The recent discovery that the
formaldehyde conjugates of
doxorubicin and
daunorubicin,
Doxoform and
Daunoform, are cytotoxic to resistant human
breast cancer cells prompted the search for hydrolytically more stable
anthracycline-
formaldehyde conjugates.
Doxoform and
Daunoform consist of two molecules of the parent
drug bound together with three methylene groups, two forming
oxazolidine rings and one binding the oxazolidines together at their 3'-amino nitrogens. The 4'-epimer of
doxorubicin, epidoxorubicin, reacts with
formaldehyde at its amino alcohol functionality to produce a conjugate,
Epidoxoform, in 59% yield whose structure consists of two molecules of epidoxorubicin bound together with three methylene groups in a 1, 6-diaza-4,9-dioxabicyclo[4.4.1]
undecane ring system. The structure was established from spectroscopic data and is consistent with products from reaction of simpler vicinal trans-
amino alcohols with
formaldehyde.
Epidoxoform hydrolyzes at pH 7.3 to an equilibrium mixture with dimeric and monomeric epidoxorubicin-
formaldehyde conjugates without release of
formaldehyde or epidoxorubicin. The hydrolysis follows the rate law (A if B) if C + D where A (
Epidoxoform) is in rapid equilibrium with B, and B is in slow equilibrium with C and D. The forward rate constant for A/B going to C+D gives a half-life of approximately 2 h at 37 degrees C. At equilibrium the mixture is stable for at least 2 days. At pH 6.0, hydrolysis proceeds with first-order kinetics to epidoxorubicin and
formaldehyde with a half-life of 15 min at 37 degrees C.
Epidoxoform and epidoxorubicin plus
formaldehyde react with the self-
complementary DNA octamer (GC)4 to yield five
drug-
DNA adducts which have structures analogous to the
doxorubicin-DNA adducts from reaction of
Doxoform with (GC)4.
Epidoxoform is 3-fold more toxic to MCF-7 human
breast cancer cells and greater than 120-fold more toxic to MCF-7/ADR resistant cells than epidoxorubicin.
Epidoxoform in equilibrium with its hydrolysis products is greater than 25-fold more toxic to resistant cells with respect to epidoxorubicin.