Chk1 (Ki = 0.9 nM); Chk1 (IC50 <1 nM); Chk2 (IC50 = 8 nM)

Prexasertib (LY2606368) inhibits ARK5 (IC50=64 nM), BRSK2 (IC50=48 nM), SIK (IC50=42 nM), and MELK (IC50=38 nM). DNA deterioration caused by LY2606368 requires both CDK2 and CDC25A[1]. Prexasertib (8-250 nM; pre-treated for 15 minutes) causes damage to DNA during the S-phase in HT-29 cells[1]. Prexasertib (4 nM; 24 hours) causes a significant change in cell cycle populations from G1 and G2-M to S-phase, along with an increase in H2AX phosphorylation[1]. Prexasertib (33 nM; for 12 hours) causes the fragmentation of chromosomes in HeLa cells. Replication stress is induced by prexasertib Mesylate Hydrate (100 nM; 0.5 to 9 hours), which also reduces the amount of RPA2 that is available for DNA binding[1].

Prexasertib (LY2606368; 1-10 mg/kg; SC; twice daily for 3 days, rest 4 days; for three cycles) inhibits the growth of tumor xenografts[1]. Prexasertib (15 mg/kg; SC) phosphorylates RPA2 (S4/S8) and H2AX (S139), inhibiting CHK1 in the blood[1].

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.

The MTS Cell Proliferation Colorimetric Assay Kit measures the anticancer effects of BMN673 and LY2606368, the proliferation inhibition effect of CHK1 ablation, and IR sensitivity. After seeding cells into 96-well cell culture plates, each well is treated according to the experiment conditions specified. After two hours of incubation, the cell viability of each well is measured using a microplate reader set to detect wavelengths of 490 nM.

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.

:Mol Cancer Ther.2015 Sep;14(9):2004-13;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 a recommended dose for stage II squamous cell carcinoma (SCC) patients in a dose expansion cohort. [1]

473.2022

C, 53.27; H, 5.75; N, 20.71; O, 20.27

2100300-72-7

1234015-54-3 (2HCl);1234015-55-4 (mesylate);1234015-52-1;1234015-57-6 (mesylate hydrate); 2100300-72-7 (lactate hydrate);

157433374

Typically exists as solid at room temperature

6

12

9

34

558

1

SQQVRJAAUOKBIG-UHFFFAOYSA-N

InChI=1S/C18H19N7O2.C3H6O3.H2O/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)10-22-171-2(4)3(5)6/h2,4-5,8,10-11H,3,6-7,19H2,1H3,(H2,22,23,24,25)2,4H,1H3,(H,5,6)1H2

5-[[5-[2-(3-Aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile lactate salt monohydrate

Prexasertib monolactate monohydrate; Prexasertib monolactate monohydrate salt; LY2606368; E4FZV27T6Z; Prexasertib lactate monohydrate; LY2606368 lactate monohydrate; LY-2606368 lactate monohydrate; 2100300-72-7; UNII-E4FZV27T6Z; Prexasertib lactate hydrate; LY-2606368; LY 2606368.

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)