Fluorinated isatin derivatives. Part 2. New N-substituted 5-pyrrolidinylsulfonyl isatins as potential tools for molecular imaging of caspases in apoptosis

Article abstract of DOI:10.1021/jm8015014

Caspases are responsible for the execution of the cell death program and are potentially suitable targets for the specific imaging of apoptosis in vivo. A series of N-1-substituted analogues of the small molecule nonpeptide caspase inhibitor (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (1), which may be useful for the development of caspase-targeted radioligands, were synthesized and their inhibition potencies were evaluated in vitro. Two of the most powerful techniques to introduce fluorine into organic compounds, viz, bromofluorination of olefins and fluorohydrin synthesis by ring-opening of epoxides, were used. Most of the target compounds are potent inhibitors of the two effector caspases-3 and -7. Furthermore, the ¹⁸F-radiolabeled model compound (S)-1-[4-(1-[¹⁸F]fluoro-2-hydroxyethyl)benzyl]-5-[1- (2-methoxymethyl-pyrrolidinyl) sulfonyl]isatin ([¹⁸F]37), a putative tracer for the noninvasive imaging of apoptosis by positron emission tomography (PET) was synthesized by nucleophilic epoxide ring-opening of its precursor 36. The radiochemistry utilized in the ¹⁸F-fluorination reverted to carrier-added [¹⁸F]Et₃N·3HF, a new fluorine-18 source for radiolabeling.

Full text of DOI:10.1021/jm8015014

3484
J. Med. Chem. 2009, 52, 3484–3495
Fluorinated Isatin Derivatives. Part 2. New N-Substituted 5-Pyrrolidinylsulfonyl Isatins as
Potential Tools for Molecular Imaging of Caspases in Apoptosis
Anil K. Podichetty,†,§ Stefan Wagner,‡,§ Sandra Schro¨er,‡ Andreas Faust,‡,§ Michael Scha¨fers,‡,§ Otmar Schober,‡,§
Klaus Kopka,‡,§ and Gu¨nter Haufe*,†,§
Organisch-Chemisches Institut and International NRW Graduate School of Chemistry, Westfa¨lische Wilhelms-UniVersita¨t, Corrensstrasse 40,
D-48149 Mu¨nster, Germany, Klinik und Poliklinik fu¨r Nuklearmedizin, UniVersita¨tsklinikum Mu¨nster, Albert-Schweitzer-Strasse 33, D-48129
Mu¨nster, Germany, European Institute of Molecular Imaging, Westfa¨lische Wilhelms-UniVersita¨t, Mendelstrasse 11, D-48149 Mu¨nster
ReceiVed NoVember 28, 2008
Caspases are responsible for the execution of the cell death program and are potentially suitable targets for
the specific imaging of apoptosis in vivo. A series of N-1-substituted analogues of the small molecule
nonpeptide caspase inhibitor (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (1), which may be useful
for the development of caspase-targeted radioligands, were synthesized and their inhibition potencies were
evaluated in vitro. Two of the most powerful techniques to introduce fluorine into organic compounds, viz,
bromofluorination of olefins and fluorohydrin synthesis by ring-opening of epoxides, were used. Most of
the target compounds are potent inhibitors of the two effector caspases-3 and -7. Furthermore, the 18F-
radiolabeled model compound (S)-1-[4-(1-[18F]fluoro-2-hydroxyethyl)benzyl]-5-[1-(2-methoxymethyl-pyr-
rolidinyl)sulfonyl]isatin ([18F]37), a putative tracer for the noninvasive imaging of apoptosis by positron
emission tomography (PET) was synthesized by nucleophilic epoxide ring-opening of its precursor 36. The
radiochemistry utilized in the 18F-fluorination reverted to carrier-added [18F]Et3N · 3HF, a new fluorine-18
source for radiolabeling.
Introduction
and hence execute the common final path of the programmed
cell death process. Apoptosis involves two different classes of
caspases, namely the initiator caspases (caspase-2, -8, -9, and
-10), whose main function is the activation of the effector
caspases, caspase-3, -6, and -7. In apoptosis, the physiological
changes (e.g., cleavage of the DNA repair enzyme poly(ADP-
ribose)polymerase-1, cytoskeleton proteins, and nuclear lami-
nins) together with the morphological changes (nuclear mem-
brane damage, membrane blebbing, and DNA strand breaks)
are brought about by the effector caspases.22,23 Caspases-1, -4,
-5, and -11 constitute the third class but are not believed to
play an active role in apoptosis.24
On the basis of the decisive role of caspases in apoptosis,
the development of structurally diverse caspase inhibitors has
been the focus of pharmaceutical drug development in recent
years. These drugs are designed to therapeutically prevent
apoptosis and rescue tissues.25 Apart from therapeutic applica-
tions, these inhibitors could also be used for molecular imaging
of caspase activity because they typically bind to activated
caspases. However, direct in vivo application of labeled peptide-
based caspase inhibitors as caspase imaging agents failed due
to poor cell permeability.26 Nonpeptidic 5-pyrrolidinylsulfonyl
isatins have been shown to selectively inhibit the downstream
caspases-3 and -7 in vitro.24,27-32 These compounds do co-
valently and reversibly bind to the active site of the activated
caspase enzymes. The C-3 carbonyl carbon of the isatin skeleton
acting as an electrophile forms a thiohemiketal with the
nucleophilic Cys thiolate function, and this in turn happens when
the dicarbonyl functionality of the isatin binds to the Cys residue
of the active site. It is for these reasons that radiolabeled
5-pyrrolidinylsulfonyl isatins were suggested as radiopharma-
ceuticals for clinical imaging of apoptosis by SPECT or
PET.32-35
Apoptosis is a natural form of cell death and is an energy
dependent and genetically controlled process without inflam-
matory response that maintains homeostasis in proliferating
tissues in adults.1,2 Dysregulation of apoptosis is linked to
increased cell proliferation or enhanced cell death, resulting in
a variety of diseases. While an increased apoptosis rate occurs
in acute myocardial infarction,3 atherosclerosis,4,5 allograft
rejection,6-9 stroke,10-12 and neurodegenerative disorders;13-15
decreased apoptosis rate can be observed during tumorige-
nesis,16-19 autoimmune diseases,20 and viral infections.21 Both
the diagnosis and therapy of such diseases demand noninvasive
and serial imaging of apoptosis in vivo, e.g., as a surrogate
marker of successful chemotherapy of tumors. Therefore,
apoptosis imaging with the scintigraphic methods like single
photon emission computed tomography (SPECTa) and positron
emission tomography (PET) would be a clinically important tool
for disease management and is still one of the greatest challenges
in modern medicine. For this, a biological target for specific
and exclusive apoptosis imaging is crucial.
The intracellular death enzyme class called caspases (cysteinyl
aspartate-specific proteases) constitute such a specific target.
During the apoptosis signaling cascade, caspases become
activated, contributing to the disassembly of the apoptotic cell
* To whom correspondence should be addressed. Phone: +49-(0)251-
83 33281. Fax: +49-(0)251-83 39772. E-mail: haufe@uni-muenster.de.
†
Organisch-Chemisches Institut and International NRW Graduate School
of Chemistry, Westfa¨lische Wilhelms-Universita¨t.
‡
Klinik und Poliklinik fu¨r Nuklearmedizin, Universita¨tsklinikum Mu¨n-
ster.
§
European Institute of Molecular Imaging, Westfa¨lische Wilhelms-
Universita¨t.
a Abbreviations: ADP, adenosine diphosphate; DMF, dimethyl forma-
mide; m-CPBA, meta-chloroperbenzoic acid; NBS, N-bromosuccinimide;
PET, positron emission tomography; SD, standard deviation; SPECT, single
photon emission computed tomography; TBAH2F3, tetrabutylammonium
dihydrogen trifluoride; TsCl, toluenesulfonic acid chloride.
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin (1)
(Scheme 1)28 was chosen as lead structure for the development
of caspase inhibitors because the cellular caspase inhibition
10.1021/jm8015014 CCC: $40.75
2009 American Chemical Society
Published on Web 05/15/2009

Fluorinated Isatin DeriVatiVes. Part 2
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3485
studies indicated that the 2-methoxymethyl compound (R )
CH3) seem to possess a higher biochemical caspase inhibition
potency in intact apoptotic cells in comparison with the
phenoxymethyl-modified compounds (R ) Ph).30 The N-1-
position in lead structure 1 offers a potential site for structural
modifications, thereby providing an excellent direction not only
in generating a library of potential caspase inhibitors but also
in targeting the potential precursors for radiolabeling. However,
our idea was to concentrate on the synthesis of fluorinated
analogues of 1 and to evaluate the influence of fluorine
introduction on the caspase inhibition potency. Our motivation
was based on the premise that introduction of a fluorine
substituent may further enhance the potency of the derivative
as compared to the lead 128 and in turn would facilitate the
identification of a target compound for radiofluorination. Ac-
cordingly, continuing our earlier work,36 different fluorinated
derivatives of 1 were synthesized with modifications at the N-1-
position and their caspase inhibition potencies for caspase-1,
-3, -6, and -7 were evaluated in vitro.
and L-proline as described in a previous work,28 its structurally
related analogues, compounds 7-22 and 28-41, were synthe-
sized by alkylation of 1 and subsequent fluorination with the
above-mentioned methods of organofluorine chemistry.
Bromofluorinations. Halofluorination of unsaturated com-
pounds is a versatile method to introduce fluorine and, in
addition, a second reactive function into organic compounds.41-46
One of the most common procedures for bromofluorination uses
N-bromosuccinimide (NBS) as the source of the bromonium
ion and different amine-HF complexes.46,47 A detailed account
of regioselectivity of bromofluorination of functionalized olefins
was provided by our research group a few years ago.48
Furthermore, Katzenellenbogen et al. have previously demon-
strated that bromofluorination could also serve as a radiochemi-
cal tool for introducing fluorine-18 into medicinally relevant
compounds and thereby facilitate their use as imaging agents
for PET.49-51
We started investigating the regio- and stereochemistry of
bromofluorination of different terminal olefins derived from lead
compound 1. To our knowledge, this is the first instance of such
methodology being applied to derivatize isatin compounds. In
consideration of aforementioned aspects, we synthesized dif-
ferent alkenyl derivatives of 1 (7, 9, 12, 15, 18, and 20) using
a variety of alkenyl halides as electrophiles. Some of the
alkenylation reagents were not commercially available and were
prepared according to Scheme 2. 2-Fluoroallyl tosylate (4)52
was synthesized in 90% yield from 2-fluoroallyl alcohol53 and
tosyl chloride using NaOH/Et2O. p-Allyloxybenzyl bromide (6)
was prepared in two steps starting from the commercially
available p-hydroxybenzyl alcohol. However, no attempts were
made to isolate intermediate 6 in pure and the crude product
obtained after flash chromatography was directly used for the
next step.
Scheme 1a
a (a) MeI, K2CO3, DMF.28
A number of these compounds after radiolabeling might also
be suitable for monitoring of apoptosis by PET. Here, our
radiofluorination methodology was focused on methods that
were suitable for the fast introduction of the [18F]fluorine
radionuclide yielding isatin variants based on fluorohydrins.
Fluorine-18 has a half-life of 110 min and a low positron energy,
resulting in a higher spatial resolution of images compared to
other light isotopes such as 11C (t1/2 ) 20 min), 13N (t1/2 ) 10
min), and 15O (t1/2 ) 2 min) used in PET.37 Thus, as a proof-
of-concept, we succeded in the synthesis of a first-in-this-series
fluorine-18 labeled model compound ([18F]37).
Scheme 2a
Results and Discussion
Chemistry. Our chemistry basically concentrated on employ-
ing fluorination techniques like bromofluorination (of precursor
olefins) and epoxide ring-opening reactions toward vicinal
fluorohydrins, which are rarely used for radiofluorination at
present but are compatible with the structural elements of
pyrrolidinylsulfonyl isatins (see discussion in the Radiochemistry
section). The nonfluorinated compounds 1, 2, and 3 were
recently identified as potent compounds with low IC50 values
for caspases-3 and -7 and were therefore, earmarked as the
standard compounds for comparing the results obtained from
our compounds.28,33,38 Accordingly, it was decided to incorpo-
rate structural variants at the N-1 position, which was proven
to tolerate structural modifications without loss of caspase
inhibition potency.28,31 Consequently, a number of substituents
with varying carbon units were attached to N-1, i.e., different
precursor olefins (compounds 7, 9, 12, 15, 18, and 20) and
precursor terminal epoxides (compounds 28, 30, 33, 36, and
38). The new fluorinated isatin analogues were then prepared
from these precursors by bromofluorination39 and epoxide ring-
opening with different amine/HF reagents.40 While the lead
compound 1 was obtained in seven steps starting from isatin
a (a) TsCl, NaOH, Et2O; (b) KOH, Allyl bromide, DMF; (c) CBr4, Ph3P,
dry CH2Cl2.
Bromofluorination of the terminal olefins (Scheme 3) afforded
the bromofluorides in 52-87% isolated yields, and their
regioisomeric and diastereomeric ratios were deduced from 19
F
NMR studies of the crude reaction mixtures. All the experiments
furnished some interesting results with respect to the regio-
chemistry of the resulting bromofluorides. Regardless of the
strong electron withdrawing (-I) effect exerted by the isatin-
sulfonamide core on the N-alkenyl moiety, which in allylic
position disfavored a potential secondary cationic center; the
regioselectivity of the bromofluorides though varied with
increasing carbon chain length was always in favor of the
secondary fluoride over the primary counterparts. While bro-
mofluorination of 9 yielded a secondary bromofluoride favoring
regioisomeric ratio of 2:1, the result was 4.5:1 in the case of
bromofluorination of compound 12. Similarly, compound 15
also yielded a secondary bromofluoride favoring regioisomeric
ratio of 12:1.

3486 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Podichetty et al.
Scheme 3a
fact that the presence of fluorine at vicinal position to the free
hydroxyl group increases the acidity of hydroxyl function and
hence influences its ability to act as hydrogen bond donor and/
or acceptor. So, when biochemically active fluorohydrins are
administered into the body, they may show enhanced activity
against their in vivo targets (enzymes/receptors) compared to
their nonfluorinated counterparts due to modified cell perme-
ability and biodistribution.63-65
The most extensively used method for the synthesis of vicinal
fluorohydrins is the anti-selective ring-opening of epoxides with
different fluorinating agents,66,67 which generally have different
selectivities with the same oxirane. Ring-opening can occur by
different reaction mechanisms, SN1 or SN2, explaining the
selectivity of the formal addition of HF. Carbenium ion-
stabilizing substituents favor an SN1-type reaction, whereas
destabilizing substituents favor an SN2 pathway.41,68 In recent
years, we systematically investigated the selectivity of different
hydrofluorinating reagents in the ring-opening of terminal and
bicyclic epoxides including first asymmetric ring-opening by
hydrofluorinating agents.69,70 As fluoride donors, Olah’s reagent,
Et3N·3HF, and KHF2 were mostly used.71,72
On the basis of the above background information, we started
investigating the regio- and stereochemistry of ring-opening of
different terminal epoxides (enantiopure and racemic) derived
from lead compound 1. The objective (as with bromofluorides)
was to synthesize nonradioactive fluorohydrins to evaluate their
caspase inhibition potency and then to select candidate com-
pounds with excellent inhibition potencies for the downstream
caspases-3 and -7, for the radiochemical resynthesis via 18F-
labeling. Accordingly, we synthesized different terminal ep-
oxides 28, 30, 33, 36, and 38 using a variety of epoxyalkyl
halides as electrophiles. Most of the alkylation reagents were
not commercially available and were prepared according to
Scheme 4.
a (a) compound 4; (b) crystallized N-bromosuccinimide, Olah’s reagent,
dry CH2Cl2; (c) allyl bromide; (d) 4-bromobut-1-ene; (e) 11-bromoundec-
1-ene; (f) 4-vinylbenzyl chloride; (g) compound 6.
Scheme 4a
In the case of olefins 7 and 18, the corresponding bromo-
fluorides 8 and 19 were obtained with 100% regioselectivity.
In case of compound 7, this could be explained by the strong
+M effect of the vinylic fluoride that stabilized the positive
charge in the R-position and hence led to the exclusive formation
of the geminal difluoride. On the other hand, compound 19 owes
its exclusive formation to the stabilization of positive charge at
the benzylic carbon. In the case of bromofluorination of 20,
the allylic oxygen destabilized the secondary cationic position
and hence a significant amount of 21 was formed in addition to
22.
Practically no diastereoselectivity (with regard to the stereo-
center in the pyrrolidine core) was witnessed in either of the
regioisomers of any of the bromofluorinated products, with the
exception of compound 16, which possessed a diastereomeric
ratio of 83:17 as observed by 19F NMR. It might be that the
long chain folds below the plane of isatin core, blocking one
face of the double bond, leading to such diastereoselection.
Considering the inhibitory activity (see section on enzyme
assays) of the synthesized bromofluorides, the above results
indicated that bromofluorination could be adopted as a powerful
tool for future radiofluorinations and efforts in this direction
are underway in our research group.
Synthesis of Fluorohydrins from Epoxides. In recent years,
fluorohydrins40,54 have attracted much attention in organofluo-
rine chemistry55 as they act as precursors in the synthesis of
biochemically active, monofluorinated analogues of natural
products56,57 such as steroids,58 amino acids,59 and carbohydr-
ates.60-62 One of the main reasons for unique biological
properties of vicinal fluorohydrins could be attributed to the
a (a) m-CPBA, CH2Cl2; (b) NBS, acetone-water (7:3); (c) powdered
NaOH; (d) SOCl2, CH2Cl2; (e) m-CPBA, CH2Cl2.
In our investigation of regioselective ring-opening of epoxides
with hydrofluorinating agents, another objective was the syn-
thesis of fluorohydrins within a short time in order to transfer
the investigated methology to 18F-radiochemistry. Keeping this
in view, most of the synthesized epoxides were initially opened
with Olah’s reagent, which predominantly yielded the regioi-
somer formed from the more stable carbocation (SN1-like
reaction), although there is no evidence for the formation of a
free cationic center.73,74 The regioisomeric and diastereomeric

Fluorinated Isatin DeriVatiVes. Part 2
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3487
ratios of resulting fluorohydrins were then deduced from 19F
NMR studies of the crude reaction mixtures (Scheme 5).
(regioisomeric ratio 2.2:1). Similarly, the racemic aromatic epoxides
36 and 38 were also opened with neat Et3N·3HF. The styrene oxide
derivative 36 yielded the secondary fluorohydrin 37 not only with
100% regioselectivity but also in considerably reduced reaction
time (1.5-2 h). This result could be attributed to the activated
benzylic position in 36 for attack by the nucleophilic fluoride.
Although up to 38% of oligomers were also detected (19F NMR),
the isolated yield of 51% was still reasonable. Likewise, the reaction
of the phenylglycidether 38 with Et3N·3HF yielded the primary
fluorohydrin 39 with more than 99% regioselectivity after 14-16
h of reaction time. Because the strong electron withdrawing (-I)
effect of the phenoxy moiety destabilized the secondary carboca-
tionic center, the incoming fluoride ion was forced to attack at the
terminal carbon in SN2 pathway, leading to the predominance of
primary fluoride 39 and increased reaction time (14-16 h).
Compound 39 was obtained in 45% yield, but 33% of oligomers
were also detected in the crude product by 19F NMR spectroscopy.
No diastereoselectivity was observed in either of the regioisomers
of any of the fluorohydrins except 29, which is diastereopure, and
34, which had a diastereomeric excess of 82% (19F NMR).
To compare the caspase inhibition potency of synthesized
fluorohydrins with the corresponding diols, two exemplary diols
were prepared (Scheme 6).
Scheme 5a
Scheme 6a
a (a) (S)-(+)-glycidyl nosylate; (b) Olah’s reagent, CH2Cl2; (c) 23; (d)
Et3N·3HF; (e) 24; (f) 25; (g) 27.
All the experiments provided some interesting results with
respect to the regioselectivity of the resulting fluorohydrins. Initially,
compound 28 was chosen as the substrate to explore the above-
mentioned chemistry. When 28 was treated with Olah’s reagent,
the results were totally surprising in the sense that the reaction
yielded the regioisomer with fluorine at the primary position
(instead of the secondary position) with 99% selectivity and 51%
isolated yield. This could be attributed to the strong electron
withdrawing (-I) effect exerted by the isatinsulfonamide core on
the N-alkyl moiety that destabilizes a potential secondary carboca-
tionic center. Owing to the strong acidity of Olah’s reagent, the
reaction also yielded about 10% of oligomers as the side products.
Because the explored reaction time (12-14 h) was too long, ring-
opening was also tried out in neat Et3N·3HF at 80-90 °C, but
the reaction time in this case was even longer (21-22 h). To reduce
the reaction time, many experiments were carried out with the main
focus on (i) optimization of Olah’s reagent amounts, (ii) varying
pyridine to HF ratios by definite addition of pyridine with an aim
to increase the nucleophilicity of fluoride, and (iii) reaction
temperature (some reactions were also carried out at 45 °C).
However, none of the above experiments could reduce the reaction
time below 8 h. These results pointed out that C3 epoxide 28 may
be unsuitable for radiofluorination. Therefore, variations of the
carbon chain length (of epoxide) on the reaction time and
regioselectivity were explored. Accordingly, reactions were carried
out with racemic C4 epoxide 30 and racemic long chain (C11)
epoxide 33. While 33 yielded the expected secondary fluorohydrin
favoring regioisomeric ratio of 30:1, fluorohydrin formation from
30 and Olah’s reagent under different reaction conditions was not
observed. Therefore, ring-opening of 30 was achieved with neat
Et3N·3HF, which provided the fluorohydrins 31 and 32 in 71%
yield. Here, the formation of the primary fluoride 32 is favored
a (a) Olah’s reagent, CH2Cl2, saturated NaHCO3 soln; (b) silica gel.
Accordingly, synthesis of enantiopure diol 40 from 28 was
realized with Olah’s reagent and saturated sodium bicarbonate
solution in 51% yield. Also, racemic diol 41 was synthesized
in 55% yield by ring-opening of the precursor epoxide 30 on a
silica gel column followed by elution of the product with 5%
MeOH/CH2Cl2.
Comprehensive scrutiny and analysis of the obtained results
in combination with the data obtained by the enzyme assays
prompted us to devise a potential radiofluorination of the racemic
styrene oxide derivative 36. Short reaction time, remarkable
regioselectivity, and excellent caspase-3 and -7 inhibition
potency (see below) were the reasons for selecting compound
36 as the precursor compound for 18F-fluorination.
Radiochemistry. The synthesis of [18F]37 was evaluated with
different fluorination reagents. Initial experiments with Kryptofix
2.2.2 (K 222) and [18F]KF resulting in [18F]K(K 222)F yielded
only traces of fluorohydrin [18F]37 under basic reaction conditions
(K2CO3). Very recently, Zhou et al.75 observed that the effective-
ness of the labeling procedure with the system K2CO3/K 222/
[18F]fluoride ion/isatin derivative depends among other parameters
on the amount of base (K2CO3) in the reaction. Under their labeling
conditions, not less than 2 mg of K2CO3 is necessary to obtain a
high yield of the labeled, ring-opened isatinate that was converted
to the corresponding isatin under acidic conditions. Our labeling
protocol uses much more K2CO3, indicating that the low radio-

3488 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Podichetty et al.
chemical yield of [18F]37 is caused by other factors. Obviously,
basic reaction conditions are not suitable to open the epoxide ring
of 36. However, the utilization of [18F]poly(hydrogen fluoride)
pyridinium ([18F](HF)n ·pyridine) prepared as previously described76
provided a radiochemical yield of up to 5% (decay corrected, d.c.)
for [18F]37. Furthermore, the radiochemical yield of [18F]37 was
increased and optimized to 7% (d.c.) when [18F]triethylamine
trihydrofluoride ([18F]Et3N·3HF) was used for radiofluorination
(Scheme 7). For the first time, a nucleophilic fluorinating reagent
generated from Et3N·3HF and [18F]F- via isotopic exchange was
successfully applied in the synthesis of the target 18F-labeled
compound [18F]37.
Enzyme Assays. The caspase inhibition potencies of all the
synthesized analogues of 5-pyrrolidinylsulfonyl isatin (compris-
ing of potential radiolabeling precursors 7, 9, 12, 15, 18, 20,
28, 30, 33, 36, and 38; the nonradioactive counterparts of
potential caspase-targeted radioligands 8, 10+11, 13+14,
16+17, 19, 21+22, 29, 31+32, 34+35, 37, and 39, together
with some important nonfluorinated derivatives 40 and 41, were
tested for their inhition potencies against caspases-1, -3, -6, and
-7 by using fluorogenic in vitro caspase inhibition assays. The
resulting caspase inhibitory values were obtained as IC50 values
by a nonlinear regression fit of the concentration-dependent
reaction rates and the in vitro results have been captured in Table
1. According to Lee et al.28,33 compounds 1, 2, and 3a are potent
caspase inhibitors. To directly compare our findings, the
inhibition potencies of compounds 1, 2, and 3a have also been
tabulated. Most of the target compounds exhibited nanomolar
binding potency for inhibiting the effector caspases, caspase-3
and caspase-7. Normally, no inhibition of caspase-1 and
caspase-6 was observed for any of the compounds.
Scheme 7a
Table 1. Caspase Inhibition Potencies of the
5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin Analogues for
Caspases-1, -3, -6, and -7 Expressed as IC50Values
IC50, nMa
compd
caspase-1
caspase-3
caspase-6
caspase-7
1
>500000
>500000
>500000
nd
84.9 ( 25.6
16.9 ( 7.7
2.4 ( 0.3
499 ( 239
1880 ( 1440
25 ( 16
26 ( 19
31 ( 13
96 ( 32
74 ( 20
34 ( 9
>500000
>500000
>500000
nd
1290 ( 466
294 ( 178
304 ( 73
266 ( 96
1660 ( 310
24 ( 11
15 ( 8
a (a) CH3CN, ∆, sonication; (b) CH3CN, ∆.
2
3a
7b,c
8b
9
In the investigations by Zhou et al.,75 the application of “no-
carrier-added” concept prevented the formation of the desired
labeled isatin substitution product in certain cases. This was
due to the consumption of [18F]fluoride caused by the nucleo-
philic attack and reversible addition of this species at the C-3
carbonyl group of the precursor that was used in a huge excess
compared to [18F]fluoride. In contrast to this, our applied carrier-
added method reverting to an excess of [18/19F]fluoride (in the
form of [18/19F]Et3N·3HF) yielded the desired labeled nucleo-
philic substitution product [18F]37. Apparently, the completely
different stoichiometric ratios of fluoride to precursor amounts
in our experiments as compared to the investigations by Zhou
et al. account for the product formation in our labeling procedure
and prevent it in the cited paper.75 Compound [18F]37 was finally
obtained in 220 min with a radiochemical purity of >95%,
showing moderate specific activities (<1 GBq/µmol) that cor-
respond with the here chosen carrier-added radiofluorination
approach. Such low specific activities may limit the application
of [18F]37 for in vivo imaging of activated caspases and
therefore, future investigations will aim at increasing the specific
activity (e.g., by the reduction of carrier addition and increase
of starting activity).
Although the radiochemical yield is low, our new fluorine-18
source ([18F]Et3N·3HF) allows us to carry out the radiosynthesis
of [18F]37 by utilizing [18F]KF. This success could be attributed
to the fact that [18F]Et3N·3HF is an acidic fluorinating agent
where the basicity and nucleophilicity of [18F]F- is considerably
reduced when compared to [18F]KF, which in turn is due to the
intermolecular hydrogen bonding. Contrary to the investigations
by Zhou et al.,75 under such conditions of reduced nucleophi-
licity and increased acidity of the fluorination reagent, the
nucleophilic addition of [18F]F-at the C-3 carbonyl group of
36 and isatinate intermediate formation by the ring-opening of
isatin are less likely to occur. The above result clearly highlights
the improved efficacy of the less basic [18F]Et3N·3HF for
radiolabeling experiments when compared to [18F]KF, as far as
the SN reactions with [18F]F- in isatin compounds are concerned.
nd
nd
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
10+11b (∼2:1)
12
50 ( 16
20 ( 7
13+14b (82:18)
15
111 ( 26
86 ( 6
16+17b (∼11:1) >500000
18
>500000
>500000
>500000
>500000
3160 ( 490
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
>500000
9.3 ( 4.3
43 ( 19
10 ( 0.5
26 ( 2
7.2 ( 2.4
16 ( 2
19b
20
0.6 ( 0.2
1.9 ( 0.2
9.0 ( 1.3
349 ( 33
68 ( 8
21+22b (∼7:3)
28
17 ( 5
29b
19 ( 12
151 ( 62
12 ( 6
30
31+32b (1:1)
214 ( 5
99 ( 32
61 ( 13
63 ( 17
1.9 ( 0.6
7.6 ( 0.1
2.4 ( 0.2
5.0 ( 0.6
43 ( 4
32b
3.6 ( 1.0
23 ( 11
47 ( 6
33
34+35b (91:9)
36
6.6 ( 1.4
80 ( 8
37b
38
42 ( 15
145 ( 5
11 ( 4
39b
40
41
275 ( 40
61 ( 12
a Values are the mean ( SD of three assays. b Nonradioactive counter-
parts of PET-compatible caspase-targeted radioligands. c Taken from ref
36.
Recently, we and Chu et al. have also modified the N-1
position of the isatins by synthesizing a variety of N-benzyl
derivatives and got encouraging results.30,31 The above results
clearly imply that the N-1 position of the 5-pyrrolidinylsulfo-
nylisatins is a potential site for modification and development
of isatins as caspase-targeted radioligands.
Discussion
The IC50-values for caspase inhibition of all compounds
revealed some striking observations. It was reported earlier that
isatin sulfonamides are highly potent and selective nonpeptide
based inhibitors of the effector caspases-3 and -7. Our results
have further reinforced the fact that 5-[1-(2-methoxymethylpyr-
rolidinyl)sulfonyl]isatins are potent inhibitors, which could be
used as radiotracers for molecular imaging of activated caspases
in apoptosis. All compounds except the gem-difluoride 8
exhibited moderate to high inhibition potencies for caspases-3

Fluorinated Isatin DeriVatiVes. Part 2
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3489
and -7. All compounds also exhibited very low potencies for
caspases-1 and -6, with the exception of compound 28, which
was a weak inhibitor of caspase-1. Among the various terminal
olefins 7, 9, 12, 15, 18, and 20, which were also the precursors
for the corresponding bromofluorides, compound 18 was the
most active against caspase-3 (IC50: 9.3 nM) and compound 20
was the most active against caspase-7, with a subnanomolar
IC50 value (0.6 nM). Similarly, having a closer look at the
derived bromofluorides 8, 10+11, 13+14, 16+17, 19, and
21+22, we observed that among this series a mixture of
regioisomeric bromofluorides 21 and 22 (∼7:3) was the most
active against both caspase-3 (IC50: 26 nM) and caspase-7 (IC50:
1.9 nM). A mixture (92:8) of compounds 16 and 17 also showed
good activity against these caspases. Because all the bromo-
fluorides possessed very good activity against caspase-3 and
-7, they provide a new class of isatin derivatives with excellent
potential for radiolabeling and might be used as caspase-targeted
radioligands for apoptosis imaging. Further efforts, i.e., to
synthesize pure isomers, are underway.
On analyzing the third series of compounds, namely the
precursor terminal epoxides 28, 30, 33, 36, and 38, we observed
that among this series, the styrene oxide derivative 36 was the
most active against both caspase-3 (IC50: 6.6 nM) as well as
caspase-7 (IC50: 1.9 nM). Similarly, comprehensive analysis of
the corresponding fluorohydrins 29, 31+32, 34+35, 37, and 39
revealed that regioisomer 32 (primary fluoride) of C4 fluoro-
hydrins was the most active against caspase-3, and compound
39 was the best inhibitor for caspase-7 within this particular
series. Generally, the N-benzyl compounds showed the lowest
IC50 values. Also, the binding potencies of the bromofluorides
and fluorohydrins were not significantly influenced by their
regiochemistry. Because all the fluorohydrins exhibited very
good activity against caspases-3 and -7, they (like bromofluo-
rides) offer a new class of isatin derivatives with tremendous
potential for radiofluorination. However, it should be noted that
the short-chain fluorohydrins 29 (17:1) and 32 (28:1) are better
inhibitors for caspase-3, while the benzyl compounds 37 (1:
11) and 39 (1:29) are much more active as inhibitors of caspase-
7. The long-chain fluorohydrins 34/35 are almost equal in their
activity for both the caspases (Table 1). Accordingly, as a proof-
of-concept, the 18F-labeled isatin [18F]37 was synthesized as a
first fluorohydrin model compound whose nonradioactive coun-
terpart 37 possessed high inhibition potency, comparable to the
activity of compounds prepared earlier.30,32
low specific activity of [18F]37, other precursors are also being
explored for immediate radiolabeling in order to establish potent
compounds that could well serve as “chemical solutions” to the
biological problem of imaging apoptosis.
Experimental Section
General Methods. Chemistry and Radiochemistry. All the
chemicals, reagents, and solvents for the synthesis of compounds
were analytical grade and used without further purification, unless
otherwise specified. 1H NMR (300.13 MHz, 400.13 MHz, 500 MHz
and, 600 MHz), 13C NMR (75.5 MHz, 100.63 MHz, 125.5 MHz,
and 150.66 MHz,) and 19F NMR (282.4 MHz) spectra were recorded
in CDCl3 with TMS for 1H NMR, CDCl3 for 13C NMR, and CFCl3
for 19F NMR as the internal standards. All chemical shift values
were recorded in ppm (δ). Exact mass analyses were conducted
on a Waters Quattro LC and a Bruker MicroTof apparatus. All
these spectroscopic and analytical investigations were done by staff
members of the Organic Chemistry Institute, University of Mu¨nster.
Silica coated aluminum foils (silica gel 60 F254) from MERCK with
0.2 mm layer thickness were used for thin layer chromatography
(TLC). Column chromatography was generally performed on silica
gel (60-120 mesh) using suitable solvent mixtures. The purity of
compounds was proved by HPLC comprised of a Knauer system
with two K-1800 pumps, an S-2500 UV detector, and a Eurospher
100 C18 reverse phase column (250 mm × 4.6 mm). Detection was
conducted at λ ) 254 nM. The solvents used were: A (water +
0.1% TFA) and B (CH3CN + 0.1% TFA). The mobile phase was
a gradient using 90:10 A/B to 20:80 A/B mixture over 30 min,
holding for 5 min and back to 90:10 A/B mixture in 5 min at a
common flow rate of 1.5 mL/min. Separation and purification of
the radiosynthesized compound [18F]37 were performed by gradient
radio-HPLC (radio-HPLC A) using a Knauer K-500 and a Latek
P-402 pump, a Knauer K-2000 UV detector (λ ) 254 nm), a
Crismatec NaI(Tl) Scintibloc 51 SP51 γ-detector, and a Nucleosil
100-10 C18 column (250 × 8 mm2). Sample injection was carried
out using a Rheodyne injector block (type 7125 incl. 1000 µL loop).
The recorded data were processed by the NINA version 4.9 software
(GE Medical SystemssFunctional Imaging GmbH).
The starting compound 1 was prepared according to a given
literature protocol,28 and 2-fluoroallyl tosylate 4 was prepared from
2-fluoroallyl alcohol according to literature.52
General Procedure for the Synthesis of N-Alkenyl Isatins.
To a solution of (S)-5-[1-(2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) in dry DMF (3 mL), anhydrous potassium carbonate
(2.5 equiv) was added under argon atmosphere and the reaction
mixture was stirred at ambient temperature for 30 min. An excess
of the alkylating agent, (2-3 equiv) was slowly added and the
reaction mixture was stirred at ambient temperature for 12-14 h
in the case of 2-fluoroallyl tosylate and simple alkyl halides but
only for 6-8 h in the case of benzylic halides. Removal of the
solvents in vacuo furnished the crude products, which were purified
by silica gel chromatography.
As far as the miscellaneous compounds were concerned, the
C3 enantiopure diol 40 was more active than its C4 racemic
counterpart 41. Interestingly, 40 was also more active than its
fluorohydrin counterpart 29.
(S)-1-(2-Fluoroallyl)-5-[1-(2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin (7). The synthesis of this compound was already
described in our earlier work.36 HPLC tR ) 24.47 min (99.8%).
(S)-1-Allyl-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (9).
(S)-5-[1-(2-Methoxymethyl-pyrrolidinyl)sulfonyl]isatin (1) (400 mg,
1.23 mmol) was converted to 9 using anhydrous K2CO3 (425 mg,
3.08 mmol) and allyl bromide (0.22 mL, 2.46 mmol), as described
in the general procedure, and stirred for 14 h. The crude product
was purified by column chromatography (ethyl acetate:cyclohexane,
9:1) to yield a deep-orange colored gummy solid. Yield: 320 mg
(71%). 1H NMR (300 MHz, CDCl3): δ 1.50-1.68 (m, 2H),
1.80-1.98 (m, 2H), 3.04-3.17 (m, 1H), 3.33-3.56 (m, 2H), 3.35
(s, 3H), 3.58 (dd, 1H, 2JHb,Ha ) 9.4 Hz, 3JHb,H ) 3.8 Hz), 3.68-3.79
(m, 1H), 4.43 (dd, 2H, 3JH,Ha ) 5.5 Hz, 4JH,Hc ) 0.6 Hz), 5.36 (m,
Conclusion
We were successful in synthesizing two new series of isatin
derivatives (the bromofluorides and fluorohydrins), and their
inhibition potencies were evaluated in vitro by biochemical
caspase inhibition assays. In line with previous findings, the
majority of synthesized 5-pyrrolidinylsulfonyl isatins exhibited
high potency for inhibiting the downstream effector caspases-3
and -7. The obtained results have added a new dimension to
the synthesis and development of nonpeptide-based inhibitors
of caspase-3 and -7 and also enabled us in expanding the range
of the existing class of such inhibitors. A potential caspase-
targeted radioligand should selectively detect one of the activated
caspases in vivo. For these reasons, as a proof-of-concept, the
radiotracer [18F]37 was synthesized as a first fluorohydrin-based
PET-compatible caspase-targeted model tracer. Aware of the
3
3
1H), 5.37 (m, 1H), 5.85 (ddt, 1H, JHa,H ) 5.5 Hz, JHa,Hb ) 17.3
3
3
Hz, JHa,Hc ) 10.3 Hz), 7.05 (d, 1H, JH,H ) 8.4 Hz), 8.04 (d, 1H,
4JH,H ) 1.9 Hz), 8.07 (dd, 1H,3JH,H ) 8.4 Hz, 4JH,H ) 1.9 Hz). 13
NMR (75 MHz, CDCl3): δ 24.1, 28.9, 42.9, 49.4, 59.1, 59.2, 74.9,
C

3490 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Podichetty et al.
111.1, 117.4, 119.4, 124.5, 129.7, 136.2, 137.4, 153.4, 157.8, 181.8.
HRMS (ESI-MicroTof): m/e 419.1242 (M + Na + CH3OH)+, calcd
for C18H24N2NaO6S 419.1247. HPLC tR ) 24.28 min (99.6%).
(S)-1-(But-3-enyl)-5-[1-(2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin (12). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-
isatin (1) (500 mg, 1.54 mmol) was converted to 12 using anhydrous
K2CO3 (532 mg, 3.85 mmol) and 4-bromo-1-butene (0.3 mL, 3.08
mmol). as described in the general procedure, and stirred for 14 h.
The crude product was purified by column chromatography
(CH2Cl2:MeOH, 98:2) to yield a deep-orange colored gummy solid.
same temperature for 30 min and then at ambient temperature for
7 h. The crude product was purified by column chromatography
(ethyl acetate:toluene 4.5:5.5) to yield a deep-orange colored gummy
solid. Yield: 139 mg (90%). 1H NMR (400 MHz, CDCl3): δ
1.64-1.66 (m, 2H), 1.84-1.91 (m, 2H), 3.07-3.11 (m, 1H),
2
3.32-3.43 (m, 2H), 3.33 (s, 3H), 3.57 (dd, 1H, JHb,Ha ) 9.4 Hz,
3
3JHb,H ) 3.8 Hz), 3.70-3.72 (m, 1H), 4.53 (dt, 2H, JH,Ha ) 5.2
Hz, 4JH,Hb+Hc ) 1.3 Hz), 4.90 (s, 2H), 5.31 (ddd, 1H, 2JHc,Hb ) 2.8
4
2
Hz,3JHc,Ha ) 10.5 Hz, JHc,H ) 1.3 Hz), 5.43 (ddd, 1H, JHb,Hc
)
3
4
3
3.1 Hz, JHb,Ha ) 17.3 Hz, JHb,H ) 1.5 Hz), 6.07 (ddd, 1H, JHa,H
) 5.3 Hz, 3JHa,Hb ) 17.2 Hz, 4JHa,Hc ) 10.5 Hz), 6.90 (d, 2H, 3JH,H
1
Yield: 531 mg (91%). H NMR (300 MHz, CDCl3): δ 1.64-1.74
) 8.5 Hz), 6.94 (d, 1H,3JH,H ) 8.3 Hz), 7.26 (d, 1H, JH,H ) 8.5
3
3
(m, 2H), 1.85-1.96 (m, 2H), 2.52 (q, 2H, JH,H ) 7.0 Hz),
3
4
Hz), 7.99 (dd, 1H, JH,H ) 8.1 Hz, JH,H ) 1.1 Hz), 8.04 (d, 1H,
4JH,H ) 1.4 Hz). 13C NMR (100 MHz, CDCl3): δ 24.1, 28.8, 44.0,
49.3, 59.1, 59.2, 68.9, 74.8, 111.2, 115.4, 117.5, 118.0, 124.5, 125.8,
129.1, 132.9, 134.0, 137.4, 153.4, 158.0, 158.7, 182.0. HRMS (ESI-
MicroTof): m/e 525.1646 (M + Na + CH3OH)+, calcd for
C25H30N2NaO7S 525.1666. HPLC tR ) 30.67 min (99.2%).
General Procedure for the Synthesis of N-(Bromofluoroalkyl)
Isatins. To a dried, argon flushed Teflon vessel with a rubber
septum, Olah’s reagent (1.1-1.2 equiv) in dry methylene chloride
(2 mL) was cooled to 0 °C. Using a syringe, a solution of terminal
olefin 7, 9, 12, 15, 18, or 20 in 5 mL of dry methylene chloride
was injected dropwise along with gentle stirring. After the addition,
crystallized N-bromosuccimide (NBS, 1-1.15 equiv) was added
portionwise (5 portions) and the reaction mixture was stirred at 0
°C for 30 min and then at ambient temperature for 3-16 h,
depending on the substrate. The reaction mixture was then cooled
to 0 °C, quenched with chilled saturated sodium bicarbonate solution
(20 mL), and the aqueous layer extracted completely with excess
CH2Cl2. The combined organic extracts were sequentially washed
with 1 N HCl solution (15 mL), followed by 5% sodium bicarbonate
solution (15 mL), dried over MgSO4, and filtered. The solvent was
then removed in vacuo to afford the crude product, which was
purified by silica gel chromatography.
3.10-3.18 (m, 1H), 3.34-3.47 (m, 2H), 3.36 (s, 3H), 3.60 (dd,
2
3
1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.8 Hz), 3.71-3.77 (m, 1H), 3.88
3
3
(t, 2H, JH,H ) 7.0 Hz), 5.08-5.19 (m, 2H), 5.85 (ddt, 1H, JHa,H
3
3
3
) 6.9 Hz, JHa,Hb ) 9.8 Hz, JHa,Hc ) 16.8 Hz), 7.06 (d, 1H, JH,H
) 8.3 Hz), 8.04 (d, 1H, 4JH,H ) 1.7 Hz), 8.10 (dd, 1H, 3JH,H ) 8.3
4
Hz, JH,H ) 1.9 Hz). 13C NMR (75 MHz, CDCl3): δ 24.1, 28.8,
31.6, 40.0, 49.4, 59.1, 59.2, 74.8, 110.5, 117.3, 118.6, 124.5, 133.4,
133.7, 137.4, 153.6, 157.8, 182.0. HRMS (ESI-MicroTof): m/e
433.1411 (M + Na + CH3OH)+, calcd for C19H26N2NaO6S
433.1415. HPLC tR ) 26.48 min (98.9%).
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-(undec-10-
enyl)isatin (15). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-
isatin (1) (500 mg, 1.54 mmol) was converted to 15 using anhydrous
K2CO3 (532 mg, 3.85 mmol) and 11-bromo-1-undecene (0.67 mL,
3.08 mmol), as described in the general procedure, and stirred for
14 h. The crude product was purified by column chromatography
(CH2Cl2:MeOH, 97:3) to yield a light yellow colored gummy solid.
1
Yield: 660 mg (90%). H NMR (400 MHz, CDCl3): δ 1.28-1.73
3
(m, 16H), 1.90-1.94 (m, 2H), 2.05 (q, 2H, JH,H ) 6.8 Hz),
3.11-3.17 (m, 1H), 3.36 (s, 3H), 3.37-3.47 (m, 2H), 3.60 (dd,
2
3
1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.8 Hz), 3.74-3.78 (m, 3H), 4.94
(dd, 1H, 2JHb,Hc ) 1.0 Hz, 3JHb,Ha ) 10.2 Hz), 5.01 (dd, 1H, 2JHc,Hb
) 1.4 Hz, 3JHc,Ha ) 17.1 Hz), 5.85 (ddt, 1H, 3JHa,H ) 6.7 Hz, 3JHa,Hb
3
3
) 10.2 Hz, JHa,Hc ) 16.9 Hz), 7.02 (d, 1H, JH,H ) 8.3 Hz), 8.04
(d, 1H, 4JH,H ) 1.5 Hz), 8.10 (dd, 1H, 3JH,H ) 8.3 Hz, 4JH,H ) 1.6
Hz). 13C NMR (100 MHz, CDCl3): δ 24.1, 26.9, 27.2, 28.8, 28.9,
29.0, 29.2, 29.3, 29.4, 33.8, 40.7, 49.4, 59.1, 59.2, 74.8, 110.4,
114.2, 117.4, 124.5, 133.6, 137.5, 139.1, 153.7, 157.8, 182.2. HRMS
(ESI-MicroTof) m/e: 531.2482 (M + Na + CH3OH)+, calcd for
C26H40NaN2O6S 531.2489. HPLC tR ) 38.73 min (98.9%).
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-(4-vinylben-
zyl)isatin (18). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-
isatin (1) (100 mg, 0.3 mmol) was converted to 18 using anhydrous
K2CO3 (106 mg, 0.77 mmol) and 4-vinylbenzyl chloride (0.09 mL,
0.617 mmol), as described in the general procedure, except that
4-vinylbenzyl chloride was added at 0 °C and the reaction mixture
stirred at the same temperature for 30 min and then at ambient
temperature for 7 h. The crude product was purified by column
chromatography (ethyl acetate:toluene 2:3) to yield a deep-orange
colored gummy solid. Yield: 112 mg (83%). 1H NMR (300 MHz,
CDCl3): δ 1.60-1.72 (m, 2H), 1.79-1.96 (m, 2H), 3.04-3.15 (m,
(S)-1-(3-Bromo-2,2-difluoropropyl)-5-[1-(2-methoxymethylpyr-
rolidinyl)sulfonyl]isatin (8). (S)-1-(2-Fluoroallyl)-5-[1-(2-meth-
oxymethylpyrrolidinyl)sulfonyl]isatin (7) (70 mg, 0.183 mmol) was
converted to 8 using Olah’s reagent (0.05 mL, 0.219 mmol) and
crystallized NBS (37.5 mg, 0.21 mmol), as described in the general
procedure, and stirred for 16 h. The crude product was purified by
column chromatography (ethyl acetate:toluene 1:1) to yield a deep-
1
orange colored gummy solid. Yield: 48 mg (55%). H NMR (400
MHz, CDCl3): δ 1.65-1.73 (m, 2H), 1.87-1.94 (m, 2H), 3.10-3.18
(m, 1H), 3.35-3.46 (m, 2H), 3.36 (s, 3H), 3.58 (dd, 1H, 2JHb,Ha
)
9.4 Hz, 3JHb,H ) 3.8 Hz), 3.69 (t, 2H, 3JH,F ) 13.3 Hz), 3.73-3.79
3
3
(m, 1H), 4.39 (t, 2H, JH,F ) 13.5 Hz), 7.19 (d, 1H, JH,H ) 8.3
4
3
Hz), 8.07 (d, 1H, JH,H ) 1.5 Hz), 8.10 (dd, 1H, JH,H ) 8.4 Hz,
4JH,H ) 1.8 Hz). 13C NMR (100 MHz, CDCl3): δ 24.1, 28.8, 29.3
2
2
(t, JC,F ) 30.7 Hz), 43.8 (t, JC,F ) 28.5 Hz), 49.3, 59.1, 59.2,
74.8, 111.6, 117.6, 119.9 (t, 1JC,F ) 247.4 Hz), 124.6, 134.7, 137.5,
152.8, 158.0, 180.4. 19F NMR (282 MHz, CDCl3): δ -100.4
3
(quintet, 2F, JH,F ) 13.3 Hz). HRMS (ESI- MicroTof): m/e
535.0314 (M + Na + CH3OH)+, calcd for C18H23BrF2N2NaO6S
535.0320. HPLC tR ) 27.28 min (99.5%).
2
1H), 3.32-3.44 (m, 2H), 3.33 (s, 3H), 3.57 (dd, 1H, JHb,Ha ) 9.4
Hz, 3JHb,H ) 3.8 Hz), 3.68-3.75 (m, 1H), 4.96 (s, 2H), 5.28-5.30
(dd, 1H, 2JHb,Hc ) 0.5 Hz, 3JHb,Ha ) 10.9 Hz), 5.75-5.78 (dd, 1H,
(S)-1-(3-Bromo-2-fluoropropyl)-5-[1-(2-methoxymethylpyrrolidi-
nyl)sulfonyl]isatin (10) and (S)-1-(2-Bromo-3-fluoropropyl)-5-[1-
(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (11) (10:1 mixture
of 10 and 11). (S)-1-Allyl-5-[1-(2-methoxymethylpyrrolidinyl)sul-
fonyl]isatin (9) (440 mg, 1.21 mmol) was converted to a mixture
of two regioisomers (10 and 11) using Olah’s reagent (0.31 mL,
1.33 mmol) and crystallized NBS (236 mg, 1.33 mmol), as
described in the general procedure, and stirred for 14 h. The crude
product was purified by column chromatography (ethyl acetate:
cyclohexane 1:5) to yield a mixture of two regioisomers as deep-
orange colored gummy solid. Yield: 293 mg (52%). 1H NMR (300
MHz, CDCl3): δ 1.54-1.90 (m, 4H), 3.01-3.10 (m, 1H), 3.27-3.61
(m, 5H), 3.30 (s, 3H), 3.52 (dd, 1H, 2JHb,Ha ) 9.6 Hz, 3JHb,H ) 3.6
Hz), 3.63-3.72 (m, 1H), 3.80-4.20 (m, 2H), 7.11-7.22 (m, 1H),
7.94-8.05 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 24.2, 29.3, 36.6
3
3
2JHc,Hb ) 0.6 Hz, JHc,Ha ) 17.6 Hz), 6.71 (dd, 1H, JHa,Hb ) 10.9
3
3
Hz, JHa,Hc ) 17.6 Hz), 6.92 (d, 1H, JH,H ) 8.3 Hz), 7.30 (d, 2H,
3JH,H ) 8.2 Hz), 7.41 (d, 2H, JH,H ) 8.2 Hz), 7.97 (dd, 1H, JH,H
) 8.9 Hz, 4JH,H ) 1.9 Hz), 8.04 (d, 1H, 4JH,H ) 1.7 Hz). 13C NMR
(75 MHz, CDCl3): δ 24.1, 28.8, 44.2, 49.3, 59.0, 59.1, 74.8, 111.3,
114.9, 117.5, 124.5, 127.0, 127.8, 133.1, 134.0, 135.9, 137.4, 137.9,
153.3, 157.8, 181.8. HRMS (ESI-MicroTof): m/e 495.1581 (M +
Na + CH3OH)+, calcd for C24H28N2NaO6S 495.1560. HPLC tR )
30.57 min (99.6%).
3
3
(S)-1-[4-(Allyloxy)benzyl]-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (20). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) (100 mg, 0.3 mmol) was converted to 20 using
anhydrous K2CO3 (106 mg, 0.77 mmol) and a crude sample of 6
(139 mg, 0.617 mmol), as described in the general procedure, except
that 6 was added at 0 °C and the reaction mixture stirred at the
2
1
(d, JC,F ) 16.7 Hz), 40.2, 49.8, 59.5, 59.7, 75.2, 90.0 (d, JC,F
)

Fluorinated Isatin DeriVatiVes. Part 2
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3491
190.4 Hz), 109.2, 117.9, 124.9, 137.9, 134.6, 153.8, 157.0, 180.5.
19F NMR (282 MHz, CDCl3): δ -180.5 (m, 1F, 2JHc,F ) 45.3 Hz,
3JHa+Hb,F ) 18.5 Hz,3JHd+He,F ) 22.8 Hz), -215.2 (m, 1F, 2JHd+He,F
) 47.8 Hz, 3JHc,F ) 19.1 Hz). HRMS (ESI-MicroTof): m/e 499.0320
(M + Na)+, calcd for C18H22BrFN2NaO5S 499.0314. HPLC tR )
26.68 min (97.3%).
134.9 (d, 5JC,F ) 1.6 Hz), 137.4, 137.6 (d, 2JC,F ) 20.6 Hz), 153.1,
157.8, 181.6. 19F NMR (282 MHz, CDCl3): δ -174.3 to -174.9
2
3
3
4
(m, 1F, JHa,F ) 46.4 Hz, JHb,F ) 16.4 Hz, JHc,F ) 23.1 Hz, JH,F
) 6.0 Hz). HRMS (ESI-MicroTof): m/e 563.0443 (M + Na)+, calcd
for C23H24BrFN2NaO5S 563.0447. HPLC tR ) 30.43 min (99.1%).
(S)-1-[4-(3-Bromo-2-fluoropropoxy)benzyl]-5-[1-(2-methoxym-
ethylpyrrolidinyl)sulfonyl]isatin (21) and (S)-1-[4-(2-Bromo-3-fluo-
ropropoxy)benzyl]-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-
isatin (22) (7:3 mixture of 21+22). (S)-1-[4-(Allyloxy)benzyl]-5-
[1-(2-methoxymethylpyrrolidinyl)-sulfonyl]isatin (20) (80 mg, 0.17
mmol) was converted to a mixture of two regioisomers (21 and
22) using Olah’s reagent (0.045 mL, 0.2 mmol) and crystallized
NBS (34.8 mg, 0.195 mmol), as described in the general procedure,
and stirred for 6 h. The crude product was purified by flash column
chromatography (ethyl acetate:cyclohexane 3.5:6.5) to yield a
mixture of two regioisomers as deep-orange colored gummy solid.
Yield (as mixture): 60 mg (62%). 1H NMR (400 MHz, CDCl3): δ
1.65-1.68 (m, 2H), 1.85-1.92 (m, 2H), 3.07-3.16 (m, 1H),
(S)-1-(4-Bromo-3-fluorobutyl)-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (13). (S)-1-(But-3-enyl)-5-[1-(2-methoxymethylpyr-
rolidinyl)sulfonyl]isatin (12) (100 mg, 0.2 mmol) was converted
to a mixture of two regioisomers (13 and 14) using Olah’s reagent
(0.067 mL, 0.29 mmol) and crystallized NBS (51.7 mg, 0.29 mmol),
as described in the general procedure, and stirred for 12 h. The
crude product was purified by column chromatography (ethyl
acetate:cyclohexane 1:1) to yield pure 13 together with its mixture
with the other regioisomer 14 as deep-orange colored gummy solids.
Yield (as mixture): 102 mg (81%). Spectral data of the secondary
1
fluoride (13) is explained below. H NMR (300 MHz, CDCl3): δ
1.65-1.73 (m, 2H), 1.87-1.95 (m, 2H), 2.15-2.27 (m, 2H),
3.12-3.18 (m, 1H), 3.36 (s, 3H), 3.37-3.47 (m, 2H), 3.51-3.60
2
3.33-3.43 (m, 2H), 3.34 (s, 3H), 3.57 (dd, 1H, JHb,Ha ) 9.4 Hz,
3
2
(m, 3H), 3.75-3.77 (m, 1H), 3.97 (t, 2H, JH,Ha+Hb ) 6.9 Hz),
3JHb,H ) 3.8 Hz), 3.58-3.75 (m, 3H), 4.22-4.38 (m, 2H, JH,F
)
3
3
4.67-4.88 (m, 1H), 7.10 (d, 1H, JH,H ) 8.3 Hz), 8.08 (d, 1H,
19.8 Hz, JH,H ) 4.2 Hz), 4.68-5.07 (m, 1H), 4.92 (s, 2H),
3
4
3
4JH,H ) 2.1 Hz), 8.13 (dd, 1H, JH,H ) 8.3 Hz, JH,H)1.4 Hz). 13C
6.90-6.94 (m, 3H), 7.29 (d, 2H, JH,H ) 8.5 Hz), 7.98-8.07 (m,
2
2
NMR (75 MHz, CDCl3): δ 24.1, 28.8, 31.3 (d, JC,F ) 20.7 Hz),
2H). 13C NMR (100 MHz, CDCl3): δ 24.1, 28.8, 29.4 (d, JC,F
)
32.5 (d, 2JC,F ) 25.0 Hz), 36.8 (d, 3JC,F ) 3.6 Hz), 49.4, 59.1, 59.2,
74.8, 89.3 (d, 1JC,F ) 176.0 Hz), 110.3, 117.4, 124.7, 134.3, 137.6,
153.1, 157.9, 181.5. 19F NMR (282 MHz, CDCl3): δ -179.2 to
-179.7 (m, 1F). HRMS (ESI-MicroTof): m/e 499.0320 (M + Na)+,
calcd for C18H22BrFN2NaO5S 499.0314. HPLC tR ) 27.62 min
(99.7%).
25.9 Hz), 43.9, 49.3, 59.1, 59.2, 67.5 (d, JC,F ) 27.4 Hz), 74.8,
111.2, 115.3, 117.5, 124.5, 126.7, 129.2, 134.0, 137.4, 153.3, 157.8,
158.2, 181.9. 19F NMR (282 MHz, CDCl3): δ -183.4 to -183.8
2
(m, 1F, 2JHc,F ) 55.6 Hz, 3JH,F ) 16.1 Hz, 3JH,F ) 19.4 Hz, 3JH,F
)
)
2
2
26.6 Hz), -216.3 to -216.8 (m, 1F, JHd,F ) 46.7 Hz, JHe,F
52.1 Hz, 3JHc,F ) 15.6 Hz). HRMS (ESI-MicroTof): m/e 623.0827
(M + Na + CH3OH)+, calcd for C25H30BrFN2NaO7S 623.0833.
HPLC tR ) 31.20 min (95.1%).
(S)-1-(11-Bromo-10-fluoroundecyl)-5-[1-(2-methoxymethylpyr-
rolidinyl)sulfonyl]isatin (16) and (S)-1-(10-Bromo-11-fluorounde-
cyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (17) (11:1
mixture of 16+17). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sul-
fonyl]-1-(undec-10-enyl)isatin (15) (100 mg, 0.21 mmol) was
converted to a mixture of two regioisomers (16 and 17) using Olah’s
reagent (0.058 mL, 0.25 mmol) and crystallized NBS (43 mg, 0.24
mmol), as described in the general procedure, and stirred for 12 h.
The crude product was purified by column chromatography (ethyl
acetate:cyclohexane 3.5:6.5) to yield a mixture of two regioisomers
General Procedure for the Synthesis of N-(Epoxyalkyl) Isatins. To
a solution of (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
(1) in 3 mL of dry DMF,2.5 equiv of anhydrous potassium
carbonate was added under argon atmosphere and the reaction
mixture was stirred for 30 min at ambient temperature. Then the
reaction mixture was cooled to 0 °C and an excess of the alkylating
agent (2-3 equiv) was slowly added. The reaction mixture was
stirred for 30 min at 0 °C and for a further 12-14 h in the case of
simple alkyl halides and for 6-8 h in the case of benzylic chlorides
and (S)-(+)-glycidyl nosylate (in the synthesis of 29). The reaction
mixture was diluted with 20 mL of ethyl acetate and, after 15 min
of stirring, was filtered. Removal of the solvents in vacuo furnished
the crude product, which was purified by silica gel chromatography.
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-[(S)-oxiran-
2-ylmethyl]isatin (28). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) (500 mg, 1.54 mmol) was converted to 28 using
anhydrous K2CO3 (532 mg, 3.85 mmol) and (S)-(+)-glycidyl
nosylate (800 mg, 3.06 mmol) as described in the general procedure
and stirred for 6 h. The crude product was purified by column
chromatography (ethyl acetate:toluene 8:2) to yield a deep-orange
colored solid. Yield: 328 mg (56%). 1H NMR (400 MHz, CDCl3):
1
as deep-orange colored gummy solid. Yield: 105 mg (87%). H
NMR (300 MHz, CDCl3): δ 1.26-1.61 (m, 18H), 1.69-1.95 (m,
2H), 3.09-3.15 (m, 1H), 3.34-3.52 (m, 4H), 3.36 (s, 3H), 3.60
2
3
(dd, 1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.8 Hz), 3.75-3.79 (m, 3H),
3
4.48-4.73 (m, 1H), 7.04 (d, 1H, JH,H ) 8.3 Hz), 8.03 (d, 1H,
4JH,H ) 1.6 Hz), 8.09 (dd, 1H, 3JH,H ) 8.3 Hz, 4JH,H ) 1.7 Hz). 13
C
3
NMR (75 MHz, CDCl3): δ 24.1, 24.7 (d, JC,F ) 4.2 Hz), 26.8,
2
27.2, 28.8, 29.0, 29.1, 29.2, 29.3, 33.3 (d, JC,F ) 20.5 Hz), 33.9
2
1
(d, JC,F ) 25.3 Hz), 40.7, 49.4, 59.1, 59.2, 74.8, 92.1 (d, JC,F
)
174.6 Hz), 110.4, 117.3, 124.5, 133.6, 137.5, 153.7, 157.7, 182.2.
19F NMR (282 MHz, CDCl3): δ -177.6 to -178.1 (m, 1F, 2JH,F
66.6 Hz, 3JH,F ) 19.9 Hz, 3JH,F ) 27.7 Hz), -209.5 to -209.9 (dt,
)
2
3
1F, JH,F ) 47.1 Hz, JH,F ) 13.9 Hz). HRMS (ESI-MicroTof):
m/e 597.1430 (M + Na)+, calcd for C25H36BrFN2NaO5S 597.1405.
HPLC tR ) 36.50 min (99.8%).
δ 1.64-1.72 (m, 2H), 1.87-1.92 (m, 2H), 2.73 (dd, 1H, 2JHd,He
)
3
4.4 Hz, JHd,Hc ) 2.6 Hz), 2.93 (t, 1H, JHe,Hc+Hd ) 4.2 Hz),
3.10-3.16 (m, 1H), 3.22-3.25 (m, 1H), 3.36 (s, 3H), 3.37-3.46
(S)-1-[4-(2-Bromo-1-fluoroethyl)benzyl]-5-[1-(2-methoxymeth-
ylpyrrolidinyl)sulfonyl]isatin (19). (S)-5-[1-(2-Methoxymethylpyr-
rolidinyl)sulfonyl]-1-(4-vinylbenzyl)isatin (18) (72 mg, 0.163 mmol)
was converted to 19 using Olah’s reagent (0.045 mL, 0.196 mmol)
and crystallized NBS (33.4 mg, 0.188 mmol), as described in the
general procedure, and stirred for 3 h. The crude product was
purified by column chromatography (ethyl acetate:cyclohexane 1:1)
to yield a deep-orange colored gummy solid. Yield: 60 mg (68%).
1H NMR (300 MHz, CDCl3): δ 1.64-1.72 (m, 2H), 1.85-1.93
2
3
(m, 2H), 3.49 (dd, 1H, JHa,Hb ) 15.2 Hz, JHa,Hc ) 6.6 Hz), 3.60
2
3
(dd, 1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.8 Hz), 3.74-3.78 (m, 1H),
2
3
4.50 (dd, 1H, JHb,Ha ) 15.2 Hz, JHb,Hc ) 2.1 Hz), 7.29 (d, 1H,
3JH,H ) 8.3 Hz), 8.06 (d, 1H, JH,H ) 1.7 Hz), 8.10 (dd, 1H, JH,H
4
3
4
) 8.3 Hz, JH,H ) 1.8 Hz). 13C NMR (100 MHz, CDCl3): δ 24.1,
28.8, 42.9, 44.8, 49.4, 49.8, 59.1, 59.2, 74.9, 111.9, 117.3, 124.5,
134.2, 137.6, 153.6, 157.9, 181.5. HRMS (ESI-MicroTof): m/e
403.0942 (M + Na)+, calcd for C17H20N2NaO6S 403.0934. HPLC
tR ) 20.50 min (100%).
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-[2-(oxiran-2-
yl)ethyl]isatin (30). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) (400 mg, 1.23 mmol) was converted to 30 using
anhydrous K2CO3 (425 mg, 3.08 mmol) and 23 (559 mg, 3.7 mmol),
as described in the general procedure, and stirred for 12 h. The
crude product was purified by column chromatography (ethyl
acetate:toluene 9:1) to yield a deep-orange colored gummy solid.
(m, 2H), 3.06-3.14 (m, 1H), 3.32-3.44 (m, 2H), 3.33 (s, 3H),
2
3.53-3.75 (m, 4H), 4.99 (s, 2H), 5.53-5.72 (ddd, 1H, JHa,F
)
)
3
3
3
46.6 Hz, JHa,Hb ) 7.1 Hz, JHa,Hc ) 4.7 Hz), 6.90 (d, 1H, JH,H
8.3 Hz), 7.30 (d, 2H, 3JH,H ) 8.2 Hz), 7.41 (d, 2H, 3JH,H ) 8.2 Hz),
7.99 (dd, 1H, 3JH,H ) 8.3 Hz, 4JH,H ) 1.9 Hz), 8.05 (d, 1H, 4JH,H
)
)
1.7 Hz). 13C NMR (75 MHz, CDCl3): δ 24.1, 28.8, 33.9 (d, 2JC,F
1
28.4 Hz), 44.1, 49.3, 59.1, 59.2, 74.8, 92.1 (d, JC,F ) 178.5 Hz),
3
111.1, 117.5, 124.6, 126.7 (d, 2C, JC,F ) 6.7 Hz), 127.9, 134.2,

3492 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Podichetty et al.
1
Yield: 301 mg (62%). H NMR (500 MHz, CDCl3): δ 1.53-1.74
MHz, CDCl3): δ 24.0, 28.8, 43.9, 44.6, 49.3, 50.0, 59.0, 59.1, 68.8,
74.8, 111.2, 115.3, 117.4, 124.4, 126.2, 129.1, 134.0, 137.3, 153.3,
157.8, 158.6, 181.9.
2
(m, 3H), 1.87-1.94 (m, 2H), 2.18-2.25 (m, 1H, JHb,Ha ) 14.4
Hz, 3JHb,H ) 7.3 Hz, 3JHb,Hc ) 3.4 Hz), 2.52-2.54 (ddd, 1H, 2JHd,He
3
4
) 4.7 Hz, JHd,Hc ) 2.6 Hz, JHd,H ) 1.2 Hz), 2.80 (dd, 1H,
2JHe,Hd ) 4.8 Hz, 3JHe,Hc ) 4.1 Hz), 3.01 (m, 1H, 3JHc,He ) 3.6 Hz,
Minor Diastereomer. 1H NMR (600 MHz, CDCl3): δ 1.65-1.69
(m, 2H), 1.85-1.91 (m, 2H), 2.77 (dd, 1H, 2JHd,He ) 4.9 Hz, 3JHd,Hc
) 2.6 Hz), 2.90-2.92 (m, 1H, 2JHe,Hd ) 4.9 Hz, 3JHe,Hc ) 4.2 Hz),
3.08-3.12 (m, 1H), 3.33-3.37 (m, 2H), 3.33 (s, 3H), 3.39-3.42
(m, 1H), 3.57 (dd, 1H, 2JHb,Ha ) 9.4 Hz, 3JHb,H ) 3.9 Hz), 3.70-3.73
3JHc,Ha ) 6.5 Hz, JHc,Hb ) 10.5 Hz), 3.12-3.18 (m, 1H), 3.36 (s,
3
3H), 3.36-3.39 (m, 1H), 3.41-3.45 (m, 1H), 3.59 (dd, 1H, 2JHb,Ha
3
) 9.4 Hz, JHb,H ) 3.9 Hz), 3.74-3.78 (m, 1H), 3.91-4.00 (m,
3
4
2
3
2H), 7.10 (d, 1H, JH,H ) 8.3 Hz), 8.06 (d, 1H, JH,H ) 1.8 Hz),
8.10 (dd, 1H,3JH,H ) 8.3 Hz, 4JH,H ) 1.9 Hz). 13C NMR (125 MHz,
CDCl3): δ 24.1, 28.8, 30.5, 37.8, 47.0, 49.3, 49.6, 59.1, 59.2, 74.8,
110.3, 117.3, 124.6, 134.0, 137.5, 153.3, 157.9, 181.7. HRMS (ESI-
MicroTof): m/e 417.1094 (M + Na)+, calcd for C18H22N2NaO6S
417.1091. HPLC tR ) 20.93 min (96.8%).
(m, 1H), 3.96 (dd, 1H, JHa,Hb ) 11.0 Hz, JHa,Hc ) 5.7 Hz), 4.24
2 3
(dd, 1H, JHb,Ha ) 11.1 Hz, JHb,Hc ) 3.2 Hz), 4.62 (s, 2H),
6.90-6.93 (m, 3H), 7.25-7.30 (m, 2H), 7.98 (dd, 1H, 3JH,H ) 8.3
Hz, JH,H ) 1.9 Hz), 8.04 (d, 1H, JH,H ) 1.9 Hz). 13C NMR (150
MHz, CDCl3): δ 24.0, 28.8, 43.9, 44.7, 49.3, 50.1, 59.0, 59.1, 68.7,
74.8, 111.2, 114.7, 117.4, 124.4, 126.2, 128.6, 134.0, 137.3, 153.3,
157.8, 158.6, 181.9. HRMS (ESI-MicroTof): m/e 541.1616 (M +
Na + CH3OH)+, calcd for C25H30N2NaO8S 541.1615. HPLC tR )
27.30 min (99.2%).
4
4
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-[9-(oxiran-2-
yl)nonyl]isatin (33). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) (200 mg, 0.61 mmol) was converted to 33 using
anhydrous K2CO3 (212 mg, 1.54 mmol) and 24 (382 mg, 1.54
mmol), as described in the general procedure, and stirred for 12 h.
The crude product was purified by column chromatography (ethyl
acetate:toluene 4.5:5.5 to yield a deep-orange colored gummy solid.
General Procedure for the Synthesis of N-(Fluorohydroxy-
alkyl) Isatins (Procedure A for the synthesis of 29 and 34+35).
To a dried, argon flushed Teflon vessel with a rubber septum, Olah’s
reagent (2-5 equiv) in dry methylene chloride (2 mL) was cooled
to 0 °C. Using a syringe, a solution of terminal epoxide 28 or 33
in 5 mL of dry methylene chloride was injected dropwise along
with gentle stirring. After the addition, the reaction mixture was
stirred at 0 °C for 30 min and then at RT for 12-16 h. The reaction
mixture was then cooled to 0 °C, quenched with chilled saturated
sodium bicarbonate solution (20 mL), and the aqueous layer
extracted completely with excess CH2Cl2. All the organic extracts
were sequentially washed with 1 N HCl solution (15 mL), followed
by 5% sodium bicarbonate solution (15 mL), dried over MgSO4,
and filtered. The solvent was then removed in vacuo to afford the
crude product, which was purified by silica gel chromatography.
General Procedure for the Synthesis of N-(Fluorohydroxy-
alkyl) Isatins (Procedure B for the synthesis of 31 + 32, 37, and
39). To a dried, argon flushed round-bottomed flask, terminal
epoxide 30, 36, or 38 was added followed by Et3N·3HF (100-200
equiv), and the reaction mixture was heated at 80-90 °C for 2-16
h (depending on the substrate) under argon atmosphere. The reaction
mixture was then cooled to 0 °C, diluted with CH2Cl2 (20 mL),
and quenched with chilled saturated sodium bicarbonate solution
(20 mL). After thoroughly shaking and separating the two layers,
the aqueous layer was extracted completely with excess CH2Cl2.
All the organic extracts were dried over MgSO4 and filtered. The
solvent was then removed in vacuo to afford the crude product,
which was purified by silica gel chromatography.
1
Yield: 260 mg (86%). H NMR (500 MHz, CDCl3): δ 1.30-1.57
(m, 14H), 1.66-1.74 (m, 4H), 1.89-1.94 (m, 2H), 2.47 (dd, 1H,
2JHd,He ) 5.1 Hz, JHd,Hc ) 2.7 Hz), 2.75 (t, 1H, JHe,Hd+Hc ) 4.4
3
Hz), 2.88-2.99 (m, 1H), 3.12-3.17 (m, 1H), 3.36 (s, 3H),
2
3.36-3.39 (m, 1H), 3.42-3.46 (m, 1H), 3.60 (dd, 1H, JHb,Ha
)
)
9.4 Hz, 3JHb,H ) 3.9 Hz), 3.63-3.78 (m, 3H), 7.02 (d, 1H, 3JH,H
4
3
8.3 Hz), 8.05 (d, 1H, JH,H ) 1.8 Hz), 8.10 (dd, 1H, JH,H ) 8.3
Hz, JH,H ) 1.9 Hz). 13C NMR (125 MHz, CDCl3): δ 24.1, 25.9,
4
26.8, 27.2, 28.8, 29.1, 29.2, 29.3, 29.4, 32.4, 40.6, 47.1, 49.3, 52.3,
59.1, 59.2, 74.8, 110.3, 117.3, 124.5, 133.7, 137.4, 153.7, 157.7,
182.1. HRMS (ESI-MicroTof): m/e 515.2198 (M + Na)+, calcd
for C25H36N2NaO6S 515.2186. HPLC tR ) 33.62 min (95.5%).
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-[4-(oxiran-2-
yl)benzyl]isatin (36). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sul-
fonyl]isatin (1) (100 mg, 0.3 mmol) was converted to 36 using
anhydrous K2CO3 (106 mg, 0.77 mmol) and 25 (156 mg, 0.925
mmol), as described in the general procedure, and stirred for 8 h.
The crude product was purified by column chromatography (ethyl
acetate:toluene 1:1) to yield a deep-orange colored gummy solid.
1
Yield: 116 mg (84%). H NMR (400 MHz, CDCl3): δ 1.62-1.70
(m, 2H), 1.84-1.93 (m, 2H), 2.79 (dd, 1H, 2JHb,Hc ) 5.4 Hz, 3JHb,Ha
2
) 2.5 Hz), 3.07-3.13 (m, 1H), 3.16 (dd, 1H, JHc,Hb ) 5.4 Hz,
3JHc,Ha ) 4.1 Hz), 3.33-3.43 (m, 2H), 3.34 (s, 3H), 3.56 (dd, 1H,
2JHb,Ha ) 9.4 Hz, JHb,H ) 3.9 Hz), 3.69-3.74 (m, 1H), 3.85 (dd,
3
3
3
1H, JHa,Hb ) 2.6 Hz, JHa,Hc ) 3.9 Hz), 4.96 (s, 2H), 6.89 (d, 1H,
3JH,H ) 8.3 Hz), 7.28-7.34 (m, 4H), 7.98 (dd, 1H, 3JH,H ) 8.3 Hz,
4JH,H ) 1.9 Hz), 8.05 (d, 1H, 4JH,H ) 1.7 Hz). 13C NMR (100 MHz,
CDCl3): δ 24.1, 28.8, 44.2, 49.3, 51.3, 51.9, 59.1, 59.2, 74.8, 111.2,
117.5, 124.6, 126.5, 127.7, 133.7, 134.2, 137.4, 138.3, 153.2, 157.8,
181.7. HRMS (ESI-MicroTof): m/e 511.1522 (M + Na +
CH3OH)+, calcd for C24H28N2NaO7S 511.1509. Because of the
instability of the product under acidic conditions in polar solvents
(TFA/MeCN/H2O), purity could not be established by HPLC.
(S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-1-[4-(oxiran-2-
ylmethoxy)benzyl]isatin (38) (3:1 mixture of 2 diastereomers). (S)-
5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin (1) (150 mg,
0.46 mmol) was converted to 38 using anhydrous K2CO3 (160 mg,
1.15 mmol) and 27 (183 mg, 0.925 mmol) as described in the
general procedure and stirred for 8 h. The crude product was purified
by column chromatography (ethyl acetate:toluene 5.5:4.5) to yield
a deep golden-yellow colored gummy solid. Yield: 187 mg (83%).
Major Diastereomer. 1H NMR (600 MHz, CDCl3): δ 1.65-1.69
(m, 2H), 1.85-1.91 (m, 2H), 2.75 (dd, 1H, 2JHd,He ) 4.9 Hz, 3JHd,Hc
) 2.6 Hz), 2.90-2.92 (m, 1H, 2JHe,Hd ) 4.9 Hz, 3JHe,Hc ) 4.2 Hz),
3.08-3.12 (m, 1H), 3.33 (s, 3H), 3.33-3.37 (m, 2H), 3.39-3.42
(m, 1H), 3.57 (dd, 1H, 2JHb,Ha ) 9.4 Hz, 3JHb,H ) 3.9 Hz), 3.70-3.73
(S)-1-[(S)-3-Fluoro-2-hydroxypropyl]-5-[1-(2-methoxymethylpyr-
rolidinyl)sulfonyl]isatin (29). (S)-5-[1-(2-Methoxymethylpyrrolidi-
nyl)sulfonyl]-1-[(S)-oxiran-2-ylmethyl]isatin (28) (90 mg, 0.236
mmol) was converted to 29 using Olah’s reagent (0.27 mL, 1.18
mmol), as described in the general procedure A, and stirred for
14 h. The crude product was purified by column chromatography
(ethyl acetate:toluene 8:2) to yield a light-orange colored solid.
1
Yield: 48 mg (51%). H NMR (300 MHz, CDCl3): δ 1.62-1.75
(m, 2H), 1.84-1.95 (m, 2H), 3.07-3.15 (m, 1H), 3.32-3.46 (m,
2H), 3.36 (s, 3H), 3.58 (dd, 1H, 2JHb,Ha ) 9.5 Hz, 3JHb,H ) 3.9 Hz),
3.70-3.77 (m, 1H), 3.86-4.01 (m, 2H), 4.24-4.34 (m, 1H),
4.38-4.44 (ddd, 1H, 2JHd,F ) 43.3 Hz, 2JHd,He ) 15.2 Hz, 3JHd,Hc
)
5.4 Hz), 4.60-4.67 (ddd, 1H, 2JHe,F ) 46.9 Hz, 2JHe,Hd ) 13.9 Hz,
3
4
3JHe,Hc ) 4.1 Hz), 7.25 (d, 1H, JH,H ) 8.3 Hz), 8.01 (d, 1H, JH,H
) 1.7 Hz), 8.07 (dd, 1H, JH,H ) 8.3 Hz, JH,H ) 1.9 Hz). 13C
3
4
3
NMR (75 MHz, CDCl3): δ 24.1, 28.8, 42.9 (d, JC,F ) 7.5 Hz),
2
1
49.4, 59.1, 59.2, 68.9 (d, JC,F ) 20.1 Hz), 74.8, 84.3 (d, JC,F
)
170.7 Hz), 111.7, 117.4, 124.4, 133.9, 137.4, 154.0, 157.8, 181.6.
19F NMR (282 MHz, CDCl3): δ -231.7 (dt, 1F, JH,F ) 46.9 Hz,
2
3JH,F ) 18.1 Hz). HRMS (ESI-MicroTof): m/e 455.1257 (M + Na
+ CH3OH)+, calcd for C18H25FN2NaO7S 455.1259. HPLC tR
)
18.82 min (98.0%).
2
3
(m, 1H), 3.92 (dd, 1H, JHa,Hb ) 11.1 Hz, JHa,Hc ) 5.8 Hz), 4.25
(S)-1-(4-Fluoro-3-hydroxybutyl)-5-[1-(2-methoxymethylpyrro-
lidinyl)sulfonyl]isatin (32). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)-
sulfonyl]-1-[2-(oxiran-2-yl)ethyl]isatin (30) (90 mg, 0.236 mmol)
was converted to a mixture of two regioisomers (31 and 32) using
2
3
(dd, 1H, JHb,Ha ) 11.0 Hz, JHb,Hc ) 3.0 Hz), 4.90 (s, 2H),
6.90-6.93 (m, 3H), 7.25-7.30 (m, 2H), 7.98 (dd, 1H, 3JH,H ) 8.3
4
4
Hz, JH,H ) 1.9 Hz), 8.04 (d, 1H, JH,H ) 1.9 Hz). 13C NMR (150

Fluorinated Isatin DeriVatiVes. Part 2
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3493
Et3N·3HF (1.85 mL, 11.4 mmol), as described in the general
procedure B, and stirred for 8 h. The crude product was purified
by column chromatography (ethyl acetate:toluene 8:2) to yield pure
32, together with its mixture with the other regioisomer 31 as deep-
orange colored gummy solids. Yield (as mixture): 48 mg (71%).
Spectral data of the primary fluoride (32) is explained below. H
NMR (300 MHz, CDCl3): δ 1.63-1.97 (m, 6H), 2.60 (s, 1H),
3.04-3.17 (m, 1H), 3.33-3.48 (m, 2H), 3.36 (s, 3H), 3.60 (dd,
(ethyl acetate:toluene 7:3) to yield a deep golden-yellow colored
gummy solid. Yield: 48 mg (45%). H NMR (300 MHz, CDCl3):
δ 1.25 (s, 1H), 1.57-1.71 (m, 2H), 1.82-1.93 (m, 2H), 3.05-3.14
1
(m, 1H), 3.30-3.45 (m, 2H), 3.34 (s, 3H), 3.58 (dd, 1H, 2JHb,Ha
)
9.4 Hz, 3JHb,H ) 3.8 Hz), 3.68-3.75 (m, 1H), 4.06 (d, 2H, 3JHa+Hb,Hc
) 4.9 Hz), 4.20-4.30 (m, 1H), 4.60-4.67 (ddd, 1H, 2JHd,F ) 47.1
Hz, 2JHd,He ) 12.7 Hz, 3JHd,Hc ) 3.0 Hz), 4.63-4.70 (ddd, 1H, 2JHe,F
) 47.1 Hz, 2JHe,Hd ) 14.1 Hz, 3JHe,Hc ) 4.6 Hz), 4.91 (s, 2H), 6.91
(d, 2H, 3JH,H ) 8.6 Hz), 6.93 (d, 1H, 3JH,H ) 8.2 Hz), 7.29 (d, 2H,
1
2
3
1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.8 Hz), 3.73-3.76 (m, 1H),
3.89-4.12 (m, 3H), 4.24-4.31 (ddd, 1H, 2JHd,F ) 47.6 Hz, 2JHd,He
3
4
3JH,H ) 9.9 Hz), 8.00 (dd, 1H, JH,H ) 8.3 Hz, JH,H ) 1.8 Hz),
8.05 (d, 1H, 4JH,H ) 1.7 Hz). 13C NMR (75 MHz, CDCl3): δ 24.1,
28.8, 43.9, 49.4, 59.1, 59.2, 67.9 (d, 3JC,F ) 6.8 Hz), 68.9 (d, 2JC,F
) 20.3 Hz), 74.8, 83.5 (d, 1JC,F ) 169.7 Hz), 111.2, 115.2, 117.5,
124.5, 126.5, 129.2, 134.0, 137.4, 153.3, 157.8, 158.4, 181.9.19F
3
2
) 15.7 Hz, JHd,Hc ) 6.3 Hz), 4.44-4.52 (ddd, 1H, JHe,F ) 46.8
2
3
3
Hz, JHe,Hd ) 12.9 Hz, JHe,Hc ) 3.5 Hz), 7.18 (d, 1H, JH,H ) 8.2
4
3
Hz), 8.05 (d, 1H, JH,H ) 1.6 Hz), 8.11 (dd, 1H, JH,H ) 8.3 Hz,
4JH,H ) 1.6 Hz). 13C NMR (75 MHz, CDCl3): δ 24.1, 28.8, 29.5
3
2
2
(d, JC,F ) 6.1 Hz), 37.2, 49.4, 59.1, 59.2, 67.5 (d, JC,F ) 19.8
NMR (282 MHz, CDCl3): δ -232.8 (dt, 1F, JHd+He,F ) 47.0 Hz,
1
3JHc,F ) 18.5 Hz). HRMS (ESI-MicroTof): m/e 561.1676 (M +
Na + CH3OH)+, calcd for C25H31FN2NaO8S 561.1677. HPLC tR
) 25.32 min (98.3%).
Hz), 74.8, 86.3 (d, JC,F ) 170.3 Hz), 110.6, 117.4, 124.6, 134.0,
137.6, 153.5, 158.3, 181.8. 19F NMR (282 MHz, CDCl3): δ -228.1
2
3
(dt, 1F, JHd+He,F ) 46.8 Hz, JHc,F ) 17.5 Hz). HRMS (ESI-
MicroTof): m/e 469.1417 (M + Na + CH3OH)+, calcd for
C19H27FN2NaO7S 469.1415. HPLC tR ) 19.57 min (97.3%).
(S)-1-(10-Fluoro-11-hydroxyundecyl)-5-[1-(2-methoxymethylpyr-
rolidinyl)sulfonyl]isatin (34). (S)-5-[1-(2-Methoxymethylpyrrolidi-
nyl)sulfonyl]-1-[9-(oxiran-2-yl)nonyl]isatin (33) (110 mg, 0.22
mmol) was converted to a mixture of two regioisomers (34 and
35) using Olah’s reagent (0.1 mL, 0.447 mmol), as described in
the general procedure A, and stirred for 10 h. The crude product
was purified by column chromatography (ethyl acetate:cyclohexane
6:4) to yield 34 as deep-orange colored gummy solid. Yield: 60
(S)-1-[(S)-2,3-Dihydroxypropyl]-5-[1-(2-methoxymethylpyrro-
lidinyl)sulfonyl]isatin (40). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)-
sulfonyl]-1-[(S)-oxiran-2-ylmethyl]isatin (28) (80 mg, 0.21 mmol)
was converted to 40 using Olah’s reagent (0.48 mL, 2.1 mmol), as
described in the general procedure A, except that after stirring the
reaction mixture at 0 °C for 20 min, it was quenched with saturated
sodium bicarbonate solution and then vigorously stirred at ambient
temperature for 2 h. The crude product was purified by column
chromatography (5% MeOH/CH2Cl2) to afford a yellow colored
1
1
gummy solid. Yield: 43 mg (51%). H NMR (300 MHz, CDCl3):
mg (52%). H NMR (300 MHz, CDCl3): δ 1.30-1.95 (m, 20H),
δ 1.46 (s, 1H), 1.59-1.70 (m, 2H), 1.83-1.94 (m, 2H), 2.05 (s,
3.09-3.17 (m, 1H), 3.34-3.48 (m, 2H), 3.36 (s, 3H), 3.60 (dd,
2
3
1H), 3.06--3.14 (m, 1H), 3.30-3.46 (m, 2H), 3.36 (s, 3H), 3.58
1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.9 Hz), 3.64-3.79 (m, 5H),
2
3
3
(dd, 1H, JHb,Ha ) 9.4 Hz, JHb,H ) 3.9 Hz), 3.66-3.74 (m, 1H),
4.46-4.68 (m, 1H), 7.02 (d, 1H, JH,H ) 8.3 Hz), 8.04 (d, 1H,
3
4JH,H ) 1.7 Hz), 8.10 (dd, 1H, 3JH,H ) 8.3 Hz, 4JH,H ) 1.8 Hz). 13
C
3.85-4.01 (m, 2H), 4.24-4.64 (m, 3H), 7.26 (d, 1H, JH,H ) 8.1
Hz), 7.99-8.06 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 24.1, 28.8,
43.0, 49.3, 59.1, 59.2, 68.7, 69.0, 74.8, 111.7, 117.4, 124.3, 133.8,
137.4, 154.1, 158.6, 181.7. HRMS (ESI-MicroTof): m/e 421.1034
(M + Na)+, calcd for C17H22N2NaO7S 421.1040. HPLC tR ) 17.68
min (99.8%).
3
NMR (75 MHz, CDCl3): δ 24.1, 24.9 (d, JC,F ) 4.1 Hz), 26.8,
2
27.2, 28.8, 29.1, 29.2, 29.3, 29.4, 30.9 (d, JC,F ) 20.4 Hz), 40.7,
2
1
49.4, 59.1, 59.2, 65.1 (d, JC,F ) 21.8 Hz), 74.8, 94.7 (d, JC,F
)
167.6 Hz), 110.4, 117.4, 124.6, 133.7, 137.5, 153.7, 157.8, 182.1.
19F NMR (282 MHz, CDCl3): δ -189.4 to -189.9 (m, 1F, JHc,F
2
3
3
3
) 49.9 Hz, JH,F ) 16.5 Hz, JH,F ) 22.9 Hz, JH,F ) 29.3 Hz).
HRMS (ESI-MicroTof): m/e 535.2248 (M + Na)+, calcd for
C25H37FN2NaO6S 535.2249. HPLC tR ) 30.75 min (100%).
(S)-1-[4-(1-Fluoro-2-hydroxyethyl)benzyl]-5-[1-(2-methoxymeth-
ylpyrrolidinyl)sulfonyl]isatin (37). (S)-5-[1-(2-Methoxymethylpyr-
rolidinyl)sulfonyl]-1-[4-(oxiran-2-yl)benzyl]isatin (36) (90 mg,
0.236 mmol) was converted to 37 using Et3N·3HF (0.27 mL, 1.18
mmol), as described in the general procedure B, and stirred for
2 h. The crude product was purified by column chromatography
(ethyl acetate:toluene 7:3) to yield a deep golden-yellow colored
(S)-1-(3,4-Dihydroxybutyl)-5-[1-(2-methoxymethylpyrrolidinyl)-
sulfonyl]isatin (41). A crude mixture of (S)-5-[1-(2-methoxymeth-
ylpyrrolidinyl)sulfonyl]-1-[2-(oxiran-2-yl)ethyl]isatin (30) (50 mg,
0.126 mmol) was converted to 41 by absorbing it on silica gel and
then loading it on a silica gel column packed in CH2Cl2:MeOH
(95:5) as the solvent system. The mass was then allowed to remain
on the column for 8 h and then eluted with CH2Cl2:MeOH (95:5)
1
to afford a yellow colored gummy solid. Yield: 29 mg (55%). H
NMR (300 MHz, CDCl3): δ 1.25 (s, 1H), 1.65-1.95 (m, 6H),
2.27-2.32 (m, 1H), 3.09-3.17 (m, 1H), 3.35-3.53 (m, 3H), 3.37
(s, 3H), 3.60 (dd, 1H, 2JHb,Ha ) 9.4 Hz, 3JHb,H ) 3.9 Hz), 3.65-3.77
(m, 3H), 3.84-3.93 (m, 1H), 4.02-4.12 (m, 1H), 7.19 (d, 1H, 3JH,H
) 8.3 Hz), 8.05 (d, 1H, 4JH,H ) 1.7 Hz), 8.11 (dd, 1H, 3JH,H ) 8.3
1
solid. Yield: 48 mg (51%). H NMR (300 MHz, CDCl3): δ 1.25
(s, 1H), 1.61-1.72 (m, 2H), 1.83-1.91 (m, 2H), 3.07-3.12 (m,
2
1H), 3.32-3.44 (m, 2H), 3.34 (s, 3H), 3.57 (dd, 1H, JHb,Ha ) 9.4
Hz, 3JHb,H ) 3.9 Hz), 3.68-3.97 (m, 3H), 4.98 (s, 2H), 5.46-5.66
4
Hz, JH,H ) 1.9 Hz). 13C NMR (75 MHz, CDCl3): δ 24.1, 28.8,
2
3
3
(ddd, 1H, JHa,F ) 48.3 Hz, JHa,Hb ) 6.8 Hz, JHa,Hc ) 3.6 Hz),
6.90 (d, 1H, 3JH,H ) 8.2 Hz), 7.30 (d, 2H, 3JH,H ) 8.2 Hz), 7.41 (d,
2H, 3JH,H ) 8.2 Hz), 8.00 (dd, 1H, 3JH,H ) 8.3 Hz, 4JH,H ) 1.9 Hz),
8.05 (d, 1H, 4JH,H ) 1.6 Hz). 13C NMR (75 MHz, CDCl3): δ 24.1,
30.4, 37.4, 49.4, 59.1, 59.2, 66.4, 68.9, 74.8, 110.7, 117.4, 124.6,
134.0, 137.6, 153.5, 158.4, 181.8. HRMS (ESI-MicroTof): m/e
467.1462 (M + Na + CH3OH)+, calcd for C19H28N2NaO8S
467.1459. HPLC tR ) 15.70 min (96.4%).
2
28.8, 44.1, 49.3, 59.1, 59.2, 66.3 (d, JC,F ) 24.5 Hz), 74.8, 92.2
Radiosynthesis of [18F]Triethylamine trihydrofluoride ([18F]Et3N-
·3HF). For the preparation of 2-deoxy-2-[18F]fluoro-D-glucose
([18F]FDG), no-carrier-added aqueous [18F]fluoride ions were
produced on an RDS 111e cyclotron (CTI-Siemens) by irradiation
of a 1.2 mL water target using 10 MeV proton beams on 97.0%
enriched [18O]water by the 18O(p,n)18F nuclear reaction. After
discharging the cyclotron target, it was rinsed with water (1.2 mL)
for injection and this rinsed batch of aqueous [18F]fluoride ions was
also used for the preparation of [18F]Et3N·3HF. This batch was
transferred to a polypropylene (PP) tube, and the water was carefully
distilled off at 120 °C in vacuo. Then, Et3N·3HF (2.0 µL, 2.0 mg,
12.5 µmol) and acetonitrile (10 µL) were added and the mixture
was heated to 60 °C for 35 min in an ultrasound bath. The solution
was used for the next step without further purification.
1
3
(d, JC,F ) 172.6 Hz), 111.1, 117.5, 124.6, 126.6 (d, 2C, JC,F
)
)
5
2
7.1 Hz), 127.8, 134.2, 134.4 (d, JC,F ) 1.4 Hz), 137.0 (d, JC,F
20.0 Hz), 137.4, 153.1, 157.8, 181.7. 19F NMR (282 MHz, CDCl3):
2
3
3
δ -186.5 (ddd, 1F, JHa,F ) 48.8 Hz, JHb,F ) 19.5 Hz, JHc,F
)
2
3
28.2 Hz), -187.4 (ddd, 1F, JHa,F ) 48.6 Hz, JHb,F ) 20.7 Hz,
3JHc,F ) 28.2 Hz). HRMS (ESI-MicroTof): m/e: 531.1565 (M +
Na + CH3OH)+, calcd for C24H29FN2NaO7S 531.1572. HPLC tR
) 23.82 min (97.7%).
(S)-1-[4-(3-Fluoro-2-hydroxypropoxy)benzyl]-5-[1-(2-methoxym-
ethylpyrrolidinyl)sulfonyl]-isatin(40). (S)-5-[1-(2-Methoxymeth-
ylpyrrolidinyl)sulfonyl]-1-[4-(oxiran-2-ylmethoxy)benzyl]isatin (38)
(90 mg, 0.236 mmol) was converted to 39 using Et3N·3HF (0.27
mL, 1.18 mmol), as described in the general procedure B, and stirred
for 16 h. The crude product was purified by column chromatography

3494 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Podichetty et al.
Radiosynthesis of (S)-1-[4-(1-[18F]Fluoro-2-hydroxyethyl)ben-
zyl]-5-[1-(2-methoxymethyl-pyrrolidinyl)sulfonyl]isatin [18F]37. The
[18F]Et3N·3HF solution was transferred to a PP tube containing
(S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-1-[4-(oxiran-2-yl)-
benzyl]isatin (36) (6.5 mg, 14.2 µmol). The tube was sealed, and
the mixture was stirred for 45 min at 120 °C. The reaction was
then quenched by adding 1 mL of acetonitrile and diluted with a
10 mL injection of water. The mixture was passed through a Waters
Sep-Pak Light C18 cartridge. The cartridge was washed with
additional 10 mL of water injection and eluted with 0.5 mL hot
DMF (warmed to 120 °C before elution). The eluate was diluted
with 0.5 mL injection of water and purified by radio-RP-HPLC
(flow ) 4 mL/min; eluents: A, CH3CN/H2O/TFA, 950/50/1; B,
CH3CN/H2O/TFA, 50/950/1; time program: eluent B from 70% to
10% in 35 min, from 10% to 70% in 5 min). The product fraction
of compound [18F]37 (retention time tR([18F]37) ) 18.6 min) was
evaporated to dryness in vacuo and redissolved in 1 mL of H2O:
EtOH (9:1 v/v). Product compound [18F]37 was obtained in a
radiochemical yield of 7%. Radiochemical purity was >95% and
the specific activity was <1 GBq/µmol at the end of synthesis,
considering a synthesis time of 220 min. The chemical identity of
[18F]37 was proven by HPLC on the above-mentioned gradient
system providing coelution of [18F]37 and its nonradioactive
counterpart 37 that was added to the product fraction beforehand.
In Vitro Enzyme Inhibition Assays (Table 1). The binding
potencies of compounds 1-22 and 28-41 were assayed for
recombinant human caspases-1, -3, -6, and -7 (Alexis Biochemicals
(Switzerland)) using their peptide-specific substrates (Alexis Bio-
chemicals (Switzerland)) Ac-YVAD-AMC (Ac-Tyr-Val-Ala-Asp-
AMC, caspase-1), Ac-DEVD-AMC (Ac-Asp-Glu-Val-Asp-AMC,
caspase-3), Ac-VEID-AMC (Ac-Val-Glu-Ile-Asp-AMC, caspase-
6), and Ac-DEVD-AMC (Ac-Asp-Glu-Val-Asp-AMC, caspase-7)
as already described.30,31 The enzymatic activity of the caspases
was determined by measuring the accumulation of the cleaved
fluorogenic product AMC (7-amino-4-methylcoumarin). Reaction
rates showing inhibitory activity of the nonradioactive model
inhibitor were measured with a Fusion universal microplate analyzer
(PerkinElmer) at excitation and emission wavelengths of 360 and
460 nm, respectively. All assays were performed at a volume of
200 µL at 37 °C in reaction buffer.30,27 Buffers contained the
nonradioactive compounds 1-22 and 28-41 in DMSO in single
doses (end concentrations 500 µM, 50 µM, 5 µM, 500 nM, 50 nM,
5 nM, 500 pM, 50 pM, or 5 pM). Recombinant caspases were
diluted into the appropriate buffer to a concentration of 0.5 units
per assay () 500 pmol substrate conversion after 60 min). After
10 min incubation time, the peptide substrates (end concentration
10 µM) were added and reacted for further 10 min. The IC50 values
were determined by nonlinear regression analysis using the
XMGRACE program (Linux software).
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Acknowledgment. This study was supported by grants from
the Deutsche Forschungsgemeinschaft (DFG), Sonderfors-
chungsbereich 656, Mu¨nster, Germany (projects A3 and B1),
and the European Institute of Molecular ImagingsEIMI,
Mu¨nster. A.K.P. is grateful to the International NRW Graduate
School of Chemistry for a stipend.
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Supporting Information Available: Experimental procedures
and spectroscopic data of starting materials and assignment of
spectroscopic data of all synthesized compounds. This material is
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