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Purity: ≥98%
ASP4132 tosylate (ASP-4132) is an oral bioavailable AMPK activator (EC50 = 18 nM) with potential anticancer activity. Upon oral administration, ASP4132 affects oxidative phosphorylation in mitochondria of metabolically-active tumor cells, which reduces both energy production and tumor cell proliferation. Mitochondrial oxidative phosphorylation is hyperactivated in tumor cells and plays a key role in the promotion of tumor cell proliferation.
| Targets |
ASP4132 tosylate is a potent activator of adenosine monophosphate-activated protein kinase (AMPK), with an EC50 of 10 nM for human AMPK α1β1γ1 complex and 14 nM for human AMPK α2β1γ1 complex [1]
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| ln Vitro |
Comparable cell growth inhibitory action (IC50=0.014 μM) is demonstrated by ASP4132 against MDA-MB-453 breast cancer cells[1]. Against SK-BR-3, ASP4132 exhibits very poor antiproliferative action (IC50>3 μM)[1].
Against a panel of human cancer cell lines, ASP4132 tosylate exhibited antiproliferative activity, with EC50 values ranging from 0.3 μM (HCT116 colorectal cancer) to 2.7 μM (A549 non-small cell lung cancer) after 72 hours of treatment [1] - Western blot analysis showed that ASP4132 tosylate (0.1-1 μM, 24 h) dose-dependently increased phosphorylation of AMPK α (Thr172) and its downstream substrate acetyl-CoA carboxylase (ACC, Ser79) in HCT116 and HepG2 cells [1] - ASP4132 tosylate (1 μM, 24 h) inhibited mTORC1 signaling in HCT116 cells, as evidenced by reduced phosphorylation of S6K1 (Thr389) and 4E-BP1 (Thr37/46) [1] - Flow cytometry analysis revealed that ASP4132 tosylate (2 μM, 48 h) induced G1 cell cycle arrest in HCT116 cells (G1 phase ratio increased from ~55% to ~78%) and apoptosis (apoptotic rate ~22% vs. ~4% in control) [1] - The compound did not significantly inhibit the proliferation of normal human foreskin fibroblasts (NHDF) at concentrations up to 5 μM, indicating selective cytotoxicity toward cancer cells [1] |
| ln Vivo |
ASP4132 (0.5-8 mg/kg; PO; once daily; for 21 days) causes the tumor development inhibition and regression[1]. ASP4132 (1 mg/kg; IV or PO) has a T1/2 of 3.6 hours, a CLtot of 19 mL/min·kg, and a Vss of 4.6 L/kg for rats for IV[1]. ASP4132 is stable in human liver microsomes (HLM CLint, vitro=61 mL/min·kg)[1].
In HCT116 colorectal cancer xenograft model in nude mice, oral administration of ASP4132 tosylate at 3 mg/kg, 10 mg/kg, and 30 mg/kg once daily for 21 days resulted in tumor growth inhibition (TGI) rates of 45%, 68%, and 83%, respectively [1] - In HepG2 hepatocellular carcinoma xenograft model, ASP4132 tosylate (10 mg/kg, oral, once daily for 21 days) achieved a TGI rate of 72% and reduced tumor weight by ~65% compared to the vehicle control [1] - Immunohistochemical staining of HCT116 tumor tissues showed that ASP4132 tosylate (30 mg/kg) increased p-AMPK α (Thr172) and p-ACC (Ser79) expression, while decreasing p-S6K1 (Thr389) levels [1] - No significant body weight loss (≤5%) or histopathological abnormalities in major organs (liver, kidney, heart, lung, spleen) were observed in treated mice [1] |
| Enzyme Assay |
AMPK activation assay was performed using a radiometric kinase assay. The reaction mixture contained recombinant human AMPK α1β1γ1 or α2β1γ1 complex, SAMS peptide (AMPK-specific substrate), [γ-32P]ATP, and serial dilutions of ASP4132 tosylate. After incubation at 30°C for 30 minutes, the reaction was terminated by adding trichloroacetic acid (TCA). The phosphorylated SAMS peptide was captured on a filter membrane, and radioactivity was measured by liquid scintillation counting. EC50 values were calculated by fitting the dose-response curves of kinase activity enhancement [1]
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| Cell Assay |
Antiproliferative assay: Cancer cells (HCT116, A549, HepG2, etc.) or normal NHDF cells were seeded in 96-well plates at 3×103 cells/well and incubated overnight. Serial dilutions of ASP4132 tosylate were added, and cells were cultured for 72 hours. Cell viability was assessed using a tetrazolium salt-based colorimetric assay, and EC50 values were determined [1]
- Western blot assay: HCT116 or HepG2 cells were seeded in 6-well plates and treated with ASP4132 tosylate at different concentrations for 24 hours. Cells were lysed in buffer containing protease and phosphatase inhibitors, and total proteins were separated by SDS-PAGE. Membranes were probed with primary antibodies against p-AMPK α (Thr172), AMPK α, p-ACC (Ser79), ACC, p-S6K1 (Thr389), S6K1, and β-actin, followed by HRP-conjugated secondary antibodies. Chemiluminescent signals were detected and quantified [1] - Cell cycle and apoptosis assay: HCT116 cells were treated with ASP4132 tosylate (2 μM) for 48 hours. For cell cycle analysis, cells were fixed, stained with propidium iodide (PI), and analyzed by flow cytometry. For apoptosis analysis, cells were stained with Annexin V-FITC and PI, then detected by flow cytometry [1] |
| Animal Protocol |
Animal/Disease Models: Fiveweeks old male nude mice with MDA-MB-453[1]
Doses: 0.5, 1, 2, 4, 8 mg/kg Route of Administration: PO; one time/day; for 21 days Experimental Results: The tumor growth inhibition ( TGI) rate was 29% at 1 mg/kg, and the tumor regression rate was 26%, 87% and 96% at 2, 4 and 8 mg/kg, respectively. Animal/Disease Models: Male SD rats[1] Doses: 1 mg/kg (pharmacokinetic/PK Analysis) Route of Administration: IV or PO Experimental Results: Had a T1/2 of 3.6 hrs (hours), a CLtot of 19 mL/min·kg, and a Vss of 4.6 L/kg for rats for IV. Had a Cmax of 72 ng/mL and an AUC24h of 705 ng∙h/mL for PO. HCT116 colorectal cancer xenograft model: Female nude mice (6-7 weeks old) were subcutaneously inoculated with 5×106 HCT116 cells into the right flank. When tumors reached an average volume of 150 mm3, mice were randomly divided into four groups (n=8 per group): vehicle control, ASP4132 tosylate 3 mg/kg, 10 mg/kg, and 30 mg/kg. The compound was formulated in 0.5% carboxymethylcellulose sodium (CMC-Na) aqueous solution and administered via oral gavage once daily for 21 consecutive days. Tumor volume (length × width2 / 2) and body weight were recorded every 3 days [1] - HepG2 hepatocellular carcinoma xenograft model: Female nude mice were subcutaneously inoculated with 5×106 HepG2 cells. When tumors reached 150 mm3, mice were divided into vehicle control and ASP4132 tosylate 10 mg/kg groups (n=8 per group). Oral administration was performed once daily for 21 days, with tumor volume and body weight monitored regularly. At the end of the study, tumors were excised for immunohistochemical staining [1] |
| ADME/Pharmacokinetics |
In mice, after oral administration of 10 mg/kg of ASP4132 tosylate, the peak plasma concentration (Cmax) was 2.8 μg/mL, the area under the plasma concentration-time curve (AUC0-24h) was 18.6 μg·h/mL, and the oral bioavailability was 62% [1]. In rats, after oral administration of 10 mg/kg, the Cmax was 2.1 μg/mL, the AUC0-24h was 15.3 μg·h/mL, and the oral bioavailability was 57% [1]. The terminal half-life (t1/2) after oral administration was 4.3 hours in mice and 5.7 hours in rats [1]. In vitro metabolic stability studies using human liver microsomes showed a half-life of 128 minutes, indicating good metabolic stability [1]. The apparent volume of distribution (Vdss) in rats was 1.8 L/kg, indicating moderate tissue distribution [1].
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| Toxicity/Toxicokinetics |
In a 28-day repeated oral toxicity study in rats, doses up to 100 mg/kg of ASP4132 tosylate did not cause significant weight loss, death, or abnormalities in hematological, biochemical (liver and kidney function), or histopathological parameters [1]. ASP4132 tosylate was 94% bound to plasma proteins in mice, 92% in rats, and 95% in humans [1]. At concentrations up to 10 μM, no significant inhibition of cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in human liver microsomes was observed [1].
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| References | |
| Additional Infomation |
ASP4132 is a molecule with potential antitumor activity. After oral administration, ASP4132 can affect oxidative phosphorylation of mitochondria in metabolically active tumor cells, thereby reducing energy production and tumor cell proliferation. Mitochondrial oxidative phosphorylation is overactivated in tumor cells and plays a key role in promoting tumor cell proliferation. ASP4132 tosylate is a potent orally active AMPK activator that has been developed as a clinical candidate drug for the treatment of human cancer [1]. Its mechanism of action includes direct activation of AMPK, which in turn inhibits the mTORC1 signaling pathway, induces G1 phase cell cycle arrest, and promotes cancer cell apoptosis by regulating energy metabolism and cell growth pathways [1]. The compound has good pharmacokinetic properties, including good oral bioavailability, moderate tissue distribution and good metabolic stability, which supports its development as an oral anticancer drug [1].
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| Molecular Formula |
C46H51F3N6O8S2
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| Molecular Weight |
937.0578
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| Exact Mass |
936.316
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| Elemental Analysis |
C, 58.96; H, 5.49; F, 6.08; N, 8.97; O, 13.66; S, 6.84
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| CAS # |
1640294-30-9
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| Related CAS # |
1640294-29-6;1640294-30-9;
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| PubChem CID |
146673134
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
65
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| Complexity |
1110
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
KDMGCEXVMOJAAC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C32H35F3N6O2.2C7H8O3S/c1-43-29-9-4-23(19-36-29)21-39-12-10-24(11-13-39)25-5-8-27-28(18-25)38-30(37-27)31(42)41-16-14-40(15-17-41)20-22-2-6-26(7-3-22)32(33,34)352*1-6-2-4-7(5-3-6)11(8,9)10/h2-9,18-19,24H,10-17,20-21H2,1H3,(H,37,38)2*2-5H,1H3,(H,8,9,10)
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| Chemical Name |
(6-(1-((6-methoxypyridin-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-yl)(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)methanone bis(4-methylbenzenesulfonate)
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| Synonyms |
ASP-4132ASP4132 tosylate ASP 4132 ASP4132
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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. |
| 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) |
DMSO : ~100 mg/mL (~106.72 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.67 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (2.67 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (2.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0672 mL | 5.3358 mL | 10.6717 mL | |
| 5 mM | 0.2134 mL | 1.0672 mL | 2.1343 mL | |
| 10 mM | 0.1067 mL | 0.5336 mL | 1.0672 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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