Synthesis, Characterization, and in silico Analysis of Cholesterol–Hydrazone Conjugates as Prodrug Models

Natural lipid-based conjugates have emerged as a strategic approach to enhance cell membrane permeability, site-specific intracellular distribution, and achieve controllable drug release. In previous studies, we prepared several lipid-based conjugates with poorly-biodegradable carbamate linkages. Herein, we replaced the carbamate with pH-responsive hydrazone linkages to explore the molecular properties and potential pH-responsive prodrug release behaviour. Accordingly, by reacting a cholesterol–hydrazine intermediate (Chol-NHNH₂) with simple aldehydes and ketones, we facilely synthesized four structurally simplified cholesterol–hydrazone conjugates (Chol–NHN=Alde–Br, Chol–NHN=Alde–OH, Chol–NHN=Keto–Br, and Chol–NHN=Keto–OH₃) as prodrug models. The purity and polarity of the synthesized conjugates was assessed by TLC, and their structures were fully characterized and confirmed by TLC, FT-IR, ¹H NMR, ¹³C NMR, and MALDI-TOF-MS. Their solubility was preliminarily investigated in various solvents. Interestingly, Chol–NHN=Keto–OH₃ formed a transparent organogel in CHCl₃, which might be attributed to the formation of intermolecular hydrogen-bonding networks. An UHPLC method was employed to evaluate the stability and degradability of these cholesterol–hydrazone conjugates under aqueous conditions (pH 4.0 and 7.0, in a THF/CH₃CN/H₂O mixture); while no significant conjugate degradation was observed by UHPLC within 4 days. Finally, in silico calculations were performed to virtually assess the molecular properties and potential pharmacological features of the cholesterol–hydrazone conjugates. The results provided insights on the natural lipid-hydrazone conjugates for potential biomedical applications.