Discovery of small molecule isozyme non-specific inhibitors of mammalian acetyl-CoA carboxylase 1 and 2

Article abstract of DOI:10.1016/j.bmcl.2009.04.091

Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

Full text of DOI:10.1016/j.bmcl.2009.04.091

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2383–2388
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of small molecule isozyme non-specific inhibitors of mammalian
acetyl-CoA carboxylase 1 and 2
a
a
a
a
Jeffrey W. Corbett a, , Kevin D. Freeman-Cook , Richard Elliott , Felix Vajdos , Francis Rajamohan ,
Darcy Kohls a, Eric Marr a, Hailong Zhang b, Liang Tong b, Meihua Tu a, Sharad Murdande a, Shawn D. Doran a,
Janet A. Houser a, Wei Song a, Christopher J. Jones a, Steven B. Coffey a, Leanne Buzon a, Martha L. Minich a,
Kenneth J. Dirico a, Susan Tapley a, R. Kirk McPherson a, Eliot Sugarman a, H. James Harwood Jr. a, William
Esler a
*
a Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
b Columbia University, New York, NY 10027, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Screening Pfizer’s compound library resulted in the identification of weak acetyl-CoA carboxylase inhib-
itors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-
based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar,
spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that
exhibited good rat pharmacokinetics were obtained from this chemotype.
Received 21 January 2009
Revised 19 April 2009
Accepted 21 April 2009
Available online 24 April 2009
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Acetyl-CoA carboxylase
ACC
Enzyme inhibitor
Spirochromanone
ACC1
ACC2
Structure based drug discovery
Diabetes
Metabolic syndrome
Type-2 diabetes mellitus (T2DM) has reached epidemic propor-
tions and threatens to become a global health scourge, currently
accounting for an estimated 5% of global deaths.1 Furthermore,
the World Health Organization (WHO) estimates that diabetes-re-
lated deaths ‘are likely to increase by more than 50% in the next 10
years without urgent action’.1 A co-morbidity associated with dia-
betes is obesity. Obesity related co-morbidities includes hyperten-
sion, dyslipidemia, coronary heart disease, stroke, some forms of
cancer, musculoskeletal disorders and other cardiovascular dis-
eases that further accelerate the morbidity and mortality associ-
ated with obesity and diabetes. Global 2005 estimates from WHO
indicate that 1.6 billion adults over the age of 15 were overweight,
with at least 400 million adults being obese. The fact that at least
20 million children under 5 years old are categorized as overweight
foretells future health problems since ‘childhood obesity is associ-
ated with a higher chance of premature death and disability in
adulthood’.2 Medicaments are now being prescribed to treat child-
hood obesity, as evidenced by the use of cholesterol reducing med-
ications in this population group.3 Even though T2DM and obesity
can be deterred through diet modification and increased physical
exercise, pharmacologic interventions continue to be sought to
treat these diseases.
A drug agent that would be expected to impact T2DM and obes-
ity would have the potential to positively impact health outcomes
for diabetics and the obese. The inhibition of acetyl-CoA carboxyl-
ase (ACC) is under investigation since modulation of its activity
poses the potential to modulate diabetes and obesity.4 ACC is one
of the enzymes responsible for modulating the rate of long chain
fatty acid biosynthesis and the rate of mitochondrial fatty acid oxi-
dation. ACC is a biotin-dependent heterodimeric protein, com-
posed of carboxyltransferase (CT), biotin carboxy carrier protein
(BCCP), and biotin carboxylase (BC) domains, whose purpose is to
synthesize malonyl-CoA (m-CoA) from acetyl-CoA in an ATP-
dependent reaction via the fixation of bicarbonate.5 The synthesis
of m-CoA is a two step process wherein the first half-reaction oc-
curs in the BC-domain and involves the ATP-dependent reaction
between bicarbonate ion and enzyme-bound biotin to afford a
* Corresponding author. Tel.: +1 860 691 1687.
E-mail address: jmc1983mac@gmail.com (J.W. Corbett).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.091

2384
J. W. Corbett et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2383–2388
biotin-carboxylate complex. The newly formed biotin-carboxylate
complex enters the CT-domain via a narrow channel between the
CT- and BC-domains, where carbon dioxide is transferred from bio-
tin carboxylate to acetyl-CoA to form m-CoA. Malonyl-CoA is then
utilized by other enzymes as the substrate for de novo fatty acid
synthesis and fatty acid chain elongation as well as being used as
an allosteric inhibitor of carnitine palmitoyltransferase (CPT1),
thereby playing a role in modulating fatty acid oxidation.
Two isoforms of mammalian ACC are known, ACC1 and ACC2
(also known as ACCa and ACCb, respectively). ACC1 is a cytosolic
enzyme that produces m-CoA that is believed to utilized in de novo
fatty acid synthesis and fatty acid chain elongation while ACC2 is
associated with the mitochondrial outer membrane where it may
play a predominate role in producing m-CoA that functions to reg-
ulate fatty acid oxidation via allosteric inhibition of CPT1. ACC1 is
found primarily in lipogenic tissues such as the liver and adipose
tissue whereas ACC2 is the predominate form found in oxidative
tissues (skeletal muscle and heart) where it plays a role in modu-
lating energy expenditure. Therefore, an ACC1/2 isozyme non-
selective inhibitor would be predicted to reduce fatty acid synthe-
sis in liver and adipose tissue while at the same time increasing
fatty acid oxidation in the liver and muscle tissues resulting in in-
creased energy expenditure, decreased whole body adiposity, con-
comitant decreased weight gain and/or weight loss, and improved
insulin sensitivity.
Figure 2. CT-domain co-crystal structure of 2 (blue) and CP-640186 (yellow).
on improving inhibitory potency without reverting to these func-
tional groups. Toward that end, a carboxylic acid scan was con-
ducted on the methylquinoline template and multiple non-
arylquinoline derivatives with rACC1 IC50 < 5
(Table 1). Compound 4 was one of the most ligand efficient (LE)
inhibitors identified, with a LE of 0.26 and rACC1 IC50 = 3.38
M.9
lM were identified
l
Screening of the Pfizer compound file and triage of the resulting
hits resulted in the identification of the micromolar screening hit 1
(rACC1 IC50 = 8910 nM, Fig. 1). Modeling of 1 suggested that it was
binding to the same CT-domain binding channel as the historic ser-
ies of ACC inhibitors, characterized by CP-640186.6,7 The hypothe-
sis was made that if compound 1 had a similar binding pose as
CP-640186, then the corresponding anthracene and 2-phenylquin-
oline amide derivatives 2 and 3 would be more potent ACC1 inhib-
itors since these groups led to improved potency in the CP-640186
series of ACC inhibitors. This was indeed the case as evidenced by
rACC1 IC50 = 325 and 842 nM for 2 and 3, respectively. CT-domain
co-crystal structures of these compounds confirmed the proposed
binding mode.8 The crystal structure of compound 2 (in blue) is
shown in Figure 2 along with an overlay of CP-640186 (in yellow,
extracted from Tong’s crystal structure),7 showing that both com-
pounds are capable of binding to the same channel within the
CT-domain. The crystal structures indicate that the anthracene
rings in CP-640186 and 2 occupy a similar region of space, the
morpholinyl-amide of CP-640186 is in approximately the same re-
gion of space as the methylquinoline and the H-bond between the
amide carbonyl and Glu-B2026 is conserved between both
compounds.
Other compounds found during the scan were m-substituted aryl
derivatives, such as pyrazoles 5 and 6.
Compounds 4 and 6 were submitted for rat PK and the data are
summarized in Table 2. The team was encouraged by modest oral
bioavailability (fraction absorbed >100%) and half-life achieved
with the methylquinoline-indazole 4. However, additional SAR on
the quinoline template failed to afford needed increases in potency
and it became apparent that the current template was unlikely to
afford sub-micromolar potency without resorting to arylquinoline
carboxylic acids.
The inability to advance the quinoline-series SAR without
resorting to undesirable carboxylic acids resulted in the need to
identify an alternate chemotype. The yeast CT-domain co-crystal
structures of compounds 2 and 3 were subsequently used as the
basis for structure-based drug design. Crystal structure of deriva-
tives 2 and 3 showed a single H-bond interaction between the pro-
tein and ligand, namely a hydrogen-bond between the amide
Table 1
Selected data from carboxylic acid scan on methylquinoline template
O
Owing to the sub-optimal physicochemical properties of the
arylquinoline and anthracene amides, the chemistry team focused
N
N
Ar
Compd
Ar
rACC1 IC50, nM10
3380
rACC1 LEa
0.26
N
O
HN
O
O
N
4
N
N
N
N
N
O
N
NH
1
CP-640186
ACC1 IC50 = 92 nM
ACC1 IC50 = 8910 nM
5
6
1270
3010
0.26
0.24
O
O
N
N
N
N
HN
N
N
3
2
ACC1 IC50 = 325 nM
Human microsome ER < 0.26
ACC1 IC50 = 842 nM
Human microsome ER = 0.68
a
LE is ligand efficiency (LE = À1.4 log IC50/number of heavy atoms).
Figure 1. Structure of HTS hit 1, CP-640186 and select analogs.

J. W. Corbett et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2383–2388
2385
Table 2
Table 3
Rat PK for selected methylquinolines
Initial SAR of spirochromanone system
O
Compd 4
Compd 6
5
N
6
7
IV dose (1 mg/kg)
CL (mL/min/kg)
Vdss (L/kg)
HN
R
44.3
2.4
34.1
2.8
O
N
8
O
PO dose (5 mg/kg)
Cmax (ng/mL)
%F
617
45%
1.7
91.5
7–23%
1.8
Compd
R
rACC1 IC50, nM
hACC2 IC50, nM14
rACC1 LE
8
7
6,7-CH3
6-CH3
163
634
400
0.32
0.30
0.30
0.29
0.29
0.28
0.27
0.26
0.26
0.23
t
½
(h)
641
9
7-CH3
850
NDa
10
11
12
13
14
15
16
6-MeO
6-Cl
562
NDa
1390
1320
1620
1830
2530
2210
2130
>1000
>1000
NDa
carbonyl and Glu-B2026. The X-ray structure of CP-640186 indi-
cated that Gly-B1958 could serve as an additional hydrogen-bond
donor (see Fig. 2).7 Therefore, an effort was undertaken to utilize
the methylquinoline template as a basis for creating de novo de-
signed templates that would be expected to make additional
hydrogen-bond interactions, while also reducing the degrees of
freedom present in the HTS series and thereby reduce entropic
penalties for binding to the enzyme.
6,8-CH3
5-MeO
5-MeO-6-Cl
7-MeO
7-Phenyl
NDa
NDa
a
ND means no data.
One of the first templates modeled was the spirochromanone
system. This ring system was utilized because it was more rigid
and modeling suggested that it had the possibility of making addi-
tional hydrogen-bond interaction, namely one between the ketone
and the protein.11 A 6-methylspirochromanone was first coupled
to the indazole carboxylic acid present in methylquinoline deriva-
tive 4 to provide 7, which had an rACC1 IC50 = 633 nM (Fig. 3).
Gratifyingly, a yeast CT-domain co-crystal structure of 7 was ob-
tained where it was found that three H-bonds were formed,
namely the conserved amide-Glu-B2026 interaction, one between
the ketone carbonyl-Gly-B1958, as in CP-640186, and a new inter-
action between indazole NH and the carboxylate of Glu-B2026
(Fig. 3).12 The benefit, if any, arising from the indazole NH-Glu-
2026 interaction was deemed to be minimal owing to the breakage
of an internal hydrogen-bond between Glu-B2026 and Arg-B1954.
The ligand efficiency improvement observed with 7 compared to 4
(LE = 0.30 vs 0.26, respectively), in conjunction with its sub-micro-
molar potency resulted in the team focusing our synthetic efforts
on this template.
Having identified a template with improved LE and having mul-
tiple hydrogen-bond interactions with the protein, the 7-indazole
carboxylic acid was held constant and an initial scan of spirochro-
manones was undertaken (Table 3). In order to access these com-
pounds a flexible synthetic route was developed. Base-catalyzed
spirocyclization reactions of 2-acetyl-3-hydroxy-aryl and -hetero-
aryl compounds with boc-protected 4-piperidone afforded the de-
sired spirochromanone systems which were then deprotected and
coupled with acids under standard conditions (Fig. 4).
The most potent analog from this work was dimethyl derivative
8 (rACC1 IC50 = 163 nM), which exhibited slightly improved po-
tency relative to mono-methyl derivative 7. Placing the methyl
group in the 7-position resulted in a compound of equivalent po-
tency as the 6-methyl derivative (compare 9 and 7), and both are
modestly lower potency and efficiency than the 6,7-dimethyl
derivative 8. The 6,8-dimethyl derivative 12 was the least potent
of the methylspirochromanone derivatives, exhibiting an
IC50 = 1.32 lM, suggesting that substitution in the 8-position is
deleterious to activity. Of the three positional isomeric methoxy
derivatives prepared (10, 13, and 15), the 6-methoxy derivative
10 exhibited the best potency. Derivatization of the 6-position
with a chlorine afforded compound 11 having decreased potency
compared to 6-methyl derivative 8. Interestingly, SAR was not al-
ways additive, as was evident by 5-methoxy-6-chloro derivative
14 being less active than either of the mono-substituted com-
pounds 11 or 13. Placement of a phenyl group in the 6-position re-
sulted in a 3.5-fold decrease in potency compared to the 6-methyl
derivative 7 (rACC1 IC50 = 2210 and 633 nM for 16 and 7, respec-
tively). Continuing to follow LE proved useful in comparison of
compounds. Compounds 15 and 16, for example, were roughly
equipotent in their ACC1 activity, but 16 was less efficient and this
helped to focus the SAR development away from the 7-position.
Confirmation of hACC2 activity was determined by testing select
analogs and these compounds were determined to be isozyme
non-selective. Significant sequence homology (>97%) exists be-
tween rat and human ACC1,13 therefore due to availability, purified
rat ACC1 was utilized in our early screening efforts.6
O
O
O
O
X
X
Y
Z
Y
Z
1. pyrrolidine, toluene
+
OH
N
2. 4M HCl Dioxane
R
N
3. RCO2H, coupling agent
Boc
O
Figure 4. General synthetic scheme for the preparation of spirochromanone
Figure 3. X-ray co-crystal structure of yeast CT-domain and spirochromanone 7.
analogs.

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J. W. Corbett et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2383–2388
Table 5
An extensive evaluation of carboxylic acids was undertaken in
SAR of 5-methoxyspirochromanones
an effort to identify compounds of increased potency and selected
results are shown in Table 4. The 6,7-dimethyl spirochromanone
was the most potent and efficient analog and this fragment was
chosen for further elaboration. Since the 5-methoxy-spirochroma-
none probed a different vector than the 6,7-dimethyl scaffold, it
was also utilized in this effort. The use of 5-carboxyindazole acids
were found to consistently afford derivatives with rACC1 potency
below ꢀ100 nM. 5-Carboxybenzimidazole derivatives were gener-
ally less potent than the indazole analogs, with the exception of
compound 24, which exhibited sub-40 nM potency against both
ACC isozymes.
Placement of an amide in the 2-position of the benzimidazole
tail-piece, as in 24, was well tolerated as evidenced by its inhibi-
tory potency and an improved LE compared to benzimidazole ana-
logs 25–27. Therefore, a series of benzimidazole and indole analogs
were made in order to more fully develop the emerging tail-piece
SAR and this data is summarized in Table 5. 2-Amidobenzimidaz-
ole 24, indole analogs 29–30 and phenyl derivative 31 suffered
from modest solubility. Therefore, heterocyclic analogs 32–36
were prepared in an effort to maintain ACC inhibitory potency
while attempting to improve overall solubility properties.
Attention was devoted to expansion of arylspiroketone SAR in
an effort to identify more soluble derivatives. In particular, a vari-
OMe O
O
N
Ar
O
Compd Ar
rACC1 IC50
(nM)
hACC2 IC50
(nM)
rACC1
LE
H
N
29
30
43
65
11
16
0.30
0.30
CONHMe
CONH2
H
N
H
N
31
32
33
34
35
16
26
16
0.30
0.29
0.30
0.28
0.27
0.30
N
H
N
12
N
N
H
N
26
19
N
N
N
H
N
60
51
N
Table 4
H
N
Expansion of tail-piece spirochromanone SAR
101
19
NDa
NDa
N
N
O
N
5
6
H
N
N
N
R
36
O
N
Ar
N
O
a
ND means no data.
Compd Ar
R
rACC1 IC50 hACC2 IC50 rACC1
(nM)
(nM)
LE
ety of 6-substituted phenylspirochromanones were prepared
(Table 6). Interestingly, it was found that the binding pocket was
tolerant of polar functionality in the 6-position, as evidenced by
the modest potency exhibited by carboxylic acid derivative 37
(rACC1 IC50 = 303 nM). Substantial improvements in ACC potency
could be achieved by building out in the vector delineated by sub-
stitution at the 6-position of the phenylspiroketone ring. Incorpo-
ration of either a pyrazole or an N-methylpyrazole in the 6-
position provided analogs with sub-10 nM rACC1 potency (e.g.,
compounds 38 and 39). The pyrazole derivatives 40 and 41 had
sub-10 nM potency against hACC2 while their rACC1 activity was
decreased ꢀ2–4-fold compared to pyrazole isomers 38 and 39,
respectively. The potent ACC inhibitory activity of analogs 43–45
indicated that it was possible to place additional heterocycles into
the 6-position.
H
N
17
18
6,7-Dimethyl 22
48
0.34
0.32
N
N
5-MeO
104
6.5
19
20
21
6,7-Dimethyl 36
NDa
164
NDa
0.34
0.30
0.34
N
H
5-MeO
220
H
N
6,7-Dimethyl 17.5
N
N
22
23
5-MeO
5-MeO
102
42
151
11
0.31
0.32
H
N
Chemistry efforts were then directed at identifying a molecule
with a similar ACC inhibitory profile but with decreased molecular
weight. To wit, the incorporation of two of the better tail-pieces
into the 7-azaspirochromanone template afforded analogs 46–48
(Table 7), with the more potent analog being N-methylpyrazole
derivative 47.
H
N
24
25
5-MeO
5-MeO
35
16
0.32
0.31
CONH2
N
H
N
216
111
N
5-Azaspirochromanone derivatives 49–53 in Table 8 were pre-
pared, but all compounds exhibited weak rACC1 inhibitory po-
tency. In contrast, the 6-aza-5-alkoxyspirochromanones 54–59
had rACC1 and hACC2 IC50’s below 100 nM for all prepared analogs
(Table 8). Compounds having a 5-isopropoxy group were found to
be the most potent ACC inhibitors (see 54 and 59).
Compound 59 was ultimately selected for additional evaluation
owing to its balanced rACC1/hACC2 inhibitory profile.15 Rat and
beagle dog pharmacokinetic data for compound 59 is provided in
Table 9. The compound exhibited moderate clearance in rat
H
N
26
5-MeO
172
126
0.31
N
H
N
27
28
5-MeO
5-MeO
663
>1000
505
0.28
0.26
N
H
N
2970
N
N
a
ND means no data.

J. W. Corbett et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2383–2388
2387
Table 6
Table 8
Summary of 6-substituted phenylspiroketone SAR
SAR of 5- and 6-azaspirochromanones
O
O
R
R1
N
R2
H
N
H
N
O
N
N
O
N
N
O
R3
O
Compd
R
rACC1 IC50 (nM)
303
hACC2 IC50 (nM)
NDa
rACC1 LE
0.29
Compd
R1/R2/R3
rACC1 IC50 (nM)
hACC2 IC50 (nM)
rACC1 LE
37
CO2H
N
49
50
51
52
53
H/Me/H
H/Me/Me
Me/Me/H
H/H/Me
154
249
366
766
1350
NDa
363
NDa
640
NDa
0.33
0.31
0.30
0.30
0.28
38
39
40
7.4
7.5
18
NDa
NDa
4.5
0.34
0.33
0.32
HN
N
N
H2N/Me/H
N
HN
R1
O
O
O
N
N
41
42
43
44
31
18
19
17
6.1
7.6
30
0.30
0.31
0.31
0.31
N
H
N
N
HN
R2
N
N
N
R3
O
O
O
54
55
56
57
58
59
iPr/H/H
Me/Me/H
iBu/H/H
Me/H/H
Et/H/H
30
34
23
86
61
12
4.5
48
0.32
0.33
0.32
0.32
0.32
0.33
39
N
98
NDa
NDa
20
N
O
45
13
6.4
0.31
iPr/H/Me
a
ND means no data.
a
ND means no data.
Table 9
(CL = 31 mL/min/kg) and low clearance in dog (3.6 mL/min/kg). The
rat oral absorption was low-to-moderate in a methylcellulose for-
mulation. A lipid emulsion formulation was tried resulting in de-
creased drug exposure, perhaps with poor drug exposure
attributable to slow diffusion of drug out of the lipid droplets. It
was found that a spray-dried dispersion (SDD) formulation im-
proved the oral bioavailability (52%) indicating that this compound
exhibited solubility limited absorption.
The strategy of identifying a compound with polarity in the
arylchromanone ring in order to identify an analog with reasonable
solubility properties, while maintaining potency at rACC1 and
hACC2, afforded compound 59. This 6-azaspiroketone derivative
possessed favorable in vitro attributes and exhibited reasonable
pharmacokinetic properties in two species, with low clearance ob-
served in beagle dog. In summary, work continues to identify iso-
zyme non-selective rACC1/hACC2 inhibitors that are highly orally
bioavailable.
Pharmacokinetic data for compound 59.
Formulation
Rat pharmacokinetic parametersa
b
t
CLb
Vdssb
%F
Cmax, poa
½
Methylcellulose
Lipid emulsion
Spray-dried dispersion
1.4 0.2 31
5
3.3 0.2 32
5
9
79 19
13
63 21
8
52
0
2
Male Beagle Dog Pharmacokinetic Dataa
40% PEG400/60% glycerol 6.1 0.5 3.6 0.4 1.6
0
NDc
NDc
a
b
c
iv dose = 1 mg/kg and po dose = 3 mg/kg.
t
in h, CL in mL/min/kg, Vdss in L/kg.
½
ND means no data.
Acknowledgment
The authors thank Bernard Fermini and Shuya Wang for hERG
patch clamp studies.
Table 7
SAR of azaspirochromanones
References and notes
O
1. Data was obtained from the World Health Organization website: http://
www.who.int/diabetes/en/.
R
2. From WHO’s website: http://www.who.int/mediacentre/factsheets/fs311/en/
index.html.
N
O
N
Ar
3. For instance, see the guidelines for management of hyperlipidemia in children
and adolescents put forward by the Kansas City Quality Improvement
Consortium: http://kcqic.org/wp-content/uploads/2008/05/2007%20KCQIC%20
Hyperlipidemia%20Guideline%20%20Children%20and%20Adolescents.pdf.
4. For recent reviews discussing acetyl-CoA carboxylase as a potential drug target,
see: (a) Harwood, H. J., Jr. Expert Opin. Cardiovasc. Renal 2005, 9, 267; (b) Tong,
L., ; Harwood, H. J., Jr. J. Cell. Biochem. 2006, 99, 1476; (c) Corbett, J. W.;
Harwood, H. J., Jr. Recent Patents Cardiovasc. Drug Disc. 2007, 2, 162.
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Chem. 1974, 249, 6657.
6. Harwood, H. J., Jr.; Petras, S. F.; Shelly, L. D.; Zaccaro, L. M.; Perry, D. A.;
Makowski, M. R.; Hargrove, D. M.; Martin, K. A.; Tracey, W. R.; Chapman, J. G.;
Magee, W. P.; Dalvie, D. K.; Soliman, V. F.; Martin, W. H.; Mularski, C. J.;
Eisenbeis, S. A. J. Biol. Chem. 2003, 278, 37099.
7. For the yeast CT-domain co-crystal structure with CP-640186, see: Zhang, H.;
Tweel, B.; Li, J.; Tong, L. Structure 2004, 12, 1683.
O
Compd R-Group(s) Ar
rACC1 IC50 (nM) rACC1 LE
N
N
N
N
N
46
975
0.21
0.30
N
H
N
H
N
N
47
24.6
N
N
H
N
48
MeO
182
0.31

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J. W. Corbett et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2383–2388
8. Vajdos, F.; Rajamohan, F.; Marr, E.; Corbett, J. W., in preparation.
9. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
10. Rat ACC1 was obtained from rat liver based upon standard procedures such as
those described by Thampy, K. G.; Wakil, S. J. J. Biol. Chem. 1985, 260, 6318. The
assay was conducted as described in Ref. 6.
2 mM EDTA, and 5% glycerol. The pooled protein was frozen and stored at
À80 °C, with no loss of activity upon thawing. For measurement of hACC2
activity and assessment of hACC2 inhibition, test compounds are dissolved in
DMSO and added to the hACC2 enzyme as a 5Â stock with a final DMSO
concentration of 1%. rhACC2 was assayed in
a Costar #3767 (Costar,
11. Subsequent to the initiation of our work, the following patent applications
published: (a) JP 2005119987; (b) WO 2007011809; (c) WO 2007011811.
12. Ascension numbers for the crystal structures are: compound 2 = 3h0j and
compound 7 = 3h0s.
13. Abu-Elheiga, L.; Jayakumar, A.; Baldini, A.; Chirala, S.; Wakil, S. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 4011.
Cambridge, MA) 384-well plate using the Transcreener ADP detection FP
assay kit (Bellbrook Labs, Madison, WI) using the manufacturers’ conditions for
a 50
7.5, 5 mM MgCl2, 5 mM tripotassium citrate, 2 mM DTT, 0.5 mg/mL BSA,
30 M acetyl-CoA, 50 M ATP, and 8 mM KHCO3. Typically, a 10 L reaction is
run for 1 h at room temperature and 10 L of Transcreener stop and detect
lM ATP reaction. The final conditions for the assay are 50 mM HEPES, pH
l
l
l
l
14. Human ACC2 inhibition is measured using purified recombinant human ACC2
(hACC2). A full length Cytomax clone of hACC2 was purchased from Cambridge
Bioscience Limited and was sequenced and subcloned into PCDNA5 FRT TO-
TOPO (Invitrogen, Carlsbad, CA). The hACC2 was expressed in CHO cells by
tetracycline induction and harvested in 5 L of DMEM/F12 with glutamine,
buffer is added and incubated for an additional 1 h. The data is acquired on an
Envision Fluorescence reader (Perkin Elmer) using a 620 excitation Cy5 FP
general dual mirror, 620 excitation Cy5 FP filter, 688 emission (S) and a 688 (P)
emission filter.
15. Spectral data for compound 59: LC/MS analysis Waters C18 column, 2.5 mL/min
flow rate, gradient 5% ACN/95% water to 95% ACN/5% water over 3 min. LC RT
2.2 min, ESI(+) 449.4, ESI(À) 447.3. Diagnostic 1H NMR signals (DMSO-d6):
12.9 (s, 1H), 8.1 (d, J = 5.8 Hz, 1H), 7.6 (s, 1H), 7.2 (s, 1H), 6.7 (d, J = 5.9 Hz), 5.4
(m, 1H), 2.8 (s, 2H), 1.3 (d, J = 6.2 Hz, 6H).
biotin, hygromycin and blasticidin with 1 lg/mL tetracycline. The conditioned
medium containing hACC2 was then applied to a Softlink Soft Release Avidin
column (Promega, Madison, WI) and eluted with 5 mM biotin. hACC2 (4 mg)
was eluted at a concentration of 0.05 mg/mL with an estimated purity of 95%.
The purified hACC2 was dialyzed in 50 mM Tris, 200 mM NaCl, 4 mM DTT,