Design, synthesis and primary activity assay of bi- or tri-peptide analogues with the scaffold l-arginine as amino-peptidase N/CD13 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.11.036

A series of bi- or tri-peptide analogues with the scaffold l-arginine were designed, synthesized and evaluated for their inhibitory activities against amino-peptidase N (APN) and metalloproteinase-2 (MMP-2). The primary activity assay showed that all the compounds exhibited higher inhibitory activities against APN than MMP-2. Within this series, compounds C6 and C7 (IC₅₀ = 4.2 and 4.3 μM) showed comparable APN inhibitory activities with the positive control bestatin (IC₅₀ = 3.8 μM).

Full text of DOI:10.1016/j.bmc.2009.11.036

Bioorganic & Medicinal Chemistry 18 (2010) 887–895
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and primary activity assay of bi- or tri-peptide analogues
with the scaffold L-arginine as amino-peptidase N/CD13 inhibitors
Jiajia Mou a, Hao Fang a, Yingzi Liu a,b, Luqing Shang a, Qiang Wang a, Lei Zhang a, Wenfang Xu a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44, Wenhuaxi Road, Ji’nan, Shandong 250012, PR China
b 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 bi- or tri-peptide analogues with the scaffold L-arginine were designed, synthesized and eval-
Received 23 September 2009
Revised 16 November 2009
Accepted 17 November 2009
Available online 22 November 2009
uated for their inhibitory activities against amino-peptidase N (APN) and metalloproteinase-2 (MMP-2).
The primary activity assay showed that all the compounds exhibited higher inhibitory activities against
APN than MMP-2. Within this series, compounds C6 and C7 (IC50 = 4.2 and 4.3
APN inhibitory activities with the positive control bestatin (IC50 = 3.8 M).
lM) showed comparable
l
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Amino-peptidase N
Metalloproteinase-2
Peptide analogue
Inhibitor
L
-Arginine
Synthesis
1. Introduction
inhibitors, bestatin is
a
representative one, which has been
launched and is now widely employed clinically as an anti-tumor
agent.9 In 2006, the 3D structure of APN has been studied accord-
ing to the co-crystal complex of APN and bestatin by Ito.10 Accord-
ingly, the interactions of bestatin with the active sites of APN can
be illustrated by a simplified diagram (see Fig. 1). It revealed that
beside the catalytic center zinc(II) ion of APN, there were three
hydrophobic binding domains, which were called S1 pocket, S10
pocket and S02 pocket, respectively. The side chains of bestatin
Amino-peptidase N (APN/CD13; EC 3.4.11.2) is a monomeric or
homodimeric type II membrane-bound zinc-dependent metallo-
proteinase, which can release neutral and basic amino acid from
the N-terminal of peptides. It is particularly noticeable that overex-
pression of APN is highly correlated with various pathological dis-
orders, such as inflammatory bowel diseases, encephalomyelitis,
multiple sclerosis, ulcerative colitis, Crohn’s disease, rheumatoid
arthritis and cancer.1–5 Because it plays key roles in extracellular
matrix degradation, tumor cell invasion and tumor angiogenesis,
APN was regarded as an attractive target for anti-cancer drug
design.
occupied S1 and S0 pocket.
1
In our previous work, we reported a series of L-arginine deriva-
tives as APN inhibitors.11 Some of them showed potential inhibi-
tory activities against APN. Especially, compound 5s, whose IC50
The matrix metalloproteinases (MMPs) are a family of structur-
ally related zinc-dependant endoproteinases that degrade and re-
model the structural protein in the extracellular matrix. MMP
gene family consists of at least 28 members, among which MMP-
2 and MMP-9 are proved to be highly correlated with cancer. Be-
cause similar to APN, MMP-2 is also a zinc-dependant metallopro-
teinase that involved in tumor invasion and metastasis. Thus in the
enzyme activity assay of our lab, both APN and MMP-2 were per-
formed so as to identify the compounds selectivity.
was 5.1 lM compared with 3.1 lM of bestatin. Considering the
side chains of this series of compounds were all aromatic acid sub-
stituents and not long enough, we designed a new series of bi- or
tri-peptide analogues, which contained amino acid and bi-peptide
Enzyme
S1
S1'
S2'
Zn2+
Up to now, several excellent reviews on natural and small mol-
O
ecule inhibitors of APN have been published.6–8 Among these
H3N
N
COOH
H
OH
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
Figure 1. Schematic diagram of the interaction of bestatin with APN.6
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.036

888
J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
analogues as side chains. Within these compounds, tri-peptide
analogues are much longer than the former compounds. We hope
that the long side chains can simultaneously occupy S01 and S20
pockets of APN and enhance the activities towards APN (Fig. 2).
for potential APNIs. And almost all the compounds showed better
APN inhibitory activities than the previous compounds.
It is worthy to mention that compound C6 and C7 (IC50 = 4.2
and 4.3
(IC50 = 3.8
lM) exhibited similar activities with bestatin
lM). That may be due to the two aromatic groups of
C6 and C7 occupying S01 and S20 hydrophobic pockets of APN suit-
ably. And compound B4, C3, C4 and C5 (IC50 = 8.4, 6.7, 4.8 and
2. Chemistry
7.2
l
M, respectively) also showed potential activities.
All the target compounds were designed and synthesized via
the route shown in Scheme 1. The synthesis of compound 3 has
been described in the previous paper. Boc protection of compound
4 led to compound 5. Compound 10 was obtained by the conden-
sation of the methyl ester 9 and Boc protected amino acids 5.
Hydrolysis of compound 10 with NaOH/MeOH led to the bi-pep-
tide 11. Compound 17 was prepared as described for compound
11. The acylation of compound 3 with Boc protected amino acids
5 or compounds 11 or compound 17 yielded compound 6, com-
pound 12 and compound 18. Then the ester groups of compound
6, compound 12 and compound 18 were treated with NHOK in
anhydrous methanol to get compounds 7, 13 and 19. The Boc group
of compound 7 and compound 13 can be easily removed by 2 N HCl
in ethyl acetate to get the target compounds.
In another way, the data shown in Table 1 suggested that the
preferred compounds against APN were, in decreasing order, C ser-
ies of compounds, B series of compounds and A series of com-
pounds. The possible reason may be due to the hydrophobic
chains of C series of compounds occupying the three hydrophobic
pockets of APN comfortably.
Among A and B series of compounds, generally speaking, com-
pounds with Boc protected amino groups showed better activities
than those with free amino group. For example, B4 and B11
(IC50 = 8.4 and 35.4 lM). This activity difference might be ex-
plained by the FlexX docking results of B4 and B11 with APN
(Fig. 3). In Figure 3, the backbone of B4 inserted into S1 pocket,
the hydroxymate group chelated with the zinc ion of APN, the iso-
butyl group occupied S01 pocket and the Boc group extended into S20
pocket. While the free amino group of B11 can not insert into S20
pocket at all.
3. Result and discussion
When it comes to C series, we can see that almost all this series
of compounds exhibited good activities. But, to some extent, the
bulk of R6 group was positively relative with APN inhibition. Com-
pound C6 and C7 with benzyl group displayed better APN inhibi-
tory activities than compound C1 and C2, which only contained
methyl group in the R6 position. This suggested that it was favor-
able to increase the bulk of R6 substituent to get better APN inhib-
itory activity.
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.
As shown in Table 1, all the compounds displayed better inhib-
itory activities towards APN as compared with MMP-2. The results,
to a certain extent, validated our strategy for rational drug design
NH
R1
H
O2N
N
N
H
N
NH2
H
O
Br
O
NHOH
R2
NH
H
O2N
N
N
N
NO2
H
H
O
NH
O
O
NHOH
H
O2N
N
N
N
N
R3
H
H
H
O
compound 5s
IC50 = 5.1 µM
O
NHOH
amino acid or organic
acid
Figure 2. (a) The docking mode of compound 5s with APN. (b) Chemical structure of compound 5s and the newly designed compounds.

J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
889
NH
O
NH
O
NH
O
a
b
O2N
O2N
H2N
N
OH
N
H
N
OH
N
N
OCH3
.HCl
H
H
H
H
NH2
NH2
NH2
2
1
3
O
O
NH
R1
H
N
c
3, d
e
R1
R1
O2N
OH
OH
NHBoc
N
N
NHBoc
H
H
NH2
O
O
OCH3
6
4
5
NH
R1
H
N
NH
R1
H
f
O2N
O2N
N
N
N
NHBoc
N
N
NH2
H
H
.HCl
O
H
H
O
O
NHOH
O
NHOH
O
7
8
O
O
R2
O
R2
R2
g
R3
h
BocHN
OH
3, d
BocHN
OCH3
OCH3
.HCl
+
OH
N
H
N
NH2
H
NHBoc
R3
O
R3
O
11
5
9
10
NH
R2
O
NH
R2
O
H
H
O2N
N
NHBoc
e
O2N
N
NHBoc
N
H
N
H
N
H
N
H
N
N
H
H
O
R3
O
R3
O
OCH3
O
NHOH
12
13
NH
R2
N
O
H
N
O2N
NH2
f
N
N
.HCl
H
H
H
O
R3
O
NHOH
14
O
O
O
R4
O
R4
g
R4
h
3, d
OCH3
OH
OCH3
.HCl
+
R5
OH
R5
N
R5
N
H
NH2
H
O
O
17
16
15
9
NH
R4
O
NH
R4
O
H
N
H
O2N
e
O2N
N
N
N
N
R5
N
N
N
R5
H
H
H
H
H
H
O
O
O
NHOH
O
OCH3
18
19
Scheme 1. Reagents and conditions: (a) fuming nitric acid, fuming sulfuric acid; (b) MeOH, HCl; (c) (Boc)2O/DCM, Et3N, 0 °C; (d) DCC, HOBt, THF, ꢀ5 °C; (e) NHOK, MeOH; (f)
HCl/EtOAC; (g) IBCF, NMM, THF, ꢀ15 °C; (h) NaOH/MeOH.
Compound C6 was the most active compound. In order to
investigate the interaction of it with APN, the docking studies
were carried out via Sybyl/Sketch module and optimized using
Powell’s method with the Tripos force field with convergence cri-
terion set at 0.05 kcal/(Å mol), and assigned with Gasteiger–Hüc-
kel method. The docking study 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 ac-
tive site. Other docking parameters implied in the program were
kept default. The docking result was shown in Figure 4. As dia-
grammed in Figure 4, the backbone of C6 extended into S1 pocket,
the hydroxymate group chelated with the zinc ion of APN, the
benzyl group occupied S01 pocket and the phenyl group occupied
S02 pocket. In addition, the hydroxyl of hydroxymate group could
form hydrogen bond with the carbonyl group of Ala262 of APN,
the amide of hydroxymate group could form hydrogen bond with
the carboxyl group of Glu264 and the amide group near hydroxy-
mate group could form hydrogen bond with the carboxyl group of
Glu298
.
It should be pointed out that although compounds C6 and C7
gave comparable APN inhibitory activities with bestatin, none of
the compounds demonstrated better potency than the positive
control. Therefore, further structural optimization should be car-
ried out towards C series.

890
J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
Table 1
The structure and inhibitory activities of the target compounds against APN and MMP-2
NH
R1
H
O2N
N
R2
N
H
N
N
H
H
O
O
NHOH
a
Compound
R1
R2
IC50
(
lM)
APN
MMP-2
A1
Boc
200.9 5.8
3464.2 4.6
A2
A3
A4
Boc
Boc
H
334.1 4.1
132.1 4.0
280.0 2.9
5684.6 5.2
1935.5 3.7
428.3 3.1
S
S
A5
A6
H
H
380.3 5.6
242.8 4.2
5308.9 7.5
4204.2 6.6
NH
R3
O
H
H
O2N
N
N
N
N
N
H
R5
H
H
O
R4
O
NHOH
a
Compound
R3
H
R4
R5
IC50
(l
M)
APN
25.2 1.4
MMP-2
B1
B2
Boc
Boc
2976.2 5.0
39.5 1.7
33.1 1.8
2895.8 5.4
446.9 3.9
B3
B4
B5
Boc
Boc
Boc
8.4 1.8
407.4 3.5
N
N
1336.1 6.6
7903.1 5.8
B6
B7
Boc
Boc
436.3 4.3
42.1 2.5
4515.2 5.1
8364.4 6.9
OH
N
N
B8
B9
H
H
H
226.2 5.1
157.7 2.6
3630.2 4.0
1710.6 3.8
B10
H
98.6 4.0
3160.1 3.6
B11
B12
H
H
35.4 1.9
513.6 3.7
N
N
363.8 6.7
8047.3 7.9
B13
B14
H
H
248.0 4.5
65.2 2.3
7586.4 7.4
8611.4 8.9
OH
N
N

J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
891
Table 1 (continued)
NH
R6
O
H
O2N
N
N
N
N
R7
H
H
H
O
O
NHOH
a
Compound
R6
R7
IC50 (lM)
APN
MMP-2
C1
C2
16.3 1.2
8967.1 6.9
1755.2 5.4
O
31.4 1.6
6.7 0.9
4.8 0.6
Cl
I
Cl
C3
C4
1776.2 4.7
630.3 3.7
Cl
Cl
C5
C6
C7
7.2 1.1
4.2 0.7
4.3 0.7
1538.5 5.6
1649.5 4.6
8135.5 7.1
Cl
Cl
Cl
NH2
O
Bestatin
3.8 0.5
253.2 3.9
N
COOH
H
OH
Mean value of three experiments and standard deviation are given.
a
Finally, although the computed information assay totally sup-
ported our assumption, the exact binding mode of the -arginine
derivatives with APN should be verified from further X-ray crystal
studies.
umn chromatography. Proton NMR spectra were determined on a
Brucker DRX spectrometer (600 MHz), d in parts per million and J
in hertz and TMS was used as an internal standard. Measurements
were made in D2O solutions. HRMS spectra were conductedby Shan-
dong Analysis and Test Center. Melting points were determined on
an electrothermal melting point apparatus (uncorrected).
L
4. Conclusions
In all, we have synthesized a new series of L-arginine derivatives
5.1.1. Methyl 2-amino-5-(3-nitroguanidino)pentanoate hydro-
as APN inhibitors. Most of the compounds showed potent activity
and selectivity against APN, in which compound C6, C7 and C4
were comparable to bestatin and could be used as lead compounds
for the development of future low molecular-weight peptidomi-
metic APN inhibitors as anti-cancer agents.
chloride (3)
The title compound was synthesized as described in our previ-
ous paper.11
5.1.2. Boc protected amino acid (5)
The title compound was synthesized as described by Haaina.12
5. Experimental section
5.1.3. (S)-2-(2-(tert-Butoxycarbonyl)-4-methylpentanamido)
acetic acid (11-B1)
5.1. Chemistry: general procedures
Compound 9 was synthesized as described by Jordis.13
To a stirred solution of compound 5 ((S)-2-(tert-butoxycarbonyl)-
4-methylpentanoic acid, 2.31 g, 10 mmol) and N-methylmorpholine
(1.34 mL, 12 mmol) in THF (30 mL) was added isobutyl chlorofor-
mate (1.60 mL, 12 mmol) at ꢀ15 °C. The mixture was stirred for
30 min. A solution of compound 9 (methyl 2-aminoacetate hydro-
All the material were commercial available. All the solvents
except fuming nitric acid and fuming sulfuric acid were distilled be-
fore use. All the reactions were monitored by thin-layer chromatog-
raphy 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 col-

892
J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
Figure 4. The docking mode of compound C6 with APN.
off. The filtrate was washed with saturated NaHCO3, 10% citric acid
and brine, and then dried with MgSO4. The solvent was evaporated
to give compound 6.
Figure 3. Comparison of the FlexX docking results of compounds B4 and B11.
5.1.6. tert-Butyl (S)-1-((S)-1-(hydroxyamino)-5-(3-nitroguani-
dino)-1-oxopentan-2-ylamino)-3-methyl-1-oxobutan-2-ylcar-
bamate (7-A1)
To a solution of compound 6-A1 (2 mmol) in 7 mL anhydrous
methanol at room temperature was added dropwise a solution of
NHOK (6 mmol) in methanol (3.4 mL). The mixture was stirred
for 12 h and the solvent was evaporated in vacuum. The residue
was purified by column chromatography (dichloromethane/meth-
anol = 20/1–5/1). Finally, compound A1 was obtained.
chloride, 1.26 g, 10 mmol) and N-methylmorpholine (1.34 mL,
12 mmol) in THF (30 mL) was added dropwise to the reaction mix-
ture. Thestirringwascontinuedfor24 hatꢀ5 °C,andthenthe solvent
was evaporated in vacuum. The residue was dissolved in EtOAC and
washed with saturated NaHCO3, 10% citric acid and brine in turn.
Then the organic phage was dried with MgSO4 and concentrated with
a rotary evaporator to give the crude product compound 10 ((S)-
methyl 2-(2-(tert-butoxycarbonyl)-4-methyl pentanamido) acetate).
Compound 10 (3.02 g, 10 mmol) was dissolved in 25 mL MeOH
and then 25 mL 2 mol/L NaOH was added in 5 min. The reaction
mixture was stirred for 5 h at room temperature. The organic phase
was evaporated off. The resulting solution was adjusted to pH 3
with 2 mol/L citric acid and compound 11-B1 precipitated as a
white solid, which was filtrated and dried in vacuum oven.
5.1.7. tert-Butyl (S)-1-((S)-1-(hydroxyamino)-5-(3-nitroguanidi-
no)-1-oxopentan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarba-
mate (A1)
Yield 72%, mp 121–124 °C. 1H NMR (D2O): 0.79–0.87 (m, 6H),
1.38 (s, 9H), 1.48–1.59 (m, 4H), 1.90–1.93 (m, 1H), 3.34–3.40 (m,
2H), 3.79–3.82 (t, J = 7.2 Hz, 1H), 4.14–4.18 (dd, J1 = 6.6 Hz,
J2 = 7.8 Hz, 1H). HRMS calcd for C16H31N7O7 (M+H)+ 434.2363,
found 434.2356.
5.1.4. (S)-2-(4-Methoxybenzamido) propanoic acid (17)
The title compound was synthesized following the procedure
described for compound 11-B1.
5.1.8. tert-Butyl (S)-1-((S)-1-(hydroxyamino)-5-(3-nitrogua-
nidino)-1-oxopentan-2- ylamino)-4-methyl-1-oxopentan-2-
ylcarbamate (A2)
5.1.5. (S)-Methyl 2-((S)-2-(tert-butoxycarbonyl)-3-methylbuta-
namido)-5-(3-nitroguanidino)pentanoate (6-A1)
A solution of DCC (4.50 g, 22 mmol) in 30 mL anhydrous THF
was added dropwise to a solution of compound 5 ((S)-2-(tert-
butoxycarbonyl)-3-methylbutanoic acid, 2.17 g, 20 mmol) and
HOBt (2.94 g, 22 mmol) in 60 mL of anhydrous THF at ꢀ5 °C for
20 h. Then the HOBt active ester was filtrated and the filtrate
would be used the next step.
Yield 73%, mp 156–159 °C. 1H NMR (D2O): 0.83–0.87 (m, 6H),
1.37 (s, 9H), 1.40–1.44 (m, 2H), 1.47–1.55 (m, 2H) 1.58–1.62 (m,
3H), 3.13–3.14 (m, 2H), 3.94–3.98 (m, 1H), 4.13–4.15 (m, 1H).
HRMS calcd for C17H33N7O7 (M+H)+ 448.2520, found 448.2506.
Compound 3 (2.70 g, 20 mmol) was suspended in 30 mL anhy-
drous THF, and TEA (2.78 mL, 20 mmol) was added. After 0.5 h,
the former filtrate was added in. The mixture was stirred at room
temperature overnight. The dicyclohexylurea (DCU) was filtrated
off and THF was evaporated in vacuum. The residues obtained
were dissolved in EtOAC and the DCU appeared again was filtrated
5.1.9. tert-Butyl (S)-1-((S)-1-(hydroxyamino)-5-(3-nitroguanidi-
no)-1-oxopentan-2-ylamino)-4-(methylthio)-1-oxobutan-2-ylc-
arbamate(A3)
Yield 64%, mp 135–137 °C. 1H NMR (D2O): 1.38 (s, 9H), 1.48–
1.58 (m, 2H), 1.69–1.84 (m, 2H), 2.02 (s, 3H), 2.40–2.48 (m, 2H),
2.67–2.70 (m, 2H), 3.18–3.19 (m, 2H), 3.97–4.00 (m, 1H), 4.06–

J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
893
4.07 (m, 1H). HRMS calcd for C16H31N7O7S (M+H)+ 466.2084, found
466.2074.
7.19 (m, 2H), 7.25–7.29 (m, 3H). HRMS calcd for C25H40N8O8
(M+H)+ 581.3047, found 581.3042.
5.1.10. (S)-2-((S)-2-Amino-3-methylbutanamido)-N-hydroxy-5-
(3-nitroguanidino) pentanamide (8-A4)
Compound A1 (1.73 g, 4 mmol) was dissolved in 20 ml 2 mol/L
HCl/EtOAC. After 30 min, the solvent was filtrated and the precip-
itate was washed with EtOAC to get compound A4.
5.1.19. tert-Butyl (S)-1-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-
ylamino)-1-oxo-3-phenylpropan-2-ylcarbamate (B4)
Yield 70%, mp 133–135 °C. 1H NMR (D2O): 0.77–0.89 (m, 6H),
1.30 (s, 9H), 1.36–1.47 (m, 4H), 1.59–1.69 (m, 3H), 2.69–2.75 (m,
1H), 2.89–2.96 (m, 1H), 3.14–3.15 (m, 2H), 4.09–4.20 (m, 2H),
4.31–4.38 (m, 1H), 7.17–7.19 (m, 2H), 7.25–7.26 (m, 3H). HRMS
calcd for C26H42N8O8 (M+H)+ 595.3204, found 595.3220.
5.1.11. (S)-2-((S)-2-Amino-3-methylbutanamido)-N-hydroxy-5-
(3-nitroguanidino)pentanamide (A4)
Yield 88%, mp 76–78 °C. 1H NMR (D2O): 0.87–0.96 (m, 6H),
1.47–1.67 (m, 4H), 2.03–2.13 (m, 1H), 3.14–3.17 (m, 2H), 3.64–
3.69 (m, 1H), 4.20–4.24 (m, 1H).
5.1.20. (S)-tert-Butyl 2-(((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-
yl)carbamoyl)pyrrolidine-1-carboxylate (B5)
HRMS calcd for C11H23N7O5 (M+H)+ 334.1833, found 334.1852.
Yield 68%, mp 139–142 °C. 1H NMR (D2O): 1.37 (s, 9H), 1.48–
1.57 (m, 4H), 1.62–1.64 (m, 3H), 1.99–2.00 (m, 1H), 2.73–2.82
(m, 1H), 2.96–3.01 (m, 1H), 3.13–3.15 (m, 2H), 3.18–3.40 (m,
2H), 4.15–4.16 (m, 2H), 4.59–4.60 (m, 1H), 7.15–7.28 (m, 5H).
HRMS calcd for C25H38N8O8 (M+H)+ 579.2891, found 579.2879.
5.1.12. (S)-2-((S)-2-Amino-4-methylpentamido)-N-hydroxy-5-
(3-nitroguanidino)pentanamide (A5)
Yield 86 %, mp 95–97 °C. 1H NMR (D2O): 0.87–0.93 (m, 6H),
1.50–1.60 (m, 4H), 1.62–1.66 (m, 2H), 1.68–1.73 (m, 1H), 3.17–
3.18 (m, 2H), 3.82–3.84 (m, 1H), 4.21–4.27 (m, 1H).
HRMS calcd for C12H25N7O5 (M+H)+ 348.1990, found 348.1985.
5.1.21. (S)-tert-Butyl 2-(((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-3-(4-hydroxyphenyl)-1-
oxopropan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (B6)
Yield 60%, mp 166–168 °C. 1H NMR (D2O): 1.37 (s, 9H), 1.50–
1.53 (m, 2H), 1.59–1.63 (m, 2H), 1.64–1.66 (m, 3H), 2.01–2.03
(m, 1H), 2.62–2.67 (m, 1H), 2.84–2.89 (m, 1H), 3.13–3.16 (m,
2H), 3.19–3.40 (m, 2H), 4.13–4.17 (m, 2H), 4.46–4.50 (m, 1H),
6.99–7.04 (m, 4H). HRMS calcd for C25H38N8O9 (M+H)+ 595.2840,
found 595.2832.
5.1.13. (S)-2-((S)-2-Amino-4-(methylthio)butanamido)-N-hydr-
oxy-5-(3-nitroguanidino)pentanamide (A6)
Yield 83%, mp 92–94 °C. 1H NMR (D2O): 1.54–1.71 (m, 4H),
1.94–2.04 (m, 2H), 2.06 (s, 3H), 2.50–2.52 (m, 2H), 3.16–3.18 (m,
2H), 3.89–3.91 (m, 1H), 4.19–4.22 (m, 1H).
HRMS calcd for C11H23N7O5S (M+H)+ 366.1554, found 366.1558.
5.1.14. (S)-Methyl 2-(2-((S)-2-(tert-butoxycarbonyl)-4-methylpen-
tanamido) acetamido)-5-(3-nitroguanidino)pentanoate (12-B1)
The title compound was synthesized as described for compound 6.
5.1.22. (S)-tert-Butyl 2-((S)-2-(((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbon-
yl)pyrrolidine-1-carboxylate (B7)
Yield 62%, mp 101–103 °C. 1H NMR (D2O): 1.37 (s, 9H), 1.44–
1.66 (m, 4H), 1.72–1.83 (m, 8H), 3.13–3.14 (m, 2H), 3.44–3.58
(m, 2H), 3.62–3.75 (m, 2H), 4.04–4.18 (m, 1H), 4.19–4.26 (m,
1H), 4.36–4.42 (m, 1H). HRMS calcd for C21H36N8O8 (M+H)+
529.2734, found 529.2728.
5.1.15. tert-Butyl (S)-1-(2-((S)-1-(hydroxyamino)-5-(3-nitrogua-
nidino)-1-oxopentan-2-ylamino)-2-oxoethylamino)-4-methyl-
1-oxopentan-2-ylcarbamate (13-B1)
The title compound was synthesized as described for compound 7.
5.1.16. tert-Butyl (S)-1-(2-((S)-1-(hydroxyamino)-5-(3-nitrogua-
nidino)-1-oxopentan-2-ylamino)-2-oxoethylamino)-4-methyl-
1-oxopentan-2-ylcarbamate (B1)
5.1.23. (S)-2-(2-((S)-2-Amino-4-methylpentanamido) acetam-
ido)-N-hydroxy-5-(3-nitroguanidino)pentanamide (14-B8)
The title compound was synthesized as described for compound
8.
Yield 63%, mp 132–134 °C. 1H NMR (D2O): 0.84–0.87 (m, 6H),
1.38 (s, 9H), 1.41–1.43 (m, 3H), 1.48–1.51 (m, 2H), 1.57–1.64 (m,
2H), 3.12–3.13 (m, 2H), 3.68–3.75 (m, 2H), 3.94–3.98 (q,
J = 7.8 Hz, 1H), 4.14–4.17 (q, J = 7.8 Hz, 1H). HRMS calcd for
C19H36N8O8 (M+H)+ 505.2729, found 505.2723.
5.1.24. (S)-2-(2-((S)-2-Amino-4-methylpentanamido)
acetamido)-N-hydroxy-5-(3-nitroguanidino)pentanamide (B8)
Yield 82%, mp 88–90 °C. 1H NMR (D2O): 0.88–0.91 (m, 6H),
1.53–1.60 (m, 4H), 1.62–1.75 (m, 3H), 3.16–3.17 (m, 2H), 3.56–
3.58 (m, 1H), 3.78–3.88 (m, 2H), 4.15–4.22 (m, 1H). HRMS calcd
for C14H28N8O6 (M+H)+ 405.2205, found 405.2216.
5.1.17. tert-Butyl (S)-1-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-1-oxopropan-2-ylamino)-
4-methyl-1-oxopentan-2-ylcarbamate (B2)
Yield 72%, mp 132–134 °C. 1H NMR (D2O): 0.84–0.86 (m, 6H),
1.16–1.17 (d, J = 7.2 Hz, 3H), 1.37 (s, 9H), 1.40–1.42 (m, 3H),
1.48–1.50 (m, 2H), 1.58–1.60 (m, 2H), 3.12–3.13 (m, 2H),
3.92–3.96 (m, 1H), 4.10–4.14 (q, J = 7.2 Hz, 1H), 4.27–4.29 (m,
1H). HRMS calcd for C20H38N8O8 (M+H)+ 519.2891, found
519.2879.
5.1.25. (S)-2-((S)-2-((S)-2-Amino-4-methylpentanamido)propa-
namido)-N-hydroxy-5-(3-nitroguanidino)pentanamide (B9)
Yield 83%, mp 96–98 °C. 1H NMR (D2O): 0.87–0.91 (m, 6H),
1.19–1.27 (m, 3H), 1.50–1.58 (m, 4H), 1.60–1.63 (m, 2H), 1.66–
1.70 (m, 1H), 3.15–3.16 (m, 2H), 3.78–3.79 (m, 1H), 4.11–4.17
(m, 1H), 4.36–4.39 (q, J = 7.2 Hz, 1H). HRMS calcd for C15H30N8O6
(M+H)+ 419.2361, found 419.2348.
5.1.18. tert-Butyl (S)-1-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-3-methyl-1-oxobutan-2-
ylamino)-1-oxo-3-phenylpropan-2-ylcarbamate(B3)
5.1.26. (S)-2-((S)-2-((S)-2-Amino-3-phenylpropanamido)-3-
methylbutanamido)-N-hydroxy-5-(3-nitroguanidino)pentana-
mide (B10)
Yield 75%, mp 116–119 °C. 1H NMR (D2O): 0.76–0.78 (m, 3H),
0.83–0.88 (m, 3H), 1.29 (s, 9H), 1.40–1.43 (m, 2H), 1.58–1.62 (m,
2H), 1.91–1.96 (m, 1H), 2.70–2.75 (m, 1H), 2.95–2.98 (m, 1H),
3.14–3.15 (m, 2H), 4.12–4.19 (m, 2H), 4.25–4.27 (m, 1H), 7.16–
Yield 86%, mp 123–125 °C. 1H NMR (D2O): 0.88–0.94 (m, 3H),
1.07–1.10 (m, 3H), 1.56–1.64 (m, 4H), 1.91–2.01 (m, 1H), 2.97–

894
J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
3.08 (m, 1H), 3.13–3.17 (m, 2H), 3.37–3.40 (m, 1H), 4.13–4.18 (m,
2H), 4.22–4.26 (m, 1H), 7.22–7.36 (m, 5H). HRMS calcd for
C20H32N8O6 (M+H)+ 481.2518, found 481.2526.
4.49 (m, 1H), 7.44–7.51 (m, 2H), 7.66–7.68 (m, 1H). HRMS calcd
for C16H21Cl2N7O6 (M+H)+ 478.1003, found 478.1027.
5.1.35. N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-2-
iodobenzamide (C3)
5.1.27. (S)-2-((S)-2-((S)-2-Amino-3-phenylpropanamido)-4-
methylpentanamido)-N-hydroxy-5-(3-nitroguanidino)pen-
tanamide (B11)
Yield 77%, mp 125–127 °C. 1H NMR (D2O): 0.84–0.88 (m, 6H),
1.42–1.58 (m, 4H), 1.91–1.94 (m, 1H), 3.14–3.15 (m, 2H), 4.13–
4.16 (m, 1H), 4.28–4.31 (m, 1H), 7.10–7.12 (m, 1H), 7.15–7.19
(m, 1H), 7.28–7.33 (m, 1H), 7.43–7.46 (m, 1H). HRMS calcd for
C18H26IN7O6 (M+H)+ 564.1062, found 564.1067.
Yield 85%, mp 121–123 °C. 1H NMR (D2O): 0.83–0.85 (m, 6H),
1.47–1.54 (m, 4H), 1.58–1.71 (m, 2H), 1.73–1.77 (m, 1H), 2.94–
2.98 (m, 1H), 3.02–3.04 (m, 1H), 3.14–3.16 (m, 2H), 4.11–4.13
(m, 2H), 4.38–4.41 (m, 1H), 7.26–7.28 (m, 5H). HRMS calcd for
C21H34N8O6 (M+H)+ 495.2674, found 495.2713.
5.1.36. 2-Chloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitrogua-
nidino)-1-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-yl)
benzamide (C4)
5.1.28. (S)-N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidi-
no)-1-oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)pyr-
rolidine-2-carboxamide (B12)
Yield 70%, mp 116–118 °C. 1H NMR (D2O): 0.84–0.90 (m, 6H),
1.36–1.60 (m, 4H), 1.71–1.83 (m, 3H), 3.14–3.15 (m, 2H), 4.16–
4.21 (m, 1H), 4.46–4.51 (m, 1H), 7.40–7.41 (m, 2H), 7.44–7.47
(m, 1H), 7.49–7.50 (m, 1H). HRMS calcd for C19H28ClN7O6 (M+H)+
486.1862, found 486.1886.
Yield 85%, mp 152–154 °C. 1H NMR (D2O): 1.55–1.69 (m, 4H),
1.71–1.84 (m, 4H), 2.29–2.30 (m, 1H), 2.70–2.83 (m, 1H), 3.03–
3.07 (m, 1H), 3.08–3.13 (m, 1H), 3.14–3.18 (m, 2H), 4.15–4.17
(m, 1H), 4.21–4.24 (m, 1H), 4.59–4.62 (m, 1H), 7.19–7.34 (m,
5H). HRMS calcd for C20H30N8O6 (M+H)+ 479.2361, found
479.2378.
5.1.37. 2,4-Dichloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-
yl) benzamide (C5)
5.1.29. (S)-N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidi-
no)-1-oxopentan-2-ylamino)-3-(4-hydroxyphenyl)-1-oxopro-
pan-2-yl)pyrrolidine-2-carboxamide (B13)
Yield 79%, mp 129–131 °C. 1H NMR (D2O): 0.89–0.90 (d,
J = 6.6 Hz, 6H), 1.46–1.58 (m, 4H), 1.63–1.69 (m, 3H), 3.14–3.15
(m, 2H), 4.16–4.20 (m, 1H), 4.46–4.52 (m, 1H), 7.41–7.42 (d,
J = 7.8 Hz, 1H), 7.49–7.51 (m, 1H), 7.67–7.69 (d, J = 7.8 Hz, 1H).
HRMS calcd for C19H27Cl2N7O6 (M+H)+ 520.1473, found 520.1498.
Yield 88%, mp 160–162 °C. 1H NMR (D2O): 1.54–1.68 (m, 4H),
1.73–1.83 (m, 4H), 2.28–2.31 (m, 1H), 2.58–2.70 (m, 1H), 2.91–
2.98 (m, 1H), 3.10–3.12 (m, 1H), 3.13–3.17 (m, 2H), 4.08–4.10
(m, 1H), 4.20–4.24 (m, 1H), 4.49–4.54 (m, 1H), 6.64–6.67 (m,
2H), 7.03–7.10 (m, 2H). HRMS calcd for C20H30N8O7 (M+H)+
495.2310, found 495.2341.
5.1.38. N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl) benzamide
(C6)
5.1.30. (S)-N-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-yl)-1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-
carboxamide (B14)
Yield 64%, mp 150–152 °C. 1H NMR (D2O): 1.33–1.63 (m, 4H),
2.94–3.00 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 1H), 3.16–3.17 (m, 2H),
3.36–3.40 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 1H), 4.15–4.21 (m, 1H),
4.70–4.76 (m, 1H), 7.14–7.17 (m, 1H), 7.24–7.27 (dd, J1 = 7.2 Hz,
J2 = 7.2 Hz, 2H), 7.36–7.37 (d, J = 7.2 Hz, 2H), 7.43–7.45 (m, 2H),
7.50–7.52 (m, 1H), 7.75–7.79 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 2H).
HRMS calcd for C22H27N7O6 (M+H)+ 486.2096, found 486.2151.
Yield 80%, mp 73–75 °C. 1H NMR (D2O): 1.45–1.68 (m, 4H),
1.70–1.90 (m, 3H), 2.08–2.14 (m, 4H), 2.25–2.34 (m, 1H), 3.15–
3.16 (m, 2H), 3.32–3.37 (m, 2H), 3.46–3.51 (m, 2H), 4.10–4.17
(m, 1H), 4.22–4.30 (m, 1H), 4.43–4.45 (m, 1H). HRMS calcd for
C16H28N8O6 (M+H)+ 429.2205, found 429.2223.
5.1.39. 2,4-Dichloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-
yl) benzamide (C7)
5.1.31. (S)-Methyl 2-((S)-2-(4-methoxybenzamido)propanam-
ido)-5-(3-nitroguanidino) pentanoate (18-C1)
The title compound was synthesized as described for compound
6.
Yield 76%, mp 141–143 °C. 1H NMR (D2O): 1.48–1.77 (m, 4H),
2.81–2.86 (m, 1H), 3.05–3.09 (m, 1H),3.11–3.16 (m, 2H), 4.17–
4.19 (m, 1H), 4.74–4.79 (m, 1H), 7.17–7.21 (m, 2H), 7.23–7.32
(m, 2H), 7.46–7.47 (m, 1H). HRMS calcd for C22H25Cl2N7O6
(M+H)+ 554.1316, found 554.1327.
5.1.32. N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-1-oxopropan-2-yl)-4-
methoxybenzamide (19-C1)
The title compound was synthesized as described for compound
7.
5.2. APN inhibition assay
IC50 values against APN were determined by using L-Leu-p-
5.1.33. N-((S)-1-((S)-1-(Hydroxyamino)-5-(3-nitroguanidino)-1-
oxopentan-2-ylamino)-1-oxopropan-2-yl)-4-
nitroanilide as substrate and microsomal amino-peptidase from
Porcine 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 temperature. The assay mixture, which contained
the inhibitor solution (concentration dependent on the inhibitor),
methoxybenzamide (C1)
Yield 68%, mp 181–183 °C. 1H NMR (D2O): 1.48–1.56 (m, 3H),
1.70–1.73 (m, 4H), 3.16–3.17 (m, 2H), 3.81 (s, 3H), 4.31–4.35 (m,
2H), 6.98–7.00 (m, 2H), 7.87–7.89 (m, 2H). HRMS calcd for
C17H25N7O7 (M+H)+ 440.1888, found 440.1876.
5.1.34. 2,4-Dichloro-N-((S)-1-((S)-1-(hydroxyamino)-5-(3-nitro-
guanidino)-1-oxopentan-2-ylamino)-1-oxopropan-2-yl)
benzamide (C2)
Yield 78%, mp 125–127 °C. 1H NMR (D2O): 1.26–1.29 (m, 3H),
1.42–1.67 (m, 4H), 3.15–3.17 (m, 2H), 4.17–4.20 (m, 1H), 4.45–
the enzyme solution (4
buffer, was adjusted to 200
l
g/mL final concentration), and the assay
L.
l

J. Mou et al. / Bioorg. Med. Chem. 18 (2010) 887–895
895
5.3. MMP-2 inhibition assay
References and notes
1. Bank, U.; Bohr, U. R.; Reinhold, D.; Lendeckel, U.; Ansorge, S.; Malfertheiner, P.;
Tager, M. Front. Biosci. 2008, 13, 3699.
2. Yamashita, M.; Kajiyama, H.; Terauchi, M.; Shibata, K.; Ino, K.; Nawa, A.;
Mizutani, S.; Kikkawa, F. Int. J. Cancer 2007, 120, 2243.
3. Shimizu, T.; Tani, K.; Hase, K.; Ogawa, H.; Huang, L.; Shinomiya, F.; Sone, S.
Arthritis Rheum. 2002, 46, 2330.
4. Reinhold, D.; Bank, U.; Täer, M.; Ansorge, S.; Wrenger, S.; Thielitz, A.; Lendeckel,
U.; Faust, J.; Neubert, K.; Brocke, S. Front. Biosci. 2008, 13, 2356.
5. Rahbar, A.; Boströ, L.; Söerberg-Naucler, C. J. Autoimmun. 2006, 26, 155.
6. Xu, W. F.; Li, Q. B. Curr. Med. Chem. Anti-Cancer Agents 2005, 5, 281.
7. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
8. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43, 305.
9. Shimazawa, R.; Takayama, H.; Hashimoto, Y., et al J. Enzyme Inhib. 1999, 14,
259.
10. Ito, K.; Nakajima, Y.; Yoshimoto, T., et al J. Biol. Chem. 2006, 281, 33664.
11. Mou, J. J.; Fang, H.; Jing, F. B.; Wang, Q.; Liu, Y. Z.; Xu, W. F., et al Bioorg. Med.
Chem. 2009, 17, 4666.
12. Haaina, G.; Lohse, A.; Buchardt, O. Angew. Chem., Int. Ed. Engl. 1995, 35,
1939.
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.
Acknowledgements
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 Nos. 30772654 and 90713041) and The Doctoral
Foundation of Ministry of Education of the People’s Republic of
China (Grant No. 20060422029).
13. Jordis, U.; Sauter, F.; Siddiqi, S. M. Indian J. Chem. Sec. B 1989, 294.