Novel leucine ureido derivatives as aminopeptidase N inhibitors. Design, synthesis and activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.11.021

Aminopeptidase N (APN/CD13) over-expressed on tumor cells and tumor microenvironment, plays critical roles in tumor invasion, metastasis and angiogenesis. Here we described the design, synthesis and preliminary activity studies of novel leucine ureido derivatives as aminopeptidase N (APN/CD13) inhibitors. The results showed that compound 7a had the most potent inhibitory activity against APN with the IC₅₀ value of 20 nM, which could be used for further anticancer agent research.

Full text of DOI:10.1016/j.ejmech.2015.11.021

European Journal of Medicinal Chemistry 108 (2016) 21e27
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel leucine ureido derivatives as aminopeptidase N inhibitors.
Design, synthesis and activity evaluation
,
Chunhua Ma a b, Jiangying Cao b, Xuewu Liang b, Yongxue Huang b, Ping Wu a, Yingxia Li a,
,
*
Wenfang Xu b, Yingjie Zhang b
a School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminopeptidase N (APN/CD13) over-expressed on tumor cells and tumor microenvironment, plays
critical roles in tumor invasion, metastasis and angiogenesis. Here we described the design, synthesis and
preliminary activity studies of novel leucine ureido derivatives as aminopeptidase N (APN/CD13) in-
hibitors. The results showed that compound 7a had the most potent inhibitory activity against APN with
the IC50 value of 20 nM, which could be used for further anticancer agent research.
© 2015 Elsevier Masson SAS. All rights reserved.
Received 11 July 2015
Received in revised form
11 November 2015
Accepted 14 November 2015
Available online xxx
Keywords:
Aminopeptidase N
Inhibitors
Leucine ureido derivatives
Synthesis
1. Introduction
To date, many natural or synthetic small APN inhibitors have
been reported. Natural APN inhibitors include Bestatin [9], Pro-
Aminopeptidase N (APN, also known as CD13; EC 3.4.11.2) is a
zinc-dependent type II membrane-bond ectopeptidase that pref-
erentially releases neutral and basic amino acids from the N-ter-
minus of oligopeptides. It has been identified as a cell surface
marker for malignant myeloid cells [1] and was over-expressed on
various mammalian tumor cells [2], such as melanoma, prostate,
ovarian, colon, renal and pancreas carcinomas, as well as tumor
microenvironment [3]. APN degrades extracellular matrix (ECM)
which is the main barrier of malignant cell dissemination to pro-
mote tumor invasion and metastasis [4]. More and more researches
indicate that APN is an important angiogenic factor and can serve as
a target for delivering drugs into tumors and inhibiting angiogen-
esis [3,5]. Moreover, APN is a mark and therapeutic target in human
liver cancer stem cells which are responsible for tumor resistance to
chemo/radiation therapy as well as tumor relapse and progression
[6e8]. Accordingly, APN is considered as an important target for
anti-tumor research. Therefore, the design and synthesis of APN
inhibitors may have clinical significance for the discovery of anti-
cancer agents.
bestin [10], AHPA-Val [11], Lapstatin [12], Amastatin [13], etc.
Synthetic small APN inhibitors include -aminoaldehydes [14],
aminophosphiric acids [15], -aminoboronic acids [16], -lysine
derivatives [17], -arginine derivatives [18], cyclic-imide derivatives
[19,20], -dicarbonyl derivatives [21] and so on. However, Bestatin
is the only marketed APN inhibitor. Low inhibitory activity is the
obstruction of APN inhibitors development. So it is necessary to
exploit more active APN inhibitors by rational drug design.
In our previous work, we have reported a series of ureido de-
rivatives [22]. The biological characterization revealed that some
compounds displayed potential inhibitory activity against APN.
a
a-
a
L
L
b
Especially, the IC50 value of compound 4k was 2.7
with 9.1 M of Bestatin. Herein, in order to find better APN in-
hibitors, Compound 4k was used as the lead compound. In our
series of -arginine derivatives, we found that compounds con-
mM compared
m
L
taining disubstituted groups on phenyl had better activities than
those containing unsubstituted or single substitution [18]. The
same tendency exists in our L-lysine derivatives [17]. Based on these
observations, a series of novel leucine ureido derivatives (7ae7k)
containing different disubstitution on the phenyl group of 4k have
been synthesized, meanwhile, some analogues (8ae8g) with
disubstituted benzyl groups replaced the phenyl group of 4k have
also been obtained (Fig. 1). The in vitro inhibitory activity was
* Corresponding author.
E-mail address: zhangyingjie@sdu.edu.cn (Y. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2015.11.021
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

22
C. Ma et al. / European Journal of Medicinal Chemistry 108 (2016) 21e27
Table 1
Structures and IC50 of 7ae7k against APN and HDACs.
Compd
R1
R2
R3
R4
IC50
(
m
M)a APN
IC50(m
M)a HDAC
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
4k
Bestatin
SAHA
Me
Et
iPr
F
Me
Me
F
Cl
OMe
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
Me
Me
F
Me
Et
iPr
F
Me
H
H
H
H
H
0.02 0.01
0.03 0.02
0.05 0.03
0.23 0.14
0.08 0.02
0.18 0.13
0.34 0.19
0.56 0.23
0.46 0.23
0.42 0.12
0.09 0.05
1.15 0.53
3.40 0.03
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Fig. 1. The new APNIs derived from compound 4k (two of R are H).
Cl
measured against APN enzyme. Additionally, the antimetastasis
effect of potent compounds was evaluated in vitro and in vivo assay.
OMe
OMe
Me
H
Cl
H
e
e
H
H
2. Chemistry
e
e
e
e
e
e
0.12 0.03
The target compounds were synthesized efficiently via the route
outlined in Scheme 1. Starting from commercially available L-
a
Mean values and standard deviations of triplicate experiments are given.
leucine, the key intermediate leucine isocyanate was obtained via
esterification and isocyanate, and then coupled with the corre-
sponding disubstituted anilines or benzylamines. Without further
purification, they were directly transformed into hydroxamic acids
as the target products.
Table 2
Structures and IC50 of 8ae8g against APN and HDACs.
3. Result and discussion
The target compounds were evaluated for their inhibitory ac-
tivities toward APN and HDACs by the spectrophotometric method
as described previously [23,24]. Similar to APN, HDACs are zinc-
dependent metalloproteinases as well and associated closely with
the invasion and metastasis of tumor [25,26]. Thereby the assay
was performed on both APN and HDACs so as to identify the
selectivity of our target compounds, all the inhibition results are
summarized in Tables 1 and 2. Bestatin was used as the positive
control for APN inhibitor, while, SAHA was used as the positive
control for HDACs inhibitor.
Compd
R1
R2
R3
R4
IC50
(
m
M)a APN
IC50(m
M)a HDAC
8a
8b
8c
8d
8e
8f
8g
4k
F
Cl
F
OMe
H
H
H
H
H
H
H
H
F
OMe
F
Me
H
F
0.69 0.25
0.58 0.14
0.33 0.21
0.46 0.19
0.42 0.17
0.44 0.16
0.07 0.02
1.15 0.53
3.40 0.03
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
Cl
H
H
H
H
H
e
e
e
F
Me
Me
e
Me
e
e
Bestatin
SAHA
e
e
e
e
e
e
0.12 0.03
a
As shown in Tables 1 and 2, it is worthy to note that these ureido
derivatives displayed dramatic selectivity towards APN over
HDACs. These results, to a certain extent, validated our strategy for
design of potential APNIs. As the above mentioned selectivity
Mean values and standard deviations of triplicate experiments are given.
against APN, the following SARs were mainly discussed about APN
inhibition.
Bioassay results indicate that all compounds exhibited better
inhibitory activity against APN than the leading compound 4k,
some of which had 10-fold or more improvement. It may be due to
the disubstitued groups on the phenyl group can enhance the
interaction with the hydrophobic region of the enzyme. The activity
of analogues with disubstituted anilines (7ae7k) is better than
analogues with disubstituted benzylamines (8ae8g). Comparing
7ae7d and 7fe7k, we could find that the compounds containing
ortho disubstituted phenyl groups have better activities than those
containing other disubstituted phenyl groups. Among compounds
7ae7d with ortho disubstitution, 7ae7c with dialkyl groups on the
benzene ring were more potent than 7d with dihalogens. And
different alkyl groups in aromatic ring also have different levels of
impacts on their activities. The data shown in Table 1 suggested
that the preferred substitutions against APN were, in decreasing
order, methyl-substitution (7a) and ethyl-substitution (7b), fol-
lowed by the isopropyl inhibitor (7c). It seems that methyl is the
most favorable group for APN and bigger bulks lead to impaired
activity. Compound with tri-substitution (7e) on the aromatic ring
Scheme 1. Reagents and conditions: (a) acetyl chloride, MeOH, reflux; (b) triphosgene,
NaHCO3, DCM, ice-bath; (c) corresponding disubstituted anilines or benzylamines,
DCM, room temperature; (d) NH2OK, MeOH, 1 h.

C. Ma et al. / European Journal of Medicinal Chemistry 108 (2016) 21e27
23
was less potent than 7a, which indicated that para-substitution was
detrimental to APN inhibition.
Furthermore, we assessed the ability of compounds 7a, 7b for
enzymatic inhibition on human APN derived from cultured ES-2
human ovarian carcinoma cells because of high APN expression.
than that of Bestatin (75.9%), which indicates that compound 7a
with promising potential in application for antimetastasis therapy
deserves further research and development in the pre-clinical
studies.
The results showed that both compounds 7a (IC50 ¼ 0.12 0.05
and 7b (IC50 ¼ 0.28 0.11 M) were still better than Bestatin
(20.12 1.26 M), which was consistent with the above trends of
m
M)
4. Conclusion
m
m
In all, one series of novel potent leucine ureido derivative as APN
inhibitors have been synthesized and evaluated. The preliminary
results showed that most of the target compounds exhibit better
inhibition than the positive control Bestatin and our lead com-
pound 4k. These results suggest that introduction of disubstitution
on ortho-position of phenyl group of 4k could result in analogs
with improved APN inhibitory activities, such as 7a which could be
used as new lead for further structure optimization in the future
APNIs exploration.
our cell-free enzyme inhibitory activity.
The anti-invasion activities of 7a and 7b were evaluated by
transwell chambers coated with Matrigel. According to the result,
ES-2 cells could freely invade and pass through Matrigel in control
group, while the invasion was significantly obstructed by bestatin,
7a and 7b. Consistent with the result of enzyme inhibitory activity,
7a and 7b were more potent than bestatin at the same concen-
tration of 150 mM (Fig. 2), which indicated their potential thera-
peutic application in overcoming cancer metastasis.
To further examine its in vivo antimetastasis effect, compound
7a was evaluated in the mouse hepatoma-22 (H22) pulmonary
metastasis model. In this test, the numbers of marcroscopic met-
astatic nodes in the lungs were counted to evaluate the antimeta-
stasis effects of tested compounds. As shown in Fig. 3, the inhibitory
rate of compound 7a (91.7%) at the dosage of 80 mg/kg is higher
5. Experiment
5.1. Chemistry: general procedures
All the materials involved were purchased from commercial
suppliers. Solvents were distilled prior to use. All the reactions were
Fig. 2. ES-2 cell invasion inhibition: (a) control; (b) Bestatin, 150
mM; (c) 7a, 150 mM; (d) 7b, 150 mM; (e) The inhibitory rates of compounds 4k, 7a, 7b and Bestatin against ES-2 cell
invasion under the concentration of 150 M. Data expressed are mean values of three independent experiments.
m

24
C. Ma et al. / European Journal of Medicinal Chemistry 108 (2016) 21e27
Fig. 3. The result of in vivo H22 cell metastasis assay.
monitored by thin-layer chromatography on 0.25 mm silica gel
plates (60 GF-254) and visualized with UV light or chloride ferric.
The products were purified by column chromatography which was
performed using 200e300 mesh silica gel. NMR spectra were
respectively.
5.2.1. (S)-2-[3-(2,6-Dimethyl-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (7a)
determined on a Brucker Avance 600 spectrometer,
d in parts per
White powder, yield 49%, mp ¼ 173e175 ꢀC; 1HNMR (600 MHz,
million and J in Hertz. TMS was used as an internal standard. ESI-
MS were determined on an API 4000 spectrometer. Measure-
ments were made in DMSO-d6 solutions. Melting points were
tested using an electrothermal melting point apparatus and were
uncorrected.
DMSO-d6): d10.72 (s, 1H), 8.85 (s, 1H), 7.57 (s, 1H), 7.05e6.98 (m,
3H), 6.25 (s, 1H), 4.17e4.13 (m, 1H), 2.13 (s, 6H), 1.64e1.57 (m, 1H),
1.44e1.35 (m, 2H), 0.90 (d, 3H, J ¼ 6.6 Hz), 0.88 (d, 3H, J ¼ 6.6 Hz),
13C-NMR (150 MHz, DMSO-d6):
d 169.2, 155.0, 135.7, 135.2, 127.5,
125.4, 48.9, 42.6, 24.1, 22.7, 22.0, 18.0; HRMS (AP-ESI): m/z: Calcd
for [C15H23N3O3þH]þ: 294.1812; Found: 294.1816 [MþH]þ.
5.2. General procedure for the synthesis of 7ae7k and 8ae8g
5.2.2. (S)-2-[3-(2,6-Diethyl-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (7b)
To a mixture of L-leucine methyl ester hydrochloride (1.8 g,
10 mmol) in saturated NaHCO3 (36 mL) and DCM (36 mL) was
added triphosgene (0.98 g, 3.3 mmol). The reaction mixture was
vigorously stirred under ice-water bath for 1 h and the organic layer
was collected. The water layer was extracted with DCM for three
times and the organic phases were combined and dried with
MgSO4. After the solvent removed under vacuum, the residue was
dissolved in DCM (20 mL). This solution was then added to corre-
sponding disubstituted anilines or benzylamines (10 mmol) in DCM
(40 mL) under ice bath. The reaction mixture was stirred at room
temperature for 12 h and then the solvent was removed under
reduce pressure. The residue was taken up with ethyl acetate
(100 mL) and washed with 1 N HCl (30 mL) and brine (30 mL). The
organic phase was dried with MgSO4. After the solvent removed,
the residue without purification was directly added to a solution of
potassium hydroxylamine (4.18 g, 28 mmol) in methanol (10 mL).
The reaction mixture was stirred at room temperature for 1 h and
then removed methanol under reduce pressure. The residue was
taken up with 1 N HCl and extracted with ethyl acetate. The organic
phase was washed with brine and dried with MgSO4. After the
solvent removed under reduce pressure, the residue was separated
by silica gel column chromatography to afford 7ae7k and 8ae8g,
White powder, yield 40%, mp ¼ 179e180 ꢀC; 1HNMR (600 MHz,
DMSO-d6): d10.73 (s,1H), 8.87 (s,1H), 7.51 (s,1H), 7.11e7.01 (m, 3H),
6.49e6.25 (m, 1H), 4.18e4.12 (m, 1H), 2.50 (q, 4H, J ¼ 7.8 Hz),
1.65e1.57 (m, 1H), 1.44e1.33 (m, 2H), 1.08 (t, 6H, J ¼ 7.8 Hz), 0.90 (d,
3H, J ¼ 6.6 Hz), 0.88 (d, 3H, J ¼ 6.6 Hz); 13C-NMR (150 MHz, DMSO-
d6):
d 169.3, 155.6, 141.7, 134.4, 126.2, 125.8, 48.8, 42.8, 24.3, 24.1,
22.7, 21.9, 14.5; HRMS (AP-ESI): m/z: Calcd for [C17H27N3O3þH]þ:
322.2125; Found: 322.2122 [MþH]þ.
5.2.3. (S)-2-[3-(2,6-Diisopropyl-phenyl)-ureido]-4-methyl-
pentanoic acid hydroxyamide (7c)
White powder, yield 48%, mp ¼ 177e178 ꢀC; 1HNMR (600 MHz,
DMSO-d6): d10.77 (s, 1H), 8.90 (s, 1H), 7.49 (s, 1H), 7.24e7.18 (m,
1H), 7.14e7.12 (m, 2H), 6.41e6.39 (m, 1H), 4.22e4.19 (m, 1H),
3.14e3.12 (m, 2H), 1.66e1.64 (m, 1H), 1.48e1.44 (m, 2H), 1.14 (d, 6H,
J ¼ 6.0 Hz), 1.12 (d, 6H, J ¼ 6.0 Hz), 0.93 (d, 3H, J ¼ 6.0 Hz), 0.90 (d,
3H, J ¼ 6.0 Hz); 13C-NMR (150 MHz, DMSO-d6):
d 169.6,155.7,146.9,
133.4, 127.3, 123.1, 48.7, 41.2, 24.6, 24.1, 22.8, 21.9, 14.5; HRMS (AP-
ESI): m/z: Calcd for [C19H31N3O3 þH]þ: 350.2438; Found: 350.2441
[MþH]þ.

C. Ma et al. / European Journal of Medicinal Chemistry 108 (2016) 21e27
25
5.2.4. (S)-2-[3-(2,6-Difluoro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (7d)
(AP-ESI): m/z: Calcd for [C15H23N3O5þH]þ 326.1710; Found:
326.1713 [MþH]þ.
White powder, yield 61%, mp ¼ 163e165 ꢀC; 1HNMR (600 MHz,
DMSO-d6):
d10.78 (s, 1H), 8.90 (s, 1H), 7.93 (s, 1H), 7.28e7.18 (m,
5.2.10. (S)-2-[3-(3-Fluoro-4-methoxy-phenyl)-ureido]-4-methyl-
pentanoic acid hydroxyamide (7j)
1H), 7.10 (d, 1H, J ¼ 7.8 Hz), 7.08 (d, 1H, J ¼ 7.8 Hz), 6.57 (d, 1H,
J ¼ 9.0 Hz), 4.18e4.06 (m, 1H), 1.61e1.51 (m, 1H), 1.44e1.36 (m, 2H),
0.90 (d, 3H, J ¼ 6.6 Hz), 0.88 (d, 3H, J ¼ 6.6 Hz); 13C-NMR (150 MHz,
White powder, yield 49%, mp ¼ 159e161 ꢀC; 1HNMR (600 MHz,
DMSO-d6): d10.80 (s, 1H), 8.90 (s, 1H), 8.57 (s, 1H), 7.45 (d, 1H,
DMSO-d6):
d
169.3, 159.0, 157.3, 154.7, 126.7, 116.2, 112.1, 112.0, 49.6,
J ¼ 14.4 Hz), 7.05 (t, 1H, J ¼ 9.0 Hz), 6.95 (d, 1H, J ¼ 8.4 Hz), 6.32 (d,
1H, J ¼ 9.0 Hz), 4.17e4.12 (m, 1H), 3.79 (s, 3H), 1.62e1.58 (m, 1H),
1.44e1.33 (m, 2H), 0.93 (d, 3H, J ¼ 6.6 Hz), 0.90 (d, 3H, J ¼ 6.6 Hz);
43.0, 24.7, 23.1, 22.6; HRMS (AP-ESI): m/z: Calcd for
[C13H17F2N3O3þH]þ 302.1311; Found: 302.1312 [MþH]þ.
13C-NMR (150 MHz, DMSO-d6):
d 174.2, 159.7, 156.3, 146.5, 139.2,
5.2.5. (S)-2-[3-(2,4,6-Trimethyl-phenyl)-ureido]-4-methyl-
pentanoic acid hydroxyamide (7e)
119.6, 118.3, 110.0, 61.4, 54.1, 47.6, 29.4, 27.9, 27.3; HRMS (AP-ESI):
m/z: Calcd for [C14H20FN3O4 þH]þ 314.1511; Found: 314.1513
[MþH]þ.
White powder, yield 40%, mp ¼ 183e175 ꢀC; 1HNMR (600 MHz,
DMSO-d6): d10.72 (s, 1H), 8.86 (s, 1H), 7.45 (s, 1H), 6.83 (s, 2H), 6.21
(m, 1H), 4.18e4.10 (m, 1H), 2.20 (s, 3H), 2.08 (s, 6H), 1.63e1.54 (m,
1H), 1.44e1.31 (m, 2H), 0.89 (d, 3H, J ¼ 6.6 Hz), 0.87 (d, 3H,
5.2.11. (S)-2-[3-(3-Chloro-4-Methyl-phenyl)-ureido]-4-methyl-
pentanoic acid hydroxyamide (7k)
J ¼ 6.6 Hz); 13C-NMR (150 MHz, DMSO-d6):
d
169.2, 155.2, 135.1,
White powder, yield 68%, mp ¼ 156e158 ꢀC; 1HNMR (600 MHz,
134.4, 133.0, 128.1, 48.9, 42.6, 24.1, 22.7, 22.0, 20.3, 17.9; HRMS (AP-
ESI): m/z: Calcd for [C16H25N3O3þH]þ 308.1969; Found: 308.1970
[MþH]þ.
DMSO-d6): d10.82 (s, 1H), 8.91 (s, 1H), 8.66 (s, 1H), 7.65 (s, 1H), 7.20
(d, 1H, J ¼ 7.8 Hz), 7.07 (d, 1H, J ¼ 8.4 Hz), 6.39 (d, 1H, J ¼ 9.0 Hz),
4.16e4.13 (m,1H), 2.25 (s, 3H),1.62e1.57 (m,1H),1.44e1.40 (m, 2H),
0.93 (d, 3H, J ¼ 6.6 Hz), 0.91 (d, 3H, J ¼ 6.6 Hz); 13C-NMR (150 MHz,
5.2.6. (S)-2-[3-(2,4-Dimethyl-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (7f)
DMSO-d6): d 169.4,154.8,139.9,133.5,131.5, 127.9, 117.8,116.6, 49.3,
42.8, 24.7, 23.2, 22.6, 19.2; HRMS (AP-ESI): m/z: Calcd for
White powder, yield 53%, mp ¼ 163e164 ꢀC; 1HNMR (600 MHz,
[C14H20ClN3O3þH]þ 314.1266; Found: 314.1264 [MþH]þ.
DMSO-d6): d10.78 (s, 1H), 8.86 (s, 1H), 7.67 (s, 1H), 7.66 (d, 1H,
J ¼ 7.2 Hz), 6.92 (s, 1H), 6.88 (d, 1H, J ¼ 7.2 Hz), 6.74 (d, 1H,
J ¼ 9.0 Hz), 4.17e4.11 (m, 1H), 2.19 (s, 3H), 2.13 (s, 3H), 1.62e1.53 (m,
1H), 1.42e1.38 (m, 2H), 0.91 (d, 3H, J ¼ 6.6 Hz), 0.88 (d, 3H,
5.2.12. (S)-2-[3-(2,6-Difluoro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8a)
White powder, yield 47%, mp ¼ 161e163 ꢀC; 1HNMR (600 MHz,
J ¼ 6.6 Hz); 13C-NMR (150 MHz, DMSO-d6):
d
168.9, 154.5, 135.2,
DMSO-d6): d10.70 (s, 1H), 8.81 (s, 1H), 7.41e7.38 (m, 1H), 7.10 (t, 2H,
130.4, 126.4, 126.3, 120.2, 48.8, 42.3, 24.1, 22.6, 22.0, 20.1, 17.6;
HRMS (AP-ESI): m/z: Calcd for [C15H23N3O3þH]þ 294.1812; Found:
294.1811[MþH]þ.
J ¼ 7.8 Hz), 6.40e6.38 (m, 1H), 6.10 (d, 1H, J ¼ 9.0 Hz), 4.37e4.23 (m,
2H), 4.08e4.04 (m, 1H), 1.54e1.50 (m, 1H), 1.37e1.28 (m, 2H), 0.89
(d, 3H, J ¼ 6.6 Hz), 0.86 (d, 3H, J ¼ 6.6 Hz); 13C-NMR (150 MHz,
DMSO-d6):
d 169.7, 157.2, 130.1, 112.0, 111.9, 110.0, 49.4, 42.9, 31.4,
5.2.7. (S)-2-[3-(2,4-Difluoro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (7g)
24.6, 23.1, 22.6; HRMS (AP-ESI): m/z: Calcd for [C14H19F2N3O3þH]þ
316.1467; Found: 316.1472 [MþH]þ.
White powder, yield 70%, mp ¼ 165e166 ꢀC; 1HNMR (600 MHz,
DMSO-d6):
d10.79 (s, 1H), 8.87 (s, 1H), 8.38 (s, 1H), 8.07e8.05 (m,
5.2.13. (S)-2-[3-(2,6-Dichloro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8b)
1H),7.25e7.23 (m, 1H), 6.98 (m, 1H), 6.87 (d, 1H, J ¼ 8.4 Hz),
4.15e4.12 (m, 1H), 1.58e1.56 (m, 1H), 1.44e1.33 (m, 2H), 0.91 (d, 3H,
J ¼ 6.6 Hz), 0.88 (d, 3H, J ¼ 6.6 Hz); 13C-NMR (150 MHz, DMSO-d6):
White powder, yield 48%, mp ¼ 175e176 ꢀC; 1HNMR (600 MHz,
DMSO-d6):
d
10.70 (s,1H), 8.81 (s,1H), 7.48 (d, 2H, J ¼ 7.8 Hz), 7.34 (t,
d
169.3, 156.7, 154.7, 151.8, 125.2, 121.4, 111.3, 104.0, 49.4, 42.7, 24.7,
1H, J ¼ 7.8 Hz), 6.26 (t, 1H, J ¼ 4.8 Hz), 6.11 (d, 1H, J ¼ 9.0 Hz),
4.50e4.40 (m, 2H), 4.09e4.03 (m, 1H), 1.53e1.48 (m, 1H), 1.37e1.28
(m, 2H), 0.87 (d, 3H, J ¼ 6.6 Hz), 0.85 (d, 3H, J ¼ 6.6 Hz); 13C-NMR
23.1, 22.5; HRMS (AP-ESI): m/z: Calcd for [C13H17F2N3O3þH]þ
302.1311; Found: 302.1312 [MþH]þ.
(150 MHz, DMSO-d6):
d 169.1, 156.6, 135.1, 134.5, 129.9, 128.5, 48.9,
5.2.8. (S)-2-[3-(2,4-Dichloro-phenyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (7h)
42.3, 39.9, 24.0, 22.5, 22.1; HRMS (AP-ESI): m/z: Calcd for
[C14H19Cl2N3O3þH]þ 348.0876; Found:348.0876 [MþH]þ.
White powder, yield 45%, mp ¼ 165e167 ꢀC; 1HNMR (600 MHz,
DMSO-d6):
d
10.77 (s, 1H), 8.85 (s, 1H), 8.23 (s, 1H), 8.16 (d, 1H,
5.2.14. (S)-2-[3-(2,4-Difluoro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8c)
J ¼ 8.4 Hz), 7.52 (s, 1H), 7.37 (d, 1H, J ¼ 8.4 Hz), 7.28 (d, 1H,
J ¼ 8.4 Hz), 4.13e4.12 (m, 1H), 1.56e1.51 (m, 1H), 1.40e1.37 (m, 2H),
0.88 (d, 3H, J ¼ 6.6 Hz), 0.85 (d, 3H, J ¼ 6.6 Hz); 13C-NMR (150 MHz,
White powder, yield 60%, mp ¼ 172e173 ꢀC; 1HNMR (600 MHz,
DMSO-d6): d10.70 (s, 1H), 8.83 (s, 1H), 7.37e7.32 (m, 1H), 7.25e7.20
DMSO-d6):
d
169.2, 154.4, 136.3, 128.9, 127.9, 125.5, 122.0, 121.7,
(m, 1H), 7.10e7.06 (m, 1H), 6.46 (t, 1H, J ¼ 6.0 Hz), 6.20 (d, 1H,
J ¼ 8.4 Hz), 4.23e4.21 (m, 2H), 4.10e4.09 (m, 1H),1.57e1.52 (m,1H),
1.40e1.33 (m, 2H), 0.90 (d, 3H, J ¼ 6.0 Hz), 0.87 (d, 3H, J ¼ 6.0 Hz);
49.5, 42.6, 24.7, 23.1, 22.5; HRMS (AP-ESI): m/z: Calcd for
[C13H17Cl2N3O3þH]þ 334.0722; Found: 334.0724 [MþH]þ.
13C-NMR (150 MHz, DMSO-d6):
d 169.2, 161.1, 159.7, 157.0, 130.2,
5.2.9. (S)-2-[3-(2,4-Dimethoxy-phenyl)-ureido]-4-methyl-
pentanoic acid hydroxyamide (7i)
123.6, 111.0, 103.4, 48.9, 42.3, 36.1, 24.2, 22.6, 22.0; HRMS (AP-ESI):
m/z: Calcd for [C14H19F2N3O3þH]þ 316.1467; Found: 316.1470
[MþH]þ.
White powder, yield 82%, mp ¼ 178e180 ꢀC; 1HNMR (600 MHz,
DMSO-d6): d10.73 (s, 1H), 8.81 (s, 1H), 7.87e7.84 (m, 2H), 6.95 (d,
1H, J ¼ 8.4 Hz), 6.55 (d, 1H, J ¼ 1.8 Hz), 6.41 (dd, 1H, J ¼ 8.4, 1.8 Hz),
4.11e4.09 (m, 1H), 3.80 (s, 3H), 3.70 (s, 3H), 1.60e1.52 (m, 1H),
1.44e1.33 (m, 2H), 0.90 (d, 3H, J ¼ 6.6 Hz), 0.87 (d, 3H, J ¼ 6.6 Hz);
5.2.15. (S)-2-[3-(2,4-Dimethoxy-benzyl)-ureido]-4-methyl-
pentanoic acid hydroxyamide (8d)
White powder, yield 66%, mp ¼ 175e177 ꢀC; 1HNMR (600 MHz,
13C-NMR (150 MHz, DMSO-d6):
d
169.7, 155.2, 154.7, 149.1, 123.1,
DMSO-d6):
d10.66 (s,1H), 8.78 (s,1H), 7.04 (d,1H, J ¼ 7.8 Hz), 6.53 (s,
119.5, 104.4, 99.1, 56.1, 55.6, 49.4, 42.6, 24.7, 23.2, 22.5; HRMS
1H), 6.45 (d, 1H, J ¼ 8.4 Hz), 6.16 (t, 1H, J ¼ 6.0 Hz), 6.13 (d, 1H,

26
C. Ma et al. / European Journal of Medicinal Chemistry 108 (2016) 21e27
J ¼ 9.6 Hz), 4.08e4.02 (m, 3H), 3.78 (s, 3H), 3.73 (s, 3H), 1.55e1.50
(m, 1H), 1.34e1.30 (m, 2H), 0.87 (d, 3H, J ¼ 6.6 Hz), 0.85 (d, 3H,
for another 30 min at 37 ꢀC. The hydrolysis of the substrate was
measured by following the change in the absorbance monitored at
405 nm with a plate reader (Varioskan, Thermo, USA).
J ¼ 6.6 Hz); 13C-NMR (150 MHz, DMSO-d6):
d 169.4, 159.4, 157.5,
157.1, 128.6, 120.1, 104.0, 98.0, 55.2, 55.0, 48.8, 42.3, 37.7, 24.0, 22.6,
22.0; HRMS (AP-ESI): m/z: Calcd for [C16H25N3O5 þH]þ 340.1794;
Found: 340.1768[MþH]þ.
5.3.3. In vitro HDACs inhibition assay
In vitro HDACs inhibitory activity assay was determined by using
Boc-Lys (acetyl)-AMC as substrate and Hela nuclear extract (mainly
containing HDAC1 and HDAC2) as enzymes in 15 mM TriseHCl (PH
5.2.16. (S)-2-[3-(3,4-Difluoro-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8e)
8.0) at 37 ꢀC. First, 10
m
L of enzymes solution was added to tested
compounds solutions at various concentrations (50 L) and incu-
bated for 5 min at 37 ꢀC. Then 40
L of substrate was added and the
mixture continued to incubate for another 30 min in the same
environment. Finally, 100 L of developer which containing trypsin
White powder, yield 61%, mp ¼ 173e174 ꢀC; 1HNMR (600 MHz,
m
DMSO-d6): d10.67 (s, 1H), 8.80 (s, 1H), 7.40e7.34 (m, 1H), 7.26e7.22
m
(m, 1H), 7.07 (s, 1H), 6.48 (t, 1H, J ¼ 6.6 Hz), 6.20 (d, 1H, J ¼ 9.0 Hz),
4.20e4.18 (m, 2H), 4.10e4.05 (m, 1H), 1.56e1.51 (m, 1H), 1.37e1.33
(m, 2H), 0.88 (d, 3H, J ¼ 6.6 Hz), 0.85 (d, 3H, J ¼ 6.6 Hz); 13C-NMR
m
and TSA was putted into the mixture. Twenty minutes later, fluo-
rescence intensity was measured at 390 nm excitation and 460 nm
emission wavelengths with a microplate reader.
(150 MHz, DMSO-d6): d 169.8, 157.8, 149.9, 148.4, 139.3, 124.0, 117.7,
116.2, 49.6, 42.9, 42.6, 24.6, 23.2, 22.6; HRMS (AP-ESI): m/z: Calcd
for [C14H19F2N3O3 þH]þ 316.1467; Found: 316.1471[MþH]þ.
5.2.17. (S)-2-(3-(3,4-Dimethylbenzyl)ureido)-N-hydroxy-4-
methylpentanamide (8f)
5.3.4. Enzyme inhibitory assay towards APN from ES-2 cells
IC50 values against APN were determined by using L-Leu-p-
nitroanilide as substrate and ES-2 human ovarian clear cell carci-
noma cells as the enzyme source. In brief, the assay was performed
in 96-well plates in 10 mM PBS, pH 7.4 as the assay buffer, at 37 ꢀC.
All solutions of inhibitors were prepared in the assay buffer with
0.2% DMSO in final concentration as the fluxing agent. Compounds
were pre-incubated with ES-2 cell suspension (1 ꢁ 105 cells per
well) for 5 min at room temperature. The assay mixture, which
White powder, yield 57%, mp ¼ 170e171 ꢀC; 1HNMR (600 MHz,
DMSO-d6):
d
10.68 (s,1H), 8.80 (s,1H), 7.05 (d,1H, J ¼ 7.8 Hz), 6.98 (s,
1H), 6.93 (d, 1H, J ¼ 7.8 Hz), 6.32 (t, 1H, J ¼ 6.0 Hz), 6.09 (d, 1H,
J ¼ 9.0 Hz), 4.12e4.06 (m, 3H), 2.18 (s, 3H), 2.17 (s, 3H),1.57e1.51 (m,
1H), 1.34 (t, 2H, J ¼ 7.2 Hz), 0.88 (d, 3H, J ¼ 6.6 Hz), 0.86 (d, 3H,
J ¼ 6.6 Hz); 13C-NMR (150 MHz, DMSO-d6):
d 169.3, 157.2, 137.7,
135.7, 134.1, 129.1, 128.1, 124.3, 48.9, 42.4, 39.9, 24.1, 22.6, 22.0, 19.2,
18.8; HRMS (AP-ESI): m/z: Calcd for [C16H25N3O3 þNa]þ 330.1788;
Found: 330.1780[MþNa]þ.
contained 20
on the inhibitors), 70
substrate solution, or the assay buffer, was adjusted to 100
m
L of the inhibitor solution (concentration dependent
L of the ES-2 cell suspension, 10 L of the
L, and
m
m
m
5.2.18. (S)-2-[3-(2,3-Dimethyl-benzyl)-ureido]-4-methyl-pentanoic
acid hydroxyamide (8g)
then incubated at 37 ꢀC for 1 h. The hydrolysis of the substrate in
the supernatant liquor after centrifugation was monitored with the
UVeVis spectrophotometer Pharmacia LKB, Biochrom 4060. The
enzyme activity inhibitory rate was calculated from the photo-
metric intensity readings, and the IC50 was determined through a
regression analysis of the concentration/inhibitory rate data.
White powder, yield 51%, mp ¼ 166e167 ꢀC; 1HNMR (600 MHz,
DMSO-d6):
d
10.66 (s, 1H), 8.77 (s, 1H), 7.37 (t, 1H, J ¼ 7.2 Hz), 7.08 (d,
1H, J ¼ 7.2 Hz), 7.07 (d, 1H, J ¼ 7.2 Hz), 7.07 (d, 1H, J ¼ 7.2 Hz), 6.36 (t,
1H, J ¼ 6.0 Hz), 6.06 (d, 1H, J ¼ 9.0 Hz), 4.21e4.17 (m, 2H), 4.10e4.05
(m, 1H), 2.23 (s, 3H), 2.13 (s, 3H),1.56e1.51 (m, 1H), 1.35e1.30 (m,
2H), 0.88 (d, 3H, J ¼ 6.6 Hz), 0.85 (d, 3H, J ¼ 6.6 Hz); 13C-NMR
5.3.5. Anti-invasion assay
(150 MHz, DMSO-d6): d 169.9, 157.8, 138.3, 136.2, 134.7, 129.7, 128.7,
The BD BioCoat™ Matrigel™ Invasion Chambers were rehy-
124.8, 49.5, 43.0, 42.9, 24.7, 23.2, 22.6,19.9, 19.4; HRMS (AP-ESI): m/
drated with 500
of the upper and lower chambers for 2 h. After the medium was
removed, 750 L of RPMI-1640 culture medium with 10% FBS were
added to the lower chambers. Target compounds in 100 L of RPMI-
1640 culture medium with 1% FBS were added to the upper
chamber at the same time. Cells in 400 L of RPMI-1640 culture
mL RPMI-1640 culture medium with 1% FBS in both
z: Calcd for [C16H25N3O3þH]þ 308.1969; Found: 308.1968 [MþH]þ.
m
5.3. Biological materials and methods
m
5.3.1. Materials
m
5.3.1.1. Chemicals. Chemicals was synthesized as mentioned above
and dissolved in dimethylsulfoxide (DMSO) or PBS and diluted to
the required concentration with culture medium when used.
medium with 1% FBS (1 ꢁ 105 cells per well) were added and
allowed to invade for 8 h at 37 ꢀC in a CO2 incubator. 8 h later,
Matrigel and cells in the upper chamber were removed with a
cotton swab. The remaining cells were fixed, stained with 0.1%
crystal violet, and photographs were taken under a microscope. ES-
2 invasion was quantified by counting the number of cells in five
random fields (ꢁ100) per insert.
5.3.1.2. Cell line and cell culture. The human leukemia cell line, ES-2
and HL-60 were obtained from the Institute of National Cancer
Research of China (Beijing, China). Cells were maintained in RPMI-
1640 supplemented with 10% (v/v) heat-inactivated fetal bovine
serum, penicillinestreptomycin (100 IU/mle100 mg/mL), 2 mM
glutamine, and 10 mM Hepes buffer at 37 ꢀC in a humid atmosphere
(5% CO2e95% air).
5.3.6. Anti-metastasis assay in vivo
Mice bearing H22 tumor were injected via the caudal vein and
randomly divided into 3 groups. The animals of the control group
were treated with the same volume of excipient, while the other
groups were given the inhibitors (7a and bestatin) by oral admin-
istration, at a dose of 80 mg/kg/day, 6 days/week for 2 weeks. The
mice were then weighed and sacrificed for autopsy immediately.
The lungs with tumor nodes were removed, weighed, and then
placed in bouin stationary solution (saturated 2,4,6-trinitrophenol
solution/formaldehyde/glacial acetic acid ¼ 15:5:1). One day later,
the metastasized nodes on the surface of lungs were counted.
5.3.2. In vitro APN enzyme assay
IC50 values against APN were determined by using L-leu-p-
nitroanilide as substrate and Microsomal aminopeptidase from
Porcine Kidney Microsomes (Sigma) as enzyme in 50 mM PBS (PH
7.2) or suspension of ES-2 cells in PBS (1 ꢁ 105/well). After adding
the detected compounds, the solution with various concentrations
was incubated with APN at 37 ꢀC for 5 min. Then the solution of
substrate was added into the above mixture, which was incubated

C. Ma et al. / European Journal of Medicinal Chemistry 108 (2016) 21e27
27
analogue from Streptomyces neyagawaensis SL-387, Biosci. Biotechnol. Bio-
Acknowledgments
chem. 60 (1996) 898e900.
ꢀ
[12] B.R. Lampret, J. Kidric, B. Kralj, L. Vitale, M. Pokorny, M. Renko, Lapstatin, a
This work was supported by National High-Tech R&D Program
of China (863 Program) (Grant No. 2014AA020523), National Nat-
ural Science Foundation of China (Grant Nos. 21302111, 81373282,
21172134).
new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits
autogenous aminopeptidases, Arch. Microbiol. 171 (1999) 397e404.
[13] D.H. Rich, B.J. Moon, S. Harbeson, Inhibition of aminopeptidases by amastatin
and bestatin derivatives. Effect of inhibitor structure on slow-binding pro-
cesses, J. Med. Chem. 27 (1984) 417e422.
[14] L. Andersson, T.C. Isley, R. Wolfenden, alpha-Aminoaldehydes: transition state
analog inhibitors of leucine aminopeptidase, Biochemistry 21 (1982)
4177e4180.
Appendix A. Supplementary data
[15] R. Grzywa, J. Oleksyszyn, First synthesis of
a
-aminoalkyl-(N-substituted)
(APN/CD13)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.11.021.
thiocarbamoyl-phosphinates: inhibitors of aminopeptidase
with the new zinc-binding group, Bioorg. Med. Chem. Lett. 18 (2008)
3734e3736.
N
[16] A.B. Shenvi, alpha.-Aminoboronic acid derivatives: effective inhibitors of
aminopeptidases, Biochemistry 25 (1986) 1286e1291.
References
[17] Q. Wang, M. Chen, H. Zhu, J. Zhang, H. Fang, B. Wang, W. Xu, Design, synthesis,
and QSAR studies of novel lysine derives as amino-peptidase N/CD13 in-
hibitors, Bioorg. Med. Chem. 16 (2008) 5473e5481.
[1] R.A. Ashmun, A.T. Look, Metalloprotease activity of CD13/aminopeptidase N
on the surface of human myeloid cells, Blood 75 (1990) 462e469.
[2] A.A. Amoscato, R.M. Sramkoski, G.F. Babcock, J.W. Alexander, Neutral surface
aminopeptidase activity of human tumor cell lines, Biochim. Biophys. Acta
(BBA) Protein Struct. Mol. Enzym. 1041 (1990) 317e319.
[3] L. Guzman-Rojas, R. Rangel, A. Salameh, J.K. Edwards, E. Dondossola, Y.-G. Kim,
A. Saghatelian, R.J. Giordano, M.G. Kolonin, F.I. Staquicini, Cooperative effects
of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells
within the tumor microenvironment, Proc. Natl. Acad. Sci. 109 (2012)
1637e1642.
[18] J. Mou, H. Fang, F. Jing, Q. Wang, Y. Liu, H. Zhu, L. Shang, X. Wang, W. Xu,
Design, synthesis and primary activity evaluation of L-arginine derivatives as
amino-peptidase N/CD13 inhibitors, Bioorg. Med. Chem. 17 (2009)
4666e4673.
[19] Q. Li, H. Fang, X. Wang, L. Hu, W. Xu, Novel cyclic-imide peptidomimetics as
aminopeptidase N inhibitors. Design, chemistry and activity evaluation. Part I,
Eur. J. Med. Chem. 44 (2009) 4819e4825.
[20] Q. Li, H. Fang, X. Wang, W. Xu, Novel cyclic-imide peptidomimetics as
aminopeptidase N inhibitors. Structure-based design, chemistry and activity
evaluation. II, Eur. J. Med. Chem. 45 (2010) 1618e1626.
[4] C. Antczak, I. De Meester, B. Bauvois, Transmembrane proteases as disease
markers and targets for therapy, J. Biol. Regul. Homeost. Agents 15 (2000)
130e139.
[21] C. Ma, X. Li, K. Jin, J. Cao, W. Xu, Novel b-dicarbonyl derivatives as inhibitors of
aminopeptidase N (APN), Bioorg. Med. Chem. Lett. 23 (2013) 4948e4952.
[22] L. Su, J. Cao, Y. Jia, X. Zhang, H. Fang, W. Xu, Development of synthetic
aminopeptidase N/CD13 inhibitors to overcome cancer metastasis and
angiogenesis, ACS Med. Chem. Lett. 3 (2012) 959e964.
[5] R. Pasqualini, E. Koivunen, R. Kain, J. Lahdenranta, M. Sakamoto, A. Stryhn,
R.A. Ashmun, L.H. Shapiro, W. Arap, E. Ruoslahti, Aminopeptidase N is a re-
ceptor for tumor-homing peptides and a target for inhibiting angiogenesis,
Cancer Res. 60 (2000) 722e727.
[6] N. Haraguchi, H. Ishii, K. Mimori, F. Tanaka, M. Ohkuma, H.M. Kim, H. Akita,
D. Takiuchi, H. Hatano, H. Nagano, CD13 is a therapeutic target in human liver
cancer stem cells, J. Clin. Investig. 120 (2010) 3326.
[7] I. Martin-Padura, P. Marighetti, A. Agliano, F. Colombo, L. Larzabal, M. Redrado,
A.-M. Bleau, C. Prior, F. Bertolini, A. Calvo, Residual dormant cancer stem-cell
foci are responsible for tumor relapse after antiangiogenic metronomic
therapy in hepatocellular carcinoma xenografts, Lab. Investig. 92 (2012)
952e966.
[23] M. Dra˛g, J. Grembecka, M. Pawełczak, P. Kafarski, a-Aminoalkylphosphonates
as a tool in experimental optimisation of P1 side chain shape of potential
inhibitors in S1 pocket of leucine-and neutral aminopeptidases, Eur. J. Med.
Chem. 40 (2005) 764e771.
[24] J. Jiao, H. Fang, X. Wang, P. Guan, Y. Yuan, W. Xu, Design, synthesis and pre-
liminary biological evaluation of N-hydroxy-4-(3-phenylpropanamido) ben-
zamide (HPPB) derivatives as novel histone deacetylase inhibitors, Eur. J. Med.
Chem. 44 (2009) 4470e4476.
[25] F.K. Hansen, S.D.M. Sumanadasa, K. Stenzel, S. Duffy, S. Meister, L. Marek,
R. Schmetter, K. Kuna, A. Hamacher, B. Mordmüller, M.U. Kassack,
E.A. Winzeler, V.M. Avery, K.T. Andrews, T. Kurz, Discovery of HDAC inhibitors
with potent activity against multiple malaria parasite life cycle stages, Eur. J.
Med. Chem. 82 (2014) 204e213.
[8] H. Nagano, H. Ishii, S. Marubashi, N. Haraguchi, H. Eguchi, Y. Doki, M. Mori,
Novel therapeutic target for cancer stem cells in hepatocellular carcinoma,
J. Hepato Biliary Pancreat. Sci. 19 (2012) 600e605.
[9] H. Suda, T. Takita, T. Aoyagi, H. Umezawa, The structure of bestatin, J. Antibiot.
29 (1976) 100e101.
[26] R. Cincinelli, L. Musso, G. Giannini, V. Zuco, M. De Cesare, F. Zunino,
S. Dallavalle, Influence of the adamantyl moiety on the activity of bipheny-
lacrylohydroxamic acid-based HDAC inhibitors, Eur. J. Med. Chem. 79 (2014)
251e259.
[10] S. Yoshida, Y. Nakamura, H. Naganawa, T. Aoyagi, T. Takeuchi, Probestin, a new
inhibitor of aminopeptidase M, produced by Streptomyces azureus MH663-
2F6. II. Structure determination of probestin, J. Antibiot. 43 (1990) 149e153.
[11] M.-C. Chung, H.-J. Lee, H.-K. Chun, C.-H. Lee, S.-I. Kim, Y.-H. Kho, Bestatin