Synthesis of new N-isobutyryl-l-cysteine/MEA conjugates: Evaluation of their free radical-scavenging activities and anti-HIV properties in human macrophages

Article abstract of DOI:10.1016/j.bioorg.2008.02.001

Four novel N-isobutyryl-l-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test

Full text of DOI:10.1016/j.bioorg.2008.02.001

Available online at www.sciencedirect.com
Bioorganic Chemistry 36 (2008) 133–140
www.elsevier.com/locate/bioorg
Synthesis of new N-isobutyryl-L-cysteine/MEA conjugates:
Evaluation of their free radical-scavenging activities and
anti-HIV properties in human macrophages
a,
b
c
a
a
*
Michael Smietana , Pascal Clayette , Patricia Mialocq , Jean-Jacques Vasseur , Joel Oiry
¨
a Institut des Biomole´cules Max Mousseron, IBMM UMR 5247 CNRS, Universite´ Montpellier 1, Universite´ Montpellier 2,
`
place Eugene Bataillon, 34095 Montpellier, France
b Laboratoire de Neurovirologie, SPI-BIO, CEA, 18 route du Panorama, B.P. 6, 92265 Fontenay aux Roses Cedex, France
c Service de Neurovirologie, CEA, DSV, 18 route du Panorama, B.P. 6, 92265 Fontenay aux Roses Cedex, France
Received 7 January 2008
Available online 25 March 2008
Abstract
Four novel N-isobutyryl-L-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The
antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test,
and deoxyribose assay) and their metal binding capacity was evaluated by the ethidium bromide fluorescence binding assay. These results
were compared with those obtained with their pro-GSH acetyl analogues recently developed in our laboratory. We observed that most of
these compounds exhibit free radical-scavenging activities similar to those of Trolox, but always superior than NAC. While none of these
new derivatives had pro-GSH activities, they displayed anti-HIV properties in human monocyte-derived macrophages infected in vitro.
The present study demonstrates that these new N-isobutyryl derivatives, which are expected to have a greater bioavailability than their
acetyl analogues, may have useful applications in HIV infection in respect to their antioxidant and anti-HIV activities.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Cysteine derivatives; Lipophilic compounds; Radical scavenging; Antioxidant activity; DNA damage; Anti-HIV activity
1. Introduction
smaller molecules such as ascorbate, glutathione, tocoph-
erol, carotenoids, polyphenols, alkaloids, and other com-
pounds. Thiol-containing compounds have an essential
role in many biochemical reactions due to their ability to
be easily oxidized and then quickly regenerated. Main rep-
resentatives are glutathione, lipoic acid, and thioredoxin,
which are synthesized de novo in mammalian cells. On
the other hand, cysteine-derived compounds such as
N-acetyl-L-cysteine (NAC) and bucillamine are synthetic
thiols which have been administered in experimental and
clinical studies for treatment of conditions associated with
oxidative stress [3,4]. N-Acetyl-L-cysteine acts as an indirect
precursor of glutathione (GSH) by raising intracellular
level of cysteine, a precursor of glutathione. NAC is being
studied and utilized in conditions characterized by
decreased GSH or oxidative stress such as in HIV infec-
tions, cancer, and heart disease [5]. These properties
Reactive oxygen species (ROS), resulting from excita-
tion or incomplete reduction of molecular oxygen, are
highly reactive, toxic oxygen moieties that affects numerous
critical cellular functions. The absence of efficient cellular
detoxification mechanisms which remove these radicals
can lead to proteins and DNA oxidation, lipid peroxida-
tion, and ultimately cell death [1,2].
To counteract these damaging radicals, antioxidant sys-
tems have evolved, including enzymes such as glutathione
peroxidase, glutathione reductase, superoxide dismutase,
S-methyl transferase, and catalase. The cellular arsenal
for scavenging ROS and toxic organic radicals also include
*
Corresponding author. Fax: +33 467042029.
E-mail address: msmietana@univ-montp2.fr (M. Smietana).
0045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2008.02.001

134
M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
prompted several research groups, including ours to inves-
tigate the antioxidant potential of different conjugated cys-
teine derivatives [6–11]. Recently, we reported that various
NAC analogues and notably N-(N-acetyl-L-cysteinyl)-S-
acetylcysteamine) 4a, increased intracellular GSH levels
in various cell lines and displayed interesting antiviral
activities [6–8]. We postulated that these compounds are
S-deacetylated upon esterase activation to their corre-
sponding dithiol derivatives which may be responsible for
in situ release of NAC and cysteamine. GSH-deficiency is
associated with impaired survival rates in HIV-infected
patients and the administration of pro-GSH drugs such
as NAC, has been shown to decrease mortality [12]. How-
ever, in the case of compound 4a and its analogues 4b–d
(Scheme 1), we were unable to correlate their pro-GSH
activities with their good anti-HIV effects. We thus sus-
pected, that, due to its sulfhydryl group, 4a may have anti-
oxidant properties of its own and therefore may function
by either directly scavenging free radicals, by inhibiting
their generation, or by chelating metals which are known
to induce free radicals formation by a variety of processes
including the Fenton reaction. In view of the importance of
antioxidants in chemoprevention of a number of degenera-
tive diseases, we decided to evaluate the antioxidant activ-
ities of N-acetyl thiols 4a–d using various antioxidant
assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPHÅ),
2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTSÅ+), deoxyribose degradation, and the ethidium bro-
mide fluorescence binding assay. Following this work,
compounds 4e–h (Scheme 1) were designed and synthesized
with the aim of producing efficient antioxidant compounds
which were expected to be much more biologically stable
than their acetyl counterparts, while keeping their antioxi-
dant potency [13,14]. The results obtained are compared
with well-known antioxidants such as cysteine, NAC,
GSH, and Trolox (a polar analogue of vitamin E). The
anti-HIV properties in human monocyte-derived macro-
phages infected in vitro of this new N-isobutyryl series were
evaluated in parallel.
2. Materials and methods
2.1. General
Unless otherwise stated, materials were purchased from
commercial suppliers and used without further purifica-
tion. Melting points were determined on a Buchi capillary
¨
melting point apparatus and are uncorrected. Nuclear
magnetic resonance spectra (1H NMR) were recorded on
a Bruker AC 250 in CDCl . Chemical shifts are reported
¨
3
in ppm and given in d units with respect to TMS, used as
an internal standard. FAB mass spectra were recorded on
a JEOL DX 300 mass spectrophotometer in the positive
ion mode, using 1:1 glycerol/thioglycerol. Specific optical
rotations were recorded on a Perkin-Elmer 241 polarime-
ter. Elemental analysis was carried out by the Service Cen-
tral de Microanalyses du CNRS de Vernaison (France).
Analytical thin-layer chromatography (TLC) was carried
out on Merck silica gel 60 F254 plates. Spots were visualized
by exposure to ultraviolet light (254 nm), iodine vapor, or
O
H
H
a
R
N
H
CO2H
Tr
R
N
H
S
R'
N
H
HCl.H2N(CH2)2S-CO-R'
O
O
O
S
S
Tr
3a : R = CH3
3b : R = CH3
3c : R = CH3
3d : R = CH3
R' = CH3
1 : R = CH3
R' = CH(CH3)2
R' = C(CH3)3
R' = C6H5
2 : R = CH(CH3)2
3e : R = CH(CH3)2 R' = CH3
3f : R = CH(CH3)2 R' = CH(CH3)2
3g : R = CH(CH3)2 R' = C(CH3)3
3h : R = CH(CH3)2 R' = C6H5
b, c
4a : R = CH3
R' = CH3
4b : R = CH3
R' = CH(CH3)2
R' = C(CH3)3
R' = C6H5
O
H
4c : R = CH3
4d : R = CH3
R
N
H
S
R'
N
H
4e : R = CH(CH3)2 R' = CH3
O
O
4f : R = CH(CH3)2 R' = CH(CH3)2
4g : R = CH(CH3)2 R' = C(CH3)3
4h : R = CH(CH3)2 R' = C6H5
HS
(a) (i) AcOEt, IBC, NMM, -15 °C, (ii) HOSu, -15 °C, (iii) NMM, -15 °C-rt, 12 h; (b) MeOH, CHCl3, mixture
of MeOH/AgNO3/pyridine, rt; (c) CHCl3, HCl 37%, rt..
Scheme 1. Synthesis of compounds 4a–h.

M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
135
by spraying with ninhydrin solution. Flash column chro-
matography was conducted with Merck silica gel 60
(230–400 mesh ASTM). All the commercial reagents and
solvents were of analytical grade and were purchased from
Aldrich or Fluka. N-Isobutyryl-L-cysteine was obtained
from Fluka. S-Acetylcysteamine hydrochloride and S-ben-
zoylcysteamine hydrochloride were prepared as previously
described [15]. UV spectra were recorded on a Varian Cary
300 Bio UV/Vis spectrometer. Fluorescence spectra were
acquired on a SML AMINCO 8100 spectrofluorometer
(slit width 4 nm for excitation and detection). Studies were
carried out at 20 °C.
added one drop at a time. The reaction mixture was then
stirred for 1 h at 0 °C and at room temperature for 12 h.
The insoluble salt was collected by filtration and washed
with EtOAc (3ꢀ 7 mL). The organic phases were pooled,
washed [water (2ꢀ 25 mL), ice-cold saturated aqueous
NaHCO3 (18 mL), water (2ꢀ 25 mL), ice-cold 1 N citric
acid (20 mL), and water (neutral pH)], dried (Na2SO4), fil-
tered, and evaporated to dryness under vacuum. The resi-
due was purified by flash column chromatography
(EtOAc/petroleum ether, 6:4) to give 3e as a white foam
(1.41 g, 67%). TLC (EtOAc/petroleum ether, 6:4)
Rf = 0.5. a20 +9.3° (c 0.97, CHCl3). 1H NMR: 1.10 (d,
D
J = 6.9 Hz, 6H, C(CH3)2), 2.18–2.36 (m, 1H, CH(CH3)2),
2.29 (overlapping s, 3H, SCOCH3); 2.50 (dd, J = 5.6,
12.8 Hz, 1H, b Ha cys), 2.72 (dd, J = 6.7, 12.8 Hz, 1H, b
Hb cys), 2.88–3.01 (m, 2H, NCH2CH2S), 3.29–3.41 (m,
2H, NCH2CH2S), 4.06–4.19 (m, 1H, a H cys), 5.82 (d,
J = 7.4 Hz, 1H, NH cys), 6.42 (t, J = 5.5 Hz, 1H, NHCH2),
7.17–7.35, 7.39–7.48 (2m, 15H, ArH). MS m/z 535
(M+H)+. Anal. Calcd for C30H34N2O3S: C, 67.42; H,
6.37; N, 5.24. Found: C, 67.05; H, 6.72; N, 5.30.
2.2. Synthesis
2.2.1. N-Isobutyryl-S-trityl-L-cysteine (2)
suspension of N-isobutyryl-L-cysteine (4.1 g,
A
21.5 mmol) and triphenylmethanol (5.6 g, 21.5 mmol) in
acetic acid (16 mL) was stirred at room temperature and
boron trifluoride diethyl etherate (4.1 mL, 32.2 mmol)
was added dropwise while maintaining the temperature at
about 20–25 °C. After the addition was complete, stirring
was continued at room temperature for 3 h and the solu-
tion was cooled to 0 °C and then added of saturated aque-
ous sodium acetate (70 mL) and water (140 mL). An
immediate colourless paste formed. After 12 h at 0 °C,
the reaction mixture was vigorously stirred and partitioned
between diethyl ether (120 mL) and ice-water (150 mL).
The organic layer was separated and the aqueous layer
was further extracted with Et2O (4ꢀ 80 mL). The com-
bined organic fractions were washed with ice-water (4ꢀ
60 mL), dried (Na2SO4), filtered, and evaporated under
to dryness under vacuum. The residue was then purified
by trituration in hexane to yield 2 (7.54 g, 81%) as gum.
TLC and NMR analysis indicated that the product was
sufficiently pure and could be used without further purifica-
tion. TLC (CH2Cl2/MeOH/AcOH, 9.4:0.6:0.07) Rf = 0.53.
a2D0 +21.4° (c 1.12, CHCl3). 1H NMR: 1.12, 1.14 (2d,
J = 2ꢀ 6.9 Hz, 2ꢀ 3H, C(CH3)2), 2.24–2.42 (m, 1H,
CH(CH3)2), 2.64–2.28 (m, 2H, CH2), 4.31–4.43 (m, 1H, a
H), 5.67–6.15 (m, 1H, CO2H), 5.90 (overlapping d, J =
7.1 Hz, 1H, NH); 7.18–7.33, 7.37–7.46 (2m, 15H, ArH).
MS m/z 867 (2M+H)+, 434 (M+H)+, 865 (2MꢁH)+, 432
(MꢁH)+. Anal. Calcd for C26H27NO3S: C, 72.06; H,
6.24; N, 3.23. Found: C, 72.24; H, 6.22; N, 3.28.
2.2.3. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-isobutyrylcys-
teamine (3f)
This compound was prepared according to the general
procedure described for 3e, using 2 (1.7 g, 3.93 mmol)
and S-isobutyrylcysteamine hydrochloride (obtained by
the procedure described for S-acetylcysteamine hydrochlo-
ride [15], mp 147–148 °C). The crude product was purified
by flash column chromatography (EtOAc/petroleum ether,
6.5:2.5) to give 3f as a colourless foam (1.66 g, 75%). TLC
(EtOAc/petroleum ether, 5:5) Rf = 0.6. a2D0 +7.9° (c 1.27,
1
CHCl3). H NMR: 1.107, 1.110, 1.159, 1.162 (4d, J = 4ꢀ
6.9 Hz, 4ꢀ 3H, 2ꢀ C(CH3)2), 2.17–2.32 (m, 1H,
CH(CH3)2: N-i-but), 2.51 (dd, J = 5.7, 12.8 Hz, 1H, b Ha
cys), 2.63–2.79 (m, 1H CH(CH3)2: S-i-but.), 2.72 (overlap-
ping dd, J = 6.8, 12.8 Hz, 1H, b Hb cys), 2.88–2.98 (m, 2H,
NCH2CH2S), 3.29–3.40 (m, 2H, NCH2CH2S), 4.07–4.19
(m, 1H, a H cys), 5.81 (d, J = 7.3 Hz, 1H, NH cys),
6.32–6.43 (m, 1H, NHCH2), 7.18–7.35, 7.39–7.47 (2m,
15H, ArH). MS m/z 563 (M+H)+. Anal. Calcd for
C32H38N2O3S2: C, 68.33; H, 6.76; N, 4.98. Found: C,
68.27; H, 6.71; N, 4.88.
2.2.4. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-pivaloylcys-
teamine (3g)
2.2.2. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-acetylcysteamine
(3e)
This compound was prepared according to the general
procedure described for 3e, using 2 (1.7 g, 3.93 mmol)
and S-pivaloylcysteamine hydrochloride (obtained by the
procedure described for S-acetylcysteamine hydrochloride
[15], mp 212–213 °C). The crude product was purified by
flash column chromatography (EtOAc/petroleum ether,
7:3) to give a colourless foam. Trituration with hexane gave
3g as a white powder (1.99 g, 88%): mp 85–88 °C. TLC
(CH2Cl2/Et2O, 6:4) Rf = 0.82. a2D0 +5.9° (c 1.02, CHCl3).
1H NMR: 1.11 (d, J = 6.9 Hz, 6H, C(CH3)2), 1.21 (s, 9H,
C(CH3)3), 2.20–2.38 (m, 1H, CH(CH3)2), 2.51 (dd,
A stirred solution of N-isobutyryl-S-trityl-L-cysteine (2,
1.7 g, 3.93 mmol) in EtOAc (20 mL) was cooled to
ꢁ15 °C and successively treated with isobutyl chlorofor-
mate (IBC, 509 lL, 3.93 mmol), 4-methylmorpholine
(NMM, 435 lL, 3.96 mmol), and then (15 min) N-
hydroxysuccinimide (HOSu, 452 mg, 3.93 mmol). After
stirring for 30 min at ꢁ15 °C, S-acetylcysteamine hydro-
chloride [15] (672 mg, 4.32 mmol) was added to the mix-
ture, followed by NMM (474 lL, 4.32 mmol), which was

136
M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
J = 5.6, 12.9 Hz, 1H, b Ha cys), 2.72(dd, J = 6.7, 12.9 Hz,
1H, b Hb cys), 2.84–2.96 (m, 2H, NCH2CH2S), 3.27–3.39
(m, 2H, NCH2CH2S), 4.03–4.17 (m, 1H, a H cys), 5.78
(d, J = 7.4 Hz, 1H, NH cys), 6.22–6.34 (m, 1H, NHCH2),
7.18–7.35, 7.38–7.48 (2m, 15H, ArH). MS m/z 577
(M+H)+. Anal. Calcd for C33H40N2O3S2: C, 68.75; H,
6.94; N, 4.86. Found: C, 68.49; H, 6.98; N, 4.93.
NHCH2). MS m/z 293 (M+H). Anal. Calcd for
C11H20N2O3S2: C, 45.20; H, 6.84; N, 9.59. Found: C,
45.22; H, 7.11; N, 9.69.
2.2.7. N-(N-Isobutyryl-L-cysteinyl)-S-isobutyrylcysteamine
(4f)
This compound was prepared from 3f (1.55 g,
2.75 mmol) according to the general procedure described
for 4e. The crude product was purified by flash column
chromatography (CH2Cl2/Et2O, 7.5:2.5) to give a colour-
less gum. Trituration with hexane gave 11 as a white pow-
der (607 mg, 69%): mp 125–127 °C. TLC (CH2Cl2/Et2O,
2.2.5. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-benzoylcys-
teamine (3h)
This compound was prepared according to the general
procedure described for 3e, using 2 (1.7 g, 3.93 mmol)
and S-benzoylcysteamine hydrochloride [15]. The crude
product was purified by flash column chromatography
(EtOAc/petroleum ether, 7:3) to give 3h as a colourless
foam (1.8 g, 77%). TLC (CH2Cl2/ Et2O, 6:4) Rf = 0.76.
5:5) Rf = 0.39. a20 ꢁ25.7° (c 1.05, CHCl3). 1H NMR:
D
1.19, 1.20 (2d, J = 2ꢀ 6.9 Hz, 2ꢀ 6H, 2ꢀ C(CH3)2), 1.62
(dd, J = 7.5, 10.3 Hz, 1H, SH), 2.41–2.54 (m, 1H,
CH(CH3)2: N-i-but.), 2.70–2.84 (m, 1H, CH(CH3)2: S-i-
but.), 2.72 (overlapping ddd, J = 6.5, 10.3, 13.7 Hz, 1H, b
Ha cys), 2.98–3.14 (m, 3H, b Hb cys, NCH2CH2S), 3.42–
3.53 (m, 2H, NCH2CH2S), 4.54–4.66 (m, 1H, a H cys),
5.81 (d, J = 7.4 Hz, 1H, NH cys), 6.74–6.85 (m, 1H,
NHCH2). MS m/z 641 (2M+H)+, 321 (M+H)+. Anal.
Calcd for C13H24N2O3S2: C, 48.75; H, 7.50; N, 8.75.
Found: C, 48.45; H, 7.82; N, 8.75.
1
a2D0 +7.8° (c 1.03, CHCl3). H NMR: 1.09 (d, J = 6.9 Hz,
6H, C(CH3)2), 2.18–2.31 (m, 1H, CH(CH3)2), 2.51 (dd,
J = 5.6, 12.9 Hz, 1H, b Ha cys), 2.74 (dd, J = 6.7,
12.9 Hz, 1H, b Hb cys), 3.07–3.25 (m, 2H, NCH2CH2S);
3.40–3.51 (m, 2H, NCH2CH2S), 4.07–4.19 (m, 1H, a H
cys), 5.74 (d, J = 7.6 Hz, 1H, NH cys), 6.35–6.45 (m, 1H,
NHCH2), 7.17–7.33, 7.38–7.47, 7.53–7.62, 7.89–7.96 (4m,
20H, ArH). MS m/z 597 (M+H)+. Anal. Calcd for
C35H36N2O3S2: C, 70.47; H, 6.04; N, 4.70. Found: C,
70.14; H, 6.10; N, 4.79.
2.2.8. N-(N-Isobutyryl-L-cysteinyl)-S-pivaloylcysteamine
(4g)
This compound was prepared from 3g (1.6 g,
2.78 mmol) according to the general procedure described
for 4e. The crude product was purified by flash column
chromatography (CH2Cl2/Et2O, 8.8:2.2) to give a colour-
less gum. Trituration with hexane gave 4g as a white pow-
der (650 mg, 70%): mp 120–122 °C. TLC (CH2Cl2/Et2O,
2.2.6. N-(N-Isobutyryl-L-cysteinyl)-S-acetylcysteamine (4e)
Compound 3e (1.367 g, 2.56 mmol) was dissolved in
MeOH (20 mL) and CHCl3 (1.4 mL). We added a mixture
of AgNO3 (522 mg, 3.07 mmol), pyridine (248 lL,
3.07 mmol) and MeOH (17 mL) at room temperature, in
the dark. The reaction mixture was then stirred for 12 h.
The resulting silver sulfide precipitate was collected by fil-
tration, washed with MeOH (2ꢀ 12 mL), CHCl3 (2ꢀ
12 mL) and rapidly dried under vacuum.
This product was then taken up in CHCl3 (17 mL),
placed in the dark under argon, and concentrated HCl
(450 lL) was added dropwise. The mixture was stirred
for 2 h at room temperature and then heated for 3 min at
30–35 °C. The reaction mixture was cooled to room tem-
perature, diluted in CHCl3 (80 mL) and the silver chloride
precipitate was removed and washed with CHCl3 (3ꢀ
12 mL). The combined filtrates were rapidly washed with
iced water (3ꢀ 10 mL), dried (Na2SO4), filtered, and evap-
orated to dryness under vacuum. The residual gum was
purified by flash column chromatography (CH2Cl2/Et2O,
7:3) to give 4e as a white solid (523 mg, 70%): mp 117–
120 °C. TLC (CH2Cl2/Et2O, 1:1) Rf = 0.44. a2D0 ꢁ36.5° (c
1.04, CHCl3). 1H NMR: 1.19, 1.20 (2d, J = 2ꢀ 6.9 Hz,
2ꢀ 3H, C(CH3)2), 1.62 (dd, J = 7.5, 10.3 Hz, 1H, SH),
2.37 (s, 3H, SCOCH3), 2.47 (hept, J = 6.9 Hz, 1H,
CH(CH3)2), 2.72 (ddd, J = 6.5, 10.3, 13.9 Hz, 1H, b Ha
cys), 3.00–3.08 (m, 2H, NCH2CH2S), 3.06 (ddd, J = 4.2,
5:5) Rf = 0.46. a20 ꢁ25.0° (c 1.04, CHCl3). 1H NMR:
D
1.194, 1.198 (2d, J = 2ꢀ 6.9 Hz, 2ꢀ 3H, C(CH3)2), 1.23
(s, 9H, C(CH3)3), 1.61 (dd, J = 7.6, 10.2 Hz, 1H, SH),
2.47 (hept app., J = 6.9 Hz, 1H, CH(CH3)2), 2.72 (ddd,
J = 6.5, 10.2, 13.8 Hz, 1H, b Ha cys), 2.95–3.13 (m, 3H,
b Hb cys, NCH2CH2S), 3.40–3.52 (m, 2H, NCH2CH2S),
4.54–4.66 (m, 1H, a H cys), 6.49 (d, J = 7.5 Hz, 1H, NH
cys), 6.73–6.88 (m, 1H, NHCH2). MS m/z 669 (2M+H)+,
335 (M+H)+. Anal. Calcd for C14H26N2O3S2: C, 50.30;
H, 7.78; N, 8.38. Found: C, 50.19; H, 7.92; N, 8.35.
2.2.9. N-(N-Isobutyryl-L-cysteinyl)-S-benzoylcysteamine
(4h)
This compound was prepared from 3h (1.6 g,
2.68 mmol) according to the general procedure described
for 4e. The crude product was purified by flash column
chromatography (CH2Cl2/Et2O, 1:1) to give a colourless
gum. Trituration with hexane gave 4h as a white powder
(550 mg, 58%): mp 127–130 °C. TLC (CH2Cl2/Et2O, 6:4)
Rf = 0.35. a20 ꢁ20.8° (c 1.06, CHCl3). 1H NMR: 1.17,
D
1.18 (2d, J = 2ꢀ 6.9 Hz, 2ꢀ 3H, C(CH3)2), 1.59 (dd,
J = 7.5, 10.3 Hz, 1H, SH), 2.44 (hept app., J = 6.9 Hz,
1H, CH(CH3)2), 2.70 (ddd, J = 6.5, 10.3, 13.8 Hz, 1H, b
Ha cys), 3.07 (ddd, J = 4.2, 7.5, 13.8 Hz, 1H, b Hb cys),
3.17–3.34 (m, 2H, NCH2CH2S), 3.53–3.64 (m, 2H,
7.5, 13.9 Hz, 1H,
b Hb cys), 3.41–3.53 (m, 2H,
NCH2CH2S), 4.61 (ddd, J = 4.2, 6.5, 7.4 Hz, 1H, a H
cys), 6.46 (d, J = 7.4 Hz, 1H, NH cys), 6.72–6.85 (m, 1H,

M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
137
NCH2CH2S), 4.62 (ddd, J = 4.2, 6.5, 8.0 Hz, 1H, a H cys),
6.47 (d, J = 8.0 Hz, 1H, NH cys), 6.80–6.91 (m, 1H,
NHCH2), 7.42–7.53, 7.56–7.65, 7.92–8.01 (3m, 5H, ArH).
MS m/z 709 (2M+H)+, 355 (M+H)+. Anal. Calcd for
C16H22N2O3S2: C, 54.23; H, 6.21; N, 7.91. Found: C,
54.20; H, 6.18; N, 7.94.
FeSO4, and 10 mM H2O2 was prepared. Various concen-
trations of the test compounds were added to the reaction
mixture before the addition of H2O2 and FeSO4. The reac-
tion was carried out at 37 °C for 10 min and terminated by
the addition of EDTA to a final concentration of 10 mM.
After incubation, 4 lL of a 1 mM EB solution was added
and fluorescence intensity was measured at 590 nm
(kex = 510 nm). The reduction in fluorescence was used
to measure the extent of DNA damage. DNA damage
was expressed as the ration ðF untreated control ꢁ F treatmentÞ=
ðF untreated control ꢁ F FeSO –H O Þ. A DNA damage ratio <1
2.3. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical
The scavenging effect of the synthesized compounds 4a–
h on the DPPH radical was evaluated according to previ-
ously published methods [16,17]. Various concentrations
of the test compounds were incubated in 1 mL of an 60%
ethanolic solution containing the DDPH radical (60 lM).
The mixture was shaken vigorously and allowed to stand
for 30 min; absorbance at 517 nm was determined, and
the percentage of activity was calculated by the following
equation: % inhibition = ((Acontrol ꢁ Atest)/Acontrol) ꢀ 100.
All tests and analyses were undertaken in triplicate and
the results averaged.
4
2
2
means that the compound inhibits the FeSO4–H2O2
induced damage, while a ratio >1 means that the com-
pound enhances the FeSO4–H2O2 induced damage.
2.7. Biological methods
Isolation of human monocyte-derived macrophages
(MDM), antiviral assay and data analysis were done as
previously reported [7].
3. Results and discussion
2.4. ABTSÅ+ radical cation scavenging assay [18]
3.1. Chemistry
The ABTS radical cation (ABTSÅ+) was prepared by
mixing ABTS stock solution (7 mM in water) with
2.45 mM potassium persulfate (final concentration). This
mixture was allowed to stand in the dark at room tem-
perature for 12–16 h before use. The ABTSÅ+ solution
was diluted with ethanol to an absorbance of 0.70 at
734 nm. Diluted ABTSÅ+ solution (0.9 mL) and 100 lL
of various concentrations of the test compounds were
mixed and measured immediately after 6 min at
734 nm. The antioxidant activity was calculated using
the above formula.
N-Acetyl derivatives 4a–d and N-isobutyryl analogues
4e–h were synthesized starting, respectively, from commer-
cially available N-acetyl-S-trityl-L-cysteine 1 or from S-trity-
lated N-isobutyryl-L-cysteine derivative 2 [6,7]. A mixed
anhydride was first formed in situ using isobutyl chlorofor-
mate (IBC) and 4-methylmorpholine (NMM) and was
allowed to react with N-hydroxysuccinimide (HOSu) in
order to generate an active O-succinimide ester. The latter
was then condensed with the appropriate S-acylcysteamine
hydrochlorides in the presence of NMM to give the expected
S-trityl derivatives in good to excellent yields. Detritylation
was performed in a two steps procedure via the correspond-
ing silver sulfides, which were treated with concentrated HCl
to give free thiols 4a–h (Scheme 1) [21].
2.5. Hydroxyl radical scavenging activity [19]
The reactions were performed in 10 mM phosphate
buffer (pH 7.4) containing 2.8 mM deoxyribose, 2.8 mM
H2O2, 12.5 mM FeCl3, 100 lM EDTA, and the test sam-
ple (0–15 mM). The reaction was started by adding ascor-
bic acid to a final concentration of 200 lM. The reaction
mixture was incubated for 1 h at 37 °C. After incubation,
1 mL of 1% thiobarbituric acid (w/v) in 0.05 M NaOH
was added followed by 1 mL of 2.8% trichloroacetic acid
(w/v). This solution was heated 20 min at 100 °C. After
cooling, the extent of deoxyribose degradation was
measured at 532 nm against a blank. The scavenging
3.2. Antioxidant activity
3.2.1. Free radical scavenging activity on 2,2-diphenyl-1-
picrylhydrazyl radical
This assay is based on the measurements of the scaveng-
ing ability of compounds towards the stable radical 2,2-
diphenyl-1-picrylhydrazyl (DPPHÅ) [22]. The disappear-
ance of this commercially available radical is measured
spectrophotometrically at 517 nm in a methanolic solution.
The antioxidant activity was expressed as the 50% inhibi-
tory concentration (IC50) based on the amount of com-
pound required for a 50% decrease of the initial DPPH
radical concentration. The radical-scavenging activities
for free thiols, range from 12 lM for 4a to 33 lM for 4e
(Table 1). Whereas 4a displays comparable IC50 to natural
antioxidants such as Trolox and ascorbic acid (11 lM
each), compounds 4b–4h activities are closer to the results
activity on hydroxyl radicals was expressed as:
%
inhibition = ((Acontrol ꢁ Atest)/Acontrol) ꢀ 100.
2.6. Ethidium bromide fluorescence binding assay
The method of Stoewe and Pruts was modified to mea-
sure DNA damage [20]. A 2 mL mixture containing 20 mM
phosphate buffer (pH 7.0), 100 lg/mL calf thymus, 50 lM

138
M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
Table 1
ter than NAC (27 lM) or ascorbic acid (22 lM). Whereas
compound 4a seemed to show a higher scavenging ability
in the DPPH assay (12 lM), this dominance was however,
not confirmed in the ABTS assay (9 lM). The results indi-
cate that the sulfhydryl group confers to these molecules
interesting free radical scavenging properties always higher
than NAC. However, these two assays do not support a
free radical scavenging ability dominance depending on
the nature of the N-protecting group of these series.
Antioxidant activities of derivatives 4a–h in DPPHÅ, ABTSÅ+, and OHÅ
radical scavenging assays
Compound
DPPHÅ IC50 ABTSÅ+
OHÅ IC50
(mM)
Calculated
logP
(lM)
IC50 (lM)
Trolox
Ascorbic acid
L-Cysteine
NAC
GSH
Bis-N,S-acetyl-
L-cysteine
11 1.9
11 1.7
25 1.5
38 3.2
32 3.7
<100
12 1.4
22 2.1
1.9 0.3
nda
3.09
ꢁ1.76
ꢁ2.35
ꢁ0.62
ꢁ3.05
ꢁ0.29
6
1.2
nda
27 3.5
11 0.4
<100
7.3 0.4
5.5 0.4
nda
3.2.3. Deoxyribose assay
4a
4b
4c
4d
4e
4f
4g
4h
a
12 0.3
22 2.1
23 1.4
22 2.7
33 2.0
27 3.1
25 3.6
26 2.1
9
3.2
7.2 0.5
8.3 0.8
8.3 0.7
7.5 0.6
9.2 0.5
9.7 0.6
9.5 0.6
11.6 1.1
ꢁ0.10
0.74
1.14
1.16
0.74
1.58
1.98
2.00
The ability of these compounds to scavenge hydroxyl
radicals (OHÅ) was assessed using the classic deoxyribose
degradation assay described by Halliwell et al. [19]. The
principle of this assay is the quantification of the 2-deoxy-
ribose degradation into malonaldehyde which is condensed
with thiobarbituric acid. The degree of hydroxyl radicals
(OHÅ) scavenging was expressed as the 50% inhibitory con-
centration (IC50) based on the amount of compound
required for a 50% decrease of the initial chromophore
absorbance at 532 nm.
Table 1 shows the effect of compounds 4a–h on deoxyri-
bose degradation induced by Fe3+/H2O2. The inhibitory
effect on sugar degradation was shown to be concentra-
tion-dependant, in the millimolar range, Trolox having
the highest apparent OHÅ radical scavenging activity
(1.9 mM). All other N-acetyl or N-isobutyryl derivatives
possessed lower scavenging activity (7.2–11.6 mM), very
similar to the ones observed with GSH (5.5 mM) and
NAC (7.3 mM).
In biochemical systems, superoxide radicals are con-
verted to hydrogen peroxides, which subsequently generate
reactive hydroxyl radicals through the Fenton reaction
with metal ions [24]. Therefore, the mode of action of these
compounds (direct OHÅ scavenging or metal chelation) was
further examined by evaluating their capacity to inhibit the
iron-driven Fenton reaction-induced damage of calf thy-
mus DNA.
10 1.4
11 2.1
8
6
6
5
7
2.8
1.1
0.7
0.2
0.4
Not determined. Values are mean of triplicate determinations SD.
obtained with cysteine (25 lM), NAC (38 lM) and GSH
(32 lM). Moreover, N-acetyl derivatives 4a–d appear to
scavenge the DPPH radical more efficiently (although
slightly) than the N-isobutyryl derivatives 4e–h.
3.2.2. ABTSÅ+ radical cation scavenging assay
In this assay the ABTS radical cation is produced chem-
ically by the oxidation of the corresponding colourless sul-
fonic acid with potassium persulfate [18]. The green-blue
ABTS radical cation has excellent spectral characteristics
is stable over a wide range of pH and is applicable to the
study of both water- and lipid-soluble antioxidants that
converts ABTSÅ+ back to the initial sulfonic acid. The dis-
appearance the ABTSÅ+ radical is measured spectrophoto-
metrically at 734 nm in a methanolic solution. The
antioxidant activity was expressed as the 50% inhibitory
concentration (IC50) based on the amount of compound
required for a 50% decrease of the initial ABTSÅ+ radical
concentration.
3.2.4. Ethidium bromide binding assay
It is important to note that the IC50 values for both
(DPPH and ABTS) of these assays are dependent on the
details on the experiments (rate at which the free radicals
are generated; use of aqueous vs. organic solvent; reaction
with the species detected by the assay) and need to be in
agreement with one another. The DPPH and ABTS assays
are generally considered to be useful for ranking the
potency of various antioxidants, and we found that the rel-
ative IC50 values determined using these assays agreed well
with one another (Table 1). It was found that all free thiols
tested were able to scavenge the ABTSÅ+ radical cation with
values in the micromolar range (5–11 lM) while protected
thiols such as bis-N-S-acetylcysteine [23] (Table 1) were
not. In this assay, almost no difference was found depend-
ing on the nature of the N-protecting group. IC50 values are
comparable with those obtained with cysteine (6 lM),
GSH (11 lM) or Trolox (12 lM) but at least two times bet-
The ethidium bromide (EB) binding assay, based on the
formation of a fluorescent complex between double-
stranded DNA and EB, was used to measure DNA damage
[20]. Damaged DNA inhibits the binding of EB to DNA
which results in a decrease in intensity of fluorescence.
Several forms of DNA damage, including strand scission,
base oxidation and base liberation, contribute to the loss
of fluorescence. Hence, the assay detects a broad range
of different DNA lesions. DNA damage was expressed
as the ratio ðF untreated control ꢁ F treatmentÞ=ðF untreated control
ꢁ
F FeSO –H O Þ. A DNA damage ratio <1 means that the com-
4
2
2
pound inhibits the FeSO4–H2O2 induced damage, while a
ratio >1 means that the compound enhances the FeSO4–
H2O2 induced damage.
The effects of compounds 4a–h to the iron-driven Fen-
ton reaction are presented in Fig. 1. In the absence of
any compound, the presence of FeSO4 and H2O2 signifi-

M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
139
1.6
1.5
1.4
1.3
1.2
1.1
1
120
100
80
60
40
20
0
0.9
0.8
NAC
4a
4b
4c
4d
4e
4f
4g
4h
NAC MEA 4a
4b
4c
4d
4e
4f
4g
4h
Fig. 1. Effect of compounds 4a–h (7 mM) on FeSO4–H2O2 induced
damage in calf thymus DNA. The level of DNA damage after treatment
was expressed as ratio of DNA damage relative to the FeSO4–H2O2
induced damage control, as described in Section 2. Values are mean of
triplicate determinations SD.
Fig. 2. Anti-HIV effects of 4a–h at a concentration of 150 lM in HIV-1/
Ba-L-infected MDM. Cumulative RT activities were used to calculate
percentages with respect to untreated control, and the results are expressed
as means SD.
Table 2
cantly damaged calf thymus DNA. Addition of 7 mM of
compounds 4a–h did not show any protection toward
DNA damage and even enhanced the FeSO4–H2O2
induced damage of calf thymus DNA, thus suggesting a
pro-oxidant effect of these derivatives. Like the Fenton
reaction, the deoxyribose assay has been used to assess
the pro-oxidant action of many compounds [25,26]. How-
ever, no pro-oxidant activity was detected with any of the
compounds tested in the deoxyribose assay. The fact that
the Fenton reaction is a potent source of hydroxyl radicals,
suggest that compounds 4a–h are bad metal chelators and
act essentially as hydroxyl radicals scavengers. The results
obtained here with N-acetyl and N-isobutyryl derivatives
are in accordance with recently published results demon-
strating that NAC could increase the level of DNA double
strand breaks instead of inhibiting the damage [27]. Look-
ing at Fig. 1, it seems however that these pro-oxidant activ-
ities might be counterbalanced by the steric hindrance
brought by large protecting groups, N-isobutyryl-S-ben-
zoyl derivative 4h showing no activity whatsoever.
It has long been proposed that the beneficial protective
effect of NAC mainly comes from its capacity to directly
scavenge radicals by means of its thiol function. Neverthe-
less, the interaction of thiols with reactive radicals can gen-
erate thiol radicals which in turn may impart a pro-oxidant
function [28]. Our results support the ability of these N-
acetyl and N-isobutyryl derivatives to scavenge free radi-
cals like DPPHÅ and ABTSÅ+ radicals and to a lesser extent
the hydroxyl radical OHÅ but enhance the Fe2+–H2O2-
dependent oxidative actions suggesting a balance between
pro- and antioxidant mechanism.
ED50, ED70, and ED90 for the anti-HIV-1/Ba-L activity of 4a, 4b, 4e, and
4f in human MDM
4a (lM)
4b (lM)
4e (lM)
4f (lM)
ED50
ED70
ED90
51
82
178
110
175
380
95
121
146
36
54
103
decided to explore the effects of these compounds in
MDM infected in vitro with the reference macrophage-tro-
pic HIV-1/Ba-L strain. As a consequence of an expected
ineffective hydrolysis of the N-isobutyryl group, none of
these derivatives were found to increase GSH level (data
not shown). However, we were pleased to find that the
introduction of the N-isobutyryl group did not seem to
affect the antiviral effects of derivatives 4e–h. In fact, the
anti-HIV-1/Ba-L activity of these thiols in human MDM
is comparable to that of the N-acetyl series, while NAC
and MEA were efficient at millimolar concentration
(Fig. 2) [7]. We therefore carried out an experiment to
determine and compare more precisely the dose-dependant
effect of compounds of the two series (Table 2). Interest-
ingly, compound 4f was found to be more efficient that
4a (formerly known as I-152) [6–8] suggesting again the
need of a not too bulky R0 group for high anti-HIV activ-
ities. These results strongly suggest that no direct correla-
tion can be drawn between pro-GSH and anti-HIV
activities of potent molecules.
4. Conclusions
In conclusion, a series of new N-isobutyryl-L-cysteine
derivatives was synthesized and their potential as antioxi-
dants was evaluated, and compared with known
pro-GSH compounds. These N-isobutyryl as well as their
N-acetyl analogues showed remarkable free radical scav-
enging abilities always better than NAC and GSH.
Moreover, both series displayed similar hydroxyl radical-
3.2.5. Anti-HIV activities in human MDM
Lipophilicity plays an important role in cell-uptake and
may be essential for dispersion of food antioxidants in bulk
oils or fats. We therefore confirmed the higher lipophilicity
of the N-isobutyryl series by calculating their respective
logP values (Table 1) [29]. On the basis of these result we

140
M. Smietana et al. / Bioorganic Chemistry 36 (2008) 133–140
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scavenging activities. As the N-isobutyryl compounds are
more lipophilic, they might produce higher levels of free
thiols in the body, reducing therefore the damage provoked
by oxidative stress. The present study support the stringent
sulfhydryl functional group requirement for maximal free
radical scavenging efficacy, and the presence of the N-iso-
butyryl group suggests that a better bioactivation of these
derivatives might play a major role in the ultimate release
of ROS scavenging active compounds. As these com-
pounds display equal or better anti-HIV activities than
already reported cysteine derivatives, they could be good
candidates to interfere with inflammatory and oxidative
disorders associated with HIV infection. Moreover, these
results suggest that anti-HIV effects of our compounds
are related to their antioxidant effects and not their pro-
GSH properties. These lipophilic analogues 4e–h may
therefore, be profitably used, in principle, as food
antioxidants.
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We thank S. Evesque and J.Y. Puy for technical assis-
tance and the CNRS for financial support.
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