High activity, high selectivity and high biocompatibility BODIPY-pyrimidine derivatives for fluorescence target recognition and evaluation of inhibitory activity

Article abstract of DOI:10.1016/j.bioorg.2021.105121

BODIPY-Pyrimidine (BP) is a highly selective, highly active, and highly biocompatible fluorescent drug, which is characterized by its own activity combined with a fluorophore. The combination of pyrimidines with good biological activity and fluorophores to obtain new compounds with both anti-tumor activity and fluorescent targeting probe functions is the focus of this research. In terms of biological activity, in vitro cytotoxicity of the compounds on four human cancer cells (HepG2, HeLa, A-459, and HCT-116) and the human normal cell line L-02 was studied. BP-4 has good antiproliferative activity, and its IC₅₀ values are 19.12 ± 2.29, 13.47 ± 3.80, 18.59 ± 7.42, 14.57 ± 2.44 and 92.48 ± 6.03 μM, respectively. Good biocompatibility with tumor cells can be observed in cell imaging. The anti-tumor mechanism of the compound was further studied by flow cytometry. After BP-2, BP-3 and BP-4 treated HeLa cells, the percentage of apoptotic cells was 19.07%, 22.09% and 27.3%, respectively. The cell cycle study found that, compared with the positive control 5-FU (48.05%), the compounds BP-2, BP-3 and BP-4 all increased the proportion of HeLa cells in the G1 phase, reaching 57.65%, 55.46% and 53.58%, respectively. In vivo bioimaging results show that all three compounds can be targeted and accurately expressed in tumor tissues. In addition, molecular docking analyzes the possible interaction between the compound and the active site of thymidylate synthase.

Full text of DOI:10.1016/j.bioorg.2021.105121

Bioorganic Chemistry 114 (2021) 105121
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
High activity, high selectivity and high biocompatibility
BODIPY-pyrimidine derivatives for fluorescence target recognition and
evaluation of inhibitory activity
,
,*
Chi Xua, Tingyu Shaob, Shihe Shaoa *, Guofan Jinb
a Digestive Disease Center, Shanghai East Hospital, Tongji University, Shanghai 200120, China
b School of Pharmacy, Jiangsu University, Zhenjiang 212013, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
BODIPY-Pyrimidine (BP) is a highly selective, highly active, and highly biocompatible fluorescent drug, which is
characterized by its own activity combined with a fluorophore. The combination of pyrimidines with good
biological activity and fluorophores to obtain new compounds with both anti-tumor activity and fluorescent
targeting probe functions is the focus of this research. In terms of biological activity, in vitro cytotoxicity of the
compounds on four human cancer cells (HepG2, HeLa, A-459, and HCT-116) and the human normal cell line L-02
was studied. BP-4 has good antiproliferative activity, and its IC50 values are 19.12 ± 2.29, 13.47 ± 3.80, 18.59 ±
BODIPY-pyrimidine
Fluorescence target recognition
Evaluation of inhibitory activity
Cell apoptosis
Cell cycle
7.42, 14.57 ± 2.44 and 92.48 ± 6.03 μM, respectively. Good biocompatibility with tumor cells can be observed
in cell imaging. The anti-tumor mechanism of the compound was further studied by flow cytometry. After BP-2,
BP-3 and BP-4 treated HeLa cells, the percentage of apoptotic cells was 19.07%, 22.09% and 27.3%, respec-
tively. The cell cycle study found that, compared with the positive control 5-FU (48.05%), the compounds BP-2,
BP-3 and BP-4 all increased the proportion of HeLa cells in the G1 phase, reaching 57.65%, 55.46% and 53.58%,
respectively. In vivo bioimaging results show that all three compounds can be targeted and accurately expressed
in tumor tissues. In addition, molecular docking analyzes the possible interaction between the compound and the
active site of thymidylate synthase.
1. Introduction
of inhibitory effect on different human cancer cell lines, indicating that
the anti-proliferation activity of pyrimidine derivatives is related to cell
Pyrimidine is an important class of nitrogen-containing heterocyclic
compounds. The existence of multiple modification sites makes it have a
wide range of biological activities, such as anti-cancer [1-4], anti-
inflammatory [4,5], antioxidant [6,7], antibacterial [8,9], antiviral
[10-12], and anti-AIDS [13,14]. Especially in anti-tumor activity, py-
rimidine derivatives show significant advantages. Although pyrimidine
derivatives have strong anti-cancer activity, they have many targets, and
the exact anti-cancer mechanism is still unclear. Studies have shown that
pyrimidine derivatives can inhibit tumor cell proliferation, block cell
cycle, and induce cell apoptosis. In the latest research on the anticancer
activity of pyrimidine derivatives, Padhy et al [15]. synthesized 5
benzimidazole compounds and pyrimidine compounds, among which
apoptosis. Liu et al. [17] synthesized a series of 5H-[1,2,4] oxadiazole
[4,5-a]pyrimidine derivatives with dual anti-cancer and antibacterial
biological activities. Compound 3f has a significant inhibitory effect on
HCT-116 cells. This shows that pyrimidine compounds have broad
research prospects in the field of anticancer.
The emerging dye BODIPY [18-21] has appeared in the public eye as
an important fluorescent labeling material [22-25] because of its stable
emission spectrum above 500 nm. Due to its high fluorescence quantum
yield, high molar absorption coefficient, strong stability and other
excellent photodynamic characteristics and low biological toxicity, it
has emerged in the field of fluorescent probes [26-32]. In 2017, Gotor
et al. [33] used quantum chemistry to design a series of pH-responsive
fluorescent probes with BODIPY as the core. The probes cover the
entire pH range and are reusable. The following year Maity et al. [34]
synthesized a BODIPY fluorescent probe with BDPS as an anchor, which
compound 5b (GI50 = 39.6 μM) has a good inhibitory effect on human
breast cancer cell lines. Huang et al. [16] synthesized a series of oxacalix
[2] arene [2] pyrimidine derivatives, which have a single concentration
* Corresponding authors.
E-mail addresses: shaoshihe2006@163.com (S. Shao), organicboron@ujs.edu.cn (G. Jin).
https://doi.org/10.1016/j.bioorg.2021.105121
Received 8 March 2021; Received in revised form 13 May 2021; Accepted 20 June 2021
Available online 24 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

C. Xu et al.
Bioorganic Chemistry 114 (2021) 105121
can effectively detect Hg2+. In addition, BODIPY has found important
applications in in vivo imaging. Chao’s [35] group designed a red
fluorescent probe composed of Ni-NTA moiety, BODIPY and an aryla-
zide. The probe can penetrate the cell membrane for live cell protein
imaging and selectively label His(6)-labeled proteins. Guixens-Gallardo
et al. [36] synthesized a series of base-modified pyrimidine-BODIPY
derivatives, dNmBdpTP combined with the recently reported trans-
porter SNTT for fluorescent staining of genomic DNA in living cells [37].
At present, the research on BODIPY mainly focuses on dyes and probes.
This research creatively connects pyrimidine and BODIPY in a
structure through ethylenediamine, and obtains a series of BODIPY-
Pyrimidine derivatives with fluorescent target recognition and tumor
suppressor activity. The original combination of pyrimidine drugs and
BODIPY, through reasonable structural modification, is expected to
obtain fluorescent drugs with high activity, high selectivity and high
biocompatibility. The synthesized compound selectively enters tumor
cells to inhibit their proliferation activity, and at the same time, the
distribution of the compound in the body is visually observed through
cell imaging to achieve precise positioning.
treated with BODIPY-NH2 overnight under alkaline conditions to obtain
the target compound BP-1 ~ BP-4 (Fig. 1). The structures of all the
compounds were confirmed by 1H NMR, 13C NMR, and MS-ESI (Sup-
plementary information).
2.2. Ultraviolet–visible absorption spectrum and fluorescence spectrum
analysis
The photophysical properties of compounds BP-1 ~ 4 have been
studied in different solvents. Figs. 2 and 3 show their UV–Vis and fluo-
rescence spectra, respectively, in different solvents, and Table 1 gives
their spectral parameters. The maximum wavelength of the absorption
spectrum of compounds BP-1 ~ 4 varies in the range of 532–540 nm
(Fig. 2). The position of the maximum absorption peak of the same
compound in different solvents does not change much, and the prop-
erties of the compound in different polar solvents are relatively stable.
However, in quantitative research, the absorption intensity of the same
concentration of compounds in different solvents varies greatly. The
absorption intensity of BP-1 in methanol is twice that of acetonitrile.
Table 2
2. Results and discussion
The emission spectra, recorded in different solvents for the com-
pounds BP-1 ~ 4 are shown in Fig. 3. The emission spectra were ob-
tained by exciting the molecules at λabs = 450 nm. And a main emission
band can be seen in the region 550–565 nm. The fluorescence intensity
of the four compounds in different solvents is strong, and the ideal
2.1. Chemistry
In presented work, we aim to design and synthesize a novel series of
biological active fluorescent target BODIPY-Pyrimidine derivatives (BP-
1 ~ BP-4). Thus, the sequence of reactions used in the synthesis of the
title compounds is shown (Schemes 1). In this design and synthesis, 4-
chloro-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester was
used as the raw material, and the original Cl was replaced by an aro-
matic amine through a substitution reaction to reduce the biological
toxicity of the compound and increase the ability of compounds to bind
fluorescence intensity can be obtained under the order of μM. The dif-
ference in the fluorescence peak wavelength of compounds in different
solvents are between 6 and 9 nm. The compounds BP-1, BP-3 and BP-4
of different solvents have the largest fluorescence absorption intensity in
the most polar DMSO solvent. The maximum fluorescence intensity of
BP-2 appears in DMSO and dichloromethane solvents.
The results of this experiment show the target compounds we syn-
thesized has strong fluorescent properties and has the potential value as
a fluorescent marker.
to DNA, and at the same time increase the π-H bond energy of the
quinoline ring and protein. The modified pyrimidine derivative is
combined with BODIPY to obtain a new compound with dual functions.
The intermediate P-1 ~ P-4 was obtained by reflux of ethyl 4-chloro-2-
(methylthio) pyrimidine-5-carboxylate and aromatic amine in acetoni-
trile solvent. The intermediate was oxidized with m-CPBA and then
The spectral values of pyrimidine derivatives are below 500 nm, and
the spectral values of the four BODIPY-Pyrimidine derivatives synthe-
sized in this study are all above 500 nm. The main reason is that the
introduction of fluoroboron pyrrole groups greatly changes the spectral
Scheme 1. Design of Organic Synthesis.
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Bioorganic Chemistry 114 (2021) 105121
Fig. 1. Photographs of compounds in sunlight and fluorescence.
Fig. 2. Effect of different solvents on ultraviolet absorption intensity.
properties of the compounds. Comparing the spectral data of compounds
BP-1 ~ 3, the length of the secondary aminoalkane chain on the py-
rimidine ring has a slight influence on the spectral properties of the
compound. For compounds BP-3 and BP-4, the presence of the methoxy
group on the benzene ring does not affect the spectral properties of the
compounds.
solvent, four BODIPY-Pyrimidine derivatives (BP-1 ~ BP-4) were dis-
solved in DMSO, and their stability was measured at room temperature
(Fig. 4). The excitation wavelength is 450 nm, and the emission wave-
lengths are 559, 564, 557, and 557 nm, respectively. The fluorescence
intensity of the four compounds in DMSO solvent hardly changes with
time. The results show that the four derivatives can exist stably in DMSO
solution.
2.3. Stability study
In order to further understand the stability of the compound in the
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Bioorganic Chemistry 114 (2021) 105121
Fig. 3. Effect of different solvents on fluorescence intensity.
Table 1
Table 2
Photophysical characteristic of BP-1 ~ BP-4 in different solvents.
Photophysical data of BP-1 ~ BP-4.
Solvent
MeOH
CH2Cl2
EtOAc
MeCN
DMSO
DMF
534
Compounds
Fluorescence quantum yield Φf
λabs max, nm
BP-
1
533
538
534
532
535
BP-1
BP-2
BP-3
BP-4
0.224
0.300
0.177
0.195
BP-
2
536
534
533
554
559
550
552
5.96
0.52
1.98
4.93
540
538
538
562
565
559
558
4.58
0.35
2.06
5.60
535
534
534
556
560
553
553
4.47
0.32
2.22
6.26
533
532
532
554
560
553
553
3.06
0.28
1.74
2.99
535
535
535
559
564
557
557
3.36
0.26
1.41
3.59
536
534
534
557
562
556
556
0.96
0.20
1.88
2.85
BP-
3
BP-
4
λem max, nm
BP-
1
BP-
2
BP-
3
BP-
4
ε
, 104 L•molꢀ 1
•cmꢀ 1
BP-
1
BP-
2
BP-
3
BP-
4
2.4. In vitro cytotoxicity activity
Four novel BODIPY-Pyrimidine derivatives were evaluated for their
cytotoxicity activities against HepG2, HeLa, A-459, HCT-116 and L-02
Fig. 4. Fluorescence stability of compounds BP-1 ~ BP-4.
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Bioorganic Chemistry 114 (2021) 105121
by MTT assay. The IC50 values of each compound for these cell lines
were calculated, and the results were summarized in Table 3, Fig. 5. 5-
Fluorouracil (5-FU) was used as a reference standard and showed IC50
values of 21.31 ± 1.15, 12.05 ± 5.38, 17.53 ± 2.21, 11.22 ± 3.10 and >
150 μM for the five cell lines respectively. Compound BP-4 displayed 1.1
folds the activity of 5-FU against HepG2 (IC50 = 19.12 ± 2.29
was equipotent to the reference drug against A-459 (IC50 = 18.59 ±
7.42 M) and HCT-116 (IC50 = 14.57 ± 2.44 M). While compound BP-2
displayed 1.2 folds the activity of 5-Fu with IC50 values of 10.04 ± 5.11
μM), and
μ
μ
μ
M against HeLa cell lines. The anticancer results profile suggested that,
synthesized compounds exhibited good anticancer activity, with some of
the findings comparable or highly potent than 5-FU.
The presence of the pyrimidine ring greatly reduces the cytotoxicity
and increases the compound’s anti-proliferative activity on tumor cells.
Comparing the activity data of compounds BP-1 and BP-2, when the
secondary amino group on the pyrimidine ring has a methylene group, it
will significantly increase the anticancer activity of the compound, but
the increase in the number of methylene groups will not significantly
increase the compound’s anti-cancer activity. In addition, the presence
of the methoxy group on the benzene ring can indeed increase the
anticancer activity of the compound.
Fig. 5. The half-maximum inhibitory concentration (IC50) of 5-FU and com-
pounds BP-1 ~ BP-4 on HepG2, HeLa, A-459, HCT-116, and L-02 cells.
2.5. Cell imaging
In order to intuitively understand whether the anti-tumor activity of
multiple compounds is related to apoptosis, we performed fluorescence
imaging of HeLa cells in the logarithmic growth phase to observe their
nuclear morphological changes (Fig. 6). After treating the cells with 2
μ
g/ml medium of different drugs for 24 h, it can be observed that the
compound enters the cell through the cell membrane, destroys the shape
of the cell nucleus, and distributes around the nucleus. Among them,
compound BP-4 has the best imaging effect. The above results indicate
that all four compounds BP-1 ~ BP-4 can enter cells through the cell
membrane and induce HeLa cell apoptosis.
2.6. Cellular mechanism of action
2.6.1. Cell apoptosis assay
The induction of apoptosis is one of the effective methods of cancer
treatment. In order to further understand the anti-proliferation effect of
newly synthesized pyrimidine derivatives on cancer cells, it was
explored whether the anti-cervical cancer activity of compounds BP-2
and BP-4 is related to cell apoptosis. As shown in Fig. 7, Q1 to Q4
represent necrotic cells, late apoptotic cells, early apoptotic cells and
non-apoptotic cells, respectively.
When HeLa cells were treated with BP-2, BP-3, BP-4 and 5-FU, the
percentages of apoptotic cells were 19.07%, 22.09%, 27.3% and 49.8%,
respectively. As a result, we could speculate that the main reason for
antiproliferative activities against cancer cells induced by BP-2 ~ 4 are
Fig. 6. Fluorescent microscope imaging of treated HCT-116 cells with BP-1 ~
BP-4 for 24 h. Scale bar is 400 units.
likely to be the cell apoptosis.
Table 3
2.6.2. Cell cycle assay
The half-maximum inhibitory concentration (IC50) of 5-FU and compounds BP-
1 ~ BP-4 on HepG2, HeLa, A-459, HCT-116, and L-02 cells.
In order to further explain the antitumor proliferation activity of the
newly synthesized pyrimidine derivatives, the cell cycle changes of the
compounds BP-2 and BP-4 were observed by flow cytometry on the
three derivatives and the positive control 5-FU. As shown in Fig. 8,
compounds BP-2, BP-3 and BP-4 all led to an increase in the percentage
of HeLa cells in G1 phase, which increased to 57.65%, 55.46% and
53.58%, respectively, compared with the positive control 5-FU
(48.05%). The percentage of cells in G2/M phase decreased from
16.21%(5-FU) to 8.70%(BP-2), 10.51%(BP-3) and 14.48%(BP-4),
respectively. The results show that the newly synthesized pyrimidine
derivatives can inhibit DNA replication and block cells in G1 phase,
thereby inhibiting cell proliferation to achieve anti-cancer effects
[38,39].
Compound
IC50
(μ
M) ± SD[a]
HepG2
HeLa
A-459
HCT-116
L-02
BP-1
BP-2
BP-3
BP-4
5-FU
30.15 ±
8.19
27.54 ±
6.97
29.40 ±
3.12
23.06 ±
7.88
68.39 ±
7.96
24.58 ±
7.23
10.04 ±
5.11
38.92 ±
4.05
16.88 ±
1.39
102.39 ±
4.77
29.27 ±
3.55
20.30 ±
6.63
26.44 ±
2.94
19.25 ±
3.17
81.07 ±
6.03
19.12 ±
2.29
13.47 ±
3.80
18.59 ±
7.42
14.57 ±
2.44
92.48 ±
6.03
21.31 ±
1.15
12.05 ±
5.38
17.53 ±
2.21
11.22 ±
3.10
>150
[a]
Data are means ± SD of triplicate experiments.
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Bioorganic Chemistry 114 (2021) 105121
Fig. 7. Apoptosis of HeLa treated with 5-FU, BP-2, BP-3 and BP-4 was analyzed by flow cytometry.
2.7. In vivo bioimaging
active site of thymidylate synthase was performed to investigate their
binding interactions and to explore their binding modes (Table 4, Figs. 9,
10).Fig. 11
BP-2, BP-3 and BP-4 with better biological activity were injected
into mice by intraperitoneal injection, and the fluorescence intensity of
the mouse subcutaneous lung tumor tissue was measured every 10 min.
Strong fluorescent signals can be observed in mouse tumor tissues at
different times.
From the docking results in Table 3, all four compounds can bind to
proteins well. The more prominent compound is BP-3, which has a
binding affinity free energy of ꢀ 8.9 kcal/mol. The binding mode of BP-3
and thymidylate synthase is mainly that the oxygen on the aliphatic
group of the pyrimidine group has a hydrogen bond interaction with Met
The fluorescence values of the mice treated with the three com-
pounds were the same at 10 min. The fluorescence intensity of the
subsequent 20 min increased significantly with the prolonging of the
action time, and the fluorescence intensity of compound BP-4 was
stronger than that of BP-3 than BP-2.
309, and the pyrimidine ring has a π-π stacking effect with Phe 80,
pyrrole ring with Trp 109 and Phe 225. In addition, there are more
hydrophobic amino acid residues around BP-3.
The F in the structure of BP-1, BP-2 and BP-4 can interact with
different amino acid residues by hydrogen bonding. Comparing the
docking results further, it can be found that both N of ethylenediamine
in the structures of BP-2 and BP-4 can interact with amino acid residues
by hydrogen bonding, resulting in more hydrogen bonds in these two
compounds than in BP-1 and BP-3. This may be the reason for the better
biological activity of BP-2 and BP-4. In addition, it can be observed that
the pyrrole ring in the structure of BP-1 and the amino acid residue Phe
225, and the benzene ring on the BP-2 pyrimidine and the amino acid
At 40 min, the compound fused with the cell flows along with the cell
membrane to flow with each other, resulting in a decrease in fluores-
cence intensity.
In vivo bioimaging results show that all three compounds can be
targeted and accurately expressed in tumor tissues (Fig. 9).
2.8. Molecular docking
residue His 256 form a π-π stacking effect.
Studies have shown that BODIPY-pyrimidine compounds inhibit cell
proliferation by inhibiting the activity of thymidylate synthase, which
leads to the synthesis of cell DNA [4,40]. Molecular modeling studies
were accomplished to investigate the possible binding mode of the
synthesized four BODIPY-pyrimidine derivatives targeting the crystal
structure of thymidylate synthase (PDB ID: 6QYQ) using AutoDock Vina
1.1.2. software. The BP-1 ~ 4 were docked into the active site of thy-
midylate synthase, using co-complex ligand as the reference with 4 A
radius. Molecular docking of BODIPY-pyrimidine derivatives to the
Although BP-4 does not form π-π accumulation with amino acid
residues, there are many hydrophobic amino acid residues around the
compound, such as Ala 111, Leu 192, Met 311, Phe 117 and so on.
3. Conclusion
In summary, a series of arylamine-modified pyrimidine anticancer
drugs with fluorescent probe function combined with BODIPY group
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Bioorganic Chemistry 114 (2021) 105121
Fig. 8. 5-FU, BP-2, BP3 and BP-4 induced cell cycle arrest at the G1 phase in HeLa were plated in six-well plates for 24 h of incubation.
Fig. 9. In vivo imaging of changes in the levels of BP-2, BP-3 and BP-4 in mice lung tumor tissues.
were synthesized, and their in vitro cytotoxicity to four human tumor
cell lines and human normal liver cell lines were determined. The
BODIPY- pyrimidine derivatives all have good anti-tumor activity and
biocompatibility. The results of in vitro cytotoxicity experiments show
that the compounds have less cytotoxicity to normal cells and have great
potential in entering the body as a fluorescent probe. At the same time,
the compounds can effectively kill tumor cells at a lower concentration,
making it have considerable potential in anti-tumor activity. The
inhibitory effect of BP-4 on HepG2 and BP-2 on HeLa is stronger than
the positive control 5-FU, and the overall anti-proliferative activity of
BP-4 is better. The introduction of the BODIPY group brings about F
with strong electronegativity, which enhances the biological activity of
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Bioorganic Chemistry 114 (2021) 105121
110/110A) was performed.
Table 4
Docking results of synthesized compounds BP-1 ~ 4 with thymidylate synthase.
4.1.2. General procedure for the synthesis of celebrex derivatives P-1 ~ P-
4
Comp.
Affinity score [kcal/
mol]
Amino acid residues [H]
Amino acid residues
[π-π]
Ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate(1.0 g, 4.30
mmol) was used as raw material, K2CO3(0.71 g, 5.16 mmol) was added
as base and aniline(0.6 g, 6.45 mmol) under stirring condition, and the
mixture was refluxed in acetonitrile (30 mL) for 8 h. The solvent was
removed by rotary evaporation, and the reaction solution was extracted
by ethyl acetate/water for three times. The combined organic layers
were dried over MgSO4, filtered, and the solvent was removed under
reduced pressure to afford P-1, which was used directly into next steps.
In a 25 mL round bottom flask, m-CPBA (0.12 g, 0.65 mmol) was
slowly added to a stirring solution of P-1 (0.08 g, 0.27 mmol) in 8 mL
dichloromethane at 30 ◦C. Reaction mixture was monitored through
thin layer chromatography (TLC) by using petroleum ether, and ethyl
acetate (6:1) as solvent system. The reaction mixture was stirred at 30 ◦C
until TLC showed the completion of reaction, (Z)-N-(1-(difluorobor-
anyl)-5-((4-ethyl-1,3,5-trimethyl-2H-4-pyrrol-2-ylidene)(phenyl)
methyl)-1H-pyrrol-2-yl)ethane-1,2-diamine [49] and triethylamine as a
base are added. After 14 h, the desired compounds were purified by
column chromatography using a mixture of petroleum ether, and ethyl
acetate (1:1).
BP-1
BP-2
ꢀ 8.5
ꢀ 8.4
Asn 226, Met 311
Asp 48, Asp 49, Arg 50,
Arg 215
Phe 225
His 256
BP-3
BP-4
ꢀ 8.9
ꢀ 8.3
Met 309
Phe 80, Trp 109, Phe
225
Arg 50, Gly 52
–
the compound to a certain extent, and with the active site of thymidine
synthase. In vivo and in vitro imaging studies have also shown an
excellent function in tumor targeting. Both BP-2 ~ BP-4 inhibit DNA
replication and block cells in G1 phase. This research applies anti-tumor
compounds to fluorescent probes at the same time, opens up another
relatively novel research direction, provides new solutions for cancer
diagnosis and treatment, and is worthy of further research.
4. Experimental section
4.1. Chemistry
4.1.1. General experimental conditions
All solvents and reagents were commercially purchased and used as
received and employed further purification. Moisture-sensitive reactions
were performed under nitrogen atmosphere. Acetonitrile was distilled
over calcium hydride. All glassware including needles and syringes were
torch flame or oven-dried and kept in a desiccator before use. The re-
action products were isolated and purified by column chromatography
(silica gel 200–230 mesh). Thin-layer chromatography (TLC) was used
to monitor the progress of the reaction. Analysis of TLC results at 254 nm
and 360 nm wavelengths under UV lamps. Characterization of the
products at each stage by NMR spectroscopy, 1H, and 13C NMR spectra
were recorded on Bruker Avance II spectrometer in CDCl3 (400 MHz for
1H and100 MHz for 13C); chemical shifts are expressed in ppm, versus
internal tetramethylsilane (TMS) = 0 for 1H and 13C. Coupling constants
(J) are given in Hz. The molecular weight by MS-ESI (Jeol LTD JMS-HX
4.1.3. Spectral data of synthetic compounds BP-1 ~ BP-4
4.1.3.1. Ethyl 2-((2-((8-ethyl-5,5-difluoro-7,9-dimethyl-10-phenyl-5H-5
l4,6l4-dipyrrolo [1,2-c:2′,1′-f][1-3]diazaborinin-3-yl)amino)ethyl)amino)-
4-(phenylamino)pyrimidine-5-carboxylate (BP-1). Carmine solid; Yield:
37%; 1H NMR: (400 MHz, CDCl3, ppm): δ 8.63 (s, 1H), 8.09 (s, 1H),
7.49–7.24 (m, 10H), 7.16–7.13 (d, J = 8.0 Hz, 1H), 5.31 (s, 1H),
4.39–4.34 (m, 2H), 3.61–3.58 (d, J = 8.0 Hz, 4H), 3.49 (s, 3H),
2.35–2.30 (m, 2H), 1.36 (s, 3H), 1.28 (s, 3H), 1.00–0.98 (t, J = 6.0 Hz,
3H); 13C NMR (100 MHz, CDCl3, ppm): δ 170.17, 166.60, 160.67,
159.97, 159.74, 157.19, 145.18, 137.71, 135.29, 134.29, 133.66,
133.03, 132.45, 129.95, 129.68, 129.49, 128.88, 128.34, 128.01,
127.96, 124.71, 122.14, 105.71, 96.83, 61.05, 43.71, 41.51, 29.27,
17.17, 14.97, 14.29, 11.55. MS (ESI) m/z calculated for C34H36BF2N7O2
Fig. 10. Predicted binding mode of compounds BP-1 and BP-2 at the binding site of thymidylate synthase (PDB ID: 6QYQ).
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Bioorganic Chemistry 114 (2021) 105121
Fig. 11. Predicted binding mode of compounds BP-3 and BP-4 at the binding site of thymidylate synthase (PDB ID: 6QYQ).
629.0371(M+H), found 629.0383.
4.0 Hz, 1H), 4.30–4.25 (m, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.80 (s, 3H),
3.62 (s, 2H), 2.91 (s, 2H), 2.42 (s, 2H), 2.34–2.30 (d, J = 8.0 Hz, 2H),
1.37 (s, 3H), 1.36 (s, 2H), 1.31–1.25 (t, J = 12.0 Hz, 3H), 1.00–0.98 (t, J
= 6.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, ppm): δ 171.30, 170.70,
161.55, 160.67, 159.70, 156.06, 148.98, 147.73, 135.25, 134.16,
133.02, 131.82, 129.81, 129.67, 129.33, 128.35, 127.98, 127.80,
120.65, 114.40, 112.15, 111.50, 105.61, 105.59, 55.89, 55.80, 44.70,
44.68, 35.04, 30.21, 17.17, 14.96, 14.29, 11.84, 11.54. MS (ESI) m/z
calculated for C38H44BF2N7O4 718.22 (M+H+), found 718.37.
4.1.3.2. Ethyl 4-(benzylamino)-2-((2-((8-ethyl-5,5-difluoro-7,9-dimethyl-
10-phenyl-5H-5
l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1-3]diazaborinin-3-yl)
amino)ethyl)amino)pyrimidine-5-carboxylate (BP-2). Darkred solid;
Yield: 31%; 1H NMR: (400 MHz, CDCl3, ppm): δ 8.53 (s, 1H), 7.99 (s,
1H), 7.42–7.35 (m, 10H), 6.40–6.39 (d, J = 4.0 Hz, 1H), 5.89–5.88 (d, J
= 4.0 Hz, 1H), 4.78 (d, J = 4.0 Hz, 2H), 4.34–4.28 (m, 2H), 3.79 (s, 2H),
3.54–3.53 (d, J = 4.0 Hz, 2H), 2.41(s, 2H), 2.39 (s, 1H), 2.35–2.30 (m,
2H), 1.40 (s, 3H), 1.00–0.98 (t, J = 6.0 Hz, 3H), 0.90–0.88 (t, J = 6.0 Hz,
3H); 13C NMR (100 MHz, CDCl3, ppm): δ 170.46, 165.99, 161.36,
159.71, 153.93, 145.10, 137.68, 135.27, 134.48, 132.99, 132.72,
130.08, 129.67, 129.63, 128.80, 128.33, 128.10, 127.96, 127.57,
127.47, 123.99, 105.75, 96.95, 60.97, 44.90, 44.58, 40.60, 17.17,
14.97, 14.24, 14.12, 11.54. MS (ESI) m/z calculated for C35H38BF2N7O2
644.27 (M+H+), found 644.16.
4.2. Fluorescence spectrum and ultraviolet spectrum test
An appropriate amount of the test compound was weighed and dis-
solved in methanol, dichloromethane, ethyl acetate, acetonitrile,
dimethyl sulfoxide and DMF to prepare solutions of different concen-
trations for use. Four compounds in different concentrations in different
solvents were detected by UV and fluorescence [41]. The ultraviolet
wavelength range is 350–700 nm. The fluorescence experiments of the
four compounds were carried out under the optical path of 10 mm and
the excitation wavelength of 450 nm, and the recorded emission spec-
trum wavelength was 400–800 nm. In absolute ethanol solution, with
reference to Rhodamine B (Φf = 0.7) standard, the fluorescence quan-
tum yields of the four compounds were measured.
4.1.3.3. Ethyl 2-((2-((8-ethyl-5,5-difluoro-7,9-dimethyl-10-phenyl-5H-5
l4,6l4-dipyrrolo [1,2-c:2′,1′-f][1-3]diazaborinin-3-yl)amino)ethyl)amino)-
4-(phenethylamino)pyrimidine-5-carboxylate (BP-3). Darkred solid;
Yield: 47%; 1H NMR: (400 MHz, CDCl3, ppm): δ 8.51 (s, 1H), 7.31–7.24
(m, 11H), 6.36 (s, 1H), 5.89 (s, 1H), 4.31–4.26 (m, 2H), 3.81–3.80 (t, J
= 4.0 Hz, 2H), 3.74–3.72 (t, J = 6.0 Hz, 2H), 3.57–3.55 (t, J = 6.0 Hz,
2H), 2.97 (s, 3H), 2.49–2.44 (t, J = 12.0 Hz, 2H), 2.37–2.32 (m, 2H),
1.37–1.35 (t, J = 6.0 Hz, 3H), 1.29 (s, 3H), 1.02–0.98 (t, J = 8.0 Hz, 3H);
13C NMR (100 MHz, CDCl3, ppm): δ 167.54, 166.69, 161.73, 161.33,
159.81, 159.44, 157.65, 139.14, 138.54, 133.10, 131.39, 129.72,
128.81, 128.75, 128.61, 128.57, 128.00, 126.85, 126.48, 126.39,
106.92, 96.17, 60.36, 42.36, 42.09, 39.22, 35.74, 17.20, 15.00, 14.34,
14.32, 11.57. MS (ESI) m/z calculated for C36H40BF2N7O2 667.35 (M +
NH+4 ), found 667.31.
Considering the solubility of the compound and the biological
toxicity of the solvent, DMSO was selected as the solvent to determine
the stability of the fluorescence intensity of the four compounds in the
solvent within 10 min.
4.3. Biological
4.3.1. Cell culture and treatment
The cytotoxic activities of the compounds were screened against 4
human tumor cell lines and human normal liver in vitro. The cell lines
HepG2 (human liver cancer cell line) [42], HeLa (human cervical cancer
cell line), A-459 (human lung cancer cell line), HCT-116 (human colon
cancer cell line) and L-02 (human normal liver cell line) were all from
the American type culture collection. HepG2, HeLa, A-459 and HCT-116
4.1.3.4. Ethyl
4-((3,4-dimethoxyphenethyl)amino)-2-((2-((8-ethyl-5,5-
difluoro-7,9-dimethyl-10-phenyl-5H-5 l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1-3]
diazaborinin-3-yl)amino) ethyl)amino)pyrimidine-5-carboxylate (BP-4).
Red solid; Yield: 22%; 1H NMR: (400 MHz, CDCl3, ppm): δ 8.48 (s, 1H),
7.44–7.28 (m, 19H), 6.43–6.42 (d, J = 4.0 Hz, 1H), 5.92–5.91 (d, J =
9

C. Xu et al.
Bioorganic Chemistry 114 (2021) 105121
were routinely cultured in RPMI-1640 (Hyclone), while L-02 were
cultured in DMEM (Hyclone). All cell lines were supplemented with 10%
fetal bovine serum and grown in a humidified atmosphere of CO2/air (5/
95%) at 37 ◦C. These cells were monitored daily and kept at 80% cell
density.
receptor interaction at atomic level. Docking simulations were per-
formed on the crystal structure of thymidylate synthase complexed with
BP-1 ~ BP-4, extracted from Protein Data Bank (PDB ID: 6QYQ). Mo-
lecular docking was performed to evaluate 2D and 3D binding patterns
of compounds BP-1 ~ BP-4 at enzyme active sites. AutoDock Vina 1.1.2.
software [47,48] was used for docking, and pymol 1.5.6 was used for
processing and analyzing the docking results.
4.3.2. Cell imaging
HeLa cells in logarithmic growth phase were treated with trypsin,
and then inoculated into 9-well plate containing round plate, and then
cultured in incubator at 5% CO2 and 37 ◦C for 24 h [43]. The original
culture solution of each well cell was changed into a culture medium
containing 3 mmol/ml (Solvent for DMSO/physiological saline = 1:9, V/
V) of different compounds, and treated for 24 h. After 24 h, wash with
PBS twice. Then add paraformaldehyde fixative, discard the fixative
after 10 min, wash it with PBS twice, and incubate DAPI in the dark. 10
min later, the staining solution was discarded and washed twice with
PBS. Anti-fluorescence quenching sealing tablets were sealed and pho-
tographed by fluorescence microscope.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
We would like to thank the scientific research foundation of Jiangsu
University (Grant No. 5501290005) for the financial supports. Thanks
again for the in vivo and in vitro biological experimental data provided
by digestive disease center, shanghai east hospital, Tongji University.
4.3.3. In vitro cytotoxicity
MTT assay [44] was used to determine the cytotoxicity of human
cancer cells and normal human liver cell lines in logarithmic growth
phase in vitro. All cells were inoculated on the 96-well plate at the rate of
106 cells/well. Then, it was treated with 1, 2, 6, 8 or 10 mmol/mL
(Solvent for DMSO/physiological saline = 1:9, V/V) compounds and 5-
FU, and the samples were detected. After 24 h, the supernatant was
dissolved in DMSO (100 mL) and shaken well (10 min). The optical
density of the sample was measured at 490 nm by microplate photom-
eter. Cell viability is expressed as percentage change of absorbance
relative to control value.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105121.
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