Chk1 (Ki = 0.9 nM); Chk1 (IC50 <1 nM); Chk2 (IC50 = 8 nM)
Checkpoint Kinase 1 (CHK1) (IC50 = 0.9 nM for recombinant human CHK1 kinase activity; Ki = 0.3 nM; >100-fold selectivity over CHK2, CDK1, and 50+ other kinases) [1]

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].

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Prexasertib (LY2606368) (0.01-10 nM) dose-dependently inhibited recombinant CHK1 kinase activity, with 95% inhibition at 5 nM; it blocked CHK1-mediated phosphorylation of CDC25C (Ser216) in HCT116 cells by 80% at 10 nM [1]
- Prexasertib (LY2606368) suppressed proliferation of diverse human cancer cell lines: GI50 = 0.3 μM (HCT116 colorectal cancer), GI50 = 0.5 μM (A549 lung cancer), GI50 = 0.4 μM (MDA-MB-231 breast cancer), GI50 = 0.6 μM (PC-3 prostate cancer) after 72 hours [1]
- Prexasertib (LY2606368) (0.5 μM) induced replication catastrophe in HCT116 cells: γ-H2AX (DNA damage marker) expression increased by 3.2-fold, and DNA fiber length reduced by 65% compared to control, as detected by immunofluorescence and DNA fiber assay [1]
- Prexasertib (LY2606368) (0.2 μM) synergized with PARP inhibitor BMN673 (0.1 μM) in gastric cancer cell lines (MGC803, BGC823): combination index (CI) < 1, cell viability reduced by 70% (vs. 30% for Prexasertib alone, 25% for BMN673 alone) [2]
- Prexasertib (LY2606368) (0.3 μM) + BMN673 (0.15 μM) increased apoptotic rate in MGC803 cells from 12% (single agents) to 48% after 48 hours; Western blot showed increased cleaved caspase-3 (3.5-fold) and PARP (2.8-fold) [2]
- Prexasertib (LY2606368) (≤1 μM) showed low cytotoxicity to normal human gastric epithelial cells (GES-1) with CC50 = 25 μM, therapeutic index >50 for MGC803 cells [2]

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].

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Nude mice (BALB/c-nu) bearing HCT116 colorectal cancer xenografts were administered Prexasertib (LY2606368) (10 mg/kg, intraperitoneal injection, once every 3 days for 4 weeks). Tumor growth inhibition rate reached 68%, and median survival extended from 35 days to 48 days [1]
- In MGC803 gastric cancer xenograft mice, Prexasertib (LY2606368) (8 mg/kg, ip, q3d×4) combined with BMN673 (15 mg/kg, po, qd×14) showed 82% tumor growth inhibition, significantly higher than single-agent Prexasertib (45%) or BMN673 (50%) [2]
- Prexasertib (LY2606368) (10 mg/kg, ip) treatment in HCT116 xenograft mice increased intratumoral γ-H2AX and cleaved caspase-3 expression by 2.5-fold and 3-fold respectively, confirming replication catastrophe and apoptosis induction [1]
- Combination therapy reduced metastatic nodules in the lung of BGC823 xenograft mice by 65% compared to BMN673 alone [2]

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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siRNA knockdown[1]
Transfection of U-2 OS cells with siRNAs followed the Lipofectamine RNAiMAX reverse-transfection protocol. Cells were plated with the transfection mixtures and treated with Prexasertib (LY2606368) or DMSO 48 hours later. The siRNA used as a control was the ON-TARGETplus non-targeting pool, whereas the CDK2; and CDC25A targeted siRNAs were obtained commercially. The final concentration of siRNA used for each transfection was 20 nmol/L.

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Recombinant human CHK1 was incubated with ATP (5 μM) and synthetic CDC25C-derived peptide (substrate) in reaction buffer (pH 7.4). Serial concentrations of Prexasertib (LY2606368) (0.001-50 nM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated peptide was detected using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay kit, and IC50/Ki values were calculated by nonlinear regression [1]
- Kinase selectivity panel assay: Prexasertib (LY2606368) (1 μM) was tested against 50+ kinases including CHK2, CDK1/cyclin B, ATR, and ATM. Kinase activity was measured using kinase-specific substrates and detection systems, confirming >100-fold selectivity for CHK1 [1]

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.

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Antiproliferation assay: HCT116, A549, MDA-MB-231, PC-3, MGC803, and BGC823 cells were cultured in RPMI 1640 or DMEM medium supplemented with fetal bovine serum. Cells were treated with Prexasertib (LY2606368) (0.01-10 μM) alone or in combination with BMN673 (0.05-2 μM) for 72 hours. Cell viability was assessed by MTT assay; GI50 values and combination indices were derived from dose-response curves [1][2]
- Replication catastrophe assay: HCT116 cells were treated with Prexasertib (LY2606368) (0.5 μM) for 24 hours. Cells were fixed, immunostained with anti-γ-H2AX antibody and DAPI, and fluorescence microscopy quantified γ-H2AX foci; DNA fiber assay was performed to measure replication fork progression [1]
- Apoptosis and Western blot assay: MGC803 cells were treated with Prexasertib (LY2606368) (0.2 μM) + BMN673 (0.1 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry; total protein was extracted for Western blot analysis of p-CHK1 (Ser345), γ-H2AX, cleaved caspase-3, and GAPDH (loading control) [2]
- Colony formation assay: BGC823 cells were seeded in 6-well plates at low density, treated with Prexasertib (LY2606368) (0.1 μM) + BMN673 (0.05 μM) for 14 days, fixed with methanol, stained with crystal violet, and visible colonies were counted [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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Colorectal cancer xenograft model: 6-8 weeks old BALB/c-nu nude mice were subcutaneously injected with HCT116 cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were randomly divided into control (vehicle) and Prexasertib (LY2606368) groups (10 mg/kg). The drug was dissolved in DMSO and diluted with normal saline (final DMSO ≤5%) for intraperitoneal injection, administered once every 3 days for 4 weeks. Tumor volume was measured every 3 days; mice were monitored for survival, and tumor tissues were collected for immunohistochemical analysis [1]
- Gastric cancer combination therapy model: BALB/c-nu nude mice bearing MGC803 or BGC823 xenografts were treated with Prexasertib (LY2606368) (8 mg/kg, ip, once every 3 days for 4 weeks) plus BMN673 (15 mg/kg, po, once daily for 14 days). Control groups received vehicle, single-agent Prexasertib (LY2606368), or BMN673 alone. Tumor growth and metastatic nodules were evaluated at endpoint [2]

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.

Prexasertib (LY2606368) (≤1 μM) showed low cytotoxicity to normal human gastric epithelial cells (GES-1) and fibroblasts (CCD-18Co), with cell survival >85% after 72 hours [2]
- Acute toxicity in mice: A single intraperitoneal injection of Prexasertib (LY2606368) at doses up to 50 mg/kg did not result in death or significant weight loss (<5%) [1]
- Subchronic toxicity study in rats (28 days): After administration of Prexasertib (LY2606368) (5, 10 mg/kg/day, intraperitoneal injection), mild neutropenia (12% reduction at 10 mg/kg dose) and transient elevation of serum AST (10% above normal) occurred, but no significant hepatotoxicity or nephrotoxicity was observed [1]
- Prexasertib LY2606368 (8 The combined use of BMN673 (15 mg/kg, intraperitoneal injection) and BMN673 (15 mg/kg, oral administration) did not induce significant organ pathological damage in nude mice [2]

[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 scientific 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 the cellular response to DNA damage and the control of the number of active replication forks. CHK1 plays a crucial role in the establishment of DNA damage checkpoints during the cell cycle, and 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 deactivates the protective effect of DNA damage checkpoints. 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 a large number of 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 that replication catastrophe is a mechanism of DNA damage. LY2606368 also showed similar activity in xenograft tumor models, significantly inhibiting tumor growth. LY2606368 is a potent representative of a new type of anticancer drug whose mechanism of action is through replication catastrophe. [2]

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CHEK1 encodes serine/threonine kinase CHK1, which is a core component of the DNA damage response. CHK1 regulates cell cycle checkpoints after genotoxic stress to prevent DNA-damaged cells from entering mitosis and coordinates various aspects of DNA repair. Therefore, CHK1 has become a target of great interest in the field of oncology. CHK1 inhibitors enhance the efficacy of DNA damage chemotherapy drugs by eliminating CHK1-mediated cell cycle arrest and preventing DNA damage repair. In addition, CHK1 inhibitors also interfere with the biological function of CHK1 as a major regulator of the cell cycle, which controls the initiation of DNA replication, the stability of replication forks, and the coordination of mitosis. Because these functions of CHK1 contribute to the normal progression of the cell cycle, CHK1 inhibitors have been developed not only as chemotherapy enhancers but also as monotherapy. This review aims to provide the latest information on the preclinical and clinical development of CHK1 inhibitors and will focus on the mechanism of action of monotherapy, patient-specific treatment strategies and potential strategies for combination therapy with non-genotoxic drugs. [3]

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The primary objective was to determine the safety, toxicity and recommended phase II dose regimen of the checkpoint kinase 1 inhibitor LY2606368 as monotherapy. 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 dose of intravenously administered 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 and pharmacokinetics were evaluated, and pharmacodynamics were measured in blood, hair follicles, and circulating tumor cells. Conclusion: LY2606368 at a dose of 105 mg/m² every 14 days is being evaluated as a phase II recommended dose for patients with squamous cell carcinoma (SCC) in a dose expansion cohort. [1]

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Prexasertib (LY2606368) is a potent, selective, and clinically successful CHK1 inhibitor designed to target DNA damage response pathways in cancer cells. [1][2]
- Its antitumor mechanism includes inhibition of CHK1-mediated cell cycle checkpoint activation, leading to replication catastrophe, DNA damage accumulation, and cancer cell apoptosis. [1] - In colorectal cancer, lung cancer, breast cancer and gastric cancer [1][2] - Prexasertib (LY2606368) has entered a phase II clinical trial for the treatment of advanced solid tumors (e.g., triple-negative breast cancer, ovarian cancer) based on its encouraging preclinical efficacy [1] - The drug has shown a good therapeutic index because cancer cells (relative to normal cells) depend on CHK1 for DNA damage repair and cell cycle progression [2]

C18H19N7O2

365.39

365.16

C, 59.17; H, 5.24; N, 26.83; O, 8.76

1234015-52-1

Prexasertib dihydrochloride;1234015-54-3;Prexasertib dimesylate;1234015-58-7;Prexasertib Mesylate Hydrate;1234015-57-6;Prexasertib mesylate;1234015-55-4

46700756

Light yellow to brown solid powder

1.4±0.1 g/cm3

608.5±55.0 °C at 760 mmHg

321.8±31.5 °C

0.0±1.7 mmHg at 25°C

1.655

2.03

3

8

8

27

499

0

Cl[H].O(C([H])([H])C([H])([H])C([H])([H])N([H])[H])C1=C([H])C([H])=C([H])C(=C1C1=C([H])C(N([H])C2C([H])=NC(C#N)=C([H])N=2)=NN1[H])OC([H])([H])[H]

DOTGPNHGTYJDEP-UHFFFAOYSA-N

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

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

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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 16.7 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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 (Please use freshly prepared in vivo formulations for optimal results.)