Synthesis and Biological Evaluation in Human Monocyte-Derived Macrophages of N-(N-Acetyl-L-cysteinyl)-S-acetylcysteamine Analogues with Potent Antioxidant and Anti-HIV Activities

Article abstract of DOI:10.1021/jm030374d

We synthesized a series of N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine (10) analogues bearing various acyl groups on thiol cysteine or cysteamine residues, to investigate the structure-activity relationship for pro-GSH and anti-HIV properties in human mac

Full text of DOI:10.1021/jm030374d

J . Med. Chem. 2004, 47, 1789-1795
1789
Syn th esis a n d Biologica l Eva lu a tion in Hu m a n Mon ocyte-Der ived Ma cr op h a ges
of N-(N-Acetyl-L-cystein yl)-S-a cetylcystea m in e An a logu es w ith P oten t
An tioxid a n t a n d An ti-HIV Activities
J oe¨l Oiry,*,† Patricia Mialocq,‡ J ean-Yves Puy,† Philippe Fretier,‡ Nathalie Dereuddre-Bosquet,§
Dominique Dormont,‡ J ean-Louis Imbach,† and Pascal Clayette*,§
Laboratoire de Chimie Organique Biomole´culaire de Synthe`se, UMR CNRS-UM II 5625, Universite´ Montpellier II Sciences et
Techniques du Languedoc, place Euge`ne Bataillon, 34095 Montpellier, France, Service de Neurovirologie, DRM/ DSV,
CEA/ CRSSA/ EPHE/ University Paris XI, 18 route du Panorama, B.P. 6, 92265 Fontenay aux Roses Cedex, France, and
SPI-BIO c/ o Service de Neurovirologie, Fontenay aux Roses Cedex, France
Received August 5, 2003
We synthesized a series of N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine (10) analogues bearing
various acyl groups on thiol cysteine or cysteamine residues, to investigate the structure-
activity relationship for pro-GSH and anti-HIV properties in human macrophages. The
S-substituents were ranked in the following order of efficacy: H g acetyl > isobutyryl > pivaloyl
> benzoyl. We found that none of these derivatives had pro-GSH or antiviral activities in vitro
higher than that of 10, but several displayed similar levels of anti-HIV activity, making them
possible candidates for use as adjuvant therapies in conjunction with HAART, for treating
neurological aspects of HIV infection.
In tr od u ction
istration of pro-GSH drugs, such as N-acetyl-L-cysteine
(NAC), has been shown to decrease mortality.18 In vitro,
GSH and NAC inhibit viral replication in human
monocyte-derived macrophages (MDM) and lympho-
cytes.19-21 Since high concentrations of GSH and NAC
are required to obtain biological effects, their efficacy
is limited due to their low bioavailability.22
We previously reported that 10 (Scheme 1), a NAC-
MEA (2-mercaptoethylamine, cysteamine) conjugate,
strongly increases GSH levels in various cell lines.23
Preliminary studies of the decomposition of 10 in cell
extracts have shown that this compound is deacetylated,
presumably upon esterase activation, to the correspond-
ing dithiol derivative, which may be a key metabolite
responsible for the in situ release of NAC and MEA.23
We therefore decided to synthesize this dithiol deriva-
tive and other 10 analogues bearing various S-acyl
groups and to carry out a structure-activity relation-
ship (SAR) analysis of this new class of antioxidants.
We report here the synthesis of this class of compounds
and their in vitro biological effects in MDM and show
that several of these compounds display potent pro-GSH
and anti-HIV effects.
Oxidative stress plays a major role in various patho-
logical disorders, including viral diseases, cardiovascular
diseases, inflammation, cancer, neurological diseases,
and septic shock.1-9 Glutathione (GSH or L-γ-glutamyl-
L-cysteinylglycine) is a tripeptide widely distributed in
mammalian cells and tissues. Its reduced form is
involved in various cell functions, including the bio-
synthesis of proteins and DNA precursors, amino acid
transport, and the maintenance of sulfhydryl redox
status. Indeed, GSH is a scavenger of NO and peroxi-
nitrite species and acts as the substrate of GSH-
peroxidases. These enzymes inactivate peroxides, which
are in turn the substrate of GSH-dehydrogenases, which
regenerate antioxidant molecules, such as ascorbate,
and of GSH-S-transferases, which detoxify endogenous
and exogenous compounds. GSH therefore plays an
important role in the intracellular antioxidant defense
system.10
HIV infection is associated with decreases in systemic
and tissue GSH contents.11,12 The molecular mecha-
nisms responsible for these decreases are unclear.
However, both oxidative stress and viral replication
seem to be involved. The deficit in GSH aggravates
oxidative stress and contributes to the pathophysiology
of HIV infection by increasing viral replication,13 CD4
T-lymphocyte apoptosis,14,15 inflammatory syndrome,16
chronic weight loss, drug toxicities, and neuroAIDS.17
Indeed, GSH deficit has been associated with lower
survival rates in HIV-infected patients and the admin-
Ch em istr y
The new compounds were synthesized from com-
mercially available N-acetyl-S-trityl-L-cysteine, com-
pound 1. Scheme 1 outlines the synthesis of pseudo-
dipeptide intermediates 6-9, which were used to obtain
the target compounds 10-13 and their corresponding
S-acyl derivatives (14-29). We recently described some
of the biological activities of 10,23 but only a summary
of experiments carried out was given. The pseudo-
dipeptides 6-9 were obtained by a two-step “one-pot”
procedure, using 1, isobutyl chloroformate, and 4-methyl-
morpholine (NMM) in EtOAc to form a mixed anhy-
dride. This anhydride was added to N-hydroxysuccin-
* To whom correspondence should be addressed. J .O.: Tel: +33 467
14 38 37. Fax: +33 467 04 20 29. E-mail: oiryj@univ-montp2.fr. P.C.:
Tel: +33 146 54 87 69. Fax: +33 146 54 77 26. E-mail: clayette@
dsvidf.cea.fr.
† Universite´ Montpellier II.
‡ CEA/CRSSA/EPHE.
§ SPI-BIO.
10.1021/jm030374d CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/02/2004

1790 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Oiry et al.
Sch em e 1a
a
Reagents and conditions: (a) (1) AcOEt, IBC, NMM, -15 °C, (2) HOSu, -15 °C, (3) 2, 3, 4, or 5, NMM, -15 °C-rt, 12 h; (b) MeOH,
CHCl3, mixture of MeOH/AgNO3/pyridine, rt; (c) CHCl3, HCl, 37%, rt.
Sch em e 2a
Ta ble 1. ED50, ED70, and ED90 for the Anti-HIV-1/Ba-L
Activity of 10, 11, 12, 18, 21, and 31 in Human MDM
10, µM
11, µM
12, µM
18, µM
21, µM
31, µM
ED50 45 ( 10 110 ( 5 145 ( 15 45 ( 5 165 ( 25 40 ( 10
ED70 80 ( 35 175 ( 15 185 ( 25 55 ( 15 205 ( 15 75 ( 10
ED90 195 ( 135 380 ( 25 270 ( 30 80 ( 30 285 ( 15 200 ( 15
Resu lts a n d Discu ssion
In human MDM, 10 has higher pro-GSH activity and
anti-HIV activity than NAC and MEA.23 In this study,
the anti-HIV effects of 10 were confirmed with effective
doses (ED50, ED70, and ED90) of 45 ( 10 µM, 80 ( 35
µM, and 195 ( 135 µM, respectively (Table 1). With the
aim of identifying analogues with more potent pro-GSH
and antiviral activities than 10, we synthesized a series
of new molecules by (1) changing the nature of the
S-acyl group on the MEA residue (11-13) and (2)
S-acylation of the free thiol groups of 10-13 (14-29,
Scheme 1). The resulting compounds, which were theo-
retically more lipophilic than 10, might be expected to
have higher pro-GSH and anti-HIV activities. We also
assessed the pro-GSH effects of 31 the major intra-
cellular metabolite of 10.
We assessed potential GSH activity by determining
GSH levels after treatment for 24 h with 10, its thioester
analogues 11-13, with 14, 15, 16, 18, 20-24, 26-28;
or with 31 the intracellular dithiol metabolite of 10
(Figure 1). The efficacy of 10 derivatives decreased with
increasing size of R′ alkyl groups (steric hindrance).
Indeed, compounds 10-13, which had a free thiol group
on a cysteine residue, displayed greater pro-GSH prop-
erties than the corresponding S-acetyl derivatives 14,
18, 22, and 26. Similarly, these S-acetyl derivatives
were more efficient than the S-pivaloyl analogues 16,
20, 24, and 28. In contrast, the presence of an R radical
on the MEA moiety had no significant effect on the pro-
GSH activity of these compounds. Nevertheless, the
a
Reagents and conditions are identical to those for Scheme 1.
imide to generate the corresponding active O-succin-
imide ester, which was finally condensed with appropri-
ate S-acylcysteamine hydrochlorides (2-5) in the pres-
ence of NMM, to give the expected compounds. We used
this mixed method to optimize the yield of coupling
reactions. S-Detritylations of 6-9 were performed with
silver nitrate-pyridine in MeOH,24 to generate the
corresponding silver sulfides, which were treated with
concentrated HCl in CHCl3 to give 10-13. Finally, these
free thiols were S-acylated with an anhydride or acid
chloride in pyridine to give the corresponding S,S′-
diacylated derivatives, 14-29.
Dithiol 31 (Scheme 2), which has been identified as a
metabolite of 10,23 was also synthesized using com-
mercially available 1 as the starting material. In a
similar sequence to that used to obtain 10-13, 1 was
coupled with an S-alkylcysteamine (S-tritylcysteamine
hydrochloride) to give 30. Removal of the S-trityl groups,
via the corresponding silver sulfides, gave the expected
compound 31.
The Experimental Section reports general synthetic
procedures and characterization of all the new com-
pounds based on the usual analytical data.

Antioxidant and Anti-HIV Cysteamine Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1791
anti-HIV effects was identified. We therefore decided
to synthesize N-isobutyryl analogues and to assess their
anti-HIV activity; these compounds should theoretically
have no pro-GSH activity.25 We also investigated the
mode of action of 10. Preliminary results showed that
10 interfered with both early and late steps in the HIV
biological cycle.
In conclusion, although none of the new derivatives
tested displayed pro-GSH and antiviral activities greater
than those of 10, several displayed similar anti-HIV
activities. The S-substituents were ranked in order of
efficacy as follows: H g acetyl > isobutyryl > pivaloyl
> benzoyl. As these compounds would be expected to
be more lipophilic than 10, they may be able to cross
barriers such as the blood-brain barrier more easily
than 10. Such compounds may therefore be of value as
adjuvant therapies to HAART (highly active anti-
retroviral therapy), targeting neurological aspects of
HIV infection by interfering with inflammatory and
oxidative processes.
F igu r e 1. Pro-GSH activity of 10 and its analogues and
derivatives at a concentration of 150 µM in uninfected MDM.
Results are expressed as means ( SD. NC: Negative control
corresponding to untreated and uninfected MDM.
Exp er im en ta l Section
Ch em ica l Meth od s. Melting points were determined on a
Bu¨chi capillary melting point apparatus and are uncorrected.
Nuclear magnetic resonance spectra (1H NMR) were recorded
on a Bru¨ker AC 250 or DRX 400 in CDCl3. Chemical shifts
are reported in ppm and given in δ units with respect to TMS,
used as an internal standard. FAB mass spectra were recorded
on a J EOL DX 300 mass spectrophotometer in the positive
ion mode, using 1:1 glycerol/thioglycerol, unless otherwise
stated. Specific optical rotations were recorded on a Perkin-
Elmer 241 polarimeter. Elemental analysis was carried out
by the Service Central de Microanalyze du CNRS de Vernaison
(France), and the results were within (0.4% of calculated
values. Analytical thin-layer chomatography (TLC) was carried
out on Merck silica gel 60 F254 plates. Spots were visualized
by exposure to ultraviolet light (254 nm), iodine vapor, or by
spraying with ninhydrin solution. Flash column chomatogra-
phy 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-Acetyl-S-trityl-L-cysteine was obtained from Bachem. S-
Acetylcysteamine hydrochloride, S-benzoylcysteamine hydro-
chloride, and S-tritylcysteamine hydrochloride were prepared
as previously described.26,27
Gen er a l Cou p lin g P r oced u r e for 1 a n d S-Acylcyste-
a m in e. N-(N-Acet yl-S-t r it yl-L-cyst ein yl)-S-a cet ylcyst e-
a m in e (6). A stirred solution of N-acetyl-S-trityl-L-cysteine
(1, 1.18 g, 2.91 mmol) in EtOAc (15 mL) was cooled to -15 °C
and successively treated with isobutyl chloroformate (IBC, 402
µL, 3.09 mmol), 4-methylmorpholine (NMM, 352 µL, 3.25
mmol), and then (15 min) N-hydroxysuccinimide (HOSu, 350
mg, 3.04 mmol). After stirring for 30 min at -15 °C, S-
acetylcysteamine hydrochloride (2,26 495 mg, 3.19 mmol) was
added to the mixture, followed by NMM (351 µL, 3.19 mmol),
which was 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 (3 × 5 mL). The organic phases were pooled,
washed [water (2 × 20 mL), ice-cold saturated aqueous
NaHCO3 (15 mL), water (2 × 20 mL), ice-cold aqueous 10%
HCl (15 mL), and water (neutral pH)], dried over Na2SO4,
collected by filtration, and evaporated to dryness under
vacuum to give 6 as a white foam (1.15 g, 78%). TLC and NMR
analysis indicated that the product was sufficiently pure and
could be used without further purification.
F igu r e 2. Anti-HIV effects of 10 and its analogues and
derivatives at a concentration of 150 µM 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.
dithiol 31 (Scheme 2) displayed stronger pro-GSH effects
than 10 (Figure 1).
With the exception of 15, 16, and 28, all the deriva-
tives presented anti-HIV activities equivalent to that
of 10 (Figure 2), with no cytotoxic events. The introduc-
tion of various R groups did not seem to affect the
antiviral effects of the thioesters 10-13. Similarly, the
HIV-1/Ba-L activity of the dithiol 31 in human MDM
was comparable to that of 10. However, S-isobutyryl (15,
23, 27) and S-pivaloyl (16, 20, 24, 28) groups on the
cysteine residue seemed to decrease antiviral activity
with respect to the corresponding free thiol precursors
(10, 11, 12, 13) and their S-acetyl derivatives (14, 18,
22, 26). We therefore carried out an experiment to
determine more precisely the dose-dependent effects of
the most effective compoundss11, 12, 18, 21, and 31s
comparing these effects with those of 10 (Table 1). We
found that none of these derivatives was more efficient
than 10 itself.
The most efficient derivatives tested appeared to be
those with R radicals that were not too bulky. The size
of the R radical seemed to be important for both pro-
GSH and anti-HIV activities. Steric interactions linked
to the size of the R radical may decrease the efficiency
of thiol release by the enzyme, therefore reducing the
ability of derivatives to increase GSH levels and to
inhibit HIV replication. As expected, the dithiol 31
increased GSH content more efficiently than 10. How-
ever, its anti-HIV activity was no higher than that of
10. Indeed, no strict correlation between pro-GSH and
An analytical sample was obtained by recrystallization from
EtOAc and petroleum ether mixture to give white crystals: mp
111-113 °C. TLC (EtOAc/petroleum ether, 9:1) Rf ) 0.41.
1
[R]20 +10.5° (c 0.8, CHCl3). H NMR: 1.90 (s, 3H, NCOCH3),
D

1792 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Oiry et al.
2.29 (s, 3H, SCOCH3), 2.48 (dd, J ) 5.7, 12.9 Hz, 1H, â Ha
cys), 2.82 (dd, J ) 6.4, 12.9 Hz, 1H, â Hb cys), 2.92-3.01 (m,
2H, NCH2CH2S), 3.32-3.42 (m, 2H, NCH2CH2S), 4.07-4.20
(m, 1H, R H cys), 5.70 (d, J ) 7.6 Hz, 1H, NH cys), 6.34 (t, J
) 5.5 Hz, 1H, NHCH2), 7.19-7.35, 7.40-7.47 (2m, 15H, ArH).
MS m/z 507 (M + H)+. Anal. (C28H30N2O3S2) C, H, N.
Gen er a l P r oced u r e for th e S-Detr ityla tion of th e
Cyst ein e R esid u e. N-(N-Acet yl-L-cyst ein yl)-S-a cet yl-
cystea m in e (10). Compound 6 (1.15 g, 2.27 mmol) was
dissolved in MeOH (18 mL) and CHCl3 (1.2 mL). We added a
mixture of AgNO3 (465 mg, 2.73 mmol), pyridine (210 µL, 2.73
mmol), and MeOH (15 mL) at room temperature, in the dark.
The reaction mixture was then stirred for 12 h. The resulting
silver sulfide precipitate was collected by filtration, washed
with MeOH (2 × 10 mL) and CHCl3 (2 × 10 mL), and rapidly
dried under vacuum.
51%): mp 92-94 °C. TLC (CH2Cl2/Et2O, 4:6) Rf ) 0.23. [R]20
D
-11.1° (c 1.08, CHCl3). 1H NMR: 1.25 (s, 9H, C(CH3)3, 2.00
(s, 3H, NCOCH3), 2.37 (s, 3H, SCOCH3), 2.94-3.12 (m, 2H,
NCH2CH2S), 3.25 (d, J ) 6.5 Hz, 2H, â CH2 cys), 3.38-3.49
(m, 2H, NCH2CH2S), 4.51 (td, J ) 6.5, 7.3 Hz, 1H, R H cys),
6.42 (d, J ) 7.3 Hz, 1H, NH cys), 6.80 (t, J ) 5.2 Hz,1H,
NHCH2). MS m/z 697 (2M + H)+, 349 (M + H)+. Anal.
(C14H24N2O4S2) C, H, N, S.
N -(N -Ac e t y l-S -b e n zo y l-L -c y s t e in y l)-S -a c e t y lc y s t e -
a m in e (17). This compound was prepared according to the
general procedure described for 14, using 10 (110 mg, 420
µmol) and benzoyl chloride (120 µL, 1.03 mmol). The crude
product was purified by flash column chromatography (CH2Cl2/
MeOH, 9.85:0.15) to give a colorless gum. Recrystallization
from CH2Cl2 and Et2O mixture gave 17 as a white powder (75
mg, 48%): mp 157-158 °C. TLC (CH2Cl2/MeOH, 9.5:0.5) Rf )
0.44. [R]20 +5.8° (c 1.03, CHCl3). 1H NMR: 2.00 (s, 3H,
D
This product was then taken up in CHCl3 (15 mL) and
placed in the dark under argon, and concentrated HCl (400
µL) was added dropwise. The mixture was stirred for 2 h at
room temperature and then heated for 2 min at 30-35 °C.
The reaction mixture was cooled to room temperature and
diluted in CHCl3 (70 mL), and the silver chloride precipitate
was removed and washed with CHCl3 (3 × 10 mL). The
combined filtrates were rapidly washed with iced water (3 ×
10 mL), dried over Na2SO4, filtered, and evaporated to dryness
under vacuum to give a white solid that was crystallized from
EtOAc and petroleum ether mixture to yield 10 (336 mg, 56%)
as white crystals. The physicochemical characteristics of this
NCOCH3), 2.33 (s, 3H, SCOCH3), 2.98-3.08 (m, 2H, NCH2-
CH2S), 3.41-3.51 (m, 2H, NCH2CH2S), 3.49 (overlapping d, J
) 6.2 Hz, 2H, CH2 cys), 4.58-4.69 (m, 1H, R H cys), 6.55 (d, J
) 7.0 Hz, 1H, NH cys), 6.79-6.90 (m, 1H, NHCH2), 7.41-7.52,
7.57-7.66, 7.92-8.01 (3m, 5H, ArH). MS m/z 737 (2M + H)+,
369 (M + H)+. Anal. (C16H20N2O4S2) C, H, N.
N -(N -Ace t yl-S-t r it yl-L-cyst e in yl)-S-isob u t yr ylcyst e -
a m in e (7). This compound was prepared according to the
general procedure described for 6, using 1 (1.82 g, 4.5 mmol)
and S-isobutyrylcysteamine hydrochloride (3, obtained by the
procedure described for S-acetylcysteamine hydrochloride,26
mp 147-148 °C). The crude product was purified by flash
column chromatography (CH2Cl2/Et2O, 7:3) to give 7 as a
colorless foam (1.92 g, 80%). TLC (EtOAc/petroleum ether, 8:2)
molecule have been described elsewhere (mp, [R]20 1H and
D
13C NMR, MS, anal.).23
Gen er a l P r oced u r e for S-Acyla tion of th e Cystein e
Resid u e. N-(N,S-Dia cetyl-L-cystein yl)-S-a cetylcystea m -
in e (14). A stirred solution of 10 (83 mg, 314 µmol) in pyridine
(1 mL) was cooled to 0 °C and treated with an excess of acetic
anhydride (90 µL, 950 µmol). The reaction mixture was stirred
for 1 h at 0 °C, allowed to warm to room temperature, and
left to stand overnight. The solution was then evaporated to
dryness under vaccum. The oily residue was taken up in
CH2Cl2 (30 mL), washed [water (2 × 20 mL), ice-cold saturated
aqueous NaHCO3 (15 mL), water (2 × 20 mL), 1 N aqueous
citric acid (15 mL), and water (neutral pH)], dried over Na2SO4,
filtered, and evaporated to dryness under vaccum. The crude
product was then recrystallized from EtOAc to give 14 as white
Rf ) 0.37. [R]20 +10° (c 1.1, CHCl3). 1H NMR: 1.16 (d, J )
D
6.9 Hz, 6H, C(CH3)2), 1.90 (s, 3H, NCOCH3), 2.49 (dd, J ) 5.7,
12.9 Hz, 1H, â Ha cys), 2.70 (hept, J ) 6.9 Hz, 1H, CH(CH3)2),
2.79 (dd, J ) 6.4, 12.9 Hz, 1H, â Hb cys), 2.88-3.01 (m, 2H,
NCH2CH2S), 3.29-3.41 (m, 2H, NCH2CH2S), 4.08-4.19 (m,
1H, R H cys), 5.76 (d, J ) 7.7 Hz, 1H, NH cys), 6.36 (t, J ) 5.5
Hz, 1H, NHCH2), 7.15-7.35, 7.38-7.52 (2m, 15H, ArH). MS
m/z 535 (M + H)+. Anal. (C30H34N2O3S2) C, H, N.
N-(N-Acetyl-L-cystein yl)-S-isobu tyr ylcystea m in e (11).
This compound was prepared from 7 (1.27 g, 2.38 mmol)
according to the general procedure described for 10. The crude
product was purified by flash column chromatography (CH2Cl2/
MeOH; 9.85:0.15) to give 11 as a colorless gum (396 mg, 57%).
crystals (73 mg, 75%): mp 153-154 °C. TLC (CH2Cl2/MeOH,
1
9.5:0.5) Rf ) 0.46. [R]20 -33.7° (c 0.8, CHCl3). H NMR: 2.02
TLC (CH2Cl2/MeOH, 9.5:0.5) Rf ) 0.45. [R]20 -26.6° (c 1.09,
D
D
1
(s, 3H, NCOCH3), 2.37, 2.39 (2s, 2 × 3H, 2 × SCOCH3), 2.93-
3.12 (m, 2H, NCH2CH2S), 3.24 (dd, J ) 7.2, 14.5 Hz, 1H, â Ha
cys), 3.31 (dd, J ) 5.2, 14.5 Hz, 1H, â Hb cys), 3.39-3.49 (m,
2H, NCH2CH2S), 4.54 (ddd, J ) 5.3, 7.2, 7.3 Hz, 1H, R H cys),
6.44 (d, J ) 7.3 Hz, 1H, NH cys), 6.83 (t, J ) 5.1 Hz,1H,
NHCH2). MS m/z 613 (2M + H)+, 307 (M + H)+. Anal.
(C11H18N2O4S2) C, H, N, S.
CHCl3). H NMR: 1.21 (d, J ) 6.9 Hz, 6H, C(CH3)2), 1.61 (dd,
J ) 7.6, 10.3 Hz, 1H, SH), 2.09 (s, 3H, NCOCH3), 2.70 (ddd, J
) 6.4, 10.3, 13.8 Hz, 1H, â Ha cys), 2.77 (hept, J ) 6.9 Hz,
1H, CH(CH3)2), 2.99-3.08 (m, 2H, NCH2CH2S), 3.09 (ddd, J
) 4.1, 7.6, 13.8 Hz, 1H, â Hb cys), 3.41-3.53 (m, 2H, NCH2-
CH2S), 4.60 (ddd, J ) 4.1, 6.4, 7.5 Hz, 1H, R H cys), 6.48 (d, J
) 7.5 Hz, 1H, NH cys), 6.68-6.88 (m, 1H, NHCH2). MS m/z
585 (2M + H)+, 293 (M + H)+. Anal. (C11H20N2O3S2) C, H, N.
N-(N-Acet yl-S-isob u t yr yl-L-cyst ein yl)-S-a cet ylcyst e-
a m in e (15). This compound was prepared according to the
general procedure described for 14, using 10 (85 mg, 322 µmol)
and isobutyryl chloride (137 µL, 1.3 mmol). The crude product
was purified by flash column chromatography (CH2Cl2/Et2O,
8:2 to 8:3) to give a white solid. Recrystallization from EtOAc
and petroleum ether mixture gave 15 as white crystals (54
N-(N,S-Dia cet yl-L-cyst ein yl)-S-isob u t yr ylcyst ea m in e
(18). This compound was prepared according to the general
procedure described for 14, using 11 (93.4 mg, 320 µmol) and
acetic anhydride. The crude product was purified by flash
column chromatography (CH2Cl2/Et2O; 4.5:5.5) to give a color-
less gum. Trituration with hexane gave 18 as a white powder
(65 mg, 61%): mp 115-117 °C. TLC (CH2Cl2/MeOH, 9.5:0.5)
Rf ) 0.58. [R]20D -20.2° (c 1.04, CHCl3). 1H NMR: 1.20 (d, J )
6.9 Hz, 6H, C(CH3)2), 2.02 (s, 3H, NCOCH3), 2.38 (s, 3H,
SCOCH3), 2.78 (hept, J ) 6.9 Hz, 1H, CH(CH3)2), 2.96-3.06
(m, 2H, NCH2CH2S), 3.19-3.36 (m, 2H, CH2 cys), 3.38-3.48
(m, 2H, NCH2CH2S), 4.47-4.60 (m, 1H, R H cys), 6.37 (d, J )
7.1 Hz, 1H, NH cys), 6.70-6.83 (m, 1H, NHCH2). MS m/z 669
(2M + H)+, 335 (M + H)+. Anal. (C13H22N2O4S2) C, H, N.
mg, 50%): mp 116-118 °C. TLC (CH2Cl2/MeOH, 9.4:0.6) Rf )
1
0.6. [R]20 -18.4° (c 0.87, CHCl3). H NMR: 1.20 (d, J ) 6.9
D
Hz, 6H, C(CH3)2, 2.00 (s, 3H, NCOCH3), 2.37 (s, 3H, SCOCH3),
2.80 (hept, J ) 6.9 Hz, 1H, CH(CH3)2, 2.93-3.12 (m, 2H,
NCH2CH2S), 3.23-3.30 (m, 2H, â CH2 cys), 3.38-3.49 (m, 2H,
NCH2CH2S), 4.46-4.57 (m, 1H, R H cys), 6.42 (d, J ) 7.3 Hz,
1H, NH cys), 6.80 (t, J ) 5.2 Hz, 1H, NHCH2). MS m/z 669
(2M + H)+, 335 (M + H)+. Anal. (C13H22N2O4S2) C, H, N, S.
N -(N -Ace t yl-S -p iva loyl-L -cyst e in yl)-S -a ce t ylcyst e -
a m in e (16). This compound was prepared according to the
general procedure described for 14, using 10 (95 mg, 360 µmol)
and pivaloyl chloride (176 µL, 1.44 mmol). The crude product
was purified by flash column chromatography (CH2Cl2/Et2O,
1:1) to give a white solid. Recrystallization from EtOAc and
petroleum ether mixture gave 16 as white crystals (64 mg,
N -(N -Ace t yl-S -isob u t yr yl-L-cyst e in yl)-S -isob u t yr yl-
cystea m in e (19). This compound was prepared according to
the general procedure described for 14, using 11 (93.4 mg, 320
µmol) and isobutyryl chloride. The crude product was purified
by flash column chromatography (CH2Cl2/Et2O, 1:1) to give a
colorless gum. Trituration with hexane gave 19 as a white
powder (70 mg, 60%): mp 99-100 °C. TLC (CH2Cl2/Et2O, 3.5:

Antioxidant and Anti-HIV Cysteamine Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1793
6.5) Rf ) 0.34. [R]20 -9.1° (c 0.88, CHCl3). 1H NMR: (1.20 (d,
1H NMR: 1.24 (s, 9H, C(CH3)3), 2.03 (s, 3H, NCOCH3), 2.38
(s, 3H, SCOCH3), 2.94-3.04 (m, 2H, NCH2CH2S), 3.18-3.36
(m, 2H, CH2 cys), 3.36-3.48 (m, 2H, NCH2CH2S), 4.48-4.60
(m, 1H, R H cys), 6.40 (d, J ) 7.5 Hz, 1H, NH cys), 6.71-6.83
(m, 1H, NHCH2). MS m/z 697 (2M + H)+, 349 (M + H)+. Anal.
(C14H24N2O4S2) C, H, N.
D
J ) 6.9 Hz, 12H, C(CH3)2), 2.01 (s, 3H, NCOCH3), 2.78 (hept,
J ) 6.9 Hz, 2H, CH(CH3)2), 2.92-3.10 (m, 2H, NCH2CH2S),
3.26 (d, J ) 6.4 Hz, 2H, CH2 cys), 3.37-3.48 (m, 2H, NCH2-
CH2S), 4.46-4.58 (m, 1H, R H cys), 6.38 (d, J ) 7.3 Hz, 1H,
NH cys), 6.73-6.83 (m, 1H, NHCH2). MS m/z 725 (2M + H)+,
363 (M + H)+. Anal. (C15H26N2O4S2) C, H, N.
N-(N-Acetyl-S-isobu tyr yl-L-cystein yl)-S-p iva loylcyste-
a m in e (23). This compound was prepared according to the
general procedure described for 14, using 12 (98 mg, 320 µmol)
and isobutyryl chloride. The crude product (gum) was tritu-
rated with hexane to give pure 23 (67 mg, 56%) as a white
powder: mp 101-102 °C. TLC (CH2Cl2/Et2O, 3.5:6.5) Rf ) 0.46.
N-(N-Acetyl-S-p iva loyl-L-cystein yl)-S-isobu tyr ylcyste-
a m in e (20). This compound was prepared according to the
general procedure described for 14, using 11 (93.4 mg, 320
µmol) and pivaloyl chloride. The crude product was purified
by flash column chromatography (CH2Cl2/Et2O; 1:1) to give a
colorless gum. Recrystallization from EtOAc and petroleum
ether mixture gave 20 as white needles (77 mg, 64%): mp
[R]20 -5.7° (c 1.05, CHCl3). 1H NMR: 1.20 (d, J ) 6.9 Hz,
D
6H, C(CH3)2), 1.25 (s, 9H, C(CH3)3), 2.01 (s, 3H, NCOCH3), 2.79
(hept, J ) 6.9 Hz, 1H, CH(CH3)2), 2.90-3.08 (m, 2H,
NCH2CH2S), 3.25 (d, J ) 6.3 Hz, 2H, CH2 cys), 3.35-3.47 (m,
2H, NCH2CH2S), 4.46-4.58 (m, 1H, R H cys), 6.38 (d, J ) 7.1
Hz, 1H, NH cys), 6.71-6.81 (m, 1H, NHCH2). MS m/z 753 (2M
+ H)+, 377 (M + H)+. Anal. (C16H28N2O4S2) C, H, N.
103-104 °C. TLC (CH2Cl2/Et2O, 1:1) Rf ) 0.27. [R]20 -8.3° (c
D
0.97, CHCl3). 1H NMR: 1.20 (d, J ) 6.9 Hz, 6H, C(CH3)2), 1.25
(s, 9H, C(CH3)3), 2.01 (s, 3H, NCOCH3), 2.77 (hept, J ) 6.9
Hz, 1H, CH(CH3)2), 2.94-3.08 (m, 2H, NCH2CH2S), 3.25 (d, J
) 6.4 Hz, 2H, CH2 cys), 3.37-3.49 (m, 2H, NCH2CH2S), 4.44-
4.57 (m, 1H, R H cys), 6.35 (d, J ) 7.3 Hz, 1H, NH cys), 6.69-
6.80 (m, 1H, NHCH2). MS m/z 753 (2M + H)+, 377 (M + H)+.
Anal. (C16H28N2O4S2) C, H, N.
N-(N-Acet yl-S-p iva loyl-L-cyst ein yl)-S-p iva loylcyst e-
a m in e (24). This compound was prepared according to the
general procedure described for 14, using 12 (104 mg, 340
µmol) and pivaloyl chloride. The crude product (gum) was
triturated with hexane to give a white powder. Recrystalliza-
tion from EtOAc and petroleum ether mixture gave 24 as white
N-(N-Acetyl-S-ben zoyl-L-cystein yl)-S-isobu tyr ylcyste-
a m in e (21). This compound was prepared according to the
general procedure described for 14, using 11 (93.4 mg, 320
µmol) and benzoyl chloride. The crude product was purified
by flash column chromatography (CH2Cl2/Et2O; 6.5:3.5) to give
a colorless gum. Trituration with hexane gave 21 as a white
crystals (80 mg, 60%): mp 109-111 °C. TLC (CH2Cl2/Et2O,
1
1:1) Rf ) 0.36. [R]20 -4.4° (c 0.91, CHCl3). H NMR: 1.24 (s,
D
18H, C(CH3)3), 2.00 (s, 3H, NCOCH3), 2.90-3.08 (m, 2H,
NCH2CH2S), 3.24 (d, J ) 6.4 Hz, 2H, CH2 cys), 3.36-3.47 (m,
2H, NCH2CH2S), 4.44-4.57 (m, 1H, R H cys), 6.38 (d, J ) 7.4
Hz, 1H, NH cys), 6.68-6.88 (m, 1H, NHCH2). MS m/z 781 (2M
+ H)+, 391 (M + H)+. Anal. (C17H30N2O4S2) C, H, N.
powder (76 mg, 60%): mp 137-138 °C. TLC (CH2Cl2/Et2O, 1:1)
1
Rf ) 0.27. [R]20 +2.8° (c 1.08, CHCl3). H NMR: 1.18 (d, J )
D
6.9 Hz, 6H, C(CH3)2), 2.02 (s, 3H, NCOCH3), 2.73 (hept, J )
6,9 Hz, 1H, CH(CH3)2), 2.96-3.06 (m, 2H, NCH2CH2S), 3.39-
3.49 (m, 2H, NCH2CH2S), 3.50 (d, J ) 6.2 Hz, 2H, CH2 cys),
4.60-4.72 (m, 1H, R H cys), 6.59 (d, J ) 7.3 Hz, 1H, NH cys),
6.85-6.97 (m, 1H, NHCH2), 7.41-7.52, 7.57-7.65, 7.93-8.01
(3m, 5H, ArH). MS m/z 793 (2M + H)+, 397 (M + H)+. Anal.
(C18H24N2O4S2) C, H, N.
N-(N-Acet yl-S-b en zoyl-L-cyst ein yl)-S-p iva loylcyst e-
a m in e (25). This compound was prepared according to the
general procedure described for 14, using 12 (104 mg, 340
µmol) and benzoyl chloride. The crude product was purified
by flash column chromatography (CH2Cl2/Et2O; 6.5:3.5) to give
a colorless gum. Trituration with hexane gave 25 as a white
N -(N -Ac e t y l-S -t r it y l-L -c y s t e in y l)-S -p iv a lo y lc y s t e -
a m in e (8). This compound was prepared according to the
general procedure described for 6, using 1 (3 g, 7.41 mmol)
and S-pivaloylcysteamine hydrochloride (4, obtained by the
procedure described for S-acetylcysteamine hydrochloride,26
mp 212-213 °C). The crude product was purified by flash
column chromatography (CH2Cl2/Et2O, 7:3) to give 8 as a
colorless foam (3.49 g, 86%). TLC (EtOAc/petroleum ether, 7:3)
powder (89 mg, 64%): mp 133-134 °C. TLC (CH2Cl2/Et2O, 1:1)
1
Rf ) 0.33. [R]20 +5.1° (c 0.98, CHCl3). H NMR: 1.21 (s, 9H,
D
C(CH3)3), 2.01 (s, 3H, NCOCH3), 2.90-3.08 (m, 2H, NCH2CH2S),
3.33-3.52 (m, 4H, NCH2CH2S, CH2 cys), 4.64-4.77 (m, 1H, R
H cys), 6.76 (d, J ) 7.4 Hz, 1H, NH cys), 7.06-7.22 (m, 1H,
NHCH2), 7.40-7.50, 7.55-7.63, 7.91-8.0 (3m, 5H, ArH). MS
m/z 821 (2M + H)+, 411 (M + H)+. Anal. (C19H26N2O4S2) C, H,
N.
Rf ) 0.5. [R]20 +8.5° (c 1.29, CHCl3). 1H NMR: 1.21 (s, 9H,
D
C(CH3)3), 1.90 (s, 3H, NCOCH3), 2.49 (dd, J ) 5.9, 12.9 Hz,
1H, â Ha cys), 2.78 (dd, J ) 6.5, 12.9 Hz, 1H, â Hb cys), 2.88-
2.98 (m, 2H, NCH2CH2S), 3.28-3.40 (m, 2H, NCH2CH2S),
4.06-4.19 (m, 1H, R H cys), 5.77 (d, J ) 7.6 Hz, 1H, NH cys),
6.27-6.41 (m, 1H, NHCH2), 7.16-7.35, 7.40-7.48 (2m, 15H,
ArH). MS m/z 549 (M + H)+. Anal. (C31H36N2O3S2) C, H, N.
N-(N-Acetyl-L-cystein yl)-S-pivaloylcysteam in e (12). This
compound was prepared from 8 (2.5 g, 4.57 mmol) according
to the general procedure described for 10. The crude product
was purified by flash column chromatography (CH2Cl2/MeOH,
9.85:0.15) to give 12 as a colorless gum (839 mg, 60%). TLC
(CH2Cl2/MeOH, 9.5:0.5) Rf ) 0.49. [R]20D -20.2° (c 0.94, CHCl3).
1H NMR: 1.24 (s, 9H, C(CH3)3), 1.61 (dd, J ) 7.6, 10.3 Hz,
1H, SH), 2.08 (s, 3H, NCOCH3), 2.70 (ddd, J ) 6.4, 10.3, 13.9
Hz, 1H, â Ha cys), 2.97-3.09 (m, 2H, NCH2CH2S), 3.08
(overlapping ddd, J ) 4.1, 7.6, 13.9 Hz, 1H, â Hb cys), 3.40-
3.52 (m, 2H, NCH2CH2S), 4.60 (ddd, J ) 4.1, 6.4, 7.8 Hz, 1H,
R H cys), 6.49 (d, J ) 7.8 Hz, 1H, NH cys), 6.69-6.82 (m, 1H,
NHCH2). MS m/z 613 (2M + H)+, 307 (M + H)+. Anal.
(C12H22N2O3S2) C, H, N.
N-(N,S-Diacetyl-L-cystein yl)-S-pivaloylcysteam in e (22).
This compound was prepared according to the general proce-
dure described for 14, using 12 (101 mg, 330 µmol) and acetic
anhydride. The crude product was purified by flash column
chromatography (CH2Cl2/Et2O, 4.5:5.5) to give a colorless gum.
Recrystallization from EtOAc and petroleum ether mixture
gave 22 as white needles (77 mg, 67%): mp 112-114 °C. TLC
(CH2Cl2/MeOH, 9.5:0.5) Rf ) 0.58. [R]20D -13.8° (c 0.94, CHCl3).
N -(N -Ac e t y l-S -t r it y l-L -c y s t e in y l)-S -b e n zo y lc y s t e -
a m in e (9). This compound was prepared according to the
general procedure described for 6, using 1 (3 g, 7.41 mmol)
and S-benzoylcysteamine hydrochloride (5).26 The crude prod-
uct was purified by flash column chromatography (CH2Cl2/
Et2O, 8.5:1.5) to give 9 as a colorless foam (2.61 g, 62%). TLC
(AcOEt/petroleum ether, 7:3) Rf ) 0.42. [R]20 +10.8° (c 1.11,
D
CHCl3). 1H NMR: 1.86 (s, 3H, NCOCH3), 2.47 (dd, J ) 5.7,
13.0 Hz, 1H, â Ha cys), 2.82 (dd, J ) 6.4, 13.0 Hz, 1H, â Hb
cys), 3.08-3.27 (m, 2H, NCH2CH2S), 3.41-3.53 (m, 2H, NCH2-
CH2S), 4.08-4.21 (m, 1H, R H cys), 5.67 (d, J ) 7.7 Hz, 1H,
NH cys), 6.34-6.46 (m, 1H, NHCH2), 7.17-7.32, 7.37-7.45,
7.54-7.61, 7.89-7.96 (4m, 20H, ArH). MS m/z 569 (M + H)+.
Anal. (C33H32N2O3S2) C, H, N.
N-(N-Acetyl-L-cystein yl)-S-ben zoylcysteam in e (13). This
compound was prepared from 9 (2.52 g, 4.44 mmol) according
to the general procedure described for 10. The crude product
was purified by flash column chromatography (EtOAc/petro-
leum ether, 1:1) to give 13 as a white solid (912 mg, 63%):
mp 128-130 °C. TLC (CH2Cl2/MeOH, 9.5:0.5) Rf ) 0.38. [R]20
D
1
-24.7° (c 1.01, CHCl3). H NMR: 1.59 (dd, J ) 7.6; 10.2 Hz,
1H, SH), 2.04 (s, 3H, NCOCH3), 2.71 (ddd, J ) 6.5, 10.2, 13.8
Hz, 1H, â Ha cys), 3.06 (ddd, J ) 4.3, 7.6, 13.8 Hz, 1H, â Hb
cys), 3.20-3.31 (m, 2H, NCH2CH2S), 3.52-3.64 (m, 2H, NCH2-
CH2S), 4.61 (ddd, J ) 4.3, 6.5, 7.4 Hz, 1H, R H cys), 6.51 (d, J
) 7.4 Hz, 1H, NH cys), 6.83-7.00 (m, 1H, NHCH2), 7.43-7.52,
7.56-7.65, 7.92-8.00 (3m, 5H, ArH). MS m/z 653 (2M + H)+,
327 (M + H)+. Anal. (C14H18N2O3S2) C, H, N.

1794 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Oiry et al.
N-(N,S-Dia cetyl-L-cystein yl)-S-ben zoylcystea m in e (26).
This compound was prepared according to the general proce-
dure described for 14, using 13 (111 mg, 340 µmol) and acetic
anhydride. The crude product was purified by flash column
chromatography (CH2Cl2/MeOH, 9.85:0.15) to give a colorless
gum. Recrystallization from EtOAc and petroleum ether
mixture gave 26 as white needles (88 mg, 70%): mp 166-168
by flash column chromatography (CH2Cl2/MeOH, 9.8:0.2) to
give 31 as a white powder (70 mg, 18%): mp 133-135 °C. TLC
(CH2Cl2/MeOH, 9.7:0.3) Rf ) 0.32. [R]20D -50.0° (c 1.2, CHCl3).
1H NMR: 1.44 (t, J ) 8.5 Hz, 1H, SH cysteamine), 1.71 (dd, J
) 7.8, 10.0 Hz, 1H, SH cys), 2.07 (s, 3H, NCOCH3), 2.62-2.82
(m, 3H, â Ha cys, NCH2CH2S), 3.06 (ddd, J ) 4.5, 7.6, 13.8
Hz, 1H, â Hb cys), 3.36-3.59 (m, 2H, NCH2CH2S), 4.53-4.66
(m, 1H, R H cys), 6.53 (d, J ) 7.0 Hz, 1H, NH cys), 6.77-6.91
(m, 1H, NHCH2). MS (NBA) m/z 445 (2M + H)+, 223 (M +
H)+. Anal. (C7H14N2O4S2) C, H, N, S.
Biologica l Meth od s. Isola tion of Mon ocyte-Der ived
Ma cr op h a ges (MDM). Human peripheral blood mononuclear
cells (PBMC) were obtained from healthy HIV-, HCV-, and
HBV-seronegative donors by Ficoll-Hypaque density gradient
centrifugation (MSL 2000, Eurobio, Les Ulis, France). Mono-
cytes were isolated from PBMC by countercurrent centrifugal
elutriation, as previously described, with a degree of enrich-
ment g95%.28 Freshly isolated human monocytes were resus-
pended in medium A: Dulbecco’s Modified Eagle Medium
(DMEM) Glutamax (Roche Products, Meylan, France) supple-
mented with 10% heat-inactivated (56 °C for 30 min) fetal calf
serum (FCS) (Roche Products), and 1% antibiotic cocktail
(penicillin, streptomycin, neomycin, PSN; InVitroGen). Cells
were dispensed (one million cells per well) into 48-well plates
(BD BioSciences, Lincoln Park, NJ ) and maintained for 7 days
in a humidified 5% CO2 atmosphere to allow their differentia-
tion into MDM. Cell culture media were endotoxin-free, as
shown by the Limulus Amebocyte Lysate test (LAL; Sigma
Chemical Co, Saint Quentin-Fallavier, France). Differences in
morphological appearance and the cell surface expression of
various molecules, such as HLA-DR and CD16, have been
described elsewhere.29
Dr u gs. NAC and MEA (Sigma), 10, and its derivatives or
analogues were immediately dissolved in dimethyl sulfoxide
(DMSO, Sigma) and stored at -80 °C. Since several of the
molecules (17, 19, 25, 29) were insoluble, they were not
subjected to biological testing. The pH of drug solutions was
adjusted to 7.0 with 3 M NaOH if necessary.
Tota l In tr a cellu la r GSH Levels. Total intracellular gluta-
thione levels were determined by the Griffith’s colorimetric
method (Cayman Chemical Company, Ann Arbor, MI)30 in
uninfected MDM, after treatment for 24 h. In parallel, we
determined the protein content of cell lysates by Bradford’s
method (BioRad, Marnes la Coquette, France). One million
uninfected MDM were treated for 24 h, washed with PBS, and
lysed by incubation with 150 µL of 0.1% Tween 20 in PBS for
1 h.
Vir u s. MDM were infected with the reference macrophage-
tropic HIV-1/BaL strain.31 This virus was propagated in vitro
in human phytohemagglutinin (PHA)-P-activated umbilical
blood mononuclear cells (UBMC). To eliminate soluble fac-
tors such as cytokines, the cell-free UMBC culture supernatant
was ultracentrifuged at 360 000g for 5 min and the pellet
resuspended in DMEM. This viral preparation was titrated
on PHA-P-activated-PBMC. The 50% tissue culture infectious
dose (TCID50) and the multiplicity of infection (moi) were
calculated using Ka¨rber’s formula32 and Poisson’s law, respec-
tively.
An tivir a l Assa y. One million MDM were pretreated for 1
h with various concentrations of compounds and infected with
10 000 TCID50 (0.01 multiplicity of infection) of the HIV-1/
Ba-L strain. The MDM were then incubated for 24 h at 37 °C,
washed once to eliminate excess virus, and fed with fresh
medium A. Twice per week we removed supernatants, which
were then frozen and stored at -20 °C for later evaluation of
viral replication. Cell culture medium and drugs were then
renewed, and cells were observed under a microscope to assess
the possible cytotoxicity of the drugs. HIV replication was
measured by quantifying reverse transcriptase (RT) activity
in cell culture supernatants. RT activity was determined using
the RT RetroSys kit, according to the manufacturer’s
(Innovagen) instructions. The kinetics of RT activity in the cell
culture supernatants of HIV-infected MDM were as previously
reported.33,34
°C. TLC (CH2Cl2/MeOH, 9.5:0.5) Rf ) 0.44. [R]20 -14.3° (c
D
0.98, CHCl3). 1H NMR: 1.99 (s, 3H, NCOCH3), 2.32 (s, 3H,
SCOCH3), 3.18-3.31 (m, 4H, NCH2CH2S, CH2 cys), 3.48-3.61
(m, 2H, NCH2CH2S), 4.49-4.62 (m, 1H, R H cys), 6.38 (d, J )
7.6 Hz, 1H, NH cys), 6.79-6.92 (m, 1H, NHCH2), 7.42-7.51,
7.55-7.64, 7.93-8.01 (3m, 5H, ArH). MS m/z 737 (2M + H)+,
369 (M + H)+. Anal. (C16H20N2O4S2) C, H, N.
N-(N-Acetyl-S-isobu tyr yl-L-cystein yl)-S-ben zoylcyste-
a m in e (27). This compound was prepared according to the
general procedure described for 14, using 13 (98 mg, 300 µmol)
and isobutyryl chloride. The crude product was purified by
flash column chromatography (CH2Cl2/Et2O, 6:4) to give a
colorless gum. Recrystallization from EtOAc and petroleum
ether mixture gave 27 as white crystals (88 mg, 74%): mp
135-136 °C. TLC (CH2Cl2/Et2O, 3:7) Rf ) 0.2. [R]20 -6.7° (c
D
1
1.2, CHCl3). H NMR: 1.17 (d, J ) 6.9 Hz, 6H, C(CH3)2), 1.97
(s, 3H, NCOCH3), 2.75 (hept, J ) 6.9 Hz, 1H, CH(CH3)2), 3.19-
3.30 (m, 4H, NCH2CH2S, CH2 cys), 3.46-3.62 (m, 2H, NCH2-
CH2S), 4.49-4.61 (m, 1H, R H cys), 6.41 (d, J ) 7.4 Hz, 1H,
NH cys), 6.85-6.96 (m, 1H, NHCH2), 7.41-7.52, 7.55-7.64,
7.90-8.02 (3m, 5H, ArH). MS m/z 793 (2M + H)+, 397 (M +
H)+. Anal. (C18H24N2O4S2) C, H, N.
N-(N-Acet yl-S-p iva loyl-L-cyst ein yl)-S-b en zoylcyst e-
a m in e (28). This compound was prepared according to the
general procedure described for 14, using 13 (98 mg, 300 µmol)
and pivaloyl chloride. The crude product was purified by flash
column chromatography (CH2Cl2/Et2O, 5.5:4.5) to give a color-
less gum. Recrystallization from EtOAc and petroleum ether
mixture gave 28 as white crystals (95 mg, 77%): mp 101-
103 °C. TLC (CH2Cl2/Et2O, 3:7) Rf ) 0.3. [R]20 -3.8° (c 1.05,
D
CHCl3). 1H NMR: 1.22 (s, 9H, C(CH3)3), 1.96 (s, 3H, NCOCH3),
3.19-3.30 (m, 4H, NCH2CH2S, CH2 cys), 3.46-3.62 (m, 2H,
NCH2CH2S), 4.47-4.58 (m, 1H, R H cys), 6.38 (d, J ) 7.2 Hz,
1H, NH cys), 6.81-6.92 (m, 1H, NHCH2), 7.42-7.52, 7.55-
7.65, 7.90-8.02 (3m, 5H, ArH). MS m/z 821 (2M + H)+, 411
(M + H)+. Anal. (C19H26N2O4S2) C, H, N.
N -(N -Ace t yl-S -b e n zoyl-L-cyst e in yl)-S -b e n zoylcyst e -
a m in e (29). This compound was prepared according to the
general procedure described for 14, using 13 (98 mg, 300 µmol)
and benzoyl chloride. The crude product was purified by flash
column chromatography (CH2Cl2/MeOH, 9.9:0.1) to give a
colorless gum. Recrystallization from EtOAc gave 29 as white
crystals (85 mg, 66%): mp 188-190 °C. TLC (CH2Cl2/MeOH,
1
9.5:0.5) Rf ) 0.66. [R]20 +4.1° (c 0.98, CHCl3). H NMR: 1.99
D
(s, 3H, NCOCH3), 3.19-3.28 (m, 2H, NCH2CH2S), 3.49 (d, J
) 6.1 Hz, 2H, CH2 cys), 3.52-3.61 (m, 2H, NCH2CH2S), 4.63-
4.71 (m, 1H, R H cys), 6.56 (d, J ) 7,2 Hz, 1H, NH cys), 6.92-
7.08 (m, 1H, NHCH2), 7.38-7.48, 7.52-7.62, 7.88-7.97 (3m,
10H, ArH). MS m/z 861 (2M + H)+, 431 (M + H)+. Anal.
(C21H22N2O4S2) C, H, N.
N-(N-Acetyl-S-tr ityl-L-cystein yl)-S-tr itylcysteam in e (30).
This compound was prepared according to the general proce-
dure described for 6, using 1 (1 g, 2.47 mmol) and S-
tritylcysteamine hydrochloride.27 Trituration of the crude
product (foam) with EtOAc at 0 °C gave pure 30 (1.26 g, 72%)
as a white powder: mp 193-195 °C. TLC (CH2Cl2/MeOH, 9.8:
1
0.2) Rf ) 0.5. [R]20 +7.2° (c 0.97, CHCl3). H NMR: 1.87 (s,
D
3H, NCOCH3), 2.33 (t, J ) 6.6 Hz, 2H, NCH2CH2S) 2.47 (dd,
J ) 6.0, 13.0 Hz, 1H, â Ha cys), 2.68 (dd, J ) 6.7, 13.0 Hz, 1H,
â Hb cys), 2.90-3.01 (m, 2H, NCH2CH2S), 3.92-4.06 (m, 1H,
R H cys), 5.74 (d, J ) 7.6 Hz, 1H, NH cys), 5.88 (t, J ) 5.5 Hz,
1H, NHCH2), 7.18-7.32, 7.34-7.46 (2m, 30H, ArH). MS (NBA)
m/z 707 (M + H)+. Anal. (C45H42N2O2S2) C, H, N.
N-(N-Acetyl-L-cystein yl)cystea m in e (31). This compound
was prepared from 30 (1.26 g, 1.78 mmol) and 2.1 mol equiv
of AgNO3 and pyridine in MeOH, according to the general
procedure described for 10. The crude product was purified

Antioxidant and Anti-HIV Cysteamine Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1795
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Da ta An a lysis. All experiments were performed in tripli-
cate and repeated with cells isolated from a second blood donor.
Results are expressed as the mean ( standard deviation (SD).
For the antiviral assay, cumulative RT activity - the sum of
RT activity obtained for each time-point in each individual cell
culture well was used to calculate percentages with respect
to untreated control and 50, 70, and 90% effective doses. To
calculate the percentages with respect to the untreated control,
the results obtained for treated MDM cultures were divided
by the untreated control value. Effective doses were deter-
mined on the basis of cumulative RT activity using computer
software (J . and T. C. Chou, Biosoft, Cambridge, UK). For the
GSH assay, intracellular GSH contents were expressed in
nanomoles per milligram of protein.
Ack n ow led gm en t. This work was supported by
grants from the Centre de Recherches de Service de
Sante´ des Arme´es Emile Parde´ (CRSSA, La Tronche,
France) and the Commissariat a` l’Energie Atomique
(CEA, Paris, France).
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