Albendazole-D3

Stepwise Synthetic Method for Albendazole-D3

Hydrolysis of Albendazole

• Objective: Remove methyl formate groups from the 2-amino position of albendazole to generate a reactive intermediate (referred to as compound 1).
• Conditions:
  • Albendazole is dissolved in a mixed solvent system of a polar solvent and water.
  • Polar solvents used include methanol, ethanol, or tetrahydrofuran.
  • An alkaline environment is created using bases such as sodium hydroxide, potassium hydroxide, or lithium hydroxide.
  • Reaction temperature is maintained between 10°C and 50°C.
  • Reaction time ranges from 4 to 24 hours.
• Process: The reaction mixture is heated and refluxed until the albendazole raw material is consumed. The mixture is then cooled, concentrated under reduced pressure to remove most of the polar solvent, filtered, washed with water and dichloromethane, and dried to isolate compound 1.

Formation of this compound via Deuterated Methylation

• Objective: Attach deuterated methyl formate groups to the amino group at the 2-position of compound 1.
• Reagents and Solvents:
  • Compound 1
  • Methyl deuterated chloroformate (deuterated methyl chloroformate)
  • Sodium bicarbonate
  • Aprotic solvent such as acetone
• Reaction Conditions:
  • The molar feed ratio of compound 1 to methyl deuterated chloroformate to sodium bicarbonate ranges from 1:0.5:3 to 1:5:3.
  • The reaction mixture is stirred at room temperature after slow addition of methyl deuterated chloroformate.
  • Thin Layer Chromatography (TLC) and Liquid Chromatography-Mass Spectrometry (LC-MS) are used to monitor the reaction progress.
  • After completion, the mixture is heated and refluxed for 12 to 16 hours.
  • The reaction mixture is cooled, filtered, washed sequentially with water, dichloromethane, and methanol, and then dried to obtain this compound.

Reaction Pathways

Two reaction pathways have been identified depending on the molar ratio of methyl deuterated chloroformate:

This flexibility in reaction pathways allows optimization based on reagent ratios to maximize yield and purity.

Summary Table of Key Reaction Parameters

Research Findings and Analytical Considerations

• The synthetic method using albendazole as raw material is advantageous for industrial scale production due to availability and cost-effectiveness.
• The use of aprotic solvents and controlled molar ratios ensures selective deuterium incorporation.
• Monitoring by TLC and LC-MS is critical to track reaction progress and confirm the formation of this compound.
• The reaction temperature and time are optimized to balance reaction completion and minimize side reactions.
• The washing steps with water, dichloromethane, and methanol remove impurities and unreacted reagents, ensuring high purity of the final product.

Types of Reactions

Albendazole-D3 undergoes various chemical reactions, including:

Oxidation: The compound can be oxidized to form sulfoxides and sulfones.

Reduction: Reduction reactions can convert this compound to its corresponding amine derivatives.

Substitution: Nucleophilic substitution reactions can introduce different functional groups into the molecule.

Common Reagents and Conditions

Oxidation: Common oxidizing agents include hydrogen peroxide and m-chloroperbenzoic acid.

Reduction: Reducing agents such as lithium aluminum hydride and sodium borohydride are used.

Substitution: Reagents like alkyl halides and acyl chlorides are employed under basic or acidic conditions.

Major Products Formed

Oxidation: Sulfoxides and sulfones.

Reduction: Amine derivatives.

Substitution: Various substituted benzimidazole derivatives.

Pharmacokinetics and Metabolism

One of the key applications of Albendazole-D3 is in pharmacokinetic studies. A recent study characterized the excretion patterns of albendazole metabolites in human saliva, utilizing this compound as an internal standard for high-performance liquid chromatography (HPLC) analysis. This method demonstrated that saliva could be a viable biological matrix for assessing compliance in mass drug administration (MDA) campaigns against soil-transmitted helminths (STHs) .

Key Findings:

• Detection Limits: The limit of detection for albendazole sulfoxide (ABZSO) was established at 0.01 µg/mL in saliva.
• Pharmacokinetic Parameters: The peak concentration was observed at 4 hours post-treatment, with a significant correlation between saliva and dried blood spot (DBS) samples (Pearson r-value: 0.907) .

Therapeutic Efficacy

Albendazole has been extensively studied for its efficacy against various parasitic infections. In a case study involving orbital cysticercosis, a three-day regimen of albendazole was shown to be curative without recurrence . The study documented ultrasonography findings that illustrated the drug's effectiveness in reducing cyst size and alleviating symptoms.

Case Study Summary:

• Participants: 10 cases of orbital cysticercosis.
• Treatment Protocol: Oral prednisolone and albendazole administered over three days.
• Outcome: Complete resolution of symptoms and cysts observed in all patients by day 30 .

Combination Therapies

Research has also explored the combination of albendazole with other antiparasitic agents. A study on the pharmacokinetics of moxidectin combined with albendazole indicated that this triple-drug therapy could enhance treatment efficacy without increasing adverse events . This approach is particularly relevant for areas with high transmission rates of filarial diseases.

Analytical Methods Development

The development of sensitive analytical methods for detecting albendazole and its metabolites continues to be a focus area. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been optimized for simultaneous determination of albendazole and its active metabolite in human plasma, using this compound as an internal standard .

Methodological Insights:

• Sample Preparation: Solid-phase extraction was utilized for efficient analyte isolation.
• Performance Metrics: The method demonstrated high sensitivity and specificity, crucial for clinical pharmacokinetic studies.

Environmental Impact Studies

Albendazole's environmental fate is also an area of concern due to its potential impact on non-target organisms. Studies investigating advanced oxidation processes (AOPs) for the degradation of albendazole have highlighted its persistence in aquatic environments, emphasizing the need for effective wastewater treatment solutions .

Albendazole-D3 exerts its effects by binding to the colchicine-sensitive site of tubulin, inhibiting its polymerization into microtubules. This disruption of microtubule formation leads to the degeneration of the intestinal cells of the parasites, ultimately causing their death. The deuterium atoms in this compound enhance the compound’s stability and metabolic profile, potentially leading to improved therapeutic outcomes.

Structural and Physicochemical Properties

Table 1: Molecular and Physicochemical Comparison

• Key Differences :
  • This compound vs. Albendazole : Deuterium substitution increases molecular weight by ~3 Da, enabling isotopic distinction in mass spectrometry .
  • This compound vs. Flubendazole-D3 : Flubendazole-D3 contains a fluorinated benzene ring, altering polarity and metabolic stability compared to this compound .
  • This compound vs. Albendazole sulfoxide-D5 : The sulfoxide metabolite has a higher oxidation state (–SO–) and additional deuterium atoms, shifting its chromatographic retention time .
    
    

Pharmacokinetic and Metabolic Studies

• This compound enables precise measurement of albendazole’s bioavailability and clearance rates. Studies show its pharmacokinetic parameters (e.g., Cₘₐₓ, AUC) correlate closely with non-deuterated albendazole, validating its use as an internal standard .
• Albendazole sulfoxide-D5 aids in quantifying the primary active metabolite, albendazole sulfoxide, which has 10-fold higher systemic exposure than the parent drug .
• Flubendazole-D3 is critical for studying flubendazole’s prolonged half-life due to fluorine-induced metabolic resistance .

Albendazole-D3 (ABZ-D3) is a deuterated form of the widely used anthelmintic drug albendazole (ABZ). It is primarily utilized in pharmacokinetic studies to trace the metabolism and distribution of albendazole and its metabolites in biological systems. This article reviews the biological activity of ABZ-D3, focusing on its pharmacological effects, metabolic pathways, and clinical implications.

1. Pharmacological Profile

Albendazole is known for its efficacy against a variety of parasitic infections, particularly those caused by soil-transmitted helminths (STH) such as Ascaris lumbricoides and Trichuris trichiura. The biological activity of ABZ-D3 can be understood through its pharmacokinetics and metabolism:

• Metabolism : After oral administration, ABZ is rapidly metabolized in the liver to its active metabolite, albendazole sulfoxide (ABZSO), which is responsible for the drug's anthelmintic action. ABZSO undergoes further conversion to albendazole sulfone (ABZSO2), which lacks significant anthelmintic activity .
• Pharmacokinetics : Studies indicate that ABZSO is approximately 70% bound to plasma proteins and is widely distributed throughout the body, detectable in urine, cerebrospinal fluid, liver, bile, cyst walls, and cyst fluids . The pharmacokinetic properties of ABZ have been characterized in various populations, including healthy subjects and patients with parasitic infections.

2.1 Anthelmintic Activity

ABZ-D3 has been evaluated for its efficacy against STH infections. A study involving a triple-dose regimen of albendazole demonstrated significant cure rates for Ascaris lumbricoides and hookworm infections, with egg reduction rates exceeding 90% . The study highlighted that children under 18 years old were particularly susceptible to STH infections due to poor sanitation practices.

2.2 Cytotoxicity Studies

Research comparing the cytotoxic effects of ABZ on various cell lines revealed that Balb/c 3T3 cells were most sensitive to ABZ, followed by FaO and HepG2 cells. The effective concentration (EC50) values indicated that Balb/c 3T3 cells had an EC50 of 0.2 µg/mL, while FaO and HepG2 cells had higher EC50 values of 1.0 µg/mL and 6.4 µg/mL, respectively . This suggests that non-metabolizing cells exhibit higher sensitivity to the parent compound compared to metabolizing hepatoma cells.

3. Metabolic Pathways

Albendazole undergoes extensive hepatic metabolism. The primary metabolic pathways include:

• Conversion to Albendazole Sulfoxide : This metabolite exhibits significant anthelmintic activity and is responsible for much of the therapeutic effect observed with albendazole treatment.
• Further Metabolism to Albendazole Sulfone : This metabolite has minimal anthelmintic activity but may contribute to the overall pharmacological profile .

The following table summarizes key metabolites and their respective activities:

4. Clinical Implications

The use of ABZ-D3 as a tracer in clinical studies has facilitated a deeper understanding of albendazole's pharmacokinetics. For instance, research has shown that ABZSO levels can be monitored in saliva as a biomarker for compliance in mass drug administration (MDA) programs targeting STH infections . This approach allows for more accurate assessments of drug efficacy and patient adherence.

5. Case Studies

Several case studies have demonstrated the effectiveness of albendazole in treating parasitic infections:

• A longitudinal study conducted among indigenous communities in Malaysia reported high prevalence rates of STH infections and evaluated the effectiveness of a triple-dose regimen of albendazole . The results indicated a significant reduction in infection rates post-treatment.
• Another clinical trial assessed the use of albendazole in combination with other antiparasitic agents, revealing enhanced efficacy compared to monotherapy .

Basic Research Questions

Q. What is the role of Albendazole-D3 as an internal standard in quantifying benzimidazole anthelmintics via LC-MS/MS?

this compound is a deuterated isotopologue used to improve analytical accuracy by correcting for matrix effects and instrument variability in liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methodologically, researchers should co-elute this compound with the target analyte (e.g., albendazole or its metabolites) to ensure identical retention times, enabling precise isotope dilution quantification. Calibration curves must validate linearity (R² > 0.99) and limits of detection (e.g., ≤1 ng/mL in plasma) .

Q. How does isotopic purity impact the reliability of this compound in pharmacokinetic studies?

Isotopic purity (>98%) is critical to avoid spectral overlap in mass spectrometry. Researchers should verify purity via nuclear magnetic resonance (NMR) or high-resolution mass spectrometry (HRMS). Impurities in deuterated standards can artificially inflate analyte concentrations, leading to erroneous pharmacokinetic parameters (e.g., AUC, C_max). Batch-specific certificates of analysis from suppliers are essential .

Q. What protocols ensure the stability of this compound in biological matrices during long-term storage?

Stability studies should replicate storage conditions (e.g., -80°C, freeze-thaw cycles) and assess degradation using spike-and-recovery experiments. For example, this compound in liver homogenates may require antioxidants (e.g., ascorbic acid) to prevent oxidation. Data should report percentage recovery (±10% deviation) and validate stability over ≥30 days .

Advanced Research Questions

Q. How can researchers resolve discrepancies in this compound recovery rates across heterogeneous tissue samples?

Tissue-specific matrix effects (e.g., lipid content in adipose vs. protein-rich liver) require optimization of extraction protocols. Solid-phase extraction (SPE) with hydrophilic-lipophilic balance (HLB) cartridges or QuEChERS methods can improve recovery. Method validation should include matrix-matched calibration standards and statistical analysis (e.g., ANOVA for inter-tissue variability) .

Q. What experimental designs address the co-elution challenges of this compound with endogenous metabolites in complex matrices?

Ultra-high-performance liquid chromatography (UHPLC) with sub-2µm particle columns (e.g., C18) enhances chromatographic resolution. Researchers should also employ dynamic multiple reaction monitoring (dMRM) to distinguish this compound from isobaric interferences. Data must include baseline separation metrics (resolution ≥1.5) and signal-to-noise ratios ≥10:1 .

Q. How do deuterium kinetic isotope effects (KIEs) influence the metabolic profiling of this compound in in vitro hepatic models?

KIEs may alter the rate of cytochrome P450-mediated oxidation (e.g., sulfoxidation of albendazole). To mitigate this, researchers should compare metabolic pathways of this compound and non-deuterated albendazole using hepatocyte incubations. Data should quantify metabolite ratios (e.g., sulfoxide/sulfone) via time-course analyses and adjust for KIEs using computational modeling (e.g., density functional theory) .

Q. Methodological Frameworks

• Literature Synthesis : Use PubMed and Web of Science with keywords: This compound, deuterated internal standards, LC-MS/MS validation. Prioritize studies reporting extraction efficiencies and inter-laboratory reproducibility .
• Experimental Validation : Adopt International Council for Harmonisation (ICH) guidelines for stability testing (Q1A) and method validation (Q2R1). Include cross-validation between LC-MS/MS and HPLC-UV to confirm robustness .
• Data Contradiction Analysis : Apply the FINER criteria (Feasible, Interesting, Novel, Ethical, Relevant) to troubleshoot inconsistent results. For example, low recovery rates may require re-evaluating SPE solvent polarity or ion suppression effects .

Q. Tables for Reference

Table 1 : Key Analytical Parameters for this compound in LC-MS/MS

Table 2 : Stability of this compound in Bovine Liver Homogenate