The antitumor activity of 4,4′-bipyridinium amphiphiles

Article abstract of DOI:10.1039/c9ra06172j

A series of 4,4′-bipyridinium amphiphiles were synthesized and their anticancer activities were further evaluated. MTT assay showed that the cytotoxicity first increased and then decreased with the growth of carbon chains (8-16 C) at both ends of bipyridy

Full text of DOI:10.1039/c9ra06172j

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The antitumor activity of 4,40-bipyridinium
amphiphiles†
Cite this: RSC Adv., 2019, 9, 33023
Senlin Wang, Hongshuai Wu, Fanghui Chen, Yu Zhang, Yuchen Zhang
*
and Baiwang Sun
A series of 4,40-bipyridinium amphiphiles were synthesized and their anticancer activities were further
evaluated. MTT assay showed that the cytotoxicity first increased and then decreased with the growth of
carbon chains (8–16 C) at both ends of bipyridyl. Specifically, compounds with saturated carbon chains
consisting of 13 carbons at both ends of bipyridyl displayed the best cell inhibitory activity with IC50
values in the low-micromolar range, which were even superior to that of cisplatin, against all the tested
human cancer cells and cisplatin-resistant A549 cancer cells in vitro. In addition, compound 6 could
evidently arrest the G2/M phase of the cell cycle in a dose-dependent manner. Moreover, this study
demonstrates the potent performance of compound 6 in cell growth inhibition and apoptosis induction
via a conceivable approach of membrane damage.
Received 8th August 2019
Accepted 26th September 2019
DOI: 10.1039/c9ra06172j
rsc.li/rsc-advances
has been widely reported that a cationic peptide mainly destroys
the cell membrane and avoids the drug resistance mechanism
of cancer cells.15 However, a variety of CAPs having cancer-
selective toxicity are peptide-based drugs that are difficult to
be developed because of poor pharmacokinetics, potential
systemic toxicity and high cost of manufacturing.16–18 Never-
theless, a small molecular cationic antibacterial peptide pos-
sessing a simple structure and synthesis approach has been
demonstrated to produce a strong antibacterial activity and
a remarkable membrane cleavage effect.19
Based on this idea, it was expected to develop a simple and
an effective method to exploit cell membrane rupture cationic
amphiphilic compounds and lay a foundation for the develop-
ment of high-efficiency and low-toxic anti-cancer drugs. In spite
of their prevalence and amphiphilic nature, the anticancer
activity of these compounds remains largely unreported. Here,
we prepared a series of 4,40-bipyridinium amphiphiles to
investigate the structure–activity relationships in the inhibition
performance of cancer.
1. Introduction
Cancer, as a serious disease, urgently needs to be eliminated
due to its high mortality despite the considerable efforts that
have been devoted to understanding cancer biology and devel-
oping available therapeutic options over the past decades.1–4
Among the current strategies for the treatment of cancer,
chemotherapy still remains a signicant approach for over-
coming cancer. Although some impressive advances have been
achieved to improve the current status of the poor control of
cancer metastasis, some drawbacks, such as severe drug resis-
tance, and extremely strong drug-induced toxic side effects5–7
exist. Also, the efficacy of existing drugs is rather limited.
Obviously, the need for discovering and developing novel and
effective therapeutic agents with high targeting, low toxicity and
no drug resistance is imminent.
Amphiphiles, from simple soaps and benzalkonium chloride
to more complex antimicrobial peptides and dendrimers, have
a privileged platform for antimicrobials.8 Additionally, a new
class of cationic bioactive peptide-based therapies have become
a research hotspot in the exploitation of antitumor drugs.9
Particularly, many natural cationic antimicrobial peptides
(CAPs) can kill tumor cells and damage tumor cell membranes,
like bacterial cell membranes, which have a negative charge.10,11
Therefore, the cell cytotoxicity of such polypeptides can be
attributed to the electrostatic and hydrophobic interactions of
cationic amphiphilic polypeptides and cell membranes.12–14 It
2. Experimental section
2.1. Materials
A549 (alveolar basal carcinoma), MCF-7 and MDA-MB-231
(breast adenocarcinoma), BEL-7402 (hepatoma), L929 (murine
broblasts) and A549/CDDP (cisplatin-resistant lung cancer)
human cell lines were obtained from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). 3-(4,5-
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189,
PR China. E-mail: chmsunbw@seu.edu.cn; Fax: +86 25 52090614; Tel: +86 25
52090614
Dimethyl-thinazol-2-yl)-2,5-diphenyl
tetrazolium
bromide
(MTT) and 4,40-bipyridine were obtained from Sigma-Aldrich.
Annexin V-FITC apoptosis detection kit was purchased from
Beyotime Biotechnology (Haimen, Jiangsu, China). Fetal bovine
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra06172j
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serum (FBS) was supplied by Shanghai Ponsure Biotechnology cells were xed by the dropwise addition of 70% ethanol over-
Co., Ltd. (Shanghai, China). RPMI-1640 medium was purchased night at 4 ꢀC. For staining, cells were centrifuged and resus-
from Gibco Laboratories (NY, USA). All other chemicals and pended in cold PBS and treated with propidium iodide (PI) for
solvents were commercially available and used without further 30 min in the dark. The samples were recorded by a ow
purication.
cytometer and the MFLT32 soware was used for data analysis.
2.2. General procedure for the synthesis of 4,40-bipyridinium
amphiphiles
2.6. Confocal cell imaging
Confocal laser scanning microscopy (CLSM) was used to qual-
itatively detect the cell interactive behaviors with compound 6.
A549 cells were seeded in the F 20 mm confocal laser dish at
a density of 1 ꢁ 105 cells per dish and cultured for 24 h. The
cells were subsequently incubated with compound 6 for 1 h. The
cells cultured with the medium alone were used as a blank
control. The medium was removed, and the cells were washed
twice with cold PBS. Subsequently, a nal concentration of 50
mg mLꢂ1 PI and a nal concentration of 5 mg mLꢂ1 DAPI were
As shown in Fig. 1, 4,40-bipyridinium amphiphiles were
prepared according to a previously published paper8 with slight
modications. Briey, the corresponding alkyl bromide was
added to a solution of 4,40-dipyridyl (0.39 g, 2.5 mmol) in
acetonitrile (40 mL). The mixture was heated to reux for 24 h,
then cooled and ltered. The ltered solid was further washed
with acetonitrile to obtain the pure product as a yellow powder.
ꢀ
2.3. In vitro cytotoxicity
added and then incubated for 20 min at 37 C. The cells were
washed twice with cold PBS and the uorescence images were
immediately obtained via CLSM.
In this study, A549, MCF-7, BEL-7402, MDA-MB-231 cancer cells
and mouse broblasts (L929) were cultured in the RPMI 1640
medium supplemented with 10% FBS and a 1% penicillin–
streptomycin solution at 37 ꢀC under 5% CO2. In brief, the cells
were seeded in 96-well plates at a density of 7 ꢁ 103 per well.
Aer 24 h of incubation, the cells were exposed to various
compounds 1–10 for 24 h at 37 ꢀC. 150 mL of the MTT solution
(5.0 mg mLꢂ1) was added to each well and incubated for 4 h at
37 ꢀC. Subsequently, the medium was replaced with 150 mL
DMSO to dissolve the resultant purple crystals. The plates were
then shaken for 15 min. Finally, the absorption at 490 nm was
measured by a microplate reader and the prism soware was
used for data analysis.
3. Results and discussion
3.1. The synthesis and characterization of 4,40-bipyridinium
amphiphiles
As shown in Fig. 1, 4,40-bipyridinium amphiphiles were
prepared according to a previously published paper.8 The ob-
tained compounds were fully characterized by 1H and 13C NMR
spectroscopy. Mass spectrometry data of the corresponding
compounds are also presented in Fig. S1–S9.† Chemical purity
was analyzed by HPLC (Fig. S10–S12 in ESI†). All the samples
were determined to possess >95% of purity. HPLC also provided
information about the increase of hydrophobicity from
compounds 1 to 9. The compounds showed a systematically
increasing retention on a C18 reverse-phase stationary phase
due to the increasing length of the chain. The lipophilicity of
the compounds were evaluated by the calculatation of Clog P
values (Table S1†).20 As expected, the Clog P values increased
from 1 (ꢂ1.25) to 9 (7.22).
2.4. Cell apoptosis
For the cell apoptosis, A549 cells were seeded in 6-well plates at
a density of 1.0 ꢁ 105 cells per well and cultured for 24 h. The
cells were treated with compound 6 (5 mM) for 24 h. The cells
were harvested, and the Annexin V-FITC apoptosis detection kit
was then used to detect and quantify the apoptosis by a ow
cytometer.
Synthesis of 1,10-dioctyl-4,40-bipyridinium dibromide (1, PQ-
8,8). Reagents used in the reaction: octyl bromide (1.93 g, 10
mmol). Yield: 0.85 g (62.4%). Mp ¼ 300–301 ꢀC (dec.); 1H NMR
2.5. Cell cycle arrest
A549 cells were seeded in 6-well plates at a density of 1 ꢁ 105 (600 MHz, DMSO-d6) d: 9.50 (d, J ¼ 7.0 Hz, 4H), 8.88 (d, J ¼
cells per well and cultured for 24 h. The cells were treated with 7.0 Hz, 4H), 4.75 (t, J ¼ 7.5 Hz, 4H), 2.02–1.96 (m, 4H), 1.34–1.24
compound 6 (5, 10, 20 and 40 mM) for 24 h. Then, the cells were (m, 20H), 0.86 (t, J ¼ 7.0 Hz, 6H); 13C NMR (150 MHz, CD3OD) d:
collected and washed with cold PBS. Aer centrifugation, the 149.88, 145.70, 126.98, 61.92, 31.50, 31.21, 28.82, 28.75, 25.86,
Fig. 1 Synthesis route of 4,40-bipyridinium amphiphiles.
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Table 1 Cytotoxicity (IC50, mM) of compounds 1–9 against human cancer cell lines after 24 h of incubation
Cytotoxicity in different cell lines (IC50, mM)
Compounds
A549
MCF-7
BEL-7402
MDA-MB-231
L929
A549/CDDP
RFa
4,40-dipyridyl
>1000
>1000
>1000
>1000
>1000
>1000
—
1
2
3
4
5
6
7
8
141.4 ꢃ 5.66
27.96 ꢃ 1.23
16.17 ꢃ 0.8
13.13 ꢃ 0.5
9.5 ꢃ 0.42
161 ꢃ 5.98
31.34 ꢃ 1.38
14.87 ꢃ 0.68
10.76 ꢃ 0.46
7.31 ꢃ 0.33
5.96 ꢃ 0.3
86.1 ꢃ 3.42
18.98 ꢃ 0.86
18.2 ꢃ 0.76
12.25 ꢃ 0.6
9.51 ꢃ 0.43
5.56 ꢃ 0.2
163.15 ꢃ 7.68
30.32 ꢃ 1.12
16.72 ꢃ 0.76
11.53 ꢃ 0.51
9.36 ꢃ 0.45
7.06 ꢃ 0.35
7.09 ꢃ 0.33
9.61 ꢃ 0.42
32.32 ꢃ 1.45
22.16 ꢃ 1.08
240.2 ꢃ 3.68
54.52 ꢃ 1.21
39.72 ꢃ 0.63
25.63 ꢃ 0.48
17.95 ꢃ 0.78
13.24 ꢃ 0.53
19.65 ꢃ 0.41
30.59 ꢃ 0.36
59.97 ꢃ 1.25
18.96 ꢃ 1.16
158.8 ꢃ 6.98
34.21 ꢃ 1.45
22.58 ꢃ 1.1
15.74 ꢃ 0.72
13.88 ꢃ 0.69
8.44 ꢃ 0.42
11.34 ꢃ 0.54
16.07 ꢃ 0.75
28.58 ꢃ 1.32
141.83 ꢃ 6.9
1.13
1.22
1.4
1.2
1.46
1.02
1.09
1.16
1.4
8.26 ꢃ 0.38
10.38 ꢃ 0.5
13.88 ꢃ 0.67
20.45 ꢃ 0.96
15.35 ꢃ 0.68
14.1 ꢃ 0.7
6.536 ꢃ 0.31
12.22 ꢃ 0.54
27.54 ꢃ 1.11
15.69 ꢃ 0.73
19.64 ꢃ 0.97
21.82 ꢃ 1.08
20.16 ꢃ 0.89
9
Cisplatin
9.24
a
RF (resistant factor) is dened as IC50 in A549/CDDP/IC50 in A549. Mean values based on three independent experiments, and the results of the
representative experiments are shown.
22.29, 13.04; low resolution mass spectrum (ESI) m/z 381.3 [(M (m, 28H), 0.85 (t, J ¼ 6.9 Hz, 6H); 13C NMR (150 MHz, CD3OD) d:
ꢂ H)+; calcd for C26H41N2: 381.33].
149.88, 145.70, 126.98, 61.92, 31.65, 31.21, 29.23, 29.15, 29.03,
Synthesis of 1,10-dinonyl-4,40-bipyridinium dibromide (2, 28.79, 25.86, 22.34, 13.07; low resolution mass spectrum (ESI)
PQ-9,9). Reagents used in the reaction: octyl bromide (2.07 g, 10 m/z 437.4 [(M ꢂ H)+; calcd for C30H49N2: 437.40].
mmol). Yield: 0.86 g (60.6%). Mp ¼ 285–286 ꢀC (dec.); 1H NMR
Synthesis of 1,10-diundecyl-4,40-bipyridinium dibromide (4,
(600 MHz, DMSO-d6) d: 9.47 (d, J ¼ 6.7 Hz, 4H), 8.86 (d, J ¼ PQ-11,11). Reagents used in the reaction: 1-bromoundecane
6.7 Hz, 4H), 4.73 (t, J ¼ 7.4 Hz, 4H), 2.02–1.95 (m, 4H), 1.32–1.25 (2.35 g, 10 mmol). Yield: 0.92 g (58.6%). Mp ¼ 286–287 ꢀC (dec.);
(m, 24H), 0.85 (t, J ¼ 6.9 Hz, 6H); 13C NMR (150 MHz, CD3OD) d: 1H NMR (600 MHz, DMSO-d6) d: 9.45 (d, J ¼ 6.5 Hz, 4H), 8.85 (d,
149.89, 145.70, 126.96, 61.92, 31.60, 31.21, 29.10, 28.94, 28.78, J ¼ 6.6 Hz, 4H), 4.72 (t, J ¼ 7.4 Hz, 4H), 2.01–1.95 (m, 4H), 1.33–
25.86, 22.32, 13.04; low resolution mass spectrum (ESI) m/z 1.23 (m, 32H), 0.85 (t, J ¼ 6.9 Hz, 6H); 13C NMR (150 MHz,
409.3 [(M ꢂ H)+; calcd for C28H45N2: 409.37].
CD3OD) d: 149.88, 145.70, 126.97, 61.92, 31.68, 31.22, 29.32,
Synthesis of 1,10-didecyl-4,4-bipyridinium dibromide (3, PQ- 29.28, 29.15, 29.07, 28.79, 25.87, 22.35, 13.07; low resolution
10,10). Reagents used in the reaction: decyl bromide (2.21 g, 10 mass spectrum (ESI) m/z 465.4 [(M ꢂ H)+; calcd for C32H53N2:
mmol). Yield: 0.92 g (61.2%). Mp ¼ 284–285 ꢀC (dec.); 1H NMR 465.43].
(600 MHz, DMSO-d6) d: 9.47 (d, J ¼ 6.7 Hz, 4H), 8.86 (d, J ¼
Synthesis of 1,10-didodecyl-4,40-bipyridinium dibromide (5,
6.7 Hz, 4H), 4.73 (t, J ¼ 7.4 Hz, 4H), 1.98 (d, J ¼ 6.4 Hz, 4H), 1.28 PQ-12,12). Reagents used in the reaction: dodecyl bromide
Fig. 2 Apoptosis of A549 cells induced by compound 6 (5 mM).
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Fig. 3 A549 cell cycle arrest after the treatment with different concentrations of 6 (5, 10, 20, 40 mM) for 24 h. (a) Cell cycle analysis examined by
flow cytometry. (b) Bar chart showing cell distributions in various cell cycles.
(2.49 g, 10 mmol). Yield: 0.89 g (54.3%). Mp ¼ 287–288 ꢀC (dec.); (m, 40H), 0.89 (t, J ¼ 7.0 Hz, 6H). 13C NMR (150 MHz, CD3OD) d:
1H NMR (600 MHz, DMSO-d6) d 9.43 (d, J ¼ 6.9 Hz, 4H), 8.83 (d, J 149.92, 145.70, 126.94, 61.93, 31.67, 31.21, 29.35, 29.35, 29.26,
¼ 6.9 Hz, 4H), 4.71 (t, J ¼ 7.4 Hz, 4H), 2.01–1.95 (m, 4H), 1.33– 29.26, 29.13, 29.07, 28.78, 25.86, 22.34, 13.04; low resolution
1.23 (m, 36H), 0.85 (t, J ¼ 7.0 Hz, 6H); 13C NMR (150 MHz, mass spectrum (ESI) m/z 521.5 [(M ꢂ H)+; calcd for C36H61N2:
CD3OD) d: 149.90, 145.69, 126.95, 61.92, 31.67, 31.21, 29.34, 521.49].
29.34, 29.26, 29.14, 29.08, 28.78, 25.86, 22.34, 13.05; low reso-
Synthesis of 1,10-ditetradecyl-4,40-bipyridinium dibromide
lution mass spectrum (ESI) m/z 493.4 [(M ꢂ H)+; calcd for (7, PQ-14,14). Reagents used in the reaction: tetradecyl bromide
C
34H57N2: 493.46].
(2.77 g, 10 mmol). Yield: 0.93 g (52.6%). Mp ¼ 287–288 ꢀC (dec.);
Synthesis of 1,10-ditridecyl-4,40-bipyridinium dibromide (6, 1H NMR (600 MHz, DMSO-d6) d: 9.41 (d, J ¼ 6.5 Hz, 4H), 8.81 (d,
PQ-13,13). Reagents used in the reaction: tridecyl bromide J ¼ 6.5 Hz, 4H), 4.69 (t, J ¼ 7.4 Hz, 4H), 2.00–1.96 (m, 4H), 1.31–
(2.63 g, 10 mmol). Yield: 0.93 g (54.4%). Mp ¼ 287–288 ꢀC (dec.); 1.23 (m, 44H), 0.86 (t, J ¼ 6.9 Hz, 6H); 13C NMR (150 MHz,
1H NMR (600 MHz, CD3OD) d: 9.29 (d, J ¼ 6.9 Hz, 4H), 8.69 (d, J CD3OD) d: 149.91, 145.70, 126.91, 61.92, 31.66, 31.21, 29.35,
¼ 6.8 Hz, 4H), 4.77–4.74 (m, 4H), 2.12–2.06 (m, 4H), 1.46–1.27
Fig. 4 The interactive behaviors of A549 cells after incubation with compound 6 (20 mM) for 1 h.
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29.35, 29.35, 29.24, 29.24, 29.12, 29.08, 28.76, 25.85, 22.32, compound 6 against A549/CDDP (cisplatin resistant cancer
13.03. cells). As shown in Table 1, the IC50 values of compounds 1–9
Synthesis of 1,10-dipentadecyl-4,40-bipyridinium dibromide against A549/CDDP cells showed the same pattern as other
(8, PQ-15,15). Reagents used in the reaction: pentadecyl cells, rst decreasing and then increasing gradually. Interest-
bromide (2.91 g, 10 mmol). Yield: 0.91 g (49.4%). Mp ¼ 287– ingly, compound 6 was also found to exhibit obvious anticancer
288 ꢀC (dec.); 1H NMR (600 MHz, CD3OD) d: 9.29 (d, J ¼ 6.8 Hz, activities against A549/CDDP cells. The IC50 values of
4H), 8.69 (d, J ¼ 6.7 Hz, 4H), 4.77–4.73 (m, 4H), 2.12–2.06 (m, compound 6 against A549/CDDP cells did not obviously change
4H), 1.44–1.27 (m, 48H), 0.89 (t, J ¼ 7.0 Hz, 6H); 13C NMR (150 (IC50: 8.44 mM). On the contrary, the corresponding IC50 values
MHz, CD3OD) d: 149.94, 145.70, 126.89, 61.94, 31.67, 31.22, of cisplatin against A549/CDDP increased to 141.83 mM. It was
29.39, 29.39, 29.36, 29.36, 29.34, 29.25, 29.14, 29.08, 28.77, gratifying to observe that compounds 1–9 had much lower
25.86, 22.34, 13.03.
resistance factors (1.02–1.46) compared to cisplatin (9.24).
Synthesis of 1,10-dihexadecyl-4,40-bipyridinium dibromide
The results indicated that the hydrophobic chains at both
(9, PQ-16,16). Reagents used in the reaction: hexadecyl bromide ends of bipyridyl had a great effect on the cytotoxicity of 4,40-
(3.05 g, 10 mmol). Yield: 0.62 g (32.6%). Mp ¼ 284–285 ꢀC (dec.); bipyridinium amphiphiles. As the carbon chains (8–16 C) at
1H NMR (600 MHz, DMSO-d6) d: 9.38 (d, J ¼ 6.9 Hz, 4H), 8.78 (d, both ends of bipyridyl grew, the cytotoxicity increased rst and
J ¼ 6.9 Hz, 4H), 4.68 (t, J ¼ 7.4 Hz, 4H), 1.98 (d, J ¼ 7.3 Hz, 4H), then decreased, which also demonstrated that the compounds
1.27 (d, J ¼ 43.1 Hz, 52H), 0.85 (t, J ¼ 7.0 Hz, 6H).
with good cytotoxicity had calculated Clog P values of 1.93 to
5.10. The compound with saturated carbon chains consisting of
13 carbons at both ends of bipyridyl displayed the best anti-
tumor activity against all four of the tested human cancer cells
3.2. In vitro cytotoxicity
To determine the effects of synthesized 4,40-bipyridinium and cisplatin resistant A549 cancer cells. Therefore, compound
amphiphiles on cell proliferation and to gain the primary 6 with a superior cytotoxicity was chosen for further study.
structure–activity relationship, the cytotoxic activity of
compounds 1–9 were evaluated by standard MTT assays on four
human cancer cell lines (A549, MCF-7, BEL-7402 and MDA-MB-
231) and non-malignant cell line L929. Cisplatin was used as the
positive control. The corresponding IC50 values (concentration
required to reduce the viability to 50%) obtained aer the
treatment with different agents are listed in Table 1. The
increase in the activity and then the decrease in the homo-
sequence is a well-known phenomenon in medicinal chem-
istry.20 The lipophilicity directly contributed to the cytotoxicity
of these complexes. In vitro evaluation results revealed that the
IC50 values of compounds 1–9 decreases rst and then increases
gradually as the calculated Clog P values increase from
compound 1 (ꢂ1.25) to compound 9 (7.22). It was much
signicant to observe that compounds 4–7, the 4,40-bipyr-
idinium amphiphiles with saturated carbon chains consisting
of 11–14 carbons at both ends of bipyridyl, possessed a higher
cytotoxicity against all test cancer cell lines than cisplatin as the
calculated Clog P values increased from 1.93 of compound 1 to
5.10 of compound 7. In particular, compound 6 showed the best
growth inhibitory activity against A549 (IC50: 8.26 mM), MCF-7
3.3. Cell apoptosis
Compound 6 was found to exhibit a broad spectrum and the
best antitumor activity against all the tested ve human cancer
cells in vitro. In view of this, compound 6 was chosen to further
evaluate the mechanism of action. The induced apoptosis
performance of compound 6 on A549 cells was performed with
FITC and PI dual-staining method (Q1, Q2, Q3 and Q4 represent
four different cell states: necrotic cells, late apoptotic or necrotic
cells, apoptotic cells and living cells, respectively). The
percentages of apoptotic cells were determined by ow cytom-
etry. A549 cells were treated with 5 mM of compound 6 for 24 h.
As shown in Fig. 2, compared to the 0.62% apoptosis induced by
the medium alone (control group), the cells incubated with
compound 6 showed a remarkable apoptosis ratio (21.59%).
This result clearly informs that compound 6 could effectively
induce apoptosis in A549 cells at the indicated concentration,
which is consistent with the results of MTT.
3.4. Cell cycle arrest
(IC50: 5.96 mM), BEL-7402 (IC50: 5.56 mM) and MDA-MB-231 To further investigate the cell cycle arrest induced by compound
(IC50: 7.06 mM) cells as the calculated Clog P value was 4.04, 6, the cell cycle distribution of A549 cells treated with
which is very suitable for the lipophilicity of the drug. The compound 6 was detected by ow cytometry under the PI
cytotoxicity of compounds against normal L929 cells was eval- staining. As shown in Fig. 3a and b, compound 6 could effec-
uated to investigate the selectivity of compounds for cancer cells tively arrest the cell cycle at the G2/M phase in a concentration-
and normal cells. As displayed in Table 1, the IC50 values of dependent manner compared to the control. Meanwhile, the
compounds 1–9 against L929 cells showed a certain cell killing percentage of untreated A549 cells in the G0/G1 phase was at
activity, but lower IC50 values against all test cancer cell lines 56.42% with only 15.33% in the G2/M phase. Aer the treatment
indicated that the compounds had a certain selectivity for with compound 6 at 10 mM, the percentage of the G2/M phase
cancer cells. This may be because the surface of cancer cells has only increased to 17.76%, while in the cells treated with
a greater electronegativity than the normal cells.10,11
compound 6 at 20 and 40 mM, the percentages of the G2/M
Drug resistance is an important therapeutic obstacle, which phase increased to 30.70% and 33.25%, respectively. These
can conne the treatment efficacy of cisplatin for some human results show that compound 6 evidently arrested the G2/M
cancer cells. Thus, we further evaluated the cytotoxicity of phase of the cell cycle in a dose-dependent manner.
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3.5. Confocal cell imaging
References
Aer incubation with compound 6 for 1 h, A549 cells exhibited
very different morphological characteristics. As shown in Fig. 2,
in the control group, their morphology stayed intact. On the
contrary, the cells incubated with compound 6 exhibited some
membrane leakage and dispersing of many membrane frag-
ments in the culture medium could be observed. The difference
in the cell membrane integrity aer drug treatments could be
clearly demonstrated by confocal imaging using nuclear dyes.
DAPI can stain all living and dead cell nuclei, while PI can only
stain dead cells or cells with large changes in membrane
permeability. As depicted in Fig. 4, compound 6 treated cells
showed a co-staining feature of PI (red) and DAPI (blue) aer
1 h. However, the cell nuclei of the control group were stained
only by DAPI and the membranes were stained by PI. The results
indicate that the treatment of cells with compound 6 could
result in large changes in membrane permeability.
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4. Conclusions
In summary, a series of 4,40-bipyridinium amphiphiles were
synthesized and their antitumor effects in vitro were further
investigated. The results indicated that the hydrophobic chains
at both ends of bipyridyl had a great effect on the cytotoxicity of
4,40-bipyridinium amphiphiles. As the carbon chains (8–16 C) at
both ends of bipyridyl grow, the cytotoxicity rst increases and
then decreases. Compounds with saturated carbon chains
consisting of 13 carbons at both ends of bipyridyl displayed the
best cell growth inhibitory activity with IC50 values in the low-
micromolar range against all four of the tested human cancer
cells and the cisplatin resistant A549 cancer cells in vitro. In
addition, compound 6 evidently arrested the G2/M phase of the
cell cycle in a dose-dependent manner. Finally, our study
demonstrated that the potent activity in the cell growth inhi-
bition and apoptosis induction of compound 6 may be related
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Conflicts of interest
17 S. Maijaroen, N. Jangpromma, J. Daduang and
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There are no conicts to declare.
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This work was nancially supported by the National Natural
Science Foundation of China (Grant No. 21628101 and
21371031), the International S&T Cooperation Program of
China (No. 2015DFG42240) and the Priority Academic Program
Development (PAPD) of Jiangsu Higher Education Institutions.
33028 | RSC Adv., 2019, 9, 33023–33028
This journal is © The Royal Society of Chemistry 2019