Bis-sulfonyl-chalcone-BODIPY molecular probes for in vivo and in vitro imaging

Article abstract of DOI:10.1016/j.molstruc.2021.130201

BODIPY derivatives have attracted much attention in cell imaging, so its excellent cell permeability has become the focus of extensive research. In this paper, different bis-sulfonyl chalcone-BODIPYs cell targeting probes were designed, synthesized and ch

Full text of DOI:10.1016/j.molstruc.2021.130201

Journal of Molecular Structure 1234 (2021) 130201
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Bis-sulfonyl-chalcone-BODIPY molecular probes for in vivo and in vitro
imaging
Zhixiang Lva, Yuling Wang d, Jinliang Zhangb, Zhou Wang c, Guofan Jind,∗
a The People’s Hospital of Danyang, Affiliated Danyang Hospital of Nantong University, Zhenjiang, 212300, P.R. China
b Zhucheng Maternal and Child Health Hospital, Zhucheng 262200, P.R. China
c College of Vanadium and Titanium, Panzhihua University, 617000, P.R. China
d School of Pharmacy, Jiangsu University, Zhenjiang 212013, P.R. China
a r t i c l e i n f o
a b s t r a c t
Article history:
BODIPY derivatives have attracted much attention in cell imaging, so its excellent cell permeability has
become the focus of extensive research. In this paper, different bis-sulfonyl chalcone-BODIPYs cell tar-
geting probes were designed, synthesized and characterized. The cell viability experiment, cell imaging,
flow cytometry, apoptosis, and in vivo experiments in mice were conducted to analyze them. The re-
sults showed that bis-methanesulfonyl chalcone-BODIPY had better activity evaluation on HeLa cells, and
Received 7 January 2021
Revised 19 February 2021
Accepted 22 February 2021
Available online 28 February 2021
the data of IC50 decreased from 79.71
3.84 to 56.10
8.51. The three compounds have amazing cell
Keywords:
Bis-sulfonyl chalcone-BODIPY
Fluorescence sensor
Target cell imaging
Cell permeability
imaging, especially compounds 5 and 6 are combined with the nucleus completely, showing strong cell
permeability. In addition, in vivo imaging of mice experiments showed strong fluorescence and poten-
tial tumor targeting. In the molecular docking simulation, compounds 5 and 6 have high affinity scores
with CDK2 of tumor cells: -8.4 and -8.6 kcal•mol−1, and there are hydrogen bond, π - π, T - π, hy-
drophobic bond, ion-π and other interactions between them, which can well bind the compounds to the
targets. These results indicate that bis-sulfonyl chalcone-BODIPYs have promising targeting capability and
biocompatibility.
Molecular docking study
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Sulfonyl group is a good leaving group [6], and its deriva-
tives have extensive anti-inflammatory and anti-tumor activities,
In recent years, the field of targeted probes has become more
and more abundant, but most probes have strong cytotoxicity
and low cell permeability. To improve the probe, researchers of-
ten introduce small molecules or groups. Chalcone is a kind of
small molecule substance widely existing in natural products. The
framework of α, β-unsaturated ketone skeleton with two benzene
rings has attracted the attention of chemists and pharmacists due
to its unique structure and wide range of physiological activities
[1–3]. The α, β-unsaturated carbonyl group in chalcone makes
it have biological activity, therefore its derivatives show impor-
tant anti-tumor, anti-cancer, antibacterial, anti-inflammatory, an-
timalaria and antiviral properties. Its derivatives can inhibit the
formation of thrombin showed the potential activity against plas-
modium falciparum [4]. It can also be used as the precursor of
flavonoids and isoflavones, or designed as the synthesizers of novel
heterocyclic compounds and a series of drug analogues [5].
so it is widely used in drug modification. Noscapine was modified
with benzenesulfonyl to improve its biocompatibility with tubu-
lin protein and its anti-proliferative activity against different tumor
cells [7]. BODIPY is modified with sulfonyl to enhance the water-
soluble and biological compatibility of the dye, which can be bet-
ter used for membrane potential imaging [8]. In addition, sulfonyl-
containing drugs such as antibacterial, anti-inflammatory, and anti-
tumor are widely used in clinical [9–11], hence, it can be seen that
the reasonable introduction of small molecules or groups in the
compound design will have an incredible effect.
Among the targeted probes, BODIPY is a kind of typical high
fluorescence dye, which involves a wide range of research fields,
such as material research, medical research, medical diagnosis and
treatment, environmental detection and so on. Compared with
other fluorescent materials, it has high fluorescence quantum yield,
good light stability, strong absorption and emission [12–14], ap-
propriate stokes shift, and even its maximum absorption peak can
reach the near-infrared region after certain modification [15–17]. It
is one of the most promising dyes to improve photodynamic ther-
apy (PDT) [18,19], making use of its good stability characteristics to
∗
Corresponding author:
E-mail address: organicboron@ujs.edu.cn (G. Jin).
https://doi.org/10.1016/j.molstruc.2021.130201
0022-2860/© 2021 Elsevier B.V. All rights reserved.

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
produce hypoxia sensors or hydroxylamine [20], lactic acid sensors
[21, 22]. Up to now, BODIPY derivatives have been proved to be
promising bioanalytical reagents for in-vivo fluorescence imaging
and diagnosis, and have been used for DNA and protein labeling
widely [23–26]. However, The BODIPY probe itself is toxic, and it
has been a long cherished wish of researchers in this field to seek
BODIPY with lower toxicity and higher cell permeability.
(E)-3-(1-(difluoroboranyl)-5-((Z)-(4-ethyl-3,5-dimethyl-2H-
pyrrol-2-ylidene)(phenyl)methyl)-2-(propylamino)-1H-pyrrol-
3-yl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-one
(4).
20
mL
acetonitrile was added into 3 (1.0 g, 2.43 mmol) and 1-(2,4-
dihydroxyphenyl) ethan-1-one (1.11 g, 7.29 mmol), 0.5 mL
piperidine was added dropwise under magnetic stirring, and
the reaction was stopped after reflux at 80°C for 2 h. After the
reaction was confirmed by TLC, the reaction solution was cooled
to room temperature and product (0.3 g, 25%)was purified by
column chromatography (petroleum ether: ethyl acetate = 8: 1).
1H NMR (400 MHz, CDCl3): δ (ppm) = 7.22 (t, J = 7.6 Hz, 2H),
7.08 (d, J = 6.8 Hz, 1H), 6.08 (d, J = 8.0 Hz, 1H), 6.11 (d, J = 8.0
Hz, 2H), 5.94 (s, 1H), 5.86 (s, 1H), 3.30 (s, 1H), 2.68 (d, J = 8.8 Hz,
6H), 2.47 (s, 2H), 2.34-2.29 (m, 2H), 1.57 (q, J = 7.2 Hz, 2H), 1.28
(d, J=5.2 Hz, 4H), 0.97 (t, J = 7.6 Hz, 3H), 0.86 (t, J = 7.6 Hz, 3H).
13C NMR (100 MHz, CDCl3): δ (ppm) = 205.7, 160.3, 158.8, 158.0,
143.3, 135.5, 135.2, 132.6, 131.3, 130.5, 129.7, 129.6, 128.2, 127.9,
125.1, 120.8, 110.2, 106.6, 45.6, 40.4, 34.1, 33.4, 29.7, 24.2, 17.2,
15.0, 11.8, 11.4, 10.8. FT-IR v/cm−1: 3443, 2923, 2360, 1600, 1437,
1373, 1334, 1221, 1125, 1043. ITMS (ESI+) m/z: [M+H]+ calcd for
C31H32BF2N3O3 544.2583, found 544.3423.
Herein, we designed different bis-sulfonyl chalcone-BODIPY flu-
orescent probes to reduce the toxicity and improve the biocompat-
ibility of target. The cytotoxicity of the three compounds to HCT-
116 and HeLa cancer cells was determined by MTT assay. In or-
der to understand its biological imaging more comprehensively, we
conducted molecular docking, cell experiment in vitro, flow cytom-
etry, apoptosis experiment and in vivo experiment in mice. In ad-
dition, the three compounds synthesized by us have the potential
properties of chalcone drugs, which will be a new breakthrough in
the field of BODIPY research.
2. Experimental section
2.1. Reagents and instrumentation
(E)-4-(3-(8-ethyl-5,5-difluoro-7,9-dimethyl-10-phenyl-
3-(propylamino)-5H-5λ4,6λ4-dipyrrolo[1,2-c:2ꢀ,1ꢀ-
f][1,3,2]diazaborinin-2-yl)acryloyl)-1,3-phenylene
All solvents are commercially purchased and further purified.
Dichloromethane and acetonitrile were distilled over calcium hy-
dride. All the reaction products were separated and purified by col-
umn chromatography (200-300 mesh silica gel). The reaction pro-
cess was detected by TLC and analyzed by UV lamp at 254 nm
and 365 nm. NMR were measured by Bruker avance II instrument
in deuterium chloroform (400 MHz for 1H and 100 MHz for 13C).
Chemical shifts are reported in ppm, versus internal tetramethyl-
silane as a standard. The mass spectrum of 4 was obtained on a
Thermo LXQ by liquid chromatgraphy-ion trap mass spectrometry,
and the high resolution mass spectrometry analysis of 5 and 6 was
performed by Micromon technical corporation, China. The infrared
of the samples was recorded by Nicolet avato-370 FT-IR analyzer
and tested by KBr tablet. UV-Vis absorption spectroscopy were
recorded by UV-2550 spectrophotometer. The fluorescence emis-
sion spectra were recorded using a Shimadzu RF-5301PCS spec-
trofluorophotometer.
dimethane-
mL
sulfonate (5). 4 (0.1 g, 0.18 mmol) was dissolved in
8
dichloromethane solution. Triethylamine (0.15 mL, 1.08 mmol) and
methylsulfonyl chloride (70 μL, 0.90 mmol) were added under
magnetic stirring at room temperature, and the reaction was
stopped after the white smoke dispersed. Orange red solid 5 (60
mg, 47%) was obtained using column separation and purification
(petroleum ether: ethyl acetate = 3: 1). 1H NMR (400MHz, CDCl3):
δ (ppm) = 7.33 (t, J = 6.0 Hz, 3H), 7.20 (d, J = 8.0 Hz, 2H), 7.15 (d,
J = 8.8 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 6.11 (s, 1H), 5.79 (s, 1H),
3.21 (s, 5H), 2.76 (s, 5H), 2.46 (s, 3H), 2.33-2.28 (dd, J = 14.8 Hz,
7.2 Hz, 2H), 1.32 (s, 3H), 0.97 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 7.2
Hz, 3H).13C NMR (100 MHz, CDCl3): δ (ppm) = 203.6, 161.0, 157.3,
147.8, 144.6, 143.5, 135.1, 134.6, 132.6, 132.1, 132.0, 130.3, 129.9,
129.8, 129.7, 128.2, 127.9, 122.5, 114.5, 111.8, 45.6, 38.5, 35.0, 32.5,
24.3, 17.1, 15.0, 11.9, 11.5, 10.8. FT-IR v/cm−1: 2928, 2363, 1593,
1460, 1373, 1215, 1182, 1122, 1023. TOF-MS (ES+) m/z: [M+H]+
calcd for C33H36BF2N3O7S2 700.2134, found 700.2139.
(E)-4-(3-(8-ethyl-5,5-difluoro-7,9-dimethyl-10-phenyl-
3-(propylamino)-5H-5λ4,6λ4-dipyrrolo[1,2-c:2ꢀ,1ꢀ-
2.2. Synthesis
f][1,3,2]diazaborinin-2-yl)acryloyl)-1,3-phenylene
dibenzene-
mL
3-chloro-8-ethyl-5,5-difluoro-7,9-dimethyl-10-phenyl-
5H-5λ4,6λ4-dipyrrolo[1,2-c:2ꢀ,1ꢀ-f][1,3,2]diazaborinine-2-
carbaldehyde (2) [27]. N,N-Dimethylformamide (5 mL) and
POCl3 (5 mL) were mixed and stirred for 5 min under the con-
dition of ice water bath and argon protection. The mixture was
continuously stirred for 30 min at room temperature. After that,
BODIPY-Cl [28] (1.5 g, 4.2 mmol) and 1,2-dichloroethane (30 mL)
were added and stirred at 50°C for 3 h. After cooling to room tem-
perature, saturated NaHCO3 aqueous solution was added slowly
under ice bath conditions. Dichloromethane and water were added
for extraction after stirring for 1 h at 25°C. Anhydrous MgSO4
was added into the organic phase and filtered, and then separated
by column chromatography (ethyl acetate: hexane = 40: 1) after
vacuum distillation to afford 2 (1.33 g, 82%).
sulfonate (6). 4 (0.1 g, 0.18 mmol) was dissolved in
8
dichloromethane solution, and triethylamine (0.15 mL, 1.08 mmol)
and benzenesulfonyl chloride (120 μL, 0.94 mmol) were dropped
under magnetic stirring at room temperature. The reaction was
stopped after the reaction was confirmed by TLC. Purplish red
solid 6 (75 mg, 50%) was obtained by column chromatography
(petroleum ether: ethyl acetate = 5: 1). 1H NMR (400MHz, CDCl3):
δ (ppm) = 8.06 (d, J = 8.0 Hz, 3H), 7.78-7.69 (m, 4H), 7.66-7.59
(m, 6H), 7.45-7.41 (m, 5H), 5.93 (s, 1H), 5.67 (s, 1H), 2.53 (s, 2H),
2.37 (d, J = 6.4 Hz, 6H), 1.51-1.43 (m, 2H), 1.39-1.28 (m, 5H), 1.04
(t, J = 7.6 Hz, 3H), 0.77 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) = 196.9, 156.4, 145.7, 144.4, 142.8, 135.3, 135.2,
135.1, 134.1, 132.3, 132.0, 131.8, 131.3, 130.1, 129.7, 129.6, 129.4,
129.4, 128.7, 128.5, 128.3, 128.3, 127.0, 122.0, 121.1, 60.4, 53.5, 46.2,
36.0, 31.5, 30.2, 23.1, 17.3, 15.0, 12.0, 11.5, 10.9. FT-IR v/cm−1: 2923,
2360, 1710, 1592, 1458, 1383, 1216, 1188, 1083. TOF-MS (ES+) m/z:
[M+H]+ calcd for C43H40BF2N3O7S2 824.2447, found 824.2446.
8-ethyl-5,5-difluoro-7,9-dimethyl-10-phenyl-3-(propylamino)-
5H-5λ4,6λ4-dipyrrolo[1,2-c:2ꢀ,1ꢀ-f][1,3,2]diazaborinine-2-
carbaldehyde (3) [27]. Acetonitrile (20 mL) and
1 drop of
propylamine were added to 2 (1.0 g, 2.6 mmol) and stirred for
4 h at room temperature. After decompression spin evaporation,
ethyl acetate and aqueous solution were added for extraction, and
anhydrous MgSO4 was added into the organic phase and filtered. 3
(1.35 g, 89%) was purified by flash chromatography (hexane: ethyl
acetate = 30:1).
2.3. Cytotoxicity determination
HCT-116, HeLa, and normal liver L-02 cells from American type
culture collection were screened for cytotoxicity in vitro. HCT-116
2

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
and HeLa cells were routinely cultured in RPMI-1640 medium,
while L-02 cells were routinely cultured in dulbecco’s modified
eagle medium (DMEM). 10% fetal bovine serum (FBS) was added
to the medium, and the cells were incubated a cell incubator at
37°C with 5% carbon dioxide. These cells were monitored daily and
maintained cell density at 80%.
Shanghai). Then they were incubated in dark at room temperature
for 25 min and detected by flow cytometry (ATTUNE NXT, Lifetech,
lnc). All test methods are in triplicate.
3. Results and discussion
The cytotoxicity of HCT-116, HeLa cells and normal lung L-02
cells were measured at logarithmic growth phase with different
compounds. All cells were inoculated into 96-well plates at a rate
of 106 cells per well. The samples were then treated with com-
pounds and berberine at different concentrations and incubated at
37°C and 5% carbon dioxide for 24 h. Add 100 mL dimethyl sulfox-
ide to the supernatant of the sample and shake well for 10 min.
The optical density of the sample was measured at 490 nm with
a microplate photometer. Cell viability was expressed by the per-
centage change of absorbance relative to the control value.
3.1. Design and synthesis of target compounds
To
synthesize
the
designed
bis-sulfonyl
chalcone-
dipyrromethene boron difluoride, we took 3-chloro-5,7-dimethyl-
6-ethyl-8-phenyl-BODIPY as the crude material (Scheme 1), cat-
alyzed by POCl3. Formyl group was introduced into the pyrrole ring
containing chlorine to get 2. The nucleophilic substitution reaction
of 2 and propylamine, obtained 3 in 89% yield with acetonitrile
as solvent. The introduction of propylamino group in this step is
expected to better increase the drug activity of the compounds.
Then, 4 was synthesized by condensation reaction of BODIPY with
aldehydes group and 1-(2,4-dihydroxyphenyl)ethan-1-one with α-
H, catalyzed by piperidine. Finally, bis-sulfonyl-chalcone-BODIPYs
were synthesized by reacting with methanesulfonyl chloride
and benzenesulfonyl chloride respectively under the catalysis of
triethylamine in dichloromethane.
2.4. Cell imaging
Cell inoculation: HeLa cells in the logarithmic growth phase
were digested with trypsin and seeded into a 6-well plate con-
taining round cap, which was cultured in an incubator at 5% CO2
and 37°C for 24 h before adhering. Drug treatment: 4, 5, 6 sam-
ples were weighed and added into dimethyl sulfoxide (C = 0.25
mmol L−1) and diluted to 2.5 μmol L−1 respectively. Dosing pro-
cess: discard the original culture medium for each well cell and re-
place it with medium containing 2.5 μmol L−1 of a different drugs.
Cell treatment: the medium was discarded and washed twice with
phosphate buffer saline (PBS) after 24 h. The fixative was added
with paraformaldehyde for 10 min, and the fixative was carefully
sucked out. Wash it with PBS twice, and incubate with DAPI dye
for 10 min under dark condition. The staining solution was dis-
carded and washed with PBS twice again. After the tablets were
sealed with anti-fluorescence quenching sealant, the samples and
nuclear changes were observed under fluorescence microscope.
3.2. Spectroscopic properties
3443 cm−1 (ν, O-H) in compound 4 is the stretching vibration
peak of phenolic hydroxyl group (Fig. 1 and Fig. S1-S3). The char-
acteristic absorption peak of B-F in BODIPY is around 1268 cm−1
1119 cm−1 and 1043 cm−1 [29]. Around 1370 cm−1 (νas, SO2), 1180
cm−1 (νs, SO2) is the symmetric and anti-symmetric stretching vi-
bration peak of R-SO2-R [30]. This indicates the successful intro-
duction of sulfonyl group to compounds 5 and 6. In addition, the
color differences of the compounds are shown in Fig. 1b. The in-
teresting thing is that compound 5 has a flake like feel of gold foil,
which reflects red and orange under light.
,
We discussed the full wavelength absorption and emission
spectra of 4, 5, 6 in dichloromethane, as shown in the support-
ing information Figure S4. The ultraviolet absorption of the three
compounds synthesized by us increases with the increase of com-
pound concentration, while the maximum ultraviolet absorption
wavelength is basically unchanged, all within the range of 540-548
nm. The concentration of the compound is proportional to its flu-
orescence. However, at the same excitation wavelength (λex = 500
nm), there is no significant change in the peak shape or emission
wavelength of the three compounds. This indicates that the intro-
duction of sulfonyl group has no obvious effect on the fluorescence
properties of the compounds.
2.5. In vivo imaging
In vivo imaging experiments were performed on a single mouse
component weighing 20 g and 7 weeks old. 5 (25 μmol L−1, 25 μL
in 1: 9 dimethyl sulfoxide/ saline, V/V) was injected on an empty
stomach for 30 min. The fluorescence intensity of subcutaneous tu-
mor tissues was then detected. In addition, the mouse experiment
meets the requirements of animal ethics (the ethics committee ap-
proval No. syxk 2018-0053).
2.6. Flow cytometry analysis
Compound 4 has a maximum ultraviolet absorption at 545 nm
and an emission maximum at 569 nm (Fig. 2a and Table 1). Com-
pound 5 has a maximum linear absorption at 548 nm and maxi-
mum emission wavelength of 569 nm (the three compounds are
all at λex = 500 nm). The maximum linear absorption of com-
pound 6 is 540 nm and the emission peak is at 566 nm. Stokes
shift of the three compounds were all around 20 nm. Under sun-
light, the three compounds were red in dichloromethane solution
and showed a very strong orange-red fluorescence under ultravio-
let light of 365 nm. In addition, the fluorescence quantum yields of
all BODIPY derivatives were evaluated in dichloromethane solution
in table 1, and the compound 6 showed a high ꢀf value.
HeLa cells cultured in 12-well petri dish were treated with 5.
Then the cell suspension was incubated with trypsin at 37°C for
12 h. It was placed in precooled 75% ethanol at 25°C below zero
overnight. Ribozyme (10 μg mL−1) and propidium iodide (15 nmol
L
−1, PI); BD Biosciences (Anolun Biotechnology Co., Ltd, Beijing)
was added to the cells for dark incubation at room temperature
for 30min. The samples were then evaluated using flow cytometry
(ATTUNE NXT, Life tech, lnc). ModFit LT 2.0 software (Verity Soft-
ware House, Inc., Topsham, ME, USA) was used for data analysis.
2.7. Apoptosis
In addition, we investigated the fluorescence stability of the
three compounds in dimethyl sulfoxide for 300 seconds (Fig. 2b).
Due to the limitation of the instrument, if the excitation wave-
length of 500 nm and the emission wavelength of 560 nm are
used, the fluorescence intensity of the compound is beyond the
measurement range of the fluorescence spectrophotometer. There-
fore, we set a relatively appropriate excitation wavelength of 430
Compound 5 was used to treat HeLa cells cultured in a 12-well
petri dish. Cell suspension was collected by trypsin at 37°C with-
out ethylene diamine tetraacetic acid (EDTA). After washing with
PBS, 250 mL binding buffer was added for resuspension. The Cell
suspension was stained with 5 μL fluorescent protein PI and 5 μL
Annexin V isothiocyanate solution (Yassen Biotechnology Co., LTD.,
3

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
Scheme 1. Synthetic route of bis-sulfonyl chalcone-BODIPY.
Fig. 1. (a)The infrared spectrogram of compounds 4, 5, 6 measured by KBr tablet. (b) Pictures of 4-6 solids in sunlight (upper) and 365 nm ultraviolet lamp (lower).
Table 1
Photophysical properties of compounds 4-6.
Compounds
λmax(abs) [nm]
Emission [nm]
Stokes shift [nm]
Stokes shift [cm−1
]
ꢀfa
4
5
6
545
548
540
569
569
566
24
21
26
774
673
850
0.22
0.25
0.31
a
Fluorescence quantum yield estimated relative to rhodamine B as the standard (ꢀf = 0.65 in ethanol).
4

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
Fig. 2. (a) The solid line is the normalized UV-Vis spectrum of compounds 4-6 in dichloromethane solution (10 μM), and the dashed line is the normalized fluorescence
spectrum of compounds (2 μM) (excitation wavelength = 500 nm). Compounds 4-6 in sunlight (left) and uv light (right) are shown in the upper right. (b) The fluorescence
stability of BODIPY derivatives 4-6 in dimethyl sulfoxide at concentrations of 2.0 μmol L−1 (excitation wavelength = 430 nm, emission wavelength = 560 nm).
Fig. 3. Stereoscopic histogram of IC50 of different compounds.
creased from 79.71 3.84 μmol L−1 to 56.10 8.51 μmol L−1 and
compound 6 to 68.47 5.34 μmol L−1 after introducing bis-sulfonyl
nm. Obviously, the three compounds remained dynamically stable
over time in the dimethyl sulfoxide solvent.
group, indicating that the introduction of sulfonyl group could im-
prove the efficacy of the compound. Among them, the effect of 5
3.3. Cytotoxicity studies
was more obvious.
Compounds 4, 5 and 6 were used to conduct the cell activity
of HCT-116, HeLa and L-02 cells, respectively (Table 2 and Fig. 3).
It can be found that the toxicity of compound 4 was relatively low
after the introduction of chalcone to BODIPY, and the IC50 value of
normal cells L-02 was 102.21. The IC50 value of compound 5 de-
3.4. Cellular imaging and in vivo imaging
Further study on the cell imaging of BODIPY derivatives (Fig. 4A
and B) shows that compounds 5 and 6 are highly overlapped with
5

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
Fig. 4. (A) HeLa cells were fixed in paraformaldehyde solution, incubated with DAPI in dark, and nuclear imaging under fluorescence microscope (blue), different compounds
at a concentration of 2.5 μmol L−1 (red) and merged image. Compound 4: a-c; Compound 5: d-f; Compound 6: g-i. (B) Magnified view of d-f cell imaging. (C) In vivo imaging
of compound 5 in mice.
Fig. 5. Cellular imaging mechanism.
6

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
Fig. 6. (a) HeLa cells were incubated with compound 5 and Berberine for 12 h, stained with PI and annexin-V / FITC, and then analyzed by flow cytometry analysis. Early
apoptotic cells are shown in the lower right quadrant. (b) Apoptosis rate of HeLa cells under Berberine. (c) The flow cytometry of HeLa cells treated with compound 5 was
analyzed using ModFit LT2.0 software. (d)The flow cytometry of berberine was used as control.
Table 2
which shows the excellent biocompatibility and targeting property
Experimental data of cytotoxicity.
of the dye.
Compounds
IC50 (μmol L−1
)
SD
Since the introduction of dimethylsulfonyl group has a greater
effect on IC50 value than benzene sulfonyl group and has more ob-
vious inhibitory effect on cancer cells, 5 was used for basic imaging
in vivo in mice in order to further enhance the follow-up biological
research. The fluorescence intensity of the subcutaneous tumor tis-
sue was detected after injection of compound 5 in mice (Fig. 4C)
30 minutes. The in vivo imaging showed a strong fluorescence in
the tumor tissue, which directly demonstrated the potential tar-
geting of the compound to tumor cells.
HCT-116
HeLa
L-02
4
64.33 8.36
60.33 4.31
74.86 2.77
36.56 7.86
79.71 3.84
56.10 8.51
68.47 5.34
27.07 5.56
102.21 5.97
61.29 10.12
78.22 9.88
188.82 8.09
5
6
Berberine
the nuclei of HeLa cells, indicating that compounds 5 and 6 com-
pletely entered the cell nucleus. However, a small part of com-
pound 4 is free from the nucleus. It may be that compound 4
contains two hydroxyl groups, which can easily form hydrogen
bonds with proteins in cells, resulting in some compounds dis-
sociating outside the nucleus (Fig. 5). For compounds 5 and 6,
when methylsulfonyl group and phenylsulfonyl group replace the
hydrogen atoms on the hydroxyl group, they enter the cell and re-
act with the amino acids in the nucleus, thus leaving the sulfonyl
group [31, 32]. Under the fluorescence microscope, they fully com-
bine with HeLa nucleus. After the introduction of double sulfonyl
group, the compound only binds to the tumor nucleus for imaging,
3.5. Flow cytometry analysis of HeLa cells apoptosis
Annexin V / PI double staining was used to identify early and
late apoptotic cells, dead cells and living cells [33–36], and the
apoptosis rate was quantified. The experiment was repeated three
times independently. The lower left quadrant (annexin V negative
- PI negative) represents normal cells (Fig. 6a and b). After com-
pound 5 treatment, the percentage of living cells was 95.07%. The
early apoptosis rate and late apoptosis rate were 0.58% and 0.58%
respectively, although the apoptotic cell rate of 3.7% was lower
than that of berberine (10.64%), it would be a breakthrough in
BODIPY probe cell experiment.
7

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
Fig. 7. molecular docking diagram of different compounds with CDK2. (a) Compound 4. (b) Compound 6. (c) Compound 5.
3.6. Compound 5 induced mitotic block in HeLa cells
3.7. Molecular docking
The effect of compound 5 on HeLa cells proliferation was in-
vestigated by flow cytometry (Fig. 6c and d). The experiment
was independently repeated three times. The average DNA con-
tent in G1 phase was 223.78, and the number of cells in G1
phase accounts for 63.12% of the total. At this time, the num-
ber of cells was the most, but the DNA content was the least.
In the S phase, DNA began to replicate to complete replication,
and the DNA content multiplied. Therefore, the S phase shows
a particularly long span in the result chart. Compared with the
cells treated with control [37], HeLa cells treated with compound
5 caused 22.76% s phase stagnation, which was higher than the
blank control. This data well demonstrated the inhibitory effect of
compound 5 on S phase in cell division. The data of S was lower
than 25.95% of berberine, which corresponded with apoptosis
experiment.
In order to elucidate the mechanism of action of these three
compounds on tumor cells, molecular docking simulations of tu-
mor target CDK2 kinases were performed (Fig. 7) [38–41]. Affin-
ity Score is one of the standard to measure the docking results
(Table 3). The binding energy of compound 5 and 6 is -8.4 and
-8.6 kcal•mol−1, respectively, which is higher than compound 4.
This indicates that the introduction of disulfonyl group increases
the affinity of CDK2, making it easier to bind to cells. However,
compounds 5 and 6 have similar binding energies, so we have an-
alyzed their main forces. The benzene ring on the BODIPY parent
nucleus in compound 5 formed a T-π accumulation with Phe 80,
and the benzene ring directly attached to the dimethyl sulfonyl
group also formed a T-π interactions with the residue Phe 82,
while compound 6 only contained one. The benzene ring directly
connected with bis-phenylsulfonyl group of compound 6 forms π
8

Z. Lv, Y. Wang, J. Zhang et al.
Journal of Molecular Structure 1234 (2021) 130201
Table 3
Interaction between residues and compounds (PDBID: 2FVD).
Entry
Affinity score
Interaction with receptor
−1
•
(PDBID 2FVD)
(kcal mol
-7.0
)
[H]
π-π
T-π
Ion-Pi
Hydrophobic bond
4
His 84
Phe 80
Asp 86
Asp 145
Lys 89
His 84
GLU12
Asp 145
Glu 8
Ala 31
Ala 144
Ile 10
Asp 145
Phe 80
5
6
-8.4
-8.6
Glu 8
Phe 80
Phe 82
Ala 144
Leu 83
Phe 80
Phe 82
Val 18
Gln 85
Gln131
Ile 10
Glu 12
Glu 162
His 84
Lys 89
Asp 86
Glu 12
His 84
Lys 9
Asp 145
Gln 131
Leu 83
Phe 80
Phe 82
Ala 31
Leu 83
Leu 134
Phe 80
Phe 82
- π stacking with Phe 80. In addition, compound 5 has more hy-
drogen bonds and ion-Pi interactions with CDK2 than compound 6,
so compound 5 may have a slightly stronger binding capacity with
cells by comparing the interactions.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130201.
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Jiangsu University (Grant No. 5501290005) for the financial sup-
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