Neuronal Nitric Oxide Synthase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6259
L-ArgNO -OH (0.319 g, 0.001 mol) in dry THF (20 mL) was cooled
2
(4.288 + 4.275) (d, 2H, J ) 6.5 Hz), 4.182 (t, 1H, J ) 6.5 Hz),
3.984-3.940 (m, 1H), 3.398-3.358 (m, 2H), 3.103 (t, 1H, J )
7.5 Hz), 3.014 (t, 1H, J ) 8 Hz), 2.092 (t, 1H, J ) 8.5 Hz), 1.986-
1.951 (m, 2H); 13C NMR (125.7 MHz, DMSO-d6) δ 175.2 (1C),
155.7 (1C), 143.9 (2C), 140.8 (2C), 138.3 (1C), 128.8 (2C), 128.2
(2C), 127.6 (2C), 127.1 (3C), 125.1 (2C), 120.1 (2C), 67.7 (1C),
65.2 (1C), 58.2 (1C), 57.8 (1C), 48.9 (1C), 46.7 (1C), 36.7 (1C).
ESI(CH3OH) m/z ) 442.6 ([M + H]+).
to -15 °C, and 4-methylmorpholine (0.111 g, 0.121 mL, 0.0011
mol) and isobutyl chloroformate (0.150 g, 0.144 mL, 0.0011 mol)
were added. The reaction mixture was stirred for 30 min, and the
free base form of 12 (0.241 g, 0.0011 mol) in anhydrous THF (5
mL) was added via a cannula. The solution was stirred for 30 min
at -15 °C and then allowed to warm to room temperature over 30
min. The reaction mixture was stirred at room temperature for 1 h,
and the solvent was evaporated in vacuo. The residue was suspended
in EtOAc (20 mL) and washed successively with 5% NaHCO3 (2
× 20 mL) and saturated NaCl (20 mL). The solution was dried
over anhydrous MgSO4, and the solvent was removed in vacuo.
The residue was purified by silica gel column chromatography
(CH2Cl2:MeOH ) 9:1) to afford 0.36 g of a colorless oil (70%):
1H NMR (500 MHz, CD3OD) δ 7.344-7.214 (m, 5H), 4.295-
4.266 (m, 1H), 4.000-3.800 (m, 1H), (3.878 + 3.852) (d, 1H, J )
13 Hz), (3.521 + 3.495) (d, 1H, J ) 13 Hz), 3.288-3.206 (m,
4H), 2.280 (t, 1H, J ) 8.5 Hz), 2.177-2.123 (m, 1H), 2.087-
2.026 (m, 1H), 1.696-1.525 (m, 4H), 1.400 (s, 9H); 13C NMR
(125.7 MHz, CD3OD) δ 179.8 (1C), 174.8 (1C), 161.1 (1C), 157.9
(1C), 139.2 (1C), 130.3 (2C), 129.6 (2C), 128.6 (1C), 80.8 (1C),
67.0 (1C), 60.2(1C), 58.9 (1C), 55.6 (1C), 41.8 (2C), 37.8 (1C),
30.8 (1C), 28.8 (3C), 26.1 (1C); ESI (CH3CN) m/z ) 521.1 ([M +
H]+), m/z ) 543.1 ([M + Na]+).
Nr-Methyl-trans-4-N-amino-L-proline-NH2 Dihydrochloride
Salt (11). 9 (0.365 g, 0.001 mol) was treated with 20% piperidine
(10 mL) in anhydrous DMF and stirred under a nitrogen atmosphere
for 2 h. TLC monitored the complete conversion of the starting
material to product. The reaction mixture was then concentrated
under reduced pressure. The resulting residue was dissolved in a
dilute HCl aqueous solution (four drops of concentrated HCl in 20
mL of H2O) and washed with hexane to remove the piperidine-
dibenzofulvene adduct and the free dibenzofulvene. The aqueous
layer was concentrated in vacuo to yield the desired white solid
(0.216 g, quantitative yield): 1H NMR (500 MHz, D2O) δ 3.921-
3.861 (m, 1H), (3.557 + 3.542, 3.536 + 3.522) (dd, 1H, J ) 7.5
Hz, J ) 7 Hz), 3.340-3.306 (m, 1H), 2.566-2.529 (m, 1H), 2.405
(s, 3H), 2.368-2.236 (m, 2H); 13C NMR (125.7 MHz, D2O) δ 176.4
(1C), 66.6 (1C), 58.7 (1C), 48.0 (1C), 39.7 (1C), 34.3 (1C); ESI
(CH3OH) m/z ) 144.2([M + H]+).
4-N-(L-ArgNO2)-Nr-methyl-trans-4-amino-L-proline-NH2 Ditri-
fluoroacetate (3). 13 (0.222 g, 0.001 mol) was dissolved in
anhydrous CH2Cl2 (1 mL), and TFA (1 mL) was added at 0 °C.
The reaction mixture was stirred for 1 h at 0 °C and 1 h at room
temperature. After the reaction was done, the solvent was evaporated
in vacuo. The residue was partitioned between water (10 mL) and
ethyl acetate (10 mL). The aqueous layer was then washed with
ethyl acetate (2 × 10 mL). The combined aqueous layers were
evaporated by high-vacuum rotary evaporation to afford a white
foamy solid (0.286 g, quantitative yield): 1H NMR (500 MHz,
D2O) δ 4.459-4.446 (m, 1H), 4.382-4.354 (m, 1H), 4.040-4.018
(m, 1H), 3.933-3.922 (m, 1H), 3.220-3.151 (m, 3H), 2.914
(s, 3H), 2.460 (m, 2H), 1.855 (m, 2H), 1.597 (m, 2H); 13C NMR
(125.7 MHz, D2O) δ 169.6 (1C), 169.4 (1C), (162.9 + 162.6) (2C,
TFA-COOH), 158.9 (1C), (119.6 + 117.5 + 115.1 + 112.8) (2C,
TFA-CF3), 67.3 (1C), 59.0 (1C), 52.8 (1C), 47.4 (1C), 40.9 (1C),
40.3 (1C), 34.6 (1C), 27.9 (1C), 23.0 (1C). Comb. Anal. (C12H24N8O4‚
3TFA) C, H, N.
Nr-Benzyl-trans-4-N-amino-L-proline-NH2 Dihydrochloride
(12). 10 (0.441 g, 0.001 mol) was treated with 20% piperidine (10
mL) in anhydrous DMF and stirred under a nitrogen atmosphere
for 2 h. After complete conversion of the starting material to product
(by TLC) the reaction mixture was concentrated under reduced
pressure. The resulting residue was dissolved in the dilute aqueous
HCl solution (four drops of concentrated HCl in 20 mL of H2O)
and washed with CH2Cl2 to remove the piperidine-dibenzofulvene
adduct and the free dibenzofulvene. The aqueous layer was
concentrated in vacuo to yield the desired white solid (0.292 g,
quantitative yield): 1H NMR (500 MHz, D2O) δ 7.471-7.414 (m,
5H), 4.650-4.614 (m, 1H), (4.504 + 4.479) (d, 1H, J ) 7.5 Hz),
(4.459 + 4.434) (d, H, J ) 7.5 Hz), 4.121-4.092 (m, 2H), 3.592-
3.540 (m, 1H), 2.757-2.708 (m, 1H), 2.595-2.533 (m, 1H); 13C
NMR (125.7 MHz, D2O) δ 168.7 (1C), 131.1 (2C), 130.8 (1C),
129.5 (2C), 128.6 (1C), 64.6 (1C), 58.7 (1C), 55.6 (1C), 46.9 (1C),
33.1 (1C); ESI (CH3OH) m/z ) 220.2 ([M + H]+).
4-N-(Nr-Boc-L-ArgNO2)-Nr-methyl-trans-4-amino-L-proline-
NH2 (13). 11 (0.324 g, 0.0015 mol) was dissolved in 0.5 M NaOH
aqueous solution (10 mL) and then was extracted with CH2Cl2 (10
mL × 2). The combined organic layers were washed with water
(10 mL) to generate the free base form of 11. A solution of
HPLC conditions for the purification of 4-N-(L-ArgNO2)-Nr-
methyl-trans-4-amino-L-proline-NH2 ditrifluoroacetate (3): col-
umn, Phenomenex Gemini 5 µm C18 110A preparative column;
column size, 250 × 21.2 mm 5 µm; injection concentration, 20
mg/mL; flow rate, 12 mL/min; detection, 254 nm; mobile phase,
H2O (0.1% TFA):60% CH3CN-40% H2O (0.08% TFA) ) 97:3;
isocratic method. The retention time was 5 min.
NR-Boc-L-ArgNO -OH (0.319 g, 0.001 mol) in dry THF (20 mL)
2
was cooled to -15 °C, and 4-methylmorpholine (0.111 g, 0.121
mL, 0.0011 mol) and isobutyl chloroformate (0.150 g, 0.144 mL,
0.0011 mol) were added. The reaction mixture was stirred for 30
min, and the free base form of 11 (0.157 mL, 0.0011 mol) in
anhydrous THF (5 mL) was added via a cannula. The solution was
stirred for 30 min at -15 °C and was then allowed to warm to
room temperature over 30 min. The reaction mixture was stirred
at room temperature for 1 h, and the solvent was evaporated in
vacuo. The residue was purified directly by silica gel column
chromatography (CH2Cl2:MeOH ) 8.5:1.5) to afford 0.39 g of a
colorless oil (88%): 1H NMR (500 MHz, CD3OD) δ 4.364-4.336
(m, 1H), 4.080-4.015 (m, 1H), 3.407-3.376 (m, 1H), 3.311-3.271
(m, 2H), 3.036-3.003 (m, 1H), 2.380 (s, 3H), 2.311 (t, 1H, J )
8.5 Hz), 2.209-2.152 (m, 1H), 2.094-2.055 (m, 1H), 1.800-1.636
(m, 4H), 1.437 (s, 9H); 13C NMR (125.7 MHz, CD3OD) δ 179.1
(1C), 174.8 (1C), 161.0 (1C), 158.0 (1C), 80.8 (1C), 68.9 (1C),
61.9 (1C), 55.6 (1C), 41.8 (1C), 41.5 (2C), 38.3 (1C), 30.8 (1C),
28.8 (3C), 26.2 (1C); ESI (CH3CN) m/z ) 445.4 ([M + H]+), m/z
) 467.3 ([M + Na]+).
4-N-(L-ArgNO2)-Nr-benzyl-trans-4-amino-L-proline-NH2 ditri-
fluoroacetate (4). 14 (0.260 g, 0.5 mmol) was dissolved in
anhydrous CH2Cl2 (1 mL), and TFA(1 mL) was added at 0 °C.
The reaction mixture was stirred for 1 h at 0 °C and for 1 h at
room temperature. After the reaction was completed, the sol-
vent was evaporated in vacuo, and the residue was dissolved in
water (10 mL). The color impurity was extracted with ethyl ace-
tate (10 mL). The aqueous layer was then washed with ethyl acetate
(2 × 10 mL), and the combined aqueous layers were evaporated
by high-vacuum rotary evaporation to afford a white foamy solid
(0.324 g, quantitative yield): 1H NMR (500 MHz, D2O) δ 7.279-
7.242 (m, 5H), 4.329-4.183 (m, 4H), 3.807-3.772 (m, 2H),
3.151-3.074 (m, 3H), 2.338-2.215 (m, 2H), 1.725 (m, 2H), 1.466
(m, 2H); 13C NMR (125.7 MHz, D2O) δ 169.5 (1C), 169.4 (1C),
(162.8 + 162.5 + 162.3 + 162.0) (2C, TFA-COOH), 158.6 (1C),
130.8 (2C), 130.4 (1C), 129.3 (2C), 128.7 (1C), (119.5 + 117.2 +
114.9 + 112.6) (2C, TFA-CF3), (64.8 + 64.7) (1C), 58.9 (1C),
57.1 (1C), (52.8 + 52.7) (1C), (47.14 + 47.09) (1C), 40.2 (1C),
34.3 (1C), 27.9 (1C), 22.9 (1C); ESI (CH3OH) m/z ) 421.3 ([M +
H]+), m/z ) 443.3 ([M + Na]+); HRMS (ES+, CH3OH) calcd for
421.2312, found 421.2319. Comb. Anal. (C18H28N8O4‚3TFA) C,
H, N.
4-N-(Nr-Boc-L-ArgNO2)-Nr-benzyl-trans-4-amino-L-proline-
NH2 (14). 12 (0.438 g, 0.0015 mol) was dissolved in 0.5 M NaOH
aqueous solution (10 mL) and then extracted with CH2Cl2 (10 mL
× 2). The combined organic layers were washed with water (10
mL) to generate the free base form of 12. A solution of NR-Boc-