Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities

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Abstract
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 conjugates was formed when the MTX .gamma.-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoracetaldehyde, 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 somewhate more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.