Discovery of oxazolo[4,5-b]pyridines and related heterocyclic analogs as novel SIRT1 activators

Article abstract of DOI:10.1016/j.bmcl.2008.11.106

SIRT1 is an NAD⁺-dependent protein deacetylase that appears to produce beneficial effects on metabolic parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to and more potent than resveratrol.

Full text of DOI:10.1016/j.bmcl.2008.11.106

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2350–2353
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of oxazolo[4,5-b]pyridines and related heterocyclic analogs
as novel SIRT1 activators
*
Jean E. Bemis , Chi B. Vu, Roger Xie, Joseph J. Nunes, Pui Yee Ng, Jeremy S. Disch, Jill C. Milne,
David P. Carney, Amy V. Lynch, Lei Jin, Jesse J. Smith, Siva Lavu, Andre Iffland,
Michael R. Jirousek, Robert B. Perni
Sirtris, A GSK Company, 200 Technology Square, Cambridge, MA 01239, USA
a r t i c l e i n f o
a b s t r a c t
SIRT1 is an NAD+-dependent protein deacetylase that appears to produce beneficial effects on metabolic
parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been
shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower
organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of
oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to
and more potent than resveratrol.
Article history:
Received 8 September 2008
Revised 26 November 2008
Accepted 26 November 2008
Available online 6 December 2008
Keywords:
Sirtuins
Ó 2009 Published by Elsevier Ltd.
SIRT1
SIRT1 activator
SIRT1 is one of seven sirtuin members of the NAD+-dependent
deacetylases identified to date,1 and has recently been linked to a
number of metabolic functions, including activation of PGC-1a, a
ulators. This SIRT1 FP assay is a coupled assay where the first
reaction is the deacetylation reaction of a p53-derived substrate
catalyzed by SIRT1 and the second reaction is cleavage by trypsin
at the newly exposed lysine residue.14 Activation in this assay is
measured both as the concentration of compound required to in-
crease the enzyme activity by 50% (EC1.5) as well as by the percent-
age maximum activation achieved at the highest doses of
compound tested (max act). One notable series of SIRT1 activators
identified through the HTS was the oxazolo[4,5-b]pyridines (OAP),
as exemplified by compounds 2 and 3 (Fig. 1). These compounds
transcriptional coactivator that may regulate glucose metabo-
lism.2,3 In addition, modulation of SIRT1 could lead to an increase
in mitochondrial biogenesis, a biological response that is similarly
produced by calorie restriction.4,5 With a net increase in mitochon-
dria, skeletal muscle and adipose tissues display an improved glu-
cose metabolism profile.6 On this basis, activation of SIRT1 could
serve as a novel approach to treat type II diabetes and other met-
abolic disorders. Additional validation for SIRT1 as a therapeutic
target has come from transgenic mice that overexpress SIRT1, as
these animals display phenotypes that are characteristic of an im-
proved metabolic profile.7 Resveratrol (1), a naturally occurring
small molecule activator of SIRT1, has been reported to extend life-
span in yeast,8 Caenorhabditis elegans, Drosophila9 and rodents.10
When administered to rodents, resveratrol has been demonstrated
to improve metabolism and glucose tolerance, as well as overall
physical performance in various stress tests.11 Recent efforts in
our laboratory have identified compounds capable of activating
SIRT1 that are structurally distinct from and significantly more po-
tent than 1.12
showed moderate activation of SIRT1, with EC1.5 = 6
max act, and 25 M, 207% max activation, respectively. Impor-
tantly, these compounds were more potent than resveratrol, 1,
(EC1.5 = 46 M, 200% max act) and were amenable to analog syn-
lM, 240%
l
l
thesis at several different points on the scaffold. The focus of the
initial analog libraries was twofold: to drive potency as well as to
increase the solubility of the OAP compounds. Fortuitously, the
attachment of solubilizing groups on specific points of the scaffold
improved the inherent activity of the compounds. For example,
substitution with the solubilizing groups amidoethyldimethyl-
amine and dimethylamine on the central phenyl ring of the OAP
provided compounds 4, 5, and 6, with 5 activating SIRT1 at submi-
A high-throughput in vitro fluorescence polarization assay was
used to screen a commercial library of small molecules, which re-
sulted in the identification of several novel scaffolds as SIRT1 mod-
cromolar levels (EC1.5 = 0.7 lM, 240% max act), Table 1. Inclusion of
the methylpiperazine moiety exhibited similar function. Com-
pound 8 has a subtle methoxy positional rearrangement, which
when combined with the methylpiperazine offers a boost in po-
tency to an EC1.5 value of 0.5
trend was lost, however, if the solubilizing group was instead
lM with 220% max activation. This
* Corresponding author. Tel.: +1 617 252 6920; fax: +1 617 252 6924.
E-mail address: jean_bemis@comcast.net (J.E. Bemis).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.11.106

J. E. Bemis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2350–2353
2351
O
O
OH
N
HN
HN
O
O
N
O
N
O
OH
N
HO
N
1
2
3
Figure 1. Resveratrol (1), and the oxazolo[4,5-b]pyridines (2 and 3).
Table 1
Table 3
Oxazolopyridines with solubilizing groups on central aryl ring
Select benzimidazole SAR
R1
R1
N
O
N
H
N
N
R2
R2
R1
R2
EC1.5
,
l
M (% max act)a
Compound
R1
R2
EC1.5, l
M (% max act)a
Compound
O
O
H
N
O
O
N
4
1.8 (330%)
0.7 (240%)
1.1 (200%)
N
N
O
N
H
N
13
H
1.7 (180%)
O
H
O
H
N
O
O
5
6
N
H
O
O
O
14
15
16
H
H
H
0.4 (595%)
0.3 (253%)
0.5 (314%)
0.4 (820%)
N
H
O
O
O
O
O
N
H
O
O
N
N
N
N
H
O
O
NH
NH
7
1.1 (240%)
0.5 (220%)
N
H
N
N
N
H
N
O
O
O
O
NH
N
N
8
N
H
17
N
H
N
O
a
a
EC1.5 values were determined from three separate titration curves. Each of the
EC1.5 values shown represents the mean of three determinations, with variations in
individual values of <15%.
EC1.5 values were determined from three separate titration curves. Each of the
EC1.5 values shown represents the mean of three determinations, with variations in
individual values of <15%.
Table 2
Ortho- and para-substituted oxazolopyridines
Table 4
Ortho-substituted azabenzimidazoles
R1
O
1
R2
R
N
N
N
N
R1
R2
EC1.5
,
l
M (% max act)a
H
N
Compound
R1
EC1.5, l
M (% max act)a
O
O
Compound
O
O
N
9
H
H
0.9 (170%)
4.4 (209%)
>100
O
O
O
H
N
H
18
4.1 (250%)
N
10
11
N
H
N
H
19
20
0.9 (273%)
0.7 (230%)
N
O
O
O
O
O
O
O
N
H
H
H
N
H
N
O
O
N
12
>100
21
0.5 (270%)
N
H
N
H
a
a
EC1.5 values were determined from three separate titration curves. Each of the
EC1.5 values shown represents the mean of three determinations, with variations in
individual values of <15%.
EC1.5 values were determined from three separate titration curves. Each of the
EC1.5 values shown represents the mean of three determinations, with variations in
individual values of <15%.

2352
J. E. Bemis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2350–2353
placed on the pyridyl ring of the OAP core; such compounds exhib-
ited no activation of SIRT1 (data not shown).
EC1.5 = 0.4 lM and sixfold activation of the enzyme. The quinazo-
line amide functionality of compound 15 imparted improved po-
tency as well. Solubilizing groups were again considered in an
effort to improve solubility, and the substitution of an aminopiper-
azine moiety on the amido-aryl ring of 17 turned the benzimid-
azole into the first high-fold SIRT1 activator with a maximum
A major breakthrough in the SAR of the OAP series was the
determination that ortho-substitution of the amide linker on the
central phenyl ring was optimal. A shift of the dimethoxyaryl
amide and the dimethylaminoarylamide from the meta- to the
ortho-positions resulted in significantly more active compounds.
activation of 820% (EC1.5 = 0.4 lM).
Compounds 9 and 10 have an EC1.5 = 0.9 and 4.4
with roughly twofold maximum activation (Table 2) as compared
with the 6 and 25 M values of their meta-counterparts, 2 and 3.
Interestingly, if the amide moiety was instead incorporated at
the para-position of the central ring, all activity was lost, as exem-
plified by compounds 11 and 12.
In addition to scanning for alternate functional groups to drive
the potency of the OAP class, other bicyclic cores were explored as
well. Appropriately substituted benzimidazoles were found to be
potent SIRT1 activators. Selected SAR from this series is shown in
Table 3. As with the oxazolopyridines, ortho-substitution of the
amide linker was preferred. Compound 14 is an analog of the
screening hit, 2, and has much improved potency with an
l
M, respectively,
A slight modification of the benzimidazole core gave rise to the
analogous azabenzimidazoles, which were also found to similarly
activate SIRT1. Compounds with tertiary amine solubilizing groups
attached to the amido-aryl ring had good potency (19 and 20,
Table 4).
l
It is interesting to note that in this series, meta- and para-sub-
stitutions of the amide linker were well tolerated, unlike the SAR
observed with the OAP and benzimidazole scaffolds (Table 5). This
divergence in the activity of seemingly similar series will be better
understood as the structural biology around SIRT1 progresses.
The synthetic routes to these bicyclic ring systems were
straightforward, facilitating the synthesis of varied analogs.13
The oxazolopyridine core was constructed through an initial con-
densation of 2-amino-3-hydroxypyridine and an aminobenzoic
acid in the presence of PPA. Subsequent amide formation was car-
ried out under microwave conditions in good yields. To incorpo-
rate solubilizing groups onto the central aryl ring, 26 was first
combined with monomethyl-5-nitroisophthalate to form the
amide, 30. Cyclization to the bicyclic system was again carried
out with PPA, and the resulting benzoic acid, 31, was reduced
and converted to the methylamine. Reduction of the nitro group
and subsequent amide formation afforded the final substituted
OAPs (Scheme 1).
The benzimidazoles were synthesized by amide couplings with
the commercially available benzimidazole-2-ylaniline, 35 (Scheme
2). The azabenzimidazole core was formed through a sulfur-medi-
ated condensation of diaminopyridine and nitrobenzaldehyde
(Scheme 3). The azabenzimidazole was then treated with SEMCl
to provide a mixture of the protected nitrogen isomers, which were
separated by column chromatography. The individual isomers
were carried forward through subsequent nitro reduction and
amide formation.
Table 5
Meta- and para-substituted azabenzimidazoles
Compound
Ar
EC1.5, l
M (% max act)a
O
22
0.5 (232%)
1.4 (324%)
2.3 (150%)
1.6 (180%)
O
HN
O
Ar
N
N
H
N
O
O
23
24
25
O
O
Ar
N
NH
N
H
N
O
O
In summary, oxazolo[4,5-b]pyridines were identified as novel
activators of SIRT1 through high-throughput screening efforts.
Diversification of the linker positions and aryl functionalities led
to compounds with submicromolar activities, such as compound
a
EC1.5 values were determined from three separate titration curves. Each of the
EC1.5 values shown represents the mean of three determinations, with variations in
individual values of <15%.
8 (EC1.5 = 0.5
lM, 220% max act). Further exploration of the bicyclic
Ar
NH2
HN
HO
HO2C
+
NH2
O
a
b
O
O
H2N
N
N
N
N
29
N
26
27
28
c
NO2
NO2
OH
NH
O
O
N
32
d
e
N
31
N
N
N
CO2H
OH
NO2
30
O
Ar
O
CO2Me
HN
NO2
O
N
34
O
N
f
g,h,i
N
N
NR1R2
NR1R2
33
Scheme 1. Reagents and conditions: (a) PPA, 180 °C, 80%; (b) ArCOCl, pyridine, microwave 160 °C, 10 min, 60–83%; (c) monomethyl 5-nitroisophthalate, HATU, DIEA, 90%; (d)
PPA, 160 °C, 80%; (e) isobutylchloroformate, NMM, then NaBH4, 68%; (f) MsCl, Et3N, CH2Cl2 then NHR1R2, 75% over two steps; (g) sodium hydrosulfide hydrate, MeOH, H2O,
88%; (h) ArCOCl, pyridine, microwave, 160 °C, 10 min, 50–75%; (i) TFA, CH2Cl2 as needed for BOC deprotection.

J. E. Bemis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2350–2353
2353
cacy in several rodent models of type 2 diabetes14 and as such offer
exciting opportunities for treatment of metabolic disease.
Ar
O
H2N
HN
N
N
N
H
N
H
References and notes
35
36
1. For a review on the sirtuins, see: Shaday, M.; Sinclair, D. Biochem. J. 2007, 404, 1.
2. Nemoto, S.; Fergusson, M. M.; Finkel, T. J. Biol. Chem. 2005, 280, 16456.
3. Rodgers, J. T.; Lerin, C.; Haas, W.; Gygi, S. P.; Spiegelman, B. M.; Puigserver, P.
Nature 2005, 434, 113.
Scheme 2. Reagents and conditions: ArCOCl, pyridine, microwave, 160 °C, 10 min,
35–80%.
4. Cohen, H. Y.; Miller, C.; Bitterman, K. J.; Wall, N. R.; Hekking, B.; Kessler, B.;
Howitz, K. T.; Gorospe, M.; de Cabo, R.; Sinclair, D. A. Science 2004, 305, 390.
5. Bordone, L.; Guarante, L. Nat. Rev. Mol. Cell. Biol. 2005, 6, 298.
6. Hollander, P. Diabetes Spectrum 2007, 20, 159.
7. Bordone, L.; Cohen, D.; Robinson, Al.; Motta, M. C.; van Veen, E.; Czopik, A.;
Steele, A. D.; Crowe, H.; Marmor, S.; Luo, J.; Gu, W.; Guarente, L. Aging Cell 2007,
6, 759.
8. Lamming, D. W.; Wood, J. G.; Sinclair, D. A. Mol. Microbiol. 2004, 53, 1003.
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Nature 2004, 430, 686.
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Prabhu, V. V.; Allard, J. S.; Lopez-Lluch, G.; Lewis, K.; Pistell, P. J.; Poosala, S.;
Becker, K. G.; Boss, O.; Gwinn, D.; Wang, M.; Ramaswamy, S.; Fishbein, K. W.;
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P.; Ingram, D. K.; de Cabo, R.; Sinclair, D. A. Nature 2006, 444, 337.
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12. Bemis, J. E; Vu, C. B; Xie, R.; Nunes, J. J.; Ng, P. Y.; Disch, J. S.; Milne, J. C.; Carney,
D. P.; Lynch, A. V.; Jin, L.; Smith, J. J.; Lavu, S.; Iffland, A.; Jirousek, M. R.; Perni, R.
B. Abstract of Papers, 235th National Meeting of the American Chemical Society,
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C.; Bemis, J. E.; Xie, R.; Vu, C. B.; Ng, P. Y.; Disch, J. S. WO2007/019345 A1, 2007.
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J.; Lynch, A. V.; Yang, H.; Galonek, H.; Israelian, K.; Choy, W.; Iffland, A.; Lavu,
S.; Medvedik, O.; Sinclair, D. A.; Olefsky, J. M.; Jirousek, M. R.; Elliott, P. J. Nature
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O2N
H2N
NH2
NH2
N
a
N
N
b,c
N
H
N
37
N
N
SEM
38
39
Ar
Ar
HN
O
O
HN
N
N
N
d
e
N
H
N
N
SEM
40
41
Scheme 3. Reagents and conditions: (a) 2-nitrobenzaldehyde, sulfur, neat, 120 °C,
52%; (b) NaH, DMF, SEMCl, 88% (mixture of regioisomers); (c) Pd/C (10%), H2 (1 bar),
EtOAc, MeOH, 72–89%; (d) ArCOCl, EtN3, CH2Cl2, 30–80%; (e) 5 N HCl/EtOH, 1:1,
70 °C, 70–90%.
core provided two additional classes of SIRT1 activators, benzimi-
dazoles and azabenzimidazoles. Within the benzimidazole series,
high-fold activators of SIRT1 were discovered, such as compound
17, showing eightfold activation of the enzyme (EC1.5 = 0.4 lM).
In total, these compounds represent the first reported SAR of small
molecule activators of SIRT1. The understanding of the SAR within
these series has led to more potent analogs which have shown effi-