Research paper
Benzoxazinone-containing 3,5-dimethylisoxazole derivatives as BET bromodomain inhibitors for treatment of castration-resistant prostate cancer

https://doi.org/10.1016/j.ejmech.2018.04.034Get rights and content

Highlights

  • Structure-based drug design yielded potent and selective BET bromodomain inhibitors.

  • BET bromodomain inhibitors suppress prostate cancer cell proliferation, migration, and invasion.

  • (R)-12 significantly suppresses the tumor growth in a 22Rv1-derived xenograft model.

  • (R)-12 is a promising lead compound for the treatment of CRPC.

Abstract

The bromodomain and extra-terminal proteins (BET) have emerged as promising therapeutic targets for the treatment of castration-resistant prostate cancer (CRPC). We report the design, synthesis and evaluation of a new series of benzoxazinone-containing 3,5-dimethylisoxazole derivatives as selective BET inhibitors. One of the new compounds, (R)-12 (Y02234), binds to BRD4(1) with a Kd value of 110 nM and blocks bromodomain and acetyl lysine interactions with an IC50 value of 100 nM. It also exhibits selectivity for BET over non-BET bromodomain proteins and demonstrates reasonable anti-proliferation and colony formation inhibition effect in prostate cancer cell lines such as 22Rv1 and C4-2B. The BRD4 inhibitor (R)-12 also significantly suppresses the expression of ERG, Myc and AR target gene PSA at the mRNA level in prostate cancer cells. Treatment with (R)-12 significantly suppresses the tumor growth of prostate cancer (TGI = 70%) in a 22Rv1-derived xenograft model. These data suggest that compound (R)-12 is a promising lead compound for the development of a new class of therapeutics for the treatment of CRPC.

Introduction

Prostate cancer, a commonly diagnosed cancer, is a leading cause of cancer-related deaths. Castration-resistant prostate cancer (CRPC), a lethal form of this disease, is largely driven by increased expression and activity of the androgen receptor (AR) [[1], [2], [3]]. AR signaling is the major driver for progression to CRPC, after which most tumors continue to rely on AR signaling [[4], [5], [6]]. The standard treatment for prostate cancer is androgen deprivation therapy (ADT). However, the disease often progresses to CRPC and eventually develops resistance to second generation drugs such as abiraterone and enzalutamide [7]. Consequently, alternative approaches to elimination of AR signaling are needed for the treatment of CRPC [8,9].

The bromodomain and extra-terminal (BET) family proteins consists of BRD2, BRD3, BRD4 and BRDT, which “read” acetyl lysines that translate chromatin status into transcription activation through RNA polymerase II. BET bromodomain inhibitors show therapeutic potential in several conditions including inflammation and oncologic diseases. Several classes of BET inhibitors with different selectivity profiles have been identified and have demonstrated therapeutic potential for various human cancers [1,[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]]. BET bromodomains are key regulators of AR-, ERG-, and c-Myc-mediated transcription in CRPC, and they have been proposed as novel epigenetic targets for the treatment of CRPC [36]. BET inhibitors such as (6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]-diazepine-6-acetic acid 1,1-dimethylethyl ester ((+)-JQ1), (1), (S)-2-(4-(4-chloro-phenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]-triazolo [4,3-a]-[1,4]diazepin-6-yl)-N-(4-hydroxyphenyl)acetamide (I-BET762), (2), and compound 3 (OTX-015) have also been identified in an alternative strategy to target AR-driven cancers (Fig. 1) [1,2,37,38]. BET bromodomain inhibition has been shown to be more efficacious than direct AR antagonism in CRPC xenograft models [2]. Several BET inhibitors, compound 2 from GlaxoSmithkline [1,20,37,39], compound 3 from Merck [29,[40], [41], [42]], ZEN003694 (structure not disclosed) from Zenith Epigenetics [43] and GS-5829 (structure not disclosed) from Gilead [44] are currently being evaluated in clinical trials as a single agent or in combination with an anti-androgen in patients with CRPC. However, reports on the efficacy in CRPC of the pharmacologic inhibition by BET bromodomain inhibitors are still limited. New, potent and specific BET bromodomain inhibitors with different chemotypes are needed in order to explore therapeutic possibilities in human diseases such as CRPC.

In this article, we report the structure-based design and biological evaluation of benzoxazinone-containing 3,5-dimethylisoxazole derivatives as new BET bromodomain inhibitors with promising therapeutic effects.

Section snippets

Design of new BET inhibitors containing a 2H-benzo[b][1,4]oxazin-3(4H)-one scaffold

Several recent reports have described the 3,5-dimethylisoxazole moiety as an acetyl-lysine mimic [12,13,16,27,[45], [46], [47]]. Our design of a new class of BET inhibitors began with analysis of the binding mode of compound 5 with in complex with BRD4(1) (PDB ID: 3SVF, Fig. 2A and B). The 3,5-dimethylisoxazole moiety binds in the acetyl lysine binding site of BRD4(1) and interacts with the conserved residues Asn140 and Tyr97 through direct hydrogen bonds and indirect hydrogen bonds via a

Conclusions

In this study, we report the structure-based design, synthesis, and evaluation of a new class of BET bromodomain inhibitors. Following a structural analysis, a series of benzoxazinone-containing 3,5-dimethylisoxazole derivatives were designed and synthesized. The most promising compound, (R)-12 binds to BRD4(1) with an IC50 of 100 nM in the AlphaScreen assay and a Kd value of 110 nM in the ITC assay. This compound also exhibits excellent selectivity over other non-BET bromodomain-containing

General chemistry

The requisite reagents and solvents were obtained from commercial suppliers and used without further purification. Flash chromatography was performed using silica gel (300–400 mesh). All reactions were monitored by TLC, using silica gel plates with fluorescence F254 and UV light visualization. 1H NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 MHz or AV-500 spectrometer at 500 MHz. 13C NMR spectra were recorded on a Bruker AV-500 spectrometer at 125 MHz. Coupling constants (J)

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgments

We gratefully acknowledge financial support from the National Natural Science Foundation of China (grant 81673357 and 21602222), the “Personalized Medicines − Molecular Signature-based Drug Discovery and Development”, Strategic Priority Research Program of the Chinese Academy of Sciences (grant No. XDA12020363), the Chinese National Programs for Key Research and Development (grant 2016YFB0201701), the Natural Science Foundation of Guangdong Province (2015A030312014), the National R&D

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