Studies Of Amine Compounds As Inhibitors Of Histone Methyllysine Readers

Epigenetic alterations relate to various human diseases like cancer, inflammation, metabolic disease, neuropsychiatric disorders. Developing small molecule inhibitors of epigenetic regulators is one of hot areas in drug discovery. So far, four of these small molecules have been approved as drugs by FDA and one of them have been approved by CFDA.Histone methyllysine reader（KMe reader） is one important class of epigenetic regulators, and there are more than 200 human KMe readers. The main functions of KMe readers are binding histone methylated lysines of chromatin and transferring signals of these marks, which could lead modification of chromatinic state, gene expression, the activation and repression of transcription, etc. However, the biological function, relationship with disease and druggability of most of KMe readers are unexplored.At the present stage, all the reported small-molecule probes of KMe readers are its corresponding inhibitors. Developing high-quality chemical probes（Selectivity>100-fold,IC50<100 n M） is one of common strategy to illuminate biological function and druggability of most of KMe readers. Small-molecule probe could flexibly perturb protein-protein interaction between histone methyllysine readers（KMe readers） and other proteins, which contributes to explore the function of KMe readers. Additionally, through the change of specific biomarker and phenotype in disease, which is generated by interaction between probe and KMe reader, we could understand the relationship between KMe reader and disease. At last, once any of KMe readers proved to have druggability, its small-molecule probe could be used as a lead compound for further research and development..At present, study of KMe reader is still in its infancy, and only inhibitors of seven KMe readers have been found. In addition, the potency and selectivity of these reported inhibitors cannot meet probe’s requirement. On the one hand we intended to improve potency and selectivity of the reported L3MBTL1 and 53BP1 inhibitors by conducting structure–activity relationships（SAR） research and hope that it could meet the needs of high quality probes; On the other hand, we would like to synthesize various fused cyclic amines and spiro cyclic amines to discover new inhibitors, in particular of those KMe readers with no reported inhibitor.In Chapter Two, we described design, synthesis and screening of 7-amino pyrimido[4,5-d]pyrimidin-4（3H）-ones as inhibitors of L3MBTL1, which is one of important KMe reader.In previous work, we replaced aromatic moiety of reported L3MBTL1 inhibitor UNC669 with pyrimidine fused rings and obtained 7-amino pyrimido[4,5-d]pyrimidin-4（3H）-one compound 501504 which has affinity for L3MBTL1（IC50 = 1.21±0.5 μM）. To investigate SAR of 501504, 41 analogues were synthesized by modifying groups of 3-, 5- and 7- positions of this scaffold. Analogues with different groups in 3-position were synthesized for investigating effect of different phenyl groups and benzyl group. Effect of various amino groups and non-amino groups in 5-position were also explored. We replaced 4-（pyrrolidin-1-yl） piperidine in 7-position of 501504 by other amino groups with similar size. The screening results showed that modification in 3-position did not improve activity. Exploring of 5-position also not generated three L3MBTL1 inhibitors that have better selective than 501504: 2-12 e [IC50 = 2.7 ± 0.2 μM, IC50（other KMe readers）/IC50（L3MBTL1） > 37], 2-12 l [IC50 = 2.2±2.1 μM, IC50（other KMe readers）/IC50（L3MBTL1）> 45] and 2-12 m [IC50 = 3.5±0.29 μM, IC50（other KMe readers）/IC50（L3MBTL1）> 29]. In addition, we conducted molecule docking by using Genetic Optimization for Ligand Docking（GOLD） program to understand why these compounds have selectivity for L3MBTL1 versus its homologous protein L3MBTL3. L3MBTL1 have a special residue PHE387 that is absences in L3MBTL3, which may be one reason that accounts for these compounds’ selectivity. When investigated 7-groups, we didn’t get any compound that have potency to L3MBTL1. However, we obtained some weak inhibitors for other KMe reader（53BP1 and PHF1）. In conclusion, we explored SAR of 7-amino pyrimido[4,5-d]pyrimidin-4（3H）-one analogues in this chapter and obtained three selective L3MBTL1 inhibitor.In Chapter Three, we summarized the common features of the binding pockets of different KMe readers and found that cyclic amino fragments could mimic ε-amino group of methyllysine. Therefore, we built a library of compounds containing fused cyclic amines or spirocyclic amines and tested their activity against different KMe readers.In this chapter, six classes of diazaspirocyclic amines were synthesized: 1,9-diazasprio[5.5]undecane, 1,8-diazasprio[5.5]undecane, 1,8-diazasprio[4.5] decane, 1,7-diazasprio[4.5]decane, 2,7-diazasprio[4.5] decane, 2,8-diazasprio[5.5]undecane. In addition, three classes fused cyclic amines were synthesized: 2,8-diazabicyclo[4.3.0]nonane, 3,8-diazabicyclo[4.3.0]nonane, 3,7-diazabicyclo[4.3.0] nonane. Each spirocyclic amine was reacted with 3-bromobenzoyl chloride or terephthaloyl dichlorideto lead to 27 spirocyclic compounds and 9 fused cyclic amine compounds. We investigated the ability of these compounds to interact with a panel of 8 KMe readers, and 7 KMe reader inhibitors were found. It is worth mentioning that these inhibitors include two PHF1 inhibitors 3-20（IC50 = 53±26 μM） and 3-52（IC50 = 39±24 μM）), since there are no any reports inhibitors for PHF1. Furthermore, we set out to investigate binding affinity of 22 chosen compounds versus a panel of 24 lysine and arginine methyltransferases（HMTs）. The activity of each enzyme was initially measured in the presence of 50 μM compound. As a result, we found two HMTs inhibitors 3-90（SETD8 IC50 = 39±11 μM） and 3-112（EZH2 IC50 = 30 μM）.In Chapter Four, we report design and synthesis of pyrimidine formamides and octahydropyrrolo[3,4-b]pyrroles as 53BP1 inhibitors based on the structure of reported 53BP1 inhibitors.Reported L3MBTL3 inhibitors L₆ and L₇, which have affinity to 53BP1 as well, both contain a moiety of phthalic diamide. We designed and synthesized 3 pyrimidine formamide as mimics of this scaffold. Additionally, we got 3 octahydropyrrolo[3,4-b]pyrrole analogues as 53BP1 inhibitors in previous works: 501798（IC50 = 38 ± 7.2 μM）, 501799（IC50 = 27 ± 1.7 μM） and 501801（IC50 = 43 ± 22 μM）. By conducted molecule docking between these compounds and reported 53BP1 inhibitor UNC2170, we learned that incorporating a hydrophobic group in octahydropyrrolo[3,4-b]pyrroles may improve activity. Based on this strategy, we synthesized 22 octahydropyrrolo[3,4-b]pyrrole analogues, and screened against different classes of KMe readers. In this study, we found a 53BP1 inhibitor with weak affinity（IC50 = 39±5 μM）. Screening of octahydropyrrolo[3,4-b]pyrrole analogues are under way.