Synthesis and Structure–Activity Relationships of Aristoyagonine Derivatives as Brd4 Bromodomain Inhibitors with X-ray Co-Crystal Research

Epigenetic regulation is known to play a key role in progression of anti-cancer therapeutics. Lysine acetylation is an important mechanism in controlling gene expression. There has been increasing interest in bromodomain owing to its ability to modulate transcription of various genes as an epigenetic ‘reader.’ Herein, we report the design, synthesis, and X-ray studies of novel aristoyagonine (benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one) derivatives and investigate their inhibitory effect against Brd4 bromodomain. Five compounds 8ab, 8bc, 8bd, 8be, and 8bf have been discovered with high binding affinity over the Brd4 protein. Co-crystal structures of these five inhibitors with human Brd4 bromodomain demonstrated that it has a key binding mode occupying the hydrophobic pocket, which is known to be the acetylated lysine binding site. These novel Brd4 bromodomain inhibitors demonstrated impressive inhibitory activity and mode of action for the treatment of cancer diseases.


Introduction
Gene transcription is regulated by multiple steps, including epigenetic modification. Epigenetic regulation is the dynamic and reversible modification of histones and DNA [1]. Among the various types of epigenetic modification [2], lysine acetylation plays a significant role in chromatin structure regulation [3]. Aberrant lysine acetylation is known to cause cancer via dysregulation of gene expression [4]. Most of the histone modifications are performed in three distinct steps: adding; removing; and reading [5,6]. The proteins involved in these steps are well studied in acetyl modification of lysine. Histone acetyltransferase (HAT) adds acetyl groups, while histone deacetylase removes acetyl groups; bromodomain reads the acetyl group [7].
Bromodomain, a conserved 110 amino acid module, is widely adapted in more than 50 cellular proteins [8]. These proteins exist in the nucleus or cytoplasm and have diverse functions and structures; they include chromatin-modifying proteins, helicases, chromatin remodelers, and transcriptional co-activators. There are several bromodomain subfamilies; Brd2, Brd3, Brd4, and the testis-specific protein Brdt belong to the bromodomain and extraterminal (BET) family [9]. BET proteins have a conserved modular structure including a In our previous study, we reported that aristoyagonine efficiently inhibited the binding of Brd4 bromodomain to acetylated histone peptide [23]. This was the first study to report effective natural product derivatives against bromodomain. In the present study, we aimed to synthesize aristoyagonine derivatives (benzo [6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one compounds) and determine their enzymatic and cellular activities. In addition, we aimed to elucidate the crystal structures of bromodomain in complex with aristoyagonine derivatives and identify the inhibitor binding modes.

Design
We previously reported that aristoyagonine exhibited Brd4 bromodomain inhibitory activity [23]. Based on the X-ray co-crystal structure (PBD code: 3MXF, human 1.6 Å) of JQ-1-bromodomain, we performed a docking study to reveal the binding mode between bromodomain and aristoyagonine. X-ray co-crystal structure showed two hydrogen bonds between bromodomain and JQ-1. The nitrogen atom of triazolodiazepine binds to the amino acid residue Asn140 via hydrogen bonding, and Tyr97 forms a hydrogen bond to the nitrogen atom of triazolodiazepine through a water molecule. The methyl group of triazole ring of JQ-1 shows a hydrophobic interaction with Val87 and Phe83; the dimethylthiophen group can be stabilized by hydrophobic interaction with Pro82 and Leu92. Molecular analysis revealed that a binding pocket was generated around the key functional site between bromodomain and inhibitor JQ-1; then, aristoyagonine was applied to the docking model. The docking model revealed that the amino acid residue Asn140 forms a direct hydrogen bond with the carbonyl oxygen of the isoindolinone moiety of aristoyagonine; it forms another hydrogen bond with Tyr97 via a water molecule. It was confirmed that the methyl group of indole is inside a hydrophobic pocket composed of Val87 and Phe83, and the fused ring forms a hydrophobic interaction with Pro82 and Leu92. Structure-based drug design in JQ-1 predicted an intra-hydrogen bonding between the nitrogen atom present in 1,3,4-triazole and hydrogen atom of diazepine-CH 2 ( Figure 2D). Therefore, JQ-1 is considered to bind the bromodomain in a similar manner as that to a four fused ring compound. Therefore, we hypothesized that benzo [6,7]oxepinoisoindole-2(1H)one, the skeleton of aristoyagonine, could be developed as a novel Brd4 inhibitor.  [6,7]oxepinoisoindol-2(1H)one scaffold derived from JQ-1.

Biological Activity
All the novel synthesized benzo [6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one derivatives were first evaluated for inhibitory activity of Brd4 bromodomain enzyme at a concentration of 10 µM. IC 50 value of iBet762 in our Brd4 enzyme assay was 0.15-0.30 µM. The structures of these compounds are presented in Tables 1-4 along with the results of alpha assay and cell cytotoxicity assay. Ty82, which expresses NUT-Brd4 fusion protein, is used for cytotoxicity assay, because the proliferation of Ty82 is dependent on Brd4 activity. Notably, three compounds (8ac, 8ad, and 8af) demonstrated an important pattern of inhibitory activity. 8ad with a bulky aromatic group at the R 3 position in the presence of 3-phenoxypropane had an IC 50 of 7.08 µM, whereas 8ac with 4-methoxyphenethyl group displayed a two-fold increased IC 50 value. Replacement of R 1 with ethoxy group (8af) instead of methoxy in 8ac afforded a weak inhibitory effect. Evaluation of the activity of these three compounds revealed that it is important for the oxygen atom present in the functional group at the R 2 position to be directed outward. Retaining ethoxy group at the R 1 position and introducing methoxy group at the R 2 and R 3 positions (8ae) slightly increased the inhibitory activity compared with 8af.
The ethoxy substituent in 8ab in the middle of the scaffold resulted in four-fold increased activity compared with 8ae, indicating the importance of size and position of functional groups on the benzo [6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one scaffold in maintaining inhibitory activity. Notably, the activity increased in 8aa (IC 50 = 0.83 µM) in which the R 3 functional group methoxy moiety was replaced with simple hydrogen atom (Table 1). From the results presented in Table 1, we confirmed that hydrogen atom, which has the smallest size, is the best group at R 2 and R 3 positions to maintain inhibitory activity. Therefore, in the second-round derivatization of compounds, hydrogen atom was substituted at R 2 and R 3 positions. Diverse derivatives were synthesized by fixing hydrogen atom at the R 2 position ( Table 2). We synthesized the derivatives of novel compounds by fixing R 2 and R 3 with hydrogen, and derivatization was performed with substitutions at R 4 position.
Compound 8ba in which isobutoxy group was introduced showed an IC 50 of 16.7 µM, but the activity dramatically increased 40-fold when the functional group was methoxy (8bc). In compound 8bd, which has a -OH group that functions as a hydrogen donor or acceptor, the IC 50 values in enzyme assay and cell cytotoxicity assay were 0.41 and 0.54 µM, respectively. Notably, the inhibitory activity was maintained in compound 8be (IC 50 = 0.86 µM), where N,N-diethylaminoethoxy group was introduced as a hydrogen bond acceptor. Compound 8bf, which has a pyridine group at a position adjacent to the oxygen atom at R 4 position, showed inhibitory activity against bromodomain (IC 50 = 0.91 µM), whereas the benzyl group-substituted compound 8bg did not.
Fluorine or trifluoromethyl group is widely used to improve metabolic stability in drug discovery and pharmaceutical science. We introduced the -CF 3 group instead of oxygen atom at R 4 position to assess the change in inhibitory activity. The replacement of methoxy group of 8bc with -CF 3 (8bb) showed no inhibitory activity. To confirm this dramatic reduction in biological activity, we synthesized R 1 = ethoxy compounds (8bj) while maintaining the same functional groups at the R 4 position. The presence of methoxy group at R 1 position (8bk) displayed an IC 50 of 0.86 µM, whereas the substitution of methoxy with trifluoromethyl group (8bj) reduced the inhibitory activity (IC 50 = 9 µM) against bromodomain. Compounds 8bh and 8bp were synthesized to evaluate the activity of derivatives containing oxygen atom at positions 3 and 4 with methylene (-CH 2 -) linkage; both the compounds exhibited modest inhibition. Through the biological evaluation of compounds in Table 2, we found that compounds with alkoxy group-including hydroxyl, methoxy, isobutoxy, and pyridinmethoxy-at R 4 position showed inhibitory activity. However, the presence of benzyloxy group at R 4 position abolished the inhibitory effect against bromodomain. The compounds presented in Table 3 were synthesized to evaluate their inhibitory activity by substituting nitrogen atom, an electron withdrawing group similar to oxygen atom and has stronger basicity, at the R 1 position. Compound 8ca in which isobutoxy group was introduced at the R 4 position showed no inhibitory activity. Compound 8cb, which had a hydroxyl group at the R 4 position, displayed weaker inhibitory effects than 8bd and 8bl, which have methoxy or epoxy groups at R 1 position. We reduced the double bond (C=C) presented in the oxepino ring located at the center of the benzo [6,7]oxepino[4,3,2cd]isoindol-2(1H)-one scaffold (8cc). The inhibitory effect of 8cc is similar to that of 8cb. From the IC 50 values of compounds 8ca-8ce in Table 3, we found that an oxygen atom is suitable for the R 1 position.
The compounds presented in Table 4 were synthesized to study the inhibitory activity on bromodomain by introducing carbon chain instead of heteroatoms at R 1 position. Considering the length of the substituent at R 1 in the compounds presented in Tables 1-3, we substituted n-propyl group at the R 1 position. Isobutoxy (8da), -CF 3 (8db), pyridinemethoxy (8dc), and benzyloxy (8dd) groups were introduced at the R 4 position. Compounds having a carbon chain at R 1 showed similar inhibitory activity to compounds having methoxy or ethoxy at R 1 position.  It is well known that c-MYC expression is controlled by the binding of bromodomain to lysine-acetylated histone [17]. Our derivatives downregulated the expression of c-MYC. As shown in Figure 3, c-MYC level was significantly decreased in 8bk-or 8bc-treated B-cell lymphoma cell line, NALM6. Our data clearly shows aristoyagonine derivatives with good activity against Brd4 have excellent cytotoxicity against Ty82.

Crystal Structures of Brd4 Bromodomain in Complex with Aristoyagonine Derivatives
To verify the specific binding of inhibitors to Brd4 bromodomain and obtain the structural information to develop further optimized inhibitors, we determined five threedimensional structures of Brd4 bromodomain-inhibitor complexes (Table S1). The overall structure of BRD4 bromodomain comprises four α-helices connected by highly variable loops, such as BC and ZA loops (Supplementary Figure S1). These two loops and their neighboring regions form a highly hydrophobic pocket, which is known to be the acetylated lysine binding pocket [9]. All electron density maps for the five compounds were found in the acetylated lysine binding pocket including JQ-1 and I-BET762 derivatives, L5S and H5C, bound bromodomain structures (Supplementary Figure S1) [9,25].

Structural Comparison of Aristoyagonine Derivatives with Other Known Brd4 Bromodomain Inhibitors
In this structural study, all five novel compounds formed a hydrogen bond with the amine group of Asn140 and oxygen atom of the carbonyl group in the core structure (benzo [6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one) of the synthesized compounds ( Figure 4). This hydrogen bond has been found in most of the bromodomain binding inhibitors, including JQ-1 and I-BET762 derivatives; Asn140 has been known as the key residue for acetylated lysine interaction [9,24]. In addition, these inhibitors form several hydrophobic interactions with Pro82, Leu92, Tyr139, and Ile146 ( Figure 4). However, the five benzo [6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one inhibitors formed fewer hydrophobic interactions with protein residues than JQ-1, which is likely due to the chemical structural planarity of inhibitors compared with JQ-1. The inhibitors adopt relatively large planar chemical structures in the core scaffold of inhibitors (Supplementary Figure S1), and the other moieties (functional groups in R 1 -R 4 ) are relatively small. In contrast, JQ-1 has a relatively small planar structure in the core of the inhibitor (13-methyl-3-thia-1,8,11,12tetraazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaene). Moreover, two other bulk moieties of JQ-1 are stretched out, fully occupying the whole acetylated lysine binding pocket and forming additional hydrophobic interactions with the protein residues Phe83, Val87, Leu94, and Asp145 around the Kac binding pocket ( Figure 4A) [9].  8ab, 8bc, 8bd, 8be, and 8bf) and JQ-1 (PDB entry:3MXF). In each figure, the residues that interacted with each inhibitor are also presented. Residues interacting with the inhibitors are indicated in green, red, blue, and purple: Hydrophilic residues are indicated in green; the residue of Asn140 forms hydrogen bond is presented in red; the water bridge in ocean blue; π-stacking is presented by purple. (B) Overlapped views of the five benzo [6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one inhibitors are presented along with the interactions between each inhibitor and protein residues. The core structure C of inhibitors and chemical moieties (R 1 -R 4 ) are shown.
In contrast to 8ab structure, the structures of 8bc, 8bf, and 8be contain an ordered water molecule forming a water-mediated interaction between the side chain amine of Gln85 and oxygen atom of 6-ether at R 4 position. The position of R 4 seems to be critical for these water-mediated interactions, considering the absence of a corresponding water molecule in 8ab, which has a methoxy group at R 3 position instead of R 4 position (Supplementary Figure S1). The formation of π-stacking interaction between phenol ring of the core structure and indole group of Trp81 is also a remarkable feature of these inhibitors. In JQ-1 and I-BET762 derivatives-bound Brd4 bromodomain structures, a methyl group (4-methyl) present at the corresponding region of the phenol ring in our inhibitors forms hydrophobic interaction with Trp81 instead of π-stacking in our inhibitors (Figure 4).

General
Unless otherwise stated, all reactions were performed under an inert (N 2 ) atmosphere. Reagents and solvents were reagent grade and purchased from Sigma-Aldrich (Milwaukee, WI, USA), Alfa Aesar (Ward Hill, MA, USA) and TCI Tokyo (Tokyo, Japan). Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany) with the indicated solvents. Thin-layer chromatography was performed using 0.25 mm silica gel plates (Merck). NMR spectra were obtained with a Bruker (Billerica, MA, USA) ultra-shield spectrometer at a 1 H frequency of 300 MHz or 500 MHz and a 13 C frequency of 125 MHz. Chemical shifts are reported in parts per million (ppm). Data for 1 H NMR are reported as follows: chemical shift (δ ppm) (integration, multiplicity, coupling constant (Hz)). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. The residual solvent peak was used as an internal reference. The mass spectra were obtained on an AcouityTM waters A06UPD9BM (Santa Clara, CA, USA), and Agilent Technologies SG12109048 (Santa Clara, CA, USA). Prior to biological testing, final compounds were confirmed to be >95% pure by UPLC chromatography using a Waters ACQUITY H-class system fitted with a C18 reversed-phase column (ACQUITY UPLC BEH C18: 2.1 mm × 50 mm, Part No. 186002350) according to the following conditions with solvents (A) H 2 O + 0.1% formic acid, (B) CH 3 CN + 0.1% formic acid, (C) MeOH + 0.1% formic acid; (I) a gradient of 95% A to 95% B over 5 min; (II) a gradient of 95% A to 95% C over 5 min.

Cell Cytotoxic Assay
For the viability experiments, Ty82 cells were seeded in 96-well plates at 30% confluency and exposed to chemicals the next day. After 72 h, WST-1 reagent was added, and absorbance at 450 nm was measured by using a Spectramax spectrophotometer (Molecular Devices, San Jose, CA, USA) in accordance with the manufacturer's instructions. The CC 50 values were calculated by using GraphPad Prism version 5 for Windows. The curves were fitted using a nonlinear regression model with a log (inhibitor) versus response formula.

Protein Expression and Purification
The Brd4 bromodomain gene (residues 44−168) was amplified by polymerase chain reaction and subcloned into the BamHI and XhoI site of pHis vector (a modified pET28b) vector. The resulting recombinant Brd4 bromodomain includes a TEV (Tobacco Etch Virus) protease cleavable polyhistidine (6×His) tag at the N-terminus.
The recombinant protein was expressed in Escherichia coli BL21(DE3) cells (Merck Millipore, Burlinton, MA, USA) cultured in Terrific Broth. After grown to an OD 600 of 0.6 at 37 • C, recombinant protein was induced with 0.1 mM isopropyl-D-thiogalactoside (IPTG) at 18 • C and cells were further cultured for about 15 h and harvested. Cells were resuspended in lysis buffer (50 mM HEPES-OH pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride) and lysed using high pressure homogenizer (PICOMAX, Seoul, Korea) for three times with 1000 bar pressure. After centrifugation at 13,000× g for 1 h, the supernatant was applied to Ni-NTA resin (GE healthcare, Chicago, IL, USA) and incubated with gentle agitation for 1 h under ice-cold condition. The Ni-NTA resin was washed using wash buffer (50 mM HEPES-OH pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, and 30 mM Imidazole) and protein was eluted with elution buffer (50 mM HEPES-OH pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, and 300 mM Imidazole). The eluted protein was incubated with TEV protease for overnight at 4 • C to remove the fusion tag. Protein was further purified using a HiLoad 16/600 Superdex 75 prep grade column (GE Healthcare, USA) with final storage buffer (10 mM HEPES-OH pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, and 10 mM dithiothreitol). Protein was concentrated to 14 mg/mL using Amicon Centrifugal Filter Units (Merck Millipore, Burlinton, MA, USA) and stored at −80 • C for crystallization.

Crystallization and Structure Solution
All crystallization experiments were performed using the sitting drop vapor diffusion method at 14 • C. Before crystallization, the Brd4 bromodomain protein stock was diluted 10 times with the final storage buffer and added with 0.7 mM inhibitor compounds (approximately 1:10 protein:compound molar ratio) and incubated at 4 • C for overnight. The mixed protein solution was reconcentrated and mixed with crystallization reservoir solution (6 M sodium formate and 10% glycerol). The protein:reservior solution ratio was 1:1 (v/v) and crystals were grown within 3 days.
Diffraction data were collected using a Dectris Pilatus3 6M CCD detector at the BL-11C beamline of Pohang Light Source (Pohang, Korea). Crystals were protected by the cryo-buffer (crystallized reservoir solution supplemented with 2.5 mM of each inhibitor compound and 30% (v/v) glycerol). The raw data were processed and scaled using the HKL2000 program suite [26]. The phase was calculated by molecular replacement with the program PHASER [27] using a Brd4 bromodomain structure (PBD entry, 2OSS) as a search model [9]. Further model building was completed with the program COOT [28] and refinement was conducted using the phenix.refine in Phenix program suite [29]. The coordinates and cif restraint files of the inhibitors were generated using the program Maestro (Schrödinger, New York, NY, USA) and the eLBOW in Phenix program suite [30,31], respectively. All data collection and refinement statistics are listed in Table S1.

Synthesis
Compounds 2a-e [32]. To a flask containing a solution of 4-hydroxybenzaldehyde (1, 10.0 g) in acetone (150 mL) were added K 2 CO 3 (1.2 eq.) and benzyl bromide (2.0 eq.). The reaction flask was stirred for 9 h at 70 • C. The solvent was removed with a rotary evaporator and transferred to a separatory funnel with H 2 O and CH 2 Cl 2 . The combined organic layers were dried over anhydrous MgSO 4 , filtered and concentrated in vacuum. The residue was purified by silica gel flash column chromatography to afford benzyloxybenzaldehyde (2). Compounds 3a-e [24]. Benzaldehyde (2, 38.5 mmol) was mixed in methylamine (40% in MeOH, 25 mL, 0.19 mol) at r.t. under a N 2 atmosphere. The mixture was stirred at r.t. for 48 h, until the benzylidene methanamine formation was completed. The methanol was removed with a rotary evaporator and the mixture was extracted with CH 2 Cl 2 from H 2 O. The combined organic layers were washed with brine, dried over MgSO 4 . Straightly, benzylidene methanamine in MeOH was carefully treated with solid NaBH 4 (2. Compounds 4a-e [24]. A stirred mixture of freshly prepared amine (3, 40.4 mmol), Pd(OAc) 2 (0.9 g, 4.05 mmol), and Cu(OAc) 2 (7.3 g, 40.5 mmol) in toluene (130 mL) was refluxed in an oil bath at 120 • C in an atmosphere of carbon monoxide (ca. 1~1.5L) containing air (corresponding to 0.3 mmol of O 2 ) delivered from a balloon for 20 h. After cooled to room temperature, the mixture was diluted with EtOAc and filtered through a short silica gel pad. The solution was concentrated in vacuo, and toluene was removed in a rotary evaporator. The residue was purified by silica gel flash column chromatography to afford 2-methylisoinolin-1-one (4).
4-(Benzyloxy)-5-methoxy-2-methylisoindolin-1-one (4a). Rf 0.23 (hexane/EtOAc = 5/1). Yield = 60.4% (6.6 g). 1  Compounds 5a-e [24]. To a solution of methylisoindolin-1-one (4, 6.70 mmol) in MeOH/EtOAc (1:1, 20 mL) was added Pd/C (10 wt %). The reaction mixture was stirred under 40 psi until the absorption of hydrogen ceased for 4 h. After the Pd/C catalyst was filtered off through a Celite pad, the solvent was removed on a rotary evaporator. The mixture was transferred to a separatory funnel with CH 2 Cl 2 . The organic layers were washed with brine, dried over MgSO 4 . The residue was treated with ether and resulting solid was collected by filtration to afford isoindolinone (5 A nitrogen-flushed microwave vial was equipped with a magnetic stirring bar and charged with 4 Å molecular sieves. The vial was flame dried for 10 min under high vacuum and purged with N 2 . After cooling to room temperature, isoindolinone (0.3 g), 2bromobenzaldehyde (2.0 eq.), CuCl (0.10 eq.), and Cs 2 CO 3 (3.0 eq.) were added sequentially. The reaction mixture was suspended in pyridine (10 mL). Then, the reaction vial was sealed and placed into a heating block at 150 • C for 24 h. The mixture was cooled to room temperature, filtered through a Celite pad, and washed with acetone. The resulting solution was concentrated with a rotary evaporator, and the residue was purified by silica gel flash column chromatography to afford dibenzoxepine lactam. 5,7-Dimethoxy-1-methylbenzo [6,7]