Synthesis and structure-activity relationship of small-molecule malonyl coenzyme A decarboxylase inhibitors

Article abstract of DOI:10.1021/jm050109n

The discovery and structure-activity relationship of first-generation small-molecule malonyl-CoA decarboxylase (MCD; CoA = coenzyme A) inhibitors are reported. We demonstrated that MCD inhibitors increased malonyl-CoA concentration in the isolated working

Full text of DOI:10.1021/jm050109n

J. Med. Chem. 2006, 49, 1517-1525
1517
Synthesis and Structure-Activity Relationship of Small-Molecule Malonyl Coenzyme A
Decarboxylase Inhibitors
Jie-Fei Cheng,*,† Mi Chen,† David Wallace,† Souvothy Tith,† Masayuki Haramura,† Bin Liu,† Chi Ching Mak,†
Thomas Arrhenius,† Sean Reily,‡ Steven Brown,‡ Vicki Thorn,‡ Charles Harmon,‡ Rick Barr,§ Jason R. B. Dyck,§
Gary D. Lopaschuk,§ and Alex M. Nadzan†
Departments of Chemistry and DiscoVery Biology, Chugai Pharma USA, LLC., 6225 Nancy Ridge DriVe, San Diego, California 92121, and
Metabolic Modulator Research Ltd. (MMRL), 2020 Research Transition Facility, UniVersity of Alberta, Edmonton, Alberta, Canada
ReceiVed February 3, 2005
The discovery and structure-activity relationship of first-generation small-molecule malonyl-CoA decar-
boxylase (MCD; CoA ) coenzyme A) inhibitors are reported. We demonstrated that MCD inhibitors increased
malonyl-CoA concentration in the isolated working rat hearts. Malonyl-CoA is a potent, endogenous, and
allosteric inhibitor of carnitine palmitoyltransferase-I (CPT-I), a key enzyme for mitochondrial fatty acid
oxidation. As a result of the increase in malonyl-CoA levels, fatty acid oxidation rates were decreased and
the glucose oxidation rates were significantly increased. Demonstration of in vivo efficacy of methyl 5-(N-
(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)morpholine-4-carboxamido)pentanoate (6u) in a pig
ischemia model indicated that MCD inhibitors may be useful for treating ischemic heart diseases.
Introduction
of CPT-I, which is more sensitive than the liver form of CPT-1
(IC50 of 0.03 vs 2.5 µM).6
The enzyme malonyl-CoA decarboxylase (MCD, EC 4.1.1.9;
CoA ) coenzyme A) catalyzes the conversion of malonyl-CoA
to acetyl-CoA and thereby regulates malonyl-CoA levels. MCD
was first purified from the uropygial glands of waterfowl and
subsequently from a number of mammalians, plants and
bacteria.1 Identification of patients with MCD deficiency led
to the cloning of a human MCD gene that is highly homologous
to the goose and rat genes.2 A single human MCD mRNA is
observed by Northern blot analysis. The highest mRNA expres-
sion levels are found in muscle and heart tissues, followed by
liver, kidney and pancreas, with detectable amounts found in
many other tissues. Recent studies indicate that MCD exists in
cytosolic, mitochondrial, and peroxisomal compartments.1h,2b,3,4
Malonyl-CoA is a potent endogenous inhibitor of carnitine
palmitoyltransferase-I (CPT-I), an enzyme essential for the
mitochondrial metabolism of long-chain fatty acids. CPT-I is
the rate-limiting enzyme in fatty acid oxidation and catalyzes
the formation of acylcarnitine, which is transported from the
cytosol across the mitochondrial membranes by acylcarnitine
translocase. Inside the mitochondria, the long-chain fatty acids
are transferred back to their CoA compounds by a complemen-
tary enzyme, CPT-II, where acyl-CoA enters the â-oxidation
pathway generating acetyl-CoA. In the liver, high levels of
acetyl-CoA lead to elevated malonyl-CoA levels, which inhibit
CPT-I, thereby preventing fatty acid oxidation and favoring fatty
acid synthesis. Conversely, low malonyl-CoA levels favor fatty
acid oxidation by allowing the transport of long-chain fatty acids
into the mitochondria. Therefore, malonyl-CoA is a central
metabolite that plays a key role in balancing fatty acid synthesis
and fatty acid oxidation.5 In skeletal and cardiac muscle, where
fatty acid synthesis rates are low, we and others hypothesized
that the role of malonyl-CoA and MCD in these tissues is to
regulate fatty acid metabolism via inhibition of the muscle form
In the heart, fatty acid oxidation is a major source of energy
and fatty acid uptake and metabolism in the heart down-regulate
glucose metabolism. The regulation of intermediary metabolism
by serum levels of fatty acid and glucose comprises the
glucose-fatty acid cycle (“Randle cycle”).7 Under ischemic
conditions, limited oxygen supply reduces both fatty acid and
glucose oxidation and reduces the amount of ATP produced by
oxidative phosphorylation in the cardiac tissues. Glycolysis
increases in an attempt to maintain ATP levels, although a
consequence of this is a buildup of lactate and a drop in
intracellular pH. Energy is spent maintaining ion homeostasis,
and myocyte cell death occurs as a result of abnormally low
ATP levels and disrupted cellular osmolarity. The problem is
complicated by a decrease in malonyl-CoA levels in the heart
due to an activation of AMP-activated protein kinase (AMPK),
which phosphylates and thus inactivates acetyl-CoA carboxylase
(ACC). This increases CPT-I activity, and fatty acid oxidation
is favored over glucose oxidation.
A number of clinical and experimental studies indicate that
shifting energy metabolism in the heart toward glucose oxidation
is an effective approach to decreasing the symptoms associated
with myocardial ischemia.8,9 Certain antianginal drugs such as
ranolazine (N-(2,6-dimethylphenyl)-2-[4-[2-hydroxy-3-(2-meth-
oxyphenoxy)propyl]piperazin-1-yl]ethanamide) inhibit fatty acid
â-oxidation and stimulate glucose oxidation.10 Trimetazidine (1-
[(2,3,4-trimethoxyphenyl)methyl]piperazine) has been shown to
specifically inhibit the long-chain 3-ketoactyl CoA thiolase (3-
KAT), an essential step in fatty acid oxidation.11 Inhibiting
CPT-I activity through increasing malonyl-CoA levels with
MCD inhibitors would result in a novel and perhaps a much
safer method, compared to other known small-molecule CPT-I
inhibitors, to the prophylaxis and treatment of ischemic heart
diseases (Figure 1).12
Two metabolic complications commonly associated with
diabetes are hepatic overproduction of ketone bodies and organ
toxicity associated with sustained elevated levels of glucose.
Malonyl-CoA inhibition of CPT-I is an important regulatory
mechanism that controls the rate of fatty acid oxidation during
* To whom correspondence should be addressed. Phone: 858-622-7029.
Fax: 858-558-9383. E-mail: jcheng@trlusa.com. Present Address: Tanabe
Research Lab USA, Inc., 4540 Towne Centre Court, San Diego, CA 92121.
† Department of Chemistry, Chugai Pharma USA, LLC.
‡ Department of Discovery Biology, Chugai Pharma USA, LLC.
§ University of Alberta.
10.1021/jm050109n CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/11/2006

1518 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Cheng et al.
Figure 1. Malonyl-CoA regulation of energy metabolism.
Scheme 1. Synthesis of p-[2-Hydroxy-(1,1,1,3,3,3-
hexafluoro-2-propyl)]aniline Derivativesa
Figure 2. Initial HTS hit.
the onset of the hypoinsulinemic-hyperglucagonemic state.
Stimulation of glucose oxidation rate via inhibition of fatty acid
oxidation may have the potential to regulate blood-glucose levels
and ameliorate some symptoms of type II diabetes. Several
irreversible and reversible CPT-I inhibitors that have been
evaluated for their ability to control blood glucose levels were
found to be invariably hypoglycemic.13 However, stimulation
of fatty acid oxidation through knockout of the ACC2 (ACCâ)14
gene or overexpression of the hepatic MCD15 protein was found
to improve insulin sensitivity, indicating the complex nature of
the metabolic pathway.
On the basis of the rationale illustrated above, we speculated
that increasing the malonyl-CoA level in tissues has potential
use in heart disease and diabetes therapy. We therefore
established a high-throughput screening assay and used it to
screen an in-house compound library employing either MCD
from rat hearts or MBP-fused human MCD protein (MBP )
maltose-binding protein).16 Using this paradigm, we discovered
a number of small-molecule MCD inhibitors, one of them
possessing the phenylhexafluoroisopropanol17 moiety (1, Figure
2). The hexafluoroisopropanol moiety of compound 1 was found
to be essential for the high in vitro MCD inhibitory activity
(vida infra). The structure-activity relationship (SAR) studies
on this initial hit led to the identification of a series of MCD
inhibitors that not only exhibit potent in vitro MCD inhibitory
activities and favorable pharmacokinetic properties but also
exhibit excellent efficacy in regulating energy metabolism in
isolated working rat hearts.
a Reagents and conditions: (a) alkyl halide, Et3N, DCM; (b) CF3COCF3‚
6H2O, molecular sieves, toluene, 60-80 °C; (c) R2COCl, poly(4-vinylpy-
ridine), DCM; or R2COOH, EDAC, DMAP, DMF; (d) R2NCO, poly(4-
vinylpyridine), DCM; (e) COCl2, DIPEA, toluene and then R2R3NH, 80-
100 °C.
alkylation of commercially available 2-(4-aminophenyl)-
1,1,1,3,3,3-hexafluoropropan-2-ol (2) or through Friedel-Craft
type alkylation of substituted aniline 3 with hexafluoroacetone
hydrate in the presence of molecular sieves or an acid such as
p-toluenesulfonic acid.18 The reaction went smoothly, giving
rise predominantly to the para-substituted product (4) in
excellent yields. Reaction of 4 with acyl chloride, anhydride,
or carboxylic acid afforded the amide derivatives 5. Similarly,
the reaction of 4 with isocyanates provided trisubstituted ureas.
The tetrasubstituted ureas were prepared in a stepwise fashion.
Intermediate 4 was first treated with phosgene/triphosgene to
provide the corresponding carbamoyl chlorides, which upon
treatment with 2 equiv of a secondary amine in toluene at 80
°C afforded the desired tetrasubstituted ureas 6 in modest to
good yields.
Chemistry
The synthesis of amide and urea derivatives of 1 is outlined
in Scheme 1. The key intermediate 4 was prepared either through
The meta-substituted isomer of the initial hit 1 was prepared
in a different fashion, as shown in Scheme 2. According to the

Malonyl-CoA Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1519
Table 1. Amide-based MCD Inhibitors
Scheme 2. Synthesis of m-[2-Hydroxy-(1,1,1,3,3,3-
hexafluoro-2-propyl)]aniline Derivativea
compd
R1
R2
IC50 (nM)a
1
-Me
-H
PhOCH2-
PhOCH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
PhCH2CH2-
i-Pr-
930
6626
4337
773
175
199
79
105
376
580
150
NAb
178
1015
394
41
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
-H
-Me
-Et
-i-Pr
-n-Bu
a Reagents and conditions: (a) PhOCH2COCl, Et3N, DCM, room temp;
(b) MeI, NaH, THF, 0 °C to room temp.
-CH2COOMe
-CH2COOH
-Bn
Scheme 3. Synthesis of p-[2-Hydroxy-(1,1,1,3,3,3-
hexafluoro-2-propyl)]benzoic Acid Amidesa
-cyclohexyl
-H
-Me
i-Pr-
-Me
3-Py-
-Me
4-Py-
-Et
i-Pr-
-n-Pr
i-Pr-
28
20
-n-Bu
i-Pr-
-CH2CH2NMe2
-CH2CH2OH
-CH2COOMe
-CH2COOH
-(CH2)4COOMe
-(CH2)4COOH
-(CH2)4-(5-1H-tetrazole)
-(CH2)4CN
-Et
i-Pr-
1147
106
34
627
26
8
8
20
37
i-Pr-
5t
i-Pr-
a Reagents and conditions: (a) R1R2NH, CDI, THF.
5u
5v
5w
5x
5y
5z
5aa
i-Pr
literature procedure,19 2-hydroxy-1,1,1,3,3,3-hexafluoropropyl-
benzene 7 was nitrated with fuming nitric acid in sulfuric acid
to provide the corresponding m-nitro derivative, which was
further reduced with iron to the desired aniline compound 8.
Acylation with phenoxyacetyl chloride followed by methylation
with MeI in the presence of NaH afforded the desired regio-
isomer 9.
i-Pr
i-Pr
i-Pr
i-Pr
-p-CNPh
-4-Py
-Et
95
a Data are reported as the mean of n g 3 determinations. SD was
generally (20% of the average. b Not active at 10 µM.
Scheme 3 illustrates the synthesis of a series of 4-(2,2,2-
trifluoro-1-hydroxy-1-trifluoromethylethyl)benzoic acid amide
derivatives 11. To distinguish these amides with the ones
mentioned above, we designated 4-(2,2,2-trifluoro-1-hydroxy-
1-trifluoromethylethyl)benzoic acid amide as reverse amides.
These reverse amides were prepared efficiently by conventional
peptide coupling methods from commercially available 4-(2,2,2-
trifluoro-1-hydroxy-1-trifluoromethylethyl)benzoic acid 10. Most
preparations did not require a chromatographic purification.
in vitro enzyme assay compared to their nonalkylated counter-
parts. Compounds 5a, 5b, and 5k inhibited MCD enzyme at
high micromolar concentrations. A methyl group at the nitrogen
atom increased potency by 6-80 times. It is well-known that
the stable conformation of an aniline amide in solution, as well
as in the crystal form, is highly dependent on the nature of the
substituents on the amide nitrogen atom, with the cis conforma-
tion being preferred in an N-alkylanilides and with the trans
conformation being preferred in a nonsubstituted acetanilide.20
It is therefore assumed that the active conformation of initial
hit 1, or similar amide compounds, is a cis conformation. The
amide carbonyl group may also play a role as a hydrogen bond
acceptor, since the corresponding tertiary amines or ether
compounds did not exhibit significant MCD inhibitory activities
(data not shown). The chain length of the alkyl group on the
nitrogen atom greatly affects the potency as well (5c-e).
N-Alkyl-N-(1,1,1,3,3,3-hexafluoro-2-hydroxypropylphenyl)-
amides generally are highly hydrophobic. To improve properties
of these hydrophobic compounds, especially the improvement
of solubility, a terminal carboxylic acid or hydroxyl group was
introduced into some compounds. This did not adversely affect
the ability of these compounds to inhibit MCD. However, a
basic group such as the N,N-dimethylamino group at the terminal
position significantly decreased the potency (5r, IC50 ) 1147
nM). Another significant observation in the amide series was
the dramatic increase in potency through introduction of
substitution at the R-position of the amide group. When the
substituent on the nitrogen atom was larger than a methyl group,
Results and Discussion
All compounds prepared were tested initially for their ability
to inhibit rat heart MCD (data not shown) as well as the soluble
form of recombinant MBP fusion protein (MBP-hMCD) as
described elsewhere and in the Experimental Section.16 An initial
attempt to replace the hexafluoroisopropanol moiety was
unsuccessful. Removal of one of two trifluoromethyl groups or
the hydroxyl group resulted in a significant or complete loss of
MCD inhibitory activity (data not shown). Replacement of the
hydroxyl group with methoxy also led to an inactive compound.
The regioisomer of initial hit 1 (e.g., compound 9) is also
inactive. The lead optimization was therefore performed in terms
of in vitro enzyme inhibitory activity, in vitro pharmaceutical
properties (solubility, liver microsome stability, and Caco-2
permeability), and ex vivo efficacy in isolated working rat hearts
using the 2-hydroxy-1,1,1,3,3,3-hexafluoro-2-propylphenyl scaf-
fold as a core structure.
As shown in Table 1, N-alkyl-N-(1,1,1,3,3,3-hexafluoro-2-
hydroxylpropylphenyl)amides exhibited a higher potency in the

1520 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Table 2. Urea-Based MCD Inhibitors
Cheng et al.
Table 3. In Vitro Data for Reversed Amides
compd
R1
R2
IC50 (µM)a
IC50
11a
11b
11c
11d
11e
11f
11g
11h
11i
PhCH2CH2
PhCH2CH2
Bn
H
2.65
1.99
1.00
1.45
0.85
1.14
0.98
0.29
0.07
compd
R1
R2
R3
(nM)a
Me
Me
Me
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
-H
-Me
-H
-Et
-Et
-Me
-Et
-n-Pr
-n-Bu
-n-pentyl
-n-hexyl
-n-heptyl
-octyl
Ph-
Ph-
-H
-H
NAb
1564
NAb
59
OCH3
-CH2CH2OCH2CH2-
-Me
-CH2CH2CH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2CH2CH2CH2-
NCCH2CH2
Me-
Et-
-(CH2)4OH 60
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
321
74
34
30
19
31
73
269
43
166
80
Et
i-Bu
i-Bu
a Data are reported as the mean of n g 3 determinations. SD was
generally (20% of the average.
-CH2CH2Ph
-CH2CH2OH
-CH2COOMe
-CH2CH2COOMe
-CH2CH2COOH
99
247
75
-CH2CH2CH2COOMe -CH2CH2OCH2CH2-
6t
6u
6v
-(CH2)3CN
-(CH2)4COOMe
-(CH2)4COOH
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
40
42
14
a Data are reported as the mean of n g 3 determinations. SD was
generally (20% of the average. b Not active at 10 µM.
Figure 3. Malonyl-CoA content in the isolated working rat hearts (n
) 8) in the absence and presence of MCD inhibitors: (1) 50 µM; (5o,
6e, 6u) 20 µM.
the effect was much greater. Taken together, our data suggest
that a small and branched aliphatic group such as an isopropyl
group on the amide side and a 4-carbon to 5-carbon substitution
on the nitrogen atom are preferred. 5-{Isobutyryl-[4-(2,2,2-
trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl]amino}-
pentanoic acid (5w) and its tetrazole analogue (5x) showed low
nanomolar IC50 values (8 nM) against MCD.
metabolized quickly in the in vitro liver microsome stability
test (data not shown).
Measurement of Malonyl-CoA Content. According to our
hypothesis illustrated in Figure 1, isolated working rat hearts
perfused with MCD inhibitors would demonstrate an elevation
of malonyl-CoA level,21 a decrease in fatty acid oxidation rates,
and an increase in glucose oxidation rates. To test the hypothesis,
a number of MCD inhibitors were selected on the basis of a
number of in vitro properties, especially in vitro potency,
solubility, and Caco-2 permeability, and were tested for their
ability to regulate energy metabolism in the isolated working
rat hearts.
The initial high-throughput screeing (HTS) hit compound 1,
with an IC50 of 0.93 µM, showed a marginal increase in
malonyl-CoA levels in the isolated working rat heart tissue at
a test concentration of 50 µM. However, amide compound 5o
and urea compound 6e, with low-nanomolar in vitro MCD
inhibitory activity, showed statistically significant increases in
malonyl-CoA concentration at 20 µM (Figure 3). The most
dramatic increase in malonyl-CoA was observed for the urea
derivative 6u, which demonstrated a 6-fold increase in MCA
levels compared to the control condition (0.05% DMSO).
Glucose Oxidation in Isolated Working Heart. A set of
MCD inhibitors was selected, and their effects on glucose
oxidation rates in the isolated working rat hearts were mea-
sured.22 These compounds exhibited 3-60 times more potent
MCD inhibitory activities than the initial hit compound 1.
Compound 1 at 50 µM caused a 100% increase in glucose
oxidation rate compared to the DMSO control. An MCD
inhibitor with low potency and low permeability such as 5h
A series of N,N′-disubstituted, trisubstituted and tetrasubsti-
tuted ureas based on the p-(1,1,1,3,3,3-hexafluoro-2-hydroxy)-
propylaniline scaffold were prepared according to Scheme 1.
As expected, tetrasubstituted ureas were generally better MCD
inhibitors compared to the corresponding trisubstituted or
disubstituted ones. N,N′-Disubstituted ureas (e.g., 6a, Table 2)
were essentially inactive against the MCD enzyme. Depending
on the substitution pattern, trisubstituted ureas showed either
poor inhibitory activity (6b) or no activity at all (6c). Most
tetrasubstituted ureas, except diaryl ones, generally exhibited
good inhibitory activity. As shown in Table 2 and seen in the
amide series, linear aliphatic chains, with up to five carbons,
on the nitrogen atom (6f-m) were preferable. Small aliphatic
substituents were also preferred, with morpholinylurea deriva-
tives (6f-v) consistently demonstrating great solubility and
metabolic stability enhancement. Terminal carboxylic acid or
other hydrophilic groups were well-tolerated, with compound
6v exhibiting the highest potency (IC50 ) 14 nM).
Reverse amides 11 (Table 3) inhibited MCD enzyme activity
in the in vitro MCD enzyme assay, but the potency was far
lower than the potency of amide compounds mentioned above.
A notable exception was those compounds with two small-
branched aliphatic chains, which exhibited a submicromolar or
nanomolar inhibition (e.g., compound 11i, IC50 ) 70 nM).
Besides the low potency, these compounds also tended to be

Malonyl-CoA Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1521
Table 4. MCD Inhibitors and Glucose Oxidation Rate in Working Rat
Hearts
activity in the isolated working rat heart, compound 6u (CBM-
300864) was chosen to be the first MCD inhibitor for proof-
of-concept studies in an in vivo pig demand-induced ischemia
model. By use of a pig model of demand-induced ischemia, 6u
significantly increased glucose oxidation rates and reduced
lactate production compared with vehicle-treated hearts, which
was accompanied by a significant increase in cardiac work
compared with controls. These data show that MCD inhibitors,
which increase myocardial malonyl-CoA levels,12,23 decrease
fatty acid oxidation and accelerate glucose oxidation in both
ex vivo rat hearts and in vivo pig hearts. This switch in energy
substrate preference improves cardiac function during and after
ischemia, suggesting that pharmacological inhibition of MCD
may be a novel approach to treating ischemic heart diseases.
solubility
(PBS,
human
liver
Caco-2
rat liver
IC50
(µM)
pH 7.4) (×10-6 microsome microsome
compd
(µM)a
cm/s)b
(%)c
(%)c
GOXd
1
5aa
5h
5n
5o
5s
5z
6e
6g
6u
6v
ranolazine
trimeta-
zidine
0.931
0.095
0.317
0.394
0.040
0.106
0.041
0.061
0.074
0.041
0.014
46
59.8
28.6
54.4
0.4
100
86
65
88
60
76
66
7
100
97
88
98
89
85
93
51
87
95
99
2.0e
2.47
1.26
2.58
2.05
1.90
2.81
2.42
2.14
3.86
2.90
1.77f
2.01g
190.6
136.2
19.3
199.5
8.7
187.3
189.3
166.9
205.6
41.0
69.1
52.0
38.7
49.4
68.2
32.9
1.7
9
1
108
Experimental Section
Chemical Methods. All commercial chemicals and solvents were
reagent grade and used without further purification unless otherwise
specified. The following solvents and reagents have been abbrevi-
ated: dichloromethane (DCM), tetrahydrofuran (THF), dimethyl
sulfoxide (DMSO), chloroform (CHCl3), and dimethylformamide
(DMF). All reactions except those in aqueous media were carried
out under an argon atmosphere or with the use of standard
techniques for the exclusion of moisture. Reactions were monitored
by thin-layer chromatography on 0.25 mm silica gel plates (Merck
Kieselgel 60 and 60 F254) and visualized with UV light, iodine
vapors, or 5% phosphomolybdic acid in EtOH. Standard workup
procedures were used for all the reactions, and solvents were dried
over MgSO4 before evaporation. The final compounds were
typically purified either by TLC on silica gel or by reversed-phase
HPLC.
a Solubility was measured using the HPLC method at pH 7.4 in PBS
containing 1% DMSO. b Caco-2 permeability was determined at Cerep using
standard protocol. c Percentage indicates the remaining of the test compound
(5 µM) after 1 h of incubation at 37 °C and 1 mg of protein/mL of
microsome. d Glucose oxidation rates (GOX) were calculated as a ratio of
test compound (20 µM) to DMSO (0.05%) control. See Experimental
Section for details. e 50 µM. f See ref 10a. g See ref 11.
tended to give a less profound effect on glucose oxidation rates.
On the other hand, compound 6v potently stimulated glucose
oxidation even though it has low permeability. The high glucose
oxidation rate induced by 6v may be ascribed to its high potency.
The effect of solubility on the glucose oxidation is less obvious
partly because of the presence of a large amount of bovine serum
albumin (BSA) in the perfusate. As can be seen in Table 4,
most of MCD inhibitors showed better efficacy in stimulating
glucose oxidation compared to ranolazine or trimetazidine.
Analytical purity was assessed by RP-HPLC using a Varian
Polaris C18-A column (0.46 mm × 100 mm) equipped with a diode
array spectrometer. The mobile phase employed 0.1% formic acid
or 0.1% trifluoroacetic acid with acetonitrile at a flow rate of 2.0
mL/min.
Conclusions
1H and 13C NMR data were recorded on a Varian Unity-400
Plus instrument. Chemical shifts were reported in parts per million
(ppm) and autocalibrated to the deuterated solvent reference peaks.
Coupling constants were reported in units of hertz (Hz). Mass
spectra were acquired in either the positive or negative ion mode
under electrospray ionization (ESI) on a Finnigan LCQ Iontrap mass
spectrometer.
On the basis of the initial hit obtained from HTS on an in-
house compound library, a series of small-molecule MCD
inhibitors with the N-alkyl(hexafluoroisopropyl)aniline scaffold
were synthesized and optimized. N-Alkyl(hexafluoroisopropyl)-
aniline amides and urea derivatives displayed potent in vitro
inhibitory activity against the soluble form MBP-fused MCD
protein. These compounds are also equally active against native
MCD (data not shown).16 The most potent inhibition was
achieved by compounds 5w and 6v in either the amide or urea
series with an IC50 of 8 and 14 nM, respectively. These potent
MCD inhibitors generally contain small aliphatic chains on the
nitrogen atom of R,R-disubstituted carboxyl groups or N,N-
disubstituted ureido functions. The small aliphatic groups are
also preferred at the R-position of the carboxyl group or on the
nitrogen atom of the ureido group.
Selected compounds were further profiled in term of malonyl-
CoA concentration and glucose oxidation rates in the isolated
working rat heart model. Compounds 5o, 6e, and 6u were
demonstrated to increase malonyl-CoA content in the rat heart
tissues compared to the control. The initial hit compound 1 was
less efficacious compared to other more potent analogues. The
ability to stimulate glucose oxidation rates in the isolated
working rat hearts was also confirmed for a large number of
compounds prepared on the basis of the initial hit 1 (cf. Table
4). A number of compounds showed 2- to 3-fold increases in
glucose oxidation rate at 10-20 µM, which are comparable to
or more potent than known metabolic modulators such as
ranolazine and trimetazidine.
In Vitro MCD Inhibitory Activity Assay. The decarboxylase
activity of MCD was measured spectrophotometrically by monitor-
ing acetyl-CoA formation using the malate dehydrogenase (MD)/
citrate synthase (CS) coupling system. The conversion of acetyl-
CoA from malonyl-CoA was assayed using a modified protocol as
previously described by Kim and Kolattukudy.24 The establishment
of the kinetic equilibrium between malate/NAD and oxaloacetate/
NADH was catalyzed by malate dehydrogenase. The enzymatic
reaction product of MCD, acetyl-CoA, shifted the equilibrium by
condensing with oxaloacetate in the presence of citrate synthase,
which resulted in a continuous generation of NADH from NAD.
The accumulation of NADH was continuously followed by
monitoring the increase of fluorescence emission at 465 nm on a
fluorescence plate reader. The fluorescence plate reader was
calibrated using authentic acetyl-CoA from Sigma. For a typical
96-well plate assay, the increase in the fluorescence emission (λex
) 340 nm and λem ) 465 nm for NADH) in each well was used to
calculate the initial velocity of human recombinant MCD. Each 50
µL assay contained 10 mM phosphate buffered saline (Sigma), pH
7.4, 0.05% Tween-20, 25 mM K2HPO4-KH2PO4 (Sigma), 2 mM
malate (Sigma), 2 mM NAD (Boehringer Mannheim), 0.786 units
of MD (Roche Chemicals), 0.028 unit of CS (Roche Chemicals),
5-10 nM hMCD, and varying amounts of MCA substrate. Assays
were initiated by the addition of MCA, and the rates were corrected
for the background rate determined in the absence of hMCD.
Isolated Working Rat Heart Assay. Isolated working hearts
from male Sprague-Dawley rats (300-350 g) are subjected to a
On the basis of the overall profile, including in vitro potency,
solubility, pharmacokinetics (data not shown), and ex vivo

1522 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Cheng et al.
60-min aerobic perfusion period.11 The working hearts were
perfused (95% O2 and 5% CO2) with a modified Krebs-Henseleit
solution containing 5 mM glucose, 100 µU/mL insulin, 3% fatty
acid-free BSA, 2.5 mM free Ca2+, and 0.4-1.2 mM palmitate. The
test compound was added 5 min before the perfusion period. DMSO
(0.05%) is used as a control.
δ 0.95 (m, 3H), 1.32 (m, 2H), 1.52 (d, 1H), 1.70 (t, 4H), 2.15 (t,
2H), 2.81 (t, 2H), 4.58 (m, 1H), 6.85 (d, 2H), 6.93 (d, 2H), 7.18
(m, 3H), 7.75 (d, 2H); ESIMS m/z 474 (M + H).
N-[4-(2,2,2-Trifluoro-1-hydroxy-1-trifluoromethylethyl)phe-
1
nyl]isobutyramide (5k). H NMR (DMSO) δ 1.07 (d, 6H), 2.57
(m, 1H), 7.55 (d, 2H), 7.70 (d, 2H), 8.52 (s, 1H), 10.0 (s, 1H);
ESIMS m/z 330.1 (M + H).
Measurement of Glucose Oxidation Rates. Samples were taken
at 10-min intervals throughout the 60-min perfusion for measure-
ments of glucose oxidation. Glucose oxidation rates were deter-
mined by the quantitative collection of 14CO2 produced by hearts
perfused with buffer containing [U-14C]glucose. Gaseous 14CO2 was
trapped in hyamine hydroxide and sampled at 10-min intervals.
After the perfusion, the 14CO2 from the perfusate was subsequently
released by injecting 1 mL of perfusate into a sealed test tube
containing 1 mL of 9 N H2SO4. The tube was sealed with a rubber
stopper attached to a scintillation vial containing a piece of filter
paper saturated with 300 µL of hyamine hydroxide. The scintillation
vials with filter papers were then removed, and Ecolite scintillation
fluid was added. Samples were counted by standard procedures as
described above. Average rates of glucose oxidation for each phase
of perfusion are expressed as nmol/min per gram of dry weight.
General Synthesis of Amide Compounds 5. 2-(p-N-Alkylphe-
nyl)hexafluoroisopropanol (0.6 mmol) and poly(4-vinylpyridine)
(204.5 mg, 1.8 mmol) are mixed in CH2Cl2 (3 mL). Acyl chloride
(0.6 mmol) is added to the suspension, and the reaction mixture is
stirred at room temperature overnight. The polymer is removed by
filtration through a pad of Celite, and the organic solvent is removed
under reduced pressure. The residue is purified by preparative TLC
(hexane/EtOAc, 7:3) to afford the desired compound.
N-Methyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
1
ethyl)phenyl]isobutyramide (5l). H NMR (CD3OD) δ 1.07 (d,
6H), 2.52 (m, 1H), 3.21 (s, 3H), 7.40 (d, 2H), 7.82 (d, 2H); ESIMS
m/z 344 (M + H).
N-Methyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]nicotinamide (5m). 1H NMR (CD3OD) δ 3.5 (s, 3H),
7.3 (m,3H), 7.6 (d, 2H), 7.7 (d, 1H), 8.4 (m, 2H); ESIMS m/z 379
(M + H).
N-Methyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]isonicotinamide (5n). 1H NMR (CD3OD) δ 3.45 (s,
3H), 7.30 (m, 4H), 7.62 (d, 2H), 8.41 (d, 2H); ESIMS m/z 379 (M
+ H).
N-Ethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyleth-
1
yl)phenyl]isobutyramide (5o). H NMR (CDCl3) δ 1.0 (d, 6H),
1.2 (t, 3H), 2.4 (m, 1H), 3.7 (q, 2H), 738 (d, 2H), 7.82 (d, 2H);
ESIMS m/z 358 (M + H).
N-Propyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]isobutyramide (5p). 1H NMR (CDCl3) δ 0.82 (t, 3H),
1.0 (d, 6H), 1.5 (m, 2H), 2.4 (m, 1H), 3.6 (t, 2H), 7.20 (d, 2H),
7.80 (d, 2H); ESIMS m/z 372 (M + H).
N-Butyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyleth-
1
yl)phenyl]isobutyramide (5q). H NMR (CDCl3) δ 0.82 (t, 3H),
2-Phenoxy-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]acetamide (5a). 1H NMR (DMSO-d6) δ 4.7 (s. 2H),
6.96 (m, 3H), 7.28 (t, 2H), 7.6 (d, 2H), 7.74 (d, 2H); ESIMS m/z
394 (M + H).
1.0 (d, 6H), 1.3 (m, 2H), 1.45 (m, 2H), 2.4 (m, 1H), 3.7 (t, 2H),
7.38 (d, 2H), 7.82 (d, 2H); ESIMS m/z 386 (M + H).
N-(2-Dimethylaminoethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]isobutyramide (5r). 1H NMR (CD3-
OD) δ 1.2 (d, 6H), 2.31 (s, 6H), 2.61 (m, 1H), 2.72 (t, 2H), 3.68
(t, 2H), 7.52 (d, 2H), 7.76 (d, 2H); ESIMS m/z 401(M + H).
N-(2-Hydroxyethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
3-Phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]propionamide (5b). 1H NMR (DMSO-d6) δ 2.62 (t,
2H), 2.88 (t, 2H), 7.15 (m, 2H), 7.25 (m, 3H), 7.55 (d, 2H), 7.66
(d, 2H); ES/MS m/z 392 (M + H).
1
romethylethyl)phenyl]isobutyramide (5s). H NMR (CDCl3) δ
N-Methyl-3-phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1.0 (d, 6H), 2.42 (m, 1H), 3.62 (t, 2H), 3.8 (t, 2H), 7.42 (d, 2H),
7.82 (d, 2H); ESIMS m/z 372 (M - H).
1
romethylethyl)phenyl]propionamide (5c). H NMR (CDCl3) δ
2.68 (t, 2H), 3.03 (t, 2H), 3.21 (s, 3H), 7.01 (m,2H), 7.25 (m, 3H),
7.66 (d, 2H), 7.76 (d, 2H); ESIMS m/z 406 (M + H).
{Isobutyryl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]amino}acetic Acid Methyl Ester (5t). 1H NMR
(CDCl3) δ 1.02 (d, 6H), 2.52 (m, 1H), 3.72 (s, 3H), 4.32 (s, 2H),
7.42 (d, 2H), 7.8 (d, 2H); ESIMS m/z 400 (M - H).
N-Ethyl-3-phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1
romethylethyl)phenyl]propionamide (5d). H NMR (CDCl3) δ
1.02 (t, 3H), 2.3 (t, 2H), 2.82 (t, 1H), 6.98 (m, 4H), 7.20 (m, 3H),
7.82 (d, 2H); ESIMS m/z 420 (M + H).
{Isobutyryl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
1
ethyl)phenyl]amino}acetic Acid (5u). H NMR (CDCl3) δ 1.02
N-Isopropyl-3-phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-tri-
fluoromethylethyl)phenyl]propionamide (5e). 1H NMR (CDCl3)
δ 1.00 (d, 6H), 2.2 (t, 3H), 2.81 (t, 2H), 4.98 (m, 1H), 6.88 (d,
2H), 6.95 (d, 2H), 7.19 (m, 3H), 7.78 (d, 2H); ESIMS m/z 434.2-
(M + H).
(d, 6H), 2.58 (m, 1H), 4.32 (s, 2H), 7.45 (d, 2H), 7.8 (d, 2H); ESIMS
m/z 386 (M - H).
5-{Isobutyryl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
ethyl)phenyl]amino}pentanoic Acid Methyl Ester (5v). 1H NMR
(CDCl3) δ 1.0 (d, 6H), 1.5 (m, 2H), 1.6 (m, 2H), 2.31 (t, 2H), 2.4
(m, 1H), 3.6 (s, 3H), 3.7 (t, 2H), 7.45 (d, 2H), 7.82 (d, 2H); ESIMS
m/z 442 (M - H).
N-Butyl-3-phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1
romethylethyl)phenyl]propionamide (5f). H NMR (CDCl3) δ
0.82 (t, 3H), 1.23 (m, 2H), 1.4 (m, 2H), 2.3 (t, 2H), 2.8 (t, 2H),
3.61 (t, 2H), 6.98 (d, 2H), 7.02 (d, 2Η), 7.2 (m, 3H), 7.7 (d, 2H);
ESIMS m/z 448 (M + H).
5-{Isobutyryl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
1
ethyl)phenyl]amino}pentanoic Acid (5w). H NMR (CDCl3) δ
1.0 (d, 6H), 1.58 (m, 4H), 2.31 (t, 2H), 2.4 (m, 1H), 3.7 (t, 2H),
7.40 (d, 2H), 7.82 (d, 2H); ESIMS m/z 428 (M - H).
N-[4-(1H-Tetrazol-5-yl)butyl]-N-[4-(2,2,2-trifluoro-1-hydroxy-
1-trifluoromethylethyl)phenyl]isobutyramide (5x). 1H NMR
(CDCl3) δ 1.0 (d, 6H), 1.52 (m, 2H), 1.78 (m, 2H), 2.4 (m, 1H),
2.91 (t, 2H), 3.72 (t, 2H), 7.38 (d, 2H), 7.82 (d, 2H); ESIMS m/z
452 (M - H).
{(3-Phenylpropionyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-tri-
fluoromethylethyl)phenyl]amino}acetic Acid Methyl Ester (5g).
1H NMR (CDCl3) δ 2.42 (t, 2H), 2.84(t, 2H), 3.71 (s, 3H), 4.3 (s,
2H), 7.0 (d, 2H), 7.2 (m, 3H), 7.26 (d, 2H), 7.76 (d, 2H); ESIMS
m/z 464 (M + H).
{(3-Phenylpropionyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-tri-
fluoromethylethyl)phenyl]amino}acetic Acid (5h). 1H NMR
(CDCl3) δ 2.42 (t, 2H), 2.84 (t, 2H), 4.32 (s, 2H), 7.0 (d, 2H), 7.18
(m, 3H), 7.22 (d, 2H), 7.76 (d, 2H); ESIMS m/z 450 (M + H).
N-Benzyl-3-phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
N-(4-Cyanobutyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1
romethylethyl)phenyl]isobutyramide (5y). H NMR (CDCl3) δ
1.0 (d, 6H), 1.62 (m, 4H), 2.42 (m, 3H), 3.72 (t, 2H), 7.40 (d, 2H),
7.82 (d, 2H); ESIMS m/z 409 (M - H).
1
romethylethyl)phenyl]propionamide (5i). H NMR (CDCl3) δ
4-Cyano-N-ethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
romethylethyl)phenyl]benzamide (5z). 1H NMR (CDCl3) δ 1.20
(s, 3H), 3.71 (q, 2H); ESIMS m/z 417 (M + H).
2.37 (t, 2H), 2.91 (t, 2H), 4.80 (s, 2H), 6.80 (d, 2H), 7.01 (d, 2H),
7.08 (m, 2H), 7.18 (d, 2H), 7.23 (m, 4H), 7.68 (d, 2H); ESIMS
m/z 482 (M + H).
N-Cyclohexyl-3-phenyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-tri-
N-Ethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyleth-
1
fluoromethylethyl)phenyl]propionamide (5j). 1H NMR (CDCl3)
yl)phenyl]isonicotinamide (5aa). H NMR (CD3OD) δ 1.20 (s,

Malonyl-CoA Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1523
3H), 3.71 (q, 2H), 7.30 (m. 4H), 7.62 (d, 2H), 8.40 (d, 2H); ESIMS
m/z 393 (M + H).
(CDCl3) δ 2.90 (t, 2H), 3.18 (t, 4H), 3.42 (t, 4H), 3.81 (t, 2H),
6.91 (d, 2H), 7.18 (m, 3H), 7.23 (d, 2H), 7.64 (d, 2H); 13C NMR
(CDCl3) δ 35.9, 46.9, 54.9, 67.1, 119.4, 122.3, 123.9, 124.6, 125.0,
125.3, 126.4, 127.4, 128.9, 129.4, 130.0, 140.3, 148.3, 160.9;
ESIMS m/z 475 (M - H).
1-Phenyl-3-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyleth-
1
yl)phenyl]urea (6a). H NMR (CD3OD) δ 7.02 (t, 1H), 7.29 (t,
2H), 7.42 (d, 2H), 7.53 (d, 2H), 7.63 (d, 2H); ESIMS m/z 379 (M
+ H).
Morpholine-4-carboxylic Acid (2-Hydroxyethyl)-[4-(2,2,2-
trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl]amide (6o).
1H NMR (CDCl3) δ 3.18 (t, 4H), 3.42 (t, 4H), 3.6 (t, 2H), 3.82 (t,
2H), 7.18 (d, 2H), 7.72 (d, 2H); ESIMS m/z 415 (M - H).
{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]amino}acetic Acid Methyl Ester
(6p). 1H NMR (CD3OD) δ 3.18 (t, 4H), 3.42 (t, 4H), 3.68 (s, 3H),
4.38 (s, 2H), 7.1 (d, 2H), 7.64 (d, 2H); ESIMS m/z 443 (M - H).
3-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]amino}propionic Acid Methyl Ester
1-Methyl-3-phenyl-1-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1
romethylethyl)phenyl]urea (6b). H NMR (CD3OD) δ 2.60 (s,
3H), 6.95 (t, 1H), 7.4 (m, 4H), 7.72 (d, 2H), 7.56 (d, 2H); ESIMS
m/z 393 (M + H).
Morpholine-4-carboxylic Acid [4-(2,2,2-Trifluoro-1-hydroxy-
1-trifluoromethylethyl)phenyl]amide (6c). 1H NMR (CD3OD) δ
3.35 (t, 4H), 3.56 (t, 4H), 7.28 (d, 2H), 7.48 (d, 2H); 13C NMR
(CD3OD) δ 45.1, 67.4, 115.6, 119.6, 120.9, 122.6, 125.3, 126.3,
128.2, 141.6, 156.9; ESIMS m/z 371 (M - H).
1
1-Ethyl-3,3-dimethyl-1-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
(6q). H NMR (CDCl3) δ 2.62 (t, 2H), 3.18 (t, 4H), 3.42 (t, 4H),
1
romethylethyl)phenyl]urea (6d). H NMR (CD3OD) δ 1.13 (t,
3.58 (s, 3H), 3.91 (t, 2H), 7.05 (d, 2H), 7.62 (d, 2H); ESIMS m/z
457 (M - H).
3H), 2.70 (s, 6H), 3.20 (q, 2H), 7.17 (d, 2H), 7.57 (d, 2H); ESIMS
m/z 359 (M + H).
3-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]amino}propionic Acid (6r). 1H NMR
(CDCl3) δ 2.52 (t, 2H), 3.18 (t, 4H), 3.42 (t, 4H), 3.82 (t, 2H),
7.15 (d, 2H), 7.62 (d, 2H); ESIMS m/z 445 (M + H).
4-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]amino}butyric Acid Methyl Ester
(6s). 1H NMR (CDCl3) δ 0.82 (m, 2H), 2.32 (t, 2H), 3.18 (t, 4H),
3.42 (t, 4H), 3.58 (s, 3H), 3.62 (t, 2H), 7.05 (d, 2H), 7.62 (d, 2H);
ESIMS m/z 471 (M - H).
1,3-Diethyl-3-(4-hydroxybutyl)-1-[4-(2,2,2-trifluoro-1-hydroxy-
1
1-trifluoromethylethyl)phenyl]urea (6e). H NMR (CD3OD) δ
1.13 (t, 3H), 1.26 (t, 3H), 1.48 (m, 2H), 1.55 (m, 2H), 3.10-3.25
(m, 6H), 3.50 (t, 2H), 7.15 (d, 2H), 7.60 (d, 2H); ESIMS m/z 431
(M + H).
Morpholine-4-carboxylic Acid Methyl-[4-(2,2,2-trifluoro-1-
hydroxy-1-trifluoromethylethyl)phenyl]amide (6f). 1H NMR
(CD3OD) δ 2.78 (t, 3H), 3.18 (t, 4H), 3.42 (t, 4H), 7.12 (d, 2H),
7.64 (d, 2H); ESIMS m/z 387 (M + H).
Morpholine-4-carboxylic Acid Ethyl-[4-(2,2,2-trifluoro-1-hy-
droxy-1-trifluoromethylethyl)phenyl]amide (6g). 1H NMR (CDCl3)
δ 1.12 (t, 3Η), 3.18 (t, 4H), 3.42 (t, 4H), 3.65 (q, 2H), 7.12 (d,
2H), 7.64 (d, 2H); ESIMS m/z 401(M + H).
Morpholine-4-carboxylic Acid Propyl-[4-(2,2,2-trifluoro-1-
hydroxy-1-trifluoromethylethyl)phenyl]amide (6h). 1H NMR
(CDCl3) δ 0.82 (t, 3H), 1.60 (m, 2H), 3.18 (t, 4H), 3.42 (t, 4H),
3.61 (t, 2H), 7.12 (d, 2H), 7.64 (d, 2H); ESIMS m/z 413 (M - H).
Morpholine-4-carboxylic Acid Butyl-[4-(2,2,2-trifluoro-1-hy-
droxy-1-trifluoromethylethyl)phenyl]amide (6i). 1H NMR (CD3-
OD) δ 0.82 (t, 3H), 1.30 (m, 2H), 1.57 (m, 2H), 3.18 (t, 4H), 3.42
(t, 4H), 3.61 (t, 2H), 7.1 (d, 2H), 7.64 (d, 2H); ES/MS m/z 427 (M
- H).
Morpholine-4-carboxylic Acid Pentyl-[4-(2,2,2-trifluoro-1-
hydroxy-1-trifluoromethylethyl)phenyl]amide (6j). 1H NMR
(CDCl3) δ 0.82 (t, 3H), 1.25 (m. 4H), 1.59 (m, 2H), 3.18 (t, 4H),
3.42 (t, 4H), 3.61 (t, 2H), 7.10 (d, 2H), 7.53 (d, 2H); 13C NMR
(CDCl3) δ 15.1, 23.4, 29.4, 30.3, 47.1, 52.7, 67.3, 122.3, 124.6,
125.3, 126.6, 128.1, 129.1, 148.1, 161.3; ESIMS m/z 443 (M +
H).
Morpholine-4-carboxylic Acid Hexyl-[4-(2,2,2-trifluoro-1-
hydroxy-1-trifluoromethylethyl)phenyl]amide (6k). 1H NMR
(CDCl3) δ 0.82 (t, 3H), 1.25 (m, 6H), 1.59 (m, 2H), 3.18 (t, 4H),
3.42 (t, 4H), 3.61 (t, 2H), 7.12 (d, 2H), 7.64 (d, 2H); 13C NMR
(CDCl3) δ 14.6, 23.4, 27.6, 29.6, 32.3, 46.9, 52.6, 67.1, 119.4, 122.1,
124.1, 124.9, 126.4, 128.9, 148.0, 160.6; ESIMS m/z 457 (M +
H).
Morpholine-4-carboxylic Acid Heptyl-[4-(2,2,2-trifluoro-1-
hydroxy-1-trifluoromethylethyl)phenyl]amide (6l). 1H NMR
(CDCl3) δ 0.82 (t, 3H), 1.25 (m, 8H), 1.59 (m, 2H), 3.18 (t, 4H),
3.42 (t, 4H), 3.61 (t, 2H), 7.12 (d, 2H), 7.64 (d, 2H); 13C NMR
(CDCl3) δ 14.9, 23.4, 28.0, 29.9, 30.0, 32.7, 47.0, 52.7, 67.1, 119.4,
122.3, 124.3, 125.1, 126.4, 128.0, 129.0, 148.0, 161.1; ESIMS m/z
471 (M + H).
Morpholine-4-carboxylic Acid (3-Cyanopropyl)-[4-(2,2,2-tri-
fluoro-1-hydroxy-1-trifluoromethylethyl)phenyl]amide (6t). 1H
NMR (CD3OD) δ 1.92 (m, 2H), 2.40 (t, 2H), 3.18 (t, 4H), 3.42 (t,
4H), 3.74 (t, 2H), 7.15 (d, 2H), 7.72 (d, 2H); 13C NMR (CD3OD)
δ 15.7, 24.9, 46.7, 50.9, 67.0, 119.4, 120.1, 122.1, 124.9, 125.0,
127.9, 128.0, 129.7, 146.9, 161.0; ESIMS m/z 438 (M - H).
5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]amino}pentanoic Acid Methyl Es-
1
ter (6u). H NMR (CDCl3) δ 1.60 (m, 4H), 2.31 (t, 2H), 3.15 (t,
4H), 3.42 (t, 4H), 3.6 (s, 3H), 3.61 (t, 2H), 7.08 (d, 2H), 7.64 (d,
2H); 13C NMR (CDCl3) δ 23.3, 29.0, 32.7, 47.0, 52.1, 52.4, 67.1,
119.4, 123.3, 124.6, 125.1, 127.1, 128.0, 129.1, 147.6, 161.1, 174.9;
ESIMS m/z 485 (M - H).
5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]amino}pentanoic Acid (6v). 1H
NMR (CDCl3) δ 1.59 (m, 4H), 2.28 (m, 2H), 3.15 (t, 4H), 3.42 (t,
4H), 3.61 (t, 2H), 7.08 (d, 2H), 7.64 (d, 2H); 13C NMR (CDCl3) δ
23.4, 29.1, 32.7, 46.9, 52.2, 67.0, 119.4, 123.3, 124.6, 125.1, 127.1,
128.0, 129.1, 147.4, 161.0, 172.1; ESIMS m/z 471 (M - H).
N-Methyl-2-phenoxy-N-[3-(2,2,2-trifluoro-1-hydroxy-1-tri-
fluoromethylethyl)phenyl]acetamide (9). A mixture of 2-(3-
aminophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (210 mg, 0.81
mmol) and triethylamine (160 µL, 0.89 mmol) in 3 mL of anhydrous
dichloromethane was added dropwise to a solution of phenoxyacetyl
chloride (145 mg, 0.85 mmol) in 3 mL of anhydrous dichlo-
romethane. The light-yellow solution was then stirred at room
temperature overnight. The reaction mixture was then washed with
water and then brine. After drying over magnesium sulfate, the
organic phase was concentrated in vacuo. The resulting white solid
was purified by preparative TLC (5% methanol, 95% chloroform)
1
to yield 260 mg (81.8%). H NMR (CDCl3) δ 4.51 (s, 2H), 6.94
(d, 2H), 7.00 (t, J ) 7.6 Hz, 1H), 7.28 (t, 2H), 7.36 (t, J ) 8 Hz,
1H), 7.46 (d, J ) 7.6 Hz, 1H), 7.67 (d, 1H), 7.85 (s, 1H); ESIMS
m/z 394 (M + H).
Sodium hydride (30 mg, 0.693 mmol) was suspended in dry THF
under an argon atmosphere. 2-Phenoxy-N-[3-(2,2,2-trifluoro-1-
hydroxy-1-trifluoromethylethyl)phenyl]acetamide (181.7 mg, 0.462
mmol) was added at 0 °C. The reaction mixture was then stirred at
room temperature for 45 min. Methyl iodide (45 µL, 0.693 mmol)
was added, and the light-yellow solution was stirred at room
temperature overnight. The reaction mixture was then diluted with
diethyl ether. The organic layer was separated, washed with water
and brine, and dried over magnesium sulfate. The solvent was
concentrated in vacuo, and the resulting colorless oil was purified
Morpholine-4-carboxylic Acid Octyl-[4-(2,2,2-trifluoro-1-hy-
droxy-1-trifluoromethylethyl)phenyl]amide (6m). 1H NMR (CD3-
OD) δ 0.82 (t, 3H), 1.25 (m, 10H), 1.59 (m, 2H), 3.18 (t, 4H),
3.42 (t, 4H), 3.61 (t, 2H), 7.12 (d, 2H), 7.64 (d, 2H); 13C NMR
(CDCl3) δ 14.9, 23.6, 28.0, 29.7, 30.2, 30.3, 32.7, 47.0, 52.7, 67.1,
116.9, 122.1, 124.3, 125.0, 126.1, 128.9, 148.0, 161.0; ESIMS m/z
483(M - H).
Morpholine-4-carboxylic Acid Phenethyl-[4-(2,2,2-trifluoro-
1
1-hydroxy-1-trifluoromethylethyl)phenyl]amide (6n). H NMR

1524 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Cheng et al.
1989, 264, 3500-3505. (h) Dyck, J. R.; Barr, A. J.; Barr, R. L.;
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Fatty Acid Oxidation. Am. J. Physiol. 1998, 275 (6, Part 2), H2122-
H2129.
by preparative TLC (5% methanol, 95% chloroform) to yield the
title compound (150 mg). H NMR (CDCl3) δ 3.22 (s, 3H), 4.51
(s, 2H), 6.6 (d, J ) 7.6 Hz, 2H), 6.83 (t, J ) 7.2 Hz, 1H), 7.12 (t,
J ) 8 Hz, 2H), 7.26 (d, 1H), 7.44 (t, J ) 8.4 Hz, 1H), 7.59 (s, 1H),
7.68 (d, J ) 7.6 Hz, 1H); ESIMS m/z 408 (M + H).
1
(2) Gao, J.; Waber, L.; Bennett, M. J.; Gibson, K. M.; Cohen, J. C.
Cloning and Mutational Analysis of Human Malonyl-coenzyme A
Decarboxylase. J. Lipid Res. 1999, 40, 178. (b) Sacksteder, K. A.;
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Fatty Acid Oxidation in Soleus Muscle. Biochem. J. 1998, 334, 233-
241. (b) Sambandam, N.; Steinmetz, M.; Chu, A.; Altarejos, J. Y.;
Dyck, J. R.; Lopaschuk, G. D. Malonyl-CoA Decarboxylase (MCD)
Is Differentially Regulated in Subcellular Compartments by 5′AMP-
Activated Protein Kinase (AMPK). Studies Using H9c2 Cells
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N-Phenethyl-4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-
1
ethyl)benzamide (11a). H NMR (CD3OD) δ 2.90 (t, 2H), 3.59
(t, 2H), 7.22 (m, 5H), 7.81 (m, 4H); ESIMS m/z 392 (M + H).
N-Methyl-N-phenethyl-4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1
romethylethyl)benzamide (11b). H NMR (CD3OD) δ 2.77 (t,
2H), 2.98 (t, 1H), 3.10 (s, 3H), 3.47 (t, 1H), 3.75 (t, 1H), 6.88 (t,
1H), 7.02 (d, 1H), 7.18 (d, 2H), 7.30 (d, 2H), 7.38 (d, 1H), 7.66
(d, 1H), 7.79 (d, 1H); ESIMS m/z 406 (M + H).
N-Benzyl-N-methyl-4-(2,2,2-trifluoro-1-hydroxy-1-trifluoro-
methylethyl)benzamide (11c). 1H NMR (CD3OD) δ 2.94 (s, 3H),
4.50 (s, 2H), 7.15 (m, 1H), 7.3 (m, 2H), 7.55 (m, 3H), 7.81 (m,
3H); ESIMS m/z 392 (M + H).
N-Methoxy-N-methyl-4-(2,2,2-trifluoro-1-hydroxy-1-trifluo-
1
romethylethyl)benzamide (11d). H NMR (CD3OD) δ 3.37 (s,
3H), 3.52 (s, 3H), 7.71 (m, 4H); ESIMS m/z 332 (M + H).
Pyrrolidin-1-yl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluorometh-
1
ylethyl)phenyl]methanone (11e). H NMR (CD3OD) δ 1.9 (m,
2H), 1.98 (m, 2H), 3.43 (t, 2H), 3.58 (t, 2H), 7.61 (d, 2H), 7.81 (d,
2H); ESIMS m/z 342 (M + H).
Morpholin-4-yl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluorometh-
1
ylethyl)phenyl]methanone (11f). H NMR (CD3OD) δ 3.4-3.8
(m, 8H), 7.52 (d, 2H), 7.81 (d, 2H); ESIMS m/z 358 (M + H).
Piperidin-1-yl-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluorometh-
1
ylethyl)phenyl]methanone (11g). H NMR (CD3OD) δ 1.7 (m,
6H), 3.38 (m, 2H), 3.7 (m, 2H), 7.46 (d, 2H), 7.81 (d, 2H); ESIMS
m/z 356 (M + H).
(7) (a) Randle, P. J. Garland, P. B.; Hales, C. N.; Newsholme, E. A.
The Glucose-Fatty Acid Cycle. Its Role in Insulin Sensitivity and
the Metabolic Disturbances of Diabetes Mellitus. Lancet 1963, 1,
785-789. (b) Randle, P. J. Regulatory Interactions between Lipids
and Carbohydrates: The Glucose Fatty Acid Cycle after 35 Years.
Diabetes Metab ReV. 1998, 14 (4), 263-283.
N-(2-Cyanoethyl)-N-ethyl-4-(2,2,2-trifluoro-1-hydroxy-1-tri-
fluoromethylethyl)benzamide (11h). H NMR (CD3OD) δ 1.15
(t, 3H), 2.88 (t, 2H), 3.38 (t, 2H), 3.78 (t, 2H), 7.46 (d, 2H), 7.81
1
(d, 2H); ESIMS m/z 369 (M + H).
N,N-Diisobutyl-4-(2,2,2-trifluoro-1-hydroxy-1-trifluorometh-
ylethyl)benzamide (11i). 1H NMR (CD3OD) δ 0.75 (d, 6H), 0.96
(d, 6H), 1.86 (m, 1H), 2.15 (m, 1H), 3.14 (d, 2H), 3.38 (d, 2H),
7.45 (d, 2H), 7.81 (d, 2H); ESIMS m/z 398 (M - H).
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Acknowledgment. We thank Prof. William Stanley at Case
Western Reserve University for helping with Figure 1. The
authors also thank Dr. Peter Simms, Cynthia Jefferies, and Aixia
Sun for their help with HPLC and MS analysis.
(10) (a) McCormack, J. G.; Barr, R. L.; Wolff, A. A.; Lopaschuk, G. D.
Ranolazine Stimulates Glucose Oxidation in Normoxic, Ischemic,
and Reperfused Ischemic Rat Hearts. Circulation 1996, 93, 135-
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zine: A Novel Metabolic Modulator for the Treatment of Angina.
Genet. Pharmacol. 1998, 30, 639-645. (c) Pepine, C. J.; Wolff, A.
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in Chronic Stable Angina Pectoris That Is Responsive to Conventional
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84, 46-50.
Supporting Information Available: HPLC and 1H NMR data
for listed compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
(11) Kantor, P. F.; Lucien, A.; Kozak, R.; Lopaschuk, G. D. The
Antianginal Drug Trimetazidine Shifts Cardiac Energy Metabolism
from Fatty Acid Oxidation to Glucose Oxidation by Inhibiting
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(12) Dyck, J. D.; Cheng, J.-F.; Stanley, W.; Barr, R.; Chandler, M. P.;
Brown, S.; Wallace, D.; Arrhenius, T.; Harmon, C.; Yang, G.;
Nadzan, A.; Lopsaschuk, G. D. Malonyl-CoA Decarboxylase Inhibi-
tion Protects the Ischemic Heart by Inhibiting Fatty Acid Oxidation
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