Design,Synthesis And Evaluation Of Small Molecules To Induce The Differentiation Of Mesenchymal Stem Cells For Cartilage Repair In Osteoarthritis

Osteoarthritis(OA)is a kind of degenerative joint disease that the clinical manifestations are articular cartilage destruction,ultimately leads to disability and even death.With the increase of population aging and obesity,and the society’s lack of attention,the incidence of OA is becoming higher and higher.OA is estimated to become the global forth most disabling disease by 2020,and the increasing incidence of OA and lack of targeted drugs will also bring a huge gap in the market.Therefore,the development of targeted drugs for OA treatment is imminent.However,the current treatment for OA is still focusing on relieving the progression of this disease through surgery,or using drugs to relieve pain and symptoms.Once the disease got further deterioration,total joint replacement surgery is the only option.In recent years,the vigorous development of disease-modifying osteoarthritis drugs(DMOADs)has made some progress in OA clinical trails,many problems still exist and make these drugs not yet appropriate for marketing.Therefore,developing a new class of OA therapeutic drugs has important research significance and huge market value.Due to the special nature of joints,the regeneration of articular cartilage is difficult once damaged.With the development of tissue engineering and regenerative medicine,promoting the selective differentiation of pluripotent mesenchymal stem cells(MSCs)into chondrocytes has been proved to promote the repair of damaged cartilage,and then achieving the purpose of treating OA.The new stem cell therapy drug Cartistem is the only OA stem cell therapy drug that achieved state approval in the world.However,due to the limitations of stem cell drugs and the heterogeneity of OA,the development of small molecule drugs for promoting the differentiation of MSCs into cartilage seems to be a better choice.Kartogenin(KGN)is the first reported small molecule drug that can promote the cartilage differentiation of MSCs and cartilage repair.It has shown excellent effects in a series of preclinical studies.However,due to the defects of the molecule itself,KGN is difficult to carry out on clinical stage.Therefore,developing a class of small molecule drugs that can promote the differentiation of MSCs into cartilage with high efficiency,low toxicity and good druggability has become a new direction for the treatment of OA.In order to develop new small molecule inducers for chondrogenesis,a preliminary screening of the internal compound library of our laboratory was implemented.Fortunately,we discovered a small molecule named compound A with the potential to induce chondrogenesis.For determining its cartilage repair ability,we designed a series of in vitro experiments to prove that Compound A has the ability to induce chondrogenesis in concentration-dependent and time-dependent manner,and the ability to induce the migration of BMSCs in a concentration-dependent manner.Subsequently,we carried out an in vivo experiment and found that Compound A can significantly reverse the process of cartilage damage in mice acute injury surgery(DMM)model with the similar efficacy of KGN.Based on the above results,we decided to consider Compound A as the hit compound.With the stragety of skeletal transition and bioelectronic isostery,we fully explored the structure-activity relationship of each functional group of the compound,and then designed and synthesized a series of N-phenylthiophene-2-formamide compounds.The structure-activity relationship shows that introducing a hydrophobic group at the3-position of the benzene ring is beneficial for the improvement of the biological activity of the compound.The introduction of substituents on the thiophene ring is also beneficial for improving the activity of the compound.Especially,the introduction of an amide bond at the 5-position of thiophene ring with amide bone and a long chain structure is beneficial for improving the activity of the compound.In general,we found a number of small molecules with the better activity than the positive compound KGN.Among them,compound 5k performed most prominently in the subsequent studies,so we decided it as our lead compound for further research.Through further verification,compound 5k was found to significantly induce chondrogenic differentiation of MSCs in vitro.In addition,compound 5k was found to significantly reverse the process of cartilage damage in mice model with the better efficancy than the positive compound KGN,and revealed the excellent cartilage repair ability in cartilage defect models both in rat and mice.For further exploring the biological mechanism,we designed and synthesized a aeries of biotin affinity probes based on Compound A and 5k.Unfortunately,we have not yet found the target protein.To our surprise,the biotin probes based on Compound A and 5k all showed better differentiation-inducing potential than the original compounds.However,due to the toxicity and the particularity of biotin motif of the compounds themselves,we considered to replace the biotin group with the intention of retaining its activity and avoiding its toxicity,then we designed and synthesized a series of new compounds.The studies had shown that these compounds have the similar differentiation-inducting potential to 8a,and compound 8g and 8l showed the most excellent biological activities.Overall,we placed emphasis on the lack of research and treatment of osteoarthritis and the focus on the small molecules of inducing MSCs differentiation for cartilage repair in this paper.Based on the hit Compound A screened in the previous stage,a series of novel N-phenylthiophene-2-carboxamides small molecule inducers for chondrogenesis with excellent activity were successfully designed and synthesized.Our lead compound 5k showed excellent ability in inducing MSCs differentiation into cartilage both in vitro and vivo,and has a significantly better cartilage repair effect than the positive compound KGN.Further exploration we found a series of Hexamido-N-phenylthiophene-2-carboxamide compounds represented by 8g and 8l,which showed superior biological activities compared to 5k in preliminary screening.However,the detail biological mechanism remains to be further studied.These series of small molecule inducers for MSCs differentiation into cartilage were expected to become potential preclinical drug candidates for cartilage repair in patients with osteoarthritis.