CVT-4325: A potent fatty acid oxidation inhibitor with favorable oral bioavailability

Article abstract of DOI:10.1016/j.bmcl.2004.09.077

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the 'Western Portion' of the molecule. SAR studies led to the discovery of CVT-4325, a potent FOXi (IC₅₀ = 380 nM, rat mitochondria) with favorable PK properties (F = 93%, t_1/2 = 13.6 h, dog). New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the 'Western Portion' of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC₅₀ = 380 nM rat mitochondria) with favorable PK properties (F = 93%, t_1/2 = 13.6 h, dog).

Full text of DOI:10.1016/j.bmcl.2004.09.077

Bioorganic & Medicinal Chemistry Letters 14 (2004) 6017–6021
CVT-4325: a potent fatty acid oxidation inhibitor with
favorable oral bioavailability
Elfatih Elzein,a Prabha Ibrahim,a Dmitry O. Koltun,a Ken Rehder,d Kevin D. Shenk,a
Timothy A. Marquart,a Bob Jiang,a Xiaofen Li,a Reina Natero,a Yuan Li,b
Marie Nguyen,b Suresh Kerwar,b Nancy Chu,c Daniel Soohoo,c Jia Hao,c
Victoria Y. Maydanik,c David A. Lustig,c Dewan Zeng,b
Kwan Leungc and Jeff A. Zablockia,*
aDepartment of Bioorganic Chemistry, CV Therapeutics, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
bDepartment of Drug Research and Pharmacological Sciences, CV Therapeutics, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
cDepartment of Pre-Clinical Development, CV Therapeutics, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
dPPD Discovery, 3500 Paramount Pkwy., Morrisville, NC 27560, USA
Received 9 August 2004; revised 26 September 2004; accepted 28 September 2004
Available online 18 October 2004
Abstract—New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the ÔWestern Por-
tionÕ of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC50 = 380nM rat mitochondria) with
favorable PK properties (F = 93%, t1/2 = 13.6h, dog).
ꢀ 2004 Elsevier Ltd. All rights reserved.
The heart has two major sources of energy, fatty acids
and glucose, that are both converted to acetyl-CoA units
and subsequently utilized to produce ATP through the
oxidative phosphorylation process.1,2 Glucose can pro-
duce as much as 13% more ATP per mole of oxygen
consumed than fatty acids.2 Therefore, during ischemic
disease where oxygen is limited, compounds that reduce
the rate of fatty acid oxidation (i.e., partial fatty acid
oxidation inhibitors, partial FOXi), and as a result, in-
crease the rate of glucose oxidation (i.e., state dependent
metabolic shift) are of potential therapeutic impor-
tance.1–3
Me
O
OH
N
N
O
N
S
N
H
Me
Me
1
"Western Portion"
Figure 1. PalmCoA oxidation inhibitor 1.
We conducted a study to investigate the structure–activ-
ity relationships (SAR) in the ÔWestern PortionÕ of our
lead molecule, 1, in order to optimize the potency with
respect to palmCoA inhibition and metabolic stability
(human liver S9, Table 1). We focused our efforts on
finding a suitable bioisosteric replacement for the amide
group by preparing various nitrogen heterocycles.
Compounds with favorable palmCoA inhibition
(IC50 < 500nM) and metabolic stability (>40% remain-
ing @ 30min) were further evaluated in pharmaco-
kinetic (PK) studies.
Compound 1 was found to be an inhibitor of 1-[14C]-
palmitoyl-CoA oxidation (PalmCoA oxidation inhibi-
tor) in mitochondria isolated from rat heart (IC50:
2.5lM),4,5 and also, it was shown to have low metabolic
stability with 21% of the parent compound remaining
after 30min incubation with human liver S9
preparations.6
All of the compounds containing an amide surrogate in
Table 1 were prepared by reacting the corresponding
chloromethyl derivative VII with the previously
described piperazine 3 as illustrated in Scheme 1.5,7
Keywords: CVT-4325; Metabolic shift; Fatty acid oxidation inhibitor.
*
Corresponding author. Tel.: +65 0384 8547; fax: +65 0858 0390;
e-mail: jeff.zablocki@cvt.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.077

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E. Elzein et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6017–6021
Table 1. Palmitoyl-CoA oxidation inhibition activity of 4–33
R
1 R2
OH
R
O
N
OH
Het
R
N
N
O
N
S
N
N
O
N
S
N
Me
Me
Compd #a
R
Heterocycle
PalmCoA
IC50 (uM)b
Human
liver S9 %
@ 30minc
Compd #a
R
R1, R2
PalmCoA
IC50 (uM)b
Human
liver S9 %
@ 30minc
O
N
N
4
5
4-tBu
4-iPr
0.18
18
31
20
21
3,4-Di-Cl
2,4-Di-Cl
H, H
H, H
0.55
0.13
30
33
N
O
0.95
N
O
N
N
6
4-Cl
2.27
0.38
0.57
1.23
6.80
3.63
1.10
8.50
4.80
11.0
1.30
48
74
39
21
66
72
25
34
49
43
40
22
23
24
25
26
27
28
29
30
31
32
4-CN
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, Me
H, Me
H, Me
4.41
1.53
1.71
1.53
6.50
4.12
13.4
13.4
0.25
0.12
0.18
56
39
—
50
31
38
42
—
43
17
30
N
O
7
4-CF3
4-tBu
4-iPr
4-OMe
4-OCF3
4-F
N
N
O
8
N
N
O
O
O
9
N
N
10
11
12
13
14
15
16
4-Cl
3-Me
N
N
4-CF3
4-tBu
4-Cl
3-Cl
N
N
N
N
N
3-F
O
N
O
N
3-CN
4-CF3
4-CF3
4-CF3
4-CF3
2,4-Di-Cl
2,4-Di-Cl
O
O
N
S
N
N
O
N
S
17
18
19
4-CF3
1.50
1.56
1.14
40
36
29
33
4-CF3
Me, Me
9.7
—
N
N
O
O
3,4-Di-Cl
2,4-Di-Cl
N
N
N
a All compounds were >95% pure by HPLC and characterized by 1H NMR, MS, and LC/MS. Some compounds were further characterized by
elemental analysis.
b Compounds were tested at six different concentrations, and IC50 values were calculated using nonlinear regression curve fitting in GraphPad (Prism
3.00 data shown are mean values (n = 3)).
c Percent of parent drug remaining after incubation with human liver S9 (2mg/mL) in the presence of NADPH for 30min. A standard was included in
every assay for the liver S9 and the data was not used unless the standard was within 5% of the historical mean.

E. Elzein et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6017–6021
6019
R
COCl
H2N
O
O
N
(a)
I
(b)
R
R
Cl
O
N
+
Cl
NH2
N
HO
Cl
II
III
N
1
OH
N
R
O
IV
(a)
N
O
NH2
(b)
R
NH2
Cl
R
+
Cl
O
N
N
O
O
Cl
2
V
VI
Cl
OH
X
Z
Y
Y
R
R
(c)
HN
N
+
N
Cl
N
Me
S
3
VII
X
OH
N
O
N
S
Z
Me
4 - 33
Scheme 1. Reagents and conditions: (a) DIEA, CH2Cl2, rt, 24h; (b) toluene, reflux, 24h; (c) R-CH2Cl, DIEA, EtOH, 80ꢁC, 24h.
The 5-phenyl-1,2,4-oxadiazoles III and the 3-phenyl-
1,2,4-oxadiazoles VI were prepared from the corre-
sponding amide oximes and acid chlorides by acylation
followed by condensation.8,9 The required chloromethyl
heterocycles VII to prepare the 2-phenyl-1,3,4-oxadiaz-
ole derivatives 12–14 (Table 1) were prepared from the
corresponding acyl hydrazides and 2-chloro-1,1,1-tri-
methoxyethane as previously described.10 Also, several
of the chloromethyl heterocycles VII required to prepare
the target compounds in Table 1 were commercially
available (including 7, 15, 16, and 17). The 2-(S)-methyl
piperazine derivatives (30–32) and 2,2-dimethylpipera-
zine derivative (33) were prepared starting with a regio-
selective Boc protection of the corresponding
piperazines (i.e., 4-N-Boc) followed by epoxide opening
and Boc deprotection to obtain the methylated analogs
of 3.11 Then, the target compounds 30–33 were obtained
through alkylation with the corresponding chloromethyl
heterocycles VII as described above.
the best combination of palmCoA inhibitory activity
and metabolic stability (Table 1). A brief exploration
of the SAR of the 2-phenyl-1,3,4-oxadiazole series with
respect to para-t-butyl, para-chloro, and para-trifluoro-
methyl (12–14) demonstrated that this series is less
active in the palmCoA assay than either 1,2,4-oxadiaz-
ole series (i.e., 3-phenyl and 5-phenyl). In general, within
the oxadiazole series explored, the following trend was
observed with respect to palmCoA inhibition: 5-phen-
yl-1,2,4-oxadiazole > 3-phenyl-1,2,4-oxadiazole > 2-
phenyl-1,3,4-oxadiazole. Based on the palmCoA inhibi-
tory activity and metabolic stability of 7, additional
heterocycles were explored with p-trifluoromethylphenyl
groups, 15–17; however, the 5-phenyl-1,2,4-oxadiazole
remained the best amide surrogate. We next explored
the two best oxadiazole series with either 3,4- or 2,4-
dichlorophenyl substitution, 18 and 20 or 19 and 21,
respectively, and once again, the 5-phenyl-1,2,4-oxadiaz-
ole series afforded the best palmCoA inhibition. Unfor-
tunately, 20 and 21 were not metabolically stable (Table
1). Further SAR on the 5-phenyl-1,2,4-oxadiazole series
22–30 demonstrates that a preference for para versus
meta substitution with respect to palmCoA inhibition
(6 vs 27, 22 vs 29, 25 vs 28). Previously within the anilide
SAR,5 a 2-(S)-methylpiperazine enhanced the inhibitory
activity in the palmCoA assay, and this trend appears to
hold in part for the 1,2,4 oxadiazole series as well
(30 > 7, 32 > 19, 31 = 21). However, the 2,2-dimethyl-
piperazine analog of 1 (Fig. 1) was very active in the
palmCoA assay (Structure not shown, IC50 = 110nM),6
and this trend did not hold for the 2,2-dimethylpipera-
zine analog 33 (Table 1). The 2-(S)-methylpiperazine
derivative 30 had high palmCoA inhibitory activity with
modest metabolic stability, and it was chosen for further
PK analysis along with the lead 7 (CVT-4325), and the
parent anilide 1.
We chose nitrogen heterocycles as amide surrogates,
since there is plenty of literature precedence. In particu-
lar, oxadiazoles have been used extensively as amide sur-
rogates in the peptide mimetic area,12–15 and even in the
adenosine field, as a 50-amide surrogate of 50-N-ethylcar-
boxamide adenosine (NECA).16 Based on previous SAR
of the anilide series,7 the para position of the 5-phenyl-
1,2,4-oxadiazoles 4–7, and the 3-phenyl-1,2,4-oxadiaz-
oles 8–11 was substituted by either a lipophilic or
electron withdrawing group (EWG). Although the
palmCoA inhibition was favorable for derivatives con-
taining a 4-t-butyl group, 4 and 8, both derivatives
had low metabolic stability (Table 1). Replacing the 4-
t-butyl group with a smaller isobutyl group lowered
the palmCoA inhibition (5 and 9). The 1,2,4-phenylox-
adiazole analogs containing a para-EWG, either chloro
(6 and 10) or trifluoromethyl (7 and 11) were found to
be more metabolically stable (liver S9, Table 1). The
para-trifluoromethyl 5-phenyl-1,2,4-oxadiazole 7 had
The PK properties of 1 in rats were not very favorable
with respect to AUC (dose adjusted), t1/2, and clearance;

6020
E. Elzein et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6017–6021
Table 2. Comparison of pharmacokinetic properties of 1, 7 [(R)-CVT-
4325], and 30
References and notes
1. Schofield, R. S.; Hill, J. A. Am. J. Cardiovasc. Drugs 2001,
1, 23.
2. McCormack, J. G.; Stanley, W. C.; Wolf, A. A. Gen.
Pharmac. 1998, 30, 639.
3. McCormack, J. G.; Barr, R. L.; Wolf, A. A.; Lopaschuk,
G. D. Circulation 1996, 93, 135.
Compound Oral
%F AUC
(ngh/mL)b IV
t1/2 (h)c Plasma
clearance
(species)
dosea
MPK
(mL/min/kg)c
1 (rat)
7 (rat)
2
2
4
2
5
19
38
93
29
30
30.5
282
1.4
3.2
54.5
22.5
12.2
19.7
4.6
7 (dog)
30 (rat)
30 (dog)
1776
253
13.6
4.0
4. Nedergaard, J.; Cannon, B. Meth. Enzymol. 1979, 55, 3.
5. Elzein, E.; Shenk, K. D.; Ibrahim, P.; Marquart, T. A.;
Kerwar, S.; Meyer, S.; Ahmed, H.; Zeng, D.; Chu, N.;
Soohoo, D.; Wong, S.; Leung, K.; Zablocki, J. A. Bioorg.
Med. Chem. Lett. 2004, 14, 973.
6. Zablocki, J.; Elzein, E.; Nudelman, G.; Marquart, T.;
Varkhedkar, V.; Ibrahim, P.; Palle, V. P.; Blackburn, B.
K. World Patent 01/62744 A2, August 30, 2001.
7. Koltun, D. O.; Marquart, T. A.; Shenk, K. D.; Elzein, E.;
Li, Y.; Nguyen, M.; Kerwar, S.; Zeng, D.; Chu, N.;
Soohoo, D.; Hao, J.; Maydanik, V. Y.; Lustig, D. A.; Ng,
K.-J.; Fraser, H.; Zablocki, J. A. Bioorg. Med. Chem. Lett.
2004, 14, 535.
8. Ibrahim, P.; Shenk, K.; Elzein, E.; Palle, V.; Zablocki, J.;
Rehder, K. World Patent 03/008411 A1, January 30, 2003.
9. Piperazine 3 (24.4g), 3-chloromethyl-5-(4-trifluoromethyl-
phenyl)1,2,4-oxadiazole (20.8g, Maybridge Plc, UK),
ethanol (400mL), and diisopropylethylamine (55.3mL)
were warmed at 70ꢁC for 16h (TLC 3:1 hexanes/ethyl
acetate). After concentration in vacuo, the reaction was
diluted with water (500mL), extracted with ethyl acetate
(2 · 500mL), and dried (MgSO4). After concentration in
vacuo (yellow powder obtained), trituration with minimal
amounts of 3:1 mixture of hexanes and ethyl acetate
resulted in a white powder, 31.5g (74% yield) of 7 (CVT-
4325). 1H NMR (400MHz, CDCl3, d): 8.28 (d, 2H,
J = 8.4Hz); 7.79 (d, 2H, J = 8.0Hz); 7.64 (d, 1H, J =
8.8Hz); 7.42 (br s, 1H); 7.00 (dd, 1H, J = 8.4, 2.4Hz);
4.30–4.20 (m, 1H); 4.09–4.02 (m, 2H); 3.83 (s, 2H); 3.50
(br s, 1H); 2.80 (s, 3H); 2.90–2.50 (m, 10H). MS (ESI+,
m/z): 534.98.
1093
65.0
a PO formulation: propylene glycol:0.1N HCl:Tween 80:0.5% carb-
oxycellulose = 20:5:0.4:74.6 (v:v:v:v). Final concentrations in for-
mulation: 2–5mg/mL.
b Dose adjusted oral AUC normalized to 1mg/kg.
c IV formulation: propylene glycol:0.1N HCl:saline water = 20:5:75
(v/v/v). Final concentration in formulation: 1mg/mL (1mg/kg dose).
and therefore, it was not further evaluated in dogs. The
lead compounds 7 and 30 were evaluated in both rat and
dog, and the results are shown in Table 2. Both amide
surrogate compounds 7 and 30 have clearly superior
PK profiles relative to anilide 1 based on comparison
of AUC, t1/2, and clearance in the rat. A closer compar-
ison of 7 and 30 demonstrated that 7 has higher oral bio-
availability in both rats and dogs. However, 30 has a low
clearance and extremely long half-life in dogs. We fur-
ther compared the rates of disappearance of 7 and 30
by liver microsomes from rats and dogs and confirmed
that while metabolism rates of these two compounds
were similar in rat liver microsomes, 30 was metaboli-
cally more stable than 7 in dog liver microsomes (74%
and 62% parent @ 30min, respectively). This small dif-
ference in the in vitro metabolism between 7 and 30 does
not account for the observed decrease in plasma clear-
ance and increase in oral bioavailability in dogs found
with 30 that maybe attributed to many factors including
a possible diminishment in N-dealkylation of the core
piperazine ring, a known route of metabolism for
ranolazine.17
10. Natero, R.; Koltun, D. O.; Zablocki, J. A. Synth.
Commun. 2004, 34(14), 1–7.
11. To a solution of 2-(S)-methylpiperazine (5.0g, 50mmol),
triethylamine (1.25g, 12.5mmol) and chloroform (30mL)
was added dropwise Boc-ON (2.0g, 8.3mmol) in chloro-
form (15mL) at 23ꢁC. After 15h, the reaction mixture was
washed with water (2 · 50mL), dried (sodium sulfate), and
purified by application of flash chromatography (metha-
nol:methylene chloride 1:10) to afford the N-4-Boc-2-(S)-
methylpiperazine. This piperazine derivative was reacted
with 5-(((R)-oxiran-2-yl)methoxy)-2-methylbenzo[d]thiaz-
ole by warming to reflux in ethanol then deprotected with
TFA.
In conclusion, the introduction of a para-trifluoro-
methyl-5-phenyl-1,2,4-oxadiazole, 7 (CVT-4325), as an
amide surrogate resulted in both good palmCoA inhibi-
tion (IC50 = 380nM) and a favorable PK profile
(F = 93%, t1/2 = 13.6h, n = 3, dog). Additional pharma-
cological studies demonstrate that 7 produces a meta-
bolic shift from fatty acids to glucose, and this finding
is disclosed elsewhere.18
12. Borg, S.; Estenne-Bouhtou, G.; Luthman, K.; Csoregh, I.;
Hesselink, W.; Hacksell, U. J. Org. Chem. 1995, 60, 3112–
3120.
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We would like to thank Dr. Brent Blackburn, and Dr.
Luiz Belardinelli, for valuable input and discussions.
Supplementary data
Supplemental materials are available online that include
the conditions of the PalmCoA assay. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2004.09.077.
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