Two Cu(II) coordination polymers: Heterogeneous catalytic Knoevenagel condensation reaction and treatment activity on atherosclerosis via regulating the expression of the COX-2 in vascular endothelial cells

Article abstract of DOI:10.1016/j.jinorgbio.2021.111464

By altering auxiliary nitrogen-donor ligands, two novel coordination polymers (CPs) containing Cu(II) formulated as [Cu_2.5(L)(trz)₂(H₂O)₂]·2H₂O (1) (Htrz = 1,2,4-triazole and H₃L = 5-(4-car

Full text of DOI:10.1016/j.jinorgbio.2021.111464

Journal of Inorganic Biochemistry 220 (2021) 111464
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Two Cu(II) coordination polymers: Heterogeneous catalytic Knoevenagel
condensation reaction and treatment activity on atherosclerosis via
regulating the expression of the COX-2 in vascular endothelial cells
,
Yong-Chang Zhaoa, Yan Zhangb, De-Ying Jiangc *, Liang Wangd, Ping Sund
a Imaging Intervention Room, 3201 Hospital of Xi’an Jiaotong University, Hanzhong, Shaanxi, China
b Internal Courage, Hiser Hospital of Qingdao City, Qingdao, Shandong, China
c Vascular Surgery, Dalian Municipal Central Hospital, Dalian, Liaoning, China
d Department of Chemistry, Capital Medical University, Beijing, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
By altering auxiliary nitrogen-donor ligands, two novel coordination polymers (CPs) containing Cu(II) formu-
lated as [Cu2.5(L)(trz)2(H2O)2]⋅2H2O (1) (Htrz = 1,2,4-triazole and H3L = 5-(4-carboxybenzyloxy)isophthalic
acid) and [Cu(HL)(Hbiz)] (2, Hbiz = benzimidazole) have been produced under the hydrothermal conditions.
The complex 2 with both acidic and basic sites was investigated as heterogeneous catalyst, which reveals highly
efficient catalytic property of the Knoevenagel condensation reaction. Dynamic changes of coagulation param-
eters during atherosclerosis was also explored via detecting activated partial thromboplastin time (APTT) and
prothrombin time (PT), and the results showed that compared with CP 2, CP 1 has a stronger improvement on
the coagulation parameters during atherosclerosis. Then, the high-sensitivity C-reactive protein and matrix
metalloproteinase-1 released by the atherosclerotic segment was detected with enzyme linked immunosorbent
assay (ELISA) detection, which also revealed that CP 1 could obviously decrease the inflammatory mediator
released by the atherosclerotic segment, but not CP 2. And, the cyclooxygenase-2 (COX-2) relative expression
level in vascular endothelial cells was detected via the real time RT-PCR. The results of the real time reverse
transcription-polymerase chain reaction (RT-PCR) indicated that CP 1 has stronger activity on inhibiting the
Notch signaling pathway than CP 2. Finally, we got this information, CP 1 has excellent application values on the
coagulation parameters during atherosclerosis via regulating the expression of the COX-2 in vascular endothelial
cells.
Coordination polymers
X-ray diffraction
Heterogeneous catalyst
Atherosclerosis
1. Introduction
Coordination polymers (CPs) composed of metal ions and organic
ligands have gained great attentions in recent years for their rich po-
Atherosclerosis is a cardiovascular disease that seriously threatens
human’s health. At present, many cardiovascular diseases are closely
related to atherosclerosis. For example, acute coronary syndrome (ACS)
is a concurrent plaque rupture or erode based on coronary atheroscle-
rosis, leading to acute or subacute reduced myocardial oxygen supply
[1,2]. Therefore, the prevention and treatment of atherosclerosis has
become the basis for the prevention of coronary heart disease. Up to
now, the cause of atherosclerosis is not very clear. Atherosclerosis is a
chronic inflammatory response, and inflammation is playing an
increasingly important role in atherosclerosis [3]. Recent studies have
shown that cyclooxygenase-2 (COX-2) is closely related to the inflam-
matory response to atherosclerosis.
tential applications in many important fields especially in catalysis and
biomedicine [4–8]. The inherent properties of CPs such as highly dense
active sites, endless selection of building blocks and robust frameworks
make them excellent catalysts. The Knoevenagel condensation is a
–
classic reaction, which provides an effective way to form C C double
bonds by the reaction of carbonyl compounds, such as aldehydes or
ketones, with compounds containing active methylene groups. It has
been widely used in the synthesis of many compounds and is a reaction
with an important application value in organic synthesis [9–11]. In
recent years, many catalysts for such reactions have emerged, among
which CPs-based catalysts have shown remarkable catalytic perfor-
mance. According to the literature, basic catalysis and bifunctional
* Corresponding author.
E-mail address: Lnjdy1977@163.com (D.-Y. Jiang).
https://doi.org/10.1016/j.jinorgbio.2021.111464
Received 26 December 2020; Received in revised form 7 April 2021; Accepted 8 April 2021
Available online 21 April 2021
0162-0134/© 2021 Elsevier Inc. All rights reserved.

Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
acid–base catalysis are two generally accepted mechanisms for CPs-
based catalysts in the Knoevenagel condensation, among which the
construction of bifunctional acid–base catalysts is more challenge
because this requires the combination of acidic and basic sites in one
catalyst [12]. The acid-base mixed-ligand approach might be a feasible
approach for building the bifunctional acid–base catalyst considering
the potential undeprotonated carboxylate groups along with the unco-
ordinated N-donor sites. On the other hand, copper is an essential trace
metal element in the human body Modern medical research shows that
copper complexes can produce antibacterial, antiviral, anti-
inflammatory, antitumor, enzyme inhibitory or chemical nuclease ac-
tivities, which are increasingly attracting the attention of researchers
[13–17]. Another drive for targeting copper(II) was due to its less toxic
nature, which can be further decreased on complexation with ligands
and thus proved promising in the development of copper complexes as
bioactive agents [18]. Considering the above aspects, in this study, two
new coordination polymers containing Cu(II) ions as nodes formulated
as [Cu2.5(L)(trz)2(H2O)2]⋅2H2O (1) (Htrz = 1,2,4-triazole and H3L = 5-
(4-carboxybenzyloxy)isophthalic acid) and [Cu(HL)(Hbiz)] (2, Hbiz =
benzimidazole) have been produced under the hydrothermal conditions,
and their structures could be modulated using different auxiliary
nitrogen-donor ligands. The complex 2 with both acidic and basic sites
was investigated as heterogeneous catalyst, which reveals the highly
efficient catalytic property of the Knoevenagel condensation reaction.
The treatment effect of CPs against the atherosclerosis was evaluated
and the particular mechanism was investigated as well. The results of
activated partial thromboplastin time (APTT) and prothrombin time
(PT) revealed that in comparison with CP 2, CP 1 has a stronger
improvement on the coagulation parameters during atherosclerosis.
Besides, the enzyme linked immunosorbent assay (ELISA) assay also
revealed that CP 1 could obviously decrease the inflammatory mediator
released by the atherosclerotic segment, but not CP 2. Following that,
the COX-2 in vascular endothelial cells was also inhibited by CP 1
instead of CP 2. Eventually, the western blot indicated that CP 1 has
stronger activity on inhibiting the Notch signaling pathway.
Table 1
The parameters of crystallography as well as the refinement for the two CPs.
Identification code
1
2
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C
40H42Cu5N12O22
C23H16CuN2O7
495.92
1360.55
293(2)
293(2)
monoclinic
P21/c
monoclinic
P21/c
11.369(2)
15.621(5)
13.2958(10)
90
14.569(2)
7.652(3)
17.996(2)
90
b/Å
c/Å
◦
α
/
β/◦
93.973(4)
90
107.9650(10)
90
γ/◦
Volume/Å3
Z
2355.7(9)
2
1908.4(8)
4
ρcalcg/cm3
1.918
1.726
μ
/mmꢀ 1
2.321
1.199
Data/restraints/
parameters
5701/6/390
3538/0/302
Goodness-of-fit on F2
1.021
1.008
Final R indexes [I ≥2
σ
(I)]
R1 = 0.0252,
0.0586
ω
R2
R2
=
=
R1 = 0.0377,
0.0714
ω
R2
R2
=
=
Final R indexes [all data]
R1 = 0.0359,
0.0622
ω
R1 = 0.0659,
0.0795
ω
Largest diff. peak/hole/e
0.34/ꢀ 0.40
0.30/ꢀ 0.33
Åꢀ 3
CCDC
2,002,951
2,002,952
0.12 g), H3L (0.35 mmol, 0.11 g), biz (41 mg, 0.35 mmol), NaOH (14 mg,
0.35 mmol) and H2O (14 mL) was sealed into the stainless steel
container of 25 mL lined with Teflon, the mixture was heated for 72 h at
150 ◦C and then cooled at 5 ◦C⋅hꢀ 1 rate to the ambient temperature. Blue
crystals with block-shape of CP 2 were harvested and cleaned by the
distilled water and then dried in the air to obtain product; the yield is
41.2% (on the basis of CuII salts). Elemental analyses calcd (%) for
C
23H16N2O7Cu (495): C, 55.70; H, 3.25; N, 5.65. Found C, 55.59; H,
3.22; N, 5.57. IR (KBr pellet, cmꢀ 1): 2965 (w), 1734 (s), 1690 (s), 1598
(s), 1460 (m), 1285 (s), 1206 (s), 1121 (m), 1057 (m), 889 (w), 765 (m),
664 (w), 531 (w).
2. Experimental
The software of crysalispro was applied to analyze the strength data
and then convert strength data into the HKL files. Via using the
diffractometer of Oxford XcaliburE we acquired the data of X-ray. The
program of SHELXS in accordance with the direct method was applied to
construct the initial skeleton pattern for the CP 1, and the program of
SHELXL-2014 in accordance with least square means was modified.
Mixing the anisotropic parameters with the non‑hydrogen atoms of CP
1. Then all of the hydrogen atom via applying the command of AFIX to
fix on the C atom geometrically they are connected to. The refinement
along with the crystallographic parameters for the two CPs are described
in detail in Table 1.
2.1. Chemicals and measurements
All chemicals were commercially available and they were used
without further purification. The H3L ligand was acquire from the Jinan
Henghua Chemical Reagent company (Jinan, China). Nitrogen,
hydrogen and carbon elements were analyzed via utilizing a analyzer of
PerkinElmer 240C. The infrared spectra from 4000 to 400 cmꢀ 1 were
recorded on the spectrometer of Bruker ALPHA utilizing pure solid
samples. Via GC contains FID detector (GC-2014C, Shimadzu, Japan)
and capillary (30 m long × 0.25 mm inner diameter, WondaCap 17), we
calculated the conversion for catalytic reaction.
2.3. Knoevenagel condensation
2.2. Preparation and characterization for [Cu2.5(L)(trz)2(H2O)2]⋅2H2O
All the experiments were carried out in a round bottom flask (10 mL)
placed in a silicone bath under nitrogen atmosphere. Typically, 10 mmol
of malononitrile as methylene compound, 2 mL of solvent (if used) and
catalyst were added at room temperature. Thereafter, the mixture was
heated up to the reaction temperature. Once the desired temperature
was achieved, 10 mmol of benzaldehyde was added. The influence of
reaction solvent and temperature was explored as listed in the Table 2. A
stirring speed of 1000 rpm was fixed in order to avoid mass transfer
limitations. Reactant and product concentrations were quantified by GC,
1H NMR and 13C NMR (Figs. S1–S9). Dodecane was used as an internal
standard.
(1) and [Cu(HL)(Hbiz)] (2)
For the synthesis of CP 1, a mixture of Cu(NO3)2⋅3H2O (0.50 mmol,
0.12 g), H3L (0.35 mmol, 0.11 g), Htrz (0.35 mmol, 0.024 g), NaOH
(0.50 mmol and 0.20 g) and H2O (14 mL) was sealed into the stainless
steel container of 25 mL lined with Teflon, the mixture was heated for
ꢀ 1
◦
◦
72 h at 150 C and then cooled at 5 C⋅h rate to the ambient tem-
perature. Blue crystals with block-shape of the CP 1 were harvested and
cleaned by the distilled water and then dried in the air to obtain product;
the yield is 36% (on the basis of CuII salts). Elemental analyses calcd (%)
for C40H42N12O22Cu5 (1360): C, 35.31; H, 3.11; N, 12.35. Found C,
35.41; H, 3.02; N, 12.13. IR (KBr pellet, cmꢀ 1): 3320 (s), 3080 (w), 1766
(s), 1649 (s), 1551 (m), 1420 (w), 1250 (s), 1200 (s), 1055 (m), 1026
(m), 910 (m), 750 (s), 650 (w).
2.4. Coagulation parameters
For the synthesis of CP 2, a mixture of Cu(NO3)2⋅3H2O (0.50 mmol,
In the process of the atherosclerosis, there was commonly combined
2

Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
Table 2
The Knoevenagel reactions catalyzed under different conditions.
Entry
Catalyst
Dose (mg)
Temperature (◦C)
Solvent
Conversion (%)
1
Blank
0
60
60
60
60
60
60
60
RT
40
50
60
60
60
60
50
50
–
18
52
46
39
82
94
>99
33
83
96
78
20
18
30
43
20
2
H3L
3
–
3
benzimidazole
2
–
4
2
3
–
5
2
5
–
6
2
8
–
7
2
10
10
10
10
10
10
10
10
10
10
–
8
2
–
9
2
–
10
11
12
13
14
15
16
2
–
2
C2H5OH
CH3COCH3
CH2Cl2
C6H5CH3
–
2
2
2
Htrz
1
–
with a dysregulated coagulation in the atherosclerotic segment, re-
flected as the reduced activated partial thromboplastin time (APTT) and
prothrombin time (PT), which were both the standard coagulation pa-
rameters. Thus, in this experiment, CPs 1 and 2 were used for indicated
treatment, and the PT, APTT values were measured. In brief, twenty
healthy New Zealand male white rabbits were utilized as the experi-
mental animals, and fed with high-fat diet, followed by treated with
hypertension on one side of the renal artery stenosis to induce the ani-
mal model of atherosclerosis. Then these two CPs were given to perform
the indicated treatment with a concentration of 5 mg/kg. All of pre-
formation in this study were authorized via Zhejiang University Animal
Ethics Committee (Hangzhou, China). Finally, animals’ blood was
collected and the PT and APTT values were measured at least three
times.
2.5. ELISA assay
The ELISA test was also carried out in this study for the content of the
high-sensitivity C-reactive protein and matrix metalloproteinase-1
released by the atherosclerotic segment into plasma. This detection
was carried out totally according to instructions’ guidance with slight
modifications. In short, New Zealand white rabbits were fed by using
high-fat diet and treated with hypertension on one side of the renal
artery stenosis to induce the animal model of atherosclerosis. Then these
two CPs were given for the indicated treatment with a concentration of
5 mg/kg. Next, the plasma of all the animals were collected and the level
Fig. 1. (a) The coordination surroundings for Cu(II) ions in CP 1. (b) The coordination diagrams of the ligand in the CP 1. (c) The single network for CP 1. (d) The
(3,8)-linked net of CP 1.
3

Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
Fig. 2. (a) The asymmetric unit of CP 2. The two-dimensional skeleton of CP 2 viewed along axis b (b) and a (c). (d) The (4)-linked two-dimensional net of CP 2.
of the high-sensitivity C-reactive protein and matrix metalloproteinase-1
was detected through the indicated ELISA kit. This experiment was
performed three times or more, and obtained results were expressed as
mean ± standard deviation.
After incubated with primary and secondary antibody, the expression of
the Notch was observed.
3. Results and discussion
3.1. Molecular structure
2.6. Real time reverse transcription-polymerase chain reaction (RT-PCR)
CP 1 was hydrothermally synthesized via Cu(NO3)2⋅6H2O reacting
with NaOH, trz and H3L, which was completely characterized via single
crystal X-ray powder diffraction (PXRD), fourier transform infrared
spectrometer (FT-IR) and the analysis of element. In accordance with the
data of crystal which harvested under the ambient temperature, the
refinement along with structural solution results indicated that the CP 1
is crystallized in monoclinic P21/c space group and CP 1’s minimum
asymmetric unit is composed of 2.5 crystallographic independent Cu(II)
centers in crystal, a L3ꢀ , two ligands aqua, two trzꢀ and two molecules of
lattice-water. As revealed in the Fig. 1a, Cu1 atom possesses a distorted
geometry of octahedron with a chromophore of CuN4O2 and it is coor-
dinated via two carboxylic acid O atoms in two H3L (O7A and O7) in
axial positions and four N atoms in four trz (N1A, N1, N4A and N4) in
equatorial plane. Cu2 atom reveals a geometry of trigonal biyramid with
one chromophore of CuN2O3 and it is coordinated via two carboxylic
acid O atoms in two H3L (O6 and O3), an O atom in one coordinated
water (O2W) and two N atoms in two trz (N5 and N2). Cu3 atom also
reveals a geometry of trigonal biyramid with one chromophore of
CuN2O3 and it is coordinated via two N atoms in two carboxylic acid O
atoms in two H3L (O2 and O1), an O atom in one coordinated water
(O1W) and two trz (N6 and N3). Cu1, Cu2 as well as symmetry-related
Cu2A are combined via four molecules of trz (N1AN2A, N1N2, N4AN5A
and N4N5) and two carboxylic groups of two ligands of H3L (O6AO7A
and O6O7) to form the linear trinuclear moiety of [Cu3N8O4]. Each of
The COX-2 relative expression in vascular endothelial cells was in-
depth measured through the real time RT-PCR according to the in-
structions of manufactures. Briefly, the New Zealand white rabbits were
fed by using high-fat diet and treated with hypertension on one side of
the renal artery stenosis at the aim of inducing the animal model of
atherosclerosis. Then these two CPs were given for the indicated treat-
ment with a concentration of 5 mg/kg. Subsequently, we isolated the
atherosclerotic segment and then extracted overall RNA via the Trizol
reagent. The RNA concentration was measured and then transcripted
into cDNA for the detection of the levels of COX-2 in the vascular
endothelial cells, gapdh was utilized as internal control gene. This
experiment was performed three times or more, and obtained results
were expressed as mean ± standard deviation.
2.7. Western blot
The western blot was implemented to detect the signaling pathway of
Notch activation level in vascular endothelial cells after indicated
treatment. This experiment was conducted according the protocols as
previous described. New Zealand white rabbits were fed by using high-
fat diet and treated with hypertension on one side of the renal artery
stenosis to induce the animal model of atherosclerosis. Then these two
CPs were given to perform the indicated treatment with a concentration
of 5 mg/kg. Then, the vascular endothelial cells of the atherosclerotic
segment were isolated for the total protein extraction. Protein samples
were loaded onto the gel of sodium dodecyl sulfate (SDS) and then
transferred onto the membranes of poly(1,1-difluoroethylene) (PVDF).
the ligand L3ꢀ with the coordination pattern μ4
-
η1
:
η1
:
η1
:
η0
:
η1
:
η1 links
two trinuclear clusters [Cu3N8O4] and a Cu3 atom in three directions in
order to generate the infinite two-dimensional layer framework
4

Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
Table 3
The reaction of distinct benzaldehyde derivatives with malononitrile catalyzed via CP 2.
Entry
Substrate
Product
Conversion (%)
1
>99
2
3
4
5
>99
>99
>99
96
6
7
8
60
88
>99
(Fig. 1b). Then the two-dimensional layers are in-depth pillared with
ligands trz to obtain the final three-dimensional skeleton (Fig. 1c). In
terms of topology, each of the trinuclear clusters [Cu3N8O4] is connected
with four ligands of L3ꢀ and four ligands of trzꢀ , which could be
regarded as an eight-linked node; each of Cu3 atom links a ligand L3ꢀ
and two ligands of trzꢀ and could be viewed as three-linked node; each
of ligand L3ꢀ links two clusters [Cu3N8O4] and a Cu3 atom, which can be
regarded as a three-linked node; whereas each of the ligand trzꢀ can be
viewed as a linear connector between the Cu3 atom and [Cu3N8O4]
clusters. Based on this simplification, CP 1 has a three-dimensional
(3,8)-linked net. The topology analysis via the program of TOPOS in-
dicates the (3,8)-linked net with topology of (53)2(58⋅64⋅78⋅84⋅94)
(Fig. 1d).
4.209(2) Å. Then a N atom of ligand biz and a carboxylic acid O atom of
ligand HL2ꢀ are combined with each Cu(II) atom of cluster [Cu2(CO2)2]
to obtain SBUs [Cu2(CO2)2O2N2] based on Cu2. In addition, the ligand
HL2ꢀ links SBUs based on Cu2 to generate a two-dimensional layer
framework (Fig. 2b and c). The unprotonated carboxybenzyloxy groups
alternately point up and down to two-dimensional tablet. The dicar-
boxyphenyl groups in the ligand of HL2ꢀ are twisted via the carbox-
ybenzyloxyl groups, and its dihedral angles is 9.1(2)◦. In the terms of
topology, each of the ligand HL2 ꢀ can be viewed as a linear connector
between SBUs based on Cu2; whereas each of SBUs based on Cu2 is
connected with four ligands HL2ꢀ and can be regarded as a four-linked
node, the entire structure for CP 2 is the extended neutral four-linked
network (Fig. 2d).
The analysis of X-ray structure exhibits that CP 2 is crystallized in
monoclinic P21/c space group and there are an absolute Cu(II) atom in
crystal, a ligand of HL2ꢀ and a biz co-ligand in CP 2’s asymmetric unit
(Fig. 2a). As exhibited in the Fig. 2a, Cu(II) atom is located in the co-
ordination geometry of tetrahedron with three carboxylic acid O atoms
(O3B, O2A and O1) of three ligands H3L and a N atom (N1) of a ligand
biz. Each of the biz ligand reveals as a terminal ligand, which binds to a
Cu(II) atom in the monodentate pattern, whereas each of ligand HL2ꢀ
links three Cu(II) atoms via two carboxylic group. And coordination
In comparison with the incomparable characteristics of CPs and the
demand of the novel generation of increased heterogeneous catalysts,
the development of the environmentally friendly Knoevenagel conden-
sation CP catalyst has become a research hotspot in past few years. The
condensation reactions of Knoevenagel are the familiar organic reaction,
–
which is mainly used to generate C C bonds and to synthesize signifi-
cant derivatives for different purposes. In accordance with the former
reports, condensation reaction of Knoevenagel is commonly catalyzed
via Lewis acid-base positions or Lewis base. In consideration with the
architecture of CP 2 involves uncoordinated nitrogen atoms in ligand
benzimidazole (Lewis base positions) and protonated carboxylic groups
(Lewis acid positions) while these active sites are absent in the CP 1, we
try to use CP 2 to catalyze this reaction. The optimum conditions of
reaction were explored with malononitrile and benzaldehyde as the
pattern for the ligand of HL2ꢀ can be expressed as the μ3
-
η1
:
η1
:
η1
:
η0
:
η0
:
η0
(Fig. 2b). It should be noted that the carboxybenzyloxy group of the
ligand is free coordination without deproton. Two corresponding Cu(II)
atoms in crystal are bridged via a pair of carboxylic acid group of ligand
HL2ꢀ to form a cluster [Cu2(CO2)2], where the distance of Cu┄Cu is
5

Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
is only 20%, which is similar to the value of the blank experiments,
indicating it shows little catalytic activity toward the Knoevenagel
condensation reaction due to the lack of uncoordinated N sites as well as
its tightly packed 3D framework structure.
In order to study the catalytic universality for CP 2, the reactions
were performed for 60 min at 60 ◦C without solvent. The reaction of a
series of malondialdehyde without functionalized benzaldehyde de-
rivatives was studied (Table 3). The results show that the conversion of
benzaldehyde with substituted electron absorption parts (such as 4-NO2,
4-CN and 2-Cl) can up to 99% (entries 2, 3, 4). However, when sub-
stituent was replaced via the electron-donating group (such as 4-CH3, 4-
CH3-O or 2-OH), conversions is respectively reduced by 96%, 60% and
80% (entries 5, 6, 7). This phenomenon is due to substituents electronic
effect. When the steric hindrance 2-naphthalene formaldehyde is used as
the substrate, the conversion rate is as high as 99% (entry 8). The results
reveal that catalytic activity for CP 2 is chiefly affected via the electronic
influence.
Fig. 3. The experiments of hot filtration (black) and Kinetic (red) catalyzed via
CP 2. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
The reaction rate was investigated via the kinetic experiment of CP 2.
We monitor the conversion rate by gas chromatography every 10 min
(Fig. 3). In first 10 min, the reaction rate was the highest. Second, the
rate kept growing rapidly, reaching 92% 40 min later. Afterward,
gradual growth was found in next 20 min, reaching 99% in 60 min. In
order to study whether it was the reaction of heterogeneous catalysis,
the crystals for CP 2 were filtered 10 min later. And then, filtrate was
stirred for another 40 min under the identical conditions. The Benzyli-
dene methacrylonitrile yield did not increase, indicating that the reac-
tion of catalysis was heterogeneous.
substrates (Table 2). First of all, blank experiment was carried out in the
inexistence of catalyst for 60 min at 60 ◦C (entry 1), and only 18% of the
product was produced. And then, in order to determine catalytic site,
H3L of 3 mg or ligand benzimidazole of 2 mg were utilized as the ho-
mogeneous catalysts, and medium conversions (52% and 46%, entry 2
and entry3) were achieved. Afterward, with catalyst amount was
enhanced from 3 mg to 5, 8 and 10 mg, the conversions respectively
enhanced from 39% to 82% and 94% as well as >99% (entries 4, 5, 6 and
7). Therefore, following reactions are carried out at distinct tempera-
tures with the identical amount of catalyst, 10 mg. At RT, only 33% of
the conversion was realized (entry 8). Via increasing the temperature to
40 ◦C, 50 ◦C and 60 ◦C, and the conversion respectively increase to 83%,
96% and >99% (entries 9, 10 and 7). At the aim of studying the effect of
solvents on the reaction, experiments were implemented in diverse
solvents for instance, acetone, ethanol, toluene and dichloromethane.
The results show that conversion were much lower than that without
solvent (entries 11, 12, 13, 14). To make a comparison, the catalytic
performances of the Htrz and CP 1 toward the Knoevenagel condensa-
tion reaction were also studied under a similar reaction condition of CP
2. As indicated in entries 15 and 16, the Htrz shows a moderate con-
version due to its three N-donor sites. However, the conversion for CP 1
3.2. CP increased the values of the PT and APTT
There was usually combined with a dysregulated coagulation in the
atherosclerotic segment during the procession of atherosclerosis. Thus,
in this experiment, after the synthesis of CPs 1 and 2, activated partial
thromboplastin time (APTT) and prothrombin time (PT) was evaluated
in this experiment. According to the resultsof Fig. 4, we can find that
there was a reduced PT and APTT value in the model groups, indicating
the coagulation was over activated in the atherosclerosis disease. While,
after CP 1 treatment, the PT and APTT values were increased signifi-
cantly, suggesting the over activated coagulation in the atherosclerotic
segment was reversed. But CP 2 showed no promotion on the dysregu-
lated coagulation.
Fig. 4. Increased values of the PT and APTT after treated by the CP. The animal model of atherosclerotic was established on New Zealand white rabbits, and one of
these two CPs was given to implement the indicated treatment with a concentration of 5 mg/kg. The values of the PT and APTT after CP treatment were determined.
Control refers to the normal animal, the model refers to the atherosclerotic animal model.
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Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
Fig. 5. Reduced content of inflammatory mediator released by the atherosclerotic segment. Atherosclerotic animal model was constructed on New Zealand white
rabbits, and one of these two CPs was given to implement the indicated treatment with a concentration of 5 mg/kg. The content of inflammatory mediator released by
the atherosclerotic segment detected via the ELISA test kit. Control refers to the normal animal, the model refers to the atherosclerotic animal model.
Fig. 6. Suppressed the COX-2 relative expression in the vascular endothelial cells after treated by the CP. The animal model of the atherosclerotic was built on New
Zealand white rabbits, and one of these two CP was given to implement the indicated treatment with a concentration of 5 mg/kg. RT-PCR was implemented to detect
the COX-2 relative expression in the vascular endothelial cells. Control refers to the normal animal, the model refers to the atherosclerotic animal model.
3.3. CP reduced the content of inflammatory mediator released by the
atherosclerotic segment
the COX-2 in vascular endothelial cells could also regulated the in-
flammatory mediator synthesis and production, so the COX-2 relative
expression in the vascular endothelial cells will be measured with real
time RT-PCR. From Fig. 6, we obtained this information, in model group,
the CP 1 possesses stronger suppression ability on reversing the COX-2
abnormal expression.
In the recent years, the high-sensitivity C-reactive protein and matrix
metalloproteinase-1 released by the atherosclerotic segment were
widely used as the indicator for the atherosclerosis evaluation. So, in this
study, the effect of these two CPs against the atherosclerosis was
detected via the ELISA assay. The results revealed in the Fig. 5 indicated
that there was an obvious enhance level of the high-sensitivity C-reac-
tive protein and matrix metalloproteinase-1 in the plasma, which could
be obviously reversed by CP 1 treatment. However, CP 2 exhibited no
influence of the inflammatory mediator releasing.
3.5. CP suppressed the Notch signaling pathway activation in the vascular
endothelial cells
As previous reported, the signaling pathway of Notch possesses a
significant function in the anti-inflammatory responses in the cells. After
CP 1 or CP 2 treatment, the signaling pathway of Notch activation level
in vascular endothelial cells was assessed by the western blot. We can
see that in consistence with previous results, in model group, the Notch
signaling pathway activation was much higher in comparison with
control group. Under CP 1 treatment, the Notch signaling pathway
activation was reduced, and CP 2 possesses no influence against
3.4. CP suppressed the relative expression of the COX-2 in vascular
endothelial cells
In the previous research we have demonstrated the outstanding in-
hibition of the CP 1 against the inflammatory mediator releasing. And
7

Y.-C. Zhao et al.
Journal of Inorganic Biochemistry 220 (2021) 111464
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jinorgbio.2021.111464.
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Author statement
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Yong-Chang Zhao and Yan Zhang conceived and designed the
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De-Ying Jiang and Liang Wang performed the experiments;
Ping Sun wrote the paper.
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Declaration of Competing Interest
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M. Peana, Kill or cure: misuse of chelation therapy for human diseases, Coord.
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The author(s) declare(s) that there is no conflict of interest regarding
the publication of this paper.
Acknowledgments
Not applicable.
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