Synthesis and study of 2-(pyrrolesulfonylmethyl)- N -arylimines: A new class of inhibitors for human glutathione transferase A1-1

Article abstract of DOI:10.1021/jm300385f

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K_i(9) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (K _i(4) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.

Full text of DOI:10.1021/jm300385f

Article
pubs.acs.org/jmc
Synthesis and Study of 2‑(Pyrrolesulfonylmethyl)‑N‑arylimines: A
New Class of Inhibitors for Human Glutathione Transferase A1‑1
Georgia E. Koutsoumpli,† Virginia D. Dimaki,§ Trias N. Thireou,‡ Elias E. Eliopoulos,‡
Nikolaos E. Labrou,† George I. Varvounis,*,§ and Yannis D. Clonis*,†
†Laboratory of Enzyme Technology and ‡Laboratory of Genetics, Department of Agricultural Biotechnology, Agricultural University
of Athens, 75 Iera Odos Street, GR-118 55 Athens, Greece
§Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, GR-451 10 Ioannina, Greece
S
* Supporting Information
ABSTRACT: Overexpression of human GSTA1-1 in tumor cells is part
of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives
as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfan-
yl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme
H binding site: the catalytic primary one accommodating shorter and
longer derivatives and the secondary one, where shorter derivatives can
occupy. Derivative 9, displaying the highest inhibition and bearing a p-
nitroarylimino moiety, and derivative 4, lacking this moiety, were studied
kinetically. Derivative 9 binds (Ki(9) = 71 4 μM) at the primary site
competitively vs CDNB. Derivative 4 binds (Ki(4) = 135 27 μM) at the
primary and secondary sites, allowing the binding of a second molecule (4
or CDNB) leading to formation of unreactive and reactive complexes,
respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may
serve as a lead structure.
inhibiting c-Jun N-terminal kinase. Furthermore, the in vitro
resistance to doxorubicin by blast cells, derived from acute
myeloid leukemia patients, has been attributed to elevated
activity of hGSTA1/A2 enzymes6 whereas a correlation in vitro
has been reported between GSTα in gastric cancer tissues and
cisplatin resistance.7 Interestingly, the death hazard of
homozygous hGSTA1*B breast cancer patients treated with
cyclophosphamide (plus other chemotherapeutic drugs) was
reduced during the first 5 years following diagnosis, compared
with homozygous hGSTA1*A individuals (hazard ratio, 0.3).9
This observation was attributed to the detoxifying role of
hepatic hGSTA1 against therapeutic metabolites of cyclo-
phosphamide.
Several synthetic drugs and prodrugs exhibiting inhibition
potency against GSTs have been proposed as strategies to
overcoming multiple drug resistance (MDR) attributed to GST
overexpression.11,12 Ethacrynic acid was one of the first
inhibitors to be utilized to sensitize cancer cells to the cytotoxic
effect of several chemotherapeutic drugs.13 Its analogues,14−16
individual compounds,11,17 and several prodrug molecules18−21
have been proposed as GST-inhibiting strategies. Furthermore,
several GSH analogues have been proposed as more specific
reversible22−24 and irreversible25 GST inhibitors, exploiting the
high affinity of GSTs for the tripeptide substrate GSH. An
alternative concept exploits the susceptibility of GSH
INTRODUCTION
■
Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of
isoenzymes that differ in their tissue-specificity expression and
distribution. They catalyze the conjugation of glutathione
(GSH) to a variety of hydrophobic endogenous and exogenous
substrates, rendering them hydrophilic and facilitating their
metabolic processing and eventual secretion from the cell.1−4
GSTs are subdivided into classes on the basis of their amino
acid sequence, whereas cytosolic ones are found as homodimers
or heterodimers.5 Each monomer has an α/β domain that
includes α1−α3 helices and a large α-helical domain
comprising helices α4−α9. The former domain contains the
GSH binding site (G-site) on top of the large α domain. A
hydrophobic pocket (H-site) lies between the two domains in
which the hydrophobic substrate binds and reacts with GSH.
Since the produced conjugates are susceptible to further
modification and eventual secretion from the cell, the GSTs are
involved in major detoxification mechanisms of the cell from
several xenobiotics and drugs. On the other hand, on the basis
of the same detoxification mechanisms, cancer cells may acquire
resistance by overexpressing GST activities,6,7 thus hampering
the effectiveness of certain chemotherapeutic drugs. This
mechanism leads to chemotherapeutic resistant tumor cells
that no longer respond appropriately to the applied therapeutic
protocol.8 A plausible mechanism by which GSTs could
contribute to drug resistance includes GST-dependent
prevention of drug-induced apoptosis via direct interaction
with signal transduction proteins, as suggested for GSTP1-19,10
Received: April 5, 2012
Published: July 31, 2012
© 2012 American Chemical Society
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Table 1. Structures and Some Properties of the New Arylmethylsulfonylpyrrole Derivatives 4−14
a
b
As in Scheme 1 and in ascending order of inhibition potency against hGSTA1-1. Found by high resolution mass spectrometry as [M + H]+
c
(calculated from the molecular formula for experimental use). Mean value of at least three enzyme activity assays (0.1 mM pyrrole derivative); error
up to 3%. Predicted octanol/water partition coefficient.
d
conjugates (products of GST catalysis) against the GSH-
degrading enzyme γ-glutamyltranspeptidase (γGT). Accord-
ingly, certain peptidase-stable GSH analogues have been put to
the test as GST inhibitors.26−29
Pyrroles have not been studied so far as GST inhibitors,
although sulfonylpyrrole derivatives (Figure SI-1 in Supporting
Information) that are part of carbocyclic−SO2−carbocyclic,
carbocyclic−SO2−heterocyclic, or aliphatic−SO2−heterocyclic
moieties have been found to exhibit a variety of important
biological activities. For example, 2-nitrophenylphenylsulfone
(Figure SI-1, 6, NPPS),30 5-chloro-3-(phenysulfonyl)indole-2-
carboxamide (Figure SI-1, 7, L-737,126),31 and ethyl 1-[(2-
amino-5-chlorophenyl)sulfonyl]-4-(hydroxymethyl)-1H-pyr-
role-2-carboxylate (Figure SI-1, 8, PAS)32,33 were lead
compounds selected as potent inhibitors of the reverse
transcriptase (RT). RT is a virus-encoded enzyme that catalyzes
the transcription of single-stranded viral genomic RNA into
double-stranded DNA, which is an essential component of the
life cycle of HIV-1 (human immunodeficiency virus)
responsible for the manifestation of AIDS. Further study of
these non-nucleoside reverse transcriptase inhibitors (NNRTs)
showed that ethyl 1-[(5-amino-2-chlorophenyl)sulfonyl]-4-
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(hydroxymethyl)-1H-pyrrole-2-carboxylate (Figure SI-1, 9)34 is
more active than (Figure SI-1, 8) and that several N5-benzyl-
N5-methyl-3-(phenylsulfonyl)-1H-pyrrole-2,5-dicarboxamide
derivatives 10 (Figure SI-1) have been patented as RT
inhibitors,35 whereas {4-[(4,5-dihydro-1,3-thiazol-2-ylmethyl)-
sulfonyl]-1H-pyrrol-3-yl}(phenyl)methanones 11 and 12 (Fig-
ure SI-1) showed excellent activity against Gram-positive
bacteria and good activity against Gram-negative bacteria.36
Herein we report on the synthesis of 11 pyrrole derivatives
4−14 and their inhibitory profile toward hGSTA1-1.
Specifically, the 2-(pyrrolesulfonylmethyl)-N-arylimine struc-
ture was investigated as an inhibitory pharmacophore against
hGSTA1-1. Following GST inhibition screening, in silico
molecular docking, and enzyme inhibition kinetics, the pyrrole
analogue identified as exhibiting the highest inhibitory potency
and structural redesign flexibility would be regarded as a new
“lead compound” useful in constructing new inhibitors and
respective prodrugs for human GSTs of medical importance.
sulfate heptahydrate and 25% ammonium hydroxide at reflux
temperature to afford 2-[(1-methyl-1H-pyrrol-2-ylsulfonyl)-
methyl]aniline (5) in 88% yield. In the last step aniline 5 was
condensed with benzaldehyde, 4-trifluoromethylbenzaldehyde,
4-dimethylaminobenzaldehyde, 4-nitrobenzaldehyde, 5-bromo-
2-methoxybenzaldehyde, 4-fluorobenzaldehyde, 2-chloroben-
zaldehyde, 4-chlorobenzaldehyde, or 1H-pyrrole-2-carbalde-
hyde by heating in dry propan-2-ol under argon containing
dry sodium sulfate and a few drops of glacial acetic acid. The
corresponding imines 6−14 were obtained in yields of 40−
1
87%. In the H NMR spectra of imines 6−14 characteristic
features are the singlet in the region 4.66−4.73 ppm, integrating
for two protons corresponding to the methylene group and the
singlet in the region 7.68−8.32 ppm and integrating for one
proton corresponding to the imine proton.
RESULTS AND DISCUSSION
■
Arylmethylsulfonylpyrrole Derivatives Screening and
in Silico Study with hGSTA1-1. In order to reveal the
inhibitory potency of the arylmethylsulfonylpyrroles against
hGSTA1-1, all compounds were subjected to experimental
screening against hGSTA1-1 activity, aided by in silico
molecular docking. It is evident (Table 1) that all compounds
sharing the arylmethylsulfonylpyrrole core structure inhibit
hGSTA1-1 activity to a different extent. Figure 1a is an in silico
presentation of the V-shaped binding area of hGSTA1-1
derived from crystallographic data of the isoenzyme structure as
a complex with the substrates ethacrynic acid and glutathione
(PDB code 1GSE). The V-shaped binding area consists of a
catalytic primary site (Figure 1a, upper area), designated as the
H-site, where hydrophobic substrates bind (CDNB, ethacrynic
acid, etc.), and of a neighboring secondary site (Figure 1a,
bottom area) where the same and other ligands may bind as
well. The hydrophilic tripeptide substrate GSH binds at the G-
site located in front of the entrances leading to the V-shaped
binding sites (Figure 1a, left area). In agreement with the
catalytic function of the enzyme, the present model predicts
proximity between the OH group of the catalytically essential
Tyr9 residue, the Cl atom of CDNB, and the sulfhydryl group
of GSH (Figure 1a and Figure SI-2). Furthermore, two
orthogonal views of space-filling models of the arylmethylsulfo-
nylpyrrole (shown in analogue 9, Figure SI-3) confirm the
advantage of choosing this core structure in terms of analogue
complementarity with the GST binding site and the shape
compliance with the V-shaped binding pocket. In more detail,
Figure 1b presents the optimal view of interaction of the core
structure present on derivative 9 as follows, going clockwise:
(A) the methylated pyrrole fits tightly in the hydrophobic
pocket formed by residues Ala12, the hydrophobic part of the
side chain of Lys205, Pro206, Pro207, and Met208; (B) the
sulfone interacts with the guanidine group of Arg13 and is
exposed to solvent; (C) the aryl ring is tightly packed between
hydrophobic Leu109, Pro110, and Tyr166; (D) a terminal p-
nitroarylimine moiety is shown (present on analogue 9),
located in the exit polar/charged channel facing the terminal
carboxylate of the substrate GSH.
CHEMISTRY
■
Compounds 4−14 (Table 1) were synthesized according to the
route depicted in Scheme 1. In the first step 1-(bromomethyl)-
Scheme 1. Synthetic Route Leading to
Arylmethylsulfonylpyrrole Derivatives 4−14
a
a
Reagents and conditions: (a) (1) NaBH4, dry i-PrOH, 0 °C, 1.5 h;
(2) 1 M NaOH in H2O, 0 °C to room temp, 2 h; (b)
2KHSO5·KHSO4·K2SO4, H2O, MeOH, room temp, 14 h; (c)
FeSO4·7H2O, 25% NH4OH, EtOH, 2 h; (d) ArCHO, dry i-PrOH,
glacial CH3CO2H, Na2SO4, argon, under reflux, 10 h.
2-nitrobenzene (1) was obtained by heating 2-nitrotoluene with
NBS and a catalytic amount of benzoyl peroxide in carbon
tetrachloride according to Kalir.37 Thiocyanation of 1-methyl-
1H-pyrrole to 1-methyl-1H-pyrrol-2-yl thiocyanate (2) took
place using ammonium thiocyanate in the presence of
molecular iodine in methanol following the procedure of
Yadav et al.38 It was found that an optimum yield of 63% was
obtained for 1-methyl-2-[(2-nitrobenzylsulfanyl)]-1H-pyrrole
(3) by reacting bromide 1 with thiocyanate 2 and sodium
borohydride in dry ethanol under argon at 0 °C and then
allowing the reaction mixture to reach room temperature.
Compound 3 is unstable but can be stored for several weeks at
≤0 °C. In the next step, sulfonylpyrrole 3 was dissolved in
methanol and oxidized by the dropwise addition of 2 equiv of
peroxymonosulfuric acid in water to give 1-methyl-2-[(2-
nitrobenzyl)sulfonyl)]-1H-pyrrole (4) in 68% yield. Reduction
of the nitro group of 4 took place in ethanol using ferrous
On the basis of the enzyme inhibition screening of the
pyrrole derivatives (Table 1), one distinguishes three groups of
inhibitory potency: a group of low inhibition (up to
approximately 30%, compounds 5 and 6), a group of medium
inhibition (approximately 35−70%, compounds 14, 11, 4, 13,
10, and 7), and a group of high inhibition (above approximately
80%, compounds 8, 12, and 9). In addition, bromosulfoph-
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Figure 1. Low energy conformations of the substrates CDNB and GSH with (a) the arylmethylsulfonylpyrrole core structure present on inhibitor 9
(b) at the binding sites of hGSTA1-1 as predicted by in silico molecular docking. All ligands are shown as ball-and-stick renditions. GSH is depicted
in magenta. Cl atoms are in green, S atoms in yellow, N atoms in blue, and O atoms in red. The figure is created using the PYMOL program, version
1.4. (a) CDNB is bound in two locations of the enzyme V-shaped binding area (gray shade): the catalytic primary (upper area; CDNB is depicted in
violet, bound in the catalytically favored orientation having the departing Cl atom opposite the reactive SH-group of GSH) and the secondary (lower
area) binding sites. (b) Binding mode of the arylmethylsulfonylpyrrole core structure of compound 9 is the following, going clockwise: (A) the CH3-
pyrrole fits in the hydrophobic pocket formed by residues Ala12, the hydrophobic part of the side chain of Lys205, Pro206, Pro207, and Met208; (B)
the sulfone interacts with the guanidine group of Arg13 and is exposed to solvent; (C) the aromatic ring is tightly packed between hydrophobic
Leu109, Pro110, and Tyr166; (D) the p-nitroarylimino moiety, present on inhibitor 9, is located in the exit polar/charged channel toward the
terminal carboxylate of GSH.
Figure 2. Inhibitors 9 and 13 (a) and inhibitors 10 and 12 (b) in the binding sites of hGSTA1-1. Inhibitors occupy parts of the primary (left area)
and secondary (right area) binding sites of the enzyme. (a) Interactions of inhibitors 9 (violet) and 13 (blue). The subsite A (part of the secondary
binding site) is located toward the bottom area where hydrogen- and halogen-bond interactions are formed with inhibitors 9 and 13, respectively.
The O atoms (in red) of the p-nitro group of inhibitor 9 form H-bond interactions with the backbone HN group (in blue) (2.9 Å) and the HO
group (in red) (3.3 Å) of Ser18. The p-Cl atom (in green) of inhibitor 13 interacts with the backbone nitrogen atom (in blue) (3.3 Å) of Ser18. (b)
Interactions of inhibitors 10 (pink) and 12 (turquoise). The subsite B (part of the secondary binding site) is located toward the upper area where a
halogen-bond interaction may be formed between the o-Cl atom (in green) of inhibitor 12 with the backbone O atom (in red) (2.9 Å) of Gly103.
The m-Br atom (in dark red) of inhibitor 10 interacts with the backbone N atom (in blue) (3.3 Å) of Ser18. Interactions are depicted by dotted
yellow lines. The figure is created using the PYMOL program, version 1.4.
thalein (BSP) was put in the test as a control inhibitor39 under
the same conditions as for the pyrrole derivatives and found to
inhibit hGSTA1-1 by approximately 63%. All but two small
analogues (4 and 5, lacking the arylimine moiety) bind
uniquely at the V-shaped deep binding site of hGSTA1-1
(Figure SI-4). One distinguishes substituents of varying
chemical nature and position. The para-substituted derivative
9 is comparatively the most potent inhibitor (90.0%, Table 1),
followed by the ortho-substituted derivative 12. The electro-
negative p-nitro group of the nitrophenylimino moiety creates
H-bond interactions with the protein’s donor groups in subsite
A formed around residues 13−18 (Figure 2a), compared with
the p-chlorine atom of analogue 13 (60.4%) (Figure 2a) or the
p-fluorine atom of analogue 11 (37.1% inhibition) forming a
weak single halogen interaction (also termed “halogen bond”,
partial charge electrostatic interaction)40,41 with the backbone
nitrogen atom (in blue) of Ser18. Analogue 13 is a stronger
inhibitor compared to 11 probably because of its elevated
hydrophobic character, as evidenced by its higher prediction
octanol/water partition coefficient QPlogPo/w (Table 1, 4.4
for inhibitor 13 and 3.8 for inhibitor 11), which generally
ranges from −2 for the most polar compounds to 6.5 for the
most hydrophobic ones.42,43 Although the two other para-
substituted derivatives (7 and 8) fail to develop strong
hydrogen bonds, they still show good inhibitory potency
(68.4% and 78.9%, respectively), probably due to their high
hydrophobic character (QPlogPo/w equals 4.9 for compound 7
and 4.6 for compound 8) favoring binding to GSTs.
Inhibitor 12 with the Cl atom in the ortho position and
inhibitor 10 with the Br atom in the meta position interact with
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Figure 3. Low energy conformations of substrate GSH and inhibitors 4 (a) and 9 (b) at the most probable binding sites of hGSTA1-1 as predicted
by in silico molecular docking. All ligands are shown as ball-and-stick renditions. The figure is created using the PYMOL program, version 1.4. (a)
Inhibitor 4 is bound in the catalytic primary (yellow ligand) and the noncatalytic secondary (turquoise ligand) binding sites. (b) Inhibitor 9 occupies
parts of the catalytic primary (upper area) and secondary (bottom area) binding sites. GSH is depicted in magenta, and the S atom is in yellow. N
atoms are in blue and O atoms in red.
Figure 4. Purely competitive inhibition kinetics of hGSTA1-1 with inhibitor 9 using CDNB as a variable substrate. (a) Lineweaver−Burk (double
reciprocal) plot of initial velocities of hGSTA1-1 vs [CDNB] (22.5−990 μM) at different concentrations of inhibitor 9 (0−60 μM). (b) Secondary
plot derived from data of plot (a). The inhibition constant Ki for inhibitor 9 is the intercept on the basis axis of plot (b). Points are the average of
three enzyme assays. The plot is created using the GraFit program, version 3.
the protein backbone, the former (12) forming a halogen-bond
interaction with the more electronegative backbone O of Gly
103 in subsite B and the latter (10) forming an interaction with
the backbone nitrogen atom of Ser 18 in subsite A (Figure 2b).
This feature, when combined with a good hydrophobic
character (QPlogPo/w equals 3.8 for compound 12 and 4.1
for compound 10), leads to fairly effective inhibition.
Compound 6, possessing no polar group on the aromatic
ring, expectedly shows much smaller inhibition. Furthermore,
the two shorter derivatives (4 and 5) lack the arylimine moiety
and differ only by the substituent of the aromatic group: a nitro
group for 4 and an amino group for 5, the latter being the
“parent” compound from which all others were synthesized
(Scheme 1). Compound 4 possessing a nitro group but without
the nitrophenylimino moiety binds in the most probable
position of binding (catalytic primary binding site) for all
arylimino-bearing analogues and with higher flexibility but
without formation of apparent H-bond interactions (Figure 3a,
yellow ligand), while a second probable binding position
toward the protein surface (part of the secondary binding site)
and closer to the entrance leading to the deep binding area is
predicted (Figure 3a, turquoise ligand), allowing the formation
of H-bond interactions with the protein (not shown).
On the basis of the experimental findings and in silico
interpretations, the arylmethylsulfonylpyrrole core structure can
be regarded as a new pharmacophore for hGSTA1-1.
Compound 9 (the most potent arylimino-bearing inhibitor)
and compound 4 (the most potent small inhibitor), both
sharing a terminal p-nitro group and the arylmethylsulfonyl-
pyrrole core structure, were chosen for further investigation by
enzyme inhibition kinetics in order to better understand their
interaction with hGSTA1-1.
Kinetic Inhibition Studies of hGSTA1-1 with the
Arylmethylsulfonylpyrroles 4 and 9. Prior to proceeding
with the kinetic inhibition experiments, control studies were
implemented with our enzyme preparation using BSP as a
known hGSTA1-1 inhibitor.39 In silico molecular docking
analysis predicted interaction of BSP with a noncatalytic
binding site of hGSTA1-1, allowing binding of CDNB in the
catalytic primary site (Figure SI-5a). In agreement with earlier
observations,39 this result was confirmed by kinetic inhibition
studies using BSP and CDNB as a variable substrate showing a
noncompetitive mode of inhibition for hGSTA1-1 (Figure SI-
5b). Under the same experimental conditions, benzylsulfonyl-
GSH has shown a competitive mode of inhibition.19
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Figure 5. Parabolic competitive inhibition kinetics of hGSTA1-1 with inhibitor 4 using CDNB as a variable substrate. (a) Lineweaver−Burk (double
reciprocal) plot of initial velocities of hGSTA1-1 against [CDNB] (22.5−990 μM) at different concentrations of inhibitor 4 (0−80 μM). (b)
Secondary plot derived from data of plot (a). Points are the average of three enzyme assays. The plot is created using the GraFit program, version 3.
(a) Inhibition Studies of hGSTA1-1 with Compound 9.
Two sets of experiments were accomplished, each using either
CDNB (22.5−990 μM) or GSH (40−2475 μM) as a variable
substrate, in the presence of compound 9 at different constant
concentrations (0, 15, 30, and 60 μM).
With CDNB as a variable substrate, compound 9 displayed a
purely competitive inhibition profile on the basis of the linearity
observed for both the double reciprocal graph (Figure 4a) and
its secondary plot (Figure 4b).44−46 This behavior implies that
inhibitor 9 competes with CDNB for the same binding site of
GST, calculated inhibition constant Ki(9) = 71 4 μM (from
Figure 4b).
GSH, and inhibitor 9 on the same hGSTA1-1 molecule.
Nonetheless, this possibility exists for the small inhibitor 4
(section b).
(b) Inhibition Studies of hGSTA1-1 with Compound 4.
Two sets of experiments were accomplished, each having either
CDNB (22.5−990 μM) or GSH (40−2475 μM) as a variable
substrate, in the presence of inhibitor 4 at different constant
concentrations (0, 10, 20, 40, 60, and 80 μM).
With CDNB as a variable substrate, for inhibitor 4 lacking
the arylimino moiety the criterion for competitive inhibition is
satisfied only on the basis of the observed linearity and the
common intercept on the vertical axis for the double reciprocal
graph of initial velocities of hGSTA1-1 vs [CDNB] at various
constant concentrations of inhibitor 4 (Figure 5a). However,
the derived secondary plot (Figure 5b) reveals a parabolic
competitive inhibition profile (S-linear I-parabolic competitive
inhibition) with the points of the plot curving upward.44,46
Therefore, it is conceivable that the binding of small molecule 4
at the large V-shaped binding site of GST would not stop or
restrict the binding of a second molecule of inhibitor 4 so that
two molecules of 4 could be bound to GST. This kinetic model
is confirmed by in silico molecular docking, predicting the
catalytic primary and noncatalytic secondary binding sites as the
two most probable locations for binding to occur (Figure 3a).
This view is strengthened by earlier observations suggesting the
existence of multiple binding sites with GSTs for a single
compound, often with different affinity and inhibitory
activity.12,39,47−49 For example, isothermal titration calorimetry
and kinetic studies of hGSTA1-1 with BSP indicated that each
subunit possesses two types of binding sites for BSP, one of
which is located at or near the active site and is capable of
binding BSP in a noncompletive manner versus CDNB.39,48
Since BSP and CDNB bind to different sites in hGSTA1-1
(Figure SI-5), it is conceivable that the inhibition caused by
BSP could be attributed to structural changes resulting in
reduced binding/catalytic ability of the substrate by the active
site.47,48 Therefore, it is reasonable to assume that binding of
inhibitor 4 to the noncatalytic secondary site of GST could
affect the catalytic function of the adjacent primary binding site,
leading to a further decline of activity and increase of inhibition
in addition to that occurring upon binding of 4 to the catalytic
site alone (Figure 2a). This is in agreement with crystallo-
graphic observations50 according to which ligand binding is
This kinetic model is supported by in silico molecular
docking suggesting that inhibitor 9 is large enough so that when
bound to GST, it restricts CDNB from binding at the catalytic
primary site of the enzyme. Figure 3b shows the V-shaped
binding site of the enzyme in complex with inhibitor 9
occupying parts of the catalytic primary and the secondary sites,
and the substrate GSH at its G binding site (left) located in
front of the entrances leading to the V-shaped binding sites.
With GSH as a variable substrate, compound 9 has shown a
mixed inhibition profile, being evident from the lines of the
primary double reciprocal plot of initial velocities of hGSTA1-1
vs [GSH] intersecting to the left of the reciprocal velocity axis
(Figure SI-6).44,45 Furthermore, the linearity observed with the
secondary plots depicting slope or intercept vs [inhibitor 9]
(Figure SI-7) is supportive of a purely mixed type of
inhibition.45 The equilibrium scheme for this type of
inhibition45,46 predicts that inhibitor 9 could bind both the
free GST (forming GST−9) and the GST−GSH complex
(forming GST−GSH−9). The latter case suggests interaction
of 9 with a site different from the GSH-binding site, that site
being partly the catalytic primary CDNB-binding site, as shown
earlier (Figures 3b and 4). In such a case, interaction of the
cosubstrate CDNB with either of complexes GST−9 and
GST−GSH−9 is restricted because of competition between
CDNB and inhibitor 9 for the same site; hence both GST−9
and GST−GSH−9 are dead-end (unreactive) complexes.
Conclusively, the large inhibitor 9, bearing the arylimino
moiety, binds at the V-shaped binding site in a manner
prohibiting simultaneous CDNB and GSH binding for catalytic
function, rendering impossible the formation of catalytic
quadruple complex, i.e., simultaneous binding of CDNB,
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sensitive to conformational changes in the C-terminal region of
hGST A1-1 and this region forms part of the active site. The
different conformations of the C-terminal region suggest that
structural cooperativity could play a role in the activity of the
enzyme.48−50 The nonlinearity observed by the secondary plot
(Figure 5b) leads to calculating the inhibition constant for
analogue 4 from a linear double reciprocal plot, exploiting the
secondary site is being occupied by inhibitor 4 rather than a
CDNB molecule, a reactive quadruple complex is formed
(Figure 7) as a result of simultaneous binding of CDNB, GSH,
Δslope function against concentration of inhibitor 4 (Ki(4)
=
135 27 μM) (Figure SI-8).45 A comparison of the inhibition
constants of compounds 4 and 9 leads to the conclusion that
compound 9 may serve as a lead structure for further
exploitation. This is supported by the substantially lower free
energy of binding (FEB, in silico calculations) for compound 9
(−9.82 kcal/mol) compared to compound 4 (−7.33 kcal/mol).
With GSH as a variable substrate, compound 4 has shown a
partially mixed inhibition profile (equivalent to S-linear I-
hyperbolic noncompetitive inhibition),46 as indicated by the
lines of the primary double reciprocal graph of initial velocities
of hGSTA1-1 vs [GSH] intersecting the left of the reciprocal
velocity axis (Figure SI-9) and the nonlinear pattern of the
derived secondary plot that curves downward to a limiting rate
(Figure 6). These findings are supportive of an inhibition
Figure 7. Quadruple catalytic complex of hGSTA1-1 with CDNB,
GSH, and inhibitor 4 as predicted by in silico molecular docking. All
ligands are shown as ball-and-stick renditions. CDNB (in violet) is
bound in the catalytic primary site, whereas inhibitor 4 (in pink) is
bound in the noncatalytic secondary site. GSH is depicted in magenta,
Cl atom in green, and S atom in yellow. N atoms are in blue and O
atoms in red. The figure is created using the PYMOL program, version
1.4.
and inhibitor 4 on the same hGSTA1-1 molecule. By
combining these findings with those obtained from using
CDNB as a variable substrate, one concludes that inhibitor 4 is
small enough to interact with the catalytic CDNB-binding site
forming a dead-end (unreactive) complex and with the
secondary site forming a catalytic (reactive) complex, in the
latter case the catalytic site being occupied by CDNB.
CONCLUDING REMARKS
■
This is the first report on pyrrole inhibitors against GSTs.
Eleven new arylmethylsulfonylpyrrole derivatives were synthe-
sized and studied with human GSTA1-1 involved in MDR. In
silico molecular modeling revealed two locations in the V-
shaped binding site where the pyrrole analogues could be
accommodated: the primary reactive one in the vicinity of
GSH’s sulfhydryl group, where both shorter and longer
analogues could be accommodated, and the nonreactive
secondary one toward the C-terminal of the glutamate of
GSH, where smaller analogues can also noncompetitively
occupy. Enzyme inhibition screening indicated the arylmethyl-
sulfonylpyrrole core structure as being a new pharmacophore
and the 2-(pyrrolesulfonylmethyl)-N-4-nitrophenylimine deriv-
ative 9 as exhibiting the highest inhibitory potency. Inhibitor 9
competitively binds partly at the catalytic primary site against
the substrate CDNB and extends its interaction also with the
secondary site, thus prohibiting simultaneous CDNB binding. A
smaller inhibitor (4) lacking the arylimino moiety can bind at
both the primary and secondary sites. The advantage of the
present new derivatives, apart from the complementarity with
the binding site elements formed by the GST side chains, is the
shape compliance with the V-shaped binding pocket. Hence,
compound 9 is regarded as a lead structure useful in designing
new inhibitors and respective prodrugs against human GSTs.
Figure 6. Secondary plot showing the partially mixed inhibition
kinetics of hGSTA1-1 with inhibitor 4 using GSH as a variable
substrate. The plot is derived from data of the Lineweaver−Burk plot
depicting initial velocities of hGSTA1-1 vs [GSH] at different inhibitor
concentrations (shown in Figure SI-9). The plot is created using the
GraFit program, version 3.
model45,46 predicting the small inhibitor 4 to bind at the free
GST and the GST−GSH complex, leading to formation of at
least two complexes, GST−CDNB−4 and GST−CDNB−
GSH−4, respectively, with a CDNB molecule possibly present
on the complexes because of use of enzyme-saturating CDNB
concentration in the respective assays. Although the GST−
CDNB−4 complex is unreactive, this model foresees break-
down of the GST−CDNB−GSH−4 complex to products at a
rate less than that in the absence of inhibitor.45,46 This could be
possible if one assumes that inhibitor 4 binds at sites different
from the GSH- and CDNB-binding sites, predicting simulta-
neous binding of three molecules (inhibitor 4 and the two
substrates) on the same GST molecule for catalytic function to
occur. This model is in agreement with in silico molecular
docking, predicting a primary and a secondary location in the
V-shaped binding site of hGSTA1-1 as the two most probable
binding locations for CDNB (Figure 1a). When the nonreactive
6808
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Journal of Medicinal Chemistry
Article
0.1% HCO2H) m/z calculated for C12H13N2O2S [M + H]+ 249.0692,
found 249.0685.
EXPERIMENTAL SECTION
■
Chemistry. Melting points were taken on a Buchi 510 apparatus
̈
1-Methyl-2-[(2-nitrobenzyl)sulfonyl)]-1H-pyrrole (4, Table
1). To a stirred solution of 2-[(2-nitrobenzyl)sulfanyl]-1H-pyrrole 3
(2.4 g, 9.7 mmol) in methanol (120 mL) was added a solution of
peroxymonosulfuric acid (5.99 g, 9.7 mmol) in water (150 mL), and
the mixture was left stirring at room temperature for 4 h. TLC
examination of the reaction mixture verified that the reaction was
incomplete, and therefore, a further portion of peroxymonosulfuric
acid (5.99 g, 9.7 mmol) in water (150 mL) was added dropwise and
stirring at room temperature continued for 6 h. The reaction mixture
was quenched with water (300 mL) and extracted with dichloro-
methane (3 × 100 mL). The combined organic extracts were washed
with brine (150 mL), dried (Na2SO4), and the solvent was evaporated
under reduced pressure. The oily residue was purified by column
chromatography (ethyl acetate/hexane, 1:2) to give the title
compound (1.84 g, 68%) as a yellow solid (microcrystals from
toluene), mp 111−112 °C. Rf = 0.2 (ethyl acetate/hexane 1:2). IR
(Nujol) vmax 3119, 2956, 1519, 1354, 1316 cm−1. 1H NMR (CDCl3) δ
3.63 (s, 3H, CH3), 4.94 (s, 2H, CH2), 6.08 (dd, 1H, J = 4.0, 2.6 Hz, H-
4), 6.53 (dd, 1H, J = 4.0, 1.8 Hz, H-3), 6.79 (t, 1H, J = 2.2 Hz, H-5),
7.66−7.45 (m, 3H, H-4′, H-5′, H-6′), 7.96 (dd, 1H, J = 8, 1.4 Hz, H-
3′). 13C NMR (CDCl3) δ 35.45, 58.99, 108.62, 120.26, 123.40, 125.21,
125.67, 129.87, 130.44, 133.20, 134.45. HRMS (ESI, MeOH, 0.1%
HCO2H) m/z calculated for C12H13N2O4S [M + H]+ 281.0591, found
281.0595.
and are uncorrected. Unless otherwise noted, reactions were carried
out under an argon atmosphere in a flame-dried, three-neck round-
bottomed flask with magnetic stirring. Organic solutions were
1
concentrated by rotary evaporation at 23−40 °C under 15 Torr. H
and 13C NMR spectra were measured in CDCl3 or DMSO-d6 on a
1
250, 400, or 500 MHz Bruker spectrometer. H chemical shifts are
̈
reported in ppm from TMS as internal standard, with residual
chloroform appearing at 7.26 ppm or DMSO-d6 at 2.50 ppm. 13C
NMR chemical shifts are reported in ppm from TMS as internal
standard, with residual chloroform (77.00 ppm) or DMSO-d6 (39.43
ppm). High resolution ESI mass spectra were measured on a Thermo
Fisher Scientific LTQ-ORBITRAP/LC−MS system. Low resolution
ESI spectra were measured with an Agilent 1100/LC−MS system. IR
spectra were acquired on a Perkin-Elmer 257 or a Perkin-Elmer GX
FTIR spectrophotometer as liquids between sodium chloride disks or
KBr disks for solids and are reported in wavenumbers (cm−1).
Analytical thin layer chromatography was performed with TLC plates
(Merck 70−230 mesh silica gel). Visualization was done under a 254
nm UV light source and generally by immersion in acidic aqueous
ethanolic vanillin solution or in potassium permanganate (KMnO4),
followed by heating using a heat gun. Purification of reaction products
was generally done by dry-column flash chromatography51 using
Merck silica gel 60 and/or flash chromatography52 using Carlo Erba
Reactifs-SDS silica gel 60. Chemicals and solvents were purchased
from Aldrich, Alfa Aesar, or Merck and were of the highest possible
purity. The purity of the synthesized compounds was checked in a
liquid chromatography−UV diode array system coupled to ion-trap
mass spectrometry with electrospray ionization interface (LC/DAD/
ESI-MSn) and found to be above 95% (indicative example in the
Supporting Information Figure SI-10). All LC−MS experiments were
performed on a quadrupole ion-trap mass analyzer (Agilent
Technologies, model MSD trap SL) retrofitted to a 1100 binary
HPLC system equipped with a degasser, autosampler, diode array
detector, and electrospray ionization source (Agilent Technologies,
Karlsruhe, Germany). All hardware components were controlled by
Agilent Chemstation software. The sample was dissolved in MeOH/
0.1% formic acid to the desired concentration. Separation was achieved
on a reverse-phase 25 cm × 4.6 mm C8 analytical column at a flow rate
of 0.4 mL min, using solvent A (MeOH/formic acid, 99.9:0.1 v/v) and
solvent B (water or CH3CN/formic acid, 99.9:0.1 v/v). The elution
was in isocratic mode (85% from solvent A for 20 or 30 min). The
UV/vis spectra were recorded in the range of 200−400 nm, and
chromatograms were acquired at 254, 280, and 302 nm.
2-[(1-Methyl-1H-pyrrol-2-ylsulfonyl)methyl]aniline (5, Table
1). To a stirred solution of 1-methyl-2-[(2-nitrobenzyl)sulfonyl)]-1H-
pyrrole 4 (2.0 g, 7.13 mmol) in hot 95% ethanol (120 mL) was added
a suspension of ferrous sulfate heptahydrate (19.9 g, 71.6 mmol) in
water (80 mL) followed by 25% ammonium hydroxide (20 mL). The
resulting mixture was heated to reflux for 2 h during which 25%
ammonium hydroxide (60 mL) was added dropwise. After cooling, the
reaction mixture was passed through a pad of Celite, and then the filter
aid was washed with small aliquots of hot ethanol. The organic solvent
was evaporated under reduced pressure, and the resulting aqueous
suspension was extracted with dichloromethane (3 × 40 mL). The
combined organic extracts were washed with brine (40 mL), dried
(Na2SO4), and the solvent was evaporated under reduced pressure.
The residue was purified by column chromatography (ethyl acetate/
hexane, 1:2) to give the title compound (1.57 g, 88%) as a pale yellow
solid (microcrystals from dichloromethane/hexane), mp 111−113 °C.
Rf = 0.2 (ethyl acetate/hexane, 1:2). IR (nujol) vmax 3455, 3377, 3128,
1
2972, 1290, 1170 cm−1. H NMR (CDCl3) δ 3.19 (s, 3H, CH3), 4.11
(br s, 2H, NH2), 4.35 (s, 2H, CH2), 6.15 (dd, 1H, J = 4, 2.6 Hz, H-4),
6.49 (dd, 1H, J = 7.6 Hz, 1.5 Hz, H-3′), 6.60 (dd, 1H, J = 7.5, 1.2 Hz,
H-5′), 6.61 (t, 1H, J = 2.2 Hz, H-5), 6.71 (dd, 1H, J = 7.4, 0.8 Hz, H-
6′), 6.89 (dd, 1H, J = 4.0, 1.8 Hz, H-3), 7.11 (dt, 1H, J = 7.9, 1.6 Hz,
H-4′). 13C NMR (CDCl3) δ 34.97, 61.24, 108.39, 113.95, 117.38,
119.14, 119.42, 125.29, 129.70, 130.07, 132.38, 146.83. HRMS (ESI,
MeOH, 0.1% HCO2H) m/z calculated for C12H15N2O2S [M + H]+
251.0849, found 251.0840.
General Procedure for the Synthesis of Imines 6−14 (Table
1). To a warm solution of 2-[(1-methyl-1H-pyrrol-2-ylsulfonyl)-
methyl]aniline 5 (1 mmol) in propan-2-ol (20 mL) under argon were
added a few drops of glacial acetic acid until pH 4−5 was obtained,
followed by anhydrous sodium sulfate (0.5 g) and benzaldehyde, 4-
trifluoromethylbenzaldehyde, 4-dimethylaminobenzaldehyde, 4-nitro-
benzaldehyde, 5-bromo-2-methoxybenzaldehyde, 4-fluorobenzalde-
hyde, 2-chlorobenzaldehyde, 4-chlorobenzaldehyde, or 1H-pyrrole-2-
carbaldehyde (1.2 mmol). The reaction mixture was heated under
reflux for 10 h. Sodium sulfate was filtered off and the solvent
evaporated under reduced pressure. To the residue was added
dichloromethane (20 mL). The resulting solution was washed with
20% aqueous sodium bisulfite (3 × 5 mL), brine (10 mL), dried
(Na2SO4) and the solvent evaporated under reduced pressure to give a
solid. The solid was purified by column chromatography (ethyl
acetate/hexane, 1:4) to afford imine 6, 7, 12, or 14 or by crystallization
to afford imine 8, 9, 10, or 11.
1-Methyl-2-[(2-nitrobenzyl)sulfanyl]-1H-pyrrole (3, Scheme
1). To a stirred solution of 1-(bromomethyl)-2-nitrobenzene 1 (6.05
g, 28 mmol) at 0 °C in dry ethanol (100 mL) under an atmosphere of
argon was added sodium borohydride (2.44 g, 64 mmol). A solution of
1-methyl-1H-pyrrol-2-yl thiocyanate 2 (4.14 g, 30 mmol) in dry
ethanol (20 mL) was added dropwise over a period of 20 min, and the
reaction mixture was left stirring at room temperature for 1.5 h. A
solution of sodium hydroxide (2 g, 50 mmol) in water (50 mL) was
added, and stirring was continued at room temperature for 2 h. The
reaction mixture was poured into water (150 mL), acidified with 1 M
HCl to pH 5−6, and extracted with dichloromethane (3 × 65 mL).
The combined organic extracts were washed with brine (100 mL),
dried (Na2SO4), and the solvent was evaporated under reduced
pressure. The oily residue was purified by column chromatography
using as eluant (dichloromethane/hexane, 1:6) to give the title
compound as a caramel oil. Yield, 4.02 g, 58%. Rf = 0.13
(dichloromethane/hexane, 1:2). IR (Nujol) vmax 3115, 2954, 1513,
1346, 859 cm−1. 1H NMR (CDCl3) δ 3.37 (s, 3H, CH3), 4.12 (s, 2H,
CH2), 6.05 (dd, 1H, J = 3.6, 2.9 Hz, H-4), 6.16 (dd, 1H, J = 3.7, 1.8
Hz, H-3), 6.72 (dd, 1H, J = 2.5, 2.0 Hz, H-5), 6.98 (dd, 1H, J = 7.1, 1.9
Hz, H-6′), 7.50 (m, 2H, H-4′, H-5′), 7.97 (dd, 1H, J = 7.1, 2.3 Hz, H-
3′). 13C NMR (CDCl3) δ 33.44, 39.67, 108.11, 118.74, 124.98, 125.43,
126.75, 127.96, 132.08, 132.86, 133.89, 148.06. HRMS (ESI, MeOH,
6809
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Journal of Medicinal Chemistry
Article
2-{[(1-Methyl-1H-pyrrol-2-yl)sulfonyl]methyl}-N-[(1E)-
(phenyl)methylene]aniline (6, Table 1). Light brown viscous oil
(0.20 g, 59%) (column chromatography). Rf = 0.43 (ethyl acetate/
dichloromethane, 2:1). IR (Nujol) νmax 3065, 2986 1628, 1313, 1109
cm−1. 1H NMR (CDCl3) δ 3.25 (s, 3H, CH3), 4.73 (s, 2H, CH2), 5.83
(t, 1H, J = 2.5 Hz, H-4), 6.31−6.38 (m, 2H, H-3, H-5), 6.87 (d, 1H, J
= 7.5 Hz, H-3′), 7.23−7.49 (m, 6H, H-4′, H-6′, H-5′, H-3″, H-4″, H-
5″), 7.70−7.79 (m, 2H, H-2″, H-6″), 7.89 (s, 1H, NCH). 13C NMR
(CDCl3) δ 35.53, 59.17, 108.09, 117.99, 120.02, 123.45, 126.31,
126.32, 128.78, 129.06, 129.75, 130.25, 131.71, 132.44, 135.93, 151.19,
158.01, 159.54. HRMS (ESI, MeOH) m/z calculated for
C19H19N2O2S [M + H]+ 339.1162, found 339.1157.
1H, H-3), 6.35 (t, 1H, J = 1.9 Hz, H-5), 6.43 (dd, 1H, J = 4.0, 1.8 Hz,
H-3), 6.83 (d, 1H, J = 8.8 Hz, H-3″), 6.90 (d, 1H, J = 7.1 Hz, H-3′),
7.25 (ddd, 1H, J = 7.4, 1.0 Hz H-5′), 7.36 (ddd, 1H, J = 7.5, 1.4 Hz, H-
4′), 7.53−7.46 (m, 2H, H-6′, H-4″), 8.07 (d,1H, J = 2.5 Hz, H-6″),
8.32 (s, 1H, NCH). 13C NMR (CDCl3) δ 35.38, 55.80, 59.15,
108.01, 113.02, 113.26, 117.97, 119.63, 123.56, 125.01, 126.10, 126.30,
129.42, 129.81, 130.04, 132.43, 135.17, 151.03, 153.49, 158.11. HRMS
(ESI, MeOH, 0.1% HCO2H) m/z calculated for C20H2079BrN2O3S [M
+ H]+ 447.0363, found 447.0363.
N-[(1E)-(4-Fluorophenyl)methylene]-2-{[(1-methyl-1H-pyr-
rol-2-yl)sulfonyl]methyl}aniline (11, Table 1). Light brown
microcrystals (0.275 g, 78%) (crystallization from methyl tert-butyl
ether/hexane), mp 93−94 °C. Rf = 0.27 (ethyl acetate/hexane, 1:2).
2-{[(1-Methyl-1H-pyrrol-2-yl)sulfonyl]methyl}-N-{(1E)-[4-
(trifluoromethyl)phenyl]methylene}aniline (7, Table 1). Light
yellow microcrystals (0.27 g, 67%) (column chromatography,
crystallization from dichloromethane/hexane), mp 140−141 °C. Rf =
0.43 (ethyl acetate/hexane, 1:2). IR (Nujol) νmax 3058, 2975, 1632,
1
IR (Nujol) νmax 3116, 2922, 1630, 1312, 1110, 1218 cm−1. H NMR
(CDCl3) δ 3.18 (s, 3H, CH3), 4.69 (s, 2H, CH2), 5.79 (dd, 1H, J = 4.0,
2.6 Hz, H-4), 6.33 (t, 1H, J = 2.2 Hz, H-5), 6.38 (dd, 1H, J = 4.0, 1.9
Hz, H-3), 6.85 (dd, 1H, J = 7.8, 1.0 Hz, H-3′), 7.12 (t, 2H, J = 8.6 Hz,
H-2″, H-6″), 7.22 (ddd 1H, J = 7.6, 1.2 Hz, H-5′), 7.34 (ddd, 1H, J =
7.6, 1.5 Hz, H-4′), 7.44 (dd, 1H, J = 7.5, 1.4 Hz, H-6′), 7.75 (dd, 2H, J
= 8.7, 5.5 Hz, H-3″, H-5″), 7.86 (s, 1H, NCH). 13C NMR (CDCl3)
δ 35.22 (CH3), 59.06 (CH2), 107.96 (C-4), 115.59 (C-3″, C-5″),
115.94 (C-3″, C-5″), 117.78 (C-3′), 119.67 (C-3), 123.15 (C-2′),
125.13 (C-2), 126.13 (C-5′), 129.43 (C-5), 130.08 (C-4′), 130,74 (C-
2″, C-6″), 130.88 (C-2″, C-6″), 132.15 (C-1″), 132.25 (C-6′), 150.79
(C-1′), 157.81 (CN), 164.59 (C-4″). HRMS (ESI, MeOH, 0.1%
HCO2H) m/z calculated for C19H18FN2O2S [M + H]+ 357.1068,
found 357.1059.
N-[(1E)-(2-Chlorophenyl)methylene]-2-{[(1-methyl-1H-pyr-
rol-2-yl)sulfonyl]methyl}aniline (12, Table 1). Yellow micro-
crystals (0.28 g, 80%) (column chromatography, crystallization from
dichloromethane/hexane), mp 124−125 °C. Rf = 0.4 (ethyl acetate/
hexane, 1:2). IR (Nujol) νmax 3123, 2929, 1620, 1308, 1110, 1052
cm−1. 1H NMR (CDCl3) δ 3.12 (s, 3H, CH3), 4.69 (s, 2H, CH2), 5.76
(t, 1H, J = 3.2 Hz, H-4), 6.32−6.33 (m, 2H, H-5, H-3), 6.88 (d, 1H, J
= 7.7 Hz, H-3′), 7.21−7.37 (m, 5H, H-4′, H-5′, H-6′, H-4″, H-6″),
7.46 (d, 1H, J = 6.6 Hz, H-5″), 8.07 (d, 1H, J = 7.07 Hz, H-3″), 8.30
(s, 1H, NCH). 13C NMR (CDCl3) δ 34.96, 59.09, 107.95, 117.70,
119.37, 123.61, 124.29, 126.37, 126.67, 128.11, 129.38, 129.73, 129.93,
132.19 (2C), 132.21, 135.64, 150.35, 154.83. HRMS (ESI, MeOH,
0.1% HCO2H) m/z calculated for C19H18ClN2O2S [M + H]+
373.0772, found 373.0761.
1
1306, 1108, 1326 cm−1. H NMR (CDCl3) δ 3.17 (s, 3H, CH3), 4.71
(s, 2H, CH2), 5.80 (dd, 1H, J = 3.9, 2.6 Hz, H-4), 6.36−3.37 (m, 1H,
H-5), 6.41 (dd, 1H, J = 4.0, 1.8 Hz, H-3), 6.91 (d, 1H, J = 7.8 Hz, H-
3′), 7.26 (ddd, 1H, J = 7.4, 1.0 Hz, H-5′), 7.38 (ddd, 1H, J = 7.5, 1.4
Hz H-4′), 7.45 (dd, 1H, J = 7.4, 1.2 Hz, H-6′), 7.70 (d, 2H, J = 8.17
Hz, H-2″, H-6″), 7.88 (d, 2H, J = 8.07 Hz, H-3″, H-5″), 7.99 (s, 1H,
NCH). 13C NMR (CDCl3) δ 35.11 (CH3), 59.15 (CH2), 108.06
(C-4), 117.67 (C-3′), 119.60 (C-3), 123.41 (C-2′), 125.08 (C-2),
125.43 (C-2″ or C-6″), 125.48 (C-2″ or C-6″), 126.64 (C-5′), 128.94
(C-3″, C-5″), 129.43 (C-5), 130.12 (C-4′), 132.35 (C-6′), 132.42 (C-
4″), 132.90 (CF3), 138.77 (C-1″), 150.31 (C-1′), 157.55 (CN).
HRMS (ESI, MeOH, 0.1% HCO2H) m/z calculated for
C20H18F3N2O2S [M + H]+ 407.1036, found 407.1026.
N-{(1E)-[4-(Dimethylamino)phenyl]methylene}-2-{[(1-meth-
yl-1H-pyrrol-2-yl)sulfonyl]methyl}aniline (8, Table 1). Pale
yellow microcrystals (0.23 g, 60%) (crystallization from dichloro-
methane/hexane), mp 152−154 °C. Rf = 0.23 (ethyl acetate/hexane,
1
1:2). IR (Nujol) νmax 3119, 2861, 2808, 1588, 1310, 1106 cm−1. H
NMR (CDCl3) δ 3.08 [s, 6H, (CH3)2N], 3.30 (s, 3H, CH3) 4.73 (s,
2H, CH2), 5.84 (dd, 1H, J = 3.9, 2.6 Hz, H-4), 6.32−6.36 (m, 2H, H-5,
H-3), 6.71 (d, 2H, J = 8.9 Hz, H-3″, H-5″), 6.81 (d, 1H, J = 7.7 Hz, H-
3′), 7.18 (ddd, 1H, J = 7.5, 1.1 Hz, H-5′), 7.31 (ddd, 1H, J = 7.6, 1.3
Hz, H-4′), 7.48 (d, 1H, J = 7.5 Hz, H-6′), 6.71 (d, 2H, J = 8.9 Hz, H-
2″, H-6″), 7.68 (s, 1H, NCH). 13C NMR (CDCl3) δ 35.49 (CH3),
40.13 [(CH3)2N], 58.85 (CH2), 107.69 (C-4), 111.27 (C-3″, C-5″),
118.04 (C-3′), 119.88 (C-3), 122.96 (C-2′), 124.06 (C-1″), 125.01
(C-2), 125.11 (C-5′), 129.56 (C-5), 129.97 (C-4′), 130.61 (C-2″, C-
6″), 132.04 (C-6′), 152.04 (C-1′), 152.48 (C-4″), 159.18 (CHN).
HRMS (ESI, MeOH, 0.1% HCO2H) m/z calculated for C21H24N3O2S
[M + H]+ 382.1584, found 382.1575.
N-[(1E)-(4-Chlorophenyl)methylene]-2-{[(1-methyl-1H-pyr-
rol-2-yl)sulfonyl]methyl}aniline (13, Table 1). Colorless micro-
crystals (0.29 g, 83%) (chloroform/hexane), mp 117−118 °C. Rf =
0.23 (ethyl acetate/hexane, 1:2). IR (Nujol) νmax 3128, 2910, 1626,
1
1312, 1108, 1088 cm−1. H NMR (CDCl3) δ 3.16 (s, 3H, CH3), 4.66
(s, 2H, CH2), 5.77 (dd, 1H, J = 3.7, 2.6 Hz, H-4), 6.29 (s, 1H, H-3),
6.34 (dd, 1H, J = 3.7, 1.7 Hz, H-5), 6.82 (d, 1H, J = 7.7 Hz, H-3′),
7.24−7.53 (m, 5H, H-4′, H-5′, H-6′, H-2″, H-6″), 7.65 (d, 2H, J = 8.4
Hz, H-3″, H-5″), 7.82 (s, 1H, NCH). 13C NMR (CDCl3) δ 35.29,
59.09, 108.03, 117.75, 119.75, 123.28, 126.36, 128.93 (2C), 129.46,
129.96 (2C), 130.12, 132.35, 134.20, 137.50, 150.67, 157.80. HRMS
(MeOH, 0.1% HCO2H) m/z calculated for C19H18ClN2O2S [M + H]+
373.0772, found 373.0759.
2-{[(1-Methyl-1H-pyrrol-2-yl)sulfonyl]methyl}-N-[(1E)-1H-
pyrrol-2-ylmethylene]aniline (14, Table 1). Pale brown micro-
crystals (0.25 g, 77%) (column chromatography, crystallization from
dichloromethane/hexane), mp 134−136 °C. Rf = 0.21 (ethyl acetate/
hexane, 1:2). IR (Nujol) νmax 3362, 3119, 2910, 1620, 1296, 1106
cm−1. 1H NMR (CDCl3) δ 3.21 (s, 3H, CH3), 4.69 (s, 2H, CH2), 5.88
(dd, 1H, J = 3.9, 2.6 Hz, H-4), 6.26 (t, 1H, J = 2.9 Hz, H-4″), 6.42 (t,
1H, J = 2.1 Hz, H-5), 6.46 (dd, 1H, J = 4.0, 1.8 Hz, H-3), 6.57 (d, 1H,
J = 2.9 Hz, H-3″), 6.83 (d, 1H, J = 7.7 Hz, H-3′), 6.88 (s, 1H, H-5″),
7.16 (ddd, 1H, J = 7.4, 0.7 Hz, H-5′), 7.32 (ddd, 1H, J = 7.6, 1.3 Hz,
H-4′), 7.40 (dd, 1H, J = 7.5, 1.0 Hz, H-6′), 7.68 (s, 1H, NCH), 9.56
(br, s, 1H, NH). 13C NMR (CDCl3) δ 34.97 (CH3), 58.89 (CH2),
107.65 (C-4), 110.28 (C-4″), 116.33 (C-3″), 117.78 (C-3′), 119.43
(C-3), 122.76 (C-2′), 122.92 (C-5″), 125.12 (C-2), 125.20 (C-4′),
129.66 (C-5), 129.97 (C-5′), 130.46 (C-2″), 132.02 (C-6′), 148.81
2-{[(1-Methyl-1H-pyrrol-2-yl)sulfonyl]methyl}-N-[(1E)-(4-
nitrophenyl)methylene]aniline (9, Table 1). Yellow microcrystals
(0.33 g, 87%) (crystallization from ethyl acetate/hexane), mp 173−
174 °C. Rf = 0.32 (ethyl acetate/hexane, 1:2). IR (Nujol) νmax 3095,
1
2988, 1602, 1524, 1346, 1310, 1106 cm−1. H NMR (CDCl3) δ 3.16
(s, 3H, CH3), 4.71 (s, 2H, CH2), 5.80 (dd, 1H, J = 4.0, 2.6 Hz, H-4),
6.39 (t, 1H, J = 2.2 Hz H-5), 6.44 (dd, 1H, J = 4.0, 1.8 Hz, H-3), 6.94
(d, 1H, J = 7.2 Hz, H-3′), 7.30 (ddd, 1H, J = 7.5, 1.0 Hz, H-5′), 7.40
(ddd, 1H, J = 7.6, 1.4 Hz, H-4′), 7.45 (dd, 1H, J = 7.6, 1.2 Hz, H-6′),
7.94 (d, 2H J = 8.8 Hz, H-2″, H-6″), 8.06 (s, 1H, NCH), 8.30 (d,
2H, J = 8.8 Hz, H-3″, H-5″). 13C NMR (CDCl3) δ 35.21 (CH3), 60.91
(CH2), 108.27 (C-4), 117.61 (C-3′), 119.66 (C-3), 123.72 (C-2′),
123.85 (C-3″, C-5″), 125.29 (C-2), 127.18 (C-5′), 129.42 (C-2″, C-
6″, C-5), 130.25 (C-4′), 132.60 (C-6′), 141.08 (C-1″), 149.25 (C-4″),
150.07 (C-1′), 156.53 (CN). HRMS (ESI, MeOH, 0.1% HCO2H)
m/z calculated for C19H18N3O4S [M + H]+ 384.1013, found 384.1010.
N-[(1E)-(5-Bromo-2-methoxyphenyl)methylene]-2-{[(1-
methyl-1H-pyrrol-2-yl)sulfonyl]methyl}aniline (10, Table 1).
Pale yellow microcrystals (0.18 g, 40%) (crystallization from
dichloromethane/hexane), mp 131−132 °C. Rf = 0.18 (ethyl
acetate/hexane, 1:2). IR (Nujol) νmax 3070, 2982, 1622, 1318, 1112,
1268, 1050 cm−1. 1H NMR (CDCl3) δ 3.29 (s, 3H, CH3), 3.89 (s, 3H,
OCH3), 4.71 (s, 2H, CH2), 5.82 (dd, 1H, J = 4.0, 2.6 Hz, H-4), 6.24 (s,
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(CN), 151.03 (C-1′). HRMS (MeOH, 0.1% HCO2H) m/z
calculated for C17H18N3O2S [M + H]+ 328.1114, found 328.1105.
Modeling and Docking: In Silico Creation of the hGSTA1-1
Structure and Docking of Pyrrole Analogues to the hGSTA1-1
Model. The structure of glutathione transferase A1-1 (hGSTA1-1) in
complex with ethacrynic acid and its glutathione conjugate (PDB code
1GSE) was processed using MGLTools 1.5.4.53 The tripeptide
substrate glutathione was added to the protein PDBQT file. Docking
of the pyrrole analogues to hGSTA1-1 was performed using AutoDock
4.0.54 Autodock free-energy scoring function is based on a linear
regression analysis, the AMBER force field,55 and a large set of diverse
protein−ligand complexes with known binding constants. The
Lamarckian genetic algorithm (LGA) search method with default
parameters was used. Limits for 5 000 000 maximum number of energy
evaluations and 50 docking runs were set. All ligands were treated as
flexible. Docking results were both visually inspected and quantita-
tively evaluated based on the estimated free energy of binding (FEB).
For identifying the possible binding sites on the protein of the
kinetically studied analogues, 4 and 9, the following procedure was
used. After docking of the pyrrole analogues, docked analogue 9 was
merged in the protein file, the grid maps were recalculated on the
resulting complex, and the competing substrate CDNB was docked on
it. Similarly, docked analogue 4 was merged with the protein, and
CDNB and another 4 molecule were docked on the complex. All
figure depicting 3D models were created using PYMOL, version 1.4.56
Expression and Purification of hGSTA1-1 from Recombinant
E. coli cells. This was based on a published method57 with
modifications. Briefly, E. coli BL21 (DE3) cells harboring plasmid
pOXO4-GSTA1 were grown in Luria−Bertani (LB) medium
containing ampicillin (100 μg/mL) and chloramphenicol (25 μg/
mL). The expression of GST was induced by addition of 1 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) when the absorbance at
600 nm was 0.6. Four hours after induction, cells (2.5 g) were
harvested by centrifugation at 8000 rpm (4 °C) for 20 min and
resuspended in 7.5 mL of phosphate buffer (20 mM, pH 7), sonicated
(13 × 5 s), and centrifuged at 13 000 rpm for 5 min. A sample of 5 mL
from the supernatant (15 mL) was loaded to an affinity
chromatography column (2 mL adsorbent) previously equilibrated
with potassium phosphate buffer (20 mM, pH 7). The affinity
adsorbent (GSH-Sepharose-CL6B) contained the tripeptide gluta-
thione immobilized to cross-linked agarose (Sepharose-CL6B) which
was previously epoxy-activated with bis-epoxirane (1,4-butanediol
diglycidyl ether) in alkaline solution. Nonadsorbed protein was washed
off with 10 mL of equilibration buffer prior to desorbing bound GST
in equilibration buffer containing 10 mM GSH (21 mL). Fractions (3
mL each) with enzyme activity were polled (547 units in 10 mL total),
concentrated (3.5 mL, nitrocellulose, cutoff of 10 kDa), diluted by
dropwise addition of glycerol to 50% (v/v) final concentration, and
stored at −20 °C for several months without appreciable loss of
activity (approximately 40 units of GST activity per mL).
GSH, DMSO (concentration in assay of 2%), and pyrrole analogue or
the control inhibitor BSP (concentration in assay of 100 μM). The
mixture was incubated at 25 °C for 5 min prior to the addition of
enzyme (15 μL, 0.5 GST unit). The observed rate was employed for
calculating the remaining activity (%), taking as 100% initial activity
value the rate observed (up to 0.2 [ΔA/min]340) after replacing the
arylmethylsulfonylpyrrole by an equal volume of buffer, thus keeping
constant the assay total volume (1 mL).
Kinetic Inhibition Studies with hGSTA1-1. Kinetic Analysis of
Inhibitors 4 and 9 Using CDNB as a Variable Substrate. Initial
velocities for the hGSTA1-1-catalyzed reaction with CDNB as variable
substrate were determined in reaction mixtures of a total volume of 1
mL (25 °C): 100 mM potassium phosphate buffer, pH 6.5, 2.5 mM
GSH, and different concentrations of CDNB (22.5−990 μM) in the
absence and the presence of inhibitor 4 (20, 40, 60, and 80 μM) or
inhibitor 9 (15, 30, and 60 μM) or the control inhibitor BSP (5, 25,
and 100 μM),
Kinetic Analysis of Inhibitors 4 and 9 Using GSH as α Variable
Substrate. Initial velocities for the hGSTA1-1-catalyzed reaction with
GSH as variable substrate were determined in reaction mixtures of a
total volume of 1 mL (25 °C): 100 mM potassium phosphate buffer,
pH 6.5, 1 mM CDNB, and different concentrations of GSH (40−2475
μM) in the absence and the presence of inhibitor 4 (10, 20, 40, 60, and
80 μM) or inhibitor 9 (15, 30, and 60 μM).
The GraFit3 computer program (Erithacus Software, Ltd., U.K.)
was used throughout for producing kinetic graphs and determining
apparent kinetic parameters/constants.
ASSOCIATED CONTENT
■
S
* Supporting Information
Bioactive arylsulfonylpyrroles (Figure SI-1), 3D models of
CDNB, GSH, BSP, and the new inhibitors with hGSTA1-1
(Figures SI-2 to SI-4 and SI-5a), enzyme inhibition kinetics
(Figures SI-6 to SI-9), HPLC−MS chromatograms of purity
analysis (Figure SI-10), and NMR (1H and13C) and HRMS
spectra of compounds 3−11. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For G.I.V.: phone, +30-26510-08382; fax, +30-26510-08799;
e-mail, gvarvoun@cc.uoi.gr. For Y.D.C.: phone, +30-210-
5294311; fax, +30-210-5294307; e-mail, clonis@aua.gr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Enzyme Assays and Screening of the Arylmethylsulfonyl-
pyrrole Derivatives for hGSTA1-1 Inhibition. GST Assay. GST
assays were performed by monitoring the formation of the conjugate
formed between CDNB and the tripeptide GSH at 340 nm (ε = 9.6
μmol mL−1 cm−1) at 25 °C. In a typical volume of 914 μL of
potassium phosphate buffer (100 mM, pH 6.5) were added 33 μL of
CDNB (30 mM) and 33 μL of GSH (75 mM). DMSO was also added
(20 μL, in place of equal volume of buffer) only for inhibition
experiments with the arylmethylsulfonylpyrroles. The mixture was
incubated at 25 °C for 5 min, prior to adding enzyme sample (typically
20 μL, 0.45 GST unit) in a total assay volume of 1 mL. Initial velocities
were determined in triplicate and were corrected for spontaneous
reaction rates when necessary. One unit of enzyme activity is defined
as the amount of enzyme that produces 1.0 μmol of product per
minute at pH 6.5 at 25 °C.
We appreciate the use of NMR and mass spectrometry facilities
funded by the Network of Research Supporting Laboratories of
the University of Ioannina, Greece, and thank Dr. V. Exarchou
for 2D NMR spectra, Dr. P. Stathopoulos for low resolution
mass spectra, and Dr. V. Boti for high resolution mass spectra.
The present work was partly supported by the action “THALIS:
Glutathione Transferases, Multifunctional Molecular Tools in
Red and Green Biotechnology” falling under the Operational
Programme (OP) “Education and Lifelong Learning (EdLL)”
and is cofinanced by the European Social Fund (ESF) and
National Resources.
ABBREVIATIONS USED
■
Screening of the Arylmethylsulfonylpyrrole Derivatives as
hGSTA1-1 Inhibitors. For enzyme inhibition screening of the
arylmethylsulfonylpyrroles (Table 1), the GST assay mixture
contained in a total assay volume of 1 mL: potassium phosphate
buffer (100 mM, pH 6.5), 33 μL of 30 mM CDNB, 33 μL of 75 mM
BSP, bromosulfophthalein; CDNB, 1-chloro-2,4-dinitroben-
zene; DMSO, dimethyl sulfoxide; NBS, N-bromosuccinimide;
GSH, glutathione; GST, glutathione S-transferase; hGSTA1-1,
human glutathione S-transferase isoenzyme A1-1; IPTG,
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