Synthesis and activity evaluation of a new bestatin derivative LYP2 as an aminopeptidase N inhibitor

Article abstract of DOI:10.1097/CAD.0b013e32833ab78a

As a ubiquitous enzyme overexpressed on the epithelium of the tumor, aminopeptidase N (APN) plays important roles in the angiogenesis and metastasis of the tumor. Bestatin as an effective inhibitor against APN is used in the ancillary treatment of various

Full text of DOI:10.1097/CAD.0b013e32833ab78a

Preclinical report 99
Synthesis and activity evaluation of a new bestatin derivative
LYP2 as an aminopeptidase N inhibitor
Yepeng Luan, Qiang Wang, Ning Liu, Jiajia Mou, Xijuan Jiao, Hao Fang,
Minyong Li and Wenfang Xu
As a ubiquitous enzyme overexpressed on the epithelium
of the tumor, aminopeptidase N (APN) plays important
roles in the angiogenesis and metastasis of the tumor.
Bestatin as an effective inhibitor against APN is used in the
ancillary treatment of various cancers. In this study, we
modified the structure of a bestatin derivative LYP reported
in our former study to provide a new bestatin derivative
LYP2 with enhanced stability. We also tested the inhibitive
activity of LYP2, which retained good efficacy in vitro and
in vivo towards APN. Anti-Cancer Drugs 22:99–103 ꢀc 2011
Wolters Kluwer Health | Lippincott Williams & Wilkins.
Anti-Cancer Drugs 2011, 22:99–103
Keywords: aminopeptidase N, A549, bestatin, 2-dimethylaminoethylamine,
ES-2, HL60, H7402, K562, PLC
Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong
University, PR China
Correspondence to Wenfang Xu, Institute of Medicinal Chemistry, School of
Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road,
250012, Ji’nan, Shandong, PR China
Tel/fax: + 86 531 88382589; e-mail: xuwenf@sdu.edu.cn
Received 5 December 2009 Revised form accepted 8 April 2010
Introduction
ancillary drug with other anti-cancer agents because of
its immunostimulant effect. As the only marketed
inhibitor of APN, the pharmacokinetics and biotransfor-
mation of bestatin have been well investigated. To
improve the water solubility of bestatin, we designed
and synthesized a derivative of bestatin (LYP) in our
earlier study [6]. However, LYP showed poor stability
because of the fragile ester bond in its structure. To
overcome this shortcoming, we optimized the structure
of LYP by substituting the ester bond with amide linkage
to give a new bestatin derivative LYP2 whose stability was
well improved. The structures of LYP and LYP2 are
showed in Fig. 1.
Aminopeptidase N (APN), a zinc-dependent metallopro-
tease, belongs to the M1 aminopeptidase family [1]. APN
is a ubiquitous enzyme distributed in many kinds of
organs, tissues and cells with the biological function of
removing free amino acid from the N terminal of various
unsubstituted biologically active peptides. APN pos-
sesses extensive substrates such as extracellular matrix,
enkephalin, etc. Especially on the epithelial of the tu-
mor, APN is overexpressed and plays key roles in the
angiogenesis and metastasis of the tumor [2]. APN can
degrade the extracellular matrix to facilitate metastasis,
which is the lethal feature of the tumor [3]. On the
other hand, neovascularization could supply nutrition and
oxygen for tumor survival. In brief, inhibition of APN
activity has been a promising avenue for the treatment of
cancers.
LYP2 was formed with bestatin and 2-dimethylami-
noethylamine through an amide bond because of which
LYP2 had an absolutely better stability than that of LYP.
Besides, LYP2 also showed good water solubility and
efficacy towards APN in vitro and in vivo, which showed
that LYP2 was a more successful reformation of bestatin
than LYP.
Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-
L-leucine, is a well-known inhibitor of APN [4,5], which
can be used in leukemia therapy directly or act as an
Fig. 1
OH
O
OH
O
H
N
H
N
N
N
N
H
2HCl
O
2HCl
NH2
O
NH2
O
LYP2
LYP
The structure of LYP and LYP2.
ꢀc
0959-4973
2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
DOI: 10.1097/CAD.0b013e32833ab78a
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100 Anti-Cancer Drugs 2011, Vol 22 No 1
Synthesis
metalloproteinase. Thus, the inhibition activity towards
MMP-2 was measured simultaneously to test the
selectivity of LYP2. MMP-2 and TNBS were purchased
from Sigma. The substrate was synthesized as described
by Vijaykumar et al. The gelatinase, substance and
inhibitor were dissolved in sodium borate (pH8.5,
The starting material is bestatin. After the protection of
the primary amine group with di-tert-butyl dicarbonate
(Boc group), coupling and removal of the Boc group, the
targeted compound LYP2 was obtained with a reasonable
overall yield as shown in the following scheme.
OH
O
OH
O
H
N
H
N
a
N
OH
OH
+
H2N
NH
Boc
O
NH2
O
OH
O
OH
O
H
N
H
N
N
N
c
N
H
b
N
H
2HCl
NH2
O
NH
O
Boc
a. (Boc)2O, NaOH; b. HOBt/DCC, dry THF; c. dry HCl in EtOAc
50 mmol/l) and incubated for 30 min at 371C, and then
0.03% TNBS was added and incubated for another
20 min; the resulting solution was detected under 450-
nm wavelength to measure absorption.
Water solubility assay
Solubility limits were estimated as the concentrations
(mmol/l) obtained in saturated solutions. Large amounts
of bestatin and LYP2 were stirred at room temperature for
1 day in 1 ml of PBS. The resulting two saturated
solutions were clarified twice by centrifugation, and the
concentrations were measured spectrophotometrically
after a 200-fold dilution in PBS using the standard curve
of absorbance as a function of concentration according to
the Lambert–Beer law.
Cell assay
The tumor cell lines we used were HL60, ES-2, K562,
A549, H7402 and PLC.
Tumor cells seeded in a 96-well microplate and incubated
at 371C with 10% fetal bovine serum in a humidified
atmosphere of 5% CO2 were exposed to different con-
centration of LYP2 and bestatin 4 h later with a solvent
plate as the blank group. After 48 h, 10 ml 0.5% MTT
was added in each well. The supernatant was separa-
ted by centrifugation (2500 rpm, 20 min) and discarded.
After adding 100 ml DMSO, the supernatant was sub-
jected to fluorometric determination (excitation, 570 nm;
emission, 630 nm). The activity was expressed as the
inhibition rate. Triplicate experiments with triplicate
samples were performed.
Biological activity assay
Enzyme inhibition assay
APN inhibition assay
The IC50 value against APN was determined by using
L-leucine-p-nitroanilide as the substrate and micro-
somal amino-peptidase from Porcine Kidney Microsomes
(Sigma, St. Louis, Missouri, USA) as the enzyme in
50 mmol/l PBS, pH 7.2 at 371C. The hydrolysis of the
substrate was monitored by following the change in
the absorbance measured at 405 nm with a UV–visible
spectrophotometer Pharmacia LKB, Biochrom 4060. All
the solutions of the inhibitors were prepared in the assay
buffer (PBS), and the pH was adjusted to 7.2 by the
addition of 0.1 mol/l HCl or 0.1 mol/l NaOH. All the
inhibitors were preincubated with APN for 30 min at
room temperature. The assay mixture, which contained
the inhibitor solution (six concentrations), substrate,
enzyme solution (4 mg/ml final concentration) and buffer
solution, was finally adjusted to 200 ml.
Anti-metastasis assay in vivo
In vivo, the efficacy of LYP2 was evaluated in H22
xenografts mice that were supplied by the Drug Analysis
Center of Shandong Academy of Sciences.
Each mouse was injected with 0.2 ml suspended solvent
of tumor cell (6 ꢁ 106) under sterile conditions. After 24 h,
the mice were randomly divided into three groups,
including the blank group, bestatin group and LYP2 group.
MMP-2 inhibition assay
Both the compounds were dissolved in 0.5% sodium
carboxymethycellulose solution with a concentration of
2 mg/ml and administrated intragastrically for 12 days.
Our laboratory also devoted efforts to find new inhi-
bitors of MMP-2, which was also a zinc-dependent
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Synthesis and activity evaluation Luan et al. 101
The mice were killed and the metastases on the lungs
were counted. The result was shown through the
inhibition rate:
at the catalytic site. In the three-dimensional structure
of APN, there are three hydrophobic pockets S1, S10, and
S20. S1 and S10 are small pockets, whereas S20 is a bigger
one. The benzene ring and isobutyl group of bestatin can
occupy the S1 and S10 pockets, respectively, leaving the
S20 pocket empty. However, in the structure of LYP2, the
N,N-dimethyl ethanolamine fragment can effectively
occupy the S20 pocket. The docking result is showed
in the Figs 2 and 3 below.
C ꢂ T
inhibition rate ¼
ꢁ 100%
C
C = control group, T = experimental group.
Results
Chemistry
Water solubility
3.08 g (10 mmol) of bestatin was stirred in 10 ml 1 mol/l
NaOH solution under an ice bath. 2.34 g (10.74 mmol) di-
tert-butyl dicarbonate dissolved in 5 ml of THF was then
added into the solution. After 2 h, the ice bath was
removed. The mixture was stirred for another 24 h. After
removing THF under vacuum, the solution was acidified
to pH 3 with saturated citric acid. The solution was
washed with 30 ml acetic ether three times. The organic
layer was collected and dried over anhydrous sodium
sulfate overnight. After evaporating the solvent, the
residue was dried in vacuum to obtain 3.93 g of white
solid (Boc-bestatin) with a yield of 99.1%.
Bestatin has poor water solubility that limits its usage,
whereas LYP2 is a double hydrochloride salt that has better
water solubility. To determine and compare the water
solubility of LYP2 and bestatin, we made standard lines of
absorbance to the concentration of LYP2 and bestatin,
respectively. The results are shown in Table 1 below.
From the table, we can see that the water solubility
of LYP2 was nearly 13-fold more than that of bestatin,
which made LYP2 suitable for a higher number of dosage
forms.
Enzyme inhibition assay
The IC50 (mmol/l) towards APN and MMP-2 is shown in
Table 2.
Three grams (7.35 mmol) of Boc-bestatin was dissolved
in 100 ml dry THF. 1.07 g (7.93 mmol) HOBt was added
into the solution under an ice bath. 1.67 g (8.03 mmol)
DCC dissolved in 50 ml dry THF was added dropwise
into the mixture. The solution was stirred for 8 h at room
temperature. 1.96 g (22 mmol) 2-dimethylaminoethyl-
amine dissolved in 20 ml dry THF were added into the
solution directly. The mixture was stirred for 24 h. After
filtration of DCU and removal of THF, the residue was
dissolved in 150 ml ethyl acetate followed by washing
with saturated sodium bicarbonate solution and brine
twice, respectively. The organic layer was collected and
dried over anhydrous sodium sulfate overnight. Removal
of the solvent followed by purification on column
chromatography by ethyl acetate/methanol (50 : 1) solu-
tion gave a colorless oil (Boc-LYP2) 2.1 g with a yield of
56.7%. 0.48 g (1 mmol) Boc-LYP2 was dissolved in 10 ml
saturated solution of hydrogen chloride in ethyl acetate.
The solution was allowed to stand at 01C overnight. 0.37 g
white solid (LYP2) was obtained after removing the
solvent under vacuum and recrystallization (ethyl ace-
tate and ether) with a yield of 96%. Melting point =
140–1421C. MS-ESI: 190.5 (M/2 + 1), 379.6 (M + 1).
1HNMR (D2O): d 0.72 (d, J = 4.2 Hz, 6H, CH (CH3)2);
d 1.44 (m, 2H, CH2CH(CH3)2); d 1.52 (m, 1H, CH2CH
(CH3)2); d 2.70 (s, 6H, N (CH3)2); d 2.75–2.96 (m, 2H,
NHCH2); d 3.10 (t, 2H, CH2N(CH3)2); d 3.34–3.47 (m,
2H, PhCH2CH); d 3.67 (m, 1H, NHCHC = O); d 4.12
(m, 1H, CH2CHNH2); d 4.14 (m, 1H, OCH); d 7.14–
7.25 (m, 5H, Ph).
Cell assay
APN is overexpressed in various tumor cells playing
important roles in the angiogenesis and metastasis of the
tumor. Accordingly, we used six tumor cell lines to test
the inhibitory activity of LYP2 towards APN. From the
results, LYP2 exhibited good inhibitive activity towards
the six tumor cell lines we had selected. The IC50 of
LYP2 is lower than that of bestatin. The results are shown
in Table 3.
Fig. 2
S2′
S1
S1′
Zn2+
The three-dimensional docking result between aminopeptidase N
(APN) and LYP2. The grey compound is bestatin whereas the other one
is LYP2.
Docking result
The program SYBYL-FlexX was performed here. APN is a
zinc-dependent metalloprotease. The zinc ion is located
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102 Anti-Cancer Drugs 2011, Vol 22 No 1
Fig. 3
Met 263
C
O
N
CA
Glu 264
Tyr 376
CB
CG
O
C
C
Glu 121
CD
O
Lys 319
N
OE1
OE2
N
CA
CA
C11
Glu 320
C10
CB
CB
CG
CD
CG
CD
2.61
NZ
C12
C9
N2
2.74
Glu 298
C7
C8
His 301
CB
CD2
OEI
OE2
O
C1
CE
NE2
C6
C
O2
03
CA
2.73
CG
ND1 CE1
CD1
3.02
Met 260
C2
N
ZN
C3
ZN 900
C49
N1
CE1
C47
LYP2
2.76
N9
OH
Tyr 381
2.37
CZ
C4
CG
N48
CB
NE2
C50
C28
CE2
C13
CD2
CE1
ND1
C5
CD2
04
CA
O
N
C
2.95
C14
CA
C
N
C16
3.27
C
N
O
CG
C15
Gly 261
CB
CA
CB
2.78
CA
O
His 297
OD2
N
C
O
N
CG
OD1
Alp 262
CA
CB
C
Arg 293
O
Asp 327
The two-dimensional view of the binding model between LYP2 and aminopeptidase N (APN).
Table 1 The UV absorption characteristic and saturated solution
concentration of LYP2 and bestatin in PBS
Table 2 IC50 of LYP2 and bestatin
LYP2
Bestatin
LYP2
Bestatin
Molecular weight
451.43
308.37
Function of the line
r2
A = 166.58c–0.0093
0.9997
A = 181.52c–0.005
0.9999
IC50 (APN, mmol/l)
IC50 (MMP-2, mmol/l)
47.4 2.6
> 1000
23.8 1.2
169.9 6.1
Solubility in PBS
479.17 0.98 mmol/l
35.70 0.86 mmol/l
APN, aminopeptidase N.
A, absorbance; c, concentration.
P < 0.01, n = 3.
P < 0.01, n = 3.
The result in vitro showed that LYP2 had a better IC50 as
compared with bestatin towards the six tumor cell lines
we had selected. From the data, we could also see that
LYP2 had a better IC50 towards tumor cells that showed
high expression of APN such as HL60, ES-2, A549 and
PLC compared with those that showed low expression of
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Synthesis and activity evaluation Luan et al. 103
Table 3 IC50 of LYP2 and bestatin (mmol/ml)
trically. The concentration of bestatin and LYP2 was
2 mg/ml, respectively. The result was shown by the
number of nodes in the lung and the inhibition rate as
shown in Figs 4 and 5.
LYP2
Bestatin
HL60
ES-2
K562
A549
H7402
PLC
0.11 0.09
0.19 0.06
1.42 0.22
0.3 0.08
0.62 0.12
0.56 0.16
0.28 0.11
0.87 0.26
NA
NA
NA
NA
LYP2 had a little better anti-metastasis effect than that of
bestatin without severe side effects towards important
organs such as the liver, kidney, spleen, which were all
in good condition after the assembly.
NA, no activity.
P = 0.005, n = 3.
Discussion
In this study, we designed and synthesized a new
derivative of bestatin for enhancing its stability. From
the in vitro and in vivo results, the efficacy of LYP2 is
better than that of bestatin in the parallel assay condition.
Considering that LYP2 is a dihydrochloride salt, its good
hydrophilicity can make it suitable for use in a higher
number of dosage forms. All of these interesting results
showed that LYP2 is a successful optimization of the
structure of bestatin.
Fig. 4
120
100
80
60
40
20
0
Blank
Bestatin
LYP2
Acknowledgements
The graph shows the number of nodes in the lungs of the LYP2 and
bestatin groups.
This study was supported by the National Nature
Science Foundation of China (Grant No. 30772654 and
36072541), the PhD Programs Foundation of Ministry
of Education of PR China (No. 20060422029), 863
National Program Foundation of Ministry of Technology
of China (No. 2007AA02Z314), Nature Science Founda-
tion of Important Research Project of China (Grant
No. 90713041) and New Drug Research and Discovery
Foundation of National Ministry of Technology of China
(No. 2009ZX09103-118).
Fig. 5
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62
61
60
59
58
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Anti-metastasis assay in vivo
In this assay, the Kunming mice transplanted with H22
liver tumor cells were used. The inhibition of metastasis
from the liver to the lungs by bestatin and LYP2 was
tested. Bestatin and LYP2 were administered intragas-
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.