Design and synthesis of heterocyclic malonyl-CoA decarboxylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.10.020

We have previously reported the discovery of small molecule inhibitors of malonyl-CoA decarboxylase (MCD) as novel metabolic modulators, which inhibited fatty acid oxidation and consequently increased the glucose oxidation rates in the isolated working ra

Full text of DOI:10.1016/j.bmcl.2005.10.020

Bioorganic & Medicinal Chemistry Letters 16 (2006) 695–700
Design and synthesis of heterocyclic malonyl-CoA
decarboxylase inhibitors
Jie-Fei Cheng,* Mi Chen, Bin Liu, Zheng Hou, Thomas Arrhenius and Alex M. Nadzan
Department of Chemistry, Chugai Pharma USA, LLC, 6275 Nancy Ridge Dr., San Diego, CA 92121, USA
Received 20 August 2005; revised 6 October 2005; accepted 7 October 2005
Available online 27 October 2005
Abstract—We have previously reported the discovery of small molecule inhibitors of malonyl-CoA decarboxylase (MCD) as novel
metabolic modulators, which inhibited fatty acid oxidation and consequently increased the glucose oxidation rates in the isolated
working rat hearts. MCD inhibitors were also shown to improve cardiac efficiency in rat and pig demand-induced ischemic models
through the mechanism-based modulation of energy metabolism. Herein, we describe the design and synthesis of a series of novel
heterocyclic MCD inhibitors with a preference for substituted imidazole and isoxazole.
Ó 2005 Elsevier Ltd. All rights reserved.
The enzyme malonyl-CoA decarboxylase (MCD, EC
4.1.1.9) 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 mammals
and bacteria.1a–g Identification of patients with MCD
deficiency led to the cloning of a human MCD gene that
is highly homologous to goose and rat.1h–k A single
human MCD mRNA is observed by Northern blot
analysis. The highest mRNA expression levels are found
in muscle and heart tissues, followed by liver, kidney,
and pancreas, with detectable amounts in all other
tissues examined. Recent work indicates that MCD
exists in both cytosolic and mitochondrial forms.1l
ies of small molecule MCD inhibitors and demonstrated
their ability in regulation of cardiac energy metabolism
in rat and pig hearts.4a,4c
Through high throughput screen on the Chugai com-
pound archive, we identified an initial hit compound
1a (CBP-33502) with moderate MCD inhibitory activi-
ties (IC50, 0.93 lM). Early structure–activity relation-
ship studies revealed that N-alkyl group played a key
role in the MCD inhibition. For example, compound
1b, which lacks a N-methyl group, showed significant
lower MCD inhibitory activity (IC50, 6.6 lM). It is well
known that the stable conformation of an aniline amide
(anilide) in solution as well as in crystal form is highly
dependent on the nature of substituents on the amide
nitrogen atom; with the cis-conformation being pre-
ferred in N-alkylanilides and trans-conformation in
non-substituted acetanilides.5 It is therefore reasonably
assumed that the active conformation of the initial hit
compound 1a is cis (as shown in 1a). Based on the
hypothesis that the conformation difference between 1a
and 1b resulted in the activity difference, we designed a
series of new MCD inhibitors 1c with a heterocycle moi-
ety in an attempt to mimic the amide conformation of 1a
(Fig. 1). Conformation overlap of 1a with substituted
imidazole or isoxazole is shown in Figure 2. 1,2-Disub-
stituted imidazoles and 3,4-disubstituted isoxazoles
seem to best fit the original conformation of 1a.
Malonyl-CoA is not only a key intermediate for fatty
acid synthesis, but also an endogenous inhibitor of car-
nitine palmytoyltransferase-1 (CPT-I)2 and a key meta-
bolic sensor.3 Through the regulation of malonyl-CoA
and acetyl-CoA levels in the system and therefore the
fatty acid synthesis and fatty acid oxidation process,
MCD plays a key role in the balance of energy intake
and expenditure. Despite the important processes
MCD is involved, the physiological role of MCD is
not clear. Recently, we developed for the first time a ser-
Keywords: Malonyl-CoA; Acetyl-CoA; Malonyl-CoA decarboxylase;
MCD.
*
Corresponding author at present address: Tanabe Research Labo-
ratories USA, Inc. 4540 Towne Centre Court, San Diego, CA 92121,
USA. Tel.: +1 858 622 7029; fax: +1 858 558 9383; e-mail:
jcheng@trlusa.com
Two different strategies have been employed to synthe-
size 3,4,5-trisubstituted isoxazole-based MCD inhibi-
tors (Schemes 1 and 2). Bromination of commercially
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.020

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J.-F. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 695–700
O
A
D
CF3
OH
CF3
O
R1
R2
N
B
O
O
N
H
F3C
CF3
OH
F3C
CF3
OH
1b
1a
(IC50:0.93 µM)
1c
A,B, D: N, O or C
(IC50: 6.6µM)
Figure 1. Five-membered heterocycles as bioisosteres of N-alkylanilide.
of this benzylbromide 3 with KCN in aqueous DMSO
under heating condition6 yielded the corresponding ben-
zonitrile 4, which upon treatment with LHMDS fol-
lowed by reaction of the resultant anion intermediate
with an ester provided the b-cyanoketone intermediate
5. 5-Aminoisoxazole compounds 6 were obtained
through cyclization of b-cyanoketones (5) with hydrox-
ylamine.7 Amide formation was accomplished by treat-
ing 6 with acyl chlorides or anhydrides.
An alternative method employing Suzuki reaction to
prepare similar isoxazole derivatives is illustrated in
Scheme 2. The commercially available 2-(4-bromo-
phenyl)-1,1,1,3,3,3-hexafluoro-propan-2-ol 8 was con-
verted into its corresponding boronate 9, which coupled
with 4-bromoisoxazole afforded the 3,4,5-trisubstituted
isoxazole derivatives 10, 13 or 12 directly. Compounds
10 and 13 possess protected ester or amino functional
groups at the 3-position of the isoxazole ring. Hydroly-
sis of ester 10 afforded the free acid, which was convert-
ed into the corresponding aldehyde intermediate 11 via
Weinreb amide. Similarly, removal of Boc-protecting
groups in 13 followed by conventional amide, urea or
sulfonamide formation provided the isoxazole deriva-
tives 14–16.
Figure 2. Overlap of 1a with 1,2-disubstituted imidazole and 3,4-
disubstituted isoxazole.
Br
CN
a
b
Scheme 3 illustrates the strategy toward synthesis of
1,2,5- or 1,2,4-trisubstituted imidazole derivatives. Syn-
thesis of key imidazole intermediate 20 was achieved
by reacting 2-bromo-3-(1-methylethoxy)-2-propenal
198 with amidine compound 18, which was in turn pre-
pared from aniline 17 and a nitrile in the presence of
AlCl3 at high temperature or in the presence of a strong
base (e.g., NaHMDS) at room temperature depending
on the nature of nitriles.9 It was found that the latter
method was useful for aromatic nitriles, especially for
heteroaryl nitriles, while the aliphatic substituted
amidines could be prepared in high yield just by simply
dissolving aniline in the nitrile compound under reflux-
ing conditions in the presence of AlCl3. Alternatively,
reaction of amidine 18 with a-haloketone provided
directly 1,2,4-trisubstituted imidazoles 26.
c
F3C
O
CF3
OH
F3C
CF3
F3C
CF3
OH
OH
3
4
2
N
O
N
O
CN
R1
NH2
R1
NHCOR2
R1
d
e
F3C
CF3
F3C
CF3
F3C
CF3
OH
OH
6
OH
7
5
Scheme 1. Reagents and conditions: (a) NBS, CCl4, 85%; (b) KCN,
DMSO/H2O, 100 °C, 78%; (c) LHDMS, THF, rt, then R1COOEt, 76–
90%; (d) NH2OH, heat, 82–90%; (e) R2COCl or (R2CO)2O, DCM,
pyridine, >90%.
With imidazole aldehyde intermediate 20 in hand; a
number of transformations were performed. For exam-
ple, the aldehyde 20 was reacted with organolithium
compounds or Grignard reagent to afford the secondary
alcohol compounds 21. Reduction of aldehyde 20
provided the corresponding alcohols 22, which was
further converted into ether derivatives (23). Reductive
available 1,1,1,3,3,3-hexafluoro-2-p-tolyl-propan-2-ol 2
with NBS in CCl4 provided predominantly the mono-
brominated derivative 3 in excellent yield. Treatment

J.-F. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 695–700
697
N
O
N O
N
O
H
EtOOC
R1
R2
R1
R1
O
d
c
F3C
CF3
F3C
CF3
F3C
CF3
OH
OH
O
O
OH
Br
B
a
b
10
12
11
N
O
N
O
F3C
CF3
R3
F3C
CF3
OH
R1
OH
N
R1
BocNH
14: R3 = R4CO-
H
e
8
9
15: R3 = R5NHCO-
-
16: R3 =R6 SO2
F3C
CF3
OH
F3C
CF3
OH
13
Scheme 2. Reagents and conditions. (a) Bis(pinacolato)diborane, Pd(dppf)Cl2, DMF, KOAc; 78%; (b) 3,5-disubstituted-4-bromoisoxazole,
Pd(dppf)Cl2, THF/H2O, NaHCO3, 39%; (c) (i) NaOH; (ii) EDAC, DMAP, MeNH(OMe); (iii) DIBAL, toluene, À78 to 0 °C, 30–40% overall yield;
(d) RMgX, THF, 0 °C to rt quantitative; or (i) Wittig reagent, benzene, reflux; (ii) Pd–C/H2, EtOH, rt; (iii) NaOHaq, dioxane, rt, 78–92%; (e) (i) TFA,
DCM, rt; (ii) R4COCl or (R4CO)2O; or, R5NCO, or R6SO2Cl, DCM, heating, 63–78%.
(R7NHCO )H
NH2
N
N
N
R8O
R6 N
R1
R1
N
N
F3C
CF3
OH
17
F3C
CF3
OH
25
F3C
CF3
OH
24
g
f
a
Oi-Pr
CHO
N
N
NH
R1
R2
R1
OHC
R1
N
Br
N
HN
c
HO
19
b
F3C
CF3
OH
F3C
CF3
F3C
CF3
OH
OH
18
20
21
h,i
d
j
R6
N
N
N
N
N
O
HO
R5
R9
R1
R1
R1
R1
N
N
e
N
F3C
CF3
F3C
CF3
F3C
CF3
F3C
CF3
OH
23
OH
22
OH
27
OH
26
Scheme 3. Reagents and conditions: (a) R1CN, AlCl3, 180 °C; or R1CN, NaHMDS, THF, rt, 85–90%; (b) K2CO3, CHCl3, H2O; 40–60%; (c)
R2MgX, THF, 0 °C, >90%; (d) NaBH4, MeOH; quantitative; (e) R5Br, t-BuOK, THF, 0 °C, 85–90%; (f) (i) R6NH2, NaBH3CN, MeOH, 75–85%; (ii)
R7NCO, DCM, rt, >90%; (g) R8ONH2, EtOH, quantitative, overall yield. (h) R9CH2PPh3Br, toluene, 80–90%; (i) H2, Pd/C, EtOH; (j) BrCH2COR6,
58–72%.
amination of 20 provided secondary amines (24) or their
corresponding tertiary amide derivatives after further
acylation. Finally, reaction with hydroxylamine afforded
the corresponding oxime or O-alkylated oximes (25).
The aldehyde intermediate could be also converted into
the alkylated derivatives 27 via Wittig reaction followed

698
J.-F. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 695–700
by further transformations (hydrogenation, hydrolysis
etc.).
equals a NH2 group, the small aliphatic (6c, 6d, 6e, 6f,
and 6k) shows a modest improvement in MCD inhibi-
tion over compounds with a phenyl or furanyl group
such as 6b, 6g, 6i, 6j, and 6n. 4-Pyridinyl group again
gave better results than the other aryl group as seen in
the amide series.4a Acylation of the amino group at
C-5 position of the isoxazole ring either improved or
exhibited similar activity. The best combination is a
small R2 group with a small aliphatic amide at C-5 posi-
tion of the isoxazole ring (7b).
All the compounds prepared above were tested for their
ability to inhibit the soluble form of recombinant human
MCD MBP fusion protein (MBP–hMCD).4b,10 Previous
SAR studies on initial hit compound 1a revealed that
removal of N-methyl group (i.e., 1b) dramatically de-
creased the activity. Amide derivatives of 1a with a small
aliphatic chain up to five carbons on the nitrogen atom
or an a,a-disubstituted small aliphatic acyl group were
shown to be excellent MCD inhibitors (unpublished re-
sults). The SAR for isoxazole derivative is parallel to the
original amide, although no significant activity improve-
ment was observed (Table 1). When R1 at C-5 position
On the other hand, MCD inhibitory activity for C-5
methyl isoxazole derivatives (12–15) was generally weak.
Only a handful of compounds with a methyl group
(12a), a trans cyanovinyl group (12b), or a 4-pyridine-
Table 1. MCD inhibitory activity of isoxazole derivativesa
O
N
R1
R2
F3C
CF3
OH
Compound
R1
R2
IC50 (lM)
6a
6b
NH2–
NH2–
4-Pyridinyl
–Ph
0.19
2.54
0.68
0.46
0.67
0.63
2.77
1.16
1.17
5.32
0.65
3.33
1.13
1.78
0.39
0.14
0.42
0.20
0.73
0.25
0.34
0.96
0.41
0.29
0.93
3.16
1.99
3.73
1.48
1.3
6c
6d
NH2–
NH2–
–Et
–i-Pr
6e
NH2–
NH2–
–n-Bu
–CH2OEt
–4-MeOPh
6f
6g
NH2–
NH2–
6h
6i
–CH2CH2COOH
–4-Hydroxylphenyl
–CH2Ph
NH2–
NH2–
6j
6k
6l
NH2–
NH2–
–CH2CH2CH2OH
–CF3
6m
6n
NH2–
NH2–
–CH2CH@CHCH3 (trans)
–3-Furanyl
–i-Pr
7a
7b
(4-CN–Ph)CONH–
n-BuCONH–
PhCH(Et)CONH–
n-BuCH(Et)CONH–
i-PrCONH–
i-PrCONH–
4-PyCONH–
Me–
–i-Pr
–i-Pr
7c
7d
–i-Pr
–Ph
7e
7f
7g
–4-Pyridinyl
–i-Pr
–COOEt
10
12a
12b
12c
12d
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
15
Me–
Me–
–Me
–trans-CH@CH–CN
–COCH2CH2CH3
–trans-CH@CH–COOEt
–NHCOCH3
–NHCONHCH2COOEt
–NHCONHPh
–NHCONHCH2COOH
–NHCONH-(c-hexane)
–NHCONHCH2CH2Ph
–NHCOCH2CH2Ph
–NHCO(i-Pr)
–NHCO(4-Py)
–NHCO(CH2)4CH3
–NHSO2–(m-CF3–Ph)
Me–
Me–
Me–
Me–
Me–
Me–
Me–
Me–
4.98
2.97
2.55
0.99
0.39
1.2
Me–
Me–
Me–
Me–
Me–
N/A
a Data are reported as means of n P 3 determinations. SD was generally 20% of the average.

J.-F. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 695–700
699
carboxylamido group (14i) at C-3 position showed IC50s
less than 0.5 lM or better activity than the initial hit 1a.
Other bulky groups at this position were not tolerated
(15).
C-5 hydroxylmethyl group (compound 21c) caused a
5-fold activity (21c, 59 nM; 22a, 321 nM) increase. An
extra methyl group at this position also increased the
activity slightly (21a), while an n-propyl (21b) is equally
effective as an isopropyl group (21c). A phenyl group
(21d), however, did not add any benefit to the activity.
Similarly, a small branched aliphatic group or 4-pyri-
dine group at C-2 position (R1) of the imidazole ring
seems to be well tolerated (Table 2). Interestingly, no
significant activity difference was observed for C-4
(26a–26d) or C-5 (22a–22d) substituted imidazoles.
Activity improvement was clearly observed when the
substitution pattern at C-5 position changes. For exam-
ple, introduction of an extra isopropyl substitution at
The effect of a long chain at C-5 position of the imidazole
ring (23a and 23b) is not as profound as that observed in
the amide or urea series.4a Even compounds with a termi-
nal tetrazole group (23c and 23d) only showed similar
activity to a simple C-5 hydroxylmethyl group, indicating
the slight different steric requirement for imidazole
Table 2. MCD inhibitory activity of imidazole derivativesa
R3
R2
N
R1
N
F3C
CF3
OH
Compound
R1
R2
R3
IC50 (nM)
21a
21b
21c
21d
22a
22b
22c
22d
23a
23b
23c
23d
24a
24b
24c
24d
24e
25a
25b
25c
25d
25e
25f
26a
26b
26c
26d
27a
27b
27c
27d
27e
27f
27g
27h
27i
s-Bu–
s-Bu–
s-Bu–
s-Bu–
s-Bu–
i-Pr–
–CH(OH)CH3
–CH(OH)(n-Pr)
–CH(OH)(i-Pr)
–CH(OH)Ph
–CH2OH
H
149
74
H
H
59
H
465
321
274
114
791
107
168
155
116
341
340
303
482
1080
152
63
H
–CH2OH
–CH2OH
H
4-Pyridinyl–
2-Pyridinyl–
i-Pr–
H
–CH2OH
–CH2O(CH2)4CN
–CH2O(CH2)5CN
H
H
i-Pr–
i-Pr–
H
CH2O(CH2)5–(5-tetrazole)
–CH2O(CH2)4–(5-tetrazole)
–CH2NH(i-Pr)
H
i-Pr–
H
s-Bu–
s-Bu–
s-Bu–
i-Pr–
H
–CH2NHCH2CH2OMe
–CH2NH–(n-Bu)
–CH2NH–(n-Bu)
–CH2N(2-morpholinylethyl)CONH (i-Pr)
@N–OH
@N–OMe
@N–OEt
@N–OPh
@N–O(i-Bu)
H
H
H
i-Pr–
i-Pr–
H
H
i-Pr–
i-Pr–
H
H
118
209
350
44
i-Pr–
i-Pr–
H
H
i-Pr–
@N–O(t-Bu)
–H
H
–COOEt
4-Pyridinyl–
s-Bu–
i-Pr–
191
318
567
243
140
20
–H
–H
–COOEt
–CF3
CH2OH
i-Pr–
i-Pr–
–H
–trans-CH@CH–CN
–cis-CH@CH–CN
–trans-CH@CH–COOMe
–CH2CH2COOMe
–CH2CH(Me)COOEt
–CH2CH2COOH
–CH2CH(Me)COOH
–CH2CH2COOH
–trans-CH@CH–COOMe
–CH2CH2COOMe
–trans-CH@CH–COOH
H
H
H
H
H
H
H
H
H
H
H
i-Pr–
i-Pr–
55
65
i-Pr–
i-Pr–
37
i-Pr–
i-Pr–
239
225
163
88
s-Bu–
s-Bu–
s-Bu–
s-Bu–
27j
27k
41
523
a Data are reported as means of n P 3 determinations. SD was generally 20% of the average.

700
J.-F. Cheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 695–700
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compounds from amides. Amine-based derivatives 24a–
24e also showed similar results. A large substitution at
C-5 position (R2) is not tolerated (e.g., 24e).
Most dramatic results were observed for oximes and
Wittig products of the intermediary aldehyde 20. O-
Methylhydroxylamine oxime (25b) and O-(t-butyl)-hy-
droxylamine oxime (25f) both gave excellent MCD inhib-
itory activities.11 Wittig reaction of 20 with (triphenyl
phosphanylidene)-acetonitrile gave rise to two products
that were stable and separable by silica gel chromatogra-
phy. The cis-product 27b is seven times more potent than
its trans counterpart 27c and is one of the most potent
compounds in the imidazole series. Other Wittig products
(27c, 27i) also exhibited potent MCD inhibitory activities.
Similarly, the terminal carboxylic acid group is tolerated,
but those compounds with terminal carboxylic acid func-
tionality tended to show lower activity as compared to
their corresponding esters.
2. McGarry, J. D.; Brown, N. F. Eur. J. Biochem. 1997, 244, 1.
3. Prentki, M.; Corkey, B. E. Diabetes 1996, 45, 273.
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A. M. J. Med. Chem. Manuscript in preparation.
In conclusion, based on the initial HTS hit 1a, a rational
design using a five-membered heterocycle to mimic the
possible active conformation of the amide moiety was
performed. As a result, a series of isoxazole and imidaz-
ole-based MCD inhibitors were synthesized and struc-
ture–activity relationship was established. Although
the isoxazole derivatives did not significantly improve
the MCD inhibitory activity over 1a, imidazole-based
derivatives generally showed good activities. Most
potent compounds were found to be those with
small-branched aliphatic group (i-Pr, s-Bu, etc.) at C-2
position of the imidazole ring. These compounds are
anticipated to have more favorable in vitro and in vivo
ADME properties.
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10. In vitro MCD inhibition assay: The decarboxylase activity
of MCD was measured spectrophotometrically by mon-
itoring acetyl-CoA formation using the malate dehydro-
genase (MD)/citrate synthase (CS) coupling system. 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 measured by the
fluorescence emission at 465 nm. Typically, the assay
system (50 lL) contains 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 MD (Roche Chem-
icals), 0.028 unit CS (Roche Chemicals), 5–10 nM MBP–
hMCD, and varying amounts of malonyl-CoA substrate.
Assays were initiated by the addition of malonyl-CoA,
and the rates were corrected for the background rate
determined in the absence of hMCD.
Acknowledgments
We thank Dr. Peter Simms, Ms. Cynthia Jeffries, and
Aixia Sun for providing analytical data (HPLC and
MS) and Mr.Õs Sean Reily, and Donald Chu for their
help with in vitro enzyme assay.
References and notes
1. (a) Kim, Y. S.; Kolattukudy, P. E. Arch. Biochem.
Biophys. 1978, 190, 234; (b) Kim, Y. S.; Kolattukudy, P.
E. Arch. Biochem. Biophys. 1978, 190, 585; (c) Kim, Y. S.;
Kolattukudy, P. E. Biochim. Biophys. Acta 1978, 531, 187;
(d) Kim, Y. S.; Kolattukudy, P. E.; Boos, A. Arch.
Biochem. Biophys. 1979, 196, 543; (e) Hunaiti, A. R.;
Kolattukudy, P. E. Arch. Biochem. Biophys. 1984, 229,
426; (f) Jang, S. H.; Cheesbrough, T. M.; Kolattukudy, P.
E. J. Biol. Chem. 1989, 264, 3500; (g) Dyck, J. R. B.; Barr,
A. J.; Barr, R. L.; Kolattukudy, P. E.; Lopaschuk, G. D.
Am. J. Physiol. 1998, 275, H2122; (h) Gao, J.; Waber, L.;
11. Two products could be detected and purified for all O-
substituted oximes. The predominant isomers were tested
and are reported in Table 2. They are presumably the
trans products. The minor isomers (<10% yield) showed
a slightly lower activity as compared to the major
products.