Chk1 0.9 nM (Ki) Chk1 <1 nM (IC50) Chk2 8 nM (IC50)
CHK1 (checkpoint kinase 1), CHK2, RSK1.

Prexasertib dimesylate (LY2606368 dimesylate) suppresses BRSK2 (IC50=48 nM), ARK5 (IC50=64 nM), SIK (IC50=42 nM), and MELK (IC50=38 nM). DNA damage caused by prexasertib dimesylate requires the presence of CDK2 and CDC25A[1]. In HeLa cells, prexasertib dimesylate (33, 100 nM) causes DNA damage during the S-phase[1]. In HT-29 cells, prexasertib dimesylate (8-250 nM; pre-treated for 15 minutes) suppresses the autophosphorylation of CHK1 (S296 ) and CHK2 (S516). In U-2 OS cells, prexasertib dimesylate (4 nM; 24 hours) induces H2AX phosphorylation and causes a significant shift in cell cycle populations from G1 and G2-M to S-phase[1]. In HeLa cells, proxasertib dimesylate (33 nM) for 12 hours fragments the chromosomes. Prexasertib dimesylate (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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Prexasertib dimesylate suppresses BRSK2 (IC50=48 nM), ARK5 (IC50=64 nM), SIK (IC50=42 nM), and MELK (IC50=38 nM). In HeLa cells, it causes DNA damage during the S-phase (33, 100 nM). In HT-29 cells, it suppresses autophosphorylation of CHK1 (S296) and CHK2 (S516). In U-2 OS cells, it induces H2AX phosphorylation and shifts cell cycle from G1 and G2-M to S-phase. It reduces RPA2 availability causing replication stress.

Tumor xenografts grow less rapidly when treated with prexasertib dimesylate (LY2606368 dimesylate; 1–10 mg/kg; SC; twice daily for three days, rest four days)[1]. Tumor xenografts treated with prexasertib dimesylate (15 mg/kg; SC) exhibit growth inhibition[1]. Inhibition of CHK1 in blood and phosphorylation of RPA2 (S4/S8) and H2AX (S139)[1].

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Tumor xenografts grow less rapidly when treated with Prexasertib dimesylate (1-10 mg/kg; SC; twice daily for three days, rest four days). Tumor xenografts treated with Prexasertib dimesylate (15 mg/kg; SC) exhibit growth inhibition, as well as inhibition of CHK1 in blood and phosphorylation of RPA2 (S4/S8) and H2AX (S139).

Assay: In vitro CHK1 kinase inhibition assay. Protocol: Recombinant CHK1 is incubated with varying concentrations of Prexasertib dimesylate (0.01-100 nM), ATP, and a specific peptide substrate. Phosphorylation is measured using a luminescent ADP-Glo or HTRF assay. Ki (0.9 nM) and IC50 are calculated. Inhibition of CHK2 and other off-target kinases (RSK1, etc.) is assessed similarly.

Cell Cycle Analysis[1]
Cell Types: HeLa cells
Tested Concentrations: 33, 100 nM
Incubation Duration: For 7 hrs (hours)
Experimental Results: Had an IC50 of 37 nM and resulted in the G2-M population received DNA damage during S-phase but continued to progress through the cell cycle into an early mitosis.

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Western Blot Analysis[1]
Cell Types: HT- 29 cells
Tested Concentrations: 8, 16, 31, 63, 125, 250 nM
Incubation Duration: Pre-treated for 15 minutes
Experimental Results: Inhibited CHK1 autophosphorylation (S296) and CHK2 autophosphorylation (S516) (IC50 of less than 31 nM) in HT- 29 cells.

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Cells: HeLa, U-2 OS, HT-29 cancer cell lines. Protocol: For mechanism studies, cells are treated with Prexasertib dimesylate (33-250 nM) for 1-24 hours. DNA damage is assessed by H2AX phosphorylation (gammaH2AX) via Western blot. Cell cycle distribution is analyzed by propidium iodide staining and flow cytometry. Replication stress is measured by RPA2 phosphorylation. Apoptosis is measured by caspase-3/7 activation or Annexin V staining.

Animal/Disease Models: Female CD-1 nu -/nu- mice (26-28 g) with Calu-6 cells[1]
Doses: 1, 3.3, or 10 mg/kg
Route of Administration: SC; twice (two times) daily for 3 days, rest 4 days; for three cycles
Experimental Results: Caused statistically significant tumor growth inhibition (up to 72.3%).

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Animal/Disease Models: Female CD-1 nu-/nu- mice (26-28 g) with Calu-6 cells[1]
Doses: 15 mg/kg (pharmacokinetic/PK Analysis)
Route of Administration: SC (200 μL)
Experimental Results: CHK1 was 7 ng/mL at 12 hrs (hours) and 3 ng/mL by 24 hrs (hours) in plasma exposures. Phosphorylation of both H2AX (S139) and RPA2 (S4/S8) was detectable at 4 hrs (hours), showing the rapid occurrence of DNA damage.

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Animal Model: Tumor xenograft models (e.g., various cancer types). Protocol: Mice bearing subcutaneous tumors (e.g., HCT-116, Calu-6) are treated with Prexasertib dimesylate via subcutaneous (SC) injection at doses of 1-15 mg/kg. Dosing schedules vary but commonly include twice daily for three consecutive days, followed by a rest period of four days (i.e., 3 days on, 4 days off) for 3-4 cycles. Tumor volume is measured twice weekly. At endpoint, tumors and blood are analyzed for PD biomarkers (p-CHK1, gammaH2AX, p-RPA2).

total of 45 patients were treated; 7 of them experienced dose-limiting toxicities (all hematologic toxicities). The maximum tolerated dose (MTD) was 40 mg/m² (regimen 1) and 105 mg/m² (regimen 2). The most common grade 3 or 4 treatment-related adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (usually <5 days). Febrile neutropenia occurred in a low rate (7%). At the MTD of each regimen, the exposure of LY2606368 in the first 72 hours (area under the curve from 0 to 72 hours) was consistent with the exposure that achieved maximum tumor response in a mouse xenograft model. Mild accumulation of LY2606368 was observed within the cycle and during the week at the maximum tolerated dose (MTD) of both dosing regimens. Two patients (4.4%) achieved partial remission; one had anal squamous cell carcinoma (SCC) and the other had head and neck SCC. The best overall response was stable disease (range: 1.2 to 6.7 months) in 15 patients (33.3%), including 6 with SCC. Conclusion: LY2606368 105 mg/m² every 14 days is being evaluated as the recommended dose for stage II patients with SCC in a dose expansion cohort.

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Prexasertib is a small molecule that is administered intravenously (IV) or subcutaneously (SC) in preclinical models. In clinical trials, Prexasertib is administered as an IV infusion (doses of 40-160 mg/m2) every 14 days. It has a short plasma half-life (1-3 hours) and is rapidly cleared, requiring intermittent dosing schedules. Plasma protein binding is moderate, and it is metabolized primarily by CYP3A4. PK profiles are dose-proportional in the studied range.

In Phase 1 clinical trials, dose-limiting toxicities (DLTs) of Prexasertib included myelosuppression (neutropenia, thrombocytopenia), fatigue, and gastrointestinal toxicity. Other common adverse events included anemia, febrile neutropenia, and increased liver enzymes. The MTD was determined to be 105 mg/m2 IV every 14 days. Preclinical toxicology studies showed similar findings in bone marrow and intestinal epithelium, consistent with on-target effects of CHK1 inhibition.

[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 a recommended dose for stage II squamous cell carcinoma (SCC) patients in a dose expansion cohort. [1]

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Prexasertib (LY2606368) was developed by Eli Lilly and has advanced into Phase 1/2 clinical trials for patients with advanced solid tumors (e.g., triple-negative breast cancer (TNBC), ovarian cancer, head and neck cancer, and small cell lung cancer). Its mechanism of inducing replication catastrophe and DNA damage is distinct from classic CHK1 inhibitors that primarily cause cell cycle arrest. Prexasertib showed promising single-agent activity in some trials, but the development may have been halted due to modest efficacy in registrational trials.

C20H27N7O8S2

557.600481271744

557.136

1234015-58-7

Prexasertib;1234015-52-1;Prexasertib dihydrochloride;1234015-54-3;Prexasertib Mesylate Hydrate;1234015-57-6;Prexasertib mesylate;1234015-55-4

137364590

Light yellow to yellow solid powder

5

14

8

37

592

0

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

HXYBEKZGRNUTBN-UHFFFAOYSA-N

InChI=1S/C18H19N7O2.2CH4O3S/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;2*1-5(2,3)4/h2,4-5,8,10-11H,3,6-7,19H2,1H3,(H2,22,23,24,25);2*1H3,(H,2,3,4)

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

Prexasertib (dimesylate); Prexasertib dimesylate; 1234015-58-7; 5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile;methanesulfonic acid; 5-((5-(2-(3-Aminopropoxy)-6-methoxyphenyl)-1H-pyrazol-3-yl)amino)pyrazine-2-carbonitrile dimethanesulfonate; LY2606368 (dimesylate); SCHEMBL20591301;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (179.34 mM)
H2O : 50 mg/mL (89.67 mM)

Solubility in Formulation 1: ≥ 3.5 mg/mL (6.28 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 35.0 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: ≥ 3.5 mg/mL (6.28 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 35.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)