L. Shang et al. / Bioorg. Med. Chem. 17 (2009) 2775–2784
2783
3.68–3.74 (m, 2H), 3.82–3.87 (m, 1H), 4.02–4.05 (m, 1H), 4.32–4.37
(J = 14.1, t, 1H), 7.32–7.41 (m, 5H).
6.1.31.
leucinamide hydrochloride (C3)
L L-
-Alanyl-N1-[(2R)-2-amino-3-phenylpropyl]-
Yield: 73%, mp = 164–166 °C. ESI-MS m/z: 335.8 (M+H)+; 1H
NMR (D2O) d 0.79–0.82 (J = 6.3, d, 3H), 0.84–0.86 (m, 4H), 1.38–
1.44 (m, 3H), 1.60–1.64 (m, 2H), 2.81–2.88 (J1 = 6.3, J2 = 13.8, dd,
6.1.24.
cysteinamide hydrochloride (B7)
L L-
-Ornithyl-N1-[(2R)-2-amino-3-phenylpropyl]-
Yield: 55%, mp = 161–164 °C, ESI-MS m/z: 368.4 (M+H)+; 1H
NMR (D2O) d 1.61–1.66 (m, 2H), 1.83–1.89 (m, 2H), 2.54–2.59
(m, 2H), 2.78–2.82 (m, 2H), 3.06–3.11 (J = 14.7, t, 2H), 3.56–3.61
(m, 2H), 3.74–3.81 (m, 1H), 4.09–4.15 (m, 1H), 4.28–4.33 (J =
14.4, t, 1H), 7.22–7.37 (m, 5H).
1H), 2.94–3.01 (J1 = 6.6, J2 = 14.1, dd, 1H), 3.32–3.38 (J1 = 4.2, J2 =
13.5, dd, 1H), 3.43–3.49 (J1 = 4.2, J2 = 12.3, dd, 1H), 3.61–3.67 (m,
1H), 4.01–4.08 (m, 1H), 4.24–4.27 (J = 9, t, 1H), 7.23–7.37 (m, 5H).
6.1.32. Glycyl-N1-[(2R)-2-amino-3-phenylpropyl]-
L-leucinamide
hydrochloride (C4)
6.1.25.
L
-Isoleucyl-N1-[(2R)-2-amino-3-phenylpropyl]-
L
-
Yield: 71%, mp = 117–119 °C. ESI-MS m/z: 321.5 (M+H)+; 1H
NMR (D2O) d 0.83–0.85 (J = 6.6, d, 6H), 1.21–1.26 (m, 1H), 1.61–
1.66 (m, 2H), 2.91–3.01 (m, 2H), 3.31–3.38 (m, 2H), 3.63–3.69
(m, 1H), 3.90 (s, 2H), 4.36–4.39 (J = 8.7, t, 1H), 7.28–7.41 (m, 5H).
cysteinamide hydrochloride (B8)
Yield: 63%, mp = 228–230 °C. ESI-MS m/z: 367.2 (M+H)+; 1H
NMR (MeOD) d 0.93–0.96 (J = 6.6, d, 3H), 1.06–1.10 (J = 14.1, t,
3H), 1.27–1.31 (m, 2H), 1.58–1.62 (m, 1H), 2.86–2.93 (m, 2H),
3.00–3.09 (m, 1H), 3.21–3.28 (J1 = 4.2, J2 = 14.1, dd, 1H), 3.36–
3.43 (J1 = 5.1, J2 = 14.4, dd, 1H), 3.51–3.59 (J1 = 5.7, J2 = 14.7, dd,
1H), 3.68–3.72 (m, 1H), 3.89–3.95 (m, 1H), 4.63–4.68 (J = 14.7, t,
1H), 7.30–7.39 (m, 5H).
6.1.33.
leucinamide hydrochloride (C5)
L L-
-Methionyl-N1-[(2R)-2-amino-3-phenylpropyl]-
Yield: 67%, mp = 126–129 °C. ESI-MS m/z: 395.4 (M+H)+; 1H
NMR (MeOD) d 0.90–1.02 (m, 6H), 1.38–1.51 (m, 1H), 1.68–1.79
(m, 2H), 2.12 (s, 3H), 2.13–2.16 (m, 2H) 2.56–2.69 (m, 2H), 3.15–
3.22 (J1 = 6.6, J2 = 13.2, dd, 1H), 3.24–3.31 (m, 1H), 3.40–3.41 (m,
1H), 3.45–3.47 (m, 1H), 3.96–4.00 (m, 1H), 4.06–4.15 (m, 1H),
4.53–4.56 (J = 9, t, 1H), 7.23–7.38 (m, 5H).
6.1.26.
cysteinamide hydrochloride (B9)
L L-
-Alanyl-N1-[(2R)-2-amino-3-phenylpropyl]-
Yield: 64%, mp = 170–173 °C. ESI-MS m/z: 353.3 (M+H)+; 1H
NMR (D2O) d 0.77–0.78 (J = 3, d, 3H), 0.92–0.94 (J = 4.5, d, 3H),
1.88–1.97 (m, 1H), 2.78–2.85 (m, 2H), 2.86–2.93 (m, 2H), 3.26–
3.33 (m, 2H), 3.34–3.40 (m, 1H), 3.58–3.61 (m, 1H), 3.70–3.76
(m, 1H), 7.18 -7.31 (m, 5H).
6.1.34.
leucinamide hydrochloride (C6)
L L-
-Tyrosyl-N1-[(2R)-2-amino-3-phenylpropyl]-
Yield: 70%, mp = 158–160 °C. ESI-MS m/z: 427.4 (M+H)+; 1H
NMR (MeOD) d 0.94–1.00 (m, 6H), 1.40–1.45 (m, 1H), 1.72–1.76
(m, 1H), 2.85–2.93 (m, 2H), 2.97–3.07 (m, 2H), 3.14–3.22 (m,
2H), 4.02–4.05 (m, 1H), 4.08–4.15 (m, 1H), 4.55–4.60 (J = 15.3, t,
1H), 6.78–6.81 (m, 2H), 7.13–7.15 (m, 2H), 7.25–7.35 (m, 5H).
6.1.27. N1-[(2R)-2-Amino-3-phenylpropyl]-N-(6-
aminohexanoyl)-L-cysteinamide hydrochloride (B10)
Yield: 72%, mp = 172–178 °C. ESI-MS m/z: 367.5 (M+H)+; 1H
NMR (MeOD) d 0.94–1.00 (m, 2H), 1.43–1.48 (m, 2H), 1.63–1.69
(m, 2H), 2.01–2.04 (J = 7.2, t, 2H), 2.32–2.35 (J = 9.6, t, 2H), 2.92–
2.95 (m, 2H), 2.96–2.98 (m, 2H), 3.21–3.26 (m, 2H), 3.31–3.34
(m, 1H), 4.39–4.43 (J = 12.3, t, 1H), 7.29–7.41 (m, 5H).
6.1.35. N1-[(2R)-2-Amino-3-phenylpropyl]-N-(6-
aminohexanoyl)-L-leucinamide hydrochloride (C7)
Yield: 63%, mp = 101–105 °C. ESI-MS m/z: 377.7 (M+H)+; 1H
NMR (MeOD) d 0.90–0.96 (m, 6H), 0.97–1.04 (m, 2H), 1.12–1.16
(m, 1H), 1.33–1.40 (m, 2H), 1.42–1.50 (m, 2H), 1.60–1.66 (m,
2H), 2.16–2.22 (J = 10.5, t, 2H), 2.97–3.04 (m, 2H), 3.07–3.12 (J =
14.7, t, 2H), 3.27–3.31 (m, 2H), 3.71–3.77 (m, 1H), 4.47–4.52 (J =
15, t, 1H), 7.27–7.41 (m, 5H).
6.1.28. N1-[(2R)-2-Amino-3-phenylpropyl]-N-(2,4-
dichlorobenzoyl)–L-cysteinamide hydrochloride (B11)
Yield: 70%, mp = 145–148 °C. ESI-MS m/z: 427.3 (M+H)+; 1H
NMR (D2O) d 2.57–2.64 (m, 2H), 2.78–2.86 (m, 1H), 2.91–2.98
(m, 1H), 3.26–3.32 (J1 = 6.6, J2 = 14.4, dd, 1H), 3.40–3.46 (J1 = 4.5,
J2 = 14.7, dd, 1H), 3.53–3.62 (m, 1H), 4.01–4.06 (J = 14.7, t, 1H),
7.21–7.33 (m, 8H).
6.2. Biological materials and methods
6.2.1. In vitro APN assay
6.1.29.
L
-Isoleucyl-N1-[(2R)-2-amino-3-phenylpropyl]-
L-
IC50 values against APN were determined as previously de-
leucinamide hydrochloride (C1)
scribed and using L-Leu-p-nitroanilide as substrate and Microsomal
Yield: 73%, mp = 153–155 °C. ESI-MS m/z: 377.8 (M+H)+; 1H
NMR (D2O) d 0.72–0.73 (J = 4.5, t, 3H), 0.76–0.78 (J = 6.3, d, 6H),
0.84–0.86 (J = 6, d, 3H), 1.03–1.08 (m, 1H), 1.31–1.37 (m, 2H),
aminopeptidase from Porcine Kidney Microsomes (Sigma) as the
enzyme in 50 mM PBS, pH 7.2, at 37 °C. The hydrolysis of the sub-
strate was monitored by following the change in the absorbance
measured at 405 nm with the UV–vis spectrophotometer Pharma-
cia LKB, Biochrom 4060. All solutions of inhibitors were prepared
in the assay buffer, and pH was adjusted to 7.5 by the addition of
0.1 M HCl or 0.1 M NaOH. All inhibitors were preincubated with
APN for 30 min at room temperature. The assay mixture, which
contained the inhibitor solution (concentration dependent on the
1.48–1.54 (m, 2H), 1.80–1.83 (m, 1H), 2.69–2.76 (J1 = 4.2, J2
=
13.5, dd, 1H), 2.87–2.92 (J1 = 4.8, J2 = 14.2, dd, 1H), 3.27–3.33 (J1
= 4.8, J2 = 13.5, dd, 1H), 3.38–3.44 (J1 = 4.5, J2 = 13.5, dd, 1H),
3.55–3.59 (m, 1H), 3.73–3.77 (m, 1H), 4.16–4.20 (J = 12, t, 1H),
7.15–7.28 (m, 5H).
6.1.30.
L
-Leucyl-N1-[(2R)-2-amino-3-phenylpropyl]-
L
-
inhibitor), the enzyme solution (4 lg/mL final concentration),
leucinamide hydrochloride (C2)
and the assay buffer, was adjusted to 200 lL.
Yield: 79%, mp = 108–112 °C. ESI-MS m/z: 377.5 (M+H)+; 1H
NMR (D2O) d 0.74–0.0.76 (J = 6.0, d, 6H), 0.81–0.84 (J = 5.7, d,
6H), 0.92–0.97 (m, 1H), 1.38–1.44 (m, 2H), 1.57–1.62 (m, 2H),
1.82–1.84 (m, 1H), 3.11–3.17 (J1 = 5.4, J2 = 13.8, dd, 1H), 3.19–
3.26 (J1 = 5.7, J2 = 14.4, dd, 1H), 3.40–3.47 (J1 = 6, J2 = 14.4,
dd, 1H), 3.52–3.58 (J1 = 6.3, J2 = 13.2, dd, 1H), 3.62–3.67 (m,
1H), 3.77–3.83 (m, 1H), 4.56–4.60 (J = 8.7, t, 1H), 7.20–7.36
(m, 5H).
6.2.2. MTT assay
HL-60 Cell was grown in RPMI1640 medium containing 10% FBS
at 37 °C in 5% CO2 humidified incubator. Cell proliferation was
determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2.5-diphe-
nyl-2H-tetrazolium bromide) assay. Briefly, cells were plated in a
96-well plate at 10,000 cells per well, cultured for 4 h in complete
growth medium, then treated with 1600, 800, 400, 200, 100 lg/mL