Design, synthesis and preliminary activity evaluation of novel L-lysine derivatives as aminopeptidase N/CD13 inhibitors

Article abstract of DOI:10.2174/092986610791306661

A novel class of L-lysine derivatives as aminopeptidase N (APN) inhibitors was designed and synthesized. Activity evaluation showed that compound C7 (IC₅₀ = 9.6 ± 1.3 μM) and C20 (IC₅₀ = 13.6 ± 1.9 μM) were equivalent to the positive control Bestatin (IC₅₀ = 11.3 ± 1.6 μM).

Full text of DOI:10.2174/092986610791306661

Protein & Peptide Letters, 2010, 17, 847-853
847
Design, Synthesis and Preliminary Activity Evaluation of Novel L-Lysine
Derivatives as Aminopeptidase N/CD13 Inhibitors
Qiang Wang1,2, Fuming Xu1, Jiajia Mou1,Jian Zhang1,Luqing Shang3,Yepeng Luan1,
Yumei Yuan1,Yingzi Liu1, Minyong Li1, Hao Fang1, Binghe Wang4 and Wenfang Xu1,*
1Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, P.R. China;
2Department of Pharmaceutics, School of Life Sciences, South-Central University for Nationalities, 708 Minyuan Road,
4
Wuhan, 430074, P.R. China; 3College of Pharmacy, Nankai University, Tian’jin, 300071, P.R. China; Department of
Chemistry, Georgia State University, Atlanta 30303, USA
Abstract: A novel class of L-lysine derivatives as aminopeptidase N (APN) inhibitors was designed and synthesized. Ac-
tivity evaluation showed that compound C7 (IC50 = 9.6 ± 1.3 μM) and C20 (IC50 = 13.6 ± 1.9 μM) were equivalent to the
positive control Bestatin (IC50 = 11.3 ± 1.6 μM).
Keywords: Lysine derivatives, Synthesis, APN; Inhibitor.
1. INTRODUCTION
pounds are proposed to bind the zinc ion with the carbonyl
group and near amino group in the active site. These interest-
ing results reminded us that this kind of zinc ion binding
groups could also be used to develop novel APN inhibitors.
Moreover, free amino group as hydrogen bond site may con-
tribute to water-solubility of the compounds.
Belonging to M1 aminopeptidase family, aminopeptidase
N (APN) is a Zn2+ dependent exopeptidase [1], which is ex-
pressed by myeloid, monocytes, epithelial cells of the intes-
tine and kidney, fibroblasts and endothelial cells, and over-
expressed on tumor cells [2]. APN plays a critical role in
physiological modification and protein degradation as well
as in the process of tumor cell metastasis [3-6].
In order to occupy pockets A and C concurrently, we
modified L-lysine with the same zinc binding group men-
tioned above to develop novel APN inhibitors as depicted in
the following scheme (Fig. 1): R1 is an aromatic ring, linked
to ꢁ-COOH of alkyl chain by amide bond. N6-amino group
was selectively protected by carbobenzoxy (Cbz) moiety. N2-
amino group was close to ꢁ-carbonyl group for chelating
with the zinc ion.
Numerous natural and synthetic small molecule inhibi-
tors against APN have been reported in the literatures since
1976 [7-16]. Our group also has reported various series of
APN inhibitors, such as 3-galloylamido-N’-substituted 2,6-
piperidinedione-N-acetamide peptidomimetics [17], L-gluta-
mine derivatives[18], AHPA (ꢀ-Amino-ꢁ-hydroxyl-phenyl-
butanoic acid) derivates and L-arginine derivatives [19, 20].
2. CHEMISTRY
In 2006, the structure of the complex of bestatin and
aminopeptidase N (EC 3.4.11.2) from Escherichia coli
(ePepN) was reported (PDB ID: 2DQM, Resolution: 1.60Å)
[21]. Then, the structure of the complex of L-lysine and
ePepN was reported in 2008 (PDB ID: 3B2X, Resolution:
1.50Å) [22]. Examining the active site of these complexes by
Sybyl, we found that bestatin and L-lysine have different
binding modes to APN. Bestatin can occupy hydrophobic
pocket A and pocket C concurrently while binding with zinc
ion, but L-lysine only occupies pocket A while binding with
zinc ion (Fig. 1).
The synthetic route of target compounds is shown in
Scheme 1. The N6-amino group was selectively Cbz-
protected, followed by the Boc-protection of the N2-amino
group to provide compound 8 N6-[(benzyloxy)carbonyl]-N2-
(tert-butoxycarbonyl)-L-lysine. Then, 20 different aromatic
amines reacted with ꢁ-COOH forming an amide bond. Fi-
nally, the N2-Boc protecting group was cleaved in HCl-
EtOAc (3 mol/L) to obtain the target compounds1.
3. RESULTS
The target compounds were assayed for the inhibitory
We also reported compounds 12i (L-phenylalanyl-N-
[(2R)-2-amino-3-phenylpropyl]-L-phenylalaninamide) and
13b ((S)-2-((S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitro-
phenyl)propanamido)-3-phenylpropanamido)-3-phenylprop-
anoic acid), both showing high APN inhibitory activities [23,
24]. An in silico docking study disclosed that these com-
activities on APN2. The biological results (Table 1) exhibited
All new compounds C1-20 provided acceptable HR-MS and 1H-NMR spectra that
1
exhibit no discernible impurities. For example, compound C7: White solid, yield 73.5
%, mp 193-195 ꢀ. HR-MS (ESI) m/z calcd for C19H25N4O3 [M+H]+ 357.1921.
+
Found: 357.1929; 1H-NMR (600 MHz, DMSO-d6) ꢀ 11.43 (s, 1H), ꢀ 8.63 (brs, 2H),
8.58 (m, 1H), ꢀ 8.39 (m, 1H), ꢀ 8.16 (s, 1H), ꢀ 8.05 (m, 1H), ꢀ 7.94 (m, 1H), ꢀ 7.31 (m,
5H), ꢀ 4.97 (s, 2H), ꢀ 4.11 (m, 1H), ꢀ 2.75 (m, 2H), ꢀ 1.86 (m, 2H), ꢀ 1.63(m, 2H), ꢀ
1.40 (m, 2H).
*Address correspondence to this author at the Department of Medicinal
Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong
250012, P.R. China; Tel/Fax: +86 531 88382264;
2
APN inhibition assay: IC50 values against APN were determined as described and
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 ꢀ. The
E-mail: xuwenf@sdu.edu.cn
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

848 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Wang et al.
Figure 1. The design of target compounds.
H2
N
H2
N
O
OH
NH2
HN Cbz
4
CO2
Cu
NH2
4
CO2
Cu
b
a
4
4
O2C
Cbz NH
N
H2N
O2C
H2N
N
4
H2
H2
6
5
4
R1
O
NH
O
OH
O
OH
e
c
H
d
H
H
Boc
Boc
N
4
N
4
N
4
N
Cbz
N
H
Cbz
H2N
Cbz
H
9
8
7
R1
O
NH
f
H
N
4
H2N
Cbz
10
Scheme 1. Reagents and conditions: (a) CuCO3Cu(OH)2, 1.2 N HCl, H2O, 80-90 ꢀ, 2 h; (b) NaHCO3, CbzCl, 25 ꢀ, 12 h; (c) EDTA SS, 25
ꢀ, 24 h; (d) (Boc)2O, THF, 1.0 N NaOH, 0 ꢀ, 12 h; (e) R1NH2, Et3N, TBTU, CH2Cl2, 25 ꢀ, 5 h; (f) HCl–EtOAc, 24 h.
that the activities of compound C7 (IC50 = 9.6 ± 1.3 μM) and
C20 (IC50 = 13.6 ± 1.9 μM) were equivalent to that of posi-
tive control bestatin (IC50 = 11.3 ± 1.6 μM).
Cell viability was assessed by MTT method3. Compound C7
showed better potency of anti-proliferation than bestatin to
HL-60 and PLC cells. But for ES-2 cell, they were equiva-
lent.
Additionally, the effects of C7 on proliferation of six
tumor cells compared with bestatin are shown in Table 2.
3 MTT assay: For example, HL-60 cell was grown in RPMI1640 medium containing 10
% FBS at 37 ꢀ in 5 % CO2 humidified incubator. Cell proliferation was determined by
the MTT (3-[4,5-dimethyl-2-thiazolyl]-2.5-diphenyl-2H-tetrazolium bromide) assay.
Briefly, cells were plated in a 96-well plate at 10,000 cells per well, cultured for 4 h in
complete growth medium, then treated with 2000, 1000, 500, 250 and 125 lg/mL of
compounds for 48 h. 0.5% MTT solution was added to each well. After further incuba-
tion for 4 h, formazan formed from MTT was extracted by adding DMSO and mixing
for 15 min. Optical density was read with an ELISA reader at 570 nm.
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 compounds 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 compounds 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 μg/mL final concentration), and the assay buffer, was adjusted to 200 μL.

Design, Synthesis and Preliminary Activity Evaluation
Protein & Peptide Letters, 2010, Vol. 17, No. 7 849
Table 1. The Inhibitory Activities of Target Compounds Against APN
R1
O
NH
O
H2N
N
H
O
Compound
R1
IC50*(μM)
F
C1
212.1±21.7
C2
C3
91.3±10.9
Cl
410.5±44.0
O
C4
>1000
S
O
C5
C6
C7
361.0±44.3
35.6±4.6
9.6±1.3
Br
N
Cl
C8
810.4±84.7
Cl
C9
277.5±54.0
>1000
C10
F
C11
C12
C13
783.8±86.4
185.1±33.9
260.5±51.3
F
O
Cl
C14
C15
475.9±57.2
460.6±43.0
F
O
C16
513.3±55.5

850 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Wang et al.
(Table 1) contd…..
Compound
R1
IC50*(μM)
C17
C18
632.4±89.2
N
215.8±31.0
224.9±29.9
C19
C20
S
13.6±1.9
11.3±1.6
N
Bestatin
*Each value represents the mean of three experiments, standard deviation is given.
Table 2. The Inhibitory Activities of Compound C7 and Bestatin Against Tumor Cell Lines
IC50*(mM)
Compound
HL-60
ES-2
K562
A549
H7402
PLC
C7
1.17±0.22
1.42±0.31
0.98±0.21
0.87±0.11
0.40±0.17
0.28±0.09
0.63±0.23
0.07±0.05
0.98±0.16
0.63±0.16
0.81±0.11
5.42±0.91
Bestatin
*Each value represents the mean of three experiments, standard deviation is given.
Figure 2. The docking result of compound C7. Bestatin in the X-ray crystal structure is showed in dark color.
In order to investigate the interaction of our target com-
pounds with APN, the representative compound C7 was con-
structed with Sybyl/Sketch module and optimized using
Powell’s method with the Tripos force field with conver-
gence criterion set at 0.05 Kcal/mol•Å, and assigned with
Gasteiger-Hückel method. The docking study was 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 pa-
rameters implied in the program were kept at default. The
docking studies proposed that the carbonyl group and amino
group of compound C7 bind to the zinc ion and form a bi-

Design, Synthesis and Preliminary Activity Evaluation
Protein & Peptide Letters, 2010, Vol. 17, No. 7 851
Figure 3. The docking result of compound C7 as suggested by LIGPLOT.
dentate chelate in the active pocket with distance of 2.3 Å
and 2.4 Å, respectively (Figs. 2,3). Furthermore, the distance
of His297 and His301 both binding to the zinc ion was 2.1 Å.
In addition, the N6-Cbz could occupy the pocket A and
R1 group for interacting with the pocket C. Compound C7
could form hydrogen bonds with His297 (< 3.3 Å) and His301
(< 2.9 Å) which are the essential amino acids of the con-
served sequence (HEXXHX18E) in the catalytic domain that
is well conserved in peptidase M1 family [25].
Figure 4. The active site of the complex of L-lysine and ePepN.
4. DISCUSSION
By examining the active site of the complex between L-
lysine and ePepN (PDB ID: 3B2X, Fig. 4), we inferred that
our target compounds might interact with APN by three steps
as showed in Fig. (5).
As shown in the results, 2 or 4-substitution of R1 could
improve the inhibitory effect weakly by comparing C1, C2,
C3, C5, C9, C12, C13 to C14 except for C10 and C16,
which demonstrated that steric effect dominated the substitu-
The association of the heterocycle N and the amide group
N in the same plane of R1 could be treated as a potent biden-
tate ZBG, both for C7 or C20, which would increase the
possibility to bind with the zinc ion when interacting with
APN in step 2 as a transition state. As a result, it might in-
tion result. Especially, 4-chloro substitution exhibited better
potency than the others. 3-substitution of R1 would decrease
the inhibitory effect by comparing C8, C11, C17 to C14.

852 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Wang et al.
Figure 5. The selectivity of the active site in APN to compounds C7, C18 and C20.
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In summary, we developed a class of novel L-Lysine
derivatives as potential APN inhibitors. Compounds C7 and
C20 exhibited similar inhibitory activities compared with
bestatin. Therefore, they could be lead compounds for us to
develop new L-lysine derivatives as APN inhibitors.
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ACKNOWLEDGEMENTS
This work was supported by National Natural Foundation
Research Grant(Grant No. 30772654; No 30672541) and Na-
tional High Technology Research and Development Program
of China (863 project; Grant No 2007AA02Z314) and Doc-
toral Foundation of Ministry of Education of the People’s
Republic of China (Grant No. 20060422029) and Shandong
Nature Science Foundation of China (Grant No. Y2004C02).
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