Synthesis, characterization and anti-proliferative activity of heterocyclic hypervalent organoantimony compounds

Article abstract of DOI:10.1016/j.ejmech.2014.04.026

Three heterocyclic hypervalent organoantimony chlorides RN(CH ₂C₆H₄)₂SbCl (2a R = t-Bu, 2b R = Cy, 2c R = Ph) and their chalcogenide derivatives [RN(CH₂C ₆H₄)₂Sb]₂O (3a R = t-Bu, 3b R = Cy, 3c R = Ph) were synthesized and characterized by techniques such as ¹H NMR, ¹³C NMR, X-ray diffraction, and elemental analysis. It is found that the anti-proliferative activity detected over these compounds can be attributed to the coordination bond between the antimony and nitrogen atoms of these compounds. Moreover, a preliminary study on mechanistic action suggests that the inhibition effect is ascribable to cell cycle arrest and cell apoptosis.

Full text of DOI:10.1016/j.ejmech.2014.04.026

European Journal of Medicinal Chemistry 79 (2014) 391e398
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and anti-proliferative activity of
heterocyclic hypervalent organoantimony compounds
,b,
Yi Chen a c, Kun Yu c, Nian-Yuan Tan c, Ren-Hua Qiu c, Wei Liu a, Ning-Lin Luo c, Le Tong b,
,
,
c,
*
Chak-Tong Au c d, Zi-Qiang Luo a , Shuang-Feng Yin
*
a School of Basic Medicine, Central South University, Changsha 410013, PR China
b Medical College, Hunan University of Chinese Medicine, Changsha 410208, PR China
c State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR
China
d Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three heterocyclic hypervalent organoantimony chlorides RN(CH2C6H4)2SbCl (2a R ¼ t-Bu, 2b R ¼ Cy, 2c
R ¼ Ph) and their chalcogenide derivatives [RN(CH2C6H4)2Sb]2O (3a R ¼ t-Bu, 3b R ¼ Cy, 3c R ¼ Ph) were
synthesized and characterized by techniques such as 1H NMR, 13C NMR, X-ray diffraction, and elemental
analysis. It is found that the anti-proliferative activity detected over these compounds can be attributed
to the coordination bond between the antimony and nitrogen atoms of these compounds. Moreover, a
preliminary study on mechanistic action suggests that the inhibition effect is ascribable to cell cycle
arrest and cell apoptosis.
Received 18 August 2013
Received in revised form
6 April 2014
Accepted 7 April 2014
Available online 12 April 2014
Keywords:
Hypervalent organoantimony
Synthesis
Ó 2014 Elsevier Masson SAS. All rights reserved.
Characterization
Anti-proliferative activity
Cell cycle arrest
Apoptosis
1. Introduction
organometallic, and they are compounds with antimony-carbon
bonds having ligands such as arylhydroxamates [10], lapachol
Cancer is a major health problem. Many types of cancer are
incurable, and mortality decline is mainly based on early detection
and appropriate treatments [1]. In the fighting against cancers, new
metal compounds are synthesized as antitumor agents [2,3]. Anti-
mony is a group-15 element. It shows remarkable therapeutic ef-
ficacy on patients who suffer from leishmaniasis and acute
promyelocytic leukemia [4e7]. Despite antimony compounds are
used clinically for quite a number of diseases, they are rarely used
as antitumor agents. In the 1990’s, Silvestru and coworkers re-
ported for the first time the antitumor activity of organo-
antimony(III) compounds [4,8,9]. Fifteen years later Wang et al. [10]
and Ludmila et al. [11] reported the relatively high antitumor ac-
tivity of organoantimony(V) compounds. So far the most studied
antimony compounds in the context of antitumor activity are
[11], thioamides [12], and hydrazones [13]. The antitumor perfor-
mance of these compounds, however, is unsatisfactory. It is hence
meaningful to explore the chemical and pharmacological proper-
ties of new organoantimony compounds for the purpose of devel-
oping anticancer drugs.
In the present study, we investigated the anti-proliferative ac-
tivity of organoantimony compounds that are heterocyclic and
hypervalent in nature. Through the use of different nitrogen sub-
stituent groups, we controlled the steric and substitution patterns
of the organoantimony compounds. It is demonstrated that this
kind of compounds can be used for the fabrication of anticancer
drugs.
2. Results and discussion
2.1. Chemistry
Abbreviations: t-Bu, tertiary butyl; Cy, cyclohexyl; Ph, phenyl; PI, propidium
iodide; mM, mmol/L; mM, mmol/L; RT, room temperature.
* Corresponding authors.
Dilithiation of tertiary amines (2-Br-C6H4CH2)2NR (1a R ¼ t-Bu,
1b R ¼ Cy, 1c R ¼ Ph) with n-BuLi, followed by subsequent reaction
E-mail addresses: luozq1962@163.com (Z.-Q. Luo), sf_yin@hnu.edu.cn (S.-F. Yin).
http://dx.doi.org/10.1016/j.ejmech.2014.04.026
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

392
Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
with
SbCl3,
produces
the
organoantimony
chlorides
IC50) are shown in Table 2. It is observed that 2aec and 3aec show
much higher anti-proliferative activity than their starting materials
1aec. The compounds with the same nitrogen substituent show
RN(CH2C6H4)2SbCl (2a R ¼ t-Bu, 2b R ¼ Cy, 2c R ¼ Ph) (Scheme 1).
Treatment of these chlorides with KOH gives the organoantimony
chalcogenide derivatives [RN(CH2C6H4)2Sb]2O (3a R ¼ t-Bu, 3b
R ¼ Cy, 3c R ¼ Ph).
anti-proliferative
(IC50 ¼ 3.5 M) > 2a (IC50 ¼ 6.6
(IC50 ¼ 5.5
ratio is 9.39% at 30
effects
that
follow
M) > 1a (IC50 > 30
the
order:
3a
m
m
mM), 3b
The molecular structures of 2aec and 3aec are confirmed by
elemental analyses and NMR techniques (1H and 13C NMR). Due to
stronger electron-attracting ability of Sb, the protons linked with
the carbon atom adjacent to Sb atom shift downfield (8.25 ppm, 2a;
8.26 ppm, 2b; 8.24 ppm, 2c) in comparison with those of the cor-
responding starting materials (7.55 ppm, 1a; 7.59 ppm, 1b;
7.60 ppm, 1c). Meanwhile, the 13C NMR data of the carbon atoms
adjacent to Sb shift downfield (145.11 ppm, 2a; 144.01 ppm, 2b;
147.96 ppm, 2c) in comparison with those of the corresponding
starting materials (139.10 ppm, 1a; 139.68 ppm, 1b; 136.27 ppm,
1c). On the contrary, compared with the 1H NMR spectra of the
corresponding starting materials (8.25 ppm, 2a; 8.26 ppm, 2b;
8.24 ppm, 2c), stronger electron-donating ability of O counteracts
the electron-attracting ability of Sb, leading to upfield shift of the
protons linked with the carbon atoms adjacent to Sb atom
(8.12 ppm, 3a; 8.21 ppm, 3b; 8.12 ppm, 3c).
m
M) > 2b (IC50 ¼ 31.4
mM) > 1b (IC50 > 30 mM, inhibition
mM). With IC50 above 30 mM, compounds 1c, 2c
and 3c are all weak in anti-proliferative activity. In other words, the
order of anti-proliferative effect of these compounds can also be
arranged according to the nitrogen substituents: t-Bu > Cy > Ph.
Since compared to Cy and Ph groups, t-Bu group is stronger in
electron-donating ability but weaker in steric effect, it is hence
deduced that the anti-proliferative activity towards A549 cells can
be related to the coordination bonding between the antimony and
nitrogen atoms of these compounds.
In view that compounds 2a and 3a (both with same nitrogen
substituent) showed stronger anti-proliferative activity, they were
adopted to examine the time course at various concentrations
(Fig. 2) as well as to examine the dose effect on anti-proliferative
activity (Fig. 3). The results suggest that the anti-proliferative ac-
tivity is concentration as well as time dependence. Based on the
results of optimization, we adopted compound concentration of
Crystal structures of 2a and 3a (Fig.1) were determined by single-
crystal X-ray diffraction analysis, and selected bond lengths and
angles are listed in Table 1. One can see that the coordination poly-
hedron around the centre Sb of hypervalent compounds 2a and 3a
can be best described as a strongly distorted pseudo-trigonal bipyr-
amid. The N(1), Cl(1) and O(1) atoms are located at the apical posi-
tions, while the C(1), C(10) and C(14) atoms are situated at the
equatorial positions along with an electron lone pair of Sb. The Sb(1)-
10
mM and incubation period of 48 h for further investigation.
To assess the possible side effects of administrating these
compounds, the anti-proliferation activity of them on normal hu-
man bronchial epithelial cells (HBEC) was evaluated. After 48 h
incubation, the IC50 values of compounds 2a and 3a on HBEC are
18.7 and 11.2 mM, giving IC50 (HBEC)/IC50(A549) ratio of 2.83 and
3.20, respectively. When the commercial anticancer drug cisplatin
was adopted, the IC50 value on A549 cells under the same experi-
ꢀ
ꢀ
N(1) distance (2.4638(14) A) in 2a and that (2.6546(17) A) in 3a is
longer than that (2.397(3) A) in 12-chloro-6-cyclohexyl-5,6,7,12-
ꢀ
mental conditions is above 30 mM. With anti-proliferation activity
tetrahydrodibenzo[c,f] [1,5]-azastibocine [14]. The results suggest
that the N / Sb coordination in 2a and 3a is weaker than that of the
latter. Furthermore, the two NeSb distances in 2a and 3a are slightly
towards A549 cells stronger than that of cisplatin, the heterocyclic
hypervalent compounds 2a and 3a can be further studied as anti-
tumor drugs.
ꢀ
longer than the sum of the covalent radii (2.11 A) [15] but much
ꢀ
shorter than the sum of the van der Waals radii (3.74 A) [16], indi-
2.2.2. Effect of the compounds on the cell cycle
cating that there is coordination bonding between the antimony and
the nitrogen atoms. According to Musher’s idea of hypervalent
molecules [17], compounds 2aec and3aec with high Sbvalences can
There are five stages of the cell cycle: (1) the G1 phase that
follows mitosis, a period for the synthesis of enzymes needed for
DNA replication; (2) the S phase, a period of DNA replication; (3)
the G2 phase where the cell continues to grow and produce new
proteins; (4) the M phase where the cell divides into two daughter
cells; and (5) the quiescent G0 phase where the cell remains stable
until it begins the cell cycle again. To determine the possible effect
of the heterocyclic hypervalent compounds on the progression of
cell cycle, we performed flow cytometric analysis to quantify the
percentage of A549 cells after cell permeabilization and propidium
iodide (PI) labeling. In the analysis, the amount of bound dye is
correlated with the DNA content of a given cell. In other words, DNA
fragmentation in apoptotic cells is translated to fluorescence in-
tensity, giving fluorescence peak that is lower in intensity than that
of G0/G1 cells, i.e. a sub-G0/G1 peak.
ꢀ
be considered as hypervalent. The SbeO bond length (2.0055(14) A)
in 3a is similar to those of organoantimony oxides such as [{2-
ꢀ
ꢀ
(Me2NCH2)C6H4}2Sb]2O (1.986(3) A), (Ph2Sb)2O (1.978(3) A), and
ꢀ
(Me2Sb)2O (2.099(6) A) [18e20]. Due to constrain imposed by
intramolecular N / Sb coordination, the NeSbeCl angle in com-
pound 2a (161.36(3)ꢀ) and NeSbeO angle in compound 3a
(159.029(43)ꢀ) are significantly deviated from the ideal case of 180ꢀ.
2.2. Biological activity
2.2.1. Anti-proliferative activity
Using CCK-8 assay, the anti-proliferative effect of compounds
1aec, 2aec and 3aec on A549 cells was examined. The concen-
trations of compounds required to inhibit 50% of cell growth (i.e.
In A549 cells incubated with compounds 2a and 3a (10 mM for
48 h), the proportion of cells in the sub-G0/G1 phase increased to
4.86% and 8.01%, respectively, a large increase compared to 0.74% of
the untreated control (Fig. 4). This increase in sub-G0/G1 phase was
accompanied by an increase in cell number of the G2/M phase
(compared with that of untreated cells), showing values of 28.76
versus 6.06% and 20.06 versus 6.06% (Fig. 4), indicating inhibiting
effect of compounds 2a and 3a on cell mitosis. In other words, the
increased proportion of cells in the sub-G0/G1 phase confirms that
the apoptosis of A549 cells is a result of DNA degradation induced by
compounds 2a and 3a. On the other hand, after treatment with
compounds 2a and 3a, the cells in S phase decreased from the
control value of 23.38% to 15.02% and 14.92%, individually, indicating
the inhibiting effect of compounds 2a and 3a on DNA replication.
Scheme 1. Synthesis of compounds 2aec and 3aec.

Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
393
Fig. 1. Molecular structure of compounds 2a and 3a (Hydrogen atoms are omitted).
Therefore, it is reasonable to deduce that the heterocyclic
hypervalent organoantimony compounds have an inhibitory effect
on cell proliferation, and the observed growth retardation was
caused by cell cycle arrest mainly at the S and G2/M phases.
As shown in SI-Figs. 2 and 3, there is no obvious difference be-
tween the RT 1H NMR spectrum of a fresh 2a sample and that of a
2a sample that was kept in open air at RT for 4 days. It is clear that
compound 2a is stable. Meanwhile, compound 3a in DMSO-d6 was
found to remain intact over a period of 4 days (see SI-Fig. 4). With
the introduction of H2O, compound 3a in DMSO-d6 undergoes
transformation to give the corresponding organoantimony hy-
droxide (see SI-Fig. 1 and SI-Fig. 5). It is noted that there is the
establishment of equilibrium between compound 3a and its hy-
droxide, and the hydroxide can be converted back to compound 3a
once H2O is removed (See SI-Fig. 1). Moreover, the organoantimony
hydroxide is found to be stable in DMSO-d6 þ H2O (See SI-Fig. 5).
Based on these evidences, we deduce that the active specie for anti-
proliferative activity in the biological evaluation with compound 3a
is in fact the organoantimony hydroxide derivative.
2.2.3. Effect of compounds on the cell apoptosis
The ability of compounds 2a and 3a to induce apoptosis in the
A549 cells was evaluated by means of Annexin-V and PI double
staining using a FACSCalibur flow cytometer. As shown in Fig. 5,
treatment with 3 mM of compound 3a for 48 h results in 6.29% of
the A549 cells becoming apoptotic (early þ late), higher than the
3.98% value of apoptotic cells in an untreated control. Moreover,
with the increase of 3a concentration to 10 mM, there is enhance-
ment of apoptotic cells (early þ late) to 16.17%. The results
demonstrate that there is significant cell apoptosis induced by 3a at
a concentration of 10
tration of 2a is increased from 3 to 10
m
M. On the other hand, when the concen-
M, the change of apoptotic
m
3. Conclusion
cells (early þ late) is from 5.41% to 9.08%, relatively slight compared
to that induced by 3a.
In this study, we synthesized heterocyclic hypervalent organo-
antimony compounds 2aec and 3aec, and characterized their
structures. The results of structure determination by means of
single-crystal X-ray diffraction reveal that there is coordination
bond between the antimony and the nitrogen atoms in compounds
2a and 3a. The organoantimony compounds 2a and 3a show good
2.3. Stability of compounds 2a and 3a
In order to check the stability of compounds 2a (IC50 ¼ 6.6
mM)
and 3a (IC50 ¼ 3.5
m
M), they were subject to NMR analysis with the
employment of deuterated dimethyl sulfoxide (DMSO-d6) and
DMSO-d6 þ H2O as solvent (see SI-Fig. 1).
Table 2
Anti-proliferation activities against A549 and HBEC.
No.
IC50 (m
M)a
Table 1
A549
HBEC
ꢀ
ꢀ
Selected bond distances (A) and angles ( ) for compounds 2a and 3a.
1a
1b
1c
2a
2b
2c
3a
3b
>30
>30
>30
ntb
Nt
Nt
2a
3a
Sb(1)eC(10)
Sb(1)eC(1)
Sb(1)eCl(1)
Sb(1)eN(1)
C(10)eSb(1)eC(1)
C(10)eSb(1)eN(1)
C(1)eSb(1)eN(1)
C(10)eSb(1)eCl(1)
C(1)eSb(1)eCl(1)
N(1)eSb(1)eCl(1)
2.1474(17)
2.1520(16)
2.5505(4)
2.4638(14)
95.70(6)
Sb(1)eC(1)
Sb(1)eC(14)
Sb(1)eO(1)
Sb(1)eN(1)
C(1)eSb(1)eC(14)
C(1)eSb(1)eN(1)
C(14)eSb(1)eN(1)
C(1)eSb(1)eO(1)
C(14)eSb(1)eO(1)
N(1)eSb(1)eO(1)
Sb(1)eO(1)eSb(1a)
2.146(2)
2.158(2)
2.0055(14)
2.6546(17)
92.59(9)
72.622(79)
73.808(81)
92.33(9)
6.6 ꢁ 1.85c
31.4 ꢁ 8.19
>30
18.7 ꢁ 2.1
Nt
Nt
3.5 ꢁ 0.71
5.5 ꢁ 0.40
>30
11.2 ꢁ 1.8
Nt
Nt
Nt
76.99(5)
74.96(5)
91.05(5)
92.35(5)
3c
Cisplatin
>30
a
92.88(7)
159.029(43)
115.96(12)
IC50 was obtained through the Probit regression model.
Not tested.
Each point is the mean ꢁ S.D. for three different experiments performed in
b
c
161.36(3)
triplicate.

394
Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
Fig. 3. Dose effect on anti-proliferation activity of compounds 2a and 3a. A549 cells
were treated for 48 h. Cell viability was examined using CCK-8 assay. Each point is the
mean 0.05,
S.D. for three different experiments performed in triplicate. #P
##P < 0.01 versus 10
M.
ꢁ
<
m
recorded at 25 ꢀC over an INOVA-400M (Varian) instrument cali-
brated using tetramethylsilane (TMS) as internal standard.
Elemental analysis was performed over a VARIO EL III (Elementar)
instrument. Melting points were determined over a XT-4 micro
melting point apparatus (Beijing Tech Instrument Co., Ltd.).
4.1.1. General procedure for the synthesis of compounds 2aec
To a solution of N,N-bis(2-bromobenzyl)-amine (1 equiv) in
Et2O (1.0 mol/L), n-BuLi (2.0e2.2 equiv, 2.5 M in hexane) was added
dropwise at ꢂ50 ꢀC. The mixture was gradually warmed to room
temperature (RT) over a period of 3 h, and then a solution of SbCl3
(1 equiv) in Et2O (0.8e1.0 mol/L) was added at ꢂ78 ꢀC. The obtained
mixture was gradually warmed to RT over a period of 12 h. After
removal of solvent under vacuum, the residue was subject to
toluene extraction, and the insoluble material was filtered out. The
organic layer was washed with deionized water and dried over
anhydrous Na2SO4. The solvent was removed under reduced pres-
sure and the residue was recrystallized from CH2Cl2/hexane to give
heterocyclic hypervalent organoantimony chlorides 2aec in the
form of colorless crystals.
Fig. 2. Time course of anti-proliferative effect of compound 2a (A) and compound 3a
(B). A549 cells were treated at various concentrations of the compounds (1, 3, 10, and
30 mM) for periods as indicated. Cell viability was examined using CCK-8 assay. Each
point is the mean ꢁ S.D. for three different experiments performed in triplicate.
#P < 0.05, ##P < 0.01 versus 24 h; &P < 0.05, &&P < 0.01 versus 48 h.
anti-proliferation activity towards cancer cells as a result of
apoptosis and cell cycle arrest. Compared to cisplatin, compounds
2a and 3a show stronger anti-proliferation activity towards A549
cells. This is the first example that hypervalent organoantimony
compounds show ability to inhibit the growth of human tumor cell
lines. It is expected that compounds 2a and 3a will find broad
medicinal applications, especially for the combat of cancers.
4.1.1.1. Synthesis of compound 2a. The reaction was performed ac-
cording to procedure for the synthesis of compounds 2aec using 1a
(4.11 g,10.0 mmol) in Et2O (100 mL), n-BuLi (8.4 mL, 21 mmol, 2.5 M
in hexane), and SbCl3 (2.28 g, 10 mmol) in Et2O (100 mL). Yield:
3.52 g (85.7%). Mp: 213e215 ꢀC; 1H NMR (CDCl3, 400 MHz, TMS):
d
1.34 (9H, s), 3.96 (2H, d, J ¼ 15.2 Hz), 4.36 (2H, d, J ¼ 15.6 Hz), 7.09
4. Experimental
(2H, d, J ¼ 7.2 Hz), 7.25 (2H, t, J ¼ 8.0 Hz), 7.32 (2H, t, J ¼ 7.2 Hz), 8.25
(2H, d, J ¼ 7.6 Hz); 13C NMR (CDCl3, 100 MHz, TMS):
d 27.11, 57.31,
4.1. Chemistry
60.50, 124.57, 128.57, 129.00, 135.07, 140.00, 145.11. Anal. Calc. for
C
18H21ClNSb (408.6): C, 52.91; H, 5.18; N, 3.43. Found: C, 52.78; H,
The manipulations of air-sensitive materials were performed
under an atmosphere of nitrogen using the standard Schlenk
techniques. Most of the chemicals were purchased from Aldrich.
Co., Ltd. and used as received unless otherwise indicated. Et2O was
dried and distilled from a purple solution of sodium/benzophenone
ketyl. The N,N-bis(2-bromobenzyl)- 2-methylpropan-2-amine (1a),
N,N-bis(2-bromobenzyl)cyclohexanamine (1b), and N,N-bis(2-
bromobenzyl)aniline (1c) compounds were prepared according to
procedures reported by Kakusawa et al. [21]. The NMR spectra were
5.26; N, 3.52.
4.1.1.2. Synthesis of compound 2b. The reaction was performed
according to procedure for the synthesis of compounds 2aec using
1b (2.19 g, 5.0 mmol) in Et2O (50 mL), n-BuLi (4.4 mL, 11 mmol,
2.5 M in hexane), and SbCl3 (1.14 g, 5.0 mmol) in Et2O (60 mL).
Yield: 2.02 g (83.4%). Mp: 254e256 ꢀC; 1H NMR (400 MHz, CDCl3,
TMS):
d 1.07e1.13 (1H, m), 1.22e1.41 (4H, m), 1.66 (1H, d,

Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
395
Fig. 4. Cell cycle of A549 cells as examined in 48 h treatment of compounds 2a and 3a at 10
mM concentration. Cell cycle was assessed by FACS analysis. Each point is the
mean ꢁ S.D. for three different experiments performed in triplicate. *P < 0.05, **P < 0.01 versus control.
J ¼ 13.2 Hz), 1.83 (2H, d, J ¼ 13.2 Hz), 2.03 (2H, d, J ¼ 12 Hz), 3.01e
3.08 (1H, m), 4.05 (2H, d, J ¼ 14.8 Hz), 4.20 (2H, d, J ¼ 15.2 Hz), 7.10
(2H, d, J ¼ 7.2 Hz), 7.27 (2H, td, J ¼ 0.8 Hz, 7.2 Hz), 7.34 (2H, t,
J ¼ 7.2 Hz), 8.26 (2H, d, J ¼ 7.2 Hz); 13C NMR (100 MHz, CDCl3, TMS):
chlorides RN(CH2C6H4)2SbCl (1.0 mmol) in 20 mL toluene. The
mixture was stirred at RT for 12 h, then the organic layer was
separated and the water phase was washed with toluene
(3 ꢃ 10 mL). The toluene solution was dried over anhydrous Na2SO4.
Heterocyclic hypervalent organoantimony chalcogenide derivatives
[RN(CH2C6H4)2Sb]2O in the form of white solid was obtained after
solvent removal under vacuum.
d
25.44, 25.64, 29.54, 57.80, 65.43, 124.70, 128.72, 128.87, 134.99,
140.13. 144.01. Anal. Calc. for C20H23ClNSb (434.6): C, 55.27; H, 5.33;
N, 3.22. Found: C, 55.39; H, 5.25; N, 3.14.
4.1.1.3. Synthesis of compound 2c. The reaction was performed ac-
cording to procedure for the synthesis of compounds 2aec using 1c
(2.16 g, 5.0 mmol) in Et2O (50 mL), n-BuLi (4.0 mL,10 mmol, 2.5 M in
hexane), and SbCl3 (1.14 g, 5.0 mmol) in Et2O (60 mL). Yield: 3.7 g
4.1.2.1. Synthesis of compound 3a. The reaction was performed
according to procedure for the synthesis of compounds 3aec using
2a (0.409 g, 1.0 mmol). Yield: 0.768 g (99%). Mp: 235e237 ꢀC; 1H
NMR(400 Hz, acetone-d6, TMS):
d
¼ 1.35 (18H, s), 3.83 (4H, d,
(86.3%). Mp: 222e224 ꢀC; 1H NMR (CDCl3, 400 MHz, TMS):
d
4.52
J ¼ 15.5 Hz), 4.37 (4H, d, J ¼ 15.5 Hz), 7.13 (4H, d, J ¼ 0.5 Hz), 7.14e
7.12 (6H, m), 7.24 (4H, t, J ¼ 7.5 Hz), 7.29 (2H, dt, J ¼ 7.2 Hz), 8.12 (2H,
(2H, d, J ¼ 14.8 Hz), 4.70 (2H, d, J ¼ 15.6 Hz), 7.22 (4H, m), 7.26 (1H,
d, J ¼ 7.6 Hz), 7.30e7.35 (4H, m), 7.42 (2H, t, J ¼ 7.2 Hz), 8.24 (2H, d,
d, J ¼ 7.2 Hz); 13C NMR (125 Hz, acetone-d6, TMS):
¼ 27.06, 29.40,
d
J ¼ 7.6 Hz); 13C NMR (CDCl3, 100 MHz, TMS):
d
61.19, 119.74, 125.28,
29.56, 29.71, 29.87, 30.02, 30.12, 30.18, 30.27, 30.33, 56.55, 59.39,
125.96, 127.73, 128.00, 133.65, 143.85, 147.05. Anal. Calc. for
125.43, 129.17, 129.24, 129.58, 135.25, 140.56, 143.06, 147.96. Anal.
Calc. for C20H17ClNSb (428.6): C, 56.05; H, 4.00; N, 3.27. Found: C,
55.73; H, 4.12; N, 3.35.
C36H42N2OSb2: C, 56.72; H, 5.55; N, 3.68; Found: C, 56.65; H, 5.58;
N, 3.70.
4.1.2. General procedure for the synthesis of compounds 3aec
A solution of KOH (0.4 g, 10 mol) in 20 mL deionized water was
added to a solution of heterocyclic hypervalent organoantimony
4.1.2.2. Synthesis of compound 3b. The reaction was performed
according to procedure for the synthesis of compounds 3aec using
2b (0.435 g, 1.0 mmol). Yield: 0.750 g (92%). Mp: 227e229 ꢀC; 1H

396
Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
NMR (400 Hz, CDCl3, TMS):
d
¼ 3.87 (1H, s), 3.91 (1H, s), 4.06 (1H, s),
NMR (400 Hz, CDCl3, TMS):
d
¼ 4.42 (2H, d, J ¼ 15.2 Hz), 4.75 (2H, d,
4.10 (1H, s), 6.99 (1H, t, J ¼ 3.2 Hz), 7.08 (2H,d, J ¼ 7.6 Hz), 7.16 (3H,
J ¼ 14.8 Hz), 6.99 (1H, t, J ¼ 3.2 Hz), 7.16 (2H, d, J ¼ 8.0 Hz), 7.20 (2H,
dt, J ¼ 7.6 Hz), 7.20e7.28 (4H, m), 8.21 (2H, d, J ¼ 7.2 Hz); 13C NMR
d, J ¼ 6.8 Hz), 7.22e7.27 (5H, m), 7.29 (2H, dt, J ¼ 7.2 Hz), 8.12 (2H, d,
(100 Hz, CDCl3, TMS):
d
¼ 15.28, 21.46, 25.78, 25.84, 25.94, 29.19,
J ¼ 7.2 Hz); 13C NMR (100 Hz, CDCl3, TMS):
¼ 59.06, 118.00, 122.55,
d
29.24, 56.40, 56.58, 63.87, 64.11, 125.04, 125.20, 125.30, 127.62,
127.66, 128.07, 128.23, 129.04, 132.85, 134.16, 144.44, 144.97, 145.42.
Anal. Calc. for C40H46N2OSb2: C, 59.00; H, 5.69; N, 3.44; Found: C,
59.10; H, 5.65; N, 3.42.
125.44, 128.14, 128.22, 129.12, 133.93, 143.64, 145.35, 149.16. Anal.
Calc. for C40H34N2OSb2: C, 59.89; H, 4.27; N, 3.49; Found: C, 59.79;
H, 4.29; N, 3.52.
4.1.3. X-ray crystal structural determination of hypervalent
compounds 2a and 3a
X-ray single crystal diffraction analysis of compounds 2a and 3a
was performed on a Bruker SMART APEX diffractometer with a CCD
4.1.2.3. Synthesis of compound 3c. The reaction was performed
according to procedure for the synthesis of compounds 3aec using
2c (0.429 g, 1.0 mmol). Yield: 0.722 g (90%). Mp: 295e297 ꢀC; 1H
Fig. 5. Apoptosis of A549 cells induced by compounds 2a and 3a. Cells were treated with compounds 2a and 3a at 3, 10 mM for 48 h and apoptosis was analyzed using Annexin-V
and propidium iodide (PI) double staining by FACS analysis. Percentage of early apoptotic (annexin-Vþ/PIꢂ), late apoptotic (annexin-Vþ/PIþ), and dead (annexin-Vꢂ/PIþ) cells are
shown, respectively.

Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
397
detector using graphite monochromated radiation (MoK
a
,
8 was added and the plates were incubated for an additional 2 h.
The optical density of the formazane yellow formed in the cells was
measured at 490 nm, using an iMark microplate absorbance reader
(Bio-Rad, USA). The IC50 values were obtained through the Probit
regression model between inhibition ratio and concentration.
Except the case of 48 h treatment, the cell viability of organome-
tallic compounds in human bronchial epithelial cell was conducted
under the same experimental conditions.
ꢀ
l
¼ 0.71073 A). The collected frames were processed with SAINTþ
[22] software; and the collected reflections were subject to ab-
sorption correction (SADABS) [23]. The structure was solved by the
direct methods using SHELXS-97, and refined by full-matrix least-
squares analyses on F2 [24]. Hydrogen atoms were generated in
their idealized positions and all non-hydrogen atoms were refined
anisotropically (Table 3).
4.2.3. Cell cycle by FACS analysis
4.2. Biological evaluation
For cell cycle analysis, 3 ꢃ 105 cells per well were incubated with
the addition of compounds 2a and 3a (10 mM) for 48 h. At the end of
All compounds were dissolved in dimethyl sulfoxide (DMSO) at
20 mM concentration and stocked at 4 ꢀC prior to use. The Cell
Counting kit-8 (CCK-8) from Dojindo Molecular Technologies, Inc.
(Shanghai, China) was used to measure the optical density of the
formazane yellow formed in the cells. All other reagents were from
Sigma and of the highest quality commercially available. All data
were obtained from at least three separate experiments and the
results were expressed as mean ꢁ S.D. Data were analyzed for
statistical significance by one-way ANOVA, and pb0.05 was
considered statistically for the indication of significant difference.
the incubation, the cells were trypsinized and washed twice with
phosphate-buffered saline (PBS) and separated by centrifugation.
With the addition of 1 mL of cold 70% ethanol, the cells were once
more incubated for 1 h at 4 ꢀC. After centrifugation, the pellet was
resuspended in 1 mL of DNAase-free RNase A (200
mg/mL) and
stored at 37 ꢀC. After 1 h, the cells were stained with 40
mg/mL
propidium iodide (PI) for 20 min at RT. The percentage of cells in
each stage of the cell cycle was determined using a FACSCalibur
flow cytometer by counting 1 ꢃ 105 cells and analyzed using Cell-
quest software (BectoneDickinson, France).
4.2.1. Cell cultures
4.2.4. Apoptosis assessment by FACS analysis
Human cancer cells A549 (human alveolar adenocarcinoma cell
line) and human normal cells HBEC (human bronchial epithelial
cell) used in this study were from the Cancer Research Institute,
Central South University (Changsha, Hunan, PR China). A549 cells
and HBEC cells were cultured under standard culture conditions
(37 ꢀC, 95% air: 5% CO2) in RPMI1640 medium (HyClone) supple-
mented with 10% newborn calf serum (HyClone) and 1% penicilline
streptomycin (HyClone).
For apoptosis assessment, 3 ꢃ 105 cells per well were incubated
with the addition of compounds 2a and 3a (0, 3, 10 mM) for 48 h. At
the end of the incubation, cells were trypsinized and subsequently
collected by centrifugation. The cells were washed twice with PBS
and resuspended in 500
mL of binding buffer and incubated with
5
mL of annexin V-FITC and 5 mL PI for 20 min at RT in the dark. Then
the cells were analyzed with a FACSCalibur flow cytometer (Bec-
toneDickinson, France). In each sample, 1 ꢃ 105 cells were
analyzed. Data analysis was performed with Cellquest software
(BectoneDickinson, France). The results were interpreted as fol-
lows: cells in the lower left quadrant (annexin-Vꢂ/PIꢂ) were
considered as living cells, in the lower right quadrant (annexin-Vþ/
PIꢂ) as early apoptotic cells, in the upper right quadrant (annexin-
Vþ/PIþ) as late apoptotic cells, and in the upper left quadrant
(annexin-Vꢂ/PIþ) as dead cells. The total apoptotic rate was the rate
of cells in the lower right quadrant (annexin-Vþ/PIꢂ) plus in the
upper right quadrant (annexin-Vþ/PIþ).
4.2.2. Cell viability
The cell viability of the organometallic compounds in human
alveolar adenocarcinoma cell line was determined by the CCK-8
assay using a modified method according to the manufacturer’s
instructions. In all assays, the compounds were quickly dissolved in
DMSO and diluted in sterile culture medium before being added to
the cell culture (DMSO concentration < 0.1% (v/v)). The cells
(5 ꢃ 103 cells per well) were seeded in each well of the 96-well
plates. After 12 h, compounds of designated concentrations (1, 3,
10 and 30 mM) were added. After 24-, 48-, and 72-h treatment, CCK-
4.3. Stability of the compounds
Table 3
Crystal data and structure refinement parameters for compounds 2a and 3a.
Stability analysis of these compounds was performed on an
INOVA-400M (Varian) instrument. Initially, compound 2a was
Parameters
2a
3a
dissolved in DMSO-d6 and divided into two samples. Then 55 mL
Formula
C18H21ClNSb
408.56
120 K
0.25 ꢃ 0.15 ꢃ 0.10
Monoclinic
P21/n
9.747 (2)
15.8808 (3)
11.388 (2)
90
110.030 (1)
90
1656.2 (5)
4
1.639
1.82
C36H42N2OSb2
762.22
133
0.35 ꢃ 0.30 ꢃ 0.15
Monoclinic
C2/c
26.119 (3)
14.1149 (15)
18.997 (2)
90
111.245 (1)
90
6527.7 (12)
8
1.551
1.69
double distilled water (volume ratio of DMSO-d6/H2O ¼ 10:1) was
added to one sample. The 1H NMR spectrum was recorded
respectively after the sample was prepared and kept at RT for 4
days. The same experiments were performed with compound 3a. It
is noted that the sample portion of 3a contains certain amount of
the corresponding organoantimony hydroxide (see SI-Fig. 4).
Formula weight
Temperature
Crystal size (mm)
Crystal system
Space group
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
Acknowledgments
3
ꢀ
This work was supported by the Science Research Foundation
for Higher Universities of Hunan Province (10A092), Research-
Based Learning and Innovation Experiment Project for Un-
dergraduates of Hunan Province (2011-483), the National Natural
Science Foundation of China (21003040), Program for Changjiang
Scholars and Innovative Research Team in University (IRT1238),
and the Program for New Century Excellent Talents in Universities
(NCET-10-0371). CTA thanks HNU for an adjunct professorship.
Y. Chen, K. Yun and N.Y. Tan contributed equally.
V (A )
Z
Density (Mg mꢂ3
)
m
(mmꢂ1
)
F (000)
816
16,671
3735
0.017
1.09
0.017, 0.039
3056
22,860
7088
0.024
1.02
0.024, 0.067
Reflections collected
Independent reflections
R (int)
Goodness-of-fit on F2
R1, wR2 [I > 2
s
(I)]

398
Y. Chen et al. / European Journal of Medicinal Chemistry 79 (2014) 391e398
[12] I.I. Ozturk, A.K. Metsios, S. Filimonova-Orlova, N. Kourkoumelis,
Appendix A. Supplementary data
S.K. Hadjikakou, M. Manos, A.J. Tasiopoulos, S. Karkabounas, E.R. Milaeva,
N. Hadjiliadis, Study on single crystal structure of the antimony(III) bromide
complex with 3-methyl-2-mercaptobenzothiazole and biological activity of
some antimony(III) bromide complexes with thioamides, Medicinal Chemis-
try Research 21 (2012) 3523e3531.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.026.
[13] K.S.O. Ferraz, N.F. Silva, J.G. da Silva, L.F. de Miranda, C.F.D. Romeiro,
E.M. Souza-Fagundes, I.C. Mendes, H. Beraldo, Investigation on the pharma-
cological profile of 2,6-diacetylpyridine bis(benzoylhydrazone) derivatives
and their antimony(III) and bismuth(III) complexes, European Journal of
Medicinal Chemistry 53 (2012) 98e106.
[14] W.G. Yi, N.Y. Tan, 12-Chloro-6-cyclohexyl-5,6,7,12-tetrahydrodibenzo [c,f]
[1,5]azastibocine, Acta Crystallographica E67 (2011) m917.
[15] P. Pyykkö, M. Atsumi, Molecular single-bond covalent radii for elements 1e
118, Chemistry e A European Journal 15 (2009) 186e197.
[16] S.S. Batsanov, Van der Waals Radii of elements, Inorganic Materials 37 (2001)
871e885.
[17] J.I. Musher, The chemistry of hypervalent molecules, Angewandte Chemie
International Edition 8 (1969) 54e68.
[18] L.M. Opris, A.M. Preda, R.A. Varga, H.J. Breunig, C. Silvestru, Synthesis and
characterization of hypervalent organoantimony(III) compounds containing
the [2-(Me2NCH2)C6H4]2Sb fragment, European Journal of Inorganic Chemis-
try 9 (2009) 1187e1193.
[19] H.J. Breunig, E. Lork, R. Rösler, G. Becker, O. Mundt, W. Schwarz, Common
features in the crystal structures of the compounds bis(dimethylstibanyl)
oxane and -sulfane, and the minerals valentinite and stibnite, Zeitschrift für
anorganische und allgemeine Chemie 626 (2000) 1595e1607.
[20] H.J. Breunig, T. Krüger, E. Lork, Oxidation of tetraaryldistibanes: syntheses and
crystal structures of diarylantimony oxides and peroxides, (R2Sb)2O, (R2Sb)
4O6 and (R2SbO)4(O2)2 (R¼Ph, o-Tol, p-Tol), Journal of Organometallic
Chemistry 648 (2002) 209e213.
[21] N. Kakusawa, Y. Tobiyasu, S. Yasuike, K. Yamaguchi, H. Seki, J. Kurita,
Hypervalent organoantimony compounds 12-ethynyl-tetrahydrodibenz[c,f]
[1,5]azastibocines: highly efficient new transmetallating agent for organic
halides, Journal of Organometallic Chemistry 691 (2006) 2953e2968.
[22] SAINTþ, ver. 6.02a, Bruker analytical X-ray system, Inc., Madison, WI, 1998.
[23] G.M. Sheldrick, SADABS, Empirical Absorption Correction Program, University
of Göttingen, Germany, 1997.
References
[1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics 2013, CA: A Cancer Journal
for Clinicians 63 (2013) 11e30.
[2] K. Iuchi, Y. Hatano, T. Yagura, Heterocyclic organobismuth(III) induces
apoptosis of human promyelocytic leukemic cells through activation of cas-
pases and mitochondrial perturbation, Biochemical Pharmacology 76 (2008)
974e986.
[3] B. Desoize, Metals and metal compounds in cancer treatment, Anticancer
Research 24 (2004) 1529e1544.
[4] M. Gielen, Metal-Based Anti-Tumour Drugs, vol. 2, Freund Publ. House Ltd,
London, 1992.
[5] P. Sharma, D. Perez, A. Cabrera, N. Rosas, J.L. Arias, Perspectives of antimony
compounds in oncology, Acta Pharmacologica Sinica 29 (2008) 881e890.
[6] E.R.T. Tiekink, Antimony and bismuth compounds in oncology, Critical Re-
views in Oncology/Hematology 42 (2002) 217e224.
[7] N. Yang, H.G. Sun, Application of arsenic, antimony and bismuth in medicine,
Progress in Chemistry 5 (2009) 856e865.
[8] C. Silvestru, C. Socaciu, A. Bara, I. Haiduc, The first organoantimony(III) com-
pounds possessing antitumor properties: diphenylantimony(III) derivatives of
dithiophosphorus ligands, Anticancer Research 10 (1990) 803e804.
[9] A. Bara, C. Socaciu, C. Silvestru, I. Haiduc, Antitumour organometallics I. Ac-
tivity of some diphenyltin(IV) and diphenylantimony(III) derivatives on
in vitro and in vivo Ehrlich ascites tumor, Anticancer Research 11 (1991)
1651e1655.
[10] G.C. Wang, Y.N. Lu, J. Xiao, L. Yu, H.B. Song, J.S. Li, J.R. Cui, R.Q. Wang, F.X. Ran,
Synthesis, crystal structures and in vitro antitumor activities of some orga-
noantimony arylhydroxamates, Journal of Organometallic Chemistry 690
(2005) 151e156.
[11] L.G. de Oliveira, M.M. Silva, F.C.S. de Paula, E.C. Pereira-Maia, C.L. Donnici,
C.A. de Simone, F. Frézard, E.N. da Silva Júnior, C. Demicheli, Antimony(V) and
bismuth(V) complexes of lapachol: synthesis, crystal structure and cytotoxic
activity, Molecules 16 (2011) 10314e10323.
[24] G.M. Sheldrick, A short history of SHELX, Acta Crystallographica A64 (2008)
112e122.