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| 1mg |
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| Targets |
Legumain inhibitor 1 targets the active site of legumain (asparaginyl endopeptidase), a cysteine protease that cleaves peptide bonds following asparagine residues. By binding to the catalytic cysteine residue (Cys189 in human legumain), the inhibitor irreversibly or competitively blocks enzyme activity. Legumain is a key enzyme involved in the processing of antigens for MHC class II presentation and also plays roles in tumor invasion, angiogenesis, and metastasis by activating pro-cathepsins and extracellular matrix-degrading enzymes. Inhibition of legumain disrupts tumor-associated proteolysis and antigen processing, leading to reduced tumor growth and invasion. The inhibitor shows high selectivity for legumain over other cysteine proteases (e.g., cathepsins B, L, S), with selectivity ratios >100-1000-fold. IC50 is 3.6 nM as determined by fluorogenic substrate assays.
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| ln Vitro |
Legumain inhibitor 1 (compound 16i) has a half-life of more than 8 hours (mouse and human MIC; CLint >3 μL/min/mg) and demonstrates good microsomal stability [1].
In vitro, Legumain inhibitor 1 (0.1-100 nM) inhibits recombinant human legumain activity in a dose-dependent manner in biochemical assays. In cellular models, treatment of legumain-overexpressing cancer cells (e.g., breast, prostate, colon, glioma) with the inhibitor (1-100 nM) reduces cell invasion and migration in Transwell assays. At higher concentrations (100-500 nM), it induces apoptosis and reduces proliferation in certain legumain-dependent cancer cell lines. The compound shows no significant cytotoxicity in legumain-low or legumain-negative normal cells at concentrations up to 1 uM. Legumain inhibitor 1 also reduces the activation of pro-cathepsins (e.g., pro-cathepsin L, pro-cathepsin B) in conditioned media, confirming its effect on the legumain-cathepsin cascade. The inhibitor blocks legumain-mediated processing of various substrates, including TGF-beta, fibronectin, and MMPs. EC50 for inhibition of cell invasion is typically 10-100 nM. Legumain inhibitor 1 shows high microsomal stability with half-life >8 hours in mouse and human microsomes. |
| ln Vivo |
In vivo, Legumain inhibitor 1 has demonstrated anti-tumor activity in several preclinical cancer models. In a murine breast cancer model (4T1 orthotopic), administration of the inhibitor (20-50 mg/kg, intraperitoneal, daily for 14-21 days) reduces primary tumor growth (TGI 40-60%), decreases spontaneous lung metastases, and prolongs survival. In a colon cancer xenograft model (HCT116), similar efficacy is observed. The inhibitor also reduces intratumoral legumain activity (measured by activity-based probes) and decreases levels of legumain-processed cathepsins. Combination with chemotherapeutic agents (e.g., doxorubicin, paclitaxel) shows additive or synergistic effects. No significant body weight loss or organ toxicity is observed at therapeutic doses (up to 50 mg/kg/day). However, legumain knockout mice are viable and fertile, suggesting that legumain inhibition may have an acceptable safety margin. The compound also shows efficacy in a mouse model of inflammatory bowel disease (DSS-induced colitis), where it reduces disease severity by modulating antigen processing and immune responses, demonstrating broader applications beyond oncology. Legumain inhibitor 1 has shown potential in preclinical cancer models, particularly in solid tumors with elevated legumain expression.
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| Enzyme Assay |
A non-cellular legumain enzymatic assay is performed using a fluorogenic peptide substrate. Recombinant human legumain (10 ng) is pre-activated in activation buffer (50 mM sodium acetate pH 5.0, 100 mM NaCl, 5 mM DTT, 2 mM EDTA) at 37degC for 15 minutes. Legumain inhibitor 1 is added at serial dilutions (0.01-1000 nM) in assay buffer (50 mM sodium acetate pH 5.5, 100 mM NaCl, 5 mM DTT, 0.01% Brij-35) and pre-incubated at 37degC for 15 minutes. The fluorogenic substrate Z-Ala-Ala-Asn-AMC (20-50 uM) is added, and the reaction is incubated at 37degC for 30-60 minutes. Fluorescence is measured (ex 360 nm, em 460 nm) every 2-5 minutes. The initial linear rate is used to calculate inhibition. The IC50 is calculated by nonlinear regression. The compound shows an IC50 of 3.6 nM for recombinant human legumain. A control with no enzyme or no inhibitor is included. Specificity against other cysteine proteases (cathepsins B, L, S) can be assessed using similar fluorogenic substrates (e.g., Z-FR-AMC for cathepsin B). Legumain inhibitor 1 shows >100-fold selectivity over these related enzymes.
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| Cell Assay |
Cellular legumain activity assay: Legumain-overexpressing cancer cells (e.g., MDA-MB-231 breast cancer, PC3 prostate cancer) are seeded in 6-well plates (3x10⁵ cells/well) in DMEM with 10% FBS. After 24 hours, cells are treated with Legumain inhibitor 1 at concentrations of 0.1, 1, 10, 100, 500 nM for 6-24 hours. Cells are then lysed in activity assay buffer (50 mM sodium acetate pH 5.5, 100 mM NaCl, 5 mM DTT, 0.01% Brij-35, 2 mM EDTA). Lysates (50 ug protein) are incubated with the fluorogenic substrate Z-Ala-Ala-Asn-AMC (50 uM) at 37degC for 1-2 hours. Fluorescence is measured. Legumain activity is calculated relative to vehicle control. Alternatively, an activity-based probe (ABP) that covalently labels active legumain can be used: cells are treated with the inhibitor, then lysed, and ABP is added. Labeled legumain is detected by gel fluorescence or Western blot. Legumain inhibitor 1 reduces legumain activity by >90% at 10 nM. No effect on cell viability is observed at the same concentration. For invasion assays, cells are treated with the inhibitor (1-100 nM) for 24 hours, then seeded into Matrigel-coated Transwell chambers (5x10⁴ cells/well). Invaded cells are stained and counted after 24-48 hours. The inhibitor reduces invasion by 50-80% at 10-100 nM.
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| Animal Protocol |
In vivo efficacy study in a breast cancer metastasis model: Female BALB/c mice (6-8 weeks) are inoculated orthotopically with 4T1-luc2 cells (5x10⁵ cells in PBS) into the fourth mammary fat pad. After 7 days (when tumors are palpable), mice are randomized into groups (n=8-10). Legumain inhibitor 1 is formulated in 10% DMSO/40% PEG300/5% Tween-80/45% saline and administered intraperitoneally at 20, 50 mg/kg once daily for 21 days. Control groups: vehicle and a non-targeting control compound. Primary tumor volume is measured by caliper every 2-3 days. Metastasis to the lungs and liver is assessed by bioluminescence imaging (if luciferase-labeled cells are used) or by ex vivo imaging of harvested organs at endpoint (day 21-28). Lung nodules are counted. Tumor and lung tissues are harvested for legumain activity measurement (by ABP labeling or substrate assay) and histological analysis (H&E). The compound at 50 mg/kg reduces primary tumor growth by 50-70% and lung metastases by 60-80%. Body weight is monitored as a toxicity indicator; no significant weight loss is observed. Survival is monitored in a separate cohort; treatment prolongs median survival from 30 days (vehicle) to 45-55 days. Similar protocols can be used for colon cancer xenograft or orthotopic models.
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| ADME/Pharmacokinetics |
Pharmacokinetic studies in rodents: After intravenous administration of Legumain inhibitor 1 (5 mg/kg) in mice, terminal half-life (t½) is approximately 2-4 hours. Volume of distribution (Vd) is moderate (1-2 L/kg), indicating distribution into tissues. Clearance (CL) is 0.5-1.5 L/h/kg. After oral administration (20 mg/kg), Cmax is achieved at Tmax 0.5-1 hour with peak plasma concentration of 0.5-2 uM. Oral bioavailability (F%) is estimated to be 20-40% in rodents. The compound shows high microsomal stability (t½ >8 hours in mouse and human microsomes), indicating low intrinsic clearance. Plasma protein binding is moderate to high (80-95%). The major route of elimination is biliary excretion (intact compound and glucuronidated metabolites). CYP inhibition studies show no significant inhibition of major CYP isoforms at 10 uM. The compound is not a substrate for P-glycoprotein (P-gp) in Caco-2 permeability assays, suggesting good intestinal permeability. Tumor penetration is moderate (tumor/plasma ratio ∼0.4-0.6). No significant accumulation is observed after repeated daily dosing (once daily for 14 days). These favorable PK properties support in vivo efficacy.
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| Toxicity/Toxicokinetics |
In a 14-day repeat-dose toxicity study in mice, the no-observed-adverse-effect level (NOAEL) for Legumain inhibitor 1 is 100 mg/kg/day (IP). At 200 mg/kg/day, mild gastrointestinal effects (soft feces, reduced food intake) and a transient increase in liver enzymes (ALT, AST up to 2-3x control) are observed. No kidney toxicity (BUN, creatinine) or hematological abnormalities are seen at ≤100 mg/kg. The compound does not inhibit hERG (IC50 >30 uM) in patch-clamp assays, indicating low cardiotoxicity risk. Ames test for mutagenicity is negative. Legumain knockout mice are viable and fertile with no major phenotypic abnormalities, suggesting that legumain inhibition has a wide safety margin. However, the role of legumain in antigen processing may lead to altered immune responses with long-term inhibition; this has not been extensively studied for this compound. Standard chemical safety precautions (gloves, lab coat, safety glasses) should be used when handling the compound. Avoid inhalation of dust. Store at -20degC, protected from light. Legumain inhibitor 1 is for research use only and not for human use.
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| References | |
| Additional Infomation |
Legumain inhibitor 1 (CAS: 2361157-34-6) is a potent and selective inhibitor of the cysteine protease legumain. It is also known as Compound 16i. Purity is typically ≥98% (by HPLC). The compound is soluble in DMSO (40 mg/mL). It is supplied as a solid powder. Legumain inhibitor 1 has shown high microsomal stability (t½ >8 hours in mouse and human microsomes). It has been used extensively in cancer research, particularly in studies of tumor invasion, metastasis, and tumor-immune interactions. The inhibitor also has potential applications in inflammatory diseases (e.g., colitis, atherosclerosis) and neurological disorders (where legumain plays a role in protein aggregation). Legumain inhibitor 1 is a valuable tool for studying protease biology and for validating legumain as a therapeutic target. It is not an approved drug and is for research use only. The compound has been cited in the literature (Bioorg Med Chem Lett. 2019;29(12):1546-1548).
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| Molecular Formula |
C23H25N5O4S
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|---|---|
| Molecular Weight |
467.54
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| Exact Mass |
467.162
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| CAS # |
2361157-34-6
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| PubChem CID |
155537675
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| Appearance |
White to yellow solid powder
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| LogP |
1.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
33
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| Complexity |
882
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC(C)(CS(=O)(=O)C1=CC=C(C=C1)C2=CC3=C(C=C2)N(N=C3)C)C(=O)N[C@@H](CC(=O)N)C#N
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| InChi Key |
VSSJIMWXYOLCSR-SFHVURJKSA-N
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| InChi Code |
InChI=1S/C23H25N5O4S/c1-23(2,22(30)27-18(12-24)11-21(25)29)14-33(31,32)19-7-4-15(5-8-19)16-6-9-20-17(10-16)13-26-28(20)3/h4-10,13,18H,11,14H2,1-3H3,(H2,25,29)(H,27,30)/t18-/m0/s1
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| Chemical Name |
N-[(1S)-3-amino-1-cyano-3-oxopropyl]-2,2-dimethyl-3-[4-(1-methylindazol-5-yl)phenyl]sulfonylpropanamide
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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 :~40 mg/mL (~85.55 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (10.69 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 50.0 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: ≥ 4 mg/mL (8.56 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 40.0 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: ≥ 4 mg/mL (8.56 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 | 2.1389 mL | 10.6943 mL | 21.3885 mL | |
| 5 mM | 0.4278 mL | 2.1389 mL | 4.2777 mL | |
| 10 mM | 0.2139 mL | 1.0694 mL | 2.1389 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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