Discovery of imidazo[1,2-6]thiazole derivatives as novel SIRT1 activators

Article abstract of DOI:10.1021/jm8012954

A series of imidazo[1,2-b]thiazole derivatives is shown to activate the NAD⁺-dependent deacetylase SIRT1, a potential new therapeutic target to treat various metabolic disorders. This series of compounds was derived from a high throughput scree

Full text of DOI:10.1021/jm8012954

J. Med. Chem. 2009, 52, 1275–1283
1275
Discovery of Imidazo[1,2-b]thiazole Derivatives as Novel SIRT1 Activators
Chi B. Vu,* Jean E. Bemis, Jeremy S. Disch, Pui Yee Ng, Joseph J. Nunes, Jill C. Milne, David P. Carney, Amy V. Lynch,
Jesse J. Smith, Siva Lavu, Philip D. Lambert, David J. Gagne, Michael R. Jirousek, Simon Schenk,† Jerrold M. Olefsky,† and
Robert B. Perni
Sirtris Pharmaceuticals, 200 Technology Square, Cambridge, Massachusetts 02139
ReceiVed September 5, 2008
A series of imidazo[1,2-b]thiazole derivatives is shown to activate the NAD+-dependent deacetylase SIRT1,
a potential new therapeutic target to treat various metabolic disorders. This series of compounds was derived
from a high throughput screening hit bearing an oxazolopyridine core. Water-solubilizing groups could be
installed conveniently at either the C-2 or C-3 position of the imidazo[1,2-b]thiazole ring. The SIRT1 enzyme
activity could be adjusted by modifying the amide portion of these imidazo[1,2-b]thiazole derivatives. The
most potent analogue within this series, namely, compound 29, has demonstrated oral antidiabetic activity
in the ob/ob mouse model, the diet-induced obesity (DIO) mouse model, and the Zucker fa/fa rat model.
Introduction
There are seven known sirtuin members (SIRT1-SIRT7) in
the histone deacetylase (HDACa) family commonly referred to
as class III HDACs, and SIRT1 is the most characterized
member of the family. Unlike classes I and II HDACs, SIRT1
and other class III HDACs utilize nicotinamide adenine di-
nucleotide (NAD+) to catalyze protein deacetylation to afford
nicotinamide and O-acetyl-ADP-ribose. Early studies have
established an important link between SIRT1 and a number of
metabolic functions that could possibly influence lifespan in
lower organisms and rodents.1 Two of the known consequences
of SIRT1 activation are up-regulation of PGC-1R and increase
in mitochondrial biogenesis,2 both of which are beneficial
biologic responses that are similarly induced by calorie
restriction.3,4 Since an increase in mitochondrial biogenesis could
lead to improved glucose metabolism in various skeletal, muscle,
and adipose tissues, activation of SIRT1 could potentially serve
Figure 1
as a novel approach to treat type II diabetes and other metabolic
significant new chemical entity that is structurally distinct from
disorders.5 Recent work has shown that transgenic mice
overexpressing SIRT1 are leaner than their control littermates
and display a significantly improved metabolic profile such as
lower levels of blood cholesterol, adipokines, insulin, and fasted
blood glucose.6 Resveratrol, a small molecule activator of
SIRT1, has been reported to extend lifespan in yeast,7 C.
elegans, Drosophila,8 and rodents.9 In rodents, resveratrol, when
dosed at 400 mg/kg over a period of 15 weeks, improves
metabolism, glucose tolerance, and their overall physical
performance in response to a rotarod or string test.2 Since
resveratrol has been shown to activate SIRT1 only at high doses,
because of its lower potency, there was an immediate need to
identify potent and selective compounds for proof-of-concept
studies. Recent high throughput screening efforts to find novel
SIRT1 activators from our laboratories have resulted in the
discovery of the oxazolopyridine derivative 1 (Figure 1), a
resveratrol.10
Discussion
In order to assess SIRT1 activation, a high throughput mass
spectrometry enzymatic assay was used. Potency was displayed
as the EC1.5, the concentration of compound required to increase
enzyme activity by 50%, and the percent maximum activation
that could be achieved at the highest doses of the compound
being tested. In this type of assay, resveratrol has an EC1.5 value
of 48 µM and a 250% maximum activation whereas the
oxazolopyridine derivative 1 has a corresponding EC1.5 value
of 11 µM and a 388% maximum activation. Preliminary work
has already shown that the potency of this lead could be
improved to a level that has not been observed before with
resveratrol.10 We have since modified the oxazolopyridine
moiety with a novel imidazo[1,2-b]thiazole ring and have noted
a similar level of SIRT1 activation (2, EC1.5 ) 7.5 µM, 380%
maximum activation). When an ortho substitution pattern was
adopted at the middle phenyl ring, an increase in potency was
observed (3, EC1.5 ) 2.5 µM, 272% maximum activation).
The imidazo[1,2-b]thiazole ring offers the opportunity to
install a variety of functional groups, such as amino derivatives,
conveniently at either the C-2 or C-3 position of the molecule.
As shown in Scheme 1, when ethyl 2-aminothiazole-4-carboxy-
* To whom correspondence should be addressed. Phone: (617)-252-6920,
extension 2129. Fax: (617)-252-6924. E-mail: cvu@sirtrispharma.com.
†
Present address: Department of Medicine, Division of Endocrinology
and Metabolism, University of CaliforniasSan Diego, 9500 Gilman Drive,
La Jolla, CA 92093.
a Abbreviations: HDAC, histone deacetylase; NAD+, nicotinamide
adenine dinucleotide; PGC-1R, peroxisome proliferator-activated receptor
coactivator-1R; DIO, diet-induced obesity.
10.1021/jm8012954 CCC: $40.75
2009 American Chemical Society
Published on Web 02/06/2009

1276 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Vu et al.
Scheme 1a
Table 1. Imidazo[1,2-b]thiazole Derivatives with Soluble Groups at the
C-2 and C-3 Positions
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
variation in individual values of <15%.
a Reagents and conditions: (a) methyl ethyl ketone, reflux (79%); (b)
LiOH, isobutyl chloroformate, DIEA, THF, NaBH4 (74%); (c) MsCl, Et3N;
NHR1R2, DIEA; (d) NaHS, MeOH, H2O, reflux; (e) RCOCl, pyridine,
microwave, 140 °C × 10 min; for NR1R2 ) Boc-piperazine, 25% TFA in
CH2Cl2.
substituted imidazo[1,2-b]thiazole derivatives of the general
structure 12.
Table 1 lists a small set of analogues that have been prepared
to compare the difference in SIRT1 activity when methylamino
derivatives were installed at either the C-2 or C-3 position of
the imidazothiazole ring. Compounds 13 and 15, with water
solubilizing groups at the C-2 position, showed SIRT1 activity
comparable to that of compound 3. Similar substitution at the
C3 position of the imidazo[1,2-b]thiazole ring also did not affect
the level of SIRT1 activation significantly (compounds 14 and
16). Introduction of these basic amino residues did, however,
increase the water solubility of these derivatives quite substan-
tially. Compound 3, for instance, had a water solubility
concentration of less than 0.1 mg/mL, whereas compound 16
had a water solubility of greater than 10 mg/mL (in pH 7.4
buffer, the kinetic solubility of 16 was still greater than 100
µM, as determined by HPLC methods). Because of its greater
water solubility, compound 16 has enabled the use of isothermal
titration calorimetry (ITC) to determine the binding energetics
of this particular class of compounds to purified SIRT1 enzyme.
In the presence of an acetylated peptide substrate, compound
16 showed protein binding in a 1:1 stoichiometry with a
dissociation constant (Kd) of 16.2 µM and an enthalpy (∆H) of
-6.1 kcal/mol.13 Interestingly, incorporation of the water soluble
amino group at the C-3 position of the imidazo[1,2-b]thiazole
ring improved the stability of the resulting molecule toward
microsomes significantly. Compound 3 has a very short half-
life in both mouse and human microsomes (mouse microsome,
t1/2 ) 5 min; human microsome, t1/2 ) 10 min),14 whereas
compound 16 showed good stability in both mouse and human
microsomes (mouse microsome, t1/2 > 300 min; human mi-
crosome, t1/2 ) 112 min). More importantly, in a mouse PK
experiment (po dosing of 100 mg/kg, iv dosing of 10 mg/kg,
saline vehicle), compound 16 showed reasonably good oral
bioavailability (F ) 40%, Cmax ) 14 µM, terminal t1/2 of 6 h,
and terminal AUC ) 45 000 ng ·h/mL).
Scheme 2a
a Reagents and conditions: (a) toluene, EtOH, H2O, [1,1′-bis(diphe-
nylphosphino)ferrocene]dichloropalladium(II) complex with CH2Cl2 (1:1),
Na2CO3, 90 °C; (b) ethyl bromopyruvate, methyl ethyl ketone, reflux; (c)
sodium hydrosulfide hydrate, MeOH, H2O, reflux; (d) 3,4,5-trimethoxy-
benzoyl chloride, pyridine, microwave, 160 °C × 10 min; (e) THF, H2O,
NaOH.
late 4 was treated with 2-bromo-1-(2-nitrophenyl)ethanone 5,
the resulting imidazo[1,2-b]thiazole ethyl ester 6 could be
obtained in good yield (79%).11 The ester group in 6 could be
reduced to the corresponding alcohol 7 by a three-step sequence
that involved hydrolysis of the ester group, followed by
formation of the mixed anhydride and treatment with NaBH4
(74% yield). In order to introduce amino derivatives at this
position, the alcohol was first converted to the corresponding
mesylate by treatment with methanesulfonyl chloride. Subse-
quent displacement with a variety of amines afforded intermedi-
ate 8. The nitro group in 8 was then reduced to the corresponding
amine and reacted with a variety of acid chlorides to form
analogues shown in the general structure 10. An analogous
synthetic sequence12 could essentially be repeated using ethyl
2-aminothiazole-5-carboxylate 11 to eventually obtain the C-2

Imidazo[1,2-b]thiazoles as SIRT1 ActiVators
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1277
Table 2. Imidazo[1,2-b]thiazole Derivatives with Varying Amide Groups
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
variation in individual values of <15%.
In terms of improving the potency of compound 16, the most
accessible position was the amide portion. We have since varied
this amide moiety and have observed a wide range of activity.
Table 2 lists a small set of piperazine derivatives where the
amide group has been changed. Unlike the trimethoxyphenyl
derivative 16, the unsubstituted phenyl derivative 19 was
significantly less potent. An aromatic group seemed be preferred
at this position, since the cyclopentyl compound 17 and the
benzyl compound 18 were not as active. However, removing
just one or two methoxy groups found in 16 (see compounds
20 and 22) did not appear to change the SIRT1 activity
significantly. Moving one of the methoxy groups to the
corresponding ortho position, as in 21, resulted in a substantial
loss of SIRT1 activity. A single methoxy group, when used in
combination with a fluoro group in either the 3,5- or 3,4-
disubstitution pattern (23 or 24, respectively), also retained most
of the SIRT1 activity. The 1,2-(methylenedioxy)benzene deriva-
tive 25 was also equipotent to the trimethoxyphenyl derivative.
When the methoxy group in 22 was replaced with the more
metabolically stable trifluoromethoxy group found in 26, or the
difluoromethylsulfide group found in 27, some improvement in
potency was observed. Interestingly, replacing the trifluo-
romethoxy phenyl group with a 2-substituted quinoline still
retained much of the SIRT1 activity (28, EC1.5 ) 0.99 µM,
440% maximum activation). The 3-substituted quinoline 30 was
slightly more active, but the 8-substituted quinoline 31 was
significantly less active. Lastly, changing the 2-quinoline group
to the corresponding 2-quinoxaline group resulted in a fairly

1278 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Vu et al.
100 mg/kg dose compared to the vehicle group. Similarly, the
plasma insulin levels were reduced significantly after 2 weeks
and remained down after this 10 week treatment (Figure 3c).17
This effect of normalizing insulin level in DIO animals is also
observed with rosiglitazone, a standard control used in the study.
No overt sign of hypoglycemia was observed with the lean
animals that were put on a regular chow diet and dosed with
29 daily for 10 weeks. Compound 29 showed no overt signs of
liver toxicity after 10 weeks of dosing. The clinical chemistry
of the DIO animals that have been dosed with 29, as measured
by the levels of AST, ALT, alkaline phosphatase, blood urea
nitrogen, creatine, was normal and showed no statistically
significant changes when compared with animals in the other
three groups. The hematology, as determined by white blood
count, red blood count, hemoglobin, and hematocrit, was also
normal for the DIO animals that were dosed with 29 daily for
10 weeks. Lastly, DIO animals that were dosed with 29 for 10
weeks showed no significant changes in body weight at the
conclusion of the study. The efficacious dose of 100 mg/kg for
compound 29 in the DIO animals essentially represents the
minimum effective dose, since animals dosed at 30 mg/kg po,
once daily, did not show a significant decrease in blood glucose
or insulin level (data not shown).
Figure 2
significant increase in potency in this enzyme assay (29, EC1.5
) 0.16 µM, 781% maximum activation). It is interesting to note
that the level of SIRT1 activation is highly dependent on the
presence of a hydrogen bond acceptor group adjacent to the
amide portion of the imidazo[1,2-b]thiazole ring. Compound
32, with a 2-susbtituted benzofuran, was still highly active.
Compound 33, with a 5-substituted benzofuran and a hydrogen
bond acceptor group more removed from the amide portion,
was significantly less active. Similarly, compound 34, with a
benzothiophene instead of a benzofuran, was significantly less
active. A significant level of SIRT1 activation was restored even
with a monocyclic pyrimidine ring as in compound 35. In order
to maintain optimal SIRT1 activation, it is important to note
the exact orientation of all the imidazo[1,2-b]thiazole analogues
shown in Tables 1 and 2. Figure 2 illustrates the significant
changes in SIRT1 activity when this important core ring is
reversed. Compound 36, with an acid group at the C-2 position
and an imidazo[1,2-b]thiazole ring in the configuration similar
to that of 16, had an EC1.5 value of 0.4 µM (305% maximum
activation). Compound 37, with the imidazo[1,2-b]thiazole ring
reversed, showed almost no SIRT1 activation (EC1.5 > 50
µM).15
In addition to being one of the more potent analogues shown
in Table 2, compound 29 also displayed significant oral
bioavailability in the mouse (F ) 50%, Cmax ) 1.0 µM, terminal
t1/2 ) 5 h, AUC ) 7900 ng ·h/mL) as well as in the rat (F )
25%, Cmax ) 0.32 µM, terminal t1/2 ) 8.4 h, AUC ) 3700 ng ·h/
mL).16 On the basis of its acceptable PK profile, compound 29
was selected for further assessment in three different efficacy
models for type 2 diabetes: the ob/ob mouse model, the diet-
induced obesity (DIO) mouse model, and the Zucker fa/fa rat
model. In the genetic model of type II diabetes, ob/ob deficient
mice were placed on a high fat diet in order to induce a diabetic
state (fasting glucose level of around 200 mg/dL). When these
animals were orally dosed with 100 mg/kg of compound 29
once daily, there was a significant reduction in fasting blood
glucose level after just 1 week of treatment. As shown in Figure
3a, a reduction of approximately 40% in blood glucose when
compared with the vehicle group was observed at the 100 mg/
kg dose. At a lower dose of 30 mg/kg po, once daily, there was
no significant reduction in fasting glucose (data not shown).
In the DIO model, mice that were on a high fat diet were
dosed po once daily with 100 mg/kg of compound 29.
Additional groups of animals used in this study included (1)
the DIO mice (high fat diet) that were dosed with the vehicle,
(2) the lean mice (regular chow) that were dosed with the
vehicle, (3) the lean mice (regular chow) that were dosed with
29 (100 mg/kg po daily), and (4) DIO mice (high fat diet) that
were dosed with rosiglitazone (5 mg/kg po, once daily), one of
the standards of care for type 2 diabetes. After 2 weeks of
dosing, there was clearly a significant decrease in the fed blood
glucose level, and that level was maintained over a 10-week
treatment period. As shown in Figure 3b, a reduction of
approximately 15-25% in blood glucose was observed at the
In the genetic Zucker fa/fa rat model, animals, in groups of
5, were dosed either with the vehicle or with 29 orally at 100
mg/kg once daily for 4 weeks. After 3 weeks, animals that were
dosed with 29 showed significantly lower fed blood glucose
level (Figure 3d, glucose AUC of vehicle-treated animals )
140 ( 5 mg·mL/dL; glucose AUC for 29-treated animals )
110 ( 3 mg·mL/dL).18 During this period, a pyruvate tolerance
test was also carried out. Figure 3e clearly showed a marked
reduction in the production of glucose, which indicated a
favorable decrease in hepatic gluconeogenesis. To further assess
the insulin sensitivity, a hyperinsulinemic-euglycemic clamp
was conducted after 4 weeks of treatment with 29. As shown
in Figure 3f, the glucose infusion rate (GIR) needed to maintain
the euglycemic state was significantly higher (approximately
35%) with animals treated with 29. Similarly, the total glucose
disposal rate (GDR) was also significantly higher, at ap-
proximately 20% (Figure 3g).19 Thus, after 4 weeks of treatment
with compound 29 (100 mg/kg po), diabetic Zucker fa/fa rats
clearly demonstrated a marked improvement in glucose homeo-
stasis and insulin sensitivity.
For the initial safety assessment, 29 was evaluated against
hERG and the IC50 was determined to be >10 µM (patch clamp
method). Compound 29 also showed no CYP inhibition against
five major isozymes (CYP1A, CYP2C19, CYP2C9, CYP2D6,
and CYP3A4) up to the highest tested concentration of 25 µM.
In order to evaluate the selectivity of 29 against other off-target
receptors, a panel consisting of 40 receptors was used.20 For
most of these receptors, 29 showed no significant inhibition at
10 µM. For five of these receptors, however, 29 did show
significant inhibition at 10 µM. The corresponding IC50 for each
of these receptors has since been determined: adrenergic R2A
(IC50 ) 2.83 µM); muscarinic M1 (IC50 ) 8.02 µM); muscarinic
M2 (IC50 ) 6.28 µM); norepinephrine transporter (IC50 ) 2.47
µM); sodium channel, site 2 (IC50 ) 3.37 µM). Compound 29
was further evaluated for mutagenicity using the tester strain
Salmonella typhimurium histidine auxotrophs TA98 and TA100.
Frameshift mutagens are known to revert TA98 strain from
auxotrophy to prototrophy, whereas TA100 strain is affected
by mutagens that cause both frameshift and base pair substitution
mutations.21 Under test conditions that either employ or do not

Imidazo[1,2-b]thiazoles as SIRT1 ActiVators
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1279
Figure 3. Efficacy studies involving compound 29: (a) diabetic mice ob/ob model (1 week); (b) mice DIO, fed plasma glucose (10 weeks); (c)
mice DIO, plasma insulin (10 weeks); (d) Zucker fa/fa rat, glucose (3 weeks); (e) Zucker fa/fa rat, pyruvate tolerance test (3 weeks); (f, g) Zucker
fa/fa rat, glucose infuse rate (GIR) and glucose disposal rate (GDR) during hyperinsulinemic-euglycemic clamp (4 weeks). The ob/ob and DIO
were carried out with 10 mice per group. The Zucker fa/fa study was done with 5 rats per group. In all efficacy studies, compound 29 was dosed
100 mg/kg po daily using a vehicle consisting of water, 2% HPMC, and 0.2% DOSS. Rosiglitazone, a positive control in the DIO study, was dosed
orally once a day at 5 mg/kg. Statistics were conducted as an ANOVA: one asterisk denotes p < 0.05; two asterisks denote p < 0.01.
microwave reactions were conducted on a Biotage Initiator
microwave synthesizer. Melting points were determined by dif-
ferential scanning calorimetry (TA Instruments DSCQ200). Unless
indicated otherwise, reagent-grade chemicals and solvents were
purchased from Aldrich, Alfa, Lancaster, or Maybridge.
employ S9 activation, no positive mutagenic response was
observed for 29 with both TA98 and TA100 strains.
Conclusion
In summary, a novel series of imidazo[1,2-b]thiazole deriva-
tives is shown to be effective SIRT1 activators. A representative
analogue within this series, compound 29, has been demon-
strated to have oral efficacy in three different rodent models of
type 2 diabetes. Even after 10 weeks of dosing in the DIO mice,
29 was able to maintain a significantly lower level of fed glucose
and insulin without affecting the body weight as well as the
overall clinical chemistry and hematology. Compound 29 has
been further demonstrated to have a reasonably clean safety
profile (negative for hERG, CYP, and Ames). Its selectivity
against undesired off-target receptors has also been determined
to be acceptable. Thus, compound 29 represents a compelling
new lead in the field of sirtuin research. Efforts aimed at
thoroughly understanding the SAR of this series are ongoing,
and the results will be disclosed in due course.
Preparation of N-(2-(Imidazo[2,1-b]thiazol-6-yl)phenyl)-3,4,5-
trimethoxybenzamide (3). Step 1. In a typical run, a solution of
2-aminothiazole (123 mg, 1.23 mmol) and 2-bromo-2′-nitroac-
etophenone (300 mg, 1.23 mmol) in methyl ethyl ketone (15 mL)
was stirred under reflux for 18 h. It was then cooled to room
temperature and filtered. The filtrate was concentrated under reduced
pressure. The resulting solids were taken up in 20 mL of EtOH,
and 5 drops of concentrated HBr were added. The reaction mixture
was stirred under reflux for 6 h. The reaction mixture was
concentrated and taken up in 20 mL of dilute aqueous NaHCO3.
The resulting solids were collected by filtration and dried to afford
300 mg of the nitro intermediate. This material was taken up in 15
mL of MeOH and 3 mL of water along with sodium hydrosulfide
hydrate (414 mg, 6.72 mmol). The reaction mixture was stirred
under reflux for 8 h. It was then cooled to room temperature and
concentrated under reduced pressure. The aqueous layer was
extracted with CH2Cl2. The combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure to afford 260
mg of 2-imidazo[2,1-b]thiazol-6-ylphenylamine (98%). ESI-MS
calcd for C11H9N3S: 215.05; found, 216 (M+ + H).
Experimental Section
General Chemistry Procedure. All proton or carbon nuclear
magnetic resonance spectra were determined in the indicated solvent
using either a Varian Unity 300 or Bruker AVANCE III 400
instrument with the appropriate internal standard. Analytical HPLC
was performed on an Agilent 1100 series HPLC equipped with a
3.5 um Eclipse XDB-C18 (4.6 mm × 100 mm) column with the
following conditions: CH3CN/H2O, modified with 0.1% formic acid
mobile phase. Gradient elution: 5% hold (2 min), 5-95% gradient
(11 min), 95-5% gradient (0.3 min), 5% hold (2.7 min), 15 min
total run time. Flow rate: 0.8 mL/min. Low resolution MS was
performed on an Agilent 1100 series LC/MSD/single quadrupole/
electrospray platform. High resolution mass spectra were obtained
with an Agilent 6210 time-of-flight LC/MS by flow injection
(solvents are water and CH3CN with 0.1% formic acid). All
preparative HPLC were carried out on a Shimadzu SIL-10AP that
has been equipped with a Waters X-Bridge C18 column. All
Step 2. 2-Imidazo[2,1-b]thiazol-6-ylphenylamine (64 mg, 0.30
mmol) was taken up in 1 mL of pyridine along with 70 mg of
3,4,5-trimethoxybenzoyl chloride (0.30 mmol). The reaction mixture
was stirred in a microwave reactor (160 °C × 10 min). The reaction
mixture was cooled to room temperature and concentrated under
reduced pressure. The resulting residue was purified by chroma-
tography (9:1 CH2Cl2/MeOH) to afford N-(2-(imidazo[2,1-b]thiazol-
6-yl)phenyl)-3,4,5-trimethoxybenzamide. 1H NMR (300 MHz,
DMSO-d6) δ 8.70 (d, 1 H, J ) 8 Hz), 8.38 (s, 1 H), 8.04 (d, 1 H
J ) 4 Hz), 7.81 (d, 1 H, J ) 8 Hz), 7.10-7.40 (m, 5 H), 3.92 (br
s, 9 H). ESI-MS calcd for C21H19N13O4S: 409; found, 410 (M+
H).
+

1280 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Vu et al.
Preparation of Ethyl 6-(2-Nitrophenyl)imidazo[2,1-b]thiazole-
3-carboxylate (6). In a typical run, ethyl 2- aminothiazole-4-
carboxylate (Combi-blocks, 2.1 g, 0.0123 mol) was taken up in
methyl ethyl ketone (25 mL) along with 2-bromo-2′-nitroacetophe-
none (3.0 g, 0.0123 mol). The reaction mixture was stirred under
reflux for 18 h. It was then cooled to room temperature and filtered
to remove some of the solids. The filtrate was concentrated under
reduced pressure to afford 3.10 g of ethyl 6-(2-nitrophenyl)imi-
dazo[2,1-b]thiazole-3-carboxylate (79%). 1H NMR (300 MHz,
DMSO-d6): δ 8.39 ppm (br s, 1 H), 8.31 (br s, 1 H), 7.92 (d, 1 H,
J ) 8 Hz), 7.82 (d, 1 H, J ) 7 Hz), 7.3-7.7 (m, 2 H), 4.4 (q, 2 H,
J ) 7 Hz), 1.37 (t, 3 H, J ) 7 Hz). ESI-MS calcd for C14H11N3O4S:
317; found, 318 (M+ + H).
Preparation of (6-(2-Nitrophenyl)imidazo[2,1-b]thiazol-3-yl)-
methanol (7). 6-(2-Nitrophenyl)imidazo[2,1-b]thiazole-3-carboxylic
acid ethyl ester (14.50 g, 0.0458 mol) was taken up in 100 mL of
THF and 100 mL of water containing 7.3 g of NaOH (4 equiv).
The reaction mixture was stirred at room temperature for 18 h. It
was then concentrated. The aqueous layer was washed once with
CH2Cl2 and then acidified with 6 N HCl. The solids were collected
by filtration and dried to afford 7.4 g of the acid intermediate. This
material (7.4 g, 0.0256 mol) was taken up in 200 mL of anhydrous
THF along with NMM (2.8 mL, 0.0256 mol) and cooled to 0 °C.
Isobutyl chloroformate (3.35 mL, 0.0256 mol) was added, and the
reaction mixture was stirred in the ice bath for 3 h. NaBH4 (0.97
g, 0.0256 mol) was added as a solution in 30 mL of water. The
reaction mixture was stirred at 0 °C for 45 min. It was then warmed
to room temperature and concentrated. The aqueous layer was
extracted with CH2Cl2. The combined organic layers were dried
(Na2SO4) and concentrated to afford the crude product. Purification
by chromatography (Isco, using a mixture of pentane/EtOAc)
afforded 5.20 g of (6-(2-nitrophenyl)imidazo[2,1-b]thiazol-3-yl)-
methanol (74%). 1H NMR (300 MHz, DMSO-d6): δ 8.14 ppm (br
s, 1 H), 7.2-7.9 (m, 4 H), 7.16 (br s, 1 H), 5.65 (t, 1 H, J ) 7 Hz),
4.6 (m, 2 H). ESI-MS, calcd for C26H29N5O4S: 275; found, 276
(M+ + H).
Preparation of N-(2-(3-((Dimethylamino)methyl)imidazo[2,1-
b]thiazol-6-yl)phenyl)-3,4,5-trimethoxybenzamide (14). Step
1. 6-(2-Nitrophenyl)imidazo[2,1-b]thiazol-3-yl)methanol (435 mg,
1.58 mmol) was dissolved in 25 mL of CH2Cl2 along with 1 equiv
of Et3N (0.330 mL). Methanesulfonyl chloride (1 equiv, 0.12 mL)
was added, and the reaction mixture was warmed to room
temperature and stirred for 15 min. It was then quenched with brine
and extracted with CH2Cl2. The combined organic layers were dried
(Na2SO4) and concentrated to afford the mesylate intermediate. This
material was taken up in 4 mL of THF along with 4 mL of a 2 N
dimethylamine solution in THF and stirred at room temperature
for 3 h. The reaction mixture was concentrated, and the resulting
residue was partitioned between CH2Cl2 and water. The organic
layer was dried (Na2SO4) and concentrated to afford essentially
quantitative yield of the product. This material was taken up in 6
mL of MeOH and 1 mL of water along with 200 mg of sodium
hydrosulfide hydrate. The resulting reaction mixture was stirred
under reflux for 6 h. It was then cooled to room temperature, diluted
with 100 mL of absolute EtOH and concentrated. The resulting
residue was taken up in 20 mL of 9:1 CH2Cl2/MeOH and filtered.
The filtrate was concentrated to afford 2-(3-((dimethylamino)m-
ethyl)imidazo[2,1-b]thiazol-6-yl)aniline.
Step 2. 2-(3-((Dimethylamino)methyl)imidazo[2,1-b]thiazol-6-
yl)aniline (0.3 mmol) was taken up in 1 mL of pyridine along with
1 equiv (70 mg) of 3,4,5-trimethoxybenzoyl chloride. The reaction
mixture was stirred in a microwave reactor (160 °C × 10 min). It
was then cooled to room temperature and concentrated. The
resulting crude product was purified by chromatography (Isco,
gradient elution, CH2Cl2 to 95% CH2Cl2, 4% MeOH, and 1% Et3N)
to afford N-(2-(3-((dimethylamino)methyl)imidazo[2,1-b]thiazol-
6-yl)phenyl)-3,4,5-trimethoxybenzamide as a light-yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 8.70 (d, 1 H, J ) 8 Hz), 8.20 (br
s, 1 H), 7.90 (d, 1 H J ) 8 Hz), 7.10-7.40 (m, 6 H), 3.90 (br s, 9
H), 3.80 (br s, 6 H), 3.20 (br s, 2 H). ESI-MS calcd for
C24H26N4O4S: 466; found, 467 (M+ + H).
Preparation of tert-Butyl 4-((6-(2-Aminophenyl)imidazo[2,1-
b]thiazol-3-yl)methyl)piperazine-1-carboxylate (Intermediate 9,
NR1R2 ) Boc-piperazine). 6-(2-Nitrophenyl)imidazo[2,1-b]thiazol-
3-yl)methanol (1.0 g, 3.64 mmol) was dissolved in 100 mL of
CH2Cl2 along with Et3N (0.51 mL, 3.64 mmol). Methanesulfonyl
chloride (0.28 mL, 3.64 mmol) was added, and the reaction mixture
was warmed to room temperature and stirred for 15 min. It was
then quenched with brine and extracted with CH2Cl2. The combined
organic layers were dried (Na2SO4) and concentrated to afford the
mesylate intermediate. This material was taken up 4 mL of CH3CN
along with Et3N (0.51 mL, 3.64 mmol) and Boc-piperazine (680
mg, 3.64 mmol) and stirred at room temperature for 24 h. The
reaction mixture was concentrated, and the resulting residue was
partitioned between CH2Cl2 and water. The organic layer was dried
(Na2SO4) and concentrated to afford essentially quantitative yield
of the product. This material was taken up in 6 mL of MeOH and
1 mL of water along with 200 mg of sodium hydrosulfide hydrate.
The resulting reaction mixture was stirred under reflux for 24 h. It
was then cooled to room temperature and concentrated under
reduced pressure. The resulting residue was diluted with 2 mL of
water and extracted with CH2Cl2. The combined organic layers were
dried (Na2SO4) and concentrated to afford 0.90 g of tert-butyl 4-((6-
(2-aminophenyl)imidazo[2,1-b]thiazol-3-yl)methyl)piperazine-1-car-
boxylate (60%). 1H NMR (300 MHz, DMSO-d6): δ 9.2 ppm (br s,
1 H), 8.7 (br s, 1 H), 8.15 (s, 1 H), 8.10 (s, 1 H), 6.8-7.8 (m, 4 H),
6.16 (br s, 2 H), 3.72 (br s, 2 H), 1.39 (br s, 9 H). ESI-MS calculated
for C21H27N5O2S: 413, found, 414 (M+ + H).
Preparation of 3,4,5-Trimethoxy-N-(2-(3-(piperazin-1-ylm-
ethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)benzamide (16). In a
typical run, tert-butyl 4-((6-(2-aminophenyl)imidazo[2,1-b]thiazol-
3-yl)methyl)piperazine-1-carboxylate (0.3 mmol) was taken up in
1 mL of pyridine along with 3,4,5-trimethoxybenzoyl chloride (70
mg, 0.3 mmol). The reaction mixture was heated in a Biotage
microwave reactor (160 °C × 10 min), cooled to room temperature,
and concentrated under reduced pressure. The resulting crude
product was purified by chromatography (Isco, gradient elution,
CH2Cl2 to 95% CH2Cl2, 4% MeOH, and 1% Et3N). The purified
product was then treated with 2 mL of 25% TFA in CH2Cl2 for
2 h. It was then concentrated and the resulting residue was triturated
with Et2O to afford 335 mg of compound 18 as the TFA salt (MS,
M+ + H ) 508). The hydrochloride salt of compound 18 could be
prepared by first dissolving the corresponding TFA salt in water,
neutralizing with NaHCO3 and extracting with CH2Cl2. The
combined organic layers were dried (Na2SO4) and concentrated to
afford the free base form of 18. This material was dissolved in
50% aqueous CH3CN along 3 mol equiv of HCl (as 1 N HCl
solution) and lyophilized to afford 18 as the HCl salt. Mp: 193.5
°C (HCl salt). 1H NMR (300 MHz, DMSO-d6) δ 9.70 (br s, 1 H),
9.0 (br s, 1 H), 8.88 (d, 1 H, J ) 8 Hz), 7.78 (d, 1 H, J ) 8 Hz),
7.71 (br s, 1 H), 7.10-7.33 (m, 4 H), 4.58 (br s, 2 H), 3.92 (br s,
9 H), 3.76 (br s, 2 H), 3.00-3.10 (m, 4H), 2.60-2.80 (m, 4 H).
13C NMR (100 MHz, DMSO-d6): δ 47.56, 50.01, 56.15, 60.16,
104.68, 111.52, 120.70, 120.93, 123.63, 126.89, 128.13, 130.13,
136.01, 140.54, 144.43, 147.75, 152.86, 164.09. High-resolution
MS calcd for C26H29N5O4S [M + H]+ 508.2018; found, 508.2039.
Preparation of N-(2-(3-(Piperazin-1-ylmethyl)imidazo[2,1-
b]thiazol-6-yl)phenyl)quinoxaline-2-carboxamide (29). Essen-
tially the same procedure as detailed in the preparation of 3,4,5-
trimethoxy-N-(2-(3-(piperazin-1-ylmethyl)imidazo[2,1-b]thiazol-6-
yl)phenyl)benzamide was employed except that 2-quinoxaloyl
1
chloride was used. Mp: dec (HCl salt), 221.4 °C (freebase). H
NMR (300 MHz, DMSO-d6) δ 9.60 (br s, 1 H), 8.88 (d, 1 H, J )
8 Hz), 8.60 (br s, 1 H), 8.50 (s, 1 H), 8.0-8.30 (m, 5 H), 7.78 (d,
1 H, J ) 8 Hz), 7.10-7.33 (m, 4 H), 3.90 (br s, 2 H), 3.00-3.10
(m, 4H), 2.60-2.80 (m, 4 H). 13C NMR (100 MHz, DMSO-d6): δ
47.49, 49.88, 111.45, 120.47, 121.84, 124.02, 127.04, 128.10,
129.20, 129.23, 131.39, 132.15, 135.39, 139.54, 143.03, 143.80,
144.36, 144.62, 147.76, 161.57. High resolution MS, calcd for
C25H23N7OS [M + H]+ 470.1763; found, 470.1753.

Imidazo[1,2-b]thiazoles as SIRT1 ActiVators
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1281
Preparation of Ethyl 6-(2-Nitrophenyl)imidazo[2,1-b]thiazole-
2-carboxylate. In a typical run, ethyl 2-aminothiazole-5-carboxylate
(Combi-blocks, 1.00 g, 5.81 mmol) was taken up in acetone (50
mL) along with 2-bromo-2′-nitroacetophenone (1.42 g, 5.81 mmol)
and stirred under reflux for 18 h. It was then filtered. The filtrate
was concentrated to afford the intermediate amide (ESI-MS, M+
+ H ) 336). This material was taken up in 20 mL of EtOH along
with 6 drops of concentrated HBr and stirred under reflux for 4 h.
The reaction mixture was cooled to room temperature and
concentrated. The resulting residue was diluted with dilute aqueous
NaHCO3. The solids were collected by filtration and dried to afford
1.5 g of ethyl 6-(2-nitrophenyl)imidazo[2,1-b]thiazole-2-carboxylate
95% CH2Cl2, 4% MeOH, and 1% Et3N). The purified product was
then treated with 2 mL of 25% TFA in CH2Cl2 for 2 h. It was then
concentrated and the resulting residue was triturated with Et2O to
afford 3,4,5-trimethoxy-N-(2-(2-(piperazin-1-ylmethyl)imidazo[2,1-
1
b]thiazol-6-yl)phenyl)benzamide as the TFA salt. H NMR (300
MHz, DMSO-d6) δ 8.70-8.80 (m, 1 H), 8.78 (br s, 1 H), 8.40 (s,
1 H), 8.05 (s, 1 H), 7.80 (d, 1 H, J ) 8 Hz), 7.10-7.33 (m, 4 H),
3.92 (br s, 6 H), 3.76 (br s, 2 H), 3.70 (s, 3 H), 3.00-3.10 (m,
4H), 2.60-2.80 (m, 4 H). ESI-MS calcd for C26H29N5O4S: 507;
found 508 (M+ + H).
Preparation of Methyl 2-(2-Aminophenyl)imidazo[2,1-b]thia-
zole-6-carboxylate (37). Step 1. Preparation of 5-(2-Nitrophe-
nyl)thiazol-2-amine (40). In a typical run, 2-amino-5-bromothiazole
monohydrobromide 38 (5.00 g, 0.0192 mol) was taken up in 40
mL of toluene, 40 mL of ethanol, and 20 mL of water. 2-Nitro-
phenylboronic acid 39 (3.2 g, 0.0192 mol) was added, along with
2.35 g of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
complexed with CH2Cl2 (1:1) and 6.10 g of anhydrous sodium
carbonate. The reaction mixture was stirred at 90 °C for 18 h. It
was then cooled to room temperature and concentrated. The
resulting residue was taken up in 500 mL of EtOAc and washed
with water (3 × 50 mL). The organic layer was filtered to remove
the black precipitate. The filtrate was extracted with dilute 1 N
HCl. The combined aqueous layers were concentrated to near
dryness. The resulting residue was purified by preparative HPLC
using a mixture of aqueous acetonitrile that has been buffered with
0.1% TFA to afford 108 mg of 5-(2-nitrophenyl)thiazol-2-amine
(MS, M+ + H ) 222). 1H NMR (300 MHz, DMSO-d6) δ 8.00 (d,
1 H, J ) 8 Hz), 7.35-7.80 (m, 3 H), 7.20 (s, 1 H), 4.90 (br s, 2
H). ESI-MS calcd for C9H7N3O2S: 221; found, 222 (M+ + H).
Step 2. Preparation of Ethyl 2-(2-Nitrophenyl)imidazo[2,1-
b]thiazole-6-carboxylate (41). 5-(2-Nitrophenyl)thiazol-2-amine
(100 mg, 0.452 mmol) was taken up in 10 mL of methyl ethyl
ketone along with 1.5 equiv of ethyl bromopyruvate. The reaction
mixture was stirred under reflux for 5 h. The reaction mixture was
cooled to room temperature and concentrated. The crude product
was purified by chromatography (Isco, gradient elution, CH2Cl2 to
9:1 CH2Cl2/MeOH) to afford 60 mg of ethyl 2-(2-nitrophenyl)imi-
(84%). ESI-MS calcd for C14H11N3O4S: 317; found, 318 (M+
H).
+
Preparation of (6-(2-Nitrophenyl)imidazo[2,1-b]thiazol-2-yl)-
methanol. Ethyl 6-(2-nitrophenyl)imidazo[2,1-b]thiazole-2-car-
boxylate (660 mg, 2.08 mmol) was dissolved in 12 mL of THF,
and NaOH (4 equiv) was added as a solution in 10 mL of water.
The reaction mixture was stirred at 50 °C for 12 h. It was then
cooled to room temperature and concentrated. The aqueous layer
was acidified to pH 5 with 6 N HCl. The solids were collected by
filtration and dried to afford essentially quantitative yield of the
acid. This material (2.08 mmol) was taken up in 20 mL of
anhydrous THF along with NMM (0.23 mL, 2.08 mmol) and cooled
in an ice bath. Isobutyl chloroformate (0.27 mL, 2.08 mmol) was
added and the reaction mixture was stirred at 0 °C for 30 min.
NaBH4 (80 mg, 2.08 mmol) was added as a solution in 5 mL of
water. The reaction mixture was stirred at 0 °C for 30 min and
then concentrated. The aqueous layer was extracted with CH2Cl2.
The combined organic layers were dried (Na2SO4) and concentrated.
Purification by chromatography (Isco, gradient elution using a
mixture of CH2Cl2 and MeOH) afforded 190 mg of (6-(2-
nitrophenyl)imidazo[2,1-b]thiazol-2-yl)methanol (33%). ESI-MS
calcd for C12H9N3O3S: 275; found, 276 (M+ + H).
Preparation of 2-(2-((Dimethylamino)methyl)imidazo[2,1-
b]thiazol-6-yl)aniline. Essentially the same procedure used during
the preparation of afford 2-(3-((dimethylamino)methyl)imidazo[2,1-
b]thiazol-6-yl)aniline was employed except that (6-(2-nitrophe-
nyl)imidazo[2,1-b]thiazol-2-yl)methanol was used as the starting
material.
1
dazo[2,1-b]thiazole-6-carboxylate. H NMR (300 MHz, DMSO-
d6) δ 8.42 (s, 1 H), 8.20 (s, 1 H), 8.00 (d, 1 H, J ) 8 Hz), 7.35-7.80
(m, 3 H), 4.2 (q, 2H, J ) 7 Hz), 1.2 (t, 3 H, J ) 7 Hz). ESI-MS
calcd for C14H11N3O4S: 317; found, 318 (M+ + H).
Preparation of N-(2-(2-((Dimethylamino)methyl)imidazo[2,1-
b]thiazol-6-yl)phenyl)-3,4,5-trimethoxybenzamide (13). 2-(2-
((Dimethylamino)methyl)imidazo[2,1-b]thiazol-6-yl)aniline (0.3
mmol) was taken up in 1 mL of pyridine along with 1 equiv (70
mg) of 3,4,5-timethoxybenzoyl chloride. The reaction mixture was
heated in a Biotage microwave reactor (160 °C × 10 min), cooled
to room temperature, and concentrated under reduced pressure. The
resulting crude product was purified by chromatography (Isco,
gradient elution, CH2Cl2 to 95% CH2Cl2, 4% MeOH, and 1% Et3N)
to afford N-(2-(2-((dimethylamino)methyl)imidazo[2,1-b]thiazol-
6-yl)phenyl)-3,4,5-trimethoxybenzamide as a light-yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 8.70 (d, 1 H, J ) 8 Hz), 8.31 (s, 1
H), 7.99 (s, 1 H), 7.80 (d, 1 H, J ) 8 Hz), 7.10-7.40 (m, 4 H),
5.80 (br s, 2 H), 4.00 (br s, 6 H), 3.92 (s, 3 H), 3.81 (s, 3 H), 3.75
Step 3. Preparation of Methyl 2-(2-Aminophenyl)imidazo[2,1-
b]thiazole-6-carboxylate. Ethyl 2-(2-nitrophenyl)imidazo[2,1-
b]thiazole-6-carboxylate (60 mg, 0.189 mmol) was taken up in 3
mL of MeOH along with sodium hydrosulfide hydrate (32 mg,
0.567 mmol) in 1 mL of water. The reaction mixture was stirred
under reflux for 1 h and monitored by LC/MS. Reduction of the
nitro group was complete at this point, and the ethyl ester group
had been exchanged to the corresponding methyl ester derivative.
The reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The aqueous layer was
extracted with CH2Cl2. The combined organic layers were dried
(Na2SO4) and concentrated to afford essentially quantitative yield
of methyl 2-(2-aminophenyl)imidazo[2,1-b]thiazole-6-carboxylate.
1H NMR (300 MHz, DMSO-d6) δ 8.42 (s, 1 H), 8.20 (s, 1 H),
7.35-7.80 (m, 3 H), 6.80 (d, 1 H, J ) 8 Hz), 3.90 (s, 3 H). ESI-
MS calcd for C13H11N3O2S: 273; found 274 (M+ + H).
Step 4. Preparation of 2-(2-(3,4,5-Trimethoxybenzamido)phe-
nyl)imidazo[2,1-b]thiazole-6-carboxylic Acid (37). Methyl 2-(2-
aminophenyl)imidazo[2,1-b]thiazole-6-carboxylate (27 mg, 0.095
mmol) was taken up in 1 mL of pyridine along with 22 mg of
3,4,5-trimethoxybenzoyl chloride. The reaction mixture was heated
in a microwave reactor (160 °C × 10 min), cooled to room
temperature, and concentrated under reduced pressure. The resulting
crude product was purified by preparative HPLC using a mixture
of aqueous acetonitrile that had been buffered with 0.1% TFA to
afford 9 mg of methyl 2-(2-(3,4,5-trimethoxybenzamido)phe-
nyl)imidazo[2,1-b]thiazole-6-carboxylate. This material was taken
up in 1 mL of THF, and sodium hydroxide (10 mg) was added as
a solution in 1 mL of water. The reaction mixture was stirred at
(s, 3 H). ESI-MS, calcd for C24H26N4O4S: 466; found, 467 (M+
H).
+
Preparation of tert-Butyl 4-((6-(2-aminophenyl)imidazo[2,1-
b]thiazol-2-yl)methyl)piperazine-1-carboxylate. Essentially the
same procedure used during the preparation of tert-butyl 4-((6-(2-
aminophenyl)imidazo[2,1-b]thiazol-3-yl)methyl)piperazine-1-car-
boxylate was employed except that (6-(2-nitrophenyl)imidazo[2,1-
b]thiazol-2-yl)methanol was used as the starting material.
Preparation of 3,4,5-Trimethoxy-N-(2-(2-(piperazin-1-ylm-
ethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)benzamide (15). 4-[6-(2-
Aminophenyl)imidazo[2,1-b]thiazol-2-ylmethyl]piperazine-1-car-
boxylic acid tert-butyl ester (0.2 mmol) was taken up in 1 mL of
pyridine along with of 3,4,5-trimethoxybenzoyl chloride (47 mg,
0.2 mmol). The reaction mixture was heated in a Biotage microwave
reactor (160 °C × 10 min), cooled to room temperature, and
concentrated under reduced pressure. The resulting crude product
was purified by chromatography (Isco, gradient elution, CH2Cl2 to

1282 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Vu et al.
room temperature for 4 h and then concentrated. The resulting crude
product was purified by preparative HPLC using a mixture of
aqueous acetonitrile that has been buffered with 0.1% TFA to afford
2-(2-(3,4,5-trimethoxybenzamido)phenyl)imidazo[2,1-b]thiazole-6-
ANOVA with comparison to control using a Dunnett’s Test. A p
value of <0.05 indicated a significant difference between groups.
ob/ob Model. ob/ob mice and heterozygous ob/+ mice were
received at 6 weeks of age (Jackson Laboratories, Bar Harbor, ME).
Mice were placed on a high fat diet (60% calories from fat, Research
Diets) for a minimum of 1 week prior to the start of a study and
remained on the high fat diet for the duration of the study. After 1
week of acclimation, all mice were weighed and blood glucose
measurements were taken. The average body weight of the ob/ob
mice used in the study were about 40-50 g. All mice were sorted
by body weight and glucose levels and then were allocated into
groups. Animals were dosed with either compound 29 (100 mg/
kg) or vehicle (2% HPMC + 0.2% DOSS) once daily by oral
gavage. Blood glucose and insulin were determined as described
above. Statistical analysis was completed using the JMP program
(version 6). Data were analyzed by a one-way ANOVA with
comparison to control using Dunnett’s test. A p value of <0.05
indicated a significant difference between groups.
Zucker fa/fa Model. Six-week old male fatty (fa/fa) Zucker (ZF)
rats (Harlan Sprague-Dawley, Inc.) were housed individually under
controlled light (12:12 light:dark) and temperature conditions. At
7 weeks of age animals were randomly assigned to receive either
compound 29 or vehicle (2% HPMC + 0.2% DOSS). The drug or
vehicle was administered by oral gavage on a daily basis (between
3-5 pm) for 4 weeks, and animals had ad libitum access to food
and water. All experimental procedures were approved by the
Animal Subjects Committee at UCSF according to NIH guidelines.
The night before beginning the drug treatment, animals were
overnight fasted (12 h). The following morning (day 1) blood
glucose concentration was measured (OneTouch Ultra, LifeScan,
Inc.) and a blood sample was taken in a heparinized capillary tube
from the tail vein. This sample was centrifuged at 13 000 rpm for
5 min, and the plasma was stored at -80 °C for analysis. This
procedure was subsequently repeated during the first 3 weeks of
drug treatment (i.e., days 8, 15, and 22). Also, in the afternoon of
day 22, a fed blood glucose measurement was taken from the tail
vein.
Zucker fa/fa Pyruvate Tolerance Test. On day 22, after the
overnight fast (and blood collection), rats were injected (1 g/kg
body weight) intraperotineal with sodium pyruvate (0.36 g/mL),
diluted with 0.9% NaCl. Blood glucose (tail vein) was measured
(OneTouch Ultra, LifeScan, Inc.) at 0, 30, 40, 50, 60, and 90 min
after injection.
Zucker fa/fa Oral Glucose Tolerance Test. On day 26 of
treatment, after a short fast (5 h), rats were orally gavaged (1 g/kg
body weight) with dextrose (Hospira, Inc.). Blood glucose (tail vein)
was measured (OneTouch Ultra, LifeScan, Inc.) at 0, 15, 30, 60,
and 90 min, and additional blood samples were also collected in
heperanized tubes at 0, 15, 30, and 60 min. These samples were
centrifuged at 13 000 rpm for 5 min, and the plasma was stored at
-80 °C for analysis of plasma insulin concentration.
Hyperinsulinemic-Euglycemic Clamp. Five days before con-
ducting clamp experiments, animals (10 weeks of age) were
chronically cannulated under single-dose anesthesia (42 mg/kg
ketamine HCl, 5 mg/kg xylazine, 0.75 mg/kg acepromazine maleate;
administered intramuscularly) in the jugular vein for infusion of
glucose, tracer, and insulin (dual cannula internal diameter [i.d.]
0.03 cm; Dow Corning Silastic, Midland, MI) and in the carotid
artery (Intramedic polyethylene tubing PE-50; Clay Adams, Becton
Dickinson, Sparks, MD) for blood sampling. All cannulae were
tunneled subcutaneously, were exteriorized at the back of the neck,
and were encased in an infusion harness (catalog no. CIH105;
Instech Laboratories, Inc.).
The evening before the hyperinsulinemic-euglycemic clamp,
animals were fasted overnight for 8 h. Ninety minutes before the
clamp, animals were weighed and infusion lines were connected.
Throughout the clamp animals were unrestrained in their standard
rat cage and had free access to water. The two infusion lines were
connected from the infusion pumps (KDS101; KD Scientific, Inc.)
to a low-torque dual channel quartz-lined swivel (catalog no. 375/
D/22QM; Instech Laboratory, Inc.), which was then connected to
1
carboxylic acid. H NMR (300 MHz, DMSO-d6) δ 10.2 (s, 1 H),
8.30 (s, 1 H), 8.20 (s, 1 H), 7.78 (d, 1 H, J ) 8 Hz), 7.30-7.35
(m, 3 H), 7.20 (br s, 2 H), 3.90 (br s, 6 H), 3.85 (s, 3 H). ESI-MS
calcd for C22H19N3O6S: 453; found, 454 (M+ + H).
Protein Expression and Purification. Human SIRT1 was
expressed in E. coli BL21Start (DE) (Invitrogen) as an N-terminal
fusion to a hexahistidine affinity tag. The protein was purified by
Ni2+ chelate chromatography followed by size exclusion and ion
exchange chromatography. The resulting protein was typically
>95% pure as assessed by SDS-PAGE. The human SIRT1-E5c
and SIRT1-A, B, D, E, F, G, H were expressed with a hexahistidine
affinity tag at the C-terminus. The truncated proteins were expressed
in E. coli BL21Start (DE3) and purified by Ni2+ chelate chroma-
tography. SIRT1-E5c was purified with an additional ion exchange
step.
SIRT1 Mass Spectrometry Assay. The mass spectrometry based
assay utilizes a 20 amino acid peptide substrate (Ac-Glu-Glu-
Lys(biotin)-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-Lys(Ac)-Nle-Ser-Thr-
Glu-Gly-Lys(MR121 or Tamra)-Glu-Glu-NH2) derived from the
sequence of p53. The peptide was N-terminally linked to biotin
and C-terminally modified with a fluorescent tag. The mass
spectrometry assay was conducted as follows: 0.5 µM peptide
substrate, 120 µM ꢀNAD+, 10 nM SIRT1, and reaction buffer (50
mM Tris-acetate, pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2,
5 mM DTT, 0.05% BSA). Reactions were incubated for 25 min at
25 °C. Test compounds were added to the reaction or vehicle
control, DMSO. After the incubation with SIRT1, 10% formic acid
with 50 mM nicotinamide (Sigma) was added to stop the reaction.
The mass spectrometry analysis was performed by Biotrove, Inc.
Determination of the mass of the substrate peptide allows for precise
determination of the degree of acetylation (i.e., substrate) compared
to deacetylated peptide (product).
Pharmacokinetic Studies. Compound 29 in vehicle (2% HPMC
+ 0.2% DOSS) was administered via oral gavage to C56BL/6 male
mice (18-22 g; 3 mice per dose group per time point) at the doses
indicated. For all in vivo studies, compound 29 was dosed as the
hydrochloride salt. Mice were sacrificed by CO2 asphyxiation, and
blood was collected at 5, 30, 120, and 360 min after dosing. Blood
was collected, and plasma was sent to Charles River Laboratories
(CRL) for drug level analysis. To determine oral bioavailability,
compound 29 in vehicle (10% ethanol/40% polyethylene glycol/
50% H2O) was administered into the tail vein of C57BL/6 male
mice (18-22 g, 3 mice per dose group per time point) at the doses
indicated. Blood was collected at the 5, 30, 120, and 360 min and
analyzed for drug levels as described above.
Compound 29 was administered via oral gavage to Sprague-
Dawley male rats (250 g, 3 rats per dose group) at 100 mg/kg in
vehicle (2% HPMC + 0.2% DOSS). Blood was collected at 0.033,
0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose and analyzed for
drug levels. To determine oral bioavailability, compound 29 was
administered into the tail vein at 10 mg/kg doses. Compound 29
was administered in 10% ethanol/ 40% polyethylene glycol/50%
H2O for iv studies. Blood was collected and analyzed as described
above.
Diet-Induced Obesity Model. Nine-week old C57BL/6 male
mice (Charles River Laboratories) were fed a high fat diet (60%
calories from fat, Research Diets) until their mean body weight
reached approximately 40 g. The mice were divided into test groups
(6-10 per group). The indicated compounds were dosed once daily
via oral gavage (100 mg/kg for compound 29, 5 mg/kg for
rosiglitazone). The vehicle used was 2% HPMC + 0.2% DOSS.
Individual mouse body weights were measured twice weekly. At
the 2, 4, 6, 8, and 10 weeks of dosing a fed glucose measure was
taken, and after 5 weeks of treatment an IPGTT was conducted on
all mice from each of the groups. After 10 weeks of treatment, an
ITT was conducted. Statistical analysis was completed using the
JMP program (version 6). Data were analyzed by a one-way

Imidazo[1,2-b]thiazoles as SIRT1 ActiVators
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1283
Le Couteur, D.; Shaw, R. J.; Navas, P.; Puigserver, P.; Ingram, D. K.;
de Cabo, R.; Sinclair, D. A. Resveratrol Improves Health and Survival
of Mice on a High-Calorie Diet. Nature 2006, 444, 337–342.
(10) Bemis, J. E.; Carney, D. P.; Disch, J. S.; Iffland, A.; Jin, L.; Jirousek,
M. R.; Lavu, S.; Lynch, A. V.; Milne, J. C.; Ng, P. Y.; Nunes, J. J.;
Smith, J. J.; Vu, C. B.; Xie, R. L.; Perni, R. B. Discovery of
Oxazolo[4,5-b]pyridines and Related Heterocyclic Analogs as Novel
SIRT1 Activators. Abstracts of Papers, 235th National Meeting of
the American Chemical Society, New Orleans, LA, April 6-10, 2008;
American Chemical Society: Washington, DC, 2008; MEDI-242.
(11) Sundberg, R. J.; Dahlhausen, D. J.; Manikumar, G.; Mavunkel, B.;
Biswa, A.; Srinivasan, V.; King, F.; Waid, P. Preparation of 2-Aryl
and 2-(Aryloxymethyl)imidazo[1,2-a]pyridines and Related Com-
pounds. J. Heterocycl. Chem. 1988, 25, 129–137.
the jugular vein cannulas. This setup allowed unrestrained move-
ments in all directions. At -60 min a priming dose of 7.5 µCi of
D-[3-3H]glucose (Perkin-Elmer, Inc.) was administered into the
carotid, and a constant-rate (0.167 µCi/min, 16.7 µL/min) infusion
of D-[3-3H]glucose was started at -70 min. Blood samples (25 µL
in duplicate) were taken at -10 and 0 min for measurement of
tracer specific activity and basal hepatic glucose production. At
time 0 min (i.e., after 60 min of tracer equilibration), glucose
(variable infusion, 50% dextrose; Hospira, Inc.) and tracer-plus-
insulin (25 (mU/kg)/min; Humulin R; Eli Lilly and Co.) infusions
were started simultaneously. Small blood samples (5 µL) were
drawn from the carotid artery at 10 min intervals and immediately
analyzed for glucose (OneTouch Ultra, LifeScan, Inc.). The glucose
infusion was adjusted to maintain blood glucose at 100 mg/dL.
Blood samples (25 µL in duplicate) were taken at 110 and 120
min for measurement of tracer specific activity. In addition, larger
blood samples (150 µL) were taken at 0 and 120 min for
determination of insulin and free fatty acid concentrations. All blood
samples were immediately centrifuged (13000g for 5 min), and
plasma was stored at -80 °C for subsequent analysis.
(12) For procedures to prepare analogues of the general structure 12, consult
the Supporting Information.
(13) For more comprehensive data on the binding energetics, functional
activity in cellular assays, and discussion on mechanism of SIRT1
activation of these imidazo[1,2-b]thiazole derivatives, see the
following: Milne, J. C.; Lambert, P. D.; Schenk, S.; Carney, D. P.;
Smith, J. J.; Gagne, D. J.; Jin, L.; Boss, O.; Perni, R. B.; Vu, C. B.;
Bemis, J. E.; Xie, R.; Disch, J. S.; Ng, P. Y.; Nunes, J. J.; Lynch,
A. V.; Yang, H.; Galonek, H.; Israelian, K.; Choy, W.; Iffland, A.;
Lavu, S.; Sinclair, D.; Olefsky, J. M.; Jirousek, M. R.; Elliott, P. J.;
Westphal, C. H. Novel Small Molecule Activators of SIRT1 as
Therapeutics for Treatment of Type 2 Diabetes. Nature 2007, 450,
712–716.
(14) The instability of C2 and C3 unsubstituted imidazo[1,2-b]thiazole
derivatives has previously been noted in the following: Trimble, L. A.;
Chauret, N.; Silva, J. M.; Nicoll-Griffith, D. A.; Li, C.-S.; Yergey,
J. A. Characterization of the in Vitro Oxidative Metabolites of the
COX-2 Selective Inhibitor L-766,112. Bioorg. Med. Chem. Lett. 1997,
7, 53–56.
(15) Compound 37 was prepared according to the sequence outlined in
Scheme 2. The aminothiazole derivative 40 was obtained by a
palladium coupling reaction between the bromide 38 and the boronic
acid 39. The imidazothiazole derivative 41 could then be obtained by
reacting 40 with ethyl bromopyruvate. Standard reduction of the nitro
group with sodium hydrosulfide hydrate followed by amide coupling
and ester hydrolysis afforded derivative 37.
(16) For both the mouse and rat PK, 29 was dosed at 100 mg/kg orally
and 10 mg/kg iv; the vehicle was water containing 2% HPMC and
0.2% DOSS.
Acknowledgment. We thank Lei Jin and Hongying Yang
for carrying out the isothermal titration calorimetry, and Can
Ozbal and William Lamarr from BioTrove, Inc. for running the
mass spectrometry samples during the determination of the EC1.5
values.
Supporting Information Available: Additional analytical data
for compounds 3, 13-16, 29, and 37. This material is available
free of charge via the Internet at http://pubs.acs.org.
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29 in a DIO study were also conducted; see ref 13 for additional details.
(18) For additional data concerning glucose metabolism and insulin
responses during an oral glucose tolerance test, see ref 13.
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(20) Panel of 40 receptors consisted of the following: ATPase, Na+/K+,
heart; cyclooxygenase COX-2; deacetylase, histone; monoamine
oxidase MAO-A; phosphodiesterase PDE4; protein tyrosine kinase,
FLT1 (VEGFR-1); adenosine A1; adenosine A2A; adrenergic R1,
nonselective; adrenergic R2A; adrenergic ꢀ1; adrenergic ꢀ2; angiotensin
AT1; calcium channel L-type, benzothiazepine; cannabinoid CB1;
cannabinoid CB2; cholecystokinin CCK1 (CCKA); cholecystokinin
CCK2 (CCKB); dopamine D1; dopamine D2S; GABAA, flunitrazepam,
central; GABAA, muscimol, central; GABAB1B; glucocorticoid;
glutamate, NMDA, agonism; histamine H1; muscarinic M1; muscarinic
M2; muscarinic M3; nicotinic acetylcholine R1, bungarotoxin; nicotinic
acetylcholine R7, bungarotoxin; opiate δ (OP1, DOP); opiate κ (OP2,
KOP); opiate µ (OP3, MOP); potassium channel [KATP]; serotonin
(5-hydroxytryptamine) 5-HT1A
; serotonin (5-hydroxytryptamine)
5-HT3; sodium channel, site 2; thyroid hormone; norepinephrine
transporter (NET).
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Carcinogens and Mutagens with the Salmonella/Mammalian Mi-
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