| Size | Price | Stock | Qty |
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| 5mg |
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| Other Sizes |
| Targets |
AEP-IN-3 targets asparagine endopeptidase (AEP, also known as legumain), a lysosomal cysteine protease that cleaves proteins after asparagine residues. In Alzheimer's disease, AEP is responsible for the cleavage of amyloid precursor protein (APP) to generate aggregation-prone Aβ fragments and the cleavage of tau to generate neurotoxic tau fragments that promote neurofibrillary tangle formation. By potently inhibiting AEP with an IC50 of 7.8 nM, AEP-IN-3 prevents the generation of these pathogenic protein fragments.
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| ln Vitro |
AEP-IN-3 shows exceptionally potent in vitro activity against AEP, with an IC50 of 7.8 ± 0.9 nM. This high potency makes it one of the most effective AEP inhibitors described in the literature. The compound's brain penetration enables direct assessment of AEP inhibition in the central nervous system. In cellular models, AEP-IN-3 effectively blocks AEP-mediated cleavage of APP and tau, reducing the production of Aβ peptides and pathogenic tau fragments.
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| ln Vivo |
The bioavailability of AEP-IN-3 (compound 18) is 83% with a T1/2[1]. AEP-IN-3 (20 mg/kg, oral, BID, for 5 days) showed significant inhibition of AEP activity in the brain of TauP301L transgenic mice[1].
In vivo activity of AEP-IN-3 has been demonstrated in transgenic mouse models of Alzheimer's disease. In FVB/N genetic background P301L mutant human tau 2N4R isoform transgenic mice (3.8 months old), AEP-IN-3 (oral administration at 20 mg/kg, twice daily with 7–8 hour intervals, for 5 consecutive days) significantly inhibited AEP activity in the brain and reduced the formation of Tau N368 fragments. The compound exhibits an oral bioavailability of 83%, supporting convenient oral administration. |
| Enzyme Assay |
Non-cell-based enzyme assays for AEP-IN-3 typically involve measuring the inhibition of recombinant AEP activity using fluorogenic peptide substrates. A standard protocol includes incubating purified AEP enzyme with a fluorogenic substrate (such as Z-Ala-Ala-Asn-AMC) in assay buffer (pH 5.5–6.0) at 37°C. AEP-IN-3 is added at various concentrations (typically 0.001–100 nM), and the release of fluorescent AMC is monitored over time using a fluorescence plate reader. IC50 values are calculated from dose-response curves, and the mode of inhibition can be determined by kinetic analysis.
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| Cell Assay |
Cellular assays for AEP-IN-3 typically involve neuronal cell lines or primary neurons expressing APP and tau. A representative protocol includes culturing cells in appropriate medium, treating with AEP-IN-3 at various concentrations (0.001–100 nM) for 24–72 hours, and measuring AEP activity using a cell-permeable fluorogenic substrate. Aβ40 and Aβ42 levels in the conditioned medium are quantified by ELISA, and p-Tau levels are assessed by Western blot using phosphorylation-specific antibodies. Cell viability is assessed to ensure that observed effects are not due to cytotoxicity.
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| Animal Protocol |
Animal/Disease Models: Mice producing the 2N4R isoform of human Tau containing the P301L mutation in a FVB/N genetic background (3.8 months old)[1]
Doses: 20 mg/kg Route of Administration: Orally, BID with an interval of 7-8 h between doses for 5 days Experimental Results: Showed significantly inhibited activity of AEP in brain, reduced formation of the Tau N368 fragment. The effect on total Tau is not significant. In vivo animal studies with AEP-IN-3 are conducted in transgenic mouse models of Alzheimer's disease, such as P301L tau transgenic mice. A typical protocol involves administering AEP-IN-3 via oral gavage at 20 mg/kg, twice daily (with 7–8 hour intervals), for 5 consecutive days. At the end of the treatment period, brain tissues are harvested for analysis of AEP activity, Tau N368 fragment levels, and total tau levels by Western blot or ELISA. Pharmacodynamic endpoints include measurement of AEP activity in brain homogenates using a fluorogenic substrate. |
| ADME/Pharmacokinetics |
Pharmacokinetic properties of AEP-IN-3 have been well characterized, with an oral bioavailability of 83%. The compound's molecular weight of 455.4 g/mol and calculated lipophilicity support its ability to cross the blood-brain barrier. The half-life (T1/2) has been determined from pharmacokinetic studies. The compound is stored as a powder at -20°C for up to 3 years and in solvent at -80°C for 1 year. Brain penetration is a critical parameter for Alzheimer's disease therapeutics, and AEP-IN-3 has been specifically designed to achieve this property.
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| Toxicity/Toxicokinetics |
As a research compound, AEP-IN-3 is not intended for human therapeutic use, and comprehensive toxicological data are limited to preclinical studies. Standard safety assessments would include cytotoxicity screening in neuronal and non-neuronal cell lines, hERG channel inhibition testing, and preliminary toxicology studies in animal models to determine maximum tolerated dose and identify potential target organs of toxicity. The compound is stored as a powder at -20°C for up to 3 years.
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| References | |
| Additional Infomation |
AEP-IN-3 (Compound 18) is a highly potent AEP inhibitor with an IC50 of 7.8 nM, making it a valuable research tool for studying the role of asparagine endopeptidase in Alzheimer's disease. Its high oral bioavailability (83%) and brain penetration enable robust in vivo studies in transgenic mouse models. The compound is referenced in the primary literature for its potent AEP inhibitory activity and its ability to reduce Tau N368 fragment formation in vivo. AEP-IN-3 represents a promising tool compound for investigating the therapeutic potential of AEP inhibition in Alzheimer's disease.
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| Exact Mass |
455.147
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| CAS # |
2978521-26-3
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| PubChem CID |
169171740
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
32
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| Complexity |
797
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| Defined Atom Stereocenter Count |
3
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| SMILES |
C#C[C@H](CC(=O)N)NC(=O)[C@@H]1C[C@H](CN1C(=O)C2(CC2)C3=CC=C(C=C3)OC(F)(F)F)F
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| InChi Key |
WXTJCPHELGTTJU-FMKPAKJESA-N
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| InChi Code |
InChI=1S/C21H21F4N3O4/c1-2-14(10-17(26)29)27-18(30)16-9-13(22)11-28(16)19(31)20(7-8-20)12-3-5-15(6-4-12)32-21(23,24)25/h1,3-6,13-14,16H,7-11H2,(H2,26,29)(H,27,30)/t13-,14-,16+/m1/s1
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| Chemical Name |
(2S,4R)-N-[(3S)-5-amino-5-oxopent-1-yn-3-yl]-4-fluoro-1-[1-[4-(trifluoromethoxy)phenyl]cyclopropanecarbonyl]pyrrolidine-2-carboxamide
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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 |
| 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 (~219.59 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.49 mM) 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; with ultrasonication.
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 and add it to 400 μL PEG300 and mix well. Then add 50 μL Tween-80 to the above system and mix well. Then continue to add 450 μL of physiological saline to make up 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 (5.49 mM) 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; with ultrasonication. 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 and add it to 900 μL of 20% SBE-β-CD saline solution and mix well. 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 (5.49 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one),clear solution; with ultrasonication. |
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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