Synthesis and biological evaluation of 2-substituted-5-(4- nitrophenylsulfonamido)benzoxazoles as human GST P1-1 inhibitors, and description of the binding site features

Article abstract of DOI:10.1002/cmdc.201400010

Glutathione-S-transferases (GSTs) are enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of glutathione (GSH) on the electrophilic center of numerous of toxic compounds and xenobiotics, including chemotherapeutic drugs. Huma

Full text of DOI:10.1002/cmdc.201400010

CHEMMEDCHEM
FULL PAPERS
DOI: 10.1002/cmdc.201400010
Synthesis and Biological Evaluation of 2-Substituted-5-(4-
nitrophenylsulfonamido)benzoxazoles as Human GST P1-
1 Inhibitors, and Description of the Binding Site Features
[a]
[b]
[a]
[a]
[b]
˘
Tugba Ertan-Bolelli, Yaman Musdal, Kayhan Bolelli, Serap Yilmaz, Yasemin Aksoy,
Ilkay Yildiz,[a] Esin Aki-Yalcin,[a] and Ismail Yalcin*[a]
Glutathione-S-transferases (GSTs) are enzymes involved in cellu-
lar detoxification by catalyzing the nucleophilic attack of gluta-
thione (GSH) on the electrophilic center of numerous of toxic
compounds and xenobiotics, including chemotherapeutic
drugs. Human GST P1-1, which is known as the most prevalent
isoform of the mammalian cytosolic GSTs, is overexpressed in
many cancers and contributes to multidrug resistance by di-
rectly conjugating to chemotherapeutics. It is suggested that
this resistance is related to the high expression of GST P1-1 in
cancers, thereby contributing to resistance to chemotherapy.
In addition, GSTs exhibit sulfonamidase activity, thereby cata-
lyzing the GSH-mediated hydrolysis of sulfonamide bonds.
Such reactions are of interest as potential tumor-directed pro-
drug activation strategies. Herein we report the design and
synthesis of some novel sulfonamide-containing benzoxazoles,
which are able to inhibit human GST P1-1. Among the tested
compounds, 2-(4-chlorobenzyl)-5-(4-nitrophenylsulfonamido)-
benzoxazole (5 f) was found as the most active hGST P1-1 in-
hibitor, with an IC50 value of 10.2 mm, showing potency similar
to that of the reference drug ethacrynic acid. Molecular dock-
ing studies performed with CDocker revealed that the newly
synthesized 2-substituted-5-(4-nitrophenylsulfonamido)benzox-
azoles act as catalytic inhibitors of hGST P1-1 by binding to
the H-site and generating conjugates with GSH to form S-(4-ni-
trophenyl)GSH (GS–BN complex) via nucleophilic aromatic sub-
stitution reaction. The 4-nitrobenzenesulfonamido moiety at
position 5 of the benzoxazole ring is essential for binding to
the H-site and for the formation of the GST-mediated GSH con-
jugate.
Introduction
Glutathione-S-transferases (GSTs) are a family of dimeric multi-
functional enzymes that play an important role in metabolism
and detoxification of numerous xenobiotics, electrophilic
chemicals (including drugs), environmental carcinogens, and
products of oxidative stress in living organisms.[1] Aside from
their detoxifying reaction (typically a conjugation reaction),
GSTs catalyze many other types of reaction, such as isomeriza-
tion and reduction involved in many biological processes, e.g.,
steroid and prostaglandin biosynthesis, tyrosine catabolism,
and cell apoptosis.[2]
lytic reactions, the activation of the sulfur atom of the G-site-
bound glutathione (GSH) to the thiolate anion (GSÀ, a strong
nucleophile) is required to generate a GS–substrate conjugate
by attack of the electrophilic center of substrates bound to the
H-site. Furthermore, the GS-conjugated compounds may be ac-
tively extruded from the cell through specialized pumps; prin-
cipally, the multidrug-resistance proteins MRP-1 and MRP-2.[3]
GSTs are found in almost all organisms, from mammals to
plants, and even in some prokaryotes. Human GSTs consist of
three families: cytosolic GSTs, mitochondrial GSTs, and microso-
mal GSTs. In mammalian cells cytosolic GSTs are further catego-
rized into seven major classes according to their amino acid se-
quence: alpha, mu, pi, theta, zeta, omega, and sigma subfami-
lies, which have been identified in dimeric forms.[4]
For the GST-mediated detoxification reactions, a substrate–
GSH conjugation (complex formation) is required. GSTs are
a family of phase II detoxification enzymes that catalyze the
conjugation of GSH to a wide variety of electrophilic com-
pounds. GSTs catalyze the conjugation of the reduced form of
glutathione (GSH) to electrophilic centers of endogenous and
exogenous hydrophobic compounds.[1] For GST-mediated cata-
Human glutathione-S-transferase pi1-1 (hGST P1-1) is
a member of the pi class subfamily of cytosolic GSTs and is
composed of two homodimer GST P1 subunits. Structural anal-
ysis indicates that hGST P1-1 is a soluble protein of 209 amino
acid residues with a relative molecular mass of 23.224 kDa.[5]
The crystal structures show that each GST subunit of the pro-
tein dimer contains an independent catalytic site composed of
two components. The first is a binding site specific for GSH or
a closely related homologue (the G-site) formed from a con-
served group of residues in the N-terminal domain of the poly-
peptide. The second component is a site that binds the hydro-
phobic substrate (the H-site), which is structurally variable
[a] Dr. T. Ertan-Bolelli, K. Bolelli, S. Yilmaz, Prof. I. Yildiz, Prof. E. Aki-Yalcin,
Prof. I. Yalcin
Department of Pharmaceutical Chemistry
Ankara University, Faculty of Pharmacy, Degol Str. Tandogan, Ankara
(Turkey)
E-mail: yalcin@ankara.edu.tr
[b] Dr. Y. Musdal, Prof. Y. Aksoy
Department of Medical Biochemistry
Hacettepe University, Faculty of Medicine, Ankara (Turkey)
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 984

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Figure 1. a) Binding sites of hGST P1-1 (PDB ID: 2GSS), GSH (shown in purple, taken from PDB ID: 6GSS), and the specific substrate (EA) (shown in yellow,
taken from PDB ID: 2GSS), shown as bound separately in H- and G-sites. b) EA and GSH conjugate (GS–EA complex) in hGST P1-1 enzyme (PDB ID: 3GSS).
(Figure 1). Residue variations in the H-site across the different
GST classes determine substrate specificity.[6]
hGST P1-1 is the most prevalent isoform of the mammalian
cytosolic GSTs. It is known that hGST P1-1 participates in a par-
ticular role in one of the mechanisms of the development of
resistance in cancer cells toward the administration of anti-
cancer agents in chemotherapy. Several synthetic drugs and
Scheme 2. GST-mediated nucleophilic aromatic substitution of CDNB with
GSH to form the GS–DNB complex.
prodrugs that show inhibitory potency against GSTs have been
proposed as strategies to overcoming multiple drug resistance
(MDR) attributed to GST overexpression. Ethacrynic acid (EA) is
one of the first specific inhibitors to be used to sensitize
cancer cells to the cytotoxic effect of several chemotherapeutic
drugs.[7–9] As shown in Figure 1, EA acts as a substrate of GST,
forming a conjugate with GSH (GS–EA complex) via Michael
addition (Scheme 1) by GST-driven catalysis.[10] The use of this
s-complex intermediate, the Meisenheimer complex
(Scheme 2).[4]
hGST P1-1 is overexpressed in many cancers and contributes
to multidrug resistance by directly conjugating to chemothera-
peutic agents including cisplatin, adriamycin, etoposide, thiote-
pa, and chlorambucil. It is suggested that this resistance is re-
lated to the high expression of hGST P1-1 in cancers such as
breast, lung, colon, pancreas, and cervix, thereby contributing
to resistance to chemotherapy.[11] Studies have shown that
hGST P1-1 levels correlate with resistance to standard chemo-
therapy and are elevated in biopsies of tumor tissues that
have become resistant to therapy after administration of anti-
cancer agents.[12,13] Consequently, inhibitors of human GST P1-
1 catalytic activity remain a potential therapeutic tool in com-
bating cancer cell resistance to drugs. To overcome this resist-
ance, specific hGST P1-1 inhibitors are in demand.
Scheme 1. Michael-type addition of EA and GS–EA complex.
inhibitor as a means of overcoming acquired multidrug resist-
ance has been thoroughly explored, reaching phase II clinical
trials. Unfortunately, due to unwanted toxic side effects, EA
was withdrawn from further trials.
hGST P1-1 appears also to regulate a c-Jun N-terminal kinase
(JNK) pathway that is part of the apoptosis control system.
Thus, hGST P1-1 defends tumor cells by direct detoxification of
anticancer drugs and by blocking apoptosis through its effects
on JNK.[7,14] Therefore, hGST P1-1 is considered a promising
target for inhibition in cancer treatment, and considerable ef-
forts have been undertaken to find specific inhibitors for this
enzyme. Furthermore, a variety of sulfonamido-containing
One of the other well-known diagnostic substrates that
forms a GST-mediated conjugate with GSH is 1-chloro-2,4-dini-
trobenzene (CDNB), which undergoes a nucleophilic aromatic
substitution (SNAr) reaction that forms a GS–DNB complex via
an addition–elimination sequence involving
a short-lived
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 985

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
compounds are apparently accommodated by the H-site of
GST and are found as substrates of GSTs. Published data clearly
show that GSTs display sulfonamidase activity by catalyzing
the GSH-mediated hydrolysis of sulfonamide bonds
(Scheme 3).[15,16] Groups capable of withdrawing sufficient elec-
hGST P1–1 in vitro inhibitory activity
Each GST subunit of the protein dimer contains an independ-
ent catalytic site composed of two components. GSH binding
site (G-site) forms from a conserved group of amino acid resi-
dues in the N-terminal domain of the polypeptide; the hydro-
phobic substrate binding site (H-site) is much more structurally
variable and is formed from residues in the C-terminal domain
(Figure 1).[6] The H-site of the pi-class enzyme is half hydropho-
bic and half hydrophilic.[40] It is reported that GSTs catalyze the
GSH-mediated hydrolysis of sulfonamide bonds to form the
corresponding amine (Figure 3 below).[15,16] Atoms and/or atom
groups positioned ortho and/or para on the phenyl ring such
as nitro, capable of withdrawing sufficient electron density
from the a-carbon atom, to which the sulfonyl group is at-
tached, are shown to activate the corresponding GST-mediated
GSH hydrolysis of sulfonamides.[15]
Scheme 3. GST/GSH-mediated hydrolysis of para-nitrobenzenesulfonamides.
tron density from the a-carbon atom to the sulfonyl group are
an absolute requirement in this enzymatic process.[15] GST-
mediated sulfonamide cleavage results in the formation of the
GS conjugate, the corresponding amine, and sulfur dioxide.
The released GS conjugate may provide a strong inhibitor
against intracellular GSTs, thus facilitating cancer chemothera-
py.[17] The sulfonamidase activity of GSTs also suggests that
prodrugs activated by GSTs might provide a strategy for tar-
geted cancer chemotherapy. The design of new sulfonamide
derivatives, which are able to undergo cleavage in tumor cells
by highly expressed intracellular GSTs such as hGST P1-1, could
be a viable approach.[17,18]
In this study, newly synthesized 2-substituted-5-(4-nitro/ami-
nophenylsulfonamido)benzoxazole derivatives were screened
for in vitro inhibition of hGST P1-1. Among all of the tested
compounds, 2-(4-chlorobenzyl)-5-(4-nitrophenyl-sulfonamido)-
benzoxazole (5 f) exhibited the most potent inhibitory activity
for hGST P1-1, with an IC50 value of 10.2 mm).[41] According to
inhibitor activity results given in Table 1, compound 2-(4-ethyl-
benzyl)-5-(4-aminophenylsulfonamido) benzoxazole (6c) dis-
played no inhibitory activity toward hGST P1-1 due to the lack
of electron-withdrawing effect of the amino group at the a-
carbon atom of the phenyl ring attached to the sulfonyl
group.
The benzoxazoles have been the aim of much research for
many years because they constitute an important class of het-
erocyclic compounds exhibiting substantial chemotherapeutic
activities.[19–35] Recently, we reported some 2,5,6-trisubstituted
benzoxazole derivatives, which exhibit antimicrobial,[27–29] anti-
viral,[30,31] antitumor activities, inhibiting eukaryotic topoisomer-
ase II in a cell-free system.[32–35] The aim of this research was to
develop new and effective hGST P1-1 enzyme inhibitors from
sulfonamido-containing benzoxazole derivatives. In this study
we synthesized some novel 2-substituted-5-(4-nitro/aminophe-
nylsulfonamido)benzoxazole derivatives and investigated their
inhibitory activities toward hGST P1-1. To describe the binding
site features of these benzoxazoles on hGST P1-1, molecular
docking studies were performed with CDocker.
Description of the binding site features
Rational approaches for finding new leads for therapeutic tar-
gets are increasingly based on three-dimensional information
about receptors. One can predict the binding conformation of
a ligand in its receptor and the affinity between the ligand and
the protein with the correct poses of ligands in the binding
Results and Discussion
Chemistry
5-(4-Nitro/aminophenylsulfonamido)benzoxazole de-
rivatives were synthesized as new structures via
a three-step route. Firstly, 5-amino-2-(4-substituted
phenyl/benzyl)benzoxazole derivatives 3a–f were ob-
tained by heating the appropriate acid with 2,4-dia-
minophenol in polyphosphoric acid (PPA).[28,36,37]
Second, 5-amino-2-(4-substituted phenyl/benzyl)ben-
zoxazole derivatives 3a–f and 4-nitrobenzenesulfonyl
chloride (4) were treated in pyridine and dichlorome-
thane to get 5-(4-nitrophenylsulfonamido)benzoxa-
zole derivatives 5a–f.[38] Finally, 2-(4-ethylphenyl)-5-(4-
aminophenylsulfonamido)benzoxazole (6c) was ob-
Scheme 4. Synthetic pathway of 5-(4-nitro/aminophenylsulfonamido)benzoxazole deriva-
tives. Reagents and conditions: a) PPA, 170–2008C, 1.5–2.5 h; b) pyridine and CH2Cl2, RT,
16 h; c) Pd-C/H2, RT, 30 min, 60.35%.
[39]
tained by reduction of the NO2 group to NH2 by
using Pd-C/H2, as shown in Scheme 4.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 986

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
well-known diagnostic substrates of hGST P1-1, CDNB revealed
hydrogen bonds with Tyr108 and Trp38; a p–p interaction
with Phe8, and a p–cation interaction with Tyr108. The most
potent hGST P1-1 inhibitor, benzoxazole derivative 5 f, revealed
hydrogen bonds with Gln51 and Tyr108 and a p–cation inter-
action with Lys44 (Figure 2). The docking scores showed that
the binding energies of EA, CDNB, and 5 f are À19.8691,
À19.2405, À18.2525 kcalmolÀ1, respectively (Table 2).
Table 1. The structures of the tested sulfonamide-containing benzoxa-
zoles and their in vitro hGST P1-1 inhibitory effects.
Compd
R1
R2
X
IC50 [mm]
5a
5b
5c
5d
5e
5 f
6c
EA
NO2
NO2
NO2
NO2
NO2
NO2
NH2
H
F
C2H5
CH3
H
–
–
–
16.67
16.67
28.57
12.5
33.33
10.2
NI[a]
As shown in Figure 3, the van der Waals contact distance be-
tween the sulfur atom of GSH and the electrophilic chlorine-
bound carbon atom of CDNB is 3.295 ꢁ. Similarly, the van der
Waals contact distance between the sulfur atom of GSH and
the electrophilic carbon atom of the 4-nitrophenyl moiety at-
tached to the sulfonyl group is 3.322 ꢁ. Notably, the van der
Waals contact distances are quite similar to each other. There-
fore, we consider that 5 f can be involved in the SNAr reaction,
like CDNB. It is also known that GSTs catalyze the GSH-mediat-
ed cleavage of sulfonamide bonds to form the corresponding
amine.[15,16] As shown in Scheme 5, the thiolate anion of GSH
can attack the electrophilic carbon atom of 4-nitrobenzene to
which the sulfonyl group is attached to start the SNAr reaction.
As shown in Scheme 5, an S-complex transition state—a Mei-
senheimer complex—initially forms, and then the S-(4-nitro-
phenyl)GSH conjugate (GS–NB complex), sulfur dioxide, and 2-
(4-chlorobenzyl)-5-aminobenzoxazole are yielded as the GST/
GSH-mediated hydrolysis products of the compound 5 f.
–
CH2
CH2
–
Cl
C2H5
10.37
[a] No inhibition.
pocket of a protein. A process is described by which two mole-
cules fit together in 3D space.
It is reported that the active site residues Tyr7 and Tyr108
play important roles for the activity of hGST P1-1.[10,42] Different
models have been proposed for the activation of GST, all high-
lighting the key role of active site residues Tyr7 and Tyr108. In
the mechanism for activation of GSH, Tyr7 acts as a general
base, promotes proton abstraction from the GSH thiol, and cre-
ates a thiolate anion with high nucleophilic reactivity.[42] Addi-
tionally, the hydroxy group of Tyr108 appears to contribute to
the catalytic mechanism as
a general acid in the conjugation
reaction of GSH with EA.[10]
In this study, newly synthe-
sized 5-(4-nitro/aminophenylsul-
fonamido)benzoxazole
deriva-
tives, CDNB, and EA were
docked into the H-site of hGST
P1-1 by using CDocker in order
to explore the mechanism for in-
hibition of hGST P1-1. EA, which
is the known specific inhibitor of
hGST P1-1, showed hydrogen
bonds with Arg13 and a H2O
molecule, and a p–p interaction
with Tyr108; these are in accord-
ance with the X-ray structure
binding features.[43] One of the
Scheme 5. hGST-P1-1-mediated S-(4-nitrophenyl)GSH conjugate (GS–NB complex) formation of 5 f via nucleophilic
aromatic substitution reaction.
Table 2. Docking results.
Compd
Ebind [kcalmolÀ1
]
Interacting Residues[a]
5a
5b
5d
5 f
EA
CDNB
À11.2459
À7.9622
Tyr7, Phe8, Val10, Arg13 (2.33 ꢁ), Trp38, Gln51, Leu52, Ile104, Tyr108 (2.34 ꢁ), Thr109, Gly205
Tyr7, Phe8, Val10, Arg13,[c] Val35, Trp38, Gln39 (2.57 ꢁ), Gln51, Leu52, Gln64, Ile104, Tyr108,[b] Gly205
Tyr7,[c] Phe8,[b,c] Val10, Trp38 (2.23 ꢁ), Gln39, Lys44, Gln51 (2.03 ꢁ), Tyr108 (2.42 ꢁ), Gly205
Tyr7, Phe8, Val10, Arg13, Val35, Trp38, Gln39, Gly41, Lys44,[c] Gln51 (2.29 ꢁ), Ile104, Tyr108 (2.29 ꢁ), Gly205
Tyr7, Phe8, Val10, Gly12, Arg13 (2.09 ꢁ), Val35, Trp38, Ile104, Tyr108,[b] Gly205, H2O (water mediated H bond with Arg13)
Tyr7, Phe8,[b] Val10, Trp38 (2.32 ꢁ), Gln51, Ile104, Tyr108[c] (2.38 ꢁ), Gly205
À13.0223
À18.2525
À19.8691
À19.2405
[a] van der Waals contact distance: <4 ꢁ; H-bonds indicated in bold text. [b] p–p interactions. [c] p-cation interactions.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 987

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Figure 2. a) Docking overlay of EA in hydrophobicity-based surface area. b) Docked position of EA with interacting residues within 3.2 ꢁ. c) Docked position
of EA: the carboxyl group hydrogen bonds with Arg13 (yellow) and a H2O molecule, the phenyl ring undergoes p–p interactions with Tyr108 (pink). d) Dock-
ing overlay of CDNB in hydrophobicity-based surface area. e) Docked position of CDNB with interacting residues within 3 ꢁ. f) Docked position of CDNB:
ortho-nitro group hydrogen bonds with Tyr108 (pink), para-nitro group hydrogen bonds with Trp38 (purple), the phenyl ring undergoes p–p interactions
with Phe8 (white), the N atom of the ortho-nitro group has p–cation interactions with Tyr108 (pink). g) Docking overlay of 5 f in hydrophobicity-based surface
area. h) Docked position of 5 f with interacting residues within 3 ꢁ. i) Docked position of 5 f: the sulfonyl group hydrogen bonds with Tyr108 (pink), the para-
nitro group hydrogen bonds with Gln51 (orange), the phenyl ring at position 2 of the benzoxazole ring undergoes p–cation interactions with Lys44 (red).
sensitizing tumor cells to anticancer drugs.[1] Consequently, it is
Conclusions
accepted that hGST P1-1 contributes directly to drug resistance
The X-ray crystallographic results published by Oakley et al. in
1997 confirmed that the well-known hGST P1-1-specific inhibi-
tor EA binds to the ligand binding site (H-site) of hGST P1-1,
while GSH binds the G-site, forming a conjugate with GSH via
conjugation reaction. These observations clearly demonstrate
that EA acts as a substrate of hGST P1-1, forming a conjugate
with GSH, where the EA–GSH conjugate also acts as an inhibi-
tor. Therefore, EA is a known inhibitor of hGST P1-1, but also
acts as a substrate.[43]
in some cell types via their catalytic activity; therefore, inhibi-
tors of hGST P1-1 remain as potential therapeutic tools. This
therapeutic strategy requires the GST-targeted agent to inhibit
catalytic function.
The development of chemotherapy-resistant tumor cells is
a significant problem encountered in cancer treatment. The
overexpression of hGST P1-1 in many cancer tissues and in
drug-resistant cell lines suggests that elevated hGST P1-1 ex-
pression may be of direct relevance not only to acquired resist-
ance, but also in natural resistance.[6] It is possible that hGST
P1-1 confers drug resistance by two distinct means: 1) direct
inactivation (detoxification) of chemotherapeutic drugs and
2) inhibition of the mitogen-activated protein (MAP) kinase
pathway.[7] Furthermore, hGST P1-1 was identified as capable
of blocking the stress-activated kinase pathway, thereby pro-
tecting cells against apoptosis in response to cellular stress
from reactive oxygen species. Therefore, hGST P1-1 is consid-
ered as a promising target for inactivation in cancer treatment,
and numerous groups have spent considerable effort finding
potent inhibitors of this enzyme.[44]
hGST P1-1 has been shown to catalyze the conjugation of
GSH with the alkylating agents chlorambucil and thiotepa, sug-
gesting that overexpression of hGST P1-1 in cells exposed to
these drugs would confer resistance. Elevated cellular levels of
hGST P1-1 have been shown to accompany resistance to vari-
ous common anticancer drugs, and the addition of the GST in-
hibitors such as EA restored sensitivity to alkylating agents in
drug-resistant cells.[1] EA has been reported to potentiate the
cytotoxic effects of chlorambucil in human colon carcinoma
cell lines and melphalan in human colon tumors.[1] A variety of
GST inhibitors were shown to modulate drug resistance by
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 988

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Figure 3. All C atoms are green for CDNB and 5 f. a) van der Waals contact distance between electrophilic carbon atom of CDNB and the S atom of GSH
(purple) is 3.295 ꢁ. b) van der Waals contact distance between the carbon atom of 5 f and the S atom of GSH (purple) is 3.322 ꢁ. c) van der Waals contact dis-
tance between the nucleophilic C atom of 5 f and the S atom of GSH (purple), and the H-bond distances with residues Tyr7 and Tyr108 in hGST P1-1 are indi-
cated.
In this work, we attempted to observe novel lead com-
pounds that act as human GST P1-1 inhibitors, available for
use as chemotherapeutic agents. For this purpose, we synthe-
sized some novel 2-substituted-5-(4-nitro/aminophenylsulfona-
mido)benzoxazole derivatives to investigate their hGST P1-1 in-
hibitory activities. Among the tested sulfonamido-containing
benzoxazole derivatives, 2-(4-chlorobenzyl)-5-(4-nitrophenylsul-
fonamido)benzoxazole (5 f) was found to be the most potent
inhibitor showing a similar potency with the reference drug
EA.
Based on previous studies by Zhao et al.[15] and Koeplinger
et al.,[16] both in 1999, the reaction of GST-mediated GSH-de-
pendent sulfonamide cleavage occurs by binding of GSH at
the G-site of GST, resulting in the formation of the thiolate
anion GSÀ. This nucleophile then attacks the (aromatic) carbon
atom linked to the sulfonyl moiety of the sulfonamide lead
substrate, which binds at the H-site of GST. Sulfonamide cleav-
age results in the formation of the GS conjugate, the corre-
sponding amine, and sulfur dioxide. The released GS conjugate
provides a strong inhibitor against intracellular GSTs, thus facili-
tating cancer chemotherapy.[15,16] In accordance with these
findings, our molecular docking results demonstrate that the
4-nitrophenylsulfonamido-containing benzoxazole derivative
5 f bind to the H-site of the enzyme and forms the S-(4-nitro-
phenyl)GSH conjugate (GS–NB complex) via SNAr reaction by
GST-mediated hydrolysis, as shown in Scheme 5.
any inhibitory activity toward hGST P1-1, as it was unable to
activate the corresponding GST-mediated GSH hydrolysis to
effect the GSH conjugation reaction, because of the electron-
donating effect of the p-amino group toward the a-carbon
atom attached to the sulfonyl group. Consequently, it was
found that groups capable of withdrawing sufficient electron
density from the a-carbon atom to the sulfonyl group are an
absolute requirement. The electrophilic substructure of the sul-
fonyl group was solely responsible for activation of the sulfo-
namide bond toward cleavage. On the other hand, the amine
portion had little or no impact on the cleavability of sulfona-
mide substrates, but influenced the catalytic rate.[15,16]
To analyze the binding site features of the sulfonamido-con-
taining benzoxazole derivatives, molecular docking studies
were performed by using CDocker, and the model for the
binding of the sulfonamide lead ligand to hGST P1-1 was con-
structed based on: 1) the fact that the GSH thiolate anion at-
tacks the a-carbon of the sulfonamide in the reaction and
2) manual fitting to obtain optimum interactions between the
sulfonamide’s hydrophobic chain and the hydrophobic resi-
dues of the H-site. The predicted modes of interaction of the
sulfonamido-containing benzoxazoles with hGST P1-1 are
shown in Figure 2g,h,i and 3b,c. It was found that the new
synthesized 2-substituted-5-(4-nitrophenylsulfonamido)benzox-
azoles bind to the H-site of hGST P1-1 and form conjugates
with GSH, generating the S-(4-nitrophenyl)GSH conjugate (GS–
BN complex) via SNAr reaction. A 4-nitrophenylsulfonamido
moiety substituted at position 5 of the benzoxazole ring is es-
In contrast, the synthesized compound 2-(4-ethylbenzyl)-5-
(4-aminophenylsulfonamido)benzoxazole (6c) did not exhibit
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 989

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
1684–1559, 1603, 1524, 1476, 1342, 1318, 1223, 1163, 1143–1063,
;
857–672 cmÀ1 1H NMR (400 MHz, [D6]DMSO): d=7.14 (dd, J=
sential for binding to the H-site and formation of the GS–NB
complex for inhibitory activity.
8.4 Hz, J=2.0 Hz, 1H), 7.43–7.51 (m, 3H), 7.70 (d, J=8.4 Hz, 1H),
8.00 (d, J=9.2 Hz, 2H), 8.21 (q, 2H), 8.37 (d, J=8.8 Hz, 2H),
10.71 ppm (s, 1H); MS (ESI): m/z (%): 412.30 (100) [MÀH]+ calcd for
C19H12FN3O5S: C 55.21, H 2.93, N 10.17, S 7.76, found: C 55.14, H
3.22, N 10.29, S 7.82.
According to the observed in vitro hGST P1-1 inhibitory ac-
tivity results, the tested compound 5 f exhibited strong hGST
P1-1 inhibition, making it a promising lead compound for fur-
ther in vitro and in vivo studies to develop new chemothera-
peutic agents.
2-(4-Ethylphenyl)-5-(4-nitrophenylsulfonamido)benzoxazole (5c):
To
a solution of 2-(4-ethylphenyl)-5-amino benzoxazole (3c)
(0.048 mmol) in CH2Cl2 (2 mL), pyridine (0.95 mmol) and 4-nitro-
benzenesulfonyl chloride were added to give compound 5c
(53.19%): mp: 217–2198C; IR (KBr): n˜ =3270, 3125, 2968, 1673–
1498, 1607, 1527, 1476, 1346, 1310, 1162, 1139–1086, 854–
Experimental Section
Chemistry
All chemicals and solvents were purchased from commercial vend-
ers and were used without purification. The progress of the reac-
tion was monitored on ready-made silica gel plates (Merck). The
melting points were measured with a capillary melting point appa-
ratus (Bꢂchi B540) and are uncorrected. Yields were calculated
after recrystallization. IR spectra were recorded on a Jasco FT/IR-
675 cmÀ1 1H NMR (400 MHz, [D6]DMSO): d=1.22 (t, 3H), 2.70 (q,
;
2H), 7.13 (dd, J=8.8 Hz, J=2.0 Hz, 1H), 7.44 (d, J=8.0 Hz, 2H),
7.50 (d, J=2.4 Hz, 1H), 7.69 (d, J=8.4 Hz, 1H), 8.01 (d, J=8.8 Hz,
2H), 8.06 (d, J=8.0 Hz, 2H), 8.37 (d, J=9.2 Hz, 2H), 10.70 ppm (s,
1H); MS (ESI): m/z (%): 422.25 (100) [MÀH]+ calcd for C21H17N3O5S:
C 59.57, H 4.05, N 9.92, S 7.57, found: C 59.50, H 4.30, N 9.95, S
7.60.
1
420 spectrometer as KBr discs. The H NMR spectra were recorded
with a Varian Mercury 400 MHz FT spectrometer, chemical shifts (d)
are reported in ppm relative to TMS, and coupling constants (J) are
reported in Hz. Mass spectra were taken on a Waters Micromass
ZQ instrument using the ESI method. Elemental analyses were per-
formed with a Leco CHNS-932 CHNS-O analyzer; results (C, H, N, S)
were within Æ0.4% of the calculated amounts.
General procedure for the preparation of 2-(4-substituted
phenyl/benzyl)-5-aminobenzoxazoles 3a–f: The derivatives were
synthesized by heating 2,4-diaminophenol dihydrochloride
(0.01 mol) with suitable acid (0.01 mol) in polyphosphoric acid
(PPA; 24 g) with stirring at 170–2008C for 1.5–2.5 h. At the end of
the reaction period, the residue was poured into an ice–water mix-
ture and neutralized with excess NaOH (10% solution), and the res-
idue was filtered and boiled with charcoal (200 mg) in ethanol and
filtered. After the evaporation of solvent in vacuo, the crude prod-
uct was obtained and recrystallized from an ethanol/water (1:3)
mixture.[28,36,37]
2-(4-Methylphenyl)-5-(4-nitrophenylsulfonamido)benzoxazole
(5d): To a solution of 2-(4-methylphenyl)-5-aminobenzoxazole (3d)
(0.048 mmol) in CH2Cl2 (2 mL), pyridine (0.95 mmol) and 4-nitro-
benzenesulfonyl chloride (0.52 mmol) were added to give com-
pound 5d (42.79%): mp: 244–2458C; IR (KBr): n˜ =3281, 3126, 1646,
1497, 1608, 1529, 1480, 1348, 1313, 1161, 1138–1086, 853–
1
669 cmÀ1; H NMR (400 MHz, [D6]DMSO): d=2.41 (s, 3H), 7.12 (dd,
J=8.4 Hz, J=2.0 Hz, 1H), 7.42 (d, J=8.4 Hz, 2H), 7.47 (d, J=2.0 Hz,
1H), 7.68 (d, J=8.8 Hz, 1H), 7.98 (d, J=8.8 Hz, 2H), 8.04 (d, J=
8.4 Hz, 2H), 8.36 (d, J=8.4 Hz, 2H), 10.69 ppm (s, 1H); MS (ESI): m/
z (%): 410.96 (100) [M+H]+ calcd for C20H15N3O5S: C 58.67, H 3.69,
N 10.26, S 7.83, found: C 58.58, H 3.79, N 10.26, S 7.85.
2-Benzyl-5-(4-nitrophenylsulfonamido)benzoxazole (5e): To a so-
lution of 2-benzyl-5-aminobenzoxazole (3e) (0.048 mmol) in CH2Cl2
(2 mL), pyridine (0.95 mmol) and 4-nitrobenzenesulfonyl chloride
(0.52 mmol) were added to give compound 5e (55.01%): mp: 145–
1468C; IR (KBr): n˜ =3063, 2861, 1568–1497, 1607, 1526, 1476, 1349,
General procedure for the preparation of 5-(4-nitrophenylsulfo-
namido)benzoxazole derivatives 5a–f: To a solution of 2-(4-sub-
stituted phenyl)-5-amino benzoxazole 3a–f (0.048 mmol), in CH2Cl2
(2 mL), 4-nitrobenzenesulfonyl chloride (0.52 mmol) was added.
The reaction mixture was stirred at room temperature for 16 h. At
the end of the reaction the residue was filtered and washed with
a saturated solution of CuSO4 and NaHCO3 in water,[38] then recrys-
tallized with a mixture of ethyl acetate/n-hexane (1:4). The crystals
were dried in vacuo. All of the compounds are new.
1310, 1166, 1114–1088, 852–668 cmÀ1
;
1H NMR (400 MHz,
[D6]DMSO): d=4.30 (s, 2H), 7.06 (dd, J=8.8 Hz, J=2.0 Hz, 1H),
7.28–7.40 (m, 5H), 7.40 (d, J=1.6 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H),
7.96 (d, J=9.2 Hz, 2H), 8.35 (d, J=8.4 Hz, 2H), 10.66 ppm (s, 1H);
MS (ESI): m/z (%): 410.22 (100) [M+H]+ calcd for C20H15N3O5S: C
58.67, H 3.69, N 10.26, S 7.83, found: C 58.32, H 3.96, N 10.26, S
7.90.
2-(4-Chlorobenzyl)-5-(4-nitrophenylsulfonamido)benzoxazole
(5 f): To a solution of 2-(4-chlorobenzyl)-5-aminobenzoxazole (3 f)
(0.048 mmol) in CH2Cl2 (2 mL), pyridine (0.95 mmol) and 4-nitro-
benzenesulfonyl chloride (0.52 mmol) were added to give the com-
pound 5 f (40.06%): mp: 179–1818C; IR (KBr): n˜ =3115, 3053,
2860–2791, 1685–1493, 1606, 1526, 1472, 1350, 1312, 1167, 1115–
2-Phenyl-5-(4-nitrophenylsulfonamido)benzoxazole (5a): To a so-
lution of 2-phenyl-5-aminobenzoxazole (3a) (0.048 mmol) in CH2Cl2
(2 mL), pyridine (0.95 mmol) and 4-nitrobenzenesulfonyl chloride
(0.52 mmol) were added to give the compound 5a (56.96%): mp:
247–2508C; IR (KBr): n˜ =3264, 3105, 1618–1554, 1606, 1532, 1481,
1059, 1085, 847–670 cmÀ1 1H NMR (400 MHz, [D6]DMSO): d=4.32
;
1346, 1326, 1157, 1108–1054, 857–668 cmÀ1 1H NMR (400 MHz,
;
(s, 2H), 7.06 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.38–7.43 (m, 5H), 7.58
(d, J=9.2 Hz, 1H), 7.95 (d, J=9.2 Hz, 2H), 8.35 (d, J=8.8 Hz, 2H),
10.66 ppm (s, 1H); MS (ESI): m/z (%): 444.18 (100) [M+H]+; 446.05
(40) [M+H+2]+ calcd for C20H14ClN3O5S: C 54.12, H 3.18, N 9.47, S
7.22, found: C 54.07, H 3.31, N 9.43, S 7.20.
[D6]DMSO): d=7.08 (dd, J=9.2 Hz, J=2.0 Hz, 1H), 7.44 (d, J=
1.6 Hz, 1H), 7.60–7.64 (m, 4H), 7.98 (d, J=8.8 Hz, 2H), 8.15 (dd, J=
7.6 Hz, J=1.6 Hz, 2H), 8.33 ppm (d, J=8.8 Hz, 2H); MS (ESI): m/z
(%): 394.25 (100) [MÀH]+ calcd for C19H13N3O5S: C 57.72, H 3.31, N
10.63, S 8.11, found: C 57.84, H 3.50, N 10.81, S 8.14.
2-(4-Fluorophenyl)-5-(4-nitrophenylsulfonamido)benzoxazole
(5b): To a solution of 2-(4-fluorophenyl)-5-aminobenzoxazole (3b)
(0.048 mmol) in CH2Cl2 (2 mL), pyridine (0.95 mmol) and 4-nitro-
benzenesulfonyl chloride (0.52 mmol) were added to give com-
pound 5b (46.01%): mp: 230–2328C; IR (KBr): n˜ =3246, 3118,
General procedure for the preparation of 2-(4-ethylphenyl)-5-(4-
aminophenylsulfonamido)benzoxazole (6c): 2-(4-Ethylphenyl)-5-
(4-nitrophenylsulfonamido)benzoxazole (5c) (0.5 mmol) in ethanol
(50 mL) was reduced by hydrogenation using H2 (40 psi) and Pd-C
(40 mg, 10%) until uptake of H2 ceased.[40] The catalyst was filtered
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 990

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
on a bed of Celite, washed with ethanol, and the filtrate was con-
centrated in vacuo. The crude product was purified by recrystalliza-
tion with a mixture of ethyl acetate/n-hexane (1:4) to obtain 6c.
The crystals were dried in vacuo. The compound is new (60.35%):
mp: 251–2548C; IR (KBr): n˜ =3407–3339, 3188, 2963, 1596–1499,
assay.[46] The molar absorption coefficient used for CDNB was
De340 =9.6 mmÀ1 cmÀ1 and for PEITC De274 =8.89 mmÀ1 cmÀ1. The
IC50 value was determined as the inhibitor concentration that gives
50% inhibition of enzyme activity. For determining the inhibition
type effected by the test compounds, varied concentrations of
GSH and CDNB were used. The compounds tested as inhibitors
were prepared freshly in 2 mm stock solutions in 96% ethanol be-
cause of their sensitivity to light.
1578, 1462, 1310, 1158, 1144–1092, 831–682 cmÀ1
;
1H NMR
(400 MHz, [D6]DMSO): d=1.22 (t, 3H), 2.70 (q, 2H), 5.96 (s, 2H),
6.52 (dd, J=7.2, J=1.6, 2H), 7.10 (dd, J=8.8, J=2.0, 1H), 7.38–7.45
(m, 5H), 7.62 (d, J=9.2, 1H), 8.06 (d, J=8.0, 2H), 9.93 ppm (s, 1H);
MS (ESI): m/z (%): 394.43 (100) [M+H]+ calcd for C21H19N3O3S: C
64.11, H 4.87, N 10.68, S 8.15, found: C 63.92, H 5.16, N 10.70, S
7.93.
Data analysis
All measurements were made in triplicate, and each point on the
graphs was given with standard deviation of the mean value. IC50
values of the compounds were determined by regression analysis
using GraphPad Prism software version 4.0. Among the all tested
compounds, 5 f 2-(4-chlorobenzyl)-5-(4-nitrophenylsulfonamido)-
benzoxazole was found as the most potent inhibitor for hGST P1–
1, with an IC50 value of ~10 mm[41] (Table 1).
Expression and purification of human GST P1-1
Recombinant hGST P1–1 was expressed in Escherichia coli strain
XL-1 Blue at 378C and purified by using S-hexylglutathione–Se-
pharose 6B.[45] E. coli XL-1 cells containing pKXHP1 plasmid were
grown overnight in 50 mL 2YT media (16 g tryptone, 10 g yeast ex-
tract, 5 g NaCl, and 100 mgLÀ1 ampicillin) and then transferred to
500 mL 2YT media and incubated at 378C in a shake incubator. In-
cubation continued until the absorbance of the culture at 600 nm
was 0.2–0.4. Then 0.2 mm isopropyl-b-d-thiogalactopyranoside
(IPTG) was added to induce the expression of hGST P1-1. The cells
were incubated for 16 h and then centrifuged at 7000 rpm (1500 g)
for 7 min. The pellets were kept at À808C for 30 min. The pellets
were resuspended in lysis buffer (10 mm Tris·HCl, 1 mm EDTA,
0.2 mm dithiothreitol (DTT) pH 7.0 and protease inhibitor cocktail,
0.2 mgmLÀ1 lysozyme) and mixed gently on ice for 30 min and
then disrupted by sonication (5ꢃ20 s pulses, amplitude 80%, pulse
on: 20; off: 40). Phenylmethanesulfonyl fluoride (170 mm) was
added, and the supernatant fraction was obtained by centrifuga-
tion at 15000 rpm (15000 g) at 58C for 1 h.
Computational methods
Molecular docking
Preparation of the enzyme: The crystal structure of hGST P1-
1 complexed with ethacrynic acid was retrieved from the Protein
Data Bank (PDB ID: 2GSS).[43] Accelrys Discovery Studio 3.5[47] soft-
ware was used for preparation of protein and ligands. The target
protein was taken, the ligand was extracted, hydrogen atoms were
added, and their positions were optimized using the all-atom
CHARMm force-field and the Adopted Basis set Newton Raphson
(ABNR) method available in Discovery Studio 3.5 protocol until the
RMSD gradient was <0.05 kcalmolÀ1 ꢁÀ2. The minimized protein
was defined as the receptor using the binding site module. The
binding site was defined from the cavity-finding method, which
was modified to accommodate all the important interacting resi-
dues in the active site of GST (H-site). The binding sphere for 2GSS
(6.64, 3.67, 26.88, 9.312) was selected from the active site using the
binding site tools.
Epoxy-activated S-hexylglutathione–Sepharose 6B affinity matrix
was used for purification of hGST P1-1. The supernatant fraction
was applied to the matrix equilibrated with binding buffer and
stirred gently for 40 min on ice. The matrix containing bound hGST
P1-1 was washed with Buffer A (10 mm Tris·HCl, pH 7.8, 1 mm
EDTA, 0.2m NaCl, 0.2 mm DTT) to eliminate nonbound proteins.
The matrix was packed on top of a Sephadex G-25 column equili-
brated with Buffer A in the cold room at 48C. The enzyme was
then eluted with Buffer B (10 mm Tris·HCl, pH 7.8, 1 mm EDTA,
0.2m NaCl, 0.2 mm DTT, 5 mm S-hexylglutathione). The fractions
containing GST activity were concentrated on ice and then dialyzed
with Buffer A without NaCl. The purity of the enzyme was deter-
mined by SDS-PAGE, applying both optimal and excessive protein
amounts for analysis to visualize possible impurities.
Preparation of ligands: Novel synthesized benzoxazole derivatives,
CDNB, and standard drug ethacrynic acid were sketched, all-atom
CHARMm force-field parameterization was assigned and then mini-
mized using the ABNR method as described above. Conformational
searches of the ligands were carried out using a simulated anneal-
ing molecular dynamics (MD) approach. The ligands were heated
at a temperature of 700 K and then annealed to 200 K.
hGST P1-1 in vitro inhibition assay
hGST P1-1 in vitro inhibitory activity of the synthesized sulfonami-
do-containing benzoxazoles was measured on a Shimadzu UV-
2501 PC spectrophotometer by measuring the initial rate of ab-
sorbance change at 340 nm with CDNB. Standard enzymatic assay
conditions consisted of 0.1m phosphate buffer (pH 6.5) containing
1 mm EDTA, 1 mm GSH, and 1 mm CDNB at 308C. The reaction
system contained 5% ethanol (from the CDNB stock solution), but
the solvent had a negligible inhibitory effect on enzyme activity.
The enzymatic reaction was obtained by subtracting the nonenzy-
matic rate from the rate measured in the presence of enzyme.[41]
Docking
The CDocker[48] method was carried out with Discovery Studio 3.5.
The protein is held rigid while the ligands are allowed to be flexi-
ble during refinement. The docking parameters were as follows:
Top Hits: 10; Random Conformations: 10; Random Conformations
Dynamics Step: 20000; Grid Extension: 8.0; Random Dynamics
Time Step: 0.002. The docking and scoring methodology was first
validated by docking of the known inhibitor, ethacrynic acid (EA).
The docked position of EA overlaps well with the crystal structure
position, with an RMSD of 0.905 ꢁ. Afterward, molecular docking
studies were performed on the new synthesized compounds.
The most potent compounds identified with CDNB were also
tested spectrophotometrically with PEITC as substrate in a standard
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 991

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[20] D. Dꢅez-Martin, N. R. Kotecha, S. V. Ley, S. Mantegani, J. C. Menendez,
H. M. Organ, A. D. White, B. J. Banks, Tetrahedron 1992, 48, 7899–7938.
[21] I. Yalcin, I. ꢆren, E. Sener, A. Akin, N. Ucartꢂrk, Eur. J. Med. Chem. 1992,
27, 401–406.
[22] I. Oren, O. Temiz, I. Yalcin, E. Sener, A. Akin, N. Ucarturk, Arzneim.-
Forsch./Drug Res. 1997, 47, 1393–1397.
[23] O. Temiz, I. Oren, E. Sener, I. Yalcin, N. Ucarturk, Farmaco 1998, 53, 337–
341.
[24] I. ꢆren, O. Temiz, I. Yalcin, E. Sener, N. Altanlar, Eur. J. Pharm. Sci. 1998,
7, 153–160.
Analysis of results
Finally, all docked poses were scored by applying Analyze Ligand
Poses sub-protocol, and binding energies were calculated by ap-
plying Calculate Binding Energy sub-protocol in Discovery Studio
3.5 by using in situ ligand minimization step (ABNR method) and
using implicit solvent model (GBMV). The lowest binding energy
was taken as the best-docked conformation of the compound for
the macromolecule.
[25] O. Temiz-Arpaci, I. Oren, N. Altanlar, Farmaco 2002, 57, 175–181.
[26] ꢆ. Temiz-Arpaci, E. Aki-Sener, I. Yalcin, N. Altanlar, Farmaco 2002, 57,
771–775.
Acknowledgements
[27] I. Yildiz-Oren, I. Yalcin, E. Aki-Sener, N. Ucarturk, Eur. J. Med. Chem. 2004,
39, 291–298.
[28] I. Yildiz-Oren, B. Tekiner-Gulbas, I. Yalcin, O. Temiz-Arpaci, E. Aki-Sener,
N. Altanlar, Arch. Pharm. 2004, 337, 402–410.
[29] ꢆ. Temiz-Arpaci, A. ꢆzdemir, I. Yalcin, I. Yildiz, E. Aki-Sener, N. Altanlar,
Arch. Pharm. 2005, 338, 105–111.
[30] A. Akbay, I. Oren, O. Temiz-Arpaci, E. Aki-Sener, I. Yalcin, Arzneim.-
Forsch./Drug Res. 2003, 53, 266–271.
This study was supported by the Research Fund of Ankara Uni-
versity, Grant No: 12B3336002 and The Scientific and Technologi-
cal Research Council of Turkey (TUBITAK) grant (Project No:
109S362). The work carried out in Bengt Mannervik’s laboratory
was supported by the Swedish Cancer Society and the Swedish
Research Council.
[31] R. K. Plemper, K. J. Erlandson, A. S. Lakdawala, A. Sun, A. Prussia, J.
Boonsombat, E. Aki-Sener, I. Yalcin, I. Yildiz, O. Temiz-Arpaci, B. P. Tekiner,
D. Liotta, J. P. Snyder, Proc. Natl. Acad. Sci. USA 2004, 101, 5628–5633.
[32] H. Lage, E. Aki-Sener, I. Yalcin, Int. J. Cancer 2006, 119, 213–220.
[33] A. Pınar, P. Yurdakul, I. Yildiz-Oren, O. Temiz-Arpaci, N. L. Acan, E. Aki-
Sener, I. Yalcin, Biochem. Biophys. Res. Commun. 2004, 317, 670–674.
[34] O. Temiz-Arpaci, B. Tekiner-Gulbas, I. Yildiz, E. Aki-Sener, I. Yalcin, Bioorg.
Med. Chem. 2005, 13, 6354–6359.
Keywords: benzoxazoles
transferase · molecular modeling · sulfonamides
·
glutathione
·
glutathione-S-
[1] N. Mathew, M. Kalyanasundaram, K. Balaraman, Expert Opin. Ther. Pat.
2006, 16, 431–444.
[2] J. D. Hayes, J. U. Flanagan, I. R. Jowsey, Annu. Rev. Pharmacol. Toxicol.
2005, 45, 51–88.
[3] I. Meijerman, J. H. Beijnen, J. H. Schellens, Cancer Treat. Rev. 2008, 34,
505–520.
[35] B. Tekiner-Gulbas, O. Temiz-Arpaci, I. Yildiz, E. Aki-Sener, I. Yalcin, SAR
QSAR Environ. Res. 2006, 17, 121–132.
˙
[36] E. S¸ener, I. YalÅın, S. ꢆzden, T. ꢆzden, A. Akın, S. Yıldız, Turk. J. Med.
Pharm. 1987, 11, 391–396.
[4] R. N. Armstrong, Chem. Res. Toxicol. 1997, 10, 2–18.
[5] T. Kano, M. Sakai, M. Muramatsu, Cancer Res. 1987, 47, 5626–5630.
[6] S. Kazemnejad, Y. Rasmi, R. Sharifi, A. Allameh, Iran. J. Biotechnol. 2006,
4, 1–16.
[7] D. M. Townsend, K. D. Tew, Oncogene 2003, 22, 7369–7375.
[8] A. Sau, F. P. Trengo, F. Valentino, G. Federici, A. M. Caccuri, Arch. Bio-
chem. Biophys. 2010, 500, 116–122.
[9] K. D. Tew, S. Dutta, M. Schultz, Adv. Drug Delivery Rev. 1997, 26, 91–104.
[10] I. Quesada-Soriano, L. J. Parker, A. Primavera, J. M. Casas-Solvas, A.
Vargas-Berenguel, C. Barꢄn, C. J. Morton, A. P. Mazzetti, M. Lo Bello,
M. W. Parker, L. Garcꢅa-Fuentes, Protein Sci. 2009, 18, 2454–2470.
[11] S. Tsuchida, K. Sato, Crit. Rev. Biochem. Mol. Biol. 1992, 27, 337–384.
[12] D. J. Waxman, Cancer Res. 1990, 50, 6449–6454.
[37] G. M. Wynne, S. P. Wren, P. D. Johnson, P. D. Price, O. De Moor, G.
Nugent, R. Storer, R. J. Pye, C. R. Dorgan, (Vastox PLC), Int. PCT Pub. No.
WO 2007/091106 A2, 2007.
[38] G. M. Wynne, S. P. Wren, P. D. Johnson, P. D. Price, O. De Moor, G.
Nugent, R. Storer, R. J. Pye, C. R. Dorgan, US Pat. Appl. No. US 2009/
075938 A1, 2009.
[39] X. Zheng, H. Oda, K. Takamatsu, Y. Sugimoto, A. Tai, E. Akaho, H. I. Ali, T.
Oshiki, H. Kakuta, K. Sasaki, Bioorg. Med. Chem. 2007, 15, 1014–1021.
[40] B. Wu, D. Dong, Trends Pharmacol. Sci. 2012, 33, 656–668.
[41] Y. Musdal, T. Ertan-Bolelli, K. Bolelli, S. Yilmaz, D. Ceyhan, U. Hagazy, B.
Mannervik, Y. Aksoy, Turk. J. Biochem. 2012, 37, 431–436.
[42] A. Karshikoff, P. Reinemer, R. Huber, R. Ladenstein, Eur. J. Biochem. 1993,
215, 663–670.
[13] J. C. Schisselbauer, R. Silber, E. Papadopoulos, K. Abrains, F. P. LaCreta,
K. D. Tew, Cancer Res. 1990, 50, 3562–3568.
[43] A. J. Oakley, J. Rossjohn, M. Lo Bello, A. M. Caccuri, G. Federici, M. W.
Parker, Biochemistry 1997, 36, 576–585.
[14] V. Adler, Z. Yin, S. Y. Fuchs, M. Benezra, L. Rosario, K. D. Tew, M. R.
Pincus, M. Sardana, C. J. Henderson, C. R. Wolf, R. J. Davis, Z. Ronai,
EMBO J. 1999, 18, 1321–1334.
[44] S. Mahajan, W. M. Atkins, Cell. Mol. Life Sci. 2005, 62, 1221–1233.
[45] R. H. Kolm, G. Stenberg, M. Widersten, B. Mannervik, Protein Expression
Purif. 1995, 6, 265–271.
[15] Z. Zhao, K. A. Koeplinger, T. Peterson, R. A. Conradi, P. S. Burton, A. Suar-
ato, R. L. Heinrikson, A. G. Tomasselli, Drug Metab. Dispos. 1999, 27,
992–998.
[46] R. H. Kolm, U. H. Danielson, Y. Zhang, P. Talalay, B. Mannervik, Biochem.
J. 1995, 311, 453–459.
[47] Discovery Studio 3.5, Accelrys Inc., 2012.
[16] K. A. Koeplinger, Z. Zhao, T. Peterson, J. W. Leone, F. S. Schwende, R. L.
Heinrikson, A. G. Tomasselli, Drug Metab. Dispos. 1999, 27, 986–991.
[17] I. Axarli, N. E. Labrou, C. Petrou, N. Rassias, P. Cordopatis, Y. D. Clonis,
Eur. J. Med. Chem. 2009, 44, 2009–2016.
[48] G. Wu, D. H. Robertson, C. L. Brooks III, M. Vieth, J. Comput. Chem. 2003,
24, 1549–1562.
[18] Y. Kodera, S. Akiyama, K. Isobe, K. Kondo, K. Ito, M. Yamauchi, H. Takagi,
Eur. J. Cancer 1994, 30A, 2158–2162.
Received: January 2, 2014
[19] M. Prudhomme, J. Guyot, G. Jeminet, J. Antibiot. 1986, 39, 934–937.
Published online on March 26, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 984 – 992 992