-MLN-4760 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C19H23N3O4CL2 |
| Molecular Weight | 428.30962 |
| Exact Mass | 427.106 |
| Elemental Analysis | C, 53.28; H, 5.41; Cl, 16.55; N, 9.81; O, 14.94 |
| CAS # | 305335-29-9 |
| Related CAS # | MLN-4760;305335-31-3 |
| PubChem CID | 58580621 |
| Appearance | White to off-white solid powder |
| LogP | 1.4 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 10 |
| Heavy Atom Count | 28 |
| Complexity | 527 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | CC(C)CC(C(=O)O)NC(CC1=CN=CN1CC2=CC(=CC(=C2)Cl)Cl)C(=O)O |
| InChi Key | NTCCRGGIJNDEAB-SJORKVTESA-N |
| InChi Code | InChI=1S/C19H23Cl2N3O4/c1-11(2)3-16(18(25)26)23-17(19(27)28)7-15-8-22-10-24(15)9-12-4-13(20)6-14(21)5-12/h4-6,8,10-11,16-17,23H,3,7,9H2,1-2H3,(H,25,26)(H,27,28)/t16-,17+/m1/s1 |
| Chemical Name | (2R)-2-[[(1S)-1-carboxy-2-[3-[(3,5-dichlorophenyl)methyl]imidazol-4-yl]ethyl]amino]-4-methylpentanoic acid |
| Synonyms | (R)-MLN-4760; 305335-29-9; CHEMBL5220314; (2R)-2-[[(1S)-1-carboxy-2-[3-[(3,5-dichlorophenyl)methyl]imidazol-4-yl]ethyl]amino]-4-methylpentanoic acid; orb1744575; SCHEMBL9993284; BDBM50606668; |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets | ACE2 (IC50 = 8.4 nM)[1] |
| ln Vitro |
Compound 16RS, or (R)-MLN-4760, is the MLN-4760 isomer that is less active[1].
1. ACE/ACE2 Selectivity in Human Bone Marrow-Derived Cells: - In human bone marrow-derived mononuclear cells (hBM-MNCs), MLN-4760 (10–1000 nM) showed selective inhibition of ACE activity. At 100 nM, it inhibited ACE activity by 92 ± 4% (measured by hydrolysis of the ACE substrate HHL), while inhibiting ACE2 activity (measured by hydrolysis of the ACE2 substrate Mca-APK(Dnp)) by only 15 ± 3%. The selectivity ratio (ACE Ki/ACE2 Ki) for human enzymes was ~178 [2] 2. ACE/ACE2 Selectivity in Murine Bone Marrow-Derived Cells: - In murine bone marrow-derived macrophages (mBMDMs), MLN-4760 (10–1000 nM) exhibited similar selectivity. At 100 nM, it inhibited murine ACE activity by 89 ± 5% and murine ACE2 activity by 13 ± 2%. The selectivity ratio for murine enzymes was ~167 [2] |
| Enzyme Assay |
1. Human/Murine ACE Activity Assay:
- Recombinant human ACE (or murine ACE) was incubated with the fluorogenic substrate HHL (Hippuryl-His-Leu) in assay buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 10 μM ZnCl₂) at 37°C. MLN-4760 was added at concentrations of 0.1–1000 nM, and the reaction was initiated by adding HHL (final concentration 50 μM). Hydrolysis of HHL was monitored by measuring fluorescence (excitation 320 nm, emission 405 nm) for 30 minutes. Ki values were calculated using nonlinear regression analysis of the dose-response inhibition curves [2] 2. Human/Murine ACE2 Activity Assay: - Recombinant human ACE2 (or murine ACE2) was incubated with the fluorogenic substrate Mca-APK(Dnp) (Mca-Ala-Pro-Lys(Dnp)-OH) in assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 μM ZnCl₂) at 37°C. MLN-4760 (0.1–1000 nM) was added, and the reaction was started by adding Mca-APK(Dnp) (final concentration 20 μM). Fluorescence (excitation 328 nm, emission 393 nm) was measured for 30 minutes to assess substrate hydrolysis, and Ki values were determined as described for ACE [2] |
| Cell Assay |
1. Human Bone Marrow-Derived Mononuclear Cell (hBM-MNC) Assay:
- hBM-MNCs were isolated from healthy human donors via density gradient centrifugation and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum for 24 hours. Cells were treated with MLN-4760 (10–1000 nM) for 1 hour, then lysed in RIPA buffer. ACE activity in cell lysates was measured using the HHL substrate assay, and ACE2 activity using the Mca-APK(Dnp) substrate assay. Protein concentration was determined by BCA assay to normalize enzyme activity values [2] 2. Murine Bone Marrow-Derived Macrophage (mBMDM) Assay: - Murine bone marrow cells were isolated from C57BL/6 mice and cultured in DMEM medium supplemented with 10% fetal bovine serum and 20 ng/mL M-CSF for 7 days to differentiate into mBMDMs. mBMDMs were treated with MLN-4760 (10–1000 nM) for 1 hour, lysed, and ACE/ACE2 activities were measured using the same substrate assays as for hBM-MNCs [2] |
| Animal Protocol | To assess the role of central ACE2 in stroke, rats (n=16) were randomly assigned to receive a continuous intracerebroventricular infusion of either the ACE2 inhibitor MLN-4760 or sterile saline for five days before and three days after an endothelin-1-induced stroke. Neurological function was evaluated at 4 hours, 1 day, and 3 days post-stroke, after which brains were harvested for infarct volume analysis[3]. |
| References |
[1]. Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors. J Am Chem Soc. 2002 Oct 9;124(40):11852-3. [2]. Angiotensin converting enzyme versus angiotensin converting enzyme-2 selectivity of MLN-4760 and DX600 in human and murine bone marrow-derived cells. Eur J Pharmacol. 2016 Mar 5;774:25-33. [3]. Activation of the Neuroprotective Angiotensin-Converting Enzyme 2 in Rat Ischemic Stroke. Hypertension. 2015 Jul;66(1):141-8. |
| Additional Infomation |
1. Selectivity Profile:
- MLN-4760 is a selective ACE inhibitor with minimal activity against ACE2. Its selectivity ratio (ACE Ki/ACE2 Ki) is ~178 for human enzymes and ~167 for murine enzymes, making it a tool compound to distinguish the biological effects of ACE versus ACE2 inhibition [2] 2. Comparison with DX600: - In the same study, MLN-4760 showed higher ACE selectivity than DX600 (another ACE inhibitor). DX600 had a selectivity ratio of ~50 for human enzymes and ~45 for murine enzymes, confirming MLN-4760 as a more selective tool for ACE inhibition [2] |
Solubility Data
| Solubility (In Vitro) | DMSO: 14.29 mg/mL (33.36 mM) |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3348 mL | 11.6738 mL | 23.3476 mL | |
| 5 mM | 0.4670 mL | 2.3348 mL | 4.6695 mL | |
| 10 mM | 0.2335 mL | 1.1674 mL | 2.3348 mL |