A mechanistic and kinetic study of the E-ring hydrolysis and lactonization of a novel phosphoryloxymethyl prodrug of camptothecin.
OBJECTIVE
This study was done to determine the E-ring hydrolysis and lactonization mechanism of a water-soluble 20-phosphoryloxymethyl (POM) prodrug of camptothecin (P-CPT). Specifically, the role of the phosphate group in facilitating E-ring hydrolysis was examined.


METHODS
Resolution between the lactone and carboxylate forms of P-CPT and camptothecin (CPT) was achieved with a RPHPLC assay using UV-visible detection. E-ring P-CPT hydrolysis and lactonization kinetics were followed using 20 mM acetate or phosphate buffer (micro = 0.15 NaCl) over the pH range of 4 to 8 at 25.0 degrees C. A kinetic solvent isotope effect (KSIE) study was used to further probe the mechanism of E-ring hydrolysis.


RESULTS
The hydrolysis and lactonization reactions followed pseudo-first-order kinetics in the approach to equilibrium. The equilibrium ratio of the open and closed forms of P-CPT was dependent on pH, with the closed form dominant at low pH and the open form dominant at high pH. Buffer concentration changes had little to no effect on the rate of P-CPT E-ring hydrolysis. The KSIE study provided an overall isotope effect of 2.47 and a proton inventory KSIE consistent with an intramolecular general base catalysis.


CONCLUSIONS
P-CPT has a pH-dependent equilibrium between the lactone and carboxylate forms similar but not identical to that of CPT. The results suggest a hydrolysis reaction mechanism that involves a single site hydrogen exchange facilitated intramolecularly by the dianionic phosphate moiety of P-CPT via either general base catalysis of the lactone ring attack by water or breakdown of the tetrahedral intermediate.
Hanson
Bradley A
BA
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA.
Schowen
Richard L
RL
Stella
Valentino J
VJ
D002166:Camptothecin / Q000737:chemistry / Q000493:pharmacokinetics*; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry / Q000493:pharmacokinetics*; D019563:Mechanics; D011355:Prodrugs / Q000737:chemistry / Q000493:pharmacokinetics*
D007783:Lactones; D011355:Prodrugs; D002166:Camptothecin