Potent biphenyl- and 3-phenyl pyridine-based inhibitors of acetyl-CoA carboxylase

Article abstract of DOI:10.1016/j.bmcl.2009.08.077

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on

Full text of DOI:10.1016/j.bmcl.2009.08.077

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5872–5876
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent biphenyl- and 3-phenyl pyridine-based inhibitors
of acetyl-CoA carboxylase
a
b
b
c
Tasir S. Haque a, , Ningning Liang , Rajasree Golla , Ramakrishna Seethala , Zhengping Ma ,
*
William R. Ewing a, Christopher B. Cooper a, Mary Ann Pelleymounter c, Michael A. Poss a, Dong Cheng c
a Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
b Department of Applied Biotechnologies, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
c Department of Discovery Biology, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2009
Revised 20 August 2009
Accepted 21 August 2009
Available online 26 August 2009
We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs)
containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are
moderately selective for either enzyme, depending on side chain substitution. Typical activities of the
most potent compounds in this class are in the low double-digit to single-digit nanomolar range in
in vitro assays using human ACC1 and ACC2 enzymes.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Carboxylase
ACC
ACC inhibitors
Biphenyl
3-Phenyl pyridine
Obesity is not only a major public health issue for the United
States,1 but also for the world in general.2 In the United States
the incidence of obesity in adults doubled (and in children and
adolescents tripled) between 1980 and 2002. In 2004, 32.2% of
adults over age 20 were classified as obese.1 Obesity is associated
with a number of serious diseases, including type 2 diabetes, coro-
nary heart disease, dyslipidemia, and hypertension.
The Acetyl-CoA Carboxylase (ACC) enzymes are potential points
of intervention for the treatment of obesity. Mammalian ACC1 and
ACC2 are large (with molecular masses of 265 kDa and 280 kDa,
respectively), functionally multicomponent enzymes that catalyze
the ATP-dependent conversion of acetyl-CoA to malonyl-CoA.3 The
ACC enzymes are believed to play pivotal roles in energy homeo-
stasis, not only through direct participation in fatty acid synthesis,
but also through regulation of fatty acid b-oxidation. Malonyl-CoA
is a key metabolite that serves as a precursor for de novo fatty acid
synthesis in the liver and adipose tissue.4 In heart, muscle, and
liver tissues, where fatty acids are utilized as a fuel source, malo-
nyl-CoA also serves as a sensor to gauge the rate of mitochondrial
fatty acid b-oxidation through an allosteric inhibition of carnitine
palmitoyl-CoA transferase (CPT-1).5
ACC2 isoform for the treatment of obesity. Intracellularly, ACC1 is
localized in the cell cytosol and is believed to be responsible for
generation of malonyl-CoA destined for fatty acid synthesis.6
ACC2 is localized at the mitochondrial membrane,7 and is believed
to be responsible for generation of the malonyl-CoA that allosteri-
cally inhibits CPT-1.6 Therefore, inhibition of ACC1 may result in
decreased fatty acid synthesis, whereas inhibition of ACC2 may
result in increased fatty acid oxidation. Inhibition of both ACC
enzymes may combine the benefits of decreased synthesis and in-
creased consumption of fatty acids, which may be required for
robust efficacy in therapeutic weight loss.8 On the other hand,
the safety profile of such a dual-isoform inhibitor is unclear. While
ACC2 knockout mice are healthy, viable, lean, and appear to exhibit
resistance to obesity induced by a high-fat diet,9 the ACC1 knock-
out in mice is embryonically lethal.10 We chose to pursue both
pan-ACC and isoform-selective inhibitors, with the ultimate goal
of evaluating the inhibitors in the appropriate rodent obesity mod-
els in the future.
A number of mammalian ACC inhibitors have been reported in
the patent and scientific literature,11 examples of which are shown
in Figure 1. Soraphen A12 is a polyketide natural product that acts
via inhibition of the eukaryotic BC domain of ACC, but lacks po-
tency against bacterial BC subunits. Pfizer reported the synthesis
and ACC inhibition of CP-640186, which displays inhibitory activ-
ity against both human ACC isoforms.13 Abbott has reported a
number of both pan-ACC and ACC2-selective inhibitors,14,15 two
There has been debate regarding the desirability of a non-selec-
tive inhibitor of the ACC isoforms versus a selective inhibitor of the
* Corresponding author. Tel.: +1 609 252 4273.
E-mail address: tasir.haque@bms.com (T.S. Haque).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.077

T. S. Haque et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5872–5876
5873
OMe
Me
Me
N
O
OMe
O
O
N
N
O
O
O
OH
OH
Me OMe
Soraphen A
CP-640186
N
O
S
O
O
S
N
NH
O
N
O
N
O
HN
O
O
A-908262
O
Abbott 9
HN
N
O
O
O
O
N
N
H
N
O
N
N
N
O
O
O
Merck/Banyu
Example 8
Pfizer 59
Figure 1. Structures of several reported ACC inhibitors.
of which are shown in Figure 1.16 Several groups have also reported
potent ACC inhibitors based on a spirochromanone core, including
Example 8 reported by Merck and Banyu Pharmaceuticals,17 re-
lated series (not shown) from Torrent Pharmaceuticals18 and Tak-
eda,19 and the independently derived Pfizer compound 59.20
Herein we disclose the discovery of ACC inhibitors based on biphe-
nyl and 3-phenyl pyridine chemotypes as shown in Figure 2. These
chemotypes are synthetically readily accessed, and provide tem-
plates that can be easily modified for building structure–activity
relationships regarding inhibitory potency and selectivity for the
two ACC enzymes.
Br
O
a
R1
O
1
2
O
O
R1
b, c, d
H
N
R2
Our initial efforts focused on exploring the right and left sides of
the biphenyl core at the same time using a positional scanning ap-
proach, holding one side constant while varying the other side. The
right-hand side of the molecule (as shown in Fig. 3) was fixed as
the acetamide, and the left-hand side was varied through coupling
of 1-(40-bromobiphenyl-4-yl)ethanone with a variety of substi-
tuted phenols under Ullmann coupling conditions (Scheme 1). Fol-
O
3
Scheme 1. Synthesis of biphenyl analogues. Reagents and conditions: (a) R1-
substituted phenol, Cs2CO3, CuI, N,N-dimethylglycine hydrochloride, 1,4-dioxane,
90 °C, 16 h; (b) NH3, Ti(OiPr)4, EtOH, 50 °C, 16 h; (c) NaBH4, 2 h, rt; (d) R2–COOH,
DIC, HOBt, DIEA, CH2Cl2, rt, 16 h.
lowing elaboration to the desired target acetamides, the
compounds were evaluated for inhibitory activity against both
human ACC1 and ACC2 enzymes.21 Data for these compounds
(3a–3n) is shown in Table 1. Substitution at both the meta and para
(3- and 4-aryl) R1 positions resulted in potent ACC inhibitors. Phen-
oxy ethers at either position were potent pan-ACC inhibitors, as
shown by 3d and 3n. Compounds containing the relatively small
ethoxy group (3a and 3l) or methoxy group (3k) at either position
resulted in loss of potency, especially against ACC1. It is interesting
to note that two compounds with alkyl substitution at the para
position displayed good to excellent selectivity for ACC1 (3g and
3i, 24-fold and >300-fold, respectively). Many of the alkoxy or aryl-
oxy-substituted compounds were approximately equipotent for
the two enzymes. However, compounds 3a, 3k, and 3l; with small
alkoxy groups at R1 at either the 3- or 4-aryl positions, exhibited
14-fold, greater than 80-fold, or 84-fold selectivity, respectively
for ACC2. These results suggest that careful selection of R1 substi-
tuent can ‘tune’ compounds of this series towards ACC1-selective,
ACC2-selective, or pan-ACC inhibition.
O
R3
R1
X
H
N
R2
R4
O
Figure 2. Core chemotype chosen for SAR exploration. X = CH, N.
O
R1
H
N
R2
O
Figure 3. Biphenyl core used for the exploration of ACC inhibition via variation at
R1 and R2.

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T. S. Haque et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5872–5876
Table 1
Human ACC1 and ACC2 inhibitory activity for biphenyl series
R1
R2
Human ACC1 IC50 (nM)
Human ACC2 IC50 (nM)
a
a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4-OCH2CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
1630
17
17
14
>30,000
81
39
35
89
140
>30,000
8580
13
41
47
2770
>30,000
188
753
2060
296
36
119
14
33
7
>30,000
114
945
113
>30,000
268
4-O(CH2)2CH3
4-OCH(CH3)2
4-OPh
4-OCH2Ph
4-(CH2)2CH3
4-CH2Phe
4-COPh
4-(CH2)2OCH3
4-(5,6,7,8-Tetrahydronaphthalen-2-ol)
3-OCH3
3-OCH2CH3
3-O(CH2)3CH3
3-OPh
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
3-O(CH2)2CH3
4-O(CH2)2CH3
373
102
12
16
CH2CH3
(CH2)2CH3
(CH2)3CH3
CH(CH3)2
CH2OCH3
CH(OH)CH3 (racemic)
Cyclopropyl
Pyrazol-4-yl
OCH2CH3
202
>30,000
>30,000
214
718
1840
550
3s
3t
3u
3v
3w
609
3100
>30,000
a
Values are the averages of 2–4 experiments, with IC50 values typically reproducible within twofold.
We concurrently explored the right-hand side of the chemo-
ing potency were the change in the torsion angle between the two
central aromatic rings. As shown by 4c, substitution at both R3 and
R4 restores some of the potency against both enzymes, especially
for ACC2. Overall, these results suggest that substitution of the cen-
tral aromatic groups in the biphenyl series could result in both
potent and selective inhibitors.
A second series of compounds designated the ‘3-phenyl pyri-
dine series’ (Fig. 4), was developed both to explore the effect of
including a heteroaromatic group in the core, and to modulate
the solubility characteristics of the molecules. We specifically
chose to explore the 3-phenyl pyridine series both to display the
nitrogen in a region similar to that found in the nitrogen of the thi-
azoles of known inhibitors A-908262 and Abbott 9 (Fig. 1), and for
the synthetic accessibility of the target molecules. Compounds in
this series were synthesized as shown in Scheme 2.
The ethers of the 3-phenyl pyridine series generally displayed
SAR trends similar to the comparable compounds from the biphe-
nyl series, with the most potent ACC2 inhibitor corresponding to
the 4-n-propoxyphenyl ether (10b, Table 3). In general the com-
pounds in the 3-phenyl pyridine series were slightly less potent
than their corresponding compounds from the biphenyl series. As
shown with compound 10a, substitution with a small 4-ethoxy
group at R1 produced a compound that displayed moderate po-
tency towards ACC2 and good selectivity (at least 100-fold) over
ACC1 (ACC1 IC50 >30,000 nM, ACC2 IC50 = 274 nM). Compounds
substituted with the optimal group for ACC2 inhibition for the
3-phenyl pyridine series (10b, R1 = 4-O(CH2)2CH3) displayed ꢀ5-
fold selectivity for ACC2 (ACC1 IC50 = 110 nM, ACC2 IC50 = 18 nM).
This is in contrast to compounds in the biphenyl series, where ana-
type, while holding the left-hand side constant as the 4-propoxy-
phenyl substituent. The data for these compounds (3o–3w) is
shown in Table 1. For the most part only small alkyl groups were
tolerated, as shown in the trend from R2 = CH3 (3b, ACC1
IC50 = 17 nM, ACC2 IC50 = 14 nM) to R2 = (CH2)3CH3 (3q, inactive
vs both ACC1 and ACC2). Cyclic groups were only moderately tol-
erated, with the pyrazol-4-yl amide (3v, with 3-O(CH2)2CH3 at
R1) displaying 17-fold selectivity for ACC1. The ethyl carbamate
3w was essentially inactive against both enzymes.
In addition to the left side and right side variations, we wanted
to explore the possibility of substitution on the two biphenyl rings
of the core. Such substitution would be expected to increase the
degree of ‘twist’ between the planes of the two phenyl groups,
and might also affect potency of the compounds via direct steric
interaction with the enzyme binding site. Three compounds were
made in this series, and are compared to the unsubstituted ana-
logue in Table 2. Substitution at R3 (4a) had no effect on potency
for either enzyme, while substitution at R4 (4b) resulted in a loss
of potency, especially against ACC1. The difference in potency
observed for substitution at R3 versus R4 suggests a direct steric
interaction with the binding site for compound 4b. One would
have expected 4a and 4b to be equipotent if the only factor affect-
Table 2
R3- and R4-substituted biphenyl compounds 4
O
R3
O
H
N
R4
O
O
R1
R3
R4
Human ACC1 IC50 (nM)
Human ACC2 IC50 (nM)
a
a
N
3c
4a
4b
4c
H
CH3
H
H
H
CH3
CH3
17
16
2490
592
33
35
303
88
H
N
R2
O
CH3
a
Values are the averages of 2–4 experiments, with IC50 values typically repro-
ducible within twofold.
Figure 4. 3-Phenyl pyridine core used for the exploration of ACC inhibition via
variation at R1 and R2.

T. S. Haque et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5872–5876
5875
I
OH
(HO)2B
+
O
a
R1
R1
+
N
N
O
N
Br
Br
5
6
7
8
O
O
R1
c, d, e
R1
b
N
H
R2
O
N
9
10
O
Scheme 2. Synthesis of 3-phenyl pyridine analogues. Reagents and conditions: (a) Cs2CO3, CuI, N,N-dimethylglycine hydrochloride, 1,4-dioxane, 90 °C, 16 h; (b) Pd[P(Ph)3]4,
K3PO4, DMF, 120 °C, 16 h; (c) NH3, Ti(OiPr)4, EtOH, 50 °C, 16 h; (d) NaBH4, 2 h, rt; (e) R2–COOH, DIC, HOBt, DIEA, CH2Cl2, rt, 16 h.
Table 3
Human ACC1 and ACC2 inhibitory activity for 3-phenyl pyridine series
R1
R2
Human ACC1 IC50 (nM)
Human ACC2 IC50 (nM)
a
a
10a
10b
10c
10d
10e
10f
10g
10h
10i
4-OCH2CH3
CH3
CH3
CH3
CH3
>30,000
110
415
49
58
69
4200
451
274
18
93
32
117
35
494
156
141
1510
881
2540
227
4-O(CH2)2CH3
4-OCH(CH3)2
3-OPh
4-OCH2Ph
CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
4-O(CH2)2CH3
CH2CH3
(CH2)2CH3
CH(CH3)2
Cyclopropyl
(CH2)3CH3
CH2O CH3
Pyrazol-4-yl
OCH2CH3
319
10j
10k
10l
>30,000
>30,000
>30,000
8150
10m
a
Values are the averages of 2–4 experiments, with IC50 values typically reproducible within twofold.
logs having the optimal R1 substitution such as 3b were equipotent
against both enzymes.
and SAR was explored at a number of points on both cores. Small
alkoxy R1 groups at both the 3- and 4-positions R1 generally affor-
ded potent ACC inhibitors with the trend of smaller alkoxy groups
providing greater ACC2 selectivity. Phenoxy substitution was also
well-tolerated at R1, yielding pan-ACC inhibitors 3d, 3n, and 10d.
At R2 there was a clear preference for methyl and ethyl amides over
larger alkyl amides, implying a relatively small binding pocket at
this position. However, the good ACC1 potency of 3v, with R2 = pyr-
azol-4-yl, suggests that small heterocycles may also be accommo-
dated at this position and could provide isoform selectivity. Both
the biphenyl and 3-phenyl pyridine cores afforded potent ACC
inhibitors. As shown with compounds 4a–4c, substitution at R3
on the central aryl groups is tolerated, suggesting an additional site
for modification to enhance potency, solubility, and PK and PD
characteristics of the two series. The most active compounds 3b,
3d, 3m, and 11a displayed low nanomolar potency for the human
ACC enzymes. The results described above suggest a variety of pos-
sibilities for further optimization of potency and/or selectivity of
the two series.
Exploration of the acyl, or ‘right-side’ SAR in the 3-phenyl
pyridine series is shown in Table 3, entries 10f–10m. Small amides
appear to be optimal at R2, with 10b and 10f (acetamide and n-pro-
pionamide, respectively) being amongst the most potent inhibitors
of either enzyme, and both displaying weak selectivity for inhibi-
tion of ACC2 (sixfold and twofold, respectively). Unlike the biphe-
nyl series, the pyrazol-4-yl amide 10l was inactive against both
ACC enzymes. The ethyl carbamate, essentially inactive against
both enzymes in the biphenyl series (3w, IC50 >3000 nM for
ACC1 and ACC2), displayed moderate potency against ACC2 and
selectivity over ACC1 in the (pyridin-3-yl)phenyl series (10m,
ACC1 IC50 = 8150 nM, ACC2 IC50 = 227 nM).
Finally, researchers at Abbott previously noted that one enan-
tiomer of their series was considerably more potent than the other
enantiomer.14 We separated and tested the two enantiomers of
10b. The results are shown in Table 4, and demonstrate that for
the 3-phenyl pyridine series the (S)-enantiomer 11a is at least
ꢀ100-fold more potent against both ACC enzymes than the (R)-
enantiomer 11b.22
References and notes
In summary, we have identified two series of ACC inhibitors
based on a biphenyl or 3-phenyl pyridine core. Compounds with
modest to good selectivity for both ACC isoforms were identified,
1. Ogden, C. L.; Carroll, M. D.; Curtin, L. R.; McDowell, M. A.; Tabak, C. J.; Flegal, K.
M. JAMA 2006, 295, 1549.
2. World Health Organization. Obesity and Overweight: Factsheet No 3011.
Geneva 2006.
3. Tong, L. Cell. Mol. Life Sci. 2005, 62, 1784.
Table 4
4. (a) Wakil, S. J.; Titchener, E. B.; Gibson, D. M. Biochim. Biophys. Acta 1958, 29,
225; (b) Thampy, K. G. J. Biol. Chem. 1989, 264, 17631.
5. McGarry, J. D.; Brown, N. F. Eur. J. Biochem. 1997, 244, 1.
6. Brownsey, R. W.; Zhande, R.; Boone, A. N. Biochem. Soc. Trans. 1997, 25, 1232.
7. Abu-Elheiga, L.; Brinkley, W. R.; Zhong, L.; Chirala, S. S.; Woldegiorgis, G.;
Wakil, S. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1444.
8. Tong, L.; Harwood, H. J., Jr. J. Cell. Biochem. 2006, 99, 1476.
9. Abu-Elheiga, L.; Matzuk, M. M.; Abo-Hashema, K. A. H.; Wakil, S. J. Science 2001,
291, 2613.
Comparison of enantiomers of 10b for 3-phenyl pyridine core
a
a
Enantiomer
Human ACC1 IC50 (nM)
Human ACC2 IC50 (nM)
11a
11b
(S)
(R)
35
>30,000
8
777
a
Values are the averages of 2–4 experiments, with IC50 values typically repro-
ducible within twofold.

5876
T. S. Haque et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5872–5876
10. Abu-Elheiga, L.; Matzuk, M. M.; Kordari, P.; Oh, W.; Shaikenov, T.; Gu, Z.; Wakil,
S. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12011.
11. For recent reviews of ACC inhibitors, see the following: Corbett, J. W.;
Harwood, H. J. Rec. Patents Cardiovasc. Drug Disc. 2007, 2, 162; Corbett, J. W.
Expert Opin. Ther. Patents 2009, 19, 943.
12. Gerth, K.; Bedorf, N.; Irschik, H.; Hofle, G.; Reichenbach, H. J. Antibiot. (Tokyo)
1994, 47, 23.
13. Harwood, H. J., Jr.; Petras, S. P.; 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.
14. Gu, Y. G.; Weitzberg, M.; Clark, R. F.; Xu, X.; Li, Q.; Zhang, T.; Hansen, T. M.; Liu,
G.; Xin, Z.; Wang, X.; Wang, R.; McNally, T.; Camp, H.; Beutel, B. A.; Sham, H. L.
J. Med. Chem. 2006, 49, 3770.
15. Gu, Y. G.; Weitzberg, M.; Clark, R. F.; Xu, X.; Li, Q.; Lubbers, N. L.; Yang, Y.; Beno,
D. W. A.; Widomski, D. L.; Zhang, T.; Hansen, T. M.; Keyes, R. F.; Waring, J. F.;
Carroll, S. L.; Wang, X.; Wang, R.; Healan-Greenberg, C. H.; Blomme, E. A.;
Beutel, B. A.; Sham, H. L.; Camp, H. S. J. Med. Chem. 2007, 50, 1078.
16. At the time we initiated the efforts described herein, the publication describing
Abbott 9 had not yet appeared.
17. Yamakawa, T.; Jona, J.; Niyama, K.; Yamada, K.; Iino, T; Ohkubo, M; Imamura,
H.; Kusunoki, J.; Yang, L. WO 2007/011811.
18. Shinde, P.; Srivastava, S. K.; Odedara, R.; Tuli, D.; Munshi, S.; Patel, J.; Zambad,
S. P.; Sonawane, R.; Gupta, R. C.; Chauthaiwale, V.; Dutt, C. Bioorg. Med. Chem.
Lett. 2009, 19, 949.
Song, W.; Jones, C. J.; Coffey, S. B.; Buzon, L.; Minich, M. L.; Dirico, K. J.; Tapley,
S.; McPherson, R. K.; Sugarman, E.; Harwood, H. J.; Esler, W. Bioorg. Med. Chem.
Lett. 2009, doi:10.1016/j.bmcl.2009.04.091.
21. The ACC inhibitors in the current paper were evaluated by the ACC/Fatty-
Acid-Synthase (FAS)-Coupled Assay according to Seethala et al. (Anal.
Biochem. 2006, 58, 257) with minimal modifications. Briefly, the assay
buffer (50 mM HEPES pH 7.5, 10 mM sodium citrate, 20 mM MgCl2, 6 mM
NaHCO3) and substrate mixture (containing 2.4
[3H] acetyl-CoA
(PerkinElmer, NET-290) and 47.6 acetyl-CoA, 100 lM NADPH and
l
M
lM
0.125 mM ATP in Assay Buffer) were all made fresh on the day of assay
from stock solutions. Human ACC1 and human ACC2 were recombinant
enzymes expressed in and purified from a bacculovirus system (Protein Expr.
Purif. 2007, 51, 11). To each well containing 0.5
DMSO as control in a 384-well phospholipid FlashPlateÒ (PerkinElmer) was
added 30 L of a solution of ACC (2–4.5 nM) and FAS (1 g/assay) enzymes
in assay buffer. After 10 min incubation, the reaction was started via
addition of 20 of substrate mixture. The reaction was carried out for
30 min at room temperature. After incubation, the reaction was quenched
lL of compound in DMSO or
l
l
a
l
l
with the addition of 10
ll of 200 mM EDTA (ꢀ33 mM final concentration).
The [3H]-palmitic acid produced was determined by counting in a TopCount
instrument (PerkinElmer). The IC50 for each compound was calculated using
a logistic 4 parameter fit equation: y = A + ((B ꢁ A)/(1+((C/x)^D))) in an in
house developed data processing program TOOLSET.
22. The identity of single enantiomers was confirmed by synthesis of the (S)-
enantiomer using chiral tert-butylsulfinimine reductive amination procedure
(Borg, G.; Cogan, D. A.; Ellman, J. A. Tetrahedron Lett. 1999, 40, 6709), and
comparison to enantiomers separated by chiral HPLC.
19. Fukatsu, K.; Kamata, M.; Yamashita, T. WO 2008/102749.
20. Corbett, J. W.; Freeman-Cook, K. D.; Elliott, R.; Vajdos, F.; Rajamohan, F.; Kohls,
D.; Marr, E.; Zhang, H.; Tong, L.; Tu, M.; Murdande, S.; Doran, S. D.; Houser, J. A.;