New annelated thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines, with potent anticancer activity, designed through VLAK protocol

Article abstract of DOI:10.1016/j.ejmech.2013.01.019

Drug design was performed through the Virtual Lock-and-Key (VLAK) protocol. This in silico approach allowed to select new annelated thienotriazolopyrimidine derivatives, potentially antitumor drugs. Starting from benzothieno[2,3-e][1,2,3]triazolo[1,5-a]py

Full text of DOI:10.1016/j.ejmech.2013.01.019

European Journal of Medicinal Chemistry 62 (2013) 416e424
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New annelated thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines,
with potent anticancer activity, designed through VLAK protocol
*
Antonino Lauria , Ilenia Abbate, Chiara Patella, Annamaria Martorana,
Gaetano Dattolo, Anna Maria Almerico
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF”, Sezione di Chimica Farmaceutica e Biologica,
Università di Palermo, Via Archirafi 32, I-90123 Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Drug design was performed through the Virtual Lock-and-Key (VLAK) protocol. This in silico approach
allowed to select new annelated thienotriazolopyrimidine derivatives, potentially antitumor drugs.
Starting from benzothieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine and Pyrido[30,20:4,5]thieno[2,3-e][1,2,3]
triazolo[1,5-a]pyrimidine core structures, new derivatives of these nuclei were designed and synthe-
sized. Three of them were selected by the Development Therapeutical Program (DTP) of the National
Cancer Institute (NCI) for the anticancer screening against a panel of 60 human tumor cell lines. The
biological results showed that the new derivatives exhibited an excellent antiproliferative activity
reaching sub-micromolar concentration. Moreover, to be evidenced their low toxicity and high potency.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 28 November 2012
Received in revised form
10 January 2013
Accepted 13 January 2013
Available online 23 January 2013
Keywords:
Annelated thienotriazolopyrimidines
Domino reactions
VLAK protocol
Developmental Therapeutics Program (DTP)
Anticancer agents
1. Introduction
The VLAK protocol consists in the building of an in silico lock for
a biological target, starting from its own ligands. In particular, the
For many decades the molecular descriptors were used in the
Quantitative StructureeActivity/Property Relationships (QSAR/
QSPR) in various field of chemistry [1e3]. One of the limits in the
application of QSAR/QSPR models is the necessity to use structures
with a common core. Thus, if a diversity set database, with useful
biological data, is available, it is not usually possible to employ
these data in such an approach, although in the past some attempt,
by using molecular descriptors and multivariate analysis, was
performed [4].
complex biological target-ligand is considered like a Lock-and-Key
system.
The lock, to be released, requires a key with a shape that is able
to fit all the lock pins, in the same way a biological target to work
needs suitable interactions with a specific ligand in the complex
formation.
In the VLAK protocol, these interactions are represented by the
molecular descriptors, the equivalent of the pins in a Lock-and-Key
system (Fig. 1).
A method to bypass this limit is to use the molecular descriptors,
rather than in QSAR, in chemometric protocols like that recently
proposed by us in Virtual Lock-and-Key (VLAK) approach [5]. This
one was defined as the “in silico son” of the famous Lock-and-Key
model of Fischer [6e8].
Thus, a structure can be considered a potential ligand of a bio-
logical target, if its molecular descriptor values fit those of the
biological target. The protocol can be applied in all the cases in
which biological data are available (inhibition of a biological target,
mechanism of action, etc).
Another feature of the VLAK protocol is the capability to identify
the molecular descriptors values which are relevant when the
enhancement of a biological activity is detected. This approach
could help in the selection of convenient substituent in the lead
optimization process.
In a previous work the molecular modeling allowed to improve
the anticancer activity of indolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimi-
dine derivatives (Fig. 2), through the insertion of selected moieties
in the core structure [9]. In fact, when the derivative 1a, inactive as
Abbreviations: ACAM, AntiCancer Mechanism of Action database; CDI, 1,10-Car-
bonyldiimidazole; DMAP, 4-DiMethylAminoPyridine; DMF, N,N-DiMethylForma-
mide; DTP, Developmental Therapeutical Program; EDCI, 1-Ethyl-3-[3-
DiMethylAminoPropyl]Carbodiimide; MG_MID, Mean Graph MIDpoint; NCI, Na-
tional Cancer Institute of Bethesda (MD, USA); QSAR/QSPR, Quantitative Structure/
Activity/Property Relationships; VLAK, Virtual Lock And Key.
* Corresponding author. Tel.: þ39 0916167210; fax: þ39 0916230923.
E-mail addresses: antonino.lauria@unipa.it, antonino.lauria@gmail.com (A. Lauria).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.019

A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
417
Fig. 3. Benzothieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine 2, Pyrido[30,20:4,5]thieno
[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine 3, and indolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimi-
dine 4 core structures.
Fig. 1. Analogy Lock-and-Key and Biological Target-Ligand.
antitumor agent, was functionalized with side chains, selected on
the basis of molecular modeling techniques, the anticancer activity
has been increased (1bed).
The first step involves 1,3-cycloaddition reactions in which the
dipolar components are the 3-azido derivatives 5 and 6, prepared
from the corresponding amino derivatives 8 and 9, respectively
(Scheme 2).
The ethyl 3-aminobenzo[b]thiophene-2-carboxylate (8) is
commercially available (CAS 34761-09-6), whereas the ethyl 3-
aminothieno[2,3-b]pyridine-2-carboxylate (9) was obtained from
2-chloro-3-cyanopyridine and ethyl thioglycolate as described in
literature [19].
They were diazotized with sodium nitrite followed by the
addition of sodium azide in acetic acid or hydrochloric acid 6 N
at ꢀ5 ꢁC, according to the classical method used for the preparation
of azides (Scheme 2).
In this work, we propose the application of the VLAK protocol, at
first to analyze the chemometric features, which are correlated
with the increase of antitumor activity of the indolo[3,2-e][1,2,3]
triazolo[1,5-a]pyrimidine derivatives 1, and then to apply these
findings in the lead optimization of new heterocyclic derivatives.
In previous works our experience in the synthesis of poly-
heterocyclic core structures led to the construction of various ring
systems [10e17]. The most common synthetic approaches regarded
the one step construction of new ring systems by starting from
ortho-amino-cyano heterocycles and N-(BisMethylthio)Methyl-
enamino Acids (BMMAs) [14], or from ortho-azido-carboxyethyl
heterocycles and acetonitrile derivatives in domino 1,3-
cycloaddition reactions [10e13], or by using the bis-cycloaddition
of dipoles on annelated pyrazine derivatives [15e17].
The azides 5 and 6 were added at room temperature to the so-
dium salt of acetonitriles 7 in dry ethanol (Scheme 1).
A particular behavior was observed in the reaction of 6 with
malononitrile (7b). Only in this case it was possible to isolate the
intermediate 3-[triazol-1-yl] compound 10 (X ¼ N, R ¼ CN) with the
amino group and carboxylate functions unreacted. The isolation of
this intermediate allowed to confirm the mechanism of these re-
actions, already proposed by us [9e12]. The subsequent intra-
molecular cyclization originated the pyrimidine ring; this process is
strictly influenced by the amount of the solvent used in the reac-
tion. In fact, smaller volume of dry ethanol allowed to isolate the
compound 10 (X ¼ N, R ¼ CN) in good yield.
2. Results and discussion
2.1. Chemometric protocol
In connection with our previous studies, devoted to the search
of heterocyclic compounds with biological activities, and with the
aim to exploit further the domino reaction involving derivatives
ortho-azido-carboxyethyl heterocyclic systems and acetonitriles,
we planned to synthesize the cores benzothieno[2,3-e][1,2,3]tri-
azolo[1,5-a]pyrimidine 2 and pyrido[30,20:4,5]thieno[2,3-e][1,2,3]
triazolo[1,5-a]pyrimidine 3 (Fig. 3).
The synthesized compounds 2 and 3 were submitted to DTP NCI
protocol and tested against a panel of 60 human tumor cell lines.
They represent the thio and aza-thio isosteres of the core indolo
[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine 4, previously synthesized by
us [12]. The core structure of type 3 is known in literature [18],
while the type 2 is a new ring system. The attention on the rings of
type 2 and 3 was driven by the interest of studying new annelated
triazolo[1,5-a]pyrimidines having a different ring fusion and con-
sequently an aromatic feature different from the previously studied
nucleus (Fig. 2).
The synthetic methodology involves a domino reaction between
the ethyl 3-azido-benzo[b]thiophene-2-carboxylate (5) or the aza
analog ethyl 3-azidothieno[2,3-b]pyridine-2-carboxylate (6) and
acetonitriles 7 (Scheme 1).
Fig. 2. Antiproliferative activity of indolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimidine
derivatives.
Scheme 1. Domino reaction leading to benzothieno[2,3-e][1,2,3]triazolo[1,5-a]pyrim-
idine 2 and Pyrido[30,20:4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine 3.

418
A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
Table 2
VLAK results on indolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimidines (aA%).
ID
bMA
A
B
C
D
E
F
1a
1b
1c
1d
72
30
31
33
36
95
84
94
28
43
36
46
31
95
74
94
74
48
41
50
68
29
26
32
Scheme 2. Synthesis of ethyl 3-azido-benzo[b]thiophene-2-carboxylate (5) and ethyl
3-azidothieno[2,3-b]pyridine-2-carboxylate (6).
a
Percentage of affinity.
b
MA, mechanism of action: A, Alkylating Agents; B, Antimitotic Agents; C,
Topoisomerase I Inhibitors; D, Topoisomerase II Inhibitors; E, RNA/DNA Antime-
tabolites; F, DNA Antimetabolites.
In the preliminary biological screenings, derivatives 2a,b and
3a,b showed poor antiproliferative activity, with a similar response
in all panels (Table 1, Supplementary material).
These results are not surprising, since analogous behavior was
already observed in the case of the indolo[3,2-e][1,2,3]triazolo[1,5-
a]pyrimidine derivatives 1 [9].
With the aim to improve the biological activities of the new
synthesized compounds, the VLAK protocol was applied to the
annelated triazolo-pyrimidines 1 to verify which chemometric
features contributed to antitumor activity increase (Fig. 2), and to
take advantage of these findings in the lead optimization of 2 and 3.
The first step of the VLAK protocol consists in the conversion of
the biological target in a “lock model” in which the keys (the can-
didate ligands) could be “fitted” [5].
In this work we considered as biological targets the mechanism
of action classes reported by the NCI in the AntiCancer Mechanism
of Action (ACAM) database [20e22].
The ACAM database consists of 121 antitumor drugs classified
according to their known mechanism of action (see Experimental
section and Supplementary material).
Applying the VLAK protocol, each set of structures, related to
a specific mechanism of action, is converted in a lock model.
The use of these models in the in silico screening of an external
database could allow the identification of new structures, poten-
tially correlated to a specific mechanism, that generates the lock
model.
Recently we successfully applied this approach in the lead
optimization of pyrrolo-pyrimidine derivatives as anticancer agents
[23].
The analysis of the VLAK results showed a predominant A% with
the mechanism of action of classes B and D, respectively Anti-
mitotic Agents and Topoisomerase II Inhibitors. The low A% values
for the derivative 1a (36 and 31) are on the line to the poor anti-
proliferative activity showed by the main core structure [9]. On the
other hand, the B and D mechanisms reached the A% mean of 91
and 86, for derivatives 1bed. These high A% values underlines the
deep influence of the side chains in the improvement of the bio-
logical activity.
On the basis of these remarks, an in house database was built
considering the structures 2 and 3, which were decorated by
appropriate substituents in positions 4 (AeL) and 5 (aef), (Fig. 4).
The selection criteria of substituents were driven by different fea-
tures: acidity, steric hindrance, and at least the guideline of the
good biological results obtained for the indolo[3,2-e][1,2,3]triazolo
[1,5-a]pyrimidine derivatives [9].
The in house database was submitted to VLAK protocol. In the
Supplementary material the output results for all derivatives
against the lock models representing each class of drugs (MAs) are
reported. Table 3 reports the best ranked derivatives, expressed in
percentage of affinity (A%), focusing on the mechanisms of action B
and D, as suggested from the results obtained for the indolo[3,2-e]
[1,2,3]triazolo[1,5-a]pyrimidines 1.
Thus, the VLAK protocol led the selection of the derivatives 2aF,
3aE, 3aK for the class B, and 3aF, for the class D.
2.2. Chemistry
Thus, for each class of drugs (Alkylating Agents, Antimitotic
Agents, Topoisomerase I Inhibitors, Topoisomerase II Inhibitors,
RNA/DNA Antimetabolites, and DNA Antimetabolites), a “lock
model” was built starting from known drugs (Experimental
section).
The derivatives of type 1aed (Fig. 2) were submitted to “locks
fitting” and the percentage of affinity (A%) for each derivative was
calculated as reported in Experimental section (Table 2).
The synthesis of the compounds 2aF, 3aE, 3aK and 3aF, selected
on the basis of the VLAK protocol, was successfully achieved
through suitable synthetic pathways, as shown in Scheme 3.
The derivatives 2aF and 3aF were synthesized in three prepar-
ative steps. The intermediates 2aB and 3aB were obtained, at first in
very low yields, by reaction of the sodium salt of 2a and 3a, gen-
erated in situ using sodium hydride as base, and ethyl 4-
bromobutyrate in dry DMF (N,N-DiMethylFormamide) at 80 ꢁC.
The use of potassium carbonate (5-fold excess of the starting ma-
terial), at 60 ꢁC, increased the yields up to 63%. Then the hydrolysis
with NaOH in a solvent mixture EtOH/H2O, quantitatively yielded
the corresponding carboxylic acids 2aC and 3aC.
Table 1
Overview of cellular growth percentage average (G%a) of annelated benzothieno
[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives 2 and 3 against the nine DTP NCI
sub-panels cancer cell lines.
PANEL
Derivative (aG%a)
2a
98.89
2b
3a
3b
Breast cancer
CNS cancer
Colon cancer
Leukemia
103.68
101.13
101.24
78.70
104.17
103.98
108.99
97.98
102.70
99.70
102.12
105.18
107.34
104.94
104.07
105.93
99.51
102.45
102.28
106.50
104.38
106.38
110.45
110.88
88.49
103.55
96.98
97.38
96.49
98.54
Melanoma
98.04
Non-small cell lung cancer
Ovarian cancer
Prostate cancer
Renal cancer
105.04
103.11
99.82
100.47
Fig. 4. Annelated thienotriazolopyrimidines in house database submitted to VLAK
protocol.
a
cellular growth percentage average.

A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
419
Table 3
Table 4
In house database virtual screening selected hits.
Overview of one dose screening for all sub-panels (G%).
ID
aMA (A%)
PANEL
Derivative (aG%a)
2aC 2aF 3aB 3aC 3aD 3aE 3aF 3aK
106 105 106 79
A
B
C
D
E
F
2aF
3aE
3aF
3aK
38
42
33
40
86
86
76
86
45
38
49
47
78
70
86
69
51
47
53
55
31
36
33
33
Breast cancer
CNS cancer
Colon cancer
Leukemia
Melanoma
Non-small cell lung cancer 91
Ovarian cancer
Prostate cancer
Renal cancer
bMean
87
87
95
93
94
ꢀ5 96
ꢀ23
ꢀ18 88
ꢀ49 82
71 79
ꢀ57 92
ꢀ26 92
ꢀ16 94
ꢀ41 99
ꢀ34 94
L21 90
105 101
99 60
97 49
99 61
80 71
87 88
99 59
ꢀ43
ꢀ37
ꢀ22
ꢀ78
14
109
98
102
103
91
88
96
98
a
A%: percentage of affinity; MA, mechanism of action: A, Alkylating Agents; B,
Antimitotic Agents; C, Topoisomerase I Inhibitors; D, Topoisomerase II Inhibitors; E,
RNA/DNA Antimetabolites; F, DNA Antimetabolites. In bold are reported the highest
A% values for the selected compounds.
92
89
82
90
110 106
7
39
109
111
106
95 103 46
98
98
76 79
91 69
ꢀ14
L23
Compounds 2aF and 3aF were suitably prepared by amidation of
the acids 2aC and 3aC with histamine, in dioxane, in the presence of
CDI (1,10-carbonyldiimidazole) as coupling agent. A significant
yield increase was obtained through the reaction with DMAP (4-
DiMethylAminoPyridine) and EDCI (1-Ethyl-3-[3-Dimethylamino-
propyl]CarbodiImide) (up to 75%) [24].
Derivative 3aE was synthesized in two steps. The reaction of 3a
and 1-bromo-3-chloropropane gave the derivative 3aD in low
yield. From this last derivative, upon heating under reflux in
dibutylamine solvent free, 3aE was obtained.
a
Cellular growth percentage average.
In bold the G% mean of compounds passed on for evaluation in the full panel of
b
60 cell lines over a 5-log dose range.
a single concentration and the culture is incubated for 48 h. End-
point determinations were made with alamar blue [28]. Results
for each tested agent are reported as the percent of growth (G%) of
the treated cells when compared to the untreated control cells.
Compounds that reduced the growth of any one of the cell lines to
approximately 32% or less are passed on for evaluation in the full
panel of 60 cell lines over a 5-log dose range.
Derivative 3aK was prepared reacting in turn the intermediate
3aD with N-methylpiperazine solvent free.
The new annelated thienotriazolopyrimidines generally
exhibited an excellent antiproliferative activity. In particular the
derivatives 2aF, 3aF, and 3aK, showed G%a mean values of ꢀ21,
69, ꢀ23, respectively (Table 4).
2.3. Biological screening
These last derivatives exhibited G%a values significantly under
the level of 32% for at least one tumor cell line. Thus, according with
the selection criteria of DTP NCI protocol, they were passed to five
dose concentrations screening.
All the new designed pyrrolo-pyrimidines 2aF, 3aE, 3aK, 3aF,
and the intermediates 2aB, 2aC, 3aB, 3aC, 3aD, were subjected to
the NCI disease-oriented human cell lines screening assay to be
evaluated for their in vitro antitumor activity.
In the five dose screening, the antitumor activity is given by
three parameters for each cell line: pGI50 value (GI50 is the molar
concentration of the compound that inhibits 50% net cell growth),
pTGI value (TGI is the molar concentration of the compound lead-
ing to total inhibition of net cell growth), and pLC50 value (LC50 is
the molar concentration of the compound that induces 50% net cell
death). Moreover, a Mean Graph MIDpoint (MG_MID) is calculated
for each of the mentioned parameters, giving an average activity
parameter overall cell lines. For the calculation of the MG_MID,
insensitive cell lines are included with the highest concentration
tested. The discovery of compounds with new selectivity patterns is
The derivatives 2aC, 2aF, 3aB, 3aC, 3aD, 3aE, 3aF, and 3aK,
passed the selection criteria adopted by DTP NCI screening data and
consequently were in vitro analyzed.
A single dose (10 mM) of the accepted compounds was tested
against a panel of approximately 60 tumor human cell lines grou-
ped in nine disease subpanels, including breast, central nervous
system, colon, leukemia, melanoma, nonsmall-cell lung, ovarian,
renal, and prostate tumors cell lines (Table 4, Supplementary
material) [25e27].
In this biological protocol, each cell line is inoculated and pre-
incubated on a microtiter plate. Test agents were then added at
Scheme 3. Reaction steps leading to derivatives 2aF, 3aE, 3aK, and 3aF selected by VLAK protocol. Reagents and conditions: (a) K2CO3, DMF, Ethyl 4-Bromobutyrate; (b) NaOH,
EtOH/H2O; (c) Dioxane, DMAP/EDCI, histamine/TEA; (d) 1-Bromo-3-Chloropropane, K2CO3, DMF; (e) 1-methylpiperazine; (f) Dibutylamine,
D
.

420
A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
Table 5
Overview of five dose screening tests results for 2aF, 3aF, 3aK derivatives (Supplementary material).
Compd
pGI50
#
pTGI
#
pLC50
#
Range
MG_MID
Range
MG_MID
Range
MG_MID
2aF
3aF
3aK
54
59
59
4.73e6.74
4.47e7.02
5.03e6.80
5.81
5.29
5.30
52
59
59
<4.00e5.78
<4.00e5.35
4.75e5.76
5.29
4.58
4.94
44
59
59
<4.00e5.35
<4.00e4.41
4.39e5.42
4.62
4.15
4.60
#Number of cell lines investigated.
one of the targets of the DTP screening program. Selectivity of
a compound with respect to a certain cell line of the screen is
characterized by a high deviation of the particular cell line
Also the thioisostere 3aF exhibited remarkable antiproliferative
effect against at least seven cell lines (GI50 in the low micromolar
range, from 3.8 ꢂ 10ꢀ7 to 9.5 ꢂ 10ꢀ8 M). In particular SF-539 (CNS,
pGI50 ¼ 6.50), CCRF-CEM (leukemia, pGI50 ¼ 6.65), SN12C (renal,
pGI50 ¼ 6.48), and OVCAR-8 (ovarian, pGI50 ¼ 7.02) cell lines
resulted the more susceptible.
parameter compared to the MG_MID value. The overview of pGI50
,
pTGI, and pLC50 values are reported in Table 5, together with the
number of human tumor cell lines investigated.
An evaluation of the data reported in Supplementary material
(Table S2) revealed that all the derivatives selected for the five
dose screening, showed a significant antiproliferative activity
against all the human tumor cell lines investigated, generally up to
sub-micromolar concentration.
If we analyze the antiproliferative profile of the tested com-
pounds from the point of view of their structural features, it is
possible to evidence as a basic chain [methylpiperazine (type K) or
histamine (type F)] is necessary for a significant increase of the
biological activity. Moreover also the core ring system resulted
crucial for the modulation of biological aspects: the benzothieno
bicycle was more effective of the isostere pyridothieno one.
Considering the MG_MID values, in the range 5.29e5.81 for
pGI50, 4.58e5.29 for pTGI, and 4.15e4.62 for pLC50 (Table 5), the
most active compound of the series resulted derivative 2aF, at both
pGI50 and pTGI level (5.81, 5.29), followed by 3aF, and 3aK. With
respect to the tumor subpanels (Table S2), 2aF resulted particularly
effective against colon cancer and leukemia ones: in fact, the cal-
culated pGI50 MG_MID values for these sub-panels (6.04 and 6.05,
respectively) were always much higher than the overall cell lines
MG_MID value (6.28). Remarkable was also the activity toward the
ovarian subpanel, with pGI50 MG_MID value ¼ 5.81. With respect to
a particular cell line, 2aF showed an excellent response against the
SF-539 (CNS, pGI50 ¼ 6.18), K-562 (leukemia, pGI50 ¼ 6.36), CAKI-1
and SN12C (renal, pGI50 ¼ 6.74 and 6.57, respectively).
4. Experimental
4.1. Computational details
The compounds of the NCI ACAM database [26e28], were drawn
and optimized in vacuo by ligprep of MAESTRO SUITE [29]. The
entries containing cations or consisting of a mix of two structures
were excluded. Thus, the starting database was constituted from
114 compounds classified in six mechanism of action: 30, Alkylat-
ing Agents; 13, Antimitotic Agents; 24, Topoisomerase I Inhibitors;
15, Topoisomerase II Inhibitors; 16, RNA/DNA Antimetabolites; 16,
DNA Antimetabolites (Supplementary material).
CODESSA PRO software was used for the molecular descriptors
calculation [30]. For all structures 172 molecular descriptors belong-
ing to different classes were calculated (Supplementary material).
In particular, for each class of drugs with known mechanism of
action (Fig. 5a), the lock model was set as a sequence of molecular
descriptor value ranges. Each range was defined as
Moreover, it has to be underlined the anticancer activity of 2aF
on the HCT-116 (colon) and OVCAR-8 (ovarian) with a pGI50 ¼ 6.47
and 6.49, respectively, and pLC50 < 4, demonstrating the high po-
tency and low toxicity of the compound. It is also of remarkable
interest in the case of 2aF, the appreciable number of cell lines with
pLC50 < 4, (19/44), a result that can give account of the low toxicity
of such a compound.
Finally, also the results of biological screenings on compound
3aF need some considerations. Although the derivative was the
least effective among those tested (MG_MID value ¼ 5.29), it
exhibited remarkable antiproliferative effect against at least seven
cell lines (GI50 in the low micromolar range, from 3.8 ꢂ 10ꢀ7 to
9.5 ꢂ 10ꢀ8 M). In particular SF-539 (CNS, pGI50 ¼ 6.50), CCRF-CEM
(leukemia, pGI50 ¼ 6.65), SN12C (renal, pGI50 ¼ 6.48), and OVCAR-8
(ovarian, pGI50 ¼ 7.02) cell lines resulted the more susceptible.
m
Dj(MA) ꢃ
average value and
When the molecular descriptor value Dj of a tested structure T
(Fig. 5c) falls within the defined range ( Dj) ¼ 1 (Fig. 5d, i.e.
Dj ꢃ
D1, D3, and Dj), otherwise
s
Dj(MA), where
mDj(MA) is the molecular descriptor
sDj(MA) is the standard deviation (Fig. 5b).
m
s
a
a
¼ 0 (i.e. D2, D4). At the end each tested
structure is converted in a binary sequence (Supplementary
material).
In the proposed protocol, it was supposed that the higher is the
number of fitted molecular descriptors (
a
¼ 1), the higher will be
3. Conclusions
the potential anticancer capability of the investigated compound.
Thus, the percentage of affinity A% (eq. (1)) was defined for each
class (MA) as:
In this work, the combined use of the chemometric protocol
Virtual Lock-and-Key (VLAK) and the synthetic procedures, well
optimized by us for the building of heterocyclic compounds,
allowed to identify annelated thienotriazolopyrimidines with
antitumor activity up to sub-micromolar concentration. The de-
X
A% ¼
a
i; jðMAÞ=Dtot*100
(1)
Where S
a
i,j(MA) is the sum of all fitted molecular descriptors for
rivative
N-(2-(1H-Imidazol-4-yl)ethyl)-4-(5-oxo-3-phenylbenzo
the MA class and Dtot is the total of the molecular descriptors used
in the VLAK protocol.
[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-4(5H)-yl)butana-
mide (2aF) revealed to be an excellent candidate as antitumor
agent. In fact, it showed antiproliferative activity against all the
fifty-six tumor cell lines tested, up to sub-nanomolar concentration.
It is also of remarkable interest the appreciable number of cell lines
with pLC50 < 4, (19/44), a result that can give account of the low
toxicity of such a compound.
4.2. Chemistry
Unless otherwise indicated, all reagents and solvents were
purchased from commercial sources and used without further

A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
421
Fig. 5. VLAK protocol applied on NCI ACAM database. MA, mechanism of action; Dj, molecular descriptors; Si, structures; T, in silico screened entry.
purification. All melting points (ꢁC) were determined on a Büchie
Tottoli capillary apparatus and are uncorrected; IR spectra were
determined in bromoform with a Jasco FT/IR 5300 spectropho-
tometer. 1H NMR, 13C NMR spectra were recorded in DMSO-d6 so-
lution, unless otherwise specified, at 200 and 50.3 MHz,
respectively, using a Bruker AC-E series 200 MHz spectrometer.
Chemical shifts values are given in ppm and referred as the internal
standard to TMS (tetramethylsilane). The following abbreviations
are used: br ¼ broad signal, s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet. The purity of all compounds screened in
biological assays was determined to be >95% by HPLC/MS analysis.
Mass spectroscopy was performed using a GCeMS Shimadzu
QP5050 with EI (75ev). Microanalyses were in agreement with
theoretical values ꢃ0.4%. Thin layer chromatography was per-
formed on precoated (0.25 mm) silica gel GF254 plates, compounds
were detected with 254 nm UV lamp. Column chromatography was
performed with Merck silica gel ASTM (230e400 mesh), or Merck
aluminum oxide 90 (70e230 mesh), and with a Biotage FLASH40i
chromatography module (prepacked cartridge system). The Ethyl
3-Aminobenzo[b]thiophene-2-carboxylate (8) is commercially
available.
dispersion in mineral oil (0.36 g, 15 mmol) and dry dimethylsulf-
oxide (DMSO) (10 mL). After stirring at room temperature for
15 min, a solution of 2-chloro-3-cyanopyridine (1.40 g, 10 mmol) in
dry DMSO (20 mL) was added dropwise. The reaction was stirred at
room temperature for further 3 h and then was poured onto stirred
water/ice. The light yellow precipitate was collected by filtration
and dried to give 9, 2.22 g. Yield 100%. Mp 183e184 ꢁC. IR: 3487,
3395, (NH2), 1689 (CO) cmꢀ1. 1H NMR
d
: 1.29 (t, J ¼ 7.1 Hz, 3H), 4.30
(q, J ¼ 7.1 Hz, 2H), 7.31 (br s, 2H), 7.46 (dd, J ¼ 4.6, 8.2 Hz, 1H), 8.54
(dd, J ¼ 1.6, 8.2 Hz, 1H), 8.68 (dd, J ¼ 1.6, 4.6 Hz, 1H). 13C NMR
d: 14.4
(q), 59.9 (t), 93.3 (s), 119.3 (d), 125.5 (s), 131.4 (d), 147.7 (s), 150.7 (d),
159.6 (s), 164.3 (s).
4.2.3. Synthesis of ethyl 3-azidothieno[2,3-b]pyridine-2-carboxylate
(6)
A
solution of ethyl 3-aminopyrido[3,2-b]thiophene-2-
carboxylate (9) (0.3 g, 1.35 mmol) in 6 N hydrochloric acid
(12 mL) was cooled in an ice bath to ꢀ5 ꢁC. Then, a solution of
sodium nitrite (0.186 g, 2.7 mmol) in water (2 mL) was added
slowly, under vigorous stirring. After 30 min sodium azide (0.35 g,
5.4 mmol) in water (2 mL) was added dropwise over 10 min. The
reaction mixture was stirred for further 3 h at room temperature
and then slowly poured onto water/ice. The precipitate was col-
lected by filtration, and dried in the absence of light to give 6, 0.22 g.
4.2.1. Synthesis of ethyl 3-azido-benzo[b]thiophene-2-carboxylate (5)
A solution of ethyl 3-amino-benzo[b]thiophene-2-carboxylate
(8) (0.50 g, 2.3 mmol) in acetic acid (6 mL), was cooled in an ice
bath to ꢀ5 ꢁC. Then, a solution of sodium nitrite (0.32 g, 4.6 mmol),
in water (2 mL), was slowly added, under vigorous stirring. After
30 min, sodium azide (0.60 g, 9.2 mmol) in water (2 mL), was added
over 10 min. The reaction mixture was stirred for further 3 h at
room temperature and then slowly poured onto water/ice. The
precipitate was collected by filtration to give 5, 0.40 g. Yield 70%.
Yield 60%. Mp 93e94 ꢁC. IR: 2127 (N3), 1705 (CO) cmꢀ1. 1H NMR
d:
1.30 (t, J ¼ 7.1 Hz, 3H), 4.30 (q, J ¼ 7.1 Hz, 2H), 7.46 (dd, J ¼ 4.5,
8.2 Hz,1H), 8.54 (dd, J ¼ 1.6, 8.2 Hz,1H), 8.68 (dd, J ¼ 1.6, 4.5 Hz,1H).
13C NMR
d: 14.41 (q), 59.9 (t), 93.3 (s), 119.3 (d), 125.5 (s), 131.4 (d),
147.7 (s), 150.7 (d), 159.6 (s), 164.3 (s). Elem. Anal. calcd. for
C10H8N3O2S: C, 48.38; H, 3.25; N, 22.75; found: C, 48.42; H, 3.27; N,
22.36.
Mp 100 ꢁC (dec). IR: 2130 (N3), 1689 (CO) cmꢀ1. 1H NMR
d: 1.30 (t,
J ¼ 7.1 Hz, 3H), 4.33 (q, J ¼ 7.1 Hz, 2H), 7.34e7.51 (m, 2H), 7.88 (d,
4.2.4. General procedure for the preparation of benzothieno[2,3-e]
[1,2,3]triazolo[1,5-a]pyrimidines 2a,b
J ¼ 8.1 Hz, 1H), 8.14 (d, J ¼ 7.7 Hz, 1H). 13C NMR
d: 14.4 (q), 59.7 (t),
122.9 (d), 123.0 (d), 123.8 (d), 128.4 (d), 131.5 (s), 133.7 (s), 138.8 (s),
149.7 (s), 164.5 (s). Elem. Anal. calcd. for C11H9N3O2S: C, 53.43; H,
3.67; N, 16.99; found: C, 53.62; H, 3.55; N, 17.30.
The appropriate acetonitrile (1.73 mmol) in dry ethanol (2 mL)
was added to a solution of 1.3 M sodium ethoxide in ethanol
(2.7 mL), at room temperature. After 15 min of stirring, the reaction
mixture was added to a solution of azido derivative 5 (0.215 g,
0.87 mmol) in dry ethanol (3 mL) and the mixture was stirred
overnight at room temperature. Evaporation of the solvent under
reduced pressure gave a crude which was purified by column
chromatography using dichloromethane/methanol 9:1 as eluent.
4.2.2. Synthesis ofethyl 3-aminothieno[2,3-b]pyridine-2-carboxylate
(9)
According to the literature procedure [19], ethyl thioglycolate
(1.2 mL, 11 mmol) was added to a stirred mixture of NaH 60%

422
A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
4.2.5. 3-Phenylbenzo[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]
pyrimidin-5(4H)-one (2a)
ethyl 4-bromobutyrate (5.0 mmol) were added. The mixture was
heated at 60 ꢁC for 16 h, cooled to room temperature, and then
slowly poured onto water/ice. The crude solid was filtered and
purified by column chromatography using dichloromethane/ethyl
acetate 95:5 as eluent.
Yellow solid, 0.18 g. Yield 65%. Mp > 300 ꢁC. IR: 3401 (NH), 1651
(CO) cmꢀ1. 1H NMR
d
: 7.21 (t, J ¼ 7.4 Hz, 1H), 7.40e7.62 (m, 4H), 7.97
(d, J ¼ 7.4 Hz, 1H), 8.33 (d, J ¼ 7.4 Hz, 1H), 8.54 (d, J ¼ 8.1 Hz, 2H). 13C
NMR d: 123.3 (d), 123.7 (d), 124.0 (d), 124.4 (d), 125.2 (d), 128.4 (d),
128.6 (d), 128.7 (s), 133.5 (s), 133.6 (s), 135.1 (s), 141.0 (s), 144.6 (s),
152.4 (s), 154.6 (s). Elem. Anal. calcd. for C17H10N4OS: C, 64.14; H,
3.17; N, 17.50; found: C, 64.13; H, 3.25; N, 17.78.
4.2.12. Ethyl 4-(5-oxo-3-phenylbenzo[4,5]thieno[2,3-e][1,2,3]
triazolo[1,5-a]pyrimidin-4(5H)-yl)butanoate (2aB)
Purified by column chromatography using dichloromethane/
petroleum ether 98:2 as eluent to afford 2aB, 0.17 g. Yield 40%. Mp
4.2.6. 5-Oxo-4,5-dihydrobenzo[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidine-3-carbonitrile (2b)
123.6e124.0 ꢁC. IR: 1725, 1677 (CO) cmꢀ1. 1H NMR (CDCl3)
d: 1.22 (t,
J ¼ 7.4 Hz, 3H), 2.07e2.15 (m, 2H), 2.41 (t, J ¼ 7.2 Hz, 2H), 4.08 (q,
J ¼ 7.4 Hz, 2H), 5.04 (t, J ¼ 7.2 Hz, 2H), 7.49e7.62 (m, 5H), 7.90 (d,
J ¼ 7.4 Hz, 1H), 8.48 (d, J ¼ 7.4 Hz, 1H), 8.60 (d, J ¼ 8.0 Hz, 2H). 13C
Yellow solid, 0.14 g. Yield 60%. Mp > 300 ꢁC. IR: 3321 (NH), 2240
(CN), 1656 (CO) cmꢀ1 1H NMR
. d: 6.64e6.81 (m, 2H), 7.18 (d,
J ¼ 8.0 Hz,1H), 7.44 (d, J ¼ 7.3 Hz,1H). 13C NMR
d
: 104.2 (s), 106.6 (s),
NMR (CDCl3) d: 14.1 (q), 23.6 (t), 31.1 (t), 55.3 (t), 60.7 (t), 115.7 (s),
114.9 (s), 123.7 (d), 123.8 (d), 124.7 (d), 129.2 (d), 134.2 (s), 141.1 (s),
149.7 (s), 152.2 (s), 155.2 (s). Elem. Anal. calcd. for C12H5N5OS: C,
53.93; H, 1.89; N, 26.20; found: C, 53.84; H, 1.93; N, 26.58.
123.4 (d), 124.3 (d), 125.0 (d), 127.0 (d), 128.6 (s), 128.8 (d), 129.5 (d),
129.9 (d), 134.1 (s), 139.2 (s), 142.1 (s), 145.9 (s), 152.0 (s), 155.5 (s),
172.2 (s). Elem. Anal. calcd. for C23H20N4O3S: C, 63.87; H, 4.66; N,
12.95; found: C, 63.61; H, 4.83; N, 12.54.
4.2.7. General procedure for the preparation of Pyrido[30,20:4,5]
thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines 3a,b
4.2.13. Ethyl 4-(5-oxo-3-phenylpyrido[30,20:4,5]thieno[2,3-e][1,2,3]
triazolo[1,5-a]pyrimidin-4(5H)-yl)butanoate (3aB)
The appropriate acetonitrile (1.73 mmol) in dry ethanol (2 mL)
was added to a solution of 1.3 M sodium ethoxide in dry ethanol
(2.7 mL), at room temperature. After 15 min of stirring, the reaction
mixture was added to a solution of azido derivative 6 (0.215 g,
0.87 mmol) in dry ethanol (3 mL) and the mixture was stirred
overnight at room temperature. Evaporation of the solvent under
reduced pressure gave a crude which was purified by column
chromatography using dichloromethane/methanol 9:1 as eluent.
Purified by column chromatography using dichloromethane/
ethyl acetate 98:2 as eluent to afford 3aB, 0.27 g. Yield 63%. Mp
109.6e110.8 ꢁC. IR: 1723, 1683 (CO) cmꢀ1 1H NMR
. d: 1.08 (t,
J ¼ 7.1 Hz, 3H), 2.06 (m, 2H), 2.41 (t, J ¼ 7.2 Hz, 2H), 3.93 (q,
J ¼ 7.1 Hz, 2H), 4.94 (t, J ¼ 6.6 Hz, 2H), 7.50e7.63 (m, 4H), 8.42 (d,
J ¼ 8.1 Hz, 2H), 8.61 (dd, J ¼ 2.0, 7.7 Hz, 1H), 8.77 (dd, J ¼ 1.6, 6.9 Hz,
1H). 13C NMR
d: 13.9 (q), 23.6 (t), 30.3 (t), 55.2 (t), 59.9 (t), 112.6 (s),
120.8 (d), 126.2 (d), 127.5 (s), 128.1 (s), 128.8 (d), 129.6 (s), 131.9 (d),
136.6 (s), 145.3 (s), 150.8 (d), 151.6 (d), 152.0 (s), 161.7 (s), 172.0 (s).
Elem. Anal. calcd. for C22H20N5O3S: C, 60.96; H, 4.42; N, 16.16;
found: C, 60.67; H, 4.51; N, 16.38.
4.2.8. 3-Phenylpyrido[30,20:4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]
pyrimidin-5(4H)-one (3a)
Yellow solid, 0.17 g. Yield 63%. Mp > 300 ꢁC. IR: 3368 (NH), 1630
(CO) cmꢀ1.1H NMR
d
: 7.25 (d, J ¼ 7.8 Hz, 1H), 7.44e7.60 (m, 3H), 7.96 (d,
J ¼ 7.8 Hz,1H), 8.52e8.75 (m, 4H).13C NMR
d
: 120.0 (d),124.1 (d),125.3
4.2.14. General procedure for the preparation of 2aC and 3aC
To a stirred suspension of the carboxyethyl derivate (2aB, 3aB)
(0.5 mmol) in dry ethanol (5 mL), a solution of potassium hydroxide
(0.13 g, 2.5 mmol) in water (2 mL) was added dropwise. The mix-
ture was stirred at room temperature for 1e8 h. The ethanol was
evaporated under reduced pressure. The residue was dissolved in
water (10 mL) and then carefully adjusted to pH ¼ 1 with 6 N hy-
drochloric acid. The precipitate was filtered off, air dried and
recrystallized from ethanol to afford the corresponding acid.
(d), 128.3 (d), 129.2 (d), 131.2 (s), 131.3 (d), 132.3 (s), 133.3 (s), 144.2 (s),
146.2 (s), 150.3 (d), 151.8 (s), 151.9 (s), 162.3 (s). Elem. Anal. calcd. for
C16H9N5OS: C, 60.18; H, 2.84; N, 21.93; found: C, 60.28; H, 2.63; N, 21.56.
4.2.9. 5-Oxo-4,5-dihydropyrido[30,20:4,5]thieno[2,3-e][1,2,3]triazolo
[1,5-a]pyrimidine-3-carbonitrile (3b)
White solid, (0.14 g). Yield 60%. Mp > 300 ꢁC. IR: 3404 (NH), 2231
(CN), 1685 (CO) cmꢀ1. 1H NMR
d
: 7.6 (dd, J ¼ 6.5, 8.1 Hz, 1H), 8.65 (dd,
J ¼ 3.2, 8.1 Hz,1H), 8.79(dd, J ¼ 3.2, 6.5 Hz,1H), 8.85(br s,1H).13C NMR
d: 104.4 (s), 105.2 (s), 114.7 (s), 120.3 (s), 128.3 (d), 131.9 (d), 149.5 (s),
4.2.15. 4-(5-Oxo-3-phenylbenzo[4,5]thieno[2,3-e][1,2,3]triazolo
[1,5-a]pyrimidin-4(5H)-yl)butanoic acid (2aC)
150.9(d),151.9 (s),152.7(s),162.2(s). Elem. Anal. calcd. for C11H4N6OS:
C, 49.25; H, 1.50; N, 31.33; found: C, 49.27; H, 1.43; N, 31.75.
The recrystallization from ethanol affords 2aC as white solid,
0.40 g. Yield 100%. Mp 219.3e220.0 ꢁC. IR: 3520e2900 (OH), 1754,
4.2.10. Ethyl 3-(5-amino-4-cyano-1H-1,2,3-triazol-1-yl)thieno[2,3-
b]pyridine-2-carboxylate (10, X ¼ N, R ¼ CN)
1679 (CO) cmꢀ1. 1H NMR
d
: 2.09e2.16 (m, 2H), 2.40 (t, J ¼ 7.4 Hz,
2H), 5.01 (t, J ¼ 7.4 Hz, 2H), 7.50e7.64 (m, 5H), 7.93 (d, J ¼ 7.4 Hz,
1H), 8.40 (d, J ¼ 7.4 Hz, 1H), 8.58 (d, J ¼ 8.0 Hz, 2H), 9.98 (br s, 1H).
By identical synthetic procedure used for 3b, reducing the sol-
vent volume (2 mL), the intermediate 10 (X ¼ N, R ¼ CN) was filtered
off, air dried and recrystallized from ethanol. Yellow solid, 0.093 g.
Yield 35%. Mp > 300 ꢁC. IR: 3550, 3420 (NH2), 2254 (CN), 1648 (CO)
13C NMR
d: 23.6 (t), 31.0 (t), 55.3 (t), 115.6 (s), 123.4 (d), 124.3 (d),
125.0 (d), 127.0 (d), 128.5 (s), 128.8 (d), 129.4 (d), 129.9 (d), 134.6 (s),
139.2 (s), 142.0 (s), 145.9 (s), 152.0 (s), 155.4 (s), 172.1 (s). Elem. Anal.
calcd. for C21H16N4O3S: C, 62.36; H, 3.99; N, 13.85; found: C, 62.44;
H, 4.16; N, 13.48.
cmꢀ1. 1H NMR
d
: 1.39 (t, J ¼ 7.0 Hz, 3H), 4.35 (q, J ¼ 7.0 Hz, 2H), 7.60
(dd, J ¼ 4.6, 7.9 Hz, 1H), 8.63 (dd, J ¼ 1.6, 7.9 Hz, 1H), 8.77 (dd, J ¼ 1.6,
4.6 Hz, 1H), 8.89 (s, 2H). 13C NMR
: 14.4 (t), 59.5 (t), 103.8 (s), 120.2
d
(d),122.5 (s),128.6 (s),131.5(d),146.7 (s),150.7 (d),152.1 (s),152.3(s),
161.3 (s), 162.3 (s). Elem. Anal. calcd. for C13H10N6O2S: C, 49.67; H,
3.21; N, 26.74; found: C, 49.88; H, 3.38; N, 26.33.
4.2.16. 4-(5-Oxo-3-phenylpyrido[30,20:4,5]thieno[2,3-e][1,2,3]
triazolo[1,5-a]pyrimidin-4(5H)-yl)butanoic acid (3aC)
The recrystallization from ethanol affords 3aC as a white solid,
0.17 g. Yield 86%. Mp 171.0e171.6 ꢁC. IR: 3490e2965 (OH), 1745,
4.2.11. General procedure for the preparation of ethyl butanoate
derivatives 2aB, 3aB
To a stirred suspension of 2a or 3a (1.0 mmol) in dry N,N-
dimethylformamide (5 mL), potassium carbonate (3.0 mmol) and
1681 (CO) cmꢀ1. 1H NMR
d
: 1.99e2.13 (m, 2H), 2.30 (t, J ¼ 6.6 Hz,
2H), 4.40 (t, J ¼ 6.6 Hz, 2H), 7.27 (dd, J ¼ 4.5, 8.4 Hz, 1H), 7.35e7.54
(m, 4H), 7.88 (dd, J ¼ 8.2 Hz, 2H), 8.65 (dd, J ¼ 1.5, 4.5 Hz, 1H), 9.33
(br s, 1H). 13C NMR
d: 24.6 (t), 30.3 (t), 53.9 (t), 99.4 (s), 118.9 (s),

A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
423
119.2 (d), 125.6 (s), 126.1 (d), 128.2 (d), 128.8 (d), 129.6 (s), 132.6 (d),
137.8 (s), 143.0 (s), 150.0 (d), 159.0 (s), 166.1 (s), 173.7 (s). Elem. Anal.
calcd. for C20H15N5O3S: C, 59.25; H, 3.73; N, 17.27; found: C, 59.19;
H, 3.56; N, 17.63.
cooling the mixture was poured onto water/ice and the precipitate
was filtered off.
4.2.23. 4-(3-(4-Methylpiperazin-1-yl)propyl)-3-phenylpyrido
[30,20:4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5(4H)-one
(3aK)
4.2.17. General procedure for the preparation of 2aF and 3aF
To a stirred suspension of the acid (2aC, 3aC) (0.44 mmol) in dry
dioxane (5 mL), DMAP (0.05 g, 0.45 mmol) and EDCI (0.17 g,
9.2 mmol) were added under nitrogen atmosphere, at 0 ꢁC. After 2 h
of stirring at room temperature, histamine (0.102 g, 9.2 mmol) was
carefully added. The reaction mixture was incubated at 50 ꢁC for
12 h. Then, the reaction solvent was evaporated under reduced
pressure and the crude was purified by column chromatography
using dichloromethane/methanol 9:1 as eluent.
Purified by alumina column chromatography using dichloro-
methane/petroleum ether 98:2 as eluent to afford 3aK, 0.10 g. Yield
85%. Mp 88.3e90.2 ꢁC. IR: 1655 (CO) cmꢀ1. 1H NMR (CDCl3)
d: 1.90e
2.03 (m, 2H), 2.14 (s, 3H), 2.28e2.43 (m,10H), 5.12 (t, J ¼ 6.8 Hz, 2H),
7.47e7.60 (m, 4H), 8.56 (d, J ¼ 8.0 Hz, 2H), 8.72 (dd, J ¼ 1.4, 7.4 Hz,
1H), 8.78 (dd, J ¼ 1.4, 4.5 Hz, 1H). 13C NMR (CDCl3)
d: 26.1 (t), 45.9
(q), 53.0 (t), 53.2 (t), 54.7 (t), 55.1 (t), 99.7 (s), 120.2 (d), 126.9 (d),
128.3 (s), 128.5 (s), 128.8 (d), 129.8 (d), 132.1 (d), 138.2 (s), 145.2 (s),
151.4 (d), 151.7 (s), 152.9 (s), 163.4 (s). Elem. Anal. calcd. for
C24H25N7OS: C, 62.72; H, 5.48; N, 21.33; found C, 62.53; H, 5.66; N,
21.62.
4.2.18. N-(2-(1H-Imidazol-4-yl)ethyl)-4-(5-oxo-3-phenylbenzo
[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-4(5H)-yl)
butanamide (2aF)
White solid, 0.13 g. Yield 60%. Mp 171.2e171.6 ꢁC. IR: 3360 (NH),
4.2.24. Procedure for the preparation of 3aE
1720,1680 (CO) cmꢀ1. 1H NMR
d
: 2.02e2.18 (m, 4H), 3.17 (t, J ¼ 7.4 Hz,
A stirred suspension of 3aD (0.10 g, 0.25 mmol) in dibutylamine
(3 mL) was heated at reflux for 2 h. After cooling the mixture was
poured into water/ice and the precipitate was filtered off and pu-
rified by alumina column chromatography, using dichloromethane/
ethyl acetate 98:2 as eluent.
2H), 3.45 (m, 2H), 4.96 (t, J ¼ 6.6 Hz, 2H), 6.72 (s, 1H), 7.49e7.89 (m,
7H), 8.16 (d, J ¼ 6.9 Hz,1H), 8.45 (d, J ¼ 6.9 Hz,1H), 8.57 (d, J ¼ 7.4 Hz,
2H). 13C NMR
d: 24.1 (t), 26.9 (t), 32.0 (t), 38.7 (t), 55.5 (t), 123.7 (d),
124.0 (d), 124.1 (d), 125.5 (d), 126.5 (d), 128.5 (s), 128.9 (d), 129.7 (d),
129.8(d),133.6 (s),134.6(d),137.0(s),137.2(s),141.0(s),151.3(s),153.9
(s),154.5 (s), 170.7 (s),171.2 (s). Elem. Anal. calcd. for C26H23N7O2S: C,
62.73; H, 4.66; N, 19.71; found: C, 62.62; H, 4.77; N, 19.35.
4.2.25. 4-(3-(Dibutylamino)propyl)-3-phenylpyrido[30,20:4,5]
thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5(4H)-one (3aE)
White solid, 0.08 g. Yield 67%. Mp 109.5e110.5 ꢁC. IR: 1643 (CO)
4.2.19. N-(2-(1H-Imidazol-4-yl)ethyl)-4-(5-oxo-3-phenylpyrido
[30,20:4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-4(5H)-yl)
butanamide (3aF)
cmꢀ1.1H NMR
d
: 0.86 (t, J ¼ 6.7 Hz, 6H),1.24e1.34 (m,10H),1.81e1.95
(m, 2H), 2.34 (t, J ¼ 6.7 Hz, 4H), 2.49 (t, J ¼ 6.7 Hz, 2H), 7.49e7.61 (m,
4H), 8.58 (dd, J ¼ 1.7, 8.2 Hz, 2H), 8.73 (dd, J ¼ 1.7, 7.9 Hz,1H), 8.81 (dd,
White solid, 0.14 g. Yield 75%. Mp 239.7e240.3 ꢁC. IR: 3363 (NH),
J ¼ 1.7, 4.6 Hz,1H). 13C NMR
d: 13.0 (q), 20.6 (t), 26.3 (t); 29.1 (t), 50.8
1727, 1685 (CO) cmꢀ1
.
1H NMR
d: 2.00e2.21 (m, 4H), 3.14 (t,
(t), 53.7 (t), 55.1 (t), 120.1 (d), 126.9 (d), 128.3 (s), 128.5 (s), 128.8 (d),
129.8 (d), 132.1 (d), 138.4 (s), 145.2 (s), 145.8 (s), 151.4 (d), 151.7 (s),
152.9 (s), 163.4 (s). Elem. Anal. calcd. for C27H33N6OS: C, 66.36; H,
6.60; N, 17.20; found C, 66.47; H, 6.84; N, 17.59.
J ¼ 7.0 Hz, 2H), 3.44 (t, J ¼ 6.5 Hz, 2H), 5.00 (t, J ¼ 6.5 Hz, 2H), 6.70 (s,
1H), 7.48 (s, 1H), 7.49e7.68 (m, 4H), 7.72 (dd, J ¼ 4.6, 8.0 Hz, 1H),
8.57 (dd, J ¼ 1.6, 8.3 Hz, 2H), 8.85e8.90 (m, 2H). 13C NMR
d: 24.5 (t),
26.9 (t), 31.1 (t), 38.8 (t), 53.7 (t), 99.4 (s), 113.0 (d), 115.8 (s), 119.2
(d), 121.4 (s), 125.5 (s), 126.1 (d), 128.2 (d), 127.8 (d), 128.4 (d), 129.6
(s), 132.5 (d), 137.6 (s), 143.0 (s), 149.9 (d), 159.0 (s), 166.1 (s), 173.6
(s). Elem. Anal. calcd. for C25H22N8O2S: C, 60.23; H, 4.45; N, 22.48;
found: C, 60.17; H, 4.51; N, 22.77.
4.3. Biology
The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and
2 mM
L-glutamine. For a typical screening experiment, cells are
4.2.20. Procedure for the preparation of 3aD
inoculated into 96 well microtiter plates in 100
ml at plating den-
To a stirred suspension of 3aA (0.62 g, 1.93 mmol) in dry N,N-
dimethylformamide (12 mL), potassium carbonate (0.8 g,
5.81 mmol) and 1-bromo-3-chloropropane (1.52 g, 9.68 mmol)
were added. The mixture was heated at 60 ꢁC for 16 h, cooled to
room temperature, and then slowly poured onto water/ice. The
precipitate was purified by column chromatography using
dichloromethane/ethyl acetate 98:2 as eluent.
sities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 ꢁC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line
at the time of drug addition (Tz). Experimental drugs are solubi-
lized in dimethylsulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate is thawed and
diluted to twice the desired final maximum test concentration with
4.2.21. 4-(3-Chloropropyl)-3-phenylpyrido[30,20:4,5]thieno[2,3-e]
[1,2,3]triazolo[1,5-a]pyrimidin-5(4H)-one (3aD)
Pale yellow solid, 0.42 g. Yield 55%. Mp 200.8e201.7 ꢁC. IR: 1645
(CO) cmꢀ1 1H NMR (CDCl3)
d
: 2.28e2.43 (m, 2H), 3.63 (t, J ¼ 6.3 Hz,
complete medium containing 50
four, 10-fold or ½ log serial dilutions are made to provide a total of
five drug concentrations plus control. Aliquots of 100 l of these
different drug dilutions are added to the appropriate microtiter
wells already containing 100 l of medium, resulting in the
required final drug concentrations.
mg/mL gentamicin. Additional
2H), 5.13 (t, J ¼ 6.3 Hz, 2H), 7.47e7.57 (m, 4H), 8.56 (d, J ¼ 8.2 Hz,
2H), 8.70 (dd, J ¼ 1.7, 6.6 Hz, 1H), 8.79 (dd, J ¼ 1.7, 4.6 Hz, 1H). 13C
m
NMR d: 31.2 (t), 41.3 (t), 53.6 (t), 100.0 (s), 115.0 (s), 120.2 (d), 127.0
(d), 128.2 (s), 128.8 (d), 130.2 (d), 132.2 (d), 139.2 (s), 145.7 (s), 151.5
(d), 151.9 (s), 152.9 (s), 163.4 (s). Elem. Anal. calcd. for C19H14ClN5OS:
C, 57.65; H, 3.56; N, 17.69; found: C, 57.76; H, 3.39; N, 17.43.
m
Following drug addition, the plates are incubated for an addi-
tional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative humidity. For
adherent cells, the assay is terminated by the addition of cold TCA.
4.2.22. Procedure for the preparation of 3aK
A
stirred suspension of 3aD (0.10 g, 0.25 mmol) in 1-
Cells are fixed in situ by the gentle addition of 50
ml of cold 50% (w/
methylpiperazine (3 mL) was heated at reflux for 2 h. After
v) TCA (final concentration, 10% TCA) and incubated for 60 min at

424
A. Lauria et al. / European Journal of Medicinal Chemistry 62 (2013) 416e424
[6] E. Fischer, Influence of the configuration on the action of the enzymes, Ber.
4
ꢁC. The supernatant is discarded, and the plates are washed five
Dtsch. Chem. Ges. 27 (1894) 2985e2993.
[7] E. Fischer, Faraday lecture on “SYNTHETICAL chemistry in its relation to
biology”, J. Chem. Soc. 91 (1907) 1747e1765.
times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 ml) at 0.4% (w/v) in 1% acetic acid is added to each well, and
[8] E. Fischer, Bedeutung der Stereochemie fur die Physilogie Hoppe-Seyler’s,
Z. Physiol. Chem. 26 (1898) 82e83.
[9] A. Lauria, C. Patella, G. Dattolo, A.M. Almerico, Design and synthesis of 4-
substituted indolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives with anti-
tumor activity, J. Med. Chem. 51 (2008) 2037e2046.
[10] A. Lauria, P. Diana, P. Barraja, A. Montalbano, G. Cirrincione, G. Dattolo,
A.M. Almerico, New tricyclic systems of biological interest. Annelated 1,2,3-
triazolo[1,5-a]pyrimidines through domino reaction of 3-azidopyrroles and
methylene active nitriles, Tetrahedron 58 (2002) 9723e9727.
plates are incubated for 10 min at room temperature. After staining,
unbound dye is removed by washing five times with 1% acetic acid
and the plates are air-dried. Bound stain is subsequently solubilized
with 10 mM trizma base, and the absorbance is read on an auto-
mated plate reader at a wavelength of 515 nm. For suspension cells,
the methodology is the same except that the assay is terminated by
fixing settled cells at the bottom of the wells by gently adding 50 ml
[11] A. Lauria, C. Patella, P. Diana, P. Barraja, A. Montalbano, G. Cirrincione,
G. Dattolo, A.M. Almerico, New tetracyclic ring system of biological interest.
Indolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimidines through domino reaction of 2-
azidoindole, Heterocycles 60 (2003) 2669e2675.
[12] A. Lauria, C. Patella, P. Diana, P. Barraja, A. Montalbano, G. Cirrincione,
G. Dattolo, A.M. Almerico, A synthetic approach to new polycyclic ring system
of biological interest through domino reaction: indolo[2,3-e][1,2,3]triazolo
[1,5-a]pyrimidine, Tetrahedron Lett. 47 (2006) 2187e2190.
of 80% TCA (final concentration, 16% TCA). Using the seven
absorbance measurements [time zero, (Tz), control growth, (C), and
test growth in the presence of drug at the five concentration levels
(Ti)], the percentage growth is calculated at each of the drug con-
centrations levels. Percentage growth inhibition is calculated as:
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢄ ꢂ 100 for concentrations for which Ti ꢅ Tz
[13] A. Lauria, I. Abbate, C. Patella, N. Gambino, A. Silvestri, G. Barone,
A.M. Almerico, Pyrazolo[3,4-d][1,2,3]triazolo[1,5-a]pyrimidine:
a new ring
system through Dimroth rearrangement, Tetrahedron Lett. 49 (2008) 5125e
5128.
[14] A. Lauria, M. Bruno, P. Diana, P. Barraja, A. Montalbano, G. Cirrincione,
G. Dattolo, A.M. Almerico, Annelated pyrrolo-pyrimidines from amino-cyano-
pyrroles and BMMAs as leads for new DNA-interactive ring systems, Bioorg.
Med. Chem. 13 (2005) 1545e1553.
[15] A. Lauria, A. Guarcello, G. Dattolo, A.M. Almerico, Bis-1,2,4-triazolo[4,3-a:3’,4’-
c]quinoxalines of pharmaceutical interest from 1,3-dipolar cycloaddition,
Tetrahedron Lett. 49 (2008) 1847e1850.
[16] A. Lauria, A. Guarcello, G. Macaluso, G. Dattolo, A.M. Almerico, Reactivity of
asymmetric benzo-condensed diazines with nitrilimine dipoles in the 1,3-
dipolar cycloaddition reactions, Tetrahedron Lett. 50 (2009) 7333e7336.
[17] A. Lauria, A. Guarcello, G. Dattolo, A.M. Almerico, Study of reactivity in the 1,3-
dipolar cycloaddition reactions leading to new triazolopyrrolopyrazine ring
systems, Synlett 14 (2010) 2067e2070.
½ðTi ꢀ TzÞ=Tzꢄ ꢂ 100 for concentrations for which Ti < Tz:
Three dose response parameters are calculated for each exper-
imental agent. Growth inhibition of 50% (GI50) is calculated from
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as meas-
ured by SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition (TGI) is
calculated from Ti ¼ Tz. The LC50 (concentration of drug resulting in
a 50% reduction in the measured protein at the end of the drug
treatment as compared to that at the beginning) indicating a net
loss of cells following treatment is calculated from [(Ti ꢀ Tz)/
Tz] ꢂ 100 ¼ ꢀ50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect is
not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum con-
centration tested.
[18] N.T. Pokhodylo, V.S. Matiychuk, N.D. Obushak, Synthesis of a new heterocyclic
system - pyrido[3’,2’:4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine, Chem.
Heterocycl. Comp. 45 (2009) 881e883.
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Tyrosine kinase inhibitors. 16. 6,5,6-tricyclic benzothieno [3,2-d]pyrimidines
and pyrimido[5,4-b]- and -[4,5-b]indoles as potent inhibitors of the epidermal
growth factor receptor tyrosine kinase, J. Med. Chem. 42 (1999) 5464e5474.
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M. Boyd, Feasibility of a high-flux anticancer drug screen utilizing a diverse
panel of human tumor cell lines in culture, J. Natl. Cancer Inst. 83 (1991) 757e
766.
Acknowledgments
Thanks are due to the NCI, Bethesda, MD (USA) for performing
the antitumor testing of the synthesized compounds.
[21] J.N. Weinstein, K.W. Kohn, M.R. Grever, V.N. Viswanadhan, L.V. Rubinstein,
A.P. Monks, D.A. Scudiero, L. Welch, A.D. Koutsoukos, A.J. Chiausa, K.D. Paull,
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through VLAK protocol: new annelated pyrrolo-pyrimidine derivatives as
antitumor agents, Eur. J. Med. Chem. 55 (2012) 375e383.
Appendix A. Supplementary material
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.019.
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M. Iwamura, Synthesis and characterization of new polyester dendrimers
from acetoacetate and acrylate, Org. Lett. 7 (2005) 525e528.
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