Design, Synthesis And Antitumor Activity Of Novel Inhibitors Based On The Ubiquitin-proteasome System (UPS)

In eukaryotic cells,Ubiquitin-Proteasome System（UPS）specifically degrades more than 80%of intracellular proteins,thereby maintaining protein homeostasis and normal life activities in the organism,and dysregulation of UPS will lead to tumorigenesis.The action mechanism of UPS includes two processes:ubiquitination of substrate protein and subsequent proteasome-mediated degradation.Ubiquitin molecules are labeled on the substrate protein through enzymatic tandem reaction by E1-E2-E3 tertiary complex to form polyubiquitin chain labelling,which are then recognized and rapidly degraded by proteasome.Bortezomib,a first proteasome inhibitor launched in 2003,displays significant antitumor effect against multiple myeloma,demonstrating the feasibility of targeting UPS for cancer therapy.Unfortunately,bortezomib exhibited more severe side effects in clinical practice due to its poor selectivity,which inhibits the 26S proteasome among the UPS,which blocks almost all UPS-mediated protein degradation.Among many UPS-related regulating proteins,ubiquitin ligases E3 are diverse with strict substrate specificity.Drugs targeting E3 ligase will have higher selectivity and lower side effects than bortezomib.Cullin-RING ligases（CRLs）,the largest subfamily of ubiquitin E3 ligases,are responsible for approximately 20%of the proteasome-dependent degradation of intracellular protein.It has been reported that CRLs are aberrantly activated in tumors.However,small molecule inhibitors directly targeting CRLs have not been reported yet.Under normal physiological conditions,CRLs are inactivated and their activation requires Neddylation modification of their backbone protein Cullin by the ubiquitin-like molecule NEDD8.Therefore,the inhibition of CRLs can be achieved indirectly by inhibiting the Neddylation pathway,which provides a new strategy for the development of anti-tumor drugs.Similar to the ubiquitination process,Neddylation is also achieved through a series of enzymatic reactions mediated by E1-E2-E3.Among them,NEDD8 activating enzyme（NAE）is the key rate-limiting enzyme during Neddylation.Accordingly,inhibition of the enzymatic activity of NAE can suppress the Neddylation signaling pathway and reduce the degradation of related proteins,thereby inhibiting tumor cell growth.MLN4924,developed by Millennium Pharmaceuticals,is the only NAE inhibitor currently in clinical trials.However,the overall response rate for MLN4924as a single agent in patients with acute myeloid leukemia（AML）or myelodysplastic syndrome（MDS）is only 17%.Therefore,it is extremely necessary to develop novel NAE small molecule inhibitors with high potency and specificity,high safety,and diverse chemotype for clinical research.Based on the co-crystal complex of MLN4924 and NAE,we first obtained structurally novel pyrimidinotriazoles compound 3-1 with comparable activity to MLN4924 through scaffold hopping strategy.Systematic medicinal chemistry optimization of the Head and Tail parts of the MLN4924 structure with aim to improve drug-like properties gave rise to compounds 3-21 and 3-29 with comparable in vitro potency and improved pharmacokinetic properties when compared to MLN4924.Compared to MLN4924,3-21 and 3-29（iv,1 mg/kg）showed 4.5-fold and 2.4-fold higher plasma exposure with AUC values of 2500 h*ng/m L and 1328 h*ng/m L,respectively,and 8-fold and 1.9-fold longer half-life time with T_1/2 values of 5.68 h and1.36 h,respectively,and 5.6-fold and 1.8-fold lower clearance with CL values of 5.33m L/min/kg and 16.5 m L/min/kg,respectively.Besides,neither 3-21 nor 3-29 at 1μM had any inhibitory effect on 468 kinds of kinases and their mutants,but significantly inhibited the Neddylation signaling pathway,indicating its high selectivity for NAE.Subcutaneous injection of 3-29 with 130 mg/kg/QD displayed significant anti-tumor effects in the mice xenograft model of HCT-116 colon cancer cells（T/C=0.20）and human leukemia MV-4-11（T/C=0.19）.However,subcutaneous injection with 20mg/kg/QD 3-21 showed significantly improved antitumor effects（T/C=0.07）than MLN4924（60 mg/kg/BID,T/C=0.37）in the mice xenograft model of HCT-116 colon cancer cells.The clinically available NAE inhibitor MLN4924 is currently administered intermittently by intravenous drip to treat patients.Because of the poor compliance of patients with intravenous drip,the development of oral effective NAE inhibitors would be beneficial to the treatment of patients.Since oral administration of 3-21 at 3 mg/kg possessed 3.9-fold higher plasma exposure（AUC=810 vs 208 h*ng/m L）and a moderate half-life（T=3.14 vs 5.50 h）when compared to MLN4924,in vivo antitumor effects of 3-21 by oral administration was explored.It is found that 3-21 showed significant anti-tumor effects in the mice xenograft model of colon cancer RKO（10mg/kg/QD,T/C=0.03）and MV-4-11（10 mg/kg/QD,T/C=0.00）,without significant body weight changes.Besides,3-21 was metabolic stable as a prototype compound in different species of hepatocytes（human,monkey,dog,rat,and mouse）without significant species differences.The safety evaluation showed that the maximum tolerated dose（MTD）of 3-21 was 0.15 mg/kg,and the biochemical indexes of male and female animals were normal.Since 3-21 is a new orally available NAE inhibitor with significant antitumor effect and good safety,this compound has been licensed out for preclinical study.At the same time,we also pursued methionine adenosine transferase 2A（MAT2A）inhibitors based on the synthetic lethal strategy with aim to obtain high activity MAT2A inhibitors,which can be combined with NAE inhibitors to achieve the synergistic and synthetic lethal antitumor effects.Synthetic lethal therapy is a phenomenon in which only the simultaneous inhibition of two non-lethal genes can lead to cell death.Therefore,it is possible to specifically kill tumor cells with less impact on normal cells by inhibiting the synthetic lethal partner of a specific mutated gene in a tumor,thus achieving precise cancer therapy.MAT2A or methylation modifying enzyme PRMT5inhibitors are under clinical trials as synthetic lethal partners of MTAP gene,and MAT2A inhibitor can indirectly inhibit the activity of PRMT5 to treat tumor.Both NAE and Prmt5 are protein post-translational modifying enzymes,but synthetic lethal therapy of NAE inhibitors together with MAT2A inhibitors has not been reported yet.Med Chem structural optimization was performed based on MAT2A inhibitors AG-270 with high potency and specificity.It was found that the core of pyrazolopyrimidinone was essential for the inhibitory activity of MAT2A after scaffold hopping.Subsequently,detailed structure-activity relationship analysis of the methoxyphenyl group at the 6-position of the core of pyrazolo-pyrimidinone delivered several compounds 4-41,4-44,and 4-49 to 4-53 with comparable molecular and cellular activity to AG-270.Among them,4-41 showed higher selectivity for HAP1/MTAP^-/-than AG-270（SI=8 vs.3.3）,and exhibited slight inhibitory effect on various tumor cells and normal cells.In SD rats,intravenous administration of 1 mg/kg 4-41displayed a moderate half-life time(T_1/2=0.87 h)and moderate clearance（CL=13.6m L/min/kg）and plasma exposure（AUC=1211 h*ng/m L）.However,plasma stability analysis showed that 4-41 will be easily metabolized by esterase into phenolic 4-33,which hampered the further clinical development of 4-31.In view of the importance of protein degradation targeting chimeras（PROTACs）technology for drug research in recent years,we tried to develop PROTACs degradation agents based on synthetic lethal target MAT2A with aim to overcome the drug resistance of synthetic lethal targets and avoid the toxic and side effects caused by excessive degradation of target proteins by PROTACs.Although different lengths of connecting chains were extensively investigated,we failed to achieve MAT2A-targeting PROTACs with obvious degradation activity.