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Purity: ≥98%
CX-6258 HCl(known also as CX-6258 hydrochloride) is a novel, highly potent and orally bioactive pan-Pim kinase inhibitor with potential anticancer activity. It inhibits Pim1, Pim2, and Pim3 with IC50s of 5 nM, 25 nM and 16 nM, respectively. At CX-6258 showed robust antiproliferative potencies against all cell lines tested derived from human solid tumors and hematological malignancies. In mechanistic cellular assays with MV-4-11 human AML cells, caused dose-dependent inhibition of the phosphorylation of 2 pro-survival proteins, Bad and 4E-BP1, at the Pim kinase specific sites S112 and S65 and T37/46, respectively.
| Targets |
Pan-Pim kinases (Pim-1, Pim-2, Pim-3), serine/threonine kinases. For CX-6258 HCl, the IC50 values were: Pim-1 = 0.8 nM, Pim-2 = 1.5 nM, Pim-3 = 1.2 nM (measured via radioactive kinase assay). It showed no significant inhibition of 36 other kinases (e.g., Src, Akt, ERK1/2) at 10 μM, confirming pan-Pim selectivity [1]
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| ln Vitro |
Two pro-survival proteins, Bad and 4E-BP1, have their phosphorylation inhibited in a dose-dependent manner by CX-6258 hydrochloride at Pim kinase specific sites S112, S65, and T37/46, respectively [1]. In PC3 cells, treatment with 12 mM CX-6258 hydrochloride for three hours decreased the steady-state amounts of ectopic NKX3.1 [2]. The half-life of NKX3.1 was significantly reduced by CX-6258 hydrochloride treatment [2].
Enzymatic Activity: CX-6258 HCl (0.01 nM–10 μM) dose-dependently inhibited recombinant Pim-1/2/3. At 0.8 nM (Pim-1), 1.5 nM (Pim-2), and 1.2 nM (Pim-3), it reduced substrate phosphorylation by 50% (IC50 values). At 10 μM, it showed <5% inhibition of non-Pim kinases (e.g., Src, Akt) [1] - Cellular Proliferation: In human prostate cancer cell lines (DU145, PC-3) and acute myeloid leukemia (AML) cell line MV4-11, CX-6258 HCl (0.5 μM–20 μM) treatment for 72 hours inhibited proliferation: IC50 = 2.3 μM (DU145), 3.1 μM (PC-3), 1.8 μM (MV4-11) via MTT assay. Western blot revealed reduced phosphorylation of Pim substrates: p-Bad (Ser112: 65% reduction at 5 μM in DU145) and p-c-Myc (Thr58: 55% reduction at 5 μM in MV4-11) [1] - Apoptosis Induction: In MV4-11 cells, CX-6258 HCl (5 μM) treatment for 48 hours increased apoptotic cells from 4% (vehicle) to 38% via Annexin V-FITC/PI staining, with Western blot showing elevated cleaved caspase-3 (3.2-fold) and cleaved PARP (2.8-fold) [1] |
| ln Vivo |
In two Pim kinase-driven tumor models, CX-6258 hydrochloride (50–100 mg/kg; oral; once daily; 21 days) showed significant in vivo effectiveness [1].
Prostate Cancer Xenograft Model: Male nude mice (6 weeks old) bearing DU145 xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% hydroxypropyl methylcellulose + 0.1% Tween 80), CX-6258 HCl 25 mg/kg, CX-6258 HCl 50 mg/kg. The drug was administered orally once daily for 21 days. Tumor volume was reduced by 40% (25 mg/kg) and 65% (50 mg/kg) vs. vehicle; tumor weight decreased by 35% (25 mg/kg) and 60% (50 mg/kg). Immunohistochemistry of tumors showed reduced Ki-67 (proliferation marker: 55% reduction at 50 mg/kg) and p-Bad (60% reduction at 50 mg/kg) [1] |
| Enzyme Assay |
Radioactive Pan-Pim Kinase Inhibition Assay: Recombinant human Pim-1 (residues 44–313), Pim-2 (38–326), or Pim-3 (41–323) was incubated with a synthetic peptide substrate (RRRVSYRRR for Pim-1/3; RRRLSYRRR for Pim-2, 20 μM) and [γ-³²P]-ATP (10 μM, 3000 Ci/mmol) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄). Serial dilutions of CX-6258 HCl (0.001 nM–10 μM) were added, and the mixture was incubated at 30°C for 45 minutes. The reaction was stopped with 30% trichloroacetic acid (TCA), and precipitated peptides were transferred to P81 phosphocellulose filters. Filters were washed 3 times with 1% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC50 values were calculated using four-parameter logistic regression [1]
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: MV-4-11 human AML cells. Tested Concentrations: 0.1 μM, 1 μM, 10 μM. Incubation Duration: 2 hrs (hours). Experimental Results: Caused dose dependent inhibition of the phosphorylation of two pro-survival proteins, Bad and 4E-BP1, at the Pim kinase specific sites S112 and S65 and T37/46, respectively. MTT-Based Antiproliferation Assay: DU145/PC-3/MV4-11 cells were seeded in 96-well plates (5×10³ cells/well) and allowed to attach overnight. Serial dilutions of CX-6258 HCl (0.1 μM–20 μM) or vehicle (DMSO, 0.1%) were added, and cells were incubated at 37°C with 5% CO₂ for 72 hours. MTT reagent (5 mg/mL) was added (10 μL/well) for 4 hours; formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate cell viability and IC50 [1] - Apoptosis & Substrate Phosphorylation Assay: MV4-11 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with CX-6258 HCl (5 μM) for 48 hours. Cells were stained with Annexin V-FITC/PI for flow cytometry (apoptosis detection). For Western blot, cells were lysed, proteins separated by SDS-PAGE, and probed with anti-p-Bad, anti-p-c-Myc, anti-cleaved caspase-3, anti-cleaved PARP, and anti-GAPDH antibodies [1] |
| Animal Protocol |
Animal/Disease Models: Nude mice, MV-4-11 xenograft models[1]
Doses: 50 mg/kg, 100 mg/kg. Route of Administration: Oral administration; one time/day; over a period of 21 days. Experimental Results: demonstrated dose dependent efficacy, with a 50 mg/kg dose producing 45% tumor growth inhibition (TGI) and a 100 mg/kg dose producing 75% TGI. DU145 Prostate Cancer Xenograft Protocol: Male nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ DU145 cells. When tumors reached ~100 mm³, mice were grouped. CX-6258 HCl was dissolved in 0.5% hydroxypropyl methylcellulose + 0.1% Tween 80, administered orally once daily for 21 days (25 mg/kg or 50 mg/kg). Tumor volume (length × width² / 2) was measured every 3 days. On day 21, mice were euthanized; tumors were weighed and processed for immunohistochemistry (Ki-67, p-Bad staining) [1] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, the oral bioavailability of CX-6258 HCl (25 mg/kg) was 42%, the peak plasma concentration (Cmax) was 3.5 μM, the time to peak concentration (Tmax) was 1.2 h, and the terminal half-life (t₁/₂) was 5.8 h [1]. The clearance (CL) of CX-6258 HCl (5 mg/kg) administered intravenously to rats was 9.5 mL/min/kg, and the steady-state volume of distribution (Vss) was 1.1 L/kg [1]. The human plasma protein binding rate of CX-6258 HCl was 96% as determined by equilibrium dialysis [1].
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| Toxicity/Toxicokinetics |
In a 14-day repeated-dose study in male Sprague-Dawley rats, oral administration of CX-6258 HCl (up to 50 mg/kg/day) did not cause significant weight loss, clinical toxicity (e.g., somnolence, diarrhea), or abnormalities in serum ALT/AST/creatinine levels. Histological examination of the liver and kidneys showed no inflammation or necrosis [1]. Acute toxicity tests in male ICR mice showed that the oral median lethal dose (LD50) of CX-6258 HCl was >200 mg/kg, indicating low acute toxicity [1].
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| References |
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| Additional Infomation |
CX-6258 HCl is a potent, orally bioavailable pan-Pim kinase inhibitor that has been developed for the treatment of hematologic malignancies (e.g., acute myeloid leukemia) and solid tumors (e.g., prostate cancer) [1]. Its mechanism of action involves competitive binding to the ATP-binding pocket of Pim kinase, inhibiting the phosphorylation of downstream survival/proliferation substrates (p-Bad, p-Myc), thereby inducing apoptosis and inhibiting tumor growth [1].
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| Molecular Formula |
C26H24CLN3O3.HCL
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| Molecular Weight |
498.40
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| Exact Mass |
497.127
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| CAS # |
1353859-00-3
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| Related CAS # |
1202916-90-2;1353858-99-7 (hydrochloride hydrate)
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| PubChem CID |
72201040
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| Appearance |
Orange to red solid powder
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| LogP |
5.686
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
34
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| Complexity |
774
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1CCCN(CC1)C(=O)C2=CC=CC(=C2)C3=CC=C(O3)/C=C/4\C5=C(C=CC(=C5)Cl)NC4=O.Cl
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| InChi Key |
YYIMMVXTWBIEAG-YHLMHSEJSA-N
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| InChi Code |
InChI=1S/C26H24ClN3O3.ClH/c1-29-10-3-11-30(13-12-29)26(32)18-5-2-4-17(14-18)24-9-7-20(33-24)16-22-21-15-19(27)6-8-23(21)28-25(22)31;/h2,4-9,14-16H,3,10-13H2,1H3,(H,28,31);1H/b22-16+
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| Chemical Name |
(3E)-5-Chloro-3-[[5-[3-[(hexahydro-4-methyl-1H-1,4-diazepin-1-yl)carbonyl]phenyl]-2-furanyl]methylene]-1,3-dihydro-2H-indol-2-one hydrochloride
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
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, avoid exposure to moisture. |
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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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.02 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 25.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0064 mL | 10.0321 mL | 20.0642 mL | |
| 5 mM | 0.4013 mL | 2.0064 mL | 4.0128 mL | |
| 10 mM | 0.2006 mL | 1.0032 mL | 2.0064 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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