2512 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Pinnen et al.
1H NMR (CDCl3) δ 1.26 (9H, s, SBut), 1.31 and 1.39 (18H, 2× s,
OBut), 1.49-1.79 (2H, m, Glu â-CH2), 2.04-2.10 (2H, m, Glu
γ-CH2), 2.17 and 2.23 (6H, 2× s, LD Ac), 2.93-3.13 (4H, m, Cys
â-CH2 and LD â-CH2), 3.70 (3H, s, OMe), 3.64-3.66 (1H, m,
Gly R-CHA), 4.03-4.09 (2H, m, Gly R-CHB and Glu R-CH), 4.48
(1H, m, LD R-CH), 4.79 (1H, m, Cys R-CH), 5.26 (1H, d, J ) 8.1
Hz, Glu NH), 6.61 (1H, d, J ) 7.05, Cys NH), 6.77 (1H, d, J )
6.3, LD NH), 6.87-7.02 (3H, m, ArH), 7.30 (1H, t, J ) 5.7 Hz,
Gly NH); 13C NMR (CDCl3) δ 20.97 and 21.07 (2× LD Ac), 25.07
(Glu â-CH2), 28.20 and 28.63 (2× OBut) 30.10 (SBut), 31.88
(Glu γ-CH2), 36.96 (LD â-CH2), 40.13 (Cys â-CH2), 43.12
(Gly R-CH2), 48.51 (SBut), 52.71 (OMe), 52.95 (Cys R-CH),
53.19 (Glu R-CH), 53.63 (LD R-CH), 80.13 and 82.18
(OBut), 123.54-141.85 (LD Ar), 156.14 (OCONH), and
168.87, 168.92, 169.17, 170.67, 171.01, 171.89, and 173.58 (7×
CO).
172.65, and 173.21(6× CO); MS (ESI) m/z 555 (M - H)-. Anal.
(C23H32N4O10S) C, H, N, S.
H-Glu-(Cys-Gly-LD-OH)-OH (2). The above-reported tetrapep-
tide 8b (1.35 g, 1.63 mmol) was dissolved in TFA (26.5 mL). After
5 h at room temperature, the solution was evaporated to dryness
and the residue was repeatedly evaporated with ether to give TFA·
H-Glu-[Cys(SBut)-Gly-LD(Ac)2-OMe]-OH (1.11 g, 86%). This
compound, obtained in good yield, was used without further
purification.
A solution of the foregoing trifluoracetate (1.11 g, 1.40 mmol)
in a mixture of n-PrOH/H2O (2:1) (30 mL) was brought to pH 8.5
with 25% aqueous NH3 and flushed with nitrogen. After 30 min,
tri-n-butylphosphine (0.34 g, 1.68 mmol) was added and the
stoppered flask was stirred at room temperature. After 1 h, the
reaction mixture was repeatedly washed with CHCl3 and the pH
of the aqueous solution was adjusted to 6.0 by use of 1 N KHSO4.
The solution was concentrated and subjected to column chroma-
tography on Sephadex LH-20 with H2O/MeOH (2:1) as eluant to
afford the corresponding reduced compound 2 (0.33 g, 49%). Rf )
0.62, n-BuOH/AcOH/H2O (4:5:1); [R]D25 -37.4° (c 1, MeOH); 1H
NMR (D2O) δ 1.90-2.01 (2H, m, Glu â-CH2), 2.26-2.37 (2H,
m, Glu γ-CH2), 2.67-2.99 (4H, m, Cys â-CH2 and LD â-CH2),
3.61 (1H, m, Glu R-CH), 3.70-3.73 (2H, m, Gly R-CH2), 4.31
(1H, m, LD R-CH), 4.51 (1H, m, Cys R-CH), 6.41-6.66 (3H, m,
ArH); 13C NMR (D2O) δ 25.25 (Cys â-CH2), 26.07 (Glu â-CH2),
31.15 (Glu γ-CH2), 35.99 (LD â-CH2), 42.44 (Gly R-CH2),
54.09 (LD R-CH), 54.18 (Glu R-CH), 55.98 (Cys R-CH), 116.32-
144.03 (LD Ar), and 170.87, 172.70, 173.39, 173.95, and 175.03
(5× CO); MS (ESI) m/z 485 (M - H)-. Anal. (C19H26N4O9S) C,
H, N, S.
Ac-LD(Ac)2-Glu-[Cys(SBut)-Gly-OMe]-OMe (8c). Yield 74%;
25
Rf ) 0.29, CHCl3/MeOH (95:5); [R]D ) -29.8° (c 1, CHCl3);
1H NMR (DMSO-d6) δ 1.26 (9H, s, SBut), 1.74 (3H, s, LD Ac),
1.78-1.82 (1H, m, Glu â-CHA), 1.93-1.98 (1H, m, Glu â-CHB),
2.14-2.19 (2H, m, Glu γ-CH2), 2.22 and 2.24 (6H, 2× s, LD Ac),
2.66-3.07 (4H, m, Cys â-CH2 and LD â-CH2), 3.59 and 3.61 (6H,
2× s, OMe), 3.78-3.80 (2H, m, Gly R-CH2), 4.23 (1H, m, Glu
R-CH), 4.48-4.56 (2H, m, LD R-CH and Cys R-CH), 7.13-7.15
(3H, m, ArH), 8.11 (1H, d, J ) 8.41 Hz, Cys NH), 8.21 (1H, d, J
) 7.81 Hz, LD NH), 8.43 (1H, t, J ) 5.71 Hz, Gly NH), 8.52 (1H,
d, J ) 7.51 Hz, Glu NH); 13C NMR (DMSO-d6) δ 20.98, 21.03,
and 23.11 (3× LD Ac), 27.59 (Glu â-CH2), 30.21 (SBut), 32.15
(Glu γ-CH2), 37.91 (LD â-CH2), 41.36 (Gly R-CH2), 43.07 (Cys
â-CH2), 48.38 (SBut), 52.20 (Glu R-CH), 52.39 and 52.59 (2×
OMe), 52.93 (Cys R-CH), 54.10 (LD R-CH), 123.72-142.15 (LD
Ar), and 168.86, 168.93, 170.02, 170.68, 171.16, 172.04, 172.12,
and 172.86 (8× CO).
Ac-LD-Glu-[Cys-Gly-OMe]-OMe (3). The reaction was per-
formed as described for tetrapeptide 1 with Ac-LD(Ac)2-Glu-[Cys-
(SBut)-Gly-OMe]-OMe (8c, 3.65 g, 5 mmol) in a mixture of
n-PrOH/H2O (2:1) (90 mL) and tri-n-butylphosphine (1.48 mL, 6
mmol) at pH 8.5 with 25% aqueous NH3 to give the reduced
tetrapeptide 3 in good yield (2.0 g, 72%). Rf ) 0.34, CHCl3/MeOH
Boc-LD(Ac)2-Glu-[Cys(SBut)-Gly-OBut]-OBut (8d). Yield 63%;
25
Rf ) 0.41, CHCl3/MeOH (97:3); [R]D ) -39.6° (c 1, CHCl3);
1H NMR (CDCl3) δ 1.30 (9H, s, SBut), 1.32, 135, and 1.36 (27H,
3× s, OBut), 1.88-1.95 (2H, m, Glu â-CH2), 2.05-2.10 (2H, m,
Glu γ-CH2), 2.19 and 2.21 (6H, 2× s, LD Ac), 2.89-3.11 (4H, m,
Cys â-CH2 and LD â-CH2), 3.71-3.78 (1H, m, Gly R- CHA), 3.98-
4.06 (1H, m, Gly R-CHB), 4.35-4.46 (2H, m, Glu R-CH and LD
R-CH), 4.68 (1H, m, Cys R-CH), 5.21 (1H, d, J ) 6.1 Hz, LD
NH), 7.01-7.20 (5H, m, Glu NH, Gly NH, and ArH), 7.47 (1H, t,
J ) 7.5 Hz, Cys NH); 13C NMR (CDCl3) δ 20.86 and 20.91 (2×
LD Ac), 27.39 (Glu â-CH2), 28.18, 28.26, and 28.51 (3× OBut),
30.10 (SBut), 32.04 (Glu γ-CH2), 37.62 (LD â-CH2), 41.40 (Cys
â-CH2), 42.26 (Gly R-CH2), 48.32 (SBut), 52.53 (Cys R-CH), 52.88
(Glu R-CH), 55.28 (LD R-CH), 80.13, 82.18, and 82.48 (3× OBut),
123.65-142.14 (LD Ar), 156.10 (OCONH), and 168.43, 168.58,
168.92, 169.52, 170.96, 172.12, and 173.26 (7× CO).
25
1
(9:1); [R]D -17.9° (c 1, MeOH); H NMR (DMSO-d6) δ 1.73
(3H, s, LD Ac), 1.77-1.80 (1H, m, Glu â-CHA), 1.96-1.99 (1H,
m, Glu â-CHB), 2.15-2.32 (2H, m, Glu γ-CH2), 2.47 (1H, s, Cys
SH), 2.62-2.80 (4H, m, Cys â-CH2 and LD â-CH2), 3.59 and 3.61
(6H, 2× s, OMe), 3.81-3.84 (2H, m, Gly R-CH2), 4.21 (1H, m,
Glu R-CH), 4.36-4.43 (2H, m, LD R-CH and Cys R-CH), 6.46-
6.63 (3H, m, ArH), 8.01 (1H, d, J ) 7.42 Hz, Glu NH), 8.41-
8.47 (2H, m, LD NH and Cys NH), 8.66 (1H, br s, Gly NH); 13C
NMR (DMSO-d6) δ 23.18 (LD Ac), 26.83 (Glu â-CH2), 27.39 (Cys
â-CH2), 31.96 (Glu γ-CH2), 37.59 (LD â-CH2), 41.32 (Gly CH2),
52.08 (Glu R-CH), 52.43 and 52.58 (2× OMe), 54.89 (Cys R-CH),
55.47 (LD R-CH), 115.79-145.44 (LD Ar), and 169.96, 170.82,
171.12, 172.13, 172.73, and 172.89 (6× CO); MS (ESI) m/z 555
(M - H)-. Anal. (C23H32N4O10S) C, H, N, S.
Ac-Glu-[Cys-Gly-LD-OMe]-OMe (1). A solution of the forego-
ing tetrapeptide dimethyl ester disulfide 8a (2.0 g, 2.74 mmol) in
a mixture of n-PrOH/H2O (2:1) (45 mL) was brought to pH 8.5
with 25% aqueous NH3 and flushed with nitrogen. After 30 min,
tri-n-butylphosphine (0.66 g, 3.29 mmol) was added and the
stoppered flask was stirred at room temperature. After 1 h the
reaction mixture was concentrated and subjected to column chro-
matography on silica gel with CHCl3/MeOH (9:1) as eluant to afford
the corresponding reduced tetrapeptide dimethyl ester 1 (0.91 g,
60%). Rf ) 0.20, CHCl3/MeOH (9:1); [R]D25 -26.4° (c 1, MeOH);
1H NMR (DMSO-d6) δ: 1.72-1.77 (1H, m, Glu â-CHA), 1.82 (3H,
s, Glu Ac), 1.88-1.96 (1H, m, Glu â-CHB), 2.18-2.20 (2H, m,
Glu γ-CH2), 2.22 (1H, s, Cys SH), 2.60-2.81 (4H, m, Cys â-CH2
and LD â-CH2), 3.33 and 3.58 (6H, 2× s, OMe), 3.67-3.70 (2H,
m, Gly R-CH2), 4.19 (1H, m, Glu R-CH), 4.29-4.35 (2H, m, LD
R-CH and Cys R-CH), 6.38-6.60 (3H, m, ArH), 8.10 (1H, d, J )
7.5 Hz, Glu NH), 8.19-8.29 (2H, m, LD NH and Cys NH), 8.75
(1H, d, J ) 3.6 Hz, Gly NH); 13C NMR (DMSO-d6) δ 22.93 (Glu
Ac), 26.75 (Glu â-CH2), 27.38 (Cys â-CH2), 31.96 (Glu γ-CH2),
37.10 (LD â-CH2), 42.27 (Gly R-CH2), 52.04 (Glu R-CH), 52.47
and 52.50 (2× OMe), 54.70 (Cys R-CH), 55.79 (LD R-CH),
116.10-145.66 (LD Ar), and 169.22, 169.30, 170.18, 170.81,
H-LD-Glu-(Cys-Gly-OH)-OH (4). Removal of Boc and But
groups from compound 8d was obtained as described for 2.
Reduction of the resulting TFA·LD-Glu-[Cys(SBut)-Gly]-OH (0.45
g, 0.65 mmol) with tri-n-butylphosphine (0.16 g, 0.78 mmol) was
performed as described for tetrapeptide 2 to afford H-LD-Glu-(Cys-
Gly)-OH (4, 0.3 g, 93%). Rf ) 0.29, n-BuOH/AcOH/H2O (4:5:1);
[R]D25 -29.4° (c 1, MeOH); 1H NMR (D2O) δ 1.74-1.83 (1H, m,
Glu â-CHA), 1.90-1.94 (1H, m, Glu â-CHB), 2.13-2.19 (2H, m,
Glu γ-CH2), 2.71-275 (2H, m, Cys â-CH2), 2.83-2.98 (2H, m,
LD â-CH2), 3.54-3.57 (2H, m, Gly R-CH2), 3.93 (1H, m, Glu
R-CH), 4.02 (1H, m, LD R-CH), 4.35 (1H, m, Cys R-CH), 6.49-
6.69 (3H, m, ArH); 13C NMR (D2O) δ 25.64 (Cys â-CH2), 27.54
(Glu â-CH2), 31.93 (Glu γ-CH2), 36.10 (LD â-CH2), 43.13 (Gly
R-CH2), 54.47 (LD R-CH), 54.87 (Glu R-CH), 55.70 (Cys R-CH),
116.67-144.41 (LD Ar), and 168.61, 171.93, 175.85, 176.01, and
177.01 (5× CO); MS (ESI) m/z 485 (M - H)-. Anal. (C19H26N4O9S)
C, H, N, S.
Animals. Male Wistar rats (n ) 100) (Harlan, UD, Italy)
weighing 250-300 g were employed. Twenty rats were assigned
to each treatment group. The animals were housed in plastic
(Makrolon) cages (five rats/cage) in a temperature-controlled room