Synthesis and characterization of polyethylene glycol-doxorubicin conjugates containing ?-thiopropanamide linkers for enhanced selective cytotoxicity against breast cancer cells

Doxorubicin (DOX) is a chemotherapeutic agent that suffers from its severe adverse effects due to non-selective cytotoxicity. This study aims to design novel beta-thiopropanamide linkers (-S-CH2-C(R)H-CONH-) for DOX conjugation to promote cancer cell selectivity and cytotoxicity. Two series of DOX conjugates with various beta-thiopropanamide linkers were synthesized and evaluated for their cytotoxic selectivity. The first series contains 7 DOX analogues with different beta-thiopropanamide linkers. The DOX analogues were synthesized using ethyl thioglycolate and acrylic acid analogues to establish a series of beta-thiopropanamide linkages with DOX, and the potential linkers were intended to be incorporated in polymer-DOX conjugates. The other series contains a polymer-DOX conjugate, in which the unsubstituted thiopropanamide (-S-CH2-CH2-CONH-, A) is selected as the linker, which conjugates DOX with methoxypolyethylene glycol (mPEG) to sustain systemic drug release, and to promote stimuli responsiveness and cancer cell selectivity. The mPEG-DOX conjugate with the unsubstituted beta-thiopropanamide linker (P-A-DOX) was synthesized using thiol-functionalized methoxypolyethylene glycol and acrylic acid to establish the beta-thiopropanamide linkage with DOX, using the mPEG-DOX (P-DOX) conjugate as the control. All DOX analogues and conjugates were structurally characterized by NMR and chemical assay. The in vitro hydrolytic stability as a function of pH and cytotoxicity tests (MDA-MB-231 and MCF-7 breast cancer cells vs MCF-10A noncancerous cells) were performed. The DOX analogue (3a) with the beta-thiopropanamide linker (where R = H) improved physicochemical stability while the P-A-DOX and P-DOX conjugates additionally exhibited sustained drug release under physiological pH conditions. Both 3a and P-A-DOX were docked in the active site of cathepsin L closer than P-DOX, which cathepsin L is a cancer enzyme that has a preference to cleave the amide bond to initiate intracellular drug release. 3a was found to enhance cancer cell selectivity. However, P-A-DOX enhanced both cancer cell cytotoxicity and selectivity and the summative effects were translated to enhance cancer cell cytotoxicity and selectivity in the following order: P-A-DOX > P-DOX > DOX. We suggest that the mPEG-DOX conjugate with the unsubstituted beta-thiopropanamide linker can serve as a selective DOX delivery system for breast cancer treatment.