1
680 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Nagasawa et al.
spectroscopy. The early fractions contained as much as 10%
N,S-bis-acetylated 3, while the later fractions showed de-
creased ester concentrations and some ester-hydrolyzed S-acetyl-
γ-L-glutamyl-L-cysteine (3) plus some other unidentified ma-
terial. The best fractions were combined and lyophilized to
give 844 mg of light yellow solid, mp 147-149 °C dec (26%
yield). The NMR spectrum showed a small doublet at about
δ 1.6 ppm. This product was treated with decolorizing carbon,
the mixture filtered, and the filtrate lyophilized again to give
COCH
2
), 3.19 (dd, J ) 14.2 Hz, J ) 7.3 Hz, 1H, SCH), 3.48
(dd, J ) 14.2 Hz, J ) 4.5 Hz, 1H, SCH), 3.82 (t, J ) 6.2 Hz,
1H, CHNH ), 4.53 (dd, J ) 7.3 Hz, J ) 4.5 Hz, 1H, CHNH);
mass spectrum (CI), m/ z (rel intensity) 130 (30), 84 (100).
The hydrochloride was prepared by treatment of a crude
sample with 1 equiv of 0.1 N aqueous HCl. This solution was
then passed through a column of RPSG to remove lipophilic
2
2 3
material by eluting with H O:CH CN (5:95). The product was
dissolved in isopropyl alcohol, and any insoluble material was
removed by filtration. Ether was added to the filtrate, and
the solids that formed after cooling to -20 °C were removed.
The filtrate was then evaporated, the resulting solids were
taken up in water, and the solution was lyophilized to give
4
91 mg (15% yield) of very pale pink solids whose NMR
spectrum showed nearly complete absence of the doublet at δ
.6 ppm. Another 1.2 g (37% yield) of less pure product was
1
1
also isolated: H NMR (D
CH CH ), 2.10 (m, 2H, CH CH), 2.36 (s, 3H, CH
H, COCH ), 3.26 (dd, J ) 14.3 Hz, J ) 7.1 Hz, 1H, SCH),
.45 (dd, J ) 14.3 Hz, J ) 4.8 Hz, 1H, SCH), 3.81 (t, J ) 6.2
), 4.20 (q, J ) 7.1 Hz, 2H, CH CH ), 4.65 (dd,
J ) 7.1 Hz, J ) 4.9 Hz, 1H, CHNH); mass spectrum (CI), m/ z
rel intensity) 130 (50), 84 (100). Anal. (C12 S) C, H,
N.
2
O) δ 1.25 (t, J ) 7.1 Hz, 3H,
2
3
2
3
CO), 2.45 (m,
the hydrochloride of 4 as a hygroscopic white solid: mp 68-
1
2
3
2
70 °C; H NMR (D
2
2 3
O) δ 2.25 (m, 2H, CH CH), 2.38 (s, 3H, CH ),
2
2.54 (m, 2H, COCH
SCH), 3.50 (dd, J ) 14.3 Hz, J ) 4.6 Hz, 1H, SCH), 4.09 (t, J
) 6.7 Hz, 1H, CHNH ), 4.6 (m, 1H, CHNH). Anal.
S‚HCl) C, H, N.
), 3.27 (dd, J ) 14.3 Hz, J ) 7.5 Hz, 1H,
Hz, 1H, CHNH
2
2
3
3
(
H
20
N
2
O
6
16 2 6
(C10H N O
N,S-Bisa cet yl-γ-L-glu t a m yl-L-cyst ein e (5). Crude γ-L-
glutamyl-L-cysteine (1b, 1.59 g, 6.36 mmol) was dissolved in
E t h yl N,S-Bisa cet yl-γ-L-glu t a m yl-L-cyst ein a t e (3).
Crude γ-L-glutamyl-L-cysteine ethyl ester (1a , 1.31 g, 4.71
mmol) was acetylated in the same manner as for 5 below using
H
2
O (20 mL), and the solution was stirred with ice bath
cooling. A solution of NaHCO (6.9 g, 82 mmol) in 80 mL of
O was added, followed by dropwise addition of acetic
3
2
.7 mL (2.9 g, 28 mmol) of acetic anhydride. The 1.53 g of
H
2
crude product obtained, along with 0.34 g from another run,
was purified by column chromatography using 105 g of C-18
reverse-phase silica gel (RPSG). The column was washed with
methanol and then with water. The column was eluted with
anhydride (3.6 mL, 3.9 g, 38 mmol) over 1 min. After 2 min a
test for free thiol was negative. The solution was acidified to
pH 2.5-3 with 6 N and then 1 N HCl. Removal of the solvent
afforded 8.3 g of white solids whose NMR spectrum showed
the presence of both S-acetyl and N-acetyl groups. The solids
were extracted with 4 × 50 mL portions of hot absolute ethanol
to give 1.71 g of product which, along with 0.36 g from another
run, was purified by flash chromatography using 100 g of silica
gel. The column was eluted with 1-propanol:0.02 N aqueous
HCl (95:5), followed by 1-propanol:0.01 N aqueous HCl (90:
H
H
2
O, followed by CH
O (10:90) to give 0.59 g of the titled compound as a
3 2 3
OH:H O (5:95), and finally with CH OH:
2
1
hygroscopic white solid: mp 66.5-68.5 °C; H NMR (D
CH ), 2.01 (s, 3H, CH CONH), 2.1
CO), 2.34 (s, 3H, CH COS), 2.4 (m, 2H,
CO), 3.21 (dd, J ) 14.2 Hz, J ) 7.4 Hz, 1H, SCH), 3.43
2
O) δ
1
.25 (t, J ) 7.2 Hz, 3H, CH
3
2
3
(m, 2H, CH CH
2
2
3
CH
2
(
2
dd, J ) 14.2 Hz, J ) 4.8 Hz, 1H, SCH), 4.18 (q, J ) 7.1 Hz,
10) to give 1.02 g of the titled compound as a hygroscopic white
1
H, CH
2
CH
3
), 4.30 (dd, J ) 9.2 Hz, J ) 4.8 Hz, 1H, CHCH
2
-
solid: mp 87-89 °C; H NMR (D
2
O) δ 2.0 (m, 2H, CH
CO), 2.37 (s, 3H,
COS), 3.14 (dd, J ) 14.1 Hz, J ) 7.5 Hz, 1H, SCH), 3.48
(dd, J ) 14.1 Hz, J ) 4.4 Hz, 1H, SCH), 4.19 (dd, J ) 9.1 Hz,
J ) 4.5 Hz, 1H, CHCH CH ), 4.46 (dd, J ) 7.5 Hz, J ) 4.3 Hz,
1H, CHCH S). Anal. (C12 S) C, H, N.
2 2
CH -
CH
2
), 4.62 (dd, J ) 7.3 Hz, J ) 4.8 Hz, 1H, CHCH
2
S); mass
CO), 2.03 (s, 3H, CH
CH
3 2
CONH), 2.3 (m, 2H, CH
+
spectrum (FAB), m/ z 363 (MH ). Anal. (C14
C, H, N.
H
22
N
2
O
7
S‚0.5H
2
O)
3
3
-(γ-L-Glu t a m yl)-2(R,S)-m et h ylt h ia zolid in e-4(R)-ca r -
2
2
boxylic Acid (7b). This product was prepared from 2(R,S)-
2
18 2 7
H N O
3
1
methylthiazolidine-4(R)-carboxylic acid (8.51 g, 57.9 mmol)
essentially as described for the preparation of 7a . The crude
product thus obtained (24.6 g of a thick oil) was purified by
ion exchange chromatography. The column packed with 400
Sta bility Stu d ies. These studies were directed toward
evaluating the stability of the S-acetylated compounds 2 and
4 in saline solution as well as in phosphate buffer at physi-
ological pH. Accordingly, 2 (and/or 4) was dissolved in 0.9%
-
g of Sigma anion exchange resin (SBR nuclear grade, OH
NaCl solution in D
2
O, and the proton NMR spectrum was
form) was washed with H
4 L) to convert it to the acetate form before charging with
the product. The column was eluted with 0.5 M AcOH and
.0 M AcOH to give 8.10 g (51% yield) of the titled compound
2 2
O (3 L), 1 M AcOH (2 L), and H O
monitored over time. There was no discernible change in the
1
(
2
spectrum of 2 (or 4) over the course of 1 / months (see
Supporting Information).
1
Compounds 2 and 4 were then dissolved in deuterated 0.1
M PO buffer, the solution was adjusted to pD 7.4, and NaCl
1
as an off-white solid: mp 190-192.5 °C dec; H NMR (D
1
2
CH
1
8
2
O) δ
CH),
CO), 3.4 (m, 2H,
), 4.8 (m, 1H, CHCO H), 5.4 (m,
); mass spectrum (CI), m/ z (rel intensity) 130 (32),
4 (100). Anal. (C10 S) C, H, N.
S-Acetyl-γ-L-glu ta m yl-L-cystein e (4). This product was
4
.52 and 1.63 (2d, J ) 6.3 Hz and J ) 6.5 Hz, 3H, CH
.2 (m, 2H, CH CHNH ), 2.7 (m, 2H, CH
S), 3.8 (m, 1H, CHNH
H, CHCH
3
was added to a concentration of 0.9%. For 4 there was no
apparent change after 1 or 2 days, but after 4 days a small
peak appeared at δ 1.90 (NaOAc) which was about 2% of the
height of the S-acetyl peak at δ 2.36. After a month this OAc
peak was only 8% of the height of the S-acetyl peak. For 2,
the OAc peak was 19% of the height of the central peak of the
stable triplet of the ethyl ester on day 1. This OAc peak grew
fairly rapidly as the S-acetyl peak shrank. After 1 month, the
OAc peak was 84% of the height of the reference peak. (see
Supporting Information).
2
2
2
2
2
2
3
16 2 5
H N O
prepared from 7b (2.10 g, 7.60 mmol) in a manner similar to
the preparation of 2. The crude 4 (ca. 2 g) thus obtained was
purified by column chromatography using 185 g of silica gel
as follows. The column was washed with 2 L of 0.001 N
aqueous HCl:1-propanol (1:10) and 500 mL of 0.001 N aqueous
HCl:1-propanol (1:20). The product was placed on the column
1
4
[
C]Glycin e In cor p or a tion in to Len s GSH in Vitr o.
1
4
The stimulation of [ C]glycine incorporation into GSH in
cultured human and rat lenses by the prodrug forms of 1a was
2
by dissolving it in H O, adding 2.0 g of silica gel, removing
2
1
evaluated using the procedure developed by Holleschau et al.
the solvent, and placing the coated silica gel on top of the
column. The flask residues were scavenged by repeating this
procedure with about 0.5 g of silica gel. The column was eluted
with 0.001 N aqueous HCl:1-propanol (1:20) and then 0.001
N aqueous HCl:1-propanol (1:10). Representative ninhydrin-
positive fractions were evaporated and the residues analyzed
by NMR spectroscopy. The best fractions were combined and
lyophilized. The early fractions contained some pyroglutamic
acid and up to about 10% of the bis-acetylated 5. The yield of
pure 4 was 0.60 g, mp 192-194.5 °C dec (27%). Another 0.70
Ack n ow led gm en t. This work was supported in part
by grants to the University of Minnesota by Allergan,
Inc., Irvine, CA, and in part by NIH Grant EY01197.
The principal investigators, H.T.N. (VA Medical Center)
and W.B.R. (University of Minnesota), were supported
by their respective institutions. H.T.N. is a Department
of Veterans Affairs Research Career Scientist and
W.B.R. is a Research to Prevent Blindness Senior
Scientific Investigator. We thank Ed Larka of the Mass
1
g (32% yield) of less pure product was also isolated: H NMR
O) δ 2.14 (m, 2H, CH CH), 2.37 (s, 3H, CH ), 2.43 (m, 2H,
(D
2
2
3