Design, synthesis and biological evaluation of novel 6-phenyl-1,3a,4,10b-tetrahydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one derivatives as potential BRD4 inhibitors

Abstract

Bromodomain-containing protein 4 (BRD4) is a key epigenetic regulator in cancer, and inhibitors targeting BRD4 exhibit great anticancer activity. By replacing the methyltriazole ring of the BRD4 inhibitor I-BET-762 with an N-methylthiazolidone heterocyclic ring, fifteen novel BRD4 inhibitors were designed and synthesized. Compound 13f had a hydrophobic acetylcyclopentanyl side chain, showing the most potent BRD4 inhibitory activity in the BRD4-BD1 inhibition assay (IC₅₀ value of 110 nM), it also significantly suppressed the proliferation of MV-4-11 cells with high BRD4 level (IC₅₀ value of 0.42 μM). Furthermore, the potent apoptosis-promoting and G0/G1 cycle-arresting activity of compound 13f were indicated by flow cytometry. As the downstream-protein of BRD4, c-Myc was in significantly low expression by compound 13f treatment in a dose-dependent manner. All the findings supported that this novel compound 13f provided a perspective for developing effective BRD4 inhibitors.

Introduction

The bromodomain and extra-terminal domain (BET) protein family includes BRD2, BRD3, BRD4 and bromodomain testis-specific protein (BRDT). The BET protein consists of two N-terminal bromodomains and one C-terminal domain (ET), and their sequences are highly conserved.¹ Among them, BRD2 functions through the E2F transcription factor pathway;² BRD3 helps in recruiting hematopoietic transcription factor, GATA1, by regulating the maturation of red blood cells, megakaryocytes and mast cells.3, 4 BRD4 is currently the most widely studied protein of BET family, and it is involved in many human diseases, including cancer, inflammation, cardiovascular disease, central nervous system (CNS) disease and human immunodeficiency virus (HIV) infection.5, 6, 7, 8 BRD4 is a critical mediator of transcriptional elongation, and it influences mitotic progression.9, 10, 11 The abnormal expression of BRD4 will cause the activation of the downstream gene c-Myc, which could lead to the occurrence of cancers such as acute leukemia, lymphoma, melanoma and so on.¹² Therefore, BRD4 has been developed as therapeutic target for research on cancer drug, and such research is applicable to the development of small molecule inhibitors.

A pair of bromodomains (BD1 and BD2) are contained in the BRD4 structure, each of which is consisted of a conserved fold and two loop regions.¹ Although BD1 and BD2 possess a high similarity in sequence of substrate-binding site, some functions of BD1 and BD2 are different. For example, BD2 have wide effects towards other acetylated substrates, whereas BD1 is inclined to only discerning H4 acetylation marks.¹³ Therefore, BD1 is the main targeted binding site for BRD4 inhibitors.14, 15 BRD4 inhibitor (+)-JQ-1 and I-BET-762 are potent BRD4 inhibitors. Provided by PDB data, the core structures of the two potent inhibitors can form essential hydrogen bonds with the residue Tyr 97 in BRD4 in Fig. 1 (PDB ID: 4c66 and 3mxf). Additionally, the co-crystal structure of BRD4 bromodomain 1 (BRD4-BD1) and histone peptide H4K8acK12ac indicates that acetylated lysine is recognized by a central hydrophobic cavity, and hydrogen bond interaction is formed between the acetylcarbonyl oxygen atom and the conserved Tyr97 via water molecules.¹⁶ Therefore, Tyr 97 residue is an important binding site for BRD4-BD1.

As shown in Fig. 2, the researchers have made some structural modifications to the BRD4 inhibitor I-BET-762. It has been reported by Hewitt, et al. that the BRD4 inhibitory activity was significantly improved when the methyltriazole ring of the lead compound I-BET-762 was substituted with methylisoxazole ring (Fig. 2A).17, 18 In addition, Li, et al. has reported that on the basis of retaining the predominant skeletal structure of benzoazepine and replacing methyltriazole with N-methylpyridone, compounds with more potent BRD4 inhibitory activity were obtained (Fig. 2B).¹⁹ Therefore, the methyltriazole ring of compound I-BET-762 could be further modified, and these modifications had little impact on target- selectivity between BRD4 and other BRD proteins. Since the acetylated lysine (KAc) binding site was the most important part in the design of BRD4 inhibitors, interacting with the acetylated lysine binding site have been used for developing several kinds of specific small molecular BRD4 inhibitors.20, 21, 22, 23, 24 It has been reported that the 2-thiazolidinone structure could well simulate the acetylated lysine structure, and it acted as an effective BRD4 mimic group.25, 26 Thus, the 2-thiazolidinone was a new strategy to replace the methyltriazole ring of compound I-BET-762, which was different from the previous studies to replace the methyltriazole ring of compound I-BET-762 with N-methylpyridone or methylisoxazole ring.

Here we synthesized a series of new scaffold compounds based on the structure of I-BET-762. The BRD proteins and their binding site were different, and a lot of research has been done on the BRD4 binding site. On the basis of this binding site, a reasonable compound design was carried out. Based on the structure-activity relationship of I-BET-762, the methyltriazole moiety was replaced with a methylthiazolidone moiety to maintain the interaction with the residue Tyr 97, which could keep selective inhibition of BRD4-BD1 instead of BRD4-BD2. Consequently, a series of biological evaluation demonstrated that compound 13f did exhibit more powerful BRD4 inhibitory activity than I-BET-762, and its activity was comparable to (+)-JQ-1. Compound 13f was shown to be potent BRD4 inhibitor with an IC₅₀ value of 110 nM, and it had an IC₅₀ value of 0.42 μM against MV-4-11 cell antiproliferation. It provided a structural perspective for developing novel effective BRD4 inhibitors.