Identification and synthesis of novel inhibitors of acetyl-CoA carboxylase with in vitro and in vivo efficacy on fat oxidation

Article abstract of DOI:10.1021/jm101179e

Acetyl CoA carboxylase isoforms 1 and 2 (ACC1/2) are key enzymes of fat utilization and their inhibition is considered to improve aspects of the metabolic syndrome. To identify pharmacological inhibitors of ACC1/2, a high throughput screen was performed w

Full text of DOI:10.1021/jm101179e

J. Med. Chem. 2010, 53, 8679–8687 8679
DOI: 10.1021/jm101179e
Identification and Synthesis of Novel Inhibitors of Acetyl-CoA Carboxylase with in Vitro and in Vivo
Efficacy on Fat Oxidation
€
Stefanie Keil,* Marco Muller, Gerhard Zoller, Guido Haschke, Katrin Schroeter, Maike Glien, Sven Ruf, Ingo Focken,
Andreas W. Herling, and Dieter Schmoll
Sanofi-Aventis Deutschland GmbH, R&D, Diabetes Division, Industriepark Hoechst, Building G 878, D-65926 Frankfurt am Main,
Germany
Received September 10, 2010
Acetyl CoA carboxylase isoforms 1 and 2 (ACC1/2) are key enzymes of fat utilization and their
inhibition is considered to improve aspects of the metabolic syndrome. To identify pharmacological
inhibitors of ACC1/2, a high throughput screen was performed which resulted in the identification of the
lead compound 3 (Gargazanli, G.; Lardenois, P.; Frost, J.; George, P. Patent WO9855474 A1, 1998) as a
moderate selective ACC2 inhibitor. Optimization of 3 led to 4m (Zoller, G.; Schmoll, D.; Mueller, M.;
Haschke, G.; Focken, I. Patent WO2010003624 A2, 2010) as a submicromolar dual ACC1/2 inhibitor of
the rat and human isoforms. 4m possessed favorable pharmacokinetic parameters. This compound
stimulated fat oxidation in vivo and reduced plasma triglyceride levels in a rodent model after
subchronic administration. 4m is a suitable tool compound for the elucidation of the pharmacological
potential of ACC1/2 inhibition.
1. Introduction
isoform unselective ACC1/2 inhibitor and describe here the
identification and optimization of a new series of ACC1/2
inhibitors with favorable pharmacokinetic parameters and in
vivo activity.
Increased ectopic fat accumulation is a risk factor for the
development of type 2 diabetes,1-23,4 indicating that a reduc-
tion of ectopic lipids could be beneficial for the treatment of
the metabolic syndrome. The inhibition of acetyl CoA carbox-
ylase (ACCa) has received special attention for this approach
and has been the focus of drug discovery programs for the
identification of ACC inhibitors.5-7 Examples of previously
described compounds include 18-10 and 25,11 (Figure 1).
Mammalian ACC catalyzes the formation of malonyl-CoA
and exists in two isoforms with distinct subcellular local-
izations.6 Although the functions of these isoforms overlap,
cytosolic ACC1 is involved predominantly in de novo lipo-
genesis, whereas mitochondrial ACC2 controls mainly fat
oxidation. In particular, the inhibition of ACC2 has been
predicted to reduce intracellular fat by the subsequent switch
to fat utilization for energy production. However, the sup-
pression of both ACC1 and ACC2 expression using antisense
oligonucleotides stimulates fat oxidation in hepatocytes more
potently than that of either ACC1 or ACC2 alone.12 This
indicates that isoform unselective inhibition of ACC is more
efficacious in stimulating fat oxidation than an ACC2-selec-
tive inhibitor. Therefore, we aimed for the identification of an
2. Chemistry
The synthesis of the first target molecules4a-4d is shown in
Scheme 1. Commercially available 3-benzyloxyphenylacetic
acid methyl ester was treated with dimethylcarbonate under
basic conditions with subsequent reduction of the ester func-
tions with diisobutylaluminiumhydride to obtain the diol 5.
Transacetalisation with 4-aminobutyraldehyde diethylacetal
and acidic catalysis yielded the dioxanyl compound 6, which
was then acetylated, and the benzyl protecting group was
removed by hydrogenation to obtain compound 7. The free
phenolic function in 7was alkylated with different alkyl halides
or reacted with different alcohols under Mitsunobu type
reaction to obtain compounds 4a-4d.
The synthesis of the target molecules 4e, 4f, and 4g is shown
in Scheme 2. Commercially available 5-benzyloxy-2-chloro-
pyridine was substituted with 2-phenyl-[1,3]dioxan-5-ol under
basic conditions to obtain dioxanyl compound 8. Opening of
the cyclic acetal under acidic conditions yielded the diol
compound 10g. Alkylation of 4-benzyloxyphenol with di-
methyl 2-chloromalonate and subsequent reduction of the
ester functions yielded the diol compound 10e. The diols 10e
and 10g were elaborated to the target molecules 4e, 4f, and 4g
in a similar manner according to Scheme 1.
*To whom correspondence should be addressed. Phone: þþ49-69-305-
80835. Fax: +496930517373. E-mail: stefanie.keil@sanofi-aventis.com.
a Abbreviations: ACC, acetyl CoA carboxylase; DEAD, diethylazo-
dicarboxylate; DIAD, diisopropylazodicarboxylate; DIBALH, diiso-
butylaluminium hydride; DMAP, 4-(dimetylamino)-pyridine; DCM,
dichloromethane; DMF, dimethylformamide; HPLC, high pressure
liquid chromatography; MW, microwave; NMP, N-methylpyrrolidon;
PPh3, triphenylphosphine; pTSOH, para-toluenesulfonic acid; RP,
reverse phase; SAR, structure-activity-relationship; THF, tetrahydro-
furan; TOTU, O-((ethoxycarbonyl)cyanomethyleneamino)-N,N,N0,N0-
tetramethyluronium tetrafluoroborate; HTS, high throughput screen.
The synthesis of the target molecules 4h-4j and 4n-4p
is shown in Scheme 3. Commercially available N-[3-(4-hydro-
xy-phenyl)-propyl]-acetamide or N-[3-(4-hydroxy-phenyl)-1-
methyl-propyl]-acetamide were reacted with pyridine or py-
rimidine halides under conditions of a nucleophilic aromatic
r
2010 American Chemical Society
Published on Web 11/17/2010
pubs.acs.org/jmc

8680 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Keil et al.
Figure 1. Structures of representative ACC inhibitors from Abbott 1 and Pfizer 2 and of the HTS screening hit 3.
Scheme 1a
a Reagents and conditions: (a) dimethylcarbonate, NaH; (b) DIBALH; (c) 4-aminobutyraldehyde diethylacetal, pTsOH, toluene, 60 °C; (d) Ac2O,
NEt3, ethyl acetate, RT; (e) H2 (3 bar)/Pd, ethanol, RT; (f) R-OH, PPh3, DEAD, THF or R-Hal, Cs2CO3.
Scheme 2a
a Reagents and conditions: (a) NaH, NMP, MW, 200 °C; (b) HCl, MeOH, RT; (c) Cs2CO3, KI, DMF, 60 °C; (d) DIBALH, toluene, 0 °C; (e) N-(4,4-
diethoxybutyl)acetamide, pTsOH, toluene, 60 °C, separation of isomeres; (f) H2 (3 bar)/Pd, ethanol, RT; (g) cyclopentylbromide, Cs2CO3, DMF, RT.
substitution reaction to obtain target compounds 4h-4j and
4n-4p.
The synthesis of the target molecules 4q-4t is shown in
Scheme 5. Racemic 17 was synthesized in a similar manner
according to Scheme 4 by use of racemic 2-(but-3-yn-2-yl)-
isoindole-1,3-dione. The amino function of 17 was then
further elaborated by known procedures to obtain target
molecules 4q-4t.
The synthesis of the target molecules 4u is shown in Scheme 6.
1,4-cis-Cyclohexandiol was reacted with 5-isopropoxy-pyridin-
2-ol under Mitsunobu type reaction under inversion of con-
figuration to obtain the 1,4-trans-cyclohexandiol derivative
18. The free hydroxyl group was alkylated with 3-bromo-2-
methyl-propene to yield compound 19. The double bond was
bishydroxylated with osmium tetroxid, the diol cleaved with
The synthesis of the target molecule 4m is shown in Scheme 4.
Commercially available 6-chloro-pyridin-3-ol and 1,4-dibromo-
benzene were reacted under copper catalysis at elevated tem-
perature to obtain compound 12. The chlorine atom was then
substituted by isopropoxide. The bromide 13 was then reacted
in a Sonogashira reaction with commercially available (S)-
2-(but-3-yn-2-yl)isoindole-1,3-dione to obtain compound 14.
The triple bond was hydrogenated and the phthalimide
protecting group removed by treatment with hydrazine. The
free amino function was then acetylated to obtain enantiomeric
pure 4m(ent-S).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8681
Scheme 3a
a Reagents and conditions: (a) NaH, NMP, MW, 220 °C.
Scheme 4a
a Reagents and conditions: (a) CuO/K2CO3, pyridine, 150 °C; (b) isopropyl alcohol, NaH, NMP, 120 °C; (c) 2-((S)-1-methyl-prop-2-ynyl)-isoindole-
1,3-dione, PPh3, DEAD, THF; (d) H2/Pd, EtOH; (e) hydrazine hydrate; (f) Ac2O, NEt3.
Scheme 5a
and rat ACC1/2 (rACC1/2). This isoform selectivity indi-
cates that 3 binds to a site within ACC that is not fully
conserved between the isoforms and between the species. The
overall sequence identity between hACC1 and hACC2 is
approximately 75% except for an extra 114 amino acids in
the N-terminus of ACC2.13 The sequence identitiy of the rat
and human orthologs amounts to 97% (isoform 1) and 90%
(isoform 2), respectively.
a Reagents and conditions: (a) 4q, 1,10-carbonyldiimazole, NEt3,
formic acid; 4r-4s, R1COCl; 4t, 17 was used instead of 16, NH3,
carbonyldiimidazole, NEt3.
Our optimization strategy had two goals: Improving
chemical synthetic accessibility of the series while optimizing
the activity profile toward obtaining potent dual ACC1/2-
inhibitors in both human and rodent. We tackled both goals
by replacing the naphthyl ring of compound 3 with a phenyl
ring and inserting an ethyl linker between ether oxygen and
the cyclopentyl moiety, essentially clipping off part of the
naphthyl ring as schematically indicated in Figure 2.
The resulting compound 4a had a lower molecular weight
than 3 and proved to be a dual ACC1/2 inhibitor, albeit weak
on rat ACC. Because of its interesting in vitro profile, we
decided to explore this scaffold further by systematically varying
different parts of the molecule in subsequent SAR studies.
3.2. SAR Studies. Our structure-activity relationship
(SAR) efforts focused initially on identifying whether either
m- or p-substitution at the phenyl ring proved to be more
promising (Tables 1, 4a-f ). As could be seen from com-
pounds 4a-d, even small hydrophobic modifications in R2
with the m-substituted phenyl ring resulted in reduced
ACC1/2 inhibition, indicating a steep SAR for this pattern
and providing less synthetic options. We therefore focused
on the p-substituted phenyl rings by introducing an oxygen
atom in position X and shortening the chain length of the
hydrophobic ether moiety in 4-position, denoted R3 (4e, 4f).
Compound 4e cis/trans indicated that the stereochemical
sodium periodate and the resulting hydroxyl function elab-
orated to an acetamide function by known procedures to obtain
target molecule 4u, which was separated into its enantiomers
on chiral phase.
The synthesis of the target molecules 4v is shown in Scheme 7.
The aromatic ring of commercially available N-[(3-(4-hydroxy-
phenyl)-1-methylpropyl]-acetamide was hydrogenated at high
pressure (110 bar) with a rhodium catalyst to obtain the cyclo-
hexyl compound 20. The free hydroxyl group of 20 was reacted
with 2-isopropoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)pyridine under copper catalysis to obtain target
compound 4v, which was separated into its isomers on chiral
phase.
3. Results and Discussion
3.1. Discovery and Optimization Strategy. Here we report
the identification, structure-activity relationship, and lead
optimization of novel dioxane derivatives resulting in potent
dual ACC1/2 inhibitors. Compound 3 (Figure 1) was dis-
covered from high-throughput screening of our internal com-
pound collection and served as a starting point for optimization.
This compound shows an IC50 value of 630 nM for human
ACC2 (hACC2) and was inactive on human ACC1 (hACC1)

8682 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Keil et al.
Scheme 6a
a Reagents and conditions: (a) PPh3, DEAD, THF; (b) 3-bromo-2-methyl-propene, NaH, DMF; (c) OsO4, NaIO4, THF/H2O, 0 °C; (d) BnNHOH
then Pd/H2; (e) LiAlH4, THF; (f) Ac2O, NEt3, DCM; (g) HPLC on chiral phase
Scheme 7a
a Reagents and conditions: (a) 100 bar H2, 5% Rh(C), 100 °C, ethanol; (b) isopropoxy-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridine,
Cu(OAc)2, 1-butylimidazole, pyridine, toluene, 100 °C; (c) HPLC on chiral phase.
Figure 2. Chemical Optimization strategy of 3.
requirements for ACC2 inhibition at the 1,3-dioxane moiety
are trans-2,5-substitution.
Modulation of the electronic properties of the aromatic
ring by replacing the phenyl group with a pyridine (4g versus
4e-trans) proved to have a positive effect on hACC1/2 and
rACC1 inhibition with no effect on rACC2.
crucial, with nitrogen in position B leading to the most active
compound 4m-(S). The introduction of a second nitrogen
atom (4n-p) drastically reduced activity on all tested ACC
isoforms. Whether these effects were due to the modulation
of properties of the aromatic ring to form π-π interactions
or whether this effect was due to a potential hydrogen-
bonding capacity of the nitrogen atom in position B remains
unclear on the basis of this data.
Modifications of R1 were also evaluated by us (4q-4t).
A small hydrophobic group like methyl gives best activities
(4m-(S)) while a cylcopropyl ring instead is well tolerated
(4s). Hydrogen in R1 (4q) or the introduction of heteroatoms
(4r, 4t) apparently lead to the loss of hydrophobic interactions
and thus reduced activity.
The sensitivity of the ACC isoform activity profile to small
changes in the chemical structure (cf 4l-(S) and 4m-(S)) is
also highlighted by the examples for the cyclohexyl linker
4u-(S) and 4v-(S). Compared with 4m-(S), the exchange of
the phenyl linker with cyclohexyl (4v-(S)) improved hACC2
and rACC2 by a factor of 2 while it simultaneously dimin-
ished rACC1 activity by a factor of 7 and hACC1 activity
remained unchanged. The exchange of a methylene with an
oxygen atom deteriorated the activity profile (4u-(S)) con-
siderably.
In relation to the previously described inhibitors 1 and 2,
both 4m-(S) and 4v-(S) inhibited human ACC1/2 signifi-
cantly more. 4m-(S) was selected for further characterization
because of its ease of synthesis and higher activity toward rat
ACC1.
In parallel to the 1,3-dioxane linker (position A) shown in
Table 1, we also explored other linker groups, such as phenyl
and cyclohexyl, for which we give examples in Table 2. These
other linker moieties had the added bonus of enabling the
easier introduction of a methyl (position R2 in Table 2) into
the acetamide group, an effect that was described as bene-
ficial for ACC inhibition by Gu et al9 and that was verified by
us (4h and 4i). We also found that the (S)-enantiomer con-
stitutes the more active compound9 as can be seen from 4j-(S)
vs 4j-(R) and 4m-(S) vs 4m-(R). Interestingly, whether the
(R)-enantiomer retains weak activity on hACC1/2 (4m-(R))
or is inactive (4j-(R)) apparently depends on the size of the
hydrophobic ether group in R3; for the smaller isopropyl
group, more activity is retained.
Smaller hydrophobic ether groups in R3 seemed to be
beneficial especially for the inhibition of rACC2 (4j, 4i, 4l)
and resulted in a good overall ACC activity profile.
Since we learned from the SAR studies in the 1,3-dioxane
part of the series that the replacement of the aromatic phenyl
ring with a heterocycle could play an important role, we more
fully evaluated such an effect by investigating different nitrogen
containing six-membered heterocycles (4j and 4k, 4l-4p). It
appeared that the exact position of the nitrogen was absolutely

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8683
Table 1. Inhibitory Activities on Human and Rat ACC1 and ACC2a
a The IC50 data are an average of at least three measurements.
3.3. Biological Characterization. 4m-(S) possessed no ac-
tivity toward pyruvate carboxylase and PPARR, γ, and δ,
respectively, and was inactive in a panel of 30 unrelated
receptors, enzymes and transporters (data not shown). 4m-(S)
dose dependently increased fat oxidation in human liver cells,
indicating cellular activity (Figure 3).
indicating a stimulation of fat oxidation by the drug in vivo.
In relation to lean controls, obese female ZDF-rats have in-
creased plasma triglyceride levels and are a model of dyslipide-
mia. Following the administration of 4m-(S) for 3 days, plasma
triglycerides decreased significantly in these animals compared
to the treatment with vehicle only, indicating an improve-
ment in dyslipidemia (Figure 6).
4m-(S) showed good plasma exposures after intravenous
and oral administration (Tables 3 and 4) to female Wistar
rats and a high bioavailability (F = 98%). The exposure in
liver tissue after oral administration was high (Table 4,
Figure 4). The liver/plasma ratios based on AUC0-inf was
calculated to be 6. Even 6 h after the oral dose of 10 mg/kg
4m-(S), the concentration in liver tissue (approximately
5.5 μmol/kg) appeared to be still higher than that required to
stimulate fat oxidation in liver cells (compare Figures 3 and 4).
To characterize the activity of 4m-(S) in vivo, we applied the
compound at a dose of 50 mg/kg to rats followed by the admin-
istration of triglycerides and analyzed the respiration by
indirect calorimetry. During the dark phase, RQ values of all
groups became increased, as this is the animals’ main feeding
period (Figure 5). The lipid load resulted in a reduced RQ
relative to the control group. 4m-(S) decreased the RQ further,
4. Conclusion
Overall we have identified in 4m-(S) a compound with good
in vitro activity toward human and rat ACC isoforms. The
compound possessed favorable pharmacokinetic parameters,
stimulated fat oxidation, and reduced plasma triglyceride
levels after subchronic administration. This compound will
be useful for further validation of ACC inhibition as a poten-
tial approach for the treatment of aspects of the metabolic
syndrome.
5. Experimental Section
5.1. General Chemistry. Solvents and other reagents were used
as received without further purification. Column chromatography

8684 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Table 2. Inhibitory Activities on Human and Rat ACC1 and ACC2a
Keil et al.
a The IC50 data are an average of at least three measurements. Enzyme potency variance was <25% for all compounds
was carried out on Merck silica gel 60 (230-400 mesh). Reversed
phase high pressure chromatography was conducted on an Abimed
Gilson instrument using a LiChrospher 100 RP-18e (5 μm)
column from Merck. Thin-layer chromatography was carried

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8685
Table 3. Pharmacokinetic Parameters of 4m(-S) in Plasma of Rats after
Intravenous Dosing (3 mg/kg)
parametera
Coh (μmol/L)
1/2 (h)
plasma
5.2
1.5
5.6
1.6
2.6
t
AUC(0-inf) (μmol ꢀ h/L)
Cl (L/h/kg)
Vdss (L/kg)
a Coh, 4 m-(S) concentration immediately after dosing; AUC(0-inf)
area under the curve; t = 0 until infinity; Cl, clearance; Vdss, volume of
distribution at steady state
,
Table 4. Pharmacokinetic Parameters of 4m(-S) in plasma and Liver of
Rats after Oral Dosing (10 mg/kg)
parametera
plasma
liver
Cmax (μmol/L or/kg)
tmax (h)
3.50
2.0
21.8
2.0
Figure 4. Pharmacokinetics of 4m-(S) in rats. Plasma concentra-
tions of 4 m-(S) after 3 mg/kg iv administration (filled circles),
plasma (filled boxes), and liver concentrations (open circles) after oral
administration of 10 mg/kg 4m-(S); values are means ( SD (n = 3).
C
6h (μmol/L or/kg)
1.59
19
1.8
7.59
120
3.0
AUC(0-inf) (μmol x h/l or/kg)
t1/2 (h)
a Cmax, maximal 4m-(S) concentration; tmax, time of Cmax; C6h
,
concentration of 4 m-(S) 6 h after oral administration; AUC(0-inf), area
under the curve; t = 0 until infinity.
Figure 3. Stimulation of fat oxidation by 4m-(S). Human hepatocytes
were incubated in the presence of [3H]palmitate and the indicated
concentration of 4m-(S). After 4 h, the release of 3H2O was
measured. Results are expressed as the fold induction of fat oxida-
tion in relation to controls (0 μM). Values are means ( SEM (n = 4)
*p < 0.05 vs 0 μM 4m-(S).
Figure 5. Lowering of RQ by 4m-(S). Rats were administered
either vehicle or 4m-(S) (50 mg/kg), followed 2 h later by application
of the lipid load. Arrows indicate time points of applications, vehicle
group (filled boxes), lipid control group (filled circles), lipid/4m-(S)
group (open circles). Values are means ( SEM (n = 8); *p < 0.05 vs
vehicle group; # p < 0.05 vs lipid control group.
out on TLC aluminum sheets with silica gel 60F254 from Merck.
Nuclear magnetic resonance (1H NMR) spectra were recorded
in DMSO-d6 on a Bruker DRX 500 or in DMSO-d6 or in CDCl3
on an Avance II 400 or INCA 400. Chemical shifts are reported
as δ values from an internal tetramethylsilane standard. HPLC-
MS method A: Waters Aquity UPLC system was equipped with
a Waters Aquity autosampler, UPLC pump and a diode array.
The HPLC column used was a Waters Aquity BEH C-18 Column
(2.1 mm ꢀ 50 mm, 1.7 μM) at a flow rate of 0.9 mL/min and at
55 °C. The HPLC eluent was coupled with a 1:3 split to a Waters
Aquity SQD single quadrupole mass spectrometer with electro-
spray ionization. Spectra were scanned from 100 to 1300 amu in
positive and negative mode. The eluents were A, water with 0.1%
formic acid, and B, acetonitrile with 0.8% formic acid. Gradient
elutionfrom5%Bto95%Bover1.1minwithafinalhold at 95% B
of 0.6 min. Total run time was 2 min. HPLC-MS method B:
Agilent series 1100 system was equipped with a degasser G1322A,
bin pump G1312A, ALS G1313A, col comp G1316A, DAD
G1315A, and MSD G1946A. The HPLC column used was a
YMC J sphere H80 (20 mm ꢀ 2 mm, 4 μM) at a flow rate of
1 mL/min. The HPLC eluent was coupled with a mass spectro-
meter with electrospray ionization. Spectra were scanned from
110 to 1000 amu in positive and negative mode. The eluents were
A, water with 0.05% trifluoro acetic acid, and B, acetonitrile.
Gradient elution from 4% B to 95%B over 2 min with a final
Figure 6. Reduction of plasma triglycerides in obese female ZDF
rats by 4m-(S). Plasma triglyceride levels in lean controls and female
obese ZDF rats treated with either vehicle or 30 mg/kg 4m-(S) for 3
days. Data are means ( SEM (n = 8), *p < 0.05 vs obese vehicle
and obese 4m-(S); **p < 0.05 vs obese vehicle.
hold at 95% B of 0.4 min. Total run time was 2.5 min. Purity and
characterization of compounds were established by a combination
ofLC-MSandNMRanalyticaltechniques.Generally, thepurity of
each compound is >95%. For selected compounds, the exact

8686 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Keil et al.
purity is given in parentheses at the end of the description of its
synthesis in the Experimental Section.
6.80 (d, 2H), 6.78 (s, 1H), 5.22 (m, 1H), 4.28 (m, 1H), 2.58 (m, 1H),
2.51 (m, 1H), 2.35 (m, 1H), 1.99 (m, 1H), 1.40 (d, 3H), 1.28 (d, 6H).
LC-MS: method B, Rt = 1.26 min, m/z 431.2 (M þ Hþ).
Reference compounds 1 (N-{(S)-3-[2-(4-isopropoxy-phenoxy)-
thiazol-5-yl]-1-methyl-prop-2-ynyl}-acetamide) and 2 (anthracen-
9-yl-[(R)-3-(morpholine-4-carbonyl)-[1,40]bipiperidinyl-10-yl]-
methanone) were obtained using previously described synthetic
procedures.
(S)-3-[4-(6-Isopropoxy-pyridin-3-yloxy)-phenyl]-1-methyl-
propylamine (16). First, 10.3 g of 15 were dissolved in 400 mL of
ethanol and 10 mL of hydrazine hydrate were added. Then the
reaction mixture was heated under reflux for two hours. The
cooled mixture was filtered through a Celite pad and the filtrate
washed with saturated NaHCO3 solution and brine and dried
over MgSO4, and then the solvent was removed in vacuo to
obtain 7.28 g of 16. 1H NMR (500 MHz, DMSO-d6) δ 8.03 (d, 1H),
7.52 (dd, 1H), 7.26 (d, 2H), 6.95 (d, 2H), 6.84 (d, 2H), 5.24
(m, 1H), 2.79 (m, 1H), 2.60 (m, 2H), 1.52 (m, 2H), 1.29 (d, 6H),
0.99 (d, 3H). LC-MS: method B, Rt = 0.74 min, m/z 301.2
(M þ Hþ).
Procedure for the Synthesis of 4m. 5-(4-Bromo-phenoxy)-2-
chloro-pyridine (12). First 39.0 g of 6-chloro-pyridin-3-ol and
177.5 g of 1,4-dibromo-benzene and were dissolved in 500 mL
pyridine. Then 100 g of potassium carbonate and 85 g of freshly
powdered CuO were added and the mixture was heated under
reflux for 3 h. The mixture was cooled to 60 °C, 400 mL ethyl
acetate were added, and the mixture was stirred for 20 min at
60 °C. Stirring was stopped and the mixture was filtered over a
350 g Celite pad. The solid was rinsed three times with 500 mL of
hot ethyl acetate. The filtrate was concentrated in vacuo. The
residual pyridine was removed by azeotropic distillation with
3 ꢀ 500 mL of toluene (1,4-dibromo benzene was codistilled or
sublimated at low vacuum <50 mbar). The black residue was
suspended in 150 mL of toluene and filtered over 350 g of silica
gel with n-heptane: ethyl acetate = 1:1. The solvent was removed in
vacuo and the residue was purified by silica gel chromatography
with the eluents n-heptane:ethyl acetate = 15:1 to obtain 38.9 g
N-{(S)-3-[4-(6-Isopropoxy-pyridin-3-yloxy)-phenyl]-1-meth-
yl-propyl}-acetamide (4m(ent-S)). First, 1.96 g of 16 was dissolved
in 150 mL of ethyl acetate and 2.0 mL of triethylamine were added.
Upon cooling in an ice bath, 0.44 mL of acetic acid anhydride
were added. The ice bath was removed and the reaction mixture
stirred at room temperature for 3 h. Then the reaction mixture
was concentrated under reduced pressure and the resulting
residue taken up in water/ethyl acetate. The water phase was
separated and extracted five times with ethyl acetate. The com-
bined organic layers were dried over MgSO4, the solvent removed in
vacuo, and the residue purified by reverse phase HPLC to obtain
1
(45%) of 12. H NMR (500 MHz, DMSO-d6) δ 8.27 (d, 1H),
7.64-7.52 (m, 4H), 7.08 (d, 2H). LC-MS: method B, Rt =
1.73 min (purity 100%); m/z 283.9, 285.9 (M þ Hþ).
1
1.56 g (97%) of 4m(ent-S). H NMR (500 MHz, DMSO-d6)
5-(4-Bromo-phenoxy)-2-isopropoxy-pyridine (13). First, 13.67
g of sodium hydride (60% in mineral oil) were added in portions
to 28 mL of isopropanol in 150 mL of N-methylpyrrolidon at
room temperature with cooling (T > 25 °C). When the gas
evolution had stopped (after about 1 h), 38.9 g of 12 dissolved in
20 mL N-methylpyrrolidon, was added via a dropping funnel
and the mixture was heated to 85 °C for 3 h. After cooling to
room temperature, 2 M HCl was added with cooling (pH 5),
followed by 200 mL of saturated NH4Cl solution. The mixture
was extracted twice with 150 mL of n-heptane/ethyl acetate =
10:1. The combined organic layers were washed three times with
100 mL of half saturated NH4Cl solution, 100 mL of brine, dried
over MgSO4, and then concentrated in vacuo. The residue was
purified by silica gel chromatography with the eluent n-heptane:
ethyl acetate = 60:1 to obtain 35.6 g (85%) of 13 as a yellowish
oil. 1H NMR (500 MHz, DMSO-d6) δ 8.07 (d, 1H), 7.61-7.54
(m, 3H), 6.97 (d, 2H), 6.85 (d, 1H), 5.23 (m, 1H), 1.30 (d, 6H).
LC-MS: method A, Rt = 1.41 min (purity 97%); m/z 308.0,
310.0 (M þ Hþ).
δ 8.03 (d, 1H), 7.80 (d, 1H), 7.52 (dd, 1H), 7.24 (d, 2H), 6.94
(d, 2H), 6.84 (d, 1H), 5.25 (m, 1H), 3.76 (m, 1H), 2.54 (m, 2H),
1.81 (s, 3H), 1.62 (m, 2H), 1.29 (d, 6H), 1.03 (d, 3H). LC-MS:
method A, Rt = 1.68 min (purity 100%), m/z 343.2 (M þ Hþ);
enantiomeric purity 99.7%, Rt = 6.569 min, stationary phase
Chiralcel OJ-H/62 250 mm ꢀ 4.6 mm, eluent n-heptane:ethanol:
methanol = 10:1:1.
Using racemic 2-(but-3-yn-2-yl)isoindole-1,3-dione and fol-
lowing the procedure as in the preparation of 4m(ent-S) gave
racemic 4m. The racemic mixture of 4m was separated on chiral
phase (Chiracel OJ/H58, n-heptane:ethanol:methanol = 10:1:1)
to give 4m(ent-S) (enantiomeric purity 100%, Rt = 6.599 min)
and 4(ent-R) (enantiomeric purity 100%, Rt = 5.211 min)
5.2. Biology. In Vitro Assays. Protein Production. Recombi-
nant ACC enzymes were expressed in baculovirus-infected High
Five insect cells as N-terminal His-tagged fusion proteins and
purified by Ni-affinity and size exclusion chromatography. The
human ACC1 and ACC2 proteins consisted of the amino acids
2-2346 and 27-2458, respectively, rat ACC1 and ACC2 included
amino acids 1-2345 and 27-2455, respectively. Bovine pyruvate
carboxylase was obtained from Sigma.
Enzymatic and Cellular Assays. ACC enzymatic activity was
determined via an ATP consumption assay. The assays were
performed in 96-well plates and included 50 mM Tris-acetate,
16 mM NaHCO3, 0.9 mg/mL BSA, 25 μM ATP, 1.14 μM
β-mercaptoethanol, 4.3 mM magnesium acetate, 0.25 mM
acetyl CoA, 1% DMSO, and 200 ng of enzyme per well. The
ACC inhibitor was added at concentrations between 0 and 10 μM.
The reaction was started by the addition of enzyme (200 ng/well)
followed by incubation for 90 min at 37 °C. Unconverted ATP
was determined using the ATP monitoring reagent (Cambrex,
Charles City, USA) according to the manufacturer’s instruc-
tions. IC50 was calculated as the inhibitor concentration that
reduced the ATP turnover by 50% relative to a sample without
inhibitor.
2-{(S)-3-[4-(6-Isopropoxy-pyridin-3-yloxy)-phenyl]-1-methyl-
prop-2-ynyl}-isoindole-1,3-dione (14). First, 12.04 g of 13, 11.7 g
of (S)-2-(but-3-yn-2-yl)isoindole-1,3-dione, 372 mg copper-
(I)iodide, and 16.3 mL of triethylamine were dissolved in 50 mL
of dimethylformamide and degassed with argon for 20 min.
Then 1.37 g of bis(triphenylphosphine)palladium(II) chloride were
added and the reaction mixture was stirred at 70 °C for 5 h. The
cooled reaction mixture was evaporated in vacuo, and the
resulting residue was purified by silica gel chromatography with
the eluent n-heptane:ethyl acetate = 1:1 to obtain 9.2 g (55%) of
14. 1H NMR (500 MHz, DMSO-d6) δ 8.05 (d, 1H), 7.97-7.90
(m, 4H), 7.55 (dd, 1H), 7.43 (d, 2H), 6.94 (d, 2H), 6.82 (d, 1H),
5.38 (m, 1H), 5.21 (m, 1H), 1.72 (s, 3H), 1.30 (d, 6H). LC-MS:
method B, Rt = 1.51 min, m/z 427.1 (M þ Hþ).
2-{(S)-3-[4-(6-Isopropoxy-pyridin-3-yloxy)-phenyl]-1-methyl-
propyl}-isoindole-1,3-dione (15). First, 9.6 g of 14 were dissolved
in 500 mL of ethanol and 200 mL of terahydrofuran. Then 4.80 g
of palladium catalyst (10% palladium on charcoal) were added
and the reaction mixture stirred at 3 bar hydrogen pressure for
3 h at room temperature. The reaction was then filtered through
a Celite pad, the catalyst was rinsed three times with 300 mL of
ethyl acetate, and then the filtrate was evaporated in vacuo to
The rate of cellular ss-oxidation of [9,10(n)-3H]palmitic acid
was measured as 3H2O release.14 HepG2 were seeded in 24-well
dishes (200000 cells/well). After attachment, the cells were
washed once with PBS and subsequently incubated in the presence
of DMEM, 5 μM (15000 Bq/ml) 3H-palmitic acid, 0.2 mg/mL BSA,
500 μM L-carnitine, 0.1 (v/v) % DMSO in the presence of glucose
(25 mM) and the ACC inhibitor. β-oxidation was initiated by
the addition of 0.5 μCi [3H]palmitate to the incubation medium.
1
obtain 9.33 g (96%) of 15 as a yellow oil. H NMR (500 MHz,
DMSO-d6) δ 7.93 (d, 1H), 7.88 (m, 3H), 7.37 (dd, 1H), 7.14 (d, 2H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8687
References
After 4 h at 37 °C, portions of the cell supernatants were loaded
onto Sep-Pak cartridges (Waters, Eschborn, Germany). Unbound
radioactivity was measured by liquid scintillation counting.
5.3. In Vivo Experiments. Pharmacokinetic Studies. Pharma-
cokinetic parameters of 4m-(S) were determined in Wistar rats
(Harlan Winkelmann, Paderborn, Germany). After intravenous
bolus administration of 3 mg/kg of 4m-(S) in 5% solutol solution,
plasma samples were taken at eight time points over 24 h. After a
single oral administration of 10 mg/kg of 4m-(S) in 10% DMSO,
plasma and tissue samples were taken at eight different time
points over 24 h. Three animals were killed at each time point.
The respective concentrations of 4m-(S) in plasma and tissues
were determined by LC-MS/MS. Quantification was performed
with help of an internal standard.
Indirect Calorimetry. The setting consisted of 17 cages, of
which 16 were used for individual housing of the animals during
the study, and one cage served as the reference cage for correc-
tions of O2 and CO2 measurements. Female Wistar rats were
accustomed to the cages at least 12 h before the start of the
measurements (day -1). O2 consumption and CO2 production
were measured every 17 min/cage for 1 min (gas analyzers:
Magnos 16 and Uras 14; ABB, Frankfurt/Main, Germany) and
recorded on a computer. Animals were treated orally with either
50 mg/kg of 4m-(S) or vehicle. Two hours later, animals were
given orally either 10 mL/kg of 20% Lipofundin MCT (B. Braun,
Melsungen, Germany) corresponding to 2 g fat/kg or vehicle
(PBS). Values are expressed as the means of liters per hour. RQ
was calculated as the quotient of CO2 production/O2 consumption,
with the value of 1 representing 100% carbohydrate oxidation and
the value of 0.7 representing 100% fat oxidation.15
In Vivo Pharmacology. Female lean (Fa/?) and obese (fa/fa)
ZDF rats [ZDF-Leprfa/Crl] (Charles River (Sulzfeld, Germany)
were maintained two per cage on a constant 12-h light/dark
cycle (light on 06:00) under controlled temperature and humidity
conditions. Water was freely accessible. They were fed with SSNIFF
pellet chow (Soest, Germany) ad libitum. At the age of eight
weeks, eight rats were treated orally for three days with 30 mg/kg
4m-(S) dissolved in 5% ethanol, 5% solutol, 90% PBS; the
control rats received the vehicle alone. Four hours after the last
administration, rats were euthanized in deep isoflurane anesthe-
sia and blood was collected from the aorta after laparotomy for
determination of triglycerides on a Hitachi 912 using the GPD-PAP
kit (Roche, Mannheim, Germany).
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Supporting Information Available: Experimental and spectro-
scopic data of test compounds 4a-4l, 4n-4v and of intermediate
compounds 5-9, 10e, 10g, 18-20. This material is available free
of charge via the Internet at http://pubs.acs.org.