3-(N-Arylsulfamoyl)benzamides, inhibitors of human sirtuin type 2 (SIRT2)

Article abstract of DOI:10.1016/j.bmcl.2012.02.089

Inhibition of sirtuin 2 (SIRT2) is known to be protective against the toxicity of disease proteins in Parkinson's and Huntington's models of neurodegeneration. Previously, we developed SIRT2 inhibitors based on the 3-(N-arylsulfamoyl)benzamide scaffold, i

Full text of DOI:10.1016/j.bmcl.2012.02.089

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2789–2793
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-(N-Arylsulfamoyl)benzamides, inhibitors of human sirtuin type 2 (SIRT2)
Soo Hyuk Choi a, , Luisa Quinti b, Aleksey G. Kazantsev b, Richard B. Silverman a,
⇑
a Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University,
Evanston, Illinois 60208-3113, United States
b MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital and Harvard Medical School, 114 16th Street, Charlestown, MA 02129, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of sirtuin 2 (SIRT2) is known to be protective against the toxicity of disease proteins in Parkin-
son’s and Huntington’s models of neurodegeneration. Previously, we developed SIRT2 inhibitors based on
the 3-(N-arylsulfamoyl)benzamide scaffold, including3-(N-(4-bromophenyl)sulfamoyl)-N-(4-bromophe-
nyl)benzamide(C2–8, 1a), which demonstrated neuroprotective effects in a Huntington’s mouse model,
but had low potency of SIRT2 inhibition. Here we report that N-methylation of 1a greatly increases its
potency and results in excellent selectivity for SIRT2 over SIRT1 and SIRT3 isoforms. Structure–activity
relationships observed for 1a analogs and docking simulation data suggest that the para-substituted
amido moiety of these compounds could occupy two potential hydrophobic binding pockets in SIRT2.
These results provide a direction for the design of potent drug-like SIRT2 inhibitors.
Received 20 January 2012
Accepted 24 February 2012
Available online 3 March 2012
Keywords:
Sirtuin 2
Neurodegenerative disease
3-(N-Arylsulfamoyl)benzamides
Sirtuin selectivity
Hydrophobic pocket
Ó 2012 Elsevier Ltd. All rights reserved.
Sirtuin 2 (SIRT2), one of seven known human sirtuins, is a
NAD+-dependent enzyme that catalyzes the deacetylation of his-
ery of more potent and highly selective SIRT2 inhibitors and
describe related structure–activity relationship (SAR) studies.
Scheme 1 shows the structures of and related synthetic routes
to analogs of 1a. Compounds 2a–d were prepared from 3-(chloro-
sulfonyl)benzoic acid (5) and the corresponding para-substituted
anilines 6 in one step. Compounds 3a–e and 4a were prepared
from 5 and 6 in two steps by consecutive amide coupling reactions.
Compounds 4b–d were synthesized from 1a in one step by selec-
tive N-alkylation using potassium carbonate and the correspond-
ing alkyl halide at 50 °C.
Figure 2 shows in vitro SIRT1, SIRT2, and SIRT3 inhibition assay
results for 1a,b and two N-methylsufonamide analogs, 2a and 4a.
Compound 1a is a weaker SIRT2 inhibitor than 1b, as reported pre-
viously.4 The potencies of 2a and 4a with SIRT2, however, are very
similar and are slightly better than that of 1b. In addition, 2a and
4a are more selective SIRT2 inhibitors than 1b; 2a and 4a are vir-
e
tone and other substrate N -acetyllysines with concomitant forma-
tion of nicotinamide and 20-O-acetyl-ADP-ribose.1 Previous studies
showed that inhibition of SIRT2 mediated neuroprotective effects
in Parkinson’s disease (PD) and Huntington’s disease (HD) mod-
els.2–4 In in vitro models of PD and HD, neuroprotective effects of
SIRT2 inhibition have been associated with changes in aggregation
of the
a-synuclein and huntingtin proteins, respectively. A previ-
ously identified inhibitor of polyglutamine aggregation, a hallmark
of many neurodegenerative diseases,5 namely C2–8 (1a, Fig. 1), has
potential as a therapeutic candidate based on its neuroprotective
effects achieved in transgenic HD models and, apparently, good
drug-like properties.6 AK-1 (1b), which has a common 3-sulfoben-
zamide scaffold to that of 1a and is neuroprotective against a-syn-
uclein toxicity,2 is a SIRT2 inhibitor that was reported to have good
selectivity for SIRT2 over SIRT1 and SIRT3.2 Most known SIRT2
inhibitors show low selectivities for SIRT1 and SIRT3, even though
their potencies are better than that of 1b.7–11 Compound 1a, how-
ever, displayed low potency as a SIRT2 inhibitor. Here we test the
feasibility of enhancing SIRT2 inhibition and selectivity of the ther-
apeutically promising structural scaffold 1a. We report the discov-
tually inactive with SIRT1 and SIRT3 up to 50 lM, while 1b shows
some inhibitory activity with SIRT1. These results suggest that sim-
ple modifications of 1a (methylation) can enhance both potency
and selectivity toward SIRT2. A subsequent SAR study, changing
the para-substituents (2) or the N-alkyl substituent (4), identified
2b as a more potent SIRT2 inhibitor (see Supplementary data
Fig. S1). Compound 2b was selective for SIRT2; at 10 lM concen-
tration 2b did not inhibit SIRT1 and inhibited SIRT3 by only 5%
(see Supplementary data Fig. S2).
Further modification of the para-substituents (3a–e, Table 1)
shows that two compounds, 3a and 3e, inhibit the SIRT2 activity
Abbreviations: SIRT, sirtuin; PD, Parkinson’s disease; HD, Huntington’s disease;
SAR, structure–activity relationship.
Corresponding author. Tel.: 847 491 5653.
E-mail addresses: r-silverman@northwestern.edu, Agman@chem.northwester-
n.edu (R.B. Silverman).
⇑
by about half at 10
lM concentration. Compounds 3a and 3e were
highly selective; there was no inhibition of SIRT1 or SIRT3 at 10
lM
Current address: Department of Chemistry, Yonsei University, Seoul, South Korea.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.02.089

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S. H. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2789–2793
Br
NO2
O
HN
HN
O
H
N
N
S
O O
S
O O
Br
AK-1
1b
C2-8
1a
Figure 1. Structures of polyglutamine aggregation inhibitor C2–8 (1a) and SIRT2
inhibitor AK-1 (1b).
R2
N
Br
O
HN
O
H3C
CH3
N
R3
N
S
S
O O
O O
R1
Br
2a R1 = R2 = Br
2b R1 = R2 = Cl
2c R1 = R2 = F
2d R1 = R2 = CF3
3a R1 = Br, R2 = Cl
3b R1 = Br, R2 = F
3c R1 = Br, R2 = CF3
3d R1 = Cl, R2 = Br
3e R1 = CF3, R2 = Br
4a R3 = methyl
4b R3 = ethyl
4c R3 = allyl
4d R3 = benzyl
HO
O
EDC
DMAP (cat.)
2a-d
R1
NHCH3
+
Cl
CH2Cl2
(2 eq)
S
O O
6a R1 = Br
6b R1 = Cl
6c R1 = F
5
6d R1 = CF3
HO
O
6, EDC
DMAP (cat.)
CH2Cl2
R1
5
+
6
CH3
N
3a-e
(3 eq)
Figure 2. Compound inhibition activities in in vitro sirtuin-catalyzed lysine
deacetylation assays. Potency and selectivity of 1a,b, 2a, and 4a have been
evaluated in dose–response assays against deacetylase activities of SIRT2, SIRT1,
and SIRT3 at indicated concentrations. Each dose has been tested in triplicate.
Compound 1b was included as a reference compound.
S
O O
7a R1 = Br
7b R1 = Cl
7c R1 = F
7d R1 = CF3
Table 1
8, K2CO3
DMF, 50 o
p-bromoaniline
In vitro SIRT2 inhibition assay results for 3a–e
1a
7a
4a
4b-d
EDC, DMAP (cat)
CH2Cl2
Compound Relative SIRT2 activitya (%) Concentration of compounds (
lM)
C
3a
3b
3c
3d
3e
54
57
76
72
55
10
50
50
10
10
8b iodoethane
8c allyl bromide
8d benzyl bromide
Scheme 1. Syntheses of 2–4.
a
Measured by the relative fluorescence observed from the SIRT2 assay.
concentration. However, more extensive testing of 3a and 3e, such
as a direct comparison of 3d and 3e as shown in Figure 3, con-
firmed 3a to be a more potent SIRT2 inhibitor than 3e. Compound
3a, therefore, was selected for testing with 2a, 2b, and 1b (Fig. 4).
Figure 4 shows that all of these analogs of 1a are more potent than
1b. It should be noted that experiments represented by Figures 2
and 4 were carried out months apart, and the values differ some-
what. Compound 1b was routinely included in assays for normali-
zation of results.
To date, the only available crystal structure of SIRT2 does not
contain any ligand molecule bound1; it is likely that the uncom-
plexed SIRT2 structure is different from that of a ligand-bound
conformation. A few computational methods have been reported
to find the active conformation of SIRT2, including those that
use energy minimization and/or molecular dynamics simula-
tions9,12–14 and a homology model constructed from the SIRT2

S. H. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2789–2793
2791
of binding interactions using a binding score would not be reli-
able, we assumed that at least a qualitative evaluation of binding
conformations of analogs of 1a with SIRT2 could be garnered. Fig-
ure 5(a) shows a putative ligand-binding site in SIRT2, proposed
previously by a comparison with the crystal structures of other
sirtuin homolog–ligand complexes.17–19 There are two potential
hydrophobic binding pockets in the active site. A small cleft be-
tween Phe119 and His187 would be a good hydrophobic binding
pocket for an aromatic ring, which could be stabilized by
p–p
interactions with the phenyl group of Phe119 and the imidazole
ring of His187. This channel has been proposed as the binding
site for the side chain of the acetylated lysine residue of a sub-
strate. There is another potential hydrophobic binding pocket sur-
rounded by residues with hydrophobic side chains Phe96, Tyr104,
Leu107, Leu112, Pro115, Ile118, Phe119, Leu134, and Leu138.
Docking simulation results predict that the two para-substituted
anilino moieties of analogs of 1a occupy the two potential hydro-
phobic pockets. Figure 5(b) shows a potential binding conforma-
tion for 4a. The two p-bromophenyl groups are bound into the
two hydrophobic pockets of SIRT2. Additionally, there is a hydro-
gen bond between the carbonyl group of 4a and the hydroxyl
group of Tyr104. Other active analogs of 1a adopted very similar
conformations in docking simulations.
Figure 3. Comparison of SIRT2 dose–response activities for 3d and 3e.
structure and other homolog–ligand complex structures.2,15 The
SIRT2 structure without any modification has been used in a
few cases.10,16 We carried out docking simulations with the origi-
nal crystal structure of SIRT2.1 Although quantitative assessment
Figure 4. SIRT2 inhibition by three analogs of 1a compared with that of 1b.
Figure 5. (a) Putative binding site of SIRT2; hydrophobic pockets are surrounded by a red dotted line. (b) Binding conformation of 4a predicted by a docking simulation with a
potential H-bond shown.

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S. H. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2789–2793
Figure 6. (a, b) Overlay of binding conformations of 4a (cyan) and 4b (magenta) from different views (c) Relative SIRT2 activity from treatment with 1a and 4a–d at 25 lM.
The inhibitory assay data for 2a–d and 3a–e suggested that the
potency might be correlated with the size of the two para-substit-
uents, R1 and R2 (Scheme 1), both of which contribute to the hydro-
phobic interactions in the purported hydrophobic pockets. It is
reasonable that there would be an optimal size for R1 or R2 that
is dependent on the size of a hydrophobic binding pocket to max-
imize hydrophobic contact. Among the four compounds with the
same R1 group (R1 = Br), 2a (R2 = Br), and 3a (R2 = Cl) showed com-
parable activities that were much higher than those of 3b (R2 = F)
and 3c (R2 = CF3). The order of van der Waals volumes for the four
R1 substituents is CF3 > Br > Cl > F. It is therefore likely that the
maximum hydrophobic contact might be achieved with an R2
group having a van der Waals volume between Cl and Br. By the
same analogy, the activities of the three compounds with the same
R2 group (R2 = Br) can be compared to derive the optimal size for
the R1 group. The activity of 2a (R1 = Br) is greater than those of
3d (R1 = Cl) and 3e (R1 = CF3), suggesting that the size of the hydro-
phobic binding pocket for the R1 group might be similar to that of
the R2-binding pocket.
comparable activities. The binding conformation of 4a in
Figure 6(b) shows that the proton of the amide moiety is exposed
to solvent, suggesting that no significant binding interaction is con-
tributed by the N-methylamide moiety of 2a.
We have demonstrated that 1a could serve as a lead scaffold for
inhibitors of SIRT2. The N-methylsulfonamide moiety of analogs of
1a increases both SIRT2 activity and selectivity, both of which are
higher than the known SIRT2 inhibitor 1b. The observed structure–
activity relationships with various R1 and R2 groups are consistent
with the binding conformation of analogs of 1a predicted by dock-
ing simulations. Both terminal aniline moieties might occupy the
two potential hydrophobic binding pockets having strict size
requirements. These observed SARs should be valuable for struc-
ture-based design of more potent SIRT2 inhibitors.
Acknowledgment
The authors are grateful to the National Institutes of Health
(5U01NS066912) for financial support of this research.
Five analogs of 1a, including 1a and 4a–d, contain the same R1
and R2 groups (R1 = R2 = Br) and are structurally different only by
the R3 substituent. Among these five compounds, only 4a
(R3 = Me) showed significant activity against SIRT2, indicating
that the N-methylsulfonamide moiety is crucial to the SIRT2
activity. Considering that the docked conformation of 1a is very
similar to that of 4a, the increased potency of 4a over 1a could
be attributed to the additional van der Waals contact between
the N-methylsulfonamide moiety of 4a and SIRT2. However, this
one additional hydrophobic interaction should not be sufficient
to explain the much greater potency of 4a. One possible explana-
tion is that the N-methyl substituent behaves as an anchor to di-
rect the adjacent para-bromoanilino group close to the channel
between Phe119 and His187, resulting in more favorable hydro-
phobic interactions.
Supplementary data
Supplementary data (experimental procedures, in vitro SIRT2
inhibition data, NMR spectra, and HRMS data) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2012.02.089.
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