ACTIVATION AND CYTOTOXICITY OF 2-ALPHA-AMINOACYL PRODRUGS OF METHOTREXATE

In an effort to improve the selectivity of the anticancer drug methotr exate (MTX), a series of potential prodrugs in which the 2-amino group was acylated with various alpha-amino acids (as well as L-pyroglutami c acid) was synthesized. Such derivatives are anticipated to be hydrol ysed to MTX by appropriate aminopeptidases localized (over-expressed n aturally or targeted as anti-tumor antibody conjugates) in the vicinit y of the tumor. The L-leucyl, L-valyl, L-isoleucyl, D-alanyl and L-pyr oglutamyl derivatives were assessed as to their suitability as prodrug s. Except for the L-pyroglutamyl compound, all derivatives decomposed slowly when incubated in phosphate buffer, pH 7.3; the formation of MT X was minimal. No major differences were observed when serum was inclu ded in the incubation medium, except for the L-leucyl compound, which was hydrolysed to MTX. The L-leucyl, L-valyl and L-isoleucyl derivativ es were hydrolysed readily to MTX by aminopeptidase M (EC 3.4.11.2), w hile the L-pyroglutamyl and D-alanyl compounds were activated by pyrog lutamate aminopeptidase (EC 3.4.19.3) (from Bacillus amyloliquefaciens ) and D-aminopeptidase (from Ochrobactrum anthropi), respectively. Whe n tested for inhibition of the target enzyme dihydrofolate reductase ( DHFR; EC 1.5.1.3), 2-L-valyl-MTX showed inhibition two orders of magni tude poorer than that given by MTX, in agreement with the expectation that acylation of the 2-amino group reduces binding to DHFR. After tre atment of this derivative with aminopeptidase M, the extent of inhibit ion correlated with the amount of MTX formed. MTX derivatives alone or in combination with the complementary peptidase were tested for cytot oxicity on murine L1210 cells in culture. The above-listed derivatives were considerably less cytotoxic than MTX, except for the L-leucyl de rivative which showed considerable cytotoxicity. When the appropriate exogenous peptidase was included, the cytotoxicity of the activated pr odrugs approached that of MTX. These results indicate that 2-L-leucyl- MTX is unsuitable as a prodrug since it is activated prematurely by se rum enzymes. Although the L-valyl and L-isoleucyl derivatives do not h ydrolyse to MTX in serum and are readily activated, they are not ideal prodrugs since they decompose under physiological conditions; the pro perties of the decomposition product will have a bearing on the ultima te suitability of these compounds. 2-L-Pyroglutamyl-MTX is the best ca ndidate prodrug, showing stability and ready activation by the appropr iate aminopeptidase.