Synthesis and structure–activity relationships of aristoyagonine derivatives as brd4 bromodomain inhibitors with x-ray co-crystal research

Article abstract of DOI:10.3390/molecules26061686

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 t

Full text of DOI:10.3390/molecules26061686

molecules
Article
Synthesis and Structure–Activity Relationships of
Aristoyagonine Derivatives as Brd4 Bromodomain Inhibitors
with X-ray Co-Crystal Research
Minjin Yoo 1,†, Tae Hyun Park 2,3,† , Miyoun Yoo 4,†, Yeongrin Kim 1,4, Joo-Youn Lee 4, Kyu Myung Lee 4
,
Seong Eon Ryu 3, Byung Il Lee 2,5,
* *
, Kwan-Young Jung 1,4,* and Chi Hoon Park 1,4,
1
Department of Medicinal Chemistry and Pharmacology, University of Science & Technology,
Daejeon 34113, Korea; yminjin10@gmail.com (M.Y.); kyr@krict.re.kr (Y.K.)
Research Institute, National Cancer Center, Goyang-si, Gyeonggi 10408, Korea; 74977@ncc.re.kr
Department of Bioengineering, Hanyang University, Seoul 04763, Korea; ryuse@hanyang.ac.kr
Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology,
2
3
4
Daejeon 34114, Korea; miyounyoo@gmail.com (M.Y.); leejy@krict.re.kr (J.-Y.L.); kmlee@krict.re.kr (K.M.L.)
Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and
Policy, Goyang-si, Gyeonggi 10408, Korea
5
*
Correspondence: bilee@ncc.re.kr (B.I.L.); krjeong@krict.re.kr (K.-Y.J.); chpark@krict.re.kr (C.H.P.);
Tel.: +82-31-920-2223 (B.I.L.); +82-42-860-7692 (K.-Y.J.); +82-42-860-7416 (C.H.P.);
Fax: +82-42-860-7160 (K.-Y.J.); +82-42-860-4246 (C.H.P.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Abstract: 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
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Yoo, M.; Park, T.H.; Yoo,
M.; Kim, Y.; Lee, J.-Y.; Lee, K.M.; Ryu,
S.E.; Lee, B.I.; Jung, K.-Y.; Park, C.H.
Synthesis and Structure–Activity
Relationships of Aristoyagonine
Derivatives as Brd4 Bromodomain
Inhibitors with X-ray Co-Crystal
Research. Molecules 2021, 26, 1686.
https://doi.org/10.3390/
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.
molecules26061686
Academic Editor: Michele R. Chierotti
Keywords: Brd4; aristoyagonine; epigenetic; crystal structure; SAR
Received: 18 February 2021
Accepted: 10 March 2021
Published: 17 March 2021
1. Introduction
Gene transcription is regulated by multiple steps, including epigenetic modification.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Epigenetic regulation is the dynamic and reversible modification of histones and DNA [
Among the various types of epigenetic modification [ ], lysine acetylation plays a signif-
icant role in chromatin structure regulation [ ]. Aberrant lysine acetylation is known to
cause cancer via dysregulation of gene expression [ ]. Most of the histone modifications
are performed in three distinct steps: adding; removing; and reading [ ]. The proteins
1].
2
3
4
5,6
involved in these steps are well studied in acetyl modification of lysine. Histone acetyl-
transferase (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 extra-
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
terminal (BET) family [9]. BET proteins have a conserved modular structure including a
Molecules 2021, 26, 1686. https://doi.org/10.3390/molecules26061686
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1686
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pair of bromodomain. It preferentially binds to
ε
-N-acetylated lysine residue of histone
through its binding pocket [10]. Subsequently, transcription and chromatin remodeling
proteins, including HATs, histone methyltransferases (HMTs), transcription initiation
factors, and ATP-dependent helicases, are recruited by Brd4 to chromatin [11]. Brd4 acts
as an important mediator of transcriptional elongation through recruiting the positive
transcription elongation factor complex (P-TEFb) [12,13].
Brd4 is fused with NUclear protein in Testis (NUT) in most of the patients with NUT
midline carcinoma (NMC) [14,15]. As Brd4-nut knockdown significantly suppresses the
proliferation of NMC cell line, Brd4-nut gene translocation is believed to be the driving
force for NMC initiation [16]. Besides NMC, Zuber et al. found that Brd4 is a potent target
for acute myeloid leukemia through RNAi screen [17]. They reported that JQ-1, which
abrogates the interaction between Brd4 bromodomain and histone peptide with acetylated
lysine, suppressed AML proliferation by considerably downregulating c-Myc protein level.
Subsequent studies found that JQ-1 is an effective anti-cancer agent in most leukemic
tumors [18]. Many novel bromodomain inhibitors have been developed in previous studies
(Figure 1) [19–22].
Figure 1. Chemical structures of representative BET bromodomain inhibitors.
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.
2. Results and Discussion
2.1. 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

Molecules 2021, 26, 1686
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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-CH2 (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.
Figure 2. Binding pocket of Brd4 bromodomain and ligand structure-based drug design (SBDD).
(
A
) Crystal structure of the bromodomain of human Brd4 in complex with the inhibitor JQ-1(PDB
code: 3MXF, human 1.6 Å). ( ) Molecular docking of designed compound of aristoyanine with
Brd4 bromodomain binding pocket. ( ) Designed docking model with Gaussian surface. ( ) Novel
B
C
D
compound benzo[6,7]oxepinoisoindol-2(1H)one scaffold derived from JQ-1.
2.2. Chemistry
Based on molecular modeling, it was found that Asn140, an important amino acid
residue of Brd4 bromodomain, forms a strong hydrogen bond with the carbonyl oxygen
of compounds. We fixed the carbonyl group on the synthesized compounds to maintain
a strong hydrogen bonding between Brd4 bromodomain and compounds. To synthesize
the designed compounds containing benzo[6,7]oxepinoisoindol-2(1H)one scaffold, we
followed the one-pot Cu-catalyzed etherification/aldol condensation cascade reaction [24].
Subunits were synthesized as described in Scheme 1. With salicylaldehyde derivatives
(
1a
the presence of excess K2CO3 yielded O-benzyl protected derivatives (2a
methylamine in the presence of anhydrous methanol solvent resulted in reductive amina-
tion, while reduction by NaBH4 yielded the desired compounds (3a ). Ring cyclization
using CO gas in the presence of Cu(OAc)2 and a catalytic amount of Pd(OAc)2 afforded
–
e
) as starting compounds, nucleophilic substitution reactions with benzyl bromide in
–e
). Treatment of
–e

Molecules 2021, 26, 1686
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2-methylisoindolin-1-one backbone (4a
–
e). Debenzylation of O-benzyl-2-methylisoindolin-
2-one performed using Pd/C in H2 atmosphere afforded the top subunits (5a
bottom subunits were synthesized from substituted 2-bromo-3-hydroxybenzaldehyde (
as the starting compound with several alkyl/aryl halides to yield the desired compound
). To obtain the final compounds, the top subunits 5a were treated by Cu-catalyzed
–e). The
6)
(
7
–e
etherification and aldol condensation with the respective bottom subunit in the presence of
CuBr and Cs2CO3.
Scheme 1. Synthesis procedure of benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one derivatives [24]. Reaction scheme.
◦
Reagents and conditions: (a) benzyl bromide, K2CO3, Acetone, 70 C, 9 h; (b) (i) 40% MeNH2 in MeOH, 48 h; (ii) NaBH4,
◦
◦
MeOH, 0 C, 18 h; (c) CO(g), Cu(OAc)2, Pd(OAc)2, Toluene, O2(g), 130 C, 18 h; (d) 10 wt % Pd/C, H2, MeOH/EtOAc = 1:1
ratio, r.t., 12 h; (e) R2—X, K2CO3, DMF, r.t., 12 h; (f) 5a–e, CuBr, Cs2CO3, 4Å molecular sieves, pyridine, 100 C, 24 h.
◦
2.3. 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. IC50 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 with a bulky aromatic group at the R3 position in the presence of 3-phenoxypropane
had an IC50 of 7.08 M, whereas 8ac with 4-methoxyphenethyl group displayed a two-fold
, 8ad, and 8af) demonstrated an important pattern of inhibitory activity.
µ
increased IC50 value. Replacement of R1 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 R2
position to be directed outward. Retaining ethoxy group at the R1 position and introducing
methoxy group at the R2 and R3 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 in-
creased 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 main-
taining inhibitory activity.

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Table 1. Inhibition effects of compounds fixed with hydrogen at R4 position.
Brd4 a
IC50(µM)
Brd4 a
IC50(µM)
Cell b
CC50(µM)
Cell b
CC50(µM)
Cpds
8aa
Structure
Cpds
Structure
0.84
0.86
3.74
NT
NT
>20
8ad
7.07
4.16
5.33
>4
8ab
8ae
8af
NT
>4
8ac
a
To measure the inhibitory effect of compounds against bromodomain binding activity, alpha-screen assay was performed. The IC50 in the
b
table was calculated with Prism6 program. To measure the Ty82 cell cytotoxic activity of compounds, MTS assay was performed. The
CC50 in the table was calculated with Prism6 program. NT = not tested.
Notably, the activity increased in 8aa (IC50 = 0.83 µ
M) in which the R3 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 R2 and R3 positions to maintain inhibitory activity. Therefore, in the
second-round derivatization of compounds, hydrogen atom was substituted at R2 and R3
positions. Diverse derivatives were synthesized by fixing hydrogen atom at the R2 position
(Table 2). We synthesized the derivatives of novel compounds by fixing R2 and R3 with
hydrogen, and derivatization was performed with substitutions at R4 position.
Compound 8ba in which isobutoxy group was introduced showed an IC50 of 16.7
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 IC50 values in enzyme assay and cell cytotoxicity assay were 0.41 and
0.54 M, respectively. Notably, the inhibitory activity was maintained in compound 8be
IC50 = 0.86 µM), where N,N-diethylaminoethoxy group was introduced as a hydrogen
µM,
(
µ
(
bond acceptor. Compound 8bf, which has a pyridine group at a position adjacent to the oxy-
gen atom at R4 position, showed inhibitory activity against bromodomain (IC50 = 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 –CF3 group instead of
oxygen atom at R4 position to assess the change in inhibitory activity. The replacement
of methoxy group of 8bc with –CF3 (8bb) showed no inhibitory activity. To confirm this
dramatic reduction in biological activity, we synthesized R1 = ethoxy compounds (8bj
)
while maintaining the same functional groups at the R4 position. The presence of methoxy
group at R1 position (8bk) displayed an IC50 of 0.86
µM, whereas the substitution of
methoxy with trifluoromethyl group (8bj) reduced the inhibitory activity (IC50 = 9 M)
µ
against bromodomain. Compounds 8bh and 8bp were synthesized to evaluate the activ-
ity of derivatives containing oxygen atom at positions 3 and 4 with methylene (–CH2–)
linkage; both the compounds exhibited modest inhibition. Through the biological evalua-
tion of compounds in Table 2, we found that compounds with alkoxy group—including
hydroxyl, methoxy, isobutoxy, and pyridinmethoxy—at R4 position showed inhibitory
activity. However, the presence of benzyloxy group at R4 position abolished the inhibitory
effect against bromodomain.

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Table 2. Inhibition effects of compounds fixed with hydrogen at R2 and R3 position.
Brd4 a
IC50(µM)
Brd4 a
IC50(µM)
Cell b
CC50(µM)
Cell b
CC50(µM)
Cpds
8ba
Structure
Cpds
8bi
Structure
16.7
>32
NT
>10
>9
NT
8bb
>20
8bj
>20
8bc
0.41
0.41
0.66
0.54
8bk
8bl
0.86
1.12
0.49
0.31
8bd
8be
8bf
0.86
0.91
0.56
0.23
8bm
8bn
0.91
0.68
0.66
0.36
8bg
>32
11.1
NT
8bo
8bp
>32
2.89
NT
8bh
2.16
3.81
a
To measure the inhibitory effect of compounds against bromodomain binding activity, alpha-screen assay was performed. The IC50 in the
b
table was calculated with Prism6 program. To measure the Ty82 cell cytotoxic activity of compounds, MTS assay was performed. The
CC50 in the table was calculated with Prism6 program. NT = not tested.
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 R1 position. Compound 8ca in which isobutoxy group
was introduced at the R4 position showed no inhibitory activity. Compound 8cb, which
had a hydroxyl group at the R4 position, displayed weaker inhibitory effects than 8bd and
8bl, which have methoxy or epoxy groups at R1 position. We reduced the double bond
(C=C) presented in the oxepino ring located at the center of the benzo[6,7]oxepino[4,3,2-
cd]isoindol-2(1H)-one scaffold (8cc). The inhibitory effect of 8cc is similar to that of 8cb
.
From the IC50 values of compounds 8ca–8ce in Table 3, we found that an oxygen atom is
suitable for the R1 position.
The compounds presented in Table 4 were synthesized to study the inhibitory ac-
tivity on bromodomain by introducing carbon chain instead of heteroatoms at R1 po-
sition. Considering the length of the substituent at R1 in the compounds presented in
Tables 1–3, we substituted n-propyl group at the R1 position. Isobutoxy (8da), –CF3 (8db),

Molecules 2021, 26, 1686
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pyridinemethoxy (8dc), and benzyloxy (8dd) groups were introduced at the R4 position.
Compounds having a carbon chain at R1 showed similar inhibitory activity to compounds
having methoxy or ethoxy at R1 position.
Table 3. Inhibition effects of compounds fixed with diethylamine at R1 position.
Brd4 a
IC50(µM)
Brd4 a
IC50(µM)
Cell b
CC50(µM)
Cell b
CC50(µM)
Cpds
8ca
Structure
Cpds
8cd
Structure
>32
4.89
5.86
>20
8
>32
7.29
>20
10
8cb
8ce
8cc
10
a
To measure the inhibitory effect of compounds against bromodomain binding activity, alpha-screen assay was performed. The IC50 in the
b
table was calculated with Prism6 program. To measure the Ty82 cell cytotoxic activity of compounds, MTS assay was performed. The
CC50 in the table was calculated with Prism6 program.
Table 4. Inhibition effects of compounds fixed with alkyl chain at R1 position.
Brd4 a
IC50(µM)
Brd4 a
IC50(µM)
Cell b
IC50(µM)
Cell b
IC50(µM)
Cpds
8da
Structure
Cpds
8dd
Structure
6.64
>32
10
9
10
8db
>20
8de
8df
2.69
4.5
8dc
1.37
1.77
>32
NT
a
To measure the inhibitory effect of compounds against bromodomain binding activity, alpha-screen assay was performed. The IC50 in the
b
table was calculated with Prism6 program. To measure the cell cytotoxic activity of compounds, MTS assay was performed The IC50 in
the table was calculated with Prism6 program. NT = not tested.
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.

Molecules 2021, 26, 1686
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Figure 3. NALM6 cells were treated with 8bk or 8bc compounds. After 18 h, cell lysates were
prepared, and western blot was conducted to see c-MYC level.
2.4. 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 three-
dimensional 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].
2.5. 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, in-
cluding 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 hydropho-
bic 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 in-
teractions 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 R1–R4) are relatively small. In contrast, JQ-1 has a
relatively small planar structure in the core of the inhibitor (13-methyl-3-thia-1,8,11,12-
tetraazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaene). Moreover, two other bulk moi-
eties 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].

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Figure 4. Binding modes of the five benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one inhibitors and
JQ-1. (A) Detailed view of binding modes of the five benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one
inhibitors (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. ( ) Overlapped views of the five benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one
B
inhibitors are presented along with the interactions between each inhibitor and protein residues. The
core structure C of inhibitors and chemical moieties (R1–R4) 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 R4 position. The position of R4 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 R3 position instead of R4 position (Supple-
mentary 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).
3. Materials and Methods
3.1. General
Unless otherwise stated, all reactions were performed under an inert (N2) 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 1H frequency of 300 MHz or 500 MHz and a 13
frequency of 125 MHz. Chemical shifts are reported in parts per million (ppm). Data for
C

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1H 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) H2O + 0.1% formic acid, (B) CH3CN + 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.
3.2. Alpha-Screen Enzyme Assay
Bromodomain was prepared as previously described. The alpha-screen assay was per-
formed in accordance with the manufacturer’s protocol (PerkinElmer, Waltham, MA, USA),
by using a buffer (50 mM HEPES, 100 mM NaCl, 0.1% BSA, pH 7.4 supplemented with
0.05% CHAPS) and OptiPlateTM-384 plate (PerkinElmer, USA). Briefly, 2.5
solution and 5 L of peptide solution [SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-
biotin] were added to 5 L of glutathione-S-transferase (GST) and His-tagged bromodomain
µL of compound
µ
µ
in OptiPlateTM-384 plate. Streptavidin-coated donor beads and anti-GST alpha-screen
acceptor beads were added under low-light condition. Plate was incubated at 25 ◦C for
60 min using a Thermomixer C (Eppendorf, Hamburg, Germany), and read using a Fusion-
Alpha
™ Multilabel Reader (PerkinElmer, USA). The alpha-screen results were confirmed
by using alpha-screen TruHit kits (PerkinElmer, USA).
3.3. Western Blot
For immunoblotting, cells were washed in PBS, lysed in 1
Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 3% β-mercaptoethanol), and boiled for 10 min.
×
sample buffer (50 mmol/L
Lysates were subjected to SDS-PAGE followed by blotting with the indicated antibodies and
detection by western blotting substrate ECL reagent (Thermo Fisher Scientific, Waltham,
MA, USA). Images were produced using a SensiQ-2000 (Lugen Sci Co., Seoul, KOREA). The
following antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA):
c-Myc (catalog no. 5605). Tubulin antibody (catalog no. T6199) was purchased from Sigma-
Aldrich. HRP-conjugated anti-mouse (catalog no. NCI1430KR), and HRP-conjugated
anti-rabbit (catalog no. NCI1460KR) antibodies were obtained from Thermo Scientific.
3.4. Cell Cytotoxic Assay
For the viability experiments, Ty82 cells were seeded in 96-well plates at 30% conflu-
ency 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 CC50
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.
3.5. 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 OD600 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-

Molecules 2021, 26, 1686
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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
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,
×
g for 1 h, the supernatant was applied to Ni-NTA resin (GE
500 mM NaCl, 5% glycerol, 5 mM
was eluted with elution buffer (50 mM HEPES-OH pH 7.5, 500 mM NaCl, 5% glycerol,
5 mM -mercaptoethanol, and 300 mM Imidazole). The eluted protein was incubated with
β-mercaptoethanol, and 30 mM Imidazole) and protein
β
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.
3.6. Crystallization and Structure Solution
All crystallization experiments were performed using the sitting drop vapor diffu-
◦
sion 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.
3.7. Synthesis
Compounds 2a–e [32]. To a flask containing a solution of 4-hydroxybenzaldehyde (1,
10.0 g) in acetone (150 mL) were add◦ed K2CO3 (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 H2O and CH2Cl2. The combined organic layers
were dried over anhydrous MgSO4, filtered and concentrated in vacuum. The residue was
purified by silica gel flash column chromatography to afford benzyloxybenzaldehyde (2).
2-(Benzyloxy)-3-methoxybenzaldehyde (2a). Rf 0.25 (hexane/EtOAc = 9/1). Yield = 82%
1
(13.0 g). H-NMR (CDCl3, 300 MHz)
δ 10.24 (1H, s, CHO), 7.40–7.32 (6H, m, Ar), 7.18–7.10
(2H, m, Ar), 5.17 (2H, s, CH2), 3.93 (3H, s, CH3); LCMS (ESI+) m/z calcd. for C15H14O3
([M + H+]) 243.09, found 243.12
2-(Benzyloxy)-3-ethoxybenzaldehyde (2b). Rf 0.25 (hexane/EtOAc = 9/1). Yield = 89%
1
(13.7 g). H-NMR (CDCl3, 300 MHz)
δ 10.26 (1H, s, CHO), 7.41–7.33 (6H, m, Ar), 7.18–7.10
(2H, m, Ar), 5.19 (2H, s, CH2), 4.18–4.11 (2H, q, J = 6.9 Hz, CH2), 1.53–1.48 (3H, t, J = 6.9 Hz,
CH3); LCMS (ESI+) m/z calcd. for C16H16O3 ([M + H+]) 256.11, found 256.17
2-(Benzyloxy)-3-methylbenzaldehyde (2c). Rf 0.29 (hexane/EtOAc = 9/1). Yield = 99%
1
(17.5 g). H-NMR (CDCl3, 300 MHz)
δ 10.26 (1H, s, CHO), 7.69 (1H, d, J = 7.7 Hz, Ar), 7.47
(1H, d, J = 7.4 Hz, Ar), 7.42–7.37 (5H, m, Ar), 7.17 (1H, t, J = 7.4 Hz, Ar), 4.96 (2H, s, CH2),
2.35 (3H, s, CH3); LCMS (ESI+) m/z calcd. for C15H14O2 ([M + H+]) 227.10, found 227.12

Molecules 2021, 26, 1686
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3-Allyl-2-(benzyloxy)benzaldehyde (2d). Rf 0.24 (hexane/EtOAc = 12/1). Yield = 84%
(13.1 g). 1H-NMR (CDCl3, 300 MHz)
δ 10.30 (1H, s, CHO), 7.78 (1H, m, Ar), 7.54 (1H,
m, Ar), 7.45–7.39 (5H, m, Ar), 7.28 (1H, m, Ar), 6.01 (1H, m, CH), 5.17 (2H, m, CH2), 5.01
(2H, s, CH2), 3.53 (2H, m, CH2); LCMS (ESI+) m/z calcd. for C17H16O2 ([M + H+]) 253.12,
found 253.14
2-(Benzyloxy)-3-(diethylamino)benzaldehyde (2e). Rf 0.28 (hexane/EtOAc = 7/3). Yield =
1
99% (14.5 g). H-NMR (CDCl3, 300 MHz)
δ 10.27 (1H, s, CHO), 7.77 (1H, d, J = 8.9 Hz, Ar),
7.49–7.35 (5H, m, Ar), 6.29 (1H, dd, J = 8.9 Hz, J = 1.86 Hz, Ar), 6.08 (1H, d, J = 2.2 Hz, Ar),
5.19 (2H, s, CH2), 3.38 (4H, q, J = 7.1 Hz, 2 CH2), 1.17 (6H, t, J = 7.1 Hz, 2 CH3); LCMS
(ESI+) m/z calcd. for C18H21NO2 ([M + H+]) 284.16, found 284.17
Compounds 3a 24]. Benzaldehyde ( , 38.5 mmol) was mixed in methylamine (40%
×
×
–e
[
2
in MeOH, 25 mL, 0.19 mol) at r.t. under a N2 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 CH2Cl2 from
H2O. The combined organic layers were washed with brine, dried over MgSO4. Straightly,
benzylidene methanamine in MeOH was carefully treated with solid NaBH4 (2.18 g,
57.8 mmol) at 0 ◦C. The reaction flask was flushed with N2 and stirred at 0 ◦C for 1 h. The
methanol was evaporated and partitioned between EtOAc and H2O. The organic layers
were collected, dried over MgSO4. The solution was evaporated in vacuum and purified
by silica gel flash column chromatography to afford methylamine derivatives (3).
1-(2-(Benzyloxy)-3-methoxyphenyl)-N-methylmethanamine (3a). Rf 0.22 (DCM/EtOAc = 9/1).
1
Yield = 95% (9.9 g). H-NMR (CDCl3, 300 MHz)
δ
7.44–7.32 (5H, m, Ar), 7.07–7.01 (1H, m,
Ar), 6.92–6.88 (2H, m, Ar), 5.05 (2H, s, CH2), 3.89 (3H, s, CH3), 3.68 (2H, s, CH2), 2.32 (3H, s,
CH3); LCMS (ESI+) m/z calcd. for C18H21NO2 ([M + H+]) 258.14, found 258.17
1-(2-(Benzyloxy)-3-ethoxyphenyl)-N-methylmethanamine (3b). Rf 0.22 (DCM/EtOAc = 9/1).
1
Yield = 97% (14.0 g). H-NMR (CDCl3, 300 MHz)
δ 7.50–7.47 (2H, m, Ar), 7.43–7.35 (3H, m,
Ar), 7.06–7.01 (1H, m, Ar), 6.90–6.87 (2H, m, Ar), 5.08 (2H, s, CH2), 4.17 (2H, q, J = 6.9 Hz,
CH2), 3.68 (2H, s, CH2), 2.34 (3H, s, CH3), 1.50 (3H, t, J = 6.9 Hz, CH3); LCMS (ESI+) m/z
calcd. for C17H21NO2 ([M + H+]) 272.16, found 272.18
1-(2-(Benzyloxy)-3-methylphenyl)-N-methylmethanamine (3c). Rf 0.26 (DCM/EtOAc = 9/1).
1
Yield = 88% (16.3 g). H-NMR (CDCl3, 300 MHz)
δ 7.53–7.39 (5H, m, Ar), 7.22–7.15 (2H,
m, Ar), 7.06 (1H, m, Ar), 4.91 (2H, s, CH2), 3.78 (2H, s, CH2), 2.42 (3H, s, CH3), 2.38 (3H, s,
CH3); LCMS (ESI+) m/z calcd. for C16H19NO ([M + H+]) 242.1, found 242.7
1-(3-Allyl-2-(Benzyloxyphenyl)-N-methylmethanamine (3d). Rf 0.27 (DCM/EtOAc = 15/1).
1
Yield = 81% (11.2 g). H-NMR (CDCl3, 300 MHz)
δ 7.47–7.32 (5H, m, Ar), 7.25 (1H, m, Ar),
7.17–7.05 (2H, m, Ar), 6.06 (1H, m, CH), 5.10 (1H, m, CH), 5.05 (1H, m, CH), 4.88 (2H, s,
CH2), 3.78 (2H, s, CH2), 3.46 (2H, m, CH2), 2.39 (3H, s, CH3); LCMS (ESI+) m/z calcd. for
C18H21NO ([M + H+]) 268.2, found 268.7
2-(Benzyloxy)-N,N-diethyl-3-((methylamino)methylaniline (3e). Rf 0.20 (DCM/EtOAc = 6/1).
Yield = 84% (12.7 g). 1H-NMR (CDCl3, 300 MHz)
δ
7.44–7.30 (5H, m, Ar), 7.10 (1H, m,
Ar), 6.23 (2H, m, Ar), 5.10 (2H, s, CH2), 3.75 (2H, s, CH2), 3.30 (4H, q, J= 7.0 Hz, 2 CH2),
CH3); LCMS (ESI+) m/z calcd. for C19H26N2O
×
2.38 (3H, s, CH3), 1.10 (6H, t, J = 7.0 Hz, 2
×
([M + H+]) 299.2, found 299.6
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 O2) 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).
1
Yield = 60.4% (6.6 g). H-NMR (CDCl3, 300 MHz)
δ
7.54 (1H, d, J = 8.2 Hz, Ar), 7.37–7.26

Molecules 2021, 26, 1686
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(5H, m, Ar), 7.03 (1H, d, J = 8.2 Hz, Ar), 5.11 (2H, s, CH2), 4.02 (2H, s, CH2), 3.96 (3H, s, CH3),
3.07 (3H, s, CH3); LCMS (ESI+) m/z calcd. for C17H17NO3 ([M + H+]) 284.1, found 284.3
4-(Benzyloxy)-5-ethoxy-2-methylisoindolin-1-one (4b). Rf 0.23 (hexane/EtOAc = 5/1).
1
Yield = 84% (12.9 g). H-NMR (CDCl3, 300 MHz)
δ
7.53 (1H, d, J = 8.2, Ar), 7.38–7.33 (5H, m,
Ar), 7.03 (1H, d, J = 8.2, Ar), 5.13 (2H, s, CH2), 4.21 (2H, q, J = 6.9 Hz, CH2), 4.05 (2H, s, CH2),
3.08 (3H, s, CH3), 1.51 (3H, t, J = 6.9 Hz, CH3); LCMS (ESI+) m/z calcd. for C18H19NO3
([M + H+]) 298.2, found 298.5
4-(Benzyloxy)-5-methyl-2-methylisoindolin-1-one (4c). Rf 0.28 (hexane/EtOAc = 5/1).
1
Yield = 62% (11.3 g). H-NMR (CDCl3, 300 MHz)
δ
7.50 (1H, d, J = 7.4 Hz, Ar), 7.41–7.37 (5H,
m, Ar), 7.29 (1H, d, J = 7.4 Hz, Ar), 5.01 (2H, s, CH2), 4.22 (2H, s, CH2), 3.12 (3H, s, CH3),
2.37 (3H, s, CH3); LCMS (ESI+) m/z calcd. for C17H17NO2 ([M + H+]) 268.2, found 268.2
5-Allyl-4-(benzyloxy)-2-methylisoindolin-1-one (4d). Rf 0.32 (hexane/EtOAc = 6/1). Yield
1
= 73% (8.9 g). H-NMR (CDCl3, 300 MHz)
δ
7.58 (1H, d, J = 7.6 Hz, Ar), 7.42–7.40 (5H, m,
Ar), 7.34 (1H, d, J = 7.6 Hz, Ar), 6.00 (1H, m, CH), 5.14 (2H, m, CH2), 5.05 (2H, s, CH2), 4.29
(2H, s, CH2), 3.53 (2H, m, CH2), 3.17 (3H, s, CH3); LCMS (ESI+) m/z calcd. for C19H19NO2
([M + H+]) 294.2, found 294.3
4-(Benzyloxy)-5-(diethylamino)-2-methylisoindolin-1-one (4e). Rf 0.18 (hexane/EtOAc = 6/4).
Yield = 32% (4.5 g). 1H-NMR (CDCl3, 300 MHz)
δ
7.46–7.41 (5H, m, Ar), 6.76 (1H, d,
J = 1.9 Hz, Ar), 6.37 (1H, d, J = 1.9 Hz, Ar), 5.16 (2H, s, CH2), 4.29 (2H, s, CH2), 3.36 (4H, q,
J = 7.0 Hz, 2 CH2), 3.19 (3H, s, CH3), 1.14 (6H, t, J = 7.0 Hz, 2
CH3); LCMS (ESI+) m/z
calcd. for C20H24N2O2 ([M + H+]) 325.2, found 325.7
Compounds 5a 24]. To a solution of methylisoindolin-1-one (
×
×
–
e
[
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 CH2Cl2. The organic layers were
washed with brine, dried over MgSO4. The residue was treated with ether and resulting
solid was collected by filtration to afford isoindolinone (5).
4-Hydroxy-5-methoxy-2-methylisoindolin-1-one (5a). Rf 0.22 (DCM/MeOH = 19/1).
Yield = 92.6% (4.2 g). 1H-NMR (CDCl3, 300 MHz)
δ 7.39 (1H, d, J = 8.1 Hz, Ar), 6.97
(1H, d, J = 8.1 Hz, Ar), 5.82 (1H, s, OH), 4.34 (2H, s, CH2), 3.96 (3H, s, CH3), 3.17 (3H, s,
CH3); LCMS (ESI+) m/z calcd. for C10H11NO3 ([M + H+]) 194.0, found 194.1
4-Hydroxy-5-ethoxy-2-methylisoindolin-1-one (5b). Rf 0.23 (DCM/MeOH = 19/1).
1
Yield = 90% (8.1 g). H-NMR (DMSO-d6, 300 MHz)
δ 7.11 (1H, d, J = 8.1 Hz, Ar), 7.05 (1H,
d, J = 8.1 Hz, Ar), 4.30 (2H, s, CH2), 4.12 (2H, q, J = 6.9 Hz, CH2), 3.02 (3H, s, CH3), 1.36 (3H,
t, J = 6.9 Hz, CH3); LCMS (ESI+) m/z calcd. for C11H13NO3 ([M + H+]) 208.1, found 208.1
4-Hydroxy-5-methyl-2-methylisoindolin-1-one (5c). Rf 0.25 (DCM/MeOH = 19/1).
1
Yield = 81% (6.1 g). H-NMR (DMSO-d6, 300 MHz)
δ 9.37 (1H, br, OH), 7.20 (1H, d, J = 7.4
Hz, Ar), 7.06 (1H, d, J = 7.4 Hz, Ar), 4.33 (2H, s, CH2), 3.05 (3H, s, CH3), 2.24 (3H, s, CH3);
LCMS (ESI+) m/z calcd. for C10H11NO2 ([M + H+]) 178.0, found 178.2
5-allyl-4-hydroxy-2-methylisoindolin-1-one (5d). Rf 0.20 (DCM/MeOH = 17/1).
Yield = 76% (4.7 g). 1H-NMR (DMSO-d6, 300 MHz)
δ 9.34 (1H, br, OH), 7.18 (1H, d,
J = 7.5 Hz, Ar), 7.09 (1H, d, J = 7.5 Hz, Ar), 4.34 (2H, s, CH2), 3.05 (3H, s, CH3), 2.61 (2H, t,
J = 7.2 Hz, CH2), 1.58 (2H, m, CH2), 0.90 (3H, t, J = 7.2 Hz, CH3); LCMS (ESI+) m/z calcd.
for C12H13NO2 ([M + H+]) 204.1, found 204.3
5-(Diethylamino)-4-hydroxy-2-methylisoindolin-1-one (5e). Rf 0.15 (DCM/MeOH = 15/1).
Yield = 38% (1.2 g). 1H-NMR (DMSO-d6, 300 MHz)
δ
9.59 (1H, br, OH), 6.38 (1H, d,
J = 1.8 Hz, Ar), 6.31 (1H, d, J = 1.8 Hz, Ar), 4.18 (2H, s, CH2), 3.32 (4H, q, J = 6.9 Hz,
CH2), 3.03 (3H, s, CH3), 1.09 (6H, t, J = 6.9 Hz, 2
CH3); LCMS (ESI+) m/z calcd. for
C13H18N2O2 ([M + H+]) 235.0, found 235.3
2
×
×
3.7.1. Alkylation of 2-Bromo-3-Hydroxybenzaldehyde (Bottom Subunit)
2-Bromo-3-hydroxybenzaldehyde (2.0 g, 8.65 mmol, 1.0 eq.), potassium carbonate
(2.4 g, 17.3 mmol) and alkyl halide (1.2 eq.) were dissolved in DMF (5 mL) and stirred

Molecules 2021, 26, 1686
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overnight at room temperature. The reaction mixture was diluted with ethyl acetate and
washed with water three times. The organic layer was concentrated and purified by column
chromatography.
2-Bromo-4-methoxy-3-(4-methoxyphenethoxy)benzaldehyde (7a). Rf 0.23 (hexane/EtOAc = 7/3).
Yield = 82.9% (2.6 g). 1H-NMR (CDCl3, 300 MHz)
δ 10.2 (1H, s, CHO), 7.75 (1H, d, J = 8.7 Hz,
Ar), 7.25 (2H, d, J = 8.5 Hz, Ar), 6.96 (1H, d, J = 8.7 Hz, Ar), 6.89 (2H, d, J = 8.5 Hz, Ar), 4.22
(2H, t, J = 7.3 Hz, CH2), 3.92 (3H, s, CH3), 3.81 (3H, s, CH3), 3.16 (2H, t, J = 7.3 Hz, CH2);
LCMS (ESI+) m/z calcd. for C17H17BrO4 ([M + H+]) 365.3, found 365.7
2-Bromo-4-methoxy-3-(3-phenoxypropoxy)benzaldehyde (7b). Rf 0.23 (hexane/EtOAc = 7/3).
Yield = 73.4% (2.3 g). 1H-NMR (CDCl3, 300 MHz)
δ
10.2 (1H, s, CHO), 7.76 (1H, d, J = 8.6 Hz
,
Ar), 7.34 (2H, m, Ar), 6.99 (4H, m, Ar), 4.33 (2H, t, J = 6.0 Hz, CH2), 4.25 (2H, t, J = 5.9 Hz
,
CH2), 3.85 (3H, s, CH3), 2.34 (2H, m, CH2); LCMS (ESI+) m/z calcd. for C17H17BrO4
([M + H+]) 365.1, found 365.3
3.7.2. Typical Procedure for Direct ‘One-Pot’ Synthesis of Dibenzoxepine Lactam
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 N2. After cooling to room temperature, isoindolinone (0.3 g), 2-
bromobenzaldehyde (2.0 eq.), CuCl (0.10 eq.), and Cs2CO3 (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]oxepino[4,3,2-cd]isoindol-2(1H)-o◦ne (8aa). Rf 0.25 (hex-
ane/EtOAc = 6/4). Yield = 31% (150 mg). Yellow solid. mp: 208–210 C; 1H-NMR (CDCl3,
300 MHz)
δ 7.37 (1H, d, J = 8.1 Hz, Ar), 6.97–6.92 (2H, m, Ar), 6.84 (1H, m, Ar), 6.61–6.58
(1H, m, Ar), 5.78(1H, s, Ar), 3.97 (3H, s, CH3), 3.91 (3H, s, CH3), 3.25 (3H, s, CH3). 13C-NMR
(125 MHz, CDCl3) δ 166.3, 152.1, 151.8, 142.6, 142.0, 137.8, 128.9, 127.8, 125.1, 123.3, 121.9,
118.9, 115.0, 113.3, 107.9, 56.9, 56.6, 25.6. LCMS (ESI+) m/z calcd. for C18H16NO4 ([M + H+])
309.3, found 309.9
7-Ethoxy-5,8-dimethoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8ab). Rf
0.25 (hexane/EtOAc = 6/4). Yield = 17% (96 mg). Light yellow solid. mp: 194–196 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 7.36 (1H, d, J = 8.2 Hz, Ar), 6.96 (1H, d, J = 8.2 Hz, Ar), 6.64
(1H, d, J = 8.7 Hz, Ar), 6.56 (1H, d, J = 8.6 Hz, Ar), 5.73 (1H, s, Ar), 4.13 (2H, q, J = 7.0 Hz,
CH2), 3.96 (3H, s, CH3), 3.85 (3H, s, CH3), 3.25 (3H, s, CH3), 1.48 (3H, t, J = 7.0 Hz, CH3).
13C-NMR (125 MHz, CDCl3)
δ 166.1, 155.2, 151.9, 148.1, 141.4, 140.6, 135.3, 127.3, 125.8,
121.9, 121.4, 118.7, 114.6, 108.3, 107.8, 69.4, 56.5, 56.1, 25.6, 15.5. LCMS (ESI+) m/z calcd. for
C20H20NO5 ([M + H+]) 353.4, found 353.9
5,8-Dimethoxy-7-(4-methoxyphenethoxy)-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-
one (8ac). Rf 0.20 (hexane/EtOAc = 7/3). Yield = 12% (84 mg). Orange solid. mp:
182–184 ◦C; 1H-NMR (CDCl3, 300 MHz)
δ 7.36 (1H, d, J = 8.2 Hz, Ar), 7.28 (2H, m, Ar),
6.96 (1H, d, J = 8.2 Hz, Ar), 6.87–6.83 (2H, m, Ar), 6.66 (1H, d, J = 8.7 Hz, Ar), 6.54 (1H, d,
J = 8.6 Hz, Ar), 5.71 (1H, s, Ar), 4.23 (2H, t, J = 7.6 Hz, CH2), 3.93 (3H, s, CH3), 3.82 (3H,
s, CH3), 3.80 (3H, s, CH3), 3.24 (3H, s, CH3), 3.18 (2H, t, J = 7.7 Hz, CH2). 13C-NMR (125
MHz, CDCl3)
δ 165.9, 158.0, 154.9, 151.8, 148.0, 141.3, 140.5, 135.1, 130.4, 129.8, 127.0, 126.0,
121.8, 121.3, 118.5, 114.5, 113.7, 108.3, 107.7, 74.4, 56.4, 56.0, 55.2, 35.5, 25.5. LCMS (ESI+)
m/z calcd. for C27H26NO6 ([M + H+]) 459.5, found 459.9.
5,8-Dimethoxy-1-methyl-7-(3-phenoxypropoxy)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one
(
8ad). Rf 0.26 (hexane/EtOAc = 7/3). Yield = 23% (94 mg). Pale yellow solid. mp:
1
189–191 ◦C; H-NMR (CDCl3, 300 MHz)
δ 7.35 (1H, d, J = 8.1 Hz, Ar), 7.30 (1H, m, Ar),
6.95–6.90 (5H, m, Ar), 6.65 (1H, d, J = 8.7 Hz, Ar), 6.51 (1H, d, J = 8.7 Hz, Ar), 5.71 (1H, s,
Ar), 4.30 (4H, m, 2 CH3), 3.89 (6H, m, 2 CH3), 3.71 (3H, s, CH3), 3.23 (3H, s, CH3), 2.31
(2H, m, CH2). LCMS (ESI+) m/z calcd. for C27H26NO6 ([M + H+]) 459.5, found 460.0
×
×

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5-Ethoxy-7,8-dimethoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8ae). Rf
0.25 (hexane/EtOAc = 6/4). Yield = 18% (90 mg). Light yellow solid. mp: 219–220 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 7.34 (1H, d, J = 8.1Hz, Ar), 6.94 (1H, d, J = 8.2 Hz, Ar), 6.67
(1H, d, J = 8.6 Hz, Ar), 6.55 (1H, d, J = 8.6 Hz, Ar), 5.72 (1H, s, Ar), 4.16 (2H, q, J = 6.9 Hz,
CH2), 3.96 (3H, s, CH3), 3.86 (3H, s, CH3), 3.23 (3H, s, CH3), 1.52 (3H, t, J = 6.9 Hz, CH3).
13C-NMR (125 MHz, CDCl3)
δ 166.1, 154.8, 151.4, 148.1, 141.6, 141.4, 135.4, 127.3, 125.8,
121.8, 121.5, 118.7, 115.8, 108.2, 107.6, 65.1, 61.4, 56.1, 25.6, 14.8. LCMS (ESI+) m/z calcd. for
C20H20NO5 ([M + H+]) 353.4, found 353.9
5-Ethoxy-8-methoxy-7-(4-methoxyphenethoxy)-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-
2(1H)-one (8af). Rf 0.24 (hexane/EtOAc = 7/3). Yield = 43% (290 mg). Light yellow solid.
mp: 189–191 ◦C; 1H-NMR (CDCl3, 300 MHz)
δ 7.30 (1H, d, J = 8.2 Hz, Ar), 7.20–7.17 (2H,
m, Ar), 6.90 (1H, d, J = 8.2 Hz, Ar), 6.84–6.81 (2H, m, Ar), 6.62 (1H, d, J = 8.6 Hz, Ar), 6.50
(1H, d, J = 8.6 Hz, Ar), 5.67 (1H, s, Ar), 4.28 (2H, t, J = 7.6 Hz, CH2), 4.14 (2H, q, J = 6.8 Hz,
CH2), 3.78–3.76 (6H, m, 2
×
CH3), 3.21 (3H, s, CH3), 3.11 (2H, t, J = 7.5 Hz, CH2), 1.47 (3H, t,
166.0, 158.0, 154.9, 151.2, 148.2, 141.5, 140.5,
J = 6.9 Hz, CH3). 13C-NMR (125 MHz, CDCl3)
δ
135.2, 130.4, 129.9, 127.0, 125.9, 121.7, 121.2, 118.4, 115.7, 113.7, 108.4, 107.6, 74.5, 65.1, 56.0,
55.2, 35.6, 25.5, 14.9. LCMS (ESI+) m/z calcd. for C28H28NO6 ([M + H+]) 473.5, found 474.0
9-Isobutoxy-5-methoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8ba). Rf
0.26 (hexane/EtOAc = 6/4). Yield = 42% (230 mg). Light yellow solid. mp: 187–189 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 7.35 (1H, d, J = 8.1 Hz, Ar), 6.96–6.93 (2H, m, Ar), 6.66 (1H,
dd, J = 8.79 Hz, 2.9 Hz, Ar), 6.53 (1H, d, J = 2.9 Hz, Ar), 5.73 (1H, s, Ar), 3.93 (3H, s, CH3),
3.68 (2H, d, J = 6.5 Hz, CH2), 3.24 (3H, s, CH3), 2.09–2.04 (1H, m, CH), 1.02 (3H, s, CH3),
1.00 (3H, s, CH3). 13C-NMR (125 MHz, CDCl3)
δ 166.2, 156.3, 151.8, 147.0, 142.3, 137.9,
128.4, 127.1, 122.9, 122.0, 118.3, 117.0, 114.9, 114.3, 107.5, 74.8, 56.7, 28.2, 25.6, 19.2 LCMS
(ESI+) m/z calcd. for C21H22NO4 ([M + H+]) 351.4, found 352.0
5-Methoxy-1-methyl-9-(trifluoromethyl)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bb).
Rf 0.27 (hexane/EtOAc = 6/4). Yield = 46% (250 mg). Light orange solid. mp: 190–191 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ
7.36–7.33 (2H, m, Ar), 7.21 (1H, s, Ar), 7.08 (1H, d, J = 8.4 Hz
,
Ar), 6.97 (1H, d, J = 8.2 Hz, Ar), 5.71 (1H, s, Ar), 3.94 (3H, s, CH3), 3.24 (3H, s, CH3). 13C-
NMR (125 MHz, CDCl3) δ 166.0, 155.9, 151.8, 141.3, 138.6, 128.3, 128.2, 127.8, 126.6, 126.2,
124.7, 122.7, 122.5, 121.8, 119.0, 115.1, 56.6, 25.6. LCMS (ESI+) m/z calcd. for C18H13F3NO3
([M + H+]) 347.3, found 347.9
5,9-Dimethoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bc). Rf 0.20
◦
(DCM/MeOH = 19/1). Yield = 23% (214 mg). Light orange solid. mp: 194–196 C;1H-
NMR (CDCl3, 300 MHz)
J = 8.7 Hz, 3.06 Hz, Ar), 6.54 (1H, d, J = 3.0 Hz, Ar), 5.76 (1H, s, Ar), 3.96 (3H, s, CH3), 3.79
(3H, s, CH3), 3.27 (3H, s, CH3). 13C-NMR (125 MHz, CDCl3)
166.1, 156.5, 151.8, 147.2,
δ 7.37 (1H, d, J = 8.1 Hz, Ar), 6.98–6.95 (2H, m, Ar), 6.69 (1H, dd,
δ
142.1, 138.0, 128.4, 126.9, 122.9, 121.9, 118.3, 116.4, 114.8, 113.6, 107.4, 56.6, 55.5, 25.5. LCMS
(ESI+) m/z calcd. for C18H16NO4 ([M + H+]) 309.3, found 309.9
9-Hydroxy-5-methoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bd). Rf
0.28 (hexane/EtOAc = 6/4). Yield = 91% (210 mg). Light yellow solid. mp: 177–179 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ
7.38 (1H, d, J = 8.3 Hz, Ar), 6.99–6.93 (2H, m, Ar), 6.58 (1H,
dd, J = 8.5 Hz, 2.9 Hz, Ar), 6.51 (1H, d, J = 3.1 Hz, Ar), 5.73 (1H, s, Ar), 4.79 (1H, br, OH),
3.96 (3H, s, CH3), 3.27 (3H, s, CH3). 13C-NMR (125 MHz, CDCl3+MeOD)
166.7, 153.8,
δ
151.9, 146.3, 137.6, 129.4, 128.2, 123.0, 121.7, 118.3, 118.1, 117.5, 115.6, 114.9, 108.3, 59.6, 25.5.
LCMS (ESI+) m/z calcd. for C17H13NO4 ([M + H+]) 295.3, found 295.9
9-(2-(Diethylamino)ethoxy)-5-methoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8be).
Rf 0.22 (hexane/EtOAc = 6/4). Yield = 34% (212 mg). Light orange solid. mp: 174–176 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 7.37 (1H, d, J = 8.1 Hz, Ar), 6.96 (2H, t, J = 6.5 Hz, Ar), 6.65
(1H, dd, J = 3.0 Hz, 8.79 Hz, Ar), 6.56–6.55 (1H, m, Ar), 5.74 (1H, s, Ar), 4.15 (2H, t, J = 6.1 Hz
,
CH2), 3.95 (3H, s, CH3), 3.26 (3H, s, CH3), 2.90 (2H, t, J = 6.0 Hz, CH2), 2.67 (4H, q, J = 7.1 Hz
,
2
×
CH2), 1.08 (6H, t, J = 7.1 Hz, 2
×
CH3). 13C-NMR (125 MHz, CDCl3)
δ
166.1, 155.7, 151.7,
147.1, 142.1, 137.9, 128.4, 126.9, 122.9, 121.8, 118.2, 117.0, 114.8, 114.3, 107.3, 66.5, 56.6, 51.5,
47.6, 25.5, 11.5. LCMS (ESI+) m/z calcd. for C23H27N2O4 ([M + H+]) 394.5, found 395.0

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5-Methoxy-1-methyl-9-(pyridine-2-ylmethoxy)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bf).
Rf 0.24 (DCM/EtOAc = 9/1). Yield = 39% (231 mg). Light yellow solid. mp: 176–178 ◦C
;
1H-NMR (CDCl3, 300 MHz) δ 8.58 (1H, dd, J = 5.8 Hz, 1.5 Hz, Ar), 7.72 (1H, td, J = 7.7 Hz,
1.7 Hz, Ar), 7.48 (1H, d, J = 7.8 Hz, Ar), 7.24–7.20 (2H, m, Ar), 6.92–6.85 (2H, m, Ar), 6.70(1H,
dd, J = 3.0 Hz, 8.7 Hz, Ar), 6.56 (1H, d, J = 3.0 Hz, Ar), 5.63 (1H, s, Ar), 5.12 (2H, s, CH2),
3.88 (3H, s, CH3), 3.18 (3H, s, CH3). 13C-NMR (125 MHz, CDCl3)
δ 166.1, 156.9, 155.3, 151.8,
149.1, 147.4, 142.0, 138.0, 136.8, 128.6, 126.9, 123.0, 122.7, 121.8, 121.3, 118.3, 117.2, 114.9,
114.6, 107.2, 70.8, 56.6, 25.6. LCMS (ESI+) m/z calcd. for C23H19N2O4 ([M + H+]) 386.4,
found 386.9
9-Benzyloxy-5-methoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-◦one (8bg). Rf
0.26 (hexane/EtOAc = 6/4). Yield = 44% (265 mg). Yellow solid. mp: 187–189 C; 1H-NMR
(CDCl3, 300 MHz)
3.00 Hz, Ar), 6.61 (1H, d, J = 3.0 Hz, Ar), 5.72 (1H, s, Ar), 5.02 (2H, s, CH2), 3.93 (3H, s, CH3),
3.25 (3H, s, CH3). 13C-NMR (125 MHz, CDCl3)
166.2, 155.8, 151.9, 147.3, 142.2, 138.1,
δ 7.41–7.33 (6H, m, Ar), 6.96–6.93 (2H, m, Ar), 6.74 (1H, dd, J = 8.7 Hz,
δ
136.7, 128.6, 128.6, 128.0, 127.4, 127.1, 123.0, 121.9, 118.5, 117.4, 114.9, 114.7, 107.4, 70.3, 56.7,
25.6. LCMS (ESI+) m/z calcd. for C24H20NO4 ([M + H0+]) 385.4, found 385.9
0
0
0
5-Methoxy-1-methyl-[1,3]dioxolo[4”,5”.4 ,5 ]benzo[1 ,2 :6,7]oxepino[4,3,2-cd]isoindol-2(1H)-
one (8bh◦). Rf 0.24 (hexane/EtOAc = 6/4). Yield = 28% (140 mg). Bright orange solid. mp:
237–239 C; 1H-NMR (CDCl3, 300 MHz)
δ 7.32 (1H, d, J = 8.2 Hz, Ar), 6.93 (1H, d, J = 8.2 Hz,
Ar), 6.59 (1H, s, Ar), 6.42 (1H, s, Ar), 5.93 (2H, s, CH2), 5.66 (1H, s, Ar), 3.90 (3H, s, CH3),
3.20 (3H, s, CH3). 13C-NMR (125 MHz, CDCl3)
δ 166.1, 151.8, 148.5, 148.1, 144.9, 136.4,
128.0, 121.7, 120.8, 118.9, 115.0, 109.4, 107.7, 104.4, 101.9, 56.6, 25.6. LCMS (ESI+) m/z calc’d
for C18H14NO5 ([M + H+]) 323.3, found 323.9
9-Isobutoxy-5-ethoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bi). Rf 0.22
(hexane/EtOAc = 6/4). Yield = 61% (323 mg). Light organe solid. mp: 178–180 ◦C; 1H-
NMR (CDCl3, 300 MHz)
J = 8.7 Hz, J = 2.9 Hz, Ar), 6.54 (1H, d, J = 2.8 Hz, Ar), 5.74 (1H, s, Ar), 4.16 (2H, q, J = 6.9 Hz
CH2), 3.68 (2H, d, J = 6.5 Hz, CH2), 3.24 (3H, s, CH3), 2.09–2.01 (1H, m, CH), 1.49 (3H,
t, J = 6.9 Hz, CH3), 1.01 (6H, d, J = 6.7 Hz, 166.2,
CH3). 13C-NMR (126 MHz, CDCl3)
δ 7.32 (1H, d, J = 8.1 Hz, Ar), 6.95–6.92 (2H, m, Ar), 6.66 (1H, dd,
,
2
×
δ
156.3, 151.3, 147.0, 142.5, 138.0, 128.5, 127.2, 122.9, 121.9, 118.3, 116.9, 116.6, 114.4, 107.4,
74.8, 65.4, 28.2, 25.6, 19.2, 14.8. LCMS (ESI+) m/z calc’d for C22H24NO4 ([M + H+]) 365.4,
found 366.0
5-Ethoxy-1-methyl-9-(trifluoromethyl)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bj).
◦
Rf 0.25 (hexane/EtOAc = 6/4). Yield = 72% (370 mg). Yellow solid. mp: 230–232 C;
1H-NMR (CDCl3, 300 MHz)
δ
7.34–26 (2H, m, Ar), 7.19 (1H, m, Ar), 7.05 (1H, d, J = 8.4 Hz,
Ar), 6.94 (1H, d, J = 8.2 Hz, Ar), 5.69 (1H, s, Ar), 4.14 (2H, q, J = 6.9 Hz, CH2), 3.22 (3H, s,
CH3), 1.49 (3H, t, J = 6.9 Hz, CH3). 13C-NMR (125 MHz, CDCl3)
166.1, 155.9, 151.3, 141.5,
δ
138.7, 128.3, 128.2, 127.5, 126.7, 126.1, 124.7, 122.7, 122.5, 121.7, 119.0, 116.5, 65.3, 25.6, 14.8.
LCMS (ESI+) m/z calcd. for C19H15F3NO3 ([M + H+]) 361.3, found 361.9
5-Ethoxy-9-methoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bk). Rf 0.22
(hexane/EtOAc = 6/4). Yield = 70% (325 mg). Light yellow solid. mp: 214–216 ◦C; 1H-
NMR (CDCl3, 300 MHz) δ 7.35 (1H, d, J = 8.1 Hz, Ar), 6.99–6.94 (2H, m, Ar), 6.70 (1H, dd,
J = 8.8 Hz, 3.03 Hz, Ar), 6.55 (1H, d, J = 3.0 Hz, Ar), 5.77 (1H, s, Ar), 4.15 (2H, q, J = 7.0 Hz,
CH2), 3.80 (3H, s, CH3), 3.27 (3H, s, CH3), 1.52 (3H, t, J = 6.9 Hz, CH3). 13C-NMR (125 MHz,
CDCl3)
δ 166.2. 156.5. 151.3. 147.1. 142.4. 138.1. 128.5. 127.1. 123.0. 121.8. 118.3. 116.5.
116.2. 113.6. 107.3. 65.4. 55.5. 25.5. 14.8. LCMS (ESI+) m/z calcd. for C19H18NO4 ([M + H+])
323.3, found 323.9
5-Ethoxy-9-hydroxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one◦(8bl). Rf 0.24
1
(hexane/EtOAc = 6/4). Yield = 31% (72 mg). Yellow solid. mp: 194–196 C; H-NMR
(CDCl3+MeOD, 300 MHz)
δ 7.26 (1H, d, J = 8.1 Hz, Ar), 6.91 (1H, d, J = 8.2 Hz, Ar), 6.84
(1H, d, J = 8.6 Hz, Ar), 6.58 (1H, dd, J = 8.6 Hz, J = 2.8 Hz, Ar), 6.43 (1H, d, J = 2.7 Hz, Ar),
5.71 (1H, s, Ar), 4.14 (2H, q, J = 6.9 Hz, CH2), 3.19 (3H, s, CH3), 1.47 (3H, t, J = 6.9 Hz, CH3).
13C-NMR (125 MHz, CDCl3+MeOD)
δ 166.8, 153.8, 151.3, 146.3, 142.5, 137.5, 128.2, 127.2,

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122.9, 121.5, 118.2, 117.4, 116.6, 115.6, 108.4, 65.5, 25.5, 14.6. LCMS (ESI+) m/z calcd. for
C18H16NO4 ([M + H+]) 309.3, found 310.0
9-(2-(Diethylamino)ethoxy)-5-ethoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one
(8bm). Rf 0.18 (DCM/MeOH = 19/1). Yield = 34% (393 mg). Light yellow solid. mp: 169–
171 ◦C; 1H-NMR (CDCl3, 300 MHz)
δ 7.34 (1H, d, J = 8.1 Hz, Ar), 6.97–6.94 (2H, m, Ar),
6.69 (1H, dd, J = 3.0 Hz, 8.82 Hz, Ar), 6.56 (1H, d, J = 3.0 Hz, Ar), 5.74 (1H, s, Ar), 4.20 (2H,
q, J = 6.9 Hz, CH2), 4.07 (2H, t, J = 6.1 Hz, CH2), 3.26 (3H, s, CH3), 2.93 (2H, t, J = 6.0 Hz,
CH2), 2.73 (4H, q, J = 7.1 Hz, 2
×
CH2), 1.53 (3H, t, J = 6.9 Hz, CH3), 1.14 (6H, t, J = 7.1 Hz,
166.2, 155.7, 151.3, 147.2, 142.4, 138.0, 128.5, 127.1,
2
×
CH3). 13C-NMR (125 MHz, CDCl3)
δ
122.9, 121.8, 118.3, 116.9, 116.5, 114.3, 107.3, 66.4, 65.4, 51.5, 47.6, 25.5, 14.8, 11.4. LCMS
(ESI+) m/z calcd. for C24H29N2O4 ([M + H+]) 408.5, found 409.0
5-Ethoxy-1-methyl-9-(pyridine-2-ylmethoxy)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bn).
Rf 0.23 (DCM/EtOAc = 6/1). Yield = 52% (307 mg). Light yellow solid. mp: 188–190 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 8.54–8.52 (1H, m, Ar), 7.66–7.63 (1H, m, Ar), 7.45–7.28 (1H, m,
Ar), 7.21–7.15 (2H, m, Ar), 6.88–6.81 (2H, m, Ar), 6.68 (1H, dd, J = 8.7 Hz, 3.0 Hz, Ar), 6.53
(1H, d, J = 3.0 Hz, Ar), 5.59 (1H, s, Ar), 5.08 (2H, s, CH2), 4.08 (2H, q, J = 6.9 Hz, CH2), 3.14
(3H, s, CH3), 1.45 (3H, t, J = 6.9 Hz, CH3). 13C-NMR (125 MHz, CDCl3)
δ 166.1, 156.8, 155.2,
151.2, 149.0, 147.4, 142.2, 138.0, 136.8, 129.4, 128.6, 123.0, 122.6, 121.2, 119.4, 118.3, 117.2,
116.4, 114.6, 107.2, 70.7, 65.3, 25.5, 14.8. LCMS (ESI+) m/z calcd. for C24H21N2O4 ([M + H+])
400.4, found 400.9
9-Benzyloxy-5-methoxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8bo). Rf
◦
0.27 (hexane/EtOAc = 6/4). Yield = 44% (391 mg). Orange solid. mp: 198–200 C; 1H-NMR
(CDCl3 300 MHz) δ 7.41–7.30 (6H, m, Ar), 6.96–6.92 (2H, m, Ar), 6.74 (1H, dd, J = 8.8 Hz,
3.00 Hz, Ar), 6.62 (1H, d, J = 3.0 Hz, Ar), 5.73 (1H, s, Ar), 5.02 (2H, s, CH2), 4.13 (2H, q,
J = 6.9 Hz, CH2), 3.24 (3H, s, CH3), 1.49 (3H, t, J = 6.9 Hz, CH3). 13C-NMR (125 MHz,
CDCl3)
δ 166.3, 155.8, 151.3, 147.4, 142.5, 138.2, 136.7, 128.7, 128.6, 128.0, 127.4, 127.3, 123.1,
121.9, 118.4, 117.3, 116.6, 114.7, 107.2, 70.3, 65.5, 25.6, 14.9. LCMS (ESI+) m/z calcd. for
C25H22NO4 ([M + H+]) 399.4, found 399.9
5-Ethoxy-1-methyl-[1,3]dioxolo[4”,5”.40,50]benzo[10,20:6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one
(
8bp). R◦f 0.23 (hexane/EtOAc = 6/4). Yield = 47% (238 mg). Bright orange solid. mp:
207–209 C; 1H-NMR (CDCl3, 300 MHz)
δ 7.32 (1H, d, J = 8.1 Hz, Ar), 6.93 (1H, d, J = 8.2 Hz,
Ar), 6.59 (1H, s, Ar), 6.43 (1H, s, Ar), 5.95 (2H, s, CH2), 5.65 (1H, s, Ar), 4.11 (2H, q, J = 6.9 Hz,
CH2), 3.90 (3H, s, CH3), 1.48 (3H, t, J = 6.9 Hz, CH3). 13C-NMR (125 MHz, CDCl3)
166.0,
δ
151.2, 148.3, 148.0, 144.8, 141.5, 136.4, 128.0, 121.6, 120.8, 118.7, 116.4, 109.4, 107.5, 104.3,
101.9, 65.3, 25.5, 14.8. LCMS (ESI+) m/z calcd. for C19H16NO5 ([M + H+]) 337.3, found 337.9
5-(Diethylamino)-9-isobutoxy-1-methyl-5-propylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one
(
8ca◦). Rf 0.27 (DCM/EtOAc = 6/1). Yield = 32% (164 mg). Light yellow solid. mp: 183–
185 C; 1H-NMR (CDCl3, 300 MHz)
δ 6.81 (1H, d, J = 8.7 Hz, Ar), 6.58–6.54 (2H, m, Ar), 6.47
(1H, d, J = 2.8 Hz, Ar), 6.17 (1H, d, J = 1.8 Hz, Ar), 5.48 (1H, s, Ar), 3.66 (2H, d, J = 6.5 Hz
CH2), 3.37 (4H, q, J = 7.0 Hz, 2 CH2), 3.21 (3H, s, CH3), 2.04 (1H, m, CH), 1.20 (6H, t,
J = 7.0 Hz, 2 CH3), 1.02 (6H, d, J = 6.7 Hz, 2 167.5,
CH3). 13C-NMR (125 MHz, CDCl3)
,
×
×
×
δ
155.9, 154.8, 151.8, 147.1, 138.4, 131.5, 128.4, 122.5, 117.0, 113.6, 103.8, 102.9, 99.6, 44.8, 28.2,
25.5, 19.2, 12.6. LCMS (ESI+) m/z calcd. for C24H29N2O3 ([M + H+]) 392.5, found 393.0
5-(Diethylamino)-9-hydroxy-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8cb).
◦
1
Rf 0.22 (DCM/EtOAc = 6/1). Yield = 91% (78 mg). Yellow solid. mp: 181–183 C; H-
NMR (CDCl3 300 MHz) 7.38 (1H, d, J = 8.3 Hz, Ar), 6.95–6.89 (2H, m, Ar), 6.58 (1H, dd,
J = 8.5 Hz, 2.94 Hz, Ar), 5.73 (1H, s, Ar), 4.79 (1H, br, OH), 3.96 (3H, s, CH3), 3.27 (3H, s,
δ
CH3). 13C-NMR (125 MHz, CDCl3+MeOD)
δ 167.7, 156.7, 154.8, 153.1, 146.7, 142.4, 138.0,
131.4, 129.6, 122.7, 118.4, 117.5, 115.0, 114.6, 113.7, 64.8, 25.6, 12.3. LCMS (ESI+) m/z calcd.
for C20H21N2O3 ([M + H+]) 336.4, found 337.0
5-(Diethylamino)-9-hydroxy-1-methyl-11,11a-dihydrobenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-
one (8cc). Rf 0.19 (DCM/EtOAc = 6/1). Yield = 38% (90 mg). Light yellow solid. mp:
165–167 ◦C; 1H-NMR (CDCl3, 300 MHz)
δ
7.12 (1H, d, J = 8.7 Hz, Ar), 6.86–6.81 (2H, m,
Ar), 6.78 (1H, d, J = 2.7 Hz, Ar), 6.64 (1H, br, OH), 6.52 (1H, d, J = 1.8 Hz, Ar), 4.44 (1H,

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dd, J = 11.2 Hz, 2.1 Hz, CH), 3.43 (4H, q, J = 7.0 Hz, 2
×
CH2), 3.30 (1H, dd, J = 2.3 Hz,
13.1 Hz, CH), 3.18 (3H, s, CH3), 2.89 (1H, t, J = 11.9 Hz, CH), 1.19 (6H, t, J = 6.9 Hz, 2 CH3).
×
13C-NMR (125 MHz, CDCl3)
δ 169.4, 152.0, 151.4, 149.8, 148.0, 133.7, 124.9, 122.4, 118.2,
118.1, 115.5, 103.3, 100.5, 60.1, 44.7, 38.2, 27.4, 12.4. LCMS (ESI+) m/z calcd. for C20H23N2O3
([M + H+]) 338.4, found 339.0
9-(Benzyloxy)-5-(diethylamino)-1-methylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8cd).
Rf 0.29 (DCM/EtOAc = 6/1). Yield = 42% (233 mg). Yellow solid. mp: 185–188 ◦C; 1H-
NMR (CDCl3, 300 MHz) δ 7.45–7.37 (5H, m, Ar), 6.87 (1H, d, J = 8.7 Hz, Ar), 6.69 (1H, dd,
J = 8.8 Hz, 3.00 Hz, Ar), 6.61–6.59 (2H, m, Ar), 6.21 (1H, d, J = 2.0 Hz, Ar), 5.52 (1H, s, Ar),
5.04 (2H, s, CH2), 3.39 (4H, q, J = 7.0 Hz, 2×CH2), 3.25 (3H, s, CH3), 1.21 (6H, t, J = 7.0 Hz,
2 δ 167.5, 155.4, 154.8, 151.9, 147.5, 138.6, 136.8, 131.6,
CH3). 13C-NMR (125 MHz, CDCl3)
×
128.7, 128.6, 128.0, 127.4, 122.6, 117.3, 113.9, 112.4, 103.5, 102.9, 99.7, 70.3, 44.8, 25.5, 12.6.
LCMS (ESI+) m/z calcd. for C27H27N2O3 ([M + H+]0) 426.5, found 427.9
0
0
0
5-(Diethylamino)-1-methyl-[1,3]dioxolo[4”,5”.4 ,5 ]benzo[1 ,2 :6,7]oxepino[4,3,2-cd]isoindol-
2(1H)-one (8ce). Rf 0.24 (DCM/EtOAc = 6/1). Yield = 21% (96 mg). Bright yellow solid. mp:
199–201 ◦C; 1H-NMR (CDCl3, 300 MHz)
δ 6.58 (1H, s, Ar), 6.48 (1H, s, Ar), 6.40 (1H, s, Ar),
6.16 (1H, s, Ar), 5.96–5.93 (2H, m, CH2), 5.42 (1H, s, Ar), 3.39 (4H, q, J = 6.8 Hz, 2
×
CH2),
167.3,
3.21 (3H, s, CH3), 1.19 (6H, t, J = 6.9 Hz, 2
×
CH3). 13C-NMR (125 MHz, CDCl3)
δ
154.3, 151.8, 148.1, 147.6, 144.3, 136.8, 131.3, 120.6, 113.0, 109.6, 104.0, 103.8, 102.9, 101.7,
99.7, 44.8, 25.5, 12.5. LCMS (ESI+) m/z calcd. for C21H21N2O4 ([M + H+]) 364.4, found 365.9
9-Isobutoxy-1-methyl-5-propylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one ◦(8da). Rf 0.27
1
(hexane/EtOAc = 6/4). Yield = 21% (63 mg). Yellow solid. mp: 196–199 C; H-NMR
(CDCl3, 300 MHz)
δ 7.30 (1H, d, J = 7.5 Hz, Ar), 7.21 (1H, d, J = 7.5 Hz, Ar), 6.89 (1H, d,
J = 8.7 Hz, Ar), 6.69 (1H, dd, J = 8.7 Hz, 3.00 Hz, Ar), 6.56 (1H, d, J = 2.9 Hz, Ar). 5.75 (1H, s,
Ar), 3.70 (2H, d, J = 6.5 Hz, CH2), 3.27 (3H, s, CH3), 2.71 (2H, t, J = 7.2 Hz, CH2), 2.10–2.05
(1H, m, CH), 1.72–1.65 (2H, m, CH2), 1.05 (3H, s, CH3), 1.03 (3H, s, CH3), 1.02–0.99 (3H,
m, CH3). 13C-NMR (125 MHz, CDCl3)
δ 166.5, 156.2, 151.3, 147.1, 138.2, 136.0, 133.2, 128.2,
126.1, 122.6, 117.4, 117.0, 114.4, 107.4, 74.8, 32.3, 28.2, 25.6, 23.6, 19.2, 14.0. LCMS (ESI+) m/z
calcd. for C23H26NO3 ([M + H+]) 363.4, found 365.0
1-Methyl-5-propyl-9-(trifluoromethyl)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8db).
Rf 0.25 (hexane/EtOAc = 6/4). Yield = 19% (100 mg). Light yellow solid. mp: 239–241 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ
7.36–7.29 (2H, m, Ar), 7.24–7.20 (2H, m, Ar), 6.99 (1H, d,
J = 8.3 Hz, Ar), 5.74 (1H, s, Ar), 3.26 (3H, s, CH3), 2.69 (2H, t, J = 7.3 Hz, CH2), 1.71–1.63 (2H,
m, CH2), 1.01 (3H, t, J = 7.3 Hz, CH3). 13C-NMR (125 MHz, CDCl3)
166.3, 156.0, 150.3,
δ
138.8, 136.1, 133.8, 128.4, 128.3, 128.2, 127.7, 126.1, 125.7, 124.7, 122.3, 118.0, 106.1, 32.3, 25.6,
23.5, 13.9. LCMS (ESI+) m/z calcd. for C20H17F3NO2 ([M + H+]) 359.3, found 359.9
1-Methyl-5-propyl-9-(pyridine-2-ylmethoxy)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8dc).
Rf 0.26 (hexane/EtOAc = 6/4). Yield = 9.3% (54 mg). Light yellow solid. mp: 189–191 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 8.61 (1H, d, J = 4.5 Hz, Ar), 7.72 (1H, td, J = 7.7 Hz, 1.62 Hz,
Ar), 7.47 (1H, d, J = 7.7 Hz, Ar), 7.28–7.16 (3H, m, Ar), 6.88 (1H, d, J = 8.7 Hz, Ar), 6.74 (1H,
dd, J = 8.7 Hz, 3.0 Hz, Ar), 6.64 (1H, d, J = 2.9 Hz, Ar), 5.71 (1H, s, Ar), 5.16 (2H, s, CH2), 3.25
(3H, s, CH3), 2.68 (2H, t, J = 7.3 Hz, CH2), 1.69–1.62 (2H, m, CH2), 0.99 (3H, t, J = 7.3 Hz
,
CH3). 13C-NMR (125 MHz, CDCl3)
δ
166.5, 156.9, 155.3, 151.1, 149.2, 147.6, 138.4, 136.8,
136.0, 133.3, 128.7, 128.2, 126.1, 122.8, 122.7, 121.2, 117.4, 117.3, 114.7, 107.1, 70.9, 32.3, 25.6,
23.6, 14.0. LCMS (ESI+) m/z calcd. for C25H23N2O3 ([M + H+]) 398.5, found 398.9
9-(Benzyloxy)-1-methyl-5-propylbenzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8dd). Rf
0.21 (hexane/EtOAc = 6/4). Yield = 21% (38 mg). Yellow solid. mp: 203–205 ◦C; 1H-NMR
(CDCl3, 300 MHz)
δ
7.44–7.36 (5H, m, Ar), 7.31 (1H, d, J = 7.6 Hz, Ar), 7.22 (1H, d, J = 7.6 Hz
,
Ar), 6.91 (1H, d, J = 8.7 Hz, Ar), 6.76 (1H, dd, J = 8.7 Hz, 2.97 Hz, Ar), 6.65 (1H, d, J = 2.9 Hz
,
Ar), 5.74 (1H, s, Ar), 5.05 (2H, s, CH2), 3.27 (3H, s, CH3), 2.71 (2H, t, J = 7.4 Hz, CH2),
1.72–1.65 (2H, m, CH2), 1.02 (3H, t, J = 7.3 Hz, CH3). 13C-NMR (125 MHz, CDCl3)
166.5,
δ
155.7, 151.2, 147.5, 138.3, 136.7, 136.0, 133.3, 128.7, 128.6, 128.2, 128.0, 127.4, 126.1, 122.7,
117.4, 114.8, 107.2, 70.4, 32.3, 25.6, 23.7, 14.0. LCMS (ESI+) m/z calcd. for C26H24NO3
([M + H+]) 397.4, found 397.9

Molecules 2021, 26, 1686
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0
0
0 0
1-methyl-5-propyl-[1,3]dioxolo[4”,5”.4 ,5 ]benzo[1 ,2 :6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8de).
Rf 0.23 (hexane/EtOAc = 6/4). Yield = 19% (93 mg). Light yellow solid. mp: 257–259 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ 7.31 (1H, d, J = 7.6 Hz, Ar), 7.20 (1H, d, J = 7.6 Hz, Ar), 6.53
(1H, s, Ar), 6.47 (1H, s, Ar), 5.96 (2H, s, CH2), 5.68 (2H, s, CH2), 3.25 (3H, s, CH3), 2.68 (2H, t,
J = 7.4 Hz, CH2), 1.67–1.65 (2H, m, CH2), 1.00 (3H, t, J = 7.3 Hz, CH3). 13C-NMR (125 MHz,
CDCl3)
δ 166.3, 150.6, 148.4, 144.7, 136.6, 135.8, 133.2, 128.0, 126.8, 120.8, 120.4, 117.7, 109.5,
107.5, 103.9, 101.9, 32.2, 25.5, 23.7, 14.0. LCMS (ESI+) m/z calcd. for C20H18NO4 ([M + H+]
)
335.3, found 335.9
1,5-Dimethyl-9-(trifluoromethyl)benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one (8df). Rf
0.27 (hexane/EtOAc = 6/4). Yield = 36% (200 mg). Light yellow solid. mp: 227–229 ◦C;
1H-NMR (CDCl3, 300 MHz)
δ
7.35 (1H, d, J = 8.4 Hz, Ar), 7.28–7.19 (3H, m, Ar), 7.01 (1H, d,
J = 8.3 Hz, Ar), 5.69 (1H, s, Ar), 3.25 (3H, s, CH3), 2.34 (3H, s, CH3). 13C-NMR (125 MHz,
CDCl3)
δ 166.2, 155.9, 150.5, 138.6, 134.3, 131.3, 128.9, 128.5, 127.1, 126.1, 125.3, 124.7, 122.5,
122.4, 117.8, 106.1, 25.5, 16.2. LCMS (ESI+) m/z calcd. for C18H13F3NO2 ([M + H+]) 331.3,
found 331.9
4. Conclusions
In this study, we found that the benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one scaf-
fold of aristoyagonine derived from natural products has inhibitory activity against Brd4
bromodomain. Based on the evaluation of inhibitory activities presented in Tables 1–4, the
substituents methoxy, ethoxy, n-propyl, or N,N-diethylaminoethox group at R1 position
showed similar inhibitory activities. These results suggest that R1 position is considered to
have a tolerance on binding pocket toward Brd4 bromodomain.
Among R2 and R3 substituents showing inhibitory activity against Brd4 bromodomain,
the most potent activity was shown by the smallest atom, hydrogen. The bulky group
at R2 position perturbed the inhibitory effect. The alkoxy substituents with methoxy,
pyridinemethoxy, and N,N-diethylaminoethoxy groups at R4 position displayed potent
activities. On the other hand, introduction of –CF3, benzyloxy, or 3,4-dioxymethylene
group at R4 position showed no activity against Brd4 bromodomain. The hydrogen bonds
between C=O of benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one and Asn140 predicted
in molecular modeling were confirmed through the results of X-ray co-crystal structure
of Brd4 bromodomain and inhibitors (8ab, 8bc, 8bf, 8be, and 8bd). Collectively, our
experiment results support these Brd4 bromodomain inhibitors with impressive mode
of action profiles. In addition, these novel small molecules are promising advanced lead
compounds for further optimization and candidates for the development of epigenetic
therapeutics that are used in the treatment of cancer diseases.
Supplementary Materials: The following are available online, Figure S1: Structure of BRD4 BD1 and
5 compounds analyzed in this study, Table S1: Data collection and refinement statistics, NMR spectra.
Author Contributions: Conceptualization, C.H.P. and K.-Y.J.; methodology, B.I.L. and K.-Y.J.; valida-
tion, M.Y. (Minjin Yoo), T.H.P., M.Y. (Miyoun Yoo), and Y.K.; formal analysis, S.E.R.; investigation,
J.-Y.L.; data curation, C.H.P., and K.M.L.; writing—original draft preparation, K.-Y.J., and C.H.P.;
writing—review and editing, B.I.L.; funding acquisition, C.H.P., K.-Y.J., and B.I.L. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by National Research Foundation of Korea (NRF-2018R1C1B6002932,
NRF-2020M2D9A2094158, 2019R1A2C1002545, and NRF-2018R1A5A2023127) and by Korea Research
Institute of Chemical Technology (BSF20-402).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The structural coordinates and X-ray diffraction data sets were de-
posited to Protein Data Bank (PDB) under accession code of 6KEC, 6KEH, 6KEI, 6KEJ, and 6KEK, re-
spectively.

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Acknowledgments: We thank the beamline staffs of Pohang Light Source (Beamline 11C) for their
assistance during the X-ray data collection.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds used in this study are available from the authors.
Abbreviations
Brd4
BET
HAT
bromodomain-containing protein 4
bromodomain and extraterminal domain
Histone acetyltransferase
P-TEFB Positive transcription elongation factor complex
HMT
NMC
AML
SBDD
Histone methyltransferases
NUT midline carcinoma
Acute myeloid leukemia
structure-based drug design
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