Synthesis and experimental validation of new designed heterocyclic compounds with antiproliferative activity versus breast cancer cell lines MCF-7 and MDA-MB-231

Article abstract of DOI:10.1155/2017/9729284

Recent drug discovery efforts are highly focused towards identification, design, and synthesis of small molecules as anticancer agents. With this aim, we recently designed and synthesized novel compounds with high efficacy and specificity for the treatment of breast tumors. Based on the obtained results, we constructed a Volsurf+ (VS+) model using a dataset of 59 compounds able to predict the in vitro antitumor activity against MCF-7 cancer cell line for new derivatives. In the present paper, in order to further verify the robustness of this model, we report the results of the projection of more than 150 known molecules and 9 newly synthesized compounds. We predict their activity versus MCF-7 cell line and experimentally verify the in silico results for some promising chosen molecules in two human breast cell lines, MCF-7 and MDA-MB-231.

Full text of DOI:10.1155/2017/9729284

Hindawi
Journal of Chemistry
Volume 2017, Article ID 9729284, 10 pages
https://doi.org/10.1155/2017/9729284
Research Article
Synthesis and Experimental Validation of New Designed
Heterocyclic Compounds with Antiproliferative Activity versus
Breast Cancer Cell Lines MCF-7 and MDA-MB-231
Vincenza Barresi,1 Carmela Bonaccorso,2 Domenico A. Cristaldi,2 Maria N. Modica,3
Nicolò Musso,1 Valeria Pittalà,3 Loredana Salerno,3 and Cosimo G. Fortuna2
1Department of Biomedical and Biotechnological Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
2Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
3Department of Drug Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
Correspondence should be addressed to Valeria Pittala`; vpittala@unict.it
Received 10 November 2016; Revised 13 February 2017; Accepted 26 February 2017; Published 2 April 2017
Academic Editor: Oliver Sutcliffe
Copyright © 2017 Vincenza Barresi et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Recent drug discovery efforts are highly focused towards identification, design, and synthesis of small molecules as anticancer
agents. With this aim, we recently designed and synthesized novel compounds with high efficacy and specificity for the treatment of
breast tumors. Based on the obtained results, we constructed a Volsurf+ (VS+) model using a dataset of 59 compounds able to predict
the in vitro antitumor activity against MCF-7 cancer cell line for new derivatives. In the present paper, in order to further verify
the robustness of this model, we report the results of the projection of more than 150 known molecules and 9 newly synthesized
compounds. We predict their activity versus MCF-7 cell line and experimentally verify the in silico results for some promising
chosen molecules in two human breast cell lines, MCF-7 and MDA-MB-231.
1. Introduction
effects. As an example, the US Food and Drug Administra-
tion (FDA) recently retired bevacizumab, approved for the
treatment of metastatic breast cancer, because of potentially
adverse side effects and minimal therapeutic benefit [7].
Drug discovery efforts are highly focused towards design
and synthesis of small molecules as anticancer agents [8].
In parallel, most efforts are devoted towards the “drug-
repositioning process” as an alternative drug development
strategy [9]. is approach gives an opportunity to find new
uses of existing drugs or molecules with a tremendous savings
of time and money.
During the years, a wide range of small molecules
based on heterocyclic ring systems have been studied for
the development of novel lead compounds in the drug
discovery paradigm. A number of the heterocyclic nuclei can
be regarded as “privileged scaffolds” [10] because they are
ubiquitous in drug molecules. Since they possess hydrogen
bond donors and acceptors in a rigid framework, they
can therefore effectively interact with target enzymes and
Despite continued research efforts, cancer remains one of the
biggest threats to human health. In the United States, it is the
second highest cause of death and is likely to soon replace
heart disease as the leading cause [1]. Breast cancer is the
most common cancer in women worldwide, with nearly 1.7
million newly diagnosed cases in 2012 (second most common
tumor overall) [2, 3]. is represents about 12% of all new
cancer cases and 25% of all cancers in women. A number
of risk factors have been identified, such as a strong family
history of breast cancer, weight gain, smoke, menopausal
hormone therapy (MHT), and alcohol consumption [4–
6]. However, the causes of the disease are not yet fully
understood. Treatment generally involves surgery, radiation,
chemotherapy, hormone therapy, and/or targeted therapy.
New anticancer agents with unique mechanisms of action
have been developed; however, many of them have not been
therapeutically useful due to low tumor selectivity or to side

2
Journal of Chemistry
R
R
R
O
O
2-OCH3
3-OCH3
4-OCH3
4-CH3
3-NO2
4-NO2
, 
, 
, 
, 
, 
, 
O
N
NH
NH
(a)
(b)
S
S−K+
S
N
N
N
N
N
N
O
N
N
N
,  4-Cl
,  4-Br
,  4-F
11–19
1
2–10
Scheme 1: Reagents and conditions: (a) ClCH COC H R, EtOH, reflux; (b) PPA, room temperature or H SO conc., 140∘C.
2
6
4
2
4
receptors via hydrogen bond interactions. ey can enhance
binding affinity and improve in vitro potency. Heterocycles
can modulate lipophilicity of the drug molecules or improve
aqueous solubility of the compounds, thus providing desired
pharmacokinetic and pharmaceutical properties [11].
by Evans et al. in the late 1980s [29], referring to scaffolds
seemingly capable of serving as ligands for a diverse array
of receptors, for example, indole, quinoline, and arylpiper-
azines.
All these 176 structures represented our training set that
is very different compared to the 59 compounds of the test
set because of the lack of the ethylenic bond that is the main
feature of the test set.
Herein, we predict the subsets activity versus MCF-7
cell line and experimentally verify the in silico results for
promising chosen molecules in two human breast cell lines,
MCF-7 and MDA-MB-231.
ese premises prompted us to design and synthesize
novel compounds with high efficacy and specificity for the
treatment of breast tumors. In 2013, this research group
reported the design and synthesis of new heterocyclic com-
pounds [12]. To fasten the lead discovery process, a Vol-
surf+ (VS+) model was constructed using a dataset of 59
compounds able to predict the in vitro antitumor activity
against MCF-7 cancer cell line for the new derivatives.
e use of Volsurf+ for in silico models generation is a
well established procedure, with proved efficacy [13, 14].
Novel compounds when tested in vitro showed a significant
agreement with in silico predictions. In the present paper,
in order to verify further the robustness of this model
and to apply the drug-repositioning concept, we report
the results of the projection of 176 derivatives. Among
them, 167 were previously synthesized for other applications
2. Material and Methods
2.1. Chemistry. e synthetic procedure for the preparation
of new compounds 2–19 is reported in Scheme 1. e
monopotassium salt of 1,5,6,7-tetrahydro-1-phenyl-6-thioxo-
4H-pyrazolo[3,4-d]pyrimidin-4-one (1) [30] reacted with
substituted phenacyl chloride at reflux in ethanol to give the
corresponding 1,5-dihydro-6-[(2-substitutedphenyl-2-ox-
oethyl)thio]-1-phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-ones
(2–10). Compounds 2–4, 6, and 7 were cyclized to the
corresponding 6-(2-substitutedphenyl)-1-phenyl-pyrazolo[3,
4-d]thiazolo[3,2-a]pyrimidin-4(1H)-ones (11–13, 15, and
16) by heating in presence of an excess of polyphosphoric
acid. Compounds 14 and 17–19 were obtained by cyclization
of derivatives 5 and 8–10 at room temperature using
concentrated sulfuric acid.
such as ꢀ1 adrenergic, 5-HT serotonergic, or endothelin
1A
receptor ligands and antiproliferative compounds active in
prostate tumor cell lines [15–25]. Moreover, 9 compounds
were designed and synthesized based on the recent liter-
ature [26–28] reporting the activity of pyrimidinketones
against MCF-7 cell line. e new compounds retain the
same heterocyclic core with respect to the literature ones,
modified by fusion with thiophene and pyrazole rings with
different substituents. Such modifications should improve
the antiproliferative activity reported in literature for similar
compounds. All the 176 structure formulas were reported
in Supporting Information (SI) (in Supplementary Material
available online at https://doi.org/10.1155/2017/9729284). e
generated dataset is made up of 7 subsets based on different
chemical scaffolds, namely, pyrrolopyrimidindione, pyrim-
idinketone, benzyloxyindole, indole, long chain arylpiper-
azines (LCAPs), quinoline, and thiophene derivatives (SI).
ese subsets share the common characteristic of being
composed of small molecules possessing a heterocyclic core.
e cores were chosen based on the idea of using privileged
scaffolds [10]. e term “privileged scaffolds” was first coined
2.1.1. Synthetic Procedures. Melting points were obtained by a
Gallemkamp apparatus provided with a digital thermometer
MFB-595 using glass capillary tubes and are uncorrected.
Elemental analyses for C, H, N, and S were carried out with
a Carlo Erba Elemental Analyzer Mod. 1108 instrument and
were within 0.4% of theoretical values. IR spectra were
obtained in KBr disks using a Perkin Elmer 1600 Series
1
FT-IR spectrometer. H NMR spectra were performed at
200 MHz with a Varian Inova Unity 200 spectrometer in
DMSO-d as solvent. Chemical shifs are reported in ꢁ
6
values (ppm) and coupling constants (J) are given in hertz

Journal of Chemistry
3
(Hz); tetramethylsilane was used as internal standard. e
purity of all synthesized compounds was checked on thin-
layer chromatography, using Merck aluminum sheet coated
2.1.7. 1,5-Dihydro-6-[[2-(3-nitrophenyl)-2-oxoethyl]thio]-1-
phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (6). White pow-
der (54%): mp 224–227∘C; IR (KBr, selected lines) cm−1 1705,
1673, 1523, 1488, 1399, 1349, 1314, 1203, 1109;1H NMR (DMSO-
d ) ꢁ 5.08 (s, 2H, CH CO), 6.90–7.15 (m, 3H, aromatic),
with silica gel 60 F and visualization by UV light at 254
254
and 366 nm of wavelength. All chemicals and solvents were
purchased from commercial vendors and were used without
further purification.
6
2
7.65–7.79 (m, 2H, aromatic), 7.85–8.10 (m, 1H, aromatic),
8.25 (m, 1H, pyrazole), 8.45–8.58 (m, 2H, aromatic), 8.65
(s, 1H, aromatic), 12.92 (s, 1H, NH). Anal. (C H N O S) C,
19 13
5
4
H, N, S.
2.1.2. General Procedure for the Synthesis of 1,5-Dihydro-6-[(2-
substitutedphenyl-2-oxoethyl)thio]-1-phenyl-4H-pyrazolo[3,4-
d]pyrimidin-4-one (2–10). A mixture of monopotassium
salt 1 (3.55 mmol) and of the appropriate phenacyl chloride
(3.55 mmol) was refluxed under stirring in ethanol (30 mL)
for 3 h. Afer cooling, the precipitate was collected, washed
with ethanol, dried, and recrystallized from a mixture
of ethanol/dioxane with the exception of compound 8
(recrystallization solvent: ethanol). Using this procedure the
following compounds were obtained.
2.1.8. 1,5-Dihydro-6-[[2-(4-nitrophenyl)-2-oxoethyl]thio]-1-
phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (7). Yellow pow-
der (53%): mp 250–251∘C dec; IR (KBr, selected lines) cm−1
1
1709, 1673, 1567, 1526, 1493, 1397, 1346, 1223, 1194; H NMR
(DMSO-d ) ꢁ 5.04 (s, 2H, CH CO), 6.90–7.18 (m, 3H,
6
2
aromatic), 7.65–7.75 (m, 2H, aromatic), 8.18–8.30 (m, 2H +
1H, aromatic + pyrazole), 8.30–8.40 (m, 2H, aromatic), 12.92
(s, 1H, NH). Anal. (C H N O S) C, H, N, S.
19 13
5
4
2.1.3. 1,5-Dihydro-6-[[2-(2-methoxyphenyl)-2-oxoethyl]thio]-
2.1.9. 6-[[2-(4-Chlorophenyl)-2-oxoethyl]thio]-1,5-dihydro-1-
1-phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (2). White
phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (8). White pow-
powder (61%): mp 220–222∘C; IR (KBr, selected lines) cm−1
der (36%): mp 250–252∘C; IR (KBr, selected lines) cm−1 1682,
1561, 1500, 1397, 1223, 1200, 1090, 984, 824; 1H NMR (DMSO-
d ) ꢁ 4.97 (s, 2H, CH CO), 6.90–7.10 (m, 2H, aromatic),
1
1688, 1644, 1595, 1569, 1384, 1309, 1168, 981, 773; H NMR
(DMSO-d ) ꢁ 3.90 (s, 3H, OCH ), 4.80 (s, 2H, CH CO),
6
3
2
6
2
6.98–7.30 (m, 5H, aromatic), 7.60–7.68 (m, 2H, aromatic),
7.80–7.95 (m, 2H, aromatic), 8.23 (s, 1H, pyrazole), 12.90 (s,
1H, NH). Anal. (C H N O S) C, H, N, S.
7.10–7.22 (m, 1H, aromatic), 7.58–7.90 (m, 4H, aromatic),
8.00–8.18 (m, 2H, aromatic), 8.23 (s, 1H, pyrazole), 12.90 (s,
1H, NH). Anal. (C H ClN O S) C, H, N, S.
20 16
4
3
19 13
4
2
2.1.4. 1,5-Dihydro-6-[[2-(3-methoxyphenyl)-2-oxoethyl]thio]-
1-phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (3). White
powder (82%): mp 247–249∘C dec; IR (KBr, selected lines)
cm−1 1684, 1595, 1560, 1519, 1426, 1395, 1260, 1223, 1166;
1H NMR (DMSO-d ) ꢁ 3.82 (s, 3H, OCH ), 4.99 (s, 2H,
2.1.10. 6-[[2-(4-Bromophenyl)-2-oxoethyl]thio]-1,5-dihydro-1-
phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (9). White pow-
der (32%): mp 254–255∘C; IR (KBr, selected lines) cm−1 1692,
1561, 1500, 1425, 1396, 1223, 1200, 984, 821; 1H NMR (DMSO-
d ) ꢁ 4.96 (s, 2H, CH CO), 6.95–7.10 (m, 2H, aromatic),
6
3
6
2
CH CO), 6.90–7.02 (m, 2H, aromatic), 7.05–7.20 (m, 1H,
7.10–7.22 (m, 1H, aromatic), 7.70–7.90 (m, 4H, aromatic),
7.95–8.10 (m, 2H, aromatic), 8.23 (s, 1H, pyrazole), 12.90 (s,
1H, NH). Anal. (C H BrN O S) C, H, N, S.
2
aromatic), 7.25–7.40 (m, 1H, aromatic), 7.45–7.60 (m, 2H,
aromatic), 7.65–7.81 (m, 3H, aromatic), 8.24 (s, 1H, pyrazole),
12.90 (s, 1H, NH). Anal. (C H N O S) C, H, N, S.
19 13
4
2
20 16
4
3
2.1.11. 1,5-Dihydro-6-[[2-(4-fluorophenyl)-2-oxoethyl]thio]-
2.1.5. 1,5-Dihydro-6-[[2-(4-methoxyphenyl)-2-oxoethyl]thio]-
1-phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (10). White
powder (71%): mp 247–250∘C; IR (KBr, selected lines) cm−1
1-phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (4). White
powder (44%): mp 252–254∘C; IR (KBr, selected lines) cm−1
1
1702, 1686, 1596, 1564, 1500, 1392, 1220, 1155, 831; H NMR
1
1686, 1600, 1572, 1512, 1423, 1398, 1311, 1294, 1174; H NMR
(DMSO-d ) ꢁ 4.98 (s, 2H, CH CO), 6.95–7.10 (m, 2H,
6
2
(DMSO-d ) ꢁ 3.91 (s, 3H, OCH ), 4.93 (s, 2H, CH CO),
aromatic), 7.10–7.22 (m, 1H, aromatic), 7.39–7.60 (m, 2H,
aromatic), 7.70–7.90 (m, 2H, aromatic), 8.10–8.20 (m, 2H,
aromatic), 8.22 (s, 1H, pyrazole), 12.89 (s, 1H, NH). Anal.
(C H FN O S) C, H, N, S.
6
3
2
6.99–7.22 (m, 5H, aromatic), 7.68–7.90 (m, 2H, aromatic),
7.99–8.15 (m, 2H, aromatic), 8.22 (s, 1H, pyrazole), 12.87 (s,
1H, NH). Anal. (C H N O S) C, H, N, S.
20 16
4
3
19 13
4
2
2.1.6. 1,5-Dihydro-6-[[2-(4-methylphenyl)-2-oxoethyl]thio]-1-
2.1.12. General Procedure for the Synthesis of 6-Substituted
Phenyl-1-phenyl-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-
4(1H)-one (11–13, 15, 16). A mixture of the suitable 1,5-
dihydro-6-[[2-(substituted phenyl)-2-oxoethyl]thio]-1-phe-
nyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (2–4, 6, and 7)
(2.46 mmol) and polyphosphoric acid (20 g) was stirred at
140∘C for 2 h. Afer cooling, the mixture was poured into cold
water, neutralized with a 10% sodium hydroxide solution.
e solid obtained was collected, washed with water, dried,
phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (5). White pow-
der (41%): mp 266–269∘C; IR (KBr, selected lines) cm−1 3070,
1729, 1547, 1529, 1509, 1482, 1426, 1394, 764; 1H NMR (DMSO-
d ) ꢁ 2.46 (s, 3H, CH ), 4.96 (s, 2H, CH CO), 6.90–7.10
6
3
2
(m, 2H, aromatic), 7.10–7.20 (m, 1H, aromatic), 7.38–7.50 (m,
2H, aromatic), 7.70–7.90 (m, 2H, aromatic), 7.90–8.10 (m, 2H,
aromatic), 8.25 (s, 1H, pyrazole), 12.89 (s, 1H, NH). Anal.
(C H N O S) C, H, N, S.
20 16
4
2

4
Journal of Chemistry
and recrystallized from dimethylformamide. Using this pro-
cedure the following compounds were obtained.
recrystallized from dioxane, with the exception of compound
18 (recrystallization solvent: ethanol/dioxane). Using this
procedure, the following compounds were obtained.
2.1.13. 6-(2-Methoxyphenyl)-1-phenyl-pyrazolo[3,4-d]thia-
zolo[3,2-a]pyrimidin-4(1H)-one (11). White powder (53%):
mp 247–249∘C; IR (KBr, selected lines) cm−1 1731, 1568, 1524,
2.1.19. 6-(4-Methylphenyl)-1-phenyl-pyrazolo[3,4-d]thia-
zolo[3,2-a]pyrimidin-4(1H)-one (14). White powder (52%):
1480, 1424, 1294, 1050, 1008, 778; 1H NMR (DMSO-d ) ꢁ 3.78
mp 273–275∘C; IR (KBr, selected lines) cm−1 1728, 1546, 1508,
6
1482, 1425, 1392, 1187, 1129, 763; 1H NMR (DMSO-d ) ꢁ 2.37
(s, 3H, OCH ), 6.98–7.45 (m, 5H + 1H, aromatic + thiazole),
3
6
7.50–7.65 (m, 2H, aromatic), 8.05–8.17 (m, 2H, aromatic), 8.35
(s, 1H, pyrazole). Anal. (C H N O S) C, H, N, S.
(s, 3H, CH ), 7.18–7.25 (m, 2H + 1H, aromatic + thiazole),
3
20 14
4
2
7.28–7.45 (m, 3H, aromatic), 7.50–7.65 (m, 2H, aromatic),
8.02–8.15 (m, 2H, aromatic), 8.33 (s, 1H, pyrazole). Anal.
(C H N OS) C, H, N, S.
2.1.14. 6-(3-Methoxyphenyl)-1-phenyl-pyrazolo[3,4-d]thia-
20 14
4
zolo[3,2-a]pyrimidin-4(1H)-one (12). White powder (47%):
mp 267–269∘C; IR (KBr, selected lines) cm−1 1724, 1587,
2.1.20. 6-(4-Chlorophenyl)-1-phenyl-pyrazolo[3,4-d]thia-
1
1546, 1521, 1480, 1399, 1242, 854, 753; H NMR (DMSO-d )
6
zolo[3,2-a]pyrimidin-4(1H)-one (17). White powder (42%):
ꢁ 3.68 (s, 3H, OCH ), 6.96–7.19 (m, 2H, aromatic), 7.21 (s,
3
mp 261–264∘C; IR (KBr) cm−1 1720, 1510, 1480, 1390,
1H, thiazole), 7.32–7.52 (m, 3H, aromatic), 7.53–7.65 (m, 2H,
aromatic), 8.02–8.12 (m, 2H, aromatic), 8.33 (s, 1H, pyrazole).
Anal. (C H N O S) C, H, N, S.
1235, 1120, 1090, 990, 745; 1H NMR (DMSO-d ) ꢁ
6
7.30 (s, 1H, thiazole), 7.38–7.50 (m, 7H, aromatic),
8.00–8.18 (m, 2H, aromatic), 8.35 (s, 1H, pyrazole). Anal.
(C H ClN OS⋅(1/2)H O) C, H, N, S.
20 14
4
2
19 11
4
2
2.1.15. 6-(4-Methoxyphenyl)-1-phenyl-pyrazolo[3,4-d]thia-
zolo[3,2-a]pyrimidin-4(1H)-one (13). White powder (60%):
mp 278–280∘C; IR (KBr, selected lines) cm−1 3065, 1731, 1543,
2.1.21. 6-(4-Bromophenyl)-1-phenyl-pyrazolo[3,4-d]thia-
1516, 1477, 1396, 1292, 1187, 764; 1H NMR (DMSO-d ) ꢁ 3.82 (s,
zolo[3,2-a]pyrimidin-4(1H)-one (18). White powder (62%):
6
mp 272–274∘C; IR (KBr, selected lines) cm−1 1706, 1576,
3H, OCH ), 6.90–7.00 (m, 2H, aromatic), 7.16 (s, 1H, thiazole),
3
1546, 1482, 1395, 1190, 1066, 1003, 768; 1H NMR (DMSO-d )
7.38–7.45 (m, 3H, aromatic), 7.50–7.60 (m, 2H, aromatic),
8.05–8.15 (m, 2H, aromatic), 8.34 (s, 1H, pyrazole). Anal.
(C H N O S) C, H, N, S.
6
ꢁ 7.30 (s, 1H, thiazole), 7.40–7.52 (m, 3H, aromatic), 7.52–7.70
(m, 4H, aromatic), 8.05–8.15 (m, 2H, aromatic), 8.35 (s, 1H,
pyrazole). Anal. (C H BrN OS) C, H, N, S.
20 14
4
2
19 11
4
2.1.16. 6-(3-Nitrophenyl)-1-phenyl-pyrazolo[3,4-d]thiazolo[3,
2-a]pyrimidin-4(1H)-one (15). Yellow powder (61%): mp
2.1.22. 6-(4-Fluorophenyl)-1-phenyl-pyrazolo[3,4-d]thia-
254–256∘C; IR (KBr, selected lines) cm−1 1716, 1546, 1505,
zolo[3,2-a]pyrimidin-4(1H)-one (19). White powder (53%):
1
1424, 1396, 1350, 1195, 1133, 787; H NMR (DMSO-d ) ꢁ
mp 286–288∘C; IR (KBr) cm−1 1725, 1530, 1510, 1482, 1395,
6
7.35–7.48 (m, 1H + 1H, aromatic + thiazole), 7.55–7.75 (m,
3H, aromatic), 7.90–8.00 (m, 1H, aromatic), 8.05–8.12 (m, 2H,
aromatic), 8.28–8.40 (m, 2H + 1H, aromatic + pyrazole). Anal.
(C H N O S) C, H, N, S.
1
1230, 820, 795, 740; H NMR (DMSO-d ) ꢁ 7.15–.35 (m,
6
2H + 1H, aromatic + thiazole), 7.35–7.95 (m, 5H, aromatic),
8.00–8.20 (m, 2H, aromatic), 8.34 (s, 1H, pyrazole). Anal.
(C H FN OS) C, H, N, S.
19 11
5
3
19 11
4
2.1.17. 6-(4-Nitrophenyl)-1-phenyl-pyrazolo[3,4-d]thiazolo[3,
2-a]pyrimidin-4(1H)-one (16). Yellow powder (74%): mp >
2.2. Computational Methods
330∘C; IR (KBr, selected lines) cm−1 1724, 1552, 1527, 1508,
1
2.2.1. VS+. VS+ is an automatic procedure which allows
converting information coded into the 3D GRID Molec-
ular Interaction Fields into 128 physicochemically relevant
descriptors [31]. e obtained descriptors concern molec-
ular size and shape, hydrophilic and hydrophobic proper-
ties, hydrogen bonding, amphiphilic moments, and critical
packing parameters. Pharmacokinetic descriptors related to
solubility, metabolic stability, and cell permeability were also
generated.
1485, 1344, 1129, 764, 746; H NMR (DMSO-d ) ꢁ 7. 3 8–7. 5 0
6
(m, 2H + 1H, aromatic + thiazole), 7.55–7.70 (m, 1H,
aromatic), 7.70–7.82 (m, 2H, aromatic), 8.10–8.15 (m, 2H,
aromatic), 8.20–8.30 (m, 2H, aromatic), 8.38 (s, 1H, pyrazole).
Anal. (C H N O S) C, H, N, S.
19 11
5
3
2.1.18. General Procedure for the Synthesis of 6-Substituted
Phenyl-1-phenyl-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-
4(1H)-one (14, 17–19). A mixture of the suitable 1,5-dihydro-
6-[[2-(substituted phenyl)-2-oxoethyl]thio]-1-phenyl-4H-
pyrazolo[3,4-d]pyrimidin-4-one (5, 8–10) (1.86 mmol) and
sulfuric acid (3.4 mL) was stirred at room temperature for 1 h.
en the solution was kept at room temperature for 4 days.
Afer cooling, the mixture was poured into cold water and
neutralized with a 10% solution of sodium hydroxide. e
solid obtained was collected, washed with water, dried, and
Surface properties such as shape, electrostatic forces,
H-bonds, and hydrophobicity influenced the interaction of
molecules with biological membranes. erefore, poten-
tial polar and hydrophobic interaction sites around target
molecules by the water (OH ), the hydrophobic (DRY), and
2
the carbonyl oxygen (O) and amide nitrogen (N1) probe [32]
were characterized by the GRID force field.

Journal of Chemistry
5
e information contained in the Molecular Interaction
Fields (MIF) is converted into a quantitative scale by calcu-
lating the volume or the surface of the interaction contours.
e VS+ procedure is as follows:
2.3.2. Treatment with Antitumor Agents and MTT Colorimet-
ric Assay. Human cancer cell line was plated in 96-well plates
“Nunclon TM Microwell TM” (Nunc) and were incubated at
37∘C. MTT assay was performed as reported by Barresi et
al. [37]. Afer 24 h, cells were treated with the compounds
(final concentration 0.01–100 ꢂM). Untreated cells were used
as controls. Microplates were incubated at 37∘C in humidified
(i) First, the interactions of OH , DRY, O, and N1 probes
2
around a target molecule generated the 3D molecular
field.
atmosphere of 5% CO , 95% air for 3 days, and then cyto-
2
toxicity was measured with colorimetric assay based on the
use of tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) [38]. e results were read
on a multiwell scanning spectrophotometer (PlateReader
AF2200, Eppendorf, Milan, Italy), using a wavelength of
570 nm. Each value was the average of six to eight wells (stan-
(ii) en, the descriptors from the 3D maps, obtained
in the first step, were calculated. ese molecular
descriptors, called VS+ descriptors, concern molec-
ular size and shape, hydrophilic and hydrophobic
regions and the balance between them, molecular
diffusion, log P, log D, “charge state” descriptors, new
3D pharmacophoric descriptors, and descriptors of
some appropriate ADME properties. e definition of
all 128 VS+ descriptors was reported elsewhere [31].
dard deviations were less than 15%). e GI value was cal-
50
culated according to NCI [39]: thus, GI is the concentration
50
of test compound where 100×(ꢃ−ꢃ00)(ꢄ−ꢃ00 = 50 (ꢃ is the
optical density of the test well afer a 72 h period of exposure
to test drug; ꢃ0 is the optical density at time zero; ꢄ is the
control optical density). e cytotoxicity effect was calculated
according to NCI when the optical density of treated cells
(iii) Finally, chemometric tools (PCA [33] and PLS [34])
are used to create relationships of the VS+ descriptor
matrix with ADME properties.
was lower than the ꢃ0 value using the following formula:
100 × (ꢃ − ꢃ00)ꢃ0 < 0.
e scheme of the VS+ programme steps and a detailed
definition of VS+ descriptors have recently been reported
[35].
e .mol2 files for 176 new molecules were generated
and classified in 7 different classes based on their different
chemical features for a better classification and identification:
pyrrolopyrimidindione, pyrimidinketones, benzyloxyindole,
indole, LCAPs, quinoline, and thiophene derivatives [15–25].
e structures of all compounds were reported in Supporting
Information (SI).
3. Results and Discussion
As a first approach we projected all the 176 compounds on
the model previously published [12]. e PLS model afer
projection of the test set is shown in Figure 1: the predicted
compounds are colored according to the different belonging
classes: pyrrolopyrimidindione in green, pyrimidinketones
in yellow, benzyloxyindole in light blue, indole in red,
LCAPs in purple, quinoline in blu, and thiophene in white.
e background of the PLS model is color-coded by the
activity values against MCF-7 cell line, using a scale from
red (active) to blue (inactive). e ꢅ1 and ꢅꢆ values refer
to the coordinates of each single compound within the
plot.
e VS+ sofware was used to perform a Structure-
Activity Relationship (QSAR) study using the default settings
for protomers and conformers generations.
Two outliers families, pyrrolopyrimidindione and thio-
phene derivatives, lying out of the confidence ellipse were
discharged, probably due to some solubility and permeability
problems.
2.3. Biological Assays
2.3.1. Human Cell Lines (MCF-7 and MDA-MB-231). Two
human breast adenocarcinoma cell lines, MCF-7 (ATCC
number: HTB-22) and MDA-MB-231 (ATCC number:
HTB26), have been cultured as previously reported [36].
MCF-7 cell line was maintained in D-MEM medium (Dul-
becco’s Modified Eagle Medium 1x; GIBCO, Cat number
31965-023 containing 1 g/L of D-glucose). MDA-MB-231 cell
line was maintained in DMEM/F12 medium (Dulbecco’s
Modified Eagle Medium Ham’s F12 Nutrient Mixture, 1x;
GIBCO, Cat. number 21331-020), 1.5 mM L-glutamine, and
MEM nonessential amino acid solution 1x (SIGMA M7145).
Each medium was supplemented with 10% fetal ovine serum
(FBS, Cat. number 10270-106, Life Technologies, Monza Italy)
and 100 U/ml of penicillin-streptomycin (Cat. number 15140-
122, Life Technologies, Monza Italy). e cell cultures were
grown and incubated at 37∘C in humidified atmosphere with
Moreover, in order to increase the predictivity power, we
performed the class model for each single subset discharging
the correspondent outliers (SI). By using this strategy, we
obtained a test set of 25 compounds that are the best
representative molecules for each class (Figure 2).
Starting from the obtained test set, we decided to
select 10 out of the 25 derivatives: 5 from active part of
the graphic, red area, and 5 from the inactive one, blue
area (Table 1). To validate our prediction we tested those
compounds in vitro against MCF-7 and MDA-MB-231 cell
lines using 5-fluorouracil (5-FU) as standard chemotherapy
agent reference (Table 1). e biological characteristics of
MCF7 and MDA-MB-231 are dissimilar: the first cell line is
a representative model of ductal breast carcinoma, oestrogen
receptor-positive, receptor tyrosine-protein kinase (erbB-2,
HER2/neu) negative and responding to endocrine therapy,
5% of CO and 95% of air. e culture medium was changed
2
twice a week.

6
Journal of Chemistry
PLS t/t (model 59 MCF7)
LV1 versus LV2 LV
10
0
−10
−20
−30
−40
−50
−60
−70
t[1]
Figure 1: PLS ꢅ)ꢅ scores plot for the 176 selected compounds.
PLS t/t (model 59 MCF7)
LV1 versus LV2 LV
12.5
10
7.5
5
2.5
0
−2.5
−5
−7.5
−10
−12.5
t[1]
Figure 2: PLS ꢅ)ꢅ scores plot for 25 derivatives (yellow points) selected based on activity prediction: active, inactive, and moderately active.
while the MDA-MB-231 is oestrogen receptor-negative unre-
sponsive to the same treatment.
4. Conclusion
e predictivity power of the model previously published
from our group was confirmed using different type of
heterocyclic compounds. ree of these derivatives showed
good antiproliferative in vitro activity against MCF-7 and
MDA-MB-231 cell lines. e MDA-MB-231 cell line repre-
sents a triple-negative breast cancer without oestrogen recep-
tor (ER), progesterone receptor (PR), and epidermal growth
factor receptor 2 (HER2), associated with strong aggres-
siveness, poor prognosis, and unresponsiveness to the usual
endocrine therapies. e antiproliferative effects observed on
MCF-7 and MDA-MB-231 are worthy of attention to develop
molecules that are able to attack the breast tumor oestrogen
receptor-positive and/or oestrogen receptor-negative. Studies
aimed at the elucidation of the above mechanism pathways
are in progress.
e antiproliferative effects observed in Table 1 for
compounds pyrimidinketone 2A, pyrimidinketone 2B, and
quinoline 7B are stronger than same similar effect observed
with the reference 5-FU in both cell lines. It is noteworthy that
these compounds, with very low t1 values in Figure 2, exhibit
a very high activity, thus confirming the robustness of the PLS
predictions from a quantitative point of view.
In Table 1, we report the experimental activity values,
expressed as log GI , for the 10 tested compounds and
50
VS+ predicted values. In particular, in red we report the 5
compounds predicted active (Table 1), while in blue we report
the remaining 5 predicted inactive. In silico predictions were
confirmed for most of the compounds tested in vitro. Two
marked compounds in the table, predicted active, resulted
inactive in the experimental test. However, it should be noted
that these two compounds showed some solubility problem
during the in vitro procedure, even if we added 1% of DMSO;
they gave flocculation; thus data relative to these compounds
are not reliable.
Conflicts of Interest
e authors declare no conflicts of interest regarding the
publication of this paper.

Journal of Chemistry
7
Table 1: Molecular structures, in vitro activity on MCF-7 and MDA-MB-231 cell lines, and in silico activity values for the ten selected
compounds and 5-FU. Activity values expressed as log GI .
50
MCF7
experimental
activity
MDA-MB-231
experimental
activity
Predicted
activity versus
MCF7
Molecule name
Molecular structure
log GI /ꢂM SD∗ log GI /ꢂM SD∗
50
50
N
O
N
S
Pyrimidinketone
2A
N
−5.20 0.7
−4.8 0.4
−6.08
−6.72
N
O
O
O
Quinoline 7B
−4.80 0.6
−4.3 0.5
N
S
O
O
N
N
S
Pyrimidinketone
2B
N
−4.90 0.8
>−4.00
−4.6 0.3
−6.21
−6.86
N
O
O
N
S
N
O
O
M LCAPs 8A
N
S
Cl
O
M Quinoline 7A
>−4.00
>−4.00
−4.8
−6.83
−3.83
S
N
O
O
OH
Indole 2B
−4.6 0.6
N
O

8
Journal of Chemistry
Table 1: Continued.
MCF7
experimental
activity
MDA-MB-231
experimental
activity
Predicted
activity versus
MCF7
Molecule name
Molecular structure
log GI /ꢂM SD∗ log GI /ꢂM SD∗
50
50
N
O
N
S
N
Pyrimidinketone
3B
N
>−4.00
NA
−4.00
O
OH
Indole 3E
Indole 3D
>−4.00
>−4.00
>−4.00
>−4.00
−3.70
−4.00
O
N
H
O
O
OH
O
N
O
H
N
N
S
N
N
Pyrimidinketone
2D
>−4.00
>−4.00
−4.00
O
N+
O
O−
5-FU
−5.40 0.7
−5.1 0.4
—
∗
Each value represents the mean SD of six to eight wells. NA, not available.
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