Chk1 (Ki = 0.49 ± 0.29 nM) [1]
Chk2 (Ki = 47 ± 9 nM) [1]
CDK1 (Ki = 9.9 μM, 20,000-fold selectivity vs Chk1) [1]

PF-477736 (128 nM) abrogates the camptothecin-induced DNA damage checkpoint in a dose-dependent manner in CA46 and HeLa cells. PF-477736 effectively abrogates the gemcitabine-induced S-phase arrest with a corresponding increase in apoptotic cell populations in HT29 cells. PF-477736 (540 nM) enhances gemcitabine-induced cytotoxicity in a time- and dose-dependent manner in HT29 cells. PF-477736 potentiates the growth-inhibitory activity of a panel of chemotherapeutic agents across a broad spectrum of p53-deficient human cancer cell lines in the MTT assay. Addition of PF-477736 (360 nM) to gemcitabine-arrested cells induces a dramatic increase in the intensity of H2AX phosphorylation, reflecting a greater number of γ-H2AX molecules near sites of DNA damage. PF-477736 (0.5 nM) selectively blocks p73 and P53 phosphorylation in presence of curcumin in HL-60 cells. PF-477736 (360 nM) suppresses docetaxel-induced phosphorylation of histone H3 (Ser10) and Cdc25C (Ser216) and potentiates apoptosis in COLO205 cells. PF-477736 (250 nM) combined with MK-1775 has marked synergistic cytotoxic activity in OVCAR-5 cells. PF-477736 (250 nM) combined with MK-1775 causes accumulation of cells with a DNA content between 2N and 4N in OVCAR-5 cells. PF-477736 (250 nM) combined with MK-1775 causes premature mitosis before the end of DNA replication, with damaged DNA leading to apoptotic cell death in OVCAR-5 cells.

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Kinase Assay: The assay is performed in a 96-well plate for 20 minutes at 30℃ in 0.1 mL of assay buffer containing 50 mM TRIS pH 7.5, 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 25 mM magnesium chloride. Assays are initiated with 1 nM of CHK1 kinase domain. The inhibition of CHK1 activity is determined by measuring initial velocities in the presence of varying concentrations of PF-477736. The data is analyzed using Enzyme Kinetic and Excel software and fit to a kinetic model for competitive inhibition to obtain a Ki value. The kinase selectivity of PF-477736 is evaluated by screening the compound at 1 μM or 10 μM against a panel 2 of about 100 protein kinases.

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Cell Assay: The IC50 assay measures the antiproliferative effects of PF-477736 on p53-defective human cancer cell lines. Cells in each line are seeded in complete medium at an exponentially growing density in 96-well assay plate and allowed to attach for 16 hours. Serial dilutions of PF-477736 are then done, and appropriate controls are added to each plate. Cells are incubated with drug for 96 hours. After incubation, MTT working stock diluted in complete medium is added to each well, and cells are incubated for 4 hours. After centrifugation and supernatant removal, DMSO is added to each well and plates are read on SpectraMax plate reader at 540 nm.

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PF-477736 2HCl inhibited Chk1 kinase activity with a Ki of 0.49 nM; it was a poor inhibitor of CDK1 (Ki = 9.9 μM) and inhibited Chk2 with Ki = 47 nM. In a panel of >100 protein kinases, only seven (VEGFR2, Aurora-A, FGFR3, Flt3, Fms, Ret, Yes) were inhibited with <100-fold selectivity. [1]
In CA46 cells (p53-mutated), PF-477736 2HCl abrogated camptothecin-induced G2 arrest with an EC50 of 45 nM (histone H3 phosphorylation assay). In HeLa cells, EC50 was 38 nM; in HT29 cells, EC50 was 42 nM. [1]
In HT29 cells, PF-477736 2HCl (360 nM, 8×EC50) combined with gemcitabine (15 nM, minimal toxicity alone) induced up to 89% increase in cell growth inhibition in a time- and dose-dependent manner (cell survival assay). PF-00477736 alone had no significant effect on cell viability. [1]
In PC3 cells, gemcitabine (30 nM) plus PF-477736 2HCl (360 nM) induced a dramatic increase in γH2AX phosphorylation, indicating increased DNA damage due to checkpoint abrogation. [1]
In COLO205 cells, PF-477736 2HCl reversed docetaxel (1 nM)-induced increase in phospho-histone H3 and increased γH2AX foci and apoptosis. At higher docetaxel concentrations (5-10 nM), docetaxel induced Chk1 phosphorylation (Ser345); PF-477736 2HCl suppressed cytoplasmic localization of phospho-Cdc25C (Ser216) and enhanced apoptosis. [2]
In HT29 cells, combination treatment (gemcitabine + PF-477736 2HCl) abrogated S-phase arrest and increased apoptotic cell death (TUNEL assay). [1]

PF-477736 (4 mg/kg i.v.) results in terminal half-life (T1/2) of 2.9 hours, AUC of 5.72 μg×hr/mL and CLp of 11.8 mL/min/kg in rats. PF-477736 dose-dependently enhances the antitumor activity of a maximum tolerated dose of gemcitabine in the Colo205 xenograft mouse model. PF-477736 (12 mg/kg) induces an increase in the phosphorylation of histone H3 (Ser10) and of phospho-histone H2AX in the Colo205 xenograft mouse model. PF-477736 (15 mg/kg i.p.) enhances docetaxel induced tumor growth inhibition and tumor growth delay in COLO205 and MDA-MB-231 xenograft models. PF 477736 (10 mg/kg once daily i.p.) combined with MK-1775 (30 mg/kg twice a day oral) leads to greater tumor growth inhibition in mice bearing OVCAR-5 xenografts.
In COLO205 human colon carcinoma xenografts, PF-477736 2HCl (40 mg/kg, i.p., q3d×4, given 24 h after gemcitabine) potentiated gemcitabine (MTD)-induced tumor growth inhibition by 75% and increased log cell kill from 0.5 to 1.25; time-to-progression enhancement ratio was 3.6-fold. No single-agent antitumor activity was observed. [1]
In COLO205 xenografts, PF-477736 2HCl (7.5 or 15 mg/kg, i.p., b.i.d. on days 1,8,15) dose-dependently enhanced docetaxel (15 or 30 mg/kg, i.p., qd) efficacy, with significant tumor growth inhibition and tumor growth delay (P<0.05). At 15 mg/kg PF-00477736 + 30 mg/kg docetaxel, 3 of 12 mice had complete remission; all tumors eventually relapsed with docetaxel alone. Body weight loss increased by an additional 5% (nadir day 21) but recovered by day 36. [2]
In MDA-MB-231 xenografts, PF-477736 2HCl (15 mg/kg) combined with docetaxel (15 mg/kg) significantly enhanced tumor growth inhibition and delay (P<0.05). [2]
Immunohistochemistry in COLO205 tumors: PF-477736 2HCl suppressed docetaxel-induced increase in phospho-histone H3 and cytoplasmic phospho-Cdc25C, and increased γH2AX foci and caspase-3, indicating enhanced apoptosis. [2]
Bioluminescence imaging (BLI) showed that PF-477736 2HCl (15 mg/kg) significantly enhanced docetaxel-induced reduction in viable tumor cells as early as 24 h after first dose (P<0.05). [2]

MTT proliferation assay: Cells seeded in 96-well plates at exponential density, allowed to attach for 16 h, then serial dilutions of PF-477736 2HCl added. After 96 h incubation, MTT working stock added for 4 h, then DMSO added, absorbance read at 540 nm. IC50 calculated by four-variable analysis. [1]
PF50 MTT assay: Fixed concentration of PF-477736 2HCl (8×EC50, 360 nM, except K562 4×EC50, 180 nM) combined with serial dilutions of DNA-damaging agents (gemcitabine, SN-38, carboplatin, doxorubicin, mitomycin C). Incubation for 96 h, then MTT assay. PF50 = IC50(agent alone)/IC50(combination). [1]
Cell survival assay: HT29 cells treated with gemcitabine (15 nM) or camptothecin (25 nM) for 16 h, then PF-477736 2HCl at varying concentrations added for 4-48 h. Drug-containing medium removed, cells incubated in drug-free medium until control cells 90% confluent (8 days), then harvested and counted. [1]
Dot blot assay (histone H3 phosphorylation): CA46 cells treated with camptothecin (50 nM, 16 h), then PF-477736 2HCl at increasing concentrations in presence of nocodazole (0.1 μg/mL) for 16 h. Cells lysed, transferred to nitrocellulose membrane, probed with anti-phospho-histone H3, detected with HRP-conjugated secondary antibody and chemiluminescence. [1]
Flow cytometry for cell cycle and apoptosis: Cells stained with bromodeoxyuridine and propidium iodide (or FITC-dUTP for apoptosis), analyzed on FACSCalibur. [1][2]
Immunofluorescence: Cells fixed, stained with antibodies against phospho-histone H3, γH2AX, phospho-Cdc25C, and DAPI. Quantified by percentage of positive cells. [2]

Dissolved in 50 nM sodium acetate buffer and 4% dextrose (pH 4); 40 mg/kg; i.v. injection Colo205 xenograft mouse model

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For COLO205 xenograft efficacy: Female nu/nu mice (5-8 weeks) implanted subcutaneously with 2×10^6 COLO205 cells in 50% Matrigel. When tumors reached 100-150 mm^3, mice were randomized. PF-477736 2HCl was formulated as a solution in 50 mM sodium acetate buffer with 4% dextrose, pH 4, and administered intraperitoneally. Gemcitabine was given i.p. q3d×4 (once every 3 days for 4 treatments). PF-477736 2HCl was given 24 h after gemcitabine, same schedule, doses 4-60 mg/kg. MTD of PF-00477736 was 40 mg/kg (based on 5-10% body weight loss and behavioral response). Tumor growth inhibition (TGI) assessed 2 days after last dose. [1]
For docetaxel combination: COLO205 tumors established similarly. Docetaxel (15 or 30 mg/kg) administered i.p. once daily on days 1, 8, and 15. PF-477736 2HCl (7.5 or 15 mg/kg) administered i.p. twice daily (0 and 6 h) on days 1, 8, and 15. Tumor volumes measured by caliper twice weekly. BLI: mice injected with D-luciferin (75 mg/kg i.p.) and imaged 10 min later with IVIS100 system. [18F]FLT-PET: mice injected with 250 μCi tracer i.v., PET/CT imaging after 60 min. [2]
For immunohistochemistry: Tumors collected at 24 or 48 h after dosing, fixed, sectioned, stained for phospho-Chk1 (Ser345), phospho-histone H3, γH2AX, phospho-Cdc25C, caspase-3. Quantified with Chromovision automated cell imaging system. [2]

In rats after i.v. administration (4 mg/kg): systemic plasma clearance was low (approximately 20% of hepatic blood flow), moderate volume of distribution (steady-state), terminal half-life = 2.9 h. [1]
In mice after i.p. administration: dose-dependent pharmacokinetics. Greater than dose-proportional increase in AUC between 20 and 40 mg/kg, suggesting saturation of elimination pathways. [1]
In combination studies with docetaxel, plasma exposure of PF-477736 2HCl and docetaxel was not altered compared to single agents (data not shown). [2]

In mice, MTD of PF-477736 2HCl alone was 40 mg/kg (i.p., q3d×4) based on 5-10% body weight loss and severity of behavioral response. [1]
When combined with MTD docetaxel (30 mg/kg), PF-477736 2HCl (15 mg/kg) caused an additional 5% body weight loss at nadir (day 21), which was reversible by day 36. At sub-MTD docetaxel (15 mg/kg), no additional toxicity was observed. [2]
No exacerbation of side effects commonly associated with cytotoxic agents was reported. [1]

Mol Cancer Ther.2008 Aug;7(8):2394-404;Clin Cancer Res.2009 Jul 15;15(14):4630-40.

PF-477736 2HCl is an ATP-competitive Chk1 inhibitor that abrogates both S-phase and G2-M checkpoints as well as the mitotic spindle checkpoint. It enhances the cytotoxicity of DNA-damaging agents (gemcitabine, carboplatin, SN-38, doxorubicin, mitomycin C) in p53-defective tumor cells but has minimal effects on normal p53-competent cells (HUVEC). [1]
At high concentrations, docetaxel activates both the DNA damage checkpoint (Chk1 phosphorylation at Ser345) and the spindle checkpoint (phospho-histone H3). PF-477736 2HCl disrupts both checkpoints, leading to mitotic catastrophe and apoptosis. [2]
Biomarkers: PF-477736 2HCl suppresses docetaxel-induced phospho-histone H3 and cytoplasmic phospho-Cdc25C, and increases γH2AX and caspase-3. [2]
The compound is currently in phase 1 clinical trials in combination with gemcitabine. [1]

C22H27CL2N7O2

492.401481866837

491.16

1247874-19-6

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

154731146

Typically exists as solid at room temperature

6

5

4

33

725

1

Cl.Cl.O=C([C@@H](C1CCCCC1)N)NC1C=C2C(NN=CC3=C(C4C=NN(C)C=4)NC(C=1)=C23)=O

RXRFKHCZZSOCOS-JQDLGSOUSA-N

InChI=1S/C22H25N7O2.2ClH/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);2*1H/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;dihydrochloride

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)