Novel inhibitors of fatty acid oxidation as potential metabolic modulators

Article abstract of DOI:10.1016/j.bmcl.2003.11.065

We describe the synthesis of novel inhibitors of fatty acid oxidation as potential metabolic modulators for the treatment of stable angina. Replacement of the 2H-benzo[d]1,3-dioxolene ring system in our initial lead 3 with different benzthiazoles, benzoxa

Full text of DOI:10.1016/j.bmcl.2003.11.065

Bioorganic & Medicinal Chemistry Letters 14 (2004) 973–977
Novel inhibitors of fatty acid oxidation as potential
metabolic modulators§
Elfatih Elzein,a,* Kevin Shenk,a Prabha Ibrahim,a Tim Marquart,a Suresh Kerwar,b
Stephanie Meyer,b Hiba Ahmed,b Dewan Zeng,b Nancy Chu,c Daniel Soohoo,c
a
Shirley Wong,c Kwan Leungc andJeff Zablocki
aDepartment of Bioorganic Chemistry, CV Therapeutics Inc., 3172 Porter Drive, Palo Alto, CA 94304, USA
bDepartment of Drug Research and Pharmacological Sciences, CV Therapeutics Inc., 3172 Porter Drive,
Palo Alto, CA 94304, USA
cDepartment of Pre-clinical Development, CV Therapeutics Inc., 3172 Porter Drive, Palo Alto, CA 94304, USA
Received8 October 2003; revised20 November 2003; accepted26 November 2003
Abstract—We describe the synthesis of novel inhibitors of fatty acid oxidation as potential metabolic modulators for the treatment
of stable angina. Replacement of the 2H-benzo[d]1,3-dioxolene ring system in our initial lead 3 with different benzthiazoles,
benzoxazoles andintroducing small alkyl substituents into the piperazine ring resultedin analogues with enhancedinhibitory
activity against 1-14[C]-palmitoyl-CoA oxidation in isolated rat heart mitochondria (6, IC50=70 nM; 25, IC50=23 nM).
# 2003 Elsevier Ltd. All rights reserved.
Ischemic heart disease (IHD) continues to be the lead-
ing cause of death in the United States.1 Chronic stable
angina, a constituent of IHD has been estimatedto
occur in at least 6.4 million Americans.2 Angina is a
result of the deficiency between myocardial oxygen
supply and demand. The primary mechanism of action
of current anti-anginal drugs (nitrates, b-adrenoceptor
antagonists, calcium channel blockers) is improvement
of myocardial oxygen balance between supply and
demand by either an increase in coronary blood flow or
a decrease in cardiac mechanical function, or both.3,4
Thus, all three major classes of anti-anginal drugs exert
their effects by altering hemodynamics. Pharmacologi-
cal agents that act by improving the efficiency of trans-
duction of biochemical energy to mechanical contractile
work have been proposedas an adjunct or alternative to
the more traditional hemodynamic therapies.5 This new
class of agents is referredto as metabolic modulators
and aimed to decrease the rate of fatty acid oxidation by
the heart and increase the oxidation of pyruvate derived
production from fatty acid oxidation to glucose oxida-
tion results in a greater ATP yieldfor a given rate of
myocardial oxygen consumption (up to 11% with com-
plete switch from fat to carbohydrate).7 This could
clearly be important in itself under conditions of limited
tissue oxygen supply as in angina. In addition, these
agents may also decrease lactate and hydrogen ion pro-
duction under low flow conditions.8,9
12,13
Ranolazine10,11 (1) andtrimetazidine
(2) are two
examples of this new class of anti-anginal drugs. Both
compounds are believed to exert their anti-anginal
effects in part by shifting myocardial energy metabolism
from fatty acids towards glucose (through inhibition of
b oxidation of fatty acids) as the substrate for produc-
tion of ATP.14À16 Studies have shown that ranolazine
partially inhibits b-oxidation of fatty acids in a dose-
dependent manner.17 Studies have also shown that a
strong correlation exists between palmitoyl-CoA oxid-
ation in isolated mitochondria and fatty acids and glu-
cose oxidation in isolated hearts.18 Therefore, inhibition
of palmitoyl-CoA oxidation in isolated mitochondria is
predictive of the effects on fatty acid and glucose oxi-
dation in isolated hearts. In this communication, we
describe the synthesis of new fatty acid oxidation inhi-
bitors (as measuredby inhibition of 1- 14[C]-palmitoyl-
CoA oxidation in rat heart mitochondria)17 as potential
6
from glucose andlactate. Switching myocardial energy
§Supplementary data associated with this article can be found at
doi:10.1016/j.bmcl.2003.11.065
* Corresponding author. Tel.: +-1-650-384-8217; fax: +1-650-858-
0390; e-mail: elfatih.elzein@cvt.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.065

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E. Elzein et al. / Bioorg. Med. Chem. Lett. 14 (2004) 973–977
(R)-epichlorohydrin 32 and 36, 5-[((2R)oxiran-2-yl)me-
thoxy]-2-methylbenzthiazole 34 and5-[((2 S)oxiran-2-
yl)methoxy]-2-methylbenzthiazole 37 were obtained,
respectively, in >95% ee using K2CO3 in acetone.23
The different 2-substituted benzoxazoles and benzthia-
24,25
zoles were preparedfollowing literature procedures.
In our discovery paradigm, IC50s were determined for
compounds that show ꢀ50% inhibition of 1-14[C]-
palmitoyl-CoA (Palm CoA) oxidation in rat heart
mitochondria at 100 mM. Compounds that display an
IC50 of <50 mM were evaluatedfor their metabolic
stability andCYP3A4 inhibition. Compounsd that
show ꢀ40% parent remaining after incubation with
human liver microsomes (liver S-9) for 30 min, no
CYP3A4 inhibition (IC50>30 mM) or have an IC50 in
Palm CoA assay <1 mM were further evaluatedfor
their oral bioavailability in rats. Our initial studies to
define the minimum structural elements that were needed
for greater Palm CoA oxidation inhibitory activity had
ledto the discovery of compound 3 (Fig. 1).26 Com-
pound 3 displayed 38% inhibition of Palm CoA oxida-
tion at 100 mM. In addition, 80% of the parent
compoundwas remaining after incubation with human
liver microsomes for 30 min. However, in subsequent
studies 3 was foundto be an inhibitor of CYP3A4
(IC50=4.3 mM). Compound 3 also showedtime depen-
Figure 1.
metabolic modulators for the treatment of ischemic
heart disease.
The synthesis of racemic analogues (4,8–15) is outlined
in Scheme 1. Reaction of epichlorohydrin with 2-sub-
stitutedbenzoxazole or benzthiazole of the general for-
mula 29 in acetone using K2CO3 as a base afforded the
corresponding epoxide 30. Opening of the epoxide with
piperazine 31 was achievedby heating the mixture in
ethanol. Studies have shown that reaction of (S) and/or
(R)-epichlorohydrin with phenols using K2CO3 in ace-
tone, predominantly proceeded via epoxide ring open-
ing andledto inversion of assignment at the C-2 chiral
center.19À22 Procedures used in the synthesis of (R)
and( S) epoxides 34 and 37 are shown in Schemes 2
and3 , respectively. Starting with the (S) and/or
dent inhibition of CYP3A4 suggesting that 3 is a
26
mechanism basedinhibitor.
We then directed our
efforts to the optimization of compound 3 with the idea
of eliminating CYP3A4 inhibition, maintaining liver S-9
stability andimproving its Palm CoA oxidation inhibitory
activity.
Studies have shown that in a 2H-benzo[d]1,3-dioxolene
ring system such as in 3, the 1,3-dioxolene subunit is
responsible for the potent mechanism basedinhibition
of CYP3A4.27 In an attempt to finda suitable bioiso-
stere for the 1,3-dioxolene subunit we replaced this
subunit with different heteroaryls as shown in Table 1.
Replacement of the 2H-benzo[d]1,3-dioxolene fused ring
system in compound 3 with 2-methylbenzthiazole as in 4
resultedin at least a 10-foldincrease in the inhibitory
activity (IC50=10 mM) comparedto 3. Compound 4
showedno significant inhibition of the CYP3A4 iso-
zyme (IC50=>30 mM), 50% parent remaining upon
incubation with liver S-9 andgoodoral bioavailability
andhalf-life in rats ( Table 3). Extending the 2-alkyl
group in 4 to ethyl andpropyl groups resultedin com-
pounds 11 and 12, that displayed IC50’s comparable to
that of 4; however, both analogues hadless liver S-9
stability relative to 4 (<30%). The 2-methylbenzthia-
zole regioisomer 9 was completely devoid of inhibitory
activity suggesting the importance of the sulfur group
being para to the 5-ether linkage. For this class of
2-alkylbenzthiazole compounds, the 2-methyl substituent
was foundto be optimal for both inhibitory activity and
liver S-9 stability. Compound 10, a benzoxazole analo-
gue of 4, displayed a significant drop in Palm CoA oxi-
dation inhibitory activity. However, introduction of a
substitutedphenyl group insteadof the 2-methyl group
in 10 restoredthe inhibitory activity andresultedin
compounds (13–15) with IC50’s comparable to that of 4.
Scheme 1.
Scheme 2.
Scheme 3.

E. Elzein et al. / Bioorg. Med. Chem. Lett. 14 (2004) 973–977
975
Among the three 2-phenylbenzoxazole compounds (13–
15) only 14 showedliver S-9 stability that satisfiedour
criteria (40% parent remaining) andthis was probably
due to the presence of the trifluoromethyl group at the
4-position blocking that important site of metabolism.
In this class of 2-substitutedbenzoxazole compounds
and considering the limited number of compounds that
were made, it seems that a 4-substituted aromatic ring
might be optimal for Palm CoA oxidation inhibitory
activity andliver S-9 stability.
The importance of the secondary hydroxyl group and
its chirality was also investigated( Table 2). Removal of
the hydroxyl group resulted in a significant loss in inhi-
bitory activity (16) relative to 4. This result suggests that
the hydrogen bond donating or/and accepting ability of
this hydroxyl group plays an important role in receptor-
ligandinteractions. The ( R) configuration at the carbon
bearing the hydroxyl group was required for the best
enhancement of inhibitory activity (compound 17 versus
compound 18). However, the (R) enantiomer was
Table 1. Fatty acidoxidation inhibition andliver S-9 stability of heteroaryl analogues
Compda
X
Y
R
% Inhibitionb
@ 100 mM
IC50 (mM)c
% Parent remaining
after 30 mind
4
8
9
10
11
12
13
14
15
N
S
S
N
N
N
N
N
N
S
N
N
O
S
Me
H
Me
Me
88
3
3
34
75
70
70
89
82
10
—
—
—
13
20
23
8
50
—
—
—
29
15
15
40
6
Et
n-Pr
Ph
4-CF3-Ph
3-CF3-Ph
S
O
O
O
13
a All compounds were >95% pure by HPLC andcharacterizedby 1H NMR, MS, LC/MS. Some compounds were further characterized by
elemental analysis.
b % Inhibition of 1-14[C]-palmitoyl-CoA oxidation in rat heart mitochondria. 95% confidence limits were generally Æ15% of the mean value.
c Concentration necessary to inhibit 50% of 1-14[C]-palmitoyl-CoA oxidation in rat heart mitochondria, IC50 values are the mean of n=3 andwere
determined using the non-linear regression curve fitting by GraphPad (prism 3.0), experiments were carried out at 6 different concentrations.
d Amount of parent drug remaining after incubation with human liver microsomes for 30 min.
Table 2. Effect of piperazine ring substitution and hydroxyl group stereochemistry on inhibition of fatty acid oxidation and liver S-9 stability
Compda
R1
R2
R3
R4
Hydroxyl group
stereochemistry
IC50 (mM)b
or % inhibitionc
@ 100 mM
% Parent remaining
after 30 mind
4
5
H
(S)-Me
Me
Me
H
H
H
Me
Me
(R)-Me
(R)-Me
(S)-Me
H
H
H
H
H
H
H
Me
Me
H
H
H
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
racemic
(R)
(R)
10
0.2
0.07
2.5
38%
4
47%
0.45
0.11
2
10
5
0.023
40%
26%
50%
<10%
21%
<30%
<30%
<27%
35%
<10%
<10%
<10%
<10%
<30%
<10%
—
6(cis)
7(cis)
16
17
18
20
21
22
23
(S)
No hydroxyl
(R)
(S)
racemic
(R)
(R)
H
H
H
H
(S)
(S)
(R)
(R)
24
25(trans)
26
27
Me
R1, R4 form CH2 bridge
R1, R4 form CH2 bridge
(S),(S)
H
(S)
—
a All compounds were >95% pure by HPLC andcharacterizedby 1H NMR, MS, LC/MS. Some compounds were further characterized by
elemental analysis.
b % Inhibition of 1-14[C]-palmitoyl-CoA oxidation in rat heart mitochondria. 95% confidence limits were generally Æ15% of the mean value.
c Concentration necessary to inhibit 50% of 1-14[C]-palmitoyl-CoA oxidation in rat heart mitochondria, IC50 values are the mean of n=3 andwere
determined using the non-linear regression curve fitting by GraphPad (prism 3.0), experiments were carried out at 6 different concentrations.
d Amount of parent drug remaining after incubation with human liver microsomes for 30 min.

976
E. Elzein et al. / Bioorg. Med. Chem. Lett. 14 (2004) 973–977
slightly less stable than the (S) enantiomer in the liver
S-9 assay. Introduction of a 2-(S) methyl group in the
piperazine ring of compound 17 (where the hydroxyl
group has the (R) configuration) as in 5 (S,R-diaster-
eomer) resultedin a substantial increase in Palm CoA
oxidation inhibitory activity (IC50=200 nM). Com-
pound 22 in which the carbon bearing the methyl group
has the (R) configuration was 10-foldless active than 5.
Compound 23 where the methyl andthe hyrdoxyl
groups have the (R) and( S) configurations respectively,
was at least 50-foldless active than 5. This result is in
agreement with our earlier finding that the (R) con-
figuration at the carbon bearing the hydroxyl group is
required for optimal inhibitory activity. In addition, the
(S) configuration of the methyl group in the piperazine
ring in conjunction with the (R) configuration of
the hydroxyl group enhanced inhibitory activity. Intro-
duction of a gem-dimethyl in 4 resultedin compound 20
that displayed a 22-fold increase in inhibitory activity
relative to 4 (IC50=450 nM). Compound 21, the (R)
enantiomer of 20 showed4-foldgreater inhibitory
activity (IC50=110 nM) comparedto that of the race-
mate 20 andthis is in accordwith the finding that the
(R) configuration of the hydroxyl group is optimal for
enhancedinhibitory activity. Introudction of a 2,6-
dimethyl group into the piperazine ring as in 6 yielded
molecules and their effect on cardiac efficiency during
ischemia will be reportedin subsequent communication.
In summary, the structure–activity relationships of our
lead 3 was investigated. Replacement of the 2H-ben-
zo[d]1,3-dioxolene ring in compound 3 with the 2-
methylbenzthiazole scaffoldresultedin a considerable
enhancement in fatty acidoxidation inhibitory activity,
significant reduction in CYP 3A4 inhibition and good
half life andoral bioavailability in rats (compound 4).
Substantial improvement in the inhibitory activity was
accomplishedthrough introduction of a methyl group
into the piperazine ring of 4. Consequently, we have
succeeded in the discovery of highly active inhibitors of
fatty acidoxidation ( 6, IC50=70 nM; 25, IC50=23 nM).
References and notes
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our secondmost active compoundwith an IC
of 70
50
nM. As anticipated, 7 (where the hydroxyl group has
the S-configuration) was less active than 6. Compound
25, the 2,5-dimethyl analogue was our most active
compoundwith an IC 50 of 23 nM. However, compound
26, a constrainedanalogue of 25 showedweak inhibitory
activity comparedto 25.
In general, compounds that incorporate at least one
methyl group in the piperazine ring displayed significant
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group couldimpart an orientation of the piperazine ring
that is favorable for binding to the receptor. However,
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ment of the metabolically labile amide moiety (e.g., in
compound 5) with different 1,2,4-oxadiazoles as amide
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Table 3. Preliminary pharmacokinetic properties of selectedpalm
CoA oxidation inhibitors in rats (values are average of n=3)
CompodsOeral d
% F
AUC
ng hr/mL
t1/2 (h)
(Mg/Kg)
4
5
6
21
13
9.7
11.2
10.5
45
5
11
13
1256
278
128
265
3
6.2
3.5
4.2
23. Caroon, J. M.; Clark, R. D.; Kluge, A. F.; Nelson, J. T.;
Strosberg, A. M.; Unger, S. H.; Michel, A. D.; Whiting,
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M.; Zeng, D.; Chu, N.; Soohoo, D.; Hao, J.; Leung, K.;
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26. Elzein, E.; Chu, N.; Soohoo, D. Unpublishedresults, CV
Therapeutics Inc., Palo Alto, CA.
andintroducing a 4-CF
group insteadof the metaboli-
3
cally labile 2,6-dimethyl groups resulted in analogue V
with IC50=180 nM, liver S-9 stability of 50%, oral bio-
availabiliy of 30% and42% in rats anddogs, respectively.
Compound 3 was foundto be a mechanism-basedinhi-
bitor of CYP3A4. It inhibits testosterone 6b-hydroxylase
activity in NADPH-and time-dependent manner and its
potency is similar to that of troleandomycin, a known
positive mechanism basedinhibitor of CYP3A4.
27. Lin, J. H.; Lu, A. Clin. Pharmacokinet. 1998, 35, 361.
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