Synthesis, structure and cytotoxicity of 3-C, N, S, Se substituted benzo[b]selenophene derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.008

Synthesis, molecular structure and cytotoxic activity of a series of 3-C, N, S, Se substituted benzo[b]selenophene derivatives on human fibrosarcoma HT-1080, mouse hepatoma MG-22A, and mouse fibroblasts 3T3 cell lines are described. The correlation betwee

Full text of DOI:10.1016/j.ejmech.2011.05.008

European Journal of Medicinal Chemistry 46 (2011) 3434e3443
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structure and cytotoxicity of 3-C, N, S, Se substituted benzo[b]
selenophene derivatives
*
Pavel Arsenyan , Edgars Paegle, Sergey Belyakov, Irina Shestakova, Elina Jaschenko, Ilona Domracheva,
Juris Popelis
Department of Medicinal Chemistry, Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006 Riga, Latvia
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, molecular structure and cytotoxic activity of a series of 3-C, N, S, Se substituted benzo[b]
selenophene derivatives on human fibrosarcoma HT-1080, mouse hepatoma MG-22A, and mouse
fibroblasts 3T3 cell lines are described. The correlation between compound LD50 3T3 fibroblast cell line
and HT-1080 morphology was shown.
Received 27 January 2011
Received in revised form
3 May 2011
Accepted 4 May 2011
Available online 13 May 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Arylation
Amination
Benzo[b]selenophene
Cytotoxic activity
Morphology
Selenium
X-ray
1. Introduction
depending on the chemical form of selenium, the nature of the
carcinogenic process, and its dosage. There was no significant
The interest of benzothiophene derivatives stems from the fact
that this molecule is found as a chromophore core for drugs.
Raloxifene and Arzoxifene are benzothiophene selective estrogen
receptor modulators (SERMs) of clinical use in postmenopausal
osteoporosis and treatment of breast cancer and potentially in
hormone replacement therapy [1e6]. Zileuton is an orally active
inhibitor of 5-lipoxygenase, and thus inhibits leukotrienes forma-
tion. It is used for the maintenance treatment of asthma. Benzo-
thiophene piperazines and piperidines are fatty acid amide
hydrolase (FAAH) inhibitors [7]. Also, it has been shown that
3-thiosubstituted benzothiophenes are serotonin receptors
modulators [8]. On the other hand, selenium has attracted great
interest as an essential element and certain diseases have been
eradicated by dietary supplementation of this element. Selenium is
essential for cell metabolism as a component of glutathione
peroxidase and other enzyme systems. Current interest lies in the
prevention of certain cancers by supplementation with selenium
[9e11]. Selenium appears to operate by several mechanisms
difference in the potency of selenate, selenite, selenium dioxide,
selenomethionine and selenocysteine to inhibit the development of
mammary tumors, drug-resistant and drug non-resistant human
ovarian tumor cells [12]. Taking into account the importance of the
selenium as a trace element [13e17]in the organism our present
investigation is connected with the elaboration of synthetic
protocols and cytotoxic activity studies in a series of 3-C, N, S, Se
substituted benzo[b]selenophene derivatives.
2. Results and discussion
2.1. Chemistry
Our synthetic strategy for the insertion of aryl and hetaryl
groups in position 3 of benzo[b]selenophene 1 was based on Suzuki,
Stille, and Sonogashira type coupling reactions. Interactions of 1
with various aryl(hetaryl)boronic acids, hetaryl stannanes and
terminal acetylenes are presented in Scheme 1. After the optimi-
zation of the reaction conditions it has been found that arylboronic
acids readily react with 3-bromobenzo[b]selenophene-2-
carboxylic acid ethyl ester (1) in the presence of in situ prepared
* Corresponding author. Tel.: þ371 9849464.
E-mail address: pavel.arsenyan@lycos.com (P. Arsenyan).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.008

P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
3435
R
2 R=H (65%)
3 R=OCH(CH3)2 (97%)
R
4 R=CN (76%)
F
COOEt
F
Se
Br
F
COOEt
Se
a
a
b or c
9
R=Ph, (b, 65%; c, 28%)
COOEt
10 R=1-cyclohexenyl
Se
(b, 41%, c, 67%)
5 (89%)
COOEt
Se
S
a
b
1
N
b
COOEt
Se
COOEt
6 (79%)
Se
S
8 (75%)
COOEt
Se
7 (79%)
Scheme 1. Synthesis of benzo[b]selenophenes 2e10. Reagents and reaction conditions: (a) aryl(hetaryl)boronic acid (2 eq.), Pd(OAc)2 (10%), tri(o-tolyl)phosphine (20%), potassium
phosphate (3.4 eq.), xylene/EtOH, 1e6 h; (b) hetaryl stannane (1.4 eq.), (Ph3P)4Pd (4%), Ph3As (7%), xylene, 110 ꢀC, 2e24 h; (c) terminal acetylene (1.46 eq.), PdCl2 (5%), Ph3P (10%),
CuI (10%), DMF/TEA, 100 ꢀC, 20 h.
palladium catalyst (Pd(OAc)2 and tri(o-tolyl)phosphine) using
potassium phosphate as a base. According to GC MS data, reaction
completed after heating in xylene/ethanol media (10:1) at 110 ꢀC in
half an hour and corresponding 3-aryl(hetaryl)benzo[b]seleno-
phenes 2e6 were obtained in good yields (65e97%). It should be
noted that almost quantitative yield was obtained in the case of
4-iso-propoxyphenyl boronic acid due to arylboronic acids with
EWG groups are more stable in basic media [18]. Molecular struc-
tures of the compounds 3 and 4 are shown in Fig.1A and B (Table 1).
Both molecules in these crystal structures are characterized by two
planar fragments: one of them is benzoselenophene system with
ester group and the second is the phenyl ring. The dihedral angles
between these fragments are equal to 70.8(4) and 65.7(5)ꢀ for 3 and
4, respectively. Despite the similarity of molecular forms and sizes,
the molecular packing in crystals of 3 and 4 is different: the crystal
lattice of 3 is characterized by monoclinic space group P 21/n, whilst
for 4 there is triclinic lattice (space group P1).
3-(5-Methylthienyl)- (7) and 3-(2-pyridyl)benzo[b]seleno-
phenes (8) were prepared using Stille coupling protocol, because
2-thiophene and 2-pyridine boronic acids are expensive and not
stable in basic media. Typically, the Stille coupling of aryl halides
with aryl stannanes is performed under anhydrous conditions in
aprotic solvents. Xylene is used as a solvent to increase the
temperature of reaction. One day heating of corresponding stan-
nanes with 1 in the presence of tetrakis(triphenylphosphino)palla-
dium(0) in xylene at 130 ꢀC leads to the formation of 7 and 8 in
medium yields. With the aim of reducing the reaction time and to
activate the catalyst, a catalytic amount (7%) of triphenylarsine was
added [24] due to the donor-acceptor AsePd bond being longer than
PePd bond. As a result desired derivatives 7 and 8 were obtained in
higher yields (75e79%) after only 2 h of heating. Then we exami-
ned introduction of terminal acetylenes using corresponding
phenyletnynyl- and cyclohexenylethynyl trimethylstannanes under
the Stille protocol. According to experimental data, it was found that
corresponding 3-(phenylethylnyl)benzo[b]selenophene 9 forms in
65% yield after 1 h of heating, but cyclohexenyl analog 10 in
moderate yield (28%). However, during interaction of terminal
acetylenes with 1 under Sonogashira type conditions using classical
(Ph3P)2PdCl2 catalyst in DMF/TEA media 3-(cyclohexenylethynyl)
benzo[b]selenophene (10) received in higher yield (67%) than phe-
nylethynyl analog 9.
With the purpose to introduce amino group inposition 3 of benzo
[b]selenophene 1 ring BuchwaldeHartwig type coupling has been
examined. The benefits of 3-N-substituted benzo[b]selenophenes
stems from the fact that these compounds could be more bioavail-
able over 3-C-substituted analogs. After reaction conditions opti-
mization Pd2dba3 as a source of palladium(0) and xantphos as an
appropriate ligand were chosen. According to our results aniline,
3- and 4-aminopyridines readily react with 1 performing reaction in
xylene at 120 ꢀC using cesium carbonate as a base. Desired 3-(phe-
nylamino)- (11), 3-(3-pyridylamino)- (12), 3-(4-pyridylamino)
benzo[b]selenophenes (13) were obtained in a very good yields,
besides, in the case of 4-pyridylamino derivative in almost quanti-
tative yield (96%) (Scheme 2). Molecular structure of compound 13 is
illustrated in Fig. 1C. The intramolecular hydrogen bond of NH∙∙∙O
type between the amino group and carbonyl oxygen atom O11 was
found in the structure of 13. The hydrogen bond length is 2.825(2) Å
(H15∙∙∙O11 ¼ 2.21 Å, NeH∙∙∙O ¼ 126ꢀ).
Notably, interaction of 1 with 2-aminopyridine under the
same reaction conditions leads to the formation of a polycyclic
derivative 14. Seems that after the routine amination in position
3 of benzo[b]selenophene ring follows electrophilic attack of the
ester group to the nitrogen atom of pyridine ring under the basic
reaction conditions, as
a result 10H-pyrido[1,2-a]benzo[b]

3436
P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
Fig. 1. ORTEP molecular structures of 3 (A), 4 (B), 13 (C), 17 (D), 18 (E), 20 (F), 22 (G), 23 (H), 24 (I), 26 (J), and 28 (K).

P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
3437
Table 1
Crystal data for the studied compounds.
3
4
13
17
18
20
22
Brutto-formula
Molecular weight
Crystal system
a, [Å]
b, [Å]
c, [Å]
C20H20O3Se
387.32
Monoclinic
11.3557(3)
7.5538(3)
21.4719(9)
90.00
94.337(1)
90.00
1836.6(1)
P 21/n
C18H13NO2Se
354.27
C16H14N2O2Se
345.26
C15H6ClF3N2OSe
401.63
C13H7N3OSe
300.18
C13H8N2OSSe
319.24
C17H14O2SSe
361.32
Triclinic
6.4543(3)
9.7467(4)
12.8454(7)
79.760(2)
78.427(2)
84.911(3)
777.86(6)
P1
Orthorhombic
7.5384(1)
19.3170(3)
20.5094(4)
90.00
90.00
90.00
2986.56(8)
P cab
Tetragonal
14.1895(4)
14.1895(4)
27.2710(9)
90.00
90.00
90.00
5490.8(2)
I 41/a
Triclinic
7.4548(2)
11.1499(4)
13.9263(5)
68.178(2)
82.042(2)
80.139(2)
1055.19(6)
P1
Monoclinic
7.3309(2)
12.2720(4)
13.3016(4)
90.00
103.607(2)
90.00
1163.09(6)
P 21/n
Triclinic
7.3643(3)
10.4661(4)
10.9983(4)
64.099(2)
80.341(2)
89.727(2)
749.49(5)
P1
a
b
g
, [ꢀ]
, [ꢀ]
, [ꢀ]
V, [Å3]
Space group
Z
m
4
2
8
16
2.969
1.943
65.0
4
4
2
, [mmꢁ1
]
2.058
1.401
55.0
6904
2.420
1.513
56.0
5818
2.520
1.536
56.0
13081
3.550
1.890
56.0
7098
3.391
1.823
56.0
5043
2.640
1.601
58.0
5617
Density (calc.) [g/cm3]
qmax for data [ꢀ]
Reflection collected
2
8506
Independent reflections 4200 (Rint ¼ 0.076) 3680 (Rint ¼ 0.030) 3592(Rint ¼ 0.029) 4982(Rint ¼ 0.066) 4856(Rint ¼ 0.034) 2764(Rint ¼ 0.032) 3943(Rint ¼ 0.030)
Reflections with I > n
Final R-factor
s
(I) 2678 (n ¼ 2)
2891 (n ¼ 3)
0.037
2806 (n ¼ 3)
0.028
2908 (n ¼ 2)
0.049
3332 (n ¼ 3)
0.034
2180 (n ¼ 3)
0.061
3127 (n ¼ 3)
0.034
0.078
wR2 index for all data
Temperature, [K]
Using programs
0.294
293
[19,20]
0.081
293
[20,21]
0.180
173
[19,20]
0.141
173
[22]
0.080
193
[19,20]
0.201
193
[20,23]
0.169
173
[20,23]
CCDC deposition number CCDC 802935
CCDC 802936
CCDC 804882
CCDC 804883
CCDC 804884
CCDC 804885
CCDC 802440
23
24
26
28
Brutto-formula
Molecular weight
Crystal system
a, [Å]
b, [Å]
c, [Å]
C17H14O2Se2
408.22
C16H13NO2SSe
362.31
C15H14N2O2SSe
C22H18O4SSe2
536.37
365.31
Monoclinic
7.4986(1)
24.4261(5)
8.9567(2)
90.00
112.780(1)
90.00
1163.09(6)
P 21/a
Triclinic
7.6940(2)
10.3688(3)
19.6867(7)
83.394(1)
79.378(1)
83.249(2)
1525.91(8)
P1
Triclinic
7.7695(2)
9.8761(4)
10.5483(4)
76.160(2)
69.384(2)
77.368(2)
727.47(4)
P1
Triclinic
10.8102(3)
11.5709(3)
17.3054(6)
104.254(1)
100.547(2)
97.577(1)
2026.7(1)
P1
a
b
g
, [ꢀ]
, [ꢀ]
, [ꢀ]
V, [Å3]
Space group
Z
m
4
2
4
4
, [mmꢁ1
]
4.847
1.777
57.0
2.727
1.654
55.0
2.625
1.604
58.0
2.640
1.601
55.0
Density (calc.) [g/cm3]
2
qmax for data [ꢀ]
Reflection collected
10342
4866
6716
13720
Independent reflections
7569(Rint ¼ 0.031)
5294 (n ¼ 3)
0.036
3307 (Rint ¼ 0.035)
2385 (n ¼ 3)
0.029
4007(Rint ¼ 0.032)
3056 (n ¼ 3)
0.031
9206(Rint ¼ 0.046)
5090 (n ¼ 3)
0.046
Reflections with I > n
Final R-factor
s(I)
wR2 index for all data
Temperature, [K]
Using programs
0.101
153
[19,20]
0.166
153
[19,20]
0.148
153
[20,23]
0.160
183
[19,20]
CCDC deposition number
CCDC 805117
CCDC 802441
CCDC 802442
CCDC 802443
selenopheno[3,2-d]pyrimidin-10-one 14 was formed in a very
good yield (87%).in a very good yield (87%). To improve a method
we made the same synthetic protocol using 6-methyl-, 4-chloro-,
and 3-chloro-4-trifluoromethyl-2-aminopyridines, the corre-
sponding fused benzo[b]selenophenes 15e17 were obtained.
Notably, reaction of 1 with 2-amino-1,4-pyridazine leads to
formation of 10H-8-pyrazino-[1,2-a]benzo[b]selenopheno[3,2-d]
pyrimidin-10-one (18) in 70% yield. Also, fused 8-selena-1,6a,13-
triaza-indeno[2,1-b]phenanthren-7-one (19) was prepared very
successfully using 8-aminonaphtyridine-1,7 in impressive yield
(74%). It should be noted that current protocol works well also
in the case of five member heterocycles. Our attempts to utilize
2-amino-5-methylthiazole and 2-aminobenzothiazole led to the
formation of the corresponding 7-methyl-9H-thiazolo[3,2-a]
benzo[b]selenopheno[3,2-d]pyrimidin-9-one (20) and 9H-benz-
thiazolo[3,2-a]benzo[b]selenopheno[3,2-d]pyrimidin-9-one (21)
in good yields (66% and 83%, correspondingly). Molecular struc-
tures of the compounds 17, 18, and 20 are given in Fig. 1D, E and F.
The condensed heterocyclic system in molecules 17, 18, and 20 is
characterized by the planar conformation. The crystal structure of
these compounds is totally different. There is intermolecular
stacking interaction between molecules in the crystal structure
20; the distance between the centroid of phenyl ring C4, C5, C6,
C7, C8, C9 and hydrogen atom H18A of methylgroup is 2.50 Å. By
means of these interactions the centrosymmetrical dimers (space
group P 21/n) are formed in the crystal structure. In the crystal
structure 18 (space group P1) there are two independent mole-
cules (a and b) in asymmetric unit; the intermolecular contact
C7a∙∙∙O11b [3.300(3) Å] can describe as a weak hydrogen bond
of C∙∙∙O type (H7a∙∙∙O11b ¼ 2.56 Å, C7aeH7a∙∙∙O11b ¼ 134ꢀ).
In the high symmetrical (space group I 41/a) crystal structure of
17 there is shortened contact (2.922(4) Å) between fluorine
atoms F23 of the centrosymmetrically connected molecules.
For the next step of our investigation we have focused on
incorporation of sulfur and selenium substituent in position 3 of
benzo[b]selenophene 1 ring (Scheme 3). As a model we chosen
thiophenol and optimized reaction conditions. Unfortunately,
according to experimental data well known copper iodide mediated

3438
P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
R
H
N
COOEt
Se
S
N
11 R=Ph (77%)
12 R=3-Py (81%)
13 R=4-Py (96%)
N
N
N
O
Se
O
Se
14 (87%)
Br
21 (83%)
COOEt
Se
R3
N
X
1
R2
S
N
R1
N
N
N
O
Se
N
N
O
15 R1=CH3 (61%)
16 R2=Cl (15%)
17 R2=CF3 R3=Cl (54%)
18 X=N (70%)
Se
20 (66%)
O
Se
19 (74%)
Scheme 2. Synthesis of benzo[b]selenophenes 11e21. Reagents and reaction conditions: arylamine (1.3 eq.), Pd2dba3 (5%), xantphos (10%), Cs2CO3 (2.7 eq.), xylene, 120 ꢀC, 20 h.
cross coupling leads to the formation of 3-phenylthiobenzo[b]
selenophene-2-carboxylic acid ethyl ester (22) in a very poor yield.
Better result was obtained using 2,2,6,6-tetramethylheptane-
3,5-dione as a ligand (21%). However, it has been found that inter-
action of thiophenol with 1 in dry acetonitrile in the presence of
freshly prepared potassium fluoride on Al2O3 and 18-crown-6
proceeds in moderate yield (47%) after overnight heating at 80 ꢀC.
The general advantage of this copper free method is an experimental
simplicity and low price of a catalyst. 3-Phenylselenylbenzo[b]
selenophene-2-carboxylic acid ethyl ester (23) was prepared under
the same reaction conditions from selenolophenol in even better
yield (64%) than corresponding sulfur analog 22. The Fig. 1G and H
illustrate the molecular structures of 22 and 23. The geometrical
parameters in the molecule 22 are near to corresponding values in
23. In 22 the C3eS15eC16 valence angle value is 104.0(1)ꢀ, in 23 the
mean value of C3eSe15eC16 angle is equal 100.3(1)ꢀ. However, the
crystal structures of 22 and 23 are not isomorphous: for 22 there is
one molecule in the asymmetric unit, while for 23 there are two
molecules connected by pseudoinversion centre.
Heterocyclic thiols also interact with 1 in the presence of KF/
Al2O3. We have been very satisfied when 3-(2-pyridylthio)benzo[b]
selenophene-2-carboxylic acid ethyl ester (24) was successfully
isolated from the reaction of 1 with 2-thiopyridine in 43% yield
under the same synthetic protocol. Besides, 3-(2-pyrimidylthio)
benzo[b]selenophene-2-carboxylic acid ethyl ester (25) and 3-[2-
(N-methyl)imidazolylthio]benzo[b]selenophene-2-carboxylic acid
Ph
H
N
Br
XPh
COOEt
S
COOEt
COOEt
Se
Se
Se
1
27
22 X=S (47%)
23 X=Se (64%)
Se
COOEt
SR
S
COOEt
Se
COOEt
24 R=2-Pyridyl (43%)
Se
28
25 R=2-Pyrimidyl (55%)
26 R=2-(N-Methylimidazolyl) (68%)
Scheme 3. Synthesis of benzo[b]selenophenes 22e28. Reagents and reaction conditions: thiol, thioamide or selenol (1.3 eq.), KF/Al2O3 (3 eq.), 18-crown-6 (30%), MeCN, 80 ꢀC, 20 h.

P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
3439
ethyl ester (26) were obtained in even higher yields (55% and 68%,
correspondingly). Molecular structures of the compounds 24 and
26 are shown in Fig. 1I and J. These molecules considerably differ by
conformations. The molecular conformation of these molecule can
be characterized by the torsion angle of C3eS15eC16eN17; this
angle is equal ꢁ18.0(3)ꢀ for 24 and 126.4(2)ꢀ for 24. The crystal
structure of 24 is isomorphous to one of 22.
Finally, we tried to incorporate thiobenzamide moiety in the
position 3 of benzo[b]selenophene 1 ring. After overnight heating
of 1 with thiobenzamide in acetonitrile in the presence of potas-
sium fluoride on Al2O3 diethyl 3,30-thiodibenzo[b]selenophene-
2-carboxylate (28) was isolated from the reaction mixture as a sole
product. Seems, that the introduction of thiobenzamide in the
position 3 of 1 proceeds very slowly, but interaction of unreacted 1
with intermediate 27 goes much faster; due to benzimido fragment
could be a good leaving group under the reaction conditions. As
a result, symmetrical sulfide 28 was obtained in 42% yield. The
Fig. 1K shows the molecular structures of 28. In this structure there
are two independent molecules in the asymmetric unit; these
molecules are connected by centre of pseudoinversion. The mean
value of C3eS1eC23 valence angle is 105.1(1)ꢀ.
effect on tumor cell lines (IC50 ¼ 2.75O3.5
m
g/mL on HT-1080 and
MG-22A), however, insertion of the additional nitrogen atom in the
molecule (compound 18) leads to the complete activity disap-
pearance. It should be noted that in the same concentrations benzo
[b]selenophenes 14e17 selectively acts against tumor and normal
mouse fibroblast (3T3) cells. Inspection of LD50 data shows that all
derivatives studied exhibit medium or low acute toxicity
(362e2216 mg/kg). The role of NO in biosystems has attracted
considerable attention in the last decade. NO is formed by enzy-
matic and non-enzymatic mechanisms. Because of its molecular
weight and high lipophilicity, NO has good diffusion properties. It
may act not only in the cell where it is produced, but also in nearby
tissues. Biologically produced NO originates from oxygen and L-
arginine in the reaction catalyzed by NO synthase. NO, a long-lived
radical with a wide range of actions, is known as a regulator of
a variety of biological processes [27]. The NO level was determined
according [28], NO release was defined using the Greyss reagent (by
NO2 concentration in the cultural medium). The yield of nitrite was
expressed as NO2 nmol/200
for 100% alive cells after CV coloration assay (benzo[b]seleno-
phenes concentration 100 g/mL). It was shown (Table 2) that
mL of cultural medium in testing plates
m
3-(5-methylthienyl)- 7 and 3-(cyclohexen-1-yl) benzo[b]seleno-
phene 10 readily increase NO concentration in the cultural medium
(TG100 ¼ 200e350%) on HT-1080 and MG-22A cell lines. However,
series of cytotoxic 3-N-polycyclic benzo[b]selenophenes 14e17 and
21 exhibit very strong NO radical protector activity on both tumor
cell lines (TG100 ¼ 2e40%).
2.2. In vitro
The results of these experiments are summarized in Table 2. The
initial 3-bromobenzo[b]selenophene
1 exhibits no cytotoxic
activity on HT-1080 and slight activity on mouse hepatoma
MG-22A tumor cell line. In a series of 3-C-substituted benzo[b]
selenophenes 2e10 3-(5-methylthienyl)benzo[b]selenophene (7)
and 3-ethynyl derivatives 9 and 10 show medium in vitro activity on
tumor cell lines. Analog 7 containing thiophene cycle shows the
2.3. Morphology
The influence of the studied benzo[b]selenophenes on the
phenotype of mouse fibroblasts 3T3 and human fibrosarcoma HT-
1080 was examined (Fig. 2). Figures in the table show the
morphological changes after 72 h at 30 ꢀC (visualization by acridine
orange). Our experimental data shows that morphology of benzo[b]
selenophenes on 3T3 cells correlates with cytotoxic data on 3T3 cell
line. Fig. 2A and B shows the morphological structure of HT-1080
and 3T3 cells, correspondingly (control). The majority of cells
were alive and the cell nucleus was a dark color. The inspection of
benzo[b]selenophenes influence on 3T3 cell line morphology let us
conclude that cytotoxic compounds 14 and 16 induce a slight
apoptosis of normal cells (Fig. 2D and F). Besides, derivatives 14 and
16 changed the HT-1080 cell phenotype and powerful cell apoptosis
with cell nucleus fragmentation was occurred (Fig. 2C and E). The
insertion of trifluoromethyl substituent into molecule (17) signifi-
cantly changed the type of action (Fig. 2G), tumor cells undergoing
mitochondrial apoptosis with the change of HT-1080 cell pheno-
type. Surprisingly, benzothiazole fragment containing derivative 21
evoke gentle apoptosis without the change of cell morphology
(Fig. 2I).
highest cytotoxicity (IC50
¼
16
mg/mL on HT-1080 and
IC50 ¼ 19.5 g/mL on MG-22A). According to our experiments
m
polycyclic derivatives 14e21 exhibit more extended activity against
HT-1080 and MG-22A tumor cell lines. Possibly, it stems from the
fact that their structure recalls known antitumoral agent Batracylin
[25,26]. In fact, 10H-pyrido[1,2-a]benzo[b]selenopheno[3,2-d]pyr-
imidin-10-one (14) as well as 4-chloro- (16) and 3-chloro-4-
trifluoromethyl-2-aminopyridine (17) show a very good cytotoxic
Table 2
In Vitro cytotoxicity in monolayer tumor cell lines [HT-1080 (human fibrosarcoma),
MG-22A (mice hepatoma), NIH 3T3 (normal mouse fibroblasts) caused by benzo[b]
selenophenesa.
a
Compound HT-TD50
1080 NO MG-TD50 22A NO NIH 3T3 LD50,
100%b
100%
TD50
mg/kg
c
1
2
3
4
5
6
7
9
10
12
14
16
17
18
19
20
21
25
14
25
9
8
6
13
350
15
200
12
5
3
2
2
20
2
40.5
40
23
15
7
12
14
350
10
200
9
896
16
2158
362
>2000
1984
881
503
349
1872
858
2313
470
c
65
c
c
c
c
c
c
c
684
100
31
81.5
c
3. Experimental section
16
76
19.5
87
15
614
120
1000
31
3.1. General
32.5
32
c
c
1H, 13C and 77Se NMR spectra were recorded on a Varian 400
Mercury spectrometer at 400, 100.3 and 39.74 MHz correspond-
ingly at 303 K in CDCl3/TMS or DMSO-d6 solution. The 1H chemical
shifts are given relative to TMS, 13C e relative to chloroform or
DMSO, and 77Se e relative to dimethyl selenide. The melting points
were determined on a “Digital melting point analyser” (Fisher), the
results are given without correction. Diffraction data were collected
on a Nonius KappaCCD diffractometer using graphite mono-
3
3
3
3
7
40
8
32
31
500
554
3.5
2.75
c
c
c
c
7
27
444
c
c
3
56
665
4
21
412
8.5
2
8
9.5
3
27
487
c
c
7
840
2216
a
TD50 - Concentration (
NO Concentration (%) (CV: coloration).
no cytotoxic effect.
m
g/mL) providing 50% cell killing effect [(CV þ MTT)/2].
b
c
chromated Mo-K
were solved by direct methods and refined by full-matrix least
a
radiation (
l
¼ 0.71073 Å). The crystal structures

3440
P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
Fig. 2. View of HT-1080 (A) and 3T3 cells (B) visualized by staining the cellular DNA with the dye acridine orange. Living cells exhibit a green color, cells with orange or yellow color
are apoptotic; (C) view of HT-1080 with 14; (D) view of 3T3 with 14; (E) view of HT-1080 with 16; (F) view of 3T3 with 16; (G) view of HT-1080 with 17; (H) view of 3T3 with 17; (I)
view of HT-1080 with 21; (J) view of 3T3 with 21.
squares. The main crystallographic data and refinement parameters
of the crystal structures are listed in Table 1. For further details, see
crystallographic data for these compounds deposited with the
Cambridge Crystallographic Data Centre as Supplementary Publi-
cations. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
3.2.3. 3-(4-Cyanophenyl)benzo[b]selenophene-2-carboxylic acid
ethyl ester (4)
Yield: 76%, mp ¼ 147e148 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.20 (3H, t, CH3, J ¼ 7.2 Hz); 4.20 (2H, q, CH2, J ¼ 7.2 Hz);
7.31e7.52 (5H, m, 5 ꢂ ArH); 7.75e7.82 (2H, m, 2 ꢂ ArH); 7.92e7.99
(1H, m, ArH). 13C NMR (100.6 MHz, CDCl3/TMS)
d
(ppm): 13.9; 61.6;
111.7; 118.7; 125.2; 125.7; 126.8; 127.4; 130.3; 131.7; 132.5; 141.4;
141.9; 142.1; 144.5; 163.1. 77Se NMR (39.74 MHz, CDCl3),
555.17. Anal. (C18H13NO2Se) C, H, N.
d (ppm):
3.2. General procedure for the preparation of benzo[b]selenophenes
2e6
3.2.4. 3-(3,4,5-Trifluorophenyl)benzo[b]selenophene-2-carboxylic
acid ethyl ester (5)
A vial charged with 1 (0.25 g, 0.753 mmol), palladium(II) acetate
(17 mg, 0.0753 mmol), tri(o-tolyl)phosphine (69 mg, 0.226 mmol)
and potassium phosphate (543 mg, 2.56 mmol) in 5 mL of xylene
under argon atmosphere was stirred for 10 min at 40 ꢀC. Then the
corresponding aryl(hetaryl)boronic acid (1.28 mmol) in abs.
ethanol (0.5 mL) was added and the resulting mixture was heated
at 110 ꢀC for 1e6 h. After usual workup crude product was purified
by flash chromatography on silica gel using the mixture petroleum
ether e ethylacetate as eluent.
Yield: 89%, mp ¼ 113e115 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.25 (3H, t, CH3, J ¼ 7.0 Hz); 4.24 (2H, q, CH2, J ¼ 7.0 Hz);
6.91e7.08 (2H, m, 2 ꢂ ArH); 7.33e7.51 (3H, m, 3 ꢂ ArH); 7.91e7.99
(1H, m, ArH). Anal. (C17H11F3O2Se) C, H.
3.2.5. 3-(Thien-3-yl)benzo[b]selenophene-2-carboxylic acid ethyl
ester (6)
Yield: 79%, mp ¼ 74e76 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.21 (3H, t, CH3, J ¼ 7.2 Hz); 4.21 (2H, q, CH2, J ¼ 7.2 Hz);
3.2.1. 3-Phenylbenzo[b]selenophene-2-carboxylic acid ethyl ester (2)
Yield: 65%, mp ¼ 68e69 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
7.14 (1H, dd, ArH, J ¼ 0.8 Hz, J ¼ 4.8 Hz); 7.31e7.43 (4H, m,
4 ꢂ ArH); 7.55e7.61 (1H, m, ArH), 7.89 (1H, d, ArH, J ¼ 8.0 Hz). 13C
d
(ppm): 1.16 (3H, t, CH3, J ¼ 7.6 Hz); 4.19 (2H, q, CH2, J ¼ 7.6 Hz);
7.28e7.52 (8H, m, 8 ꢂ ArH); 7.90e7.96 (1H, m, ArH). 13C NMR
NMR (100.6 MHz, CDCl3/TMS)
d (ppm): 14.0; 61.3; 124.4; 124.5;
125.0; 125.5; 127.2; 127.5; 129.2; 132.1; 135.4; 141.7; 141.8; 142.8;
(100.6 MHz, CDCl3/TMS)
d (ppm): 13.9; 61.2; 124.9; 125.5; 127.1;
163.6. 77Se NMR (39.74 MHz, CDCl3),
(C15H12O2SSe) C, H.
d (ppm): 547.23. Anal.
127.6; 127.8; 128.0; 129.3; 131.7; 136.3; 142.0; 142.9; 147.0; 163.7. 77Se
NMR (39.74 MHz, CDCl3), (ppm): 544.65. Anal. (C17H14O2Se) C, H.
d
3.2.2. 3-(4-Isopropoxyphenyl)benzo[b]selenophene-2-carboxylic
acid ethyl ester (3)
3.3. General procedure for the preparation of benzo[b]selenophenes
7e10 (Method B)
Yield: 97%, mp ¼ 125e126 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.18 (3H, t, CH3, J ¼ 6.8 Hz); 1.38 (6H, d, 2 ꢂ CH3,
J ¼ 6.0 Hz); 4.18 (2H, q, CH2, J ¼ 6.8 Hz); 4.61 (1H, sept, CH,
J ¼ 6.0 Hz); 6.93e7.00 (2H, m, 2 ꢂ ArH); 7.22e7.27 (2H, m, 2 ꢂ ArH);
7.28e7.34 (1H, m, ArH); 7.35e7.41 (1H, m, ArH); 7.47e7.51 (1H, m,
ArH); 7.86e7.91 (1H, m, ArH). 13C NMR (100.6 MHz, CDCl3/TMS)
A vial charged with 1 (0.25 g, 0.753 mmol), tetrakis(-
triphenylphosphino)palladium(0) (35 mg, 0.03 mmol), tripheny-
larsine (16 mg, 0.053 mmol) and corresponding stannane
(1.05 mmol) in 5 mL of xylene under argon atmosphere was stirred
for 2e24 h at 110ꢀ C. After usual workup crude product was purified
by flash chromatography on silica gel using the mixture petroleum
ether e ethylacetate as eluent.
d
(ppm): 14.0; 22.1; 61.2; 69.8; 115.1; 124.8; 125.5; 127.0; 127.7;
127.9; 130.7; 131.2; 141.9; 143.1; 147.0; 157.7; 163.8. Anal.
(C20H20O3Se) C, H.

P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
3441
3.3.1. 3-(5-Methylthien-2-yl)-benzo[b]selenophene-2-carboxylic
acid ethyl ester (7)
3.5. General procedure for the preparation of benzo[b]selenophenes
11e21
Yield: 79%, mp ¼ 90e91 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.27 (3H, t, CH3, J ¼ 7.2 Hz); 2.56 (3H, d, CH3,
A vial charged with 1 (0.25 g, 0.753 mmol), Pd2dba3 (34 mg,
0.037 mmol), xantphos (39 mg, 0.075 mmol) and arylamine
(0.979 mmol) in 5 mL of xylene was barbotated with Ar for 15 min.
Then cesium carbonate (0.663 g, 2.03 mmol) was added and reac-
tion mixture was heated for 20 h at 120ꢀ C. After usual workup
crude product was purified by flash chromatography on silica gel
using the mixture petroleum ether e ethylacetate as eluent.
J ¼ 0.8 Hz); 4.27 (2H, q, CH2, J ¼ 7.2 Hz); 6.81e6.84 (1H, m, ArH);
6.91 (1H, d, ArH, J ¼ 3.6 Hz); 7.35e7.44 (2H, m, ArH); 7.72e7.75
(1H, m, ArH); 7.88e7.91 (1H, m, ArH). 13C NMR (100.6 MHz,
CDCl3/TMS)
d (ppm): 14.0; 15.4; 61.4; 125.0; 125.3; 125.4; 125.5;
127.2; 127.6; 128.2; 133.0; 133.5; 139.4; 141.0; 141.4; 142.9;
163.3. 77Se NMR (39.74 MHz, CDCl3),
(C16H14O2SSe) C, H.
d (ppm): 551.74. Anal.
3.5.1. 3-Phenylaminobenzo[b]selenophene-2-carboxylic acid ethyl
ester (11)
3.3.2. 3-(2-Pyridyl)-benzo[b]selenophene-2-carboxylic acid ethyl
ester hydrochloride (8)
Yield: 77%, mp ¼ 63e64 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
Free base was converted to hydrochloride salt after purification
d
(ppm): 1.37 (3H, t, CH3, J ¼ 6.8 Hz); 4.33 (2H, q, CH2, J ¼ 6.8 Hz);
using HCl solution in dry methanol. Yield: 75%, mp ¼ 156e157 ꢀC.
6.97e7.06 (3H, m, 3 ꢂ ArH); 7.11e7.17 (1H, m, ArH); 7.20e7.26 (2H,
m, 2 ꢂ ArH); 7.30e7.36 (1H, m, ArH); 7.41e7.45 (1H, m, ArH);
7.77e7.82 (1H, m, ArH); 8.79 (1H, br s, NH). 13C NMR (100.6 MHz,
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 1.07 (3H, t, CH3, J ¼ 7.2 Hz);
4.14 (2H, q, CH2, J ¼ 7.2 Hz), 7.47 (2H, d, 2 ꢂ ArH, J ¼ 3.6 Hz);
7.52e7.60 (1H, m, ArH); 7.90e8.01 (2H, m, 2 ꢂ ArH); 8.29 (1H, d,
ArH, J ¼ 8.0 Hz); 8.39e8.50 (1H, m, ArH); 8.94 (1H, d, ArH,
CDCl3/TMS)
d (ppm): 14.4; 61.0; 108.3; 121.2; 123.0; 123.8; 126.3;
127.5; 127.6; 129.0; 135.1; 140.2; 143.0; 148.3; 166.4. Anal.
(C17H15NO2Se) C, H, N.
J ¼ 4.8 Hz). 13C NMR (100.6 MHz, DMSO-d6)
d (ppm): 13.6; 61.6;
125.2; 125.8; 126.4; 126.5; 127.2; 127.7; 140.7; 141.7; 162.3. Anal.
(C16H14ClNO2Se) C, H, N.
3.5.2. 3-(Pyrid-3-ylamino)benzo[b]selenophene-2-carboxylic acid
ethyl ester (12)
3.3.3. 3-Phenylethynylbenzo[b]selenophene-2-carboxylic acid ethyl
ester (9)
Yield: 81%, mp ¼ 174e175 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.39 (3H, t, CH3, J ¼ 7.2 Hz); 4.36 (2H, q, CH2, J ¼ 7.2 Hz);
Yield: 65%, mp ¼ 68e70 C. 1H NMR (400 MHz, CDCl3/TMS)
7.15 (1H, dd, ArH, J ¼ 4.4 Hz, J ¼ 8.4 Hz); 7.18e7.23 (2H, m, 2 ꢂ ArH);
7.36e7.40 (2H, m, 2 ꢂ ArH); 7.84 (1H, dt, ArH, J ¼ 1.4 Hz, J ¼ 8.4 Hz);
8.29 (1H, dd, ArH, J ¼ 1.4 Hz, J ¼ 4.8 Hz); 8.38 (1H, d, ArH,
J ¼ 2.8 Hz); 8.72 (1H, br s, NH). 13C NMR (100.6 MHz, CDCl3/TMS)
d
(ppm): 1.45 (3H, t, CH3, J ¼ 7.2 Hz); 4.45 (2H, q, CH2, J ¼ 7.2 Hz);
7.38e7.56 (5H, m, 5 ꢂ ArH); 7.66e7.73 (2H, m, 2 ꢂ ArH); 7.87e7.91
(1H, m, ArH); 8.17e8.21 (1H, m, ArH). 13C NMR (100.6 MHz, CDCl3/
TMS)
d
(ppm): 14.4; 61.7; 84.4; 97.7; 122.9; 125.4; 125.6; 126.4;
d (ppm): 14.3; 61.3; 110.9; 123.4; 124.3; 126.5; 126.9; 127.0; 124.8;
127.0; 127.6; 128.4; 128.9; 131.9; 137.7; 141.2; 142.0; 163.2. Anal.
(C19H14O2Se) C, H.
127.9; 134.6; 139.6; 140.2; 142.5; 143.8; 146.7; 166.2. 77Se NMR
(39.74 MHz, CDCl3), (ppm): 474.42. Anal. (C16H14N2O2Se) C, H, N.
d
3.3.4. 3-(Cyclohex-1-enylethynyl)benzo[b]selenophene-2-
carboxylic acid ethyl ester (10)
3.5.3. 3-(Pyrid-4-ylamino)benzo[b]selenophene-2-carboxylic acid
ethyl ester (13)
Yield: 41%, mp ¼ 54e55 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
Yield: 96%, mp ¼ 136e138 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.42 (3H, t, CH3, J ¼ 7.2 Hz); 1.62e1.78 (4H, m, 2 ꢂ CH2);
d
(ppm): 1.37 (3H, t, CH3, J ¼ 7.2 Hz); 4.34 (2H, q, CH2, J ¼ 7.2 Hz);
2.16e2.24 (2H, m, CH2); 2.34e2.40 (2H, m, CH2); 4.41 (2H, q, CH2,
J ¼ 7.2 Hz); 6.40e6.47 (1H, m, CH); 7.38e7.55 (2H, m, ArH);
7.82e7.89 (1H, m, ArH); 8.04e8.10 (1H, m, ArH). 13C NMR
6.73 (2H, d, ArH, J ¼ 5.2 Hz); 7.27e7.32 (1H, m, ArH); 7.39e7.44 (1H,
m, ArH); 7.58 (1H, d, ArH, J ¼ 4.0 Hz); 7.86 (1H, d, ArH, J ¼ 4.0 Hz);
8.31 (1H, d, ArH, J ¼ 5.2 Hz); 8.40 (1H, br s, NH). 13C NMR
(100.6 MHz, CDCl3/TMS)
d
(ppm): 14.4; 21.5; 22.3; 26.0; 29.1; 61.6;
(100.6 MHz, CDCl3/TMS) d (ppm): 14.3; 31.5; 112.6; 115.9; 124.5;
82.0; 100.1; 120.8; 125.2; 125.5; 127.0; 127.1; 127.5; 136.6; 137.1;
141.1; 142.1; 163.3. Anal. (C19H18O2Se) C, H.
126.4; 127.0; 127.9; 135.3; 140.0; 144.2; 150.0; 150.3; 165.5. ESI-
MS: 347.3. Anal. (C16H14N2O2Se) C, H, N.
3.5.4. 11-Selena-5,9a-diazabenzo[b]fluoren-10-one (14)
3.4. General procedure for the preparation of benzo[b]selenophenes
9e10 (Method C)
Yield: 87%, mp ¼ 222e223 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d (ppm): 7.14e7.15 (1H, m, ArH), 7.55e7.57 (2H, m, ArH), 7.67e7.68
(1H, m, ArH), 7.69e7.78 (1H, m, ArH), 7.95e7.97 (1H, m, ArH),
A vial charged with palladium(II) chloride (7 mg, 0.037 mmol),
triphenylphosphine (20 mg, 0.075 mmol) and copper(I) iodide
(14 mg, 0.075 mmol) in 5 mL of DMF was barbotated with Ar for
15 min at 40 ꢀC. Then 1 (0.25 g, 0.753 mmol), terminal acethylene
(1.1 mmol), and triethylamine (1 mL) was added to the reaction
mixture. After 20 h of heating at 100ꢀ C and usual workup crude
product was purified by flash chromatography on silica gel using
the mixture petroleum ether e ethylacetate as eluent.
8.46e8.49 (1H, m, ArH), 9.08e9.10 (1H, m, ArH). 13C NMR
(100.6 MHz, CDCl3/TMS) d (ppm): 114.5; 125.3; 126.1; 126.4; 126.5;
129.8; 134.4; 137.0; 142.0; 149.6; 155.7; 156.9. ESI-MS: 300.9. Anal.
(C14H8N2OSe) C, H, N.
3.5.5. 9-Methyl-11-selena-5,9a-diazabenzo[b]fluoren-10-one (15)
Yield: 61%, mp ¼ 203e205 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 3.13 (3H, s, CH3); 6.62e6.63 (1H, m, ArH); 7.36e7.38 (1H, m,
ArH); 7.48e7.53 (3H, m, ArH); 7.88e7.91 (1H, m, ArH); 8.35e8.38
3.4.1. 3-Phenylethynylbenzo[b]selenophene-2-carboxylic acid ethyl
(1H, m, ArH). 13C NMR (100.6 MHz, CDCl3/TMS)
d
(ppm): 25.1; 117.6;
ester (9)
125.2; 125.4; 125.9; 126.4; 129.5; 133.6; 136.9; 142.0; 143.1; 152.3;
155.5; 159.5. ESI-MS: 315.2. Anal. (C15H10N2OSe) C, H, N.
Yield: 28%.
3.4.2. 3-(Cyclohex-1-enylethynyl)benzo[b]selenophene-2-
carboxylic acid ethyl ester (10)
Yield: 67%.
3.5.6. 11-Selena-5,9a-diaza-8-chlorobenzo[b]fluoren-10-one (16)
Yield: 17%, mp>230 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 7.55e7.67 (3H, m, 3 ꢂ ArH), 7.72e7.76 (1H, m, ArH),

3442
P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
7.95e8.01 (1H, m, ArH), 8.45e8.51 (1H, m, ArH), 9.11e9.14 (1H, m,
ArH). 13C NMR (100.6 MHz, CDCl3/TMS)
(ppm): 123.2; 124.2;
125.5; 126.2; 126.4; 127.4; 130.0; 135.8; 136.8; 142.1; 147.9; 154.8;
3.6.2. 3-Phenylselenobenzo[b]selenophene-2-carboxylic acid ethyl
ester (23)
d
Yield: 64%, mp ¼ 60e61 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
156.7. ESI-MS: 335.0.
d
(ppm): 1.35 (3H, t, CH3, J ¼ 7.2 Hz); 4.36 (2H, q, CH2, J ¼ 7.2 Hz);
7.10e7.18 (3H, m, 3 ꢂ ArH); 7.25e7.31 (3H, m, 3 ꢂ ArH); 7.32e7.38
3.5.7. 6-Chloro-8-trifluoromethyl-11-Selena-5,9a-diazabenzo[b]
fluoren-10-one (17)
(1H, m, ArH); 7.84e7.89 (1H, m, ArH); 7.91e7.96 (1H, m, ArH). 13C
NMR (100.6 MHz, CDCl3/TMS)
d (ppm): 14.2; 61.9; 125.2; 125.5;
Yield: 54%, mp ¼ 215e217 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
126.6; 127.3; 129.0; 129.2; 129.3; 130.8; 132.1; 137.2; 142.1; 143.0;
163.2. Anal. (C17H14O2Se2) C, H.
d
(ppm): 7.56e7.60(2H,m,ArH),7.88(1H,d,J¼ 2, 0Hz,ArH),7.94e7.97
(1H, m, ArH), 8.52e8.55 (1H, m, ArH), 9.30e9.31(1H, m, ArH).13CNMR
(100.6 MHz, CDCl3/TMS) (ppm): 116.9; 117.3; 117.4; 121.0; 123.7;
d
3.6.3. 3-(Pyrid-2-ylthio)benzo[b]selenophene-2-carboxylic acid
ethyl ester (24)
124.9; 125.7; 126.3; 126.7; 128.3; 130.4; 132.4; 136.6; 142.5; 146.0;
155.3; 156.2. ESI-MS: 403.0. Anal. (C15H6ClF3N2OSe) C, H, N.
Yield: 43%, mp ¼ 124e125 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.28 (3H, t, CH3, J ¼ 7.2 Hz); 4.31 (2H, q, CH2, J ¼ 7.2 Hz);
3.5.8. 11-Selena-5,7,9a-triazabenzo[b]fluoren-10-one (18)
6.76e6.80 (1H, m, ArH); 6.92e6.98 (1H, m, ArH); 7.33e7.46 (3H, m,
3 ꢂ ArH); 7.89e7.94 (1H, m, ArH); 7.99e8.04 (1H, m, ArH);
Yield: 70%, mp>230 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 7.61e7.65 (2H, m, ArH), 7.99e8.03 (1H, m, ArH), 8.13 (1H,
8.34e8.38 (1H, m, ArH). 13C NMR (100.6 MHz, CDCl3/TMS)
d (ppm):
d, J ¼ 9.2 Hz, ArH), 8.53e8.57 (1H, m, ArH), 8.75 (1H, d,
J ¼ 12,4 Hz, ArH), 9.25 (1H, s, ArH). ESI-MS: 302.0. Anal.
(C13H7N3OSe) C, H, N.
14.1; 61.9; 119.9; 121.1; 125.5; 125.6; 127.5; 127.6; 130.3; 136.6;
140.5; 141.5; 142.4; 149.5; 159.8; 162.8. Anal. (C17H13NO2SSe)
C, H, N.
3.5.9. 8-Selena-1,6a,13-triaza-indeno [2,1-b]phenanthren-7-one
3.6.4. 3-(2-Pyrimidylthio)benzo[b]selenophene-2-carboxylic acid
ethyl ester (25)
(19)
Yield: 74%, mp>230 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm):
Yield: 55%, mp ¼ 95e97 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
7.28 (1H, d, J ¼ 7.6 Hz, ArH), 7.57e7.64 (2H, m, 2 ꢂ ArH), 7.76 (1H,
dd, J1 ¼ 4.4 Hz, J2 ¼ 8.0 Hz, ArH), 7.97e8.03 (1H, m, ArH), 8.13 (1H,
dd, J1 ¼1.6 Hz, J2 ¼ 8.0 Hz, ArH), 8.80e8.86 (1H, m, ArH), 8.97 (1H, d,
J ¼ 8.0 Hz, ArH), 9.20 (1H, dd, J1 ¼1.6 Hz, J2 ¼ 4.4 Hz, ArH). ESI-MS:
352.0. Anal. (C17H9N3OSe) C, H, N.
d
(ppm): 1.28 (3H, t, CH3, J ¼ 7.2 Hz); 4.30 (2H, q, CH2, J ¼ 7.2 Hz);
6.95 (1H, m, ArH); 7.36e7.51 (2H, m, 2 ꢂ ArH); 7.89e7.98 (1H, m,
ArH); 8.02e8.11 (1H, m, ArH); 8.43 (2H, d, 2 ꢂ ArH, J ¼ 4.6 Hz). 13C
NMR (100.6 MHz, CDCl3/TMS)
d (ppm): 14.1; 61.7; 117.0; 125.3;
125.6; 127.0; 127.4; 129.1; 140.2; 141.3; 142.5; 157.4; 162.7; 171.5.
77Se NMR (39.74 MHz, CDCl3),
(C15H12N2O2SSe) C, H, N.
d
(ppm): 588.14. Anal.
3.5.10. 7-Methyl-9H-thiazolo [3,2-a]benzo[b]selenopheno [3,2-d]
pyrimidin-9-one (20)
Yield: 66%, mp>230 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm):
3.6.5. 3-[2-(N-Methylimidazolylthio)]benzo[b]selenophene-2-
carboxylic acid ethyl ester (26)
2.90 (3H, d, J ¼ 1.2 Hz, CH3), 6.46 (1H, d, J ¼ 1.2 Hz, ArH), 7.49e7.56
(2H, m, 2 ꢂ ArH), 7.89e7.95 (1H, m, ArH), 8.30e8.36 (1H, m, ArH).
Yield: 68%, mp ¼ 105e106 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
13C NMR (100.6 MHz, CDCl3/TMS)
d
(ppm): 18.7; 105.5; 125.3;
d
(ppm): 1.41 (3H, t, CH3, J ¼ 7.6 Hz); 3.56 (3H, s, CH3); 4.40 (2H, q,
125.8; 126.4; 129.3; 135.8; 136.3; 141.9; 155.4; 159.0; 162.6. ESI-
MS: 320.9. Anal. (C13H8N2OSSe) C, H, N.
CH2, J ¼ 7.6 Hz); 6.89 (1H, s, CH); 7.07 (1H, s, CH); 7.33e7.40 (2H, m,
ArH); 7.83e7.85 (1H, m, ArH); 8.05e8.08 (1H, m, ArH). 13C NMR
(100.6 MHz, CDCl3/TMS)
d (ppm): 14.3; 33.8; 61.9; 123.3; 125.5;
3.5.11. 9H-Benzthiazolo [3,2-a]benzo[b]selenopheno [3,2-d]
127.5; 127.6; 129.7; 132.0; 134.9; 138.1; 141.2; 141.8; 162.9. Anal.
(C15H14N2O2SSe) C, H, N.
pyrimidin-9-one (21)
Yield: 83%, mp>230 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d (ppm):
7.49e7.60 (4H, m, 4 ꢂ ArH), 7.70e7.75 (1H, m, ArH), 7.94e7.99 (1H,
m, ArH), 8.37e8.43 (1H, m, ArH), 9.14e9.19 (1H, m, ArH). ESI-MS:
357.1. Anal. (C16H8N2OSSe) C, H, N.
3.6.6. Diethyl 3,3’-thiodibenzo[b]selenophene-2-carboxylate (28)
Yield: 42%, mp ¼ 128e129 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.29 (6H, t, 2 ꢂ CH3, J ¼ 7.2 Hz); 4.28 (4H, q, 2 ꢂ CH2,
J ¼ 7.2 Hz); 7.18e7.25 (2H, m, 2 ꢂ ArH); 7.28e7.35 (2H, m, 2 ꢂ ArH);
3.6. General procedure for the preparation of benzo[b]selenophenes
22e28
7.73e7.77 (2H, m, 2 ꢂ ArH); 7.79e7.83 (2H, m, 2 ꢂ ArH). 13C NMR
(100.6 MHz, CDCl3/TMS) d (ppm): 14.2; 61.8; 125.2; 125.7; 126.6;
127.3; 133.9; 134.5; 140.8; 141.7; 162.9. Anal. (C22H18O4SSe2) C, H.
A vial charged with 1 (0.25 g, 0.753 mmol), KF/Al2O3 (0.7 g) thiol
(1.1 mmol), and 18-crown-6 (0.05 g, 0.225 mmol) in 5 mL of
acetonitrile was heated for 20 h at 80 ꢀC. Then solvent was evap-
orated under reduced pressure and crude product was purified by
flash chromatography on silica gel using the mixture petroleum
ether e ethylacetate as eluent.
3.7. In vitro cytotoxicity assay
Monolayer tumor cell line: HT-1080 (human fibrosarcoma), MG-
22A (mice hepatoma), and NIH 3T3 (normal mouse fibroblasts)
were cultured in standard medium DMEM (Dulbecco’s modified
Eagle’s medium) without an indicator (“Sigma”) supplemented
with 10% heat-inactivated fetal bovine serum (“Sigma”). After the
ampoule was thawed the cells from 1 to 4 passages were used.
About 2e5.104 cells/mL (depending on line nature) were placed in
96-well plates immediately after compounds were added to the
wells. The control cells without test compounds were cultured on
separate plate. The plates were incubated for 72 h, 37 ꢀC, 5% CO2.
The number of surviving cells was determined using tri(4-
dimethylaminophenyl)methyl chloride (Crystal Violet) or 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide (MTT),
3.6.1. 3-Phenylthiobenzo[b]selenophene-2-carboxylic acid ethyl
ester (22)
Yield: 47%, mp ¼ 71e72 ꢀC. 1H NMR (400 MHz, CDCl3/TMS)
d
(ppm): 1.32 (3H, t, CH3, J ¼ 7.2 Hz); 4.33 (2H, q, CH2, J ¼ 7.2 Hz);
7.06e7.18 (5H, m, 8 ꢂ ArH); 7.32e7.7.42 (2H, m, ArH); 7.88e7.89
(1H, m, ArH); 7.96e7.99 (1H, m, ArH). 13C NMR (100.6 MHz,
CDCl3/TMS)
d (ppm): 14.2; 61.9; 125.3; 125.6; 125.7; 127.4; 127.6;
127.7; 128.9; 132.7; 136.7; 139.2; 141.4; 142.3; 162.8. Anal.
(C17H14O2SSe) C, H.

P. Arsenyan et al. / European Journal of Medicinal Chemistry 46 (2011) 3434e3443
3443
[2] D.A. Bradley, A.G. Godfrey, C.R. Schmid, Tetrahedron Lett. 40 (1999)
5155e5159.
[3] T.A. Grese, S. Cho, D.R. Finley, A.G. Godfrey, C.D. Jones, C.W. Lugar III,
M.J. Martin, K. Matsumoto, L.D. Pennington, M.A. Winter, M. Dee Adrian,
H.W. Cole, D.E. Magee, D. Lynn Phillips, E.R. Rowley, L.L. Short, A.L. Glasebrook,
H.U. Bryant, J. Med. Chem. 40 (1997) 146e167.
[4] M.J. Martin, T.A. Grese, A.L. Glasebrook, K. Matsumoto, L.D. Pennington,
D. Lynn Phillips, L.L. Short, Bioorg. Med. Chem. Lett. 7 (1997) 887e892.
[5] A.D. Palkowitz, A.L. Glasebrook, K. Jeff Thrasher, K.L. Hauser, L.L. Short, D. Lynn
Phillips, B.S. Muehl, M. Sato, P.K. Shetler, G.J. Cullinan, T.R. Pell, H.U. Bryant,
J. Med. Chem. 40 (1997) 1407e1416.
[6] C.R. Schmid, J.P. Sluka, K.M. Duke, Tetrahedron Lett. 40 (1999) 675e678.
[7] D.S. Johnson, K. Ahn, S. Kesten, S.E. Lazerwith, Y. Song, M. Morris, L. Fay,
T. Gregory, C. Stiff, J.B. Dunbar Jr., M. Liimatta, D. Beidler, S. Smith,
T.K. Nomanbhoy, B.F. Cravatt, Bioorg. Med. Chem. Lett. 19 (2009) 2865e2869.
[8] J. Kehler, K. Juhl, B. Norgaarg, US2009/0192213 A1, July 30 (2009).
[9] M. Hill, J. Meat, Eur. J. Clin. Nutr. 56 (2002) S36eS41.
and the concentration of nitric oxide (NO) was determined
according to [28]. MTT-test: after incubating with preparations
culture medium was removed and 200
10 mM HEPES was added in each well of the plate, than 20
(2 mg/mL in HBSS) was added. After incubation (3 h, 37 ꢀC, 5% CO2)
the medium with MTT was removed and 200 L DMSO and 25 mL
mL fresh medium with
m
L MTT
m
glycine buffer pH 10.5 were added at once to each sample. The
samples were tested at 540 nm on Anthos HT II photometer. CV-
test: after incubating with preparations cell culture was removed
and 100 ml 1% glutaraldehide in HBSS was added to each well. After
incubation (15 min) the HBSS with glutaraldehide the samples
washed off H2O (1 time) and 0.05% crystal violet were added. After
incubation with dye (15 min) the samples washed off H2O (3 times)
and citrate buffers pH 4.2 and ethanol (1:1) was added. The samples
were tested at 540 nm.
[10] M.A. Moyad, Urology 59 (2002) 9e19.
[11] J.E. Spallholz, Bull. Selenium-Tellurium 05 (2001) 1e6.
[12] P.B. Caffrey, G.D. Frenkel, Cancer Lett. 121 (1997) 177e180.
[13] E. Lukevics, P. Arsenyan, I. Shestakova, I. Domracheva, I. Kanepe, S. Belyakov,
J. Popelis, O. Pudova, Appl. Organometal. Chem. 16 (2002) 228e234.
[14] E. Lukevics, P. Arsenyan, K. Rubina, I. Shestakova, I. Domracheva, A. Nesterova,
J. Popelis, O. Pudova, Appl. Organometal. Chem. 16 (2002) 235e238.
[15] P. Arsenyan, K. Rubina, I. Shestakova, I. Domracheva, Eur. J. Med. Chem. 42
(2007) 635e640.
[16] P. Arsenyan, I. Shestakova, K. Rubina, I. Domracheva, A. Nesterova, K. Vosele,
O. Pudova, E. Lukevics, Eur. J. Pharmacol. 465 (2003) 229e235.
[17] P. Arsenyan, K. Rubina, I. Shestakova, E. Abele, R. Abele, I. Domracheva,
A. Nesterova, J. Popelis, E. Lukevics, Appl. Organometal. Chem. 17 (2003)
825e830.
[18] D.G. Hall (Ed.), Boronic Acids. Structure, Properties, and Preparation of Boronic
Acid Derivatives. Overview of Their Reactions and Applications, Wiley-VCH
Verlag GmbH, 2005.
[19] A. Altomare, M. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, A. Guagliardi,
A. Moliterni, R. Spagna, J. Appl. Cryst. 32 (1999) 115e119.
3.8. Morphology assay
The change in cell morphology caused by benzo[b]selenophenes
was investigated under inverted fluorescence microscope Nikon
ECLIPSE TE 300. Acridine orange stain (Sigma) was used. Stain
solution: 10
mg/mL acridin orange in phosphate buffered saline
(PBS) pH 7.4. After incubating with benzo[b]selenophenes 72 h,
37 ꢀC, 5% CO2 the adherent cells were stained in 96-wells cell
culture plate (Sarsted AG). Cell culture mediums were removed and
40
mL stain solution was added in each well. After 2 min the dye was
removed and samples once washed with PBS. Then 40
mL PBS was
added to the samples and they were investigated under microscope
control. Chromatin condensation in apoptotic cells was visualized
by staining the cellular DNA with the dye acridine orange [29].
Living cells stained green, apoptotic cells orange or yellow, and
necrotic cells red (Fig. 2).
[20] S. Mackay, W. Dong, C. Edwards, A. Henderson, C.J. Gilmore, N. Stewart,
K. Shankland, A. Donald, maXus, Integrated Crystallography Software. Bruker-
Nonius and University of Glasgow, 2003.
[21] A.F. Mishnev, S.V. Belyakov, Krystallografiya 33 (1988) 835e837.
[22] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112e122.
[23] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, M.C. Burla, G. Polidori,
M. Camalli, J. Appl. Cryst 27 (1994) 435e436.
[24] P. Arsenyan, M. Ikaunieks, S. Belyakov, Tetrahedron Lett. 48 (2007) 961e964.
[25] C.M. Martinez-Vitturo, D. Domingues, Tetrahedron Lett. 48 (2007)
1023e1026.
[26] J. Guillaumel, S. Leonce, A. Pierre, P. Renard, B. Pfeiffer, P.B. Arimondo,
C. Monneret, Eur. J. Med. Chem. 41 (2006) 379e386.
[27] G. Bauer, S. Dormann, I. Engelmann, A. Schulz, M. Sarah, Reactive oxygen
species and apoptosis. in: R.G. Cameron, G. Feuer (Eds.), Apoptosis and its
Modulation by Drugs. Springer, Berlin, 2000, pp. 275e318.
Acknowledgment
The financial support for this work provided by ESF project Nr.
2009/0197/1DP/1.1.1.2.0/09/APIA/VIAA/014 and Latvian Council of
Science (09.1588) is gratefully acknowledged.
References
[28] D.J. Fast, R.C. Lynch, R.W. Leu, J. Leucocyt. Biol. 52 (1992) 255e261.
[29] A.S. Loweth, N.G. Morgan, Methods for the study of NO-induced apoptosis in
cultured cells. in: M.A. Titheradge (Ed.), Methods in Molecular Biology, Nitric
Oxide Protocols, vol. 100. Humana Press Inc., Totowa, 1998, pp. 311e320.
[1] J.A. Dodge, C.W. Lugar, S. Cho, J.J. Osborne, D.L. Phillips, A.L. Glasebrook,
C.A. Frolik, Bioorg. Med. Chem. Lett. 7 (1997) 993e996.