Discovery and structure-activity relationship studies of N6-benzoyladenine derivatives as novel BRD4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.01.022

Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that bind to acetylated lysines in histones. Among them, BRD4 is a candidate target molecule of therapeutic agents for diverse diseases, including cancer and inflammatory disease.

Full text of DOI:10.1016/j.bmc.2015.01.022

Bioorganic & Medicinal Chemistry 23 (2015) 953–959
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structure–activity relationship studies of
N6-benzoyladenine derivatives as novel BRD4 inhibitors
⇑
Tomomi Noguchi-Yachide , Taki Sakai, Yuichi Hashimoto, Takao Yamaguchi
Institute of Molecular and Cellular, Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that bind to acetylated
lysines in histones. Among them, BRD4 is a candidate target molecule of therapeutic agents for diverse
diseases, including cancer and inflammatory disease. As a part of our continuing structural development
studies of thalidomide to obtain a broad spectrum of biological modifiers based on the ‘multi-template’
approach, in this work we focused on BRD4-inhibitory activity, and discovered that N6-benzoyladenine
derivatives exhibit this activity. Structure–activity relationship studies led to N6-(2,4,5-trim-
ethoxybenzoyl)adenine (29), which exhibits potent BRD4 bromodomain1 inhibitory activity with an
Received 19 December 2014
Revised 14 January 2015
Accepted 14 January 2015
Available online 28 January 2015
Keywords:
BRD4
Bromodomain
Benzoyladenine
Structure–activity relationship
IC50 value of 0.427
BRD4 inhibitors.
l
M. N6-Benzoyladenine appears to be a new chemical scaffold for development of
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
positive transcription elongation factor b (P-TEFb), thereby stimu-
lating G1 gene transcription and promoting cell cycle progression
Bromodomains target epigenetic alterations, specifically,
-acetyl-lysine residues in histones, and serve as regulators of tran-
to S phase.5,6 These activities have potential value for the treat-
ment of cancer. BRD4 inhibitors also have potential as anti-inflam-
matory agents.4 BRD4 is a coactivator for transcriptional activation
e
scriptional activity and chromatin remodeling.1 The human gen-
ome encodes 61 bromodomains in 46 diverse proteins, including
histone acetyltransferases (HATs) and chromatin remodeling fac-
tors.2 These bromodomains have been classified into eight major
families based on structure and sequence-based identity.2 The
bromodomain and extra-terminal domain (BET) protein family,
belonging to Family II, consists of four members (BRD2, BRD3,
BRD4 and BRDT), and they control expression of genes related to
regulation of various physiological functions, including inflamma-
tion, apoptosis, cell proliferation, cell cycle, pancreatic b cell func-
tion, and adipogenesis.3–8 Therefore, BET family members have
been receiving increasing attention as candidate therapeutic
targets for inflammatory diseases, cancer, diabetes, obesity, cardio-
vascular diseases and Alzheimer’s disease.9–13 Selective BET inhib-
itors I-BET762 and RVX-208 (Fig. 1) are already under clinical trial
for treatment of nuclear protein in testis (NUT) midline carcinoma
(NMC) and atherosclerosis, respectively.14,15
of NF-
lysine of RelA, one of the subunits of NF-
plex, and enhances the RNA polymerase II-mediated expression
of NF-
B-dependent inflammatory genes, including TNF-a 5,20
j
B, which mediates inflammation, via binding to acetyl-
jB transcriptional com-
j
.
Thus, there is great interest in discovering new structural scaf-
folds for BRD4 inhibitors. In this connection, we have proposed the
‘multi-template approach’ to develop compounds with diverse bio-
logical activities by structural development of thalidomide,21 and
we have developed a range of biological modifiers, including
anti-angiogenic agents, cyclooxygenase (COX) inhibitors and
nuclear receptor ligands.22–26 Thalidomide is an immunomodula-
tory and anti-inflammatory agent that was withdrawn from the
market in the 1960s due to serious teratogenicity,21,27 but subse-
quently re-introduced as a therapeutic agent for multiple myeloma
and complications of leprosy, though its action mechanism
remains unclear.27,28 Recent research indicates that thalidomide
is a multi-target agent, and might be a lead compound for anti-
inflammatory and/or anti-cancer agents. Therefore, we focused
here on the bromodomain as a potential new target. Initial screen-
ing of thalidomide and its derivatives revealed that N6-benzoylad-
enine showed the desired activity. Subsequent structure–activity
studies led us to N6-(2,4,5-trimethoxybenzoyl)adenine (29) as a
potent BRD4 bromodomain1 inhibitor. Our findings indicate that
Among the BET family, BRD4 binds to acetyl-lysine residues in
the tail of histones H3 and H4.2,16 It induces increased expression
of MYC target genes and has been reported to promote transcrip-
tion of the c-MYC oncogene itself.10,17–19 In addition, BRD4 recruits
⇑
Corresponding author. Tel.: +81 3 5841 7875; fax: +81 3 5841 8495.
E-mail address: noguchi@iam.u-tokyo.ac.jp (T. Noguchi-Yachide).
http://dx.doi.org/10.1016/j.bmc.2015.01.022
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

954
T. Noguchi-Yachide et al. / Bioorg. Med. Chem. 23 (2015) 953–959
Figure 1. BET bromodomain inhibitors.
N6-benzoyladenine is a promising chemical scaffold for developing
novel BRD4 inhibitors with smaller molecular weight than previ-
ously reported typical BET inhibitors.
Table 1
BRD4-inhibitory activity of N6-(2-substituted benzoyl)adenines
2. Results and discussion
We investigated the inhibitory effects of our compounds on
BRD4 bromodomain1 using a commercially available assay kit
(BRD4 bromodomain 1 TR-FRET assay kit, Cayman). BRD4-inhibi-
tory activity of test compounds was expressed as IC50 values,
which were estimated from the sigmoidal dose–response curves
using R software and Origin software (Fig. 2 and Tables 1–3;
Compound
R
IC50 (lM) or
(): inhibitory rate at 100 lM
(+)-JQ1
I-BET151
RVX208
—
—
—
0.008
0.107
0.645
‘N.A.’ means that no activity was observed at 100
pounds that did not achieve 50% inhibition at 300
bition at 100 M is shown in parenthesis).
l
M; for com-
lM, the % inhi-
N-Benzoyladenine (4)
H
34.2
5.60
16.0
20.0
29.7
(40.4%)
(39.0%)
13
14
15
16
17
18
NMe2
OMe
OnPr
SMe
Me
l
First, we evaluated the BRD4-inhibitory activities of thalido-
mide (1) and three analogs (2–4) from our chemical library.
Compounds 2 and 3 are phthalimide derivatives containing an
acetyl-lysine bioisostere instead of the glutarimide moiety of tha-
lidomide (1). Compound 4 can be regarded as a ring-opened analog
of N-heteroaromatic phthalimide structure. Although thalidomide
(1) and its derivatives (2, 3) were inactive, N6-benzoyladenine
Br
100 lM, that is, all of them showed weaker activity than N6-ben-
(4) showed moderate activity with an IC50 value of 34.2 lM
zoyladenine (4). The decreased activity of N6-benzoyl-9-methylad-
enine (7) might indicate importance of the 9NH group, which could
act as a hydrogen bond donor. Of course, various tautomers of N6-
benzoyladenine (4) may exist, so it is possible that any nitrogen
atom in the adenine moiety could be both a hydrogen bond donor
and a hydrogen bond acceptor. Decreased activity of 4-(benzoyla-
mino)benzimidazole (8) suggested that one or both of the 1,3-dini-
trogen atoms of the adenine skeleton might be important as a
hydrogen bond acceptor(s). Weaker activity of 1-benzoyl-4-(ben-
zoylamino)benzimidazole (9) could be attributed to substitution
of the 9NH group with the 9-benzoyl group and the absence of
nitrogen atoms at the 1- and 3-positions. However, it is
(Table 1). Next, we examined the inhibitory activities of N6-benzy-
ladenine (5) and its analog trans-zeatin (6) to investigate the struc-
tural requirements for BRD4-inhibitory activity, but both of them
were inactive, suggesting that the benzoyl moiety (including the
carbonyl group) is essential for the activity. As discussed later,
the basicity (or hydrogen bond acceptor ability) of the carbonyl
oxygen in the benzoyl moiety, as well as its planar structure, might
be important for the activity.
Next, we evaluated the activity of six benzoylated heteroaro-
matics, 7–12, to investigate the structural requirements of the ade-
nine (heteroaromatic) moiety of N6-benzoyladenine (4). As shown
in Figure 3, none of compounds 7–12 achieved 50% inhibition at
Figure 2. BRD4-inhibitory activity of thalidomide and its analogs.

T. Noguchi-Yachide et al. / Bioorg. Med. Chem. 23 (2015) 953–959
955
Table 2
Next, we investigated the substituent effect at the benzoyl
group. The activities of N6-(2-substituted benzoyl)adenines (13–
18) are shown in Table 1. The BRD4-inhibitory activity of the com-
pounds decreased in the following order: NMe2 (13) > OMe
(14) > OnPr (15) > SMe (16) > Me (17) > Br (18). Compounds 13–
16 showed more potent activity than N6-benzoyladenine (4). On
the other hand, N6-(2-methylbenzoyl)adenine (17) and N6-(2-bro-
mobenzoyl)adenine (18) showed weaker activity than N6-benzoy-
ladenine (4). A possible explanation of these results is that (i) a
substituent with hydrogen bond acceptor ability at the 2-position
of the benzoyl group, and/or (ii) enhancement of basicity (or
hydrogen bond acceptor ability) of the carbonyl oxygen in the ben-
zoyl moiety contribute(s) to the inhibitory activity.
Effect of substitution site on BRD4-inhibitory activity
Compound
R1
R2
R3
IC50 (lM) or
(): inhibitory rate at 100 lM
(+)-JQ1
I-BET151
RVX208
—
—
—
—
—
—
—
—
—
0.008
0.107
0.645
For further structure–activity relationship studies, we investi-
gated the positional effect of substitution on the benzoyl moiety.
In accordance with the case of 2-substitution (vide supra, com-
pounds 14 and 18, Table 1), bromo-substituted compounds (20,
22) were less potent inhibitors than N6-benzoyladenine (4), while
methoxy-substituted compounds (19, 21) showed moderate activ-
ities (Table 2). The enhancing effect of a methoxy substituent on
the BRD4-inhibitory activity decreased in the order of: ortho-
N-Benzoyladenine (4)
H
H
H
34.2
14
18
OMe
Br
H
H
H
H
16.0
(39.0%)
19
20
H
H
OMe
Br
H
H
63.0
(44.5%)
21
22
H
H
H
H
OMe 34.5
Br (36.7%)
(14: IC50 value of 16.0
lM) > para- (21: IC50 value of
34.5 M) > meta- (19: IC50 value of 63.0
l
l
M). The observed prefer-
Table 3
Effect of multiple substitution on BRD4-inhibitory activity
ence for ortho/para-methoxy substitution over meta-methoxy or
bromo substitution implies the importance of the basicity (or
hydrogen bond acceptor character) of the carbonyl oxygen in the
benzoyl moiety for the activity (vide supra). This interpretation
prompted us to investigate the efficacy of multiple substitution
(Table 3).
Thus, di- and tri-methoxy derivatives 23–29 were prepared and
evaluated. Among compounds 23–26, which possess an additional
methoxy substituent on N6-(2-methoxybenzoyl)adenine (14), only
N6-(2,5-dimethxybenzoyl)adenine (25) is more potent than N6-(2-
Compound
R
IC50 (lM) or
(): inhibitory rate at 100 lM
methoxybenzoyl)adenine (14), having an IC50 value of 7.2 lM
(Table 3). The other three derivatives were less potent than 14,
and their activity decreased in the following order: 23 (2,3-dime-
(+)-JQ1
I-BET151
RVX208
—
—
—
0.008
0.107
0.645
thoxy) > 24
(2,4-dimethoxy) > 26
(2,6-dimethoxy).
Thus,
N-Benzoyladenine (4)
H
34.2
introduction of a methoxy group at the p-position regarding to
the 2-position, that is, the 5-position, is the most effective, imply-
ing a role of the basicity (or electron-donating nature) of the oxy-
gen atom in the 2-methoxy group.
Other dimethoxy derivatives with different substitution pat-
terns, that is, compounds 27, 28, were also prepared and evaluated.
N6-(3,4-Dimethoxybenzoyl)adenine (27) was more potent than
N6-(2-methoxybenzoyl)adenine (14), having an IC50 value of
23
24
25
26
27
28
2,3-OMe
2,4-OMe
2,5-OMe
2,6-OMe
3,4-OMe
3,5-OMe
78.6
(40.8%)
7.20
(11.5%)
7.80
17.9
29
2,4,5-OMe
0.427
7.8
l
M. N6-(3,5-Dimethoxybenzoyl)adenine (28) was also moder-
M), having a potency comparable
ately active (IC50 value of 17.9
l
noteworthy that even simple analogs, 10–12, retained weak BRD4-
inhibitory activity (Fig. 3).
to that of N6-(2-methoxybenzoyl)adenine (14) and superior to that
of N6-benzoyladenine (4).
Figure 3. BRD4-inhibitory activity (%) of N6-benzoyladenine derivatives at 100 lM.

956
T. Noguchi-Yachide et al. / Bioorg. Med. Chem. 23 (2015) 953–959
Figure 4. Graphical illustration30 of the structure–activity relationship for methoxy-substituted derivatives.
The results indicate that 2,5-dimethoxy and 3,4-dimethoxy sub-
tetramethylsilane (0.00 ppm) or CDCl3 (77.16 ppm) for 13C NMR.
Data are reported as follows: chemical shift, multiplicity (s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), coupling
constants (Hz), integration. Fast atom bombardment mass (FAB-
MS) spectra were recorded on a JEOL JMA-HX110 mass spectrom-
eter with m-nitrobenzyl alcohol as the matrix. High-resolution
mass spectrum was recorded using a Bruker micrOTOF II mass
spectrometer.
stitution patterns are preferred. The latter substitution pattern can
be considered as the same as 4,5-dimethoxy substitution, because
the 3- and 5-positions are both meta to the N-carbonyladenyl moi-
ety. Therefore, we next focused on the 2,4,5-trimethoxy derivative
(Fig. 4). The structure–activity relationships for methoxy-substi-
tuted derivatives (14, 19, 21, 23–29) are graphically illustrated in
Figure 4, based on the method similar with that of reported by
Zhang et al.30 In Figure 4, nodes represent substitution sites or site
combinations that are scaled in size/color density according to the
potency of BRD4-inhibitory activity. As expected, N6-(2,4,5-trim-
ethoxybenzoyl)adenine (29) was the most potent inhibitor (IC50
4.1.1. N6-Benzoyl-9-methyladenine (7)
Sodium hydride (60 wt % in oil, 24 mg, 0.75 mmol) was added to
a solution of N6-benzoyladenine (150 mg, 0.63 mmol) in anhydrous
DMF (2.0 mL), and the mixture was stirred for 5 min at room tem-
value of 0.427 lM) among the N6-benzoyladenine derivatives
prepared in this paper (7–29), being 80-fold more potent than
N6-benzoyladenine (4). Its potency is comparable to those of the
reported inhibitors I-BET151 and RVX208 (Fig. 1).10,29
perature. Iodomethane (47 lL, 0.75 mmol) was added, and the
resulting suspension was stirred for 14 h at room temperature.
Brine was added, and the resulting mixture was extracted with
AcOEt. The combined organic layer was dried over Na2SO4 and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography (MeOH/CHCl3, 1:40) to afford the
product 7 (84 mg, 53%) as a white solid. The same compound was
also alternatively synthesized by benzoylation of 9-methylade-
nine.31 Mp 78.5–80.5 °C; 1H NMR (500 MHz, CDCl3) d 3.84 (s, 3H),
7.42 (dd, J = 7.8, 7.8 Hz, 2H), 7.51 (tt, J = 1.3, 7.5 Hz, 1H), 7.93 (s,
1H), 7.97 (d, J = 7.5 Hz, 2H), 8.71 (s, 1H), 9.52 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 30.0, 122.9, 128.0, 128.7, 132.7, 133.7, 143.6,
149.5, 152.4, 152.6, 165.0; FAB-MS m/z 254 (M+H)+.
3. Conclusions
Our continuing investigations of thalidomide derivatives based
on the ‘multi-template approach’ led to the discovery that N6-
benzoyladenine derivatives exhibit BRD4-inhibitory activity.
Structure–activity relationship studies led to the identification of
N6-(2,4,5-trimethoxybenzoyl)adenine (29) as
a potent small-
molecular BRD4 inhibitor. N6-Benzoyladenine appears to be a
new chemical scaffold for development of BRD4 inhibitors.
4.1.2. 4-(Benzoylamino)benzimidazole (8) and 1-Benzoyl-4-
(benzoylamino)benzimidazole (9)
4. Experimental section
4.1. General chemistry
Benzoyl chloride (87 lL, 0.75 mmol) was added to a solution of
4-amino-1,3-benzodiazole32 (100 mg, 0.75 mmol) in anhydrous
pyridine (1.0 mL) at 0 °C, and the mixture was stirred for 12 h
at room temperature. After removal of the solvent, the residue
was purified by silica gel column chromatography (MeOH/CHCl3,
1:20 to 1:10) to afford the products 8 (28 mg, 16%) and 9 (46 mg,
18%), each as a white solid. Compound 8: mp 183–185 °C; 1H
NMR (500 MHz, CDCl3/CD3OD, 99:1) d 7.20 (dd, J = 8.0, 8.0 Hz,
1H), 7.26 (d, J = 8.0 Hz, 1H), 7.43 (dd, J = 7.5, 7.5 Hz, 2H), 7.50 (t,
J = 7.0 Hz, 1H), 7.88 (s, 1H), 7.98 (d, J = 7.5 Hz, 2H), 8.03 (d,
J = 7.5 Hz, 1H), 9.51 (br s, 1H); 13C NMR (125 MHz, CDCl3/CD3OD,
99:1) d 109.5, 113.1, 123.6, 127.4, 127.9, 128.7, 131.5, 132.0,
134.5, 135.7, 139.9, 166.8; FAB-MS m/z 238 (M+H)+. Compound
9: mp 152–154 °C; 1H NMR (500 MHz, CDCl3) d 7.46 (dd, J = 8.0,
8.0 Hz, 1H), 7.51 (dd, J = 7.5, 7.5 Hz, 2H), 7.55–7.62 (m, 3H),
7.70 (t, J = 7.5 Hz, 1H), 7.79–7.83 (m, 3H), 8.00 (d, J = 8.5 Hz,
2H), 8.16 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 9.13 (br s, 1H); 13C
All chemical reagents and solvents were purchased from
Sigma–Aldrich Co., LLC, Kanto Chemical Co., Inc., Tokyo Chemical
Industry Co., Ltd and Wako Pure Chemical Industries, Ltd, and used
without further purification. Moisture-sensitive reactions were
performed under an atmosphere of argon, unless otherwise noted,
and monitored by thin-layer chromatography (TLC, Merck silica gel
60 F254 plates). Bands were visualized using UV light or by applica-
tion of appropriate reagents followed by heating. Flash chromatog-
raphy was carried out with silica gel (Silica gel 60N, 40–50 lm
particle size) purchased from Kanto Chemical Co., Inc. Melting
points (Mp) were determined by using a MP-J3 melting point appa-
ratus (Yanaco). NMR spectra were recorded on a JEOL JNM-GX500
(500 MHz) spectrometer, operating at 500 MHz for 1H NMR and at
125 MHz for 13C NMR. Proton and carbon chemical shifts are
expressed in d values (ppm) relative to internal tetramethylsilane
(0.00 ppm) or residual CHCl3 (7.26 ppm) for 1H NMR, and internal
NMR (125 MHz, CDCl3)
d 110.7, 114.6, 127.0, 127.3, 128.9,

T. Noguchi-Yachide et al. / Bioorg. Med. Chem. 23 (2015) 953–959
957
129.2, 129.7, 130.4, 132.2, 132.2, 132.8, 133.5, 134.4, 134.7, 141.8,
4.1.4.5. N6-(4-Methoxybenzoyl)adenine (21).
Pale yellow
165.7, 167.1; FAB-MS m/z 342 (M+H)+.
solid (42%): mp 233–236 °C; 1H NMR (500 MHz, CDCl3/CD3OD,
100:1) d 3.93 (s, 3H), 7.06 (d, J = 8.6 Hz, 2H), 7.33 (s, 1H), 8.06 (d,
J = 8.6 Hz, 2H), 8.38 (s, 1H), 8.74 (s, 1H); FAB-MS m/z 270 (M+H)+.
4.1.3. 4-(Benzoylamino)pyrimidine (11) and 4-
(Dibenzoylamino)pyrimidine (12)
Compound 11 was prepared by a modification of a literature
4.1.4.6. N6-(4-Bromobenzoyl)adenine (22).
Brown solid
method.33 Triethylamine (220
lL, 1.6 mmol) and benzoyl chloride
(42%): mp >300 °C (reached maximum measurement limit); 1H
NMR (500 MHz, DMSO-d6) d 7.78 (d, J = 8.3 Hz, 2H), 8.03 (d,
J = 8.3 Hz, 2H), 8.49 (s, 1H), 8.72 (s, 1H); FAB-MS m/z 318 (M+H)+.
(122
lL, 1.1 mmol) were added to a solution of 4-aminopyrimidine
(50 mg, 0.53 mmol) in anhydrous DMF (0.5 mL), and the mixture
was stirred for 6 h at room temperature. The precipitate was col-
lected by filtration and washed with hexane, and the filtrate was
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (AcOEt/hexane, 1:3 to 1:2)
to afford the products 11 (54 mg, 51%) and 12 (25 mg, 16%), each
as a white solid. Compound 11: mp 135.5–137 °C; 1H NMR
4.1.4.7. N6-(2,6-Dimethoxybenzoyl)adenine (26).
White
solid (1%): mp 204–206 °C; 1H NMR (500 MHz, CDCl3) d 3.81 (s,
6H), 6.69 (d, J = 8.6 Hz, 2H), 7.51 (t, J = 8.6 Hz, 1H), 7.99 (s, 1H),
8.54 (s, 1H); FAB-MS m/z 300 (M+H)+.
(500 MHz, CDCl3)
d
7.47 (dd, J = 8.8, 8.8 Hz, 2H), 7.57 (t,
4.1.4.8. 2-(Methylthio)benzoic acid.
This compound was
J = 7.5 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 8.32 (dd, J = 1.5, 6.0 Hz,
1H), 8.61 (d, J = 5.5 Hz, 1H), 8.66 (d, J = 1.0 Hz, 1H), 9.40 (br s,
1H); 13C NMR (125 MHz, CDCl3) d110.6, 127.6, 129.0, 133.0,
133.5, 157.6, 158.4, 158.6, 166.8; FAB-MS m/z 200 (M+H)+. Com-
pound 12: mp 132.5–133.5 °C; 1H NMR (500 MHz, CDCl3) d 7.37
(dd, J = 7.8, 7.8 Hz, 4H), 7.42 (dd, J = 1.5, 5.5 Hz, 1H), 7.50 (tt,
J = 1.3, 7.5 Hz, 2H), 7.75 (dd, J = 1.4, 8.3 Hz, 4H), 8.73 (d,
J = 6.0 Hz, 1H), 8.90 (d, J = 1.0 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 116.9, 129.0, 129.4, 133.3, 134.1, 158.2, 158.9, 160.7, 172.4;
FAB-MS m/z 304 (M+H)+.
prepared by means of a literature method.34 Thiosalicylic acid
(300 mg, 2.0 mmol) and K2CO3 (809 mg, 5.9 mmol) were dissolved
in anhydrous acetone (20 mL). Iodomethane (134 lL, 2.2 mmol)
was slowly added to the solution, and the mixture was stirred
for 20 h. The solvent was removed and the residue was taken up
in water and acidified to pH ꢀ1 with 2 N HCl. The precipitated
white solid was washed with H2O and dried to afford the product
(260 mg, 79%) as a white solid. 1H NMR (500 MHz, DMSO-d6) d 2.38
(s, 3H), 7.19 (dd, J = 7.4, 7.4 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.52–
7.55 (m, 1H), 7.89 (dd, J = 1.4, 7.7 Hz, 1H); FAB-MS m/z 168 (M)+.
4.1.4. General synthetic procedure (Procedure A) for N6-
benzoyladenine derivatives 14, 18–22 and 26
4.1.5. General synthetic procedure (Procedure B) for N6-
benzoyladenine derivatives 13, 16, 23–25, and 28
N,N-Diisopropylethylamine (5.0 equiv) or triethylamine
(5.0 equiv) was added to a suspension of adenine (1.0 equiv) in
anhydrous DMF (0.4 M). To this mixture was added the appropri-
ate benzoyl chloride (1.2 equiv), and the resulting mixture was
stirred at 110–130 °C. After completion of the reaction, the mixture
was diluted with water and extracted with AcOEt. The combined
organic layer was washed with brine and dried over Na2SO4. After
removal of the solvent, the residue was purified by recrystalliza-
tion (MeOH/CH2Cl2) or by silica gel column chromatography
(MeOH/CHCl3) to afford the product. Compound 20 was purified
as follows: after completion of the reaction, the mixture was
diluted with water, and the precipitate was collected by filtration
and washed with water and AcOEt to afford the product.
The appropriate benzoic acid (1.2 equiv), 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (1.2 equiv) and
4-(dimethylamino)pyridine (1.2 equiv) were added to a suspension
of adenine (1.0 equiv) in anhydrous DMF (0.6 M), and the mixture
was stirred for 10 h at 130 °C. The resulting mixture was diluted
with water and extracted with AcOEt. The combined organic layer
was washed with brine and dried over Na2SO4. After removal of the
solvent, the residue was purified by silica gel column chromatogra-
phy (MeOH/CHCl3) to afford the product.
4.1.5.1. N6-[2-(Dimethylamino)benzoyl]adenine (13). White
solid (12%): mp 220–222 °C; 1H NMR (500 MHz, DMSO-d6) d 2.82
(s, 6H), 7.29 (dd, J = 7.4, 7.4 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.60–
7.63 (m, 1H), 8.04 (dd, J = 1.7, 7.4 Hz, 1H), 8.46 (s, 1H), 8.66 (s, 1H);
FAB-MS m/z 283 (M+H)+.
4.1.4.1. N6-(2-Methoxybenzoyl)adenine (14).
Pale yellow
solid (22%): mp 235–237 °C; 1H NMR (500 MHz, DMSO-d6) d 4.08
(s, 3H), 7.23 (dd, J = 7.5, 7.5 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.69–
7.72 (m, 1H), 8.01 (dd, J = 1.7, 8.0 Hz, 1H), 8.55 (s, 1H), 8.74 (s,
1H), 11.07 (br s, 1H), 12.45 (br s, 1H); FAB-MS m/z 270 (M+H)+.
4.1.5.2. N6-[2-(Methylthio)benzoyl]adenine (16).
Pale yel-
low solid (12%): mp 127–130 °C; 1H NMR (500 MHz, CDCl3) d
2.56 (s, 3H), 7.32–7.35 (m, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.56 (ddd,
J = 1.5, 7.7, 7.7 Hz, 1H), 7.82 (dd, J = 1.5, 7.7 Hz, 1H), 8.36 (s, 1H),
8.73 (s, 1H), 9.62 (br s, 1H), 11.62 (br s, 1H); FAB-MS m/z 286
(M+H)+.
4.1.4.2. N6-(2-Bromobenzoyl)adenine (18).
Brown solid
(42%): mp 223–225 °C; 1H NMR (500 MHz, CDCl3/CD3OD, 100:1)
d 7.43–7.51 (m, 2H), 7.69 (d, J = 7.4 Hz, 1H), 7.73 (d, J = 7.4 Hz,
1H), 8.39 (s, 1H), 8.75 (s, 1H); FAB-MS m/z 318 (M+H)+.
4.1.5.3. N6-(2,3-Dimethoxybenzoyl)adenine (23).
White
solid (25%): mp 208–211 °C; 1H NMR (500 MHz, DMSO-d6) d 3.88
(s, 3H), 3.90 (s, 3H), 7.25 (dd, J = 8.0, 8.0 Hz, 1H), 7.32 (dd, J = 1.7,
8.0 Hz, 1H), 7.39 (d, J = 6.9 Hz, 1H), 8.48 (s, 1H), 8.68 (s, 1H),
11.24 (br s, 1H), 12.35 (br s, 1H); FAB-MS m/z 300 (M+H)+.
4.1.4.3. N6-(3-Methoxybenzoyl)adenine (19).
Brown solid
(45%): mp 237–240 °C; 1H NMR (500 MHz, DMSO-d6) d 3.85 (s,
3H), 7.22 (dd, J = 2.3, 8.0 Hz, 1H), 7.47 (dd, J = 7.7, 7.7 Hz, 1H),
7.68–7.70 (m, 2H), 8.48 (s, 1H), 8.72 (s, 1H); FAB-MS m/z 270
(M+H)+.
4.1.5.4. N6-(2,4-Dimethoxybenzoyl)adenine (24).
White
solid (1.5%): mp 267–270 °C; 1H NMR (500 MHz, CDCl3) d 3.92 (s,
3H), 4.14 (s, 3H), 6.60 (d, J = 2.3 Hz, 1H), 6.71 (dd, J = 2.3, 8.6 Hz,
1H), 8.26 (d, J = 8.6 Hz, 1H), 8.30 (d, J = 1.1 Hz, 1H), 8.80 (s, 1H),
10.75 (br s, 1H), 11.81 (br s, 1H); FAB-MS m/z 300 (M+H)+.
4.1.4.4. N6-(3-Bromobenzoyl)adenine (20).
White solid
(44%): mp >300 °C (reached maximum measurement limit); 1H
NMR (500 MHz, DMSO-d6) d 7.53 (dd, J = 8.0, 8.0 Hz, 1H), 7.86 (d,
J = 8.0 Hz, 1H), 8.08 (d, J = 7.4 Hz, 1H), 8.51 (s, 1H), 8.28 (s, 1H),
8.73 (s, 1H), 11.66 (br s, 1H), 12.38 (br s, 1H); FAB-MS m/z 318
(M+H)+.
4.1.5.5. N6-(2,5-Dimethoxybenzoyl)adenine (25).
White
solid (21%): mp 213–216 °C; 1H NMR (500 MHz, CDCl3) d 3.88 (s,

958
T. Noguchi-Yachide et al. / Bioorg. Med. Chem. 23 (2015) 953–959
3H), 4.13 (s, 3H), 7.06 (d, J = 8.8 Hz, 1H), 7.18 (dd, J = 3.1, 8.8 Hz,
1H), 7.81 (d, J = 3.1 Hz, 1H), 8.32 (d, J = 1.1 Hz, 1H), 8.82 (s, 1H),
11.01 (br s, 1H), 11.76 (br s, 1H); FAB-MS m/z 300 (M+H)+.
format by exciting the test compounds at 320 nm and reading
emissions at 615 and 665 nm, using a 100 s delay and a 400
read window in Envision (PerkinElmer). Data analysis was per-
formed using the TR-FRET ratio (665 nm emission/615 nm
emission).
l
ls
4.1.5.6. N6-(3,5-Dimethoxybenzoyl)adenine (28).
White
solid (43%): mp 242–245 °C; 1H NMR (500 MHz, CDCl3) d 3.89 (s,
6H), 6.73 (t, J = 2.3 Hz, 1H), 7.09 (s, 1H), 7.10 (s, 1H), 8.36 (s, 1H),
8.81 (s, 1H), 9.01 (br s, 1H), 11.62 (br s, 1H); FAB-MS m/z 300
(M+H)+.
Acknowledgements
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and a grant
from The Japan Society for the Promotion of Science.
4.1.6. General synthetic procedure (Procedure C) for N6-
benzoyladenine derivatives 15, 17, 27, and 29
The appropriate benzoic acid (1.0 equiv) and 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (1.2 equiv)
were added to a suspension of adenine (1.0 equiv) in anhydrous
DMF (0.6 M), and the mixture was stirred at 100–130 °C. After
completion of the reaction, the solvent was removed under
reduced pressure. The residue was then purified by silica gel col-
umn chromatography (MeOH/CHCl3) and by washing with CH2Cl2/
hexane (1:1 or 1:0) to afford the product.
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White
solid (20%): mp 266.5–268 °C; 1H NMR (500 MHz, CDCl3/CD3OD,
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161.4, 164.4; FAB-MS m/z 298 (M+H)+.
4.1.6.2. N6-(2-Methylbenzoyl)adenine (17).
White solid
(15%): mp 197–197.5 °C; 1H NMR (500 MHz, CDCl3/CD3OD, 99:1)
d 2.54 (s, 3H), 7.26–7.33 (m, 2H), 7.44 (dd, J = 7.5, 7.5 Hz, 1H),
7.61 (dd, J = 1.0, 7.5 Hz, 1H), 8.34 (s, 1H), 8.48 (s, 1H), 9.93 (br s,
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4.1.6.3. N6-(3,4-Dimethoxybenzoyl)adenine (27).
White
solid (25%): mp 223.5–224.5 °C; 1H NMR (500 MHz, CDCl3) d 3.98
(s, 3H), 3.98 (s, 3H), 6.98 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 2.5 Hz,
1H), 7.62 (dd, J = 2.5, 8.5 Hz, 1H), 8.36 (s, 1H), 8.76 (s, 1H), 9.15
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White
solid (23%): mp 247.5–249.5 °C; 1H NMR (500 MHz, CDCl3) d 3.94
(s, 3H), 3.99 (s, 3H), 4.14 (s, 3H), 6.59 (s, 1H), 7.74 (s, 1H), 8.30
(s, 1H), 8.79 (s, 1H), 10.85 (br s, 1H), 11.80 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 56.5, 56.5, 57.2, 96.5, 110.7, 113.3, 113.8,
143.7, 143.9, 144.2, 152.6, 154.1, 154.7, 162.4, 164.1; FAB-MS m/
z 330 (M+H)+; HRMS (ESI) Calcd. For C15H15N5O4Na: 352.1016,
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