Daclatasvir

Method 1: Condensation-Ring Closure Approach

This method, detailed in Patent CN106432204A, involves four stages:

• Condensation of 4,4'-di(2-chloroacetyl)biphenyl with Boc-L-proline :
  The reaction proceeds in acetonitrile with diisopropylethylamine (DIPEA) as a base, yielding a bis-ester intermediate. The solvent’s polarity facilitates rapid nucleophilic substitution, achieving >90% conversion at 25–30°C.

• Imidazole formation via ammonium acetate cyclization :
  Heating the ester with ammonium acetate in toluene at 95–100°C induces cyclodehydration. The ammonium ion acts as a dual acid catalyst and nitrogen source, forming the imidazole ring in 15 hours.

• Boc deprotection with HCl :
  Treatment with concentrated hydrochloric acid in methanol removes the tert-butoxycarbonyl (Boc) group, generating a primary amine hydrochloride salt. This step achieves 85% yield with 99.68% purity after recrystallization.

• Final coupling with Moc-L-valine :
  The amine intermediate reacts with N-methoxycarbonyl-L-valine (Moc-L-valine) using carbodiimide coupling agents, yielding daclatasvir. Purification via column chromatography ensures >99% chiral purity.

Method 2: Bromoacetyl-Based Pathway

Patent CN106496199A optimizes the process using brominated precursors:

• Synthesis of 4,4'-bis(2-bromoacetyl)biphenyl :
  Biphenyl undergoes Friedel-Crafts acylation with bromoacetyl bromide in chloroform catalyzed by AlCl₃. This method suppresses monosubstituted byproducts (<2%) through precise stoichiometry and low-temperature (0–5°C) conditions.

• Proline coupling and cyclization :
  Bromoacetyl groups exhibit higher reactivity than chloro analogues, reducing the coupling time with Boc-L-proline to 3 hours. Subsequent cyclization with ammonium acetate in toluene achieves 92% yield due to improved leaving-group ability.

• Deprotection and final assembly :
  Hydrochloric acid-mediated Boc removal followed by Moc-L-valine condensation completes the synthesis. The bromo route reduces overall steps by eliminating intermediate purification, achieving a 40% cost reduction compared to chloro-based methods.

Method 3: Continuous Flow Microreactor Technology

A novel approach (described in LookChem articles) employs micro-electro-flow reactors for ultrafast C–C coupling:

• Electrochemical biphenyl acylation :
  Biphenyl and chloroacetyl chloride react in a microreactor under an electric field (10 V), achieving 95% conversion in 5 minutes. The continuous flow design eliminates oxidants and reduces waste by 70%.

• Tandem cyclization-deprotection :
  Integrated reactor modules enable sequential imidazole formation (via ammonium acetate) and Boc deprotection in a single pass, cutting processing time from 20 hours to 2 hours.

Comparative Analysis of Synthetic Routes

Method 2’s bromoacetyl pathway offers superior yields and scalability, whereas Method 3’s flow chemistry approach promises sustainability but requires further scale-up validation.

Critical Challenges and Solutions

• Byproduct Formation in Acylation :
  Monosubstituted byproducts during biphenyl acylation are mitigated using excess AlCl₃ (1.5 equiv) and slow reagent addition.

• Imidazole Ring Instability :
  The imidazole intermediate is hygroscopic; anhydrous toluene and inert atmospheres prevent decomposition during cyclization.

• Chiral Purity Maintenance : Enantiomeric excess (>99.5%) is ensured via crystallization-driven dynamic resolution using L-tartaric acid.

Q. What is the mechanistic basis of Daclatasvir's antiviral activity against HCV?

this compound targets the HCV NS5A protein, a multifunctional phosphoprotein critical for viral RNA replication and virion assembly. Computational modeling reveals that it binds to NS5A's domain I, disrupting interactions required for replication complex formation and viral RNA synthesis. This inhibition occurs at picomolar to nanomolar concentrations across HCV genotypes 1–6. Methodological insights include molecular docking studies and in vitro assays measuring EC50 values .

Q. What analytical methods are validated for quantifying this compound in biological matrices?

Reverse-phase liquid chromatography with photodiode array detection (RP-LC-PDA) and ionic liquid-based dispersive liquid-liquid microextraction (DLLME) are robust for bioanalysis. Key validation parameters include linearity (0.05–7.5 µg/mL), precision (RSD <2%), and recovery rates (98.4–99.5%). Advanced electrochemical nanosensors using cobalt nanoparticles and multi-walled carbon nanotubes (MWCNTs) achieve detection limits of 8.82 × 10⁻¹⁰ M, suitable for pharmacokinetic studies .

Q. How do CYP3A4-mediated drug interactions influence this compound dosing strategies?

this compound is a CYP3A4 substrate, necessitating dose adjustments with enzyme inducers (e.g., efavirenz) or inhibitors (e.g., ritonavir-boosted atazanavir). Pharmacokinetic modeling recommends increasing the dose to 90 mg with efavirenz (a CYP3A4 inducer) and reducing to 30 mg with strong inhibitors. Clinical trials (ALLY-2) validate these adjustments through measured plasma concentrations and sustained virologic response (SVR) rates .

Advanced Research Questions

Q. How can pharmacokinetic-pharmacodynamic (PK/PD) modeling predict this compound's in vivo efficacy?

Integrative PK/PD models combine plasma concentration data, viral load dynamics, and tissue-specific drug distribution. For example, a mathematical model estimated active tissue concentrations at ~9% of plasma levels (95% CI: 1–29%), aligning with clinical SVR outcomes. Advanced simulations using Blue Gene/Q supercomputers refine half-life estimates (4x shorter than prior models) and identify dual mechanisms: blocking viral RNA replication and assembly .

Q. What methodologies identify this compound resistance mutations in HCV genotypes?

Resistance profiling involves:

• Phylogenetic analysis of NS5A sequences to detect baseline polymorphisms (e.g., Y93H in genotype 4d).
• In vitro resistance selection using subgenomic replicons to measure fold-changes in EC50 (e.g., ≥1000-fold resistance in rare variants).
• Clinical correlation through trials (e.g., ALLY-3) assessing SVR rates in patients with preexisting resistance-associated substitutions (RASs) .

Q. How do protein binding and hepatic transporters affect this compound's pharmacokinetics?

this compound exhibits 99% protein binding, primarily to albumin, and relies on OCT1 transporters for hepatocyte uptake. In vitro studies show negligible renal excretion (6.6% in urine), with hepatic clearance via CYP3A4 metabolism. Population pharmacokinetic models account for OCT1 polymorphisms (e.g., reduced exposure in OCT1 poor transporters) to optimize dosing in cirrhotic patients .

Q. What experimental designs optimize this compound combination therapy trials?

Key considerations include:

• Randomization by HCV genotype/subtype and prior treatment history.
• Endpoint selection : SVR at 12 weeks (SVR12) as the primary efficacy marker.
• Ribavirin stratification to mitigate anemia risk while maintaining efficacy (e.g., 98% SVR12 in genotype 1 with sofosbuvir ± ribavirin).
• Resistance monitoring using deep sequencing to track RAS emergence .

Q. How can in vitro models evaluate this compound's activity against non-HCV viruses?

SARS-CoV-2 inhibition studies employ:

• Cell-based assays (Vero E6, pneumocytes) to measure EC50 (0.6–1.1 µM).
• Molecular docking into viral RNA (PDB: 6XRZ) or RdRp (PDB: 7BV2) to predict binding affinity.
• Melting curve assays to assess RNA stability under drug exposure .

Q. How does NS5A genetic heterogeneity impact this compound's clinical efficacy?

While baseline NS5A polymorphisms (e.g., L31M in genotype 4a) are common (>80%), only 1.6% confer high-level resistance (EC50 ≥1000-fold). Clinical trials show no significant SVR12 reduction in these patients, suggesting minimal clinical impact. Subtype-specific efficacy is validated in genotype 3 (89% SVR12) and genotype 4 (100% SVR12 in 4a) cohorts .

Q. What computational approaches elucidate this compound-NS5A binding and resistance?

Advanced molecular dynamics simulations and free-energy perturbation calculations map this compound's interactions with NS5A domain I. These models predict resistance mechanisms (e.g., Y93H disrupting hydrogen bonding) and guide second-generation NS5A inhibitor design to broaden genotype coverage and reduce resistance risk .