Synthesis and pharmacological screening of several aroyl and heteroaroyl selenylacetic acid derivatives as cytotoxic and antiproliferative agents

Article abstract of DOI:10.3390/molecules14093313

The synthesis and cytotoxic activity of a series of twenty six aroyl and heteroaroyl selenylacetic acid derivatives of general formula Ar-CO-Se-CH2-COOH or Heterar-CO-Se-CH₂-COOH are reported. The synthesis was carried out by reaction of acyl chlorides with sodium hydrogen selenide, prepared in situ, and this led to the formation of sodium aroylselenides that subsequently reacted with --bromoacetic acid to produce the corresponding selenylacetic acid derivatives. All of the compounds were tested against a prostate cancer cell line (PC-3) and some of the more active compounds were assessed against a panel of four human cancer cell lines (CCRF-CEM, HTB-54, HT-29, MCF-7) and one mammary gland-derived non-malignant cell line (184B5). Some of the compounds exhibited remarkable cytotoxic and antiproliferative activities against MCF-7 and PC-3 that were higher than those of the reference compounds doxorubicin and etoposide, respectively. For example, in MCF-7 when Ar=phenyl, 3,5-dimethoxyphenyl or benzyl the TGI values were 3.69, 4.18 and 6.19 -M. On the other hand, in PC-3 these compounds showed values of 6.8, 4.0 and 2.9 -M. Furthermore, benzoylselenylacetic acid did not provoke apoptosis nor did it perturb the cell cycle in MCF-7.

Full text of DOI:10.3390/molecules14093313

Molecules 2009, 14, 3313-3338; doi:10.3390/molecules14093313
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Pharmacological Screening of Several Aroyl and
Heteroaroyl Selenylacetic Acid Derivatives as Cytotoxic and
Antiproliferative Agents
Carmen Sanmartín 1,*, Daniel Plano 1, Enrique Domínguez 1, María Font 2, Alfonso Calvo 3,
Celia Prior 3, Ignacio Encío 4 and Juan Antonio Palop 1,*
1
Synthesis Section, Department of Organic and Pharmaceutical Chemistry, University of Navarra,
Irunlarrea, 1, E-31008 Pamplona, Spain
2
Molecular Modeling Section, Department of Organic and Pharmaceutical Chemistry, University of
Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain
3
Oncology Division, Center for Applied Medical Research, CIMA, University of Navarra, Pío XII,
53, E-31008 Pamplona, Spain
4
Department of Health Science, Public University of Navarra, Avda. Barañain, s/n, E-31008
Pamplona, Spain
* Authors to whom correspondence should be addressed; E-mails: sanmartin@unav.es (C.S.),
jpalop@unav.es (J.A.P.)
Received: 1 July 2009; in revised form: 4 August 2009 / Accepted: 27 August 2009 /
Published: 1 September 2009
Abstract: The synthesis and cytotoxic activity of a series of twenty six aroyl and
heteroaroyl selenylacetic acid derivatives of general formula Ar-CO-Se-CH2-COOH or
Heterar-CO-Se-CH2-COOH are reported. The synthesis was carried out by reaction of acyl
chlorides with sodium hydrogen selenide, prepared in situ, and this led to the formation of
sodium aroylselenides that subsequently reacted with α-bromoacetic acid to produce the
corresponding selenylacetic acid derivatives. All of the compounds were tested against a
prostate cancer cell line (PC-3) and some of the more active compounds were assessed
against a panel of four human cancer cell lines (CCRF-CEM, HTB-54, HT-29, MCF-7)
and one mammary gland-derived non-malignant cell line (184B5). Some of the compounds
exhibited remarkable cytotoxic and antiproliferative activities against MCF-7 and PC-3
that were higher than those of the reference compounds doxorubicin and etoposide,
respectively. For example, in MCF-7 when Ar = phenyl, 3,5-dimethoxyphenyl or benzyl

Molecules 2009, 14
the TGI values were 3.69, 4.18 and 6.19 μM. On the other hand, in PC-3 these compounds
3314
showed values of 6.8, 4.0 and 2.9 μM. Furthermore, benzoylselenylacetic acid did not
provoke apoptosis nor did it perturb the cell cycle in MCF-7.
Keywords: selenylacetic acid; cytotoxicity; apoptosis
Introduction
Cancer is currently one of the leading causes of death worldwide and could become the most
common cause in the future [1]. Our understanding of the biology of cancer has undoubtedly improved
in the last decade. One characteristic of cancer cells is their highly proliferative nature. Consequently,
inhibition of proliferative pathways is considered an effective strategy to fight cancer and a great deal
of attention has recently been paid to the discovery and development of new, more selective anticancer
agents. The results of numerous studies indicate that selenium is an essential trace element [2,3] with
structural and enzymatic roles for viral suppression and against AIDS. This element is also implicated
in delaying the aging process. Selenium deficiency has been linked to a number of disorders such as
heart disease, diabetes and cancer. Selenium compounds have attracted renewed interest as
chemopreventive agents for human cancer on the basis of the pioneering intervention study carried out
by Clark and Combs [4]. Later epidemiological and clinical intervention studies support the protective
role of selenium against the development of prostate cancer [5-7]. More recently, mounting evidence
indicates that Se compounds inhibit neoplasm of lung, colon, mammary gland, sarcoma and
glioblastoma [8,9]. In addition, selenium supplementation [10-12] could provide significant
therapeutic potential for patients with gastrointestinal, liver and head and neck cancers.
Furthermore, literature reports have consistently shown that the dose and chemical forms of
selenium [13,14] are determinant factors in anticancer activity, with organic Se compounds being
more bioavailable than the inorganic forms.
Several chemical forms are under investigation for their potential as candidate drugs and these
include methylseleno derivatives such as methylselenocysteine [15], p-xylylbis(methylselenide) [16]
and methylseleninic acid [17], compounds with selenium in the heterocyclic ring as in selenazolidines
[18], ebselen [19], D-501036 [20], BBSKE [21] or the presence of a selenide function in a symmetrical
structure such as diphenyl-, dimethyl-, dibenzylselenide [22-24], bis(3-methyl-4-pyridyl)selenide [25]
and di(3-indolyl)selenide [26].
On the other hand, the metabolism of selenium compounds is critical for activity and at least two
selenium metabolite pools, hydrogen selenide and methylselenol [27,28], that induce distinct types of
antiangiogenesis, apoptosis, and cell cycle responses, have been implicated as active metabolites for
the anticancer effects.
Based on the interesting and widely observed antitumoral activity of selenium compounds and our
own experience in this field [29,30], we report here the synthesis, biological evaluation and molecular
modelling study of a group of selenylacetic acid derivatives 1–26, (Figure 1) as potential agents in
cancer therapy.

Molecules 2009, 14
3315
Figure 1. General formula of compounds.
R: aryl, substituted aryl, arylalkyl, heteroaryl.
Among the strategies adopted for the design of these structures, particular consideration was given
to the following approaches:
(a) The use of the selenide function due to the facile scission of Se from the organic moiety in these
types of compounds. In addition, this proposed preliminary hypothesis concerning the action
mechanism of these derivatives, related to the possible scission of Se from the organic moiety,
allow us to determine the bond order. According to the Molecular Orbital Theory, the bond order
(b.o.) is equivalent to the number of electrons in the antibonding molecular orbital minus the
number of electrons in the bonding molecular orbital divided by two. This parameter can be taken
as a quantitative descriptor for the bond strength and can be related to the aforementioned Se
scission.
(b) The appropriate choice of the substituent on the phenyl ring was made according to a synthetic
accessibility and with the aim of assessing the following aspects:
(b.1) Influence of the substituents on the Se charge and bond stability, as well as on the acidic
character of the derivatives, expressed as pKa.
(b.2) Modulation of the electronic distribution over the aromatic moiety with a variety of electron-
donating and electron-withdrawing substituents, placed at different position with respect to the
lateral chain.
(b.3) Molecular volume, conformational behaviour and hydrophobic character (expressed as
AlogP), considering the presence of the keto moiety, the aromatic/heteroaromatic ring, the
methylene bridge and the carboxylic moiety as the structural basic pattern.
Results and Discussion
Chemistry
The synthesis of the title compounds 1–26 was carried out as depicted in Scheme 1. In the first step,
sodium hydrogen selenide was produced by reaction of powdered grey selenium with metallic
hydrides in an appropriate medium. In the second step, the aroyl or heteroaroyl chloride was reacted
with sodium hydrogen selenide to produce the intermediate sodium aroyl selenides. The last step
involved the formation of selenylacetic acids 1–26 by treatment of this intermediate with α-
bromoacetic acid. This synthesis was based on a published procedure with modifications [31]
following our own protocol.

Molecules 2009, 14
3316
Scheme 1. Synthesis of compounds 1–26.
The compounds were obtained in yields from 3 to 78%. The analytical data are shown in Table 1.
The purity levels of compounds 1–26 were assessed by TLC and elemental analyses and their
structures were identified from spectroscopic data (IR, 1H NMR). The IR spectra of these compounds
revealed intense absorption bands in the ranges 1,720–1,700 cm–1 and 1,690–1,665 cm–1, assigned to
carboxyl and carbonyl groups, respectively. The frequencies of these absorptions varied according to
the substitution pattern on the arylidene ring. As expected, a decrease in the carbonyl absorption
frequencies was observed for compounds having electron-donating groups such as methylenedioxy,
trimethoxy or tert-butyl attached to the benzylidene ring in comparison to electron-withdrawing
groups (chloro, bromo).
Biological Evaluation
Cytotoxic activity in PC-3
Initially, the new compounds 1–26 were evaluated for their in vitro cytotoxic activity against a
human prostate cancer cell line (PC-3, ATCC, Manassas, VA, USA) using the MTT assay [32]. We
selected this cell line because there are numerous clinical trials that show the activity of selenium
compounds in the reduction of prostate cancer [5,9,15,16]. The results are tabulated as IC50 values. All
experiments were independently performed at least three times and the values were calculated after 72
hours exposure (drug concentrations of 2, 5, 7 and 10 μM). The results are shown in Table 2.
The maximum cytotoxic activity was exhibited by compounds 1, 2, 11, 14 and 21. By comparing
the activities of compounds with those of methylseleninic acid and etoposide, both effective drugs
against prostate cancer that have been employed as controls for cytotoxic assays, the results revealed
that three of the new compounds −1, 11, 14 − were more active than methylseleninic acid and
etoposide and that five were better than etoposide. It is noteworthy that the introduction of a new
methoxy groups in the 4-position in 14 to give 15 results in suppression of the antitumour activity on
the PC-3 cell line. These findings, along with the observed antitumour activity of compound 14,
suggest that the activity of the compounds depends on the location of the methoxy substituents on the
phenyl ring. With the aim of evaluating the preliminary structure-activity relationships to gain an
understanding of the activity shown by such compounds as antitumorals, we carried out a molecular
modelling study on the aforementioned derivatives.

Molecules 2009, 14
3317
Table 1. Physical constants for compounds 1–26.
Yield
(%)
75
78
62
19
53
19
M. p.
(ºC)
Ref.
R
Recryst. Solvent
CHN
Anal (%) Calcd/Found
phenyl
4-cyanophenyl
4-(trifluoromethyl)phenyl
4-chlorophenyl
83a
Toluene
Toluene
Toluene
Toluene
Toluene
C9H8O3Se
C10H7NO3Se
C10H7F3O3Se
C9H7ClO3Se
C10H1003Se
C13H16O3Se
C, 44.44/44.47; H, 3.29/3.24; N, 0.00/0.00.
C, 44.77/44.85; H, 2.61/2.67; N, 5.22/4.98.
C, 38.58/38.73; H, 2.25/2.15; N, 0.00/0.00.
C, 38.95/38.71; H, 2.54/2.43; N, 0.00/0.00.
C, 44.69/44.95; H, 3.89/3.89; N, 0.00/0.00.
C, 52.18/52.04; H, 5.39/5.31; N, 0.00/0.05.
1
2
3
4
5
6
147a
108a
135-136b,c
92a
4-methylphenyl
4-(tert-butyl)phenyl
99-103b,c
Chloroform
Chloroform/Carbon
tetrachloride
Carbon tetrachloride
Carbon tetrachloride
Carbon tetrachloride
Toluene
Hexane
Carbon tetrachloride
Toluene
Ethanol
Carbon tetrachloride
Toluene
Carbon tetrachloride
Ether / hexane
Methanol
4-methoxyphenyl
16
104-107b
C10H10O4Se
C, 43.97/43.49; H, 3.69/3.52; N, 0.00/0.00.
7
2-chlorophenyl
2-bromophenyl
2-iodophenyl
44
32
3
123-125b
124-128b
105-108b
74a
C9H7ClO3Se
C9H7BrO3Se
C9H7IO3Se
C, 38.95/38.97; H, 2.54/2.52; N, 0.00/0.06.
C, 33.57/33.14; H, 2.19/2.02; N, 0.00/0.20.
C, 29.29/29.14; H, 1.91/1.92; N, 0.00/0.05.
C, 46.69/46.87; H, 3.89/3.89; N, 0.00/0.00.
C, 47.93/48.06; H, 4.57/4.47; N, 0.00/0.18.
C, 33.67/33.56; H, 2.20/1.84; N, 0.00/0.06.
C, 43.56/43.70; H, 3.96/3.88; N, 0.00/0.00.
C, 43.26/43.32; H, 4.24/3.97; N, 0.00/0.11.
C, 41.83/41.56; H, 2.81/2.67; N, 0.00/0.02.
C, 53.24/52.99; H, 3.41/3.25; N, 0.00/0.00.
C, 56.90/56.88; H, 4.33/4.11; N, 0.00/0.14.
C, 39.36/39.39; H, 2.89/2.78; N, 5.74/5.67.
C, 39.36/39.57; H, 2.89/2.76; N,5.74/5.63.
C, 34.47/34.68; H, 2.15/2.17; N, 5.01/5.26.
C, 41.51/41.28; H, 4.12/3.90; N, 4.40/4.24.
C, 33.75/33.45; H, 2.43/2.54; N, 0.00/0.07.
C, 34.30/34.18; H, 2.47/2.49; N, 1.43/1.37.
C, 49.00/49.33; H, 3.08/3.13; N, 4.76/4.75.
C, 49.00/48.95; H, 3.08/3.23; N, 4.76/4.66.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
benzyl
48
11
17
69
14
47
37
16
11
15
C9H10O3Se
2-phenylethyl
3,5-dichlorophenyl
3,5-dimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4-methylendioxyphenyl
naphthyl
65-69b
C11H12O3S ¼ H2O
C9H6Cl2O3Se·½ H2O
C11H12O5Se
108-109b
117a
107-110b
106-113b
130a
C12H14O6Se
C10H8O5Se
C13H10O3Se
C16H14O3Se
C8H7NO3Se
C8H7NO3Se
C8H6ClNO3Se
C11H13NO3SSe
C7H6O3SSe
C7H6N2O3Se
C12H9NO3Se
C12H9NO3Se
diphenylmethyl
4-pyridyl
127-130b
119-121b,c
147-150b,c
157a
3-pyridyl
3-(2-chloropyridyl)
3-(2-propylthiopyridyl)
2-thienyl
Toluene
36
33
10
6
109-111b
82-84b
Carbon tetrachloride
Carbon tetrachloride
Isopropanol
pyrazinyl
2-quinolyl
3-quinolyl
138-140b
131-132b,c
187-189b,c
Toluene
Chloroform
9
a Determined by differential scanning calorimetry; b Determined by thermomicroscopy; c Fusion with degradation.

Molecules 2009, 14
Table 2. Cytotoxic activities of compounds 1–26 against the PC-3 cell line.
3318
PC-3 cell line
Compound
R
IC50 (μM)
1
phenyl
4-cyanophenyl
4-(trifluoromethyl)phenyl
4-chlorophenyl
4-methylphenyl
4-tert-butylphenyl
4-methoxyphenyl
2-chlorophenyl
2-bromophenyl
2-iodophenyl
6.8
10.0
NEa
NE
2
3
4
5
NE
6
NE
7
8
NE
NE
9
NE
10
NE
11
benzyl
2.9
12
2-phenylethyl
NE
13
3,5-dichlorophenyl
3,5-dimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4-methylenedioxyphenyl
naphthyl
NE
14
4.0
15
NE
16
NE
17
NE
18
diphenylmethyl
4-pyridyl
NE
19
NE
20
3-pyridyl
NE
21
3-(2-chloropyridyl)
3-(2-propylthiopyridyl)
2-thienyl
10.0
NE
22
23
NE
24
pyrazinyl
NE
25
2-quinolyl
NE
26
3-quinolyl
NE
MSAb
8.38 [33]
13.6 ± 2.2 [34]
Etoposide
a NE = no effect is observed; b methylseleninic acid.
Molecular modelling
From the molecular modelling point of view, three approaches were applied in an effort to gain an
insight into the structural requirements for activity in these compounds (Figure 2).

Molecules 2009, 14
Figure 2. Overview of structural variations carried out for the studied derivatives: (a) aryl
3319
or heteroaryl monocyclic derivatives, (b) aryl or heteroaryl bicyclic derivatives.
(a)
(b)
Firstly, it was planned to obtain some descriptive parameter at a molecular level, such as the log of
the partition coefficient expressed as ALogP and the molecular volume, particularly the volume of the
aromatic moiety, that would allow discrimination between active and inactive compounds.
A second approach was carried out. This was based on the pharmacophoric hypothesis that an
active compound can be characterised by a set of chemical functions with a certain spatial arrangement
and molecular shape that could be related to the observed biological activity but would not be
associated with the inactive compounds. Thus, the presence of certain chemical functions is not only
necessary, but these functions must be exposed in a manner that can be recognised by the associated
binding site — a property that requires specific conformational behaviour [35]. In agreement with this
approach, a compound can be considered as a collection of multiple instances, one for each
conformation. An active compound contains at least one active instance, while an inactive compound
does not contain any active instance. On the assumption that all of the active compounds bind to the
same site, we would expect their binding conformations to have similar shapes. For this second

Molecules 2009, 14
3320
approach it is necessary to carry out a study of the conformational behaviour of the compounds and
subsequently select and analyse the supposed bioactive conformations of the compounds.
The third approach considered here concerned the structural characteristics of designed molecules,
especially the presence of the selenium atom, the proposed structural variations related to the length of
the chain, which acts like a bridge between the keto group and the aromatic or heteroaromatic moiety,
and the electronic characteristics of the groups located in these rings. These structural variations led us
to carry out a study from the mechano-quantic point of view in order to obtain data on parameters such
as the value of the partial charge on the selenium, the energy and distribution of the HOMO and
LUMO orbitals as well as the electronic density (ED) and molecular electrostatic potential (MEP)
distribution. The aim was to try to relate these values with the biological activity of our derivatives.
The calculations were performed on a Dell Precision 380 workstation, provided with the software
package Discovery Studio v1.7 [36], and on a SGI Virtu VS100 workstation, provided with
MOPAC2009 [37] and Mercury [38] software packages.
Once the models for the compounds had been constructed, the initial geometries were fully
minimized to an energy gradient below 10–3 kcal mol–1Å–1. The minimum energy conformers were
superimposed, with the ring moiety taken as adjusting atoms. The effectiveness of the superimposed
models was evaluated in terms of the Root Mean Square (RMS) values obtained. The energy
differences between the different conformations analysed for each trajectory were in the range
2–5 kcal.
Analysis of the resulting data (Tables 3 and 4) enabled the following general observations to be
made in an attempt to correlate the data with the cytotoxic activity levels found for the compounds on
the PC-3 cell line.
1. The permissible maximum volume for the aromatic fragment is approximately 111–112 Å3.
Thus, the bicyclic compounds (naphthyl, diphenylmethyl and quinolyl derivatives) and the monocyclic
derivatives with voluminous substituents are inactive.
2. In relation to the conformational behaviour, it can be observed that, in general, in the
conformational trajectory of active compounds the number of extended conformations is greater than
that in the inactive ones, with a better general superposition of the conformations, and the total volume
of the conformational trajectory is also greater (rms data not shown for the sake of brevity). An
example is shown in Figure 3.
3. With respect to the ALogP98 values determined on the preliminary geometries obtained after the
first minimization (Table 3), although a direct correlation could not be established, the resulting data
did shed some light on the biological data, since the active products have a value between 1.518
(compound 21, IC50 = 10.0 μM) and 1.829 (compound 11, the most active compound, IC50 = 2.9 μM).
As expected, some structural modifications give rise to a marked reduction in the AlogP values, as is
the case for compounds 19 and 20, derived from pyridine, with a value of 0.644, or for compound 24,
derived from pyrazine, with a negative value for this parameter. On the other hand, it can be seen that
the compounds with an AlogP greater than 1.90 are inactive in this assay.

Molecules 2009, 14
3321
Table 3. Molecular descriptors obtained for the analyzed compounds (aryl or heteroaryl monocyclic derivatives) a.
O
R6
Y
b
n
d
Se
a
R5
R4
e
c
OH
z
O
X
R2
R3
Ref. X Y Z n R2 R3
R4
R5 R6 AlogP Volb
D)c IC50(M)e
1
2
3
4
5
6
7
8
9
C C C 0
C C C 0
C C C 0
C C C 0
C C C 0
C C C 0
C C C 0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.795 68.58 3.465
6.8
10.0
NE
NE
NE
NE
NE
NE
NE
NE
2.9
CN
CF3
Cl
1.673 82.16 2.865
2.737 92.64 1.137
2.459 83.19 2.628
2.281 82.76 4.430
3.195 125.55 4.584
1.778 89.51 5.337
2.459 82.97 2.874
2.543 91.02 4.032
2.373 100.49 3.259
1.829 82.73 3.418
CH3
C(CH3)3
OCH3
H
C C C 0 Cl
C C C 0 Br
H
H
H
10 C C C 0
11 C C C 1
I
H

Molecules 2009, 14
3322
Table 3. Cont.
Ref.
12
13
14
15
16
19
20
21
22
23
24
X Y Z n
C C C 2
C C C 0
C C C 0
C C C 0
C C C 0
C C N 0
N C C 0
N C C 0
R2
H
H
H
H
H
H
H
Cl
R3
H
Cl
R4
H
H
R5
H
Cl
R6 AlogP Volb
D)c IC50(M)d
H
H
H
H
H
H
H
H
H
2.286 93.29
3.123 98.15
1.762 111.05 4.591
1.745 131.49 5.090
1.563 88.78
0.644 64.45
0.644 64.64
1.518 78.98
4.243
NE
2.441
NE
OCH3
OCH3 OCH3 OCH3
-O-CH2-O-
H
OCH3
4.0
NE
H
H
H
H
H
4.352
1.081
3.543
4.094
NE
NE
NE
10.0
NE
H
H
H
H
-
H
H
H
N C C 0 S(CH2)2CH3
2.597 126.66 4.964
2-thienyl
1.520 64.02
-0.078 60.35
3.315
NE
N N C 0
H
-
-
H
-
-
H
-
-
H
-
-
-
-
-
3.096
NE
MSA
Etoposide
-
-
-
-
-
-
-
-
-
-
-
-
-
-
8.38
13.6 ±2.2
a General structure for the analysed compounds showing the bonds (a–e) selected for the conformational analysis. b Volume (average
value obtained from the lowest energy conformations) of the cyclic fragment in Å 3. c Dipolar moment (in Debyes) calculated for the
representative low-energy. d Cytotoxic activity in PC-3 cell line, NE= no effect.

Molecules 2009, 14
3323
Table 4. Molecular descriptors obtained for the analyzed compounds (aryl or heteroaryl
bicyclic derivatives) a.
O
c
a
Se
R
OH
d
b
O
Ref.
17
18
R
AlogP Volb
2.703 106.65 3.759
diphenylmethyl 3.324 144.34 3.632
D)c IC50(M)d
naphthyl
NE
NE
25
26
2-quinolyl
3-quinolyl
2.409 102.41 4.451
1.981 102.22 2.685
NE
NE
MSA
Etoposide
-
-
-
-
-
-
-
-
8.38
13.6 ±2.2
a
General structure for the analysed compounds showing the bonds (a–d) selected for the
b
conformational analysis. Volume (average value obtained from the lowest energy conformations)
of the cyclic fragment in Å 3. Dipolar moment (in Debyes) calculated for the representative low-
c
energy conformation. d Cytotoxic activity in PC-3 cell line, NE= no effect.
Figure 3. Conformational models for some representative compounds: (a) active
compounds, left 11; right 1; (b) inactive compounds: left 12; right 6. Carbon in green;
hydrogen in grey; oxygen in red; selenium in violet.
(a)
(b)

Molecules 2009, 14
3324
4. With respect to the length of the bridging chain between the aromatic moiety and the keto group,
the introduction of a methylene fragment significantly improves the activity (IC50 = 6.8 μM for
compound 1 and IC50 = 2.9 μM for compound 11; Table 3 and Figure 4c). However, the introduction
of a second methylene leads to the disappearance of the activity. This change could be related to the
conformational behaviour. For example, in compound 12 the preferred conformations are the folded
ones (see Figure 4c).
Figure 4. Influence of the alkyl chain and ring on the HOMO 0 (a), LUMO 0 (b)
distribution, valuated as descriptive parameters, showed on a low-energy representative
conformation (c). Carbon in cyan; hydrogen in white; oxygen in red; selenium in violet;
sulphur in yellow.
(a)
(b)
(c)
1
11
12
23

Molecules 2009, 14
3325
5. As regards the dipolar moment, a correlation could not be established and great variability was
observed for this parameter, even within the conformational trajectory of each individual compound.
6. With respect to the descriptor parameters obtained by quantum chemistry semi-empirical
calculations, the structural variations that lead to a negative value for the partial charge on the
selenium lead to the loss of activity. Concerning the distribution of the frontier molecular orbitals (see
4a, 4b, 5a and 5b), it was also found that in the most active compounds the HOMO orbital is
preferentially located on the selenium, with a significant contribution from carbons of the aromatic
nucleus. In general it can be observed that the presence of substituents in the 4-position brings about
the displacement of the HOMO towards the ring, with the contribution of selenium decreasing along
with the activity. As an example, the distribution of this orbital for compounds 1 (IC50 = 6.8 μM,
Figure 4a) and 2 (IC50 = 10.0 μM, Figure 5a) can be compared with that of compound 7, which
contains a methoxy group and is inactive. Nevertheless a correlation with the data of energy for orbital
HOMO and LUMO, nor with the gap (LUMO-HOMO energy) cannot be established (Tables 5 and 6).
Figure 5. Influence of the substituents placed in 4-position on the HOMO 0 (a), LUMO 0
(b) distribution, valuated as descriptive parameters, showed on a low-energy representative
conformation (c). Carbon in cyan; hydrogen in white; oxygen in red; selenium in violet;
nitrogen in blue; chlorine in green.
(a)
(b)
(c)
2
4
7

Molecules 2009, 14
3326
Table 5. Mechano-quantic descriptive parameters (semiempirical: PM6) obtained for the analyzed compounds (aryl or heteroaryl monocyclic
derivatives).
O
R6
Y
b
a
Se
c
R5
R4
n
OH
z
O
X
R2
R3
Bond order
b
IC50
Ref.
X
Y
Z
n
R2
R3
R4
R5
R6
pKab
HOMOc LUMOd
EL-M
Q_See
a
c
(M)f
6.8
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
C
C
C
0
0
0
0
0
0
0
0
0
0
1
2
0
0
0
0
0
0
0
0
H
H
H
H
H
H
H
Cl
Br
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
OCH3
OCH3
H
CN
CF3
Cl
CH3
C(CH3)3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2,119
2,057
2,148
2,188
2,184
2,182
2,196
2,360
2,346
2,388
2,370
2,378
1,858
2,791
2,431
2,083
1,985
2,230
2,145
2,486
2,181
1,813
1,8315
1,8478
1,8492
1,7914
1,8230
1,8256
1,8159
1,8508
1,8611
1,8721
1,8419
1,8360
1,8482
1,9205
1,8021
1,8413
1,7891
1,7775
1,8279
1,8722
1,7872
1,7829
0,9068
0,9050
0,9048
0,9659
0,9037
0,9027
0,8940
0,9320
0,9172
0,9069
0,9742
0,9799
0,9123
0,9126
0,9528
0,9068
0,9918
0,9840
0,9540
0,8969
0,9417
1,0108
0,9618
0,9608
0,9608
0,9624
0,9613
0,9611
0,9592
0,9673
0,9524
0,9626
0,9841
0,9622
0,9626
0,9673
0,9633
0,9620
0,9766
0,9767
0,9686
0,9628
0,9598
0,9746
-9,210
-9,422
-9,399
-9,382
-9,002
-9,108
-9,039
-9,258
-9,295
-8,864
-9,439
-9,485
-9,410
-9,275
-8,770
-9,223
-9,546
-9,483
-9,368
-8,871
-9,204
-9,406
-1,552
-2,022
-1,894
-1,666
-1,487
-1,403
-1,208
-1,384
-1,244
-1,582
-0,936
-1,138
-1,961
-1,063
-1,329
-1,630
-1,717
-1,663
-1,712
-1,585
-1,696
-2,030
7,658
7,400
7,505
7,716
7,515
7,705
7,831
7,874
8,051
7,282
8,503
8,347
7,449
8,212
7,441
7,593
7,829
7,820
7,656
7,286
7,508
7,376
0.0577
0.0778
-0.0303
-0.0394
0.0491
0.0485
0.0433
0.0292
0.0134
-0.0182
0.0681
0.0580
-0.0331
0.0693
0.0652
0.0720
0.0825
0.0712
0.0129
-0.0093
-0.0438
0.1126
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
19
20
21
22
23
24
10.0
NE
NE
NE
NE
NE
NE
NE
NE
2.9
H
H
H
H
H
H
NE
NE
4.0
OCH3
OCH3
H
H
H
OCH3
-OCH2O-
NE
NE
NE
NE
10.0
NE
NE
NE
H
H
Cl
H
H
H
H
-
H
H
H
H
H
S(CH2)2CH3
2-thienyl
H
N
N
C
0
H
H
H
-

Molecules 2009, 14
3327
Table 6. Mechano-quantic descriptive parameters (semiempirical: PM6) obtained for the
analyzed compounds (aryl or heteroaryl bicyclic derivatives) a.
O
c
b
Se
R
OH
a
O
Bond order
b
IC50
EL-
Ref.
17
R
pKab
HOMOc LUMOd
Q_See
a
c
(M)f
NE
M
Naphtyl
2,655 1,8432 0,8921 0,9578
-9,076
-9,456
-9,214
-9,222
-1,647
-1,110
-1,918
-1,881
7,429 0.0644
8,346 0.0598
7,296 0.0918
7,341 0.0619
18 Diphenylmethyl 2,592 1,8829 0,9492 0,9689
NE
NE
NE
25
26
2-quinolyl
3-quinolyl
2,306 1,7884 1,0012 0,9606
2,375 1,8453 0,8972 0,9589
a
Notes for Tables 5-6: General structure for the analyzed compounds showing the bonds (a-b)
selected for the bond order determination. b pKa value calculated for the low-energy representative
c
conformation HOMO 0 orbital energy, in eV, calculated for the low-energy representative
d
conformation LUMO 0 orbital energy, in eV, calculated for the low-energy representative
e
conformation Charge on Selenium atom, value obtained for the low-energy representative
conformation. fCytotoxic activity in PC-3 cell line, NE= no effect.
7. With respect to the bond order (Tables 5 and 6) a correlation with the activity cannot be
established, which contradicts our starting hypothesis related to the stability of the C-Se bond.
8. In relation to the acidic character evaluated by the pKa values (Tables 5 and 6), the values of pKa
for the most active compounds oscillate between 2.05 and 2.80, although the proposed structural
modifications apparently do not affect in a significant manner this parameter.
In the present state of our research on these selenium derivatives, we do not try to propose a
pharmacophore applying only a conformacional analysis. We have carried out a conformacional
analysis in order to obtain data about the conformacional behavior of these molecules, with a high
degree of conformational freedom, and to sample what are the preferred conformations, between that
we hoped to find the bioactive one.
Cytotoxic activity in CCRF-CEM, HTB-54, HT-29, MCF-7 and 184B5
In order to investigate the effect of the active compounds in more detail, we examined the activity
on proliferation in other cancer cell lines. The most active compounds in PC-3 were tested, along with
the reference compound doxorubicin, for cytotoxic and antiproliferative activities. Tests were carried
out in the Department of Health Science, Public University of Navarra against a panel of four human
tumour cell lines: lung (HTB-54), colon (HT-29), leukaemia (CCRF-CEM) and breast adenocarcinoma
(MCF-7). Cytotoxicity assays were performed based on the reactivity of MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-tetrazolium bromide], as described by NCI [39]. The results are expressed as GI50
values, i.e., the concentration that reduces by 50% the growth of treated cells with respect to untreated
controls, TGI, the dose that completely inhibits cell growth, and LD50, the concentration that kills 50%
of the cells. The cytotoxic effect of each substance was tested at five different doses between 0.01 and

Molecules 2009, 14
3328
100 μM, or at lower levels when the GI50 was less than 10 nM. Mean GI50, TGI, and LD50 values are
summarized in Table 7. Doxorubicin was used as a control. As guidance with regard to selectivity, all
of the compounds were further examined for toxicity in a mammary gland cell culture derived from
non-malignant cells (184B5). Drug concentrations ranged from 0.01 to 100 μM.
a
c
Table 7. Cytotoxic activities (average GI50 , TGIb and LD50 values) for compounds
against tumour cell lines.
Comp.
Cell lines
HT-29
C.Pa (μM)
CCRF-CEM
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.033
0.071
0.29
HTB-54
0.58
MCF-7
0.09
184B5
1.97
22.50
76.73
2.29
20.14
78.91
1.05
9.64
72.34
0.0009
8.21
62.55
0.05
1.89
7.20
nd
1
bGI50
cTGI
dLD50
GI50
TGI
LD50
GI50
TGI
LD50
GI50
TGI
LD50
GI50
TGI
LD50
GI50
TGI
LD50
7.39
55.98
>100
8.95
42.47
>100
1.90
3.69
58.01
2.64
2
11
9.91
51.95
>100
16.19
72.19
>100
5.99
6.54
>100
11.14
>100
>100
16.82
>100
>100
11.79
>100
>100
<0.01
1.25
23.12
0.006
4.18
53.49
1.57
14
47.42
>100
7.53
6.19
52.65
0.003
8.27
21
60.94
>100
nde
89.21
0.88
Doxorub.
nd
>100
nd
3.45
nd
>100
nd
c
a Cytotoxic parameters ; b Dose that inhibits 50% of cell growth; Dose that completely
inhibits cell growth; d Dose that kills 50% of cells; e n.d.: not determined.
The data show that the compounds under investigation influenced tumour cell growth differently
depending on the cell line. The human breast adenocarcinoma cell line (MCF-7) was most sensitive to
the antiproliferative effects of the investigated compounds. In particular, 1 (GI50 = 0.09 μM), 11
(GI50 = 0.006 μM), 14 (GI50 = 1.57 μM ) and 21 (GI50 = 0.003 μM) were strongly antiproliferative.
Compounds 1, 11 and 21 were 10, 147 and 293 times more active, respectively, than standard
doxorubicin (GI50 = 0.88 μM). Besides, if we compare the TGI values for these compounds
(TGI = 3.69, 4.18, 6.19 and 8.27 μM, respectively), all of them are lower than that of doxorubicin. In
addition, 11 and 21 exhibited a better antitumoral profile than paclitaxel (GI50 = 0.010 ± 0.04 and TGI
= 20 ± 1.9 μM) and vinorelbine (GI50 = 5 ± 1.3 and TGI = 100 ± 10.2 μM), both drugs that are widely
used in clinic. The least sensitive cell line was CCRF-CEM leukemia with cytotoxic parameters
>100 μM for all the compounds. HT-29 and HTB-54 showed moderate levels of susceptibility to the
compounds with the exceptions of 1 (GI50 = 7.39 and 0.58 μM) and 2 (GI50 = 8.95 and 1.90 μM).

Molecules 2009, 14
3329
As an example, curves with the original data from which the GI50, TGI and LD50 values for
compounds 1 and 2 were calculated are shown in Figure 6. Fortunately, the LD50 in 184B5 is higher
than in MCF-7 and the selectivity indexes are 1.3 and 3.4, respectively (184B5/MCF-7 LD50 ratio).
Figure 6. Cytotoxic effects of 1 and 2 on CCRF-CEM, HTB-54, HT-29, MCF-7 and
184B5 cells. Cells were incubated in the presence of each compound at the indicated
concentration for 72 hours. Cytotoxicity was then determined by a colorimetric microassay
based on the use of MTT. Data are expressed as percentage growth ± SEM from at least 3
independent experiments performed in quadruplicate.
It is evident that compound 1 displayed the most prominent antiproliferative activity against all
treated cell lines compared to the untreated control. This compound is therefore a representative
candidate for these compounds for preliminary studies in order to explore a possible mechanism of
action.
Apoptosis
Mounting evidence indicates that apoptosis is a critical mechanism for cancer prevention by Se
compounds [40,41]. For this reason, we investigated whether apoptosis was involved in cell growth
inhibition in the MCF-7 cells by 1.
The apoptotic status of the cells after 48 hours of treatment with 25 μM of the corresponding
compound was determined using the Apo-Direct kit (BD Pharmingen) [42] based on the TUNEL
technique. Camptothecin was used as a positive control. The results obtained are shown in Figure 7.
As can be seen, for compound 1 the induction of cell death was independent of the apoptotic process.
Effects on cell cycle progression
Cell cycle arrest is one of the targets of many anticancer drugs, including doxorubicin and
camptothecin. In an effort to ascertain whether compound 1 could affect cell cycle progression in
MCF-7 cells, these cells were treated with 25 M of the corresponding compound for 48 hours and
cell cycle progression was determined by flow cytometry analysis [42]. As shown in Figure 8, DNA
flow cytometric analysis indicated that treatment of the cells with compound 1 did not induce any
specific phase arrest of the cell cycle.

Molecules 2009, 14
Figure 7. MCF-7 cells were incubated either with 25 µM of the indicated compound or
3330
vehicle (control cells) for 48 hours. The results are presented as the mean ± SEM of three
independent experiments (duplicate wells). * p < 0.01 with respect to the control.
Figure 8. Cell cycle phase distribution of MCF-7 cells after 48 hours of treatment with 25
M of the indicated compound or vehicle (control). Results are expressed as percentages
of total cell counts. Each bar represents mean ± SEM of three independent experiments
(duplicate wells).

Molecules 2009, 14
3331
Conclusions
In summary, we have synthesized a series of 26 aroyl and heteroaroyl selenylacetic acids and
evaluated the cytotoxicity against a prostate cancer cell line (PC-3). Five compounds (1, 2, 11, 14 and
21) showed interesting activity and three of them (1, 11 and 14) were more powerful than etoposide
and methylseleninic acid. Exploration of the SAR study suggested that the permissible maximum
volume for the aromatic fragment is approximately 111–112 Å. A study of the ALogP data indicated
that the compounds with values much greater than 1.90 were inactive. In terms of the length of the
bridging chain between the aromatic moiety and the keto group, the presence of a methylene fragment
improves the activity. We also tested some selected compounds against a panel of four human tumoral
cell lines (CCRF-CEM, HTB-54, HT-29, MCF-7) and one mammary gland-derived non-malignant cell
line (184B5). Compounds 1, 11 and 21 exhibited excellent growth inhibition activity against MCF-7,
with values in the nanomolar range, and moderate values in HTB-54 and HT-29. However, these
compounds did not show inhibitory potency against CCRF-CEM. Comparable activity was observed
on two of the cell lines studied, one a hormone-independent prostate (PC-3) and the other a hormone-
dependent MCF-7 breast cancer cell line. This could suggest that the mode of action is to a large extent
unrelated to the expression of the androgen receptor (PC-3 negative) and oestrogen receptor (MCF-7
positive). Compound 1, which exhibited the best profile, was examined as an apoptotic agent and cell
cycle modulator in MCF-7. The preliminary results revealed that the mechanism of action is
independent of apoptosis and additional studies are required to determine the mechanism by which this
compound exerts its activity. This work is currently in progress in our laboratory. The results reported
here open up new perspectives for future investigations into the synthesis of new compounds of this
family and these are potentially useful for the modulation of pharmacological potential with enhanced
and selective anticancer activity.
Experimental
General
Melting points were determined by differential scanning calorimetry with a Perkin-Elmer DSC
Diamond and by thermomicroscopy with a Mettler FP82+FP80 apparatus (Greifense, Switzerland) and
have not been corrected. The 1H NMR spectra (Table 8) were recorded on a Bruker 400 UltrashieldTM
(Rheinstetten, Germany), using TMS as the internal standard (Table 8). The IR spectra (Table 8) were
performed on a Thermo Nicolet FT-IR Nexus in KBr pellets (Table 8). Elemental microanalyses were
carried out on vacuum-dried samples (Table 1) using an Elemental Analyser (LECO, CHN-900
Elemental Analyser). Silica gel 60 (0.040–0.063 mm) 1.09385.2500 (Merck KGaA, 64271 Darmstadt,
Germany) was used for Column Chromatography and Alugram SIL G/UV254 (Layer: 0.2 mm)
(Macherey-Nagel GmbH & Co. KG. Postfach 101352. D-52313 Düren, Germany) was used for Thin
Layer Chromatography. Chemicals were purchased from E. Merck (Darmstadt, Germany), Scharlau
(F.E.R.O.S.A., Barcelona, Spain), Panreac Química S.A. (Montcada i Reixac, Barcelona, Spain),
Sigma-Aldrich Química, S.A. (Alcobendas, Madrid, Spain), Acros Organics (Janssen
Pharmaceuticalaan 3a, 2440 Geel, België), Maybridge (Cambridge, CB5 8BZ, UK) and Lancaster
(Bischheim-Strasbourg, France).

Molecules 2009, 14
3332
Table 8. Spectroscopic data for compounds 1–26.
Ref.
1
IR (KBr, υ in cm-1) 1H NMR (400 MHz, δ/ppm, J in Hz)
1702, 1678
DMSO-d6, 3.81 (s, 2H, Se-CH2-COOH), 7.59 (m, 2H, H3 + H5), 7.75 (tt, 1H, H4, J4-
5=J4-3=7.9, J4-6=J4-2=1.2), 7.90 (dd, 2H, H2 + H6, J6-5=J2-3=8.4), 12.80 (br s, 1H,
COOH).
2
2232, 1681
1694, 1712
1697, 1686
1691
CDCl3, 3.91 (s, 2H, Se-CH2-COOH), 7.81 (d, 2H, H3 + H5, J3-2=J5-6=8.7), 8.00 (d, 2H,
H2 + H6).
3
CDCl3 3.91 (s, 2H, Se-CH2-COOH), 7.78 (dd, 2H, H3+ H5, J3-2=J5-6=8.1, J3-CF3=J5-
CF3=0.5), 8.02 (dd, 2H, H2 + H6, J2-CF3=J6-CF3=0.6).
4
DMSO-d6, 3.81 (s, 2H, Se-CH2-COOH), 7.66 (d, 2H, H3+H5; J3-2= J5-6=8.5), 7.91 (d,
2H, H2+H6), 12.89 (br s, 1H, COOH).
5
CDCl3, 2.43 (s, 3H, CH3), 3.85 (s, 2H, Se-CH2-COOH), 7.29 (d, 2H, H3+ H5, J3-2=J5-6=
8.1), 7.81 (d, 2H, H2 + H6).
6
1715, 1666
1707, 1692
1707, 1688
1703, 1684
1701, 1677
1694, 1685
1708, 1686
1699, 1667
1714, 1696
1703, 1671
1701, 1675
CDCl3, 1.36 (s, 9H, C-(CH3)3), 3.85 (s, 2H, Se-CH2-COOH), 7.51 (d, 2H, H3+H5, J3-2=
J5-6=8.7), 7.86 (d, 2H, H2+H6).
7
CDCl3, 3.83 (s, 2H, Se-CH2-COOH), 3.91 (s, 3H, CH3O), 6.97 (d, 2H, H3+H5, J3-2=
9.0), 7.90 (d, 2H, H2+H6).
8
CDCl3, 3.89 (s, 2H, Se-CH2-COOH), 7.37-7.43 (m, 1H, H5), 7.49-7.50 (m, 2H,
H4+H3), 7.76-7.79 (m, 1H, H6).
9
CDCl3, 3.89 (s, 2H, Se-CH2-COOH), 7.40 (tt, 1H, H5, J5-6=1.8, J5-4=7.5 J5-3=8.9), 7.44
(tt, 1H, H4, J4-3=1.4 Hz, J4-6=7.6), 7.70 (dd, 1H, H3), 7.72 (dd, 1H, H6).
CDCl3, 3.90 (s, 2H, Se-CH2-COOH), 7.23 (dt, 1H, H5, J5-3=1.5, J5-6=7.9, J5-4=7.7), 7.48
(dt, 1H, H4, J4-6=0.4, J4-3=7.7), 7.69 (dd, 1H, H3); 8.00 (dd, 1H, H6).
CDCl3, 3.62 (s, 2H, Se-CH2-COOH), 3.90 (s, 2H, Ar-CH2-COSe), 7.32 (m, 2H, H3 +
H5), 7.38 (m, 3H, H2 + H4 + H6).
10
11
12
13
14
15
16
CDCl3, 3.03 (s, 4H, Ar-CH2-CH2-COSe + Ar-CH2-CH2-COSe), 3.69 (s, 2H, Se-CH2-
COOH), 7.20-7.26 (m, 3H, H3+H4+H5), 7.30-7.34 (m, 2H, H2+H6).
CDCl3, 3.84 (s, 2H, Se-CH2-COOH), 7.57 (s, 1H, H4), 7.70 (s, 2H, H2+H6), 8.44 (br s,
1H, COOH).
CDCl3, 3.85 - 3.86 (s + s, 8H, 2 OCH3 + Se-CH2-COOH, JCH2-Se=72.2), 6.72 (dt, 1H,
H4, J4-2=J4-6=0.8 Hz, J4-OCH3=2.3), 7.04 (dd, 2H, H2 + H6, J2-OCH3=J6-OCH3=2.2).
CDCl3, 3.86 (s, 2H, Se-CH2-COOH), 3.94 (s, 6H, 3,5-diCH3O), 3.95 (s, 3H, 4-CH3O),
7.16 (s, 2H, H2).
DMSO-d6, 3.76 (s, 2H, Se-CH2-COOH), 6.18 (s, 2H, O-CH2-O), 7.09 (dd, 1H, H5, J5-
2= 0.9 Hz, J5-6=7.8), 7.34 (dd, 1H, H2, J2-6=2.9), 7.55 (dd, 1H, H6), 12.79 (br s, 1H,
COOH).
17
18
19
20
21
1719, 1671
1720, 1693
1717, 1659
1713, 1673
1724, 1690
CDCl3, 3.92 (s, 2H, Se-CH2-COOH), 4.40 (br s, 1H, COOH), 7.63 (m, 2H, H4+ H5),
7.91 (m, 3H, H3 + H6 + H7), 8.00 (d, 1H, H8, J8-7=8.1), 8.48 (s, 1H, H2).
CDCl3, 3.68 (s, 2H, Se-CH2-COOH), 5.23 (s, 1H, (Ph)2-CH-COSe), 7.31-7.40 (m, 10
H, 2H2, 2H6, 2H3, 2H5, 2 H4).
DMSO–d6
, 3,87 (s, 2H, Se-CH2-COOH), 7,78 (td, 2H, H3+H5), 8,85 (td, 2H,
H2+H6).
DMSO-d6, 3.87 (s, 2H, Se-CH2-COOH), 7.61-7.65 (m, 1H, H5), 8.26-8.30 (m, 1H, H4),
8.87-8.90 (m, 1H, H6), 9.02-9.03 (m, 1H, H2), 12.84 (br s, 1H, COOH).
DMSO-d6, 3.87 (s, 2H, Se-CH2-COOH), 7.63 (ddd, 1H, H5, J5-4=7.7, J5-6=4.8, J5-Cl
1.1), 8.24 (ddd, 1H, H4, J4-Cl=1.1), 8.63 (ddd, 1H, H6, J6-Cl=1.1), 12.90 (br s, 1H,
COOH).
=

Molecules 2009, 14
3333
Table 18. Cont.
22
1700, 1668
DMSO-d6, 0.97 (t, 3H, CH3), 1.58-1.70 (m, 2H, S-CH2-CH2-CH3), 3.11 (dt, 2H, S-
CH2-CH2-CH3), 3.81 (s, 2H, Se-CH2-COOH), 7.32 (dd, 1H, H4, J4-5=4.8, J4-6=7.8),
7.97 (dt, 1H, H5, J5-6=1.7), 8.29 (dd, 1H, H6), 12.89 (br s, 1H, COOH).
23
24
25
1712, 1643
1703, 1674
1714, 1690
DMSO-d6, 3.80 (s, 2H, Se-CH2-COOH), 7.30 (dd, 1H, H4, J4-5=3.9 Hz, J4-3=4.9 Hz),
8.02 (dd, 1H, H5, J5-3=1.1 Hz), 8.16 (dd, 1H, H3), 12.82 (bs, 1H, COOH).
DMSO-d6, 3.74 (s, 2H, Se-CH2-COOH), 8.87 (dd, 1H, H6, J6-3=1.2 Hz, J6-5=2.4 Hz),
9.05 (dd, 1H, H5, J5-3=0.2 Hz), 9.06 (dd, 1H, H3), 12.73 (br s, 1H, COOH).
DMSO-d6, 3.73 (s, 2H, Se-CH2-COOH), 7.82 (t, 1H, H7, J7-6=7.5, J7-8=8.0), 7.94 (t,
1H, H6, J6-5=8.3), 7.97 (d, 1H, H5), 8.15 (d, 1H, H8), 8.19 (d, 1H, H3, J3-4=8.4), 8.66 (d,
1H, H4), 12.68 (br s, 1H, COOH).
26
1709, 1675
DMSO-d6, 3,91 (s, 2H, Se-CH2-COOH), 7,77 (t, 1H, H6, J6-7=7,6 ), 7,98 (t, 1H, H7),
8,13 (d, 1H, H5, J5-8=8,3), 8,31 (d, 1H, H8), 9,07 (d, 1H, H4, J4-2=1,8), 9,23 (d, 1H,
H2),12,75 (bs, 1H, COOH).
General procedure for the preparation of selenylacetic acids 1–6, 8–26
A solution of sodium borohydride (1.00 g, 26.3 mmol) in distilled water (12.5 mL) was added to a
stirred suspension of grey selenium (1.00 g, 12.6 mmol) in distilled water (12.5 mL) at room tempera-
ture. The reaction mixture was stirred until an almost colourless solution of NaHSe was formed. The
acyl chloride (12.6 mmol) was added in small portions and the reaction mixture was magnetically stirred
for 1 hour. A yellow solution was formed and the bromoacetic acid (1.75 g, 12.6 mmol) was added.
Within 30 minutes a solid was formed. The product was filtered off and washed with distilled water
(3 × 25 mL). The products were recrystallised from an appropriate solvent (Table 1). The following
compounds were synthesized according to this procedure: (Benzoylselenyl)acetic acid (1). (4-
cyanobenzoylselenyl)acetic acid (2). (4-trifluoromethylbenzoylselenyl)acetic acid (3). (4-
chlorobenzoylselenyl)acetic acid (4). (4-methylbenzoylselenyl)acetic acid (5). (4-tert-butylbenzoyl-
selenyl)acetic acid (6). (2-chlorobenzoylselenyl)acetic acid (8). (2-bromobenzoylselenyl)acetic acid
(9). (2-iodobenzoylselenyl)acetic acid (10). Phenylacetoylselenylacetic acid (11). (3-phenyl-
propanonylselenyl)acetic acid (12). (3,5-dichlorobenzoylselenyl)acetic acid (13). (3,5-dimethoxy-
benzoylselenyl)acetic acid (14). (3,4,5-trimethoxybenzoylselenyl)acetic acid (15). (3,4-methylenedioxy-
benzoylselenyl) acetic acid (16). (2-naphthoylselenyl)acetic acid (17). (diphenylacetylselenyl)acetic
acid (18). (4-pyridoylselenyl)acetic acid (19). (3-pyridoylselenyl)acetic acid (20). (3-(2-chloro)-
pyridoylselenyl)acetic acid (21). (3-(2-propylthio)pyridoylselenyl)acetic acid (22). (2-thienoylselenyl)-
acetic acid (23). (pyrazinoylselenyl)acetic acid (24). (2-quinoloylselenyl)acetic acid (25). (3-
quinoloylselenyl)acetic acid (26).
Procedure for the preparation of (4-methoxyphenylselenyl)acetic acid (7)
An equivalent of grey-powder selenium was suspended in tetrahydrofuran (20 mL) and two
equivalents of lithium aluminium hydride were added slowly. The carbonyl chloride was added to the
reaction mixture and the mixture was stirred for 1 h. The suspension was filtered and the solid was
discarded. An equivalent of bromoacetic acid was added to the liquid filtrate. After 20 min the mixture
was poured onto 500 g of ice. The resulting solid was filtered off, washed and recrystallised (Table 1).

Molecules 2009, 14
3334
Cytotoxic activity in PC-3
PC-3 cells were seeded in 96-well plates (Millipore, Eschborn, Germany) at a density of 5 × 103
cells per well. The samples were incubated at 37 ºC under 5% CO2 overnight prior to the addition of
the compounds. Compounds were diluted in complete medium. After 3 days of incubation, 10 μL MTT
solution (5 mg/mL in PBS) was added to the cells in each well and these were stored for an additional
4 h at 37 ºC. The absorbance of formazan at λ = 570 nm was measured on a Polarstar Galaxy plate
reader (BMG LabTechnologies GmbH, Offenburg, Germany). The percentage of viable cells was
calculated to obtain IC50-values.
PC-3 are human tumorigenic and metastatic prostate cancer cells and these were obtained from
American Type Culture Collection (ATCC, Manassas, VA, USA) (passage 36). The cells were
cultured under standard conditions (Dulbecco’s RPMI 1640 medium, with GlutamaxTM 1, Invitrogen
supplemented with 10% fetal bovine serum, Fetalclone III, SH30109.03, HYCLONE and 1%
Penicillin-Streptomycin, Invitrogen, Carlsbad, CA, USA).
Molecular modelling
The initial computational work was performed on a Dell Precision 380 workstation, provided with
the software package Discovery Studio v1.7. The three-dimensional models of the studied compounds
were constructed, in the vacuum phase, using atoms and structural fragments from the Viewer module
(Discovery Studio) and using the Dreiding force field [43]. Once the models were constructed, a
preliminary conformational analysis was carried out. The applied protocol (Diverse Conformational
Generation integrated in the Pharmacophore protocol Discovery Studio) can be summed up as follows:
(a) Initial construction of the model and first minimization by application of the Dreiding minimize
protocol (Steepest descent algorithm with a convergence criterion of 10–6). The AlogP98 [44,45]
descriptor (an implementation of the atom-based ALogP method) was calculated for each compound.
(b) Application of the BEST routine for conformation generation (First: Conjugate-gradient
minimization in torsion space; second: conjugate-gradient minimization in Cartesian space; third:
Quasi_Newton minimization in Cartesian space). (c) Elimination of those conformations whose
relative energy is greater than 10 Kcal/mol at a global minimum. (d) Analysis of conformational
trajectory and selection of representative lowest energy conformations. Root mean square (rms)
deviations of the structures were monitored. The energy differences between the different
conformations analysed for each trajectory were in the range 2–5 Kcal.
For each of the compounds, ten lowest energy conformations were selected and a new minimization
cycle was applied. The volumes of the ring moiety and the whole molecule were calculated for each of
the new representative low-energy conformations selected.
The mechano-quantic analysis of the conformations obtained in the previous step was carried out
with the package Mopac2009, PM6 [46] (or PM3 [47] for halogen and/or sulfur-containing
derivatives) semi-empirical approaches, with the geometry optimized using an eigenvector following
algorithm. The energy and distribution of the HOMO and LUMO orbitals, the selenium charge, the
electronic density (ED) and molecular electrostatic potential (MEP) distribution and the Dipolar
moment were obtained for each of the conformations obtained. The data corresponding to the

Molecules 2009, 14
3335
representative lowest energy conformation for each compound was selected and used in the
establishment of the preliminary structure-activity relationships.
Cytotoxic activity in CCRF-CEM, HTB-54, HT-29, MCF-7 and 184B5
The cytotoxic effects of each substance were tested at five different doses between 0.01 and
100 μM. Each substance was initially dissolved in DMSO at a concentration of 0.1 M, and serial
dilutions were prepared using culture medium. The plates with cells from the different lines, to which
medium containing the substance under test were added, were incubated for 72 h at 37 °C in a
humidified atmosphere containing 5% CO2. European Collection of Cell Cultures (ECACC) or
American Type Culture Collection (ATCC) provided human tumour cell lines. Four cell lines were
used: one human lymphocytic leukemia (CCRF-CEM) and three human solid tumours, one colon
carcinoma (HT-29), one lung carcinoma (HTB-54) and one breast adenocarcinoma (MCF-7). CCRF-
CEM, HT-29 and HTB-54 cells were grown in RPMI 1640 medium (Life Technologies, Carlsbad, CA,
USA) supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin, 100
μg/mL streptomycin and 10 mM HEPES buffer (pH = 7.4). MCF-7 cells were grown in EMEM
medium (Clonetics) supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100 units/mL
penicillin and 100 μg/mL streptomycin. 184B5 cells were grown Hams F-12/DMEM (50:50)
supplemented as described by Li et al. [48]. Cytotoxicity was then determined by the MTT method.
Results are expressed as GI50 values, the concentration that reduces by 50% the growth of treated cells
with respect to untreated controls, TGI, the dose that completely inhibits cell growth, and LD50, the
concentration that kills 50% of the cells. Data were obtained from at least three independent
experiments performed in quadruplicate.
Apoptosis and cell cycle
For breast adenocarcinoma MCF-7 cells, the apoptotic status and cell cycle analysis of the cells
were determined using the Apo-Direct kit (BD Pharmingen), based on the TUNEL technique, under
the conditions described by the manufacturer.
Acknowledgements
The authors wish to express their gratitude to the University of Navarra Research Plan (Plan de
Investigación de la Universidad de Navarra, PIUNA) and CAN Foundation for financial support for
this project.
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Sample Availability: Samples of the compounds 1–26 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).