Phenolic thio- and selenosemicarbazones as multi-target drugs

Article abstract of DOI:10.1016/j.ejmech.2015.02.037

A series of isosteric phenolic thio- and selenosemicarbazones have been obtained by condensation of naturally-occurring phenolic aldehydes and thio(seleno)semicarbazides. Title compounds were designed as potential multi-target drugs, and a series of struc

Full text of DOI:10.1016/j.ejmech.2015.02.037

European Journal of Medicinal Chemistry 94 (2015) 63e72
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Phenolic thio- and selenosemicarbazones as multi-target drugs
a
a
,
*
a
b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
~
Veronica Calcatierra , Oscar Lopez , Jose G. Fernandez-Bolanos , Gabriela B. Plata ,
b
ꢀ
ꢀ
Jose M. Padron
a
ꢀ
Departamento de Química Organica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain
BioLab, Instituto Universitario de Bio-Organica “Antonio Gonzalez” (IUBO-AG), Centro de Investigaciones Biomedicas de Canarias (CIBICAN), Universidad
b
ꢀ
ꢀ
ꢀ
ꢀ
de La Laguna, c/ Astrofísico Francisco Sanchez 2, E-38206 La Laguna, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of isosteric phenolic thio- and selenosemicarbazones have been obtained by condensation of
naturally-occurring phenolic aldehydes and thio(seleno)semicarbazides. Title compounds were designed
as potential multi-target drugs, and a series of structure-activity relationships could be established upon
Received 1 October 2014
Received in revised form
13 January 2015
Accepted 19 February 2015
Available online 21 February 2015
their in vitro assays: antioxidant activity, a-glucosidase inhibition and antiproliferative activity against six
human tumor cell lines: A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small
cell lung), T-47D (breast) and WiDr (colon). For the antiradical activity, selenium atom and 2 or 3
phenolic hydroxyl groups proved to be essential motifs; remarkably, the compound with the most potent
Keywords:
activity, with a trihydroxyphenyl scaffold (EC50 ¼ 4.87 1.57
hydroxytyrosol, a potent antioxidant present in olive oil (EC50 ¼ 13.80 1.41
the thiosemicarbazones was found to be strong non-competitive inhibitor of
1.6
marketed for the treatment of type-2 diabetes. Most of the synthesized compounds also exhibited
relevant antiproliferative activities; in particular, seleno derivatives showed GI50 values lower than
m
M) was found to be stronger than natural
M). Furthermore, one of
-glucosidase
11.4 M),
Phenolic compounds
Thio(seleno)semicarbazones
Antioxidants
ROS scavengers
Glycosidase inhibitors
Antiproliferative agents
m
a
a
(Ki ¼ 9.6
m
M), with an 8-fold increase in activity compared to acarbose (Ki ¼ 77.9
m
6.0
potent antiproliferative agents than 5-fluorouracil or cisplatin.
© 2015 Elsevier Masson SAS. All rights reserved.
mM for all the tested cell lines; N-naphthyl mono- and dihydroxylated derivatives behaved as more
1. Introduction
has also been reported for thiosemicarbazones. Some other rele-
vant biological properties associated to these Schiff bases include
antimicrobial activity [9] and inhibition of xanthine oxidase [10]
involved in the catabolism of purines to give uric acid.
Schiff bases are attractive and versatile scaffolds with relevant
applications in several areas, including asymmetric catalysis [1],
chemosensors [2], photochemical switches [3], live cell imaging [4],
or pharmacology [5], among others. Remarkably, triapine™, a
simple 3-aminopyridinyl-based thiosemicarbazone has progressed
through Phase I and II clinical trials and is currently a promising
pharmaceutical drug exhibiting potent anticancer activity against
an ample panel of human tumors [6]. Furthermore, potent in vitro
antiparasitic activity against the protozoan Toxoplasma gondii [7],
the agent causing toxoplasmosis disease, and Trypanosoma cruzi
[8], provoking the Chagas disease (or American trypanosomiasis)
Herein we report the preparation of a series of phenolic thio-
semicarbazones and their selenium isosters; we have analyzed the
influence of the number and position of the phenolic hydroxyl
groups, the nature of the chalcogen atom, and the type of N-aryl
substituent on the antioxidant and antiproliferative activities, and
on glycosidase inhibition, with the aim of developing potential
multi-target drugs. Such approach is the basis of an emerging and
promising area within Medicinal Chemistry research coined as
polypharmacology [11], targeted at the treatment of complex
multifactorial diseases, which require the simultaneous modula-
tion of a network of targets.
Abbreviations: AAPH, 2,20-azobis(2-amidinopropane) dihydrochloride; DPPH,
2,2-diphenyl-1-picrylhydrazyl, free radical; EC50, half maximal Effective Concen-
tration; FTC, Ferric Thio Cyanate method; GI50, Growth Inhibition of 50%; NCI,
National Cancer Institute; ROS, Reactive Oxygen Species; TBARS, thiobarbituric acid
reactive substances.
2. Results and discussion
2.1. Chemistry
* Corresponding author.
E-mail address: osc-lopez@us.es (O. Lopez).
ꢀ
ꢀ
Taking into consideration that Shiff bases are endowed with a
http://dx.doi.org/10.1016/j.ejmech.2015.02.037
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

64
V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
series of biological activities mainly due to their redox and
chelating capacities, we envisioned the possibility of combining a
phenolic scaffold with thio- or selenosemicarbazone moieties
(Fig. 1). The strong antioxidant properties associated to both
phenolic and organoselenium compounds are widely known [12].
Furthermore, these families of compounds have also attracted a
considerable pharmaceutical interest [13]. Therefore, an enhance-
ment of the bio-activities in the target compounds might take place
via synergic effects when both functionalities are simultaneously
present.
2.2.1. Antiradical activity
The antiradical activity of title compounds was evaluated using
the DPPH method [19]. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a
stable free-radical with a strong absorbance at 515 nm; in the
presence of an antioxidant, the absorbance is consequently reduced
upon formation of the corresponding hydrazine by H-donation. The
antiradical activity was estimated by calculating EC50, that is, the
concentration of antioxidant required to scavenge 50% of the initial
free radical. Data depicted in Table 1 suggest that an increase in the
number of phenolic hydroxyl groups leads to
a significant
The synthetic route for accessing phenolic thio- and selenose-
micarbazones is illustrated in Scheme 1. Thus, derivatives 10e20
were obtained from moderate to almost quantitative yields
(28e93%) by acid-promoted condensation of a series of naturally-
occurring phenolic aldehydes (p-hydroxybenzaldehyde 1, 3,4-
dihydroxybenzaldehyde or protocatechuic aldehyde 2, 2,4,5-
trihydroxybenzaldehyde 3, and 3,4,5-trihydroxybenzaldehyde 4)
with commercially-available thiosemicarbazide 5 or its selenium
improvement of the antiradical activity, especially when changing
from one to two hydroxyls. Concerning the starting aldehydes, p-
hydroxybenzaldehyde 1 shows no antiradical activity at a concen-
tration of 250
an EC50 value of 6.57 0.79
remarkable for thiosemicarbazones 10e11 (90.10
9.27 1.87 M) and selenosemicarbazones 14e15 (27.80 1.61
vs. 8.77 2.23 M).
m
M, whereas 3,4-dihydroxybenzaldehyde 2 exhibits
M. The same tendency was especially
13.46 vs.
m
m
mM
m
analogs 8 (R ¼ Ph) [14] and 9 (R ¼
zides were, in turn, obtained from the corresponding aryl iso-
selenocyanates 6e7 [15] by addition of hydrazine monohydrate. 13
NMR spectrum of the hitherto unknown 4-( -naphthyl)selenose-
micarbazide 9 showed the presence of the characteristic selenoxo
group, with a resonance of 178.4 ppm.
Compounds 9e20 were isolated by column chromatography, or
directly by recrystallization from the crude reaction media to give
highly crystalline colored solids; spectral data fully confirmed the
structures of these Schiff bases. Thus, 1H NMR spectra of 10e20
depicted the presence of the imine-type proton, located at
7.92e8.33 ppm, and NH protons involved in strong hydrogen
bonding with the solvent (DMSO-d6), as deduced from their
remarkable deshielding (11.55e12.08 ppm). The resonance of both,
the thioxo and selenoxo groups in 13C NMR showed no appreciable
differences (roughly 173e175 ppm).
a
-naphthyl). Selenosemicarba-
The much lower EC50 values obtained for monohydroxylated de-
rivatives 10,14 and 17 in comparison with p-hydroxybenzaldehyde is
a clear evidence of the positive effect of incorporating a thioxo or
selenoxogroup for the antiradicalactivityof thetested compounds. In
this context, selenosemicarbazide 9, lacking phenolic hydroxyl
C
a
groups, exhibited an EC50 value of 6.65 0.67
as that obtained for the most potent compound, the trihydroxy
selenosemicarbazone 16 (4.87 1.57 M), and the most potent
aldehyde 3 (5.97 0.41 M). Moreover, derivatives 9,11,13,15,16,19,
mM, virtually the same
m
m
and 20 turned out to be stronger antiradical agents (up to a three-fold
increase) than hydroxytyrosol (2-(30,40-dihydroxyphenyl)ethanol), a
natural polyphenol especially abundant in olive tree [20], and
partially responsible for the nutraceutical properties of extra virgin
olive oil [21]. This simple phenolic derivative, a potent antioxidant, is
usuallytakenasareferencecompound(EC50 ¼13.80 1.41
mM)when
measuring the antiradical properties of naturally-occurring and
synthetic polyphenols.
2.2.2. Hydrogen peroxide scavenging
2.2. Antioxidant activity
Hydrogen peroxide is an important ROS in the cellular metabolic
pathway, being especially abundant in mitochondria, in a process
regulated by nitric oxide [22]. The capacity of scavenging H2O2 by
the prepared compounds was evaluated following the methodol-
ogy reported by Bahorun et al. [23]. In this assay, phenol red indi-
cator is subjected to oxidation with H2O2 in a process promoted by
horseradish peroxidase to furnish a chromophore with a strong
absorption at 610 nm. Reduction of the absorbance in the presence
of an antioxidant agent is directly correlated with the scavenging
activity of such compound.
Compounds 9e20 were tested as antioxidant agents against
Reactive Oxygen Species (ROS), a series of highly reactive in-
termediates produced in mitochondrial respiratory processes, or
induced by exogenous agents, and whose accumulation in tissues is
responsible for the oxidative stress, a cellular state with deleterious
effects on virtually all biomolecules [16]. Oxidative stress has been
demonstrated to play a predominant role in chronic inflammation
and in a plethora of degenerative processes, like cell aging, cardiac
damage, Parkinson's and Alzheimer's diseases [17]. Furthermore,
increased generation of ROS coming from mitochondrial dysfunc-
tion also constitutes a common and decisive feature in the initial
stages of many types of cancer [18].
Thio- and seleno derivatives 9e20, together with starting al-
dehydes 1e4 were tested at a final concentration of 100 mM; the
percentage of inhibition at this concentration is depicted in Table 1.
In this assay, the most potent derivatives turned out to be the thi-
osemicarbazides 10e13, with scavenging activities of roughly 80%.
Furthermore, the thioxo and selenoxo groups proved to be again
important motifs; thus, p-hydroxybenzaldehyde 1 lacked anti-H2O2
activity at the tested concentration, whereas thio- and seleno-
derivatives 10, 14 and 17, also with one single phenolic hydroxyl
moiety, showed 53.1e79.5% scavenging activity at the same con-
centration. In general, the seleno-isosters presented reduced ac-
tivity, in particular the N-naphthyl derivatives, the latter case
possibly due to steric hindrance.
In particular, derivatives 9e20 were evaluated as scavengers of
free radicals, H2O2 and alkyl peroxides, and the results are depicted
in Table 1.
2.2.3. Lipid peroxidation inhibition (Ferric Thiocyanate Method,
FTC)
High concentration of ROS provokes degradation of lipid bi-
layers, particularly at the doubly allylic positions of
Fig. 1. General structure of the targeted phenolic thio(seleno)semicarbazones.

V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
65
Scheme 1. Preparation of phenolic thio- and selenosemicarbazones 10e20.
Table 1
Antioxidant activity of compounds 9e20 compared to starting aldehydes 1e4.
Compound
Assay
(OH)n
X
Ar
DPPH method (EC50
,
m
M)
%H2O2 scavenginga
%Lipid peroxidation inhibitionb
Aldehydes
1
2
3
4
4-Hydroxy
e
e
e
e
e
e
e
e
>250
ec
ec
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
6.57 0.79
5.97 0.41
15.35 0.70
78.3 1.1
80.3 1.2
79.1 1.1
35.7 2.2
56.1 2.0
ec
9
e
e
e
6.65 0.67
56.9 0.6
21.9 2.0
Thiosemicarbazones
10
11
12
13
4-Hydroxy
S
S
S
S
Ph
Ph
Ph
Ph
90.10 13.46
9.27 1.87
13.9 1.23
7.0 0.65
79.5 0.2
79.3 0.9
ec
5.5 2.4
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
72.0 1.9
56.8 2.2
59.9 1.5
80.0 0.9
Selenosemicarbazones
14
15
16
17
18
19
20
4-Hydroxy
Se
Se
Se
Se
Se
Se
Se
Ph
Ph
Ph
27.80 1.61
8.77 2.23
4.87 1.57
21.67 2.31
18.17 2.19
10.70 0.73
8.87 1.47
72.5 0.2
69.0 0.6
64.0 0.8
53.1 0.7
46.3 0.7
4.5 0.4
31.0 2.5
66.5 1.6
65.7 1.5
23.3 2.2
72.0 1.6
43.0 5.0
24.3 3.1
3,4-Dihydroxy
3,4,5-Trihydroxy
4-Hydroxy
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
C
C
C
C
10H7
10H7
10H7
10H7
38.1 1.1
a
At 100
m
M.
At 0.74 mM.
No activity at the tested concentration.
b
c
polyunsaturated fatty acid residues, what therefore affects the
physical properties of cell membranes, like permeability [24].
Furthermore, lipid peroxidation is also quite a frequent process in
breast cancer, due to the abundant adipose tissue surrounding the
mammary gland, and the pro-oxidant environment generated [18].
The lipid peroxidation process was herein reproduced following
the procedure reported by Olszewska et al. [25], using linoleic acid
as the model compound and AAPH (2,20-azobis(2-amidinopropane)
dihydrochloride), a water-soluble azo compound, as a thermal free
radical initiator to induce the lipid degradation. The subsequent
alkyl peroxides generated oxidize Fe(II) to Fe(III) which in turn
furnishes a brick-red color complex with thiocyanate anion,
detectable by UVeVis spectroscopy.
All the compounds (tested at a final concentration of 0.74 mM)
exhibited inhibition of lipid peroxidation, except for 4-
hydroxybenzaldehyde 1, and 3,4,5-trihydroxybenzaldehyde 4,

66
V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
(Table 1). Especially remarkable are di- and trihydroxy thio(seleno)
semicarbazones 11 (72.0 6.25%), 15 (66.4 6.1%), 16 (65.6 5.9%)
and 18 (71.9 5.6%), which behaved as more potent alkyl peroxide
scavengers than the corresponding initial aldehydes (35.8 2.2%
2.4. a-Glucosidase inhibition
Carbohydrate-processing enzymes are ubiquitous biocatalysts
involved in a series of relevant processes like sugar, glycolipid and
glycoprotein metabolic routes, molecular recognition, or cell wall
formation [30]; one of the most widely-studied families of this kind
of enzymes are glycosidases, that catalyze the hydrolysis and for-
mation of glycosidic bonds. Defective glycosidases are responsible
for the development of certain metabolic disorders and diseases,
like lysosomal storage disorders (e.g. Gaucher or Fabry diseases) or
type-2 diabetes [26]. Nowadays it is widely accepted that tumor
cells are featured with aberrant glycosidase activities [31] and
therefore the corresponding glycosidases can be a promising anti-
cancer therapeutic target.
and 56.1
5.1%). These results show that not only the catechol
framework is important for this activity, but the thioxo and sele-
noxo groups present in the corresponding Schiff base architectures
provoke a substantial enhancement (up to a two-fold increase).
2.3. Pro-oxidant activity
It has been reported that some polyphenols exert both, antiox-
idant and pro-oxidant activities on a concentration-dependent
manner [26]. Although it is usually considered as a deleterious ef-
fect, the pro-oxidant properties of a substance, that is, the gener-
ation of ROS, might be useful, for instance, in certain anticancer
therapies [27]. In this context, the potential pro-oxidant activities of
thio- and selenosemicarbazones has been analyzed, and the results
are depicted in Table 2.
For this purpose, a Fenton reaction model [28] containing
FeCl3eEDTAeH2O2 was used; under these conditions, hydroxyl
radicals are generated, provoking the degradation of 2-
deoxyribose. The extent of such degradation was evaluated by
treatment with thiobarbituric acid to give the so called thio-
barbituric acid reactive substances (TBARS) that can be measured
spectrophotometrically at 532 nm.
The solvent used for each sample was DMSO; as it can be
observed, the control experiment shows a low absorbance value,
what can be attributed to the scavenging properties of DMSO [29].
This value is increased when in the control experiment, DMSO is
replaced by water.
For all the tested compounds, selenosemicarbazide 9, thio-
semicarbazones 10, 12, and selenosemicarbazones 14, 15, 18 and
20, a second measurement was accomplished without deoxyri-
bose, in order to take into consideration possible interactions of
the above compounds with TBARS. For all of them, a clear pro-
oxidant effect was observed at high concentrations (0.83 mM),
showing absorbance values higher than the control experiment.
This effect was particularly significant for monohydroxylated thio-
and selenosemicarbazones 10 and 14, 3,4-dihydroxy N-phenyl
selenosemicarbazone 15 and 3,4,5-trihydroxy-N-naphthyl sele-
nosemicarbazone 20. Moreover, for derivative 18, the same study
Stimulated by the fact that some plant extracts have shown
hypoglycemic properties, attributed to the
exerted by their phenolic constituents [32], we decided to test
compounds 9e20, and aldehydes 1e4 against -glucosidase from
baker's yeast (Table 3). For this purpose, p-nitrophenyl- -gluco-
pyranoside was used as the model substrate; the formation of the
corresponding p-nitrophenolate in a buffered medium (pH 6.8) was
followed at 400 nm. Initially, title compounds were tested at a final
a-glucosidase inhibition
a
a-D
concentration of 500 mM using a substrate concentration (250 mM)
equal to the calculated value of the enzymatic MichaeliseMenten
constant (KM, 0.25 0.03 mM), in order to calculate the percentage
of inhibition. Under these conditions, phenolic aldehydes did not
show any inhibition, and selenosemicarbazide 9, only 14% of inhi-
bition. Nevertheless, at the same concentration, all thio- and sele-
nosemicarbazones exhibited a percentage of inhibition against a-
glucosidase ranging from 52 to 100%. For all these compounds, the
type of inhibition and the corresponding inhibition constants (Ki's)
were calculated. Accordingly, the LineweavereBurk plot (or double
reciprocal plot) (1/v vs. 1/[S]) was used, where substrate concen-
tration ranged from 1/4 Km to 4 Km at a fixed inhibitor concentra-
tion (2e3 different inhibitor concentrations).
As depicted in Table 3, three different types of inhibition were
detected: uncompetitive (the inhibitor only binds the ES complex),
mixed (the inhibitor binds both E and ES complex with two
different inhibition constants, Kia and Kib, respectively), and non-
competitive, an especial case for the mixed inhibition, where Kia
and Kib are the same. Fig. 2aec shows, as an example, the three
modes of inhibition for compounds 12 (non-competitive), 16
(mixed) and 17 (uncompetitive).
was conducted on
a range of concentrations from 0.83 to
It is especially remarkable the activity found for the trihy-
droxylated thiosemicarbazone 12, a potent non-competitive in-
0.083 mM; as it can be observed, the pro-oxidant activity varies in
concentration-dependent mode, being more pronounced at
higher concentrations.
hibitor, with Kia ¼ Kib ¼ 9.6
exhibited an 8-fold increase in activity against baker's yeast
glucosidase compared to the pseudo-tetrasaccharide acarbose
mM. It is noteworthy that compound 12
a
-
Table 2
(Ki ¼ 77.9 11.4
mM) [33], a pharmaceutical drug marketed by Bayer
Pro-oxidant activity of thio- and selenosemicarbazonesa.
(Glucobay®) for reducing the post-prandial hyperglycemia associ-
ated to type-2 diabetes (non-insulin dependent).
Compound
Abs
Abs' (without deoxyribose)
Asample ꢀ A0sample
Derivatives 16e19, although featured with a smaller potency
Control (DMSO)
Control (H2O)
9
10
12
14
15
18
0.038
0.155
0.231
0.108
0.230
0.161
0.191
0.180
0.111b
0.060c
0.039d
0.262
e
e
compared to 12, also turned out to be moderate to good
sidase inhibitors, with inhibition constants ranging from 30.3 2.1
to 78.4 13.9 M. To the best of our knowledge, no previous reports
a-gluco-
e
e
0.167
0.014
0.163
0.074
0.094
0.101
0.044
0.019
0.002
0.165
0.064
0.094
0.067
0.087
0.097
0.079
0.067
0.041
0.037
0.097
m
on glycosidase inhibition exerted by thio- or selenosemicarbazones
can be found in literature.
2.5. Antiproliferative activity
The in vitro antiproliferative activity of compounds 1e4, 10e20
was evaluated using the protocol of the National Cancer Institute
(NCI) of the United States [34]. Compound 9 could not be tested due
to the lack of solubility under the experimental conditions. As a
model, a panel of six human solid tumor cell lines was used, namely
20
a
At 0.83 mM.
At 0.42 mM.
At 0.17 mM.
At 0.083 mM.
b
c
d

V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
67
Table 3
-Glucosidase inhibition of compounds 10e20 compared to starting aldehydes 1e4.
a
a
b
Compound
(OH)n
%Inhibition at 500
m
M
Kia
Kib
Type of inhibition
X
Ar
Aldehydes
1
2
3
4
4-Hydroxy
e
e
e
e
e
S
S
S
S
Se
Se
Se
Se
Se
Se
Se
e
N.I.c
N.I.c
N.I.c
N.I.c
14%
52%
68%
100%
70%
62%
81%
95%
100%
95%
95%
98%
e
e
e
e
e
e
e
e
e
e
e
e
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
e
e
e
e
e
e
e
9
e
e
Thiosemicarbazones
10
11
12
13
14
15
16
17
18
19
20
4-Hydroxy
Ph
Ph
Ph
Ph
Ph
Ph
Ph
213.0 172
134.4 8.5
Uncompetitive
Mixed
Non-competitive
Mixed
Non-competitive
Non-competitive
Mixed
Uncompetitive
Mixed
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
4-Hydroxy
3,4-Dihydroxy
3,4,5-Trihydroxy
4-Hydroxy
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
707.6 105.5
9.6 1.6
297.0 15.4
335.8 138.1
201.2 33.7
30.3 2.1
e
86.2 4.7
Selenosemicarbazones
78.4 13.9
59.0 9.2
69.8 18.2
76.8 9.2
70.3 19.9
C
C
C
C
10H7
10H7
10H7
10H7
57.8 19.0
33.0 2.4
153.6 24.1
Mixed
Mixed
a
Inhibition constant (
m
M) for the EeI binding.
M) for the EeSeI binding.
b
Inhibition constant (
m
c
No inhibition at 500 mM concentration.
A549 (non-small cell lung), HBL-100, (breast), HeLa (cervix),
SW1573 (non-small cell lung), as drug sensitive lines, T-47D
(breast) and WiDr (colon) as drug resistant lines.
exhibited a better antiproliferative profile against HBL-100, HeLa,
SW1573, T-47D and WiDr cell lines than 5-fluorouracil and cisplatin
[35], two widely-used chemotherapeutic agents (Table 4).
Data concerning the antiproliferative activity of the tested
Compound 17 is especially relevant, with a 6.5-, 19- and 21-fold
increase compared to 5-fluorouracil (HeLa, T-47D and WirD), and a
6- and 11-fold increase (T-47D, WirD), compared to cisplatin.
Therefore, these results, although preliminary, show the po-
tential of phenolic selenosemicarbazones, for becoming lead com-
pounds in the search for more potent anticancer drugs.
compounds are depicted in Table 4, and are expressed as GI50
,
that is, the concentration of the compound that inhibits 50% of
the tumor cell growth. Analysis of such data allowed us to
establish a series of structure-activity relationships. Thus, incor-
poration of a thio- or selenosemicarbazone scaffold to the
phenolic framework affords a very significant increase in the
antiproliferative activity, as within phenolic aldehydes, only
3,4,5-trihydroxybenzaldehyde 4 showed relevant activity against
3. Conclusions
A549 cell line (GI50 ¼ 7.30 2.40
m
M). There are also remarkable
In conclusion, phenolic N-aryl thio- and selenosemicarbazones
10e20 constitute attractive templates for the development of
multi-target drugs as an attempt to address multiple biological
targets in the treatment of complex diseases, like cancer. Relevant
activity differences in terms of the nature of the chalcogen atom
(sulfur or selenium), the number and position of the phenolic
hydroxyl groups, and the aromatic N-substituent. In this context,
selenosemicarbazones 14e20 were found to be stronger anti-
proliferative agents than the corresponding thio-isosters 10e13;
the difference (up to a 35-fold increase) is particularly significant
for monohydroxylated derivatives.
in vitro activities (antioxidant, a-glucosidase inhibition, and anti-
proliferative) were found for the synthesized compounds, where
some structure-activity relationships could be established
regarding the number and position of phenolic hydroxyl groups,
the nature of the chalcogen atom and the type of the aromatic N-
substituent. Improved activities were found in comparison with
marketed drugs, like acarbose (anti-diabetic), 5-fluorouracil,
cisplatin (anticancer agents), or natural compounds like phenolic
hydroxytyrosol (antioxidant found in olive oil).
It is also remarkable that for thiosemicarbazones 10e12, and
lines A549, HBL-100, SW1573 and T-47D, the order of activity was
found to be 10 < 11 < 12, what clearly indicates the importance of
the phenolic skeleton, and the positive effect of adding phenolic
hydroxyl groups.
Regarding selenium counterparts, most of them showed quite a
potent antiproliferative activity. Thus, derivatives 15e20 exhibited
4. Experimental section
GI50 values lower than 6.0
particular, N-naphthyl mono- and dihydroxylated derivatives 17
and 18 had values lower than 3.3 M (1.3e2.5 M, 1.1e3.2 M,
respectively) for all the lines considered.
mM for all the tested cell lines. In
4.1. Materials and methods
m
m
m
4.1.1. General procedures
It is also noticeable the higher potency associated to N-naphthyl
derivatives 17 and 18 when compared to N-phenyl counterparts 14
and 15, bearing the same phenolic skeleton. Accordingly, the nature
of the N-aryl substituent is another important factor when
designing a lead anticancer phenolic Schiff base.
Melting points were recorded on an Electrothermal apparatus
and are uncorrected. Optical rotations were measured with a Jasco
P-2000 polarimeter. 1H (300.1 and 500.1 MHz) and 13C (75.5 and
125.7 MHz) NMR spectra were recorded on Bruker Avance-300 and
Avance-500 spectrometers (with a TCI cryoprobe for 13C NMR
spectra) at rt. The assignments of 1H and 13C signals were
confirmed by homonuclear COSY and heteronuclear 2D correlated
It isalsoworth mentioning that N-naphthyl selenosemicarbazones
17 and 18, the derivatives featured with the strongest activity,

68
V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
Fig. 2. Double reciprocal plots for compounds 12 (a) (non-competitive), 16 (b) (mixed) and 17 (c) (uncompetitive).
spectra, respectively. Mass spectra (CI and LSI) were recorded on
Micromass AutoSpec-Q mass spectrometer with a resolution of
1000 or 10,000 (10% valley definition). For LSI spectra, ions were
produced by a beam of xenon atoms and Csþ ions, respectively,
using thioglycerol or o-nitrobenzyl alcohol as matrix and NaI as
additive. TLCs were performed on aluminum pre-coated sheets (E.
Merck Silica gel 60 F254); spots were visualized by UV light, and by
charring with 10% vanillin in EtOH containing 1% of H2SO4, or with
3% ninhydrin in EtOH. Column chromatography was performed
(HPLC-grade, 1.17 mL) was added the DMSO solution of the tested
compounds (30 L, 7 different concentrations), or pure DMSO
(30 L) as the control. The corresponding mixtures were kept in the
dark at rt for 30 min and then, the absorbance was measured at
515 nm against a blank. Plotting the values of DPPH remaining vs.
antioxidant concentration allows a straight line from which the
EC50 was calculated. All the measurements were carried out in
triplicate. The remaining DPPH concentration was calculated using
the expression:
m
m
using E. Merck Silica Gel 60 (40e63 mm). The antioxidant assays
were performed in a Hitachi U-2900 spectrophotometer with a
thermostated cuvette holder, using PS cuvettes.
Asample
Acontrol
%DPPH remaining ¼
ꢁ 100
Asample and Acontrol refer to the absorbances at 515 nm of DPPH in
the sample and control solutions, respectively.
4.1.2. Antioxidant assays
4.1.2.1. Free radical scavenging (DPPH method). The antiradical ac-
tivity of the thio- and seleno derivatives was measured using
commercially-available free radical DPPH, following the procedure
4.1.2.2. H2O2 scavenging activity. H2O2 scavenging activity was
reported by Prior et al. [19]. To a 60
mM methanolic solution of DPPH
measured using the procedure reported by Bahorun et al. [23]. To a

V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
69
Table 4
Antiproliferative activity (GI50) against human solid tumor cells of compounds 10e20 compared to starting aldehydes 1e4a.
Compound
(OH)n
Cell line
A549
X
Ar
HBL-100
SW1573
HeLa
T-47D
WiDr
Aldehydes
1
2
3
4
4-Hydroxy
e
e
e
e
e
e
e
e
>100
42 26
>100
>100
>100
>100
72 7.8
>100
54 7.5
>100
>100
49 3.1
>100
>100
>100
>100
43 5.8
>100
>100
>100
39 5.6
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
7.3 2.4
38 7.6
16 2.1
Thiosemicarbazones
Selenosemicarbazones
10
11
12
13
4-Hydroxy
S
S
S
S
Ph
Ph
Ph
Ph
34b
1.6 0.4
1.2 0.3
9.3 1.9
43 1.8
35 3.7
29 8.1
71 4.0
35 2.3
24 9.1
30 11
49 4.8
42 11
7.0 0.6
19 2.7
32 1.7
88 16
25 10
10 3.3
61 13
61 12
27 6.1
30 5.3
>100
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
14
15
16
17
18
19
20
4-Hydroxy
Se
Se
Se
Se
Se
Se
Se
Ph
Ph
Ph
1.4 0.7
2.6 0.5
1.5 0.3
1.3 0.01
1.1 0.2
1.7 0.2
1.7 0.2
ec
13 3.1
3.8 0.7
2.0 0.4
2.1 0.3
2.6 0.2
4.0 1.2
2.4 0.6
5.5 2.3
1.9 0.2
19 2.0
5.8 1.3
3.9 0.3
2.3 0.3
3.2 0.5
5.0 1.1
2.9 0.2
4.3 1.6
3.4 0.7
3.7 0. 6
3.3 0.4
2.1 0.2
2.2 0.3
2.2 0.3
3.0 0.3
2.3 0.5
15 4.7
2.0 0.3
7.2 0.8
3.7 0.4
2.9 0.6
2.5 0.4
2.5 0.4
3.5 0.6
2.7 0.5
47 18
16 2.9
3,4-Dihydroxy
3,4,5-Trihydroxy
4-Hydroxy
3,4-Dihydroxy
2,4,5-Trihydroxy
3,4,5-Trihydroxy
5-Fluorouracil [31]
Cisplatin [31]
3.2 0.6
3.7 1.6
2.3 1.7
2.8 0.7
5.2 1.6
3.6 1.0
49 6.7
26 5.6
C
C
C
C
10H7
10H7
10H7
10H7
ec
15 2.3
a
Values are expressed in mM, and are means of two to three experiments standard deviation.
Only one experiment.
Not tested.
b
c
solution of 3 ꢁ 10ꢀ3% H2O2 (100
m
m
L), 0.1 M NaCl (100
L) was added a 10.0 mM DMSO
L), or pure DMSO (100 L)
mL) and 0.1 M
Acontrol ꢀ Asample
phosphate buffer (pH 7.4, 700
%Inhibition ¼
ꢁ 100
Acontrol
solution of the tested compound (100
m
m
instead of the sample, as a control. The corresponding mixture was
incubated in the dark at 37 ꢂC for 20 min. Then, a solution con-
taining phenol red (0.2 mg/mL) and horseradish peroxidase
(0.1 mg/mL) in 0.1 M phosphate buffer (pH 7.4, 1.0 mL) was added.
The corresponding solution was kept at 37 ꢂC during 15 min. Then,
Acontrol and Asample refer to the absorbance of the control and
sample solutions, respectively.
4.1.3. Pro-oxidant activity
The analysis of the pro-oxidant activity [28] was accomplished
by using a modification of the original procedure reported [36] by
Halliwell et al. for determining the concentration of hydroxyl rad-
icals. A mixture of 0.1 M phosphate buffer (pH 7.4, 0.14 mL),
33.6 mM 2-deoxyribose (0.1 mL), 33.6 mM H2O2 (0.1 mL), 0.27 mM
FeCl3 (0.1 mL), 1.2 mM EDTA (in phosphate buffer, 0.1 mL), sample
(0.1 mL) and water (0.56 mL) was heated at 37 ꢂC for 1 h. The FeCl3
and EDTA solutions were premixed prior to the addition to the
mixture. After that, 1% (w/v) TBA in 0.05 M NaOH (1.0 mL) and 2.8%
(w/v) trichloroacetic acid (1.0 mL) were added and the corre-
sponding mixture was heated at 100 ꢂC in a water bath for 15 min.
Finally, after cooling down to rt, the absorbance was measured at
532 nm against the appropriate blank solution. For the control
experiment, the sample solution was replaced with pure DMSO
(0.1 mL).
1 M NaOH (100 mL) was added, and the mixture was kept at rt for
10 min; after that, the absorbance was read at 610 nm against a
blank. The results are expressed as percentages of reduction of
H2O2:
Acontrol ꢀ Asample
%Reduction H2O2 ¼
ꢁ 100
Acontrol
Acontrol and Asample refer to the absorbance in the control and
sample solutions, respectively.
4.1.2.3. Lipid peroxidation assay (ferric thiocyanate method, FTC).
Inhibition of the lipid peroxidation was measured using the ferric
thiocyanate method (FTC), following the procedure reported by
Olszewska et al. [25].
4.1.4.
a-Glucosidase inhibition
To a 10.0 mM DMSO solution (37 mL) of the tested compounds
The enzymatic inhibition assay was accomplished following the
method reported by Bols and co-workers [37]. The percentage of
inhibition was measured by preparing two 1.2-mL samples in PS
cuvettes containing 0.1 M phosphate buffer (pH 6.8) and p-nitro-
and 1.3% (w/v) methanolic linoleic acid (175
0.2 M phosphate buffer (pH 7.0, 175 L) in a screw-cap vial was
added AAPH in buffer (55.3 mM, 25
prepared by adding pure DMSO (37
mL) in H2O (88 mL) and
m
mL). The control solution was
m
L), instead of the sample. The
phenyl-a-
D-glucopyranoside at a concentration equal to the value
vial was incubated at 50 0.1 ꢂC for 24 h in the dark. After that, an
of Km. DMSO or inhibitor solution (500
mM final concentration in
aliquot (30
2.7:1H2OeDMSO mixture (1.1 mL), and a 10% aqueous solution of
NH4SCN (30 L) and 20 mM FeCl2 in 3.5% HCl (30 L) were added;
m
L) of the reaction mixture was dissolved in a
DMSO) plus water were added up to a constant volume of 1.14 mL
(control or inhibitor solution, respectively). Reaction was started by
m
m
adding 60 m
L of properly diluted enzyme solution at 25 ꢂC and the
after 3 min of incubation at rt, the absorbance was measured at
546 nm against the corresponding blank. The results are expressed
as the percentage of lipid peroxidation inhibition:
formation of the p-nitrophenolate was monitored for 125 s by
measuring the increase of absorbance at 400 nm. Initial rates were
calculated from the slopes of both plots (Abs vs. t) and were used for

70
V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
calculating the percentage of inhibition:
DMSO-d6) d 178.4 (CS), 136.2, 133.6, 130.1, 128.0, 126.3, 125.9 (x2),
125.8, 125.2, 123.0 (AreC); CI-MS m/z 265 ([M]þ, 9%); HRCI-MS
vo ꢀ v
calcd. for C11H11N3Se ([M]þ):265.0118, found: 265.0111.
%Inhibition ¼
ꢁ 100
vo
4.2.2. General procedure for the preparation of thio- and
selenosemicarbazones 10e20
To a solution of the aryl thio- or selenosemicarbazide 5, 8 or 9
(0.4 mmol) in EtOH (4 mL) were added the corresponding aldehyde
vo and v refer to reaction rates for the enzyme and enzyme plus
inhibitor solutions.
DMSO concentration was maintained at 5% (v/v) of the total
assay mixture; under these conditions, no appreciable effect of
(0.4 mmol), AcOH (50 mL, 3.0 mmol) and water (0.5 mL), and the
DMSO was observed on a-glucosidase activity. When a percentage
corresponding mixture was refluxed, or heated at 45 ꢂC (com-
pounds 18 and 20) under inert atmosphere for 24 h. After that, the
solvent was removed in vacuo and the residue was purified as
indicated in each case.
of inhibition over 50% was observed, the mode of inhibition and the
inhibition constants (Ki) were determined. In this case, a similar
assay was conducted, but using 5 different substrate concentra-
tions, ranging from 0.25 to 4.0 Km at a fixed inhibitor concentration;
2e3 different inhibitor concentrations were used for estimating the
mode of inhibition. For this purpose, the LineweavereBurk, or
double reciprocal plot, was used (1/v vs. 1/[S]).
4 . 2 . 2 .1. 1 - ( 3 0, 4 0- D i hy d ro x y b e n z yl i d e n e ) - 4 - p h e n yl - 3 -
thiosemicarbazone (11). 3,4-Dihydroxybenzaldehyde (55.2 mg,
0.4 mmol) and 4-phenylthiosemicarbazide (67 mg, 0.4 mmol) were
used. Column chromatography (cyclohexane/1:1 cyclo-
hexaneeEtOAc) afforded 11 as a yellow solid. Yield: 63 mg, 55%; mp
190 ꢂC (dec.) (Et2O); Rf: 0.30 (1:2 EtOAcecyclohexane). 1H NMR
4.1.5. Antiproliferative activity
The human solid tumor cell lines A549, HBL-100, HeLa, SW1573,
T-47D and WiDr were used herein. Cells were maintained in 25 cm3
culture flasks in RPMI 1640 supplemented with 5% heat inactivated
(300 MHz, DMSO-d6)
d 11.60 (s, 1H, NH), 9.93 (s, 1H, AreNH), 9.53,
9.00 (2s, 1H each, 2OH), 8.00 (s, 1H, N]CH), 7.60 (m, 2H, AreHo,
fetal calf serum and 2 mM L
-glutamine in a 37 ꢂC, 5% CO2, 95%
Ph), 7.37 (m, 2H, AreHm, Ph), 7.36 (d,1H, J3 ,6 ¼ 2.0 Hz, H-20), 7.23
0
0
humidified air incubator. Exponentially growing cells were trypsi-
nized and re-suspended in antibiotic containing medium (100 units
penicillin G and 0.1 mg of streptomycin per mL). Single cell sus-
pensions displaying >97% viability by trypan blue dye exclusion
were subsequently counted. After counting, dilutions were made to
give the appropriate cell densities for inoculation onto 96-well
microtiter plates. Cells were inoculated in a volume of 100 mL per
well at densities of 10,000 (A549, HBL-100, HeLa and SW1573),
15,000 (T-47D), and 20,000 (WiDr) cells per well, based on their
doubling times.
Compounds to be tested were dissolved in DMSO at an initial
concentration of 40 mM; derivative 9 was not soluble under the
experimental conditions, so it was discarded. Control cells were
exposed to an equivalent concentration of DMSO (0.25% v/v,
negative control). Each agent was tested in triplicate at different
0
0
0
(m, 1H, AreHp, Ph), 7.14 (dd, 1H, J5 ,6 ¼ 8.3 Hz, H-6 ), 6.79 (d, 1H, H-
50); 13C-RMN (75.5 MHz, DMSO-d6)
d
173.1 (CS), 148.2, 145.6 (C-30,
C-40), 145.2 (AreC), 139.8 (C]N), 128.0, 126.4, 125.6, 125.2, 120.9
(AreC), 115.5, 114.3 (C-20, C-50); CI-MS m/z 287 ([M]þ, 24%); HRCI-
MS calcd. for C14H13N3O2S ([M]þ): 287.0728, found: 287.0724.
4.2.2.2. 4-Phenyl-1-(30,40,50-trihydroxybenzylidene)-3-
thiosemicarbazone (13). 3,4,5-Trihidroxybenzaldehyde (61.6 mg,
0.4 mmol) and 4-phenylthiosemicarbazide (67 mg, 0.4 mmol) were
used. Column chromatography (cyclohexane/2:1 cyclo-
hexaneeEtOAc) afforded 13 as a yellow solid. Yield: 58 mg, 48%; mp
158 ꢂC (dec.) (Et2O); Rf: 0.40 (1:1 EtOAcecyclohexane); 1H NMR
(300 MHz, DMSO-d6)
d 11.56 (s, 1H, NH), 9.88 (s, 1H, AreNH), 9.02
(s, 2H, 2OH), 8.70 (s, 1H, OH), 7.92 (s, 1H, N]CH), 7.59 (m, 2H,
AreHo), 7.35 (m, 2H, AreHm), 7.18 (m, 1H, AreHp), 6.79 (s, 2H, H-20,
dilutions in the range of 1e100 mM. The drug treatment was started
H-60); 13C NMR (125.7 MHz, DMSO-d6)
d
175.2 (CS), 146.2 (C-30, C-
on day 1 after plating. Drug incubation times were 48 h, after which
time cells were precipitated with 25 mL ice-cold TCA (50% w/v) and
fixed for 60 min at 4 ꢂC. Then the sulforhodamine B (SRB) assay was
performed [38]. The optical density (OD) of each well was
measured at 492 nm, using BioTek's PowerWave XS Absorbance
Microplate Reader. Values were corrected for background OD from
wells only containing medium.
50), 144.2 (C-40), 139.1 (N]C), 136.0 (AreC ipso), 128.1 (x2) (AreCm),
125.2 (x2) (AreCo), 125.0þ(AreC), 124.2 (AreC), 107.0 (x2) (C-20, C-
60); CI-MS m/z 303 ([M] , 13%); HRCI-MS calcd for C14H13N3O3S
([M]þ): 303.0678, found: 303.0664.
4.2.2.3. 1-(40-Hydroxybenzylidene)-4-phenyl-3-selenosemicarbazone
(14). 4-Hydroxybenzaldehyde (48.8 mg, 0.4 mmol) and 4-
phenylselenosemicarbazide (8) (66.9 mg, 0.4 mmol) were used.
Column chromatography (cyclohexane/2:1 cyclohexaneeAcOEt)
afforded 14 as a brown solid. Yield: 53 mg, 42%; mp 136 ꢂC (dec.)
(Et2O); Rf: 0.59 (3:1 Et2O-cyclohexane); 1H NMR (300 MHz, DMSO-
4.1.6. Statistical analysis
For antioxidant and glycosidase inhibition assays, all tests were
run in triplicate. Values are expressed as the confidence interval,
which was calculated for P ¼ 0.95 using the Student's t-distribution.
For antiproliferative assays, the median and standard deviation
(SD) for 2ꢀ3 measurements were calculated.
d6) d 11.95 (s, 1H, NH), 10.32 (s, 1H, OH), 9.96 (s, 1H, AreNH), 8.18 (s,
1H, N]CH), 7.75 (m, 2H, H-20, H-60), 7.53 (m, 2H, AreHo), 7.37 (m,
2H, AreHm), 7.23 (m, 1H, AreHp), 6.81 (m, 2H, H-30, H-50); 13C NMR
(75.5 MHz, DMSO-d6)
d
173.3 (CS), 159.7 (C-40), 144.7 (AreC ipso),
4.2. Chemistry
139.9 (N]C), 129.7 (x2) (C-20, C-60), 128.0 (x2) (AreCm), 126.6 (x2)
(AreC), 125.7 (C-10), 124.8 (AreCp), 115.6 (x2) (AreC); CI-MS m/z
320 ([M þ H]þ, 10%); HRCI-MS calcd for C14H14N3O80Se ([M þ H]þ):
320.0302; found: 320.0302.
4.2.1. 4-a-Naphthylselenosemicarbazide (9)
A
solution of
a
-naphthyl isoselenocyanate 7 (500 mg,
2.15 mmol) and 85% hydrazine monohydrate (0.10 mL, 2.15 mmol)
in CH2Cl2 (25 mL) was stirred in the dark at rt and under inert at-
mosphere for 1 h. Removal of the solvent and crystallization (Et2O)
afforded 9 as a white solid. Yield: 522 mg, 92%; mp > 210 ꢂC (Et2O);
4 . 2 . 2 . 4 . 1 - ( 3 0, 4 0- D i hy d ro x y b e n z yl i d e n e ) - 4 - p h e nyl - 3 -
selenosemicarbazone (15). 3,4-Dihydroxybenzaldehyde (55.2 mg,
0.4 mmol) and 4-phenylselenosemicarbazide (8) (66.9 mg,
0.4 mmol) were used. Column chromatography (cyclohexane/1:2
cyclohexaneeEtOAc) afforded 15 as a yellow solid. Yield: 96 mg,
72%; mp 119 ꢂC (dec.) (Et2O); Rf: 0.59 (3:1 Et2Oꢀcyclohexane); 1H
Rf: 0.46 (EtOAc); 1H NMR (300 MHz, DMSO-d6)
d 9.66 (s, 1H, NH),
7.97e7.94 (m, 1H, AreH), 7.88e7.83 (m, 2H, AreH), 7.59e7.48 (m,
5H, 4AreH, AreNH), 6.55 (brs, 2H, NH2); 13C NMR (125.7 MHz,

V. Calcatierra et al. / European Journal of Medicinal Chemistry 94 (2015) 63e72
71
NMR (300 MHz, DMSO-d6)
d
11.90 (s, 1H, NH), 10.27 (s, 1H, AreNH),
(m, 1H, H-8 naphth), 7.89 (brd, 1H, J5,6 ¼ 7.7 Hz, H-5 naphth),
7.86e7.83 (m,1H, AreH), 7.57e7.51 (m, 4H, AreH), 7.41 (s,1H, H-60),
9.55, 9.02 (2brs, 1H each, 2OH), 8.12 (s, 1H, N]CH), 7.54 (m, 2H,
AreHo, Ph), 7.37 (m, AreHm, Ph), 7.35 (d, 1H, J2 ,6 ¼ 02.2 Hz, H-20),
6.35 (s, 1H, H-30); 13C NMR (125.7 MHz, DMSO-d6)
d 174.5 (CSe),
0
0
7.22 (m, 1H, Ar-Hp, Ph), 7.13 (dd, 1H, J5 ,6 ¼ 8.2 Hz, H-6 ), 6.78 (d, 1H,
151.3,149.6 (C-20, C-40),142.7 (N]C),138.7,136.8,133.7,130.6,127.9,
126.9, 126.7, 126.0, 125.4, 123.4, 113.3, 110.7, 103.2 (AreC).
0
0
H-50); 13C NMR (75.5 MHz, DMSO-d6)
d 173.1 (CSe), 148.2, 145.6 (C-
30, C-40), 145.2 (AreC), 139.9 (C]N), 128.0, 126.4, 12þ5.6, 125.2, 120.9
(AreC), 115.5, 114.3 (C-20, C-50); CI-MS m/z 335 ([M] , 2%); HRCI-MS
calcd for C14H13N3O820Se ([M]þ): 335.0173, found: 335.0180.
4.2.2.9. 4-(
selenosemicarbazone (20). 3,4,5-Trihydroxybenzaldehyde (61.6 mg,
0.4 mmol) and 4- -naphthylselenocarbazide (9) (105.7 mg,
a
-Naphthyl)-1-(30,40,50-trihydroxybenzylidene)-3-
a
4.2.2.5. 4-Phenyl-1-(30,40,50-Trihydroxybenzylidene)-3-
selenosemicarbazone (16). 3,4,5-Trihydroxybenzaldehyde (68.9 mg,
0.4 mmol) and 4-phenylselenosemicarbazide (8) (66.9 mg,
0.4 mmol) were used. Column chromatography (cyclohexane/1:2
cyclohexaneeEtOAc) afforded 16 as an orange solid. Yield: 39 mg,
28%; mp 148 ꢂC (dec.) (Et2O); Rf: 0.27 (1:1 EtOAc-cyclohexane); 1H-
0.4 mmol) were used. Recrystallization from Et2O afforded 20 as an
orange solid. Yield: 148 mg, 93%; mp 153 ꢂC (dec.) (Et2O); Rf: 0.56
(5:1 Et2O-cyclohexane); 1H NMR (300 MHz, DMSO-d6)
d 11.98 (s,
1H, NH), 10.55 (s, 1H, AreNH), 9.00 (s, 2H, 2OH), 8.76 (s, 1H, OH),
8.08 (s, 1H, N]CH), 7.99e7.96 (m, 1H, H-8 naphth), 7.90 (brd, 1H,
J5,6 ¼ 7.7 Hz, H-5 Naphth), 7.86e7.83 (m, 1H, AreH), 7.57e7.49 (m,
4H, AreH), 6.86 (s, 2H, H-20, H-60); 13C NMR (125.7 MHz, DMSO-d6)
RMN (300 MHz, DMSO-d6) d 11.85 (s, 1H, NH), 10.22 (s, 1H, AreNH),
d
175.1 (CSe), 146.1 (x2) (C-30, C-50), 145.5 (C-40), 136.7 (N]C), 136.2,
9.07e8.70 (m, 3H, 3OH), 8.03 (s, 1H, N]CH), 7.54 (m, 2H, AreHo,
Ph), 7.36 (m, 2H, AreHm, Ph), 7.22 (m, 1H, AreHp, Ph), 6.81 (s, 2H,
133.7, 130.5, 128.0, 127.0, 126.7, 126.0 (x2), 125.4, 124.3þ, 123.4
(AreC), 107.3 (x2) (C-20, C-60); LSI-MS m/z 402 ([M þ H] , 5%);
HRLSI-MS calcd for C18H16N3O830Se ([M þ H]þ): 402.0357, found:
402.0363.
H-20, H-60); 13C NMR (125.7 MHz, DMSO-d6)
d 173.0 (CSe), 146.1,
145.9, 145.6 (C-30, C, 40, C-50), 139.9 (C]N), 136.2 (AreC ipso), 128.0
(x2) (AreCm), 126.2 (x2) (AreCo), 125.6 (AreC), 124.1 (AreC), 107.2
(x2) (C-20, C-60); CI-MS m/z 353 ([M þ H]þ, 8%); HRCI-MS calcd for
C
14H14N3O830Se ([M]þ): 352.0200, found: 352.0193.
Acknowledgments
4.2.2.6. 1-(40-Hydroxybenzylidene)-4-(
selenosemicarbazone (17). 4-Hydroxybenzaldehyde (48.8 mg,
0.4 mmol) and 4- -naphthylselenocarbazide (9) (105.7 mg,
0.4 mmol) were used. Column chromatography (cyclohexane/1:2
cyclohexaneeEtOAc) afforded 17 as a yellow solid. Yield: 64 mg,
43%; mp 153 ꢂC (dec.) (Et2O); Rf: 0.44 (1:2 EtOAcecyclohexane); 1H
a
-naphthyl)-3-
ꢀ
ꢀ
We thank the Direccion General de Investigacion of Spain
(CTQ2008-02813, CTQ2011-28417-C02-01, CEI10/00018), the Junta
de Andalucía (FQM134), the Instituto de Salud Carlos III (PI11/
00840), the European Regional Development Fund (FEDER), and
the EU Research Potential (FP7-REGPOT-2012-CT2012-31637-
IMBRAIN), for financial support. G.B.P. also thanks the Obra Social
a
ꢀ
NMR (300 MHz, DMSO-d6)
d
12.08 (s, 1H, NH), 10.61 (s, 1H, OH),
La Caixa-Fundacion Caja Canarias for a predoctoral grant.
00 00
9.94 (s, 1H, AreNH), 8.23 (s, 1H, N]CH), 7.98 (dd, 1H, J6 ,8 ¼ 3.1 Hz,
J7 ,8 ¼ 6.2 Hz, H-800), 7.91 (brd, 1H, J5 ,6 ¼ 8.0 Hz, H-500), 7.84 (dd,
00 00
00 00
Appendix A. Supplementary data
1H, JH,H ¼ 3.4 Hz, JH,H ¼ 6.2 Hz, AreH), 7.78 (m, 2H, H-20, H-60),
13
7.58e7.49 (m, 4H, AreH, naphthyl), 6.79 (m, 2H, H-30, H-50);
C
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.02.037.
NMR (125.7 MHz, DMSO-d6) d
175.3 (CSe), 169.6 (C-40), 144.5 (C-100),
136.7 (N]C), 133,7, 130.6 (AreC), 129.7 (x2) (C-20, C-60), 127.9, 127.0,
126.8, 126.0 (x2), 125.4, 125.0, 123.5 (AreC), 115.6 (x2) (C-30, C-50);
LSI-MS m/z 370 ([M þ H]þ, 5%); HRLSI-MS calcd for C18H16N3O80Se
([M þ H]þ): 370.0459, found: 370.0460.
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