B2 and B1 Receptor Antagonist Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1479
chilled to 0 °C under nitrogen; to this solution was added, by
dropwise addition, a solution of benzyl 2-bromoacetate (7.93
mL, 50 mmol) in 50 mL of THF. The reaction mixture was
allowed to warm to room temperature and then stirred for ca.
15 h. The solvent was removed in vacuo, and the residue was
taken up in 200 mL of CH2Cl2 and washed with 100 mL of
10% Na2CO3 solution. The Na2CO3 solution was extracted
twice with 50 mL of CH2Cl2. All CH2Cl2 layers were combined,
dried over anhydrous MgSO4, and concentrated in vacuo. The
residue was treated with 26 mL of 4 N HCl in dioxane, volatiles
were removed in vacuo, and the residue was triturated with
cold, anhydrous diethyl ether. The solid was collected and
dried for several hours under reduced pressure to yield 15 g
of the hydrochloride salt contaminated with traces of cyclo-
hexylamine hydrochloride. This mixture was dissolved in 300
mL of CH2Cl2, and the solution was washed with 100 mL of
10% Na2CO3 solution. The aqueous solution was back-
extracted with CH2Cl2. All CH2Cl2 layers were combined,
dried over MgSO4, and dried under high vacuum (∼1 Torr) to
provide 11.63 g (91%) of the title compound as an oil: 1H NMR
(CDCl3) δ 1.0-1.32 (m, 4H), 1.55-1.78 (m, 4H), 1.83 (m, 2H),
2.41 (tt, J ) 10, 4 Hz, 1H), 3.48 (s, 2H), 5.17 (s, 2H), 7.36 (s,
5H); 13C NMR (DMSO) δ 24.62, 25.82, 33.08, 48.02, 56.12,
66.28, 128.15, 128.36, 135.47, 142.53.
Boc-N-cycloh exylglycin e Ben zyl Ester . N-Cyclohexy-
lglycine benzyl ester (11.0 g, 44.4 mmol) was dissolved in 44.4
mL of dioxane, and 44.4 mL of 1 N NaOH solution was added
followed by di-tert-butyl dicarbonate (10.68 g, 48.8 mmol).
Stirring was continued for ca. 15 h, after which volatiles were
removed by rotary evaporation. The resulting residue was
partitioned between 100 mL of water and 100 mL EtOAc. The
layers were separated, and the aqueous layer was acidified to
pH 3 with 5% KHSO4 solution. The aqueous layer was
extracted with EtOAc, and all EtOAc solutions were combined,
washed with saturated NaHCO3 solution and saturated NaCl
solution, and dried over MgSO4. Removal of the solvent by
rotary evaporation and further drying under high vacuum
provided 14.50 g (94%) of the title compound as an oil: 1H
NMR (CDCl3) δ 0.95-1.55 (m, 14H), 1.63 (m, 1H), 1.77 (m,
4H), 3.70-4.1 (m, 3H), 5.16 (s, 2H), 7.36 (s, 5H).
Boc-N-cycloh exylglycin e. Boc-N-cyclohexylglycine ben-
zyl ester (14.0 g, 40 mmol) was dissolved in 240 mL of
anhydrous EtOH, flushed with dry nitrogen, and combined
carefully under inert atmosphere with 1.5 g of 10% palladium
on carbon. Using a Parr apparatus, the nitrogen atmosphere
was replaced with hydrogen (43 psi), and the mixture was
shaken over 24 h at room temperature. The mixture was
purged with nitrogen, and the catalyst and solids were
removed by filtration through a pad of Celite. The filtrate was
concentrated by rotary evaporation, and the residue was taken
up in 300 mL of EtOAc. The EtOAc layer was extracted with
150 mL of 1 N NaOH solution. The basic solution was acidified
in an ice bath to pH 3 with 1 N HCl. The acid solution was
extracted with EtOAc (3 × 100 mL). This solution was washed
with saturated NaCl solution and concentrated in vacuo to a
colorless oil which was further dried under high vacuum. The
resulting glass/solid was triturated with hexane, which after
filtration and drying provided the title compound (8.89 g, 87%)
as a colorless powder: mp 103-104 °C (uncorrected); 1H NMR
(CDCl3) δ 1.07 (m, 1H), 1.10-1.55 (m, 4H), 1.43 (s, 9H), 1.55-
1.9 (m, 5H), 3.67-4.13 (m, 3H), 11.33 (br s, 1H); 13C NMR
(CDCl3) δ 25.43, 25.68, 28.20, 30.91, 44.03, 44.26, 44.36, 44.42,
80.64, 80.77, 126.32, 126.42, 128.5, 128.56, 128.80, 138.79,
138.93, 155.97, 175.32, 176.02. Anal. (C15H21NO4) C, H, N.
Nr-Boc-N-Cycloh exylm eth ylglycin e. The title compound
was prepared as described for Boc-N-cyclohexylglycine: 1H
NMR (CDCl3) δ 0.84-1.8 (m, 20H), 3.04-3.2 (m, 2H), 3.9-
4.04 (rotamers, 2H), 11.44 (br s, 1H); 13C NMR (CDCl3) δ 25.67,
25.78, 26.25, 26.36, 28.13, 28.19, 28.24, 30.59, 30.63, 30.72,
36.85, 37.08, 37.69, 45.53, 49.08, 49.69, 54.47, 54.62, 80.17,
80.37, 155.46, 156.37, 174.68, 175.06. Anal. (C14H25NO4) C,
H, N.
Nr-Boc-N-ben zylglycin e. NaOH (0.3 g, 7.57 mmol) dis-
solved in a minimum amount of water was added to a stirred
solution of NR-Boc-N-benzylglycine ethyl ester (0.74 g, 2.52
mmol) in MeOH (5.0 mL). The reaction mixture was stirred
at room temperature for 18 h. MeOH was evaporated in vacuo,
and the resulting residue was dissolved in water. The aqueous
layer was extracted with CHCl3 (2 × 50 mL) and chilled to 0
°C; then the pH was adjusted to 2.0 with 1 N HCl, and the
solution was then extracted with EtOAc. The EtOAc layer was
then washed with brine, dried (MgSO4), and evaporated in
vacuo to yield 1.18 g (88.0%) of the title compound: 1H NMR
(CDCl3) δ 1.49 (s, 9H), 3.82 (s, 1H), 3.98 (s, 1H), 4.52 (s, 1H),
4.56 (s, 1H), 7.2-7.4 (m, 5H), 11.5 (br s, 1H); 13C NMR (CDCl3)
δ 28.19, 28.26, 47.43, 47.57, 50.85, 51.49, 80.92, 81.10, 127.46,
127.52, 128.08, 128.6, 136.98, 137.16, 155.58, 155.99, 175.40,
175.70. Anal. (C14H19NO4) C, H, N.
Nr-Boc-D-cyclop en tylglycin e. D-Cyclopentylglycine was
synthesized by the method of Dunn.44 D-Cyclopentylglycine
(3.93 g, 27.5 mmol) and NaOH (27.5 mL, 1 M solution) were
added to a mixture of 150 mL of dioxane and 75 mL of H2O.
The solution was chilled to 0 °C, and di-tert-butyl dicarbonate
(6.60 g, 30.2 mmol) was added. The reaction mixture was
warmed to room temperature and stirred for ca. 15 h. Volatiles
were removed in vacuo, and the residue was taken up in water
and basified, using 5% NaOH, to pH 9. The aqueous layer
was extracted three times with EtOAc, and the combined
extracts were washed with brine, dried over anhydrous Na2-
SO4, and concentrated in vacuo to yield 5.44 g (81.5%) of NR-
Boc-cyclopentylglycine as a glass: 1H NMR (CDCl3) δ 1.15-
1.9 (m, 8H), 1.45 (s, 9H), 2.25 (m, 1H), 3.99 (m, 0.32H), 4.25
(m, 0.68H), 5.01 (d, J ) 7.0 Hz, 0.68H), 6.27 (d, J ) 7.0 Hz,
0.32H), NH and R-H yield two signals arising from rotamers.
Gen er a l P r oced u r e for Solid -P h a se Syn th esis of P ep -
tid es: P ep tid es En d in g in C-Ter m in a l Ar gin in e. All
amino acids were protected at the R-nitrogen with the tert-
butoxycarbonyl group. Serine was protected as the benzyl
ether. The guanidine of arginine was protected with the tosyl
group. No side chain protection was employed for hydrox-
yproline.
Resin P r ep a r a tion . PAM resin (generally 0.20-2 g;
Bachem) prederivatized with NR-Boc-Ng-p-tosyl-L-arginine
(∼0.25-0.75 equiv/g) was charged in a vessel designed for
manual solid-phase peptide synthesis.45 The resin was treated
with 25 mL of dry CH2Cl2, with agitation provided by nitrogen
bubbling for 1 min. The solution was drained away, and
washing with CH2Cl2 was repeated (2 × 25 mL). A similar
wash was repeated with dimethylformamide (3 × 25 mL)
followed by a wash with CH2Cl2 (3 × 25 mL).
Dep r otection . The washed resin was treated with 25 mL
of a 1:1 mixture of TFA in CH2Cl2. Agitation was maintained
for 5 min by nitrogen bubbling; then the solvent was filtered
away. The resin was again treated with 25 mL of a 1:1
mixture of TFA in CH2Cl2, agitation was maintained for 25
min by nitrogen bubbling, and then the solvent was filtered
away.
54.03, 54.18, 56.13, 80.36, 80.47, 154.81, 176.83. Anal. (C13H23
NO4) C, H, N.
-
Nr-Boc-N-cyclop en tylglycin e. The title compound was
prepared as described for Boc-N-cyclohexylglycine. The result-
ing compound was recrystallized from EtOAc/hexane to yield
1.09 g (47.0%) of the title compound as a colorless solid: 1H
NMR (CDCl3) δ 1.2-1.72 (m, 15H), 1.76-1.96 (m, 2H), 3.64-
Neu tr a liza tion . The resin was washed sequentially with
CH2Cl2 (3 × 25 mL), DMF (2 × 25 mL), and again with CH2-
Cl2. The resin was washed with a solution of 10% (v/v)
diisopropylethylamine in CH2Cl2 (3 × 25 mL). To remove
traces of base, the resin was washed with CH2Cl2 (3 × 25 mL).
P r oced u r e for Cou p lin g w ith N-Boc-a m in o Acid HOBt
Ester s. Four equivalents of the Boc-protected amino acids
were dissolved in a minimum amount of DMF followed by 4
equiv of HOBt (1-hydroxybenzotriazole monohydrate). To this
was added 4 equiv of DCC (dicyclohexylcarbodiimide). The
reaction mixture was stirred at room temperature for 1 h. The
4.0 (br s, 2H), 4.1-4.6 (m, 1H), 10.36 (br s, 1H). Anal. (C12H21
NO4) C, H, N.
-
Nr-Boc-N-p h en eth ylglycin e. The title compound was
prepared as described for Boc-N-cyclohexylglycine: 1H NMR
(CDCl3) δ 1.45 (s, 9H), 2.8-2.92 (m, 2H), 3.44-3.6 (m, 2H),
3.8, 3.92 (rotamers, 2H), 7.12-7.36 (m, 5H), 11.32 (br s, 1H);
13C NMR (CDCl3) δ 34.65, 35.01, 49.15, 49.69, 50.37, 50.55,