Synthesis and evaluation of novel dual BRD4/HDAC inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.04.043

Epigenetic regulation of gene expression via histone acetylation modulates many cellular processes, including apoptosis, the cell cycle, cell growth and differentiation, and inhibitors are promising drug candidates. We have previously developed inhibitors

Full text of DOI:10.1016/j.bmc.2017.04.043

Accepted Manuscript
Synthesis and evaluation of novel dual BRD4/HDAC inhibitors
Seika Amemiya, Takao Yamaguchi, Yuichi Hashimoto, Tomomi Noguchi-
Yachide
PII:
S0968-0896(17)30764-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.043
BMC 13713
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Accepted Date:
7 April 2017
28 April 2017
Please cite this article as: Amemiya, S., Yamaguchi, T., Hashimoto, Y., Noguchi-Yachide, T., Synthesis and
evaluation of novel dual BRD4/HDAC inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.04.043
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Synthesis and evaluation of novel dual BRD4/HDAC inhibitors
Seika Amemiya, Takao Yamaguchi, Yuichi Hashimoto, Tomomi
Noguchi-Yachide*
Institute of Molecular and Cellular, Biosciences, the University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan
* Corresponding author.
T. Noguchi-Yachide
Institute of Molecular & Cellular Biosciences, The University of Tokyo
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
E-mail: noguchi@iam.u-tokyo.ac.jp
Tel: +81.3.5841.7875
Fax: +81.3.5841.8495
Graphical abstract

Keywords: BET bromodomain; BRD4; HDAC; Benzoyladenine; Structure-activity
relationship; Polypharmacology
Abstract
Epigenetic regulation of gene expression via histone acetylation modulates many
cellular processes, including apoptosis, the cell cycle, cell growth and differentiation, and
inhibitors are promising drug candidates. We have previously developed inhibitors of
BRD4, which recognizes acetylated lysine residue on histones and recruits transcription
elongation factor to the transcription start site, while inhibitors of histone deacetylase
(HDAC), which catalyzes the removal of acetyl groups on histones, are already in clinical
use for cancer treatment. Based on the idea that polypharmacological agents with
multiple targets would have a more robust action, we set out to develop dual
BRD4/HDAC inhibitors. Here, we describe the design and synthesis of
N6-[2-(7-hydroxyamino-7-oxoheptyloxy)benzoyl]adenine (5d) as a BRD4/HDAC dual
inhibitor. This compound showed HL-60 cell growth-inhibitory and apoptosis-inducing
activity, as well as all-trans retinoic acid (ATRA)-induced HL-60 cell
differentiation-enhancing activity, and c-MYC production-inhibitory activity.

Interestingly, it also showed growth-inhibitory activity towards BRD4 inhibitor-resistant
cells.
1. Introduction
Polypharmacology has been receiving increasing attention as a novel strategy in drug
development over the past decade. In contrast to the conventional one-drug/one-target
philosophy,1, 2) polypharmacology aims to develop drugs with multiple related targets in
order to obtain more robust effects on diseases involving dysfunction of complex
biological networks, such as cancers and inflammatory diseases. This approach addresses
some of the issues that can occur with multidrug chemotherapy (polypharmacy), such as
drug-drug interaction and poor medication compliance.3)
We have previously developed BRD4 inhibitors and reported their structure-activity
relationships. 4, 5) BRD4 is the most extensively studied member of the bromodomain and
extra-terminal domain (BET) family proteins, which consist of BRD2, BRD3, BRD4 and
BRDT. BRD4 recognizes acetylated lysine residue (Kac) on histones via its tandem
N-terminal bromodomains and also interacts with positive transcription elongation factor
b (P-TEFb), a heterodimer of cyclin-dependent kinase 9 (CDK9) and its regulator cyclin
T, through its C-terminal domain.6, 7) BRD4 recruits P-TEFb to the transcription start site,
leading to transcription elongation through phosphorylation of the carboxyl-terminal of
RNA polymerase II.8) Moreover, BRD4 can act as a super-enhancer, driving expression
of oncogenes, such as the c-MYC oncogene, in tumor cells and facilitating transcription.9)
In addition, BRD4 was recently reported to promote elongation of noncoding enhancer
RNAs (eRNAs), in a manner dependent on interaction between the bromodomain and
Kac on histones.10) Thus, BRD4 is involved in modulation of a wide range of biological
functions that are potentially relevant to the treatment of cancers, including apoptosis, the
cell cycle, growth, proliferation, differentiation and invasion. Consequently, BET
bromodomains are promising therapeutic targets for cancers, and many BET inhibitors
with Kac-mimetic moieties (Fig. 1), including (+)-JQ1, I-BET762, OTX-015, CPI-0610,
RVX-208, have been reported since the first inhibitor was developed in 2010.11) Some of
them are in clinical trials for the treatment of cancers such as multiple myeloma (MM)
and nuclear protein in testis (NUT) midline carcinoma (NMC).12, 13)

On the other hand, histone deacetylases (HDACs) are involved in regulating histone
acetylation. The HDAC superfamily consists of eleven zinc-containing amidehydrolases,
which are classified into four major classes (class I, IIa, IIb and IV), and seven sirtuins
(SIRT1-7), which are referred to as class III and are NAD-dependent amidehydrolases.
By removing acetyl groups on histones, HDACs play key roles in the epigenetic
regulation of gene expression, modulating many cellular processes, including apoptosis,
the cell cycle, growth and differentiation.14, 15) In acute promyelocytic leukaemia (APL)
and acute myeloid leukaemia (AML), HDACs are aberrantly recruited to promoters
through physical association with oncogenic DNA-binding fusion proteins and
HDAC-containing repressor complexes, resulting in repression of specific target
genes.16-18) Numerous HDAC inhibitors have been reported (Fig. 1) as candidate drugs in
the field of oncology. Among them, vorinostat (SAHA; Fig. 1) was approved by the U.S.
Food and Drug Administration (FDA) for the treatment of the cutaneous manifestations
of cutaneous T cell lymphoma (CTCL).19)
Since both BRD4 and HDAC are related to epigenetic regulation of gene expression via
histone acetylation, and are associated with similar biological phenotypes related to
cancer,20) there is a great interest in developing dual BRD4/HDAC inhibitors. Here, we
report the design and synthesis of dual BRD4/HDAC inhibitors. We also examined their
effects on cancer cells in order to assess their potential utility as candidate
polypharmacological drugs for treatment of cancer.

Figure 1. Chemical structures of typical BET and HDAC inhibitors
2. Results and discussion
We have reported N6-benzoyladenine as a new chemical scaffold for development of
BRD4 inhibitors and examined its structure-activity relationship (SAR).4, 5) Subsequently,
related BET bromodomain inhibitors with a 9H-purine moiety were reported to interact
with the conserved asparagine (Asn140) in the Kac-binding pocket of BRD4 via purine
nitrogen.21) Thus, we considered that the N6-benzoyladenine moiety serves as a mimic of
Kac. In our previous work, we found that compounds with an electron-donating
substituent at the 2-position of the benzoyl group possess more potent BRD4-inhibitory
activity than those with an electron-accepting substituent, and
N6-(2-phenoxybenzoyl)adenine and N6-(2-pentyloxybenzoyl)adenine (6) retained the
inhibitory activity.4, 5) These observations imply that there is unoccupied space between
the 2-position of the benzoyl group of N6-benzoyladenine derivatives and BRD4 in the
Kac-binding pocket. On the other hand, the SAR of HDACs has been extensively studied.
Many HDAC inhibitors consist of a zinc-binding group, which chelates the zinc ion at the
bottom of the HDAC active site, such as a hydroxamic group coupled via a spacer moiety
to a cap group, which occupies a position between the hydrophobic residues at the
entrance of the HDAC active site.22) Based on these findings, we designed and
synthesized 5a-5e as candidate dual BRD4/HDAC inhibitors (Scheme 1). These
compounds, in which N6-benzoyladenine as a BRD4-binding moiety is connected to a
hydroxamic group as an HDAC-binding moiety by a flexible alkyl linker, were prepared

as shown in Scheme 1. Briefly, benzyl 2-hydroxybenzoates were alkylated to afford
1a-1e. Deprotection of the benzyl group afforded 2a-2e, which were condensed with
adenine in DMA to give 3a-3e. After de-esterification of 3a-3e, the compounds were
condensed with THP-protected hydroxylamine to afford 4a-4e. Deprotection of the THP
group gave the desired compounds 5a-5e.
Scheme 1. Synthesis of compounds 5a-5e. Reaction conditions: (a) alkyl bromide,
K2CO3, MeCN, 54%-quant.; (b) H2, Pd/C, EtOH, 64-99%; (c) adenine, EDCI·HCl, DMA,
5-25%; (d) LiOH·H2O, THF, H2O; (e) NH2OTHP, HOBt·H2O, EDCI·HCl, DMF, 20-87%
(2 steps); (f) p-TsOH·H2O, MeOH, 24-81%.
The BRD4- and HDAC-inhibitory activities of these compounds were investigated
using commercially available assay kits, BRD4 bromodomain 1 TR-FRET assay kit
(Cayman) and Fluor de Lys®-Green HDAC Assay kit (Enzo Life Sciences). (+)-JQ1,
which is a pan-BET bromodomain inhibitor, and vorinostat, which is a pan-HDAC
inhibitor, were used as positive controls. Compounds 5a-5e, which were prepared to
evaluate the influence of linker length, showed both BRD4- and HDAC-inhibitory
activities (Table 1). Among them, 5d with a hexyl linker exhibited the most potent
BRD4/HDAC dual-inhibitory activities.
Table 1. BRD4- and HDAC-inhibitory activities of 5a-5e.

Next, we investigated the cell growth-inhibitory activity, all-trans retinoic acid
(ATRA)-induced cell differentiation-enhancing activity, and c-MYC
production-inhibitory activity in order to evaluate the potential of 5d as an anti-cancer
agent.23-31) In addition, we evaluated the suitability of 5d for polypharmacology by using
BRD4 inhibitor-resistant cells.
Firstly, we investigated the growth-inhibitory activity of 5d towards a human
promyelocytic leukemia cell line, HL-60, in comparison with
N6-(2-pentyloxybenzoyl)adenine (6), which we have previously reported as a BRD4
inhibitor.5) As shown in Table 2, (+) JQ1, vorinostat, 6, and 5d all showed HL-60 cell
growth-inhibitory activity. The activities decreased in the following order: (+)-JQ1 >
vorinostat > 6 > 5d.
Table 2. HL-60 cell growth-inhibitory activity and differentiation-inducing activity
Next, we also examined whether 5d induces apoptosis. We double-stained HL-60 cells
with Annexin V-FITC and PI, and detected apoptotic cells using flow cytometry. Annexin

V-positive cells increased after incubation with 5d for 24 h, and 5d showed
time-dependent apoptosis-inducing activity (Fig. 2D-F). We confirmed the expression of
cleaved caspase 3 and cleaved poly ADP-ribose polymerase (PARP) as indicators of
apoptosis by western blot analysis. Compounds 6 and 5d both showed time-dependent
cleavage of caspase 3 and PARP (Figure 3). Overall, the results of flow cytometry and
western blot analysis indicated that 6 and 5d cause growth inhibition followed by
apoptosis.
Figure 2. Detection of apoptotic cells by Annexin V and PI double staining

Figure 3. Western blot analysis of caspase 3 and PARP
Secondly, we investigated the effect of 5d on ATRA-induced HL-60 cell differentiation.
ATRA was selected because it is used in differentiation therapy for APL, and is an
endogenous active form of vitamin A that is generally present in normal serum. HL-60
cells were incubated in the presence of test compound with 0.6 nM ATRA, which is
approximately equal to the endogenous retinoic acid concentration in human plasma.
(+)-JQ1, vorinostat, 6, and 5d all enhanced ATRA-induced cell differentiation, and the
activities decreased in the following order: (+)-JQ1 > vorinostat > 6 > 5d (Table 2).
Thirdly, we investigated c-MYC production-inhibitory activity. As shown in Table 3,
(+)-JQ1, vorinostat, 6, 5d, and 5e showed c-MYC production-inhibitory activity, while
5a-5c did not. The activities decreased in the following order: (+)-JQ1 > vorinostat > 6 >
5e > 5d. The reason why 5a-5c were inactive may be that these compounds have low
permeability across the cell membrane. Overall, the results in Fig. 2, Fig. 3, and Table 2
suggest that dual BRD4/HDAC inhibitor 5d is a promising candidate agent for treatment
of cancer.
Table 3. c-MYC production-inhibitory activity

Finally, we evaluated the growth-inhibitory effect of 5d on K-562, T-47D, and OVCAR-5
cell lines, which are (+)-JQ1-resistant, and on HL-60, MCF-7, MDA-MB-231, and
A2780 cell lines, which are (+)-JQ1-sensitive.25, 32-34) As shown in Table 4, (+)-JQ1 and 6
showed weaker growth-inhibitory activity towards leukemia K-562 cells compared with
leukemia HL-60 cells, while vorinostat and 5d showed equivalent activities towards both
K-562 and HL-60 cells. Similar results were observed with ovarian cancer cells,
OVCAR-5 cells and A2780 cells. On the other hand, 5d showed weaker activity in breast
cancer cells, MCF-7 cells and MDA-MB-231 cells, but showed comparable activity to
(+)-JQ1 in T-47D cells. Thus, 5d is effective against BRD4 inhibitor-resistant cells.
Table 4. Growth-inhibitory activities towards various cancer cell lines
3. Conclusion
We designed and synthesized novel dual BRD4/HDAC inhibitors 5a-5e based on the
structure-activity relationships of N6-benzoyladenine derivatives. Among these
compounds, 5d showed the most potent BRD4/HDAC dual-inhibitory activities, and also
possessed HL-60 cell growth-inhibitory activity, HL-60 cell differentiation-inducing

activity, and c-MYC production-inhibitory activity. Moreover, it showed a significant
growth-inhibitory effect on BRD4-resistant cells. These results suggest that
polypharmacology may be an effective strategy for the treatment of cancers.
4. Experimental Section
4.1. General chemistry
1H-NMR spectra were recorded on a JEOL JNM-ECA-500 (500 MHz) spectrometer.
Chemical shift values were expressed in d values (ppm) relative to internal
tetramethylsilane (0.00 ppm), residual CHCl3 (7.26 ppm), CHD2OD (3.31 ppm), or
C2HD5SO (2.49 ppm) for 1H-NMR, and internal tetramethylsilane (0.00 ppm),
chloroform-d1 (77.16 ppm), methanol-d4 (49.00 ppm), or dimethylsulfoxide-d6 (39.50
ppm) for 13C-NMR. Electrospray ionization (ESI) and fast atom bombardment (FAB)
mass spectra were recorded on a Bruker micrOTOF II mass spectrometer. Melting points
were determined by using a MP-J3 melting point apparatus (Yanako). Thin layer
chromatography (TLC) was performed on silica gel 60 F254 plates (Merck). Flash
column chromatography was performed on silica gel 60 (spherical, particle size 40-100
ꢀm; Kanto).
4.1.1. Synthesis
General synthetic procedure for benzyl 2-(ethoxy-oxoarkyloxy)benzoates
The corresponding alkyl bromide (2.0 eq) and potassium carbonate (2.0 eq) were added
to a solution of benzyl 2-hydroxybenzoate (1.0 eq) in CH3CN, and the mixture was
refluxed. After completion of the reaction, the residue was purified by silica gel column
chromatography (EtOAc/Hexane) to afford the product.
4.1.1.1. Benzyl 2-(4-ethoxy-4-oxobutyloxy)benzoate (1a)
Colorless oil (quant.): 1H-NMR (500 MHz, CDCl3) δ 1.24 (t, J=6.9 Hz, 3H), 2.05-2.11
(m, 2H), 2.47 (t, J=7.5 Hz, 2H), 4.07 (t, J=6.3 Hz, 2H), 4.12 (q, J=7.5 Hz, 2H), 5.34 (s,
2H), 6.93-6.97 (m, 2H), 7.30-7.33 (m, 1H), 7.35-7.38 (m, 2H), 7.41-7.46 (m, 3H), 7.82 (d,
J=7.5 Hz, 1H); ESI-TOF-HRMS (M+Na)+ calculated for C20H22O5Na 365.1359; found
365.1373.

4.1.1.2. Benzyl 2-(5-ethoxy-5-oxopentyloxy)benzoate (1b)
Colorless oil (94 %): 1H-NMR (500 MHz, CDCl3) δ 1.24 (t, J=6.9 Hz, 3H), 1.72-1.85
(m, 4H), 2.30 (t, J=7.5 Hz, 2H), 4.02 (t, J=6.3 Hz, 2H), 4.11 (q, J=7.5 Hz, 2H), 5.34 (s,
2H), 6.92-6.97 (m, 2H), 7.32-7.38 (m, 3H), 7.41-7.45 (m, 3H), 7.82 (d, J=7.5 Hz, 1H);
ESI-TOF-HRMS (M+Na)+ calculated for C21H24O5Na 379.1516; found 379.1499.
4.1.1.3. Benzyl 2-(6-ethoxy-6-oxohexyloxy)benzoate (1c)
Colorless oil (86 %): 1H-NMR (500 MHz, CDCl3) δ 1.25 (t, J=6.9 Hz, 3H), 1.42-1.48
(m, 2H), 1.60-1.66 (m, 2H), 1.75-1.81 (m, 2H), 2.26 (t, J=8.0 Hz, 2H), 4.00 (t, J=6.3 Hz,
2H), 4.12 (q, J=7.5 Hz, 2H), 5.34 (s, 2H), 6.92-6.96 (m, 2H), 7.30-7.33 (m, 1H),
7.35-7.38 (m, 2H), 7.40-7.45 (m, 3H), 7.81 (d, J=7.5 Hz, 1H); ESI-TOF-HRMS (M+Na)+
calculated for C22H26O5Na 393.1672; found 393.1698.
4.1.1.4. Benzyl 2-(7-ethoxy-7-oxoheptyloxy)benzoate (1d)
Colorless oil (quant.): 1H-NMR (500 MHz, CDCl3) δ 1.24 (t, J=6.9 Hz, 3H), 1.31-1.36
(m, 2H), 1.40-1.46 (m, 2H), 1.57-1.64 (m, 2H), 1.74-1.80 (m, 2H), 2.27 (t, J=7.4 Hz, 2H),
4.00 (t, J=6.9 Hz, 2H), 4.12 (q, J=7.5 Hz, 2H), 5.34 (s, 2H), 6.93-6.96 (m, 2H), 7.30-7.33
(m, 1H), 7.36 (dd, J=7.5, 7.5 Hz, 2H), 7.40-7.45 (m, 3H), 7.81 (d, J=8.0 Hz, 1H);
ESI-TOF-HRMS (M+Na)+ calculated for C23H28 O5Na 407.1829; found 407.1845.
4.1.1.5. Benzyl 2-(8-ethoxy-8-oxooctyloxy)benzoate (1e)
Colorless oil (54%): 1H-NMR (500 MHz, CDCl3) δ 1.25 (t, J=7.5 Hz, 3H), 1.28-1.34 (m,
4H), 1.37-1.45 (m, 2H), 1.58-1.64 (m, 2H), 1.74-1.78 (m, 2H), 2.27 (t, J=7.5 Hz, 2H),
4.00 (t, J=6.9 Hz, 2H), 4.12 (q, J=6.9 Hz, 2H), 5.34 (s, 2H), 6.93-6.96 (m, 2H), 7.30-7.38
(m, 3H), 7.41-7.45 (m, 3H), 7.81 (d, J=8.0 Hz, 1H); ESI-TOF-HRMS (M+Na)+
calculated for C24H30O5Na 421.1985; found 421.1995.
General synthetic procedure for 2-(ehoxy-oxoarkyloxy)benzoic acids
Palladium carbon (10 %) was added to a solution of the corresponding benzyl benzoate in
EtOH. The mixture was stirred under a hydrogen atmosphere. After completion of the
reaction, the mixture was filtered through a pad of celite. After removal of the solvent, the
residue was purified by silica gel column chromatography (EtOAc/Hexane) to afford the
product.

4.1.1.6. 2-(4-ethoxy-4-oxobutyloxy)benzoic acid (2a)
White solid (63 %): 1H-NMR (500 MHz, CDCl3) δ 1.25 (t, J=7.5 Hz, 3H), 2.21-2.27 (m,
2H), 2.53 (t, J=6.9 Hz, 2H), 4.15 (q, J=7.4 Hz, 2H), 4.32 (t, J=6.3 Hz, 2H), 7.05 (d, J=7.5
Hz, 1H), 7.13 (dd, J=7.5, 8.6 Hz, 1H), 7.55 (dd, J=7.5, 8.3 Hz, 1H), 8.18 (d, J=7.5 Hz,
1H); ESI-TOF-HRMS (M+Na)+ calculated for C13H16O5Na 275.0890; found 275.0885.
4.1.1.7. 2-(5-ethoxy-5-oxopentyloxy)benzoic acid (2b)
Yellow oil (90 %): 1H-NMR (500 MHz, CDCl3) δ 1.23 (t, J=6.9 Hz, 3H), 1.78-1.84 (m,
2H), 1.92-1.97 (m, 2H), 2.39 (t, J=6.9 Hz, 2H), 4.11 (q, J=7.5 Hz, 2H), 4.24 (t, J=6.3 Hz,
2H), 7.01 (d, J=8.0 Hz, 1H), 7.10 (dd, J=7.5, 8.0 Hz, 1H), 7.52 (dd, J=6.9, 7.5 Hz, 1H),
8.14 (d, J=8.0 Hz, 1H); ESI-TOF-HRMS (M+Na)+ calculated for C14H18O5Na 289.1046;
found 289.1073.
4.1.1.8. 2-(6-ethoxy-6-oxohexyloxy)benzoic acid (2c)
Yellow solid (87 %): 1H-NMR (500 MHz, CDCl3) δ 1.24 (t, J=6.9 Hz, 3H), 1.50-1.56
(m, 2H), 1.68-1.74 (m, 2H), 1.92-1.95 (m, 2H), 2.33 (t, J=7.5 Hz, 2H), 4.12 (q, J=6.9 Hz,
2H), 4.25 (t, J=6.3 Hz, 2H), 7.02 (d, J=8.0 Hz, 1H), 7.11 (dd, J=7.5, 8.0 Hz, 1H), 7.53 (dd,
J=7.5, 7.5 Hz, 1H), 8.16 (d, J=6.3 Hz, 1H); ESI-TOF-HRMS (M-H)+ calculated for
C15H19O5 279.1227; found 279.1229.
4.1.1.9. 2-(7-ethoxy-7-oxooctyloxy)benzoic acid (2d)
Yellow oil (99 %): 1H-NMR (500 MHz, CDCl3) δ 1.24 (t, J=7.5 Hz, 3H), 1.39-1.45 (m,
2H), 1.48-1.54 (m, 2H), 1.63-1.69 (m, 2H), 1.90-1.95 (m, 2H), 2.31 (t, J=7.5 Hz, 2H),
4.12 (q, J=6.9 Hz, 2H), 4.24 (t, J=6.9 Hz, 2H), 7.03 (d, J=8.6 Hz, 1H), 7.13 (d, J=6.9, 7.5
Hz, 1H), 7.54 (dd, J=7.5, 8.3 Hz, 1H), 8.18 (d, J=8.0 Hz, 1H); ESI-TOF-HRMS (M+Na)+
calculated for C16H22O5Na 317.1359; found 317.1348.
4.1.1.10. 2-(8-ethoxy-8-oxooctyloxy)benzoic acid (2e)
Brown solid (92 %): 1H-NMR (500 MHz, CDCl3) δ 1.24 (t, J=7.5 Hz, 3H), 1.31-1.43 (m,
4H), 1.45-1.53 (m, 2H), 1.60-1.66 (m, 2H), 1.86-1.95 (m, 2H), 2.29 (t, J=6.9 Hz, 2H),
4.11 (q, J=6.9 Hz, 2H), 4.24 (t, J=6.3 Hz, 2H), 7.03 (d, J=8.6 Hz, 1H), 7.12 (d, J=7.4, 7.5
Hz, 1H), 7.54 (dd, J=7.5, 7.5 Hz, 1H), 8.17 (d, J=7.5 Hz, 1H); ESI-TOF-HRMS (M-H)-

calculated for C17H23O5 307.1540; found 307.1543.
General synthetic procedure for N6-benzoyl/heteroarylcarbonyladenine derivatives
Benzoic acid or heteroaryl carboxylic acid (1.0 eq) and EDCI·HCl (1.2 eq) were added to
a suspension of adenine (1.0 eq) in anhydrous DMA, and the mixture was stirred at
100°C-130 °C. After completion of the reaction, the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatography (MeOH/CHCl3)
and by washing with EtOAc and/or CH2Cl2/hexane (2:1 or 1:1 or 1:0) to afford the
product.
4.1.1.11. N6-[2-(4-Ethoxy-4-oxobutyloxy)benzoyl]adenine (3a)
White solid (22 %): 1H-NMR (500 MHz, DMSO-d6) δ 1.08 (t J=6.9 Hz, 3H), 2.04-2.14
(m, 2H), 2.55 (t, J=7.5 Hz, 2H), 3.97 (q, J=6.9 Hz, 2H), 4.23 (t, J=6.3 Hz, 2H), 7.13 (dd,
J=8.0, 8.1 Hz, 1H), 7.24 (d, J=8.0 Hz, 1H), 7.58 (dd, J=7.7, 8.0 Hz, 1H), 7.93 (d, J=7.5
Hz, 1H), 8.43 (s, 1H), 8.60 (s, 1H), 11.00 (s, 1H), 12.34 (brs, 1H); ESI-TOF-HRMS
(M+Na)+ calculated for C18H19N5O4Na 392.1329; found 392.1335.
4.1.1.12. N6-[2-(5-Ethoxy-5-oxopentyloxy)benzoyl]adenine (3b)
White solid (5 %): 1H-NMR (500 MHz, CDCl3) δ 1.19 (t, J=7.5 Hz, 3H), 1.93-1.99 (m,
2H), 2.05-2.10 (m, 2H), 2.46 (t, J=7.5 Hz, 2H), 4.07 (q, J=7.5 Hz, 2H), 4.27 (t, J=6.3 Hz,
2H), 7.04 (d, J=8.1 Hz, 1H), 7.13 (dd, J=6.9, 8.0 Hz, 1H), 7.55 (dd, J=7.6, 7.9 Hz, 1H),
8.23 (d, J=8.1 Hz, 1H), 8.55 (s, 1H), 8.74 (s, 1H), 11.15 (brs, 1H); ESI-TOF-HRMS
(M+Na)+ calculated for C19H21N5O4Na 406.1486; found 406.1507.
4.1.1.13. N6-[2-(6-Ethoxy-6-oxohexyloxy)benzoyl]adenine (3c)
White solid (25 %): 1H-NMR (500 MHz, DMSO-d6) δ 1.08 (t, J=7.5 Hz, 3H), 1.44-1.62
(m, 4H), 1.79-1.91 (m, 2H), 2.18-2.27 (m, 2H), 3.93 (q, J=7.5 Hz, 2H), 4.21 (t, J=6.3 Hz,
2H), 7.13 (dd, J=7.5, 8.0 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H), 7.59 (dd, J=7.7, 7.8 Hz, 1H),
7.98 (d, J=8.0 Hz, 1H), 8.44 (s, 1H), 8.62 (s, 1H), 11.00 (brs, 1H), 12.32 (brs, 1H);
ESI-TOF-HRMS (M+Na)+ calculated for C20H23N5 O4Na 420.1642; found 420.1659.
4.1.1.14. N6-[2-(7-Ethoxy-7-oxoheptyloxy)benzoyl]adenine (3d)
White solid (16 %): 1H-NMR (500 MHz, CDCl3) δ 1.23 (t, J=7.5 Hz, 3H), 1.43-1.49 (m,

2H), 1.58-1.69 (m, 4H), 2.04-2.08 (m, 2H), 2.30 (t, J=7.5 Hz, 2H), 4.11 (q, J=7.5 Hz, 2H),
4.25-4.28 (m, 2H), 7.05 (d, J=8.1 Hz, 1H), 7.11 (d, J=6.9, 7.5 Hz, 1H), 7.57 (dd, J=6.9,
7.5 Hz, 1H), 8.21 (d, J=7.5 Hz, 1H), 9.11 (s, 1H), 9.57 (s, 1H), 11.63 (brs, 1H);
ESI-TOF-HRMS (M+Na)+ calculated for C21H25N5 O4Na 434.1799; found 434.1788.
4.1.1.15. N6-[2-(8-Ethoxy-8-oxooctyloxy)benzoyl]adenine (3e)
White solid (13 %): 1H-NMR (500 MHz, DMSO-d6) δ 1.10 (t, J=6.9 Hz, 3H), 1.15-1.23
(m, 2H), 1.24-1.36 (m, 2H), 1.37-1.54 (m, 4H), 1.79-1.90 (m, 2H), 2.17 (t, J=7.5 Hz, 2H),
3.97 (q, J=6.9 Hz, 2H), 4.21 (t, J=6.3 Hz, 2H), 7.13 (dd, J=7.5, 7.5 Hz, 1H), 7.26 (d,
J=8.6 Hz, 1H), 7.59 (dd, J=7.5, 8.0 Hz, 1H), 7.98 (d, J=7.5 Hz, 1H), 8.44 (s, 1H), 8.61 (s,
1H), 11.00 (brs, 1H), 12.33 (brs, 1H); ESI-TOF-HRMS (M+Na)+ calculated for
C22H27N5O4Na 448.1955; found 448.1983.
General synthetic procedure for THP-protected hidroxamic acids
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
1-hydroxybenzotriazole monohydrate
hydrochloride
(1.2
(1.2
eq),
and
eq)
o-(tetrahydropyran-2-yl)hydroxylamine (1.5 eq) were added to a solution of the
corresponding carboxylic acid (1.0 eq) in DMF and the mixture was stirred at room
temperature. After completion of the reaction, the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatography (MeOH/CHCl3)
to afford the product.
4.1.1.16.
N6-(2-{4-[(tetrahydro-2H-pyran-2-yl)oxy]amino-4-oxobutyloxy}benzoyl)adenine
(4a)
1
White solid (2 steps, 20 %): H-NMR (500 MHz, CDCl3) δ 1.38-1.58 (m, 3H),
1.62-1.80 (m, 3H), 2.22-2.34 (m, 2H), 2.44-2.56 (m, 2H), 3.35-3.41 (m, 1H), 3.75-3.82
(m, 1H), 4.26 (t, J=5.2 Hz, 2H), 4.87 (s, 1H), 7.02 (d, J=8.6 Hz, 1H), 7.06 (dd, J=7.5, 8.0
Hz, 1H), 7.50 (dd, J=7.7, 8.3 Hz, 1H), 8.10 (d, J=7.5 Hz, 1H), 8.43 (s, 1H), 8.72 (s, 1H);
ESI-TOF-HRMS (M+Na)+ calculated for C21H24N6O5Na 463.1700; found 463.1698.
4.1.1.17.
N6-(2-{5-[(tetrahydro-2H-pyran-2-yl)oxy]amino-5-oxopentyloxy}benzoyl)adenine

(4b)
1
White solid (2 steps, 72 %): H-NMR (500 MHz, CDCl3) δ 1.43-1.60 (m, 3H),
1.65-1.82 (m, 3H), 1.91-1.99 (m, 2H), 2.05-2.10 (m, 2H), 2.29 (t, J=6.3 Hz, 2H), 3.51 (d,
J=10.9 Hz, 1H), 3.88 (t, J=9.7 Hz, 1H), 4.23 (t, J=6.3 Hz, 2H), 4.88 (s, 1H), 7.02 (d, J=8.6
Hz, 1H), 7.09 (dd, J=7.5, 7.5 Hz, 1H), 7.53 (dd, J=6.9, 7.5 Hz, 1H), 8.18 (d, J=7.5 Hz,
1H), 8.51 (brs, 1H), 8.73 (s, 1H); ESI-TOF-HRMS (M+Na)+ calculated for
C22H26N6O5Na 477.1857; found 477.1859.
4.1.1.18.
N6-(2-{6-[(tetrahydro-2H-pyran-2-yl)oxy]amino-6-oxohexyloxy}benzoyl)adenine
(4c)
1
White solid (2 steps, 87 %): H-NMR (500 MHz, CDCl3) δ 1.45-1.79 (m, 10H),
1.99-2.07 (m, 2H), 2.13 (t, J=6.9 Hz, 2H), 3.48-3.55 (m, 1H), 3.87-3.91 (m, 1H), 4.24 (t,
J=6.3 Hz, 2H), 4.85 (brs, 1H), 7.05 (d, J=8.0 Hz, 1H), 7.12 (dd, J=6.9, 7.4 Hz, 1H), 7.56
(dd, J=7.8, 8.3 Hz, 1H), 8.22 (d, J=8.0 Hz, 1H), 8.83 (s, 1H), 8.95 (s, 1H);
ESI-TOF-HRMS (M+Na)+ calculated for C23H28N6O5Na 491.2013; found 491.2042.
4.1.1.19.
N6-(2-{7-[(tetrahydro-2H-pyran-2-yl)oxy]amino-7-oxoheptyloxy}benzoyl)adenine
(4d)
White solid (2 steps, 54 %): 1H-NMR (500 MHz, CDCl3) δ 1.46-1.77 (m, 12H), 2.00
(brs, 2H), 2.21 (brs, 2H), 3.56 (d, J=11.2 Hz, 1H), 3.97 (brs, 1H), 4.16 (brs, 2H), 4.98 (brs,
1H), 6.97 (d, J=8.0 Hz, 1H), 7.05 (brs, 1H), 7.49 (brs, 1H), 8.13 (d, J=6.9 Hz, 1H), 8.66 (s,
1H), 8.77 (s, 1H), 9.75 (brs, 1H), 11.07 (brs, 1H); ESI-TOF-HRMS (M+Na)+ calculated
for C24H30N6O5Na 505.2170; found 505.2183.
4.1.1.20.
N6-(2-{8-[(tetrahydro-2H-pyran-2-yl)oxy]amino-8-oxooctyloxy}benzoyl)adenine
(4e)
1
White solid (2 steps, 49 %): H-NMR (500 MHz, CDCl3) δ 1.26-1.36 (m, 2H),
1.36-1.46 (m, 2H), 1.47-1.84 (m, 10H), 1.92-2.14 (m, 2H), 2.05-2.17 (m, 2H), 3.53-3.60
(m, 1H), 3.85-4.02 (m, 1H), 4.02 (t, J=6.3 Hz, 2H), 4.95 (s, 1H), 7.02 (d, J=8.0 Hz, 1H),
7.09 (dd, J=7.5, 8.0 Hz, 1H), 7.53 (dd, J=7.7, 8.3 Hz, 1H), 8.20 (d, J=7.5 Hz, 1H), 8.50 (s,

1H), 8.76 (s, 1H), 11.11 (s, 1H); ESI-TOF-HRMS (M+Na)+ calculated for C25H32N6O5Na
519.2326; found 519.2330.
General synthetic procedure for N6-[2-(hydroxyamino-oxoarkyloxy)bezoyl]adenines
p-toluenesulfuric acid (0.4 eq) was added to a solution of the corresponding
THP-protected hydroxamic acids (1.0 eq) in MeOH and the mixture was stirred at room
temperature. After completion of the reaction, the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatography (MeOH/CHCl3)
to afford the product.
4.1.1.21. N6-[2-(4-hydroxyamino-4-oxobutyloxy)benzoyl]adenine (5a)
White solid (24 %): m.p. 214.0-215.0 ºC; 1H-NMR (500 MHz, DMSO-d6) δ 2.03-2.12
(m, 2H), 2.20 (t, J=6.9 Hz, 2H), 4.20 (t, J=6.3 Hz, 2H), 7.14 (dd, J=7.5, 8.0 Hz, 1H), 7.24
(d, J=8.6 Hz, 1H), 7.59 (dd, J=6.9, 8.6 Hz, 1H), 7.96 (d, J=7.5 Hz, 1H), 8.45 (brs, 1H),
8.63 (s, 1H), 8.69 (brs, 1H), 10.34 (brs, 1H), 11.00 (brs, 1H), 12.34 (brs, 1H);
ESI-TOF-HRMS (M-H)- calculated for C16H15N6O4 355.1149; found 355.1153.
4.1.1.22. N6-[2-(5-hydroxyamino-5-oxopentyloxy)benzoyl]adenine (5b)
White solid (81 %): m.p. 168.0-169.0 ºC; 1H-NMR (500 MHz, DMSO-d6) δ 1.65-1.78
(m, 2H), 1.78-1.94 (m, 2H), 2.02 (t, J=6.9 Hz, 2H), 4.22 (t, J=6.3 Hz, 2H), 7.14 (dd, J=7.5,
7.5 Hz, 1H), 7.26 (d, J=8.0 Hz, 1H), 7.60 (dd, J=6.9, 8.6 Hz, 1H), 7.98 (d, J=8.0 Hz, 1H),
8.45 (s, 1H), 8.65 (brs, 2H), 10.33 (s, 1H), 11.04 (s, 1H), 12.33 (brs, 1H);
ESI-TOF-HRMS (M-H)- calculated for C17H17N6O4 369.1306; found 369.1303.
4.1.1.23. N6-[2-(6-hydroxyamino-6-oxohexyloxy)benzoyl]adenine (5c)
White solid (30 %): m.p. 193.0-194.0 ºC; 1H-NMR (500 MHz, DMSO-d6) δ 1.44-1.58
(m, 4H), 1.83-1.89 (m, 2H), 1.92 (t, J=6.8 Hz, 2H), 4.21 (t, J=6.3 Hz, 2H), 7.14 (dd, J=6.9,
7.5 Hz, 1H), 7.26 (d, J=8.6 Hz, 1H), 7.59 (dd, J=7.7, 7.7 Hz, 1H), 7.99 (d, J=8.0 Hz, 1H),
8.45 (s, 1H), 8.65 (brs, 2H), 10.31 (s, 1H), 11.02 (s, 1H), 12.34 (brs, 1H);
ESI-TOF-HRMS (M-H)- calculated for C18H19N6O4 383.1462; found 383.1463.
4.1.1.24. N6-[2-(7-hydroxyamino-7-oxoheptyloxy)benzoyl]adenine (5d)

White solid (36 %): m.p. 193.0-195.0 ºC; 1H-NMR (500 MHz, DMSO-d6) δ 1.26-1.32
(m, 2H), 1.44-1.51 (m, 4H), 1.83-1.86 (m, 2H), 1.89 (t, J=7.5 Hz, 2H), 4.22 (t, J=6.3 Hz,
2H), 7.14 (dd, J=8.0, 8.0 Hz, 1H), 7.26 (d, J=8.6 Hz, 1H), 7.60 (dd, J=7.7, 7.8 Hz, 1H),
8.00 (d, J=8.1 Hz, 1H), 8.45 (s, 1H), 8.65 (s, 1H), 10.29 (brs, 1H), 11.01 (brs, 1H), 12.38
(brs, 1H); ESI-TOF-HRMS (M+Na)+ calculated for C19H22N6O4Na 421.1595; found
421.1614.
4.1.1.25. N6-[2-(8-hydroxyamino-8-oxooctyloxy)benzoyl]adenine (5e)
White solid (64 %): m.p. 155.0-157.0 ºC; 1H-NMR (500 MHz, CD3OD) δ 1.28-1.35 (m,
2H), 1.41-1.47 (m, 2H), 1.56-1.64 (m, 4H), 1.97-2.01 (m, 2H), 2.04 (t, J=7.5 Hz, 2H),
4.29 (t, J=6.3 Hz, 2H), 7.14 (dd, J=7.4, 8.1 Hz, 1H), 7.23 (d, J=8.6 Hz, 1H), 7.60 (dd,
J=7.8, 8.0 Hz, 1H), 8.21 (d, J=8.0 Hz, 1H), 8.49 (s, 1H), 8.64 (s, 1H); ESI-TOF-HRMS
(M-H)- calculated for C20H23N6O4 411.1775; found 411.1752.
4.2 BRD4 bromodomain1 TR-FRET assay
BRD4-inhibitory activity was evaluated by means of europium-based LANCE TR-FRET
(time-resolved fluorescence resonance energy transfer) assay using a BRD4
bromodomain 1 TR-FRET assay kit (No. 600520, Cayman). (+)-JQ1 (No. 11187,
Cayman) was used as a positive control. Plates were read in the time-resolved format with
excitation at 320 nm and emission at 615 and 665 nm, using a 100 ꢀs delay and a 400 ꢀs
reading window on an Envision (PerkinElmer). Data analysis was performed using the
TR-FRET ratio (665 nm emission/615 nm emission).
4.3 Fluor de Lys®-Green HDAC Assay
HDAC-inhibitory activity was evaluated using a Fluor de Lys®-Green HDAC assay kit
(BML-AK530, Enzo Life Sciences). SAHA (No. 10009929, Cayman) was used as a
positive control. Plates were read with excitation at 485 nm and emission at 535 nm on an
Envision (PerkinElmer).
4.4 Cell culture
HL-60 cells, K-562 cells, OVCAR-5 cells, A2780 cells, and T-47D cells were cultured in

RPMI 1640 supplemented with 10 % FBS and penicillin/streptomycin mixture, and
MCF-7 cells were cultured in D-MEM (High Glucose) supplemented with 10 % FBS and
penicillin/streptomycin mixture at 37 °C in a humidified incubator (5 % CO2 in air).
4.5 HL-60 cells or K-562 cells growth-inhibiting activity assay
HL-60 cells or K-562 cells were seeded at a density of 1×105 cells/mL in 24-well plates
and treated with test compounds or DMSO. After incubation for 3 days, the living cells
were counted.
4.6 Annexin V assay
HL-60 cells were seeded at a density of 4×105 cells/mL (0 h or 24 h), or 1×105 cells/mL
(48 h) in 24-well plates and treated with test compounds or DMSO. After incubation for 0
h, 24 h, or 48 h, the cells were suspended in 1×binding buffer [Annexin V-FITC
Apoptosis Detection Kit, BioVision (No. K101-100)] at a density of 1×106 cells/mL, and
then Annexin V-FITC and PI were added according to the supplier’s protocol. After
incubation for 5 min at room temperature, the cells were analyzed by flow cytometry
using BD FACSCantoTMII.
4.7 Western blot analysis
Cell lysates were boiled in SDS sample buffer at 100 °C for 5 min and then cooled on ice
for 10 min, and subjected to SDS-PAGE using SuperSepTM Ace 5-12% or 10-20%. The
proteins were transferred to PVDF membrane. After blocking with 5% skim milk, the
membrane was probed with the primary antibody (anti-PARP, anti-caspase 3, or
anti-α-tubulin) and washed three times with TBS-T buffer. Then, the membrane was
probed with appropriate secondary antibody (anti-rabbit or anti-mouse) and washed three
times with TBS-T buffer. The immunoblots were visualized by enhanced
chemiluminescence with ImmobilonTM Western Chemiluminescent HRP substrate.
4.8 HL-60 cell differentiation-inducing activity assay
HL-60 cells were seeded at a density of 1×105 cells/mL in 24-well plates and treated with
test compounds or DMSO. After incubation for 1 h, the cells were treated with ATRA or
DMSO and incubated for 3 days. PBS containing 0.2 % NBT and 20 nM TPA in a 1:1

(v/v) ratio was added, and incubation was continued at 37 °C for 30 min. NBT-positive
cells were counted. After Wright-Giemsa staining, cell differentiation was
morphologically evaluated by microscopy.
4.9 c-MYC production-inhibiting activity assay
HL-60 cells were seeded at a density of 5×105 cells/mL in 60 mm dishes and treated with
test compounds or DMSO. After incubation for 24 h, the cells were lysed with lysis buffer
at a density of 4×106 cells/mL and sonicated using a Bioruptor®, Diagnode. The c-MYC
levels were measured by using Human c-MYC (Total) ELISA kit (No. KH02041, Enzo
Life Technologies) according to the supplier’s protocol.
4.10 WST-1 assay
OVCAR-5 cells or T-47D cells were seeded at a density of 5×104 cells/mL, and A2780
cells or MCF-7 cells were seeded at a density of 2×104 cells/mL respectively. After
incubation for 1 day, the cells were treated with test compounds or DMSO. After
incubation for 3 days, cell growth-inhibitory activity was determined by using WST-1
[Cell Counting Kit, Dojindo (No. CK01)], according to the supplier’s protocol.
Acknowledgements
The work described in this paper was partially supported by Grants-in-Aid for Scientific
Research from The Ministry of Education, Culture, Sports, Science and Technology,
Japan, and a grant from The Japan Society for the Promotion of Science. We are grateful
to Dr. Yuki Okada for the sonication device, Bioruptor®, and to Dr. Yutaka Enomoto,
and Dr. Eiko Saijyo-Oikawa for their help with flow-cytometric detection.
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