Design,Synthesis And Biological Activity Evaluation Of New Pyrimidinone Based USP7 Inhibitors

Ubiquitin-specific protease 7(USP7),the deubiquitinating enzyme found in all eukaryotes,removes the ubiquitin molecule from proteins and reverses the ubiquitin mediated process.USP7 is well known for its interaction with the tumor suppressors murine double minute 2(MDM2)and p53 protein through the USP7-MDM2-p53 pathway.This network plays a crucial role in the regulation of p53 protein which controls cell growth,apoptosis,cell proliferation,immune response and cell cycle progression.Overexpression of USP7 and MDM2 levels appeared to induce p53 inactivation and promote tumorigenesis.USP7 inhibition led to extensive developments of USP7 inhibitors that could induce MDM2 deubiquitination and p53 reactivation resulting in cell cycle arrest and apoptosis in cancer cells.As a result,USP7 inhibitors have been at the forefront of therapeutic development for the past thirty years,particularly in the treatment of cancer.The present work focuses on the synthesis and evaluation of new pyrimidinone based USP7 inhibitors containing a pyrimidin-4(3H)-one ring bound to the 4hydroxypiperidine core.By using molecular docking Autodock 4.0,new analogs that showed good potential inhibitory activity against USP7 were designed,synthesized and evaluated.The research work is summarized as follows:1.Compound 2-1 was chosen as a lead compound,and structural modifications were mainly focused on the C-6 position of the pyrimidin-4(3H)-one ring and the N-1 position of the piperidine ring.A Suzuki coupling technique was used to make a nucleophilic substitution at the C-6 position of the pyrimidin-4(3H)-one ring,and an ethylbenzene ring(2-6)or a methoxybenzene ring(2-7)was added.2.The N-1 of the piperidine ring was subject to two different types of modifications:(i)an amide bond side chain was attached via the addition of carboxylic acids derivatives giving intermediates(2-9a～2-9d)and(ii)for the first time in this type of inhibitors,a carbamoyl bond was introduced through a one-pot synthesis with oxalyl chloride and aromatic amines derivatives or substituted aniline derivatives to obtain compounds 2-9e～2-9j.3.The final step consisted of an epoxide opening reaction through a nucleophilic substitution mechanism.Compounds 2-6 or 2-7 reacted with intermediates 2-9a～29j to obtain series Ⅰ(Ⅰ-1～Ⅰ-10)and series Ⅱ(Ⅱ-1～Ⅱ-10).1H NMR,13C NMR,and MS analysis were used to characterize all the synthesized compounds.4.The activities against USP7 catalytic domain of the target compounds were assessed.Compared to the control drug,all the compounds bearing a carbamoyl bond showed no or very low activity within the measured concentration of 50 μM.Compounds I1 and Ⅱ-1 were observed to have moderate effects on USP7 with an inhibition rate of 47.39%and 58.72%,respectively.Compared to the other compounds,these results indicate that the chiral methyl group,present in Ⅰ-1 and Ⅱ-1,maintained their stability and contributed to enhance their activity.5.The structure-activity relationships of the target compounds revealed that(R)-3phenylbutylamide side chain had a significant impact on USP7 activity.Other structural modifications such as the introduction of the carbamoyl linkage on the N1 of piperidine ring and the attachment of the p-metoxybenzene group or an ethylbenzene group at the C-6 of the pyrimidinone group strongly influenced the overall potency of the inhibitors.To obtain a possible correlation of enzymatic USP7 inhibition,all the newly synthesized analogs of series Ⅰ and Ⅱ were subjected to molecular docking studies.Our studies found that the most active compounds(Ⅰ-1 and Ⅱ-1)interacted with USP7 catalytic domain through hydrogen bonding,alkyl interactions and van der Waals(vdW)interactions with Cys 223,Phe 409,Tyr 465,Ala 221,His 464,Val 296,Leu 406.As a result,these interactions are significant for their potent inhibitory effects on USP7.The novel USP inhibitors described here could be useful as mechanistic probes for USP7 research.