Synthesis of 7-halogenated isatin sulfonamides: Nonradioactive counterparts of caspase-3/-7 inhibitor-based potential radiopharmaceuticals for molecular imaging of apoptosis

Article abstract of DOI:10.1021/jm500718e

N-Alkylated (S)-7-halogen-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatins were developed as a new group of nonradioactive reference compounds for future radiotracers. Inhibitor potency studies of these compounds suggest that the binding pockets readily accommodate both the 7-halogen substituents and aliphatic side chains (methyl to n-butyl) as well as some ω-fluorinated analogues (3-fluoropropyl and 4-fluorobutyl) at the isatin nitrogen. Indeed, compared to the halogen free parent compounds, some 7-halogenated derivatives exhibited slightly improved inhibitory potencies with IC₅₀ values up to 2.6 nM (caspase-3) and 3.3 nM (caspase-7), respectively. Moreover, the 7-position of isatin, a potential cytochrome P450 hydroxylation site, was substituted by I, Br, Cl, and F to potentially enhance the metabolic stability of isatin sulfonamides. As an example, the radiotracer [¹⁸F]39 that was produced by ¹⁹F/¹⁸F isotope exchange was shown to be stable in human blood serum after incubation at 37 °C for at least 90 min.

Full text of DOI:10.1021/jm500718e

Article
pubs.acs.org/jmc
Synthesis of 7‑Halogenated Isatin Sulfonamides: Nonradioactive
Counterparts of Caspase-3/‑7 Inhibitor-Based Potential
Radiopharmaceuticals for Molecular Imaging of Apoptosis
†
,‡,∇
Stefan Wagner, Klaus Kopka, Otmar Schober, Michael Schaf
ers,§,∥,⊥
§
§,●
§,⊥
Panupun Limpachayaporn,
̈
,†,∥,⊥
and Gu
†
̈
nter Haufe*
Organisch-Chemisches Institut, Westfal
International NRW Graduate School of Chemistry, Westfal
nster, Germany
r Nuklearmedizin, Universitat
European Institute for Molecular Imaging, Westfal
̈
ische Wilhelms-Universitat
̈
Mu
̈
nster, Corrensstraße 40, D-48149 Mu
̈
nster, Germany
‡
̈
ische Wilhelms-Universitat
̈
Munster, Wilhelm-Klemm-Straße 10,
̈
D-48149 Mu
̈
§
Klinik fu
̈
̈
sklinikum Mu
̈
nster, Albert-Schweitzer-Campus 1, Gebau
̈
de A1, D-48149 Mu
̈
nster, Germany
∥
̈
ische Wilhelms-Universitat
̈
Munster, Waldeyerstraße 15, D-48149 Mu
̈
̈
nster,
Germany
⊥
Cells-in-Motion Cluster of Excellence, Westfal
̈ ̈
̈ ̈
ische Wilhelms-Universitat Munster, Waldeyerstraße 15, D-48149 Munster, Germany
*
S Supporting Information
ABSTRACT: N-Alkylated (S)-7-halogen-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatins were developed as a
new group of nonradioactive reference compounds for future
radiotracers. Inhibitor potency studies of these compounds
suggest that the binding pockets readily accommodate both the
7-halogen substituents and aliphatic side chains (methyl to n-
butyl) as well as some ω-fluorinated analogues (3-fluoropropyl
and 4-fluorobutyl) at the isatin nitrogen. Indeed, compared to the
halogen free parent compounds, some 7-halogenated derivatives
exhibited slightly improved inhibitory potencies with IC50 values
up to 2.6 nM (caspase-3) and 3.3 nM (caspase-7), respectively.
Moreover, the 7-position of isatin, a potential cytochrome P450
hydroxylation site, was substituted by I, Br, Cl, and F to
18
potentially enhance the metabolic stability of isatin sulfonamides. As an example, the radiotracer [ F]39 that was produced by
19
18
F/ F isotope exchange was shown to be stable in human blood serum after incubation at 37 °C for at least 90 min.
1
. INTRODUCTION
infections, neurodegenerative disorders, developmental defects,
and tumorigenesis.
Although apoptosis consists of a complex network of
numerous biological pathways, the process is mainly controlled
by a set of intracellular cell death enzymes, so-called caspases
1
Apoptosis, or the programmed type I cell death, is the regulated
destruction of overproduced or potentially dangerous and
damaged cells without inflammatory responses. It is a highly
conserved biological process through the evolution from hydra,
nematodes, insects, and fishes to mammals and humans, and it
plays vital roles in the development of cells, morphological
(
cysteinyl-aspartate-specific proteases), which are responsible1
for specific cleavage of peptide bonds after aspartate residues.
More than a dozen caspases have been characterized; however,
it was demonstrated that inhibition of the executing caspases-3
and -7 alone is enough to slow down or even block
1,2
changes, and metamorphosis in multicellular organisms.
Apoptosis is activated either by extrinsic binding of the CD95
ligand (CD95L) to the CD95 cell surface receptor (death
receptor), resulting in intracellular death signaling (death
receptor pathway), or by DNA damage and release of
cytochrome c from the mitochondria (mitochondrial path-
way). The apoptotic cell death is a counter process to mitosis.
The equilibrium of both processes strictly maintains the cell
number, so-called “homeostasis”, as well as the size and shape
of organisms.
3
programmed cell death. Thus, targeting the executing caspases
might enable novel procedures to be discovered for prevention,
treatment, diagnosis, and therapy monitoring of the above-
mentioned diseases by molecular imaging using positron
emission tomography (PET) or single-photon emission
computed tomography (SPECT). Moreover, it allows specific
visualization of apoptosis at the molecular level, leading to a
1
1
,2
Dysregulation of apoptosisdown- or
upregulatedcan lead to a loss of balance, which results in
the induction of pathological processes and a variety of diseases
such as autoimmune and cardiovascular diseases, persistent
Received: May 8, 2014
©
XXXX American Chemical Society
A
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1
1
12
greater understanding of the mystery of the type-I cell death in
more detail. Therefore, a specific imaging agent (radiotracer) is
highly desirable, which possesses high affinity toward the target
enzymes and is radiolabeled with a suitable positron emitter or
a gamma emitter.
substituted phenoxy, pyridinylhydroxy, triazolyl, alkoxy, and
14,15
fluoroalkoxy groups.
In contrast, introduction of functional
groups such as methoxy, PEG yloxy (PEG = tetraethylene
glycol), fluoro, gem-difluoro, and trifluoromethyl groups at the
4-position and an extra methoxymethyl group at the 5-position
of the pyrrolidine part resulted in an increase of the IC50 values
to hundreds of nanomolar to micromolar level, and some
compounds were even inactive. Modification of the cysteine
binding site (3-keto group) resulted in a loss or dramatic drop
except for the introduction of Michael
acceptors in the place of the carbonyl group.
4
4
(
S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin (1)
and (S)-N-methyl-5-[1-(2-phenoxymethylpyrrolidinyl)-
sulfonyl]isatin (2) were identified by screening as potent and
16
selective inhibitors of caspases-3 and -7 with K values in the
i
8,14
3
of binding affinity,
nanomolar scale (Figure 1).
17,18
Since N-benzyl isatin analogues including fluorinated and
methoxybenzyl ones exhibited excellent affinity, some were
developed as radiotracers. For instance, the most recent isatin-
20
1
8
19 18
20 18
6,21 11
based radiotracers [ F]3, [ F]4a, [ F]4b,
[ C]5,
18
22
and [ F]6 (Figure 2) were prepared and their dynamics in
Figure 1. Chemical structures and the K values of (S)-5-[1-(2-
i
methoxymethylpyrrolidinyl)sulfonyl]isatin (1) and (S)-N-methyl-5-[1-
(
2-phenoxymethylpyrrolidinyl)sulfonyl]isatin (2) toward caspases-3
and -7. In the structure of 2, interactions with the enzyme’s pockets
3
studied by X-ray cocrystallization are highlighted.
The excellent enzyme−inhibitor interaction was confirmed
by X-ray cocrystal studies of the complex of recombinant
human caspase-3 with isatin sulfonamide 2 (Figure 1, right). It
was revealed that the cysteine-163 of the enzyme binds the
ligand covalently at the 3-keto group to form a tetrahedral
thiohemiketal intermediate. While the N-methyl group
Figure 2. Most recent isatin-based radiotracers for molecular imaging
of apoptosis using PET.
6
,19−22
putatively interacts with the S -pocket of the enzyme, the
1
pyrrolidine moiety incorporates within the S -pocket and the 2-
vivo was investigated in mice using PET. However, the N-
benzyl group was shown to be cleaved from the isatin core
structure and the compounds are hydroxylated at the isatin
aromatic ring according to electrochemical and microsoma4l
oxidation in the metabolic stability test using EC/MS-ESI.
2
phenoxymethyl group of the pyrrolidine is accommodated in
3
the enzyme’s S -pocket. Furthermore, recent metabolic
3
stability studies using electrochemical and microsomal
oxidation revealed a hydroxylation at the aromatic ring of the
4
18
isatin core structure. This might occur at the electronically
This might result in a loss of F-activity leading to suboptimal
most appropriate 7-position.
PET imaging.
After the inhibitory properties of isatin sulfonamides have
been disclosed, various structural modifications have been
realized in the past decade to extend the chemical library and to
study the structure−activity relationships (SAR). Moreover, the
development of isatin sulfonamides as PET and SPECT
imaging agents for the visualization of apoptosis was
We assumed that some metabolic challenges of isatin
sulfonamides could be overcome by blocking the potential
aromatic hydroxylation site and replacing the exposed benzyl
substituent at the isatin nitrogen with alkyl groups. There was
earlier evidence from our group that a 7-brominated analogue,
(S)-7-bromo-5-{2-[difluoro(methoxy)methyl]pyrrolidine-1-yl-
sulfonyl}-1-propylindoline-2,3-dione (9), not only maintained
the binding potency toward caspases-3 and -7 in the nanomolar
scale, but even increased it. In the fluoro-desulfurization of 7
using 1,3-dibromo-5,5-dimethylhydantoin (DBH) and pyridine·
5
,6
proposed. Thus, numerous analogues of isatin sulfonamide
including fluorinated ones were synthesized. N-Substitution of
2
-methoxymethyl and 2-phenoxymethyl pyrrolidinyl and
7,8
azetidinyl sulfonyl isatins with functionalized alkyl, bromo-
fluoroalkyl, fluorohydroxyalkyl,
and triazole groups
9
9,10
5,7,8,11,12
11
23
benzyl,
pyridyl,
9HF, two products showing high binding potencies for
1
2,13
was confirmed to improve the
caspases-3 and -7, respectively, were obtained. Compound 9
was even more potent than the parent 8 or the lead compound
1 (Scheme 1).
biological activities against caspases-3 and -7 in vitro. In most
cases, the compounds exhibited selective activities against
caspases-3 and -7 with IC50 values in the nanomolar range.
Compounds in the 2-phenoxymethyl class, such as 2, tended to
express superior inhibitory activities in vitro, while the 2-
methoxymethyl versions, such as 1, inhibited the enzymes more
5,14
Herein, we report the synthesis of a series of new N-alkyl-7-
halogenated isatin sulfonamides, which are expected to be
metabolically more stable, when the potential hydroxylation site
is blocked by introduction of a halogen atom (I, Br, Cl, or F)
and when the benzyl group at the isatin nitrogen is replaced by
alkyl or fluoroalkyl substituents. In addition, the influence of
the 7-halogen on the inhibition potency toward caspases-1, -3,
-6, and -7 will be studied systematically. Moreover, the 7-fluoro
5
efficiently in vivo. Therefore, 1 was selected as lead compound
for further structural alterations. High binding affinities in the
nanomolar range were maintained when 2-methoxymethyl or 2-
phenoxymethyl groups of isatin sulfonamides were replaced by
B
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1
4
Scheme 1. Fluoro-desulfurization of 7 To Form Isatins 8 and 9 and Their in Vitro Binding Affinities toward Caspases-3 and -7
Scheme 2. Synthetic Routes towards 7-Halogenated Isatin Sulfonamides
Scheme 3. Preparation of 7-Iodo and 7-Bromo Isatin Analogues 10−16 and 18−24
1
4
and 7-iodo analogues might allow future labeling with fluorine-
8 and iodine-124 for PET application and with iodine-123 and
131 for SPECT apoptosis imaging in vivo. Furthermore, for a
model compound, F/ F isotope exchange and blood serum
stability tests were carried out.
The two doublet H NMR signals with meta-coupling ( J
=
H,H
1
-
1.3−1.6 Hz) of 4-CH and 6-CH were observed for both
13
compounds. In addition, C NMR signals of 7-C bearing either
iodine (10) or bromine (18) were detected at δ = 78.1 and
106.2 ppm (δ = 112.7 ppm for 7-CH of 1), respectively.
As mentioned above, N-substitution significantly improved
the affinities. Thus, various aliphatic chains, such as methyl,
ethyl, propyl, 3-fluoropropyl, butyl, 4-fluorobutyl, and 4-
hydroxybutyl groups, were attached to the 7-iodinated and 7-
brominated isatins 10 and 18 to yield the corresponding
derivatives 11−16 and 19−24, as listed in Table 1. The N-
methylation took place using MeI and NaH in DMF after
stirring at 0 °C for 30−60 min and at room temperature for
another 30 min to give products 11 and 19 in 82% and 81%
yields, respectively. N-Propylation of 18 using propyl bromide
and K CO in DMF at room temperature needed 4 days to
1
9
18
2. RESULTS AND DISCUSSION
2.1. Chemistry. A series of 7-halogenated isatin sulfona-
mides was prepared following two methodologies depending
on the halogen atom to be attached: (i) direct bromination and
iodination of the lead compound 1 and (ii) coupling of 7-
chloro- and 7-fluoroisatin-5-sulfonyl chloride with 2-methox-
ymethylpyrrolidine as depicted in Scheme 2.
The syntheses of the 7-iodinated and 7-brominated series
7
started from the lead compound 1, and iodine or bromine was
attached to the aromatic ring first by direct electrophilic
aromatic substitution and the N-substitutions were performed
subsequently (Scheme 3). In analogy to a literature
2
3
obtain 21 in good yield. For other N-alkylations, the reaction
efficacy was therefore enhanced using Cs CO as base and
26
3
2
27
2
4
microwave irradiation (at 90−100 °C for 2−10 min). The
desired products 12−16 and 20, 22−24 were obtained within a
short time with moderate to excellent yields (Table 1).
In contrast to the mentioned N-alkylations, the 7-iodinated
and 7-brominated (S)-N-(4-hydroxybutyl)-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatins 17 and 25 were
obtained in two consecutive steps by coupling of 10 and 18
with 4-bromobutyl pivalate using Cs CO in DMF under
precedent, the direct iodination was successful by treatment
of 1 with H IO and I under strong acidic conditions to give
5
6
2
the 7-iodoisatin 10 in 72% yield, while using NaI and NaClO
2
25
analogously to a literature procedure did not lead to
formation of the desired product. Similarly to a literature
1
4
protocol, a bromine atom was attached to the 7-position of
the aromatic ring using DBH in the presence of concentrated
H SO to give 18 in 65% yield. The structures of the new
2
4
2
3
1
13
products were confirmed by H and C NMR spectroscopy.
microwave irradiation followed by acidic hydrolysis as shown in
C
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Table 1. In Vitro Inhibitory Potencies of New 7-Halogenated Isatin Sulfonamides 10−17, 18−25, 31−33, and 37−39 with
Standard Deviation of Three Independent Experiments Expressed as IC Values
5
0
Inhibitory potencies IC50 (nM)
caspase-3 caspase-6
84.9 ± 25.6 >500,000
Inhibitor
15
X
R
Yield [%]
caspase-1
>500,000
caspase-7
H
H
H
H
H
H
I
H
47
82
59
65
86
70
72
82
79
51
89
57
84
45
65
81
62
72
59
65
95
42
60
97
95
43
82
78
1,290 ± 466
304 ± 73
319 ± 60
58 ± 15
8.1 ± 1.1
28 ± 6
a5
Me
Et
n.d.
n.d
a
2.4 ± 0.3
183 ± 48
5.3 ± 3.8
29 ± 13
n.d.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
5
b
n.d.
5
c
n-C H
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3
7
5
d
n-C H
n.d.
4
9
5
e
n-C H F
41 ± 17
n.d.
4
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
1
2
3
7
8
9
H
>500,000
1,190 ± 194
210 ± 44
124 ± 10
17.4 ± 1.6
20.1 ± 0.3
50.7 ± 0.9
33.2 ± 3.6
>500,000
353 ± 80
133 ± 41
26.2 ± 12.6
46.9 ± 4.3
81.4 ± 24.6
31.5 ± 6.2
2.6 ± 0.7
>500,000
57.7 ± 15.3
n.d.
>500,000
I
Me
Et
n.d.
10,500 ± 2,020
20.1 ± 10.0
42.4 ± 9.8
907 ± 844
123 ± 13
68.1 ± 0.5
279 ± 11
>500,000
I
n.d.
I
n-C H
n.d.
3
7
I
n-C H
>500,000
>500,000
>500,000
>500,000
n.d.
>500,000
28,500 ± 6,710
>500,000
>500,000
n.d.
4
9
I
n-C H F
3 6
I
n-C H F
4 8
I
n-C H OH
4
8
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
Cl
F
H
Me
Et
n.d.
n.d.
553 ± 55
135 ± 35
157 ± 53
3.3 ± 0.3
20.1 ± 9.1
5.95 ± 2.55
22.7 ± 1.3
>500,000
n.d.
n.d.
n-C H
n.d.
n.d.
3
7
n-C H
n.d.
n.d.
4
9
n-C H F
n.d.
n.d.
3
6
n-C H F
n.d.
n.d.
4
8
n-C H OH
>500,000
>500,000
>500,000
2,590 ± 383
>500,000
9,970 ± 4,250
7,430 ± 3,520
>500,000
>500,000
>500,000
>500,000
>500,000
78,900 ± 69,700
12,900 ± 5,160
4
8
H
n-C H
263 ± 104
55.7 ± 30.6
6,620 ± 234
9.7 ± 5.4
4
9
b
n-C H F
4.8 ± 2.3
>500,000
4
8
H
F
n-C H
2.9 ± 0.8
41.0 ± 19.0
4
9
F
n-C H F
39.3 ± 11.2
4
8
a
b
n.d. = not determined. Determined using a different bach of caspase-3.
Scheme 4. Synthetic Route to 7-Iodinated and 7-Brominated (S)-N-(4-hydroxybutyl)-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatins 17 and 25
1
Scheme 4. The N-(4-hydroxybutyl)isatins 17 and 25 were
formed in 45% and 42% yields (over two steps), respectively.
Differing from the synthesis of the 7-iodinated and 7-
brominated isatin sulfonamides 11−17 and 19−25, the 7-
chloro and 7-fluoroisatin analogues 31−33 and 37−39 were
constructed commencing from the commercially available 7-
chloro- and 7-fluoroisatins 28 and 34, as depicted in Scheme 5.
and 6-positions in H NMR spectra revealed the addition at the
5-position for both compounds. Subsequent chlorination using
POCl resulted in the formation of 7-chloro and 7-fluoro-5-
3
chlorosulfonyl isatins 30 and 36 in 80% and 66% yields,
respectively. The couplings of 30 and 36 with 2-methox-
ymethylpyrrolidine were performed in the presence of
diisopropyl ethyl amine (DIPEA) to give isatin sulfonamides
7
13
Similar to the preparation of the parent system 1, 28 and 34
31 and 37 in 60% and 43% yields. The C NMR spectra
were sulfonated using oleum followed by treatment with
NaOH, giving the 7-halogenated sodium sulfonates 29 and 35
in quantitative yields. The meta-coupling of protons at the 4-
indicated signals of 7-C bearing chlorine (δ = 118.9 ppm) and
fluorine (δ = 146.7 ppm), respectively. Since the N-butyl- and
N-fluorobutyl-7-iodo and 7-bromoisatins exhibited superior
D
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Scheme 5. Synthetic Pathways to N-Substituted 7-Chloro 32, 33 and 7-Fluoroisatin Sulfonamides 38, 39
3
0
potencies toward caspases-3 and -7 over the shorter chains (see
section 2.2), compounds 31 and 37 were N-alkylated only with
n-butyl and 4-fluorobutyl groups using similar conditions as
described in the 7-iodine and 7-bromine series to obtain the
target compounds 32, 33, 38, and 39 in good to excellent yields
bimolecular nucleophilic substitution of sulfonate esters
18
using no-carrier-added [ F]fluoride should be suitable for
I labeling, the iodinated
precursors can be isotopically exchanged with radioactive
123
124
these inhibitors. For
I and
3
1,32
123
124
iodides.
Alternatively, the
I and
I labeled tracers
(
Table 1).
.2. In Vitro Caspase Inhibition Potencies. The
should be available by iododemetalation reactions as
5
,33
2
precedented in the literature.
In order to get the first information on stability, compound
inhibitory potencies of all synthesized 7-halogenated isatin
sulfonamides toward caspases-3 and -7 were evaluated in vitro
and compared to those of the lead compound 1. Some selected
compounds were additionally evaluated toward caspases-1 and
19
18
39 was chosen for F/ F isotope exchange and the subsequent
blood serum stability test.
2.3. Radiosynthesis and Serum Stability Test. One
18
-
6. The IC50 values in nanomolar scale are listed in Table 1.
possible method for [ F]-radiolabeling is the isotope exchange
either in the aromatic position by nucleophilic aromatic
All N-unsubstituted 7-halogenated isatins 10, 18, 31, and 37
28
30
did not inhibit caspases-3 and -7 on the 500 μM scale, except
compound 37, which showed no caspase-3 inhibition and
reduced caspase-7 inhibition with IC50 = 6.62 μM. However,
after N-substitution, the inhibition potencies toward caspases-3
and -7 were improved significantly to nanomolar level. These
findings revealed that N-substitution has a crucial impact on
binding of 7-halogenated isatins. It was also observed that 7-
iodo and 7-bromo N-substituted isatin sulfonamides with n-
propyl and in particular n-butyl, 4-fluorobutyl, and 4-
hydroxybutyl groups showed considerably superior potencies
over the shorter chain analogues. Thus, n-butyl and 4-
fluorobutyl groups were selected to be appropriate candidates
for N-substitutions of 7-chloro- and 7-fluoroisatins.
substitution or in the aliphatic position by an S 2 process.
N
Therefore, compound 39 is an attractive candidate offering
1
8
both possibilities. As a result, its reaction with K[ F]/K in
222
18
DMF at 130 °C for 10 min provided [ F]39 with 0.2% (d.c.)
radiochemical yield (r.y.) and 99% radiochemical purity by
either one or the other of the mentioned reactions (Scheme 6).
1
8
Scheme 6. Radiosynthesis of [ F]39
Among the compounds in this class, the 7-fluoro derivative
3
2
1
8 was among the most potent inhibitor for caspase-3 (IC50 =
.9 nM), i.e. 10 times more potent than the parent compound
d. The 7-bromo compound 22 was most potent against
caspase-7 (3.3 nM), which means 2.5 more potent than 1d.
Furthermore, compound 38 was equally as potent as
compound 1d with respect to caspase-7. Compared to the
lead compound 1, this is a 20- and 400-fold improvement,
respectively. In contrast, the binding potencies toward caspases-
Unfortunately, all further variations of the solvent,
stoichiometry, reaction time, and temperature did not improve
the r.y. The radio-HPLC is shown in Figures S1 and S2 in the
Supporting Information (SI).
18
The in vitro stability study of [ F]39 was carried out using
1
and -6 were weak, in most cases with IC50 > 500 μM.
human blood serum. After the long-term incubation for up to
90 min at 37 °C, only the parent compound [ F]39 was
detected by radio-HPLC as shown in Figure 3.
1
8
Therefore, only a selection of compounds was assayed for
caspases-1 and -6.
According to the above results, several compounds, such as
Oxidative metabolism tests using electrochemistry/mass
spectrometry of compounds 24, 33, and 39 showed
monooxygenation in the heterocyclic part of the isatin core
and dehydrogenation of the pyrrolidine ring as major processes.
Oxygenation of the benzene moiety, which was a major process
in electrochemical and liver microsome oxidation in similar 7-
1
2−17, 23, 24, 33, 38, and 39, which possess high potencies to
the target enzymes and contain fluorine and/or iodine, can be
considered as potential radiotracers for PET after labeling with
fluorine-18 or iodine-124, and for SPECT when labeled with
1
8
19
28
iodine-123. Besides F for F isotope exchange, also
2
9
4
aromatic substitution of iodonium salts and aliphatic
unsubstituted isatins, was not observed. The methodological
E
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improved in vivo stability is predicted. The in vitro metabolic
18
19 18
stability of compound [ F]39 prepared by F/ F isotope
exchange revealed the resistance to the hydrolytic metabolism
after incubation in human blood serum at 37 °C for at least 90
min.
4
. EXPERIMENTAL SECTION
4.1. Materials and Methods. All reagents and solvents for
reactions were analytical grade and were used as delivered without
further purification. 1-Bromo-4-fluorobutane was purchased from
Apollo Scientific. 1-Bromo-3-fluoropropane was prepared by treating
3
-bromopropanol with DAST in dry THF. 4-Bromobutyl pivalate was
obtained by reaction of 4-bromobutanol and pivaloyl chloride in the
presence of TEA in dry DCM. The lead compound (S)-5-[1-(2-
8
methoxymethylpyrrolidinyl)sulfonyl]isatin (1) was synthesized using
7
the protocol published by Lee et al. For reactions under anhydrous
conditions, the glassware was heated under vacuum and flushed with
argon gas prior to use. The reactions were performed under argon
atmosphere. To characterize the synthesized compounds, melting
points (if the products were solid at r.t.), NMR, and ESI-MS were
1
13
exploited. H NMR (300 and 400 MHz), C NMR (75 and 100
MHz), and 19F NMR (282 MHz) spectra were recorded in CDCl3,
CD CN, or DMSO-d with TMS and CHCl as the internal standards
3
6
3
1
13
for H NMR, and CDCl , CD CN, or DMSO-d for C NMR, and
3
3
6
1
9
CFCl for F NMR. DEPT and two-dimensional NMR techniques
3
(
COSY, HMQC, and HMBC) were used to assign the signals of some
complex structures. All chemical shifts were indicated in ppm. Exact
mass analyses were recorded with a MicroTOF apparatus. All
spectroscopic and analytical investigations were performed by staff
Figure 3. Radio-HPLC chromatograms of a typical quality control
members of Organisch-Chemisches Institut, Westfal
̈
ische Wilhelms-
18
18
(
(
°
QC) of a [ F]39 batch (top) and of the in vitro stability of [ F]39
Universitat Munster. Thin layer chromatography (TLC) analyses were
̈
̈
t = 11.38−11.67 min) after incubation in human blood serum at 37
performed on silica coated aluminum foils (Silica Gel 60 F254) with
0.02 mm layer thickness from Merck. Silica gel (60−120 mesh) used
for column chromatography was obtained from Merck. The purity of
all compounds tested in vitro or in vivo was proved by HPLC to be
>95%. Reactions under microwave irradiation were performed in
sealed glass vessels using a CEM Discovery microwave machine.
R
C after 10 min (middle) and 90 min (bottom) measured at analytical
HPLC. Radiometabolites or decomposition products were not
observed. The full radio-HPLC chromatograms are attached to the
SI (Figure S3−S6).
4
.2. General Procedure for N-Alkylation using Microwave
developments and detailed results of this study will be reported
elsewhere.
Irradiation. In a microwave compatible glass vessel, a stirred solution
of isatin sulfonamides (1 equiv) dissolved in dry DMF (3−5 mL) was
treated with 60% NaH (1.5 equiv in mineral oil) or Cs CO (2−3
3
4
2
3
3. CONCLUSION
equiv) under argon atmosphere at room temperature. After stirring for
1
0 min, the yellow mixture turned blue-purple. Methyl iodide or the
A complete series of new 7-halogenated and N-alkylated isatin
sulfonamides was synthesized by direct electrophilic aromatic
substitution (for iodine and bromine) or starting from
commercially available 7-halogenated isatins (for chlorine and
fluorine). Inhibitor potency studies of these compounds suggest
that the binding pockets readily accommodate both the 7-
halogen substituents and aliphatic side chains (methyl to n-
butyl) as well as some analogues fluorinated in the terminal
position (3-fluoropropyl and 4-fluorobutyl) at nitrogen in the
respective alkyl bromide (3−10 equiv) was added slowly into the blue
mixture. Subsequently, the vessel was sealed and the mixture was
stirred at 90−100 °C for 2−10 min using microwave irradiation
(
maximum power = 150 W). Then, the precipitate was removed by
filtration through Celite, and the filtrate was concentrated under
reduced pressure. Finally, the residue was purified by flash column
chromatography to obtain the corresponding final N-substituted isatin
sulfonamides.
4.3. (S)-7-Iodo-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-
isatins. 4.3.1. (S)-7-Iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
1
-position (interacting with the S pocket). Most of these
24
1
sulfonyl]isatin (10). Similar to a literature procedure, a mixture of
compounds exhibited excellent binding potencies toward
caspases-3 and -7 in the nanomolar range, similar to the earlier
reported parent compounds with hydrogen in the 7-position.
Although attachment of halogens at this position did increase
the inhibitory activities only in some cases, these compounds
provide the possibility to be labeled with positron emitters
7
the lead compound 1 (162 mg, 0.50 mmol, 1.00 equiv), H IO (50
mg, 0.22 mmol, 0.44 equiv), and I (102 mg, 0.40 mmol, 0.80 equiv)
was treated with a solution of concentrated H SO (0.70 mL) and
5
6
2
2
4
H O (0.42 mL) in acetic acid (2.10 mL) at room temperature. The
2
resulting mixture was stirred at 90 °C under microwave irradiation for
10 min. The reaction mixture was allowed to cool down to room
temperature, quenched with sat. aq. Na S O (30 mL), and extracted
with EtOAc (3 × 30 mL). The combined organic phase was washed
with H O (20 mL) and brine (20 mL). After removal of the solvent
under reduced pressure, the product was purified by flash column
chromatography (silica gel, 40% EtOAc in toluene) to obtain a yellow
solid (162 mg, 72%). Mp. 195 °C. H NMR (400 MHz, CDCl ): δ =
.50 (br s, 1H), 8.39 (d, J = 1.6 Hz, 1H), 8.03 (d, J = 1.3 Hz,
(fluorine-18 and iodine-124) and gamma emitters (iodine-123
2
2
3
and -131) for PET and SPECT imaging of apoptosis.
Compounds 12−17, 23, 24, 33, 38, and 39 were discovered
to be appropriate candidates for these purposes. Moreover, the
fluoroalkyl groups at nitrogen might provide another position
for fluorine-18 labeling. Furthermore, the 7-halogenated isatin
compounds are expected to resist a potential cytochrome P450
metabolic hydroxylation at this position, and hence, an
2
1
3
4
4
8
H,H H,H
2
3
1H), 3.84−3.75 (m, 1H), 3.54 (dd, J = 9.5 Hz, J = 4.0 Hz, 1H),
3.47−3.38 (m, 1H), 3.40 (dd, J = 9.8 Hz, J = 3.7 Hz, 1H), 3.36
H,H
H,H
2
3
H,H
H,H
F
dx.doi.org/10.1021/jm500718e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
s, 3H), 3.20−3.12 (m, 1H), 2.00−1.84 (m, 2H), 1.80−1.64 (m, 2H)
°C for 10 min under microwave irradiation as described in the general
procedure (section 4.2). The residue was purified by flash column
13
ppm. C NMR (100 MHz, CDCl ): δ = 182.0, 157.9, 154.0, 145.0,
3
1
36.0, 124.1, 118.7, 78.1, 74.7, 59.4, 59.2, 49.3, 28.8, 24.2 ppm. HRMS
chromatography (silica gel, 30% EtOAc in cyclohexane) to obtain a
+
1
(
ESI+, MeOH): m/z = 472.9637, [M + Na] ; calcd. 472.9639 for
yellow solid (80 mg, 89%). Mp. 99 °C. H NMR (300 MHz, CDCl ):
3
4
4
C H IN O S + Na.
δ = 8.47 (d, J = 1.9 Hz, 1H), 7.99 (d, J = 1.9 Hz, 1H), 4.19 (t,
14
15
2
5
H,H H,H
3
2
3
4
.3.2. (S)-N-Methyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
JH,H = 7.5 Hz, 2H), 3.85−3.74 (m, 1H), 3.54 (dd, J = 9.5 Hz, J
H,H
H,H
sulfonyl]isatin (11). Under argon, a stirred solution of (S)-7-iodo-5-
=
4.0 Hz, 1H), 3.46−3.34 (m, 2H), 3.36 (s, 3H), 3.22−3.10 (m, 1H),
3
[
1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (10) (25 mg, 55.5
2
2
=
.01−1.83 (m, 2H), 1.83−1.65 (m, 4H), 1.46 (sextet, J = 7.3 Hz,
H,H
3
13
μmol, 1.00 equiv) in dry DMF (3 mL) at 0 °C was treated with 60%
NaH (7 mg, 167 μmol, 3.00 eq. in mineral oil). After stirring for 10
min, MeI (10.4 μL, 167 μmol, 3.00 equiv) was added to the stirred
mixture at the same temperature. The resulting reaction mixture was
stirred further at 0 °C for 1 h and at room temperature for another 30
min. The solvent was completely removed under reduced pressure,
and the residue was purified by flash column chromatography (silica
gel, 25% EtOAc in toluene) to furnish an orange solid (21 mg, 82%).
H), 0.99 (t, J = 7.3 Hz, 3H) ppm. C NMR (75 MHz, CDCl ): δ
H,H
3
181.4, 158.4, 153.7, 149.3, 135.4, 123.8, 119.9, 74.7, 72.5, 59.3, 59.2,
9.3, 40.5, 31.7, 28.8, 24.2, 19.6, 13.8 ppm. HRMS (ESI+, MeOH): m/
z = 529.0252 [M + Na] , 561.0517 [M + Na + MeOH] ; calcd.
4
+
+
5
29.0265 for C H IN O S + Na, 561.0527 for C H IN O S + Na +
18 23 2 5 18 23 2 5
MeOH.
4 . 3 . 6 . ( S ) - N - ( 3 - F l u o r o p r o p y l ) - 7 - i o d o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (15). (S)-7-Iodo-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (10) (80 mg, 178 μmol,
1.00 equiv) was converted to (S)-N-(3-fluoropropyl)-7-iodo-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (15) by stirring with
1
4
Mp. 140 °C. H NMR (400 MHz, CDCl ): δ = 8.47 (d, J = 1.9 Hz,
3
H,H
4
1
3
H), 7.99 (d, J = 1.8 Hz, 1H), 3.84−3.75 (m, 1H), 3.73 (s, 3H),
H,H
2 3
.54 (dd, J = 9.5 Hz, J = 4.0 Hz, 1H), 3.45−3.35 (m, 1H), 3.39
H,H
H,H
2
3
(
1
dd, J = 9.6 Hz, J = 3.4 Hz, 1H), 3.36 (s, 3H), 3.21−3.10 (m,
Cs CO3 (117 mg, 355 μmol, 2.00 equiv) and 1-bromo-3-
H,H
H,H
2
1
3
H), 2.00−1.83 (m, 2H), 1.80−1.65 (m, 2H) ppm. C NMR (100
fluoropropane (75 mg, 533 μmol, 3.00 equiv) in dry DMF (5 mL)
at 95 °C for 10 min under microwave irradiation as described in the
general procedure (section 4.2). The residue was purified by flash
column chromatography (silica gel, 25% EtOAc in cyclohexane) to
MHz, CDCl ): δ = 181.2, 158.3, 153.9, 149.0, 135.7, 123.7, 119.6,
3
7
4.7, 72.7, 59.4, 59.2, 49.3, 29.9, 28.8, 24.2 ppm. HRMS (ESI+,
+
MeOH): m/z = 486.9786, [M + Na] ; calcd. 486.9795 for
C H IN O S + Na.
1
obtain a sticky orange-yellow solid (52 mg, 57%). H NMR (300
15
17
2
5
4
4
4
.3.3. (S)-N-Ethyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
MHz, CDCl ): δ = 8.48 (d, J = 1.9 Hz, 1H), 8.00 (d, J = 1.9
3 H,H H,H
2
3
3
sulfonyl]isatin (12). (S)-7-Iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (10) (25 mg, 55.5 μmol, 1.00 equiv) was converted to
Hz, 1H), 4.62 (dt, J = 46.9 Hz, J = 5.6 Hz, 2H), 4.43 (t, J
=
H,H
H,F
H,H
2
3
7.4 Hz, 2H), 3.85−3.75 (m, 1H), 3.54 (dd, J = 9.5 Hz, J = 4.0
H,H
H,H
(
(
S)-N-ethyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
12) by stirring with Cs CO (54 mg, 167 μmol, 3.00 equiv) and ethyl
Hz, 1H), 3.47−3.34 (m, 2H), 3.37 (s, 3H), 3.23−3.11 (m, 1H), 2.31−
13
2
3
2.11 (m, 2H), 2.00−1.84 (m, 2H), 1.84−1.65 (m, 2H) ppm. C NMR
bromide (12.4 μL, 167 μmol, 3.00 equiv) in dry DMF (3 mL) at 95 °C
for 10 min under microwave irradiation as described in the general
procedure (section 4.2). The residue was purified by flash column
chromatography (silica gel, 20% EtOAc in toluene) to yield an orange-
yellow solid (21 mg, 79%). Mp. 117 °C. H NMR (400 MHz, CDCl ):
(75 MHz, CDCl ): δ = 181.0, 158.6, 153.3, 149.2, 135.6, 123.9, 120.0,
3
1
3
81.5 (d, J = 166.4 Hz), 74.7, 72.6, 59.4, 59.1, 49.3, 37.9 (d, J
4.6 Hz), 30.5 (d, J = 19.7 Hz), 28.8, 24.2 ppm. F NMR (282
=
C,F
C,F
2
19
C,F
MHz, CDCl ): δ = −221.5 (s, 1F) ppm. HRMS (ESI+, MeOH): m/z
3
1
+
+
3
= 533.0027 [M + Na] , 565.0285 [M + Na + MeOH] ; calcd.
533.0014 for C H FIN O S + Na, 565.0276 for C H FIN O S +
4
4
δ = 8.42 (d, J = 1.9 Hz, 1H), 7.93 (d, J = 1.9 Hz, 1H), 4.25 (q,
H,H
H,H
17 20
2
5
17 20
2
5
3
2
3
JH,H = 7.1 Hz, 2H), 3.78−3.70 (m, 1H), 3.48 (dd, J = 9.5 Hz, J
Na + MeOH.
H,H
H,H
2
3
=
4.0 Hz, 1H), 3.39−3.31 (m, 1H), 3.34 (dd, J = 9.5 Hz, J
=
4 . 3 . 7 . ( S ) - N - ( 4 - F l u o r o b u t y l ) - 7 - i o d o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (16). (S)-7-Iodo-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (10) (80 mg, 178 μmol,
1.00 equiv) was converted to (S)-N-(4-fluorobutyl)-7-iodo-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (16) by stirring with
H,H
H,H
3
1
.3 Hz, 1H), 3.30 (s, 3H), 3.16−3.04 (m, 1H), 1.94−1.79 (m, 2H),
3
1
3
.75−1.61 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H) ppm. C NMR (100
H,H
MHz, CDCl ): δ = 181.5, 158.3, 153.5, 149.3, 135.4, 123.8, 120.0,
7
MeOH): m/z = 500.9946, [M + Na] ; calcd. 500.9952 for
3
4.7, 72.3, 59.4, 59.2, 49.3, 35.9, 28.8, 24.2, 15.0 ppm. HRMS (ESI+,
+
Cs CO (117 mg, 355 μmol, 2.00 equiv) and 1-bromo-4-fluorobutane
2 3
C H IN O S + Na.
(82 mg, 533 μmol, 3.00 equiv) in dry DMF (5 mL) at 95 °C for 10
min under microwave irradiation as described in the general procedure
(section 4.2). The residue was purified by flash column chromatog-
16
19
2
5
4
.3.4. (S)-N-Propyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (13). (S)-7-Iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (10) (25 mg, 55.5 μmol, 1.00 equiv) was converted to
raphy (silica gel, 10% EtOAc in toluene) to obtain a brown-yellow
1
(
S)-N-propyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-
solid (78 mg, 84%). Mp. 111 °C. H NMR (300 MHz, CDCl
3
): δ =
4
4
isatin (13) by stirring with Cs CO (54 mg, 167 μmol, 3.00 equiv) and
8.48 (d, JH,H = 1.9 Hz, 1H), 8.00 (d, JH,H = 1.9 Hz, 1H), 4.52 (dt,
2
3
2
3
3
1-bromopropane (15 μL, 167 μmol, 3.00 equiv) in dry DMF (3 mL)
JH,F = 47.4 Hz, J
= 5.5 Hz, 2H), 4.26 (t, J
= 7.4 Hz, 2H),
= 4.0 Hz, 1H),
H,H
H,H
2
3
at 95 °C for 10 min under microwave irradiation as described in the
general procedure (section 4.2). The residue was purified by flash
column chromatography (silica gel, 20% EtOAc in toluene) to obtain a
3.85−3.75 (m, 1H), 3.54 (dd, J
= 9.5 Hz, J
H,H
H,H
3.46−3.33 (m, 2H), 3.36 (s, 3H), 3.22−3.11 (m, 1H), 2.03−1.57 (m,
8H) ppm. 13C NMR (75 MHz, CDCl ): δ = 181.2, 158.4, 153.4, 149.3,
3
1
1
sticky orange-yellow solid (14 mg, 51%). H NMR (400 MHz,
135.6, 123.9, 119.9, 83.2 (d, J = 165.8 Hz), 74.7, 72.5, 59.4, 59.2,
C,F
4
4
2
3
CDCl ): δ = 8.47 (d, J = 1.9 Hz, 1H), 7.99 (d, J = 1.9 Hz, 1H),
49.3, 40.2, 28.8, 27.3 (d, J = 20.3 Hz), 26.0 (d, J = 4.4 Hz), 24.2
3
H,H
H,H
C,F C,F
3
3
3
19
4
1
3
.15 (t, J = 7.7 Hz, 2H), 3.80 (tt, J = 7.3 Hz, J = 3.8 Hz,
ppm. F NMR (282 MHz, CDCl ): δ = −217.9 (s, 1F) ppm. HRMS
H,H
H,H
H,H
3
2
3
+
H), 3.54 (dd, J = 9.6 Hz, J = 4.0 Hz, 1H), 3.46−3.37 (m, 1H),
(ESI+, MeOH): m/z = 547.0166 [M + Na] , 579.0430 [M + Na +
H,H
H,H
2
3
+
.40 (dd, J = 9.9 Hz, J = 2.9 Hz, 1H), 3.36 (s, 3H), 3.22−3.10
MeOH] ; calcd. 547.0170 for C H FIN O S + Na, 579.0432 for
H,H
H,H
18 22
2
5
3
(
m, 1H), 2.00−1.85 (m, 2H), 1.81−1.68 (m, 2H), 1.77 (sextet, J
=
C H FIN O S + Na + MeOH.
H,H
18 22 2 5
3
13
7
.5 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H) ppm. C NMR (100 MHz,
4 . 3 . 8 . ( S ) - N - ( 4 - H y d r o x y b u t y l ) - 7 - i o d o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (17). Under argon, a
stirred solution of (S)-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (10) (60 mg, 0.133 mmol, 1.00 equiv) in dry DMF
H,H
CDCl ): δ = 181.4, 158.4, 153.7, 149.3, 135.5, 123.8, 119.9, 74.7, 72.4,
3
59.4, 59.2, 49.3, 42.0, 28.8, 24.2, 23.1, 10.5 ppm. HRMS (ESI+,
+
MeOH): m/z = 515.0108, [M + Na] ; calcd. 515.0108 for
C H IN O S + Na.
(4 mL) was treated with Cs CO (87 mg, 0.267 mmol, 2.00 equiv) at
2 3
17
21
2
5
4
.3.5. (S)-N-Butyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
ambient temperature. After stirring for 10 min, the yellow mixture
turned to purple-blue and 4-bromobutyl pivalate (63 mg, 0.267 mmol,
2.00 equiv) was added to this mixture. The reaction mixture was
stirred in a sealed glass vessel at 100 °C for 10 min using microwave
irradiation, and then the solvent was removed completely under
reduced pressure. The residue was dissolved in 1,4-dioxane (2 mL),
sulfonyl]isatin (14). (S)-7-Iodo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (10) (80 mg, 178 μmol, 1.00 equiv) was converted to
(
S)-N-butyl-7-iodo-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
14) by stirring with Cs CO (117 mg, 355 μmol, 2.00 equiv) and 1-
bromobutane (57 μL, 533 μmol, 3.00 equiv) in dry DMF (5 mL) at 95
(
2
3
G
dx.doi.org/10.1021/jm500718e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
treated with 4 M HCl (1 mL), and stirred at 85 °C for 15 h. The
mixture was stirred in a sealed glass vessel at 100 °C for 3 min using
microwave irradiation. After that the solvent was removed completely
under reduced pressure and the residue was purified by flash column
resulting mixture was neutralized by dropwise addition of sat. aq.
NaHCO until no more gas evolved and extracted with EtOAc (3 × 10
3
mL). The combined organic phase was concentrated under reduced
pressure, and the residue was purified by flash column chromatography
chromatography (silica gel, 40% EtOAc in toluene) to obtain a yellow
1
solid (20 mg, 62% yield). Mp. 145 °C. H NMR (400 MHz, CDCl ):
3
4
4
(
silica gel, 70% EtOAc in toluene) to obtain the required product as a
δ = 8.22 (d, J = 1.8 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H), 4.26 (q,
H,H H,H
1
3
2
3
yellow wax (31 mg, 45% yield). H NMR (300 MHz, CDCl ): δ =
8
JH,H = 7.1 Hz, 2H), 3.84−3.76 (m, 1H), 3.54 (dd, J = 9.5 Hz, J
2 3
3
H,H
H,H
4
4
3
.48 (d, J = 1.9 Hz, 1H), 8.00 (d, J = 1.9 Hz, 1H), 4.26 (t, J
7.4 Hz, 2H), 3.85−3.75 (m, 1H), 3.73 (t, J = 6.2 Hz, 2H), 3.55
dd, J = 9.5 Hz, J = 4.0 Hz, 1H), 3.47−3.34 (m, 1H), 3.39 (dd,
= 4.0 Hz, 1H), 3.46−3.36 (m, 1H), 3.39 (dd, J = 9.5 Hz, J
=
H,H
H,H
H,H
H,H
H,H
3
2
3
=
(
7.2 Hz, 1H), 3.36 (s, 3H), 3.17 (dt, J = 9.8 Hz, J = 7.3 Hz, 1H),
H,H
H,H H,H
2
3
3
H,H
H,H
2.00−1.84 (m, 2H), 1.81−1.68 (m, 2H), 1.39 (t, J = 7.1 Hz, 3H)
ppm. C NMR (100 MHz, CDCl ): δ = 181.5, 158.1, 150.6, 142.5,
H,H
2
3
13
JH,H = 9.5 Hz, J = 7.2 Hz, 1H), 3.37 (s, 3H), 3.22−3.11 (m, 1H),
H,H
3
13
2
1
4
5
.02−1.63 (m, 8H) ppm. C NMR (75 MHz, CDCl ): δ = 181.3,
58.5, 153.5, 149.3, 135.4, 123.8, 120.0, 74.7, 72.6, 62.1, 59.4, 59.2,
135.3, 123.2, 120.2, 103.9, 74.7, 59.4, 59.1, 49.3, 36.9, 28.8, 24.2, 14.9
3
+
ppm. HRMS (ESI+, MeOH): m/z = 455.0062 [M + Na] , 487.0331
+
9.3, 40.4, 29.2, 28.8, 26.4, 24.2 ppm. HRMS (ESI+, MeOH): m/z =
[M + Na + MeOH] ; calcd. 455.0070 for C16
H19BrN
2
O
5
S + Na,
+
+
45.0217 [M + Na] , 577.0482 [M + Na + MeOH] ; calcd. 545.0214
487.0332 for C16H19BrN O S + Na + MeOH.
2 5
for C H IN O S + Na, 577.0476 for C H IN O S + Na + MeOH.
4.4.4. (S)-N-Propyl-7-bromo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (21). Under argon, a stirred solution of (S)-7-bromo-5-
[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (18) (45 mg, 0.112
mmol, 1.00 equiv) in dry DMF (3 mL) was treated with anhydrous
K CO (31 mg, 0.223 mmol, 2.00 equiv). After stirring for 30 min, the
18
23
2
6
18 23
2
6
4
.4. (S)-7-Bromo-5-[1-(2-methoxymethylpyrrolidinyl)-
s u l f o n y l ] i s a t i n s . 4 . 4 . 1 . ( S ) - 7 - B r o m o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (18). According to a
14
modified literature procedure, a stirred solution of (S)-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (1) (250 mg, 0.771 mmol,
2
3
7
yellow solution turned to a purple-blue mixture and then 1-
bromopropane (30 μL, 0.335 mmol, 3.00 equiv) was added. The
reaction mixture was stirred at room temperature for 4 days (or until
the starting material was completely disappeared). The solvent was
removed under reduced pressure, and the crude product was purified
by flash column chromatography (silica gel, 40% EtOAc in toluene) to
1
.00 equiv) in dry DCM (200 mL) was cooled to 0 °C and conc.
H SO (30 drops) was added. After stirring for 5 min, the solution was
2
4
treated slowly with DBH (441 mg, 1.54 mmol, 2.00 equiv) at the same
temperature. The resulting solution was stirred at 0 °C for 20 min and
refluxed in the dark for 3 h. Then it was allowed to cool to room
temperature before water (50 mL) was added. The organic phase was
separated and the aq. phase was extracted with DCM (3 × 100 mL).
1
yield a sticky red-yellow solid (36 mg, 72% yield). H NMR (400
4
4
MHz, CDCl ): δ = 8.22 (d, J = 1.8 Hz, 1H), 7.99 (d, J = 1.8
3
H,H
H,H
3
The combined organic layer was dried over MgSO and concentrated
Hz, 1H), 4.15 (t, J = 7.8 Hz, 2H), 3.85−3.77 (m, 1H), 3.55 (dd,
4
H,H
2
3
2
under reduced pressure. The residue was purified by flash column
JH,H = 9.5 Hz, J = 4.0 Hz, 1H), 3.48−3.37 (m, 1H), 3.40 (dd, J
H,H
H,H
3
2
chromatography (silica gel, 40% EtOAc in toluene) to obtain the title
= 9.5 Hz, J = 7.2 Hz, 1H), 3.37 (s, 3H), 3.19 (dt, J = 9.9 Hz,
H,H H,H
1
3
product as a yellow solid (202 mg, 65% yield). Mp. 158 °C. H NMR
JH,H = 7.3 Hz, 1H), 2.01−1.87 (m, 2H), 1.85−1.69 (m, 2H), 1.80
4
3 3 13
(
7
9
400 MHz, CDCl ): δ = 8.74 (s, 1H), 8.17 (d, J = 1.6 Hz, 1H),
(sextet, J
= 7.6 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H) ppm.
H,H
C
3
H,H
H,H
4
2
.96 (dd, J = 1.6 Hz, 1H), 3.78−3.69 (m, 1H), 3.49 (dd, J
=
NMR (100 MHz, CDCl ): δ = 181.4, 158.3, 150.7, 142.5, 135.3, 123.2,
H,H
H,H
3
3
2
.5 Hz, J = 4.0 Hz, 1H), 3.40−3.30 (m, 1H), 3.33 (dd, J = 9.5
120.2, 104.0, 74.7, 59.4, 59.1, 49.3, 43.1, 28.9, 24.2, 23.0, 10.8 ppm.
H,H
H,H
+
Hz, JH,H = 7.2 Hz, 1H), 3.30 (s, 3H), 3.16−3.06 (m, 1H), 1.94−1.78
HRMS (ESI+, MeOH): m/z = 467.0245, 469.0232 [M + Na] ; calcd.
13
(
1
m, 2H) 1.74−1.60 (m, 2H) ppm. C NMR (100 MHz, CDCl ): δ =
467.0247, 469.0227 for C H BrN O S + Na.
3
17 21
2
5
81.2, 157.9, 150.8, 139.4, 135.7, 123.4, 118.9, 106.2, 74.7, 59.4, 59.1,
4.4.5. (S)-N-Butyl-7-bromo-5-[1-(2-methoxymethylpyrrolidinyl)-
s u l f o n y l ] i s a t i n ( 2 2 ) . ( S ) - 7 - B r o m o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (18) (30 mg, 74.4 μmol,
1.00 equiv) was converted to (S)-N-butyl-7-bromo-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (22) by stirring with
Cs CO (73 mg, 0.223 mmol, 3.00 equiv) and 1-bromobutane (24
4
9.3, 28.8, 24.2 ppm. HRMS (ESI+, MeOH): m/z = 424.9776,
+
426.9753 [M + Na] ; calcd. 424.9777, 426.9757 for C H BrN O S +
14
15
2
5
Na.
4
.4.2. (S)-N-Methyl-7-bromo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (19). Under argon, a stirred solution of (S)-7-bromo-5-
1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (18) (30 mg, 74.4
2
3
[
μL, 0.223 mmol, 3.00 equiv) in dry DMF (3 mL) at 100 °C for 3 min
under microwave irradiation as described in the general procedure
(section 4.2). The residue was purified by flash column chromatog-
raphy (silica gel, 20% EtOAc in toluene) to obtain the required
μmol, 1.00 equiv) in dry DMF (2 mL) was cooled to 0 °C and treated
with a 60% NaH dispersion in mineral oil (4.46 mg, 112 μmol, 1.50
equiv). After stirring for 10 min, the solution turned blue-purple and
MeI (6.95 μL, 112 μmol, 1.50 equiv) was added dropwise. The
reaction mixture was stirred at 0 °C for 30 min and at ambient
temperature for 30 min. The solvent was removed completely under
reduced pressure, and the crude was purified by flash column
chromatography (silica gel, 35% EtOAc in toluene) to furnish the
desired product as an orange-yellow solid (25 mg, 81% yield). Mp. 111
1
product as a yellow solid (20 mg, 59% yield). Mp. 104 °C. H NMR
4
4
(400 MHz, CDCl ): δ = 8.15 (d, J = 1.8 Hz, 1H), 7.91 (d, J =
3
H,H
H,H
3
1.8 Hz, 1H), 4.11 (t, J = 7.6 Hz, 2H), 3.78−3.70 (m, 1H), 3.48
H,H
2
3
(dd, J = 9.5 Hz, J = 4.0 Hz, 1H), 3.40−3.31 (m, 1H), 3.33 (dd,
H,H
H,H
2
3
2
JH,H = 9.5 Hz, J = 7.2 Hz, 1H), 3.30 (s, 3H), 3.11 (dt, J = 9.8
H,H
H,H
3
Hz, J
= 7.2 Hz, 1H), 1.94−1.80 (m, 2H), 1.76−1.62 (m, 2H),
H,H
1
4
3
3
°
C. H NMR (400 MHz, CDCl ): δ = 8.20 (d, J = 1.8 Hz, 1H),
3 H,H
1.73−1.62 (m, 2H), 1.37 (sextet, J = 7.5 Hz, 2H), 0.92 (t, J
=
H,H
H,H
4
13
7
.96 (d, J = 1.8 Hz, 1H), 3.83−3.75 (m, 1H), 3.70 (s, 3H), 3.53
7.4 Hz, 3H) ppm. C NMR (100 MHz, CDCl ): δ = 181.4, 158.2,
H,H
3
2
3
(
dd, J = 9.5 Hz, J = 4.0 Hz, 1H), 3.45−3.36 (m, 1H), 3.39 (dd,
150.7, 142.5, 135.3, 123.2, 120.3, 104.0, 74.7, 59.4, 59.1, 49.3, 41.5,
H,H
H,H
2
3
JH,H = 9.9 Hz, J = 7.2 Hz, 1H), 3.35 (s, 3H), 3.21−3.11 (m, 1H),
31.6, 28.8, 24.2, 19.8, 13.7 ppm. HRMS (ESI+, MeOH): m/z =
H,H
1
3
+
+
1
.99−1.84 (m, 2H), 1.79−1.65 (m, 2H) ppm. C NMR (100 MHz,
483.0370 [M + Na] , 515.0633 [M + Na + MeOH] ; calcd. 483.0383
for C H BrN O S + Na, 515.0646 for C H BrN O S + Na +
CDCl ): δ = 181.1, 158.2, 151.0, 142.2, 135.4, 123.0, 119.9, 104.5,
3
18 23
2
5
18 23
2
5
7
4.7, 59.4, 59.1, 49.3, 29.9, 28.8, 24.2 ppm. HRMS (ESI+, MeOH): m/
MeOH.
+
z = 438.9941, 440.9913 [M + Na] , 471.0193, 473.0171 [M + Na +
MeOH] ; calcd. 438.9934, 440.9914 for C H BrN O S + Na,
4 . 4 . 6 . ( S ) - N - ( 3 - F l u o r o p r o p y l ) - 7 - b r o m o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (23). (S)-7-Bromo-5-[1-
(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (18) (50 mg, 0.124
mmol, 1.00 equiv) was converted to (S)-N-(3-fluoropropyl)-7-
bromo-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (23) by
+
15
17
2
5
4
71.0196, 473.0176 for C H BrN O S + Na + MeOH.
4
15 17 2 5
.4.3. (S)-N-Ethyl-7-bromo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (20). Under argon, a stirred solution of (S)-7-bromo-
-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (18) (30 mg, 74.4
5
stirring with Cs CO3 (80 mg, 0.248 mmol, 2.00 equiv) and 1-
2
μmol, 1.00 equiv) in dry DMF (3 mL) was treated with a 60% NaH
dispersion in mineral oil (4.5 mg, 112 μmol, 1.50 equiv). After stirring
for 10 min, the yellow solution turned to blue-purple mixture and 1-
bromoethane (17 μL, 223 μmol, 3.00 equiv) was added. The reaction
bromo-3-fluoropropane (52 mg, 0.372 mmol, 3.00 equiv) in dry DMF
(4 mL) at 90 °C for 5 min under microwave irradiation as described in
the general procedure (section 4.2). The residue was purified by flash
column chromatography (silica gel, 20% EtOAc in toluene) to obtain
H
dx.doi.org/10.1021/jm500718e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
the required product as an orange-yellow oil (37 mg, 65% yield). H
nate (29). A dried round-bottom flask containing oleum (3.1 mL) was
cooled to −20 °C, and 7-chloroisatin (28) (2.00 g, 11.0 mmol, 1.00
equiv) was slowly added in portions over 10 min. The suspension was
allowed to stir below −10 °C for 30 min and then was heated to 70 °C
and further stirred for 45 min. The reaction mixture was cooled and
poured on ice (3 g). The mixture was neutralized using 20% aq.
NaOH solution (30−40 mL) to approximately pH 7.5 and completely
evaporated under reduced pressure. The resulting dry crude product
was suspended in MeOH (30 mL) and stirred for 30 min. The
obtained suspension was filtered and the solid was washed with
MeOH (30 mL). The combined filtrate was evaporated under reduced
pressure to afford an orange-red solid (3.09 g, 99% yield). The product
was used in the next step without further purification. Mp. >300 °C.
4
NMR (400 MHz, CDCl ): δ = 8.24 (d, J = 1.8 Hz, 1H), 8.00 (d,
= 1.8 Hz, 1H), 4.61 (dt, J = 46.9 Hz, J = 5.5 Hz, 2H), 4.41
t, J = 7.3 Hz, 2H), 3.86−3.78 (m, 1H), 3.55 (dd, J = 9.5 Hz,
3
H,H
4
2
3
J
H,H
3
H,F H,H
2
(
H,H
H,H
3
2 3
JH,H = 4.0 Hz, 1H), 3.47−3.39 (m, 1H), 3.40 (dd, J = 9.5 Hz, J
H,H
H,H
2
3
=
7.1 Hz, 1H), 3.36 (s, 3H), 3.19 (dt, J = 9.1 Hz, J = 7.0 Hz,
H), 2.29−2.13 (m, 2H), 2.02−1.85 (m, 2H), 1.83−1.68 (m, 2H)
ppm. C NMR (100 MHz, CDCl ): δ = 181.0, 158.4, 150.4, 142.5,
35.7, 123.3, 120.3, 104.0, 81.6 (d, J = 166.4 Hz), 74.7, 59.4, 59.1,
9.3, 38.9 (d, J = 4.4 Hz), 30.5 (d, J = 19.7 Hz), 28.9, 24.2 ppm.
F NMR (282 MHz, CDCl ): δ = −221.5 (s, 1F) ppm. HRMS (ESI+,
MeOH): m/z = 485.0148, 487.0130 [M + Na] , 517.0413, 519.0391
H,H H,H
1
13
3
1
1
4
C,F
3
2
C,F
C,F
1
9
3
+
+
[
M + Na + MeOH] ; calcd. 485.0153, 487.0133 for C H BrFN O S
17 20 2 5
1
4
H NMR (400 MHz, DMSO-d ): δ = 11.55 (s, 1H), 7.78 (d, J =
+
Na, 517.0415, 519.0395 for C H BrFN O S + Na + MeOH.
4
6
H,H
17
20
2
5
4
13
1
.5 Hz, 1H), 7.57 (d, J = 1.5 Hz, 1H) ppm. C NMR (100 MHz,
H,H
. 4 . 7 . ( S ) - N - ( 4 - F l u o r o b u t y l ) - 7 - b r o m o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (24). (S)-7-Bromo-5-[1-
2-methoxymethylpyrrolidinyl)sulfonyl]isatin (18) (50 mg, 0.124
mmol, 1.00 equiv) was converted to (S)-N-(4-fluorobutyl)-7-bromo-
-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (24) by stirring
with Cs CO3 (80 mg, 0.248 mmol, 2.00 equiv) and 1-bromo-4-
fluorobutane (40 μL, 0.372 mmol, 3.00 equiv) in dry DMF (3 mL) at
DMSO-d
): δ = 183.3, 160.0, 147.8, 144.0, 134.1, 120.0, 119.4, 115.5
6
+
(
ppm. HRMS (ESI+, MeOH): m/z = 305.9206 [M + Na] , 337.9480
[M + Na + MeOH] ; calcd. 305.9210 for C
+
H ClNO SNa + Na,
3 5
8
5
337.9473 for C ClNO SNa + Na + MeOH. HRMS (ESI-, MeOH):
8
H
3
5
−
−
m/z = 259.9429 [M-Na] , 291.9691 [M − Na + MeOH] ; calcd.
2
259.9426 for C
MeOH.
H
ClNO
S−Na, 291.9688 for C H ClNO S − Na +
8 3 5
8
3
5
1
00 °C for 2 min under microwave irradiation as described in the
general procedure (section 4.2). The residue was purified by flash
column chromatography (silica gel, 30% EtOAc in toluene) to obtain
the required product as a yellow solid (56 mg, 95% yield). Mp. 114 °C.
4.5.2. 5,7-Dichlorosulfonyl Isatin (30). To a suspension of sodium
7-chloro-2,3-dioxoindoline-5-sulfonate (29) in sulfolane (14.5 mL)
was added POCl (2.9 mL, 31.7 mmol, 4.50 equiv), dropwise at 40 °C.
3
1
4
H NMR (400 MHz, CDCl ): δ = 8.29 (d, J = 1.8 Hz, 1H), 8.05
d, J = 1.8 Hz, 1H), 4.58 (dt, J = 47.4 Hz, J = 5.7 Hz, 2H),
The reaction mixture was stirred at 60 °C for 4 h followed by cooling
to 0 °C and very slow quenching with ice−water (50 mL). The
precipitate was filtered and washed with cold water (20−30 mL). The
resulting solid was suspended in EtOAc (30 mL) and washed with
water (3 × 10 mL) and brine (20 mL). The orange solution was dried
3
H,H
4
2
3
(
4
H,H H,F H,H
3
2
.31 (t, J = 7.6 Hz, 2H), 3.92−3.83 (m, 1H), 3.61 (dd, J = 9.5
H,H
H,H
3
2
Hz, J = 4.0 Hz, 1H), 3.53−3.45 (m, 1H), 3.46 (dd, J = 9.5 Hz,
H,H
H,H
3
2
3
JH,H = 7.2 Hz, 1H), 3.43 (s, 3H), 3.25 (dt, J = 9.8 Hz, J = 7.1
H,H
H,H
over MgSO and concentrated under reduced pressure to obtain a
Hz, 1H), 2.05−1.92 (m, 4H), 1.95−1.76 (m, 2H), 1.88−1.76 (m, 2H)
4
ppm. 13C NMR (100 MHz, CDCl ): δ = 181.2, 158.3, 150.5, 142.5,
yellow solid (1.58 g, 80% yield). The product was used in the next step
3
1
1
without further purification. Mp. 178 °C. H NMR (300 MHz,
1
4
35.5, 123.2, 120.3, 104.0, 83.2 (d, J = 165.8 Hz), 74.7, 59.4, 59.1,
9.3, 41.2, 28.8, 27.5 (d, J = 20.3 Hz), 26.0 (d, J = 4.2 Hz), 24.2
ppm. F NMR (282 MHz, CDCl ): δ = −218.6 (s, 1F) ppm. HRMS
ESI+, MeOH): m/z = 499.0317, 501.0302 [M + Na] , 531.0578,
33.0558 [M + Na + MeOH] ; calcd. 499.0309, 501.0289 for
C H BrFN O S + Na, 531.0571, 533.0551 for C H BrFN O S +
C,F
4
2
3
DMSO-d ): δ = 11.52 (s, 1H), 7.72 (d, J = 1.5 Hz, 1H), 7.51 (d,
6
H,H
C,F
C,F
4
13
19
JH,H = 1.5 Hz, 1H) ppm. C NMR (75 MHz, DMSO-d ): δ = 183.4,
6
3
+
(
5
160.2, 147.9, 144.2, 134.1, 120.0, 119.6, 115.7 ppm. HRMS (ESI+,
MeOH): m/z = 329.9809 [M − Cl + MeO + Na + MeOH]+; calcd.
329.9810 for C H Cl NO S−Cl + MeO + Na + MeOH.
8 3 2 4
+
18
22
2
5
18 22
2
5
Na + MeOH.
. 4 . 8 . ( S ) - N - ( 4 - H y d r o x y b u t y l ) - 7 - b r o m o - 5 - [ 1 - ( 2 -
Note: In the ESI-MS measurement MeOH was used as the solvent,
which reacted with product 30 to form the corresponding methyl
sulfonate. The latter compound was detected in the ESI-MS spectrum.
4.5.3. (S)-7-Chloro-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-
isatin (31). Under argon, a stirred solution of N-Boc-2-methox-
ymethylpyrrolidine (699 mg, 3.25 mmol, 1.00 equiv) in dry DCM
(17.5 mL) was cooled to 0 °C and treated slowly with TFA (2.40 mL,
32.5 mmol, 10.00 equiv). The resulting solution was stirred at 0 °C for
30 min and at ambient temperature for 2 h. Then, the solution was
poured into cooled 10% aq. NaOH (50 mL) and extracted with DCM
4
methoxymethylpyrrolidinyl)sulfonyl]isatin (25). Under argon, a
stirred solution of (S)-7-bromo-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (18) (50 mg, 0.124 mmol, 1.00 equiv) in dry DMF (4
mL) was treated with Cs CO (80 mg, 0.248 mmol, 2.00 equiv) at
2
3
ambient temperature. After stirring for 10 min, the yellow mixture
turned to purple-blue and 4-bromobutyl pivalate (59 mg, 0.248 mmol,
2
.00 equiv) was added. This mixture was stirred in a sealed glass vessel
at 100 °C for 8 min using microwave irradiation, and then the solvent
was removed completely under reduced pressure. The residue was
dissolved in 1,4-dioxane (1.5 mL), treated with 4 M HCl (0.80 mL),
and stirred at 85 °C for 15 h. The resulting mixture was neutralized by
(3 × 50 mL). The solution was dried over MgSO and concentrated
4
under reduced pressure to obtain the deprotected pyrrolidine as a
colorless oil. A solution of this pyrrolidine and DIPEA (1.13 mL, 6.49
dropwise addition of sat. aq. NaHCO until no more gas evolved and
mmol, 2.00 equiv) in CHCl
solution of 5,7-dichlorosulfonyl isatin (30) (1.00 g, 3.57 mmol, 1.10
equiv) in CHCl /THF (1:1, 46 mL) at room temperature. It was
3
(10 mL) was added dropwise to a stirred
3
extracted with EtOAc (3 × 5 mL). The combined organic phase was
concentrated under reduced pressure, and the residue was purified by
flash column chromatography (silica gel, 90% EtOAc in toluene) to
obtain the required product as a yellow gummy-solid (25 mg, 42%
3
stirred at this temperature for 1 h, and the solvent was removed under
reduced pressure. The crude product was purified by flash column
chromatography (silica gel, 60% EtOAc in cyclohexane) to furnish a
1
4
yield). H NMR (400 MHz, CDCl ): δ = 8.22 (d, J = 1.8 Hz, 1H),
3
H,H
4
3
1
7
(
4
.98 (d, J = 1.8 Hz, 1H), 4.24 (t, J = 7.6 Hz, 2H), 3.85−3.77
m, 1H), 3.72 (t, J = 6.3 Hz, 2H), 3.55 (dd, J = 9.5 Hz, J =
.0 Hz, 1H), 3.48−3.37 (m, 1H), 3.40 (dd, J = 9.5 Hz, J = 7.2
Hz, 1H), 3.37 (s, 3H), 3.18 (dt, J = 9.8 Hz, J = 7.2 Hz, 1H),
yellow solid (700 mg, 60% yield). Mp. 172 °C. H NMR (300 MHz,
H,H
H,H
3
2
3
4
CD
CN): δ = 9.64 (br s, 1H), 8.04 (d, JH,H = 1.7 Hz, 1H), 7.84 (d,
H,H
H,H
H,H
3
2
3
4
2
3
JH,H = 1.7 Hz, 1H), 3.80−3.66 (m, 1H), 3.46 (dd, JH,H = 9.6 Hz, JH,H
H,H
H,H
2
3
2 3
H,H
= 4.1 Hz, 1H), 3.38−3.27 (m, 1H), 3.36 (dd, J = 9.5 Hz, J
=
H,H
H,H
H,H
1
1
1
4
4
.99−1.90 (m, 2H), 1.94−1.84 (m, 2H), 1.82−1.72 (m, 2H), 1.73−
7.1 Hz, 1H), 3.30 (s, 3H), 3.23−3.11 (m, 1H), 1.90−1.47 (m, 4H)
.63 (m, 2H) ppm. 13C NMR (100 MHz, CDCl ): δ = 181.3, 158.3,
ppm. C NMR (75 MHz, CD CN): δ = 183.2, 159.6, 151.6, 137.2,
13
3
3
50.6, 142.5, 135.4, 123.2, 120.3, 104.0, 74.7, 62.2, 59.4, 59.1, 49.3,
134.7, 123.1, 120.4, 118.9, 75.7, 60.4, 59.3, 50.3, 29.3, 24.8 ppm.
+
1.5, 29.4, 28.9, 26.4, 24.2 ppm. HRMS (ESI+, MeOH): m/z =
HRMS (ESI+, MeOH): m/z = 381.0289 [M + Na] , 413.0546 [M +
+
+
97.0359, 499.0345 [M + Na] , 529.0611, 531.0596 [M + Na +
Na + MeOH] ; calcd. 381.0282 for C H ClN O S + Na, 413.0545
14
15
2
5
+
MeOH] ; calcd. 497.0352, 499.0332 for C H BrN O S + Na,
for C H ClN O S + Na + MeOH.
18
23
2
6
14 15 2 5
5
29.0615, 531.0595 for C H BrN O S + Na + MeOH.
4.5.4. (S)-N-Butyl-7-chloro-5-[1-(2-methoxymethylpyrrolidinyl)-
s u l f o n y l ] i s a t i n ( 3 2 ) . ( S ) - 7 - C h l o r o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (31) (100 mg, 0.279
18
23
2
6
4
.5. (S)-7-Chloro-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatins. 4.5.1. Sodium 7-chloro-2,3-dioxoindoline-5-sulfo-
I
dx.doi.org/10.1021/jm500718e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mmol, 1.00 equiv) was converted to (S)-N-butyl-7-chloro-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (32) by stirring with
resulting crude product was suspended in EtOAc (30 mL) and
warmed to 40 °C (for complete dissolution). The solution was cooled
to ambient temperature and washed with water (3 × 10 mL) and brine
Cs CO3 (182 mg, 0.557 mmol, 2.00 equiv) and 1-bromobutane
2
(
298 μL, 2.79 mmol, 10.00 equiv) in dry DMF (4 mL) at 95 °C for 10
min under microwave irradiation as described in the general procedure
section 4.2). The crude product was purified by flash column
(10 mL). The organic phase was dried over MgSO and evaporated to
4
dryness to yield a yellow-brown solid (1.30 g, 66% yield). Mp. 199 °C.
1
3
(
H NMR (300 MHz, DMSO-d ): δ = 11.68 (s, 1H), 7.73 (dd, J
=
6
H,F
4
13
chromatography (silica gel, 30% EtOAc in cyclohexane) to obtain a
9.8 Hz, J = 1.4 Hz, 1H), 7.70−7.66 (m, 1H) ppm. C NMR (75
H,H
1
1
yellow solid (112 mg, 97% yield). Mp. 103 °C. H NMR (300 MHz,
MHz, CDCl3): δ = 183.0, 159.2, 146.5 (d, J = 248.9 Hz), 143.5 (d,
C,F
4
4
3
2
2
CDCl ): δ = 8.03 (d, J = 1.8 Hz, 1H), 7.93 (d, J = 1.8 Hz, 1H),
JC,F = 3.1 Hz), 138.1 (d, J = 13.9 Hz), 122.3 (d, J = 19.1 Hz),
3
H,H
H,H
C,F C,F
3
2
4
.15 (t, J = 7.4 Hz, 2H), 3.85−3.73 (m, 1H), 3.55 (dd, J = 9.5
3
4
19
H,H
H,H
119.2 (d, J = 4.0 Hz), 118.0 (d, J = 3.3 Hz) ppm. F NMR
C,F C,F
3
Hz, J = 4.0 Hz, 1H), 3.48−3.34 (m, 1H), 3.43−3.35 (m, 1H), 3.36
H,H
(282 MHz, DMSO-d ): δ = −133.0 (s, 1F) ppm. HRMS (ESI+,
6
(
1
s, 3H), 3.24−3.10 (m, 1H), 2.04−1.64 (m, 4H), 1.84−1.64 (m, 2H),
+
+
MeOH): m/z = 281.9852 [M + Na] , 314.0110 [M + Na + MeOH] ;
3
3
13
.43 (sextet, J = 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H) ppm. C
H,H H,H
calcd. 281.9848 for C H FNO S + Na, 314.0111 for C H FNO S +
9
6
5
9
6
5
NMR (75 MHz, CDCl ): δ = 181.5, 158.1, 149.3, 139.2, 134.9, 122.6,
3
Na + MeOH.
1
20.0, 117.5, 74.7, 59.3, 59.1, 49.3, 42.0, 31.6, 28.8, 24.2, 19.8, 13.7
Note: 5-Chlorosulfonyl-7-fluoroisatin was not stable in MeOH
under the ESI-MS measurement. Methyl 7-fluoroisatin-5-sulfonate was
exclusively observed in the MS-spectrum.
+
ppm. HRMS (ESI+, MeOH): m/z = 437.0913 [M + Na] , 469.1177
+
[
M + Na + MeOH] ; calcd. 437.0908 for C H ClN O S + Na,
18 23 2 5
4
69.1171 for C H ClN O S + Na + MeOH.
4
18 23 2 5
4.6.3. (S)-7-Fluoro-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-
isatin (37). Under argon, a stirred solution of N-Boc-2-methox-
ymethylpyrrolidine (200 mg, 0.929 mmol, 1.00 equiv) in dry DCM (5
mL) was treated dropwise with TFA (690 μL, 9.29 mmol, 10.00
equiv) at 0 °C. The solution was stirred at 0 °C for 30 min and at
ambient temperature for 2 h. Then, the resulting mixture was poured
into ice-cooled 10% aq. NaOH (25 mL) and extracted with DCM (3 ×
. 5 . 5 . ( S ) - N - ( 4 - F l u o r o b u t y l ) - 7 - c h l o r o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (33). (S)-7-Chloro-5-[1-
2-methoxymethylpyrrolidinyl)sulfonyl]isatin (31) (100 mg, 0.279
mmol, 1.00 equiv) was converted to (S)-N-(4-fluorobutyl)-7-chloro-5-
1-(2-methoxymethyl-pyrrolidinyl)sulfonyl]isatin (33) by stirring with
(
[
Cs CO3 (182 mg, 0.557 mmol, 2.00 equiv) and 1-bromo-4-
2
fluorobutane (299 μL, 2.79 mmol, 10.00 equiv) in dry DMF (4
mL) at 95 °C for 10 min under microwave irradiation as described in
the general procedure (section 4.2). The crude product was purified by
flash column chromatography (silica gel, 50% EtOAc in cyclohexane)
2
5 mL). The combined organic phase was dried over MgSO , and
4
solvent was removed under reduced pressure. The residue was taken
up in CHCl (3.0 mL) and treated with DIPEA (324 μL, 1.86 mmol,
3
2
.00 equiv). The above solution was added dropwise to a stirred
1
to obtain a yellow solid (115 mg, 95% yield). Mp. 115 °C. H NMR
solution of 5-chlorosulfonyl-7-fluoroisatin (36) (367 mg, 1.39 mmol,
4
4
(
400 MHz, CDCl ): δ = 8.03 (d, J = 1.8 Hz, 1H), 7.93 (d, J
=
3
H,H
H,H
1.50 equiv) in CHCl /THF (1:1, 18 mL) at room temperature. The
2
3
3
1
.8 Hz, 1H), 4.51 (dt, J = 47.4 Hz, J = 5.7 Hz, 2H), 4.21 (t,
H,F H,H
reaction mixture was stirred at this temperature for 1 h and then
concentrated under reduced pressure. The crude product was purified
by flash column chromatography (silica gel, 65% EtOAc in
3
2
3
JH,H = 7.4 Hz, 2H), 3.85−3.75 (m, 1H), 3.54 (dd, J = 9.5 Hz, J
H,H
H,H
2
3
=
7
4.0 Hz, 1H), 3.49−3.34 (m, 1H), 3.39 (dd, J = 9.5 Hz, J
=
H,H
H,H
.3 Hz, 1H), 3.36 (s, 3H), 3.24−3.12 (m, 1H), 2.04−1.64 (m, 8H)
cyclohexane) to obtain a yellow solid (138 mg, 43% yield). Mp. 174
13
ppm. C NMR (100 MHz, CDCl ): δ = 181.3, 158.2, 149.0, 139.2,
1
3
°C. H NMR (300 MHz, DMSO-d ): δ = 11.99 (br s, 1H), 8.01 (dd,
6
1
1
4
35.2, 122.7, 120.1, 117.5, 83.3 (d, J = 165.6 Hz), 74.7, 59.4, 59.1,
9.3, 41.7, 28.8, 27.5 (d, J = 20.2 Hz), 26.0 (d, J = 4.2 Hz), 24.2
3
4
4
C,F
JH,F = 9.5 Hz, J = 1.6 Hz, 1H), 7.66 (d, J = 1.6 Hz, 1H), 3.81−
H,H
H,H
2
3
2
3
C,F
C,F
3
.68 (m, 1H), 3.44 (dd, J = 9.5 Hz, J = 3.9 Hz, 1H), 3.37−3.30
19
H,H
H,H
ppm. F NMR (282 MHz, CDCl ): δ = −219.1 (s, 1F) ppm. HRMS
2
3
(
m, 1H), 3.31−3.25 (m, 1H), 3.28 (s, 3H), 3.14 (dt, J = 10.2 Hz,
+
H,H
(
ESI+, MeOH): m/z = 455.0821 [M + Na] , 487.1080 [M + Na +
3
13
J
= 7.1 Hz, 1H), 1.90−1.65 (m, 2H), 1.65−1.43 (m, 2H) ppm. C
+
H,H
MeOH] ; calcd. 455.0814 for C H ClFN O S + Na, 487.1076 for
1
18
22
2
5
NMR (75 MHz, DMSO-d ): δ = 181.7, 159.2, 146.7 (d, J = 250.3
Hz), 141.2 (d, J = 13.6 Hz), 131.5 (d, J = 4.0 Hz), 123.0 (d,
JC,F = 20.2 Hz), 120.6 (d, J = 4.5 Hz), 119.0 (d, J = 3.1 Hz),
4.4, 58.7, 58.4, 48.9, 28.1, 23.5 ppm. F NMR (282 MHz, DMSO-
6
C,F
C H ClFN O S + Na + MeOH.
2
3
18
22
2
5
C,F
C,F
4
.6. (S)-7-Fluoro-5-[1-(2-methoxymethylpyrrolidinyl)-
2
3
4
C,F
C,F
sulfonyl]isatins. 4.6.1. Sodium 7-Fluoroisatin-5-sulfonate (35).
Oleum (4.0 mL) was cooled to −20 °C and treated portionwise
with 7-fluoroisatin (34) (2.00 g, 12.1 mmol, 1.00 equiv) over 10 min.
The suspension was stirred vigorously below −10 °C for 45 min and
then at 70 °C for 1 h. After cooling to r.t., the mixture was neutralized
with 20% aq. NaOH to approximately pH 7.5 and concentrated to
dryness under reduced pressure. The residue was suspended in MeOH
19
7
d ): δ = −130.5 (s, 1F) ppm. HRMS (ESI+, MeOH): m/z = 365.0570
6
+
+
[
M + Na] , 397.0830 [M + Na + MeOH] ; calcd. 365.0578 for
C H FN O S + Na, 397.0840 for C H FN O S + Na + MeOH.
14 15
2
5
14 15
2
5
4
.6.4. (S)-N-Butyl-7-fluoro-5-[1-(2-methoxymethylpyrrolidinyl)-
s u l f o n y l ] i s a t i n ( 3 8 ) . ( S ) - 7 - F l u o r o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (37) (100 mg, 0.292
mmol, 1.00 equiv) was converted to (S)-N-butyl-7-fluoro-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (38) by stirring with
(40 mL) and stirred for 30 min. The precipitate was filtered and
washed with MeOH (10 mL). The combined filtrate was evaporated
1
to obtain a red-colored solid (quantitative). Mp. >300 °C. H NMR
3
Cs CO3 (190 mg, 0.584 mmol, 2.00 equiv) and 1-bromobutane
2
(
300 MHz, DMSO-d ): δ = 11.65 (br s, 1H), 7.64 (dd, J = 10.0 Hz,
6
H,F
13
4
4
(313 μL, 2.92 mmol, 10.00 equiv) in dry DMF (4.0 mL) at 95 °C for
10 min under microwave irradiation as described in the general
procedure (section 4.2). The residue was purified by flash column
JH,H = 1.3 Hz, 1H), 7.50 (d, J = 1.4 Hz, 1H) ppm. C NMR (75
MHz, DMSO-d ): δ = 182.8, 159.4, 146.1 (d, J = 247.4 Hz), 143.8
d, J = 3.0 Hz), 137.4 (d, J = 13.7 Hz), 121.4 (d, J = 18.8
Hz), 119.7 (d, J = 3.9 Hz), 117.2 (d, J = 3.1) ppm. F NMR
H,H
1
6
C,F
3
2
2
(
C,F
C,F
C,F
19
3
4
chromatography (silica gel, 40% EtOAc in cyclohexane) to afford a
C,F
C,F
1
yellow waxy solid (95 mg, 82% yield). H NMR (300 MHz, CDCl ): δ
3
(
282 MHz, DMSO-d ): δ = −132.9 (s, 1F) ppm. HRMS (ESI+,
6
3
4
4
+
+
= 7.85 (dd, JH,F = 11.2 Hz, JH,H = 1.5 Hz, 1H), 7.85 (d, JH,H = 1.5
MeOH): m/z = 289.9511 [M + Na] , 321.9769 [M + Na + MeOH] ;
3
5
Hz, 1H), 3.91 (td, J = 7.4 Hz, J = 1.6 Hz, 2H), 3.82−3.73 (m,
H,H
H,F
calcd. 289.9511 for C H FNO SNa + Na, 321.9774 for
8
3
5
2
3
1
H), 3.56 (dd, J = 9.5 Hz, J = 3.9 Hz, 1H), 3.48−3.37 (m, 1H),
H,H
H,H
C H FNO SNa + Na + CH OH. HRMS (ESI-, MeOH): m/z =
8
3
5
3
−
−
3.43−3.35 (m, 1H), 3.36 (s, 3H), 3.22−3.10 (m, 1H), 2.03−1.64 (m,
2
43.9725 [M − Na] , 275.9988 [M − Na + MeOH] , calcd. 243.9710
for C H FNO S, 275.9973 for C H FNO S + CH OH.
3
4
J
H), 1.81−1.64 (m, 2H), 1.42 (sextet, J = 7.3 Hz, 2H), 0.98 (t,
13
H,H
8
3
5
8
3
5
3
3
= 7.3 Hz, 3H) ppm. C NMR (75 MHz, CDCl ): δ = 181.4 (d,
3
H,H
4
.6.2. 5-Chlorosulfonyl-7-fluoroisatin (36). A stirred suspension of
4J = 3.0 Hz), 157.5, 147.2 (d, J = 253.1 Hz), 140.5 (d, J = 8.8
1
2
sodium 7-fluoroisatin-5-sulfonate (35) (2.00 g, 7.49 mmol, 1.00 equiv)
C,F
C,F
C,F
3
2
Hz), 134.7 (d, J = 4.1 Hz), 125.5 (d, J = 22.8 Hz), 120.3 (d,
in sulfolane (15 mL) was treated dropwise with POCl (3.07 mL, 33.7
C,F
C,F
3
4
3
JC,F = 3.4 Hz), 120.0 (d, J = 2.8 Hz), 74.7, 59.3, 59.1, 49.4, 42.7 (d,
mmol, 4.50 equiv) at 40 °C over 10 min. The reaction mixture was
then heated at 60 °C for 4 h followed by cooling to 0 °C and very slow
quenching with ice−water (30 mL). The mixture was stirred at 0 °C
for 30 min, filtered, and washed with a small amount of water. The
C,F
4JC,F = 4.8 Hz), 30.8 (d, JC,F = 2.6 Hz), 28.8, 24.2, 19.9, 13.6 ppm.
5
19
F
NMR (282 MHz, CDCl ): δ = −130.7 (s, 1F) ppm. HRMS (ESI+,
3
+
+
MeOH): m/z = 421.1197 [M + Na] , 453.1456 [M + Na + MeOH] ;
J
dx.doi.org/10.1021/jm500718e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
calcd. 421.1204 for C H FN O S + Na, 453.1466 for C H FN O S
10% CH CN in water (0.1% TFA) over 2 min, with λ = 254 nm and a
18
23
2
5
18 23
2
5
3
−
1
+
Na + MeOH.
. 6 . 5 . ( S ) - N - ( 4 - F l u o r o b u t y l ) - 7 - fl u o r o - 5 - [ 1 - ( 2 -
methoxymethylpyrrolidinyl)sulfonyl]isatin (39). (S)-7-Fluoro-5-[1-
2-methoxymethylpyrrolidinyl)sulfonyl]isatin (37) (100 mg, 0.292
mmol, 1.00 equiv) was converted to (S)-N-(4-fluorobutyl)-7-fluoro-5-
1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (39) by stirring with
flow rate of 1.0 mL·min . The recorded data of both HPLC-systems
4
were processed by the GINA Star software (Raytest Isotopenmessg-
18
̈
erate GmbH). No-carrier-added aqueous [ F]fluoride was produced
(
on a RDS 111e cyclotron (CTI-Siemens) by irradiation of a 2.8 mL
water target using 10 MeV proton beams on 97.0% enriched
1
8
18
18
[
[ O]H O by the O(p,n) F nuclear reaction. After the production
2
18
Cs CO3 (190 mg, 0.584 mmol, 2.00 equiv) and 1-bromo-4-
of [ F]fluoride, the 2.8 mL water target was unloaded. To remove
residual [ F]fluoride activity from the target, it was rinsed with 2.0 mL
2
18
fluorobutane (314 μL, 2.92 mmol, 10.00 equiv) in dry DMF (4.0
mL) at 95 °C for 10 min under microwave irradiation as described in
the general procedure (section 4.2). The residue was purified by flash
column chromatography (silica gel, 50% EtOAc in cyclohexane) to
of water. These rinsing water batches were used for the radiosyntheses.
18
4.9. Radiosynthesis of [ F]39 by Isotopic Exchange. In a
computer controlled TRACERLab Fx Synthesizer the batch of
FDG
1
18
afford a yellow waxy solid (95 mg, 78% yield). H NMR (400 MHz,
aqueous [ F]fluoride ions (336−12780 MBq) from the rinsing water
was passed through an anion exchange resin (Sep-Pak Light Waters
Accell Plus QMA cartridge, preconditioned with CO
ions). [ F]Fluoride ions were eluted from the resin with a mixture of
3
4
CDCl ): δ = 7.86 (dd, J = 11.1 Hz, J = 1.5 Hz, 1H), 7.85 (d,
3
H,F
H,H
4
2
3
2−
J
(
= 1.5 Hz, 1H), 4.50 (dt, J = 47.6 Hz, J = 5.6 Hz, 2H), 3.97
as counter-
H,H
3
H,F
H,H
3
2
18
t, J = 6.7 Hz, 2H), 3.81−3.73 (m, 1H), 3.55 (dd, J = 9.5 Hz,
H,H
H,H
3
2
3
JH,H = 3.9 Hz, 1H), 3.47−3.38 (m, 1H), 3.40 (dd, J = 9.8 Hz, J
36 μL of 1 M K CO , 200 μL of water for injection, and 800 μL of
H,H
H,H
2
3
=
2.5 Hz, 1H), 3.36 (s, 3H), 3.21−3.12 (m, 1H), 2.02−1.64 (m, 8H)
DNA-grade CH CN containing 23.8 mg (63 μmol) of Kryptofix2.2.2
3
13
4
18
ppm. C NMR (100 MHz, CDCl ): δ = 181.2 (d, J = 3.1 Hz),
(K222) in the reactor. Subsequently, the aqueous K[ F]F/K222
solution was carefully evaporated to dryness in vacuo. An amount of
2.1 mg (5.3 μmol) of precursor 39 in 0.5 mL of dry DMF was added,
and the mixture was heated at 130 °C for 10 min. After cooling to 55
3
C,F
1
2
1
57.5, 147.2 (d, J = 252.9 Hz), 140.3 (d, J = 8.8 Hz), 135.0 (d,
C,F C,F
3
2
4
JC,F = 4.1 Hz), 125.5 (d, J = 22.7 Hz), 120.3 (d, J = 3.4 Hz),
C,F
C,F
3
1
1
20.0 (d, J = 2.8 Hz), 83.2 (d, J = 165.5 Hz), 74.7, 59.4, 59.1,
C,F C,F
4
2
°
C, 10 mL of water was added and the mixture was passed through a
4
2.4 (d, J = 4.8 Hz), 49.3, 28.8, 27.5 (d, J = 20.2 Hz), 25.0 (dd,
JC,F = 4.3 Hz, J = 2.7 Hz), 24.2 ppm. F NMR (282 MHz,
C,F
C,F
19
3
5
Waters Sep-Pak C18 Light cartridge (preconditioned with 10 mL of
ethanol and 10 mL of water). The cartridge was washed with 10 mL of
water and eluted with 0.5 mL of DMF that was preheated to 90 °C.
After addition of 0.5 mL of water, the raw product solution was
purified by gradient-radio-HPLC-system A (method A). The product
C,F
CDCl ): δ = −131.0 (s, 1F), −219.5 (s, 1F, CH F) ppm. HRMS (ESI
3
2
+
+
, MeOH): m/z = 439.1108 [M + Na] , 471.1368 [M + Na +
MeOH] ; calcd. 439.1110 for C H F N O S + Na, 471.1372 for
+
18
22
2
2
5
C H F N O S + Na + MeOH.
18
22
2
2
5
18
18
fraction of compound [ F]39 (retention time t ([ F]39) = 10.47
4.7. In Vitro Enzyme Inhibition Assay. The inhibitory activities
R
min) was evaporated to a volume of 40 μL in vacuo at <50 °C. Product
toward caspases of the target isatin sulfonamides 10−25, 31−33, and
18
compound [ F]39 was obtained in a radiochemical yield (r.y.) of
3
7−39 are determined as IC values. The recombinant human
50
1
8
≤
(
0.2% (decay-corrected based on cyclotron-derived [ F]fluoride ions
caspases-1, -3, -6, and -7, including their peptide-specific substrate Ac-
YVAD-AMC (Ac-Tyr-Val-Ala-Asp-AMC) for caspase-1, Ac-DEVD-
AMC (Ac-Asp-Glu-Val-Asp-AMC) for caspases-3 and -7, and Ac-
VEID-AMC (Ac-Val-Glu-Ile-Asp-AMC) for caspase-6, were purchased
d.c.)) in 93 min. The target compound was isolated in radiochemical
purities of >99%. The radiochemical identity of [ F]39 was proven by
coinjection and coelution of [ F]39 and 39 on analytical radio-HPLC
18
18
5,11
B (method B, retention time t = 11.38−11.67 min). The
from Alexis Biochemicals (Switzerland). As already described,
R
18
radiochemical purities of [ F]39 were determined with the same
reaction rates showing inhibitory potencies of the inhibitors were
assessed by measuring the accumulation of the cleaved fluorogenic
product 7-amino-4-methylcoumarin (AMC) with a Fusion universal
microplate analyzer (PerkinElmer) at excitation and emission
wavelengths of 360 and 460 nm, respectively. All assays were
HPLC-system and -method.
18
4
.10. Stability Test of the Radiotracer [ F]39 in Human
18
Serum. The serum stability of radioligand [ F]39 was evaluated by
incubation in human serum at 37 °C for up to 90 min. An aliquot of
18
5
[ F]39 (20 μL, 5.8 MBq) was added to a sample of human serum
performed at a volume of 200 μL at 37 °C in reaction buffer. Buffers
(
200 μL), and the mixture was incubated at 37 °C. Samples of 20 μL
contained the target compounds 10−25, 31−33, and 37−39 in
each were taken after periods of 10, 30, 60, and 90 min and quenched
DMSO (1−5%) in single doses (end concentrations 500 μM, 50 μM,
in MeOH/CH Cl (1:1 (v/v), 100 μL) followed by centrifugation for
2
2
5
μM, 500 nM, 50 nM, 5 nM, 500 pM, 50 pM, or 5 pM). Recombinant
2
min. The clear solution was analyzed by analytical radio-HPLC.
caspases were diluted into the appropriate buffer to a concentration of
.5 units per assay (=500 pmol substrate conversion after 60 min).
0
ASSOCIATED CONTENT
After 10 min incubation time, the peptide substrates (end
concentration 10 μM) were added and reacted for further 10 min.
The IC50 values were determined by nonlinear regression analysis
using the XMGRACE program (Linux software).
■
*
S
Supporting Information 1
Radio-HPLC chromatograms, H, 13C, 19F NMR spectral data
1
13
19
with assignment of signals and copies of H, C, F NMR
spectra of all inhibitors. This material is available free of charge
via the Internet at http://pubs.acs.org.
4
.8. General Procedure for Radiochemistry. The radio-
syntheses were carried out on a modified PET tracer radiosynthesizer
TRACERLab FxFDG, GE Healthcare). The recorded data were
(
processed by the TRACERLab Fx software (GE Healthcare).
Separation and purification of the radiosynthesized compounds were
performed on the following semipreparative radio-HPLC-system A: K-
AUTHOR INFORMATION
Corresponding Author
Phone: +49 251 83 33281. Fax: +49 251 83 39772. E-mail:
haufe@uni-muenster.de.
Present Addresses
(P.L.) Department of Chemistry, Faculty of Science,
Silpakorn University, Sanam Chandra Palace Campus, Nakhon
Pathom 73000, Thailand.
■
5
00 and K-501 pump, K-2000 UV detector (Herbert Knauer GmbH),
*
NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec), and an ACE 5 AQ
column (250 mm × 10 mm). Method A started with a linear gradient
from 30% to 90% CH CN in water (0.1% TFA) over 30 min, holding
3
∇
for 5 min, followed by a linear gradient from 90% to 30% CH CN in
3
water (0.1% TFA) over 5 min, with λ = 254 nm and a flow rate of 5.5
−
1
mL·min . Radiochemical purities were determined using the
analytical radio-HPLC-system B: Two Smartline 1000 pumps and a
Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-
●
(
K.K.) Division of Radiopharmaceutical Chemistry, German
Cancer Research Center, Im Neuenheimer Feld 280, D-69120
Heidelberg, Germany.
̈
detector (Raytest Isotopenmessgerate GmbH), and a Nucleosil 100-5
C-18 column (250 mm × 4 mm). Method B started with a linear
gradient from 10% to 100% CH CN in water (0.1% TFA) over 15
Notes
3
min, holding for 3 min, followed by a linear gradient from 100% to
The authors declare no competing financial interest.
K
dx.doi.org/10.1021/jm500718e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
sulfonamide analogues as potent caspase-3 inhibitors: Synthesis, in
vitro activity, and molecular modeling studies. J. Med. Chem. 2005, 48,
ACKNOWLEDGMENTS
Financial support by the Deutsche Forschungsgemeinschaft
Collaborative Research Center, SFB 656, projects B1, A3, and
Z5), the International Graduate School of Chemistry Mu
GSC-MS), and the Development and Promotion of Science
and Technology talent project (DPST), Thailand, is gratefully
acknowledged. We thank Sandra Schroer and Wiebke
Gottschlich, European Institute for Molecular Imaging
EIMI), for executing the enzyme inhibition assays and Mark
■
7
(
637−7647.
(
12) Smith, G.; Glaser, M.; Perumal, M.; Nguyen, Q. D.; Shan, B.;
̈
nster
Arstad, E.; Aboagye, E. O. Design, synthesis, and biological
characterization of a caspase 3/7 selective isatin labeled with 2-
(
18
[ F]fluoroethylazide. J. Med. Chem. 2008, 51, 8057−8067.
(13) Jiang, Y.; Hansen, T. V. Isatin 1,2,3-triazoles as potent inhibitors
against caspase-3. Bioorg. Med. Chem. Lett. 2011, 21, 1626−1629.
̈
(
̈
14) Podichetty, A. K.; Wagner, S.; Faust, A.; Schafers, M.; Schober,
(
O.; Kopka, K.; Haufe, G. Fluorinated isatin derivatives. Part 3. New
side-chain fluoro-functionalized pyrrolidinyl sulfonyl isatins as potent
caspase-3 and -7 inhibitors. Fut. Med. Chem. 2009, 1, 969−989.
P. Waller for the abstract image.
ABBREVIATIONS USED
■
(
̈
15) Limpachayaporn, P.; Schafers, M.; Schober, O.; Kopka, K.;
CD95, cell death receptor 95; DBH, N,N′-dibromo-5,5-
dimethylhydanthoin; d.c., decay corrected; DIPEA, diisopropy-
lethyl amine; EC/MS, electrochemistry/mass spectrometry;
K222, Kryptofix 222; MW, microwave irradiation; QC, quality
control; r.y., radiochemical yield; TEA, triethyl amine
Haufe, G. Synthesis of new fluorinated, 2-substituted 5-pyrrolidinyl-
sulfonyl isatin derivatives as caspase-3 and caspase-7 inhibitors:
Nonradioactive counterparts of putative PET-compatible apoptosis
imaging agents. Bioorg. Med. Chem. 2013, 21, 2025−2030.
̈
(16) Limpachayaporn, P.; Schafers, M.; Schober, O.; Riemann, B.;
Kopka, K.; Haufe, G. Influence of 4- or 5-substituents on the
pyrrolidine ring of 5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
derivatives on their inhibitory activities towards caspases-3 and -7. Eur.
J. Med. Chem. 2013, 64, 562−578.
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