Thiazolidine prodrugs as protective agents against γ-radiation-induced toxicity and mutagenesis in V79 cells

Article abstract of DOI:10.1021/jm010162l

Representatives of two classes of thiazolidine prodrug forms of the well-known radioprotective agents L-cysteine, cysteamine, and 2-[(aminopropyl)amino]ethanethiol (WR-1065) were synthesized by condensing the parent thiolamine with an appropriate carbonyl donor. Inherent toxicity of the prodrugs was assessed in V79 cells using a clonogenic survival assay. Protection against radiation-induced cell death was measured similarly after exposure to 0-8 Gy γ (¹³⁷Cs) radiation. Antimutagenic activity was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. All thiazolidine prodrugs exhibited less toxicity than their parent thiolamines, sometimes dramatically so. Protection against radiation-induced cell death was observed for the 2-alkylthiazolidine, 2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl) thiazolidine (RibCyst), which produced a protection factor at 8 Gy of 1.8; the cysteine analogue, 2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl) thiazolidine-4(R)-carboxylic acid (RibCys), was less active. RibCyst also exhibited excellent antimutational activity, rivaling that of WR-1065. The 2-oxothiazolidine analogues showed little activity in either determination under the conditions tested, perhaps due to their enhanced chemical and biochemical stability.

Full text of DOI:10.1021/jm010162l

J . Med. Chem. 2001, 44, 2661-2666
2661
Th ia zolid in e P r od r u gs a s P r otective Agen ts a ga in st γ-Ra d ia tion -In d u ced
Toxicity a n d Mu ta gen esis in V79 Cells
†
†
‡
,†
Britta H. Wilmore, Pamela B. Cassidy, Raymond L. Warters, and J eanette C. Roberts*
Departments of Medicinal Chemistry and Radiation Oncology, University of Utah, Salt Lake City, Utah 84112
Received April 9, 2001
Representatives of two classes of thiazolidine prodrug forms of the well-known radioprotective
agents L-cysteine, cysteamine, and 2-[(aminopropyl)amino]ethanethiol (WR-1065) were syn-
thesized by condensing the parent thiolamine with an appropriate carbonyl donor. Inherent
toxicity of the prodrugs was assessed in V79 cells using a clonogenic survival assay. Protection
1
37
against radiation-induced cell death was measured similarly after exposure to 0-8 Gy γ ( Cs)
radiation. Antimutagenic activity was determined at the hypoxanthine-guanine phosphoribo-
syltransferase (HGPRT) locus. All thiazolidine prodrugs exhibited less toxicity than their parent
thiolamines, sometimes dramatically so. Protection against radiation-induced cell death was
observed for the 2-alkylthiazolidine, 2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine
(
RibCyst), which produced a protection factor at 8 Gy of 1.8; the cysteine analogue, 2(R,S)-D-
ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid (RibCys), was less active.
RibCyst also exhibited excellent antimutational activity, rivaling that of WR-1065. The
2
-oxothiazolidine analogues showed little activity in either determination under the conditions
tested, perhaps due to their enhanced chemical and biochemical stability.
In tr od u ction
Sch em e 1
Cysteine and cysteamine have been recognized as
radioprotective agents for decades.1,2 To extend the
original observations, an extensive survey of compounds,
mainly thiolamines and their derivatives, was initiated
3
by the Walter Reed Army Hospital in the 1950s. A
promising radioprotective agent that emerged from this
effort was WR-1065 (2-[(aminopropyl)amino]ethane-
thiol), a derivative of cysteamine.
Unfortunately, the use of many thiolamines as radio-
protectors is limited by the undesired side effects
observed at protective doses, as well as limitations in
Sch em e 2
4
-9
oral activity.
To avoid these problems, we applied a
prodrug approach, wherein the parent agent is chemi-
cally modified to overcome a biological barrier to its
1
0,11
effective use.
Our prodrug strategy involves incorporating the par-
ent thiolamine into a thiazolidine ring structure, which
effectively masks the reactive thiol group, reduces
potential side effects, and alters key physicochemical
properties.1
2,13
For the current studies, representatives
of two classes of thiazolidine analogues were synthesized
and evaluated. The 2-alkylthiazolidines (Scheme 1) were
designed to release the free thiolamine by nonenzymatic
ring opening and hydrolysis.1
2,14,15
Preliminary inves-
1
6,17
tigation of such radioprotectors has been promising.
In contrast, the 2-oxothiazolidines (Scheme 2) were
designed to be inherently more stable, releasing the
parent thiolamine only upon enzymatic cleavage, if at
all. This category was modeled after the cysteine pro-
drug, 2-oxothiazolidine-4(R)-carboxylic acid (OTZ, Pro-
cysteine, “OxoCys”), which is known to release cysteine
after biotransformation by the enzyme, 5-oxopro-
linase.
We constructed thiazolidine prodrug forms of cysteine,
cysteamine, and WR-1065 to explore the radioprotective
and antimutagenic activity of these derivatives. The
thiazolidine analogues were tested in cultured V79 cells
for (1) their inherent toxicity, (2) the ability to protect
cells from radiation-induced cell death, and (3) the
ability of selected agents to reduce radiation-induced
1
8-20
*
To whom correspondence should be addressed at Department of
Medicinal Chemistry, University of Utah, 30 South 2000 East, Room
2
9
01, Salt Lake City, UT 84112. Phone: 801/581-3598. Fax: 801/585-
119. E-mail: jroberts@deans.pharm.utah.edu.
†
Department of Medicinal Chemistry.
‡
Department of Radiation Oncology.
1
0.1021/jm010162l CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/30/2001

2
662 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Wilmore et al.
mutagenesis at the hypoxanthine-guanine phosphori-
bosyltransferase (HGPRT) locus. Antimutagenic activity
may be particularly advantageous for radioprotectors
employed during the radiotherapy of cancer. Patients
can develop secondary tumors as a result of mutations
brought on by the radiation treatment, a complication
becoming more prevalent as patients survive for longer
periods of time after treatment. The ability to decrease
therapy-induced mutagenesis in normal tissue, and
hence treatment-derived secondary neoplasias, could
significantly enhance the long-term clinical success of
radiation treatments.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the 2-alkylthiazolidine
analogues of cysteine (RibCys, 1) and cysteamine (Rib-
Cyst, 2), depicted in Scheme 1, involves the condensa-
tion of the aminothiol with D-ribose, which proceeds in
good yield in either water or methanol. The chemistry
of this reaction was originally investigated in the 1930s
and is well documented.1
2,15,21-24
Various synthetic
methods were employed in the attempted synthesis of
the corresponding alkylthiazolidine analogue of WR-
1
065, but none was successful, presumably due to the
proximity of the nucleophilic primary amine and its
effect on thiazolidine ring stability.
Upon nonenzymatic ring opening and hydrolysis, the
2
-alkylthiazolidines release the parent thiolamine, along
with an equimolar amount of the aldehyde used in
1
4,15
prodrug construction.
Ribose was chosen as the
carbonyl donor to eliminate possible toxicity associated
1
2
with the release of the aldehyde component.
The synthesis of the 2-oxothiazolidine analogues of
cysteamine and WR-1065 is outlined in Scheme 2.
OxoCyst (3) was prepared by the reaction of cysteamine
with carbonyl diimidazole and also served as an inter-
mediate in the synthesis of the WR-1065 analogue,
OxoWR (5). The preparation of the latter involved
alkylation of OxoCyst with 3-bromopropylphthalimide,
followed by deprotection of the primary amine with
NaBH4.
F igu r e 1. Effect of parent thiolamines and their thiazolidine
prodrugs on clonogenic survival of V79 cells. The average of
at least three separate experiments, each run in triplicate, is
depicted; error bars represent standard deviation. (A) Toxicity
of cysteine (O), RibCys (1, 9), and OxoCys (2). (B) Toxicity of
cysteamine (O), RibCyst (2, 9), and OxoCyst (3, 2). (C) Toxicity
of WR-1065 (O) and OxoWR (5, 2).
The 2-oxothiazolidines were designed to be enzymati-
cally cleaved by 5-oxoprolinase, similar to a correspond-
ing prodrug of cysteine, OxoCys.18,19 However, com-
pounds with the molecular features present in the novel
analogues (lack of the carboxyl group, N-alkylation) may
not be substrates for this enzyme.20 Agents that are not
subject to either enzymatic or nonenzymatic biotrans-
formation may provide further information regarding
the ability of intact thiazolidines themselves to act as
radioprotectors without the prerequisite release of
thiolamine. This property has been suggested in previ-
toxic at or above 10 mM. The dramatic toxicity at lower
concentrations of WR-1065 (∼0.5 mM), the character-
istic so-called “paradoxical toxicity” of thiol com-
4
,5,16
pounds,
was also observed.
The agents were then studied for their ability to
protect V79 cells from radiation-induced cell death,
again using a clonogenic survival assay. Figure 2
provides the results of these assays, and Table 1 lists
the protection factors (PF) and dose modification factors
(DMF) derived from these experiments. None of the
thiazolidines in either the cysteine series (Figure 2A),
the cysteamine series (Figure 2B), or the WR-1065
series (Figure 2C) provided the level of radioprotection
observed upon treatment with the parent thiolamines.
However, RibCyst (2) exhibited the greatest radiopro-
tective activity under the conditions tested (PF ) 1.8
at 8 Gy).
2
5,26
ous reports.
prolinase.
V79 cells are known to contain 5-oxo-
2
7
Biologica l Eva lu a tion . As seen in Figure 1, each
thiazolidine was less toxic to the V79 cells than its
parent thiolamine, sometimes dramatically so. The
thiazolidines of cysteine and cysteamine exhibited es-
sentially no toxicity in these studies (Figure 1A,B),
whereas the thiazolidine analogue of WR-1065 produced
noticeable toxic effects, with a decrease in surviving
fraction (SF) to <0.8 at 25 mM. In addition, the dose-
response curve of the prodrug was very different from
that of the parent drug (Figure 1C). WR-1065 was very

Thiazolidine Prodrugs as Protective Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2663
Ta ble 1. Protection Factors (PF) and Dose Modification
Factors (DMF) of the Parent Thiolamines and Their
Thiazolidine Prodrugs
a
a
DMFb
agent (mM)
PF at 4 Gy
PF at 8 Gy
cysteine (10)
RibCys, 1 (25)
OxoCys (25)
cysteamine (10)
RibCyst, 2 (25)
OxoCyst, 3 (25)
WR-1065 (4)
1.4
1.0
0.9
1.5
1.3
1.3
1.8
0.8
2.7
1.6
1.4
3.8
1.8
1.3
4.1
0.8
2.2
1.5
1.3
2.9
1.3
1.1
4.4
0.9
OxoWR, 5 (10)
a
PFs are calculated as the ratio of surviving fraction of cells in
the presence of the agent to that in its absence at the respective
radiation doses. b DMFs are calculated as the ratio of the terminal
slope of the survival curve of the cells in the presence of the agent
to that in its absence.
F igu r e 3. HGPRT mutation frequency in V79 cells receiving
radiation alone (control) or radiation plus treatment with 10
mM cysteamine (Cyst), 25 mM RibCyst (2), 25 mM OxoCyst
(3), or 4 mM WR-1065 (WR). Each point represents the average
of two experiments with five data points per experiment; error
bars represent standard deviation. ANOVA analysis was used
to identify results which differ significantly (p < 0.05) from
the control samples, indicated by the asterisk (*) symbol.
tion. However, the 2-alkylthiazolidine analogue, RibCyst
(2), displayed excellent activity, providing protection
similar to WR-1065. The parent thiolamine, cysteamine,
produced intermediate results, which were statistically
indistinguishable from controls. None of the agents
themselves increased mutation rates above background
F igu r e 2. Radioprotection by the parent thiolamines and
their thiazolidine prodrugs in V79 cells. Each point represents
the average of at least three experiments, each run in
triplicate; error bars represent standard deviation. ANOVA
analysis was used to identify results which differ significantly
(data not shown).
(
p < 0.05) from the control samples (at 8 Gy), indicated by the
Con clu sion s
asterisk (*) symbol. The media control in each panel is
represented by 9. (A) Protection by 10 mM cysteine (O), 25
mM RibCys (1, 2), and 25 mM OxoCys (0). (B) Protection by
The studies discussed here illustrate that thiazo-
lidines are less toxic in vitro than their parent thiol-
amines. The 2-oxothiazolidines appeared to be largely
inactive as radioprotective agents under the conditions
examined. In contrast, the 2-alkylthiazolidines, particu-
larly RibCyst, were much more promising. RibCyst also
displayed excellent antimutagenic activity, which ri-
valed that of WR-1065, a well-accepted standard of
1
0 mM cysteamine (O), 25 mM RibCyst (2, 2), and 25 mM
OxoCyst (3, 0). (C) Protection by 4 mM WR-1065 (O) and 10
mM OxoWR (5, 0).
Due to the superior performance of RibCyst in the
radioprotection experiments, the cysteamine series was
tested for the ability to protect cells from radiation-
induced mutagenesis. The hypoxanthine-guanine phos-
phoribosyltransferase (HGPRT) system monitors the
ability of the cells to utilize purine salvage pathways
and can be used to detect the induction of point
mutations and small deletions.28,29 The results of the
HGPRT antimutation studies are shown in Figure 3.
Irradiation at 4 Gy increased the frequency of mutation
3
0,31
radioprotection and antimutagenesis;
OxoCyst was
again inactive. Such antimutagenic activity may be
beneficial in cancer radiotherapy, suppressing the for-
mation of treatment-derived secondary tumors. Coupled
with the reduced inherent toxicity, a significant en-
hancement in the therapeutic index of radiation treat-
ment may result if an agent such as RibCyst is admin-
istered in conjunction with radiation therapy.
6
by approximately 30 mutants per 10 cells over back-
ground levels. The 2-oxothiazolidine, OxoCyst (3), ex-
hibited no protection against radiation-induced muta-
The activity associated with the 2-alkylthiazolidine
analogues suggests that a similar prodrug form of WR-

2
664 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Wilmore et al.
1
065 may also provide radioprotection and/or antimu-
thiazolidine-2-one (4, 12 g, 0.043 mol) in a 6:1 solution of
2
1
-propanol:water (425 mL). After being stirred at reflux for
6 h, the mixture was cooled slightly and glacial acetic acid
tation with reduced toxicity. Alternative schemes are
being pursued to synthetically access this potentially
interesting prodrug form.
(45 mL) was added. After an additional 4 h at reflux, the
solvent was removed in vacuo, and the resulting solid was
washed with ether (700 mL). The crude product was purified
in three portions on Dowex (50W ×8) cation-exchange resin
Exp er im en ta l Section
(
18 × 3 cm), washing with water (500 mL) and 1 N HCl (500
Ch em istr y. All reagents were purchased from Sigma
Chemical Co. (St. Louis, MO), Aldrich Chemical Co. (Milwau-
kee, WI), or Fisher Scientific (Pittsburgh, PA). WR-1065 was
a gift from the Drug Synthesis and Chemistry Branch,
Developmental Therapeutics Program, Division of Cancer
Treatment, National Cancer Institute. Melting points were
determined on a Laboratory Devices USA Mel-Temp II melting
point apparatus and are uncorrected. Nuclear magnetic reso-
nance (NMR) spectra were obtained on either an IBM Instru-
ments 200 MHz or a Varian Unity 500 MHz FT-NMR
spectrometer, as indicated. High-resolution mass spectral
mL), and eluting the product, as the hydrochloride salt, with
N HCl (500 mL). Yield: 4.8 g (55%); mp 78-82 °C. HRMS
6
+
+
(
FAB ): m/z ) 161.07447 (M + 1, theoretical ) 161.07486).
1
H NMR (DMSO-d
.39 (t, 2H, J ) 3 Hz), 3.95-4.00 (m, 4H), 4.31 (t, 2H, J ) 8,
6
.5 Hz), 8.89 (br s, 3H) ppm. C NMR (DMSO-d , 125 MHz):
6
, 500 MHz): δ 2.49 (t, 2H, J ) 7.5 Hz),
3
6
13
δ 25.0, 25.3, 36.4, 41.5, 48.1, 171.1 ppm. Anal. C, H, N.
Biologica l Eva lu a tion . Gen er a l Cell Cu ltu r e P r oce-
d u r es. V79 Chinese hamster lung fibroblasts (V79-4) were
obtained from American Type Culture Collection (Manassas,
VA). The following supplies were purchased from Sigma
Chemical Co. (St. Louis, MO): Dulbecco’s modified minimum
essential media (D-MEM), Hanks’ balanced salt solution,
trypsin (1:250), phosphate buffered saline (PBS), antibiotic/
antimycotic solution (100X), and OxoCys. Serum was obtained
from HyClone Laboratories (Logan, UT). V79 cells were grown
in D-MEM containing 3.7 g/L sodium bicarbonate, 10% Fetal
Clone I serum, and 0.1% antibiotic/antimycotic solution. Cells
(HRMS) analyses were carried out on a Finnegan MAT 95 in
the Department of Chemistry at the University of Utah.
Elemental analysis was conducted by Galbraith Laboratories
(Knoxville, TN). Thin-layer chromatography (TLC) was carried
out using Whatman (Clifton, NJ ) flexible backed 60 Å silica
gel plates, with a layer thickness of 0.25 mm. Whatman silica
gel (60 Å, 230-400 mesh) was used for sample purification
using column chromatography.
were maintained in a humidified 5% CO
7 °C.
The number of cells was determined using a Coulter
2
-air atmosphere at
2
(R,S)-D-r ib o-(1′,2′,3′,4′-Tet r a h yd r oxyb u t yl)t h ia zoli-
3
d in e-4(R)-ca r boxylic Acid (RibCys, 1) a n d 2(R,S)-D-r ibo-
(
1′,2′,3′,4′-Tetr a h yd r oxybu tyl)th ia zolid in e (RibCyst, 2)
Counter, model Z1 (Beckman Coulter, Fullerton, CA) particle
counter. Colonies were stained using a solution of 2.5 g of
powdered crystal violet in 900 mL of methanol and 100 mL of
(Scheme 1). RibCys and RibCyst were synthesized and char-
1
6
acterized as described. RibCyst was stored under argon at
15 °C but occasionally displayed evidence of decomposition,
-
1
0% formalin; colonies consisting of at least 50 cells were
such as discoloration and development of a slight odor, which
was remedied by recrystallization from methanol.
counted using the Manostat Colony Counting System (Manostat
Corp., New York, NY).
Th ia zolid in -2-on e (OxoCyst, 3) (Scheme 2). The synthetic
Irradiation of cells was carried out in a Shepherd Mark I
irradiator (1 Cs source) at a dose rate of 7.5 Gy/min.
strategy for OxoCyst was based on the methods of d’Ischia et
37
3
2
al. 1,1′-Carbonyl diimidazole (30.0 g, 0.185 mol) was dissolved
with heating) in acetonitrile (300 mL), which had been
degassed and flushed with argon. To this solution were added
8-crown-6 (0.7 g, 0.025 mol), 2-aminoethanethiol hydrochlo-
Data analysis was conducted using GraphPad Prism (v. 2.0,
San Diego, CA).
(
1
Clon ogen ic Su r viva l Assa y of Dr u g Toxicity. Each well
of a six-well culture plate was seeded with enough cells in 3
mL of media to yield at least 50 colonies after treatment, and
the cells were incubated for 16 h to allow attachment to the
plate surface. The media was then replaced with 5 mL of
freshly made solutions of each drug in media at various
concentrations (pH 7-7.5). The cells were incubated with the
treatment solutions for 3 h, and then the media was removed
and the cells were washed with Hanks’ buffered salt solution
for 15 min at room temperature. Fresh media (3 mL) was
added, and the plates were incubated 4 d longer to allow colony
development. The colonies were stained and counted, and the
surviving fraction (SF) was calculated by dividing the number
of cells retaining colony forming ability after drug treatment
by those receiving media alone. These values were normalized
for comparison (controls, SF ) 1). The plating efficiency of
untreated V79 cells was approximately 60%.
ride (20.1 g, 0.185 mol), and potassium carbonate (26 g, 0.19
mol). The mixture was heated to reflux and stirred for 18 h.
Water (100 mL) was added, and the mixture was refluxed for
an additional 4 h. The solution was cooled slightly, and the
solvent was removed in vacuo. Ethyl acetate (500 mL) was
added to the resulting solid, and the insoluble salts were
removed by filtration. The filtrate was dried, and the crude
product was purified on silica gel, eluting with 50% ethyl
acetate in hexane. Yield: 13 g (68%); mp 48-50 °C; R
f
) 0.22
+
(
(
2:1 ethyl acetate:hexane). HRMS (FAB ): m/z ) 104.01752
+
1
M
3
+ 1, theoretical ) 104.01701). H NMR (CDCl , 500
MHz): δ 3.37 (t, 2H, J ) 8, 6.5 Hz), 3.59 (t, 2H, J ) 8, 6.5
1
3
Hz), 6.75 (br s, 1H) ppm. C NMR (CDCl
3.4, 176.7 ppm. Anal. C, H, N.
-(N-P h t h a loyl-3-a m in op r op yl)t h ia zolid in -2-on e (4)
Scheme 2). To a solution of thiazolidin-2-one (3, 6.4 g, 0.062
3
, 125 MHz): δ 30.0,
4
3
(
mol) in acetonitrile (100 mL) was added N-(3-bromopropyl)-
phthalimide (26.0 g, 0.093 mol), potassium carbonate (17 g,
P r otection a ga in st Ra d ia tion -In d u ced Cell Dea th .
Sufficient cells were plated in each well of a 6-well plate to
yield at least 50 colonies after treatment. After overnight
incubation, the media was removed and replaced with 5 mL
of freshly prepared treatment solutions (pH 7-7.5) at a drug
concentration in the relatively nontoxic range (derived from
Figure 1). A minimum of three wells per study served as media
controls.
0
.12 mol), and 18-crown-6 (1.6 g, 0.0061 mol). The reaction
mixture was stirred at reflux for 16 h, and then the acetonitrile
was removed in vacuo. The crude product was redissolved in
0
.5 M potassium chloride (60 mL), and the product was
extracted with ethyl acetate (3 × 75 mL). The combined
organic fractions were washed with a saturated aqueous
solution of sodium chloride (50 mL), dried over sodium sulfate,
and concentrated in vacuo. The resulting solid was recrystal-
Cells were incubated for 3 h, then irradiated at doses of 0,
1
, 2, 4, or 8 Gy. After irradiation, treatment solutions were
lized from ethyl acetate (150 mL). Yield: 13 g (72%); mp 121-
removed, and cells were washed for 10 min at room temper-
ature with Hanks’ buffered salt solution. Fresh media was
added, and the plates were incubated for 4 d to allow colony
development. The colonies were then stained and counted. The
ratio of colonies per cells plated in treated versus untreated
samples represented the SF. These values were normalized
for comparison (controls, SF ) 1).
1
1
3
24 °C. H NMR (DMSO-d
6
, 200 MHz): δ 1.68-1.91 (m, 2H),
.22-3.35 (m, 4H), 3.48-3.65 (m, 4H), 7.75-7.88 (m, 4H) ppm.
-(3-Am in op r op yl)th ia zolid in -2-on e (5) (Scheme 2). The
removal of the phthalimide protecting group was based on the
3
3
3
procedure of Osby et al. Sodium borohydride (8.1 g, 0.21 mol)
was added to a suspension of 3-(N-phthaloyl-3-aminopropyl)-

Thiazolidine Prodrugs as Protective Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2665
P r otection a ga in st Ra d ia tion -In d u ced Mu ta gen esis.
Assays of mutagenesis at the hypoxanthine-guanine phospho-
ribosyltransferase (HGPRT) locus in V79 cells were based on
(4) Held, K. D.; Sylvester, F. C.; Hopcia, K. L.; Biaglow, J . E. Role
of Fenton Chemistry in Thiol-Induced Toxicity and Apoptosis.
Radiat. Res. 1996, 145, 542-553.
2
8,29
(5) Held, K. D.; Biaglow, J . E. Mechanisms for the Oxygen Radical-
Mediated Toxicity of Various Thiol-Containing Compounds in
Cultured Mammalian Cells. Radiat. Res. 1994, 139, 15-23.
previously reported methods.
After removing preexisting
HGPRT mutants by growth in HAT media (hypoxanthine (0.1
mM), aminopterin (0.4 µM), and thymidine (0.016 mM); Gibco
BRL, Rockville, MD) for 1 d, cells were allowed to recover for
(6) Munday, R. Toxicity of Thiols and Disulphides: Involvement of
Free-Radical Species. Free Radical Biol. Med. 1989, 7, 659-673.
6
to 9 d, subculturing every 3 d prior to treatment.
(7) Olney, J . W.; Zorumski, C.; Price, M. T.; Labruyere, J . L-Cysteine,
a Bicarbonate-Sensitive Endogenous Excitotoxin. Science 1990,
240, 596-599.
6
The cells were then trypsinized and counted. Cells (2 × 10 )
2
were seeded into each of a series of fresh 25 cm flasks, each
in 5 mL of media. For each experiment, sufficient flasks were
prepared to investigate both irradiated and nonirradiated
samples of each drug studied, as well as of controls, which
received no drug treatment. The flasks were incubated for 24
h to allow cell adhesion to the flask surface.
(
8) J eitner, T. M.; Delikatny, E. J .; Bartier, W. A.; Capper, H. R.;
Hunt, N. H. Inhibition of Drug-Naive and -Resistant Leukemia
Cell Proliferation by Low Molecular Weight Thiols. Biochem.
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9) Glatt, H.; Protic-Samljic, M.; Oesch, F. Mutagenicity of Gluta-
thione and Cysteine in the Ames Test. Science 1983, 220, 961-
962.
(
The following day, media was removed from cell cultures
and was replaced with fresh media (in control samples) or
freshly prepared drug solutions (pH 7). Two flasks were
prepared for each drug treatment group. One flask from each
drug treatment group was irradiated at 4 Gy. Cells were
incubated 3 h longer in the treatment solutions, then washed
with Hanks’ buffered salt solution. The cells were trypsinized
and counted. For each sample, three wells of a six-well plate
were seeded with 200-250 cells for plating efficiency deter-
(10) Albert, A. Chemical Aspects of Selective Toxicity. Nature 1958,
182, 421-423.
11) Sinkula, A. A.; Yalkowsky, S. H. Rationale for Design of
Biologically Reversible Drug Derivatives: Prodrugs. J . Pharm.
Sci. 1975, 64, 181-210.
(
(
12) Roberts, J . C.; Nagasawa, H. T.; Zera, R. T.; Goon, D. J . W.
Prodrugs of L-Cysteine as Protective Agents against Acetami-
nophen-Induced Hepatotoxicity. 2-(Polyhydroxyalkyl)- and 2-(Poly-
acetoxyalkyl)thiazolidine-4(R)-carboxylic Acids. J . Med. Chem.
1987, 30, 1891-1896.
mination. Additionally, fresh cultures were initiated with
6
1
.5 × 10 cells each.
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Biodistribution of [ S]-Cysteine and Cysteine Prodrugs: Poten-
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After 5 d, colonies in the six-well plates were stained and
counted to determine the surviving fraction of each treatment
group.
The cultures were grown for a total of 6 d, subculturing at
(
14) Pesek, J . J .; Frost, J . H. Decomposition of Thiazolidines in Acidic
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3
d. After this time, cells were trypsinized and counted. For
each sample, each well of five six-well plates was seeded with
4
8
× 10 cells, each in media containing 6-thioguanine (6TG)
at a concentration of 5 µg/mL. In addition, three wells per
sample were seeded with 200 cells each, in media without 6TG,
for plating efficiency determinations.
(
All culture plates were incubated for 5 d to allow colony
growth, then stained and counted.
(
Mutation frequencies for each sample were calculated as
the number of mutant colonies per number of cells plated. (The
latter value was corrected for cell survival rates, based on the
plating efficiency determinations.) Mutation frequencies were
6
expressed as mutants per 10 surviving cells. The background
mutation frequency of untreated cells (6 mutants per 106
surviving cells) was subtracted from the value obtained for
each treatment sample.
(
19) Williamson, J . M.; Boettcher, B.; Meister, A. Intracellular
Cysteine Delivery System that Protects against Toxicity by
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982, 79, 6246-6249.
Ack n ow led gm en t. Partial financial support from
the Armed Forces Radiobiology Research Institute is
gratefully acknowledged. Underlying support for Uni-
versity of Utah facilities from NIH grants RR13030,
RR06262, and RR14768 and NSF grant DBI-0002806
and the Huntsman Cancer Institute is appreciated.
B.H.W. received additional financial support from the
American Chemical Society, Division of Medicinal Chem-
istry, and Hoechst Marion Roussel; the NIH Biological
Chemistry Training Grant; the Huntsman Cancer In-
stitute; and the American Foundation for Pharmaceuti-
cal Education. We also thank the Drug Synthesis and
Chemistry Branch, Developmental Therapeutics Pro-
gram, Division of Cancer Treatment, National Cancer
Institute, for providing the WR-1065.
(
20) Williamson, J . M.; Meister, A. New Substrates of 5-Oxo-L-
prolinase. J . Biol. Chem. 1982, 257, 12039-12042.
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(
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