Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach

Article abstract of DOI:10.1021/jm500777s

Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD⁺-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the d

Full text of DOI:10.1021/jm500777s

Article
pubs.acs.org/jmc
Discovery of Potent and Selective Sirtuin 2 (SIRT2) Inhibitors Using a
Fragment-Based Approach
Huaqing Cui, Zeeshan Kamal, Teng Ai, Yanli Xu, Swati S. More, Daniel J. Wilson, and Liqiang Chen*
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street SE, Minneapolis, Minnesota 55455,
United States
S
* Supporting Information
ABSTRACT: Sirtuin 2 (SIRT2) is one of the sirtuins, a family
of NAD+-dependent deacetylases that act on a variety of
histone and non-histone substrates. Accumulating biological
functions and potential therapeutic applications have drawn
interest in the discovery and development of SIRT2 inhibitors.
Herein we report our discovery of novel SIRT2 inhibitors
using a fragment-based approach. Inspired by the purported
close binding proximity of suramin and nicotinamide, we
prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene−benzamides and −nicotinamides.
Biochemical evaluation of these two series provided structure−activity relationship (SAR) information, which led to the design of
(5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2
activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-
tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed
that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor
against NAD+. Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel
structure merits further exploration.
treat diverse diseases. Activation of SIRT1 has been proposed
as a potential therapy for aging-related diseases such as
metabolic syndromes and neurodegenerative diseases, including
Alzheimer’s and Huntington’s diseases.7 In contrast, inhibition
of SIRT2 may hold promise as a treatment for Parkinson’s
disease and other disorders.7 SIRT2 inhibitors have been shown
to rescue α-synuclein-induced toxicity and reduce cell death in
cellular and Drosophila models of Parkinson’s disease.8 More
recently, SIRT2 has been shown to be a key player in
transcription regulation signaling initiated by bacterial infection,
suggesting that blocking host SIRT2 may be a viable approach
to fight bacterial infection.9 Intriguingly, both tumor-suppress-
ing and tumor-prompting roles have been proposed for
SIRT1−3.10 Even though the precise roles of sirtuins in cancer
pathogenesis remain to be revealed, it is clear that they depend
on the affected tissues and the stage of cancer progression.
Further studies are needed to unravel the therapeutic
applications of sirtuins in not only cancers but also other
diseases, a challenging task that can be facilitated by the
availability of potent and selective sirtuin inhibitors.
INTRODUCTION
■
Sirtuins are commonly classified as class-III histone deacetylases
(HDACs) that use NAD+ as a cofactor to remove an acetyl
group from the lysine residue, whereas classical HDACs are
zinc-dependent. There are seven human sirtuins (SIRT1−
SIRT7) with distinctive subcellular localizations and enzymatic
functions.1 SIRT1 is mainly a nuclear protein, while SIRT2
predominantly resides in the cytoplasm. SIRT3−5 are
mitochondrial, but SIRT6 and SIRT7 are localized to the
nucleus. As depicted in Figure 1, SIRT1−3 remove the acetyl
group from the acetylated lysine residue of substrate 1 in the
presence of NAD+, releasing substrate 2 bearing a lysine, 2′-O-
acetyl-ADP-ribose (3), and its regioisomer 3′-O-acetyl-ADP-
ribose.2 Nicotinamide (4) (Figure 1), another product of
sirtuin-catalyzed reactions, is a physiological, albeit weak,
inhibitor of sirtuins.3 In contrast to SIRT1−3, SIRT4 lacks
deacetylase activity but catalyzes ADP-ribosylation. Even
though SIRT5 was identified as a deacetylase, it has recently
been reported to primarily possess demalonylation and
desuccinylation activities.4 SIRT6 catalyzes both deacetylation
and ADP-ribosylation. SIRT7, the least-studied sirtuin, has
recently been shown to deacetylate histone H3K18.5
In addition to nicotinamide, a wide range of chemical
structures have been reported as sirtuin inhibitors,11 some of
which are depicted in Figure 2. As examples of initial inhibitors,
sirtinol (5),12 cambinol (6),13 and splitomicin (7)14 all feature a
β-naphthol moiety and have been subjected to structural
Sirtuins deacetylate histone as well as numerous cellular
substrates, and they have been implicated in a wide range of
biological functions, including chromatin modification, gene
transcription, metabolism, mitosis, stress response, DNA
damage repair, and cell survival.1b,6 Consequently, modulation
of sirtuins has emerged as a promising therapeutic strategy to
Received: May 19, 2014
© XXXX American Chemical Society
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Figure 1. Deacetylation catalyzed by sirtuins.
Figure 2. Examples of sirtuin inhibitors.
Figure 3. Selected SIRT2 inhibitors.
optimization. EX-527 (8),15 one of the most potent and
selective SIRT1 inhibitors, has recently been reported to
alleviate pathology of Huntington’s disease.16 Suramin (9)17 is
slightly selective against SIRT1 over SIRT2 and SIRT3, but it
suffers from high molecular weight and polarity.
Because of SIRT2’s potential therapeutic applications, there
has been a growing interest in the discovery of potent and
selective SIRT2 inhibitors (Figure 3). AGK2 (10) with its
modest activity and selectivity has been shown to mitigate α-
synuclein toxicity and prevent against dopaminergic cell death
in multiple models of Parkinson’s disease.8 Similarly, AK-1 (11)
and its analogues have been evaluated for their therapeutic
application in Huntington’s disease and have been subjected to
extensive structural modifications.18 Ro31-8220 shows sub-
micromolar inhibition against SIRT2, but its antikinase activity
excludes its use as a selective SIRT2 inhibitor.19 Tenovins,
which are represented by tenovin-6 (12) and stem from a high-
throughput screening for activators of p53, exhibit modest anti-
SIRT2 activity.20 In contrast, excellent anti-SIRT2 activity has
been accomplished with macrocyclic peptides equipped with a
mechanism-based warhead,21 but the peptidic nature limits
their applications. More recently, substituted chroman-4-one 13
was shown to be a low-micromolar inhibitor of SIRT2 with
high selectivity for SIRT2 over SIRT1 and SIRT3.22
Compound 14, based on an anilinobenzamide chemotype,
displays submicromolar inhibitory activity and an excellent
selectivity profile.23 A new series of thieno[3,2-d]pyrimidine-6-
carboxamides, as exemplified by compound 15, were reported
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Figure 4. Fragment-based approach toward the design of sirtuin inhibitors.
to possess remarkable low-nanomolar activity against SIRT1−3
but only modest isozyme selectivity.24 Aiming to identify
SIRT2 inhibitors with improved potency and selectivity, we
have used fragment-based ligand design to discover novel
chemotypes, as discussed in this report.
RESULTS AND DISCUSSION
■
Design and Biochemical Evaluations. Fragment-based
drug design has emerged as a powerful strategy to explore
chemical space and identify novel leads for further
optimization.25 The fragment-based approach generally in-
volves screening of a relatively small library of fragments and
subsequent generation of new hit/lead compounds through
structural manipulations, including linking, merging, and
growth. The identification of novel scaffolds leads to further
iterative optimization of activity, selectivity, and drug-like
properties.
Figure 5. Fragments based on naphthylamide sulfonic acids.
Our design of fragments was inspired by close examination of
a crystal structure of SIRT5 in complex with suramin, which
mainly occupies the peptide substrate-binding site with one
sulfonate group protruding into the nicotinamide-binding
site.17a It has been shown that the naphthalene portion (circled
in Figure 4) is close to the binding region proposed for
nicotinamide. On the basis of the fact that suramin displays
inhibitory activities against SIRT1, SIRT2, and SIRT5,17 it is
reasonable to speculate that it binds to sirtuins in a similar
fashion, which may allow for a fragment-based approach to the
discovery of novel sirtuin inhibitors. To this end, we planned to
assemble a focused library of fragments containing structural
features from either suramin or nicotinamide. More impor-
tantly, because of the close binding proximity of these two
known inhibitors, we envisioned that we could generate
fragments by linking nicotinamide or its analogues to the
structural elements extracted from suramin. Because of
suramin’s high molecular weight and polarity, we decided to
first focus on a significantly simplified naphthylamine moiety to
which a sulfonate group is attached at various positions (16,
Figure 4). To enhance structural diversity, the naphthylamine
moiety was further acylated with various benzoyl chlorides to
give substituted benzamides, a functionality that is also present
in suramin. As a result, in this naphthylamide sulfonic acid
series, a sulfonic acid is attached at position 2 (19−24, Figure
5), 4 (25−30), or 8 (31−36). Since suramin contains 4-methyl
and 3-benzamido substituents, we decided to explore 4-methyl
and 3-nitro substituents, which were installed individually or in
combination. Furthermore, the 3-nitro group was reduced to
give a 3-amino group, which if necessary could be further
derivatized for structure−activity relationship (SAR) studies.
In the other set of fragments, we decided to investigate
compounds in which nicotinamide is connected to naphthalene
at position 1 or 2 (17, Figure 4). More specifically,
nicotinamide was attached at the para and meta positions (in
relation to the primary amide) to give fragments 37−48 and
49−58, respectively (Figure 6). Simple linkers such as a
nitrogen, oxygen, or sulfur atom were used. To investigate the
importance of the nicotinamide nitrogen atom, analogous
fragments based on benzamide were also included. We
anticipated that screening of these two sets of fragments
would provide necessary SAR information, allowing us to link
selected fragments to generate inhibitors represented by the
general structure 18 (Figure 4).
These two series of fragments were tested against human
recombinant SIRT1−3 using AMC-tagged deacetylation
substrates. The naphthylamide sulfonic acids generally
exhibited marginal inhibition when tested at 100 μM (Table
S1 in the Supporting Information). However, there are two
noticeable observations. First, the position of sulfonic acid does
not affect the activity against SIRT1−3. Second, compounds
23, 29, and 35, all of which share 4-Me and 3-NO2 groups,
appeared to show modest inhibition at 100 μM, suggesting that
these substituents could be explored in constructing advanced
inhibitors on the basis of the information gathered on the
fragments.
The naphthalene−benzamides and −nicotinamides were also
tested against SIRT1−3 at 100 μM. Fragments 37−42, in
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against SIRT1−3. Nevertheless, fragments 40 and 42 displayed
more than 50% inhibition of SIRT2, suggesting that linking
nicotinamide to naphthalene through an oxygen or sulfur atom
might be productive.
In comparison, fragments 49−53, in which benzamide or
nicotinamide is attached to naphthalene via the meta position,
displayed improved activity (Table 2). Examination of this
Table 2. Inhibitory Activities of Meta-Substituted Fragments
Based on Naphthalene−Benzamide or Naphthalene−
a
Nicotinamide
% inhibition at 100 μM or IC50 (μM)
compound
X
Y
SIRT1
NA
SIRT2
SIRT3
NA
49
50
51
52
53
54
55
56
57
58
NH
NH
O
CH
N
65
5
b
b
b
33.6
4.30
84
12.3
Figure 6. Fragments based on naphthalene−benzamide or naph-
thalene−nicotinamide.
CH
N
47
5
1
49
3
b
b
b
b
b
b
O
13.5
26.9
NA
40.9
35
6.29
12.1
46
2.14
27.9
NA
9.00
35
S
N
NH
NH
O
CH
N
1
2
which the benzamide or nicotinamide is attached at position 2
of naphthalene via the position para to the primary amide
functionality, generally showed very weak inhibition (Table 1).
Similarly, fragments 43−48 in which the linkage happens at
position 1 also generally exhibited weak inhibitory activity
b
b
b
9.70
66
CH
N
7
5
b
b
b
b
b
b
O
4.89
30.3
7.57
9.80
16.6
30.1
S
N
a
Inhibitory activities were determined as described in SIRT1−3
Biochemical Assays in the Experimental Section. NA indicates <10%
inhibition. The percentages of inhibition at 100 μM were determined
in duplicate, and the averages are reported. The IC50 values were
determined in a 10-dose mode with a 3-fold serial dilution starting at
Table 1. Inhibitory Activities of Para-Substituted Fragments
Based on Naphthalene−Benzamide or Naphthalene−
a
Nicotinamide
b
200 μM in singlet. IC50 value.
series revealed two SAR trends. First, fragments containing a
nicotinamide moiety are more active than those containing a
benzamide moiety, highlighting the importance of the nitrogen
atom. Second, an ether linkage as seen in 51 and 52 generally
affords improved inhibitory activity against SIRT1−3. For
fragments 54−58, similar trends can be observed. However,
there is no significant difference in terms of the position of the
naphthalene (position 2 or 1) through which the nicotinamide
moiety is attached.
Biochemical evaluation of these two sets of fragments
revealed that an ether linkage to the meta position of
nicotinamide conferred promising low-micromolar activity.
Furthermore, a benzamide substituted with groups such as 4-
Me and 3-NO2 and coupled to the naphthylamine is potentially
beneficial. To take advantage of these productive chemical
features, we designed compounds 59−68 (Figure 7). Since
examination of fragments 49−58 did not reveal whether
position 2 or 1 is preferred, we opted to synthesize and evaluate
compounds linked at either position.
% inhibition at 100 μM
compound
X
Y
SIRT1
SIRT2
SIRT3
37
38
39
40
41
42
43
44
45
46
47
48
NH
NH
O
CH
N
NA
NA
NA
15
3
4
6
4
2
6
47
4
20
16
61
41
73
NA
41
27
33
NA
NA
44
CH
N
O
31
4
2
S
CH
N
20
6
6
NA
42
S
31
5
1
1
0
4
NH
NH
O
CH
N
NA
19
22
5
3
2
4
38
CH
N
NA
NA
NA
40
10
O
39
S
CH
N
24 19
47
NA
42
We first explored compounds 59−62, in which the
attachment site is at position 2. Since benzamide bearing 4-
Me and 3-NO2 substituents showed promising activity in the
naphthylamide sulfonic acid series, we first examined
compound 59, which gratifyingly possessed a submicromolar
IC50 value against SIRT1 and SIRT2 and a slightly lower
S
4
3
3
a
Inhibitory activities were determined as described in SIRT1−3
Biochemical Assays in the Experimental Section. NA indicates <10%
inhibition. The percentages of inhibition at 100 μM were determined
in duplicate, and the averages are reported.
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Figure 7. Linking of naphthylamide fragments and nicotinamide to give (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives.
a
Table 3. Inhibitory Activities of (5-Benzamidonaphthalen-1/2-yloxy)nicotinamide Derivatives
IC50 (μM)
compound
R1
R2
SIRT1
SIRT2
SIRT3
59
Me
NO2
H
0.788
22.4
3.82
11.7
100
0.869
0.436
0.489
0.811
0.978
0.0483
0.0774
0.434
0.955
0.558
10.5
4.37
18.3
22.9
10.4
232
60
H
61
Me
H
62
H
NO2
NO2
H
63
Me
64
H
12.0
18.7
64.0
15.4
70.7
73.0
0.210
135
44.2
33.5
19.3
32.5
33.6
6.20
128
65
Me
H
66
H
NO2
67
X = N, Y = CH
68
X = CH, Y = N
4
−
−
−
−
−
−
8 (EX-527)
10 (AGK2)
1.94
1.56
52.8
a
Inhibitory activities were determined as described in SIRT1−3 Biochemical Assays in the Experimental Section. IC50 values were determined in a
10-dose mode with a 3-fold serial dilution starting at 200 μM in singlet.
activity against SIRT3 (Table 3). These results prompted us to
initiate a preliminary SAR study on the benzamide moiety.
Removal of both the 4-Me and 3-NO2 groups, as seen in
compound 60, slightly increased the anti-SIRT2 activity but
significantly improved the isozyme selectivity. Reinstitution of
the 4-Me (compound 61) or 3-NO2 (compound 62) group
conferred no gain in either anti-SIRT2 activity or selectivity
compared with compound 60.
The corresponding position-1 analogues 63−66 were also
investigated. In comparison with compound 59, its analogue 63
showed very similar anti-SIRT2 activity but markedly enhanced
selectivity (>100-fold). Removing the 4-Me and 3-NO2 groups
from compound 63 afforded compound 64, which showed a
20-fold increase in anti-SIRT2 activity (48 nM) and excellent
selectivity for SIRT2 over SIRT1 (248-fold) or SIRT3 (915-
fold). Reintroducing the 4-Me group as seen in compound 65
slightly reduced the activity against SIRT2 (77 nM) without
significantly affecting the activity against SIRT1 or SIRT3.
Compound 66, in which the 3-NO2 group was added, also
showed reduced anti-SIRT2 activity.
Our preliminary SAR study on compound 64 and its
analogues indicated that substitutions such as 4-Me and 3-NO2
are not optimal. In an attempt to explore modifications that
impose minimal steric hindrance, we prepared compounds 67
and 68, in which the phenyl ring was replaced with a pyridine
ring. Compound 67 showed an anti-SIRT2 activity 20-fold
lower than that of compound 64, while its activity against
SIRT1 and SIRT3 did not change significantly. A reduction in
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Figure 8. Compound 64 increased the acetylation level of α-tubulin in MCF-7 cells. The samples were analyzed using antibodies against acetylated
α-tubulin and total tubulin. (A) MCF-7 cells were treated with or without compound 64 (1 μM) for varied periods of time without TSA. (B) MCF-7
cells were treated with various concentrations of compound 64 for 5 h without TSA. (C) MCF-7 cells were treated with or without compound 64 (1
μM) for varied periods of time, and TSA (50 nM) was added to deplete the non-sirtuin histone deacetylase activity. (D) MCF-7 cells were treated
with varied concentrations of compound 64 for 5 h, and TSA (50 nM) was added to deplete the non-sirtuin histone deacetylase activity.
anti-SIRT2 activity was also observed for compound 68, albeit
to a lesser extent. Taken together, these initial SAR studies
indicate that structural modifications centered on the phenyl
ring are not well-tolerated. Investigation of an extensive scope
of modifications is required in order to pinpoint the structural
features that confer improved activity and selectivity.
SIRT2 in cells and consequently increases the acetylation level
of α-tubulin, thus establishing compound 64 as a bona fide
SIRT2 inhibitor.
Isozyme Selectivity. We determined the Km values for
SIRT1−3 with respect to the peptide substrate and NAD+
(Table 4). In order to accurately assess the isoform selectivity
Cellular Activity of Compound 64. Using a fragment-
based approach as described above, we identified the novel
SIRT2 inhibitor 64, whose nanomolar anti-SIRT2 activity and
excellent selectivity warranted further biological investigation.
First we examined whether compound 64 increased the
acetylation of α-tubulin, a well-characterized cellular substrate
of SIRT2.26 To this end, MCF-7 cells were incubated with
compound 64 and harvested, and the acetylation level of α-
tubulin was assessed by Western blot analysis. In the first study,
we incubated MCF-7 cells with or without 1 μM compound 64
for different periods of time (0, 1, 3, 5, and 8 h). Western blot
analysis showed that the level of acetylated α-tubulin changed
in a time-dependent manner, with a peak level at the time point
of 5 h (Figure 8). Accordingly, we set the incubation time at 5 h
and varied the concentration of inhibitor 64 (0, 0.2, 0.5, 1, and
20 μM). Western blot analysis indicated that acetylated α-
tubulin increased in a concentration-dependent manner.
Since α-tubulin can also be deacetylated by Zn-dependent
HDAC6,27 we used trichostatin A (TSA), a pan-HDAC
inhibitor, in order to eliminate the deacetylation activity of
the Zn-dependent HDAC family. On the basis of the fact that
TSA does not inhibit SIRT2, it is expected that the change in
the acetylated α-tubulin level predominately stems from the
change in SIRT2 enzymatic activity. In the presence of TSA (50
nM), when MCF-7 cells were treated with 1 μM compound 64,
the level of acetylated α-tubulin changed in a time-dependent
manner (0, 1, 3, 5, and 7 h), reaching a peak level at the time
point of 5 h (Figure 8). When MCF-7 cells were incubated with
different concentrations of compound 64 (0, 0.2, 0.5, 1, and 20
μM) for 5 h, the level of acetylated α-tubulin increased in a
concentration-dependent manner. These studies clearly dem-
onstrate that compound 64 inhibits the enzymatic activity of
Table 4. Kinetic Parameters for SIRT1−3 and Inhibitory
Activities of Compound 64 against SIRT1−3
SIRT1
SIRT2
SIRT3
a
Km peptide (μM)
452.8 22.4
297.4 14.3
254.8 30.4
a
Km NAD+ (μM)
264.2 16.9
201.2 25.9
334.9 22.9
b
peptide conc. (μM)
76
50
43
b
NAD+ conc. (μM)
66
50
83
c
compd 64 IC50 (μM)
compd 64 Kai pp (μM)
52.7 4.5
−
29.7 5.8
c
−
−
0.131 0.025
−
−
compd 64 Ki peptide
0.045 0.013
d
(μM)
compd 64 Ki NAD+
−
0.165 0.013
−
d
(μM)
a
Kinetic parameters of SIRT1−3 were determined as described in
Determination of Kinetic Parameters of SIRT1−3 in the Experimental
b
Section. Concentrations of the peptide and NAD+ used, as described
in Selectivity of Compound 64 in the Experimental Section.
c
Inhibitory activities of compound 64 were determined as described
d
in Selectivity of Compound 64 in the Experimental Section. Ki values
for compound 64 were determined as described in Determination of
Inhibition Modality by Compound 64 against SIRT2 in the
Experimental Section.
of compound 64, we used the same peptide substrate
(Ac)RHKK(Ac)-AMC, whose concentrations were maintained
at a 1:6 ratio with respect to the Km values. Similarly, the
concentrations of NAD+ were fixed at a 1:4 ratio with respect to
the Km values measured for SIRT1−3. Under these conditions,
the IC50 values of compound 64 were determined to be 52.7
and 29.7 μM for SIRT1 and SIRT3, respectively. Since
compound 64 appeared to be a tight-binding inhibitor of
SIRT2, we used Morrison’s equation to determine its Kai pp value
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Figure 9. Inhibition of SIRT2 by compound 64. Kai pp is plotted against the concentration of either (A) the peptide substrate (varied from 0.04 to 1.2
mM with NAD+ fixed at 0.2 mM) or (B) NAD+ (varied from 0.03 to 1 mM with the peptide substrate held at 0.3 mM). Various concentrations of
compound 64 were used. Compound 64 is a competitive inhibitor with respect to the peptide with a Ki value of 0.045
0.013 μM and a
noncompetitive inhibitor with respect to NAD+ with a Ki value of 0.165 0.013 μM and an α value of 0.51 0.07.
Figure 10. Potential binding modes and interactions of compound 64 docked into the crystal structure of human SIRT2 (PDB entry 1j8f). (A) Two
binding modes of compound 64 in the active site of SIRT2 (surface representation). (B) Potential interactions between compound 64 and SIRT2 in
the first binding mode (magenta). (C) Potential interactions between compound 64 and SIRT2 in the second binding mode (green).
(131 nM). This study indicated that compound 64 is indeed a
selective inhibitor of SIRT2 over SIRT1 and SIRT3, displaying
about 400-fold and 200-fold selectivity, respectively.
lines is disappointing, low cytotoxicity is desired if SIRT2
inhibitors are used to treat other medical conditions such as
Parkinson’s disease and bacterial infection.
Mode of Inhibition. In order to characterize the inhibition
mode of compound 64, we determined the Kai pp values of
compound 64 against the peptide substrate and NAD+ under
tight-binding conditions (for the primary data, see Figure S1 in
the Supporting Information). A plot of Kiapp versus the
concentration of peptide substrate (Figure 9A) indicated that
compound 64 is a competitive inhibitor against the peptide
substrate. Furthermore, a plot of Kai pp versus the concentration
of NAD+ (Figure 9B) suggested that compound 64 is a
noncompetitive inhibitor with respect to NAD+. Nevertheless,
we cannot entirely rule out the possibility that compound 64 is
an uncompetitive inhibitor because the data can potentially be
fit to an uncompetitive inhibition modality under tight-binding
conditions.
Madin−Darby Canine Kidney (MDCK) Permeability.
The potential application of SIRT2 inhibitors in Parkinson’s
disease requires inhibitors to traverse the blood−brain barrier
(BBB). The probability that an entity traverses this barrier is
widely accepted to be represented in its ability to traverse a
monolayer of the MDCK II continuous cell line, with the side
facing the ISF being the “basolateral” (B) side and that facing
the blood being the “apical” (A) side. The apparent
permeability coefficient (Papp) for an inhibitor would then be
∫ t0(dQ/dt)/(A × C0), where dQ/dt is the rate obtained from
the slopes of plots of amount transported versus time, A is the
total area subject to transport, and C0 is the initial
A→B
concentration in the donor compartment. P
for compound
app
B→A
64 was 3.84 × 10−6 cm·s−1, while P
was 4.35 × 10−6 cm·s−1.
app
Cytotoxicity. Inhibition of SIRT2 has been proposed as a
potential therapy for various cancers.10a To investigate the
anticancer application of compound 64, we assessed its
cytotoxicity in three human cancer cell lines, namely, breast
cancer (MCF-7), human prostate cancer (DU 145), and
chronic myelogenous leukemia (K562) cell lines. Compound
64 possesses CC50 values of 30.6, 33.3, and 26.2 μM against the
MCF-7, DU 145, K562 cell lines, respectively. While the
modest anticancer activity of compound 64 in these three cell
The low magnitudes of these two coefficients and their
relatively similar values foretell compound 64’s poor ability to
translocate in either direction across the BBB, thus requiring
further structural optimization. They also indicate that there is a
lack of or negligible contribution from an active (ATP-utilizing)
or electrostatically driven transporter to facilitate transport in
either direction.
Computational Modeling. To explore potential binding
modes for these newly identified SIRT2 inhibitors, the
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a
Scheme 1
a
Reagents and conditions: (a) Na2CO3, H2O, toluene. (b) H2, Pd/C, MeOH.
a
Scheme 2
a
Reagents and conditions: (a) Pd(OAc)2, rac-BINAP, Cs2CO3, dioxane, 100 °C. (b) Pd(OAc)2, tert-butyl Xphos, K3PO4, toluene, 100 °C. (c)
Pd2(dba)3, Xantphos, DIPEA, dioxane, 100 °C. (d) Ammonia solution. For compound 88: (i) NaOH, DMF/MeOH, 100−110 °C; (ii) oxalyl
chloride, DMF/CH2Cl2; (iii) NH3/H2O. (e) Cs2CO3, DMA, toluene, 130 °C.
representative compound 64 was docked into human SIRT2
(PDB entry 1j8f),28 which revealed two major potential binding
modes (Figure 10). In the first binding mode (magenta), the
nicotinamide moiety interacts with the C site of the NAD+
binding pocket, which nicotinamide riboside normally occupies.
The binding configuration is reminiscent of that observed for
nicotinamide in the X-ray crystal structure of Sir2Tm (PDB
entry 1yc5).29 In this binding mode, the nicotinamide moiety
forms two hydrogen bonds involving its primary amide
functionality, the side-chain carboxylate of Asp 170, and the
backbone NH of Ile 169. The naphthalene moiety is involved in
potential π−π interactions with Phe 96 and Phe 119. The
attached benzamide moiety extends into a mainly hydrophobic
pocket, forming a π−π interaction with Tyr 104 and a hydrogen
bond with Tyr 104’s phenolic functionality. Interestingly,
engagement of the same hydrophobic pocket has been
suggested for SIRT2 inhibitors based on the 3-(N-
arylsulfamoyl)benzamide scaffold.18d
In the second binding mode (green), the nicotinamide
moiety of compound 64 interacts with the C site of the NAD+
binding pocket in a manner almost identical to that observed in
the first binding mode (magenta). Nevertheless, the naph-
thalene moiety adopts a configuration that is perpendicular to
that seen in the first binding mode, and it is implicated in π−π
interactions with Phe 119 and His 187. Consequently, the
attached benzamide moiety extends into the peptide substrate
binding channel, engaging an additional π−π interaction with
Phe 235. Our studies of the inhibition mode of compound 64
revealed that it is a competitive and most likely noncompetitive
inhibitor in relation to the peptide substrate and NAD+,
respectively. As a result, we prefer the second binding mode, in
which compound 64 occupies the peptide substrate channel.
Nevertheless, we cannot rule out the first binding mode, in
H
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a
Scheme 3
a
Reagents and conditions: (a) Pd(OAc)2, rac-BINAP, Cs2CO3, dioxane, 100 °C. (b) Pd(OAc)2, tert-butyl Xphos, K3PO4, toluene, 100 °C. (c)
Pd2(dba)3, Xantphos, DIPEA, dioxane, 100 °C. (d) Ammonia solution. (e) CuI, picolinic acid, K3PO4, DMSO, 90 °C.
a
Scheme 4
a
Reagents and conditions: (a) DIPEA, CH3CN and then NaOH, H2O/THF/MeOH. For the synthesis of compound 117, nicotinic acid (107),
EDC, HOBt, CH2Cl2 and then NaOH, H2O/THF/MeOH. (b) Bromide 103, CuI, picolinic acid, K3PO4, DMSO, 90 °C.
which compound 64 extends into the hydrophobic pocket and
might induce a conformational change in the adjacent peptide
substrate channel. A recent study has shown that SIRT2
possesses the largest active site among all the human sirtuins.30
Compound 64, with its naphthalene moiety occupying the
upper portion of peptide substrate channel, is relatively bulky.
Therefore, its higher molecular volume may be tolerated only
by SIRT2, accounting for compound 64’s selectivity toward
SIRT2 over SIRT1 and SIRT3. We expect that this spatial
feature of SIRT2 can be further exploited to enhance the
isozyme selectivity.
75 in an aqueous mixture gave benzamides 19, 20, 21, and 23,
respectively (Scheme 1). The latter two fragments were
reduced to afford the corresponding anilines 22 and 24.
Analogously, anilines 70 and 71 were converted into fragments
25−36.
The synthesis of the naphthalene−benzamides and −nic-
otinamides generally involved C−heteroatom coupling to give
methyl esters, which underwent ammonolysis to produce the
corresponding primary amides. As shown in Scheme 2,
palladium-catalyzed C−N coupling32 between 2-naphthylamine
(76) and bromide 82 afforded methyl ester 84, which was
treated with ammonia solution to give the desired primary
amide.37 In a similar manner, C−O33 and C−S34 couplings of
naphthols (77 and 80) and naphthalenethiols (78 and 81) were
Chemistry. The synthesis of the naphthylamide sulfonic
acid fragments was carried out according to literature
procedures.31 Acylation of aniline 69 with acyl chlorides 72−
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constants are first-order. High-resolution mass spectra were recorded
on an Agilent TOF II TOF/MS instrument equipped with either an
ESI or APCI interface. Analysis of sample purity was performed on a
Varian Prepstar SD-1 HPLC system with a Phenomenex Gemini C18
column (5 μm, 4.6 mm × 250 mm). HPLC conditions were the
following: solvent A = water, solvent B = MeCN or MeOH; flow rate
= 1.0 mL/min; elution with a gradient of 40% to 95% MeCN/water or
50 to 95% MeOH/water in 15 min. Purity was determined by the
absorbance at 254 nm. All of the tested compounds had a purity of
≥95%.
Methyl 4-(Naphthalen-2-ylamino)benzoate (84). A mixture of
2-naphthylamine (76) (200 mg, 1.3 mmol), bromide 82 (200 mg, 0.97
mmol), Pd(OAc)2 (21 mg, 0.094 mmol), rac-BINAP (38 mg, 0.14
mmol) ,and Cs2CO3 (440 mg, 1.3 mmol) in dioxane (2 mL) was
heated at 100 °C overnight. The reaction mixture was filtered through
a pad of Celite, and the filtrate was concentrated in vacuo. The residue
was purified by flash column chromatography (hexanes/EtOAc) to
give compound 84 as a brown solid (88 mg, 33%). 1H NMR (CDCl3,
600 MHz) δ 7.95 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.79
(d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.59−7.56 (m, 1H), 7.46
(dd, J = 7.2, 7.2 Hz, 1H), 7.38 (dd, J = 7.5, 7.5 Hz, 1H), 7.30 (dd, J =
8.7, 2.1 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.19 (s, 1H), 3.89 (s, 3H).
HRMS (ESI+) calcd for C18H16NO2 (M + H)+ 278.1181, found
278.1184.
Methyl 6-(Naphthalen-2-ylamino)nicotinate (85). In a manner
similar to that described for the preparation of compound 84, 2-
naphthylamine (76) (400 mg, 2.70 mmol) and chloride 83 (326 mg,
1.90 mmol) underwent a C−N coupling reaction to give compound
85 as a brownish solid (82 mg, 15%). 1H NMR (CDCl3, 600 MHz) δ
8.89 (d, J = 1.8 Hz, 1H), 8.09 (dd, J = 8.7, 1.5 Hz, 1H), 7.88 (d, J = 2.4
Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.78 (d, J
= 8.4 Hz, 1H), 7.48 (ddd, J = 8.4, 7.8, 1.0 Hz, 1H), 7.44−7.41 (m,
2H), 6.90 (d, J = 2.4 Hz, 1H), 3.90 (s, 3H). HRMS (ESI+) calcd for
C17H15N2O2 (M + H)+ 279.1134, found 279.1129.
used to prepare methyl esters 85−91, which except for 88 were
then converted into primary amides. Ammonolysis of methyl
ester 88 proved to be extremely difficult, probably because of its
low solubility in water or MeOH. As a result, it was hydrolyzed
to form the corresponding acid, which was activated with oxalyl
chloride and subsequently treated with ammonia to give amide
43. In contrast, amides 40, 42, 46, and 48 were directly
obtained through nucleophilic displacement of chloride 92 by
naphthols and naphthalenethiols.
Analogously, palladium-catalyzed C−heteroatom couplings
were employed to prepare methyl esters 95−102, from which
primary amides 49−58 with the exceptions of 52 and 57 were
obtained (Scheme 3). Amides 52 and 57 were directly prepared
by copper-mediated C−O coupling35 performed on bromide
103.
The synthesis of (5-benzamidonaphthalen-1/2-yloxy)-
nicotinamide derivatives 59−68 began with selective acylation
of the aniline functionality in 104 and 105. This transformation
was accomplished by acylation of both aniline and phenol with
excess acyl chloride or nicotinic acid (107) in the presence of
peptide coupling reagents followed by selective hydrolysis of
the phenol ester (Scheme 4). The phenols obtained were
subjected to copper-mediated C−O coupling35 with bromide
103 to give amides 59−68 in very low yields.
CONCLUSIONS
■
We have reported our discovery of novel SIRT2 inhibitors
using a fragment-based approach. Taking advantage of the
purported close binding proximity of suramin and nicotina-
mide, we designed two fragment series, namely, the
naphthylamide sulfonic acids and the naphthalene−benzamides
and −nicotinamides. Biochemical testing of these two series
allowed us to identify and subsequently combine chemical
features, leading to (5-benzamidonaphthalen-1/2-yloxy)-
nicotinamide derivatives 59−68, among which compound 64
exhibited superior anti-SIRT2 activity and excellent selectivity
for SIRT2 over SIRT1 and SIRT3. Inside cells, compound 64
also inhibited SIRT2 and increased the acetylation level of α-
tubulin, a well-established SIRT2 substrate, in both concen-
tration- and time-dependent manners. Kinetic studies under
tight-binding conditions revealed that compound 64 is a
competitive inhibitor against the peptide substrate and most
likely a noncompetitive inhibitor versus NAD+. Molecular
docking of compound 64 into the active site of SIRT2
suggested that it can occupy the peptide substrate channel,
offering clues for further structural modifications. The excellent
activity and selectivity displayed by compound 64 indicate that
its core (5-benzamidonaphthalen-1-yloxy)nicotinamide struc-
ture holds promise for further chemical modifications, which
are currently in progress and will be reported in due course.
Methyl 4-(Naphthalen-2-yloxy)benzoate (86).36 A mixture of
2-naphthol (77) (300 mg, 2.08 mmol), bromide 82 (430 mg, 2.00
mmol), Pd(OAc)2 (8.9 mg, 0.0040 mmol), tert-butyl XPhos (25 mg,
0.0059 mmol), and K3PO4 (840 mg, 3.96 mmol) in toluene (3 mL)
was heated at 100 °C for 16 h. The reaction mixture was diluted with
EtOAc and filtered through a pad of Celite, and the filtrate was
concentrated in vacuo. The residue was purified by flash column
chromatography (hexanes/EtOAc) to give compound 86 as a white
1
solid (382 mg, 69%). H NMR (CDCl3, 600 MHz) δ 8.01 (d, J = 9.0
Hz, 2H), 7.84 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.72 (d, J
= 7.8 Hz, 1H), 7.48−7.40 (m, 3H), 7.23 (dd, J = 8.7, 2.1 Hz, 1H), 7.02
(d, J = 9.0 Hz, 2H), 3.88 (s, 3H). HRMS (ESI+) calcd for C18H15O3
(M + H)+ 279.1021, found 279.1025.
Methyl 4-(Naphthalen-2-ylthio)benzoate (87).36 A mixture of
2-naphthalenethiol (78) (300 mg, 1.87 mmol), bromide 82 (400 mg,
1.86 mmol), Pd2(dba)3 (42 mg, 0.046 mmol), Xantphos (54 mg, 0.094
mmol), and DIPEA (0.62 mL, 3.74 mmol) in dioxane (6 mL) was
heated at 100 °C overnight. The reaction mixture was diluted with
EtOAc and filtered through a pad of Celite, and the filtrate was
concentrated in vacuo. The residue was purified by flash column
chromatography (hexanes/EtOAc) to give compound 87 as a white
solid (10.6 mg, 2%). 1H NMR (CDCl3, 600 MHz) δ 8.03 (s, 1H), 7.90
(d, J = 8.4 Hz, 2H), 7.87−7.83 (m, 2H), 7.82−7.78 (m, 1H), 7.53 (dd,
J = 4.5, 4.5 Hz, 2H), 7.48 (dd, J = 8.4, 1.8 Hz, 1H), 7.27−7.23 (m,
2H), 3.89 (s, 3H). HRMS (ESI+) calcd for C18H15OS (M + H)+
279.0844, found 279.0849.
EXPERIMENTAL SECTION
■
Chemical Synthesis. Commercial reagents were used as provided,
unless otherwise indicated. An anhydrous solvent dispensing system (J.
C. Meyer) using two packed columns of neutral alumina was used for
drying of THF, Et2O, and CH2Cl2, while two packed columns of
molecular sieves were used to dry DMF. Solvents were dispensed
under argon. Flash chromatography was performed with Ultra Pure
silica gel (Silicycle) with the indicated solvent system. NMR spectra
were recorded on a Varian 600 MHz spectrometer with Me4Si or
signals from residual solvent as the internal standard for 1H. Chemical
shifts are reported in parts per million, and signals are described as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brs
(broad singlet), or dd (doublet of doublets). Values given for coupling
Methyl 4-(Naphthalen-1-ylamino)benzoate (88).37 In a
manner similar to that described for the preparation of compound
84, 1-naphthylamine (79) (400 mg, 2.70 mmol) and bromide 82 (400
mg, 1.90 mmol) underwent a C−N coupling reaction to give
1
compound 88 as a brownish solid (321 mg, 61%). H NMR (CDCl3,
600 MHz) δ 7.97 (d, J = 7.8 Hz, 1H), 7.91−7.87 (m, 3H), 7.74−7.70
(m, 1H), 7.54−7.45 (m, 4H), 6.84 (d, J = 8.4 Hz, 2H), 6.18 (brs, 1H),
3.86 (s, 3H). HRMS (ESI+) calcd for C18H16NO2 (M + H)+ 278.1181,
found 278.1181.
J
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Methyl 6-(Naphthalen-1-ylamino)nicotinate (89). In a manner
similar to that described for the preparation of compound 84, 1-
naphthylamine (79) (400 mg, 2.70 mmol) and chloride 83 (326 mg,
1.90 mmol) underwent a C−N coupling reaction to give compound
89 as a brownish solid (65 mg, 12%). 1H NMR (CDCl3, 600 MHz) δ
8.84 (d, J = 1.8 Hz, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.97 (dd, J = 9.0, 2.4
Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.58−7.49
(m, 4H), 7.36 (brs, 1H), 6.49 (d, J = 2.4 Hz, 1H), 3.88 (s, 3H). HRMS
(ESI+) calcd for C17H15N2O2 (M + H)+ 279.1134, found 279.1128.
Methyl 4-(Naphthalen-1-yloxy)benzoate (90). In a manner
similar to that described for the preparation of compound 86, 1-
naphthol (80) (300 mg, 2.08 mmol) and bromide 82 (430 mg, 2.00
mmol) underwent a C−O coupling reaction to give compound 90 as a
pale solid (370 mg, 66%). 1H NMR (CDCl3, 600 MHz) δ 8.02 (d, J =
8.4 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 1H), 7.67
(d, J = 8.4 Hz, 1H), 7.49 (dd, J = 7.5, 7.5 Hz, 1H), 7.43 (dd, J = 7.8,
7.8 Hz, 1H), 7.40 (dd, J = 7.8, 7.8 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H),
6.98 (d, J = 8.4 Hz, 2H), 3.86 (s, 3H). HRMS (ESI+) calcd for
C18H15O3 (M + H)+ 279.1021, found 279.1023.
Methyl 3-(Naphthalen-1-ylamino)benzoate (99). In a manner
similar to that described for the preparation of compound 84, 1-
naphthylamine (79) (1.00 g, 6.98 mmol) and bromide 93 (1.20 g, 5.58
mmol) underwent a C−N coupling reaction to give compound 99 as a
brown solid (292 mg, 19%). 1H NMR (CDCl3, 600 MHz) δ 8.00 (d, J
= 2.4 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 1.8, 1.8 Hz, 1H),
7.62 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.52−7.45 (m, 2H),
7.42 (dd, J = 7.8, 7.8 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.28 (dd, J =
7.8, 7.8 Hz, 1H), 7.11 (dd, J = 7.8, 2.1 Hz, 1H), 6.00 (brs, 1H), 3.88 (s,
3H). HRMS (ESI+) calcd for C18H16NO2 (M + H)+ 278.1181, found
278.1181.
Methyl 5-(Naphthalen-1-ylamino)nicotinate (100). In a
manner similar to that described for the preparation of compound
84, 1-naphthylamine (79) (400 mg, 2.79 mmol) and bromide 94 (400
mg, 1.85 mmol) underwent a C−N coupling reaction to give
compound 100 as a white solid (346 mg, 67%). 1H NMR (CDCl3, 600
MHz) δ 8.71 (d, J = 1.8 Hz, 1H), 8.46 (d, J = 2.4 Hz, 1H), 7.97 (d, J =
8.4 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.76−7.74 (m, 1H), 7.71 (d, J =
8.4 Hz, 1H), 7.56−7.49 (m, 2H), 7.46 (dd, J = 7.5, 7.5 Hz, 1H), 7.40
(d, J = 7.2 Hz, 1H), 6.01 (s, 1H), 3.90 (s, 3H). HRMS (ESI+) calcd for
C17H15N2O2 (M + H)+ 279.1134, found 279.1131.
Methyl 3-(Naphthalen-1-yloxy)benzoate (101). In a manner
similar to that described for the preparation of compound 86, 1-
naphthol (80) (300 mg, 2.08 mmol), and bromide 93 (430 mg, 2.00
mmol) underwent a C−O coupling reaction to give compound 101 as
a white solid (166 mg, 30%). 1H NMR (DMSO-d6, 600 MHz) δ 9.47
(s, 1H), 8.34−8.28 (m, 1H), 8.26 (d, J = 0.6 Hz, 1H), 7.94 (d, J = 7.2
Hz, 1H), 7.92−7.85 (m, 2H), 7.63 (dd, J = 7.5, 7.5 Hz, 1H), 7.56−
7.50 (m, 3H), 7.46 (d, J = 9.0 Hz, 1H), 3.89 (s, 3H). HRMS (ESI+)
calcd for C18H15O3 (M + H)+ 279.1021, found 279.1021.
Methyl 5-(Naphthalen-1-ylthio)nicotinate (102). In a manner
similar to that described for the preparation of compound 87, 1-
naphthalenethiol (81) (120 mg, 0.75 mmol) and bromide 94 (160 mg,
0.74 mmol) underwent a C−S coupling reaction to give compound
102 as a pale solid (4.3 mg, 2%). 1H NMR (CDCl3, 600 MHz) δ 8.94
(d, J = 1.8 Hz, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.32 (dd, J = 5.4, 4.2 Hz,
1H), 8.02 (dd, J = 2.4, 2.4 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.93−
7.89 (m, 1H), 7.80 (dd, J = 7.2, 1.2 Hz, 1H), 7.56−7.52 (m, 2H), 7.49
(dd, J = 8.1, 7.5 Hz, 1H), 3.88 (s, 3H). HRMS (ESI+) calcd for
C17H14NO2S (M + H)+ 296.0745, found 296.0745.
Methyl 4-(Naphthalen-1-ylthio)benzoate (91). In a manner
similar to that described for the preparation of compound 87, 1-
naphthalenethiol (81) (300 mg, 1.87 mmol) and bromide 82 (400 mg,
1.87 mmol) underwent a C−S coupling reaction to give compound 91
1
as a pale solid (192 mg, 35%). H NMR (CDCl3, 600 MHz) δ 8.29
(dd, J = 8.4, 1.2 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.91 (dd, J = 8.4,
1.2 Hz, 1H), 7.85 (dd, J = 7.5, 0.9 Hz, 1H), 7.81 (ddd, J = 9.0, 2.1, 2.1
Hz, 2H), 7.58−7.48 (m, 3H), 7.05 (ddd, J = 8.8, 1.8, 1.8 Hz, 2H), 3.85
(s, 3H). HRMS (ESI+) calcd for C18H15O2S (M + H)+ 295.0793,
found 295.0795.
Methyl 3-(Naphthalen-2-ylamino)benzoate (95). In a manner
similar to that described for the preparation of compound 84, 2-
naphthylamine (76) (450 mg, 3.14 mmol) and bromide 93 (530 mg,
2.46 mmol) underwent a C−N coupling reaction to give compound
95 as a brownish solid (424 mg, 62%). 1H NMR (CDCl3, 600 MHz) δ
7.79 (s, 1H), 7.78−7.74 (m, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.62 (ddd, J
= 7.2, 1.6, 1.6 Hz, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 7.5, 7.5
Hz, 1H), 7.38−7.32 (m, 3H), 7.24 (dd, J = 9.0, 2.4 Hz, 1H), 5.97 (brs,
1H), 3.89 (s, 3H). HRMS (ESI+) calcd for C18H16NO2 (M + H)+
278.1181, found 278.1182.
Methyl 5-(Naphthalen-2-ylamino)nicotinate (96). In a manner
similar to that described for the preparation of compound 84, 2-
naphthylamine (76) (200 mg, 1.40 mmol) and bromide 94 (200 mg,
0.92 mmol) underwent a C−N coupling reaction to give compound
4-(Naphthalen-2-ylamino)benzamide (37). A mixture of
methyl ester 84 (13 mg, 0.047 mmol) in strong ammonium hydroxide
solution (Fisher Scientific) (3 mL) in a sealed tube was heated at 55
°C for 16 h. After concentration in vacuo, the reaction residue was
purified by flash column chromatography (EtOAc/hexanes) to give
compound 37 as a brown solid (7.0 mg, 57%). 1H NMR (CDCl3, 600
MHz) δ 7.81 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.75 (d, J =
9.0 Hz, 2H), 7.57 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 7.2, 7.2 Hz, 1H),
7.38 (dd, J = 7.2, 7.2 Hz, 1H), 7.30 (dd, J = 8.7, 3.1 Hz, 1H), 7.11 (d, J
= 9.0 Hz, 2H), 6.12 (s, 1H). HRMS (ESI+) calcd for C17H15N2O (M +
H)+ 263.1184, found 263.1185.
1
96 as a brown solid (176 mg, 69%). H NMR (CDCl3, 600 MHz) δ
8.78 (d, J = 1.2 Hz, 1H), 8.61 (d, J = 3.0 Hz, 1H), 8.03 (dd, J = 2.4, 2.4
Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.71 (d, J
= 8.4 Hz, 1H), 7.50 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 7.2, 7.2 Hz, 1H),
7.38 (dd, J = 7.2, 7.2 Hz, 1H), 7.27−7.24 (m, 1H), 6.01 (s, 1H), 3.94
(s, 3H). HRMS (ESI+) calcd for C17H15N2O2 (M + H)+ 279.1134,
found 279.1138.
Methyl 3-(Naphthalen-2-yloxy)benzoate (97). In a manner
similar to that described for the preparation of compound 86, 2-
naphthol (77) (300 mg, 2.08 mmol) and bromide 93 (430 mg, 2.00
mmol) underwent a C−O coupling reaction to give compound 97 as a
6-(Naphthalen-2-ylamino)nicotinamide (38). In a manner
similar to that described for the preparation of compound 37, methyl
ester 85 (20 mg, 0.072 mmol) in MeOH (2 mL) was treated with
ammonium hydroxide solution (3 mL) at 80 °C to give compound 38
1
clear oil (184 mg, 33%). H NMR (CDCl3, 600 MHz) δ 8.16 (d, J =
8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.71 (s,
1H), 7.66 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 7.5, 7.5 Hz, 1H), 7.49 (dd,
J = 7.8, 7.8 Hz, 1H), 7.40 (dd, J = 7.8, 7.8 Hz, 2H), 7.23 (dd, J = 7.8,
2.4 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 3.88 (s, 3H). HRMS (ESI+)
calcd for C18H15O3 (M + H)+ 279.1021, found 279.1025.
Methyl 5-(Naphthalen-2-ylthio)nicotinate (98). In a manner
similar to that described for the preparation of compound 87, 2-
naphthalenethiol (78) (120 mg, 0.75 mmol) and bromide 94 (160 mg,
0.74 mmol) underwent a C−S coupling reaction to give compound 98
as a pale solid (2.6 mg, 1%). 1H NMR (CDCl3, 600 MHz) δ 9.04 (d, J
= 2.4 Hz, 1H), 8.69 (d, J = 1.8 Hz, 1H), 8.19 (dd, J = 2.4, 2.4 Hz, 1H),
7.95 (d, J = 1.2 Hz, 1H), 7.86−7.81 (m, 2H), 7.78 (dd, J = 6.0, 3.6 Hz,
1H), 7.55−7.50 (m, 2H), 7.42 (dd, J = 8.4, 1.8 Hz, 1H), 3.91 (s, 3H).
HRMS (ESI+) calcd for C17H14NO2S (M + H)+ 296.0745, found
296.0743.
1
as a pale solid (4.9 mg, 26%). H NMR (CD3OD, 600 MHz) δ 8.73
(d, J = 2.4 Hz, 1H), 8.24 (d, J = 2.4 Hz, 1H), 8.02 (dd, J = 8.7, 2.7 Hz,
1H), 7.80 (d, J = 8.4 Hz, 1H), 7.79−7.76 (m, 2H), 7.58 (dd, J = 8.4,
2.4 Hz, 1H), 7.43 (ddd, J = 8.4, 7.8, 1.2 Hz, 1H), 7.34 (ddd, J = 8.4,
7.8, 1.4 Hz, 1H), 6.90 (dd, J = 8.4, 0.6 Hz, 1H). HRMS (ESI+) calcd
for C16H14N3O (M + H)+ 264.1137, found 264.1139.
4-(Naphthalen-2-yloxy)benzamide (39). In a manner similar to
that described for the preparation of compound 37, methyl ester 86
(92 mg, 0.33 mmol) in MeOH (2.5 mL) and dioxane (3.5 mL) was
treated with 7 N NH3/MeOH solution (4 mL) at 62 °C to give
1
compound 39 as a white solid (14 mg, 16%). H NMR (CDCl3, 600
MHz) δ 7.88 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.81 (d, J =
9.0 Hz, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 7.2, 7.2 Hz, 1H),
7.45 (dd, J = 7.2, 7.2 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.27−7.24 (m,
K
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1H), 7.08 (d, J = 9.0 Hz, 2H). HRMS (ESI+) calcd for C17H14NO2 (M
+ H)+ 264.1025, found 264.1027.
6-(Naphthalen-1-yloxy)nicotinamide (46). In a manner similar
to that described for the preparation of compound 40, 1-naphthol
(80) (500 mg, 3.47 mmol) was treated with chloride 92 (532 mg, 3.40
mmol) to give compound 46 as a pale solid (149 mg, 16%). 1H NMR
(CD3OD, 600 MHz) δ 8.58 (d, J = 2.4 Hz, 1H), 8.27 (dd, J = 8.7, 2.7
Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.82 (d, J
= 8.4 Hz, 1H), 7.58−7.50 (m, 2H), 7.46 (ddd, J = 8.4, 7.8, 1.2 Hz,
1H), 7.27 (dd, J = 7.8, 0.6 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H). HRMS
(ESI+) calcd for C16H13N2O2 (M + H)+ 265.0977, found 265.0980.
4-(Naphthalen-1-ylthio)benzamide (47). In a manner similar to
that described for the preparation of compound 37, methyl ester 91
(900 mg, 3.06 mmol) was treated with 7 N NH3/MeOH solution (2
mL) at 45 °C and then ammonium hydroxide solution (2 mL) at 92
6-(Naphthalen-2-yloxy)nicotinamide (40). A solution of 2-
naphthol (77) (250 mg, 1.73 mmol), chloride 92 (266 mg, 1.70
mmol), and Cs2CO3 (1.65 g, 5.07 mmol) in DMA (5 mL) and toluene
(5 mL) was heated at 130 °C. After the reaction was complete, the
reaction mixture was poured in EtOAc and H2O. The resultant
mixture was extracted with EtOAc, and the organic layer was washed
with brine, dried over Na2SO4 and filtered. The filtrate was
concentrated in vacuo, and the residue was purified by flash column
chromatography to give compound 40 as a brown solid (100 mg,
1
22%). H NMR (CDCl3, 600 MHz) δ 8.59 (s, 1H), 8.19 (d, J = 7.8
Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.81 (d, J
= 8.4 Hz, 1H), 7.60 (s, 1H), 7.52−7.45 (m, 2H), 7.29 (d, J = 9.0 Hz,
1H), 7.03 (d, J = 9.0 Hz, 1H). HRMS (ESI+) calcd for C16H13N2O2
(M + H)+ 265.0977, found 265.0981.
1
°C to give compound 47 as a pale solid (8.4 mg, 1%). H NMR
(CDCl3, 600 MHz) δ 8.29 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 7.8 Hz,
1H), 7.91 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 6.6 Hz, 1H), 7.59 (d, J = 7.8
Hz, 2H), 7.56−7.48 (m, 3H), 7.08 (d, J = 8.4 Hz, 2H). HRMS (ESI+)
calcd for C17H14NOS (M + H)+ 280.0796, found 280.0797.
6-(Naphthalen-1-ylthio)nicotinamide (48). In a manner similar
to that described for the preparation of compound 40, 1-
naphthalenethiol (81) (360 mg, 2.25 mmol) was treated with chloride
92 (234 mg, 1.49 mmol) to give compound 48 as a yellowish solid
(213 mg, 51%). 1H NMR (CDCl3, 600 MHz) δ 8.78 (s, 1H), 8.31 (d,
J = 8.4 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 6.6 Hz, 1H),
7.94 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.59−7.52 (m, 3H),
6.60 (d, J = 9.0 Hz, 1H). HRMS (ESI+) calcd for C16H13N2OS (M +
H)+ 281.0749, found 281.0756.
3-(Naphthalen-2-ylamino)benzamide (49). In a manner similar
to that described for the preparation of compound 37, methyl ester 95
(102 mg, 0.368 mmol) was treated with 7 N NH3/MeOH solution (6
mL) at 95 °C to give compound 49 as a brown solid (38 mg, 39%). 1H
NMR (CDCl3, 600 MHz) δ 7.77 (dd, J = 9.0, 9.0 Hz, 2H), 7.68 (d, J =
8.4 Hz, 1H), 7.59−7.57 (m, 1H), 7.48−7.46 (m, 1H), 7.43 (dd, J =
7.5, 7.5 Hz, 1H), 7.38−7.33 (m, 2H), 7.33−7.29 (m, 2H), 7.26−7.23
(m, 2H), 5.99 (brs, 2H). HRMS (ESI+) calcd for C17H15N2O (M +
H)+ 263.1184, found 263.1185.
5-(Naphthalen-2-ylamino)nicotinamide (50). In a manner
similar to that described for the preparation of compound 37, methyl
ester 96 (20 mg, 0.072 mmol) was treated with ammonium hydroxide
solution (3 mL) at 50 °C to give compound 50 as a white solid (9.3
mg, 50%). 1H NMR (DMSO-d6, 600 MHz) δ 8.76 (s, 1H), 8.56−8.51
(m, 2H), 8.12 (s, 1H), 7.96 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.80 (d, J
= 7.2 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.56−7.51 (m, 2H), 7.42 (dd,
J = 7.5, 7.5 Hz, 1H), 7.34−7.29 (m, 2H). HRMS (ESI+) calcd for
C16H14N3O (M + H)+ 264.1137, found 264.1134.
4-(Naphthalen-2-ylthio)benzamide (41). In a manner similar to
that described for the preparation of compound 37, methyl ester 87
(84 mg, 0.28 mmol) in MeOH (5 mL) was treated with ammonium
hydroxide solution (5 mL) at 50 °C to give compound 41 as a white
1
solid (21 mg, 26%). H NMR (DMSO-d6, 600 MHz) δ 8.04 (s, 1H),
7.93−7.87 (m, 4H), 7.77 (d, J = 8.4 Hz, 2H), 7.53−7.51 (m, 2H), 7.41
(dd, J = 5.4, 1.8 Hz, 1H), 7.31 (br, 1H), 7.26 (d, J = 9.0 Hz, 2H).
HRMS calcd for C17H14NOS (M + H)+ 280.0791, found 280.0796.
6-(Naphthalen-1-ylthio)nicotinamide (42). In a manner similar
to that described for the preparation of compound 40, 2-
naphthalenethiol (78) (250 mg, 1.56 mmol) was treated with chloride
92 (266 mg, 1.70 mmol) to give compound 42 as a yellowish solid
(126 mg, 29%). 1H NMR (CDCl3, 600 MHz) δ 8.79 (s, 1H), 8.17 (s,
1H), 7.94−7.84 (m, 4H), 7.62−7.54 (m, 3H), 6.94 (d, J = 8.4 Hz,
1H). HRMS (ESI+) calcd for C16H13N2OS (M + H)+ 281.0749, found
281.0742.
4-(Naphthalen-1-ylamino)benzamide (43). A mixture of
methyl ester 88 (380 mg, 1.3 mmol) in 1 N NaOH (14 mL),
MeOH (5 mL), and DMF (5 mL) in a sealed tube was heated at 100−
110 °C until the reaction was complete. The reaction mixture was then
acidified with 2 N HCl, and the precipitate formed was filtered,
washed, and dried to give the corresponding acid of compound 88
(346 mg), which was used for the next reaction. To a solution of acid
(25 mg, 0.11 mmol) in DMF (1 mL) and CH2Cl2 (2 mL) was added
oxalyl chloride (16 mg, 0.13 mmol) dropwise, and the resultant
mixture was allowed to stir at rt for 30 min and then quenched with
ammonium hydroxide solution (1 mL). After dilution with EtOAc (20
mL), the organic layer was washed with brine and dried over Na2SO4.
After filtration and concentration in vacuo, the residue was purified by
flash column chromatography to give compound 43 as a yellow solid
1
3-(Naphthalen-2-yloxy)benzamide (51). In a manner similar to
that described for the preparation of compound 37, methyl ester 97
(61 mg, 0.22 mmol) in MeOH (5 mL) and dioxane (2 mL) was
treated with 7 N NH3/MeOH solution (5 mL) at 95 °C to give
(14 mg, 50%). H NMR (CD3OD, 600 MHz) δ 7.98 (d, J = 8.4 Hz,
1H), 7.90 (d, J = 8.4 Hz, 1H), 7.71 (dd, J = 6.3, 2.7 Hz, 1H), 7.69 (d, J
= 7.8 Hz, 2H), 7.54−7.51 (m, 1H), 7.51−7.48 (m, 1H), 7.47−7.44 (m,
2H), 6.87 (d, J = 8.4 Hz, 2H), 6.15 (s, 1H). HRMS (ESI+) calcd for
C17H15N2O (M + H)+ 263.1184, found 263.1190.
1
compound 51 as a white solid (21 mg, 37%). H NMR (CDCl3, 600
MHz) δ 8.13 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.66 (d, J =
8.4 Hz, 1H), 7.55−7.50 (m, 2H), 7.50−7.46 (m, 2H), 7.42−7.36 (m,
2H), 7.19−7.16 (m, 1H), 6.99 (dd, J = 7.8, 1.2 Hz, 1H), 6.04 (brs,
1H), 5.88 (brs, 1H). HRMS (ESI+) calcd for C17H14NO2 (M + H)+
264.1025, found 264.1024.
6-(Naphthalen-1-ylamino)nicotinamide (44). In a manner
similar to that described for the preparation of compound 37, methyl
ester 89 (54 mg, 0.19 mmol) was treated with ammonium hydroxide
solution (2.5 mL) to give compound 44 as a white solid (26 mg, 52%).
1H NMR (CDCl3, 600 MHz) δ 8.66 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H),
7.92 (d, J = 7.2 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.80 (d, J = 8.4 Hz,
1H), 7.56 (dd, J = 6.3, 6.3 Hz, 2H), 7.54−7.49 (m, 3H), 7.08 (s, 1H),
6.54 (d, J = 8.4 Hz, 1H). HRMS (ESI+) calcd for C16H14N3O (M +
H)+ 264.1137, found 264.1132.
5-(Naphthalen-2-yloxy)nicotinamide (52). A mixture of phenol
77 (173 mg, 1.2 mmol), bromide 103 (201 mg, 1.0 mmol), CuI (19
mg, 0.10 mmol), picolinic acid (25 mg, 0.20 mmol), and K3PO4 (424
mg, 2.0 mmol) in DMSO (2.0 mL) in a sealed tube was evacuated and
backfilled with argon. The reaction mixture was heated at 90 °C for 24
h and then diluted with EtOAc (10 mL) and water (1 mL), and the
aqueous layer was extracted with EtOAc (2 × 10 mL). The combined
organic layers were dried over Na2SO4 and concentrated, and the
residue was purified by preparative TLC (5% MeOH/CH2Cl2) to give
compound 52 as a white solid (15 mg, 5%). 1H NMR (DMSO-d6, 600
MHz) δ 8.87 (d, J = 1.2 Hz, 1H), 8.61 (d, J = 2.4 Hz, 1H), 8.20 (brs,
1H), 8.03 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.4
Hz, 1H), 7.84 (dd, J = 2.1, 2.1 Hz, 1H), 7.66 (brs, 1H), 7.56−7.51 (m,
2H), 7.49 (dd, J = 7.5, 7.5 Hz, 1H), 7.38 (dd, J = 8.7, 2.7 Hz, 1H).
4-(Naphthalen-1-yloxy)benzamide (45). In a manner similar to
that described for the preparation of compound 37, methyl ester 90
(83 mg, 0.30 mmol) in MeOH (2.5 mL) and dioxane (1.5 mL) was
treated with 7 N NH3/MeOH solution (4 mL) at 60 °C to give
1
compound 45 as a white solid (12 mg, 15%). H NMR (CDCl3, 600
MHz) δ 8.04 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.79 (d, J =
8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 7.8, 7.8 Hz, 1H),
7.48 (dd, J = 7.8, 7.8 Hz, 1H), 7.45 (dd, J = 7.8, 7.8 Hz, 1H), 7.09 (d, J
= 7.2 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H). HRMS (ESI+) calcd for
C17H14NO2 (M + H)+ 264.1025, found 264.1025.
L
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HRMS (ESI+) calcd for C16H13N2O2 (M + H)+ 265.0977, found
265.0979.
40% EtOAc/hexanes) to give compound 108 as a pale solid (195 mg,
20%). 1H NMR (DMSO-d6, 600 MHz) δ 10.58 (s, 1H), 9.84 (s, 1H),
8.66 (s, 1H), 8.30 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.70
(d, J = 7.8 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 7.8, 7.8 Hz,
1H), 7.35 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.11 (dd, J =
9.0, 2.4 Hz, 1H), 2.62 (s, 3H). HRMS (ESI+) calcd for C18H15N2O4
(M + H)+ 323.1032, found 323.1028.
5-(Naphthalen-2-ylthio)nicotinamide (53). In a manner similar
to that described for the preparation of compound 37, methyl ester 98
(1.3 mg, 0.0044 mmol) was treated with 7 N NH3/MeOH solution (2
mL) at 80 °C to give compound 53 as a pale solid (1.1 mg, 89%). 1H
NMR (CDCl3, 600 MHz) δ 8.81 (d, J = 1.8 Hz, 1H), 8.66 (d, J = 3.0
Hz, 1H), 8.01 (s, 1H), 7.98 (s, 1H), 7.86−7.82 (m, 2H), 7.79 (dd, J =
6.0, 3.9 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.55−7.52 (m, 2H). HRMS
(ESI+) calcd for C16H13N2OS (M + H)+ 281.0749, found 281.0745.
3-(Naphthalen-1-ylamino)benzamide (54). In a manner similar
to that described for the preparation of compound 37, methyl ester 99
(30 mg, 0.11 mmol) was treated with 7 N NH3/MeOH solution (7
mL) at 95 °C to give compound 54 as a light-brown solid (7 mg,
N-(6-Hydroxynaphthalen-1-yl)benzamide (109). In a manner
similar to that described for the preparation of compound 108,
compound 104 (100 mg, 0.63 mmol) was treated with acyl chloride 72
(194 mg, 1.38 mmol) followed by hydrolysis to give compound 109 as
1
a white solid (48 mg, 30%). H NMR (DMSO-d6, 600 MHz) δ 10.32
(s, 1H), 9.78 (s, 1H), 8.07 (d, J = 7.2 Hz, 2H), 7.84 (d, J = 9.6 Hz,
1H), 7.64−7.60 (m, 2H), 7.56 (dd, J = 7.5, 7.5 Hz, 2H), 7.42 (dd, J =
8.1, 8.1 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H),
7.10 (dd, J = 9.3, 2.1 Hz, 1H). HRMS (ESI+) calcd for C17H14NO2 (M
+ H)+ 264.1025, found 264.1030.
N-(6-Hydroxynaphthalen-1-yl)-4-methylbenzamide (110). In
a manner similar to that described for the preparation of compound
108, compound 104 (1.00 g, 6.1 mmol) was treated with acyl chloride
73 (1.70 mL, 12.8 mmol) followed by hydrolysis to give compound
110 as a brown solid (1.60 g, 94%). 1H NMR (DMSO-d6, 600 MHz) δ
10.23 (s, 1H), 9.77 (s, 1H), 7.98 (d, J = 7.8 Hz, 2H), 7.82 (d, J = 9.6
Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 7.8, 7.8 Hz, 1H), 7.36
(d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H),
7.10 (dd, J = 9.0, 2.4 Hz, 1H), 2.41 (s, 3H). HRMS (ESI+) calcd for
C18H16NO2 (M + H)+ 278.1181, found 278.1189.
1
24%). H NMR (CDCl3, 600 MHz) δ 8.00 (d, J = 8.4 Hz, 1H), 7.88
(d, J = 8.4 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.53−7.46 (m, 2H),
7.45−7.41 (m, 2H), 7.40 (d, J = 7.2 Hz, 1H), 7.29 (dd, J = 7.8 Hz,
1H), 7.24 (d, J = 7.2 Hz, 1H), 7.07 (dd, J = 8.1, 2.1 Hz, 1H), 6.02 (s,
1H). HRMS (ESI+) calcd for C17H15N2O (M + H)+ 263.1184, found
263.1187.
5-(Naphthalen-1-ylamino)nicotinamide (55). In a manner
similar to that described for the preparation of compound 37, methyl
ester 100 (20 mg, 0.072 mmol) was treated with ammonium
hydroxide solution (2 mL) at 90 °C to give compound 55 as a
yellow solid (7.8 mg, 41%). 1H NMR (DMSO-d6, 600 MHz) δ 8.56 (s,
1H), 8.46 (d, J = 1.8 Hz, 1H), 8.44 (d, J = 2.4 Hz, 1H), 8.11 (d, J = 7.8
Hz, 1H), 8.04 (s, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.69−7.66 (m, 1H),
7.66 (d, J = 7.8 Hz, 1H), 7.57−7.50 (m, 2H), 7.49−7.43 (m, 2H), 7.38
(d, J = 7.8 Hz, 1H). HRMS (ESI+) calcd for C16H14N3O (M + H)+
264.1137, found 264.1141.
N-(6-Hydroxynaphthalen-1-yl)-3-nitrobenzamide (111). In a
manner similar to that described for the preparation of compound 108,
compound 104 (1.00 g, 6.1 mmol) was treated with acyl chloride 74
(2.40 g, 12.8 mmol) followed by hydrolysis to give compound 111 as a
3-(Naphthalen-1-yloxy)benzamide (56). In a manner similar to
that described for the preparation of compound 37, methyl ester 101
(92 mg, 0.33 mmol) in MeOH (5 mL) was treated with ammonium
hydroxide solution (5 mL) at rt to give compound 56 as a brown solid
1
yellow solid (1.80 g, 96%). H NMR (DMSO-d6, 600 MHz) δ 10.70
(s, 1H), 9.81 (s, 1H), 8.90 (s, 1H), 8.51 (d, J = 7.8 Hz, 1H), 8.47 (d, J
= 8.4 Hz, 1H), 7.90−7.84 (m, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.44 (dd,
J = 7.5, 7.5 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.19 (s, 1H), 7.12 (d, J =
9.0 Hz, 1H). HRMS (ESI+) calcd for C17H13N2O4 (M + H)+
309.0875, found 309.0878.
N-(5-Hydroxynaphthalen-1-yl)-4-methyl-3-nitrobenzamide
(112). In a manner similar to that described for the preparation of
compound 108, compound 105 (480 mg, 3.02 mmol) was treated
with acyl chloride 75 (0.93 mL, 6.4 mmol) followed by hydrolysis to
give compound 112 as a tan solid (737 mg, 76%). 1H NMR (DMSO-
d6, 600 MHz) δ 10.59 (s, 1H), 10.21 (s, 1H), 8.67 (s, 1H), 8.31 (d, J =
8.4 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.2 Hz, 1H), 7.58
(d, J = 7.8 Hz, 1H), 7.48 (dd, J = 7.8, 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz,
1H), 7.34 (dd, J = 7.8, 7.8 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 2.62 (s,
3H). HRMS (ESI+) calcd for C18H15N2O4 (M + H)+ 323.1032, found
323.1031.
1
(31 mg, 36%). H NMR (CD3OD, 600 MHz) δ 8.31−8.26 (m, 1H),
8.12−8.10 (m, 1H), 7.87−7.84 (m, 1H), 7.83−7.79 (m, 2H), 7.57 (dd,
J = 7.8, 7.8 Hz, 1H), 7.51−7.46 (m, 3H), 7.41 (d, J = 9.0 Hz, 1H).
HRMS (ESI+) calcd for C17H14NO2 (M + H)+ 264.1025, found
264.1027.
5-(Naphthalen-1-yloxy)nicotinamide (57). In a manner similar
to that described for the preparation of compound 52, phenol 80 (173
mg, 1.2 mmol) was treated with bromide 103 (201 mg, 1.0 mmol) to
1
give compound 57 as a pale solid (5.2 mg, 2%). H NMR (CD3OD,
600 MHz) δ 8.77 (d, J = 1.8 Hz, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.04
(d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.81 (dd, J = 2.7, 2.1 Hz,
1H), 7.78 (d, J = 8.4 Hz, 1H), 7.59−7.51 (m, 2H), 7.49 (dd, J = 7.8,
7.8 Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H). HRMS (ESI+) calcd for
C16H13N2O2 (M + H)+ 265.0977, found 265.0978.
N-(5-Hydroxynaphthalen-1-yl)benzamide (113). In a manner
similar to that described for the preparation of compound 108,
compound 105 (1.00 g, 6.28 mmol) was treated with acyl chloride 72
(1.53 mL, 13.2 mmol) followed by hydrolysis to give compound 113
as a tan solid (1.30 g, 79%). 1H NMR (DMSO-d6, 600 MHz) δ 10.33
(s, 1H), 10.18 (s, 1H), 8.11−8.05 (m, 3H), 7.62 (d, J = 7.2, 7.2 Hz,
1H), 7.58−7.53 (m, 3H), 7.47 (dd, J = 7.5, 7.5 Hz, 1H), 7.42 (d, J =
8.4 Hz, 1H), 7.32 (dd, J = 8.1, 8.1 Hz, 1H), 6.90 (d, J = 7.2 Hz, 1H).
HRMS (ESI+) calcd for C17H14NO2 (M + H)+ 264.1025, found
264.1025.
N-(5-Hydroxynaphthalen-1-yl)-4-methylbenzamide (114). In
a manner similar to that described for the preparation of compound
108, compound 105 (480 mg, 3.02 mmol) was treated with acyl
chloride 73 (0.84 mL, 6.38 mmol) followed by hydrolysis to give
compound 114 as a tan solid (500 mg, 60%). 1H NMR (CD3OD, 600
MHz) δ 8.20 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.56 (d, J =
6.6 Hz, 1H), 7.49−7.44 (m, 2H), 7.37 (d, J = 7.8 Hz, 2H), 7.32 (dd, J
= 7.8, 7.8 Hz, 1H), 6.86 (d, J = 7.2 Hz, 1H), 2.45 (s, 3H). HRMS
(ESI+) calcd for C18H16NO2 (M + H)+ 278.1181, found 278.1188.
N-(5-Hydroxynaphthalen-1-yl)-3-nitrobenzamide (115). In a
manner similar to that described for the preparation of compound 108,
compound 105 (480 mg, 3.02 mmol) was treated with acyl chloride 74
5-(Naphthalen-1-ylthio)nicotinamide (58). In a manner similar
to that described for the preparation of compound 37, methyl ester
102 (2.3 mg, 0.0078 mmol) in MeOH (0.5 mL) and dioxane (0.5 mL)
was treated with ammonium hydroxide solution at 80 °C to give
compound 58 as a light-yellow solid (2.1 mg, 96%). 1H NMR
(CD3OD, 600 MHz) δ 8.77 (brs, 1H), 8.33−8.28 (m, 2H), 8.05 (d, J
= 8.4 Hz, 1H), 8.01−7.96 (m, 3H), 7.88 (d, J = 6.6 Hz, 1H), 7.60−
7.54 (m, 4H). HRMS (ESI+) calcd for C16H13N2OS (M + H)+
281.0749, found 281.0751.
N-(6-Hydroxynaphthalen-1-yl)-4-methyl-3-nitrobenzamide
(108). To a suspension of compound 104 (480 mg, 3.02 mmol) in dry
CH3CN (30 mL) were added DIPEA (0.52 mL, 3.0 mmol) and then
acyl chloride 75 (0.45 mL. 3.1 mmol) dropwise, and the resulting
mixture was allowed to stir at rt for 48 h. The reaction mixture was
poured into a cold solution of water (250 mL) and 1 N HCl (5 mL),
and the solid formed was filtered. After a quick column separation, the
fractions were combined and concentrated. To the residue were then
added THF (6 mL), MeOH (3 mL), and 1 N NaOH (6 mL), and the
resulting mixture was allowed to stir at rt overnight. After
concentration, the residue was dissolved in water (20 mL) and
acidified with 1 N HCl to pH ≈ 2. The solid formed was filtered,
washed, air-dried, and purified by flash column chromatography (0−
M
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(1.18 g, 6.36 mmol) followed by hydrolysis to give compound 115 as a
tan solid (410 mg, 44%). H NMR (CD3OD, 600 MHz) δ 8.93 (s,
1H), 8.49 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.24 (d, J = 8.4
Hz, 2H), 7.83 (dd, J = 7.8, 7.8 Hz, 1H), 7.61 (d, J = 6.6 Hz, 1H),
7.51−7.46 (m, 2H), 7.35 (dd, J = 8.1, 8.1 Hz, 1H), 6.88 (d, J = 7.2 Hz,
1H). HRMS (ESI+) calcd for C17H13N2O4 (M + H)+ 309.0875, found
309.0872.
7.5 Hz, 1H), 8.48 (d, J = 7.6 Hz, 1H), 8.21 (s, 1H), 8.11 (d, J = 9.2 Hz,
1H), 7.88 (t, J = 7.9 Hz, 1H), 7.86 (s, 2H), 7.66 (s, 1H), 7.62 (d, J =
2.4 Hz, 1H), 7.60−7.59 (m, 2H), 7.42 (dd, J = 9.0, 2.4 Hz, 1H).
HRMS (ESI) calcd for C23H16N4O5 (M + H)+ 429.1193, found
429.1203.
5-((5-(4-Methyl-3-nitrobenzamido)naphthalen-1-yl)oxy)-
nicotinamide (63). In a manner similar to that described for the
preparation of compound 59, compound 112 (360 mg, 1.12 mmol)
was treated with bromide 103 (225 mg, 1.12 mmol) to give compound
63 as a light-tan solid (12.6 mg, 2%). 1H NMR (CD3OD, 600 MHz) δ
8.79 (d, J = 1.8 Hz, 1H), 8.68 (s, 1H), 8.52 (d, J = 3.0 Hz, 1H), 8.26
(d, J = 7.8 Hz, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H),
7.84 (dd, J = 2.4, 2.4 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.8
Hz, 1H), 7.59 (dd, J = 7.8, 7.8 Hz, 1H), 7.55 (dd, J = 7.8, 7.8 Hz, 1H),
7.18 (d, J = 7.2 Hz, 1H), 2.68 (s, 3H). HRMS (ESI+) calcd for
C24H19N4O5 (M + H)+ 443.1355, found 443.1349.
1
N-(5-Hydroxynaphthalen-1-yl)isonicotinamide (116). In a
manner similar to that described for the preparation of compound
108, compound 105 (1.00 g, 6.28 mmol) was treated with acyl
chloride 106 (1.62 g, 13.2 mmol) followed by hydrolysis to give
1
compound 116 as a brown solid (1.50 g, 90%). H NMR (DMSO-d6,
600 MHz) δ 10.60 (s, 1H), 10.22 (s, 1H), 8.82 (d, J = 5.4 Hz, 2H),
8.11 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 4.2 Hz, 2H), 7.59 (d, J = 7.2 Hz,
1H), 7.48 (dd, J = 7.8, 7.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.34 (dd,
J = 7.8, 7.8 Hz, 1H), 6.91 (d, J = 7.2 Hz, 1H). HRMS (ESI+) calcd for
C16H13N2O2 (M + H)+ 265.0977, found 265.0977.
5-((5-Benzamidonaphthalen-1-yl)oxy)nicotinamide (64). In a
manner similar to that described for the preparation of compound 59,
compound 113 (526 mg, 2.0 mmol) was treated with bromide 103
(562 mg, 2.8 mmol) to give compound 64 as a brownish solid (13 mg,
N-(5-Hydroxynaphthalen-1-yl)nicotinamide (117). In a man-
ner similar to that described for the preparation of compound 108,
compound 105 (1.00 g, 6.28 mmol) was treated with nicotinic acid
(107) (1.55 g, 12.6 mmol) in the presence of EDC (3.74 g, 19.5
mmol) and HOBt (1.83 g, 12.6 mmol) followed by hydrolysis to give
1
2%). H NMR (DMSO-d6, 600 MHz) δ 10.51 (s, 1H), 8.84 (s, 1H),
8.63 (d, J = 2.5 Hz, 1H), 8.19 (s, 1H), 8.10 (d, J = 7.3 Hz, 2H), 8.02
(d, J = 8.4 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.74 (s, 1H), 7.72 (d, J =
7.2 Hz, 1H), 7.64−7.54 (m, 6H), 7.21 (d, J = 7.5 Hz, 1H). HRMS
(ESI) calcd for C23H17N3O3 (M + H)+ 384.1343, found 384.1351.
5-((5-(4-Methylbenzamido)naphthalen-1-yl)oxy)-
nicotinamide (65). In a manner similar to that described for the
preparation of compound 59, compound 114 (320 mg, 1.15 mmol)
was treated with bromide 103 (230 mg, 1.15 mmol) to give compound
1
compound 117 as a tan solid (610 mg, 37%). H NMR (DMSO-d6,
600 MHz) δ 10.52 (s, 1H), 10.21 (s, 1H), 9.23 (s, 1H), 8.79 (d, J = 4.8
Hz, 1H), 8.42−8.38 (m, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.62−7.56 (m,
2H), 7.50−7.44 (m, 2H), 7.34 (dd, J = 7.8, 7.8 Hz, 1H), 6.91 (d, J =
7.8 Hz, 1H). HRMS (ESI+) calcd for C16H13N2O2 (M + H)+
265.0977, found 265.0980.
5-((5-(4-Methyl-3-nitrobenzamido)naphthalen-2-yl)oxy)-
nicotinamide (59). A mixture of compound 108 (103 mg, 0.32
mmol), bromide 103 (67 mg, 0.33 mmol), CuI (8.2 mg, 0.043 mmol),
picolinic acid (10 mg, 0.084 mmol), and K3PO4 (202 mg, 0.95 mmol)
in DMSO (1.0 mL) in a sealed tube was evacuated and backfilled with
argon. The reaction mixture was heated at 90 °C for 44 h and then
diluted with water (20 mL), and the resulting mixture was extracted
with EtOAc (2 × 40 mL). The combined organic layers were
concentrated, and the residue was purified by flash column
chromatography (0−10% MeOH/CH2Cl2) to give compound 59 as
a pale solid (12.7 mg, 9%). 1H NMR (CD3OD, 600 MHz) δ 8.80 (d, J
= 1.2 Hz, 1H), 8.66 (s, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.25 (d, J = 7.8
Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.90 (dd, J = 2.4, 2.4 Hz, 1H), 7.80
(d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.60−7.52 (m, 3H), 7.35
(dd, J = 9.0, 2.7 Hz, 1H), 2.66 (s, 3H). HRMS (ESI+) calcd for
C24H19N4O5 (M + H)+ 443.1355, found 443.1357.
1
65 as a brown solid (21 mg, 5%). H NMR (DMSO-d6, 600 MHz) δ
10.42 (s, 1H), 8.84 (s, 1H), 8.63 (d, J = 2.2 Hz, 1H), 8.18 (s, 1H), 8.01
(d, J = 7.6 Hz, 3H), 7.89 (d, J = 8.5 Hz, 1H), 7.73 (s, 1H), 7.70 (d, J =
7.2 Hz, 1H), 7.64 (s, 1H), 7.61 (t, J = 7.9 Hz, 1H), 7.55 (t, J = 8.1 Hz,
1H), 7.38 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.3 Hz, 1H), 2.41 (s, 3H).
HRMS (ESI) calcd for C24H19N3O3 (M + H)+ 398.1499, found
398.1501.
5-((5-(3-Nitrobenzamido)naphthalen-1-yl)oxy)nicotinamide
(66). In a manner similar to that described for the preparation of
compound 59, compound 115 (374 mg, 1.2 mmol) was treated with
bromide 103 (341 mg, 1.7 mmol) to give compound 66 as a brownish
1
solid (25 mg, 5%). H NMR (DMSO-d6, 600 MHz) δ 10.88 (s, 1H),
8.92 (s, 1H), 8.86 (s, 1H), 8.64 (d, J = 2.5 Hz, 1H), 8.54 (d, J = 7.6
Hz, 1H), 8.50 (d, J = 8.2 Hz, 1H), 8.20 (s, 1H), 8.05 (d, J = 8.4 Hz,
1H), 7.93−7.88 (m, 2H), 7.76−7.73 (m, 2H), 7.65 (d, J = 7.2 Hz,
2H), 7.57 (t, J = 7.9 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H). HRMS (ESI)
calcd for C23H16N4O5 (M + H)+ 429.1193, found 429.1193.
5-((5-Benzamidonaphthalen-2-yl)oxy)nicotinamide (60). In a
manner similar to that described for the preparation of compound 59,
compound 109 (410 mg, 1.6 mmol) was treated with bromide 103
(442 mg, 2.2 mmol) to give compound 60 as a brownish solid (8 mg,
5-((5-(Isonicotinamido)naphthalen-1-yl)oxy)nicotinamide
(67). In a manner similar to that described for the preparation of
compound 59, compound 116 (610 mg, 2.31 mmol) was treated with
bromide 103 (643 mg, 3.20 mmol) to give compound 67 as a tan solid
1
1%). H NMR (DMSO-d6, 600 MHz) δ 10.46 (s, 1H), 8.59 (s, 1H),
8.63 (d, J = 1.8 Hz, 1H), 8.19 (s, 1H), 8.09−8.06 (m, 3H), 7.83−7.80
(m, 2H), 7.65−7.60 (m, 3H), 7.56−7.54 (m, 4H), 7.41 (d, J = 9.1 Hz,
1H). HRMS (ESI) calcd for C23H17N3O3 (M + H)+ 384.1343, found
384.1353.
1
(9 mg, 1%). H NMR (CD3OD, 600 MHz) δ 8.84−8.77 (m, 3H),
8.54 (s, 1H), 8.12 (d, J = 9.0 Hz, 1H), 8.06−8.02 (m, 2H), 7.93 (d, J =
8.4 Hz, 1H), 7.85 (s, 1H), 7.74 (d, J = 6.0 Hz, 1H), 7.62 (dd, J = 8.4,
8.4 Hz, 1H), 7.58 (dd, J = 8.4, 8.4 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H).
HRMS (ESI+) calcd for C22H17N4O3 (M + H)+ 385.1301, found
385.1297.
5-((5-(4-Methylbenzamido)naphthalen-2-yl)oxy)-
nicotinamide (61). In a manner similar to that described for the
preparation of compound 59, compound 110 (548 mg, 2.0 mmol) was
treated with bromide 103 (562 mg, 2.8 mmol) to give compound 61
as a brownish solid (100 mg, 12%). 1H NMR (DMSO-d6, 600 MHz) δ
10.38 (s, 1H), 8.87 (s, 1H), 8.62 (d, J = 2.6 Hz, 1H), 8.20 (s, 1H), 8.06
(d, J = 9.1 Hz, 1H), 8.00 (d, J = 7.9 Hz, 2H), 7.84 (s, 1H), 7.82 (t, J =
4.4 Hz, 1H), 7.66 (s, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.56 (d, J = 5.1 Hz,
2H), 7.40 (dd, J = 9.2, 2.5 Hz, 1H), 7.37 (d, J = 7.9 Hz, 2H), 2.41 (s,
3H). HRMS (ESI) calcd for C24H19N3O3 (M + H)+ 398.1499, found
398.1508.
N-(5-((5-Carbamoylpyridin-3-yl)oxy)naphthalen-1-yl)-
nicotinamide (68). In a manner similar to that described for the
preparation of compound 59, compound 117 (610 mg, 2.31 mmol)
was treated with bromide 103 (643 mg, 3.20 mmol) to give compound
68 as a tan solid (8 mg, 1%). 1H NMR (CD3OD, 600 MHz) δ 9.25 (s,
1H), 8.80−8.76 (m, 2H), 8.53 (s, 1H), 8.52−8.48 (m, 1H), 8.11 (d, J
= 7.8 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.84 (s, 1H), 7.74 (d, J = 6.6
Hz, 1H), 7.68−7.63 (m, 1H), 7.63−7.56 (m, 2H), 7.20 (d, J = 7.8 Hz,
1H). HRMS (ESI+) calcd for C22H17N4O3 (M + H)+ 385.1301, found
385.1306.
In Vitro Activity of Compound 64. The human breast cancer cell
line MCF-7 (ATCC number HTB-22) was purchased from the
National Cancer Institute. MCF-7 cells were cultured in RPMI
medium and treated with varied concentrations of compound 64 for
5-((5-(3-Nitrobenzamido)naphthalen-2-yl)oxy)nicotinamide
(62). In a manner similar to that described for the preparation of
compound 59, compound 111 (612 mg, 2.0 mmol) was treated with
bromide 103 (562 mg, 2.8 mmol) to give compound 62 as a yellowish
1
solid (120 mg, 14%). H NMR (DMSO-d6, 600 MHz) δ 10.84 (s,
1H), 8.92 (s, 1H), 8.88 (s, 1H), 8.23 (d, J = 2.6 Hz, 1H), 8.53 (d, J =
N
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varied periods of time. In selected experiments, MCF-7 cells were
simultaneously treated with 50 nM TSA to abolish the activity of non-
sirtuin HDAC members. The cells were harvested and immediately
lysed with SDS-PAGE loading buffer. The cell lysate was separated
with SDS-PAGE, and the proteins were then transferred to the PVDF
membrane, which was used for Western blot analysis to detect
acetylated tubulin or whole tubulin. Western blot analysis was
performed using the following antibodies: antiacetylated tubulin
antibody (Sigma, T6793), HRP-conjugated anti-mouse antibody
(Sigma, A5278). The expression of whole regular tubulin (Sigma,
T9026) was used to normalize the loading amount of cell extraction of
each sample.
performed as for SIRT2 and yielded 3.6 and 8.1 mg/L of culture for
SIRT1 and SIRT3, respectively.
Determination of Kinetic Parameters of SIRT1−3. All of the
reactions were carried out under initial-velocity conditions. Reactions
were run in 384-well black nonbinding surface plates (Corning) in a
final reaction volume of 50 μL. Reactions were performed in assay
buffer (50 mM HEPES, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2, 1 mM DTT, and 1 mg/mL BSA) containing either 0.5 μM
SIRT1, 0.4 μM SIRT2, or 1 μM SIRT3 as determined by the Bradford
assay (BioRad). Reactions were performed with either a fixed
concentration of NAD+ (1 mM) and variable peptide (Ac)RHKK-
(Ac)-AMC (0.00137−1 mM) or with a fixed concentration of peptide
(1 mM) and variable NAD+ (0.00137−1 mM). In the case of the
peptide, reactions were also performed without enzyme to allow for
background subtraction at each concentration of the peptide substrate.
Reactions were initiated by the addition of the peptide substrate and
incubated at 37 °C for 30 min. The reactions were then developed by
the addition of 10 μL of quenching buffer (12 mM nicotinamide and
30 000 BAEE/mL trypsin in water) followed by a further 20 min of
incubation at 37 °C. The fluorescence intensity at 460 nm (relative to
DMSO controls) was measured using excitation at 355 nm; this could
be converted into product concentration using a standard curve for
AMC under then reaction conditions. Velocities were fit to the
Michaelis−Menten model using GraphPad Prism.
Selectivity of Compound 64. To confirm the selectivity of
compound 64 for SIRT2 over SIRT1 and SIRT3, reactions were
performed as above. For SIRT1, the reactions contained 0.06 μM
enzyme, 0.066 mM NAD+, 0.076 mM peptide, and 0.045−100 μM 64
(3-fold dilution). For SIRT2, reactions contained 0.2 μM enzyme, 0.05
mM NAD+, 0.05 mM peptide, and 0.031−2 μM 64 (2-fold dilution)
with an additional set containing 100 μM 64 as a negative control. For
SIRT3, reactions contained 0.4 μM enzyme, 0.083 mM NAD+, 0.043
mM peptide, and 0.045−100 μM 64 (3-fold dilution). For each
enzyme, the concentrations of peptide and NAD+ were maintained at a
constant ratio with respect to the individual Km values of peptide and
NAD+ (6 and 4 times below Km, respectively). Assays were quenched
and developed as above with the addition of 100 μM 64 to each
reaction (except for the negative control, which already contained 100
μM 64) to correct for the effect of the compound on the fluorescence
at high concentrations. The data were analyzed using either the
Morrison equation (SIRT2) or the four-parameter dose response
equation (SIRT1 and -3) in GraphPad Prism. This analysis was also
used to determine the active-site concentration of SIRT2.
SIRT1−3 Biochemical Assays. The biochemical assays against
human SIRT1−3 were performed by Reaction Biology Corporation
(RBC) (Malvern, PA, USA; http://www.reactionbiology.com). Full-
length human SIRT1 (GenBank accession no. NM012238, MW = 82
kDa), full-length human SIRT2 (GenBank accession no. NM012237,
MW = 43 kDa), and catalytically active human SIRT3 (GenBank
accession no. NM012239, amino acids 102−399, MW = 32.7 kDa)
were expressed in Escherichia coli and purified. The peptide substrate
for SIRT1−3 was the fluorogenic 7-amino-4-methylcoumarin (AMC)-
labeled peptide Ac-Arg-His-Lys-Lys(Ac)-AMC. The assays were
performed in a buffer containing 50 mM Tris-HCl (pH 8.0), 137
mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mg/mL BSA, which was
added before use. The testing involved a two-step reaction. First, 50
μM AMC-labeled substrate with an acetylated lysine side chain was
incubated with 91 nM SIRT1 (6 μL reaction volume) for 2 h at 30 °C
to produce the deacetylated substrate. The concentrations of SIRT2
and SIRT3 were 233 and 917 nM, respectively. Second, the
deacetylated substrate was digested with a mixture of developer (6
μL) to release AMC, which was detected at λex = 360 nm/λem = 460.
Each compound was dissolved in DMSO (final concentration 1% or
less, which does not significantly affect the enzymatic assay),
sequentially diluted, and used for testing. Background fluorescence
produced by compounds, if detected, was subtracted. The percentages
of enzyme activity (relative to DMSO controls) were calculated. To
determine the IC50 values, compounds were tested in a 10-dose mode
with a 3-fold serial dilution starting at 200 μM (200 μM−10.2 nM) in
singlet. The IC50 values were calculated from the resulting sigmoidal
dose−response curves using GraphPad Prism. Suramin was used as a
reference compound for SIRT1 as well as SIRT2, and nicotinamide
was used for SIRT3.
Expression and Purification of SIRT1−3 for Selectivity and
Mode of Inhibition Studies. The full-length human SIRT2 gene
(accession no. NM012237) was cloned into the expression vector
pReceiver-B01 (GeneCopoeia). The recombinant human SIRT2
contains a His6 affinity purification tag at the N-terminus of the
protein. The plasmid was transformed into E. coli BL21 (DE3) cells
and cultured to an optical density at 600 nm (OD600) of 0.6. The E.
coli was induced with 0.4 mM isopropyl β-D-thiogalactoside (IPTG)
and cultured for 16 h at 18 °C. The cultures were harvested by
centrifugation, and the cell pellets were stored at −80 °C. The cell
pellets were resuspended in 40 mL binding buffer (50 mM HEPES,
pH 8.0, 300 mM NaCl, and 10 mM imidazole). Cells were sonicated
four times on ice using a Branson Sonifier 250 (power = 8, 30% duty
cycle, 2 min) and centrifuged at 45000g for 10 min at 4 °C. The
protein was recovered from the cleared lysate by the addition of 0.5
mL of His60 resin (Clontech) followed by incubation at 4 °C for 1 h
with constant rotation. The resin was recovered by pouring the lysate
into a gravity column. The column was washed using 15 mL of wash
buffer (50 mM HEPES, pH 8.0, 300 mM NaCl, and 20 mM
imidazole) to remove all of the unbound proteins. Then the bound
protein was eluted with 2.5 mL of elution buffer (50 mM HEPES, pH
8.0, 300 mM NaCl, and 500 mM imidazole). The SIRT2 protein was
desalted into storage buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl,
and 10% glycerol) using PD-10 columns (GE Healthcare) and stored
at −80 °C. The yield of the protein was approximately 5.7 mg/L.
Plasmids for the expression of SIRT1 (SIRT1.1, plasmid 13735) and
SIRT3 (SIRT3L-12, plasmid 13736) were obtained from Addgene and
transformed into E. coli BL21 (DE3). Expression and purification were
Determination of Inhibition Modality by Compound 64
against SIRT2. The mode of inhibition assay was performed using the
peptide substrate (Ac)RHKK(Ac)-AMC. The assay was determined in
50 μL of assay buffer as described for the determination of kinetic
parameters, and 100 nM recombinant SIRT2 protein was used [as
determined by active-site titration (data not shown)]. The
concentration of NAD+ was either held constant at 0.2 mM with
variable peptide concentrations (0.044, 0.075, 0.133, 0.150, 0.400,
0.600, 1.20 mM) or varied (0.0312, 0.0625, 0.125, 0.250, 0.500, and
1.00 mM) with a constant peptide concentration (0.3 mM).
Compound 64 was varied from 0.0625 to 2.0 μM (2-fold serial
dilution) with 100 μM 64 included as a negative control. Reactions
were initiated by the addition of the peptide substrate and incubated at
37 °C for 20 min. The reactions were then developed as above. The
fluorescence intensity at 460 nm was measured using excitation at 355
nm. The value of Kai pp was calculated by using GraphPad Prism
software to fit the data to the Morrison equation with the enzyme
concentration constrained to that determined by active-site titration.
Secondary plots of Kai pp as a function of the substrate concentration
were used to determine binding modality by fitting to the Cheng−
Prusoff equations and selecting the mode that afforded the best fit.
Cytotoxicity of Compound 64. Compound 64 was dissolved in
DMSO to give a 100 mM stock solution. For the cell viability assay,
the human breast cancer cell line MCF-7 (ATCC number HTB-22),
human prostate cancer cell line DU 145 (ATCC number HTB-81),
and human chronic myelogenous leukemia K562 (ATCC number
CCL-243) were used. They were seeded at 5000 cells/well in 200 μL
O
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Journal of Medicinal Chemistry
Article
of DMEM containing 10% FCS and allowed to adhere for 24 h prior
to use. For each well, a different concentration of compound 64 was
added, and the contents were mixed and incubated for another 48 h.
Then 20 μL of alamarBlue reagent (Pierce, cat no. 88951) was added
to each well, and the cells were incubated at 37 °C for another 2−4 h
and read under a plate reader (excitation at 530 nm, emission at 590
nm). The CC50 value is the concentration of compound 64 reducing
the growth of mammalian cancer cells by 50%.
ACKNOWLEDGMENTS
■
This research was supported by the Center for Drug Design in
the Academic Health Center of the University of Minnesota to
L.C. The University of Minnesota Supercomputing Institute
provided all of the necessary computational resources.
ABBREVIATIONS USED
Directional Transport across MDCK II Cell Monolayers. The
transport experiments were performed under conditions described
previously.38 Briefly, Madin−Darby Canine Kidney (MDCK II) cells
were seeded onto six-well Transwell semipermeable inserts at a density
of 3.0 × 105 cells/well and allowed to grow to confluency (typically 3
■
SIRT2, sirtuin 2; NAD+, nicotinamide adenine dinucleotide;
SAR, structure−activity relationship; AMC, 7-amino-4-methyl-
coumarin; MDCK, Madin−Darby Canine Kidney; BBB,
blood−brain barrier; A → B, from the apical to basolateral
side; B → A, from the basolateral to apical side; Papp, apparent
permeability coefficient.
days). A transepithelial electrical resistance of 291
8 Ω·cm2 was
observed across the monolayers tested. Mannitol transport across the
monolayers was used as an indication of tight junctions. For the
experiment, the cells were washed and preincubated with assay buffer
at 37 °C for 30 min before the addition of assay buffer containing the
SIRT2 inhibitor (25 μM in assay buffer containing 2% DMSO) to the
donor side (apical side, 1.5 mL; basolateral side, 2.6 mL). Inhibitor-
free assay buffer was added to the receiver side, and 100 μL samples
were drawn from the receiver side at 0, 15, 30, 45, 60, 90, 120, 150,
and 180 min. The volume of the sample aliquots was replenished
immediately with fresh assay buffer. Additionally, samples were drawn
from the donor side at 0 and 180 min. All of the aliquots were
analyzed by HPLC using the Agilent Infinity LC system.
REFERENCES
■
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HPLC Assay. UV detection at 254/280 nm; column, Varian
Microsorb C18, 5 μm, 4.6 mm × 250 mm; solvent system, linear
gradient 30−90% solvent B in 10 min [solvent A = 10 mM KH2PO4;
solvent B = acetonitrile]; flow rate, 1.0 mL/min.
Assay Buffer. NaCl (7.13 g), NaHCO3 (2.10 g), glucose (1.8 g),
HEPES (2.38 g), KCl (0.224 g), MgSO4 (0.295 g), CaCl2 (0.206 g),
and K2HPO4 (0.070 g) in 1000 mL of H2O adjusted to pH 7.4.
The apparent permeability coefficient (Papp) was calculated using
the following equation:
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dQ
( dt )
P
=
app
A × C0
where dQ/dt is the rate obtained from the slopes of plots of amount
transported versus time, A is the surface area of the monolayer, and C0
is the initial drug concentration in the donor compartment.
Molecular Modeling. The modeling study was carried out using
the Schrodinger modeling package.39 The solved X-ray crystallo-
̈
graphic structure of human SIRT2 (PDB entry 1j8f, apo structure)28
was taken from the Protein Data Bank. The missing residues were
added to the X-ray structure using Prime, and the energy minimization
was performed using the OPLS 2005 force field. SIRT2 inhibitors
generated by LigPrep were docked into the Glide grid encompassing
the active site of SIRT2. Docking was carried out in both standard SP
and XP modes. Structural visualization and representation were
performed with PyMOL.40
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ASSOCIATED CONTENT
* Supporting Information
Inhibitory activities of the fragments based on naphthylamide
sulfonic acids, chemical synthesis of compounds 19−36, and
inhibition of SIRT2 by 64 for the determination of its
inhibition modality. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel.: 612-624-2575. Fax: 612-624-8154. E-mail: chenx462@
umn.edu.
■
Notes
The authors declare no competing financial interest.
P
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