Development of pyrazoline-based derivatives as aminopeptidase N inhibitors to overcome cancer invasion and metastasis

Article abstract of DOI:10.1039/d1ra03629g

Aminopeptidase N is considered as a promising anti-tumor target due to its role in tumor invasion, metastasis and angiogenesis. In this report, a new series of pyrazoline-based derivatives were designed, synthesized and evaluated for biological activities

Full text of DOI:10.1039/d1ra03629g

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PAPER
Development of pyrazoline-based derivatives as
aminopeptidase N inhibitors to overcome cancer
invasion and metastasis†
Cite this: RSC Adv., 2021, 11, 21426
ab
a
Chunlong Zhao,a Hang Dong,a Qifu Xua and Yingjie Zhang
*
Jiangying Cao,
Aminopeptidase N is considered as a promising anti-tumor target due to its role in tumor invasion,
metastasis and angiogenesis. In this report, a new series of pyrazoline-based derivatives were designed,
synthesized and evaluated for biological activities. The structure–activity relationships of these
pyrazoline-based derivatives were also discussed in detail. Among them, compound 2k, with 2,6-
dichloro substitution, showed the best APN inhibitory activity, of which the IC50 value was two orders of
magnitude lower than that of the positive control bestatin. At the same concentration of 100 mM, the in
vitro anti-invasion activity of compound 2k was also significantly better than that of bestatin. Moreover,
compound 2k could effectively prevent the pulmonary metastasis of mice H22 hepatoma cells in vivo,
supporting its further research and development as an antitumor agent.
Received 9th May 2021
Accepted 6th June 2021
DOI: 10.1039/d1ra03629g
rsc.li/rsc-advances
angiogenesis, in response to hypoxia or angiogenic growth
factor stimulation.9 Impaired neovascularizations were found
in APN-null mice.10 What's more, APN was considered as one
member of surface markers of liver cancer stem cells.11 The
combination of APN inhibitor bestatin and cytotoxic drug 5-Fu
demonstrated synergistic anti-hepatoma effect, by inhibiting
hepatoma stem cells and normal hepatoma cells, respectively.12
All these ndings support the rationale that APN is a promising
antitumor target.
Introduction
Aminopeptidase N (APN, CD13), a type II glycoprotein, is
a member of the superfamily of the zinc-dependent M1
aminopeptidase.1,2 APN is mainly found in liver, the renal brush
border, intestines, epithelial cells, endothelial cells and so on.3
As an exopeptidase, the enzyme preferentially hydrolyzes the
neutral or basic amino acid residues from the N-terminals of
oligopeptides.4 Besides cleaving biological peptides, APN also
functions as a signaling molecule and a receptor of coronavirus,
so consequently is designated to be a “moonlighting enzyme”.5
Enhanced APN expression was observed in many tumor cells,
such as melanoma, colon, thyroid, renal, breast, lung and liver
cancers, and was associated with poor prognosis.6 Metastasis is
a main cause of chemotherapeutical failure and tumor recur-
rence. Basement membrane is the main barrier of the invasion
and metastasis of tumor. APN could hydrolyze the type-IV
collagen, the main ingredient of basement membrane, and
promote tumor metastasis.7 Moreover, compared with the
normal vessels, up-regulated APN expression is exclusively
found in the angiogenic vasculatures.8 The cooperative effects
of APN on both tumor cells and nonmalignant stromal cells
within the tumor microenvironment promoted the
Structurally, APN, composed of 967 amino acids, can be
divided into four parts: a short intracellular tail, a trans-
membrane anchor, a small serine-/threonine-rich extracellular
stalk and a large ectodomain. The X-ray crystal structure of APN-
bestatin complex revealed that the catalytic site existing in the
ectodomain contained a catalytic zinc ion and three hydro-
phobic pockets (S1, S01 and S02).13,14 To date, many natural
products and synthetic small molecules were reported as APN
inhibitors (APNIs). Natural products include bestatin,15 pro-
bestin,16 amastatin,17 AHPA-Val,18 and so on. Synthetic APNIs
contain amino-benzosuberone derivative,19 avone derivative,20
aminophosphinic derivative,21 ureido peptidomimetics,22 and
so on. The structure–activity relationships (SARs) of potent
APNIs revealed that zinc binding group (ZBG) and hydrophobic
group interacting with at least one of three hydrophobic pockets
were essential for the activities against APN.23 In our previous
work, compound 1, one pyrazoline-based hydroxamate deriva-
tive, exhibited much more potent inhibitory activity against APN
than the positive control bestatin.24 In order to extend the SARs
of this series of compounds and nd more potent compounds,
derivatization was focused on the terminal phenyl group of
compound 1, leading to compound 2 (Fig. 1). In the structure of
compound 2, the introduction of the hydrophobic substituents
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, 44 West Wenhua Road, Jinan, Shandong, 250012, P. R. China.
E-mail: zhangyingjie@sdu.edu.cn; Fax: +86 531 88382009; Tel: +86 531 88382009
bSchool of Pharmacology, Shandong University of Traditional Chinese Medicine, Jinan
250355, P. R. China
† Electronic supplementary information (ESI) available: The spectrum data of
compounds 4b–4n, 5b–5n, 6b–6n and 2b–2n. See DOI: 10.1039/d1ra03629g
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Fig. 1 The design strategy of novel APNIs.
on the terminal phenyl group or the replacement of the phenyl bestatin and compound 1 as the positive controls. The results
group with naphthyl group may enhance the hydrophobic are listed in Table 1. Compound 2o with the hydrazide group
interaction with APN.25 Besides, one compound with the was over-100 fold less potent than its corresponding hydrox-
hydrazide group was also synthesized to investigate the effect of amate analog 2d, indicating the important role of the hydrox-
the ZBG on APN inhibition.
amate group as ZBG in APN inhibition. Comparing compounds
2a–2f, it was easily observed that compounds with mono-
substituent in the ortho position exhibited better activities
against APN than the counterparts with mono-substituent in
the meta or para position (2a vs. 2b, 2c; 2d vs. 2e, 2f). Therefore,
compounds (2g–2i) with various mono-substituents in the ortho
position were synthesized and evaluated. Among the ortho
mono-substituted analogs (2a, 2d, 2g–2i), compound 2i with
ortho chlorine substitution exhibited the best APN inhibitory
activity, with the IC50 value of 0.10 ꢀ 0.01 mM. The inhibitory
order of compounds containing ortho mono-substituents was 2i
(2-Cl) > 2g (2-CH3) > 2f (2-F), 2a (2-Br) > 2d (2-OCH3), suggesting
that the hydrophobicity and size of substituents both impact
the inhibitory activities against APN. Moreover, compounds 2j–
2l were synthesized to explore the effects of dual-substituents of
the phenyl group on APN inhibition. Compound 2k with 2,6-
dichlorine substitution showed better APN inhibitory activity
than the counterparts 2j and 2l, which possessed 2,6-diuorine
substitution and 2,4-dichlorine substitution, respectively.
Moreover, 2,6-dual substituted analogues 2j and 2k showed
Results and discussion
Chemistry
The target compounds 2 were synthesized according to the
procedures listed in Scheme 1. Firstly, 2-hydroxybenzaldehyde 3
reacted with corresponding ketone to give the chalcone deriv-
atives 4 by the Claisen–Schmidt condensation. Subsequently,
compounds
4 underwent cyclization with semicarbazide
hydrochloride to form the pyrazoline-based intermediates 5,
which further reacted with methyl bromoacetate to give
compounds 6. Finally, the methyl ester groups of 6 were
transformed to hydroxamate groups by NH2OK to give target
compounds 2a–2n. Besides, compound 6d reacted with hydra-
zine hydrate led to compound 2o.
Inhibitory activities of the target compounds against APN
All the newly synthesized pyrazoline-based derivatives were
rstly evaluated for their inhibitory activities against APN using
Scheme 1 Reagents and conditions: (a) substituted acetophenone for 4a–4l, 1-(naphthalen-1-yl)ethan-1-one for 4m, 1-(naphthalen-2-yl)
ethan-1-one for 4n, KOH, 80% EtOH, 25 ꢁC, 48 h; (b) semicarbazide hydrochloride, NaOH, EtOH, 78 ꢁC, 5 h; (c) DMF, NaH, methyl bromoacetate,
25 ꢁC, 12 h; (d) NH2OK, MeOH, 25 ꢁC, 0.5 h; (e) 6d, 80% hydrazine hydrate, MeOH, 65 ꢁC, 6 h.
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Table 1 The structures and IC50 values of target compounds against
porcine APN
2g–2k exhibited comparable or improved APN inhibitory activ-
ities relative to the parent compound 1, supporting our design
strategy. Notably, the most potent one 2k (IC50 ¼ 0.064 ꢀ 0.01
mM) was over 2-fold more potent than compound 1 (IC50 ¼ 0.15
ꢀ 0.02 mM) and over 140-fold more potent than the positive
control bestatin (IC50 ¼ 9.0 ꢀ 0.5 mM).
Inhibitory activities of 2k against the proliferation of tumor
cells in vitro
Compound 2k with the best APN inhibitory activity was selected
for further anti-proliferative evaluation against six tumor cell
lines (U937, K562, PLC/PRF/5, PC-3, ES-2, HepG2). The results
are listed in Table 2. Though better than bestatin, compound 2k
showed only moderate anti-proliferative activities against the
selected tumor cell line, indicating the low cytotoxicity of 2k
against tumor cells.
Anti-invasion assay in vitro
Compd
R1
APN IC50 (mM)a
APN could hydrolyze the basement membrane to promote the
invasion and metastasis of tumor cells. In this research, the
transwell chambers coated with matrigel that could mimic the
basement membrane were used to evaluate the anti-invasion
effects of selected compounds in vitro. As shown in Fig. 2,
compound 2k could inhibit the ES-2 cells invasion in
a concentration dependent manner. It was remarkable that 10
mM of 2k exhibited comparable, if not better, anti-invasion
activity than that of 100 mM of bestatin. At the same concen-
tration of 100 mM, the anti-invasion effect of 2k was signicantly
better than that of bestatin. Note that the compound concen-
trations used in anti-invasion assay were lower than the IC50
values against ES-2 cells shown in Table 2, conrming the anti-
invasion effects were not due to cytotoxicity against ES-2 cells.
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
Bestatin
H
2-Br
3-Br
4-Br
2-OCH3
3-OCH3
4-OCH3
2-CH3
2-F
0.15 ꢀ 0.02
0.15 ꢀ 0.03
0.26 ꢀ 0.05
2.1 ꢀ 0.2
0.27 ꢀ 0.01
0.32 ꢀ 0.03
12.5 ꢀ 0.8
0.11 ꢀ 0.01
0.15 ꢀ 0.01
0.10 ꢀ 0.01
0.12 ꢀ 0.01
0.064 ꢀ 0.01
0.42 ꢀ 0.03
0.21 ꢀ 0.04
2.5 ꢀ 0.1
2-Cl
2,6-diF
2,6-diCl
2,4-diCl
—
—
—
30.2 ꢀ 1.9
9.0 ꢀ 0.5
—
Anti-metastasis assay in vivo
a
Assays were performed in triplicate; data are shown as mean ꢀ SD.
Encouraged by the in vitro anti-invasion effect of 2k, we further
evaluated the in vivo anti-metastasis effect of 2k using the mice
H22 hepatoma cell pulmonary metastasis model. The meta-
static nodes on the surface of pulmonary lobes were counted
and the inhibition rate was calculated. As shown in Fig. 3, at the
dose of 60 mg kgꢂ1 dꢂ1, bestatin and 2k demonstrated similar
in vivo anti-metastasis effects, both of which could obviously
decrease the numbers of pulmonary lobes. The potent in vivo
anti-metastasis effect of 2k was in line with its potent in vitro
anti-invasion activity shown in Fig. 2. Besides, in the mice
groups treated by bestatin and 2k, no signicant body weight
slightly better APN inhibition than the ortho mono-substituted
counterparts 2h and 2i, respectively. Replacement of the
terminal phenyl group of compound 1 using naphthyl group led
to compounds 2m and 2n. Compound 2m with 1-naphthyl
group was over 10-fold more potent than 2n with 2-naphthyl
group. However, the APN inhibitory activity of 2m was inferior
to those of compounds with terminal phenyl group, including
compounds 1, 2a and 2g–2k. Satisfactorily, compounds 2a and
Table 2 In vitro anti-proliferative activities of selected compounds
IC50 (mM)a
Compd
U937
K562
PLC/PRF/5
PC-3
ES-2
HepG2
2k
Bestatin
49.2 ꢀ 3.8
15.6 ꢀ 2.2
105.5 ꢀ 4.7
56.1 ꢀ 3.1
141.3 ꢀ 3.8
101.4 ꢀ 4.9
>500
>500
>500
>500
>500
>500
a
Assays were performed in triplicate; data are shown as mean ꢀ SD.
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Fig. 2 Effect of compound 2k against ES-2 cell invasion. (A) Representative images of ES-2 cell invasion treated with bestatin or compound 2k.
(B) The inhibition rates of tested compounds against ES-2 cells invasion. Data are expressed as the mean ꢀ standard deviation for triplicate
experiments. *P < 0.05, 2k (100 mM) vs. bestatin (100 mM).
Fig. 3 Anti-metastasis potency of tested compounds using mice H22 hepatoma cell pulmonary metastasis model. (A) Picture of pulmonary
lobes with metastasis nodes. (B) Inhibition rate of the tested compounds against metastasis. Data are expressed as mean ꢀ SD. *P < 0.05, treated
group vs. control group. (C) Mean body weights of mice monitored every two days.
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loss was observed (Fig. 3C), suggesting no systematic toxicities,
which were in line with the marginal in vitro cytotoxicity against
tumor cells shown in Table 2. All experiments involving labo-
ratory animals were performed with the approval by the insti-
tutional guidelines of Animal Care and Use Committee at
Shandong University.
3-(2-Bromophenyl)-5-(2-hydroxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (5a)
To a solution of 4a (6.04 g, 20 mmol) in ethanol (100 mL) was
added semicarbazide hydrochloride (2.68 g, 24 mmol) and
sodium hydroxide (3.60 g, 90 mmol). Aer stirring at 78 C for
ꢁ
5 h, water (100 mL) was added into the mixture and 10% HCl
solution was used to adjust pH to 6. The white solid formed
from the mixture was ltered off and washed with EtOAc to give
4.67 g of compound 5a. Yield: 65%, mp: 218.4–220.2 ꢁC. 1H
NMR (400 MHz, DMSO-d6): d 10.01 (s, 1H), 7.70–7.68 (m, 2H),
7.43 (td, J ¼ 7.6 Hz, J ¼ 1.3 Hz, 1H), 7.33 (td, J ¼ 7.7 Hz, J ¼
1.8 Hz, 1H), 7.02 (td, J ¼ 7.7 Hz, J ¼ 1.7 Hz, 1H), 6.89 (dd, J ¼
7.6 Hz, J ¼ 1.7 Hz, 1H), 6.80 (dd, J ¼ 8.1 Hz, J ¼ 1.2 Hz, 1H), 6.67
(t, J ¼ 7.4 Hz, 1H), 6.46 (s, 2H), 5.56 (dd, J ¼ 11.9 Hz, J ¼ 4.8 Hz,
1H), 3.91 (dd, J ¼ 17.7 Hz, J ¼ 11.9 Hz, 1H), 3.04 (dd, J ¼ 17.7 Hz,
J ¼ 4.8 Hz, 1H).
Conclusion
In summary, a new series of APN inhibitors with pyrazoline
scaffold was designed, synthesized and evaluated. In the enzy-
matic assay against APN, except 2f, all the compounds with the
hydroxamate moiety displayed better activities than the positive
control bestatin. Satisfactorily, compounds 2a, 2g, 2h, 2i, 2j and
2k showed similar or better APN inhibitory activities relative to
their lead compound 1. Moreover, compound 2k with the best
APN inhibitory activity exhibited promising in vitro anti-
invasion and in vivo anti-metastasis potencies, suggesting its
prospect as anti-invasion and anti-metastasis lead.
Compounds 5b–5n were prepared in a similar manner as
described for compound 5a.
Methyl 2-(2-(3-(2-bromophenyl)-1-carbamoyl-4,5-dihydro-1H-
pyrazol-5-yl) phenoxy)acetate (6a)
Materials and methods
Chemistry: general procedures
To a solution of compound 5a (3.59 g, 10 mmol) in DMF (30 mL)
was added 60% sodium hydride (0.48 g, 12 mmol) gradually.
The mixture was stirred at 25 ꢁC for 5 minutes and methyl
bromoacetate (2.26 g, 15 mmol) was added. Aer stirring at
25 ꢁC for 12 h, the mixture was poured into ice-cold water. Then
the mixture was extracted with EtOAc (100 mL ꢃ 3), washed
with brine (50 mL ꢃ 3) and dried over MgSO4. Filtration and
concentration gave the crude product, which was further puri-
ed by column chromatography (DCM/MeOH) to give 2.80 g of
compound 6a as a white solid. Yield: 65%, mp: 144.4–146.2 ꢁC.
1H NMR (400 MHz, DMSO-d6): d 7.71–7.66 (m, 2H), 7.44 (t, J ¼
7.6 Hz, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H), 7.21 (t, J ¼ 7.6 Hz, 1H), 7.01–
6.92 (m, 3H), 6.50 (s, 2H), 5.63 (dd, J ¼ 12.0 Hz, J ¼ 5.0 Hz, 1H),
4.93–4.85 (m, 2H), 3.94 (dd, J ¼ 17.8 Hz, J ¼ 12.0 Hz, 1H), 3.68 (s,
3H), 3.10 (dd, J ¼ 17.8 Hz, J ¼ 5.0 Hz, 1H).
All the commercially available materials were used without
further purication otherwise noted. All reactions were moni-
tored by thin-layer chromatography (TLC) on 0.25 mm silica gel
plates (60 GF-254) and the product spots were visualized by UV
light, ferric chloride and iodine vapor. The purication of the
products was conducted by column chromatography and
recrystallization. Melting points determined on an electro-
thermal melting point apparatus were not corrected. Using TMS
as an internal standard, 1H-NMR and 13C-NMR spectra were
determined on a Brucker DRX spectrometer and the values of
chemical shis were described as d in parts per million and J in
Hertz. HRMS were conducted by Shandong Analysis and Test
Center.
Compounds 6b–6n were prepared in a similar manner as
described for compound 6a.
(E)-1-(2-Bromophenyl)-3-(2-hydroxyphenyl)prop-2-en-1-one
(4a)
3-(2-Bromophenyl)-5-(2-(2-(hydroxyamino)-2-oxoethoxy)
phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (2a)
To a solution of compounds 3 (6.10 g, 50 mmol) and 1-(2-
bromophenyl)ethan-1-one (11.88 g, 60 mmol) in ethanol (200 Potassium hydroxide (28.00 g, 509 mmol) was dissolved in
mL) was added potassium hydroxide aqueous solution (25.20 g, distilled methanol (70 mL) at 0 ꢁC. The mixture was added into
ꢁ
ꢁ
450 mmol, in 50 mL water) at 0 C. Aer stirring at 25 C for hydroxylamine hydrochloride (23.35 g, 343 mmol) in distilled
48 h, the mixture was poured into ice-cold water and 1N HCl methanol (120 mL) gradually. Then the mixture was stirred at
solution was added to neutralize the excess base. The formed 0 ꢁC for 40 min. The formed precipitate was ltered out to give
precipitate was ltered off and further puried by column a fresh methanol solution of potassium hydroxylamine as the
chromatograph (DCM/MeOH ¼ 100 : 5) to give 8.61 g of effective agent for hydroxamate formation. Compound 6a
compound 4a as a yellow solid. Yield: 57%, mp: 140.2–142.2 ꢁC. (0.86 g, 2 mmol) was dissolved in the above solution (8 mL).
1H NMR (400 MHz, DMSO-d6): d 10.33 (s, 1H), 7.75 (d, J ¼ 7.8 Hz, Aer stirring at 25 ꢁC for 0.5 h, the mixture was poured into
1H), 7.70 (d, J ¼ 7.8 Hz, 1H), 7.61 (d, J ¼ 16.3 Hz, 1H), 7.56–7.50 water (50 mL) and 10% HCl solution was added to adjust pH to
(m, 2H), 7.49–7.44 (m, 1H), 7.29 (t, J ¼ 8.5 Hz, 1H), 7.20 (d, J ¼ 6. The formed precipitate was ltered off and dried under
16.3 Hz, 1H), 6.92 (d, J ¼ 7.8 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 1H).
vacuum to give 0.45 g of compound 2a. White solid, yield: 52%,
Compounds 4b–4n were prepared in a similar manner as mp: 168.4–170.2 C. H NMR (400 MHz, DMSO-d6): d 10.76 (s,
1
ꢁ
described for compound 4a.
1H), 9.01 (s, 1H), 7.72–7.66 (m, 2H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.34
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(td, J ¼ 7.7 Hz, J ¼ 1.8 Hz, 1H), 7.23 (t, J ¼ 7.5 Hz, 1H), 7.04–7.02
(m, 1H), 6.97–6.93 (m, 2H), 6.51 (s, 2H), 5.77 (dd, J ¼ 12.0 Hz, J ¼
5.1 Hz, 1H), 4.64–4.54 (m, 2H), 3.94 (dd, J ¼ 18.0 Hz, J ¼ 12.0 Hz,
1H), 3.15 (dd, J ¼ 18.0 Hz, J ¼ 5.1 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6): d 164.7, 155.3, 154.5, 151.8, 134.3, 133.0, 131.4,
131.4, 131.2, 128.6, 128.2, 126.0, 121.6, 121.2, 112.4, 66.5, 55.1,
44.5; HRMS (AP-ESI) m/z [M + H]+ calcd for C18H18BrN4O4:
433.0511, found: 433.0516.
In vitro anti-invasion assay
The carbonate membrane coated with matrigel in the upper
chamber of transwell insert (BD BioCoat™ Matrigel™ Invasion
Chambers) was rehydrated with RPMI-1640 culture medium (500
mL) containing 1% FBS for 2 h. Aer removing the medium, the
upper and lower chambers were added the RPMI-1640 culture
medium (100 mL) with 1% FBS containing the tested compounds
and the RPMI-1640 culture medium (750 mL) with 10% FBS,
respectively. Then, ES-2 cells suspending in the RPMI-1640
culture medium containing 1% FBS (400 mL, 2.5 ꢃ 105 cells per
mL) were added into the upper chambers. The system was placed
at 37 ꢁC in 5% CO2 for 8 h. The ES-2 cells could invade the
matrigel and migrate from the upper surface of the carbonate
membrane into the lower surface of that. ES-2 cells on the upper
surface of the carbonate membrane were erased by cotton swabs.
Then, ES-2 cells on the lower surface were xed with methanol,
stained with 0.1% crystal violet, of which the photographs were
taken under an inverted microscope. The average numbers of ES-
2 cells in ve random elds (100ꢃ) per well were counted. The
inhibition rate of ES-2 cells invasion was calculated as follows: (A
ꢂ B)/A ꢃ 100%, A means the number of ES-2 cells in the control
group, and B means the number of ES-2 cells in the treated group.
The experiments were repeated three times.
Compounds 2b–2n were prepared in a similar manner as
described for compound 2a.
Preparation of 5-(2-(2-Hydrazinyl-2-oxoethoxy) phenyl)-3-(2-
methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (2o)
To a solution of compound 6d (0.77 g, 2 mmol) in hot methanol
(10 mL) was added 80% hydrazine hydrate (0.5 g, 8 mmol). Aer
stirred at 65 ꢁC for 6 h, the solvent was evaporated and the
residue was washed with water. The solid was dried under
vacuum to give 0.36 g of compound 2o. White solid, yield: 47%,
1
mp: 156.6–158.2 ꢁC. H NMR (400 MHz, DMSO-d6): d 9.26 (s,
1H), 7.91 (dd, J ¼ 7.8 Hz, J ¼ 1.8 Hz, 1H), 7.41–7.37 (m, 1H),
7.22–7.18 (m, 1H), 7.07 (d, J ¼ 7.8 Hz, 1H), 7.00–6.90 (m, 4H),
6.48 (s, 2H), 5.71 (dd, J ¼ 12.0 Hz, J ¼ 5.0 Hz, 1H), 4.64–4.56 (m,
2H), 4.34 (s, 2H), 3.85 (dd, J ¼ 18.5 Hz, J ¼ 12.0 Hz, 1H), 3.34 (s,
3H), 3.11 (dd, J ¼ 18.5 Hz, J ¼ 5.0 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6): d 167.0, 158.1, 155.4, 154.5, 151.2, 131.9, 131.6,
129.2, 128.4, 125.8, 121.5, 120.9, 112.7, 112.4, 66.9, 56.1, 54.7,
45.2; HRMS (AP-ESI) m/z [M + H]+ calcd for C19H22N5O4:
384.1672, found: 384.1663.
In vivo anti-metastatic assay
All animal procedures were performed in accordance with the
Guidelines for Care and Use of Laboratory Animals of Shandong
University and approved by the Animal Ethical and Welfare
Committee (AEWC, China). The mice hepatoma H22 cell line was
our laboratory-owned and 6 week old male Kunming mice were
purchased from Center for New Drugs Evaluation of Shandong
University, China. Firstly, H22 cells suspension (0.1 mL, 7.5 ꢃ 107
mLꢂ1) was injected via tail vein to establish a mice H22 pulmonary
metastasis model. Subsequently, the mice were randomly divided
into treatment and control groups. Treatment groups received
60 mg kgꢂ1 dꢂ1 of compound 2k or bestatin intraperitoneally for
12 days, while the control group received an equal volume of PBS
intraperitoneally for the same period. The body weights were
monitored every two days. Finally, the mice were sacriced and the
lungs were removed and xed in Bouin's solution. Aer 24 h, the
metastasis nodes on the surface of pulmonary lobes were counted.
Biological evaluation
Enzymatic inhibition assay against APN in vitro
In the enzymatic inhibition assay against APN in vitro, L-Leu-p-
nitroanilide was used as substrate and soluble APN from
porcine kidney microsomes (Biocol) was used as enzyme.
50 mM PBS with pH 7.2 was used as buffer solution. Briey,
inhibitors (40 mL), PBS (145 mL), substrate (5 mL, 16 mmol Lꢂ1
)
and APN solution (10 mL, 0.15 IU mLꢂ1) were added into 96-well
plates. The mixture was incubated at 37 ꢁC for 30 min. The
optical density resulting from the hydrolysis product p-nitro-
anilide was measured at 405 nm with a plate reader (Varioskan,
Thermo, USA).
Statistical analysis
Anti-proliferation assay
The statistical signicance of differences between groups was
assessed by Student's t test. P < 0.05 was taken as statistical
signicance.
The anti-proliferative activities of the selected compounds were
evaluated using the MTT method. All the tumor cell lines were
cultured in RPMI-1640 medium with 10% FBS at 37 C in 5%
ꢁ
CO2 humidied incubator. Firstly, cells (100 mL) were plated in
96-well plates and allowed to grow for 4 h. Then different
concentrations of inhibitors (100 mL) were added. Aer incu-
bation for 48 h, MTT solution (20 mL per well, 5 mg mLꢂ1) was
added and the mixture was incubated for additional 4 h.
Subsequently, the medium were poured off and DMSO (200 mL)
was added to dissolve the formed formazan. Aer shaking for
Conflicts of interest
There are no conicts to declare.
Acknowledgements
15 min, the optical density values were measured using a plate This work was supported by Natural Science Foundation of
reader at 570 nm (Varioskan, Thermo, USA).
Shandong Province (Grant No. ZR2018QH007), Key Research
© 2021 The Author(s). Published by the Royal Society of Chemistry
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and Development Program of Shandong Province 13 A. Addlagatta, L. Gay and B. W. Matthews, Biochemistry,
(2017CXGC1401), Young Scholars Program of Shandong
University (YSPSDU, Grant No. 2016WLJH33).
2008, 47, 5303–5311.
14 A. H. Wong, D. Zhou and J. M. Rini, J. Biol. Chem., 2012, 287,
36804–36813.
15 H. Suda, T. Takita, T. Aoyagi and H. Umezawa, J. Antibiot.,
1976, 29, 100–101.
Notes and references
1 C. Antczak, I. D. Meester and B. Bauvois, BioEssays, 2001, 23,
251–260.
2 N. D. Rawlings and A. J. Barrett, Nucleic Acids Res., 1999, 27,
325–331.
16 T. Aoyagi, S. Yoshida, Y. Nakamura, Y. Shigihara,
M. Hamada and T. Takeuchi, J. Antibiot., 1990, 43, 143–148.
17 D. H. Rich, B. J. Moon and S. Harbeson, J. Med. Chem., 1984,
27, 417–422.
3 B. Bauvois and D. Dauzonne, Med. Res. Rev., 2006, 26, 88–
130.
4 N. M. Hooper, FEBS Lett., 1994, 354, 1–6.
5 P. Mina-Osorio, Trends Mol. Med., 2008, 14, 361–371.
18 M. C. Chung, H. J. Lee, H. K. Chun, C. H. Lee, S. I. Kim and
Y. H. Kho, Biosci., Biotechnol., Biochem., 1996, 60, 898–900.
19 G. Revelant, M. Al-Lakkis-Wehbe, M. Schmitt, S. Alavi,
C. Schmitt, L. Roux, M. Al-Masri, N. Schifano-Faux,
C. Maiereanu, C. Tarnus and S. Albrecht, Bioorg. Med.
Chem., 2015, 23, 3192–3207.
¨
6 M. Wickstrom, R. Larsson, P. Nygren and J. Gullbo, Cancer
Sci., 2011, 102, 501–508.
7 I. Saiki, H. Fujii, J. Yoneda, F. Abe, M. Nakajima, T. Tsuruo
and I. Azuma, Int. J. Cancer, 1993, 54, 137–143.
20 B. Bauvois, M. L. Puiffe, J. B. Bongui, S. Paillat, C. Monneret
and D. Dauzonne, J. Med. Chem., 2003, 46, 3900–3913.
21 P. Kokkala, A. Mpakali, F. X. Mauvais, A. Papakyriakou,
8 S. V. Bhagwat, J. Lahdenranta, R. Giordano, W. Arap,
R. Pasqualini and L. H. Shapiro, Blood, 2001, 97, 652–659.
9 L. Guzman-Rojas, R. Rangel, A. Salameh, J. K. Edwards,
E. Dondossola, Y. Kim, A. Saghatelian, R. J. Giordano,
M. G. Kolonin, F. I. Staquicini, E. Koivunen, R. L. Sidman,
W. Arap and R. Pasqualiniet, Proc. Natl. Acad. Sci. U. S. A.,
2012, 109, 1637–1642.
10 R. Rangel, Y. Sun, L. Guzman-Rojas, M. G. Ozawa, J. Sun,
R. J. Giordano, C. S. Van Pelt, P. T. Tinkey, R. R. Behringer,
R. L. Sidman, W. Arap and R. Pasqualini, Proc. Natl. Acad.
Sci. U. S. A., 2007, 104, 4588–4593.
I.
Daskalaki,
I.
Petropoulou,
S.
Kavvalou,
M. Papathanasopoulou, S. Agrotis, T. M. Fonsou, P. van
Endert, E. Stratikos and D. Georgiadis, J. Med. Chem., 2016,
59, 9107–9123.
22 J. Y. Cao, J. Zang, X. J. Kong, C. L. Zhao, T. Chen, Y. Y. Ran,
H. Dong, W. F. Xu and Y. J. Zhang, Bioorg. Med. Chem., 2019,
27, 978–990.
23 S. A. Amin, N. Adhikari and T. Jha, J. Med. Chem., 2018, 61,
6468–6490.
24 J. Y. Cao, J. Zang, C. H. Ma, X. G. Li, J. N. Hou, J. Li,
Y. X. Huang, W. F. Xu, B. H. Wang and Y. J. Zhang,
ChemMedChem, 2018, 13, 431–436.
25 L. Su, J. Y. Cao, Y. P. Jia, X. N. Zhang, H. Fang and W. F. Xu,
ACS Med. Chem. Lett., 2012, 3, 959–964.
11 N. Haraguchi, H. Ishii, K. Mimori, T. Tanaka, M. Ohkuma,
H. M. Kim, H. Akita, D. Takiuchi, H. Hatano, H. Nagano,
G. F. Barnard, Y. Doki and M. Mori, J. Clin. Invest., 2010,
120, 3326–3339.
12 M. Yamashita, H. Wada, H. Eguchi, H. Ogawa, D. Yamada,
T. Noda, T. Asaoka, K. Kawamoto, K. Gotoh, K. Umeshita,
Y. Doki and M. Mori, Int. J. Oncol., 2016, 49, 89–98.
21432 | RSC Adv., 2021, 11, 21426–21432
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