Design, synthesis, and biological activity of a novel series of human sirtuin-2-selective inhibitors

Article abstract of DOI:10.1021/jm3002108

Selective inhibitors of human sirtuin 2 (SIRT2), a deacetylase, are candidate therapeutic agents for neurodegenerative diseases such as Parkinson's disease and Huntington's disease as well as potential tools for elucidating the biological functions of SIRT2. On the basis of homology models of SIRT1 and SIRT2, we designed and prepared a series of 2-anilinobenzamide analogues. Enzyme assays using recombinant SIRT1 and SIRT2 revealed that 3'-phenethyloxy-2- anilinobenzamide analogues such as 33a and 33i are potent and selective SIRT2 inhibitors, showing more than 3.5-fold greater SIRT2-inhibitory activity and more than 35-fold greater SIRT2-selectivity compared with AGK2 (3), a previously reported SIRT2-selective inhibitor. Compound 33a also induced a dose-dependent selective increase of α-tubulin acetylation in human colon cancer HCT116 cells, indicating selective inhibition of SIRT2 in the cells. These 3'-phenethyloxy-2-anilinobenzamide derivatives represent an entry into a new class of SIRT2-selective inhibitors.

Full text of DOI:10.1021/jm3002108

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Activity of a Novel Series of Human
Sirtuin-2-Selective Inhibitors
Takayoshi Suzuki,*,†,‡ Mohammed Naseer Ahmed Khan,‡ Hideyuki Sawada,†,‡ Erika Imai,‡
Yukihiro Itoh,† Katsura Yamatsuta,§ Natsuko Tokuda,§ Jun Takeuchi,§ Takuya Seko,§
Hidehiko Nakagawa,‡ and Naoki Miyata*,‡
†Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 13 Taishogun Nishitakatsukasa-cho, Kita-ku, Kyoto
603-8334, Japan
‡Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
§Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai Shimamoto-cho, Mishima-gun, Osaka 618-8585, Japan
S
* Supporting Information
ABSTRACT: Selective inhibitors of human sirtuin 2 (SIRT2),
a deacetylase, are candidate therapeutic agents for neuro-
degenerative diseases such as Parkinson’s disease and
Huntington’s disease as well as potential tools for elucidating
the biological functions of SIRT2. On the basis of homology
models of SIRT1 and SIRT2, we designed and prepared a
series of 2-anilinobenzamide analogues. Enzyme assays using
recombinant SIRT1 and SIRT2 revealed that 3′-phenethyloxy-
2-anilinobenzamide analogues such as 33a and 33i are potent
and selective SIRT2 inhibitors, showing more than 3.5-fold
greater SIRT2-inhibitory activity and more than 35-fold
greater SIRT2-selectivity compared with AGK2 (3), a previously reported SIRT2-selective inhibitor. Compound 33a also
induced a dose-dependent selective increase of α-tubulin acetylation in human colon cancer HCT116 cells, indicating selective
inhibition of SIRT2 in the cells. These 3′-phenethyloxy-2-anilinobenzamide derivatives represent an entry into a new class of
SIRT2-selective inhibitors.
only as tools for probing the biological functions of SIRT2 but
also as candidate therapeutic agents with potentially few side
INTRODUCTION
■
Reversible acetylation of protein lysine residues, which is tightly
controlled by histone acetyltransferases and histone deacety-
lases (HDACs), is important in the regulation of various
fundamental life processes such as gene expression and cell
cycle progression.1 Thus far, 18 HDAC family members have
been identified and they can be divided into two categories, i.e.,
zinc-dependent enzymes (HDAC1−11) and NAD+-dependent
enzymes (SIRT1−7).2 SIRT2 is unique in that it deacetylates
histone in nucleus and nonhistone proteins such as α-tubulin,
p65, and FOXO1 in cytoplasm, and it is involved in
microtubule stabilization, gene expression, and cell cycle
progression.3 Furthermore, recent studies have indicated that
changes of SIRT2 are associated with several diseases, especially
neurodegenerative diseases. Green et al. reported that
nicotinamide, a SIRT2 inhibitor, increased acetylated α-tubulin,
reduced Thr 231-phosphorylated tau, and restored cognition in
Alzheimer’s disease transgenic mice.4 Outeiro et al. reported
that inhibition of SIRT2 rescued α-synuclein toxicity and
modified inclusion morphology in a cellular model of
Parkinson’s disease.5 In addition, SIRT2 inhibition has been
reported to reduce neuronal cholesterol reduction and to be
neuroprotective in cellular models of Huntington’s disease.6
Therefore, SIRT2-selective inhibitors are of great interest not
effects.
Although many SIRT inhibitors have been found so far by us
and other groups,7 including salermide (1)8 and EX-527 (2)
(Chart 1),9 most lack SIRT2 selectivity. It has been reported
that SIRT2 is selectively inhibited by AGK2 (3)5 and
compounds 4,10 5,11 and 6 (Chart 1).12 Among these
compounds, AGK2 (3) is the most widely used and most
potent SIRT2-selective inhibitor.
We recently described a series of 2-anilinobenzamide
analogues, including compounds 7a and 7b (Chart 2), which
act as SIRT1 inhibitors.13 Following these findings, we
performed further investigation of 2-anilinobenzamide ana-
logues, seeking to find SIRT2-selective inhibitors more potent
and selective than AGK2 (3). We describe here the design,
synthesis, and biological activity of a series of 2-anilinobenza-
mide derivatives.
Received: January 26, 2012
© XXXX American Chemical Society
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Chart 1. Examples of SIRT Inhibitors
and phenethyl compound 10. Treatment of benzyl bromide 35
with triphenylphosphine yielded benzyltriphenylphosphonium
bromide 36. Wittig reaction between 4-bromobenzaldehyde
and the phosphonium ylide generated from 36 afforded a
mixture of (Z)-4-bromostilbene 37 and (E)-4-bromostilbene
38, which were separated by silica gel flash column
chromatography. Buchwald−Hartwig reaction between 2-
aminobenzamide 39 and (Z)- or (E)-4-bromostilbene using
2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl as a ligand
gave the desired styryl compounds 8 and 9. The double bond
of 9 was hydrogenated to yield phenethyl analogue 10.
The routes for the synthesis of 4-substituted amide
compounds 11−14 are illustrated in Scheme 2. Condensation
of amines 42−45 with an appropriate acid chloride 40 or 41
gave amides 46−49. The nitro group of compounds 46−49
was hydrogenated to afford anilines 50−53. Buchwald−
Hartwig reaction of 50−53 with methyl 2-bromobenzoate
yielded compounds 54−57. Methyl esters 54−57 were
hydrolyzed under alkaline conditions to give benzoic acids
58−61. Benzamides 11−14 were obtained by the coupling of
benzoic acids 58−61 and ammonia in the presence of EDCI
and HOBt.
Chart 2. 2-Anilinobenzamide-Based SIRT1 Inhibitors
CHEMISTRY
■
The routes used for the synthesis of compounds 8−34, which
were prepared for this study, are shown in Schemes 1−6.
Scheme 1 shows the preparation of 4-styryl compounds 8, 9,
a
Scheme 1
The preparation of 4-substituted anilide compounds 15−21
is shown in Scheme 3. Buchwald−Hartwig reaction of amine 39
with 4-bromonitrobenzene gave 2-(4-nitrophenylamino)-
benzamide 62. The nitro group of 62 was reduced to amine
(63), and the product was acylated to afford anilides 15−17,
64, and 19−21. Removal of the Boc group of compound 64
using TFA yielded the desired 4-piperidinyl compound 18.
Scheme 4 shows the preparation of 3-substituted amide
analogues 22−29. Acid chlorides 65−67 were reacted with
amines 68−71 to give corresponding amides 72, 74, 76, 78,
and 79. Treatment of amides 72, 74, and 76 with methyl iodide
in the presence of sodium hydride yielded the N-methylated
compounds 73, 75, and 77. Bromides 72−79 were converted
into compounds 22−29 by means of Buchwald−Hartwig
reaction.
Scheme 5 illustrates the synthesis of 3-substituted ether
compounds 30−32 and 33a. Alkylation of phenols 80 and 81
gave compounds 82−85, which were converted into com-
pounds 30−32 and 33a by means of Buchwald−Hartwig
reaction.
a
Reagents and conditions: (a) PPh3, CH2Cl2, reflux, 93%; (b) 4-
bromobenzaldehyde, LiOH, iPrOH, reflux, 45% for 37, 42% for 38;
(c) 37 or 38, Pd2dba3, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl
biphenyl, K2CO3, tert-BuOH, reflux, 58% for 8, 13% for 9; (d) H2, Pd/
C, MeOH, 60 °C, 99%.
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a
Scheme 2
a
Reagents and conditions: (a) Et3N, THF, 0 °C to room temp, 46−89%; (b) H2, Pd/C, EtOH, room temp, 84−94%; (c) methyl 2-bromobenzoate,
Pd2dba3, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl biphenyl, K2CO3, tert-BuOH, reflux, 82−85%; (d) LiOH, H2O, MeOH, THF, room temp,
78−90%; (e) NH4Cl, Et3N, EDCI, HOBt, THF, room temp, 62−74%.
a
Scheme 3
a
Reagents and conditions: (a) 4-bromonitrobenzene, Pd2dba3, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl biphenyl, K2CO3, tert-BuOH, reflux,
82%; (b) Pd/C, MeOH, room temp, 95%; (c) Ac2O, Et3N, DMAP, THF, 38%; (d) RCOOH, PyBOP; Et3N, CH2Cl2 or DMF, room temp, 35−99%;
(e) TFA, room temp, 95%.
The synthesis of compounds 33b−p and 34 is outlined in
Scheme 6. Catalytic reduction of O-benzyl compound 32 gave
3-hydroxyl compound 86. Compounds 33b−p and 34 were
obtained by the treatment of 86 with an appropriate alkyl
bromide in the presence of K2CO3 in acetone.
over SIRT1. In particular, benzyloxy compound 7t displayed
relatively strong SIRT2 inhibition (IC50 = 33 μM), whereas it
was almost inactive against SIRT1 (13% inhibition at 100 μM).
Compounds modified at the amide moiety (7ab−ad) or the
amino moiety (7ae−ag) did not inhibit SIRT2 at concen-
trations up to 100 μM.
The preliminary structure−selectivity relationship study
showed that compounds with a relatively bulky group at the
meta position (7b, 7e, and 7h) tend to inhibit both SIRT1 and
SIRT2, and compounds bearing a substituent at the para
position (7n−y) tend not to inhibit SIRT1 (Table 1). To
explore the origin of the SIRT2 selectivity of compound 7t, we
initially performed a binding mode study of SIRT1 inhibitor 7b
and SIRT2-selective inhibitor 7t with homology models of
SIRT1 and SIRT2, respectively. The binding mode studies were
performed using the software packages Glide 3.5 and
MacroModel 8.1. Inspection of the simulated SIRT1/
compound 7b complex shows that the NH2 group of 7b
RESULTS AND DISCUSSION
■
We initially screened 2-anilinobenzamide derivatives13 for
SIRT2- and SIRT1-inhibitory activity with an in vitro assay
using recombinant SIRT2 and SIRT1, aiming to find potent
and selective SIRT2 inhibitors. The results are summarized in
Table 1. Among meta-substituted 2-anilinobenzamides 7b−m,
phenyl analogue 7b, phenoxy analogue 7e, and methyl acrylate
analogue 7h showed relatively potent SIRT2 inhibition
although they also inhibited SIRT1. On the other hand, several
para-substituted 2-anilinobenzamides such as methyl 7o,
methoxy 7s, and benzyloxy 7t inhibited SIRT2 preferentially
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a
Scheme 4
a
Reagents and conditions: (a) Et3N, CH2Cl2, 0 °C to room temp, 73−85%; (b) NaH, CH3I, DMF, 0 °C to room temp, 82−86%; (c) 39, Pd2dba3, 2-
dicyclohexylphosphino-2′,4′,6′-triisopropyl biphenyl, K2CO3, tert-BuOH, reflux, 25−63%.
a
Scheme 5
a
Reagents and conditions: (a) R-Br, K2CO3, acetone or DMF, 60 °C, 30−63%; (b) 39, Pd2dba3, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl
biphenyl, K2CO3, tert-BuOH, reflux, 13−36%.
a
Scheme 6
a
Reagents and conditions: (a) H2, Pd/C, EtOH, room temp, 73%; (b) R-PhCH2(CH2)nBr, K2CO3, acetone, reflux, 9−72%.
forms hydrogen bonds with the backbone carbonyls of Phe 413
and Val 412, while the CO group of 7b forms a hydrogen bond
with guanidine of Arg 446 in the active site of SIRT1 (Figure
1). An intramolecular hydrogen bond is observed between the
CO and NH group of 7b, which suggests the importance of the
conformation of the inhibitor.13b The phenyl group of 7b is
located in the hydrophobic pocket formed by Ile 360, Pro 419,
and Phe 422. In addition, Met 426 is positioned near the para
position of the anilino group of 7b. Steric hindrance between
Met 426 and the substituent at the para position of 7o−z may
be the reason why these compounds do not inhibit SIRT1
(Table 1). In the simulated SIRT2/compound 7t complex, the
NH group and the CO group of 7t form hydrogen bonds with
Gln 267 (Figure 2). As is the case with SIRT1/7b complex, an
intramolecular hydrogen bond is observed in the SIRT2/7t
complex, suggesting the importance of the CONH2 group and
the NH group of the inhibitor. Val 233 is positioned around the
para position of the anilino group of 7t. A steric clash between
Val 233 and the bulky group at the para position of 7q and 7r
may be the reason why these compounds do not inhibit SIRT2
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a
Table 1. SIRT2- and SIRT1-Inhibitory Activities of Compounds 7a−7ag
IC50 (μM)
compd
R1
R2
X
SIRT2
>100
SIRT1
7a
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
OH
Ph
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
56 3.2
52 2.1
68 6.4
7b
7c
3-Ph-Ph
3-Me-Ph
3-CF3-Ph
3-OPh-Ph
33 3.6
72 2.1
100 9.9
25 3.8
>100
b
7d
7e
NE
58 3.7
>100
7f
3-COOMe-Ph
3-COOH-Ph
3-(E)-CHCHCOOMe-Ph
3-(E)-CHCHCOOH-Ph
3-CH2CH2COOMe-Ph
3-CH2CH2COOH-Ph
3-CH2OH-Ph
3-CH2CH2OH-Ph
4-Cl-Ph
7g
7h
7i
>100
>100
32 1.6
>100
65 5.0
58 5.4
b
7j
93 4.4
>100
NE
7k
7l
93 7.4
60 3.2
72 6.0
56 4.9
>100
>100
7m
7n
7o
7p
7q
7r
>100
>100
4-Me-Ph
60 5.4
69 9.6
>100
b
4-CF3-Ph
NE
b
4-tert-Bu-Ph
4-Ph-Ph
NE
>100
>100
>100
>100
7s
4-OMe-Ph
4-OBn-Ph
80 5.0
33 6.6
93 4.3
83 7.8
>100
7t
b
7u
7v
4-CN-Ph
NE
4-COOMe-Ph
4-COOH-Ph
4-SMe-Ph
>100
b
7w
7x
NE
b
>100
NE
7y
4-OPh-Ph
>100
>100
>100
7z
2-Naph
>100
b
7aa
7ab
7ac
7ad
7ae
7af
7ag
Bn
>100
NE
Ph
>100
68 5.4
>100
NHMe
NMe2
NH2
NH2
NH2
Ph
>100
Ph
>100
>100
Ph
>100
>100
Ph
S
>100
78 3.8
75 6.5
Ph
NMe
>100
a
b
Values are means of at least three experiments. NE = Not evaluated.
Figure 1. Calculated docking model of compound 7b (ball-and-stick)/SIRT1.
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Figure 2. Calculated docking model of compound 7t (ball-and-stick)/SIRT2.
(Table 1). In addition, the phenyl ring of the benzyloxy group
Table 2. SIRT2- and SIRT1-Inhibitory Activities of
Compounds 8−21
a
of 7t is positioned in the hydrophobic pocket delineated by Phe
96, Phe 119, Ile 232, Phe 235, and Val 266, which is larger and
deeper than the corresponding pocket of SIRT1 (Figure 1).
On the basis of the SIRT inhibition data shown in Table 1,
we selected compound 7t as a lead compound. We initially
focused on the s-cis conformation of the benzyloxyphenyl part
of 7t in the active site of SIRT2 (Figure 2). From the calculated
docking model of 7t/SIRT2, we hypothesized that the s-cis
structure of 7t is an active conformer in which the benzyl group
can interact with hydrophobic amino acid residues (Figure 3).
IC50 (μM)
compd
R
SIRT2
SIRT1
7t
OCH2Ph
33 6.6
20 2.4
>300
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8
(Z)-CHCHPh
(E)-CHCHPh
CH2CH2Ph
9
10
11
12
13
14
15
16
17
18
19
20
21
88 2.2
69 8.7
23 1.5
>300
CON(CH3)Ph
CONHPh
N(CH3)COPh
NHCOPh
17 1.5
77 9.9
>100
NHCOCH3
NHCOCH(CH3)2
NHCOcyclohexyl
NHCO-4-piperidinyl
NHCO-2-Py
25 3.5
53 8.2
>100
Figure 3. Predicted conformation of compound 7t in the active site of
SIRT2.
NHCO-3-Py
38 5.6
>100
On the basis of this hypothesis, we designed and synthesized
cis-olefin analogue 8 and N-methyl amide analogues 11 and 13
bearing an N-methyl benzanilide structure, which has been
reported to display a “cis” conformation.14 Compounds 9, 10,
11, and 14 were prepared as reference compounds. The SIRT-
inhibitory data for compounds 8−14 are shown in Table 2. In
accordance with the data shown in Table 1, these para-
substituted analogues did not inhibit SIRT1 at concentrations
up to 100 μM. As expected, while trans-olefin analogue 9 did
not inhibit SIRT2 (IC50 > 300 μM), cis-olefin analogue 8
inhibited SIRT2 more potently than 7t (IC50 = 20 μM). The
SIRT2-inhibitory activity of the reduced compound 10 was
greater than that of trans-olefin 9 and less than that of cis-olefin
8. These results support the hypothesis that s-cis structure of 7t
is an active conformer. However, unexpectedly, the N-methyl
benzanilides 11 and 13 were less active than the reference
compounds 12 and 14, respectively. The reason for the weak
activity of 11 and 13 in comparison with 12 and 14 is unclear
NHCO-4-Py
a
Values are means of at least three experiments.
but might be related to steric repulsion between the methyl
group of 11 or 13 and an amino acid residue such as Val 233
(Figure 2). The CONH amide of 12 and 14 displays cis
conformation in the active site of SIRT2. Among compounds
8−14, compound 14 showed the most potent SIRT2-inhibitory
activity (IC50 = 17 μM). We therefore chose compound 14 for
further study. We investigated the effect of replacement of the
phenyl group of compound 14 with a methyl group (15), bulky
alkyl groups (16−18), and pyridine rings (19−21), but the
changes merely resulted in retention (compounds 17 and 20)
or decrease (compounds 15, 16, 18, 19, and 21) of the activity.
We next turned our attention to the meta-substituted 2-
anilinobenzamides. The results of the molecular modeling
suggested that the hydrophobic pocket of SIRT2 is larger than
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that of SIRT1 (Figures 1 and 2). The calculation results also
suggested that compounds with a lipophilic group more bulky
than the phenyl group (7b) and phenoxy group (7e) at the
meta position of 2-anilinobenzamide would not be able to bind
to SIRT1, which has a smaller hydrophobic pocket, whereas
they would be able to bind to SIRT2, which has a larger
hydrophobic pocket, thereby resulting in selective inhibition of
SIRT2. We thus prepared compounds bearing a substituent at
the meta-position of 2-anilinobenzamide, focusing on benzene-
containing amides and ethers, and evaluated their ability to
inhibit SIRT2 and SIRT1 (Table 3). In the amide series, some
Table 4. SIRT2- and SIRT1-Inhibitory Activities of
Compounds 33a−33q
a
IC50 (μM)
compd
R
SIRT2
SIRT1
Table 3. SIRT2- and SIRT1-Inhibitory Activities of
Compounds 22−34
33a
33b
33c
33d
33e
33f
H
1.0 0.12
1.8 0.30
1.8 0.42
1.6 0.18
3.1 0.30
2.1 0.39
1.4 0.26
2.0 0.64
0.57 0.12
2.7 0.082
1.5 0.15
2.0 0.057
0.83 0.11
1.1 0.18
3.4 0.10
1.2 0.26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
a
2-CH3
3-CH3
4-CH3
2-CF3
3-CF3
4-CF3
2-F
33g
33h
33i
3-F
33j
4-F
IC50 (μM)
33k
33l
2-Cl
compd
R
SIRT2
SIRT1
>100
3-Cl
22
23
24
25
26
27
28
29
7e
30
31
32
33a
34
CONHPh
83 3.2
>100
33m
33n
33o
33p
4-Cl
CON(CH3)Ph
CONHCH2Ph
CON(CH3)CH2Ph
NHCOPh
>100
2-Br
39 5.4
40 2.2
57 7.8
>100
>100
3-Br
>100
4-Br
a
>100
Values are means of at least three experiments.
N(CH3)COPh
NHCOCH2Ph
N(CH3)COCH2Ph
OPh
>100
74 5.7
>100
>100
We compared the SIRT2-inhibitory activity and selectivity of
>100
compounds 33a and 33i with those of AGK2 (3), a
representative previously reported SIRT2-selective inhibitor.5
As shown in Table 5, compounds 33a and 33i showed SIRT2-
25 3.8
24 5.0
30 4.1
54 6.9
1.0 0.12
4.3 0.59
58 3.7
210 4.1
>300
OCH2cyclohexyl
OCH2CH(CH3)2
OCH2Ph
>300
Table 5. SIRT1- and SIRT2-Inhibitory Activities of AGK2
(3), Compounds 33a and 33i
a
OCH2CH2Ph
OCH2CH2CH2Ph
>300
>300
a
IC50 (μM)
selectivity
Values are means of at least three experiments.
SIRT1/
SIRT2
SIRT3/
SIRT2
compd
SIRT2
SIRT1
SIRT3
3
3.5 0.30
1.0 0.12
0.57 0.12
30 0.40
>300
91 2.7
>300
8.6
26
of the compounds (22, 24, 25, 26, and 28) showed SIRT2-
selective inhibition, but the SIRT2-inhibitory activity was
weaker than that of 7e. In the ether series, compounds with
an aliphatic substituent (30 and 31) displayed SIRT2-inhibitory
activity similar to that of 7e. However, compound 30 also
inhibited SIRT1, although its activity was relatively weak.
Compounds with a phenyl group (32−34) were found to be
SIRT2-selective inhibitors. Especially, a pronounced inhibitory
effect (IC50 = 1.0 μM) was observed with phenethyl ether 33a,
which was 25-fold more active than 7e. SIRT2 inhibition was
distinctly dependent on the methylene chain length, with n = 0
(7e), n = 1 (32), and n = 3 (34) resulting in less potent
inhibitors.
We further synthesized several derivatives of 33a and tested
them in SIRT inhibition assays. As shown in Table 4, most of
the compounds showed potent and selective inhibitory activity
against SIRT2. While methyl or trifluoromethyl compounds
33b−g showed slightly reduced SIRT2-inhibitory activity as
compared with parent compound 33a, two of the halogenated
compounds (compounds 33i and 33m) inhibited SIRT2 with
IC50 values in the submicromolar range.
33a
33i
>300
>530
>300
>530
>300
>300
a
Values are means of at least three experiments.
inhibitory activity 3.5- and 6.1-fold more potent than that of
AGK2 (3), respectively. Furthermore, AGK2 (3) inhibited
SIRT1 and SIRT3 with IC50 values of 30 and 91 μM,
respectively, whereas compounds 33a and 33i did not inhibit
either SIRT1 or SIRT3 at concentrations up to 300 μM,
showing high selectivity for SIRT2 over both SIRT1 and
SIRT3. Thus, compounds 33a and 33i are more potent and
more selective SIRT2 inhibitors than AGK2 (3) in these
enzyme assays.
Next, we evaluated the interaction between SIRT2 and
compound 33a using Biacore surface plasmon resonance (SPR)
analysis. Recombinant SIRT2 was immobilized onto CM5
sensor chip, over which a compound solution was passed.
Dose-dependent SPR responses were recorded for a series of
concentrations of compound 33a as shown in Supporting
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(1H NMR) and carbon nuclear magnetic resonance spectra (13C
NMR) were recorded on JEOL JNM-LA500, JEOL JNM-A500, or
BRUKER AVANCE600 spectrometers in the indicated solvent.
Chemical shifts (δ) are reported in parts per million relative to the
internal standard tetramethylsilane. Elemental analysis was performed
with a Yanaco CHN CORDER NT-5 analyzer, and all values were
within 0.4% of the calculated values, indicating >95% purity. High-
resolution mass spectra (HRMS) were recorded on a JEOL JMS-
SX102A mass spectrometer. GC-MS analyses were performed on a
Shimadzu GCMS-QP2010. Purity testing was done by means of
analytical HPLC on a Shimadzu instrument equipped with an Inertsil
ODS-3 column (4.6 mm × 150 mm, GL Science) eluted at 1 mL/min
with Milli-Q water and CH3CN. All tested compounds were ≥95%
pure. Reagents and solvents were purchased from Aldrich, Tokyo
Kasei Kogyo, Wako Pure Chemical Industries, and Kanto Kagaku and
used without purification. Flash column chromatography was
performed using silica gel 60 (particle size 0.046−0.063 mm) supplied
by Merck.
Information Figure S1, which clearly indicated its direct
interactions with SIRT2.
To examine whether compounds 33 selectively inhibit SIRT2
in cells, we performed a cellular assay using Western blot
analysis. Because SIRT2 and SIRT1 are known to catalyze the
deacetylation of α-tubulin3b and p53,15 respectively, the effect
of the inhibitors on the acetylation levels of α-tubulin and p53
in HCT116 cells were analyzed. As can be seen in Figure 4,
(Z)-2-(4-Styrylphenylamino)benzamide (8). Steps 1 and 2:
Preparation of (Z)-4-Bromostilbene (37) and (E)-4-Bromostilbene
(38). To a well-stirred solution of benzyl bromide 35 (0.40 mL, 3.34
mmol) in anhydrous CH2Cl2 (30 mL) was added triphenylphosphine
(1.31 g, 5.01 mmol). The reaction mixture was refluxed for 12 h and
then allowed to cool to room temperature. The solvent was removed
in vacuo to afford the crude salt as a white powder. The powder was
rinsed with Et2O until no PPh3 was detected by TLC in the ethereal
filtrate, affording 1.35 g (93%) of benzyltriphenylphosphonium
bromide 36 as a white powder.
Figure 4. Western blot detection of acetylated α-tubulin and p53 levels
in HCT116 cells after 8 h treatment with AGK2 (3), EX-527 (2), and
compound 33a. Each value means the ratio of acetylated α-tubulin
amount to total α-tubulin or acetylated p53 amount to total p53.
To a stirred solution of 36 (760 mg, 1.76 mmol) in isopropyl
alcohol (7 mL) was added lithium hydroxide (100 mg, 2.4 mmol).
After 15 min, 4-bromobenzaldehyde (296 mg, 1.60 mmol) was further
added. The reaction mixture was refluxed for 1 h, allowed to cool to
room temperature, and then quenched with water and extracted with
AcOEt. The organic layer was separated, washed with brine, and dried
over Na2SO4. Filtration, concentration of the filtrate in vacuo, and
purification of the residue by silica gel flash column chromatography
(AcOEt/n-hexane = 1/20) gave 205 mg (45%) of 37 and 190 mg
(42%) of 38. 37: 1H NMR (CDCl3, 500 MHz, δ, ppm) 7.34 (2H, d, J
= 7.6 Hz), 7.25−7.16 (5H, m), 7.10 (2H, d, J = 7.6 Hz), 6.63 (1H, d, J
compound 33a induced a dose-dependent increase of α-tubulin
acetylation, and its effect was greater than that of AGK (3).
Furthermore, while EX-527 (2), a previously reported SIRT1-
selective inhibitor,9 caused selective acetylation of p53, the level
of acetylated p53 was not elevated in the presence of 33a.
These results indicated that the compound 33a selectively
inhibits SIRT2 in preference to SIRT1 in cells.
Compound 33a was also selective against zinc-dependent
HDACs (IC50 > 30 μM). In addition, compound 33a was
weakly potent against CYP450s such as 3A4 or 2D6 (IC50 > 10
μM). Furthermore, compound 33a displayed a certain level of
stability in human liver microsomes (64% remaining after 15
min incubation of 0.5 mg 33a).
1
= 12.5 Hz), 6.50 (1H, d, J = 12.5 Hz). 38: H NMR (CDCl3, 500
MHz, δ, ppm) 7.53−7.45 (2H, m), 7.40−7.34 (2H, m), 7.30−7.24
(5H, m), 7.10 (1H, d, J = 16.4 Hz), 7.03 (1H, d, J = 16.4 Hz).
Step 3: Preparation of (Z)-2-(4-Styrylphenylamino)benzamide
(8). A mixture of 2-aminobenzamide 39 (71 mg, 0.52 mmol), 37 (100
mg, 0.38 mmol) obtained above, Pd2dba3 (32 mg, 0.033 mmol), 2-
dicyclohexylphosphino-2′-4′,6′-triisopropylbiphenyl (83 mg, 0.17
mmol), and K2CO3 (131 mg, 0.95 mmol) in tert-BuOH (3 mL) was
refluxed for 15 h. The reaction mixture was diluted with AcOEt,
washed with 1 N aqueous HCl and brine, and dried over Na2SO4.
Filtration, concentration of the filtrate in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/3 to 1/2) gave 70 mg (59%) of 8 as a white solid: mp 137−139 °C.
1H NMR (DMSO-d6, 500 MHz, δ, ppm) 10.0 (1H, broad s), 8.06
(1H, broad s), 7.70 (1H, dd, J = 8.0, 1.5 Hz), 7.49 (broad s, 1H),
7.35−7.26 (6H, m), 7.24−7.20 (1H, m), 7.16 (2H, d, J = 8.5 Hz) 7.02
(2H, d, J = 8.5 Hz), 6.81 (1H, t, J = 6.7 Hz), 6.56 (1H, d, J = 12.2 Hz),
6.52 (1H, d, J = 12.2 Hz). 13C NMR (CD3OD, 125 MHz, δ, ppm)
171.19, 144.22, 140.69, 137.21, 132.09, 129.82, 129.71, 129.33, 128.39,
128.37, 128.33, 127.67, 127.07, 126.17, 118.59, 118.15, 117.98. MS
(EI) m/z 314 (M+). HRMS calcd for C21H18N3O, 314.142; found,
314.142; purity 96.2% (HPLC).
CONCLUSION
■
We have designed and synthesized novel SIRT2-selective
inhibitors based on homology models of SIRT1 and SIRT2. 3′-
Phenethyloxy-2-anilinobenzamides such as compounds 33a and
33i exhibited potent and selective SIRT2 inhibition in enzyme
assays, showing more than 3.5-fold greater SIRT2-inhibitory
activity and more than 10-fold greater SIRT2-selectivity over
SIRT1 and SIRT3, as compared with AGK2 (3), the most
potent and selective SIRT2 inhibitor among previously
reported SIRT inhibitors. Preliminary SAR study and molecular
modeling suggested that the terminal phenethyl group of 33 is
essential for the SIRT2 selectivity, and the methylene linker
length is important for potent inhibition of SIRT2. In cellular
assays, compound 33a induced a selective increase of α-tubulin
acetylation, suggesting that it is a cell-active SIRT2-selective
inhibitor. Thus, compounds 33 are potent and selective SIRT2
inhibitors, and should be useful as tools for probing the biology
of SIRT2.
(E)-2-(4-Styrylphenylamino)benzamide (9). Compound 9 was
prepared from 38 obtained above and 39 using the procedure
described for 8 (step 3) in 13% yield as colorless crystals; mp 203−206
°C. 1H NMR (CDCl3, 600 MHz, δ, ppm) 9.61 (1H, s), 7.50 (2H, d, J
= 7.8 Hz), 7.48 (1H, t, J = 7.8 Hz), 7.47 (2H, d, J = 8.4 Hz), 7.39 (1H,
d, J = 8.4 Hz), 7.35 (2H, t, J = 7.8 Hz), 7.32 (1H, t, J = 7.2 Hz), 7.24
(1H, d, J = 7.8 Hz), 7.20 (2H, d, J = 8.4 Hz), 7.08 (1H, d, J = 16.2
Hz), 7.02 (1H, d, J = 16.2 Hz), 6.78 (1H, t, J = 7.2 Hz). MS (EI) m/z:
EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a Yanagimoto
micro melting point apparatus or a Buchi 545 melting point apparatus
and were left uncorrected. Proton nuclear magnetic resonance spectra
■
̈
H
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Journal of Medicinal Chemistry
Article
1
314 (M+). HRMS calcd for C21H18N2O, 314.141; found, 314.142;
purity 95.5% (HPLC).
a colorless solid; mp 202−204 °C. H NMR (CDCl3, 500 MHz, δ,
ppm) 9.53 (1H, s), 7.45 (1H, dd, J = 1.5, 7.8 Hz), 7.34 (1H, dt, J = 1.2,
8.2 Hz), 7.23−7.20 (5H, m), 7.16 (1H, t, J = 7.8 Hz), 7.06 (2H, d, J =
8.5 Hz), 6.96 (2H, d, J = 8.5 Hz), 6.80 (1H, dt, J = 1.2, 7.8 Hz), 3.49
(3H, s). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.38, 170.22, 145.44,
144.82, 142.92, 132.85, 130.58, 129.19, 128.90, 128.25, 126.84, 126.31,
118.68, 118.139, 117.19, 116.42, 38.59. MS (EI) m/z 345 (M+).
HRMS calcd for C21H19N3O2, 345.147; found, 345.147; purity 99.0%
(HPLC).
2-(4-Phenethylphenylamino)benzamide (10). To a solution of
9 (42 mg, 0.130 mmol) obtained above in MeOH (14 mL) was added
10% Pd/C (8.4 mg). The reaction mixture was stirred for 4 h at 60 °C
under a H2 atmosphere and filtered through Celite. The filtrate was
concentrated in vacuo, and the residue was purified by silica gel flash
column chromatography (n-hexane/AcOEt = 3/2) to give 41 mg
(99%) of 10 as a white solid: mp 112−114 °C. 1H NMR (CDCl3, 500
MHz, δ, ppm) 9.49 (1H, s), 7.45 (1H, d, J = 8.0 Hz), 7.30−7.26 (7H,
m), 7.25−7.16 (4H, m), 6.72 (1H, t, J = 8.0 Hz), 2.91 (4H, m). MS
(EI) m/z: 316 (M+). HRMS calcd for C21H20N2O, 316.157; found,
316.160; purity 97.5% (HPLC).
4-(2-Carbamoylphenylamino)-N-methyl-N-phenylbenza-
mide (11). Step 1: Preparation of N-Methyl-4-nitro-N-phenyl-
benzamide (46). To a mixture of N-methyl-4-nitroaniline 42 (0.40
mL, 3.70 mmol) and triethylamine (2.00 mL, 14.8 mmol) in
anhydrous THF (10 mL) was added 4-nitrobenzoyl chloride 40 (1
g, 5.5 mmol) in one portion at 0 °C. The resulting mixture was stirred
for 30 min at 0 °C, allowed to warm to room temperature, and further
stirred for 4 h. The solvent was removed in vacuo, and the residue was
diluted with AcOEt, washed with saturated aqueous Na2CO3 and
brine, and dried over Na2SO4. Filtration, concentration of the filtrate in
vacuo and purification of the residue by recrystallization from AcOEt/
n-hexane gave 460 mg (49%) of 46 as a yellow solid. 1H NMR
(CDCl3, 500 MHz, δ; ppm) 8.02 (2H, d, J = 8.5 Hz), 7.44 (2H, d, J =
8.5 Hz), 7.29−7.23 (2H, m), 7.20 (1H, t, J = 7.3 Hz), 7.03 (2H, d, J =
7.3 Hz), 3.52 (3H, s, 3H). MS (GC) m/z: 256 (M+).
Compounds 12−14 were prepared from an appropriate acid
chloride (40 or 41) and aniline (43, 44, or 45) using the procedure
described for 11.
4-(2-Carbamoylphenylamino)-N-phenylbenzamide (12).
Yield 18%; colorless crystals; mp 230−232 °C. H NMR (CD3OD,
500 MHz, δ, ppm) 7.79 (2H, d, J = 8.8 Hz), 7.62 (1H, dd, J = 1.5, 7.8
Hz), 7.54 (2H, d, J = 7.6 Hz), 7.41 (1H, d, J = 7.6 Hz), 7.32 (1H, dt, J
= 1.2, 7.6 Hz), 7.25 (2H, t, J = 8.2 Hz), 7.13 (2H, d, J = 8.8 Hz), 7.03
(1H, t, J = 7.6 Hz), 6.86 (1H, dt, J = 1.2, 7.3 Hz). 13C NMR (CD3OD,
125 MHz, δ, ppm) 174.02, 168.39, 147.37, 144.40, 140.05, 133.39,
130.41, 130.35, 129.75, 125.38, 122.34, 121.69, 121.24, 118.69, 118.21.
MS (EI) m/z 331 (M+). HRMS calcd for C20H17N3O2, 331.132;
found, 331.1323. Anal. (C20H17N3O2·1/4H2O) C, H, N.
1
N-(4-(2-Carbamoylphenylamino)phenyl)-N-methylbenza-
mide (13). Yield 31%; colorless crystals; mp 245−247 °C. H NMR
1
(CDCl3, 500 MHz, δ, ppm) 9.53 (1H, broad s), 7.46 (1H, dd, J = 1.5,
7.9 Hz), 7.35−7.30 (2H, m), 7.28−7.22 (2H, m), 7.22−7.17 (3H, m),
7.07 (2H, d, J = 8.5 Hz), 6.96 (2H, d, J = 8.5 Hz), 6.77 (1H, dt, J = 1.5,
7.9 Hz), 3.49 (3H, s). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.51,
170.69, 145.91, 139.69, 139.37, 136.06, 132.97, 129.52, 128.68, 128.30,
127.81, 127.75, 121.39, 118.00, 116.10, 115.38, 38.47. MS (EI) m/z
345 (M+). HRMS calcd for C21H19N3O2, 345.148; found, 345.148;
purity 97.8% (HPLC).
Step 2: Preparation of 4-Amino-N-methyl-N-phenylbenzamide
(50). A mixture of 46 (256 mg, 1.00 mmol) and 10% Pd/C (25 mg) in
EtOH (5 mL) was stirred under H2 for 2 h at room temperature. The
reaction mixture was filtered through Celite. The filtrate was
concentrated in vacuo, and the residue was recrystallized from
N-(4-(2-Carbamoylphenylamino)phenyl)benzamide (14).
Yield 44%; colorless crystals; mp 209−211 °C. H NMR (CD3OD,
1
1
AcOEt/n-hexane to afford 204 mg (90%) of 50 as a white solid. H
NMR (CDCl3, 500 MHz, δ, ppm) 7.24 (2H, d, J = 7.9 Hz), 7.19−7.10
(3H, m), 7.05 (2H, dd, J = 1.5, 7.9 Hz), 6.41 (2H, d, J = 8.5 Hz), 3.75
(2H, broad s), 3.47 (3H, s). MS (GC) m/z: 226 (M+).
500 MHz, δ, ppm) 7.93 (2H, d, J = 8.8 Hz), 7.67−7.61 (3H, m), 7.57
(1H, t, J = 7.6 Hz), 7.51 (2H, t, J = 7.6 Hz), 7.32−7.27 (2H, m), 7.18
(2H, d, J = 8.8 Hz), 6.78 (1H, dt, J = 1.5, 7.6 Hz). 13C NMR (CD3OD,
125 MHz, δ, ppm) 174.47, 168.75, 147.26, 139.77, 136.34, 134.61,
133.58, 132.80, 130.29, 129.62, 128.57, 123.68, 122.22, 118.95, 118.49,
116.10. MS (EI) m/z 331 (M+). HRMS calcd for C20H17N3O2,
331.132; found, 331.133. Anal. (C20H17N3O2·1/3H2O) C, H, N.
2-(4-Acetamidophenylamino)benzamide (15). Step 1: Prep-
aration of 2-(4-Nitrophenylamino)benzamide (62). Compound 62
was prepared from 39 and 4-bromonitrobenzene using the procedure
described for 8 (step 3) in 82% yield. 1H NMR (CDCl3, 500 MHz, δ,
ppm) 9.97 (1H, s), 8.16 (2H, dd, J = 2.0, 7.0 Hz), 7.57−7.55 (2H, m),
7.46 (1H, t, J = 7.0 Hz), 7.20 (2H, dd, J = 2.0, 7.5 Hz), 7.02 (1H, t, J =
7.0 Hz).
Step 3: Preparation of Methyl 2-{4-(Methylphenylcarbamoyl)-
phenylamino}benzoate (54). Compound 54 was prepared from 50
obtained above and methyl 2-bromobenzoate using the procedure
described for 8 (step 3) in 85% yield. 1H NMR (CDCl3, 500 MHz, δ;
ppm) 9.47 (1H, broad s), 7.94 (1H, dd, J = 1.5, 8.0 Hz), 7.32 (1H, dt,
J = 1.8, 8.2 Hz), 7.28−7.23 (5H, m), 7.15 (1H, t, J = 7.3 Hz), 7.06
(2H, d, J = 8.8 Hz), 7.00 (2H, d, J = 8.8 Hz), 6.77 (1H, dt, J = 1.2, 8.2
Hz), 3.87 (3H, s), 3.52 (3H, s). MS (EI) m/z: 360 (M+).
Step 4: Preparation of 2-{4-(Methylphenylcarbamoyl)-
phenylamino}benzoic Acid (58). To a solution of 54 (150 mg, 0.41
mmol) obtained above in THF (6 mL) and MeOH (2 mL) was added
an aqueous solution (2 mL) of lithium hydroxide monohydrate (175
mg, 4.10 mmol), and the resulting solution was stirred overnight at 50
°C. After removal of organic solvents in vacuo, the remaining aqueous
phase was acidified with 2 N HCl and the precipitate was extracted
with AcOEt. The organic layer was washed with brine and dried over
Na2SO4. Filtration, concentration of the filtrate in vacuo, and
purification of the residue by recrystallization from AcOEt/n-hexane
Step 2: 2-(4-Aminophenylamino)benzamide (63). Compound 63
was prepared from 62 obtained above using the procedure described
for 11 (step 2) in 95% yield. H NMR (CDCl3, 500 MHz, δ; ppm);
9.32 (1H, s), 7.21 (1H, t, J = 7.0 Hz), 7.04 (2H, dd, J = 2.0, 6.5 Hz),
6.98 (1H, dd, J = 1.0, 8.5 Hz), 6.69 (2H, dd, J = 2.0, 6.5 Hz), 6.63 (1H,
t, J = 7.0 Hz).
1
Step 3: 2-((4-Acetamidophenyl)amino)benzamide (15). To a
solution of 63 (50.0 mg, 0.220 mmol) obtained above, DMAP (13.4
mg, 0.110 mmol) and Et3N (33.4 mg, 0.330 mmol) in THF (3 mL)
was added acetic anhydride (34 mg, 0.330 mmol). The mixture was
stirred at room temperature for 3 h and then poured into 2 N aqueous
HCl, and the whole was extracted with AcOEt. The AcOEt layer was
separated, washed with water and brine, and dried over Na2SO4.
Filtration, concentration of the filtrate in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/1) gave 23 mg (38%) of 15 as a gray powder. The powder was
recrystallized from AcOEt/n-hexane to give 18 mg of 15 as a gray
powder; mp 181−183 °C. 1H NMR (CDCl3, 500 MHz, δ, ppm) 9.50
(1H, s), 7.45 (1H, d, J = 7.5 Hz), 7.44 (2H, d, J = 7.0 Hz), 7.27 (1H, t,
J = 7.0 Hz), 7.23 (1H, d, J = 7.0 Hz), 7.17 (2H, d, J = 8.5 Hz), 7.11
(1H, s), 6.73 (1H, t, J = 7.0 Hz), 2.18 (3H, s). 13C NMR (DMSO-d6,
125 MHz, δ, ppm) 171.29, 167.76, 145.87, 136.15, 134.22, 132.12,
1
gave 115 mg (80%) of 58 as a yellow solid. H NMR (CDCl3, 500
MHz, δ; ppm) 9.33 (1H, broad s), 8.01 (1H, dd, J = 1.8, 8.2 Hz), 7.35
(1H, dt, J = 1.5, 7.3 Hz), 7.31−7.24 (4H, m), 7.18 (1H, t, J = 7.3 Hz),
7.08 (2H, d, J = 8.5 Hz), 7.02 (2H, d, J = 8.5 Hz), 6.79 (1H, t, J = 8.2
Hz), 3.51 (3H, s). MS (EI) m/z: 346 (M+).
Step 5: Preparation of 4-(2-Carbamoylphenylamino)-N-methyl-
N-phenylbenzamide (11). A mixture of 58 (100 mg, 0.29 mmol)
obtained above, NH4Cl (77 mg, 1.44 mmol), Et3N (202 μL, 1.44
mmol), EDCI (164 mg, 0.85 mmol), and HOBt·H2O (131 mg, 0.85
mmol) in THF (5 mL) was stirred at room temperature for 15 h. The
reaction mixture was poured into water, and the whole was extracted
with AcOEt. The AcOEt layer was separated, washed with water and
brine, and dried over Na2SO4. Filtration, concentration of the filtrate in
vacuo, and purification of the residue by silica gel flash column
chromatography (AcOEt/n-hexane = 1/1) gave 63 mg (63%) of 11 as
I
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Journal of Medicinal Chemistry
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129.19, 121.16, 120.10, 116.83, 116.22, 113.71, 23.77. MS (EI) m/z:
269 (M+). HRMS calcd for C15H15N3O2, 269.116; found, 269.117;
purity 99.6% (HPLC).
N-(4-2-Carbamoylphenylaminophenyl)isonicotinamide (21).
Yield 75%; mp 249−252 °C. 1H NMR (DMSO-d6, 500 MHz, δ, ppm)
10.46 (1H, s), 10.52 (1H, s), 8.79 (2H, dd, J = 2.0, 4.5 Hz), 7.86 (2H,
dd, J = 1.5, 4.5 Hz), 7.72 (3H, m), 7.22 (2H, d, J = 8.0 Hz), 7.71 (1H,
d, J = 8.0 Hz), 7.57 (1H, t, J = 5.0 Hz), 8.29 (1H, t, J = 8.0 Hz), 7.73
(2H, d, J = 8.0 Hz), 7.71 (1H, d, J = 8.0 Hz), 7.57 (1H, t, J = 5.0 Hz),
7.31 (1H, t, J = 8.5 Hz), 7.21 (1H, dd, J = 1.5, 8.5 Hz), 6.77 (1H, t, J =
7.0 Hz). MS (EI) m/z: 332 (M+). HRMS calcd for C19H16N4O2,
332.127; found, 332.128; purity 96.3% (HPLC).
2-(4-Isobutyramidophenylamino)benzamide (16). To a sol-
ution of 63 (100 mg, 0.44 mmol) obtained above, isobutyric acid (32
mg, 0.360 mmol), PyBop (281 mg, 0.540 mmol), and Et3N (89 mg,
0.880 mmol) in dry CH2Cl2 (5 mL) was stirred at room temperature
for 23 h. The reaction mixture was poured into aqueous saturated
NaHCO3, and the whole was extracted with AcOEt. The AcOEt layer
was separated, washed with water and brine, and dried over Na2SO4.
Filtration, concentration of the filtrate in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/2) gave 93 mg (87%) of 16 as a white powder. The powder was
recrystallized from AcOEt/n-hexane to give 45 mg of 16 as a white
Compounds 22, 24, 26, 28, and 29 were prepared from an
appropriate acid chloride (65−67) and amine (68−71) using the
procedure described for 11 (step 1) and 8 (step 3).
2-(3-N-Phenylbenzamidoamino)benzamide (22). Yield 45%;
1
mp 182−184 °C. H NMR (CDCl3, 500 MHz, δ, ppm) 9.66 (1H, s),
1
7.79 (1H, broad s), 7.73 (1H, t, J = 2.1 Hz), 7.64 (2H, d, J = 7.9 Hz),
7.50 (1H, dd, J = 1.2, 7.8 Hz), 7.47 (1H, td, J = 1.5, 7.5 Hz), 7.41 (1H,
t, J = 7.6 Hz), 7.30−7.38 (5H, m), 7.15 (1H, t, J = 7.6 Hz), 6.83 (1H,
dt, J = 1.5, 8.2 Hz);. 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.52,
165.53, 145.47, 142.22, 137.92, 136.36, 133.14, 129.80, 129.12, 128.36,
124.57, 123.98, 120.59, 120.12, 119.04, 118.58, 116.80, 115.81. MS
(EI) m/z: 331 (M+). HRMS (EI) m/z: calcd for C20H17N3O2,
331.132; found, 331.132. Anal. (C20H17N3O2) C, H, N.
powder; mp 227−230 °C. H NMR (DMSO-d6, 500 MHz, δ, ppm)
10.54 (1H, s), 10.05 (1H, s), 8.38 (2H, d, J = 9.0 Hz), 8.19 (2H, d, J =
9.0 Hz), 7.71 (1H, dd, J = 1.5, 8.0 Hz), 7.32 (1H, t, J = 7.0 Hz), 7.23
(1H, d, J = 8.5 Hz), 7.19 (2H, d, J = 9.0 Hz), 6.77 (1H, t, J = 7.0 Hz).
13C NMR (DMSO-d6, 125 MHz, δ, ppm) 171.25, 163.38, 148.97,
145.36, 140.61, 137.42, 133.15, 132.13, 129.02, 123.44, 121.64, 120.48,
117.26, 116.74, 114.11. MS (EI) m/z: 376 (M+). HRMS calcd for
C20H16N4O4, 376.117; found, 376.117; purity 95.6% (HPLC).
2-(3-N-Benzylbenzamidoamino)benzamide (24). Yield 37%;
2-(4-Cyclohexanecarboxamidophenylamino)benzamide
(17). Compound 17 was prepared from 63 obtained above and
cyclohexanecarboxylic acid using the procedure described for 16 in
38% yield; mp 207−209 °C. 1H NMR (DMSO-d6, 500 MHz, δ, ppm)
9.95 (1H, s), 9.75 (1H, s), 7.69 (1H, dd, J = 1.5, 8.0 Hz), 7.56 (2H, d, J
= 9.0 Hz), 7.27 (1H, t, J = 7.5 Hz), 7.12 (1H, d, J = 8.5 Hz), 7.05 (2H,
d, J = 9.0 Hz), 6.72 (1H, d, J = 7.5 Hz), 1.80−1.17 (11H, m). 13C
NMR (DMSO, 125 MHz, δ, ppm) 173.83, 171.31, 145.94, 136.02,
134.42, 132.13, 129.20, 121.22, 120.15, 116.79, 116.16, 113.66, 29.07,
25.31, 25.15. MS (EI) m/z: 337 (M+). Anal. (C20H23N3O2) C, H, N.
N-(4−2-Carbamoylphenylaminophenyl)piperidine-4-car-
boxamide Trifluoroacetic Acid Salt (18·TFA). Step 1: Preparation
of tert-Butyl 4-((4−2-Carbamoylphenylaminophenyl)carbamoyl)-
piperidine-1-carboxylate (64). Compound 64 was prepared from
63 and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid using the
1
mp 194−196 °C. H NMR (CDCl3, 500 MHz, δ, ppm) 9.62 (1H, s),
7.66 (1H, s), 7.46 (1H, d, J = 7.9 Hz), 7.40−7.33 (m, 8H), 7.32−7.28
(2H, m), 6.81 (1H, t, J = 7.5 Hz), 6.35 (1H, broad s), 4.65 (2H, d, J =
5.5 Hz). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.52, 167.17,
148.48, 145.67, 141.99, 138.10, 135.75, 133.12, 129.58, 128.83, 128.31,
127.95, 127.67, 123.86, 120.65, 119.34, 118.36, 115.1, 44.20. MS (EI)
m/z: 345 (M+). HRMS (EI) m/z: calcd for C21H19N3O2, 345.148;
found, 345.147. Anal. (C21H19N3O2·1/10H2O) C, H, N.
2-(3-Benzamidophenylamino)benzamide (26). Yield 18%; mp
158−160 °C. 1H NMR (CDCl3, 500 MHz, δ, ppm) 9.54 (1H, s), 7.86
(2H, d, J = 8.2 Hz), 7.75 (1H, broad s), 7.60 (1H, t, J = 2.3 Hz), 7.58−
7.53 (1H, m), 7.52−7.46 (3H, m), 7.43 (1H, d, J = 8.2 Hz), 7.33 (2H,
dt, J = 1.2, 7.2 Hz), 7.29 (2H, d, J = 7.9 Hz), 6.99 (1H, td, J = 2.1, 7.2
Hz), 6.79 (1H, dt, J = 1.2, 8.2 Hz). 13C NMR (CDCl3, 125 MHz, δ,
ppm) 171.54, 165.65, 145.89, 142.26, 138.86, 135.00, 133.04, 131.88,
129.89, 128.83, 128.27, 126.99, 118.05, 117.28, 116.47, 115.95, 114.29,
112.47. MS (EI) m/z: 331 (M+). HRMS (EI) m/z: calcd for
C20H17N3O2, 331.132; found, 331.132. Anal. (C20H17N3O2·1/5H2O)
C, H, N.
1
procedure described for 16 in 35% yield. H NMR (DMSO-d6, 500
MHz, δ, ppm) 9.96 (1H, s), 9.86 (1H, s), 7.68 (1H, dd, J = 1.5, 8.0
Hz), 7.54 (2H, d, J = 9.0 Hz), 7.27 (1H, t, J = 7.5 Hz), 7.12 (1H, d, J =
8.0 Hz), 6.89 (2H, d, J = 8.5 Hz), 6.72 (1H, t, J = 7.0 Hz), 1.40 (9H,
s).
2-(3-2-Phenylacetamidophenylamino)benzamide (28). Yield
Step 2: N-(4-2-Carbamoylphenylaminophenyl)piperidine-4-car-
boxamide Trifluoroacetic Acid Salt (18·TFA). A solution of 64 (26
mg, 0.06 mmol) obtained above in TFA (2 mL) was stirred at room
temperature for 1 h. The reaction mixture was concentrated in vacuo,
and the residue was recrystallized from CHCl3/MeOH to give 19 mg
(95%) of 18·TFA as a gray powder; mp 207−209 °C. 1H NMR
(DMSO-d6, 500 MHz, δ, ppm) 9.99 (1H, s), 9.97 (1H, s), 7.69 (1H, d,
J = 7.5 Hz), 7.55 (2H, d, J = 8.5 Hz), 7.28 (1H, t, J = 7.5 Hz), 7.14−
7.11 (3H, m), 6.74 (1H, t, J = 6.3 Hz), 2.93−1.82 (9H, m). MS (EI)
m/z: 338 (M+). HRMS calcd for C19H21N4O2, 338.174; found,
338.174; purity 98.7% (HPLC).
1
18%; mp 159−161 °C. H NMR (CDCl3, 500 MHz, δ, ppm) 9.46
(1H, s), 7.45 (1H, dd, J = 1.2, 7.5 Hz), 7.40 (2H, t, J = 7.5 Hz), 7.36−
7.28 (6H, m), 7.20 (1H, t, J = 7.9 Hz), 7.20 (1H, d, J = 7.9 Hz), 6.99
(1H, broad s), 6.94 (1H, d, J = 7.5 Hz), 6.78−6.73 (1H, m), 3.74 (2H,
s). 13C NMR (CDCl3, 150 MHz, δ, ppm) 171.53, 168.95, 145.94,
142.05, 138.57, 134.34, 133.01, 129.68, 129.55, 129.32, 128.23, 127.77,
117.95, 117.16, 116.32, 115.80, 113.99, 112.39, 44.96. MS (EI) m/z:
345 (M+). HRMS (EI) m/z: calcd for C21H19N3O2, 345.148; found,
345.148; purity 99.0% (HPLC).
2-(3-N-Methyl-2-phenylacetamidophenylamino)benzamide
1
(29). Yield 45%; mp 173−175 °C. H NMR (CDCl3, 500 MHz, δ,
Compounds 19−21 were prepared from 63 and an appropriate
carboxylic acid using the procedure described for 16.
ppm) 9.63 (1H, s), 7.49 (1H, dd, J = 1.2, 7.5 Hz), 7.34−7.27 (2H, m),
7.25−7.16 (5H, m), 7.10 (2H, d, J = 7.3 Hz), 6.97−6.93 (1H, m), 6.82
(1H, t, J = 7.5 Hz), 6.77 (1H, d, J = 7.3 Hz), 3.53 (2H, s), 3.26 (3H,
s). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.46, 170.92, 145.29,
144.96, 142.89, 135.50, 133.02, 130.40, 129.06, 128.36, 128.34, 126.57,
121.18, 119.92, 118.98, 118.55, 116.75, 115.77, 40.99, 37.52. MS (EI)
m/z: 359 (M+). HRMS (EI) m/z: calcd for C22H21N3O2, 359.163;
found, 359.163; purity 97.1% (HPLC).
N-(4-2-Carbamoylphenylaminophenyl)picolinamide (19).
Yield 19%; mp 231−233 °C. 1H NMR (DMSO-d6, 500 MHz, δ,
ppm) 10.60 (1H, s), 10.00 (1H, s), 8.75−8.74 (1H, m), 8.07 (1H, t, J
= 8.0 Hz), 7.87 (2H, dd, J = 2.5, 9.0 Hz), 7.71−7.67 (2H, m), 7.31
(1H, t, J = 7.5 Hz), 7.21 (1H, d, J = 8.3 Hz), 7.17 (2H, dd, J = 2.0, 9.0
Hz), 6.76 (1H, t, J = 7.0 Hz). MS (EI) m/z: 332 (M+). Anal.
(C19H16N4O2) C, H, N.
N-(4-2-Carbamoylphenylaminophenyl)nicotinamide (20).
Yield 97%; mp 177−179 °C. 1H NMR (DMSO-d6, 500 MHz, δ,
ppm) 10.41 (1H, s), 10.04 (1H, s), 9.11 (1H, d, J = 1.5 Hz), 8.76 (1H,
dd, J = 1.5, 5.0 Hz), 8.29 (1H, t, J = 8.0 Hz), 7.73 (2H, d, J = 8.0 Hz),
7.71 (1H, d, J = 8.0 Hz), 7.57 (1H, dd, J = 1.5, 5.0 Hz), 7.31 (1H, t, J =
8.5 Hz), 7.21 (1H, d, J = 8.5 Hz), 6.76 (1H, t, J = 8.0 Hz). MS (EI) m/
z: 332 (M+). Anal. (C19H16N4O2) C, H, N.
2-(3-N-Methyl-N-phenylbenzamidoamino)benzamide (23).
Step 1: Preparation of 3-Bromo-N-methyl-N-phenylbenzamide
(73). To a solution of 72 (300 mg, 1.08 mmol) in DMF (4 mL)
was added NaH (87 mg, 60% in oil, 2.16 mmol), and the mixture was
stirred at room temperature for 30 min. MeI (101 μL, 1.62 mmol) was
then added dropwise at 0 °C, and stirring was continued at 0 °C for 1
h and at room temperature for 2 h. The reaction was quenched with
saturated aqueous NH4Cl, and the whole was extracted with AcOEt.
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The AcOEt layer was separated, washed with water and brine, and
dried over Na2SO4. Filtration, concentration of the filtrate in vacuo,
and purification of the residue by silica gel flash column
chromatography (AcOEt/n-hexane = 1/3) gave 270 mg (86%) of
73 as a colorless solid. 1H NMR (CDCl3, 500 MHz, δ, ppm) 7.50 (1H,
t, J = 1.8 Hz), 7.35 (1H, d, J = 7.9 Hz), 7.27−7.24 (2H, m), 7.20−7.11
(2H, m), 7.03 (2H, d, J = 7.6 Hz), 7.00 (1H, t, J = 7.9 Hz), 3.50 (3H,
s).
(1H, s), 6.58 (1H, dd, J = 2.0, 8.0 Hz), 3.73 (2H, d, J = 6.5 Hz), 1.87−
1.84 (2H, m), 1.78−1.74 (4H, m), 1.31−1.28 (3H, m). MS (EI) m/z:
324 (M+). HRMS calcd for C20H25N2O2, 324.184; found, 324.184;
purity 95.9% (HPLC).
2-(3-Benzyloxyphenylamino)benzamide (32). Yield 31%; mp
105−107 °C. 1H NMR (CDCl3, 600 MHz, δ, ppm) 9.51 (1H, s), 7.46
(1H, dd, J = 1.5, 8.0 Hz), 7.43 (2H, d, J = 9.0 Hz), 7.39 (2H, t, J = 7.5
Hz), 7.33 (1H, t, J = 2.5 Hz), 7.28 (1H, dd, J = 1.5, 7.0 Hz), 7.26 (1H,
s), 7.21 (1H, t, J = 7.5 Hz), 6.85 (1H, t, J = 2.5 Hz), 6.80 (1H, dd, J =
1.5, 8.0 Hz), 6.76 (1H, t, J = 7.0 Hz), 6.66 (1H, dd, J = 1.5, 7.5 Hz),
5.03 (2H, s). MS (EI) m/z: 318 (M+). HRMS calcd for C20H18N2O2,
318.137; found, 318.137; purity 97.4% (HPLC).
S t e p 2 : P r e p a r a t i o n o f 2 - ( 3 - N - M e t h y l - N -
phenylbenzamidoamino)benzamide (23). Compound 23 was
prepared from 73 and 39 using the procedure described for 8 (step
1
3) in 53% yield; mp 157−159 °C. H NMR (CDCl3, 500 MHz, δ,
ppm) 9.45 (1H, s), 7.43 (1H, dd, J = 1.5, 7.8 Hz), 7.28 (2H, t, J = 7.8
Hz), 7.21−7.12 (4H, m), 7.06 (2H, d, J = 8.5 Hz), 7.01 (2H, dd, J =
1.5, 7.5 Hz), 6.76−6.70 (2H, m), 3.49 (3H, s). 13C NMR (CDCl3, 125
MHz, δ, ppm) 171.50, 170.56, 145.79, 144.91, 140.70, 137.06, 133.02,
129.24, 129.07, 128.19, 126.77, 126.35, 123.03, 122.79, 120.70, 117.82,
116.08, 115.16, 38.37. MS (EI) m/z: 345 (M+). HRMS (EI) m/z:
calcd for C21H19N3O2, 345.148; found, 345.148. Anal.
(C21H19N3O2·1/3H2O) C, H, N.
2-((3-Phenethoxyphenyl)amino)benzamide (33a). Yield 6%;
mp 129−131 °C. 1H NMR (CDCl3, 600 MHz, δ; ppm) 9.49 (1H, s),
7.47 (1H, d, J = 8.4 Hz), 7.36 (1H, t, J = 8.0 Hz), 7.34−7.22 (3H, m),
7.19 (1H, d, J = 8.5 Hz), 6.82−6.74 (4H, m), 6.58 (1H, d, J = 8.5 Hz),
4.16 (2H, t, J = 7.0 Hz), 3.09 (2H, t, J = 7.0 Hz). MS (EI) m/z: 332
(M+). HRMS calcd for C21H20N2O2, 332.152; found, 332.153; purity
98.7% (HPLC).
2-{3-(3-Fluorophenethyloxy)phenylamino}benzamide (33i).
Step 1: Preparation of 2-(3-Hydroxyphenylamino)benzamide (86).
A solution of 32 (150 mg, 0.47 mmol) in EtOH (4 mL) and 10% Pd/
C (30 mg) was vigorously stirred under hydrogen gas at room
temperature for 6 h. After complete disappearance of the starting
material, the reaction mixture was filtered through Celite and the
filtrate was concentrated in vacuo. The residue was purified by silica
gel flash column chromatography (AcOEt/n-hexane = 2/1) to give 86
Compounds 25 and 27 were prepared from 74 and 76, respectively,
using the procedure described for 23.
2-(3-N-Methyl-N-benzylbenzamidoamino)benzamide (25).
1
Yield 45%; mp 60−62 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.60 (1H, s), 9.56 (1H, s), 7.45 (2H, m), 7.37−7.28 (14H, m), 7.20−
7.16 (6H, m), 7.10−7.05 (2H, m), 6.80−6.76 (2H, m), 4.75 (2H, s),
4.55 (2H, s), 3.01 (3H, s), 2.88 (3H, s). 13C NMR (CDCl3, 125 MHz,
δ, ppm) 171.53, 133.04, 129.61, 129.49, 128.87, 128.73, 128.29,
128.17, 127.61, 126.82, 122.05, 121.03, 120.53, 119.24, 118.49, 118.22,
115.72, 55.21, 5.78, 37.00, 33.10. MS (EI) m/z: 359 (M+). HRMS (EI)
m/z: calcd for C22H21N3O2, 359.163; found, 359.163; purity 98.2%
(HPLC).
1
(78 mg, 73%) as a colorless solid. H NMR (CDCl3, 500 MHz, δ,
ppm) 9.45 (1H, s), 7.46 (1H, dd, J = 1.5, 7.8 Hz), 7.38 (1H, d, J = 8.5
Hz), 7.30 (1H, dt, J = 1.5, 8.2 Hz), 7.15 (1H, t, J = 8.2 Hz), 6.82−6.75
(2H, m), 6.72 (1H, t, J = 2.1 Hz), 6.51 (1H, dd, J = 1.5, 8.2 Hz), 5.55
(broad s, 1H).
2-(3-N-Methylbenzamidophenylamino)benzamide (27).
Yield 53%; mp 178−180 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
Step 2: 2-{3-(3-Fluorophenethyloxy)phenylamino}benzamide
(33i). To a stirred solution of 86 (100 mg, 0.430 mmol) obtained
above and potassium carbonate (191 mg, 1.31 mmol) in acetone (10
mL) was added portionwise 3-fluorophenethyl bromide (185 μL, 1.31
mmol). The mixture was refluxed for 12 h, allowed to cool to room
temperature, and then filtered. The filtrate was concentrated, and the
residue was purified by silica gel flash column chromatography
(AcOEt/n-hexane = 1/3 to 1/2) to give 33i as a colorless solid, which
was recrystallized from CHCl3/n-hexane to give a colorless solid (76
1
9.49 (1H, s), 7.41 (1H, dd, J = 1.8, 7.8 Hz), 7.38 (2H, dd, J = 1.5, 7.8
Hz), 7.32 (1H, tt, J = 1.2, 7.2 Hz), 7.28 (2H, td, J = 1.5, 8.2 Hz), 7.21
(1H, t, J = 8.0 Hz), 7.07 (1H, dt, J = 1.5, 8.5 Hz), 6.90−6.74 (1H, m),
6.84−6.80 (2H, m), 6.73 (1H, dt, J = 0.9, 8.2 Hz), 6.40 (1H, d, J = 8.5
Hz), 3.49 (3H, s). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.47,
170.46, 145.82, 145.44, 141.99, 135.95, 133.04, 130.18, 129.70, 128.92,
128.19, 127.90, 120.08, 119.49, 119.41, 118.13, 116.31, 115.07, 38.27.
MS (EI) m/z: 345 (M+). HRMS (EI) m/z: calcd for C21H19N3O2,
345.148; found, 345.148. Anal. (C21H19N3O2·1/10H2O) C, H, N.
2-(3-Isobutoxyphenylamino)benzamide (31). Step 1: Prep-
aration of 1-Iodo-3-isobutoxybenzene (83). A mixture of 3-
iodophenol 81 (5.00 g, 22.7 mmol) and 1-bromo-2-methylpropane
(3.10 g, 22.7 mmol) in acetone (10 mL) was refluxed for 18 h. The
reaction mixture was poured into water and extracted with AcOEt. The
AcOEt layer was separated, washed with water and brine, and dried
over Na2SO4. Filtration, concentration of the filtrate in vacuo, and
purification of the residue by silica gel flash column chromatography
(AcOEt/n-hexane = 1/10) gave 2.90 g (46%) of 83 as a colorless oil.
1H NMR (CDCl3, 500 MHz, δ, ppm) 7.26 (2H, m), 6.98 (1H, t, J =
1
mg, 50%); mp 114−116 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.49 (1H, s), 7.47 (1H, dd, J = 1.5, 7.8 Hz), 7.37 (1H, dd, J = 1.2, 8.5
Hz), 7.32−7.27 (2H, m), 7.20 (1H, t, J = 8.2 Hz), 7.05 (1H, d, J = 7.6
Hz), 7.00 (1H, td, J = 1.8, 8.2 Hz), 6.93 (1H, ddt, J = 0.9, 2.4, 8.5 Hz),
6.81 (1H, dd, J = 1.5, 7.5 Hz), 6.79−6.75 (2H, m), 6.57 (1H, ddd, J =
0.9, 1.5, 8.5 Hz), 4.15 (2H, t, J = 7.0 Hz), 3.08 (2H, t, J = 7.0 Hz). 13C
1
NMR (CDCl3, 150 MHz, δ, ppm) 171.58, 162.80 (d, JC−F = 244.2
3
Hz),159.67, 146.14, 142.62, 140.88 (d, JC−F = 7.5 Hz), 132.95,
3
130.00, 129.86 (d, ′JC−F = 7.9 Hz), 128.26, 124.64, 124.62, 117.76,
2
2
116.18, 115.91 (d, JC−F = 19.3 Hz), 113.81, 113.37 (d, ′JC−F = 21.0
Hz), 108.94, 107.477, 68.19, 35.48. MS (EI) m/z: 350 (M+). HRMS
calcd for C21H19FN2O2, 350.143; found, 350.142. Anal.
(C21H19FN2O2) C, H, N.
8.0 Hz), 6.85 (1H, m), 3.68 (2H, d, J = 7.0 Hz), 2.06 (1H, m), 1.01
(6H, m, J = 7.0 Hz).
Compounds 33b−h, 33j−p, and 34 were prepared from 86 and an
appropriate alkyl bromide using the procedure described for 33i (step
2).
Step 2: Preparation of 2-(3-Isobutoxyphenylamino)benzamide
(31). Compound 31 was prepared from 83 and 39 using the procedure
described for 8 (step 3) in 13% yield; mp 77−79 °C. 1H NMR
(CDCl3, 500 MHz, δ; ppm) 9.48 (1H, s), 7.48 (1H, dd, J = 1.5, 8.0
Hz), 7.38 (1H, dd, J = 1.0, 8.5 Hz), 7.31 (1H, t, J = 7.5 Hz), 7.20 (1H,
t, J = 8.0 Hz), 6.80 (1H, d, J = 8.0 Hz), 6.80−6.75 (2H, m), 6.58 (1H,
dd, J = 2.0, 8.0 Hz), 3.70 (2H, d, J = 6.5 Hz), 2.07 (1H, m), 1.01 (6H,
d, J = 6.5 Hz). MS (EI) m/z: 284 (M+). HRMS calcd for C17H20N2O2,
284.153; found, 284.153; purity 96.1% (HPLC).
2-{3-(2-Methylphenethyloxy)phenylamino}benzamide
1
(33b). Yield 30%; mp 120−122 °C. H NMR (CDCl3, 500 MHz, δ,
ppm) 9.48 (1H, s), 7.45 (1H, dd, J = 1.5, 7.9 Hz), 7.36 (1H, dd, J =
0.9, 8.5 Hz), 7.30 (1H, dd, J = 1.5, 7.0 Hz), 7.23−7.18 (2H, m), 7.17−
7.13 (3H, m), 6.80 (1H, dd, J = 1.5, 7.9 Hz), 6.78−6.73 (2H, m), 6.58
(1H, dd, J = 2.4, 8.0 Hz), 4.13 (2H, t, J = 7.3 Hz), 3.10 (2H, t, J = 7.3
Hz), 2.37 (3H, s). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.58,
159.81, 146.19, 142.59, 136.49, 136.23, 132.95, 130.32, 129.98, 129.52,
128.24, 126.66, 126.10, 117.70, 116.14, 115.85, 113.72, 108.90, 107.49,
67.73, 33.04, 19.49. MS (EI) m/z: 346 (M+). HRMS calcd for
C22H22N2O2, 346.168; found, 346.168; Anal. (C22H22N2O2) C, H, N.
2-{3-(3-Methylphenethoxy)phenylamino}benzamide (33c).
Compounds 30, 32, and 33a were prepared from 3-bromophenol
80 or 3-iodophenol 81 using the procedure described for 31.
2-(3-Cyclohexylmethoxyphenylamino)benzamide (30). Yield
1
13%; mp 123−125 °C. H NMR (CDCl3, 500 MHz, δ, ppm) 9.48
(1H, s), 7.46 (1H, dd, J = 1.5, 8.0 Hz), 7.38 (1H, d, J = 7.5 Hz), 7.19
(1H, t, J = 8.0 Hz), 6.79 (1H, dd, J = 1.5, 8.0 Hz), 6.76 (1H, m), 6.75
1
Yield 43%; mp 109−111 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
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9.48 (1H, s), 7.46 (1H, dd, J = 1.5, 7.5 Hz), 7.37 (1H, dd, J = 1.2, 8.5
Hz), 7.29 (1H, dt, J = 1.2, 7.2 Hz), 7.23−7.16 (2H, m), 7.10 (2H, s),
7.06 (1H, t, J = 7.6 Hz), 6.80 (1H, d, J = 6.4 Hz), 6.79−6.74 (2H, m),
6.58 (1H, ddd, J = 0.9, 2.4, 7.2 Hz), 4.14 (2H, t, J = 7.3 Hz), 3.05 (2H,
t, J = 7.3 Hz), 2.34 (3H, s). MS (EI) m/z: 346 (M+). HRMS calcd for
C22H22N2O2, 346.168; found, 346.168. Anal. (C22H22N2O2·1/5H2O)
C, H, N.
7.31 (1H, dd, J = 1.5, 7.0 Hz), 7.25−7.21 (2H, m), 7.19 (1H, t, J = 8.2
Hz), 7.00 (2H, t, J = 8.5 Hz), 6.81 (1H, dd, J = 2.1, 7.8 Hz), 6.77 (1H,
dd, J = 1.5, 7.0 Hz), 6.76−6.73 (1H, m), 6.57 (1H, dd, J = 2.4, 7.2 Hz),
4.13 (2H, t, J = 6.8 Hz), 3.05 (2H, t, J = 6.8 Hz). 13C NMR (CDCl3,
1
125 MHz, δ; ppm) 171.57, 161.75 (d, JC−F = 238.7 Hz),159.74,
3
146.19, 142.61, 134.00, 132.94, 130.42 (d, JC−F = 8.3 Hz), 129.99,
3
128.26, 117.74, 116.16, 115.84, 115.24 (d, JC−F = 22.2 Hz), 113.78,
108.97, 107.53, 68.59, 34.97. MS (EI) m/z: 350 (M+). HRMS calcd for
C21H19FN2O2, 350.143; found, 350.143. Anal. (C21H19FN2O2) C, H,
N.
2-{3-(4-Methylphenethyloxy)phenylamino}benzamide
1
(33d). Yield 69%; mp 189−191 °C. H NMR (CDCl3, 500 MHz, δ,
ppm) 9.47 (1H, s), 7.45 (1H, dd, J = 1.5, 7.8 Hz), 7.36 (1H, dd, J =
1.2, 8.5 Hz), 7.29 (1H, dt, J = 1.2, 7.0 Hz), 7.21−7.15 (3H, m), 7.12
(2H, d, J = 8.0 Hz), 6.79 (1H, dd, J = 1.5, 7.8 Hz), 6.78−6.73 (2H, m),
6.57 (1H, ddd, J = 0.9, 1.5, 7.5 Hz), 4.13 (3H, t, J = 7.3 Hz), 3.05 (3H,
t, J = 7.3 Hz), 2.33 (3H, s). 13C NMR (CDCl3, 125 MHz, δ, ppm)
171. 59, 159.83, 146.21, 142.56, 136.01, 135.09, 132.94, 129.95,
129.17, 128.88, 128.24, 117.68, 116.13, 115.85, 113.72, 108.99, 107.56,
68.82, 35.33, 21.04. MS (EI) m/z: 346 (M+). HRMS calcd for
C22H22N2O2, 346.168; found, 346.167. Anal. (C22H22N2O2) C, H, N.
2-{3-(2-Trifluoromethylphenethyloxy)phenylamino}-
benzamide (33e). Yield 18%; mp 89−91 °C. 1H NMR (CDCl3, 500
MHz, δ, ppm) 9.48 (1H, d, J = 7.6 Hz), 7.65 (1H, d, J = 7.8 Hz),
7.51−7.45 (3H, m), 7.36 (1H, dd, J = 0.9, 8.5 Hz), 7.34−7.28 (2H,
m), 7.20 (1H, t, J = 8.2 Hz), 6.81 (1H, dd, J = 2.1, 7.8 Hz), 6.78−6.74
(2H, m), 6.57 (1H, dd, J = 2.1, 8.5 Hz), 4.17 (2H, t, J = 6.8 Hz), 3.28
(2H, t, J = 6.8 Hz). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.58,
159.67, 146.19, 142.64, 136.79, 132.95, 132.01, 131.74, 130.01, 128.25,
126.64, 126.07, 126.03, 117.75, 116.18, 115.86, 113.83, 108.89, 107.59,
67.96, 32.50. MS (EI) m/z: 400 (M+). HRMS calcd for
C22H19F3N2O2, 400.140; found, 400.140. Anal. (C22H19F3N2O2·1/
3H2O) C, H, N.
2-{3-(2-Chlorophenethyloxy)phenylamino}benzamide (33k).
Yield 28%; mp 118−120 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
1
9.48 (1H, s), 7.46 (1H, dd, J = 1.8, 8.0 Hz), 7.38−7.34 (2H, m), 7.32
(1H, dd, J = 2.1, 7.0 Hz), 7.30 (1H, dt, J = 1.5, 7.0 Hz), 7.23−7.16
(3H, m), 6.80 (1H, dd, J = 1.8, 7.0 Hz), 6.78−6.73 (2H, m), 6.59 (1H,
dd, J = 2.4, 8.0 Hz), 4.18 (2H, t, J = 7.0 Hz), 3.23 (2H, t, J = 7.0 Hz).
13C NMR (CDCl3, 125 MHz, δ, ppm) 171.57, 159.70, 146.18, 142.59,
135.79, 134.21, 132.95, 131.32, 129.98, 129.52, 128.23, 128.04, 126.84,
117.71, 116.14, 115.85, 113.75, 108.95, 107.54, 66.73, 33.53. MS (EI)
m/z: 366, 368 (M+). HRMS calcd for C21H1935ClN2O2, 366.114;
found, 366.112; calcd for C21H1937ClN2O2, 368.111; found, 368.109.
Anal. (C21H19ClN2O2) C, H, N.
2-{3-(3-Chlorophenethyloxy)phenylamino}benzamide (33l).
1
Yield 65%; mp 129−131 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.49 (1H, s), 7.46 (1H, dd, J = 1.2, 7.8 Hz), 7.36 (1H, dd, J = 1.2, 8.5
Hz), 7.31 (1H, dd, J = 1.5, 7.0 Hz), 7.29−7.27 (1H, m), 7.24−7.19
(2H, m), 7.18−7.15 (2H, m), 6.81 (1H, dd, J = 1.8, 7.0 Hz), 6.79−
6.74 (2H, m), 6.57 (1H, dd, J = 1.5, 7.0 Hz), 4.15 (2H, t, J = 6.7 Hz),
3.06 (2H, t, J = 6.7 Hz). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.55,
159.65, 146.15, 142.62, 140.38, 134.20, 132.95, 135.01, 129.70, 129.14,
128.25, 127.21, 126.69, 117.76, 116.17, 115.85, 113.83, 108.93, 107.48,
68.16, 35.42. MS (EI) m/z: 366, 368 (M+). HRMS calcd for
C21H1935ClN2O2, 366.114; found, 366.113; calcd for C21H1937ClN2O2,
368.111; found, 368.109. Anal. (C21H19ClN2O2·1/10H2O) C, H, N.
2-{3-(4-Chlorophenethoxy)phenylamino}benzamide (33m).
2-{3-(3-Trifluoromethylphenethyloxy)phenylamino}-
1
benzamide (33f). Yield 26%; mp 90−92 °C. H NMR (CDCl3, 500
MHz, δ, ppm) 9.49 (1H, s), 7.54 (1H, broad s), 7.50 (1H, d, J = 8.2
Hz), 7.46 (2H, dd, J = 1.2, 7.6 Hz), 7.42 (1H, t, J = 7.6 Hz), 7.36 (1H,
dd, J = 1.2, 8.2 Hz), 7.30 (1H, dt, J = 1.5, 7.0 Hz), 7.19 (1H, t, J = 8.0
Hz), 6.81 (1H, dd, J = 1.5, 7.5 Hz), 6.79−6.70 (2H, m), 6.57 (1H, dd,
J = 2.4, 8.0 Hz), 4.17 (2H, t, J = 6.7 Hz), 3.14 (2H, t, J = 6.7 Hz). 13C
NMR (CDCl3, 125 MHz, δ, ppm) 171.60, 159.60, 146.11, 142.64,
139.35, 132.94, 132.43, 130.02, 128.87, 128.26, 125.80, 123.40, 123.37,
123.0, 117.79, 116.20, 115.86, 113.84, 108.89, 107.43, 68.08, 35.56.
MS (EI) m/z: 400 (M+). HRMS calcd for C22H19F3N2O2, 400.140;
found, 400.141. Anal. (C22H19F3N2O2) C, H, N.
1
Yield 41%; mp 192−194 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.45 (1H, s), 7.46 (1H, dd, J = 1.5, 7.8 Hz), 7.36 (1H, dd, J = 0.9, 8.2
Hz), 7.32−7.26 (3H, m), 7.23−7.16 (3H, m), 6.81 (1H, dd, J = 1.5,
7.8 Hz), 6.79−6.72 (2H, m), 6.56 (1H, dd, J = 2.4, 8.2 Hz), 4.13 (2H,
t, J = 7.0 Hz), 3.05 (2H, t, J = 7.0 Hz). 13C NMR (CDCl3, 125 MHz, δ,
ppm) 171.56, 159.68, 146.15, 142.61, 136.83, 132.94, 132.29, 130.35,
129.99, 128.57, 128.25, 117.76, 116.17, 115.83, 113.80, 108.93, 107.48,
68.31, 35.11. MS (EI) m/z: 366, 368 (M+). HRMS calcd for
C21H1935ClN2O2, 366.114; found, 366.112; calcd for C21H1937ClN2O2,
368.111; found, 368.112. Anal. (C21H19ClN2O2·1/10H2O) C, H, N.
2-{3-(2-Bromophenethyloxy)phenylamino}benzamide
2-{3-(4-Trifluoromethylphenethyloxy)phenylamino}-
1
benzamide (33g). Yield 11%; mp 179−181 °C. H NMR (CDCl3,
500 MHz, δ, ppm) 9.49 (1H, s), 7.56 (2H, d, J = 8.2 Hz), 7.46 (1H,
dd, J = 1.5, 7.9 Hz), 7.40 (2H, d, J = 7.9 Hz), 7.36 (1H, dd, J = 0.9, 8.2
Hz), 7.30 (1H, dt, J = 1.5, 7.2 Hz), 7.19 (1H, t, J = 8.2 Hz), 6.81 (1H,
dd, J = 1.5, 7.9 Hz), 6.79−6.70 (2H, m), 6.56 (1H, dd, J = 2.7, 8.2 Hz),
4.18 (2H, t, J = 6.5 Hz), 3.14 (2H, t, J = 6.5 Hz). 13C NMR (CDCl3,
125 MHz, δ, ppm) 171.56, 159.61, 146.13, 142.65, 142.56, 132.94,
130.03, 129.34, 128.27, 125.39, 125.335, 125.31, 117.80, 116.20,
115.84, 113.85, 108.90, 107.46, 67.99, 35.57. MS (EI) m/z: 400 (M+).
HRMS calcd for C22H19F3N2O2, 400.140; found, 400.140; purity
99.2% (HPLC).
1
(33n). Yield 24%; mp 118−120 °C. H NMR (CDCl3, 500 MHz, δ,
ppm) 9.48 (1H, s), 7.55 (1H, dd, J = 1.2, 7.9 Hz), 7.46 (1H, dd, J =
1.5, 7.5 Hz), 7.37 (1H, dd, J = 0.9, 7.3 Hz), 7.32 (1H, dt, J = 1.8, 7.9
Hz), 7.30−7.27 (2H, m), 7.19 (1H, t, J = 8.2 Hz), 7.10 (1H, dt, J = 1.5,
7.5 Hz), 6.80 (1H, dd, J = 1.8, 7.5 Hz), 6.78−6.74 (2H, m), 6.60 (1H,
dd, J = 2.4, 8.2 Hz), 4.18 (2H, t, J = 7.0 Hz), 3.23 (2H, t, J = 7.0 Hz).
13C NMR (CDCl3, 125 MHz, δ, ppm) 171.58, 159.70, 146.17, 132.95,
132.85, 131.33, 131.25, 129.98, 128.29, 128.24, 128.20, 127.49, 127.40,
117.72, 115.90, 115.86, 113.74, 108.97, 107.53, 66.81, 36.01. MS (EI)
m/z: 410, 412 (M+). HRMS calcd for C21H1979BrN2O2, 410.063;
found, 410.062; calcd for C21H1981BrN2O2, 412.061; found, 412.062.
Anal. (C21H19BrN2O2·1/10H2O) C, H, N.
2-{3-(2-Fluorophenethyloxy)phenylamino}benzamide (33h).
1
Yield 21%; mp 113−115 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.48 (1H, s), 7.45 (1H, dd, J = 1.5, 7.6 Hz), 7.36 (1H, dd, J = 0.9, 7.6
Hz), 7.32−7.27 (2H, m), 7.24−7.16 (2H, m), 7.08 (1H, dt, J = 1.2, 6.3
Hz), 7.05 (1H, dt, J = 1.2, 8.5 Hz), 6.80 (1H, d, J = 1.5, 6.3 Hz), 6.78−
6.73 (2H, m), 6.57 (1H, dd, J = 1.8, 8.0 Hz), 4.16 (2H, t, J = 7.0 Hz),
3.13 (2H, t, J = 7.0 Hz). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.57,
2-{3-(3-Bromophenethoxy)phenylamino}benzamide (33o).
1
Yield 31%; mp 133−135 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.49 (1H, s), 7.47 (1H, dd, J = 1.5, 7.8 Hz), 7.44 (1H, t, J = 2.1 Hz),
7.36 (2H, d, J = 7.8 Hz), 7.30 (1H, dt, J = 1.2, 7.0 Hz), 7.23−7.15 (3H,
m), 6.82−6.76 (1H, m), 6.78 (1H, t, J = 0.9 Hz), 6.75 (1H, t, J = 2.1
Hz), 6.57 (1H, ddd, J = 0.9, 1.8, 8.2 Hz), 4.14 (2H, t, J = 7.0 Hz), 3.05
(2H, t, J = 7.0 Hz). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.55,
159.64, 146.14, 142.62, 140.70, 132.96, 132.05, 130.00, 129.62, 128.24,
127.68, 122.47, 117.76, 116.18, 115.85, 113.84, 108.93, 107.48, 100.57,
68.16, 35.38. MS (EI) m/z: 410, 412 (M+). HRMS calcd for
C21H1979Br N2O2, 410.063; found, 410.062; calcd for C21H19N2O281Br,
412.061; found, 412.061. Anal. (C21H19N2O2B·1/4H2O) C, H, N.
1
161.25 (d, JC−F = 243.7 Hz), 159.71, 146.18, 142.58, 132.94, 131.41,
3
3
129.97, 128.27 (d, JC−F = 10.2 Hz), 125.00 (d, ′JC−F = 16.4 Hz),
4
2
124.05 (d, JC−F = 4.1 Hz), 124.03, 117.70, 116.00 (d, ′JC−F = 37.0
2
Hz), 115.28 (d, JC−F = 22.6 Hz), 115.12, 113.76, 108.93, 107.55,
67.17, 29.16. MS (EI) m/z: 350 (M+). HRMS calcd for C21H19FN2O2,
350.143; found, 350.143. Anal. (C21H19FN2O2) C, H, N.
2-{3-(4-Fluorophenethyloxy)phenylamino}benzamide (33j).
1
Yield 72%; mp 163−165 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
9.49 (1H, s), 7.46 (1H, dd, J = 1.5, 7.2 Hz), 7.36 (1H, d, J = 8.5 Hz),
L
dx.doi.org/10.1021/jm3002108 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2-{3-(4-Bromophenethoxy)phenylamino}benzamide (33p).
Yield 16%; mp 154−156 °C. H NMR (CDCl3, 500 MHz, δ, ppm)
both the protein and the ligand allowed to move, using Macromodel
8.1 software. The conformation of compounds 7b and 7t in the active
site was minimized by MM calculation based upon the OPLS-AA force
field with parameters set as follows; Solvent, water; method, LBFGS;
max no. of iterations, 10000; converge on, gradient; convergence
threshold, 0.05.
1
7.47 (1H, dd, J = 1.5, 8.0 Hz), 7.43 (2H, d, J = 8.0 Hz), 7.37−7.29
(2H, m), 7.19 (1H, t, J = 8.3 Hz), 7.16 (2H, d, J = 8.5 Hz), 6.81 (1H,
d, J = 7.5 Hz), 6.77 (1H, t, J = 8.5 Hz), 6.74 (1H, m), 6.62−6.60 (1H,
m), 6.56 (1H, dd, J = 2.0, 8.0 Hz), 4.13 (2H, t, J = 6.8 Hz), 3.04 (2H, t,
J = 6.8 Hz). 13C NMR (CDCl3, 125 MHz, δ, ppm) 171.76, 159.67,
146.14, 142.62, 137.37, 132.94, 131.63, 130.08, 129.04, 128.25, 117.76,
115.84, 114.02, 113.80, 109.22, 108.92, 107.48, 68.22, 35.17. MS (EI)
m/z: 410, 412 (M+). HRMS calcd for C21H1979BrN2O2, 410.063;
found, 410.063; calcd for C21H19N2O281Br, 412.061; found, 412.061;
purity 97.0% (HPLC).
ASSOCIATED CONTENT
* Supporting Information
■
S
Results of elemental analysis of compounds 12, 14, 17, 19, 20,
22−24, 26, 27, 33b−f, and 33h−o. This material is available
free of charge via the Internet at http://pubs.acs.org.
Biology. SIRT Enzyme Assays. SIRT activity assay was performed
using SIRT fluorimetric drug discovery kits (AK-555, AK-556, and AK-
557, BIOMOL Research Laboratories), according to the supplier’s
protocol. SIRT (human, recombinant) (15 μL/well), NAD+ (1 mM),
and various concentrations of samples were incubated at 37 °C for 60
min, and Fluor de Lys-SIRT substrate (25 μM) was added to the
mixture. Reactions were stopped after 60 min by adding Fluor de Lys
Developer II with nicotinamide, which stops further deacetylation.
Then, 45 min after addition of this developer, the fluorescence of the
wells was measured on a fluorometric reader with excitation set at 360
nm and emission detection set at 460 nm. The value of % inhibition
was calculated from the fluorescence readings of inhibited wells
relative to those of control wells. The compound concentration
resulting in 50% inhibition was determined by plotting log[Inh] versus
the logit function of % inhibition. IC50 values were determined by
means of regression analysis of the concentration/inhibition data.
Western Blot Analysis. HCT116 human colon cancer cells were
purchased from American Type Culture Collection (ATCC, Manassas,
VA, U.S.A.) and cultured in McCoy’s 5A culture medium containing
penicillin and streptomycin, which was supplemented with fetal bovine
serum as described in the ATCC instructions. HCT-116 cells (5 ×
105) were treated for 8 h with 20 μM etoposide and 10 μM AGK2 (3),
10 μM EX-527 (2), and 1 μM and 10 μM 33a in 10% FBS-
supplemented McCoy’s 5A medium and then collected and extracted
with SDS buffer. Protein concentrations of lysates were determined
using a Bradford protein assay kit (Bio-Rad Laboratories), with which
equivalent amounts of protein from each lysate were resolved in 15%
SDS-polyacrylamide gels and then transferred onto nitrocellulose
membranes (Bio-Rad Laboratories). After having been blocked for 30
min with Tris-buffered saline (TBS) containing 3% skim milk, the
transblotted membrane was incubated overnight at 4 °C with
acetylated p53 antibody (Cell Signaling) (1:500 dilution), p53
antibody (Calbiochem) (1:500 dilution), acetylated α-tubulin antibody
(SIGMA) (1:2000 dilution), or α-tubulin antibody (SIGMA) (1:2500
dilution) in TBS containing 3% skim milk. The membrane was probed
with the primary antibody and then washed twice with water,
incubated with goat antirabbit (for acetylated p53) or antimouse (for
p53, α-tubulin, and acetylated α-tubulin) IgG-horseradish peroxidase
conjugates (1:2500 dilution) for 2 h at room temperature, and again
washed twice with water. The immunoblots were visualized by
enhanced chemiluminescence.
Molecular Modeling. Homology models for SIRT1 and SIRT2
based on the crystal structure of Hst2 (PDB code 1Q17) were built
using the homology modeling procedure of the Full Automatic
Modeling System (FAMS). Although the X-ray crystal structure of
SIRT2 has been reported,16 we used the crystal structure of Hst2
complexed with an NAD+ analogue17 for homology modeling, because
the reported SIRT2 structure is an apo-structure, not a substrate-
bound structure, which is not suitable for our drug design of inhibitors
competitive with the acetylated lysine substrate.13b The homology
models of SIRT1 and SIRT2 were used as the target structures for
docking. Protein preparation, receptor grid generation, and ligand
docking were performed using the software Glide 3.5. Compounds 7b
and 7t were docked into the active site of the protein. The standard
precision mode of Glide was used to determine favorable binding
poses, which allowed the ligand conformation to be flexibly explored
while holding the protein as a rigid structure during docking. The
predicted complex structure was then fully energy-minimized, with
AUTHOR INFORMATION
Corresponding Author
*Phone and fax: +81-52-836-3407. E-mail: suzukit@koto.kpu-
m.ac.jp (T.S.); miyata-n@phar.nagoya-cu.ac.jp (N.M.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the JST A-STEP program
(T.S.) and by a Grant-in-Aid for Scientific Research from the
Japan Society for the Promotion of Science (M.N., A.K.).
ABBREVIATIONS USED
SIRT, sirtuin; HDAC, histone deacetylase; SPR, surface
plasmon resonance
■
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