Design,Synthesis And Bioactivity Study Of Imidazo[4,5-c] Quinoline And β-Amino Alcohol Derivatives

For many years,malignant tumor has been a serious threat to human health and life safety.Studies have shown that high frequency of activation mutations in the genes encoding PI3K,AKT and mTOR in cancer patients leads to increased activity of this pathway,which promotes the growth,survival and angiogenesis of tumor cells.PI3K/AKT/mTOR signaling pathway is involved in the proliferation,growth,cell transformation,angiogenesis,glucose metabolism and DNA repair of malignant tumors,and plays a very important role in the occurrence and development of malignant tumors.At present,pharmacists are working on novel small molecule inhibitors targeting key kinases in the PI3K/AKT/mTOR signaling pathway.It has been reported in the literature that the imidazo[4,5-c]quinoline framework is a specific structure that acts on ATP site associated kinases,and this chemical structure has been used and optimized as a novel regulator of the PI3K/mTOR pathway.NVP-BEZ235,a dual inhibitor of PI3K/mTOR,and its derivatives BGT-226and PF-04979064 were developed by pharmaceutical chemists.Although these compounds have strong kinase inhibitory and cell inhibitory activities,they still have problems such as poor water solubility,unsatisfactory metabolokinetic properties,adverse reactions and toxicity.Therefore,it is of practical significance to develop small molecule protein kinase inhibitors with good water solubility,strong activity and lower toxicity.Hexocyclic complexes such as pyridine,pyrimidine,quinoline and quinazoline are common structural features of kinase inhibitors in which the Nitrogen atom acts as the key hydrogen bond receptor and binds to amino acid residues located in the hinge region of protein kinase.In this study,several aromatic heterocyclic substituents,such as benzene,pyridine,quinoline and pyrazole,were introduced into the C-8 region of the imidazo[4,5-c]quinoline skeleton to assess the importance of their antitumor activities.The N-1 site is also a variable structural modification site,and different substituents such as benzene ring and piperidine ring are introduced.Four series of analogues based on the framework structure of imidazolinone[4,5-c]quinoline were designed and synthesized,and their inhibitory activity of protein kinase and anti-tumor cell proliferation in vitro were measured.It is expected to synthesize PI3K/mTOR small molecule inhibitors with novel structure and better activity,establish a compound library,and study the structure-activity relationship of the compounds containing this skeleton structure.In this paper,thirty-two derivatives containing imidazo[4,5-c]quinoline skeleton structure were designed and synthesized.Their structures were confirmed by ¹H NMR,¹³C NMR and HRMS.The compounds were all new and had not been reported in literature.The results showed that most of the compounds had good activities in luciferase reporter gene assay,and the IC₅₀values on HIF-1αin tumor cells under hypoxia condition were less than 100 nmol/L.Among them,compound 17d(IC₅₀=28.3 nmol/L)with“2,3’-bipyridyl”at the C-8 position and compound 28a(IC₅₀=35.5nmol/L)with“4-（1-acetylpiperidine-4-yl）phenyl”at the N-1 position had excellent activities.MTT assay was used to determine the effects of the compounds on the activity of Hep3b cells under normal oxygen conditions.The results showed that none of the compounds exhibited significant cytotoxicity,suggesting that the inhibition of HIF-1αtranscriptional activity was not caused by the toxicity of the compounds to cells.Meanwhile,the inhibitory activities of mTOR and PI3Kαkinases in vitro were detected.All compounds exhibited certain inhibitory activities against both kinases.Compounds 17a,17d,17f,28a～28e,36a～36e and 44a～44e exhibited"double"kinase inhibitory activities against mTOR kinase(IC₅₀<10 nmol/L)and PI3Kαkinase(IC₅₀<100 nmol/L).Then,17d,the compound with the best HIF-1αinhibition effect,was further evaluated by Western-blot assay,Ed U assay,cell migration ability and colony cell formation assay,which showed good activities.In addition,molecular docking experiment showed that both 17d and 28a could well interact with the binding pocket of PI3Kγreceptor protein,which may play an anti-tumor role by inhibiting the related receptor protein.Compounds 17d and 28a showed good bioactivities,which provided a good starting point for the exploration and development of PI3K/mTOR dual inhibitor.At present,further experimental studies on druggability is being carried out.The structure-activity relationship analysis,combined with the comparison of HIF-1αinhibition activity,showed that the introduction of C-8 electron-absorbing fluorine atom was positively correlated with the inhibition activities of the compounds.When“2,3’-bipyridyl”or“2,4’-bipyridyl”is substituted at C-8 position,its activity is stronger than that of“（6-phenyl）pyridyl”.When substituted by“2,3’-bipyridyl”,its activity was higher than that of“2,4’–bipyridyl”.In addition,the activity of compounds introducing bicyclic substituents at C-8 site was higher than that introducing tricyclic substituents,suggesting that appropriate reduction in the volume of substituents could improve the activities of the compounds,which might be more conducive to matching with the active pocket of the receptor target protein.Different substituents were introduced at N-1 site,and the general structure activity relationship was-COCH₃>-COCH₂OH>-SO₂CH₃>-COCH（CH₃）OH>4-triazole formyl.In addition,the introduction of triazole,pyrano,oxazine or cyclobutyl on the N-1 of piperidyl,1,2,3,6-tetrahydropyridine-4-yl or 8-azabicyclic[3.2.1]oct-2-ene-3-yl in the parent structure has a negative effect on HIF-1αinhibition activity,resulting in a decrease in HIF-1αinhibition activity.Immunosuppressant is a kind of drug commonly used in the treatment of rejection of transplantation and autoimmune diseases.Most of the immunosuppressants currently used in clinical practice have low selectivity,and long-term use will reduce the body’s normal immune response and induce infection.FTY720 is a new type of immunosuppressant developed in recent years.In this study,FTY720 was modified to reduce its toxicity and improve its physical and chemical properties,and further determined as the necessary group of immunosuppressive activity.In this paper,five newβ-amino alcohol derivatives containing ether or thioether at C-7 position were synthesized.The structure of the target compound was confirmed by¹H NMR,¹³C NMR and HRMS.Their excitatory activity of Sphingosine1-phosphate receptor type 1（S1P₁）were evaluated by[³⁵S]GTPγS binding assay.All the compounds showed stimulative effect on S1P₁receptor,among which compound54(EC₅₀=0.698μmol/L)had the best activity,which may be developed as a safer and more effective immunosuppressive drug for autoimmune diseases and organ transplantation.At the same time,the chiral separation of the racemes ofβ-amino alcohols 87～89 described in our earlier work was performed with chiral separation reagent.High purity optically active isomers were obtained.The activity of dextral isomers was slightly better than that of left-handed isomers in all the compounds,but there was no significant difference between them.It is suggested that the synthesizedβ-aminoalcohol derivative dextrorotal may be a more active S1P₁agonist immunosuppressant,but further biological activity experiments are needed to confirm this.