br The di Cu salt

The di-Cu salt of 5, which was prepared from the corresponding di-Li salt of 5, was reacted with the corresponding methoxyiodobenzenes under Ullmann-type coupling conditions to give diaryl-m-carborane derivatives 4a and 4b (Scheme 2).13 To investigate the steric and electronic effects of a B-iodinated m-carborane cage on the anti-cancer activity, we designed and synthesized 9-iodo-m-carborane derivative (2 g) (Scheme 3). Compound 2 g was
Scheme 2. Synthesis of diaryl-m-carborane derivatives 4. Reagents and condi-tions: a) n-BuLi, dimethoxyethane, then CuCl, pyridine, Ar-I, 37–56%. 
Compound X Y R1 R2 R3 R4 R5 GI50 (μM)a 1a CH(OH) H H CH3O CH3O CH3O H 8.6 2a CH2 H H CH3O CH3O CH3O H 6.5 2b CH2 H CH3O H H H CH3O 34 2c CH2 H H CH3O CH3O H H inactive 2e CH2 H H CH3O OH H H 15 2f CH2 H H H NO2 H H inactive 2g CH2 I H CH3O CH3O CH3O H 12 3a CO H H CH3O CH3O CH3O H 4.8 4a
H CH3O H CH3O H H 45 COL
a GI50 values represent the average of at least three individual measurements (n = 3).
synthesized by the same method as that described for the synthesis of 2a using 9-iodo-m-carborane (6)14 instead of 5.
2.2. Biological evaluation of synthesized compounds 2–4
First, the cell growth inhibitory activities of the synthesized com-pounds at specific concentrations (100 μM−0.1 μM) were evaluated using MDA-MB-453 cells.15 Table 1 summarizes the GI50 values of the test compounds. COL, CA-4, and paclitaxel (PTX), a tubulin depoly-merization inhibitor, 16 were used positive controls. Compound 2a showed more potent cell growth inhibitory activity than lead com-pound 1a. It seems that the hydroxyl groups of 1a negatively affect the growth of MDA-MB-453 cells. Dimethoxy derivative 2b showed much weaker anti-cancer activity than 2a, and 2c was inactive. Compound 2e, which has a methoxy and a hydroxyl group, also exhibited weaker activity than 1a. The results suggested that a decrease of the number of methoxy groups on the benzene ring significantly reduced the cell growth inhibitory activity. On the other hand, CORM-3 2f, containing nitro group with the different physicochemical properties from methoxy group, was inactive. 9-Iodo-m-carborane derivative 2 g showed weaker activity than the parent compound 2a. Therefore, in-troduction of iodine atom at the 9 position of the carborane cage ne-gatively affected cell growth inhibitory activity. Benzoyl derivative 3a having a GI50 value of 4.8 μM showed the most potent anti-cancer ac-tivity among the synthesized compounds. Diaryl-m-carborane deriva-tive 4a exhibited much weaker anti-cancer activity than 1a, although it has a trimethoxyphenyl moiety. It is reasonable that dimethoxy
Fig. 3. Tubulin polymerization inhibitory activity of 2a and 3a at 10 μM and 20 μM. DAPI emission at 450 nm (λex = 360 nm) was measured in intervals of 1 min. The fluorescence intensity represents the average of two experiments (n = 2).
derivative 4b had very weak anticancer activity and could be con-sidered inactive. We suggested that the linking group X played an im-portant role in flexibility control rather than in some interactions with the target protein.
2.3. Tubulin polymerization inhibitory activity of selected compounds 2a and 3a
As the most potent derivatives 2a and 3a have a trimethoxyphenyl moiety as observed in various tubulin inhibitors,17 one of the target proteins for both compounds was expected to be tubulin. Therefore, we next examined the effect of 2a and 3a on tubulin polymerization using a fluorescence assay kit (Cytoskeleton). Tubulin polymerization was measured by means of the fluorescence indicator 4′,6-diamidino-2-phenylindole (DAPI), which binds to polymerized tubulin with en-hancement of fluorescence intensity. Fig. 3 summarizes the time-course analysis of tubulin polymerization in the presence or absence of the test compounds. COL and CA-4 were used as positive controls for tubulin polymerization inhibition. Furthermore, PTX was used as a positive control for tubulin stabilization. This assay system showed that COL and CA-4 were potently inhibited tubulin polymerization process with at the concentration of 10 μM and that PTX was stabilized that process at the same concentration as COL and CA-4. Unfortunately, 2a showed no tubulin polymerization inhibitory activity even at the concentration of 20 μM. Thus, it was determined that the target molecule of 2a was not tubulin. On the other hand, 3a inhibited tubulin polymerization at 20 μM, although weak inhibition was observed at 10 μM. This suggested at least that one of the target proteins of 3a is tubulin. However, the concentration of 3a for tubulin polymerization inhibition was not the same as that for cell growth inhibition. Therefore, compound 3a would act on tubulin as well as other target molecules.