Impurity of Sacubitril
Neprilysin (NEP), a zinc-dependent metallopeptidase. Sacubitril is a potent NEP inhibitor that can be used for the research of heart failure. The (2S,4S)-isomer is an impurity and stereoisomer of sacubitril. Unlike the active sacubitril, this stereoisomer may have reduced or altered NEP inhibitory activity. It is primarily used as an analytical reference standard rather than a pharmacological tool.

(2S,4S)-Sacubitril is a stereoisomer produced from Sacubitril [1].

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In vitro studies of (2S,4S)-Sacubitril are primarily focused on its characterization as an impurity rather than its pharmacological activity. The compound's NEP inhibitory activity may be reduced compared to the active sacubitril stereoisomer due to the altered stereochemistry at the C-2 and C-4 positions. It is used as a reference standard in analytical method development and quality control assays for sacubitril-containing pharmaceutical products.

In vivo studies of (2S,4S)-Sacubitril are limited as the compound is used primarily as a reference standard and impurity rather than as a pharmacological agent. Its in vivo effects, if any, would likely be related to its potential NEP inhibitory activity, which may be reduced compared to the active sacubitril isomer. The compound is not used therapeutically and is not intended for in vivo pharmacological studies.

Sacubitril was recently approved by the Food and Drug Administration for use in combination with valsartan for the treatment of patients with heart failure with reduced ejection fraction. As a prodrug, sacubitril must be metabolized (hydrolyzed) to its active metabolite sacubitrilat (LBQ657) to exert its intended therapeutic effects. Thus, understanding the determinants of sacubitril activation will lead to the improvement of sacubitril pharmacotherapy. The objective of this study was to identify the enzyme(s) responsible for the activation of sacubitril, and determine the impact of genetic variation on sacubitril activation. First, an incubation study of sacubitril with human plasma and the S9 fractions of human liver, intestine, and kidney was conducted. Sacubitril was found to be activated by human liver S9 fractions only. Moreover, sacubitril activation was significantly inhibited by the carboxylesterase 1 (CES1) inhibitor bis-(p-nitrophenyl) phosphate in human liver S9. Further incubation studies with recombinant human CES1 and carboxylesterase 2 confirmed that sacubitril is a selective CES1 substrate. The in vitro study of cell lines transfected with wild-type CES1 and the CES1 variant G143E (rs71647871) demonstrated that G143E is a loss-of-function variant for sacubitril activation. Importantly, sacubitril activation was significantly impaired in human livers carrying the G143E variant. In conclusion, sacubitril is selectively activated by CES1 in human liver. The CES1 genetic variant G143E can significantly impair sacubitril activation. Therefore, CES1 genetic variants appear to be an important contributing factor to interindividual variability in sacubitril activation, and have the potential to serve as biomarkers to optimize sacubitril pharmacotherapy[1].

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In vitro enzyme assays for (2S,4S)-Sacubitril measure its inhibition of neprilysin (NEP) activity, if applicable. The assay uses recombinant human NEP and a fluorogenic substrate (e.g., Mca-RPPGFSAFK(Dnp)-OH). The compound is serially diluted in assay buffer and pre-incubated with the enzyme. The reaction is initiated by adding substrate, and fluorescence is measured over time at appropriate excitation/emission wavelengths (Ex/Em ~320/405 nm). IC50 values are calculated from dose-response curves. The (2S,4S)-isomer is typically compared to the active sacubitril isomer to assess stereochemical effects on NEP inhibition.

In vitro cell-based assays for (2S,4S)-Sacubitril are not typically performed as the compound is used as a reference standard rather than a pharmacological tool. If performed, cells expressing NEP or cells treated with NEP substrates could be used to assess the compound's effects on NEP activity. However, the primary use of this compound is in analytical chemistry applications including method development, method validation, and quality control.

In vivo animal studies for (2S,4S)-Sacubitril are not typically performed as the compound is used as an analytical reference standard. In vivo pharmacokinetic and pharmacodynamic studies are conducted with the active sacubitril isomer for heart failure research. The (2S,4S)-isomer impurity may be detected and quantified in biological samples as part of bioanalytical method development for sacubitril and its metabolites.

Pharmacokinetic properties of (2S,4S)-Sacubitril would be expected to be similar to those of sacubitril given their structural similarity. Sacubitril is a prodrug that is converted to its active form sacubitrilat, which inhibits NEP. The compound has a molecular weight of 411.49 and a molecular formula of C24H29NO5. Standard pharmacokinetic parameters are determined for sacubitril and its active metabolite in preclinical and clinical studies. The (2S,4S)-isomer is not used therapeutically.

Toxicological data for (2S,4S)-Sacubitril are not extensively documented as the compound is used as a reference standard and impurity. Toxicity studies are conducted with the active sacubitril isomer for its therapeutic use in heart failure. The (2S,4S)-isomer impurity is controlled in pharmaceutical products to ensure quality and safety. Standard safety precautions for handling chemical compounds apply.

[1]. Synthesis, Isolation, and Analysis of Stereoisomers of Sacubitril. Org. Process Res. Dev. 2019, 23, 1, 102-107.

[2]. Sacubitril Is Selectively Activated by Carboxylesterase 1 (CES1) in the Liver and the Activation Is Affected by CES1 Genetic Variation. Drug Metab Dispos. 2016 Apr;44(4):554-9.

(2S,4S)-Sacubitril is a research-grade reference standard and impurity of sacubitril. It is the (2S,4S)-stereoisomer of sacubitril and is used for analytical method development, method validation, and quality control during commercial production of sacubitril-containing pharmaceuticals. The compound has a molecular formula of C24H29NO5 and molecular weight of 411.49. Purity is typically ≥95%. Sacubitril is a potent NEP inhibitor used for the research of heart failure. Not approved for clinical use as a therapeutic agent.

C24H29NO5

411.490767240524

411.204

149709-63-7

Sacubitril;149709-62-6

122172973

Colorless to light yellow ointment

1.2±0.1 g/cm3

656.9±55.0 °C at 760 mmHg

351.1±31.5 °C

0.0±2.1 mmHg at 25°C

1.549

3.96

2

5

12

30

550

2

O(CC)C([C@@H](C)C[C@@H](CC1C=CC(C2C=CC=CC=2)=CC=1)NC(CCC(=O)O)=O)=O

PYNXFZCZUAOOQC-UWJYYQICSA-N

InChI=1S/C24H29NO5/c1-3-30-24(29)17(2)15-21(25-22(26)13-14-23(27)28)16-18-9-11-20(12-10-18)19-7-5-4-6-8-19/h4-12,17,21H,3,13-16H2,1-2H3,(H,25,26)(H,27,28)/t17-,21-/m0/s1 Create Date: 2016-10-08

4-[[(2S,4S)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoic acid

(2S,4S)-Sacubitril; 149709-63-7; 4-(((2S,4S)-1-([1,1'-Biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoic acid; 4-[[(2S,4S)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoic acid; [1,1'-Biphenyl]-4-pentanoic acid, gamma-[(3-carboxy-1-oxopropyl)amino]-alpha-methyl-, alpha-ethyl ester, (alphaS,gammaS)-; 3-{[(2S,4S)-1-{[1,1'-biphenyl]-4-yl}-5-ethoxy-4-methyl-5-oxopentan-2-yl]carbamoyl}propanoic acid; LCZ 696 Impurity C; UTU8FGY2BV;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)