Discovery of bicyclic pyrazoles as class III histone deacetylase SIRT1 and SIRT2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.04.068

Abstract A series of bicyclic pyrazole carboxamides was synthesized and tested for inhibitory activity against the class III deacetylase sirtuin enzymes. Moderate to low micromolar inhibitory activities were obtained against SIRT1 and SIRT2. These bicycli

Full text of DOI:10.1016/j.bmcl.2015.04.068

Accepted Manuscript
Discovery of Bicyclic Pyrazoles as Class III Histone Deacetylase SIRT1 and
SIRT2 Inhibitors
Eric Therrien, Guillaume Larouche, Natalie Nguyen, Jubrail Rahil, Anne-Marie
Lemieux, Zuomei Li, Marielle Fournel, Theresa P. Yan, Anne-Julie Landry,
Sylvain Lefebvre, James J. Wang, Kyle MacBeth, Carla Heise, Aaron Nguyen,
Jeffrey M. Besterman, Robert Déziel, Amal Wahhab
PII:
S0960-894X(15)00400-X
http://dx.doi.org/10.1016/j.bmcl.2015.04.068
BMCL 22651
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 February 2015
15 April 2015
20 April 2015
Please cite this article as: Therrien, E., Larouche, G., Nguyen, N., Rahil, J., Lemieux, A-M., Li, Z., Fournel, M.,
Yan, T.P., Landry, A-J., Lefebvre, S., Wang, J.J., MacBeth, K., Heise, C., Nguyen, A., Besterman, J.M., Déziel, R.,
Wahhab, A., Discovery of Bicyclic Pyrazoles as Class III Histone Deacetylase SIRT1 and SIRT2 Inhibitors,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.04.068
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Discovery of Bicyclic Pyrazoles as Class III Histone Deacetylase
SIRT1 and SIRT2 Inhibitors
Eric Therrien,a,*,† Guillaume Larouche,a Natalie Nguyen,a Jubrail Rahil,b Anne-Marie
Lemieux,b Zuomei Li,c Marielle Fournel,c Theresa P. Yan,c Anne-Julie Landry,c Sylvain
Lefebvre,c James J. Wang,c Kyle MacBeth,d Carla Heise,d Aaron Nguyen,d Jeffrey M.
Besterman,c Robert Déziel,a and Amal Wahhaba
aDepartment of Medicinal Chemistry, MethylGene Inc., 7150 rue Frederick-Banting, Montreal, Qc., Canada H4S 2A1
bDepartment of Lead Discovery, MethylGene Inc., 7150 rue Frederick-Banting, Montreal, Qc., Canada H4S 2A1
cDepartment of Molecular Biology, MethylGene Inc., 7150 rue Frederick-Banting, Montreal, Qc., Canada H4S 2A1
dCelgene Inc., 4550 Towne Centre Court, San Diego, CA 92121
Received Month XX, 2015; Accepted Month XX, 2015 [BMCL RECEIPT]
This manuscript is dedicated to Professor Stephen Hanessian on the occasion of his 80th birthday.
Abstract—A series of bicyclic pyrazole carboxamides was synthesized and tested for inhibitory activity against the class III deacetylase
sirtuin enzymes. Moderate to low micromolar inhibitory activities were obtained against SIRT1 and SIRT2. These bicyclic pyrazole
compounds represent a new class of sirtuin inhibitors with a preference for SIRT1 over SIRT2. ©2014 Elsevier Science Ltd. All rights
reserved. Keywords: Sirtuin; SIRT; inhibitor; pyrazole; docking.
13
The histone deacetylase (HDAC) family of enzymes is
known to catalyze the removal of an acetyl group from
lysine residues at the N-terminal tails of histone
proteins. Class I (HDAC1-3 and 8), class IIa
(HDAC4-7 and 9), and class IIb (HDAC6 and 10)
HDACs remove acetyl groups through a zinc-
mediated hydrolysis.1,2 However, class III, also known
as sirtuins are NAD-dependent histone deacetylases
that catalyze the deacetylation of acetylated lysine
resulting in the formation of 2’- and 3’-O-acetyl-ADP-
ribose3,4 and free nicotinamide. Silent information
regulator 2 (Sir2) family of proteins (sirtuins)
comprises five homologues in yeast (ySir2 and HST1-
4) and seven in humans (SIRT1-7).5-7
Activators of SIRT1, such as resveratrol and a
structurally unrelated small molecule SRT2104, are
also very well studied and show promising results in
clinical trials.14,15 SIRT2 was originally linked to age-
associated diseases such as Huntington’s disease,
Alzheimer’s disease, and diabetes,16,17 mainly due to
its cytosolic localization. Recently, SIRT1 and SIRT2
have been considered as targets for cancer
chemotherapy.18-22
SIRT1, the most studied of all sirtuins, has been
implicated in several cellular processes such as
metabolism, fat mobilization, differentiation, and
muscle development.8-10 SIRT1 has also been
demonstrated to deacetylate different transcription
factors such as p53, the FOXO family, and NF-κB.11-
_________________
*Corresponding author. Tel.: +1 514 554 3410; E-mail address:
eric@molecularforecaster.com (E. Therrien)
† Current address. Molecular Forecaster Inc., 969 Marc-Aurèle
Figure 1. Known sirtuin inhibitors and activators.
Fortin, Laval, Qc., Canada H7L 6H9.

Among the few known inhibitors of sirtuins23-25 (Fig.
1), only a limited number exhibit inhibitory activity
against human subtypes. Besides the common and
unselective inhibitor nicotinamide,26 sirtinol27 was one
of the first synthetic small molecule inhibitors of
SIRT1. Although sirtinol is a micromolar inhibitor of
yeast Sir2p enzyme and human SIRT2, it is a weak
inhibitor of SIRT1.28,29 The sirtuin inhibitor cambinol,
which was identified as active against both human
SIRT1 and SIRT2 enzymes, showed antitumor activity
in vitro and in mouse xenograft models.30
2). Acylation of the bis-enolate of methyl 2-
oxocycloheptanecarboxylate and subsequent
treatment with hydrazine hydrate yielded the
corresponding hexahydrocycloheptapyrazole 7.
Conversion of the methyl carboxylate 7 to the
carboxamide was achieved under the usual
5
9
conditions. Similarly, the hexahydrocyclooctapyrazole
analogue 10 was also synthesized from keto-ester 6.
Several indoles were identified from
a high-
throughput screen as selective inhibitors of SIRT1.31
The most potent analogue, EX-527 has a reported IC50
of 0.1 µM against SIRT1 and inhibits the
deacetylation of p53 at a concentration of 1 µM.31
More recently, low nanomolar pan-inhibitors of
SIRT1, SIRT2, and SIRT3 (e.g. compound 31) were
discovered through affinity screening using encoded
library technology (ELT).32
o
Scheme 2. Reagents and conditions: (a) NaH, n-BuLi, THF 0 C then
4-Cl-PhCOCl; (b) NH2NH2.H2O, THF; (c) NH3 7N in MeOH, 95 oC
(sealed tube).
Ring modifications were also expanded to include
various substitutions. Thus, mono- and di-substituted
cyclohexanones 11 were converted to their
corresponding keto-ester analogues 12 (Scheme 3).
Acylation of the dianion derived from 12 with a
selection of acyl chlorides or methyl carboxylates
provided the 1,3-diketone intermediates (not shown in
Scheme 3) which were further transformed to
pyrazoles 13a-g using the method described above.
The gem-dimethyl regioisomer 14 was also
synthesized based on this procedure starting from 3,3-
dimethylcyclohexanone. The tricyclic analogue 17
was made from 4,5-benzo-cycloheptanone carboxylate
16 which was synthesized as described in the
literature.34
At the time of this investigation, we envisaged EX-
527 as a starting point for the design of novel small
molecule sirtuin inhibitors. Our initial efforts were
based on identification of a replacement for the central
core of the molecule. Hence, we utilized a pyrazole
scaffold in lieu of an indole. We herein describe the
synthesis and biological evaluation of an
unprecedented series of bicyclic pyrazole derivatives
as SIRT1 and SIRT2 inhibitors.
The synthesis of these pyrazoles was achieved via the
dianion of the commercially available ethyl 2-
oxocyclohexanecarboxylate 1 which was acylated
with either the desired acyl chloride or the methyl
carboxylate
and
the
resulting
1,3-diketone
intermediate33 was reacted with hydrazine hydrate in
THF to provide the tetrahydroindazole 2 (Scheme 1).
The ethyl ester 2 was then converted to the
carboxamide 3 in the presence of a 7N solution of
ammonia in methanol upon heating in a sealed tube.
The methyl amide 4 was obtained from ester 2 using a
solution of methylamine in methanol.
Scheme 3. Reagents and conditions: (a) NaH, KH, diethyl carbonate,
THF, 65 oC; (b) NaH, n-BuLi, THF 0 oC then R1COCl or R1CO2Me; (c)
NH2NH2.H2O, THF (d) NH3 7N in MeOH, 95 oC (sealed tube); (e)
o
Ethyl 3-oxo-4-(triphenylphosphoranylidene)butyrate, THF, 120 C; (f)
H2, Pd/C, EtOH.
The inhibitory activities of SIRT1 and SIRT2 for
different aryl and heteroaryl substituted bicyclic
pyrazoles, compounds 3a-w, are presented in Table 1.
The inhibitory activities were tested in a fluorescence-
based assay using Cbz-Lys(Ac)-AMC as a substrate.35
Attempts to separate the two enantiomers of
compound 3b by chiral HPLC36 failed to provide the
required separation level to yield optically pure
material. Therefore, the compounds were tested as
o
Scheme 1. Reagents and conditions: (a) NaH, n-BuLi, THF 0 C then
R1COCl or R1CO2Me; (b) NH2NH2.H2O, THF; (c) NH3 7N in MeOH,
95 oC (sealed tube); (d) MeNH2, MeOH, 95 oC (sealed tube).
In order to further explore the chemical space of the
active site of the enzyme, we modified the ring size on
the bicyclic pyrazole scaffold. Thus, the 7- and 8-
membered ring analogues were synthesized (Scheme
2

their racemic mixtures of stereoisomers. In general,
the bicyclic pyrazoles displayed a moderate degree of
selectivity for SIRT1 over SIRT2 and did not show
any inhibition of class I or class II HDACs (data not
shown).37
similar activity to that of phenyl 3a. Analogues
bearing fused bicyclic heteroaryl rings attached at the
2-position such as 2-benzothiophene 3o, 2-benzofuran
3p, 2-benzothiazole 3q and N-methyl-2-indole 3r
proved to be tolerated replacements of the 4-
chlorophenyl group. Although these compounds did
not show increased enzymatic inhibitory activities
against SIRT1 compared to the 4-chlorophenyl
analogue 3b, they showed moderate activity against
SIRT2 with IC50 of 5.3-9.0 µM (3o-r). However,
further substitutions (3t-u) and different attachment
points (3v-w) led to weaker inhibitors against both
enzymes.
The unsubstituted phenyl ring (3a) provided a weak
inhibitor of SIRT1 (showing only partial inhibition at
20 µM). Systematic variation of the position of the
chlorine on the phenyl ring resulted in the 4-
chlorophenyl pyrazole 3b being the most active
among analogues 3b-d showing IC50 of 1.0 µΜ for
SIRT1. Equipotent inhibitor was obtained with the
3,4-dichlorophenyl analogue 3e, whereas the
regioisomer 3f was devoid of activity. The
replacement of the 3-chlorine of 3e with fluorine gave
a slightly weaker inhibitor 3g. Exchange of the para-
chlorine on the phenyl ring with different groups (3h-
l) reduced considerably the inhibitory activity against
SIRT1. Both the N-methylamide 4b and the ethyl
carboxylate 2g showed no inhibition at 20 µM,
indicating an essential requirement for the primary
carboxamide.
Table 2 lists SIRT inhibitory activities for different
ring modifications of aryl and heteroaryl bicyclic
pyrazoles. The seven-membered ring compound 9 was
2-fold less potent against SIRT1 than the
corresponding tetrahydroindazole 3b, whereas the
eight-membered ring analogue 10 showed no
inhibitory activity. All attempts to prepare the
unprecedented five-membered ring derivative failed.
Further modifications on the ring led to compound 13a
with increased activity against both SIRT enzymes.
Unlike modifications of pyrazoles 3, every variation of
the R1 group of 13a (compounds 13b-e) was
detrimental to the activity against both enzymes.
Mono-phenyl 13f and fused benzyl compound 17
showed only partial inhibition while spiro ketal 13g
and regioisomer gem-dimethyl analogue 14 were
devoid of activity.
Table 1. SIRT inhibitory activities for different aryl and heteroaryl
substituted bicyclic pyrazoles.
SIRT1
SIRT2
IC50 µM
IC50 µM
Compounds
R1
(% inh @ 20 (% inh @ 20
µM)a,b
µM)a,b
EX-527c
3a
3b
4b
3c
-
0.5
6.5
Phenyl
(44%)
1.0
NI
4-Chlorophenyl
(43%)
NI
4-Chlorophenyl
NI
Table 2. SIRT inhibitory activities for different ring modifications of
aryl and heteroaryl bicyclic pyrazoles.
3-Chrorophenyl
(27%)
NI
NI
3d
3e
2-Chrorophenyl
(11%)
14.8
NI
SIRT1
SIRT2
3,4-Dichlorophenyl
2,4-Dichlorophenyl
4-Chloro-3-fluorophenyl
4-Chloro-3-fluorophenyl
4-Bromophenyl
1.2
IC50 µM IC50 µM
(% inh @ (% inh @
20 µM)a,b 20 µM)a,b
Compounds
R1
R2
R3
3f
(24%)
2.6
3g
2g
3h
3i
(44%)
NI
NI
9
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
-
-
-
-
2.1
(28%)
0.8
6.8
(22%)
1.7
1.9
(47%)
NI
10
4-Fluorophenyl
(42%)
6.2
13a
13b
13c
13d
13e
13fc
13g
14
Me Me
3j
4-Methylphenyl
(36%)
NI
3,4-Dichlorophenyl Me Me
4-Bromophenyl Me Me
N-Methyl-2-indole Me Me
(23%)
(56%)
NI
(28%)
(42%)
NI
3k
3l
4-Methoxyphenyl
4-Trifluoromethylphenyl
2-Thiophene
NI
NI
NI
3m
3n
3o
3p
3q
3r
(47%)
11.0
1.7
NI
2-Benzothiophene
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
Me Me
(53%)
(59%)
NI
(67%)
(62%)
NI
4-Bromo-2-thiophene
2-Benzothiophene
2-Benzofuran
NI
Ph
H
O
5.3
O
3.1
9.0
-
-
-
NI
NI
2-Benzothiazole
2.2
8.3
17
-
(42%)
(50%)
aValues are means of three experiments. All compounds are racemic.
N-Methyl-2-indole
2-Indole
2.7
7.8
bNI
diastereoisomers.
=
no inhibition (less than 20% at 20 µM). cMixture of
3s
(46%)
(31%)
(38%)
NI
(43%)
(51%)
(33%)
NI
3t
5-Chloro-N-methyl-2-indole
5-Bromo-2-benzothiophene
N-Methyl-3-indole
3-Benzothiophene
3u
3v
3w
In addition to the inhibitory activity of both SIRT1
and SIRT2, we evaluated the ability of the bicyclic
pyrazoles to inhibit the endogenous deacetylase
activity of p53 in Namalwa cells (human, blood,
lymphoma, Burkitt) using an ELISA-based assay
(Table 3).38 The compounds were ranked by their
IC2xB, which is defined as the inhibitor concentration
needed to enhance p53 deacetylation to twice the basal
level. In general, compounds that showed low
(48%)
(44%)
aValues are means of three experiments. All compounds are racemic.
bNI
diastereoisomers.
=
no inhibition (less than 20% at 20 µM). cMixture of
Replacement of the 4-chlorophenyl substituent with
different heteroaryl moieties led to bicyclic pyrazoles
3m-w. The 2-thiophene 3m gave a compound with
3

micromolar activity against SIRT1 enzyme in vitro
inhibited p53 deacetylation in the cells. Compounds
3b, 3e, 3h, 3q, 9, and 13a showed p53 deacetylation
with IC2xB from 0.5 to 1.5 µM in accord with their
enzymatic potency. Although a correlation was
observed between p53 deacetylation and SIRT1
inhibition, further investigation is required to help
substantiate that the cellular effects are due to Sirt1
inhibiton.
crystallized EX-527 analogue (compound 35, PDB ID:
4I5I) obtained from docking experiments using the
automated docking software FITTED.39,40 All docked
inhibitors showed a similar binding mode when the
(R)-isomers were considered.
Key interactions of the inhibitor with the active site of
the enzyme are the hydrogen bonds of the
carboxamide with residues Asp348 and Ile347, the
pyrazole N-H with the residue Asn346, and the
hydrophobic contacts of the aryl group with amino
acid residues Phe273, Phe297, Ile279, Ile316, and
Ile347 (not shown). One of the two bridging water
molecules was displaced during the docking41 to
accommodate the pyrazole in 13a which occupies a
slightly shifted position as compared with compound
35 in the X-ray structure, although a very well
organized hydrogen bond network was conserved.
Table 3. SIRT1 enzymatic activity and in vitro p53 deacetylation
inhibitory activity of bicyclic pyrazoles.a
p53 deacetylation,
IC2xB, µMc
SIRT1
IC50, µM
0.5
Compounds
EX-527b
0.6
3b
3e
0.5
1.0
1.2
1.2
3h
3q
9
1.5
1.9
0.9
2.2
0.8
2.1
13a
1.2
0.8
In conclusion, we have discovered a new class of
sirtuin inhibitors based on the bicyclic pyrazole
scaffold. Structure-activity relationships led to the
identification of a few compounds that are low
micromolar inhibitors of SIRT1. However, there was
little tolerance for substitution both on the phenyl ring
and on the tetrahydrocyclohexane moiety. The
simplicity of these molecules and their relative ease of
synthesis should lead to further optimization of this
new class of sirtuin inhibitors.
aValues are means of three experiments. bInhibitory activity was
c
determined in our laboratories. This is an in-house method developed
to rank the ability of our SIRT1 inhibitors to inhibit p53 deacetylation.
The value reported is the measured concentration at twice the basal
level (basal: the lowest detected fluorescence value in Rfu).
Preliminary investigation of the pharmacokinetic
profile of 3b and 13a, indicated that both compounds
were stable in mouse plasma. In rats, 3b had a
clearance of 1.8 (L/h)/kg and a half-life of 1.1 hours.
The bicyclic pyrazole 13a had a lower clearance (0.3
(L/h)/kg) and a longer half-life (5.1 hours). Both 3b
and 13a had moderate oral bioavailability of 20% and
23%, respectively.
Acknowledgments
The authors like to thank Dr. Arkadii Vaisburg for his
comments and corrections to this manuscript.
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38. Namalwa cells (50,000 cells/well) were plated in 96
well culture plates in a final volume of 100 µl. Test
compounds were dissolved in DMSO and serial-
dilutions were carried out with media mixture for
final compound concentrations of 0.005 to 50 µM and
DMSO concentration of 0.95%. Plates were
incubated for 3 hours at 37 ºC under a 5% CO2
atmosphere. Cells were lysed for 30 min with 10 µl of
10X Lysis Buffer, and endogenous p53 protein
present in 25 µl of cell lysate was captured by a p53
antibody (Santa-Cruz Cat# SC-126 DO-1) coated into
the microwells of 96 well black plates (Nunc
Maxisorp). A 25 µl of an acetylated-lysine382-p53
antibody (Cell Signaling Cat#2525) was added to
detect the acetylated-lysine on p53 protein during an
overnight incubation. After a washing step, 50 µl of
an anti-mouse HRP antibody (Sigma #A-0545) was
used to recognize the bound detection antibody; and
after another washing step, 50 µl of Amplex Red
solution was added to develop the reaction. Following
a 15 min incubation period, fluorescence reading was
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5

the fluorescence was proportional to the quantity of
the acetylated p53 protein
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FITTED.
6