Enhancement of cell uptake and antitumor activity of selenadiazole derivatives through interaction and delivery by serum albumin

Article abstract of DOI:10.1039/c6ra28801d

Serum albumin is an important carrier in the transport of endogenous and exogenous substances across cell membranes. Small molecule drugs could bind to serum albumin to various extents after intravenous injection, which may affect the bioavailability, metabolism, pharmacological and toxicological potency. Organic selenium (Se) compounds exhibit favorable biocompatibility and low toxicity that attracts increased attention from researchers. Herein, a series of selenadiazole derivatives (4a-e) have been synthesized and their cytotoxicity towards various kinds of cells were evaluated. The results suggested that isopropyl benzo[c][1,2,5]selenadiazole-5-carboxylate (4d) exhibited a broad inhibition spectrum on the proliferation of tested cancer cells, with a relatively higher anticancer activity than other derivatives. We found that the differences in their anticancer activities were attributed to the various binding abilities of the Se compounds toward BSA. The bimolecular quenching constant (K_q), apparent quenching constant (K_SV), effective binding constant (K_A) and binding site number (n) were obtained through fluorescence quenching calculations, which indicated that all compounds could efficiently bind to BSA molecules and the fluorescence quenching mechanism was mainly a static quenching procedure. Moreover, serum albumin was found to interact with selenadiazole derivatives (4a-e), thus promoting the cellular uptake and anticancer activity of the Se compounds. Taken together, this study demonstrated that the synthetic selenadiazole derivatives exhibited high anticancer activity and cellular uptake through delivery by human serum.

Full text of DOI:10.1039/c6ra28801d

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Enhancement of cell uptake and antitumor activity
of selenadiazole derivatives through interaction
and delivery by serum albumin†
Cite this: RSC Adv., 2017, 7, 16721
a
a
a
b
c
Shulin Deng,‡ Delong Zeng,‡ Yi Luo, Jianfu Zhao, Xiaoling Li, Zhennan Zhaoa
*
a
*
and Tianfeng Chen
Serum albumin is an important carrier in the transport of endogenous and exogenous substances across
cell membranes. Small molecule drugs could bind to serum albumin to various extents after intravenous
injection, which may affect the bioavailability, metabolism, pharmacological and toxicological potency.
Organic selenium (Se) compounds exhibit favorable biocompatibility and low toxicity that attracts
increased attention from researchers. Herein, a series of selenadiazole derivatives (4a–e) have been
synthesized and their cytotoxicity towards various kinds of cells were evaluated. The results suggested
that isopropyl benzo[c][1,2,5]selenadiazole-5-carboxylate (4d) exhibited a broad inhibition spectrum on
the proliferation of tested cancer cells, with a relatively higher anticancer activity than other derivatives.
We found that the differences in their anticancer activities were attributed to the various binding abilities
of the Se compounds toward BSA. The bimolecular quenching constant (Kq), apparent quenching
constant (KSV), effective binding constant (KA) and binding site number (n) were obtained through
fluorescence quenching calculations, which indicated that all compounds could efficiently bind to BSA
molecules and the fluorescence quenching mechanism was mainly a static quenching procedure.
Moreover, serum albumin was found to interact with selenadiazole derivatives (4a–e), thus promoting
the cellular uptake and anticancer activity of the Se compounds. Taken together, this study
demonstrated that the synthetic selenadiazole derivatives exhibited high anticancer activity and cellular
uptake through delivery by human serum.
Received 29th December 2016
Accepted 2nd March 2017
DOI: 10.1039/c6ra28801d
rsc.li/rsc-advances
has been focused on the application of organoselenium
compounds in chemoprevention and chemotherapy. Recently,
Introduction
a large number of potent organoselenium compounds have
been designed and synthesized which showed greater chemo-
preventive efficacy,8 such as selenocyanate,9 selenobetaine and
selenium analogues of amino acids.10–13 In addition, the
synthesis of heterocyclic compounds with a Se atom in the ring
is a fascinating eld owing to their pharmacological potential
and fascinating chemical properties.5,14–17 For instance, a large
amounts of selenazolo derivatives, like ebselen, 6-bromo[1,2,5]
Although people's living standard has been improved, cancer is
still one of the serious diseases threatening human health.1 In
the past decades, a large number of agents have been synthe-
sized and investigated for their effects on cancer chemo-
prevention and chemotherapy. Particularly, selenium (Se), an
essential trace element that is fundamentally important to
humans,2,3 has been veried as a potential cancer chemo-
preventive and chemotherapeutic agent by various epidemio-
logical, preclinical and clinical studies.4,5 Comparing with
inorganic selenium, organoselenium compounds show several
advantages, such as higher bio-absorption, higher anticancer
activity and lower cytotoxicity.6,7 Therefore, growing attention
selenadiazolo[3,4-b]pyridine,
4-methyl-1,2,3-selenadiazole-5-
carboxylic acid amides and 6-phenyl-7(6H)-isoselenazolo[4,3-d]
pyrimidone, have been synthesized and found to exhibit anti-
tumor activities in vitro and in vivo.14,18
However, the anticancer mechanisms of the selenium
compounds remain elusive, which limits their clinical devel-
opment. Serum albumin, a globular heart-shaped protein
composed of three structurally similar domains (I, II and III), is
a 583-amino acid residue monomer with a molecular weight of
66.4 kDa. And each domain consists of two subdomains (A and
B) stabilized by 17 pairs of disulde bridges.19,20 Human serum
albumin (HSA)21 is a major constituent of blood plasma, having
many physiological functions like transportation facilitation or
aDepartment of Chemistry, Jinan University, Guangzhou 510632, China. E-mail:
tchentf@jnu.edu.cn; tlxlli@jnu.edu.cn
bThe First Affiliated Hospital of Jinan University, Guangzhou, China
cInstitute of Food Safety and Nutrition, Jinan University, Guangzhou, China
† Electronic supplementary information (ESI) available. CCDC 1516438, 1516447
and 1517017. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c6ra28801d
‡ These authors contributed equally to the work.
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delivery of several endogenous and exogenous compounds.22,23
It is well known that small molecule drugs are capable to bind
with HSA,24 which may have impacts on their bioavailability and
antitumor potency. Especially, the binding affinity of drugs
toward BSA and HSA affect the rate of the drug release to sites of
catabolism or pharmacological action.25 Generally, BSA is
chosen as a target protein molecule due to its low cost, ready
availability and unique ligand-binding properties.26 The studies
on drug's binding to BSA provide the information about struc-
tural features that could determine the therapeutic effective-
ness of drugs, which become an important research eld in
chemistry, life science and clinical medicine.
Scheme 1 Synthetic scheme for the production of selenadiazole
derivatives (4a–e).
The quantum yield of uorescence was decreased because
the uorophore was induced by a variety of molecular interac-
tions with a quencher molecule.27 Fluorescence quenching can
be dynamic, resulting from collisional encounters between the
uorophore and the quencher, or static, resulting from the
formation of a ground-state complex between the uorophore
and the quencher.28 In both cases, molecular contact is required
between the uorophore and the quencher for uorescence
quenching. A number of studies on the interaction of small
molecules or drugs with BSA have been recently reported29–31
and elucidation of the molecular aspects of the binding
continues to be of great importance in the terms of under-
standing protein structure–function and designing drug.
In the present study, we have synthesized a series of sele-
nadiazole derivatives (4a–e), and investigated for their under-
lying anticancer actions, including interactions toward BSA,
cellular uptake and anticancer activities. The results showed
that, BSA molecules could easily interact with these ve sele-
nadiazole, and then quench their intrinsic uorescence effi-
ciently. Interestingly, all the emission maximum of tyrosine
residues have a slight blue shi and the slight red shi of all
tryptophan (Trp) residues uoresce, which indicated that the
polarity around the tyrosine (Tyr) residues increased and the
hydrophobicity decreased, while the tryptophan residues'
hydrophobicity was increased. Among these selenadiazole
derivatives, compound 4d with isopropyl group could be easily
internalized by cells due to its high binding affinity toward BSA,
Fig. 1 Crystal structures of compounds 4c, 4d and 4e.
2a, and one human normal cells HK-2 by MTT assay32 aer
72 h treatment. From the Table 1, we found 4d showed broad
inhibition on the tested cancer cells with IC50 values signi-
cantly lower than those of its analogues, indicating higher
cytotoxic effects of 4d on cancer cells.
These results suggest that, the as-synthesized selenadiazole
derivatives demonstrate comparable anticancer efficacy with
the previously reported compounds. For instance, Xie et al. re-
ported that 4 biphenyl selenadiazole derivatives with IC50 on
A375 cells ranging from 23.3 mM to 66.6 mM had a radiation
sensitization activity.5 Yang et al. reported 7 biphenylselena-
diazole derivatives with IC50 on bladder cancer cells ranging
from 1.48 mM to 78.67 mM had activities of inducing apoptosis
and inhibition of migration and invasion.17 Liu et al. reported
biphenylselenadiazole derivatives with a IC50 at about 30 mM
against HepG2 cells had the ability to reverse the drug resis-
tance induced by hyperglycemia.16 Taken together, these studies
conrm the anticancer application potential of selenadiazole
derivatives.
Binding property of the selenadiazole derivatives (4a–4e) to
the serum albumins
thus enhancing the anticancer efficacy toward MCF-7 cancer Next the binding of 4a–e to BSA was investigated by uorescence
cells. Taken together, this study demonstrates that, the spectroscopy. BSA excited at 296 nm and showed emission at
synthetic selenadiazole derivatives could be further developed 348 nm, which is mainly attributed to the presence of two
as effective and safe anticancer agent for cancer chemotherapy. tryptophan residues (Trp-134 and Trp-213), that processed
intrinsic uorescence.33,34 Trp-134 in the rst domain is located
on the surface of the hydrophilic region of the protein while
Results and discussion
Trp-213 in the second domain is located within a hydrophobic
Synthesis and in vitro anticancer activities of selenadiazole
binding pocket.35,36 The absorption spectra of 4a–4e were
derivatives (4a–e)
showed in Fig. S19.† The maximum absorption of 4a–4e were
The synthesis of selenadiazole derivatives (4a–e) was outlined in around 320 nm, which will not interfere on the observation the
Scheme 1. The crystal structures of 4c, 4d and 4e (Fig. 1) were binding property of these compounds to serum albumin by
obtained. Crystal data and structure renement data of the using uorescence quenching assay. The uorescence quench-
three compounds were listed in ESI Table S1.† Crystallographic ing spectra of BSA (1.0 mM) in Tris–HCl buffer with increasing
data structures of compound 4c, 4d and 4e have been deposited concentration of compounds 4a–e were shown in Fig. 2A–E. The
to the Cambridge Crystallographic Data Centre with CCDC uorescence intensity was attenuated gradually with the
1516447, 1516438 and 1517017. The in vitro anticancer activities increasing concentration of each compound, which displayed
of 4a–e were rstly screened against a panel of ve human that there was a binding interaction of compounds to BSA and
cancer cell lines, including A549, A375, MCF-7, HepG2, Neuro- the energy transfer occurred. Generally, the uorescence
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Table 1 Growth inhibition of 4a–e against human cancer and normal cell lines
IC50 (mM)
Cell linesa
4a
4b
4c
4d
4e
A549
A375
MCF-7
HepG2
Neuro-2a
HK-2b
125.2 ꢂ 7.36
69.14 ꢂ 2.71
59.78 ꢂ 2.77
27.81 ꢂ 4.33
68.54 ꢂ 2.57
118.23 ꢂ 4.7
47.90 ꢂ 7.56
76.40 ꢂ 2.39
69.71 ꢂ 2.78
193.61 ꢂ 3.59
115.06 ꢂ 4.14
122.17 ꢂ 3.29
156.61 ꢂ 5.53
151.13 ꢂ 3.99
217.61 ꢂ 2.71
109.85 ꢂ 5.00
78.0 ꢂ 8.96
117.63 ꢂ 1.68
46.3 ꢂ 1.61
63.71 ꢂ 1.65
33.2 ꢂ 8.31
141.97 ꢂ 10.19
143.85 ꢂ 11.51
224.63 ꢂ 8.51
103.28 ꢂ 2.21
105.83 ꢂ 5.19
208.31 ꢂ 3.74
72.45 ꢂ 9.87
51.82 ꢂ 9.46
136.74 ꢂ 10.77
a
b
Cancer cells were exposed to different concentration of SeDs for 72 h and then cell viability was measured by MTT assay. Normal cells.
quenching can be dynamic or static.35 Firstly, as a hypothetical derived from these plots, Kq could be calculated from the ratio
dynamic quenching process, the data of uorescence intensities of KSV and s0 showed value of 1012 order of magnitude. The
were analyzed by using the Stern–Volmer equation:37
corresponding plot expressions were shown in Table 2.
For dynamic quenching, the maximum value of the scat-
tering collision quenching constant Kdiff is 2.0 ꢁ 1010 l molꢀ1
F0/F ¼ 1 + Kqs0[Q] ¼ 1 + KSV[Q]
(1)
sꢀ1 39 Since the value of the biomolecular quenching is higher, it
.
where F0 and F are the uorescence intensities in the absence
and presence of the quencher, respectively. Kq is the quenching
rate constant. KSV is the dynamic quenching rate constant, s0 is
the average lifetime of the protein without the quencher, which
is of the order of 10ꢀ8 s (ref. 38) and [Q] is the concentration of
compounds. The plot of F0/F versus [Q] was linear (Fig. 2F–J),
indicating one type of quenching. The values of KSV could be
can be assumed that the complex formation between the
quencher and the protein is a static mechanism. The static
quenching mechanism was further conrmed by the tempera-
ture dependence of the quenching; KSV values decreased with
increasing temperature for static quenching while the reverse is
observed for dynamic quenching.40,41 The uorescence data
were analyzed at two different temperatures. Seen from the
Table 2, the Stern–Volmer quenching constant KSV and the
quenching rate constant Kq values decreased with increasing
temperature, suggesting that the quenching mechanism was
complex formation rather than dynamic collision. In other
words, the uorescence quenching of BSA induced by these
compounds was caused by specic complex formation.
Analysis of binding equilibria
From Fig. 2K–O, the equilibrium constants (KA) and the binding
site numbers (n) could be calculated by using the Double
logarithm equation:42
lg[(F0 ꢀ F)/F] ¼ lg KA + n lg[Q]
(2)
Apparently, the KA and n could be measured, respectively,
from the intercept and slope obtained through plotting lg[(F0 ꢀ
F)/F] against lg[Q], shown in Table 2. There was a strong binding
force between 4a–e and BSA. In addition, the KA value of these
complexes demonstrated that the binding affinity of 4d to BSA
was stronger. The values of n were equal to about 1, illustrating
that there was one class of binding site for the compounds in
the neighborhood of the tryptophan residue.43
Types of interaction force between BSA with selenadiazole
derivatives
Fig. 2 (A–E) Fluorescence emission spectra of BSA in the presence of
various concentrations of 4a–e, curves from (A to I) correspond to 4a–
e concentrations of 0, 1, 2, 3, 4, 5, 6, 7 and 8 mM, respectively (T ¼ 298
K, pH ¼ 7.4 and lex ¼ 296 nm); (F–J) Stern–Volmer plot for the binding
of 4a–e with BSA at 298 K and 303 K; (K–O) The Double logarithm plot
of 4a–e at 298 K and 303 K.
Following, studies were also carried out to analyse the modes of
interaction between BSA and the as-synthesized selenadiazole
derivatives. Utilizing KA, the free energy change (DG) was
calculated from the relationship:
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Table 2 Quenching constants (KSV and Kq), binding constants and binding site numbers of the interaction of 4a–e with BSA at two different
temperaturea
Eqn (1)
Eqn (2)
System
Temperature (K)
KSV (L molꢀ1
)
Kq (L molꢀ1 sꢀ1
)
KA (L molꢀ1
)
n
BSA + 4a
BSA + 4b
BSA + 4c
BSA + 4d
BSA + 4e
298
303
298
303
298
303
298
303
298
303
8.42 ꢁ 104
6.43 ꢁ 104
7.24 ꢁ 104
6.00 ꢁ 104
8.35 ꢁ 104
7.12 ꢁ 104
11.81 ꢁ 104
9.54 ꢁ 104
11.35 ꢁ 104
6.38 ꢁ 104
8.42 ꢁ 1012
6.43 ꢁ 1012
7.24 ꢁ 1012
6.00 ꢁ 1012
8.35 ꢁ 1012
7.12 ꢁ 1012
11.81 ꢁ 1012
9.54 ꢁ 1012
11.35 ꢁ 1012
6.38 ꢁ 1012
3.87 ꢁ 104
7.94 ꢁ 104
3.84 ꢁ 104
4.07 ꢁ 104
3.18 ꢁ 104
1.21 ꢁ 105
3.27 ꢁ 105
3.47 ꢁ 105
1.91 ꢁ 105
1.01 ꢁ 105
0.935
1.0209
0.9578
0.9707
0.9239
1.0335
1.0866
1.10
1.052
1.0367
a
These values were obtained respectively using Stern–Volmer plots, Double logarithm plots and Van't Hoff plots of the interaction of 4a–e with BSA
(compounds concentrations from 0 ꢁ 10–6 mol lꢀ1 to 8 ꢁ 10–6 mol lꢀ1).
process. From Table 3, we can deduce that the acting forces of
DG ¼ ꢀRT ln KA
(3)
BSA-4a, 4b, 4c system and the BSA-4d, 4e system were mainly
hydrophobic interactions and hydrogen bonding, van der Waals
force respectively.
When the temperature change is not very large, the enthalpy
change (DH) of a system can be regarded as a constant.43 Under
these conditions, both the enthalpy change (DH) and entropy
change (DS) can be evaluated from the following equations:
Effect of selenadiazole derivatives (4a–e) on BSA synchronous
uorescence spectra
ln(K2/K1) ¼ (1/T1 ꢀ 1/T2)DH/R
DG ¼ DH ꢀ TDS
(4)
(5)
Synchronous uorescence spectra can provide much valuable
information about the microenvironment around uorogens in
biomolecules.46,47 For protein molecules, when the l ¼ 15 nm is
xed, the spectrum characteristic of tyrosine residues could be
observed, and when l ¼ 60 nm is done, the spectrum charac-
teristic of tryptophan residues could be obtained.48 In Fig. 3, ve
courses the uorescence intensities for l ¼ 15 nm (upper) and l
¼ 60 nm (nether) are both quenched more and more severely
with increasing compounds concentration, which further
demonstrated uorescence quenching in the binding process.
Furthermore, all the emission maximum of tyrosine residues
have a slight blue shi and a slight red shi of all tryptophan
residues uoresce, which indicated that the polarity around the
tyrosine residues increased and the hydrophobicity decreased,49
while the tryptophan residues' hydrophobicity was increased. In
conclusion, these ve compounds affect the microenvironment
of Tyr and Trp residues through binding to BSA molecules with
different accessibility.50
In this study, the results (Table 3) showed that the binding of
compounds 4a–e to BSA is a spontaneous process, as indicated
by the negative DG values. From the values of DG, it demon-
strated that compound 4d bound more easily with BSA mole-
cules. Essentially, there are four main types of noncovalent
interactions existing in ligand binding to proteins, which are
hydrogen bonds, van der Waals forces, hydrophobic and elec-
trostatic interactions.44,45 According to the principles summa-
rized by Ross and Subramanian, for drug protein interactions,
the positive values of DH and DS indicate that hydrophobic
interactions play a major role in the binding process. While, the
negative values of DH and DS suggest that hydrogen bonding
and van der Waals force play a crucial role in the binding
Table 3 Thermodynamic parameters of the interaction of 4a–e with
BSA at different temperatures
Selenadiazole derivatives (4a–e) inhibit cancer cell growth by
enhanced cellular uptake and induced apoptosis
System
T (K)
DGꢃ (kJ molꢀ1
)
DHꢃ (kJ molꢀ1
)
DSꢃ (J molꢀ1
)
Cellular uptake is a complicated biological process that asso-
ciates with the interaction between extracellular materials and
the plasma membrane or the receptor proteins.51,52 The efficacy
of cellular uptake is a critical factor that affects the selenadia-
zole derivative agent activities and determines their successful
clinical application. Therefore, in this study, we have examined
the uptake of selenadiazole derivatives (4a–e) in MCF-7 cell in
the presence or absence of serum. The stability of BSA in DMEM
medium with or without 4a–4b was detected by measuring the
size distribution. As shown in Fig. S20,† the size of BSA was
BSA + 4a
BSA + 4b
BSA + 4c
BSA + 4d
BSA + 4e
298
303
298
303
298
303
298
303
298
303
ꢀ26.17
ꢀ28.42
ꢀ26.15
ꢀ26.73
ꢀ25.69
ꢀ23.68
ꢀ31.46
ꢀ32.13
ꢀ31.07
ꢀ29.03
543.61
543.61
354.44
354.44
200.63
200.63
ꢀ336.80
ꢀ336.80
ꢀ498.81
ꢀ498.81
1.73
1.73
1.28
1.28
0.76
0.76
ꢀ1.02
ꢀ1.02
ꢀ1.55
ꢀ1.55
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Fig. 4 Inhibition rate of 4a–e. (A–E) The inhibitory effects of 4a–e on
MCF-7 cells after 72 h incubation with or without BSA in DMEM culture
medium. (F–J) The inhibitory effects of 4a–e on MCF-7 cells after 72 h
incubation with or without HSA in DMEM culture medium. Significant
difference between treatment and control groups is indicated at the P
< 0.05 (*) or P < 0.01 (**) level.
Fig. 3 Synchronous fluorescence spectra of BSA in the presence of
various concentrations of 4a–e, concentrations from 0 to 6 mM. Dl ¼
15 nm (A–E); Dl ¼ 60 nm (F–J) (T ¼ 298 K, pH ¼ 7.4).
stable at around 4 nm in the absent or present of 4a–4b for up to
72 h. These results demonstrate the stability of BSA during drug normal cells (Fig. 5B and C). Moreover, as shown in Fig. 4F–J
delivery. As shown in Fig. 5A, the absorption of selenium value and 5D, similar results can be gained when BSA was replaced by
of 4d containing an isopropyl substituent in MCF-7 cells was HSA. To further conrm the selective growth inhibition of 4d on
about 5.7 mM/106 cells, which was higher than that of 4a (4.45 cancer cells, we performed a co-cultured assay. As shown in
mM/106 cells), 4b (4.59 mM/106 cells), 4c (4.91 mM/106 cells), 4e Fig. 6A and B, the viabilities of A549 cells were signicantly
(5.22 mM/106 cells). Consistently, comparing with 4a, 4b, 4c and decreased when the cells were treated with 4d, while the
4e, 4d showed a higher absorption value and a dramatic viabilities of HK-2 cells have no obvious change.
enhancement in the anticancer efficacy (Fig. 4A–E). The viabil-
To compare the importance of interaction with BSA on the
ities of cells treated with different concentrations of the sele- cellular uptake and anti-tumour activity of selenadiazole
nadiazole derivatives were more than 80% in basal culture derivatives and other cancer drugs, the IC50 of a wildly used
medium without FBS, but the viabilities decreased signicantly anti-cancer drug, cisplatin, and 3 selenadiazole derivatives 4c–
when BSA (25 mM or 50 mM) was added to the media. These 4e which showed relative higher activity were detected by MTT
results indicate that BSA as a drug carrier of selenadiazole assay. As shown in Fig. 6C, although cisplatin exhibited lower
derivatives effectively promote cell uptake and enhance anti- IC50 then all the selenadiazole derivatives, the values were not
tumor activity against cancer cells. Further determination of the affected by the present or absent of BSA. On the contrary, 4c–4e
cellular uptake of 4d in A549 cells and HK-2 cells shown that shown signicantly reduced IC50 when BSA was added. In
uptake of 4d by cancer cells were much higher than that by addition, the selenadiazole derivatives demonstrate high
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To explore the mechanism of cell death induced by selena-
diazole derivatives aer uptake by cancer cells, we measured the
cell cycle distribution and apoptosis of MCF-7 cells aer treated
with 4d. The result of PI-staining and ow cytometric analysis
(Fig. 7) shown that 4d induced the increase of sub-G1 fraction of
MCF-7 cells in a dose-dependent manner, suggesting anti-
tumor activity of 4d may be caused by the induction of
apoptosis.
Conclusions
In conclusion, we have synthesized ve selenadiazole deriva-
tives (4a–e), and compared their anticancer activities in
vitro. Based on these results, the isopropyl benzo[c][1,2,5]
selenadiazole-5-carboxylate (4d) exhibited better anticancer
activities than others. The studies of the interaction with BSA
suggested that these ve compounds can bind to BSA molecules
easily, and then quench their intrinsic uorescence efficiently.
The binding parameters further illustrated that all compounds
can efficiently bind to BSA molecules. We speculated that
a series of complexes were formed between compounds 4a–e
and BSA by hydrophobic interactions, hydrogen bond, and van
der Waals force. Interestingly, 4d exhibited not only better
capacity of binding to BSA, but also higher anticancer activities.
The biological signicance of this work is that albumin serves
as a carrier molecule for selenadiazole derivatives to enhance
their antitumor activity, which may have an inuence on the
elds of pharmacology and clinical medicine.
Fig. 5 Cellular uptake of 4a–e. (A–C) Different cellular uptake of 4a–e
(200 mM) on MCF-7, A549 and HK-2 cells treated with or without BSA
in DMEM culture medium for 6 h. (D) Different cellular uptake of 4a–e
(200 mM) on MCF-7 cells treated with or without HSA in DMEM culture
medium for 6 h. Significant difference between treatment and control
groups is indicated at the P < 0.05 (*) or P < 0.01 (**) level.
Materials and methods
Materials
Fig. 6 (A) The represent images of HK-2 (green) and A549 (red) cells
treated with 4d for indicated time. (B) The viabilities of HK-2 and A549
cells treated with 4d in (A) were calculated by normalizing to untreated
control group. Bars ¼ 400 mm (C) The IC50 of 4c–e and cisplatin (cis)
on MCF-7 cells after 72 h incubation with or without BSA in DMEM
culture medium. Significant difference between groups with or
without BSA is indicated at the P < 0.05 (*) or P < 0.01 (**) level.
Methanol, ethanol, propanol, isopropyl, n-butanol, chloroform,
sodium hydroxide, sodium sulfate anhydrous, hydrochloric
acid were purchased from Guangzhou chemical reagent factory
and selenium dioxide, 3,4-diaminobenzoic acid, BSA and HSA
were purchased from J&K Chemical Company. Thiazolyl blue
tetrazolium bromide (MTT), DMSO, DMEM media were
purchased from sigma. The solutions of BSA and HSA were
prepared in 0.01 M Tris–HCl buffer of pH 7.4 containing 0.01 M
NaCl. BSA and HSA solutions were prepared based on their
molecular weights of 65 000 and 66 000, respectively. The water
used in all experiments was ultrapure by a Mili-Q water puri-
cation system from Milipore.
selectivity between cancer (A549) and normal (HK-2) cells,
which is an important advantage for the future clinical appli-
cation. Taken together, these results fully showed that BSA (or
HSA) as a drug carrier could greatly enhance the absorption of
selenadiazole derivatives to signicantly enhance their anti-
tumor activity and selectivity.
Synthesis and characterization of selenadiazole derivatives
(4a–e)
The synthetic route for SeDs (4a–4e) was elucidated in Scheme
1. The rst step, 10 mmol of 3,4-diamino-benzoic acid was
dissolved in 250 ml hydrochloric acid solution (HCl : H2O ¼
1 : 5) in a 500 ml ask. 10 mmol SeO2 which dissolved in 20 ml
of hot distilled water was dropping into the ask through
a constant pressure funnel stirred for 2 h. Aer that, the reacted
solution was ltrated and the obtained solids were washed with
water for several times until the pH value reached to 7. Then the
products were dried at 60 degree to get the pure benzo[1,2,5]
Fig. 7 Quantitative analysis of cell cycle distribution and apoptotic
cells induced by different concentrations of compound 4d by PI-flow
cytometric analysis in MCF-7 cells.
16726 | RSC Adv., 2017, 7, 16721–16729
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selenadiazole-5-carboxylic acid. Yield: 95%. In the second step, 1H), 7.95 (d, 1H), 8.45 (s, 1H), 5.91 (m, 1H), 1.37 (d, 6H). 13C
10 mmol of benzo[1,2,5]selenadiazole-5-carboxylic acid dis- NMR (125 MHz, DMSO, d, ppm): 22.09, 69.53, 123.94, 125.83,
solved with 50 ml CHCl3 was placed in a 100 ml three-necked 127.85, 131.03, 159.46, 161.16, 165.24. Elemental analysis calc.
ask, which was provided with a stirrer. Then the correspond- (%) for C10H10N2O2Se: C, 44.62; H, 3.74; N, 10.41; found (%): C,
ing alcohol was added slowly and dropwise under stirring and 44.59; H, 3.77; N, 10.40.
ꢃ
the temperature at 50 C. And small amounts of sulfuric acid
were added as a catalyst. The reaction mixture was stirred for
another 4 h. The solvent was removed in vacuum distillation,
then, another 300 ml 1 mol/LNaSO4 solution was added to wash
the crude product. Aer that, the crude products were ltrated,
and the pure product was obtained through recrystallization
from corresponding alcohols. Yield: about 65%.
Butyl benzo[c][1,2,5]selenadiazole-5-carboxylate
The compound 4e was prepared according to a general
procedure. ESI-MS (in CH3OH): m/z calcd for C11H12N2O2Se (M
1
+ H)+ 285.0; found 285.2. H NMR (500 MHz, DMSO, d, ppm):
7.95 (d, 1H), 7.96 (d, 1H), 8.44 (s, 1H), 1.45 (m, 2H), 1.74 (m,
2H), 4.05 (t, 2H), 0.95 (t, 3H). 13C NMR (125 MHz, DMSO, d,
ppm): 14.08, 19.22, 30.63, 65.54, 123.96, 125.86, 127.78,
130.64, 159.41, 161.14, 165.75. Elemental analysis calc. (%) for
Benzo[c][1,2,5]selenadiazole-5-carboxylic acid
The compound 2 was prepared according to a general proce-
dure. ESI-MS (in CH3OH): m/z calcd for C7H4N2O2Se (M ꢀ H)ꢀ
226.9; found 227.0. 1H NMR (500 MHz, DMSO, d, ppm): 7.96 (d,
1H), 7.94 (d, 1H), 8.42 (s, 1H), 13.18 (s, 1H). 13C NMR (125 MHz,
DMSO, d, ppm): 123.73, 125.86, 128.31, 131.66, 159.63, 161.18,
167.33. Elemental analysis calc. (%) for C7H4N2O2Se: C, 37.03;
H, 1.78; N, 12.34; found (%): C, 37.00; H, 1.77; N, 12.35.
C
11H12N2O2Se: C, 46.65; H, 4.27; N, 9.89; found (%): C, 46.62;
H, 4.28; N, 9.88.
Cell line and cell culture
All human cell lines used in this research, including A549
(adenocarcinoma human alveolar basal epithelial cells),
MCF-7 (breast adenocarcinoma cells), Neuro-2a (neuroblas-
toma cells), HepG2 (hepatocellular carcinoma cells), A375
(human melanoma cells), HK-2 (human renal proximal
tubule cells), were purchased from American Type Culture
Collection (Manassas, VA). All the cell lines were incubated in
DMEM media supplemented with penicillin (100 units per
ml), streptomycin (50 units per ml) and fetal bovine serum
(10%) at 37 ꢃC in a humidied incubator with 5% CO2
atmosphere.
Methyl benzo[c][1,2,5]selenadiazole-5-carboxylate
The compound 4a was prepared according to a general proce-
dure. ESI-MS (in CH3OH): m/z calcd for C8H6N2O2Se (M + H)+
243.0; found 243.2. 1H NMR (500 MHz, DMSO, d, ppm): 7.95 (d,
1H), 7.96 (d, 1H), 8.45 (s, 1H), 3.93 (s, 3H). 13C NMR (125 MHz,
DMSO, d, ppm): 53.20, 124.02, 126.02, 127.83, 130.46, 159.41,
161.15, 166.30. Elemental analysis calc. (%) for C8H6N2O2Se: C,
39.85; H, 2.51; N, 11.62; found (%): C, 39.82; H, 2.53; N, 11.61.
MTT assay
Ethyl benzo[c][1,2,5]selenadiazole-5-carboxylate
The anticancer activity of selenadiazole derivatives on cells were
evaluated by MTT assay as reported.32 Data was expressed as the
percentage of MTT reduction relative to the absorbance of
control cells. Cell viability assays included the addition of
different concentrations BSA (or HSA) in DMEM (the DMEM
used in these comparative experiments did not contain fetal
bovine serum).
The compound 4b was prepared according to a general proce-
dure. ESI-MS (in CH3OH): m/z calcd for C9H8N2O2Se (M + H)+
257.0; found 257.2. 1H NMR (500 MHz, DMSO, d, ppm): 7.95 (d,
1H), 7.96 (d, 1H), 8.45 (s, 1H), 4.37 (d, 2H), 1.37 (t, 3H). 13C NMR
(125 MHz, DMSO, d, ppm): 14.58, 61.92, 123.97, 125.91, 127.83,
130.70, 159.43, 161.16, 165.76. Elemental analysis calc. (%) for
C9H8N2O2Se: C, 42.37; H, 3.16; N, 10.98; found (%): C, 42.35; H,
3.17; N, 10.95.
Cellular uptake of selenadiazole derivatives (4a–4e)
MCF-7 cells (8 ꢁ 104 cells per ml, 8 ml) were seeded in 10 cm cell
culture dishes and pre-cultured for 24 h. Aer the 10 cm cell
culture dishes were washed twice lightly, the cells were then
incubated in DMEM with or without BSA (or HSA) containing
200 mM SeDs (4a–4e) for another 6 h, respectively. Cellular
uptake was determined by atomic uorescence spectrometry of
intracellular selenium content obtained.
Propyl benzo[c][1,2,5]selenadiazole-5-carboxylate
The compound 4c was prepared according to a general proce-
dure. ESI-MS (in CH3OH): m/z calcd for C10H10N2O2Se (M + H)+
271.0; found 271.3. 1H NMR (500 MHz, DMSO, d, ppm): 7.96 (d,
1H), 7.97 (d, 1H), 8.45 (s, 1H), 1.78 (m, 2H), 4.30 (t, 2H), 1.01 (t,
3H). 13C NMR (125 MHz, DMSO, d, ppm): 10.84, 22.04, 67.28,
124.02, 125.90, 127.83, 130.69, 159.44, 161.17, 165.81.
Elemental analysis calc. (%) for C10H10N2O2Se: C, 44.62; H, 3.74;
N, 10.41; found (%): C, 44.60; H, 3.77; N, 10.40.
Selenadiazole derivatives (4a–4e)–protein interactions
Based on preliminary experiments, the BSA concentrations were
xed at 1 mM and the drug concentration was varied from 0 to 8
Isopropyl benzo[c][1,2,5]selenadiazole-5-carboxylate
The compound 4d was prepared according to a general proce- mM for BSA. Fluorescence spectra were recorded at two
dure. ESI-MS (in CH3OH): m/z calcd for C10H10N2O2Se (M + H)+ temperatures (298 and 303 K) in the range of 290–450 nm upon
271.0; found 271.2. 1H NMR (500 MHz, DMSO, d, ppm): 7.94 (d, excitation at 296 nm for BSA.
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Acknowledgements
This work was supported by Natural Science Foundation
of China (21271002, 21371076), Science Foundation for
Distinguished Young Scholars of Guangdong Province
(S2013050014667), YangFan Innovative & Entepreneurial
Research Team Project (201312H05), Guangdong Special
Support Program and Guangdong Frontier Key Technological
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