Synthesis of alkoxy-substituted diaryl compounds and correlation of ring separation with inhibition of tubulin polymerization: differential enhancement of inhibitory effects under suboptimal polymerization reaction conditions

Abstract

A number of cytostatic compounds (2-4, 7, and 8), which can be described as "diaryl", inhibit tubulin polymerization, cause cells to accumulate in mitotic arrest, and competitively inhibit the binding of colchicine to tubulin. They differ, however, in the separation of the two aryl moieties. To attempt to understand this variability we prepared a series of analogues modeled on 3 and 4 ("benzodioxole series") and on 7 and 8 ("combretastatin series") which differed only in the number of methylene units (ranging from none to four) separating the aryl moieties. These compounds were evaluated for their effects on tubulin polymerization, colchicine binding, and the growth of L1210 murine leukemia cells. In terms of inhibitory effects on tubulin polymerization, for the combretastatin series there was an optimal separation of the two phenyl rings by a two-carbon bridge (compound 24), with progressively decreasing inhibitory activity when the separation was by one carbon (20), three carbons (25), or four carbons (28) (the biphenyl analogue 16 was inactive). The benzodioxole series, however, did not permit us to generalize this finding, because the least active agents prepared (39 and 40) had a two-carbon bridge, while those with one- (5 and 6) and three-carbon (46 and 47) bridges were nearly equivalent in potency. Submicromolar IC50 values for inhibition of L1210 cell growth were only obtained for compounds 20 (IC50, 0.2 microM), 24 (0.07 microM), and 25 (0.4 microM). While evaluating the effects of these agents on tubulin polymerization, we noted with the combretastatin series and with several standard agents that apparent potency (in terms of IC50 values) was always lower if the reaction was performed at 30 degrees C, with 0.25 mM MgCl2, than at 37 degrees C, with 1.0 mM MgCl2. This enhancement of IC50 values in the former system as compared with the latter was particularly dramatic for the less active agents (e.g., 28) as compared with the more active (e.g. 24).

2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.

“…2A. These suboptimal conditions also yielded significantly lower IC50 values than were obtained with the standard condition for agents that could be examined under both conditions (12). Such a suboptimal condition was useful with 2ME.…”

“…This could assist in the rational design of additional chemotherapeutic agents. Excluding alkyl derivatives of benzimidazole methyl carbamate (23) 12), but it may be useful to discuss these §The critical concentration in the standard system is 0.10 mg/ml. The interactions of highly active colchicine analogs with tubulin have been studied in some detail.…”

“…Purified bovine brain tubulin and H2-CSA4 were prepared as described (11,12). Monosodium glutamate (2.0 M) was adjusted to pH 6.6 with HCl.…”

“…Tubulin polymerization was followed in Gilford recording spectrophotometers equipped with electronic temperature controllers. A drug-tubulin preincubation (0.24-ml reaction mixture) without GTP was performed for 15 (12). Reaction mixtures contained 1.0 M monosodium glutamate (pH 6.6), 0.1 M glucose 1-phosphate, albumin (0.5 mg/ml), 1.0 mM MgCl2, 1.0 mM GTP, and tubulin, [3H]colchicine, and inhibitors as indicated.…”

Cytotoxicity of New Stilbenoids from Pholidota chinensis and Their Spin‐Labeled Derivatives

“…The presence of two C-atoms between two aromatic rings, like in combretastatin A, is thought to be a standard feature essential to their activities [18] [19]. Our previous study of natural dibenz[b,f]oxepins also supported this inference [20].…”