Design and synthesis of carbamate-linked ester prodrugs selectively activated by carboxylesterase 1 with enhanced stability against intestinal hydrolysis

Ester-based prodrugs are often prematurely hydrolyzed by intestinal carboxylesterases, leading to reduced bioavailability and potential gastrointestinal (GI) toxicity. To overcome this limitation, a series of diester, carbonate-linked ester, and carbamate-linked ester prodrugs were synthesized and evaluated using human liver microsomes (HLM), human intestinal microsomes (HIM), and recombinant carboxylesterases (CES1A1 and CES2A1). Under strongly acidic conditions (pH 1.2), all prodrugs remained highly stable (>98% remaining after 6 h). In human plasma, however, diester prodrugs showed relatively rapid degradation (approximately 50% remaining after 6 h), whereas carbonate- and carbamate-linked esters displayed enhanced stability. Diester and carbonate-linked ester prodrugs were hydrolyzed by both HLM and HIM, indicating non-selective activation. By contrast, several carbamate-linked ester prodrugs showed high HLM/HIM selectivity, with CES1A1/CES2A1 hydrolysis rate ratios exceeding 50. Docking simulations showed favorable active-site binding of carbamate-linked ester prodrugs to CES1A1 but not to CES2A1, supporting the selectivity of CES1A1. These findings suggest that carbamate-based prodrug strategies can be employed to improve intestinal stability and enable controlled systemic activation, potentially reducing off-target effects and enhancing oral drug delivery. This strategy may also be applicable to liver- or lung-targeted prodrug design.