Synthesis and in vitro anti-HIV activity in human monocyte-derived macrophages of 2-oxothiazolidine-4(R)-carboxylic acid derivatives

Article abstract of DOI:10.1021/jm980289j

Oxidative stress and glutathione (GSH) deficit may play an important role in HIV infection pathogenesis, and oral administration of GSH- replenishing drugs such as N-acetylcysteine (NAC) and 2-oxothiazolidine-4(R)- carboxylic acid (OTC) may be associated

Full text of DOI:10.1021/jm980289j

© Copyright 1999 by the American Chemical Society
Volume 42, Number 23
November 18, 1999
Articles
Syn th esis a n d in Vitr o An ti-HIV Activity in Hu m a n Mon ocyte-Der ived
Ma cr op h a ges of 2-Oxoth ia zolid in e-4(R)-ca r boxylic Acid Der iva tives
J oe¨l Oiry,*,† J ean-Yves Puy,† Patricia Mialocq,‡ Pascal Clayette,*,‡,§ Philippe Fretier,‡ Philippe J accard,‡
Nathalie Dereuddre-Bosquet,‡,§ Dominique Dormont,‡ and J ean-Louis Imbach†
Laboratoire de Chimie Bio-Organique, CNRS UMR 5625, Universite´ Montpellier II, Sciences et Techniques du Languedoc,
place Euge`ne-Bataillon, 34095 Montpellier Cedex 5, France, CEA, Service de Neurovirologie, DSV/ DRM, CRSSA, 60-68 avenue
de la Division Leclerc, B.P. 6, 92265 Fontenay aux Roses Cedex, France, and SPI-BIO, 2 rue du Buisson aux Fraises, Z.I. de la
Bonde, 91741 Massy Cedex, France
Received May 8, 1998
Oxidative stress and glutathione (GSH) deficit may play an important role in HIV infection
pathogenesis, and oral administration of GSH-replenishing drugs such as N-acetylcysteine
(NAC) and 2-oxothiazolidine-4(R)-carboxylic acid (OTC) may be associated with an increased
survival rate of HIV-infected patients. Nevertheless, beneficial effects of these molecules are
restricted in vivo by the high concentrations that are necessary to obtain biological effects,
rapid extracellular metabolization, and low availability and plasma concentrations. We
synthesized OTC derivatives that are more lipophilic than OTC and theoretically able to
overcome these limitations and to generate, in addition to cysteine, other substrates of the
γ-glutamyl cycle. Their antiviral effects were investigated in human HIV-1/Ba-L-infected
monocyte-derived macrophages. In our experimental conditions, OTC exhibited anti-HIV-1
effects and little cytotoxicity at high doses. None of the nine tested derivatives showed higher
cytotoxicity than OTC, nor anti-HIV-1/Ba-L activity.
In tr od u ction
precursors, amino acid transport, and maintenance of
sulfhydryl redox status. It also plays a central role in
the antioxidant defense system, protecting cells against
free radicals and toxic compounds of endogenous and
exogenous origin.1
The production of highly reactive oxygen intermedi-
ates (ROI) is counter-balanced by antioxidants in physi-
ological conditions, and oxidative stress results from an
imbalance between pro- and anti-oxidant molecules.
Glutathione (GSH) is a cysteine-containing tripeptide
(γ-glutamylcysteinylglycine) found in all eukaryotic
cells. It is synthesized and degraded intracellularly,
mainly via its reduced form. GSH participates in several
cell functions, such as synthesis of proteins and DNA
HIV-infected individuals have decreased levels of
acid-soluble thiols, particularly GSH, in tissue, e.g. lung
and central nervous system (CNS), in plasma, and in
CD4+ T lymphocytes and monocytes.2-5 This GSH
deficit and the oxidative stress that follows may par-
ticipate in human immunodeficiency virus (HIV)6 infec-
tion pathogenesis, by enhancing viral replication, CD4+
T lymphocyte deficiency,7 inflammatory syndrome, apo-
ptosis, chronic weight loss, and drug toxicities and by
decreasing immune cell proliferative response and im-
mune functions.8,9 HIV replication is probably potenti-
* To whom correspondence should be addressed. Chemistry: J .O.:
phone, +33 (0)4 67 14 38 37; fax, +33 (0)4 67 04 20 29; e-mail,
oiryj@crit.univ-montp2.fr. Biology: P.C.: phone, +33 (0)1 46 54 87 69;
fax, +33 (0)1 46 54 77 26; e-mail, clayette@dsvidf.cea.fr.
† Universite´ Montpellier II.
‡ CEA.
§ SPI-BIO.
10.1021/jm980289j CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/02/1999

4734 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Oiry et al.
Sch em e 1a
ated through NF-κB transcription factor activation10 and
subsequent induction of inflammatory cytokines, e.g.
tumor necrosis factor R (TNF-R).11
Administration of N-acetylcysteine (NAC) increases
GSH levels and is associated with decreased mortality
in HIV-infected patients.12 In vitro, GSH and NAC
inhibit viral replication in monocyte-derived macroph-
ages (MDM) and/or lymphocytes.13-15 These compounds
suppress the activation of the transcription factor NF-
κB. However, the direct inhibition of the reverse tran-
scriptase (RT) activity may be an alternative mecha-
nism.16 As a consequence, beneficial effects may be
expected by compensating GSH levels in HIV-infected
patients. Nevertheless, the efficiency of GSH and NAC
is restricted by the high concentrations that are neces-
sary to obtain biological effects,11 their rapid oxidation
and hydrolysis by intestinal and hepatic γ-glutamyl-
transferase,17 and their low oral bioavailability.18,19
Moreover, the rate of intracellular GSH synthesis is also
limited by the availability of cysteine. 2-Oxothiazolidine-
4-carboxylic acid (OTC, procysteine) is converted to
cysteine by the intracellular 5-oxoprolinase enzyme20
and is able to overcome these limitations. Indeed, oral
OTC administration results in effective intracellular
delivery of cysteine and increased cellular GSH.20-22
Nevertheless, in the light of the short half-life of OTC,23
OTC derivatives were synthesized, and their in vitro
antiretroviral activity was investigated in primary
cultures of HIV-1/Ba-L-infected MDM. Theoretically,
these derivatives are more lipophilic and therefore could
be less susceptible than OTC to extracellular metabo-
lization. They could generate, in addition to cysteine,
one or two other substrates of the γ-glutamyl cycle, i.e.
glycine and glutamic acid, and so could facilitate GSH
synthesis. Antiviral effects on macrophages were evalu-
ated because these cells or related cells such as micro-
glia are the major source of ROI and have a powerful
antioxidant system which is supported by efficient
uptake of cysteine.24 They are one of the main HIV
targets involved in the inflammatory process and AIDS
encephalopathy25,26 and alter GSH level in response to
HIV infection.27
a
Reagents: (a) TEA, acetone, then acetyl chloride, 0 °C to rt;
(b) phenacyl bromide, TEA, DMF, rt; (c) Boc-Glu(OSu)-OBut, TEA,
DMF and DMAP for 4, rt to 50 °C; (d) AcOH, H2O, Zn, rt; (e) 4 M
HCl/AcOH, 0 °C.
DMAP. It is noteworthy that the use of DMAP is
essential to maximize the yield of this coupling. The
phenacyl ester was removed, according to the previously
described method,33,34 by adding a severalfold molar
excess of zinc powder at room temperature to a solution
of 4 in dilute aqueous acetic acid. The resulting pseudo-
dipeptide 5 was then deprotected at 0 °C by treatment
with 4 M HCl in glacial acetic acid to give pure 6 as the
hydrochloride salt.
Ch em istr y
The syntheses of compounds 2, 3, 5-7, and 9 are
outlined in Scheme 1. Compound 2 has been synthesized
previously by heating of 1 with acetic anhydride in
52.1% yield, but two other byproducts [2-oxo-3-(2′-
oxothiazolidin-4′(R)-ylcarbonyl)thiazolidine-4(R)-carbox-
ylic acid and 1,4,5,8-tetraoxo-2,6-dithia-4a,8a-diazaper-
hydro-s-indacene] were also isolated by fractional
recrystallizations.28 To increase the yield of 2, and also
to simplify isolation, we introduced the N-acyl group by
treatment of 1 in acetone, with acetyl chloride in the
presence of TEA. Thus, 2 was easily obtained after flash
chromatography and crystallization in 82% yield.
For the synthesis of 6, we used the phenacyl ester as
a temporary protecting carboxyl function of 1, to avoid
the side reactions during subsequent coupling. Thus, 3
was obtained by reaction of phenacyl bromide with 1 in
the presence of TEA according to a classical method.29-32
The preparation of 4 was accomplished in DMF for 2
days at 50 °C, by coupling of 3 with active ester Boc-L-
Glu(OSu)-OBut in the presence of TEA and catalytic
In a similar sequence to that described above, 9 was
prepared from OTC ethyl ester 7 which was obtained,
according to a published procedure, by one-pot reac-
35
tion of L-cysteine ethyl ester with 1,1′-carbonyldiimida-
zole. Briefly, 7 was coupled with Boc-L-Glu(OSu)-OBut
in the presence of TEA to give 8. N,O-protecting groups
of glutamyl residue were then removed to afford the
expected compound as the hydrochloride salt.
The synthetic strategy for pseudo-dipeptides 10 and
11 and pseudo-tripeptide 13 is illustrated in Scheme 2.
Condensation of 1 with Gly-OBut‚HCl in DMF and TEA
by the DCC/HOBt method36 gave the desired derivative
10. Removal of the tert-butyl protecting group with 4
M HCl in glacial acetic acid (30 min/0 °C, 2.5 h/room
temperature) followed by lyophilization and trituration
with ether afforded pure 11. Through a reaction similar
to that used for the preparation of 9, 10 was coupled in
acetonitrile with Boc-L-Glu(OSu)-OBut in the presence
of TEA (1 h/0 °C, 12 h/room temperature) to give 12.

Synthesis and Anti-HIV Activity of OTC Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4735
Sch em e 2a
the reference compounds (Table 1). Neither the chemical
modifications nor the delivery of additional substrates
to the γ-glutamyl cycle increased cytotoxicity or antiviral
activity. On the contrary, compound 13, which theoreti-
cally released most γ-glutamyl cycle substrates by
generating OTC, glycine, and glutamic acid, increased
viral replication (cumulative RT activity: compound 13
159 ( 45% of untreated control vs untreated control 100
( 12%).
Taken together, these data do not support the use of
these GSH-replenishing drugs as monotherapy in HIV
infection. Nevertheless, these drugs are of value in
adjuvant therapy of HAART,43 because of their probable
beneficial effects in restoration of the immune system
and their ability to attenuate HIV-associated inflam-
matory disorders and to increase survival rate of HIV-
infected patients.12 Consequently, synthesis and anti-
viral evaluation of new cyclized derivatives with greater
bioavailability are now in progress.
a
Reagents: (a) HOBt, DCC, DMF, 0 °C, then Gly-OBut‚HCl,
TEA/DMF, 0 °C to rt; (b) 4
M HCl/AcOH, 0 °C to rt; (c)
Boc-Glu(OSu)-OBut, TEA, acetonitrile, 0 °C to rt.
Final compound 13 was isolated, as the hydrochloride
salt, after removal of Boc and But protecting groups of
glycyl and glutamyl residues, as already described.
Exp er im en ta l Section
Ch em ica l Meth od s. Melting points were determined in a
Bu¨chi capillary melting point apparatus, and are uncorrected.
IR spectra were recorded on a Beckman Acculab 4 spectro-
photometer; absorbances are reported in ν (cm-1). NMR spectra
were recorded on a Varian EM 390 spectrometer unless
otherwise stated, in which case a Bruker AC 250 instrument
was used. Chemical shifts are reported in ppm and given in δ
units relative to TMS as internal standard. Specific optical
rotations were recorded on a Perkin-Elmer 241 polarimeter.
Elemental analyses were performed by the Service de Micro-
analyse de l’Ecole Nationale Supe´rieure de Chimie de Mont-
pellier and were within ( 0.4% of calculated values. Analytical
thin-layer chromatography (TLC) was carried out on Merck
silica gel 60 F254 plates. Spots were visualized by ultraviolet
light (254 nm), by iodine vapor, or by spraying with ninhydrin
solution. Flash column chromatography was conducted with
Merck silica gel 60 (230-400 mesh ASTM). All the solvents
used were dried and purified in the usual manner. OTC was
from Aldrich. Amino acid derivatives were from Bachem.
3-Acetyl-2-oxoth ia zolid in e-4(R)-ca r boxylic Acid (2). A
stirred solution of 2-oxothiazolidine-4(R)-carboxylic acid (OTC,
1; 1.47 g, 10 mmol) and triethylamine (TEA; 3.63 mL, 26 mmol)
in acetone (50 mL) was cooled to 0 °C and then treated
dropwise with 0.782 mL of acetyl chloride (11 mmol). The
reaction mixture was stirred for 1 h at 0 °C and at room
temperature over 12 h. The alkaline solution was then reduced
to an oil by evaporation in vacuo. This oil was taken up in
water (10 mL), acidified to pH 3 with 2 M HCl, and extracted
with ethyl acetate (5 × 30 mL). The combined extracts were
dried over Na2SO4, filtered, and evaporated to dryness in
vacuo. The crude product was then purified by flash column
chromatography (eluent: CH2Cl2/MeOH/AcOH, 9.5:0.4:0.08)
and crystallized from ethyl acetate/petroleum ether to afford
1.56 g (82%) of 2 as white crystals: Rf 0.6 (CH2Cl2/MeOH/
AcOH, 8:2:1); mp 153-154 °C; [R]D20 ) -140.5° (c 1.8 acetone/
H2O, 1/1) [lit.28 mp 152-154 °C; [R]D20 ) -136.8° (c 1.8 acetone/
H2O, 1/1)]; 1H NMR (CDCl3) 2.52 (s, 3H, acetyl CH3), 3.41 (dd,
J ) 1.9, 11.8 Hz, 1H, H5′), 3.68 (dd, J ) 8.8, 11.8 Hz, 1H, H5),
5.23 (dd, J ) 1.9, 8.8 Hz, 1H, H4), 9.03 (br, 1H, CO2H).
2-Oxoth ia zolid in e-4(R)-ca r boxylic Acid P h en a cyl Es-
ter (3). To a stirred solution of 1 (1.61 g, 11 mmol) in DMF
(30 mL) were added R-bromoacetophenone (phenacyl bromide;
3.28 g, 16.5 mmol) and TEA (1.53 mL, 11 mmol). A white
precipitate formed within a few minutes and the suspension
was stirred overnight at room temperature. The reaction
mixture was filtered and the filtrate was reduced to an oil by
evaporation in vacuo. This oil was diluted with ethyl acetate
(200 mL) and washed with water (3 × 100 mL). The organic
phase was dried over Na2SO4, filtered, and evaporated to
dryness in vacuo. The crude product was then crystallized from
Biologica l Resu lts a n d Discu ssion
The redox imbalance or oxidative stress associated
with HIV infection may be one of the mechanisms by
which this retrovirus promotes its own replication.
Moreover, this oxidative stress plays a part in patho-
genesis and in survival impairment in HIV disease.12
This supports the in vitro evaluation of effects on cell
survival and viral replication of nine OTC derivatives.
The anti-HIV effects of compounds 3, 5-7, 9-11, and
13 were compared to those of OTC and those of
compound 2 to those of OTC and NAC. This N-acetyl-
ated OTC derivative compound is able to generate NAC
(Scheme 1).
HIV-1/Ba-L replicated efficiently in MDM (Figure 1)
as previously described.37 Cell death was observed in
these HIV-1/Ba-L-infected MDM cultures when viral
replication was maximal between 10 and 14 days after
infection.37 In these experimental conditions, OTC and
NAC induced slight cytotoxicity in uninfected cells when
used at high doses, 20 and 15 mM, respectively. These
two molecules dose-dependently inhibited HIV-1 repli-
cation (Figures 1, 2) and favored cell survival of HIV-
1/Ba-L-infected MDM. The ED50 was 7.5 and 2 mM for
NAC and OTC, respectively. These results are in ac-
cordance with those of Ho et al.38,39 and Lioy et al.40 As
also observed by Ho et al.,38,39 the synthesis of HIV
provirus was reduced by NAC (60% inhibition with 0.5
mM NAC compared with untreated control). On the
other hand, no inhibition of RT activity was observed
in an acellular system (data not shown). NAC signifi-
cantly increased HIV replication in one experiment
(Figure 3A,B). These deleterious effects may be related
to an increased HIV particle production, i.e. 15 mM NAC
dose (Figure 3A). Nottet et al.41 associated these effects
with hydroxyl radical overexpression and Chen et al.42
with cell-cell interactions. In our experiment with this
blood cell donor, contaminating lymphocytes persisted
in MDM cultures and may have favored this increase
in viral replication.
None of the new tested derivatives showed higher
cytotoxicity nor higher anti-HIV-1/Ba-L activity than

4736 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Oiry et al.
F igu r e 1. Antiviral activity of NAC and OTC in HIV-1/Ba-L-infected MDM. Results are expressed as mean ( SD and are
representative of three (OTC) or four (NAC) blood donors.
2-Oxo-3-[r-[N-(ter t-bu toxyca r bon yl)-O-(ter t-bu tyl)]-L-
γ-glu ta m yl]th ia zolid in e-4(R)-ca r boxylic Acid P h en a cyl
Ester (4). To a stirred solution of 3 (2.65 g, 10 mmol) in DMF
(80 mL) were added N-(tert-butoxycarbonyl)-L-glutamic acid
R-tert-butyl γ-N-succinimidyl ester (4 g, 10 mmol), TEA (1.39
mL, 10 mmol), and 4-(dimethylamino)pyridine (DMAP; 50 mg)
at room temperature. The mixture was then heated for 2 days
at 50 °C and concentrated to dryness in vacuo. After cooling,
the resulting precipitate was filtered and the filtrate was then
concentrated to dryness in vacuo. The residual paste was
diluted with CH2Cl2 (300 mL) and washed consecutively with
water, ice-cold saturated aqueous sodium bicarbonate, water,
1 N aqueous citric acid, and water (neutral pH). The organic
phase was dried over Na2SO4, filtered, and evaporated to
dryness in vacuo. The crude product (oil) was then purified
by flash column chromatography [eluent: stepwise gradient
of ether (40-70%) in petroleum ether] and crystallized from
ether/petroleum ether to afford 2.69 g (65%) of 4 as white
needles: Rf 0.4 (ethyl acetate/petroleum ether, 3:7); mp 83-
20
85 °C; [R]D ) -87.7° (c 1.14 CHCl3); IR (Nujol mull) 3380,
1760, 1710, 1690, 1500, 1450, 1365, 1230, 1170; 1H NMR
(CDCl3) 1.42, 1.45 (2s, 18H, t- butyl H), 1.76-1.93, 2.13-2.30
(2m, 2H, â-Glu CHa, CHb), 2.91-3.08 (m, 2H, γ-Glu CH2),
3.72-3.80 (m, 2H, H5, 5′), 4.15-4.28 (m, 1H, R-Glu CH), 5.10
(d, J ) 8 Hz, 1H, NH), 5.27 (d, J ) 16.3 Hz, 1H, phenacyl
CHa), 5.27-5.36 (m, 1H, H4 overlapping with the dd at 5.27),
5.65 (d, J ) 16.3 Hz, 1H, phenacyl CHb), 7.43-7.53, 7.57-
7.66, 7.84-7.92 (3m, 5H, ArH). Anal. (C26H34N2O9S) C, H, N.
F igu r e 2. Dose-effect of antiviral activity of NAC and OTC
in HIV-1/Ba-L-infected MDM. Cumulative RT activities were
considered to calculate the percentages of untreated control.
Results are expressed as mean ( SD and are representative
of three (OTC) or four (NAC) blood donors.
ethyl acetate/petroleum ether to afford 2.62 g (90%) of 3 as
white needles: Rf 0.6 (ethyl acetate/petroleum ether, 7:3); mp
2-Oxo-3-[r-[N-(ter t-b u t oxyca r b on yl)-O-(ter t-b u t yl)]-L-
γ-glu ta m yl]th ia zolid in e-4(R)-ca r boxylic Acid (5). A solu-
tion of 4 (1.5 g, 2.72 mmol) in a mixture of acetic acid (230
mL) and water (40 mL) was stirred at room temperature and
Zn powder (22.1 g, 0.34 mol) was then added in one portion.
The reaction, followed by TLC, was over in 2 h. The reaction
mixture was filtered and the filtrate was evaporated to dryness
in vacuo. The crude product was diluted with aqueous 0.1 M
20
124-125 °C; [R]D ) -29.9° (c 1.17 CHCl3); IR (Nujol mull)
3200, 1755, 1675, 1655, 1445, 1205; 1H NMR (CDCl3) 3.78 (dd,
J ) 7.7, 11.4 Hz, 1H, H5), 3.83 (dd, J ) 5.5, 11.4 Hz, 1H, H5′),
4.64 (ddd, J ) 0.9, 5.5, 7.7 Hz, 1H, H4), 5.39 (d, J ) 16.3 Hz,
1H, phenacyl CHa), 5.54 (d, J ) 16.3 Hz, 1H, phenacyl CHb),
6.16 (br, 1H, NH), 7.46-7.53, 7.59-7.66 and 7.86-7.91 (3m,
5H, ArH). Anal. (C12H11NO4S) C, H, N.

Synthesis and Anti-HIV Activity of OTC Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4737
F igu r e 3. Deleterious effects of NAC on HIV-1/Ba-L replication in MDM (A, kinetic of viral replication; B, dose-effect). Results
are expressed as mean ( SD. In panel B, cumulative RT activities were considered to calculate the percentages of untreated
control.
Ta ble 1. Antiviral Activity of OTC Derivatives in HIV-1/
ice-cold anhydrous ether (100 mL), and the ether phase was
then decanted. This procedure of washing with ether was
performed three times. The crude product was then dried in a
vacuum desiccator containing KOH pellets and P2O5 to afford
639 mg (91%) of 6 as an analytically pure white powder: Rf
Ba-L-Infected MDMa
percent of untreated control (%)
compd
at 0.5 mM
at 2 mM
20
untreated control
NAC
OTC, 1
100 ( 7
100 ( 12
88 ( 5
0.4 (acetonitrile/water, 7:3); mp 184-186 °C; [R]D ) -77.1°
(c 0.7 MeOH); IR (Nujol mull) 3230-2460, 1725, 1715, 1690,
1585, 1515, 1390, 1230; 1H NMR (D2O) 2.24-2.48 (m, 2H,
â-Glu CH2), 3.14-3.41 (m, 2H, γ-Glu CH2), 3.58 (dd, J ) 2.0,
11.9 Hz, 1H, H5′), 3.92 (dd, J ) 9.0, 11.9 Hz, 1H, H5), 4.19 (t,
J ) 6.7 Hz, 1H, R-Glu CH), 5.34 (dd, J ) 2.0, 9.0 Hz, 1H, H4).
Anal. (C9H12N2O6S‚HCl) C, H, N, Cl.
81 ( 16
69 ( 12
37 ( 17
34 ( 21
2
3b
4c
5
102 ( 26
83 ( 15
86 ( 39
6
2-Oxot h ia zolid in e-4(R)-ca r b oxylic Acid E t h yl E st er
(7). The synthesis of 7 was carried out as described by D’Ischia
7
8c
9
35
37 ( 10
104 ( 15
45 ( 29
et al:
yield 90% (oil); Rf 0.4 (ether/petroleum ether, 8:2);
[R]D ) -52.8° (c 0.89 CHCl3) [lit.35 [R]D ) -55.9° (c 2.1
CHCl3)]; 1H NMR (CDCl3) 1.31 (t, J ) 7.1 Hz, 3H, ethyl CH3),
3.62 (dd, J ) 5.5, 11.3 Hz, 1H, H5′), 3.71 (dd, J ) 7.8, 11.3
Hz, 1H, H5), 4.27 (q, J ) 7.1 Hz, 2H, ethyl CH2), 4.42 (ddd, J
) 0.8, 5.5, 8.6 Hz, 1H, H4), 6.03 (br, 1H, NH).
20
20
10
11
12c
13
159 ( 45
a
Cumulative RT activities were considered to calculate the
percentages of untreated control. Results are expressed as mean
2-Oxo-3-[r-[N-(ter t-b u t oxyca r b on yl)-O-(ter t-b u t yl)]-L-
γ-glu ta m yl]th ia zolid in e-4(R)-ca r boxylic Acid Eth yl Es-
ter (8). This compound was prepared from 7 (2.3 g, 13.1 mmol)
in DMF (60 mL), N-(tert-butoxycarbonyl)-L-glutamic acid
R-tert-butyl γ-N-succinimidyl ester (4.6 g, 11.5 mmol), and TEA
(1.83 mL, 13.1 mmol), following the same procedure as
described for 4. The pure product was obtained as a colorless
b
( SD and are representative of two blood donors. Compound 3
was cytotoxic at 2 mM and was tested at 0.5 mM. c Compounds 4,
8, and 12 were insoluble in solvent concentrations compatible with
cell culture.
EDTA disodium salt dihydrate (150 mL) and extracted with
ethyl acetate (3 × 80 mL). The organic phases were pooled,
dried over Na2SO4, filtered, and evaporated to dryness in
vacuo. The crude product was then crystallized from ethyl
acetate/petroleum ether to afford 970 mg (82%) of 5 as white
crystals: Rf 0.5 (CH2Cl2/MeOH/AcOH, 8.8:0.7:0.5); mp 172-
20
paste (2.7 g, 51%): Rf 0.69 (ether/petroleum ether, 7:3); [R]D
) -61.2° (c 0.98 CHCl3); IR (film) 3380, 1740, 1720, 1705, 1690,
1500, 1370, 1220, 1150; H NMR (CDCl3) 1.29 (t, J ) 7.1 Hz,
1
3H, ethyl CH3), 1.42, 1.45 (2s, 18H, tert-butyl H), 1.75-1.92,
2.13-2.31 (2m, 2H, â-Glu CHa, CHb), 2.89-3.06 (m, 2H, γ-Glu
CH2), 3.35 (dd, J ) 1.7, 11.7 Hz, 1H, H5′), 3.67 (dd, J ) 8.6,
11.7 Hz, 1H, H5), 4.16-4.36 (m, 3H, ethyl CH2, R-Glu CH),
5.10 (d, J ) 8.1 Hz, 1H, Glu NH), 5.19 (dd, J ) 1.7, 8.6 Hz,
1H, H4). Anal. (C20H32N2O8S) C, H, N.
20
174 °C dec; [R]D ) -61.4° (c 1.14 CHCl3); IR (Nujol mull)
3350, 3330-2480, 1755, 1740, 1690, 1660, 1535, 1390, 1220,
1150; 1H NMR (DMSO-d6 + D2O) 1.33, 1.35 (2s, 18H, tert-butyl
H), 1.65-2.0 (m, 2H, â-Glu CH2), 2.70-2.97 (m, 2H, γ-Glu
CH2), 3.32 (dd, J ) 1.0, 12 Hz, 1H, H5′), 3.62-3.86 (m, 2H,
H5, R-Glu CH), 5.05 (dd, J ) 1.0, 4.7 Hz, 1H, H4). Anal.
(C18H28N2O8S) C, H, N.
2-Oxo-3-(L-γ-glu tam yl)th iazolidin e-4(R)-car boxylic Acid
Eth yl Ester Hyd r och lor id e (9). This compound was pre-
pared from 8 (1.62 g, 3.52 mmol) in 19 mL of 4 M HCl in glacial
acetic acid, following the same procedure as described for 6,
2-Oxo-3-(L-γ-glu tam yl)th iazolidin e-4(R)-car boxylic Acid
Hyd r och lor id e (6). A solution of 5 (970 mg, 2.24 mmol) in 7
mL of 4 M HCl in glacial acetic acid was stirred at 0 °C under
nitrogen. The reaction, followed by TLC, was over in 3 h. The
hydrochloride was precipitated from the mixture by adding
to afford 1 g (83%) of 9 as analytically pure white crystals: Rf
20
0.6 (CH2Cl2/MeOH/AcOH, 8:2:1); mp 150-152 °C; [R]D
)

4738 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
-80.6° (c 0.98 MeOH); IR (Nujol mull) 3400-2450, 1735, 1715,
Oiry et al.
mmol) in 15 mL of 4 M HCl in glacial acetic acid was stirred
at 0 °C under nitrogen for 30 min and at room temperature.
The reaction, followed by TLC, was over in 2 h. The mixture
was then worked up as described for 6. After drying, the crude
hydrochloride was recrystallized from methanol and ether to
1
1700, 1690, 1500, 1370, 1220; H NMR (D2O) 1.30 (t, J ) 7.1
Hz, 3H, ethyl CH3), 2.16-2.37 (m, 2H, â-Glu CH2), 3.05-3.34
(m, 2H, γ-Glu CH2), 3.53 (dd, J ) 2.0, 12.0 Hz, 1H, H5′), 3.85
(dd, J ) 8.9, 12.0 Hz, 1H, H5), 4.06 (t, J ) 6.7 Hz, 1H, R-Glu
CH), 4.32 (q, J ) 7.1 Hz, 3H, ethyl CH2), 5.34 (dd, J ) 2.0, 8.9
Hz, 1H, H4). Anal. (C11H16N2O6S‚HCl) C, H, N, Cl.
afford 1.22 g (80%) of 13 as white flakes: Rf 0.6 (2-propanol/
20
ammonium hydroxide 30%, 7:3); mp 175-178 °C; [R]D
)
-104.3° (c 0.92 MeOH); IR (Nujol mull) 3330-2470, 1725,
1715, 1690, 1660, 1550, 1515, 1390, 1220; 1H NMR (D2O) 2.25-
2.44 (m, 2H, â-Glu CH2), 3.17-3.31 (m, 2H, γ-Glu CH2), 3.52
(dd, J ) 2.4, 11.9 Hz, 1H, H5′), 3.94 (dd, J ) 9.2, 11.9 Hz, 1H,
H5), 4.07-4.24 (m, 3H, Gly CH2, R-Glu CH), 5.37 (dd, J ) 2.4,
9.2 Hz, 1H, H4). Anal. (C11H15N3O7S‚HCl) C, H, N, Cl.
N-(2-Oxoth ia zolid in -4(R)-ylca r bon yl)glycin e ter t-Bu -
tyl Ester (10). To a cold (0 °C) stirred solution of 1 (3 g, 34
mmol) in DMF (200 mL) were added 1-hydroxybenzotriazole
(HOBt; 4.8 g, 35.5 mmol) and N,N′-dicyclohexylcarbodiimide
(DCC; 7 g, 34 mmol). After 30 min of stirring at 0 °C, glycine
tert-butyl ester hydrochloride (7 g, 41,8 mmol) was added to
the mixture, followed by the dropwise addition of TEA (8 mL,
57 mmol) in DMF (10 mL). Stirring was continued at 0 °C for
2 h and at 60 °C for 48 h. After cooling, the mixture was
worked up as described for 4. The crude product was purified
by flash column chromatography [eluent: stepwise gradient
of MeOH (0-2%) in CH2Cl2] to afford 4.1 g (46%) of 10 as a
white solid. TLC [Rf 0.4 (CH2Cl2/MeOH, 9.5:0.5)] and NMR
analysis indicated that the product was over 95% pure and
can be used in the next step without further purification.
Biologica l Meth od s. 1. Mon ocyte-Der ived Ma cr op h a ge
Isola tion . Human peripheral blood mononuclear cells (PBMC)
were obtained from healthy HIV-, HCV-, and HBV-seronega-
tive donors by Ficoll-Hypaque density gradient centrifugation
(MSL 2000, Eurobio, Les Ulis, France). Monocytes were
isolated from PBMC by countercurrent centrifugal elutriation
as previously described with an enrichment degree g 95%.37,44
Freshly isolated human monocytes were resuspended in
medium A: RPMI 1640 medium (Roche Diagnostics, Man-
nheim, Germany) supplemented with 10% heat-inactivated (56
°C for 30 min) fetal calf serum (FCS) (Roche Diagnostics), 2
mM L-glutamine (Roche Diagnostics), and 1% triantibiotic
mixture (penicillin, streptomycin, neomycin, PSN; Life Tech-
nologies, Grand Island, NY). Cells were distributed (1 million/
well) into 48-well plates (Becton Dickinson Labware, Lincoln
Park, NJ ) and maintained for 7 days in a humidified 5% CO2
atmosphere to allow their differentiation in MDM.
2. Dr u gs. NAC and OTC (1) from Sigma (Saint Quentin-
Fallavier, France) were solubilized in RPMI 1640 or dimethyl
sulfoxide (DMSO; Sigma). The OTC derivatives were solubi-
lized in RPMI 1640 (5, 7, 10, 13) or DMSO (2, 6, 9, 11, 13)
and stored at -80 °C. Solubilization of several molecules was
difficult (3) or impossible (4, 8, 12). The pH of drug solutions
was adjusted to 7.0 using 3 M NaOH when necessary.
An analytical sample was obtained, by recrystallization from
ethyl acetate and petroleum ether, as white crystals: mp 106-
20
107 °C; [R]D ) -84.9° (c 0.73 CHCl3); IR (Nujol mull) 3280,
1730, 1680, 1660, 1540, 1360, 1220, 1150; 1H NMR (CDCl3)
1.45 (s, 9H, t- butyl H), 3.60 (dd, J ) 4.8, 11.4 Hz, 1H, H5′),
3.76 (dd, J ) 8.7, 11.4 Hz, 1H, H5), 3.89 (dd, J ) 5.2, 18.0 Hz,
1H, Gly CHa), 4.0 (dd, J ) 5.7, 18.0 Hz, 1H, Gly CHb), 4.38
(dd, J ) 4.8, 8.7 Hz, 1H, H4), 6.84 (br, 1H, NH), 7.03 (br, 1H,
Gly NH). Anal. (C10H16N2O4S) C, H, N.
N-(2-Oxot h ia zolid in -4(R)-ylca r b on yl)glycin e (11). A
solution of 10 (2.5 g, 9.61 mmol) in 13 mL of 4 M HCl in glacial
acetic acid was stirred at 0 °C under nitrogen for 30 min and
at room temperature. The reaction, followed by TLC, was over
in 3 h. The mixture was then lyophilized. The residual paste
was dissolved in distilled water (30 mL), washed with CH2Cl2
(2 × 20 mL), and again lyophilized to afford a gum. This was
triturated with anhydrous ether to yield pure 11 as an
analytically pure white powder (1.89 g, 96%): Rf 0.6 (2-
propanol/ammonium hydroxide 30%, 7.5:2.5), 0.45 (acetoni-
trile/H2O, 8:2); mp 165-167 °C; [R]D20 ) -87.4° (c 0.87 MeOH);
IR (Nujol mull) 3450-2450, 1715, 1660, 1640, 1550, 1535,
1400, 1210; 1H NMR (D2O) 3.66 (dd, J ) 4.8, 11.6 Hz, 1H, H5′),
4.01 (dd, J ) 9.1, 11.5 Hz, 1H, H5), 4.16 (s, 2H, Gly CH2), 4.74
(dd, J ) 4.8, 9.1 Hz, 1H, H4). Anal. (C6H8N2O4S) C, H, N.
N-[2-Oxo-3-[r-[N-(ter t-bu toxyca r bon yl)-O-(ter t-bu tyl)]-
L-γ-glu t a m yl]t h ia zolid in -4(R)-ylca r b on yl]glycin e ter t-
Bu tyl Ester (12). To a cold (0 °C) stirred solution of 10 (3.36
g, 12.9 mmol) in acetonitrile (150 mL) were added N-(tert-
butoxycarbonyl)-L-glutamic acid R-tert-butyl γ-N-succinimidyl
ester (5.2 g, 13 mmol) and TEA (1.8 mL, 12.9 mmol). The
solution was stirred for 1 h at 0 °C, and at room temperature
for 12 h. The mixture was then worked up as described for 4.
The crude product was purified by flash column chromatog-
raphy [eluent: stepwise gradient of ether (0-6%) in CH2Cl2]
to afford 3.52 g (50%) of 12 as a colorless paste. TLC [Rf 0.65
(CH2Cl2/MeOH, 9.65:0.35)] and NMR analysis indicated that
the product was over 95% pure and can be used in the next
step without further purification.
3. Vir u s. MDM were infected with reference macrophage-
tropic HIV-1/Ba-L strain.45 This virus was propagated in vitro
using human phytohemagglutinin (PHA)-P-activated umbilical
blood mononuclear cells (UBMC). To eliminate soluble factors
such as cytokines, the cell-free UMBC culture supernatant was
ultracentrifuged at 360000g for 5 min and resuspended in
RPMI 1640. This viral preparation was titrated using PHA-
P-activated PBMC; 50% tissue culture infectious doses (TCID50)
and multiplicity of infection (moi) were calculated using
46
Ka¨rber’s formula and Poisson’s law, respectively.
4. An tivir a l Assa y. One million MDM were pretreated for
1 h with different concentrations of compounds and infected
with 10 000 TCID50 (0.01 moi) of the HIV-1/Ba-L strain; 24 h
later, MDM were washed once to eliminate excess virus and
fed with fresh medium A. Twice a week, supernatants were
removed and stored at -20 °C in order to measure viral
replication. Cell culture medium and drugs were then renewed,
and cells were microscopically observed to assess possible drug-
induced cytotoxicity.
5. Assa y of Vir a l Rep lica tion . HIV replication was
assessed by the assay of RT activity in cell culture superna-
tants, as described elsewhere.47
6. Da ta An a lysis. All experiments were repeated with cells
isolated from a second blood donor. Results are expressed as
the mean of RT activity ( standard deviation (SD). Cumulative
RT activities were used to calculate the percentages of
untreated control and 50% effective doses (ED50). To calculate
the percentages of untreated control, results obtained in
cultures with treated MDM were normalized to the untreated
control value. The ED50 was calculated using cumulative RT
activity and microcomputer software (J . and T. C. Chou,
Biosoft, Cambridge, U.K.).
An analytical sample was obtained, by crystallization from
20
CH2Cl2 and hexane, as white crystals: mp 67-69 °C; [R]D
)
-73.7° (c 1.14 CHCl3); IR (Nujol mull) 3290, 3265, 1730, 1710,
1700, 1680, 1540, 1500, 1360, 1230, 1150; 1H NMR (CDCl3)
1.42, 1.45 (2s, 27H, t- butyl H), 1.76-2.0, 2.20-2.37 (2m, 2H,
â-Glu CHa, CHb), 2.90-3.04 (m, 2H, γ-Glu CH2), 3.60 (dd, J
) 7.6, 11.1 Hz, 1H, H5), 3.66 (dd, J ) 1.7, 11.1 Hz, 1H, H5′),
3.89 (dd, J ) 4.8, 18.3 Hz, 1H, Gly CHa), 4.0 (dd, J ) 5.3, 18.3
Hz, 1H, Gly CHb), 4.16-4.29 (m, 1H, R-Glu CH), 5.08-5.16
(m, 1H, Glu NH overlapping with the dd at 5.18), 5.18 (dd, J
) 1.7, 7.6 Hz, 1H, H4), 6.76-6.83 (m, 1H, Gly NH). Anal.
(C24H39N3O9S) C, H, N.
Refer en ces
N-[2-Oxo-3-(L-γ-glu ta m yl)th ia zolid in -4(R)-ylca r bon yl]-
glycin e Hyd r och lor id e (13). A solution of 12 (2.26 g, 4.14
(1) Meister, A.; Anderson, M. E. Glutathione. Annu. Rev. Biochem.
1983, 52, 711-760.

Synthesis and Anti-HIV Activity of OTC Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4739
(2) Staal, F. J . T.; Ela, S. W.; Roederer, M.; Anderson, M. T.;
Herzenberg, L. A.; Herzenberg, L. A. Glutathione deficiency and
human immunodeficiency virus infection. Lancet 1992, 339,
909-912.
(3) Aukrust, P.; Svardal, A. M.; Mu¨ller, F.; Lunden, B.; Berge, R.
K.; Ueland, P. M.; Froland, S. S. Increased levels of oxidized
glutathione in CD4+ lymphocytes associated with disturbed
intracellular redox balance in human immunodeficiency virus
type 1 infection. Blood 1995, 86, 258-267.
(4) Castagna, A.; Le Grazie, C.; Accordini, A.; Giulidori, P.; Cavalli,
G.; Bottiglieri, T.; Lazzarin, A. Cerebrospinal fluid S-adenosyl-
methionine (SAMe) and glutathione concentrations in HIV
infection: effect of parenteral treatment with SAMe. Neurology
1995, 45, 1678-1683.
(23) Kalayjian, R. C.; Skowron, G.; Emgushov, R.-T.; Chance, M.;
Spell, S. A.; Borum, P. R.; Webb, L. S.; Mayer, K. H.; J ackson,
J . B.; Yen-Lieberman, B.; Story, K. O.; Rowe, W. B.; Thompson,
K.; Goldberg, D.; Trimbo, S.; Lederman, M. M. A phase I/II trial
of intravenous L-2-oxothiazolidine-4-carboxylic acid (procysteine)
in asymptomatic HIV-infected subjects. J . Acquired Immune Def.
Syndr. 1994, 7, 369-374.
(24) Auger, M. J .; Ross, J . A. The biology of the macrophage. In The
macrophage; Lewid, C. E., McGee, J . O. Eds.; Oxford University
Press: New York, 1992; pp 1-74.
(25) Collman, R.; Hassan, N. F.; Walker, B.; Godfrey, B.; Cutilli, J .;
Freidman, H.; Douglas, S. D.; Nathanson, N. Infection of
monocyte-derived macrophages with human immunodeficiency
virus type 1 (HIV-1). J . Exp. Med. 1989, 170.
(5) Helbling, B.; Von Overbeck, J .; Lauterburg, B. H. Decreased
release of glutathione into the systemic circulation of patients
with HIV infection. Eur. J . Clin. Invest. 1996, 26, 38-44.
(6) Barre´-Sinoussi, F.; Chermann, J . C.; Rey, F.; Chamaret, S.;
Gruest, J .; Dauguet, C.; Axler-blin, C.; Vezinet-Brun, F.; Rouzi-
oux, C.; Rozenbaum, W.; Montagnier, L. Isolation of T-lympho-
tropic retrovirus from a patient at risk for acquired immunode-
ficiency syndrome (AIDS). Science 1983, 220, 868-871.
(7) Eck, H.-P.; Mertens, T.; Rosokat, H.; Fa¨tkenheuer, G.; Pohl, C.;
Schrappe, M.; Daniel, V.; Na¨her, H.; Petzoldt, D.; Drings, P.;
Dro¨ge, W. T4+ cell numbers are correlated with plasma glutamate
and cystine levels: association of hyper glutamatæmia with
immunodeficiency in diseases with different ætiologies. Intern.
Immunol. 1992, 4, 7-13.
(8) Pace, G. W.; Leaf, C. D. The role of oxidative stress in HIV
disease. Free Radical Biol. Med. 1995, 19, 523-528.
(9) Marchetti, P.; Decaudin, D.; Macho, A.; Zamzami, N.; Hirsch,
T.; Susin, S. A.; Kroemer, G. Redox regulation of apoptosis:
impact of thiol oxidation status on mitochondrial function. Eur.
J . Immunol. 1997, 27, 289-296.
(26) Gendelman, H. E.; Orenstein, J . M.; Baca, L. M.; Weiser, B.;
Burgers, H.; Kalter, D. C.; Meltzer, M. S. The macrophage in
persistence and pathogensis of HIV infection. AIDS 1989, 3,
475-495.
(27) Rœderer, M.; Ela, S. W.; Staal, F. J . T.; Herzenberg, L. A.;
Herzenberg, L. A. N-Acetylcysteine: a new approach to anti-
HIV therapy. AIDS Res. Hum. Retroviruses 1992, 8, 209-217.
(28) Kaneko, T.; Shimokobe, T.; Ota, Y.; Toyokawa, E.; Inui, T.; Shiba,
T. Synthesis and properties of 2-oxothiazolidine-4-carboxylic acid
and its derivatives. Bull. Chem. Soc. J pn. 1964, 37, 242-244.
(29) Sheehan, J . C.; Daves, G. D., J r. Facile alkyl-oxygen ester
cleavage. J . Org. Chem. 1964, 29, 2006-2008.
(30) Stelakatos, G. C.; Paganou, A.; Zervas, L. New methods in
peptide synthesis. Part III. Protection of carbonyl group. J .
Chem. Soc. 1966, 1191-1199.
(31) Taylor-Papadimitriou, J .; Yovanidis, C.; Paganou, A.; Zervas, L.
New methods in peptide synthesis. Part V. On R- and γ-diphenyl-
methyl and phenacyl esters of L-glutamic acid. J . Chem. Soc.
1967, 1830-1836.
(32) Yang, C. C.; Merrifield, R. B. The â-phenacyl ester as
a
(10) Mihm, S.; Ennen, J .; Pessara, U.; Kurth, R.; Dro¨ge, W. Inhibition
of HIV-1 replication and NF-κB activity by cysteine and cysteine
derivatives. AIDS 1991, 5, 497-503.
(11) Israe¨l, N.; Gougerot-Pocidalo, M. A. Oxidative stress in human
immunodeficiency virus infection. Cell. Mol. Life Sci. 1997, 53,
864-970.
(12) Herzenberg, L. A.; De Rosa, S. C.; Dubs, J . G.; Rœderer, M.;
Anderson, M. T.; Ela, S. W.; Deresinski, S. C.; Herzenberg, L.
A. Glutathione deficiency is associated with impaired survival
in HIV disease. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1967-
1972.
temporary protecting group to minimize cyclic imide formation
during subsequent treatment of aspartyl peptides with HF. J .
Org. Chem. 1976, 41, 1032-1041.
(33) Hendrickson, J . B.; Kandall, C. The phenacyl protecting group
for acids and phenols. Tetrahedron Lett. 1970, 5, 343-344.
(34) Sheh, L.; Chen, B. L.; Chen, C. F. Synthesis of cyclic peptide
homologues of glutathione as potential antitumor agents. Int.
J . Pept. Res. 1990, 35, 55-62.
(35) D’Ischia, M.; Prota, G.; Rotteveel, R. C.; Westerhof, W. A facile
synthesis of 2-oxo-thiazolidines of biological interest. Synth.
Commun. 1987, 17, 1577-1585.
(13) Kalebic, T.; Kinter, A.; Poli, G.; Anderson, M. E.; Meister, A.;
Fauci, A. Suppression of human immunodeficiency virus expres-
sion in chronically infected monocytic cells by glutathione,
glutathione ester, and N-acetylcysteine. Proc. Natl. Acad. Sci.
U.S.A. 1991, 88, 986-990.
(14) Bergamini, A.; Capozzi, M.; Ghibelli, L.; Salanitro, A.; Milanese,
G.; Wagner, T.; Beninati, S.; Pesce, C. D.; Amici, C.; Rocchi, G.
Cystamine potently suppresses in vitro HIV replication in
acutely and chronically infected human cells. J . Clin. Invest.
1994, 93, 2251-2257.
(15) Bergamini, A.; Ventura, A.; Mancino, G.; Capozzi, M.; Placido,
R.; Salanitro, A.; Cappannoli, L.; Faggioli, E.; Stoler, A.; Rocchi,
G. In vitro inhibition of the replication of human immunodefi-
ciency virus type 1 by â-mercaptoethylamine (Cysteamine). J .
Infect. Dis. 1996, 174, 214-218.
(16) Kameoka, M.; Okada, Y.; Tobiume, M.; Kimura, T.; Ikuta, K.
Intracellular glutathione as a possible direct blocker of HIV type
1 reverse transcription. AIDS Res. Hum. Retroviruses 1996, 12,
1635-1638.
(17) Witschi, A.; Reddy, S.; Stofer, B.; Lauterburg, B. H. The systemic
availability of oral glutathione. Eur. J . Clin. Pharmacol. 1992,
43, 667-669.
(18) Borgstroem, L.; Kagedal, B.; Paulsen, O. Pharmacokinetics of
N-acetylcysteine in man. Eur. J . Clin. Pharmacol. 1986, 34, 77-
82.
(19) Burgunder, J . M.; Varriale, A.; Lauterburg, B. H. Effects of
N-acetylcysteine on plasma cysteine and glutathione following
paracetamol administration. Eur. J . Clin. Pharmacol. 1989, 36,
127-131.
(20) Williamson, J . M.; Meister, A. Stimulation of hepatic glutathione
formation by administration of L-2-oxothiazolidine-4-carboxy-
late, a 5-oxo-L-prolinase substrate. Proc. Natl. Acad. Sci. U.S.A.
1981, 78, 936-939.
(36) Ko¨nig, W.; Geiger, R. New method for the synthesis of peptides:
Activation of the carboxyl group with dicyclohexylcarbodiimide
by using 1-hydroxybenzotriazoles as additives. Chem. Ber. 1970,
103, 788-798.
(37) Dereuddre-Bosquet, N.; Clayette, P.; Martin, M.; Benveniste, O.;
Fretier, P.; J accard, P.; Vaslin, B.; Lebeaut, A.; Dormont, D. Lack
of IL-10 expression in monocyte-derived macrophages in re-
sponse to in vitro infection by HIV-1 isolates. AIDS Res. Hum.
Retroviruses 1997, 13, 9961-966.
(38) Ho, W.-Z.; Douglas, S. D. Glutathione and N-acetylcysteine
suppression of human immnodeficiency virus replication in
human monocyte/macrophages in vitro. AIDS Res. Hum. Ret-
roviruses 1992, 8, 1249-1253.
(39) Ho, W. Z.; Starr, S. E.; Sison, A.; Douglas, S. D. L-2-oxothiazo-
lidine-4-carboxyclic acid inhibits human immunodeficiency virus
type 1 replication in mononuclear phagocytes and lymphocytes.
Clin. Diagn. Lab. Immunol. 1997, 4, 352-357.
(40) Lioy, J .; Ho, W.-Z.; Cutilli, J . R.; Polin, R. A.; Douglas, S. D. Thiol
suppression of human immunodeficiency virus type 1 replication
in primary cord blood monocyte-derived macrophages in vitro.
J . Clin. Invest. 1993, 91, 495-498.
(41) Nottet, H. S. L. M.; van Asbeck, S.; de Graaf, L.; de Vos, N. M.;
Visser, M. R.; Verhoef, J . Role for oxygen radicals in self-
sustained HIV-1 replication in monocyte-derived macrophages:
enhanced HIV-1 replication by N-acetylcysteine. J . Leukocyte
Biol. 1994, 56, 702-707.
(42) Chen, P.; Bauer, G.; Mitchell, J .; Factor, R.; Markham, R.;
Schwartz, D. H. N-acetyl-cysteine and L-2-oxathiazolidine-4-
carboxylic acid enhance contact-dependent growth of HIV in
resting peripheral blood mononuclear cells (PBMC) in vitro and
increase recovery of HIV from human-PBMC SCID mice. AIDS
1997, 11, 33-41.
(43) Magnani, M.; Fraternale, A.; Casabianca, A.; Schiavano, G.;
Chiarantini, L.; Palamara, A.; Ciriolo, M.; Rotilio, G.; Garaci,
E. Antiretroviral effect of combined zidovudine and reduced
glutathione therapy in murine AIDS. AIDS Res. Hum. Retro-
viruses 1997, 13, 1093-1099.
(21) Porta, P.; Aebi, S.; Summer, K.; Lauterburg, B. H. L-2-oxoxthia-
zolidine-4-carboxylic acid, a cysteine prodrug: pharmacokinetics
and effects on thiols in plasma and lymphocytes in human. J .
Pharmacol. Exp. Ther. 1991, 257, 331-334.
(22) Boesgaard, S.; Aldershvile, J .; Poulsen, H. E.; Loft, S.; Anderson,
M. E.; Meister, A. Nitrate tolerance in vivo is not associated with
depletion of arterial or venous thiol levels. Circ. Res. 1994, 74,
115-120.
(44) Fidgor, C. G.; Leemans, J . M. M.; Bont, W. S.; Vries, J . E. Theory
and practice of centrifugal elutriation (CE). Factors influencing
the separation of human blood cells. Cell. Biophys. 1983, 5, 105-
118.

4740 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Oiry et al.
(45) Gartner, S.; Markovits, P.; Markovitz, D. M.; Kaplan, M. H.;
Gallo, R. C.; Popovic, M. The role of mononuclear phagocytes in
HTLV-III/LAV infection. Science 1986, 233, 215-219.
(46) Ka¨rber, G. Beitag Zur Kollektiven Behandlung Pharmakolo-
gisher Reihenvesuche. Arch. Exp. Pathol. Pharmakol. 1931, 162,
956-959.
(47) Rey, F.; Spire, B.; Dormont, D.; Barre´-Sinoussi, F.; Montagnier,
L.; Chermann, J . C. Characterization of RNA dependent DNA
polymerase of a new human T-lymphotropic retrovirus (lym-
phadenopathy associated virus). Biochem. Biophys. Res. Com-
mun. 1984, 121, 126-133.
J M980289J