In vitro inhibition of human erythrocyte glutathione reductase by some new organic nitrates

Article abstract of DOI:10.1016/j.bmcl.2009.04.087

Glutathione reductase (GR), is responsible for the existence of GSH molecule, a crucial antioxidant against oxidative stress reagents. The antimalarial activities of some redox active compounds are attributed to their inhibition of antioxidant flavoenzyme

Full text of DOI:10.1016/j.bmcl.2009.04.087

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3661–3663
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro inhibition of human erythrocyte glutathione reductase
by some new organic nitrates
_
*
*
Murat Sßentürk , Oktay Talaz, Deniz Ekinci, Hüseyin Çavdar , Ömer Irfan Küfreviog˘lu
Atatürk University, Faculty of Science, Department of Chemistry, Erzurum, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Glutathione reductase (GR), is responsible for the existence of GSH molecule, a crucial antioxidant against
oxidative stress reagents. The antimalarial activities of some redox active compounds are attributed to
their inhibition of antioxidant flavoenzyme glutathione reductase, and inhibitors are therefore expected
to be useful for the treatment of malaria. Twelve organic nitrate derivatives were synthesized and treated
with human erythrocyte GR. The molecules were identified as strong GR inhibitors and novel antimalaria
candidates.
Received 13 March 2009
Revised 14 April 2009
Accepted 17 April 2009
Available online 24 April 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Organic nitrates
Gulutathione reductase
Inhibition
Cancer
Antimalaria agent
For many years, Nitroglycerin (NTG) and other organic nitrates
have been the mainstay of cardiovascular therapy1 and of particu-
lar benefit in the treatment of diangina pectoris,2 unstable angina3
and the early stages of acute myocardial infarction.4 They have
shown real efficacy in coronary atherosclerosis, hypercholesterol-
emia or other blood vessel wall disorders involving endothelial
dysfunction5,6 and reduced vasodilator capacity of the coronary
arteries7 (Fig. 1).
roles for the intraerythrocyte growth of parasites, protecting them
from oxidative stress.10c,11,12 Since nitroaromatic and quinoidal
compounds may efficiently inhibit GR from various sources,13–15 it
is necessary to assess the relative importance of redox cycling and
GR inhibition in their antiplasmodial activity.10c,14–18
In our work, toward the discovery of novel GR inhibitors, we
synthesized novel organic nitrate derivatives (Fig. 3). Compounds
were evaluated for their ability to inhibit human erythrocyte GR.
Glutathione reductase (GR; NADPH: oxidized glutathione oxi-
doreductase, EC 1.6.4.2), a flavoprotein, is an important enzyme
which catalyzes the conversion of oxidized glutathione into the re-
duced form. GR enables several vital functions of the cell, such as
the detoxification of free radicals and reactive oxygen species as
well as protein and DNA biosynthesis, by maintaining a high ratio
of GSH/GSSG.8,9 It is a target enzyme for antimalarial and antitu-
mor drugs,10 and studies on the enzyme are therefore important
for drug development.10c
One may expect an increase in the antiplasmodial activity of
nitroaromatic and quinoidal compounds with their redox potential.
However, the antimalarial activity of some redox active compounds,
such as 10-arylizoalloxazines11 and methylene blue,12 was also
attributed to their inhibition of antioxidant flavoenzyme glutathi-
one reductase, which catalyzes the reduction of glutathione disul-
fide (GSSG) at the expense of NADPH. It is assumed that both
human erythrocyte and Plasmodium falciparum GR play important
Inhibition is reported as IC50 and Ki (lM) and the results are the
average of at least three independent experiments (Fig. 2).
The rationale of investigating nitro compounds as GR inhibitors is
due to the fact that the simple molecules have been shown to be
inhibitor of human GR.12a Grellier et al. showed the antiplasmodial
activity of a series of homologous nitroaromatic compounds,19
O NO2
ONO2
N
O
S
O
O
O
H
N
-O
N
O-
H
+
NH3
O
O NO2
NTG
GSNO
O
O2N O
O-
O
O
N
H
N+
O
O
ISDN
N
O NO2
NR
* Corresponding authors. Tel.: +90 442 2314414 (M.Sß.); +90 442 2314379 (H.Ç.).
E-mail addresses: senturkm36@gmail.com (M. Sßentürk), hcavdar43@hotmail.
com (H. Çavdar).
NTG: Nitroglycerin, GSNO: S-Nitrosoglutathione, ISDN: Isosorbide dinitrate, NR: Nicorandil
Figure 1. Structure of some organic nitrates.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.087

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M. Sßentürk et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3661–3663
ONO2
Bi(NO3)3
OH
OH
O+ Bi(ONO2)2
-O-NO2
O
OH
ONO2
ONO2
13
14
1
2
5
3
OH
O
H2O
Bi(ONO2)2
OH
OH
O
ONO2
ONO2
ONO2
ONO2
ONO2
1
15
Br
4
7
6
Figure 3. Preparation of organic nitrates.
OH
HO
ONO2
HO
OH
ONO2
Compounds 1–12 behaved as strong inhibitors for GR, with Ki
values in the range of 11.7–44.3 M. trans-(1S(R),8S(R),Z)-8-
hydroxycyclooct-4-enyl nitrate 5 was relatively ineffective GR
inhibitor (KI: 44.3 M), similarly to the structurally related com-
pounds 1 and 2 (KI: 43.7 M). A second group of derivatives,
l
O2NO
OH
O2NO
ONO2
8
9
l
ONO2
l
OH
OH
ONO2
HO
HO
including 3, 4 and 6–12, showed stronger inhibitory activity as
compared to the previously mentioned organic nitrates, with KI
values of 11.7–25.6 lM, (Table 1). Thus, the nature of the groups
ONO2
in ortho- and para- to the cyclic OH moiety strongly influences
GR inhibitory activity of the molecules. It is also interesting to
note that hydroxybicyclo [2.2.1]heptan-2-yl nitrate derivatives
11–12 were much better hGR inhibitors as compared to the cor-
responding trans-(1S(R),2S(R))-2-hydroxycyclohexyl nitrate (2)
and trans-(1S(R),8S(R),Z)-8-hydroxycyclooct-4-enyl nitrate (5)
from which they were prepared. Kinetic investigations (Linewe-
aver–Burke plots, data not shown) indicate that similarly to
some antibiotics and analgesic drugs,22–24 all the investigated or-
ganic nitrates act as noncompetitive inhibitors. These results
suggest that protein sulphydryl groups are the target for inhibi-
tion by organic nitrates. The differences in the kinetics of inacti-
vation of trypanothione reductase and glutathione reductase
could reflect differences in the crystal structures of the disul-
phide-binding sites of the enzymes. The active site of trypanothi-
ONO2
10
11
Figure 2. Structure of the investigated molecules.
12
which were either strong or weak inhibitors of erythrocyte GR. For
this purpose, that study selected the derivatives of 2-(59-nitrofuryl-
vinyl)-quinoline-4-carbonic acid, which possess a broad spectrum
of bactericidal and antiparasitic activities,20 and inhibit yeast
GR14a and Trypanosoma congolense trypanothione reductase.14c
The purification of human erythrocyte GR was performed using
simple two-step method by 20,50-ADP Sepharose-4B affinity gel
chromatography and Sephadex G-200 gel filtration chromatogra-
phy.21,22 Human erythrocyte glutathione reductase was purified,
2633-fold with a specific activity of 26.33 EU mgꢀ1 and overall
yield of 32..21–26 b-hydroxy nitrates were synthesized using ring-
opening reaction of epoxides with Bi(NO3)3ꢁ5H2O, which was used
both as catalyst and reagent.27 Some of the nitrate derivatives have
previously been synthesized by other groups as well.28 Inhibitory
effects of organic nitrates on the enzyme activity were tested un-
der in vitro conditions; Ki values were calculated from Linewe-
aver–Burk graphs29 and given in Table 1.
one reductase is wider and possesses
a hydrophobic and
negatively charged region31 that accommodates the spermidine
moiety of its substrate,32 whereas that of glutathione reductase
is much narrower and contains a positively charged and hydro-
philic region33 that interacts with the glycine carboxylates of
glutathione disulphide.34 Molecular modelling studies show that
in glutathione reductase, the disulphide-binding site is narrower,
mainly due to Arg-A330, Arg-A20 and Asn-A100, whereas in try-
panothione reductase, residues that are found at equivalent posi-
tions are hydrophobic and generally smaller. In both pockets,
there is a His near to the phenyl ring of the drug which could
serve to stabilise this aromatic moiety, while a Tyr close to the
melaminyl ring may form electrostatic interactions with the con-
siderable number of nitrogens. These additional noncovalent
We report here the first study on the inhibitory effects of the
organic nitrates 1–12 on human erythrocyte GR. The previous
reports by Becker et al.11c,12 investigated other nitro derivatives
(including arylizoalloxazines) by using Beutler’s method, monitor-
ing GR inhibition.30 Data of Table 1 show inhibition of hGR by com-
pounds 1–12.
Table 1
Ki and IC50 values obtained from regression analysis graphs for GR in the presence of different inhibitors concentrations
Inhibitor
IC50 value (
lM)
Ki value (lM)
Inhibition type
trans-(1S(R),6S(R))-6-Hydroxycyclohex-3-enyl nitrate (1)
trans-(1S(R),2S(R))-2-Hydroxycyclohexyl nitrate (2):
trans-(R(S))-2-Hydroxy-1-phenylethyl nitrate (3):
trans-(1S(R),2S(R))-2-Hydroxycyclooctyl nitrate (4):
23
21
16
14.2
29
35.1
43.7
25.6
21.9
44.3
21.5
18.8
18.4
17.9
17.4
13.1
11.7
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
trans-(1S(R),8S(R),Z)-8-Hydroxycyclooct-4-enyl nitrate (5)
(1S(R),2S(R),5R(S),6R(S))-5-Bromo-9-oxabicyclo[4.2.1] nonan-2-yl nitrate (6)
9(R(S))-Hydroxy-1,2,3,4-tetrahydro-1,4-methano-naphthalen-2(R(S)-yl nitrate (7)
(1R(S),2R(S),4R(S),5R(S))-2,5-Dihydroxycyclo-hexane-1,4-diyl dinitrate (8)
(1S(R),3S(R),4S(R),6S(R))-4,6-Dihydroxycyclo-hexane-1,3-diyl dinitrate (9)
(1R(S),2R(S),3S(R),4S(R))-2,3-Dihydroxycyclo-hexane-1,4-diyl dinitrate (10)
(2S(R),7R(S))-7-Hydroxybicyclo[2.2.1] heptan-2-yl nitrate (11)
(2R(S),7R(S))-7-Hydroxybicyclo[2.2.1]heptan-2-yl nitrate (12)
11
10.3
8.71
8.1
7.17
7.13
6.81

M. Sßentürk et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3661–3663
3663
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interactions could thus serve to stabilise the initial monothioar-
sane enzyme inhibitor complex.33–35
Cunningham et al. demonstrated relatively weaker inhibitory
activity of phenylarsenoxide and arsenite against both enzymes
in their study.35 However, non-covalent interactions per se are
insufficient to allow inhibition by analogues such as sodium melar-
sen or p-[(4,6-diamino-s-triazin-2-yl)amino] benzoic acid ethyl es-
ter that lack the trivalent arsenic atom. The most striking
difference between glutathione reductase and trypanothione
reductase is the 13-fold lower Ki involving the time-dependent
rearrangement to form the dithioarsane adduct.35 Comparison of
the models of trypanothione reductase and glutathione reductase
with the arsenical bound to the disulphide exchange thiol would
suggest that the charge-transfer thiol is indeed less accessible in
glutathione reductase due to the narrower active-site cleft.35
As mentioned above, the active site of GR is narrow and pos-
itively charged, and it might therefore be inhibited because of
the interaction with the hydroxyl groups on organic nitrates.
Contrarily, the active site of typanothion reductase is wider
and negatively charged. This may allow organic nitrates to bind
this enzyme with positively charged nitrogen atoms in a wider
region, and to inhibit typanothion reductase at lower concentra-
tions. Therefore, we propose that the organic nitrates used in
this study, which are also cyclitol derivatives, may be potential
antimalaria drugs.
Organic nitrates 1–12 used in this study affect the activity of
human erythrocyte GR due to the presence of the different func-
tional groups (–OH, –ONO2 and –Br) present in their mono and
bicyclic scaffold. Our findings here indicate thus another class of
possible GR inhibitors of interest, in addition to the well-known
chloroquine and aminoquinoline derivatives bearing bulky in their
molecules. Indeed, some new organic nitrates investigated here
showed effective hGR inhibitory activity, in the low micromolar
range, by the Beutler’s method30 which usually gives KI-s an order
of magnitude higher. These findings point out that substituted ni-
tro compounds may be used as leads for generating potent GR
inhibitors. This approach may also be useful in the design and
exploitation of trypanocidal quinones and nitroaromatics.
16. Henderson, G. B.; Ulrich, P.; Fairlamb, A. H.; Rosenberg, I.; Pereira, M.; Sela, M.;
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22. Detailed procedures for enzyme purification can be found in: Senturk, M.;
Kufrevioglu, O. I.; Ciftci, M. J. Enzym. Inhib. Med. Chem. 2008, 23, 144. To
determine Ki constants in the media with inhibitor, the substrate (GSSG)
concentrations were 0.015, 0.04, 0.07, 0.10, and 0.15 mM. Inhibitor solution
was added into the reaction medium, resulting in three different fixed
concentrations of inhibitors in 1 ml of total reaction volume. Lineweaver–
Burk graphs) were drawn using 1/V versus 1/[S] values, and Ki constants were
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10.0) on a computer (Student t-test; n: 3).
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2036; (b) Senturk, M.; Kufrevioglu, O. I.; Ciftci, M. J. Enzym. Inhib. Med. Chem.
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14, 1027.
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27. Detailed synthetic procedures for the preparation of all derivatives can be
found in: Cavdar, H.; Saracoglu, N. Eur. J. Org. Chem. 2008, 27, 4615.
trans-(1S(R),2S(R))-2-Hydroxycyclohexyl nitrate (2): The product 2 was obtained
from cyclohexenepoxide (1.00 g, 10.21 mmol) and Bi(NO3)3ꢁ5H2O (4.95 g,
10.21 mmol) as described above by typical procedure in 5 min. The residue
was submitted to column chromatography (silica gel, 30 g) eluting with ethyl
acetate/hexane (10:90). While the first elution gave the unidentified product(s)
(500 mg), the last eluate provided the product 2 as colourless oil (1.04 g, 63%).
1H NMR (200 MHz, CDCl3) d 4.77 (ddd, J = 13.6, 9.1, 4.6 Hz, CHONO2, 1H), 3.61
(ddd, J = 13.6, 9.1, 4.6 Hz, CHOH, 1H), 2.89–2.84 (m, OH, 1H), 2.20–2.10 (m,
CH2, 2H), 1.80–1.50 (m, CH2, 2H), 1.49–1.20 (m, CH2, 4H); 13C NMR (50 MHz,
CDCl3) d 89.21, 72.41, 35.06, 30.70, 25.73, 25.43; IR (CH2Cl2, cmꢀ1) 3573, 3400,
3039, 2908, 2854, 1631, 1557, 1440, 1348, 1314, 1277, 1213, 1101, 1071, 1040,
996, 973, 867, 755. Anal. Calcd for C6H11NO4: C, 44.72; H, 6.88; N, 8.69. Found:
C, 44.91; H, 6.58; N, 8.60.
trans-(R(S))-2-Hydroxy-1-phenylethyl nitrate (3): The product 3 (colourless oil,
520 mg, 98%) was prepared as described above by typical procedure for 20 min
by starting Styrene epoxide (350 mg, 2.89 mmol) and Bi(NO3)3ꢁ5H2O (1.40 g,
2.89 mmol). 1H NMR (400 MHz, CDCl3) d 7.40–7.36, (m, aryl, 5H), 5.93 (dd,
J = 8.4, 3.9 Hz, CHONO2, 1H), 3.97 (dd, J = 12.6, 8.4 Hz, A part of AB system,
CH2OH, 1H), 3.86 (dd, J = 12.6, 3.9 Hz, A part of AB system, CH2OH, 1H); 13C
NMR (100 MHz, CDCl3) d 134.73, 129.68, 129.23, 126.88, 86.16, 64.18; IR
(CH2Cl2, cmꢀ1) 3451, 3064, 3032, 2926, 1727, 1706, 1632, 1603, 1575, 1496,
1454, 1413, 1374, 1277, 1255, 1135, 1072, 1027, 753. Anal. Calcd for C8H9NO4:
C, 52.46; H, 4.95; N, 7.65. Found: C, 52.80; H, 4.99; N, 7.77.
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