Novel L-arginine derivatives as aminopeptidase N inhibitors: design, chemistry, and pharmacological evaluation

Article abstract of DOI:10.1007/s00044-017-1999-2

Considering the important roles played in tumor, aminopeptidase N has been an appealing target for anti-tumor drug development. Here, a serial of novel aminopeptidase N inhibitors with L-arginine scaffold were designed, synthesized and evaluated for aminopeptidase N inhibitory activities. The preliminary anti-enzyme activity assay demonstrated that compounds 5e, 5h, 11e, 11g, and 11h showed comparable activities with the positive control bestatin, an approved aminopeptidase N inhibitor. In vitro anti-proliferation assay, compound 5f showed excellent activities against four kinds of tumor cells which overexpress aminopeptidase N. In vivo anti-metastasis assay, compounds 5f and 11g exhibited better activities than bestatin. So 5f and 11g should be lead compounds as novel aminopeptidase N inhibitors for further development.

Full text of DOI:10.1007/s00044-017-1999-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1999-2
ORIGINAL RESEARCH
Novel L-arginine derivatives as aminopeptidase N inhibitors:
design, chemistry, and pharmacological evaluation
Jiajia Mou1 Yepeng Luan2 Danghui Chen1 Qiang Wang3
●
●
●
Received: 31 March 2017 / Accepted: 20 July 2017
© Springer Science+Business Media, LLC 2017
Abstract Considering the important roles played in tumor,
aminopeptidase N has been an appealing target for anti-
tumor drug development. Here, a serial of novel amino-
peptidase N inhibitors with L-arginine scaffold were
designed, synthesized and evaluated for aminopeptidase N
inhibitory activities. The preliminary anti-enzyme activity
assay demonstrated that compounds 5e, 5h, 11e, 11g, and
11h showed comparable activities with the positive control
bestatin, an approved aminopeptidase N inhibitor. In vitro
anti-proliferation assay, compound 5f showed excellent
activities against four kinds of tumor cells which over-
express aminopeptidase N. In vivo anti-metastasis assay,
compounds 5f and 11g exhibited better activities than bes-
tatin. So 5f and 11g should be lead compounds as novel
aminopeptidase N inhibitors for further development.
Introduction
Aminopeptidase N (APN/CD13; EC 3.4.11.2) is a zinc-
dependent metalloproteinase with the capacity of cleaving
neutral or basic amino acids from the N-terminus of oli-
gopeptides. Overexpression of APN is significantly corre-
lated with various pathological disorders, such as
inflammatory bowel diseases, encephalomyelitis, multiple
sclerosis, ulcerative colitis, Crohn’s disease, rheumatoid
arthritis and especially cancers (Bank et al. 2008; Yamashita
et al. 2007; Shimizu et al. 2002; Reinhold et al. 2008;
Rahbar et al. 2006). Tumor cells which overexpress APN
are highly invasive via degrading extracellular matrix (Fujii
et al. 1995; Ishii et al. 2001). On another hand, angiogenesis
sprout from microvascular endothelial cells where APN is
expressed plays a central role in tumor growth, invasion,
and metastasis (Tomanek and Schatteman 2000). In case the
mRNA of the APN was knocked out, the level of angio-
genesis decreased dramatically (Fukasawa et al. 2006).
Satisfactory curative chemotherapeutic agents with high
affinity and selectivity are expected to be rationally
designed against specific tumor-associated targets. Given
the fact that APN is overexpressed on endothelial cells and
the surface of many kinds of tumor cells and plays a vital
role in tumorigenesis, it is reasonable to develop potential
APN inhibitors to block its enzymatic activity for cancer
therapeutic purpose (Minaosorio 2008).
●
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Keywords L-arginine derivatives Aminopeptidase N
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●
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Synthesis Anti-enzyme activity Anti-proliferation activity
Anti-metastasis activity
* Jiajia Mou
moujiajia66@163.com
Up to now, a wealth of inhibitors of APN with potency in
different range has been reported from natural resource or
artificially synthesis (Xu and Li 2005). Among these inhi-
bitors, bestatin is a representative which has been launched
and is now clinically employed as an anti-tumor agent
(Shimazawa et al. 1999). In 2006, the 3D structure of APN
has been disclosed according to the co-crystal complex of
APN and bestatin by Ito et al. (2006). The interaction mode
1
Department of Medicinal Chemistry, School of Chinese Materia
Medica, Tianjin University of Traditional Chinese Medicine, 312,
Anshanxi Road, Tianjin 300193, China
2
3
Department of Medicinal Chemistry, College of Pharmacy,
Qingdao University, Qingdao, Shandong 266021, China
College of Pharmacy, South-central University for Nationalities,
Wuhan, Hubei 430074, China

Med Chem Res
between bestatin and the active sites of APN can be illu-
strated by a simplified diagram (see Fig. 1). It revealed that
beside the catalytic center zinc(II) ion, there were three
hydrophobic binding pockets called S1 pocket, S1′ pocket
and S2′ pocket respectively. The carbonyl group in amide of
bestatin chelated with zinc(II) ion. The side chains of bes-
tatin could occupy S1 and S1′ pockets.
The specificity of the S1-binding pocket suggests that the
preferred amino acid is, in decreasing order, L-arginine, L-
lysine, L-alanine, L-tyrosine, and L-phenylalanine (Addla-
gatta et al. 2008). The crystal structures of Escherichia coli
amino-peptidase N (e PepN) in complex with L-arginine, L-
lysine, L-phenylalanine, L-tyrosine, and L-tryptophan
showed that all of the amino acids bind with their backbone
atoms close to the active-site zinc ion and their residues
occupy the S1 pocket of e PepN. In 2008, our group
designed and synthesized a series of L-lysine derivatives by
computer-aided drug design and found that some com-
pounds exhibited potential APN inhibitory activities (Fig. 2)
(Wang et al. 2008). Accordingly, we choose L-arginine as
lead compound to develop potential APN inhibitors. In fact,
L-arginine is very similar with L-lysine in structure, so good
results are expected.
First, compounds with aromatic rings as side chains were
designed and synthesized. The aromatic rings are antici-
pated to plunge in S1′ pocket of APN. But it is reported that
the analogs of bestatin, for example, amastatin (Aoyagi
et al. 1978) and probestatin (Aoyagi et al. 1990) (Fig. 2),
showed better activity against APN, which may be due to
that both of them have long side chains that can interact
with S1′ pocket and S2′ pocket simultaneously. With this
fact in mind, we designed and synthesized another series of
compounds with di-peptide mimics as side chains. The
compounds we designed are showed in Fig. 3.
Chemistry
Enzyme
S1
S1'
S2'
+
Zn2
Two kinds of L-arginine derivatives were designed and
synthesized via the routine outlined in Scheme 1. The
guanido of compound 1 was protected by nitro group to get
compound 2. The free carboxyl of compound 2 was then
methylated under acetyl chloride to give compound 3. The
acylation of compound 3 with acyl chloride or carboxylic
acid led to compounds 4a–h. Compound 8 was obtained by
the condensation of amino acid methyl ester 6 and car-
boxylic acids 7. Hydrolysis of compound 8 with NaOH/
MeOH led to di-peptide mimics 9. The acylation of com-
pound 3 with compound 9 yielded compounds 10a–h.
Finally the ester groups of 4a–h and 10a–h were treated
with NH2OK in anhydrous methanol to get the target
compounds 5a–h and 11a–h.
O
H3N
N
H
COOH
OH
Fig.1 Schematic diagram of the interaction of bestatin with APN (Xu
and Li 2005)
Fig. 2 Structures of amastatin and probestatin
Fig. 3 The structures of L-lysine
derivative and L-arginine
derivative
C

Med Chem Res
Scheme 1 Reagents and
conditions: a fuming nitric acid,
fuming sulfuric acid; b
CH3COCl, MeOH; c Et3N,
TBTU, CH2Cl2; d NH2OK,
MeOH; e IBCF, NMM, THF,
-15 °C; f NaOH/MeOH; g DCC,
HOBt, THF, -5 °C
Result and discussion
The possible reason maybe that R3 benzene ring was
designed to occupy S2′ pocket, which could accommodate a
moderate size group, but not a bulky one.
According to the data in Table 1, we could also make a
conclusion that compounds 5e, 5h, 11e, 11g, and 11h had
comparable activities with the positive control, bestatin.
According to the former scheme, two series of compounds
were obtained, di-peptide derivatives of L-arginine 5a–h
and tri-peptide derivatives of L-arginine 11a–h.
Anti-enzyme activity in vitro
Anti-proliferation activity in vitro
All the target compounds were evaluated for their inhibitory
activities toward APN. The inhibition results were sum-
marized in Table 1. Bestatin was used as positive control.
As shown in Table 1, all the compounds exhibited
appropriate activities with IC50 ranging from 4.2 to 31.4 µM
(bestatin IC50 = 3.8 µM). Compounds 11a–h have better
activities than compounds 5a–h. This result coincides with
our design principle.
Among the compounds 5a–h, the phenyl ring with two
electron-withdrawing groups (5e, 5g, and 5h) exhibited
better activities than the counterparts with only one sub-
stituent (5a, 5b, 5c, and 5d). A reasonable explanation is that
the phenyl ring with two electron-withdraing groups can
well accommodate the S1′ pocket. It is worthy of mentioning
that compound 5f showed exceptional moderate activity
against APN. The reason is speculated to be that the large
naphthalene ring could intensively occupy the S1′ pocket.
About compounds 11a–h which have increasingly bulky
substituents on R2, the activities were ameliorated (11g and
11h). The reason is seemingly like compound 5f. While R3
benzene ring with one electron-withdrawing group exhibited
better activities than the ones with two substituents (11a and
11b, 11e and 11f). These results differ from compounds 5a–h.
The compounds with desirable anti-enzyme activities were
selected for anti-proliferation assay in vitro against four
tumor cell lines HL-60, A549, ES-2, and PLC which
overexpress APN with bestatin as the positive control. The
results were shown in Table 2.
Almost all the selected compounds showed anti-proliferation
activities towards tumor cells which highly express APN,
except for 11a to ES-2 (IC50 = 22.78 µM) and 11g to HL-60
(IC50 = 30.93 µM). All tested compounds exhibited better
activities towards PLC cell than that of bestatin. Some com-
pounds displayed better activity against other three tumor cells
than bestatin. For example, compounds 5f, 5h to HL-60, 5f to
A549, 11g to ES-2. In all the tested compounds, 5f possessed
the best activities towards all the four kinds of tumor cells.
Although 5f does not posses the best anti-enzyme activity,
suitable geometric configuration and cell permeability should
be attributed to its better potency in cellular level.
Anti-metastasis activity against H22 tumor cell in vivo
Compounds 5f, 11d, 11g, and 11h were finally chosen as
candidates to carry out the anti-metastasis assay against H22

Med Chem Res
Table 1 The structures and inhibitory activities of the target compounds against APN in vitro
NH
H
O2N
N
R1
N
H
N
H
O
O
NHOH
Compd.
R1
IC50/µM (APN)a
5a
20.4 1.1
Cl
I
5b
21.9 1.5
5c
16.5 2.2
15.9 2.1
O
5d
NO2
Cl
5e
5.3 1.2
Cl
5f
14.6 0.8
5g
8.3 0.8
5h
5.1 0.6
NO2
Br

Med Chem Res
Table 1 continued
NH
R2
O
H
N
O2N
N
H
N
N
H
R3
H
O
O
NHOH
Compd.
R2
R3
IC50/µM (APN)
11a
16.3 1.2
O
11b
31.4 1.6
Cl
Cl
11c
11d
15.8 1.1
6.7 0.9
I
Cl
11e
11f
4.8 0.6
7.2 1.1
Cl
Cl
Cl
11g
11h
4.2 0.7
4.3 0.7
Cl

Med Chem Res
Table 1 continued
Compd.
R2
R3
IC50/µM (APN)
Bestatin
3.8 0.1
NH2
O
N
H
COOH
OH
a
Values are means of three experiments, standard deviation is given.
Table 2 Anti-proliferation
Compd.
IC50/µMa
activities of selected compounds
against several tumor cell lines
in vitro
HL-60
A549
ES-2
PLC
5e
3.92 0.21
0.11 0.09
0.73 0.12
6.83 0.19
3.91 0.17
4.32 0.18
2.87 0.18
30.93 0.43
1.24 0.13
1.16 0.13
1.37 0.14
9.80 0.22
0.90 0.10
5.41 0.20
22.78 0.45
1.19 0.13
1.03 0.10
3.48 0.13
0.66 0.11
3.98 0.16
0.87 0.10
1.20 0.14
0.87 0.10
1.85 0.15
1.14 0.16
0.36 0.06
0.90 0.08
0.57 0.08
0.31 0.06
0.31 0.05
5.19 0.38
5f
0.71 0.09
2.03 0.16
1.37 0.13
1.78 0.15
10.30 0.45
1.33 0.13
2.89 0.21
2.89 0.23
0.83 0.11
5h
11a
11d
11e
11f
11g
11h
Bestatin
a
Values are means of three experiments, standard deviation is given
tumor cell in vivo. CMC-Na was taken as blank control,
while bestatin was used as positive control. The results were
listed in Table 3 and Fig. 4. Four tested compounds did not
induce body weight loss in mice (p < 0.05) which implied
low toxicity. The compounds 5f and 11g showed better
activities than bestatin, out of which 11g significantly
inhibit H22 tumor cell metastasis (p < 0.01).
included as the active site. Other docking parameters in the
program were kept default.
From Fig. 5, we can see that the hydroxymate of com-
pound 5f could chelate with the zinc ion of APN, the
residue of nitro-arginine inserted into S1 pocket and the
aromatic ring stretched into the S1′ pocket. Compound 5f
could adapt to the active site of APN suitablely.
While about the binding mode of 11g showed in Fig. 6,
alike with the 5f, the hydroxymate group also can interact
with the zinc ion of APN, the residue of nitro-arginine
inserted into S1 pocket, the phenyl group at R2 plunged in
S2′ pocket and the terminal phenyl group occupied S1′
pocket, which is slightly different from our design principle.
But it does occupy all the three hydrophobic pockets of
APN.
Although the computed information supported our
design principle and experimental results, the exact binding
modes of our target compounds with APN should be ver-
ified from further X-ray crystallography studies.
Because of their effective activities, compounds 5f and
11g could be used as new lead compounds for the devel-
opment of future low molecular-weight peptidomimetic
APN inhibitors as anti-cancer agents.
Conclusion
In this work, we reported the development of a serial of
peptidomimetic L-arginine derivatives as novel APN inhi-
bitors. Compounds 5e, 5h, 11e, 11g, and 11h showed
comparable activities to bestatin in anti-enzyme activity
assay in vitro. While in anti-proliferation assay in vitro,
compound 5f exhibited potent activity. Finally, in anti-
metastasis activity assay in vivo, compounds 5f and 11g
presented potential activities. Considering that compounds
5f and 11g showed good activities in all three assays, the
presumed binding modes of these two compounds with
APN were predicted by Sybyl/FlexX module (Figs. 5
and 6). The residues within a radius of 7.0 Å around bestatin
in the co-crystal structure (PDB code: 2DQM) were

Med Chem Res
Table 3 Anti-metastasis activities of four compounds against H22 tumor cell in vivo
Compd.
Mice of survived (n)
Body weighta (g)
Lung weighta (g)
Metastasized nodes on lung surfacea (n)
Inhibition rate (%)
CMC-Na
5f
13
10
11
10
11
10
26.03 5.04
25.22 2.83
23.76 3.89
25.06 2.87
26.80 2.68
23.70 2.71
0.223 0.067
0.213 0.055
0.208 0.057
0.234 0.099
0.219 0.058
0.215 0.068
83.9 11.9
32.3 5.2
61.8 7.6
24.0 3.1
49.4 9.9
35.2 9.9
/
61.50
26.34
71.39
41.12
58.04
11d
11g
11h
Bestatin
a
Values are means of survived mice, standard deviation is given
Fig. 4 a The number of
metastasized nodes on mice lung
surface after administrating the
corresponding compound by
A 100
B
100
80
60
40
20
80
Compound
bestatin
60
oral. b The inhibition rate of
each compound compared with
positive control bestatin
40
20
0
n
i
d
g
h
5f
0
t
11
11
11
a
t
Blank
5f
11d 11g 11h
Compound
es
b
Compound
Experimental part
Chemistry
Yield 85.8%, mp 105–108 °C. 1H NMR (D2O) δ:
1.52–1.69 (m, 4H, CH2CH2), 3.14–3.17 (m, 2H, CH2),
4.30–4.34 (m, 1H, CH), 7.38–7.47 (m, 4H, H-3, H-4, H-5,
H-6). 13C NMR (D2O) δ: 168.7 (C, HONHC=O), 167.7 (C,
C=O), 163.2 (C, C=NH), 133.8 (CH, C-4), 132.4 (C, C-1),
132.2 (C, C-2), 129.0 (CH, C-3), 128.8 (CH, C-6), 127.3
(CH, C-5), 50.3 (CH, C-2′), 36.4 (CH2, C-5′), 28.9 (CH2, C-
3′), 24.2 (CH2, C-4′). ESI-MS m/z: 373.3 (M + H)+. Anal.
calcd for C13H17ClN6O5: C 41.89, H 4.60, N 22.55. Found
C 41.72, H 4.49, N 22.63.
All the materials were commercial available. All the sol-
vents except fuming nitric acid and fuming sulfuric acid
were distilled before using. All the reactions were mon-
itored by thin-layer chromatography on 0.25 mm silica gel
plates (60GF-254) and visualized with UV light or chloride
ferric. 200–300 mesh silica gel was used in column chro-
matography. Melting points were determined on an elec-
trothermal melting point apparatus (uncorrected). IR spectra
were recorded on Brucker VECTOR 22 spectrometer. ESI-
MS were determined on an API 4000 spectrometer. Proton
and carbon NMR spectra were determined on a Brucker
DRX spectrometer (600 MHz), δ in parts per million and J
in Hertz and TMS was used as an internal standard. Mea-
surements were made in D2O solutions. Elemental analysis
were performed on Vario EI III elemental analyzer. High
resolution mass spectroscopy (HRMS) spectra were con-
ducted by Shandong Analysis and Test Center.
Compounds 5b–h were synthesized with the same
procedure.
N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopentan-2-
yl)-2-iodobenzamide (5b)
1
Yield 79.8%, mp 76–80 °C. H NMR (D2O) δ: 1.54–1.69
(m, 4H, CH2CH2), 3.15–3.19 (m, 2H, CH2), 4.27–4.30 (m,
1H, CH), 7.13–7.46 (m, 4 H, H-3, H-4, H-5, H-6). 13C
NMR (D2O) δ: 168.8 (C, HONHC=O), 167.6 (C, C=O),
163.0 (C, C=NH), 142.4 (C, C-1), 137.6 (CH, C-3), 133.7
(CH, C-4), 129.1 (CH, C-6), 127.6 (CH, C-5), 92.1 (C, C-
2), 50.4 (CH, C-2′), 36.1 (CH2, C-5′), 29.1 (CH2, C-3′),
24.3 (CH2, C-4′). ESI-MS m/z: 465.3 (M + H)+. Anal.
calcd for C13H17IN6O5: C 33.64, H 3.69, N 18.10. Found C
33.48, H 3.66, N 18.02.
2-Chloro-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-yl)benzamide (5a)
The title compound was synthesized as described in our
previous paper (Mou et al. 2009).

Med Chem Res
S1
S1
S1’
S1’
11g
5f
Zinc ion
Zinc ion
S2’
Fig. 6 The docking mode of compound 11g with APN
Fig. 5 The docking mode of compound 5f with APN
N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopentan-2-
yl)-3-methoxybenzamide (5c)
168.7 (C, HONHC=O), 167.6 (C, C=O), 163.3 (C,
C=NH), 139.3 (C, C-4), 133.6 (C, C-2), 130.5 (CH, C-3),
130.4 (C, C-1), 130.2 (CH, C-6), 127.3 (CH, C-5), 50.4
(CH, C-2′), 36.0 (CH2, C-5′), 29.2 (CH2, C-3′), 24.2 (CH2,
C-4′). ESI-MS m/z: 407.4 (M + H)+. Anal. calcd for
C13H16Cl2N6O5: C 38.34, H 3.96, N 20.64. Found C 38.23,
H 4.04, N 20.52.
1
Yield 85.6%, mp 68–71 °C. H NMR (D2O) δ: 1.57–1.62
(m, 2H, CH2), 1.69–1.75 (m, 2H, CH2), 3.16–3.18 (m, 2H,
CH2), 3.81 (s, 3H, O–CH3), 4.30–4.38 (m, 1H, CH),
7.08–7.11 (m, 1H, H-4), 7.34–7.39 (m, 1H, H-5), 7.45–7.50
(m, 2H, H-2, H-6). 13C NMR (D2O) δ: 168.7 (C, HON-
HC=O), 167.6 (C, C=O), 163.1 (C, C=NH), 160.6 (C, C-
3), 135.4 (C, C-1), 129.6 (CH, C-5), 119.5 (CH, C-6), 117.7
(CH, C-4), 111.6 (CH, C-2), 55.8 (CH3), 50.3 (CH, C-2′),
36.0 (CH2, C-5′), 29.0 (CH2, C-3′), 24.2 (CH2, C-4′). ESI-
MS m/z: 369.3 (M + H)+. Anal. calcd for C14H20N6O6: C
45.65, H 5.47, N 22.82. Found C 45.54, H 5.38, N 22.74.
N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopentan-2-
yl)-2-naphthamide (5f)
1
Yield 75.4%, mp 72–74 °C. H NMR (D2O) δ: 1.60–1.73
(m, 2H, CH2), 1.77–1.88 (m, 2H, CH2), 3.17–3.20 (m, 2H,
CH2), 4.38–4.43 (m, 1H, CH), 7.58–7.80 (m, 4H, H-5, H6,
H7, H8), 7.98–8.05 (m, 3H, H-1, H-3, H-4). 13C NMR
(D2O) δ: 168.8 (C, HONHC=O), 167.6 (C, C=O), 163.4
(C, C=NH), 135.5 (C, C-8a), 134.6 (C, C-2), 132.5 (C, C-
4a), 129.2 (CH, C-8), 128.4 (CH, C-1), 128.3 (C, C-4),
128.2 (CH, C-6), 127.5 (CH, C-5), 126.8 (CH, C-7), 124.5
(CH, C-3), 50.3 (CH, C-2′), 36.1 (CH2, C-5′), 29.2 (CH2, C-
3′), 24.3 (CH2, C-4′). ESI-MS m/z: 389.4 (M + H)+. Anal.
calcd for C17H20N6O5: C 52.57, H 5.19, N 21.64. Found C
52.28, H 5.07, N 21.46.
N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopentan-2-
yl)-4-nitrobenzamide (5d)
Yield 91.3%, mp 148–150 °C. 1H NMR (D2O) δ: 1.51–1.54
(m, 2H, CH2), 1.69–1.76 (m, 2H, CH2), 3.14–3.16 (m, 2H,
CH2), 4.31–4.36 (m, 1H, CH), 8.09–8.11 (d, J = 8.4 Hz,
2H, H-3, H-5), 8.29–8.32 (d, J = 8.4 Hz, 2H, H-2, H-6). 13
C
NMR (D2O) δ: 168.7 (C, HONHC=O), 167.5 (C, C=O),
163.5 (C, C=NH), 151.7 (C, C-4), 140.4 (C, C-1), 128.6
(CH, C-2), 128.5 (CH, C-6), 121.6 (CH, C-5), 121.0 (CH,
C-3), 50.4 (CH, C-2′), 36.0 (CH2, C-5′), 29.2 (CH2, C-3′),
24.2 (CH2, C-4′). ESI-MS m/z: 384.3 (M + H)+. Anal.
calcd for C13H17N7O7: C 40.73, H 4.47, N 25.58. Found C
40.46, H 4.39, N 25.61.
(S)-N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopentan-
2-yl)-3,4-dinitro benzamide (5g)
Yield 78.1%, mp 146–148 °C. 1H NMR (D2O) δ: 1.51–1.57
(m, 2H, CH2), 1.75–1.81 (m, 2H, CH2), 3.17–3.18 (m, 2H,
CH2), 4.41 (t, J = 7.2 Hz, 1H, CH), 8.89–8.95 (m, 2H, H-5,
H-6), 9.11 (s, 1H, H-2). 13C NMR (D2O) δ: 168.8 (C,
HONHC=O), 167.6 (C, C=O), 163.7 (C, C=NH), 146.8
(C, C-4), 143.5 (C, C-3), 141.3 (C, C-1), 134.4 (CH, C-6),
123.4 (CH, C-2), 122.1 (CH, C-5), 50.2 (CH, C-2′), 36.2
(CH2, C-5′), 29.0 (CH2, C-3′), 24.3 (CH2, C-4′). ESI-MS m/
z: 429.4 (M + H)+. Anal. calcd for C13H16N8O9: C 36.45,
H 3.77, N 26.16. Found C 36.53, H 3.82, N 26.12.
2,4-Dichloro-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-yl)benzamide (5e)
Yield 83.4%, mp 154–156 °C. 1H NMR (D2O) δ: 1.61–1.64
(m, 2H, CH2), 1.73–1.77 (m, 2H, CH2), 3.16–3.18 (m, 2H,
CH2), 4.26–4.33 (m, 1H, CH), 7.43–7.51 (m, 2H, H-3, H-
5), 7.67–7.68 (d, J = 1.8 Hz, 1H, H-6). 13C NMR (D2O) δ:

Med Chem Res
(S)-4-Bromo-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-yl)-3-nitrobenzamide (5h)
2-Chloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-
nitroguanidino)-1-oxopentan-2-ylamino)-3-methyl-1-
oxobutan-2-yl)benzamide (11c)
Yield 69.3%, mp 147–150 °C. IR (KBr, cm−1) σ: 3382,
2939, 2868, 1637, 1608, 1528, 1334, 1264, 1144. 1H NMR
(D2O) δ: 1.54–1.56 (m, 2H, CH2), 1.73–1.76 (m, 2H, CH2),
3.16–3.18 (m, 2H, CH2), 4.34–4.36 (m, 1H, CH), 7.44 (d, J
= 9 Hz, 1H, H-5), 7.95 (d, J = 9 Hz, 1H, H-6), 8.52 (s, 1H,
H-2). 13C NMR (D2O) δ: 168.6 (C, HONHC=O), 167.4 (C,
C=O), 163.6 (C, C=NH), 151.6 (C, C-3), 135.4 (CH, C-6),
134.3 (C, C-1), 132.5 (CH, C-5), 124.4 (CH, C-2), 121.3
(C, C-4), 50.4 (CH, C-2′), 36.1 (CH2, C-5′), 29.1 (CH2, C-
3′), 24.3 (CH2, C-4′). ESI-MS m/z: 462.4 (M + H)+. Anal.
calcd for C13H16BrN7O7: C 33.78, H 3.49, N 17.29. Found
C 33.60, H 3.42, N 17.35.
Yellow solid, yield 77.4%, mp123–125 °C. 1H NMR (D2O)
δ: 0.87–0.96 (m, 6H, 2CH3), 1.42–1.59 (m, 4H, CH2CH2),
1.90–1.95 (m, 1H, H-3′), 3.14–3.15 (m, 2H, CH2),
4.13–4.21 (m, 1H, H-2″), 4.33–4.34 (m, 1H, H-2′),
7.39–7.40 (m, 2H, H-3, H-4), 7.43–7.46 (m, 1H, H-5),
7.49–7.50 (m, 1H, H-6). 13C NMR (D2O) δ: 172.1 (C, C′
=O), 169.0 (C, HONHC=O), 167.9 (C, C=O), 164.2 (C,
C=NH), 134.3 (CH, C-4), 132.4 (C, C-2), 132.3 (C, C-1),
130.0 (CH, C-3), 129.4 (CH, C-6), 127.4 (CH, C-5), 59.1
(CH, C-2′), 50.3 (CH, C-2″), 36.7 (CH2, C-5″), 32.1 (CH,
C-3′), 29.5 (CH2, C-3″), 24.7 (CH2, C-4″), 17.5 (2CH3).
HRMS calcd for C18H26ClN7O6 (M + H)+ 472.1706 found
472.1712.
N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-1-oxopropan-2-yl)-4-
methoxybenzamide (11a)
N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-2-
iodobenzamide (11d)
The title compound was synthesized as described in our
previous paper (Mou et al. 2010).
Yield 68.4%, mp 181–183 °C. 1H NMR (D2O) δ:
1.48–1.56 (m, 3H, CH3), 1.70–1.73 (m, 4H, CH2CH2),
3.16–3.17 (m, 2H, CH2), 3.81 (s, 3H, O–CH3), 4.31–4.35
(m, 2H, H-2′, H-2″), 6.98–7.00 (m, 2H, H-3, H-5),
7.87–7.89 (m, 2H, H-2, H-6). 13C NMR (D2O) δ: 171.7 (C,
C′=O), 168.8 (C, HONHC=O), 167.5 (C, C=O), 164.3 (C,
C-4), 163.4 (C, C=NH), 128.4 (CH, C-2), 128.4 (CH, C-6),
126.3 (C, C-1), 114.6 (CH, C-3), 114.4 (CH, C-5), 55.8
(CH3, O–CH3), 50.0 (CH, C-2″), 49.4 (CH, C-2′), 36.2
(CH2, C-5″), 29.3 (CH2, C-3″), 24.3 (CH2, C-4″), 17.7
(CH3). HRMS calcd for C17H25N7O7 (M + H)+ 440.1888
found 440.1876.
Yield 77.1%, mp 125–127 °C. 1H NMR (D2O) δ: 0.84–0.88
(m, 6H, 2CH3), 1.42–1.58 (m, 4H, CH2CH2), 1.91–1.94 (m,
1H, H-3’), 3.14–3.15 (m, 2H, CH2), 4.13–4.16 (m, 1H, H-
2″), 4.28–4.31 (m, 1H, H-2′), 7.10–7.12 (m, 1H, H-4),
7.15–7.19 (m, 1H, H-5), 7.28–7.33 (m, 1H, H-6), 7.43–7.46
(m, 1H, H-3). 13C NMR (D2O) δ: 171.9 (C, C′=O), 169.3
(C, HONHC=O), 167.6 (C, C=O), 164.0 (C, C=NH),
142.3 (C, C-1), 138.0 (CH, C-3), 134.3 (CH, C-4), 129.7
(CH, C-6), 127.9 (CH, C-5), 92.4 (C, C-2), 59.5 (CH, C-2′),
50.1 (CH, C-2″), 36.3 (CH2, C-5″), 31.6 (CH, C-3′), 29.0
(CH2, C-3″), 24.9 (CH2, C-4″), 17.4 (2CH3). HRMS calcd.
for C18H26IN7O6 (M + H)+ 564.1062 found 564.1067.
Compounds 11b–h were synthesized with the same
procedure.
2-Chloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-
nitroguanidino)-1-oxopentan-2-ylamino)-4-methyl-1-
oxopentan-2-yl) benzamide (11e)
2,4-Dichloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-
nitroguanidino)-1-oxopentan-2-ylamino)-1-oxopropan-2-
yl) benzamide (11b)
Yield 70.3%, mp 116–118 °C. 1H NMR (D2O) δ: 0.84–0.90
(m, 6H, 2CH3), 1.36–1.60 (m, 4H, CH2CH2), 1.71–1.83 (m,
3H, CHCH2), 3.14–3.15 (m, 2H, CH2), 4.16–4.21 (m, 1H,
H-2″), 4.46–4.51 (m, 1H, H-2′), 7.40–7.41 (m, 2H, H-3, H-
4), 7.44–7.47 (m, 1H, H-5), 7.49–7.50 (m, 1H, H-6). 13C
NMR (D2O) δ: 172.3 (C, C′=O), 169.3 (C, HONHC=O),
167.7 (C, C=O), 163.2 (C, C=NH), 134.0 (CH, C-4), 132.3
(C, C-1), 132.3 (C, C-2), 130.2 (CH, C-3), 129.0 (CH, C-6),
127.0 (CH, C-5), 51.1 (CH, C-2′), 50.1 (CH, C-2″), 41.1
(CH2, C-3′), 36.2 (CH2, C-5″), 29.4 (CH2, C-3″), 24.7 (CH2,
C-4″), 22.8 (2CH3), 22.1 (CH, C-4′). HRMS calcd. for
C19H28ClN7O6 (M + H)+ 486.1862 found 486.1886.
Yield 77.8%, mp 125–127 °C. 1H NMR (D2O) δ: 1.26–1.29
(m, 3H, CH3), 1.42–1.67 (m, 4H, CH2CH2), 3.15–3.17 (m,
2H, CH2), 4.17–4.20 (m, 1H, H-2″), 4.45–4.49 (m, 1H, H-
2′), 7.44-7.51 (m, 2H, H-3, H-5), 7.66–7.68 (m, 1H, H-6).
13C NMR (D2O) δ: 172.7 (C, C′=O), 169.2 (C, HON-
HC=O), 167.8 (C, C=O), 163.4 (C, C=NH), 140.3 (C, C-
4), 138.4 (C, C-2), 131.3 (C, C-1), 130.6 (CH, C-3), 130.4
(CH, C-6), 127.4 (CH, C-5), 50.1 (CH, C-2′), 50.0 (CH, C-
2″), 36.8 (CH2, C-5″), 29.6 (CH2, C-3″), 24.8 (CH2, C-4″),
17.9 (CH3). HRMS calcd for C16H21Cl2N7O6 (M + H)+
478.1003 found 478.1027.

Med Chem Res
2,4-Dichloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-
nitroguanidino)-1-oxopentan-2-ylamino)-4-methyl-1-
oxopentan-2-yl) benzamide (11f)
130.5 (CH, C-3), 130.3 (C, C-1), 128.6 (CH, 3′-C-3′), 128.6
(CH, 3′-C-5′), 127.9 (CH, 3′-C-2′), 127.9 (CH, 3′-C-6′), 127.5
(CH, C-5), 126.5 (CH, 3′-C-4′), 53.9 (CH, C-2′), 49.7 (CH,
C-2″), 37.9 (CH2, C-3′), 36.1 (CH2, C-5″), 29.5 (CH2, C-3″),
24.9 (CH2, C-4″). HRMS calcd. for C22H25Cl2N7O6 (M + H)+
554.1316 found 554.1327.
Yield 79.2%, mp 129–131 °C. 1H NMR (D2O) δ: 0.89–0.90
(d, J = 6.6 Hz, 6H, 2CH3), 1.46–1.58 (m, 4H, CH2CH2),
1.63–1.69 (m, 3H, CHCH2), 3.14–3.15 (m, 2H, CH2),
4.16–4.20 (m, 1H, H-2″), 4.46–4.52 (m, 1H, H-2′),
7.41–7.42 (d, J = 7.8 Hz, 1H, H-5), 7.49–7.51 (m, 1H, H-
3), 7.67–7.69 (d, J=7.8 Hz, 1H, H-6). 13C NMR (D2O) δ:
172.1 (C, C′=O), 169.0 (C, HONHC=O), 167.6 (C, C=O),
163.9 (C, C=NH), 139.0 (C, C-4), 133.3 (C, C-2), 130.7
(CH, C-6), 130.5 (CH, C-3), 130.3 (C, C-1), 127.5 (CH, C-
5), 51.6 (CH, C-2′), 50.1 (CH, C-2″), 41.5 (CH2, C-3′), 36.0
(CH2, C-5″), 29.3 (CH2, C-3″), 24.5 (CH2, C-4″), 22.5
(2CH3), 22.2 (CH, C-4′). HRMS calcd for C19H27Cl2N7O6
(M + H)+ 520.1473 found 520.1498.
Biological materials and methods
Chemicals
Chemicals were synthesized as mentioned above and dis-
solved in dimethylsulfoxide (DMSO) or PBS and diluted to
the required concentration with culture medium when used.
Cell line and cell culture
HL-60, A549, ES-2, and PLC cell line were obtained from
the Institute of National Cancer Research of China (Beijing,
China). Cells were maintained in RPMI-1640 supplemented
with 10%(v/v) heat–inactivated fetal bovine serum (FBS),
penicillin–streptomycin (100 IU/ml–100 mg/ml), 2 mM
glutamine, and 10 mM Hepes buffer at 37 °C in a humid
atmosphere (5% CO2–95% air).
N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)
benzamide (11g)
Yield 63.8%, mp 150–152 °C. 1H NMR (D2O) δ: 1.33–1.63
(m, 4H, CH2CH2), 2.94–3.00 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz,
1H, CH2C6H5), 3.16–3.17 (m, 2H, CH2), 3.36–3.40 (dd, J1
= 7.2 Hz, J2 = 7.2 Hz, 1H, CH2C6H5), 4.15–4.21 (m, 1H,
H-2”), 4.70–4.76 (m, 1H, H-2′), 7.14–7.17 (m, 1H, H-4′),
7.24-7.27 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 2H, H-2′, H-6′),
7.36–7.37 (d, J = 7.2 Hz, 2H, H-3′, H-5′), 7.43–7.45 (m, 2H,
H-3, H-5), 7.50–7.52 (m, 1H, H-4), 7.75–7.79 (dd, J1 = 7.2
Hz, J2 = 7.2 Hz, 2H, H-2, H-6). 13C NMR (D2O) δ: 171.9
(C, C′=O), 169.3 (C, HONHC=O), 167.6 (C, C=O), 163.5
(C, C=NH), 139.3 (C, 3′-C-1′), 134.3 (C, C-1), 132.0 (CH,
C-4), 128.5 (CH, C-3), 128.5 (CH, C-5), 128.4 (CH, 3′-C-
3′), 128.4 (CH, 3′-C-5′), 127.8 (CH, 3′-C-2′), 127.8 (CH, 3′-
C-6′), 127.7 (CH, C-6), 127.3 (CH, C-2), 126.0 (CH, 3′-C-
4′), 53.6 (CH, C-2′), 50.0 (CH, C-2″), 37.5 (CH2, C-3′), 36.3
(CH2, C-5″), 29.1 (CH2, C-3″), 24.3 (CH2, C-4″). HRMS
calcd. for C22H27N7O6 (M + H)+ 486.2096 found 486.2151.
Animal subject
The mouse bearing hepatoma H22 tumor cell line was from
Department of Pharmacology, Shandong University.
Kuming mice were purchased from Center for New Drugs
Evaluation of Shandong University.
In vitro anti-APN assay
IC50 values against APN were determined by using L-Leu-p-
nitroanilide as substrate and microsomal aminopeptidase from
Porcine Kidney Microsomes (Sigma) as the enzyme in 50
mM PBS, pH 7.2, at 37 °C Lejczak et al. (1989). The
hydrolysis of the substrate was monitored by following the
change in the absorbance measured at 405 nm with the
UV–Vis spectrophotometer Pharmacia 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 the inhibitors were preincubated with APN
for 30 min at room temperature. The assay mixture, which
contained the inhibitor solution (concentration dependent on
the inhibitor), the enzyme solution (4 mg/ml final concentra-
tion), and the assay buffer, was adjusted to 200 μl.
2,4-Dichloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-
nitroguanidino)-1-oxopentan-2-ylamino)-1-oxo-3-
phenylpropan-2-yl) benzamide (11h)
1
Yield 76.4%, mp 141–143 °C. H NMR (D2O) δ: 1.48–1.77
(m, 4H, CH2CH2), 2.81–2.86 (m, 1H, CH2C6H5), 3.05–3.09
(m, 1H, CH2C6H5), 3.11–3.16 (m, 2H, CH2), 4.17–4.19 (m,
1H, H-2″), 4.74–4.79 (m, 1H, H-2’), 7.17–7.21 (m, 1H, H-4′),
7.23–7.32 (m, 2H, H-2′, H-6′), 7.38–7.40 (m, 2H, H-3′, H-5′),
7.46–7.48 (m, 1H, H-5), 7.50 (s, 1H, H-3), 7.67 (d, J = 7.2
Hz, 1H, H-6). 13C NMR (D2O) δ: 172.3 (C, C′=O), 169.2 (C,
HONHC=O), 167.9 (C, C=O), 163.9 (C, C=NH), 139.6 (C,
3′-C-1′), 139.0 (C, C-4), 133.3 (C, C-2), 130.7 (CH, C-6),
In vitro anti-proliferaion assay
All cell lines were maintained in RPMI-1640 medium
containing 10% FBS at 37 °C in a 5% CO2 humid

Med Chem Res
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Acknowledgements This work was supported by National Nature
Science Foundation of China (Grant No. 21502142) and Tianjin
Research Program of Application Foundation and Advanced Tech-
nology (No.15JCQNJC14100) and Program for Changjiang Scholars
and Innovative Research Team in University (IRT_14R41). The
authors thank South-central University for Nationalities for supporting
of CADD softwares.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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