Design, synthesis and primary activity evaluation of l-arginine derivatives as amino-peptidase N/CD13 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.04.056

A series of l-arginine derivatives were designed, synthesized and assayed for their activities against amino-peptidase N (APN)/CD13 and metalloproteinase-2 (MMP-2). The results showed that most compounds exhibited high inhibitory activities against APN and low activities against MMP-2. Within this series, two compounds 5q and 5s (IC₅₀ = 5.3 and 5.1 μM) showed similar inhibitory activities compared with bestatin (IC₅₀ = 3.8 μM), which could be used as novel lead compounds for the future APN inhibitors development as anticancer agents.

Full text of DOI:10.1016/j.bmc.2009.04.056

Bioorganic & Medicinal Chemistry 17 (2009) 4666–4673
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and primary activity evaluation of L-arginine derivatives
as amino-peptidase N/CD13 inhibitors
Jiajia Mou a, Hao Fang a, Fanbo Jing b, Qiang Wang a, Yingzi Liu a,c, Huawei Zhu a, Luqing Shang a,
Xuejian Wang a, Wenfang Xu a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44, Wenhuaxi Road, Ji’nan, Shandong 250012, PR China
b Pharmacy Department of the Affilated Hospital, Qingdao University, Qingdao, Shandong 266003, PR China
c Shandong Police College, Ji’nan, Shandong 250014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of L-arginine derivatives were designed, synthesized and assayed for their activities against
Received 29 March 2009
Revised 24 April 2009
Accepted 25 April 2009
Available online 3 May 2009
amino-peptidase N (APN)/CD13 and metalloproteinase-2 (MMP-2). The results showed that most com-
pounds exhibited high inhibitory activities against APN and low activities against MMP-2. Within this
series, two compounds 5q and 5s (IC50 = 5.3 and 5.1
with bestatin (IC50 = 3.8 M), which could be used as novel lead compounds for the future APN inhibitors
development as anticancer agents.
lM) showed similar inhibitory activities compared
l
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Amino-peptidase N
Metalloproteinase-2
Inhibitors
L-Arginine derivatives
Synthesis
1. Introduction
tal complex. The binding sites of APN with bestatin can be divided
to three parts. Part 1 is a hydrophobic pocket (S1); part 2 is the zinc
binding group (ZBG); part 3 is another hydrophobic pocket in the
other side (S01).
With the help of computer-aided molecular design, in 2008, our
group designed and synthesized a series of L-lysine derivatives and
Amino-peptidase N (APN/CD13; EC 3.4.11.2) is a zinc-depen-
dent metalloproteinase that cleaves neutral or basic amino acids
from the N-terminus of oligopeptides. Scientist found that outside
the hematopoietic system, APN was widely expressed on various
kinds of cells. For example, epithelial cells of the intestine and kid-
ney, hepatocytes, osteoclasts, endometrial cells, fibroblasts, endo-
thelial cells, bone marrow stromal cells and on neuronal synaptic
membranes. APN is involved in many physiology and pathology
processes such as hydrolysis of nutrients, inactivation of bioactive
peptides, binding of corona viruses, mediating cytomegalovirus
infection and antigen presentation. Furthermore, APN has been a
target for anti-tumor agents due to its important functions in
ECM degradation, tumor cells invasion and tumor angiogenesis.1–3
In the past two decades, several APN inhibitors have been
found. For example, Bestatin, Probestin, Amastatin, Actinonin,
Phebestin, Lapstatin, AHPA-Val, Leuhistin, Curcumin and Pasmma-
plin A.4–13 Among them, bestatin has been launched and is now
widely employed clinically as an anti-tumor agent.14 In 2006, the
3D structure of APN has been studied according to the co-crystal
complex of APN and bestatin by Kiyoshi.15 Our group has studied
the binding site and catalytic domain of APN based on this co-crys-
found that some compounds exhibited potential APN inhibitory
activities16 (Fig. 1). Also in 2008, the crystal structures of Esche-
richia coli amino-peptidase N (e PepN) in complex with
L-arginine,
L
-lysine, -phenylalanine, -tyrosine and -tryptophan have been
L
L
L
determined to understand the structural basis for APN’s hydrolysis
pattern. The result showed that all these amino acids bind with
their backbone atoms close to the active-site zinc ion and their side
chain occupying the S1 pocket of e PepN. And the specificity of the
S1-binding pocket suggest that the preferred amino acid is, in
decreasing order, arginine, lysine, tyrosine and phenylalanine.17
Additionally, arginine is similar with lysine in structure except
the guanidinium group and the longer carbon chain. According to
the former reasons, we choose L-arginine as the starting material
in order to get more efficient and potential APN inhibitors. The
compounds designed are showed in Figure 1.
2. Chemistry
All the target compounds were designed and synthesized via
the route shown in Scheme 1. The guanidinium group of
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.056

J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
4667
Aromatic ring, aliphlic, etc
Cl
Cl
O
R
O
NH
O2N
NH
HN
NH
NHOH
O
N
N
H
H
O
O
NHOH
O
L-lysine derivative
L-Aginine derivative
IC50 =13.2 µM
Figure 1. The structure of L-lysine derivative and L-aginine derivative.
compound 1 was protected by nitro group to get compound 2.
Compound 2 was then esterificated with methanol under HCl
atmosphere to get compound 3. The acylation of compound 3 with
acyl chloride, carboxylic acid or sulfochloride led to compounds
4a–w, 6a,b. Finally the ester groups of 4a–w, 6a,b were treated
with NHOK in anhydrous methanol to get the target compounds
5a–w, 7a,b.
against MMP-2.16,18 So
for APN inhibitors.
Comparing 5a–d and 5e–w, we could find that the compounds
contained substituted phenyl groups have better activities than
those contained unsubstituted phenyl groups.
L
-arginine derivatives are more suitable
Among compounds 5e–w, we can conclude that the compounds
contained two substituted groups on phenyl group have more po-
tent activities than others. The most active compounds 5q and 5s
both contain bi-substituted phenyl group. It is worth mentioning
that the 2,4-dichlorobenzyl moiety of 5q was also effective in the
3. Results and discussion
precious L
-lysine derivatives.16 The possible reason might be that
All the inhibition results were listed in Table 1. Similar to APN,
MMP-2 is also a zinc-dependant metalloproteinase that involved in
tumor invasion and metastasis. Thus the assay was performed on
both of APN and MMP-2 so as to identify the compounds selectiv-
ity. Bestatin was used as the positive control.
Almost all the compounds except 5a, 5c, 5i, 5v and 7b showed
better activities against APN than MMP-2. For example, 5q with
an IC50 (MMP-2)/ IC50 (APN) ratio equals to 65.8, while 5s equals
to 46.3. The result, to a certain extent, confirmed our strategy for
designing APN inhibitors. This possibly descript from the differ-
ences between the structures of two enzymes, leading to different
requirements for their respective inhibitors. APN is a membrane-
bound zinc exopeptidase that catalyzed the removal of NH2-ter-
minal amino acid from the peptide, while MMP-2 is a zinc-depen-
dent endopeptidase that could cut the peptide to parts from the
the 2,4-dichloro substituted benzene ring could accommodate
the hydrophobic site of APN suitably, suggesting it is a potential
moiety for APN inhibitors.
From compounds 7a,b, we could confirm that the compounds
contained sulfonyl groups showed equal activities against APN
and MMP-2. That coincides with the fact that some sulfonyl groups
containing compounds our group had synthesized before showed
better MMP-2 inhibitory activities.19,20
The most active compounds are 5q and 5s (IC50 = 5.3 and
5.1
(IC50 = 3.8
(IC50 = 15.9, 14.6 and 16.5
l
M), which have comparable activity with bestatin
M). 5o, 5t and 5l also have considerable activities
M, respectively).
l
l
In order to investigate the interaction of our compounds with
APN, the most active compound 5s was constructed with Sybyl/
Sketch module and optimized using Powell’s method with the Tri-
pos force field with convergence criterion set at 0.05 kcal/(Å mol),
and assigned with Gasteiger–HÜckel method. The docking study
specific amino acid residue of the peptide. The former L-lysine
derivatives and 3-phenylpropane-1, 2-diamine derivatives de-
signed by our group to inhibit APN all showed better activities
NH
O
NH
O
NH
O
a
b
O2N
O2N
H2N
N
OH
N
N
OH
N
H
N
OCH3
.HCl
H
H
H
H
NH2
NH2
NH2
2
1
3
R1 Cl
O
NH
O
NH
O
,c
e
O2N
O2N
N
N
H
OCH3
N
H
N
NHOH
H
H
R
NH
R
NH
R2 OH
O
,d
or
O
O
4a-w
5a-w
NH
O
NH
O
O2N
O2N
e
3,c
N
N
OCH3
R3
Cl
N
N
NHOH
H
H
S
H
H
R3
NH
R3
NH
O
O
S
S
O
O
O
O
6a, b
7a, b
Scheme 1. Reagents and conditions: (a) fuming nitric acid, fuming sulfuric acid; (b) MeOH, HCl; (c) Et3N, THF, 0 °C; (d) Et3N, TBTU, CH2Cl2; (e) NHOK, MeOH.

4668
J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
Table 1
The structure and inhibitory activities of compounds against APN and MMP-2
NH
O
O2N
N
N
NHOH
H
H
R
NH
O
a
Compounds
R
IC50
(lM)
IC50(MMP-2)/IC50(APN)
APN
MMP-2
5a
5b
5c
5d
5e
5f
1662.6 4.9
331.8 2.8
1008.0 5.9
80.6 1.2
20.4 1.1
21.9 1.5
221.5 2.6
78.4 2.5
202.2 2.9
43.4 1.3
42.8 1.9
16.5 2.2
215.1 3.3
443.4 2.7
8.7 3.9
0.05
4.9
0.04
8.6
7.3
8.0
9.3
3.2
0.4
2.3
14.6
9.1
1.1
1.1
1642.9 8.4
36.7 1.6
692.8 5.7
149.6 2.8
176.0 2.4
2067.2 23.5
253.2 3.1
89.2 2.9
Cl
I
5g
5h
5i
O
Cl
O2N
5j
101.6 2.1
623.1 4.5
150.8 3.0
237.6 3.6
483.6 3.9
5k
5l
O
5m
5n
Cl
Br

J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
4669
Table 1 (continued)
a
Compounds
R
IC50
(l
M)
IC50(MMP-2)/IC50(APN)
APN
MMP-2
5o
5p
5q
15.9 2.1
60.2 2.2
5.3 1.2
203.6 4.6
322.5 3.2
348.5 3.6
12.8
5.4
O2N
O
65.8
Cl
Cl
Cl
5r
33.5 1.7
193.6 2.9
5.8
Cl
O2N
Br
5s
5t
5.1 0.6
236.3 4.5
85.5 2.6
345.5 3.8
46.3
5.9
14.6 0.8
26.9 1.3
O
5u
12.8
O
O
5v
270.6 3.4
41.1 1.8
0.2
2.6
O
S
O
5w
85.7 2.1
226.4 2.6
NH
O
O2N
N
N
NHOH
H
H
R3
NH
S
O
O
a
Compounds
R3
IC50
(l
M)
IC50(MMP-2)/IC50(APN)
APN
MMP-2
7a
60.2 1.8
108.8 2.3
3.8 0.1
100.4 3.9
28.9 2.4
162.0 4.8
1.7
7b
0.3
NH2
O
Bestatin
42.6
N
COOH
H
OH
a
Mean value of three experiments and standard deviation are given.

4670
J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
see the backbone of 5s inserted to S1 pocket, the hydroxymate of
5s interacted with the zinc ion of APN and the 4-bromo-3-nitro
benzamide side chain extended to S01 pocket.
For a further and detail understanding of the binding mode of 5s
with APN, a 2D picture was also created with the program LIGPLOT. In
Figure 3, we can see the backbone of 5s could form hydrophobic
contacts with Glu121, Met260 and Tyr376 of S1 pocket and form
hydrogen bond with Glu121 by the imine of guanidinium group.
The two oxygen atoms of hydroxymate chelated with the zinc
ion of APN. The carbonyl of amide in R position could form hydro-
gen bond with Gly261 and Ala262 of S01 pocket. The R substituted
side chain of 5s could form hydrophobic contact with Gly261 of S10
pocket. While, the nitro group at the aromatic ring could form
hydrogen bonds with Arg783and Arg825
Although the computed information partially supported our
assumption, the exact binding mode of the -arginine derivatives
.
L
with APN should be obtained from further X-ray crystal studies.
Figure 2. The docking mode of compound 5s with APN. Zinc ion is shown as pale
sphere.
4. Conclusion
performed using Sybyl/FlexX module, the residues in a radius of
7.0 Å around Bestatin in the co-crystal structure (PDB code:
2DQM) were selected as the active site. Other docking parameters
implied in the program were kept default. From Figure 2, we can
In all, we have synthesized a new series of L-aginine derivatives
as APN inhibitors. Most of the compounds showed potent activity
and selectivity against APN, in which 5q and 5s were comparable
to bestatin and could be used as lead compounds for the develop-
Figure 3. The docking result of 5s with APN showed by LIGPLOT. Compound 5s is shown in violet.

J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
4671
ment of future low molecular-weight peptidomimetic APN inhibi-
tors as anticancer agents.
Methyl 2-(4-bromobenzamido)-5-(3-nitroguanidino)pentanoate
(4n): (1.87 g, 90.0%).
Methyl 2-(3,4-dichlorobenzamido)-5-(3-nitroguanidino)pentano-
ate (4r): (1.78 g, 87.5%).
Methyl2-(4-bromo-3-nitrobenzamido)-5-(3-nitroguanidino)pent-
anoate (4s): (1.85 g, 80.1%).
Methyl 2-(2-naphthamido)-5-(3-nitroguanidino)pentanoate (4t):
(1.61 g, 83.4%).
5. Experimental
5.1. Chemistry: general procedures
All the material were commercial available. All the solvents ex-
cept fuming nitric acid and fuming sulfuric acid were distilled be-
fore use. All the reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates (60GF-254) and visu-
alized with UV light or chloride ferric. 200–300 mesh silica gel was
used in column chromatography. Proton NMR spectra were deter-
mined on a Brucker DRX spectrometer (300 MHz), d in parts per
million and J in hertz and TMS was used as an internal standard.
Measurements were made in D2O solutions. ESI-MS were deter-
mined on an API 4000 spectrometer. Elemental analysis for com-
pound was performed using an elementar vario EL III CN
analyzer (Germany). Melting points were determined on an elec-
trothermal melting point apparatus (uncorrected).
Methyl 2-(3,4-dimethoxybenzamido)-5-(3-nitroguanidino)pent-
anoate (4u): (1.67 g,84.4%).
Methyl 5-(3-nitroguanidino)-2-(3,4,5-trimethoxybenzamido)pent-
anoate (4v): (1.80 g, 84.3%).
Methyl 5-(3-nitroguanidino)-2-(thiophene-2-carboxamido)pent-
anoate (4w): (1.44 g, 84.0%).
5.1.4. Methyl 5-(3-nitroguanidino)-2-(3-
phenylpropanamido)pentanoate (4b)
Compound 3 (1.35 g, 5 mmol) and triethylamine (1 equiv) were
mixed in 30 ml anhydrous DCM under ice-bath. 3-Phenylpropionyl
chloride (1.2 equiv) dissolved in DCM was added dropwise. The
resulting solution was stirred for 2 h and then washed with satu-
rated Na2CO3, 10% citric acid and brine. After lying for a while,
white solid would precipitate in DCM. The solid was filtered and
washed with DCM, then dried in vacuum drying oven. Finally
1.69 g product was obtained, yield 92.7%.
5.1.1. 2-Amino-5-(3-nitroguanidino)pentanoic acid (2)
The title compound was prepared as described by Hashimoto
et al.21 from compound 1.
Compounds 4d, 4o, 4p and 4q were synthesized following the
procedure described above.
Methyl 2-benzamido-5-(3-nitroguanidino)pentanoate (4d):
(1.43 g, 85.1%).
Methyl 2-(4-nitrobenzamido)-5-(3-nitroguanidino)pentanoate
(4o): (1.74 g, 91.3%).
5.1.2. Methyl 2-amino-5-(3-nitroguanidino)pentanoate
hydrochloride (3)
The title compound was prepared as described by Jordis22 from
compound 2.
5.1.3. Methyl 5-(3-nitroguanidino)-2-(2-phenylacetamido)-
pentanoate (4a)
Methyl 2-(4-methoxybenzamido)-5-(3-nitroguanidino)pentano-
ate (4p): (1.65 g, 89.9%).
Phenylacetic acid (0.68 g, 5 mmol) and trimethylamine
(3 equiv) were dissolved in 30 ml anhydrous dichloromethane
(DCM). To this stirring solution was added TBTU (1.3 equiv) fol-
lowed by compound 3. The resulting solution was stirred for 6 h
and then washed with saturated Na2CO3, 10% citric acid and brine.
Lying for a while, the white solid precipitated in DCM was filtered
and washed with DCM, then dried in vacuum drying oven. Finally,
1.60 g product was obtained, yield 90.8%, mp 115–117 °C. ESI-MS
m/z: 352.4 (M+H)+; 1H NMR (D2O): 1.58–1.65 (m, 2H), 1.71–1.76
(m, 2H), 3.13–3.14 (m, 2H), 3.47 (s, 2H), 3.62 (s, 3H), 4.22–4.26
(m, 1H), 7.20–7.30 (m, 5H).
Methyl 2-(2,4-dichlorobenzamido)-5-(3-nitroguanidino)pent-
anoate (4q): (1.68 g, 90.3%).
5.1.5. Methyl 2-(4-methylphenylsulfonamido)-5-(3-
nitroguanidino)pentanoate (6a)
Compound 6a was synthesized following the procedure of com-
pound 4b. Finally, 1.72 g product was got, yield 88.9%.
Compound 6b was synthesized following the procedure of com-
pound 6a.
Methyl 5-(3-nitroguanidino)-2-(phenylsulfonamido)pentano-
ate (6b): (1.62 g, 87.0%).
Compounds 4c, 4e-n and 4r-w were synthesized following the
procedure described above.
Methyl 2-cinnamamido-5-(3-nitroguanidino)pentanoate (4c):
(1.46 g, 80.7%).
Methyl 2-(2-chlorobenzamido)-5-(3-nitroguanidino)pentano-
ate (4e): (1.67 g,89.9%).
Methyl 2-(2-iodobenzamido)-5-(3-nitroguanidino)pentanoate (4f):
(2.02 g, 87.2%).
Methyl 2-(2-methylbenzamido)-5-(3-nitroguanidino)pentano-
ate (4g): (1.62 g, 92.3%).
Methyl 2-(2-methoxybenzamido)-5-(3-nitroguanidino)pentanoate
(4h): (1.61 g, 87.7%).
Methyl 2-(3-chlorobenzamido)-5-(3-nitroguanidino)pentanoate
(4i): (1.69 g, 91.0%).
5.1.6. N-Hydroxy-5-(3-nitroguanidino)-2-(2-phenylacetamido)-
pentanamide (5a)
To a solution of compound 4a (0.71 g, 2 mmol) in 7 ml anhy-
drous methanol at room temperature was added dropwise a solu-
tion of NHOK (6 mmol) in methanol (3.4 ml). The mixture was
stirred for 12 h and the solvent was evaporated in vacuum. The res-
idue was purified by column chromatography (dichloromethane/
methanol = 20/1–5/1). Finally, 059 g 5a was obtained. White solid,
yield 83.4%, mp 76–79 °C. ESI-MS m/z: 353.5 (M+H)+; 1H NMR
(D2O): 1.51–1.77 (m, 4H), 3.00–3.13 (m, 2H), 3.41–3.50 (m, 2H),
4.09–4.17 (m, 1H), 7.18–7.32 (m, 5H). Anal. Calcd for
C14H20N6O5: C, 47.72; H, 5.72; N, 23.85. Found: C, 47.62; H, 5.65;
N, 23.75.
Methyl 2-(3-nitrobenzamido)-5-(3-nitroguanidino)pentanoate
(4j): (1.76 g, 92.1%).
Compounds 5b–w and 7a,b were synthesized following the pro-
cedure described above.
Methyl 2-(3-methylbenzamido)-5-(3-nitroguanidino)pentano-
ate (4k): (1.61 g, 91.5%).
Methyl 2-(3-methoxybenzamido)-5-(3-nitroguanidino)pentano-
ate (4l): (1.65 g, 89.7%).
Methyl 2-(4-chlorobenzamido)-5-(3-nitroguanidino)pentanoate
(4m): (1.69 g, 91.1%).
5.1.6.1. N-Hydroxy-5-(3-nitroguanidino)-2-(3-phenylpropa-
namido)pentanamide (5b). White solid, yield 78.8%, mp 161–
162 °C. ESI-MS m/z: 367.3 (M+H)+; 1H NMR (D2O):1.31–1.62 (m,
4H), 2.42–2.50 (m, 2H), 2.76–2.79 (t, J = 7.5 Hz, 2H), 3.12–3.16
(m, 2H), 4.12–4.18 (m, 1H), 7.16–7.29 (m, 5H). Anal. Calcd for

4672
J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
C15H22N6O5: C, 49.19; H, 6.05; N, 22.94. Found: C, 49.01; H, 5.99; N,
22.88.
5.1.6.10. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-3-methylbenzamide (5k). White solid, yield 86.5%, mp
176–179 °C. ESI-MS m/z: 353.5 (M+H)+; 1H NMR (D2O):1.49–1.54
(m, 2H), 1.71–1.75 (m, 2H), 2.36 (S, 3H), 3.16–3.18 (d, J = 5.4 Hz,
2H), 4.30–4.38 (m, 1H), 7.33–7.35 (d, J = 4.8 Hz, 2H), 7.66–7.71
(m, 2H). Anal. Calcd for C14H20N6O5: C, 47.72; H, 5.72; N, 23.85.
Found: C, 47.55; H, 5.78; N, 23.66.
5.1.6.2. 2-Cinnamamido-N-hydroxy-5-(3-nitroguanidino)pen-
tanamide (5c). White solid, yield 70.4%, mp 296–298 °C. ESI-MS
m/z: 365.3 (M+H)+; 1H NMR (D2O):1.53–1.57 (m, 2H), 1.73–1.77
(m, 2H), 3.28–3.35 (m, 2H), 4.01–4.04 (m, 1H), 6.76–6.84 (d,
J = 16 Hz, 1H), 7.38–7.48 (m, 4H), 7.52–7.58 (m, 2H). Anal. Calcd
for C15H20N6O5: C, 49.45; H, 5.53; N, 23.07. Found: C, 49.26; H,
5.49; N, 23.15.
5.1.6.11. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-3-methoxybenzamide (5l). White solid, yield 85.6%,
mp 68–71 °C. ESI-MS m/z: 369.3 (M+H)+; 1H NMR (D2O): 1.57–
1.62 (m, 2H), 1.69–1.75 (m, 2H), 3.16–3.18 (m, 2H), 3.81 (s, 3H),
4.30–4.38 (m, 1H), 7.08–7.11 (m, 1H), 7.34–7.39 (m, 1H), 7.45–
7.50 (m, 2H). Anal. Calcd for C14H20N6O6: C, 45.65; H, 5.47; N,
22.82. Found: C, 45.54; H, 5.38; N, 22.74.
5.1.6.3. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)benzamide (5d). White solid, yield 73.5%, mp 76–
79 °C. ESI-MS m/z: 339.5 (M+H)+; 1H NMR (D2O): 1.54–1.58 (m,
2H), 1.66–1.75 (m, 2H), 3.15–3.18 (m, 2H), 4.31–4.39 (m, 1H),
7.44–7.56 (m, 5H). Anal. Calcd for C13H18N6O5: C, 46.15; H, 5.36;
N, 24.84. Found: C, 46.03; H, 5.22; N, 24.68.
5.1.6.12. 4-Chloro-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-
1-oxopentan-2-yl)benzamide (5m). White solid, yield 82.1%,
mp 67–72 °C. ESI-MS m/z: 373.3 (M+H)+; 1H NMR (D2O): 1.49–
1.52 (m, 2H), 1.69–1.73 (m, 2H), 3.16–3.18 (d, J = 5.1 Hz, 2H),
4.29–4.36 (m, 1H), 7.53–7.56 (d, J = 8.4 Hz, 2H), 7.90–7.93 (d,
J = 8.4 Hz, 2H). Anal. Calcd for C13H17ClN6O5: C, 41.89; H, 4.60; N,
22.55. Found: C, 41.48; H, 4.79; N, 22.56.
5.1.6.4. 2-Chloro-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-yl)benzamide (5e). White solid, yield 85.8%, mp
105–108 °C. ESI-MS m/z: 373.3 (M+H)+; 1H NMR (D2O): 1.52–1.69
(m, 4H), 3.14–3.17 (m, 2H), 4.30–4.34 (m, 1H), 7.38–7.47 (m,
4H). Anal. Calcd for C13H17ClN6O5: C, 41.89; H, 4.60; N, 22.55.
Found: C, 41.72; H, 4.49; N, 22.63.
5.1.6.13. 4-Bromo-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-
1-oxopentan-2-yl)benzamide (5n). Jacinth solid, yield 84.4%,
mp 147–149 °C. ESI-MS m/z: 417.4 (M+H)+; 1H NMR (D2O):1.50–
1.52 (m, 2H), 1.68–1.78 (m, 2H), 3.16–3.18 (m, 2H), 4.28–4.36
(m, 1H), 7.67–7.80 (d, J = 8.4 Hz, 2H), 7.82–7.85 (d, J = 8.4 Hz, 2H).
Anal. Calcd for C13H17BrN6O5: C, 37.42; H, 4.11; N, 20.14. Found:
C, 37.18; H, 4.24; N, 19.93.
5.1.6.5. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-2-iodobenzamide (5f). Jacinth solid, yield 79.8%, mp
76–80 °C. ESI-MS m/z: 465.3 (M+H)+; 1H NMR (D2O): 1.54–1.69
(m, 4H), 3.15–3.19 (m, 2H), 4.27–4.30 (m, 1H), 7.13–7.46 (m,
4H). Anal. Calcd for C13H17IN6O5: C,33.64; H, 3.69; N, 18.10. Found:
C, 33.48; H, 3.66; N, 18.02.
5.1.6.14. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-4-nitrobenzamide (5o). White solid, yield 91.3%, mp
148–150 °C. ESI-MS m/z: 384.3 (M+H)+; 1H NMR (D2O):1.51–1.54
(m, 2H), 1.69–1.76 (m, 2H), 3.14–3.16 (m, 2H), 4.31–4.36 (m,
1H), 8.09–8.11 (d, J = 8.4 Hz, 2H), 8.29–8.32 (d, J = 8.4 Hz, 2H). Anal.
Calcd for C13H17N7O7: C, 40.73; H, 4.47; N, 25.58. Found: C, 40.46;
H, 4.39; N, 25.61.
5.1.6.6. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-2-methylbenzamide (5g). White solid, yield 89.9%,
mp 201–203 °C. ESI-MS m/z: 353.5 (M+H)+; 1H NMR (D2O): 1.48–
1.52 (m, 2H), 1.68–1.73 (m, 2H), 2.27 (s, 3H), 3.16–3.17 (d,
J = 5.4 Hz, 2H), 4.12–4.18 (m, 1H), 7.22–7.33 (m, 4H). Anal. Calcd
for C14H20N6O5: C, 47.72; H, 5.72; N, 23.85. Found: C, 47.51; H,
5.60; N, 23.81.
5.1.6.15. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-4-methoxybenzamide (5p). White solid, yield 86.7%,
mp 156–159 °C. ESI-MS m/z: 369.2 (M+H)+; 1H NMR (D2O):1.50–
1.52 (m, 2H), 1.69–1.71 (m, 2H), 3.16–3.19 (m, 2H), 3.81(s, 3H),
4.31–4.33 (m, 1H), 6.97–7.00 (d, J = 8.7 Hz, 2H), 7.86–7.89 (d,
J = 8.7 Hz, 2H). Anal. Calcd for C14H20N6O6: C, 45.65; H, 5.47; N,
22.82. Found: C, 45.55; H, 5.42; N, 22.80.
5.1.6.7. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-2-methoxybenzamide (5h). Jacinth solid, yield 85.3%,
mp 173–175 °C. ESI-MS m/z: 369.3 (M+H)+; 1H NMR (D2O): 1.52–
1.63 (m, 2H), 1.67–1.76 (m, 2H), 3.14–3.22 (m, 2H), 3.92 (S, 3H),
4.38–4.45(m, 1H), 7.04–7.08 (t, J = 7.2 Hz, 1H), 7.17–7.20 (d,
J = 7.8 Hz, 1H),7.48–7.54 (m, 1H), 7.82–7.85 (d, J = 7.8 Hz, 1H). Anal.
Calcd for C14H20N6O6: C, 45.65; H, 5.47; N, 22.82. Found: C, 45.48;
H, 5.43; N, 22.67.
5.1.6.16. 2,4-Dichloro-N-(1-(hydroxyamino)-5-(3-nitroguanidi-
no)-1-oxopentan-2-yl)benzamide (5q). White solid, yield 83.4%,
mp 154–156 °C. ESI-MS m/z: 407.4 (M+H)+; 1H NMR (D2O):1.61–
1.64 (m, 2H), 1.73–1.77 (m, 2H), 3.16–3.18 (m, 2H), 4.26–4.33
(m, 1H), 7.43–7.51 (m, 2H), 7.67–7.68 (d, J = 1.8 Hz, 1H). Anal.
Calcd for C13H16Cl2N6O5: C, 38.34; H, 3.96; N, 20.64. Found: C,
38.23; H, 4.04; N, 20.52.
5.1.6.8. 3-Chloro-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-yl)benzamide (5i). White solid, Yield 80.0%, mp
77–79 °C. ESI-MS m/z: 373.2 (M+H)+; 1H NMR (D2O): 1.55–1.76
(m, 4H), 3.15–3.17 (m, 2H), 4.32–4.34 (m, 1H), 7.47–7.96 (m,
4H). Anal. Calcd for C13H17ClN6O5: C, 41.89; H, 4.60; N, 22.55.
Found: C, 41.63; H, 4.53; N, 22.47.
5.1.6.17. 3,4-Dichloro-N-(1-(hydroxyamino)-5-(3-nitroguanidi-
no)-1-oxopentan-2-yl)benzamide (5r). White solid, yield 86.4%,
mp 130–134 °C. ESI-MS m/z: 407.5 (M+H)+; 1H NMR (D2O):1.50–
1.52 (m, 2H), 1.63–1.76 (m, 2H), 3.16–3.18 (d, J = 5.7 Hz, 2H),
4.29–4.36 (m, 1H), 7.75–7.78 (d, J = 8.4 Hz, 1H), 7.85–7.88 (dd,
J1 = 8.4 Hz, J2 = 2.1 Hz, 1H), 8.17–8.18 (s, d, J = 2.1 Hz, 1H). Anal.
Calcd for C13H16Cl2N6O5: C, 38.34; H, 3.96; N, 20.64. Found: C,
38.19; H, 3.94; N, 20.69.
5.1.6.9. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-3-nitrobenzamide (5j). Yellow solid, yield 83.1%, mp
149–151 °C. ESI-MS m/z: 384.3 (M+H)+; 1H NMR (D2O): 1.52–
1.54 (m, 2H), 1.73–1.75 (m, 2H), 3.15–3.17 (m, 2H), 4.35–4.37
(m, 1H), 7.72–7.77 (t, J = 7.8 Hz, 1H), 8.32–8.37 (m, 2H), 8.68 (s,
1H). Anal. Calcd for C13H17N7O7: C, 40.73; H, 4.47; N, 25.58. Found:
C, 40.55; H, 4.53; N, 25.61.

J. Mou et al. / Bioorg. Med. Chem. 17 (2009) 4666–4673
4673
5.1.6.18. 4-Bromo-N-(1-(hydroxyamino)-5-(3-nitroguanidino)-
1-oxopentan-2-yl)-3-nitrobenzamide (5s). Brown solid, yield
69.3%, mp 147–150 °C. ESI-MS m/z: 462.4 (M+H)+; 1H NMR
(D2O):1.54–1.56 (m, 2H), 1.73–1.76 (m, 2H), 3.16–3.18 (m, 2H),
4.34–4.36 (m, 1H), 7.42–7.45 (d, J = 9 Hz, 1H), 7.93–7.96 (d,
J = 9 Hz, 1H), 8.52 (s, 1H). Anal. Calcd for C13H16BrN7O7: C, 33.78;
H, 3.49; N, 17.29. Found: C, 33.60; H, 3.42; N, 17.35.
Kidney Microsomes (Sigma) as enzyme in 50 mM PBS, pH 7.2, at
37 °C. 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 the solutions
of the inhibitors were prepared in the assay buffer, and the 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 tempera-
ture. The assay mixture, which contained the inhibitor solution (con-
5.1.6.19. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-2-naphthamide (5t). Yellow solid, yield 75.4%, mp 72–
74 °C. ESI-MS m/z: 389.4 (M+H)+; 1H NMR (D2O): 1.60–1.73 (m,
2H), 1.77–1.88 (m, 2H), 3.17–3.20 (m, 2H), 4.38–4.43 (m, 1H),
7.58–7.80 (m, 4H), 7.98–8.05 (m, 3H). Anal. Calcd for C17H20N6O5:
C, 52.57; H, 5.19; N, 21.64. Found: C, 52.28; H, 5.07; N, 21.46.
centration dependent on the inhibitor), the enzyme solution (4
mL final concentration), and the assay buffer, was adjusted to 200
l
l
g/
L.
5.3. MMP-2 inhibition assay
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-
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.
5.1.6.20. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-3,4-dimethoxybenzamide (5u). White solid, yield
90.0%, mp 148–151 °C. ESI-MS m/z: 399.3 (M+H)+; 1H NMR
(D2O):1.49–1.53 (m, 2H), 1.71–1.77 (m, 2H), 3.16–3.18 (m, 2H),
3.80 (s, 6H), 4.34–4.36 (m, 1H), 6.99–7.02 (d, J = 8.4 Hz, 1H),
7.48–7.54(m, 2H). Anal. Calcd for C15H22N6O7: C, 45.22; H, 5.57;
N, 21.10. Found: C, 45.01; H, 5.49; N, 21.00.
Acknowledgments
5.1.6.21. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)-3,4,5-trimethoxybenzamide (5v). White solid, yield
85.2%, mp 101–104 °C. ESI-MS m/z: 429.5 (M+H)+; 1H NMR
(D2O):1.47–1.56 (m, 2H), 1.68–1.72 (m, 2H), 3.14–3.17 (m, 2H), 3.70
(s, 9H), 4.35–4.40 (m, 1H), 7.25 (s, 2H). Anal. Calcd for C16H24N6O8:
C, 44.86; H, 5.65; N, 19.62. Found: C, 44.49; H,5.56; N, 19.46.
This work was supported by National High Technology Re-
search and Development Program of China (863 Project, Grant
No. 2007AA02Z314) and National Natural Science Foundation of
China (Grant No. 30772654 and No. 90713041) and The Doctoral
Foundation of Ministry of Education of the People’s Republic of
China (Grant No. 20060422029).
5.1.6.22. N-(1-(Hydroxyamino)-5-(3-nitroguanidino)-1-oxopen-
tan-2-yl)thiophene-2-carboxamide (5w). White solid, yield
78.6%, mp 160–162 °C. ESI-MS m/z: 345.4 (M+H)+; 1H NMR
(D2O):1.50–1.69 (m, 2H), 1.71–1.80 (m, 2H), 3.16–3.18 (d,
J = 5.4 Hz, 2H), 4.29–4.31 (m, 1H), 7.13–7.16 (t, J = 4.8 Hz, 1H),
7.76–7.77 (d, J = 5.4 Hz, 1H), 7.82–7.85 (d, J = 3.0 Hz, 1H). Anal. Calcd
for C11H16N6O5S: C, 38.37; H, 4.68; N, 24.41. Found: C, 38.12; H, 4.62;
N, 24.30.
References and notes
1. Nathalie, L.; Cynthia, M. C.; Emmanuel, R.; Ann, B.; Bernard, P. R.; Marie-Claude,
F. Z. Biochemistry 1998, 37, 686.
2. Florence, N.; Nathalie, L.; Sophie, D. N.; René, L. K.; Laurent, B.; Hui, X. C.; Marie-
Claude, F. Z.; Bernard, P. R. FEBS Lett. 2000, 467, 81.
3. Xu, W. F.; Li, Q. B. Curr. Med. Chem., Anti-Cancer Agents 2005, 5, 281.
4. Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M. Antibiotics 1976, 29,
97.
5.1.6.23. N-Hydroxy-5-(3-nitroguanidino)-2-(phenylsulfonami-
do)pentanamide (7a). White solid, yield 86.6%, mp 59–61 °C.
ESI-MS m/z: 375.3 (M+H)+; 1H NMR (D2O): 1.15–1.23 (m, 2H),
1.30–1.49 (m, 2H), 2.98–3.08 (m, 2H), 3.52–3.57 (m, 1H), 7.54–
7.61(m, 2H), 7.76–7.89 (m, 3H). Anal. Calcd for C12H18N6O6S: C,
38.50; H, 4.85; N, 22.45. Found: C, 38.29; H, 4.76; N, 22.63.
5. Aoyagi, T.; Yoshida, S.; Nakamura, Y.; Shigihara, Y. Antibiotics 1990, 43,
143.
6. Rich, D. H.; Moon, B. J.; Harbeson, S. J. Med. Chem. 1984, 27, 417.
7. Umezawa, H.; Aoyagi, T.; Tanaka, T.; Suda, H. Antibiotics 1985, 38, 1629.
8. Nagai, M.; Kojima, F.; Naganawa, H. Antibiotics 1997, 50, 82.
9. Lampret, B. R.; Kidrie, J.; Kralj, B. Arch. Microbiol. 1999, 171, 397.
10. Chung, M. C.; Lee, H. J.; Chun, H. K. Biosci., Biotechnol., Biochem. 1996, 60,
898.
11. Aoyagi, T.; Yoshida, S.; Matsuda, N. Antibiotics 1991, 44, 573.
12. Egan, M. E.; Pearson, M.; Weiner, S. A., et al Science 2004, 304, 600.
13. Shim, J. S.; Lee, H.; Shin, J. Cancer Lett. 2004, 203, 163.
14. Shimazawa, R.; Takayama, H.; Hashimoto, Y., et al J. Enzyme Inhib. 1999, 14,
259.
15. Ito, K.; Nakajima, Y.; Yoshimoto, T., et al J. Biol. Chem. 2006, 281,
33664.
16. Wang, Q.; Chen, M. Y.; Zhu, H. W.; Zhang, J.; Fang, H.; Wang, H. B.; Xu, W. F.
Bioorg. Med. Chem. 2008, 16, 5473.
5.1.6.24. N-Hydroxy-2-(4-methylphenylsulfonamido)-5-(3-
nitroguanidino)pentanamide (7b). Orange solid, yield 82.2%,
mp 64–66 °C. ESI-MS m/z: 389.3 (M+H)+; 1H NMR (D2O): 1.13–
1.35 (m, 2H), 1.38–1.49 (m, 2H), 2.29–2.36 (m, 3H), 2.99–3.06 (q,
J = 7.2 Hz, 2H), 3.50–3.54 (m, 1H), 7.11–7.13 (d, J = 8.1 Hz, 1H),
7.32–7.35 (d, J = 8.1 Hz, 1H),7.46–7.49 (d, J = 8.1 Hz, 1H), 7.63–
7.66 (d, J = 8.1 Hz, 1H). Anal. Calcd for C13H20N6O6S: C, 40.20; H,
5.19; N, 21.64. Found: C, 40.06; H, 5.23; N, 21.58.
17. Addlagatta, A.; Gay, L.; Matthews, B. W. Biochemistry 2008, 47, 5303.
18. Shang, L. Q.; Wang, Q.; Fang, H.; Mu, J. J.; Wang, X. J.; Yuan, Y. M.; Wang, B. H.;
Xu, W. F. Bioorg. Med. Chem. 2008, 16, 6663.
19. Li, Y. L.; Xu, W. F. Bioorg. Med. Chem. 2004, 12, 5171.
20. Li, X.; Li, Y. L.; Xu, W. F. Bioorg. Med. Chem. 2006, 15, 1287.
21. Hashimoto, S.; Okada, K.; Sakakibara, R.; Fujii, S. U.S. Patent [11] 3, 799, 988
March 26, 1974.
5.2. APN inhibition assay
IC50 values against APN were determined by using
anilide as substrate and microsomal amino-peptidase from Porcine
L-Leu-p-nitro-
22. Jordis, U.; Sauter, F.; Siddiqi, S. M. Indian J. Chem. Sec. B 1989, 294.