Sofosbuvir

Nucleophilic Substitution and Thioglycoside Formation

The synthesis of sofosbuvir’s thioglycoside derivatives relies on nucleophilic substitution reactions between heterocyclic bases and α-bromo-sugars. In a representative method, pyridine- and pyrimidine-based thioglycosides (3a , 3b , 10 , 14a , 14b , 18a , 18b ) were synthesized via coupling thiol-containing heterocycles (1 , 13 , 17 ) with brominated sugars (2a , 2b , 9 ) in dichloromethane or THF . X-ray crystallography confirmed the thioglycosidic linkage through sulfur rather than nitrogen, critical for antiviral activity .

Deacetylation of intermediates (3a , 14a , etc.) using methanolic ammonia yielded unprotected thioglycosides (5a , 15a , etc.), which underwent regioselective phosphoramidate coupling. Grignard reagents like isopropyl magnesium chloride generated alkoxide anions, enabling efficient nucleophilic attack on phosphorochloridate precursors . This step’s regioselectivity was pivotal for minimizing byproducts.

Phosphoramidate Coupling Techniques

Phosphoramidate formation, a cornerstone of this compound synthesis, involves reacting deprotected nucleosides with phosphorochloridate derivatives. A patented method bypasses traditional Grignard reagents by employing metallic salts (e.g., MgCl₂, LiCl) at room temperature, enhancing industrial feasibility. For example, reacting compound III with II in THF using MgCl₂ achieved >99% purity with a 98:2 this compound-to-salt ratio .

Alternative approaches utilized aluminum chloride (AlCl₃) and pyridine in dichloromethane at 20°C, yielding 97.5% this compound after extraction and crystallization . Comparative studies highlight AlCl₃’s role in activating the phosphorochloridate electrophile, though MgCl₂ offers milder conditions .

Industrial-Scale Processes Using Metallic Salts

A breakthrough in this compound manufacturing emerged from room-temperature reactions avoiding cryogenic conditions (−20°C to −15°C). Patent US20180022774A1 details a scalable method:

• Deprotection : Tribenzoyl cytidine derivative (V ) was refluxed in 80% acetic acid, yielding IV (85–90% yield).

• Hydrolysis : IV treated with ammonia/methanol produced III .

• Coupling : III reacted with IIa in THF/MgCl₂, followed by SFC chiral resolution (20% MeOH/CO₂) to isolate this compound .

Table 1: Industrial Process Optimization

This method’s eco-friendliness and cost-efficiency stem from eliminating hazardous reagents and energy-intensive cooling.

Novel Intermediates and Hydrolysis Methods

WO2015097605A1 introduced crystalline intermediate 3a , characterized by X-ray diffraction (Figure 1), streamlining this compound synthesis:

• Condensation : Compound 4 and 5 formed 3 in dimethoxyethane.

• Hydrolysis : 3 treated with acetic acid/1,4-dioxane yielded 2 .

• Deprotection : 2 deprotected under acidic conditions (HCl/MeOH) afforded this compound .

Table 2: Key Intermediates and Properties

This route’s innovation lies in intermediates’ crystallinity, simplifying purification and ensuring batch consistency.

Comparative Analysis of Synthesis Routes

Efficiency : The MgCl₂-mediated process outperforms AlCl₃-based methods in scalability (20 kg vs. 5 kg batches).
Cost : Avoiding Grignard reagents reduces raw material costs by ~30% .
Purity : SFC resolution achieves >99.9% enantiomeric excess, critical for FDA compliance .

Table 3: Method Comparison

Sofosbuvir undergoes various chemical reactions, including:

Oxidation: This compound can be oxidized to form its active metabolite, GS-461203, which is a triphosphate.

Reduction: Reduction reactions are not commonly associated with this compound.

Substitution: This compound can undergo substitution reactions, particularly involving the fluorine atom on the furanose ring.

Common reagents and conditions used in these reactions include organic solvents, Lewis acids, and alkali . The major products formed from these reactions include the active triphosphate form of this compound and other intermediates used in its synthesis .

Treatment of Chronic Hepatitis C

Sofosbuvir is primarily indicated for the treatment of chronic hepatitis C across all genotypes. Its effectiveness has been demonstrated in various clinical trials:

• Efficacy Across Genotypes : this compound-based regimens have shown high SVR rates, particularly in genotypes 1 and 2, with studies reporting SVR rates exceeding 90% in treatment-naïve patients .
• Challenges with Genotype 3 : Despite its overall effectiveness, genotype 3 infections present challenges, with lower SVR rates reported, especially in patients with cirrhosis .

Special Populations

This compound has been evaluated for use in special populations, including those with advanced chronic kidney disease (CKD):

• Patients with CKD : A systematic review indicated that this compound-based regimens are safe and effective for patients with stage 4–5 CKD. The pooled SVR rate was found to be 99%, highlighting its utility even in patients with significant renal impairment .
• Co-infection Scenarios : this compound is also effective in patients co-infected with HIV or those who have previously failed other DAA therapies, demonstrating its versatility in complex clinical situations .

Real-World Efficacy

A study involving a cohort of decompensated cirrhotic patients treated with this compound/Velpatasvir reported an impressive SVR12 rate of 94.4%. This highlights the drug's effectiveness even in challenging patient populations .

Phase III Trials

In a Phase III trial conducted in Japan, all participants receiving an all-oral regimen containing this compound achieved undetectable HCV levels by week four, underscoring its rapid action against the virus .

Safety Profile

This compound is generally well-tolerated, but some adverse events have been reported:

• Common Adverse Events : Fatigue, headache, and anemia are frequently observed but are typically manageable. Serious adverse events occur at a rate of approximately 9% in clinical settings .
• Monitoring Recommendations : Close monitoring of renal function is advised for patients with CKD during treatment to mitigate risks associated with potential renal impairment .

Comparative Effectiveness

A systematic review comparing this compound-based regimens with and without ribavirin found that while adding ribavirin may enhance SVR rates in some cases, it also increases the risk of serious adverse events . This underscores the importance of tailoring treatment regimens to individual patient needs.

Sofosbuvir is a prodrug that is metabolized into its active form, GS-461203, within the body . This active form inhibits the HCV NS5B RNA-dependent RNA polymerase by acting as a defective substrate, thereby terminating the viral RNA chain and preventing replication . The inhibition of this enzyme is crucial for the suppression of HCV replication and the achievement of a sustained virologic response .

Structural Analogues: Tenofovir Alafenamide

Sofosbuvir shares a phosphoramidate side chain with tenofovir alafenamide (TAF), a prodrug for HIV/hepatitis B treatment. Both require intracellular activation to their triphosphate forms. However, this compound targets RdRp, while TAF inhibits reverse transcriptase. This compound exhibits higher specificity for HCV NS5B, with an IC50 of 0.12 µM against HCV RdRp compared to TAF’s weaker RdRp affinity .

This compound Derivatives Against SARS-CoV-2 RdRp

Recent in silico studies identified this compound derivatives with enhanced binding to SARS-CoV-2 RdRp (Table 1):
Table 1: Pharmacokinetic and Binding Properties of this compound and Derivatives

Derivatives 3 and 4 showed superior binding energy (-7.89 to -8.12 kcal/mol) compared to this compound (-7.46 kcal/mol), with compound 4 exhibiting the highest solubility and permeability . Halogen substitutions (e.g., chlorine, iodine) in derivatives altered solubility but retained comparable absorption and metabolism profiles to this compound .

Nucleotide Analogs: Ribavirin and Remdesivir

Ribavirin: A guanosine analog with broad antiviral activity. Unlike this compound, ribavirin requires concurrent interferon for HCV efficacy (SVR: 44–51% vs. This compound’s 78–97% in genotype 2/3 ). Against ZIKV, this compound’s selectivity index (SI: 184–1,191) was 30-fold higher than ribavirin (SI: 6–40) . Remdesivir: An adenosine analog targeting RdRp in Ebola and SARS-CoV-2. This compound showed comparable SARS-CoV-2 RdRp binding (-7.46 kcal/mol) to remdesivir (-7.1 kcal/mol) . However, remdesivir’s clinical efficacy in COVID-19 is better established.

Table 2: Antiviral Efficacy Across Viruses

Pharmacokinetic and Pharmacodynamic Comparisons

• Solubility/Permeability : this compound’s derivatives (e.g., compound 4) surpass it in solubility (-2.84 vs. -2.271 log(mol/L)) and Caco2 permeability (58.2% vs. 28.7%) .
• Metabolism : this compound and TAF both require hepatic activation but target distinct enzymes .
• Drug-Drug Interactions : this compound has fewer interactions than ribavirin, which causes hemolytic anemia .

Clinical Outcomes in Viral Infections

• HCV: this compound/ribavirin achieved 78–97% SVR in genotype 2/3, outperforming peginterferon/ribavirin (67%) .
• COVID-19 : this compound/daclatasvir reduced mortality to 5.7% vs. 19.2% with lopinavir/ritonavir .
• ZIKV : this compound inhibited replication in neuroepithelial cells (EC50: 2.6–5.2 µM) with minimal cytotoxicity .

Basic Research Questions

Q. What validated analytical methods are available for quantifying sofosbuvir in pharmaceutical formulations or biological matrices?

• Methodology : Reverse-phase high-performance liquid chromatography (RP-HPLC) is widely used, with parameters optimized via Quality by Design (QbD) approaches. For example, a QbD-based method optimized mobile phase ratio (e.g., acetonitrile:buffer), buffer pH (e.g., phosphate buffer at pH 4.5–6.5), and column type (C18 or phenyl-hexyl) using Design-Expert® software. This method achieved retention times of 2.5–4.5 minutes, peak tailing <2, and theoretical plates >5,000 .
• Validation : Include specificity, linearity (e.g., 10–100 µg/mL), precision (RSD <2%), and robustness against pH or flow-rate variations .

Q. How are clinical trials for this compound-based regimens designed to assess efficacy against HCV genotypes?

• Design : Trials often use randomized, controlled designs with stratification by genotype (e.g., genotype 2 vs. 3), cirrhosis status, and prior treatment history. For example, the VALENCE trial extended treatment duration for genotype 3 from 12 to 24 weeks after interim analysis showed improved sustained virologic response (SVR) rates (85% vs. 56% in cirrhotic patients) .
• Endpoints : Primary endpoints include SVR at 12 weeks post-treatment (SVR12), analyzed via intention-to-treat. Secondary endpoints may address adverse events (e.g., headache, fatigue) and subgroup efficacy .

Q. What statistical tools are employed to analyze this compound clinical trial data?

• Tools : SPSS or R for descriptive statistics (mean, SD) and inferential tests (Chi-square, paired t-tests). For example, a study comparing dual (this compound + ribavirin) vs. triple therapy (with peginterferon) used a p-value ≤0.05 to define significance in SVR rates .

Advanced Research Questions

Q. How can multivariate chemometric models resolve challenges in quantifying this compound in combination therapies (e.g., with ledipasvir)?

• Approach : Genetic algorithm-partial least squares (GA-PLS) and artificial neural networks (ANN) improve precision in spectral data analysis. For instance, GA-PLS applied to UV-Vis spectra (200–400 nm) of this compound/ledipasvir mixtures achieved recovery rates of 98–102% with RSD <1.5% .
• Validation : Cross-validate models using synthetic mixtures and real-world samples, ensuring minimal interference from excipients or metabolites .

Q. What experimental designs optimize this compound-loaded lipid nanocarriers for enhanced bioavailability?

• Design : A 2³ factorial design evaluates factors like lipid concentration (X1), cholesterol content (X2), and centrifugation speed (X3). Responses such as drug entrapment efficiency (DEE >80%) and particle size (<200 nm) are modeled via Design-Expert® software. For example, high X1 and low X3 maximized DEE while minimizing particle size .
• Characterization : Use dynamic light scattering (DLS) for size distribution and in vitro release studies in simulated intestinal fluid (pH 6.8) .

Q. How do researchers address contradictory efficacy data for this compound across HCV genotypes?

• Analysis : Stratify data by genotype and cirrhosis status. Genotype 3 patients show lower SVR rates (68% with cirrhosis vs. 91% without) compared to genotype 2 (93% SVR). Meta-analyses or pooled data from trials like FISSION and NEUTRINO can reconcile discrepancies by adjusting for covariates (e.g., viral load, IL28B polymorphisms) .

Q. What advanced bioanalytical techniques quantify this compound metabolites (e.g., GS-331007) in plasma?

• Technique : Online SPE-LC coupled to Q-Exactive HRMS enables sensitive detection (LOQ: 1 ng/mL) using t-MS² or t-SIM modes. This method validated this compound’s pharmacokinetics, including its short half-life (0.4 hours) and metabolite GS-331007’s prolonged half-life (27 hours) .

Q. How can QbD principles improve robustness in this compound assay development?

• Implementation : Define critical quality attributes (CQAs) like retention time and peak symmetry. Use risk assessment (e.g., Ishikawa diagrams) to prioritize factors (mobile phase, column temperature). A 14-run experimental design identified optimal conditions (e.g., pH 5.0, 70:30 acetonitrile:buffer) with robustness confirmed via intermediate precision testing (RSD <1.5%) .

Q. Methodological Frameworks

Q. How to formulate a research question for this compound studies addressing gaps in HCV management?

• Process : Start with systematic reviews to identify gaps (e.g., optimizing regimens for genotype 4). Narrow questions using PICOT criteria: Population (HCV genotype 4 patients), Intervention (this compound/velpatasvir), Comparison (historical interferon-based therapies), Outcome (SVR12), Time (12 weeks). Sub-questions may explore resistance-associated substitutions (RASs) or cost-effectiveness in low-resource settings .

Q. What strategies mitigate bias in this compound clinical trials with open-label designs?

• Controls : Use blinded endpoint adjudication committees and centralized PCR testing for HCV RNA. For example, the SYNERGY trial achieved 95% SVR12 in genotype 4 patients by masking laboratory analysts to treatment allocation .