Design,Synthesis And Biological Activity Evaluation Of Inhibitors Of DCN1-UBC12 Protein-Protein Interaction And Steroidal Pyridines

We have designed,synthesized and biologically evaluated the compounds originally deriving from a hit which was discovered from our in-house molecular library and functioned by disturbing DCN1-UBC12 interaction.At the same time,the structural modification of D ring of pregnenolone resulted in a series of novel steroidal pyridines,all of which were further evaluated for their antitumor activity.In addition,we described a Rh(Ⅲ)-catalyzed C7-selective C-H activation/annulation of indolines with 1,3-enynes to efficiently construct various privileged 1,7-fused indolines.Part one of this dissertation is on the design,synthesis and biological evaluation of inhibitors of DCN1-UBC12 protein-protein interaction.Ubiquitin proteasome system(UPS)plays a vital role in the maintenance of cellular homeostasis via regulating the elimination of misfolded or damaged proteins.Cullin RING ubiquitin ligases(CRLs),the largest family proteins of UPS,are responsible for the degradation of approximately 20%of cellular proteins and are imperative for the normal cellular physiology and various human diseases.The activation of CRLs requires neddylation of cullins,involving DCN1-UBC12 protein-protein interaction,MLN4924 inhibits the NAE activity effectively,but as NAE functions upstream of the neddylation pathway,complete inhibition of NAE inevitably leads to the accumulation of the substrates for all CRLs and causes cytotoxicity.Consequently,it will be of great value to develop small molecules that can selectively modulate neddylation of individual CRL members.Thus,the inhibition of DCN1-UBC12 protein-protein interaction to regulate the degradation of proteins becomes a promising approach for the treatment of human disease.Based on our previous work,we identified a hit compound Ⅱ-1a(IC50=1881 nM,FP assay)via screening our in-house compound library.Extensive structure-activity relationship(SAR)investigations lead to the discovery of several potent inhibitors with IC50 values less than 50 nM.SARs studies suggest that:i)the P1 regions could tolerate a large substituent;ⅱ)the sulfydryl moiety on the P2 is necessary for the activity;ⅲ)employing a piperidyl group as a linker may benefit potency improvement.Metabolic stability study in liver microsomes indicated that compound Ⅱ-6x exhibits moderate stability in human,rat and mouse liver microsomes.Compound Ⅱ-6r,Ⅱ-6u and Ⅱ-6x demonstrated an excellent selectivity for DCN1 against DCN3.Docking was performed to reveal the binding models ofⅡ-6u in the binding site of UBC12,the results suggested that the hydrophobic interactions between compound and DCN1 are helpful for their binding.Based on DI591,an inhibitor of DCN1,we designed and synthesized a set of DCN1 heterobifunctional molecules,with its biological evaluation indicating that these compounds failed to induce the degradation of DCN1 in U2OS cells line(the Roman number before compound No.refers to the chapter where the compound appears).In the second part,a series of new steroidal pyridines have been synthesized through the base-promoted three-component reaction and preliminarily evaluated for their antiproliferative activity against different types of cancer cell lines.SARs studies showed that the heterocyclic rings attached to the 4-position of the pyridine ring were preferred over the phenyl rings for the activity.Among these compounds,compound III-3j exhibited good growth inhibition activity against all the tested cancer cells,especially for PC-3 cells with an IC50 value of 1.55 μM.Further mechanistic studies revealed that compound Ⅲ-3j inhibited colony formation,migration and evasion of PC-3 cells in a concentration-dependent manner as well as induced apoptosis of PC-3 cells possibly through the mitochondria-related apoptotic pathways.Caspase-3/-9 and PARP were activated,leading to the apoptosis of PC-3 cells.For the AR+-sensitive prostate cancer cell line LNCaP,the most potent compound available was less potent than abiraterone with the IC50 value of 8.482 and 3.290 pM,respectively.Thus,compound Ⅲ-3j could be used as a starting point for the development of new steroidal heterocycles with improved anticancer potency and selectivity.The synthesized steroidal pyridines containing the-OEt(ethoxy)and-CN(cyano)groups could be used as tools for further modifications in the construction of the steroid library.In the third part,we described the construction of 1,7-fused indolines,aiming at establishing a drug-like compound library.Given the great application of 1,7-fused indolines in medicinal chemistry,especially in the field of protein ubiquitination and neddylation inhibition,we developed a Rh(Ⅲ)-catalyzed C7-selective C-H activation/annulation of indolines with 1,3-enynes to efficiently access various privileged 1,7-fused indolines which bear an all-carbon quaternary stereogenic carbon center.Notably,the resulting product can be readily transformed into 1,7-fused indoles,further widening the C-7 derivatization of indoles and highlighting the synthetic utility of this methodology.In conclusion,these works described above threw light on ⅰ)the strategies of target-based drug discovery that were employed for structural optimization in the pursuit of highly potent DCN1 inhibitors;ⅱ)the tactic of phenotypic drug discovery in the revealing of a series of anti-cancer agents with the novel structure of steriod and ⅲ)one original approach for the synthesis of structurally diverse 1,7-fused indoline derivatives and the establishment of a small molecule library thereof.