Benzoylurea Derivatives As A Novel Class Of β-tubulin Ligands: Design & Synthesis, Anticancer Activity And Structure-Activity Relationships

3-(α-Bromopropionylamino) benzoylurea (JIMB01) is a novelβ-tubulin ligand withsmall molecule weight (Chinese patent). The structure of the compound is shown in Figure 1.In the present study, we take JIMB01 as a leading compound for SAR analysis. On the baseof our previous knowledge of the 3-haloacylamino benzoylurea (3-HBU) series, the structuralmodification in this study was focused on the substitutions on the 6- or 5-positions of thearomatic ring, or alteration of the aromatic ring, or attachment of a various formylurea chainsat the 1-position of the aromatic ring. With this strategy, 46 new analogues were designed andsynthesized. The biological activity of the compounds was evaluated as well in the CEMhuman leukemic cells therefore the structure-activity relationship (SAR) of this group ofchemical entities was elucidated accordingly. In addition, the 3-D pharmacophore model ofthe compounds was constructed using computer-assistant program.(1) In despite of the structural change in the substitutions of aromatic ring, alteration of the aromatic ring or formylurea chain attachment, the order of anticancer activity was as following, -CH₍₂₎I≧ -CH₍₂₎Br > -CHBrCH₍₃₎ >> -CH₍₂₎Cl, consistent with the previous observations.(2) The phenyl ring is essential for the activity; and the P-Ï€electron-cloud interaction between the aromatic ring and formyl group is essential; formyl group is auxiliary for the activity.(3) The substitution on the 6-position of the phenyl ring largely affected the anticancer activity of the compounds. 45 and 47 beating 6-fluoro, an isosteric to hydrogen atom, showed the highest potency. 86 and 87 with methylation on the 6-position exhibited a substantial decrease in anticancer activities.(4) The substitutions at the 5-position of the phenyl ring showed very minor effect on the anticancer activity.(5) The N-aromatic substituted benzoylurea derivatives, such as those with 3,4,5 -trimethoxyphenyl (164) and pyridyl (184), showed potent activity in liquid and solid tumor cell lines. Introduction of an aromatic ring (especially pyridine) at the N-end improved the solubility, and therefore offers a potential of making soluble salts.(6) N-substituted phenylurea analogues (3, 11, 16) showed moderate anticancer activities for leukemia, but poor activities for the solid tumor lines.(7) The 3-dimensional pharmcophore models of this series compounds binding atβ-tubulin were analyzed using Catalyst software computation. The optimal pharmacophore model includes two hydrogen-bonding acceptors and two hydrophobic cores. The results were closely correlated with that from biological experiments. The correlation coefficient was 0.98 showing a good prediction. The 3-D pharmcophore model could be the base to design novelβ-tubulin inhibitors.Out ofthe46compounds, ten(3, 14, 16, 45, 46, 47, 54, 64, 164, 184)exhibitedpotent activities in the CEM cells with IC50 values of≦ 1.0 uM, four of which (45, 47, 164,184) showed strong activities in Bel-7402 cells with IC₍₅₀₎ values≦2.05μM. Furthermore,compounds 45 and 47 displayed potential anticancer activities in 9 human tumor cell lines,including CEM leukemia, Daudi lymphoma, Bel-7402 hepatoma, DU-145 prostate, MCF-7breast, PC-3 prostate, DND-1 melanoma, LOVO colon and MIA -Paca pancreatic cancercells with IC₍₅₀₎ values within the range of 0.11-0.30μM.The anticancer efficacy of 45 and 47 was then evaluated in human hepatoma (Bel-7402)in nude mice. The tumor-bearing mice were treated with 45 at the dose of 5 (ip, q2d) or 10mg/kg (ip, q2d), and the inhibition rate was 37% and 52%, respectively. Similarly, the micetreated with 47 at 5 mg/kg (ip, q2d) or 10 mg/kg (ip, q2d) showed a tumor inhibition of 62%or 86% in size, respectively. The average body weight of the mice had no obvious changeafter the treatment. Toxic effects of this regimen were not observed during the treatmentcourse.With respect to the parent compound IAABu, 47 at equal molar concentration showed ahigher tumor inhibitory effect in vivo (57% inhibition by IAABu vs 86% by 47). In addition,the LD₍₅₀₎ of 47 (ip) in the mice was 40.7 mg/kg, higher than that of parent compound IAABu(LD₍₅₀₎, 31 mg/kg). As compound 47 bears fluorine at the 6-position, it presumably possesses alonger halftime (T_(1/2)) in vivo as compared with its parent compound. This could be part of theexplanation for the increased anticancer efficacy of 47 in vivo.Mechanism studies showed that the compound 47 interfered with the microtubuledynamics selectively at the assembly phase, similar to that of vincristine, and had no effect onthe disassembly process. 47 interrupted the formation of mitotic spindles, suspend the cells atM-phase, and then lead the tumor cells to apoptosis after promoting bcl-2 phosphorylation.The 46 new analogues in this study were not previously reported. The physical andchemical information of the compounds has been confirmed by MS,⁽¹⁾HNMR and IR. Thecompounds have been patented in China with the application number of 200610083214.5. Weconsider the compound 47 a promising candidate for preclinieal anticancer investigation.