Synthesis of new fluorinated, 2-substituted 5-pyrrolidinylsulfonyl isatin derivatives as caspase-3 and caspase-7 inhibitors: Nonradioactive counterparts of putative PET-compatible apoptosis imaging agents

Article abstract of DOI:10.1016/j.bmc.2013.01.011

Downstream caspases-3 and -7 are essential to execute the programmed type I cell death (apoptosis). In order to better understand their role, specific inhibitors of these enzymes are required, which after radiolabeling can be applied to non-invasively visualize and monitor apoptotic pathways in vivo using Positron Emission Tomography (PET). Therefore, 2-methoxyethyl-, 2-methoxypropyl-, 2-ethoxymethyl-, 2-(2-fluoroethoxymethyl)-, and 2-(2,2,2-trifluoroethoxymethyl)pyrrolidinyl analogues of (S)-5-[1-(2- methoxymethylpyrrolidinyl)sulfonyl]isatin (2) were prepared and their in vitro binding affinities towards caspases-1, -3, -6 and -7 were evaluated and compared to that of the lead structure 2. While the inhibition potencies against caspases-1 and -6 were in the micromolar range, all synthesized compounds exhibited excellent and selective inhibition of caspases-3 and -7 in the nanomolar range up to IC₅₀ = 4.79 nM and 7.47 nM, respectively. These highly potent 2-substituted analogues of 2 might be developed as anti-apoptosis agents and some selected fluorinated inhibitors might be useful as potential PET radiotracers for apoptosis imaging after ¹⁸F-labeling.

Full text of DOI:10.1016/j.bmc.2013.01.011

Bioorganic & Medicinal Chemistry 21 (2013) 2025–2036
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new fluorinated, 2-substituted 5-pyrrolidinylsulfonyl
isatin derivatives as caspase-3 and caspase-7 inhibitors:
Nonradioactive counterparts of putative PET-compatible
apoptosis imaging agents
Panupun Limpachayaporn a,b, Michael Schäfers c, Otmar Schober d, Klaus Kopka d, Günter Haufe a,c,
⇑
a Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany
b International NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Straße 10, D-48149 Münster, Germany
c European Institute for Molecular Imaging (EIMI), Westfälische Wilhelms-Universität Münster, Mendelstraße 11, D-48149 Münster, Germany
d Klinik für Nuklearmedizin, Universitätsklinikum Münster, Albert-Schweitzer-Campus 1, Gebäude A1, 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:
Downstream caspases-3 and -7 are essential to execute the programmed type I cell death (apoptosis). In
order to better understand their role, specific inhibitors of these enzymes are required, which after radio-
labeling can be applied to non-invasively visualize and monitor apoptotic pathways in vivo using
Positron Emission Tomography (PET). Therefore, 2-methoxyethyl-, 2-methoxypropyl-, 2-ethoxymethyl-,
2-(2-fluoroethoxymethyl)-, and 2-(2,2,2-trifluoroethoxymethyl)pyrrolidinyl analogues of (S)-5-[1-(2-
methoxymethylpyrrolidinyl)sulfonyl]isatin (2) were prepared and their in vitro binding affinities
towards caspases-1, -3, -6 and -7 were evaluated and compared to that of the lead structure 2. While
the inhibition potencies against caspases-1 and -6 were in the micromolar range, all synthesized com-
pounds exhibited excellent and selective inhibition of caspases-3 and -7 in the nanomolar range up to
IC50 = 4.79 nM and 7.47 nM, respectively. These highly potent 2-substituted analogues of 2 might be
developed as anti-apoptosis agents and some selected fluorinated inhibitors might be useful as potential
PET radiotracers for apoptosis imaging after 18F-labeling.
Received 18 November 2012
Revised 8 January 2013
Accepted 9 January 2013
Available online 16 January 2013
Keywords:
Apoptosis
Caspase inhibitor
Isatin sulfonamide
Fluorinated pyrrolidine
Radiotracer
Positron Emission Tomography (PET)
Molecular imaging
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
death receptor (death receptor pathway) or by DNA damage fol-
lowed by mitochondrial induction of the cell death program (mito-
Apoptosis or the programmed type I cell death is a complex reg-
ular mechanism to get rid of excess, potentially dangerous and
damaged cells leading to apoptotic bodies which become cleared
without activation of the immune system. Apoptosis plays a vital
role in physiological cellular processes such as control of shape
and size of tissue, cell homeostasis, fetal development and mainte-
nance of the immune system. Dysregulation of apoptosis is in-
chondrial pathway). Signals from both paths cumulate in the
activation of executing caspases driving the apoptotic process.1 It
was proven that inhibition of the executing caspases-3 and -7
slows down or even stops apoptosis.2 Therefore, targeting execut-
ing caspases might be a sustainable approach for prevention, ther-
apy and monitoring of diseases where apoptosis is involved.
Isatin sulfonamides were discovered by screening methods and
described as potent, non-peptide, selective inhibitors of caspases-3
and -7. Superior to peptide-based inhibitors, this class of com-
pounds not only possesses higher metabolic stability and cell per-
meability, but it also differentiates apoptosis from necrosis, since
caspases-3 and -7 are only involved in apoptosis.2 The X-ray co-
crystal study of human recombinant caspase-3 and the inhibitor
(S)-N-methyl-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin
(1) revealed their configuration and binding modes. Cysteine163 of
the enzyme binds covalently at the 3-carbonyl group of the isatin
to form a tetrahedral thiohemiacetal intermediate.2 Therefore,
modification at this position generally leads to a decrease or loss
of biological activity,3 except the substitution by Michael addition
acceptors which leads to irreversible caspase inhibitors.4,5
volved in
a
wide variety of diseases, such as cancer,
neurodegenerative and developmental defects, atherosclerosis,
autoimmune and cardiovascular diseases including Alzheimer’s,
Parkinson’s and Huntington’s diseases.1 The apoptotic pathway is
strictly controlled by a set of intracellular enzymes called caspases
(cysteinyl aspartate-specific proteinases). In the human being, over
a dozen of caspases have been discovered and classified as initiat-
ing (e.g., caspases-8, -9 and -10) and executing caspases (e.g., casp-
ases-3, -6 and -7). In principle there exist two biological pathways
that initiate apoptosis either by a specific protein binding to the
⇑
Corresponding author. Tel.: +49 251 83 33281; fax: +49 251 83 39772.
E-mail address: haufe@uni-muenster.de (G. Haufe).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.011

2026
P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
executing caspases-3 and -7.6 Therefore, (S)-5-[1-(2-meth-
oxymethylpyrrolidinyl)sulfonyl]isatin (2) was chosen as lead com-
pound for further developments towards apoptosis imaging
agents.
cysteine163
binding site
O
S2-pocket
O O
S
O
N
N
Recently, we and others have published applications of N-ben-
zyl derivatives of isatin sulfonamides as PET radiotracers for spe-
cific molecular imaging of apoptosis.9–11 However, our recent
metabolite identification study using electrochemical oxidation
coupled to liquid chromatography/mass spectrometry analysis
(EC-LC/MS) proved oxidative metabolism leading in part to the
cleavage of the benzyl group from the isatin core and secession
of the methoxymethyl group from the pyrrolidine part.12 There-
fore, 18F-labeling at the N-benzyl group could lead to a loss of
the radiolabel if applied in vivo resulting in a poor PET signal in
the target tissue. To overcome these limitations, we envisioned
an N-substitution by a saturated alkyl chain and replacement of
the methoxymethyl group of the pyrrolidine ring with longer
chains including fluorinated ones, which are expected to improve
their metabolic stability without loss of caspase inhibition potency.
We found that substituent variations, for instance replacement
of the 2-methoxymethyl group of the pyrrolidine moiety with a tri-
R2
R1O
S1-pocket
S3-pocket
1 (R1 = Ph, R2 = Me)
2 (R1 = Me, R2 = H)
Figure 1. General structure of isatin sulfonamides with assigned interactions with
enzyme’s sub-pockets.
O O
S
O O
S
O
O
N
N
O
O
N
N
H
H
MeO
PhO
2
3
fluoromethoxymethyl,
a methoxydifluoromethyl, a trifluoro-
IC50 (caspase-3): 84.9 nM
IC50 (caspase-7): 1290 nM
IC50 (caspase-3): 16.9 nM
IC50 (caspase-7): 294 nM
methyl,13 a phenoxymethyl,6,7,14 a fluorophenoxymethyl group,15
a triazole15 or a pyridine ring,5,14 still maintain high binding poten-
cies towards caspases-3 and -7. Thus, this position might be toler-
ant for chain elongation and introduction of fluorine(s).
Figure 2. Structure and caspase inhibition potencies (IC50) towards caspases-3 and
-7 of (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (2) and (S)-5-[1-(2-
phenoxymethylpyrrolidinyl)sulfonyl]isatin (3).2,6
Here, we report on the synthesis and evaluation of the in vitro
caspase inhibition potencies of the aforementioned pyrrolidine
side-chain modified isatin sulfonamide derivatives. In addition, in
all cases butyl and 4-fluorobutyl groups were attached at the isatin
nitrogen in order to compare the influence of the new substituents
with the 2-methoxymethyl parent compounds. 2-Methoxyethyl,
2-methoxypropyl, 2-ethoxymethyl, 2-(2-fluoroethoxymethyl) and
the 2-(2,2,2-trifluoroethoxymethyl) functionalities were attached
at the 2-position of the pyrrolidine ring. This study will allow us
to further explore the environment of the enzymes’ S3-pocket
and improvement of the pharmacological properties of this class
of non-peptide caspase inhibitors is expected. Moreover, we sug-
gest the new fluorinated potential inhibitors as non-radioactive
counterparts of putative PET radiotracers for apoptosis imaging.
Substituents at the isatin nitrogen may reach into the S1-pocket
of the enzyme, while the pyrrolidine moiety interacts with its
hydrophobic S2-pocket and substituents at the 2-position of the
pyrrolidine ring fit into the enzyme’s S3-pocket. Figure 1 represents
the general structure of isatin sulfonamides and interactions
with highlighted sub-pockets of the enzyme as described in the
literature.2
Compounds of this group attracted our interest some years ago
and (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (2) and
(S)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin (3) were
identified as lead compounds. Both molecules exhibited good inhi-
bition potencies towards caspases-3 and -7 in vitro as depicted in
Figure 2.2,6
2. Results and discussion
2.1. Chemistry
N-Substituted derivatives of both 2 and 3 showed even lower
IC50 values in vitro in the nanomolar to subnanomolar range.3,7,8
In most of the cases, the N-substituted derivatives of 2-phenoxy-
methyl isatin 3 indicated better inhibition potencies than those
of the 2-methoxymethyl version 2. In contrast, in vivo cell culture
studies revealed that N-substituted analogues of 2 have a signifi-
cantly higher potency than the 2-phenoxy series in inhibiting the
The structure of isatin-based inhibitors consists of the isatin
core and a pyrrolidine ring bridged by a sulfonyl group. This class
of compounds was prepared by coupling of 5-chlorosulfonyl isatin
O O
S
O
Cl
O
N
H
O O
S
O O
S
O
O
alkyl bromides
Cs2CO3, DMF
5-chlorosulfonyl isatin
N
N
+
O
O
MW 95 °C
10 min
N
N
Boc
H
N
R2
R2 = n-butyl, 4-fluorobutyl
R1
2
R1
(R1 = OMe)
R1
lead compound
2-substituted
N-Boc-pyrrolidines
Scheme 1. General synthesis of 2-substituted pyrrolidinyl sulfonyl isatin derivatives (MW = microwave irradiation).

P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
2027
O
ref. [18]
25%
BH3 SMe2
OH
MeI, NaH
THF
OH
OH
OMe
N
Boc
N
Boc
N
Boc
N
Boc
THF
57%
74%
4
5
6
7
O O
O
O
S
O
O
S
N
N
R2-Br, Cs2CO3
1. TFA
O
O
N
N
DMF, Microwave
2. 5-chlorosulfonyl
isatin, DIPEA
H
R2
OMe
OMe
62%
8
9 (R2 = n-Bu) 73%
10 (R2 = 4-F-Bu) 99%
Scheme 2. Synthesis of N-Boc-
inyl)sulfonyl]isatins (10).
L-homoprolinol methyl ether (7) and subsequent preparation of (S)-N-butyl- (9) and (S)-N-(4-fluorobutyl)-5-[1-(2-methoxyethylpyrrolid-
OH
OH
OMe
Boc2O, TEA
DCM
NaH, MeI
THF
100%
N
H
N
Boc
N
Boc
82%
11
12
13
O O
O O
S
O
O
S
N
N
R2-Br, Cs2CO3
1. TFA
O
O
N
N
2. 5-chlorosulfonyl
isatin, DIPEA
DMF, Microwave
H
R2
OMe
OMe
15 (R2 = n-Bu) 73%
16 (R2 = 4-F-Bu) 90%
82%
14
Scheme 3. Synthetic pathway towards rac-N-butyl- (15) and rac-N-(4-fluorobutyl)-5-[1-(2-methoxypropylpyrrolidinyl)sulfonyl]isatins (16).
and 2-substituted N-Boc-pyrrolidines under basic conditions simi-
lar to the synthesis described for the lead structure 2.7 According to
findings of previous work,3,6–8 the analogues were subsequently
N-substituted with alkyl or fluoroalkyl groups in order to enhance
their inhibition potencies. The general synthetic plan is depicted in
Scheme 1.
The preparation of 5-chlorosulfonyl isatin was smoothly accom-
plished starting from commercial available isatin in two steps, that
is, sulfonation using oleum and chlorination with POCl3.7 The pyr-
were inserted between the ring and the oxygen atom leading to
2-methoxyethyl (8, 9, 10) and 2-methoxypropyl derivatives (14,
15, 16). The synthetic sequences are illustrated in Schemes 2 and 3.
The 2-methoxyethyl isatin derivatives were prepared by cou-
pling of 5-chlorosulfonyl isatin and N-Boc-
ether (7). The preparation of the essential pyrrolidine component
7 was accomplished in several steps starting from N-Boc- -prolinol
L-homoprolinol methyl
L
(4) as shown in Scheme 2. Conversion of 4 to the acid 5 was per-
formed smoothly in four steps as described by Andries.18 Applying
a protocol by Hermet,19 the chain elongation was successful by
mesylation followed by substitution with cyanide. Acidic hydroly-
sis of the nitrile was accompanied by Boc-deprotection. Therefore,
the resulting free amine had to be re-protected with Boc resulting
in the carboxylic acid 5. Subsequent reduction of 5 was performed
using BH3ÁSMe2 to obtain the desired alcohol 6 in satisfactory yield.
Finally, the alcohol was methylated using MeI and NaH to furnish
the methyl ether 7 in good yield.
To obtain the isatin sulfonamide inhibitors 9 and 10, the ether 7
was Boc-deprotected using TFA and the formed amine was coupled
to 5-chlorosulfonyl isatin in the presence of DIPEA in 62% yield
(over two steps). N-Alkylation of obtained 8 was successful using
1-bromobutane and 1-bromo-4-fluorobutane in the presence of
Cs2CO3 under microwave irradiation to yield (S)-N-butyl- (9) and
(S)-N-(4-fluorobutyl)-5-[1-(2-methoxyethylpyrrolidinyl)sulfo-
nyl]isatins (10) in 73% and 99% yields, respectively (Scheme 2).
The racemic bishomoproline methyl ether (13) was prepared in
two steps commencing from racemic bishomoprolinol (11). Boc-
protection of 11 using Boc2O and TEA led to N-Boc-bishomoprolinol
rolidine part was synthesized starting from N-Boc-L-prolinol (4)
using different routes or bishomoprolinol (11) depending on the
desired substituents. Prior to coupling, 2-substituted N-Boc-pyrrol-
idines were first deprotected under acidic conditions using TFA in
DCM followed by reaction with 5-chlorosulfonyl isatin in the pres-
ence of DIPEA to obtain the corresponding isatin-based inhibitors.7
Subsequent N-alkylation of the isatin, especially with n-butyl and
4-fluorobutyl groups, enhanced the inhibition potencies of the
compounds as proved earlier.3 The incorporation of these substit-
uents was performed using 1-bromobutane or 1-bromo-4-fluo-
robutane in DMF in the presence of Cs2CO3. The advantages of
microwave irradiation16 and the effect of the cesium ion17 were
exploited to elevate the reaction, nevertheless resulting in good
to excellent yields.
In order to explore the environment of the S3-pocket of the tar-
get enzymes, the 2-position of the pyrrolidine ring was modified by
chain length elongation and their structure–activity relationships
were evaluated compared to the lead compound 2. Instead of the
2-methoxymethyl chain, one and two more methylene groups

2028
P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
O
O
O O
S
O
O
R2-Br
R3
NaH
S
X
R3
N
N
Cs2CO3
1. TFA
O
O
OH
O
N
N
N
Boc
N
Boc
2. 5-chlorosulfonyl
isatin, DIPEA
DMF
Microwave
DMF
X = Br, OTs
H
R2
OCH2R3
18 (R3 = CH3) 80%
OCH2R3
17 (R3 = CH3) 61%
19 (R2 = n-Bu) 76%
4
(R2 = 4-F-Bu) 77%
23 (R2 = n-Bu) 91%
24
20
21 (R3 = CH2F) 69%
25 (R3 = CF3) 69%
22 (R3 = CH2F) 68%
(R2 = 4-F-Bu) 89%
27 (R2 = n-Bu) 85%
28
3
26
(R = CF3) 59%
(R2 = 4-F-Bu) 84%
Scheme 4. Synthesis of fluorinated N-butyl- (19, 23, 27) and N-(4-fluorobutyl)-5-[1-(2-ethoxymethylpyrrolidinyl)sulfonyl]isatins (20, 24, 28).
(12) in 82% yield, after methylation the desired ether 13 was
obtained quantitatively (Scheme 3).
isatins expressed superior inhibition potencies for caspases-3 and
-7 in the nanomolar range down to IC50 = 4.79 nM and 7.47 nM,
respectively. Notably, the 2-methoxypropyl derivatives 14–16
belong to the weakest inhibitors within this class, despite consid-
ering its racemic nature. We also found that, in the majority of
cases, the N-alkylated compounds were significantly more affine
than the N–H parents, in which, with exception of compounds 20
and 28, the affinities for the N-butyl compounds are comparable
with those of the N-4-fluorobutyl analogues. Since the environ-
ments of the S2-pocket of caspases-3 and -7 are very similarly
surrounded by Tyr204, Trp206 and Phe256, poor selectivity was
observed for most of the new compounds, except for (S)-5-[1-(2-
After Boc-deprotection and coupling with the isatin component,
the inhibitor 14 was synthesized in 82% yield (over two steps).
N-Butylation and N-fluorobutylation of 14 using the appropriate
bromides and Cs2CO3 under microwave irradiation generated
rac-N-butyl- (15) and rac-N-(4-fluorobutyl)-5-[1-(2-meth-
oxypropylpyrrolidinyl)sulfonyl]isatins (16) in 73% and 90% yields,
respectively (Scheme 3).
Additional modifications at the 2-position of the pyrrolidine
moiety were derived from N-Boc-L-prolinol (4). The 2-methoxy-
methyl group in the lead compound was replaced by ethoxy-
methyl, fluoroethoxymethyl and trifluoroethoxymethyl functions.
This provides the possibility to introduce fluorine(s) into the
molecule, which delivers the non-radioactive counterparts of puta-
tive 18F-labeled radiotracers. The substitutions were performed
smoothly in one step using ethyl bromide, 1-fluoro-2-tosylethane
and 1,1,1-trifluoro-2-tosylethane under basic conditions in
DMF to obtain (S)-N-Boc-2-ethoxymethyl- (17), which is a
ethoxyethylpyrrolidinyl)sulfonyl]isatin (18) showing
a 85-fold
selectivity for caspase-7, which was 5.7 times more selective for
caspase-7 than the lead compound 2.
3. Conclusion
2-Methoxyethyl-, 2-methoxypropyl-, 2-ethoxymethyl-, 2-(2-
fluoroethoxymethyl)- and 2-(2,2,2-trifluoroethoxymethyl)pyrro-
lidinyl sulfonyl isatin derivatives were synthesized and their
caspase inhibition potencies for caspases-1, -3, -6 and -7 were
evaluated. Most of the synthesized compounds were inactive
against caspase-6, while their activities towards caspase-1, unex-
pectedly, were proven to be in the two-digit micromolar scale.
Anyhow, the chosen modifications of the pyrrolidine 2-methoxy-
methyl group maintained the characteristic inhibition properties
of 2 and other isatin sulfonamides towards caspases-3 and -7 in
the nanomolar range. In most cases even a slight improvement
of activities was observed. These findings confirm that longer
side chains at the 2-position, including fluorinated ones, do
interact efficiently with the environment of the corresponding
enzymes’ sub-pocket. Finally, the new fluorinated isatin sulfona-
mides 10, 16, 23 and 24 can be suggested as potent candidates
for 18F-labeling for application as PET radiotracers for apoptosis
imaging.
constitutional isomer of L-homoproline methyl ether (7), (S)-N-
Boc-2-(2-fluoroethoxymethyl)- (21) and (S)-N-Boc-2-(2,2,2-triflu-
oroethoxymethyl)pyrrolidines (25) in 61–69% yields, respectively
(Scheme 4).
After connecting the unprotected pyrrolidines 17, 21 and 25
with 5-chlorosulfonyl isatin, the potential inhibitors (S)-5-[1-
(2-ethoxymethylpyrrolidinyl)sulfonyl]isatin (18), (S)-5-{1-[2-
(2-fluoroethoxymethyl)pyrrolidinyl]sulfonyl}isatin (22) and
(S)-5-{1-[2-(2,2,2-trifluoroethoxymethyl)pyrrolidinyl]sulfonyl}-
isatin (26) were formed in 80%, 68% and 59% yields, respectively.
The n-butyl and 4-fluorobutyl substitutions of the resulting inhib-
itors 18, 22 and 26 were accomplished using 1-bromobutane and
1-bromo-4-fluorobutane in the presence of Cs2CO3 and DMF under
microwave irradiation. The expected N-butyl and N-4-fluorobutyl
derivatives 19, 20, 23, 24, 27 and 28 were obtained in 76–91%
yields as given in Scheme 4.
2.2. Caspase inhibition potencies
All synthesized target isatin sulfonamides were tested in vitro
as inhibitors of caspases-1, -3, -6 and -7. The resulting inhibition
potencies are listed in Table 1.
4. Experimental section
4.1. Materials and methods
The lead compound 2 is a selective inhibitor of caspases-3 and
-7 as displayed by IC50-values of 84.9 nM and 1290 nM, respec-
tively,6 while it was much less active towards caspase-1 and inac-
tive towards caspase-6. Similarly, most of the compounds in the
present series exhibited quite weak activities towards caspase-1
and two of them (10 and 19) also towards caspase-6 in the micro-
molar range. The best result for caspases-1 and -6 inhibitory poten-
All chemicals, reagents and solvents for reactions were analyti-
cal grade and used as delivered without further purification. 1-Bro-
mo-4-fluorobutane was purchased from Apollo Scientific, United
Kingdom. rac-Bishomoprolinol (11) was a gift of Dr. Ivan Kondra-
tov, National Ukrainian Academy of Sciences, Kiev. Concerning
reactions under dry conditions, glassware was heated under
vacuum and flushed with argon gas prior to use. The reactions
were performed under argon atmosphere. To characterize the
cies has been found for compound 10 with IC50 = 7.34
lM and
1.03 M, respectively. The 2-substituted pyrrolidinyl sulfonyl
l

P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
2029
Table 1
Caspase inhibition potencies of the synthesized isatin sulfonamides towards caspases-1, -3, -6 and -7 expressed as IC50-values (nM)
O O
S
O
N
O
N
R2
R1
Inhibitor
R1
R2
Yield (%)
Caspase inhibition potencies IC50 (nM)
Caspase-1
Caspase-3
Caspase-6
Caspase-7
26
36
8
OMe
OPh
H
H
H
—
—
>15,000
>10,000
84.9 25.6
16.9 7.7
>500,000
>500,000
>500,000
>500,000
1030 1020
>500,000
>500,000
>500,000
>500,000
2610 2540
>500,000
>500,000
>500,000
>500,000
>500,000
>500,000
>500,000
1290 466
294 178
540 133
24.6 13.1
12.3 6.6
125 34
CH2OMe
CH2OMe
CH2OMe
CH2CH2OMea
CH2CH2OMea
CH2CH2OMea
OEt
62
73
99
82
73
90
80
76
77
68
91
89
59
85
84
12,100 2330
15,500 1040
7340 3760
11,600 2780
>500,000
>500,000
>500,000
18,400 649
>500,000
18,400 3710
24,700 824
45,800 23,700
14,700 881
25,900 3460
30,700 3820
349
9
9
n-Butyl
4-Fluorobutyl
H
n-Butyl
4-Fluorobutyl
H
n-Butyl
4-Fluorobutyl
H
n-Butyl
4-Fluorobutyl
H
94.3 15.2
92.3 38.6
1580 392
10
14
15
16
18
19
20
22
23
24
26
27
28
170
2
515 55
184 18
33.7 4.0
13.2 5.7
64.6 15.0
218 91
1330 100
39.6 15.7
9.68 0.26
1420 259
7.47 2.09
265 42
1120 78
25.6 1.4
365 99
346 167
4.79 0.33
74.3 27.7
1060 129
46.8 1.2
149 81
OEt
OEt
OCH2CH2F
OCH2CH2F
OCH2CH2F
OCH2CF3
OCH2CF3
OCH2CF3
n-Butyl
4-Fluorobutyl
a
Racemic mixture of (2R)- and (2S)-substituted pyrrolidinyl inhibitors.
compounds synthesized, melting point (if the products were solid
at rt), NMR and ESI-MS were exploited. 1H NMR (300 MHz and
400 MHz), 13C NMR (75 MHz and 100 MHz) and 19F NMR
(282 MHz) spectra were recorded by Bruker machines in CDCl3,
CD3CN or DMSO-d6 with TMS for 1H NMR, CDCl3, CD3CN or
DMSO-d6 for 13C NMR and CFCl3 for 19F NMR as the internal stan-
dards. DEPT and two dimensional NMR techniques (COSY, HMQC
and HMBC) were recorded to characterize some complex struc-
tures. All chemical shifts were indicated in ppm. Exact mass anal-
50 lM, 5 lM, 500 nM, 50 nM, 5 nM, 500 pM, 50 pM, or 5 pM).
Recombinant caspases were diluted into the appropriate buffer to
a concentration of 0.5 units per assay (=500 pmol substrate conver-
sion after 60 min). After 10 min incubation time, the peptide
substrates (end concentration 10 lM) were added and reacted
for further 10 min. The IC50 values were determined by nonlinear
regression analysis using the XMGRACE program (Linux software).
4.3. General procedures
yses were recorded with
a Bruker MicroTOF apparatus. All
spectroscopic and analytical investigations were performed by
staff members of the Organic Chemistry Institute, University of
Münster. Thin layer chromatography (TLC) analyses were per-
formed 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. Reac-
tions under microwave irradiation were performed in sealed glass-
vessel using CEM Discovery microwave machine.
4.3.1. General procedure A: coupling of the pyrrolidine and
isatin moieties
A stirred solution of N-Boc protected pyrrolidine (1.00 mmol) in
dry DCM (2.2 mL) at 0 °C under argon atmosphere was treated
dropwise with TFA (10.00 mmol). Stirring was continued at 0 °C
for 30 min and at ambient temperature for 2 h. Subsequently, the
solution was poured to ice-cooled 10% aq NaOH (15 mL) and ex-
tracted with DCM (3 Â 15 mL). The combined organic phase was
dried over MgSO4 and concentrated in vacuo to obtain a colorless
oil as the corresponding unprotected pyrrolidine. After that a solu-
tion of the free pyrrolidine and DIPEA (2.00 mmol) in CHCl3
(1.1 mL) was added dropwise to a stirred solution of 5-chlorosulfo-
nyl isatin (1.50 mmol) in CHCl3/THF (1:1, 19 mL) at room temper-
ature. The resulting solution was stirred at room temperature for
1 h and the solvent was completely removed in vacuo. The crude
product was purified by flash column chromatography to obtain
the corresponding inhibitors.
4.2. In vitro enzyme inhibition assays
The inhibitory activities towards caspases of the target isatin
sulfonamides 8–10, 14–16, 18–20, 22–24 and 26–28 are indicated
as IC50 values. The recombinant human 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 from
AlexisÒ Biochemicals (Switzerland). As already described,6,14
reaction rates showing inhibitory potencies of the inhibitors were
assessed by measuring the accumulation of the cleaved fluorogenic
product AMC (7-amino-4-methylcoumarin) with a Fusion univer-
sal microplate analyzer (Perkin Elmer) at excitation and emission
wavelengths of 360 and 460 nm, respectively. All assays were
4.3.2. General procedure B: N-alkylation using microwave
irradiation
In a microwave compatible glass-vessel, a stirred solution of
isatin sulfonamides (1.00 mmol) dissolved in dry DMF (15 mL)
was treated with Cs2CO3 (2.00 mmol) under argon atmosphere at
room temperature. After stirring for 10 min, the yellow mixture
turned blue-purple. The alkylbromide (10.00 mmol) was added
slowly into the blue mixture. Subsequently the vessel was sealed
and the mixture was stirred at 95 °C for 10 min using microwave
performed at a volume of 200
fers contained the target compounds 8–10, 14–16, 18–20, 22–24
and 26–28 in DMSO in single doses (end concentrations 500 M,
l
L at 37 °C in reaction buffer.6 Buf-
l

2030
P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
3
5
irradiation (maximum power = 150 W). Then, the precipitate was
removed by filtration through CeliteÒ and the filtrate was concen-
trated in vacuo. Finally the residue was purified by flash column
chromatography to obtain the corresponding final N-substituted
isatin sulfonamides.
4-CH), 7.13 (dd, JH,H = 8.3 Hz, JH,H 0.6 Hz, 1H, 7-CH), 3.71–3.62
(m, 1H, 12-CH), 3.46–3.38 (m, 2H, 17-CH2), 3.38–3.29 (m, 1H, 15-
CHa), 3.28 (s, 3H, 19-CH3), 3.24–3.10 (m, 1H, 15-CHb), 2.09–1.97
(m, 1H, 16-CHa), 1.85–1.71 (m, 1H, 14-CHa), 1.71–1.57 (m, 1H,
16-CHb), 1.66–1.47 (m, 2H, 13-CH2), 1.54–1.41 (m, 1H, 14-CHb)
ppm. 13C NMR (100 MHz, CD3CN): d = 184.0 (3-CO), 159.9 (2-CO),
154.2 (8-CN), 138.3 (6-CH), 133.5 (5-CSO2), 124.8 (4-CH), 119.1
(9-CCO), 113.9 (7-CH), 70.3 (17-CH2), 59.4 (12-CH), 58.7 (19-
CH3), 50.0 (15-CH2), 36.9 (16-CH2), 31.5 (13-CH2), 24.7 (14-CH2)
ppm. HRMS (ESI+, MeOH): m/z = 361.0825 [M+Na]+, 393.1084
[M+Na+MeOH]+; calcd 361.0829 for C15H18N2O5S+Na, 393.1091
for C15H18N2O5S+Na+MeOH.
4.4. Synthetic procedures and characterization
4.4.1. (S)-N-tert-Butyloxycarbonyl-2-methoxyethylpyrrolidine
(7)
4
3
11 12 13
2
5
10
OMe
9
4.5.1. (S)-N-Butyl-5-[1-(2-methoxyethylpyrrolidinyl)sulfonyl]-
9
N 1
isatin (9)
8
6
7
O
O
9
O O
O
A solution of (S)-N-tert-butyloxycarbonyl-2-hydroxyethylpyrr-
olidine (6) (0.91 g, 4.21 mmol, 1.00 equiv) in dry THF (25 mL) un-
der argon atmosphere was cooled to À10 °C and treated with
60% NaH (252 mg, 6.31 mmol, 1.50 equiv). After stirring for
15 min, MeI (0.39 mL, 6.31 mmol, 1.50 equiv) was added dropwise
to the reaction mixture. Stirring was continued at this temperature
for 30 min, at 0 °C for 2 h and at room temperature overnight. Then
water (60 mL) was added and the mixture was extracted with
EtOAc (3 Â 80 mL). The combined organic phase was dried over
MgSO4 and the solvent was removed completely in vacuo. The res-
idue was purified by flash column chromatography (silica gel, 30%
EtOAc in cyclohexane) to obtain a colorless oil (0.72 g, 3.13 mmol,
74%). 1H NMR (400 MHz, CDCl3): d = 4.00–3.70 (m, 1H, 2-CH), 3.47–
3.19 (m, 2H, 5-CH2), 3.41 (t, 3JH,H = 6.6 Hz, 2H, 11-CH2), 3.32 (s, 3H,
13-CH3), 2.19–1.89 (m, 1H, 10-CHa), 2.01–1.85 (m, 1H, 3-CHa),
1.93–1.75 (m, 2H, 4-CH2), 1.77–1.67 (m, 1H, 3-CHb), 1.56 (m, 1H,
10-CHb), 1.46 (s, 9H, 9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d = 154.6 (6-CO), 79.0 (8-CO), 70.6 (11-CH2), 58.6 (13-CH3), 55.1
(2-CH), 46.1 (5-CH2), 34.4 (10-CH2), 30.9 (3-CH2), 28.5 (3C, 9-
CH3), 23.2 (4-CH2) ppm. HRMS (ESI+, MeOH): m/z = 230.1751
[M+H]+, 252.1568 [M+Na]+, calcd 230.1751 for C12H23NO3+H,
252.1570 for C12H23NO3+Na.
4
7
15
11
3
S
5
9
8
10
N
2
14
O
12
6
1
N
13
16
17
20
21
22
18OMe
19
23
(S)-5-[1-(2-Methoxyethylpyrrolidinyl)sulfonyl]isatin
(8)
(100 mg, 0.296 mmol, 1.00 equiv) was converted to (S)-N-butyl-
5-[1-(2-methoxyethylpyrrolidinyl)sulfonyl]isatin (9) using Cs2CO3
(193 mg, 0.592 mmol, 2.00 equiv), dry DMF (5.0 mL) and 1-bromo-
butane (0.32 mL, 3.00 mmol, 10.00 equiv) under microwave irradi-
ation as described in the general procedure B. The crude product
was purified by flash column chromatography (silica gel, 45%
EtOAc in cyclohexane) to obtain an orange-yellow wax (85 mg,
0.215 mmol, 73%). 1H NMR (400 MHz, CDCl3): d = 8.07 (dd,
3JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 8.01 (d, JH,H = 2.0 Hz, 1H,
4-CH), 7.06 (d, JH,H = 8.3 Hz, 1H, 7-CH), 3.83–3.69 (m, 1H, 12-
CH), 3.78 (t, JH,H = 7.4 Hz, 2H, 20-CH2), 3.54–3.43 (m, 2H, 17-
4
4
3
3
CH2), 3.54–3.39 (m, 1H, 15-CHa), 3.35 (s, 3H, 19-CH3), 3.15 (dt,
2JH,H = 9.9 Hz, JH,H = 7.2 Hz, 1H, 15-CHb), 2.20–2.05 (m, 1H, 16-
3
CHa), 1.91–1.80 (m, 1H, 14-CHa), 1.77–1.66 (m, 2H, 21-CH2),
1.76–1.62 (m, 2H, 13-CHa), 1.74–1.65 (m, 1H, 16-CHb), 1.67–1.56
(m, 1H, 14-CHb), 1.44 (sextet, JH,H = 7.4 Hz, 2H, 22-CH2), 0.99 (t,
3
4.5. (S)-5-[1-(2-Methoxyethylpyrrolidinyl)sulfonyl]isatin (8)
3JH,H = 7.4 Hz, 3H, 23-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d = 182.3 (3-CO), 157.8 (2-CO), 153.7 (8-CN), 137.5 (6-CH), 133.6
(5-CSO2), 124.5 (4-CH), 117.3 (9-CCO), 110.5 (7-CH), 69.6 (17-
CH2), 58.6 (19-CH3), 58.5 (12-CH), 49.0 (15-CH2), 40.5 (20-CH2),
36.0 (16-CH2), 31.2 (13-CH2), 29.2 (21-CH2), 24.1 (14-CH2),
20.1 (22-CH2), 13.6 (23-CH3) ppm. HRMS (ESI+, MeOH):
m/z = 417.1446 [M+Na]+, 449.1707 [M+Na+MeOH]+; calcd 417.1455
for C19H26N2O5S+Na, 449.1717 for C19H26N2O5S+Na+MeOH.
O O
O
4
7
15
11
3
S
5
9
8
10
N
2
14
O
12
6
1
N
13
16
17
H
18OMe
19
4.5.2. (S)-N-(4-Fluorobutyl)-5-[1-(2-methoxyethylpyrrolidinyl)-
sulfonyl]isatin (10)
(S)-N-tert-Butyloxycarbonyl-2-methoxyethylprrolidine (7) was
converted to (S)-5-[1-(2-methoxyethylpyrrolidinyl)sulfonyl]isatin
(8) as described in the general procedure A. Boc-deprotection
of (S)-N-tert-butyloxycarbonyl-2-methoxyethylpyrrolidine (7)
(0.40 g, 1.75 mmol, 1.00 equiv) to (S)-2-methoxyethylpyrrolidine
was performed using TFA (1.34 mL, 17.5 mmol, 10.00 equiv) and
dry DCM (6.0 mL). The resulting product in CHCl3 (3.0 mL) was
coupled with 5-chlorosulfonyl isatin (0.64 g, 2.62 mmol,
1.50 equiv) using DIPEA (0.59 mL, 3.50 mmol, 2.00 equiv) and
CHCl3/THF (1:1, 34 mL). The crude product was purified by flash
column chromatography (silica gel, 80% EtOAc in cyclohexane) to
obtain a yellow powder (0.37 g, 1.08 mmol, 62%). Mp 130 °C. 1H
NMR (400 MHz, CD3CN): d = 9.36 (br s, 1H, 1-NH), 7.99 (dd,
O O
O
4
7
15
11
3
S
5
9
8
10
N
2
14
O
12
6
1
N
13
16
17
20
21
22
18OMe
19
23 F
(S)-5-[1-(2-Methoxyethylpyrrolidinyl)sulfonyl]isatin (8)
(100 mg, 0.296 mmol, 1.00 equiv) was converted to (S)-N-(4-fluo-
robutyl)-5-[1-(2-methoxyethylpyrrolidinyl)sulfonyl]isatin (10)
4
4
3JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 7.87 (d, JH,H = 1.9 Hz, 1H,

P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
2031
using Cs2CO3 (193 mg, 0.592 mmol, 2.00 equiv), dry DMF (5.0 mL)
and 1-bromo-4-fluorobutane (0.32 mL, 3.00 mmol, 10.00 equiv)
under microwave irradiation as described in the general procedure
B. The crude product was purified by flash column chromatography
(silica gel, 65% EtOAc in cyclohexane) to obtain an orange-yellow
wax (121 mg, 0.293 mmol, 99%). 1H NMR (300 MHz, CDCl3):
(15 mL) under argon atmosphere at À10 °C was treated with NaH
(152 mg, 3.80 mmol, 1.50 equiv, 60% in mineral oil). After stirring
for 15 min, MeI (315 lL, 5.06 mmol, 2.00 equiv) was added drop-
wise to the mixture. The resulting mixture was stirred below
À5 °C for 30 min, at 0 °C for 2 h and room temperature for 20 h.
Then, the mixture was cooled to 0 °C, before it was quenched with
satd aq NH4Cl until no H2 evolved anymore. Subsequently, water
(15 mL) was added and the aq phase was extracted with EtOAc
(3 Â 40 mL). The combined organic phase was washed with brine
(1 Â 20 mL) and dried over MgSO4. After removal of the solvent,
the residue was purified by flash column chromatography (silica
gel, 20% EtOAc in cyclohexane) to obtain a colorless oil (618 mg,
2.54 mmol, 100%). 1H NMR (400 MHz, CDCl3): d = 3.87–3.67 (m,
3
4
d = 8.06 (dd, JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 8.00 (dd,
5
3
4JH,H = 1.9 Hz, JH,H = 0.5 Hz, 1H, 4-CH), 7.10 (dd, JH,H = 8.3 Hz,
5JH,H = 0.5 Hz, 1H, 7-CH), 4.53 (dt, JH,F = 47.5 Hz, JH,H = 5.3 Hz,
2
3
3
2H, 23-CH2), 3.85 (t, JH,H = 7.0 Hz, 2H, 20-CH2), 3.73 (tt,
3
3JH,H = 8.5 Hz, JH,H = 4.8 Hz, 1H, 12-CH), 3.54–3.39 (m, 1H, 15-
3
CHa), 3.48 (t, JH,H = 6.6 Hz, 2H, 17-CH2), 3.35 (s, 3H, 19-CH3),
3.21–3.08 (m, 1H, 15-CHb), 2.20–2.05 (m, 1H, 16-CHa), 1.99–1.54
(m, 9H, 13-CH2, 14-CH2, 16-CHb, 21-CH2, 22-CH2) ppm. 13C NMR
(75 MHz, CDCl3): d = 182.1 (3-CO), 157.9 (2-CO), 153.5 (8-CN),
137.5 (6-CH), 133.6 (5-CSO2), 124.5 (4-CH), 117.3 (9-CCO), 110.6
3
1H, 2-CH), 3.44–3.22 (m, 2H, 5-CH2), 3.37 (t, JH,H = 6.7 Hz, 1H,
12-CH2), 3.33 (s, 3H, 14-CH3), 1.99–1.85 (m, 1H, 3-CHa), 1.90–
1.74 (m, 2H, 4-CH2), 1.84–1.63 (m, 1H, 10-CHa), 1.70–1.60 (m,
1H, 3-CHb), 1.61–1.50 (m, 2H, 11-CH2), 1.46 (s, 9H, 9-CH3), 1.43–
1.27 (m, 1H, 10-CHb) ppm. 13C NMR (100 MHz, CDCl3): d = 154.7
(6-CO), 78.9 (8-CO), 72.8 (12-CH2), 58.5 (14-CH3), 57.1 (2-CH),
46.3 (5-CH2), 31.0 (10-CH2), 30.3 (3-CH2), 28.6 (3C, 9-CH3), 26.5
(11-CH2), 23.4 (4-CH2) ppm. HRMS (ESI+, MeOH): m/z = 266.1724
[M+Na]+, 509.3547 [2M+Na]+; calcd 266.1727 for C13H25NO3+Na,
509.3561 for 2(C13H25NO3)+Na.
1
(7-CH), 83.3 (d, JC,F = 165.4 Hz, 23-CH2), 69.6 (17-CH2), 58.6 (19-
CH3), 58.5 (12-CH), 49.0 (15-CH2), 40.2 (20-CH2), 36.0 (16-CH2),
2
31.1 (13-CH2), 27.6 (d, JC,F = 20.1 Hz, 22-CH2), 24.0 (14-CH2),
3
23.5 (d, JC,F = 4.1 Hz, 21-CH2) ppm. 19F NMR (282 MHz, CDCl3):
d = À219.8 (s, 1F, 23-CH2F) ppm. HRMS (ESI+, MeOH):
m/z = 435.1361 [M+Na]+, 467.1622 [M+Na+MeOH]+; calcd 435.1360
for C19H25FN2O5S+Na, 467.1623 for C19H25FN2O5S+Na+MeOH.
4.5.3. rac-N-tert-Butyloxycarbonyl-2-(3-hydroxypropyl)pyrroli-
dine (12)
4.5.5. rac-5-{1-[2-(3-Methoxypropyl)pyrrolidinyl]sulfonyl}-
isatin (14)
4
3
11
OH
12
O O
O
2
5
10
4
9
15
11
3
S
5
9
N1
9
N
10
2
14
8
6
O
7
O
12
6
O
1
9
8
N
13
16
17
7
H
A
stirred solution of 2-(3-hydroxypropyl)pyrrolidine (11)
18
19
(500 mg, 3.87 mmol, 1.00 equiv) in DCM (20 mL) was treated with
TEA (1.62 mL, 11.6 mmol, 3.00 equiv) followed by addition of
Boc2O (929 mg, 4.26 mmol, 1.10 equiv) at room temperature and
stirred further for 22 h. Then, the reaction was acidified to pH 2
with 15% aq KHSO4 (ꢀ10 mL). Subsequently, water (10 mL) was
added, the organic phase was separated and the aq phase was ex-
tracted with EtOAc (3 Â 15 mL). The collected organic phase was
dried over MgSO4 and concentrated in vacuo. The crude product
was purified by flash column chromatography (silica gel, 50%
EtOAc in cyclohexane) to obtain colorless oil (726 mg, 3.17 mmol,
82%). 1H NMR (400 MHz, CDCl3): d = 3.92–3.70 (m, 1H, 2-CH),
20
MeO
rac-N-tert-Butyloxycarbonyl-2-(3-methoxypropyl)pyrrolidine
(13) was converted to rac-5-{1-[2-(3-methoxypropyl)pyrrolidi-
nyl]sulfonyl}isatin (14) as described in the general procedure A.
Boc-deprotection of rac-N-tert-butyloxycarbonyl-2-(3-methoxy-
propyl)pyrrolidine (13) (518 mg, 2.13 mmol, 1.00 equiv) to 2-(3-
methoxypropyl)pyrrolidine was performed using TFA (1.6 mL,
21.3 mmol, 10.00 equiv) and dry DCM (7.0 mL). The resulting prod-
uct in CHCl3 (3.5 mL) was coupled with 5-chlorosulfonyl isatin
3
3.66 (t, JH,H = 6.3 Hz, 2H, 12-CH2), 3.43–3.23 (m, 2H, 5-CH2), 2.61
(785 mg, 3.20 mmol, 1.50 equiv) using DIPEA (742 lL, 4.26 mmol,
(br s, 1H, 12-COH), 1.99–1.60 (m, 4H, 3-CH2, 4-CH2), 1.65–1.27
(m, 4H, 10-CH2, 11-CH2), 1.46 (s, 9H, 9-CH3) ppm. 13C NMR
(100 MHz, CDCl3): d = 154.8 (6-CO), 79.1 (8-CO), 62.5 (12-CH2),
56.8 (2-CH), 46.3 (5-CH2), 30.8 (10-CH2), 30.4 (3-CH2), 29.3 (11-
CH2), 28.5 (3C, 9-CH3), 23.4 (4-CH2) ppm. HRMS (ESI+, MeOH):
m/z = 252.1567 [M+Na]+, 481.3247 [2M+Na]+; calcd 252.1570 for
2.00 equiv) and CHCl3/THF (1:1, 40 mL). The crude product was
purified by flash column chromatography (silica gel, 80% EtOAc
in cyclohexane) to obtain a yellow gum (616 mg, 1.75 mmol,
82%) as the desired product. 1H NMR (400 MHz, CDCl3): d = 9.68
3
4
(s, 1H, 1-NH), 8.03 (dd, JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH),
4
3
7.99 (d, JH,H = 1.8 Hz, 1H, 4-CH), 7.22 (d, JH,H = 8.3 Hz, 1H, 7-CH),
C
12H23NO3+Na, 481.3248 for 2(C12H23NO3)+Na.
3
3
3.64 (tt, JH,H = 7.7 Hz, JH,H = 4.3 Hz, 1H, 12-CH), 3.43 (t,
3JH,H = 6.3 Hz, 2H, 18-CH2), 3.43–3.33 (m, 1H, 15-CHa), 3.16 (dt,
4.5.4. rac-N-tert-Butyloxycarbonyl-2-(3-methoxypropyl)pyrroli-
2JH,H = 10.3 Hz, JH,H = 7.1 Hz, 1H, 15-CHb), 3.36 (s, 3H, 20-CH3),
3
dine (13)
1.90–1.77 (m, 2H, 14-CHa, 16-CHa), 1.72–1.50 (m, 6H, 13-CH2,
14-CHb, 16-CHb, 17-CH2) ppm. 13C NMR (100 MHz, CDCl3):
d = 182.6 (3-CO), 159.1 (2-CO), 152.8 (8-CN), 137.6 (6-CH), 133.7
(5-CSO2), 124.7 (4-CH), 117.7 (9-CCO), 113.5 (7-CH), 72.6 (18-
CH2), 60.7 (12-CH), 58.6 (20-CH3), 49.1 (15-CH2), 33.0 (16-CH2),
30.8 (13-CH2), 26.1 (17-CH2), 24.1 (14-CH2) ppm. HRMS
(ESI+, MeOH): m/z = 375.0987 [M+Na]+, 407.1247 [M+Na+MeOH]+;
14
4
3
13
11
OMe
12
2
10
5
9
N
1
9
8
6
7
O
O
9
calcd
375.0985
for
C16H20N2O5S+Na,
407.1247
for
A stirred solution of N-tert-butyloxycarbonyl-2-(3-hydroxypro-
pyl)pyrrolidine (12) (580 mg, 2.53 mmol, 1.00 equiv) in dry THF
C16H20N2O5S+Na+MeOH.

2032
P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
4.5.6. rac-N-Butyl-5-{1-[2-(3-methoxypropyl)pyrrolidinyl]-
NMR (75 MHz, CDCl3): d = 182.1 (3-CO), 157.8 (2-CO), 153.4 (8-
sulfonyl}isatin (15)
CN), 137.5 (6-CH), 133.9 (5-CSO2), 124.4 (4-CH), 117.3 (9-CCO),
110.5 (7-CH), 83.3 (d, JC,F = 165.5 Hz, 24-CH2), 72.5 (18-CH2),
1
60.6 (12-CH), 58.6 (20-CH3), 49.0 (15-CH2), 40.2 (21-CH2), 33.0
O O
O
2
4
7
(16-CH2), 30.8 (13-CH2), 27.6 (d, JC,F = 20.0 Hz, 23-CH2), 26.2
15
11
3
S
5
9
8
3
10
N
2
(17-CH2), 24.1 (14-CH2), 23.5 (d, JC,F = 4.1 Hz, 22-CH2) ppm.
14
O
19F NMR (282 MHz, CDCl3): d = À219.9 (s, 1F, 24-CH2F) ppm.
HRMS (ESI+, MeOH): m/z = 449.1519 [M+Na]+, 481.1781
[M+Na+MeOH]+; calcd 449.1517 for C20H27FN2O5S+Na, 481.1779
for C20H27FN2O5S+Na+MeOH.
12
6
1
N
13
16
17
21
22
23
18
19
24
20
MeO
4.5.8. (S)-N-tert-Butyloxycarbonyl-2-ethoxymethylpyrrolidine
(17)
rac-5-{1-[2-(3-Methoxypropyl)pyrrolidinyl]sulfonyl}isatin (14)
(100 mg, 0.284 mmol, 1.00 equiv) was converted to rac-N-butyl-
5-{1-[2-(3-methoxypropyl)pyrrolidinyl]sulfonyl}isatin (15) using
dry DMF (5 mL), Cs2CO3 (185 mg, 0.568 mmol, 2.00 equiv) and
4
3
1-bromobutane (304
lL, 2.84 mmol, 10.00 equiv) under micro-
13
11
O
12
2
wave irradiation as described in the general procedure B. The crude
product was purified by flash column chromatography (silica gel,
40% EtOAc in cyclohexane) to obtain a yellow wax (85 mg,
0.208 mmol, 73%) as the desired product. 1H NMR (400 MHz,
10
5
9
N1
6
9
8
7
O
O
9
3
4
CDCl3): d = 8.06 (dd, JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 8.01
4
3
(d, JH,H = 1.8 Hz, 1H, 4-CH), 7.05 (d, JH,H = 8.3 Hz, 1H, 7-CH), 3.78
(t, JH,H = 7.4 Hz, 2H, 21-CH2), 3.66 (tt, JH,H = 8.2 Hz, JH,H = 4.3 Hz,
3
3
3
A suspension of NaH (397 mg, 9.94 mmol, 2.00 equiv, 60% in
mineral oil) in dry THF (10 mL) was cooled to 0 °C under argon
atmosphere and then treated slowly with a solution of N-Boc-L-
1H, 12-CH), 3.47–3.36 (m, 3H, 15-CHa, 18-CH2), 3.34 (s, 3H, 20-
2
3
CH3), 3.17 (dt, JH,H = 10.2 Hz, JH,H = 7.2 Hz, 1H, 15-CHb), 1.92–
1.77 (m, 2H, 14-CHa, 16-CHa), 1.77–1.51 (m, 6H, 13-CH2, 14-CHb,
16-CHb, 17-CH2), 1.71 (quintet, 3JH,H = 7.4 Hz, 2H, 22-H2), 1.43 (sex-
prolinol (4) (1.00 g, 4.97 mmol, 1.00 equiv) in dry THF (10 mL) fol-
lowed by addition of 1-bromoethane (1.11 mL, 14.9 mmol,
3.00 equiv). The resulting mixture was stirred at 0 °C for 30 min
and at ambient temperature for 20 h. It was quenched at 0 °C by
addition of satd aq NH4Cl until no H2 evolved anymore. Water
(20 mL) was added and two phases were separated. After that
the aq phase was extracted with EtOAc (3 Â 30 mL) and the com-
bined organic phase was washed with water (1 Â 30 mL), brine
(1 Â 30 mL) and dried over MgSO4. The solvent was removed in va-
cuo and the residue was purified by flash column chromatography
(silica gel, 10% EtOAc in cyclohexane) to obtain a colorless oil
(690 mg, 3.01 mmol, 61% yield) as the pure desired product. 1H
NMR (400 MHz, CDCl3): d = 4.06–3.80 (m, 1H, 2-CH), 3.63–3.44
(m, 1H, 10-CHa), 3.60–3.42 (m, 2H, 12-CH2), 3.39–3.25 (m, 2H,
5-CH2), 3.36–3.19 (m, 1H, 10-CHb), 2.01–1.70 (m, 4H, 3-CH2,
3
3
tet, JH,H = 7.4 Hz, 2H, 23-CH2), 0.99 (t, JH,H = 7.4 Hz, 3H, 24-CH3)
ppm. 13C NMR (100 MHz, CDCl3): d = 182.3 (3-CO), 157.8 (2-CO),
153.7 (8-CN), 137.4 (6-CH), 133.9 (5-CSO2), 124.4 (4-CH), 117.4
(9-CCO), 110.4 (7-CH), 72.5 (18-CH2), 60.6 (12-CH), 58.6 (20-
CH3), 49.0 (15-CH2), 40.5 (21-CH2), 33.0 (16-CH2), 30.8 (13-CH2),
29.2 (22-CH2), 26.2 (17-CH2), 24.1 (14-CH2), 20.1 (23-CH2), 13.6
(24-CH3) ppm. HRMS (ESI+, MeOH): m/z = 431.1609 [M+Na]+,
463.1869 [M+Na+MeOH]+; calcd 431.1611 for C20H28N2O5S+Na,
463.1873 for C20H28N2O5S+Na+MeOH.
4.5.7. rac-N-(4-Fluorobutyl)-5-{1-[2-(3-methoxypropyl)pyrroli-
dinyl]sulfonyl}isatin (16)
3
4-CH2), 1.47 (s, 9H, 9-CH3), 1.18 (t, JH,H = 7.0 Hz, 3H, 13-CH3) ppm.
13C NMR (100 MHz, CDCl3): d = 154.5 (6-CO), 79.1 (8-CO), 71.3
(10-CH2, rotamer A), 70.7 (10-CH2, rotamer B), 66.5 (12-CH2),
56.4 (2-CH), 46.8 (5-CH2, rotamer B), 46.3 (5-CH2, rotamer A),
28.7 (3-CH2, rotamer A), 28.5 (3C, 9-CH3), 27.9 (3-CH2, rotamer
B), 23.7 (4-CH2, rotamer B), 22.8 (4-CH2, rotamer A), 15.2 (13-
CH3) ppm. HRMS (ESI+, MeOH): m/z = 252.1576 [M+Na]+; calcd
252.1570 for C12H23NO3Na.
O O
O
4
15
11
3
S
5
9
N
10
2
14
O
12
6
1
8
N
13
16
17
7
21
22
23
18
24 F
MeO19
20
rac-5-{1-[2-(3-Methoxypropyl)pyrrolidinyl]sulfonyl}isatin (14)
(100 mg, 0.284 mmol, 1.00 equiv) was converted to rac-N-(4-fluo-
robutyl)-5-{1-[2-(3-methoxypropyl)pyrrolidinyl]sulfonyl}isatin
(16) using dry DMF (5 mL), Cs2CO3 (185 mg, 0.568 mmol,
4.5.9. (S)-5-[1-(2-Ethoxymethylpyrrolidinyl)sulfonyl]isatin (18)
O O
O
4
7
2.00 equiv) and 1-bromo-4-fluorobutane (305 lL, 2.84 mmol,
15
11
3
S
5
9
8
N
12
10
2
10.00 equiv) under microwave irradiation as described in the gen-
eral procedure B. The crude product was purified by flash column
chromatography (silica gel, 50% EtOAc in cyclohexane) to obtain
a thick yellow oil (109 mg, 0.256 mmol, 90%) as the desired prod-
14
O
6
1
N
13
16
H
O17
18
3
uct. 1H NMR (300 MHz, CDCl3): d = 8.07 (dd, JH,H = 8.3 Hz,
19
4
4JH,H = 1.9 Hz, 1H, 6-CH), 8.01 (d, JH,H = 1.9 Hz, 1H, 4-CH), 7.08 (d,
2
3
3JH,H = 8.3 Hz, 1H, 7-CH), 4.53 (dt, JH,F = 47.5 Hz, JH,H = 5.3 Hz,
3
2H, 24-CH2), 3.84 (t, JH,H = 7.0 Hz, 2H, 21-CH2), 3.70–3.59 (m, 1H,
(S)-N-tert-Butyloxycarbonyl-2-ethoxymethylpyrrolidine (17) was
converted to (S)-5-[1-(2-ethoxymethylpyrrolidinyl)sulfonyl]isatin
(18) as described in the general procedure A. Boc-deprotection of
12-CH), 3.47–3.36 (m, 3H, 15-CHa, 18-CH2), 3.35 (s, 3H, 20-CH3),
2
3
3.16 (dt, JH,H = 10.4 Hz, JH,H = 7.2 Hz, 1H, 15-CHb), 1.96–1.50 (m,
12H, 13-CH2, 14-CH2, 16-CH2, 17-CH2, 22-CH2, 23-CH2) ppm. 13C

P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
2033
(S)-N-tert-butyloxycarbonyl-2-ethoxymethylpyrrolidine (17)
(400 mg, 1.74 mmol, 1.00 equiv) to (S)-2-ethoxymethylpyrrolidine
was performed using TFA (1.30 mL, 17.4 mmol, 10.00 equiv) and
dry DCM (10 mL). The resulting product in CHCl3 (5.0 mL) was cou-
pled with 5-chlorosulfonyl isatin (643 mg, 2.62 mmol, 1.50 equiv)
using DIPEA (608 mL, 3.49 mmol, 2.00 equiv) and CHCl3/THF (1:1,
30 mL). The residue was purified by flash column chromatography
(silica gel, 5% MeOH in DCM) to obtain a yellow solid (473 mg,
1.40 mmol, 80%). Mp 164 °C. 1H NMR (300 MHz, DMSO-d6):
4.5.11. (S)-N-(4-Fluorobutyl)-5-[1-(2-ethoxymethylpyrrolidinyl)-
sulfonyl]isatin (20)
O O
O
4
7
15
11
3
S
5
9
8
N
12
10
2
14
O
6
1
N
13
16
O17
3
4
20
d = 11.45 (s, 1H, 1-NH), 8.02 (dd, JH,H = 8.3 Hz, JH,H = 2.0 Hz, 1H,
21
18
19
4
3
6-CH), 7.77 (d, JH,H = 1.9 Hz, 1H, 4-CH), 7.09 (d, JH,H = 8.3 Hz, 1H,
22
7-CH), 3.72–3.60 (m, 1H, 12-CH), 3.54–3.24 (m, 5H, 15-CHa,
23 F
2
3
16-CH2, 18-CH2), 3.06 (dt, JH,H = 10.1 Hz, JH,H = 7.6 Hz, 1H,
15-CHb), 1.89–1.65 (m, 2H, 13-CHa, 14-CHa), 1.61–1.43 (m, 2H,
13-CHb, 14-CHb), 1.11 (t, JH,H = 7.0 Hz, 3H, 19-CH3) ppm. 13C
3
The preparation of (S)-N-(4-fluorobutyl)-5-[1-(2-ethoxymethyl-
pyrrolidinyl)sulfonyl]isatin (20) was performed successfully start-
ing from (S)-5-[1-(2-ethoxymethylpyrrolidinyl)sulfonyl]isatin (18)
(100 mg, 0.296 mmol, 1.00 equiv) using dry DMF (4 mL), Cs2CO3
(193 mg, 0.591 mmol, 2.00 equiv) and 1-bromo-4-fluorobutane
NMR (75 MHz, DMSO-d6): d = 182.9 (3-CO), 159.4 (2-CO), 153.6
(8-CN), 136.7 (6-CH), 130.8 (5-CSO2), 123.0 (4-CH), 118.1
(9-CCO), 112.7 (7-CH), 72.3 (16-CH2), 65.8 (18-CH2), 58.8 (12-
CH), 49.0 (15-CH2), 28.3 (13-CH2), 23.5 (14-CH2), 15.0 (19-CH3)
ppm. HRMS (ESI+, MeOH): m/z = 361.0838 [M+Na]+, 393.1102
[M+Na+MeOH]+; calcd 361.0829 for C15H18N2O5S+Na, 393.1091
for C15H18N2O5S+Na+MeOH.
(317 lL, 2.96 mmol, 10.00 equiv) under microwave irradiation as
described in the general procedure B. The resulting residue was
purified by flash column chromatography (silica gel, 35% EtOAc
in toluene) to obtain a yellow solid (94 mg, 0.228 mmol, 77%).
3
4.5.10. (S)-N-Butyl-5-[1-(2-ethoxymethylpyrrolidinyl)sulfonyl]-
isatin (19)
Mp 128 °C. 1H NMR (400 MHz, CDCl3): d = 8.09 (dd, JH,H = 8.3 Hz,
4
4JH,H = 1.9 Hz, 1H, 6-CH), 8.04 (d, JH,H = 1.9 Hz, 1H, 4-CH), 7.06 (d,
2
3
3JH,H = 8.3 Hz, 1H, 7-CH), 4.52 (dt, JH,F = 47.4 Hz, JH,H = 5.5 Hz,
3
2H, 23-CH2), 3.85 (t, JH,H = 7.1 Hz, 2H, 20-CH2), 3.78–3.71 (m, 1H,
12-CH), 3.64 (dd, JH,H = 9.4 Hz, JH,H = 3.8 Hz, 1H, 16-CHa), 3.58–
2
3
O O
O
4
7
15
3.45 (m, 2H, 18-CH2), 3.48–3.41 (m, 1H, 15-CHa), 3.38 (dd,
11
3
S
5
9
8
3
2JH,H = 9.4 Hz, JH,H = 8.0 Hz, 1H, 16-CHb), 3.15–3.06 (m, 1H,
N
12
10
2
14
O
15-CHb), 1.99–1.82 (m, 2H, 13-CHa, 14-CHa), 1.94–1.82 (m, 2H,
21-CH2), 1.89–1.73 (m, 2H, 22-CH2), 1.75–1.60 (m, 2H, 13-CHb,
6
1
N
13
16
O17
3
21
14-CHb), 1.19 (t, JH,H = 7.0 Hz, 3H, 19-CH3) ppm. 13C NMR
20
18
19
(100 MHz, CDCl3): d = 182.0 (3-CO), 157.8 (2-CO), 153.5 (8-CN),
22
23
137.6 (6-CH), 133.8 (5-CSO2), 124.6 (4-CH), 117.4 (9-CCO),
1
110.4 (7-CH), 83.3 (d, JC,F = 165.5 Hz, 23-CH2), 72.7 (16-CH2),
66.8 (18-CH2), 59.4 (12-CH), 49.4 (15-CH2), 40.2 (20-CH2), 28.9
2
(13-CH2), 27.6 (d, JC,F = 20.1 Hz, 22-CH2), 24.0 (14-CH2), 23.5
The preparation of (S)-N-butyl-5-[1-(2-ethoxymethylpyrrolidi-
nyl)sulfonyl]isatin (19) was achieved successfully starting from
(S)-5-[1-(2-ethoxymethylpyrrolidinyl)sulfonyl]isatin (18)
(100 mg, 0.296 mmol, 1.00 equiv) using dry DMF (4 mL), Cs2CO3
(d, JC,F = 4.0 Hz, 21-CH2), 15.2 (19-CH3) ppm. 19F NMR (282 MHz,
CDCl3): d = À219.9 (s, 1F, 23-CH2F) ppm. HRMS (ESI+, MeOH):
m/z = 435.1360 [M+Na]+, 467.1620 [M+Na+MeOH]+; calcd 435.1360
for C19H25FN2O5S+Na, 467.1623 for C19H25FN2O5S+Na+MeOH.
3
(193 mg, 0.591 mmol, 2.00 equiv) and 1-bromobutane (316 lL,
2.96 mmol, 10.00 equiv) under microwave irradiation as described
in the general procedure B. The resulting crude product was puri-
fied by flash column chromatography (silica gel, 25% EtOAc in tol-
uene) to obtain a yellow solid (89 mg, 0.226 mmol, 76%). Mp
4.5.12. (S)-N-tert-Butyloxycarbonyl-2-(2-fluoroethoxymethyl)-
pyrrolidine (21)
3
125 °C. 1H NMR (300 MHz, CDCl3): d = 8.09 (dd, JH,H = 8.3 Hz,
4
4JH,H = 1.9 Hz, 1H, 6-CH), 8.04 (d, JH,H = 1.9 Hz, 1H, 4-CH), 7.04 (d,
4
3
13
3JH,H = 8.3 Hz, 1H, 7-CH), 3.80–3.70 (m, 1H, 12-CH), 3.78 (t,
11
O
12
2
10
F
2
3
3JH,H = 7.3 Hz, 2H, 20-CH2), 3.64 (dd, JH,H = 9.4 Hz, JH,H = 3.8 Hz,
5
9
N1
6
9
1H, 16-CHa), 3.58–3.45 (m, 2H, 18-CH2), 3.49–3.40 (m, 1H, 15-
8
7
2
3
CHa), 3.38 (dd, JH,H = 9.4 Hz, JH,H = 8.0 Hz, 1H, 16-CHb), 3.12 (dt,
O
O
9
2JH,H = 9.5 Hz, JH,H = 6.6 Hz, 1H, 15-CHb), 2.00–1.83 (m, 2H, 13-
3
CHa, 14-CHa), 1.79–1.61 (m, 2H, 21-CH2), 1.79–1.59 (m, 2H, 13-
3
CHb, 14-CHb), 1.43 (sextet, JH,H = 7.4 Hz, 2H, 22-CH2), 1.19 (t,
3
3JH,H = 7.0 Hz, 3H, 19-CH3), 0.99 (t, JH,H = 7.3 Hz, 3H, 23-CH3)
Under argon atmosphere, a stirred solution of (S)-N-tert-butyl-
oxycarbonyl-2-hydroxymethylpyrrolidine (4) (1.50 g, 7.45 mmol,
1.50 equiv) in dry DMF (35 mL) was cooled to À10 °C and treated
with 60% NaH (397 mg, 9.94 mmol, 2.00 equiv). After stirring for
10 min, 1-fluoro-2-tosyloxyethane (1.08 g, 4.97 mmol, 1.00 equiv)
was added to the mixture. It was stirred below À5 °C for 4 h, at
0 °C for 4 h and room temperature for 18 h. Then, it was quenched
by dropwise addition of water until no H2 evolved anymore and
concentrated in vacuo. The residue was taken up in water
ppm. 13C NMR (75 MHz, CDCl3): d = 182.2 (3-CO), 157.8 (2-CO),
153.8 (8-CN), 137.5 (6-CH), 133.6 (5-CSO2), 124.5 (4-CH), 117.4
(9-CCO), 110.4 (7-CH), 72.7 (16-CH2), 66.8 (18-CH2), 59.4 (12-
CH), 49.4 (15-CH2), 40.5 (20-CH2), 29.2 (21-CH2), 28.9 (13-CH2),
24.0 (14-CH2), 20.1 (22-CH2), 15.2 (19-CH3), 13.6 (23-CH3) ppm.
HRMS (ESI+, MeOH): m/z = 417.1452 [M+Na]+, 449.1711
[M+Na+MeOH]+; calcd 417.1455 for C19H26N2O5S+Na, 449.1717
for C19H26N2O5S+Na+MeOH.

2034
P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
(50 mL) and extracted with DCM (3 Â 50 mL). The combined or-
ganic phase was dried over MgSO4 and concentrated in vacuo. Sub-
sequently, the crude product was purified by flash column
chromatography (silica gel, 20% EtOAc in cyclohexane) to obtain
a colorless oil (845 mg, 3.42 mmol, 69%). 1H NMR (400 MHz,
4.5.14. (S)-N-Butyl-5-{1-[2-(2-fluoroethoxymethyl)pyrrolidinyl]-
sulfonyl}isatin (23)
O
O
O
4
7
15
3
11
5
9
8
S
2
3
N
10
12
16
2
CDCl3): d = 4.53 (dt, JH,F = 47.8 Hz, JH,H = 4.2 Hz, 2H, 13-CH2),
4.05–3.83 (m, 1H, 2-CH), 3.80–3.60 (m, 2H, 12-CH2), 3.69–3.57
(m, 1H, 10-CHa), 3.51–3.31 (m, 1H, 10-CHb), 3.43–3.21 (m, 2H,
5-CH2), 2.00–1.75 (m, 4H, 3-CH2, 4-CH2), 1.47 (s, 9H, 9-CH3) ppm.
13C NMR (100 MHz, CDCl3): d = 154.6 (6-CO, rotamer B), 154.5 (6-
14
O
6
1
N
13
O17
20
21
22
18
19
23
1
CO, rotamer A), 83.1 (d, JC,F = 169.3 Hz, 13-CH2), 79.3 (8-CO, rot-
F
amer B), 79.2 (8-CO, rotamer A), 72.3 (10-CH2, rotamer B), 71.5
2
(10-CH2, rotamer A), 70.3 (d, JC,F = 19.6 Hz, 12-CH2), 56.4 (2-CH),
(S)-5-{1-[2-(2-Fluoroethoxymethyl)pyrrolidinyl]sulfonyl}isatin
(22) (100 mg, 0.281 mmol, 1.00 equiv) was converted to (S)-N-bu-
tyl-5-{1-[2-(2-fluoroethoxymethyl)pyrrolidinyl]sulfonyl}isatin
(23) using dry DMF (5 mL), Cs2CO3 (183 mg, 0.561 mmol,
46.9 (5-CH2, rotamer B), 46.4 (5-CH2, rotamer A), 28.7 (3-CH2, rot-
amer A), 28.5 (3C, 9-CH3), 28.0 (3-CH2, rotamer B), 23.7 (4-CH2, rot-
amer B), 22.9 (4-CH2, rotamer A) ppm. 19F NMR (282 MHz, CDCl3):
d = À223.6 (s, 13-CH2F, rotamer A), À223.9 (s, 13-CH2F, rotamer B)
ppm. HRMS (ESI+, MeOH): m/z = 270.1479 [M+Na]+; calcd
270.1476 for C12H22FNO3+Na.
2.00 equiv) and 1-bromobutane (300 lL, 2.81 mmol, 10.00 equiv)
as described in the general procedure B. The crude product was
purified by flash column chromatography (silica gel, 45% EtOAc
in cyclohexane) to obtain a yellow solid (105 mg, 0.255 mmol,
91%). Mp 101 °C. 1H NMR (300 MHz, CDCl3): d = 8.08 (dd,
4
4
3JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 8.02 (d, JH,H = 1.8 Hz, 1H,
4.5.13. (S)-5-{1-[2-(2-Fluoroethoxymethyl)pyrrolidinyl]-
sulfonyl}isatin (22)
3
4-CH), 7.06 (d, JH,H = 8.3 Hz, 1H, 7-CH), 4.65–4.44 (m, 2H, 19-
CH2), 3.84–3.66 (m, 4H, 12-CH, 16-CHa, 18-CH2), 3.78 (t,
3JH,H = 7.3 Hz, 2H, 20-CH2), 3.51 (dd, JH,H = 9.3 Hz, JH,H = 7.4 Hz,
1H, 16-CHb), 3.49–3.40 (m, 1H, 15-CHa), 3.19–3.04 (m, 1H, 15-
CHb), 2.05–1.60 (m, 4H, 13-CH2, 14-CH2), 1.82–1.60 (m, 2H, 21-
CH2), 1.43 (sextet, 3JH,H = 7.3 Hz, 2H, 22-CH2), 0.99 (t, 3JH,H = 7.3 Hz,
3H, 23-CH3) ppm. 13C NMR (75 MHz, CDCl3): d = 182.2 (3-CO),
157.8 (2-CO), 153.8 (8-CN), 137.5 (6-CH), 133.4 (5-CSO2), 124.4
2
3
O O
O
4
7
15
3
11
5
9
8
S
2
N
10
12
14
O
6
1
N
13
16
H
O17
1
(4-CH), 117.3 (9-CCO), 110.6 (7-CH), 83.0 (d, JC,F = 169.1 Hz, 19-
18
19
2
CH2), 73.5 (16-CH2), 70.5 (d, JC,F = 19.5 Hz, 18-CH2), 59.2 (12-
CH), 49.4 (15-CH2), 40.5 (20-CH2), 29.2 (21-CH2), 28.8 (13-CH2),
24.1 (14-CH2), 20.1 (22-CH2), 13.6 (23-CH3) ppm. 19F NMR
(282 MHz, CDCl3): d = À223.6 (s, 1F, 19-CH2F) ppm. HRMS (ESI+,
MeOH): m/z = 435.1356 [M+Na]+, 467.1621 [M+Na+MeOH]+; calcd
F
(S)-N-tert-Butyloxycarbonyl-2-(2-fluoroethoxymethyl)pyrroli-
dine (21) was converted to (S)-5-{1-[2-(2-fluoroethoxymethyl)pyr-
rolidinyl]sulfonyl}isatin (22) as described in the general procedure
A. Boc-deprotection of (S)-N-tert-butyloxycarbonyl-2-(2-fluoroeth-
oxymethyl)pyrrolidine (21) (350 mg, 1.42 mmol, 1.00 equiv) to (S)-
2-(2-fluoroethoxymethyl)pyrrolidine was performed using TFA
(1.1 mL, 14.2 mmol, 10.00 equiv) and dry DCM (4.0 mL). The result-
ing product in CHCl3 (2.0 mL) was coupled with 5-chlorosulfonyl
435.1360
for
C
19H25FN2O5S+Na,
467.1623
for
C19H25FN2O5S+Na+MeOH.
4.5.15. (S)-N-(4-Fluorobutyl)-5-{1-[2-(2-fluoroethoxymethyl)-
pyrrolidinyl]sulfonyl}isatin (24)
O O
O
4
7
15
3
11
N
5
9
8
S
2
isatin (523 mg, 2.13 mmol, 1.50 equiv) using DIPEA (495
lL,
10
14
O
12
2.84 mmol, 2.00 equiv) and CHCl3/THF (1:1, 28 mL). The crude
product was purified by flash column chromatography (silica gel,
3% MeOH in DCM) to obtain a yellow solid (344 mg, 0.965 mmol,
68%). Mp 173 °C. 1H NMR (400 MHz, CDCl3): d = 9.27 (br s, 1H,
6
1
N
13
16
O17
20
21
22
18
19
23 F
3
4
1-NH), 8.02 (dd, JH,H = 8.4 Hz, JH,H = 2.0 Hz, 1H, 6-CH), 7.91 (d,
F
4JH,H = 2.0 Hz, 1H, 4-CH), 7.12 (d, JH,H = 8.4 Hz, 1H, 7-CH), 4.52
(dt, JH,F = 48.0 Hz, JH,H = 4.0 Hz, 2H, 19-CH2), 3.79–3.58 (m, 3H,
3
2
3
(S)-5-{1-[2-(2-Fluoroethoxymethyl)pyrrolidinyl]sulfonyl}isatin
(22) (100 mg, 0.281 mmol, 1.00 equiv) was converted to (S)-N-
(4-fluorobutyl)-5-{1-[2-(2-fluoroethoxymethyl)pyrrolidinyl]-
sulfonyl}isatin (24) using dry DMF (5 mL), Cs2CO3 (183 mg,
2
3
12-CH, 18-CH2), 3.62 (dd, JH,H = 9.8 Hz, JH,H = 3.9 Hz, 1H, 16-
2
3
CHa), 3.48 (dd, JH,H = 9.7 Hz, JH,H = 7.3 Hz, 1H, 16-CHb), 3.40–
3.28 (m, 1H, 15-CHa), 3.21–3.06 (m, 1H, 15-CHb), 1.93–1.45
(m, 4H, 13-CH2, 14-CH2) ppm. 13C NMR (100 MHz, CDCl3):
d = 184.0 (3-CO), 159.8 (2-CO), 154.3 (8-CN), 138.4 (6-CH), 133.5
(5-CSO2), 124.9 (4-CH), 119.1 (9-CCO), 113.9 (7-CH), 84.3 (d,
0.561 mmol, 2.00 equiv) and 1-bromo-4-fluorobutane (301 lL,
2.81 mmol, 10.00 equiv) as described in the general procedure B.
The crude product was purified by flash column chromatography
(silica gel, 50% EtOAc in cyclohexane) to obtain a yellow solid
(107 mg, 0.249 mmol, 89%). Mp 116 °C. 1H NMR (400 MHz, CDCl3):
d = 8.08 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 8.01
1JC,F = 166.0 Hz, 19-CH2), 74.3 (16-CH2), 71.3 (d, JC,F = 19.1 Hz,
2
18-CH2), 60.4 (12-CH), 50.4 (15-CH2), 29.4 (13-CH2), 24.8 (14-
CH2) ppm. 19F NMR (282 MHz, CDCl3): d = À222.2 (s, 1F, 19-CH2F)
ppm. HRMS (ESI+, MeOH): m/z = 379.0734 [M+Na]+, 411.0995
[M+Na+MeOH]+; calcd 379.0734 for C15H17FN2O5S+Na, 411.0997
for C15H17FN2O5S+Na+MeOH.
4
3
(d, JH,H = 1.9 Hz, 1H, 4-CH), 7.09 (d, JH,H = 8.3 Hz, 1H, 7-CH),
3
4.65–4.42 (m, 4H, 19-H, 23-CH2), 3.84 (t, JH,H = 7.0 Hz, 2H,
20-CH2), 3.81–3.72 (m, 1H, 12-CH), 3.81–3.66 (m, 2H, 18-CH2),

P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
2035
2
3
3.74–3.66 (m, 1H, 16-CHa), 3.51 (dd, JH,H = 9.5 Hz, JH,H = 7.5 Hz,
oxymethyl)pyrrolidinyl]sulfonyl}isatin (26) as described in the
general procedure A. Boc-deprotection of (S)-N-tert-butyloxycar-
bonyl-2-(2,2,2-trifluoroethoxymethyl)pyrrolidine (25) (220 mg,
0.777 mmol, 1.00 equiv) to (S)-2-(2,2,2-trifluoroethoxymethyl)pyr-
rolidine was performed using TFA (595 lL, 7.77 mmol,
10.00 equiv) and dry DCM (2.0 mL). The resulting product in CHCl3
1H, 16-CHb), 3.47–3.39 (m, 1H, 15-CHa), 3.17–3.08 (m, 1H, 15-
CHb), 2.01–1.63 (m, 8H, 13-CH2, 14-CH2, 21-CH2, 22-CH2) ppm.
13C NMR (100 MHz, CDCl3): d = 182.0 (3-CO), 157.9 (2-CO), 153.5
(8-CN), 137.5 (6-C), 133.6 (5-CSO2), 124.5 (4-CH), 117.4 (9-CCO),
1
110.6 (7-CH), 83.3 (d, JC,F = 165.4 Hz, 23-CH2), 83.0 (d,
1JC,F = 169.1 Hz, 19-CH2), 73.5 (16-CH2), 70.5 (d, JC,F = 19.4 Hz,
(1.0 mL) was coupled with 5-chlorosulfonyl isatin (286 mg,
2
18-CH2), 59.2 (12-CH), 49.4 (15-CH2), 40.2 (20-CH2), 28.8 (13-
1.16 mmol, 1.50 equiv) using DIPEA (271 lL, 1.55 mmol,
2
CH2), 27.6 (d, JC,F = 20.1 Hz, 22-CH2), 24.1 (14-CH2), 23.5 (d,
2.00 equiv) and CHCl3/THF (1:1, 15 mL). The crude product was
purified by flash column chromatography (silica gel, 5% MeOH in
DCM) to obtain a yellow solid (179 mg, 0.456 mmol, 59%). Mp
139 °C. 1H NMR (400 MHz, CD3CN): d = 9.28 (br s, 1H, 1-NH), 8.02
3JC,F = 4.1 Hz, 21-CH2) ppm. 19F NMR (282 MHz, CDCl3):
d = À219.8 (s, 1F, 23-CH2F), À223.6 (s, 1F, 19-CH2F) ppm. HRMS
(ESI+, MeOH): m/z = 453.1270 [M+Na]+, 485.1535 [M+Na+MeOH]+;
3
4
4
calcd
453.1266
for
C19H24F2N2O5S+Na,
485.1528
for
(dd, JH,H = 8.3 Hz, JH,H = 2.0 Hz, 1H, 6-CH), 7.91 (d, JH,H = 1.9 Hz,
3
3
C19H24F2N2O5S+Na+MeOH.
1H, 4-CH), 7.13 (d, JH,H = 8.3 Hz, 1H, 7-CH), 3.98 (q, JH,F = 9.1 Hz,
2H, 18-CH2), 3.78–3.68 (m, 2H, 12-CH, 16-CHa), 3.63 (dd,
3
4.5.16. (S)-N-tert-Butyloxycarbonyl-2-(2,2,2-trifluoroethoxy-
methyl)pyrrolidine (25)
2JH,H = 8.7 Hz, JH,H = 6.3 Hz, 1H, 16-CHb), 3.40–3.31 (m, 1H, 15-
2
3
CHa), 3.14 (dt, JH,H = 10.4 Hz, JH,H = 7.1 Hz, 1H, 15-CHb), 1.90–
1.75 (m, 2H, 13-CHa, 14-CHa), 1.68–1.48 (m, 2H, 13-CHb, 14-CHb)
ppm. 13C NMR (100 MHz, CD3CN): d = 183.9 (3-CO), 159.8 (2-CO),
154.3 (8-CN), 138.3 (6-CH), 133.2 (5-CSO2), 125.4 (q,
1JC,F = 278.5 Hz, 19-CF3), 124.9 (4-CH), 119.1 (9-CCO), 113.9 (7-
13
CF3
4
3
11
10 O
12
2
5
9
N1
6
9
2
CH), 75.5 (16-CH2), 69.0 (q, JC,F = 33.5 Hz, 18-CH2), 60.0 (12-CH),
8
7
O
50.3 (15-CH2), 29.2 (13-CH2), 24.7 (14-CH2) ppm. 19F NMR
(282 MHz, CD3CN): d = À74.8 (s, 3F, 19-CF3) ppm. HRMS (ESI+,
MeOH): m/z = 415.0546 [M+Na]+, 447.0809 [M+Na+MeOH]+; calcd
O
9
Under argon atmosphere, a stirred solution of (S)-N-tert-butyl-
oxycarbonyl-2-(hydroxymethyl)pyrrolidine (4) (300 mg,
415.0546
for
C
15H15F3N2O5S+Na,
447.0808
for
1.49 mmol, 1.30 equiv) in dry DMF (7 mL) was cooled to À10 °C
and treated with 60% NaH (92 mg, 2.29 mmol, 2.00 equiv). After
stirring for 10 min, 1,1,1-trifluoro-2-tosyloxyethane (292 mg,
1.15 mmol, 1.00 equiv) was added to the mixture. It was stirred be-
low À5 °C for 2 h, at 0 °C for 2 h and room temperature for 16 h.
The reaction mixture was quenched by dropwise addition of water
(10 mL) and it was extracted with cyclohexane (3 Â 30 mL). The
combined organic phase was washed with water (1 Â 30 mL), brine
(1 Â 30 mL) and dried over MgSO4. After removal of solvent in va-
cuo, the residue was purified by flash column chromatography (sil-
ica gel, 10% EtOAc in cyclohexane) to obtain a colorless oil (224 mg,
0.791 mmol, 69%). 1H NMR (400 MHz, CDCl3): d = 3.99–3.81 (m,
C
15H15F3N2O5S+Na+MeOH.
4.5.18. (S)-N-Butyl-5-{1-[2-(2,2,2-trifluoroethoxymethyl)-
pyrrolidinyl]sulfonyl}isatin (27)
O O
O
15
13
4
7
11
3
S
5
9
8
N
12
2
10
14
O
6
N
1
16
3
O17
20
1H, 2-CH), 3.83 (q, JH,F = 8.7 Hz, 2H, 12-CH2), 3.77–3.44 (m, 2H,
21
22
18
19
10-CH2), 3.41–3.25 (m, 2H, 5-CH2), 2.00–1.74 (m, 4H, 3-CH2, 4-
CH2), 1.46 (s, 9H, 9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d = 154.7 (6-CO, rotamer A), 154.4 (6-CO, rotamer B), 124.0 (q,
1JC,F = 279.7 Hz, 13-CF3), 79.6 (8-CO, rotamer B), 79.4 (8-CO, rot-
amer A), 73.5 (10-CH2, rotamer B), 72.9 (10-CH2, rotamer A), 68.6
23
F3C
(S)-5-{1-[2-(2,2,2-Trifluoroethoxymethyl)pyrrolidinyl]sulfonyl}
isatin (26) (60 mg, 0.153 mmol, 1.00 equiv) was converted to (S)-
N-butyl-5-{1-[2-(2,2,2-trifluoroethoxymethyl)pyrrolidinyl]sul-
fonyl}isatin (27) using dry DMF (2.2 mL), Cs2CO3 (100 mg,
2
(q, JC,F = 33.9 Hz, 12-CH2), 56.3 (2-CH), 46.9 (5-CH2, rotamer A),
46.5 (5-CH2, rotamer B), 28.7 (3-CH2, rotamer B), 28.5 (3C, 9-
CH3), 27.9 (3-CH2, rotamer A), 23.8 (4-CH2, rotamer A), 22.9 (4-
CH2, rotamer B) ppm. 19F NMR (282 MHz, CDCl3): d = À74.8 (s,
13-CF3, rotamer B), À75.0 (s, 13-CF3, rotamer A) ppm. HRMS
(ESI+, MeOH): m/z = 306.1279 [M+Na]+; calcd 306.1287 for
0.306 mmol, 2.00 equiv) and 1-bromobutane (164 lL, 1.53 mmol,
10.00 equiv) as described in the general procedure B. The crude
product was purified by flash column chromatography (silica gel,
40% EtOAc in cyclohexane) to obtain a yellow solid (58 mg,
0.129 mmol, 85%). Mp 113 °C. 1H NMR (300 MHz, CDCl3): d = 8.08
(dd, JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 8.02 (d, JH,H = 1.8 Hz,
1H, 4-CH), 7.06 (dd, JH,H = 8.3 Hz, JH,H = 0.5 Hz, 1H, 7-CH), 3.89
C12H20F3NO3+Na.
4.5.17. (S)-5-{1-[2-(2,2,2-Trifluoroethoxymethyl)pyrrolidinyl]-
sulfonyl}isatin (26)
3
4
4
3
5
3
(q, JH,F = 8.6 Hz, 2H, 18-CH2), 3.86–3.70 (m, 2H, 12-CH, 16-CHa),
3
2
3.78 (t, JH,H = 7.4 Hz, 2H, 20-CH2), 3.65 (dd, JH,H = 8.9 Hz,
3JH,H = 6.9 Hz, 1H, 16-CHb), 3.51–3.41 (m, 1H, 15-CHa), 3.17–3.06
(m, 1H, 15-CHb), 2.03–1.61 (m, 6H, 13-CH2, 14-CH2, 21-CH2), 1.43
(sextet, JH,H = 7.3 Hz, 2H, 22-CH2), 0.99 (t, JH,H = 7.3 Hz, 3H, 23-
CH3) ppm. 13C NMR (75 MHz, CDCl3): d = 182.2 (3-CO), 157.7 (2-
CO), 153.9 (8-CN), 137.5 (6-CH), 133.1 (5-CSO2), 124.5 (4-CH),
O O
O
15
13
4
7
11
3
S
5
9
8
N
2
10
14
O
12
3
3
6
1
N
16
H
O17
18
1
123.8 (q, JC,F = 280.0 Hz, 19-CF3), 117.4 (9-CCO), 110.6 (7-CH),
74.7 (16-CH2), 68.8 (q, JC,F = 34.1 Hz, 18-CH2), 59.0 (12-CH), 49.5
F3C19
2
(S)-N-tert-Butyloxycarbonyl-2-(2,2,2-trifluoroethoxymethyl)-
pyrrolidine (25) was converted to (S)-5-{1-[2-(2,2,2-trifluoroeth-
(15-CH2), 40.5 (20-CH2), 29.2 (21-CH2), 28.7 (13-CH2), 24.1 (14-
CH2), 20.1 (22-CH2), 13.6 (23-CH3) ppm. 19F NMR (282 MHz,

2036
P. Limpachayaporn et al. / Bioorg. Med. Chem. 21 (2013) 2025–2036
CDCl3): d = À74.8 (s, 3F, 19-CF3) ppm. HRMS (ESI+, MeOH):
m/z = 471.1170 [M+Na]+, 503.1436 [M+Na+MeOH]+; calcd 471.1172
for C19H23F3N2O5S+Na, 503.1434 for C19H23F3N2O5S+Na+MeOH.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft
(Collaborative Research Center, SFB 656, projects B1 and A3), the
International Graduate School of Chemistry Münster (GSC-MS)
and the Development and Promotion of Science and Technology
talent project (DPST), Thailand, is gratefully acknowledged. We like
to thank Sandra Schröer and Wiebke Gottschlich for enzyme inhi-
bition assays. We are grateful to Dr. Ivan Kondratov, National
Ukrainian Academy of Sciences, Kiev, for providing bishomoprolin-
ol and Amke Trentmann for practical assistance.
4.5.19. (S)-N-(4-Fluorobutyl)-5-{1-[2-(2,2,2-
trifluoroethoxymethyl)pyrrolidinyl]sulfonyl}isatin (28)
O O
O
15
13
4
7
11
3
S
5
9
8
N
2
10
14
O
12
6
N
1
16
References and notes
O17
20
21
22
18
19
1. Hengartner, M. O. Nature 2000, 407, 770.
23 F
2. 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.; 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.
3. Podichetty, A. K.; Faust, A.; Kopka, K.; Wagner, S.; Schober, O.; Schäfers, M.;
Haufe, G. Bioorg. Med. Chem. 2009, 17, 2680.
4. Chu, W.; Rothfuss, J.; Chu, Y.; Zhou, D.; Mach, R. H. J. Med. Chem. 2009, 52, 2188.
5. Wang, Q.; Mach, R. H.; Reichert, D. E. J. Chem. Inf. Model. 2009, 49, 1963.
6. 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.
F3C
(S)-5-{1-[2-(2,2,2-Trifluoroethoxymethyl)pyrrolidinyl]sulfonyl}
isatin (26) (60 mg, 0.153 mmol, 1.00 equiv) was converted to (S)-
N-(4-fluorobutyl)-5-{1-[2-(2,2,2-trifluoroethoxymethyl)pyrrolid-
inyl]sulfonyl}isatin (28) using dry DMF (2.2 mL), Cs2CO3 (100 mg,
0.306 mmol, 2.00 equiv) and 4-fluoro-1-bromobutane (164 lL,
1.53 mmol, 10.00 equiv) as described in the general procedure B.
The crude product was purified by flash column chromatography
(silica gel, 50% EtOAc in cyclohexane) to obtain a yellow solid
(60 mg, 0.129 mmol, 84%). Mp 130 °C. 1H NMR (400 MHz, CDCl3):
7. Lee, D.; Long, S. A.; Murray, J. H.; Adams, J. L.; Nuttall, M. E.; Nadeau, D. P.; Kikly,
K.; Winkler, J. D.; Sung, C. M.; Ryan, M. D.; Levy, M. A.; Keller, P. M.; DeWolf, W.
E. J. Med. Chem. 2001, 44, 2015.
8. Podichetty, A. K.; Wagner, S.; Schröer, S.; Faust, A.; Schäfers, M.; Schober, O.;
Kopka, K.; Haufe, G. J. Med. Chem. 2009, 52, 3484.
3
4
9. Zhou, D.; Chu, W.; Rothfuss, J.; Zeng, C.; Xu, J.; Jones, L.; Welch, M. J.; Mach, R. H.
Bioorg. Med. Chem. Lett. 2006, 16, 5041.
d = 8.08 (dd, JH,H = 8.3 Hz, JH,H = 1.9 Hz, 1H, 6-CH), 8.02 (d,
3
4JH,H = 1.9 Hz, 1H, 4-CH), 7.08 (d, JH,H = 8.3 Hz, 1H, 7-CH), 4.52
10. Faust, A.; Wagner, S.; Law, M. P.; Hermann, S.; Schnöckel, U.; Keul, P.; Schober,
O.; Schäfers, M.; Levkau, B.; Kopka, K. Q. J. Nucl. Med. Mol. Imaging 2007, 51, 67.
11. Zhou, D.; Chu, W.; Chen, D. L.; Wang, Q.; Reichert, D. E.; Rothfuss, J.; D’Avignon,
A.; Welch, M. J.; Mach, R. H. Org. Biomol. Chem. 2009, 7, 1337.
12. Baumann, A.; Faust, A.; Law, M. P.; Kuhlmann, M. T.; Kopka, K.; Schäfers, M.;
Karst, U. Anal. Chem. 2011, 83, 5415.
(dt, 2JH,F = 47.6 Hz, 3JH,H = 5.5 Hz, 2H, 23-CH2), 3.89 (q, 3JH,F = 8.7 Hz,
2
2H, 18-CH2), 3.87–3.82 (m, 2H, 20-CH2), 3.82 (dd, JH,H = 9.1 Hz,
3JH,H = 3.6 Hz, 1H, 16-CHa), 3.78–3.70 (m, 1H, 12-CH), 3.65 (dd,
3
2JH,H = 9.2 Hz, JH,H = 7.0 Hz, 1H, 16-CHb), 3.49–3.43 (m, 1H, 15-
CHa), 3.16–3.07 (m, 1H, 15-CHb), 2.04–1.56 (m, 8H, 13-CH2, 14-
CH2, 21-CH2, 22-CH2) ppm. 13C NMR (100 MHz, CDCl3): d = 182.0
(3-CO), 157.8 (2-CO), 153.6 (8-CN), 137.5 (6-CH), 133.3 (5-CSO2),
13. Podichetty, A. K.; Wagner, S.; Faust, A.; Schäfers, M.; Schober, O.; Kopka, K.;
Haufe, G. Future Med. Chem. 2009, 1, 969.
14. Chu, W. H.; Zhang, J.; Zeng, C. B.; Rothfuss, J.; Tu, Z. D.; Chu, Y. X.; Reichert, D. E.;
Welch, M. J.; Mach, R. H. J. Med. Chem. 2005, 48, 7637.
15. Smith, G.; Glaser, M.; Perumal, M.; Nguyen, Q. D.; Shan, B.; Arstad, E.; Aboagye,
E. O. J. Med. Chem. 2008, 51, 8057.
16. Kappe, C. O.; Dallinger, D. Nat. Rev. Drug Disc. 2006, 5, 51.
17. Galli, C. Org. Prep. Proced. Int. 1992, 24, 287.
18. Andries, J.; Lemoine, L.; Le Bars, D.; Zimmer, L.; Billard, T. Eur. J. Med. Chem.
2011, 46, 3455.
19. Hermet, J. P.; Porter, D. W.; Dearden, M. J.; Harrison, J. R.; Koplin, T.; O’Brien, P.;
Parmene, J.; Tyurin, V.; Whitwood, A. C.; Gilday, J.; Smith, N. M. Org. Biomol.
Chem. 2003, 1, 3977.
1
124.5 (4-CH), 123.8 (q, JC,F = 279.4 Hz, 19-CF3), 117.4 (9-CCO),
1
110.6 (7-CH), 83.3 (d, JC,F = 165.5 Hz, 23-CH2), 74.7 (16-CH2),
2
68.8 (q, JC,F = 34.0 Hz, 18-CH2), 59.1 (12-CH), 49.5 (15-CH2), 40.3
2
(20-CH2), 28.8 (13-CH2), 27.6 (d, JC,F = 20.1 Hz, 22-CH2), 24.1
3
(14-CH2), 23.5 (d, JC,F = 4.1 Hz, 21-CH2) ppm. 19F NMR (282 MHz,
CDCl3): d = À74.8 (s, 3F, 19-CF3), À219.9 (s, 1F, 23-CH2F) ppm.
HRMS (ESI+, MeOH): m/z = 489.1081 [M+Na]+, 521.1341
[M+Na+MeOH]+; calcd 489.1078 for C19H22F4N2O5S+Na, 521.1340
for C19H22F4N2O5S+Na+MeOH.