Design,Synthesis And Mechanism Study Of Novel EZH2 Covalent Inhibitors And Microtubule Polymerization Inhibitors Containing Pyridine-2(H)-One

The structure of pyridone includes a hydrogen bond acceptor and a hydrogen bond donor,and it can be substituted at all sites except the carbonyl group.These characteristics make pyridone used as a side chain fragment or skeleton structure in a variety of bioactive molecules.In this paper,three novel inhibitors containing pyridine-2（H）-ketone were designed and synthesized,which further expanded the application of pyridione in medicinal chemistry.They are briefly described as follows:PartⅠDesign,Synthesis and Mechanism Study of Paralog-Selective EZH2Covalent InhibitorsIn recent years,the incidence of malignant tumors is still on the rise worldwide.Although there are surgical treatment,radiotherapy,drug therapy and some other treatments,the occurrence and development of some tumors have been controlled.In general,the current situation of malignant tumor treatment is not satisfactory.Researchers have found that epigenetic events are a key driver of tumor development and play an important role in tumor genesis,migration and invasion.Targeting epigenetic regulators provides us with a new cancer treatment strategy.EZH2 is a member of the histone lysine methyltransferase family,and its classical biological function is to act as the core catalytic subunit of PRC2 to methylate of 27lysine on histone H3 and increase the level of H3K27Me3,thus silencing the expression of various tumor suppressor genes.More and more studies have shown that the methylation substrates of EZH2 include GATA4,AR/AR related proteins,STAT3,etc.,as well as histones.Meanwhile,in addition to the methylation function,EZH2 has been reported to play other important biological functions by forming complexes with a variety of proteins,including EZH2-Rela-Relb complex,EZH2-ER-β-catenin complex,etc.EZH1 is a homologous protein of EZH2,sharing 96%sequence identity in their catalytic SET domains.The classic biological function of EZH1 is also to act as the catalytic subunit of PRC2 to methylate histones.Additionally,EZH1 plays an important role in the maintenance of stem cell multifunctionality,heart development and regeneration,and innate immunity.Although some EZH2 inhibitors have shown good anti-tumor activity in vivo and in vitro and even clinically,they were widely used as chemical probes to study the biological functions of EZH2 or PRC2.However,EZH2 and EZH1 are highly homologous in the SET domain,and most EZH2 inhibitors also have high inhibitory activity against EZH1.When the biological functions of EZH2 instead of EZH2/EZH1are studied separately,the dual inhibitory effects of these compounds may lead to erroneous conclusions and cannot be effective chemical probes due to the unique biological functions of EZH1 and EZH2.At the same time,when these inhibitors inhibit EZH2 to exert anti-tumor effects,the inhibition of EZH1 may bring potential toxic side effects.Therefore,the existing EZH2/EZH1 dual inhibitors cannot be satisfied either as chemical probes or clinical applications,and the development of paralog-selective EZH2 inhibitors is urgently needed.By aligning the SET domain sequence of EZH2 and EZH1,we found that there was a unique cysteine at position 663 of EZH2,and there was no similar high reactivity amino acid near this position of EZH1.Meanwhile,the crystal structure of the human PRC2 complex showed that Cys663 was located near the SAM pocket of EZH2,and no mutation has been reported.Therefore,we hypothesized that targeting Cys663 as a covalent coupling site might achieve a higher selectivity for EZH2 than EZH1.By analyzing the co-crystal structures of multiple compounds complexed with PRC2,we found that indole derivatives were more suitable for introducing acrylamide,which was easy to synthesize and was inert to glutathione,as an‘electrophilic warhead’.Therefore,we designed and synthesized the indole derivatives A1.However,this compound did not exhibit EZH2^WTinhibitory activity(IC₅₀>10μM).We speculated that the indole ring occupied a large space,and the introduction of acrylamide group made the compound more steric hindrance,which could not enter SAM pocket,resulting in the loss of activity.Furthermore,we reduced the indole ring to a six-membered benzene ring and a five-membered pyrazole ring to obtain compounds of class B and C.Compared with the indole derivatives,the steric hindrance of these two types of compounds is smaller.And the increased flexibility of these compounds would make them easier to form a covalent bond through the Michael addition.In the study of class B derivatives,we obtained compound B2 with low micromolar inhibitory activity against EZH2^WT.The structure-activity relationship study of class B compounds mainly investigates the influence of different structures of pyridine 2（H）-one and different substituents at the C5 of the benzene ring on the inhibitory activity.Among the 27 compounds,SKLB-03176 exhibited the best inhibitory activity against EZH2^WT with an IC₅₀ of 47 n M.The inhibitory activity of SKLB-03176 was about 4 times higher than that of its reversible analog SKLB-03176’,and the inhibitory activity of SKLB-03176 increased in a time dependent manner.SAM competition experiment showed that after prolonging the incubation time of SKLB-03176 with protein,it covalently bound to EZH2,and the activity was no longer affected by the concentration of SAM.Furthermore,mass spectrometry proved that SKLB-03176 covalently binds to EZH2 and formed a single modified adduct.SKLB-03176 could inhibit EZH2 methylation in a concentration-dependent and time-dependent manner in cells,and it could continuously inhibit the expression of H3K27Me3 within 48 hours after washing-out.SKLB-03176 could inhibit a variety of EZH2 mutants,suggesting that it has a certain therapeutic potential for EZH2mutation-driven tumors.However,its inhibitory activity against EZH2 was only about50 times better than that against EZH1.We speculated that other groups of SKLB-03176（such as pyridinone,hydrophilic side chain group,etc.）could form some hydrogen bonds and hydrophobic interactions with EZH1 to maintain the activity without covalent bonds.The unique spatial conformation of this type of structure made the covalent bond not play a decisive role in homologous selectivity.In the study of class C derivatives,we obtained compound C1 with submicromolar inhibitory activity against EZH2^WT.Then,the optimal compound SKLB-0335 was obtained by SAR study of pyrazole derivatives.Its reversible analog SKLB-0335’showed no inhibitory activity,and the activity of SKLB-0335 was significantly increased with prolonged incubation time.Similarly,the inhibitory activity of SKLB-0335 did not change with SAM concentration after prolonged incubation time.SKLB-0335 displayed an extremely slow off-rate(k_off)of 0.00347min^-1,which resulted in a residence time half-life(t_1/2)of 199.8 min.The MS analyses revealed that SKLB-0335 effectively covalently bound to EZH2 protein.Notably,SKLB-0335 could continuously abolish H3K27Me3 and did not affect the m RNA expression of EZH1-related genes in cells.SKLB-0335 exhibited excellent selectivity,and did not exhibit inhibitory activity against 7 histone methyltransferases,including G9a and SETD8.Meanwhile,SKLB-0335 had no effect on more than 40 kinases targeted by covalent inhibitors(IC₅₀>100μM).More importantly,its paralog-selectivity to EZH2/EZH1 was more than 1500 times,which was the best among the reported compounds.SKLB-0335 significantly inhibit the proliferation of two lymphoma cells in vitro.In this section,we designed and synthesized two classes of EZH2 covalent inhibitors that have rarely been reported and illustrated their mechanism of action.Both covalent inhibitors showed good in vitro activity and could continuously inhibit EZH2 methylated histones after washing-out.SKLB-0335 showed excellent paralog-selectivity and had no effect on EZH1 downstream genes.SKLB-0335 could be used as a paralog-selective chemical probe to study the unique effects of EZH2.At the same time,SKLB-0335 had no inhibitory activity on a variety of histone methyltransferases and kinases,less potential toxic and side effects,and a good prospect of application development.In addition,its unique covalent mode of action allowed it to be linked to biotin and then used to discover unknown EZH2 interacting proteins and methylated substrate proteins through mass spectrometry and biochemical experiments.PartⅡDesign,Synthesis and Anti-tumor Activity of Purine-Aminomethyl-Pyridone Derivatives as Novel Microtubule Polymerization InhibitorsMicrotubules are one of the main components of the cytoskeleton,which play an important role in the maintenance of cell morphology,intracellular material transport,and the formation of spindles in the process of cell division and proliferation.Targeted microtubule drugs have been verified for clinical effectiveness,and they have always been a hot field for oncology drug development.The existing microtubule polymerization inhibitors mostly have the disadvantages of high toxicity,drug resistance,poor solubility,low bioavailability,and complex structure.Therefore,it is still necessary to develop novel microtubule inhibitors which are safe,effective and easy to synthesize.Microtubule polymerization inhibitors targeting colchicine site have the advantages of overcoming drug resistance mediated by P-gp andβIII-tubulin overexpression,small molecular weight and antiangiogenic effect.In order to obtain novel microtubule polymerization inhibitors with simple structure,we focused on these compounds owning concise double-ring core structure targeting colchicine site.In this study,we designed and synthesized 64 novel 3-（（（9H-purin-6-yl）amino）methyl）-4,6-dimethylpyridin-2（1H）-one derivatives using a bioelectronic isosteric strategy.The optimal compounds SKLB0533 and SKLB0565 were obtained through structure-activity relationship studies.Both SKLB0533 and SKLB0565 could significantly inhibit the proliferation of various tumor cells,with an optimal inhibitory effect on the CRC cell lines.Both compounds could effectively inhibit tubulin aggregation in vitro.Immunofluorescence assay also showed that these compounds can destroy the microtubule network and inhibit the polymerization of microtubules in cells.Molecular docking showed that the two compounds were bound in the same pocket as Colchicine,and the binding modes were similar.The pyridine-2（1H）-one structure could form a hydrogen bond with Asp251.SKLB0533 and SKLB0565 could arrest HCT116 and SW620 cells in the G2/M phase,induce cell apoptosis,and inhibit the formation of HUVECs tubules.In the HCT116 xenograft tumor model,SKLB0565 exhibited a slightly worse anti-tumor activity than SKLB0533.SKLB0533 could effectively inhibit the colorectal tumor growth（80 mg/kg orally administered once a day for 28 days,TGI:71.6%）,which was superior to Capecitabine.During the treatment period,the body weight of the mice did not change significantly.After the treatment,the haematological and serum biochemical values were comparable with those of the vehicle group,and the pathological morphology of the main organs did not change significantly,which preliminarily showed that SKLB0533 is safe.Furthermore,SKLB0533 showed no significant inhibitory activity on 420 kinase（1μM）and EZH2（10μM）.In this section,we designed and synthesized a class of novel microtubule polymerization inhibitors,which exhibited good anti-tumor activity in vivo and in vitro.SKLB0533 did not exhibit obvious inhibitory activity against more than 400 kinases and EZH2,suggesting that it has good target selectivity.And no obvious side effects were found in the treatment in vivo.SKLB0533 was expected to be developed into a safe,effective and easy-to-synthesize preclinical drug candidate due to its excellent antitumor activity and target selectivity.