S. Ray et al. / Tetrahedron 62 (2006) 7274–7283
7281
in vacuo to about 15–20 mL, cooled in an ice-water
bath, covered with a layer of ethyl acetate (about 30 mL),
and acidified with a dilute solution of KHSO to pH 2–3
4.2.13. Boc-Ile(1)-Val(2)-Tyr(3)-OMe 5. Boc-Ile(1)-
Val(2)-OH 14 (1.65 g, 5 mmol) in DMF (10 mL) was cooled
in an ice-water bath. H-Tyr-OMe was isolated from the cor-
responding methyl ester hydrochloride (2.31 g, 10 mmol) by
neutralization, subsequent extraction with ethyl acetate and
concentration to 10 mL, and it was added to the reaction
mixture, followed immediately by DCC (1.03 g, 5 mmol)
and HOBt (0.675 g, 5 mmol). The reaction mixture was
stirred for three days. The reaction mixture was then taken
in ethyl acetate (60 mL) and the DCU was filtered off. The
organic layer was washed with 2 M HCl (3ꢂ50 mL), brine
(2ꢂ50 mL), 1 M sodium carbonate (3ꢂ50 mL), and brine
(2ꢂ50 mL) and then dried over anhydrous sodium sulfate
and evaporated in vacuo to yield peptide 5 as a white solid.
Purification was done by silica gel column (100–200 mesh)
using 3:1 ethyl acetate/toluene as eluent.
4
(
Congo red). The aqueous phase was extracted with ethyl
acetate and this operation was done repeatedly. The ethyl
acetate extracts were pooled, washed with water and dried
over anhydrous Na SO , and evaporated in vacuo to obtain
2
4
1
2 as a solid material.
Yield¼2.2 g (9.5 mmol, 95%). Elemental Analysis Calcd
for C H NO (231): C, 57.14; H, 9.09; N, 6.06.
21
1
1
4
ꢀ
Found: C, 56.9; H, 8.9; N, 5.9%. Mp 65–67 C, lit. mp
ꢀ
6
6–69 C.
4
8
.2.11. Boc-Ile(1)-Val(2)-OMe 13. Boc-Ile-OH 12 (1.84 g,
mmol) was dissolved in dichloromethane (DCM) (10 mL)
in an ice-water bath. H-Val-OMe was isolated from the cor-
responding methyl ester hydrochloride (2.68 g, 10 mmol) by
neutralization, subsequent extraction with ethyl acetate and
concentration to 10 mL, and it was added to the reaction
mixture, followed immediately by di-cyclohexylcarbodi-
imide (DCC) (1.64 g, 8 mmol). The reaction mixture was
allowed to come to room temperature and stirred for 24 h.
DCM was evaporated, residue was taken in ethyl acetate
Yield¼2.23 g (4.4 mmol, 89%); R ¼0.64 (25% toluene/
f
ꢀ
ethyl acetate); mp 110–112 C; IR (KBr): 3488, 3313,
ꢁ
1
20
1739, 1692, 1645, 1522 cm ; [a]D ꢁ35 (c 0.5, CH OH);
3
1
H NMR (300 MHz, CDCl ) d 6.92 (ring Hs of Tyr(3), 2H,
3
d, J¼9 Hz), 6.74 (ring Hs of Tyr(3), 2H, d, J¼8.4 Hz);
6.60 (Tyr(3) NH, 1H, d, J¼8.7 Hz); 6.45 (Val(2) NH, 1H,
d, J¼8.1 Hz); 5.01 (Ile(1) NH, 1H, d, J¼9 Hz); 4.82
a
a
(
60 mL), and dicyclohexylurea (DCU) was filtered off.
(C H of Tyr(3), 1H, m); 4.23 (C H of Val(2), 1H, m); 3.94
a
The organic layer was washed with 2 M HCl (3ꢂ50 mL),
brine (2ꢂ50 mL), 1 M sodium carbonate (3ꢂ50 mL), and
brine (2ꢂ50 mL) and then dried over anhydrous sodium
sulfate, and evaporated in vacuo to yield 13 as a solid
compound.
(C H of Ile(1), 1H, m); 3.72 (–OCH , 3H, s); 3.10–2.94
3
b
b
(C Hs of Tyr(3), 2H, m); 2.12–2.05 (C H of Val(2), 1H,
m); 1.95–1.90 (C H of Ile(1), 1H, m); 1.44 (Boc–CH s,
9H, s); 1.28 (C Hs of Ile(1), 2H, m); 0.91–0.87 (C Hs of
b
3
g
g
g
d
Val(2) and C Hs and C Hs of Ile(1), 12H, m); (found: C,
1.34; H, 8.10; N, 8.06. C H N O (507) requires C,
61.53; H, 8.09; N, 8.28%); ESI-MS m/z (%): 508.4 (100)
(M+H) , 509.4 (30) (M+2H) , Mcalcd¼507.
4.2.14. Boc-Phe(1)-OH 15. See Ref. 17.
6
2
6 41 3 7
Yield¼2.4 g (7 mmol, 87%); R ¼0.65 (ethyl acetate);
f
ꢀ
20
D
1
+
+
mp 78–82 C; [a] ꢁ38.8 (c 0.69, CH OH); H NMR
3
(
300 MHz, CDCl ) d 6.35 (Val(2) NH, 1H, d, J¼7.5 Hz);
3
a
5
1
2
1
.03 (Ile(1) NH, 1H, d, J¼6.6 Hz); 4.54 (C H of Val(2),
a
H, m); 3.94 (C H of Ile(1), 1H, m); 3.73 (–OCH , 3H, s);
3
b
b
.15 (C H of Val(2), 1H, m); 1.86 (C H of Ile(1), 1H, m);
.67–1.62 (C Hs of Ile(1), 2H, m); 1.42 (Boc–CH s, 9H,
4.2.15. Boc-Phe(1)-Val(2)-OMe 16. Boc-Phe-OH (2.65 g,
10 mmol) was dissolved in dichloromethane (DCM) (10 mL)
in an ice-water bath. H-Val-OMe was isolated from the cor-
responding methyl ester hydrochloride (3.34 g, 20 mmol) by
neutralization, subsequent extraction with ethyl acetate and
concentration to 10 mL, and it was added to the reaction
mixture, followed immediately by di-cyclohexylcarbodi-
imide (DCC) (2.06 g, 10 mmol). The reaction mixture was
allowed to come to room temperature and stirred for 24 h.
DCM was evaporated, residue was taken in ethyl acetate
(60 mL), and dicyclohexylurea (DCU) was filtered off.
The organic layer was washed with 2 M HCl (3ꢂ50 mL),
brine (2ꢂ50 mL), 1 M sodium carbonate (3ꢂ50 mL), and
brine (2ꢂ50 mL) and then dried over anhydrous sodium
sulfate, and evaporated in vacuo to yield 16 as a white
solid.
g
3
g
g
d
s); 0.94–0.89 (C Hs of Val(2), C Hs and C Hs of Ile(1),
2H, m); (found: C, 59.0; H, 8.21; N, 8.03. C H N O
7 32 2 5
1
1
(
344) requires C, 59.30; H, 9.30; N, 8.14%).
4
1
.2.12. Boc-Ile(1)-Val(2)-OH 14. Boc-Ile(1)-Val(2)-OMe
3 (2.23 g, 6.5 mmol), MeOH (20 mL), and 2 M NaOH
(
10 mL) were added and the progress of saponification
was monitored by thin layer chromatography (TLC). The
reaction mixture was stirred. After 10 h methanol was re-
moved under vacuo, the residue was taken in 50 mL of water
and washed with diethyl ether (2ꢂ50 mL). Then the pH of
the aqueous layer was adjusted to 2 using 1 M HCl and it
was extracted with ethyl acetate (3ꢂ50 mL). The extracts
were pooled, dried over anhydrous sodium sulfate, and evap-
orated in vacuo to yield 14 as a waxy solid.
Yield¼3.5 g (9.2 mmol, 92%); R ¼0.76 (ethyl acetate); mp
f
1
ꢀ
Hs of Phe(1), 5H, m); 6.36 (Val(2) NH, 1H, d,
1
Yield¼1.8 g (5.5 mmol, 84%); H NMR (300 MHz,
58–60 C; H NMR (300 MHz, CDCl ) d 7.32–7.19 (ring
3
(
CD ) SO) d 12.46 (–COOH, 1H, b); 7.70 (Val(2) NH, 1H,
3
2
d, J¼6 Hz); 6.69 (Ile(1) NH, 1H, d, J¼9 Hz); 4.08
J¼8.4 Hz); 5.01 (Phe(1) NH, 1H, d, J¼8.1 Hz); 4.45
a
a
a
a
(
C H of Val(2), 1H, m); 3.79 (C H of Ile(1), 1H, m);
(C H of Val(2), 1H, m); 4.35 (C H of Phe(1), 1H, m);
3.69 (–OCH , 3H, s); 3.07 (C Hs of Phe(1), 2H, d,
b
b
b
2
1
0
.44 (C H of Val(2), 1H, m); 1.97 (C H of Ile(1), 1H, m);
.60 (C Hs of Ile(1), 2H, m); 1.3 (Boc–CH s, 9H, s); 1.03–
3
g
b
J¼6 Hz); 2.13–2.04 (C H of Val(2), 1H, m); 1.42 (Boc–
3
g
d
g
g
.96 and 0.82–0.70 (C Hs and C Hs of Ile(1) and C Hs of
CH s, 9H, s); 0.88–0.83 (C Hs of Val(2), 6H, m); (found:
3
Val(2), 12H, m); (found: C, 58.01; H, 9.13; N, 8.34.
C H N O (330) requires C, 58.18; H, 9.09; N, 8.48%).
C, 63.2; H, 7.6; N, 7.03. C H N O (378) requires C,
0 30 2 5
63.49; H, 7.94; N, 7.4%).
2
1
6 30 2 5