M. Marastoni et al. / European Journal of Medicinal Chemistry 40 (2005) 445–451
449
6.2. Chemistry
6.2.1.8. Reduction of ethyl ester (f). The hydrazide ester inter-
mediate (0.4 mmol) in THF (3 ml) was added dropwise to a
cold solution of LiAlH4 (0.5 mmol) in THF (3 ml). The mix-
ture was stirred for 2 h at RT and then evaporated. The resi-
due was suspended in H2O (5 ml) and extracted with AcOEt
(2 × 20 ml); the organic phase was dried, filtered and evapo-
rated. The residue was triturated with Et2O, centrifugated and
the resulting solid was collected and used for the final con-
densation.
6.2.1. General procedure for compounds 17–28
6.2.1.1. Introduction of Boc (a). Phenylhydrazine or benzyl-
hydrazine (2 mmol) and (Boc)2 O (2.2 mmol) were dissolved
in 20 ml of a mixture H2O/tert-butanol. NMM (2.2 mmol)
were added, and the resultant mixture was stirred for 2 h at
0 °C and further 8 h at room temperature (RT). Following
ilution with a saturated solution of NaCl, the mixture was
extracted with AcOEt (2 × 50 ml). The combined extracts
were washed with brine, dried over Na2SO4, filtered and con-
centrated under reduced pressure. The resultant oil was used
without any further purification for the next step.
1
6.2.1.9. H NMR data of compounds. Hmb-Met-N(Ph)-
1
N(CH2-OH)-rPro-Boc (41). H NMR (CDCl3): d 1.29 (s,
9H), 1.58–1.77 (m, 4H), 1.94 (s, 3H), 2.23–2.51 (m, 7H),
3.28 (bs, 1H), 3.74 (t, 2H), 4.12 (m, 1H), 4.49 (m, 1H), 5.27
(s, 2H), 5.54 (bs, 1H), 6.67–7.19 (m, 8H), 7.63 (s, 1H).
Hmb-Met-N(Bzl)-N(CH2-OH)-rPro-Boc (42). 1H NMR
(CDCl3): d 1.24 (s, 9H), 1.54–1.86 (m, 4H), 2.03 (s, 3H),
2.17–2.29 (m, 7H), 2.74 (bs, 1H), 3.27 (t, 2H), 4.08–4.37 (m,
4H), 5.13 (bs, 1H), 5.42 (s, 2H), 7.03–7.32 (m, 8H), 7.81 (s,
1H).
6.2.1.2. Coupling with HBTU (b, g). The hydrazide compo-
nent (1 mmol), NMM (1 mmol) and HBTU (1 mmol) were
added to a solution of Fmoc-Xaa-OH or Boc-Pro-OH
(1 mmol) in DMF (3 ml) at 0 °C. The reaction mixture was
stirred for 1 h at 0 °C and 18 h at RT; then the solution was
diluted with AcOEt (70 ml) and washed consecutively with
HCl 0.1 N, brine, NaHCO3 and brine. The organic phase was
dried (MgSO4), filtered and evaporated to dryness. The resi-
due was treated with Et2O and resulting solid separated by
centrifugation.
1
Hmb-Met-N(Ph)-N(CH2-CH2-OH)-rPro-Boc (43). H
NMR (CDCl3): d 1.19 (s, 9H), 1.47–1.79 (m, 4H), 1.98 (s,
3H), 2.13–2.25 (m, 7H), 2.81 (bs, 1H), 3.35 (t, 2H), 3.71 (t,
2H), 4.15–4.36 (m, 2H), 4.98 (bs, 1H), 5.35 (t, 2H), 6.95–
7.18 (m, 8H), 8.01 (s, 1H).
Hmb-Met-N(Bzl)-N(CH2-CH2-OH)-rPro-Boc (44). 1H
NMR (CDCl3): d 1.11 (s, 9H), 1.57–1.83 (m, 4H), 1.95 (s,
3H), 2.33–2.47 (m, 7H), 3.01 (bs, 1H), 3.44 (t, 2H), 3.59 (t,
2H), 4.05–4.35 (m, 4H), 5.05 (bs, 1H), 5.46 (t, 2H), 7.12–
7.40 (m, 8H), 8.04 (s, 1H).
6.2.1.3. Fmoc removal (c.1). The Fmoc group was cleaved
from 5–10 with a solution of 20% piperidine in DMF for
25 min.After evaporation the residue was triturated with Et2O,
centrifugated and the resulting solid was collected and dried.
Hmb-Leu-N(Ph)-N(CH2-OH)-rPro-Boc (45). 1H NMR
(CDCl3): d 0.99 (d, 6H), 1.08 (s, 9H), 1.58–1.95 (m, 7H),
2.02 (bs, 1H), 2.33 (s, 3H), 3.29 (t, 2H), 4.25 (m, 1H), 4.39
(m, 1H), 4.96 (bs, 1H), 5.34 (s, 2H), 7.03–7.41 (m, 8H), 8.05
(s, 1H).
6.2.1.4. Coupling with WSC/HOBt (c.2). To a solution of the
3-hydroxy-2-methyl benzoic acid (1.5 mmol) in DMF (5 ml)
were added the amine component (1.5 mmol), HOBt
(1.5 mmol) and WSC (1.5 mmol) in this order at 0 °C. The
reaction mixture was stirred for 1 h at 0 °C and 12 h at RT;
then the solution was diluted withAcOEt (100 ml) and worked
up as described in Section 6.2.1.2.
Hmb-Leu-N(Bzl)-N(CH2-OH)-rPro-Boc (46). 1H NMR
(CDCl3): d 1.05 (d, 6H), 1.15 (s, 9H), 1.55–1.65 (m, 4H),
1.67–1.78 (m, 3H), 1.99 (bs, 1H), 2.30 (s, 3H), 3.33 (t, 2H),
4.40–4.52 (m, 4H), 5.03 (bs, 1H), 5.51 (s, 2H), 7.10–7.57 (m,
8H), 8.02 (s, 1H).
1
6.2.1.5. TFA deprotection (d.1; e.1). Boc was removed by
treating intermediates 7, 8 with aqueous 90% TFA (1:10, w/v)
for 30–40 min. After evaporation, the residue was worked up
as described in Section 6.2.1.3.
Hmb-Leu-N(Ph)-N(CH2-CH2-OH)-rPro-Boc (47). H
NMR (CDCl3): d 0.98 (d, 6H), 1.13 (s, 9H), 1.50–1.81 (m,
7H), 1.99 (bs, 1H), 2.42 (s, 3H), 2.76 (d, 2H), 3.44 (d, 2H),
4.29–4.40 (m, 2H), 4.97 (bs, 1H), 5.45 (t, 2H), 7.07–7.49 (m,
8H), 7.99 (s, 1H).
1
6.2.1.6. N-Acylation (d.2). TEA (2 mmol) and ethyl chloro-
formate (1 mmol) were added to a solution of hydrazide inter-
mediate (1 mmol) in DMF (10 ml). The resultant mixture
was stirred for 2 h at RT and refluxed for 1 h; after evapora-
tion the residue was diluted with AcOEt (50 ml) and washed
with H2O (2 × 10 ml). The organic phase was dried (MgSO4),
filtered, evaporated to dryness and the residue was used for
the following reduction.
Hmb-Leu-N(Bzl)-N(CH2-CH2-OH)-rPro-Boc (48). H
NMR (CDCl3): d 0.99 (d, 6H), 1.08 (s, 9H), 1.78–1.84 (m,
3H), 2.03–2.11 (m, 7H), 2.28–2.35 (m, 3H), 3.24 (d, 2H),
3.52 (d, 2H), 4.38 (m, 1H), 4.47 (s, 2H), 4.55 (m, 1H), 5.04
(bs, 1H), 6.93–7.27 (m, 8H), 7.97 (s, 1H).
Hmb-Thr-N(Ph)-N(CH2-OH)-rPro-Boc (49). 1H NMR
(CDCl3): d 1.03 (m, 3H), 1.24 (s, 9H), 1.38 (m, 2H), 1.82 (m,
2H), 2.14 (bs, 2H), 2.42 (s, 3H), 3.20 (t, 2H), 4.14 (m, 1H),
4.33–4.46 (m, 2H), 4.88 (s, 2H), 5.11 (bs, 1H), 6.99–7.28 (m,
8H), 8.12 (s, 1H).
6.2.1.7. N-Alkylation (e.2). The reaction was conducted as
described in Section 6.2.1.6. using ethyl bromoacetate as alky-
lating agent.
Hmb-Thr-N(Bzl)-N(CH2-OH)-rPro-Boc (50). 1H NMR
(CDCl3): d 1.08 (m, 3H), 1.27 (s, 9H), 1.78–1.84 (m, 4H),