Synthesis, radiolabeling and initial in vivo evaluation of [ 11C]KSM-01 for imaging PPAR-α receptors

Article abstract of DOI:10.1016/j.bmcl.2012.08.010

Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated nuclear receptor transcription factor that regulates the fatty acid β-oxidation. An in vitro assay identified the p-methoxy phenyl ureido thiobutyric acid derivative KSM-01 (IC₅₀ = 0.28 ± 0.09 nM) having a higher affinity to activate PPAR-α than the PPAR-α agonist GW7647 (IC₅₀ = 0.46 ± 0.19 nM). In this study, we report the synthesis and initial in vivo evaluation of [¹¹C]KSM-01. The radiosynthesis was carried out by first alkylating the corresponding p-phenol precursor with [¹¹C]MeI in DMF using NaOH, followed by deprotection of the t-butyl ester group by TFA, yielding [¹¹C]KSM-01. SUV analysis of dynamic micro PET/CT imaging data showed that [¹¹C]KSM-01 accumulation was ～2.0-fold greater in cardiac-specific PPAR-α overexpressing transgenic mice compared to wild-type littermates. The post-PET biodistribution studies were consistent with these results and demonstrated 2.5-fold greater radiotracer uptake in the heart of transgenic mice compared to the wild-type littermates. These results demonstrate the potential utility of PPAR-α agonists as PET radiopharmaceuticals.

Full text of DOI:10.1016/j.bmcl.2012.08.010

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6233–6236
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, radiolabeling and initial in vivo evaluation of [11C]KSM-01
for imaging PPAR-a receptors
Kiran Kumar Solingapuram Sai a, Kun-eek Kil a, Zhude Tu a, Wenhua Chu a, Brian N. Finck b,
Justin M. Rothfuss a, Kooresh I. Shoghi a, Michael J. Welch a,z, Robert J. Gropler a, Robert H. Mach a,
⇑
a Division of Radiological Sciences, Washington University School of Medicine, 510 S Kingshighway Blvd, St. Louis, MO 63110, USA
b Division of Geriatrics and Nutritional Science, Washington University School of Medicine, 660 S. Euclid Ave., Box 8031, St. Louis, MO 63110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Peroxisome proliferator-activated receptor alpha (PPAR-
scription factor that regulates the fatty acid b-oxidation. An in vitro assay identified the p-methoxy phe-
nyl ureido thiobutyric acid derivative KSM-01 (IC50 = 0.28 0.09 nM) having a higher affinity to activate
a
) is a ligand-activated nuclear receptor tran-
Received 17 May 2012
Revised 20 July 2012
Accepted 1 August 2012
Available online 9 August 2012
PPAR-a than the PPAR-a agonist GW7647 (IC50 = 0.46 0.19 nM). In this study, we report the synthesis
and initial in vivo evaluation of [11C]KSM-01. The radiosynthesis was carried out by first alkylating the
corresponding p-phenol precursor with [11C]MeI in DMF using NaOH, followed by deprotection of the
t-butyl ester group by TFA, yielding [11C]KSM-01. SUV analysis of dynamic micro PET/CT imaging data
Keywords:
Peroxisome proliferator-activated receptor
showed that [11C]KSM-01 accumulation was ꢀ2.0-fold greater in cardiac-specific PPAR-
a overexpressing
alpha (PPAR-
b-Oxidation
PET imaging
a)
transgenic mice compared to wild-type littermates. The post-PET biodistribution studies were consistent
with these results and demonstrated 2.5-fold greater radiotracer uptake in the heart of transgenic mice
compared to the wild-type littermates. These results demonstrate the potential utility of PPAR-
as PET radiopharmaceuticals.
a agonists
Cardiomyopathy
Ureido thioisobutyric acid (TiBA)
Ó 2012 Elsevier Ltd. All rights reserved.
Peroxisome proliferator-activated receptors (PPAR) are ligand-
activated transcription factors which belong to the nuclear recep-
tor gene family.1–3 PPARs bind to endogenous ligands including
eicosanoids, free fatty acids, leukotrienes, and prostaglandins and
for endothelial cell activation in the arterial wall during
atherosclerotic disease progression.10,11 PPAR-
a agonists may be
involved in prevention of HDL cholesterol accumulation espe-
cially in cardiocytes and could affect the body weight by regulat-
ing fatty acid oxidation, thus also playing a potentially important
role in diabetic cardiomyopathy.11,12 Hypolipidemic fibrate drugs
are classified into three subtypes: PPAR-
a, PPAR-b/d and PPAR-
c 1,3
.
Ligand-activated PPARs bind to the retinoid X receptor (RXR)
to form heterodimer complexes that trigger PPAR-response ele-
ments (PPRE) which modulate lipid, glucose, or cholesterol associ-
ated metabolic pathways, depending on the nature of ligand.3,4
are an important class of PPAR-
which are considered highly selective in vivo activators of
hepatic PPAR- in rodents, often do not express the same level
a ligands; however, fibrates
a
PPAR-
a
plays a vital role in regulating cellular fatty acid b-oxida-
of selectivity in humans; many are also only moderately selec-
tion and ketogenesis and is activated by a wide range of fibrate
drugs; this activation induces proliferation of peroxisomes. PPAR-
tive for PPAR-
a
over the PPAR-
c
and PPAR-d subtypes.3 Attempts
to identify more potent PPAR-
a
ligands have led to synthesis of
a
is highly expressed in tissues with significant breakdown of fatty
ureidofibrates that are active at lower concentrations in rodent
models of hyperlipidemia. The ureido thioisobutyric acid (TiBA)
derivative GW9578 was observed to be a more potent and selec-
acids including liver, heart, brown adipose tissue, kidney and intes-
tine.5–8 Due to its critical role in lipid metabolism, drugs which can
modulate PPAR-
a
are being evaluated as targeted therapeutic
tive PPAR-a agonist with lipid-lowering activity when compared
strategies against cardiovascular diseases including type 2 diabetes
to traditional fenofibrate derivatives.13 However, difficulties in
mellitus, lipodistropy, and atherosclerosis.9
handling GW9578, which is a viscous oil, and its poor selectivity
PPAR-
a
agonists have been studied extensively as therapeutic
for human PPAR-
onstrated ꢀ200-fold selectivity towards human PPAR-
PPAR-
and PPAR-d.14 Considerable literature evidence suggests
that sufficient PPAR- density exists in cardiac cells for the eval-
uation of agonists in imaging studies.10,12 Transgenic mice with
cardiac-specific overexpression of PPAR- display a phenotype
similar to that of human diabetic cardiomyopathy. Furthermore,
a
led to development of GW7647, which dem-
candidates for atherosclerosis because they down-regulate pro-
duction of the adhesion molecule VCAM-1, which is responsible
a
over
c
a
⇑
Corresponding author. Tel.: +1 314 362 8538; fax: +1 314 362 0039.
E-mail address: rhmach@mir.wustl.edu (R.H. Mach).
Deceased.
a
z
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.010

6234
K. K. Solingapuram Sai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6233–6236
H
CO2
S
F
S
CO2H
O
O
N
N
N
N
H
H
F
GW9578
GW7647
MeO
S
CO2 H
CO2H
O
O
HN
MeO
N
N
N
O
H
O
GW7845
KSM-01
Figure 1. Structures of the ureido-TiBA PPAR-a agonists.
several diabetic metabolic abnormalities, including higher fatty
acid and lower glucose uptake were observed in a transgenic
mouse model with cardiac-specific overexpression of PPAR-
agonist in connection with metabolic syndromes like dyslipidemia
and atherosclerosis.14
GW7647 was used as a lead in designing a PET tracer by
replacing the cyclohexyl group with a methoxyphenyl group; we
synthesized the -2, -3, and -4-methoxyphenyl isomers of tert-
butyl-2-(4-(2-(1-(4-cyclohexylbutyl)-3-methoxyphenyl)ureido)
a 10,12,15
.
Previous attempts have been made to measure PPAR-a activity
using PPRE luciferase transgenic mice through in vivo and ex vivo
bioluminescence imaging.16 However, to date, no PET tracer has
ethyl)phenylthio)-2-methylpropanoate as potential PPAR-a radio-
been reported for the in vivo imaging of PPAR-
PPAR- agonist as a PET radiotracer would thus become a pivotal
tool to fill critical gaps in understanding the pathogenesis of dia-
betic cardiomyopathy triggered by PPAR- . The ureido-TiBA deriv-
ative GW7647 (Fig. 1) has been investigated as potent PPAR-
a
in the heart. A
tracers. The structure–activity relationships derived through
in vitro binding assay results indicated the p-methoxyphenyl ure-
idothiobutyric derivative KSM-01 (Fig. 1) IC50 = 0.28 0.09 nM had
the highest affinity to activate PPAR-a among the -o,-m,-p-
methoxyphenyl isomers. This report describes the synthesis,
a
a
a
t
SH
S
CO2 Bu
a
b
SH
d
HO
HO
S
HOOC
c
2
3
5
1
t
t
S
CO2 Bu
CO2 Bu
Cl
H2N
4
t
e
S
CO2 Bu
f
N
H
6
S
CO2H
O
MeO
N
H
N
KSM-03
2-OMe-phenylisocyanate
3-OMe-phenylisocyanate KSM-02
4-OMe-phenylisocyanate KSM-01
Scheme 1. Reagents and conditions: (a) LiAlH4, THF, 67%; (b) a-bromoisobutyrate, KOH, EtOH, 94%; (c) PPh3, C2Cl6, 95%; (d) KCN, BH3.THF, 78%; (e) 4-cyclohexyl-1-
bromobutane, DIPEA, 68%; (f) 2/3/4-methoxy-phenylisocyanate, TFA. Yields: KSM-03, 65%; KSM-02, 85%; KSM-01, 90%.

K. K. Solingapuram Sai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6233–6236
6235
Table 1
MHC-PPARα
WT
In vitro binding affinities of ureido-TiBAs for PPAR-
a
Heart
Compound
IC50 (nM)
KSM-01
KSM-02
KSM-03
GW7647
0.28 0.09
0.59 0.22
1.93 0.99
0.46 0.19
Heart
radiolabeling and initial in vivo microPET evaluation of [11C]KSM-
01 to image PPAR-
mice.
a in cardiac-specific PPAR-a overexpressing
Max
0
The syntheses of KSM-01, 02, 03 are shown in Scheme 1 and de-
scribed in detail in the online Supplemental Methods. Reduction of
4-mercaptobenzoic acid 1 with LiAlH4 in THF resulted in the pri-
mary alcohol 2.17 The sulfur group on 2 was alkylated with
a-bro-
moisobutyrate in KOH-EtOH13 to give compound 3 after which the
hydroxyl group in 3 was converted to the corresponding chloro
analog 4 using PPh3 in CCl4.18 Cyanation and reductive amination
of compound 4 using KCN and BH3ÁTHF, respectively gave the
amine analog 5.19 Further reaction with 4-cyclohexyl-1-bromobu-
tane20 in DIPEA and THF gave the secondary amine 6,21 which was
coupled with 2-methoxy, 3-methoxy, or 4-methoxy phenylisocya-
nate followed by TFA-assisted t-butyl ester deprotection to give
analogs KSM-03, KSM-02 and KSM-01, respectively.13 The precur-
sor KSM-01A for 11C-radiolabeling was obtained from the second-
ary amine 6 coupled with 1,11-dicarbonyl imidazole followed by
4-amino phenol in THF.13
Figure 2. Representative Focus 220 microPET image of [11C]KSM-01 in male MHC-
PPAR- transgenic and wild type (WT) mice. The images were summed from
0–60 min after iv injection of 170
Ci [11C]KSM-01.
a
l
purity of [11C]KSM-01 was >98% and was confirmed by co-elution
with non-radioactive KSM-01. The chemical purity of [11C]KSM-01
determined by the HPLC UV mass was >97%. The calculated radio-
chemical yield was ꢀ19% and the final product had a specific activ-
ity of 987 mCi/lmol (decay-corrected to end of synthesis).
The uptake of [11C]KSM-01 was compared in transgenic mice
with cardiac-specific overexpression of PPAR- (PPAR- +/+) and
wild type littermates (PPAR-
a
a
a
À/À) through microPET imaging
PPAR-
assessed to identify the most potent PPAR-
beta-lactamase reporter-gene under control of the PPAR-
sponse element, a cell-based assay developed by Invitrogen was
used to determine the IC50 values of the novel PPAR- ligands. No-
vel PPAR- compounds were measured for their ability to inhibit
reporter gene activity. The assay utilizing GeneBLAzer PPAR-
a
binding affinity of KSM-01, KSM-02 and KSM-03 was
agonist. Using a
re-
and post-PET biodistribution studies as described in the online
a
Supplemental Methods. Transgenic mice were produced as previ-
a
ously described.10 PPAR-
a protein levels are approximately 15-fold
more abundant in the cardiac ventricles of these transgenic ani-
mals when compared with wild-type littermates; this model has
a
a
been used to evaluate the role of PPAR-
a mediated lipid metabo-
a
lism in the development of diabetic cardiomyopathy.10
UAS-bla HEK293T cells is described in detail in the online Supple-
mental Methods. The fluorescence intensity was measured using a
Victor3 plate reader after addition of the LiveBLAzer™-FRET B/G
(CCF4-AM) substrate. Concentration-response titration points for
each compound were fitted to the Hill equation yielding concentra-
tions of half-maximal inhibition (IC50) and maximal response (effi-
cacy) values. The IC50 values of the analogs are shown in
Table 1.16,21 Compound KSM-01 showed higher potency towards
Dynamic PET imaging was performed for 0–60 min post intra-
venous injection of [11C]KSM-01. Standard uptake values (SUVs)
analysis of the PET data revealed a twofold greater accumulation
of radioactivity in PPAR-
a overexpressing transgenic mice
(0.68 0.007) over the control animals (0.37 0.09). Although liver
uptake is significant, microPET images show higher [11C]KSM-01
accumulation in the heart of PPAR-
mice compared to wild-type mice (Fig. 2).
a overexpressing transgenic
PPAR-
PPAR-
a
a
compared to the two isomers and the previously reported
agonist GW7647, suggesting that KSM-01 could be a suit-
A post-PET biodistribution study was also conducted and the re-
sults are presented in Table 2. Radiotracer accumulation was
observed to be high in liver tissue, which is a primary organ for
able PPAR-a PET imaging agent.
The radiochemical synthesis of [11C]KSM-01 is described in de-
tail in the online Supplemental Methods. It was achieved by first
alkylating the corresponding p-phenol precursor KSM-01A with
PPAR-
a
expression5–8 in both transgenic and control animals:
%ID/g = 55.031 4.926 and 60.699 0.774, respectively. Addition-
11C]MeI in DMF using NaOH at 90 °C for 5 min and then deprotect-
ally, the radiotracer demonstrated ꢀ2.5-fold greater distribution
[
in the cardiac tissue of PPAR-
a overexpressing transgenic mice
ing the t-butyl ester group with trifluoroacetic acid at 90 °C for
3 min as depicted in Scheme 2. The total time required for the syn-
thesis of [11C]KSM-01, including [11C]MeI production, purification
and formulation was approximately 50 min. The radiochemical
(1.09 0.246) when compared to the wild type littermates
(0.479 0.007) (Fig. 3). The biodistribution results were consistent
with the microPET imaging data.
11CH3
O
t
HO
S
CO2 Bu
S
CO2H
O
O
N
H
N
N
H
N
a,b
Scheme 2. Reagents and conditions: (a) [11C]MeI, 5 N NaOH, DMF, 90 °C, 5 min; (b) TFA, 90 °C, 3 min.

6236
K. K. Solingapuram Sai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6233–6236
Table 2
the Small Animal Imaging Core for conducting the rodent studies.
This research was funded by HL13851.
Post-PET biodistribution of [11C]KSM-01 in non-target tissues of male MHC PPAR-
a
overexpressing transgenic (TG) and wild type mice (WT). Results are expressed as
%ID/g standard deviation
Supplementary data
Organ
TG
WT
Blood
Lung
Liver
Spleen
Kidney
Muscle
0.42 0.14
2.65 0.34
55.30 4.92
1.33 0.29
2.58 0.56
0.36 0.15
0.42 0.10
2.11 0.31
60.69 0.77
1.55 0.14
2.01 0.21
0.35 0.18
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.010.
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Figure 3. Post-PET uptake of [11C]KSM-01 in cardiac tissue (%ID/g) of male MHC-
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a
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In summary, the ureido TiBAC analog KSM-01, a PPAR-a agonist
with high potency (IC50 = 0.28 nM) was synthesized and [11C]-radi-
olabeled for the first time. MicroPET imaging data comparing
transgenic mice which selectively over-express PPAR-
heart with wild-type littermates indicate the potential utility of
PPAR- agonists as PET radiopharmaceuticals. Although liver up-
take is high due to the normal expression of PPAR- , elevated car-
diac uptake can be clearly visualized in PPAR- over-expressing
a in the
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Acknowledgments
The authors thank Washington University School of Medicine
Cyclotron Facilities for carbon-11 methyl iodide production and