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

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 ⁴and 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^–1and 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 IC₅₀values. 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

83^a

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

147^a

108^a

135-136^b,c

92^a

4-methylphenyl

4-(tert-butyl)phenyl

99-103^b,c

Chloroform

Chloroform/Carbon

tetrachloride

Carbon tetrachloride

Toluene

Hexane

Carbon tetrachloride

Toluene

Ethanol

Carbon tetrachloride

Toluene

Carbon tetrachloride

Ether / hexane

Methanol

4-methoxyphenyl

16

104-107^b

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

123-125^b

124-128^b

105-108^b

74^a

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

65-69^b

C₁₁H₁₂O₃S ¼ H₂O

C₉H₆Cl₂O₃Se·½ H₂O

C₁₁H₁₂O₅Se

108-109^b

117^a

107-110^b

106-113^b

130^a

C₁₂H₁₄O₆Se

C₁₀H₈O₅Se

C₁₃H₁₀O₃Se

C₁₆H₁₄O₃Se

C₈H₇NO₃Se

C₈H₆ClNO₃Se

C₁₁H₁₃NO₃SSe

C₇H₆O₃SSe

C₇H₆N₂O₃Se

C₁₂H₉NO₃Se

diphenylmethyl

4-pyridyl

127-130^b

119-121^b,c

147-150^b,c

157^a

3-pyridyl

3-(2-chloropyridyl)

3-(2-propylthiopyridyl)

2-thienyl

Toluene

36

33

10

6

109-111^b

82-84^b

Carbon tetrachloride

Isopropanol

pyrazinyl

2-quinolyl

3-quinolyl

138-140^b

131-132^b,c

187-189^b,c

Toluene

Chloroform

9

^aDetermined by differential scanning calorimetry; ^bDetermined by thermomicroscopy; ^cFusion 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

NE^a

NE

2

3

4

5

NE

6

NE

7

8

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

MSA^b

8.38 [33]

13.6 ± 2.2 [34]

Etoposide

^aNE = no effect is observed; ^bmethylseleninic 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^–3kcal 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 R₂R₃

R₄

R₅R₆AlogP Vol^b

D)^cIC₅₀(M)^e

1

2

3

4

5

6

7

8

9

C C C 0

H

1.795 68.58 3.465

6.8

10.0

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

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 N 0

N C C 0

R₂

H

Cl

R₃

H

Cl

R₄

H

R₅

H

Cl

R₆AlogP Vol^b

D)^cIC₅₀(M)^d

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

OCH₃OCH₃OCH₃

-O-CH₂-O-

H

OCH₃

4.0

NE

H

4.352

1.081

3.543

4.094

NE

10.0

NE

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

^aGeneral structure for the analysed compounds showing the bonds (a–e) selected for the conformational analysis. ^bVolume (average

value obtained from the lowest energy conformations) of the cyclic fragment in Å ³. ^cDipolar moment (in Debyes) calculated for the

representative low-energy. ^dCytotoxic 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 Vol^b

2.703 106.65 3.759

diphenylmethyl 3.324 144.34 3.632

D)^cIC₅₀(M)^d

naphthyl

NE

25

26

2-quinolyl

3-quinolyl

2.409 102.41 4.451

1.981 102.22 2.685

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. ^dCytotoxic 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

pKa^b

HOMO^cLUMO^d

E_L-M

Q_Se^e

a

c

(M)^f

6.8

C

N

C

N

C

0

1

2

0

H

Cl

Br

I

H

Cl

OCH₃

H

CN

CF₃

Cl

CH₃

C(CH₃)₃

OCH₃

H

Cl

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

0,9624

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

2.9

H

NE

4.0

OCH₃

H

OCH₃

-OCH₂O-

NE

10.0

NE

H

Cl

H

-

H

S(CH₂)₂CH₃

2-thienyl

H

N

C

0

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

E_L-

Ref.

17

R

pKa^b

HOMO^cLUMO^d

Q_Se^e

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

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. ^bpKa 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 GI₅₀was less than 10 nM. Mean GI₅₀, TGI, and LD₅₀values 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, TGI^band LD₅₀values) for compounds

against tumour cell lines.

Comp.

Cell lines

HT-29

C.P^a(μM)

CCRF-CEM

>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

16.82

>100

11.79

>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

nd^e

89.21

0.88

Doxorub.

nd

>100

nd

3.45

nd

>100

nd

c

^aCytotoxic parameters ; ^bDose that inhibits 50% of cell growth; Dose that completely

inhibits cell growth; ^dDose that kills 50% of cells; ^en.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 LD₅₀values for

compounds 1 and 2 were calculated are shown in Figure 6. Fortunately, the LD₅₀in 184B5 is higher

than in MCF-7 and the selectivity indexes are 1.3 and 3.4, respectively (184B5/MCF-7 LD₅₀ratio).

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 Ultrashield^TM

(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, J_4-

₅=J_4-3=7.9, J_4-6=J_4-2=1.2), 7.90 (dd, 2H, H₂+ H₆, J_6-5=J_2-3=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, J_3-2=J_5-6=8.7), 8.00 (d, 2H,

H₂+ H₆).

3

CDCl₃3.91 (s, 2H, Se-CH₂-COOH), 7.78 (dd, 2H, H₃+ H₅, J_3-2=J_5-6=8.1, J_3-CF3=J_5-

_CF3=0.5), 8.02 (dd, 2H, H₂+ H₆, J_2-CF3=J_6-CF3=0.6).

4

DMSO-d₆, 3.81 (s, 2H, Se-CH₂-COOH), 7.66 (d, 2H, H₃+H₅; J_3-2= J_5-6=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+ H_5,J_3-2=J_5-6=

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, J_3-2=

J_5-6=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, J_3-2=

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, J_5-6=1.8, J_5-4=7.5 J_5-3=8.9), 7.44

(tt, 1H, H₄, J_4-3=1.4 Hz, J_4-6=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, J_5-3=1.5, J_5-6=7.9, J_5-4=7.7), 7.48

(dt, 1H, H₄, J_4-6=0.4, J_4-3=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, J_CH2-Se=72.2), 6.72 (dt, 1H,

H₄, J_4-2=J_4-6=0.8 Hz, J_4-OCH3=2.3), 7.04 (dd, 2H, H₂+ H₆, J_2-OCH3=J_6-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, J_5-

₂= 0.9 Hz, J_5-6=7.8), 7.34 (dd, 1H, H₂, J_2-6=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, J_8-7=8.1), 8.48 (s, 1H, H₂).

CDCl_3,3.68 (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, J_5-4=7.7, J_5-6=4.8, J_5-Cl

1.1), 8.24 (ddd, 1H, H₄, J_4-Cl=1.1), 8.63 (ddd, 1H, H₆, J_6-Cl=1.1), 12.90 (br s, 1H,

COOH).

=

Molecules 2009, 14

3333

Table 18. Cont.

22

1700, 1668

DMSO-d_6,0.97 (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, J_4-5=4.8, J_4-6=7.8),

7.97 (dt, 1H, H₅, J_5-6=1.7), 8.29 (dd, 1H, H₆), 12.89 (br s, 1H, COOH).

23

24

25

1712, 1643

1703, 1674

1714, 1690

DMSO-d_6,3.80 (s, 2H, Se-CH₂-COOH), 7.30 (dd, 1H, H₄, J_4-5=3.9 Hz, J_4-3=4.9 Hz),

8.02 (dd, 1H, H₅, J_5-3=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, J_6-3=1.2 Hz, J_6-5=2.4 Hz),

9.05 (dd, 1H, H₅, J_5-3=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, J_7-6=7.5, J_7-8=8.0), 7.94 (t,

1H, H₆, J_6-5=8.3), 7.97 (d, 1H, H₅), 8.15 (d, 1H, H₈), 8.19 (d, 1H, H₃, J_3-4=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, J_6-7=7,6 ), 7,98 (t, 1H, H₇),

8,13 (d, 1H, H₅, J_5-8=8,3), 8,31 (d, 1H, H₈), 9,07 (d, 1H, H₄, J_4-2=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% CO₂overnight 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 GI₅₀values, 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.

References

1. Varmus, H. The new era in cancer research. Science 2006, 312, 1162–1165.

2. Jariwalla, R.J.; Gangapurkar, B.; Nakamura, D. Differential sensitivity of various human tumour-

derived cell types to apoptosis by organic derivatives of selenium. Br. J. Nutr. 2009, 101,

182–189.

Molecules 2009, 14

3336

3. Latreche, L.; Chavatte, L. Selenium incorporation into selenoproteins, implications in human

health. In 10th International Symposium on Metal Ions in Biology and Medicine, Bastia, France,

2008; pp. 731–737.

4. Clark, L.C.; Combs, G.F. Selenium-compounds and the prevention of cancer-research needs and

public health implications. J. Nutr. 1986, 116, 170–173.

5. Sonn, G.A.; Aronson, W.; Litwin, M.S. Impact of diet on prostate cancer: A review. Prostate

Cancer Prostatic Dis. 2005, 8, 304–310.

6. El-Bayoumy, K.; Sinha, R.; Pinto, J.T.; Rivlin, R.S. Cancer chemoprevention by garlic and garlic-

containing sulfur and selenium compounds. J. Nutr. 2006, 136, 864S–869S.

7. Gonzalez, C.A.; Salas-Salvado, J. The potential of nuts in the prevention of cancer. Br. J. Nutr.

2006, 96, S87–S94.

8. Sharma, A.K.; Sharma, A.; Desai, D.; Madhunapantula, S.V.; Huh, S.J.; Robertson, G.P.; Amin,

S. Synthesis and anticancer activity comparison of phenylalkyl isoselenocyanates with

corresponding naturally occurring and synthetic isothiocyanates. J. Med. Chem. 2008, 51,

7820–7828.

9. Sanmartín, C.; Plano, D.; Palop, J.A. Selenium compounds and apoptotic modulation: A new

perspective in cancer therapy. Mini Rev. Med. Chem. 2008, 8, 1020–1031.

10. Bardia, A.; Tleyjeh, I.M.; Cerhan, J.R.; Sood, A.K.; Limburg, P.J.; Erwin, P.J. Montori, V.M.

Efficacy of antioxidant supplementation in reducing primary cancer incidence and mortality:

Systematic review and meta-analysis. Mayo Clin. Proc. 2008, 83, 23–34.

11. Bjelakovic, G.; Nikolova, D.; Simonetti, R.G.; Gluud, C. Systematic review: Primary and

secondary prevention of gastrointestinal cancers with antioxidant supplements. Alimen.

Pharmacol. Ther. 2008, 28, 689–703.

12. Connelly-Frost, A.; Poole, C.; Satia, J.A.; Kupper, L.L.; Millikan, R.C.; Sandler, R.S. Selenium,

folate, and colon cancer. Nutr. Cancer 2009, 61, 165–178.

13. Smith, M.L.; Lancia, J.K.; Mercer, T.I.; Ip, C. Selenium compounds regulate p53 by common and

distinctive mechanisms. Anticancer Res. 2004, 24, 1401–1408.

14. Rikiishi, H. Apoptotic cellular events for selenium compounds involved in cancer prevention. J.

Bioenerg. Biomembr. 2007, 39, 91–98.

15. Hurst, R.; Elliot, R.M.; Goldson, A.J.; Fainweather-Tait, S.J. Se-methylselenocysteine alters

collagen gene and protein expression in human prostate cells. Cancer Lett. 2008, 269, 117–126.

16. Pinto, J.T.; Sinha, R.; Papp, K.; Facompre, N.D.; Desai, D.; El-Bayoumy, K. Differential effects

of naturally occurring and synthetic organoselenium compounds on biomarkers in androgen

responsive and androgen independent human prostate carcinoma cells. Int. J. Cancer 2007, 120,

1410–1417.

17. Singh, U.; Null, K.; Sinha, R. In vitro growth inhibition of mouse mammary epithelial tumor cells

by methylseleninic acid: Involvement of protein kinases. Mol. Nutr. Food Res. 2008, 52,

1281–1288.

18. Poerschke, R.L.; Franklin, M.R.; Moos, P.J. Modulation of redox status in human lung cell lines

by organoselenocompounds: Selenazolidines, selenomethionine, and methylseleninic acid.

Toxicol. Vitro 2008, 22, 1761–1767.

19. Talas, Z.S.; Ozdemir, I.; Yilmaz, I.; Gok, Y. Antioxidative effects of novel synthetic

organoselenium compound in rat lung and kidney. Ecotoxicol. Environ. Saf. 2009, 72, 916–921.

20. Shiah, H.S.; Lee, W.S.; Juang, S.H.; Hong, P.C.; Lung, C.C.; Chang, C.J.; Chou, K.M.; Chang,

Molecules 2009, 14

3337

F.Y. Mitochondria-mediated and p53-associated apoptosis induced in human cancer cells by a

novel selenophene derivative, D-501036. Biochem. Pharmacol. 2007, 73, 610–619.

21. Xing, F.; Li, S.L.; Ge, X.Y.; Wang, C.; Zeng, H.H.; Li, D.; Dong, L. The inhibitory effect of a

novel organoselenium compound BBSKE on the tongue cancer Tca8113 in vitro and in vivo. Oral

Oncol. 2008, 44, 963–969.

22. Barbosa, N.B.D.; Nogueira, C.W.; Guecheva, T.N.; Bellinaso, M.D.; Rocha, J.B.T. Diphenyl

diselenide supplementation delays the development of N-nitroso-N-methylurea-induced mammary

tumors. Arch. Toxicol. 2008, 82, 655–663.

23. Xiao, H.; Parkin, K.L. Induction of Phase II enzyme activity by various selenium compounds.

Nutr. Cancer 2006, 55, 210–223.

24. Rosa, R.M.; Picada, J.D.; Saffi, J.; Henriques, J.A.P. Cytotoxic, genotoxic, and mutagenic effects

of diphenyl diselenide in Chinese hamster lung fibroblast. Mut. Res. 2007, 628, 87–98.

25. Zhao, G.H.; Lin, H.K.; Zhu, S.R.; Sun, H.W.; Chen, Y.T. Dinuclear palladium(II) complexes

containing two monofunctional [Pd(en)(pyridine)Cl]⁺units bridged by Se or S. Synthesis,

characterization, cytotoxicity and kinetic studies of DNA-binding. J. Inorg. Biochem. 1998, 70,

219–226.

26. Abele, E.; Popelis, J.; Shestakova, I.; Domracheva, I.; Arsenyan, P.; Lukevics, E. Synthesis and

cytotoxicity of di(3-indolyl) selenide derivatives. Khimiya Geterotsiklicheskikh Soedinenii 2004,

6, 868–872.

27. Zeng, H.W.; Briske-Anderson, M.; Idso, J.P.; Hunt, C.D. The selenium metabolite methylselenol

inhibits the migration and invasion potential of HT1080 tumor cells. J. Nutr. 2006, 136,

1528–1532.

28. Ohta, Y.; Suzuki, K.T. Methylation and demethylation of intermediates selenide and

methylselenol in the metabolism of selenium. Toxicol. Appl. Pharmacol. 2008, 226, 169–177.

29. Plano, D.; Sanmartín, C.; Moreno, E.; Prior, C.; Calvo, A.; Palop, J.A. Novel potent

organoselenium compounds as cytotoxic agents in prostate cancer cells. Bioorg. Med. Chem. Lett.

2007, 17, 6853–6859.

30. Plano, D.; Moreno, E.; Font, M.; Encío, I.; Palop, J.A.; Sanmartín, C. Synthesis and biological

evaluation of selenadiazole derivatives as anticancer agents. Bioorg. Med. Chem. 2009,

(Submitted for publication).

31. De Athayde, P.F.; De Souza, A.G.; De Morais, S.A.; Botelho, J.R.; Barbosa, J.M.; Miller, J.; Lira,

B.F. Synthesis and characterization of three new organo-selenium compounds. A convenient

synthesis of aroylselenoglycolic acids. Arkivoc 2004, 6, 22–26.

32. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to

proliferation and cytotoxicity assays. J. Inmunol. Methods 1983, 65, 55–63.

33. Dong, Y.; Zhang, H.; Hawthorn, L.; Ganther, H.E.; Ip, C. Delineation of the molecular basis for

selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array. Cancer

Res. 2003, 63, 52–59.

34. Van Brussel, J.P.; Oomen, M.A.; Vosselbeld, P.J.M.; Wiemer, E.A.C.; Sonneveld, P.; Mickisch,

G.H.J. Identification of multidrug resistance-associated protein 1 and glutathione as multidrug

resistance mechanisms in human prostate cancer cells: chemosensitization with leukotriene D4

antagonists and buthionine sulfoximine. BJU Int. 2004, 93, 1333–1338.

Molecules 2009, 14

3338

35. Font, M.; Ardaiz, E.; Cordeu, L.; Cubedo, E.; García-Foncillas, J.; Sanmartín, C.; Palop, J.A.

Structural characteristics of novel symmetrical diaryl derivatives with nitrogenated functions.

Requirements for cytotoxic activity. Bioorg. Med. Chem. 2006, 14, 1942–1948.

36. Accerlys Software Inc. Discovery Studio Modeling Environment Release 1.7. Accerlys Software

Inc.: San Diego, USA, 2007.

37. MOPAC MOPAC2009, Stewart, J.P. Stewart Computational Chemistry, Colorado Springs, CO,

USA, http://OpenMOPAC.net, 2008.

38. Macrae, C.F.; Edgington, P.R.; McCabe, P.; Pidcock, E.; Shields, G.P.; Taylor, R.; Towler, M.;

Van de Streek, J. Mercury: Visualization and analysis of crystal structures. J. Appl. Crystallogr.

2006, 39, 453–457.

39. http://dtp.nci.nih.gov/branches/btb/ivclsp.html, accessed on 27 June 2009.

40. Philchenkov, A.; Zavelevich, M.; Khranovskaya, N.; Surai, P. Comparative analysis of apoptosis

induction by selenium compounds in human lymphoblastic leukemia MT-4 cells. Exp. Oncol.

2007, 29, 257–261.

41. Naithani, R. Organoselenium compounds in cancer chemoprevention. Mini Rev. Med. Chem.

2008, 8, 657–668.

42. Fimognari, C.; Nüsse, M.; Cesari, R.; Iori, R.; Cantelli-Forti, G.; Hrelia, P. Growth inhibition,

cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane.

Carcinogenesis 2002, 23, 581–586.

43. Mayo, S.L.; Olafson, B.D.; Goddard, W.A. Dreiding: A generic force field for molecular

simulations. J. Phys. Chem. 1990, 94, 8897–8909.

44. Viswanadhan, V.N.; Ghose, A.K.; Revankar, G.R.; Robins, R.K. Atomic physicochemical

parameters for three-dimensional structure-directed quantitative structure–activity relationships.

Additional parameters for hydrophobic and dispersive interactions and their application for an

automated superposition of certain naturally-occurring nucleoside antibiotics. J. Chem. Inf.

Comput. Sci. 1989, 29, 163–172.

45. Ghose, A.K.; Viswanadhan, V.N.; Wendoloski, J.J. Prediction of hydrophobic (lipophilic)

properties of small organic molecules using fragmental methods: An analysis of ALOGP and

CLOGP methods. J. Phys. Chem. A 1998, 102, 3762–3772.

46. Stewart J.J.P.; Optimization of parameters for semiempirical methods V: Modification of NDDO

approximations and application to 70 elements. J. Mol. Model. 2007, 13, 1173–1213.

47. Stewart, J.J.P. Optimization of parameters for semi-empirical methods I-Method. J. Comp. Chem.

1989, 10, 209–220.

48. Li, Y.Z.; Pan, J.; Li, J.L.; Lee, J.H.; Tunkey, C.; Saraf, K.; Garbe, J.C.; Whitley, M.Z.; Jelinsky,

S.A.; Stampfer, M.R.; Haney, S.A. Transcriptional changes associated with breast cancer occur as

normal human mammary epithelial cells overcome senescence barriers and become immortalized.

Mol. Cancer 2007, 6, Article 7.

Sample Availability: Samples of the compounds 1–26 are available from the authors.

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/).

Article Doi

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

DOI: 10.3390/molecules14093313

Source and publish data:

Authors:

Article abstract of DOI:10.3390/molecules14093313

Full text of DOI:10.3390/molecules14093313

Products guided by the article

R&D Labs maybe for 704892-29-5

Relevant to this article

Hot Product