Synthesis and biological evaluation of the mitochondrial complex 1 inhibitor 2-[4-(4-fluorobutyl)benzylsulfanyl]-3-methylchromene-4-one as a potential cardiac positron emission tomography tracer

Article abstract of DOI:10.1021/jm0701831

A series of fluorinated chromone analogs with IC₅₀ values ranging from 9 to 133 nM for the mitochondrial complex I (MC-I) has been prepared. A structure-activity relationship (SAR) study of the most potent fluorinated chromone analog 10 demonstrated the linkage heteroatom preference of the side chain region of the molecule while maintaining potent MC-I inhibitory activity. Tissue distribution studies 30 min after [¹⁸F]10 administration to Sprague-Dawley (SD) rats demonstrated high uptake of the radiotracer from the blood pool into the myocardium (2.24% ID/g), kidney (1.93% ID/g), and liver (2.00% ID/g). After 2 h about 66% of the activity in the myocardium at 30 min had been retained, whereas ～70% had been cleared from the liver and kidney. MicroPET images of SD rats after [¹⁸F]10 administration allowed easy assessment of the myocardium through 60 min with minimal lung or liver interference.

Full text of DOI:10.1021/jm0701831

4304
J. Med. Chem. 2007, 50, 4304-4315
Synthesis and Biological Evaluation of the Mitochondrial Complex 1 Inhibitor
2-[4-(4-Fluorobutyl)benzylsulfanyl]-3-methylchromene-4-one as a Potential Cardiac Positron
Emission Tomography Tracer
Heike Radeke,* Kelley Hanson, Padmaja Yalamanchili, Megan Hayes, Zhi-Qin Zhang, Michael Azure, Ming Yu,
Mary Guaraldi, Mikhail Kagan, Simon Robinson, and David Casebier
Research and DeVelopment, Bristol-Myers Squibb Medical Imaging, 331 Treble CoVe Road, North Billerica, Massachusetts 01862
ReceiVed February 16, 2007
A series of fluorinated chromone analogs with IC50 values ranging from 9 to 133 nM for the mitochondrial
complex 1 (MC-I) has been prepared. A structure-activity relationship (SAR) study of the most potent
fluorinated chromone analog 10 demonstrated the linkage heteroatom preference of the side chain region of
the molecule while maintaining potent MC-I inhibitory activity. Tissue distribution studies 30 min after
[18F]10 administration to Sprague-Dawley (SD) rats demonstrated high uptake of the radiotracer from the
blood pool into the myocardium (2.24% ID/g), kidney (1.93% ID/g), and liver (2.00% ID/g). After 2 h
about 66% of the activity in the myocardium at 30 min had been retained, whereas ∼70% had been cleared
from the liver and kidney. MicroPET images of SD rats after [18F]10 administration allowed easy assessment
of the myocardium through 60 min with minimal lung or liver interference.
Introduction
Recent in vitro studies with 125I-iodorotenone (2, Figure 1)
demonstrated the superior extraction and retention of that agent
versus 99mTc-sestamibi.19 Rotenone (1) is a natural product that
is widely used as an insectide, acaricide, and miticide.20,21 It is
a potent inhibitor of NADH:ubiquinone oxidoreductase com-
plex.22,23 This highly conserved 46-subunit heterocomplex is
the first component in the electron transport chain (ETC) and
is referred to as mitochondrial complex 1 (MC-I).24 The ETC
is embedded in the inner mitochondrial membrane and consists
of four membrane-bound enzyme complexes, which generate
the proton gradient that drives ATP synthesis and thereby the
energy supply of the cell.25 Cardiac tissue, because of its high-
energy expenditure, has a very high mitochondrial content and
is therefore an appealing target for the development of cardiac
PET tracers.26
Numerous classes of MC-I inhibitors have been reported in
addition to 1.27-31 Inhibitors of MC-I include piericidin A,
ubicidin-3, rollinisatatin-1 and 2 (bullatacin), capsaicin, an-
nonaceous acetogenins,32,33 pyridaben, fenpyroximate, tebufen-
pyrad, substituted quinolones and quinolines, synthetically
simplified deguelin compounds,34 and hybrid structures of MC-I
and MC-III inhibitors (“chromone derivatives”).35 The chromone
derivative 6 is of particular interest because of its structural
similarity to fenazaquin (3), pyridaben (4), and tebufenpyrad
(5, Figure 2). MC-I inhibitors 3-6 all possess (a) a hydrophobic
heterocyclic “headpiece”, for example a chromone core as 6;
(b) a p-tert-butylphenyl moiety as the “side chain”; and (c) a
heteroatom-containing linker, connecting a and b.
Coronary artery disease (CAD)a is currently the leading cause
of morbidity and mortality in developed nations.1 In the
management of CAD, single-photon emission computed to-
mography (SPECT) based myocardial perfusion imaging agents
(MPIA) has become an important tool used by cardiologists in
the evaluation of regional myocardial blood flow and viability
under rest or stress conditions.2-4 The most widely used SPECT
MPIA in clinical settings are 201Tl or 99mTc complexes, which
are characterized by rapid myocardial extraction and by cardiac
uptake proportional to blood flow.5-7
More recently, positron emission tomography (PET) imaging
has emerged as an alternative approach to evaluating myocardial
blood flow by use of positron-emitting radionuclides.8,9 Al-
though SPECT constitutes the primary modality for assessing
myocardial perfusion in hospital settings, PET provides several
advantages over SPECT imaging.10,11 These include higher
special resolution, accurate attenuation correction, and the ability
to quantify myocardial perfusion in absolute terms (milliliters
per gram per minute) due to the physics inherent in the decay
of positron-emitting radionuclides.12
Currently, three tracers are used for the assessment of
myocardial perfusion with cardiac PET: 82RbCl, 13NH3, and
H215O.13-18 Ammonia and water (13NH3 and H215O) are
produced using a cyclotron via the 16O(p, R)13N and 15N(p, n)15O
reactions, respectively, whereas 82RbCl is produced from a
-
82
Sr-82Rb generator. Unfortunately, the majority of currently
available PET radiotracers are short-lived (<20 min). Therefore,
production of 13NH3, and H215O must be in close proximity to
a camera facility, which requires significant investment. The
development of extractable perfusion imaging agents containing
positron emitters with a sufficient half-life (e.g., 18F, 76Br), so
that commercial production and distribution would be feasible,
would therefore be highly desirable.
Our goal was to investigate radiolabeled MC-I inhibitors as
potential MPIA. Therefore, PET radionuclide 18F was incorpo-
rated into an analog of 6 without adversely affecting the
inhibitory properties of the parent structure. The incorporation
of the radioisotope was carried out by nucleophilic displacement
to ensure high specific activity of the resultant radiotracer. The
most amenable site for chemical modification within the
chromone structure was determined to be the side chain.
* Corresponding author. Phone: (978) 671-8151. Fax: (978) 436-7500.
E-mail: heike.radeke@bms.com.
Results and Discussion
Structure-Activity Relationships. Initial efforts were di-
rected toward modification of the side chain of 6 for future
a Abbreviations: CAD, coronary artery disease; SPECT, single-photon
emission computed tomography; PET, positron emission tomography; ETC,
electron transport chain.
10.1021/jm0701831 CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/15/2007

MC-I Inhibitor as Potential Cardiac PET Tracer
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4305
Chemistry. The syntheses of all chromone analogs discussed
above are shown in Scheme 1. In general, these analogs were
prepared by linking the chromone core to the aromatic moiety
of the side chain via either Mitsunobu coupling (for the
preparation of S-chromone analogs 7 and 10) or displacement
of a sulfoxide/sulfone (for the preparation for O-chromone
analogs 8 and 11 and N-chromone analogs 9 and 12).
S-Chromone analog 7 and intermediate 19 were prepared by
reacting known chromone core 16 with the appropriately
functionalized benzyl alcohol in THF in the presence of
triphenylphosphine and diisopropyl azodicarboxylate (DIAD).
It should be noted that intermediate 19 was formed exclu-
sively under these Mitsunobu conditions when diol 18 was used.
The corresponding toluenesulfonate ester 20 of compound 19
was formed by reaction with TsCl, DMAP, and TEA in
anhydrous DCM. Displacement of the tosylate leaving grouping
with potassium fluoride in the presence of Kryptofix222 in
acetonitrile at 90 °C afforded the desired S-chromone analog
10.
Figure 1. The natural product rotenone, a known MC-I inhibitor.
fluorine incorporation. Replacement of the tert-butyl moiety in
the side chain with an n-butyl moiety yielded compound 7,
which is equal in inhibitory potency to parent chromone 6, with
an IC50 value of 52 nM (Table 1). Variation of the heteroatom
in the linker of 7 yielded compounds 8 and 9, with inhibitory
activities of 60 and 57 nM, respectively, demonstrating that
changes in the heteroatom of the linker are tolerated well.
For the synthesis of O-chromone analogs, alkylation of
sulfone 21 with the appropriate functionalized benzyl alcohol
in the presence of NaH in DMF yielded the O-chromone analog
8 and intermediate 25 in good yield. Sulfone 21 was obtained
by oxidizing chromone core 16 with m-chloroperbenzoic acid
(m-CPBA). Deprotection of the alcohol functionality of reaction
intermediate 24 with TBAF in THF followed by the prior
established reaction sequence of tosylation and the subsequent
fluorination renders O-chromone analog 11.
Simultaneously, other known MC-I inhibitors were screened
for activity. Rotenone (1) and pyridaben (4) were found to be
more potent MC-I inhibitors than chromones (7-9), whereas
fenazaquin (3) was less potent. Since replacement of the tert-
butyl group of the side chain with an n-butyl group did not
disrupt inhibitory activity against MC-I, the next phase of the
program was incorporation of a fluorine atom into the side chain.
Chromone analogs containing a primary fluorine atom in the
side chain were prepared, which exhibited IC50 values ranging
from 9 to 133 nM (Table 2). The most potent analogs, 10 and
11, exhibited IC50 values of 9 and 33 nM, respectively,
increasing 2-5-fold in potency compared to their nonfluorinated
counterparts 7 and 8 in Table 1. Conversely, the fluorinated
analog 12,with an IC50 value of 133 nM, was approximately
2.5-fold less active then the nonfluorinated 9 counterpart in
Table 1. Due to the additional space required to accommodate
the longer side chain, incorporation of sulfur or oxygen atoms
in the linker as in analogs 10 and 11 is preferred because of the
ability to orient the side chain for optimal binding with MC-I.
The hydrogen-donating ability of the amine functionality in
analog 12 may be detrimental to interactions with MC-I.
Additionally, the amine linkage imparts a rigidity and a dramatic
difference in orientation of the side chain due to the conjugation
of the NH functionality with the chromone headpiece, com-
pounding a less favorable interaction with MC-I.
Further SAR studies of the most potent chromone analog 10
were pursued. In the literature it has been suggested that
radiotracers containing primary 18F labels may be metabolically
deflourinated in vivo at a faster rate than secondary or aromatic
18F.36,37 However, primary aliphatic 18F atoms which are â to a
heteroatom, e.g., [18F]CH2CH2O-R, have been reported to be
metabolized at a slower rate.38 This has been termed the
â-heteroatom effect. Analogs 13-15 in Table 3 were prepared
wherein an oxygen atom was positioned in the side chain to
take advantage of the â-heteroatom effect. MC-I inhibitory
activities of these analogs range from 15 to 197 nM. Compound
13, which contained a secondary fluorine atom in addition to
the â-heteroatom effect, exhibited a potent inhibitory activity
of 16 nM. Extension of the alkyl side chain from four carbons
to seven afforded compound 14, which had significantly less
inhibitory activity (IC50 ) 197 nM). The four-carbon side chain
is optimal (by length or volume) for MC-I inhibition. Finally,
replacement of a methylene unit with an oxygen atom in the
original n-butyl side chain provides compound 15, which is the
most potent compound in this series (IC50 ) 15 nM).
Next, N-chromone analogs 9 and intermediate 29 were
obtained using modified alkylation conditions. Here, sulfoxide
26 reacted with the appropriate functionalized benzyl amine at
50 °C in acetonitrile to afford final compound 9 and reaction
intermediate 29. Again, formation of the toluenesulfonate ester
of the alcohol moiety of intermediate 29 with TsCl, DMAP,
and TEA in dichloromethane followed by displacement of the
leaving group using previous fluorination conditions renders the
desired N-chromone analog 12.
When chromone analogs 10-12 were prepared according to
the reaction sequence shown in Scheme 1, the required
functionalized benzyl alcohol or amine used as reaction partners
in Mitsunobu reactions or alkylations had to be synthesized
separately. Scheme 2 shows the preparation of benzyl alcohols
18 and 23 and benzyl amine 28 required in the preparation of
10, 11, and 12. Sonogashira coupling of methyl 4-bromoben-
zoate with 3-butyn-1-ol in the presence of palladium chloride,
triphenylphosphine, and a catalytic amount of copper iodide in
diethylamine yielded desired intermediate 30, which was
reduced with hydrogen at 50 psi using palladium on carbon as
a catalyst to afford key intermediate 31.39 Reduction of the ester
functionality of 31 with LAH in THF afforded diol 18 in
high yield. To obtain desired benzyl alcohol 23, protection of
the alcohol moiety of 31 with TBDMS-Cl followed by LAH
reduction in THF was carried out. The functionalized benzyl
amine substrate 28 was obtained in a similar fashion. Alkylation
of 4-iodobenzyl bromide with phthalic anhydride in the presence
of cesium carbonate in DMF introduced the nitrogen moiety
masked as a protecting group. Sonogashira coupling of inter-
mediate 32 with 3-butyn-1-ol in the presence of palladium
chloride, triphenylphosphine, and a catalytic amount of copper
iodide in diethylamine yielded desired intermediate 33, which
was reduced with hydrogen at 50 psi in the presence of
palladium on carbon to yield 34. Deprotection of the nitrogen
functionality in 34 with hydrazine in refluxing butanol afforded
the desired functionalized benzyl amine 28 used in Scheme 1.

4306 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Radeke et al.
Figure 2. Various structure classes of MC-I inhibitors.
Table 1. Binding Affinity (IC50 Determined in Vitro) and Lipophilicity
(clog D7.4) of Nonfluorinated Chromone Derivatives and Known MC-I
Inhibitors
analog 13, the appropriate benzyl alcohol 36 was obtained in
two steps. Alkylation of 4-hydroxy benzyl alcohol with chlo-
roacetone in the presence of potassium carbonate in acetone
afforded the ketone intermediate 35, which was reduced with
sodium borohydride in methanol to the diol 36. Mitsunobu
coupling of 36 with 16 afforded analog 37 as the only product.
Formation of the toluenesulfonate ester of 37 with TsCl, DMAP,
and TEA in DCM followed by fluorination with KF and
Kryptofix222 in acetonitrile afforded final analog 13. For analog
15 the appropriate benzyl alcohol 37 was obtained in one step:
alkylation of 1,4-benzenedimethanol with bromofluoroethane
in the presence of KHMDS in THF/toluene. Coupling under
Mitsunobu conditions with chromone 16 yielded 15. Last, analog
14 was prepared via alkylation of intermediate 19 with bro-
mofluoroethane in the presence of NaH in DMF.
compd
X
n
IC50 (nM)
clog D7.4
7
8
9
1
3
4
6
S
O
NH
1
1
1
52
60
57
16
90
8
6.3
5.7
5.5
4.7
5.5
4.7
5.9
52
In Vitro Experiments. The inhibitory activities of analogs
1, 3, 4, 6, and 10-15 for the MC-I enzyme were determined
by measuring the rate of NADH oxidation in the presence of
decyl ubiquinone at 340 nm with a UV-vis spectrophotometer
over 120 s.40 Submitochondrial particles (SMP) were isolated
from bovine heart mitochondria41 according to the method of
Lester and Matsuno-Yagi et al.42 The SMP were used as a
suspension and each determination was referred to a concurrent
rotenone standard, ensuring MC-I activity in the SMP prepara-
tion.
Table 2. Binding Affinity (IC50 Determined in Vitro) and Lipophilicity
(clog D7.4) of Fluorinated Chromone Derivatives
compd
X
n
IC50 (nM)
clog D7.4
10
11
12
S
O
NH
1
1
1
9
33
133
5.6
5.0
4.8
Lipophilicity. log D7.4 is a partition coefficient for charged
species between octanol and water at pH ) 7.4. In general, the
log DpH is defined as log DpH ) log(∑aiorg/∑aiwater), where aiorg
is the concentration of the ith microspecies in octanol and aiwater
is the concentration of the ith microspecies in water. The
computed lipophilicity, clog D7.4, was calculated with ACD/
LogD Suite Software (Advanced Chemistry Development Inc.,
Toronto, Canada) for all the compounds in this study (Table
1-3).
Table 3. Binding Affinity (IC50 Determined in Vitro) and Lipophilicity
(clog D7.4) of Additional Fluorinated S-Chromone Derivatives
Radiosynthesis. Compound 10 manifests the highest inhibi-
tion constant within the series and was therefore chosen as a
candidate for radiolabeling with the PET isotope 18F. Nucleo-
philic radiofluorination methods require activated leaving
groups, such as sulfonates (e.g., tosylates, mesylate), in order
to generate alkyl fluoride containing radioligands.45 Primary
sulfonates are often the preferred leaving group because of their
ease of displacement with nucleophilic 18F versus secondary
sulfonate leaving groups, which often eliminate under fluorina-
tion conditions to yield undesired olefin byproducts. With this
in mind, the [18F]10 was prepared with 6% chemical yield
(decay corrected) by the reaction of tosylate precursor 20 with
Kryptofix222/K18F complex and potassium carbonate in aceto-
compd
R
IC50 (nM)
clog D7.4
13
14
15
OCH2CH(CH3)F
CH2(CH2)3OCH2CH2F
CH2OCH2CH2F
16
197
15
4.7
5.1
4.2
Compounds containing variations in the side chain of
S-chromone analog 10 were prepared in order to investigate
the effect of a heteroatom-containing alkyl side chain on the
current MC-I inhibition activity of 10. Analogs 13 and 15 were
prepared using Mitsunobu conditions, which required prior
synthesis of the appropriate functionalized benzyl alcohol. For

MC-I Inhibitor as Potential Cardiac PET Tracer
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4307
Scheme 1. Synthesis of Chromone Analogsa
a Reagents: (a) PPh3, DIAD, THF, rt; (b) NaH, DMF, rt; (c) TBAF, THF, rt; (d) ACN, 50 °C; (e) TsCl, DMAP, TEA, DCM, rt; (f) KF, K222, ACN,
90 °C.
Table 4. Biodistribution of [18F]10a and 99mTc-Sestamibib in
Spraque-Dawley Rats
Ci/mmol, depending upon the quantity of 19F present in the
system.
[18F]10
In Vivo Studies. Biodistribution Study of [18F]10 and
99mTc-Sestamibi in Spraque-Dawley (SD) Rats. Table 4
shows the results of the biodistribution study carried out with
99mTc-sestamibi:
organ
30 min
120 min
30 min
[18F]10 in SD rats. The widely used clinical SPECT MPIA, 99m
-
blood
heart
lung
0.11 ( 0.02
2.24 ( 0.27
0.32 ( 0.07
2.00 ( 0.31
0.41 ( 0.05
1.93 ( 0.14
0.36 ( 0.04
0.06 ( 0.02
0.26 ( 0.03
0.06 ( 0.01
1.52 ( 0.19
0.12 ( 0.03
0.60 ( 0.12
0.09 ( 0.08
0.59 ( 0.08
0.74 ( 0.06
0.09 ( 0.01
0.17 ( 0.02
0.02 ( 0.00
1.59 ( 0.11
0.51 ( 0.02
0.82 ( 0.08
0.89 ( 0.07
2.52 ( 0.39
0.22 ( 0.02
0.05 ( 0.01
-
Tc-sestamibi, was used as a reference compound for this study.
High uptake of radiotracer [18F]10 from the blood pool into the
myocardium was observed at 30 min postinjection (2.24% ID/
g; ∼20:1 heart:blood pool ratio). During this 30 min time frame,
uptake of [18F]10 was also elevated in liver and kidney tissue,
2.00% and 1.93% ID/g, respectively. Retention of [18F]10 in
the heart was good and 66% of the activity at 30 min was present
at 120 min. In contrast, levels in the liver and kidney decreased
by 120 min to approximately 30% of the activity at 30 min.
[18F]10 had heart levels at 30 min after administration 40%
higher than 99mTc-sestamibi. Both radiotracers exhibit a blood:
heart ratio >20:1, which should be more than sufficient to obtain
clear images of the myocardium using SPECT or PET imaging
technology. In addition, heart:lung ratio of [18F]10 compared
to 99mTc-sestamibi was significantly improved, 1:7 versus 1:3,
respectively. Low uptake of the radiotracer in lung tissue
suggests an excellent signal: noise ratio in future imaging
liver
spleen
kidney
femur
muscle
brain
a Data are expressed as the %ID/g ( SD with five animals per data point.
b Data are expressed as the %ID/g ( SD with six animals per data point.
nitrile at 90 °C for 30 min followed by preparative HPLC
separation of the reaction mixture. The appropriate fractions
were concentrated and analyzed for radiochemical yield and
purity. The radioactivity of the final product was on average
25 mCi with radiochemical purity >99% when prepared with
500 mCi [18F]fluoride, with a total time of synthesis of 90 min.
The specific activity of the material ranged from 750 to 2000

4308 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Scheme 2. Synthesis of Side Chains 18, 23, and 28a
Radeke et al.
a Reagents: (a) PPh3, PdCl2, CuI, 3-butyn-1-ol, DEA; (b) H2, Pd/C, EtOH; (c) TBDMS-Cl, imidazole, DMF; (d) LAH, THF; (e) phthalic anhydride,
Cs2CO3, DMF; (f) hydrazine, BuOH, reflux.
Figure 3. Blood clearance curve of [18F]10.
Figure 5. Images of a rat heart at two different time points.
corresponds to approximately 7% and 15% of injected dose at
30 and 120 min postinjection, respectively.43 The overall ratio
of radioactivity measured in the femur versus cardiac tissue
decreased from 1:6 femur:heart at 30 min postinjection to 1:2
femur:heart after 120 min for [18F]10. It should be noted that
99mTc-sestamibi exhibited a 1:7 femur:heart at 30 min postin-
jection as well. It has been our experience that more defluori-
nation is seen with some compounds in the rat than occurs in
higher species.
Figure 4. Time-activity curves from rat imaging data of blood,
myocardium, and liver tissue.
In summary, the biodistribution data of [18F]10 is superior
to that of 99mTc-sestamibi with regards to uptake of the
radiotracer in the myocardium. Furthermore, the organ selectiv-
ity of [18F]10 is comparable to that of 99mTc-sestamibi and
therefore represents a suitable agent for pursuing PET imaging
studies.
Imaging Studies in SD Rats. Dynamic PET imaging studies
carried out on SD rats complemented the results obtained in
the above distribution study. The radiotracer [18F]10 ac-
cumulated within minutes after the injection of the animal in
cardiac and liver tissue, as is depicted in Figure 5. Time-activity
studies with [18F]10. However, both [18F]10 and 99mTc-sestamibi
exhibit low selectivity toward liver tissue (1:1 and 1:2,
respectively). The level of radiotracer [18F]10 in the blood
initially decreased rapidly (within 5-7 min; Figure 3) to well
below that of the heart and was then maintained throughout
the remainder of the study (120 min).
With regards to the previous concern of in vivo defluorination
and subsequent accumulation of free fluoride in bone, periodic
measurements of the femur detected an approximate 2-fold
increase of free fluoride from 30 to 120 min for [18F]10, which

MC-I Inhibitor as Potential Cardiac PET Tracer
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4309
All study protocols were approved by the Institutional Animal Care
and Use Committee.
plots generated from the rat imaging studies show a steady blood
level from 5 to 55 min postinjection, whereas heart levels
decreased slightly and liver levels decreased markedly (Figure
4). These findings are consistent with those of the biodistribution
study. Images of the rat heart show sufficient clearance of the
radiotracer from the liver was observed by 25-35 min postin-
jection to obtain good myocardial images (Figure 5) and was
complete by 55-60 min postinjection. No interference of the
radiotracer from lung uptake was detected, as demonstrated by
the high-quality images obtained from this study. However,
some in vivo bone uptake of 18F could be detected in images
after 30 min postinjection.
Synthesis of [2-(4-Butyl)benzylsulfanyl]-3-methylchromen-4-
one (7). To a solution of 2-mercaptochromen-4-one (16, prepared
according to the method of Lindell et al.)35 (0.5 g, 2.6 mmol) and
(4-butylphenyl)methanol (0.64 g, 3.90 mmol) dissolved in anhy-
drous THF (20 mL) were added solid PPh3 (1.02 g, 3.90 mmol)
and DIAD (757 µL, 3.90 mmol). After complete addition the
reaction mixture was stirred at room temperature for 20 h. The
reaction mixture was concentrated to yield a crude yellow oil. The
crude material was treated with diethyl ether (10 mL), and the
precipitated phosphine byproducts were removed via filtration. The
filtrate was concentrated to yield a yellow oil, which was purified
using silica gel chromatography (4:1 pentane:EtOAc) followed by
HPLC purification using a Phenomenex Luna C-18 (2) column (10
µm, 250 × 21.2 mm, gradient method of 40-60% B over 25 min,
where B ) 90% ACN in water using 0.1% TFA as a modifier and
A ) water using 0.1% TFA as a modifier) with a flow rate of 20
mL/min to afford compound 7 (571 mg, 1.69 mmol, 65%). 1H NMR
(CDCl3, 600 MHz): δ 8.18 (dd, J ) 8.0, 1.3 Hz, 1H), 7.60 (ddd,
J ) 8.4, 7.3, 1.8 Hz, 1H), 7.34-7.40 (m, 2H), 7.29 (d, J ) 8.0 Hz,
2H), 7.13 (d, J ) 8.0 Hz, 2H), 4.36 (s, 2H), 2.57 (t, J ) 7.8 Hz,
2H), 2.04 (s, 3H), 1.53-1.59 (m, 2H), 1.31 (m, 2H), 0.90 (t, J )
7.8 Hz, 3H). 13C NMR (CDCl3, 150 MHz): δ 175.4, 162.2, 156.7,
142.6, 133.4, 132.8, 128.9, 128.7, 126.2, 125.0, 122.7, 117.2, 116.8,
35.3, 35.2, 33.5, 22.3, 13.9 10.8. HRMS calcd for C21H22O2S:
339.1413, found 339.1416.
Synthesis of 2-(4-Butylbenzyloxy)-3-methylchromen-4-one (8).
Solid NaH (10.8 mg, 0.45 mmol) was placed in a reaction flask
and cooled at 0 °C in an ice bath while a solution of (4-butylphenyl)-
methanol (81.2 mg, 0.50 mmol) in dry THF (1 mL) was added
dropwise with stirring. After complete addition, the reaction mixture
was stirred at 0 °C for an additional hour. Solid 2-methanesulfinyl-
3-methylchromen-4-one (21) (100 mg, 0.45 mmol) was added. After
complete addition, the reaction mixture was stirred at room
temperature for 6 h. The reaction mixture was cooled to 0 °C and
quenched with methanol. The crude material was concentrated to
yield a solid, which was redissolved in ACN/H2O (3.5 mL, 1:1).
HPLC purification using a Phenomenex Luna C-18 (2) column (10
µm, 250 × 21.2 mm, gradient method of 40-100% B over 15 min,
where B ) 90% ACN in water using 0.1% TFA as a modifier and
A ) water using 0.1% TFA as a modifier) with a flow rate of 20
mL/min afforded compound 8 (71 mg, 0.22 mmol, 49%). 1H NMR
(DMSO-d6, 600 MHz): δ 8.00 (dd, J ) 7.8, 1.7 Hz, 1H), 7.75
(ddd, J ) 8.5, 7.3, 1.9 Hz, 1H), 7.66 (d, J ) 8.0 Hz, 1H), 7.48 (d,
J ) 8.0 Hz, 2H), 7.45 (d, J ) 8.0 Hz, 1H), 7.24 (d, J ) 8.0 Hz,
2H), 5.53 (s, 2H), 2.58 (t, J ) 7.8 Hz, 2H), 1.83 (s, 3H), 1.50-
1.57 (m, 2H), 1.26-1.32 (m, 2H), 0.88 (t, J ) 7.3 Hz, 3H). 13C
NMR (DMSO-d6, 150 MHz): δ 176.7, 162.2, 152.2, 142.9, 133.0,
132.6, 128.5, 128.3, 125.4, 125.0, 121.7, 117.3, 97.2, 70.6, 34.5,
33.0, 21.7, 13.7, 7.2. HRMS calcd for C21H22O3: 323.1641, found
323.1642.
Conclusion
A series of fluorinated chromone derivatives with high affinity
for MC-I have been prepared. SAR studies have shown that
chromone analogs containing a sulfur atom in the linker moiety
such as 10 exhibit the highest binding affinities for the MC-I
complex versus oxygen- or nitrogen-containing chromone
analogs 11 and 12, respectively. In addition, introduction of an
oxygen atom in the alkyl side chain of 10 does not disrupt MC-I
inhibition, provided the chain length remains at four atoms. In
vivo evaluation of [18F]10 demonstrated high uptake of the
radiotracer by 30 min postinjection and minimal wash-out over
2 h. Initial high uptake of the radiotracer in the liver was seen
in biodistribution and imaging studies. However, clearance of
the radiotracer from the liver was sufficient by 25 min
postinjection to afford high-quality images of the heart. Some
in vivo bone uptake of 18F was observed after [18F]10
administration in both in vivo studies. Biodistribution studies
indicated higher myocardium uptake of [18F]10 than 99mTc-
sestamibi, while maintaining a similar nontarget organ distribu-
tion. Future imaging studies will be directed toward investigating
in vivo defluorination of [18F]10 in other animal species in
addition to chromone derivatives 13-15. Furthermore, ad-
ditional studies will be directed toward determining the linearity
of myocardial extraction of [18F]10 at high blood flow rates.
Experimental Section
General Methods and Materials. All reagents used in synthesis
were commercial products and were used without further purifica-
tion unless otherwise indicated. All solvents used were ACS or
1
HPLC grade. H NMR spectra were obtained in a Bruker DRX
600 MHz FTNMR spectrometer in CDCl3 unless otherwise
indicated. Chemical shifts are reported as δ values (parts per
million) relative to the solvent peak. Coupling constants are reported
in hertz. The multiplicity is defined by an s (singlet), d (doublet),
t (triplet), br (broad), or m (multiplet).
Synthesis of 2-(4-Butylbenzylamino)-3-methylchromen-4-one
(9). To a solution of 2-methanesulfinyl-3-methylchromen-4-one (26)
(100 mg, 0.45 mmol) in ACN (5 mL) was added 4-butyl-
benzylamine (78 mg, 0.50 mmol). The reaction was stirred in a
50 °C oil bath overnight under N2 atmosphere. The reaction mixture
was allowed to cool to room temperature, concentrated to yield a
yellow oil, and redissolved in ACN/H2O (3.5 mL, 1:1). HPLC
purification using a Phenomenex Luna C-18 (2) column (10 µm,
250 × 21.2 mm, gradient method of 60-90% B over 30 min, where
B ) 90% ACN in water using 0.1% TFA as a modifier and A )
water using 0.1% TFA as a modifier) with a flow rate of 20 mL/
High-performance liquid chromatography (HPLC) analysis and
purification were performed on a Varian/PrepStar Model SD-1 at
220 and 254 nm. The instrument was equipped with the ProStar/
Dynamax.24 software program on a Dell/Optiplex GX 1 computer
system. Water used in the preparation of the mobile phases was
purified using a Millipore/Milli-Q Gradient A10 system. Flash
chromatography was conducted using silica gel 230-400 mesh (60
Å, Merck). LCMS and TOF analysis were carried out on an Agilent
LC/MSD instrument (model G1969A), which is connected to an
1100 Series HPLC system. The clog D7.4 values were calculated
using ACD/LogD Suite software (Advanced Chemistry Develop-
ment Inc., Toronto, Canada). For radiolabeling studies, 18F was
obtained from PETNET Pharmaceutical Services, Cummings Park,
Woburn, MA, and the synthesis was carried out in Wheaton vials
using a customized robotic synthesis platform. Biodistribution
studies were measured in an autogamma counter (Wallac Wizard
1480), while cardiac imaging was carried out using a microPET
camera (Focus220, CTI Molecular Imaging, Inc. Knoxville, TN).
1
min afforded compound 9 (81 mg, 0.25 mmol, 55%). H NMR
(DMSO-d6, 300 MHz): δ 7.95 (t, J ) 6.1 Hz, 1H), 7.92 (dd, J )
7.8, 1.8 Hz, 1H), 7.57 (ddd, J ) 8.4, 6.6, 1.3 Hz, 1 H), 7.41 (d, J
) 8.3 Hz, 1 H), 7.37-7.29 (m, 3H), 7.26 (d, J ) 8.3 Hz, 2 H),
4.55 (d, J ) 6.2 Hz, 2 H), 2.52 (m, 2H), 1.88 (s, 3H), 1.56-1.46
(m, 2H), 1.33-1.21 (m, 2H), 0.86 (t, J ) 7.3 Hz, 3H). 13C NMR
(DMSO-d6, 150 MHz): δ 172.6, 160.5, 152.1, 141.1, 136.7, 131.4,

4310 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Radeke et al.
128.3, 127.2, 124.7, 124.4, 122.1, 116.4, 92.0, 44.1, 34.4, 33.1,
21.7, 13.7, 7.7. HRMS calcd for C21H23NO2: 322.1801, found
322.1800.
2-methanesulfonyl-3-methylchromen-4-one (21) (0.92 g, 3.84 mmol)
dissolved in dry DMF (20 mL) was added dropwise and the
resultant mixture was stirred at room temperature overnight. The
next day, the mixture was cooled to 0 °C and quenched with water
(10 mL). The aqueous layer was separated and extracted with ethyl
acetate (3 × 50 mL). All combined organic layers were dried over
Na2SO4 and concentrated to yield an oil. The crude material was
partially purified using silica gel chromatography (1:1 hexanes:
EtOAc) to yield 2-{4-[4-(tert-butyldimethylsilanyloxy)butyl]-
phenyloxy}-3-methylchromen-4-one (24) in addition to a minor
amount of {4-[4-(tert-butyldimethylsilanyloxy)butyl]phenyl}methanol.
HRMS calcd for C27H36O4Si: 453.2455, found 453.2457.
To a solution of the above product mixture (258 mg, 0.57 mmol)
dissolved in anhydrous THF (5 mL) was added a solution of TBAF
(1.0 M solution in THF, 1.15 mL, 1.15 mmol). After complete
addition, the reaction was stirred at room temperature for 1 h and
then quenched with water (5 mL). The aqueous layer was separated
and extracted with ethyl acetate (3 × 10 mL). All combined organic
layers were dried over Na2SO4 and concentrated to yield an oil.
The crude material was partially purified using silica gel chroma-
tography (1:2 hexanes:EtOAc) to afford compound 25, which was
contaminated with 4-(4-hydroxymethylphenyl)butan-1-ol (40 %mol
according to 1H NMR), in moderate yield (101 mg of impure
product mixture, 23.3% yield of impure product). 1H NMR (CDCl3,
600 MHz): δ 8.21 (dd, J ) 8.1, 1.4 Hz, 1H), 7.61 (m, 1H), 7.41-
7.37 (m, 4H), 7.23 (d, J ) 8.2 Hz, 2H), 5.44 (s, 2H), 3.67 (m, 2H),
2.65 (m, 2H), 1.99 (s, 3H), 1.72-1.68 (m, 2H), 1.64-1.60 (m,
2H). 13C NMR (CDCl3, 150 MHz): δ 178.7, 162.3, 152.7, 143.2,
132.5, 132.3, 128.8, 128.2, 126.1, 125.1, 122.6, 116.6, 99.0, 70.7,
62.7, 35.3, 32.3, 27.4, 7.2. HRMS calcd for C21H22O4: 339.1590,
found 339.1591.
Synthesis of 2-[4-(4-Fluorobutyl)benzyloxy]-3-methylchromen-
4-one (11). To a solution of 2-[4-(4-hydroxybutyl)benzyloxy]-3-
methylchromen-4-one (25) (101 mg, 0.30 mmol) dissolved in
anhydrous DCM (3.0 mL) were added TsCl (68 mg, 0.36 mmol),
DMAP (55 mg, 0.45 mmol), and TEA (0.050 mL, 0.36 mmol).
The reaction mixture was stirred at room temperature for 20 h.
The reaction mixture was diluted with water (3 mL), and the
aqueous layer was separated and extracted with ethyl acetate (3 ×
10 mL). All combined organic layers were dried over Na2SO4 and
concentrated to yield an oil. The crude material was purified using
silica gel chromatography (4:1 pentane:EtOAc) to yield toluene-
4-sulfonic acid 4-[4-(3-methyl-4-oxo-4H-chromen-2-yloxymethyl)-
phenyl]butyl ester 38 (75.2 mg, 0.15 mmol, 51%). 1H NMR (CDCl3,
600 MHz): δ 8.22 (dd, J ) 8.2, 1.8 Hz, 1H), 7.77 (d, J ) 8.4 Hz,
2H). 7.61 (m, 1H), 7.41-7.33 (m, 6H), 7.16 (d, J ) 8.0 Hz, 2H),
5.44 (s, 2H), 4.04 (t, J ) 5.9 Hz, 2H), 2.59 (t, J ) 6.8 Hz, 2H),
2.44 (s, 3H), 2.00 (s, 3H), 1.68 (m, 4H). 13C NMR (CDCl3, 150
MHz): δ 178.2, 161.8, 152.2, 144.2, 142.0, 132.7, 132.2, 131.9,
129.3, 128.3, 127.7, 127.4, 125.6, 124.7, 122.1, 116.1, 98.5, 70.1,
69.8, 34.3, 27.9, 26.5, 21.1, 6.7. HRMS calcd for C28H28O6S:
515.1498, found 515.1493.
Synthesis of 2-[4-(4-Hydroxybutyl)benzylsulfanyl]-3-methyl-
chromen-4-one (19). To a solution of 2-mercaptochromen-4-one
(16) (1.52 g, 7.90 mmol) and 4-(4-hydroxymethylphenyl)butan-1-
ol (18) (1.90 g, 9.90 mmol) dissolved in anhydrous THF (80 mL)
were added solid PPh3 (3.11 g, 11.90 mmol) and DIAD (2.30 mL,
11.90 mmol). After complete addition, the reaction mixture was
stirred at room temperature for 20 h. The reaction mixture was
diluted with water (80 mL). The aqueous layer was separated and
extracted with ethyl acetate (3 × 100 mL). All combined organic
layers were dried over Na2SO4 and concentrated to yield an oil.
The crude material was purified using silica gel chromatography
(1:1 pentane:EtOAc) to yield compound 19 (1.29 g, 3.64 mmol,
1
46%). H NMR (DMSO-d6, 600 MHz): δ 8.01 (dd, J ) 7.9, 2.2
Hz, 1H), 7.78 (ddd, J ) 8.6, 7.0, 1.6 Hz, 1H), 7.70 (d, J ) 8.5 Hz,
2H), 7.49-7.44 (m, 2H), 7.38 (d, J ) 8.1 Hz, 2H), 7.15 (d, J )
8.0 Hz, 2H), 4.50 (s, 2H), 3.37 (t, J ) 6.5 Hz, 2H), 2.55-2.49 (m,
2H), 1.91 (s, 3H), 1.60-1.49 (m, 2H), 1.43-1.34 (m, 2H). 13C
NMR (DMSO-d6, 150 MHz): δ 173.8, 161.9, 156.0, 141.6, 134.3,
133.5, 128.8, 128.5, 125.4, 125.2, 121.8, 117.5, 115.9, 60.4, 34.5,
34.1, 32.0, 27.3, 10.2. HRMS calcd for C21H22O3S: 355.1362, found
355.1363.
Synthesis of 2-[4-(4-Fluorobutyl)benzylsulfanyl]-3-methyl-
chromen-4-one (10). To a solution of 2-[4-(4-hydroxybutyl)-
benzylsulfanyl]-3-methylchromen-4-one (19) (300 mg, 0.85 mmol)
dissolved in anhydrous DCM (8.0 mL) were added TsCl (194 mg,
1.01 mmol), DMAP (124 mg, 1.01 mmol), and TEA (0.213 mL,
1.52 mmol). The reaction mixture was stirred at room temperature
for 3 h and was then diluted with water (4 mL). The aqueous layer
was separated and extracted with ethyl acetate (3 × 10 mL). All
combined organic layers were dried over Na2SO4 and concentrated
to yield an oil. The crude material was purified using silica gel
chromatography (1:1 pentane:EtOAc) to yield 20 (280 mg, 0.55
1
mmol, 65%). H NMR (CDCl3, 300 MHz): δ 8.20 (dd, J ) 7.8,
1.70 Hz, 1H), 7.80-7.76 (m, 2H), 7.62 (ddd, J ) 8.6, 6.9, 1.6 Hz,
1H), 7.41-7.29 (m, 6H), 7.08 (d, J ) 8.2 Hz, 2H), 4.38 (s, 2H),
4.03 (t, J ) 6.0 Hz, 2H), 2.55 (t, J ) 7.2 Hz, 2H), 2.45 (s, 3H),
2.05 (s, 3H), 1.65 (m, 4H). 13C NMR (CDCl3, 150 MHz): δ 175.5,
162.1, 156.7, 144.9, 141.5, 134.1, 133.4, 133.0, 130.0, 129.1, 129.0,
128.1, 126.5 125.3, 122.9, 117.5, 117.0, 70.5, 35.3, 34.9, 28.6, 27.2,
21.8, 10.8. HRMS calcd for C28H28O5S2: 509.1450, found 509.1441.
To a solution of 20 (10 mg, 0.020 mmol) in anhydrous ACN
(0.2 mL) were added KF (2.28 mg, 0.04 mmol) and Kryptofix222
(14.8 mg, 0.04 mmol). After complete addition, the reaction mixture
was heated at 90 °C for 25 min. The resultant mixture was allowed
to cool to room temperature and diluted with water (1 mL). The
aqueous layer was separated and extracted with ethyl acetate (3 ×
2 mL). All combined organic layers were dried over Na2SO4 and
concentrated to yield an oil. Purification by HPLC using a
Phenomenex Luna C-18 (2) column (10 µm, 250 × 21.2 mm,
gradient method of 20-80% B over 30 min, where B ) 90% ACN
in water using 0.1% TFA as a modifier and A ) water using 0.1%
TFA as a modifier) with a flow rate of 20 mL/min afforded
compound 10 (3.3 mg, 0.01 mmol, 46%). 19F NMR (CDCl3, 564
To a solution of 38 (20 mg, 0.04 mmol) in anhydrous ACN (0.5
mL) were added KF (4.72 mg, 0.08 mmol) and Kryptofix222 (30.6
mg, 0.08 mmol). After complete addition, the reaction mixture was
heated at 90 °C for 15 min. The reaction mixture was cooled to
room temperature and diluted with water (1 mL). The aqueous layer
was separated and extracted with ethyl acetate (3 × 5 mL). All
combined organic layers were dried over Na2SO4 and concentrated
to yield an oil. Purification by HPLC using a Phenomenex Luna
C-18 (2) column (10 µm, 250 × 21.2 mm, isocratic method of
70% B over 30 min, where B ) 90% ACN in water using 0.1%
TFA as a modifier and A ) water using 0.1% TFA as a modifier)
with a flow rate of 20 mL/min afforded compound 11 (6.8 mg,
0.02 mmol, 13.6%).19F NMR (CDCl3, 564 MHz): δ -218.72 (m,
1
MHz): δ -218.67 (m, 1F). H NMR (CDCl3, 600 MHz): δ 8.18
(dd, J ) 8.0, 1.3 Hz, 1H), 7.60 (ddd, J ) 8.5, 6.7, 1.3 Hz, 1H),
7.39-7.35 (m, 4H), 7.13 (d, J ) 8.2 Hz, 2H), 4.47 (m, 1H), 4.39
(t, J ) 5.7 Hz, 1H), 4.36 (s, 2H), 2.63 (t, J ) 7.5 Hz, 2H), 2.03 (s,
3H), 1.73-1.67 (m, 4H). 13C NMR (CDCl3, 150 MHz): δ 175.3,
162.0, 156.5, 141.7, 133.8, 132.7, 128.8 (2C), 126.2, 125.2, 122.6,
117.3, 116.8, 84.4 (83.3), 35.1, 35.0, 29.9 (29.8), 26.9, 10.5. HRMS
calcd for C21H21FO2S: 357.1319, found 357.1324.
Synthesis of 2-[4-(4-Hydroxybutyl)benzyloxy]-3-methyl-
chromen-4-one (25). Solid NaH (37 mg, 1.5 mmol) was placed in
a reaction flask and cooled to 0 °C in an ice bath. A solution of
{4-[4-(tert-butyldimethylsilanyloxy)butyl]phenyl}methanol (23) (377
mg, 1.28 mmol) in dry DMF (23 mL) was added to the reaction
flask dropwise with stirring. After complete addition, the reaction
mixture was stirred at 0 °C for an additional hour. A solution of
1
1F). H NMR (CDCl3, 600 MHz): δ 8.21 (dd, J ) 8.2, 1.7 Hz,
1H), 7.59 (m, 1H), 7.39 (m, 4H), 7.21 (d, J ) 8.0 Hz, 2H), 5.43 (s,
2H), 4.48 (m,1H), 4.40 (t, J ) 5.6 Hz, 1H), 2.67 (t, J ) 7.8 Hz,
2H), 1.98 (s, 3H), 1.76-1.69 (m, 4H); 13C NMR (CDCl3, 150
MHz): δ 178.7, 162.3, 152.2, 142.9, 132.6, 132.3, 128.8, 128.2,

MC-I Inhibitor as Potential Cardiac PET Tracer
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4311
126.1, 125.2, 122.6, 116.1, 99.0, 84.4 (83.3), 70.6, 35.1, 23.0 (29.9),
26.9, 7.2. HRMS calcd for C21H21FO3: 341.1547, found 341.1547.
Synthesis of 2-[4-(4-Hydroxybutyl)benzylamino]-3-methyl-
chromen-4-one (29). A solution of 2-[4-(4-hydroxybutyl)benzyl]-
isoindole-1,3-dione (34) (964 mg, 3.12 mmol) and hydrazine (215
µL, 6.86 mmol) in n-butanol (59 mL) was heated at reflux for 1 h.
Upon cooling to room temperature, a precipitate formed which was
removed by filtration and washed with n-butanol. The filtrate was
then concentrated in vacuo to obtain 4-(4-aminomethylphenyl)butan-
1-ol (28) as a yellow solid (0.47 g, 2.61 mmol, 84%), which was
used in the next step without any further purification.
in acetone (120 mL) was added iodomethane (807 µL, 12.93 mmol).
The reaction was stirred under nitrogen atmosphere at room
temperature for 16 h and then was concentrated to yield a crude
oil. The residue was taken up in water (50 mL) and adjusted to pH
7 with 5% HCl. The resulting aqueous layer was extracted with
ethyl acetate (3 × 200 mL). The organic layer was then washed
with water (1 × 200 mL) and brine (1 × 200 mL), dried over
sodium sulfate, and concentrated to yield 3-methyl-2-methylsulfa-
nylchromen-4-one as a yellow solid (1.95 g, 9.45 mmol, 80%),
which was taken on to the next step without further purification.
1H NMR (DMSO-d6, 300 MHz): δ 8.03-7.99 (m, 1H), 7.77-
7.71 (m, 1H), 7.62 (d, J ) 8.5 Hz, 1H), 7.48-7.43 (m, 1H), 2.69
(d, J ) 0.8 Hz, 3H), 1.94 (d, J ) 0.8 Hz, 3H). 13C NMR (DMSO-
d6, 75 MHz): δ 174.0, 163.2, 156.4, 133.7, 125.8, 125.6, 122.3,
118.0, 115.4, 13.3, 10.5. HRMS calcd for C11H10O2S: 207.04743,
found 207.04743.
To a solution of 2-methanesulfinyl-3-methylchromen-4-one (26)
(0.35 g, 1.78 mmol) in ACN (37 mL) were added 4-(4-aminom-
ethylphenyl)butan-1-ol (28) (0.47 g, 2.13 mmol) and DMF (18 mL).
The reaction was heated with stirring at 50 °C in an oil bath
overnight under N2 atmosphere. The reaction mixture was allowed
to cool to room temperature and concentrated to yield an oil.
Purification of the oil by silica gel chromatography (100% EtOAc)
To a solution containing 3-methyl-2-methylsulfanylchromen-4-
one (0.97 g, 4.70 mmol) dissolved in DCM (19 mL) and cooled at
0 °C in an ice bath was added mCPBA (2.03 g, 11.75 mmol)
dissolved in DCM (19 mL). The resultant mixture was stirred for
2 h at 0 °C and then warmed up to room temperature overnight.
The next day, the reaction mixture was filtered and the resulting
filtrate was washed with 1 N NaOH (2 × 100 mL), dried over
sodium sulfate, and concentrated to obtain compound 21 as a white
solid (0.84 g, 3.53 mmol, 75%), which was taken on to the next
1
afforded the desired product 29 (120 mg, 0.32 mmol, 18%). H
NMR (CDCl3, 600 MHz): δ 8.21 (d, J ) 7.7 Hz, 1H), 7.53 (t, J
) 8.3 Hz, 1H), 7.35 (t, J ) 7.4 Hz, 1H), 7.31 (d, J ) 8.3 Hz, 1H),
7.29 (d, J ) 7.9 Hz, 2H), 7.21 (d, J ) 7.9 Hz, 2H), 4.83 (br t, 1H),
4.66 (m, 2H), 3.67 (m, 2H), 2.67 (t, J ) 7.7 Hz, 2H), 1.98 (s, 3H),
1.74-1.69 (m, 2H), 1.64-1.60 (m, 2H), 1.24 (t, J ) 5.4 Hz, 1H).
13C NMR (DMSO-d6/CDCl3, 150 MHz): δ 174.0, 160.3, 152.2,
141.4, 135.5, 130.7, 128.2, 126.9, 125.0, 123.8, 122.3, 115.8, 92.7,
61.5, 44.6, 34.8, 31.8, 27.2, 7.3. HRMS calcd for C21H23NO3:
380.1751, found 338.1753.
1
step without further purification. H NMR (CDCl3, 600 MHz): δ
8.08 (dd, J ) 8.0, 1.4 Hz, 1H,), 7.90 (m, 1H), 7.75 (d, J ) 8.0 Hz,
1H), 7.56 (m, 1H), 3.52 (s, 3H), 2.30 (s, 3H). 13C NMR (DMSO-
d6, 150 MHz): δ 177.3, 156.2, 154.6, 135.3, 126.3, 125.3, 121.5,
119.8, 118.5, 41.2, 8.5. HRMS calcd for C11H10O4SNa: 261.0192,
found 261.0196.
Synthesis of 2-[4-(4-Fluorobutyl)benzylamino]-3-methyl-
chromen-4-one (12). To a solution of 2-[4-(4-hydroxybutyl)-
benzylamino]-3-methylchromen-4-one (29) (100 mg, 0.30 mmol)
in DCM (37 mL) cooled at 0 °C in an ice bath were added TsCl
(68 mg, 0.36 mmol), DMAP (43.4 mg, 0.36 mmol), and TEA (62
µL, 0.44 mmol). The resultant slurry was stirred overnight under
N2 atmosphere, slowly warming to room temperature. The reaction
mixture was concentrated and purified by flash column chroma-
tography (3:1 hexane:EtOAc increasing gradient to 100% EtOAc)
to yield toluene-4-sulfonic acid 4-{4-[(3-methyl-4-oxo-4H-chromen-
2-ylamino)methyl]phenyl}butyl ester 39 as an oil (45 mg, 0.091
Synthesis of 2-Methanesulfinyl-3-methylchromen-4-one (26).
To a solution containing 3-methyl-2-methylsulfanylchromen-4-one
(1.95 g, 9.45 mmol) in DCM (75 mL) at 0 °C was added m-CPBA
(2.0 g, 11.82 mmol) and the resultant mixture was stirred for 2 h.
Upon consumption of the starting material, the solids were removed
by filtration, and the resulting filtrate was washed with cold 5%
sodium carbonate (1 × 150 mL), water (1 × 150 mL), and saturated
sodium bisulfate (1 × 150 mL). The organic layer was dried over
sodium sulfate and concentrated to obtain the desired product as a
light yellow solid (1.74 g, 7.84 mmol, 83%), which was taken on
to the next step without further purification. 1H NMR (CDCl3, 300
MHz): δ 8.21-8.17 (m, 1H,), 7.75-7.69 (m, 1H), 7.60-7.57 (m,
1H), 7.46-7.40 (m, 1H), 2.97 (s, 3H), 2.23 (s, 3H). 13C NMR
(CDCl3, 150 MHz): δ 177.2, 160.8, 156.3, 134.5, 126.1, 125.8,
122.5, 120.9, 118.1, 37.9, 9.3. HRMS calcd for C11H10O3S:
223.04234, found 223.04217.
1
mmol, 31%). H NMR (CDCl3, 600 MHz): δ 8.19 (dd, J ) 8.1,
1.1 Hz, 1H), 7.53 (m, 1H), 7.36-7.29 (m, 4H), 7.22 (d, J ) 7.9
Hz, 2H), 4.83 (br t, 1H), 4.67 (d, J ) 5.6 Hz, 2H), 3.68 (m, 2H),
2.67 (t, J ) 7.7 Hz, 2H), 1.98 (s, 3H), 1.73-1.69 (m, 2H), 1.64-
1.60 (m, 2H), 1.24 (t, J ) 5.4 Hz, 1H). 13C NMR (CDCl3, 75
MHz): δ 178.6, 162.6, 152.6, 140.7, 135.5, 132.3, 129.0, 128.6,
126.1, 125.6, 122.6, 116.6, 98.3, 84.1 (81.9), 68.9, 36.3, 35.0, 30.8
(30.6), 28.7, 27.4, 7.2. HRMS calcd for C28H29NO5S: 492.1839,
found 492.1846.
Synthesis of 4-(4-Hydroxybut-1-ynyl)benzoic Acid Methyl
Ester (30). Compound 30 was prepared according to the method
of Taylor et al.39 To a stirred solution of methyl 4-bromobenzoate
(13.4 g, 0.62 mmol) in diethyl amine (200 mL) were added
palladium chloride (0.55 g, 3.06 mmol) and triphenylphosphine
(0.16 g, 0.62 mmol). The solution was degassed, and copper iodide
(0.12 g, 0.62 mmol) and 3-butyn-1-ol (4.34 g, 62 mmol) were added.
The reaction mixture was stirred at room temperature for 48 h,
during which time an additional 0.5 mol % palladium chloride,
1.0 mol % triphenyl phosphine, and 12 mol % 3-butyn-1-ol were
added. Once the reaction was complete as judged by LCMS, the
reaction mixture was concentrated and the crude material was taken
up in a slurry of silica gel and EtOAc. The organic solvent was
removed and the remaining dried silica gel was packed in a fritted
funnel. Extensive washes with a hexane:ethyl acetate mixture (1:
4) followed by ethyl acetate (100%) washes yielded compound 30
To a solution of 39 (24 mg, 0.049 mmol) in ACN (1.1 mL) was
added Kryptofix222 (37 mg, 0.098 mmol) followed by KF (6 mg,
0.098 mmol). The reaction was heated at 90 °C for 30 min with
stirring under nitrogen atmosphere. The reaction was then cooled
to room temperature, filtered, and injected directly onto the
preparative HPLC coloum chromatography (Luna, 10 µm, C18, 250
× 21.2 mm 10 micro, 60% B isocratic method over 40 min, where
B ) 90% ACN in water using 0.1% TFA as a modifier and A )
water with 0.1% TFA as the modifier). The desired fractions were
collected and neutralized to pH 7.6 and then lyophilized. The
material was repurified by flash column chromatography (3:1
hexanes: EtOAc) to obtain the desired product 12 (0.3 mg, < 2%).
19F NMR (CDCl3, 564 MHz): δ -178.2 (m, 1F). 1H NMR (CDCl3,
300 MHz): δ 8.21-8.17 (m, 1H), 7.53-7.48 (m, 1H), 7.35-7.28
(m, 4H), 7.19 (d, J ) 9.0 Hz, 2H), 4.88 (br t, 1H), 4.65 (d, J ) 6.0
Hz, 2H), 4.52 (m, 1H), 4.37 (m, 1H), 2.66 (m, 2H), 1.96 (s, 3H),
1.76-1.67 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ 175.0, 160.2,
152.8, 141.9, 135.3, 131.4, 129.0, 127.6, 125.9, 124.6, 122.9, 116.2,
93.4, 85.0 (82.77), 45.5, 35.0, 30.1 (29.8), 27.0 (26.9), 7.6. HRMS
calcd for C21H22FNO2: 340.1707, found 340.1701.
1
(11.9 g, 0.58 mmol, 94%). H NMR (CDCl3, 300 MHz): δ 7.97
(d, J ) 8.58 Hz, 2H), 7.47 (d, J ) 8.7 Hz, 2H), 3.92 (s, 3H), 3.83
(t, J ) 6.4 Hz, 2H), 2.73 (t, J ) 6.3 Hz, 2H). 13C NMR (CDCl3,
75 MHz): δ 166.8, 131.8, 129.6, 129.5, 128.4, 90.0, 82.0, 61.2,
1
52.4, 24.1. H NMR data corresponds to published data.
Synthesis of 2-Methanesulfonyl-3-methylchromen-4-one (21).
To a solution containing 2-mercapto-3-methylchromen-4-one (16)
(2.26 g, 11.76 mmol) and potassium carbonate (1.62 g, 11.76 mmol)
Synthesis of 4-(4-Hydroxybutyl)benzoic Acid Methyl Ester
(31). To a stirring solution of 4-(4-hydroxybut-1-ynyl)benzoic acid
methyl ester (30) (6.29 g, 0.031 mol) in ethanol (60 mL) was added

4312 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Radeke et al.
palladium on carbon (5 g, 10% on carbon), and the reaction mixture
pressurized to 50 psi under an atmosphere of hydrogen. After 20
h, the reaction mixture was filtered through Celite to remove the
catalyst and the filtrate was concentrated to afford compound 31
3-butyn-1-ol (417 µL, 5.51 mmol). The reaction was stirred at room
temperature overnight under nitrogen atmosphere. The reaction
mixture was concentrated and purified by flash column chroma-
tography (2:1 hexane:EtOAc) to yield compound 33 as a yellow
solid (0.76 g, 2.49 mmol, 45% yield). 1H NMR (CDCl3, 600
MHz): δ 7.86 (m, 2H), 7.72 (m, 2H), 7.36 (s, 4H), 4.83 (s, 2H),
3.80 (m, 2H), 2.68 (t, J ) 6.0 Hz, 2H), 1.78 (t, J ) 6.6 Hz, 2H).
13C NMR (CDCl3, 150 MHz): δ 167.6, 136.4, 134.6, 131.5, 131.4,
127.5, 123.2, 122.3, 88.7, 80.6, 59.7, 40.6, 23.2. HRMS calcd for
C19H15NO3Na: 328.0944, found 328.0947.
Synthesis of 2-[4-(4-Hydroxybutyl)benzyl]isoindole-1,3-dione
(34). To a solution of 2-[4-(4-hydroxybut-1-ynyl)benzyl]isoindole-
1,3-dione (33) (2 g, 6.55 mmol) in EtOH/EtOAc (3:1, 163 mL)
was added palladium on carbon (1.04 g, 10 wt %). The reaction
was stirred at room temperature overnight under 50 psi of hydrogen.
The reaction was monitored by 1H NMR for conversion to product.
Upon completion, the reaction mixture was filtered through Celite,
washed with EtOAc, and concentrated to obtain compound 34 as
a yellow oil (1.88 g, 6.14 mmol, 93% yield). 1H NMR (DMSO-d6,
600 MHz): δ 7.90-7.85 (4H, m), 7.21 (d, J ) 8.0 Hz, 2H), 7.14
(d, J ) 7.8 Hz, 2H), 6.51 (br s, 1H), 4.73 (s, 2H), 3.38 (t, J ) 6.6
Hz, 2H), 2.53 (t, J ) 7.5 Hz, 2H), 1.57-1.52 (m, 2H), 1.42-1.37
(m, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 167.7, 141.5, 134.5,
133.9, 131.5, 128.4, 127.3, 60.4, 34.5, 32.0, 27.3.
Synthesis of 1-(4-Hydroxymethylphenoxy)propan-2-ol (36).
To a suspention of 4-hydroxybenzyl alcohol (1 g, 8.06 mmol) and
K2CO3 (1.34 g, 9.68 mmol) in acetone (80 mL) was added
chloroacetone (0.771 mg, 9.68 mmol). After complete addition, the
reaction mixture was heated at reflux overnight. The reaction
mixture was concentrated to yield a crude oil, which was partitioned
between EtOAc (100 mL) and water (100 mL). The aqueous layer
was separated and extracted with EtOAc (2 × 150 mL). Combined
organic layers were dried over Na2SO4 and concentrated. The
resulting crude material was partially purified using silica gel
chromatography (4:1 pentane:EtOAc to 1:1 pentane:EtOAc) to yield
1-(4-hydroxybenzyloxy)propan-2-one (35) in addition to 4-hy-
droxybenzyl alcohol (10% mol present according to 1H NMR, 981
mg of product mixture obtained). 1H NMR (DMSO-d6, 600
MHz): δ 7.32-7.29 (m, 2H), 6.90-6.85 (m, 2H), 4.54 (s, 2H,),
2.28 (s, 3H), 13C NMR (CDCl3, 75 MHz): δ 201.0, 157.6, 134.5,
129.0, 114.8, 73.4, 65.1, 26.8.
To a solution of the partially purified 35 (1.26 g) dissolved in
MeOH (60 mL) was added solid NaBH4 (0.32 g, 8.39 mmol). After
complete addition, the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with water
(30 mL), the layers were separated, and the aqueous layer was
extracted with ethyl acetate (3 × 50 mL). All combined organic
layers were dried over Na2SO4 and concentrated to yield 36 as an
oil (1.24 g, 6.81 mmol, 98% yield). 1H NMR (DMSO-d6, 600
MHz): δ 7.21 (m, 2H), 6.87 (m, 2H), 5.00 (br s, 1H), 4.8 (br s,
1H), 4.41 (s, 2H,), 3.95-3.91 (m, 1H), 3.82-3.73 (m, 2H), 1.14
(d, J ) 6.32 Hz, 3H). 13C NMR (DMSO-d6, 150 MHz): δ 157.6,
134.4, 127.9, 114.1, 73.2, 64.5, 62.5, 20.1.
1
(5.67 g, 0.027 mol, 89%). H NMR (CDCl3, 600 MHz): δ 7.96
(d, J ) 8.1 Hz, 2H), 7.26 (d, J ) 8.6 Hz, 2H), 3.91 (s, 3H), 3.68
(t, J ) 6.6 Hz, 2H), 2.71 (t, J ) 7.5 Hz, 2H), 1.76-1.71 (m, 2H),
1.64-1.59 (m, 2H). 13C NMR (CDCl3, 75 MHz): δ 167.2, 147.9,
129.8, 128.4, 127.8, 62.7, 52.0, 35.7, 32.2, 27.2.
Synthesis of 4-(4-Hydroxymethylphenyl)butan-1-ol (18). To
a stirred solution of 4-(4-hydroxybutyl)benzoic acid methyl ester
(31) (2.24 g, 0.01 mol) in THF (100 mL) was added dropwise a
solution of lithium aluminum hydride (8.0 mL, 1 M in THF). After
complete addition the reaction mixture was stirred at room
temperature for 6 h. The reaction mixture was quenched with water
(50 mL), the layers were separated, and the aqueous layer was
extracted with ethyl acetate (3 × 150 mL). All combined organic
layers were dried over Na2SO4 and concentrated to afford compound
18 (1.90 g, 0.01 mol, 98%). 1H NMR (CDCl3, 600 MHz): δ 7.27
(d, J ) 8.3 Hz, 2H), 7.17 (d, J ) 8.0 Hz, 2H), 4.64 (s, 2H), 3.64
(t, J ) 6.4 Hz, 2H), 2.64 (t, J ) 7.6 Hz, 2H), 1.71-1.66 (m, 2H),
1.62-1.57 (m, 2H). 13C NMR (CDCl3, 75 MHz): δ 141.9, 138.4,
128.6, 127.2, 65.3, 62.8, 35.3, 32.3, 27.5.
Synthesis of {4-[4-(tert-Butyldimethylsilanyloxy)butyl]phenyl}-
methanol (23). To a solution of 4-(4-hydroxybutyl)benzoic acid
methyl ester (31) (300 mg, 1.44 mmol) in DMF (4 mL) was added
imidazole (147 mg, 2.16 mmol) followed by TBDMS-Cl (324 mg,
2.16 mmol). The reaction was stirred at room temperature for 2 h,
with monitoring by TLC (3:1 hexane:EtOAc). Upon consumption
of the starting material, the reaction was diluted with EtOAc (25
mL) and washed with water (3 × 50 mL) and saturated sodium
bicarbonate (1 × 50 mL). The organic layer was dried over sodium
sulfate and concentrated down to obtain 4-[4-(tert-butyldimethyl-
silanoxy)butyl]benzoic acid methyl ester (360 mg, 1.12 mmol,
1
77%). H NMR (DMSO-d6, 600 MHz): δ 7.87 (d, J ) 8.3 Hz,
2H), 7.32 (d, J ) 8.6 Hz, 2H), 3.83 (s, 3H), 3.50 (t, J ) 6.5 Hz,
2H), 2.66 (t, J ) 7.5 Hz, 2H), 1.66-1.61 (m, 2H), 1.49-1.45 (m,
2H), 0.85 (s, 9H), 0.01 (s, 6H). 13C NMR (DMSO-d6, 150 MHz):
δ 166.1, 148.0, 129.1, 128.6, 127.2, 62.1, 51.9, 34.7, 31.7, 26.8,
25.7, 17.8, -5.4.
To a stirred solution of 4-[4-(tert-butyldimethylsilanoxy)butyl]-
benzoic acid methyl ester (670 mg, 2.18 mmol) in THF (22 mL)
was added dropwise a solution of lithium aluminum hydride (2.18
mL, 1 M in THF). After complete addition, the reaction mixture
was stirred at room temperature for 3 h. The reaction mixture was
quenched with water (50 mL), the layers were separated, and the
aqueous layer was extracted with ethyl acetate (3 × 150 mL). All
combined organic layers were dried over Na2SO4 and concentrated
1
to afford compound 23 (586.8 mg, 2.00 mmol, 92%). H NMR
(CDCl3, 600 MHz): δ 7.29 (d, J ) 8.0 Hz, 2H), 7.19 (d, J ) 8.0
Hz, 2H), 4.67 (s, 3H), 3.64 (t, J ) 6.3 Hz, 2H), 2.65 (t, J ) 7.9
Hz, 2H), 1.74-1.64 (m, 2H), 1.62-1.52 (m, 2H), 0.91 (s, 9H),
0.06 (s, 6H). 13C NMR (DMSO-d6, 150 MHz): δ 142.4.0, 138.5,
128.8, 127.3, 65.4, 63.2, 35.6, 32.6, 27.9, 26.2, 18.6, -5.1.
Synthesis of 2-[4-(4-Hydroxybut-1-ynyl)benzyl]isoindole-1,3-
dione (33). To a solution of 4-iodobenzyl bromide (9.04 g, 30.4
mmol) in DMF (316 mL) were added phthalimide (4.47 g, 30.4
mmol) and cesium carbonate (14.86 g, 45.6 mmol). The reaction
was stirred at room temperature overnight under nitrogen atmo-
sphere. The reaction mixture was diluted with water (500 mL) and
2-(4-iodobenzyl)isoindole-1,3-dione precipitated out of solution. The
white solid was collected by filtration and washed with water to
afford the desired compound (9.50 g, 26.15 mmol, 86% yield),
which was used in the next step without additional purification.
1H NMR (CDCl3, 600 MHz): δ 7.83 (m, 2H), 7.71 (m, 2H), 7.63
(m, 2H), 7.17 (m, 2H), 4.77 (s, 2H). 13C NMR (CDCl3, 150 MHz):
δ 168.1, 138.0, 136.2, 134.5, 132.2, 130.8, 123.7, 93.7, 41.3.
To a slurry of 2-(4 iodobenzyl)isoindole-1,3-dione (2 g, 5.51
mmol), triphenylphosphine (14.4 mg, 0.055 mmol), and palladium
chloride (5 mg, 0.028 mmol) in diethylamine (20 mL) were added
DMF (4 mL) and copper iodide (11 mg, 0.055 mmol) followed by
Synthesis of 2-[4-(4-Hydroxypropoxy)benzylsulfanyl]-3-me-
thylchromen-4-one (37). To a solution of 2-mercapto-3-methyl-
chromen-4-one (16) (115.4 mg, 0.60 mmol), 1-(4-hydroxymeth-
ylphenoxy)propan-2-ol (36) (131.3 mg, 0.72 mmol) and PPh3 (236.4
mg, 0.90 mmol) dissolved in THF (6 mL) was added DIAD (174.6
µL, 0.90 mmol) dropwise. After complete addition, the reaction
was stirred at room temperature overnight. The reaction mixture
was concentrated in vacuo to yield a yellow oil, which was purified
using silica gel chromatography (3:2 pentane:EtOAc) to obtain
compound 37 (190 mg, 0.534 mmol, 89%).1H NMR (CDCl3, 300
MHz): δ 8.18 (dd, J ) 8.03,1.63 Hz, 1H), 7.61 (ddd, J ) 8.6, 6.9,
1.6 Hz, 1H), 7.40-7.30 (m, 4H), 6.86 (d, J ) 8.7 Hz, 2H), 4.35 (s,
2H), 4.22-4.12 (m, 1H), 3.91 (dd, J ) 9.2, 3.3 Hz, 1H), 3.77 (dd,
J ) 9.3, 7.6 Hz, 1H), 2.04 (s, 3H), 1.27 (d, J ) 6.4 Hz, 3H). 13C
NMR (CDCl3, 150 MHz): δ 174.8, 161.5, 157.7, 156.0, 132.3,
129.6, 128.2, 125.7, 124.6, 122.1, 116.8, 116.3, 114.4, 72.9, 65.7,
34.4, 18.3, 10.1. HRMS calcd for C20H20O4S: 357.1155, found
357.1157.

MC-I Inhibitor as Potential Cardiac PET Tracer
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4313
Synthesis of 2-[4-(2-Fluoropropoxy)benzylsulfanyl]-3-meth-
ylchromen-4-one (13). To a solution of 2-[4-(4-hydroxypropoxy)-
benzylsulfanyl]-3-methylchromen-4-one (37) (345 mg, 0.97 mmol)
dissolved in anhydrous DCM (10.0 mL) were added TsCl (222 mg,
1.16 mmol), DMAP (178 mg, 1.45 mmol), and TEA (0.162 mL,
1.16 mmol). The reaction mixture was stirred at room temperature
for 20 h. The reaction mixture was diluted with water (10 mL),
and the aqueous layer was separated and extracted with ethyl acetate
(3 × 20 mL). All combined organic layers were dried over Na2-
SO4 and concentrated to yield an oil. The crude material was
purified using silica gel chromatography (4:1 pentane:EtOAc) to
yield toluene-4-sulfonic acid 1-methyl-[4-(3-methyl-4-oxo-4H-
chromen-2-ylsulfanylmethyl)phenoxy]ethyl ester (387 mg, 0.75
mmol, 78%).1H NMR (CDCl3, 600 MHz): δ 8.21 (dd, J ) 8.2,1.5
Hz, 1H), 7.78 (d, 2H, J ) 8.3 Hz), 7.62 (m, 1H), 7.39 (m, 2H),
7.31 (m, 2H), 7.30 (m, 2H), 6.67 (m, 2H), 4.87 (m, 1H), 4.35 (s,
2H), 4.00 (m, 1H), 2.89 (m, 1H), 2.44 (s, 3H), 2.05 (s, 3H), 1.41
(m, 3H). 13C NMR (CDCl3, 150 MHz): δ 175.5, 162.0, 157.9,
156.7, 144.9, 134.3, 133.0, 130.2, 129.9, 129.1, 128.1, 126.5, 125.3,
122.9, 117.7, 117.0, 115.0, 70.1, 35.1, 21.8, 18.0, 10.8. HRMS calcd
for C27H26O6S: 511.1243, found 511.1247.
To a solution of toluene-4-sulfonic acid 1-methyl-[4-(3-methyl-
4-oxo-4H-chromen-2-ylsulfanylmethyl)phenoxy]ethyl ester (100
mg, 0.20 mmol) in anhydrous ACN (2.8 mL) were added KF (22.8
mg, 0.39 mmol) and Kryptofix222 (146.8 mg, 0.39 mmol). After
complete addition, the reaction mixture was heated at 90 °C for 30
min. The reaction mixture was cooled to room temperature and
concentrated to yield a dark orange oil. The crude material was
redissolved in DCM (2.0 mL) and purified using a preparative TLC
plate (4:1 hexane:EtOAc) to obtain compound 13 (43.0 mg, 0.12
mmol, 60%).19F NMR (DMSO-d6, 564 MHz): δ -178.2 (m, 1F).
1H NMR (DMSO-d6, 600 MHz): δ 8.00 (dd, J ) 8.3,1.6 Hz, 1H),
7.77 (m, 1H), 7.72 (d, J ) 8.3 Hz, 1H), 7.47 (m, 1H), 7.40 (d, J )
8.8 Hz, 2H), 6.91 (d, J ) 8.8, 2H), 5.02-4.91 (m, 1H), 4.50 (s,
2H), 4.41-3.98 (m, 2H), 1.91 (s, 3H), 1.35 (dd, J ) 6.6, 23.7 Hz,
3H). 13C NMR (DMSO-d6, 150 MHz): δ 173.8, 161.9, 157.5, 156.0,
133.5, 131.7, 130.2, 129.3, 125.4, 125.1, 121.8, 117.5, 116.0, 115.0,
114.6, 89.0 (87.9), 70.4 (70.2), 33.9, 16.8, 10.2. HRMS calcd for
C20H19FO3: 359.1111, found 359.1114.
Synthesis of 2-{4-[4-(2-Fluoroethoxy)butyl]benzylsulfanyl}-
3-methylchromen-4-one (14). To a solution of 2-[4-(4-fluorobutyl)-
benzylsulfanyl]-3-methylchromen-4-one (10) (50 mg, 0.15 mmol)
dissolved in DMF (1.0 mL) was added solid NaH (4.26 mg, 0.18
mmol). After 15 min of stirring at room-temperature, 1-bromo-2-
fluoroethane (13.18 µL, 0.18 mmol) was added, and the reaction
was stirred at room temperature overnight. The reaction mixture
was quenched with water (0.5 mL), the layers were separated, and
the aqueous layer was extracted with ethyl acetate (3 × 2 mL). All
combined organic layers were dried over Na2SO4 and concentrated
to yield a crude oil. Purification by HPLC using a Phenomenex
Luna C-18 (2) column (10 µm, 250 × 21.2 mm, gradient method
of 35-95% B over 33 min, where B ) 90% ACN in water using
0.1% TFA as a modifier and A ) water using 0.1% TFA as a
modifier) with a flow rate of 20 mL/min afforded compound 14
(10.2 mg, 0.025 mmol, 6.0%). 19F NMR (DMSO-d6, 564 MHz):
δ -212.53 (m, 1F). 1H NMR (CDCl3, 300 MHz): δ 8.18 (dd, J )
6.0, 9.0 Hz, 1H, m), 7.61-7.55 (m, 1H), 7.40-7.32 (m, 2H), 7.21
(d, J ) 6.0 Hz, 2H), 7.14 (d, J ) 6.0 Hz, 2H), 4.55 (t, J ) 6.0 Hz,
1H), 4.44 (t, J ) 6.0 Hz, 2H), 4.37 (d, J ) 9.0 Hz, 1H), 2.74-2.63
(m, 4H), 1.97 (s, 3H), 1.85-1.80 (m, 4H). HRMS calcd for C19H15-
NO3Na: 401.1581, found 401.1583.
Synthesis of 2-[4-(2-Fluoroethoxymethyl)benzylsulfanyl]-3-
methylchromen-4-one (15). To a stirred solution of 1,4-benzene-
dimethanol (81.5 g, 0.59 mol) in DMF (236 mL) was added
dropwise a solution of KHMDS (0.5 M in toluene, 283.2 mL, 0.14
mol). After complete addition, the reaction mixture was stirred at
room temperature for 2 h. 1-Bromo-2-fluoroethane (8.80 mL, 0.12
mol) was added dropwise and the reaction was stirred at room
temperature overnight. The reaction mixture was poured over ice,
and the organic layer was separated and concentrated in vacuo to
yield an oily solid. The crude material was redissolved in a minimal
amount of toluene and cooled at -20 °C for several hours to
precipitate excess 1,4-benzenedimethanol from the solution, which
was removed via filtration. The filtrate collected was washed
multiple times with water to remove remaining 1,4-benzene-
dimethanol and the resultant organics were concentrated to yield a
yellow oil. The remaining excess 1,4-benzenedimethanol was
removed via liquid-liquid extraction over 24 h using water and
DCM to obtain [4-(2-fluoroethoxymethyl)phenyl]methanol as a
1
white solid (10.4 g, 56.5 mmol, 47%). H NMR (DMSO-d6, 600
MHz): δ 7.29 (m, 4H), 5.15 (m, 1H), 4.59 (m, 1H), 4.51-4.48
(m, 5H), 3.68 (m, 1H), 3.63 (m, 1H).
To a solution of 2-mercapto-3-methylchromen-4-one (80.9 mg,
0.42 mmol), 4-(2-fluoroethoxymethyl)phenyl]methanol (93 mg, 0.51
mmol), and PPh3 (165 mg, 0.63 mmol) dissolved in THF (1.0 mL)
was added DIAD (122.4 µL, 0.63 mmol) dropwise. After complete
addition, the reaction was stirred at room temperature overnight.
The reaction mixture was concentrated in vacuo to yield a yellow
oil, which was purified using silica gel chromatography (4:1
pentane:EtOAc) to obtain the desired product mixed with DIAD
byproducts. Repurification via silica gel chromatography (4:1 DCM:
hexane to 100% DCM) afforded the desired compound in low yield
1
(3.5 mg, 9.75 mmol, 23%). H NMR (CDCl3, 600 MHz): δ 8.18
(dd, J ) 8.0, 2.2 Hz, 1H), 7.60 (ddd, J ) 8.7, 7.1, 1.4 Hz, 1H),
7.39-7.35 (m, 4H), 7.31 (d, J ) 8.5 Hz, 2H), 4.60 (m, 1H), 4.56
(s, 2H), 3.72 (m, 1H), 3.67 (m, 1H), 2.03 (s, 3H). 13C NMR (150
MHz, CDCl3): δ 175.3, 161.6, 156.5, 137.5, 136.0, 132.8, 128.9,
128.1, 126.2, 125.0, 122.6, 117.4, 116.8, 83.6 (82.5), 72.9, 69.4
(69.3), 35.1, 10.6. HRMS calcd for C20H19FO3S: 359.1111, found
359.1112.
Preparation of Submitochondrial Particles from Bovine
Hearts. Bovine heart mitochondria were prepared as described by
Lester et al.41 A brief description of the procedure follows: bovine
heart was minced and 200 g of ground heart tissue was suspended
in 400 mL of 0.25 M sucrose, 0.01 M Tris-Cl, 1 mM Tris-succinate,
and 0.2 mM ethylenediaminetetraacetic acid (EDTA) and homog-
enized in a Waring blender. The homogenate was centrifuged for
20 min at 1200g and the supernatant was centrifuged for 15 min at
26 000g, resulting in a mitochondrial pellet. The protein concentra-
tion of the mitochondrial samples as measured by a Bio-Rad Protein
Assay Kit (Bio-Rad Life Science Research, Hercules, CA) was
adjusted to 20 mg/mL using 0.25 M sucrose, 10 mM Tris-acetate
pH 7.5, 1.5 mM adenosine triphosphate (ATP), and 10 mM MgCl2.
The samples were stored at -80 °C.
Bovine submitochondrial particles (SMPs) were prepared from
mitochondria as described by Matsuno-Yagi et al.43 Isolated bovine
heart mitochondria were sonicated in batches of 15 mL for 1 min
with a digital Branson sonifier (Branson, Danbury, CT) at 70%
maximum output in an ice bath. The sonicated suspension was
centrifuged at 16 000g for 10 min, and the supernatant was
centrifuged at 150 000g for 45 min at 4 °C. The submitochondrial
pellet was resuspended in buffer containing 0.25 M sucrose, 10
mM Tris-acetate, pH 7.5. The protein concentration was determined
using the Bio-Rad Protein Assay Kit (Bio-Rad Life Science
Research, Hercules CA), and the samples were stored at -80 °C,
at a concentration of 20 mg/mL.
Submitochondrial Particle (Bovine) Catalytic Activity and
Compound Inhibition Assay. The procedure for determining the
catalytic activity of submitochondrial particles was adapted from
Satoh et al.40 NADH-DB reductase activity was measured using a
stirred cuvette in a spectrophotometer (Hewlett-Packard, Houston
TX) at 37 °C, as the rate of NADH oxidation at 340 nm (ꢀ ) 5.4
mM-1 cm-1) for 120 s. The final volume of the reaction was 2.5
mL, containing 50 mM K2HPO4 (pH 7.4), 0.4 µM antimycin A,
and 2 mM KCN. The final SMP concentration was 45 µg/mL. The
enzyme reaction was initiated by the addition of 100 µM decyl
ubiquinone (DB) and 50 µM NADH. Inhibitors at varying
concentrations were preincubated with the reaction mixture contain-
ing SMPs for 4 min prior to initiation of the reaction. The IC50
value was determined as the concentration of the inhibitor required

4314 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Radeke et al.
for 50% inhibition of NADH oxidation. The IC50 value was
calculated using GraphPad Prism Version 4 (GraphPad, San Diego,
CA).
intravenously. Blood samples were collected from these rats by
the bleeding tail veins at 1, 3, 5, 7, 10, 45, and 90 min or puncturing
the heart at the time of tissue harvesting at 120 min after the
injection. Rats (n ) 3/each time point) were sacrificed in a CO2
chamber and blood samples were counted in an autogamma counter
(Wallac Wizard 1480, Perkin-Elmer Life and Analytical Sciences,
Shelton, CT) for radioactivity. The radioactivity measurements were
decay-corrected, and the blood clearance of [18F]10 was expressed
as percent injected dose per gram (%ID/g).
Procedure for the Radiosynthesis of 18F-Labeled S-Chromone
10. A 500 mCi sample of aqueous 18F was made by the 18O(p,n)18F
reaction (PETNET Pharmaceutical Services, Cummings Park,
Woburn, MA) and applied to a previously activated MP1 anion
exchange resin (Bio-Rad) cartridge. This cartridge was placed into
an elution loop contained within a remote control radiosynthesis
system. The radioactivity was eluted from the cartridge and collected
into a silanized 25 mL pear-shaped flask by the addition of 1 mL
of a solution prepared as follows: K2CO3 (15 mg) was dissolved
in deionized water (1 mL), and Kryptofix222 (90 mg) was dissolved
in anhydrous acetonitrile (4 mL); the two solutions were combined,
and the appropriate aliquot was used for elution of the column.
The eluate from the MP1 anion exchange resin was then concen-
trated to dryness by applying a gentle stream of heated He and a
slight vacuum. An aliquot of acetonitrile (0.5 mL) was added and
the solvent was again removed by the same vacuum heated He
procedure to ensure removal of all water. The material was
reconstituted with acetonitrile (0.5 mL). The solvated 18F with the
remaining constituents (K, CO32-, K222) was transferred to a
conical bottomed 5 mL Wheaton vial containing 3.0 mg of tosylate
precursor 20 (3.0 mg) dissolved in acetonitrile (0.5 mL). The vial
and contents were heated at 90 °C for 30 min. The reaction solution
was transferred to a 25 mL pear-shaped flask and diluted with water
(18.5 mL). The resultant mixture was passed through a Sep Pak
C18 cartridge, rinsed with water (5 mL). The Sep Pak C18 cartridge
was then eluted with acetonitrile (3 mL) and the column dried with
N2(g). The collected acetonitrile fraction was purified via HPLC
(Phenomenex LUNA C-18 column 250 × 10 mm, 5 µm particle
size, 100 Å pore, mobile phases A ) 90/10 H2O/CH3CN, B )
CH3CN, both containing 0.1% TFA as stabilizer; gradient 0-100/
15 sustained at 100% B to 20 min, flow rate 2.5 mL/min). The
18F-chromone analog 10 eluted from the column in 16-17 min
and was collected as a single fraction. The solvent was then
concentrated via rotary evaporation. Upon drying, the contents of
the flask were reconstituted with a 10% ethanol solution. The final
product yield was on average 25 mCi, with a radiosynthesis and
purification time of 90 min. Aliquots of this material were used
for biodistribution and PET imaging studies in rats. The specific
activity of the material ranged from 750 to 2000 Ci/mmol depending
upon the quantity of 19F present in the system.
Cardiac Imaging with [18F]10 in Rats. Cardiac imaging was
performed in anesthetized (pentobarbital, 50 mg/kg ip) rats (300-
400 g, male, SD) using a microPET camera (Focus220, CTI
Molecular Imaging, Inc. Knoxville, TN), which provides 95
transaxial slices in 22 cm operational field of view. After the animal
was positioned for cardiac imaging, about 1 mCi of [18F]10 (range
of specific activity: 750-2000 Ci/mmol) was injected intravenously
into the rat via a catheter precatheterized in femoral vein. Image
acquisition was started 5 min postinjection and performed for the
next 2 h in dynamic mode (10 min × 12 frames). The images were
reconstructed using OSEM2D algorithm with 256 × 256 matrix at
zoom 2, no attenuation correction. Image visualization, ROI
placement, and quantification were performed using ASIPRO
software developed by the manufacturer.
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