1098 Chem. Res. Toxicol., Vol. 15, No. 8, 2002
Yoshioka et al.
500 NMR spectrometer at 202.5 and 125.7 MHz, respectively.
methyl ester‚HCl (vide supra) in 6 mL of pyridine, and stirred
at room temperature for 2.5 h. The reaction mixture was
concentrated in vacuo; the residue was dissolved in CH2Cl2 and
washed with 1 N HCl and then H2O and dried over Na2SO4.
Evaporation of the organic solvent gave a solid (91%), which
was purified by recrystallization from a CH3OH/H2O mixture.
A 6 mL solution of HBr in CH3CO2H (30%, w/v) was added to
the compound (700 mg) and the reaction proceeded at room
temperature for 40 min, followed by addition of (C2H5)2O to give
a precipitate (97% yield). The compound (560 mg) was then
hydrolyzed with 1 N NaOH in 5 mL aqueous CH3OH to give
the title compound, which was neutralized by addition of 1 N
HCl, followed by purification by using AG1X8 ion-exchange resin
(acetate form; 1.0 × 15 cm) with elution with H2O. After
lyophilization, the solid (76% yield, overall yield 67%) was
obtained as a fine powder (recrystallized from aqueous C2H5-
OH): mp 124 °C (decomp.); 1H NMR (D2O) δ 1.37 (m, 2H, γ-H),
1.67 (m, 2H, δ-H), 1.81 (m, 1H, â -H1), 1.89 (m, 1H, â-H2), 2.97
(t, 2H, J ) 7.5 Hz, ꢀ-H), 4.18 (dd, 1H, J ) 5.2 and 7.8 Hz, R-H);
13C NMR (D2O) δ 22.7 (γ), 27.0 (δ), 31.5 (â), 39.8 (ꢀ), 55.9 (R),
161.0 (CONH), 178.5 (CO2H); MS, m/z 347 (MH+).
(6) Bis(N6-L-lysyl)oxa lyl Am id e. To 3.0 mmol of L-Lys in
3.0 mL of 1 N NaOH (3.0 mmol), 190 µL (0.5 eq) of diethyl
oxalate was added. The reaction was stirred for 15 min in an
ice-water bath and then at room temperature. After 1.5 h at
room temperature, 2.5 mL of 1 N HCl was added; the solid was
collected and dried to give the product (40% yield): mp 280 °C
(decomp.); 1H NMR (D2O-DCl) δ 1.50 (m, 2H, γ-H), 1.61 (m, 2H,
â-H), 1.99 (m, 2H, δ-H), 3.31 (t, 2H, J ) 6.7 Hz, ꢀ-H), 4.13 (t,
1H, J ) 6.3 Hz, R-H); 13C NMR (D2O-DCl) 22.1 ppm (γ), 28.2
ppm (â), 29.8 ppm (δ), 39.5 ppm (ꢀ), 53.2 ppm (R), 161.4 ppm
(CONH), 172.3 ppm (CO2H); MS, m/z 347 (MH+).
31P NMR showed a peak at -0.50 ppm (using 85% H3PO4 as an
external standard) and 13C NMR showed peaks at 161.0 (J CP
10.3 Hz) and 164.4 ppm (J CP ) 7.3 Hz) (using dioxane as an
external standard).
)
Syn th esis of Lys Der iva tives. (1) N6-Tr ich lor oa cetyl-L-
Lys. To 5.0 mmol of L-Lys in 2.5 mL of 2 N NaOH (5.0 mmol),
1.0 mL (1.5 equiv) of ethyl trichloroacetate was added in one
portion and the reaction was stirred for 2 h at room temperature.
The solid that was formed was collected, washed with CH3CN,
and then dried to give the product (23% yield, fine needles from
aqueous C2H5OH): mp 204 °C (decomp.); 1H NMR (D2O) δ 1.51
(m, 2H, γ-H), 1.72 (m, 2H, â-H), 1.91 (m, 2H, δ-H), 3.41 (t, 2H,
J ) 6.9 Hz, ꢀ-H), 3.76 (t, 1H, J ) 6.3 Hz, R-H); 13C NMR (D2O)
22.4 ppm (γ), 28.2 ppm (δ), 30.8 ppm (â), 41.4 ppm (ꢀ), 55.3 ppm
(R), 92.3 ppm (COCCl3), 165.1 ppm (COCl3), 175.4 ppm (CO2H);
MS, m/z 291/293/295/297 (MH+).
(2) N2-Tr ich lor oa cetyl-L-Lys. N6-Carbobenzyloxy (CBZ)-L-
Lys benzyl ester‚HCl was prepared (28) by heating N6-CBZ-L-
Lys (9.81 g, 35.0 mmol) and p-toluenesulfonic acid (6.7 g, 35
mmol) in a mixture of 100 mL of toluene and 66 mL of benzyl
alcohol for 20 h, using a Dean-Stark trap. The yield was 65%.
To 1.5 mmol of N6-CBZ-L-Lys benzyl ester in 8 mL of CH2Cl2
and 0.8 mL of (C2H5)3N, trichloroacetyl chloride (0.22 mL, 1.3
equiv) was added; the reaction was stirred at room temperature
for 50 min. The reaction mixture was diluted with CH2Cl2 and
washed sequentially with 1 N HCl, 1 N NaOH, and H2O and
dried over Na2SO4. Evaporation of the organic solvent gave a
pale yellow syrup (92%). The compound was then hydrolyzed
with 1 NaOH in aqueous CH3OH (200 mL, room temperature,
1 h) to the corresponding carboxylic acid derivative (80%), which
was treated with 4 mL of HBr in CH3CO2H (30%, w/v) for 1 h,
followed by the addition of (C2H5)2O to give N2-trichloroacetylLys‚
HBr (91%, overall yield 67%). Neutralization with NaHCO3
followed by purification using a Sep-Pak Vac 35 cm3 cartridge
(C18, 10 g, Waters) gave free base (fine powder from aqueous
C2H5OH): mp 191 °C, (decomp); 1H NMR (D2O) δ 1.42 (m, 2H,
γ-H), 1.66 (m, 2H, δ-H), 1.83 (m, 1H, â-H1), 1.96 (m, 1H, â-H2),
2.98 (t, 2H, J ) 7.6 Hz, ꢀ-H), 4.20 (dd, 1H, J ) 5.1 and 8.1 Hz,
R-H); 13C NMR (D2O) δ 22.7 (γ), 27.0 (δ), 31.2 (â), 39.9 (ꢀ), 57.2
(R), 92.2 (CCl3), 164.2 (COCCl3), 178.0 (CO2H); MS m/z 291/293/
295/297 (MH+).
(7) (N2,N6-L-Lysyl)oxa lyl Am id e. N2-Ethoxycarbonylcarbo-
nyl-N6-CBZ-L-Lys methyl ester was prepared in the following
procedure: ethyl chlorooxoacetate (0.27 mL, 1.2 equiv) was
added to N6-CBZ-L-Lys methyl ester‚HCl (2.0 mmol) in
a
mixture of 8 mL of CH2Cl2 and 1.0 mL (C2H5)3N at 0 °C, and
the reaction was stirred for 1.5 h, yielding a pale yellow syrup
after concentration in vacuo (0.86 g, 91% yield). N2-CBZ-L-Lys
methyl ester (2.3 mmol) (28, 30) was added to 1.6 mmol of N2-
ethoxycarbonylcarbonyl-N6-CBZ-L-Lys methyl ester and the
reaction stirred at room temperature for 2 days. The reaction
mixture was evaporated and the residue was dissolved in CHCl3
and washed with 0.5 N HCl and dried over Na2SO4. Concentra-
tion in vacuo and purification using silica gel flash chromatog-
raphy give the corresponding oxalyl amide (57%). To 500 mg of
the compound, 4 mL of HBr in CH3CO2H (30%, w/v) was added
and the reaction kept at room temperature for 1 h followed by
addition of (C2H5)2O. After removal of the (C2H5)2O by decanta-
tion, the precipitate was extracted with H2O and NaOH was
added (to pH ∼10) and the reaction was allowed to stand at
room temperature to give sodium salt of the title compound.
Neutralization followed by purification by using a Sep-Pak Vac
35 cm3 cartridge (C18, 10 g, Waters) gave the product in 66%
yield (overall yield 38%) (fine powder from aqueous C2H5OH):
(3) N6-Oxa lyl-L-Lys. The compound was synthesized accord-
ing to a literature procedure (29) (yield 45%, fine needles from
aqueous 2-propanol): mp 182 °C (decomp.); 1H NMR (D2O) δ
1.42 (m, 2H, γ-H), 1.58 (m, 2H, â-H), 1.90 (m, 2H, δ-H), 3.24 (t,
2H, J ) 6.7 Hz, ꢀ-H), 3.92 (t, 1H, J ) 6.5 Hz, R-H); 13C NMR
(D2O) 22.2 ppm (γ), 28.4 ppm (â), 30.2 ppm (δ), 39.6 ppm (ꢀ),
54.1 ppm (R), 163.8 ppm (COCO2H), 165.5 ppm (COCO2H), 173.8
ppm (CO2H); MS m/z 219 (MH+).
(4) N2-Oxa lyl-L-Lys. Ethyl chlorooxoacetate (0.27 mL, 1.2
equiv) was added to 2.0 mmol of N6-CBZ-L-Lys benzyl ester HCl
(vide supra) dissolved in 8 mL of CH2Cl2 [and 1 mL of (C2H5)3N]
and stirred at 0 °C for 1.5 h. The reaction mixture was diluted
with CH2Cl2 and washed with 1 N HCl, 1 N NaOH, and then
H2O and dried with Na2SO4. Evaporation of the organic solvent
gave a pale yellow syrup (91%). The compound was then
hydrolyzed with 0.7 g of NaOH in a mixture of 5 mL of (CH3)2-
NCHO and 10 mL (1 h, room temperature) to give the corre-
sponding dicarboxylic acid derivative (82%), which was then
treated with 6 mL of HBr in CH3CO2H (30%, w/v) for 1.5 h
followed by addition of (C2H5)2O to give the HBr salt of the title
compound in quantitative yield (overall yield 75%). Neutraliza-
tion with NaHCO3 yielded the free base (prisms from aqueous
1
mp 242 °C (decomp.); H NMR (D2O) δ 1.43 (m, 4H, γ-H), 1.68
(m, 4H, δ-H), 1.89 (m, 4H, â-H), 3.01 (t, 1H, J ) 7.4 Hz, ꢀ-H),
3.33 (dt, 1H, J ) 2.4, 6.8 Hz, ꢀ′-H), 3.74 (t, 1H, J ) 5.7 Hz, R′-
H), 4.22 (dd, 1H, J ) 5.0 and 8.0 Hz, R-H); 13C NMR (D2O):
22.3 and 22.7 (γ), 26.7 and 28.4 (δ), 30.7 and 31.5 (â), 39.8 and
39.9 (ꢀ), 55.3 and 55.8 (R), 161.1 and 161.5 (CONH), 175.4 and
178.6 (CO2H); MS, m/z 347 (MH+).
Syn t h esis of Or n it h in e (Or n ) Der iva t ives. (1) N5-
Tr ich lor oa cetyl-L-Or n . To 5.0 mmol of l-Orn‚HCl in 5.0 mL
of 2 N NaOH (10 mmol), 1.0 mL (1.5 equiv) of ethyl trichloro-
acetate was added in one portion and stirred for 1.5 h at room
temperature. After the reaction mixture was neutralized by
adding 3 N HCl, the reaction mixture was concentrated in vacuo
to give a white solid. After cooling, the solid was collected and
then dried to give the product (36% yield, fine needles from
aqueous C2H5OH): mp 202 °C (decomp.); 1H NMR (D2O) δ 1.67
(m, 2H, γ-H), 1.88 (m, 2H, â-H), 3.37 (t, 2H, J ) 6.8 Hz, δ-H),
1
C2H5OH) mp 225 °C (decomp.); H NMR (D2O) δ 1.43 (m, 2H,
γ-H), 1.67 (m, 2H, δ-H), 1.85 (m, 1H, â -H1), 1.96 (m, 1H, â-H2),
2.97 (t, 2H, J ) 7.4 Hz, ꢀ-H), 4.36 (dd, 1H, J ) 4.9 and 8.8 Hz,
R-H); 13C NMR (D2O) δ 22.7 (γ), 26.8 (δ), 30.8 (â), 39.8 (ꢀ), 53.6
(R), 165.0 (COCO2H), 165.9 (COCO2H), 176.4 (CO2H); MS m/z
219 (MH+).
(5) Bis(N2-L-lysyl)oxa lyl Am id e. Oxalyl chloride (0.13 mL,
0.5 eq) was added in portions to 3.0 mmol of N-CBZ-L-Lys