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| 25mg |
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Purity: ≥98%
ML241 HCl is a novel and potent p97 inhibitor which inhibits p97 ATPase with an IC50 value of 100 nM. In a dual-reporter cell line, ML241 restricted the breakdown of a p97-dependent proteasome substrate but not a p97-independent one. Additionally, ML241 hampered the endoplasmic reticulum-associated degradation pathway (ERAD). Because it promotes the proteasome's degradation of ubiquinated proteins and aids in the maturation of autophagosomes, AAA ATPase p97 is essential for preserving protein homeostasis in eukaryotic cells. There is a chance that ML241 can treat cancer.
| Targets |
p97 ATPase ( IC50 = 0.11 μM ); UbG76V–GFP ( IC50 = 3.5 μM )
ML241 hydrochloride is a strong p97 modulator with an IC50 value of 100 nM that inhibits p97 ATPase. With a Ki of 0.35 μM, ML241 hydrochloride inhibits p97 in relation to ATP. About 170 species of rest examined were not significantly inhibited by ML241 hydrochloride (20 μM). UbG76V-GFP is stabilized by ML241 hydrochloride, with an IC50 of 3.5 μM[1]. After 24 hours and 72 hours, respectively, the GI50s of ML241 hydrochloride on cytotoxic HCT15 and SW403 cells were 53 and 33 μM and 13 and 12 μM [2]. |
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| ln Vitro |
ML241 hydrochloride is a strong p97 modulator with an IC50 value of 100 nM that inhibits p97 ATPase. With a Ki of 0.35 μM, ML241 hydrochloride inhibits p97 in relation to ATP. About 170 species of rest examined were not significantly inhibited by ML241 hydrochloride (20 μM). UbG76V-GFP is stabilized by ML241 hydrochloride, with an IC50 of 3.5 μM[1]. After 24 hours and 72 hours, respectively, the GI50s of ML241 hydrochloride on cytotoxic HCT15 and SW403 cells were 53 and 33 μM and 13 and 12 μM [2].
ML241 inhibits p97 ATPase activity with an IC50 of approximately 0.1 μM and is competitive with ATP (Ki = 0.35 μM). It inhibits degradation of the p97-dependent reporter UbG76V–GFP with an IC50 of 3.5 μM, but is more than 10-fold less potent at stabilizing the p97-independent proteasome substrate ODD-Luc. ML241 impairs the endoplasmic-reticulum-associated degradation (ERAD) pathway, as shown by accumulation of TCRα-GFP and CFTR reporters, but does not induce accumulation of LC3-II, indicating no effect on autophagy. Unlike ML240, ML241 does not induce cleavage of PARP or activation of caspases 3 and 7, and exhibits weak antiproliferative activity in colon cancer cell lines HCT15 and SW403 (IC50 values in the range of 10–50 μM after 72 h). In the NCI-60 cell line panel, ML241 showed a slight decrease in overall growth rate but did not decrease tumor cell density. Kinase profiling revealed that ML241 (20 μM) did not appreciably inhibit labeling of any of the ~170 kinases tested. Binding affinity screening against 43 CNS-relevant targets showed that ML241 has significant binding affinity for only eight targets, with only three having Ki < 1 μM. |
| Enzyme Assay |
p97 ATPase inhibition assay: The assay was performed in a buffer containing 50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP, and 0.01% Triton X-100 to prevent compound colloid formation. ATPase activity was measured using a biomol green reagent after incubation with the compound.
Mechanism of inhibition was determined by evaluating ATP hydrolysis rates at different ATP concentrations, showing that ML241 inhibits p97 competitively with respect to ATP. |
| Cell Assay |
HeLa cells that are stable in their expression of ODD-luciferase are seeded (5000 cells/well) onto a 96-well white solid bottom plate and allowed to grow for 16 hours. Following a one-hour treatment with DMEM containing MG132 (4 μM), cells are twice washed with 100 μL PBS. In the well, cycloheximide (50 μg/mL), ML241, and DMEM containing 2.5% FBS are added. One of the four 96-well plates that have been prepared is removed from the incubator at each time interval (70, 90, 120, or 150 minutes). Each well holds 50 μL of medium. Luciferin (50 μL of 1 mg/mL in PBS) is added and incubated for 5 minutes at room temperature with 500 rpm shaking. The Synergy HT Microplate Reader uses an integration time of 0.1 ms to determine luminosity intensity[2].
UbG76V–GFP degradation assay: A dual-reporter stable HeLa cell line expressing UbG76V–GFP and ODD-Luc was used. Cells were treated with compounds, and reporter stabilization was measured. ODD-Luc degradation assay: Western blot analysis was performed in parallel to confirm that compounds did not interfere with luciferase activity measurement. Antiproliferative activity: Colon cancer cell lines (HCT15 and SW403) were treated with compounds for 24 or 72 h, and viability was assessed. Caspase activity assay: Caspase 3/7 activity was measured in cell extracts after treatment with compounds. Western blot analysis: Cells were fractionated into cytosolic, nuclear plus membrane, and insoluble fractions, and proteins were detected by immunoblotting. ERAD and autophagy pathway evaluation: Cells expressing TCRα-GFP, CFTR, or LC3 were treated with compounds, and protein levels were analyzed by immunoblotting. |
| ADME/Pharmacokinetics |
Water solubility: 28 μg/mL at pH 5.0, 0.13 μg/mL at pH 6.2, and 0.20 μg/mL at pH 7.4. PAMPA permeability: 1164 × 10⁻⁶ cm/s at pH 5.0 (containing 20% CH₃CN), 2504 × 10⁻⁶ cm/s at pH 6.2, and 2278 × 10⁻⁶ cm/s at pH 7.4. Plasma protein binding rate: >99.9% at concentrations of 1 μM and 10 μM in both human and mouse plasma. Plasma stability: 100% residue remained in both human and mouse plasma after 3 hours.
Liver microsomal stability: 18.9% of the liver microsomal residue remained in human plasma after 1 hour, and 2.5% remained in mouse plasma. |
| Toxicity/Toxicokinetics |
Hepatotoxicity: In Fa2N-4 immortalized human hepatocytes, LC50 > 50 μM. Off-target kinase activity: Minimal inhibition of approximately 170 kinases at a concentration of 20 μM. Central nervous system receptor binding: Significant binding affinity to 8 of 43 central nervous system targets, with only 3 having Ki < 1 μM.
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| References | |
| Additional Infomation |
ML241 is a probe compound developed through structure-activity relationship studies based on a quinazoline skeleton discovered in high-throughput screening (HTS). It is a highly potent and selective p97 ATPase inhibitor, but unlike its analogue ML240, it does not induce apoptosis or impair autophagy. ML241 exhibits a favorable off-target effect profile against kinases and central nervous system targets, making it an effective tool for studying the function of p97 in cellular processes.
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| Molecular Formula |
C23H25CLN4O
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| Molecular Weight |
408.93
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| Exact Mass |
408.171
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| Elemental Analysis |
C, 67.56; H, 6.16; Cl, 8.67; N, 13.70; O, 3.91
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| CAS # |
2070015-13-1
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| Related CAS # |
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| PubChem CID |
119081413
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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 |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
497
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| Defined Atom Stereocenter Count |
0
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| SMILES |
Cl.O1C2C=CC=CC=2N(CC1)C1=NC(=C2C(CCCC2)=N1)NCC1C=CC=CC=1
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| InChi Key |
DYHMHNNBOLCULH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H24N4O.ClH/c1-2-8-17(9-3-1)16-24-22-18-10-4-5-11-19(18)25-23(26-22)27-14-15-28-21-13-7-6-12-20(21)27;/h1-3,6-9,12-13H,4-5,10-11,14-16H2,(H,24,25,26);1H
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| Chemical Name |
N-benzyl-2-(2,3-dihydro-1,4-benzoxazin-4-yl)-5,6,7,8-tetrahydroquinazolin-4-amine;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 (6.11 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 (6.11 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 (6.11 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 | 2.4454 mL | 12.2270 mL | 24.4541 mL | |
| 5 mM | 0.4891 mL | 2.4454 mL | 4.8908 mL | |
| 10 mM | 0.2445 mL | 1.2227 mL | 2.4454 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.
ML240 and ML241 impair the endoplasmic-reticulum-associated degradation (ERAD) pathway, and only ML240 impairs the autophagy pathway and induces apoptosis. |
Michaelis–Menten plots for inhibition of p97 ATPase activity by a) ML240 and b) ML241.ChemMedChem.2013 Feb;8(2):297-312. |
ML240 induces activation of caspases 3 and 7 and apoptosis.ChemMedChem.2013 Feb;8(2):297-312. |
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