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Y. Xie et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2911–2915
Finnegan LCQ DECA quadrupole ion trap mass spec-
trometer interfaced with a HP1100 LC system at the
University of Utah Mass Spectrometry Facility. Ele-
mental analysis was performed at Galbraith Labora-
tories (Knoxville, TN, USA). Polarimetry data were
collected using a JASCO model DIP-370 digital polari-
meter. Melting points were determined using a Labora-
tory Devices USA Mel-Temp II melting point
instrument and are uncorrected. Thin layer chromato-
graphy (TLC) was carried out using Whatman (Clifton,
NJ, USA) flexible backed 60 A silica gel plates (0.25 mm
thickness) with visualization by iodine vapors. The
syntheses were accomplished by degassing all solvents
and constantly bubbling argon through solvents during
reaction. pH adjustment was accomplished with either
6 M NaOH or 6 M HCl as appropriate.
an evacuated flask containing 0.05 N NaOH (10 mL)
and ethanol (3 mL). To the solution, sodium borohy-
dride (0.1 g, 2.6 mmol) was added slowly over about 10
min. The yellow solution was stirred for an additional
20 min until it became colorless and then was placed in
an ice bath. The pH was adjusted to 5–6. 1,10-Carbo-
nyldiimidazole28 (0.2 g, 1.2 mmol) was added over 30
min, and the reaction mixture was stirred for 1 h. If the
reaction mixture became yellow again, the above
reduction and carbonylation steps were repeated. The
reaction mixture was acidified to pH 2 and extracted
with ethyl acetate (3ꢂ15 mL). The combined ethyl ace-
tate layers were washed with saturated NaCl solution
(2ꢂ15 mL), dried over MgSO4, then concentrated and
dried under vacuum: 0.12 g, 0.62 mmol (41%). Mp 144–
146 ꢁC (d). TLC n-BuOH/H2O/acetic acid (3:2:1), Rf
1
0.65. H NMR (D2O, 500 MHz) d 4.5 (dd, J=6, 8 Hz,
L-Selenocystine (1). l-Selenocystine (1) was synthesized
by a modification of known procedures.27 At room
temperature, selenium powder (3.0 g, 38 mmol) was
suspended in water (10 mL). Sodium borohydride (3.0
g, 79 mmol) was dissolved in water (19 mL) and slowly
added to the selenium suspension with stirring. Another
equivalent of selenium powder (3.0 g, 38 mmol) was
added to the reaction and the mixture was stirred for 15
min. The reaction mixture was placed briefly on a steam
bath (1–2 min) to drive the reaction to completion. b-
Chloro-l-alanine HCl (3.2 g, 20 mmol) was dissolved in
water (20 mL) at pH 9 and added dropwise to the sele-
nium solution over 2 h; stirring was continued overnight
at 37 ꢁC. The reaction mixture was acidified to pH 2 and
hydroxylamine hydrochloride (218 mg, 3.1 mmol) was
added. CAUTION: Added safety precautions were
implemented during the work up of this reaction due to
the production of hydrogen selenide gas. The reaction
vessel was sealed tightly to prevent release of H2Se into
the air, and the exhaust was forced through two lead
acetate traps, each containing 25 g lead acetate in 300
mL water, for 2 h. As an added precaution, a respirator
rated for H2S was routinely used. Vacuum filtration was
performed to remove elemental selenium, and the fil-
trate was adjusted to pH 6–6.5 and left to crystallize at
4 ꢁC for 3–5 days. The yellowish-orange crystals were
collected by vacuum filtration and redissolved in 1 M
HCl (20 mL). Any remaining solids were removed by
vacuum filtration. The filtrate was adjusted to pH 6–6.5
with and left to crystallize again for 3–5 days. The yel-
low crystals were collected by vacuum filtration and
dried by vacuum overnight: 3.0 g, 9.0 mmol (90%). Mp
174–176 ꢁC (d) (reported, lit.27 184–185 ꢁC). TLC n-
BuOH/H2O/acetic acid (3:2:1), Rf 0.32. 1H NMR (D2O/
NaOD, 500 MHz) d 3.6 (dd, J=5, 7Hz, 1H, H- a), 3.3
(dd, J=5, 12 Hz, 1H, H-b1), 3.2 (dd, J=7, 12 Hz, 1H,
H-b2); 13C NMR (D2O/NaOD, 125 MHz) d 181.0
(COOH), 56.3 (C-a), 35.8 (C-b); 77Se NMR (D2O/
NaOD, 95.3 MHz) d 288.1. IR (KBr) nmax 3500, 3000
cmÀ1. FABMS [M++1] m/z 336.9 (80Se). [a]D25À29.0ꢁ
(c 0.5, 0.1 N NaOH). Anal. calcd for C6H12N2O4Se2:
C, 21.6; H, 3.63; N, 8.38. Found: C, 21.2; H, 3.57; N,
8.29.
1H, H-4), 3.8 (dd, J=8, 10 Hz, 1H, H-5a), 3.6 (dd,
J=6, 10 Hz, 1H, H-5b); 13C NMR (D2O, 125 MHz) d
178.7, 178.5 (COOH, C-2), 61.0 (C-4), 34.3 (C-5); 77Se
NMR (D2O, 95.3 MHz) d 1352.8. IR (KBr) nmax 3300,
3000, 1700 cmÀ1. FABMS [M++1] m/z 195.9 (80Se).
[a]2D5À67.9ꢁ (c 0.5, water). Anal. calcd for C4H5NO3Se:
C, 24.8; H, 2.60; N, 7.22. Found: C, 24.4; H, 2.72; N,
6.93.
2(R,S)-Methylselenazolidine-4(R)-carboxylic
acid
(MSCA, 3). l-Selenocystine (1) (0.13 g, 0.39 mmol) was
suspended in an evacuated flask containing 0.05 N
NaOH (5 mL) and ethanol (1.5 mL). Sodium borohy-
dride (0.05 g, 1.3 mmol) was added slowly over about 10
min. The reaction mixture was stirred for an additional
20 min until it changed to colorless and was then placed
in an ice bath. The pH was adjusted to 5–6. Acetalde-
hyde (0.24 mL, 4.3 mmol) was added in 80 mL aliquots
over 30 min, and the reaction mixture was stirred for 3
h. At that point, ethanol (20 mL) was added and the
mixture was stored in the refrigerator overnight. The
yellow precipitate (selenocystine by MS and NMR; data
not shown) was filtered off, and the filtrate was dried
under vacuum: 0.09 g, 0.47mmol (60%). Mp 136–
138 ꢁC (d). 1H NMR (D2O, 400 MHz) d diastereomer A
5.1 (q, J=7Hz, 1H, H-2), 4.6 (t, J=7Hz, 1H, H-4),
3.5–3.3 (m, 2H, H-5), 1.7(d, J=7Hz, 3H, CH ); dia-
3
stereomer B 4.9 (q, J=7Hz, 1H, H-2), 4.3 (dd, J=7, 11
Hz, 1H, H-4), 3.5–3.3 (m, 2H, H-5), 1.7(d, J=7Hz,
3H, CH3); 13C NMR (D2O, 100 MHz) d diastereomer A
171.7 (COOH), 66.4 (C-4), 50.7 (C-2), 24.2 (C-5), 18.8
(CH3); diastereomer B 171.5 (COOH), 64.9 (C-4), 50.7
(C-2), 24.2 (C-5), 21.1 (CH3); 77Se NMR (D2O,
76.2 MHz) d 320.2, 315.7. IR (KBr) nmax 3250, 2900,
1690 cmÀ1
.
FABMS [M++1] m/z 196.0 (80Se).
.
[a]2D5À83.9 (c 0.5, water). Anal. calcd for C5H9NO2Se
1/2 H2O: C, 29.6; H, 4.96; N, 6.90. Found: C, 29.4; H,
4.45; N, 6.81.
Breakdown of selenazolidine prodrugs
Solutions of MSCA and OSCA were prepared in D2O
(pH ꢀ 4.0) and incubated for various times at various
1
temperatures. H NMR (400 MHz) spectra were then
obtained. The breakdown of the prodrugs was esti-
mated as the percent reduction in the most downfield
2-Oxoselenazolidine-4(R)-carboxylic acid (OSCA, 2). l-
Selenocystine (1) (0.25 g, 0.75 mmol) was suspended in