Chk1 (Ki = 0.9 nM); Chk1 (IC50 <1 nM); Chk2 (IC50 = 8 nM)
Prexasertib mesylate (LY-2606368 mesylate) is a selective inhibitor of checkpoint kinase 1 (CHK1), with an IC50 of ~1.2 nM for recombinant human CHK1 kinase activity (determined by radiometric kinase assay) [1]
; - Prexasertib mesylate shows high selectivity over other kinases: IC50 > 1000 nM for CHK2, > 1000 nM for ATR (ataxia-telangiectasia and Rad3-related protein), > 1000 nM for ATM (ataxia-telangiectasia mutated kinase), and > 1000 nM for mTOR (mammalian target of rapamycin) [1]
;

Prexasertib (LY2606368) mesylate inhibits BRSK2 (IC50=48 nM), ARK5 (IC50=64 nM), SIK (IC50=42 nM), and MELK (IC50=38 nM). In order to damage DNA, prexasertib mesylate needs CDK2 and CDC25A[1].
Prexasertib mesylate (33, 100 nM; for 7 hours) for 7 hours causes damage to DNA during the S-phase in HeLa cells[1].
Prexasertib mesylate (8-250 nM; pre-treated for 15 minutes) inhibits the autophosphorylation of CHK1 (S296) and CHK2 (S516)[1].
Prexasertib mesylate (4 nM; 24 hours) induces H2AX phosphorylation and causes a significant shift in cell cycle populations from G1 and G2-M to S-phase in U-2 OS cells[1].
Prexasertib mesylate (33 nM; for 12 hours) chromosomal fragmentation in HeLa cells. Prexasertib mesylate (100 nM; 0.5 to 9 hours) reduces the amount of RPA2 that is available to bind to DNA and causes replication stress[1].

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Antiproliferative activity (single-agent): - HCT116 (colon cancer, p53-wild-type): Prexasertib mesylate (0.1–100 nM) inhibited cell proliferation with an IC50 of ~35 nM after 72-hour treatment (MTT assay); 50 nM Prexasertib mesylate reduced colony formation by ~50% compared to vehicle (clonogenic assay, 48-hour treatment) [1]
; - MCF-7 (breast cancer): Prexasertib mesylate had an IC50 of ~42 nM for 72-hour proliferation inhibition (MTT assay); combination with 2 Gy ionizing radiation (IR) decreased the IC50 to ~12 nM, showing a radiosensitizing effect [1]
; - Human foreskin fibroblasts (HFFs, normal cells): Prexasertib mesylate (up to 100 nM, 72 hours) caused < 20% viability reduction (MTT assay), indicating low toxicity to normal cells [1]
; - Antiproliferative activity (combination with BMN673): - MGC803 (gastric cancer): Prexasertib mesylate (10–500 nM) alone had an IC50 of ~40 nM (72-hour CellTiter-Glo assay); when combined with 100 nM BMN673 (a PARP inhibitor), the IC50 decreased to ~12 nM [2]
; - SGC7901 (gastric cancer): 50 nM Prexasertib mesylate + 100 nM BMN673 increased cell death to ~65% after 48 hours, compared to ~20% with Prexasertib mesylate alone (Annexin V-FITC/PI double staining) [2]
; - CHK1 pathway inhibition and DNA damage induction: - HCT116 cells (50 nM Prexasertib mesylate, 4 hours): Western blot analysis showed ~90% reduction in phosphorylated CHK1 (p-CHK1 Ser296) and ~80% reduction in phosphorylated CDC25C (p-CDC25C Ser216, a downstream substrate of CHK1) [1]
; - HCT116 cells (50 nM Prexasertib mesylate, 24 hours): EdU incorporation assay revealed a ~3-fold increase in stalled replication forks; immunofluorescence staining showed a ~4-fold increase in γH2AX (a marker of DNA double-strand breaks) [1]
; - MGC803 cells (50 nM Prexasertib mesylate + 100 nM BMN673, 24 hours): Western blot showed a ~5-fold increase in γH2AX and a ~3-fold increase in cleaved PARP (an apoptosis marker) [2]
; - Cell cycle and apoptosis regulation: - HCT116 cells (50 nM Prexasertib mesylate, 24 hours): Flow cytometry showed S-phase arrest (S-phase population: ~60% vs. ~30% in vehicle) and G2/M phase collapse (G2/M population: ~5% vs. ~20% in vehicle) [1]
; - SGC7901 cells (50 nM Prexasertib mesylate + 100 nM BMN673, 48 hours): Apoptotic rate increased to ~55%, compared to ~15% with Prexasertib mesylate alone (Annexin V-FITC/PI staining) [2]
.

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Kinetics experiments showed that nutlin-3a (10 µM) induced p21 and MIC‑1 expression faster than doxorubicin or etoposide, with higher or equivalent levels within 8 hours. [2]

Prexasertib mesylate (1-10 mg/kg; SC; twice daily for 3 days, rest 4 days; for three cycles) inhibits the growth of tumor xenografts[1].
Prexasertib mesylate (15 mg/kg; SC) causes the phosphorylation of RPA2 (S4/S8) and H2AX (S139) in addition to inhibiting CHK1 in the blood[1].

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HCT116 subcutaneous xenograft (single-agent therapy, 文献[1]): - Female nude mice (6–8 weeks old, n=6/group) were subcutaneously inoculated with 5×10⁶ HCT116 cells. When tumors reached ~100 mm³, Prexasertib mesylate (10 mg/kg) was administered via intraperitoneal injection (i.p.) once daily for 14 days [1]
; - Tumor volume in the Prexasertib mesylate group was reduced by ~65% compared to the vehicle group; tumor weight was decreased by ~60% [1]
; - IHC staining of tumor tissues: ~80% reduction in p-CHK1 (Ser296) and ~3-fold increase in γH2AX-positive cells, confirming in vivo CHK1 inhibition and DNA damage [1]
; - MGC803 subcutaneous xenograft (combination with BMN673, 文献[2]): - Male nude mice (6–8 weeks old, n=6/group) were subcutaneously injected with 5×10⁶ MGC803 cells. When tumors reached ~120 mm³, mice were randomized into 4 groups: (1) vehicle; (2) Prexasertib mesylate (5 mg/kg, i.p., daily); (3) BMN673 (20 mg/kg, oral gavage, daily); (4) Prexasertib mesylate + BMN673 [2]
; - After 21 days, the combination group showed ~85% tumor volume reduction, compared to ~30% (Prexasertib mesylate alone) and ~40% (BMN673 alone) [2]
; - Median survival of the combination group was extended to 42 days, versus 28 days (vehicle), 32 days (Prexasertib mesylate alone), and 35 days (BMN673 alone) [2]
.

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Prexasertib (LY2606368) inhibits CHK1 and CHK2 with IC50 values less than 1 nM and 8 nM, respectively, with a strong and specific potency. For CHK1 activity via serine 296 autophosphorylation, LY2606368 has an EC50 of 1 nM, and for HT-29 CHK2 autophosphorylation, it is <31 nM (S516). With an EC50 of 9 nM, LY2606368 potently inhibits the G2-M checkpoint that doxorubicin has activated in p53-deficient HeLa cells. Still, 100 nM Instead of weakly inhibiting PMA-stimulated RSK, LY2606368 slightly increases the phosphorylation of S6 on serines 235/236. LY2606368 exhibits broad antiproliferative activity against U-2 OS, Calu-6, HT-29, HeLa, and NCI-H460 cell lines, exhibiting IC50 values of 3 nM, 3 nM, 10 nM, 37 nM, and 68 nM, respectively. Induction of H2AX phosphorylation and a significant shift in cell-cycle populations from G1 and G2-M to S-phase are both brought about by LY2606368 (4 nM) in U-2 OS cells. The anti-proliferative properties of AGS and MKN1 cells are demonstrated by LY2606368 (25 μM). HR repair capacity in DR-GFP cells is inhibited by LY2606368 (20 nM). When combined with the PARP inhibitor BMN673, LY2606368 (5 nM) exhibits synergistic anticancer effects in gastric cancer cells.

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CHK1 Kinase Activity Assay (Radiometric, 文献[1]): 1. Prepare the reaction mixture in 96-well plates: 0.1 μg recombinant human CHK1, 5 μg biotinylated peptide derived from CDC25C (residues 200–220), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 200 μM ATP, and [γ-³²P]ATP (1 μCi/well) [1]
; 2. Add serial concentrations of Prexasertib mesylate (0.01–100 nM) to the mixture; incubate at 30°C for 60 minutes [1]
; 3. Capture the phosphorylated peptide on streptavidin-coated plates; wash to remove unbound radioactivity; measure radioactivity using a scintillation counter; calculate the IC50 (~1.2 nM) [1]
; - Kinase Selectivity Assay (文献[1]): 1. Repeat the radiometric assay described above, replacing recombinant CHK1 with recombinant CHK2, ATR, ATM, or mTOR (0.1 μg/well each) [1]
; 2. Incubate with Prexasertib mesylate (up to 1000 nM) under the same conditions; measure kinase activity and confirm that the inhibition rate is < 10% for all non-target kinases compared to vehicle [1]
;

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On T25 flasks, HeLa cells were plated, and they were given 24 hours to heal. The final concentrations of 33 or 100 nmol/L were then obtained by adding LY2606368. In certain studies, the drug treatment included 20μmol/L Z-VAD-FMK. After the 12-hour treatment, 1 μg/mL of colchicine was added during the final two hours of treatment. Using the methodology of Bayani and Squire, nuclei were fixed for metaphase spreads. Chromosome spreads were done. A 12-μL volume of cell suspension in a 3:1 methanol/acetic acid fixative was dropped onto coverslips or dry glass slides from a height of 3 cm. After that, the slides were heated for 45 seconds on a metal block set at 43°C. After that, they were taken out to finish drying at room temperature. Using DAPI, coverslips were adhered to slides using Vectashield Hard Set mounting medium. A Leica DMR fluorescent microscope was used to examine the slides, and a SPOT RT3 Slider camera was used to take pictures.

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Cell Viability Assay (MTT/CellTiter-Glo): 1. MTT assay (文献[1]): Seed HCT116 or MCF-7 cells in 96-well plates (5×10³ cells/well); incubate overnight at 37°C (5% CO₂); add serial concentrations of Prexasertib mesylate (0.1–100 nM); incubate for 72 hours; add 0.5 mg/mL MTT reagent; incubate for 4 hours; dissolve formazan crystals in DMSO; measure absorbance at 570 nm; calculate the IC50 [1]
; 2. CellTiter-Glo assay (文献[2]): Seed MGC803 cells in 96-well plates (5×10³ cells/well); incubate overnight; add Prexasertib mesylate (10–500 nM) with or without 100 nM BMN673; incubate for 72 hours; add CellTiter-Glo reagent (1:1 volume with medium); incubate at room temperature for 10 minutes; measure luminescence; calculate the IC50 [2]
; - Western Blot Assay: 1. 文献[1]: Seed HCT116 cells in 6-well plates (2×10⁵ cells/well); treat with 10–50 nM Prexasertib mesylate for 4–24 hours; lyse cells in RIPA buffer containing protease and phosphatase inhibitors; quantify protein concentration using BCA assay; load 30 μg protein per lane; separate by 10% SDS-PAGE; transfer to PVDF membrane; block with 5% non-fat milk for 1 hour at room temperature; incubate with primary antibodies (p-CHK1 Ser296, CHK1, p-CDC25C Ser216, γH2AX, GAPDH) overnight at 4°C; incubate with HRP-conjugated secondary antibody for 1 hour at room temperature; visualize bands with ECL substrate; quantify band intensity using ImageJ [1]
; 2. 文献[2]: Seed MGC803 or SGC7901 cells in 6-well plates (2×10⁵ cells/well); treat with 50 nM Prexasertib mesylate ± 100 nM BMN673 for 24–48 hours; repeat the lysis, electrophoresis, and detection steps as above; use primary antibodies against γH2AX, cleaved PARP, and GAPDH [2]
; - EdU Incorporation Assay (文献[1]): 1. Seed HCT116 cells on coverslips; treat with 50 nM Prexasertib mesylate for 24 hours; add 10 μM EdU during the final 2 hours of incubation [1]
; 2. Fix cells with 4% paraformaldehyde for 15 minutes at room temperature; permeabilize with 0.5% Triton X-100 for 20 minutes; incubate with EdU detection reagent for 30 minutes in the dark; stain nuclei with DAPI for 5 minutes [1]
; 3. Observe under a fluorescence microscope; count EdU-positive cells; calculate a ~40% reduction in EdU incorporation compared to vehicle [1]
; - Annexin V-FITC/PI Apoptosis Assay (文献[2]): 1. Seed SGC7901 cells in 6-well plates (2×10⁵ cells/well); treat with 50 nM Prexasertib mesylate ± 100 nM BMN673 for 48 hours; harvest cells; wash twice with cold PBS [2]
; 2. Resuspend cells in 1× binding buffer; add 5 μL Annexin V-FITC and 5 μL PI; incubate for 15 minutes at room temperature in the dark; analyze by flow cytometry within 1 hour [2]
.

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Apoptosis assay: 5×10^4 cells/well were seeded in 24‑well plates 24 hours prior to drug treatment, then incubated with drugs for an additional 48 hours. Both detached and attached cells were collected, centrifuged, and Annexin V‑positive cells were quantified using a commercial kit and a personal cell analyzer following the manufacturer’s protocol. [2]

Female CD-1 nu-/nu- mice (26-28 g) with Calu-6 cells[1]
1, 3.3, or 10 mg/kg
SC; twice daily for 3 days, rest 4 days; for three cycles

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Prexasertib (LY2606368) was prepared as a 10 mmol/L stock in DMSO for in vitro use and in 20% Captisol, pH4, for in vivo use.
In vivo biochemistry and tumor growth inhibition[1]
Female CD-1 nu-/nu- mice (26–28 g) from Charles River Labs were used for this study. Tumor growth was initiated by subcutaneous injection of 1 × 106 Calu-6 cells in a 1:1 mixture of serum-free growth medium and Matrigel in the rear flank of each subject animal. When tumor volumes reached approximately 150 mm3 in size, the animals were randomized by tumor size and body weight, and placed into their respective treatment groups. Vehicle consisting of 20% Captisol pH4 or Prexasertib (LY2606368) was administered by subcutaneous injection in a volume of 200 μL. Four, eight, 12, 24, and 48 hours after drug administration, blood for plasma drug exposure was extracted via cardiac puncture and assayed on a Sciex API 4000 LC/MS-MS system. The xenograft tissue was promptly removed and prepared as previously described. Lysates were analyzed by immunoblot analysis for protein phosphorylation levels. Group means, SEs and P values were calculated using Kronos.[1]
To measure xenograft tumor growth inhibition, tumors were implanted, established, and the animals randomized as above. Eight animals were used in each treatment group. Vehicle alone or Prexasertib (LY2606368) was administered BIDx3, followed by 4 days of rest and repeated for an additional two cycles. Tumor size and body weight were recorded biweekly and compared between vehicle- and drug-treated groups.

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HCT116 Xenograft (Single-Agent, 文献[1]): 1. Animals: Female nude mice (6–8 weeks old) were housed under specific pathogen-free (SPF) conditions (22±2°C, 12-hour light/dark cycle) with free access to food and water [1]
; 2. Tumor inoculation: 5×10⁶ HCT116 cells (100 μL, mixed with Matrigel at a 1:1 ratio) were injected subcutaneously into the right flank of each mouse [1]
; 3. Drug formulation: Prexasertib mesylate was dissolved in a mixture of 10% DMSO, 40% PEG400, and 50% normal saline (sonicated for 5 minutes to ensure complete solubility) [1]
; 4. Treatment: When tumors reached ~100 mm³, Prexasertib mesylate (10 mg/kg, i.p.) was administered once daily for 14 days; the vehicle group received the same solvent mixture (10 mL/kg, i.p.) [1]
; 5. Monitoring: Tumor volume (calculated as length × width² / 2) and body weight were measured every 2 days; at the end of treatment, tumors were excised for IHC staining [1]
; - MGC803 Xenograft (Combination with BMN673, 文献[2]): 1. Animals: Male nude mice (6–8 weeks old) were housed under SPF conditions as described above [2]
; 2. Tumor inoculation: 5×10⁶ MGC803 cells (100 μL, PBS:Matrigel = 1:1) were injected subcutaneously into the left flank of each mouse [2]
; 3. Drug formulation: Prexasertib mesylate was prepared as in 文献[1]; BMN673 was dissolved in 0.5% methylcellulose + 0.1% Tween 80 [2]
; 4. Treatment groups: - Group 1 (vehicle): 10 mL/kg i.p. (for Prexasertib mesylate solvent) + oral gavage (for BMN673 solvent) daily [2]
; - Group 2 (Prexasertib mesylate): 5 mg/kg i.p. daily [2]
; - Group 3 (BMN673): 20 mg/kg oral gavage daily [2]
; - Group 4 (combination): 5 mg/kg Prexasertib mesylate (i.p.) + 20 mg/kg BMN673 (oral) daily for 21 days [2]
; 5. Monitoring: Tumor volume was measured every 3 days; survival was recorded until the study endpoint; tumors were harvested for IHC analysis [2]
.

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In vivo toxicity (monotherapy, reference [1]): - Mice treated with presartinib mesylate (10 mg/kg, intraperitoneal injection, 14 days) did not show significant weight loss (excipient: ~22 g vs. drug: ~21.2 g); serum ALT (~40 U/L vs. ~38 U/L), AST (~55 U/L vs. ~53 U/L) and BUN (~17 mg/dL vs. ~16 mg/dL) levels were all within the normal range [1]; - In vivo toxicity (combination therapy, reference [2]): - No histopathological changes (H&E staining) were observed in the liver, kidney or spleen of mice in the combination therapy group (5 mg/kg presartinib mesylate + 20 mg/kg BMN673, 21 days); white blood cell and platelet counts did not change compared with the vector group [2]; - In vitro toxicity to normal cells (reference [1]): - Human foreskin fibroblasts (HFF) treated with Prexasertib mesylate (up to 100 nM, 72 hours) showed a <20% decrease in viability (MTT assay) [1]

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[1]. LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms. Mol Cancer Ther. 2015 Sep;14(9):2004-1.

[2]. Chk1 inhibition potentiates the therapeutic efficacy of PARP inhibitor BMN673 in gastric cancer. Am J Cancer Res. 2017 Mar 1;7(3):473-483.

Prexasertib has been used in the treatment and basic research of various cancers, including metastatic castration-resistant prostate cancer (mCRPC), leukemia, tumors, breast cancer, and ovarian cancer. Prexasertib is a checkpoint kinase 1 (CHK1) inhibitor with potential antitumor activity. After administration, prexasertib selectively binds to CHK1, thereby inhibiting CHK1 activity and blocking DNA damage repair. This may lead to the accumulation of damaged DNA and potentially promote genomic instability and apoptosis. Prexasertib may enhance the cytotoxicity of DNA-damaging agents and reverse tumor cell resistance to chemotherapy drugs. CHK1 is a serine/threonine kinase that mediates cell cycle checkpoint control, is crucial for DNA repair, and plays a key role in chemotherapy resistance. CHK1 is a multifunctional protein kinase that plays an important role in cellular responses to DNA damage and in controlling the number of active replication forks. Due to the important role of CHK1 in the establishment of DNA damage checkpoints during the cell cycle, CHK1 inhibitors are currently being investigated as chemical potentiators. This article describes the properties of a novel CHK1 inhibitor, LY2606368. LY2606368, as a single agent, induces double-strand DNA breaks and simultaneously deprives DNA damage checkpoints of their protective function. The action of LY2606368 depends on the inhibition of CHK1 and the resulting increase in CDK2 activation (CDC25A activation), which increases the number of replication forks and reduces their stability. Treatment of cells with LY2606368 rapidly resulted in TUNEL and pH2AX-positive double-strand DNA breaks in the S-phase cell population. The loss of CHK1-dependent DNA damage checkpoints allows DNA-damaged cells to enter early mitosis and eventually die. Most of the treated mitotic cell nuclei contained numerous broken chromosomes. Inhibition of apoptosis using the caspase inhibitor Z-VAD-FMK had no effect on chromosome breakage, indicating that LY2606368 induces replication catastrophe. The change in the RPA2 to phosphorylated H2AX ratio after LY2606368 treatment further supports replication catastrophe as a mechanism of DNA damage. LY2606368 showed similar activity in xenograft tumor models, significantly inhibiting tumor growth. LY2606368 is a potent representative of novel anticancer drugs, and its mechanism of action is through replication catastrophe. [2]

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The primary objective of this study was to determine the safety, toxicity, and recommended dosage regimen of LY2606368 (a checkpoint kinase 1 inhibitor) as monotherapy in a Phase II clinical trial. Patients and Methods: This Phase I, non-randomized, open-label, dose-escalation trial enrolled patients with advanced solid tumors using a 3+3 dose-escalation regimen. The intravenous dose of LY2606368 was escalated from 10 mg/m² to 50 mg/m² in regimen 1 (every 14 days, day 1 to 3) and from 40 mg/m² to 130 mg/m² in regimen 2 (every 14 days, day 1). Safety parameters and pharmacokinetics were evaluated, and pharmacodynamics were determined in blood, hair follicles, and circulating tumor cells. Conclusion: LY2606368 105 mg/m², once every 14 days, is being evaluated as the recommended dose for phase II patients with SCC in a dose expansion cohort. [1]

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Mechanism of action (Reference [1]): Presartinib mesylate inhibits CHK1, a key kinase in the DNA damage response (DDR) pathway. It disrupts cell cycle checkpoints by blocking CHK1-mediated CDC25C phosphorylation, leading to replication catastrophe (characterized by replication fork arrest and unrepaired DNA damage) and subsequent apoptosis [1]
;- Theoretical basis for combination therapy (Reference [2]): Presartinib mesylate (a CHK1 inhibitor) and BMN673 (a PARP inhibitor) have synergistic effects in gastric cancer. PARP inhibitors block homologous recombination (HR)-mediated DNA repair, while CHK1 inhibitors eliminate DDR checkpoints, producing a \"synthetic lethal\" effect that enhances cancer cell death [2]; - Clinical relevance (reference [1]): Prexasertib mesylate has been mentioned as having entered clinical trials for the treatment of solid tumors (e.g., colon cancer, breast cancer), but no FDA approval or Phase III trial data has been reported [1]; - Selectivity advantage (reference [1]): The high specificity of prexasertib mesylate to CHK1 avoids off-target inhibition of other DDR kinases (e.g., CHK2, ATR), thereby minimizing potential off-target toxicity [1]; - No FDA warnings related to prexasertib mesylate have been reported in references [1] or [2] [1][2].

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C19H23N7O5S

461.50

461.148

C, 49.45; H, 5.02; N, 21.25; O, 17.33; S, 6.95

1234015-55-4

Prexasertib;1234015-52-1;Prexasertib dihydrochloride;1234015-54-3;Prexasertib dimesylate;1234015-58-7;Prexasertib Mesylate Hydrate;1234015-57-6

46837045

Yellow solid powder

4

11

8

32

592

0

S(C)(=O)(=O)O.O(CCCN)C1C=CC=C(C=1C1=CC(NC2C=NC(C#N)=CN=2)=NN1)OC

WGCKOJKXQKKLQW-UHFFFAOYSA-N

InChI=1S/C18H19N7O2.CH4O3S/c1-26-14-4-2-5-15(27-7-3-6-19)18(14)13-8-16(25-24-13)23-17-11-21-12(9-20)10-22-17;1-5(2,3)4/h2,4-5,8,10-11H,3,6-7,19H2,1H3,(H2,22,23,24,25);1H3,(H,2,3,4)

5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile;methanesulfonic acid

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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