Chem.-Biol. Interactions, 18 (1977) 163--178 163 © Elsevier/North-Holland ScientificPublishers, Ltd. THIOLYTIC CLEAVAGE OF THE ANTI-TUMOUR COMPOUND 4'-(9-ACRIDINYLAMINO)-METHANESULPHON-m-ANISIDINE (m-AMSA, NSC 156 303) IN BLOOD WILLIAM ROBERT WILSON, BRUCE F. CAIN and BRUCE C. BAGULEY Department of Cell Biology, Universityof Auckland, Auckland and Cancer Chemotherapy Laboratory, P.O. Box 1724, Auckland (New Zealand) (Received April 5th, 1977) (Accepted April 30th, 1977) SUMMARY 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA), a compound with a broad spectrum of experimental anti-tumour activity, was found to nave a short biological half-life in mice bearing L1210 leukaemia. The fate of m-AMSA [3H]-labelled in the acridine nucleus, was determined following injection into mice. There was rapid formation of covalent adducts with plasma proteins. Adducts were also formed in freshly isolated blood samples following incubation at 37°C, and were found to be highly fluorescent. The formation of adducts was accompanied by a decrease in the free thiol con- centration in plasma, and the concomitant addition of radioactivity from [3H] acridine nuclei. Acid or alkaline hydrolysis of the plasma protein adduct liberated acridone, while digestion with a protease produced unstable fluore- scent compounds. A comparison of the rates of acid hy.drolysis of the adducts and of model compounds suggested that the adducts were produced as a result of nucleophilic attack at the C-9 position of m-AMSA by protein thiol groups. The side chain of m-AMSA was liberated as 4-amino-3-meth- oxymethanesulphonanilide. Several congeners of m-AMSA were shown to form similar or identical adducts both in vivo and in vitro, and at rates which correlated with their reactivity towards simple organic thiols. INTRODUCTION The finding that a number of 4'-(9-acridinylamino)methanesulphonanilide Abbreviations: AMSA, 4'-(9-acridinylamino)methanesulphonanilide; m-AMSA, 4°-(9-acri - dinylamino)methanesulphon-m-ani~idide; o-AMSA, 4'-(9-acridinylamino)methanesulphon- o-anisidide: 3°-F-AMSA, 4~-(9-acridinylamino)-3'-fluoromethanesulphonanilide; 3'-Me- AMSA, 4°-(9-acridinylamJno)3°-methylmethanesulphonanilide; m-[3H]AMSA, m-AMSA labelled in the acridine nucleus; TLC, thin-layer chromatography; PBS, phosphate- buffered saline.