Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy) thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors

Article abstract of DOI:10.1021/jm060484v

A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC₅₀ < 20 nM and > 1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high ACC2 activity and lowered muscle malonyl-CoA dose-dependently in acute rodent studies, whereas (R)-enantiomer 9o was weak and had no effect on the malonyl-CoA level.

Full text of DOI:10.1021/jm060484v

3770
J. Med. Chem. 2006, 49, 3770-3773
Synthesis and Structure-Activity Relationships
of N-{3-[2-(4-Alkoxyphenoxy)thiazol-5-yl]-1-
methylprop-2-ynyl}carboxy Derivatives as
Selective Acetyl-CoA Carboxylase 2 Inhibitors
Yu Gui Gu,* Moshe Weitzberg, Richard F. Clark,
Xiangdong Xu, Qun Li, Tianyuan Zhang,
T. Matthew Hansen, Gang Liu, Zhili Xin, Xiaojun Wang,
Rongqi Wang, Teresa McNally, Heidi Camp,
Bruce A. Beutel, and Hing L. Sham
Metabolic Disease Research, Global Pharmaceutical Research and
DeVelopment, Abbott Laboratories, 200 Abbott Park Road,
Abbott Park, Illinois 60064
Figure 1. Structures of ACC inhibitors.
reported to increase fatty acid oxidation in mouse muscle cells
C2C12 and in rat muscle strips ex vivo. It also acutely lowered
mCoA levels in rat liver and muscles and increased whole-body
fatty acid oxidation. Thus far, the most potent ACC inhibitor
reported is the natural product soraphen A (Figure 1),12 which
is also a nonselective ACC inhibitor with single-digit-nanomolar
potency. To our knowledge, there have been no isoform-
selective ACC inhibitors reported thus far. Although a nonselec-
tive ACC inhibitor may be beneficial in maximizing the whole-
body fatty acid metabolism by simultaneously inhibiting the fatty
acid synthesis in lipogenic tissues and increasing fatty acid
oxidation in oxidative tissues,13 genetic studies have demon-
strated that ACC1 homozygous knockout mice are embryoni-
cally lethal and the safety of chronic inhibition of ACC1 is
unknown.14 On the other hand, ACC2 homozygous knockout
mice are healthy and fertile and exhibit a favorable metabolic
phenotype of increased fatty acid oxidation, increased thermo-
genesis, reduced hepatic triglyceride content, and decreased body
weight despite increased food intake.15 In addition, ACC2-/-
mice are resistant to high-fat diet-induced obesity and insulin
resistance and demonstrate increased fatty acid and glucose
oxidation in adipose tissue. ACC2 is therefore an attractive target
for the treatment of obesity-induced type 2 diabetes.16,17 Here,
we report the discovery of a novel class of ACC2 selective
inhibitors.
ReceiVed April 25, 2006
Abstract: A structurally novel acetyl-CoA carboxylase (ACC) inhibitor
is identified from high-throughput screening. A preliminary structure-
activity relationship study led to the discovery of potent dual ACC1/
ACC2 and ACC2 selective inhibitors against human recombinant ACC1
and ACC2. Selective ACC2 inhibitors exhibited IC50 < 20 nM and
>1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high
ACC2 activity and lowered muscle malonyl-CoA dose-dependently in
acute rodent studies, whereas (R)-enantiomer 9o was weak and had no
effect on the malonyl-CoA level.
The incidence of type 2 diabetes has dramatically increased
over the past decade. There is ample evidence to support a strong
correlation between insulin resistance and the development of
type 2 diabetes mellitus. At the cellular level, an increase in
ectopic fat storage in nonadipose tissues such as muscle, liver,
and pancreas has been implicated as a strong predictor of the
development of insulin resistance and type 2 diabetes.1,2
Although it is unclear how increased intracellular lipid content
exacerbates whole-body insulin sensitivity, it has been suggested
that increased levels of long-chain fatty acyl-CoAs, ceramide,
or diacylglycerol, whose contents are proportional to the
accumulation of intramyocellular triglyceride, antagonize the
metabolic actions of insulin, reduce muscle glucose uptake, and
inhibit hepatic glucose production.2,3
Compound 3 (A-80040, Figure 1), an initial high-throughput
screening (HTS) hit from our drug sample room, showed an
IC50 of 80 nM against hACC2 and an IC50 of 1.0 µM against
hACC1 enzyme. This compound originated from our internal
5-lipoxygenase inhibitor program several years ago. The hy-
droxyurea moiety in 3, while essential for 5-lipoxygenase
inhibitory activity,18 was thought to be a principal cause of the
poor pharmacokinetic and toxicity properties associated with
the molecule. An initial structure-activity relationship (SAR)
study revealed that the hydroxyurea group is not required for
ACC inhibitory activity. Urea (9a) and acetamide (9b) (Table
1) replacements for the hydroxyurea resulted in improved ACC2
potency profiles relative to 3. In addition, while the isoform
selectivity of 9b is comparable to that of HTS hit 3, 9a showed
>1000-fold ACC2 selectivity. These encouraging initial findings
led us to focus on this series for lead optimization aimed at
identifying potent and selective ACC2 inhibitors.
Acetyl-CoA carboxylase4 (ACC) catalyzes the biotin-depend-
ent carboxylation of acetyl-CoA to form malonyl-CoA5,6
(mCoA), a key intermediate metabolite that regulates the rate
of fatty acid metabolism. mCoA is a known substrate for fatty
acid synthase for de novo lipogenesis and also an allosteric
inhibitor of carnitine palmitoyltransferase 1, a mitochondrial
outer membrane protein that shuttles long-chain fatty acyl-CoAs
into the mitochondria for oxidation.7 Therefore, ACC inhibition
is expected to lower mCoA levels, resulting in reduced fatty
acid synthesis, increased fatty acid oxidation, and consequently
improved insulin sensitivity. In rodents and humans, there are
two isoforms of ACC encoded by distinct genes. ACC1, a 265
kDa cytosolic protein, is highly expressed in lipogenic tissues
(liver and adipose), whereas the 280 kDa ACC2 isoform is
primarily expressed in oxidative tissues (muscle, heart, and
liver).8,9 Recently, there has been increasing interest in the
discovery of ACC inhibitors for the treatment of metabolic
syndrome, obesity, and type 2 diabetes.10 Harwood and co-
workers reported a class of potent and nonselective inhibitors
exemplified by 1 (CP-640186, Figure 1).11 Compound 1 was
A general synthesis for N-[3-(2-phenoxythiazol-5-yl)-1-
methylprop-2-ynyl]carboxy derivatives 9 is shown in Scheme
1. The various para-substituted phenols 4 (X ) CH, Y ) O)
employed in Scheme 1 are commercially available or were
synthesized via simple chemical transformations. Selective
displacement of 2,5-dibromothiazole 5 with 4 in the presence
* To whom correspondence should be addressed. Phone: (847) 937-
4792. Fax: (847) 938-3403. E-mail: yu-gui.y.gu@abbott.com.
10.1021/jm060484v CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/07/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3771
Scheme 1a
Table 1. ACC Inhibitory Activitya
a Reagents and conditions: (a) K2CO3 (1.2 equiv), DMF, 10 min at 180
°C in microwave reactor or 4 h heating at 135 °C, 50-75%; (b) Pd(Ph3P)2Cl2
(5% equiv), CuI (2% equiv), Et3N (5 equiv), THF, reflux, 3 h, 60-80%;
(c) NH2NH2 (10 equiv), CH2Cl2/EtOH (10:1), reflux, 3 h; (d) Ac2O (3 equiv)
(or Cl3CCONdCdO for 9a, MeNdCdO for 9m, MeOCOCl for 9n-p),
Et3N (10 equiv), room temp, 3 h, 35-70% (two steps).
following steps b-d in Scheme 1. The enantiomerically pure
9o and 9p were synthesized by starting with chiral 3-butyn-2-
ol. Reaction of (R)- or (S)-3-butyn-2-ol with phthalimide under
Mitsunobu conditions produced the corresponding (S)- or (R)-
enantiomers 7, respectively. The reaction proceeded with
complete inversion of the chiral center, and the final products
were obtained with >98.5% enantiomeric excess and high yield.
The two enantiomers have distinct retention times under chiral
HPLC conditions, allowing the determination of enantiomeric
excess.
Recombinant human ACC1 and ACC2, expressed in HEK293
and baculovirus/Sf9 systems, respectively, were utilized for the
measurement of ACC activity. To increase the expression and
solubility of ACC2 protein, the N-terminal transmembrane
domain (1-275 aa’s of ACC2) was replaced with the corre-
sponding ACC1 sequence (1-133 aa’s). The enzymatic assay
measures ACC-mediated incorporation of [14C]CO2 into malo-
nyl-CoA. Compound 1 was reported as a dual inhibitor against
rat ACC1 and ACC2 with virtually equal potency (ACC1 IC50
) 53 nM and ACC2 IC50 ) 61 nM).22 Under our assay
conditions, it exhibited similar potency against recombinant
human ACC2 with IC50 ) 38 nM. The activity against human
ACC1 (IC50 ) 410 nM), however, was significantly weaker
(Table 1).
a The IC50 data are an average of at least three measurements.
of potassium carbonate at an elevated temperature produced
2-phenoxy-5-bromothiazoles 6 (X ) CH, Y ) O). Sonogashira
coupling19 of 6 with phthalimide-protected propargylamine 7,
which was prepared from the corresponding alcohol via a
Mitsunobu20 reaction with phthalimide, gave rise to 8 (X ) CH,
Y ) O). Removal of the phthalimide protecting group followed
by derivatization of the resulting primary amine provided 9 (X
) CH, Y ) O).
Similarly, 9g and 9h were synthesized starting with 6-iso-
propoxypyridin-3-ol and 4-isopropoxybenzenethiol, respectively,
following the reaction sequence in Scheme 1. Compound 10
was prepared by replacing 5 with 2,4-dibromothiazole according
to Scheme 1. The Ullmann coupling reaction of 4-isopro-
poxyphenol with 1,4-dibromobenzene using Evans’ conditions21
provided biphenyl ether 11, which was then converted to 12 by
SAR studies were carried out by systematically varying the
different parts of the molecule, and the results are summarized
in Table 1. In general, an ether or thioether linker on the left
side of the molecule is optimal for high ACC2 potency (9a-d
vs 9e-f). A large lipophilic group on the left side of the
molecule appears to increase ACC1 potency (9b,d vs 9c,e). It
is interesting that 9f is the only ACC1-selective compound
identified, suggesting that the H-bonding acceptor ability of ether
or thioether linkers may play an important role in ACC2
inhibitory activity for this class of compounds. Replacement of
the phenyl group with a pyridine had little effect on ACC2
activity but reduced ACC2 selectivity (9c vs 9g). The linkage
point of the thiazole ring seems to be critical for ACC2 potency.
While 9c is a potent and selective ACC2 inhibitor, regioisomer

3772 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Table 2. Pharmacokinetic Profile of 9na
Letters
species
dose (mg kg-1
5.0
)
F (%)
Vss (L kg-1
)
Clp (L h-1 kg-1
0.20
)
oral t1/2 (h)
>12
oral AUC (µg h mL-1
)
Cmax (µg mL-1
)
Tmax (h)
rat (n ) 3)
80.4
5.0
18.9
0.90
2.0
a The vehicle used for pharmacokinetic study: 10% DMSO in PEG-400.
10 is inactive against both ACC1 and ACC2. This dramatic
activity difference is difficult to understand, given the fact that
replacement of thiazole with a para-substituted phenyl resulted
in a potent dual inhibitor (12). A methyl group in the propargyl
position is optimal for ACC2 activity. Removing this methyl
group or replacing it with a larger substituent resulted in
significant loss of ACC2 potency (9c vs 9i-j). The terminal
carbonyl group is also important for ACC activity. Propargyl-
amine analogue 9l exhibited much weaker ACC potency than
the corresponding acyl derivatives 9a and 9b. Methylurea 9m
and methyl carbamate 9n analogues are potent and selective
ACC2 inhibitors. Finally, while (S)-enantiomer 9p is a potent
and selective ACC2 inhibitor, the corresponding (R)-enantiomer
(9o) is 40-fold weaker in ACC2 activity. Differences in ACC
activity were also observed between the dual ACC inhibitor 1
and its enantiomer.11
It is interesting that ACC2 selectivity is affected by changes
in various positions of the molecule. For example, while 9b is
only modestly selective, ACC2 selectivity can be dramatically
enhanced by replacing either the acetamide with urea (9a) or
the phenyl with an isopropyl group (9c). Conversely, replace-
ment of the thiazole moiety of 9c with a phenyl group resulted
in the loss of ACC2 selectivity (12). A similar loss of ACC2
selectivity was observed when the phenyl of 9c was replaced
with a pyridyl group (9g).
Figure 2. Effects on mCoA levels in muscle and liver of Sprague-
Dawley rats (n ) 6) after treatment with active enantiomer 9p and
inactive enantiomer 9o. A mixture of 70% PEG-400/QS water was used
as vehicle. Animals were free-fed overnight, and food was removed 1
h before dosing orally with 9o and 9p at 10 and 50 mg/kg. Two hours
later, they were given a glucerna/cornstarch meal challenge. After an
additional hour, the animals were sacrificed and tissues were harvested
for mCoA measurement. The plasma drug levels were comparable for
9o (1.33 and 5.33 µg/mL at 10 and 50 mg/kg, respectively) and 9p
(1.54 and 4.90 µg/mL at 10 and 50 mg/kg, respectively) treated animals.
2 diabetic/obese patients. Because ACC1-/- mice are embryoni-
cally lethal, it is foreseeable that selective ACC2 inhibitors may
provide superior safety profiles relative to nonselective ACC
inhibitors.
In general, this class of compounds exhibits good oral
pharmacokinetic properties in rodents, characterized by high
volume of distribution, low clearance, long half-life, and high
bioavailability. Table 2 shows the pharmacokinetic profile of
9n.
Acknowledgment. We thank Dr. Xueheng Cheng, Hua
Tang, Lan Gao, Barbara Cool, Ning Cao, Lemma Kifle, Dr.
David Beno, Sherry Carroll, Bob Dickinson, Dr. Bradley Zinker,
and Dr. Xiaolin Zhang for technical support.
Since ACC catalyzes the synthesis of mCoA, mCoA levels
are expected to be lower when animals are treated with a small-
molecule ACC inhibitor. In addition, since ACC2 is the
predominant isoform in muscle, an ACC2-selective inhibitor
should have more profound mCoA-lowering effects in muscle
than liver tissues as reported in the ACC2 knockout mice.15
The enantiomeric pair of inhibitors 9o and 9p was selected for
acute in vivo mCoA study in Sprague-Dawley rats. As shown
in Figure 2, active enantiomer 9p dose-dependently lowered
mCoA in muscle (36% and 54% reduction at 10 and 50 mg/kg,
respectively). A less robust but statistically significant reduction
in mCoA levels in liver (26%) was also observed at 50 mg/kg
dose, whereas there was no statistically significant effect on
liver mCoA levels at 10 mg/kg. Not surprisingly, inactive
enantiomer 9o had no effects on mCoA levels in muscle or in
liver. A similar mCoA lowering effect was observed in muscle
tissues of diet-induced obese mice when treated with active
enantiomer 9p (data not shown).
In conclusion, a class of structurally novel ACC inhibitors
was discovered from HTS. A preliminary SAR study led to the
identification of several potent and selective ACC2 inhibitors.
A representative ACC2-selective inhibitor from this class
demonstrated dose-dependent mCoA lowering in muscle tissues
of rodent models. The correlation of in vitro potency with acute
mCoA lowering between active and inactive enantiomers (9p
and 9o) indicates mechanism-based effects. Since mCoA plays
a critical role in modulating lipid metabolism, ACC inhibition
is expected to increase fatty acid oxidation and overall energy
expenditure and ultimately increase insulin sensitivity in type
Supporting Information Available: Experimental procedures
for the synthesis of the compounds in Table 1, characterization data
for all final compounds and key intermediates, detailed protocol
of human ACC1 and ACC2 assays, and LC/MS results for malonyl-
CoA. This material is available free of charge via the Internet at
http://pubs.acs.org.
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