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XL413 xHCl (XL-413; BMS863233; XL 413; BMS-863233), the hydrochloride salt of XL413, is a novel and selective cell division cycle 7 homolog (CDC7) kinase inhibitor with potential antitumor activity. It exhibits 63-, 12-, and 35-fold selectivity over CK2, Pim-1, and pMCM2, respectively, and inhibits CDC7 with an IC50 of 3.4 nM.
Physicochemical Properties
| Molecular Formula | C14H13CL2N3O2 | |
| Molecular Weight | 326.1779 | |
| Exact Mass | 325.038 | |
| Elemental Analysis | C, 58.04; H, 4.17; Cl, 12.24; N, 14.50; O, 11.04 | |
| CAS # | 1169562-71-3 | |
| Related CAS # | XL413 monohydrochloride;2062200-97-7;XL413;1169558-38-6 | |
| PubChem CID | 135564632 | |
| Appearance | Off-white to light yellow solid powder | |
| LogP | 4.29 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 1 | |
| Heavy Atom Count | 20 | |
| Complexity | 456 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | ClC1C([H])=C([H])C2=C(C=1[H])C1=C(C(N([H])C([C@]3([H])C([H])([H])C([H])([H])C([H])([H])N3[H])=N1)=O)O2.Cl[H] |
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| InChi Key | UNDKJUKLBNARIZ-FVGYRXGTSA-N | |
| InChi Code | InChI=1S/C14H12ClN3O2.ClH/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);1H/t9-;/m0./s1 | |
| Chemical Name | 8-chloro-2-[(2S)-pyrrolidin-2-yl]-3H-[1]benzofuro[3,2-d]pyrimidin-4-one;hydrochloride | |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Cdc7 (IC50 = 3.4 nM); PIM1 (IC50 = 42 nM); CK2 (IC50 = 215 nM) | ||
| ln Vitro |
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| ln Vivo |
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]. 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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| Enzyme Assay | 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. | ||
| Cell Assay |
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. 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. 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. 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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| Animal Protocol |
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| References |
[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. |
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| Additional Infomation |
XL413 is a benzofuropyrimidine that is 3,4-dihydro[1]benzofuro[3,2-d]pyrimidine substituted by (2S)-pyrrolidin-2-yl, oxo and chloro groups at positions 2, 4, and 8, respectively. It is a potent ATP competitive inhibitor of Cdc7 kinase (IC50 = 3.4 nM) and exhibits anticancer properties. It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an antineoplastic agent. It is a benzofuropyrimidine, an organochlorine compound and a member of pyrrolidines. BMS-863233 has been investigated for the treatment of Refractory Hematologic Cancer. CDC7 Kinase Inhibitor BMS-863233 is an orally bioavailable cell division cycle 7 homolog (CDC7) kinase inhibitor with potential antineoplastic activity. CDC7 kinase inhibitor BMS-863233 binds to and inhibits the activity of CDC7, which may result in the inhibition of DNA replication and mitosis, the induction of tumor cell apoptosis, and the inhibition of tumor cell proliferation in CDC7-overexpressing tumor cells. CDC7, a serine-threonine kinase overexpressed in a variety of tumor cell types, plays an essential role in the initiation of DNA replication by activating origins of replication. CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead 3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model.[1] Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.[2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0658 mL | 15.3290 mL | 30.6579 mL | |
| 5 mM | 0.6132 mL | 3.0658 mL | 6.1316 mL | |
| 10 mM | 0.3066 mL | 1.5329 mL | 3.0658 mL |