Design, synthesis, and biological evaluation of anthracycline-formaldehyde conjugates

The anthracycline antitumor drug, doxorubicin has been utilized for decades as a broad spectrum chemotherapeutic. Recent literature evidence documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. Targeting a doxorubicin-formaldehyde conjugate specifically to cancer cells may provide a more efficacious chemotherapeutic. The design, synthesis and biological evaluation of a doxorubicin-formaldehyde conjugate targeted to breast cancer cells, is presented. The formaldehyde is incorporated in a masked form as an N-Mannich base linkage between doxorubicin and salicylamide. The salicylamide triggering molecule, previously developed to release an acyclic doxorubicin-formaldehyde metabolite, is tethered via derivatized hydroxylamine ether to a penta-peptide targeting group, acyclic- or cyclic-CNGRC which targets aminopeptidase N associated with tumor cell angiogenesis. The lead compounds, acyclic- and cyclic-CNGRC-DoxSF, showed approximately equal activity to clinical doxorubicin in both in vitro cell studies and in vivo mouse xenograft experiments. These results led to the abandonment of an acyclic doxorubicin-formaldehyde derived conjugate and the pursuit of cyclic doxorubicin-formaldehyde conjugates such as Doxoform.; Doxoform, a dimeric doxorubicin-formaldehyde conjugate with two oxazolidine rings each created with an equivalent of formaldehyde and linked together by a third equivalent of formaldehyde, inhibits the growth of tumor cells at one to four orders of magnitude lower concentration than doxorubicin. We now hypothesize that Doxoform is highly cytotoxic because it serves as a prodrug of a monomeric doxorubicin oxazolidine, Doxazolidine, that directly cross-links DNA. Doxazolidine inhibits the growth of MCF-7, MCF-7/Adr, MDA-MB-435 breast and DU-145 prostate cancer cells as well as Doxoform and at two to four orders of magnitude lower concentration than doxorubicin. Further, Doxazolidine inhibits the growth of these same tumor cell lines at one to four orders of magnitude lower concentration than doxsaliform that serves as a prodrug for an acyclic doxorubicinformaldehyde conjugate. Both Doxazolidine and Doxoform are taken up by multidrug resistant MCF-7/Adr cells three to four-fold better than doxorubicin. A molecular model suggests that Doxazolidine with its daunosamine in a chair conformation can cross-link DNA by direct reaction with a G-base in a tautomeric form with ring opening of the oxazolidine. These results point to Doxoform being a prodrug for Doxazolidine and Doxazolidine being the reactive species that directly cross-links DNA to trigger tumor cell death. Future drug designs will target Doxazolidine to an activating enzyme overexpressed by the cancer cell.