QSAR study on the contribution of log P and Es to the in vitro antiprotozoal activity of glutathione derivatives

Article abstract of DOI:10.1021/jm000502n

A series of N-S-blocked glutathione monoester and diester derivatives based on N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione were evaluated for activity against the pathogenic parasites Trypanosoma brucei brucei, Trypanosoma cruzi, and Leishmania d

Full text of DOI:10.1021/jm000502n

2976
J . Med. Chem. 2001, 44, 2976-2983
QSAR Stu d y on th e Con tr ibu tion of Log P a n d Es to th e in Vitr o An tip r otozoa l
Activity of Glu ta th ion e Der iva tives
Sylvie Daunes and Claudius D’Silva*
Department of Chemistry & Materials, Faculty of Science and Engineering, The Manchester Metropolitan University,
J ohn Dalton Building, Chester Street, Manchester, M1 5GD, U.K.
Howard Kendrick, 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 November 27, 2000
A series of N-S-blocked glutathione monoester and diester derivatives based on N-benzyloxy-
carbonyl-S-(2,4-dinitrophenyl)glutathione were evaluated for activity against the pathogenic
parasites Trypanosoma brucei brucei, Trypanosoma cruzi, and Leishmania donovani in vitro.
Only monoesters 7-9 with a log P value of >2.7 were active inhibitors of T.b. brucei bloodstream
form trypomastigotes. Diester compounds 10-15 and 17-27 in most cases were better inhibitors
of T.b. brucei than monoester compounds, and some displayed high activity against T. cruzi
14 and L. donovani 17, 19, 29. Compounds 14, 24, and 25 were the most active compounds
identified against T.b. brucei having ED50 values of <0.4 µM. Analysis of the inhibition data
(ED50) vs calculated log P and Es values provided evidence to support membrane penetration
and steric factors as the key component in the activity of these compounds. The optimum values
for log P and Es determined were 5.8 and -0.70, respectively. A QSAR equation relating log-
(1/ED50) vs log P and Es was determined and interpreted within the proposed mechanism of
activity for these compounds.
In tr od u ction
of these compounds have proved to be inactive against
trypanosomes in vitro.6-8 The poor success of rational
drug design studies to produce compounds active in vitro
against these types of protozoa led us to adopt a lead
directed approach to identify potential antiparasitic
compounds. In a recent paper9 we reported the identi-
fication of several glutathione derivatives with activity
against Trypanosoma and Leishmania. A structure-
function study on S-bromobenzylglutathione derivatives
identified the antiparasitic activity to be exclusively
associated with N,S-blocked glutathione diester deriva-
tives.9 The nature of the N and S groups was found to
contribute to the compounds’ selectivity.9 Glutathione
diesters, due to their ease of membrane penetration and
hydrolysis by nonspecific esterases to free acids, have
been proposed as chemical delivery systems (CDS) for
glutathione into cells.10,11
Trypanosomiasis and leishmaniasis are important
parasitic diseases in humans and domestic animals
throughout the tropical and subtropical world. These
infections pose serious health problems to the countries
in these regions as well as damage to their economies.1,2
The chemotherapy of these diseases is deficient as many
drugs have either poor efficacy, toxic side effects, or are
ineffective in the late chronic stages of the disease.
Parasites display a large number of unique metabolic
pathways not present in other mammalian cells. One
general approach to the development of novel antipara-
sitic drugs is to identify key differences in metabolism
between the host and pathogen and to exploit them in
the design of selective toxic agents. Thiol metabolism
of trypanosomatids3 is characterized by a dependence
on the hexapeptide trypanothione (N1,N8-bis(glutathio-
nyl)spermidine) (T(SH)2), an antioxidant replacing glu-
tathione (GSH), the major antioxidant in other eukary-
otic cells. The importance of trypanothione as an
antiprotozoal drug target is highlighted by the fact that
some trypanocidal drugs, notably the arsenicals (melar-
soprol)4 and difluoromethylornithine (eflornithine),5 may
work by interfering with the metabolism or synthesis
of this hexapeptide. The central role of trypanothione
in Trypanosoma and Leishmania parasites make thiol-
dependent enzymes potential targets for the develop-
ment of chemotherapeutic drugs. Despite the successful
inhibition of enzymes such as trypanothione reductase6
and glutathionylspermidine synthetase7,8 in situ, some
The nature of the ester group controls the hydrophobic
character of derivatives and its ability to cross cell
membranes, while the conformation and structure of the
group may control its susceptibility to hydrolysis, due
to its fit with the binding sites of esterases and as a
consequence its receptor interactions. To evaluate the
role of hydrophobicity and conformation on the Trypa-
nosoma brucei brucei activity of glutathione derivatives,
we synthesized a series of glutathione mono 3-9 and
diester derivatives 10-32 of increasing hydrophobicity
and steric variability as shown in Scheme 1. We present
here a QSAR analysis of these results using the phys-
icochemical parameters log P (an index of hydrophobic-
ity) and Es (Taft’s steric parameter) to quantify the roles
* To whom correspondence should be addressed. Tel: 00 44 161
2471416. Fax: 00 44 161 2476357. E-mail: C.DSilva@mmu.ac.uk.
10.1021/jm000502n CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/08/2001

Antiprotozoal Activity of Glutathione Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2977
Sch em e 1
Ta ble 1. Monoester Derivatives
played by each parameter to the in vitro activity of these
compounds.
ED50 (µM)
ED50 (µM)
L. donovani
ED50 (µM)
T. cruzi
cpd
T.b. brucei
log P
3
4
5
6
7
8
9
naa
na
na
>30
>30
>30
na
na
na
na
na
na
na
1.32
1.82
2.31
2.23
2.8
Ch em istr y
S-(2,4-Dinitrophenyl)glutathione 1 and N-benzyloxy-
carbonyl-S-(2,4-dinitrophenyl) glutathione 2 were pre-
pared by standard procedures12 and used as the starting
materials for the synthesis of a series of mono- and
diester derivatives 3-32. Monoesters and diesters were
synthesized using known methodology in the litera-
ture.9,12,13 The synthesis of monoesters 3-9 was under-
taken using S-(2,4-dinitrophenyl)glutathione 1 as the
starting material with 1 mol equiv of thionyl chloride.
An intimate mixture of mono- and diester glutathione
derivatives was obtained which on reaction with ben-
zylchloroformate was converted into the corresponding
N-carbobenzyloxy derivatives and separated by pre-
parative thin-layer chromatography (PTLC).9 This pro-
cedure (A) allowed the synthesis of monoesters 3-9 and
diesters 10-13 (see Scheme 1). The remaining diesters
14-32 (see Scheme 1) were obtained by the reaction of
N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione 2
with the appropriate alcohols and thionyl chloride
(procedure B).
na
20.0
20.0
10.26
na
20.0
2.72
2.8
>30 (12)f
>30 (42)f
a
na ) not active at ∼30 µmol.
glutathione 1-butyl glutamate 9 was also isolated in
small quantities in addition to the diester 13.
The inhibitory activities of N-benzyloxycarbonyl-S-
(2,4-dinitrophenyl) glutathione monoesters 3-9 were
investigated against T.b. brucei, Leishmania donovani,
and Trypanosoma cruzi (see Table 1). Low grade activity
was observed against T.b. brucei with compounds 7-9
and against L. donovani with 8. These results indicate
that monoesters based on N-benzyloxycarbonyl-S-(2,4-
dinitrophenyl)glutathione with a log P value of >2.7 are
probably sufficiently hydrophobic to enter parasite cells.
Diester compounds 13, 14, 29, 30 (Table 2) and 20,
24, 25 (Table 3) showed significant inhibitory activity
against T.b. brucei (>µM). Of the linear glutathione
diester derivatives (see Table 2), compound 14, the
pentyl diester (log P ∼ 6.04), proved the most active with
an ED50 value of 0.38 µM against T.b. brucei and 6.7
µM against T. cruzi. For the heteroatom substituted
diesters 28-30, the most active linear diesters against
T.b. brucei (ED50 ∼ 0.50 µΜ) were the chloroethyl and
chloropropyl diesters 29 and 30, respectively. However,
the toxicity of these types of compounds to KB cells was
high, as expressed by the small relative toxicity values
(KB/T. brucei) of less than unity (see Table 2), except
in the case of 29.
Resu lts a n d Discu ssion
The synthesis of monoesters 3-9 did not proceed as
expected, and the major product formed was the diesters
10-13, contaminated with a small quantity of mo-
noester (<20%). The latter required a significant amount
of purification to achieve homogeneity. This result was
contrary to studies on the use of this technique in the
preparation of S-bromobenzylglutathione monoesters13
and probably a result of the poor solubility of 1 in the
alcohols used resulting in the initially formed mo-
noesters being immediately converted to diesters. The
synthesis of S-(2,4-dinitrophenyl)glutathione 1-butyl
glycinate 7 proved an anomaly, as S-(2,4-dinitrophenyl)-
In the branched series, compounds 20, 24, and 25
displayed the highest activity against T.b. brucei (0.25
µM, 0.18 µΜ, and 0.21 µM, respectively). The inhibitory

2978 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Ta ble 2. Linear Diester Derivatives
Daunes et al.
ED50 (µM)
T.b. brucei
ED50 (µM)
L. donovani
ED50 (µM)
T. cruzi
ED50 (µM)
KB cells
rel. tox
KB/T.b.
c
cpd
log P
Es
10
11
12
13
14
15
16
28
29
30
31
32
16.0 ( 0.64
3.5 ( 0.1
6.0 ( 0.16
1.07 ( 2.1
0.38
5.8 ( 0.32
na
21.8 ( 0.03
0.49 ( 0.06
1.05
>30
na
na
na
na
17.9
272
205
1.1
78
34
89
789
18.8
2.11
3.09
4.07
5.06
6.04
7.02
8.99
2.97
3.6
0.0
>30
-0.07
-0.36
-0.39
-0.41e
-0.43e
>30
>30 (36)f
>30
95.2
>300
109.3
95.3
15.2
49.5
0.39
0.38
0.70
6.7 ( 2.9
>30 (36)f
na
>30 (9.0)f
>30 (11)f
>30 (26)f
9.92 ( 1 × 10-4
>30
>30 (8.0)f
T/+a
0.99
101
0.8
0.12
0.07
>30 (35)f
28.0 ( 2.1
4.58
5.56
1.56
3.05
10.04
>30
20
a
b
d
T/+: toxic to macrophages (∼20 µmol)/inhibition of parasites. na ) not active at ∼30 µmol. c Taft steric factor. Calculated value
of Es using equation shown in Appendix. e Graphically extrapolated values from a plot of Es vs number of carbon atoms for the series of
linear esters (Me, Et, Pr, Bu). f The percent (%) inhibition, in parentheses, at 30 µΜ is expressed as the percent (%) decrease in viable
parasites relative to the control.
Ta ble 3. Branched Diester Derivatives
ED50 (µM)
T.b. brucei
ED50 (µM)
L. donovani
ED50 (µM)
T. cruzi
ED50 (µM)
KB cells
rel. tox
KB/T.b.
c
cpd
log P
Es
17
18
19
20
21
22
23
24
25
26
27
20 ( 0.33
2.0
7.8
na
na
269
>300
245
43.2
393
13.5
150
24
173
118
46
3.93
4.91
5.75
6.73
5.89
6.87
7.71
5.67
6.65
5.89
6.87
-0.47
-1.13
-1.36
-1.59d
-1.15
-1.37
-1.8d
-0.51
-0.67
-1.1
>30 (36)
13.4 ( 0.15
>30 (8.0)e
>30 (25)e
>30 (6.0)e
>30 (70)e
29.6 ( 1.3
T/+a
6.0 ( 0.08
0.25 ( 0.02
3.34 ( 0.4
5.2 ( 0.44
34.8
0.18 ( 0.004
0.21 ( 0.04
3.74 ( 0.03
2.06 ( 0.05
<30 (64)e
>30 (31)e
>30 (17)e
>30 (21)e
T/+a
178
>300
50.5
51.0
213
>9
>30 (30)e
T/+a
280
243
57
>30 (17)e
>30 (12)e
T/+a
T/+a
211
102
-1.3
a
b
d
T/+: toxic to macrophages (∼20 µmol)/inhibition of parasites. na ) not active at ∼30 µmol. c Taft steric factor. Calculated value
of Es using the equation shown in the Appendix. e The percent (%) inhibition, in parentheses, at 30 µΜ is expressed as the percent (%)
decrease in viable parasites relative to the control.
activity of 24 (0.18 µM; log P ∼ 5.67) and 25 (0.21 µM;
log P ∼ 6.65) against T.b. brucei (Table 3) was margin-
ally better than 14 (0.38 µM; log P ∼ 6.04), the best
linear diester (see Table 2). However, 25 displayed
toxicity against macrophages used in the assay of these
parasites (see Table 3). The branched diester compounds
17 and 19 exhibited low activity against T.b. brucei but
a high inhibitory activity against L. donovani (7.8 and
13.4 µM, respectively), indicating a major difference in
the inhibitory behavior of these compounds. The assay
of L. donovani and T. cruzi is undertaken in murine
macrophages, so the inhibition pattern observed prob-
ably reflects differences both in the penetration of host
macrophage and the intracellular parasite.
A large proportion of branched diesters tested (see
Table 3) showed some degree of toxicity against mac-
rophages not seen in KB cells which may reflect differ-
ences in the phase I metabolism of branched chain
esters between different cell types14 and the toxicity of
the hydrolysis product generated.
(1/ED50) and log P, but the fit in this case was better.
The optimum value for log P (log Po) was estimated from
the graphical plot to be 5.8 in the case of T.b. brucei.
This value was consistent with compounds possessing
good membrane penetration, with log P values in the
range ∼4-7,14,16 and also indicates that the T.b. brucei
activity of these glutathione diester derivatives was
controlled to some degree by their ability to enter cells,
as in the case of the anaesthetic ethers.17 To address
the poor fit of the linear diesters 10 and 11 and their
branched counterparts 19, 21, 22, and 26 to the para-
bolic dependence, the QSAR analysis of these data was
evaluated in addition to the hydrophobicity descriptor
(log P), two steric descriptors, molar refractivity (MR),
and Taft’s steric factor (Es).18,19. Inclusion of MR ap-
peared to be nonbeneficial, but this was not the case
for Es. Due to the absence of Es values for the ester
group 20 and 23 in the literature,18,19 a value for this
parameter was derived using multiple regression analy-
sis (see Appendix and Table 3).
To identify the nature of the relationship between log-
(1/ED50) and log P for T.b. brucei, a plot was undertaken
on the data in Tables 2 and 3. The plot was found to be
predominantly parabolic in nature with an optimum
value for log P in the range 5.0-6.0. However, diesters
19, 21, and 26, and to a lesser extent 10 and 11,
exhibited a poor fit to the plot, indicating a more
complex relationship between log(1/ED50) and log P,
which involved other descriptors. The inhibition of
Trypanosoma brucei rhodesiense15 by compounds 10-
32 showed a similar parabolic dependence between log-
In the case of the pentyl 14 and hexyl 15 esters, Es
values were obtained by graphical extrapolation of the
nonlinear plot of ED50 vs Es, for linear diesters. Due to
the limitations of the SciQSAR program permitting the
evaluation of only one user derived descriptor, analysis
of log P and Es was not possible with this package. The
QSAR equation determined was therefore derived by
multiple linear regression analysis using the LINEST
function in Microsoft Excel with the descriptors log P,
Es, and SciQSAR calculated descriptors (see Appendix).
The best linear QSAR equation derived by a rigorous

Antiprotozoal Activity of Glutathione Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2979
process of elimination, using a set of 17 compounds,
excluding the outliers 17, 19, and 20, was eq 1 with an
R2 ) 0.84 and F ) 12.0.
Exp er im en ta l Section
Precursors and solvents were purchased commercially and
purified appropriately. GSH, DMAP, and CbzCl were obtained
from the Avocado Chemical Co. Ltd. (U.K). Glutathione
derivatives 1, 9, 11, 12, 14, 15 and 25 were prepared as
previously described.9 Compounds 3-9 and 11 were synthe-
sized using literature procedure A9 and 14, 17-24, and 26-
32 using procedure B.9 5,5′-Dithiobis{N-[3-(dimethylamino)-
propyl]-2-nitrobenzamide}‚HCl salt21 was synthesized and
assayed against trypanothione reductase as described previ-
ously by standard assay on microtiter plates.21
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e (2) was prepared by the modification of a previously
reported method.12 To a stirred solution of S-(2,4-dinitrophe-
nyl)glutathione (1) (2 g; 4.2 mmol) in MeOH were added
dropwise Et3N (0.88 mL; 6.34 mmol) and benzylchloroformate
(0.9 mL; 6.34 mmol). After 20 h the solution was evaporated,
acidified (pH 2), extracted (Et2O), dried (MgSO4), and evapo-
rated in vacuo. The residue was triturated with Et2O and dried
(P2O5) to give a yellow product (2.3 g; 90% yield): mp 120-
125 °C; 1H NMR (D6MSO) δ 9.0 (s, ArH3), 8.7 (m, glyNH), 8.5
(2H, m, cysNH and ArH5), 8.1 (d, ArH6), 7.8 (d, GluNH), 7.3-
7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.65 (m, GluCH), 4.1 (m, HN-
CH-CH2), 4.0 (d, glyCH2), 3.6 (dd, CH2-S), 2.2 (t, CH2CO), 2.0
(td, GluCH2CHCOO).
log(1/ED50) ) -2.57 log P + 1.87Es + 0.20 MW -
0.002W - 102 (1)
The equation consisted of two types of descriptors,
hydrophobicity (log P) and steric factors (Es, MW, W).
The WienI (W) descriptor is a topological parameter20
and a measure of the branching of a molecule, the value
being larger for extended molecules than compact ones.
Equation 1 indicates that the therapeutic activity of
these compounds decreases with increasing values of
log P and Es, the latter a result of the overall negative
character of these parameters (the log P coefficient is
negative (see eq 1), while Es values are predominantly
negative (see Appendix), thus these two parameters
render the overall equation negative in character). This
result is consistent with there being a maximum value
for log P and Es. A plot of log(1/ED50) vs Es also showed
a parabolic dependence between the parameters with
an optimum value in this case of -0.70 for Es. The
significance of log P and Es may be interpreted within
the proposed mechanism9 for the delivery of glutathione
diesters into cells involving diester delivery (related to
log P and Es) and hydrolysis to free acid and monoester
by nonspecific esterases (related to Es). HPLC studies15
have confirmed the fate of these diesters in T.b. brucei
cells by the isolation of monoester and diacid, in the cell.
Trypanothione reductase, the primary target for the
design of antiparasitic drugs, was again tested and
eliminated in this case by evaluation of compounds 1,
2, 7, and 13 (Scheme 1) against enzyme isolated from
Crithidia fasciculata using 5,5′-dithiobis{N-[3-(dimethy-
lamino)propyl]-2-nitrobenzamide}‚HCl salt21 as a sub-
strate as previously reported.21
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e m eth yl glycin a te (3) was prepared using procedure A
from S-(2,4-dinitrophenyl)glutathione methyl ester (1 g, 1.97
mmol), MeOH (30 mL), Et3N (0.42 mL, 2.96 mmol), and
benzylchloroformate (0.42 mL, 2.96 mmol) and isolated by
PTLC (MeOH/CHCl3; 1:3) as pale yellow crystals (48 mg; 4%
1
yield): mp 128-130 °C; H NMR (D6MSO) δ 9.1 (t, glyNH),
9.0 (d, ArH3), 8.7 (d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.3-
7.4 (m, Ar-5H), 6.7 (d, GluNH), 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, CH3OCO), 2.25 (t, CH2CO), 2.0 (t, GluCH2CHCOO).
Anal. (C25H27N5O12S‚H2O) N; C: calcd, 46.86; found, 46.30;
H: calcd, 5.48; found, 5.03.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e eth yl glycin a te (4) was prepared using procedure A from
S-(2,4-dinitrophenyl)glutathione ethyl ester (1 g, 1.92 mmol),
MeOH (30 mL), Et3N (0.40 mL, 2.9 mmol), and benzylchloro-
formate (0.40 mL, 2.9 mmol) and isolated by PTLC (MeOH/
CHCl3; 1:3) as pale yellow crystals (72 mg; 6% yield): mp 105-
The pharmacological value of the compounds identi-
fied in this study is dependent on their specificity which
is a consequence of three main factors: (a) differences
in transport properties between parasite and host
resulting in delivery of compound into the cell (depend-
ent on log P and Es in the former case (see text) and
possibly Es, in the case of KB cell (see Tables 2 and 3));
(b) differences in metabolism of the peptide into its
several active forms9 with or without the formation of
toxic byproducts (see Table 3); (c) differences in recogni-
tion by the target or targets responsible for cell death,
present in the parasitic cell with respect to the host.
The relative toxicity values given in Tables 2 and 3 are
a suitable indicator of specificity for compounds 13 <
29 < 20 < 25 < 24 < 14. The difference in relative
toxicity values (KB/T.b.) for the above compounds fall
in a range between 80 and 800, clearly indicating that
by optimization of structures to exploit subtle differ-
ences in characteristics between parasite and host it is
possible to develop therapeutically useful compounds
based on glutathione. While branched glutathione di-
esters have been proposed as therapeutic agents for use
in cancer chemotherapy,11 this is not the case here and
both linear diesters 13, 14, and 29 and branched diester
derivatives 20, 24, and 25 show a good therapeutic value
based on their specificity.
1
110 °C; H NMR (D6MSO) δ 9.1 (t, glyNH), 9.0 (d, ArH3), 8.7
(d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.3-7.4 (m, Ar-5H),
6.7 (d, GluNH), 5.1 (s, CH2Ar), 4.65 (td, GluCH),4.2 (q, CH2-
CH3) 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7 (dd, CH2-S),
2.25 (t, CH2CO), 2.0 (t, GluCH2CHCOO), 1.2 (t, CH3-CH2);
FABMS m/z 636 ((M + H)+, 18), 658 ((M + Na)+, 37), 680 ((M
+ 2Na)+, 7). HRFABMS Calcd for C26H30N5O12S 636.1611,
Found 636.1611. Anal. (C26H29N5O12SNa‚3H2O) C, H; N: calcd,
9.65. found, 8.64.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d ieth yl ester (11) was prepared using procedure A from
S-(2,4-dinitrophenyl)glutathione diethyl ester (767 mg; 61%
yield): mp 100-105 °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 (d, CH2Ar), 4.65 (td, GluCH),
4.2 (q, 2 × CH2-CH3), 4.1 (td, HN-CH-CH2), 3.95 (d, glyCH2),
3.7(dd, CH2-S), 2.25 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.2
(t, 2 × CH3-CH2); FABMS m/z 664 ((M + H)+, 100), 686 ((M +
Na)+, 80). HRFABMS Calcd for C28H34N5O12S 664.1924, Found
664.1931. Anal. (C28H33N5O12S‚3H2O) N; C: calcd, 46.86;
found, 46.33; H: calcd 5.48; found, 5.03.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e 1-p r op yl glycin a te (5) was prepared using procedure A
from S-(2,4-dinitrophenyl)glutathione n-propyl ester (i.e., a )
(1 g, 1.9 mmol), MeOH (30 mL), Et3N (0.40 mL, 2.82 mmol),
benzylchloroformate (0.40 mL, 2.82 mmol) and isolated by
PTLC (MeOH/CHCl3; 3:17) as pale yellow crystals (24 mg; 2%
1
yield): mp 130-132 °C; H NMR (D6MSO) δ 9.3 (t, glyNH),
9.0 (d, ArH3), 8.7 (d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.3-
7.4 (m, Ar-5H), 6.4 (d, GluNH), 5.1 (s, CH2Ar), 4.65 (td,

2980 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Daunes et al.
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), 2.0 (t, GluCH2-
CHCOO), 1.6 (q, CH3-CH2-CH2), 1.0 (t, 2 × CH3-CH2). Anal.
(C27H31N5O12S‚5H2O) C; H: calcd, 5.59; found, 5.16; N: calcd,
9.47; found, 8.92.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-bu tyl ester (18) was prepared using procedure A
from S-(2,4-dinitrophenyl)glutathione di-2-butyl ester (171.5
mg; 26.2% yield): mp 120-122 °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 (d, CH2Ar), 4.7 (m,
CH), 4.65 (td, GluCH), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2),
3.7 (dd, CH2-S), 2.25 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.5
(td, 2 × CH2-CH3), 1.2 (d, 2 × CH3-CH), 0.9 (t, 2 × CH3-CH2);
FABMS m/z 720 ((M + H)+, 50), 742 ((M + Na)+, 100). Anal.
(C32H41N5O12S) C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e 2-p r op yl glycin a te (6) was prepared using procedure A
from S-(2,4-dinitrophenyl)glutathione 2-propyl ester (1 g, 1.9
mmol), MeOH (30 mL), Et3N (0.40 mL, 2.82 mmol), and
benzylchloroformate (0.40 mL, 2.82 mmol) and isolated by
PTLC (MeOH/CHCl3; 3:17) as pale yellow crystals (229 mg;18%
yield): mp135-138 °C; 1H NMR (D6MSO) δ 9.1 (1H, t, glyNH),
9.0 (1H, d, ArH3), 8.7 (1H, d, cysNH), 8.4 (1H, dd, ArH5), 8.0
(1H, d, ArH6), 7.3-7.4 (5H, m, Ar), 6.7 (1H, d, GluNH), 5.1
(2H, s, CH2Ar), 5.0 (1H, m, CH(CH3)2), 4.65 (1H, td, GluCH),
4.0(1H, td, HN-CH-CH2), 3.95 (2H, d, glyCH2), 3.7(2H, dd, CH2-
S), 2.25 (2H, t, CH2CO), 2.0 (2H, t, GluCH2CHCOO), 1.2 (6H,
d, 2 × CH3); FABMS m/z 650 ((M + H)+, 18), 672 ((M + Na)+,
100), 694 ((M + 2Na)+, 42). Anal. (C27H31N5O12SNa‚H2O) C,
H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d ip en tyl ester (14) was prepared using procedure B from
N-benzyloxycarbonyl-S-(2,4-dinitrophenyl) glutathione (2) (0.3
g, 0.41 mmol), n-pentanol (25 mL), and thionyl chloride (0.085
mL, 0.97 mmol) and isolated by trituration with Et2O (2×) as
1
pale yellow crystals (0.24 g; 89% yield): mp 115-120 °C; H
NMR (D6MSO) 9.0 (1H, s, ArH3), 8.7 (1H, t, glyNH), 8.5 (1H,
d, cysNH), 8.4 (1H, m, ArH5), 8.0 (1H, d, ArH6), 7.7 (1H, d,
GluNH), 7.3-7.4 (5H, m, Ar), 5.1 (2H, d, CH2Ar), 4.65 (1H,
td, GluCH), 4.1(4H, t, 2 × CH2OCO), 4.0 (1H, td, HN-CH-CH2),
3.95 (2H, d, glyCH2), 3.7(2H, dd, CH2-S), 2.3 (2H, t, CH2CO),
2.0 (2H, td, GluCH2CHCOO), 1.6 (4H, t, 2 × CH2-CH2-OCO),
1.3 (8H, m, 2 × (CH2)2), 0.9 (6H, t, 2 × CH3-CH2); FABMS m/z
748.3 ((M +H)+, 35), 770.3 ((M +Na)+, 100). Anal. (C34H45N5O12S‚
H2O) C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-p r op yl ester (17) was prepared using procedure A
from S-(2,4-dinitrophenyl)glutathione di-2-propyl ester (175.3
1
mg; 14% yield): mp 130-135 °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), 5.1 (d, CH2Ar), 5.0(m,
2 × CH(CH3)2), 4.65 (td, GluCH), 4.0 (td, HN-CH-CH2), 3.95
(d, glyCH2), 3.7(dd, CH2-S), 2.25 (t, CH2CO), 2.0 (td, GluCH2-
CHCOO), 1.2 (d, 4 × CH3); CIMS m/z 709 ((MNH4)+, 20), 692
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-3-m eth yl-2-bu tyl ester (19) was prepared using
procedure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)
glutathione (2) (0.2 g, 0.33 mmol), 3-methyl-2-butanol (10 mL),
and thionyl chloride (0.06 mL, 0.724 mmol) and isolated by
trituration with Et2O (2×) (0.17 g; 70% yield): mp 112-115
((MH)+, 30), 493 ((MH)+ - C6H3N2O4S), 100), 558 ((MH)+
-
C8H7O2), 30). HRFABMS Calcd for C30H38N5O12S 692.2237,
Found 692.2226. Anal. (C30H37N5O12S‚H2O) C, H, N.
1
°C; H NMR (D6MSO) δ 9.0 (1H, s, ArH3), 8.8 (1H, t, glyNH),
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e 1-bu tyl glycin a te (7) was prepared using procedure A
from a mixture of S-(2,4-dinitrophenyl)glutathione butyl esters
(0.5 g, 0.91 mmol), MeOH (20 mL), Et3N (0.20 mL, 1.3 mmol),
and benzylchloroformate (0.20 mL, 1.3 mmol) and separated
from 9 and 13 by PTLC (MeOH/CHCl3: 3/7) as pale yellow
crystals (100.3 mg; 17% yield): mp 173-175 °C; 1H NMR (D6-
MSO) δ 9.1 (t, glyNH), 9.0 (d, ArH3), 8.7 (d, cysNH), 8.4 (dd,
ArH5), 8.0 (d, ArH6), 7.3-7.4 (m, Ar), 6.7 (d, GluNH), 5.1 (s,
CH2Ar), 4.65 (td, GluCH), 4.1 (t, CH2-OCO), 4.0 (td, HN-CH-
CH2), 3.95 (d, glyCH2), 3.7(dd, CH2-S), 2.25 (t, CH2CO), 2.0 (t,
GluCH2CHCOO), 1.6 (m, CH3-CH2-CH2), 1.15 (m, CH2-CH3),
0.95 (t, CH3-CH2). Anal. (C28H33N5O12S) C, H, N.
8.6 (1H, d, cysNH), 8.5 (1H, m, ArH5), 8.1 (d, ArH6), 7.8 (d,
GluNH), 7.3-7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.7 (m, GluCH
and 2 × CH3-CH-OCO), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2),
3.7 (dd, CH2-S), 2.3 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.8
(m, 2 × CH(CH3)2), 1.2 (d, 2 × (CH3-CH-OCO)), 0.9 (d, 2 ×
CH(CH3)2); FABMS m/z 748 ((M + H)+, 70), 770 ((M + Na)+,
85). HRFABMS Calcd for C34H46N5O12S 748.2863, Found
748.2883. Anal. (C34H45N5O12S‚H2O) C; H: calcd, 6.19; found,
5.64; N: calcd, 9.15; found, 9.67.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-4-m eth yl-2-p en tyl ester (20) was prepared using
procedure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)-
glutathione (2) (0.2 g, 0.33 mmol), 4-methyl-2-pentanol (10
mL), and thionyl chloride (0.06 mL, 0.72 mmol) and isolated
by trituration with Et2O (2×) as pale yellow crystals (0.16 g;
63% yield): mp 103-105 °C; 1H NMR (D6MSO) δ 9.0 (s, ArH3),
8.8 (t, glyNH), 8.6 (d, cysNH), 8.5 (dd, ArH5), 8.1 (d, ArH6),
7.8 (d, GluNH), 7.3-7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.8 (m,
GluCH and 2 × CH3-CH-OCO), 4.1 (m, HN-CH-CH2), 3.95 (d,
glyCH2), 3.6 (dd, CH2-S), 2.3 (2H, m, CH2CO), 2.0 (m, GluCH2-
CHCOO), 1.8 (m, 2 × CH(CH3)2), 1.2 (m, 2 × (CH3-CH-OCO)
and 2 × CH2-CH(CH3)2), 0.9 (d, 2 × CH(CH3)2); FABMS m/z
776 ((M + H)+, 45), 798 ((M + Na)+, 100). Anal. (C36H49N5O12S‚
H2O) C, H; N: calcd, 8.83; found, 9.34.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-p en tyl ester (21) was prepared using procedure B
from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione (2)
(0.3 g, 0.49 mmol), 2-pentanol (15 mL) and thionyl chloride
(0.09 mL, 1.08 mmol) and isolated by trituration with Et2O
(2×) as pale yellow crystals (0.25 g; 69% yield): mp 123-125
°C; 1H NMR (D6MSO) δ 9.0 (s, ArH3), 8.7 (m, glyNH), 8.5 (m,
cysNH and ArH5), 8.1 (d, ArH6), 7.8 (d, GluNH), 7.3-7.4 (m,
Ar-5H), 5.1 (s, CH2Ar), 4.9 (m, 2 × CH3-CH-OCO), 4.7 (m,
GluCH), 4.1 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.6 (dd, CH2-
S), 2.3 (t, CH2CO), 2.0 (m, GluCH2CHCOO), 1.8 (m, 2 × CH2-
CH), 1.4 (m, 2 × CH2-CH3), 1.2 (m, 2 × CH3-CH), 1.0 (m, 2 ×
CH3-CH2); ESIMS m/z 748 ((M + H)+, 75), 770 (M + Na)+,
746 (M - N)-, 782 (M - Cl)-. Anal. (C34H45N5O12S) C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e 1-bu tyl glu ta m a te (9) was prepared using procedure A
from a mixture of S-(2,4-dinitrophenyl) butyl esters (0.5 g, 0.91
mmol), MeOH (20 mL), Et3N (0.20 mL, 1.3 mmol), and
benzylchloroformate (0.20 mL, 1.3 mmol) and separated from
7 and 13 by PTLC (MeOH/CHCl3: 3/7) as pale yellow crystals
(20.0 mg; 4% yield): mp 160-165 °C; 1H NMR (D6MSO) δ 9.1
(t, glyNH), 9.0 (d, ArH3), 8.6 (m, cysNH, ArH5), 8.0 (d, ArH6),
7.6 (d, GluNH), 5.1 (s, CH2Ar), 4.65 (m, GluCH), 4.1 (t, CH2-
OCO), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7(m, CH2-S),
2.25 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.6 (m, CH3-CH2-
CH2), 1.15 (m, CH2-CH3), 0.95 (t, CH3-CH2); FABMS m/z 664
((M + H)+, 65), 686 ((M + Na)+, 70). Anal. (C28H33N5O12S‚
H2O): C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e 2-bu tyl glycin a te (8) was prepared using procedure A
from S-(2,4-dinitrophenyl)glutathione 2-butyl ester (0.5 g, 0.91
mmol), MeOH (20 mL), Et3N (0.20 mL, 1.3 mmol), and
benzylchloroformate (0.20 mL, 1.3 mmol) and isolated by PTLC
(MeOH/CHCl3; 1:9) as pale yellow crystals. (42 mg; 7.0%
1
yield): mp 130-135 °C; H NMR (D6MSO) δ 9.1 (t, glyNH),
9.0 (d, ArH3), 8.7 (d, cysNH), 8.4 (dd, ArH5), 8.0 (d, ArH6), 7.3-
7.4 (m, Ar), 6.6 (d, GluNH), 5.1 (s, CH2Ar), 4.7 (m, CH), 4.65
(td, GluCH), 4.0 (td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7 (dd,
CH2-S), 2.25 (t, CH2CO), 2.0 (td, GluCH2CHCOO), 1.5 (td, CH2-
CH3), 1.2 (d, CH3-CH), 0.9 (t, CH3-CH2); FABMS m/z 686 ((M
+ Na)+, 10), 708 ((M + 2Na)+, 5). Anal. (C28H33N5O12SNa) C,
N; H: calcd, 4.85; found, 5.67.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-h exyl ester (22) was prepared using procedure B
from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl glutathione (2)

Antiprotozoal Activity of Glutathione Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2981
(0.2 g, 0.33 mmol), 2-hexanol (20 mL), and thionyl chloride
(0.15 mL, 1.65 mmol). Removal of the solvent gave an oily
residue which on trituration with petroleum ether (40:60) gave
a yellow solid which was purified by PTLC (CHCl3/AcOEt; 5/5)
(0.157 g, 62% yield): mp 125-128 °C; 1H NMR (D6MSO) δ 9.0
(s, ArH3), 8.75 (m, glyNH), 8.5 (m, cysNH and ArH5), 8.1 (d,
ArH6), 7.8 (d, gluNH), 7.5 (m, Ar-5H), 5.1 (s, CH2Ar), 4.9 (m,
2 × CH3-CHOCO), 4.8 (m, gluCH), 4.2 (m, HN-CH-CH2), 4.0
(d, glyCH2), 3.8-3.7 (dd, CH2-S), 2.3 (m, CH2CO), 2.0 (m,
gluCH2CHCOO), 1.6 (m, 2 × CH2-CH), 1.4 (m, 4 × CH2), 1.2
(d, 2 × CH3-CH), 1.0 (m, 2 × CH3-CH2); FABMS m/z 776.4((M
+ H)+, 85), 798.4 ((M + Na)+, 100); Anal. (C36H49N5O14S) C,
N; H: calcd, 6.37; found, 5.91.
thionyl chloride (0.15 mL, 1.65 mmol). Removal of the solvent
gave an oily residue which on trituration with Et2O gave a
yellow solid which was purified by PTLC (CHCl3/MeOH; 9/1)
(0.14 g, 61% yield): mp 125-128 °C; 1H NMR (D6MSO) δ 8.95
(s, ArH3), 8.8 (t, glyNH), 8.6 (m, cysNH and ArH5), 8.1 (d,
ArH6), 7.9 (d, gluNH), 7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.6-4.8
(m, 2 × CH2OCO and gluCH), 4.3-4.5 (m, 2 × CH2F), 4.25
(m, gluCH2OCO), 4.2 (m, HN-CH-CH2), 4.0 (d, glyCH2), 3.6
(dd, CH2-S), 2.3 (m, CH2CO), 2.0 (m, gluCH2CHCOO); FABMS
m/z 700.24 ((M + H)+,100), 722.23 ((M + Na)+, 45); Anal.
(C28H31N5O12SF2) N; C: calcd, 47.39; found, 46.94; H: calcd,
4.45; found, 3.97.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-ch lor oeth yl ester (29) was prepared using proce-
dure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glu-
tathione (2) (0.4 g, 0.66 mmol), chloroethanol (20 mL), and
thionyl chloride (0.15 mL, 1.65 mmol) and isolated by tritu-
ration with Et2O (2×) as pale yellow crystals (0.38 g; 75%
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-3-p en tyl ester (23) was prepared using procedure B
from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione (2)
(0.3 g, 0.49 mmol), 3-pentanol (15 mL), and thionyl chloride
(0.09 mL, 1.08 mmol) and isolated by trituration with Et2O
(2×) as pale yellow crystals (0.15 g; 43% yield): mp 153-155
°C; 1H NMR (D6MSO) δ 9.0 (s, ArH3), 8.7 (m, glyNH), 8.5 (m,
cysNH and ArH5), 8.1 (d, ArH6), 7.9 (d, GluNH), 7.4 (m, Ar-
5H), 5.1 (s, CH2Ar), 4.8 (m, 2 × CH3-CH2-CH-OCO and
GluCH), 4.1 (m, HN-CH-CH2), 4.0 (d, glyCH2), 3.6 (dd, CH2-
S), 2.4 (m, CH2CO), 2.0 (m, GluCH2CHCOO), 1.6 (m, 4 × CH2-
CH), 0.9 (t, 4 × CH3-CH2); FABMS m/z 748 ((M + H)+, 100),
1
yield): mp 123-125 °C; H NMR (D6MSO) δ 8.95 (s, ArH3),
8.7 (m, glyNH), 8.5 (m, cysNH and ArH5), 8.0 (d, ArH6), 7.8
(d, GluNH), 7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.7 (m, GluCH),
4.3 (t, 2 × CH2OCO), 4.1 (m, HN-CH-CH2), 3.9 (d, glyCH2),
3.8 (t, 2 × CH2Cl), 3.6 (dd, CH2-S), 2.3 (m, CH2CO), 2.0 (m,
GluCH2CHCOO); FABMS m/z 732 ((M + H)+, 60), 754 ((M +
Na)+, 100). HRFABMS Calcd for C28H32N5O12SCl2 732.1145,
Found 732.1144. Anal. (C28H31N5O12SCl2) C, H, N.
770 ((M + Na)+, 55). HRFABMS Calcd for C34H46N5O12
748.2863, Found 748.2840. Anal. (C34H45N5O12S) C, H, N.
S
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-3-ch lor op r op yl ester (30) was prepared using pro-
cedure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)-
glutathione (2) (0.15 g, 0.25 mmol), 3-chloro-1-propanol (15
mL), and thionyl chloride (0.11 mL, 1.23 mmol) and isolated
by trituration with Et2O (2×) as pale yellow crystals (0.14 g;
72% yield): mp 110-115 °C; 1H NMR (D6MSO) δ 8.95 (s,
ArH3), 8.7 (m, glyNH), 8.5 (m, cysNH and ArH5), 8.1 (d, ArH6),
7.8 (d, GluNH), 7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.7 (m, GluCH),
4.3 (t, 2 × CH2OCO), 4.1 (m, HN-CH-CH2), 3.9 (d, glyCH2),
3.8 (t, 2 × CH2Cl), 3.6 (dd, CH2-S), 2.3 (m, CH2CO), 2.1 (t, 2 ×
CH2-CH2Cl), 2.0 (m, GluCH2CHCOO); FABMS m/z 760 ((M +
H)+, 100), 782 ((M + Na)+, 60). Anal. (C30H35N5O12SCl2‚H2O)
C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-4-ch lor obu tyl ester (31) was prepared using proce-
dure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glu-
tathione (2) (0.3 g, 0.49 mmol), 4-chloro-1-butanol (20 mL), and
thionyl chloride (0.22 mL, 2.4 mmol) and isolated by trituration
with Et2O (2×) as pale yellow crystals (0.19 g; 50% yield): mp
215-220 °C; 1H NMR (D6MSO) δ 9.0 (s, ArH3), 8.8 (m, glyNH),
8.5 (m, cysNH and ArH5), 8.1 (d, ArH6), 7.8 (d, GluNH), 7.4
(m, Ar-5H), 5.1 (s, CH2Ar), 4.7 (m, GluCH), 4.1 (t, 2 × CH2-
OCO), 4.0 (m, HN-CH-CH2 and glyCH2), 3.8 (t, 2 × CH2Cl),
3.6 (dd, CH2-S), 2.3 (m, CH2CO), 2.0 (m, GluCH2CHCOO), 1.8
(m, 2 × CH2-CH2-CH2Cl); FABMS m/z 788 ((M + H)+, 100),
810 ((M + Na)+, 92). HRFABMS Calcd for C32H39N5O12SCl2
787.1692, Found 787.1673. Anal. (C32H39N5O12SCl2) C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d icyclop en tyl ester (24) was prepared using procedure
B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione
(2) (0.5 g, 0.82 mmol), cyclopentanol (25 mL), and thionyl
chloride (0.18 mL, 2.05 mmol) and isolated by trituration with
Et2O (2×) as pale yellow crystals (0.18 g; 67% yield): mp 110-
1
112 °C; H NMR (D6MSO) δ 8.9 (s, ArH3), 8.6 (t, glyNH), 8.4
(m, cysNH and ArH5), 8.0 (d, ArH6), 7.7 (d, GluNH), 7.3-7.4
(m, Ar-5H), 5.1 (s, CH2Ar), 4.6 (m, GluCH and 2 × CHcPt), 4.0
(td, HN-CH-CH2), 3.95 (d, glyCH2), 3.7(dd, CH2-S), 2.3 (t, CH2-
CO), 2.0 (m, GluCH2CHCOO), 1.4 (m, 2 × (CH2-CH-CH2)), 1.2
(m, 2 × (CH2)3 cPt); ESIMS m/z 744 (M + H)+, 761 (M + NH4)+,
766 (M + Na)+, 742 (M-H)-, 778 (M-Cl)-. Anal. (C34H41N5O12S‚
H2O) H, N; C: calcd, 53.60; found, 54.13.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-3-h exyl ester (26) was prepared using procedure B
from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl) glutathione (2)
(0.3 g, 0.49 mmol), 3-hexanol (15 mL), and thionyl chloride
(0.09 mL, 1.08 mmol) and isolated by trituration with Et2O
(2×) as pale yellow crystals (0.21 g; 57% yield): mp 135-138
°C; 1H NMR (D6MSO) δ 9.0 (s, ArH3), 8.7 (m, glyNH), 8.5 (m,
cysNH and ArH5), 8.1 (d, ArH6), 7.8 (d, GluNH), 7.4 (m, Ar-
5H), 5.1 (s, CH2Ar), 4.9 (m, 2 × CH3-CH2-CH-OCO and
GluCH), 4.1 (m, HN-CH-CH2), 3.95 (d, glyCH2), 3.6 (dd, CH2-
S), 2.3 (m, CH2CO), 2.0 (m, GluCH2CHCOO), 1.7 (m, 4 × CH2-
CH), 1.3 (m, 2 × CH2-CH3), 0.9 (q, 4 × CH3-CH2). FABMS m/z
776 ((M + H)+, 75), 798 ((M + Na)+, 70). Anal. (C36H49N5O12S)
C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-m eth oxyeth yl ester (32) was prepared using
procedure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)-
glutathione (2) (0.3 g, 0.49 mmol), 2-methoxyethanol (20 mL),
and thionyl chloride (0.22 mL, 2.4 mmol) and isolated by
trituration with Et2O/petroleum ether/CHCl3 as a yellow solid
which was purified by PTLC (CHCl3/MeOH; 9/1) (0.21 g, 58%
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-5-m eth yl-2-h exyl ester (27) was prepared using
procedure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)-
glutathione (2) (0.4 g, 0.65 mmol), 5-methyl-2-hexanol (20 mL),
and thionyl chloride (0.12 mL, 1.44 mmol) and isolated by
trituration with Et2O (2×) as pale yellow crystals (0.35 g; 68%
1
1
yield): mp 145-148 °C; H NMR (D6MSO) δ 8.95 (s, ArH3),
yield): mp 112-115 °C; H NMR (D6MSO) δ 8.95 (s, ArH3),
8.7 (m, glyNH), 8.5 (m, cysNH and ArH5), 8.1 (d, ArH6), 7.7
(d, GluNH), 7.3-7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.8 (m, 2 ×
CH3-CH-OCO), 4.7 (m, GluCH), 4.1 (m, HN-CH-CH2), 3.95 (m,
glyCH2), 3.5 (dd, CH2-S), 2.3 (m, CH2CO), 2.0 (m, GluCH2-
CHCOO), 1.6 (m, 2 × CH-CH2-CH2 and 2 × CH(CH3)2), 1.2
(m, 2 × CH2-CH-CH3 and 2 × (CH3-CH)), 0.9 (m, 4 × CH3-
CH); FABMS m/z 809 ((M + H)+, 40), 826 ((M + Na)+, 10).
Anal. (C38H53N5O12S) C, H, N.
N-Ben zyloxyca r b on yl-S-(2,4-d in it r op h en yl)glu t a t h i-
on e d i-2-flu or oeth yl ester (28) was prepared using proce-
dure B from N-benzyloxycarbonyl-S-(2,4-dinitrophenyl glu-
tathione (2) (0.2 g, 0.33 mmol), 2-fluoroethanol (20 mL), and
8.8 (m, glyNH), 8.5 (m, cysNH and ArH5), 8.1 (d, ArH6), 7.8
(d, GluNH), 7.4 (m, Ar-5H), 5.1 (s, CH2Ar), 4.7 (m, GluCH),
4.3 (t, 2 × CH2OCO), 4.2 (m, HN-CH-CH2), 4.0 (m, glyCH2),
3.7 (m, CH2-S), 3.6 (m, 2 × CH2O-), 3.3 (s, 2 × OCH3) 2.3 (m,
CH2CO), 2.0 (m, GluCH2CHCOO); ESIMS m/z 746 (M + Na)+,
758 (M + Cl)-. Anal. (C36H49N5O12S) C, N; H: calcd, 5.16;
found, 5.63.
Log P , MR. Theoretical values of log Kow (log P) were
calculated using an interactive demo program established at
the Environmental Service Center, Syracuse Research Cor-
poration (SRC), in which structures were entered in SMILES
notation (limited to a 100 characters). The program estimated

2982 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Daunes et al.
the octanol/water partition coefficient (log P) using an atom/
fragment contribution method developed at SRC.22 Calculated
values of MR were undertaken using an online demo program
(CMR) established at Daylight Chemical Information Systems,
Inc.,23 in which structures were entered in SMILES notation.
Qu a n tita tive Str u ctu r e-Activity Regr ession (QSAR).
Data analysis was undertaken using SciQSAR a module of
Alchemy 2000 (Tripos). This program searched Alchemy 2000
for low energy molecular conformations and used the informa-
tion to calculate several molecular descriptors. These descrip-
tors were used in the programs regression approach for
predicting QSAR parameters. The program allowed the cre-
ation of regression equations derived from a specific set of
molecules or to alter existing equations by the addition of new
agents (e.g., log P) to a group of molecules. Statistical regres-
sion analysis of data was undertaken using the LINEST curve
fitting routine provided with Excel (Microsoft Corporation).
F igu r e 1.
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. Trypanosoma brucei brucei
(strain S427) bloodstream form trypomastigotes were main-
tained in HMI-18 medium24,25 with 20% heat inactivated foetal
calf serum (HIFCS) (Harlan Sera-Lab., Crawley, U.K.) at 37
°C in 5% CO2-air mixture. Leishmania donovani (strain
MHOM/ET/67/L82) was maintained routinely in special patho-
gen free (SPF) female Golden hamsters by serial passage every
6 to 8 weeks. Trypanosoma 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 Assa ys. T.b. br u cei. 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
Company, NJ). Appropriate controls with pentamidine isothion-
ate (Aventis, U.K.) 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 the
Alamar Blue assay26 on day three.
F igu r e 2. Experimental and calculated Es values.
B. Stein of the EPSRC Mass Spectrometry service
center, Swansea, for FABMS, HRFABMS, and ESIMS
measurements. The authors thank the Biological Sci-
ences Department (MMU) and the Microbiology Section
for access to their facilities.
L. d on ova n i a n d T. cr u zi. 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 being washed, cells
were dispensed into 16-well Lab-tek tissue culture slides (Nunc
Inc., IL) at 4 × 104/well in a volume of 100 mL of RPMI-1640
medium (Sigma-Aldrich Company 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.27
Eva lu a tion of th e Cytotoxicity of Glu ta th ion e Der iva -
tives in Vitr o. Cytotoxicity testing on KB cells (human oral
pharyngeal carcinoma) was evaluated using the Almar Blue
assay.28 Cell cultures were grown in RPMI medium with 10%
calf foetal serum (CFS) at 37 °C in a 5% CO2-air mixture in
a humidified incubator. Plates were incubated with compound
for 3 days at 37 °C in a 5% CO2-humidified air mixture prior
to determination of activity.
Ap p en d ix
E xt r a p ola t ion of Ta ft ’s St er ic F a ct or . QSAR
analysis was made on the 25 methyl esters listed in
Figure 1 with known experimental values,18 and the
regression equation was used to calculate values for the
unknown branched esters. The Es values calculated for
the unknown linear structures using this equation were
much larger than expected, and their values were
obtained by graphical extrapolation of the experimental
observed trend between Es vs number of carbon atoms
for the series of linear esters (Me, Et, Pr, and Bu); see
Figure 2.
Es ) 120 + 1.59(X1) - 0.72(Polar) + 0.0218(MW) +
29.31(ABSQon) - 219(MaxQp) + 192(MaxNeg)
RMSD ) 0.19;
multiple correlation coefficient R2 ) 0.95
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