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

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].

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].

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.

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.

Forty-five patients were treated; seven experienced dose-limiting toxicities (all hematologic). The maximum-tolerated doses (MTDs) were 40 mg/m(2) (schedule 1) and 105 mg/m(2) (schedule 2). The most common related grade 3 or 4 treatment-emergent adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (typically < 5 days). Febrile neutropenia incidence was low (7%). The LY2606368 exposure over the first 72 hours (area under the curve from 0 to 72 hours) at the MTD for each schedule coincided with the exposure in mouse xenografts that resulted in maximal tumor responses. Minor intra- and intercycle accumulation of LY2606368 was observed at the MTDs for both schedules. Two patients (4.4%) had a partial response; one had squamous cell carcinoma (SCC) of the anus and one had SCC of the head and neck. Fifteen patients (33.3%) had a best overall response of stable disease (range, 1.2 to 6.7 months), six of whom had SCC. Conclusion: An LY2606368 dose of 105 mg/m(2) once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC.

[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 trials studying the treatment and basic science of mCRPC, Leukemia, Neoplasm, breast cancer, and Ovarian Cancer, among others. Prexasertib is an inhibitor of checkpoint kinase 1 (chk1) with potential antineoplastic activity. Upon administration, prexasertib selectively binds to chk1, thereby preventing activity of chk1 and abrogating the repair of damaged DNA. This may lead to an accumulation of damaged DNA and may promote genomic instability and apoptosis. Prexasertib may potentiate the cytotoxicity of DNA-damaging agents and reverse tumor cell resistance to chemotherapeutic agents. Chk1, a serine/threonine kinase, mediates cell cycle checkpoint control and is essential for DNA repair and plays a key role in resistance to chemotherapeutic agents.

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CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.[2]

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The primary objective was to determine safety, toxicity, and a recommended phase II dose regimen of LY2606368, an inhibitor of checkpoint kinase 1, as monotherapy. Patients and methods: This phase I, nonrandomized, open-label, dose-escalation trial used a 3 + 3 dose-escalation scheme and included patients with advanced solid tumors. Intravenous LY2606368 was dose escalated from 10 to 50 mg/m(2) on schedule 1 (days 1 to 3 every 14 days) or from 40 to 130 mg/m(2) on schedule 2 (day 1 every 14 days). Safety measures and pharmacokinetics were assessed, and pharmacodynamics were measured in blood, hair follicles, and circulating tumor cells. Conclusion: An LY2606368 dose of 105 mg/m(2) once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC.[1]

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.)