Design,synthesis And Biological Evaluation Of Tubulin Inhibitors As Antitumor Agents Targeting The Colchicine Binding Site

Microtubules are formed by the polymerization of a-and β-tubulin heterodimers and serve as an essential component of the cytoskeleton.The dynamic characteristics of microtubule polymerization/depolymerization play important roles in multiple functions in cells.Interfering with the tubulin dynamics can effectively inhibit tumor cell mitosis and tumor growth.Therefore,microtubule has become an effective target for the development of antitumor agents.Currently,the paclitaxel site and vinblastine site binding agents are widely used in clinic as antimitotic agents,however,these drugs have some neurotoxicity and myelosuppression related toxicity.In addition,the appearance of drug resistance,especially the multidrug resistance due to the overexpression of the P-glycoprotein(P-gp),limits their antitumor efficiency and affects their long-term uses.Moreover,these MTAs usually have low water solubility and other chemical cosolvents are needed,and that may cause other issues like allergic reactions in patients.On the other hand,the colchicine site binding agents are generally not the substrate of MDR efflux pump and can overcome the multidrug resistance mediated by P-gp.In addition,the colchicine site binding agents also serve as vascular disrupting agents.In previous study,our group designed a series of novel tubulin inhibitors based on the crystal structures of the colchicine site and the privileged indenopyrazole structures,and discovered the indenopyrazole derivative LL01 as a potent tubulin inhibitor.LL01 was not a P-gp substrate and still effective for multidrug-resistant tumor cells.LL01 showed good in vitro and in vivo antitumor efficiency and pharmacokinetics.To overcome the shortcomings of MTAs used in clinic,this study used LL01 as a lead to find more potent tubulin polymerization inhibitors targeting the colchicine binding site that have high water solubility,low toxicity and are effective for multidrug-resistant tumor cells.The main contents are divided into the following three parts:Part 1.To improve the water solubility of the indenopyrazole derivatives and to funrther investigate the structure-activity relationships(SARs)of indenopyrazoles as the tubulin inhibitors,in this study,we made systematic modifications at the phenolic 6-and 7-positions,and the aniline at the 3-position of the indenopyrazole core.The synthesis of indenpyrazole derivatives was started from 5,6-dihydroxy-l-indenone.After the phenolic hydroxyl groups were selectively protected or substituted,the indenone derivative was treated with LiHMDS or NaH and reacted with a variety of isothiocyanates via the nucleophilic addition to give the respective thioamide intermediate that underwent cyclization with hydrazine hydrate followed by methylation at the indenopyrazole NH to form the 1-methyl-1,4-dihydroindeno[1,2-c]pyrazoles.Further removing the phenolic hydroxyl protecting group and the subsequent introducing substituents and through other transformations,a series of novel indenopyrazole derivatives were synthesized.Based on the different phenolic protective groups(TBS,MOM or their combinations)and the different sequences in introducing the substituents at the 6-or 7-positions,five synthetic routes were designed,a total of 33 indenopyrazole derivatives were synthesized and a series of high active indenopyrazole derivatives were identified.The SARs investigations showed that the substitution of the 6-phenolic hydroxyl with an ethyl was essential for maintaining the activity,while a methyl,a propyl and a larger substituent at this position may affect the binding with tubulin,resulting in a significant reduction or loss of activity.The substituent at the 7-phenolic hydroxyl must be of proper length and contain a hydrogen bond donor,such as a terminal amide,hydroxyl,or amino group,to maintain hydrogen bindings with Ser178a of the a-tubulin subunits.The aniline at the indenpyrazole 3-position interacted with the zone III at the colchicine binding site.A substituent at the meta-position of the aniline was essential for the binding,while a substituent at the para-position of the aniline was detrimental for the activity.The/meta-substituent must contain a heteroatom as a hydrogen bond aceptor,such as an ethoxy,a propionyl or an ester group,to interact with Tyr202β and Asnl67β.For the ester derivatives,the ethyl ester was the best choice,followed by the methyl ester,while an isopropyl ester greatly decreased the activity.Moreover,the replacement of the 3-ethoxyaniline by a 6-ethoxypyridine-2-amine was well tolerated.The hydrochloride of 7-(3-aminopryoxy)-3-(3-ethoxyphenylamino)indenpyrazole derivative 1D08 and 7-(3-aminopryoxy)-3-(6-ethoxypyridine-2-amino)indenpyrazole analogue ID25 had good water solublility(>1 mg/mL).ID08 and ID25 inhibited tubulin polymerization with the IC50 value of 3.65 and 3.13 μM,respectively.They showed strong anti-proliferation activities for a variety of tumor cells including those with taxol resistant with the GI50 ranging from 3 to 15 nM.At the cellular level,ID08 and ID25 could interfere with microtubule and microfilament formation.The EBI competition assay proved that they could target the colchicine binding site.ID08 and ID25 arrested the tumor cells in G2/M phase and induced tumor cell apoptosis.In addition,ID08 could inhibit tumor cell migration and the phosphorylation of cMET and Akt kinases.In HCT116 and HepG2 xenograft mouse models,ID08 or ID25 could effectively inhibit tumor growth more than 50%at the dose of 25 mg/kg by oral administration.In HepG2 xenograft mouse models,ID08 effectively reduced the tumor growth by 54%and 68%at the dose of 5 mg/kg or 10 mg/kg via the intravenous injection for once every two days for 21 days,without obviously effect on animal weights.Therefore,ID08 and ID25 are potential drug leads or candidates for further systematic preclinical evaluation as antitumor agents.Part 2.In this study,the benzofuropyrazole derivatives are designed by the bioisosteric replacement of the methelene at the 4-position of indenpyrazole with an oxygen atom.The 6-methoxybenzofuran-3-one was first prepared by the Hoesch reaction of resorcinol with chloroacetonitrile,followed by the intramolecular cyclization in the present of a weak base and the subsequent methylation of the 6-hydroxyl group.After the treatment of 6-methoxybenzofuran-3-one with LiHMDS,the nucleophilic addition with meta-substituted phenyl isothiocyanates generated the thioamide intermediates which underwent cyclization with hydrazine hydrate to give the benzofuropyrazole derivatives,respectively.In this process,in addition to the formation of the benzofuropyrazole derivatives,the pyrazole derivatives were accompanied due to the partial cleavage of the furan ring,and they were not easily to separate for purification.Thus,only five benzofuropyrazole derivatives and five pyrazole derivatives were synthesized and evaluated for their antitumor cell proliferative activities.All these five benzofuropyrazole derivatives were not active against the breast tumor MCF-7 cell.Only compound BF01 with a m-ethoxyaniline at the 3-position of the benzofuropyrazole potently inhibited the leukemia K562 and the lung tumor A549 cell proliferation with the GI50 values of 0.26 and 0.19 μM,respectively,while the other benzofuropyrazoles showed very weak activity toward these two tumor cells.In contrast,all the five pyrazole analogues were active against these three tumor cells.When the aniline at the pyrazole 3-position was substituted at the meta-position with a methyl ester,the resulting compound PY02 was the most active,followed by those with a substituent of ethoxy,cyano,N-methylformamide,and formamide.PY02 inhibited the K562,MCF-7,and A549 cell growth with GI50 values of 0.021,1.7 and 0.69 μM,respectively.PY02 can inhibit the tubulin polymerization with an IC50 of 7.30 μM.Due to the difficulty in the synthesis and purification of these compounds,further systematic investigation was not continued.However,the the discovery of PY02 as a moderate tubulin inhibitor provided a basis for the development of indazole derivatives as the next generation tubulin inhibitors.Part 3.The pyrazole derivative PY02 is a moderate tubulin polymerization inhibitor.In order to further improve its potency,we applied the comformation-ristriction drug design strategy to merge the benzene ring and the pyrazole moiety in PY02 into the indazole motif,and introduced the 3,4,5-trimethoxyphenyl moiety,a common pharmacophore found in natural tubulin colchicine binding site ligands such as colchicine,CA-4 and podophyllotoxin,into the indazole 3-position bridged by a NH group.To synthesis of these novel indazole analogues,2-fluorobenzoic acid or different-substituted-2-fluorobenzoic acid was first reacted with a variety of anilines in the presence of HATU and triethylamine in DMF to give the respective 2-fluorobenzamide intermediate,which was then converted into the thioamide intermediate by Lawson reagent,and the resulting thioamide was finally reacted with hydrazine hydrate.A total of 23 indazole derivatives were prepared and evaluated for their growth inhibitory activity against human hepatoma HepG2,human colon cancer HCT116,and non-small cell lung cancer A549 cells.The SARs showed that the substitutions at the 6-position of the indazole ring were preferred than at other positions,the methyl and methoxy were the best choices.In addition,the 3,4,5-trimethoxyphenyl at the 3-position of the indazole ring was essential for maintaining the activity.When the 3,4,5-trimethoxyphenyl was replaced by other alkoxy-substituted phenyls,the activity decreased obviously.Among these indazole derivatives,the 6-methylindazole derivative IZ03 and the 6-methoxyindazole derivative IZ06 showed potent anti-proliferative activity against these three cells as well as the human colon cancer HT29 and SW620 cells and the taxol-resistant A549 cells,with the GI50 ranging from 8 to 96 nM.IZ03 and IZ06 targeted the colchicine binding site,inhibited tubulin polymerization,disrupted the tubulin network in tumor cells,arrested tumor cells in G2/M phase and induced tumor cell apoptosis.Moreover,IZ03 and IZ06 also showed vascular disrupting effects.In kinase assay,IZ03 and IZ06 showed little off-target effects against cancer-related kinases.In HCT116 xenograft mouse models,IZ06 suppressed the tumor growth 58.9%at an orally dose of 25 mg/kg without affect:ing the animal weight and was more potent than IZ03 and the positive control 5-FU,indicating it may be a promising lead or drug candidate for future developments.In summary,this study made a systematic investigation on the SARs of the indenopyrazoles as tubulin polymerization inhibitors.The indenopyrazoles ID08 and ID25 are potent MTAs and have high water solubility and are effective against the multidrug resistance tumor cells,thus overcoming the common shortcomings for most of the MTAs.Based on the investigations of benzofuropyrazole derivatives and the discovery of pyrazole derivative PY02 as a moderate tubulin inhibitor,a series of indazole derivatives were further designed,synthesized and discovered the indazole derivative IZ06 as a potent MTA.These highly potent tubulin inhibitors,especially the water soluble indenopyrazoles ID08 and ID25,displayed good in vitro and in vivo antitumor efficiency and will continue to be studied on their pharmacokinetic,toxicological and other preclinical systematic investigations to provide a base for their clinical applications as antitumor agents.