942 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Rawale et al.
H-P h e-Th r (Bn )-Leu -OBn (19). It was prepared as de-
scribed for dipeptide 16 from tripeptide 17 (4 g, 6.06 mmol).
The tripeptide 19 (2.8 g, 82%) was obtained as a thick oil: TLC,
Rf 0.71 (S2); HPLC (S12), flow rate 0.6 mL/min, tR 6.6 min, 96%
gave tripeptide 29 (5.31 g, 77%): mp 50-51 °C; Rf 0.62 (S8);
1H NMR δ 8.40 (bs, 1H), 7.79 (d, 1H), 7.33 (s, 5H), 6.80 (d,
1H), 5.08 (s, 2H), 4.36 (m, 1H), 3.94-3.73 (m, 3H), 1.62 (bs,
2H), 1.34 (s, 9H), 1.04 (m, 2H), 0.83-0.77 (m, 14H); ESI-MS
492 (M + H).
1
purity); H NMR δ 8.32 (d, 1H), 8.27 (d, 1H), 7.31-7.20 (m,
15H), 5.06 (dd, 2H), 4.47-4.36 (m, 5H), 3.90 (t, 1H), 3.69 (bs,
1H), 3.02 (m, 1H), 2.68 (m, 1H), 1.58-1.50 (m, 4H), 1.03 (d,
3H), 0.83 (dd, 6H); ESI-MS 560 (M + H).
Boc-Ile-Leu -Gly-OH (31). Tripeptide 29 (2.0 g, 4.07 mmol)
was hydrogenated in ethanol (25 mL) over Pd-C (10%, 0.2 g)
as described for dipeptide 22 to give tripeptide 31 (1.4 g,
85%): mp 108-109 °C; Rf 0.69 (S1); HPLC (S13, flow rate 0.8
H-Va l-OBn (20). Free base was released from the corre-
sponding hydrochloride as described for H-Leu-OBn (13) as a
1
mL/min, tR 7.9, purity 98.5%); H NMR δ 8.18 (bs, 1H), 7.79
1
thick oil: Rf 0.7 (S7); H NMR δ 7.33 (m, 5H), 5.12 (dd, 2H),
(d, 1H), 6.82 (d, 1H), 4.36 (m, 1H), 3.77-3.62 (m, 3H), 1.63
(bs, 2H), 1.34 (s, 9H), 1.03 (m, 2H), 0.85-0.77 (m, 14H); ESI-
MS 402 (M + H).
3.13 (d, 1H), 1.83 (m, 1H), 1.69 (bs, 2H), 0.80 (dd, 6H); EI-MS
207 (M + H).
Boc-P h e-Va l-OBn (21). The reaction was performed as
described for dipeptide 14 with Boc-Phe-OH (18, 5.0 g, 18.84
mmol) and H-Val-OBn (20, 5.49 g, 26.35 mmol) to give
Boc-Ile-Leu -Gly-P h e-Va l-P h e-Th r (Bn )-Leu -OBn (32).
To a solution of tripeptide 31 (0.460 g, 1.02 mmol) in THF (20
mL) was added HOBT (0.14 g, 1.02 mmol) at 0 °C followed by
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCl, 0.196 g, 1.02 mmol). The reaction mixture was stirred
for 30 min. A cooled solution of pentapeptide 24 (0.46 g, 0.57
mmol) in DMF (10 mL) was then added. The stirring was
continued at 0 °C for 2 h and at room temperature for 3 days.
The mixture was worked up as described for the dipeptide 14.
Chromatography of the crude product in solvent S8 provided
octapeptide 32 (0.45 g, 66%): mp 177-179 °C; Rf 0.57 (S8);
HPLC (S14, flow rate 0.6 mL/min, tR 9.20, 91% purity); 1H NMR
δ 8.11 (t, 2H), 8.03 (d, 1H), 7.98 (t, 1H), 7.92 (bs, 2H), 7.79 (d,
1H), 7.32-7.11 (m, 20H), 6.78 (d, 1H), 5.05 (dd, 2H), 4.70 (m,
1H), 4.55 (m, 1H), 4.47-4.36 (m, 4H), 4.27 (m, 1H), 4.13 (m,
1H), 3.89 (m, 2H), 3.59 (m, 2H), 2.80 (m, 4H), 1.34 (s, 9H),
1.56 (m, 3H), 1.05 (d, 3H), 0.83-0.77 (m, 26H); ESI-MS (NaCl)
1,211 (M + Na).
H-Ile-Leu -Gly-P h e-Va l-P h e-Th r -Leu -OH (9). Octapep-
tide 32 (0.2 g, 4.07 mmol) was hydrogenated in AcOH (50 mL)
over Pd-C (10%, 0.4 g) for 6 h. The catalyst was filtered off
and the solvent evaporated. The residue was dissolved in TFA
(10 mL), and the solution was stirred at room temperature
for 1 h. The solvent was evaporated, and residue (0.14 g) was
purified by HPLC using Phenomenex Aqua semipreparative
column (10 × 250 mm, S16, flow rate 2.4 mL/min, tR 7.4 min)
to give octapeptide 9 (95 mg, 62%): mp 124-126 °C; Rf 0.63
(S7); HPLC (S15, tR 4.2 min, flow rate 0.8 mL/min, purity 92%);
1H NMR δ 12.60 (bs, 1H), 8.43 (bs, 1H), 8.29 (d, 1H), 8.20 (bs,
1H), 8.07 (bs, 3H), 7.86 (bs, 1H), 7.20-7.14 (b, 10H), 4.79 (d,
1H), 4.61 (bs, 1H), 4.38 (m, 2H), 4.24-4.16 (m, 3H), 3.90 (bs,
1H), 3.76 (m, 1H), 3.61 (bs, 2H), 2.98 (m, 2H), 2.75 (m, 2H),
1.90 (m, 1H), 1.75 (m, 1H), 1.61-1.43 (m, 6H), 1.01 (bs, 3H),
0.86-0.72 (m, 26H); ESI-MS 909 (M + H).
1
dipeptide 21 (7 g, 81%): mp 68-69 °C; Rf 0.48 (S8); H NMR
δ 8.17 (d, 1H), 7.38 (m, 10H), 6.95 (d, 1H), 5.11 (dd, 2H), 4.24
(t, 2H), 2.88 (m, 1H), 2.67 (t, 1H), 2.06 (m, 1H), 1.26 (s, 9H),
0.86 (m, 6H); EI-MS 454 (M).
Boc-P h e-Va l-OH (22). Boc-Phe-Val-OBn (21, 4.0 g, 8.81
mmol) was hydrogenated in a Parr apparatus at 40 psi for 2
h in ethanol (50 mL) over Pd-C (10%, 0.42 g). The catalyst
was filtered off, and the solvent was evaporated. The residue
was partitioned between aqueous NaHCO3 (100 mL) and ether
(2 × 50 mL). The mixture (pH 3) was extracted with ethyl
acetate (2 × 100 mL), and the organic phase was washed with
water, dried (Na2SO4), and evaporated to give the dipeptide
22 (2.9 g, 90%); mp 77-78 °C; Rf 0.58 (S7); HPLC (S12, flow
1
rate 0.6 mL/min, tR 5.6 min, purity 97%); H NMR δ 7.88 (d,
1H), 7.23 (m, 5H), 6.96 (d, 1H), 4.23-4.14 (m, 2H), 2.93 (m,
1H), 2.71 (q, 1H), 2.05 (m, 1H), 1.26 (s, 9H), 0.87 (m, 6H); FAB-
MS (KCl) 403 (M + K).
Boc-P h e-Va l-P h e-Th r (Bn )-Leu -OBn (23). The reaction
of dipeptide 22 (2.5 g, 7.01 mmol) with tripeptide 19 (2.8 g,
5.00 mmol) was performed as described for dipeptide 14.
Chromatography in solvent (S8) furnished pentapeptide 23 (4.4
g, 70%): mp 80-81 °C, Rf 0.66 (S2), HPLC (S14, flow rate 0.6
mL/min, tR 11.4 min, purity 90%); 1H NMR δ 8.19 (d, 1H), 8.12
(d, 1H, 8.02 (d, 1H) and 7.61 (d, 1H), 7.32-7.10 (m, 20H), 7.00
(d, 1H), 5.00 (dd, 2H), 4.70 (m, 1H), 4.47-4.36 (m, 6H), 4.21-
4.11 (m, 2H), 3.89 (t, 1H), 3.00 (m, 1H), 2.87-2.63 (m, 2H),
1.90 (m, 1H), 1.53 (m, 2H), 1.25 (s, 9H), 1.05 (d, 3H), 0.82 (dd,
6H), 0.73 (m, 6H); ESI-MS (NaCl) 928 (M + Na).
H-P h e-Va l-P h e-Th r (Bn )-Leu -OBn (24). Deprotection of
pentapeptide 23 (0.5 g, 0.55 mmol) was performed as described
for dipeptide 14 to afford free pentapeptide 24 (0.4 g, 90%):
mp 147-148 °C; Rf 0.55 (S2); HPLC (S14, flow rate 0.6 mL/
Meth yl N-[â-D-Glu cop yr a n u r on a te-3-n itr oben zyloxy-
car bon yl]-Gly-Ile-Leu -Gly-P h e-Val-P h e-Th r -Leu -OH (11b).
A solution of pentafluorophenylester 10 (136 mg, 0.22 mmol)
in THF (10 mL) was added to a mixture of octapeptide 9 (100
mg, 0.11 mmol) and NEt3 (15 µL, 0.11 mmol) in DMF (10 mL)
with stirring at room temperature. The stirring was continued
for 40 h, the solvents were evaporated, and residue was
chromatographed (three times) using CHCl3:MeOH (3:2) to
obtain compound 11b (68 mg, 46%) which was further purified
by semipreparative HPLC (see octapeptide 9, S16, flow rate
3.4 mL/min) to give 52 mg (34%) of 11b: HPLC (S16, flow rate
1
min, tR 6.3 min, purity 96%); H NMR δ 8.25 (d, 1H), 8.14 (d,
1H), 8.02 (d, 1H) and 7.89 (d, 1H), 7.30-7.17 (m, 20H), 5.05
(dd, 2H), 4.68 (m, 1H), 4.47-4.36 (m, 6H), 4.16 (m, 2H), 3.90
(t, 1H), 2.95 (m, 1H), 2.75 (m, 2H), 1.89 (m, 1H), 1.52 (m, 4H),
1.05 (d, 3H), 0.82 (dd, 6H), 0.75-0.63 (dd, 6H); ESI-MS 806
(M + H).
H-Gly-OBn (25). Free base was released from the corre-
sponding hydrochloride as described in the workup procedure
1
for dipeptide 16 as a thick oil: Rf 0.50 (S1); H NMR δ 7.38-
7.30 (m, 5H), 5.11 (s, 2H), 3.87 (m, 2H), 1.89 (bs, 2H): ESI-MS
165 (M + H).
1.0 mL/min, tR 13.1, purity 86%); mp 140-142 °C; [R]26 11°
D
(c 0.1, MeOH); Rf 0.49 /CHCl3:MeOH:NH4OH (4:6:0.1)/; 1H
NMR δ 8.11 (bs, 4H), 8.03 (bs, 1H), 7.98 (d, 1H), 7.93 (d, 1H),
7.88 (d, 1H), 7.83 (bs, 1H), 7.61 (d, 1H),7.53 (bs, 1H), 7.41 (d,
1H), 7.26-7.11 (m, 10H), 5.54-5.50 (m, 2H), 5.30 (m, 2H), 4.99
(s, 2H), 4.66 and 4.52 (2m, 3H), 4.35 (bs, 1H), 4.25 (bs, 2H),
4.14-4.00 (m, 5H), 3.63 (s, 3H), 3.54 (d, 1H), 3.15 (d, 1H), 3.05
(m, 5H), 2.88-2.60 (m, 7H), 1.86 (m, 1H), 1.63 (m, 1H), 1.54
(m, 1H), 1.39 (m, 2H), 1.03 (m, 3H), 0.98 (d, 3H), 0.86-0.74
(m, 23H); Negative ESI-MS (NH4OH) 1,349 (M - H).
N-[â-D-(Glu cop yr a n u r on ic a cid )-3-n itr oben zylca r bon -
yl]-Gly-Ile-Leu -Gly-P h e-Va l-P h e-Th r -Leu -OH (2a ). A mix-
ture of compound 11b (20 mg, 0.014 mmol) and Ba(OH)2‚8H2O
(4.6 mg, 0.014 mmol) in MeOH (10 mL) was stirred at room
temperature for 3 h. Dowex 50 (H(+) form, 25 mg) was then
added, and the mixture was filtered. The filtrate was evapo-
Boc-Leu -Gly-OBn (26). It was prepared as described for
dipeptide 14 from Boc-Leu-OH (27, 5 g, 20.05 mmol) and Gly-
OBn (25, 4.13 g, 25.06 mmol). Chromatography afforded
dipeptide 26 as a thick oil (7.0 g, 87%): Rf 0.90 (S8); 1H NMR
δ 8.26 (t, 1H), 7.32 (m, 5H), 6.89 (d, 1H), 5.09 (s, 2H), 3.91-
3.82 (m, 3H), 1.60 (m, 1H), 1.34 (s, 11H), 0.81 (m, 6H); FAB-
MS (KCl) 417 (M + K).
H-Leu -Gly-OBn (28). Deprotection of 26 (8 g, 21.16 mmol)
was performed as described for dipeptide 14 to give 28 (5.88
1
g, 85%): Rf 0.51 (S6); H NMR δ 8.60 (bs, 1H), 7.34 (s, 5H),
5.10 (s, 2H), 3.91 (ddd, 2H), 3.40 (t, 1H), 1.69 (bs, 2H), 1.44
(m, 1H), 1.30 (m, 2H), 0.83 (m, 6H); ESI-MS 279 (M + H).
Boc-Ile-Leu -Gly-OBn (29). This tripeptide was prepared
as described for dipeptide 14 from Boc-Ile-OH (30, 2.50 g, 10.80
mmol) and dipeptide 28 (2.5 g, 8.99 mmol). Chromatography