Structure-activity study on the in vitro, antiprotozoal activity of glutathione derivatives

Article abstract of DOI:10.1021/jm990259w

A series of N-, S-, and COOH-blocked glutathione derivatives were evaluated against the pathogenic parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani in vitro, to identify the determinants necessary for activity and for further devel

Full text of DOI:10.1021/jm990259w

2072
J . Med. Chem. 2000, 43, 2072-2078
Brief Articles
Str u ctu r e-Activity Stu d y on th e in Vitr o An tip r otozoa l Activity of Glu ta th ion e
Der iva tives
Claudius D’Silva* and Sylvie Daunes
Department of Chemistry & Materials, Manchester Metropolitan University, Faculty of Science and Engineering,
J ohn Dalton Building, Chester Street, Manchester M1 5GD, U.K.
Peter Rock, Vanessa Yardley, and Simon L. Croft
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London WC1E 7HT, U.K.
Received J uly 14, 1999
A series of N-, S-, and COOH-blocked glutathione derivatives were evaluated against the
pathogenic parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani in
vitro, to identify the determinants necessary for activity and for further development into an
active lead structure. The results show that N,S-blocked glutathione diesters are the most
effective inhibitors of T. brucei with structures 14-16 being the most active, 14 having an
IC50 ∼ 1.9 µM. The toxicity effects observed for glutathione derivatives 12, 14, and 16 have
been correlated to the K562 antileukemic activity of these compounds and their inhibitory
effects on the glyoxalase system of the host. Diester compounds based on S-2,4-dinitrophenyl-
glutathione (17-22) were found to be significantly better inhibitors of T. brucei with ED50’s in
the range 16-0.19 µM. Compounds 19 and 20 were the two best inhibitors, with an ED50 of
∼1.07 and 0.19 µM, respectively; however 20 displayed toxicity in parasitic assays. Monoesters,
monoamides, and diamides tested generally exhibited low in vitro activity. The compounds
did not inhibit glutathionylspermidine synthetase and trypanothione reductase enzyme targets
in the unique trypanothione pathway of these parasites. Diester compounds per se were
considered to be ineffective inhibitors of trypanothione metabolism suggesting that these
compounds might act as prodrugs, being hydrolyzed in situ into a variety of glutathione
derivatives which include combinations of monoesters, free acids, and amines, some of which
are inhibitors of trypanothione metabolism.
In tr od u ction
sleeping sickness the only effective compounds are ones
which create oxidative stress by altering trypanothione
levels,1,2 such as melarsoprol, which produces fatal side
effects in ∼5% of treated cases,3 or DFMO, an inhibitor
of ornithine decarboxylase, an essential enzyme in
spermidine synthesis and a precursor in trypanothione
biosynthesis.
The effects of trypanosomiasis and leishmaniasis are
a serious problem to the health and economics of
developing countries. Current chemotherapy is in-
adequate. One approach to the development of new
drugs is to identify differences in metabolism between
the host and pathogen and exploit these differences to
design toxic agents. Trypanothione, like glutathione in
the host, plays a central role in the metabolism of
trypanosomatids acting both as a source of reducing
equivalents and as a defense against oxidative stress.1
Drugs effective against African trypanosomiasis1,2 in
humans and animals are few and include suramin (in
human and camels), pentamidine (in humans), and
berenil (in cattle), the arsenicals melarsoprol (in hu-
mans) and cymelarsan (in cattle), and eflornithine (D,L-
R-difluoromethylornithine (DFMO); in humans only).
The ionic nature of suramin and the diamidines however
limits their usefulness as they are not therapeutically
active in the late CNS stage of this disease as they
cannot cross the blood-brain barrier. In late-stage
A key difference in the metabolism of the trypanoso-
matids1,4 is the replacement of the tripeptide gluta-
thione (GSH) and its dependent enzymes in the host
by an analogous family utilizing trypanothione (N1,N8-
bis(glutathionyl)spermidine, T(SH)2). Alternative che-
motherapeutic strategies to find less toxic drugs include
the inhibition of trypanothione reductase with trypano-
thione mimics5 and glutathionylspermidine synthetase
(GSS) with glutathione-modified substrate analogues.6
Despite the successful inhibition of these enzymes in
situ these compounds are inactive against trypanosomes
in vitro.5,6 The inability of rational drug design studies
to produce compounds active in vitro against parasitic
cells led us to adopt a lead directed approach to identify
potential antiparasitic compounds.7 We present here
structure-activity results aimed at identifying the
* Corresponding author. Tel: 00 44 161 2471416. Fax: 00 44 161
2476357. E-mail: C.DSilva@mmu.ac.uk.
10.1021/jm990259w CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/21/2000

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10 2073
esters of N-acetyl-S-benzyloxycarbonylglutathione 15,
and N,S-dibenzyloxycarbonylglutathione 16 showing
considerable activity with ED50’s of 1.9, 3.23 and 17 µM,
respectively. Compounds 14 and 16 had activity against
L. donovani and T. cruzi, but in both cases toxicity to
host macrophages was also observed. Examination of
the data obtained for compounds 1-16 (Table 1) with
T. brucei indicates that diester compounds such as 14-
16 generally show higher activity relative to diamides
13, monoamides 11, and monoesters 10. The nature of
the ester group also appears important if we compare
the dimethyl 12 versus the dibutyl esters 14, with the
latter showing greater activity against T. brucei and
low-grade activity against L. donovani.
necessary determinants required of glutathione ana-
logues to act as toxic agents against trypanosomes in
vitro.
The presence and nature of the N-blocking group
appear important with respect to toxicity against
amastigotes especially if we compare 7, 12, and 15.
Replacement of the formyl group of 7 by an N-acetyl
group (15) increases the activity of this compound
against T. brucei while substitution with a benzyloxy-
carbonyl group (16) produces a lower degree of inhibi-
tion relative to 15 and toxicity effects. This result is not
surprising in view of the inhibitory activity of the de-
esterified derivatives of 14-16 against enzymes of the
mammalian glyoxalase system (GLI and GLII)10,14,15
which are in the order 14 > 16 > 15.10,15 Inhibition of
the glyoxalase system by S-bromobenzylglutathione
diester derivatives has been investigated as a strategy
to antileukemic agents.18 The K562 antileukemic prop-
erties of diesters 1-16 (see Table 1) correlate well with
the toxicity results observed for compounds 12, 14 and
16 in parasitic assays. This result prevents us from
improving the efficacy of the compounds by extrapolat-
ing data obtained in the study of glutathione diester
antitumor agents,18 due to their toxicity and general
nonselectivity in their mode of action.
Ch em istr y
Glutathione ester derivatives 1-10 and 12-13 were
prepared using known methodology published in the
literature.9,11,12,21 Compound 11 was synthesized by the
(dimethylamino)pyridine (DMAP) catalyzed acylation of
2 with benzyl chloroformate (CbzCl)9 in a manner
similar to 10. Derivatives 14 and 16 were synthesized
by the esterification of 9 and N,S-dibenzyloxycarbonyl-
glutathione (mp lit.8 105-107 °C) with their respective
alcohols according to literature procedures.11 The N-
acetyl derivative 15 was synthesized by DMAP acyl-
ation9 of S-benzyloxycarbonylglutathione dimethyl ester
with acetic anhydride in a manner similar to 8.9
S-Benzyloxycarbonylglutathione dimethyl ester was
prepared by cleavage of the Cbz group of 16 with HBr/
AcOH.11
S-2,4-Dinitrophenylglutathione derivatives 17-19, 21
and 22 were synthesized by triethylamine (TEA) cata-
lyzed N-acylation of a mixture of S-2,4-dinitrophenyl-
glutathione mono- and diesters with CbzCl9 and chro-
matographic separation. The mixture of mono- and
diesters was prepared by SOCl2 esterification of S-2,4-
dinitrophenylglutathione in the appropriate alcohol as
described for 3;11 however in this case monoesters11 were
not obtained as the exclusive product but a mixture of
mono- and diesters was isolated. The monoesters were
prepared for use in another study. Compound 20 was
prepared in a manner similar to that described for 10,
12, 14 and 16 by esterification of N-benzyloxycarbonyl-
S-2,4-dinitrophenylglutathione with cyclohexanol ac-
cording to literature procedures.11 S-Bromobenzylgluta-
thione and S-2,4-dinitrophenylglutathione derivatives
were primarily chosen as the basis of these investiga-
tions due to their previously reported efficacy against
a range of glutathione-dependent enzymes.12-15,22
The nature of the S-blocking group appears important
if we compare 12 with 16 as the latter compound with
an S-benzyloxycarbonyl group has higher activity against
T. brucei. However both the S-bromobenzyl and S-
benzyloxycarbonyl groups are good inhibitors of the
glyoxalase system10,12-15,21 and so potentially have the
ability to generate toxicity in parasitic assays depending
on the nature of groups attached at the N-site. S-2,4-
Dinitrophenylglutathione is a weak inhibitor of GLI (Ki
∼ 0.77 mM21) but a moderate inhibitor of glutathione
reductase (Ki50% ∼ 30 µM22), an enzyme structurally
related to trypanothione reductase, and therefore pos-
sibly a better starting material for the synthesis of
antiparasitic compounds. The antiparasitic activities of
diesters 17-21 (see Table 2) are significantly better
especially in the cases of 19 and 20, with activities in
the submicromolar range against T. brucei. The opti-
mum length for the ester group appears to lie between
a butyl group and a hexyl group. Precedence for the
improvement in activity of butyl esters (19) has been
reported for a series of alkyl ester prodrugs based on
ibuprofren.23 Compound 20, the cyclohexyl diester, is
one of the most active compounds as expected with an
ED50 ∼ 0.21 µM and the most toxic based on the
pharmacological responses of glutathione-based anti-
cancer agents.18 However the cytotoxicity in this case
may be associated with the nature of the cyclohexyl
group and its metabolism, as the K562 antileukemic
Resu lts a n d Discu ssion
Glutathione derivatives 1-16 (see Table 1) were
evaluated for antiparasitic activity against bloodstream
form Trypanosoma brucei trypomastigotes and both
Leishmania donovani and Trypanosoma cruzi amastig-
otes in mouse peritoneal macrophages. The inhibition
of parasitic growth observed was expressed as a per-
centage of the untreated control at concentrations up
to ∼30 µM (see Table 1). One of the 16 compounds
evaluated, 2, showed moderate activity against T.
brucei, with another 3, the dibutyl esters of N-benzyl-
oxycarbonyl-S-bromobenzylglutathione 14, the dimethyl

2074 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10
Brief Articles
Ta ble 1. Biological Properties of Glutathione Derivatives against T. brucei, L. donovani, and T. cruzi
compound
R2
ED50 (µM)
T. bruceia
% inhibition (µM)
ID50 (µM)
R1
R3
R4
L. donovanib
T. cruzib
K562
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
BrBz
Cbz
H
H
H
H
OH
OH
OH
OMe
NH2
OH
OMe
OH
OH
OH
OH
OMe
NH2
OBu
OMe
OMe
OH
>30 (40)a
<30 (60)a
>30 (20)a
na
na
na
na
n.a
na
8.7
4.0
na
na
na
na
na
na
T/0
na
68
na
na
na
na
na
na
11
12
na
na
na
na
T/0
na
T/+
na
T/100
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
16.7
>50
16.7
>50
1.26
NH2
OMe
ÃΜe
NH2
OH
OMe
OH
OH
OMe
NH2
OMe
NH2
OBu
OMe
OMe
H
HCO
HCO
CH3CO
Cbz
Cbz
Cbz
Cbz
Cbz
Cbz
CH3CO
Cbz
na
>30 (40)a
>30 (40)a
na
na
>30 (40)a
>30 (20)a
1.9 ( 0.04
3.2 ( 2.4
17 ( 0.66
Cbz
T/100
a
The percent (%) inhibition, in parentheses, at 30 µM is expressed as the percent (%) decrease in viable parasites relative to the
b
control. T/100, toxic to macrophages/100% inhibition of parasites; T/0, toxic to macrophages/0% inhibition of parasites; T/+, toxic to
macrophages/inhibition of parasites; na, not active at ∼30 µM.
Ta ble 2. Biological Properties of S-2,4-Dinitrophenylglutathione Derivatives against T. brucei, L. donovani, and T. cruzi
compound
R2
ED50 (µM)
ID50 (µM)
R1
R3
R4
T. brucei
L. donovani
T. cruzi
na
K562
17
18
19
20
21
22
2,4-DNP
2,4-DNP
2,4-DNP
2,4-DNP
2,4-DNP
2,4-DNP
Cbz
Cbz
Cbz
Cbz
Cbz
Cbz
OMe
OPr
OBu
OcHx
OHx
OOct
OMe
OPr
OBu
OcHx
OHx
OOct
16.2 ( 0.64
6 ( 0.16
1.07 ( 2.1
0.19 ( 0.04
5.8 ( 0.32
na
>30
>30
>30
T/+a
>50
>50
23.4
17.6
21.8
15.9
>50
24.7
na
na
3.0a ( 0.11
<50
na
a
T/+, toxic to macrophages (∼20 µM)/inhibition of parasites; na, not active at ∼30 µM.
activity of 20 is not significantly different from that of
17-19 and 22. Comparison of compound 14 with 19
indicates that the presence of the S-2,4-dinitrophenyl
group enhances the T. brucei activity of 19 by ∼20-fold
relative to the S-bromobenzyl group.
disulfide.16 Compounds 1-3, 8, 9, 12-16 and 19 were
tested at a relatively high concentration of 100 µM as
inhibitors of trypanothione reductase using 5,5′-dithio-
bis{N-[3-(dimethylamino)propyl]-2-nitrobenzamide} as
a colorimetric substrate as described by Davioud-
Charvet.25 No loss in trypanothione reductase activity
was observed suggesting that the target of these com-
pounds was another trypanothione dependent enzyme.
The mechanism of action of these compounds is
consistent with the proposed mechanism reported for
cancer cells treated with GSH esters where diesters
were found to be the most effective compounds for
delivery of GSH to cells due to their partial de-esteri-
fication to free acid and monoester in situ by nonspecific
esterases, within 1 h of addition.17 These compounds (c;
see Scheme 1) were considered prodrugs17-20 functioning
primarily by increasing the membrane penetration of
glutathione derivatives prior to cleavage into three
possible glutathione derivatives (d , f, g; Scheme 1), of
which only two were observed17 (f, g), as expected.
However in this case seven derivatives (a -g; Scheme
1) are possible based on the current understanding of
detoxification mechanisms, which may account for the
diverse range of therapeutic activities found with these
compounds.26 Of these potential derivatives only four
or five types (b, e, f, g, and possibly c) are expected to
Based on the current understanding of glutathione
chemistry and the determinants required for gluta-
thione11-15 and trypanothione2,5,6 to bind to their re-
spective enzymes, we speculate that compounds 14-
21 are ineffective as inhibitors of trypanothione2 or
glutathione enzymes.11-15 Diesters, per se, lack the
minimum requirements for binding, the presence of a
free R-Glu-COOH group.12,13 To be effective as inhibit-
ors these diester compounds would have to undergo
partial or complete de-esterification by nonspecific es-
terases on entry into the parasite. The N-blocked
monoesters or diacids are discounted as inhibitors of the
two enzymes responsible for trypanothione biosynthesis,
glutathionylspermidine synthetase6,24 and trypanothione
synthetase,24 due to the requirement of the former for
a glutathione derivative with a free R-amino group6 and
the latter for an intact glutamyl residue. This leaves
trypanothione reductase as the final candidate, known
to bind derivatives of glutathione lacking a γ-Glu group
or a free COOH group as in the case of N-benzyloxy-
carbonyl-L-cysteinylglycyl-3-methylaminopropylamide

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10 2075
Sch em e 1
be observed in reality depending on the nature of the
groups R2, R3, and R4, located on the glutathione
inhibitor (see Scheme 1). A preliminary study on four
of these glutathione derivatives (b, e, f, g) linked to
sensors27,28 tested against ammonium sulfate fraction-
ated cell extracts of the insect trypanosome Crithidia
fasciculata indicates that two of the four inhibitors (f
and e) were strongly recognized by proteins present in
the 40% and 60-100% (NH4)2SO4 extracts, respectively.
In both cases, up to 76% of the recognition could be
reversed by the addition of inhibitor to the solution. The
40% extract contains proteins of high molecular weight
associated with trypanothione biosynthesis (e.g. GSS
and TSS24) and the latter low molecular weight proteins
(<30 kDa) associated with the trypanosomal peroxidase
cycle (e.g. tryparedoxin peroxidase29 and the trypare-
doxins30,31), while the 40-60% (NH4)2SO4 extract con-
tains the enzyme trypanothione reductase32 which
showed no binding to e, consistent with enzymic studies.
The recognition by these different classes of proteins for
particular inhibitors is consistent with the known
binding preferences of enzymes associated with trypan-
othione biosynthesis and to some extent tryparedoxins
in view of the latter’s relationship to glutaredoxin31 and
ribonucleotide reductase which is known to be inhibited
by glutathione derivatives33 such as e or g. The recogni-
tion of f by a different type of proteins to e may explain
the higher inhibitory activity of compound 15 containing
an N-acetyl group than a benzyloxycarbonyl group
(Cbz). The N-acetyl group is readily deacylated by
amidases, and therefore this compound as a monoester
could possibly inhibit a different parasitic target (e.g.
GSS6,24 or TRS24) to that inhibited by Cbz-protected
glutathiones (10-14, 16) where de-esterification occurs
more readily than deacylation and the resulting com-
pounds resemble b or e (see Scheme 1).
balance of three factors: a, delivery of the compound
into the cell; b, cleavage of the peptide by nonspecific
esterases and amidases in the cytosol into several
inhibitory forms; c, recognition by the target or targets,
inhibition, and cell death.
The activity of compounds 15, 17-19, 21 and 22
clearly suggests that N,S-blocked GSH diester deriva-
tives fulfill many of the above requirements for in vitro
activity, and optimization of their structure to utilize
the prodrug mechanisms proposed to generate the
therapeutically most important active structure of those
considered should eventually lead to a useful chemo-
therapeutic agent.
Con clu sion
Glutathione derivatives have been shown for the first
time to possess significant antiparasitic activity in vitro
with only a few compounds showing selectivity against
T. brucei. A limited structure-activity study indicates
that N,S-blocked glutathione diesters are active against
T. brucei parasites and that N-acetyl-S-benzyloxycar-
bonylglutathione dimethyl ester 15 and the N,S-ben-
zyloxycarbonyl-S-2,4-dinitrophenylglutathione diester
derivatives 17-19 and 21 represent lead structures
possessing minimal toxicity which potentially could be
developed further to yield a therapeutically active agent
for the treatment of trypanosomiasis or leishmaniasis.
Exp er im en ta l Section
GSH, DMAP and CbzCl were obtained from the Avocado
Chemical Co. Ltd. (U.K.). Silica gel 60 F234 and alumina TLC
plates were purchased from BDH Chemicals (Poole, Dorset,
U.K.). NMR spectra were obtained on a J EOL 270 MHz FT-
NMR spectrometer using TMS as an internal standard and
FAB mass spectra on a VG Autospec instrument using cesium
bombardment at 25kV in a matrix of 3-nitrobenzyl alcohol
(NOBA). S-(4-Bromobenzyl)glutathione (1) was synthesized by
the method of Vince et al.21 and S-2,4-dinitrophenylglutathione
Based on this reasoning the overall mode for the in
vitro action of these compounds can be related to the

2076 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10
Brief Articles
by the method of Sokolovsky et al.8 Glutathione derivatives
2-10, 12, and 13 were synthesized by previously reported
methods.11
and extracted into EtOAc, washed H2O, dried (MgSO4 ) and
evaporated in vacuo to give the monoester for use elsewhere.
An analytical sample of monosubstituted and disubstituted
products were prepared by purification using preparative thin-
layer chromatography (PTLC).
P r oced u r e B. This was prepared by SOCl2 (2.2 equiv)
esterification of N-benzyloxycarbonyl-S-2,4-dinitrophenyl-
glutathione in the appropriate alcohol as described for 12.11
N-Ben zyloxyca r b on yl-S-(4-b r om ob en zyl)glu t a t h ion e
glycin a m id e (11) was prepared by N-acylation of 2 (0.5 g;
1.0 mmol) with the CbzCl/DMAP salt (2 equiv) according to
literature procedures9 and recrystallized from CH3CN to
1
recover colorless crystals (0.24 g; 40%): mp 178-182 °C; H
NMR (D6MSO) δ 8.34 (t, Gly-NH, J ) 4.6), 8.2 (d, Cys-NH, J
) 9.3), 7.5 (d, Ar-m-2H, J ) 9.3), 7.35 (s, Ar-5H), 7.3 (d, Ar-
o-2H, J ) 9.3), 7.2 (s, NH), 5.0 (s, O-CH2-Ar), 4.5 (m, CH),
4.0 (m, CH), 3.7 (s, Ar-CH2-S), 3.6 (m, Gly-CH2), 2.7 (m, CH),
2.5 (m, CH2), 2.2 (t, 2H, γ-Glu-CH2), 1.8-2.0 (m, 2H, γ-Glu-
CH2); FABMS m/z 609 ((M + H)+, 40), 633 ((M + Na)+, 32);
HRFABMS calcd for C25H30 N4O7SBr 609.1018, found 609.1017.
Anal. Calcd for C25H29N4O7SBr‚1/2H2O: C, 48.6; H, 4.7; N, 9.0.
Found: C, 49.14; H, 4.41; N, 8.49.
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e d im eth yl ester (17) was prepared using procedure
A from a mixture (1 g) of S-(2,4-dinitrophenyl)glutathione
methyl mono- and diester and isolated by PTLC (MeOH/
CHCl3; 1:3) as pale yellow crystals (0.78 g; 63% yield): mp
142-145 °C; 1H NMR (D6MSO) δ 9.0 (d, ArH3), 8.7 (t, glyNH),
8.5 (d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.7 (d, GluNH),
7.3-7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.65 (td, GluCH), 4.2 (td,
HN-CH-CH2), 3.95 (d, glyCH2), 3.7 (dd, CH2-S), 3.6 (s, 2 ×
CH3OCO), 2.25 (t, CH2CO), 2.0 (td, GluCH2CHCOO); CIMS
m/z 653 ((M + NH4)+, 20), 636 ((MH)+, 35), 436 ((M -
C6H3N2O4S), 100), 502 ((M - C8H7O2), 50); HRFABMS calcd
for C26H30N5O12S 636.3802, found 636.3802. Anal. Calcd for
N-Ben zyloxyca r b on yl-S-(4-b r om ob en zyl)glu t a t h ion e
d i-n -bu tyl ester (14) was synthesized by SOCl2 (0.093 g; 0.72
mmol) esterification of N-benzyloxycarbonyl-S-(4-bromoben-
zyl)glutathione (0.2 g; 0.33 mmol) (9) in n-butanol (15 mL)
using literature procedures as described for 12.11 The solvent
was removed by azeotropic vacuum distillation with water to
give an oily residue which was titurated with Et2O to afford
colorless crystals (0.13 g; 56% yield): mp 110-115 °C; 1H NMR
(D6MSO) δ 8.8 (m, glyNH), 8.2 (d, cysNH, J ) 9.9), 7.8 (d,
GluNH, J ) 9.9), 7.6 (d, 2 × Ar-m-H, J ) 9.1), 7.4 (m, Ar-5H),
7.3 (d, 2 × Ar-o-H, J ) 9.1), 5.1 (s, CH2Ar), 4.65 (m, GluCH),
4.1 (t, 2 × CH2-OCO, J ) 7.2), 4.0 (d, glyCH2, J ) 6.5), 3.7 (s,
S-CH2), 3.6 (m, HN-CH-CH2), 2.8 (dd, CHa-S, J ) 6.6, J )
0.3), 2.7 (dd, CHb-S, J ) 6.6, J ) 0.3), 2.3 (t, CH2CO, J ) 5.7),
2.0 (m, GluCH2CHCOO), 1.6 (m, 2 × CH2-CH2-CH3), 1.4 (m,
2 × CH2-CH3), 1.0 (t, 2 × -CH2-CH3, J ) 9.6). Anal. Calcd for
C
26H29N5O12S: C, 49.13; H, 4.60; N, 11.02. Found: C, 49.54; H,
4.57; N, 10.56.
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e d ip r op yl ester (18) was prepared using procedure A
from a mixture (1 g) of S-(2,4-dinitrophenyl)glutathione n-
propyl mono- and diester and isolated by PTLC (MeOH/CHCl3;
3:17) as pale yellow crystals (0.83 g; 64% yield): mp 110 °C;
1H NMR (D6MSO) δ 9.0 (d, ArH3), 8.7 (t, glyNH), 8.5 (d,
cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6),7.7 (d, GluNH), 7.3-7.4
(m, Ar-5H), 5.1 (s, CH2Ar), 4.65 (td, GluCH), 4.1 (q, 2 × CH3-
CH2-CH2), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7(dd,
CH2-S), 2.25 (t, CH2CO), 1.6 (q, 2 × CH3-CH2-CH2), 2.0 (td,
GluCH2CHCOO), 1.0 (t, 2 × CH3-CH2); CIMS m/z 709 ((M -
NH4)+, 30), 692 ((MH)+, 50), 493 ((MH)+ - C6H3N2O4S), 80),
664 ((M - C2H4), 40), 464 (M - (C2H4 + C6H3N2O4S), 100);
HRFABMS calcd for C30H38N5O12S 692.2237, found 692.2234.
Anal. Calcd for C30H37N5O12S (Mr 691.2159): C, 52.1; H, 5.35;
N, 10.13. Found: C, 52.04, H, 5.33; N, 10.25.
C
33H44N3O8SBr‚3/2H2O: C, 52.93; H, 6.28; N, 5.6. Found: C,
52.77; H, 6.47; N, 6.19.
N-Acetyl-S-(ben zyloxyca r bon yl)glu ta th ion e d im eth yl
ester (15) was synthesized by cleavage of the Cbz group from
compound 16 (1.5 g; 2.5 mmol) with HBr/AcOH as described
for 7,11 followed by acetylation with acetic anhydride/DMAP
(4 equiv) as described for 8,9 to afford colorless crystals (0.38
g), purified by PTLC (1:10; MeOH/CHCl3) to afford an analyti-
cal sample (0.11 g; 8.7%): mp 193-195 °C; 1H NMR (D6MSO)
δ 8.46 (t, Gly-NH, J ) 5.1), 8.26 (d, 2 × NH, J ) 7.1), 7.43 (s,
Ar-5H), 5.28 (s, O-CH2-Ar), 4.58 (m, CH), 4.26 (m, CH), 3.86
(m, Gly-CH2), 3.66 (s, 2 × OCH3), 2.96 (m, CH2), 2.26 (t, 2H,
γ-Glu-CH2), 1.88 (m,s, 5H, γ-Glu-CH2, -COCH3); FABMS m/z
512 ((M + H)+, 67), 534 ((M + Na)+, 100), 1023 ((2M + H)+,
3), 1045 ((2M + Na)+, 5). Anal. Calcd for C22H29N3O9S: C,
51.65; H, 5.72; N, 8.22. Found: C, 51.51; H, 5.69; N, 8.27.
N,S-Diben zyloxyca r bon ylglu ta th ion e d im eth yl ester
(16) was synthesized by SOCl2 (1.6 g; 12.5 mmol) esterification
of N,S-dibenzyloxycarbonylglutathione8 (1 g; 1.7 mmol) in
MeOH (20 mL) as described for 1211 to afford colorless crystals
on recrystallization from methanol (0.7 g; 66.7% yield): mp
167-168 °C; 1H NMR (CDCl3) δ 7.35 (s, 2 × ArH), 5.25 (s,
SCO2-CH2-Αr), 5.1 (s, Αr-CH2-S), 4.7 (m, CH), 4.0 (d, Ar
-CH2-S), 3.7 (s, 2 × OCH3), 3.3 (m, CH), 2.1 (m, Gly-CH2,
γ-Glu-CH2CH2). Anal. Calcd for C28H33N3O10S: C, 55.71; H,
5.51; N, 6.96. Found: C, 55.7; H, 5.7; N, 6.9.
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e Diester s. P r oced u r e A. A mixture of S-(2,4-dinitro-
phenyl)glutathione mono- and diesters was prepared by SOCl2
esterification of S-(2,4-dinitrophenyl)glutathione with the ap-
propriate alcohol using literature procedures as described for
3.11 The crude mixture of mono- and diesters on evaporation
of solvent was triturated with diethyl ether and the residue
N-acylated with CbzCl/TEA (1.1 equiv) in MeOH containing
a catalytic amount of DMAP. After stirring overnight at 55
°C, the solution was evaporated in vacuo and the residue
triturated with Et2O to remove the monoester. The residue
left was extracted into AcOEt, washed water, dried (MgSO4 )
and evaporated in vacuo and then retriturated with Et2O to
give the diester. The Et2O layer containing the crude mono-
ester was acidified to pH 4 with dilute hydrochloric acid (10%)
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e d ibu tyl ester (19) was prepared using procedure A
from a mixture (0.5 g) of S-(2,4-dinitrophenyl)glutathione
n-butyl mono- and diester and isolated by PTLC (EtOAc/
CHCl3; 1:1) as pale yellow crystals (0.179 g; 28% yield): mp
1
115 °C; H NMR (D6MSO) δ 9.0 (d, ArH3), 8.7 (t, glyNH), 8.5
(d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.7 (d, GluNH), 7.3-
7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.65 (td, GluCH), 4.1 (t, 2 ×
CH2-OCO), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7 (dd,
CH2-S), 2.25 (t, CH2CO), 2 (td, GluCH2CHCOO), 1.6 (m, 2 ×
CH3-CH2-CH2), 1.15 (m, 2 × CH2-CH3), 0.95 (t, 2 × CH3-
CH2); FABMS m/z 720 ((M + H)+, 61), 742 ((M + Na)+, 80).
Anal. Calcd for C32H41N5O12S‚1/2H2O: C, 52.72; H, 5.76; N, 9.61.
Found: C, 52.54; H, 5.95; N, 10.25.
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e d icycloh exyl ester (20) was prepared using proce-
dure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)gluta-
thione (0.5 g, 0.82 mmol) in cyclohexanol (25 mL). Azeotropic
removal of the solvent with water gave an oily residue which
on trituration with Et2O gave a hygroscopic yellow solid which
was purified by PTLC (MeOH/CHCl3; 1:9) (0.4 g; 63% yield):
mp 135-137 °C; 1H NMR (D6MSO) δ 8.9 (s, ArH3), 8.6 (t,
glyNH), 8.4 (m, cysNH & ArH5), 8.0 (d, ArH6), 7.7 (d, GluNH),
7.3-7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.6 (m, GluCH & 2 ×
CHcHx), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7 (dd, CH2-
S), 2.3 (t, CH2CO), 2.0 (m, GluCH2CHCOO), 1.4 (m, 2 × (CH2-
CH-CH2)), 1.2 (m, 2 × (CH2)3 cHx); FABMS m/z 772 (M + H)+,
794 (M + Na)+; HRFABMS calcd for C36H46N5O12S 772.2863,
found 772.2827. Anal. Calcd for C36H45N5O12S‚2H2O: C, 53.52;
H, 6.11; N, 8.67. Found: C, 53.23; H, 5.52; N, 8.68.
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e d ih exyl ester (21) was prepared using procedure A
from a mixture (0.5 g, 0.89 mmol) of S-(2,4-dinitrophenyl)-
glutathione n-hexyl mono- and diester and isolated by PTLC
(MeOH/CHCl3; 1:9) as pale yellow solid (0.56 g; 94% yield):

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10 2077
mp 130-132 °C; 1H NMR (D6MSO) δ 9.0 (d, ArH3), 8.7 (t,
glyNH), 8.5 (d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.7 (d,
GluNH), 7.3-7.4 (m, Ar), 5.1 (s, CH2Ar), 4.65 (td, GluCH), 4.1
(t, 2 × CH2OCO), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7
(dd, CH2-S), 2.3 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.6 (t,
2 × CH2-CH2-OCO), 1.3 (m, 2 × (CH2)3), 0.9 (t, 2 × CH3-
CH2); FABMS m/z 776 ((M + H)+, 75), 798 ((M + Na)+, 100);
HRFABMS calcd for C36H50N5O12S 776.3176, found 776.3664.
Anal. Calcd for C36H49N5O12S: C, 55.72; H, 6.37; N, 9.03.
Found: C, 55.75; H, 6.58; N, 9.00.
prior to determination of activity by the use of a tetrazolium
salt colorimetric assay35,37 on day 5.
Tr yp a n oth ion e Red u cta se: Isola tion a n d Assa y. Tryp-
anothione reductase was isolated as previously described32
from Crithidia fasciculata grown on Schneider’s insect medium
(Sigma-Aldrich Co. Ltd., Dorset, U.K.), containing 0.0002%
streptomycin sulfate, FMN (2.4 mg/L) and 10% heat inacti-
vated CFS (Sigma-Aldrich Co. Ltd., Dorset, U.K.). All enzy-
matic and nonenzymatic reactions were conducted in a flat-
bottomed 96-well microtiter plates (Sterilin) in a total volume
of 100 µL. The plates on incubation at room temperature were
then read using a 410 nm filter in a Dynatech MR5000
multiscan microplate reader connected to a Star Multipart LC-
10 printer. Inhibition studies were undertaken on compounds
1-3, 8, 9 and 12-16 using a one-spot enzyme inhibition assay
as previously described25 with 5,5′-dithiobis{N-[3-(dimethyl-
amino)propyl]-2-nitrobenzamide}‚HCl salt25 as the colorimetric
substrate. The reaction was initialized by the addition of 80
µL of enzyme solution containing 42 × 10-4 U of trypanothione
reductase and the reaction stopped after 10 min by the
addition of 20 µL of acetonitrile. Measurements were under-
taken against suitable controls, with and without enzyme,
NADPH, or substrate.
N -B e n zy lo x y c a r b o n y l-S -(2,4-d in it r o p h e n y l)g lu t a -
th ion e d ioctyl ester (22) was prepared using procedure A
from a mixture (0.5 g, 0.81 mmol) of S-(2,4-dinitrophenyl)-
glutathione n-octyl mono- and diester and isolated by PTLC
(MeOH/CHCl3; 1:9) as a pale yellow solid (0.57 g; 82% yield):
1
mp 144 °C; H NMR (D6MSO) δ 9.0 (d, ArH3), 8.7 (t, glyNH),
8.5 (d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.7 (d, GluNH),
7.3-7.4 (m, ArH), 5.1 (s, CH2Ar), 4.65 (td, GluCH), 4.1 (t, 2 ×
CH2OCO), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7 (dd,
CH2-S), 2.3 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.6 (t, 2 ×
CH2-CH2-OCO), 1.3 (m, 2 × (CH2)5), 0.9 (t, 2 × CH3-CH2);
FABMS m/z 832 ((M + H)+, 60), 854 ((M + Na)+, 100). Anal.
Calcd for C40H57N5O12S: C, 57.75; H, 6.91; N, 8.42. Found: C,
57.29; H, 7.10; N, 8.27.
5,5′-Dithiobis{N-[3-(dimethylamino)propyl]-2-nitrobenz-
amide}‚HCl salt was prepared in a manner similar to the
literature25 and obtained as a pale yellow amorphous powder:
ESIMS m/z 565 ([MH]+, 15), 283 (100); HRESIMS calcd for
E va lu a t ion of t h e P a r a sit ic Act ivit y of Glu t a t h ion e
Der iva tives in Vitr o. P a r a sites: T. brucei (strain S427)
bloodstream form trypomastigotes were maintained in HMI-
18 medium34,35 with 20% heat inactivated foetal calf serum
(HIFCS) (Harlan Sera-Lab, Crawley, U.K.) at 37 °C in 5%
CO2-air mixture. L. donovani (strain MHOM/ET/67/L82) was
maintained routinely in special pathogen free (SPF) female
golden hamsters by serial passage every 6-8 weeks. T. cruzi
(strain MHOM/BR/OO/Y) trypomastigotes were derived from
MDCK fibroblasts in Dulbecco’s modified Eagle medium (Life
Technologies Ltd., Paisley, Scotland) with 10% HICFS at 37
°C in a 5% CO2-air mixture.
In vitr o a ssa ys: T. brucei: All compounds were tested in
triplicate in a 3-fold dilution series from a top concentration
of 30 µM. Parasites were diluted to 2 × 105/mL and added in
equal volumes to the test compounds in 96-well, flat bottom
Microtest III tissue culture plates (Becton Dickinson and Co.,
NJ). Appropriate controls with pentamidine isethionate (Rhone-
Poulenc-Rorer) as the positive were set up in parallel. Plates
were maintained for 3 days at 37 °C in a 5% CO2-air mixture.
Compound activity was determined by the use of a tetrazolium
salt colorimetric assay37 on day 3.
C
24H33N6O6S2 [MH]+ 565.1903, found 565.1905.
Ack n ow led gm en t. This investigation received fi-
nancial support from the UDNP/World Bank/WHO
Special Programme for Research and Training in Tropi-
cal Diseases (TDR). The authors also thank EPSRC for
support under Grant GR/F 20937 and Dr. Ballantine
and B. Stein of the EPSRC Mass Spectrometry Service
Center, Swansea, for FABMS and HRFABMS measure-
ments. The Christie Paterson Institute for Cancer
Research, Manchester, is thanked for toxicity testing
of compounds and H. Kendrick (LSHTMS) for statistical
analysis of the data. The authors also thank the
Biological Sciences Department (MMU) and Microbiol-
ogy Section for access to their facilities and 21C Tech-
nology Ltd. (Cheshire) for use of their sensor.
Refer en ces
L. donovani and T. cruzi: Peritoneal macrophages were
harvested from female CD1 mice (Charles Rivers Ltd., Mar-
gate, U.K.) by peritoneal lavage 24 h after starch (Merck Ltd.,
Leics, U.K.) induced recruitment. After washing cells were
dispensed into 16-well Lab-tek tissue culture slides (Nunc Inc.,
IL) at 4 × 104/well in a volume of 100 µL of RPMI-1640
medium (Sigma-Aldrich Co. Ltd., Dorset, U.K.) and 10%
HIFCS. After 24 h, macrophages were infected at a ratio of
10:1 (4 × 105/well) with L. donovani amastigotes or 5:1 (2 ×
105/well) with T. cruzi trypomastigotes. Infected macrophages
were then maintained in the presence of drug in a 3-fold
dilution series in quadruplicate for 5 days in the case of L.
donovani and 3 days with T. cruzi. Drug activity was evaluated
from the percentages of macrophages cleared of amastigotes
in treated cultures. Sodium stibogluconate (NaSbv) (Glaxo-
Wellcome, Dartford, U.K.) and nifurtimox (Bayer, U.K.) were
used as the respective controls.36
Eva lu a tion of th e Cytotoxicity of Glu ta th ion e Der iva -
tives in Vitr o. Cytotoxicity testing on K562 (human, chronic
myelogenous leukemia) was undertaken independently on our
behalf by the Christie Patterson Institute for Cancer Research,
Manchester. The cytotoxicity of drugs were evaluated using
the semi automated MMT assay developed by the National
Cancer Institute (NCI), Maryland, based on the original work
of Mosmann.37 Cell cultures were grown in RPMI medium with
10% calf fetal serum (CFS) at 37 °C in a 5% CO2-air mixture
in a humidified incubator. Plates were incubated with com-
pound for 5 days at 37 °C in a 5% CO2-humidified air mixture
(1) Barrett, M. P.; Fairlamb, A. H. The Biochemical Basis of
Arsenical-Diamidine Crossresistance in African Trypanosomes.
Parasitol. Today 1999, 15 (4), 136-140.
(2) Krauth-Siegel, R. L.; Coombs, G. H. Enzymes of Parasite Thiol
Metabolism as Drug Targets. Parasitol. Today 1999, 15 (10),
404-409.
(3) Hajduk, S. L.; Englund, P. T.; Smith, D. H. In Tropical and
Geographic Medicine; Warren, K. S., Mahmoud, A. A. F., Eds.;
McGraw-Hill: New York, NY, 1989; pp 268-281; Tropical
Disease Science at Work (World Health Organisation, Geneva,
Switzerland, 1986).
(4) Fairlamb, A. H.; Cerami, A. Metabolism and functions of
trypanothione in the kinetoplastid. Annu. Rev. Microb. 1992, 46,
695-729.
(5) Kosower, E. M.; Radkowsky, A. E.; Fairlamb, A. H.; Croft, S.
L.; Neal, R. A. Bimane cyclic esters, possible stereologues of
trypanothione as antitrypanosomal agents. Bimanes 29. Eur. J .
Med. Chem. 1995, 30, 659-671.
(6) DeCraecker, S.; Verbruggen, C.; Rajan, P. K.; Smith, K.; Hae-
mers, A.; Fairlamb, A. H. Characterization of the peptide
substrate specificity of glutathionylspermidine synthetase from
Crithidia fasciculata. Mol. Biochem. Parasitol. 1997, 84, 25-
32.
(7) D’Silva, C.; Daunes, S. Glutathione peptides as anti-parasitic
drugs. Symposium abstracts, 1st COST-B9 Congress on Anti-
protozoal Chemotherapy, Sierra Nevada, Granada, Spain, May
31-J une 3, 1998.
(8) Sokolovsky, M.; Wilchek, M.; Patchornik, A. On the synthesis
of cysteine peptides. J . Am. Chem. Soc. 1964, 86, 1202-1206.
(9) D’Silva C.; Timari A. A.; Douglas K. T. Synthesis of N-acyl
glutathione derivatives by (dimethylamino)pyridine catalysis.
Biochem. J . 1982, 207, 329-332.

2078 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 10
Brief Articles
(10) Douglas K. T.; Timari A. A.; D’Silva, C.; Gohel D. I. Role of the
N-terminus of glutathione in the action of yeast glyoxalase I.
Biochem. J . 1982, 207, 323-329.
(11) D’Silva, C. Synthesis of carboxy-residue-modified coenzyme
derivatives as probes to the mechanism of glutathione enzymes.
Biochem. J . 1990, 271, 167-169. D’Silva, C. Correction. Biochem.
J . 1990, 3, 483.
(12) D’Silva C. Reinvestigation of the roles of the carboxyl groups of
glutathione with yeast glyoxalase I: Implications to the mech-
anism and coenzymic role of glutathione. FEBS Lett. 1986, 202,
240-244.
(13) D’Silva C. Inhibition and Recognition Studies on the glutathione
binding site of Equine liver glutathione-S-transferase. Biochem.
J . 1990, 271, 161-165.
(14) Norton, S. J .; Elia, A. C.; Chyan, M. K.; Gillis, G.; Frenzel, C.;
Princpato, G. B. Inhibitors and inhibition studies of mammalian
glyoxalase II. Biochem. Trans. 1993, 21, 545-549.
(15) Al-Timari, A.; Douglas, K. T. Inhibition by glutathione deriva-
tives of bovine liver glyoxalase II (hydroxyacylglutathione hy-
drolase) as a probe of the N- and S-sites for substrate binding.
Biochim. Biophys. Acta 1986, 870, 219-225.
(16) El-Waer, A. F.; Smith, K.; McKie, J . H.; Benson, T.; Fairlamb,
A. H.; Douglas, K. T. The glutamyl binding site of trypanothione
reductase from Crithidia fasciculata: enzyme kinetic properties
of γ-glutamyl modified substrate analogues. Biochim. Biophys.
Acta 1993, 1203, 93-98.
(17) Thornalley, P. J .; Edwards, L. G.; Kang, Y.; Wyatt, C.; Davies,
N.; Ladan, M. J .; Double, J . Antitumour activity of S-p-
bromobenzylglutathione cyclopentyl diester in vitro and in vivo.
Biochem. Pharmacol. 1996, 51, 1365-1372.
(18) Thornalley, P. J .; Ladan, M. J .; Ridgway, J . S.; Kang, Y.
Antitumour Activity of S-(p-Bromobenzyl)glutathione diesters
in Vitro: A Structure-Activity Study. J . Med. Chem. 1996, 39,
3409-3411.
(19) Levy, E. J .; Anderson, M. E.; Meister, A. Transport of glutathione
diethyl esters in human cells. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 9171-9175.
(25) Davioud-Charvet, E.; Becker. K.; Landry, V.; Gromer, S.; Loge′,
C.; Sergheraert, C.; Synthesis of 5,5′-Dithiobis(2-nitrobenz-
amides) as Alternative Substrates for Trypanothione Reductase
and Thioredoxin Reductase: A Microtitre Colorimetric Assay for
Inhibitor Screening. Anal. Biochem. 1999, 268, 1-8.
(26) S-Blocked Glutathiones. U.K. Patent filed Sept 1,1997, Pub. no.
GB2332676A.
(27) D’Silva, C. Receptor based Biosensors. In Biosensors (Current
Topics in Biophysics); Frangopol, P. T., Sanduloviciu, M., Eds.;
Iasy University Press: Iasy, Romania, 1995; Vol. 5, pp 1-19.
(28) Barnes, C.; D’Silva. C.; J ones, J . P.; Lewis, T. J . The Theory of
operation of Piezoelectric Crystal Sensors for Biochemical Ap-
plications. Sensors Actuators A. 1992, 31, 159-163.
(29) Montemartin, M.; Nogoceke, E.; Singh, M.; Steinert, P.; Flohe,
L.; Kalisz, H. M. Sequence analysis of the Tryparedoxin Per-
oxidase Gene from Crithidia fasciculata and its Functional
Expression in Escherichia coli. J . Biol. Chem. 1998, 273, 4864-
4871.
(30) Gommel, D. U.; Nogoceke, E.; Morr, M.; Kiess, M.; Kalisz, H.
M.; Flohe, L. Catalytic characteristics of tryparedoxin. Eur J .
Biochem. 1997, 248, 913-918.
(31) Ludemann, H.; Dormeyer, M.: Sticherling, C.; Stallmann, D.;
Follmann, H.; Krauth-Siegel, R. L. Trypanosoma brucei trypare-
doxin, a thioredoxin-like protein in African trypanosomes. FEBS
Lett. 1998, 431, 381-385.
(32) Shames, S. L.; Fairlamb, A. H.; Cerami, A.; Walsh, C. T.
Purification and Characterisation of Trypanothione Reductase
from Crithidia fasciculata a Newly Discovered Member of the
family of Disulphide-Containing Flavoprotein Reductases. Bio-
chemistry 1986, 25, 3519-3526.
(33) Hoog, J .-O.; Holmgren, A.; D’Silva, C.; Douglas, K. T.; Seddon,
A. P. Glutathione Derivatives as inhibitors of Glutaredoxin and
ribonucleotide Reductase from Escherichia coli. FEBS Lett. 1982,
138, 59-61.
(34) Hirumi-H.; Hirumi-K. Continuous cultivation of Trypanosoma
(20) Minhas, H. M.; Thornalley, P. J . Comparison of the delivery of
reduced glutathione into P388D1 cells by reduced glutathione
and its mono and di-ethyl ester derivatives. Biochem. Pharmacol.
1995, 49, 1475-1482.
brucei blood stream forms in
a medium containing a low
concentration of serum proteins without feeder cell layers. J .
Parasitol. 1989, 75, 985-989.
(21) Vince, R.; Daluge; S.; Wadd. W. B. Studies on the inhibition of
glyoxalase I by S-substituted glutathiones. J . Med. Chem. 1971,
14, 402-405.
(35) Ellis, J . A.; Fish, W. R.; Sileghem, M.; McOdimba, F.
A
colorimetric assay for trypanosome viability and metabolic
function. Vet. Parasitol. 1993, 50, 143-149.
(36) Neal, R. A.; Croft, S. L. An in vitro system for determining the
activity of compounds against the intracellular amastigote form
of Leishmania donovani. J . Antimicrob. Chemother. 1984, 14,
463-475.
(37) Mossmann, T. Rapid Colorimetric assay for cellular growth and
Survival. Application to Proliferation and Cytotoxicity Assay.
J . Immunol. Methods 1983, 63, 55-66.
(22) Blizer, M.; Krauth-Siegel, R. L.; Schirmer, R. H.; Akerboom, T.
P. M.; Sies, H and Schulz, G. E. Eur. J . Biochem. 1984, 138,
373-378.
(23) Bansal, A. K.; Dubey, R.; Khar, R. K. Quantitation of Activity
of Alkyl ester Prodrugs of Ibuprofen. Drug Dev. Ind. Pharm.
1994, 20 (12), 2025-2034.
(24) Smith, K.; Nadeau, K.; Bradley, M.; Walsh, C.; Fairlamb, A. H.
Purification of glutathionylspermidine and trypanothione syn-
thetases from Crithidia fasciculata. Protein Sci. 1992, 1, 874-
883.
J M990259W