Design,Synthesis And Biological Evaluation Of Theophylline Derivatives As BRD4 Inhibitors

Epigenetics and classical genetics are relative.Epigenetics refers to the heritable changes in gene expression caused by non-DNA sequence changes without altering the DNA sequence,while the genetics refers to the heritable changes in gene expression caused by changes of DNA sequences.The involved main changes are histone modifications,chromatin remodeling and DNA methylation and so on.Histone is the basic structural protein of chromatin,and the acetylation modification as one of the posttranslational modifications of histone is gradually becoming the focus on current research.The chromatin remodeling caused by the acetylation modification of histone plays an important role in gene expression.The BET?bromodomain and extra teminal?protein family is one of the eight families of bromodomains?BRDs?.It also has the characteristic of specific binding to histone acetylated lysine?KAc?,and is distinguished from other families in that the four members as BRD2,BRD3,BRD4,and BRDT of BET family all have two highly conserved BRDs,such as BRD4?1?and BRD4?2?.Analysis of the binding crystal structure of the protein with the inhibitors reveals that the binding pockets exist at the end of the four parallel?-helical bundles,and the inhibitors as the mimics of KAc form a hydrogen bond with the 140-position asparagine residue?Asn140?at the hydrophobic pocket,and also form a water-mediated hydrogen bond with the97-position tyrosine residue?Tyr97?.In addition,two hydrophobic regions are formed between the loops and the helix,named ZA channel and WPF region.The two pockets are effective regions for the selectivity of BRD4 small molecule inhibitors.Currently widely known BET inhibitor structures are mainly divided into four categories as triazoles?such as JQ1 and I-BET762?,isoxazoles?such as I-BET151?,pyridone or pyrimidone?such as PFI-1 and RVX-208?and others.These inhibitors are used for the treatment of various diseases such as inflammation,malignancy,and cardiovascular diseases and so on.Therefore,the BET family proteins,which represent BRDs,gradually become one of the hot drug targets.The main works are as the following three aspects:1.Select BRD4?1??PDB:4ZC9?protein as the target in the PDB database,design eight potentially active series of compounds,and use software to evaluate the molecular docking and scoring function of the designed compounds.The results show that the structures of eight series of compounds can all interact with the hydrophobic pocket and therefore may be good BRD4 inhibitors.Then we choose theophylline compounds as the optimal to synthesize.At the same time,the ligand molecules database of Chemdiv and Targetmol were screened and 20 compounds with the best screening results were purchased from the agent company,and then the biological activity test of the compounds was performed.2.The compounds with the best scoring results for the designed theophylline series compounds were synthesized:firstly,the intermediate of 8-chlorotheophylline was synthesized in the water by theophylline reacting with N-chlorosuccinimide?NCS?.Then,the aryl carbon-nitrogen bond formation reaction was carried out using the theophylline and 8-Chlorophylline to react with?,?-p-dibromobenzyl chloride to yield 2-3 and 2-4 at conditions of DMF,K₂CO₃,and 30?.In the synthesis of target products using hydroquinone and 4-methylsulphonylphenol as starting materials,the reaction failed due to different reasons.The results of the late activity test showed that the products of this series had no inhibitory activity and thus we gave up the synthesis of these target products.In addition,the synthesis process of the key intermediate3-methylxanthine was studied.The reaction conditions as the reaction solvent,the reaction temperature,the reaction time,the demethylation reagent and the riato of raw materials to improve the yield was investigated.Then the optimal conditions were determined.3.The bioactivity of a total of 22 compounds including compounds synthesized and screened was tested using the time-resolved fluorescence energy transfer?TR-FRET?method.The results showed that theophylline,the compound with1,3-dimethyl purine as the parent ring,had no inhibitory activity,which may be due to the fact that one of the methyl groups affected the hydrogen bond formation between carbonyl oxygen atom and Asn140/Tyr97.The 3-methylxanthine derivatives have good inhibitory activity.The compound with best bioactivity among these compounds is compound 2-26 with the value of IC50 as 8.304?M targeting BRD4.It can be determined that after this series of compounds are modified and optimized,the inhibitory activity is likely to decrease from micromolar to nanomolar,becoming good BRD4 inhibitors.