Cdc7 (IC50 = 3.4 nM); PIM1 (IC50 = 42 nM); CK2 (IC50 = 215 nM)
CDC7 kinase (IC50 = 3.3 nM, biochemical assay)[1]

XL413 suppresses Colo-205 cells' ability to proliferate (IC50 = 2685 nM), reduces their viability (IC50 = 2142 nM), and activates caspase 3/7 (EC50 = 2288 nM). In soft agar, XL413 also dramatically suppresses the anchorage-independent growth of colo-205 (IC50 = 715 nM)[1]. When applied to tumors, XL413 has cytotoxic effects, with an IC50 of 22.9 µM in HCC1954 cells and 1.1 µM in Colo-205 cells—but not in HCC1954 cells. At an IC50 of 22.7 nM, XL413 is a potent DDK inhibitor when used in vitro.

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Compound XL413 demonstrated potent inhibition of CDC7-mediated phosphorylation of MCM2 in multiple cell lines. In the MDA-MB-231T cell line, the pMCM2 IC50 was 118 nM, and in the Colo-205 cell line, the pMCM2 IC50 was 140 nM.[1]
Flow cytometry analysis of Colo-205 cells treated with XL413 for 24 hours showed a dose-dependent accumulation of cells in the late S and G2 phases of the cell cycle, consistent with impaired DNA synthesis.[1]
Prolonged treatment (3 days) of Colo-205 cells with XL413 inhibited cell proliferation (IC50 = 2685 nM), decreased cell viability (IC50 = 2142 nM), and elicited caspase 3/7 activity (EC50 = 2288 nM).[1]
Compound XL413 significantly inhibited the anchorage-independent growth of Colo-205 cells in soft agar (IC50 = 715 nM).[1]

In mice, XL413 (100 mg/kg, po) exhibits acceptable plasma exposures and good PK characteristics. All dosages of XL413 (10, 30, or 100 mg/kg, po) are well tolerated, and no appreciable reduction in body weight occurs[1].

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XL413 (BMS-863233; 100 mg/kg, p.o.) hydrochloride exhibits good PK characteristics and excellent plasma exposures in mice. At all dosages, XL413 (10, 30, or 100 mg/kg, p.o.) hydrochloride is well tolerated and does not cause appreciable weight loss[1].

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Multiple-dose studies of compound 14 (XL413) in a Colo-205 xenograft model demonstrates significant anti-tumor efficacy. Tumor bearing mice were administered 14 orally at doses of 10, 30, or 100 mg/kg once daily (qd) for 14 days (Fig. 5). Two alternate dosing regimens were also examined in this study: a dose of 30 mg/kg administered twice-daily (bid) and a dose of 100 mg/kg administered every-other day (q2d). Compound 14 (XL413) was well tolerated at all the doses and regimens examined, with no significant body weight loss observed. Only modest tumor growth inhibition (36%) was observed for the 10 mg/kg qd dosage, but significant tumor growth inhibition (83%) was observed at the 30 mg/kg qd dose. More impressively, significant tumor growth regression (32%) was observed if dosed twice-daily at 30 mg/kg. The ED50 is estimated at 13 mg/kg.

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In a Colo-205 xenograft model, oral administration of XL413 showed dose-dependent target modulation, with 70% inhibition of phosphorylated MCM2 detected at a dose of 3 mg/kg. The ED50 for target modulation was calculated to be < 3 mg/kg.[1]
Multiple-dose studies in the Colo-205 xenograft model demonstrated significant anti-tumor efficacy. Oral dosing at 30 mg/kg once daily (qd) for 14 days resulted in 83% tumor growth inhibition. Dosing at 30 mg/kg twice-daily (bid) resulted in significant tumor regression (32%). The estimated ED50 for efficacy was 13 mg/kg. The compound was well tolerated at all doses and regimens tested.[1]

For five minutes, 20 ng of purified human DDK is pre-incubated with DDK inhibitors at escalating concentrations. Next, in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT, 10 µCi (γ)-32P ATP and 1.5 µM cold ATP are added, and the mixture is incubated for 30 minutes at 30°C. The proteins are autoradiographed on HyBlot CL film and SDS-PAGEd after being denatured in 1X Laemmli buffer at 100°C. DDK's auto-phosphorylation is a measure of its kinase activity. Using ImageJ, 32P-labeled bands are quantified, and GraphPad is used to compute the IC50 values.

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The biochemical kinase assay for CDC7 used N-terminally Myc-tagged human CDC7 and N-terminally His-tagged human ASK co-expressed and purified. Kinase activity and compound inhibition were determined using a luciferase-luciferin-coupled chemiluminescence assay, measuring the percentage of ATP utilized after the kinase reaction. The final assay conditions were 6 nM CDC7/ASK, 1 μM ATP, 50 mM Hepes pH 7.4, 10 mM MgCl₂, 0.02% BSA, 0.02% brij 35, 0.02% tween 20, and 1 mM DTT. Reactions were incubated at room temperature for 1-2 hours.[1]

Analysis of cell viability [2]
There are 2500 cells plated in each well of 96-well plates used for assays. Cells undergo treatment with small molecule inhibitors after 24 hours, and they are then incubated at 37°C for 72 hours. Next, the cells undergo lysis, and the CellTiter-Glo assay is employed to quantify the ATP content, which serves as a marker of metabolically active cells. Utilizing GraphPad software, IC50 values are determined. 100,000 cells are plated per well in six-well plates used for assays. Small molecule inhibitors are applied to the cells after a day, and they are then cultured for different lengths of time. Trypsinized cells are suspended in 5 milliliters of phosphate-buffered saline. After mixing 30 µL of this suspension with 30 µL of CellTiter-Glo reagent, it is incubated at room temperature for 10 minutes. The EnVision 2104 Multilabel Reader and the BioTek Synergy Neo Microplate Reader are used to measure luminosity.

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Analysis of Caspase 3/7 activity [2]
5,000 cells per well were plated in a 96 well plate. After 24 hours, cells were treated with small molecule inhibitors and incubated for 24 hours at 37°C. Caspase 3/7 activity and viable cell number were then measured using the Caspase-Glo 3/7 assay and CellTiter-Glo assay, respectively. The “Caspase activity per cell” was obtained by normalizing total Caspase activity to cell number.

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Immunoblot Analysis [2]
Whole cell extracts were prepared by re-suspending the pellets in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris HCl, pH 8) containing protease inhibitors (100 µM PMSF, 1 mM Benzamide, 2.5 µg/ml Pepstatin A, 10 µg/ml Leupeptin, and 10 µg/ml Aprotinin) and phosphatase inhibitors (1 mM each NaF, Na3VO4 and Na4P2O7). Protein concentration was measured using the BCA protein assay kit according to manufacturer's protocol. Equal amounts of protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Transfer efficiency and equal loading was confirmed by Ponceau S staining. Following primary and secondary antibody treatments, proteins were visualized using SuperSignal West Pico solutions.

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Thermal Stability Shift Assay (TSA) [2]
All reactions were incubated in a 10 µl final volume and assayed in 96-well plates using 20 x SYPRO Orange (Invitrogen) and 200 µg/ml purified DDK. Reactions were incubated with inhibitor compounds on ice for 30 minutes. Compounds from four kinase inhibitor libraries were screened at 20 µM for Tm increases with a total DMSO concentration of 2% or less. Thermal melting experiments were carried out using the StepOnePlus Real-Time PCR System melt curve program with a ramp rate of 1°C and temperature range of 15°C to 85°C. Subsequent TSAs on the 12 hits obtained were carried out as above but in triplicate and using a 200-fold range of inhibitor concentrations. Data analysis was performed as described. Melting temperatures (Tm) were calculated by fitting the sigmoidal melt curve to the Boltzmann equation using GraphPad Prism, with R2 values of >0.99. The difference in Tm values calculated for reactions with and without compounds is ΔTm.

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An endogenous MCM2 phosphorylation fixed cell immunofluorescence assay was conducted. MDA-MB-231T cells were seeded in 96-well plates, treated with serial dilutions of the test compound for 4 hours, then fixed and permeabilized. Cells were incubated overnight with a primary antibody against phosphorylated MCM2 (Ser40/Ser41), followed by a fluorescently labeled secondary antibody. The pMCM2 signal intensity was read on a high-content imaging system. IC50 values were determined by comparing signal intensity in compound-treated wells versus DMSO-treated control wells.[1]
Cell proliferation was measured by BrdU incorporation assay, cell viability was assayed using a luminescent cell viability assay kit, and apoptosis was measured using a homogeneous caspase-3/7 assay kit.[1]

3, 100 mg/kg
Colo-205 xenograft model

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For pharmacodynamic and efficacy studies in Colo-205 xenograft models, athymic nude mice bearing tumors were administered XL413 orally (by gavage) as a solution/suspension. Dosing regimens included single doses of 3, 10, 30, and 100 mg/kg for pharmacodynamic analysis (samples taken 4 hours post-dose), and multiple doses of 10, 30, or 100 mg/kg once daily (qd) for 14 days for efficacy. Additional regimens tested were 30 mg/kg twice-daily (bid) and 100 mg/kg every-other-day (q2d). Body weight was monitored for tolerability.[1]

In a complete rat pharmacokinetic study, after oral administration of 3 mg/kg to rats, the Cmax of XL413 was 8.61 μM, the AUC (PO) was 75 μM·h, the clearance (CL) was 117 mL/h·kg, the steady-state volume of distribution (Vss) was 0.55 L/kg, the half-life (T1/2) was 2.32 h, and the oral bioavailability (F) was 95%. [1] In a rat pharmacokinetic study, after oral administration of 100 mg/kg, the plasma drug concentrations at 1 h and 4 h were 141 μM and 81 μM, respectively. [1]

XL413 exhibits weak inhibitory activity against major cytochrome P450 isoenzymes: IC50 > 50 μM for CYP3A4, 2C9, 2D6, and 2C19; IC50 = 6.9 μM for CYP1A2. [1] It is inactive against hERG potassium channels (IC50 > 30 μM). [1] In repeated-dose efficacy studies in mice (up to 100 mg/kg daily for 14 days), no significant weight loss was observed, indicating good tolerability at effective doses. [1]

[1]. Discovery of XL413, a potent and selective CDC7 inhibitor. Bioorg Med Chem Lett. 2012 Jun 1;22(11):3727-31.

[2]. The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds. PLoS One. 2014 Nov 20;9(11):e113300.

XL413 is a benzofuranopyrimidine compound with the structure 3,4-dihydro[1]benzofurano[3,2-d]pyrimidine, substituted at positions 2, 4, and 8 with (2S)-pyrrolidine-2-yl, oxo, and chlorine, respectively. It is a potent competitive inhibitor of Cdc7 kinase ATP (IC50 = 3.4 nM) and has anticancer properties. It can be used as an EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitor and an antitumor drug. It is a benzofuranopyrimidine compound, belonging to organochlorine compounds and also pyrrolidine compounds.
BMS-863233 has been studied for the treatment of refractory hematologic malignancies.
CDC7 kinase inhibitor BMS-863233 is an orally bioavailable cyclin 7 homolog (CDC7) kinase inhibitor with potential antitumor activity. The CDC7 kinase inhibitor BMS-863233 binds to CDC7 and inhibits its activity, which may lead to inhibition of DNA replication and mitosis, induction of tumor cell apoptosis, and inhibition of the proliferation of CDC7-overexpressing tumor cells. CDC7 is a serine/threonine kinase that is overexpressed in a variety of tumor cell types and plays a crucial role in the initiation of DNA replication by activating the replication origin.

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CDC7 is a serine/threonine kinase that has been shown to be essential for the initiation and maintenance of DNA replication. Upregulation of CDC7 has been detected in a variety of tumor cell lines, and inhibition of CDC7 leads to cell cycle arrest. In this paper, we disclose the discovery of a highly potent and selective CDC7 inhibitor, XL413 (14), which has entered a phase I clinical trial. Starting from the lead compound 3 described above, we optimized its potency and selectivity for CDC7 to demonstrate that it can induce CDC7-dependent cell cycle arrest in vitro and inhibit tumor growth in vivo in a Colo-205 xenograft model. [1]

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Cdc7-Dbf4 kinase, or DDK (Dbf4-dependent kinase), initiates DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells, and it has become an emerging chemotherapeutic target because DDK inhibitors can induce apoptosis in a variety of cancer cells but not in normal cells. PHA-767491 and XL413 are two of a number of highly potent DDK inhibitors with low nanomolar IC50 values for purified kinases. Although XL413 is highly selective for DDK, its activity in cell lines has not been well characterized. We determined the antiproliferative and pro-apoptotic effects of XL413 on a range of tumor cell lines and compared it with that of PHA-767491, whose activity has been well characterized. Both compounds are potent biochemical inhibitors of DDK, but surprisingly, their activities differed greatly in different cell lines. Unlike PHA-767491, XL413 showed significant antiproliferative activity against only one of the ten cell lines tested. Since XL413 failed to effectively inhibit DDK in multiple cell lines, the bioavailability of this compound may be limited. In order to find other potential DDK inhibitors, we also tested the cross-reactivity of about 400 known kinase inhibitors with DDK using DDK thermostability variation analysis (TSA). We identified 11 compounds that could significantly stabilize DDK. Some of these compounds inhibited DDK with potency comparable to PHA-767491, including Chk1 and PKR kinase inhibitors, but their chemical skeletons were very different from those of known DDK inhibitors. Taken together, these data suggest that several known kinase inhibitors cross-react with DDK, and also highlight the potential to design more specific and bioactive DDK inhibitors as chemotherapeutic agents. [2] XL413 is a potent, selective, ATP-competitive CDC7 kinase inhibitor that was discovered through a structure-based optimization approach. It is a benzofuran pyrimidinone derivative. [1]
Its antitumor mechanism is related to the arrest of tumor cells in the late S phase to G2 phase of the cell cycle. This phenotype indicates that CDC7 is inhibited, ultimately leading to apoptosis. [1]
Based on its promising preclinical results, XL413 has entered Phase I clinical trials. [1]

C₁₄H₁₂CLN₃O₂

289.72

289.062

1169558-38-6

XL413 monohydrochloride;2062200-97-7;XL413 hydrochloride;1169562-71-3

135564632

Typically exists as Off-white to light yellow solid at room temperature

3.488

2

4

1

20

456

1

ClC1=CC=C2C(C(N=C([C@@H]3CCCN3)NC4=O)=C4O2)=C1

JJWLXRKVUJDJKG-VIFPVBQESA-N

InChI=1S/C14H12ClN3O2/c15-7-3-4-10-8(6-7)11-12(20-10)14(19)18-13(17-11)9-2-1-5-16-9/h3-4,6,9,16H,1-2,5H2,(H,17,18,19)/t9-/m0/s1

8-chloro-2-[(2S)-pyrrolidin-2-yl]-3H-[1]benzofuro[3,2-d]pyrimidin-4-one

XL413; 1169558-38-6; BMS-863233; XL-413; 1169562-71-3;

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.)