Folate analogues. 33. Synthesis of folate and antifolate poly-gamma-glutamates by [(9-fluorenylmethoxy)oxy]carbonyl chemistry and biological evaluation of certain methotrexate polyglutamate polylysine conjugates as inhibitors of the growth of H35 hepatoma cells.
Representative examples of folate and antifolate poly-gamma-glutamyl metabolites were synthesized via the [(9-fluorenylmethoxy)oxy]carbonyl (Fmoc) chemistry using the KH polyamide resin. Polyglutamate yields were consistently better in all cases compared to the previous Merrifield method, and the crude products were obtained in greater than 85% purity. The symmetrical anhydride (7) derived from alpha-tert-butyl N-Fmoc-L-glutamate (6) was used for the initial coupling of the first glutamate residue to the KH resin and also for subsequent chain elongation. The alpha-tert-butyl protective groups were not labile under the conditions used for the cleavage of the finished peptide from the resin. A series of poly-gamma-glutamyl metabolites of methotrexate (MTX) with a chain length ranging from two to five glutamyl residues were synthesized and coupled with poly(L-lysine) having an average molecular weight of 27,000 and 52,000. Each conjugate was tested for its ability to inhibit the growth of wild type (H35) and MTX transport resistant (H35R) strains of hepatoma cells in culture, the latter having a 100-fold reduced sensitivity to MTX. 4-Amino-4-deoxy-N10-methylpteroylglutamyl-gamma-glutamylpoly (L-lysine) conjugate [MTX(G2)-poly-L-Lys-52000] and MTX(G4)-poly-L-Lys-52000 were among the most active (I50 = 8.0 and 10 nM against H35 cells) MTX-polylysines synthesized to date, and they were somewhat more inhibitory to the transport resistant cells. MTX(G5)-poly-L-Lys-52000 was approximately 1000 times more effective than MTX(G5)-poly-D-Lys-52000 in inhibiting the growth of H35R hepatoma cells in culture, indicating that internal cleavage of the gamma-glutamate chain of the conjugate with subsequent release of MTX or shorter chain polyglutamates of MTX is unlikely to be an important determinant of MTX-polyglutamate polylysine cytotoxicity. The results indicate that MTX-polyglutamate poly(L-lysine) conjugates are taken up by the cells independently of MTX and probably via endocytosis.
Abraham
A
A
Drug Development Laboratory, University of South Alabama Cancer Center, Mobile.
Nair
M G
MG
Kisliuk
R L
RL
Gaumont
Y
Y
Galivan
J
J
D000964:Antimetabolites, Antineoplastic / Q000138:chemical synthesis* / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D055598:Chemical Phenomena; D002621:Chemistry; D002627:Chemistry, Physical; D004351:Drug Resistance; D005110:Extracellular Space / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms; D008727:Methotrexate / Q000031:analogs & derivatives*; D011099:Polyglutamic Acid; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D005493:Folic Acid Antagonists; D011099:Polyglutamic Acid; D008727:Methotrexate