Design And Synthesis Of Pyrimidine-fused Diazepine Derivatives As Selective L3MBTL3Inhibitors

Epigenetics is a term that refers to the phenomenon of heritable phenotypic traitswithout altering the underlying DNA sequence itself. Epigenetic regulation plays animportant role during the cellular differentiation and development of an organism;epigenetic changes are responsible for aging mechanisms and response to theenvironment. Major epigenetic regulation involves DNA methylation andpost-translational modifications. The latter include acetylation, methylation,ubiquitylation and phosphorylation, the dysregulation of which has been shown tocontribute to various diseases. Epigenetic regulation becomes an emerging area ofbiological science owing to its therapeutic value.Histone methylation occurs mainly on histone lysine and arginine residues.Immense signaling potential of the histone methylation system attributes to differentsites of methylation and distinct methylation states of mono-, di-or trimethylation.Methyllysine binding proteins, referred as readers, recognize various levels of lysinemethylation, which in turn leads to downstream signaling.MBT domains selectively bind to monomethylated and dimethylated lysineversus unmethylated and trimethylated lysine. Studies suggest that the biologicalconsequences of MBT domain inhibition may be functionally associated withdedifferentiation, re-expression of silenced genes and cellular reprogramming. Smallmolecules that inhibit MBT containing proteins will be of great value.Based on the structure of MBT inhibitors and the co-crystal structure with MBTproteins, we designed a series of molecules maintaining the piperidino-pyrrolidineamide, side-chain of UNC669, including N-substituted tetrahydropyridine carboxamide derivatives, N-substituted pyrrolidine carboxamide derivatives,N-substituted3-aminocyclohexane carboxamide derivatives and pyrimidine-fuseddiazepine derivatives.32molecules were synthesized through8synthetic routes.Using Methyl Nicotinate and Methyl Isonicotinate as starting materials, wesynthesized14compounds via substitution, reduction, hydrolization, amide formation,debenzylation, and amide or urea formation reactions; using Boc-Proline and3-aminocyclohexanecarboxylic acid as starting materials, we synthesized12compounds via amide formation, deprotection, and amide or urea formation reactions;using pyrimidine-4,6-diol as starting material, we synthesized6Pyrimidine-fusedDiazepine compounds via2synthetic routes in6and7steps, respectively. Evaluationon these compounds in an Alpha Screen assay (IC50) identified the pyrimidine-fuseddiazepine derivatives (2-36a-d) as potent and selective inhibitors of the L3MBTL3methyllysine reader protein. And finally2-36c was chosen to lead our further design.Our next efforts were focused on optimization of site6and9of2-36c toimprove the potency and selectivity of the inhibitor based on the feature of2-36c andthe structure of L3MBTL3protein. We synthesized51Pyrimidine-fused Diazepinederivatives and screened them. We found4inhibitors with their IC50for L3MBTL3less than1μM.2-51b and2-51d with IC50of0.37μM and0.18μM, respectively, are30and89times more selective for L3MBTL3over L3MBTL1;2-67a and2-67c withIC50of0.12μM and0.73μM, respectively, are167and62times more selective forL3MBTL3over L3MBTL1. In addition to L3MBTL1, these compounds are moreselective for L3MBTL3against many reader proteins, such as MBTD1、53BP1、CBX7-F、Jarid1A、PHF2. The results indicate that N-substituted aminophenyl on site6may interact with L3MBTL3by π-π interaction and hydrogen bond; andN-substituted aminopropyl on site9may interact with L3MBTL3by hydrogen bond.This will be extremely helpful for the future research to develop more potent andselective L3MBTL3inhibitors.