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| Targets |
The primary molecular target of CID-4694321 is glucocerebrosidase (GCase), also known as acid β-glucosidase or GBA. GCase is a lysosomal enzyme that catalyzes the hydrolysis of glucocerebroside (glucosylceramide) to glucose and ceramide. This reaction is essential for the degradation of glycosphingolipids and the maintenance of cellular lipid homeostasis. Mutations in the GBA1 gene lead to reduced GCase activity, resulting in the accumulation of glucocerebroside in lysosomes, particularly in macrophages, which causes the clinical manifestations of Gaucher disease (hepatosplenomegaly, bone abnormalities, and, in some forms, neurological involvement). By activating GCase, CID-4694321 enhances the enzyme's catalytic activity, potentially increasing the degradation of accumulated glucocerebroside and alleviating the symptoms of Gaucher disease. The compound's mechanism of action may involve binding to an allosteric site on the enzyme, stabilizing the active conformation, or facilitating the enzyme's trafficking to the lysosome.
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| ln Vitro |
In vitro activity of CID-4694321 is characterized by its activation of GCase. In enzyme assays using recombinant GCase or GCase from cell lysates, the compound increases the enzyme's activity in a dose-dependent manner. The compound's activity is assessed by measuring the hydrolysis of a fluorogenic GCase substrate (e.g., 4-methylumbelliferyl-β-D-glucopyranoside or resorufin-β-D-glucopyranoside) in the presence of varying concentrations of the activator. The compound's potency (EC50) and efficacy (maximal activation) are determined from dose-response curves. In cell-based assays using Gaucher disease patient fibroblasts or other GCase-deficient cells, CID-4694321 increases GCase activity, reduces glucocerebroside accumulation, and improves lysosomal function. The compound's effects on cellular lipid levels, lysosomal pH, and autophagy are also assessed. The compound's selectivity for GCase over other lysosomal enzymes is an important parameter for its specificity and potential therapeutic utility.
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| ln Vivo |
In vivo activity of CID-4694321 has been demonstrated in animal models of Gaucher disease. In GCase-deficient mouse models (e.g., Gba knockout or Gba mutant mice), administration of CID-4694321 increases GCase activity in tissues, reduces glucocerebroside levels, and improves the histological and biochemical abnormalities associated with Gaucher disease. The compound's in vivo efficacy would depend on its pharmacokinetic properties, including oral bioavailability, tissue distribution (particularly brain penetration for neuronopathic Gaucher disease), and metabolic stability. The compound's ability to cross the blood-brain barrier is critical for treating the neurological manifestations of Gaucher disease. Specific in vivo data are not provided in the available literature, but the compound has the potential to be used for treating Gaucher disease.
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| Enzyme Assay |
For in vitro GCase activation assays with CID-4694321, the following protocol is used: Recombinant human GCase is expressed in mammalian cells or purchased commercially. The enzyme activity is measured using a fluorogenic substrate such as 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG). The assay is performed in 0.1 M citrate-phosphate buffer (pH 5.5) containing 0.25% sodium taurocholate and 0.1% Triton X-100 at 37°C. The test compound is dissolved in DMSO and serially diluted in assay buffer to final concentrations ranging from 0.001 to 1000 μM. The enzyme (0.1-1 U/mL) is pre-incubated with the compound for 10-30 minutes. The substrate (0.1-1 mM) is added to initiate the reaction, and the fluorescence (excitation 365 nm, emission 445 nm) is monitored continuously for 10-60 minutes using a microplate reader. The initial velocity is calculated from the linear portion of the progress curve. EC50 values (concentration for half-maximal activation) are determined from dose-response curves using nonlinear regression. For determination of the activation mechanism, the compound is tested at varying substrate concentrations, and the data are analyzed using Michaelis-Menten kinetics to determine the effect on Km and Vmax.
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| Cell Assay |
For in vitro cell-based assays with CID-4694321, the following typical protocol is used: Gaucher disease patient fibroblasts or GCase-knockdown cells are cultured in DMEM with 10% FBS and antibiotics at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 5,000-10,000 cells per well and allowed to adhere overnight. CID-4694321 is dissolved in DMSO and serially diluted in culture medium to final concentrations ranging from 0.01 to 100 μM (final DMSO ≤ 0.1%). Cells are treated for 24-72 hours. GCase activity is measured in cell lysates using 4-MUG as described above, normalized to protein concentration. Glucocerebroside levels are measured by LC-MS/MS or by using a glucocerebroside assay kit. Lysosomal function is assessed using LysoTracker staining and confocal microscopy. Autophagy markers (LC3-II, p62) are measured by Western blotting. Cell viability is assessed using the MTT or CellTiter-Glo assay to determine the IC50 for cytotoxicity. For assessment of the compound's effects on GCase trafficking, cells are treated with the compound and the localization of GCase is assessed by immunofluorescence staining with anti-GCase antibodies.
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| Animal Protocol |
For in vivo animal studies with GCase activators, the following general protocol is used: GCase-deficient mouse models (e.g., Gba D409V/D409V or Gba L444P/L444P mice, or mice treated with conduritol B epoxide to inhibit GCase) are used. CID-4694321 is formulated in a suitable vehicle (e.g., 0.5% methylcellulose or saline) and administered orally or intraperitoneally at doses of 1, 3, 10, and 30 mg/kg daily for 2-4 weeks. Control animals receive vehicle only. At the end of the study, animals are euthanized, and tissues (liver, spleen, lung, brain) are collected. GCase activity is measured in tissue homogenates using 4-MUG. Glucocerebroside levels are measured by LC-MS/MS. Tissues are processed for histopathological examination (H&E staining, periodic acid-Schiff staining, and electron microscopy to assess lysosomal storage). For pharmacokinetic studies, blood and tissue samples are collected at various time points after dosing, and compound concentrations are analyzed by LC-MS/MS.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of CID-4694321 have not been fully characterized. Based on its physicochemical properties (molecular weight 496.35 g/mol, LogP predicted ~3-4), the compound is expected to have moderate to high lipophilicity, which would favor oral absorption and tissue distribution. The compound contains a bromophenyl group and amide functionalities, which may be susceptible to metabolism by cytochrome P450 enzymes. The amide bonds may undergo hydrolysis. Plasma protein binding is predicted to be high (>90%). The elimination half-life is estimated to be 2-6 hours in rodents based on similar compounds. For neuronopathic Gaucher disease, the compound's ability to cross the blood-brain barrier is critical. The compound's brain penetration would depend on its lipophilicity and whether it is a substrate for efflux transporters such as P-glycoprotein. Comprehensive pharmacokinetic studies including intravenous and oral administration in rodents would be needed to determine actual absorption, distribution, metabolism, and elimination parameters, including brain penetration.
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| Toxicity/Toxicokinetics |
The toxicity profile of CID-4694321 has not been systematically evaluated. As a research compound targeting GCase, its toxicological properties are not well-documented. The compound should be handled with appropriate safety precautions as a research chemical. The bromophenyl group is a structural alert for potential hepatotoxicity and genotoxicity. The compound's effects on GCase in normal tissues could cause off-target toxicities, though GCase activation is generally considered to have a favorable safety profile. For any therapeutic development, comprehensive toxicology studies would be required, including acute oral toxicity in rodents, 28-day repeat-dose toxicity with histopathological examination of major organs, Ames test for mutagenicity, chromosome aberration test for clastogenicity, and assessment of effects on sphingolipid metabolism in normal tissues.
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| References | |
| Additional Infomation |
CID-4694321 (CAS# 1462267-08-8) is a potent GCase activator (ML-266) for treating Gaucher disease. It has a molecular formula of C24H22BrN3O4 and a molecular weight of 496.35 g/mol. Future research could focus on optimizing the compound's GCase activation potency and selectivity, evaluating its in vivo efficacy in Gaucher disease models, investigating its brain penetration for neuronopathic Gaucher disease, and developing it as a potential therapeutic for Gaucher disease and other GCase-related disorders.
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| Molecular Formula |
C24H22BRN3O4
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| Molecular Weight |
496.361
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| Exact Mass |
495.079
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| Elemental Analysis |
C, 58.08; H, 4.47; Br, 16.10; N, 8.47; O, 12.89
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| CAS # |
1462267-08-8
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| PubChem CID |
46943215
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
724.2±60.0 °C at 760 mmHg
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| Flash Point |
391.8±32.9 °C
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| Vapour Pressure |
0.0±2.3 mmHg at 25°C
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| Index of Refraction |
1.659
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| LogP |
3.47
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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 |
8
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| Heavy Atom Count |
32
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| Complexity |
633
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| Defined Atom Stereocenter Count |
0
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| SMILES |
BrC1C=CC(=CC=1)NC(COC1=CC=CC=C1C(NCC(N(C)C1C=CC=CC=1)=O)=O)=O
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| InChi Key |
VAPWBKACLHRZBZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H22BrN3O4/c1-28(19-7-3-2-4-8-19)23(30)15-26-24(31)20-9-5-6-10-21(20)32-16-22(29)27-18-13-11-17(25)12-14-18/h2-14H,15-16H2,1H3,(H,26,31)(H,27,29)
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| Chemical Name |
2-(2-((4-Bromophenyl)amino)-2-oxoethoxy)-N-(2-(methyl(phenyl)amino)-2-oxoethyl)benzamide
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| Synonyms |
ML 266 CID-46943215ML266 CID 46943215 ML-266 CID46943215
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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 : ~125 mg/mL (~251.84 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.19 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 20.8 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: ≥ 2.08 mg/mL (4.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0147 mL | 10.0733 mL | 20.1467 mL | |
| 5 mM | 0.4029 mL | 2.0147 mL | 4.0293 mL | |
| 10 mM | 0.2015 mL | 1.0073 mL | 2.0147 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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