Download Synthesis, DNA-binding and antiproliferative activity of N

Cent. Eur. J. Chem. • 7(3) • 2009 • 569-575
DOI: 10.2478/s11532-009-0058-3
Central European Journal of Chemistry
Synthesis, DNA-binding and antiproliferative
activity of N-(Nitrogen heterocyclic)
norcantharidin acylamide acid
Research Article
Wen-Zhong Zhu , Rui-Ding Hu
Xiao-Liang Zheng 3
1,2
, Qiu-Yue Lin , Xiao-Xia Wang ,
1,2*
1,2
1,2
1
Zhejiang Key Laboratory for Reaction Chemistry on Solid Surfaces,
Zhejiang Normal University,
Jinhua 321004,China
2
College of Chemical and Life Science,
Zhejiang Normal University,
Jinhua 321004, China
3
Institute of Matria Medica,
Zhejiang Academy of Medical Sciences,
Hangzhou 310013, China
Received 24 December 2008; Accepted 09 March 2009
Abstract:Two novel norcantharidin acylamide acids (HL1=N-pyrimidine norcantharidin acylamide acid, C12H13N3O4; HL2=N-pyridine
norcantharidin acylamide acid, C13H14N2O4) were synthesized by a reaction of norcantharidin(NCTD) with 2-aminopyrimidine and
2-aminopyridine, respectively. Their structures were characterized by elemental analysis, IR, UV and 1 H NMR. Fluorescence titration
and viscosity measurements indicated that HL1, HL2 and HL3 (HL3=N-phenyl norcantharidin acylamide acid, C14H15NO4) can bind calf
thymus DNA via partial intercalation. The liner Stern–Volmer quenching constant Ksv values for HL1, HL2 and HL3 were 2.05 × 104 L mol-1,
1.15 × 104 L mol-1 and 8.30×103 L mol-1, respectively. Two compounds containing heterocycle of HL1 and HL2 have been found to
cleave pBR322 plasmid DNA at physiological pH and temperature. The test of antiproliferation activity showed that the compounds
had moderate to strong antiproliferative ability against the tested cell lines except of HL3 against the SMMC7721 cell line. The results
indicated that the heterocycle attached to the norcantharidin was favorable to antiproliferative activity. This result was consistent with
the DNA binding experiment.
Keywords: Norcantharidin • Pyrimidine • Pyridine • DNA binding • Antiproliferative activity
© Versita Warsaw and Springer-Verlag Berlin Heidelberg.
1. Introduction
Many clinically successful anticancer drugs are
themselves either naturally occurring molecules or have
been developed from their synthetic analogs. Great
interest is currently being focused on developing natural
products due to their interesting anticancer activities
[1-4]. Cantharidin has been used as a medicinal agent
and shown to be active in cervical, leukaemia, bladder
and colon cancer cell lines [5-7]. Although cantharidin
is cytotoxic to cancer cells and stimulatory on the bone
marrow, the renal toxicity of this drug has prevented its
use in mainstream oncology. Norcantharidin (Fig. 1a),
the demethylated analogue of cantharidin, also
possesses anticancer activity and stimulates the bone
marrow, but its nephrotoxicity weakens significantly
[8]. Many derivatives of norcantharidin have been
synthesized and some of them have good anti-tumor
activity in vitro [9-10].
To improve the action of drugs with DNA in cell
and assess the effect of varying the aromatic ring and
hydrophilicity of acylamide on the DNA-binding structure,
* E-mail: [email protected]
569
Synthesis, DNA-binding and antiproliferative
activity of N-(Nitrogen heterocyclic)
norcantharidin acylamide acid
Figure 1.
Structures of NCTD(a), HL1(b), HL2(c) and HL3(d).
two new norcantharidin acylamide acid HL1 (Fig. 1b) and
HL2 (Fig. 1c) were synthesized. DNA-binding properties
of the compounds were studied by luminescent spectra
and viscosity measurement. DNA cleavages of the
compounds are also demonstrated. Furthermore, the
antiproliferative activities of the compounds toward
human hepatoma cells SMMC7721 and human lung
cancer cells A549 in vitro were also evaluated with MTT
cell viability assay.
2. Experimental Procedures
2.1 Reagents and Instruments
Norcantharidin (NCTD, C8H8O4) was obtained from
Suzhou Sunray Pharmaceutical Co. 2-aminopyrimidine,
2-aminopyridine and aniline were purchased from
Sinopharm Chemical Reagent Co. Aniline and THF
(Analytical Reagent) were distilled before use. MTT
(3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium
bromide) was purchased from the Sigma Company.
Calf thymus DNA (ct-DNA, Huamei Co.) was dissolved
in 50 mmol L−1 NaCl, 5 mmol L−1 Tris–HCl (pH 7.4).
Solutions of ct-DNA gave ratios of UV-vis absorbance
of 1.8 at 260 and 280 nm, indicating that the DNA was
sufficiently free of protein [11]. Plasmid DNA (pBR322)
purchased from Shanghai Biologic Engineering Co. was
diluted to 25 μg mL−1. Double-distilled water was used
to prepare buffer solutions. Human hepatoblastoma
cells (SMMC7721) and human lung cancer cells (A549)
were purchased from Shanghai Institute of Cell Bank,
Shanghai, China.
Elemental analyses of C, H and N were carried out
in a Vario EL III elemental analyzer. The melting points
of all compounds were determined by the capillary
method on a Büchi 510 melting point apparatus and
were uncorrected. Infrared spectra were recorded as
KBr pellet by using a NEXUS-670 FT-IR spectrometer.
Ultraviolet-visible spectra were obtained on a UV-2501PC
spectrophotometer. 1H NMR was run on a Brucker
Avance 400MHZ NMR spectrometer using trimethylsilyl
(TMS) as standard internal reference. The chemical
shifts are reported in ppm (δ scale). Fluorescence
emission spectra were recorded with a Perkin-Elmer
570
LS-55 spectrofluorometer. Agarose gel electrophoresis
was performed on PowerPac Basic electrophoresis
apparatus (BIO-RAD). Gel image formation were
obtained on UNIVERSAL HOOD 11-S.N. (BIO-RAD
Laboratories). Sheldon CO2 culture box and East-China
Electronic tube Factory DG3022A ELISA instruments
were used to perform antiproliferative activity.
2.2 Preparation of the N-pyrimidine norcantharidin
acylamide
acid
(HL1=(1S,4R)-3(pyrimidine-2-ylcarbamoyl)-7-oxa-bicyclo
[2.2.1] heptane-2-carboxylc acid)
30 mL THF solution containing 2-aminopyrimidine
(2.36 g, 25 mmol) was added dropwise to another THF
solution containing norcantharidin (4.12 g, 25 mmol).
The mixture was stirred for 10 hours at room temperature
and a white precipitate was formed. The precipitate
was filtered and washed with acetonitrile. Then the
compound was recrystallized from 95% ethanol,
and dried for 2 hours in a vacuum. Yield 88%, mp:
104-106oC. Anal. Calcd. for HL1 (C12H13N3O4): C, 54.75;
H, 4.98; N, 15.96. Found: C, 54.23; H, 5.26; N, 15.38. IR
(KBr pellet, cm-1): ν(N-H) 3339(s); ν(C=O) (COOH) 1703(s);
ν(C=O) (CONH) 1669(s); ν(pyrimidine, C=N) 1627(s);
ν(C-O-C) 1170(m). UV-Vis: 293.5 nm, 220.5 nm.
1
H NMR (DMSO-d6), δ (ppm): 6.52-6.56(m, 3H, ArH); 4.66-4.67(m, 2H, C1C4-H); 2.90(d, 2H, C2C3-H);
1.48-1.56(m, 4H, C5C6-H).
2.3 Preparation of the N-pyridine norcantharidin
acylamide acid (HL2 =(1S,4R)-3-(pyridine2-ylcarbamoyl)-7-oxa-bicyclo
[2.2.1]
heptane-2-carboxylic acid)
HL2 was prepared in a manner similar to that of HL1 except
for 2-aminopyridine instead of 2-aminopyrimidine. Yield
92%, mp: 147-148 oC. Anal. Calcd. for HL2 (C13H14N2O4):
C, 59.54; H, 5.38; N, 10.68. Found: C, 59.60; H,
5.43; N, 9.90. IR (KBr pellet, cm-1): ν(N-H) 3316 (s);
ν(C=O) (COOH) 1714(s); ν(C=O) (CONH) 1662(s); ν(pyridine,
C=N) 1622(s); ν(C-O-C) 1172(m). UV-Vis: 299.4 nm,
229.6 nm. 1H NMR (DMSO-d6), δ (ppm): 6.42-7.88
(m, 4H, Ar-H), 4.64-4.65(m, 2H, C1C4-H), 2.90(d, 2H,
C2C3-H), 1.47-1.55(m, 4H, C5C6-H).
W-Z. Zhu et al.
2.4 Preparation of the N-phenyl norcantharidin
acylamide acid (HL3= (1S,4R)-3-Phenyl
carbamoyl-7-oxabicyclo [2.2.1] heptane2-carboxylic acid)
HL3 was prepared by using a method that is similar
to that found in literature [12]. Anal. Calcd. for HL3
(C14H15NO4): C, 64.36;H, 5.78; N, 5.36. Found: C,
64.77; H, 5.98; N, 5.09. IR (KBr pellet, cm-1): ν(N-H)
3302 (s); ν(C=O) (COOH) 1720(s); ν(C=O) (CONH) 1681(s);
ν(benzene) 1601(s); ν(C-O-C) 1170(m). UV-Vis:
240.8 nm. 1H NMR(DMSO-d6), δ(ppm): 11.96(s, 1H,
COOH); 9.65(s, 1H, NH); 6.99-7.53 (m, 5H, Ar-H);
4.62-4.79(m, 2H, C1C4-H), 2.90(d, 2H, C2C3-H),
1.49-1.61(m, 4H, C5C6-H).
2.5 DNA binding and cleavage experiments
Fluorescence quenching experiments were carried out
by adding different concentrations (0 - 108 μmol L-1)
ct-DNA solution (3.74 × 10-4 mol L-1) to the samples
containing 40 μmol L-1 compounds solution and TrisHCl buffer (PH 7.4). After incubation for 4 hours at 4°C,
fluorescence measurements were excited at 280 nm
and emission was observed between 300 and 500 nm.
Viscosity experiments were conducted on an
Ubbelohde viscometer, immersed in a thermostatic
water-bath maintained to 30°C. Each compound was
introduced into a DNA solution (3.74 × 10-4 mol L-1) by
microsyringe. The average values of three replicated
measurements were used to evaluate the viscosity of
the samples. Data is represented as (η/η0)1/3 vs. the ratio
of the concentration of the compound to DNA, where
η and η0 are the viscosity of DNA in the presence and
absence of compounds, respectively. Viscosity was
calculated from the observed flow time of DNA solution
in the presence of compounds (t), corrected from the
flow time of buffer alone (t0), η=(t- t0) / t0.
The compounds were incubated with the pBR322
plasmid DNA in order to understand whether any
interaction occurred. The incubation time lasted for 3 h at
37°C until 0.25% bromophenol blue and 1 mmol L-1 EDTA
were added. The DNA cleavage products were subjected
to electrophoresis on 1.0% agarose gel containing
0.5 μg mL-1 ethidium bromides. The gels were run at
110 V for 1.2 h in Tris-borate - ethylenediaminetetraacetic
acid (TBE) buffer and the bands were photographed.
2.6 Antiproliferative activity evaluation
Growth of cells in the exponential phase were assayed
in 96-well plates by adding 100 μL stock solution directly
to culture wells. After the cells were seeded for 24 h,
the HL and NCTD were added. Then the cells were
incubated for 72 h, followed by adding 100 μL MTT
(1 mg mL-1, dissolved in DMEM nutrient solution) into
each well for 4 h at 37°C. Later, the liquid in each well
was discarded and then 150 μL acidifying isopropanol
(containing 0.04 mol L-1 HCl) was added. The mixture
was placed in the dark for 30 min. The inhibition rate
and IC50 were calculated [13].
3. Results and Discussion
3.1 Characterization of HL1 and HL2
The acylamide acids were prepared by direct reaction
of norcantharidin with appropriate mole ratios of
2-aminopyrimidine, 2-aminopyridine and aniline in THF.
The yields were good. The desired compounds were
separated from solution by suction filtration and purified
by recrystallization with ethanol. The compounds have
been visibly characterized through UV-vis and IR
spectral analysis.
The main UV-Vis absorption spectra of the
compounds at 220.5 nm (HL1) and 229.6 nm (HL2) are
ascribed to the 1La(π-π*) transition of the pyrimidine
and pyridine. The bands at 293.5 nm (HL1) and
299.4 nm (HL2) are ascribed to the 1Lb (π-π*) transition
of the pyrimidine and pyridine [14], respectively.
IR absorption spectra of the compounds have
been tested. The bands at 3339 cm -1 and 3316 cm-1
are assigned to the N-H stretching vibration of HL1 and
HL2, respectively. The amide I band consists mainly
of ν(C=O), and the amide II and III bands arise from
δ(N-H) as well as from ν(C-N), although these modes
are coupled to one another [15]. So the bands at
1669 cm-1 (HL1) and 1662 cm-1 (HL2) are the amide
I band that consist of ν(C=O), and the bands at 1541 cm-1
(HL1), 1540 cm-1(HL2) are amide II bands that arise
from δ(N-H). Meanwhile, at the 1243 ~ 1245 cm-1 bands
of amide III from ν(C-N) are observed. The bands at
1703 cm-1 and 1714 cm-1 belong to the ν(C=O) in carboxyl
of HL1 and HL2, respectively. The compounds exhibits
one sharp band at 1627 cm-1(HL1) and 1622 cm-1(HL2)
due to ν(C=N) of heterocyclic stretching vibration.
3.2.DNA binding and cleavage studies
3.2.1 Fluorescence spectral studies
The interaction mechanism of compounds molecules
with DNA can be studied by fluorescence spectra. When
the compounds combine with DNA, their emission
spectra and lifetime of excited state will change. The
change of the fluorescence spectra will indicate that
the compounds combined with DNA in a certain form.
It is reported that if the emission of the compounds
decreases obviously with the amount of DNA increasing,
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Synthesis, DNA-binding and antiproliferative
activity of N-(Nitrogen heterocyclic)
norcantharidin acylamide acid
Figure 2.
Fluorescence spectra of the combination HL1(a), HL2(b) and HL3(c) in the absence and presence of increasing amounts DNA,
λex = 280 nm, λem = 300 – 500 nm. Arrows show the intensity changes upon increasing concentration of the DNA.
For(a) [HL1]=40 μmol L-1, [DNA]/10-5 mol L-1: (1): 0; (2): 1.8; (3): 3.6; (4): 5.4; (5): 7.2; (6): 9.0; (7): 10.8. For(b) [HL2]=40 μmol L-1 ,
[DNA]/10-5 mol L-1: (1): 0; (2): 1.8; (3): 3.6; (4): 5.4; (5): 7.2; (6): 9.0; (7):10.8. For(c) [HL3]=40 μmol L-1, [DNA]/10-5 mol L-1: (1): 0; (2): 1.8;
(3): 3.6; (4): 5.4; (5): 7.2; (6): 9.0; (7):10.8.
the compounds interact with DNA by intercalation fully
[16] or partially [17].
The reduction in emission intensity of HL with
increasing ct-DNA concentration is shown in Fig. 2. In
the absence of DNA, HL1, HL2 and HL3 emit intense
luminescence in Tris-HCl buffer at ambient temperature,
with a maximum appearing at 362 nm (HL1) Fig. 2a,
355 nm (HL2) Fig. 2b and 348 nm (HL3) Fig. 2c,
respectively. When DNA is present the intensity
of the emission for HL1, HL2 and HL3 all decrease.
This phenomenon of the luminescence property of
compounds in presence of DNA strongly supports
that the compounds bind to double-stranded DNA by
intercalation. The stacking of the compound with the
base pairs of DNA leads to transfer of electron and
energy from the aromatic ring of compound to the base
pairs [18] and the nitrogen atoms of aromatic ring in HL1
and HL2 play a beneficial role in the accumulation. This
maybe relative to the hydrogen bonds formed between
nitrogen atoms of heterocycle and base pairs of the
DNA. As a result, HL1 containing pyrimidine can stack
with base pairs of DNA more easily than HL2 containing
pyridine. According to the Stern–Volmer equation [19]:
I0/I = 1+ Ksv [Q], where I0 and I represent the fluorescence
572
intensities in the absence and presence of the DNA,
respectively. [Q] is the concentration of DNA. The Ksv
values for HL1, HL2 and HL3 are 2.05 × 104 L mol-1,
1.15 × 104 L mol-1 and 8.30 × 103 L mol-1, respectively. It
can be found that the luminescence reduce of HL1 was
more obviously than that of HL2 and HL3.
3.2.2 Viscosity study
To further confirm the interaction mode of the
compounds with DNA, a comparative viscosity study
between the NCTD and the HL were carried out
(Fig. 3). Hydrodynamic measurements which are
sensitive to length increase (for example, viscosity,
sedimentation) are regarded as the least ambiguous
and the most critical tests of binding mode in solution
in the absence of crystallographic structure data or
NMR [20]. A classical intercalative mode thinks that
significant increase in viscosity of DNA solution is due
to increase in lengthening the DNA helix, as base pairs
are separated to accommodate the binding compound.
However, a partial and/or nonclassical intercalation
of compounds may bend (or kink) the DNA helix,
resulting in the decrease of its effective length and,
concomitantly, its viscosity [21]. As shown in Fig. 3, the
W-Z. Zhu et al.
(η/η0)1/3
effects of HL and NCTD on the viscosity of ct-DNA at
30.0oC were investigated. It is found that the presence
of NCTD has no obvious effect on the relative viscosity
of DNA, whereas the HL decreases the relative viscosity
of DNA. The NCTD can not insert the DNA double helix,
because of the rigid non-planarity of the six-membered
ring and the hindering effect of ether oxygen atom in
NCTD. HL molecule which contained planer aromatic
ring could insert into DNA base pairs. The reduced
degree of DNA viscosity for HL1 is stronger than that for
HL2 and HL3. This result demonstrates that the increased
number of nitrogen atoms of heterocycle could form
more hydrogen bonds with base pairs of DNA, which
facilitates intercalation of compounds to DNA.
Fluorescent titration experiment and viscosity
measurement suggest that the compounds may bind
to DNA by partial intercalation. The binding ability with
DNA decreased in following order: HL1, HL2, and HL3.
3.2.3 Gel eletrophoresis studies
The cleavage reaction on plasmid DNA can be monitored
by agarose gel electrophoresis. When circular plasmid
DNA is subjected to electrophoresis, relatively fast
migrations will be observed for the intact supercoil form
(Form I). If scission occurs on one strand (nicking), the
supercoil will partial relax to generate a slower-moving
open circular form (Form II) [22].
The ability of NCTD and HL in effecting DNA
cleavage has been investigated by gel electrophoresis
using supercoiled pBR322 DNA (Fig. 4 and Table 1).
Initially, in the untreated pBR322 plasmid DNA (lane 1)
does not show apparent cleavage of DNA. The cleavage
efficiency of HL1 (Fig. 4a, lane 4) and HL2 (Fig. 4b,
lane 4) can cleave 68% and 43% the supercoiled plasmid
DNA (Form I) to partial nicked circular DNA (Form II
at 400 μM, respectively. In contrast to HL1 and HL2,
NCTD and HL3 (Table 1) cannot cleave the supercoiled
plasmid DNA. And the observed DNA cleavage activity
of HL1 and HL2 are believed to form monofunctional
adducts with Guanine. The monofunctional adducts
could close to form initially bifunctional interstrand GC
adducts that can evolve into interstrand GG adducts.
When bifunctional interstrand adducts is formed, planar
compounds containing nitrogen heterocycle, HL1 and
HL2 will be positioned along the helix axis so that they
will push apart adjacent base pairs [23]. All these results
are well consistent with spectroscopic and viscosity
measurement.
3.3 Antiproliferative activity evaluation
r(CCompound/CDNA)
Figure 3. Effect of increasing amounts of compounds on the relative
viscosities of ct-DNA at 30oC. [DNA] = 3.72 × 10-4 mol L-1,
r = [compound]/[DNA]. (1): r = 0; (2): r = 0.09;
(3): r = 0.18; (4): r = 0.27; (5): r = 0.36; (6): r = 0.45;
(7): r = 0.54; (8): r = 0.63, respectively.
The antiproliferative activity of HL and NCTD against two
kinds of tumor cells (human hepatoma cells SMMC7721
and human lung cancer cells A549) were studied, the
results of which are listed in Fig. 5. From the figure, the
average inhibitory ratio against SMMC7721 (Fig. 5a)
and A549 (Fig. 5b) cancer cell lines increases with the
Figure 4. Electrophoretic separation of pBR322 DNA induced by HL1(a) and HL2(b), [DNA] = 3 µg mL-1. lane 1: DNA alone; lane 2: DNA+compound
(100 µM); lane 3: DNA+compound (200 µM), lane 4: DNA+ compound (400 µM).
573
Synthesis, DNA-binding and antiproliferative
activity of N-(Nitrogen heterocyclic)
norcantharidin acylamide acid
Table 1.
Comparison of the pBR322 DNA cleavage efficiency at different concentrations of NCTD and HL.
Compounds
Form / %
DNA control
NCTD
HL
1
HL2
HL
3
Figure 5.
DNA + Compound
(100 µM)
DNA + Compound
(200 µM)
DNA + Compound
(400 µM)
77
I
81
80
80
II
19
20
20
23
I
81
71
75
32
II
19
29
25
68
I
81
76
67
57
II
19
24
33
43
I
81
78
74
68
II
19
22
26
32
Effect of the NCTD and compounds at different concentrations for 72 hours on the proliferation of tumor cell. All assays were performed
in triplicate for three independent experiments. (a): SMMC7721; (b): A549. Data are expressed as mean±S.D. (n = 3).
Table 2. 72 h IC50 (μM) values of test compounds in SMMC7721 and A549 cells lines. Data represent mean±S.D. All assays were performed in
triplicate for three independent experiments.
Compounds
a
IC50 (μM)a
NCTD
HL
HL2
HL3
SMMC7721
115.5 ± 9.5
84.2 ± 4.1
111.0 ± 5.7
> 200
A549
88.7 ± 12.8
126.8 ± 9.4
190.0 ± 9.3
163.6 ± 33.4
1
IC50 (μM) was given as the concentration at 50% inhibition of cell growth.
increasing of the concentration of HL. Generally, the
anti-proliferative activities of HL against the SMMC7721
cell lines are stronger than toward A549. The order of
anti-proliferative activity against the tumor cell lines
in the range of tested concentration (25 – 200 μM)
is: HL1>HL2>HL3. As shown in Fig. 5a, at higher dose
(200 μM), the inhibition rates of HL1 (88.3 ± 3.1%) and
HL2 (85.4 ± 1.2%) are much higher than that of NCTD
(66.1 ± 3.2%) against SMMC7721 cell lines, respectively.
In the range of tested concentration, HL3 almost showed
no anti-proliferative activity against the SMMC7721
cell lines, whereas as the dose is increased, HL3 have
obvious inhibition against A549.
The concentration of the compounds for 50%
inhibition (IC50) on the SMMC7721 and A549 cell
lines were determined and the results were listed in
Table 2. As shown in Table 2, IC50 of the HL1 and
HL2 were lower (84.2 ± 4.1 μM and 111.0 ± 5.7 μM,
respectively) compared with NCTD (115.5 ± 9.5 μM) for
574
the SMMC7721 cell lines. The results indicated that the
nitrogen-heterocycle attached to the norcantharidin is
favorable for antiproliferative activity. It is consistent with
the results of the DNA binding experiment. Therefore,
we think that it is more likely that the molecular target
contributing to this antiproliferative activity of this
compound is not only the proteins [24], but also DNA.
4. Conclusions
Two novel norcantharidin acylamide acids of nitrogenheterocycle were synthesized and characterized. The
HL can bind to DNA by partial intercalation binding
modes. The compounds of HL1 and HL2 have been
found that they not only bind to ct-DNA but also cleave
pBR 322 DNA at physiological pH and temperature.
The results showed that HL1 and HL2 have stronger
anti-proliferative activity to SMMC7721 cells than to A549
W-Z. Zhu et al.
cells. The inhibition activity of HL1 (IC50 = 84.2 ± 4.1 μM)
are much higher than that of NCTD (IC50 = 115.5 ± 9.5 μM)
against SMMC7721 cell lines. So HL1 seems to be well
suited for further work aiming at the development of new
anticancer agents. These observations suggested that
the structure of side chains attached to NCTD plays an
important role in the binding extent with DNA and in the
anti-proliferative effect against cancer cells. However,
further studies are necessary.
Acknowledgments
The authors would like to thank the financial support from
the Natural Science Foundation of Zhejiang Province,
China (No. Y407301).
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