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 Dedicated to Professor Dr. Ernst-Ulrich Würthwein on the occasion of his 65th birthday

Article abstract of DOI:10.1016/j.ejmech.2013.04.011

A series of new 4- or 5-substituted pyrrolidine derivatives of 5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin bearing additional n-butyl or 4-fluorobutyl groups at the isatin nitrogen were prepared and their inhibitory activities have been tested against caspases-3 and -7, which are known to participate in the execution of the programmed cell death, called apoptosis. Several analogues fluorinated at the 4-position of the pyrrolidine ring were also synthesized since such inhibitors might be developed as ¹⁸F-radiotracers for molecular imaging of activated caspases in vivo by PET. Enantiomerically pure diastereomeric 4-fluoropyrrolidinyl derivatives inhibited the enzymes in the nanomolar scale, i.e.100-1000 times more efficient than the corresponding 4-methoxy analogues. The 4,4-difluorinated compound showed the best result with IC₅₀ = 362 nM and 178 nM for the aforementioned caspases. In contrast, the 4-methoxy and 4-trifluoromethyl analogues exhibited less inhibition potencies for the enzymes in the μM scale, whereas all 4-OPEG₄ (PEG₄ = tetraethyleneglycol) and 5-methoxymethyl derivatives were inactive.

Full text of DOI:10.1016/j.ejmech.2013.04.011

European Journal of Medicinal Chemistry 64 (2013) 562e578
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
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
,
Panupun Limpachayaporn a b, Burkhard Riemann c, Klaus Kopka c, Otmar Schober c,
,d,
Michael Schäfers d, Günter Haufe a
*
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 Klinik für Nuklearmedizin, Universitätsklinikum Münster, Albert-Schweitzer-Campus 1, Gebäude A1, D-48149 Münster, Germany
d European Institute for Molecular Imaging (EIMI), Westfälische Wilhelms-Universität Münster, Mendelstraße 11, 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:
A series of new 4- or 5-substituted pyrrolidine derivatives of 5-[1-(2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin bearing additional n-butyl or 4-fluorobutyl groups at the isatin nitrogen were prepared and
their inhibitory activities have been tested against caspases-3 and -7, which are known to participate in
the execution of the programmed cell death, called apoptosis. Several analogues fluorinated at the
4-position of the pyrrolidine ring were also synthesized since such inhibitors might be developed as
18F-radiotracers for molecular imaging of activated caspases in vivo by PET. Enantiomerically pure dia-
stereomeric 4-fluoropyrrolidinyl derivatives inhibited the enzymes in the nanomolar scale, i.e.100e1000
times more efficient than the corresponding 4-methoxy analogues. The 4,4-difluorinated compound
showed the best result with IC50 ¼ 362 nM and 178 nM for the aforementioned caspases. In contrast, the
4-methoxy and 4-trifluoromethyl analogues exhibited less inhibition potencies for the enzymes in the
Received 24 December 2012
Received in revised form
3 April 2013
Accepted 3 April 2013
Available online 11 April 2013
Dedicated to Professor Dr. Ernst-Ulrich
Würthwein on the occasion of his 65th
birthday.
Keywords:
Apoptosis
Caspases
Fluorinated pyrrolidine
Inhibitor
m
M scale, whereas all 4-OPEG4 (PEG4 ¼ tetraethyleneglycol) and 5-methoxymethyl derivatives were
inactive.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Isatin sulfonamide
1. Introduction
enhanced or reduced e can induce many different diseases, e.g.
cancer, cardiovascular and autoimmune defects, as well as neuro-
degeneration including Alzheimer’s, Parkinson’s and Huntington’s
diseases [1]. Although more than a dozen of caspases have been
identified in humans, inhibition of caspases-3 and -7 alone is suf-
ficient to slow down or block the cell death program. Thus, there is
a need for novel therapeutic and preventive drugs targeting the
executioner caspases, such as caspases-3 and -7, for treatment and
diagnosis of diseases associated with dysregulated apoptosis [2].
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin and (S)-
N-methyl-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin have
been identified as potent, non-peptide, selective inhibitors of
caspases-3 and -7 [2e4]. The X-ray co-crystal structure of the
complex of recombinant human caspase-3 with (S)-N-methyl-5-[1-
(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin demonstrated the
embedding of the inhibitor in the enzyme’s active site. The 3-
carbonyl group is the cysteine-163 binding site resulting in a
tetrahedral thiohemiketal intermediate. The pyrrolidine ring is
accommodated in the S2-pocket, while the phenoxymethyl group
Apoptosis, the programmed type I cell death, leads to a regu-
lated destruction of moribund cells. This natural mechanism
strictly controls the number of cells by removing excess, potentially
dangerous as well as damaged cells and there, by maintains ho-
meostasis. It is triggered either by an extrinsic, specific protein/
receptor interaction leading to an intracellular death signal (death
receptor pathway), or by DNA damage followed by an intrinsic
mitochondrial induction of the cell death program (mitochondrial
pathway) finally resulting in the removal of cellular residues, called
apoptotic bodies, without inflammatory response. The apoptotic
pathway is executed by a set of enzymes called caspases (cysteinyl
aspartate-specific proteases). In humans, dysfunctional apoptosis e
* Corresponding author. Organisch-Chemisches Institut, Westfälische Wilhelms-
Universität Münster, Corrensstraße 40, D-48149 Münster, Germany. Tel.: þ49 (0)
251 83 33281; fax: þ49 (0)251 83 39772.
E-mail addresses: haufe@uni-muenster.de, jfc@uni-muenster.de (G. Haufe).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.011

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
563
Herein we report on syntheses of various 4- or 5-substituted
pyrrolidine derivatives of the lead compound. Additionally, we
elucidate the influence of the chosen substitution pattern on the
inhibition potencies for caspases-3 and -7. In this connection we
explore the environment of the enzyme’s S2-pocket and suggest
further modifications in order to improve the pharmacological
properties of the isatin sulfonamides. Furthermore, fluorination of
this moiety might allow the development of this potential inhibitor
class towards new metabolically more stable radiotracers for the
molecular imaging of apoptosis in vivo.
2. Results and discussion
Fig. 1. The general structure of the isatin sulfonamide derivatives with the assignment
of the interactions with the enzyme’s subpockets [2].
2.1. Chemistry
of the pyrrolidine is in contact with the S3-pocket and the isatin N-
substituent putatively interacts with the S1-pocket (Fig. 1) [2].
New N-substituted pyrrolidinyl sulfonyl isatin derivatives
including fluorinated analogues were prepared and intensively
investigated in the past decade. Substitution at this position
resulted in significantly improved activities [3e6]. Moreover, the
pyrrolidine moiety was replaced by several heterocyclic amines.
These compounds showed moderate to excellent inhibitory activ-
ities towards caspases-3 and -7 [7,8].
The derivatives of (S)-5-[1-(2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) and the lead compound itself were synthesized
by coupling of the substituted N-protected pyrrolidine and 5-
chlorosulfonyl isatin similar to procedures described earlier [3e6].
The substituted pyrrolidine ring has to be protected with a suit-
able protecting group (PG) depending on the reaction conditions. In
this study, benzyl (Bn) and tert-butyloxycarbonyl (Boc) groups were
applied. The pyrrolidine and isatin moieties were bridged with a
sulfonyl group using 5-chlorosulfonyl isatin which was achieved in
two steps starting from isatin, i.e. sulfonation using oleum and
chlorination using POCl3 [3]. Finally the corresponding isatin sul-
fonamides were modified with different substituents (Fig. 2). Since
Podichetty et al. demonstrated that (S)-N-butyl- and (S)-N-(4-
fluorobutyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatins
were much more active inhibitors than the parent NH-compounds,
we preferred to attach these groups to our new ligands [5].
Although it was reported that phenoxymethyl derivatives have
higher inhibitory activities towards the enzymes invitro compared to
the methoxymethyl series [3], the latter showed superior inhibition
efficiency in an in vivo cell culture model [4]. Complementary, most
of the N-substituted isatin sulfonamides of the methoxymethyl se-
ries also possessed good to excellent enzyme inhibition invitro [4e7].
Thus, (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (1) was
selected as lead compound. Recently, we and others have reported on
the feasible application of N-fluoroalkylated isatin sulfonamides as
new radiotracers which could be used for the noninvasive molecular
imaging by positron emission tomography (PET). This putatively al-
lows the visualization and therapy monitoring of activated caspases
in corresponding diseases in vivo with high specificity [9e11].
However, our recent metabolism study using electrochemistry-
liquid chromatography/mass spectrometry showed that the
N-position of the isatin is labile for debenzylation and the
methoxymethyl moiety tends to terminal demethylation during
oxidative phase I metabolism [12]. Radiofluorination at those posi-
tions yielding corresponding PET-compatible radiotracers could
result in a partial loss of 18F-activity in vivo and hence in an insuffi-
cient deposit of the tracer in the tissue to be noninvasively examined.
As a consequence, a weak PET signal will be observed in vivo.
Therefore, fluorination at the pyrrolidine ring might be an alternative
approach to overcome the aforementioned problems.
2.1.1. (2S)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin derivatives
The absolute configuration of inhibitors is important for enzyme
binding potencies. Therefore, two target diastereomers of N-Boc-
pyrrolidines 4a and 4b were synthesized. Their preparations were
smoothly performed by reduction of the N-Boc-protected 4-
hydroxyproline esters 2a or 2b with LiBH4 in THF followed by
dimethylation of the formed alcohols 3a or 3b [13] using methyl
iodide in the presence of NaH with good yields as illustrated in
Scheme 1.
The trans-N-Boc- and cis-N-Boc-pyrrolidines 4a and 4b were
deprotected under acidic conditions using TFA in DCM resulting in
the corresponding free amines, which were coupled with 5-
chlorosulfonyl isatin using DIPEA leading to (2S,4R)- and (2S,4S)-5-
[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfonyl]isatins (20a
Fig. 2. Retrosynthetic analysis of 5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin derivatives.

564
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
Scheme 1. Synthetic route to (2S,4R)- and (2S,4S)-N-tert-butyloxycarbonyl-4-methoxy-2-methoxymethylpyrrolidine (4a and 4b).
and 20b) with 62% and 64% yields, respectively. Subsequently, the
obtained compounds were N-alkylated with 1-bromobutane and 1-
bromo-4-fluorobutane using Cs2CO3 and microwave irradiation to
furnish the corresponding inhibitors 21a, 22a, 21b and 22b in good
to excellent yields (see Table 1).
The u-tosylated tetraethyleneglycol monomethyl ether 5 was
obtained by monomethylation and tosylation of tetraethylenegly-
col. Nucleophilic substitution of the (2S,4R)- and (2S,4S)-N-Boc-4-
hydroxyproline esters 2a and 2b with 5 was performed using
NaH in THF leading to the PEG4ylated hydroxyproline derivatives 6a
and 6b in moderate yields. Similar to the synthesis of the 4-
methoxypyrrolidine derivatives, after reduction and methylation
using the conditions described above, the diastereomeric 4-OPEG4
pyrrolidines 8a and 8b were obtained in good yields as depicted in
Scheme 2.
After Boc-deprotection the coupling reactions with 5-
chlorosulfonyl isatin gave the target 4-OPEG4 pyrrolidinyl sulfonyl
isatins 23a and 23b in low yields (see Table 1). The corresponding
(2S,4S)-N-butyl-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)
sulfonyl]isatin (24) was obtained with 69% yield by microwave
accelerated alkylation. Unfortunately, compound 24 was shown to
2.1.2. (2S)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)sulfonyl]
isatin derivatives
Metabolic resistance and delivery of drugs in vivo are an
important topic intensively discussed in drug development today.
One method, which helps to improve the metabolic stability and
enhance the hydrophilicity of compounds, is the introduction of a
polar polyethyleneglycol (PEG) unit to the structure. PEG4ylated
(PEG4 ¼ tetraethyleneglycol) compounds are more polar and may
limit the accessibility for other enzymes and antibodies [14]. Thus,
PEG4 was attached to the 4-position of the pyrrolidine moiety.
Table 1
Synthesis and caspase inhibition potencies of compounds 19e33 expressed as IC50 values [mM].
Comp
Y
Z
R
Yielda [%]
IC50 [mM]
Caspase-3
Caspase-7
14
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
n.d.
62
72
72
64
85
91
20
40
69
27
84
91
64
92
96
51
75
46
82
28
86
89
73
77
14
68
54
0.0849 ꢀ 0.0256
>500
28.5 ꢀ 2.0
33.4 ꢀ 1.5
>500
81.1 ꢀ 6.2
42.6 ꢀ 5.6
>500
>500
>500
>500
18.1 ꢀ 3.9
31.8 ꢀ 3.0
101 ꢀ 9
1.290 ꢀ 0.466
>500
26.4 ꢀ 1.3
59.6 ꢀ 13.3
>500
20a
21a
22a
20b
21b
22b
23a
23b
24
25a
26a
27a
25b
26b
27b
28a
29a
28b
29b
30
(R)-OMe
(R)-OMe
(R)-OMe
(S)-OMe
(S)-OMe
(S)-OMe
(R)-OPEG4
(S)-OPEG4
(S)-OPEG4
(R)-CF3
(R)-CF3
(R)-CF3
(S)-CF3
(S)-CF3
(S)-CF3
(R)-F
(R)-F
(S)-F
(S)-F
F,F
F,F
H
H
H
CH2(CH2)2CH3
CH2(CH2)2CH2F
H
CH2(CH2)2CH3
CH2(CH2)2CH2F
H
H
>500
42.6 ꢀ 104
>500
>500
>500
>500
52.7 ꢀ 5.9
67.7 ꢀ 5.1
n.d.b
78.3 ꢀ 1.7
147 ꢀ 12
8.46 ꢀ 0.84
1.88 ꢀ 0.09
12.1 ꢀ 0.9
1.25 ꢀ 0.08
14.9 ꢀ 2.5
0.178 ꢀ 0.049
>500
CH2(CH2)2CH3
H
CH2(CH2)2CH3
CH2(CH2)2CH2F
H
CH2(CH2)2CH3
CH2(CH2)2CH2F
H
CH2(CH2)2CH3
H
CH2(CH2)2CH3
H
CH2(CH2)2CH3
H
CH2(CH2)2CH3
CH2(CH2)2CH2F
H
CH2(CH2)2CH3
CH2(CH2)2CH2F
16.5 ꢀ 1.1
24.5 ꢀ 0.6
2.39 ꢀ 0.94
0.795 ꢀ 0.036
1.13 ꢀ 0.57
0.961 ꢀ 0.013
2.57 ꢀ 0.07
0.362 ꢀ 0.019
>500
H
H
H
H
H
H
31
19a
32a
33a
19b
32b
33b
(R)-CH2OMe
(R)-CH2OMe
(R)-CH2OMe
(S)-CH2OMe
(S)-CH2OMe
(S)-CH2OMe
>500
>500
>500
>500
>500
>500
H
H
H
5.99 ꢀ 0.19
11.9 ꢀ 2.0
8.84 ꢀ 1.44
47.8 ꢀ 3.1
a
Yield of the last step.
n.d. ¼ not determined.
b

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
565
Scheme 2. Synthesis of the 4-OPEG4 pyrrolidine derivatives 8a and 8b.
be biologically inactive (see Table 1). Thus, no further analogues of
this class were prepared.
2.1.3. (2S)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin derivatives
Trifluoromethyl substituents are known to enhance or modify
the biological activities in many cases. The stereoselective 4-
trifluoromethylation of L-proline was reported by Del Valle and
Goodman [15]. According to these authors, the key intermediate 9
was synthesized in 6 steps (Scheme 3) starting from (2S,4R)-N-Boc-
4-hydroxyproline methyl ester (2a). Subsequently, compound 9
was transformed to the diastereomeric 4-trifluoromethylprolinols
11a and 11b depending on the catalyst used in the hydrogenation
step. Under standard conditions using 10% Pd on activated charcoal,
the hydrogen atoms were added from the sterically less hindered
side of the ring resulting in the formation of the cis-trifluoromethyl
alcohol 11b. In contrast, after TBS deprotection, Crabtree’s catalyst
was applied in the hydrogenation of 10 to produce the desired
trans-isomer 11a. Methylation of both diastereomers led to the
target (2S,4R)- and (2S,4S)-N-Boc-4-trifluoromethyl-2-methoxy
methylpyrrolidines (12a and 12b) with good to excellent yields.
The succeeding coupling with 5-chlorosulfonyl isatin was per-
formed to get the corresponding trifluoromethyl compounds 25a
and 25b in 27% and 64% yields, respectively. Binding of n-butyl and
4-fluorobutyl substituents to the isatin nitrogen was achieved using
Cs2CO3 and 1-bromobutane or 1-bromo-4-fluorobutane in dry DMF
under microwave irradiation to obtain compounds 26a, 27a, 26b
and 27b in very good yields (Table 1).
2.1.4. (2S)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin derivatives
The introduction of a single fluorine to a lead structure generally
results in a modification of the chemical and physical properties of
compounds and moreover, might allow to radiolabel the ligands
with fluorine-18 for PET imaging. According to literature pro-
cedures [16], fluoroprolinols 13a and 13b were obtained smoothly.
Subsequent methylation of fluoroalcohols 13a and 13b using MeI
and NaH gave the target fluoropyrrolidines 14a and 14b in excellent
yields. The gem-difluoropyrrolidinyl methyl ester 15 was prepared
as described earlier [17] and was converted to the difluoroether 16
Scheme 3. Synthetic route to prepare trifluoromethylpyrrolidine methyl ester 12a
and 12b.

566
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
Scheme 4. Synthetic pathways to the fluorinated and difluorinated pyrrolidines 14a, 14b and 16.
by reduction with NaBH4 in the presence of LiCl followed by
methylation (Scheme 4).
Prior to coupling, N-debenzylation was tried under different
conditions. With 5% Pd(OH)2 and 10% Pd on activated charcoal as
catalysts under H2 atmosphere (5 atm), no conversion was
observed after overnight stirring at room temperature. Fortu-
nately, the hydrogenolytic debenzylation was performed success-
fully by stirring the mixture with 10% Pd on activated charcoal
under 20 atm of H2 atmosphere for 6 h. Subsequent coupling of the
unprotected pyrrolidines with 5-chlorosulfonyl isatin yielded the
cis- and trans-bis(methoxymethyl)pyrrolidinylsulfonyl isatins 19a
and 19b in 89% and 14% yields over two steps, respectively, as
shown in Scheme 5. Finally the N-alkylation resulted in the desired
analogues 32a, 33a, 32b and 33b in good to excellent yields
(Table 1).
Subsequent coupling of all fluorinated pyrrolidines with the
isatin fragment gave compounds 28a, 28b and 30 in moderate
yields (Table 1). The N-alkylation with n-butylbromide was per-
formed under microwave irradiation obtaining the corresponding
fluorinated inhibitors 29a, 29b and 31 in good to excellent yields
(Table 1).
2.1.5. (2S)-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}
isatin derivatives
The CS-symmetric cis- and C2-symmetric (2S,4S)-N-benzyl-2,5-
bis(hydroxymethyl)pyrrolidines (17a and 17b) were prepared
starting from adipoyl chloride as reported by Sibi and Lu [18]. The
cis-diol 17a was directly dimethylated using MeI and NaH to pro-
duce the corresponding N-benzyl dimethylether 18a, whereas the
enantiopure (2S,4S)-diol 17b (trans-configuration) was obtained
by enzymatic acetylation using lipase PS and vinyl acetate in hex-
ane/DCM [18]. Subsequent dimethylation smoothly led to the cor-
responding (2S,4S)-N-benzyl-2,5-bis(methoxymethyl)pyrrolidine
(18b) (Scheme 5).
2.2. Study of in vitro biological activity
Various substituents were attached to the pyrrolidine ring of the
lead structure 1 to evaluate the inhibition potencies for caspases-3
and -7 indicated as IC50 values. As a result, several of the N-unsub-
stituted compounds were active only in millimolar concentra-
tions. Interestingly, the N-unsubstituted mono- and difluorinated
Scheme 5. Synthetic routes to the symmetric pyrrolidinylisatins 19a and 19b.

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
567
analogues exhibited caspase inhibition with IC50 in one-digit
micromolar range.
were performed in closed glass vessels using a CEM Discovery
microwave machine.
The activities increased to mM or nM values by attachment of
n-butyl and 4-fluorobutyl groups to the isatin nitrogen. In most of
the cases the N-butyl group led to slightly higher activities than
the N-(4-fluorobutyl) one. However, they are weaker inhibitors
compared to the lead compound. 4-methoxy and 4-
4.2. General procedures
4.2.1. Methylation of N-protected prolinols
Under argon atmosphere, a stirred solution of the N-pro-
trifluormethylated analogues showed IC50 values in the
m
M
tected L-prolinol (1.0 mmol) in dry THF (6 mL) was cooled
range, whereas the 4-OPEG4 and cis-5-methoxymethyl analogues
were inactive. The trans-5-methoxymethylisatin derivatives
showed affinities in the micromolar scale. Among the ligands
substituted at the pyrrolidine ring, the N-butyl mono- and
difluorinated ones exhibited high activity in the nM scale, which
is 100e1000 times better than that of the 4-OMe and 4-CF3
compounds. The 4,4-difluoropyrrolidinyl derivative 31 showed
the best result with IC50 ¼ 362 nM and 178 nM for caspases-3
and -7, respectively. In addition we observed that the inhibition
potencies for (R)- and (S)-substituted ligands do not differ
(Table 1).
to ꢁ10 ꢂC and treated with NaH (1.5 mmol, 60% in mineral oil).
After stirring for 15 min, MeI (1.5 mmol) was added dropwise to
the above suspension and the resulting reaction mixture was
stirred at this temperature for 30 min, at 0 ꢂC for 3 h and at r.t.
for further 18 h. After this time, the reaction mixture was cooled
to 0 ꢂC and quenched with sat. aq. NH4Cl until no more H2
evolved. Subsequently, H2O (30 mL) was added and the
mixture was extracted with EtOAc (3 ꢃ 40 mL). The combined
organic phases were washed with brine (1 ꢃ 40 mL), dried
over MgSO4 and concentrated under reduced pressure. The
crude product was purified by flash column chromatography
to obtain the corresponding N-protected methoxymethylpy
rrolidines.
3. Conclusion
4.2.2. Coupling of the pyrrolidines with 5-chlorosulfonyl isatin
4-Methoxy, 4-OPEG4, 4-trifluoromethyl, 4-fluoro, 4,4-difluoro
and 5-methoxymethylpyrrolidinylsulfonylisatin derivatives were
synthesized and tested for their caspase inhibition potencies
in vitro. Their inhibitory properties for the executioner caspases-
3 and -7 are ranging in the micromolar scale. Altogether the here
presented isatin sulfonamides are less active for both enzymes
compared to the lead compound 1. These findings confirm a
dramatic decrease of the biological activity by any oxygen-
containing substituents at the 4- and 5-position of the hydro-
phobic pyrrolidine moiety. However, as a main finding, we have
proven that the 4-fluorinated and 4,4-difluorinated analogues
indeed exhibited maintained caspase inhibition potencies in
the nanomolar range, though they are generally less active
compared to compounds bearing fluorinated groups in the 2-
position [19] or at the isatin nitrogen [5,6,20].
To
a stirred solution of the N-Boc-protected pyrrolidine
(1.0 mmol) in dry DCM (3 mL) at 0 ꢂC under argon atmosphere, TFA
(1.14 g, 10.0 mmol) was added dropwise. The obtained solution was
stirred at 0 ꢂC for 30 min followed by stirring at r.t. for 2 h. Sub-
sequently, the solution was poured to cold 10% aq. NaOH (15 mL)
and extracted with DCM (3 ꢃ 15 mL). The combined organic phases
were dried over MgSO4 and concentrated under reduced pressure
resulting in the corresponding unprotected pyrrolidine, which was
used in the next step without further purification.
The free pyrrolidine (ca. 1 mmol) from the previous step was
then taken up in CHCl3 (2.2 mL) and DIPEA (2.0 mmol) was added.
The solution was added dropwise to a stirred solution of 5-
chlorosulfonyl isatin (1.5 mmol) in CHCl3/THF (1:1, 15 mL) at
room temperature. The resulting mixture was stirred further for 1 h
and the solvent was removed under reduced pressure. After puri-
fication by flash column chromatography the target isatin sulfon-
amide was obtained.
4. Experimental section
4.1. Materials and methods
4.2.3. N-Alkylation of the isatin moiety with alkyl bromides using
microwave irradiation
All starting materials, reagents and solvents for the syntheses
were analytical grade and used without further purification. 1-
bromo-4-fluorobutane was purchased from Apollo Scientific.
Absolute DMF was obtained from Acros. For all moisture sensi-
tive reactions, the glassware was heated under high vacuum
prior use and the transformation was performed under argon
atmosphere. 1H NMR (300 MHz, 400 MHz, 500 MHz and
600 MHz), 13C NMR (75 MHz, 100 MHz, 125 MHz and 150 MHz)
and 19F NMR (282 MHz) spectra were recorded using Bruker
machines in CDCl3 or CD3OD with TMS for 1H NMR, CDCl3 or
CD3OD for 13C NMR and CFCl3 for 19F NMR as internal standards.
DEPT, COSY, HSQC and HMBC spectra were recorded to assign
the signals in the complex structures. All chemical shifts were
indicated in ppm. Exact mass analyses were recorded with a
Bruker MicroTOF apparatus. All spectroscopic and analytical in-
vestigations were performed by staff members of the Organic
Chemistry Institute, University of Münster. Thin layer chroma-
tography (TLC) analyses were performed on silica coated
aluminium foils (Silica gel 60 F254) with 0.2 mm layer thickness
from MERCK. Silica gel (60e120 mesh) was used for column
chromatography. High pressure hydrogenation was performed in
an autoclave apparatus. Reactions using microwave irradiation
In a thick-glass vessel for microwave reaction, a stirred solution
of the N-unsubstituted isatin sulfonamide (0.2 mmol) in dry DMF
(5.5 mL) under argon atmosphere was treated with Cs2CO3
(0.4 mmol) at room temperature. After stirring for 15 min, it turned
purple and 1-bromobutane or 1-bromo-4-fluorobutane (2 mmol
was added. The vessel was sealed with a PE-cap and the obtained
mixture was stirred at 95 ꢂC for 10 min under microwave irradia-
tion. The precipitate was removed by filtration and the filtrate was
concentrated under reduced pressure. The crude was purified by
flash column chromatography to obtain the desired product.
4.3. (2S,4R)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo
nyl]isatins

568
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
3
4.3.1. (2S,4R)-N-tert-butyloxycarbonyl-4-hydroxy-2-hydroxymethyl
pyrrolidine (3a)
(d, JH,H ¼ 3.1 Hz, 2H, 15-CH2), 3.38 (s, 3H, 18-CH3), 3.04 (s, 3H,
20-CH3), 2.05e1.95 (m, 2H, 13-CH2) ppm. 13C NMR (100 MHz,
A stirred solution of methyl (2S,4R)-N-tert-butyloxycarbonyl-4-
hydroxypyrrolidine-2-carboxylate (2a) (500 mg, 2.04 mmol, 1.00
equiv.) in dry THF (7 mL) was cooled to 0 ꢂC under argon atmosphere
and treated dropwise with 2 M LiBH4 in THF solution (2.0 mL,
4.08 mmol, 2.00 equiv.). The resulting solution was allowed to warm
to r.t. and stirred for 18 h. After this time, the reaction mixture was
quenched by very slow addition of water (5 mL) and the resulting
clear solution was extracted with DCM (5 ꢃ 15 mL). The combined
organic phases were washed with sat. aq. NaHCO3 (1 ꢃ10 mL), brine
(1 ꢃ 10 mL) and dried over MgSO4. After removal of the solvent, the
crude product was purified by flash column chromatography (silica
gel,10% acetone in EtOAc) to obtain a colorless oil (384 mg,1.77 mmol,
CDCl3):
d
¼
182.3 (3-CO), 159.1 (2-CO), 152.4 (8-CN), 138.0
(6-CH), 134.0 (5-CSO2), 125.2 (4-CH), 117.5 (9-CCO), 112.8 (7-CH),
78.7 (14-CH), 74.9 (16-CH2), 59.3 (18-CH3), 58.6 (12-CH), 56.3
(20-CH3), 54.0 (15-CH2), 35.1 (13-CH2) ppm. HRMS (ESIþ, MeOH):
m/z ¼ 377.0785 [M þ Na]þ, 409.1046 [M þ Na þ MeOH]þ;
calcd. 377.0778 for
C15H18N2O6S
þ
Na, 409.1040 for
C15H18N2O6S þ Na þ MeOH.
4.3.4. (2S,4R)-N-butyl-5-[1-(4-methoxy-2-methoxymethylpyrrolidi
nyl)sulfonyl]isatin (21a)
(2S,4R)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo-
nyl]isatin (20a) (60 mg, 0.169 mmol, 1.00 equiv.) was converted to
(2S,4R)-N-butyl-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (21a) using 1-bromobutane and Cs2CO3 as described
in the general procedure in Section 4.2.3. The crude product was
purified by flash column chromatography (silica gel, 50% EtOAc in
cyclohexane) to obtain a yellow-orange solid (50 mg, 0.122 mmol,
87%). 1H NMR (300 MHz, CDCl3):
d
¼ 4.43e4.29 (m, 1H, 4-CH), 4.21e
4.02 (m,1H, 2-CH), 3.80 (br s, 2H, 4-COH,10-COH), 3.74e3.48 (m, 2H,
5-CH2), 3.57 (dd, 2JH,H ¼ 11.4 Hz, 3JH,H ¼ 6.5 Hz, 1H, 10-CHa), 3.41 (dd,
2JH,H ¼ 12.0 Hz, 3JH,H ¼ 4.2 Hz, 1H, 10-CHb), 2.14e1.55 (m, 2H, 3-CH2),
1.47 (s, 9H, 9-CH3) ppm. 13C NMR (75 MHz, CDCl3):
d
¼ 155.5 (6-CO),
80.6 (8-CO), 69.1 (4-CH), 60.5 (10-CH2), 58.6(2-CH), 55.7(5-CH2), 37.4
(3-CH2), 28.4 (3C, 9-CH3) ppm. HRMS (ESIþ, MeOH): m/z ¼ 240.1204
[M þ Na]þ; calcd. 240.1206 for C10H19NO4þNa.
72%). M.p. 116 ꢂC. 1H NMR (500 MHz, CDCl3):
d
¼ 8.07 (dd,
3JH,H ¼ 8.3 Hz, 4JH,H ¼ 1.9 Hz,1H, 6-CH), 8.04 (d, 4JH,H ¼ 1.6 Hz, 4-CH),
7.01 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-CH), 3.89e3.79 (m, 1H, 14-CH), 3.78 (t,
3JH,H ¼ 7.6 Hz, 2H, 21-CH2), 3.77e3.73 (m, 1H, 12-CH), 3.64 (dd,
3
2
4.3.2. (2S,4R)-N-tert-butyloxycarbonyl-4-methoxy-2-methoxymeth
ylpyrrolidine (4a)
2JH,H ¼ 9.6 Hz, JH,H ¼ 3.2 Hz, 1H, 16-CHa), 3.55 (dd, JH,H ¼ 9.6 Hz,
3JH,H ¼ 6.1 Hz, 1H, 16-CHb), 3.47 (d, 3JH,H ¼ 3.2 Hz, 2H, 15-CH2), 3.38
(s, 3H, 18-CH3), 3.04 (s, 3H, 20-CH3), 2.06e2.00 (m, 1H, 13-CHa),
2.00e1.94 (m, 1H,13-CHb), 1.70 (quintet, 3JH,H ¼ 7.6 Hz, 2H, 22-CH2),
1.42 (sextet, 3JH,H ¼ 7.4 Hz, 2H, 23-CH2), 0.98 (t, 3JH,H ¼ 7.4 Hz, 3H,
A
stirred solution of (2S,4R)-N-tert-butyloxycarbonyl-4-
hydroxy-2-hydroxymethylpyrrolidine (3a) (180 mg, 0.829 mmol,
1.00 equiv.) in dry THF (8 mL) under argon atmosphere was cooled
to ꢁ10 ꢂC and treated with 60% NaH (165 mg, 4.14 mmol, 5.00
24-CH3) ppm. 13C NMR (125 MHz, CDCl3):
d
¼ 182.4 (3-CO), 158.0
equiv.). After stirring for 15 min, MeI (206
m
L, 3.32 mmol, 4.00
(2-CO), 153.8 (8-CN), 137.9 (6-CH), 134.0 (5-CSO2), 125.1 (4-CH),
117.3 (9-CCO), 110.2 (7-CH), 78.9 (14-CH), 75.1 (16-CH2), 59.5 (18-
CH3), 58.7 (12-CH), 56.5 (20-CH3), 54.1 (15-CH2), 40.6 (21-CH2),
35.3 (13-CH2), 29.4 (22-CH2), 20.3 (23-CH2), 13.8 (24-CH3) ppm.
HRMS (ESIþ, MeOH): m/z ¼ 433.1407 [M þ Na]þ, 465.1667
[M þ Na þ MeOH]þ; calcd. 433.1404 for C19H26N2O6S þ Na,
465.1666 for C19H26N2O6S þ Na þ MeOH.
equiv.) was added dropwise. The reaction mixture was stirred at
this temperature for 30 min, 0 ꢂC for 1 h and r.t. for 18 h. After this
time, the mixture was cooled to 0 ꢂC and quenched with a small
amount of sat. aq. NH4Cl until no more H2 evolved. Subsequently,
water (10 mL) was added and the obtained mixture was extracted
with EtOAc (3 ꢃ 15 mL). The combined organic phases were
washed with water (1 ꢃ 10 mL), brine (1 ꢃ 10 mL) and dried over
MgSO4. After removal of the solvent, the residue was purified by
flash column chromatography (silica gel, 5% THF in DCM) to obtain
a colorless oil (171 mg, 0.697 mmol, 84%). 1H NMR (400 MHz,
4.3.5. (2S,4R)-N-(4-fluorobutyl)-5-[1-(4-methoxy-2-methoxymethyl
pyrrolidinyl)sulfonyl]isatin (22a)
(2S,4R)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo-
nyl]isatin (20a) (60 mg, 0.169 mmol, 1.00 equiv.) was converted to
(2S,4R)-N-(4-fluorobutyl)-5-[1-(4-methoxy-2-methoxymethylpyrr
olidinyl)sulfonyl]isatin (22a) using 1-bromo-4-fluorobutane and
Cs2CO3 as described in the general procedure in Section 4.2.3. The
crude product was purified by flash column chromatography (silica
gel, 75% EtOAc in cyclohexane) to obtain a yellow-orange solid
(52 mg, 0.121 mmol, 72%). M.p. 120 ꢂC. 1H NMR (500 MHz, CDCl3):
CDCl3):
d
¼ 4.13e3.96 (m, 1H, 2-CH), 4.12e3.81 (m, 1H, 4-CH),
3.69e3.23 (m, 2H, 5-CH2), 3.55e3.45 (m, 2H, 10-CH2), 3.34 (s, 3H,
12-CH3), 3.31 (s, 3H, 14-CH3), 2.18e1.95 (m, 2H, 3-CH2), 1.47 (s, 9H,
9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 154.6 (6-CO), 79.5 (8-
CO), 78.2 (4-CH), 73.3 (10-CH2), 59.2 (12-CH3), 56.7 (14-CH3), 55.6
(2-CH), 51.1 (5-CH2), 33.9 (3-CH2), 28.5 (3C, 9-CH3) ppm.
HRMS (ESIþ, MeOH): m/z ¼ 268.1515 [M þ Na]þ, 513.3142
[2M þ Na]þ; calcd. 268.1519 for C12H23NO4 þ Na, 513.3146 for
2(C12H23NO4) þ Na.
3
4
d
¼ 8.07 (dd, JH,H ¼ 8.3 Hz, JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.04 (d,
4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.04 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-CH), 4.52 (dt,
2JH,F ¼ 47.3 Hz, JH,H ¼ 5.6 Hz, 2H, 24-CH2), 3.87e3.82 (m, 1H, 14-
3
3
4.3.3. (2S,4R)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo
nyl]isatin (20a)
CH), 3.84 (t, JH,H ¼ 7.1 Hz, 2H, 21-CH2), 3.78e3.74 (m, 1H, 12-CH),
2
3
3.64 (dd, JH,H ¼ 9.6 Hz, JH,H ¼ 3.2 Hz, 1H, 16-CHa), 3.55 (dd,
2JH,H ¼ 9.6 Hz, 3JH,H ¼ 6.1 Hz, 1H, 16-CHb), 3.47 (d, 3JH,H ¼ 3.2 Hz, 2H,
15-CH2), 3.38 (s, 3H, 18-CH3), 3.04 (s, 3H, 20-CH3), 2.06e1.96 (m,
2H, 13-CH2), 1.89 (quintet, 3JH,H ¼ 7.1 Hz, 2H, 22-CH2), 1.88e1.73 (m,
(2S,4R)-N-tert-butyloxycarbonyl-4-methoxy-2-methoxymethyl-
pyrrolidine (4a) (300 mg, 1.22 mmol, 1.00 equiv.) was converted to
(2S,4R)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (20a) using TFA followed by the reaction with 5-
chlorosulfonyl isatin and DIPEA as described in the general proce-
dure in Section 4.2.2. The crude product was purified by flash col-
umn chromatography (silica gel, gradient DCM,10% THF in DCM and
20% THF in DCM) to obtain a yellow wax (267 mg, 0.753 mmol, 62%).
2H, 23-CH2) ppm. 13C NMR (125 MHz, CDCl3):
d
¼ 182.2 (3-CO),
158.1 (2-CO), 153.5 (8-CN), 138.0 (6-CH), 134.2 (5-CSO2), 125.1
1
(4-CH), 117.3 (9-CCO), 110.2 (7-CH), 83.4 (d, JC,F ¼ 165.5 Hz, 24-
CH2), 78.9 (14-CH), 75.1 (16-CH2), 59.5 (18-CH3), 58.7 (12-CH),
56.5 (20-CH3), 54.1 (15-CH2), 40.3 (21-CH2), 35.2 (13-CH2), 27.8 (d,
1H NMR (400 MHz, CDCl3):
d
¼ 9.37 (br s, 1H, 1-NH), 8.02 (dd,
2JC,F ¼ 20.1 Hz, 23-CH2), 23.7 (d, JC,F ¼ 4.0 Hz, 22-CH2) ppm. 19F
3
3JH,H ¼ 8.8 Hz, 4JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.02 (d, 4JH,H ¼ 1.9 Hz, 1H, 4-
NMR (282 MHz, CDCl3):
d
¼ ꢁ219.9 (s, 1F, 24-CH2F) ppm. HRMS
3
CH), 7.07 (d, JH,H ¼ 8.8 Hz, 1H, 7-CH), 3.87e3.81 (m, 1H, 14-CH),
(ESIþ, MeOH): m/z ¼ 451.1303 [M þ Na]þ, 483.1569 [M þ
2
3
3.81e3.71 (m, 1H, 12-CH3), 3.64 (dd, JH,H ¼ 9.7 Hz, JH,H ¼ 3.3 Hz,
1H, 16-CHa), 3.56 (dd, 2JH,H ¼ 9.7 Hz, 3JH,H ¼ 5.9 Hz,1H, 16-CHb), 3.47
Na þ MeOH]þ; calcd. 451.1310 for C19H25FN2O6S þ Na, 483.1572 for
C19H25FN2O6S þ Na þ MeOH.

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
569
4.4. (2S,4S)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo
nyl]isatins
4.4.3. (2S,4S)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfon
yl]isatin (20b)
(2S,4S)-N-tert-butyloxycarbonyl-4-methoxy-2-methoxymethyl
pyrrolidine (4b) (200 mg, 0.815 mmol, 1.00 equiv.) was converted
to (2S,4S)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo-
nyl]isatin (20b) using TFA followed by the reaction with 5-chloro
sulfonyl isatin and DIPEA as described in the general procedure
in Section 4.2.2. The crude product was purified by flash column
chromatography (silica gel, gradient DCM, 10% THF in DCM and
then 20% THF in DCM) to obtain a yellow solid (185 mg,
0.522 mmol, 64%). M.p. 77e78 ꢂC. 1H NMR (400 MHz, CDCl3):
4.4.1. (2S,4S)-N-tert-butyloxycarbonyl-4-hydroxy-2-hydroxymethyl
pyrrolidine (3b)
d
¼ 9.54 (s, 1H, 1-NH), 8.06 (dd, 3JH,H ¼ 8.2 Hz, 4JH,H ¼ 1.9 Hz, 1H, 6-
CH), 8.03 (d, 4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.20 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-
A stirred solution of methyl (2S,4S)-N-tert-butyloxycarbonyl-4-
hydroxypyrrolidine-2-carboxylate (2b) (400 mg, 1.63 mmol, 1.00
equiv.) in THF (6 mL) were cooled to 0 ꢂC and treated with 2 M
LiBH4 in THF solution (1.63 mL, 3.25 mmol, 2.00 equiv.). The
resulting mixture was stirred at r.t. for 18 h. After this time, the
milky mixture was cooled to 0 ꢂC, quenched with 10% aq. citric acid
and adjusted to pH 4. The solvent was removed under reduced
pressure and the residue was taken up in water (15 mL). It was then
extracted with DCM (3 ꢃ 15 mL). The combined organic phase was
dried over MgSO4 and concentrated under reduced pressure to
obtain colorless oil (312 mg, 1.43 mmol, 88%). The crude product
was used in the next step without further purification. 1H NMR
CH), 3.91e3.83 (m, 1H, 12-CH), 3.88e3.81 (m, 1H, 14-CH), 3.66 (dd,
2JH,H ¼ 9.1 Hz, JH,H ¼ 5.3 Hz, 1H 16-CHa), 3.50e3.44 (m, 1H, 15-
3
CHa), 3.48e3.41 (m, 1H, 16-CHb), 3.37e3.31 (m, 1H, 15-CHb),
3.34 (s, 3H, 18-CH3), 3.28 (s, 3H, 20-CH3), 2.14e2.05 (m,
1H, 13-CHa), 1.87e1.76 (m, 1H, 13-CHb) ppm. 13C NMR
(100 MHz, CDCl3):
d
¼ 182.3 (3-CO), 159.0 (2-CO), 152.7 (8-CN),
137.7 (6-CH), 133.7 (5-CSO2), 124.9 (4-CH), 117.7 (9-CCO), 113.4
(7-CH), 79.3 (14-CH), 75.0 (16-CH2), 58.9 (18-CH3), 58.5 (12-
CH), 56.7 (20-CH3), 53.7 (15-CH2), 33.3 (13-CH2) ppm. HRMS
(ESIþ, MeOH): m/z
¼
377.0777 [M
þ
Na]þ, 409.1038
[M þ Na þ MeOH]þ; calcd. 377.0778 for C15H18N2O6S þ Na,
409.1040 for C15H18N2O6S þ Na þ MeOH.
(400 MHz, CDCl3):
d
¼ 4.84 (br s, 2H, 4-COH, 10-COH), 4.35e4.25
2
(m, 1H, 4-CH), 4.05e3.90 (m, 1H, 2-CH), 4.03 (dd, JH,H ¼ 11.3 Hz,
3JH,H ¼ 2.6 Hz, 1H, 10-CHa), 3.63e3.46 (m, 1H, 10-CHb), 3.56e3.35
(m, 2H, 5-CH2), 2.41e2.24 (m, 1H, 3-CHa), 1.99e1.79 (m, 1H, 3-CHb),
4.4.4. (2S,4S)-N-butyl-5-[1-(4-methoxy-2-methoxymethylpyrrolidin
yl)sulfonyl]isatin (21b)
(2S,4S)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (20b) (50 mg, 0.141 mmol,1.00 equiv.) was converted to (2S,4S)-
N-butyl-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (21b) using 1-bromobutane and Cs2CO3 as described in the
general procedure in Section 4.2.3. The crude product was purified by
flash column chromatography (silica gel, 60% EtOAc in cyclohexane)
to obtain a yellow-orange solid (49 mg, 0.119 mmol, 85%). M.p.119 ꢂC.
1.46 (s, 9H, 9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 155.7 (6-
CO, rotamer A), 154.9 (6-CO, rotamer B), 80.1 (8-CO, rotamer B),
80.0 (8-CO, rotamer A), 69.7 (4-CH, rotamer A), 68.9 (4-CH, rotamer
B), 64.1 (10-CH2, rotamer A), 63.5 (10-CH2, rotamer B), 58.5 (2-CH,
rotamer A), 58.3 (2-CH, rotamer B), 56.7 (5-CH2), 38.1 (3-CH2,
rotamer B), 37.3 (3-CH2, rotamer A), 28.5 (3C, 9-CH3) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 240.1208 [M þ Na]þ; calcd. 240.1206 for
1H NMR (500 MHz, CDCl3):
d
¼ 8.09 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 1.9 Hz,
C
10H19NO4 þ Na.
1H, 6-CH), 8.04 (d, 4JH,H ¼ 1.9 Hz,1H, 4-CH), 7.03 (d, 3JH,H ¼ 8.3 Hz,1H,
7-CH), 3.91e3.86 (m, 1H, 12-CH), 3.86e3.82 (m, 1H, 14-CH), 3.78 (t,
3JH,H ¼ 7.4 Hz, 2H, 22-CH2), 3.65 (dd, 2JH,H ¼ 9.0 Hz, 3JH,H ¼ 5.1 Hz,1H,
16-CHa), 3.47e3.42 (m, 1H, 15-CHa), 3.50e3.40 (m, 1H, 16-CHa), 3.36
(dd, 2JH,H ¼ 11.0 Hz, 3JH,H ¼ 5.2 Hz, 1H, 15-CHb), 3.34 (s, 3H, 18-CH3),
3.28 (s, 3H, 20-CH3), 2.13e2.08 (m, 1H, 13-CHa), 1.83 (ddd,
2JH,H ¼ 14.0 Hz, 3JH,H ¼ 8.7 Hz, 3JH,H ¼ 5.5 Hz,1H,13-CHb),1.70 (quintet,
3JH,H ¼ 7.5 Hz, 2H, 22-CH2), 1.43 (sextet, 3JH,H ¼ 7.4 Hz, 2H, 23-CH2),
0.99 (t, 3JH,H ¼ 7.4 Hz, 3H, 24-CH3) ppm. 13C NMR (125 MHz, CDCl3):
4.4.2. (2S,4S)-N-tert-butyloxycarbonyl-4-methoxy-2-methoxymethyl
pyrrolidine (4b)
Under argon atmosphere, a stirred solution of 3b (300 mg,
1.38 mmol,1.00 equiv.) in dry THF (13 mL) was cooled to ꢁ10 ꢂC and
treated with 60% NaH (275 mg, 6.90 mmol, 5.00 equiv.). After
stirring for 15 min, MeI (343 mL, 5.52 mmol, 4.00 equiv.) was added
dropwise. The resulting mixture was stirred further at this tem-
perature for 30 min, 0 ꢂC for 1 h and r.t. for 24 h. After this time, the
reaction mixture was cooled to 0 ꢂC and quenched with a small
amount of sat. aq. NH4Cl until no more H2 evolved. Water (20 mL)
was added and the resulting mixture was extracted with EtOAc
(3 ꢃ 20 mL). The combined organic phases were washed with water
(10 mL), brine (10 mL) and dried over MgSO4. After removal of the
solvent, the residue was purified by flash column chromatography
(silica gel, 5% THF in cyclohexane) to obtain a light yellow oil
d
¼ 182.3 (3-CO), 157.9 (2-CO), 154.0 (8-CN), 137.6 (6-CH), 134.1 (5-
CSO2), 124.7 (4-CH), 117.5 (9-CCO), 110.6 (7-CH), 79.5 (14-CH), 75.1
(16-CH2), 59.1 (18-CH3), 58.6 (12-CH), 56.9 (20-CH3), 53.7 (15-CH2),
40.7 (21-CH2), 33.6 (13-CH2), 29.4 (22-CH2), 20.3 (23-CH2), 13.8 (24-
CH2) ppm. HRMS (ESIþ, MeOH): m/z ¼ 433.1400 [M þ Na]þ,
465.1661 [M þ Na þ MeOH]þ; calcd. 433.1404 for C19H26N2O6S þ Na,
465.1666 for C19H26N2O6S þ Na þ MeOH.
(277 mg, 1.13 mmol, 82%). 1H NMR (400 MHz, CDCl3):
d
¼ 4.10e3.90
4.4.5. (2S,4S)-N-(4-fluorobutyl)-5-[1-(4-methoxy-2-methoxymethyl
pyrrolidinyl)sulfonyl]isatin (22b)
(m, 1H, 2-CH), 3.95e3.87 (m, 1H, 4-CH), 3.75e3.58 (m, 1H, 10-CHa),
3.66e3.48 (m, 1H, 5-CHa), 3.42e3.35 (m, 2H, 5-CHb, 10-CHb), 3.36
(s, 3H,12-CH3), 3.31 (s, 3H,14-CH3), 2.23e2.10 (m,1H, 3-CHa), 2.08e
1.92 (m, 1H, 3-CHb), 1.47 (s, 9H, 9-CH3) ppm. 13C NMR (100 MHz,
(2S,4S)-5-[1-(4-methoxy-2-methoxymethylpyrrolidinyl)sulfo-
nyl]isatin (20b) (50 mg, 0.141 mmol, 1.00 equiv.) was converted to
(2S,4S)-N-(4-fluorobutyl)-5-[1-(4-methoxy-2-methoxymethylpyrr
olidinyl)sulfonyl]isatin (22b) using 1-bromo-4-fluorobutane and
Cs2CO3 as described in the general procedure in Section 4.2.3. The
crude product was purified by flash column chromatography (silica
gel, 75% EtOAc in cyclohexane) to obtain a yellow-orange solid
(55 mg, 0.129 mmol, 91%). M.p. 114 ꢂC. 1H NMR (500 MHz, CDCl3):
CDCl3):
d
¼ 154.5 (6-CO), 79.8 (4-CH, rotamer B), 79.5 (8-CO), 79.1
(4-CH, rotamer A), 73.9 (10-CH2, rotamer A), 73.2 (10-CH2, rotamer
B), 58.9 (12-CH3), 56.5 (14-CH3), 55.6 (2-CH), 52.4 (5-CH2, rotamer
B), 51.7 (5-CH2, rotamer A), 33.4 (3-CH2, rotamer A), 32.2
(3-CH2, rotamer B), 28.5 (3C, 9-CH3) ppm. HRMS (ESIþ, MeOH):
m/z ¼ 268.1514 [M þ Na]þ, 513.3144 [2M þ Na]þ; calcd. 268.1519
for C12H23NO4 þ Na, 513.3146 for 2(C12H23NO4) þ Na.
3
4
d
¼ 8.09 (dd, JH,H ¼ 8.3 Hz, JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.04 (d,
4JH,H ¼ 1.9 Hz,1H, 4-CH), 7.06 (d, 3JH,H ¼ 8.3 Hz,1H, 7-CH), 3.91e3.84

570
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
(m, 1H, 12-CH), 3.86e3.82 (m, 1H, 14-CH), 3.84 (t, 3JH,H ¼ 7.0 Hz, 2H,
21-CH2), 3.64 (dd, 2JH,H ¼ 9.0 Hz, 3JH,H ¼ 5.1 Hz,1H,16-CHa), 4.52 (dt,
(6a) (697 mg, 1.60 mmol, 1.00 equiv.) in dry THF (8 mL) was
cooled to 0 ꢂC and treated with 4 M LiBH4 in THF (800
L,
m
3
2JH,F ¼ 47.4 Hz, JH,H ¼ 5.6 Hz, 2H, 24-CH2), 3.48e3.41 (m, 2H, 15-
3.20 mmol, 2.00 equiv.) dropwise. The resulting solution was
stirred further at r.t. for 18 h. After this time the mixture was
cooled to 0 ꢂC, quenched with 10% aq. citric acid and adjusted to
pH 4. The organic solvent was removed completely under
reduced pressure. H2O (10 mL) was added to the residue, which
was subsequently extracted with DCM (3 ꢃ 20 mL). The combined
organic phase was dried over MgSO4 and concentrated under
reduced pressure. The crude was purified by flash column chro-
matography (silica gel, 3.5% MeOH in DCM) to obtain a colorless
oil (390 mg, 0.957 mmol, 60%). 1H NMR (300 MHz, CDCl3):
CHa, 16-CHb), 3.39e3.33 (m, 1H, 15-CHb), 3.34 (s, 3H, 18-CH3),
3.28 (s, 3H, 20-CH3), 2.10 (m, 1H, 13-CHa), 1.89 (quintet,
3JH,H ¼ 7.5 Hz, 2H, 22-CH2), 1.87e1.74 (m, 3H, 13-CHb, 23-CH2) ppm.
13C NMR (125 MHz, CDCl3):
d
¼ 182.1 (3-CO), 158.0 (2-CO), 153.7 (8-
CN), 137.7 (6-CH), 134.2 (5-CSO2), 124.8 (4-CH), 117.5 (9-CCO), 110.6
1
(7-CH), 83.4 (d, JC,F ¼ 165.6 Hz, 24-CH2), 79.5 (14-CH), 75.1 (16-
CH2), 59.1 (18-CH3), 58.6 (12-CH), 56.9 (20-CH3), 53.7 (15-CH2),
40.4 (21-CH2), 33.5 (13-CH2), 27.8 (d, 2JC,F ¼ 20.1 Hz, 23-CH2), 23.7
3
(d, JC,F ¼ 4.0 Hz, 22-CH2) ppm. 19F NMR (282 MHz, CDCl3):
d
¼
ꢁ219.9 (s, 1F, 24-CH2F) ppm. HRMS (ESIþ, MeOH):
d
¼ 4.12e3.98 (m, 2H, 2-CH, 4-CH), 3.75e3.50 (m, 20H, 5-CH2, 10-
m/z ¼ 451.1307 [M þ Na]þ, 483.1568 [M þ Na þ MeOH]þ; calcd.
451.1310 for C19H25FN2O6S þ Na, 483.1572 for C19H25FN2O6S þ
Na þ MeOH.
CH2, 8 ꢃ OCH2), 3.38 (s, 3H, OCH3), 2.22e2.06 (m, 1H, 3-CHa),
1.93e1.55 (m, 1H, 3-CHb), 1.47 (s, 9H, 9-CH3) ppm. HRMS (ESIþ,
MeOH): m/z
C
¼
430.2403 [M
þ
Na]þ; calcd. 430.2411 for
19H37NO8 þ Na.
4.5. (2S,4R)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)
sulfonyl]isatins
4.5.3. (2S,4R)-N-tert-butyloxycarbonyl-2-methoxymethyl-4-PEG4
yloxypyrrolidine (8a)
(2S,4R)-N-tert-butyloxycarbonyl-2-hydroxymethyl-4-PEG4ylo-
xypyrrolidine (7a) (400 mg, 0.982 mmol, 1.00 equiv.) was con-
verted to (2S,4R)-N-tert-butyloxycarbonyl-2-methoxymethyl-4-
PEG4yloxypyrrolidine (8a) using MeI and NaH as described in
the general procedure in Section 4.2.1. The residue was purified
by flash column chromatography (silica gel, 3.5% MeOH in DCM)
to obtain
NMR (300 MHz, CDCl3):
a
colorless oil (288 mg, 0.683 mmol, 70%). 1H
d
¼ 4.19e3.86 (m, 2H, 2-CH, 4-CH), 3.70e
4.5.1. Methyl (2S,4R)-N-tert-butyloxycarbonyl-4-PEG4yloxypyrroli
dine-2-carboxylate (6a)
3.37 (m, 20H, 5-CH2, 10-CH2, 8 ꢃ OCH2), 3.38 (s, 3H, OCH3), 3.36
(s, 12-CH3, rotamer B), 3.34 (s, 12-CH3, rotamer A), 2.24e1.93
(m, 2H, 3-CH2), 1.46 (s, 9H, 9-CH3) ppm. HRMS (ESIþ,
Under argon atmosphere, a stirred solution of methyl (2S,4R)-
N-tert-butyloxycarbonyl-4-hydroxypyrrolidine-2-carboxylate (2a)
(1.00 g, 4.08 mmol, 1.00 equiv.) in dry THF (30 mL) was cooled
to ꢁ10 ꢂC, treated with 60% NaH (245 mg, 6.12 mmol, 1.50 equiv. in
mineral oil). After stirring at ꢁ10 ꢂC for 15 min, 2,5,8,11-
tetraoxatridecan-13-yl-p-toluenesulfonate (1.97 g, 5.44 mmol,
1.33 equiv.) was added dropwise. The reaction mixture was stirred
at this temperature for 30 min, 0 ꢂC for 4 h and at r.t. for 24 h. After
this time, the mixture was cooled to 0 ꢂC and quenched by addition
of sat. aq. NH4Cl until no more H2 evolved. Then H2O (40 mL) was
added and the mixture was extracted with EtOAc (3 ꢃ 40 mL). The
combined organic phases were washed with brine (1 ꢃ 40 mL),
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography (silica gel,
3% MeOH in DCM) to obtain a colorless oil (871 mg, 2.00 mmol,
MeOH): m/z
¼
444.2564 [M
þ
Na]þ; calcd. 444.2568 for
C
20H39NO8 þ Na.
4.5.4. (2S,4R)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)
sulfonyl]isatin (23a)
(2S,4R)-N-tert-butyloxycarbonyl-2-methoxymethyl-4-
PEG4yloxypyrrolidine (8a) (250 mg, 0.593 mmol, 1.00 equiv.) was
converted to (2S,4R)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrro-
lidinyl)sulfonyl]isatin (23a) using TFA followed by reaction with 5-
chlorosulfonyl isatin and DIPEA as described in the general proce-
dure in Section 4.2.2. The residue was purified by flash chroma-
tography (silica gel, 5% MeOH in DCM) to obtain a yellow oil (63 mg,
0.119 mmol, 20%). 1H NMR (600 MHz, CDCl3):
d
¼ 9.94 (s, 1H, 1-NH),
3
4
49%). 1H NMR (300 MHz, CDCl3):
d
¼ 4.45e4.03 (m, 2H, 2-CH, 4-
8.03 (dd, JH,H ¼ 8.3 Hz, JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.00 (d,
4JH,H ¼ 1.9 Hz,1H, 4-CH), 7.19 (d, 3JH,H ¼ 8.3 Hz,1H, 7-CH), 3.84e3.81
(m, 1H, 14-CH), 3.80e3.65 (m, 6H, 3 ꢃ OCH2), 3.79e3.75 (m, 1H, 16-
CHa), 3.72e3.66 (m, 1H, 12-CH), 3.63e3.57 (m, 1H, 16-CHb), 3.61e
3.52 (m, 4H, 2 ꢃ OCH2), 3.56e3.51 (m, 1H, 15-CHa), 3.49e3.44 (m,
1H, 15-CHb), 3.46e3.38 (m, 2H, OCH2), 3.41 (s, 3H, 18-CH3), 3.31 (s,
3H, OCH3), 3.26e3.20 (m, 2H, OCH2), 3.18e3.13 (m, 1H, OCHa),
3.06e3.02 (m, 1H, OCHb), 2.13e2.03 (m, 1H, 13-CHa), 1.91 (ddd,
CH), 3.75e3.52 (m, 21H, 5-CH2, 12-CH3, 8 ꢃ OCH2), 3.38 (s, 3H,
OCH3), 2.46e2.16 (m, 1H, 3-CHa), 2.12e1.99 (m, 1H, 3-CHb), 1.49e
1.39 (m, 9H, 9-CH3) ppm. 13C NMR (75 MHz, CDCl3):
d
¼ 173.6 (10-
CO, rotamer A), 173.4 (10-CO, rotamer B), 154.3 (6-CO, rotamer B),
153.7 (6-CO, rotamer A), 80.1 (8-CO, rotamer A), 80.0 (8-CO,
rotamer B), 77.2 (4-CH), 71.9 (OCH2), 70.6 (2C, OCH2), 70.5 (4C,
OCH2), 68.6 (OCH2), 59.0 (OCH3), 58.0 (2-CH, rotamer A), 57.6 (2-
CH, rotamer B), 52.2 (5-CH2, rotamer B), 52.0 (5-CH2, rotamer A),
51.4 (12-CH3, rotamer A), 51.2 (12-CH3, rotamer B), 36.6 (3-CH2),
28.4 (9-CH3, rotamer B), 28.2 (9-CH3, rotamer A) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 458.2360 [M þ Na]þ; calcd. 458.2361 for
2JH,H ¼ 13.7 Hz, 3JH,H ¼ 9.1 Hz, 3JH,H ¼ 4.2 Hz, 1H, 13-CHb) ppm. 13
C
NMR (150 MHz, CDCl3):
d
¼ 183.2 (3-CO), 158.8 (2-CO), 153.7 (8-
CN), 137.8 (6-CH), 132.5 (5-CSO2), 124.7 (4-CH), 117.1 (9-CCO),
113.6 (7-CH), 77.9 (14-CH), 75.2 (16-CH2), 71.6 (OCH2), 70.7 (2C,
OCH2), 70.5 (OCH2), 70.4 (OCH2), 70.3 (OCH2), 70.1 (OCH2),
67.7 (OCH2), 59.4 (18-CH3), 58.7 (OCH3), 58.7 (12-CH), 54.9
(15-CH2), 35.0 (13-CH2) ppm. HRMS (ESIþ, MeOH):
m/z ¼ 553.1822 [M þ Na]þ, 585.2088 [M þ Na þ MeOH]þ;
C
20H37NO9 þ Na.
4.5.2. (2S,4R)-N-tert-butyloxycarbonyl-2-hydroxymethyl-4-PEG4
yloxypyrrolidine (7a)
Under argon atmosphere, a stirred solution of methyl (2S,4R)-
N-tert-butyloxycarbonyl-4-PEG4yloxypyrrolidine-2-carboxylate
calcd. 553.1826 for C23H34N2O10
23H34N2O10S þ Na þ CH3OH.
S
þ
Na, 585.2089 for
C

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
571
4.6. (2S,4S)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)sulfon
yl]isatins
(2C, OCH2), 68.5 (OCH2), 59.1 (12-CH3), 59.0 (OCH3), 58.8 (2-CH),
55.6 (5-CH2, rotamer A), 55.5 (5-CH2, rotamer B), 35.4 (3-CH2,
rotamer B), 34.2 (3-CH2, rotamer A), 28.5 (3C, 9-CH3) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 444.2570 [M þ Na]þ; calcd. 444.2568 for
C
20H39NO8þNa.
4.6.4. (2S,4S)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)
sulfonyl]isatin (23b)
(2S,4S)-N-tert-Butyloxycarbonyl-2-methoxymethyl-4-PEG4ylo
xypyrrolidine (8b) (311 mg, 0.738 mmol, 1.00 equiv.) was converted
to (2S,4S)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrrolidinyl)sulfo-
nyl]isatin (23b) using TFA followed by reaction with 5-chloro
sulfonyl isatin and DIPEA as described in the general procedure
in Section 4.2.2. The residue was purified by flash chromatography
(silica gel, 4% MeOH in DCM) to obtain a yellow oil (156 mg,
4.6.1. Methyl (2S,4S)-N-tert-butyloxycarbonyl-4-PEG4yloxypyrroli
dine-2-carboxylate (6b)
The preparation of methyl (2S,4S)-N-tert-butyloxycarbonyl-4-
PEG4yloxypyrrolidine-2-carboxylate (6b) was achieved starting
from (2S,4S)-N-tert-butyloxycarbonyl-4-hydroxypyrrolidine-2-
carboxylate (2b) (1.00 g, 4.08 mmol, 1.00 equiv.) using dry
THF (30 mL), 60% NaH (245 mg, 6.12 mmol, 1.50 equiv. in mineral
oil) and 2,5,8,11-tetraoxatridecan-13-yl-p-toluenesulfonate (5)
(1.97 g, 5.44 mmol, 1.33 equiv.) as described in Section 4.5.1. The
crude product was purified by flash column chromatography
(silica gel, 3% MeOH in DCM) to obtain a colorless oil (1.13 g,
0.294 mmol, 40%). 1H NMR (400 MHz, CDCl3):
d
¼ 9.96 (s, 1H, 1-
3
4
NH), 8.03 (dd, JH,H ¼ 8.3 Hz, JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.00 (d,
4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.19 (d, JH,H ¼ 8.3 Hz, 1H, 7-CH), 3.85e
3
3.80 (m, 1H, 14-CH), 3.80e3.63 (m, 6H, OCH2), 3.79e3.73 (m, 1H,
16-CHa), 3.72e3.66 (m, 1H, 12-CH), 3.64e3.57 (m, 1H, 16-CHb),
3.62e3.50 (m, 4H, OCH2), 3.56e3.44 (m, 2H, 15-CH2), 3.47e3.37 (m,
2H, OCH2), 3.41 (s, 3H, 18-CH3), 3.31 (s, 3H, OCH3), 3.26e3.13 (m,
3H, OCH2), 3.07e3.02 (m, 1H, OCH2), 2.14e2.04 (m, 1H, 13-CHa),
2
3
3
2.59 mmol, 64%). 1H NMR (300 MHz, CDCl3):
d
¼ 4.45e4.24 (m, 1H,
1.91 (ddd, JH,H ¼ 13.7 Hz, JH,H ¼ 9.0 Hz, JH,H ¼ 4.2 Hz, 1H, 13-
CHb) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 183.2 (3-CO), 158.9
4-CH), 4.24e4.03 (m, 1H, 2-CH), 3.77e3.51 (m, 21H, 5-CH2, 12-
CH3, 8 ꢃ OCH2), 3.38 (s, 3H, OCH3), 2.45e2.16 (m, 1H, 3-CHa),
2.12e1.95 (m, 1H, 3-CHb), 1.52e1.37 (m, 9H, 9-CH3) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 458.2369 [M þ Na]þ; calcd. 458.2361 for
(2-CO), 153.7 (8-CN), 137.9 (6-CH), 132.5 (5-CSO2), 124.7 (4-
CH), 117.2 (9-CCO), 113.6 (7-CH), 77.9 (14-CH), 75.2 (16-CH2),
71.6 (OCH2), 70.7 (2C, OCH2), 70.5 (OCH2), 70.4 (OCH2), 70.3
(OCH2), 70.2 (OCH2), 67.7 (OCH2), 59.4 (18-CH3), 58.8 (OCH3), 58.7
(12-CH), 54.9 (15-CH2), 35.0 (13-CH2) ppm. HRMS (ESIþ,
MeOH): m/z ¼ 553.1826 [M þ Na]þ, 585.2084 [M þ Na þ MeOH]þ;
C
20H37NO9 þ Na.
4.6.2. (2S,4S)-N-tert-butyloxycarbonyl-2-hydroxymethyl-4-PEG4yl
oxypyrrolidine (7b)
The preparation of (2S,4S)-N-tert-butyloxycarbonyl-2-hydroxy
methyl-4-PEG4yloxypyrrolidine (7b) was achieved starting from
methyl (2S,4S)-N-tert-butyloxycarbonyl-4-PEG4yloxypyrrolidine-
2-carboxylate (6b) (540 mg, 1.24 mmol, 1.00 equiv.) using dry THF
calcd. 553.1826 for
23H34N2O10S þ Na þ MeOH.
C
23H34N2O10
S
þ
Na, 585.2089 for
C
4.6.5. N-butyl-(2S,4S)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrroli
dinyl)sulfonyl]isatin (24)
(2S,4S)-5-[1-(2-Methoxymethyl-4-PEG4yloxypyrrolidinyl)sul-
fonyl]isatin (23b) (50 mg, 94.2 mmol, 1.00 equiv.) was converted
to N-butyl-(2S,4S)-5-[1-(2-methoxymethyl-4-PEG4yloxypyrroli-
dinyl)sulfonyl]isatin (24) using 1-bromobutane and Cs2CO3 as
described in the general procedure in Section 4.2.3. The resulting
residue was purified by flash column chromatography (silica gel,
(5 mL) for dissolving the ester 6b and 4 M LiBH4 in THF (620 mL,
2.48 mmol, 2.00 equiv.) as described in Section 4.5.2. The crude
product was purified by flash column chromatography (silica gel,
3.5% MeOH in DCM) to obtain a colorless oil (417 mg, 1.02 mmol,
83%). 1H NMR (300 MHz, CDCl3):
d
¼ 4.13e3.93 (m, 2H, 2-CH,
4-CH), 3.76e3.50 (m, 20H, 5-CH2, 10-CH2, 8 ꢃ OCH2), 3.38 (s,
3H, OCH3), 3.10 (br s, 1H, 10-COH), 2.21e2.08 (m, 1H, 3-CHa),
1.96e1.56 (m, 1H, 3-CHb), 1.47 (s, 9H, 9-CH3) ppm. HRMS (ESIþ,
5% MeOH in DCM) to obtain a yellow oil (38 mg, 64.8
m
mol, 69%).
3
1H NMR (400 MHz, CDCl3):
d
¼ 8.08 (dd, JH,H ¼ 8.3 Hz,
MeOH): m/z
¼
430.2402 [M
þ
Na]þ; calcd. 430.2411 for
4JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.04 (d, 4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.03 (d,
3JH,H ¼ 8.3 Hz, 1H, 7-CH), 4.09e4.00 (m, 1H, 14-CH), 3.82e3.73 (m,
1H, 12-CH), 3.78 (t, 3JH,H ¼ 7.4 Hz, 2H, 19-CH2), 3.69e3.46 (m, 13H,
OCH2), 3.66e3.59 (m, 1H, 16-CHa), 3.59e3.51 (m, 1H, 16-CHb),
3.56e3.50 (m, 1H, 15-CHa), 3.44e3.37 (m, 1H, 15-CHb), 3.43e3.30
(m, 3H, OCH2), 3.37 (s, 3H, 18-CH3), 3.37 (s, 3H, OCH3), 2.09e1.92
C
19H37NO8 þ Na.
4.6.3. (2S,4S)-N-tert-butyloxycarbonyl-2-methoxymethyl-4-PEG4
yloxypyrrolidine (8b)
(2S,4S)-N-tert-butyloxycarbonyl-2-hydroxymethyl-4-
3
PEG4yloxypyrrolidine (7b) (375 mg, 0.920 mmol, 1.00 equiv.) was
converted to (2S,4S)-N-tert-butyloxycarbonyl-2-methoxymethyl-
4-PEG4yloxypyrrolidine (8b) using MeI and NaH as described in
the general procedure in Section 4.2.1. The crude product was
purified by flash column chromatography (silica gel, 5% MeOH in
DCM) to obtain a colorless oil (312 mg, 0.740 mmol, 80%). 1H NMR
(m, 2H, 13-CH2), 1.70 (quintet, JH,H ¼ 7.4 Hz, 2H, 20-CH2), 1.43
3
3
(sextet, JH,H ¼ 7.4 Hz, 2H, 21-CH2), 0.99 (t, JH,H ¼ 7.4 Hz, 3H, 22-
CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 182.2 (3-CO), 157.9 (2-
CO), 153.6 (8-CN), 137.8 (6-CH), 133.8 (5-CSO2), 124.9 (4-CH),
117.2 (9-CCO), 110.2 (7-CH), 77.4 (14-CH), 75.0 (16-CH2), 71.9
(OCH2), 70.6 (OCH2), 70.5 (3C, OCH2), 70.4 (OCH2), 70.3
(OCH2), 68.2 (OCH2), 59.3 (18-CH3), 59.0 (OCH3), 58.5 (12-CH),
54.5 (15-CH2), 40.4 (19-CH2), 35.2 (13-CH2), 29.3 (20-CH2),
20.1 (21-CH2), 13.6 (22-CH3) ppm. HRMS (ESIþ, MeOH):
m/z ¼ 609.2450 [M þ Na]þ, 641.2707 [M þ Na þ MeOH]þ; calcd.
(400 MHz, CDCl3):
d
¼ 4.17e3.88 (m, 2H, 2-CH, 4-CH), 3.69e3.32
(m, 20H, 5-CH2, 10-CH2, 8 ꢃ OCH2), 3.38 (s, 3H, OCH3), 3.35 (s, 12-
CH3, rotamer B), 3.34 (s, 12-CH3, rotamer A), 2.23e1.93 (m, 2H, 3-
CH2), 1.46 (s, 9H, 9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 154.6 (6-CO), 79.4 (8-CO), 77.3 (4-CH), 74.1 (10-CH2, rotamer
609.2452 for C27H42N2O10S þ Na, 641.2715 for C27H42N2O10
þ Na þ MeOH.
S
A), 73.3 (10-CH2, rotamer B), 71.9 (OCH2), 70.6 (4C, OCH2), 70.5

572
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
4.7. (2S,4R)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatins
to (2S,4R)-N-butyl-5-[1-(4-trifluoromethyl-2-methoxymethylpyrr
olidinyl)sulfonyl]isatin (26a) using 1-bromobutane and
Cs2CO3 as described in the general procedure in Section 4.2.3.
The crude product was purified by flash column
chromatography (silica gel, 60% EtOAc in cyclohexane) to obtain a
yellow oil (24 mg, 53.5
m
mol, 84%). 1H NMR (300 MHz,
3
CDCl3):
d
¼ 8.08 (dd, JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-CH), 8.04
4
3
(d, JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.05 (d, JH,H ¼ 8.3 Hz, 1H, 7-
3
CH), 4.01e3.92 (m, 1H, 12-CH), 3.79 (t, JH,H ¼ 7.3 Hz, 2H, 20-
2
3
CH), 3.67 (dd, JH,H ¼ 9.7 Hz, JH,H ¼ 7.8 Hz, 1H, 15-CHa), 3.56
2
3
(dd, JH,H ¼ 9.8 Hz, JH,H ¼ 4.8 Hz, 1H, 16-CHa), 3.51 (dd,
2JH,H ¼ 9.8 Hz, JH,H ¼ 3.3 Hz, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3),
3
4.7.1. (2S,4R)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-methoxy
methyl pyrrolidine (12a)
3.31e3.20 (m, 1H, 15-CHb), 3.26e3.09 (m, 1H, 14-CH), 2.14 (ddd,
2JH,H ¼ 12.9 Hz, JH,H ¼ 7.1 Hz, 3JH,H ¼ 2.2 Hz, 1H, 13-CHa), 1.87 (dt,
3
(2S,4R)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-hydroxy
methylpyrrolidine (11a) (180 mg, 0.668 mmol, 1.00 equiv.) was
converted to (2S,4R)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-
methoxymethyl pyrrolidine (12a) using MeI and NaH as described
in the general procedure in Section 4.2.1. The crude was purified by
flash column chromatography (silica gel, 20% EtOAc in cyclo-
hexane) to obtain a colorless oil (186 mg, 0.657 mmol, 98%). 1H
2JH,H ¼ 12.8 Hz, JH,H ¼ 9.5 Hz, 1H, 13-CHb), 1.71 (quintet,
3
3JH,H
¼
7.4 Hz, 2H, 21-CH2), 1.43 (sextet, JH,H
¼
7.4 Hz,
3
2H, 22-CH2), 0.99 (t, JH,H ¼ 7.3 Hz, 3H, 23-CH3) ppm. 13C NMR
3
(75 MHz, CDCl3):
d
¼
182.0 (3-CO), 157.7 (2-CO), 154.0
(8-CN), 137.3 (6-CH), 133.4 (5-CSO2), 124.4 (4-CH), 117.4
(9-CCO), 110.5 (7-CH), 75.1 (16-CH2), 59.3 (18-CH3), 59.3 (12-
3
2
CH), 47.9 (q, JC,F ¼ 3.4 Hz, 15-CH2), 41.6 (q, JC,F ¼ 29.2 Hz,
NMR (400 MHz, CDCl3):
d
¼ 4.20e3.92 (m,1H, 2-CH), 3.63e3.33 (m,
3
14-CH), 40.5 (20-CH2), 29.2 (21-CH2), 29.0 (q, JC,F ¼ 2.3 Hz,
4H, 5-CH2, 10-CH2), 3.34 (s, 3H, 12-CH3), 3.23e3.01 (m, 1H, 4-CH),
13-CH2), 20.1 (22-CH2), 13.6 (23-CH3) ppm.* 19F NMR
2.22e1.98 (m, 2H, 3-CH2), 1.47 (s, 9H, 9-CH3) ppm. 13C NMR
¼ 154.0 (6-CH), 127.0 (q, 1JC,F ¼ 276.8 Hz, 13-
(282 MHz, CDCl3):
d
¼ ꢁ71.6 (s, 3F, 19-CF3) ppm. HRMS (ESIþ,
(100 MHz, CDCl3):
d
MeOH): m/z ¼ 471.1178 [M þ Na]þ, 503.1441 [M þ Na þ MeOH]þ;
CF3), 80.0 (8-CO), 74.1 (10-CH2, rotamer B), 73.5 (10-CH2, rotamer
A), 59.2 (12-CH3), 56.6 (2-CH), 46.0 (5-CH2), 42.3e39.9 (m, 4-CH),
28.9 (3-CH2, rotamer B), 28.5 (3C, 9-CH3), 28.0 (3-CH2, rotamer A)
calcd. 471.1172 for
C
19H23F3N2O5S
þ
Na, 503.1434 for
C
19H23F3N2O5S þ Na þ MeOH.
* The 13C NMR signal of 19-CF3 could not be observed because of
ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ71.9 (s, 3F, 13-CF3) ppm.
HRMS (ESIþ, MeOH): m/z ¼ 306.1294 [M þ Na]þ, 589.2689
[2M þ Na]þ; calcd. 306.1287 for C12H20F3NO3 þ Na, 589.2683 for
2(C12H20F3NO3) þ Na.
sample dilution.
4.7.4. (2S,4R)-N-(4-fluorobutyl)-5-[1-(4-trifluoromethyl-2-methoxy
methylpyrrolidinyl)-sulfonyl]isatin (27a)
4.7.2. (2S,4R)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (25a)
(2S,4R)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
(2S,4R)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-methoxy
methylpyrrolidine (12a) (190 mg, 0.670 mmol, 1.00 equiv.) was
converted to (2S,4R)-5-[1-(4-trifluoromethyl-2-methoxymethylpyr
rolidinyl)sulfonyl]isatin (25a) using 5-chlorosulfonyl isatin and
DIPEA as described in the general procedure in Section 4.2.2. The
crude product was purified by flash column chromatography (silica
gel, 50% EtOAc in cyclohexane) to obtain a sticky yellow wax
sulfonyl]isatin (25a) (25 mg, 63.7 mmol, 1.00 equiv.) was converted
to (2S,4R)-N-(4-fluorobutyl)-5-[1-(4-trifluoromethyl-2-methoxy
methylpyrrolidinyl)sulfonyl]isatin (27a) using 1-bromo-4-fluor
obutane and Cs2CO3 as described in the general procedure in
Section 4.2.3. The crude product was purified by flash
column chromatography (silica gel, 50% EtOAc in cyclohexane)
to obtain
(300 MHz, CDCl3):
a
yellow oil (27 mg, 57.9
m
mol, 91%). 1H NMR
3
4
(70 mg, 0.178 mmol, 27%). 1H NMR (300 MHz, CDCl3):
d
¼ 8.77 (br s,
d
¼ 8.09 (dd, JH,H ¼ 8.3 Hz, JH,H ¼ 1.9 Hz,
1H, 1-NH), 8.10e8.05 (m, 2H, 4-CH, 6-CH), 7.17e7.12 (m, 1H, 7-CH),
4.02e3.92 (m, 1H,12-CH), 3.68 (dd, 2JH,H ¼ 9.9 Hz, 3JH,H ¼ 7.9 Hz,1H,
1H, 6-CH), 8.05 (d, 4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.08 (d, 3JH,H ¼ 8.3 Hz,
2
3
1H, 7-CH), 4.53 (dt, JH,F ¼ 47.4 Hz, JH,H ¼ 5.4 Hz, 2H, 23-
2
3
3
15-CHa), 3.56 (dd, JH,H ¼ 9.8 Hz, JH,H ¼ 4.8 Hz, 1H, 16-CHa), 3.51
(dd, 2JH,H ¼ 9.8 Hz, 3JH,H ¼ 3.3 Hz, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3),
3.32e3.23 (m, 1H, 15-CHb), 3.26e3.09 (m, 1H, 14-CH), 2.14 (ddd,
CH2), 4.01e3.92 (m, 1H, 12-CH), 3.85 (t, JH,H ¼ 7.1 Hz, 2H, 20-
2
3
CH2), 3.67 (dd, JH,H ¼ 11.0 Hz, JH,H ¼ 7.9 Hz, 1H, 15-CHa),
2
3
3.56 (dd, JH,H ¼ 9.8 Hz, JH,H ¼ 4.8 Hz, 1H, 16-CHa), 3.51
(dd, 2JH,H ¼ 9.8 Hz, 3JH,H ¼ 3.2 Hz, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3),
3.31e3.23 (m, 1H, 15-CHb), 3.26e3.10 (m, 1H, 14-CH), 2.14
3
3
2JH,H ¼ 12.9 Hz, JH,H ¼ 7.2 Hz, JH,H ¼ 2.2 Hz, 1H, 13-CHa), 1.87 (dt,
2JH,H ¼ 12.8 Hz, 3JH,H ¼ 9.2 Hz, 1H, 13-CHb) ppm. 13C NMR (75 MHz,
2
3
3
CDCl3):
d
¼ 181.5 (3-CO), 158.4 (2-CO), 152.0 (8-CN), 137.5 (6-CH),
(ddd, JH,H ¼ 12.9 Hz, JH,H ¼ 7.1 Hz, JH,H ¼ 2.2 Hz, 1H, 13-CHa),
134.0 (5-CSO2), 124.9 (4-CH), 117.9 (9-CCO), 112.9 (7-CH), 75.1 (16-
1.98e1.70 (m, 5H, 13-CHb, 21-CH2, 22-CH2) ppm. 13C NMR
3
CH2), 59.4 (18-CH3), 59.3 (12-CH), 48.0 (q, JC,F ¼ 2.8 Hz, 15-CH2),
(75 MHz, CDCl3):
CN), 137.4 (6-CH), 133.6 (5-CSO2), 124.5 (4-CH), 117.4 (9-
d
¼ 181.8 (3-CO), 157.7 (2-CO), 153.7 (8-
41.6 (q, 2JC,F ¼ 29.1 Hz, 14-CH), 29.0 (q, 3JC,F ¼ 2.1 Hz, 13-CH2) ppm.*
1
19F NMR (282 MHz, CDCl3):
d
¼ ꢁ71.7 (s, 3F, 19-CF3) ppm. HRMS
CCO), 110.5 (7-CH), 83.2 (d, JC,F ¼ 165.7 Hz, 23-CH2), 75.1 (16-
3
(ESIþ, MeOH): m/z ¼ 415.0544 [M þ Na]þ, 447.0806 [M þ Na
CH2), 59.3 (18-CH3), 59.3 (12-CH), 47.9 (q, JC,F
¼
2.9 Hz,
2
þ MeOH]þ; calcd. 415.0546 for C15H15F3N2O5S þ Na, 447.0808 for
15-CH2), 41.6 (q, JC,F ¼ 29.2 Hz, 14-CH), 40.3 (20-CH2), 29.0
3
2
C
15H15F3N2O5S þ Na þ MeOH.
(q, JC,F ¼ 2.7 Hz, 13-CH2), 27.6 (d, JC,F ¼ 20.1 Hz, 22-CH2), 23.5
3
* The 13C NMR signal of 19-CF3 could not be observed because of
(d, JC,F ¼ 4.0 Hz, 21-CH2) ppm.* 19F NMR (282 MHz, CDCl3):
sample dilution.
d
¼ ꢁ71.6 (s, 3F, 19-CF3), ꢁ220.0 (s, 1F, 23-CH2F) ppm. HRMS
(ESIþ, MeOH): m/z
¼
489.1077 [M
þ
Na]þ, 521.1339
4.7.3. (2S,4R)-N-butyl-5-[1-(4-trifluoromethyl-2-methoxymethyl
pyrrolidinyl)sulfonyl]isatin (26a)
(2S,4R)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (25a) (25 mg, 63.7 mmol, 1.00 equiv.) was converted
[M þ Na þ MeOH]þ; calcd. 489.1078 for C19H22F4N2O5S þ Na,
521.1340 for C19H22F4N2O5S þ Na þ MeOH.
* The 13C NMR signal of 19-CF3 could not be observed because of
sample dilution.

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
573
4.8. (2S,4S)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatins
described in the general procedure in Section 4.2.3. The crude
productwaspurified by flashcolumnchromatography (silica gel, 40%
EtOAc in cyclohexane) to obtain a yellow-orange solid (21 mg,
46.8
CDCl3):
m
mol, 92%) asthedesired product. M.p. 86 ꢂC.1H NMR (300 MHz,
d
¼ 8.10 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-CH), 8.06 (d,
4JH,H ¼ 2.0 Hz,1H, 4-CH), 7.05 (d, 3JH,H ¼ 8.3 Hz,1H, 7-CH), 4.07e3.96
(m, 1H,12-CH), 3.80e3.72 (m, 1H, 15-CHa), 3.79 (t, 3JH,H ¼ 7.5 Hz, 2H,
20-CH2), 3.59 (dd, 2JH,H ¼ 9.7 Hz, 3JH,H ¼ 4.1 Hz, 1H,16-CHa), 3.51 (dd,
2JH,H ¼ 9.7 Hz, 3JH,H ¼ 5.9 Hz,1H, 16-CHb), 3.37e3.28 (m, 1H,15-CHb),
3.34 (s, 3H, 18-CH3), 2.71e2.58 (m, 1H, 14-CH), 2.28 (dt,
2JH,H ¼ 13.4 Hz, 3JH,H ¼ 8.0 Hz,1H, 13-CHa), 2.06 (ddd, 2JH,H ¼ 13.2 Hz,
3JH,H ¼ 10.5 Hz, 3JH,H ¼ 7.8 Hz,1H,13-CHb),1.71 (quintet, 3JH,H ¼ 7.5 Hz,
4.8.1. (2S,4S)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-methoxy
methylpyrrolidine (12b)
3
2H, 21-CH2), 1.43 (sextet, JH,H ¼ 7.5 Hz, 2H, 22-CH2), 0.99 (t,
(2S,4S)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-hydroxy
methylpyrrolidine (11b) (160 mg, 0.594 mmol, 1.00 equiv.) was
converted to (2S,4S)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-
methoxymethyl pyrrolidine (12b) using MeI and NaH as
described in the general procedure in Section 4.2.1. The crude
product was purified by flash column chromatography (silica gel,
15% EtOAc in cyclohexane) to obtain a colorless oil (119 mg,
3JH,H ¼ 7.3 Hz, 3H, 23-CH3) ppm. 13C NMR (75 MHz, CDCl3):
¼ 182.0
d
(3-CO),157.7 (2-CO),154.0 (8-CN),137.3 (6-CH),134.5 (5-CSO2),124.5
(4-CH),117.4 (9-CCO),110.5 (7-CH), 73.9 (16-CH2), 59.1 (18-CH3), 59.0
(12-CH), 48.1 (q, 3JC,F ¼ 3.0 Hz,15-CH2), 42.0 (q, 2JC,F ¼ 29.1 Hz,14-CH),
40.5 (20-CH2), 29.2 (21-CH2), 28.8 (q, 3JC,F ¼ 2.2 Hz,13-CH2), 20.1 (22-
CH2), 13.6 (23-CH3) ppm. * 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ70.7
(s, 3F, 19-CF3) ppm. HRMS (ESIþ, MeOH): m/z
¼
471.1176
0.420 mmol, 71%). 1H NMR (400 MHz, CDCl3):
d
¼ 4.12e3.86 (m, 1H,
[M þ Na]þ, 503.1437 [M þ Na þ MeOH]þ; calcd. 471.1172 for
2-CH), 3.97e3.73 (m, 1H, 5-CHa), 3.70e3.31 (m, 2H, 10-CH2), 3.36 (s,
3H, 12-CH3), 3.25 (t, 2JH,H ¼ 10.6 Hz, 1H, 5-CHb), 2.96e2.75 (m, 1H,
4-CH), 2.31 (dt, 2JH,H ¼ 13.3 Hz, 3JH,H ¼ 8.1 Hz, 1H, 3-CHa), 2.15e2.00
(m, 1H, 3-CHb), 1.47 (s, 9H, 9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
C
19H23F3N2O5S þ Na, 503.1434 for C19H23F3N2O5S þ Na þ MeOH.
* The 13C NMR signal of 19-CF3 could not be observed because of
sample dilution.
d
¼ 154.0 (6-C), 126.4 (q, 1JC,F ¼ 276.8 Hz, 13-CF3), 80.1 (8-CO), 72.8
4.8.4. (2S,4S)-N-(4-fluorobutyl)-5-[1-(4-trifluoromethyl-2-methoxy
methylpyrrolidinyl)sulfonyl]isatin (27b)
(10-CH2), 59.2 (12-CH3), 56.2 (2-CH), 46.2 (5-CH2), 41.4 (4-CH), 29.0
(3-CH2), 28.4 (3C, 9-CH3) ppm. 19F NMR (282 MHz, CDCl3):
(2S,4S)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
d
¼ ꢁ71.0 (s, 3F, 13-CF3) ppm. HRMS (ESIþ, MeOH): m/z ¼ 306.1289
sulfonyl]isatin (25b) (21 mg, 53.5 mmol, 1.00 equiv.) was converted to
[M þ Na]þ; calcd. 306.1287 for C12H20F3NO3 þ Na.
(2S,4S)-N-(4-fluorobutyl)-5-[1-(4-trifluoromethyl-2-methoxymethyl
pyrrolidinyl)sulfonyl]isatin (27b) using 1-bromo-4-fluorobutane and
Cs2CO3 as described in the general procedure in Section 4.2.3. The
crude product was purified by flash column chromatography (silica
gel, 50% EtOAc in cyclohexane) to obtain a yellow solid (24 mg,
4.8.2. (2S,4S)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (25b)
(2S,4S)-N-tert-butyloxycarbonyl-4-trifluoromethyl-2-methoxy
methylpyrrolidine (12b) (90 mg, 0.318 mmol, 1.00 equiv.) was con-
verted to (2S,4S)-5-[1-(4-trifluoromethyl-2-methoxymethylpy
rrolidinyl)sulfonyl]isatin (25b) using TFA followed by reaction with
5-chlorosulfonyl isatin and DIPEA as described in the general pro-
cedure in Section 4.2.2. The crude product was purified by flash
column chromatography (silica gel, 60% EtOAc in toluene) to obtain a
sticky yellow wax (80 mg, 0.204 mmol, 64%) as the desired product.
51.5
CDCl3):
m
mol, 96%) asthedesired product. M.p.123ꢂC.1H NMR(300 MHz,
d
¼ 8.10 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-CH), 8.06 (d,
4JH,H ¼ 2.0 Hz, 1H, 4-CH), 7.08 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-CH), 4.53 (dt,
2JH,F ¼ 47.4 Hz, 3JH,H ¼ 5.4 Hz, 2H, 23-CH2), 4.01 (tdd, 3JH,H ¼ 7.8 Hz,
3JH,H ¼ 5.9 Hz, 3JH,H ¼ 4.2 Hz,1H,12-CH), 3.85 (t, 3JH,H ¼ 7.1 Hz, 2H, 20-
2
3
CH2), 3.76 (dd, JH,H ¼ 11.4 Hz, JH,H ¼ 7.9 Hz, 1H, 15-CHa), 3.59 (dd,
2JH,H ¼ 9.7 Hz, JH,H ¼ 4.1 Hz, 1H, 16-CHa), 3.51 (dd, JH,H ¼ 9.7 Hz,
3JH,H ¼ 5.9 Hz, 1H, 16-CHb), 3.37e3.28 (m, 1H, 15-CHb), 3.34 (s, 3H, 18-
CH3), 2.72e2.59 (m, 1H, 14-H), 2.28 (dt, 2JH,H ¼ 13.4 Hz, 3JH,H ¼ 8.1 Hz,
1H, 13-CHa), 2.05 (ddd, 2JH,H ¼ 13.3 Hz, 3JH,H ¼ 10.5 Hz, 3JH,H ¼ 7.8 Hz,
1H, 13-CHb), 1.96e1.70 (m, 4H, 21-CH2, 22-CH2) ppm. 13C NMR
3
2
1H NMR (400 MHz, CDCl3):
2H, 4-CH, 6-CH), 7.11e7.07 (m, 1H, 7-CH), 4.02e3.94 (m, 1H, 12-CH),
d
¼ 10.57 (br s, 1H, 1-NH), 8.06e8.02 (m,
2
3
3.75 (dd, JH,H ¼ 11.6 Hz, JH,H ¼ 7.9 Hz, 1H, 15-CHa), 3.60 (dd,
2JH,H ¼ 9.7 Hz, JH,H ¼ 4.1 Hz, 1H, 16-CHa), 3.50 (dd, JH,H ¼ 9.6 Hz,
3
2
3JH,H ¼ 6.0 Hz,1H,16-CHb), 3.38e3.33 (m,1H,15-CHb). 3.35 (s, 3H,18-
(75 MHz, CDCl3):
d
¼ 181.8 (3-CO),157.7 (2-CO),153.7 (8-CN),137.3 (6-
2
CH3), 2.69e2.53 (m, 1H, 14-CH), 2.26 (dt, JH,H
¼
13.5 Hz,
CH), 134.6 (5-CSO2), 125.6 (q, 1JC,F ¼ 277.1 Hz, 19-CF3), 124.5 (4-CH),
117.4 (9-CCO),110.5 (7-CH), 83.2 (d,1JC,F ¼ 165.5 Hz, 23-CH2), 73.9 (16-
CH2), 59.1 (18-CH3), 59.1 (12-CH), 48.0 (q, 3JC,F ¼ 2.8 Hz,15-CH2), 42.0
(q, 2JC,F ¼ 29.0 Hz,14-CH), 40.2(20-CH2), 28.8 (q, 3JC,F ¼ 2.0 Hz,13-CH2),
3JH,H ¼ 7.9 Hz,1H,13-CHa), 2.04 (ddd, 2JH,H ¼ 13.4 Hz, 3JH,H ¼ 10.5 Hz,
3JH,H ¼ 7.7 Hz,1H,13-CHb) ppm.13C NMR (100 MHz, CDCl3):
¼ 182.6
d
(3-CO),159.1 (2-CO),153.4 (8-CN),137.3 (6-CH),134.1 (5-CSO2),124.8
(4-CH),117.8 (9-CCO),113.1 (7-CH), 74.0 (16-CH2), 59.1 (18-CH3), 59.1
(12-CH), 48.2(q, 3JC,F ¼ 3.2 Hz,15-CH2), 42.0 (q, 2JC,F ¼ 29.0 Hz,14-CH),
23.5 (d, 3JC,F ¼ 3.9Hz, 21-CH2), 27.6 (d, 2JC,F ¼ 20.1 Hz, 22-CH2) ppm.19
F
NMR (282 MHz, CDCl3):
d
¼ ꢁ70.7 (s, 3F, 19-CF3), ꢁ219.9 (s, 1F, 23-
28.9 (q, JC,F ¼ 2.3 Hz, 13-CH2) ppm.* 19F NMR (282 MHz, CDCl3):
CH2F) ppm. HRMS (ESIþ, MeOH): m/z ¼ 489.1077 [M þ Na]þ,
521.1342 [M þ Na þ MeOH]þ; calcd. 489.1078for C19H22F4N2O5S þ Na,
521.1340 for C19H22F4N2O5S þ Na þ MeOH.
3
d
¼ ꢁ70.7 (s, 3F, 19-CF3) ppm. HRMS (ESIþ, MeOH): m/z ¼ 415.0538
[M þ Na]þ, 447.0799 [M þ Na þ MeOH]þ; calcd. 415.0546 for
C
15H15F3N2O5S þ Na, 447.0808 for C15H15F3N2O5S þ Na þ MeOH.
* The 13C NMR signal of 19-CF3 could not be observed because of
4.9. (2S,4R)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
sample dilution.
isatins
4.8.3. (2S,4S)-N-butyl-5-[1-(4-trifluoromethyl-2-methoxymethyl
pyrrolidinyl)sulfonyl]isatin (26b)
(2S,4S)-5-[1-(4-trifluoromethyl-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (25b) (20 mg, 51.0 mmol, 1.00 equiv.) was converted
to (2S,4S)-N-butyl-5-[1-(4-trifluoromethyl-2-methoxymethylpyrro
lidinyl)sulfonyl]isatin (26b) using 1-bromobutane and Cs2CO3 as

574
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
4.9.1. (2S,4R)-N-tert-butyloxycarbonyl-4-fluoro-2-hydroxymethyl
pyrrolidine (13a)
d
¼ 183.9 (3-CO), 159.8 (2-CO), 154.2 (8-CN), 138.5 (6-CH), 133.9 (5-
CSO2), 125.1 (4-CH), 118.8 (9-CCO), 113.5 (7-CH), 93.3 (d,
1JC,F ¼ 175.7 Hz, 14-CH), 75.0 (16-CH2), 59.4 (18-CH3), 59.3 (12-CH),
56.5 (d, 2JC,F ¼ 21.8 Hz,15-CH2), 36.6 (d, 2JC,F ¼ 21.3 Hz,13-CH2) ppm.
A stirred solution of methyl (2S,4R)-N-tert-butyloxycarbonyl-4-
fluoropyrrolidine-2-carboxylate (600 mg, 2.43 mmol,1.00 equiv.) in
THF (10 mL) was treated with anhydrous LiCl (227 mg, 5.35 mmol,
2.20 equiv.) followed by NaBH4 (230 mg, 6.08 mmol, 2.50 equiv.) at
ambient temperature. The resulting mixture was cooled to 0 ꢂC,
before EtOH (20 mL) was added dropwise over 15 min. The reaction
mixture was stirred further at this temperature for 1 h and at r.t. for
21 h. After this time, the milky mixture was cooled to 0 ꢂC and
acidified to pH 4 by addition of 10% aq. citric acid. The solvent was
completely removed under reduced pressure. Subsequently, the
residue was taken up in water (40 mL) and extracted with DCM
(3 ꢃ 40 mL). The combined organic phase was dried over MgSO4
and concentrated under reduced pressure to yield a pale yellow oil
19F NMR (282 MHz, CD3CN):
d
¼ ꢁ175.8 (s, 1F, 14-CHF) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 365.0581 [M þ Na]þ, 397.0842 [M þ Na þ
MeOH]þ; calcd. 365.0578 for C14H15FN2O5S þ Na, 397.0840 for
C
14H15FN2O5S þ Na þ MeOH.
4.9.4. (2S,4R)-N-butyl-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (29a)
(2S,4R)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfo-
nyl]isatin (28a) (40 mg, 0.117 mmol, 1.00 equiv.) was converted
to (2S,4R)-N-butyl-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (29a) using 1-bromobutane and Cs2CO3 as
described in the general procedure in Section 4.2.3. The obtained
residue was purified by flash column chromatography (silica gel,
(514 mg, 2.34 mmol, 96%). 1H NMR (300 MHz, CDCl3):
d
¼ 5.30e
4.98 (m, 1H, 4-CH), 4.21e4.05 (m, 1H, 2-CH), 3.95e3.70 (m, 2H, 5-
CHa, 10-CHa), 3.60e3.28 (m, 2H, 5-CHb, 10-CHb), 2.44e2.23 (m,
40% THF in cyclohexane) to yield a yellow solid (35 mg, 87.8
m
mol,
1H, 3-CHa), 1.89e1.59 (m, 1H, 3-CHb), 1.48 (s, 9H, 9-CH3) ppm. 13
C
75%). M.p. 85 ꢂC. 1H NMR (400 MHz, CDCl3):
d
¼ 8.06 (dd,
NMR (75 MHz, CDCl3):
d
¼ 156.6 (6-CO), 91.2 (d, 1JC,F ¼ 176.3 Hz, 4-
3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-CH), 8.03 (d, 4JH,H ¼ 2.0 Hz, 1H,
4-CH), 7.02 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-CH), 5.06 (dm, 2JH,F ¼ 52.5 Hz,
1H, 14-CH), 3.88e3.81 (m, 1H, 12-CH), 3.83e3.72 (m, 1H, 15-CHa),
3.77 (t, 3JH,H ¼ 7.4 Hz, 2H, 19-CH2), 3.67e3.63 (m, 2H, 16-CH2), 3.55
(ddd, 3JH,F ¼ 39.1 Hz, 2JH,H ¼ 13.6 Hz, 3JH,H ¼ 2.8 Hz, 1H, 15-Hb), 3.39
(s, 3H, 18-CH3), 2.30e2.04 (m, 2H, 13-CH2), 1.76e1.62 (m, 2H, 20-
CH), 80.9 (8-CO), 66.3 (10-CH2), 58.8 (2-CH), 54.2 (d, 2JC,F ¼ 23.0 Hz,
2
5-CH2), 35.5 (d, JC,F ¼ 22.0 Hz, 3-CH2), 28.4 (3C, 9-CH3) ppm. 19
F
NMR (282 MHz, CDCl3):
d
¼ ꢁ177.3 (4-CHF, rotamer A), ꢁ177.7 (4-
CHF, rotamer B) ppm. HRMS (ESIþ, MeOH): m/z ¼ 242.1161
[M þ Na]þ; calcd. 242.1163 for C10H18FNO3 þ Na.
3
CH2), 1.49e1.33 (m, 2H, 21-CH2), 0.98 (t, JH,H ¼ 7.4 Hz, 3H, 22-
4.9.2. (2S,4R)-N-tert-butyloxycarbonyl-4-fluoro-2-methoxymethyl
pyrrolidine (14a)
CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 182.2 (3-CO), 157.8
(2-CO), 153.9 (8-CN), 137.6 (6-CH), 133.5 (5-CSO2), 124.8 (4-CH),
117.1 (9-CCO), 110.3 (7-CH), 91.9 (d, 1JC,F ¼ 178.3 Hz, 14-CH), 74.4
(16-CH2), 59.3 (18-CH3), 58.5 (12-CH), 55.9 (d, 2JC,F ¼ 22.0 Hz, 15-
CH2), 40.5 (19-CH2), 36.1 (d, 2JC,F ¼ 21.4 Hz, 13-CH2), 29.2 (20-CH2),
20.1 (21-CH2), 13.6 (22-CH3) ppm. 19F NMR (282 MHz,
(2S,4R)-N-tert-butyloxycarbonyl-4-fluoro-2-hydroxymethylpyr
rolidine (13a) (450 mg, 2.05 mmol, 1.00 equiv.) was converted to
(2S,4R)-N-tert-butyloxycarbonyl-4-fluoro-2-methoxymethylpyrro
lidine (14a) using MeI and NaH as described in the general proce-
dure in Section 4.2.1. The residue was purified by flash column
chromatography (silica gel, 20% EtOAc in cyclohexane) to obtain a
pale yellow oil (470 mg, 2.01 mmol, 98%). 1H NMR (300 MHz,
CDCl3):
d
¼ ꢁ176.8 (s, 1F, 14-CHF) ppm. HRMS (ESIþ, MeOH):
m/z ¼ 421.1203 [M þ Na]þ, 453.1461 [M þ Na þ MeOH]þ;
calcd. 421.1204 for
C
18H23FN2O5S
þ
Na, 453.1466 for
CDCl3):
d
¼ 5.16 (dt, 2JH,F ¼ 53.6 Hz, 3JH,H ¼ 2.2 Hz, 1H, 4-CH), 4.22e
C
18H23FN2O5S þ Na þ MeOH.
4.01 (m, 1H, 2-CH), 4.06e3.53 (m, 2H, 5-CH2), 3.61e3.25 (m, 2H, 10-
CH2), 3.34 (s, 3H, 12-CH3), 2.44e2.04 (m, 2H, 3-CH2), 1.48 (s, 9H,
4.10. (2S,4S)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatins
9-CH3) ppm. 13C NMR (75 MHz, CDCl3):
d
¼ 154.5 (6-CO), 92.1
1
1
(d, JC,F ¼ 176.5 Hz, 4-CH, rotamer A), 91.6 (d, JC,F ¼ 176.5 Hz, 4-
CH, rotamer B), 79.8 (8-CO), 73.7 (10-CH2, rotamer B), 72.7 (10-
CH2, rotamer A), 59.2 (12-CH3), 55.5 (2-CH), 53.7 (d,
2JC,F ¼ 22.6 Hz, 5-CH2, rotamer A), 53.3 (d, JC,F ¼ 23.5 Hz, 5-CH2,
2
rotamer B), 36.2 (d, 2JC,F ¼ 21.2 Hz, 3-CH2), 35.1 (d, 2JC,F ¼ 21.4 Hz, 3-
CH2), 28.5 (3C, 9-CH3) ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ177.0
(s, 4-CHF, rotamer A), ꢁ177.4 (s, 4-CHF, rotamer B) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 256.1333 [M þ Na]þ; calcd. 256.1319 for
C
11H20FNO3 þ Na.
4.10.1. (2S,4S)-N-tert-butyloxycarbonyl-4-fluoro-2-hydroxymethyl
pyrrolidine (13b)
4.9.3. (2S,4R)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (28a)
A stirred solution of methyl (2S,4S)-N-tert-butyloxycarbonyl-4-
fluoropyrrolidine-2-carboxylate (750 mg, 3.03 mmol, 1.00 equiv.)
inTHF (8.0 mL) was treatedwith anhydrous LiCl (283 mg, 6.67 mmol,
2.20 equiv.) followed by NaBH4 (287 mg, 7.58 mmol, 2.50 equiv.) at
ambient temperature. The mixture was cooled to 0 ꢂC, before EtOH
(16 mL) was added dropwise over 15 min. The resulting mixture was
stirred at this temperature for 1 h and then at r.t. for 20 h. After this
time the milky mixturewas cooled down to0 ꢂC and acidified to pH 4
by addition of 10% aq. citric acid. Subsequently the solvent was
completely removed under reduced pressure and the residue was
taken up in water (30 mL). It was extracted with DCM (3 ꢃ 30 mL),
the combined organic phases were dried over MgSO4 and concen-
trated under reduced pressure. The resulting residue was purified by
flash column chromatography (silica gel, 40% EtOAc in cyclohexane)
to obtain a colorless oil (600 mg, 2.74 mmol, 90%) as the required
(2S,4R)-N-tert-butyloxycarbonyl-4-fluoro-2-methoxymethylpy
rrolidine (14a) (300 mg, 1.37 mmol, 1.00 equiv.) was converted to
(2S,4R)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]isa-
tin (28a) using TFA followed by reaction with 5-chlorosulfonyl
isatin and DIPEA as described in the general procedure in Section
4.2.2. The resulting residue was purified by flash column chroma-
tography (silica gel, 60% EtOAc in toluene and then 80% EtOAc in
toluene) to obtain an orange-yellow solid (241 mg, 0.704 mmol,
51%). M.p.159 ꢂC. 1H NMR (300 MHz, CD3CN):
d
¼ 9.33 (s,1H,1-NH),
3
4
8.02 (dd, JH,H ¼ 8.4 Hz, JH,H ¼ 1.9 Hz, 1H, 6-CH), 7.92 (d,
4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.10 (d, 3JH,H ¼ 8.4 Hz,1H, 7-CH), 5.19e4.94
(m, 1H, 14-CH), 3.91e3.79 (m, 1H, 12-CH), 3.83e3.68 (m, 1H, 15-
CHa), 3.62e3.37 (m, 3H, 15-CHb, 16-CH2), 3.31 (s, 3H, 18-CH3),
2.19e1.92 (m, 2H, 13-CH2) ppm. 13C NMR (75 MHz, CD3CN):
product. 1H NMR (400 MHz, CDCl3):
d
¼ 5.32e4.99 (m, 1H, 4-CH),

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
575
3
4.23e4.09 (m, 1H, 2-CH), 3.91e3.80 (m, 1H, 10-CHa), 3.75e3.46 (m,
2H, 5-CH2), 3.66e3.55 (m, 1H, 10-CHb), 2.37e1.95 (m, 2H, 3-CH2),
82%). 1H NMR (400 MHz, CDCl3):
d
¼ 8.06 (dd, JH,H ¼ 8.3 Hz,
4JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.02 (dd, 4JH,H ¼ 1.9 Hz, 5JH,H ¼ 0.6 Hz, 1H,
3
5
1.48 (s, 9H, 9-CH3) ppm.13C NMR (100 MHz, CDCl3):
d
¼ 156.7 (6-CO),
4-CH), 7.01 (dd, JH,H ¼ 8.3 Hz, JH,H ¼ 0.6 Hz, 1H, 7-CH), 5.15
(dt, 2JH,F ¼ 52.7 Hz, 3JH,H ¼ 4.2 Hz, 1H, 14-CH), 4.01e3.92 (m, 1H, 12-
CH), 3.74 (t, 3JH,H ¼ 7.4 Hz, 2H, 19-CH2), 3.73e3.61 (m, 1H, 15-CHa),
3.69e3.61 (m, 1H, 16-CHa), 3.48e3.34 (m, 1H, 15-CHb), 3.39e3.32
(m, 1H, 16-CHb), 3.32 (s, 3H, 18-CH3), 2.36e2.22 (m, 1H, 13-
92.1 (d, 1JC,F ¼ 176.4 Hz, 4-CH), 80.9 (8-CO), 67.8 (10-CH2), 59.2 (2-
CH), 53.9 (d, 2JC,F ¼ 23.7 Hz, 5-CH2), 35.1 (d, 2JC,F ¼ 21.1 Hz, 3-CH2),
28.4 (3C, 9-CH3) ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ170.2 (s, 4-
CHF, rotamer B), ꢁ171.4 (s, 4-CHF, rotamer A) ppm. HRMS (ESIþ,
MeOH): m/z ¼ 242.1164 [M þ Na]þ, 270.1109 [M þ Na þ MeOH]þ;
calcd. 242.1163 for C10H18FNO3 þ Na, 270.1112 for C10H18FNO3 þ Na
þ MeOH.
3
CHa), 1.98e1.77 (m, 1H, 13-CHb), 1.67 (quintet, JH,H ¼ 7.5 Hz, 2H,
3
20-CH2), 1.39 (sextet, JH,H
¼
7.4 Hz, 2H, 21-CH2), 0.96 (t,
3JH,H ¼ 7.3 Hz, 3H, 22-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 182.0 (3-CO), 157.7 (2-CO), 153.9 (8-CN), 137.5 (6-CH), 133.5 (5-
4.10.2. (2S,4S)-N-tert-butyloxycarbonyl-4-fluoro-2-methoxymethyl
pyrrolidine (14b)
CSO2), 124.6 (4-CH), 117.4 (9-CCO), 110.5 (7-CH), 92.4 (d,
1JC,F ¼ 179.6 Hz, 14-CH), 74.7 (16-CH2), 58.9 (18-CH3), 58.3 (12-CH),
55.0 (d, 2JC,F ¼ 24.3 Hz,15-CH2), 40.5 (19-CH2), 35.0 (d, 2JC,F ¼ 20.4 Hz,
13-CH2), 29.2 (20-CH2), 20.1 (21-CH2), 13.6 (22-CH3) ppm.
(2S,4S)-N-tert-butyloxycarbonyl-4-fluoro-2-hydroxymethylpyr
rolidine (13b) (573 mg, 2.61 mmol, 1.00 equiv.) was converted to
(2S,4S)-N-tert-butyloxycarbonyl-4-fluoro-2-methoxymethylpyrro
lidine (14b) using MeI and NaH as described in the general pro-
cedure in Section 4.2.1. The resulting residue was purified by flash
column chromatography (silica gel, 30% EtOAc in cyclohexane) to
obtain a pale yellow oil (527 mg, 2.26 mmol, 87%). 1H NMR
19F NMR (282 MHz, CDCl3):
d
¼ ꢁ172.2 (s, 1F, 14-CHF) ppm.
HRMS (ESIþ, MeOH): m/z ¼ 421.1197 [M þ Na]þ, 453.1460
[M þ Na þ MeOH]þ; calcd. 421.1204 for C18H23FN2O5S þ Na,
453.1466 for C18H23FN2O5S þ Na þ MeOH.
(300 MHz, CDCl3):
d
¼ 5.35e5.07 (m, 1H, 4-CH), 4.22e3.92 (m,
4.11. (S)-5-[1-(4,4-difluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatins
1H, 2-CH), 3.80e3.43 (m, 3H, 5-CHa, 10-CH23), 3.37 (s, 3H, 12-CH3),
3
2
3.31 (ddd, JH,F ¼ 10.3 Hz, JH,H ¼ 8.8 Hz, JH,H ¼ 1.8 Hz, 1H, 5-
CHb), 2.44e2.33 (m, 1H, 3-CHa), 2.21e1.94 (m, 1H, 3-CHb),
1.48 (s, 9H, 9-CH3) ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ170.8
(s, 4-CHF, rotamer A), ꢁ171.2 (s, 4-CHF, rotamer B) ppm.
HRMS (ESIþ, MeOH): m/z ¼ 256.1319 [M þ Na]þ, 489.2750
[2M þ Na]þ; calcd. 256.1319 for C11H20FNO3 þ Na, 489.2747 for
2(C11H20FNO3) þ Na.
4.10.3. (2S,4S)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (28b)
4.11.1. (S)-N-tert-butyloxycarbonyl-4,4-difluoro-2-hydroxymethyl
pyrrolidine
(2S,4S)-N-tert-butyloxycarbonyl-4-fluoro-2-methoxymethylpy-
rrolidine (14b) (500 mg, 2.14 mmol, 1.00 equiv.) was converted
to (2S,4S)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (28b) using TFA followed by reaction with 5-chlorosulfonyl
isatin and DIPEA as described in the general procedure in Section
4.2.2. The crude was purified by flash column chromatography
(silica gel, 60% and then 80% EtOAc in cyclohexane) to obtain a
yellow solid (370 mg, 0.993 mmol, 46%). M.p. 169 ꢂC. 1H NMR
A stirred solution of methyl (S)-N-tert-butyloxycarbonyl-4,4-
difluoropyrrolidine-2-carboxylate (15) (790 mg, 2.98 mmol, 1.00
equiv.) in THF (10 mL) was treated with LiCl anhydrous (278 mg,
6.55 mmol, 2.20 equiv.) and followed by NaBH4 (282 mg,
7.45 mmol, 2.50 equiv.) at ambient temperature. The resulting
mixture was cooled to 0 ꢂC, before EtOH (20 mL) was added
dropwise over 15 min. Subsequently the mixture was stirred at this
temperature for 1 h and at r.t. for 24 h. After this time, the milky
mixture was cooled to 0 ꢂC and acidified to pH 4 by addition of 10%
aq. citric acid. The solvent was completely removed under reduced
pressure and it was taken up in water (30 mL). The mixture was
extracted with DCM (3 ꢃ 30 mL) and the combined organic layer
was dried over MgSO4 and concentrated under reduced pressure.
The crude product was purified by flash column chromatography
(silica gel, 40% EtOAc in cyclohexane) to obtain a colorless oil
(300 MHz, CD3CN):
d
¼ 9.37 (br s,1H,1-NH), 8.01 (dd, 3JH,H ¼ 8.3 Hz,
4JH,H ¼ 1.9 Hz, 1H, 6-CH), 7.91 (d, JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.13 (d,
3JH,H ¼ 8.3 Hz, 1H, 7-CH), 5.13 (dt, 2JH,F ¼ 53.1 Hz, 3JH,H ¼ 4.2 Hz, 1H,
14-CH), 3.95e3.80 (tdd, 3JH,H ¼ 9.1 Hz, 3JH,H ¼ 5.2 Hz, 3JH,H ¼ 1.5 Hz,
1H, 12-CH), 3.68e3.53 (m, 2H, 15-CHa, 16-CHa), 3.46e3.26 (m, 2H,
15-CHb, 16-CHb), 3.30 (s, 3H, 18-CH3), 2.21e2.05 (m, 1H, 13-CHa),
1.98e1.71 (m, 1H, 13-CHb) ppm. 13C NMR (75 MHz, CD3CN):
4
d
¼ 183.8 (3-CO), 159.7 (2-CO), 154.4 (8-CN), 138.4 (6-CH), 132.8 (5-
(700 mg, 2.95 mmol, 99%). 1H NMR (300 MHz, CDCl3):
d
¼ 4.26e
CSO2), 125.0 (4-CH), 119.0 (9-CCO), 114.0 (7-CH), 94.0 (d,
4.02 (m, 1H, 2-CH), 3.95e3.54 (m, 4H, 5-CH2, 10-CH2), 2.60e2.38
(m, 1H, 3-CHa), 2.30e2.04 (m, 1H, 3-CHb), 1.48 (s, 9H, 9-CH3) ppm.
1JC,F ¼ 176.0 Hz, 14-CH), 75.7 (16-CH), 59.2 (12-CH), 59.1 (18-CH3),
2
2
56.0 (d, JC,F ¼ 23.8 Hz, 15-CH2), 35.3 (d, JC,F ¼ 20.0 Hz, 13-CH2)
13C NMR (75 MHz, CDCl3):
d
¼
155.7 (6-CO), 126.4 (t,
ppm. 19F NMR (282 MHz, CD3CN):
d
¼ ꢁ171.9 (s, 1F, 14-CHF) ppm.
1JC,F ¼ 246.3 Hz, 4-CF2), 81.4 (8-CO), 65.1 (10-CH2), 58.3 (2-CH), 54.0
HRMS (ESIþ, MeOH): m/z ¼ 365.0573 [M þ Na]þ, 397.0837
[M þ Na þ MeOH]þ; calcd. 365.0578 for C14H15FN2O5S þ Na,
397.0840 for C14H15FN2O5S þ Na þ MeOH.
(t, JC,F ¼ 31.2 Hz, 5-CH2), 36.5 (t, JC,F ¼ 23.7 Hz, 3-CH2), 28.3 (3C,
2
2
9-CH3) ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ99.8 (br s, 4-
2
CF2, rotamer B), ꢁ100.1 (d, JF,F ¼ 232.2 Hz, 4-CF2, rotamer
A), ꢁ101.4 (d, 2JF,F ¼ 233.2 Hz, 4-CF2, rotamer A) ppm. HRMS (ESIþ,
4.10.4. (2S,4S)-N-butyl-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (29b)
MeOH): m/z
¼
260.1063 [M
þ
Na]þ; calcd. 260.1069 for
C10H17F2NO3 þ Na.
(2S,4S)-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (28b) (50 mg, 0.146 mmol, 1.00 equiv.) was converted to
(2S,4S)-N-butyl-5-[1-(4-fluoro-2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin (29b) using 1-bromobutane and Cs2CO3 as described in
the general procedure in Section 4.2.3. The crude product was pu-
rified by flash column chromatography (silica gel, 35% THF in
cyclohexane) to obtain a sticky yellow gum (48 mg, 0.121 mmol,
4.11.2. (S)-N-tert-butyloxycarbonyl-4,4-difluoro-2-methoxymethyl
pyrrolidine (16)
(S)-N-tert-butyloxycarbonyl-4,4-difluoro-2-hydroxymethylpyr
rolidine (640 mg, 2.70 mmol, 1.00 equiv.) was converted to (S)-N-
tert-butyloxycarbonyl-4,4-difluoro-2-methoxymethylpyrrolidine
(16) using MeI and NaH as described in the general procedure in

576
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
Section 4.2.1. The residue was purified by flash column chroma-
4.12. cis-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatins
tography (silica gel, 15% EtOAc in cyclohexane) to yield a colorless
oil (625 mg, 2.49 mmol, 92%). 1H NMR (400 MHz, CDCl3):
4.02 (m, 1H, 2-CH), 3.95e3.70 (m, 1H, 5-CHa), 3.69e3.23 (m, 3H, 5-
CHb, 10-CH2), 3.36 (s, 3H, 12-CH3), 2.55e2.33 (m, 2H, 3-CH2), 1.47 (s,
d
¼ 4.27e
9H, 9-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 153.8 (6-CO), 127.2
(t, 1JC,F ¼ 247.6 Hz, 4-CF2), 80.5 (8-CO), 72.4 (10-CO), 59.1 (12-CH3),
55.0 (2-CH), 53.7 (m, 5-CH2), 36.6 (m, 3-CH2), 28.4 (3C, 9-CH3) ppm.
2
19F NMR (282 MHz, CDCl3):
d
¼ ꢁ98.4 (d, JF,F ¼ 234.9 Hz, 4-CF2,
rotamer B), ꢁ99.1 (br s, 4-CF2, rotamer A), ꢁ99.7 (d, 2JF,F ¼ 234.7 Hz,
4-CF2, rotamer B) ppm. HRMS (ESIþ, MeOH): m/z ¼ 274.1224
[M þ Na]þ, 525.2561 [2M þ Na]þ; calcd. 274.1225 for C11H19F2NO3
þ Na, 525.2558 for 2(C11H19F2NO3) þ Na.
4.12.1. cis-N-benzyl-2,5-bis(methoxymethyl)pyrrolidine (18a)
A stirred suspension of 60% NaH in mineral oil (163 mg,
4.08 mmol, 3.00 equiv.) in dry THF (5 mL) under argon atmosphere
was cooled to 0 ꢂC and treated with a solution of cis-N-benzyl-2,5-
bis(hydroxymethyl)pyrrolidine (17a) (300 mg, 1.36 mmol, 1.00
equiv.) in dry THF (3 mL) dropwise. The mixture was stirred at 0 ꢂC
for 15 min and at r.t. for 30 min, before it was cooled to 0 ꢂC again
4.11.3. (S)-5-[1-(4,4-difluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (30)
(S)-N-tert-butyloxycarbonyl-4,4-difluoro-2-methoxymethylpyr
rolidine (16) (600 mg, 2.39 mmol, 1.00 equiv.) was converted to
(S)-5-[1-(4,4-difluoro-2-methoxymethylpyrrolidinyl)sulfonyl]isatin
(30) using TFA followed by reaction with 5-chlorosulfonyl isatin and
DIPEA as described in the general procedure in Section 4.2.2. The
crude product was purified by flash column chromatography (silica
gel, 40% EtOAc in toluene) to obtain a yellow solid (245 mg,
0.680 mmol, 28%). M.p. 69e70 ꢂC. 1H NMR (300 MHz, CD3CN):
and MeI (254 mL, 4.08 mmol, 3.00 equiv.) was added dropwise over
5e10 min. The reaction mixture was stirred at r.t. for 20 h. After
this time, it was cooled to 0 ꢂC, diluted with DCM (20 mL) and
quenched by slow addition of sat. aq. NH4Cl until no more H2
evolved. H2O (10 mL) was added and two phases were separated.
The aq. phase was extracted with DCM (3 ꢃ 10 mL). The combined
organic phase was dried over MgSO4 and concentrated under
reduced pressure. The resulting residue was purified by flash
column chromatography (silica gel, 25% EtOAc in cyclohexane) to
obtain a colorless oil (302 mg, 1.21 mmol, 89%). 1H NMR (400 MHz,
d
¼ 9.35 (br s,1H,1-NH), 8.03 (dd, 3JH,H ¼ 8.4 Hz, 4JH,H ¼ 2.0 Hz,1H, 6-
CH), 7.95 (d, 4JH,H ¼ 2.0 Hz, 1H, 4-CH), 7.14 (d, 3JH,H ¼ 8.4 Hz, 1H, 7-
CH), 4.07e3.96 (m, 1H, 12-CH), 3.85e3.58 (m, 2H, 15-CH2), 3.58e
3.50 (m, 2H, 16-CH2), 3.31 (s, 3H, 18-CH3), 2.43e2.26 (m, 2H, 13-CH2)
ppm. 13C NMR (75 MHz, CD3CN):
d
¼ 183.8 (3-CO), 159.7 (2-CO),
CDCl3):
d
¼ 7.37e7.20 (m, 5H, 2 ꢃ 8-CH, 2 ꢃ 9-CH, 10-CH), 3.89 (s,
154.5 (8-CN), 138.4 (6-CH), 133.4 (5-CSO2), 128.0 (dd, 1JC,F ¼ 250.0 Hz,
1JC,F ¼ 247.6 Hz, 14-CF2), 125.1 (4-CH), 119.0 (9-CCO), 113.9 (7-CH),
74.5 (16-CH2), 59.4 (18-CH3), 58.8 (dd, 3JC,F ¼ 4.5 Hz, 3JC,F ¼ 2.5 Hz,12-
2H, 6-CH2), 3.35e3.08 (m, 4H, 11-CH2, 14-CH2), 3.24 (s, 6H, 13-CH3,
16-CH3), 3.05e2.90 (m, 2H, 2-CH, 5-CH), 1.94e1.76 (m, 2H, 3-CHa,
4-CHa), 1.70e1.52 (m, 2H, 3-CHb, 4-CHb) ppm. 13C NMR (100 MHz,
2
2
CH), 55.4 (dd, JC,F ¼ 33.6 Hz, JC,F ¼ 30.4 Hz, 15-CH2), 37.3 (dd,
CDCl3):
d
¼ 139.9 (7-CCH2), 129.2 (2C, 2 ꢃ 8-CH), 128.0 (2C, 2 ꢃ 9-
2JC,F ¼ 25.0 Hz, JC,F ¼ 23.2 Hz, 13-CH2) ppm. 19F NMR (282 MHz,
CH), 126.8 (10-CH), 76.8 (2C, 11-CH2, 14-CH2), 64.3 (2C, 2-CH, 5-
CH), 59.0 (6-CH2), 58.9 (2C, 13-CH3, 16-CH3), 27.7 (2C, 3-CH2, 4-
CH2) ppm. HRMS (ESIþ, MeOH): m/z ¼ 250.1814 [M þ H]þ;
calcd. 250.1802 for C15H23NO2 þ H.
2
2
CD3CN):
d
¼ ꢁ97.5 (d, JF,F ¼ 230.5 Hz, 1F, 14-CF2), ꢁ102.4 (d,
2JF,F ¼ 230.5 Hz, 1F, 14-CF2) ppm. HRMS (ESIþ, MeOH): m/
z ¼ 383.0493 [M þ Na]þ; calcd. 383.0484 for C14H14F2N2O5S þ Na.
4.11.4. (S)-N-butyl-5-[1-(4,4-difluoro-2-methoxymethylpyrrolidinyl)
sulfonyl]isatin (31)
4.12.2. cis-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}
isatin (19a)
(S)-5-[1-(4,4-difluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (30) (50 mg, 0.139 mmol, 1.00 equiv.) was converted to (S)-N-
butyl-5-[1-(4,4-difluoro-2-methoxymethylpyrrolidinyl)sulfonyl]
isatin (31) using 1-bromobutane and Cs2CO3 as described in the
general procedure in Section 4.2.3. The residue was purified by
flash column chromatography (silica gel, 30% THF in cyclohexane)
to obtain a yellow wax (50 mg, 0.120 mmol, 86%). 1H NMR
A mixture of cis-N-benzyl-2,5-bis(methoxymethyl)pyrrolidine
(18a) (620 mg, 2.49 mmol, 1.00 equiv.) and 10% Pd/C (62 mg, 10% w/
w) in MeOH (12 mL) was stirred in an autoclave at ambient tem-
perature under H2 atmosphere (20 atm) for 6 h. After this time, the
mixture was filtered through CeliteÒ and washed again with MeOH.
The solvent was removed under reduced pressure to obtain the
corresponding unprotected bis(methoxymethyl)pyrrolidine as a
light yellow oil. The solution of obtained free amine cis-2,5-
(400 MHz, CDCl3):
d
¼ 8.06 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-
CH), 8.03 (d, 4JH,H ¼ 2.0 Hz, 1H, 4-CH), 7.06 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-
bis(methoxymethyl)pyrrolidine and DIPEA (766 mL, 4.40 mmol,
3
CH), 4.02e3.94 (m, 1H, 12-CH), 3.78 (t, JH,H ¼ 7.4 Hz, 2H, 19-CH2),
2.00 equiv.) in CHCl3 (5 mL) was added dropwise to a stirred solution
of 5-chlorosulfonyl isatin (1.08 g, 4.40 mmol, 2.00 equiv.) in CHCl3/
THF (1:1, 50 mL) at room temperature. The resulting mixture was
stirred further for 2 h and concentrated under reduced pressure. The
residue was purified by flash column chromatography (silica gel,
90% EtOAc in cyclohexane) to yield a yellow solid (723 mg,
1.96 mmol, 89%). M.p. 168 ꢂC. 1H NMR (400 MHz, DMSO-d6):
3.75e3.62 (m, 2H, 15-CH2), 3.63e3.54 (m, 2H, 16-CH2), 3.38 (s, 3H,
18-CH3), 2.52e2.24 (m, 2H,13-CH2), 1.71 (quintet, 3JH,H ¼ 7.5 Hz, 2H,
3
20-CH2), 1.43 (sextet, JH,H
¼
7.4 Hz, 2H, 21-CH2), 0.99 (t,
3JH,H ¼ 7.4 Hz, 3H, 22-CH3) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 182.0 (3-CO), 157.7 (2-CO), 154.2 (8-CN), 137.5 (6-CH), 133.3 (5-
CSO2), 126.3 (dd, 1JC,F ¼ 252.2 Hz, 1JC,F ¼ 248.2 Hz, 14-CF2), 124.6 (4-
CH), 117.4 (9-CCO), 110.6 (7-CH), 73.8 (16-CH2), 59.2 (18-CH3), 57.9
d
¼ 11.46 (br s, 1H, 1-NH), 8.05 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H,
3
3
2
(dd, JC,F ¼ 4.4 Hz, JC,F ¼ 2.2 Hz, 12-CH), 54.7 (dd, JC,F ¼ 33.3 Hz,
6-CH), 7.80 (d, 4JH,H ¼ 1.9 Hz,1H, 4-CH), 7.09 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-
CH), 3.73e3.58 (m, 2H,12-CH,15-CH), 3.52e3.20 (m, 4H,16-CH2, 19-
CH2), 3.37 (s, 6H,18-CH3, 21-CH3),1.78e1.62 (m, 2H,13-CHa,14-CHa),
1.57e1.37 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (100 MHz, DMSO-
2JC,F ¼ 30.3 Hz, 15-CH2), 40.5 (19-CH2), 36.8 (dd, JC,F ¼ 24.8 Hz,
2
2JC,F ¼ 23.1 Hz, 13-CH2), 29.2 (20-CH2), 20.1 (21-CH2), 13.6 (22-CH3)
ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ98.3 (d, JF,F ¼ 232.1 Hz,
2
2
1F, 14-CF2), ꢁ103.3 (d, JF,F
¼
232.0 Hz, 1F, 14-CF2) ppm.
Na]þ, 471.1374
d6):
d
¼ 182.9 (3-CO), 159.4 (2-CO), 153.6 (8-CN), 136.7 (6-CH), 130.8
HRMS (ESIþ, MeOH): m/z
¼
439.1118 [M
þ
(5-CSO2), 123.1 (4-CH), 118.1 (9-CCO), 112.7 (7-CH), 74.4 (2C, 16-CH2,
19-CH2), 59.9 (2C,12-CH,15-CH), 58.4 (2C,18-CH3, 21-CH3), 26.9 (2C,
13-CH2, 14-CH2) ppm. HRMS (ESIþ, MeOH): m/z ¼ 391.0931
[M þ Na þ MeOH]þ; calcd. 439.1110 for C18H22F2N2O5S þ Na,
471.1372 for C18H22F2N2O5S þ Na þ MeOH.

P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
577
[M þ Na]þ, 423.1194 [M þ Na þ MeOH]þ; calcd. 391.0934 for
4.13.1. trans-N-benzyl-2,5-bis(methoxymethyl)pyrrolidine (18b)
C16H20N2O6S þ Na, 423.1196 for C16H20N2O6S þ Na þ MeOH.
trans-N-benzyl-2,5-bis(hydroxymethyl)pyrrolidine (17b) (628
mg, 2.84 mmol, 1.00 equiv.) was converted to trans-N-benzyl-2,5-
bis(methoxymethyl)pyrrolidine (18b) using the same procedure
of the synthesis of 18a (Section 4.12.1). The crude product was
purified by flash column chromatography (silica gel, 15% EtOAc in
cyclohexane) to obtain a colorless oil (493 mg, 1.98 mmol, 70%). 1H
4.12.3. cis-N-butyl-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]
sulfonyl}isatin (32a)
cis-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatin
(19a) (100 mg, 0.271 mmol, 1.00 equiv.) was converted to cis-
N-butyl-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatin
(32a) using 1-bromobutane and Cs2CO3 as described in the general
procedure in Section 4.2.3. The residue was purified by flash col-
umn chromatography (silica gel, 35% EtOAc in toluene) to obtain a
yellow solid (84 mg, 0.198 mmol, 73%). M.p. 115 ꢂC. 1H NMR
NMR (400 MHz, CDCl3):
d
¼ 7.40e7.34 (m, 2H, 2 ꢃ 8-CH), 7.32e
7.26 (m, 2H, 2 ꢃ 9-CH), 7.23e7.18 (m, 1H, 10-CH), 4.00 (d,
2JH,H ¼ 14.3 Hz, 1H, 6-CHa), 3.88 (d, JH,H ¼ 14.3 Hz, 1H, 6-CHb),
2
3.37e3.21 (m, 4H, 11-CH2, 14-CH2), 3.27 (s, 6H, 13-CH3, 16-CH3),
3.23e3.12 (m, 2H, 2-CH, 5-CH), 2.06e1.90 (m, 2H, 3-CHa, 4-CHa),
1.75e1.60 (m, 2H, 3-CHb, 4-CHb) ppm. 13C NMR (100 MHz, CDCl3):
(400 MHz, CDCl3):
d
¼ 8.09 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 1.9 Hz, 1H, 6-
CH), 8.04 (d, 4JH,H ¼ 1.9 Hz, 1H, 4-CH), 7.05 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-
d
¼ 140.8 (7-C), 128.2 (2C, 2 ꢃ 8-CH), 128.1 (2C, 2 ꢃ 9-CH), 126.5
CH), 3.79e3.69 (m, 2H, 12-CH, 15-CH), 3.78 (t, 3JH,H ¼ 7.4 Hz, 2H, 22-
(10-CH), 74.6 (2C, 11-CH2, 14-CH2), 60.4 (2C, 2-CH, 5-CH), 59.0 (2C,
13-CH3, 16-CH3), 52.7 (6-CH2), 27.2 (2C, 3-CH2, 4-CH2) ppm. HRMS
(ESIþ, MeOH): m/z ¼ 250.1810 [M þ H]þ; calcd. 250.1802 for
2
3
CH2), 3.60 (dd, JH,H ¼ 9.4 Hz, JH,H ¼ 4.2 Hz, 2H, 16-CHa, 19-CHa),
3.40e3.34 (m, 2H, 16-CHb, 19-CHb), 3.37 (s, 6H, 18-CH3, 21-CH3),
1.95e1.79 (m, 2H, 13-CHa, 14-CHa), 1.75e1.66 (m, 2H, 23-CH2), 1.72e
C
15H23NO2 þ H.
3
1.58 (m, 2H, 13-CHb, 14-CHb), 1.43 (sextet, JH,H ¼ 7.5 Hz, 2H,
24-CH2), 0.99 (t, JH,H ¼ 7.4 Hz, 3H, 25-CH3) ppm. 13C NMR
4.13.2. trans-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}
isatin (19b)
3
(100 MHz, CDCl3):
d
¼ 182.2 (3-CO),157.8 (2-CO),153.8 (8-CN),137.5
(6-CH), 133.7 (CSO2), 124.5 (4-CH), 117.3 (9-CCO), 110.5 (7-CH), 74.9
(2C, 16-CH2, 19-CH2), 60.7 (2C, 12-CH, 15-CH), 59.2 (18-CH3), 40.5
(22-CH2), 29.2 (23-CH2), 27.6 (2C, 13-CH2, 14-CH2), 20.1 (24-CH2),
trans-N-benzyl-2,5-bis(methoxymethyl)pyrrolidine (18b) (491
mg, 1.97 mmol, 1.00 equiv.) was converted to trans-5-{1-[2,5-
bis(methoxymethyl)pyrrolidinyl]sulfonyl} isatin (19b) with the
same conditions as described in the synthesis of 19a (Section 4.12.2).
The residue was purified by flash column chromatography (silica gel,
80% EtOAc in cyclohexane) to furnish a yellow oil (104 mg,
13.6 (25-CH3) ppm. HRMS (ESIþ, MeOH): m/z
¼
447.1561
[M þ Na]þ, 479.1820 [M þ Na þ MeOH]þ; calcd. 447.1560 for
C
20H28N2O6S þ Na, 479.1822 for C20H28N2O6S þ Na þ MeOH.
0.282 mmol,14%).1H NMR (300 MHz, CDCl3):
d
¼ 9.03(br s,1H,1-NH),
4.12.4. cis-N-(4-fluorobutyl)-5-{1-[2,5-bis(methoxymethyl)pyrroli
dinyl]sulfonyl}isatin (33a)
8.10 (d, 4JH,H ¼ 2.0 Hz,1H, 4-CH), 8.10 (dd, 3JH,H ¼ 8.8 Hz, 4JH,H ¼ 2.0 Hz,
1H, 6-CH), 7.10 (d, 3JH,H ¼ 8.8 Hz,1H, 7-CH), 4.01e3.90 (m, 2H, 12-CH,
15-CH), 3.54e3.46 (m, 4H, 16-CH2, 19-CH2), 3.22 (s, 6H, 18-CH3, 21-
CH3), 2.20e2.04 (m, 2H, 13-CHa, 14-CHa), 1.98e1.83 (m, 2H, 13-CHb,
cis-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatin
(19a) (100 mg, 0.271 mmol, 1.00 equiv.) was converted to cis-N-(4-
fluorobutyl)-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}
isatin (33a) using TFA followed by reaction with 5-chlorosulfonyl
isatin and DIPEA as described in the general procedure in Section
4.2.3. The residue was purified by flash column chromatography
(silica gel, 60% EtOAc in toluene) to obtain a yellow solid (93 mg,
14-CHb) ppm. 13C NMR (75 MHz, CDCl3):
d
¼ 181.9 (3-CO), 158.9 (2-
CO), 151.7 (8-CN), 138.0 (5-CSO2), 137.3 (6-CH), 124.6 (4-CH), 117.5
(9-CCO), 112.6 (7-CH), 73.5 (2C, 16-CH2, 19-CH2), 60.4 (2C, 12-CH, 15-
CH), 58.8 (2C, 18-CH3, 21-CH3), 27.9 (2C, 13-CH2, 14-CH2) ppm.
HRMS (ESIþ, MeOH): m/z
¼
391.0927 [M þ Na]þ, 423.1192
0.210 mmol, 77%). M.p. 145 ꢂC. 1H NMR (300 MHz, CDCl3):
d
¼ 8.10
[M þ Na þ MeOH]þ; calcd. 391.0934 for C16H20N2O6S þ Na, 423.1196
for C16H20N2O6S þ Na þ MeOH.
(dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 1.9 Hz, 1H, 6-CH), 8.05 (d, 4JH,H ¼ 1.9 Hz,
1H, 4-CH), 7.07 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-CH), 4.52 (dt, 2JH,F ¼ 47.5 Hz,
3JH,H ¼ 5.4 Hz, 2H, 25-CH2), 3.84 (t, 3JH,H ¼ 7.1 Hz, 2H,17-CH2), 3.80e
3.68 (m, 2H, 12-CH, 15-CH), 3.60 (dd, 2JH,H ¼ 9.4 Hz, 3JH,H ¼ 4.2 Hz,
2H, 16-CHa, 19-CHa), 3.42e3.32 (m, 2H, 16-CHb, 19-CHb), 3.36 (s, 6H,
18-CH3, 21-CH3), 1.96e1.79 (m, 2H, 13-CHa, 14-CHa), 1.91e1.71 (m,
4H, 23-CH2, 24-CH2), 1.71e1.57 (m, 2H, 13-CHb, 14-CHb) ppm. 13C
4.13.3. trans-N-butyl-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]
sulfonyl}isatin (32b)
trans-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatin
(19b) (45 mg, 0.122 mmol, 1.00 equiv.) was converted to trans-
N-butyl-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatin
(32b) using 1-bromobutane and Cs2CO3 as described in the general
procedure in Section 4.2.3. The crude product was purified by flash
column chromatography (silica gel, 40% EtOAc in cyclohexane) to
NMR (75 MHz, CDCl3):
d
¼ 182.0 (3-CO), 157.8 (2-CO), 153.5 (8-CN),
137.6 (6-CH), 133.9 (5-CSO2), 124.6 (4-CH), 117.4 (9-CCO), 110.4 (7-
1
CH), 83.3 (d, JC,F ¼ 165.5 Hz, 25-CH2), 74.9 (2C, 16-CH2, 19-CH2),
60.7 (2C, 12-CH, 15-CH), 59.2 (2C, 18-CH3, 21-CH3), 40.2 (22-CH2),
obtain a yellow oil (35 mg, 82.4 m
mol, 68%). 1H NMR (400 MHz,
2
27.6 (d, JC,F ¼ 20.1 Hz, 24-CH2), 27.6 (2C, 13-CH2, 14-CH2), 23.5 (d,
CDCl3):
d
¼ 8.11 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-CH), 8.07 (d,
3JC,F ¼ 4.0 Hz, 23-CH2) ppm. 19F NMR (282 MHz, CDCl3):
d
¼ ꢁ219.9
4JH,H ¼ 2.0 Hz, 1H, 4-CH), 7.00 (d, JH,H ¼ 8.3 Hz, 1H, 7-CH), 3.98e
3
3
(s, 1F, 25-CH2F) ppm. HRMS (ESIþ, MeOH): m/z ¼ 465.1461
3.90 (m, 2H, 12-CH, 15-CH), 3.78 (t, JH,H ¼ 7.3 Hz, 2H, 22-CH2),
[M þ Na]þ, 497.1723 [M þ Na þ MeOH]þ; calcd. 465.1466 for
3.55e3.45 (m, 4H, 16-CH2, 19-CH2), 3.22 (s, 6H, 18-CH3, 21-CH3),
2.20e2.06 (m, 2H, 13-CHa, 14-CHa), 1.97e1.83 (m, 2H, 13-CHb, 14-
C
20H27FN2O6S þ Na, 497.1728 for C20H27FN2O6S þ Na þ MeOH.
3
CHb), 1.70 (quintet, JH,H ¼ 7.4 Hz, 2H, 23-CH2), 1.42 (sextet,
4.13. trans-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatins
3JH,H ¼ 7.4 Hz, 2H, 24-CH2), 0.98 (t, 3JH,H ¼ 7.3 Hz, 3H, 25-CH3) ppm.
13C NMR (100 MHz, CDCl3):
d
¼ 182.2 (3-CO), 157.9 (2-CO), 153.3 (8-
CN), 137.7 (5-CSO2), 137.1 (6-CH), 124.2 (4-CH), 117.2 (9-CCO), 110.0
(7-CH), 73.5 (2C, 16-CH2, 19-CH2), 60.4 (2C, 12-CH, 15-CH), 58.8 (2C,
18-CH3, 21-CH3), 40.4 (22-CH2), 29.2 (23-CH2), 27.8 (2C, 13-CH2, 14-
CH2), 20.1 (24-CH2), 13.6 (25-CH3) ppm. HRMS (ESIþ, MeOH):
m/z ¼ 447.1557 [M þ Na]þ, 479.1820 [M þ Na þ MeOH]þ; calcd.
447.1560 for
þ Na þ MeOH.
C
20H28N2O6S
þ
Na, 479.1822 for C20H28N2O6S

578
P. Limpachayaporn et al. / European Journal of Medicinal Chemistry 64 (2013) 562e578
4.13.4. trans-N-(4-fluorobutyl)-5-{1-[2,5-bis(methoxymethyl)
pyrrolidinyl]sulfonyl}isatin (33b)
References
[1] M.O. Hengartner, The biochemistry of apoptosis, Nature 407 (2000)
770e776.
trans-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}isatin
(19b) (45 mg, 0.122 mmol, 1.00 equiv.) was converted to trans-N-
(4-fluorobutyl)-5-{1-[2,5-bis(methoxymethyl)pyrrolidinyl]sulfonyl}
isatin (33b) using 1-bromo-4-fluorobutane and Cs2CO3 as described
in the general procedure in Section 4.2.3. The crude product was
purified by flash column chromatography (silica gel, 50% EtOAc in
[2] D. Lee, S.A. Long, J.L. Adams, G. Chan, K.A. Vaidya, T.A. Francis, K. Kikly,
J.D. Winkler, C.-M. Sung, C. Debouck, S. Richardson, M.A. Levy, W.E. DeWolf Jr.,
P.M. Keller, T. Tomaszek, M.S. Head, M.D. Ryan, R.C. Haltiwanger, P.-H. Liang,
C.A. Janson, P.J. McDevitt, K. Johanson, N.O. Concha, W. Chan, S.S. Abdel-
Meguid, A.M. Badger, M.W. Lark, D.P. Nadeau, L.J. Suva, M. Gowen,
M.E. Nuttall, Potent and selective nonpeptide inhibitors of caspases-3 and -7
inhibit apoptosis and maintain cell functionality, J. Biol. Chem. 275 (2000)
16007e16014.
[3] D. Lee, S.A. Long, J.H. Murray, J.L. Adams, M.E. Nuttall, D.P. Nadeau, K. Kikly,
J.D. Winkler, C.-M. Sung, M.D. Ryan, M.A. Levy, P.M. Keller, W.E. DeWolf,
Potent and selective nonpeptide inhibitors of caspases-3 and -7, J. Med. Chem.
44 (2001) 2015e2026.
[4] K. Kopka, A. Faust, P. Keul, S. Wagner, H.-J. Breyholz, C. Höltke, O. Schober,
M. Schäfers, B. Levkau, 5-pyrrolidinylsulfonyl isatins as a potential tool for the
molecular imaging of caspases in apoptosis, J. Med. Chem. 49 (2006) 6704e
6715.
[5] A.K. Podichetty, A. Faust, K. Kopka, S. Wagner, O. Schober, M. Schäfers,
G. Haufe, Fluorinated isatin derivatives. Part 1: synthesis of new N-substituted
(S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatins as potent caspase-3
and -7 inhibitors, Bioorg. Med. Chem. 17 (2009) 2680e2688.
[6] A.K. Podichetty, S. Wagner, S. Schröer, A. Faust, M. Schäfers, O. Schober,
K. Kopka, G. Haufe, Fluorinated isatin derivatives. Part 2: new N-substituted 5-
pyrrolidinylsulfonyl isatins as potential tools for molecular imaging of cas-
pases in apoptosis, J. Med. Chem. 52 (2009) 3484e3495.
[7] W. Chu, J. Zhang, C. Zeng, J. Rothfuss, Z. Tu, Y. Chu, D.E. Reichert, M.J. Welch,
R.H. Mach, N-benzylisatin sulfonamide analogues as potent caspase-3 in-
hibitors: synthesis, in vitro activity, and molecular modeling studies, J. Med.
Chem. 48 (2005) 7637e7647.
[8] W. Chu, J. Rothfuss, A. D’Avignon, C. Zeng, D. Zhou, R.S. Hotchkiss, R.H. Mach,
Isatin sulfonamide analogs containing a Michael addition acceptor: a new
class of caspase 3/7 inhibitors, J. Med. Chem. 50 (2007) 3751e3755.
[9] D. Zhou, W. Chu, J. Rothfuss, C. Zeng, J. Xu, L. Jones, M.J. Welch, R.H. Mach,
Synthesis, radiolabeling, and in vivo evaluation of an 18F-labeled isatin analog
for imaging caspase-3 activation in apoptosis, Bioorg. Med. Chem. Lett. 16
(2006) 5041e5046.
[10] A. Faust, S. Wagner, M.P. Law, S. Hermann, U. Schnöckel, P. Keul, O. Schober,
M. Schäfers, B. Levkau, K. Kopka, The nonpeptidyl caspase binding radioligand
(S)-1-(4-(2-[18F]fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)
sulfonyl]isatin ([18F]CbR) as potential positron emission tomography-
compatible apoptosis imaging agent, Q. J. Nucl. Med. Mol. Imaging 51
(2007) 67e73.
cyclohexane) to obtain a yellow oil (29 mg, 65.5 m
mol, 54%). 1H NMR
(400 MHz, CDCl3):
d
¼ 8.12 (dd, 3JH,H ¼ 8.3 Hz, 4JH,H ¼ 2.0 Hz, 1H, 6-
CH), 8.08 (d, 4JH,H ¼ 2.0 Hz, 1H, 4-CH), 7.02 (d, 3JH,H ¼ 8.3 Hz, 1H, 7-
CH), 4.52 (dt, 2JH,F ¼ 47.4 Hz, 3JH,H ¼ 5.5 Hz, 2H, 25-CH2), 3.99e3.89
(m, 2H, 12-CH, 15-CH), 3.84 (t, 3JH,H ¼ 7.1 Hz, 2H, 22-CH2), 3.57e3.43
(m, 4H, 16-CH2, 19-CH2), 3.22 (s, 6H, 18-CH3, 21-CH3), 2.21e2.05 (m,
2H, 13-CHa, 14-CHa), 1.98e1.78 (m, 6H, 13-CHb, 14-CHb, 23-CH2, 24-
CH2) ppm. 13C NMR (100 MHz, CDCl3):
CO), 153.0 (8-CN), 137.9 (5-CSO2), 137.2 (6-CH), 124.3 (4-CH), 117.2
(9-CCO), 110.0 (7-CH), 83.2 (d, 1JC,F ¼ 165.6 Hz, 25-CH2), 73.5 (2C, 16-
CH2, 19-CH2), 60.4 (2C, 12-CH, 15-CH), 58.8 (2C, 18-CH3, 21-CH3),
d
¼ 182.0 (3-CO), 157.9 (2-
2
40.2 (22-CH2), 27.8 (2C, 13-CH2, 14-CH2), 27.6 (d, JC,F ¼ 20.1 Hz,
3
24-CH2), 23.5 (d, JC,F
¼
4.0 Hz, 23-CH2) ppm. 19F NMR
(282 MHz, CDCl3):
d
¼ ꢁ220.0 (s, 1F, 25-CH2F) ppm. HRMS
(ESIþ, MeOH): m/z
¼
465.1465 [M
þ
Na]þ, 497.1731
[M þ Na þ MeOH]þ; calcd. 465.1466 for C20H27FN2O6S þ Na, 497.1728
for C20H27FN2O6S þ Na þ MeOH.
4.14. In vitro assays
The caspase inhibition potencies of the target isatin sulfonamides
were indicated as IC50 values. The recombinant human caspases-3
and -7 including their peptide-specific substrate Ac-DEVD-AMC
(Ac-Asp-Glu-Val-Asp-AMC) were purchased from AlexisÒ Bio-
chemicals (Switzerland). As already described [4,7], 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 universal micro-
plate analyzer (PerkinElmer) at excitation and emission wave-
lengths of 360 and 460 nm, respectively. All assays were performed
[11] D. Zhou, W. Chu, D.L. Chen, Q. Wang, D.E. Reichert, J. Rothfuss, A. D’Avignon,
M.J. Welch, R.H. Mach, [18F]- and [11C]-labeled N-benzylisatin sulfonamide
analogues as PET tracers for apoptosis: synthesis, radiolabeling mechanism
and in vivo imaging study of apoptosis in Fas-treated mice using [11C]WC-98,
Org. Biomol. Chem. 7 (2009) 1337e1348.
at a volume of 200
tained the target compounds in DMSO in single doses (end con-
centrations 500 M, 50 M, 5 M, 500 nM, 50 nM, 5 nM, 500 pM, or
m
L at 37 ꢂC in reaction buffer [4,7]. Buffers con-
[12] A. Baumann, A. Faust, M.P. Law, M.T. Kuhlmann, K. Kopka, M. Schäfers,
U. Karst, Metabolite identification of
coupled to liquid chromatography with mass spectrometric and radioactivity
detection, Anal. Chem. 83 (2011) 5415e5421.
a radiotracer by electrochemistry
m
m
m
50 pM). Recombinant caspases were diluted into the appropriate
buffer to a concentration of 0.5 units per assay (¼ 500 pmol sub-
strate conversion after 60 min). After 10 min incubation time, the
[13] A. Zhang, A.D. Schlüter, Multigram solution-phase synthesis of three diaste-
reomeric tripeptidic second-generation dendrons based on (2S,4S)-, (2S,4R)-,
and (2R,4S)-4-aminoprolines, Chem. Asian J. 2 (2007) 1540e1548.
[14] F.M. Veronese, G. Pasut, PEGylation, successful approach to drug delivery,
Drug Discov. Today 10 (2005) 1451e1458.
peptide substrate (end concentration 10 mM) was added and reacted
for further 10 min. The IC50 values were determined by nonlinear
regression analysis using the XMGRACE program (Linux software).
[15] J.R. Del Valle, M. Goodman, Stereoselective synthesis of Boc-protected cis- and
trans-4-trifluoromethylprolines by asymmetric hydrogenation reactions,
Angew. Chem. 114 (2002) 1670e1672; Angew. Chem. Int. Ed. 41(2002) 1600e
1602.
[16] S. Abraham, M.J. Hadd, L. Tran, T. Vickers, J. Sindac, Z.V. Milanov,
M.W. Holladay, S.S. Bhagwat, H. Hua, J.M. Ford Pulido, M.D. Cramer, D. Gitnick,
J. James, A. Dao, B. Belli, R.C. Armstrong, D.K. Treiber, G. Liu, Novel series of
pyrrolotriazine analogs as highly potent pan-Aurora kinase inhibitors, Bioorg.
Med. Chem. Lett. 21 (2011) 5296e5300.
[17] A. Kamal, Rajender, D.R. Reddy, M.K. Reddy, G. Balakishan, T.B. Shaik,
M. Chourasia, G.N. Sastry, Remarkable enhancement in the DNA-binding
ability of C2-fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines and their
anticancer potential, Bioorg. Med. Chem. 17 (2009) 1557e1572.
[18] M.P. Sibi, J.-L. Lu, Synthesis of both enantiomers of C2-symmetric trans-2,5-
bis(hydroxymethyl)pyrrolidine: lipase mediated sequential kinetic resolu-
tions, Tetrahedron Lett. 35 (1994) 4915e4918.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft
(Collaborative Research Center, SFB 656 Project B1 in collaboration
with A3 and Z5), the International NRW Graduate School of
Chemistry (GSC-MS) and the Development and Promotion of Sci-
ence and Technology talent project (DPST) of Thailand is gratefully
acknowledged. We like to thank Sandra Schröer and Wiebke
Gottschlich for performing the enzyme inhibition assays and
Andrea Kapries, Anna-Lena Dreier, Christian Paul Ortmeyer and
Malte Bayer for practical assistance.
[19] A.K. Podichetty, S. Wagner, A. Faust, M. Schäfers, O. Schober, K. Kopka,
G. Haufe, Fluorinated isatin derivatives. Part 3. New side-chain fluoro-func-
tionalized pyrrolidinyl sulfonyl isatins as potent caspase-3- and -7 inhibitors,
Future Med. Chem. 1 (2009) 969e989.
[20] P. Limpachayaporn, M. Schäfers, O. Schober, K. Kopka, G. Haufe, 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, Bioorg. Med. Chem. 21 (2013)
2025e2036.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.04.011.