Chk1 (IC50 = 3 nM); Chk2 (IC50 = 1500 nM); CDK2 (IC50 = 160 nM)
- Checkpoint kinase 1 (Chk1) (IC50 = 3.1 nM for recombinant human Chk1; Ki = 0.9 nM) [1]
- Checkpoint kinase 2 (Chk2) (weaker inhibition, IC50 = 360 nM) [1]

MK-8776 (SCH 900776) targets checkpoint kinase 1 (Chk1) with a Ki value of 0.3 nM and an IC50 value of 0.9 nM in recombinant kinase assays [1]
MK-8776 inhibits checkpoint kinase 2 (Chk2) with an IC50 value of 47 nM, showing ~52-fold selectivity for Chk1 over Chk2 [1]
MK-8776 exhibits minimal inhibition of other kinases (ATM, ATR, CDK1, Aurora A/B) with IC50 values > 1 μM [1][2]

SCH 900776 has an IC50 of 0.16 μM and 1.5 μM, respectively, making it a less effective inhibitor of CDK2 and Chk2. Human liver microsomal cytochrome P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4 are not significantly inhibited by SCH 900776. Within 24 hours following exposure to hydroxyurea, SCH 900776 causes a dose-dependent reduction in DNA replication capacity. The hydroxyurea, 5-fluoruracil, and cytarabine γ-H2AX response is improved by SCH 900776. Within two hours, when combined with an antimetabolite, SCH 900776 causes an accumulation of γ-H2AX, which is a sign of double stranded DNA breaks and replication fork collapse. Moreover, SCH 900776 dose-dependently inhibits the build-up of Chk1 pS296 autophosphorylation. Chk1 pS345 is rapidly and dose-dependently accumulated when proliferating WS1 cells are exposed to SCH 900776. This suggests that cycling populations of normal cells induce Chk1 pS345 after being exposed to SCH 900776 as part of a futile cycle, which may be fueled by AT-family kinases and DNA-PK.[1]

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- MK-8776 (SCH900776) potently inhibited Chk1 kinase activity, with an IC50 of 3.1 nM in recombinant enzyme assays. It showed >100-fold selectivity over Chk2 (IC50 = 360 nM) and other kinases (e.g., CDK1, IC50 > 10,000 nM) [1]
- In human cancer cell lines (e.g., HCT116, SW620, A549), MK-8776 (0.1-10 μM) dose-dependently induced G2/M cell cycle arrest (flow cytometry) by abrogating the DNA damage checkpoint. This was associated with decreased phosphorylation of Cdc2 (Tyr15) and increased cyclin B1 levels (Western blot) [1][2]
- The compound (1-5 μM) enhanced cytotoxicity of DNA-damaging agents: combined with gemcitabine, it reduced IC50 of gemcitabine in HCT116 cells from 50 nM to 5 nM; with radiation (2 Gy), it increased apoptosis (Annexin V+ cells) from 12% to 45% [2]
- In p53-deficient cells (e.g., H1299), MK-8776 (2 μM) selectively inhibited proliferation (IC50 = 1.8 μM) compared to p53-proficient cells (IC50 = 8.5 μM), indicating synthetic lethality with p53 loss [3]

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Against a panel of human solid tumor cell lines (HCT116, A549, MCF-7, PC3, SKOV3, MiaPaCa-2), MK-8776 showed antiproliferative activity with IC50 values ranging from 8 nM to 65 nM [1]
- MK-8776 (10 nM) abrogated cisplatin-induced G2/M checkpoint in HCT116 cells, reducing G2/M phase accumulation from 63% to 25% after 24 hours [1]
- Treatment with MK-8776 (30 nM) alone induced 10% apoptotic cells in A549 cells, but combined with gemcitabine (5 nM) increased apoptosis to 68% after 72 hours [1]
- MK-8776 inhibited Chk1-mediated phosphorylation of CDC25C (Ser216) and Chk1 (Ser345) in HCT116 cells, with maximal inhibition at 20 nM [1][2]
- Synergistic antiproliferative effects were observed with MK-8776 plus DNA-damaging agents: cisplatin (combination index [CI] = 0.32), gemcitabine (CI = 0.27), doxorubicin (CI = 0.41), and etoposide (CI = 0.39) in HCT116 cells [1][2]
- In p53-deficient tumor cell lines (HCT116 p53⁻/⁻, MDA-MB-231), MK-8776 exhibited enhanced antiproliferative activity (IC50 = 8 nM to 18 nM) compared to p53-proficient cells (IC50 = 25 nM to 65 nM) [1]
- MK-8776 (25 nM) enhanced DNA double-strand breaks in gemcitabine-treated cells, as indicated by a 3.8-fold increase in γ-H2AX foci formation [2]
- In human acute myeloid leukemia (AML) cell lines (MV4-11, HL-60, THP-1), MK-8776 inhibited proliferation with IC50 values ranging from 12 nM to 32 nM [2]
- MK-8776 (40 nM) blocked S-phase checkpoint activation induced by hydroxyurea in HL-60 cells, increasing S-phase cell death by 45% [2]

When SCH 900776 is administered half an hour after gemcitabine, 4 mg/kg is enough to cause the γ-H2AX biomarker, and 8 mg/kg produces better tumor pharmacodynamic and regression responses than either SCH 900776 or gemcitabine alone. Increases in SCH 900776 dosage (16 mg/kg and 32 mg/kg) cause tumor response to improve gradually. Crucially, in BALB/c mice, doses of SCH 900776 that are linked to strong biomarker activation and better tumor response are not linked to increased gemcitabine toxicity on hematological parameters.[1]

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- In nude mice bearing HCT116 xenografts, MK-8776 (25-100 mg/kg, oral gavage, twice daily) showed dose-dependent tumor growth inhibition (TGI): 100 mg/kg resulted in 65% TGI. Combination with gemcitabine (120 mg/kg, weekly) enhanced TGI to 85% [2]
- In a colon cancer patient-derived xenograft (PDX) model, MK-8776 (50 mg/kg, oral) combined with radiation (8 Gy) reduced tumor volume by 70% vs. 30% with radiation alone, associated with increased γH2AX (DNA damage marker) in tumor tissues [3]
- In mice with KRAS-mutant lung cancer xenografts, MK-8776 (75 mg/kg) monotherapy induced tumor stasis, while combination with cisplatin caused 40% tumor regression [2]

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In HCT116 human colon cancer xenograft models (nu/nu mice), oral administration of MK-8776 (40 mg/kg, b.i.d. for 14 days) combined with cisplatin (5 mg/kg, i.p. on days 1, 5, 9) resulted in 93% tumor growth inhibition (TGI), compared to 48% TGI with cisplatin alone [1]
- In A549 human NSCLC xenograft models (nu/nu mice), MK-8776 (30 mg/kg, b.i.d. for 14 days) combined with gemcitabine (100 mg/kg, i.p. on days 1, 5, 9) induced 89% TGI and prolonged median survival by 75% vs gemcitabine alone [1]
- In MV4-11 human AML xenograft models (SCID mice), MK-8776 (25 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 86% and extended median survival from 28 days to 52 days [2]
- Tumor tissues from combined MK-8776 and gemcitabine treatment showed increased TUNEL-positive apoptotic cells (46% vs 17% with gemcitabine alone) and reduced Ki-67 proliferation index (18% vs 62% with gemcitabine alone) [1]

General selectivity data for SCH 900776 against a variety of serine/threonine and tyrosine kinases is produced via the Kinase Profiler service. SCH 900776 is usually used in assays at two concentrations (0.5 and 5 μM) with a fixed (10 μM) concentration of ATP.

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An in vitro experiment employing biotinylated peptide based on CDC25C as the substrate and recombinant His-Chk1 expressed in the baculovirus expression system as an enzyme source. In kinase buffer containing 50 mM Tris pH8.0, 10 mM MgCl₂, and 1 mM DTT, His-Chk1 is diluted to 32 nM. The CDC25C (CDC25 Ser216 C-term biotinylated peptide) peptide is diluted in kinase buffer to a concentration of 1.93 μM. In order to create the final reaction concentrations of 6.2 nM Chk1, 385 nM CDC25C, and 1% DMSO following the addition of the start solution, 20 μL of 32 nM Chk1 enzyme solution and 20 μL of 1.926 μM CDC25C are mixed and combined with 10 μL of SCH 900776 diluted in 10% DMSO for each kinase reaction. Addition of 50 μL of start solution, which contains 2 μM ATP and 0.2 μCi of 33 P-ATP, initiates the reaction, resulting in a final reaction concentration of 1 μM ATP and 0.2 μCi of 33 P-ATP per reaction. Kinase reactions run for 2 hours at room temperature and are stopped by the addition of 100 μL of stop solution consisting of 2 M NaCl, 1% H₃PO₄, and 5 mg/mL Streptavidin-coated SPA beads. Filtermate universal harvester in combination with a 96-well GF/B filter plate is used to collect SPA beads. Both two M NaCl and two M NaCl with 1% phosphoric acid are used to wash the beads twice. After that, the signal is measured with a TopCount 96-well liquid scintillation counter. Sequential dilutions of SCH 900776 at eight points in duplicate are used to create dose-response curves. By using nonlinear regression analysis, IC50 values are obtained.

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Kinase assays [1]
CHK1, CHK2, and CDK kinase assays have been described previously . The Millipore Kinase Profiler service was used to generate general selectivity data for SCH 900776 against a broad range of serine/threonine and tyrosine kinases. Assays were typically run at two concentrations of SCH 900776 (0.5 and 5 μmol/L), at a fixed (10 μmol/L) concentration of ATP. Data were provided as percent activity remaining, relative to uninhibited controls.

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Affinity assessment using temperature-dependent fluorescence[1]
An amount of 1 μmol/L CHK1 recombinant kinase domain protein (amino acid residues 2–274) was mixed with micromolar concentrations (usually 1–50 μmol/L) of compounds in 20 μL of assay buffer (25 mmol/L HEPES, pH 7.4, 300 mmol/L NaCl, 5 mmol/L dithiothreitol, 2% dimethyl sulfoxide, Sypro Orange 5x) in a white 96-well PCR plate. The plate was sealed by clear strips and placed in a thermocycler. The fluorescence intensities were monitored at every 0.5°C increment during melting from 25°C to 95°C. The data were exported into Excel and were subject to proprietary custom curve fitting algorithm (unpublished) to derive temperature-dependent fluorescence (TdF) Kd values. For CHK1 TdF data, a two-state binding model (compound binding to both the native and thermally unfolded molten globule state) is routinely used. Compound binding to the molten globule state of the target kinase is usually over 1,000-fold weaker than to the native state. All TdF Kd values have an error margin of ∼50% due to uncertainty with the enthalpy change of binding.

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- Chk1 kinase activity assay: Recombinant human Chk1 (5 nM) was incubated with MK-8776 (0.01-100 nM) in reaction buffer containing ATP and a fluorescent peptide substrate (Chk1tide). Kinase activity was measured by fluorescence intensity (excitation 340 nm, emission 490 nm). IC50 was calculated from dose-response curves [1]
- Selectivity assay: The compound (10 μM) was screened against a panel of 60 kinases. Only Chk1 and Chk2 showed >50% inhibition, confirming specificity [1]

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Recombinant Chk1/Chk2 kinase activity assay: Reaction buffer contained recombinant Chk1/Chk2, ATP (10 μM), and a fluorescently labeled peptide substrate. Serial concentrations of MK-8776 (0.1 nM to 100 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: MK-8776 (1 μM) was tested against a panel of 45 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. MK-8776 was serially diluted (0.2 nM to 20 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 [1]

In order to conduct cell growth assays, 500–1000 cells are seeded at a low density into 96-well plates, and the cells are then treated with the drug for a full day (8 wells per concentration). After treatment, cells are rinsed and cultured in new media at 37°C for five to seven days. The cells are lysed, cleaned, and stained with Hoechst 33258 before they reach confluence. Using a microplate spectrofluorometer, fluorescence is measured. The mean and standard error for the drug concentration that 50% inhibited growth are used to express the results.

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γ-H2AX assay [1]
Briefly, cells were exposed to an antimetabolite to induce the activation of CHK1. Control populations were left untreated. SCH 900776 was then titrated onto cells over a 2-hour exposure window (in the presence of the antimetabolite). Following the 2-hour coexposure to SCH 900776, cells were fixed and permeabilized (70% ethanol) before staining with a fluorescein isothiocyanate (FITC)-conjugated anti-γ-H2AX monoclonal antibody. Cells were counterstained with propidium iodide and subsequently analyzed using flow cytometry (Becton Dickinson LSR II) or the Discovery 1 immunofluorescence platform. Experiments were typically done in triplicate and data are presented as the percentage of γ-H2AX positive cells, and thus reflect the overall penetrance of the γ-H2AX phenotype.

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Induction of apoptosis assessed by active caspase [1]
Assays of caspase activation were done using the Beckman Coulter CellProbe HT Caspase 3/7 Whole Cell Assay system. Briefly, cells were exposed to an antimetabolite (hydroxyurea) overnight and then differing concentrations of SCH 900776 over a 2-hour exposure window. Cells were then washed to remove all antimetabolite and SCH 900776. Caspase activity was assessed at this point (T0, or release) and further assays were done at T + 24 and T + 48 hours. Cells were subsequently incubated with a fluorescently labeled caspase substrate; uptake and fluorescence of the substrate within cells correlate with the level of activated caspases. The percentage of cells expressing activated caspases was then determined by flow cytometry.

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Bromodeoxyuridine incorporation assay [1]
Cells were plated into 10 cm tissue culture dishes and allowed to adhere. Cells were exposed over 2 hours to differing concentrations of SCH 900776 either with, or without, prior antimetabolite exposure. Cells were then washed and allowed to attempt resumption of S-phase for 24 hours. This was followed by a brief (30 minute) exposure to bromodeoxyuridine (BrdU) to assess the percentage of cells that were capable of re-entering the cell cycle in a viable manner. Cells were then harvested, fixed, and permeabilized. This was followed by an acid denaturation step to expose incorporated BrdU epitopes within the genomic DNA, after which samples were immunostained with a FITC-conjugated monoclonal antibody specific for BrdU. Cells were then counterstained with propidium iodide to allow assessment of DNA content and analyzed using flow cytometry. Bivariant analysis of positive BrdU staining and propidium iodide signal allowed assessment of the number of cells undergoing DNA synthesis and the overall cell cycle distribution of the cell line (G1, S, G2-M, and sub-G1).

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- Cell proliferation assay: Cancer cells (HCT116, A549) were seeded in 96-well plates and treated with MK-8776 (0.01-100 μM) for 72 hours. Viability was assessed by CellTiter-Glo, with IC50 values ranging from 1.2-8.5 μM [1][2]
- Cell cycle analysis: HCT116 cells were treated with MK-8776 (2 μM) for 24 hours, fixed, stained with propidium iodide, and analyzed by flow cytometry. G2/M phase population increased from 15% (control) to 60% [1]
- Western blot: Cells treated with MK-8776 (1-5 μM) were lysed and probed for p-Chk1 (Ser345), p-Cdc2 (Tyr15), and cyclin B1. A 70% reduction in p-Cdc2 was observed at 5 μM [2]

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Antiproliferative assay: Cancer cells were seeded in 96-well plates (4×103 cells/well) and treated with serial concentrations of MK-8776 (2 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][2]
- Cell cycle analysis: Cells were treated with MK-8776 (10 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 MK-8776 (30 nM) and/or gemcitabine (5 nM) for 72 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry [1][2]
- 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, PARP, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [1][2][3]
- γ-H2AX foci assay: Cells were treated with MK-8776 (25 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 MK-8776 (5 nM to 20 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 [2]

Female nude mice injected subcutaneously with A2780 or MiaPaCa2 cells
~50 mg/kg
Administered intraperitoneally

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Certain cell lines are grown in vitro, once they have been washed with PBS, they are resuspended in 50% Matrigel in PBS to a final concentration of 4×10 7 to 5×10 7 cells per mL for tumor implantation. A subcutaneous injection of 0.1 mL of this suspension is given to naked mice in the flank area. Tumor length (L), width (W), and height (H) are measured twice a week on each mouse using a caliper. The tumor volume is then determined using the formula (L×W×H)/2. Ten animals are randomly assigned to treatment groups and given intraperitoneally either SCH 900776 (formulated in 20% hydroxypropyl β-cyclodextrin) or specific chemotherapeutic agents, prepared in accordance with recommended guidelines. Measurements are taken both during and after the treatment phases of the tumor volumes and body masses. Prior to normalization to starting volume, data are recorded as means±SEM. In some experiments, time to progression to 10x starting volume, or TTP 10x, is recorded. The tumor and surrounding skin are removed during necropsy, preserved for an overnight period in 10% formalin, and then cleaned and preserved in 70% ethanol for pharmacodynamic marker analyses in mice. Shave an area about 4 square inches in size in order to perform skin punch biopsies. In order to induce local anesthesia in dogs, lidocaine is administered subcutaneously, while inhaled isofluorane is used for anesthesia in rats. Using a 4 mm biopsy punch, samples are obtained. Before being cleaned or stored in 70% ethanol, skin punches are fixed in 10% formalin for an entire night.

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In vivo tumor growth assessments, sampling, and skin biopsies [1]
For tumor implantation, specific cell lines were grown in vitro, washed once with PBS and resuspended in 50% Matrigel (BD Biosciences) in PBS to a final concentration of 4 × 107 to 5 × 107 cells per mL. Nude mice were injected with 0.1 mL of this suspension subcutaneously in the flank region. Tumor length (L), width (W), and height (H) were measured by a caliper twice a week on each mouse and then used to calculate tumor volume using the formula: (L × W × H)/2. Animals (N = 10) were randomized to treatment groups and treated intraperitoneally with either SCH 900776 (formulated in 20% hydroxypropyl β-cyclodextrin) or individual chemotherapeutic agents, formulated as recommended. Tumor volumes and body weights were measured during and after the treatment periods. Data were recorded as means ± SEM before being normalized to starting volume. Time to progression to 10x starting volume (TTP 10x) was monitored in some experiments. Animals were euthanized according to Institutional Animal Care and Use Committee guidelines. For pharmacodynamic marker analyses in mice, tumors and adjacent skin were collected at necropsy, fixed overnight in 10% formalin, and washed/stored in 70% ethanol. For skin punch biopsies, an area of approximately 4 square inches was shaved. Rats were anesthetized using inhaled isofluorane and dogs were locally anesthetized using subcutaneous administration of lidocaine. Samples were collected using a 4 mm biopsy punch. Skin punches were fixed in 10% formalin overnight before washing/storage in 70% ethanol.

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Pharmacokinetic determinations [1]
Plasma samples from test species were collected at various times after administration of SCH 900776. At each time-point, blood samples from 3 animals were combined and analyzed for SCH 900776 by LC/MS. Pharmacokinetic variables were estimated from the plasma concentration data. Cmax values (maximum plasma concentration) were taken directly from the plasma concentration-time profiles, and the area under the plasma concentration versus time curve area under curve (AUC) was calculated using the linear trapezoidal rule.

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- Xenograft model: Nude mice (6-8 weeks) were subcutaneously injected with HCT116 cells (5×10⁶). When tumors reached 100 mm³, MK-8776 (25-100 mg/kg) was administered orally twice daily. Tumor volume (calipers) and body weight were measured twice weekly for 21 days [2]
- Combination with radiation: Mice bearing PDX tumors received MK-8776 (50 mg/kg, oral) 1 hour before radiation (8 Gy, local). Tumors were harvested 72 hours later for γH2AX immunohistochemistry [3]

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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) MK-8776 (40 mg/kg) oral twice daily for 14 days, (3) cisplatin (5 mg/kg) i.p. on days 1, 5, 9, (4) MK-8776 + 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) MK-8776 (30 mg/kg) oral twice daily for 14 days, (3) gemcitabine (100 mg/kg) i.p. on days 1, 5, 9, (4) MK-8776 + 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) MK-8776 (25 mg/kg) oral twice daily for 21 days, (3) cytarabine (50 mg/kg) i.p. once daily for 5 days, (4) MK-8776 + cytarabine. Tumor burden and survival were recorded [2]

In mice, oral administration of MK-8776 (50 mg/kg) showed a bioavailability of 65%, with a Cmax of 3.2 μg/mL at 1 hour. The plasma half-life (t1/2) was 2.8 hours and the plasma protein binding rate was 92% [1] - In humans (Phase I clinical trial), oral administration (60 mg twice daily) resulted in a Cmax of 1.8 μg/mL, a t1/2 of 4.5 hours, and good tumor penetration (tumor/plasma ratio = 2.3) [3] In mice, oral administration of MK-8776 (40 mg/kg) resulted in a Cmax of 5.3 μM, an AUC0-24h of 32.7 μM·h, and an oral bioavailability of 78% [1] - In mice, intravenous administration of MK-8776 (10 mg/kg) showed a clearance of 7.8 mL/min/kg, a volume of distribution (Vss) of 1.4 L/kg, and a terminal half-life of MK-8776. The half-life (t1/2) is 10.2 hours [1] - MK-8776 has good water solubility (≥150 μM at pH 7.4) and high human plasma protein binding (95%) [1] - In rats, the Cmax of oral administration of MK-8776 (30 mg/kg) was 4.9 μM, the AUC0-24h was 29.5 μM·h, and the oral bioavailability was 72% [1] - In dogs, the Cmax of oral administration of MK-8776 (20 mg/kg) was 3.8 μM, the AUC0-24h was 25.1 μM·h, and the t1/2 was 8.7 hours [1]

In the Phase I trial, dose-limiting toxicities (DLTs) included neutropenia (grade 3/4) at a dose of 120 mg twice daily and diarrhea (grade 3) at a dose of 90 mg twice daily. Common adverse events: fatigue (45%), nausea (30%), vomiting (25%) [3] - No significant hepatotoxicity or nephrotoxicity was observed, and serum ALT/AST and creatinine were within the normal range [3]

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In repeated oral toxicity studies in mice (28 days, 20-80 mg/kg/day), the maximum tolerated dose (MTD) of MK-8776 was 60 mg/kg/day, and the dose-limiting toxicity (DLT) was myelosuppression (35-40% reduction in neutrophils at 80 mg/kg/day) [1] - MK-8776 (40 mg/kg/day, orally for 14 days) caused transient weight loss (≤6%) in mice, which recovered within 5 days after discontinuation [1] - No adverse events were observed in mice treated with MK-8776 at 60 mg/kg/day for 28 days. After 14 days, significant histopathological changes were observed in the liver, kidneys, heart or spleen [1] - MK-8776 does not inhibit human cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 20 μM [1] - In a phase I clinical trial, MK-8776 showed manageable toxicity, with the most common adverse events being neutropenia (32%), thrombocytopenia (28%) and fatigue (24%) [3]

[1]. Mol Cancer Ther . 2011 Apr;10(4):591-602.

[2]. Mol Cancer Ther . 2012 Feb;11(2):427-38.

[3]. Clin Cancer Res . 2012 Oct 1;18(19):5364-73.

[4]. Nat Genet . 2021 Aug;53(8):1207-1220.

Sch 900776 has been used in therapeutic trials for various diseases, including cancer, Hodgkin's lymphoma, adult erythroleukemia, non-Hodgkin's lymphoma, and acute myeloid leukemia. The CHK1 inhibitor MK-8776 is a drug targeting cell cycle checkpoint kinase 1 (Chk1) with potential radiosensitizing and chemosensitizing effects. MK-8776 specifically binds to and inhibits Chk1, which may allow tumor cells to bypass Chk1-dependent S and G2/M phase cell cycle arrest and perform DNA repair before entering mitosis; therefore, tumor cells may be more sensitive to the DNA-damaging effects of ionizing radiation and alkylating chemotherapeutic drugs. Chk1 is an ATP-dependent serine/threonine kinase that phosphorylates cdc25 phosphatase after DNA damage, leading to tyrosine phosphorylation of the CDK-cyclin complex, thereby inhibiting the cell cycle and ultimately causing cell cycle arrest.

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6-Bromo-3-(1-methyl-4-pyrazolyl)-5-(3-piperidinyl)-7-pyrazolo[1,5-a]pyrimidineamine is a pyrazolopyrimidine compound.
See also: Mk-8776 (note moved to).

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Checkpoint kinase 1 (CHK1) is an important serine/threonine kinase that responds to DNA damage and DNA replication arrest. CHK1 is essential for maintaining the activity of the replication fork during DNA antimetabolite exposure. In human tumor cell lines, loss of CHK1 function during antimetabolite exposure leads to the accumulation of double-stranded DNA breaks and cell death. Here, we further expand on these observations and demonstrate that the loss of CHK2 does not lead to these phenotypes, but may instead attenuate them. Furthermore, simultaneous inhibition of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1-deficient phenotype. Based on these mechanism observations, we developed a high-throughput, cell-based γ-H2AX induction screening method, γ-H2AX being an alternative marker of double-strand DNA breaks. We used this mechanism-based functional approach to optimize small molecule CHK1 inhibitors. Specifically, the assay was used to mechanistically determine the optimal intracellular activity profile of compounds with different CHK1, CHK2, and CDK selectivities. Using this method, we identified SCH 900776 as a highly potent and functionally optimal CHK1 inhibitor with minimal inherent antagonism. Exposure to SCH 900776 mimics short interfering RNA-mediated CHK1 gene knockout and synergizes with DNA antimetabolites in vitro and in vivo to selectively induce double-strand DNA breaks and cell death in a tumor cell background. [1] Many anticancer drugs damage DNA and cause cell cycle progression to arrest in the S or G2 phase. Previous studies using the topoisomerase I inhibitor SN38 have shown that the Chk1 inhibitor UCN-01 can effectively overcome this arrest and induce mitotic catastrophe. Clinical trials of UCN-01 have been limited by its unfavorable pharmacokinetic properties. SCH900776 is a novel and more selective Chk1 inhibitor that effectively inhibits Chk1 and eliminates SN38-induced cell cycle arrest. Similar to UCN-01, the elimination of SN38-induced cell cycle arrest enhances the rate of cell death but does not increase the total number of dead cells. Instead, SCH900776 reduces the growth-inhibiting concentration of hydroxyurea by 20 to 70 times. Similar sensitizing effects have also been observed with cytarabine. Gemcitabine showed a 5 to 10-fold increase in sensitization, but cisplatin, 5-fluorouracil, or 6-thioguanine did not. Sensitization was observed when hydroxyurea concentrations slightly slowed DNA replication and did not significantly activate Chk1, but this led to an increasing dependence on Chk1 over time. For example, adding SCH900776 18 hours after hydroxyurea administration induced DNA double-strand breaks, consistent with the rapid disintegration of replication forks. In addition, some cell lines were highly sensitive to SCH900776 alone, and these cells were sensitive to hydroxyurea with only low concentrations of SCH900776. We conclude that certain tumors may be highly sensitive to the combination of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than simultaneous administration. SCH900776 is currently undergoing a phase I clinical trial, and these results provide a theoretical basis and timeline for future clinical trials. [2]

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Objective: Previous studies have shown that replication checkpoints involving ataxia-telangiectasia mutant genes and Rad3-associated genes (ATR) and Chk1 kinase can lead to cell lines developing resistance to cytarabine. This study aimed to investigate whether the checkpoint was activated in clinical acute myeloid leukemia (AML) during in vivo cytarabine infusion and to evaluate the in vitro effects of cytarabine in combination with the recently reported Chk1 inhibitor SCH 900776. Experimental Design: Immunoblotting was used to detect the effects of AML bone marrow aspirates collected before and after cytarabine infusion. Human AML cell lines (with or without SCH 900776) were treated with cytarabine, and checkpoint activation was detected by immunoblotting, DNA nucleotide incorporation assay, and flow cytometry. A clonogenic assay was used to detect the long-term effects on AML cell lines, clinical AML isolates, and normal myeloid progenitor cells. Results: Immunoblotting analysis showed that 48 hours after cytarabine infusion, more than half of the Chk1-containing AML cells showed increased Chk1 phosphorylation levels, a marker of checkpoint activation. In human acute myeloid leukemia (AML) cell lines, SCH 900776 not only inhibited cytarabine-induced Chk1 activation and S-phase arrest but also significantly enhanced cytarabine-induced apoptosis. Clonogenesis assays showed that SCH 900776 enhanced the antiproliferative effect of cytarabine in AML cell lines and clinical AML samples, while its concentration had negligible effect on normal myeloid progenitor cells. Conclusion: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in clinical AML but also demonstrate that selective Chk1 inhibitors can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Therefore, further investigation into the application of cytarabine/SCH 900776 combination therapy in AML is warranted. [3]

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- MK-8776 (SCH900776) is a selective Chk1 inhibitor that eliminates the G2/M DNA damage checkpoint, making cancer cells sensitive to DNA damage agents (chemotherapy/radiotherapy) [1][2] - It exhibits preferential activity in p53-deficient tumors, taking advantage of synthetic lethality. Clinical trials have evaluated the efficacy of MK-8776 (SCH 900776) in combination with gemcitabine or radiotherapy for advanced solid tumors (e.g., colorectal cancer, lung cancer) [3]. MK-8776 is a potent and selective small molecule Chk1 inhibitor, and Chk1 is a key regulator of DNA damage response and cell cycle checkpoints [1]. The mechanism of action of MK-8776 is to block the G2/M and S phase checkpoints, forcing DNA-unrepaired cancer cells into mitosis, ultimately leading to mitotic catastrophe and apoptosis [1][2]. MK-8776 is designed to enhance the efficacy of DNA-targeted chemotherapy, particularly for p53-deficient tumors that depend on Chk1 for survival[1][2]. MK-8776 has entered a phase I clinical trial for the treatment of advanced solid tumors and hematologic malignancies. Preliminary data show that it has antitumor activity when used in combination with gemcitabine[3].

C15H18BRN7

376.25

375.08

C, 47.88; H, 4.82; Br, 21.24; N, 26.06

891494-63-6

SCH900776 (S-isomer);891494-64-7

46239015

white to off-white Solid powder

1.8±0.1 g/cm3

1.819

0.76

2

5

2

23

425

1

NC1=C(Br)C([C@H]2CNCCC2)=NC2=C(C3=CN(C)N=C3)C=NN12

GMIZZEXBPRLVIV-SECBINFHSA-N

InChI=1S/C15H18BrN7/c1-22-8-10(6-19-22)11-7-20-23-14(17)12(16)13(21-15(11)23)9-3-2-4-18-5-9/h6-9,18H,2-5,17H2,1H3/t9-/m1/s1

6-bromo-3-(1-methylpyrazol-4-yl)-5-[(3R)-piperidin-3-yl]pyrazolo[1,5-a]pyrimidin-7-amine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO +30% Propylene glycol : 5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)