New benzothieno[3,2-d]-1,2,3-triazines with antiproliferative activity: Synthesis, spectroscopic studies, and biological activity

Article abstract of DOI:10.1016/j.bmcl.2014.06.007

New benzothieno[3,2-d]-1,2,3-triazines, together with precursors triazenylbenzo[b]thiophenes, were designed, synthesized and screened as anticancer agents. The structural features of these compounds prompted us to investigate their DNA binding capability

Full text of DOI:10.1016/j.bmcl.2014.06.007

Accepted Manuscript
New Benzothieno[3,2-d]-1,2,3-triazines with Antiproliferative Activity: Syn-
thesis, Spectroscopic Studies, and Biology Activity
Antonino Lauria, Alessia Alfio, Riccardo Bonsignore, Carla Gentile, Annamaria
Martorana, Giuseppe Gennaro, Giampaolo Barone, Alessio Terenzi, Anna
Maria Almerico
PII:
S0960-894X(14)00635-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.06.007
BMCL 21726
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
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14 April 2014
2 June 2014
4 June 2014
Please cite this article as: Lauria, A., Alfio, A., Bonsignore, R., Gentile, C., Martorana, A., Gennaro, G., Barone,
G., Terenzi, A., Almerico, A.M., New Benzothieno[3,2-d]-1,2,3-triazines with Antiproliferative Activity: Synthesis,
Spectroscopic Studies, and Biology Activity, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.06.007
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New Benzothieno[3,2-d]-1,2,3-triazines with
Antiproliferative Activity: Synthesis,
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Spectroscopic Studies, and Biology Activity
Antonino Lauria,* Alessia Alfio, Riccardo Bonsignore, Carla Gentile, Annamaria Martorana, Giuseppe
Gennaro, Giampaolo Barone, Alessio Terenzi, Anna Maria Almerico

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
New Benzothieno[3,2-d]-1,2,3-triazines with Antiproliferative Activity: Synthesis,
Spectroscopic Studies, and Biology Activity
Antonino Lauria,* Alessia Alfio, Riccardo Bonsignore, Carla Gentile, Annamaria Martorana, Giuseppe
Gennaro, Giampaolo Barone, Alessio Terenzi, Anna Maria Almerico
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università di Palermo - Via Archirafi, 32 - 90123 Palermo, Italy.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
New benzothieno[3,2-d]-1,2,3-triazines, together with precursors triazenylbenzo[b]thiophenes,
were designed, synthesized and screened as anticancer agents. The structural features of these
compounds prompted us to investigate their DNA binding capability through UV-Vis absorption
titrations, circular dichroism, and viscometry, pointing out the occurrence of groove-binding.
The derivative 3-(4-methoxy-phenyl)benzothieno[3,2-d]-1,2,3-triazin-4(3H)-one showed the
highest antiproliferative effect against HeLa cells and was also tested in cell cycle perturbation
experiments. The obtained results assessed for the first time the anticancer activity of
benzothieno[3,2-d]-1,2,3-triazine nucleus, and we related it to its DNA-binding properties.
Keywords:
Benzothienotriazines
Antiproliferative Activity
Spectroscopic Studies
Cell-cycle analysis
VLAK
2009 Elsevier Ltd. All rights reserved.
NO
NC
N
N
Fused heterocyclic systems are frequently focus of interest in
medicinal chemistry. Due to the presence of heteroatoms, these
small molecules, generally aromatic, could mime endogenous
substrates displaying a wide spectrum of biological effects. The
1,2,3-triazine scaffold represents a versatile lead structure with
N
N
N
N
N
N
O
S
N
1
2
4
multitude
of
interesting
applications
in
numerous
O
N
O
O
N
pharmacological fields. Tubercidin, is a clear example of 1,2,3-
triazine derivative with potent antimicrobial activity.1 Fusion of
heterocyclic nuclei like indole, thiophene and pyrazolo rings to
the 1,2,3-triazine core generates series of derivatives with
enhanced biological activities (e.g. antitumor,2–4 antimicrobial,5
antiviral,6 analgesic properties7).
N
N
S
S
N
N
N
N
N
N
N
N
O
Ph
Ph
3
3
HN
N
HN
2
N
N
3c
3a
3b
Figure 1. Designed heterocyclic compounds with wide spectra of
In this light, our research interests are focused on planning
heterocyclic compounds endowed with anticancer activity.8
Therefore, the synthesis of designed compounds leads to the
screening of new small molecules able to inhibit cancer cell
proliferation. Among planned structures, characterized by the
presence of annelated triazine ring and extended aromaticity,
derivatives of the nuclei indolo[1,2-c]-1,2,3-benzotriazine 1 and
pyrrolo[2,1-c]-1,2,4-triazine 2 (Figure 1) displayed a good
antitumor effect at micromolar concentration.4,9 Recently, also
heterocyclic compounds 3a,b, both embedded with properly
decorated triazine nucleus and sulfur atom in the skeleton,
demonstrated excellent in vitro and in vivo antiproliferative
activity.10 In the same way, indolo[3,2-e]-1,2,3-triazolo[1,5-
a]pyrimidine derivatives of type 3c, designed as DNA interactive
agents, have shown good antiproliferative activity against a wide
panel of human tumor cell lines.11
antiproliferative activity.
On the basis of these data, we decided to investigate the
benzothieno[3,2-d]-1,2,3-triazine core 4, which presents three
important requirements: the 1,2,3-triazine nucleus, the
polyaromatic system, and the sulfur atom. This choice was driven
by the opportunity of combining two different structural features
present in several DNA anticancer drugs: the 1,2,3-triazine core
and the aromaticity of polyheterocyclic moieties, enhanced by the
presence of the isosteric sulfur atom. In particular, the 1,2,3-
triazine ring may undergo ring-opening with the release of
alkylating species, and the planar heterocyclic scaffold could
electrostatically bind DNA.12 Moreover, as below reported, the
synthesis of the 1,2,3-triazine nucleus was achieved by
cyclization of the corresponding triazene derivatives. In this
regard, several triazene structural analogues showed antibacterial
and antifungal effects,13 in addition to an interesting antitumor

activity as DNA alkylating agents.14 Indeed, even though few
papers described the synthesis of the benzothieno[3,2-d]-1,2,3-
triazine core,15 only the anti-hyperlipidemic properties of few
tetrahydro derivatives have been reported,16–18 pointing out the
lack of data as anticancer candidate drug.
absorption band, in a region where DNA does not absorb is
noticeable in 10a (389 nm), 10b (392 nm), 10c (394 nm),10e
(398 nm), 11a (340 nm), 11b (348 nm), 11c (364 and 339 nm),
11d (342 nm) and 11e (333 nm). The linear trend of the band
intensities (Amax) with the compounds molar concentration shows
that the Lambert-Beer law is nicely fulfilled in the concentration
ranges used for the interaction studies.
In this paper we have explored a synthetic strategy
leading for the first time to benzothieno[3,2-d]-1,2,3-triazine
compounds 11. DNA binding experiments and biological assays
as antiproliferative agents against HeLa cells, on synthesized
derivatives and their triazenes precursors, were carried out.
Moreover, we have analysed the effect of the derivative with the
highest antiproliferative activity on cell cycle. Finally, in silico
studies, through the chemometric protocol VLAK (Virtual Lock-
and-Key),19 were also performed.20
Among linear derivatives, only compounds 10a and 10e show
modifications in their UV-Vis spectra in presence of increasing
amounts of B-DNA. In detail, absorption band at 389 nm for 10a
shows hypochromism of about 12% whereas the one sited at 398
nm for 10e displays hypochromism of about 11%. Furthermore, a
red shift of about 10 nm and 4 nm is noticeable respectively for
10a and 10e.
10a - ctDNA
10b - ctDNA
A well known synthetic route was applied to obtain the
benzothieno[3,2-d]-1,2,3-triazine-4(3H)-one derivatives of type
11 (Scheme 1).21,22
10c - ctDNA
10e- ctDNA
Figure 2. Absorption spectra of 10a-c and 10e in presence of increasing
amounts of ct-DNA. [10a]=17.0 μM, [DNA]=0.0-85.0 μM. [10b]=14.6 μM,
[DNA]=0.0-74.0 μM. [10c]=18.4 μM, [DNA]=0.0-48.3 μM. [10e]=18.5 μM,
[DNA]=0.0-74.0 μM. The arrows indicate the change upon DNA addition.
11a - ctDNA
11b - ctDNA
Scheme 1. Synthesis of properly decorated benzothieno[3,2-d]-1,2,3-triazines
11a-e.
The key intermediate ethyl 3-aminobenzo[b]thiophene-2-
carboxylate 7 was obtained, in excellent yield, by reaction of 2-
fluorobenzonitrile 5, ethyl thioglycolate 6, and sodium hydride,
using dry dimethyl sulfoxide (DMSO) as reaction solvent
(Scheme 1). Diazotization of 7 in acetic acid and consequent
coupling of the resulting diazonium salt 8 with primary amines
9a-e,23,24 allowed the isolation of the ethyl 3-(3-phenyltriaz-1-
enyl)benzo[b]thiophene-2-carboxylates 10a-e. Finally, title
benzothieno[3,2-d]-1,2,3-triazin-4(3H)-ones 11a-e were achieved
by intramolecular cyclization of triazenes 10, heating the reaction
mixture at 80 °C under basic condition.
11c - ctDNA
11d - ctDNA
11e- ctDNA
The benzothieno[3,2-d]-1,2,3-triazine compounds of type 11
and their precursors 10 were tested to verify the DNA binding
ability, through different techniques: UV-Vis absorption
spectroscopy, circular dichroism (CD), and viscosity studies,
using calf thymus DNA (ct-DNA). These methodologies were
recently applied by us in the DNA binding studies of heterocyclic
compounds and metal-complexes.25 Compound 10d was found to
be slightly insoluble in buffered aqueous solutions at the
DMSO/ethanol percentage used in all experiments, giving
scattering during spectroscopic titration and therefore it was not
further investigated. All tested compounds share absorption
bands at about 250 and 280 nm; furthermore, a characteristic
Figure 3. Absorption spectra of 11a-e in presence of increasing amounts of
ct-DNA. [11a]=27 μM, [DNA]=0.0-159.6 μM. [11b]=60 μM, [DNA]=0.0-
302.75 μM. [11c]=51.4 μM, [DNA]=0.0-222.7 μM. [11d]=54 μM,
[DNA]=0.0-149 μM. [11e]=29.1 μM, [DNA]=0.0-87.8 μM. The arrows
indicate the change upon DNA addition.

In each titration of triazine derivatives (Figure 3), with the
exception of 11b, the characteristic spectrum is modified by the
addition of increasing amounts of ct-DNA, showing a clear
hypocromic effect.
Table 1. DNA-binding constant (Kb) and binding
stoichiometry obtained using equation (1).
Cpd
10a
10e
11a
11c
11d
11e
Kb (M-1)
9.87×105
4.14×106
6.67×105
1.23×105
9.72×104
9.84×105
n
2.53
2.43
1.23
1.49
0.66
0.95
In details, the absorption band of 11a at 338 nm shows
hypochromism of about 85%, 11c at 339 nm shows
hypochromism of about 25%; the absorption band of 11d at 342
nm shows hypochromism of about 28%; the absorption band of
11e at 333 nm shows hypochromism of about 29%.
On the other hand, the band at 346 nm of 11b is not modified
by the presence of increasing amounts of DNA, suggesting thus
the absence of any perturbation of its electronic structure (Figure
3). Interestingly, the lack of interaction between 11b and DNA
might be due to the presence of Cl and F in the derivative, which
increases the lipophilic properties of the molecule.
CD is a useful technique to monitor changes in the DNA
structure following drug–DNA interactions, since its CD signals,
characterized by a positive band centered at 275 nm and a
negative band at 240 nm (black solid lines in Figure 5 and 6),27
are sensitive to little variations of the DNA chiral conformation.28
In order to obtain the DNA-binding constant (Kb) and
stoichiometry (n) of the tested compounds, the results reported in
Figure 2 and 3 have been analyzed by means of the McGhee-von
Hippel equation (1):26
For such a reason, we recorded CD titrations of DNA
solutions with increasing amounts of binders. As shown in Figure
5 and Figure 6, results demonstrate that CD spectra of DNA
never changes drastically as it would be if binders were acting as
intercalators.29,30 Only compounds 10a, 11c and 11d induce slight
changes in the CD spectrum of DNA, whereas nothing occurs
with compounds 10e, 11a and 11e (Figure 5 and 6).
r/L = Kb -nr) { (1-nr) / [1-(n-1)r] }(n-1)
(1)
where r is the ratio between the concentration of bound ligand
and DNAphosphate, L is the concentration of free ligand, Kb is the
DNA-binding constant and n is the number of DNAphosphate
binding sites occupied by a bound ligand.
Results of data fitting are shown in Figure 4 and summarized
in Table 1. As evident, linear derivatives 10a,e interact with B-
DNA with a stoichiometry that is almost two-fold than that one
of cyclic derivatives 11a, 11c-e. Moreover, obtained binding
constants are higher for linear than those of cyclic derivatives.
This is in agreement with the statistical treatment of McGhee-von
Hippel, by which the available DNA-binding sites, after ligand
binding, are as less as much sites are bound, since each bound
molecule hinders more than one free site.
ctDNA - 10a
ctDNA - 10e
Figure 5. Circular dichroism spectra of ct-DNA (black line) in presence of
increasing amounts of 10a and 10e. [DNA]=53 μM; [10a]=0.0-35.00 μM.
[DNA]=53 μM, [10e]=0.0-35.00 μM. The arrows indicate the change upon
binder addition. R1 = [Binder]/[DNA].
ctDNA - 11a
ctDNA - 11c
ctDNA - 11d
ctDNA - 11e
Figure 6. Circular dichroism spectra of ct-DNA (black line) in presence of
increasing amounts of 10a and 10c-e. [DNA]=53 μM, [11a]=0.0-41.27 μM.
[DNA]=53 μM, [11c]=0.0-24.96 μM. [DNA]=53 μM, [11d]=0.0-35.04 μM.
[DNA]=53 μM, [11e]=0.0-35.04 μM. The arrows indicate the change upon
binder addition. R1 = [Binder]/[DNA].
Figure 4. Scatchard plots of the absorption titrations shown in Figures 3: 10a,
black squares; 10e, red circles; 11a, blue up triangles; 11c, purple down
triangles; 11d, green diamonds; 11e, orange pentagons. Solid lines show the
fits to McGhee-von Hippel equation (1). DNA-binding constants (Kb) and
binding stoichiometries (n) are reported in Table 1.
In details, the DNA dichroic band at 275 nm decreases in the
presence of 11c and it increases at the higher molar ratios studied

for compounds 10a and 11d. Moreover CD induced bands (ICD)
in the range 300-400 point out that 11c and 11d act as new CD
chromophores after interacting with polynucleotide. These results
allow us to argue that no deep conformational changes in the
DNA helical structure occur due to interaction with the
synthesized compounds, as it is referred in literature for typical
groove binders.31
noting that synthetized triazines and their triazene precursors in
our experimental setup result more potent antiproliferative
agents, against HeLa cell line, then the drugs DTIC and TMZ.
Indeed, when tested in a concentration range from 50 to 1 µM,
neither DTIC nor its triazene precursor TMZ showed
antiproliferative effects (GI50>50M).
The derivative 11d, which showed the highest
antiproliferative effect, was also tested in cell cycle perturbation
experiments to evaluate its potential influence on cell-cycle
distribution. Flow cytometric analysis was performed after 12 h
and 24 h of incubation in order to detect the shifts in cell cycle
distribution before a significant amount of cells underwent
apoptosis. The working concentrations of compound 11d were
fixed at 1x and 2x its GI50 value at 24 h.
It is known that molecules able to intercalate DNA could
induce an unwinding of the duplex supercoils and elongation of
the DNA, giving rise to changes in its hydrodynamic properties,
especially an increase of its solution viscosity.32,33 In the absence
of more detailed structural data (X-ray crystallography or NMR),
viscosity experiments provide a critical test for intercalative
binding.34–36 Thus, viscosity measurements were carried out in
order to definitively exclude the intercalation mechanism for our
compounds.
As a matter of fact, the relative viscosity of a 5.3×10-5 M DNA
solution is not significantly affected by the presence of all the
compounds investigated (see Figure S1 in supplementary
material). Therefore, these results suggest the absence of DNA
intercalation and stress the conclusion that compounds 10a, 10e,
11a, 11c-e act as groove binders. On the contrary, no interaction
is observed for 10b, 10c, and 11b.
The histograms in Figure 5 represent the percentage of cells in
the respective cell cycle phase (G1, S, and G2/M), along with the
percentage of cells in the sub-G1 (dead cells) obtained by flow
cytometry. Untreated HeLa cells showed a normal diploid
distribution presenting fast proliferation characteristics, with S +
G2/M phase cells accounting for over 35% of the total cells.
Strong suppression of the G2/M phase with greater accumulation
of cells in the G0/G1 was observed after 11d exposure. The
effect is concentration-dependent and in our experimental
conditions treatment with 2xGI50 (8.8 µM) induced cell cycle
arrest as a result of 12 h treatment whereas after cell exposure to
1xGI50 (4.4 µM) cell cycle arrest was observed after 24 h
treatment. Moreover, after 24 h treatment with compound 11d at
2xGI50, a significant increase in the cell population in the sub-
G1 phase was observed, which is indicative of apoptotic cells.
The derivatives 11 and, due to the interesting antiproliferative
activity showed by triazenes compounds in our previous
findings,37 the intermediate triazenes 10, were tested using MTT
based cell viability assay against HeLa cancer cell line.
Dacarbazine (DTIC), an alkylating triazine employed in the
treatment of various malignancies,38 and its triazene precursor
temozolomide (TMZ), used against malignant brain tumors,39
were tested as internal reference compounds. All synthesized
compounds showed concentration-dependent and time-dependent
cytotoxicity, but the antiproliferative effect was achieved with
diverse efficacy. The GI50 values of selected compounds tested
at 24h and 48h are shown in Table 2. In general, triazenes 10
showed poor antiproliferative effects and displayed GI50 values
> 20 µM at 24 h and > 10 µM at 48 h, whereas, in most cases, the
corresponding triazines 11 were more active. This may indicate
that the thiophene ring, unlike what is observed for pyrrole
derivatives,37 makes the triazene moiety more stable and
consequently less active in biological environment.
Table 2. Cytotoxicity of triazines 11 and triazenes 10
expressed as GI50 values against HeLa cell line.
Cpd
GI50±SE (µM)
Cpd
GI50±SE (µM)
24 h
48 h
24 h
48 h
10a
10b
10c
10d
10e
49.34 ± 3.42
31.36 ± 2.81
42.08 ± 4.13
49.09 ± 3.91
22.76 ± 1.97
26.09 ± 2.23
12.87 ± 0.89
18.27 ± 2.37
17.39 ± 1.17
14.44 ± 1.05
11a
11b
11c
11d
11e
18.57± 1.52 9.79 ± 0.91
6.35 ± 0.74
5.32 ± 0.47
4.37 ± 0.83
2.45 ± 0.22
1.88 ± 0.23
1.24 ± 0.63
20.69 ±1.31 9.44 ± 0.73
Among triazenes, derivative 10e is the most active, but no
significant difference in GI50 values was observed between this
compound and the corresponding triazine 11e. Among triazines
11b, 11c, and 11d showed very strong cytotoxic effect with GI50
at 24 h in the low micromolar range and at 48 h in the
micromolar range. Finally, as determined by the LDH (lactate
dehydrogenase) assay, all the tested compounds in the
concentration range 1-50 µM did not affect the plasma membrane
integrity of the HeLa cell line (data not shown). It is worth
Figure 5. Effects of 11d derivative at 2x and 1x of its GI50 value on the cell
cycle distribution of HeLa cells following 12 h and 24 h treatment. Results
are expressed as the mean of two independent experiments, performed in
duplicate ±SE. Statistical analyses were performed using the Student’s t test
to determine the differences between the datasets. * denote significant
differences (p < 0.05) from untreated cells (control)

The specific arrest of cells in the G1 phase suggests that cell
exposure to 11d can lead to perturbation, and consequent
prevention, of DNA synthesis. Our results from spectroscopic
studies suggest that DNA-binding ability of 11d is correlated to
capacity of this derivative to act as minor groove binder. DNA-
interaction, even only reversible, can cause inhibition of DNA-
dependent functions, including DNA-replication, by interference
with the catalytic activity of important regulatory proteins, such
as DNA-helicase and topoisomerase I and II.40,41 Inhibition of
DNA-Topoisomerase-I by groove ligands has already been
shown and the ability of these DNA-binders to inhibit the
enzyme was attributed to direct interactions with DNA at the
minor groove.42
Table 3. VLAK results on benzothieno[3,2-d]-1,2,3-triazine
derivatives
MA (A%)*
Compound
11a
A
B
C
D
E
F
2. 55
8. 58
14. 57
20. 59
26. 59
3. 44
9. 43
15. 46
21. 48
27. 49
4. 39
5. 48
6. 53
7. 53
11b
10. 38
16. 51
22. 53
28. 47
11. 48
17. 49
23. 51
29. 54
12. 53
18. 54
24. 56
30. 57
13. 53
19. 54
25. 56
31. 55
11c
11d
11e
*MA, mechanism of action: A%, percentage of affinity; A, Alkylating
Agents; B, Antimitotic Agents; C, Topoisomerase Inhibitors; D,
I
On the other hand, arrest of cells in the G1 phase is thought to
be an important cellular defense mechanism that prevents
replication of damaged DNA,43 and we cannot exclude the
possibility that cell exposure to 11d may produce DNA-damage.
As shown in literature, DNA damage can results also by non
covalent-interactions after cell exposure to minor groove
binders.44,45
Topoisomerase II Inhibitors; E, RNA/DNA Antimetabolites; F, DNA
Antimetabolites.
Benzothieno[3,2-d]-1,2,3-triazines of type 11 are well
classified prevalently as alkylating agents (A). This trend,
although not sharp, suggests that the antiproliferative activity of
the benzothieno[3,2-d]-1,2,3-triazine derivatives could be related
also to DNA interactions and consequent DNA damage and
interference with DNA synthesis.
Moreover, despite spectroscopic studies suggest no direct
alkylating ability of 11d, as is the case for classical minor groove
ligands,46 the possibility that in cellular environment 11d may
produce adducts with alkylating capacity, similarly to what
reported for other minor groove binders, cannot be excluded.47
It is evident the spectroscopic studies give an account of the
first step of the DNA interaction of the proposed compounds with
a minor groove binding mode. Instead, the cell cycle analysis,
performed on the most active compound 11d, leads to
hypothesize an antiproliferative mechanism which could involve
covalent interactions, as in part confirmed by the in silico
analysis VLAK. In fact, strong suppression of the G2/M phase
with greater accumulation of cells in the G0/G1 was observed.
The specific arrest of cells in the G1 phase suggests that cell
exposure to 11d can lead to consequences as dramatic as DNA
damage with perturbation of DNA synthesis. Moreover, a
significant increase in the cell population in the sub-G1 phase
was observed, which is indicative of apoptotic cells. G1 arrest can
also be compatible with DNA-Topoisomerase-I inhibition and
potential disturbances of replication or DNA damage.59
Recent in silico approaches for the design of heterocyclic
compounds with biological activity48,49 allowed us to study
specific biological targets involved in carcinogenic processes,50–52
as well as to improve the antiproliferative activity of different
classes of heterocyclic compounds.48,53–55
The new benzothieno[3,2-d]-1,2,3-triazines, proposed herein,
were submitted to the in silico protocol VLAK,19 with the aim to
get information on the expected mechanism of action.
The first step of the VLAK protocol is the conversion of the
mechanism of action or the biological target in a “lock model” in
which the keys (the structures) are “fitted”.19
The NCI AntiCancer Agent Mechanism (ACAM) Database
was chosen as source of biological data for the construction of
the VLAK “lock models”. It consists of a repository of structures
with anticancer activity and a reasonably well-known mechanism
of action. Drug screening data are available for each structure as
measurement of their growth inhibition ability over a panel of
about 60 human tumour cell lines, and all tested molecules are
explicitly designed as a training set for neural network and
multivariate analysis.56,57
Acknowledgments
This work was in part supported by FFR2012 - University of
Palermo grant.
References and notes
1. Kumar, R.; Singh, A.; Singh, J.; Singh, H.; Roy, R. K.; Chaudhary, A.
Mini-Rev. Med. Chem. 2014, 14, 72.
2. Garuti, L.; Roberti, M.; Rossi, T.; Castelli, M.; Malagoli, M. Eur. J.
Med. Chem. 1998, 33, 43.
3. Ali, M. M.; Mahmoud, A. E.; Rashad, A. E. Asian J. Pharm. Clin. Res.
2011, 4, 118.
4. Cirrincione, G.; Almerico, A. M.; Barraja, P.; Diana, P.; Lauria, A.;
Passannanti, A.; Musiu, C.; Pani, A.; Murtas, P.; Minnei, C.;
Marongiu, M. E.; La Colla, P. J. Med. Chem. 1999, 42, 2561.
5. Saravanan, J.; Mohan, S.; Roy, J. J. Eur. J. Med. Chem. 2010, 45,
4365.
In particular, this database is made up by 121 anticancer drugs
classified according to their mechanism of action, which can be
considered like the biological target in the protocol. The NCI
ACAM Database was chosen because it was demonstrated that
molecular descriptors, by means of multivariate analysis, are able
to discriminate the drugs action mechanisms.58
The VLAK protocol, through a calculation procedure detailed
in the supplementary material, assigned to benzothieno[3,2-d]-
1,2,3-triazine compounds a percentage of affinity (A%) with
respect to six mechanisms of action, including alkylating agents,
antimitotic agents, topoisomerase I inhibitors, topoisomerase II
inhibitors, RNA/DNA antimetabolites, and DNA antimetabolites
(Table 3).
6. Migawa, M. T.; Drach, J. C.; Townsend, L. B. J. Med. Chem. 2005, 48,
3840.
7. Viswanatha, G. L.; Akinapally, N.; Shylaja, H.; Nandakumar, K.;
Srinath, R.; Janardhanan, S. Maced. J. Med. Sci. 2011, 4, 131.
8. Barone, G.; Terenzi, A.; Lauria, A.; Almerico, A. M.; Leal, J. M.;
Busto, N.; García, B. Coord. Chem. Rev. 2013, 257, 2848.
9. Diana, P.; Barraja, P.; Lauria, A.; Montalbano, A.; Almerico, A. M.;
Dattolo, G.; Cirrincione, G. Eur. J. Med. Chem. 2002, 37, 267.
10. Lauria, A.; Patella, C.; Abbate, I.; Martorana, A.; Almerico, A. M. Eur.
J. Med. Chem. 2013, 65, 381.
11. Lauria, A.; Patella, C.; Dattolo, G.; Almerico, A. M. J. Med. Chem.
2008, 51, 2037.

12. Lauria, A.; Diana, P.; Barraja, P.; Almerico, A. M.; Cirrincione, G.;
Dattolo, G. J. Heterocycl. Chem. 2000, 37, 747.
13. Nesterova, I. N.; Velezheva, V. S.; Alekseeva, L. M.; Padeiskaya, E.
N.; Radkevich, T. P.; Baklanova, O. V.; Golovanova, E. A. Pharm.
Chem. J. 1990, 24, 813.
14. Viviani, S.; Bonadonna, G.; Santoro, A.; Bonfante, V.; Zanini, M.;
Devizzi, L.; Soncini, F.; Valagussa, P. J. Clin. Oncol. Off. J. Am. Soc.
Clin. Oncol. 1996, 14, 1421.
L. Mol. Pharmacol. 1993, 44, 1064.
41. Drozdowska, D.; Rusak, M.; Miltyk, W.; Midura-Nowaczek, K. Arch.
Pharm. (Weinheim) 2009, 342, 87.
42. Bugreev, D. V.; Vasyutina, E. L.; Ryabinin, V. A.; Sinyakov, A. N.;
Buneva, V. N.; Nevinsky, G. A. Antisense Nucleic Acid Drug Dev.
2001, 11, 137.
43. Smith, M. L.; Fornace Jr., A. J. Mutat. Res. Genet. Toxicol. 1996, 340,
109.
15. Beck, J. R.; Yahner, J. A. J. Org. Chem. 1976, 41, 1733.
16. Beck, J. R.; Yahner, J. A. J. Org. Chem. 1973, 38, 2450.
17. Viswanatha, G. L.; Janaki Priyadarshini, B.; Hanumanthappa, S.;
Rangappa, S.; Janardhanan, S. Oman Med. J. 2012, 27, 388.
18. W. Schneller, S.; W. Clough, F. Heterocycles 1975, 3, 135.
19. Lauria, A.; Tutone, M.; Almerico, A. M. Eur. J. Med. Chem. 2011, 46,
4274.
44. Turner, P.; Denny, W. Curr. Drug Targets 2000, 1, 1.
45. Zihlif, M.; Catchpoole, D. R.; Stewart, B. W.; Wakelin, L. P. G.
Cancer Genomics - Proteomics 2010, 7, 323.
46. Hurley, L.; Reynolds, V.; Swenson, D.; Petzold, G.; Scahill, T. Science
1984, 226, 843.
47. Fedier, A.; Fowst, C.; Tursi, J.; Geroni, C.; Haller, U.; Marchini, S.;
Fink, D. Br. J. Cancer 2003, 89, 1559.
20. Lauria, A.; Terenzi, A.; Martorana, A.; Gennaro, G.; Barone, G.;
Almerico, A. M. Lett. Drug Des. Discov. 2014, 11, 15.
21. Neunhoeffer, H. Chemistry of 1,2,3-triazines and 1,2,4-triazines,
tetrazines, and pentazines; The Chemistry of heterocyclic
compoundsꢀ ; v. 33; Wiley: New York,1978.
22. Chattaway, F. D.; Walker, A. J. J. Chem. Soc. Resumed 1927, 323.
23. Day, B. F.; Campbell, T. W.; Coppinger, G. M. J. Am. Chem. Soc.
1951, 73, 4687.
48. Lauria, A.; Abbate, I.; Gentile, C.; Angileri, F.; Martorana, A.;
Almerico, A. M. J. Med. Chem. 2013, 56, 3424.
49. Lauria, A.; Tutone, M.; Barone, G.; Almerico, A. M. Eur. J. Med.
Chem. 2014, 75, 106.
50. Lauria, A.; Ippolito, M.; Fazzari, M.; Tutone, M.; Di Blasi, F.;
Mingoia, F.; Almerico, A. M. J. Mol. Graph. Model. 2010, 29, 72.
51. Lauria, A.; Ippolito, M.; Almerico, A. M. Comput. Biol. Chem. 2009,
33, 386.
24. Vaughan, K. Can. J. Chem. 1972, 50, 1775.
52. Lauria, A.; Ippolito, M.; Almerico, A. M. J. Mol. Graph. Model. 2009,
27, 712.
53. Lauria, A.; Patella, C.; Abbate, I.; Martorana, A.; Almerico, A. M. Eur.
J. Med. Chem. 2012, 55, 375.
25. Lauria, A.; Bonsignore, R.; Terenzi, A.; Spinello, A.; Giannici, F.;
Longo, A.; Almerico, A. M.; Barone, G. Dalton Trans. 2014, 43, 6108.
26. McGhee, J. D.; von Hippel, P. H. J. Mol. Biol. 1974, 86, 469.
27. Eriksson, M.; Nordén, B. In Methods in Enzymology; Jonathan B.
Chaires, M. J. W., Ed.; Drug-Nucleic Acid Interactions; Academic
Press, 2001; Vol. Volume 340, pp. 68–98.
28. Waring, M. J. J. Mol. Biol. 1965, 13, 269.
29. Silvestri, A.; Barone, G.; Ruisi, G.; Anselmo, D.; Riela, S.; Liveri, V.
T. J. Inorg. Biochem. 2007, 101, 841.
30. Barone, G.; Gambino, N.; Ruggirello, A.; Silvestri, A.; Terenzi, A.;
Turco Liveri, V. J. Inorg. Biochem. 2009, 103, 731.
31. Terenzi, A.; Barone, G.; Piccionello, A. P.; Giorgi, G.; Guarcello, A.;
Portanova, P.; Calvaruso, G.; Buscemi, S.; Vivona, N.; Pace, A. Dalton
Trans. 2010, 39, 9140.
54. Lauria, A.; Abbate, I.; Patella, C.; Martorana, A.; Dattolo, G.;
Almerico, A. M. Eur. J. Med. Chem. 2013, 62, 416.
55. Mingoia, F.; Di Sano, C.; Di Blasi, F.; Fazzari, M.; Martorana, A.;
Almerico, A. M.; Lauria, A. Eur. J. Med. Chem. 2013, 64, 345.
56. Weinstein, J. N.; Kohn, K. W.; Grever, M. R.; Viswanadhan, V. N.;
Rubinstein, L. V.; Monks, A. P.; Scudiero, D. A.; Welch, L.;
Koutsoukos, A. D.; Chiausa, A. J.; Et, A. Science 1992, 258, 447.
57. Van Osdol, W. W.; Myers, T. G.; Paull, K. D.; Kohn, K. W.;
Weinstein, J. N. JNCI J. Natl. Cancer Inst. 1994, 86, 1853.
58. Lauria, A.; Ippolito, M.; Almerico, A. M. QSAR Comb. Sci. 2009, 28,
387.
32. Cohen, G.; Eisenberg, H. Biopolymers 1969, 8, 45.
33. Reynisson, J.; Schuster, G. B.; Howerton, S. B.; Williams, L. D.;
Barnett, R. N.; Cleveland, C. L.; Landman, U.; Harrit, N.; Chaires, J.
B. J. Am. Chem. Soc. 2003, 125, 2072.
59. Antony, S.; Agama, K. K.; Miao, Z.-H.; Hollingshead, M.; Holbeck, S.
L.; Wright, M. H.; Varticovski, L.; Nagarajan, M.; Morrell, A.;
Cushman, M.; Pommier, Y. Mol. Pharmacol. 2006, 70, 1109.
34. Lerman, L. S. Proc. Natl. Acad. Sci. U. S. A. 1963, 49, 94.
35. Dougherty, G.; Pilbrow, J. R. Int. J. Biochem. 1984, 16, 1179.
36. Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochemistry
(Mosc.) 1992, 31, 9319.
37. Diana, P.; Barraja, P.; Lauria, A.; Almerico, A. M.; Cirrincione, G.;
Loi, A. G.; Musiu, C.; Pani, A.; La Colla, P.; Marongiu, M. E. Eur. J.
Med. Chem. 1999, 34, 353.
Supplementary Material
Experimental methodologies and NMR spectra are available.
38. Serrone, L.; Zeuli, M.; Sega, F. M.; Cognetti, F. J. Exp. Clin. Cancer
Res. CR 2000, 19, 21.
39. Friedman, H. S.; Kerby, T.; Calvert, H. Clin. Cancer Res. 2000, 6,
2585.
40. Bachur, N. R.; Johnson, R.; Yu, F.; Hickey, R.; Applegren, N.; Malkas,