NAC/MEA conjugate: A new potent antioxidant which increases the GSH level in various cell lines

Article abstract of DOI:10.1016/S0960-894X(01)00171-8

I-152 is a prodrug of NAC and MEA with potent pro-GSH effects in human macrophages, astrocytes and lymphocytes. This molecule could be of interest in HIV infection in respect to its antioxidant and anti-HIV activities, but also in other diseases to counte

Full text of DOI:10.1016/S0960-894X(01)00171-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1189–1191
NAC/MEA Conjugate: A New Potent Antioxidant Which
Increases the GSH Levelin Various CelLines
Joel Oiry,a,* Patricia Mialocq,b Jean Y. Puy,a Philippe Fretier,b
Pascal Clayette,b Dominique Dormontb and Jean L. Imbacha
a
´
`
´
Laboratoire de Chimie Organique Biomoleculaire de Synthese, UMR 5625 CNRS-UM II, Universite Montpellier II,
`
Sciences et Techniques du Languedoc, place Eugene Bataillon, 34095 Montpellier Cedex 5, France
bService de Neurovirologie, CEA/DSV/DRM, CRSSA, EPHE, 60–68 avenue de la Division Leclerc, BP 6,
92265 Fontenay aux Roses Cedex, France
Received 9 February 2001; revised 7 March 2001; accepted 7 March 2001
Abstract—I-152 is a prodrug of NAC and MEA with potent pro-GSH effects in human macrophages, astrocytes and lymphocytes.
This molecule could be of interest in HIV infection in respect to its antioxidant and anti-HIV activities, but also in other diseases to
counteract oxidative stress, that is, inflammation, cardiovascular diseases, and neurodegenerative diseases. # 2001 Elsevier Science
Ltd. All rights reserved.
It is now well established that antioxidants could be
valuable in various clinical situations in which oxidative
stress plays a role in pathological disorders (e.g., viral
diseases, cardiovascular diseases, inflammation, cancer,
neurological diseases, septic shock, etc.).1À12 Hence, our
interest in this study was in the design of new potent
antioxidant molecules. In this respect, we would like to
report herewith on an NAC/MEA13 conjugate com-
pound, I-152, which seems to be a highly potent anti-
oxidant by increasing the glutathione (GSH) content in
various cell lines. This research is in continuation of our
previous work in the area of radical scavengers and
other pro-GSH compounds.14À16
and the substrate of GSH-S-transferase that detoxifies
endogenous and exogenous compounds.17
The synthesis of I-152 is outlined in Scheme 1 and was
performed starting from commercially available N-ace-
tyl-S-trityl-l-cysteine 1 and S-acetylcysteamine hydro-
chloride 2.18
The pseudo-dipeptide 3 was obtained by formation in
situ of the mixed anhydride using 1, isobutyl chloro-
formate (IBC) and N-methylmorpholine (NMM). This
anhydride was then added to N-hydroxysuccinimide
(HOSu) to form the OSu active ester which was finally
coupled with 2 in the presence of NMM. After treat-
ment of the reaction mixture, the S-trityl derivative 3
Glutathione, or l-g-glutamyl-l-cysteinylglycine, is an
anionic tripeptide, widely distributed in animals. It is
involved in various cell functions such as the detoxi-
fication of xenobiotics, the immune response, inflam-
matory processes and the biosynthesis of proteins and
DNA, and it plays an important role in cell defense
against oxidative stress. GSH is an intrinsic efficient
scavenger of nitric oxide and peroxinitrite, but it is
also the substrate of GSH peroxidases that inactivate
peroxides, the substrate of GSH-dehydrogenase that
regenerates antioxidant molecules such as ascorbate,
Scheme 1. Reagents and conditions: (a) (1) AcOEt, IBC, NMM,
À15 ꢀC, 15 min; (2) HOSu, À15 ꢀC, 15 min; (3) 2, NMM, À15 ꢀC–rt,
12 h; (b) MeOH, CHCl3, mixture of MeOH/AgNO3/pyridine, rt, 12 h;
(c) CHCl3, HCl 37%, rt, 2 h.
*Corresponding author. Tel.: +33-4-6714-3837; fax: +33-4-6754-
9610; e-mail: oiryj@univ-montp2.fr
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00171-8

1190
J. Oiry et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1189–1191
was isolated. This crude product was over 95% pure
and can be used in the next step without further pur-
ification. S-Trityl protecting group was then removed,
via formation of the corresponding silver sulfide 4,
according to the described method.19 Compound I-152
was isolated after recrystallization from ethyl acetate
and petroleum ether.20
at high I-152 doses is probably the consequence of a
negative feedback of GSH on g-glutamylcysteinyl
synthetase, the first step in GSH synthesis.24,25 I-152 is
also efficient to increase GSH in human spleen and BAL
macrophages, in human astrocytes, major cells also
involved in defense against oxidative stress in the central
nervous system, and in PBLs susceptible to oxidative
stress-induced apoptosis and particularly in human
immunodeficiency virus (HIV) infection (Fig. 2).
I-152 is a prodrug expected to liberate after metaboli-
zation, NAC and MEA, two potential pro-GSH com-
pounds which may act as part of the glutathione (GSH/
GSSG) redox system.21,22 Hence, intracellular total
GSH level was measured in primary human macro-
phage-derived monocytes (MDMs) and other human
cell lines (spleen and broncho-alveolar (BAL) macro-
phages, astrocytes, and lymphocytes). Peripheral blood
mononuclear cells (PBMCs) of healthy blood donors
were obtained by Ficoll-Hypaque density gradient cen-
trifugation, and monocytes were separated from peri-
pheral blood lymphocytes (PBLs) using countercurrent
centrifugal elutriation. Monocytes were then differ-
entiated in MDM during 7 days of culture. Spleens and
broncho-alveolar lavages were generously provided by
Pr. T. de Revel (Hopital D’Instruction des Armees
Percy, Clamart, France) and M. Humbert (Hopital
Antoine Beclere, Clamart, France), respectively; spleen
and BAL macrophages were obtained by adherence as
previously described.23 Embryonic astrocytes were gen-
erously provided by Pr. M. Tardieu (Hopital de Bicetre,
Kremlin Bicetre, France). All these cells were cultivated
in DMEM Glutamax cell culture medium supplemented
with 10% heat-inactivated (+56 ꢀC, 45 min) fetal calf
serum, 1% triantibiotic mixture (penicillin, streptomy-
cin, and neomycin). After 24 h treatment, cells were
lysed and GSH level was determined using Griffith’s
method (Cayman, Ann Arbor, MI, USA) and protein
level using Bradford’s method (BioRad, Hercules, CA,
USA).
The toxicity of I-152 was appreciated in vitro (neutral-red
uptake) and in vivo. In MDMs, 50% cytotoxic concentra-
tions (CC50) were respectively equal to >5mM at 24 h of
treatment and 635 mM at 12 days.26 In OF1 mice, the
intraperitoneal LD50 (30 days) was equal to 700 mg/kg.27
HIV infection is associated with systemic and tissular
decreases in the GSH content.28,29 This decreased GSH
content may reduce the survival of HIV-infected
patients,30 perhaps by contributing to diverse disorders,
that is, CD4+ T cell apoptosis,24 neuroAIDS31 and
enhancing HIV replication. The anti-HIV effects of pro-
GSH compounds such as NAC and l-2-oxo-thiazoli-
dine-4-carboxylic acid confirm the relationships between
viral replication and GSH.32,33 Hence, anti-HIV effects
of I-152 were explored in MDM infected with the
reference macrophage-tropic HIV-1/Ba-l strain. In
these experimental conditions, I-152 significantly
decreased viral replication in MDMs, with a 50% effec-
tive dose (ED50) equal to 3–50 mM regarding the multi-
plicity of infection (moi, Table 1).
On the basis of such preliminary data, we decided to
explore the behavior of I-152 in cell extracts as a model
for intracellular metabolization.
The compound was incubated in total cell extracts of T
CD4+ lymphoid CEM-SS cell line and of MDMs. The
structure of the metabolites was determined by HPLC/
MS coupling and coinjection with authentic synthesized
samples. Surprisingly, we observed a rapid transposition
of I-152 (CEM-SS: T1/2=14 min, c=10À4 M) giving rise
In human MDM, I-152 increased GSH level in a dose-
dependent manner. At 250 mM, GSH content increased
by 165% (Fig. 1A), whereas NAC and MEA, alone or
in equimolar combination, had no significant effect at
the same dose (Fig. 1B). The decrease in GSH content
Figure 1. (A) Dose-effect of I-152 on intracellular GSH level in 24 h
treated MDMs; (B) comparison between effects of I-152 and other
pro-GSH drugs at 250 mM on intracellular GSH content (GSH dosa-
ges 24 h after the initiation of treatment). Experiments were performed
in triplicate and results are representative of five independent experi-
ments (n=15).
Figure 2. (A) Effects of I-152 on intracellular GSH level in human
spleen macrophages, (B) in human macrophages obtained from
broncho-alveolar lavages, (C) in human peripheral blood lymphocytes,
(D) in human embryonic astrocytes. Experiments were performed in
triplicate and results are representative of at least three independent
experiments (n=9).

J. Oiry et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1189–1191
1191
Table 1. Anti-HIV activity of I-152 in HIV-1/Ba-l-infected MDMsa
10. Rahman, I.; MacNee, W. Free Radical Biol. Med. 2000,
28, 1405.
11. Tang, A. M.; Smit, E. J. Acquir. Immune Defic. Syndr.
2000, 25 (Suppl. 1), S12.
12. Treitinger, A.; Spada, C.; Verdi, J. C.; Miranda, A. F.;
Oliveira, O. V.; Silveira, M. V.; Moriel, P.; Abdalla, D. S. Eur.
J. Clin. Invest. 2000, 30, 454.
13. NAC: N-acetyl-l-cysteine, MEA: cysteamine, 2-mercap-
toethylamine.
14. Oiry, J.; Pue, J. Y.; Fatome, M.; Sentenac-Roumanou, H.;
Lion, C.; Imbach, J. L. Eur. J. Med. Chem. 1992, 27, 809.
15. Oiry, J.; Pue, J. Y.; Laval, J. D.; Fatome, M.; Imbach, J. L.
Eur. J. Med. Chem. 1995, 30, 47.
Multiplicity of infection (moi)
0.001 (mM)
0.01 (mM)
ED50
ED70
ED90
3
15
75
50
90
190
aThese results are representative of six independent experiments per-
formed in triplicate.
16. Oiry, J.; Puy, J. Y.; Mialocq, P.; Clayette, P.; Fretier, P.;
Jaccard, P.; Dereuddre-Bosquet, N.; Dormont, D.; Imbach, J.
L. J. Med. Chem. 1999, 42, 4733.
17. Meister, A. Methods Enzymol. 1995, 251, 3.
18. Wieland, T.; Bokelmann, E. Ann. Chem. 1952, 516, 20.
19. Zervas, L.; Photaki, I. J. Am. Chem. Soc. 1962, 84, 3887.
20. I-152: mp 122–124 ꢀC; [a]D20=À40ꢀ (c 0.87, CHCl3); 1H
NMR (400 MHz, CDCl3) d 1.60 (dd, J=7.6, 10.3 Hz, 1H, SH),
2.07 (s, 3H, NCOCH3), 2.36 (s, 3H, SCOCH3), 2.70 (ddd,
J=6.5, 10.3, 13.9 Hz, 1H, b Ha cys), 3.03 (t, J=6.3 Hz, 2H,
NCH2CH2S), 3.06 (ddd, J=4.3, 7.6, 13.9 Hz, 1H, b Hb cys),
3.46 (td, J=6.0, 6.3 Hz, 2H, NCH2CH2S), 4.59 (ddd, J=4.3,
6.5, 7.9 Hz, 1H, a H cys), 6.52 (d, J=7.9 Hz, 1H, NH cys),
6.75–6.90 (m, 1H, NHCH2); 13C NMR (100.6 MHz, CHCl3) d
23.2 (NCOCH3), 26.6 (b CH2 cys), 28.4 (NCH2CH2S), 30.6
(SCOCH3), 39.7 (NCH2CH2S), 54.1 (a CH cys), 169.8
(NCOCH3), 170.4 (CO cys), 196.4 (SCOCH3); MS: (FAB+/
G-T) m/z 529 [2M+H]+, 265 [M+H]+. Anal.: C9H16N2O3S2:
C, H, N.
Scheme 2. I-152 transposition in CEM-SS and MDM cell extracts.
to the corresponding fully acetylated derivative 5 and
the expected dithiol 6 (Scheme 2). An identical result
was observed in MDMs. This transposition is solvent-,
temperature-, and concentration-dependent which
seems to indicate an intermolecular process.
It is noteworthy that the dithiol derivative 6 may play a
crucial role in the I-152 mode of action as the key moi-
ety of the NAC has been assumed to be related to the
thiol function.34 In addition, we have also shown in a
complementary experiment that incubation of 5 in CEM-
SS cell extracts gives rise to I-152 formation (T1/2=27
min, c=10À4 M) presumably upon esterase activation.
21. Selig, C.; Nothdurft, W.; Fliedner, T. M. J. Cancer Res.
Clin. Oncol. 1993, 119, 346.
22. Wartenberg, A. A. In Concise Texbook of Medicine, 2nd
ed.; Kochar, M. S., Kutty, K., Lennon, E. J., Eds.; Elsevier:
New York, 1960; pp 135–160.
23. Rimaniol, A. C.; Haik, S.; Martin, M.; Le Grand, R.;
Boussin, F. D.; Dereuddre-Bosquet, N.; Gras, G.; Dormont,
D. J. Immunol. 2000, 164, 5430.
24. Dobmeyer, T. S.; Findhammer, S.; Dobmeyer, J. M.; Klein,
S. A.; Raffel, B.; Hoelzer, D.; Helm, E. B.; Kabelitz, D.; Rossol,
R. Free Radical Biol. Med. 1997, 22, 775.
25. Richman, P. G.; Meister, A. J. Biol. Chem. 1975, 250,
1422.
In conclusion, I-152 is a potent pro-GSH compound as
compared to NAC, MEA or other molecules of the
same family. These pro-GSH effects are observed in
different cell lines suggesting the high potential of this
new molecule. The biological interest of I-152 is con-
firmed by the significant decrease of HIV replication in
MDMs, in absence of cytotoxicity. Altogether, these
data suggest that I-152 could be of great interest in
pharmacology but also in cosmetology or other
domains involving oxidative stress.
26. CC50 values are means of three independent experiments
performed in triplicate.
27. The toxicity was evaluated by a Probit analysis of the
LD50, the dose range being determined in a preliminary study.
Five groups of 10 mice were then injected with different doses
within this range.
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