Oxadiazole-carbonylaminothioureas as SIRT1 and SIRT2 inhibitors

Article abstract of DOI:10.1021/jm800639h

A new inhibitor for human sirtuin type proteins 1 and 2 (SIRT1 and SIRT2) was discovered through virtual database screening in search of new scaffolds. A series of compounds was synthesized based on the hit compound (3-[[3-(4-tert-butylphenyl)1,2,4-oxadiazole-5-carbonyl]ammo]-1-[3- (trifluoromethyl)phenyl]thiourea). The most potent compound in the series was nearly as potent as the reference compound (6-chloro-2,3,4,9-tetrahydro-1H- carbazole-1-carboxamide).

Full text of DOI:10.1021/jm800639h

J. Med. Chem. 2008, 51, 4377–4380
4377
Oxadiazole-carbonylaminothioureas as SIRT1
and SIRT2 Inhibitors
Tero Huhtiniemi,*,† Tiina Suuronen,‡ Valtteri M. Rinne,†
Carsten Wittekindt,†,§ Maija Lahtela-Kakkonen,†
Elina Jarho,† Erik A. A. Walle´n,†,| Antero Salminen,‡
Antti Poso,† and Jukka Leppa¨nen†
Figure 1
the binding pocket is restricted by the conformation of the
flexible loop and the side chains of the residues in the flexible
loop, especially by the residue F273, which is located closest
to the deacetylation site.
Department of Pharmaceutical Chemistry, UniVersity of Kuopio,
P.O. Box 1627, 70211 Kuopio, Finland, Department of Neurology,
UniVersity of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland
The NAD+-nicotinamide exchange reaction,19,20 the reverse
catalysis with binding of nicotinamide to ADP-ribose and NAD+
resynthesis, underlies the inhibition of sirtuins by nicotinamide
at the nicotinamide binding site.1 We suggest that the binding
of a high affinity inhibitor at this site, such as compound 1 or
2,4,16 may prevent the productive binding and subsequent
cleavage of NAD+. Napper et al. have also proposed that
compound 1 binds after nicotinamide release and inhibits the
release of one or both of the products, 2-O-acetyl-ADP-ribose
and deacetylated peptide.4 However, more kinetic analyses are
needed to clarify the possible interaction of these inhibitors with
the reaction intermediates or the products.
The synthetic route to the oxadiazole-carbonylaminothioureas
and the oxadiazole-carbonylaminoureas is presented in Scheme
1. Arylnitriles 3-7 were reacted with hydroxylamine in aqueous
ethanol to obtain hydroxyamidines 8-12. Ethyl chloroacetate
was added dropwise to the hydroxylamidines in dry pyridine/
dichloromethane solution to yield compounds 13-17. Stirring
these compounds with hydrazine monohydrate in ethanol gave
compounds 18-22, respectively. The products 2, 23-35 were
obtained by adding the appropriate isothiocyanate or isocyanate
in dry dimethylformamide (DMF) dropwise to a solution of
compounds 18-22 in dry DMF. After stirring the mixtures
overnight at room temperature (rt), the solvent was evaporated
and the products were precipitated from toluene or ethanol/water.
Schemes 2 and 3 present the synthetic route for the replace-
ments of the carbonylaminothiourea moiety of the hit structure.
In Scheme 2, Boc-glycine 36 was activated with ethyl chloro-
formate and coupled with the appropriate aniline to form
compounds 37 and 38. The Boc protecting group was removed,
and the resulting intermediate products 39 and 40 were coupled
with compounds 13 and 15 to yield compounds 41 and 42,
respectively. In Scheme 3, glutaric anhydride and the hydroxya-
midines 8 and 10 were reacted to form the 4-aryl-[1,2,4]oxa-
diazol-5-yl]-butyric acids 43 and 44. Activation with thionyl
chloride to the corresponding acid halide and subsequent
coupling with the appropriate aniline gave compounds 45 and
46. The reference compound 1 was synthesized as a racemate
as described in the literature.4
ReceiVed May 28, 2008
Abstract: A new inhibitor for human sirtuin type proteins 1 and 2
(SIRT1 and SIRT2) was discovered through virtual database screening
in search of new scaffolds. A series of compounds was synthesized
based on the hit compound (3-[[3-(4-tert-butylphenyl)1,2,4-oxadiazole-
5-carbonyl]amino]-1-[3-(trifluoromethyl)phenyl]thiourea). The most
potent compound in the series was nearly as potent as the reference
compound (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide).
Sirtuins have pathogenetic roles in cancer, diabetes, heart
failure, neurodegeneration, and aging, and they are currently
under investigation as new potential targets for drug discovery.1,2
Several inhibitors of SIRT1a and SIRT2 have been reported,3
including nicotinamide, EX-527 analogues (compound 1 in
Figure 1),4 splitomicin analogues,5–7 sirtinol analogues,8
cambinol,9 2-anilinobenzamides,10 aristoforin,11 and compounds
reported by our group.12–15
We have reported a comparative model of SIRT1 and a
binding mode for analogues of compound 1.16 This model of
SIRT1 was applied for virtual database screening of novel
inhibitors, which share a similar binding site with compound
1. In this study, we report a novel hit compound (compound 2
(SPB 00466) in Figure 1 and Table 1) and a series of analogues.
The hit compound possesses an oxadiazole-carbonylaminothio-
urea backbone, which is new for SIRT1 and SIRT2 inhibitors,
and it inhibits SIRT1 at 192 µM and SIRT2 at 57 µM level
(IC50).
The proposed binding mode of 2 in the active site of SIRT1
is presented in Figure 2. The docking was performed with
GOLD program, which is based on a genetic algorithm.17 The
results were visualized using the Sybyl 7.1 software package.18
The tert-butylphenyl group of 2 binds at the deacetylation site
of the acetylated lysine substrate in close contact with H363.
The surrounding area consists of several hydrophobic residues,
giving rise to hydrophobic interactions. The oxadiazole-carbo-
nylaminothiourea backbone is bound in a pocket surrounded
by a flexible loop (residues from 269 to 295). The oxadiazole
ring acts as an H-bond acceptor forming an H-bond to the
backbone NH of I347. The NH of the amino-thiourea group
donates an H-bond to the carboxyl group of D348. The size of
The in vitro assays for SIRT1 and SIRT2 activity were
performed using the protocol of McDonagh et al.21 based on
the release of nicotinamide from NAD+ during the reaction.
The radioactively labeled nicotinamide was detected by thin
layer chromatography technique22 because the optic properties
of some compounds disturb the use of fluorescent techniques.
The detection of radioactively labeled nicotinamide indicates
the magnitude of enzymatic activity indirectly, hence, the
deacetylation of one acetylated substrate is in a stoichiometric
relation with the cleavage of one NAD+ to nicotinamide and
O-acetyl-ADP-ribose.22 The poor solubility of the compounds
* To whom correspondence should be addressed. Phone: (358) 17
163693. Fax (358) 17 162456. E-mail: tero.huhtiniemi@uku.fi.
†
Department of Pharmaceutical Chemistry, University of Kuopio.
Department of Neurology, University of Kuopio.
Present address: COSMOlogic GmbH & Co. KG, Burscheider Strasse
‡
§
515, D-51381, Leverkusen, Germany.
|
Present address: Division of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Helsinki, PO Box 56, 00014, Helsinki, Finland.
a Abbreviations: SIRT, human sirtuin type protein; sirtuin, Sir2 (silent
information regulator 2).
10.1021/jm800639h CCC: $40.75
2008 American Chemical Society
Published on Web 07/22/2008

4378 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Letters
Table 1. SIRT1 and SIRT2 Activity Assay Results for Compounds 1, 2, 23-35, 41, 42, 45, and 46 (95% Confidence Intervals for IC50 Given in
Parentheses).
inhibition at 200 µM ( SDa (%)
IC50 (µmol/L)b
compd
R1
X
R2
SIRT1
SIRT2
SIRT1
SIRT2
c
1
2
3 (1-5)
59 ( 3
70 ( 2
18 ( 15
14 ( 7
-2 ( 5
22 ( 10
57 ( 16
12 ( 10
54 ( 6
10 ( 3
29 ( 5
-22 ( 1
4 ( 3
79 (45-140)
87 ( 2
89 ( 15
39 ( 2
38 ( 10
29 ( 3
7 ( 2
4-t-Bu-Ph
4-t-Bu-Ph
4-t-Bu-Ph
4-t-Bu-Ph
4-t-Bu-Ph
4-t-Bu-Ph
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
Ph
S
S
O
O
S
O
S
O
S
S
S
S
S
S
3-CF3-Ph
4-CF3-Ph
3-NO2-Ph
Ph
Ph
n-Bu
3-CF3-Ph
3-CF3-Ph
4-CF3-Ph
4-F-Ph
4-CF3-Ph
4-F-Ph
4-CF3-Ph
4-CF3-Ph
3-CF3-Ph
Ph
3-CF3-Ph
Ph
192 (104-354)
325 (153-689)
57 (26-125)
109 (67-177)
23
24
25
26
27
28
29
30
31
32
33
34
35
41
42
45
46
56 ( 4
51 ( 4
71 ( 3
35 ( 3
8 ( 11
22 ( 2
-4 ( 26
71 ( 4
18 ( 10
17 ( 9
0 ( 4
13 (5-37)
113 (64-200)
257 (179-395)
74 (47-115)
318 (140-723)
Ph
3-pyridyl
3-(Boc-NH)-Ph
4-t-Bu-Ph
Ph
4-t-Bu-Ph
Ph
69 ( 19
20 ( 1
28 ( 8
17 ( 9
0 ( 12
168 (98-289)
129 (72-232)
5 ( 14
a SD, standard deviation, (n ) 2-3). b IC50 were determined with the NAD+ based assay21 for compounds that had over 50% inhibition at 200 µM for
SIRT1 or SIRT2 (repeated at least three times). c 1 was tested as a racemate.
Scheme 1a
Figure 2. The binding mode of compound 2 in the SIRT1 model.
The yellow dashed lines represent H-bond between the ligand and the
residues at the active site. The green color visualizes the surface of the
binding pocket.
a Reagents and conditions: (a) hydroxylamine hydrochloride (1.5 equiv),
sodium hydroxide (1.5 equiv), 80% ethanol/water, rt, 1-2 d; (b) ethyl
2, 23-35 presented difficulties to determine the IC50 values with
high concentration samples. The IC50 values were determined
for all compounds, which had over 50% inhibition at the
concentration of 200 µM.
chloroacetate (1.2 equiv), pyridine/dichloromethane, 0 °C-rt, 2-24 h; (c)
hydrazine monohydrate (5 equiv), ethanol, rt, 1-24 h; (d) the appropriate
isocyanate or isothiocyanate, dimethylformamide, 0 °C-rt, 1 d.
The inhibitory activity of the hit compound 2 was determined
in vitro for SIRT1 (IC50 ) 192 µM) and for SIRT2 (IC50 ) 57
µM). These in vitro results were also confirmed by using a more
physiological probe than the Biomol probe. The deacetylation
of acetylated R-tubulin isolated from rat hippocampal stem cells
were determined in vitro. The inhibitory activity of the hit
compound 2 was demonstrated in this assay with SIRT1 at 200
µM and with SIRT2 at 50 µM concentration level (Supporting
Information).
The synthesized compounds 1, 2, 23-35, 41, 42, 45, and 46
and their inhibitory activities for SIRT1 and SIRT2 are presented
in Table 1. The most potent SIRT1 inhibitor in the series was
compound 28 (IC50 ) 13 µM). It was 15 times more potent
than the hit compound 2 (IC50 ) 192 µM). In addition,
compound 28 was almost as potent as the reference compound
1 (IC50 ) 3 µM), which is the most potent known SIRT1
inhibitor. Interestingly, compound 28 was the only compound
in the series showing weak to moderate selectivity for SIRT1

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4379
Scheme 2a
The intermediates 18-22, which lack the R2 substituent, were
also tested at the concentration of 200 µM, but they did not
show inhibitory activity (not included in Table 1).
Compounds 41, 42, 45, and 46 were synthesized to study
the importance of the carbonylaminothiourea part of the
backbone by replacing it with either a carbamoyl-methyl-amide
or a butyramide linker. These compounds showed only weak
inhibitory activities and confirmed the importance of carbony-
laminothiourea part of the scaffold for the inhibitory activity.
In conclusion, a new oxadiazole-carbonylaminothiourea scaf-
fold with inhibitory activity for SIRT1 and SIRT2 was found
through virtual screening. The series of compounds based on
this scaffold showed that each of the modified positions R1,
R2, and X were important for the inhibitory activity. For
example, replacement of aryl substituent with phenyl at either
position (R1 or R2) was not tolerated. At the R1 position, bulky
and lipophilic substituents were preferred over smaller or more
polar substituents such as phenyl and pyridyl. Modifications at
the R2 position indicated that the trifluoromethyl substituent of
the phenyl group is important for the inhibitory activity.
However, no specific binding has been observed for the
trifluoromethyl group in the binding model. In addition to its
high electronegativity, the trifluoromethyl group is known to
increase the lipophilicity of the compounds and therefore the
affinity of compounds in the hydrophobic binding sites, which
may lead to increased inhibitory activity of these compounds.
Modifications at the X positions indicated that the compounds
based on the oxadiazole-carbonylaminothiourea scaffold were
more potent than the compounds based on the oxadiazole-
carbonylaminourea scaffold. The relatively high acidity of the
NH thiourea protons compared to urea protons respectively is
correlated with a strong hydrogen-bonding donor capability, thus
providing efficient anchoring points of complementary func-
tional groups such as carboxyl group of D348 in the proposed
binding mode (Figure 2). The most potent compound 28 was
nearly as potent as the previously reported reference compound
1.
a Reagents and conditions: (a) (i) triethylamine (1.1 equiv), ethyl
chloroformate (1.1 equiv), dichloromethane, -20 °C, 30 min.; (ii) triethy-
lamine (1.1 equiv), the appropriate aniline (1 equiv), rt, 1 d. (b) 25%
Trifluoroacetic acid/dichloromethane, 0 °C-rt, 2 h. (c) Compd 13 or 15
(1.1 equiv), triethylamine (1.1 equiv), methanol, 40 °C, 2-24 h.
Scheme 3a
a Reagents and conditions: (a) glutaric anhydride, few drops of DMF,
microwave 100 °C. (b) (i) Thionyl chloride (2.2 equiv), benzene, 60 °C,
1 h; (ii) the appropriate aniline (1.1 equiv), benzene, 0 °C-rt, 30 min.
similar to compound 1, while the other compounds had a weak
selectivity for SIRT2. Compound 2 (IC50 ) 57 µM) was the
most potent SIRT2 inhibitor in the series. Compounds 2, 28
(IC50 ) 113 µM), and 30 (IC50 ) 74 µM) and the reference
compound 1 (IC50 ) 79 µM) are equipotent inhibitors for
SIRT2.
Acknowledgment. This study was financially supported by
Finnish Cultural Foundation. We thank Tiina Koivunen and Erja
Kivioja for technical assistance.
Compounds 2, 28 (X ) S, R2 ) 3-CF3-Ph), and 23, 30, 32,
34, 35 (X ) S, R2 ) 4-CF3-Ph) and 31, 33 (X ) S, R2 )
4-F-Ph) were synthesized to study the modifications at the R1
position. 4-tert-butylphenyl, 1-naphthyl, and 3-(Boc-amino)-
phenyl groups were preferred as R1 substituents and showed
active compounds (23, 30, and 35). However, the compounds
with the phenyl and 3-pyridyl groups as R1 substituents had a
strongly decreased (32) or no detectable (34) inhibitory activity.
Compounds 28, 29 (R1 ) 1-naphthyl, R2 ) 3-CF3-Ph) and
25, 26 (R1 ) 4-t-Bu-Ph, R2 ) Ph) were synthesized to study
the modifications at the X position, but the results were not
consistent. Compound 28 with the thiourea group showed a
preference for SIRT1 over SIRT2 (IC50 ) 13 µM and 113 µM,
respectively). Compound 29 with the urea group was an
equipotent SIRT2 inhibitor (IC50 ) 257 µM, 51% at 200 µM)
but clearly a weaker SIRT1 inhibitor (12% inhibition at 200
µM) as compared to compound 28. Compounds 2, 23, 26 (R1
) 4-t-Bu-Ph, X ) S), 28, 30, 31 (R1 ) 1-naphthyl, X ) S),
24, 25, 27 (R1 ) 4-t-Bu-Ph, X ) O), and 32, 33 (R1 ) Ph, X
) S) were synthesized to study the modifications at the R2
position. The para and meta substituted (trifluoromethyl)-phenyl
groups (2 and 23, 28 and 30) were preferred over phenyl (26)
or 4-fluorophenyl group (31) at the R2 position. However, all
combinations with different R1 and X groups were not made.
Supporting Information Available: Database screening pro-
cedure, detailed experimental procedures for the syntheses, NMR
spectra, ESI-MS results, and elemental analysis data for the new
compounds and in vitro assay for SIRT1 and SIRT2 activities are
described. This material is available free of charge via the Internet
at http://pubs.acs.org.
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