Design, synthesis and preliminary activity evaluation of novel 3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as aminopeptidase N/CD13 inhibitors

Article abstract of DOI:10.3109/14756366.2012.656622

Aminopeptidase N (APN/CD13) over expressed on tumour cells, plays a critical role in tumour invasion, metastasis and tumour angiogenesis. In this article, we described the design, synthesis and preliminary activity studies of novel 3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as APN inhibitors. The in vitro enzymatic inhibitions on APN from porcine kidney showed that compound 7e had the most potent inhibitory activity against APN with the IC50 value to 1.26±0.01 μM, which is better than that of bestatin (IC50=2.55±0.11 μM). In addition, compound 7e also showed better inhibitory activity against APN on human ovary clear cell carcinoma cell ES-2 than bestatin with the IC50 value to 30.19±1.02 μM versus 60.61±0.1 μM. Compound 7e could be used as the lead compound in the future for anti-cancer agent research.

Full text of DOI:10.3109/14756366.2012.656622

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 545–551
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.656622
RESEARCH ARtIClE
Design, synthesis and preliminary activity evaluation of novel
3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as
aminopeptidase N/CD13 inhibitors
Xiaopan Zhang, Lei Zhang, Jian Zhang, Jinhong Feng, Yumei Yuan, Hao Fang, and Wenfang Xu
Department of Medicinal Chemistry, School of Pharmacy, Shan Dong University, Ji’nan, China
Abstract
Aminopeptidase N (APN/CD13) over expressed on tumour cells, plays a critical role in tumour invasion, metastasis
and tumour angiogenesis. In this article, we described the design, synthesis and preliminary activity studies of novel
3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as APN inhibitors. The in vitro enzymatic inhibitions on APN
from porcine kidney showed that compound 7e had the most potent inhibitory activity against APN with the IC
value to 1.26 0.01 μM, which is better than that of bestatin (IC50 =2.55 0.11 μM). In addition, compound 7e als5o0
showed better inhibitory activity against APN on human ovary clear cell carcinoma cell ES-2 than bestatin with the
IC value to 30.19 1.02 μM versus 60.61 0.1 μM. Compound 7e could be used as the lead compound in the future
fo5r0anti-cancer agent research.
Keywords: Aminopeptidase N inhibitors, 3-amino-2-hydroxyl-3-phenylpropanoic acids derivatives, structure-
based drug design
Introduction
olivoreticuli in 197611, and now it is widely used in clinical
Aminopeptidase N (APN; EC 3.4.11.2) is a zinc-dependent
type II membrane-bond ectopeptidase and belongs to the
M1 family of MA clan of peptidase1,2. is enzyme consists
of 967 amino acids with a short N-terminal cytoplasmic
domain, a single transmembrane part and a large cellular
ectodomain containing the active site3,4, which can pref-
erentially release neutral or basic amino acids from the
N-terminal of peptides5,6. APN is widely distributed in the
body of mammalian. It can be expressed on the surface
of different cells such as myeloid progenitors and mono-
cytes, epithelial cells of the intestine and kidney, synaptic
membranes in the central nervous system, fibroblasts,
endothelial cells, epithelial cells7–10 and so on. APN is
highly expressed on tumour cells and plays a critical role in
tumour invasion, metastasis and angiogenesis. erefore,
the design and synthesis of APN inhibitors may be a clini-
cal significance for the discovery of anti-cancer agents.
Bestatin (AHPA-Leu), a well-known APN inhibitor,
was first isolated from the culture filtrate of Streptomyces
practise for the treatment of adult acute non-lymphocytic
leukemia. Since then, many APN inhibitors such as
probestin12, prebestin12, AHPA-Val13 and amatatin14 have
been developed. Judging from the structure of these
compounds, we can see that they all contain the key unit
3-amino-2-hydroxy acyl scaffold, which is important for
their biological activity for that it can chelate the Zn2+ in
the active site of metal-dependent enzymes15.
e interaction of bestatin with the active site of APN
showed that the hydroxyl group and carbonyl group of
bestatin belong to the zinc-binding group (ZBG) to che-
late the Zn2+ in the active site of APN. e phenyl group
can insert into the S1 pocket of APN and the Leu residue
can insert into the S1′ pocket, while the free amino group
can interact with Glu 350. In our previous work, we have
designed a series of novel chloramphenicol amine deriv-
atives as APN inhibitors, but the result was not satisfied,
most compounds showed less activity than bestatin16,
the most active compound 13b is a pseudotripeptide
Address for Correspondence: Wenfang Xu, Department of Medicinal Chemistry, School of Pharmacy, ShanDong University, 44, West Culture
Road, Ji’nan 250012, ShanDong, China. Tel.: +86-531-88382264; Fax: +86-531-88382264; E-mail: wfxu@yahoo.cn
(Received 06 September 2011; revised 25 November 2011; accepted 07 January 2012)
545

546 X. Zhang et al.
(2R, 3S)-3-amino-2-hydroxyl-3-phenylpropanoic acid
hydrochloride 1 was protected by (Boc)2O to get the key
intermediate compound 2. Compound 2 can be coupled
with different methyl ester of amino acids or methyl ester
of dipeptides by classical EDCI/HOBt method to provide
compounds 3a–3o. e esters of compounds 3a–3o were
hydrolysed to carboxylic acid by NaOH/H2O in methanol,
and then converted into the target compounds 5a–5o by
deprotecting the Boc group. Compounds 7a–7h contain-
ing hydroxymate group were obtained from compounds
3a–3h by reacting with NH2OK in methanol and then
cleaving the Boc group.
with the IC50 value to 7.1 μM, which is still less than that
of bestatin (IC50 = 3.00 μM). Recently, our group has syn-
thesised a series of novel L-isoserine derivatives as APN
inhibitors, most of these compounds showed moderate
activities, the most active compound 14b (IC50 = 12.2 μM)
showed still less inhibitory activity toward APN than that
of bestatin (IC50 = 7.3 μM)17,18
.
Taxol, a well-known anti-cancer drug, also has the
3-amino-2-hydroxy acyl scaffold ((2R, 3S)-3-amino-2-
hydroxy-3-phenylpropanoic acid residue). Compared
with L-isoserine, (2R, 3S)-3-amino-2-hydroxy-3-phenyl-
propanoic acid is much more similar with the residue of
bestatin (2S, 3R)-3-amino-2-hydroxy-4-phenylbutyl acid
except for the chirality and one carbon. ere is a phenyl
group in C3 of (2R, 3S)-3-amino-2-hydroxy-3-phenylpro-
panoic acid, which may be beneficial for APN interac-
tion. So, we used bestatin and 14b as the lead compound,
(2R, 3S)-3-amino-2-hydroxy-3-phenylpropanoic acid as
the key scaffold, by coupling with different amino acids
or other functional groups, and we got a series of novel
bi-peptide and tri-peptide derivatives with 3-amino-2-
hydroxy-3-phenylpropanoic acid scaffold . Considering
the best ZBG hydroxamate group may be good for APN
inhibitors, we also converted the carboxyl group of some
compounds into hydroxamate group (Figure 1).
As shown in Scheme 2, compounds 9a–9d were
obtained from compound 4a by coupling it with different
substituted organic aromatic amines and then cleaving
the Boc group.
NH2
O
OH
OH
H
N
N
R1
R2
H
OH
bestatin
O
NH2
O
O
O
O
R1= side chain of amino acid
R2= -OH;
H
N
H2N
N
H
OH
-NHOH;
Results and discussion
OH
O
14b
amino acid;
organic amino
Chemistry
e target compounds were synthesised via the
route outlined in Schemes 1 and 2. e commercial
Figure 1. Novel aminopeptidase N inhibitors with 3-amino-2-
hydroxy-3-phenylpropanoic acid scaffold.
OH
O
OH
O
H
H
N
d
N
OH
c
OH
NH3
Cl
5a-5h
O
R1
OH
O
HN
O
R1
H
Boc
N
O
4a-4h
e
HN
O
R1
b
Boc
OH
OH
O
O
3a-3h
a
OH
H
OH
H
d
N
N
OH
OH
NHOH
NHOH
H3N
Cl
O
R1
HN
O
R1
.HCl
O
HN
O
NH2
Boc
Boc
b
1
2
6a-6h
7a-7h
OH
O
R3
H
N
OH
O
R3
OH
O
R3
H
N
O
H
N
OH
d
c
N
OH
N
H
N
HN
O
R2
O
H
H
HN
O
R2
O
Boc
3i-3o
NH3
Cl
O
R2
5i-5o
O
Boc
4i-4o
Scheme 1. Reagents and conditions: (a) (Boc)2O, NaOH, THF; (b) EDCI, HOBT, DCM, NH2-AA-COOCH3; (c) NaOH, CH3OH; (d) 3N HCl-
EtOAc; (e) NH2OK, CH3OH;
b
a
O
O
O
Boc
O
O
Boc
O
N
N
H2N
N
N
N
.HCl
H
H
H
H
H
OH
HN
OH
HN
OH
4a
OH
R4
R4
8a-8d
9a-9d
Scheme 2. Reagents and conditions: (a) EDCI, HOBT, DCM, NH2R4; (b) 3N HCl-EtOAc
Journal of Enzyme Inhibition and Medicinal Chemistry

Design, synthesis and preliminary activity evaluation 547
between them is that MMP-2 is an endopeptidase, while
APN is an exopeptidase. All the target compounds were
assayed for the inhibitory activities on APN and MMP-2
to observe the selectivity.
As shown in Table 1, the results showed that most com-
pounds had moderate MMP inhibitory activity, which
was consistent with our early report. But the result of APN
inhibition was not satisfied, most compounds did not
show expected APN inhibition, and some compounds
Preliminary in vitro evaluation and SAR discussion
e newly synthesised 3-amino-2-hydroxyl-3-phenyl-
propanoic acid derivatives were assayed for the enzy-
matic inhibitions both on APN from porcine kidney
(Microsomal; Sigma-Aldrich, St. Louis, MO, USA) and
matrix metalloproteinases-2 (MMP-2). e results are
listed in Table 1. Similar to APN, MMP-2 is also a zinc-de-
pendent metalloproteinase, which is involved in the pro-
cess of tumour invasion and metastasis19. e difference
Table 1. e structures and in vitro APN inhibitory activities of the target compounds.
IC50a(μM)
Compound
R1
R2
APN
MMP-2
5a
5b
5c
5d
5e
−CH2CH(CH3)2
−CH(CH3)2
−CH3
−OH
−OH
−OH
−OH
−OH
351.71 0.70
NAb
214.87 0.49
287.42 0.93
273.08 0.25
264.1 0.5
342.42 0.45
NAb
−CH(CH3)CH2CH3
197.57 11.02
254.11 0.25
5f
−OH
−OH
−OH
NAb
NAb
497.15 0.91
407.25 1.89
5g
5h
5i
NAb
561.79 2.55
690.44 1.92
590.39 10.61
5j
512.91 23.91
424.18 1.18
185.90 0.36
5k
NAb
5l
NAb
NAb
(Continued)
© 2013 Informa UK, Ltd.

548 X. Zhang et al.
Table 1. (Continued).
IC50a(μM)
Compound
5m
R1
R2
APN
NAb
MMP-2
NAb
5n
5o
104.53 0.21
NAb
NAb
NAb
7a
7b
7c
7d
7e
−CH2CH(CH3)2
−CH(CH3)2
−CH3
−CH(CH3)CH2CH3
−NHOH
−NHOH
−NHOH
−NHOH
−NHOH
56.71 0.01
76.95 0.43
NAb
82.89 2.67
1.26 0.01
64.60 0.79
128.12 0.14
292.22 2.08
207.11 1.32
212.97 1.35
7f
−NHOH
−NHOH
−NHOH
44.00 2.32
249.42 0.94
298.79 1.48
7g
172.87 1.99
7h
9a
157.64 0.58
NAb
292.35 1.97
NAb
9b
362.76 0.04
NAb
9c
NAb
NAb
NAb
NAb
9d
Bestatin
2.55 0.11
163.48 1.13
aValues are means of three experiments and standard derivation.
bbNA, no activity.
(such as 5b, 5d, 5f and so on) even had no APN inhibitory
activity except for the hydroxamic acid derivatives 7a–7h.
Of all these compounds, compound 7e showed to be the
best APN inhibitor with the IC50 value to 1.26 μM, which
was much better than that of the positive control bestatin
(IC50 =2.55 μM). By analysing the results, we supposed
that these compounds may not have the same interac-
tion model with bestatin by using the hydroxyl group
and carbonyl group to chelate the zinc ion but using the
hydroxamic acid group, this hypothesis could be verified
by the docking results (Figure 2). If the phenyl group of
3-amino-2-hydroxyl-3-phenylpropanoic acid could form
Journal of Enzyme Inhibition and Medicinal Chemistry

Design, synthesis and preliminary activity evaluation 549
hydrophobic interaction with the S1 pocket as bestatin, the
distance of the hydroxyl group and carbonyl group to the
zinc ion may be unsuitable because of the lack of one car-
bon, this may explain why compounds 5a–5o and 9a–9d
had poor APN inhibitory activity. But compounds 7a–7h
showed very good APN inhibitory activity because of the
introduction of the strong ZBG hydroxamic acid group.
Comparing compounds 7a–7h, we can know that
the APN inhibitory activities were different with the
substitutions in R1. Compounds 7a and 7b with the Leu
and Val in R1 had better APN inhibitory activity than
the others (such as 7c, 7d, 7g and 7h), this may suggest
that the Leu and Val in R1 is good for APN inhibition.
Compound 7e with phenylglycine in R1 had the best
APN inhibition with the IC50 value to 1.26 0.01 μM.
Compound 7f with phenylalanine in R1 also had good
APN inhibition but not better than compound 7e with
the IC50 value to 44.00 2.32 μM. Comparing with com-
pounds 7e and 7f, we can know that the length of the
α-carbon atom to the aromatic ring may be important
for APN inhibition. Compound 7g with a hydroxyl
group in the para-position of the phenyl group had less
inhibitory activity than compound 7f, this may suggest
Figure 2. e FlexX docking result of 7e with the active site of
aminopeptidase N (PDB: 2DQM).
Figure 3. e docking result of 7e with aminopeptidase N showed by LIGPLOT.
© 2013 Informa UK, Ltd.

550 X. Zhang et al.
Figure 5. In vitro anti-proliferative activities of compound 7e and
bestatin against ES-2 cells. e bars indicate means standard
deviation (n=3).
Figure 4. Inhibition of aminopeptidase N (APN)/CD13 activity
on ES-2 induced by 7e or bestatin. OVCA cells (1×105) seeded
in 96-well plates were exposed to various concentrations of 7e
or bestatin at 37°C, and the substrate L-leucine-p-nitroanilide
was added. APN/CD13 activity was estimated by measuring
absorbance at 405nm using a microplate reader after 60min of
incubation. e inhibition rate was calculated by comparing to
the control (without drug exposure). e bars indicate means
standard deviation (n=3).
hydrogen bond with His297 and His301 at the distance of
2.70 Å and 2.84 Å.
Although the computed information partially sup-
ported our assumption, the exact binding mode of the
3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives
with APN should be obtained from further X-ray crystal
studies.
that the additional hydroxyl group may be unsuitable
for APN interaction.
APN is highly expressed on the surface of human
ovary clear cell carcinoma cell ES-220; to study the
inhibitory activity of the target compounds toward
human APN, we also tested the in vitro enzymatic
inhibitions of compound 7e toward APN on ES-2.
e results showed that compound 7e is also a dose-
dependent APN inhibitor in the concentration range
of 0.39–400 μM (Figure 4). e IC50 value of compound
7e is 30.19 1.02 μM, which is still better than that of
bestatin (IC50= 60.61 0.1 μM).
Additionally, the effects of compound 7e on the pro-
liferation of human ovary clear cell carcinoma cell ES-2
compared with bestatin were further assessed by using
MTT assay, which were shown in Figure 5. e results
showed that the anti-proliferative effects of compound
7e against ES-2 cell were slightly better than that of
bestatin with the IC50 value of 0.51 0.08 mM versus
0.57 0.04 mM.
Compound 7e showed the most inhibitory activ-
ity toward APN. To investigate the interaction of these
compounds with APN, the docking studies of the rep-
resentative compound 7e were carried out via Sybyl/
Sketch module and optimised using Powell’s method
with the Tripos force field with convergence criterion
set at 0.05 kcal/ (Å mol) and assigned with Gasteiger–
Hückel method. e docking study performed using
Sybyl7.3/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.
e docking result was shown in Figure 2. As shown in
Figure 2, 3-amino-2-hydroxyl-3-phenylpropanoic acid
scaffold of compound 7e inserted into the S1 pocket, and
the hydroxymate group chelated with the zinc ion in the
active site, while the phenyl group of L-phenylglycine
moiety interacted with the S1′ pocket. For a further and
detail understanding of the binding mode of 7e with
APN, a two-dimensional picture was also created with
the program LIGPLOT, which was shown in Figure 3; we
can see that the phenyl group of 3-amino-2-hydroxyl-3-
phenylpropanoic acid could form hydrophobic interac-
tion with the S1 pocket formed by Arg825, Tyr376, Met260 and
Tyr381. e free amino group could form hydrogen bond
with Arg825 at the distance of 2.64 Å. e phenyl group
of L-phenylglycine moiety could form hydrophobic
interaction with the S1′ pocket formed by Arg293, Asp372
and His297. e two oxygen atoms of hydroxamic acid
could chelate with the zinc ion of APN. Additionally,
the hydroxyl group of hydroxamic acid could also form
Conclusion
In summary, we have described the synthesis and prop-
erties of a series of novel (2R,3S)-3-amino-2-hydroxyl-
3-phenylpropanoic acid derivatives as APN inhibitors.
Because of the lack of one carbon as compared with
3-amino-2-hydroxyl-4- phenylbutyl acid, most of the
target compounds did not show expected APN inhibi-
tory activities, but the introduction of hydroxamate
group to the compounds could greatly improve the
inhibitory activities. e most effective compound 7e,
not only had better inhibitory activity against APN
both from porcine kidney and human but also showed
Journal of Enzyme Inhibition and Medicinal Chemistry

Design, synthesis and preliminary activity evaluation 551
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slightly better anti-proliferative effects toward ES-2
cells than that of bestatin. Compound 7e could be used
as a lead compound for further development of small
molecular peptidomimetic APN inhibitors. ough the
preliminary enzymatic results are not very encourag-
ing, it still provides useful clues for further APN inhibi-
tors exploitation.
Supplementary data
Supplementary data (Experimental details, and spectro-
scopic data for all the compounds and procedures for the
activity assays were provided.).
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Declaration of interest
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is work was supported by National High Technology
Research and Development Program of China (863 proj-
ect; Grant No. 2007AA02Z314), National Nature Science
Foundation of China (No. 90713041 and No. 30772654)
and National Science and Technology Major Project
(Grant No. 2009ZX09103-118).
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