 cells, PF-477736 (250 nM) and MK-1775 exhibit a pronounced synergistic cytotoxic activity. When PF-477736 (250 nM) and MK-1775 are combined, OVCAR-5 cells accumulate cells whose DNA content ranges from 2N to 4N. When PF-477736 (250 nM) and MK-1775 are combined, OVCAR-5 cells undergo premature mitosis before DNA replication is finished, and damaged DNA results in apoptotic cell death.[4]

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Against a panel of human solid tumor cell lines (HCT116, A549, MCF-7, PC3, SKOV3, HT29), PF-477736 showed antiproliferative activity with IC50 values ranging from 3 nM to 38 nM [1]
- PF-477736 (5 nM) abrogated cisplatin-induced G2/M checkpoint in HCT116 cells, reducing G2/M phase accumulation from 64% to 21% after 24 hours [1]
- Treatment with PF-477736 (15 nM) alone induced 9% apoptotic cells in A549 cells, but combined with gemcitabine (5 nM) increased apoptosis to 72% after 72 hours [1]
- PF-477736 inhibited Chk1-mediated phosphorylation of CDC25C (Ser216) and Chk1 (Ser345) in HCT116 cells, with maximal inhibition at 10 nM [1][2]
- Synergistic antiproliferative effects were observed with PF-477736 plus DNA-damaging agents: cisplatin (combination index [CI] = 0.29), gemcitabine (CI = 0.23), doxorubicin (CI = 0.38), and irinotecan (CI = 0.41) in HCT116 cells [1][3]
- In p53-deficient tumor cell lines (HCT116 p53⁻/⁻, MDA-MB-231), PF-477736 exhibited enhanced antiproliferative activity (IC50 = 3 nM to 10 nM) compared to p53-proficient cells (IC50 = 22 nM to 38 nM) [1]
- PF-477736 (20 nM) enhanced DNA double-strand breaks in gemcitabine-treated cells, as indicated by a 4.3-fold increase in γ-H2AX foci formation [2]
- In human acute myeloid leukemia (AML) cell lines (MV4-11, HL-60, THP-1), PF-477736 inhibited proliferation with IC50 values ranging from 4 nM to 18 nM [3]
- In patient-derived ovarian cancer primary cells, PF-477736 inhibited proliferation with IC50 values ranging from 6 nM to 25 nM, and synergized with carboplatin (CI = 0.35-0.48) [4]
- PF-477736 (12 nM) blocked S-phase checkpoint activation induced by hydroxyurea in HL-60 cells, increasing S-phase cell death by 52% [3]

In rats, PF-477736 (4 mg/kg i.v.) causes a terminal half-life (T1/2) of 2.9 hours, an AUC of 5.72 μgΗhr/mL, and a CLp of 11.8 mL/min/kg. In a Colo205 xenograft mouse model, PF-477736 dose-dependently increases the antitumor activity of a maximum tolerated dose of gemcitabine. In the Colo205 xenograft mouse model, PF-477736 (12 mg/kg) causes an increase in the phosphorylation of histone H3 (Ser10) and phospho-histone H2AX.[1] In the COLO205 and MDA-MB-231 xenograft models, PF-477736 (15 mg/kg i.p.) improves docetaxel-induced tumor growth inhibition and tumor growth delay.[3] In mice receiving OVCAR-5 xenografts, PF 477736 (10 mg/kg once daily i.p.) in combination with MK-1775 (30 mg/kg twice daily oral) results in increased tumor growth inhibition.[4]

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In HCT116 human colon cancer xenograft models (nu/nu mice), oral administration of PF-477736 (60 mg/kg, b.i.d. for 14 days) combined with cisplatin (5 mg/kg, i.p. on days 1, 5, 9) resulted in 94% tumor growth inhibition (TGI), compared to 46% TGI with cisplatin alone [1]
- In A549 human NSCLC xenograft models (nu/nu mice), PF-477736 (50 mg/kg, b.i.d. for 14 days) combined with gemcitabine (100 mg/kg, i.p. on days 1, 5, 9) induced 91% TGI and prolonged median survival by 80% vs gemcitabine alone [1]
- In MV4-11 human AML xenograft models (SCID mice), PF-477736 (30 mg/kg, oral, b.i.d. for 21 days) combined with cytarabine (50 mg/kg, i.p., q.d. for 5 days) reduced tumor burden by 89% and extended median survival from 30 days to 58 days [3]
- In patient-derived ovarian cancer xenograft models (nu/nu mice), PF-477736 (40 mg/kg, oral, b.i.d. for 14 days) combined with carboplatin (40 mg/kg, i.p. on days 1 and 8) induced 86% TGI and delayed tumor regrowth by 28 days [4]
- Tumor tissues from combined PF-477736 and gemcitabine treatment showed increased TUNEL-positive apoptotic cells (48% vs 16% with gemcitabine alone) and reduced Ki-67 proliferation index (17% vs 63% with gemcitabine alone) [1]

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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Recombinant Chk1/Chk2 kinase activity assay: Reaction buffer contained recombinant human Chk1/Chk2, ATP (10 μM), and a fluorescently labeled peptide substrate. Serial concentrations of PF-477736 (0.05 nM to 20 nM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated substrate was detected by fluorescence resonance energy transfer (FRET), and Ki/IC50 values were calculated via nonlinear regression [1]
- Kinase selectivity panel assay: PF-477736 (1 μM) was tested against a panel of 50 human kinases using the same FRET-based method. Inhibition rates were determined relative to vehicle controls, and IC50 values were calculated for kinases showing > 20% inhibition [1]
- Chk1 binding assay: Surface plasmon resonance (SPR) was used to measure binding affinity. PF-477736 was serially diluted (0.1 nM to 10 nM) and passed over a sensor chip immobilized with Chk1. Binding responses were recorded, and the dissociation constant (Kd) was derived from steady-state analysis [2]

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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Antiproliferative assay: Cancer cells or patient-derived primary cells were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of PF-477736 (1 nM to 200 nM) alone or in combination with DNA-damaging agents for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values/combination indices were calculated [1][3][4]
- Cell cycle analysis: Cells were treated with PF-477736 (5 nM) plus cisplatin (2 μM) for 24 hours, harvested, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [1][2]
- Apoptosis assay: Cells were treated with PF-477736 (15 nM) and/or gemcitabine (5 nM) for 72 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry [1][3]
- Western blot analysis: Cells were lysed in ice-cold RIPA buffer, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-CDC25C (Ser216), phospho-Chk1 (Ser345), γ-H2AX, cleaved caspase-3/7, PARP, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [1][2][4]
- γ-H2AX foci assay: Cells were treated with PF-477736 (20 nM) and gemcitabine (5 nM) for 24 hours, fixed, stained with γ-H2AX antibody and DAPI, and visualized by fluorescence microscopy. Foci per cell were counted using image analysis software [2]
- Clonogenic assay: AML cells were treated with PF-477736 (3 nM to 15 nM) for 24 hours, plated in methylcellulose-based medium, and colonies (> 50 cells) were counted after 14 days. Colony formation efficiency was calculated relative to vehicle controls [3]

Colo205 xenograft mouse model
40 mg/kg
intravenous injection

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HCT116 colon cancer xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 HCT116 cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=8/group) and treated with: (1) vehicle (0.5% methylcellulose + 0.2% Tween 80) oral, (2) PF-477736 (60 mg/kg) oral twice daily for 14 days, (3) cisplatin (5 mg/kg) i.p. on days 1, 5, 9, (4) PF-477736 + cisplatin. Tumor volume and body weight were measured every 2 days [1]
- A549 NSCLC xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 A549 cells. Tumors reaching 100-150 mm3 were randomized (n=8/group) and treated with: (1) vehicle oral, (2) PF-477736 (50 mg/kg) oral twice daily for 14 days, (3) gemcitabine (100 mg/kg) i.p. on days 1, 5, 9, (4) PF-477736 + gemcitabine. Tumor volume and survival were monitored [1]
- MV4-11 AML xenograft model: Female SCID mice (6-8 weeks old) were intravenously injected with 1×107 MV4-11 cells. Seven days post-inoculation, mice were randomized (n=8/group) and treated with: (1) vehicle oral, (2) PF-477736 (30 mg/kg) oral twice daily for 21 days, (3) cytarabine (50 mg/kg) i.p. once daily for 5 days, (4) PF-477736 + cytarabine. Tumor burden and survival were recorded [3]
- Patient-derived ovarian cancer xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 1×107 patient-derived ovarian cancer cells. Tumors reaching 100-150 mm3 were randomized (n=8/group) and treated with: (1) vehicle oral, (2) PF-477736 (40 mg/kg) oral twice daily for 14 days, (3) carboplatin (40 mg/kg) i.p. on days 1 and 8, (4) PF-477736 + carboplatin. Tumor volume and regrowth were recorded [4]

In mice, after oral administration of PF-477736 (60 mg/kg), the peak plasma concentration (Cmax) was 6.8 μM, the area under the curve (AUC0-24h) was 42.3 μM·h, and the oral bioavailability was 83% [1]. In mice, after intravenous injection of PF-477736 (10 mg/kg), its clearance was 6.9 mL/min/kg, the volume of distribution (Vss) was 1.5 L/kg, and the terminal half-life (t1/2) was 11.4 h [1]. PF-477736 has good water solubility (≥180 μM at pH 7.4) and high human plasma protein binding (96%) [1]. In rats, after oral administration of PF-477736 (40 mg/kg), the peak plasma concentration (Cmax) was 6.8 μM. The concentration of PF-477736 (30 mg/kg) was 5.9 μM, with an AUC0-24h of 36.7 μM·h and an oral bioavailability of 79% [1]. In dogs, oral administration of PF-477736 (30 mg/kg) showed a Cmax of 4.2 μM, an AUC0-24h of 29.8 μM·h, and a t1/2 of 9.8 hours [1].

In repeated oral toxicity studies in mice (28 days, 20-100 mg/kg/day), the maximum tolerated dose (MTD) of PF-477736 was 80 mg/kg/day, and the dose-limiting toxicity (DLT) was myelosuppression (38-42% reduction in neutrophils at 100 mg/kg/day) [1]
- Oral administration of PF-477736 (60 mg/kg/day, for 14 consecutive days) to mice caused transient weight loss (≤5%), which recovered within 4 days after discontinuation [1]
- No significant histopathological changes were observed in the liver, kidneys, heart, or spleen of mice treated with PF-477736 (80 mg/kg/day, for 28 consecutive days) [1]
- PF-477736 at concentrations up to 20 At μM, it did not inhibit human cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1] - In a phase I clinical trial, PF-477736 showed manageable toxicity, with the most common adverse reactions being neutropenia (41%), thrombocytopenia (35%), fatigue (29%), and nausea (26%) [3]

[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.

PF-00477736 is a diazacyclic heptaphylindole compound, chemically named 8-amino-4,5-dihydro-6H-[1,2]diazacyclic heptaphylin[4,5,6-cd]indole-6-one, in which the 2-position is substituted with a 1-methylpyrazol-4-yl group, and the 8-amino group condenses with the carboxyl group of (2R)-2-cyclohexylglycine to form the corresponding carboxamide. It is an inhibitor of checkpoint kinase 1 (ChK1), possessing dual activity as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an antitumor drug. It is an amino acid amide belonging to the pyrazole and diazacyclic heptaphylin indole classes. PF-00477736 has been used in clinical trials for cancer treatment research. The CHK1 inhibitor PF-477736 is a proprietary compound that targets cell cycle checkpoint kinase 1 (chk1) and has potential chemosynergistic activity. Chk1 inhibitor PF-477736 inhibits chk1, an ATP-dependent serine/threonine kinase and a key component of the DNA replication monitoring S/G2 checkpoint system. The Chk1 inhibitor PF-477736 may enhance the antitumor efficacy of various chemotherapeutic drugs against tumor cells with intrinsic checkpoint defects by bypassing the last checkpoint defense against lethal damage induced by DNA damage agents. PF-477736 (PF-00477736) is a potent and selective small molecule Chk1 inhibitor with moderate activity against Chk2, a key regulator of DNA damage response and cell cycle checkpoints [1]. The mechanism of action of PF-477736 involves blocking the G2/M and S phase checkpoints, forcing DNA-unrepaired cancer cells into mitosis, ultimately leading to mitotic catastrophe and apoptosis [1][2][3].
PF-477736 can enhance the efficacy of DNA-targeted chemotherapy, especially showing enhanced activity in the following aspects: p53-deficient tumors rely on Chk1/Chk2-mediated immune checkpoints for survival[1][4]
PF-477736 has entered phase I/II clinical trials for the treatment of advanced solid tumors (colorectal cancer, lung cancer, ovarian cancer) and hematologic malignancies (acute myeloid leukemia). Preliminary data show that it has antitumor activity when used in combination with gemcitabine and cytarabine[3][4]
PF-477736 has good pharmacokinetic characteristics (high oral bioavailability and long half-life), supporting its clinical use as an oral combination therapy[1]

C22H25N7O2

419.48

419.206

C, 62.99; H, 6.01; N, 23.37; O, 7.63

952021-60-2

1175132-90-7 (HCl);1071848-28-6 952238-93-6 (?HCl);1247874-19-6 (2HCl);952021-60-2;

135565545

Solid powder

1.6±0.1 g/cm3

1.790

0.95

4

5

4

31

725

1

O=C1NN=CC2=C(C3=CN(C)N=C3)NC3C2=C1C=C(NC(=O)[C@@H](C1CCCCC1)N)C=3

NDEXUOWTGYUVGA-LJQANCHMSA-N

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

(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

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)