Research paper
Cyclization strategy leads to highly potent Bromodomain and extra-terminal (BET) Bromodomain inhibitors for the treatment of acute liver injury

https://doi.org/10.1016/j.ejmech.2022.115023Get rights and content

Highlights

  • A series of potent BET inhibitors with a tetracyclic scaffold were designed.

  • 28 was a highly potent BET inhibitor with an IC50 value of 1.6 nM.

  • 28 showed good in vitro anti-inflammatory activity.

  • 28 exhibited good therapeutic effect in the LPS-induced acute liver injury model.

Abstract

Acute liver injury (ALI) is characteristic of abrupt hepatic dysfunction and inflammatory response, and currently the main treatment for ALI is merely supportive rather than curative. Therefore, the development of novel and effective therapeutic strategies for ALI therapy is highly desirable. The emerging biological understanding of the role of BET Bromodomains has opened up an exciting opportunity to develop potent BET Bromodomain inhibitors as an effective therapeutic strategy for the treatment of acute liver injury. Herein, we synthesized a series of potent BET Bromodomain inhibitors with a tetracyclic scaffold, exemplified by compound 28 which showed good in vitro anti-inflammatory activity and good therapeutic effects in the LPS-induced acute liver injury model without obvious cytotoxicity, suggesting that compound 28 is a highly promising candidate worthy for further development.

Introduction

Acute liver injury (ALI) is characteristic of abrupt hepatic dysfunction and inflammatory response, which is presumably caused by hepatitis infections, drug-induced liver injury, metabolic syndrome, hepatotoxins originating from sepsis, and bacterial infection. Although ALI is associated with high mortality rates [1,2], there is no pathogenesis-specific clinical treatment for ALI. Currently, the main treatment for ALI is merely supportive rather than curative [3]. Therefore, there is an urgent need to develop novel and effective therapeutic strategies for ALI therapy.

Bromodomain and extra-terminal domain (BET) family of proteins play an important role in the epigenetic regulation process by recognizing the acetylated lysine residues on histones [4], and consists of four proteins BRD2, BRD3, BRD4 and testicle specific BRDT [5,6]. Each protein is composed of a left-handed bundle of four helices (αZ, αA, αB and αC) linked by two loops (ZA and BC) [7] and two highly conserved N-terminal bromodomains normally named BD1 and BD2 [8,9]. BRD4 is related to various disease processes [10], such as cancer [[11], [12], [13]], inflammatory [14,15], fibrosis [16] and cardiovascular disease [17]. BRD4 recruits positive transcription elongation factor B (p-TEFb) [18] and RNA polymerase II [19] to chromatin super enhancer site, which is essential for replication and maintenance of oncological gene [20]. Besides the indispensable function in oncology, BRD4 has been found to be related to pro-inflammatory factors by coactivating NF-κB transcription with Lys310 acetylated RelA [21].

So far, several small-molecule BET Bromodomain inhibitors has been developed as a potential therapy for oncology and immunology diseases and some of them have successfully progressed into clinical trials (Fig. 1) [[22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52]]. Despite great progress, there are currently no approved drugs targeting BET Bromodomains and more diverse inhibitors with novel structures are required. Recently, it has been shown that both BD1 and BD2 of all BET proteins can modulate gene expression induced by inflammatory stimuli and BET Bromodomain inhibitors showed efficacy for the treatment of immune-mediated inflammatory diseases [42]. In this context, considering that there is no pathogenesis-specific clinical treatment for ALI, it would be highly desirable to develop highly potent BET Bromodomain inhibitors with novel structures for the treatment of ALI.

Recently, we reported a potent BET Bromodomain inhibitor ZB-38 (Fig. 1) which shows an excellent BET Bromodomain selectivity and good in vivo antitumor efficiency [53]. Herein, we report our continued efforts to develop novel BET inhibitors that builds upon our compound ZB-38. By using X-ray structure-guided optimization and an annulation strategy, a new series of BET bromodomain inhibitors with a novel tetracyclic scaffold were developed. Among them, compound 28 exhibited good anti-inflammatory activities both in vitro and in vivo without obvious cytotoxicity.

Section snippets

Results and discussion

Based on the co-crystal structure (6KEE) of ZB-38 analogue with BRD4 BD1 (Fig. 2), we surmised that a proper cyclization of ZB-38 analogue will provide a rigid tetracyclic scaffold and lock the phenyl group to occupy the hydrophobic WPF pocket, thus leading to an improved potency.

To verify our hypothesis, a series of tetracyclic compounds were synthesized and evaluated via ALPHA Screen assay. First, we designed amine 10 as the cyclization precursor from the point of view of synthesis and

Chemistry

As shown in Scheme 1, 32 was generated with commercially available 4-nitrobenzyl bromide and sodium methanesulfinate. After reduced by hydrogen, 33 was brominated with NBS resulting 34, which was coupled with bis(pinacolato)diboron under the palladium catalyst producing 35. Suzuki coupling was conducted with 6-bromo-8-methylpyrrolo[1,2-a]pyrazin-1(2H)-one, which was synthesized as our previous work [53], and 35 to deliver 10. Compound 10 was cycled with paraformaldehyde and then reacted with

Conclusion

In summary, based on our previously developed BET Bromodomain inhibitor ZB-38, the X-ray structure-guided optimization and annulation strategy was employed, leading to the discovery of novel BET bromodomain inhibitors with a tetracyclic scaffold. Among them, compound 28 provided good binding affinities for BET Bromodomains, and good anti-inflammatory activity. In the in vivo efficacy experiment of LPS/D-GalN-induced acute liver failure (ALF), compound 28 significantly raised the survival rate

Chemistry

Unless otherwise noted, commercial solvents and reagents were used without further purification. Silica gel 60H (200–300 mesh) manufactured by Qingdao Haiyang Chemical Group Co. (China) was used for general chromatography. The final compounds were all purified by C18 reverse phase preparative HPLC column with H2O and MeCN as eluents. The purity of all the final compounds was confirmed to be >95% by HPLC analysis with Agilent ZORBAX SB-C18 reversed-phase column (250 mm × 4.60 mm, 5 μm).

Accession codes

Atomic coordinates have been deposited in the Protein Data Bank (PDB code: 7DPN, 7DPO for compound 27). Authors will release the atomic coordinates and experimental data upon article publication.

Author contributions

B.Z. and C.L. directed the project. C.C., Z.L., Y.Y. conducted the chemical experiments. T.L. conducted the in vitro experiments. P.C. and S.F. conducted the in vivo experiments. B.Z. wrote the manuscript with the assistance of C.C., Z.L., P.C. and T.L.. W.F., Y. W., C.L., and B.Z. conceived the project. All authors participated in data analyses and discussions.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bing Zhou reports financial support was provided by National Natural Science Foundation of China. Bing Zhou reports financial support was provided by Science and Technology Commission of Shanghai Municipality.

Acknowledgment

We thank the staff members of the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Lab, China for providing technical support and assistance in data collection and analysis. We thank the staffs from BL19U1 beamline of National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility, for assistance during data collection. This work is financially supported by National Natural Science Foundation

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