Synthesis, radiolabeling, and in vivo evaluation of an 18F-labeled isatin analog for imaging caspase-3 activation in apoptosis

Article abstract of DOI:10.1016/j.bmcl.2006.07.045

A non-peptide-based isatin sulfonamide analog, WC-II-89, was synthesized and its inhibition toward recombinant human caspase-3 and other caspases was determined. This compound showed high potency for inhibiting caspase-3 and -7, and high selectivity again

Full text of DOI:10.1016/j.bmcl.2006.07.045

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5041–5046
18
Synthesis, radiolabeling, and in vivo evaluation of an F-labeled
isatin analog for imaging caspase-3 activation in apoptosis
Dong Zhou, Wenhua Chu, Justin Rothfuss, Chenbo Zeng,
Jinbin Xu, Lynne Jones, Michael J. Welch and Robert H. Mach*
Division of Radiological Sciences, Washington University School of Medicine, 510 South Kingshighway Boulevard,
St. Louis, MO 63110, USA
Received 13 June 2006; revised 12 July 2006; accepted 13 July 2006
Available online 7 August 2006
Abstract—A non-peptide-based isatin sulfonamide analog, WC-II-89, was synthesized and its inhibition toward recombinant
human caspase-3 and other caspases was determined. This compound showed high potency for inhibiting caspase-3 and -7, and high
selectivity against caspases-1, -6, and -8. [18F]WC-II-89 was synthesized via a nucleophilic substitution of the corresponding mesy-
late precursor in high yield and radiochemical purity. Biodistribution studies using [18F]WC-II-89 revealed higher uptake in liver and
spleen of cycloheximide-treated rats, an animal model of apoptosis, relative to control animals. Western blot analysis confirmed the
presence of activated caspase-3 in the liver and spleen of cycloheximide-treated animals. MicroPET imaging studies revealed a high
uptake of the radiotracer in the liver of a cycloheximide-treated rat relative to the untreated control. These data suggest that
[18F]WC-II-89 is a potential radiotracer for imaging caspase-3 activation in tissues undergoing apoptosis.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Apoptosis, or programmed cell death, is critical for the
normal development and function of multicellular
organisms as a common and universal mechanism of cell
death.1 It is a conserved process that is mediated by the
activation of a series of cysteine aspartyl-specific prote-
ases termed caspases. The abnormal regulation of cellu-
lar death via apoptosis is believed to play a key role in a
variety of human diseases, such as ischemia-reperfusion
injury (stroke and myocardial infarction), cardiomyopa-
thy, neurodegeneration (Alzheimer’s Disease, Parkin-
son’s Disease, Huntington’s Disease, and ALS), sepsis,
Type I diabetes, fulminant liver disease, and allograft
rejection.2,3 In addition, the beneficial effect of many
drugs, especially antitumor drugs, can be attributed to
their activation of the apoptotic process.4–9 Therefore,
the development of a non-invasive imaging procedure
that can study the process of apoptosis in a variety of
disease states, and monitor the ability of a drug to either
induce or halt apoptosis, would be of tremendous value
to the research and clinical community.
in vivo imaging techniques that measure the change
in tissue and cellular function at the molecular level.
The agents used so far for imaging apoptosis in vivo
are mostly based on Annexin V,10 which is a 36-kDa
protein that binds selectively with high affinity to exter-
nalized phosphatidylserine. Phosphatidylserine is nor-
mally found only on the interior of the cell
membrane but translocates to the exterior of the cell
membrane in the early stages of apoptosis. However,
since the externalization of phosphatidylserine also oc-
curs in necrosis, a further step, the propidium iodide
exclusion test,11 is required to discriminate between
apoptosis and necrosis in vitro. Although this test is
routinely used to distinguish apoptosis from necrosis
using ex vivo techniques such as flow cytometry, it can-
not be applied to in vivo techniques such as PET and
SPECT. In addition, the slow clearance of radiolabeled
Annexin V from blood generally requires imaging stud-
ies be conducted 4–6 h after administration of the radi-
otracer. Although this long time interval between
radiotracer injection and image acquisition is compati-
ble with the radionuclides used in SPECT imaging
studies, it is not suitable for the short half-life radio-
nuclides used in PET.
Positron emission tomography (PET) and single
photon emission computed tomography (SPECT) are
Keywords: Caspase-3; Apoptosis; Positron emission tomography.
*
An alternative strategy for measuring and imaging cells
and tissues undergoing apoptosis is the use of antibodies
Corresponding author. Tel.: +1 3143628538; fax: +1 3143620039;
e-mail: rhmach@mir.wustl.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.045

5042
D. Zhou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5041–5046
directed toward enzymes that are intrinsic to the bio-
chemical pathways of apoptosis. The enzymes responsi-
ble for the regulation and execution of apoptosis are the
caspases, which exist as inactive zymogens (pro-caspas-
es) in the cytosol and become activated when cells re-
ceive an apoptotic signal. Caspases activated early in
the process of apoptosis are known as initiator caspases;
the function of the initiator caspases is to activate the
executioner caspases, which are responsible for the pro-
teolytic cleavage of proteins that are necessary for cellu-
lar function. The initiator caspases are caspases-6, -8,
and -9, and the executioner caspases are caspase-3 and
-7. Cellular apoptosis can be initiated via two different
pathways, extrinsic and intrinsic.11 Since the intrinsic
and extrinsic pathways involve the activation of cas-
pase-3, immunohistochemical staining for activated cas-
pase-3 provides an unambiguous method for measuring
and imaging apoptosis. Furthermore, small molecule
inhibitors of caspase-3 that can be radiolabeled with
either carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/
2 = 110 min) have the potential for imaging apoptosis
with PET under a variety of pathological conditions.
This imaging procedure also has the capability to image
chemotherapy-induced apoptosis and capacity to pro-
vide an imaging-based procedure for measuring a posi-
tive response to treatment in cancer patients.
product, 7-amino-4-methylcoumarin (7-AMC), as previ-
ously reported.12 The IC50 values from the enzyme as-
says are shown in Table 1. WC-II-89 shows high
potency for inhibiting caspase-3 and -7, with IC50 values
at least 150-fold higher versus the initiator caspases-1,
-6, and -8. This caspase-inhibitory profile suggests that
WC-II-89 is a potential radiotracer for imaging apopto-
sis with PET when labeled with fluorine-18.
Starting from 10, the [18F]WC-II-89 was synthesized by
the nucleophilic substitution of the mesylate group with
[18F]fluoride ion using the radiochemical procedure out-
lined in the Scheme.14 The incorporation yield was more
than 70% and the synthesis time was less than 100 min.
[18F]WC-II-89 was confirmed by the co-elution with
non-radioactive standard WC-II-89 on an analytical
HPLC system. The radiochemical purity of [18F]WC-
II-89 was 99% and the specific activity was determined
as ꢀ1500 mCi/lmol at the end of synthesis.15
The evaluation of [18F]WC-II-89 as a radiotracer for
imaging caspase-3 activation was determined using a
well-characterized animal model of chemically induced
apoptosis.16,17 This model, which uses the protein syn-
thesis inhibitor, cycloheximide (CHX), was previously
used in the evaluation of radiolabeled Annexin V ana-
logs.18 Tissue morphology and TUNEL staining studies
have shown that cycloheximide induces apoptosis in rat
liver in both a dose-dependent and time-dependent man-
ner. Within 3 h of treatment with 1.5, 3, or 10 mg of
cycloheximide per kilogram of body weight, apoptosis
was induced in rat liver.16,17 Therefore, we chose 3 h
treatment of 5 mg/kg to induce the maximum apoptosis
in rat liver, expecting a high level of caspase-3
activation.
Recently, we reported the synthesis of a number of non-
peptide-based isatin sulfonamide analogs,12 many of
which displayed nanomolar potency for inhibiting cas-
pase-3 and caspase-7, while exhibiting a low potency
for inhibiting the initiator caspases, caspase-1, -6, and
-8. In this letter, we describe the synthesis of a new isatin
sulfonamide analog, WC-II-89, that is suitable for
radiolabeling with fluorine-18, and the biodistribution
of [18F]WC-II-89 in an animal model of apoptosis. We
also report the first microPET imaging study directly
measuring caspase-3 activation in tissues undergoing
apoptosis using [18F]WC-II-89.
The biodistribution results of [18F]WC-II-89 in normal
and cycloheximide-treated male Sprague–Dawley rats19
are shown in Table 2 and Figure 1. In general, the initial
uptake was higher for CHX-treated rats than control
rats. However, the difference between control and treat-
ed rats was reduced with time with the exception of the
liver and spleen. At 1 h after injection (Fig. 1), the up-
take in liver and spleen for the treated rats was 94 and
184% higher than the control animals at 1-h post-iv
injection of the radiotracer. The increase in uptake of
[18F]WC-II-89 in the cycloheximide-treated versus con-
trol animals is consistent with chemically induced apop-
tosis and caspase-3 activation. Since the isatin analogs
are competitive inhibitors of caspase-3,12 [18F]WC-II-
89 binds to the activated form of caspase-3 in tissues
undergoing apoptosis, which explains the slower wash-
out of radioactivity from the liver and spleen of the
cycloheximide-treated animals. The results of the biodis-
tribution study also revealed a very low uptake of radio-
activity in bone, indicating that defluorination is not a
concern with this radiotracer. The result of the biodistri-
bution study implies that [18F]WC-II-89 is a potential
PET radiotracer for imaging caspase-mediated
apoptosis.
The synthesis of WC-II-89 and its precursor for 18F-la-
beling, 10, is shown in the Scheme 1. O-Alkylation of
methyl 4-hydroxybenzoate 1 was achieved by conversion
to the corresponding sodium salt (sodium hydride in
THF at 0 ꢁC) followed by addition of 1-bromo-2-fluoroe-
thane to give compound 2, which was reduced by LiAlH4
in ether to afford the alcohol, 3. The hydroxyl group of 3
was then converted to the corresponding bromo analog 4
via treatment with CBr4 and Ph3P in CH2Cl2. 1-(2-
Bromoethoxy)-4-(bromomethyl)benzene 6 was obtained
by bromination of 5 with NBS in CCl4. The N-Boc group
of 7 was removed with TFA and the secondary amine was
coupled with 5-chlorosulfonylisatin in THF using trieth-
ylamine as an acid scavenger to produce 5-(2-phenoxy-
methyl-pyrrolidine-sulfonyl)-1H-2,3-dione, 8. The isatin
nitrogen was alkylated by treatment of 8 with sodium hy-
dride in DMF at 0 ꢁC followed by addition of 4 or 6 to
give compounds WC-II-89 and 9, respectively. Com-
pound 9 was then heated to reflux with silver methanesul-
fonate in acetonitrile to generate the precursor 10.13
Inhibition of recombinant human caspase-3 and other
caspases by WC-II-89 was assessed using a fluorescent
Western blot studies were carried out to measure cas-
pase-3 levels in control and cycloheximide-treated rats20

D. Zhou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5041–5046
5043
F
a,b
H3COOC
OH
H3COOC
O
2
1
F
F
c
d
O
O
HO
Br
3
4
Br
Br
e
O
H3C
O
Br
6
5
O
O
O
Boc
S
N
N
f,g
h,i
O
N
H
O
O
7
8
O
O
O
O
N
O
S
O
S
N
j
O
O
N
N
X = Br
O
O
X
O
OMs
O
10
WC-II-89: X = F
9: X = Br
O
O
O
S
N
[
18F]WC-II-89
O
k
N
10
O
18F
O
Scheme 1. Reagents: (a) NaH, THF; (b) BrCH2CH2F; (c) LiAlH4, ethyl ether; (d) CBr4, Ph3P, CH2Cl2; (e) NBS, CCl4; (f) TFA, CH2Cl2;
(g) 5-sulfonylisatin Chloride, Et3N; (h) NaH, DMF; (i) 4 or 6; (j) AgOMs, acetonitrile; (k) [18F]KF, kryptofix[2,2,2].
Table 1. Inhibition of WC-II-89 for caspases-1, -3, -6, -7, and -8
studies correlate very well to the biodistribution data of
liver, spleen, and fat at 1-h post-iv injection of the radi-
otracer as shown in Figure 1. The good correlation
between caspase-3 activity and biodistribution of
[18F]WC-II-89 in the cycloheximide-treated rats
establishes the basis for imaging apoptosis using
[18F]WC-II-89.
IC50 (nM)
Caspase-6
Caspase-1 Caspase-3
>50,000 9.7 1.3
Caspase-7 Caspase-8
> 50,000
3700 390 23.5 3.5
in order to correlate caspase-3 activity to the biodistri-
bution results. Western blot analysis of spleen, liver,
and fat for both control and treated rats is shown in Fig-
ure 2. The level of cleaved caspase-3 in the spleen and
liver of the treated rats is much higher than that of the
control animals, which is consistent with cyclohexi-
mide-induced apoptosis. There was no cleaved cas-
pase-3 in the Western blot of the fat tissues from both
control and treated rats. The results of the Western blot
The microPET images of the liver region at 10–60 min
post-iv injection of [18F]WC-II-89 are shown in Figure
3.21 The animal receiving a 3 h pre-treatment of cyclo-
heximide displayed a higher uptake of [18F]WC-II-89
in the liver versus the control animal. Figure 4 shows
the tissue–time activity curves from the microPET imag-
ing study. Although the early imaging frames (0–3 min)
suggest there is approximately a 20% increase in blood

5044
D. Zhou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5041–5046
Table 2. Biodistribution of [18F]WC-II-89 in normal and cycloheximide-treated (5 mg/kg, 3 h pre-treated) male Sprague–Dawley rats (200–250 g)a
5 min
1 h
2 h
Blood
Lung
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
2.70 0.21
3.66 0.40
1.42 0.34
0.112 0.010
0.165 0.012
0.178 0.032
0.229 0.026
0.378 0.059
0.733 0.118
0.150 0.048
0.427 0.052
0.093 0.012
0.123 0.020
0.534 0.073
0.547 0.048
0.077 0.008
0.104 0.018
0.149 0.019
0.143 0.014
0.132 0.018
0.116 0.009
0.063 0.007
0.074 0.004
0.081 0.012
0.115 0.016
0.156 0.015
0.219 0.026
0.058 0.010
0.107 0.026
0.043 0.003
0.063 0.007
0.178 0.037
0.230 0.047
0.033 0.004
0.064 0.002
0.071 0.008
0.086 0.014
0.154 0.056
0.128 0.027
***
*
ns
*
2.08 0.23
3.13 0.26
Liver
***
***
*
*
*
*
4.02 0.45
1.14 0.08
2.24 0.41
Spleen
Thymus
Kidney
Muscle
Fat
0.231 0.070
0.382 0.104
1.245 0.136
1.184 0.082
0.143 0.009
0.094 0.000
0.119 0.022
0.087 0.028
0.445 0.036
0.661 0.059
ns
ns
**
***
ns
ns
ns
ns
Bone
a Student’s t-test, p > 0.05; not significant (ns); *p < 0.05; **p < 0.01; ***p < 0.001.
0.8
blood flow. Caspase-3 activation in the cycloheximide-
treated versus control animal was also confirmed by
Western blot analysis of the rat livers following comple-
tion of the microPET imaging study (data not shown).
The treated:control ratios from the microPET study
(Fig. 4 inset) are also identical with the 2-fold increase
in uptake of [18F]WC-II-89 observed in the biodistribu-
tion study (Fig. 1).
94% Increase
p-value 0.0003
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
184% Increase
p-value 0.0001
1hr.control
1hr.treated
No Change
p-value 0.60
To summarize, radiotracers for imaging apoptosis have
been traditionally based on radiolabeled Annexin V,
some of which have gone to clinical trials.10 Although
some of these studies have been encouraging, Annexin
V cannot distinguish apoptosis and necrosis in vivo
since externalized-phosphatidylserine occurs in both
pathways of cellular death. Therefore, there is a need
to develop radiotracers that are specific for imaging cell
death via apoptosis (programmed cell death) using
PET.18 Since the activation of the executioner caspases,
caspase-3 and -7, occurs late in apoptosis, radiolabeled
inhibitors of caspase-3 and -7 represent a novel strategy
for discerning programmed cell death from necrosis. A
previous study reported the synthesis and carbon-11
radiolabeling of an isatin analog having a modest poten-
cy for inhibiting caspase-3.22 However, no in vivo data
were reported in this meeting abstract, and the selectiv-
liver
spleen
fat
Figure 1. Selected biodistribution of [18F]WC-II-89 in control and
cycloheximide (5 mg/kg) 3 h pre-treated male Sprague–Dawley rats
(200–250 g). The data represent tracer uptake 1 h post-iv injection of
the radiotracer.
flow to the cycloheximide-treated liver, the higher peak
accumulation of [18F]WC-II-89 in the treated rat liver
versus the control animal is consistent with drug-in-
duced caspase-3 activation. The normal rat liver also
displayed a faster washout of radioactivity than the
cycloheximide-treated liver, which is consistent with
drug-induced caspase-3 activation versus differences in
Figure 2. Western blot study of control and treated (5 mg/kg, 3 h pre-treated) male Sprague–Dawley rats (200–250 g).

D. Zhou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5041–5046
5045
Figure 3. Whole-body MicroPET images of [18F]WC-II-89 distribution in a control rat (left) and cycloheximide-treated rat (right). Images were
summed from 10 to 60 min after iv injection of ꢀ150 lCi [18F]WC-II-89.
Acknowledgments
This work was supported by HL13851, EB 001729, and
CA121952. The authors thank Nicole M. Fettig, Jerrel
Rutlin, and Lori Strong for animal handling and micro-
PET imaging.
References and notes
1. Jacobson, M. D.; Weil, M.; Raff, M. C. Cell 1997, 88, 347.
2. Reed, J. C. Nat. Rev. Drug Disc. 2002, 1, 111.
3. Rodriguez, I.; Matsuura, K.; Ody, C.; Nagata, S.; Vassalli,
P. J. Exp. Med. 1996, 184, 2067.
4. White, E. Gene Dev. 1996, 10, 1.
5. Ashkenazi, A.; Dixit, V. M. Science 1998, 281, 1305.
6. Evan, G.; Littlewood, T. Science 1998, 281, 1317.
7. Green, D. R.; Reed, J. C. Science 1998, 281, 1309.
8. Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312.
9. Dive, C.; Hickman, J. A. Br. J. Cancer 1991, 64, 192.
10. Review see Lahorte, C. M. M.; Vanderheyden, J.; Stein-
Figure 4. Tissue time–activity curves (mean percentage of injected dose
per cube centimeter) of rat liver. Cycloheximide-treated rat (s);
metz, N.; Van De Wiele, C.; Dierckx, R. A.; Slegers, G.
Eur. J. Nucl. Med. Mol. Imaging 2004, 31, 887.
control rat (h). Inset graph shows the ratio of the liver uptake of the
cycloheximide-treated versus control liver.
11. Neuss, M.; Crow, M. T.; Chesley, A.; Lakatta, E. G.
Cardiovasc. Drugs Ther. 2001, 15, 507.
12. Chu, W.; Zhang, J.; Zeng, C.; Rothfuss, J.; Tu, Z.; Chu,
Y.; Reichert, D. E.; Welch, M. J.; Mach, R. H. J. Med.
Chem. 2005, 48, 7637.
13. 1-[4-(2-Fluoroethoxy)-benzyl]-5-(2-phenoxymethyl-pyrrol-
ity of this compound for caspase-3 versus other caspases
was not mentioned. Our group previously reported the
idine-1-sulfonyl)- 1H -indole-2,3-dione (WC-II-89). A
synthesis and in vitro potency of a number of isatin-
based analogs having a high potency for inhibiting the
solution of 8 (97 mg, 0.25 mmol) in DMF (3 mL) was
added 60% NaH (10 mg, 0.25 mmol) at 0 ꢁC. The mixture
executioner caspases, caspase-3 and -7, relative to casp-
ases-1, -6, and -8.12 In this letter, we have shown that
was stirred for 5 min, then 4 (250 mg) was added. The
mixture was stirred for 10 min. at 0 ꢁC, ethyl acetate
WC-II-89 binds to caspase-3 and -7 with high affinity
and specificity versus caspases-1, -6, and -8. Biodistribu-
tion studies of [18F]WC-II-89 revealed a higher uptake in
the liver and spleen of rats treated with cycloheximide, a
well-established murine model of chemically induced
apoptosis. Western blot analysis confirmed this uptake
was related to caspase-3 activation. Our results have
demonstrated for the first time that apoptosis can be
measured and imaged by PET using 18F-labeled cas-
pase-3 inhibitors such as [18F]WC-II-89. We are current-
ly evaluating [18F]WC-II-89 in additional animal models
of apoptosis.
(50 mL) was added, washed with water (30 mL), NaCl
(30 mL), and dried over Na2SO4. After evaporation of the
ethyl acetate, the crude product was purified with ether to
afford 74 mg (55%) of WC(II)-89 as a yellow solid, mp
164.0–164.8 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.01 (s,
1H), 7.95 (d, J = 8.1 Hz, 1H), 7.28-7.21 (m, 4H), 6.95–6.80
(m, 6H), 4.86 (s, 2H), 4.75 (dt, J = 47.4 Hz, J = 4.2 Hz,
2H), 4.20 (dt, J = 28.5 Hz, J = 4.2 Hz, 2H), 4.15 (m, 1H),
3.92 (m, 2H), 3.49 (m, 1H), 3.22 (m, 1H), 2.02 (m, 2H),
1.78 (m, 2H). Anal. Calcd for C28H27FN2O6S: C, 62.44; H,
5.05; N, 5.20. Found: C, 62.50; H, 5.11; N, 5.12. 1-[4-(2-
Bromoethoxy)-benzyl]-5-(2-phenoxymethyl-pyrrolidine-1-
sulfonyl)- 1H-indole-2,3-dione (9) was prepared according

5046
D. Zhou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5041–5046
to the same procedure for compound WC-II-89 except
mediated liver apoptosis. At set time-points following
radiopharmaceutical injection, rats were again anesthe-
tized and euthanized. Target and non-target organs were
removed, weighed, and the radioactivity was counted
using a Beckman Gamma 8000 well counter. Standard
dilutions of the injected dose were counted along with the
samples and uptake was calculated and reported as
percent injected dose per gram (%ID/g).
using compound 6, purified with hexane–ether (1:2) to
afford 587 mg (68%) of 9 as a yellow solid, mp 164.1–
164.9 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.05 (s, 1H), 8.01
(dd, J = 8.1 Hz, J = 2.1 Hz, 1H), 7.32–7.25 (m, 4H), 6.70–
6.84 (m, 6H), 4.91 (s, 2H), 4.32 (t, J = 6.0 Hz, 2H), 4.20
(m, 1H), 3.97 (m, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.55 (m,
1H), 3.26 (m, 1H), 2.07 (m, 2H), 1.83 (m, 2H). Anal. Calcd
for C28H27BrN2O6SÆ0.25 H2O: C, 55.68; H, 4.59; N, 4.64.
Found: C, 55.66, 4.28; N, 4.54. Methanesulfonic acid 2-
{4-[2,3-dioxo-5-(2-phenoxymethyl-pyrrolidine-1-sulfonyl)-
2,3-dihydro- indol-1-ylmethyl]-phenoxy}-ethyl ester (10).
A solution of 9 (300 mg, 0.5 mmol) and AgOMs (1.01 g,
5.0 mmol) in CH3CN (10 mL) was heated to reflux
overnight. After evaporation of the solvent, the crude
product was purified with ether to afford 228 mg (74%) of
10 as a yellow solid, mp 151.8–152.6 ꢁC. 1H NMR
(300 MHz, CDCl3) d 8.05 (s, 1H), 8.01 (dd, J = 8.1 Hz,
J = 1.8 Hz, 1H), 7.33–7.25 (m, 4H), 7.00–6.84 (m, 6H),
4.90 (s, 2H), 4.60 (t, J = 4.8 Hz, 2H), 4.27 (t, J = 4.8 Hz,
2H), 4.20 (m, 1H), 3.97 (m, 2H), 3.54 (m, 1H), 3.26 (m,
1H), 3.11 (s, 3H), 2.06 (m, 2H), 1.83 (m, 2H). Anal. Calcd
for C29H30N2O9S2: C, 56.66; H, 4.92; N, 4.56. Found: C,
56.74; H, 4.88; N, 4.67.
20. Mature male Sprague–Dawley rats (Charles River Labo-
ratories, Inc., Wilmington, MA) were anesthetized with 1–
2% isoflurane in oxygen and treated rats were injected via
tail vein with 5 mg/kg CHX/saline solution to activate
caspase-mediated apoptosis. Rats were euthanized three
hours post-treatment and the organs of interest were
immediately snap-frozen in liquid nitrogen, then stored at
À80 ꢁC until analysis. Whole organs were homogenized in
ice-cold T-PERꢂ protein extraction buffer (Pierce Bio-
technology, Rockford, IL) containing 5 mM DTT, 2 mM
EDTA, and Completeꢂ protease inhibitor cocktail tablets
(Roche Diagnostics Co., Indianapolis, IN). The fully
homogenized samples were then sonicated on ice, centri-
fuged at 4 ꢁC at 14,000g for fifteen minutes, and the
protein-containing supernatant was collected. Forty
micrograms of protein from each sample was analyzed
using standard immunoblotting techniques. Caspase-3 was
probed with anti-caspase-3 antibody (Cell Signaling
Technology, Danvers, MA) at 1:1000 dilution and horse-
radish peroxidase-conjugated goat anti-rabbit IgG (Cell
Signaling Technology, Danvers, MA) at 1:3000. Actin was
resolved using anti-b-actin antibody (Cell Signaling Tech-
nology, Danvers, MA) at 1:1000 dilution and the same
secondary antibody as mentioned above. SuperSignalꢂ
WestDura extended duration substrate (Pierce Biotech-
nology, Rockford, IL) was used for detection.
14. Yoo, J.; Dence, C. S.; Sharp, T. L.; Katzenellenbogen, J.
A.; Welch, M. J. J. Med. Chem. 2005, 48, 6366.
15. HPLC conditions for purification of [18F]WC-II-89: All-
tech Ecosoil C18 250 · 10 mm, 10 l; 25% acetonitrile, 45%
methanol, and 30% 0.1 M ammonium formate buffer (pH
4.5); 5 mL/min, 251 nm; tR = 15 min.
16. Ledda-Columbano, G. M.; Coni, P.; Faa, G.; Manenti,
G.; Columbano, A. Am. J. Pathol. 1992, 140, 545.
17. Faa, G.; Ledda-Columbano, G. M.; Ambu, R.; Congiu,
T.; Conti, P.; Riva, A.; Columbano, A. Liver 1994, 14,
270.
21. MicroPET imaging studies were performed using
a
18. Yagle, K. J.; Eary, J. F.; Tait, J. T.; Grierson, J. R.; Link,
J. M.; Lewellen, B.; Gibson, D. F.; Krohn, K. A. J. Nucl.
Med. 2005, 46, 658.
MicroPET Focus 220 and MicroPET Focus 120 scanner
(Siemens/CTI, Knoxville, TN). A control and cyclohexi-
mide-treated (5 mg/kg, 3 h pre-treated) rat were anesthe-
tized and a catheter inserted in the jugular vein. Each rat
was then placed in the scanner and, following a transmis-
sion scan, was injected with ꢀ150 lCi [18F]WC-II-89 for a
one hour dynamic imaging session. MicroPET images
were reconstructed with OSEM-2D data analysis software
package (Siemens/CTI, Knoxville, TN).
19. All animal studies in this letter were performed in
accordance with the regulations of the Washington
University Institutional Animal Care and Use Committee.
Mature male Sprague–Dawley rats from Charles River
Laboratories were briefly anesthetized with 1–2% isoflu-
rane in oxygen. Each rat received 10–15 lCi [18F]WC-II-
89 via the tail vein. Treated rats also received 5 mg/kg
cycloheximide in saline via the tail vein three hours prior
to radiotracer administration in order to induce caspase-
22. Faust, A.; Wagner, S.; Keul, P.; Schober, O.; Levkau, B.;
Schaefers, M.; Kopka, K. J. Labelled Compd. Radiopharm.
2005, 48, S260 (abstract).