Zanubrutinib

Structural Features of Zanubrutinib

This compound ((S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide) contains a tetrahydropyrazolopyrimidine core with two chiral centers. The (S)-enantiomer exhibits superior BTK inhibition, necessitating asymmetric synthesis or resolution to achieve ≥99% enantiomeric excess (ee).

Key Synthetic Challenges

• Chiral Purity : Early routes suffered from racemization during pyrimidine ring reduction, requiring multiple resolution steps.

• Nitrile Hydrolysis : Controlled conversion of the cyano group to carboxamide without side reactions.

• Hazardous Reagents : Traditional methods used acryloyl chloride and dichloromethane (DCM), complicating industrial safety protocols.

WO2018033853 Patent Methodology

The foundational route involved 12 steps, with critical issues in chiral resolution:

• Pyrazole-Pyrimidine Ring Formation : Condensation of 5-amino-3-bromo-1H-pyrazole-4-carbonitrile (II) with N-Boc-(E)-4-(3-(dimethylamino)acryloyl)piperidine (III) in DCM/acetic acid (9:1) at 60°C.

• Sodium Borohydride Reduction : Generated racemic tetrahydropyrimidine intermediate (IV) with 50% ee.

• Chiral Resolution : Sequential tartaric acid crystallizations (D- followed by L-dibenzoyl) increased ee to 92%, necessitating further purification.

Limitations :

• Cumulative yield of 4% due to multiple resolution steps.

• High solvent consumption (DCM, ethanol).

WO2023062504A1 Patent: Single-Resolution Process

This method introduces the phenoxyphenyl group via Suzuki coupling late in the synthesis, avoiding intermediate racemization:

Key Steps :

• Delayed Suzuki Reaction : Coupling of 2-bromo intermediate (VII) with 4-phenoxyphenylboronic acid in acetonitrile/methanol (2:1) at 80°C.

• Single L-DBTA Resolution : Achieves 99.85% ee in one step by leveraging steric hindrance from the phenoxyphenyl group.

Advantages :

• Yield increased to 28% (7-fold improvement).

• Eliminates DCM usage in final steps.

Computer-Aided Synthesis Planning (CASP)

The ChemRxiv study identified a 10-step route using renewable feedstocks and asymmetric catalysis:

Highlights :

• Enzymatic Desymmetrization : Lipase-catalyzed acetylation of diol intermediates (98% ee).

• Electrochemical Amination : Replaces acryloyl chloride with CO₂-derived reagents.

Outcomes :

• 45% overall yield.

• 100% reduction in organochlorine solvents.

Solvent System Optimization

• Toluene/Acetic Acid : Preferred for initial condensation (≥60°C) due to improved regioselectivity.

• Ethanol Replacement : Cyclopentyl methyl ether (CPME) reduced borohydride decomposition in CASP routes.

Catalytic Asymmetric Hydrogenation

Pilot-scale trials using Ir-(S)-Binap catalysts achieved 98% ee in pyrimidine reduction, bypassing resolution steps.

Continuous Flow Nitrile Hydrolysis

Microreactor systems with NaOH/isopropanol at 120°C completed nitrile-to-amide conversion in 8 minutes (vs. 12 hours batch) .

Q. How can researchers assess BTK inhibition by zanubrutinib in preclinical models?

Methodological Answer:

• Use kinase activity assays (e.g., ADP-Glo™ Kinase Assay) to measure BTK enzymatic inhibition.
• Validate target engagement via Western blotting for phosphorylated BTK (p-BTK) in B-cell lines (e.g., MCL cell lines).
• Employ xenograft models to evaluate tumor growth suppression, correlating plasma drug levels with p-BTK reduction. Ensure reproducibility by detailing protocols for dosing, sampling intervals, and control groups .

Q. What are key considerations for dose optimization in early-phase this compound trials?

Methodological Answer:

• Conduct phase I dose-escalation studies using a 3+3 design to identify the maximum tolerated dose (MTD).
• Monitor pharmacokinetic (PK) parameters (e.g., Cmax, AUC) and pharmacodynamic (PD) markers (e.g., BTK occupancy in peripheral blood).
• Reference long-term safety data (e.g., 35.2-month follow-up) to predefine adverse event thresholds, such as neutropenia or atrial fibrillation rates .

Q. How should researchers validate biomarkers predictive of this compound response?

Methodological Answer:

• Perform genomic profiling (e.g., whole-exome sequencing) on tumor samples to identify mutations (e.g., TP53) associated with resistance.
• Use multivariate Cox regression to correlate baseline biomarker levels (e.g., serum CXCL13) with progression-free survival (PFS).
• Standardize assays across labs using guidelines from Clinical Laboratory Improvement Amendments (CLIA) to ensure reproducibility .

Q. How can conflicting efficacy data between this compound and other BTK inhibitors be reconciled in cross-trial comparisons?

Methodological Answer:

• Apply matching-adjusted indirect comparison (MAIC) to adjust for differences in patient demographics (e.g., prior therapy lines, ECOG scores).
• Stratify analyses by risk factors (e.g., high-risk MCL subtypes) and trial design variables (e.g., follow-up duration).
• Critically evaluate endpoints (e.g., ORR vs. PFS) and censoring rules, as variations may bias outcomes .

Q. What statistical methods address censored data in this compound survival analyses?

Methodological Answer:

• Use Kaplan-Meier estimators with log-rank tests for unadjusted survival comparisons.
• For multivariate analysis, apply Cox proportional hazards models with time-dependent covariates (e.g., treatment discontinuation).
• Address non-proportional hazards using restricted mean survival time (RMST) or parametric survival models (e.g., Weibull) .

Q. How can real-world evidence (RWE) be integrated with clinical trial data for this compound?

Methodological Answer:

• Link electronic health records (EHRs) with trial databases using unique patient identifiers, ensuring compliance with GDPR/HIPAA.
• Apply propensity score matching to balance confounding variables (e.g., comorbidities) between RWE and trial cohorts.
• Validate findings through sensitivity analyses (e.g., varying inclusion criteria) .

Q. What strategies mitigate heterogeneity in patient responses during this compound trials?

Methodological Answer:

• Pre-stratify randomization by molecular subtypes (e.g., SOX11+ vs. SOX11– MCL).
• Use adaptive trial designs to enrich subgroups showing early response signals.
• Report subgroup analyses with Bonferroni correction to control for type I error .

Q. How should meta-analyses reconcile differing endpoints across this compound trials?

Methodological Answer:

• Harmonize endpoints using standardized criteria (e.g., Lugano 2014 for lymphoma).
• Perform individual participant data (IPD) meta-analysis to pool raw datasets, enabling uniform endpoint definitions.
• Quantify heterogeneity via I² statistics and explore sources using meta-regression .

Q. What ethical considerations are critical when recruiting R/R MCL patients for this compound trials?

Methodological Answer:

• Obtain informed consent detailing risks (e.g., bleeding, infections) and alternatives (e.g., CAR-T therapy).
• Establish Data Safety Monitoring Boards (DSMBs) to review interim safety data.
• Follow ICH-GCP guidelines for vulnerable populations (e.g., elderly patients with comorbidities) .

Q. How can reproducibility be ensured in this compound preclinical studies?

Methodological Answer:

• Publish detailed protocols for animal models (e.g., NOD-SCID mice), including dosing schedules and endpoint criteria.
• Share raw data and code in public repositories (e.g., GitHub) for independent validation.
• Adopt BLISS (Biological Loopholes for Experimental Standardization) guidelines to minimize batch effects .