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The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with ␤-tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified ␤-tubulin that had reacted with 3-(chloromethyl-[ Tubulin interacts with a wide variety of small ligands, including divalent cations, guanine nucleotides, and antimitotic drugs. Perhaps because of its instability, its tendency to polymerize into pleomorphic structures, and/or its microheterogeneity, tubulin or tubulin-ligand complexes have not been crystallized. A polymeric site for paclitaxel (1), probably on the ␤-subunit (2, 3), has been defined in zinc-induced sheets of antiparallel protofilaments. Efforts to define ligand binding sites have generally involved induction of covalent interactions between tubulin and specific ligands. Usually photoreactive derivatives of the ligand were used (e.g. Refs. 2-5), but "direct photoaffinity" labeling, in which a covalent bond between ligand and tubulin forms under UV irradiation, has also been a valuable technique (6 -8). In most cases the target amino acids in the covalent interaction between protein and ligand were not identified, with localization of the reactive site limited to the tubulin subunit or a peptide fragment.No ligand has attracted more attention than colchicine (9), whose interaction with tubulin permitted the initial isolation of the protein (10). Photoaffinity (4, 5) and direct photoaffinity (7, 11) studies have provided evidence that both the ␣-and ␤-subunits participate in forming the binding site, as has genetic evidence (12-14). In only one of these studies (11) was label localized to specific peptide fragments of tubulin, and the region of colchicine that reacted with the protein was uncertain.We have taken a different approach from concern that use of bulky photoaffinity groups introduces a paradox into the technique. Adequate affinity of such compounds for tubulin implies that the reactive group has been introduced at a position in the ligand of secondary importance for the binding reaction. Thus, covalent reaction(s) may occur at some distance from the binding site itself. While this problem does not arise with the direct photoaffinity method, the precise ligand-peptide reaction has only been established with GTP (6). We decided to explore the use of small, chemically reactive substituents introduced at defined positions in colchicinoids and allocolchicinoids (15, 16). Schmitt and Atlas (17) had used an analog derivatized with the bromoacetyl group in the B ring side chain, but they found extensive nonspecific covalent reactions between their analog and tubulin. We used the isothiocyanate and chloroacetyl groups (16), and introduced them into the side chain, at position C-9 of t...

The aS,7S-absolute configuration of (-)-colchicine (1) and (-)-N-acetylcolchinol methyl ether (3, NCME) suggested on the basis of 'H NMR data and negative Cotton effects at about 260 nm (EtOH) is firmly established by an X-ray analysis of urea 5, a compound derived from 3. Binding of these compounds to tubulin requires an as-configuration of the biaryl system.

This pelper is cleclicclted lo Dt-. Zdetzc.k (Drtztzy) Vtllet~tcl otr the oc.cnsiotl of his 65th birtlzclc~y 0 . BoyL. A. B~ossr, H. J. C. YEH, E. HAMEI-, B. WEGRZYNSKI. and V . TOOME. Can. J. Chcm. 70, 1237 (1992). Trimethoxy-substituted dihydrodibcnzocycloheptenes 4-7, required for a structure-activity study measuring the inhibition of tubulin polymerization in vitro, were synthesized by four different routes: ( I ) Synthesis of 4 was achieved from 2,3-di~nethoxybenzaldehyde via biphenyl aldehyde 17, chain lengthening to propionic acid 20, acid-catalyzed cyclization toward kctone 21, and removal of the carbonyl group. Mapping and full characterization of the colchicine binding site on tubulin remains a challenging and multifaceted research project (1, 2). Modification of natural (-)-(as, 7 s ) colchicine 1 or the parent compound N-acetylcolchinol methyl ether 2 offers a possibility to gain valuable infor~nation on how these lnolecules bind to tubulin and inhibit its polymerization. Phenolic congeners of colchicine 1, especially I-demethylcolchicine, were found to be less potent than '~u t h o r to whom correspondence may be addressed.colchicine itself (1-4) as inhibitors of tubulin polymerization, with activity partially restored on esterification ( 5 , 6). After the finding of the high potency of 6,7-dihydro-l,2,3,9-tetramethoxy-5H-dibenzo[u,c]cycloheptene (DBCH) 3 (7~11, the systematic synthesis of its deinethoxy derivatives 4-7 was undertaken in order to investigate the contribution of each aromatic oxygen atom in the binding process. The acetamido group in 1 and 2 is not required for binding to tubulin, as indicated by the high activity of compound 3 (7u). The investigation included a synthesis of the optically active amides 8 a , 0 , with the acetamido group at C ( 5 ) . Determination of their absolute configurations, a primary factor in tubulin binding ability, was made by ' H NMR and CD. Can. J. Chem. Downloaded from www.nrcresearchpress.com by 34.212.246.108 on 05/11/18For personal use only.

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Synthesis of deaminocolchinyl methyl ether 9 was achieved from tetramethoxy-substituted biphenyl-2-carbaldehyde 12 via tricyclic ketone 20 and 5,6-didehydro congener 11. Compound 9 was identical in every respect with material prepared from colchicine uiu 6,7-didehydro congener 10. Measuring inhibition of tubulin polymerization in vitro showed compounds 4, 5, and 9-11 of the alloseries of colchicinoids to be particularly potent inhibitors.Introduction. -A large number of synthetic and natural compounds bind to the protein tubulin and, in so doing, inhibit its polymerization in vitro [l]. This property accounts for many of the effects observed when cells are treated with this class of agents, most notably failure of mitotic spindle formation and dissolution of the interphase microtubule network. This disruption of microtubules is probably responsible for the therapeutic properties of antimitotic agents. The majority of compounds which inhibit tubulin polymerization also, despite remarkably diverse structures, competitively inhibit the binding of radiolabeled colchicine to tubulin. This implies that, despite their structural diversity, they share features that allow them to bind at a common site on the protein. The most thoroughly studied member of this class of drugs is colchicine ( = ( S ) -N-(5,6,7,9-tetrahydro-l,2,3,l0-tetramethoxy-9-oxobenzo[a]heptalen-7-yl)acetamide; 1, R' = CH,, R2 = CH,CONH, R3 = CH,O).Because of the antineoplastic potential of agents which inhibit microtubule assembly, a thorough understanding of the colchicine binding site is important. One approach to this problem is evaluation of antitubulin properties of analogs of active drugs. For example, virtually all colchicinoids summarized in structure 1, as well as the dehydro analog 2 [2], inhibit tubulin polymerization and the growth of tumor cells in vitro and, in some cases, in vivo. More drastic changes in the colchicine molecule have surprisingly little apparent effect on most of these inhibitory properties. Complete elimination of ring B yields an antimitotic compound binding at the colchicine site (2-methoxy-5-(2',3',4-trimethoxyphenyl)cyclohepta-2,4,6-trien-l-one (3)) [3] [4]. Conversion of the 7-membered 2-substituted cycloheptatrienone moiety (ring C) to a 6-membered phenyl ring yields compounds more potent than colchicine as polymerization inhibitors (allocolchicine (4) and N-acetylcolchinyl methyl ether 5) [3] [5].

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1%-Chloroacetates of 2-demethylthiocolchicine 7 (specific activity 55.0 mCi/mmol, radiochemical yield 26.1%), and of 3demethylthiocolchicine 8 (specific activity 55.0 mCi/mmol, radiochemical yield 5.7%) were synthesized and found to covalently bind with high specificity to the P-subunit of tubulin. The 1%-chloroacetate of N-acetylcolchinol 5 (specific activity 56.0 mCi/mmol, radiochemical yield 7.8%) and the 1"-isothiocyanate 6 (specific activity 50.0 mCi/mmol, radiochemical yield 32%) were also prepared and found to react covalently with tubulin but in a nonspecific manner. With the radiolabelled chloroacetates 7 and 8 two compounds are now available to further characterize the colchicine binding site on the p subunit of tubulin. 2 94 0. Boy6 et al. colchicine binding site. itself with tubulin,7 reactive moieties have been introduced in the B-ring side chain at position C(7). Except for direct photoaffinity reaction of colchicine 1 R1= H, Rz = NHCWH3 3 R1 =NCS, Rz= NHCOCH3 4 Rl = H, Rz = NCS 2 RI=Rz=OCH3 5 R1= 014COCH~Cl Rz = W H 3 6 R, = NCS, Rz = Oi4CH3 7 R1= 14COCHzC1. Rz = CH, 8 R1= CH3. Rz = 14COCHfll