Two-Step Coupling Procedure for Avizafone Dihydrochloride

The foundational synthesis of this compound dihydrochloride begins with 5-chloro-2-methyl-aminobenzophenone and (S)-2-(2,6-bis(((benzyloxy)carbonyl)amino)hexanamido)acetic acid. The process involves: Buy Pro-Diazepam (Avizafone HCl)

• Formation of the Benzodiazepine Intermediate : Reacting 5-chloro-2-methyl-aminobenzophenone with (S)-2-(2,6-bis(((benzyloxy)carbonyl)amino)hexanamido)acetic acid under vacuum, facilitated by a double-needle transfer apparatus. Dry ether serves as the primary solvent, with rigorous washing to isolate intermediate compounds.
• Lyophilization for Salt Formation : The precipitated intermediate is dissolved in distilled water, subjected to ethyl acetate extraction, and lyophilized for 65 hours to yield this compound dihydrochloride at 58% efficiency. This step ensures the removal of residual solvents and stabilizes the hydrochloride salt.

Critical parameters influencing yield include solvent purity (dry ether >99.9%), reaction temperature (25°C ± 2°C), and vacuum pressure (0.1–0.5 mbar). Impurities such as unreacted starting materials are minimized through triple ethyl acetate washes.

Enzymatic Synthesis for Enantiomeric Control

A patent-pending method utilizes recombinant human aminopeptidase B to synthesize S-avizafone dihydrochloride with 99% enantiomeric excess. Key steps include:

• Gene Cloning : Inserting the aminopeptidase B gene (RNPEP) into a pQE-30 Xa vector, followed by transformation into Escherichia coli JM109 for enzyme production.
• Enzymatic Coupling : The purified enzyme catalyzes the stereospecific coupling of 5-chloro-2-methylaminobenzophenone with a lysine derivative in aqueous buffer (pH 7.4, 32°C). This method avoids racemization, critical for maintaining pharmacological activity.

This approach achieves superior stereochemical purity compared to traditional chemical synthesis, albeit with higher production costs due to enzyme purification requirements.

Industrial Production and Scalability

Batch Reactor Optimization

Industrial-scale synthesis employs 500–1,000 L batch reactors to accommodate the exothermic nature of the coupling reaction. Key considerations include:

• Temperature Control : Jacketed reactors maintain temperatures at 25°C ± 1°C to prevent thermal degradation.
• Solvent Recovery Systems : Closed-loop distillation units reclaim ethyl acetate, reducing waste and costs.

A representative production batch yields 385 kg of this compound dihydrochloride per 1,000 kg of starting material, aligning with laboratory-scale efficiencies.

Purification and Quality Control

Post-synthesis purification involves:

• Crystallization : The crude product is recrystallized from a methanol-water mixture (7:3 v/v) to remove hydrophobic impurities.
• Chromatographic Polishing : High-performance liquid chromatography (HPLC) with a Zorbax XDB Eclipse C18 column (4.6 × 150 mm, 5 µm) resolves residual benzophenone derivatives, achieving >99.5% purity.

Table 1: Comparative Analysis of this compound Synthesis Methods

Characterization and Analytical Validation

Structural Confirmation via Spectroscopy

• Nuclear Magnetic Resonance Spectroscopy : ¹H and ¹³C spectra confirm the presence of characteristic benzodiazepine peaks (δ 7.2–7.8 ppm for aromatic protons) and lysine methylene groups (δ 1.2–1.6 ppm).
• Mass Spectrometry : High-resolution electrospray ionization (HR-ESI-MS) verifies the molecular ion peak at m/z 431.2 [M+H]⁺, consistent with this compound’s molecular formula.

Stability-Indicating Assays

Forced degradation studies under acidic (0.1 M hydrochloric acid), basic (0.1 M sodium hydroxide), and oxidative (3% hydrogen peroxide) conditions reveal this compound’s susceptibility to hydrolysis. HPLC analyses quantify degradation products, ensuring method robustness for shelf-life determinations.

Avizafone vs. Diazepam