Novel 3-phenylpropane-1,2-diamine derivates as inhibitors of aminopeptidase N (APN)

Article abstract of DOI:10.1016/j.bmc.2008.10.025

Aminopeptidase N (APN) is an essential peptidase involved in the process of tumor invasion and metastasis. Here we describe a novel class of inhibitor with 3-phenylpropane-1,2-diamine as scaffold to APN. Preliminary activity evaluation with enzyme inhibition studies showed that compound 12i exhibited potent and selective inhibitory activity towards APN with the IC₅₀ value 15.5 ± 1.2 μM.

Full text of DOI:10.1016/j.bmc.2008.10.025

Bioorganic & Medicinal Chemistry 16 (2008) 9984–9990
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 3-phenylpropane-1,2-diamine derivates as inhibitors
of aminopeptidase N (APN)
Luqing Shang a, Qiang Wang a, Hao Fang a, Jiajia Mu a, Xuejian Wang a, Yumei Yuan a, Binghe Wang b,
Wenfang Xu a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, PR China
b Department of Chemistry, Georgia State University, Atlanta 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminopeptidase N (APN) is an essential peptidase involved in the process of tumor invasion and metas-
tasis. Here we describe a novel class of inhibitor with 3-phenylpropane-1,2-diamine as scaffold to APN.
Preliminary activity evaluation with enzyme inhibition studies showed that compound 12i exhibited
Received 26 August 2008
Revised 9 October 2008
Accepted 10 October 2008
Available online 14 October 2008
potent and selective inhibitory activity towards APN with the IC50 value 15.5 1.2 lM.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Aminopeptidase N inhibitor
3-Phenylpropane-1,2-diamine derivates
Synthesis
1. Introduction
of AHPA-Val inserted deeply into the extended hydrophobic
pocket.16
Aminopeptidase N (APN) is a Zn2+-dependent exopeptidase that
is expressed by monocytes, myeloid, epithelial cells of the intestine
and kidney, fibroblasts, endothelial cells and tumor cells.1 Being
responsible for the cleavage the N-amino group from polypeptide
chains, APN plays a key role in physiological processing and degra-
dation of peptides and proteins, and alterations in their activity
have been associated with various pathological disorders.2–5
Several excellent reviews on natural and small molecule inhib-
itors of APN have been published.6–8 Of these inhibitors, the best
well known is Bestatin, which was isolated by Umezawa et al. in
a search for low molecular weight inhibitors for hydrolytic en-
zymes on the surfaces of cells.9
Our group has studied the binding site and catalytic domain of
APN based on the co-crystal complex of APN and Bestatin reported
by Kiyoshi,11 the binding site of the APN with Bestatin can be di-
vided to three part, part A is a hydrophobic pocket which interact
with phenyl group of Bestatin; part B is the zinc binding group
(ZBG) and part C is another hydrophobic pocket in other side. Addi-
tionally, part C can be divided into pocket C and pocket C0 in de-
tails. However, Bestatin did not show well interaction with part
C.6,14,18
In our ongoing work, we wanted to develop a novel scaffold
based on the AHPA structure and can occupy the part C. Addition-
ally, different building block (such as the residue group of amino
acid or carboxylic acid) will be introduced to interact with the
hydrophobic pocket A of APN. In this article, we described the syn-
thesis and enzymatic evaluation of the 3-phenylpropane-1,2-dia-
mine derivates and docking studies of the interaction are also
discussed.
In recent years, the 3D-structures of APN have been investi-
gated by the X-ray crystallographic studies on the co-crystal of
the enzyme and various inhibitors.10–12 Our group had reported
some new APN inhibitors, such as 3-galloylamido-N0-substituted
2,6-piperidinedione-N-acetamide peptidomimetics,13
L
-lysine deri-
vates,14
L
-iso-glutamine derivatives,15 and AHPA (b-amino-
a
-hy-
droxyl-phenylbutanoic acid) derivates.16 In the previous study,
we reported that one AHPA derivative, AHPA-Val, exhibited better
inhibitory activity against APN than that of Bestatin. The docking
studies showed that the amino group and carbonyl group of
AHPA-Val were chelated to the zinc ion of APN and the aryl ring
2. Chemistry
The target compounds were synthesized efficiently following
the procedures as shown in Scheme 1. The starting material com-
pound
was prepared from
1
(2R)-2-[(tert-butoxycarbonyl)amino]-3-phenyl-propano
D
-phenylalanine according to the literature.17
The hydroxyl group of compound 1 was converted to mesyl
group by methanesulfonyl chloride and then reacted with sodium
* Corresponding author.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.025

L. Shang et al. / Bioorg. Med. Chem. 16 (2008) 9984–9990
9985
A
O
A
O
R
N
H
H2N
N
H
COOH
NH2
OH
Zn2+
C
amino acid or
organic acid
Bestatin
Zn2+
C
B
B
azide to generate azide 3. The key intermediate 4 was obtained by
the disoxidation of compound 3.
site, such as amino, hydroxyl and thiol. However, all of these com-
pounds showed a little less activity than that of Bestatin. This phe-
nomenon may result from the difference of the ZBG and the active
conformation between the Bestatin and the target compounds we
designed. Additionally, the effects of 8f, 8g, 8j, and 12i on the HL-
60 cell proliferation compared with Bestatin were shown in Fig. 1.
Cell viability was assessed by MTT method. Compound 12i and 8j
exhibited the inhibitory effect of cell proliferation with IC50 values
of 0.63 0.08 mM and 1.09 0.12 mM, respectively, which showed
better potency than that of Bestatin with IC50 value of
1.65 0.09 mM. These result suggested that compound 12i exhibit
the most potent and selective inhibitory activity towards APN with
the IC50 value in micromole range. It is necessary to investigate the
structure of tripeptide analogue compound 12i which interact
favorably with APN.
In order to investigate the interaction of our target compound
with APN, the most active compound 12i was built and docked into
the active site of APN (PDB code: 2DQM) using Sybyl7.0. The bind-
ing studies showed that the carbonyl group and amino group of
compound 12i bind to the zinc ion and form a five-membered ring
in the active pocket with distance of 2.04 Å and 1.97 Å, respec-
tively. According to the structure, the C0 pocket of APN contains
tyrosine residues. This gives favorable interactions with phenyl
group of the backbone because of the increase of hydrophobic con-
tact area and possible interactions between the benzene ring of 12i
and the C0 pocket residues. Additionally, the terminal benzene ring
of the substituent inserted to the pocket A and another phenyl
group of the substituent R2 interact with the pocket C (Fig. 2a).
Compound 12i also formed hydrogen bonds with His297
(<2.98 Å), Glu298 (<3.19 Å) and His301 (<2.38 Å) which are the
essential amino acids of the conserved sequence (HEXXHX18E) in
the catalytic domain that is well conserved in peptidase M1 fam-
ily.19 In addition, the carbonyl group of compound 12i could inter-
act with the hydroxyl group of Tyr381 by hydrogen bond which
would be benefit to stabilize of the reaction intermediate with
the zinc ion11 (Fig. 2b)
The Boc-amino acids 6 were activated with isobutyl chlorofor-
mate and N-methylmorpholine and then coupled with compound
4 to yield 7a–7j. The Boc-protecting group can be easily removed
by 3 N HCl in ethyl acetate to give hydrochloride salts 8a–8j. Com-
pounds 12a–12j were synthesized similar to 8a–8j.
3. Results and discussion
The preliminary pharmacological studies have been investi-
gated on enzymatic inhibition of APN and MMP-2 and the result
listed in Table 1. Similar to APN, MMP-2 is also a zinc-dependent
metalloproteinase involved in the process of tumor invasion and
metastasis. MMP-2 is an endopeptidase which is different with
APN, an exopeptidase. To observe the selectivity of these target
compounds against two enzyme family, the target compounds
were assayed for the inhibitory activities on APN and MMP-2.
As shown in the enzyme assay results, most compounds exhib-
ited a better inhibitory activityon MMP-2than that of APN. This phe-
nomenon was also shown in our previous studies 3-galloylamido-
N0-substituted-2,6-piperidinedione-N-acetamide peptidomimet-
ics13 and -lysine derivates.14 This result possibly resulted from the
L
differences between the structure of active site of APN and MMP-2.
According to the binding site of APN (PDB code: 1HS6) and MMP-2
(PDB code: 1HOV, 1CK7, and 1QIB), Wang et al.14 showed that the ac-
tive site of APN was more deeper than the active site of MMP-2,
which may lead to the difficulty to access the active site of APN com-
pared with the binding with MMP-2 in most compounds.
According to the result of enzyme inhibition, compounds 8f, 8g,
8j, and 12i exhibited most active toward APN among these target
compounds. The IC50 values for these APN inhibitors range be-
tween 15 and 50 lM. Interestingly, these four compounds also
showed more potent toward APN than that against MMP-2. The
structure in R1 or R2 position contains the essential functional
group which could interact with the zinc ion in the APN catalytic
OH
NHBoc
1
NH2
NHBoc
4
c
b
OMs
NHBoc
N3
NHBoc
a
2
3
O
O
O
O
O
R1
R1
N
H
f
d
g
R1
4
, e
N
H
R1
2HCl
OH
OH
OH
NH2
8
NH2
NHBoc
7
NHBoc
NHBoc
6
NH2
5
O
O
O
N
H
R2
f
4
N
R2
, h
R2
10
Cl
R2
9
HCl
H
NH2
12
NHBoc
11
Scheme 1. Reagents and conditions: (a) MsCl/THF, 0 °C; (b) NaN3/DMF; (c) Mg/MeOH; (d) (Boc)2O/DCM, Et3N, 0 °C; (e) isobutyl chloroformate, N-methylmorpholine, THF,
ꢀ15 °C; (f) HCl/EtOAc; (g) oxalyl chloride/DCM, 0 °C; (h) Et3N, THF, 0 °C.

9986
L. Shang et al. / Bioorg. Med. Chem. 16 (2008) 9984–9990
Table 1 (continued)
Table 1
The structures and IC50 values of target compounds
a
Compound
R1
IC50
(l
M)
APN
MMP-2
1
OH
OH
12h
491.8 2.7
328.5 1.9
2
2
a
Compound
R1
H
IC50 (lM)
APN
MMP-2
NH2
H
N
8a
8b
>1000
23.5 1.2
12i
12j
15.5 1.2
182.9 2.7
O
142.4 5.3
476.0 1.9
170.2 2.3
151.4 3.6
8c
NO2
204.5 3.5
162.0 4.8
100.7 3.4
8d
8e
NH2
164.2 2.3
118.7 1.5
99.7 2.2
NO2
Bestatin
3.1 0.6
112.1 3.5
S
a
Each value represents the mean of three experiments, standard deviation is
given.
8f
33.7 1.1
50.5 0.9
201.9 2.9
219.4 2.8
OH
8g
NH2
4
2
0
8h
8i
CH3
>1000
771.9 3.9
802.3 6.5
78.8 1.8
O
>1000
NH2
8j
21.1 0.8
R2
Bes
8f
8g
8j
12i
12a
12b
NH2
408.6 4.6
164.3 3.2
11.2 0.8
99.7 2.6
Compound
NH2
Figure 1. Effects of Bestatin and compounds 8f, 8g, 8j, and 12i on the HL-60 cell line
proliferation. Each column represents the mean with SE of five independent
experiments.
12c
12d
12e
289.5 2.7
595.8 2.8
>1000
546.8 3.4
211.3 3.4
632.4 3.9
4. Conclusions
In summary, we demonstrated a new family of APN inhibitor
origin with the scaffold 3-phenylpropane-1,2-diamine. Most of
the compounds possess potent activity toward APN and the most
potent compound, 12i, exhibited good enzymatic inhibition and
selectivity to APN. This feature may offer one lead compound and
could be used to design more potent APN inhibitors in the future.
5. Experimental
12f
>1000
>1000
221.0 2.3
5.1. Synthetic methods and spectroscopic details
Unless specified otherwise, all starting material, reagents and
solvents were commercially available. All reactions except those
in aqueous media were carried out by standard techniques for the
exclusion of moisture. All reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates (60GF-254) and visu-
Cl
12g
>1000
HN
Cl

L. Shang et al. / Bioorg. Med. Chem. 16 (2008) 9984–9990
9987
Figure 2. (a) The docking result of 12i (Bestatin in the X-ray crystal is showed in red); (b) The docking result of 12i showed by LIGPLOT.
alized with UV light, or iodine vapor. Proton NMR spectra were
determined on a Brucker DRX spectrometer operating at 400 MHz
for 1H, d in parts per million and J in Hertz, using TMS as an internal
standard. ESI-MS were determined on an API 4000 spectrometer.
Measurements were made in D2O, CD3OD or DMSO-d6 solutions.
Melting points were determined on an electrothermal melting
point apparatus and were uncorrected. Anhydrous reactions were
carried out in over-dried glassware under a nitrogen atmosphere.
5.1.1. (2R)-2-[(tert-Butoxycarbonyl)amino]-3-phenylpropyl
methanesulfonate (2)
Compound 1 (5 g, 20 mmol) was dissolved in anhydrous THF
(20 mL) and Et3N (3.03 g, 30 mmol) was added to the solution. A
solution of methanesulfonyl chloride (3.4 g, 30 mmol) in anhy-
drous THF (10 mL) was added dropwise to the mixture. The reac-
tion mixture was stirred for 4 h. The mixture was poured to the
cold water and white solid was precipitated to give crude product

9988
L. Shang et al. / Bioorg. Med. Chem. 16 (2008) 9984–9990
(R)-2-(tert-butoxycarbonyl)-3-phenylpropyl
methanesulfonate
5.1.6. (2S)-2-Amino-3-methyl-N-[(2R)-2-amino-3-phenyl-
(5.52 g yield 84.3%), which was of suitable purity to use directly
in next reaction.
propyl]propionamide hydrochl-oride (8c)
Yield: 72.45%, mp = 190–193.5 °C, ½a D25
ꢁ
+55.0° (c 1, MeOH). ESI-
MS m/z: 250.3 (M+H)+; 1H NMR (D2O) d 0.91–0.95 (J = 12, d, 6H),
2.12–2.17 (m, 1H), 1.65–1.73 (m, 1H), 2.80–2.88 (J1 = 6, J2 = 15,
dd, 1H), 2.97–3.04 (J1 = 6, J2 = 15, dd, 1H), 3.31–3.40 (J1 = 9,
J2 = 18, dd, 1H), 3.57–3.63 (J1 = 6, J2 = 15, dd, 1H), 3.60–3.63 (m,
1H), 3.71–3.73 (J = 6, d, 1H), 7.23–7.37 (m, 5H).
5.1.2. (2R)-2-[(tert-Butoxycarbonyl)amino]-3-phenyl-
propylamine (4)
A mixture of sulfonate 2 (10 g, 30 mmol) and NaN3 (3.95 g,
60 mmol) in anhydrous DMF (80 mL) was stirred at 60 °C for
10 h. The mixture was poured to the cold water and extracted with
ethyl acetate. The organic layer was washed with water and brine,
dried over Na2SO4 and concentrated in vacuum. The residue was
purified by flash column chromatography to give on silica gel
(Petroleum/EtOAc 10:1) to afford compound 3 (2R)-tert-butyl (2-
azidomethyl-1-benzylethyl) carbamate.
The azide 3 (4 g, 0.015 mol) was hydrogenated on Mg (1.08 g,
0.045 mol) in MeOH at ordinary pressure and 0 °C for 1 h. MeOH
was removed under reduced pressure and the residue was diluted
with water. The aqueous solution was extracted efficiently with
ether and the ether layer was washed with brine, dried over
Na2SO4 and concentrated in vacuum to give product 4 (2R)-2-
[(tert-butoxycarbonyl)amino]-3-phenyl-propylamine. ESI-MS m/z:
251.1 (M+H)+; 1H NMR (MeOD) d 1.41(s, 9H), 2.59–2.65 (J1 = 6,
J2 = 12, dd, 1H), 2.71–2.81 (m, 3H), 3.79 (m, 1H), 7.18–7.32 (m, 5H).
5.1.7. (2S)-2,6-Amino-N-[(2R)-2-amino-3-phenylpropyl]caproyl-
amide hydrochloride (8d)
Yield: 78.48%, mp = 221.4–223 °C, ½a D25
ꢁ
+61.6° (c 1, MeOH). ESI-
MS m/z: 280.3 (M+H)+; 1H NMR (D2O) d 1.36–1.41 (m, 2H), 1.59–
1.64 (m, 2H), 1.77–1.82 (m, 2H), 2.80–2.87 (J1 = 6, J2 = 15, dd, 1H),
2.87–2.90 (J = 9, t, 2H), 2.96–3.03 (J1 = 6, J2 = 15, dd, 1H), 3.34–
3.40 (m, 1H), 3.55–3.62 (J1 = 6, J2 = 15, dd, 1H), 3.62–3.68 (J1 = 6,
J2 = 15, dd, 1H), 3.89–3.93 (J1 = 12, t, 1H), 7.23–7.37 (m, 5H).
5.1.8. (2S)-2-Amino-4-methylthio-N-[(2R)-2-amino-3-phenyl-
propyl]butyramide hydrochloride (8e)
Yield: 72.41%, mp = 82–84.5 °C, ½a D25
ꢁ
+19.2° (c 1, MeOH). ESI-MS
m/z: 283.4 (M+H)+; 1H NMR (D2O) d 2.01 (s, 3H), 2.05–2.09 (m, 2H),
2.50–2.55 (J = 15, t, 2H), 2.80–2.87 (J1 = 6, J2 = 15, dd, 1H), 2.92–
3.00 (J1 = 6, J2 = 15, dd, 1H), 3.47–3.53 (m, 1H), 3.55–3.610 (J1 = 6,
J2 = 12, dd, 1H), 3.62–3.69 (J1 = 6, J2=12, dd, 1H), 3.62–3.66 (J = 12,
t, 1H), 7.22–7.37 (m, 5H).
5.1.3. 2-[(tert-Butoxycarbonyl)amino]-N-[(2R)-2-[(tert-
butoxycarbonyl)amino]-3-phenylpropyl] acetamide (7a)
To a stirred solution of tert-butoxycarbonyl-2-amino-acetic
acid (1.54 g, 8.80 mmol) and N-methylmorpholine (1.05 mL,
9.60 mmol) in THF (15 mL) was added isobutyl chloroformate
(1.23 mL, 9.60 mmol) at ꢀ15 °C. The mixture was stirred for
30 min at the same temperature. A solution of compound 4
(2R)-2-[(tert-butoxycarbonyl)amino]-3-phenyl-propylamine
(2.00 g, 8.00 mmol) in THF (20 mL) was added dropwise to the
reaction mixture. The stirring was continued for 1 h at ꢀ15 °C
and then remove the cooling bath. The reaction was continued
for 4 h and the mixture was filtrated. After filtration, the filtrate
was concentrated with a rotary evaporator. The residue was dis-
solved in EtOAc and washed with 5% NaHCO3, 10% citric acid,
and brine in turn. The EtOAc solution was dried over Na2SO4
and concentrated with a rotary evaporator to afford crude prod-
uct. The crude product was recrystallized by EtOAc to afford
pure title compound 7a. Yield: 67.54%, mp = 103–105 °C. ESI-
MS m/z: 408.3 (M+H); 1H NMR (DMSO-d6) d 1.22 (s, 9H), 1.38
(s, 9H), 2.58–2.61 (m, 1H), 2.67–2.74 (J1 = 5.4, J2 = 13.8, dd, 1H),
3.01–3.07 (m, 1H), 3.16–3.19 (m, 1H), 3.50–3.56 (m, 2H), 3.66–
3.68 (m, 1H) 7.17–7.27 (m, 5H).
5.1.9. (2S)-2-Amino-3-hydroxyl-N-[(2R)-2-amino-3-phenyl-
propyl]propionamide hydrochloride (8f)
Yield: 78.61%, mp = 111–114 °C, ½a D25
ꢁ
+19.5° (c 1, MeOH). ESI-
MS m/z: 238.4(M+H)+; 1H NMR (D2O) d 2.80–2.88 (J1 = 6, J2 = 13.5,
dd, 1H), 2.96–3.03 (J1 = 6, J2 = 12.6, dd, 1H), 3.47–3.49 (J = 6, d,
2H), 3.64–3.69 (m, 1H), 3.85–3.90 (m, 2H), 4.03–4.06 (J = 9, t,
1H), 7.23–7.37 (m, 5H).
5.1.10. (2S)-2,5-Amino-N-[(2R)-2-amino-3-phenylpropyl]-
valeramide hydrochloride (8g)
Yield: 68.73%, mp = 130–133.3 °C, ½a D25
ꢁ
+16.8° (c 1, MeOH). ESI-
MS m/z: 265.2 (M+H)+; 1H NMR (D2O) d 1.64–1.72 (m, 2H), 1.82–
1.88 (m, 2H), 2.81–2.88 (J1 = 6.6, J2 = 15, dd, 1H), 2.89–2.96 (J1 = 6,
J2 = 14.4, dd, 1H), 3.01–3.03 (m, 2H), 3.34–3.40 (m, 1H), 3.57–
3.63 (m, 2H), 3.93–3.99 (J = 15, t, 1H), 7.23–7.36 (m, 5H).
5.1.11. (2S)-2-Amino-N-[(2R)-2-amino-3-phenylpropyl]-
propionamide hydrochloride (8h)
Yield: 67.54%, mp = 161–163.5 °C, ½a D25
ꢁ
+28.4° (c 1, MeOH). ESI-
MS m/z: 222.4(M+H)+; 1H NMR (D2O) d 1.42–1.46 (J = 12, d, 3H),
2.82–2.89 (J1 = 6, J2 = 14.4, dd, 1H), 2.96–3.03 (J1 = 5.4, J2 = 15, dd,
1H), 3.41–3.50 (m, 2H), 3.64–3.71 (m, 1H), 4.01–4.08 (m, 1H),
7.24–7.39 (m, 5H).
5.1.4. 2-Amino-N-[(2R)-2-amino-3-phenylpropyl] acetamide
hydrochloride (8a)
Compound 7a (0.8 g, 1.96 mmol) was dissolved in 20 mL HCl–
EtOAc (3 mol/L). After 30 min, the solvent was filtrated and the
precipitate was washed with EtOAc to get 0.32 g of compound 8a
5.1.12. N-[(2R)-2-Amino-3-phenylpropyl]-L-glutamamide
hydrochloride (8i)
as a white solid. Yield: 78.66%, mp = 195.5–197.8 °C, ½a D25
ꢁ
+26.8°
(c 1, MeOH). ESI-MS m/z: 208.3 (M+H)+; 1H NMR (D2O) d 2.81–
2.89 (J1 = 6, J2 = 15, dd, 1H), 2.94–3.01 (J1 = 6, J2 = 15, dd, 1H),
3.38–3.48 (J1 = 4.5, J2 = 15, dd, 1H), 3.49–3.56 (J1 = 4.5, J2 = 15, dd,
1H), 3.62–3.73 (m, 1H), 3.80 (s, 2H), 7.23–7.37 (m, 5H).
Yield: 70.53%, mp = 167–171 °C, ½a D25
ꢁ
+24.6° (c 1, MeOH). ESI-
MS m/z: 279.4 (M+H)+; +H NMR (D2O) d 1.98–2.07 (m, 2H), 2.35–
2.40 (J = 15, t, 2H), 2.83–2.89 (J1 = 6.3, J2 = 14.1, dd, 1H), 2.96–3.03
(J1 = 6, J2 = 14.7, dd, 1H), 3.38–3.45 (J1 = 4.8, J2 = 14.4, dd, 1H),
3.54–3.61 (J1 = 4.5, J2 = 14.7, dd, 1H), 3.63–3.67 (J = 12, t, 1H),
3.95–3.99 (m, 1H), 7.24–7.38 (m, 5H).
5.1.5. (2S)-2-Amino-4-methyl-N-[(2R)-2-amino-3-phenyl-
propyl]valeramide hydrochloride (8b)
Yield: 75.46%, mp = 165.5–168 °C, ½a D25
ꢁ
+53.8° (c 1, MeOH). ESI-
5.1.13. (2S)-2-Amino-3-thiol-N-[(2R)-2-amino-3-phenyl-
MS m/z: 264.0 (M+H)+; 1H NMR (D2O) d 0.86–0.87 (J = 3d, 6H),1.52–
1.63 (m, 2H), 1.65–1.73 (m, 1H), 2.81–2.88 (J1 = 6, J2 = 15, dd, 1H),
2.94–3.01 (J1 = 6, J2 = 15, dd, 1H), 3.36–3.43 (J1 = 6, J2 = 15, dd,
1H), 3.49–3.56 (J1 = 6, J2 = 15, dd, 1H), 3.64–3.74 (m, 1H), 3.92–
3.94 (J = 6, t, 1H), 7.22–7.36 (m, 5H).
propyl]propionamide hydrochloride (8j)
Yield: 61.28%, mp = 156–158 °C, ½a D25
ꢁ
+30.3° (c 1, MeOH). ESI-
2.83–2.89 (J1 = 5.4,
MS m/z: 254.3(M+H)+; 1H NMR (D2O)
d
J2 = 13.2, dd, 1H), 2.92–2.99 (J1 = 5.1, J2 = 12.6, dd, 1H), 3.08–3.15
(J1 = 6, J2 = 13.5, dd, 1H), 3.27–3.32 (J1 = 5.7, J2 = 12.6, dd, 1H),

L. Shang et al. / Bioorg. Med. Chem. 16 (2008) 9984–9990
9989
3.42–3.44 (J = 5.7, d, 2H), 3.47–3.52 (m, 1H), 4.10–4.12 (J = 6.3, t,
3.34(J = 6, d, 1H) 3.40–3.42 (J = 6, d, 1H), 3.59–3.62 (m, 3H), 7.24–
1H), 7.25–7.37 (m, 5H).
7.39 (m, 10H).
5.1.14. (3S)-3-[(tert-Butoxycarbonyl)amino]-N-[(2R)-2-[(tert-
butoxycarbonyl)amino]-3-phenylpropyl]propionamide hydro-
chloride (11a)
N-(tert-Butoxycarbonyl)-b-alanine (2.00 g, 10.58 mmol) was
dissolved in anhydrous CH2Cl2 (30 mL). The solution of oxalyl chlo-
ride (2.01 g, 15.87 mmol) in CH2Cl2 was added dropwise sur-
rounded with ice bath. After 4 h, CH2Cl2 was removed in vacuum
to afford compound 10 (2.07 g).
5.1.20. N-[(2R)-2-Amino-3-phenylpropyl]-4-phenylbutanamide
hydrochloride (12f)
Yield: 71.34%, mp = 102–104.5 °C, ½a D25
ꢁ
+48.7° (c 1, MeOH). ESI-
MS m/z: 297.5 (M+H)+; 1H NMR (D2O) d 1.73–1.90 (m, 2H), 2.11–
2.15 (J = 12, t, 2H), 2.50–2.55 (J = 12, t, 2H), 2.77–2.84 (J1 = 6.3,
J2 = 14.1, dd, 1H), 2.86–2.93 (J1 = 6.6, J2 = 14.1, dd, 1H), 3.21–3.28
(J1 = 6, J2 = 14.7, dd, 1H), 3.32–3.40 (J1 = 6, J2 = 14.7, dd, 1H), 3.54–
3.58 (m, 1H), 7.15–7.34 (m, 10H).
Compound 4 (2.00 g, 8.00 mmol) was dissolved in anhydrous
THF (40 mL) and Et3 N (1.33 mL). After 10 mL THF with 1.99 g com-
pound 13 was added dropwise at 0 °C and stirred for 4 h, THF was
removed under reduced pressure and the residue was diluted with
EtOAc. The organic phase was washed with 5% NaHCO3, 10% citric
acid, and brine in turn. The EtOAc solution was dried over Na2SO4
and concentrated with a rotary evaporator to afford crude product.
The crude product was recrystallized by EtOAc to afford pure title
compound 11a. Yield: 63.82%, mp = 121–123 °C. ESI-MS m/z: 422.3
(M+H)+; 1H NMR (DMSO-d6) d 1.31 (s, 9H), 1.36 (s, 9H), 2.21–2.26
(m, 2H), 2.53–2.61 (J1 = 5.1, J2 = 13.5, dd, 1H), 3.01–3.07 (m, 1H),
2.67–2.73 (J1 = 5.4, J2 = 13.5, dd, 1H), 3.11–3.15 (m, 4H), 3.68–
3.70 (m, 1H) 7.17–7.28 (m, 5H).
5.1.21. N-[(2R)-2-Amino-3-phenylpropyl]-2-{2-[(2,6-dichloro-
phenyl)amino]phenyl} acetamide hydrochloride (12g)
Yield: 76.83%, mp = 85–87 °C, ½a D25
ꢁ
+20.0° (c 1, MeOH). ESI-MS
m/z: 428.5 (M+H)+; 1H NMR (D2O) d 2.81–2.88 (J1 = 3, J2 = 15, dd,
1H), 2.95–3.01 (J1 = 3, J2 = 12, dd, 1H), 3.22–3.30 (m, 2H), 3.67 (s,
2H), 4.00–4.06 (m, 1H), 6.27–6.30 (J = 6, d, 1H), 6.83–6.87 (J = 12,
1H), 7.02–7.07 (J = 15, t, 1H), 7.17–7.19 (J = 6, d, 1H), 7.23–7.30
(m, 6H), 7.50–7.53 (J = 9, d, 2H).
5.1.22. (2E)-N-[(2R)-2-Amino-3-phenylpropyl]-3-(3,4-hydroxy)-
acrylamide hydrochloride (12h)
Yield: 55.37%, mp = 143–145 °C, ½a D25
ꢁ
+21.6° (c 1, MeOH). ESI-
MS m/z: 313.4 (M+H)+; 1H NMR (D2O) d 2.82–2.98 (m, 2H), 2.98–
3.05 (m, 2H), 3.72–3.74 (m, 1H), 6.37–6.42 (J = 15, d, 1H), 6.74–
6.79 (J = 15, d, 1H), 7.21–7.38 (m, 8H).
5.1.15. (3S)-3-Amino-N-[(2R)-2-amino-3-phenylpropyl]pro-
pionamide hydrochloride (12a)
Compound 11a (0.8 g, 1.96 mmol) was dissolved in 20 mL HCl–
EtOAc (3 mol/L). After 30 min, the solvent was filtrated and the
precipitate was washed with EtOAc to get 0.39 g of compound
5.1.23.
L
-Phenylalanyl-N-[(2R)-2-amino-3-phenylpropyl]-
L
-phenyl-
alaninamide hydrochloride (12i)
12a as a white solid. Yield: 85.68%, mp = 179–181 °C, ½a D25
ꢁ
+54.9°
Yield: 69.54%, mp = 90–93 °C, ½a D25
ꢁ
+40.9° (c 1, MeOH). ESI-MS
(c 1, MeOH). ESI-MS m/z: 222.3 (M+H)+; 1H NMR (D2O) d 2.57–
2.62 (J = 15, t, 2H), 2.82–2.89 (J1 = 6, J2 = 15, dd, 1H), 2.93–3.00
(J1 = 6, J2 = 15, dd, 1H), 3.13–3.18 (J = 15, t, 2H), 3.32–3.39 (J1 = 6,
J2 = 15, dd, 1H), 3.44–3.50 (J1 = 6, J2 = 15, dd, 1H), 3.62–3.69 (m,
1H), 7.23–7.37 (m, 5H).
m/z: 445.5 (M+H)+; 1H NMR (D2O) d 2.75–2.95 (m, 5H), 3.10–3.16
(m, 2H), 3.28–3.34 (m, 2H), 4.00–4.06 (J1 = 4.5, J2 = 12, dd, 1H),
4.52–4.54 (m, 1H), 7.27–7.31 (m, 15H).
5.1.24. N-[(2R)-2-Amino-3-phenylpropyl]-3,5-dinitrobenz-
amide hydrochloride (12j)
5.1.16. 6-Amino-N-[(2R)-2-amino-3-phenylpropyl]caproyl-
amide hydrochloride (12b)
Yield: 49.48%, mp = 205–207 °C, ½a D25
ꢁ
+39.1° (c 1, MeOH). ESI-
MS m/z: 345.1 (M+H)+; 1H NMR (D2O) d 2.85–2.92 (J1 = 5.4,
J2 = 13.8, dd, 1H), 2.99–3.06 (m, 1H), 3.40–3.47 (m, 1H), 3.55–
3.60 (m, 2H), 7.26–7.36 (m, 5H), 8.97 (s, 1H), 9.03 (s, 2H).
Yield: 65.42%, mp = 135–137.3 °C, ½a D25
ꢁ
+46.2° (c 1, MeOH). ESI-
MS m/z: 264.2 (M+H)+; 1H NMR (D2O) d 1.24–1.32 (m, 2H), 1.45–
1.62 (m, 4H), 2.15–2.20 (J = 15, t, 2H), 2.80–2.86 (J1 = 3, J2 = 12,
dd,1H), 2.86–2.91 (J = 15, t, 2H), 2.94–3.01 (J1 = 3, J2 = 12, dd, 1H),
3.31–3.38 (J1 = 3, J2 = 12, dd, 1H), 3.41–3.48 (J1 = 3, J2 = 12, dd,
1H), 3.62–3.68 (m, 1H), 7.22–7.37 (m, 5H).
5.2. Biological materials and methods
5.2.1. In vitro APN assay
5.1.17. N-[(2R)-2-Amino-3-phenylpropyl]-3-phenylpropan-
amide hydrochloride (12c)
IC50 values against APN were determined as previously de-
scribed and using L-Leu-p-nitroanilide as substrate and Microsomal
Yield: 77.37%, mp = 184.8–188.3 °C, ½a D25
ꢁ
+23.5° (c 1, MeOH).
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
ESI-MS m/z: 283.3 (M+H)+; 1H NMR (D2O) d 2.46–2.51 (J = 14.4, t,
2H), 2.55–2.62 (J1 = 6, J2 = 14.4, dd, 1H), 2.63–2.70 (J1 = 6.3,
J2 = 15, dd, 1H), 2.79–2.84 (J = 15, t, 2H), 3.17–3.24 (J1 = 6, J2 = 15,
dd, 1H), 3.24–3.31 (J1 = 6, J2 = 14.4, dd, 1H), 3.38–3.45 (m, 1H),
7.08–7.18 (m, 5H), 7.22–7.33 (m, 5H).
5.1.18. 1 (2E)-N-[(2R)-2-Amino-3-phenylpropyl]-3-phenylacryl-
amide hydrochloride (12d)
Yield: 67.89%, mp = 191–195 °C, ½a D25
ꢁ
+16.8° (c 1, MeOH). ESI-MS
inhibitor), the enzyme solution (4 lg/mL final concentration),
and the assay buffer, was adjusted to 200 lL.
m/z: 281.3 (M+H)+; 1H NMR (MeOD) d 3.00–3.03 (J = 9, d, 2H), 3.49–
3.56 (J1 = 3.9, J2 = 9.6, dd, 1H), 3.58–3.65 (J1 = 3.6, J2 = 8.4, dd, 1H),
3.67–3.73 (m, 1H), 7.68–7.73 (J = 15, d, 1H), 7.31–7.63 (m, 11H).
5.2.2. In vitro MMP-2 assay
5.1.19. N-[(2R)-2-Amino-3-phenylpropyl]-2-phenylacetamide
Gelatinase A (MMP-2) and TNBS were purchased from Sigma,
and the substance was synthesized as described by Vijaykumar
et al. The gelatinase, substance, and inhibitor were dissolved in so-
hydrochloride (12e)
Yield: 73.65%, mp = 67–69.5 °C, ½a D25
ꢁ
+13.0° (c 1, MeOH). ESI-MS
m/z: 269.5 (M+H)+; 1H NMR (MeOD) d 2.92–2.95 (d, 2H), 3.32–

9990
L. Shang et al. / Bioorg. Med. Chem. 16 (2008) 9984–9990
dium borate (pH 8.5, 50 mmol/L) and incubated for 30 min at 37 °C,
and then 0.03% TNBS was added and incubated for another 20 min,
the resulting solution was detected under 450 nm wavelength to
gain absorption.
Development Program of China (863 project; Grant No.
2007AA02Z314).
References and notes
5.2.3. MTT assay
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We thank Chunhua Ma and Huawei Zhu for their contributions
to this work. This work was supported by National Nature Science
Foundation of China (Grant Nos. 30772654 and 36072541) and the
Ph.D. Programs Foundation of Ministry of Education of PR China
(No. 20060422029) and National High Technology Research and
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