Synthesis and biological activity of novel organoselenium derivatives targeting multiple kinases and capable of inhibiting cancer progression to metastases

Article abstract of DOI:10.1016/j.ejmech.2011.12.006

The present study reports synthesis and biological activity of novel benzoisoselenazolone compounds derived from ebselen and conjugated to a sugar molecule. Cell proliferation assay using cancer cells combined with in vitro biochemical assays revealed that benzoisoselenazolone 2d, 5a, and 6a exerted anti-proliferative activity, which correlated with selective in vitro inhibition of focal adhesion kinase, AKT-1, and protein kinase C-α. Active molecules were able to significantly inhibit cell migration and invasion in vitro compared to cells treated with the vehicle alone or ebselen. Moreover, in vivo anticancer activity focusing on lead compound 2d and using an invasive human breast cancer orthotopic mouse model revealed a potent anti-metastatic activity at well-tolerated doses. In summary, these novel benzoisoselenazolones we report herein target multiple kinases with established roles in cancer progression and possess anti-invasive and anti-metastatic activity in preclinical models supporting a potential for therapeutic application for human disease.

Full text of DOI:10.1016/j.ejmech.2011.12.006

European Journal of Medicinal Chemistry 48 (2012) 143e152
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of novel organoselenium derivatives targeting
multiple kinases and capable of inhibiting cancer progression to metastases
,
b
,
,
,
,
,b
Krikor Bijian a 1, Zhongwei Zhang c 1, Bin Xu a b, Su Jie a b, Bo Chen c, Shengbiao Wan c, JianHui Wu a
**
,
Tao Jiang c , Moulay A. Alaoui-Jamali
,
a,b,
*
a The Segal Cancer Center and Lady Davis Institute of the Sir Mortimer Jewish General Hospital, Montreal, Quebec, Canada
b Departments of Medicine, Oncology, and Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
c Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study reports synthesis and biological activity of novel benzoisoselenazolone compounds
derivedfromebselenandconjugatedto asugarmolecule. Cell proliferationassay usingcancercellscombined
with in vitro biochemical assays revealed that benzoisoselenazolone 2d, 5a, and 6a exerted anti-proliferative
activity, which correlated with selective in vitro inhibition of focal adhesion kinase, AKT-1, and protein kinase
Received 1 August 2011
Received in revised form
25 November 2011
Accepted 3 December 2011
Available online 9 December 2011
C-a. Active molecules were able to significantly inhibit cell migration and invasion in vitro compared to cells
treated with the vehicle alone orebselen. Moreover, invivo anticanceractivity focusingon lead compound 2d
and using an invasive human breast cancer orthotopic mouse model revealed a potent anti-metastatic
activity at well-tolerated doses. In summary, these novel benzoisoselenazolones we report herein target
multiple kinases with established roles in cancer progression and possess anti-invasive and anti-metastatic
activity in preclinical models supporting a potential for therapeutic application for human disease.
Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Organoselenium-saccharide derivative
Ebselen
Cancer
Invasion
Metastasis
1. Introduction
range of cancer cell types, as well as endothelial and immune cells,
and to inhibit the activity of drug resistance mechanisms to poten-
Discovery of novel therapeutic agents for advanced invasive
cancers is at the forefront of preclinical and clinical research. A wide
range of small molecules belonging to both synthetic and naturally
derived molecules and targeting a wide range of extracellular
receptors, intracellularcellsignalling molecules, cellcytoskeleton, as
well as tissue microenvironment are under development. Of interest
to this study, selenium (Se)-containing molecules are emerging as
exciting candidate therapeutic agents due to their newfound ability
to modulate multiple physiological functions implicated in cancer
development. For instance, Se-containing molecules have been
shown to exert anti-proliferative and proapoptotic effects on a wide
tiate chemotherapy/radiotherapy efficacy [1e3]. Moreover, inhibi-
tion of angiogenic factors by seleno-compounds has been reported
to result in inhibition of neo-vessel formation “angiogenesis”, a key
process essential for cancer progression and dissemination [4e6].
Several selenium-containing derivatives have also been reported to
efficiently target key cancer cell signaling mechanisms such as
calcium-insensitive nitric oxide synthase (iNOS), Akt3 kinase, and
the mitogen-activated protein kinase (MAPK) signaling [2]; histone
deacetylases [7]; and melanin biosynthesis by melanocytes [8].
The impact of Se-containing molecules on multiple targets is not
surprising since several kinases are regulated by selenium [9].
Using high-throughput screening of chemical libraries against
kinases associated with cancer cell invasion, we initially identified
the Se-containing molecule ebselen (1) as a potential inhibitor.
Abbreviations: BSZ, benzisoselenazolone fragment; FA, focal adhesion; FAK,
focal adhesion kinase; PKC-a, protein kinase C.
* Corresponding author. The Segal Cancer Center and Lady Davis Institute of the
Sir Mortimer Jewish General Hospital, 3755 Cote-Ste-Catherine, Montreal, Quebec,
Canada H3T 1E2.
** Corresponding author. School of Medicine and Pharmacy, Ocean University of
China, 5 Yushan Road, Qingdao 266003, China.
E-mail addresses: jiangtao@ouc.edu.cn (T. Jiang), moulay.alaoui-jamali@mcgill.
ca (M.A. Alaoui-Jamali).
1
Ebselen (1)
These authors contributed equally to this manuscript.
0223-5234/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.006

144
K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
Ebselen, 2-phenylbenzo[d] [1,2]selenazol-3(2H)-one, a synthetic
1-aminosubstitute derivatives following the respective literature
procedures [31,32], which were then coupled with 2-(chloroseleno)
benzoyl chloride followed by deprotection of eOH group to
generate 3ae3d in a good yield of 48e57.9%.
The general method for the synthesis of compounds of series II
is shown in Scheme 2. The synthesis of compounds 6a and 6b were
obtained by an efficient cyclization reaction starting from 2-
(chloroseleno)benzoyl chloride and the OH-protected amino-
saccharide-butyramide derivatives such as 3-amino-N-(2,3,4,6-
compound identified in 1984, was found to exhibit GSH-
peroxidase-like activity in vitro [d](2H) [10]. Ebselen exerts a wide
spectrum of biological activities, ranging from anti-oxidant, cyto-
protective, neuroprotective, and anti-inflammatory. Ebselen func-
tions in part as a glutathione peroxidase mimicking agent and free
radical/peroxynitrite scavenging agent. Also, ebselen inhibits other
enzymes such as cyclooxygenases, lipoxygenases, and indoleamine
2,3-dioxygenase, which play a broad functional role in cancer sig-
nalling, as well as the regulation of the immune response [11e16].
This broad target specificity could explain why clinical applications
of ebselen have been limited to neurological diseases such as for the
treatment of ischemic stroke [17,18]. Due to its modest anticancer
activity which is in part due to its lower uptake by cancer cells
and limited biodistribution [19], we undertook additional modifi-
cations to the parent ebselen molecule to improve its intracellular
uptake and efficacy. In prior studies, we have observed that
conjugation to sugar greatly improves uptake and biodistribution
of benzoisoselenazolone compounds in cancer cells, as compared
to unconjugated molecules, which is in agreement with other
studies [20e22]. In this report, we describe the synthesis of novel
benzoisoselenazolone-fragment containing (BSZ) compounds
derived from ebselen, which demonstrate anticancer properties,
including inhibition of focal adhesion kinase (FAK), AKT-1, and
tetra-O-acetyl-
b-
D-glucopyranosyl)-butyramide (4a), and 3-
amino-N-(hepta-O-acetyl-
b-D
-lactopyranose)-butyramide (4c) in
a good yield, which was carried out at 0 ꢀC to r.t. and catalyzed by
DCC and DMC. In this method, compound 10 can be obtained in
high yield by the reaction between the OH-protected amino-
saccharide and 4-(benzyloxycarbonylamino)butanoic acid followed
by hydrolysis to remove the benzyloxycarbonyl protection group
from the amino-group and were then directly used in the next step
without any further purification (see experimental section).
The general method for the synthesis of compounds of series III
is shown in Scheme 3. Ring closure reaction was accomplished by
the treatment of 2-(chloroseleno)benzoyl chloride with 7 to afford
2-glucopyranosyl-oxyethyl-benzo[d] [1,2]selenazol-3(2H)-one (22)
in a yield of 27% through a step of deprotection by methanol
sodium. In this method, compound 7 can be obtained in high
yield by the reaction of penta-acetylglucose and benzyl 2-
hydroxyethylcarbamate followed by hydrolysis to remove the
benzyloxycarbonyl protection group from the amino-group in the
yields of 62% for step a and 97% for step b.
protein kinase C-a (PKC-a), which have been shown to play a crit-
ical role in cell survival and invasive signalling [23e30].
A series of organoselenium analogues derived from ebselen and
conjugated to sugar molecules were synthesized in multistep
reactions as described in the Experimental section. Three series of
sugar-modified derivatives of benzoisoselenazol-3(2H)-ones were
designed and synthesized in the current study. They are benzoi-
soselenazolones connected with carbohydrate directly (I), benzoi-
soselenazolones connected with carbohydrate by amide (II) and
benzoisoselenazolones connected with carbohydrate by oxygen
glycoside (III).
3. Results and discussion
3.1. Effect on cell proliferation and in vitro targets
As shown in Table 1, the inhibitory activity (IC50) of the various
compounds tested on the proliferation of the breast cancer cell
I
II
III
2. Chemistry
line MDA-231 revealed that molecules 2bed, 5a, 5b and 6a are the
most active with IC50 in the range of 9.8e25
mM while 3bed and 9
The general method for the synthesis of compounds of series I is
shown in Scheme 1. We started with different kinds of sugars
such as glucose, galactose, lactose and maltose to prepare their
are inactive (IC50 > 100 M). Further screening of these molecules
using our in vitro kinase assays revealed that compounds showing
significant anti-proliferative activity, e.g. 2d, 5a and 6a, were also
m
Scheme 1. Reagents and conditions: (a) 2-(chloroseleno)benzoyl chloride, Et3N, CHCl3, 0 ꢀC to rt, 4 h, 50e65%; (b) MeONa/CH3OH, rt, 1 h, 94e98%.

K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
145
OAc
R2
OAc
R2
OAc
R2
O
O
H
N
O
a
R1
O
H
N
R1
NH2
OAc
b
R1
AcO
O
AcO
N
H
c
A O
OAc
NH2
O
OAc
O
4a-4c
c
OAc
R2
OH
R2
O
C
O
C
O
O
d
H
N
H
R1
HO
R1
AcO
N
N
N
OAc
OH
Se
Se
O
O
R1 = OAc, R2 = H
5a
6a
R1 = OH, R2 = H
OAc
AcO
OH
HO
O
OAc
O
R1 =
5b
R2 = H
,
6b
O
R =
HO
1
O
AcO
R2 = H
,
HO
Scheme 2. Reagents and conditions: (a) 4-(benzyloxycarbonylamino)butanoic acid, DCC, DCM, 0 ꢀC to rt, 5 h; (b) H2, Pd/C, CH3OH/CHCl3 (v/v ¼ 1:1), 4 h; (c) 2-(chloroseleno)
benzoyl chloride, Et3N, THF, 0 ꢀC to rt, 4 h, 42e52%; (d) MeONa/CH3OH, rt, 1 h, 92e97%.
OAc
OAc
OAc
O
O
a
O
AcO
AcO
O
O
AcO
AcO
b
OAc
OAc
AcO
AcO
O
N
H
O
OAc
NH2
OAc
7
c
OH
OAc
O
O
O
O
HO
HO
d
O
AcO
O
N
AcO
OH
N
OAc
Se
Se
9
8
Scheme 3. Reagents and conditions: (a) benzyl 2-hydroxyethylcarbamate, BF3. Et2O, DCM, 0 ꢀC, 4 h, 62%; (b) H2, Pd/C, CH3OH/CHCl3 (v/v ¼ 1:1), 4 h, 97%; (c) 2-(chloroseleno)benzoyl
chloride, Et3N, THF, 0 ꢀC to rt, 4 h, 30%; (d) MeONa/CH3OH, rt, 1 h, 90%.
able to inhibit AKT-1, PKC-
compared to ebselen (1) at 1
a
and FAK kinase activity by 55e95%, as
M final concentration, while no
signalling, which are essential for cell-substrate generating forces
needed for the process of cancer cell migration and invasion.
Central to FA signalling is the FAK, which is targeted by the BSZ
compounds. FAK is activated by integrins as well as numerous
growth factor receptors [34,35]. FAK also serves as a scaffolding
protein mediating multiple proteineprotein interactions critical for
m
inhibitory effect was observed on several other kinases, including
Src, EGFR, Aurora B, IGF-R, VEGFR, and ErbB2/Her 2 kinases
(Tables 1 and 2). The cellular inhibitory activity of our most active
BSZ, namely compound 2d, was demonstrated on FAK phosphor-
ylation at the critical phospho-site Y397 (Fig. 1A) and FAK kinase
activity in intact cells (Fig. 1B). As noted, a concentration of 10
was able to inhibit FAK phosphorylation (Fig. 1A), whereas inhi-
bition of FAK kinase activity was obtained at 10e50 M (Fig. 1B).
Higher concentrations were found to be toxic to the cells. To rule-
out potential “off target” effects of the BSZs, due to their structural
similarities to ebselen, which have been shown to inhibit enzymes
such as lipooxygenases [33] and to act as an anti-oxidant, in vitro
studies revealed that the BSZ compounds with inhibitory activity
mM
Table 1
Inhibitory activity on MDA-231 cell proliferation and in vitro FAK kinase inhibition of
BSZ compounds.
m
Cpd
Inhibition of cell
proliferation (IC50
% FAK inhibition
(1 M)
:m
M))
m
1
42.3
21.0
20.0
9.8
>100
>100
>100
18.9
25
0
2b
2c
2d
3b
3c
3d
5a
5b
6a
6b
8
69.3
54.9
80.1
7.4
9.9
1.3
71.1
55.9
51.9
13.9
51.9
50.9
against FAK, AKT-1 and PKC-a had no significant activity on lip-
ooxygenase (using Lipoxygenase Screening Assay Kit, Cayman
Chemical), nor do they inhibit free radical formation (using the
CM-H2DCFDA-based assay for the detection of reactive oxygen
species) (data not shown). Together, this data support specificity
toward the triple kinases but the mechanism of this selectivity
and its occurrence in in-vivo conditions deserves further
investigations.
20.4
53
35
A crucial early event by which cancer cells acquire autonomous
motile properties is driven by focal adhesion (FA) and survival
9
>100

146
K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
Table 2 (continued)
Table 2
Kinase inhibition profile of compounds 2d, 5a, 6a and ebselen (1) at 1
concentration in vitro.
mM final
Compound name
Kinase tested
% Inhibition; IC50 [mM]
EPHA1
FGFR1
FLT3
FYN
HER2
IKK-ε
INSR
LYN
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
22
1
25
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Compound name
Kinase tested
% Inhibition; IC50 [mM]
2d
AKT-1
FAK
63; 0.83
89; 0.42
95; 0.22
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
67; 0.80
69; 0.92
77; 0.74
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
PKC-
a
IGF1R
ABL1
Aurora A
Aurora B
CDK1/Cyclin B
DDR1
EGFR
EPHA1
FGFR1
FLT3
FYN
HER2
IKK-ε
INSR
MET
P38-g
PAK1
PDGFR-
PIM1
b
PLK4
RPS6KA4
SGK3
SRC
SYK
TIE1
TRKB
VEGFR2
YES1
AKT-1
FAK
LYN
MET
P38-
g
PAK1
PDGFR-
PIM1
1
b
PKC-a
PLK4
IGF1R
ABL1
RPS6KA4
SGK3
SRC
SYK
TIE1
TRKB
VEGFR2
YES1
AKT-1
FAK
Aurora A
Aurora B
CDK1/Cyclin B
DDR1
EGFR
EPHA1
FGFR1
FLT3
FYN
HER2
IKK-ε
INSR
5a
PKC-
a
IGF1R
ABL1
Aurora A
Aurora B
CDK1/Cyclin B
DDR1
EGFR
EPHA1
FGFR1
FLT3
FYN
HER2
IKK-ε
INSR
LYN
MET
P38-
g
PAK1
PDGFR-
PIM1
b
PLK4
RPS6KA4
SGK3
SRC
SYK
TIE1
TRKB
VEGFR2
YES1
LYN
MET
P38-
g
PAK1
PDGFR-
PIM1
b
cancer cell survival. For instance, the non-catalytic domain of FAK
interacts with phosphatidylinositol 3-kinase (PI3-K) secondary to
FAK autophosphorylation at Y397, which serves as a binding site for
the SH2 domains of PI3-K, which can act as a second lipid
messenger regulating protein kinase C (PKC) family members, as
well as the phosphoinositide-dependent serine/threonine protein
kinase AKT-1. These interactions are critical for cell migration and
PLK4
RPS6KA4
SGK3
SRC
SYK
TIE1
TRKB
VEGFR2
YES1
cell survival signaling [35,36]. Not surprising, FAK, PKC-a, and AKT-
1 are upregulated/hyperactivated in invasive cancers [23,37e39],
and inhibition of specific components of these networks, e.g. using
RNA interference or small molecules, prevented cancer cell prolif-
eration, invasion and/or metastasis formation [23e30]. However,
approaches targeting these proteins individually have been proven
of limited efficacy due in part to compensatory signalling loops that
can overcome a single target inhibition. As such, the triple kinase
inhibitory profile of these BSZs is novel and has great biological and
pharmacological implications. In order to speculate on the binding
6a
AKT-1
FAK
60; 0.88
55; 0.96
74; 0.73
0
0
0
0
0
0
0
PKC-
a
IGF1R
ABL1
Aurora A
Aurora B
CDK1/Cyclin B
DDR1
EGFR

K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
147
to vehicle treated (DMSO) control samples. Ebselen (1) alone had
no effect on MDA-231 cell invasion (Fig. 3).
Compound 2d
0.1 10 :µM
A
0
1
3.3. Efficacy in vivo
-Y397-FAK
The ability of these compounds to inhibit breast cancer cell
metastasis is also investigated in a highly invasive mouse model of
human breast cancer in vivo. MDA-231-M2 cells, a highly invasive
human breast cancer cell line, were implanted into the mammary
fat pad of female SCID mice. Once tumors become palpable, mice
were randomised into experimental groups and then treated with
vehicle, or 60 mg/kg of compound 2d or ebselen (1) administered
by intraperitoneal (IP) route 3 times a week for a total of 9
administrations. 60 mg/kg was very well tolerated with no body
weight loss or apparent toxicity. 40 days after the initiation of
treatment, mice were sacrificed and primary tumors and lungs
metastases were examined as described in methods. As demon-
strated in Fig. 4, compound 2d at 60 mg/kg induced a significant
inhibition of tumor growth and lung metastases, as compared to
vehicle- or ebselen (1)-treated mice. Therapeutic activity was also
observed at lower doses while higher doses were not tested. On the
other hand, ebselen (1) treatment did not significantly differ from
the vehicle treated controls. In summary, the BSZ compounds
reported here represent a novel class of kinase inhibitors with
biological activity against invasive breast cancer.
-FAK
B
100,0
75,0
50,0
25,0
0,0
*
**
Ebselen (1)
50µM
1
10
50
Compound 2d [µ M]
4. Experimental section
Fig. 1. Compound 2d inhibits FAK phosphorylation and kinase activity in intact cells.
A) representative western blot analysis of total cell extracts of MDA-231 cells treated
with increasing concentrations of compound 2d, as indicated. Inhibition of FAK acti-
4.1. Chemistry
vation, as demonstrated by FAK (Tyr-397) phosphorylation is observed at 10
compound 2d. B) FAK was immunoprecipated from lysates of MDA-231 cells treated
with indicated concentration of compound 2d and were incubated with [
32P]-ATP in
order to monitored for FAK kinase activity. Significant inhibition of FAK kinase activity
was observed at 10 M (*P < 0.05) and 50 M (**P < 0.01) of compound 2d, as compared
to untreated controls (N ¼ 4). Ebselen was unable to inhibit FAK kinase activity at 50 M.
mM of
4.1.1. General methods
g-
Solvents were purified in a conventional manner. Thin-layer
chromatography (TLC) was performed on precoated E. Merck
silica-gel 60 F254 plates. Flash-column chromatography was per-
formed on silica gel (200e300 mesh, Qingdao, China). Melting
point is determined on a Mitamura-Riken micro-hot stage and is
not corrected. 1H NMR and 13C NMR spectra were obtained on a Jeol
JNM-ECP600 spectrometer with tetramethylsilane (Me4Si) as the
internal standard, and chemical shifts were recorded in values.
Mass spectra were recorded on a Q-TOF Global mass spectrometer.
All reagents and solvents were purchased from Sinopharm Chem-
ical Reagent Co., Ltd. The intermediate compounds 4a, 4c [40], 7
[41] and 2-(chloroseleno)benzoyl chloride [42] were prepared
according to the literature. According to the reference, 2-(chlor-
oseleno)benzoyl chloride was prepared from the intermediate of
2,20-bisselanyldibenzoic acid which was prepared in a good yield
from the diozotization of o-aminobenzoic acid with a basic solution
of Na2Se2, which was in turn obtained from the reaction of sele-
nium and hydrazine hydrate in the basic solution under the
protection of nitrogen. Then, a solution of 2,20-bisselanyldibenzoic
acid (20 g, 50 mmol) in thionyl chloride (80 mL) was refluxed for 3 h
and the unreacted thionyl chloride was removed by vacuum
distillation. The crude solid was extracted three times by hexane
and after hexane removal, the solid product was recrystallized by
vacuum distillation from diethyl ether to give a yellow solid (18 g)
in the yield of 60.5%, mp: 60e62 ꢀC.
m
m
m
mode of these compounds to each of these kinases, through
molecular modelling studies we demonstrated the predictive
binding mode of compound 2d in the ATP-binding pocket of the
AKT-1, FAK and PKC-a kinase domains (Fig. 2).
3.2. Effect on cell motility and invasion
The effect of some of the compounds listed above on cell
motility was investigated using the wound healing motility assay.
In this study, cells were grown on coverslips and then wounded by
cell scraping using a micropipette tip. Cells were allowed to migrate
and heal the wound. Photomicrographs were taken at each time
point in order to examine the wound healing areas, which were
measured and quantified. As shown in Fig. 3A, compounds 2d, 5a
and 6a were able to inhibit MDA-231 cell motility by approximately
50%, as compared to vehicle (DMSO) treated control sample,
thereby suggesting a possible impairment in the cell’s motility
machinery. This is in contrast to ebselen (1), which was unable to
significantly inhibit MDA-231 cell motility.
Similarly, cell invasion experiments were performed using the
Boyden chamber assay where serum starved MDA-231 cells were
placed into the upper compartment of the Boyden chamber, treated
with BSZ molecules or ebselen (1). Cells were then allowed to
invade through a Matrigel matrix for 48 h. As shown in Fig. 3B,
compounds 2d, 5a and 6a were able to significantly inhibit cell
invasion revealed by an approximate 50%inhibition of the number
of cancer cells able to invade through the matrigel at BSZ concen-
trations that had no apparent direct cytotoxic effects, as compared
4.1.2. 2-(2,3,4,6-Tetra-O-acetyl-1-deoxy-
benzo[d] [1,2] selenazol-3 (2H)-one (2a)
b
-
D
-glucopyranosyl)-
1-amino-2,3,4,6-tetra-O-acetyl-1-deoxy-b-
D-glucopyranose
(690 mg, 2 mmol) was dissolved in dry CHCl3 (10 mL). Then solu-
tions of 2-(chloroseleno)benzoyl chloride (500 mg, 2 mmol) in dry
CHCl3 (10 mL) and triethylamine (0.60 mL, 4 mmol) in dry CHCl3
(10 mL) were added dropwise to the solution at 0 ꢀC over a period of
30 min. The reaction mixture was allowed to stir for an additonal

148
K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
Fig. 2. Predicted binding mode of compound 2d in the ATP-binding pocket of the AKT-1, FAK and PKC-a kinase domains (aec). Compound 2d was colored according to the atomic
coloring scheme (C in green, O in red and N in blue). The side chains that are within 3 Å of the docked ligand are shown in orange sticks. Hydrogen bonds are indicated by dash lines.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4 h at rt. The organic layer was washed successively with water and
aqueous NaHCO3 and dried over Na2SO4, and the solvent was
removed under reduced pressure to afford a crude product. The
purification of the residue by column chromatography gave the title
4.1.4. 2-(2,3,6,20,30,40,60-hepta-O-acetyl-1-deoxy-
b-D-malcopyra-
nosyl)-benzo[d][1,2]selenazol-3(2H)-one (2c)
Following the procedure for the preparation of compound 2a
but substituting 1-amino-2,3,4,6-tetra-O-acetyl-1-deoxy-b- -glu-
copyranose with 1-amino-2,3,6,20,30,40,60-hepta-O-acetyl-1-deoxy-
b-
-maltopyranose provided the title compound in 52% yield. 1H
D
compound as a yellow powder (630 mg, 60.3%). 1H NMR (600 MHz,
0
CDCl3):
d
8.05e7.40 (m, 04H, ArH), 5.94 (d, 1H, J ¼ 9.2 Hz, C1 eH),
D
0
0
5.37 (t, 1H, J ¼ 9.1 Hz C2 eH), 5.17 (m, 2H, C4 -H, C3 eH), 4.25 (dd,
NMR (600 MHz, CDCl3):
d
8.05e7.44 (m, 4H, ArH), 5.96 (d, 1H,
0
0
0
0
1H, J ¼ 4.7, 12.5 Hz, C6 -H1), 4.15 (dd, 1H, J ¼ 2.2, 12.5 Hz, C6 eH2),
J ¼ 9.2 Hz, C1 eH), 5.46 (m, 2H, C3 -H, C100eH), 5.39 (dd, 1H, J ¼ 9.7,
3.96 (m, 1H, C5 eH), 2.09e1.92 (4s, 12H, Ac-H). 13C NMR (150 MHz,
10.5 Hz, C300eH), 5.09 (dd, 1H, J ¼ 9.7, 10.1 Hz, C400eH), 5.06 (t, 1H,
0
0
CDCl3):
d
170.6, 169.9, 169.5, 169.4, 167.7, 138.7, 132.9, 129.1, 126.4,
J ¼ 9.2 Hz, C2 eH), 4.89 (dd, 1H, J ¼ 4.1, 10.5 0Hz, C200eH), 4.53e4.11
0
0
126.2, 124.2, 80.7, 74.5, 73.2, 71.4, 67.9, 61.7, 20.7, 20.6, 20.5, 20.4.
(m, 5H, C4 -H, C6 -H, C600eH), 3.98 (m, 1H, C5 -H, C500eH), 2.14e1.90
ESIMS m/z 530.0 [M þ H]þ.
(7s, 21H, Ac-H). 13C NMR (150 MHz, CDCl3):
d 170.6, 170.5, 170.4,
169.9, 169.8, 169.4, 167.5, 138.5, 132.9, 129.1, 126.4, 126.3, 124.1, 95.7,
80.4, 75.7, 74.8, 72.5, 72.1, 69.9, 69.3, 68.6, 67.9, 62.6, 61.4, 20.9, 20.8,
20.7, 20.6, 20.4. ESIMS m/z 818.1 [M þ H]þ.
4.1.3. 2-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-D-galcopyranosyl)-
benzo[d][1,2] selenazol-3 (2H)-one (2b)
Following the procedure for the preparation of compound 2a
but substituting 1-amino-2,3,4,6-tetra-O-acetyl-1-deoxy-b- -glu-
copyranose with 1-amino-2,3,4,6-tetra-O-acetyl-1-deoxy-b- -gal-
acopyranose provided the title compound in 65% yield. 1H NMR
D
4.1.5. 2-(2,3,6,20,30,40,60-hepta-O-acetyl-1-deoxy-
b-D-lactopyra-
nose)-benzo[d][1,2]selenazol-3(2H)-one (2d)
Following the procedure for the preparation of compound
2a but substituting 1-amino-2,3,4,6-tetra-O-acetyl-1-deoxy-b-
glucopyranose with 1-amino-2,3,6,20,30,40,60-hepta-O-acetyl-1-
D
(600 MHz, CDCl3):
d
8.05e7.44 (m, 4H, ArH), 5.92 (d, 1H, J ¼ 9.1 Hz,
D-
0
0
C10eH), 5.50 (d, 1H, J ¼ 3.2 Hz C4 eH), 5.35 (dd, J ¼ 9.2, 10.1 Hz 1H,
0
0
C2 eH), 5.22 (dd, 1H, J ¼ 3.2, 10.1 Hz, C3 eH), 4.20e4.11 (m, 3H, C5 -
deoxy-b-D-lactopyranose provided the title compound in 52%
H, C6 eH), 2.22e1.94 (4s, 12H, Ac-H). 13C NMR (150 MHz, CDCl3):
170.4, 170.0, 169.9, 169.5, 167.5, 138.8, 132.9, 129.0, 126.3, 126.2,
yield. 1H NMR (600 MHz, CDCl3):
d
8.05e7.44 (m, 4H, ArH), 5.91 (d,
0
0
0
0
d
1H, J ¼ 8.7 Hz, C1 eH), 5.39 (m, 2H, C3 -H, C400eH), 5.15 (m, 2H, C2 -
H, C200eH), 4.99 (dd, 1H, J ¼ 3.3, 10.6 Hz, C300eH), 4.53 (d, 1H,
J ¼ 8.3 Hz, C100eH), 4.51 (d, 1H, J ¼ 12.4 Hz, C600eH), 4.17e4.08 (m,
124.2, 81.2, 73.3, 71.3, 68.9, 67.1, 61.3, 20.7, 20.6, 20.5, 20.5. ESIMS m/
z 530.0 [M þ H]þ.

K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
149
4.1.6. 2-(1-Deoxy-b-D-glucopyranosyl)-benzo[d][1,2]selenazol-3
(2H)-one (3a)
Inhibition of cell motility
A
75
50
25
0
Compound 2a (270 mg, 0.5 mmol) was dissolved in methanol-
chloroform (20 mL, v/v ¼ 1:1). Then freshly prepared sodium
methoxide (0.63 M, 1 mL) was added to the solution at 0 ꢀC. After
stirring at rt for 1 h, the resulting precipitate was filtrated and
washed with methanol, and the solvent was removed under
reduced pressure to obtain title compound as a yellow powder
(170 mg, 96%). 1H NMR (600 MHz, CD3OD) :
d
7.92e7.42 (m, 4H,
12 HOURS
24 HOURS
6a
0
Ar0H), 5.53 (d, 1H, J ¼ 8.7 Hz, C1 eH), 3.86 (dd, 1H, J ¼ 2.4, 12.40Hz,
1
2d
5a
0
C60-H1), 3.66 (dd, 1H, J ¼ 5.9,011.9 Hz, C6 eH2), 3.49 (m, 3H, C2 -H,
C3 -H, C5 eH), 3.35 (m, 1H, C4 eH). 13C NMR (150 MHz, DMSO-d6):
0
d
167.1, 139.8, 131.9, 128.2, 127.6, 125.8, 82.4, 79.6, 77.4, 74.3, 70.0,
Inhibition of cell invasion
B
61.0. ESIMS m/z 362.0 [M þ H]þ.
50
25
0
4.1.7. 2-(1-Deoxy-b-D-galcopyranosyl)-benzo[d][1,2]selenazol-
3(2H)-one (3b)
Following the procedure for the preparation of compound 3a
but substituting 2a with 2b provided the title compound in 94%
yield. 1H NMR (600 MHz, CD3OD):
d 7.94e7.42 (m, 4H, ArH), 5.50 (d,
1H, J ¼ 8.7 Hz, C10eH), 3.95 (d, 1H, J ¼ 3.3 Hz, C40eH), 3.86 (dd, 1H,
J ¼ 8.7, 9.2 Hz, C20eH), 3.78e3.72 (m, 3H, C60-H, C50eH), 3.65 (dd,
1H, J ¼ 3.3, 9.6 Hz, C30eH). 13C NMR (150 MHz, DMSO-d6):
d 166.9,
1
2d
5a
6a
139.9, 131.8, 128.2, 127.5, 125.9, 125.7, 82.8, 77.8, 74.1, 71.3, 68.3,
Compound [10µM]
60.6. ESIMS m/z: 362.0 [M þ H]þ.
Fig. 3. EBZ compounds inhibit MDA-231 cell motility and invasion. Cell migration (A)
and invasion (B) were analyzed by the wound healing and Boyden chamber assays
4.1.8. 2-(1-Deoxy-b-D-malcopyranosyl)-benzo[d][1,2]selenazol-3
(2H)-one (3c)
Following the procedure for the preparation of compound 3a
but substituting 2a with 2c provided the title compound in 98%
respectively. A) MDA-231 cells treated with the indicated EBZ compounds at 10 mM,
were wounded and monitored for 12 and 24 h to determine the rate of migration into
the scratched area. Bar graph represents the mean ꢁ SD of five independent experi-
ments. B) invasion of MDA-231 cells treated with the indicated EBZ compounds at
10 mM was analyzed using the Boyden chamber assay as described in the experimental
section. Bar graph represents the mean number of invaded cells. Results are expressed
yield. 1H NMR (600 MHz, CD3OD):
d
7.97e7.45 (m, 4H, ArH), 5.56 (d,
1H, J ¼ 8.7 Hz, C1 eH), 5.23 (d, 1H, J ¼ 4.1 Hz, C100eH), 3.91 (dd, 1H,
0
as the mean ꢁ SD of five independent experiments and five fields per condition.
0
J ¼ 1.4, 12.4 Hz, C6 eH), 3.84e3.79 (m,03H, Mal-H), 3.72e3.62 (m,
0
0
0
3H, C6 -H, C600eH), 3.91 (m, 3H, C4 -H, C5 -H, C500eH), 2.17e1.92 (7s,
5H, Mal-H), 3.60 (t, 1H, J ¼ 8.7 Hz, C2 eH), 3.46 (dd, 1H, J ¼ 3.7,
9.7 Hz, C200eH), 3.29 (t, 1H, J ¼ 9.6 Hz, C300eH). 13C NMR (150 MHz,
21H, Ac-H). 13C NMR (150 MHz, CDCl3):
d
170.4, 170.3, 170.2, 170.1,
DMSO-d6): d 167.1, 139.9, 131.9, 128.1, 127.6, 125.9, 125.8, 100.9, 82.3,
169.7,169.5,169.0,167.6,138.6,132.9,129.1,126.4,126.3,124.2,101.1,
80.6, 75.9, 75.4, 73.1, 71.7, 70.9, 70.8, 69.0, 66.6, 61.8, 60.8, 20.9, 20.8,
20.7, 20.7, 20.6, 20.5. ESIMS m/z 818.3 [M þ H]þ.
79.4, 77.7, 77.1, 73.8, 73.5, 73.3, 72.5, 69.9, 60.8, 60.5, 59.8. ESIMS m/z
524.1 [M þ H]þ.
Fig. 4. Compound 2d inhibits cancer progression in a preclinical orthotopic breast cancer model. MDA-231-M2 cells were implanted into the mammary fat pad of 6 SCID mice per
condition as described in the experimental section. Once the tumor became palpable, mice were treated with vehicle, compound 2d or ebselen (1) at 60 mg/kg IP for a total of 9
injections. Mice were then subjected to autopsy and lung metastases and primary tumor volume were examined. Graphs show the mean % inhibition of lung metastases (*, P < 0.01)
(left panel), and primary tumor volume ꢁ SEM (*, P < 0.01), as compared to vehicle controls. Bottom panel shows representative lungs with metastatic nodules.

150
K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
4.1.9. 2-(1-Deoxy-
b-
D-laccopyranosyl)-benzo[d][1,2]selenazol-3
(205 mg, 94%). 1H NMR( 600 MHz, DMSO-d6):
d 8.39 (d, 1H,
(2H)-one (3d)
J ¼ 9.4 Hz, N-H), 8.05e7.40 (m, 4H, ArH), 4.96 (d, 1H, J ¼ 5.0 Hz,
Following the procedure for the preparation of compound 3a
but substituting 2a with 2d provided the title compound in 96%
eOH), 4.86 (d, 1H, J ¼ 6.0 Hz, eOH)， 4.85 (d, 1H, J ¼ 5.4 Hz, eOH),
0
4.69 (dd, 1H, J ¼ 8.8, 9.3 Hz, C1 eH), 4.47 (dd, 1H, J ¼ 5.4, 6.1 Hz,
yield. 1H NMR (600 MHz, CD3OD):
d
7.97e7.46 (m, 4H, ArH), 5.57 (d,
eOH), 3.68 (m, 2H, a-CH2), 3.61e3.00 (m, 6H, Glu-H)，2.12 (m, 2H,
1H, J ¼ 8.7 Hz, C1 eH), 4.41 (d, 1H, J ¼ 7.8 Hz, C100eH), 3.93 (dd, 1H,
g-CH2), 1.80 (m, 2H, b d 171.9,
-CH2). 13C NMR (150 MHz, DMSO-d6):
0
0
0
J ¼ 2.3, 12.4 Hz, C6 -H1), 3.87 (dd, 1H, J ¼ 4.1, 12.4 Hz, C6 eH2),
3.82e3.59 (m, 8H, Lac-H), 3.59 (dd, 1H, J ¼ 7.8, 9.6 Hz, C200eH), 3.51
(dd, 1H, J ¼ 3.3, 9.6 Hz, C300eH). 13C NMR (150 MHz, DMSO-d6):
166.4, 139.2, 131.4, 128.0, 127.3, 125.8, 125.7, 79.5, 78.5, 77.5, 72.5,
69.9, 42.8, 32.3, 25.8. ESIMS m/z 447.1 [M þ H]þ.
d
167.8, 132.6, 132.2, 131.9, 129.9, 126.1, 103.9, 80.6, 80.3, 76.7, 75.7,
4.1.13. 4-(benzo[d] [1,2] selenazol-3 (2H)-one-2-yl)-N-(1-deoxy-
-laccopyranosyl)-butyramide (6b)
Following the procedure for the preparation of compound 6a but
substituting 5a with 5b provided the title compound in 97% yield. 1H
NMR (600 MHz, DMSO-d6):
b-
75.6, 73.2, 71.6, 70.6, 68.1, 60.4, 60.3. ESIMS m/z 524.1 [M þ H]þ.
D
4.1.10. 4-(benzo[d][1,2]selenazol-3(2H)-one-2-yl)-N-(2,3,4,6-tetra-
O-acetyl-1-deoxy-
2-amino-N-(2,3,4,6-tetra-O-acetyl-
amide (4a) was prepared from hydrolysis of N-[2-oxo-2-[(2,3,4,6-
tetra-O-acetyl- -glucopyranosyl)amino] butyl]-Carbamic phe-
nylmethyl ester, with H2/Pd-C which was in turn obtained from
4-(benzyloxycarbonylamino)butanoic acid and 1-amino-2,3,4,6-
b
-
D
-glucopyranosyl)-butyramide (5a)
d
8.46 (d, 1H, J ¼ 9.1 Hz, N-H), 8.08e7.41
b
-D-glucopyranosyl)- butyr-
(m, 4H, ArH), 5.13 (d,1H, J ¼ 4.6 Hz,eOH), 5.04 (d,1H, J ¼ 5.5 Hz,eOH),
0
4.81 (d,1H, J ¼ 5.0 Hz, eOH), 4.74 (t,1H, J ¼ 9.1 Hz, C1 eH), 4.70 (d,1H,
b
-
D
J ¼ 1.3 Hz, eOH), 4.66 (t, 1H, J ¼ 5.0 Hz, eOH), 4.56 (t,1H, J ¼ 5.9 Hz,
eOH), 4.53 (d, 1H, J ¼ 4.6 Hz, eOH),4.19 (d, 1H, J ¼ 7.3 Hz, C100eH),
3.67e3.09 (m,13H, Lac-H, a-CH2), 2.12 (m, 2H, g-CH2),1.80 (m, 2H, b-
tetra-O-acetyl-
b-
D
-glucopyranose as starting material in 90% yield
CH2). 13C NMR (150 MHz, DMSO-d6):
d 172.0,166.4,139.2,131.5,128.0,
according to the method of Sawaki M [40]. Then, to a solution of
compound 4a (800 mg, 1.8 mmol)) in dry THF (30 mL) was added
a solution of 2-(chloroseleno)benzoyl chloride (500 mg, 2 mmol) in
dry THF (10 mL) and a solution of triethylamine (0.6 mL, 4 mmol) in
dry THF (10 mL) dropwise at 0 ꢀC over a period of 30 min. The
reaction mixture was continued stiring for an additional 4 h at
room temperature. When the reaction was completed as detected
by TLC, the reaction mixture was filtered and the organic layer was
washed with water and aqueous NaHCO3 and dried over anhydrous
Na2SO4. After removing the dryer by filtration, the solvent was
removed under reduced pressure to afford a crude product. The
purification of the crude product by column chromatography to
afford yellow power, the title compound, 530 mg in 48% yield. 1H
127.3, 125.9, 125.8, 80.6, 79.2, 76.4, 75.6, 75.5, 73.2, 72.1, 70.5, 68.2,
60.4, 60.3, 42.8, 32.4, 25.9. ESIMS m/z 609.1 [M þ H]þ.
4.1.14. 2-(2-(2,3,4,6-Tetra-O-acetyl-
b-D-glucopyranosyl) ethyl)-
benzo[d][1,2] selenazol-3 (2H)-one (8)
Compound 7 can be obtained in high yield by reaction of penta-
acetylglucose and benzyl 2-hydroxyethylcarbamate followed by
hydrolysis to remove benzyloxycarbonyl protection group for
amino-group according to the literature [41]. Then compound 7
(0.76 g, 1.9 mmol) was dissolved in dry THF (20 mL). A solution of 2-
(chloroseleno)benzoyl chloride (0.52 g, 2 mmol) in dry THF (10 mL)
and a solution of triethylamine 0.6 mL in dry THF (10 mL) were
added dropwise at 0 ꢀC over a period of 30 min. And the reaction
was continued for an additional 2 h at room temperature. When the
reaction was completed, purification of the crude reaction product
by column chromatography to afford yellow power (540 mg, 50%).
NMR (600 MHz, CDCl3):
d 8.05e7.43 (m, 4H, ArH), 6.99 (d, 1H,
0
0
0
J ¼ 9.2 Hz,0N-H), 5.27 (m, 2H, C3 -H, C4 eH), 5.07 (m, 1H C2 eH), 4.97
0
(m, 1H, C1 eH), 4.27 (dd, 1H, J ¼ 4.6, 12.4 Hz, C6 -H1), 4.08 (dd, 1H,
J ¼ 2.3, 12.4 Hz, C6 eH2), 3.92 (m, 1H, C5 eH), 3.80 (m, 2H,
a
b
-CH2),
-CH2).
1H NMR (600 MHz, CDCl3):
d 8.05e7.43 (m, 4H, ArH), 5.21 (t, 1H,
0
0
2.24 (m, 2H,
g
-CH2), 2.01e2.08 (4s, 12H, Ac-H), 1.77 (m, 2H,
J ¼ 9.1 Hz, C40eH), 5.12 (m, 2H, C20-H, C30eH), 4.57 (d, 1H, J ¼ 7.8 Hz,
13C NMR (150 MHz, CDCl3):
d
172.7, 170.7, 170.0, 169.6, 167.7, 137.8,
C10eH), 4.26 (m, 1H, -CH2), 3.99 (m, 1H, C60-
b-CH2), 4.15 (m, 1H, a
132.3, 129.1, 127.1, 126.4, 124.1, 78.2, 73.6, 73.1, 70.7, 68.2, 61.7, 43.7,
H1), 3.84 (m, 1H, C60-H2), 3.73 (m, 1H, C50eH), 2.08e1.95 (4s, 12H,
33.0, 26.2, 20.8, 20.8, 20.7. ESIMS m/z 615.1 [M þ H]þ.
Ac-H). 13C NMR (150 MHz, CDCl3):
d
170.6, 170.2, 169.4, 167.5, 139.0,
132.1,128.7,126.5,126.1,123.7,100.5, 72.8, 71.9, 71.2, 68.9, 68.3, 61.8,
4.1.11. 4-(benzo[d][1,2]selenazol-3(2H)-one-2-yl)-N-(2,3,6,20,30,40,
60-hepta-O-acetyl-1-deoxy-
b-D-laccopyranosyl)-butyramide (5b)
44.5, 20.7, 20.6. ESIMS m/z 574.1 [M þ H]þ.
Following the procedure for the preparation of compound
4.1.15. 2-(2-(1-Deoxy-b-D-glucopyranosyl) ethyl)-benzo [d] [1,2]
5a but substituting 4a with 4c and the starting material with
selenazol-3 (2H)-one (9)
1-amino-2,3,6,20,30,40,60-hepta-O-acetyl-1-deoxy-
b
-
D
-laccopyranose
Compound 8 (286 mg, 0.5 mmol) was dissolved in methanol-
chloroform (20 mL, v/v ¼ 1:1). Then freshly prepared sodium
methoxide (0.63 M, 1 mL) was added to the solution at 0 ꢀC. After
stirring at rt for 1 h, the resulting precipitate was filtrated and
washed with methanol, and the solvent was removed under
reduced pressure to obtain title compound as a yellow powder
provided the title compound in 42% yield.1H NMR (600 MHz, CDCl3):
d
8.03e7.43 (m, 4H, ArH), 7.11 (d, 1H, J ¼ 9.1 Hz, 0N-H), 5.35 (d, 1H,
J ¼ 2.3 Hz, C400eH), 5.27 (t, 1H, J ¼ 9.1 Hz, C1 eH), 5.24 (t, 1H,
J ¼ 9.1 Hz, C100eH), 5.10e3.72 (m, 13H, Lac-H,
a-CH2), 2.22 (m, 2H,
g
b
-CH2), 2.16e1.97(7s, 21H, Ac-H), 2.00 (m, 1H,
b
eCH2e1),1.26(m,1H,
eCH2e2). 13C NMR (150 MHz, DMSO-d6):
d
172.7,170.9,170.5,170.3,
(182 mg, 90%). 1H NMR (600 MHz, CD3OD):
d 7.93e7.45 (m, 4H,
170.2, 169.6, 169.1, 167.8, 137.9, 132.3, 129.1, 127.1, 126.5, 124.2, 101.1,
78.1, 76.1, 74.5, 72.8, 71.1, 71.0, 70.8, 69.1, 66.7, 62.0, 61.0, 43.7, 33.0,
26.3, 21.0, 20.9, 20.8, 20.7, 20.7, 20.6. ESIMS m/z 903.2 [M þ H]þ.
ArH), 4.35 (d, 1H, J ¼ 7.8 Hz, C10eH), 4.15 (m, 3H, C60-H1,
b-CH2),
3.86 (m, 1H, a
-CH2), 3.65 (m, 1H, C60-H2), 3.36e3.22 (m, 4H, C20-H,
C30-H, C40-H, C50eH). 13C NMR (150 MHz, DMSO-d6):
d 166.6, 140.1,
131.4, 127.6, 127.2, 125.7, 125.6, 103.2, 77.1, 76.7, 73.5, 70.1, 67.9, 61.1,
4.1.12. 4-(benzo[d][1,2] selenazol-3 (2H)-one-2-yl)-N-(1-deoxy-
-glucopyranosyl)-butyramide (6a)
Compound 5a (300 mg, 0.5 mmol) was dissolved in methanol-
b-
39.8. ESIMS m/z 406.0 [M þ H]þ.
D
4.2. Biological studies
chloroform (20 mL, v/v ¼ 1:1). Then freshly prepared sodium
methoxide (0.63 M, 1 mL) was added to the solution at 0 ꢀC. After
stirring at rt for 1 h, the resulting precipitate was filtrated and
washed with methanol, and the solvent was removed under
reduced pressure to obtain title compound as a yellow powder
4.2.1. Cell culture
The metastatic cell variant MDA231-M was established from
metastatic lung nodules induced in vivo by the parental breast
carcinoma cells MDA-231 engineered to overexpress ErbB-2 and

K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
151
implanted into the mammary fat pad of Scid mice [34]. These cells
were cultured in DMEM (Life Technologies, Rockville, MD) sup-
plemented with 10% fetal bovine serum (FBS), 1 mM sodium
Cell invasion experiments were performed with 8-
m
m porous
chambers coated with Matrigel (Becton Dickinson). Briefly, serum
starved cells were placed into the upper compartment (30,000
cells) of the Boyden chamber with or without indicated compounds
and the chambers were then placed into 24-well culture dishes
pyruvate, 1% (v/v) non-essential amino acids, 100
M 2-
mercaptoethanol, and penicillin/streptomycin. Cells were main-
tained in culture at 37 ꢀC in an atmosphere of 5% CO2.
containing 400
mL of DMEM 0.2% BSA with 10% serum (lower
compartment). Cells were allowed to invade through the Matrigel
membrane for 48 h. The invasive cells underneath the membrane
were fixed and stained. Filters were viewed under bright-field 40ꢂ
objective and the counting was performed for at least three fields in
each sample [34]. In all cases, DMSO treated samples were
normalized to 0% inhibition.
4.2.2. Cell proliferation
Exponentially growing cells (1 ꢂ 103) were seeded in 96
well plates and incubated for 24, 48 and 72 h. Cell survival was
evaluated using the metabolic MTT assay by removing culture
media and replacing with fresh media containing 1 mg/mL of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
dissolved in PBS. Following 3e4 h incubation at 37 ꢀC, the medium
containing MTT was removed and 200 L dimethyl sulfoxide
(DMSO) was added to dissolve the insoluble reduced MTT formazan
precipitate into a colored solution, followed by 25 L of glycine/
NaCl buffer (0.1 M glycine, 0.1 M NaCl pH 10.5). Once the formazan
crystals dissolved, the absorbance was determined at 570 nm using
a microplate reader. As yellow MTT is reduced to purple formazan
in the mitochondria of living cells, the assay allows discrimination
between viable and non-viable cells. The concentration that
inhibits 50% of cell growth (IC50) for each compound was deter-
mined graphically [34].
bromide
4.2.6. In vivo tumorigenic and invasion studies in mice
m
The ability of compound 2d to inhibit breast cancer cell
metastasis was further investigated in a highly invasive mouse
model of human breast cancer in vivo. In vivo studies were
approved by the McGill Animal Care Committee (Protocol number
4101) and were conducted in accordance with institutional and
Canadian Federal Guidelines. Female Scid mice were obtained from
Charles River Laboratories, St. Zotique, QC, Canada. Briefly, 1.5
million MDA-231-M2 cells, a highly invasive human breast cancer
cell line, were injected into the mammary fat pad of female SCID
mice. Once a palpable tumor was observed, animals were treated
with vehicle, compound 2d or ebselen (1) 3 times a week (60 mg/kg
IP, 9 total injections). Animals were sacrificed on day 40, lungs were
fixed in 10% Bouin’s fixative, and lung surface metastases were
counted using a stereomicroscope. 6 mice were used per condition.
For data analysis, an unpaired Student t test was used to compare
tumor growth between control and chemical-treated groups.
ANOVA was used to compare groups, and significance was set at
P < 0.05 level [34].
m
4.2.3. In vitro kinase activity
Human recombinant full-length FAK was incubated in kinase
buffer containing ATP and substrate (Poly Glu:Tyr) for 4 h at room
temperature with or without the presence of the compounds at
various concentrations. Remaining ATP in solution was then
quantified utilizing the Kinase-Glo-luminescence kit (Promega).
Moreover, the inhibitory activity of these compounds against
a multitude of related kinases was also analyzed through Invi-
trogen’s SelectScreenÒ kinase profiling service [43].
4.2.7. Docking studies
Crystal structures of AKT1, FAK and PKC-
a in complex with their
4.2.4. Cellular FAK kinase assay
inhibitors (PDB entries: 3MVH, 2JK0 and 3IW4, respectively) were
utilized in the docking study after the bound inhibitors were taken
out. Compound 2d was docked into the ATP-binding pocket of
MDA231 cells were serum starved overnight, treated with each
compound for 2 h and then stimulated with 20 ng/mL EGF. Control
without EGF and no treatment was included to confirm FAK acti-
vation. After 30min, cells were washed with cold PBS and then
lysed in a lysis buffer (50 mM HEPES; 150 mM NaCL; 10 mM MgCl2;
0.5 mM EGTA; 0.1% Triton X-100; 10% glycerol; 0.5 mM DTT; 1 mM
Na3VO4; 1 mM PMSF; 5 mg/mL aprotinin; and 5 mg/mL leupeptin)
and immunoprecipitated with a polyclonal anti-FAK antibody (C-
20; Santa Cruz Biotechnologies). The resulting immunocomplex
was used for FAK kinase assay using a kinase buffer (10% glycerol,
20 mM Hepes, 10 mM MgCl2, 10 mM MnCl2, and 100 mM NaCl)
AKT1, FAK and PKC-a, respectively, using software Gold v3.2 (The
Cambridge Crystallographic Data Centre, Cambridge, U.K.) by
Goldscore function and standard default parameter settings [44].
The ligand is treated as fully flexible, whereas the proteins were
kept rigid except that each Ser, Thr and Tyr hydroxyl group was
allowed to rotate to optimize hydrogen-bonding to the ligand,
whereas other parts of the protein were kept rigid.
containing 3 mCi/nmol [g-
32P]-ATP (4500 Ci/mmol) as previously
Acknowledgements
described [23,34]. Reactions were stopped with SDS-PAGE sample
buffer and boiling for 5 min. Samples were resolved by SDS-PAGE
electrophoresis and excised bands were used to quantify the
radioactivity using scintillation counting. The data shown repre-
sents 2 duplicate experiments for each condition.
This work was supported by the Canadian Institutes of Health
Research and the Quebec Breast Cancer Foundation (MAAJ) and Key
International S&T Cooperation Project of Ministry of Science and
Technology of the People’s Republic of China and Natural Science
Foundation of China (21171154) (JT).
4.2.5. Cell motility and invasion
The effect of each compound on cell motility was investigated
using the wound healing motility assay. In this study, cells were
grown on sterile cover slips for 24 h and were then wounded by cell
scraping using a micropipette tip. Cultures were washed and then
incubated with fresh culture media at 37 ꢀC with or without the
References
[1] S. Cao, F.A. Durrani, Y.M. Rustum, Selective modulation of the therapeutic
efficacy of anticancer drugs by selenium containing compounds against
human tumor xenografts, Clinical Cancer Research 10 (2004) 2561e2569.
[2] S.V. Madhunapantula, D. Desai, A. Sharma, S.J. Huh, S. Amin, G.P. Robertson,
presence of EBZ compounds (10 mM final concentration) for the
PBISe,
a novel selenium-containing drug for the treatment of malignant
melanoma, Molecular Cancer Therapeutics 7 (2008) 1297e1308.
[3] L. Wang, J.N. Fu, J.Y. Wang, C.J. Jin, X.Y. Ren, Q. Tan, J. Li, H.W. Yin, K. Xiong,
T.Y. Wang, X.M. Liu, H.H. Zeng, Selenium-containing thioredoxin reductase
inhibitor ethaselen sensitizes non-small cell lung cancer to radiotherapy, Anti-
Cancer Drugs 22 (2011) 732e740.
indicated time periods. Cells were allowed to migrate and heal the
wound. Photomicrographs were taken at each time point in order
to examine the wound healing areas. Remaining areas were
measured and displayed as a bar graph [34].

152
K. Bijian et al. / European Journal of Medicinal Chemistry 48 (2012) 143e152
[4] C. Jiang, W. Jiang, C. Ip, H. Ganther, J. Lu, Selenium-induced inhibition of
[24] V.M. Golubovskaya, R. Finch, W.G. Cance, Direct interaction of the N-terminal
domain of focal adhesion kinase with the N-terminal transactivation domain
of p53, The Journal of Biological Chemistry 280 (2005) 25008e25021.
[25] J.M. Haughian, E.M. Reno, A.M. Thorne, A.P. Bradford, Protein kinase C alpha-
dependent signaling mediates endometrial cancer cell growth and tumori-
genesis, International Journal of Cancer 125 (2009) 2556e2564.
[26] D. Kim, S. Kim, H. Koh, S.O. Yoon, A.S. Chung, K.S. Cho, J. Chung, Akt/PKB
promotes cancer cell invasion via increased motility and metalloproteinase
production, Faseb J 15 (2001) 1953e1962.
[27] E. Kurenova, L.H. Xu, X. Yang, A.S. Baldwin Jr., R.J. Craven, S.K. Hanks, Z.G. Liu,
W.G. Cance, Focal adhesion kinase suppresses apoptosis by binding to the
death domain of receptor-interacting protein, Molecular and Cellular Biology
24 (2004) 4361e4371.
angiogenesis in mammary cancer at chemopreventive levels of intake,
Molecular Carcinogenesis 26 (1999) 213e225.
[5] C. Jiang, H. Ganther, J. Lu, Monomethyl seleniumespecific inhibition of MMP-2
and VEGF expression: implications for angiogenic switch regulation, Molec-
ular Carcinogenesis 29 (2000) 236e250.
[6] A. Bhattacharya, M. Seshadri, S.D. Oven, K. Toth, M.M. Vaughan, Y.M. Rustum,
Tumor vascular maturation and improved drug delivery induced by methyl-
selenocysteine leads to therapeutic synergy with anticancer drugs, Clinical
Cancer Research 14 (2008) 3926e3932.
[7] N. Karelia, D. Desai, J.A. Hengst, S. Amin, S.V. Rudrabhatla, J. Yun, Selenium-
containing analogs of SAHA induce cytotoxicity in lung cancer cells, Bio-
organic & Medicinal Chemistry Letters 20 (2000) 6816e6819.
[8] E.H. Lee, Y.J. Lim, S.K. Ha, T.H. Kang, M. Koketsu, C. Kang, S.Y. Kim, J.H. Park,
[28] P. Liu, H. Cheng, T.M. Roberts, J.J. Zhao, Targeting the phosphoinositide 3-
kinase pathway in cancer, Nature Reviews 8 (2009) 627e644.
Inhibitory effects of 5-chloroacetyl-2-piperidino-1,3-selenazole,
a novel
selenium-containing compound, on skin melanin biosynthesis, The Journal of
Pharmacy and Pharmacology 62 (2010) 352e359.
[9] C. Sanmartin, D. Plano, M. Font, J.A. Palop, Kinase regulation by sulfur and
selenium containing compounds, Current Cancer Drug Targets 11 (2011)
496e523.
[10] A. Wendel, M. Fausel, H. Safayhi, G. Tiegs, R. Otter, A novel biologically active
seleno-organic compoundeII. Activity of PZ 51 in relation to glutathione
peroxidase, Biochemical Pharmacology 33 (1984) 3241e3245.
[11] M. Maiorino, A. Roveri, M. Coassin, F. Ursini, Kinetic mechanism and substrate
specificity of glutathione peroxidase activity of ebselen (PZ51), Biochemical
Pharmacology 37 (1988) 2267e2271.
[29] W.G. Roberts, E. Ung, P. Whalen, B. Cooper, C. Hulford, C. Autry, D. Richter,
E. Emerson, J. Lin, J. Kath, K. Coleman, L. Yao, L. Martinez-Alsina, M. Lorenzen,
M. Berliner, M. Luzzio, N. Patel, E. Schmitt, S. LaGreca, J. Jani, M. Wessel,
E. Marr, M. Griffor, F. Vajdos, Antitumor activity and pharmacology of
a selective focal adhesion kinase inhibitor, PF-562,271, Cancer Research 68
(2008) 1935e1944.
[30] I. Tanjoni, C. Walsh, S. Uryu, A. Tomar, J.O. Nam, A. Mielgo, S.T. Lim, C. Liang,
M. Koenig, C. Sun, N. Patel, C. Kwok, G. McMahon, D.G. Stupack,
D.D. Schlaepfer, PND-1186 FAK inhibitor selectively promotes tumor cell
apoptosis in three-dimensional environments, Cancer Biology & Therapy 9
(2010) 764e777.
[12] H. Masumoto, R. Kissner, W.H. Koppenol, H. Sies, Kinetic study of the reaction
of ebselen with peroxynitrite, FEBS Letters 398 (1996) 179e182.
[13] S.T. Miorelli, R.M. Rosa, D.J. Moura, J.C. Rocha, L.A. Lobo, J.A. Henriques, J. Saffi,
Antioxidant and anti-mutagenic effects of ebselen in yeast and in cultured
mammalian V79 cells, Mutagenesis 23 (2008) 93e99.
[14] M.J. Parnham, E. Graf, Seleno-organic compounds and the therapy of
hydroperoxide-linked pathological conditions, Biochemical Pharmacology 36
(1987) 3095e3102.
[15] M.J. Parnham, S. Kindt, A novel biologically active seleno-organic compounde
III. Effects of PZ 51 (Ebselen) on glutathione peroxidase and secretory activi-
ties of mouse macrophages, Biochemical Pharmacology 33 (1984)
3247e3250.
[16] A.C. Terentis, M. Freewan, T.S. Sempertegui Plaza, M.J. Raftery, R. Stocker,
S.R. Thomas, The selenazal drug ebselen potently inhibits indoleamine 2,3-
dioxygenase by targeting enzyme cysteine residues, Biochemistry 49 (2010)
591e600.
[17] I. Saito, T. Asano, K. Sano, K. Takakura, H. Abe, T. Yoshimoto, H. Kikuchi,
T. Ohta, S. Ishibashi, Neuroprotective effect of an antioxidant, ebselen, in
patients with delayed neurological deficits after aneurysmal subarachnoid
hemorrhage, Neurosurgery 42 (1998) 269e277 discussion 277e268.
[18] T. Yamaguchi, K. Sano, K. Takakura, I. Saito, Y. Shinohara, T. Asano,
H. Yasuhara, Ebselen in acute ischemic stroke: a placebo-controlled, double-
blind clinical trial. Ebselen Study Group, Stroke; A Journal of Cerebral Circu-
lation 29 (1998) 12e17.
[31] P. Boullanger, M. Jouineau, B. Bouammali, D. Lafont, G. Descotes, The use of N-
alkoxycarbonyl derivatives of 2-amino-2-deoxy-D-glucose as donors in
glycosylation reactions, Carbohydrate Research 202 (1990) 151e164.
[32] H. Weidmann, D. Tartler, P. Stockl, L. Binder, H. Honig, [Derivatives of D-
glucosamine. 12. Reactions of 2-acylamido-1,3,4,6-tetra-o-benzoyl-2-desocy-
alpha-D-glucopyranoses with hydrogen bromide], Carbohydrate Research 29
(1973) 135e140.
[33] M. Walther, H.G. Holzhutter, R.J. Kuban, R. Wiesner, J. Rathmann, H. Kuhn, The
inhibition of mammalian 15-lipoxygenases by the anti-inflammatory drug
ebselen: dual-type mechanism involving covalent linkage and alteration of
the iron ligand sphere, Molecular Pharmacology 56 (1999) 196e203.
[34] N. Benlimame, Q. He, S. Jie, D. Xiao, Y.J. Xu, M. Loignon, D.D. Schlaepfer,
M.A. Alaoui-Jamali, FAK signaling is critical for ErbB-2/ErbB-3 receptor
cooperation for oncogenic transformation and invasion, The Journal of Cell
Biology 171 (2005) 505e516.
[35] D.J. Sieg, C.R. Hauck, D. Ilic, C.K. Klingbeil, E. Schaefer, C.H. Damsky,
D.D. Schlaepfer, FAK integrates growth-factor and integrin signals to promote
cell migration, Nature Cell Biology 2 (2000) 249e256.
[36] S.M. Frisch, K. Vuori, E. Ruoslahti, P.Y. Chan-Hui, Control of adhesion-
dependent cell survival by focal adhesion kinase, The Journal of Cell Biology
134 (1996) 793e799.
[37] J.H. Kang, D. Asai, R. Toita, H. Kitazaki, Y. Katayama, Plasma protein kinase C
(PKC){alpha} as a biomarker for the diagnosis of cancers, Carcinogenesis 30
(2009) 1927e1931.
[19] G. Mugesh, W.W. du Mont, H. Sies, Chemistry of biologically important
synthetic organoselenium compounds, Chemical Reviews 101 (2001)
2125e2179.
[20] B.G. Davis, M.A. Robinson, Drug delivery systems based on sugar-
macromolecule conjugates, Current Opinion in Drug Discovery & Develop-
ment 5 (2002) 279e288.
[21] T.E. McCann, N. Kosaka, M. Mitsunaga, P.L. Choyke, J.C. Gildersleeve,
H. Kobayashi, Biodistribution and excretion of monosaccharide-albumin
conjugates measured with in vivo near-infrared fluorescence imaging, Bio-
conjugate Chemistry 21 (2010) 1925e1932.
[38] M. Lahn, G. Kohler, K. Sundell, C. Su, S. Li, B.M. Paterson, T.F. Bumol, Protein kinase
C alpha expression in breast and ovarian cancer, Oncology 67 (2004) 1e10.
[39] K.M. Nicholson, N.G. Anderson, The protein kinase B/Akt signalling pathway in
human malignancy, Cellular Signalling 14 (2002) 381e395.
[40] M. Sawaki, T. Takeda, Y. Ogihara, S. Shibata, Selective Cleavage of the Amido
linkage of Glucopyranosylamine derivatives by Ion-Exchange Resin treatment,
Chemical & Pharmaceutical Bulletin 32 (1984) 3698e3701.
[41] N. Teng, J. Sadoff, A. Bhattacharjee, Lipid
a analog/immunogenic carrier
conjugates, the use thereof as vaccines and polyvalent hyperimmune gam-
maglobulins, 1992.
[22] Z. Zhongwei, R. Sumei, W. Shengbiao, L. Wei, J. Tao, Synthesis of d-glucos-
amine-modified benzo[d][1,2]selenazol-3-(2H)-one derivatives, Synthetic
Communications 40 (2010) 3438e3446.
[42] N. Kamigata, I. H., I. A., K. M., Photochemical reaction of 2-aryl-1,2-benziso-
selenazol-3(2H)-ones, Bulletin of the Chemical Society of Japan 59 (1986)
2179e2183.
[23] E. Behmoaram, K. Bijian, S. Jie, Y. Xu, A. Darnel, T.A. Bismar, M.A. Alaoui-
[43] http://www.invitrogen.com/etc/medialib/en/filelibrary/Services/Discovery-
Research/PDFs.Par.89800.File.dat/SSBK-ZLYTE-Assay-Conditions-and-
Protocols.pdf.
[44] J.W. Nissink, C. Murray, M. Hartshorn, M.L. Verdonk, J.C. Cole, R. Taylor, A new
test set for validating predictions of protein-ligand interaction, Proteins 49
(2002) 457e471.
Jamali, Focal adhesion kinase-related proline-rich tyrosine kinase
2 and
focal adhesion kinase are co-overexpressed in early-stage and invasive
ErbB-2-positive breast cancer and cooperate for breast cancer cell tumori-
genesis and invasiveness, The American Journal of Pathology 173 (2008)
1540e1550.