Biochemical Pharmacology, Vol 35, No. 22. pp. 4013-4018. 1986. Printed in Great Britain. 00062952/86 $3.00 + 0.00 Pergamon Journals Ltd. zyxwvut MORPHOLINO DERIVATIVES OF BENZYL- BENZODIOXOLE, A STUDY OF STRUCTURAL REQUIREMENTS FOR DRUG INTERACTIONS AT THE COLCHICINE/PODOPHYLLOTOXIN BINDING SITE OF TUBULIN JANENDRA K. BATRA,* LEONARD JURD~ and ERNEST HAMEL*$ *Laboratory of Pharmacology and Experimental Therapeutics, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and twestern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Berkeley, CA 94710, U.S.A. zyxwvutsrqponmlkjihgfedcbaZYX (Receiued 21 January 1986; accepted 18 April 1986) Abstract-We performed a structure-activity evaluation of the effects of methoxy substituents in the benzyl moiety of a series of morpholinyl Mannich base derivatives of 6-benzyl-1,3-benzodioxol-5-01 (“morpholino compounds”) on the ability of these compounds to inhibit tubulin polymerization in vitro. Structurally these agents are most similar to the natural product podophyllotoxin and, like podophyllotoxin, they inhibited in uitro tubulin polymerization, tubulin-dependent GTP hydrolysis, and the binding of colchicine to tubulin. The benzyl ring (C ring) of these compounds appeared to be analogous to the trimethoxybenzene ring (E ring) of podophyllotoxin (with its methoxy substituents at the 3’, 4’ and 5’ positions), but the morpholino compound superficially most similar to podophyllotoxin (with 3’, 4’ and 5’ methoxy substituents) was the least active in the series. The most potent methoxv- substituted morpholino compounds bear these substituents either at the 2’ and 4’ positions (NSC 37027’1) or at the 2’. 4’ and 6’ positions (NSC 381577). NSC 370277 and NSC 381577 were essentiallv identical i in their inhibitory effects on tubulin polymerization, but the latter compound was considerably more effective as an inhibitor of the binding of colchicine to tubulin. The most active of the monomethoxy substituted compounds bore this group at position 4’. A number of compounds with alternate substituents at this position (in particular, alkyl-substituted amines) also had significant in vitro inhibitory effects on tubulin polymerization. Although the morpholino compounds appear to possess only limited cytotoxicity, these findings suggest possible modifications of the antimitotic benzyl-benzodioxole compounds de- scribed previously [Batra et al., Molec. Pharmac. 27, 94 (1985)] to enhance their antineoplastic activity. Among the many functions of microtubules in euca- ryotic cells is their participation in cell division, for they form the framework of the mitotic spindle. As a consequence, virtually all antimitotic drugs interfere with normal formation of microtubules. This generally occurs through a specific interaction with their major component, the protein tubulin. Although of all antimitotic agents only the vinca alkaloids have a well-defined role in the therapy of neoplastic diseases, a great deal of attention has also been directed at colchicine and its interactions with tubulin. Indeed, the essentially irreversible binding of colchicine to tubulin was an important property exploited in the initial purification of tubulin from mammalian brain [l]. Subsequently a large number of antimitotic compounds have been discovered to have specific interactions with tubulin which include interference with the binding of colchicine to the t Address correspondence to: Dr. E. Hamel, Building 37: Room 6D28. National Institutes of Health, Bethesda. MD 20892. $ Abbreviations: MTPT. 2-methoxy-5-(2’,3’.4’-tri- methoxyphenyl)tropone; MAPS. microtubule-associated proteins; and Mes, 2-(N-morpholino)ethanesulfonate. protein. The most potent agents include the natural products podophyllotoxin [2,3], steganacin [4], and combretastatin [5], and the following synthetic com- pounds: a large number of benzimidazole carbamates including nocodazole [6,7]; the 1-deaza-7,8-di- hydropteridine carbamate NSC 181928 [8]; the bicyclic compound MTPT§ [9]; 3-(l-anilinoethyl- idene)-5-benzylpyrrolidine-2,Cdione (TN-16) [lo]; and a number of derivatives of benzyl-benzodioxole WI. The benzyl-benzodioxole compounds have been of particular interest to us because of their apparent structural analogy to podophyllotoxin, steganacin and colchicine (see Fig. 1) and because their synthesis is relatively facile. This permits a detailed exam- ination of structure-function relationships which should provide insights into key features of the colchicine/podophyllotoxin binding site on tubulin and may assist in the design of optimally active antineoplastic drugs. In this report we describe in vitro interactions of a new series of benzyl-benzo- dioxole derivatives [12] with tubulin. This series of compounds all bear a morpholino substituent on the benzylic bridge carbon between the benzodioxole and phenyl rings (“morpholino compounds”, see Fig. 1). 4013