Design And Synthesis Of Small Antitumor Agents Acting On The Colchicine Site Of β-Tubulin

Malignant tumors represent one of the most common human diseases that seriously threaten the life of the people.Internationally,there are more than 10 million new cancer cases and more than 7 million cancer-related deaths reported each year,making it urgent to take efficacious measures to prevent,detect and treat tumor.Antitumor agents have been one of the major methods to combat against this fatal disease,prolonging the life time and improving the life quality of the patients.But searching for the effective drugs with simple structure and proper physiochemical features is always the pursuing goal of many researchers.As an important cellular component in all the eukaryotic cells,microtubules have many key biological functions,including intracellular transport,morphogenesis,motility and cell division.Structurally,microtubule is composed ofα-βtubulin heterodimers.Targeting at microtubules by inhibiting or inducing the assembly form its subunits(α-βtubulin) by several agents will result in the apoptosis of the cell.Tumor cells acquire unlimited replicative potential and extremely dependent upon microtubule and are vulnerable to the drugs that bind at microtubules.Additionally,proliferating endothelial cells that form neovasculature in the tumor are also sensitive to tubulin-binding agents.These findings suggest that microtubules will continue to be among the most promising cancer targets for the development of new anticancer drugs.According to the different binding areas,the microtubule binding agents can be mainly divided into three classes:the taxol-site agents,the vinblastin-site agents and the colchicine-site agents.But in general,the molecular structures of the last class compounds are much simpler than those binding bind in the other two domains.The simplicity of these molecules offers promise for the rational design of antitubulin agents.Hitherto,many diverse colchicine-site inhibitors(CSI) have been discovered(i.e.,Indonacin,CA-4,E7010 and Curacin A),which have proven or potential utility as antitumor drugs.Although large number of compounds has been reported,the research method on the inhibitors still focuses on the traditional protocols,e.g.the random screening or the structural modification.The research based on the structures of the protein or the inhibitors was rarely revealed.Furthermore,despite of bearing high activity,the further application of these found agents was interfered by the poor water solubility,severe site effects and acquired drug resistance.For the above reasons,the colchicine site on the microtubule was selected as the target to find novel antitumor agents in this study.By analyzing the structure of the binding site and simulating the binding modes with its agents,the properties of the sub-regions of the site,the key binding residues and potential binding areas were determined.Derived from the active conformations and the available structural-activity relationship of the reported inhibitors,the structural model of the agents was built,and the other structural factors affecting the binding affinity were also proposed.Based on the above results,a systemic research platform from the views of the receptor and the ligand was constructed.Directed by this platform,several novel scaffolds were designed and then selected on the principle of synthesis easiness and binding energies.Next,proper synthesis routes and modification were selected.Finally,in vitro biological test was conducted on all the compounds.Ⅰ.The research of the colchicine binding site on the microtubules1.The properties of the subregions of the binding siteThe multiple copy simultaneous search(MCSS) methodology was used to explore the binding site.The results showed that the binding site could be divided into three hydrophobic pockets(Ⅰ,ⅡandⅢ) and three polar regions(Ⅳ,ⅤandⅥ).The pocketⅠandⅡare the main distribution areas for the hydrophobic probes,but inⅡthe aromatic groups are superior to others,and preferred the vertical conformations. PocketⅢlocated in the deep bottom of the binding site,only the groups with smaller size were accepted but with higher energy.The regionⅣandⅤwere responsible for the almost the polar groups,while the latter favorite the hydrogen bond acceptor groups. Several polar probes were found inⅥ,but with higher energies.The electrostatic potential of the binding site was revealed by Delphi,which showed that nearly all the site was nearly neutral,while the bottom of theⅠandⅥwere exhibited as negative charge,the regionⅣandⅤwere represented as positive charge.This result was in accordance to the finding of MCSS,and to the conformation of the colchicines in the crystal structure. 2.The binding modes between the tubulin and the inhibitorsSimulated in the docking software AutoDock and Affinity,the binding modes were studied.The active conformations showed the agents mainly depended on the hydrophobic interaction with the pocketⅠandⅡ,and the hydrogen bond with the regionⅣin binding with tubulin.The larger hydrophobic part of the agent located in the pocketⅠ,with the hydrogen bond acceptor(O) or the aromatic ring polar bonding;with the -SH of theβ-Cys241.The planar hydrophobic part inserted in the pocketⅡ,hydrogen bonding with theα-Ser178 andα-Thr181.The soft docking study was further applied in the Affinity moduler on the conformations obtained from AutoDock,to investigate the movement of the residues lineing the site.Only the conformations of the side chains of the major binding residuesβ-Cys241,β-Leu248,α-Thr179,α-Val181andβ-Asn352 had changed much,due to the motion of neighbouring groups of the ligands.3.The key binding residues in the binding site and the potential binding areaAccording to binding energy and the number of copies of the distributed groups in the MCSS calculation,theβ-Cys241 in the pocketⅠand theα-Ser178 andα-Thr181 in the regionⅣmight be necessary for binding.By docking simulation,tire above three residues were almost involved in the binding with the agents.So they were identified as the key residues.Though not involved in binding,the polar regionⅤwas suggested as a potential binding area by the distribution of hydrogen bond acceptor groups in the MCSS.While the cluster of the backbone-NH in V and the enclosed broad space also present its influential role in binding.The other two unoccupied pocketsⅢandⅥwere just under the important hydrophobic pocketⅠ.They might provide some new strategy for modification on the groups locating in the pocketⅠ.Ⅱ.The structural features of the colchicine site inhibitors 1.Proposition of the two dimensional structural features and the structural scaffoldHinted by the common groups in some of the inhibitors,a coarse structural feature was proposed.According to the properties,the common groups were expanded to the other groups.Then the structural feature was modified,which is:two hydrophobic groups (presented as part A and B) were connected by a bridge part that is composed of 1-4 atoms.One group may be similar to TMP,the other should similar to 2-methoxyphenol. The structural scaffold was also supposed,furthermore three detained scaffolds were provided.2.Construction of the three dimensional pharmacophoreAccording to their binding conformations,the structures of the inhibitors were divided into three parts,namely A,B and the bridge between them.The pharmacophore of the inhibitor(the hydrophobic centers H1 and H2,the hydrophobic group H3,the hydrogen-bond acceptors A1 and A2 and the polar atom P) was built.In the pharmacophore,H1 and A1 located in the hydrophobic pocketⅠ,polar bond could be found between A1 andβ-Cys241;H2 and H3 was buried in pocketⅡ;P was in polar regionⅣby hydrogen bonding withα-Ser178 orα-Thr181;A2 was in the vicinity ofβ-Ala250 in the regionⅤ,but no obvious interaction was identified between them.Another hydrogen bond acceptor A3 was proposed between A2 and regionⅤ.3.The other structural factors affected the binding affinity of the agentsBy combining the reported modification results and computer simulation,the structural factors affected the activity were enclosed.The factors included the volume size of H1,and the planarity of H2,while the bridge part should be in rigid form to maintain parts A and B to be in the same side of the bridge(in cis-conformation).A potential hydrogen-bond acceptor A3 was proposed between A2 and the loop area forming the regionⅤ.Ⅲ.Design of lead compoundsBased on the above results,the lead compounds were designed.Firstly,according to the properties of the structural elements of the pharmacophore,the corresponding proper groups were selected.But their positions in the site were adjusted in MCSS.Then diverse linkage parts were designed to link the elements by LUDI screening.By optimization and docking,the binding conformations of diverse lead scaffolds were simulated.At last,the final structure was determined by the binding conformation,the binding energy,the LUDI score points and the numbers of hydrogen bonds.During design,the following factors were taken in account:complied with the pharmacophore that composed of six elements; complied with the structural model that the scaffold should contain the A,B and the bridge part;the linkage part should keep the groups in theⅠandⅡin the cis-conformation;the hydrophilic groups was introduced to interacted with the key residues,especial the residues in potential binding areaⅤ;the synthetic accessibility and the modification margin.Finally, the imidazolones were selected as the lead compound.Ⅳ.Synthesis and structural modification of the lead compoundBased on the idea that the structural model was composed of three parts(A,B and the bridge),the lead compound could be synthesized by these three parts sequentially.That is, the bridge part was firstly added to the part A,and then the part B was added.In planning the synthetic route,the following factors were considered:the reaction condition should be as mild as possible;the reagent should be available;the structural modification should be easily managed.Modification was conducted on part B.The position,the number,the volume size and the electronic acceptor/donor of the substituents on the aromatic were all studied.Finally,more than 20 compounds were synthesized,which were also revealed to be firstly reported by searching the database.Furthermore,the synthesized mthods of the key intermediate was modified.Ⅴ.Biological assayCytotoxicity activity,antivascular activity and Tubulin polymerization assays were conducted to explore the effect of diverse substituents on activity with MTT method.The results showed that nearly all the compounds could inhibit the growth the six tumor cell types effectively.The electron donor groups and the hydrogen bond donor groups could enhance the activity.Also the substituents in the m-position were advantageous than the p-position.