Novel aminopeptidase N inhibitors with improved antitumor activities

Article abstract of DOI:10.2174/1570180812666150611190608

A series of aminopeptidase N (APN) inhibitors were designed and synthesized. Enzyme inhibitory, docking and antiproliferative studies were performed to evaluate the derived molecules. Molecule D15, with IC₅₀ values of 10.9 μM, showed the best performance in the APN enzymatic inhibition assay. The binding pattern of molecule D9 and D15 in the active site of APN was predicted by docking studies. Hydrophobic and H-bond interactions were discovered to make key roles in the ligand-receptor bindings. Compared with the previous C7, several molecules such as D9, D14 and D15, exhibited significantly improved activities in inhibiting the growth of HL-60, ES-2, A549 and PLC cell lines.

Full text of DOI:10.2174/1570180812666150611190608

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98
Letters in Drug Design & Discovery, 2016, 13, 98-106
Novel Aminopeptidase N Inhibitors with Improved Antitumor Activities
Qiang Wang1,2,*, Qiao Shi1 and Lu Huang3
1182 Minyuan Road, Hongshan District, Wuhan, Hubei Province, P.R.China, 430074
2Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology
and Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA
3BGI Wuhan Pharmaceutical Co. Ltd, Wuhan, Hubei Province, P.R.China, 430075
Abstract: A series of aminopeptidase N (APN) inhibitors were designed and synthesized. Enzyme in-
hibitory, docking and antiproliferative studies were performed to evaluate the derived molecules.
Molecule D15, with IC50 values of 10.9 μM, showed the best performance in the APN enzymatic inhi-
bition assay. The binding pattern of molecule D9 and D15 in the active site of APN was predicted by
docking studies. Hydrophobic and H-bond interactions were discovered to make key roles in the ligand-receptor bindings.
Compared with the previous C7, several molecules such as D9, D14 and D15, exhibited significantly improved activities
in inhibiting the growth of HL-60, ES-2, A549 and PLC cell lines.
Keywords: Aminopeptidase N, inhibitor, structural modification, L-lysine based, docking.
INTRODUCTION
APN, structural modifications were performed on the lysine
based structures. Hydroxamic acid group was introduced to
improve the binding affinity of designed molecules to the
zinc ion of APN. In order to enhance interactions between
the new compounds and surrounding residues, various aro-
matic groups were introduced to the N2 and N6 (or N5)-
amino group (Fig. 1). Molecular docking studies were per-
formed to predict the binding patterns of synthesized mole-
cules in the active site of APN. Antiproliferative assays
against a group of tumor cells such as HL-60, ES-2, K562,
A549, H7402 and PLC were applied to evaluate the growth
inhibition potency of the derived compounds.
Aminopeptidase N (APN, also known as CD13) is a Zn2+
dependent metalloproteinase widely expressed in the surface
of human renal cells, intestinal brush border cells, synaptic
membranes in central systems, etc [1, 2]. Multiple roles of
APN have been discovered, such as functional enzyme, anti-
gen presentation protein and virus receptor [3]. Notably,
APN overexpressed in tumor cells has been associated with
the proliferation [4], invasion [5, 6] and metastasis [7] of
different human cancers. Moreover, APN also plays impor-
tant role in the promotion of angiogenesis for tumor tissues
[8].
APN inhibitors have been developed for the treatment of
various tumors. The US Food and Drug Administration
(FDA) approved APN inhibitor, Bestatin [9], is capable of
inhibiting the growth of several tumor cells (such as ES-2,
HL-60, A549 and K562 cells), and now is clinically used in
the treatment with acute adult nonlymphocytic leukemia. In
the recent years, a number of APN inhibitors derived from
natural sources or synthetic approaches [10-13] have been
reported to have potent antitumor activities.
CHEMISTRY
If R1 and R2 are the same substituent groups, the syn-
thetic route described in (Scheme 1) will be applied. The α-
COOH groups of L-lysine or L-ornithine were protected by
ester; aromatic substituent groups were then introduced to
the N2 and N6 (or N5)-amino group; target compounds (D1-
D15) were derived by treating the corresponding intermedi-
ates (c1-c15) with NH2OK in methanol.
In our previous work, a series of L-lysine based APN in-
hibitors were synthesized and tested for the APN enzymatic
inhibition and antitumor activities [14]. Molecule C7 (Table
1), with enzymatic inhibition activity of 10.0 μM (compared
with Bestatin of 11.3 μM) and HL-60, PLC cell growth inhi-
bition activities of 1.17 and 0.81 mM (compared with Bes-
tatin of 1.42 mM and 5.42 mM), exhibited the best perform-
ance in the activity assay. In development of more potent
If R1 and R2 are different, the target molecules will be
synthesized via (Scheme 2). In order to selectively protect
the N6 - amino group using Cbz, the α-COOH and N2 -
amino groups of L-lysine were firstly protected by cupric
ion. The R1 groups were introduced to the N2 - amino group,
and R2 groups were coupled to the N6 - amino group after the
methyl esterification of α-COOH group, and N6-
deprotection. Introduction of hydroxamic group by treating
corresponding esters with NH2OK afforded the target com-
pounds (D16-D23). An additional N-deprotection on the R1
group was performed to generate compound D24.
*Address correspondence to this author at the Department of Chemistry,
Center for Diagnostics and Therapeutics, and Center for Biotechnology and
Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA;
Tel: +86-27-67841196; E-mail: 3056010@mail.scuec.edu.cn
1875-628X/16 $58.00+.00
©2016 Bentham Science Publishers

Novel Aminopeptidase N Inhibitors with Improved Antitumor Activities
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1 99
Table 1. Enzymatic inhibition activities of synthesized molecules (IC50, µM) a
O
NHOH
H
N
R1
N
H
R2
n
Compound
D1
n
R1
R2
IC50 (µM)
O
O
4
>100
O
O
D2
D3
D4
D5
4
4
4
4
>100
>100
O
O
Cl
Cl
Cl
O
Cl
Cl
Cl
O
89.6 1.9
23.4 1.2
O
O
O
O
O
O
O
O
D6
D7
4
4
>100
>100
O
O
O
O
O
O
O
O
O
O
D8
4
>100
O
O
O
O
O
O
D9
3
3
11.9 1.3
>100
O2N
Cl
O2N
Cl
D10
O
O
D11
3
>100

100 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1
Table 1. contd…
Wang et al.
Compound
D12
n
R1
R2
IC50 (µM)
O
O
3
>100
>100
O
O
O
O
O
O
D13
3
O
O
O
S
O
O
S
O
D14
D15
3
3
96.6 2.6
10.9 1.3
O
S
O
S
O
O
O
O
D16
D17
4
4
>100
>100
Cl
Cl
O
O
S
O
Cl
Cl
O
O
D18
D19
D20
4
4
4
23.9 1.4
21.2 1.9
20.4 2.1
S
O
Cl
O
S
O
O
S
O
O2N
O
O
S
O
Br
O
O
O
S
O
Cl
D21
D22
D23
4
4
4
48.3 2.5
28.3 3.7
67.8 2.3
O
O2N
Cl
Cl
Cl
O
O
S
O

Novel Aminopeptidase N Inhibitors with Improved Antitumor Activities
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1 101
Table 1. contd…
Compound
D24
n
R1
R2
IC50 (µM)
O
O
4
13.5 0.9
O
NH2
O
N
H
N
O
C7
10.0 1.1
13.7 1.2
N
H
NH2
O
Bestatin
a
Each value is the mean of three experiments.
tions were monitored by TLC with 0.25 mm silica gel plates
(60GF-254). UV light and ferric chloride were used to visu-
alize the spots. ESI-MS was determined on an API 4000
O
NHR
O
NHOH
H
H
N
N
R1
H2N
Cbz
N
H
R2
1
spectrometer. H NMR spectra were recorded on a Bruker
3
3-4
DRX spectrometer at 600 MHz, using TMS as an internal
standard. Melting points were determined uncorrected on an
electrothermal melting point apparatus.
Fig. (1). Design of new APN inhibitors based on the previous C
series (lysine based).
Methyl L-lysinate Dihydrochloride (b, n = 4)
EXPERIMENT SECTION
Materials and Methods
L-lysine (a) (14.60 g, 100 mmol) was dissolved in 150
mL of MeOH, anhydrous HCl gas was added until the solu-
tion changed from clarification to turbidity. Then, the solvent
was evaporated with the desired compound b (17.10 g, 87 %
All commercially available starting materials, reagents
and solvents were used without further purification. All reac-
OH
NH
O
OH
NH2
O
O
O
O
O
2
1
3
H
N
n
H
NH2
n
R1
R1
N
HCl
H2N
H2N
N
H
R2
N
H
R2
n
n
c1-c15
D1-D15
a
b
R1 = R2
n = 3 or 4
o
Scheme (1). Synthetic route of target compound D1-D15. Reagents and conditions: (1) MeOH, HCl, 25 C, 6 h; (2) R1Cl, CH2Cl2, ice-salt
bath, 5 h; (3) NH2OK, MeOH, 25 oC.
H2
H2
O
OH
NH2
N
HN Cbz
CO2
N
NH2
CO2
2
1
Cbz
Cu
4
Cu
4
4
4
CO2
NH
N
H2N
CO2
H2N
N
4
H2
H2
O
O
O
O
OH
O
O
3
4
5
H
N
4
H
H
N
N
R1
HCl
H2N
O
Cbz
H2N
Cbz
N
H
Cbz
4
4
e
OH
NH
f16-24
8
O
O
O
O
6
7
8
9
H
N
H
R1
R1
N
R1
NH2
4
N
H
R2
N
H
R2
N
H
4
4
D24
D16-D23
c16-23
g16-g23
Scheme (2). Synthetic route of target compound D16-D24. Reagents and conditions: (1) CuCO3Cu(OH)2, 1.2 N HCl, H2O, 80-90 oC, 2 h; (2)
o
o
o
NaHCO3, CbzCl, H2O, 25 C, 12 h; (3) EDTA SS, 25 C, 24 h; (4) MeOH, HCl, 25 C,6 h; (5) R1Cl, CH2Cl2, ice-salt bath, 5 h or R1OH,
o
o
o
Et3N, TBTU, CH2Cl2, 25 C, 5 h; (6) H2, Pd/C, 25 C, 24h; (7) R2Cl, CH2Cl2, ice-salt bath, 5 h or R2OH, Et3N, TBTU, CH2Cl2, 25 C, 5 h;
(8) NH2OK, MeOH, 25 oC, 12h; (9) HCl-EtOAc, 25 oC, 8 h.

102 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1
Wang et al.
yield) being recrystallized as white solid by ester. Mp: 200-
203 ºC.
Other compounds (D2 – D15) were prepared in the same
procedure as described above.
Molecule methyl L-ornithinate dihydrochloride (b, n = 3)
were prepared in the same procedure as described above.
White solid, yield 94 %, Mp: 212ºC.
N1-hydroxy-N2,N6-bis(4-methylbenzoyl)-L-lysinamide
(D2)
White solid, 0.26 g, yield 26 %, mp 141-143 ºC. MS (E-
Methyl N2,N6-bis(2-methylbenzoyl)-L-lysinate (c1)
1
SI) m/z [M+H]+ 398.5; H-NMR (600 MHz, DMSO-d6) δ
10.72 (s, 1H), δ 8.88 (s, 1H), δ 8.41 (m, 2H), δ 7.81 (d, J =
7.8 Hz, 2H), δ 7.75 (d, J = 7.8 Hz, 2H), δ 7.27 (d, J = 3.6 Hz,
2H), δ 7.26 (d, J = 3.6 Hz, 2H), δ 4.33 (m, 1H), δ 3.21 (m,
2H), δ 2.36 (d, J = 5.4 Hz, 6H), δ 1.89 (m, 2H), δ 1.38 (m,
2H), δ 1.19 (m, 2H), δ 1.06 (m, 2H)
To a solution of 2-Methylbenzoic acid (1.36 g, 10 mmol)
in DCM (50 mL), Et3N (1.12 g, 11 mmol) and TBTU (3.60 g,
11 mmol) were added in turn. After 20 min, compound b (n =
4, 1.17 g, 5 mmol) and Et3N (0.50 g, 5 mmol) were added.
The reaction solution was stirred at room temperature for 8 h.
Then, the solvent was evaporated with the residue being taken
up in EtOAc (50 mL). the EtOAc solution was washed with
saturated citric acid (3 × 20 mL), NaHCO3 (3 × 20 mL), and
brine (3 × 20 mL), dried over MgSO4, and evaporated under
vacuum. The desired compound c (1.50 g, 76 % yield) was
derived by crystallization in EtOAc/petroleum ether (1/4) as
white powder. ESI-MS: m/z: 397.8 [M+H]+.
N2,N6-bis(2,4-dichlorobenzoyl)-N1-hydroxy-L-lysinamide
(D3)
White solid, 0.18g, yield 18 %, mp 151-154 ºC. MS (E-
1
SI) m/z [M+H]+ 506.4; H-NMR (300 MHz, DMSO-d6) δ
10.78 (s, 1H), δ 8.95 (s, 1H), δ 8.52 (m, 1H), δ 8.33 (s, 1H),
δ 7.67 (s, 2H), δ 7.46 (m, 4H), δ 4.17 (m, 1H), δ 3.18 (m,
2H), δ 1.57 (m, 2H), δ 1.32 (m, 2H), δ 0.89 (m, 2H)
Other compounds (f16 – f24, c2 – c23) were prepared in
the same procedure as described above.
N2,N6-bis(4-chlorobenzoyl)-N1-hydroxy-L-lysinamide
(D4)
Synthesis of intermediate e has been described in our
previous work [14].
White solid, 0.20 g, yield 20 %, mp 137-140 ºC. MS (E-
1
Methyl N2-benzoyl-L-lysinate (g16)
SI) m/z [M+H]+ 438.6; H-NMR (600 MHz, DMSO-d6) δ
10.98 (s, 1H), δ 9.25 (s, 1H), δ 8.46 (d, J = 8.4 Hz, 1H), δ
8.01 (s, 1H), δ 7.50 (d, J = 7.8 Hz, 2H), δ 7.46 (d, J = 7.8 Hz,
2H), δ 7.39 (d, J = 3.0 Hz, 2H), δ 7.37 (d, J = 3.0 Hz, 2H), δ
4.46 (m, 1H), δ 3.33 (m, 2H), δ 1.58 (m, 2H), δ 1.45 (m, 2H)
To a solution of f16 (1.00 g, 2.6 mmol) in MeOH (20
mL), 0.10 g of Pt/C was added. In hydrogen atmosphere, the
reaction solution was stirred at room temperature for 24 h.
Then, the solvent was filtered and evaporated. The residue
(0.64 g, 98 % yield) was used for the next step without fur-
ther purification. ESI-MS: m/z: 251.3 [M+H]+.
N1-hydroxy-N2,N6-bis(4-methoxybenzoyl)-L-lysinamide
(D5)
Compounds (g16 – g23) were prepared in the same pro-
cedure as described above.
White solid, 0.21 g, yield 21 %, mp 147-149 ºC. MS (E-
1
SI) m/z [M+H]+ 430.3; H-NMR (600 MHz, DMSO-d6) δ
Preparation of NH2OK methanol solution: 28.10 g of
KOH was dissolved in 70 mL of methanol and the precipi-
tate was filtered out. Then the solution was added into the
suspension of 23.50 g of hydroxylamine hydrochloride in
120 mL of methanol dropwise. The precipitate was filtered
out and the filtrate was stored at room temperature.
10.61 (s, 1H), δ 9.07 (s, 1H), δ 8.47 (d, J = 8.4 Hz, 1H), δ
7.91 (d, J = 8.4 Hz, 1H), δ 7.54 (m, 2H), δ 7.44 (m, 2H), δ
7.09 (m, 2H), δ 7.01 (m, 2H), δ 3.83 (m, 6H), δ 3.68 (m,
1H), δ 2.40 (m, 2H), δ 1.88 (m, 2H), δ 1.23 (m, 2H), δ 1.09
(m, 2H)
N1-hydroxy-N2,N6-bis(2-methoxybenzoyl)-L-lysinamide
N1-hydroxy-N2,N6-bis(2-methylbenzoyl)-L-lysinamide
(D6)
(D1)
White solid, 0.20 g, yield 20 %, mp 131-134 ºC. MS (E-
1
SI) m/z [M+H]+ 430.5; H-NMR (600 MHz, DMSO-d6) δ
Compound c1 (1.00 g, 1.6 mmol) was dissolved in 28 mL
of NH2OK (1.12 g, 48 mmol) methanol solution. After 2 h,
the solvent was evaporated under vacuum. The residue was
acidified with saturated citric acid, and then extracted with
EtOAc (3 × 20 mL). The organic layers were combined,
washed with brine (3 × 20 mL) and dried over MgSO4. The
crude residue was purified by flash-chromatography eluting
with petroleum ether/EtOAc (4:1 v/v). White solid, 0.18g,
yield 18 %, mp 134-136 ºC. MS (ESI) m/z [M+H]+ 398.8;
1H-NMR (600 MHz, DMSO-d6) δ 10.69 (s, 1H), δ 8.94 (s,
1H), δ 8.44 (d, J = 8.4 Hz, 1H), δ 7.69 (s, 1H), δ 7.30 (m,
8H), δ 4.05 (m, 1H), δ 2.36 (m, 6H), δ 1.98 (m, 2H), δ 1.24
(m, 2H), δ 1.14 (m, 2H), δ 0.90 (m, 2H).
10.70 (s, 1H), δ 8.94 (s, 1H), δ 8.36 (d, J = 8.4 Hz, 1H), δ
7.93 (d, J = 8.4 Hz, 1H), δ 7.61 (m, 2H), δ 7.48 (m, 2H), δ
7.39 (m, 2H), δ 7.32 (m, 2H), δ 4.18 (m, 1H), δ 3.73 (m,
6H), δ 2.39 (m, 2H), δ 1.86 (m, 2H), δ 1.25 (m, 2H), δ 1.08
(m, 2H)
N1-hydroxy-N2,N6-bis(3-methoxybenzoyl)-L-lysinamide
(D7)
White solid, 0.25 g, yield 25 %, mp 124-126 ºC. MS (E-
1
SI) m/z [M+H]+ 430.4; H-NMR (600 MHz, DMSO-d6) δ
10.87 (s, 1H), δ 8.88 (s, 1H), δ 8.20 (d, J = 8.4 Hz, 1H), δ

Novel Aminopeptidase N Inhibitors with Improved Antitumor Activities
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1 103
7.96 (d, J = 8.4 Hz, 1H), δ 7.90 (d, J = 7.8 Hz, 1H), δ 7.70
(d, J = 7.8 Hz, 1H), δ 7.66 (m, 2H), δ 7.56 (m, 2H), δ 7.47
(d, J = 7.2 Hz, 1H), δ 7.42 (t, J = 2.4 Hz, 1H), δ 4.62 (m,
1H), δ 3.90 (m, 6H), δ 2.41 (m, 2H), δ 1.88 (m, 2H), δ 1.24
(m, 2H) δ 1.06 (m, 2H)
1.8 Hz, J2 = 8.7 Hz, 2H), δ 8.11 (dt, J1 = 8.7 Hz, J2 = 15.4
Hz, 2H), δ 7.89 (s, 1H), δ 4.35 (m, 1H), δ 3.17 (m, 2H), δ
1.63 (m, 2H), δ 1.18 (m, 2H)
N1-hydroxy-N2,N5-bis[(4-methylphenyl)sulfonyl]-L-
ornithinamide (D14)
N1-hydroxy-N2,N6-bis(3,4,5-trimethoxybenzoyl)-L-
lysinamide (D8)
Yellowish solid, 0.27 g, yield 27 %, mp 154-156 ºC. MS
(ESI) m/z [M+H]+ 456.4; 1H-NMR (600 MHz, DMSO-d6) δ
10.53 (s, 1H), δ 8.83 (s, 1H), δ 7.91 (d, J = 9.0 Hz, 1H), δ
7.62 (d, J = 6.6 Hz, 4H), δ 7.45 (t, J = 6.0 Hz, 1H), δ 7.38 (d,
J = 7.8 Hz, 2H), δ 7.32 (d, J = 7.8 Hz, 2H), δ 3.47 (dd, J1 =
7.8 Hz, J2 = 7.2 Hz, 1H), δ 2.53 (m, 2H), δ 2.37 (d, J = 12.6
Hz, 6H), δ 1.41 (m, 2H), δ 1.29 (m, 2H)
White solid, 0.24 g, yield 24 %, mp 141-143 ºC. MS (E-
1
SI) m/z [M+H]+ 550.1; H-NMR (600 MHz, DMSO-d6) δ
11.18 (s, 1H), δ 9.04 (s, 1H), δ 8.67 (s, 1H), δ 8.09 (d, J =
8.4 Hz, 1H), δ 7.09 (s, 4H), δ 4.53 (m, 1H), δ 3.76 (m, 18H) ,
δ 2.38 (m, 2H), δ 1.86 (m, 2H), δ 1.21 (m, 2H), δ 1.02 (m,
2H)
N1-hydroxy-N2,N5-bis(phenylsulfonyl)-L-ornithinamide
(D15)
N1-hydroxy-N2,N5-bis(3-chlorobenzoyl)-L-ornithinamide
(D10)
Yellowish solid, 0.19 g, yield 19 %, mp 169-171 ºC. MS
(ESI) m/z [M+H]+ 428.4; 1H-NMR (600 MHz, DMSO-d6) δ
10.55 (s, 1H), δ 8.85 (s, 1H), δ 8.02 (d, J = 8.4 Hz, 1H), δ
7.74 (d, J = 7.2 Hz, 1H), δ 7.57 (m, 10H), δ 3.50 (m, 1H), δ
3.33 (m, 2H), δ 2.56 (m, 2H), δ 1.43 (m, 2H)
White solid, 0.17 g, yield 17 %, mp 149-151 ºC. MS (E-
1
SI) m/z [M+H]+ 424.3; H-NMR (300 MHz, DMSO-d6) δ
10.66 (s, 1H), δ 8.79 (s, 1H), δ 8.42 (d, J = 8.4 Hz, 1H), δ
8.01 (s, 1H), δ 7.88 (dt, J1 = 2.1 Hz, J2 = 16.2 Hz, 2H), δ
7.78 (m, 2H), δ 7.57 (m, 2H), δ 7.46 (dt, J1 = 3.0 Hz, J 2= 7.7
Hz, 2H), δ 4.52 (t, J = 7.5 Hz, 1H), δ 3.45 (m, 2H), δ 1.98
(m, 2H), δ 1.75 (m, 2H)
N2-benzoyl-N6-(2,4-dichlorobenzoyl)-N1-hydroxy-L-
lysinamide (D16)
White solid, 0.14 g, yield 14 %, mp 144-147 ºC. MS
(ESI) m/z [M+H]+: 438.4; 1H-NMR (300 MHz, DMSO-d6) δ
10.73 (s, 1H), δ 10.17 (s, 1H), δ 8.48 (m, 2H), δ 7.88 (d, J =
7.2 Hz, 1H), δ 7.66 (m, 1H), δ 7.53 (m, 1H), δ 7.43 (m, 5H),
δ 7.54 (m, 5H), δ 4.33 (m, 1H), δ 3.20 (m, 2H), δ 1.72 (m,
2H), δ 1.50 (m, 2H), δ 1.39 (m, 2H)
N1-hydroxy-N2,N5-bis(2-methylbenzoyl)-L-ornithinamide
(D11)
White solid, 0.26 g, yield 26 %, mp 148-150 ºC. MS (E-
1
SI) m/z [M+H]+ 384.4; H-NMR (300 MHz, DMSO-d6) δ
10.62 (s, 1H), δ 8.90 (s, 1H), δ 8.51 (d, J = 7.8 Hz, 1H), δ
8.40 (s, 1H), δ 7.33 (m, 8H), δ 4.49 (m, 1H), δ 3.45 (m, 2H),
δ 1.92 (m, 2H), δ 1.72 (m, 2H)
N6-(2,4-dichlorobenzoyl)-N1-hydroxy-N2-(phenylsulfonyl)
-L-lysinamide (D17)
N1-hydroxy-N2,N5-bis(4-methylbenzoyl)-L-ornithinamide
(D12)
White solid, 0.18 g, yield 18 %, mp 162-164 ºC. MS
(ESI) m/z [M+H]+: 474.2; 1H-NMR (600 MHz, DMSO-d6) δ
10.56 (s, 1H), δ 8.82 (s, 1H), δ 8.42 (d, J = 8.4 Hz, 1H), δ
8.01 (d, J = 8.4 Hz, 2H), δ 7.97 (d, J = 8.4 Hz, 1H), δ 7.91 (t,
J = 10.8 Hz, 1H), δ 7.75 (d, J = 7.8 Hz, 1H), δ 7.59 (m, 1H),
δ 7.53 (m, 2H), δ 3.48 (m, 1H), δ 1.37 (m, 2H), δ 1.22 (m,
2H), δ 1.07 (m, 2H), δ 0.96 (m, 2H)
White solid, 0.22 g, yield 22 %, mp 156-158 ºC. MS (E-
1
SI) m/z [M+H]+ 384.5; H-NMR (600 MHz, DMSO-d6) δ
10.69 (s, 1H), δ 8.86 (brs, 1H), δ 8.40 (t, J = 6.4 Hz, 2H), δ
7.80 (d, J = 7.8 Hz, 2H), δ 7.73 (d, J = 7.8 Hz, 2H), δ 7.26
(d, J = 3.6 Hz, 2H), δ 7.24 (d, J = 3.6 Hz, 2H), δ 4.35 (m,
1H), δ 3.25 (dd, J1 = 6.0 Hz, J2 = 7.2 Hz, 2H), δ 2.35 (d, J =
5.4 Hz, 6H), δ 1.74 (m, 2H), δ 1.55 (m, 2H)
N6-{[(4-chlorobenzyl)oxy]carbonyl}-N1-hydroxy-N2-
(phenylsulfonyl)-L-lysinamide (D18)
N1-hydroxy-N2,N5-bis(3,4,5-trimethoxybenzoyl)-L-
ornithinamide (D13)
White solid, 0.23 g, yield 23 %, mp 158-160 ºC. MS
(ESI) m/z [M+H]+ 454.6; 1H-NMR (600 MHz, DMSO-d6) δ
10.57 (s, 1H), δ 8.83 (s, 1H), δ 8.46 (m, 1H), δ 7.90 (d, J =
8.4 Hz, 1H), δ 7.83 (d, J = 8.4 Hz, 2H), δ 7.72 (d, J = 8.4 Hz,
2H), δ 7.54 (t, J = 15.0 Hz, 2H), δ 7.42 (t, J = 15.6 Hz, 2H),
δ 3.79 (m, 1H), δ 2.30 (m, 3H), δ 1.89 (m, 2H), δ 1.44 (m,
2H), δ 1.27 (m, 2H), δ 1.10 (m, 2H)
White solid, 0.31 g, yield 31 %, mp 161-163 ºC. MS (E-
1
SI) m/z [M+H]+ 536.6; H-NMR (600 MHz, DMSO-d6) δ
11.19 (s, 1H), δ 8.16 (s, 1H), δ 7.97 (d, J = 8.4 Hz, 1H), δ
7.68 (d, J = 8.4 Hz, 1H), δ 7.51 (t, J = 7.2 Hz, 1H), δ 7.39
(dt, J1 = 1.2 Hz, J2 = 8.4 Hz, 1H), δ 7.24 (d, J = 4.8 Hz, 2H),
δ 4.53 (m, 1H), δ 3.83 (m, 12H), δ 3.70 (m, 6H), δ 3.31 (m,
2H), δ 2.51 (m, 2H), δ 1.22 (m, 2H)
N1-hydroxy-N6-[(4-nitrophenyl)sulfonyl]-N2-(phenylsulfo
nyl)-L-lysinamide (D19)
N1-hydroxy-N2,N5-bis(4-nitrobenzoyl)-L-ornithinamide
(D9)
White solid, 0.26 g, yield 26 %, mp 170-172 ºC. MS
(ESI) m/z [M+H]+ 487.5; 1H-NMR (600 MHz, DMSO-d6) δ
10.58 (s, 1H), δ 9.18 (s, 1H), δ 8.42 (d, J = 8.4 Hz, 2H), δ
8.01 (dd, J1 = 9.0 Hz, J2 = 8.4 Hz, 2H), δ 7.92 (t, J = 7.2 Hz,
Yellowish solid, 0.14 g, yield 14 %, mp 165-167 ºC. MS
(ESI) m/z [M+H]+ 446.3; 1H-NMR (300 MHz, DMSO-d6) δ
11.82 (s, 1H), δ 9.73 (s, 1H), δ 9.16 (s, 1H), δ 8.23 (dd, J1 =

104 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1
Wang et al.
1H), δ 7.75 (d, J = 7.2 Hz, 2H), δ 7.59 (t, J = 7.2 Hz, 1H), δ
7.54 (t, J = 7.2 Hz, 2H), δ 3.48 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz,
1H), δ 2.62 (m, 2H), δ 1.36 (m, 2H), δ 1.04 (m, 2H)
APN Inhibition Assay
IC50 values against APN (Sigma Inc.) were determined as
described and using L-Leu-p-nitroanilide (Sigma Inc.) as
substrate. All solutions of compounds were prepared in the
assay buffer and preincubated with APN for 30 min at room
temperature. The assay mixture, which contained the inhibi-
tor solution (concentration dependent on the inhibitor), the
enzyme solution (4 μg/mL final concentration), and the assay
N6-(4-bromobenzoyl)-N1-hydroxy-N2-[(4-methylphenyl)
sulfonyl]-L-lysinamide (D20)
White solid, 0.13 g, yield 13 %, mp 147-149 ºC. MS
(ESI) m/z [M+H]+ 498.3; 1H-NMR (600 MHz, DMSO-d6) δ
10.72 (s, 1H), δ 8.89 (s, 1H), δ 8.42 (, H), δ 7.93 (d, J = 7.8
Hz, 1H), δ 7.85 (d, J = 7.8 Hz, 2H), δ 7.74 (d, J = 7.8 Hz,
2H), δ 7.55 (t, J = 15.6 Hz, 2H), δ 7.44 (t, J = 15.6 Hz, 2H),
δ 3.74 (m, 1H), δ 2.34 (m, 3H), δ 1.88 (m, 2H), δ 1.43 (m,
2H), δ 1.25 (m, 2H), δ 1.06 (m, 2H)
o
buffer, was adjusted to 200 μL and incubated at 37 C for 30
min. The hydrolysis of the substrate was monitored by detec-
tion the changes in the absorbance measured at 405 nm with
a UV-Vis spectrophotometer.
Molecular Docking Study
N2-(3-chlorobenzoyl)-N1-hydroxy-N6-[(4-methylphenyl)
sulfonyl]-L-lysinamide (D21)
The docking procedure was performed using Maestro
v7.5 (schrodinger Inc, supported by Shanghai Institute of
Materia Medica Chinese Academy of Sciences). Molecule
D15 sketched by Maestro were ionized and assigned OPLS
2005 force field by LigPrep. The structure of human APN
derived from the protein database (PDB entry: 4FYR) was
was prepared by removal of waters and ligands, assignment
of OPLS 2005 force field using Maestro. The active site was
defined as a cubic box centered by zinc ion by Glide dock,
and the side length of the box is 20 Å. Extra precision was
applied in the docking process, other parameters were set as
default.
White solid, 0.23 g, yield 23 %, mp 141-143 ºC. MS
(ESI) m/z [M+H]+ 453.5; 1H-NMR (600 MHz, DMSO-d6) δ
10.59 (s, 1H), δ 8.82 (s, 1H), δ 8.45 (d, J = 8.4 Hz, 1H) , δ
7.97 (s, 1H), δ 7.89 (m, 1H), δ 7.77 (m, 1H), δ 7.39 (d, J =
7.8 Hz, 2H), δ 7.31 (d, J = 7.8 Hz, 2H), δ 4.22 (m, 1H), δ
3.44 (m, 2H), δ 2.36 (d, J = 12.6 Hz, 6H), δ 2.27 (m, 2H), δ
1.44 (m, 2H), δ 1.18 (m, 2H)
N2-(2,4-dichlorobenzoyl)-N1-hydroxy-N6-(4-nitrobenzoyl)
-L-lysinamide (D22)
White solid, 0.18 g, yield 18 %, mp 131-133 ºC. MS
(ESI) m/z [M+H]+ 483.4; 1H-NMR (600 MHz, DMSO-d6) δ
10.82 (s, 1H), δ 10.11 (s, 1H), δ 8.97 (s, 1H), δ 8.29 (d, J =
8.4 Hz, 1H), δ 7.58 (d, J = 8.4 Hz, 1H), δ 7.31 (t, J = 8.4 Hz,
1H), δ 7.22 (t, J = 8.4 Hz, 1H), δ 7.17 (t, J = 8.4 Hz, 1H), δ
7.14 (d, J = 8.4 Hz, 1H), δ 7.05 (t, J = 7.8 Hz, 1H), δ 6.85 (d,
J = 9.0 Hz, 1H), δ 4.62 (m, 1H), δ 2.94 (m, 2H), δ 2.39 (m,
2H), δ 1.57 (m, 2H), δ 1.14 (m, 2H)
MTT Assay
For example, HL-60 cell was grown in RPMI1640 me-
dium containing 10 % FBS at 37 in 5 % CO2 humidified
incubator. Cell proliferation was determined by the MTT
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 incubation 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.
N2-(3-chlorobenzoyl)-N1-hydroxy-N6-(phenylsulfonyl)-L-
lysinamide (D23)
White solid, 0.19 g, yield 19 %, mp 153-155 ºC. MS
(ESI) m/z M+Na]+ 474.2; 1H-NMR (600 MHz, DMSO-d6) δ
10.61 (s, 1H), δ 9.20 (s, 1H), δ 8.85 (s, 1H), δ 8.40 (m, 1H),
δ 8.02 (d, J = 9.0 Hz, 1H), δ 7.78 (d, J = 8.4 Hz, 2H), δ 7.68
(d, J = 8.4 Hz, 1H), δ 7.61 (t, J = 7.8 Hz, 1H), δ 7.56 (t, J =
7.8 Hz, 1H), δ 7.47 (dd, J1 = 6.0 Hz, J2 = 7.2 Hz, 1H), δ 7.40
(J = 8.4 Hz,), δ 3.52 (m, 1H), δ 3.05 (m, 2H), δ 1.47 (m, 2H),
δ 1.32 (m, 2H), δ 1.56 (m, 2H)
RESULTS AND DISCUSSIONS
Enzyme Inhibitory Assays
In the enzymatic inhibition assay (Table 1), molecule D9
(IC50 = 11.9 1.3 μM) and D15 (IC50 =10.9 1.3 μM) dis-
played improved inhibitory activities compared with the
positive control (Bestatin, IC50 =13.7 1.2 μM). Compound
D24 (IC50 =13.5 0.9 μM) showed nearly equal activities
with Bestatin against the APN enzyme. The sulfonyl substi-
tuted molecules (D14, D15, D18-D21) with mean IC50 value
of 58.0 1.9 μM, exhibited more potent activities than mole-
cules with carbonyl R groups. It suggests that the introduc-
tion of sulfonyl groups can improve the inhibitor-APN inter-
actions. Moreover, the nitro substitution at the phenyl ring of
the R2 groups also makes significant contributions to the
enhancement of the inhibitory activities. Compared with
L-phenylalanyl-N6-[(benzyloxy)carbonyl]-N1-hydroxy-L-
lysinamide hydrochloride (D24)
White solid, 0.62 g, yield 76 %, mp 139-141 ºC. MS
(ESI) m/z [M+H]+ 443.5; 1H-NMR (600 MHz, DMSO-d6) δ
10.72 (s, 1H), δ 8.92 (s, 1H), δ 8.81 (d, J = 8.4 Hz, 1H), δ
8.11 (brs, 2H), δ 7.34 (m, 10H), δ 4.99 (s, 2H), δ 4.16 (dd, J1
= 8.4 Hz, J2 = 14.4 Hz, 1H), δ 3.79 (t, J = 7.2 Hz, 1H), δ 2.95
(m, 2H), δ 1.63 (m, 2H), δ 1.38 (m, 2H), δ 1.20 (m, 2H), δ
0.90 (m, 2H).

Novel Aminopeptidase N Inhibitors with Improved Antitumor Activities
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1 105
D9 and D15 share similar binding patterns in the catalytic
site with hydroxamic acid groups binding to the zinc ion
with a bidentate chelating pattern. Hydrophobic interactions
formed between the aromatic rings of the ligands and sur-
rounded residues play significant role in the ligand-receptor
interactions. There is π-π stacking interaction between the
phenyl ring of Phe472 and R1 of molecule D9 and D15. Hy-
drogen bond (H-bond) interactions also make contributions
to the binding of ligands to the active site of APN. Multiple
H-bond interactions between the sulfonamide groups and
adjacent amino acids (Gly352, Arg381, His388, Glu418 and
Tyr477) were deduced to be the reason of good enzymatic
inhibition activities of sulfonyl substituted molecules. H-
bond interaction formed between the nitro group in the R2
position of D9 and OE2 of Glu380 was considered to the
explanation of the higher potency of nitro substituted mole-
cules. The hydroxamic acid group also strengthens the
ligand-receptor binding by H-bond interactions with His388,
Glh389 and Tyr477.
Fig. (2). Surface (a) and hydrogen bond (b) representation of mole-
cule D9 and D15 in the active site of APN. a: the surface repre-
sented by hydrophobic mode, was colored from blue to brown with
the increment of hydrophobicity; D9 (with white carbon atoms) and
D15 (with green carbon atoms) are represented as ball and stick
model; b: the magenta ball is the zinc ion, and the dotted lines rep-
resent the formed H-bonds.
other molecules, compound D9, D19 and D22 display re-
duced IC50 values of 11.9 1.3 μM, 21.2 1.9 μM and
28.3 3.7 μM, respectively. Introduction of nitro group in the
R1 position also enhances the APN inhibitory activities, such
as D9. Linkers in the R groups might improve the enzyme
inhibitory activities from the SAR of molecule D24. Al-
though several molecules are potent in the inhibition of
APN, the activity improvement of the present molecules than
the previous reported lysine based molecules is not signifi-
cant. The difference between the activities of molecules L-
lysine or L-ornithine structures is also not obvious.
Cell Based Assays
HL-60, ES-2, K562, A549, H7402 and PLC cell lines
was selected for the anti-proliferation assay (Table 2). Mole-
cule C7 and Bestatin were used for the positive control. In
inhibition of HL-60 cell proliferation, molecule D9 (IC50
0.54 0.12 mM), D14 (IC50 = 0.44 0.15 mM), D15 (IC50
=
=
0.62 0.17 mM) and D23 (IC50 = 0.30 0.07 mM) displayed
improved activities than C7 (IC50 = 1.17 0.22 mM) and Bes-
tatin (IC50 = 1.42 0.31 mM). Only molecule D23 (IC50
=
Docking Studies
0.91 0.22 mM) were less potent than Bestatin in the ES-2
cell inhibition assay. The most potent compound D19,
showed ES-2 cell inhibition activity of 0.32 0.11 mM in the
current assay. Molecule D9 (IC50 = 0.17 0.06 mM), D15
(IC50 = 0.32 0.13 mM) and D21 (IC50 = 0.09 0.04 mM)
Molecular docking was performed to predict the binding
pattern of molecule D9 and D15 in the active site of APN.
There is a good spatial matching between the docked struc-
ture of ligands and the surface of APN active site (Fig. 2).
Table 2. Antiproliferative activities of synthesized molecules (IC50, mM) a.
Compound
HL-60
ES-2
K562
A549
H7402
PLC
C20
D9
5.50 0.41 0.84 0.12
0.54 0.12 0.67 0.13
0.44 0.15 0.59 0.18
0.62 0.17 0.58 0.16
3.88 0.37 0.32 0.11
2.11 0.33 0.63 0.14
0.30 0.07 0.91 0.22
NA
NA
NA
NA
NA
NA
NA
0.52 0.11 7.38 0.89 0.29 0.09
0.17 0.06 5.51 0.76 0.80 0.27
0.44 0.13 3.30 0.36 0.57 0.19
0.32 0.13 2.39 0.29 0.58 0.14
0.42 0.15 5.52 0.43 0.15 0.06
0.09 0.04 0.39 0.17 0.46 0.17
0.88 0.21 3.84 0.41 0.57 0.13
D14
D15
D19
D21
D23
D24
Mean
C7
1.22 0.29 0.72 0.26 2.63 0.39 0.39 0.11 0.77 0.22 0.70 0.33
1.83 0.24 0.66 0.17 NA 0.40 0.12 3.64 0.44 0.52 0.17
1.17 0.22 0.98 0.21 0.40 0.17 0.63 0.23 0.98 0.16 0.81 0.11
1.42 0.31 0.87 0.11 0.28 0.09 0.07 0.05 0.63 0.16 5.42 0.91
Bestatin
aEach value is the mean of three experiments.
NA: Not available.
Mean: The mean values of the assayed D series molecules.

106 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 1
Wang et al.
were capable of effectively inhibiting the growth of A549
cells compared with other molecules. Only molecule D21
(IC50 = 0.39 0.17 mM) exhibited smaller IC50 values than
R&D Program in the 12th Five Year Plan of China (No.
2012BAI27B06), Students Innovation Research and Training
Project of South-Central University For Nationalities (No.
KYCX130506Z), Education Research Project of South-
Central University For Nationalities (No. 20110020) and
National Pharmaceutical Experimental Teaching Demonstra-
tion Center of South-Central University For Nationalities.
both C7 (IC50 = 0.98 0.16 mM) and Bestatin (IC50
=
0.63 0.16 mM) in inhibition the growth of H7402 cells.
Compared with C7 and Bestatin, all the investigated D series
molecules showed enhanced activities in inhibition of the
proliferation of PLC cells. Molecule D19 was the most po-
tent inhibitor of PLC cells with IC50 values of 0.15 0.06
mM.
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CONCLUSION
Structural modification was performed in discovery of
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The current work was supported by the Chinese National
Science Fund (No. 21102185), National Key Technology
Received: January 19, 2015
Revised: May 28, 2015
Accepted: June 16, 2015