In order to increase the retention of
drug activity, regiospecific coupling has been used to synthesize conjugates of
methotrexate (MTX, 1) with normal rabbit
IgG (NRG) and a mouse anti-human
renal cancer monoclonal
IgG (Dal K-20). MTX gamma-methyl
ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic
acid (2) with suitable
glutamic acid derivatives. The MTX gamma-methyl
ester (4) was then converted to the corresponding
hydrazide 6. An
amide-linked conjugate was formed when the MTX gamma-
hydrazide (6) was converted to reactive acylating species 7 by using tert-
butyl nitrite or
trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native
IgG. A
hydrazone-linked conjugate was formed when MTX gamma-
hydrazide (6) was reacted directly with
IgG that had first been oxidized with
periodate to form polyaldehyde
IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of
dihydrofolate reductase and of colony formation by human
renal cancer (Caki-1) cells than were control nonregiospecific conjugates.