Novel metal complexes of naphthalimide-cyclam conjugates as potential multi-target receptor tyrosine kinase (RTK) inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.07.068

A novel series of metal complexes of naphthalimide-cyclam conjugates were synthesized and their in vitro antitumor activities were evaluated. The newly-synthesized compounds showed huge diversity of antiproliferative potency due to variety of metal ions and length of alkyl chains, among which the Zn(II) and Cr(III) complexes exhibited comparable antiproliferative activities with amonafide via multiple tyrosine kinase inhibition. Further research revealed that the representative compound 8a displayed broad-spectrum antiproliferative activity against 15 cancer cell lines with average IC₅₀ value 10.18 ± 3.25 μM, and effective antiangiogenic activity on human microvascular endothelial cells (HMEC-1). In brief, metal complexes of naphthalimide-cyclam conjugates were firstly designed and synthesized as multi-target tyrosine kinase inhibitors and proved of their antitumor capacities.

Full text of DOI:10.1016/j.ejmech.2014.07.068

European Journal of Medicinal Chemistry 85 (2014) 207e214
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel metal complexes of naphthalimideecyclam conjugates as
potential multi-target receptor tyrosine kinase (RTK) inhibitors:
Synthesis and biological evaluation
,
*
Shaoying Tan a, Kun Han b, Qiang Li a, Linjiang Tong b, Yiqi Yang a, Zhuo Chen a
,
,
, *
Hua Xie b *, Jian Ding b, Xuhong Qian a, Yufang Xu a
a State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, Shanghai Key Laboratory of New Drug Design, School of
Pharmacy, East China University of Science and Technology, Shanghai 200237, China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of metal complexes of naphthalimideecyclam conjugates were synthesized and their
in vitro antitumor activities were evaluated. The newly-synthesized compounds showed huge diversity of
antiproliferative potency due to variety of metal ions and length of alkyl chains, among which the Zn(II)
and Cr(III) complexes exhibited comparable antiproliferative activities with amonafide via multiple
tyrosine kinase inhibition. Further research revealed that the representative compound 8a displayed
broad-spectrum antiproliferative activity against 15 cancer cell lines with average IC50 value
Received 19 March 2014
Received in revised form
2 July 2014
Accepted 20 July 2014
Available online 22 July 2014
10.18 3.25 mM, and effective antiangiogenic activity on human microvascular endothelial cells (HMEC-
Keywords:
1). In brief, metal complexes of naphthalimideecyclam conjugates were firstly designed and synthesized
as multi-target tyrosine kinase inhibitors and proved of their antitumor capacities.
© 2014 Elsevier Masson SAS. All rights reserved.
Metal complexes
Naphthalimide
Cyclam
RTK inhibitors
Antiproliferation
Antiangiogenesis
1. Introduction
point mutations and drug resistance according to the clinical data
which reveals that patients responded to targeted therapies
Receptor tyrosine kinases (RTKs) are a superfamily of trans-
membrane proteins which play critical roles in the transduction of
extracellular signals to the cytoplasm. They are tightly regulated in
normal cells. Aberrantly activated RTKs are implicated with tumor
growth, progression and metastasis by modulating downstream
signaling pathways [1e3]. Tyrosine kinase inhibitors are verified of
their effective antitumor activities, and are approved in clinical use.
And more of them able to target RTKs are in clinical trials. For
example, Gefitinib (trade name Iressa) is an EGFR inhibitor
approved for the treatment of patients with breast, lung and other
cancers [4,5]. Nevertheless, single RTK inhibitors tempted to induce
initially when treated with specific tyrosine kinase inhibitors, but
most of them relapsed later [6]. Moreover, redundant and adapt-
able molecular pathways between individual patients and within
tumors in the same patients thwart the efficiency of treatment
targeting a single RTK.
Tyrosine kinase inhibitors which modulate a limited number of
tyrosine kinases are therapeutically more useful by overcoming
drug resistance and crippling the alternative pathways of cancer
cells [7,8]. Taking the multi-target RTK inhibitor sorafenib for
example, it could simultaneously inhibit vascular endothelial
growth factor receptor 2 (VEGFR-2), PDGFR-b, FMS-like tyrosine
kinase 3 (Flt-3), Raf kinase and stem cell factor receptor (c-kit)) [9].
The success of dasatinib [6] (Bcr/Abl and Src) and sunitinib (VEGFR-
1, -2, and-3, PDGFR-a, and -b, Flt-3 and colony stimulating factor 1
receptor (CSF-1R)) also supports the importance of multi-target
RTK inhibitors [10,11].
Angiogenesis, or new blood vessel formation, is limited in
healthy adults, but crucial in sustaining tumor growth and metas-
tasis [12]. Therefore, developing angiogenesis inhibitors is a
Abbreviation: CSF-1R, colony stimulating factor 1 receptor; Cyclam, 1,4,8,11-
tetraazamacrocycle; DDQ, 2,3-dichloro-5,6-dicyano-benzoquinone; Flt-3, FMS-like
tyrosine kinase 3; HMEC, human microvascular endothelial cells; RTK, Receptor
Tyrosine Kinase; VEGFR2, vascular endothelial growth factor receptor 2.
* Corresponding authors.
E-mail
addresses: chenzhuo@ecust.edu.cn,
chenzhuo@mail.ecust.edu.cn
(Z. Chen), hxie@simm.ac.cn (H. Xie), yfxu@ecust.edu.cn (Y. Xu).
http://dx.doi.org/10.1016/j.ejmech.2014.07.068
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

208
S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
recommendable antitumor strategy as few side effects might be
expected [13]. RTKs, or VEGFR, FGFR and PDGFR in detail, are
involved as therapeutic targets which regulate the angiogenic
process in growing tumors [14,15]. For example, the small-molecule
tyrosine kinase inhibitors sunitinib and sorafinib exhibits clinical
efficacy in patients with highly-vascularized renal cell cancer
[16,17].
Cyclam (1,4,8,11-tetraazamacrocycle) is one of the most widely-
used macrocyclic polyamines and attracts continuous attention due
to its biological properties and its importance in coordination
chemistry. Cyclam derivatives and their metal-ion complexes are
applied in medical trials for diagnosis and the treatment of AIDs
[18,19]. Medical interest of cyclam derivatives in the treatment of
tumors is also emerging. For example, Silbert et al. developed two
new lipophilic cyclam derivatives with IC50 values below 10 mM
Scheme 2. Synthesis of target metal complexes. (a) Corresponding metal salt, CH3OH,
r.t., 6 h.
against cancer cell line L1210 and demonstrated that the intro-
duction of lipophilic substitution could improve the ability of
antiproliferative activity [20]. Paola and cooperators certificated
that the positive antitumor activity could probably be ascribed to
an increased lipophilicity of cyclam for the improvement of trans-
portation across cell membrane [21]. On the other hand, cyclam can
complex various transition metal ions with high kinetic and ther-
modynamic stability because of its strong affinity [22] which was
disadvantageous for selective metal chelating, but benefit for
medical application. It is reported that cyclam could chelate with
Zn(II) and Cu(II) to inhibit cancer cells growth [23e25].
5,6-dicyano-benzoquinone (DDQ), tetrabutyl ammonium bromide
and triphenylphosphine. Then compounds 4ae4d were obtained
from cyclam condensed with 3ae3d in CHCl3 (see Scheme 2).
The metal complexes (5ae5e, 6ae6e, 7ae7e and 8ae8e) were
obtained with a mild, high-yield one-step method by compounds
4ae4d condensed with Zn(ClO4)2, Ni(ClO4)2, Cu(ClO4)2, Co(ClO4)2,
and Cr(ClO4)3 in methanol, respectively.
Previously, we reported our work on developing naphthalimide
derivatives as angiogenesis-related RTK inhibitors [26]. Naph-
thalimide derivatives were widespread of its antitumor potency,
but were seldom studied of their RTK inhibition activity before
[27e29]. Herein, naphthalimideecyclam conjugates were designed
to improve their antitumor capacities and further functionalized
with various lipophilic alkyls and complexed with Zn(II), Cu(II),
Ni(II), Co(II), Cr(III). We demonstrated that these novel compounds,
especially Zn(II) and Cr(III) complexes, were proved to possess
comparable antiproliferative activities with amonafide due to
multi-target RTK inhibition activities. The representative com-
pound 8a exhibited dual effects in vitro antiproliferation and
antiangiogenesis.
2.2. In vitro cytotoxic activity of metal complexes
Firstly, the antiproliferative activity of naphthlimideecyclam
conjugates 4ae4d and their metal complexes 5ae5e, 6ae6e, 7ae7e
and 8ae8e were evaluated against human breast carcinoma cell
lines MDA-MB-231 and MDA-MB-435, human ducal breast
epithelial tumor cell line T47D and human breast epithelial cell line
HBL-100. As shown in Table 1, the antiproliferative activities of the
target compounds were affected by the length of alkyl chains and
variety of metal ions. Normally speaking, the derivatives with long
alkyl chains inhibited growth of cancer cells more significantly than
their short alkyl chain analogues, such as 4d v.s. 4b and 8a v.s. 6a,
against the tested cell lines. The results were in accordance with
our previous work [26,30]. Meanwhile, the introduction of different
metal ions led to the diversity of the antiproliferative activity. Zn(II),
Cr(III)-cyclam complexes generally reduced cell proliferation better
than the derivatives of other metal ions and exhibited comparable
activity with amonafide. Taking 8ae8e for example, the IC50 value
of 8a and 8d against MDA-MB-231 were at single-digital micro-
molar range, while other metal complexes were 2e8 folds less
potent than 8a and 8d. Most of the metal complexes showed better
cytotoxic activities against MDA-MB-231 and MDA-MB-435 than
T47D. As for the normal human breast cell line HBL-100, the tested
2. Results and discussions
2.1. Chemistry
Compounds 4ae4d were synthesized in four steps from 4-
bromo-naphathalimide as shown in Scheme 1. Firstly, 4-bromo-
naphathalimide was refluxed in ethanol with corresponding pri-
mary amino to obtain 1ae1d. Compounds 1ae1d were reacted
with ethanol amine under Ullmann's condition to get 2ae2d, which
further were converted to 3ae3d in the existence of 2,3-dichloro-
Scheme 1. Synthesis of target compounds 4ae4d. (a) Corresponding primary amines, ethanol, reflux, 3 h; (b) ethanol amine, methoxyethanol, reflux, 4 h (c) DDQ, PPh3, (n-
butyl)4NBr, CH2Cl2, r.t., 12 h; (d) cyclam, KI, CHCl3, reflux, 6 h.

S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
209
Table 1
ELISA experiment. According to Table 2, the naphthalimideecyclam
conjuagate 4d could inhibit 4 of the tested 14 RTKs, namely ErbB4,
c-Src, Abl, c-Met. Its Cr3þ complex 8d exhibited wider and stronger
RTK inhibition activities against 5 of the tested 14 RTKs, which were
ErbB4, c-Src, Abl, c-Met and IGF1R. What's more, the Zn2þ complex
8a could effectively inhibit not only RTK which would be concerned
with tumor proliferation and so on, like Flt-1, EGFR, ErbB4, c-Src,
Abl, c-Met, and IGF1R, but also angiogenesis-related RTKs,
including VEGFR2 and FGFR1 [31,32].
Antiproliferative activity of parent compounds and cyclam fused naphthalimides
metal complexes.
2.4. Antiproliferative activity of 8a
compound R ¼
M ¼
Antiproliferative activity (IC50
, mM)
MDA-MB-231 MDA-MB-435 T47D HBL-100
As demonstrated above, 8a exhibited broad-spectrum RTK in-
hibition activities as expected and potent antitumor activity to the
tested three human breast cancer cell lines. We further evaluated
compound 8a as potential antitumor drug. Firstly, we studied the
antiproliferative activity of 8a against fifteen human cancer cell
lines from various tissues in vitro, including one human promye-
locytic leukemia cell line (HL 60), two human hepatocellular car-
cinoma cell lines (SMMC-7H, BEL-7402), two human lung
adenocarcinoma cell lines (SGC-7901, A549), two human breast
adenocarcinoma cell lines (MDA-MB-468, MDA-MB-231, MCF-7),
one human cervical carcinoma cell line (Hela), one human
pancreatic carcinoma cell line (BXPC-3), one human ovarian car-
cinoma cell line (SKOV3), one human colon carcinoma cell line
(HCT-116), one human caucasian prostate adenocarcinoma cell line
(PC-3), one human nasopharyngeal carcinoma cell line (KB), one
human epidermoid carcinoma cell line (A431) and one human
kidney carcinoma cell line (786-O). As shown in Fig. 1, 8a could
inhibit the growth of most of the cancer cells potently, with IC50
4a
5a
5b
5c
5d
5e
4b
6a
6b
6c
6d
6e
4c
7a
7b
7c
7d
7e
4d
8a
8b
8c
8d
8e
CH3
/
12.66
14.47
>100
34.94
9.53
17.89
23.97
>100
35.67
10.04
43.37
23.04
33.16
>100
35.16
14.60
49.39
8.50
39.43
90.93
>100
92.04
26.02
86.79
36.23
41.36
>100
34.81
22.33
36.23
25.08
22.64
21.96
57.03
7.33
N.D.a
>50
N.D.
N.D.
>50
N.D.
N.D.
>50
N.D.
N.D.
>50
N.D.
N.D.
>50
N.D.
N.D.
31.58
N.D.
N.D.
>50
N.D.
N.D.
>50
N.D.
N.D.
CH3 Zn(ClO4)2
CH3 Cu(ClO4)2
CH3 Ni(ClO4)2
CH3 Cr(ClO4)3
CH3 Co(ClO4)2
41.17
23.09
19.24
>100
30.26
10.79
38.89
8.78
C4H9
/
C4H9 Zn(ClO4)2
C4H9 Cu(ClO4)2
C4H9 Ni(ClO4)2
C4H9 Cr(ClO4)3
C4H9 Co(ClO4)2
C8H17
/
C8H17 Zn(ClO4)2
C8H17 Cu(ClO4)2
C8H17 Ni(ClO4)2
C8H17 Cr(ClO4)3
C8H17 Co(ClO4)2
8.08
22.56
40.11
56.18
9.09
51.19
35.43
6.50
39.06
6.73
8.00
63.42
10.72
6.53
53.42
5.10
78.08
10.32
22.33
72.44
18.87
9.23
35.27
19.88
19.32
17.56
>100
30.41
68.63
19.07
C
C
C
C
C
C
12H25
/
12H25 Zn(ClO4)2
12H25 Cu(ClO4)2
12H25 Ni(ClO4)2
12H25 Cr(ClO4)3
12H25 Co(ClO4)2
values ranging from 4.86 to 15.30
average. For example, the IC50 values were 4.86 1.50
BEL-7402 and 5.47 0.65 M against MDA-MB-468. Meanwhile,
the antiproliferative activities against 786-O and SMMC-7721 were
relatively poor (the IC50 value 15.30 2.63 M and 14.13 0.84 M,
mM, and 10.18
3.25
mM on
m
M against
m
69.56
5.36
Amonafide
a
m
m
N.D. : not determined.
respectively). The result indicated that 8a was a potential broad-
spectrum antiproliferative agent.
compounds (5a, 5d, 6a, 6d, 7a, 7d, 8a and 8d), exhibited 1e7 folds
less potent cytotoxic activities against normal cell line in contrast to
cancer cell lines. The results revealed that these compounds
exhibited certain selective against tumor cell lines.
2.5. Anti-angiogenesis activity of 8a
2.5.1. Inhibition of migration and tube formation
According to the results of RTK inhibition activities, the repre-
sentative metal ion complex 8a could simultaneously inhibit
2.3. Receptor tyrosine kinase (RTK) inhibitory activity
VEGFR2, PDGFR
a and b, and FGFR, which were involved in the
Furthermore, representative compounds 8a and 8d and their
precursor 4d were evaluated of their RTK inhibition activities by
process of angiogenesis. To investigate whether 8a could inhibit the
migration of HMEC-1 cells (human microvascular endothelial cell
line), transwell Boyden chamber assay was performed. As illus-
trated in Fig. 2a, about 44% of the migrated HMEC-1 cells were
Table 2
inhibited in the present of 5 mM 8a, which indicated that 8a could
Inhibitory activity of 4d, 8a and 8d to tyrosine kinases.
effectively inhibit the migration of cancer cells. Further research
Tyrosine kinase
IC50
(m
M) SD
4d
8a
8d
Flt-1
VEGFR-2
PDGFR-
PDGFR-
RET
>10.0
>10.0
>10.0
>10.0
>10.0
4.07 1.56
6.00 1.15
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
a
b
EGFR
ErbB2
ErbB4
c-Src
>10.0
>10.0
3.35 1.21
>10.0
0.80 0.59
0.42 0.05
0.32 0.10
>10.0
0.21 0.14
1.75 0.49
2.68 0.73
>10.0
>10.0
0.92 0.72
0.47 0.11
7.44 1.23
>10.0
0.42 0.21
>10.0
0.61 0.12
0.87 0.32
0.52 0.08
>10.0
0.07 0.03
0.61 0.23
>10.0
ABL
EPH-A2
c-Met
IGF1R
FGFR1
>10.0
Fig. 1. Antitumor effect of 8a in 15 cancer cell lines.

210
S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
Fig. 2. Inhibitory effect of 8a on HMEC-1 cells migration and tube formation activities. a) Representative images showing inhibition of migration, b) Representative images showing
inhibition of tube formation.
about the inhibitory effect to the formation of functional tubes was
performed in the HMEC-1 cells. The results revealed that the
functional tubes were nearly completely destroyed when exposed
their potential antitumor capacities from both antiproliferative and
antiangiogenic activities as multi-target RTK inhibitors.
in 5 mM 8a.
4. Experimental section
2.5.2. Down-regulation of downstream signaling pathways by 8a
Furthermore, we evaluated the effect of 8a on the key down-
stream signaling pathways of RTK by western blotting, including
AKT and Erk. As shown in Fig. 3, compound 8a caused little change
All the chemical regents and solvents were analytic grade. All
the synthesized fused naphthalimideecyclam conjugates were
confirmed by 1H NMR, 13C NMR and HRMS, while the metal com-
plexes were verified by HRMS and HPLC except the Cr(III) com-
plexes. 1H NMR and 13C NMR were measured on a Bruker AV-400
at the concentration of 1.25
dependent decrease in phosphorylation of AKT in HMEC-1 cells at
the concentration of 5 M and 10 M. In detail, phosphor-AKT and
Erk declined after exposure to 5 M 8a and became undetectable at
mM and 2.5 mM. It induced dose-
m
m
m
exposure to 10 mM 8a.
3. Conclusion
In all, to develop novel multi-target RTK inhibitors, a series of
metal complexes of naphthalimideecyclam conjugates were
designed and synthesized. The newly-synthesized compounds
exhibited structureeactivity relationship due to the diversity of
length of alkyl chains and complexes of metal ions. Among the
target compounds, derivatives with long alkyl chains and their
Zn(II), Cr(III)-complexes exhibited effective antiproliferative effect.
Furthermore, 8a, as representative compound, was evaluated as a
potential multi-target RTK inhibitor. It was certified to be a broad-
spectrum antiproliferative agent against 15 cancer cell lines with an
average IC50 value at 10.18 3.25 mM. It was also proved of effective
antiangiogenic activity on human microvascular endothelial cells
(HMEC-1). Further research certified that 8a could down-regulate
downstream signaling pathway of RTKs. In summary, novel metal
complexes of napthalimideecyclam conjugates were verified of
Fig. 3. Induction of the down-regulated Erk1/2 (Thr 202/204) and AKT (Ser 473)
signaling pathways induced in HMEC-1 cells in the present of 8a.

S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
211
spectrometer (in CDCl3 and DMSO-d6, TMS as an internal standard).
HRMS were collected in the Center of Analysis & Test of East China
University of Science and Technology. Analytical HPLC was per-
4.1.2.2. 6-(2-Hydroxyethylamino)-2-butyl-1H-benzo[de]isoquino-
line-1,3(2H)-dione (2b). Yellow solid, yield: 87%. 1H NMR (400 MHz,
CDCl3):
d
8.58 (d, J ¼ 7.2 Hz, 1H), 8.46 (d, J ¼ 8.4 Hz, 1H), 8.16 (d,
formed on
a
HewlettePackard 1100 system chromatograph
J ¼ 8.4 Hz, 1H), 7.64 (t, J ¼ 8.4 Hz, 1H), 6.79 (d, J ¼ 8.4 Hz, 1H), 4.17 (t,
J ¼ 7.6 Hz, 2H), 4.09 (t, J ¼ 5.2 Hz, 2H), 3.60 (t, J ¼ 5.2 Hz, 1H),
1.74e1.70 (m, 2H), 1.49e1.43 (m, 2H), 0.99 (t, J ¼ 7.6 Hz, 3H).
MS(ESI) calcd for C18H21N2O3 [MþH]þ 312.1, found: 312.1.
equipped with photodiode array detector. SRB was purchased from
Sigma Aldrich (St. Louis, MO, USA); all medium and FBS from Gibco
(Grand Island, NY, USA); Matrigel from BD Biosciences (San Jose, CA,
USA); antibodies to extracellular signal-regulated kinase (Erk) and
AKT from Cell Signaling Technology (Danvers, MA, USA); antibody
to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from
KangChen Bio-tech (Shanghai); secondary antibodies from Calbio-
chem (San Diego, CA, USA).
4.1.2.3. 6-(2-Hydroxyethylamino)-2-octyl-1H-benzo[de]isoquino-
line-1,3(2H)-dione (2c). Yellow solid, yield: 84%. 1H NMR (400 MHz,
CDCl3):
d
8.57 (d, J ¼ 7.2 Hz, 1H), 8.45 (d, J ¼ 8.4 Hz, 1H), 8.13 (d,
J ¼ 8.4 Hz, 1H), 7.62 (t, J ¼ 7.6 Hz, 1H), 6.74 (d, J ¼ 8.4 Hz, 1H), 4.16 (t,
J ¼ 8.0 Hz, 2H), 4.09 (t, J ¼ 5.2 Hz, 2H), 3.60 (t, J ¼ 5.2 Hz, 2H),
1.77e1.70 (m, 2H), 1.47e1.28 (m, 10H), 0.89 (t, J ¼ 7.2 Hz, 3H);
MS(ESI) calcd for C22H29N2O3 [MþH]þ 369.2, found: 369.2.
4.1. Synthesis
4.1.1. General procedure for the preparation of 1ae1d
6-bromobenzo[de]-isochromene-1,3-dione (1.94 g, 7.00 mmol)
was dissolved in 20 ml ethanol. Then corresponding primary amine
(7.70 mmol) was added, and the mixture was stirred at 60 ꢀC for
5e6 h. The mixture was cooled to room temperature and evapo-
rated in vacuum to obtain the residue. Then the residue was puri-
fied on silica gel chromatography (PE: EA ¼ 10:1, V/V) to provide
1ae1d.
4.1.2.4. 6-(2-Hydroxyethylamino)-2-dodecyl-1H-benzo[de]isoquino-
line-1,3(2H)-dione (2d). Yellow solid, yield: 84%. 1H NMR (400 MHz,
CDCl3):
d
8.53 (d, J ¼ 7.2 Hz, 1H), 8.41 (d, J ¼ 8.4 Hz, 1H), 8.11 (d,
J ¼ 8.0 Hz, 1H), 7.59 (t, J ¼ 8.0 Hz, 1H), 6.71 (d, J ¼ 8.4 Hz, 1H), 4.15 (t,
J ¼ 7.6 Hz, 2H), 4.09 (t, J ¼ 5.2 Hz, 2H), 3.58 (t, J ¼ 5.2 Hz, 2H),
1.76e1.69 (m, 2H), 1.45e1.26 (m, 18H), 0.89 (t, J ¼ 7.2 Hz, 3H);
MS(ESI) calcd for C26H37N2O3 [MþH]þ 425.3, found: 425.3.
4.1.1.1. 6-Bromo-2-methyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
4.1.3. General procedure for the preparation of 3ae3d
(1a). White solid, yield: 90%. 1H NMR (400 MHz, CDCl3):
d
8.68 (d,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.41 g, 1.80 mmol)
and Triphenyl Phosphine (0.47 g, 1.80 mmol) were dissolved in
10 ml dry CHCl3. Then the mixture of tetrabutyl ammonium bro-
mide (0.58 g, 1.80 mmol), 2ae2d (1.50 mmol, respectively) and
10 ml dry CHCl3 was dropped. 6 h later, the solvent was concen-
trated by vacuum and 3ae3d were purified on silica gel chroma-
tography (CH2Cl2).
J ¼ 7.2 Hz, 1H), 8.59 (d, J ¼ 8.4 Hz, 1H), 8.44 (d, J ¼ 8.0 Hz, 1H), 8.06
(d, J ¼ 7.6 Hz, 1H), 7.86 (t, J ¼ 8.4 Hz, 1H), 3.57 (s, 3H); MS(ESI) calcd
for C13H9BrNO2 [MþH]þ 289.0, found: 289.0.
4.1.1.2. 6-Bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1b). White solid, yield: 80%. 1H NMR (400 MHz, CDCl3)
d 8.66 (d,
J ¼ 7.2 Hz, 1H), 8.56 (d, J ¼ 8.8 Hz, 1H), 8.41 (d, J ¼ 8.0 Hz, 1H), 8.04
(d, J ¼ 8.0 Hz, 1H), 7.85 (t, J ¼ 8.0 Hz, 1H), 4.19 (t, J ¼ 7.6 Hz, 1H),
1.77e1.69 (m, 2H),1.51e1.42 (m, 2H),1.00 (t, J ¼ 7.2 Hz, 3H); MS(ESI)
calcd for C16H15BrNO2 [MþH]þ 331.0, found: 331.0.
4.1.3.1. 6-((2-Bromoethyl)amino)-2-methy-1H-benzo[de]isoquino-
line-1,3(2H)-dione (3a). Orange solid, yield: 75%. 1H NMR
(500 MHz, CDCl3):
d
8.63 (d, J ¼ 7.3 Hz, 1H), 8.50 (d, J ¼ 7.2 Hz, 1H),
8.15 (d, J ¼ 8.3 Hz,1H), 7.69 (t, J ¼ 8.0 Hz,1H), 6.75 (d, J ¼ 6.9 Hz,1H),
3.89 (t, J ¼ 5.8 Hz, 2H), 3.76 (t, J ¼ 5.7 Hz, 2H), 3.54 (s, 3H); MS(ESI)
calcd for C15H14BrN2O2 [MþH]þ 332.0, found: 332.0.
4.1.1.3. 6-Bromo-2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1c). White solid, yield: 85%. 1H NMR (400 MHz, CDCl3):
d 8.68 (d,
J ¼ 7.2 Hz, 1H), 8.59 (d, J ¼ 7.6 Hz, 1H), 8.44 (d, J ¼ 8.0 Hz, 1H), 8.06
(d, J ¼ 8.0 Hz, 1H), 7.87 (t, J ¼ 7.6 Hz, 1H), 4.18 (t, J ¼ 8.0 Hz, 2H),
1.78e1.71 (m, 2H), 1.47e1.29 (m, 10H), 0.89 (t, J ¼ 7.2 Hz, 3H);
MS(ESI) calcd for C20H23BrNO2 [MþH]þ 387.1, found: 387.1.
4.1.3.2. 6-((2-Bromoethyl)amino)-2-butyl-1H-benzo[de]isoquino-
line-1,3(2H)-dione (3b). Orange solid, yield: 68%. 1H NMR
(400 MHz, CDCl3):
d
8.62 (d, J ¼ 7.2 Hz, 1H), 8.49 (d, J ¼ 8.4 Hz, 1H),
8.15 (d, J ¼ 8.4 Hz,1H), 7.68 (t, J ¼ 7.6 Hz, 1H), 6.76 (d, J ¼ 8.4 Hz, 1H),
4.18 (t, J ¼ 7.6 Hz, 2H), 3.89 (t, J ¼ 6.0 Hz, 2H), 3.77 (t, J ¼ 6.0 Hz, 1H),
1.77e1.69 (m, 2H), 1.51e1.41 (m, 2H), 0.99 (t, J ¼ 7.2 Hz, 3H). MS
(ESI) calcd for C18H20BrN2O2 [MþH]þ 375.1, found: 375.0.
4.1.1.4. 6-Bromo-2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1d). White solid, yield: 85%. 1H NMR (400 MHz, CDCl3):
d 8.68 (d,
J ¼ 7.2 Hz, 1H), 8.59 (d, J ¼ 8.4 Hz, 1H), 8.44 (d, J ¼ 7.6 Hz, 1H), 8.06
(d, J ¼ 8.0 Hz, 1H), 7.87 (t, J ¼ 7.6 Hz, 1H), 4.18 (t, J ¼ 7.6 Hz, 2H),
1.78e1.71 (m, 2H), 1.47e1.27 (m, 18H), 0.90 (t, J ¼ 7.2 Hz, 3H);
MS(ESI) calcd for C24H31BrNO2 [MþH]þ 443.1, found: 443.2.
4.1.3.3. 6-((2-Bromoethyl)amino)-2-octyl-1H-benzo[de]isoquinoline-
1,3(2H)-dione (3c). Orange solid, yield: 85%. 1H NMR (400 MHz,
CDCl3):
d
8.63 (d, J ¼ 6.4 Hz, 1H), 8.50 (d, J ¼ 8.4 Hz, 1H), 8.16 (d,
4.1.2. General procedure for the preparation of 2ae2d
J ¼ 7.6 Hz, 1H), 7.69 (t, J ¼ 8.0 Hz, 1H), 6.77 (d, J ¼ 8.4 Hz, 1H), 5.60
(br, 1H), 4.17 (t, J ¼ 7.6 Hz, 2H), 3.89 (q, J ¼ 4.4 Hz, 2H), 3.77 (t,
J ¼ 6.0 Hz, 2H), 1.78e1.70 (m, 2H), 1.47e1.28 (m, 10H), 0.89 (t,
J ¼ 7.2 Hz, 3H); MS(ESI) calcd for C22H28BrN2O2 [MþH]þ 431.1,
found: 431.1.
1ae1d (6 mmol) were dissolved in 50 ml 2-methoxyethanol,
respectively. Then ethanol amine (24 mmol) was added and the
mixture was refluxed for 6 h. 150 ml water was added and solid was
precipitated immediately. The crude production was obtained by
suction filtration and was purified on silica gel chromatography
(CH2Cl2: MeOH ¼ 50:1e20:1, V/V).
4.1.3.4. 6-((2-Bromoethyl)amino)-2-dodecyl-1H-benzo[de]isoquino-
line-1,3(2H)-dione (3d). Orange solid, yield: 83%. 1H NMR
4.1.2.1. 6-(2-Hydroxyethylamino)-2-methyl-1H-benzo[de]isoquino-
(400 MHz, CDCl3):
d
8.63 (d, J ¼ 7.2 Hz, 1H), 8.50 (d, J ¼ 8.4 Hz, 1H),
line-1,3(2H)-dione (2a). Yellow solid, yield: 90%. 1H NMR (400 MHz,
8.16 (d, J ¼ 8.0 Hz,1H), 7.69 (t, J ¼ 8.4 Hz,1H), 6.77 (d, J ¼ 8.4 Hz,1H),
5.60 (br, 1H), 4.17 (t, J ¼ 7.6 Hz, 2H), 3.89 (t, J ¼ 5.6 Hz, 2H), 3.77 (t,
J ¼ 6.0 Hz, 2H), 1.78e1.70 (m, 2H), 1.45e1.27 (m, 18H), 0.90 (t,
J ¼ 7.2 Hz, 3H), MS(ESI) calcd for C26H36 BrN2O2 [MþH]þ 487.2,
found: 487.2.
CD3COCD3):
d
8.59 (d, J ¼ 8.4 Hz, 1H), 8.50 (d, J ¼ 7.6 Hz,1H), 8.37 (d,
J ¼ 8.4 Hz, 1H), 7.68 (t, J ¼ 8.0 Hz,1H), 6.89 (d, J ¼ 8.4 Hz,1H), 3.92 (t,
J ¼ 5.6, 2H), 3.62 (t, J ¼ 5.6, 2H), 3.42 (s, 3H); MS(ESI) calcd for
C
15H15N2O3 [MþH]þ 270.1, found: 270.1.

212
S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
4.1.4. General procedure for the preparation of 4ae4d
HRMS (ESI) calcd for
515.2094.
C
25H35N6O2Zn [M]þ 515.2107, found:
1,4,8,11-Tetraazacyclotetradecane (0.10 g, 0.50 mmol) was dis-
solved in 10 ml dry CHCl3. Then the mixture of 3ae3d (0.20 mmol,
respectively) and 15 ml dry CHCl3 was dropped in r.t. under the
argon. 3 days later, the solvent was concentrated by vacuum and
4ae4d was purified on silica gel chromatography (CH2Cl2:MeO-
H:Ammonium Hydroxide ¼ 100:15:2).
4.1.5.2. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
methyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Cu(II)(ClO4)2 (5b).
HRMS (ESI) calcd for C25H35N6O2Cu [M]þ 514.2112, found: 514.2114.
4.1.5.3. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
methyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Ni(II)(ClO4)2 (5c).
HRMS (ESI) calcd for
509.2154.
4.1.4.1. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
C
25H35N6O2Ni [M]þ 509.2169, found:
methyl-1H-benzo [de]isoquinoline-1,3(2H)-dione (4a). Orange solid,
yield: 50%. 1H NMR (400 MHz, CD3OD):
d 8.24e8.18 (m, 2H), 8.05 (d,
J ¼ 8.4 Hz, 1H), 7.40 (t, J ¼ 8.4 Hz,1H), 6.59 (d, J ¼ 8.4 Hz,1H), 3.50 (t,
J ¼ 7.2, 2H), 3.34 (s, 3H), 2.78e2.53 (m, 18H), 1.79e1.71 (m, 4H); 13
C
4.1.5.4. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
methyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Co(II)(ClO4)2 (5e).
HRMS (ESI) calcd for C25H34N6O2Co [M]þ 509.2070, found:
509.2061.
NMR (100 MHz, CD3OD):
d 164.90, 164.37, 150.86, 134.32, 130.67,
129.58, 127.81, 123.98, 121.81, 120.38, 107.94, 103.68, 54.35, 53.48,
50.74, 50.47, 49.15, 48.34, 46.69, 40.19, 27.40, 25.33, 24.05; HRMS
(ESI) calcd for C25H37N6O2 [MþH]þ 453.2978, found: 453.2968.
4.1.5.5. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
butyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Zn(II)(ClO4)2 (6a).
HRMS (ESI) calcd for C28H41N6O2Zn [M]þ 557.2577, found: 557.2557.
4.1.4.2. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
butyl-1H-benzo [de]isoquinoline-1,3(2H)-dione (4b). Orange solid,
yield: 66%. 1H NMR (400 MHz, CDCl3):
d
9.09 (d, J ¼ 8.0 Hz, 1H), 8.58
(d, J ¼ 6.8 Hz,1H), 8.43 (d, J ¼ 8.4 Hz,1H), 7.80 (t, J ¼ 6.0 Hz,1H), 7.66
(t, J ¼ 7.6 Hz, 1H), 6.68 (d, J ¼ 8.8 Hz, 1H), 4.17 (t, J ¼ 7.6 Hz, 2H), 3.79
(d, J ¼ 5.6 Hz, 2H), 3.09e3.03 (m, 4H), 2.97 (br, 2H), 2.89 (br, 2H),
2.73 (t, J ¼ 4.8 Hz, 2H), 1.77e1.69 (m, 2H), 1.49e1.43 (m, 2H), 0.98 (t,
4.1.5.6. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
butyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Cu(II)(ClO4)2 (6b).
HRMS (ESI) calcd for
556.2579.
C
28H41N6O2Cu [M]þ 556.2582, found:
J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 164.8, 164.2, 150.4,
134.5, 131.3, 130.1, 128.9, 124.4, 122.8, 120.7, 109.2, 103.5, 51.2, 51.0,
50.8, 50.6, 47.2, 46.8, 46.4, 46.0, 45.1, 40.1, 40.0, 30.3, 25.8, 25.2, 20.5,
13.9; HRMS (ESI) calcd for C28H43N6O2 [MþH]þ 495.3448, found:
495.3454.
4.1.5.7. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
butyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Ni(II)(ClO4)2 (6c).
HRMS (ESI) calcd for C28H41N6O2Ni [M]þ 551.2639, found: 551.2618.
4.1.5.8. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
4.1.4.3. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione-Co(II)(ClO4)2
(6e).
octyl-1H-benzo [de]isoquinoline-1,3(2H)-dione (4c). Orange solid,
HRMS (ESI) calcd for
551.2544.
C
28H40N6O2Co [M]þ 551.2545, found:
yield: 65%. 1H NMR (400 MHz, CD3OD):
d 8.31e8.28 (m, 2H), 8.16 (d,
J ¼ 8.4 Hz, 1H), 7.45 (t, J ¼ 8.0 Hz,1H), 6.66 (d, J ¼ 8.8 Hz,1H), 3.99 (t,
J ¼ 7.2 Hz, 2H), 3.50 (t, J ¼ 6.4 Hz, 2H), 2.77e2.51 (m,18H),1.77e1.62
(m, 6H), 1.31e1.23 (m, 10H), 0.85 (t, J ¼ 7.2 Hz, 3H); 13C NMR
4.1.5.9. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
octyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Zn(II)(ClO4)2 (7a).
(100 MHz, CD3OD):
d 164.39, 163.81, 150.74, 134.30, 130.56, 129.56,
HRMS (ESI) calcd for
613.3208.
C
32H49N6O2Zn [M]þ 613.3203, found:
127.73, 123.87, 121.88, 120.25, 108.07, 103.62, 54.53, 53.67, 50.78,
50.60, 49.31, 48.51, 46.76, 40.25, 39.68, 31.64, 29.14, 29.05, 27.88,
27.56, 26.94, 25.40, 22.36, 13.20; HRMS (ESI) calcd for C32H51N6O2
[MþH]þ 551.4074, found: 551.4070.
4.1.5.10. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
2-octyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Cu(II)(ClO4)2 (7b).
HRMS (ESI) calcd for
607.3248.
C
32H49N6O2Cu [M]þ 607.3265, found:
4.1.4.4. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (4d). Orange solid,
yield: 62%. 1H NMR (400 MHz, CD3OD):
d 8.33e8.29 (m, 2H), 8.18 (d,
4.1.5.11. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
J ¼ 8.4 Hz, 1H), 7.47 (t, J ¼ 8.0 Hz, 1H), 6.67 (d, J ¼ 8.8 Hz,1H), 4.00 (t,
J ¼ 7.2 Hz, 2H), 3.52 (t, J ¼ 6.4 Hz, 2H), 2.78e2.53 (m,18H),1.77e1.63
(m, 6H), 1.31e1.19 (m, 18H), 0.85 (t, J ¼ 7.2 Hz, 3H); 13C NMR
2-octyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Ni(II)(ClO4)2 (7c).
HRMS (ESI) calcd for
612.3205.
C
32H49N6O2Ni [M]þ 612.3208, found:
(100 MHz, CD3OD):
d 164.40, 163.83, 150.74, 134.33, 130.58, 129.60,
127.77, 123.89, 121.92, 120.28, 108.12, 103.62, 54.49, 53.69, 50.81,
50.61, 49.31, 48.51, 46.75, 40.28, 39.70, 31.73, 29.44, 29.21, 29.15,
27.92, 27.51, 26.95, 25.40, 22.41, 13.25; HRMS (ESI) calcd for
4.1.5.12. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-dione-Co(II)(ClO4)2 (7e).
C
32H48N6O2Co [M]þ 607.3165, found:
C
36H59N6O2[MþH]þ 607.4700, found: 607.4688.
HRMS (ESI) calcd for
607.3150.
4.1.5. General procedure for the preparation of 5ae5e, 6ae6e,
7ae7e and 8ae8e
4.1.5.13. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione-Zn(II)(ClO4)2
(8a). HRMS (ESI) calcd for C36H57N6O2Co [M]þ 669.3829, found:
669.3809.
4ae4d (0.05 mmol) were dissolved in 2 ml CH3OH, respectively,
then six different salts in CH3OH were added, respectively. 10 min
to 2 h later, the solid was precipitated. The crude production was
obtained by suction filtration and was washed twice with CH3OH to
get purity 5ae5e, 6ae6e, 7ae7e and 8ae8e.
4.1.5.14. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione-Cu(II)(ClO4)2
(8b). HRMS (ESI) calcd for C36H57N6O2Cu [M]þ 668.3834, found:
668.3820.
4.1.5.1. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-2-
methyl-1H-benzo [de]isoquinoline-1,3(2H)-dione-Zn(II)(ClO4)2 (5a).

S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
213
4.1.5.15. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione-Ni(II)(ClO4)2
(8c). HRMS (ESI) calcd for C36H57N6O2Ni [M]þ 663.3891, found:
663.3881.
100 mL of color development solution (0.03% H2O2 and 2 mg/mL o-
phenylenediamine in 0.1 mol/L citrate buffer, pH 5.4) was added
and the plate was incubated at room temperature until color
emerged. The reaction was terminated by the addition of 50 mL of
2 mol/L H2SO4, and then the plate was read using a multi-well
spectrophotometer (MAX190™) at 490 nm. The inhibition rate
(%) was calculated using the following equation: inhibition
rate ¼ [1 ꢁ (A490 treated/A490control)] ꢂ 100%.
4.1.5.16. 6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)amino)-
2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione-Co(II)(ClO4)2
(8e). HRMS (ESI) calcd for C36H56N6O2Co [M]þ 663.3791, found:
663.3774.
4.5. Cell migration assay
4.2. Cell lines and cell culture
[34] HMEC-1s migration was determined in a transwell Boyden
chamber (Costar, MA, USA). Firstly, cell suspension (5 ꢂ 105 cells/
mL) with different concentrations of 8a or control was added to the
upper compartment of the chamber. Secondly, 20% FBS MCDB131
medium and the same concentrations of 8a or control were added
to the lower compartment. After incubation 6 h at 37 ꢀC, the inhi-
MDA-MB-231, MDA-MB-435, T47D, MDA-MB-468, MCF-7, A549,
HeLa, BXPC-3, SKOV3, HCT-116, PC-3, KB, A431, 786-O and HMEC-1
were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). SMMC-7721, BEL-7402 and HL60 were ob-
tained from the Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences (Shanghai, China). SGC-7901 was obtained
from Renji Hospital (Shanghai, China). They were maintained in
strict accordance with the supplier's instructions and established
procedures.
bition
of
migration
was
calculated:
inhibition
of
migration ¼ [1 ꢁ (Acompound ꢁ Ablank)/(Acontrol ꢁ Ablank)] ꢂ 100%.
4.6. Tube formation assay
4.3. In vitro cytotoxicity assays of the 4ae4d, 5ae5e, 6ae6e,
7ae7e and 8ae8e
[35] Matrigel (75 m
L wellꢁ1) was used to coat 96-well plates and
solidify after 8 h incubation at 37 ꢀC. For investigation of neovessel
disruption, HMEC-1s (1 ꢂ 105 cells/mL) were seeded on Matrigel
and left to align for 24 h. The formed capillary-like structures or
cords were exposed to 8a, and then examined with an inverted
phase contrast microscope (DP70, Olympus, Japan). The number of
the tubes was quantified from five random fields. The inhibition of
MTT or SRB assay was applied to evaluate the in vitro cytotox-
icity of target compounds. Cancer cells were plated and incubated
for 24 h in 96-well plates. The cells were then treated with
increasing concentration of compounds 4ae4d, 5ae5e, 6ae6e,
7ae7e, 8ae8e and amonafide for a further 72 h. For HL60 cell line,
tube
formation
was
calculated:
inhibition
of
tube
20
well. The cells were then incubated for an additional 4 h, after
which 100 L of “triplex solution” (10% SDS-5% isobutanol-HCl,
mL (5 mg/mL in 0.9% brine) of MTT (Sigma) was added to each
formation ¼ [1 ꢁ (tubescompound/tubescontrol)] ꢂ 100%.
m
4.7. Western blot analysis
12 mM) was added, and the cells were incubated overnight at 37 ꢀC.
The plates were read at 570 nm on the scanning multiwell spec-
trophotometer (MAX190™). The inhibition rate on cell prolifera-
tion was calculated as: inhibition rate ¼ (1 ꢁ A570 treated/A570
control) ꢂ 100%. The IC50 values were obtained by the Logit method.
Each experiment was repeated in triplicate. For the rest cell lines,
cells were then fixed with 10% trichloroacetic acid and stained with
sulforhodamine B (Sigma). Sulforhodamine B in the cells was dis-
solved in 10 mM TriseHCl and was measured at 515 nm using a
multiwell spectrophotometer (MAX190™, Molecular Devices,
Sunnyvale, USA). The inhibition rate on cell proliferation was
calculated as follows: inhibition rate ¼ (1 ꢁ A515 treated/A515
control) ꢂ 100%. IC50 values were determined from the results of at
least three independent tests and calculated from the inhibition
curves.
HMEC-1s cells were plated into 6-well plate (3 ꢂ 105 cells/well)
and incubated for 24 h. The cells were then exposed to corre-
sponding compounds for 12 h for analysis of Erk and AKT signal
transduction pathways. Whole-cell lysates were collected and
boiled for 10 min in 2 ꢂ SDS sample buffer, subjected to 10% SDS-
PAGE, and transferred to nitrocellulose (Amersham Life Sciences).
The blot was blocked in blocking buffer (5% non-fat dry milk/1%
Tween-20 in TBS) for 1 h at room temperature, and then incubated
with primary antibodies (anti-phospho-AKT (1:500), anti-AKT
(1:5000), anti-phospho-Erk (1:2000), anti-Erk (1:2000) and anti-
GAPDH (1:10,000)) in blocking buffer for 2 h at room tempera-
ture. The bands were then visualized using horseradish peroxidase-
conjugated secondary antibodies (1:2000) followed by ECL (Pierce
Biotech, Rockford, IL).
4.4. Protein kinase assays
Acknowledgment
[33] Tyrosine kinase activity was determined by an enzyme-
linked-immunosorbent assay (ELISA). Fifty microliters of 10 mmol/
L ATP solution diluted in reaction buffer [50 mmol/L HEPES, pH 7.4,
20 mmol/L MgCl2, 0.1 mmol/L Na3VO4, and 1 mmol/L DTT] was
added to each well of 96-well plates pre-coated with 2.5 mg/well
poly (Glu, Tyr)4:1 (Sigma) as a substrate and then added increasing
concentrations of tyrosine kinase. After incubation for 1 h at 37 ꢀC,
the plate was washed three times with phosphate buffered saline
This work is financially supported by the National Basic
Research Program of China (973 Program, 2010CB126100), the
National High Technology Research and Development Program of
China (863 Program, 2011AA10A207), the National Natural Science
Foundation of China (grants 21236002 and grants 21302054), the
National Science & Technology Pillar Program, the Shanghai Com-
mittee of Science and Technology (Grant 13ZR1453100) and the
Fundamental Research Funds for the Central Universities.
containing 0.1% Tween20 (PBST). After adding 100
mL of anti-
phosphotyrosine (PY99; 1:1000 dilution) antibody, another 0.5 h
incubation at 37 ꢀC, the plate was washed three times and goat
Appendix A. Supplementary data
anti-mouse IgG horseradish peroxidase (100 mL of a 1:2000 dilu-
tion) diluted in PBST containing 5 mg/mL BSA was added. The plate
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.068.
was reincubated at 37 ꢀC for 0.5 h and washed as before. Finally,

214
S. Tan et al. / European Journal of Medicinal Chemistry 85 (2014) 207e214
[17] B. Escudier, T. Eisen, W.M. Stadler, C. Szczylik, S. Oudard, M. Siebels, S. Negrier,
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