PAPER
Improved Preparation of JPM-OEt
243
1H NMR (CDCl3): d = 4.28 (dq, J = 7.0 Hz, 2.1 Hz, 4 H), 3.67 (s, 2
H), 1.32 (t, J = 7.1 Hz, 6 H).
13C NMR (CDCl3): d = 166.56, 62.28, 52.08, 14.18.
pressure. Traces of trifluoroacetic acid were removed by repeated
co-evaporation with toluene, affording the free carboxylic acid as a
viscous pale yellow oil. Neat isobutylchloroformate (2.32 mL,
17.89 mmol) was added at –40 °C to a solution of the carboxylic
acid and N-methyl morpholine (1.95 mL, 17.78 mmol) in anhyd
THF (100 mL). One minute after addition, a solution of tyramine
hydrochloride (3.09 g, 17.78 mmol) and Et3N (2.47 mL, 17.78
mmol) in DMF (40 mL) was added dropwise over a period of 2 min.
Then, the cooling bath was removed and the reaction mixture was
allowed to stir for 4 h, at which time it was filtered and evaporated.
The residue was dissolved in EtOAc (300 mL), washed with 1 N
HCl (3 × 100 mL), sat. NaHCO3 (3 × 100 mL), H2O, and concen-
trated to afford a golden yellow oil (6.9 g). After initial purification
by flash chromatography (CH2Cl2–MeOH, 9:1), further purification
was performed with a second flash column, eluting first with
CH2Cl2 and then 3% MeOH in CH2Cl2. The appropriate fractions
were collected and concentrated to yield a white foam which upon
recrystallization from CH2Cl2–Et2O–petroleum ether (bp 40–60 °C)
afforded JPM-OEt (5.30 g, 13.5 mmol, 76%) as a white solid;
Rf 0.35 (CH2Cl2–MeOH, 9:1).
HRMS: m/z [M + H]+ calcd for C8H13O5 : 189.0763; found:
+
189.0648.
Synthesis of Diethyl (2S,3S)-(+)-2,3-Epoxysuccinate (4) via L-
Arginine Method
A solution of L-arginine (33.24 g, 0.1908 mol) in warm water (50–
60 °C; 92 mL) was added gradually over a 45 min period to a stirred
solution of ( )-trans-epoxysuccinic acid (11) (1 equiv) in MeOH
(368 mL). The mixture was then allowed to stand at r.t. for 5 h be-
fore standing overnight at 4 °C. The precipitate was filtered, washed
with MeOH–H2O (4:1, 75 mL) and then recrystallized from
MeOH–H2O (425 mL, 2:1) to give 12 (26.0 g) as fine, brilliant crys-
tals after drying under vacuum overnight. Subsequently, 95%
H2SO4 (25.0 g) was added dropwise to 12 in absolute EtOH (300
mL). The mixture was refluxed for 5 h, during which it turned into
a colorless solution. The solvent was evaporated to a minimum (ca
50 mL remaining) and the colorless residue was poured into ice-wa-
ter (200 mL). The aqueous layer was extracted with CH2Cl2 (4 ×
100 mL), and the combined organics were washed with sat.
NaHCO3, dried (MgSO4), and evaporated, giving the title com-
pound (13.9 g, 73.9 mmol, 78% yield based on single enantiomeric
isolation) as a colorless oil that required no further purification.
1H NMR (DMSO-d6): d = 9.12 (s, 1 H), 8.57 (d, J = 8.2 Hz, 1 H),
8.10 (t, J = 5.9 Hz, 1 H), 6.95 (d, J = 8.2 Hz, 2 H), 6.63 (d, J = 8.6
Hz, 2 H), 4.31–4.23 (m, 1 H), 4.22–4.12 (m, 2 H), 3.70 (d, J = 1.7
Hz, 1 H), 3.58 (d, J = 1.5 Hz, 1 H), 3.28–3.09 (m, 2 H), 2.56 (t, J =
7.0 Hz, 2 H), 1.55–1.44 (m, 1 H), 1.44–1.35 (m, 2 H), 1.23 (t, J =
7.0 Hz, 3 H), 0.86 (d, J = 6.4 Hz, 3 H), 0.82 (d, J = 6.4 Hz, 3 H).
Synthesis of Ethyl (2S,3S)-(p-Nitrophenyl)-oxirane-2,3-dicar-
boxylate (6)
13C NMR (DMSO-d6): d = 170.70, 166.93, 164.20, 155.35, 129.29,
129.10, 114.82, 61.47, 52.88. 51.19, 51.12, 41.09, 40.50, 34.17,
24.24, 22.92, 21.69, 13.97.
Diethyl epoxysuccinate 4 (6.95 g, 0.0369 mol) was dissolved in ab-
solute EtOH (100 mL) and placed in an ice-bath. A solution of KOH
(2.44 g, 0.0369 mol) in absolute EtOH (50 mL) was added dropwise
over 15 min to the colorless solution. The reaction mixture was then
allowed to stir at 0 °C for 3 h, and then 2 h at r.t. After evaporation
of solvent in vacuo, H2O (100 mL) was added to the resulting white
solid and the solution was washed once with CH2Cl2 (30 mL). The
aqueous layer was acidified with concd HCl (3.5 mL) and NaCl (30
g) was added. After extraction with EtOAc (4 × 100 mL), the or-
ganic layers were collected, dried (MgSO4), and concentrated to af-
ford the crude half ester (5) as a colorless oil. Next, a solution of
DCC (7.84 g, 0.038 mmol) in EtOAc (20 mL) was added dropwise
to a solution of crude compound 5 and p-nitrophenol (5.14 g, 0.0369
mol) at 0 °C. During addition, the solution developed a white pre-
cipitate. After the reaction was stirred at r.t. overnight, the mixture
was filtered and the precipitate was washed with EtOAc (2 ×). The
filtrate was concentrated and the resulting residue was recrystal-
lized from EtOAc–hexanes to afford 6 (6.85 g, 66%) as off-white
crystals; Rf 0.24 (EtOAc–hexanes, 1:4).
ESI–MS: m/z = 393.1 [M + H]+, 415.2 [M + Na]+.
+
HRMS: m/z [M + H]+ calcd for C20H29N2O6 : 393.2026; found:
393.1947.
N-[(L-trans-3-Carboxyoxiran-2-yl)carbonyl]-L-leucyl-3-(p-hy-
droxyphenyl)ethylamide, Sodium Salt [JPM-565 Na+ Salt (2)]
NaOH (1 N, 3.9 mL) was added to a solution of JPM-OEt (1.50 g,
3.82 mmol) in EtOH (20 mL). The solution was stirred at r.t. for 4
h and concentrated under reduced pressure. The solid was redis-
solved in MeOH (20 mL) and ice-cold Et2O was added to precipi-
tate the product. The sodium salt was filtered, washed twice with
Et2O and dried in vacuo to afford the sodium salt of JPM-565, 2
(1.20 g, 3.11 mmol, 81%) as a cream-colored solid.
1H NMR (DMSO-d6): d = 8.54 (d, J = 8.6 Hz, 1 H), 8.20 (t, J = 5.1
Hz, 1 H), 6.86 (d, J = 8.6 Hz, 2 H), 6.56 (d, J = 8.6 Hz, 2 H), 4.29–
4.20 (m, 1 H), 3.27 (d, J = 2.0 Hz, 1 H), 3.25–3.05 (m, 2 H), 2.97
(d, J = 2.0 Hz, 1 H), 2.54 (q, J = 6.7 Hz, 2 H), 1.54–1.31 (m, 3 H),
0.84 (d, J = 6.3 Hz, 3 H), 0.80 (d, J = 6.3 Hz, 3 H).
1H NMR (CDCl3): d = 8.31 (d, J = 9.4 Hz, 2 H), 7.37 (d, J = 9.4 Hz,
2 H), 4.34 (dq, J = 7.1, 4.3 Hz, 2 H), 3.96 (d, J = 1.6 Hz, 1 H), 3.88
(d, J = 1.6 Hz, 1 H), 1.38 (t, J = 7.1 Hz, 3 H).
13C NMR (CDCl3): d = 166.3, 164.8, 154.5, 146.0, 125.7, 122.3,
63.1, 53.0, 52.2, 14.6.
13C NMR (DMSO-d6): d = 171.13, 169.01, 166.95, 157.30, 129.15,
127.44, 115.25, 54.86, 52.19, 51.01, 41.12, 40.40, 34.04, 24.30,
22.95, 21.71.
+
HRMS: m/z [M + H]+ calcd for C18H25N2O6 : 365.1713; found:
365.1634.
N-{[L-trans-3-(Ethoxycarbonyl)oxiran-2-yl]carbonyl}-L-leucyl-
3-(p-hydroxyphenyl)ethylamide [JPM-OEt (3)]
Labeling Experiments
A solution of leucine t-butyl ester hydrochloride (3.98 g, 17.78
mmol) and diisopropylethylamine (3.1 mL, 17.78 mmol, 1.0 equiv)
in CH2Cl2 (40 mL) was added dropwise to a solution of nitrophenyl
ester 6 (5.0 g, 17.78 mmol) in EtOAc (50 mL). After stirring over-
night, the reaction mixture was diluted with Et2O (200 mL), subse-
quently washed with H2O, 2% NaOH (6 × 50 mL), H2O (3 × 100
mL) and brine, dried (MgSO4) and concentrated under reduced
pressure to afford 7 as a colorless oil. The t-butyl ester 7 was dis-
solved in CH2Cl2 (50 mL) and triisopropylsilane (2.5 mL), H2O (2.5
mL) and trifluoroacetic acid (50 mL) were added. The colorless so-
lution was stirred for 4 h at r.t. and then concentrated under reduced
Cells of the EL4 mouse lymphoma cell line were treated for 30 min
with equal amounts of I-125 labeled compound 2 and 3, either with
or without a 30 min pretreatment with 50 mM JPM-OEt (3). Cell ly-
sates were prepared using glass beads in a buffer of pH 5.5 (50 mM
acetate buffer, 2 mM DTT and 5 mM MgCl2), and treated in similar
fashion as the whole cells. Samples were quenched by dilution with
4 × SDS sample buffer, boiled and run on a 12.5% SDS-PAGE gel.
Labeled proteins were visualized by exposure of the gel to a phos-
phorimaging screen and scanning on a typhoon scanner.
Synthesis 2005, No. 2, 240–244 © Thieme Stuttgart · New York