Design, synthesis, and biological characterization of a caspase 3/7 selective isatin labeled with 2-[18f]fluoroethylazide

Article abstract of DOI:10.1021/jm801107u

Imaging of programmed cell death (apoptosis) is important in the assessment of therapeutic response in oncology and for diagnosis in cardiac and neurodegenerative disorders. The executioner caspases 3 and 7 ultimately effect cellular death, thus providing

Full text of DOI:10.1021/jm801107u

J. Med. Chem. 2008, 51, 8057–8067
8057
Design, Synthesis, and Biological Characterization of a Caspase 3/7 Selective Isatin Labeled
with 2-[18F]fluoroethylazide
Graham Smith,† Matthias Glaser,‡ Meg Perumal,† Quang-De Nguyen,† Bo Shan,‡ Erik Årstad,‡ and Eric O. Aboagye*,†
Molecular Therapy Group, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, United
Kingdom, MDx DiscoVery (part of GE Healthcare) at Hammersmith Imanet Ltd, Hammersmith Hospital, Du Cane Road, London W12 0NN,
United Kingdom
ReceiVed June 26, 2008
Imaging of programmed cell death (apoptosis) is important in the assessment of therapeutic response in
oncology and for diagnosis in cardiac and neurodegenerative disorders. The executioner caspases 3 and 7
ultimately effect cellular death, thus providing selective molecular targets for in vivo quantification of
apoptosis. To realize this potential, we aimed to develop 18F-labeled isatin sulfonamides with high metabolic
stability and moderate lipophilicity while retaining selectivity and affinity for caspase 3/7. A small library
of isatins modified with fluorinated aromatic groups and heterocycles was synthesized. A lead compound
incorporating 2′-fluoroethyl-1,2,3-triazole was identified with subnanomolar affinity for caspase 3. “Click
labeling” provided the 18F-labeled tracer in 65 ( 6% decay-corrected radiochemical yield from 2-[18F]fluo-
roethylazide. The compound showed high stability in vivo with rapid uptake and elimination in healthy
tissues and tumor. The novel 18F-labeled isatin is a candidate radiotracer for further preclinical evaluation
for imaging of apoptosis.
Introduction
key signaling proteins (e.g., Akt, Ras), nuclear skeletal proteins
(e.g., actin, fodrin, lamins), and cell cycle regulators (e.g.,
p27Kip1).6
Apoptosis or programmed cell death (PCD) is the most
prevalent cell death pathway and proceeds via a highly regulated,
energy conserved mechanism.1 In the healthy state, apoptosis
plays a pivotal role in controlling cell growth. Dysregulation
of this process has been implicated in a number of disease states
such as cancer, autoimmunity, neurodegeneration, ischemia, and
transplant rejection.2,3 Noninvasive imaging of apoptosis will
therefore be of immense value for early assessment of response
to therapeutic intervention and can provide new insight into
devastating pathological processes. Early monitoring of the
efficacy of cancer therapy is of particular interest. Apoptosis is
regulated by both intrinsic (via mitochondria) and extrinsic
(activation of death receptors) signaling networks that control
a family of enzymes known as caspases (cysteine aspartate
specific proteases).4,5 The pathways activate “initiator” caspases
8 (extrinsic) or 9 (intrinsic), which in turn cleave the inactive
pro-caspases 3, 6, and 7 into the active “executioner” caspases
3, 6, and 7.1 The executioner caspases ultimately effect cellular
death through cleavage of cellular proteins, which occurs
adjacent to aspartate residues in a highly selective manner. The
proteins cleaved include DNA repair enzymes (e.g., PARPa),
Following a high throughput screen, Lee and co-workers
identified the isatin 1 as an inhibitor of caspase 3 (Chart 1).7
Structural optimization led to the discovery of the highly potent
sulfonamide 2 (2.5 nM). Realizing the potential of radiolabeled
isatins for imaging of apoptosis, the groups of Kopka and Mach
independently developed the 18F-labeled fluoroethyl phenyl ether
3 as a putative tracer for positron emission tomography (PET)
imaging of activated caspase 3 levels.8,9 Recently, Mach and
co-workers reported that the uptake of [18F]3 increases in the
liver and spleen of rats treated with cycloheximide, a model
for chemically induced apoptosis.10 To further characterize this
class of compounds, we investigated the biological properties
of a radioiodinated analogue [125I]4.9 The results indicated poor
biological stability and led us to design a new series of isatins.
Here we report the development and characterization of a novel
18F-labeled isatin with improved metabolic profile, reduced
lipophilicity, and subnanomolar affinity for capsase 3.
Results
Chemistry. The isatins 2, 4, and 5 were synthesized as
described in the literature.7–9 Compound 6 was obtained by
alkylation of 5-[1-(pyrrolidinyl)sulfonyl]isatin7 with 4-fluo-
robenzyl bromide. The isatin 5 was chosen as lead compound,
and modifications were made to the left side ether moiety and
at the N-1 position with the aim to improve the biological
stability while retaining selectivity and affinity for caspase 3.
To achieve this, we incorporated fluorine groups into the left
side phenyl ether group and also investigated the tolerance of
heterocycles in this position. Of particular interest was the
tolerance to 1,2,3-triazoles, as this group is reportedly inert to
metabolic degradation11,12 and can be readily labeled with
fluorine-18.12–14 The target compounds were synthesized by
condensation of functionalized pyrrolidines with 5-chlorosul-
fonylisatin and subsequent alkylation of the isatin nitrogen using
potassium carbonate/DMF (Scheme 1).
* To whom correspondence should be addressed. Phone: +44-208-383-
3759. Fax: +44-208-383-1783. E-mail: eric.aboagye@imperial.ac.uk.
†
Molecular Therapy Group, Faculty of Medicine, Imperial College
London, Hammersmith Hospital.
‡
MDx Discovery (part of GE Healthcare) at Hammersmith Imanet Ltd,
Hammersmith Hospital.
a Abbreviations: PARP, poly (ADP-ribose) polymerase; DMF, N,N-
dimethylformamide; BOC, tert-butoxycarbonyl; DIAD, diisopropyl azodi-
carboxylate; DCM, dichloromethane; TFA, trifluoroacetic acid; TEA,
triethylamine; THF, tetrahydrofuran; CDDP, cis-dichlorodiammine plati-
num(II); RIPA, radio-immunoprecipation assay; SDS, sodium dodecyl
sulfate; PVDF, polyvinylidene fluoride; HRP, horseradish peroxidase;
DMSO, dimethyl sulfoxide; CCPMA, corrected counts per minute average;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; EDTA, eth-
ylenediamminetetraacetic acid; BCA, bicinchoninic acid.
10.1021/jm801107u CCC: $40.75
2008 American Chemical Society
Published on Web 12/02/2008

8058 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smith et al.
Chart 1
Scheme 1a
a Reagents and conditions: (a) phenol/fluoro-substituted phenol/4-tetrahydropyran, NaH, DMF, 80°C, 17 h; (b) 4(3H)-pyrimidone, PPh3, DIAD, DCM, rt,
48 h; (c) propargyl bromide, KOH, DMF, rt, 18 h; (d) TFA, DCM, 0°C, 1 h; (e) 5-chlorosulfonylisatin, TEA, THF/DCM, rt, 19 h; (f) 4-fluorobenzyl
bromide, K2CO3, DMF, rt, 2 h; (g) propargyl bromide, K2CO3, DMF, rt, 2 h; (h) 2-fluoroethyl azide, CuSO4, L-ascorbic acid, DMF, rt, 2 h.
Reaction of commercially available phenols as well as
4-hydroxytetrahydropyran with tosylate 7 provided the pyrro-
lidines 9a-f in good yield. 4(3H)-Pyrimidone was coupled with
the alcohol 8 using a modification of the procedure reported by
Wipf et al.15 to furnish the pyrrolidine 9g in 69% yield.
O-Alkylation of 8 with propargyl bromide afforded the corre-
sponding ether 9h in 67% yield. Deprotection of the BOC
protected pyrrolidines 9a-h with trifluoroacetic acid followed
by conjugation with 5-chlorosulfonylisatin provided the sul-
fonamides 10a-h in moderate to good yields. Subsequent
treatment of 10b-h with 4-fluorobenzyl bromide under basic
conditions yielded the target compounds 11b-h, whereas
treatment of 10a and 10d with propargyl bromide provided the
alkynes 12 and 13, respectively. The triazoles 14-16 were
prepared by copper catalyzed cycloaddition of 2-fluoroethylazide
with the respective alkyne precursors 11h, 12, and 13. Somewhat
surprisingly, the isatin scaffold decomposed on heating at 90
°C in the presence of the copper sulfate and ascorbic acid,
resulting in poor yields of the target triazoles 14-16. The issue
was partly resolved by increasing the copper sulfate concentra-
tion from 5% to 50% relative to the alkyne precursor, carrying
out the reaction at ambient temperature and reducing the reaction
time to 1 h, which provided the triazoles 14-16 in yields of
48-57%.
Radiochemistry. The triazole 15 was labeled by copper
catalyzed cycloaddition of 2-[18F]fluoroethylazide ([18F]17) with
the alkyne precursor 13. [18F]17 was prepared by reaction of
[18F]fluoride with the corresponding tosylate precursor 18 and
was purified by distillation as previously described (Scheme 2).14
Investigation of the impact of the catalytic system, temper-
ature, and pH on the radiochemical yield revealed poor thermal
stability of [18F]15, with the highest yields obtained at room
temperature. Addition of sodium phosphate buffer (pH 6.0, 250
mM)) to the reaction mixture dramatically improved the
radiochemical yield, whereas only minor differences were
observed for the two catalytic systems investigated (copper
powder or copper sulfate/ascorbate). The optimal conditions
were found to be 30 min reaction time at room temperature in
the presence of a slight excess of copper sulfate relative to the
alkyne precursor 13. This provided the triazole [18F]15 in 65 (
6% (n ) 26, decay-corrected from [18F]17) isolated radiochemi-
cal yield with a radiochemical purity of >99% after purification

2-[18F]Fluoroethyazide-Labeled Isatins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8059
Scheme 2a
a Reagents: (a) [18F]KF, Kryptofix[2,2,2], K2CO3, acetonitrile, 80°C, 15 min; (b) CuSO4, sodium ascorbate, phosphate buffer pH 6.0, rt, 30 min, 13.
Figure 1. Typical preparative HPLC trace of reaction mixture containing [18F]15 (left) and corresponding trace after formulation of [18F]15 (right)
(top: radioactivity channel; bottom: UV channel at 254 nm).
by HPLC. A typical HPLC chromatogram for the product
mixture is shown in Figure 1. As the procedure was carried out
manually with low amounts of radioactivity, only modest
specific activity (1.2 GBq/µmol) was achieved at end of
synthesis. The identity of [18F]15 was confirmed by coelution
with the nonradioactive reference compound. The purified
[18F]15 was formulated by solid-phase extraction with an
efficiency of 91 ( 6% (n ) 26, decay-corrected). The
radiosynthesis including formulation of [18F]15 took three hours
in total.
Enzyme Assays. The affinities of the novel fluorinated isatins
11b-h and 14-16 for different activated caspases 1, 3, 6, 7,
and 8 were measured by a fluorimetric in vitro caspase inhibition
assay similar to that described by Kopka and co-workers.9
Inhibition of recombinant human caspases was assessed by
measuring the accumulation of a fluorogenic product, 7-amino-
4-methylcoumarin (7-AMC). Compounds 2 and 4-6 were
included as reference compounds. The results are summarized
in Table 1. The data are the average for two runs, each done in
duplicate, at 9 concentrations ranging from 5 pM to 500 µM;
the drug concentration required to inhibit each enzyme by 50%
(EC50) was obtained from nonlinear regression analysis of the
inhibition profiles. In this assay, the affinity of the iodinated
isatin 4 was 59.9 and 25.3 nM for caspase 3 and 7, respectively.
Substituting the iodine for fluorine as in compound 5 led to a
slight increase in affinity for both caspase 3 and 7. The
importance of the left side ether group was clearly demonstrated
by analysis of isatin 6, which with 199.5 nM affinity for caspase
3 was 4-fold less potent than isatins 4 and 5. Fluorine
substituents on the phenyl ether were well tolerated and, for
the isatins 11b-c, the affinity for caspase 3 increased 2-fold as
compared with nonfluorinated phenyl ether 4. The difluorinated
isatins 11d-e were even more potent, with an affinity of 12.4
and 10.4 nM for caspase 3, respectively. Similar affinities were
found for the tetrahydropyran 11f, and replacement of the phenyl
ether with a pyrimidinyl group as in 11g led to a further increase
in potency, with affinities of 5.5 and 2.3 nM for caspase 3 and
7, respectively. Gratifyingly, introduction of 2′-fluoroethyl-1,2,3-
triazole on either side of the molecule led to a sharp increase in
potency, with measured caspase 3 affinities of 16.7 and 12.6
nM for the triazoles 14 and 16, respectively. Of the compounds
tested, the triazole 15 was by far the most potent, with affinities
of 0.5 and 2.5 nM for caspase 3 and 7, respectively. All the
compounds tested were poor substrates for caspases 1, 6, and 8
(EC50 > 5000 nM).
Biodistribution and Metabolic Stability. Radioiodination
of isatin 4 afforded the opportunity for facile and rapid
assessment of the suitability of this compound class as apoptosis
imaging agents. To achieve this, isatin 4 was radiolabeled with
iodine-125 (t1/2 ) 59.9 days) by iodo-destannylation using the
method of Kopka.9 The radiotracer [125I]4 was injected intra-
venously into mice, and its tissue distribution was measured in
the major organs at selected time points from 2 to 60 min after
injection. The radiotracer distributed rapidly to the major organs
and was also rapidly eliminated (Figure 2). The in vivo stability
of [125I]4 in mice was determined in plasma and liver at selected
time points from 2 to 60 min after tracer injection. The
radiotracer [125I]4 was completely eliminated from plasma within
10 min and represented <15% (n ) 3) of the radioactivity
detected in the liver at this point of time (chromatograms

8060 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Table 1. Caspase Inhibition Profile for Selected Compounds
Smith et al.
R
R′
caspase EC50 (nM)a
cLog Pb
3
7
1/6/8
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
2
4
5
6
11b
11c
11d
11e
11f
11g
13
14
15
16
phenoxymethyl
phenoxymethyl
phenoxymethyl
H
4-fluorophenoxymethyl
3-fluorophenoxymethyl
2,4-difluorophenoxymethyl
3,5- difluorophenoxymethyl
4-tetrahydropyranyloxymethyl
pyrimidin-4-yloxymethyl
2,4-difluorophenoxymethyl
phenoxymethyl
benzyl
41.8
59.9
50.5
199.5
26.1
17.0
12.4
10.4
10.7
5.5
29.4
25.3
19.8
78.6
8.0
13.5
13.0
16.8
14.4
2.3
3.65
4.68
3.70
1.93
3.83
3.80
3.87
3.94
1.87
2.06
2.42
1.38
1.55c
1.55
4-iodobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
2-propynyl
50.1
16.7
0.5
60.4
28.2
2.2
1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-ylmethyl
1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-ylmethyl
4-fluorobenzyl
2,4-difluorophenoxymethyl
1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-ylmethoxymethyl
12.6
18.3
a Each value is an average of two independent measurements each obtained from a fit to a 9-concentration level data set (r2 > 0.9). b Values calculated
using ACD/Chemsketch Laboratories 10.02. c Measured Log P ) 1.61.
the substituted carbon but also reduces metabolic attack on
neighboring nonsubstituted carbons by exerting a strong electron
withdrawing effect.16,17 If aromatic hydroxylation indeed is the
major metabolic pathway for degradation of the isatins inves-
tigated, the 2,4-difluorophenyl ether containing 15, the most
potent compound in the enzyme assay, should be significantly
more stable in vivo compared to unsubstituted phenyl ethers
(e.g., 4). We tested this hypothesis, first by assessing the
behavior of 15 and [18F]15 in cell-based assays and then by
analyzing the stability of [18F]15 over time in mice.
We assessed the cellular activity of the most active compound
in the enzyme assay (isatin 15) against the previously analyzed9
iodo compound 4. Figure 4 shows that preincubation of RIF-1
cells with isatin 15, but not 4, disrupted the induction of caspase
3 cognate target PARP by cisplatin (CDDP), demonstrating
cellular activity of 15. To further establish the binding of istain
15 under conditions of drug-induced apoptosis, we then evalu-
ated uptake of radiolabeled [18F]15 in RIF-1 cells. The results
are summarized in Figure 5 and show a statistically significant
increase in uptake of [18F]15 of 1.5-fold for treated compared
to untreated cells (p ) 0.04).
Following the findings of the cellular assays using 15 and
[18F]15, the radiotracer [18F]15 was injected intravenously into
tumor-bearing mice and its tissue distribution was measured in
selected tissues at 2, 15, and 60 min after injection. Furthermore,
the in vivo metabolic stability of the radiotracer was determined
in plasma, liver, and urine samples. Figure 6 shows that [18F]15
distributed rapidly to tissues and was also rapidly eliminated.
High localization of [18F]15-derived radioactivity was seen in
kidney, urine, and liver, suggesting the importance of both renal
and hepatic routes of elimination. However, even in these
tissues, rapid elimination of radioactivity was seen. Importantly,
from an imaging standpoint, the rapid clearance of [18F]15 in
untreated tumors and heart should facilitate measurement of
increased binding associated with caspase activation in these
tissues. Bone uptake was low, suggesting an absence of in vivo
defluorination and hence metabolic stability of the 2′-fluoroethyl-
1,2-3-triazole moiety. Tumor uptake studies were also carried
out in control and CDDP treated RIF-1 tumor bearing mice (24
h after treatment) to assess the effect of CDDP treatment on
[18F]15 tumor uptake in vivo. Figure 7 shows an increase in
[18F]15-derived radioactivity by 2.9-fold in CDDP treated
Figure 2. Tissue distribution of [125I]4 in untreated nontumor bearing
mice at 2, 10, 30, and 60 min. Data are mean ( SEM; n ) 3 mice per
time point. There was no 2 min urine sample.
supplied in Supporting Information). Within 30 min after
injection, the parent [125I]4 was fully metabolized in the liver,
with a single, polar metabolite constituting the majority of the
radioactivity detected. Consistent with these findings, we
observed rapid clearance in all tissues investigated, and from
10 min onward, the majority of the injected radioactivity was
excreted in the urine (Figure 2).
Organ distribution studies pointed to a metabolic pathway
other than deiodination, as radioactivity in the stomach, which
is known to accumulate radioiodide, was low. To identify the
main site of metabolism, we compared the metabolic fate of
compounds 47 and 58 (Chart 1) to that of 6 after in vitro
incubation with murine liver S9 fraction. As shown in Figure
3, the metabolic profiles of isatins 4 and 5 were similar, with
near complete degradation observed after 60 min incubation.
In contrast, the isatin 6 was markedly more stable and remained
largely intact at this time. While no attempts were made to
identify the nature of the metabolites, the results indicate that
the isatin scaffold is relatively stable, whereas the phenyl ether
group in 4 and 5 undergoes rapid breakdown consistent with
aromatic hydroxylation.
It is well-known that the introduction of fluorine to aromatic
groups not only blocks P450-catalyzed ring hydroxylation of

2-[18F]Fluoroethyazide-Labeled Isatins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8061
Figure 3. Representative HPLC profiles of isatins incubated with mouse liver S9 fractions. Upper row shows, from left to right, isatin 4, 5, and
6 as they appear at zero time. Lower row shows, from left to right, isatin 4, 5, and 6 after incubation for 60 min.
Figure 4. Cellular activity of isatin 15 and 4 assessed by Western
blot analysis. Inhibitory activity of isatin 15 and 4 on the caspase 3
cognate target PARP. RIF-1 cells were treated for 15 min with the
caspase inhibitors Z-VAD-FMK (100 µM), isatin 15 and 4, prior to
apoptosis induction with CDDP (100 µM) for 48 h. The ∆ PARP
immunoblot band corresponds to the endogenous 89 kDa large fragment
of PARP resulting from caspase 3 cleavage. R tubulin was used as a
loading control.
Figure 6. Biodistribution of [18F]15 in RIF-1 tumor bearing mice
between 2 and 60 min. Data are mean ( SEM; n ) 3-6 mice per
time point.
Figure 5. Uptake profile of [18F]15 in RIF-1 cells treated with vehicle
or CDDP. RIF-1 cells were treated with vehicle (0.1% DMSO) or
CDDP (100 µM) for 48 h. The cells were then incubated with [18F]15
for 1 h, washed and analyzed for radioactivity. Data are expressed as
decay-corrected counts per min averaged per milligram of total cellular
protein. Data are mean ( SEM done in triplicate.
compared to control tumor, suggesting that [18F]15 is promising
enough to warrant further evaluation as an in vivo caspase-3/7
imaging agent.
Figure 7. Uptake of [18F]15 in RIF-1 tumor treated with vehicle
(control: 50% DMSO) or CDDP (10 mg/kg single dose). The [18F]-
derived radioactivity levels at 60 min post radiotracer injection were
analyzed and expressed as a ratio to that of blood. Data are mean (
SEM and n ) 8 mice per group.
In keeping with our design goals, radio-HPLC analysis
showed that [18F]15 was relatively more stable to metabolic
degradation (Figure 8), producing a single polar metabolite peak
in plasma and liver. The parent compound was still present in
plasma at 60 min and was the dominant peak. In contrast, the
metabolite was the dominant peak in liver samples at all time
points studied. Urine radioactivity comprised mainly of the
metabolite (traces provided in supplementary section). The
proportions of parent radiotracer in plasma and liver at the
selected time points, together with the apparent extraction
efficiency are summarized in Table 2. Following a rapid
decrease, the rate of in vivo metabolism appeared to plateau

8062 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smith et al.
Figure 8. In vivo metabolism of [18F]15 assessed in plasma by radio-HPLC. Top left, [18F]15 standard; top right, 2 min plasma; bottom left, 15
min plasma; bottom right, 60 min plasma.
Table 2. In Vivo Metabolism of [18F]15 at Selected Time Points
caspases as potential probes for studying caspase-dependent
apoptosis, as these should be more specific than membrane
Showing the Proportion of [18F]15 Present in Plasma and Liver Extractsa
time
(min)
parent
recovery
parent
recovery
(liver)c
activity for apoptosis. The suitability of imaging probes based
on the caspase-3-specific DEVD (Asp-Glu-Gly-Asp) peptide
sequence (optimum substrate sequence)23 has been tested in one
study.24 The highly polar nature of the DEVD sequence,
however, resulted in poor cellular uptake, making the peptide-
based strategy less attractive for use in PET imaging. Further
attachment of a Tat sequence resulted in increased caspase-3-
independent unidirectional cellular uptake;24 again this property
made it unsuitable for imaging. The groups of Kopka and Mach
have independently developed 18F-labeled fluoroethyl phenyl
ether isatins as putative tracers for imaging caspase activation
by PET.8,9 The mechanism of action of isatins involves
formation of an intracellular enzyme-inhibitor complex with
caspase 3 (and 7) through covalent binding to the enzyme active
site of the activated caspase.25,26 The dicarbonyl functionality
of the isatin is essential to its mechanism of action; it binds to
the cysteine residue of the active site, forming a thiohemiketal
via the electrophilic C-3 carbonyl carbon of the isatin and the
nucleophilic cysteine thiolate functionality.26
(plasma)b
(plasma)b
(liver)c
2
15
60
86.1 ( 3.7
61.3 ( 5.9
64.8 ( 7.0
92.1 ( 3.4
76.8 ( 5.2
50.9 ( 3.7
35.7/43.4
9.7/10.2
27.0/29.7
79.7/86.9
67.1/68.9
46.4/65.2
a The extracts were analyzed by radio-HPLC. The efficiencies of
extraction of radioactive analytes from the samples are reported below.
b Average of 3 independent studies per time point. c Individual results from
independent studies. Individual results from two samples are reported for
liver. Proportion [18F]15 in plasma and liver were calculated by comparison
of [18F]15 peak to total radioactivity present on chromatogram; recovery
was calculated by comparing the amount of radioactivity extracted from
the tissue sample to the radioactivity in an equivalent volume of tissue
plasma or homogenate.
between 15 and 60 min. There was also a time-dependent
increase in radioactivity in the residual pellets after plasma
extraction, suggesting an increase in binding to plasma proteins.
We do not know if this is due to nonspecific binding or related
to binding of the isatin to a specific recognition site on plasma
proteins.
Our initial studies with [125I]4 suggested that structural
optimization was necessary to develop novel radiotracers which
are stable in vivo. Comparison of the metabolic profile of isatins
4-6 in a murine liver S9 in vitro assay pointed to the (S)-2-
phenoxymethyl moiety as the major site of metabolism. This
led us to design a focused library of isatins incorporating
heterocycles and fluorine substituted aromatic groups in this
position. Motivated by the need for efficient labeling chemistry,
increased polarity, and metabolic stability, we incorporated 2′-
fluoroethyl-1,2,3-triazole in the N-1 position as well as on the
left side ether position of the isatin 5-sulfonamide scaffold. In
total 10 novel compounds were synthesized and evaluated for
caspase affinity. Introduction of fluorine to the (S)-2-phenoxym-
ethyl moiety led to a 3-fold increase in caspase 3 affinity, with
3-substituted 11c being slightly more potent than the 4-substi-
tuted 11b. Addition of a second fluorine group was well tolerated
with the 2,4 disubstituted 11d and the 3,5 disubstituted 11e being
equipotent to 11c. According to the molecular modeling studies
reported by Chu and co-workers, the (S)-2-phenoxymethyl
moiety interacts with the S3 binding domain through π-π
Discussion
We have developed a high affinity and metabolically stable
2-[18F]fluoroethylazide-conjugated isatin sulfonamide for use in
studying caspase 3/7 activation and apoptosis in vivo. The isatin
[18F]15 meets several of the design goals of a good imaging
agent, including subnanomolar affinity for caspase 3, high
selectivity for target, potential for efficient radiolabeling to
generate high specific radioactivity product, moderate lipophi-
licity, and high metabolic stability. Apoptosis is one of the major
mechanisms of cell death and hence an important target for
imaging therapeutic response in cancer. Beyond cancer, it has
a role in the evaluation of pathophysiology of neurodegeneration,
acute myocardial infarction, and chronic heart failure. Of the
probes available for imaging cell death, radiolabeled annexin
V has received the most attention.18,19 With regards to mem-
brane interacting probes, a number of N,N-didansyl cysteine,
dansyl ethylfluoroalanine, and malonic acid derivatives have also
been developed to image apoptosis.20–22
More recently there has been a growing interest in the
development of compounds that bind to activated executioner

2-[18F]Fluoroethyazide-Labeled Isatins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8063
binding with Phe381.8 The improved affinity of the fluorinated
derivatives could be due to more favorable π stacking or
hydrogen bonding with other groups in the S3 domain such as
Ser381.27 The 6-fold increase in potency of the tetrahydropyran
11f as compared to the (S)-2-phenoxymethyl 5 is surprising
given that this ring is fully saturated, however, according to
the model of Chu, the oxygen atom in the tetrahydropyran
moiety would be expected to form a strong hydrogen bond with
the hydroxyl group of Ser381.8 The pyrimidyl derivative 11g
should benefit from both favorable π-π binding with Phe381
and hydrogen bonding with Ser381, which explains the high
potency of this compound. The potency of the fluoroethyltriazole
16 was similar to that of the fluorinated aryl ether 11b-e, which
is surprising given the considerable smaller size and higher
polarity of this group. Lee7 denoted the binding domain around
the isatin nitrogen as a hydrophilic pocket, and most groups
have since included benzyl moieties in this position. It was
therefore highly unexpected that the triazole 14 is 4-fold more
potent than the benzyl derivative 5, which suggests the presence
of hydrophilic groups in this binding domain. Gratifyingly,
combining the fluoroethyltriazole group on the isatin nitrogen
with the 2,4-difluorinated aryl ether moiety to give 15 led to a
sharp increase in caspase 3 affinity (EC50 ) 0.5 nM). Analysis
of the cellular inhibition profile indicates that 15 is more potent
than 4. This correlates with the findings in a previous study by
Kopka et al.,9 which also reported differences in the cellular
caspase-3 inhibition profile for different isatin analogues. Isatin
15 has a cellular inhibitory potency in the 50-100 µM range,
comparable to the most active compounds reported by Kopka
et al.9 The basis of this finding is not clearly understood.
Furthermore, the increased uptake of [18F]15 under conditions
of drug induced apoptosis in cells and tumors supports the view
that [18F]15 warrants further evaluation as an in vivo apoptosis
marker.
Our studies have highlighted the need to consider metabolic
stability as well as labeling efficiency early in the discovery of
probes. “Click labeling” of the isatin precursor 13 with
2-[18F]fluoroethylazide proceeded in high radiochemical yields,
and the resulting triazole [8F]15 demonstrated high metabolic
stability, with no indication of defluorination in vivo. The modest
specific activity (1.2 GBq/µmol) was a concern for us, given
that [18F]15 is acting as a binding molecule (as distinct from a
substrate). However, our results indicated that the specific
activity was sufficient for the initial evaluation of [18F]15. As
we progress into more sophisticated in vivo models or clinical
development, this issue will be fully addressed. The rapid
clearance of [18F]15 in the heart and untreated tumors is a clear
advantage for imaging of caspase 3/7 activity in these tissues.
The overall low values for uptake in untreated tumor in the
biodistribution study (Figure 6) is likely the result of the low
levels of apoptosis (approximately 1%) in the RIF-1 tumor
model.28 In contrast, the high localization of [18F]15-derived
radioactivity in liver will preclude the use of liver models of
apoptosis in the preclinical evaluation of this tracer, as results
from such models will be difficult to interpret.10
The synthesis of a focused library of isatins led to identifica-
tion of an 1,2,3-triazole conjugated compound with subnano-
molar affinity for caspase-3. “Click labeling” with 2-[18F]flu-
oroethylazide proceeded in high radiochemical yields, and the
resulting triazole demonstrated high metabolic stability, with
no indication of defluorination in vivo. The rapid clearance of
the 18F-labeled isatin from heart and untreated tumor suggests
that the compound is suitable for imaging of apoptosis in these
tissues.
Experimental Section
Chemistry. Reagents and solvents were purchased from Sigma-
Aldrich (Gillingham, United Kingdom) and used without further
purification. Potassium hydroxide and potassium carbonate were
stored in a vacuum desiccator over phosphorus pentoxide. All
reactions were carried out under argon unless otherwise stated.
Petroleum ether refers to the fraction that is distilled between 40
and 60 °C. Automated flash chromatography was performed on a
CombiFlash Companion machine (Companion Presearch Ltd.),
using RediSep 4 or 12 g normal phase silica cartridges (flow rate
12 or 26 mL/min). Manual flash chromatography was carried out
using Davisil neutral silica (60 Å, 60-200 µm, Fisher Scientific,
Loughborough, UK), solvent mixtures are quoted as volume/
1
volume. H NMR spectra were obtained on a Bruker Avance 600
MHz NMR machine and spectra are referenced to residual solvent.
Coupling constants (J) are given in Hertz (Hz). GC-MS data was
acquired under electron ionization using an Agilent 6890N system.
Mass spectra were obtained in positive electrospray ionization mode
on a WatersMicromass LCT Premier or a Finnigan MAT 900 XLT
machine. Melting points were determined in capillary tubes on a
Stuart Scientific SMP1 melting point apparatus and are uncorrected.
Solvent mixtures for thin layer chromatography (TLC) are quoted
as volume/volume and samples were developed on aluminum
backed neutral silica plates (0.2 mm thickness) (Fluka, Seelze,
Germany). Purity analysis for compounds 2, 4, 5, 6, 10b-10h,
11b-11h, 12, 13, 14, 15, and 16 was evaluated by analytical HPLC;
compounds 9b-9h were analyzed by GC-MS with only a single
peak observed in the GC trace. Tabulated values of purity can be
found in the Supporting Information, and the purity of all
compounds was greater than 95%. [18F]Fluoride was produced by
a cyclotron (GE PETrace) using the 18O(p,n)18F nuclear reaction
with 16.4 MeV proton irradiation of an enriched [18O]H2O target.
HPLC Methods. (a) Purity analysis of nonradioactive com-
pounds was carried out on an Agilent 1100 series system with Laura
3 software (Lablogic, Sheffield, UK) with a Phenomenex Luna 50
mm × 4.6 mm (3 µm) HPLC column attached and a mobile phase
of 0.1 M ammonium formate and methanol/acetonitrile (1.8:1 v/v),
gradient (50% organic for 1 min; 50 f 90% organic in 14 min;
90% organic for 4 min; 90 f 50% organic phase for 4 min) flow
rate 1 mL/min, and wavelength 254 nm.
(b) Preparative radio-HPLC was carried out on a Beckman
System Gold equipped with a Bioscan Flowcount FC-3400 PIN
diode detector (Lablogic) and a linear UV-200 detector (wavelength
254 nm). A Phenomenex Onyx C18 100 mm × 10 mm HPLC
column and a mobile phase of water and methanol/acetonitrile (1.8:1
v/v), gradient 45 f 90% organic in 20 min, and flow rate 3 mL/
min.
(c) Analytical radio-HPLC was carried out as above but using a
Bioscan Flowcount FC3200 sodium iodide/PMT gamma detector
(Lablogic), a linear UV-200 detector (wavelength 254 nm), and a
Phenomenex Luna 50 mm × 4.6 mm (3 µm) column with a mobile
phase of water and methanol/acetonitrile (1.8:1 v/v), gradient 60
f 90% organic in 20 min, flow rate 1 mL/min.
Conclusion
We have designed, synthesized, and radiolabeled a novel
isatin 5-sulfonamide that has potential utility for imaging
caspase-dependent cell death. The approach described here
involved design of a focused library of compounds based on
metabolic stability, affinity, and ease of radiolabeling consid-
erations. This strategy permitted isotopic radiolabeling of high
affinity compounds discovered from screening such a library.
Synthesis. The following compounds were synthesized according
to established literature procedures and 1H NMR spectral data was
consistent with reported values: (S)-1-benzyl-5-(2-phenoxymethyl-
pyrrolidine-1-sulfonyl)isatin 2, (S)-1-(4-iodobenzyl)-5-(2-phenoxym-
ethyl-pyrrolidine-1-sulfonyl)isatin 4, and (S)-1-(4-fluorobenzyl)-5-
(2-phenoxymethyl-pyrrolidine-1-sulfonyl)isatin 5.

8064 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smith et al.
1
The radiotracer [125I]4 was synthesized using the method of
Kopka9 by GE Healthcare Ltd. (Little Chalfont, Buckinghamshire,
UK) and supplied as a solution in ethanol (∼37 MBq/mL) with
91.9% radiochemical purity and specific activity of approximately
2000 Ci/mmol.
) 240.1597 (M + H)+. Calcd for C13H22NO3 240.1594. H NMR
(600 MHz, CDCl3) δ 4.13 (s, 2 H), 3.96-3.88 (m, 1 H), 3.64 (dd,
J ) 9 Hz, J ) 3.6 Hz, 1 H), 3.49-3.22 (m, 3 H), 2.40 (s, 1 H),
1.94-1.78 (m, 4 H), 1.46 (s, 9 H). TLC (I2) Rf ) 0.67 (1:1 hexanes/
ethyl acetate).
1-(4-Fluorobenzyl)-5-(pyrrolidine-1-sulfonyl)isatin (6). To an
ice-cold, stirred solution of 5-pyrrolidine-1-sulfonylisatin7 (0.14 g,
0.5 mmol) in dry DMF (8 mL) was added sodium hydride (40 mg,
1 mmol). After 30 min, 4-fluorobenzyl bromide (0.38 g, 2 mmol)
was added and the mixture allowed warm to room temperature.
After 19 h, the orange solution was poured onto 10% aq NH4Cl
(25 mL) and extracted with DCM (3 × 15 mL). Following
concentration in vacuo, the residue taken up in diethyl ether (10
mL) and washed with water (3 × 10 mL) and dried over Na2SO4.
Chromatography (diethyl ether/hexanes) afforded the title compound
as an orange gum (83 mg, 43%). HRMS (ESI) ) 389.0988 (M +
H)+. Calcd for C19H18FN2O4S 389.0971. 1H NMR (600 MHz,
CDCl3) δ 8.05 (d, J ) 1.5 Hz, 1H), 7.99 (dd, J ) 8.4 Hz, J ) 1.5
Hz, 1 H), 7.35-7.32 (m, 2 H), 7.09-7.06 (m, 2 H), 6.91 (d, J )
8.4 Hz, 1 H), 4.94 (s, 2 H), 3.25-3.23 (4 H, m), 1.84-1.79 (4 H,
m). TLC (UV254) Rf ) 0.63 (4:1 ethyl acetate/hexanes). HPLC tR
) 6.83 min.
(S)-5-(2-(4-Fluorophenoxymethyl)-pyrrolidine-1-sulfonyl)isatin
(10b). To a stirred solution of 9b (0.15 g, 0.5 mmol) in dry DCM
(4 mL) cooled in an ice bath was added TFA (0.6 mL, 10 mmol).
After 1 h, bulk solvent was removed in vacuo and the residue
remaining taken up in dry DCM (8 mL) and cooled in an ice bath.
Dry triethylamine (1.5 mL) was then added followed by 5-chlo-
rosulfonylisatin7 (0.16 g, 0.65 mmol) in dry THF (4 mL) and the
solution was then stirred. After 19 h, bulk solvent was removed in
vacuo and redissolved in DCM (10 mL), washed with water (2 ×
10 mL) and then brine (1 × 10 mL) and dried over Na2SO4.
Chromatography (hexanes/ethyl acetate) gave the desired product
as an orange solid (104 mg, 51%); mp: 205-207 °C. HRMS (ESI)
1
) 405.0941 (M + H)+. Calcd for C19H18FN2O5S 405.0920. H
NMR (600 MHz, CDCl3) δ 8.10 (s,1 H), 8.08 (dd, J ) 8.4 Hz, J
) 1.8 Hz, 1 H), 8.00 (br, 1 H), 7.00 (d, J ) 7.8 Hz, 1 H), 6.99-6.95
(m, 2 H), 6.83-6.81 (m, 2 H), 4.17 (dd, J ) 9.6 Hz, J ) 3.6 Hz,
1 H), 3.98-3.95 (m, 1 H), 3.91 (dd, J ) 9 Hz, J ) 7.8 Hz, 1 H),
3.54-3.50 (m, 1 H), 3.24-3.19 (m, 1 H), 2.10-1.99 (m, 2 H),
1.87-1.77 (m, 2 H). TLC (UV254) Rf ) 0.27 (2:1 ethyl acetate/
hexanes). HPLC tR ) 7.83.
(S)-tert-Butyl 2-(4-Fluorophenoxymethyl)pyrrolidine-1-car-
boxylate (9b). To a stirred solution of 4-fluorophenol (0.27 g, 2.4
mmol) in dry DMF (10 mL) was added sodium hydride (60% w/w
in mineral oil) (0.11 g, 2.8 mmol). After 30 min, ((S)-1-(tert-
butoxycarbonyl)pyrrolidin-2-yl)toluene-4-sulfonate 77 (0.71 g, 2.0
mmol) in dry DMF (5 mL) was then added and the mixture heated
to 80 °C for 17 h. The reaction was allowed to cool to room
temperature and poured over 1 M NaOH (25 mL) and extracted
with DCM (3 × 15 mL). The combined organic fractions were
reduced under vacuum and diethyl ether (20 mL) added and then
washed with 1 M NaOH (1 × 20 mL), water (1 × 20 mL), and
then brine (1 × 20 mL) and dried over Na2SO4. Chromatography
(hexanes/ethyl acetate) gave the product as a colorless oil (0.36 g,
61%). HRMS (ESI) ) 296.1654 (M + H)+. Calcd for C16H23FNO3
(S)-1-(4-Fluorobenzyl)-5-(2-(4-fluorophenoxymethyl)-pyrro-
lidine-1-sulfonyl)isatin (11b). To a stirred solution of 10b (40 mg,
0.1 mmol) in dry DMF (3 mL) was added potassium carbonate
(21 mg, 0.15 mmol) followed by 4-fluorobenzyl bromide (76 mg,
0.4 mmol). After 2 h, TLC indicated complete conversion of 10b
and the solution was poured onto 10% aq NH4Cl (10 mL) and
extracted with DCM (3 × 10 mL). Combined organic fractions
concentrated in vacuo and taken up in diethyl ether (10 mL), washed
with water (2 × 10 mL) and then brine (1 × 10 mL) and dried
over Na2SO4. Chromatography (hexanes/ethyl acetate) gave the title
compound as an orange gum (34 mg, 66%). HRMS (ESI) )
513.1306 (M + H)+. Calcd for C26H23F2N2O5S 513.1296. 1H NMR
(600 MHz, CDCl3) δ 8.04 (d, J ) 1.8 Hz, 1 H), 7.97 (dd, J ) 8.4
Hz, J ) 1.8 Hz, 1 H), 7.33-7.30 (m, 2 H), 7.09-7.05 (m, 2 H),
6.96-6.92 (m, 2 H), 6.86 (d, J ) 8.4 Hz, 1 H), 6.81-6.77 (m, 2
H), 4.92 (d, J ) 15.6, 1 H), 4.91 (d, J ) 15.6, 1 H), 4.14 (dd, J )
9.6 Hz, J ) 3.6 Hz, 1 H), 3.95-3.92 (m, 1 H), 3.88 (dd, J ) 9.6
Hz, J ) 7.2 Hz, 1 H), 3.51-3.47 (m, 1 H), 3.20-3.15 (m, 1 H),
2.06-1.93 (m, 2 H), 1.83-1.73 (m, 2 H). TLC (UV254) Rf ) 0.61
(2:1 ethyl acetate/hexanes). HPLC tR ) 12.25 min.
1
296.1656. H NMR (600 MHz, CDCl3) δ 6.97-6.93 (m, 2 H),
6.88-6.84 (m, 2 H), 4.18-4.03 (m, 2 H), 3.94-3.73 (m, 1 H),
3.46-3.32 (m, 2 H), 2.07-1.81 (m, 4 H), 1.47 (s, 9 H). TLC
(UV254) Rf ) 0.51 (2:1 hexanes/ethyl acetate).
(S)-tert-Butyl 2-(Pyrimidin-4-yloxymethyl)pyrrolidine-1-car-
boxylate (9g). To a stirred solution of N-tert-butoxycarbonyl-L-
prolinol (0.81 g, 4 mmol) 8 in dry DCM (10 mL) was added
triphenylphosphine (5.24 g, 20 mmol) followed by 4(3H)-pyrimi-
done (0.77 g, 8mmol). The solution was cooled in an ice bath and
DIAD (3.24 g, 16 mmol) added dropwise over 10 min. After 48 h,
GC-MS indicated complete conversion of 8 and the reaction mixture
was poured onto water (30 mL), the organic fraction collected, and
the aqueous phase washed with further DCM (2 × 20 mL). The
combined organic fractions were washed with 1 M NaOH (2 × 15
mL) and then brine (1 × 15 mL) and dried over Na2SO4. Removal
of bulk solvent yielded an orange gum and addition of hexanes/
diethyl ether (1:1) resulted in formation of a precipitate of
triphenylphosphine oxide, which was removed by filtration. Chro-
matography (ethyl acetate) afforded the desired product as a
colorless oil (0.77 g, 69%). HRMS (ESI) ) 280.1655 (M + H)+.
(S)-1-(2-Propynyl)-5-(2-phenoxymethyl-pyrrolidine-1-sulfo-
nyl)isatin (12). To a solution of (S)-5-(2-phenoxymethyl-pyrroli-
dine-1-sulfonyl)isatin 10a7 (0.39 g, 1mmol) in dry DMF (10 mL)
was added potassium carbonate (0.21 g, 1.5 mmol) followed by
propargyl bromide (80 wt. % in toluene) (0.14 g, 1.2 mmol). After
2 h, TLC indicated complete conversion of 10a and the solution
was poured onto 10% aq NH4Cl (20 mL) and washed with DCM
(3 × 10 mL). The combined organic fractions were then reduced
in vacuo and the residue taken up in diethyl ether (10 mL) and
washed with water (2 × 10 mL) and then brine (1 × 10 mL) and
dried over Na2SO4. Chromatography (hexanes/ethyl acetate) gave
the product as an orange gum. Recrystallization from ethyl acetate/
hexanes furnished the product as an orange solid (0.28 g, 66%);
mp: 90-92 °C. HRMS (ESI) ) 425.1185 (M + H)+. Calcd for
1
Calcd for C14H22N3O3 280.1656. H NMR (600 MHz, CDCl3) δ
8.71 (s, 1 H), 8.39 (d, J ) 6 Hz, 1 H), 6,69 (d, J ) 6 Hz, 1 H),
4.45-3.91 (m, 3 H), 3.40-3.33 (m, 2 H), 2.00-1.82 (m, 4 H),
1.42 (s, 9 H). TLC (UV254) Rf ) 0.49 (ethyl acetate).
1
C22H21N2O5S 425.1171. H NMR (600 MHz, CDCl3) δ 8.12 (dd,
J ) 8.4 Hz, J ) 1.8 Hz, 1 H), 8.05 (d, J ) 1.8 Hz, 1 H), 7.25-7.21
(m, 2 H), 7.16 (d, J ) 8.4 Hz, 1 H), 6.96-6.93 (m, 1 H), 6.81 (d,
J ) 9 Hz, 2 H), 4.56 (dd, J ) 18 Hz, J ) 2.4 Hz, 1 H), 4.53 (dd,
J ) 18 Hz, J ) 2.4 Hz, 1 H), 4.19 (dd, J ) 9.6 Hz, J ) 3 Hz, 1
H), 4.07-4.01 (m, 1 H), 3.97 (dd, J ) 9.6 Hz, J ) 7.2 Hz, 1 H),
3.55-3.51 (m, 1 H), 3.33-3.28 (m, 1 H), 2.36 (t, J ) 2.4 Hz, 1
H), 2.09-1.99 (m, 2 H), 1.89-1.75 (m, 2 H). TLC (UV254) Rf )
0.54 (2:1 ethyl acetate/hexanes). HPLC tR ) 9.00 min.
(S)-tert-Butyl 2-(2-Propynyloxymethyl)pyrrolidine-1-carboxy-
late (9h). To a stirred solution of 8 (0.40 g, 2 mmol) in dry DMF
(10 mL) was added potassium hydroxide (0.56 g, 10 mmol),
followed by dropwise addition of propargyl bromide (80 wt. % in
toluene) (0.48 g, 4 mmol) over 5 min. After 18 h, the reaction
mixture was poured onto water (30 mL) and washed with DCM (3
× 15 mL). The combined organic fractions concentrated in vacuo
and the liquid remaining taken up in diethyl ether (15 mL) and
washed with water (2 × 10 mL) and then brine (1 × 10 mL) and
dried over Na2SO4. Chromatography (hexanes/ethyl acetate) gave
the desired product as a colorless oil (0.32 g, 67%). HRMS (ESI)
(S)-1-((1-(2-Fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2-
phenoxymethyl-pyrrolidine-1-sulfonyl)isatin (14). To a stirred
solution of 12 (138 mg, 0.3 mmol) in dry DMF (3 mL) was added

2-[18F]Fluoroethyazide-Labeled Isatins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8065
copper sulfate (38 mg, 0.15 mmol) in water (0.2 mL) followed by
ascorbic acid (53 mg, 0.3 mmol) in water (0.2 mL) and then
2-fluoroethylazide14 (33 mg, 0.36 mmol) in dry DMF (1.5 mL)
and the mixture left to stir under argon. After 2 h, TLC indicated
reaction completion and mixture was poured onto 10% aq NH4Cl
(12 mL) and extracted with DCM (3 × 10 mL) and dried over
Na2SO4. Chromatography (hexanes/ethyl acetate) afforded an orange
solid (26 mg, 51%); mp: 165-167 °C. HRMS (ESI) ) 514.1557
(M + H)+. Calcd for C24H25FN5O5S 514.1560. 1H NMR (600 MHz,
CDCl3) δ 8.07 (dd, J ) 8.4 Hz, J ) 1.8 Hz, 1 H), 8.01 (d, J ) 1.8
Hz, 1 H), 7.76 (s, 1 H), 7.47 (d, J ) 8.4 Hz, 1 H), 7.23-7.19 (m,
2 H), 6.92 (t, J ) 7.8 Hz, 1 H), 6.82 (d, J ) 7.8 Hz, 2 H), 5.03 (d,
J ) 15.6 Hz, 1 H), 5.02 (d, J ) 15.6 Hz, 1 H), 4.79 (dt, J ) 46.2
Hz, J ) 4.8 Hz, 2 H), 4.66 (dt, J ) 27 Hz, J ) 4.8 Hz, 2 H), 4.17
(dd, J ) 9.6 Hz, J ) 3.6 Hz, 1 H), 4.00-3.97 (m, 1 H), 3.93 (dd,
J ) 9 Hz, J ) 7.2 Hz, 1 H), 3.53-3.49 (m, 1 H), 3.27-3.24 (m,
1 H), 2.08-1.97 (m, 2 H), 1.86-1.75 (m, 2 H). TLC (UV254) Rf )
0.56 (ethyl acetate). HPLC tR ) 7.93 min.
Radiochemistry. Under an atmosphere of nitrogen, a buffered
solution (sodium phosphate buffer, pH 6.0, 250 mM) of sodium
ascorbate (50 µL, 8.7 mg, 43.2 µmol) was added to a Wheaton
vial (1 mL) containing an aqueous solution of copper(II) sulfate
(50 µL, 1.7 mg pentahydrate, 7.0 µmol). After one min, a solution
of alkyne 13 (3.0 mg, 6.5 µmol) in DMF (25 µL) was added
followed by distilled [18F]-2-fluoroethylazide (185-740 MBq) in
acetonitrile (100 µL). The mixture was left at room temperature
for 30 min, water (15 µL) was added and the resulting mixture
was purified by preparative radio-HPLC. The isolated HPLC
fraction was diluted with water (5 mL) and loaded onto a SepPak
C18-light cartridge (Waters) that had been conditioned with ethanol
(5 mL) and water (10 mL). The cartridge was subsequently flushed
with water (5 mL) and [18F]15 eluted with ethanol (0.1 mL
fractions). The product fraction was diluted with PBS to provide
an ethanol content of 10-15% (v/v).
the final concentration of DMSO in all wells was 5% of the total
volume. Recombinant caspases were used at 0.5 units per assay
(∼500 pmol substrate converted per h). All reagents except the
peptide substrate were preincubated for 10 min. The peptide
substrate (final concentration 10 µM) was then added, and the plate
was incubated for a further 30 min; 30 min was selected after initial
linearity study where reaction was assessed after 10, 30, 60, or 90
min. Respective control wells contained all reaction components
without enzyme. The amount of 7-AMC produced was measured
on a fluorescence microplate reader (Victor2; Perkin-Elmer Life
sciences) at excitation and emission wavelengths of 355 and 460
nm, respectively. The concentration of isatin that inhibits the caspase
activity by 50% (EC50) was estimated by nonlinear regression
analysis using GraphPad Prism (Version 4.0 for Windows, Graph-
Pad Software, San Diego, CA). All the isatins were analyzed in
duplicate; the assays were repeated once.
In Vitro Mouse Liver S9 Metabolism Studies. Liver S9
metabolism studies were performed as previously described.30
Mouse livers were rapidly excised and kept at 4 °C throughout the
experiment. Tissues were weighed and homogenized in an equiva-
lent volume of 50 mM Tris-150 mM KCl-HCl buffer (pH 7.4) using
an Ultra-Thurrax homogenizer (IKA, Staufen, Germany). To obtain
the S9 fraction, the homogenate was centrifuged (10000g) for 30
min to remove nuclei, mitochondria, and cell debris. Protein
concentration of the S9 fraction was determined by a commercial
BCA protein assay kit (Perbio Science, Cheshire, UK). S9 fractions
were stored at -80 °C for up to 6 weeks. Selected unlabeled isatins
4, 5, and 6 (10 mM, 10 µL) were incubated with the S9 fraction
(33.26 mg/mL, 20 µL) and 0.5 mM nicotinamide adenine dinucle-
otide phosphate (reduced form) in air for 60 min at 37 °C in 0.1
mM Tris-HCl buffer (pH 7.4) in a total volume of 1 mL. Control
incubation samples contained no isatin. The reactions were
terminated by addition of ice-cold acetonitrile (2 mL); the samples
were then immediately placed on dry ice prior to extraction and
HPLC analysis (below).
Log P Determination. Calculated Log P values for all com-
pounds were determined using ACD/Chemsketch Laboratories
10.02. For [18F]15, lipophilicity was also assessed by measuring
the octanol-water partition coefficient using the method of Barthel
et al.29 Briefly, [18F]15 (∼180 µCi in 25 µL ethanol) was diluted
to a final volume of 100 µL with water (stock solution). Aliquots
of stock solution (10 µL) were added to water (490 µL) and octan-
1-ol (Aldrich anhydrous grade) (500 µL). The solutions were then
shaken vigorously for 10 min then centrifuged (13201g, 20 °C, 30
min). Following centrifugation, portions (200 µL) of the water and
octanol layers were carefully removed and analyzed on a Cobra II
Auto-Gamma counter (Packard Instruments, Meriden, CT) and
compared. The octanol-water partition coefficient was obtained
by dividing the octanol containing radioactivity by the water
containing radioactivity and the log10 of the ratio calculated.
Caspase Enzyme Inhibition Assays. Recombinant human
caspases 1, 3, 6, 7, and 8 and their peptide-specific substrates9 were
purchased from Biomol International, UK. Inhibition of the
recombinant caspases by nonradioactive isatins was assessed using
a fluorometric assay that measures the accumulation of a fluorogenic
product, 7-amino-4-methylcoumarin (7-AMC). All assays were
performed in 96-well plates at a volume of 200 µL per well. The
assays were performed at 37 °C in an appropriate reaction buffer
as described below for each caspase. For caspase 1, the buffer
comprised of 0.1% CHAPS, 100 mM NaCl, 5 mM 2-mercaptoet-
hanol, 100 mM HEPES (pH 7.4), 2 mM EDTA, 10% sucrose, and
10 µM of the peptide substrate Ac-YVAD-AMC. For caspase 3:
20 mM HEPES (pH 7.4), 10% sucrose, 100 mM NaCl, 0.1%
CHAPS, 2 mM EDTA, and 10 µM Ac-DEVD-AMC. For caspase
6: 20 mM HEPES (pH 7.4), 10% sucrose, 100 mM NaCl, 0.1%
CHAPS, 2 mM EDTA, and 10 µM Ac-VEID-AMC. For caspase
7: 20 mM HEPES (pH 7.4), 10% sucrose, 5 mM 2-mercaptoethanol,
100 mM NaCl, 0.1% CHAPS, 2 mM EDTA, 10 µM Ac-DEVD-
AMC. For caspase 8: 20 mM HEPES (pH 7.4), 10% sucrose, 100
mM NaCl, 0.1% CHAPS, 2 mM EDTA, and 10 µM Ac-IETD-
AMC. The buffers contained nonradioactive isatins in DMSO at a
final concentration of 500, 50, 5 µM; 500, 50, 5nM; 500, 50, 5pM;
Western Blot Analysis. RIF-1 cells were incubated for 15 min
with the caspase inhibitors Z-VAD-FMK (Biomol International,
UK), isatin 15, and 4, and subject to apoptosis with CDDP for 48 h.
Protein samples were prepared by lysing RIF-1 cells in RIPA buffer
(Invitrogen Ltd., Paisley, UK) supplemented with protease and
phosphatase inhibitor cocktails (Sigma-Aldrich Company Ltd.,
Dorset, UK). Equal amounts of protein (30 µg) were denaturated
in sample buffer, subjected to SDS-polyacrylamide gel electro-
phoresis on 4-12% gels (Invitrogen Ltd., Paisley, UK) and
transferred to PVDF membranes (GE Healthcare Life Sciences,
Buckinghamshire, UK). The membranes were immunoblotted with
specific primary antibodies, horseradish peroxidase-conjugated
secondary antibodies, and visualized by enhanced chemilumines-
cence (GE Healthcare Life Sciences, Buckinghamshire, UK). The
following antibodies were used: a mouse monoclonal anticleaved
PARP (Cell Signaling Technology Inc., Danvers, MA), a mouse
monoclonal antialpha-tubulin, and a goat antimouse HRP (Santa
Cruz Biotechnology Inc., Santa Cruz, CA).
In Vitro [18F]15 Uptake and Caspase 3 Activation Assays.
Cells were plated in triplicate in 12-well plates 2 or 3 days prior to
the experiments and treated with CDDP (Sigma, UK) or vehicle
(0.1% DMSO) at the indicated concentration and time. On the day
of the experiment, ∼10 µCi/well of [18F]15 was added and allowed
to accumulate into cells for 60 min at 37 °C. Cells were collected,
washed, and resuspended in 400 µL of PBS. Then 260 µL of each
sample were transferred in counting tubes and fluorine-18 radio-
activity was immediately determined using a Packard Cobra II
gamma counter (Perkin-Elmer, UK). BCA Protein assay (Pierce,
UK) was performed for all samples and data are normalized and
expressed as CCPMA/mg of protein.
In Vivo Biodistribution and Metabolism Studies. The radia-
tion-induced murine fibrosarcoma (RIF-1) tumor cells31 were
maintained in RPMI 1640 medium (Invitrogen Ltd., Paisley, UK)
supplemented with 10% fetal calf serum (BioWhittaker Europe Ltd.,
Verviers, Belgium), 2 mM L-glutamine, 100 U/mL penicillin, 100
µg/mL streptomycin, and 0.25 µg/mL fungizone (Gibco, UK) at

8066 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smith et al.
37 °C in a humidified incubator with 5% CO2. All animal work
was done by licensed investigators in accordance with the United
Kingdom’s “Guidance on the Operation of Animals (Scientific
Procedures) Act 1986” (HMSO, London, United Kingdom, 1990)
and in full compliance with government regulations and guidelines
on the welfare of animals in experimental neoplasia.32 Tumors were
established in mice by subcutaneous injection of 5 × 105 cells on
the back of male C3H/hej mice (Harlan, Bicester, Oxfordshire, UK).
Tumor growth was monitored every two days using electronic
calipers, and tumor volume estimated using the equation (π/6) ×
L × W × D (L ) length, W ) width, and D ) depth). Animals
were selected for biodistribution studies of [18F]15 when the tumors
reached ∼100-150 mm3; biodistribution of [125I]4 was done in
nontumor-bearing mice. Mice were injected intravenously via the
lateral vein with 0.08-0.13 mL of radioactivity (∼0.37 MBq of
total volume of HPLC injectate was recorded and an aliquot (0.1
mL) removed for counting. The radioactivity in the 0.1 mL aliquot
and the pellet were then analyzed by gamma counting (Packard
Instruments).
Acknowledgment. This work was funded by Cancer Re-
search UK programme grant C2536/A5708 and UK Medical
Research Council core funding grant U.1200.02.005.00001.01.
We thank Dr Harold Toms, Queen Mary and Westfield College,
for NMR assistance, and the EPSRC National Mass Spectros-
copy Service Centre (Swansea, UK) for carrying out some of
the mass spectral analysis.
Supporting Information Available: Experimental and analytical
data for compounds 9c-9f, 10c-10h, 11c-11 h, 13, 15, and 16.
Further HPLC chromatograms relating to the metabolism studies,
tables of purity data, HPLC chromatograms, and 1H NMR spectra
for compounds 2, 4, 5, 6, 11b-h, 13, 14, 15, and 16. This material
is available free of charge via the Internet at http://pubs.acs.org.
[
125I]4 or 3.7 MBq of [18F]15) dissolved in phosphate buffered
saline. At selected times after injection (2-60 min), mice were
sacrificed by exsanguination via cardiac puncture under general
anesthesia (isofluorane inhalation). Aliquots of heparinized blood
were rapidly centrifuged (2000g for 5 min) to obtain plasma. The
radioactivity contained in tissues was determined by gamma-
counting on a Cobra II Auto-Gamma counter (Packard Instruments,
Meriden, CTA) and expressed as a percentage of injected dose per
gram of tissue (%ID/g). A minimum of three mice were used for
each time point. All animals were treatment-na¨ıve.
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