Identification and further development of thiazolidinones spiro-fused to indolin-2-ones as potent and selective inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase B

Article abstract of DOI:10.1016/j.tet.2011.04.026

Tuberculosis continues to be a major cause of morbidity and mortality throughout the world. Protein tyrosine phosphatases from Mycobacterium tuberculosis are attractive targets for developing novel strategies in battling tuberculosis due to their role in

Full text of DOI:10.1016/j.tet.2011.04.026

Tetrahedron 67 (2011) 6713e6729
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Identification and further development of thiazolidinones spiro-fused
to indolin-2-ones as potent and selective inhibitors of Mycobacterium
tuberculosis protein tyrosine phosphatase B
,
b
a
,
b
a
b,c
Viktor V. Vintonyak a, Karin Warburg a , Bjorn Over , Katja Hubel , Daniel Rauh
,
€
€
,b,
Herbert Waldmann a
*
a Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany
b
€
Technische Universitat Dortmund, Chemical Biology, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany
c Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis continues to be a major cause of morbidity and mortality throughout the world. Protein
tyrosine phosphatases from Mycobacterium tuberculosis are attractive targets for developing novel
strategies in battling tuberculosis due to their role in the intracellular survival of M. tuberculosis in
various infection models. Here, we report on the identification and further development of thiazolidi-
nones spiro-fused to indolin-2-ones as a new class of potent and selective inhibitors of M. tuberculosis
protein tyrosine phosphatase B. Detailed structureeactivity relationship (SAR) studies revealed that
a nitro-substituted 2-oxoindole core together with a dihalogenated anilide and a halogenated N-benzyl
moiety are essential for strong inhibitory activity against MptpB (M. tuberculosis protein tyrosine
phosphatase B). Small structural modification of the identified compounds led to significant improve-
ment of compound solubility and cell permeability retaining inhibitory activity in the micromolar range.
The configuration of the spiro-center was found to be crucial for the inhibitory activity and the sepa-
ration of the racemate revealed the R-(ꢀ)-enantiomers as the biologically active component. The re-
Received 15 February 2011
Received in revised form 4 April 2011
Accepted 7 April 2011
Available online 14 April 2011
Keywords:
Chemical biology
Enzyme inhibitors
Medicinal chemistry
Molecular modeling
ported MptpB inhibitors show excellent selectivity against
a selected panel of protein tyrosine
phosphatases, including MptpA (M. tuberculosis protein tyrosine phosphatase A), PTP1B (protein tyrosine
phosphatase 1B), SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase), PTPN2,
h-PTP
b (human protein tyrosine phosphatase b), and VHR (Vaccinia virus VH1-related dual-specific
protein phosphatase) and further highlight the identified thiazolidinones spiro-fused to indolin-2-ones
as a promising class of new compounds that might prove useful for chemical biology research to dis-
sect MptpB function and eventually foster the development of next generation antibiotics.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
of host proteins that are involved in the interferon signaling, which
represents a crucial pathway of the immune system.2 The genetic
Tuberculosis (TB) continues to be a major cause of morbidity and
mortality throughout the world. According to the World Health
Organization, one-third of the world’s population is infected with
Mycobacterium tuberculosis and about 35 million people are
expected to die from TB in the first 20 years of this century.1 Be-
cause of the increasing occurrence of drug-resistant mycobacteria
and the need of the extended use of current drugs, new targets and
drugs for therapeutic interventions are in high demand. M. tuber-
culosis protein tyrosine phosphatase A (MptpA) and MptpB are two
enzymes secreted by growing mycobacteria and believed to me-
diate M. tuberculosis survival in macrophages by dephosphorylation
knock-out of MptpB suppresses growth of M. tuberculosis in acti-
vated macrophages and guinea pigs and suggests that MptpA/B
could qualify as potential drug targets in the treatment of TB.3 The
importance of MptpB to the intracellular survival of M. tuberculosis
was recently confirmed by two independent studies in which
specific inhibitors directed against MptpB were shown to impair
mycobacterial survival in murine macrophages.4 Since MptpB has
no direct human orthologues, inhibitors active against MptpB
might not only prove useful as probe molecules in chemical biology
research to dissect the functional role of phosphatases in cell in-
vasion, but also offer unique opportunities for the development of
new antitubercular drugs.
Compounds with anti MptpA and MptpB activity have been
* Corresponding author. Tel.: þ49 (0) 231 133 2400; fax: þ49 (0) 231 133 2499;
e-mail address: herbert.waldmann@mpi-dortmund.mpg.de (H. Waldmann).
successfully identified and characterized in vitro.5 In particular, the
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.04.026

6714
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
H
O3S
H
N
O
S
S
O
S
H
O
F
C
3
O
Ph
N
N
O
N
O
F
Ph
O
O
l
C
Ph
H
O
1
3
( )
S
MT
2
O
I
0.36 µM
Ki 0.22 µM
C50
I
0.44 µM
C50
F
O
O
H
N
N
O
O
O
N
N
N
N
N
O
S
N
O
NH
H
O
H
O
N
4
O
H
O
O
4
5
Ki 0.15 µM
0.64 µM
I
1.26 µM
C50
I
C50
Fig. 1. Structures of selected MptpB inhibitors, IC50 or Ki values are given.
application of biology-oriented synthesis (BIOS) approaches resul-
ted in the identification of indole derivative 1 (Fig. 1), as potent and
specific inhibitor of MptpB.6 Recently Alber et al. reported the de-
velopment of a potent and selective (oxalylamino-methylene)-
thiophene sulfonamide inhibitor for MptpB (2) (OMTS).7 Compound
2 shows an IC50 value of 440ꢁ50 nM and >60-fold selectivity for
MptpB over six human PTPs. Application of a substrate-based frag-
ment approach resulted in the discovery of the isoxazole 3, which is
one of the most potent MptpB inhibitors known from literature to
date (Ki of 220 nM).8 Using the structure of isoxazole 3 as a starting
point, Yao et al. recently synthesized and screened a click chemistry-
based library of MptpB inhibitors and identified 4 as the most active
can be expected to display exceedingly poor cell permeability and
low oral bioavailability. Indeed, compound 4 is not cell permeable
or its cell permeability cannot be determined.9 After all, the dis-
covery of potent cell permeable and orally bioavailable MptpB in-
hibitors is a challenging task of medicinal chemistry research. New
inhibitor classes with good selectivity profiles and improved
pharmacological properties are therefore in high demand.
Recently we communicated on the identification of the indolin-
2-on-3-spirothiazolidinones as a novel class of potent and selective
inhibitors of MptpB.10 In the present study we present detailed
systematic structureeactivity relationship (SAR) studies, in-
vestigations of the inhibition profiles, and biochemical evaluation
toward the mode of action of the early discovered compounds as
well as molecular docking study.
representative with a Ki value of 0.15 mM for MptpB but only mod-
erate selectivity when screened against PTP1B, TCPTP (T-cell protein
tyrosine phosphatase), Yop-H (Yersinia protein tyrosine phospha-
tase), and LMW-PTP (low molecular weight protein tyrosine phos-
phatase).9 Recently Zhou et al. identified a potent and selective
MptpB inhibitor 5 with significant cellular activity, from a combi-
natorial library of bidentate benzofuran salicylic acid derivatives
assembled by click chemistry.4b
2. Results and discussion
Using enzyme activity assays we screened a library of 40,000
compounds to discover inhibitors of MptpB.6a In a 384-well HTS
format we used p-nitrophenyl phosphate (p-NPP) as the substrate
and identified 6e9 as a series of compounds that perturbed sub-
strate hydrolysis by MptpB in the mid-micromolar range (Fig. 2).
Remarkably, the aforementioned inhibitors all contain highly
polar acidic groups that are highly ionized at physiologic pH and
F
OMe
O
Cl
F
MeO
O
O
O
O
N
H2N
N
O
S
SO2
O
SO2
O
O
O
N
O
N
N
N
H
F
F
NO2
NO2
6
7
8
9
IC50 20 2.5 µM
IC50 29 3.1 µM
IC50 22 2.4 µM
IC50 30 3.4 µM
Fig. 2. Structures and IC50 values of primary hits derived from HTS against MptpB.

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6715
Interestingly, 6 and 7 showed excellent selectivity against a se-
lected panel of PTP (Cdc25A, PTP1B, MptpA, VHR, PP1 (protein
phosphatase 1), SHP-2, VE-PTP (vascular endothelial protein tyrosine
phosphatase), PTPN2) and, to the best of our knowledge, were not
reported as phosphatase inhibitors before. Moreover, 6 and 7 contain
a 2-oxindole moiety, which is found in a large number of natural
products with broad spectrum biological activity. We concluded that
these compounds might represent biologically prevalidated starting
points for further compound development as their underlying scaf-
foldsweremostlikelyselectedbyevolutionassuitablecorestructures
for protein binding.11,12 We focused on the indolin-2-on-3-
spirothiazolidinone scaffold to develop further compound libraries
with improved affinities and to investigate the relevance of the con-
figuration of the spiro-center for biological activity.
indolin-2-on-3-spirothiazolidinones 12, 16, and 17. The obtained
sulfides 12, 16, and 17 were readily oxidized to the corresponding
sulfones 13, 18, and 19 with 5 equiv of mCPBA. Remarkably, the
reaction of 12ac with a slight excess of mCPBA (1.1 equiv) in CHCl3
at 0 ꢂC resulted in exclusive formation of sulfoxide 14 as a single
diastereomer (dr>20:1). In order to improve the solubility of the
final compounds, the tert-butyl ester of 19b was removed and the
obtained acid 20 was subsequently coupled to different aliphatic
amines using standard techniques to provide the corresponding
amides 21aed in excellent yield.
2.2. In vitro characterization of first generation substituted
indolin-2-on-3-spirothiazolidinone
To explore how chemical modifications of the indolin-2-on-3-
spirothiazolidinones are tolerated by MptpB, we measured en-
zyme inhibition by using p-NPP as the substrate and observing the
release of p-nitrophenol at 405 nm (Table 1). We observed a sig-
nificant (up to 23-fold) increase in potency in the measured IC50
values when compared to the primary screening hits 6 and 7.
Briefly, the kinetics clearly demonstrate that (a) the halogenated N-
benzyl substituent on the 1-position of the oxoindole is needed to
retain activity while (b) a nitro group in the 5-position of the
oxoindole core significantly enhances potency against MptpB.
One of the trends observed in the indolin-2-on-3-spirothia
zolidinone series was the crucial role of the substituted N-benzyl
fragment for activity against MptpB (Table 1). Compounds lacking
any substituent at the 2-oxindole nitrogen (18aef) or bearing ali-
phatic alkyl groups (19, 20, and 21) were inactive. Another impor-
tant trend was observed for substituents in the 2-oxidole core.
Much to our surprise, the introduction of a nitro group in the 5-
position of 2-oxindole core (13y) enhanced potency (17-fold)
against MptpB while its replacement with a methoxy (13z) or
2.1. Design and synthesis of first generation library based on
the primary hits
Given the moderate activities of the identified primary hits 6
and 7 in inhibiting phosphatase activity, we set out to improve
potency against MptpB and to obtain proper SAR of the newly
identified inhibitor scaffold. We followed a classical medicinal
chemistry approach and identified the anilide substituent of the
thiazolindinone, the N-alkyl substituent on the 2-oxo-indole as
well as the thiazolidinone moiety and the 2-oxo-indole inhibitor
core as suitable positions for chemical modifications (Fig. 3).
Our synthesis commenced with the alkylation of commercially
available 1H-indole-2,3-diones 10 and 10a with different alkyl and
benzyl halides and provided
a small focused library of N-
substituted isatins 11 and 15 (Scheme 1). Condensation of 11 and 15
and the unprotected isatin 10 with various anilines resulted in the
formation of isatin-3-imines, which subsequently were directed to
cyclocondensation with mercaptoacetic acid to provide substituted
R1
F
F
O
F
F
O
O
b) modification of
2-oxo-indole core
a) modification of
N
the anilide moiety
N
N
SO2
O
R2
SO2
O
SO2
O
N
N
N
F
F
F
6
d) synthesis of
sulfoxide and
sulfide analogues
c) modification
of N-alkyl fragment
F
F
F
F
F
F
O
O
O
N
N
N
N
SO2
O
SOn
O
SO2
O
and
N
N
R4
n = 0, sulfide
F
n = 1, sulfoxide
R3
R4 = H, Alk
Fig. 3. Potential positions for modification of primary hit 6.

6716
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
O
N
O
R1
R2
O
R2
N
R2
a
b
S
O
O
N
O
(80–95%)
(45–82%)
N
H
10
(R2 = H, Me, F,
OMe, NO2, Cl)
R3
R3
O
11 (R3 = 2-F, 3-F, 4-Me,
12
3-CF3, 4-NO2)
F
F
O
O
R1
R1
R2
N
S
N
N
R2
S
SO2
O
O
c
d
O
O
N
N
N
(87%)
(75–94%)
12ac R1 = 3,4-diF
R2 = H
F
R3 = 3-F
R3
R3
14
12
13
d.r. > 20:1
O
O
O
O
R1
R2
a
b
(52–80%)
N
O
S
N
(75–81%)
N
O
H
R4
N
10a
R4
15a,
R4 = Me
16
(R4 = H)
15b, R4 = CH2CO2tBu
17 (R4 = Me, CH2CO2tBu)
F
F
(85-93%
O
c
F
F
O
O
f
R1
R2
N
N
SO2
O
SO2
O
N
(65–87%)
SO2
O
N
N
N
CH2CO2R
O
R4
19b R = tBu
NR'R''
e
18 (R4 = H)
19
(95%)
(R4 = Me, CH2CO2tBu)
20, R = H
21a R'R''NH = N-Me piperazine
21b R'R''NH = morpholine
21c R'R''NH = N-(2-Hydroxyethyl)piperazine
21d R'R''NH = propargylamine
Scheme 1. Preparation of the first generation library based on the primary hit 6. Reagents and conditions: (a) NaH, DMF, 0 ꢂC, then ArCH2Br or AlkeHal; (b) (1) Ar0NH2, EtOH, reflux,
6 h; (2) mercaptoacetic acid, toluene, reflux, 16 h; (c) mCPBA (5 equiv), CHCl3, rt, 24 h; (d) mCPBA (1.1 equiv), CHCl3, 0 ꢂC, 1 h; (e) TFA, DCM, 0 ꢂC, 2 h; (f) R0R00NH, HBTU, HOBt, DIPEA,
DMF.
methyl group (13aa) resulted in inactive compounds. Additionally,
the halide substitution pattern of the anilide fragment turned out to
be important as well. Compounds possessing two fluorine or
fluorine and chlorine atoms in the meta- and para-position of the
anilide fragment (6, 13g, 13o, 13p, 13x, 13y, and 13ab) are preferred
over analogues bearing mono- (13aed, 13h, 13n, and 13w) or dia-
lkylsubstituents. The sulfide intermediate 12 did not show any in-
2.3. Synthesis of the second generation indolin-2-on-3-
spirothiazolidinones
To build on the initial SAR of indolin-2-on-3-spirothiazolidinone
substituted at the 1- and 30-positions and the finding that a nitro
group at the 5-position is crucial for affinity, we set out to further
explore this position. For this purpose, carbomethoxy, sulfonamide,
and trifluoromethoxy groups were chosen. Substituted isatins
25e30 were prepared as illustrated in Scheme 2. Preparation of 24a
hibitory activity, while sulfoxide 14 (IC50, 13.3
twice as active, as the corresponding sulfone 6.
mM) was almost

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6717
Table 1
IC50 values of indolin-2-on-3-spirothiazolidinones are shown for MptpBa
O
O
Ar1
Ar1
N
N
R2
R2
SO2
O
SO2
O
N
N
R4
R3
IC50
Compd
Ar1
R2
H
R3 or R4
,
m
M
Compd
Ar1
R2
H
R3 or R4
IC50
, mM
F
6
R3¼3-F
20.0ꢁ2.5
13v
R3¼3-F
n.a.
F
MeO
7
H
H
H
H
H
H
H
H
R3¼3-F
29.0ꢁ3.1
n.a.
13w
13x
13y
13z
H
R3¼3-F
R3¼4-NO2
R3¼3-F
R3¼3-F
R3¼3-F
R3¼4-NO2
R3¼3-F
R4¼H
n.a.
MeO
F
F
F
F
F
F
13a
13b
13c
13d
13e
13f
13g
R3¼4-Me
R3¼4-Me
R3¼2-F
H
22.4ꢁ1.8
1.2ꢁ0.2
n.a.
F
F
F
F
F
F
F
n.a.
NO2
OMe
Me
Cl
n.a.
R3¼3-CF3
R3¼3-F
n.a.
13aa
13ab
14b
n.a.
32.8ꢁ2.4
35.5ꢁ3.3
22.8ꢁ2.7
21.1ꢁ3.1
13.3ꢁ2.2
n.a.
F
F
R3¼3-F
H
Cl
Cl
R3¼3-F
18a
H
F
13h
13i
H
H
R3¼3-F
R3¼3-F
n.a.
n.a.
18b
18c
H
H
R4¼H
R4¼H
n.a.
n.a.
F3C
F
MeO
F
O
F
F
F
O
F
F
13j
F
H
R3¼3-F
29.4ꢁ2.1
18d
OMe
R4¼H
n.a.
F
Br
F
F
13k
13l
H
H
R3¼3-F
R3¼3-F
n.a.
n.a.
18e
18f
F
R4¼H
R4¼H
n.a.
n.a.
F
F
F
O
S
O
H2N
NO2
(continued on next page)

6718
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
Table 1 (continued )
Compd
Ar1
R2
R3 or R4
IC50
,
mM
Compd
Ar1
R2
R3 or R4
IC50, mM
O
S
O
H2N
13m
13n
H
H
R3¼3-F
R3¼3-F
n.a.
n.a.
19a
19b
H
H
R4¼Me
n.a.
n.a.
F
OtBu
F
O
F
F
MeO2C
13o
H
H
H
R3¼4-Me
R3¼3,4-diF
R3¼3-F
27.4ꢁ3.7
22.5ꢁ1.9
28.0ꢁ3.1
19c
19d
20
H
H
H
R4¼Me
n.a.
n.a.
n.a.
F
F
F
13p
R4¼Me
F
F
F
H2NO2S
OH
13q
13r
O
N
F
F
Cl
Cl
N
O
H
H
H
R3¼3-F
24.8ꢁ2.2
32.2ꢁ3.2
33.0ꢁ2.8
21a
21b
21c
H
H
H
n.a.
n.a.
n.a.
O
F
F
F
F
N
13s
13t
R3¼3-CF3
R3¼2-F
O
F
F
F
F
N
N
OH
O
F
F
F
F
H
N
13u
F
R3¼3-F
n.a.
21d
H
n.a.
O
n.a¼not active (no inhibition up to a concentration of 100
mM).
a
All IC50 values were calculated from at least three independent measurements.
Sulfoxide.
b
was started from ethyl 4-aminobenzoate 22a using reported pro-
cedures.13
-Isonitrosoacetanilide, obtained by reaction of ethyl 4-
(Scheme 3). Derivatives 40 and 45, bearing a tert-butyl acetate were
subjected to acid mediated deprotection. This transformation de-
livered carboxylic acids 46 and 47.
The newly synthesized indolin-2-on-3-spirothiazolidinones
36e47 were investigated for their inhibitory effects on MptpB
(Tables 2 and 3).
a
aminobenzoate, hydroxylamine hydrochloride and chloral hydrate,
was subsequently cyclized into isatin-5-carboxylic acid 23a and the
carboxylic function was then transformed into the corresponding
methyl ester 24a. In a similar way, starting with 4-amino-
benzenesulfonamide 22b isatin 23b could be prepared in respect-
able yield. In order to enlarge our building blocks library,
commercially available isatins 10 as well as synthesized derivatives
23b and 24a were alkylated with different benzyl bromides.
Methoxy substituted derivatives 27 were selected for further
derivatisation. Demethylation mediated by boron tribromide
afforded 5-hydroxy isatins 29, which afterward were subjected to
alkylation with tert-butyl bromoacetate. The tert-butyl group could
be easily removed on the final step of the synthesis to provide
corresponding hydroxyacetic acid derivatives.
N-Benzylated isatins were then converted into the corre-
sponding isatin-3-imines and subsequently cyclized into indolin-2-
on-3-spirothiazolidinone 31e35 following established procedures
outlined above. Since the sulfoxide 14 showed higher activity
compared to the corresponding sulfone 6, we decided to further
explore this SAR and synthesized a small focused library of sulf-
oxides. Consequently, the obtained sulfides 31e35 were converted
into the corresponding sulfones 36e40 and sulfoxides 41e45
Our initial SAR (Table 1) showed that introduction of a nitro
group into the 5-position of the 2-oxindole core (13y,
IC50¼1.2ꢁ0.2
mM) significantly enhances the inhibitory activity. The
measured IC50 values for the second generation of compounds
further highlight this trend.
We next set out to investigate the role of the substituted N-
benzyl fragment and synthesized 13 analogues of 13y, displaying
different substituents in the phenyl ring (Scheme 3). IC50 values
showed that replacement of the fluorine with chlorine (39an) or
a trifluoromethyl group (39ao) as well as relocation of the fluorine
to the para-position of the phenyl ring did not improve potency
when compared to 13y. Surprisingly, substitution of the para-
fluorine with chlorine (39l) or bromine (39ah) resulted in two and
threefold increase in potency and highlight that the strength of the
interactions attenuates in the order Br>Cl>F. This observation is
perfectly in line with characteristics discovered within small model
halogen-bonded systems.14 Replacement, of the para-fluorine with
a cyano group (39al) was well tolerated and resulted in a slight

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6719
O
O
O
R2
R2
a
b
O
O
O
R2 = CO2H
(65%)
N
N
NH2
H
H
22a R2 = CO2Et
23a R2 = CO2H (51%)
24a
22b R2 = SO2NH2
23b R2 = SO2NH2 (45%)
O
O
25a R2 = CO2Me, R3 = 4-F
25b R2 = CO2Me, R3 = 4-Cl
R2
R2
c
O
O
26
R2 = SO2NH2, R3 = 3-F
(51–93%)
N
N
H
27a R2 = OMe, R3 = 4-F
27b R2 = OMe, R3 = 4-Cl
28
10
(R2 = H, Me, F, OMe,
NO2, Cl, OCF3)
23b R2 = SO2NH2
R2 = H, Me, F, Cl, OCF3, NO2
R3 = 4-F, 4-Cl, 4-Br, 4-CN, 4-Ph,
4-OMe, 3-Cl, 3-CF3, 2-Cl-6-F
R3
24a
R2 = CO2Me
O
O
O
MeO
HO
O
e
d
O
O
O
N
N
N
(75-81%)
(85-89%)
O
O
R3
R3
R3
30a
29a
R3 = F
R3 = F
30b R3 = Cl
27a
R3 = F
29b R3 = Cl
27b R3 = Cl
Scheme 2. Synthesis of an isatin library. Reagents and conditions: (a) (1) CCl3(OH)2, NH2OH$HCl, H2SO4; (2) H2SO4(concd), 90 ꢂC; (b) EDC, DMAP, MeOH, rt; (c) NaH, DMF, 0 ꢂC, then
ArCH2Br; (d) BBr3, DCM, 0 ꢂC; (e) NaH, DMF, 0 ꢂC, then BrCH2CO2tBu.
improvement in potency when compared to 39g. On the other
hand, introduction of para-hydroxy (39ai) and methoxy groups
(39ag) resulted in a two to sixfold decrease in activity. Notably, the
replacement of the N-benzyl fragment by 5-methylisoxazole (39av)
was tolerated but did not improve potency of the compound when
compared to 39g. To further explore the role of the dihalogenated
anilide fragment for MptpB inhibitory activity we synthesized an-
alogues with different substituents in this part of the molecule and
found that the substitution of the meta-fluorine with chlorine was
well tolerated. On the other hand, introduction of alkyl substituents
in the anilide moiety resulted in complete loss of inhibitory activity
(Table 2). From these results we concluded that a nitro-substituted
2-oxoindole core in combination with a dihalogenated anilide
moiety, as well as a halogenated N-benzyl fragment are pre-
requisites for strong inhibitory activity against MptpB.
moiety and a halogen-substituted N-benzyl fragment are essential
for high inhibitory activity of the described sulfoxides. Since sulf-
oxides bearing alkyl residues in para-position of the anilide frag-
ment and different polar substituents in the 2-oxindole core also
show significant inhibitory activity, one can speculate that such
a high tolerance to the nature of substituents in the sulfoxide series
is due to the presence of the highly polarized SeO bond, which may
play a crucial role as hydrogen bond acceptor to interact with the
active site of MptpB. However, further studies to investigate the
sulfoxides including co-crystallization experiments with target
phosphatases failed due to their instability in solution. We hy-
pothesize that the instability results from ring opening of the
thiazolidinone to form the sulfenic acid, which is likely to de-
compose further.
2.5. Additional SAR study in the sulfone series
2.4. SAR trends in the sulfoxide series
To further explore the SAR around the lead structure 13y, we
reduced the nitro group in the 5-position to the resulting amine 48,
which was then converted to the corresponding acetamide 49a and
carbamate 49b (Scheme 4). In addition, we were interested in de-
termining the role of the amide carbonyl for inhibitory activity
against MptpB. To this end, sulfone 13y was efficiently converted
into the corresponding bis-thioamide 50 by refluxing with Law-
esson’s reagent in toluene. Biological evaluation showed that
transformation of the nitro derivative 13y into its corresponding
amine 48, acetamide 49a, and carbamate 49b is not tolerated by
MptpB and leads to a decrease in activity (Table 4). We attribute the
moderate activity of the thioamide derivative 50 to the importance
of carbonyl groups in 13y, which may serve as hydrogen bond
acceptors.
As a general trend in the sulfoxide series we observed a high
tolerance toward the nature of substituents in the anilide moiety
and the 2-oxoindole core. Similar to the sulfones, the most active
sulfoxides possessed a nitro group in the 5-position of the 2-
oxoindole core and a dihalogenated anilide moiety (Table 3).
However, this time derivatives bearing an alkyl-substituted anilide
retained activity. Much to our surprise, replacement of the nitro
group by an ester, sulfonamide or trifluoromethoxy group but not
by a methoxy group was well tolerated in most cases. Similarly,
replacement of the nitro group with halogens (F, Cl) or hydroxy-
acetic acid resulted in a two to threefold decrease in activity. In
conclusion and similar to the investigated sulfones, a nitro-
substituted 2-oxindole core together with a dihalogenated anilide

6720
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
O
O
R1
R2
R1
R2
O
N
N
R2
S
SO2
O
a
b
O
O
N
N
N
(47–84%)
(73–93%)
R3
R3
R3
25–30
31–35
36–40
O
O
R1
R2
R1
36 41
,
R
R
R
R
2 = CO2Me, R3 = 4-F, 4-Cl
2 = SO2NH2, R3 = 3-F
N
N
37, 42
38, 43
c
R2
S
S
O
2 = OMe, R3 = 4-F, 4-Cl
2 = H, Me, F, Cl, OCF3, NO2
39, 44
O
O
(75–89%)
R3 = 4-F, 4-Cl, 4-Br, 4-CN, 4-Ph,
4-OMe, 3-Cl, 3-CF3, 2-Cl-6-F
2 = OCH2CO2tBu, R3 = 4-F, 4-Cl
N
N
40, 45
R
R3
R3
31 35
–
41 45
–
O
O
O
R1
O
R1
R1
N
N
N
d
d
O
O
O
SOn
O
SO2
O
S
O
O
(88–92%)
(91–95%)
N
N
N
O
O
HO
O
HO
R3
R3
46 R3 = F, Cl
R3
47 R3 = F, Cl
40
n = 2
45 n = 1
Scheme 3. Preparation of the second generation indolin-2-on-3-spirothiazolidinones library. Reagents and conditions: (a) (1) ArNH2, EtOH, reflux, 6 h; (2) mercaptoacetic acid,
toluene, reflux, 16 h; (b) mCPBA (5 equiv), CHCl3, rt, 24 h; (c) mCPBA (1.1 equiv), CHCl3, 0 ꢂC, 1 h; (d) TFA, DCM, 0 ꢂC, 2 h.
In order to improve the physicochemical properties of our in-
hibitors we decided to introduce a carboxylic group into the thia-
zolidinone ring system. Condensation of isatins 28 with
3,4-difluoroaniline delivered isatin-3-imines, which afterward
were directly subjected to cyclocondensation with mercapto-
succinic acid to provide substituted spiro[indoline-3,20-thiazoli-
dine]-2,40-diones 51. In order to avoid difficulties during
purification we replaced mCPBA by OxoneÒ in the oxidation step.15
All synthesized compounds were evaluated for inhibitory ac-
tivity against MptpB (Tables 4 and 5) In addition, sulfones 13y, 39av,
39g, 39l, 39ah, 39an, 39ao and the acetic acid containing analogue
52, were subjected to PAMPA (parallel artificial membrane per-
meability) assays to determine cell membrane permeability.
Introduction of a carboxylic function into the thiazolidinone ring
does not alter biological activity and significantly increases com-
pound solubility and cell permeability. Remarkably, despite rela-
of the configuration of the spiro-center on the inhibitory activity
against MptpB, we separated the 10 most potent sulfones into their
pure enantiomers by means of preparative high-performance liq-
uid chromatography with chiral stationary phase (see Experimental
section for details) and determined their IC50 values (Table 6).
In all cases the (R)-(ꢀ)-enantiomers were 10e20 times more
potent than their corresponding (S)-(þ) antipodes. The most active
compounds displayed IC50 values in the nanomolar range (Table 6).
The absolute configuration was assigned using combination of ex-
perimental circular dichroism (CD) investigations with quantum
chemical CD calculations.10
2.7. Synthesis of structural analogues in order to determine
the binding mode
One of the key structural features of the indolin-2-on-3-
spirothiazolidinones is the presence of highly acidic methylene
protons in the thiazolidinone ring. This position is activated by both
the carbonyl and the sulfone/sulfoxide function, respectively. In
polar solvents one can assume rapid formation of an equilibrium
between the ‘amide’- (A) and ‘enol’-form (B) (with a shift in favor of
the ‘amide’-form A) (Fig. 4). We hypothesized, that due to its acidic
nature, the ‘enol’-form (B) might play a crucial role in recognizing
tively moderate solubility (103
cell permeability (46%).
mM), sulfone 39av shows excellent
2.6. Separation of enantiomers
All indolin-2-on-3-spirothiazolidinone discussed above were
synthesized as racemates. In order to shed light onto the relevance

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6721
Table 2
IC50 values of indolin-2-on-3-spirothiazolidinones are shown for MptpBa
O
N
Ar1
N
R2
SO2
O
R3
Compd
Ar1
R2
R3
IC50
,
mM
Compd
Ar1
R2
R3
IC50, mM
F
F
F
36a
CO2Me
4-F
18.1ꢁ2.1
14.7ꢁ1.7
16.4ꢁ1.9
39x
NO2
4-F
n.a.
F
F
F
Cl
36b
37
CO2Me
4-Cl
4-F
39y
39z
NO2
NO2
4-F
4-F
6.5ꢁ1.0
F
SO2NH2
n.a.
38a
38b
OMe
OMe
4-F
4-F
n.a.
39aa
39ab
NO2
NO2
4-F
4-F
30.5ꢁ2.4
13.7ꢁ1.8
HO
F
F
22.1ꢁ2.1
F
F
38c
39a
39b
39c
39d
39e
39f
39g
39h
39i
OMe
NO2
F
4-Cl
4-F
4-F
4-F
4-Cl
4-Cl
4-F
4-F
4-F
4-F
4-F
4-Cl
4-Cl
24.2ꢁ2.3
n.a.
39ac
39ad
39ae
39af
39ag
39ah
39ai
H
4-F
n.a.
Cl
NO2
OCF3
OCF3
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
4-Br
1.8ꢁ0.4
22.3ꢁ2.4
20.5ꢁ1.9
22.1ꢁ2.1
1.1ꢁ0.3
8.2ꢁ1.9
6.4ꢁ0.8
4.8ꢁ0.5
3.1ꢁ0.4
7.5ꢁ0.7
2.1ꢁ0.6
3.2ꢁ0.5
F
F
F
F
F
F
F
F
F
F
F
F
n.a.
4-F
F
F
F
F
F
F
F
F
F
F
F
Me
NO2
F
n.a.
4-Cl
n.a.
4-OMe
4-Br
n.a.
F
F
F
F
F
F
F
H
32.3ꢁ2.1
3.6ꢁ0.8
27.7ꢁ2.3
n.a.
4-OH
3,4,5-Tri-F
3,4-Di-F
4-CN
F
F
F
F
F
F
F
NO2
F
39aj
39ak
39al
Me
Cl
39j
28.5ꢁ2.5
29.7ꢁ3.2
1.8ꢁ0.4
39am
39an
39ao
2-Cl, 6-F
3-Cl
39k
39l
H
NO2
3-CF3
(continued on next page)

6722
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
Table 2 (continued )
Compd
Ar1
R2
R3
IC50
,
m
M
Compd
Ar1
R2
R3
IC50, mM
F
F
F
39m
F
4-Cl
4-Cl
4-F
4-F
4-F
4-F
4-F
24.2ꢁ2.8
28.0ꢁ2.2
n.a.
39ap
39aq
39ar
39as
39at
39au
39av
NO2
NO2
NO2
NO2
NO2
NO2
NO2
4-Ph
19.8ꢁ1.5
2.7ꢁ0.4
4.7ꢁ0.4
3.3ꢁ0.3
3.9ꢁ0.8
2.7ꢁ0.7
4.0ꢁ0.4
F
F
F
F
F
F
F
F
Cl
39n
Cl
H
H
H
H
H
3-Cl
F
MeO
MeO
Cl
Cl
Cl
Cl
39o
3,4,5-Tri-F
3,4-Di-F
3-CF3
39p
39q
39r
39s
n.a.
n.a.
nPr
27.7ꢁ1.9
n.a.
4-Cl
nBu
F
N
O
F
F
F
Cl
39t
39u
39v
H
H
H
4-F
4-F
4-F
29.3ꢁ2.3
28.2ꢁ2.5
n.a.
40a
40b
46a
OCH2-CO2tBu
OCH2-CO2tBu
OCH2-CO2H
4-Cl
4-F
31.4ꢁ2.7
n.a.
F
F
F
F
4-Cl
20.9ꢁ1.9
F
39w
NO2
4-F
18.4ꢁ2.1
46b
OCH2-CO2H
4-F
22.5ꢁ2.4
F
Bold values represents highlight IC50 values < 10
mM.
n.a¼not active (no inhibition up to a concentration of 100
m
M). We observed that replacement of the nitro group by methyl ester (36a,b), sulfonamide (37), methoxy (38b,c) or
trifluoromethoxy (39ae,af) groups led to loss of inhibitory activity. Introduction of halogens (39h,j), methyl groups (39i), and hydroxyacetate residues (40a,b, 46a,b) into the 2-
oxindole core had a detrimental effect on the inhibitory activity. The obtained analogues displayed the same or even lower activity compared to the unsubstituted sulfone 6.
From the above in vitro data it can be concluded that the nitro-substituted 2-oxindole fragment is essential for the high inhibitory activity against MptpB.
a
All IC50 values were calculated from at least three independent measurements.
Table 3
IC50 values of indolin-2-on-3-spirothiazolidinones are shown for MptpBa
Compd
Ar1
R2
R3
IC50
,
m
M
Compd
Ar1
R2
Cl
R3
IC50, mM
F
41a
CO2Me
4-F
6.0ꢁ1.0
5.6ꢁ1.1
4.5ꢁ0.6
44q
4-Cl
7.6ꢁ1.2
8.9ꢁ1.4
n.a.
F
F
41b
41c
CO2Me
CO2Me
4-F
4-F
44r
44s
Cl
H
4-Cl
4-F
F
MeO
MeO

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6723
M
Table 3 (continued )
Compd
Ar1
R2
R3
IC50
,
m
M
Compd
Ar1
R2
R3
IC50, m
F
41d
CO2Me
CO2Me
CO2Me
SO2NH2
4-Cl
4-Cl
4-Cl
4-F
5.1ꢁ0.5
5.5ꢁ1.5
6.2ꢁ0.9
6.9ꢁ1.6
44t
H
H
F
4-F
12.7ꢁ1.6
25.9ꢁ2.1
25.0ꢁ2.7
30.2ꢁ2.6
F
F
F
F
41e
41f
44u
44v
44w
4-Me
3-F
F
F
F
F
42a
H
2-F
F
42b
42c
SO2NH2
SO2NH2
4-F
4-F
8.3ꢁ2.4
3.1ꢁ0.7
44x
44y
H
H
3-F
27.5ꢁ2.4
18.2ꢁ2.3
F
4-NO2
F
43a
OMe
OMe
4-Cl
4-F
14.1ꢁ2.2
21.0ꢁ2.5
44z
NO2
NO2
4-Cl
4-Cl
5.4ꢁ0.8
2.9ꢁ0.5
F
F
43b
44aa
F
43c
OMe
OMe
H
4-Cl
3-F
4-F
18.3ꢁ1.8
24.0ꢁ2.1
10.5ꢁ1.3
44ab
44ac
44ad
NO2
NO2
NO2
4-Cl
4-Cl
4-Br
1.5ꢁ0.4
2.2ꢁ0.3
2.6ꢁ0.5
F
F
Cl
43d
F
F
F
F
F
44a
44b
44c
NO2
F
4-F
4-F
2.7ꢁ0.8
44ae
44af
NO2
4-Br
4-F
3.8ꢁ0.5
6.1ꢁ0.7
10.9ꢁ1.3
OCF3
F
44d
44e
H
4-Cl
4-Cl
4-F
22.1ꢁ2.3
3.1ꢁ0.6
26.1ꢁ2.2
44ag
44ah
44ai
OCF3
NO2
NO2
4-F
13.1ꢁ0.5
1.4ꢁ0.4
9.3ꢁ0.9
NO2
H
4-F
F
F
F
F
F
F
F
44f
4-OMe
F
F
F
44g
NO2
F
4-F
4-F
3-F
4-Cl
4-Cl
4-F
4-F
5.1ꢁ1.1
12.3ꢁ1.3
4.3ꢁ0.4
4.5ꢁ0.6
22.7ꢁ2.3
8.7ꢁ1.8
12.6ꢁ1.7
44aj
45a
45b
45c
45d
47a
47b
NO2
4-OMe
4-F
14.3ꢁ2.6
8.7ꢁ0.7
28.7ꢁ2.5
9.2ꢁ0.6
8.3ꢁ0.6
9.6ꢁ0.7
8.7ꢁ1.1
F
44h
OCH2eCO2tBu
OCH2eCO2tBu
OCH2eCO2tBu
OCH2eCO2tBu
OCH2eCO2H
OCH2eCO2H
F
44i
NO2
NO2
H
4-F
F
44j
4-F
F
F
F
44k
44l
4-Cl
4-F
F
F
NO2
NO2
44m
4-F
(continued on next page)

6724
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
Table 3 (continued )
Compd
Ar1
R2
R3
IC50
,
m
M
Compd
Ar1
R2
R3
IC50, mM
Cl
44n
NO2
NO2
4-F
4-F
4.3ꢁ0.5
1.5ꢁ0.5
47c
47d
OCH2eCO2H
OCH2eCO2H
4-F
3.9ꢁ0.6
8.2ꢁ1.0
F
F
44o
44p
4-Cl
F
NO2
4-F
1.2ꢁ0.2
47e
OCH2eCO2H
4-Cl
3.0ꢁ0.5
Bold values represents highlight IC50 values < 10
mM.
n.a¼not active (no inhibition up to a concentration of 100
m
M).
a
All IC50 values were calculated from at least three independent measurements.
F
F
F
F
F
F
O
N
O
S
N
c
a
N
N
O2N
H2N
SO2
O
SO2
O
O2N
SO2
S
(79%)
(75%)
N
N
Ar
Ar
Ar
50
13y
b
48
Ar = 3-F-C6H4
MeO
F
F
O
N
S
Lawesson's
reagent
P S
N
H
N
S
P
S
OMe
R
SO2
O
O
49a R = Me (89%)
Ar
49b R = OMe (92%)
F
F
O
O
OH
OH
F
F
O
N
O
O
O2N
e
d
N
O
O2N
S
O2N
SO2
O
N
(69–77%)
(65–74%)
O
N
N
R
R
R
28
(R = 4-F, 4-Cl, 4-Br,
3-Cl, 3-CF3)
52
d.r. = 15:1
51
d.r. = 1:1
Scheme 4. Synthesis of amine 48, derivatives 49, 50 and carboxylic acids 52. Reagents and conditions: (a) HCO2NH4, Pd/C, EtOH, reflux, 3 h; (b) RCOCl, pyridine, rt, 12 h; (c)
Lawesson’s reagent, toluene, reflux, 2 h; (d) 1) 3,4-difluoroaniline, EtOH, reflux, 6 h; (2) mercaptosuccinic acid, toluene reflux, 16 h; (e) OxoneÒ (5 equiv), MeOH/H2O (1:1), rt, 24 h.
the charged active center of the phosphatase. In order to prove this
Table 4
IC50 values of spiro[indoline-3,20-thiazolidine]-2,40-diones are shown for MptpBa
assumption we decided to introduce two methyl groups into the
thiazolidinone ring to inactivate tautomerization and to freeze the
‘amide’-form.
The synthesis of 53 was straightforward and started with the
reaction of isatins 11h, 28b, and 28g with different anilines, fol-
lowed by cyclocondensation of obtained isatin-3-imines with 2-
mercapto-2-methylpropionic acid (Scheme 5). Oxidation of the
obtained sulfides delivered the sulfone 53a and the sulfoxides 53b,
53c, and 53d.
Compd
Structural feature
IC50, mM
13y
48
49a
49b
50
Nitro
Amine
Acetamide
Carbamate
Thioamide
1.2ꢁ0.2
31.4ꢁ2.5
33.1ꢁ3.2
34.7ꢁ2.8
9.6ꢁ1.4
Bold values represents highlight IC50 values < 10
mM.
a
All IC50 values were calculated from at least three independent measurements.

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6725
Table 5
O
O
IC50 values, solubility, and cell-permeability data of indolin-2-on-3-
R1
R2
R1
R2
O
spirothiazolidinones 13, 39, and 52a
N
N
R2
S
SOn
O
O
a
b or c
O
N
N
N
(64–76%)
(85–92%)
R3
R3
R3
11h 28b 28g
,
54
53a
,
, n = 2 (Cond. b)
53b 53d
–
n = 1 (Cond. c)
Scheme 5. Synthesis of dimethylated analogues 53. Reagents and conditions: (a) (1)
ArNH2, EtOH, reflux, 6 h; (2) 2-mercapto-2-methylpropionic acid, toluene, reflux, 16 h;
(b) mCPBA (5 equiv), CHCl3, 0 ꢂC, 1 h; (c) mCPBA (1.1 equiv), CHCl3, rt, 1 h.
Compd
R
IC50
(
m
M) Solnb Compd
M)
R
IC50
(
m
M) Solnb Fluxc (%)
M)
(m
(m
13y
39g
39l
39ah
39an
39ao
3-F
4-F
4-Cl
1.2ꢁ0.2
3.6ꢁ0.8
1.8ꢁ0.6
157
205
225
250
240
139
39av
52a
52b
52c
52d
52e
d
4.0ꢁ0.4
4.8ꢁ0.6
2.9ꢁ0.2
103
430
412
376
425
421
46
13
20
20
22
21
4-F
4-Cl
strong hydrogen bonds to surrounding amino acid residues of the
active site (Table 7).
4-Br 1.1ꢁ0.3
4-Br 2.6ꢁ0.2
3-Cl
2.1ꢁ0.6
3-Cl
2.3ꢁ0.2
Table 7
3-CF3 3.2ꢁ0.4
3-CF3 2.4ꢁ0.3
IC50 values of spiro[indoline-3,20-thiazolidine]-2,40-dione are shown for MptpBa
Bold values represents highlight IC50 values < 10 mM.
a
All IC50 values were determined from at least three independent measurements.
b
c
Kinetic solubility as determined by a direct UV assay.16
Cell permeability determined via a parallel artificial membrane permeability
assay (PAMPA). Flux (%) denotes concentration (test well)/concentration (control
well)ꢃ100.
Table 6
IC50a values of enantiomerically pure 13y and 39 are shown for MptpBa
Compd
R1
R2
R3
IC50, mM
53a
53b
53c
53d
13y
44b
44aa
44ab
NA
Et
i-Pr
t-Bu
NA
Et
NA
F
Cl
Cl
NA
F
Me
Me
Me
Me
H
H
H
H
n.a.
n.a.
n.a.
n.a.
1.2ꢁ0.2
2.7ꢁ0.8
2.9ꢁ0.5
1.5ꢁ0.4
i-Pr
t-Bu
Cl
Cl
Bold values represents highlight IC50 values < 10 mM.
n.a¼not active (no inhibition up to a concentration of 100
mM).
a
Compd
R1
R2
IC50, mM.
All IC50 values were calculated from at least three independent measurements.
(S)-(þ)
(R)-(ꢀ)
2.8. Determination of the mode of inhibition of MptpB by the
identified inhibitors
13y
39g
F
F
F
F
F
F
Cl
Cl
Cl
Cl
3-F
4-F
3.9ꢁ0.6
5.8ꢁ1.1
2.9ꢁ0.5
8.8ꢁ0.6
9.6ꢁ1.4
7.9ꢁ0.4
8.9ꢁ0.8
11ꢁ1.2
9.1ꢁ0.4
9.5ꢁ0.5
0.32ꢁ0.05
0.52ꢁ0.08
0.28ꢁ0.06
1.3ꢁ0.1
39ah
39al
39an
39ao
39aq
39as
39at
39au
4-Br
4-CN
3-Cl
3-CF3
3-Cl
3,4-Di F
3-CF3
4-Cl
To gain deeper insight into the mode of MptpB inhibition by the
identified spiro-fused indol-2-one-thiazolidinones and to further
investigate the proposed role of the ‘enol’-form of the thiazolidi-
none ring as a phosphate mimic, we subjected R-(ꢀ)-13y, R-
(ꢀ)-39g, R-(ꢀ)-39ah, and R-(ꢀ)-39ao to detailed kinetic analysis
according to Dixon and LineweavereBurk and determined in-
hibitory IC50 values at different substrate concentrations. Thereby
we could clearly show that R-(ꢀ)-13y, R-(ꢀ)-39g, R-(ꢀ)-39ah, and
0.94ꢁ0.12
0.38ꢁ0.06
0.51ꢁ0.08
1.3ꢁ0.2
1.4ꢁ0.2
1.24ꢁ0.15
a
All IC50 values were calculated from at least three independent measurements.
The doubly methylated derivatives 53 showed a complete loss in
inhibitory activity when tested against MptpB and highlight that
the presence of the ‘enol’-form B (Fig. 4) might indeed play a central
role possibly as phosphate mimic or to allow for the formation of
R-(ꢀ)-39a with Ki values between 0.2 and 0.8
mM are potent and
substrate competitive inhibitors, which confirms our hypothesis,
that the thiazolidinone ring might indeed serve as a crucial element
and binds close to the phosphate binding site (Fig. 4).
O
O
HO
phosphate
mimic
Ar1
Ar1
Ar1
N
N
N
R
SOn
O
R
SOn
O
R
SOn
O
N
N
N
Ar2
Ar2
Ar2
53
A
B
Fig. 4. Tautomerization of spiro-fused indol-2-one-thiazolidinones.

6726
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
Fig. 5. Proposed binding mode of spiro-fused indol-2-one-thiazolidinone to the active site of MptpB. (A) The R-(ꢀ)-enantiomer of 13y was docked to the complex crystal structure
of OMTS (2) in MptpB. The surface of the protein (white) and the van der Waals radii of the inhibitor (pink colored mesh) indicate good shape complementarity. The proposed
binding mode of 13y indicates the sulfone and the tautomeric oxygen of the thiazolidinone ring within hydrogen bonding distance (red dotted lines) to amino acids of the
phosphate binding site (K164, D165, and R166). The oxoindole forms favorable catione
p interactions with the side chain of R166 and the nitro group at the 5-position of the
oxoindole core forms water-mediated (W382) hydrogen bonds to the side chains of D129 and R136 and helps to explain why the nitro but not the amine significantly enhances
potency against MptpB (Table 1). The dihalogenated anilide and the halogenated N-benzyl moiety of the inhibitor are located in a hydrophobic, aromatic sub-pocket flanked by
L232, L227, F98, and Y125. (B) Structural alignment of the modeled R-(ꢀ)-13y-MptpB complex with MptpB in complex with phosphate (PDB code: 1YWF) and OMTS (thin blue
sticks) (PDB code: 2OZ5) shows the sulfone of 13y and the carboxylate of 2 in a position close to the catalytic cysteine (C160) and isostructural to that of the phosphate (large
spheres).
Table 8
Overview of the relative phosphatase activity (%) after addition of the compound (50
compounds
mM), where green fill marks no activity, yellow slight activity, and red is a sign for an active

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6727
At least the reversibility, which goes along with the competitive
binding mode, marks an important precondition for the de-
velopment of new, desired antitubercular drugs.
further purification. mCPBA was purified according to the reported
procedure. Drysolvents werepurchasedasanhydrous reagents from
commercial suppliers. 1H and 13C NMR spectra were recorded on
a Bruker Avance DRX 400 spectrometer at 400 MHz and 100 MHz,
2.9. Molecular modeling studies
respectively.1H chemical shifts are reported in
d (ppm) as s (singlet),
d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (mul-
tiplet) and br s (broad singlet) and are referenced to the residual
solvent signal: CDCl3 (7.26), DMSO-d6 (2.50). 13C spectra are refer-
enced to the residual solvent signal: CDCl3 (77.0) or DMSO-d6 (39.0).
All final compounds were purified to >95% purity, as determined by
high-performance liquid chromatography (HPLC). Purity was mea-
sured using Agilent 1200 Series HPLC systems with UV detection at
To get deeper insights into the possible binding mode of spiro-
fused indol-2-one-thiazolidinones in MptpB and to better un-
derstand the impact of the configuration of the spiro-center and the
‘enol’-form of the thiazolidinone for binding, we used molecular
docking. The crystal structures of MptpB in complex with phosphate
(PDB code: 1YWF) and an inhibitor (PDB code: 2OZ5) are known from
literature and highlight structural elements that are crucial for ligand
binding. We docked the ‘enol’-form of both enantiomers of 13y to the
rigid protein structure of the inhibitor bound form of MptpB (PDB
code: 2OZ5). Only the R-(ꢀ)-enantiomer of 13y showed a convincing
bindingmodewithdirectcontactstoaminoacidsoftheactivesiteand
an overall good shape complementarity (Fig. 5). The binding mode of
R-(ꢀ)-13y revealed a number of polar interactions and indicates that
the thiazolidinone ring indeed serves as a phosphate mimic.
210 nm (System: Agilent Eclipse XDB-C18 4.6ꢃ150 mm, 5
mM,
10e100% CH3CN in H2O, with 0.1% TFA, for 15 min at 1.0 mL/min).
High resolution electrospray ionization mass spectra (ESIeFTMS)
were recorded on a Thermo LTQ Orbitrap (high resolution mass
spectrometer from Thermo Electron) coupled to an ‘Accela’ HPLC
System supplied with a ‘Hypersil GOLD’ column (Thermo Electron).
Analytical TLC was carried out on Merck 60 F245 aluminum-backed
silica gel plates. Compounds were purified by column chromatog-
raphy using Baker silica gel (40e70 mm particle size). Melting points
€
2.10. Investigation of the selectivity profile of MptpB
inhibitors
were determined using a Buchi Melting Point B-540 and are un-
corrected. Preparative chiral HPLC was conducted on a Dionex HPLC
system (UltiMate 3000 preparative) with a CHIRALPAKÒ IC column
In order to compare the phosphatase inhibitor selectivity of our
newly developed spiro-fused indol-2-one-thiazolidinones, we per-
formed phosphatase profiling for the 25 most potent inhibitors
against a panel of six different phosphatases including MptpA, PTP1B,
(cellulose tris(3,5-dichlorophenylcarbamate) immobilized on 5
silica gel) and iso-hexane/CH2Cl2/EtOH, 60:39.2:0.8 as an eluent and
monitored by UV at
recorded on a JASCO J-815 CD spectropolarimeter using 5 mm cu-
vette made from Quartz SUPRASILÒ.
mm
l
¼254 nm. Circular dichroism spectra were
SHP-2, PTPN2, h-PTP
b, VHR and measuring the remaining enzyme
activity at a concentration of 50
mM (Table 8). Of particular interest
was the finding that all tested compounds showed excellent selec-
tivity in favor of MptpB.
4.1.1. General procedure for the preparation of N-alkyl isatins 11, 16,
17, and 25e28 (method A). NaH (60% dispersion in mineral oil,
240 mg, 6.0 mmol) was added to a solution of isatin 10, 23b or 24a
(5.00 mmol) in DMF (25 mL) at 0 ꢂC. The mixture was stirred for
10 min at 0 ꢂC and then the corresponding benzyl bromide or alkyl
halide (5.25 mmol) was added. The mixture was stirred at room
temperature for 8 h and then poured into 120 mL of ice cold water
whereupon an orange (brown) solid precipitates. The solid was
filtered, washed with water, and dried in vacuo. The crude material
was recrystallized from MeOH or purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH, 98:2) to provide the corre-
sponding N-alkyl isatin.
3. Conclusion
In conclusion, indolin-2-on-3-spirothiazolidinones were identi-
fied as a novel class of potent and selective MptpB inhibitors. Detailed
systematicstructureeactivityrelationship(SAR)studiesrevealedthat
a nitro-substituted 2-oxoindole core, a dihalogenated anilide moiety,
and a halogenated N-benzyl fragment are essential for strong in-
hibitory activity against MptpB. Introduction of the carboxyl function
into the thiazolidinone ring led to significant improvement of the
solubility and cell permeability of compounds without affecting the
inhibitory activity. It was found that the configuration of the spiro-
center plays a crucial role for the inhibitory activity against MptpB, R-
(ꢀ)-enantiomers are active in the nanomolar range, and are generally
10e20 times more potent than the corresponding S-(þ)-antipodes. In
addition, synthesis of structural analogues and extensive kinetic
studies showed that (a) the indolin-2-on-3-spirothiazolidinones are
reversible andcompetitiveMptpBinhibitorsand(b)the‘enol’-formof
the thiazolidinone ring is involved in hydrogen bonding with the
enzyme. Modeling studies indeed suggested that the R-(ꢀ)-enantio-
mer fits best to the active site of MptpB and that the thiazolidinone
ring system serves as a phosphomimic. Finally, all identified MptpB
inhibitors showed excellent selectivity against a panel of other pro-
tein tyrosine phosphatases, including MptpA, PTP1B, SHP-2, PTPN2,
4.1.2. Generalprocedureforthepreparationofindolin-2-on-3-spirothia-
zolidinones 12, 16, 17, 31e35, and 54 (method B).
4.1.2.1. Synthesis of isatin-3-imine. A mixture of substituted isatin
(0.3 mmol) and the appropriate aromatic amine (0.36 mmol) was
refluxed in absolute EtOH (5 mL) for 6 h. EtOH was removed in vacuo
and the obtained isatin-3-imine was used directly in the next step.
4.1.2.2. Cyclization into indolin-2-on-3-spirothiazolidinone. A
mixture of isatin-3-imine and mercaptoacetic acid (or 2-mercapto-
2-methylpropanoic acid) (35 mL, 0.48 mmol) in 6 mL of absolute
toluene was refluxed for 16 h and the water formed was removed
by azeotropic distillation. The reaction mixture was cooled, diluted
with 20 mL of EtOAc, washed with 1 M aqueous HCI, saturated
NaHCO3 solution, dried over MgSO4, and concentrated in vacuo. The
crude product was purified by column chromatography on silica gel
(CH2Cl2/MeOH, 99:1) to provide corresponding indolin-2-on-3-
spirothiazolidinone as an amorphous solid. The resulting sulfides
were further oxidized without characterization.
h-PTP
b, and VHR. Consequently, indolin-2-on-3-spirothiazolidinones
may be a promising starting point for the development of novel an-
tibiotic agents with activity against M. tuberculosis.
4. Experimental
4.1. Chemistry
4.1.3. General procedure for the oxidation of indolin-2-on-3-
spirothiazolidinones into sulfones 13, 18, 19, 36e40, and 53a
(method C). To a cooled (0 ꢂC) solution of an appropriate sulfide
(0.1 mmol) in 3 mL of CHCl3 was added mCPBA (86 mg, 0.5 mmol,
Unless otherwise noted, all reagents and solvents were pur-
chased from Acros, Fluka, Sigma, Aldrich or Merck and used without

6728
V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
5 equiv). The resulting solution was allowed to reach room tem-
perature and stirred for 24 h. Then the reaction mixture was diluted
with 15 mL of EtOAc, washed with 15% NaHSO3 solution (2ꢃ20 mL),
saturated NaHCO3 solution (2ꢃ20 mL), dried over MgSO4, and
concentrated in vacuo. The crude product was purified by column
chromatography on silica gel (CH2Cl2/MeOH, 99:1) to provide the
corresponding sulfone as an amorphous solid.
in dry pyridine (1 mL) was added an appropriate acid chloride
(0.075 mmol). After the mixture was stirred at room temperature
for 4 h, H2O (5 mL) was added and the obtained emulsion was
extracted with EtOAc (3ꢃ10 mL). The combined organic layers were
washed with saturated NaHCO3 solution (15 mL), dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
flash chromatography (CH2Cl2/MeOH, 97:3) to give amide 49 as
a colorless amorphous solid.
4.1.4. General procedure for the oxidation of indolin-2-on-3-
spirothiazolidinones into sulfoxides 14, 41e45, and 53bed (method
D). To a cooled (0 ꢂC) solution of an appropriate sulfide (0.1 mmol)
in 3 mL of CHCl3 was added mCPBA (1.1 equiv) and the reaction
mixture was stirred at this temperature for 1 h. Then the reaction
mixture was diluted with 15 mL of EtOAc, washed with saturated
NaHCO3 solution (2ꢃ20 mL), dried over MgSO4, and concentrated
in vacuo. The crude product was purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH, 98:2) to provide the corre-
sponding sulfoxide as an amorphous solid.
4.1.9. General procedure for the preparation of acids 52 (method I).
4.1.9.1. Synthesis of indolin-2-on-3-spirothiazolidinones 51. A mix-
ture of substituted isatin 28 (0.3 mmol) and 3,4-difluoroaniline
(37 mL, 0.36 mmol) was refluxed in absolute EtOH (5 mL) for 6 h.
EtOH was removed in vacuo and the obtained isatin-3-imine was
redissolved in 10 mL of absolute toluene. Mercaptosuccinic acid
(74 mg, 0.48 mmol) was added to the obtained solution and the
reaction mixture was refluxed for 16 h with azeotropic removal of
water. The reaction mixture was cooled to room temperature, di-
luted with 20 mL of EtOAc, washed with 1 M aqueous HCI
(2ꢃ20 mL), water (2ꢃ20 mL), brine (2ꢃ20 mL), dried over MgSO4,
and concentrated in vacuo. The crude product was purified by
column chromatography on silica gel (CH2Cl2/MeOH/HOAc,
95:5:0.5) to provide corresponding acid 51, which was used directly
in the next step.
4.1.5. General procedure for the preparation of amides 21aed
(method E). To a solution of acid 20 (42 mg, 0.1 mmol, 1 equiv) in
dry DMF (1.5 mL) were added an appropriate aliphatic amine
(0.2 mmol, 2 equiv), HBTU (76 mg, 0.2 mmol, 2 equiv), HOBt (27 mg,
0.2 mmol, 2 equiv), and N,N-diisopropylethylamine (136
mL,
0.8 mmol, 8 equiv). After the mixture was stirred at room tem-
perature for 4 h, H2O (5 mL) was added and the obtained emulsion
was extracted with EtOAc (3ꢃ15 mL). The combined organic layers
were washed with saturated NaHCO3 solution (20 mL), dried over
MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by flash chromatography (CH2Cl2/MeOH, 98:2/97:3) to give
the corresponding amide as a colorless amorphous solid.
4.1.10. Oxidation of acid 51 into the corresponding sulfone 52. A
mixture of acid 51 (0.2 mmol) and OxoneÒ (2KHSO4$KHSO4$K2SO4;
738 mg, 1.2 mmol) in 50% methanol/water (20 mL) was stirred at
room temperature for 24 h. The reaction mixture was concentrated
under reduced pressure to dryness, and the residue was taken up in
EtOAc (30 mL). The ethyl acetate layer was washed with 5% citric
acid solution (2ꢃ15 mL), water (2ꢃ15 mL), and brine (2ꢃ15 mL)
before drying over sodium sulfate. The filtrate was concentrated
under reduced pressure and the residue was purified by flash
chromatography (CH2Cl2/MeOH/HOAc, 95:5:0.5) to give acid 52 as
a colorless amorphous solid.
4.1.6. General procedure for the preparation of tert-butyl esters 30
(method F).
4.1.6.1. Demethylation of 5-methoxy isatin. A solution of an appropri-
ate isatin 27 (4 mmol) in CH2Cl2 (20 mL) was treated with BBr3
(8.0 mL, 1.0 M in CH2Cl2, 8.0 mmol, 2 equiv) at ꢀ5 ꢂC. The reaction was
stirred for 2 h at 0 ꢂC before a saturated solution of NaOAc (25 mL) was
added. After separation of the layers, the aqueous phase was extracted
twice with CH2Cl2 (2ꢃ25 mL). The combined organic layers were
washed with H2O, saturated NaCl solution, dried over MgSO4, filtered,
and concentrated in vacuo. Flash chromatography (CH2Cl2/MeOH,
97:3/19:1) afforded phenol 29 as an orange amorphous solid.
4.2. Proteinbiochemistry
4.2.1. Phosphatase expression and purification. MptpA/B, h-PTPb,
VHR, SHP-2, and PTP1B were cloned, expressed, and purified as
recently described.17e19 GST-labeled phosphatase PTPN2 was pur-
chased from Stratagene (Amsterdam, NL).
4.1.6.2. Alkylation of phenol 29 with tert-butyl bromoaceta-
te. NaH (60% dispersion in mineral oil, 144 mg, 3.6 mmol) was
added to a solution of phenol 29 (3.00 mmol) in DMF (10 mL) at
0 ꢂC. The mixture was stirred for 10 min at 0 ꢂC and then tert-butyl
bromoacetate (0.53 mL, 3.3 mmol) was added. The mixture was
stirred at room temperature for 3 h and then poured into 50 mL of
ice cold water. The obtained emulsion was extracted twice with
EtOAc (2ꢃ50 mL). The combined organic layers were washed with
H2O, saturated NaCl solution, dried over MgSO4, filtered, and con-
centrated in vacuo. Flash chromatography (CH2Cl2/MeOH, 98:2)
afforded tert-butyl ester 30 as an orange amorphous solid.
4.2.2. Kinetic assays for IC50 and Ki determinations.
4.2.2.1. MptpB inhibition assay/IC50 determination. M. tuberculosis
protein tyrosine phosphatase B was dissolved in 25 mM HEPES/
50 mM NaCl/Na2ꢃEDTA 2.5 mM/NP-40 0.025%/DTE 2 mM/1%
DMSO buffer at a concentration of 50 nM. Kinetic analysis at 37 ꢂC
using the substrate 4-nitrophenol phosphate at pH 7.2 and
monitoring the increase of p-NP (4-Nitrophenol) at 405 nm gave
KM and Kcat values of (2.3ꢁ0.3) mM and
5
sꢀ1 (Kcat
mL. The reaction
/
KM¼2300 sꢀ1 Mꢀ1). The reaction volume was 100
was started by the addition of p-nitrophenol phosphate (20 mM
stock solution, 10
mL) to 90 mL of a solution containing MptpB
(preincubated with inhibitor for 15 min). A Tecan InfiniteÒ 200
plate reader was used to measure the absorbance of the samples
at 405 nm. The reaction velocity was determined from the slope of
the absorbance change at 405 nm and related to control values in
the absence of the inhibitor. IC50 values were calculated from
linear extrapolations of reaction velocity as a function of the
logarithm of the concentration. All IC50 values were calculated
from at least three independent determinations. The dissociation
constant Ki, as well as the differentiation between competitive
and non-competitive inhibition were determined using the
method described by Dixon.20
4.1.7. General procedure for the deprotection of tert-butyl esters 40
and 45 (method G). To a cooled (0 ꢂC) solution of tert-butyl ester 40
or 45 (0.1 mmol) in 1 mL of dry CH2Cl2 was added 1 mL of TFA. The
reaction mixture was stirred at 0 ꢂC for 2 h and all volatiles were
removed in vacuo. The crude product was purified by column
chromatography on silica gel (CH2Cl2/MeOH/HOAc, 95:5:0.5) to
provide the corresponding acid as a colorless amorphous solid.
4.1.8. General procedure for the preparation of derivatives 49
(method H). To a solution of amine 48 (24 mg, 0.05 mmol, 1 equiv)

V.V. Vintonyak et al. / Tetrahedron 67 (2011) 6713e6729
6729
3. Singh, R.; Rao, V.; Shakila, H.; Gupta, R.; Khera, A.; Dhar, N.; Singh, A.; Koul, A.;
Singh, Y.; Naseema, M.; Narayanan, P. R.; Paramasivan, C. N.; Ramanathan, V. D.;
Tyagi, A. K. Mol. Microbiol. 2003, 50, 751e762.
4. (a) Beresford, N. J.; Mulhearn, D.; Szczepankiewicz, B.; Liu, G.; Johnson, M. E.;
Fordham-Skelton, A.; Abad-Zapatero, C.; Cavet, J. S.; Tabernero, L. J. Antimicrob.
Chemother. 2009, 63, 928e936; (b) Zhou, B.; He, Y.; Zhang, X.; Xu, J.; Luo, Y.;
Wang, Y.; Franzblau, S. G.; Yang, Z.; Chan, R. J.; Liu, Y.; Zheng, J.; Zhang, Z. Y. Proc.
Natl. Acad. Sci. U.S.A. 2010, 107, 4573e4578.
4.2.2.2. MptpA, PTP1B, SHP-2, PTPN2, h-PTPb, VHR inhibition as-
say. All buffered solutions contained 2 mM DTE (added on the day
of the experiment from 200 mM stock) and the detergent NP-40
(0.025% v/v). The buffers consisted of Tris (50 mM), NaCl
(50 mM), and EDTA (0.1 mM) in the case of h-PTPb; HEPES (25 mM),
NaCl (50 mM), and EDTA (2.5 mM) in the case of PTP1B, PTPN2,
MptpA, and SHP-2; and MOPS (25 mM) and EDTA (5 mM) in the
5. For recent reviews see: (a) Vintonyak, V. V.; Waldmann, H.; Rauh, D. Bioorg.
Med. Chem. 2011, 19, 2145e2155; (b) Vintonyak, V. V.; Antonchick, A. P.; Rauh,
D.; Waldmann, H. Curr. Opin. Chem. Biol. 2009, 13, 272e283; (c) Bialy, L.;
Waldmann, H. Angew. Chem., Int. Ed. 2005, 44, 3814e3839.
case of VHR. The final concentration of the enzymes was 1.6
(PTP1B), 0.7 g/mL (PTPN2), 5.0 g/mL,180 nM (MptpA),10.0
(SHP-2), 0.13 g/mL (h-PTP ), and 15.6
m
m
g/mL
g/mL
m
m
m
€
€
b
m
g/mL (VHR). Each reaction
6. (a) Noren-Muller, A.; Reis-Correa, I., Jr.; Prinz, H.; Rosenbaum, C.; Saxena, K.;
Schwalbe, H. J.; Vestweber, D.; Cagna, G.; Schunk, S.; Schwarz, O.; Schiewe, H.;
Waldmann, H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10606e10611; (b) Correa, I.
was performed in triplicate from identical manual dilutions.
€
€
R., Jr.; Noren-Muller, A.; Ambrosi, H. D.; Jakupovic, S.; Saxena, K.; Schwalbe, H.;
Kaiser, M.; Waldmann, H. Chem.dAsian. J. 2007, 2, 1109e1126.
7. Grundner, C.; Perrin, D.; Hooft van Huijsduijnen, R.; Swinnen, D.; Gonzalez, J.;
Gee, C. L.; Wells, T. N.; Alber, T. Structure 2007, 15, 499e509.
8. Soellner, M. B.; Rawls, K. A.; Grundner, C.; Alber, T.; Ellman, J. A. J. Am. Chem. Soc.
2007, 129, 9613e9615.
4.3. Molecular modeling
€
Docking studies were performed using the Schrodinger soft-
ware package.21 The receptor was prepared with the Protein
Preparation Wizard and ligands were docked using Glide in XP
mode without any constraints.22,23 Following settings differed from
the default parameters. For H-bond assignment using exhaustive
sampling the ligands from the crystal structure and surrounding
waters were kept. Finally, two important waters were kept for re-
ceptor grid generation. For the latter, the ligand diameter midpoint
9. Tan, L. P.; Wu, H.; Yang, P. Y.; Kalesh, K. A.; Zhang, X.; Hu, M.; Srinivasan, R.; Yao,
S. Q. Org. Lett. 2009, 11, 5102e5105.
10. Vintonyak, V. V.; Warburg, K.; Kruse, H.; Grimme, S.; Hubel, K.; Rauh, D.;
€
Waldmann, H. Angew. Chem., Int. Ed. 2010, 49, 5902e5905.
11. Koch, M. A.; Wittenberg, L. O.; Basu, S.; Jeyaraj, D. A.; Gourzoulidou, E.; Re-
inecke, K.; Odermatt, A.; Waldmann, H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
16721e16726.
12. Koch, M. A.; Schuffenhauer, A.; Scheck, M.; Wetzel, S.; Casaulta, M.; Odermatt,
A.; Ertl, P.; Waldmann, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17272e17277.
13. Zhou, L.; Liu, Y.; Zhang, W.; Wei, P.; Huang, C.; Pei, J.; Yuan, Y.; Lai, L. J. Med.
Chem. 2006, 49, 3440e3443.
ꢀ
box was enlarged to 12ꢃ12x12 A. Figures were prepared with
PYMOL (www.pymol.org).24
14. Lu, Y.; Shi, T.; Wang, Y.; Yang, H.; Yan, X.; Luo, X.; Jiang, H.; Zhu, W. J. Med. Chem.
2009, 52, 2854e2862.
Acknowledgements
15. It was found that oxidation of sulfides 51 with 6 equiv of oxoneÒ in the MeOH/
H2O (1:1) mixture resulted in formation of one major diastereomer 52.
16. Avdeef, A. In Pharmacokinetic Optimization in Drug Research; Testa, B., van de
This research was supported by the Max-Planck-Gesellschaft,
the Fonds der Chemischen Industrie, and the German Federal
Ministry for Education (Grant No. BMBF 01GS08102).
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Experimental procedures, analytical data and biochemical
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