Discovery of 1,8-acridinedione derivatives as novel GCN5 inhibitors via high throughput screening

Article abstract of DOI:10.1016/j.ejmech.2018.02.005

The general control nonrepressed protein 5 (GCN5) plays a crucial role in many biological processes. Dysregulation of GCN5 has been closely related to various human diseases, especially cancers. Hence, the exploitation of small molecules targeting GCN5 is

Full text of DOI:10.1016/j.ejmech.2018.02.005

Accepted Manuscript
Discovery of 1,8-acridinedione derivatives as novel GCN5 inhibitors via high
throughput screening
Huan Xiong, Jie Han, Jun Wang, Wenchao Lu, Chen Wang, Yu Chen, Fulin Lian,
Naixia Zhang, Yu-Chih Liu, Chenhua Zhang, Hong Ding, Hualiang Jiang, Wencong
Lu, Cheng Luo, Bing Zhou
PII:
S0223-5234(18)30127-2
10.1016/j.ejmech.2018.02.005
EJMECH 10182
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 January 2018
Revised Date: 31 January 2018
Accepted Date: 3 February 2018
Please cite this article as: H. Xiong, J. Han, J. Wang, W. Lu, C. Wang, Y. Chen, Fulin Lian, N. Zhang, Y.-
C. Liu, C. Zhang, H. Ding, H. Jiang, W. Lu, C. Luo, B. Zhou, Discovery of 1,8-acridinedione derivatives
as novel GCN5 inhibitors via high throughput screening, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.02.005.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Discovery of 1,8-acridinedione derivatives as novel
GCN5 inhibitors via high throughput screening
Huan Xionga,b,#, Jie Hanc,e,#, Jun Wangc,d,#, Wenchao Luc,e, Chen Wangc,e, Yu
Chenc,e，Fulin Lianc, Naixia Zhangc, Yu-Chih Liuf, Chenhua Zhangf, Hong Dingc,
Hualiang Jiangc, Wencong Lua,*, Cheng Luoc,e,*, and Bing Zhoub,*
aDepartment of Chemistry, College of Sciences, Shanghai University, 99 Shangda Road, Shang-
hai 200444, China
bDepartment of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
cCAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
201203, China
dJiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of
Chinese Medicine,138 Xianlin Road, Nanjing 210023, China
eUniversity of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
fIn Vitro Biology, Shanghai ChemPartner Life Science Co., Ltd., #5 Building, 998 Halei Road,
Shanghai 201203, China
* Corresponding authors.
# These authors contributed equally.
1

ACCEPTED MANUSCRIPT
ABSTRACT: The general control nonrepressed protein 5 (GCN5) plays a crucial role in many biological
processes. Dysregulation of GCN5 has been closely related to various human diseases, especially cancers.
Hence, the exploitation of small molecules targeting GCN5 is essential for drug design and academic
research. Based on the amplified luminescent proximity homogeneous assay screen methodology, we
performed high throughput screening and discovered a novel GCN5 inhibitor DC_G16 with 1,8-acridinedione
scaffold. Structure optimization led to the identification of a highly potent inhibitor, namely DC_G16-11 with
the half-maximal inhibitory concentration (IC50) value of 6.8 µM. The binding between DC_G16-11 and GCN5
was demonstrated by NMR and SPR with a KD of 4.2 µM. It could also inhibit proliferation and induce cell
cycle arrest and apoptosis in cancer cells while it presented minimal effects on normal cells. Herein,
DC_G16-11 could be applied as a validated chemical probe for GCN5-related biological function research
and presented great potential for clinical disease treatment.
Highlights
ò
ò
A potent GCN5 inhibitor with a new scaffold was identified via high throughput screening.
The direct binding interactions between compounds and GCN5 were demonstrated by
NMR and SPR.
ò
ò
Further chemical optimization led to the identification of DC_G16-11 with improved activ-
ity in vitro.
The detailed binding mode between DC_G16-11 and GCN5 and structure-activity rela-
tionship was disclosed by molecular docking.
Keywords
Epigenetics; Histone acetyltransferase; GCN5; High throughput screening; 1,8-acridinedione
derivative
2

ACCEPTED MANUSCRIPT
1. Introduction
Histone acetylation, one of the most important post-translational modifications in epigenetics, plays a vital
role in chromatin remodeling and gene transcription [1, 2]. The acetylation level of histones is altered by
the synergy effects of histone acetyltransferases and deacetylases. Disfunction of them leads to aberrant
cellular processes and multiple diseases [3, 4]. The general control nonrepressed protein 5 (GCN5),
originally identified in yeast, shows a preference for H3K14, while it can also acetylate H3K8 and H4K16
to a weaker extent [5-7]. Besides histones, many transcription factors can be also acetylated by GCN5
such as p53 and PGC-1α [8, 9]. Many studies show that GCN5 is a critical regulator in cell cycle and
metabolic processes, which is closely related to cancer progression and metabolic disorders [10-12].
GCN5 expression is elevated in colon cancer and inhibition of GCN5 blocks the proliferation of colon
cancer cells [13]. Overexpression of transcription factors such as E2F1 was tightly regulated by GCN5 in
non-small-cell lung cancer [14]. GCN5 also takes part in the activation of the oncogenic EGF signaling
pathway in many cancer subtypes [15]. In leukemic cells, GCN5 acetylates and stabilizes chimeric tran-
scription factor E2A-PBX1, which contributes to cell transformation [16]. Collectively, the oncogenic role
of GCN5 has been evidenced in the literature making it an attractive therapeutic target for chemical in-
tervention.
Several HAT inhibitors with different scaffolds have been reported and generally, they can be clas-
sified as bi-substrate inhibitors, natural products and synthetic compounds. Bi-substrate inhibitors are
synthesized peptide CoA conjugates. In 2002, Poux and colleagues developed the H3-(Me)CoA-20 as
the potent inhibitor for tetrahymena GCN5 with the IC50 value of 500 nM [17]. However, the lack of stabi-
lization and cell permeability of bi-substrate inhibitors limit their further development in drug design [18].
Natural products such as garcinol and anacardic acid have been reported to decrease the acetylation
level in a variety of cell models [19]. Nonetheless, due to their polypharmacology effects at cellular level,
it’s difficult to clarify the precise mechanism of action of natural products [20]. Synthetic compounds have
unique advantages of significantly low production cost and easy drug delivery. Hence, many synthesized
small molecules were developed as GCN5 inhibitors. γ-Butyrolactone MB-3 is the first reported
small-molecule inhibitor of GCN5 with IC50 value of 100 µM [21]. Isothiazolone and its derivatives were
identified as pan-HAT inhibitors that presented broad inhibition of HATs [22, 23]. Although many of these
compounds have been developed to underscore the therapeutic potential for HATs, none of them has
entered into clinic trails. Recently, many breakthroughs have been made in the development of small
molecular inhibitors for p300 [24, 25]. Instead, the researches on GCN5 inhibitors still lag behind.
Therefore, the exploitation of potent and selective GCN5 inhibitors with novel scaffolds is essential for
drug design in GCN5-related mechanism studies.
Herein, we identified a novel GCN5 inhibitor
via high throughput screening based on the
DC_G16
amplified luminescent proximity homogeneous assay. The inhibitory activity of
was validated by
DC_G16
radioactive acetylation assay. Both SPR and NMR studies gave convincing evidence of the direct binding
between GCN5 and . The design and optimization based on that scaffold resulted in the dis-
DC_G16
with improved potency in vitro. Molecular docking studies proposed that
covery of
DC_G16-11
occupied the pocket of H3 substrate in a competitive manner. Further studies at cellular level
DC_G16-11
showed that
reduced the acetylation in H3K14 and inhibited cell growth in MV4-11 cell lines
DC_G16-11
while it presented minimal effects on normal cells supporting its potential use for clinical translation.
2. Results and Discussion
2.1 Discovery of hit compound DC_G16 through HTS
3

ACCEPTED MANUSCRIPT
Nowadays, high throughput screening has developed rapidly and becomes a powerful tool for pharma-
ceutical research in both academia and industry [26]. The ALPHAScreen is an efficient and reliable
method for studies in enzyme kinetics and protein-protein interactions and has been widely applied in
epigenetic-related research [27]. In this study, a high throughput screening platform based on ALPHAS-
creen assay was established to screen in-house compound library containing about 20,000 diverse
compound collections (
). As shown in
, the primary screening was carried out at
Figure 1a
Figure S1
concentration of 100 µM. The compounds with inhibition rate higher than 70% at 100 µM were selected
for further validation. The remaining 47 hit compounds were re-purchased and re-tested in a
dose-dependent assay leading to the identification of eight candidate compounds with significant
dose-dependent inhibitory activity for GCN5. Then, in order to rule out the possibility of false positives
commonly observed in ALPHAScreen assays, radioactive acetylation assays were performed to validate
the compounds’ inhibitory activity. Among them, DC_G18, DC_G31, DC_G7, DC_G16 and DC_G11
presented inhibitory activity with IC50 value of 2.9 µM, 20.0 µM, 8.6 µM, 34.7 µM and 19.0 µM, respec-
tively and were kept for follow-up NMR studies (
). Both Carr-Purcell-Meiboom-Gill
Figure 1b, Figure 2a
(CPMG) and saturation transfer difference (STD) in NMR studies revealed that only
could bind
DC_G16
to the catalytic domain of GCN5 (
). The direct binding between
and GCN5 was
Figure2b, 2c
DC_G16
), which was in ac-
further measured quantitatively using SPR with a K value of 26.2 µM (
Figure 2d
). Hence, we identified
D
cordance with the inhibitory activity in radioactive acetylation assay (
Figure 2a
as a hit compound for follow-up optimization.
DC_G16
2.2. Chemistry
2.2.1. Synthesis
A series of derivatives were designed and synthesized for structure optimization based on DC_G16. The
synthetic routes employed to prepare the new derivatives 6a-6h are depicted in Scheme 1 and Scheme
2. The key intermediates 4a-4e were prepared through a modified Hantzsch reaction. Condensation of
1,3-cyclohexanedione (1a) / dimedone (1b) with corresponding aromatic aldehydes (2a-2e) and ammo-
nium acetate (3) in the presence of ionic liquid in anhydrous ethanol afforded the desired intermediates
4a-4e in acceptable yields. The ionic liquid was prepared according to the literature procedure [28]. In the
next step, advanced intermediates 4a-4e and 4f were reacted with 5a-5c in the presence of potassium
hydroxide in anhydrous DMSO to obtain the target compounds 6a-6h. The synthetic route for the prep-
aration of the target compounds 9a-9c is illustrated in Scheme 3. The synthesis of key intermediates
8a-8b was carried out by treatment of 4-hydroxybenzaldehyde and 1,3-cyclohexanedione dissolved in
dry THF with appropriate amine in the presence of piperidine, according to the literature [28]. The syn-
thesis of the intermediate 8c via one-pot multicomponent condensation of 1,3-cyclohexanedione,
α-naphthylamine and 4-hydroxybenzaldehyde in butanol is shown in Scheme 2 [29]. Intermediates 8a-8c
were further derivatized by a similar procedure as described above with 4-(bromomethyl)benzoic acid to
give the target compounds 9a-9c.
2.2.2. Structure-activity relationship studies and selectivity profiling
For the convenience of description, we divided the general scaffold into three parts: H, M and T, referred
to head, middle and tail regions, respectively (
). With the hit compound
) in hand, a series of
Figure 3a
(6a
derivatives were obtained by chemical synthesis and tested (
) to get a deeper understanding of
Table 1
the structure activity relationship (SAR). Firstly, replacement of the dichloro group in the M region (R2 and
R3) with the methoxy group and hydrogen in the compound
led to decreaseof its inhibitory activity
6b
4

ACCEPTED MANUSCRIPT
(
, entry 2) indicating the essential role of halogen atoms in the M region in the compounds.
Table 1
Therefore, with the R2 and R3 in the M region substituted with one halogen group, we conducted slight
modification on 1,3-cyclohexanedione to make T region more hydrophobic. Intriguingly, the resulting
compound
performed a little improved inhibitory activity (
Table 1
, entry 3) than . Encouraged by the
6a
6c
result, we initiated a couple of changes in the T region to explore whether the number of rings in the T
region restrict compounds activity. Bicyclic derivatives and , and tetracyclic derivative were
9a
9b
9c
, entry 4-6). Combined
synthesized as illustrated in
and showed similar weak activity (
Scheme 2
Table 1
( , entry 7, 8), we concluded that both H
4c Table 1
with the inhibitory activity of the intermediates
and
6h
and T regions were important for GCN5 inhibitory activity. To further investigate the SAR in H region, we
found that replacement of the carboxyl group (R5) by hydrogen ( ) and addition of fluorine as R4 ( ) led
6d
6e
to a significant decrease in their inhibitory activity which underlined the importance of carboxyl group
, entry 9,10). Taken together, we retained substituent groups based on the structure of and
(
Table 1
6c
, entry 11). exhibited
add another bromo group as R2 to obtain the compound
(
6f (DC_G16-11) Table 1
6f
Figure 3b
more than 5-fold increase in inhibitory activity versus
with an IC50 value of 6.8 µM (
).
6a
Meanwhile,
presented good selectivity against p300, EZH2 and G9a with inhibition rate of 21%, 7%
6f
and -8% at 33 µM. Taken together,
and the negative compound
were selected for further evalua-
6e
6f
tion.
2.3. Binding assay based on SPR
Given the prominent potency of
, surface plasmon resonance (SPR) based binding assay
DC_G16-11
was performed to quantitatively determine the binding affinity between
and GCN5.The re-
DC_G16-11
sults demonstrated the binding between
and GCN5 with a K value of 4.2 µM (
).
Figure 4a
DC_G16-11
D
Compared with
,
presented a higher binding affinity with GCN5, which was con-
DC_G16 DC_G16-11
sistent with inhibitory activity measured by radioisotope assays.
2.4. Binding mode analysis
To further disclose the molecular mechanism of inhibitory activity of
, we employed molecular
DC_G16-11
docking studies to investigate the binding mode between GCN5 and the inhibitors. A putative binding
pose of
was generated using Glide program with XP mode. The results revealed that
DC_G16-11
occupied the substrate H3 peptide pocket in GCN5 which may account for its in vitro activity
DC_G16-11
(
). The carboxyl group in the H region formed a hydrogen bond to the carbonyl group of Y212
Figure 4b
which clearly explained the differences in inhibitory activity between
,
and . The lack of
6d
DC_G16-11 6e
carboxyl group in
and
significantly diminished GCN5 inhibition activity as is presented in
.
6e
6d
Table 1
In addition, another two hydrogen bonds were observed between
and the sidechain of K242,
DC_G16-11
). Besides polar interactions, a hydrophobic environment sur-
Y135 in GCN5 catalytic pocket (
Figure 4c
rounded by V175, F176, Y244, Y212, Y240 and Y135 stabilized the
conformations in pocket
DC_G16-11
(
). The main difference between
and
DC_G16 DC_G16-11
in the substitution of Br instead of Cl
Figure 4c
unexpectedly improved the activity of compound. An assumption was proposed that the halogen bond
[30] was formed between E173, the most important catalytic residue in GCN5, and halogen in M region
∠
with d(Br···O) = 2.9 Å and
(C–Br···O) = 130°. The intensity of Br···O is stronger than Cl···O, which
may explain the improvement in inhibitory activity from
to .
DC_G16 DC_ G16-11
2.5. Cell-based assays
5

ACCEPTED MANUSCRIPT
To explore the cellular activity of
in cancer cells, we measured cell viability of MV4-11 leu-
DC_G16-11
kemia cells for 3 days by CellTiter-Glo luminescent assays. The results showed that
could
DC_G16-11
induce a dose-dependent cell growth inhibition with the IC50 value of 24.7 µM which presented minimal
effect on HUV-EC-C and MRC5 normal cells growth ( ). As expected, the negative
Figure 5a, Figure S3
could not retard the proliferation of MV4-11 ( ). Then western blot assay was
compound
6e
Figure S2a
conducted to evaluate the acetylation patterns in MV4-11. The results showed that the acetylation level of
H3K14 was significantly reduced by treatment of for 24 h compared to negative control
DC_G16-11
), which demonstrated the on-target behavior of
compound
(
,
. Ad-
DC_G16-11
6e Figure 5b Figure S2b
ditionally, treatment of could significantly led to cell cycle arrest in G0/G1 phase and induced
DC_G16-11
apoptosis in a dose-dependent manner in MV4-11 cells (
).
Figure 5c-d
3. Conclusion
Acetyltransferase, which transfers acetyl from Ac-CoA to histone and non-histone substrates, has been
considered as a significant regulatory factor in epigenetic modification. GCN5, one of the members in
GNAT subfamily, has got more and more attention for its global acetylation activity. However, in stark
contrast to p300/CBP, the development of the selective GCN5 inhibitor has progressed quite slowly. In
this study, we identified
as a potential GCN5 inhibitor by ALPHAScreen-based high throughput
DC_G16
screening. Radioactive acetylation assays confirmed the inhibitory activity with an IC50 value of 34.7 µM.
Biophysical methods including NMR and SPR demonstrated the direct binding between and
DC_G16
GCN5. Based on the scaffold, a series of derivatives were designed and synthesized to evaluate their
inhibition activity. Among them, presented the best inhibitory activity against GCN5 with an
DC_G16-11
IC50 value of 6.8 µM. The binding affinity was also validated by SPR with a KD value of 4.2 µM. Further
molecular docking studies proposed that occupied substrate H3 pocket which accounted for
DC_G16-11
its inhibitory activity in vitro and provided atomic-resolution interpretation of structure activity relationship.
On the cellular level, could retard the proliferation of MV4-11 cell lines while it showed no
DC_G16-11
effects on normal cells. Meanwhile, the treatment of
reduced the acetylation level on H3K14
DC_G16-11
and led to cell cycle arrest and apoptosis, which validated the biological activity of this new scaffold
compounds in leukemic models. Taken together, represents the novel scaffold which de-
DC_G16-11
serves further structure optimization and lays a solid foundation for future drug design and GCN5-related
mechanism studies.
4. Method
4.1. Plasmid construction, protein expression and purification
Sequence encoding the HAT domain of yeast GCN5 (residues 99-262) was cloned in pRSET vector and
transformed into Escherichia coli BL21 (DE3). The cells were grown in LB medium at 37 °C for 4~6 h
until the OD600 reached 0.8. Cells were induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside
and cultured overnight at 16 °C before harvest by centrifugation. Cell pellets were re-suspended and
lysed in lysis buffer (50 mM HEPES pH 8.0, 500 mM NaCl and 20 mM imidazole) followed by centrifuga-
tion at 18,000 rpm. Then supernatant was loaded onto HisTrap FF column (GE Healthcare). The protein
was eluted from the column with an increasing concentration of imidazole in lysis buffer (20-500 mM) and
further purified by SP FF column cation-exchange (GE Healthcare) and Superdex200 column (GE
Healthcare) in final buffer containing 20 mM HEPES (pH 8.0), 150 mM NaCl, 1 mM DTT. The purified
protein was concentrated to 10 mg/mL and flash-frozen in liquid nitrogen for further experiments.
6

ACCEPTED MANUSCRIPT
4.2. High throughput screening based on amplified luminescent proximity homogeneous assay
In order to discover potential GCN5 inhibitors, high throughput screening was performed based on am-
plified luminescent proximity homogeneous assay. Compounds from in house compound library were
diluted and incubated with 5 nM protein in optimized assay buffer for 30 min in 384-well plate. Then 2 µM
AC-CoA and 100 nM H3 peptide (1-23) tagged with biotin were added and reacted for 30 min at room
temperature. Subsequently, the detection reagent compromising of acceptors beads coupled with an-
ti-H3K14AC and donor beads was transferred to the assay plate. The whole system was incubated for 60
min at room temperature before amplified chemiluminescent signal at 570 nm was collected on the mul-
tilabel reader. All data were analyzed in Graphpad Prism 6.0. Detailed information about high throughput
screening and assay development is provided in Supplementary material (
,
).
Table S1 Figure S1
4.3. Radioactive acetylation assay
Briefly, the compounds in a range of concentrations were pre-incubated with protein for 15 min and
added to reactions containing peptide and [3H]-Ac-CoA in optimized Tris buffer. Subsequently, 25 L of
µ
the mix solution per well was transferred to Flashplate from assay plate. After 60 min of incubation at
37°C, reaction was stopped by cold Ac-CoA .Then the whole reaction system was read on Microbeta.
The data was analyzed by GraphPad Prism 6.0 to obtain IC50 values.
4.4. NMR assay
The interaction between compounds and yGCN5 was investigated by a Carr-Purcell-Meiboom-Gill
(CPMG) and saturation transfer difference (STD) experiments as described before [31]. Compounds and
yGCN5 protein were diluted to 200 µM and 5 µM, respectively, in assay buffer (20 mM NaH2PO4, 20 mM
Na2HPO4, 100 mM NaCl, pH 7.4) containing 5% DMSO. All NMR assays were carried out on 600 MHz
Bruker Avance III spectrometer with a cryo-probe (Bruker Biospin, Germany).
The solvent-suppressed 1D-1H CPMG (cpmgPr1d) was acquiredby [RD-90 -(τ-180 -τ)n-ACQ] pulse
°
°
sequence. A 54.78 dB pulse during the recycle delay (RD) of 4 s was employed to avoid water resonance.
90°pluselength was adjusted to around 11.82 µs. Finally, we collected 4 dummy scans and 64 free in-
（
）
duction decays FID into 64K acquisition point with a spectral width of 12 KHz (20 ppm) and an acqui-
sition time (AQT) of 2.73 s. 128 scans in totally 20 min acquisition time were used in STD experiment
including the acquisition time of 1.71 s, 4 dummy scans, relaxation delay of 3 s and a 40 dB pulse with
the irradiation frequency at 0.25 ppm or -1000 ppm, alternatively.
4.5. Surface Plasmon Resonance (SPR) based binding assay
The SPR binding assays were performed on a Biacore T200 instrument (GE Healthcare). GCN5 protein
was covalently coupled on CM5 chip according to standard procedure [32]. The whole system was
equilibrated overnight first with HBS buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% (v/v) surfac-
tant P20, and 0.25% (v/v) dimethyl sulfoxide). Compounds were diluted with HBS buffer and then in-
jected at a flow of 30 µL/min for 60 s to contact, followed by disassociation for 300 s. The equilibrium
dissociation constants of the compounds were determined by Biacore T200 evaluation software (GE
Healthcare).
4.6. Chemistry
All solvents were purchased from commercially available sources and used without purification (HPLC or
analytical grade). Anhydrous solvents were purchased from Sigma-Aldrich and stored under nitrogen
7

ACCEPTED MANUSCRIPT
atmosphere with activated molecular sieves. All reagents were purchased from Sigma Aldrich and Alfa
Aesar and used without further purification, unless otherwise indicated. All products were characterized
by their NMR and MS spectra. 1H NMR and 13C NMR spectra were obtained on Bruker Avance 400 or
500 instruments at 400/100 MHz or 500/125 MHz, respectively. Chemical shifts were reported in parts
per million (ppm, δ) downfield from tetramethylsilane (TMS) as the internal standard and coupling con-
stants (J values) were reported in Hertz (Hz). Proton coupling patterns were described as broad (b),
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). Reactions were monitored by thin-layer
chromatography or UPLC-MS analysis. Column chromatography (petroleum ether/ethyl acetate) was
performed on silica gel (200-300 mesh). Yields were not optimized.
4.6.1. General procedure A for synthesis of 1,8-acridinedione intermediates 4a-4e
The catalyst ionic liquid was prepared according to the literature procedure [28]. To a mixture of
1,3-cyclohexanedione/dimedone (2 mmol), arylaldehyde (1 mmol), and ammonium acetate (3 mmol) in 5
mL of anhydrous ethanol, and 0.02 g of ionic liquid was added and stirred at 80°C for 4 h. After comple-
tion of the reaction (TLC monitoring), the mixture was cooled to room temperature. The resulting precip-
itate was filtered off and washed with ethanol before drying to a ord the pure 1,8-acridinedione inter-
mediates in good yields (50-80%) which were submitted to the following step without further purification
[33].
4.6.1.1. 9-(3,5-Dichloro-4-hydroxyphenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione
(4a)
Compound 4a was prepared using 3,5-dichloro-4-hydroxybenzaldehyde (191 mg, 1 mmol, 1.0 eq),
1,3-cyclohexanedione (224 mg, 2 mmol, 2.0 eq) and ammonium acetate (231 mg, 3 mmol, 3.0 eq) ac-
cording to the general procedure A to give the desired product (240 mg, yield 63.5%) as an offwhite sol-
id. 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 7.01 (s, 2H), 4.77 (s, 1H), 2.56-2.43 (m, 4H), 2.25-2.17
(m, 4H), 1.96-1.86 (m, 2H), 1.85-1.72 (m, 2H). One NH was not seen.
4.6.1.2.
9-(4-Hydroxy-3-methoxyphenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione
(4b)
Compound 4b was prepared using 4-hydroxy-3-methoxybenzaldehyde (152 mg, 1 mmol, 1.0 eq),
1,3-cyclohexanedione (224 mg, 2 mmol, 2.0 eq) and ammonium acetate (231 mg, 3 mmol, 3.0 eq) ac-
cording to the general procedure A to give the desired product (245 mg, yield 72.3%) as an off-white
solid. 1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.57 (s, 1H), 6.74 (d, J = 1.8 Hz, 1H), 6.55 (d, J = 8.1
Hz, 1H), 6.46 (dd, J = 8.1, 1.8 Hz, 1H), 4.83 (s, 1H), 3.67 (s, 3H), 2.57-2.42 (m, 4H), 2.26-2.15 (m, 4H),
1.96-1.86 (m, 2H), 1.85-1.72 (m, 2H).
4.6.1.3
9-(3-Bromo-4-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-
dione (4c)
Compound 4c was prepared using 3-bromo-4-hydroxybenzaldehyde (201 mg, 1 mmol, 1.0 eq),
5,5-dimethyl-1,3-cyclohexanedione (280 mg, 2 mmol, 2.0 eq) and ammonium acetate (231 mg, 3 mmol,
3.0 eq) according to the general procedure A to give the desired product (370 mg, yield 83.3%) as a
light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 7.18 (d, J = 2.1 Hz, 1H), 6.92 (dd, J = 8.3,
2.1 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 4.67 (s, 1H), 4.36 (s, 1H), 2.44 (d, J = 17.0 Hz, 2H), 2.31 (d, J = 17.0
Hz, 2H), 2.17 (d, J = 16.1 Hz, 2H), 1.99 (d, J = 16.1 Hz, 2H), 1.00 (s, 6H), 0.88 (s, 6H);13C NMR (126
MHz, DMSO) δ 194.39, 151.69, 149.20, 139.63, 131.72, 127.69, 115.66, 111.30, 108.30, 50.21, 32.16,
31.81, 29.10, 26.39; UPLC-MS m/z (ESI, positive) found [M+H]+ 444.33, >99% pure.
8

ACCEPTED MANUSCRIPT
4.6.1.4. 9-(3-Bromo-4-hydroxyphenyl)-3, 4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (4d)
Compound 4d was prepared using 3-bromo-4-hydroxybenzaldehyde (201 mg, 1 mmol, 1.0 eq),
1,3-cyclohexanedione (224 mg, 2 mmol, 2.0 eq) and ammonium acetate (231 mg, 3 mmol, 3.0 eq) ac-
cording to the general procedure A to give the desired product (331 mg, yield 85.3%) as an off-white
solid.1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 9.45 (s, 1H), 7.18 (d, J = 2.1 Hz, 1H), 6.90 (dd, J = 8.3,
2.1 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 4.77 (s, 1H), 2.57-2.41 (m, 4H), 2.23-2.17 (m, 4H), 1.94-1.87 (m,
2H), 1.82-1.72 (m, 2H).
4.6.1.5.
9-(3,5-Dibromo-4-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,
5H)-dione (4e)
Compound 4e was prepared using 3,5-dibromo-4-hydroxybenzaldehyde (280 mg, 1 mmol, 1.0 eq),
5,5-dimethyl-1,3-cyclohexanedione (280 mg, 2 mmol, 2.0 eq) and ammonium acetate (231 mg, 3 mmol,
3.0 eq) according to the general procedure A to give the desired product (458 mg, yield 87.5%) as a
yellow solid.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 7.21 (s, 2H), 4.67 (s, 1H), 2.45 (d, J = 17.1 Hz,
2H), 2.35 (d, J = 17.1 Hz, 2H), 2.19 (d, J = 16.2 Hz, 2H), 2.01 (d, J = 16.2 Hz, 2H), 1.01 (s, 6H), 0.89 (s,
6H).
4.6.2. General procedure B for synthesis of hexahydroquinoline intermediates (8a-8b)
To
a solution of the 4-hydroxybenzaldehyde (2d, 4 mmol) in THF (10 mL) was added
1,3-cyclohexanedione/5,5-dimethyl-1,3-cyclohexanedione (4 mmol) followed by 3 drops of piperidine.
The mixture was stirred at reflux under nitrogen atmosphere for 4 h. The solvent was concentrated in
vacuo, and the residue was dissolved in ethanol (10 mL). 3-Aminocrotono-analogue (7a-7b, 4 mmol) was
added to the solution, and the reaction mixture was stirred at reflux under nitrogen overnight. The re-
sulting precipitate was filtered off and washed with ethanol before drying to a ord the desired com-
pounds. The crude product was pure enough to be used without further purification [34].
4.6.2.1.
4-(4-Hydroxyphenyl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile
(8a)
Compound 8a was prepared using 4-hydroxybenzaldehyde (488 mg,
4
mmol, 1.0 eq),
1,3-cyclohexanedione (448 mg, 4 mmol, 1.0 eq) and (E)-3-aminobut-2-enenitrile (328 mg, 4 mmol, 1.0 eq)
according to the general procedure B to give the desired product (472 mg, yield 42.1%) as an off-white
solid. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.26 (s, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.5
Hz, 2H), 4.34 (s, 1H), 2.54 – 2.47 (m, 2H),2.25- 2.18 (m, 2H), 2.06 (s, 3H), 1.96-1.89 (m, 1H), 1.86-1.76
(m, 1H).
4.6.2.2. Methyl
4-(4-hydroxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (8b)
Compound 8b was prepared using 4-hydroxybenzaldehyde (488 mg,
4
mmol, 1.0 eq),
5,5-dimethyl-1,3-cyclohexanedione (560 mg, 4 mmol, 1.0 eq) and methyl (E)-3-aminobut-2-enoate (460
mg, 4 mmol, 1.0 eq) according to the general procedure B to give the desired product (480 mg, yield
35.2%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 9.02 (s, 1H), 6.91 (d, J = 8.5
Hz, 2H), 6.55 (d, J = 8.5 Hz, 2H), 4.74 (s, 1H), 3.52 (s, 3H), 2.40 (d, J = 17.0 Hz, 1H), 2.26 (s, 3H), 2.25(d,
J = 17.0 Hz, 1H) , 2.15 (d, J = 16.1 Hz, 1H), 1.96 (d, J = 16.1 Hz, 1H), 1.00 (s, 3H), 0.84 (s, 3H).
4.6.2.3. Synthesis of naphthalene-acridone intermediate 8c
A mixture of α-naphthylamine (286 mg, 2 mmol, 1.0 eq), 1,3-cyclohexanedione (224 mg, 2 mmol, 1.0
eq) and 4-hydroxybenzaldehyde (244 mg, 2 mmol, 1.0 eq) in 20 mL of butanol was heated at reflux under
9

ACCEPTED MANUSCRIPT
nitrogen atmosphere for 6 h. The precipitate was filtered off and washed with ethanol before drying to
a ord the desired compound 8c [29]. 1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 9.07 (s, 1H), 8.45 (d,
J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.56 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.51 – 7.47 (m, 1H), 7.45 (d,
J = 8.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.01 – 6.95 (m, 2H), 6.58 – 6.51 (m, 2H), 5.11 (s, 1H), 2.94-2.85
(m, 1H), 2.73-2.63 (m, 1H), 2.33-2.18 (m, 2H), 2.03-1.95 (m, 1H), 1.93 – 1.83 (m, 1H).
4.6.3. General procedure C for synthesis of target compounds 6a-6h, 9a-9c
Potassium hydroxide (6 eq) was ground in a mortar until powdered, then added to a 25 mL Erlenmeyer
flask containing 5 mL of dimethyl sulfoxide. The resulting mixture was stirred for 5 min, after which time 1
eq of phenolic hydroxyl intermediates 4a-4f, 8a-8c was added. The mixture was stirred an additional 15
min, after which time 1 eq of benzyl bromide derivatives 5a-5c was added. The reaction was stirred at
room temperature for about 4h. Distilled water was added to the brown reaction mixture, and concen-
trated aqueous hydrochloric acid was added until the pH of the solution was about 5. The mixture was
diluted with EtOAc and the aqueous layer was separated and extracted with EtOAc. The organic layers
were combined, washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography to give the target compounds.
4.6.3.1.
4-((2,6-Dichloro-4-(1,8-dioxo-1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)phenoxy)methyl)benz
oic acid (6a)
Compound 6a was prepared from 4a (189 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (106 mg, yield
41.3%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.01 (s, 1H), 9.59 (s, 1H), 7.98 (d, J = 8.2
Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.19 (s, 2H), 5.01 (s, 2H), 4.86 (s, 1H), 2.63-2.46 (m, 4H), 2.29-2.16 (m,
4H), 1.97-1.87 (m, 2H), 1.87-1.74 (m, 2H);13C NMR (126 MHz, DMSO-d6) δ 194.90, 167.02, 152.06,
148.01, 145.61, 141.10, 130.53, 129.42, 128.04, 127.97, 127.78, 111.20, 73.87, 36.62, 32.30, 26.28,
20.75; UPLC-MS m/z (ESI, positive) found [M+H]+ 512.28, >99% pure.
4.6.3.2.
4-((4-(1,8-Dioxo-1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)-2-methoxyphenoxy)methyl)benzo
ic acid (6b)
Compound 6b was prepared from 4b (170 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (84 mg, yield
35.6%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 9.43 (s, 1H), 7.95 (d, J = 8.3,
2H), 7.52 (d, J = 8.3 Hz, 2H), 6.84 (d, J = 2.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.57 (dd, J = 8.4, 2.0 Hz,
1H), 5.07 (s, 2H), 4.88 (s, 1H), 3.70 (s, 3H), 2.51 – 2.41 (m, 4H), 2.24-2.19 (m, 4H), 1.95-1.87 (m, 2H),
1.85 – 1.72 (m, 2H).13C NMR (126 MHz, DMSO-d6) δ 194.89, 167.10, 151.16, 148.41, 145.62, 142.59,
140.74, 129.44, 127.26, 119.00, 113.50, 112.45, 112.24, 69.46, 55.44, 36.83, 31.36, 26.34, 20.86;
UPLC-MS m/z (ESI, positive) found [M+H]+ 474.36, >95% pure.
4.6.3.3.
4-((2-Bromo-4-(3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)phen
oxy)methyl)benzoic acid (6c)
Compound 6c was prepared from 4c (222 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (154 mg, yield
53.1%) as a light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.29 (s, 1H), 7.18 (d, J = 2.1
Hz, 1H), 6.92 (dd, J = 8.3, 2.1 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 4.67 (s, 1H), 2.44 (d, J = 17.0 Hz, 2H),
2.31 (d, J = 17.0 Hz, 2H), 2.17 (d, J = 16.1 Hz, 2H), 1.99 (d, J = 16.1 Hz, 3H), 1.00 (s, 6H), 0.87 (s,
10

ACCEPTED MANUSCRIPT
6H).13C NMR (126 MHz, DMSO) δ 194.40, 167.07, 152.11, 149.41, 141.77, 141.51, 132.05, 129.44,
127.80, 127.05, 113.44, 111.08, 110.18, 69.41, 54.91, 50.16, 32.17, 29.04, 26.45; UPLC-MS m/z (ESI,
positive) found [M+H]+ 578.28, >95% pure.
4.6.3.4.
4-((4-(3-Cyano-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinolin-4-yl)phenoxy)methyl)benzoic
acid (9a)
Compound 9a was prepared from 8a (140 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (80 mg, yield
38.7%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 9.48 (s, 1H), 7.95 (d, J = 8.1
Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 5.15 (s, 2H), 4.37 (s,
1H), 2.45 – 2.49 (m, 2H),2.25- 2.16 (m, 2H), 2.04 (s, 3H), 1.95-1.86 (m, 1H), 1.86-1.72 (m, 1H); 13C NMR
(126 MHz, DMSO-d6) δ 194.59, 167.06, 156.86, 151.30, 145.91, 142.30, 138.56, 130.11, 129.47, 128.29,
127.28, 119.89, 114.54, 108.94, 86.05, 68.59, 36.89, 36.63, 26.28, 20.69, 17.60; UPLC-MS m/z (ESI,
positive) found [M+H]+ 415.34, >99% pure.
4.6.3.5.
4-((4-(3-(Methoxycarbonyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinolin-4-yl)phenoxy)
methyl)benzoic acid (9b)
Compound 9b was prepared from 8b (170 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (60 mg, yield
25.3%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 9.06 (s, 1H), 7.94 (d, J = 8.3 Hz,
2H), 7.52 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.10 (s, 2H), 4.80 (s, 1H),
3.52 (s, 3H), 3.00 (s, 1H), 2.40 (d, J = 17.0 Hz, 1H), 2.27 (s, 3H), 2.26(d, J = 17.0 Hz, 1H) , 2.16 (d, J =
16.1 Hz, 1H), 1.97 (d, J = 16.1 Hz, 1H), 0.99 (s, 3H), 0.83 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ
194.27, 167.36, 167.08, 156.25, 149.23, 145.02, 142.30, 140.25, 130.16, 129.41, 128.25, 127.28, 114.05,
110.19, 103.43, 68.52, 50.62, 50.25, 34.73, 32.12, 29.10, 26.49, 18.29; UPLC-MS m/z (ESI, positive)
found [M+H]+ 476.78, >99% pure.
4.6.3.6. 4-((4-(8-Oxo-7,8,9,10,11,12-hexahydrobenzo[c]acridin-7-yl)phenoxy)methyl)benzoic
acid (9c)
Compound 9c was prepared from 8c (170 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (65 mg, yield
27.2%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.93 (s, 1H), 9.30 (s, 1H), 8.46 (d, J = 8.5 Hz,
1H), 7.96 – 7.88 (m, 2H), 7.86 – 7.78 (m, 1H), 7.61 – 7.53 (m, 1H), 7.53 – 7.43 (m, 4H), 7.28 (d, J = 8.4
Hz, 1H), 7.14 – 7.07 (m, 2H), 6.85 – 6.78 (m, 2H), 5.18 (s, 1H), 5.07 (s, 2H), 2.29-2.23 (m, 2H), 2.03-1.96
(m, 3H), 1.94-1.80 (m, 1H); 13C NMR (126 MHz, DMSO-d6) δ 193.87, 156.28, 153.43, 141.21, 132.23,
130.56, 129.61, 129.39, 128.09, 127.89, 127.14, 125.63, 122.60, 122.13, 121.22, 121.12, 114.36, 108.66,
68.54, 36.77, 31.24, 26.94, 21.04; UPLC-MS m/z (ESI, positive) found [M+H]+ 476.32, >99% pure.
4.6.3.7. 4-((2-Bromophenoxy)methyl)benzoic acid (6h)
Compound 6h was prepared from 2-bromophenol (4f, 173 mg, 1 mmol, 1.0 eq) and 4-(bromomethyl)
benzoic acid (5a, 215 mg, 1 mmol, 1.0 eq) following the general procedure C to give the target product
(192 mg, yield 62.5%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 8.2 Hz, 2H), 7.54-7.48
(m, 3H), 7.19 (ddd, J = 8.3, 7.4, 1.6 Hz, 1H), 6.87 (dd, J = 8.3, 1.4 Hz, 1H), 6.81 (td, J = 7.4, 1.4 Hz, 1H),
5.16 (s, 2H) (One COOH was not seen); 13C NMR (126 MHz, DMSO-d6) δ 167.03, 154.20, 141.67,
133.05, 129.49, 128.98, 127.05, 122.41, 114.24, 111.20, 69.36.
11

ACCEPTED MANUSCRIPT
4.6.3.8.
9-(4-(Benzyloxy)-3-bromophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,
5H)-dione (6d)
Compound 6d was prepared from 4c (222 mg, 0.5 mmol, 1.0 eq) and benzyl bromide (5b, 86 mg, 0.5
mmol, 1.0 eq) following the general procedure C to give the target product (199 mg, yield 74.5%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 7.46 – 7.41 (m, 2H), 7.41 – 7.35 (m, 2H), 7.35 –
7.28 (m, 2H), 7.07 (dd, J = 8.5, 2.1 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 5.09 (s, 2H), 4.72 (s, 1H), 2.45 (d, J
= 17.1 Hz, 2H), 2.33 (d, J = 17.1 Hz, 2H), 2.17 (d, J = 16.2 Hz, 2H), 2.00 (d, J = 16.2 Hz, 2H), 1.01 (s, 6H),
0.88 (s, 6H); 13C NMR (126 MHz, DMSO-d6) δ 194.40, 152.29, 149.38, 141.31, 136.76, 132.00, 128.40,
127.81, 127.34, 113.38, 111.10, 110.17, 69.93, 50.16, 32.17, 32.06, 29.05, 26.45; UPLC-MS m/z (ESI,
positive) found [M+H]+ 534.34, >99% pure.
4.6.3.9.
9-(3-Bromo-4-((3-fluorobenzyl)oxy)phenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione
(6e)
Compound 6e was prepared from 4d (194 mg, 0.5 mmol, 1.0 eq) and 1-(bromomethyl)-3-fluorobenzene
(5c, 95 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (135 mg, yield
54.3%) as an off-white solid.1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 7.48-7.40 (m, 1H), 7.32 (d, J =
2.1 Hz, 1H), 7.30 – 7.24 (m, 2H), 7.19-7.12 (m, 1H), 7.05 (dd, J = 8.5, 2.1 Hz, 1H), 6.97 (d, J = 8.5 Hz,
1H), 5.14 (s, 2H), 4.81 (s, 1H), 2.58 – 2.51 (m, 4H), 2.26 – 2.15 (m, 4H), 1.95-1.87 (m, 2H), 1.84-1.70 (m,
2H); 13C NMR (126 MHz, DMSO-d6) δ 194.81, 152.09, 151.40, 141.73, 139.78, 131.92, 130.55, 127.69,
123.06, 114.65, 113.87, 113.70, 113.61, 112.07, 110.38, 69.08, 36.70, 31.45, 26.28, 20.80; UPLC-MS
m/z (ESI, positive) found [M+H]+ 496.25, >99% pure.
4.6.3.10.
4-((2,6-Dibromo-4-(3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)p
henoxy)methyl)benzoic acid (6h)
Compound 6h was prepared from 4e (261 mg, 0.5 mmol, 1.0 eq) and 4-(bromomethyl)benzoic acid (5a,
108 mg, 0.5 mmol, 1.0 eq) following the general procedure C to give the target product (205 mg, yield
62.5%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 13.03 (s, 1H), 9.45 (s, 1H), 7.97 (d, J = 8.4 Hz,
2H), 7.63 (d, J = 8.4 Hz, 2H), 7.36 (s, 2H), 4.99 (s, 2H), 4.76 (s, 1H), 2.45 (d, J = 17.1 Hz, 2H), 2.35 (d, J
= 17.1 Hz, 2H), 2.19 (d, J = 16.2 Hz, 2H), 2.01 (d, J = 16.2 Hz, 2H), 1.01 (s, 6H), 0.89 (s, 6H); 13C NMR
(126 MHz, DMSO-d6) δ 194.50, 167.03, 150.01, 149.58, 146.42, 140.98, 131.83, 130.53, 129.38, 127.84,
116.95, 110.33, 73.49, 50.04, 32.72, 32.23, 28.97, 26.39; UPLC-MS m/z (ESI, positive) found [M+H]+
656.28, >99% pure.
4.7. Molecular docking
The molecular docking was carried out using the Glide program in Maestro v9.2.The crystal structure of
yGCN5 (PDB 1YGH) was used as target for docking. The protein was optimized by the Protein prepara-
tion Wizard module in Maestro. A receptor grid box was defined as a 30 Å × 30 Å × 30 Å space region
centered on E173.The ligands were prepared by LigPrep module in Maestro v9.2 for the generation of all
stereo isomers and different protonation states. Extra-precision (XP) mode was used to perform the
molecular docking. The final pose for ligand was selected according to the Glide scoring function
(G-Score).
4.8. Cell proliferation assays
12

ACCEPTED MANUSCRIPT
MV4-11 cell lines were purchased from ATCC and were maintained in RPMI-1640 medium (Life
Technologies) supplemented with 10% FBS and 1% PS (Life Technologies).The cells were cultured in a
humidified atmosphere at 37 °C with 5% CO2 in a CO2 incubator. For cell proliferation assay, the cells
were plated in 96-well plate (Corning) in total volume of 100 µL at a density of 104/well and then treated
with compounds dissolved in DMSO at indicated concentration for 72 h. CellTiter-Glo luminescent assays
(Promega) were used to determine the fraction of viable cells according to the manufacturer’s instruction.
The plates were read on a multilabel reader (Envision, PerkinElmer). All treatments were performed in
triplicate. All data were analyzed in Graphpad Prism 6.0.
4.9. Cell cycle and Apoptosis assay
For 24 h cell cycle analysis, cells were seeded in 6-well plate (Corning) at a density of 5×105/mL and
treated with indicated concentration of compounds or DMSO. After 24 h, cells were harvested and
washed three times by 1 mL PBS. Then 70 %(v/v) ethanol was used to re-suspend and fix the sample at
4 °C overnight. Afterwards, the cells were washed twice by PBS again to remove ethanol and stained by
PI/Rnase Staining Buffer (BD Pharmingen) at room temperature for further analysis. For 72 h apoptosis
assay, the cells were cultured in 6-well plate and treated with compounds as mentioned above. After 72 h
incubation, the cells were collected and apoptosis was measured using Annexin V-FITC Apoptosis De-
tection Kit (Vazyme Biotech) as the manufacturer instructed. All samples were measured by flow cytom-
etry using a LSR II cytometer (BD Pharmingen).
4.10. Western blot
For 24 h acetylation pattern analysis, the cells were cultured in 6-well plate and treated with indicated
concentration of compounds or DMSO. 20 µM SAHA was added to each well before compound treatment.
After 24 h, the cells were harvested and lysed in 1XSDS cell lysis buffer and boiled for 10 min. Protein
samples were subjected to 4%-16% SDS-polyacrylamide gradient gel and transferred to nitrocellulose
membranes. The membranes were blocked in 5% (w/v) no fat dry milk for 1 h and incubated with primary
antibody at 4 °C overnight .Then the membranes were washed three times by TBST at room tempera-
ture. The blots were probed by HRP-conjugated anti-rabbit or anti-mouse IgG antibody and detected by
chemiluminescent HRP substrate.
SUPPLEMENTARY MATERIAL
Details on additional experimental procedures, synthesis description, Figures S1-S3 and Table S1. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
cluo@simm.ac.cn; zhoubing@simm.ac.cn; wclu@shu.edu.cn.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge financial support from the National Natural Science Foundation of China
(81625022, 21472208 and 81430084 to C.L.)
13

ACCEPTED MANUSCRIPT
14

ACCEPTED MANUSCRIPT
Figures and tables
Scheme 1. Synthesis of 1,8-acridinedione derivatives
.
Scheme 2. Synthesis of compound 6h
.
Scheme 3. Synthesis of hexahydroquinoline derivatives.
15

ACCEPTED MANUSCRIPT
Table 1: Structures and radioisotope activity for synthesized derivatives
Entry
1
Compound
Structure
IC50 (µM)
6a
34.7
(DC_G16)
2
3
4
5
6
6b
6c
9a
9b
9c
24%@33 µM
16
34%@33 µM
31
34
7
8
6h
4c
81
18%@33 µM
16

ACCEPTED MANUSCRIPT
9
6d
15%@33 µM
26%@33 µM
6.8
10
11
6e
6f
(DC_G16-11)
17

ACCEPTED MANUSCRIPT
Figure 1. ALPHAScreen-based high throughput screening procedures and GCN5 radioactive
a) Flowchart of hierarchical high throughput screening for GCN5 inhibitors.
acetylation assays results.
b) Inhibitory activities of eight potential hit compounds determined by radioactive acetylation assays.
18

ACCEPTED MANUSCRIPT
Figure 2. Evaluation of hit compound DC_G16 by biochemical and biophysical assays in vitro. a)
The inhibitory activity of DC_G16 determined in GCN5 radioisotope assay. b) STD NMR experiments of 5
µM GCN5 in the presence of 200 µM DC_G16. c) Dose-dependent CPMG spectra for 200 µM DC_G16
(red) in the presence of 5 µM GCN5 (blue), 2 µM GCN5 (green) and 1 µM GCN5 (aqua). d) SPR-based
binding assay of DC_G16 with GCN5.The compound was prepared at concentration of 19.2 µM, 21.2 µM,
25.6 µM, 34.1 µM, 41.3 µM, and 50 µM, respectively.
19

ACCEPTED MANUSCRIPT
a) Decomposition diagrams of 1,8-acridinedione scaffold. The scaffold was divided into three
Figure 3.
parts: H (head region), M (middle region) and T (tail region) which were highlighted in red, green and blue
ellipses, respectively. b) The structure and inhibitory activity against GCN5 of
acetylation assay.
based on
DC_G16-11
20

ACCEPTED MANUSCRIPT
a) SPR based
Figure 4. The interaction and putative binding mode between DC_G16-11 and GCN5.
binding assay of
with GCN5.The compound was prepared at concentration of 0.9 µM,1.2 µM,
DC_G16-11
1.7 µM, 2.4 µM, 4.7 µM and 6.5 µM, respectively. b) The alignment of H3 peptide binding site of tetra-
hymena GCN5 (PDB ID: 1QSN) and predicted binding conformation of . H3 peptide is de-
DC_G16-11
is depicted as magenta sticks. GCN5 is shown in white surface.
picted as green sticks, while
DC_G16-11
c) Close-up view of the key interactions stabilizing
in H3 peptide binding pocket.
DC_G16-11
DC_G16-11
is depicted as magenta sticks, the surrounding key residues are shown in slate sticks and labelled. Hy-
drogen bonds are shown as yellow dot lines. GCN5 is shown in white surface. d) A putative halogen bond
between bromine atom and oxygen atom. Bromine atom, oxygen atom and carbon atom involved in
halogen bond are shown in green, yellow and orange ball, respectively, the measurement results of dis-
tance between Br and O and angle of C–Br···O are shown in red.
sticks. E173 is shown in slate sticks.
is depicted as magenta
DC_G16-11
21

ACCEPTED MANUSCRIPT
Figure 5. Evaluation of compound DC_G16-11 at cellular level. a) Cell proliferation inhibition assay
for DC_G16-11. b) Acetylation pattern analysis of H3K14 by western blot co-treated with 20 µM SAHA.
MV4-11 cells were treated with 0 µM, 25 µM, 50 µM and 100 µM DC_G16-11 for 24 h. c) 24 h cell cycle
analysis. MV4-11 cells were treated with 0 µM, 12.5 µM and 25 µM DC_G16-11 for 24 h. d) 72 h cell
apoptosis analysis. MV4-11 cells were treated with 0 µM, 20 µM and 40 µM DC_G16-11 for 72 h.
22

ACCEPTED MANUSCRIPT
Reference
[1] J.A. Latham, S.Y. Dent, Cross-regulation of histone modifications, Nat. Struct. Mol. Biol., 14
(2007) 1017-1024.
[2] B.D. Strahl, C.D. Allis, The language of covalent histone modifications, Nature, 403 (2000)
41-45.
[3] C. Choudhary, C. Kumar, F. Gnad, M.L. Nielsen, M. Rehman, T.C. Walther, J.V. Olsen, M.
Mann, Lysine acetylation targets protein complexes and co-regulates major cellular functions,
Science, 325 (2009) 834-840.
[4] C.H. Arrowsmith, C. Bountra, P.V. Fish, K. Lee, M. Schapira, Epigenetic protein families: a
new frontier for drug discovery, Nat. Rev. Drug Discovery, 11 (2012) 384-400.
[5] M.H. Kuo, J.E. Brownell, R.E. Sobel, T.A. Ranalli, R.G. Cook, D.G. Edmondson, S.Y. Roth,
C.D. Allis, Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines,
Nature, 383 (1996) 269-272.
[6] P.A. Grant, A. Eberharter, S. John, R.G. Cook, B.M. Turner, J.L. Workman, Expanded lysine
acetylation specificity of Gcn5 in native complexes, J. Biol. Chem., 274 (1999) 5895-5900.
[7] R.L. Schiltz, C.A. Mizzen, A. Vassilev, R.G. Cook, C.D. Allis, Y. Nakatani, Overlapping but
distinct patterns of histone acetylation by the human coactivators p300 and PCAF within
nucleosomal substrates, J. Biol. Chem., 274 (1999) 1189-1192.
[8] N.A. Barlev, L. Liu, N.H. Chehab, K. Mansfield, K.G. Harris, T.D. Halazonetis, S.L. Berger,
Acetylation of p53 activates transcription through recruitment of coactivators/histone
acetyltransferases,Mol. Cell, 8 (2001) 1243-1254.
[9] C. Lerin, J.T. Rodgers, D.E. Kalume, S.H. Kim, A. Pandey, P. Puigserver, GCN5
acetyltransferase complex controls glucose metabolism through transcriptional repression of
PGC-1alpha, Cell Metab., 3 (2006) 429-438.
[10] C.D. Tavares, K. Sharabi, J.E. Dominy, Y. Lee, M. Isasa, J.M. Orozco, M.P. Jedrychowski,
T.M. Kamenecka, P.R. Griffin, S.P. Gygi, P. Puigserver, The Methionine Transamination
Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5
Acetyltransferase and the PGC-1alpha Transcriptional Coactivator, J. Biol. Chem., 291 (2016)
10635-10645.
[11] L. Howe, D. Auston, P. Grant, S. John, R.G. Cook, J.L. Workman, L. Pillus, Histone H3
specific acetyltransferases are essential for cell cycle progression, Genes Dev., 15 (2001)
3144-3154.
[12] M. Sakai, T. Tujimura-Hayakawa, T. Yagi, H. Yano, M. Mitsushima, H. Unoki-Kubota, Y.
Kaburagi, H. Inoue, Y. Kido, M. Kasuga, M. Matsumoto, The GCN5-CITED2-PKA signalling
module controls hepatic glucose metabolism through a cAMP-induced substrate switch, Nat.
Commun., 7 (2016) 13147.
[13] Y.W. Yin, H.J. Jin, W. Zhao, B. Gao, J. Fang, J. Wei, D.D. Zhang, J. Zhang, D. Fang, The
Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1
Transcription Factors in Human Colon Cancer, Gene Expression, 16 (2015) 187-196.
[14] L. Chen, T. Wei, X. Si, Q. Wang, Y. Li, Y. Leng, A. Deng, J. Chen, G. Wang, S. Zhu, J.
Kang, Lysine acetyltransferase GCN5 potentiates the growth of non-small cell lung cancer via
promotion of E2F1, cyclin D1, and cyclin E1 expression, J. Biol. Chem., 288 (2013)
14510-14521.
23

ACCEPTED MANUSCRIPT
[15] P. Cheung, K.G. Tanner, W.L. Cheung, P. Sassone-Corsi, J.M. Denu, C.D. Allis,
Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal
growth factor stimulation, Mol. Cell, 5 (2000) 905-915.
[16] T. Holmlund, M.J. Lindberg, D. Grander, A.E. Wallberg, GCN5 acetylates and regulates
the stability of the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia, Leukemia, 27
(2013) 578-585.
[17] A.N. Poux, M. Cebrat, C.M. Kim, P.A. Cole, R. Marmorstein, Structure of the GCN5
histone acetyltransferase bound to a bisubstrate inhibitor, Proc. Natl. Acad. Sci. U. S. A., 99
(2002) 14065-14070.
[18] O.D. Lau, T.K. Kundu, R.E. Soccio, S. Ait-Si-Ali, E.M. Khalil, A. Vassilev, A.P. Wolffe, Y.
Nakatani, R.G. Roeder, P.A. Cole, HATs off: selective synthetic inhibitors of the histone
acetyltransferases p300 and PCAF, Mol. Cell, 5 (2000) 589-595.
[19] F.D. Piaz, A. Vassallo, O.C. Rubio, S. Castellano, G. Sbardella, N. De Tommasi, Chemical
biology of histone acetyltransferase natural compounds modulators, Mol. Diversity, 15 (2011)
401-416.
[20] S.D. Furdas, S. Kannan, W. Sippl, M. Jung, Small molecule inhibitors of histone
acetyltransferases as epigenetic tools and drug candidates, Arch. Pharm. (Weinheim, Ger.),
345 (2012) 7-21.
[21] M. Biel, A. Kretsovali, E. Karatzali, J. Papamatheakis, A. Giannis, Design, synthesis, and
biological evaluation of a small-molecule inhibitor of the histone acetyltransferase Gcn5,
Angew. Chem., 43 (2004) 3974-3976.
[22] L. Stimson, M.G. Rowlands, Y.M. Newbatt, N.F. Smith, F.I. Raynaud, P. Rogers, V.
Bavetsias, S. Gorsuch, M. Jarman, A. Bannister, T. Kouzarides, E. McDonald, P. Workman,
G.W. Aherne, Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity,
Mol. Cancer Ther., 4 (2005) 1521-1532.
[23] J.M. Gajer, S.D. Furdas, A. Grunder, M. Gothwal, U. Heinicke, K. Keller, F. Colland, S.
Fulda, H.L. Pahl, I. Fichtner, W. Sippl, M. Jung, Histone acetyltransferase inhibitors block
neuroblastoma cell growth in vivo, Oncogenesis, 4 (2015) e137.
[24] E.M. Bowers, G. Yan, C. Mukherjee, A. Orry, L. Wang, M.A. Holbert, N.T. Crump, C.A.
Hazzalin, G. Liszczak, H. Yuan, C. Larocca, S.A. Saldanha, R. Abagyan, Y. Sun, D.J. Meyers,
R. Marmorstein, L.C. Mahadevan, R.M. Alani, P.A. Cole, Virtual ligand screening of the
p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor,
Chem. Biol. (Oxford, U. K.), 17 (2010) 471-482.
[25] L.M. Lasko, C.G. Jakob, R.P. Edalji, W. Qiu, D. Montgomery, E.L. Digiammarino, T.M.
Hansen, R.M. Risi, R. Frey, V. Manaves, B. Shaw, M. Algire, P. Hessler, L.T. Lam, T. Uziel, E.
Faivre, D. Ferguson, F.G. Buchanan, R.L. Martin, M. Torrent, G.G. Chiang, K. Karukurichi, J.W.
Langston, B.T. Weinert, C. Choudhary, P. de Vries, J.H. Van Drie, D. McElligott, E. Kesicki, R.
Marmorstein, C. Sun, P.A. Cole, S.H. Rosenberg, M.R. Michaelides, A. Lai, K.D. Bromberg,
Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours,
Nature, 550 (2017) 128-132.
[26] R. Macarron, M.N. Banks, D. Bojanic, D.J. Burns, D.A. Cirovic, T. Garyantes, D.V. Green,
R.P. Hertzberg, W.P. Janzen, J.W. Paslay, U. Schopfer, G.S. Sittampalam, Impact of
high-throughput screening in biomedical research, Nat. Rev. Drug Discovery, 10 (2011)
188-195.
24

ACCEPTED MANUSCRIPT
[27] J. Xing, W. Lu, R. Liu, Y. Wang, Y. Xie, H. Zhang, Z. Shi, H. Jiang, Y.C. Liu, K. Chen, H.
Jiang, C. Luo, M. Zheng, Machine-Learning-Assisted Approach for Discovering Novel
Inhibitors Targeting Bromodomain-Containing Protein 4, J. Chem. Inf. Model., 57 (2017)
1677-1690.
[28] Erratum: Borderud SP, Li Y, Burkhalter JE, Sheffer CE and Ostroff JS. Electronic cigarette
use among patients with cancer: Characteristics of electronic cigarette users and their
smoking cessation outcomes. Cancer. doi: 10.1002/ cncr.28811, Cancer, 121 (2015) 800.
[29] V. Nadaraj, S.T. Selvi, S. Mohan, Microwave-induced synthesis and anti-microbial
activities of 7,10,11,12-tetrahydrobenzo[c]acridin-8(9H)-one derivatives, Eur. J. Med. Chem.,
44 (2009) 976-980.
[30] R. Wilcken, M.O. Zimmermann, A. Lange, A.C. Joerger, F.M. Boeckler, Principles and
applications of halogen bonding in medicinal chemistry and chemical biology, J. Med. Chem.,
56 (2013) 1363-1388.
[31] W.Y. Zhang, W.C. Lu, H. Jiang, Z.B. Lv, Y.Q. Xie, F.L. Lian, Z.J. Liang, Y.X. Jiang, D.J.
Wang, C. Luo, J. Jin, F. Ye, Discovery of alkyl bis(oxy)dibenzimidamide derivatives as novel
protein arginine methyltransferase 1 (PRMT1) inhibitors, Chem. Biol. Drug Des., 90 (2017)
1260-1270.
[32] S. Chen, Y. Wang, W. Zhou, S. Li, J. Peng, Z. Shi, J. Hu, Y.C. Liu, H. Ding, Y. Lin, L. Li, S.
Cheng, J. Liu, T. Lu, H. Jiang, B. Liu, M. Zheng, C. Luo, Identifying novel selective
non-nucleoside DNA methyltransferase 1 inhibitors through docking-based virtual screening, J.
Med. Chem., 57 (2014) 9028-9041.
[33] R.D. Bland, T.L. Clarke, L.B. Harden, Rapid infusion of sodium bicarbonate and albumin
into high-risk premature infants soon after birth: a controlled, prospective trial, Am. J. Obstet.
Gynecol., 124 (1976) 263-267.
[34] G.B. Arhancet, S.S. Woodard, K. Iyanar, B.L. Case, R. Woerndle, J.D. Dietz, D.J. Garland,
J.T. Collins, M.A. Payne, J.R. Blinn, S.I. Pomposiello, X. Hu, M.I. Heron, H.C. Huang, L.F. Lee,
Discovery of novel cyanodihydropyridines as potent mineralocorticoid receptor antagonists, J.
Med. Chem., 53 (2010) 5970-5978.
25