Design And Synthesis Of Anti-Fibrotic Drugs Based On Collagen Prolyl-4-Hydroxylase In Kidney

The feature of fibrosis is the abnormal deposition of extracellular matrix,and collagen is the main component of extracellular matrix.The content of collagen accounts for about one-third of the total protein in the human body and three-quarters of the dry weight of human skin.The biological synthesis of collagen includes a series of post-translational modification of procollagen.The modification process requires the participation of five enzymes,including three collagenases and two collagen glycosyltransferases.Among the three types of collagen hydroxylase,collagen prolyl-4-hydroxylase(C-P4H)can convert(2S)-proline in the procollagen into(2S,4R)-4-hydroxyproline that is necessary for the stability of the triple helix conformation of mature collagen.Therefore,C-P4H is considered to be one of the important targets for the treatment of fibrotic diseases.At present,the research of C-P4H inhibitors is still in its infancy and there are many common problems in the previously reported C-P4H inhibitors such as:bad cell activity,strong off-target effects,and high cytotoxicity.Therefore,we firstly summarized the structure-activity relationship of the previously reported C-P4H inhibitors.And we designed and synthesized new small molecule compounds,which based on this summary,in order to find new C-P4H inhibitors that have better cell activity.Secondly,the organ-targeting ability of C-P4H inhibitors was one of the keys to the successful development of C-P4H inhibitors.Therefore,this article also studied the connection fragments of renal targeting vectors.The research work of this project mainly includes the following parts:1.Design,synthesis and cell activity screening of the compoundsIn the initial stage of the design of the compounds,the ortho modification strategy of carboxyl group was proposed,which was based on the recognition of the crystal structure complex formed by the C-P4H protein and the known C-P4H inhibitor.The new 4-methoxy-2,2'-bipyridine skeleton structure was designed by introducing a methoxy group at the ortho position of the one-sided carboxylic acid of[2,2'-bipyridine]-5,5'-dicarboxylic acid,which was based on ortho modification strategy of carboxyl group and the structure-activity relationship of C-P4H inhibitors summarized in the literature.Then 21 4-methoxy-2,2'-bipyridine derivatives were synthesized and their anti-fibrotic cell activities were evaluated in HSC-T6 cells.The results showed that:15 of the compounds had different levels of cellular activity.It provides a clear direction for structural optimization for the further design of C-P4H inhibitors with better cell activity.In the depth stage of the design of the compounds.A strategy was proposed which is based on the 4-methoxy-2,2'-bipyridine skeleton,bioisosterism and the ortho or meta modification of one-sided carboxyl group.We planned to use the diaza heterocycles(pyrazine,pyrimidine,imidazole)to substitute 4-methoxypyridine structural fragment to design the new skeleton structures.Considering that pyrimidine has a variety of biological activities,so we mainly investigated the novel2-(2-pyrimidine)pyrimidine skeleton containing pyrimidine ring and synthesized 49new small molecule compounds,and then their anti-fibrotic cell activities were evaluated in HSC-T6 cells.The results of the study showed that the antifibrotic activity of 15 compounds on HSC-T6 was stronger than that of pirfenidone,2,4-PDCA and Bipy55'DC.Among them,ethyl 6-(5-(p-tolylcarbamoyl)pyrimidin-2-yl)nicotinate(3-12q)and ethyl 6-(5-((3,4-difluorophenyl)carbamoyl)pyrimidin-2-yl)nicotinate(3-12u)had the best inhibitory activity on HSC-T6,with IC₅₀ of 45.69?M and 45.81?M,respectively.Finally,we were based on the C-P4H protein inhibitory activity and cell activity of 2-(2-pyridyl)imidazole skeleton,the structure-activity relationship summarized in the literature and the ortho or meta modification strategy of carboxyl group.Then,we designed and modified three series of imidazonaphthyridine skeleton structures to synthesize 60 new small molecule compounds and evaluated the anti-fibrotic cell activity of the compound in HSC-T6 cells.Unfortunately,these compounds have bad cellular activity and bad solubility.The main goal of this thesis is to enhance the cellular activity of the designed compounds because C-P4H inhibitors are generally known to have no cellular activity.In the design,synthesis and cellular selection stage of the compounds,131 novel compounds were synthesized.Among them,2-(2-pyridyl)pyrimidine derivatives(3-12q and 3-12u)showed better anti-fibrotic cell activity.It is proved that the ortho or meta modification scheme of carboxyl group of unilateral pyridine ring is feasible and the results lay a foundation for further research.2.Anti-fibrosis mechanism of compounds 3-12q and 3-12uWe analyzed the cell activity screening results of 131 compounds,and finally selected compounds 3-12q and 3-12u to study the anti-fibrosis mechanism.In this paper,the Sirus Red Staining experiment was used to test the inhibition of compounds3-12q and 3-12u on total collagen;the ELISA experiment was used to test the effect of compounds 3-12q and 3-12u on type I collagen;the content of hydroxyproline was used to determine the inhibition of compounds 3-12q and 3-12u on C-P4H enzyme.The results showed that:1.Compounds 3-12q and 3-12u can reduce the content of total collagen in a dose-dependent manner,and are significantly better than the positive drug pirfenidone.2.Compounds 3-12q and 3-12u can significantly reduce the content of type I collagen,and the effect of compound 3-12q is slightly stronger than compound 3-12u.3.Design,synthesis and in vitro release of glutamine conjugated fragment for renal targeting vectorC-P4H protein is widely expressed in most organs of the human body.The delivery of C-P4H inhibitors to the kidney without affecting the function of other normal organs has become the key of the study of C-P4H inhibitors that can be used in the treatment of renal fibrotic diseases.So this article will study the kidney-targeted carrier ligation fragments.The N⁵-phenylglutamine can be quickly recognized and transformed by the abundant?-glutamyl transpeptidase in kidney.Because of this,we designed and synthesized the fully modified glutamine renal targeting carrier linking fragment,and investigated its ability to release active ingredients of the drug in vitro.The results of the study show that the compound S4682-Gln can stably exist in the PBS buffer solution,and the compound S4682-Gln releases part of the C-P4H inhibitor S4682 in the presence of only?-glutamyl transpeptidase,indicating that the designed new fully modified glutamine carrier structure linking fragment has potential renal targeting effect.However,further experiments found that this compound is unstable in plasma.The research results lay the foundation for further research on kidney-targeted C-P4H inhibitor.In summary,the main goal of this thesis is to enhance the cellular activity of C-P4H inhibitors.Firstly,we proposed the ortho or meta modification scheme of unilateral pyridine ring carboxyl group,which based on the recognition of the reported binding model of C-P4H protein and C-P4H inhibitors.Secondly,we designed five new interrelated skeleton structures and successfully synthesized 131new small molecule compounds,which was based on the ortho or meta modification scheme of unilateral pyridine ring carboxyl group and the structure-activity relationship of all the reported C-P4H inhibitors.These compounds expand the structure types of C-P4H inhibitors.The anti-fibrotic cell activity of the compounds was evaluated by HSC-T6 cells,and two of the compounds(3-12q and 3-12u)showed better cell activity.And they can reduce the content of type I collagen and total collagen in HSC-T6 cells in a dose-dependent manner.In addition,the study of glutamine kidney targeting carrier linking fragments also laid the foundation for the renal targeting of C-P4H inhibitors.However,the further biological mechanism of the compounds and the specific inhibition rate of the compounds on C-P4H protein are still in progress.