Novel fluorine-18 labeled 5-(1-pyrrolidinylsulfonyl)-7-azaisatin derivatives as potential PET tracers for in vivo imaging of activated caspases in apoptosis

Article abstract of DOI:10.1016/j.bmc.2015.07.014

The programmed type I cell death, defined as apoptosis, is induced by complex regulated signaling pathways that trigger the intracellular activation of executioner caspases-3, -6 and -7. Once activated, these enzymes initiate cellular death through cleavage of proteins which are responsible for DNA repair, signaling and cell maintenance. Several radiofluorinated inhibitors of caspases-3 and -7, comprising a moderate lipophilic 5-(1-pyrrolidinylsulfonyl)isatin lead structure, are currently being investigated for imaging apoptosis in vivo by us and others. The purpose of this study was to increase the intrinsic hydrophilicity of the aforementioned lead structure to alter the pharmacokinetic behavior of the resulting caspase-3 and -7 targeted radiotracer. Therefore, fluorinated and non-fluorinated derivatives of 5-(1-pyrrolidinylsulfonyl)-7-azaisatin were synthesized and tested for their inhibitory properties against recombinant caspases-3 and -7. Fluorine-18 has been introduced by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) of an alkyne precursor with 2-[¹⁸F]fluoroethylazide. Using dynamic micro-PET biodistribution studies in vivo the kinetic behavior of one promising PET-compatible 5-pyrrolidinylsulfonyl 7-azaisatin derivative has been compared to a previously described isatin based radiotracer.

Full text of DOI:10.1016/j.bmc.2015.07.014

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel fluorine-18 labeled 5-(1-pyrrolidinylsulfonyl)-7-azaisatin
derivatives as potential PET tracers for in vivo imaging of activated
caspases in apoptosis
a,
⇑
b
b
a
a
Christopher M. Waldmann , Sven Hermann , Andreas Faust , Burkhard Riemann , Otmar Schober ,
Michael Schäfers
a
a,b,d
c,d
a,
, Günter Haufe , Klaus Kopka
Department of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1, D-48149 Münster, Germany
European Institute for Molecular Imaging (EIMI), University of Münster, Waldeyerstraße 11, D-48149 Münster, Germany
Organic Chemistry Institute, University of Münster, Corrensstrasse 40, D-48149 Münster, Germany
b
c
d
Cells-in-Motion Cluster of Excellence (EXC 1003—CiM), University of Münster, Waldeyerstraße 15, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The programmed type I cell death, defined as apoptosis, is induced by complex regulated signaling path-
ways that trigger the intracellular activation of executioner caspases-3, -6 and -7. Once activated, these
enzymes initiate cellular death through cleavage of proteins which are responsible for DNA repair, signal-
ing and cell maintenance. Several radiofluorinated inhibitors of caspases-3 and -7, comprising a moderate
lipophilic 5-(1-pyrrolidinylsulfonyl)isatin lead structure, are currently being investigated for imaging
apoptosis in vivo by us and others. The purpose of this study was to increase the intrinsic hydrophilicity
of the aforementioned lead structure to alter the pharmacokinetic behavior of the resulting caspase-3 and
Received 29 April 2015
Revised 1 July 2015
Accepted 9 July 2015
Available online xxxx
Keywords:
Apoptosis
-7 targeted radiotracer. Therefore, fluorinated and non-fluorinated derivatives of 5-(1-pyrrolidinylsul-
7
-Azaisatin
fonyl)-7-azaisatin were synthesized and tested for their inhibitory properties against recombinant cas-
pases-3 and -7. Fluorine-18 has been introduced by copper(I)-catalyzed azide–alkyne cycloaddition
Caspase inhibitor
Click-chemistry
18
18
F-radiolabeling
Positron emission tomography (PET)
(CuAAC) of an alkyne precursor with 2-[ F]fluoroethylazide. Using dynamic micro-PET biodistribution
studies in vivo the kinetic behavior of one promising PET-compatible 5-pyrrolidinylsulfonyl 7-azaisatin
derivative has been compared to a previously described isatin based radiotracer.
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Apoptosis is a form of programmed cell death in multicellular
-7, once activated, irrevocably initiate cellular death through cleav-
age of proteins which are responsible for DNA repair, signaling and
cell maintenance. Therefore, these enzymes are suitable in vivo
biomarkers of living apoptotic cells and tissues.
1
organisms. In adult individuals cell homeostasis is achieved when
the rate of mitotic cell division is balanced by cell death. However,
apoptosis failure can contribute to profound pathologies such as
tumor growth and autoimmune diseases whereas unwanted apop-
In 2000, Lee et al. described the discovery of 1-methyl-5-ni-
troisatin as a selective, non-peptidic inhibitor of recombinant cas-
5
pase-3 showing an K
i
value of 0.5
l
M. In order to substitute the
2
tosis occurs in many neurodegenerative disorders. Apoptotic cell
metabolically susceptible nitro group (S)-N-methyl-5-[1-(2-phe-
noxylmethylpyrrolidinyl)sulfonyl]isatin was developed which
retained high selectivity and inhibitory potency towards execu-
tioner caspases-3 and -7. X-ray crystallographic analysis of an
enzyme-inhibitor complex revealed the binding mode of the inhi-
bitor. It has been shown that the nucleophilic cysteine thiolate
within the active site binds the carbonyl group in position 3 of
the isatin covalently under formation of a tetrahedral thiohemiac-
etal. Selectivity towards caspases-3 and -7 is achieved by
hydrophobic interactions between the pyrrolidine-moiety of the
death is induced by complex regulated signaling pathways trig-
gered by either activation of death receptors (extrinsic pathway)
or mitochondria (intrinsic pathway).3 Both pathways activate the
intracellular enzyme class of cysteinyl aspartate-specific proteases,
4
in short caspases. Among these the executioner caspases-3, -6 and
⇑
Corresponding author at present address: Crump Institute for Molecular
Imaging, Department of Molecular and Medical Pharmacology, David Geffen School
of Medicine at UCLA, 570 Westwood Plaza, Los Angeles, CA 90095, United States.
Tel.: +1 310 794 1731; fax: +1 310 206 8975.
2
inhibitor and the S binding pocket of the enzyme.
Based on these findings, structure–activity relationship studies
were performed leading to an extensive compound library of
E-mail address: cwaldmann@mednet.ucla.edu (C.M. Waldmann).
Present address: Radiopharmaceutical Chemistry, German Cancer Research
Center (dkfz), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
http://dx.doi.org/10.1016/j.bmc.2015.07.014
0
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Waldmann, C. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.014

2
C. M. Waldmann et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
-(1-pyrrolidinylsulfonyl)isatin derivatives.6–11 Mainly, side chains
as starting materials.5 Therefore, isatin is sulfonated in position 5
using sulfur trioxide. The resulting sulfonate is converted into the
corresponding sulfonyl chloride and subsequently coupled with an
appropriate (S)-proline derivative to yield the inhibitor’s core
structure. Initially we attempted to adapt this methodology in
order to synthesize an inhibitor of caspase-3 and -7 with a 7-aza-
isatin core. However, forming the sulfonate of 7-azaisatin in posi-
tion 5 under various conditions was unsuccessful. This could be
explained in part by the low electron density at the pyridine moi-
ety of the molecule. In a next attempt to prepare the sulfonyl chlo-
with different functionality were introduced either at position N1
of isatin or at position 2 of the pyrrolidine leading to highly selec-
tive inhibitors with K (or IC50) values in the low nanomolar range.
i
To enable the use of 5-(1-pyrrolidinylsulfonyl)isatin derivatives
for imaging of activated caspases in apoptosis, we and others intro-
8
,12–21
duced fluorine-18 to various sites of the inhibitor (Fig. 1).
Fluorine-18 is a low energy positron emitter with a half life of
110 min making it an ideal substituent to label molecular imag-
ꢀ
ing probes for positron emission tomography (PET). PET-compati-
ble derivatives of isatin derived caspase inhibitors show potential
ride of 7-azaisatin we adapted
a known one-pot synthesis
22,24
in monitoring apoptosis during cancer therapy.
sequence starting with previously described 5-bromo-1-methyl-
2
7,28
A possible drawback of 5-(1-pyrrolidinylsulfonyl)isatin based
apoptosis imaging agents concerns their pharmacokinetic behavior,
which is mainly controlled by the moderate lipophilicity of the core
skeletal structure. Biodistribution studies of fluorine-18 labeled
derivatives in both rodents and humans often reveal a lower renal
excretion compared to routinely used radiotracers such as
7-azaisatin (1) (Scheme 1).
The reaction sequence is composed of a bromine lithium
exchange reaction by using 2.0 equiv tert-butyllithium (t-BuLi) at
ꢁ80 °C in tetrahydrofuran (THF) (1), conversion of the resulting
2
aryllithium with gaseous SO to an arylsulfinate (2) and subse-
quent oxidation to the desired sulfonyl chloride with N-chlorosuc-
cinimide (NCS) at room temperature in dichloromethane (DCM)
(3). Analysis of the resulting complex mixture revealed that com-
pound 2 was not formed under these conditions. In another
attempt the reactive carbonyl function in position 3 of 7-azaisatin
was protected as 1,3-dioxane beforehand, but still the desired pro-
duct could not be isolated (reaction sequence not shown).
1
8
18
23
2
-deoxy-2-[ F]fluoro-
D
-glucose ([ F]FDG).
The predominant
clearance via the hepatobiliary route implicates a rather maintained
circulation of the radiotracer over time which potentially supports
undesired oxidative metabolism of any corresponding radiotracer.
2
5
Generally, prolonged biodistribution of radiotracers and its metabo-
lites can increase unspecific binding to off-targets which in turn
leads to an undesired low signal-to-noise ratio and higher back-
ground signal. Therefore, we intended to increase the inherent
hydrophilicity of the isatin-based apoptosis imaging agents by
envisioning second generation caspase-3 and -7 inhibitors based
on a 7-azaisatin sulfonamide skeletal structure (Fig. 2).
Here, we report on the synthesis and in vitro evaluation of the
caspase inhibition potencies of non-fluorinated and fluorinated
derivatives of 5-(1-pyrrolidinylsulfonyl)-7-azaisatin. The fluorine-
Therefore, we designed a new synthetic strategy starting with
commercially available 5-bromo-7-azaindole (Scheme 2). This
strategy aims at the formation of a 5-(1-pyrrolidinylsulfonyl)-
7-azaindole skeletal structure and subsequent oxidation of the 7-
azaindole moiety to 7-azaisatin. In order to prepare two N1-
alkylated non-fluorinated 5-(1-pyrrolidinylsulfonyl)-7-azaisatin
derivatives, position N1 was alkylated either with methyl iodide
or n-propyl bromide, respectively. The two different alkyl
substituents were chosen because of their favorable properties in
1
8 labeled substituent is introduced by copper(I)-catalyzed
26
7,9
azide–alkyne cycloaddition (CuAAC). This study will allow us to
evaluate the impact of the hydrophilicity of the skeletal structure
on the pharmacokinetic behavior of the corresponding radiotracer.
regard to inhibition potency and cellular uptake. As shown in
Scheme 2 we were able to prepare the 5-(1-pyrrolidinylsulfonyl)-
7-azaindole derivatives 6 and 7 in a modified four step sequence.
In previous protocols usually P2 equiv t-BuLi are used for a com-
2
9
plete halogen–lithium exchange reaction. The first equivalent is
used for the exchange, and the second reacts with the produced
t-BuBr to form isobutene, isobutane, and lithium bromide. We
found that the use of just one equiv t-BuLi is sufficient and
improves the reaction sequence in terms of reproducibility, atom
2
2
. Results and discussion
.1. Chemistry
According to Lee et al. 5-(1-pyrrolidinylsulfonyl)isatin based
3
0
efficiency and higher yields. Furthermore, we were able to
caspase inhibitors can be synthesized from isatin and (S)-proline
O
O
O
O
S
O
O
O
S
O
O
S
N
O
N
O
N
N
O
N
n
N
N
O
N
O
O
N
O
18F
O
18F
F
18F
F
[18
[18
F]WC-IV-3 (n = 1)
F]WC-II-89 (n = 2)
[18
F]AF110
[18F]ICMT-11
O
O
S
O
O
S
O
O
N
O
N
O
N
N
MeO
18F
OMe
HO
18F
(
S)-N-[(3R)-4-[ 18F]Fluoro-3-hydroxybutyl]-
5-[1-(2-methoxymethyl)pyrrolidinyl]-sulfonyl}isatin
(S)-N-(4-Methoxybenzyl)-{5-[1-(2-
[18F]fluoromethyl)pyrrolidinyl]sulfonyl}isatin
{
8,12,13,15,16,19,20
[18F]ICMT-11 was previously
Figure 1. Selected 5-(1-pyrrolidinylsulfonyl)isatin based apoptosis imaging agents currently tested in preclinical studies.
subjected to biodistribution and radiation dosimetry studies in humans.
2
3
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C. M. Waldmann et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
O
O
O
O
O
to yield alkyne 13, CuAAC-precursor 14 was prepared by oxidation
with CrO as described before. The cycloaddition reaction of pre-
cursor 14 with 2-fluoroethylazide (15) to yield desired inhibitor
was accomplished under optimized ‘click’-conditions but
4
S
5
9
8
3
S
N
N
2
3
O
O
6
N 1
N
N
7
1
6
O
O
resulted in poor yields in several attempts.
A
B
It has been shown that in case of 5-(1-pyrrolidinylsul-
0
fonyl)isatin based inhibitors of caspase-3 and -7 a 2 -fluoroethyl-
Figure 2. Core structures of hitherto existing inhibitors based on isatin (A) and
envisioned 7-azaisatin based inhibitors of caspases-3 and -7 (B).
1,2,3-triazole side chain can also be attached in position 2 of the
pyrrolidine moiety without compromising on the inhibitory activ-
ity. Therefore, we planned to prepare a comparable 7-azaisatin
8
1
) t-BuLi, -80 °C, THF
derivative of type 20 (Scheme 4). In anticipation of higher inhibi-
tion potencies, we chose to attach the n-propyl substituent at N1
(see Table 1 in Section 2.2). The 7-azaindole skeletal structure 18
was prepared following the optimized one-pot synthesis condi-
tions in 69% yield. Alkyne functionalized pyrrolidine 17 was pre-
O
O
S
O
O
2) SO2(g), -75 °C
Br
Cl
O
O
N
N
N
N
3) NCS, RT, DCM
1
2
8
pared according to literature precedent. In this case the
Scheme 1. Attempted one-pot synthesis of sulfonyl chloride 2.
3
oxidation reaction with CrO gave a higher yield of 67% of
CuAAC-precursor 19 compared to corresponding precursor 14.
Additionally, despite similar reaction conditions, cycloaddition of
alkyne 19 with 2-fluoroethylazide (15) gave considerably higher
yields of inhibitor 20 (68% yield) compared to corresponding inhi-
bitor 16 (23% yield).
replace gaseous SO
bis(sulfur dioxide) (DABSO). This bench-stable colorless solid is
easier to handle and allows a fully controlled dosing of SO equiv-
alents. After formation of the arylsulfinate by reacting DABSO with
the organolithium compound the sulfonyl chloride was prepared
with NCS. 2-Methoxymethyl-substituted pyrrolidine 5 was pre-
pared according to literature procedure and used as an amine for
the sulfonamide formation.7 Using this new protocol compounds
2
with 1,4-diazabicyclo-[2.2.2]octane (DABCO)-
31
2
2
.2. Caspase inhibition potencies
All synthesized non-fluorinated and fluorinated 7-azaisatin sul-
fonamides were tested in vitro as inhibitors of caspases-1, -3, -6
6
and 7 were synthesized in overall yields of 40% and 37%, respec-
7
and -7 according to a published protocol. The resulting inhibition
3
tively. Both compounds were easily oxidized by the use of CrO to
form desired 7-azaisatin derivatives 8 and 9 in fair yields.
potencies are listed in Table 1. All tested inhibitors expressed
inhibition potencies against effector caspases-3 and -7 in the
nanomolar range (IC50 = 17–501 nM). A notable exception is the
IC50 (caspase 3) = 17,300 nM of compound 20. Non-fluorinated inhibi-
tor 8 exhibits a moderate inhibition potential of 429 nM (caspase
In order to enable the incorporation of 2-fluoroethylazide (15)
1
8
18
or 2-[ F]fluoroethylazide ([ F]15) via CuAAC we conducted the
synthesis of alkyne precursor 14 as depicted in Scheme 3.
Starting with N-triisopropylsilyl (N-TIPS) protected 5-bromo-7-
azaindole (10) we were able to prepare the 5-(1-pyrrolidinylsul-
fonyl)-7-azaindole skeletal structure 11 via an optimized four step
one-pot synthesis in 64% yield. The silyl protecting group was
smoothly removed with tetra-n-butylammonium fluoride (TBAF)
under standard conditions. After propargylation of compound 12
3
) and 501 nM (caspase 7) which is comparable, yet lower, to the
known corresponding isatin based inhibitor in which the nitrogen
in position 7 is replaced by a CH-group (IC50 (caspase 3) = 2 nM and
7
IC50 (caspase 7) = 304 nM). The differences in the inhibition potencies
of 7-azaisatin inhibitor 8 and its previously described isatin coun-
7
terpart could in part be explained by their differing hydrophilicity.
O
O
O
O
O
Br
1. t-BuLi (1 equiv), THF, -80 °C
S
S
2
3
4
. DABSO, -70 °C
N
CrO3
N
O
N
R
N
N
R
N
R
. NCS, DCM, RT
.
N
N
AcOH/H2O, RT
O
O
O
+ Hünig´s base
N
H
3
4
(R = Me)
6 (R = Me) 40%
7 (R = n-Pr) 37%
8 (R = Me) 53%
9 (R = n-Pr) 54%
(R = n-Pr)
5
Scheme 2. Synthesis of fluorine-free 5-(1-pyrrolidinylsulfonyl)-7-azaisatin derivatives 8 and 9.
O
O
O O
Br
1. t-BuLi, THF, -80 °C
1. NaH
S
S
2
. DABSO, -70 °C
N
N
2. Propargyl bromide
TBAF
THF
N
N
N
TIPS
N
H
3
4
. NCS, DCM, RT
N
N
DMAc
78%
TIPS
. 5 + Hünig´s base
O
O
84%
10
11
12
64%
F
N3
1
5
O
O
S
O
O
O
O
O
O
O
S
S
CuSO4/Na-Ascorbate
N
N
CrO3
AcOH/H O
N
O
N
F
N
N
DMF/H2O
N
N
2
N
14
N
N
O
O
O
N
48 %
23 %
13
16
Scheme 3. Synthesis of CuAAC-precursor 14 and fluorinated 5-(1-pyrrolidinylsulfonyl)-7-azaisatin derivative 16.
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4
C. M. Waldmann et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
O
O
S
O
O
O
Br
1. t-BuLi, THF, -80 °C
. DABSO, -70 °C
S
2
CrO3
N
N
O
N
N
3. NCS, DCM, RT
N
AcOH/H O
N
N
2
N
4
.
O
O
O
+ Hünig´s base
4
N
H
18
67%
19
17
69%
F
N3
15
O
O
O
F
S
CuSO4/Na-Ascorbate
N
O
N
N
DMF/H2O
N
N
O
N
68 %
20
Scheme 4. Synthesis of CuAAC-precursor 19 and fluorinated 5-(1-pyrrolidinylsulfonyl)-7-azaisatin derivative 20.
Table 1
Caspase inhibition potencies of synthesized 7-azaisatin sulfonamides towards caspases-1, -3, -6 and -7 expressed as IC50 values (nM)
O
O
O
S
N
O
N
N
R2
R1
O
Inhibitor
R1
R2
Caspase inhibition potencies IC50 (nM)
Caspase-1
Caspase-3
429 ± 374
83 ± 10
21 ± 2
17,300 ± 3110
Caspase-6
Caspase-7
8
9
Me
Me
Me
n-Pr
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
501 ± 25
75 ± 12
97 ± 13
17 ± 2
1
2
6
0
Me
[1-(2-Fluoroethyl)-1,2,3-triazol-4-yl]methyl
n-Pr
[1-(2-Fluoroethyl)-1,2,3-triazol-4-yl]methyl
The hydrophilicity of a compound can be indicated by its calcu-
lated octanol/water partition coefficient (clogD) which is 0.28 for
the isatin- and ꢁ1.39 for the 7-azaisatin derivative, respectively,
showing the considerable impact of the 7-azaisatin moiety on
the water solubility. Moreover, a modified receptor–ligand interac-
tion of phenyl and pyridyl moieties due to increased electron den-
sity in 7-position might contribute to the decreased potency. This
argument is encouraged by our previous finding that the potency
of N-alkyl isatins halogenated at the 7-position drops as compared
to the corresponding unsubstituted compounds.9 Thus, increased
electron density in the 7-position seems to result in modified
receptor–ligand interactions. The n-propyl substituent of com-
pound 9 increases the inhibition potencies against caspases-3
and -7. The IC50 values of the already known analog isatin com-
pound are in the same order of magnitude (IC50 (caspase 3) = 5 nM
and IC50 (caspase 7) = 58 nM). This is also true for a series of 7-halo
more, the inhibition potencies of the corresponding analog isatin-
16 (see Supporting information) are higher (IC50 (caspase 3) = 2 nM
and IC50 (caspase 7) = 2 nM). Notably, compound 16 comprises the
lowest clogD value of ꢁ1.88 of all compounds tested indicating
its comparably high water solubility. The highest caspase-7 inhibi-
tion potency is observed for compound 20, however, there is an
unexpected drop of the IC50 value against caspase-3 to
17,300 nM. A comparable isatin sulfonamide analog which corre-
sponds to the 7-azaisatin derivative 20 could not be found in
literature.
2.3. Radiosynthesis
Radiosynthesis is accomplished using the same CuAAC method-
ology as described for the preparation of the nonradioactive
azaisatin derivatives 16 and 20 (Section 2.1). Prosthetic group
9
18
18
isatin derivatives. Fluorinated 7-azaisatin compound 16 is a
potent inhibitor of caspases-3 and -7 which is underlined by the
corresponding IC50 values 21 nM and 97 nM, respectively. Once
2-[ F]fluoroethylazide ([ F]15) was synthesized by automated
nucleophilic radiofluorination of 2-azidoethyl-4-methylbenzene-
sulfonate (21) and could be isolated by distillation in 60 ± 6% decay
N3
Tos
21
K(K222)[18F]F
CH3CN, 84°C, 15 min
N3
O
O
O
O
O
O
18F
S
S
[18
F]15
N
N
O
O
18F
N
CuSO4/Na-ascorbate
DMF/H2O, RT, 12 min
N
N
N
N
N
O
O
N
1
4
[18
F]16
Scheme 5. Radiosynthesis of [18F]16 by CuAAC of a corresponding alkyne precursor 14 with 2-[18F]fluoroethylazide [18F]15.
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C. M. Waldmann et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
3
2
formulation [18F]20 was prepared in 21 ± 0% rcy (d.c., n = 2) in
91 ± 9 min from the end of radionuclide production.
Radiochemical purity was >99% and the determined specific activ-
corrected radiochemical yields (d.c. rcy, n = 6) (Scheme 5). The
cycloaddition to yield desired compound [18F]16 was conducted
outside the automated radiosynthesizer by reacting alkyne precur-
1
8
sor 14 with [ F]15 in the presence of copper sulfate and sodium
ities were in the range of 0.2–0.3 GBq/lmol. The logD7.4 value was
ascorbate. After purification by semipreparative HPLC and subse-
determined to be ꢁ0.01 (clogD = ꢁ0.49).
1
8
quent formulation, [ F]16 was obtained in an overall rcy of
1 ± 5% (d.c., n = 5) in 94 ± 12 min from the end of radionuclide
production. Radiochemical purity was 94 ± 5% and the determined
specific activities were in the range of 0.2–9.5 GBq/ mol. In order
to determine the octanol/water partition coefficient at physiologi-
1
2.4. In vivo biodistribution studies
l
For biodistribution studies in wild-type mice (C57Bl/6, 11–
12 weeks, 19–21 g body weight), we have chosen the most promis-
ing 7-azaisatin derived caspase-3 and -7 inhibitor [ F]16. Dynamic
PET scans in combination with computed tomography (CT) were
used to obtain a series of images reflecting the radiotracer distribu-
tion at different time frames after injection (Fig. 3). Quantitative
analysis (Fig. 4) revealed a fast washout of the radioactivity from
1
8
18
cal pH (logD7.4) [ F]16 was formulated in phosphate buffered sal-
3
3
ine (PBS). The measurement revealed a logD7.4 value of ꢁ1.32
which is in agreement with the calculated value of ꢁ1.88.
1
8
Compound [ F]20 was prepared by using the same synthetic
1
8
protocol as for [ F]16 starting with alkyne precursor 19. After
Figure 3. Biodistribution of radioactivity in an adult C57/BL6 mouse after intravenous injection of [18F]16 visualized by small-animal PET. Maximum intensity projections
MIP, first row) of selected time frames depict rapid hepatobiliary and renal excretion of [18F]16. Whole-body horizontal slices from fused PET/CT imaging (one selected slice,
(
second row) allow for better anatomical correlation and localization of radioactivity. Labeled structures are as follows: lung (lu), heart (ht), liver (lv), gallbladder (gb), small
bowel (sb), kidneys (kd), and urinary bladder (bl).
Figure 4. Representative time–activity concentration curves illustrating the rapid clearance of [18F]16 from the blood due to hepatobiliary and renal tracer elimination.
Control region of interest (ROI) in reference tissues (thymus, lung, muscle) show low levels of radioactivity according to their blood fraction but no tracer accumulation.
Please cite this article in press as: Waldmann, C. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.014

6
C. M. Waldmann et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Acknowledgments
Elimination characteristics of [18F]AF110 and [18F]16 within 90 min
Tracer
cLogD
Elimination
Hepatobiliary
These investigations were supported by the Deutsche
Forschungsgemeinschaft (DFG), Collaborative Research Centre
(SFB) 656 ‘Molecular Cardiovascular Imaging’, Projects A3, B1 and
Z2.
Total (% injected
activity)
Renal
(%)
(%)
1
1
8
F]AF110a
[
[
2.20
ꢁ1.88
63 ± 3
82 ± 2
96 ± 1
68 ± 1
4 ± 1
32 ± 1
8
b
F]16
a
n = 3), biodistribution data of [18F]AF110 in Supporting information.
n = 2).
Supplementary data
(
(
b
Supplementary data (synthetic procedures, spectroscopic data,
in vitro caspase inhibition experiments, in vivo data, and copies
of NMR spectra) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.014.
the blood, resulting in a blood activity comparable to background
levels within 10 min. The tracer is rapidly eliminated via liver and
kidneys and accumulates in gallbladder and bladder, respectively.
In order to examine the influence of the more hydrophilic 7-azaisatin
skeletal structure on the pharmacokinetic behavior in vivo, we
compared the distribution data obtained from previously
References and notes
1
2
3
.
.
.
Kerr, J. F.; Wyllie, A. H.; Currie, A. R. Br. J. Cancer 1972, 26, 239.
Brunner, T.; Mueller, C. Essays Biochem. 2003, 39, 119.
Hengartner, M. O. Nature 2000, 407, 770.
18
described isatinsulfonamide [ F]AF110 (IC50 (caspase 3) = 36 nM and
4. Li, J.; Yuan, J. Oncogene 2008, 27, 6194.
1
2
18
5.
Lee, D.; Long, S. A.; Adams, J. L.; Chan, G.; Vaidya, K. S.; Francis, T. A.; Kikly, K.;
Winkler, J. D.; Sung, C. M.; Debouck, C.; Richardson, S.; Levy, M. A.; DeWolf, W.
E., Jr.; Keller, P. M.; Tomaszek, T.; Head, M. S.; Ryan, M. D.; Haltiwanger, R. C.;
Liang, P. H.; Janson, C. A.; McDevitt, P. J.; Johanson, K.; Concha, N. O.; Chan, W.;
Abdel-Meguid, S. S.; Badger, A. M.; Lark, M. W.; Nadeau, D. P.; Suva, L. J.;
Gowen, M.; Nuttall, M. E. J. Biol. Chem. 2000, 275, 16007.
IC50 (caspase 7) = 93 nM) and [ F]16 (Table 2).
These results demonstrate an explicit difference in the elimina-
tion characteristics of both radiotracers. Within the observed time
span of 90 min, 63 ± 3% injected activity of [18F]AF110 were elim-
inated via the organs of excretion (see Supporting information). In
comparison, clearance of 7-azaisatin derivative [18F]16 was more
efficient with a total of 82 ± 2% eliminated activity, implying a
shorter retention time of the tracer in the organism. Notably, the
fraction of tracer excreted via the kidneys was more pronounced
6.
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.
7. Kopka, K.; Faust, A.; Keul, P.; Wagner, S.; Breyholz, H. J.; Höltke, C.; Schober, O.;
Schäfers, M.; Levkau, B. J. Med. Chem. 2006, 49, 6704.
8.
Smith, G.; Glaser, M.; Perumal, M.; Nguyen, Q. D.; Shan, B.; Arstad, E.; Aboagye,
E. O. J. Med. Chem. 2008, 51, 8057.
9. Podichetty, A. K.; Faust, A.; Kopka, K.; Wagner, S.; Schober, O.; Schäfers, M.;
1
8
18
using [ F]16 (32 ± 1%) as compared to [ F]AF110 (4 ± 1%). The
reasons for the observed differences can be manifold. The overall
higher hydrophilicity of 7-azaisatin derivative [18F]16 might con-
tribute to the increased renal elimination and faster washout.
Haufe, G. Bioorg. Med. Chem. 2009, 17, 2680.
10. Podichetty, A. K.; Wagner, S.; Faust, A.; Schäfers, M.; Schober, O.; Kopka, K.;
Haufe, G. Future Med. Chem. 2009, 1, 969.
11. Limpachayaporn, P.; Schäfers, M.; Schober, O.; Kopka, K.; Haufe, G. Bioorg. Med.
Chem. 2013, 21, 2025.
1
8
18
Also, it is likely that [ F]16, as it is the case for [ F]AF110, forms
12. Faust, A.; Wagner, S.; Law, M. P.; Hermann, S.; Schnöckel, U.; Keul, P.; Schober,
metabolites in vivo that have a strong effect on the observed activ-
O.; Schäfers, M.; Levkau, B.; Kopka, K. Q. J. Nucl. Med. Mol. Imaging 2007, 51, 67.
13. Zhou, D.; Chu, W.; Chen, D. L.; Wang, Q.; Reichert, D. E.; Rothfuss, J.; D’Avignon,
2
5
ity distribution.
A.; Welch, M. J.; Mach, R. H. Org. Biomol. Chem. 2009, 7, 1337.
1
1
1
4. Podichetty, A. K.; Wagner, S.; Schröer, S.; Faust, A.; Schäfers, M.; Schober, O.;
Kopka, K.; Haufe, G. J. Med. Chem. 2009, 52, 3484.
5. Chen, D. L.; Zhou, D.; Chu, W.; Herrbrich, P. E.; Jones, L. A.; Rothfuss, J. M.; Engle,
J. T.; Geraci, M.; Welch, M. J.; Mach, R. H. Nucl. Med. Biol. 2009, 36, 651.
6. Chen, D. L.; Zhou, D.; Chu, W.; Herrbrich, P. E.; Engle, J. T.; Griffin, E.; Jones, L. A.;
Rothfuss, J. M.; Geraci, M.; Hotchkiss, R. S.; Mach, R. H. Nucl. Med. Biol. 2012, 39,
3
. Conclusion
PET-compatible fluorine-18 labeled 5-(1-pyrrolidinylsul-
fonyl)isatin derivatives are considered as putative in vivo apoptosis
imaging agents. In order to influence the pharmacokinetic behavior
of this class of radiotracers, we introduced a second generation
lead structure in which the isatin moiety is replaced by 7-azaisatin.
The syntheses of non-fluorinated and fluorinated 5-(1-pyrro-
lidinylsulfonyl)-7-azaisatin derivatives are described. All synthe-
sized substances were tested for their inhibitory activity against
caspases-1, -3, -6 and -7. While not being as potent as their isatin
counterparts, the 7-azaisatin derivatives revealed satisfactory IC50
values against caspases-3 and -7 in the nanomolar scale. PET-com-
patible fluorine-18 labeled tracers were prepared in a semi-auto-
mated radiosynthesis based on the CuAAC strategy. Compared to
a previously described isatin PET tracer, the novel 7-azaisatin PET
tracer exhibits an increased renal excretion in rodents and is
cleared more efficiently from all organs investigated. These results
demonstrate that 5-(1-pyrrolidinylsulfonyl)-7-azaisatin deriva-
tives have the potential to be further developed as PET tracers
for imaging of activated caspases in apoptosis. In future work, we
plan to investigate whether or not the caspase-3 and -7 inhibitors
described in this study can accumulate over time in living apop-
totic cells with upregulated caspase-3 activity. Furthermore, com-
1
37.
7. Nguyen, Q. D.; Challapalli, A.; Smith, G.; Fortt, R.; Aboagye, E. O. Eur. J. Cancer
012, 48, 432.
1
2
18. Limpachayaporn, P.; Riemann, B.; Kopka, K.; Schober, O.; Schäfers, M.; Haufe, G.
Eur. J. Med. Chem. 2013, 64, 562.
9. Limpachayaporn, P.; Wagner, S.; Kopka, K.; Hermann, S.; Schäfers, M.; Haufe, G.
1
J. Med. Chem. 2013, 56, 4509.
20. Krause-Heuer, A. M.; Howell, N. R.; Metesic, L.; Dhand, G.; Young, E. L.; Burgess,
L.; Jiang, C. D.; Lengkeek, N. A.; Fookes, C. J. R.; Pham, T. Q.; Sobrio, F.; Greguric,
I.; Fraser, B. H. Med. Chem. Commun. 2013, 4, 347.
1. Limpachayaporn, P.; Wagner, S.; Kopka, K.; Schober, O.; Schäfers, M.; Haufe, G.
J. Med. Chem. 2014, 57, 9383.
22. Nguyen, Q. D.; Smith, G.; Glaser, M.; Perumal, M.; Arstad, E.; Aboagye, E. O.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16375.
3. Challapalli, A.; Kenny, L. M.; Hallett, W. A.; Kozlowski, K.; Tomasi, G.; Gudi, M.;
Al-Nahhas, A.; Coombes, R. C.; Aboagye, E. O. J. Nucl. Med. 2013, 54, 1551.
24. Witney, T. H.; Fortt, R. R.; Aboagye, E. O. PLoS One 2014, 9, e91694.
5. Baumann, A.; Faust, A.; Law, M. P.; Kuhlmann, M. T.; Kopka, K.; Schäfers, M.;
Karst, U. Anal. Chem. 2011, 83, 5415.
6. Glaser, M.; Årstad, E. Bioconjugate Chem. 2007, 18, 989.
27. Mader, M. M.; Shih, C.; Considine, E.; De Dios, A.; Grossman, C. S.; Hipskind, P.
A.; Lin, F. S.; Lobb, K. L.; Lopez, B.; Lopez, J. E.; Cabrejas, L. M. M.; Richett, M. E.;
White, W. T.; Cheung, Y. Y.; Huang, Z. P.; Reilly, J. E.; Dinn, S. R. Bioorg. Med.
Chem. Lett. 2005, 15, 617.
28. Tatsugi, J.; Tong, Z. W.; Tsuchiya, Y.; Ito, T. ARKIVOC 2009, 132.
9. Seebach, D.; Neumann, H. Chem. Ber. 1974, 107, 847.
30. Waldmann, C.; Schober, O.; Haufe, G.; Kopka, K. Org. Lett. 2013, 15, 2954.
1. Woolven, H.; González-Rodríguez, C.; Marco, I.; Thompson, A. L.; Willis, M. C.
Org. Lett. 2011, 13, 4876.
2. Schrigten, D.; Breyholz, H. J.; Wagner, S.; Hermann, S.; Schober, O.; Schäfers,
M.; Haufe, G.; Kopka, K. J. Med. Chem. 2012, 55, 223.
3. Prante, O.; Hocke, C.; Löber, S.; Hübner, H.; Gmeiner, P.; Kuwert, T.
Nuklearmedizin 2006, 45, 41.
2
2
2
2
2
1
8
18
pounds [ F]16 and [ F]AF110 will be tested in experimental
3
3
3
models of increased apoptosis. We anticipate that the more effi-
1
8
cient clearance of [ F]16 will result in a higher signal-to-noise
ratio as well as a decreased background signal when compared
1
8
to [ F]AF110.
Please cite this article in press as: Waldmann, C. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.014