Product name: Piperacillin EP Impurity N
Synonym name: L-Piperacillin
Catalogue No.: BPI012
Cas No.:
Formula: C23H27N5O7S
Mol Weight: 517.56
Botanical Source:
Type of Compound:
Purity: 95%~99%
Analysis Method: HPLC-DAD or/and HPLC-ELSD
Identification Method: Mass, NMR
Packing: Brown vial or HDPE plastic bottle
Storage: Store in a well closed container, protected from air and light. Put into refrigerate or freeze for long term storage.
Whenever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20℃. Generally, these will be useable for up to two weeks.
The product could be supplied from milligrams to grams
Inquire for bulk scale.
For Reference Standard and R&D, Not for Human Use Directly.
Storage conditions：Short-term storage at 2~8℃, long-term storage at -20 ~ -80℃
153.4000
.3000
-1.2000
No
.3500
No
Negative
Piperacillin EP impurity N, also known as L-piperacillin, is a specific optical isomer impurity that may occur during the production or storage of the broad-spectrum semi synthetic penicillin antibiotic - piperacillin. In the quality control system of the European Pharmacopoeia (EP), it is explicitly listed as one of the impurities that requires strict monitoring. Piperacillin itself is an important weapon in clinical anti infective treatment, especially with strong activity against Gram negative bacteria (such as Pseudomonas aeruginosa) and anaerobic bacteria. It is often used in combination with β - lactam inhibitors (such as tazobactam) to deal with complex hospital infections. As an impurity, the presence of piperacillin EP impurity N, although usually present in extremely small amounts, may affect the effectiveness, safety, and stability of the main drug. Therefore, in-depth research on it is an important link to ensure drug quality, understand drug degradation pathways, and explore new chemical entities.
From the perspective of medicinal chemistry, piperacillin belongs to acylpenicillin, and its molecular structure contains a D-configuration alpha amino group. The impurity N of piperacillin EP is speculated to be its corresponding L-type optical isomer. In biological systems, targets such as enzymes and receptors often exhibit high stereoselectivity, which means that D-configured and L-configured molecules may exhibit vastly different biological activities, metabolic pathways, and toxicity. Therefore, the study of L-piperacillin is not only related to the "quality control" proposition of impurity control, but also extends to the core field of "structure-activity relationship" in basic pharmacology. Although there is currently relatively limited detailed research literature on this impurity alone, based on its structural association with piperacillin and the commonality of penicillin drugs, we can conduct a systematic scientific analysis and prospect based on its known target information.
The exact molecular formula and molecular weight data of impurity N in piperacillin EP have not been disclosed yet, but as an optical isomer of piperacillin (C23H27N5O7S), its molecular formula should be the same, with a molecular weight of approximately 517.56 g/mol. Its core structure inevitably contains the iconic β - lactam ring of penicillin, which is a key pharmacophore that binds to bacterial targets and exerts antibacterial activity. The difference from piperacillin lies in the stereochemistry of the alpha carbon atom in the side chain: piperacillin is in the D-configuration, while impurity N is in the L-configuration.
This change in three-dimensional configuration, although not altering the atomic composition and connection order, can profoundly affect the three-dimensional spatial morphology of molecules, thereby altering their physicochemical properties. For example, key pharmacological parameters such as the polar surface area (TPSA) and lipid water partition coefficient (LogP) of molecules may undergo subtle but potentially biologically significant changes. Typically, the transport, recognition, and metabolism of L-configured amino acid derivatives in organisms may differ from that of D-configured derivatives. Piperacillin itself has amphiphilic properties (containing both acidic carboxyl groups and basic amino groups), and its LogP value is low, indicating strong hydrophilicity, which is beneficial for its distribution in body fluids but not conducive to penetrating cell lipid membranes. The conformational changes of impurity N may slightly affect its interaction with water molecules, thereby potentially altering its solubility and stability in different pH environments.
In addition, the β - lactam ring itself is sensitive to acids, bases, heat, and nucleophiles (such as hydroxyl groups), and is prone to ring opening reactions leading to deactivation. Impurity N, as a degradation product in the production process or storage, may be generated through racemization of chiral centers or other chemical transformations accompanied by instability of the β - lactam ring. Therefore, the confirmation of its structure and comprehensive characterization of its physicochemical properties (such as optical rotation, pKa, stability in different solvents and pH) are important aspects of pharmaceutical analytical chemistry and stability research.
It should be clarified that the impurity N in piperacillin EP does not directly originate from any plant. It is a chemical impurity generated entirely through chemical synthesis or semi synthetic processes, related to the synthesis of the antibiotic piperacillin. Therefore, it does not have a traditional history of medicinal plants or a background in folk applications.
However, tracing its' ancestors', we can turn our attention to the origin of penicillin - microorganisms. In 1928, Alexander Fleming discovered penicillin, which originated from Penicillium purpureum（Penicillium notatum）This fungus. This is the first β - lactam antibiotic discovered by humans from secondary metabolites of microorganisms in nature, ushering in a revolution in anti infective treatment. Natural penicillin (such as penicillin G) is extracted from fungal fermentation broth, but it has disadvantages such as narrow antibacterial spectrum, lack of acid and enzyme resistance.
Piperacillin is a semi synthetic product based on the chemical modification of penicillin core (6-aminopenicillanic acid, 6-APA). 6-APA was initially mass-produced through microbial fermentation, and then chemists introduced specific groups (such as piperazine formyl and ethyl in piperacillin) into its side chains, giving it a wider antibacterial spectrum, stronger anti pseudomonas activity, and slightly higher stability against β - lactases. Therefore, the "root" of piperacillin and its impurity N can be regarded as natural products of microorganisms, but it itself is the crystallization of modern pharmaceutical chemical engineering, reflecting the modern drug development path from natural products to structural optimization and synthetic manufacturing.
Although piperacillin EP impurity N is not expected to have significant antibacterial therapeutic effects as an impurity itself, its structure determines that it may still interact with a range of bacterial targets. The database information lists 7 relevant targets that are highly concentrated and specific towards bacterial cell wall synthesis, which is the core mechanism for all β - lactam antibiotics (including penicillins, cephalosporins, carbapenems, etc.) to exert bactericidal effects.
Core mechanism of action: Inhibit bacterial cell wall synthesis
Bacteria, especially Gram positive and Gram negative bacteria, have a tough peptidoglycan (also known as mucin) cell wall outside the cell membrane in order to maintain their morphology and resist internal high osmotic pressure. Peptidoglycan is a network structure formed by cross-linking short peptide side chains of polysaccharide chains (alternating between N-acetylglucosamine and N-acetylmuramic acid). The final step of its biosynthesis, cross-linking reaction, is catalyzed by a class of enzymes located on the cell membrane, which are collectively referred to as penicillin due to their ability to covalently bind with penicillin Penicillin binding protein。
PBPs have various enzymatic activities, mainly including:
1. Transpeptidase activity Catalyze the peptide bond cleavage between D-alanyl-D-alanine at the end of the pentapeptide side chain on a peptidoglycan chain, releasing one terminal D-alanine. At the same time, the acyl group formed after cleavage is connected to the amino group of the glycine pentapeptide bridge (or other amino acids) on another chain, forming cross-linking.Bacterial DD transpeptidase It is the key PBP for executing this function.
2. Carboxypeptidase activity Catalytic removal of D-alanine at the end of the pentapeptide side chain without crosslinking may play a role in regulating crosslinking density.
3. Transglycosyltransferase activity Catalytic elongation of polysaccharide chains.
The mode of action of piperacillin and its impurity N：
The β - lactam ring of piperacillin (D-configuration) is highly similar in spatial structure to the D-alanyl-D-alanine dipeptide at the end of the peptidoglycan pentapeptide side chain (a precursor for bacterial synthesis of peptidoglycan). When piperacillin enters the bacterial interstitium (Gram negative bacteria) or contacts the cell wall (Gram positive bacteria), its highly active β - lactam ring acts as a "pseudo substrate" and undergoes irreversible acylation with the serine residue in the active center of PBP, forming a stable covalent complex. This process is like a wrong key (piperacillin) jamming the lock (active center of PBP), permanently rendering it inactive.
After PBP is inhibited, the cross-linking reaction of bacterial cell walls is hindered. However, the balance between cell wall synthesis (polysaccharide chain extension) and autolytic enzyme (autolysin) activity is disrupted. In continuously growing bacteria, autolytic enzyme activity is relatively enhanced, leading to cell wall defects and fragility. Under the action of high internal osmotic pressure, water continuously infiltrates, and bacteria eventually expand, crack, and die Bactericidal effect。
Speculation on the Activity of N (L-configuration) Impurity in Piperacillin EP：
For impurity N, its key β - lactam ring still exists, so theoretically it can still react with the active serine of PBP. However, changes in the L-configuration of its side chains may have the following effects:
* Affinity and reaction rate The active center pocket of PBP evolved to recognize D-alanyl-D-alanine (D-configuration). The side chains of the L-configuration may not be able to perfectly match the specific binding sites of the active pocket, resulting in a significant decrease in their initial binding affinity (reversible complex formation) with PBP, which in turn affects the speed and efficiency of subsequent acylation reactions. Therefore, the antibacterial activity of impurity N is likely to be much lower than that of piperacillin, or even completely inactivated.
* Sensitivity to β - lactaseβ - lactase is a antibiotic resistant enzyme produced by bacteria, which can hydrolyze the β - lactam ring to inactivate it. Enzyme active centers also exhibit stereoselectivity. Impurity N may become a poor or ineffective substrate for β - lactase due to its different configurations, or it may unexpectedly become a weak inhibitor of the enzyme. This requires experimental verification.
* Association with related diseases The relevant diseases listed in the database (bacterial infections, respiratory infections, sepsis, urinary tract infections, abdominal infections, skin and soft tissue infections) are the indications for the clinical application of piperacillin. Impurity N itself is not used to treat these diseases, but if its content exceeds the standard in the drug, on the one hand, it may dilute the effective concentration of the main drug due to low activity; On the other hand, its potential metabolites or immunogenicity (risk of triggering allergic reactions) that are different from the main drug may affect the safety of medication. For example, penicillin impurities are sometimes associated with allergic reactions, and the allergenicity of impurities with different structures may vary.
The independent "pharmacological" evaluation of piperacillin EP impurity N is more meaningful in understanding its properties as a chemical entity from a medicinal chemistry perspective, rather than developing it into a new drug. We analyze based on known parameters of piperacillin and general rules for drug design.
Lipinski's Rule of Five This is a set of empirical rules used to predict the oral bioavailability of small molecules. Piperacillin (molecular weight~517.6) has exceeded the upper limit of "molecular weight<500"; Its structure contains multiple hydrogen bond donors (amino, amide NH) and acceptors (carboxyl, amide carbonyl, β - lactam carbonyl), and the number of hydrogen bond donors is likely to be greater than 5, and the number of hydrogen bond acceptors is greater than 10, which also exceeds the rule limit (donor<5, acceptor<10); Calculating LogP (cLogP) values is usually low (hydrophilic). Therefore, piperacillin itself does not comply with the Lipinski Five Rules, which is consistent with its actual administration method (intravenous or intramuscular injection) - it is not designed as an oral medication. As its isomer, impurity N also does not comply with the rules for oral medication.
Analysis of Key Medicinal Parameters：
* Topological polarity surface area The TPSA value of piperacillin is relatively high (usually>200 Å ²), which is the main reason for its strong hydrophilicity and difficulty in penetrating the blood-brain barrier (BBB). The configuration change of impurity N has little effect on TPSA, and it is expected to still have high TPSA characteristics.
* Blood-brain barrier penetrability The high TPSA, low fat solubility, and potential substrate properties of efflux transporters (such as P-glycoprotein) collectively determine that piperacillin and its impurity N are difficult to effectively penetrate the intact blood-brain barrier, and have limited efficacy in the treatment of central nervous system infections.
* Solubility and permeability Belongs to compounds with high solubility and low permeability (BCS classification III or IV). Need to rely on injection administration to achieve effective systemic exposure.
* Metabolism and toxicity As a β - lactam class, its main metabolic pathways may include β - lactam ring opening, side chain metabolism, etc. The difference in stereoisomers of impurity N may result in different metabolic rates and products compared to piperacillin, and potential toxicity (such as neurotoxicity and nephrotoxicity that may occur at extremely high doses) or immunogenicity needs to be specifically evaluated. The main safety concern of penicillin is allergic reactions, where impurities as haptens may bind to proteins and trigger immune responses.
Comprehensive Assessment Piperacillin EP impurity N does not possess the physicochemical properties to become an ideal oral medication. Its development value lies not in becoming a new antibacterial drug, but in serving as:
1. Quality control reference material It is necessary to strictly monitor its content in piperacillin raw materials and preparations to ensure the purity of the drug.
2. Metabolic and toxicological research reference standards Used to study the effects of different configurations on the fate and safety of drug substances.
3. Chemical and Biological Tool Molecules Used to study the selective mechanism of PBP enzyme towards substrate stereoconfiguration.
At present, public research on impurity N in piperacillin EP mainly focuses on the field of drug analysis. Pharmacopoeias of various countries (such as EP, USP) and Good Manufacturing Practice (GMP) require qualitative and quantitative control of known impurities in active pharmaceutical ingredients and formulations. Therefore, the research status of this impurity is mainly reflected in:
* Establishment of analytical methods Develop efficient and sensitive chromatographic methods (such as high-performance liquid chromatography (HPLC), especially chiral HPLC) to separate and quantitatively detect impurity N in piperacillin, ensuring its content is below the specified safety threshold (such as 0.1% or lower).
* Structural confirmation and characterization By means of mass spectrometry (MS), nuclear magnetic resonance (NMR), and optical rotation measurement, the chemical structure of L-piperacillin was confirmed, and its basic physicochemical properties were studied.
* Stability indicator method Study the conversion of piperacillin into degradation products such as impurity N under stress conditions such as light, high temperature, and high humidity, providing a basis for the formulation of drug packaging, storage conditions, and expiration dates.
There is a severe lack of publicly available literature on its independent and in-depth pharmacological, toxicological, or pharmacokinetic studies. This conforms to the conventional pattern of impurity research: the primary goal is to control risks rather than develop applications.
Future research directions and prospects：
1. In depth chiral pharmacology research Although the expected activity is low, through in vitro biochemical experiments (such as determining its inhibition constant Ki against purified PBP or β - lactase) and microbiological experiments (minimum inhibitory concentration MIC determination), the activity differences caused by changes in its stereoconfiguration can be accurately quantified, providing precise data for the study of antibiotic structure-activity relationships.
2. Comprehensive assessment of impurity safety Under the premise of ethical and regulatory compliance, conduct more comprehensive toxicology screening, especially immunotoxicology research, to assess the risk of potential allergens and provide a basis for developing more scientific impurity limits.
3. Potential roles in drug resistance research Explore whether this impurity can serve as a weak inhibitor or "bait" for β - lactase, interfering with the hydrolysis of the main drug by antibiotic resistant enzymes in bacteria. Although unlikely, it still holds value as a fundamental research.
4. Synthetic Chemistry and Process Optimization Studying its generation pathway (such as racemization conditions) can help optimize the synthesis and purification methods of piperacillin, reduce the production of this impurity from the source, and improve drug quality.
In summary, piperacillin EP impurity N is a typical drug-related impurity with clear structural characteristics. Studying it is the cornerstone of ensuring the safety and effectiveness of clinical medication, and also a unique window for a deeper understanding of the precise mechanism of action of β - lactam antibiotics. In today's increasingly severe antibiotic resistance, even research on impurities may bring new insights into the design and quality control of future antibiotics from unexpected perspectives.
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