Chk1 (IC50 = 3 nM); CDK2 (IC50 = 0.16 μM)

SCH 900776 is a less effective inhibitor of CDK2 and Chk2, with IC50 values of 0.16 μM and 1.5 μM, respectively. The human liver microsomal isoforms of cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A4 are not significantly inhibited by SCH 900776. Twenty-four hours after exposure to hydroxyurea, SCH 900776 causes a dose-dependent loss of DNA replication capacity. SCH 900776 increases the hydroxyurea, 5-fluoruracil, and cytarabine γ-H2AX response. When SCH 900776 is combined with an antimetabolite, it causes γ-H2AX to accumulate in less than two hours, 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. Following exposure to SCH 900776, cycling populations of normal cells induce Chk1 pS345 as part of a futile cycle, possibly driven by AT-family kinases and DNA-PK. This rapid, dose-dependent accumulation of Chk1 pS345 is associated with exposure of proliferating WS1 cells to SCH 900776.[1]

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]

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 ³³P-ATP, initiates the reaction, resulting in a final reaction concentration of 1 μM ATP and 0.2 μCi of ³³P-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.

---

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.

---

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.

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.

---

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.

---

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). Percentages of each population at each concentrati

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

---

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.

---

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.

Furthermore, CHK1 pS345 positive cells were detected in skin punch biopsies taken from mice at SCH 900776 doses ≤25 mg/kg (75 mg/m2), in rats dosed IV at 5 and 10 mg/kg (30 and 60 mg/m2) and from dogs dosed IV at 2.5 and 5 mg/kg (45 and 89 mg/m2; Supplementary Fig. S10A to C). These data and the associated plasma exposures (pharmacokinetics) are summarized in Table 4 and comprise a pharmacological audit trail of SCH 900776 activity in three relevant preclinical species.

[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.

6-bromo-3-(1-methyl-4-pyrazolyl)-5-(3-piperidinyl)-7-pyrazolo[1,5-a]pyrimidinamine is a pyrazolopyrimidine.
See also: Mk-8776 (annotation moved to).

---

Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds. [1]

---

Many anticancer agents damage DNA and arrest cell-cycle progression primarily in S or G(2) phase of the cell cycle. Previous studies with the topoisomerase I inhibitor SN38 have shown the efficacy of the Chk1 inhibitor UCN-01 to overcome this arrest and induce mitotic catastrophe. UCN-01 was limited in clinical trials by unfavorable pharmacokinetics. SCH900776 is a novel and more selective Chk1 inhibitor that potently inhibits Chk1 and abrogates cell-cycle arrest induced by SN38. Like UCN-01, abrogation of SN38-induced arrest enhances the rate of cell death but does not increase overall cell death. In contrast, SCH900776 reduced the growth-inhibitory concentration of hydroxyurea by 20- to 70-fold. A similar magnitude of sensitization was observed with cytarabine. A 5- to 10-fold sensitization occurred with gemcitabine, but no sensitization occurred with cisplatin, 5-fluorouracil, or 6-thioguanine. Sensitization occurred at hydroxyurea concentrations that marginally slowed DNA replication without apparent activation of Chk1, but this led to dependence on Chk1 that increased with time. For example, when added 18 hours after hydroxyurea, SCH900776 induced DNA double-strand breaks consistent with rapid collapse of replication forks. In addition, some cell lines were highly sensitive to SCH900776 alone, and these cells required lower concentrations of SCH900776 to sensitize them to hydroxyurea. We conclude that some tumors may be very sensitive to the combination of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than concurrent treatment. SCH900776 is currently in phase I clinical trials, and these results provide the rationale and schedule for future clinical trials.[2]

---

Purpose: Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design: AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results: Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted.[3]

C₁₅H₁₈BRN₇

376.25

375.08

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

891494-64-7

SCH900776;891494-63-6

16224745

white to off-white Solid powder

1.8±0.1 g/cm3

1.819

0.76

2

5

2

23

425

0

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

GMIZZEXBPRLVIV-VIFPVBQESA-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-/m0/s1

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

MK 8776; MK8776; MK-8776; SCH900776; 891494-64-7; SCH900776 S-isomer; SCH900776 (S-isomer); (S)-6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(piperidin-3-yl)pyrazolo[1,5-A]pyrimidin-7-amine; 6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-((3S)-piperidin-3-yl)pyrazolo(1,5-a)pyrimidin-7-amine; UNII-99Y1V29WVE; 99Y1V29WVE; MK-8776 S-isomer; SCH 900776; SCH-900776

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)

DMSO: ~75 mg/mL (~199.3 mM)

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.

---

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.

---

View More

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.

---

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

---

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