Synthesis and evaluation of the cytotoxic activity of Furanaphthoquinones tethered to 1H-1,2,3-triazoles in Caco-2, Calu-3, MDA-MB231 cells

Article abstract of DOI:10.1016/j.ejmech.2018.07.018

Naphthoquinones and 1,2,3-triazoles are structural pharmacophore that is known to impart several cancer cells. This work shows a synthetic methodology to obtain hybrid molecules involving naphthoquinone and triazol scaffold as multiple ligands. A simple a

Full text of DOI:10.1016/j.ejmech.2018.07.018

Accepted Manuscript
Synthesis and evaluation of the cytotoxic activity of Furanaphthoquinones tethered to
1H-1,2,3-triazoles in Caco-2, Calu-3, MDA-MB231 cells
Dora C.S. Costa, Gabriella Silva de Almeida, Vitor Won-Held Rabelo, Lucio Mendes
Cabral, Plínio Cunha Sathler, Paula Alvarez Abreu, Vitor Francisco Ferreira, Luiz
Cláudio Rodrigues Pereira da Silva, Fernando de C. da Silva
PII:
S0223-5234(18)30574-9
10.1016/j.ejmech.2018.07.018
EJMECH 10553
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 May 2018
Revised Date: 28 June 2018
Accepted Date: 7 July 2018
Please cite this article as: D.C.S. Costa, G.S. de Almeida, V.W.-H. Rabelo, L.M. Cabral, Plí.Cunha.
Sathler, P. Alvarez Abreu, V.F. Ferreira, L. Cláudio Rodrigues Pereira da Silva, F.d.C. da Silva,
Synthesis and evaluation of the cytotoxic activity of Furanaphthoquinones tethered to 1H-1,2,3-triazoles
in Caco-2, Calu-3, MDA-MB231 cells, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.07.018.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Evaluation of the Cytotoxic Activity of
Furanaphthoquinones Tethered to 1H-1,2,3-Triazoles in
Caco-2, Calu-3, MDA-MB231 Cells
Dora C. S. Costa,a Gabriella Silva de Almeida,b Vitor Won-Held Rabelo,c Lucio
Mendes Cabral,b Plínio Cunha Sathler,b Paula Alvarez Abreu,c Vitor Francisco
Ferreira,d,* Luiz Cláudio Rodrigues Pereira da Silva,b,* Fernando de C. da Silvaa,*
a Universidade Federal Fluminense, Departamento de Química Orgânica, Instituto de
Química, Campus do Valonguinho, Niterói, RJ, CEP 24020-150, Brazil.
b
Universidade Federal do Rio de Janeiro, Laboratório de Tecnologia Industrial
Farmacêutica (LabTIF), Faculdade de Farmácia, Rio de Janeiro, RJ, CEP 21941-902,
Brazil.
c
Universidade Federal do Rio de Janeiro, Laboratório de Modelagem Molecular e
Pesquisa em Ciências Farmacêuticas (LaMCiFar), NUPEM, Campus Macaé, RJ, CEP
27965-045, Brazil.
d
Universidade Federal Fluminense, Departamento de Tecnologia Farmacêutica,
Faculdade de Farmácia, Niterói, RJ, CEP 24241-002, Brazil.
Corresponding authors
Biological Assays: Luiz Cláudio Rodrigues Pereira da Silva, Universidade Federal do
Rio de Janeiro, Laboratório de Tecnologia Industrial Farmacêutica (LabTIF), Faculdade
de
Farmácia,
Rio
de
Janeiro,
RJ,
21941-902,
Brazil;
E-mail:
luizclaudio@pharma.ufrj.br

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Chemistry: Fernando de Carvalho da Silva, Universidade Federal Fluminense,
Departamento de Química Orgânica, Instituto de Química, Campus do Valonguinho,
CEP 24020-150, Niterói-RJ, Brazil; E-mail: gqofernando@vm.uff.br
Vitor Francisco Ferreira, Universidade Federal Fluminense, Departamento de
Tecnologia Farmacêutica, Faculdade de Farmácia, Niterói, RJ, CEP 24241-002, Brazil.
vitorferreira@id.uff.br
Abstract: Naphtoquinones and 1,2,3-triazoles are structural pharmacophore that
is known to impart several cancer cells. This work shows a synthetic methodology to
obtain hybrid molecules involving naphthoquinone and triazol scaffold as multiple
ligands. A simple and efficient synthetic route was used to prepare a series of sixteen
compounds being eight 2-(1-aryl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2
b]furan-4,5-diones and eight 2-(1-aryl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-
b]furan-4,9-diones. These compounds were tested in MDA-MB231, Caco-2 and Calu-3
human cancer cells, and among them 7a was the most selective compound on Caco-2
cells, the most sensitized cell line in this study. In silico study suggest that the blockage
of topoisomerase I and IIα may be one of the mechanisms of action responsible for the
cytotoxic effect of 7a in Caco-2 cells.
Keywords: quinones; triazole; cancer; anticancer agents; molecular modelling;
topoisomerase.

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1. Introduction
There are several synthetic and natural low molecular weight naphthoquinones
with many applications in various scientific and technological fields [1]. In this sense,
quinones have already been described with a diversity of bioactivities such as anticancer
[1], molluscicides [2,3], antiparasitics [4-6], antimalarial [7], leischmanicides [8], anti-
inflammatory [9], antifungal [10], antimicrobial [11] and trypanocidal [12].
On the other hand, heterocyclic rings are considered the pillars of medicinal
chemistry because they are incorporated in the molecular structure of most of the drugs
available in the market. From the earliest days of medicinal chemistry, these rings were
known to be the pharmacophore in many compounds with biological activities. Taylor
and colleagues estimated that about six new ring systems are created per year and some
of them are incorporated into new drugs. According to those authors, 1,2,4 and 1,2,3-
triazoles are among the 100 most commonly used ring systems in small molecules listed
in the FDA orange book [13].
Although the scaffold quinones and 1,2,3-triazoles are of major importance
separately, molecules containing these two nuclei only have been studied in recent years
against various diseases showing that this molecular combination have great potential
synergism, i.e., are more bioactive when coupled than in the isolated form (e.g. the
naphthoquinone-1,2,3-triazole conjugates 1 [ 14 ] and 2 [ 15 ] with good antitumor
activity.

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Fig. 1. Examples of hybrids naphthoquinone-1,2,3-triazole with high activities in cancer
cells
As part of our research program on the synthesis of biologically active quinones,
we became interested in the synthesis and antitumor evaluation of hybrids
naphthoquinone-1,2,3-triazoles. Among the aforementioned hybrids, a series of eight 2-
(1-phenyl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (6a-h) and
eight 2-(1-phenyl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-4,9-dione (7a-
h) were synthesized and screened in vitro against MDA-MB231, Caco-2 and Calu-3
human cancer cell lines and also evaluated their cell viability on Vero cells.
2. Experimental Section
2.1. Chemistry
The reagents were purchased from Sigma-Aldrich Brazil and were used without
further purification. Column chromatography was performed with silica gel 60 (Merck
70-230 mesh). Analytical thin layer chromatography was performed with silica gel
plates (Merck, TLC silica gel 60 F254), and the plates were visualized using UV light.
The indicated yields refer to homogeneous materials purified by chromatography and

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confirmed by spectroscopic techniques. Melting points were obtained on a Thermo
scientific 9100 apparatus and were uncorrected. Infrared spectra were collected using
KBr pellets on a Perkin-Elmer model 1420 FT-IR spectrophotometer, and the spectra
were calibrated relative to the 1601.8 cm-1 absorbance of polystyrene. 1H and 13C NMR
were recorded at room temperature using a Varian Mercury 300 or Varian Mercury 400
MHz, in the DMSO-d6. Chemical shifts (δ) are given in ppm and coupling constants (J)
in Hertz. The chemical shift data were reported in units of δ (ppm) downfield from
solvent, and the solvent was used as an internal standard; coupling constants (J) are
reported in hertz and refer to apparent peak multiplicities. High-resolution mass spectra
(HRMS) were recorded on a MICROMASS Q-TOF mass spectrometer (Waters).
2.1.1. General procedure for the preparation of 4a-h and 5a-h
To a stirred suspension of 2-hydroxy-1,4-naphthoquinone 5 (1.3 mmol, 1.0 eqv.)
in freshly dried acetonitrile, 0.48 g of NaHCO3 was added at once, in an ice bath and
sodium chloride at 0-5 ºC. This mixture was stirred for 15 min. and then the appropriate
4-vinyl-1H-1,2,3-triazoles 4 (1.1 equiv.) was added at once, at the same temperature.
After that, 1.75 g of ceric ammonium nitrate (CAN) was added slowly and reaction
mixture was stirred until total consumption of the starting material. During the reaction
time, it was allowed that the bath temperature rose up spontaneously to room
temperature. The acetonitrile was removed under reduced pressure and the resulting
crude was purified via silica flash chromatography, using a gradient mixture of
chloroform/ethyl acetate, as eluent to obtain 1,4-isomer 6a-h as a yellow solid and the
1,2-isomer 7a-h as an orange solid.

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2.1.1.1. 2-(1-phenyl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-4,9-dione
(6a). Yellow solid. m.p. 219-220 ºC. IR (KBr, cm-1): ν 1681, 1624 (C-H Ar), 1593,
1503, 1389, 1241, 1200, 1056, 956, 832, 757, 718, 685. 1H NMR (DMSO-d6, 500 MHz)
δ ppm: 3.53 (dd, J 17.0 and 8.0 Hz, 1H), 3.66 (dd, J 17.0 and 10.8 Hz, 1H), 6.32 (dd, J
10.8 and 8.0 Hz, 1H), 7.51 (t, J 7.5 Hz, 1H), 7.61 (t, J 8.0 Hz, 2H), 7.81-7.89 (m, 2H),
7.90 (d, J 7.5 Hz, 2H), 8.05-7.97 (m, 2H), 9.09 (s, 1H). 13C NMR (DMSO-d6, 125 MHz
APT) δ ppm: 32.7, 79.0, 120.7, 123.1, 124.5, 126.0, 126.3, 129.3, 130.3, 131.8, 133.0,
133.8, 134.9, 137.0, 146.9, 159.3, 177.6, 182.0. HRMS-ESI m/z [M + H]+ calcd for
C20H14N3O3: 344.10297; found: 344.10297. ∆ = 0 ppm.
2.1.1.2. 2-(1-phenyl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione
(7a). Orange solid. m.p. 208-209 ºC. IR (KBr, cm-1): ν 1697, 1650, 1614, 1571, 1489,
1274, 1250, 1219, 853, 755, 723, 687. 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.41 (dd,
J 15.4 and 7.5 Hz, 1H), 3.54 (dd, J 15.4 and 10.2 Hz, 1H), 6.44 (dd, J 10.2 and 7.5 Hz,
1H), 7.50-7.53 (m, 1H), 7.60-7.65 (m, 3H), 7.69 (td, J 7.8 and 1.1 Hz, 1H), 7.77 (td, J
7.8 and 1.1 Hz, 1H), 7.93-7.88 (m, 2H), 7.98 (dd, J 7.8 and 1.1 Hz, 1H), 9.12 (s, 1H).
13C NMR (DMSO-d6, 125 MHz APT) δ ppm: 31.5, 79.9, 115.0, 120.1, 122.5, 124.2,
126.8, 128.6, 128.8, 129.8, 130.5, 132.0, 134.6, 136.4, 146.4, 167.4, 174.8, 180.3.
HRMS-ESI m/z [M + H]+ calcd for C20H14N3O3: 344.10297; found: 344.10297. ∆ = 0
ppm.
2.1.1.3. 2-(1-(4-tolyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-4,9-dione
(6b). Yellow solid. m.p. 254-255 ºC. IR (KBr, cm-1): ν 1679, 1622, 1585, 1520, 1389,
1
1389, 1238, 1189, 1055, 955, 855, 815, 720, 620. H NMR (DMSO-d6, 500 MHz) δ
ppm: 2.38 (s, 3H), 3.52 (dd, J 17.0 and 7.9 Hz, 1H), 3.65 (dd, J 17.0 and 10.9 Hz, 1H),
6.31 (dd, J 10.9 and 7.9 Hz, 1H), 7.40 (d, J 8.5 Hz, 1H), 7.77 (d, J 8.5 Hz, 2H), 7.80-

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13
7.88 (m, 2H), 7.99-8.03 (m, 2H), 9.03 (s, 1H). C NMR (DMSO-d6, 125 MHz APT) δ
ppm: 20.4, 32.1, 78.5, 120.0, 122.4, 123. 9, 125.4, 125.7, 130.1, 131.2, 134.2, 133.3,
134.3, 138.5, 146.3, 158.7, 177.0, 181.4. HRMS-ESI m/z [M + H]+ calcd for
C21H16N3O3: 358.11862; found: 358.11862. ∆ = 0 ppm.
2.1.1.4. 2-(1-(p-tolyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione
(7b). Orange solid, m.p.: 239-240 ºC. IR (KBr, cm-1): ν 1694, 1646, 1613, 1570, 1517,
1
1486, 1345, 1255, 1211, 1143, 1076, 996, 972, 876, 816, 771, 725, 703, 660. H NMR
(DMSO-d6, 500 MHz) δ ppm: 3.38 (s, 3H), 3.40 (dd, J 15.3 and 7.5 Hz, 1H), 3.53 (dd, J
15.3 and 10.5 Hz, 1H), 6.43 (dd, J 10.5 and 7.5 Hz, 1H), 7.40 (d, J 8.3 Hz, 2H), 7.62-
13
7.74 (m, 3H), 7.79 (d, J 8.3 Hz, 2H), 7.97 (dd, J 8.0 and 1.0 Hz, 1H) 9.07 (s, 1H). C
NMR (DMSO-d6, 125 MHz APT) δ ppm: 21.0, 32.1, 80.5, 115.6, 120.6, 122.9, 124.8,
127.4, 129.2, 130.7, 131.1, 132.6, 134.7, 135.2, 139.1, 146.8, 168.0, 175.4, 180.9.
HRMS-ESI m/z [M + H]+ calcd for C21H16N3O3: 358.11862; found: 358.11862. ∆ = 0
ppm.
2.1.1.5.
2-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-
b]furan-4,9-dione (6c). Yellow solid, m.p. 247-248 ºC. IR (KBr, cm-1): ν 1682, 1639,
1626, 1594, 1519, 1390, 1373, 1284, 1253, 1241, 1192, 1174, 1035, 958, 830, 722, 620.
1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.52 (dd, J 17.0 and 8.0 Hz, 1H), 3.64 (dd, J
17.0 and 10.5 Hz, 1H), 3.83 (s, 3H), 6.30 (dd, J 10.5 and 8.0 Hz, 1H), 7.14 (d, J 9.0 Hz,
2H), 7.80 (d, J 9.0 Hz, 2H), 7.82-7.88 (m, 2H), 7.99-8.02 (m, 2H), 8.98 (s, 1H). 13C
NMR (DMSO-d6, 125 MHz APT) δ ppm: 32.1, 55.5, 78.5, 114.8, 121.8, 122.5, 123.9,
125.4, 125.7, 130.0, 131.2, 132.2, 133.3, 134.3, 146.1, 158.7, 159.4, 177.0, 181.4.
HRMS-ESI m/z [M + H]+ calcd for C21H17N3O4: 374.111353; found: 374.111353. ∆ = 0
ppm.

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2.1.1.6.
2-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-
b]furan-4,5-dione (7c). Orange solid, m.p. 191-192 ºC. IR (KBr, cm-1): ν 1699, 1653,
1
1619, 1590, 1572, 1519, 1411, 1283, 1261, 1218, 1043, 992, 843, 827, 778. H NMR
(DMSO-d6, 500 MHz) δ ppm: 3.40 (dd, J 15.2 and 7.4 Hz, 1H), 3.53 (dd, J 15.2 and
10.5 Hz, 1H), 3.83 (s, 3H), 6.42 (dd, J 10.5 and 7.4 Hz, 1H), 7.14 (d, J 9.0 Hz, 1H),
7.64 (dd, J 7.5 and 1.5 Hz, 1H), 7.69 (td, J 7.8 and 1.3 Hz, 1H), 7.76 (td, J 7.8 and 1.3
Hz, 1H), 7.81 (d, J 9.0 Hz, 2H), (dd, J 7.5 and 1.5 Hz, 1H , 1H), 9.01 (s, 1H). 13C NMR
(DMSO-d6, 125 MHz APT) δ ppm: 31.5, 55.4, 80.0, 114.8, 114.9, 121.8, 122.3, 124.1,
126.8, 128.8, 129.7, 130.5, 131.6, 134.6, 146.0, 159.3, 167.4, 174.8, 180.3. HRMS-ESI
m/z [M + H]+ calcd for C21H17N3O4: 374.111353; found: 374.111353. ∆ = 0 ppm.
2.1.1.7.
2-(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-
4,9-dione (6d). Yellow solid, m.p. 246 ºC. IR (KBr, cm-1): ν 1681, 1625, 1594, 1531,
1504, 1459, 1343, 1244, 1190, 1254, 1020, 922, 955, 855, 833, 748, 720, 662. 1H NMR
(DMSO-d6, 500 MHz) δ ppm: 3.52 (dd, J 16.9 and 7.9 Hz, 1H), 3.68 (dd, J 16.9 and 7.9
Hz, 1H), 6.35 (dd, J 10.8 and 7.9 Hz, 1H), 7.81-7.89 (m, 2H), 7.99-8.03 (m, 2H), 8.23
13
(d, J 9.0 Hz), 8.46 (d, J 9.0 Hz), 9.30 (s, 1H). C NMR (DMSO-d6, 125 MHz APT) δ
ppm: 32.2, 78.2, 120.7, 122.9, 123.8, 125.3, 125.6, 131.2, 132.4, 133.2, 134.3, 140.6,
146.8, 147.0, 158.6, 176.9, 181.3. HRMS-ESI m/z [M + Na]+ calcd for C20H13N4NaO5:
411.0699; found: 411.0700. ∆ = 0.2 ppm.
2.1.1.8.
2-(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-b]furan-
4,5-dione (7d). Orange solid, m.p. 243-244 ºC. IR (KBr, cm-1): ν 1698, 1649, 1608,
1573, 1536, 1505, 1449, 1404, 1348, 1319, 1294, 1251, 1217, 1140, 1112, 1082, 1053,
1
1025, 889, 854, 832, 768, 747, 682, 657. H NMR (DMSO-d6, 500 MHz) δ ppm: 3.40

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(dd, J 15.5 and 7.5 Hz, 1H), 3.56 (dd, J 15.5 and 7.5 Hz, 1H), 6.48 (dd, J 10.5 and 7.5
Hz, 1H), 7.64 (dd, J 7.5 and 1.0 Hz 1H), 7.68-7.72 (m, 1H), 7.76-7.79 (m, 1H), 7.98
(dd, J 7.5 and 1.0 Hz, 1H), (8.24 (d, J 9.3 Hz), 8.46 (d, J 9.3 Hz), 9.33 (s, 1H). 13C
NMR (DMSO-d6, 125 MHz APT) δ ppm: 31.6, 79.6, 114.9, 120.7, 123.0, 124.2, 125.4,
126.8, 128.6, 130.5, 132.0, 134.7, 140.6, 146.8, 147.0, 167.4, 174.8, 180.3. HRMS-ESI
m/z [M + H]+ calcd for C20H13N4O5: 389.08805; found: 389.08867. ∆ = 0.2 ppm.
2.1.1.9. 2-(1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-
4,9-dione (6e). Yellow solid, m.p. 248–249 ºC. IR (KBr, cm-1): ν 1681, 1624 (C-H Ar),
1582, 1513, 1454, 1371, 1328, 1283, 1237, 1190, 1098, 1054, 1022, 994, 955, 889, 831,
720, 653, 617. 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.52 (dd, J 17.0 and 7.9 Hz, 1H),
3.65 (dd, J 17.0 and 10.7 Hz, 1H), 6.32 (dd, J 10.7 and 7.9 Hz, 1H), 7.46 (t, J 8.8 Hz,
13
2H), 7.80-7.88 (m, 2H), 7.93-7.96 (m, 2H), 7.99-8.03 (m, 2H), 9.06 (s, 1H). C NMR
(DMSO-d6, 125 MHz APT) δ ppm: 32.7, 79.0, 117.2 (d, J 23.3 Hz), 123.1 (d, J 8.8 Hz),
123.4, 124.5, 125.0, 126.3, 131.8, 133.0, 133.5 (d, J 2.9 Hz), 133.9, 134.9, 147.0, 159.3,
162.3 (d, J 244.5 Hz), 177.6, 182.0. 19F RMN (DMSO-d6, 470 MHz) δ ppm: -109.7 (bs,
1F). HRMS-ESI m/z [M + H]+ calcd for C20H13FN3O3: 362.09355; found: 362.09354. ∆
= 0 ppm.
2.1.1.10. 2-(1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-b]furan-
4,5-dione (7e). Orange solid, m.p. 209-210 ºC. IR (KBr, cm-1): ν 1698, 1653 (C-H Ar),
1618, 1587, 1514, 1489, 1453, 1408, 1357, 1278, 1219, 1160, 1081, 1032, 993, 860,
1
833, 773, 720, 696, 657. H NMR (DMSO-d6, 500 MHz) δ ppm: 3.40 (dd, J 15.3 and
7.5 Hz, 1H), 3.54 (dd, J 15.3 and 10.3 Hz, 1H), 6.44 (dd, J 10.3 and 7.5 Hz, 1H), 7.46
(t, J 8.8 Hz, 2H), 7.64 (dd, J 7.5 and 0.5 Hz, 1H), 7.70 (td, J 7.5 and 1.1 Hz, 1H), 7.77
13
(td, J 8.3 and 1.1 Hz, 1H), 7.94-7.99 (m, 3H), 9.09 (s, 1H). C NMR (DMSO-d6, 125

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MHz APT) δ ppm: 32.1, 80.4, 115.5, 117.2 (d, J 23.3 Hz), 123.2 (d, J 8.8 Hz), 123.3,
124.8, 127.4, 129.2, 131.1, 132.6, 133.5 (d, J = 2.9 Hz), 135.2, 147.0, 162.3 (d, J 244.7
Hz), 168.0, 175.4, 180.9. 19F RMN (DMSO-d6, 470 MHz) δ ppm: -109.7-109.7 (m, 1F).
HRMS-ESI m/z [M + H]+ calcd for C20H13FN3O3: 362.09355; found: 362.09354. ∆ = 0
ppm.
2.1.1.11. 2-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-
4,9-dione (6f). Yellow solid, m.p. 262-263 ºC. IR (KBr, cm-1): ν 1681, 1624 (C-H Ar),
1582, 1502, 1438, 1371, 1329, 1283, 1240, 1191, 1092, 1055, 1022, 955, 890, 857, 828,
721, 653. 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.51 (dd, J 17.0 and 7.8 Hz, 1H), 3.65
(dd, J 17.0 and 11.0 Hz, 1H), 6.32 (dd, J 11.0 and 7.8 Hz, 1H), 7.68 (d, J 9.0 Hz, 2H),
13
7.81-7.89 (m, 2H), 7.95 (d, J 9.0 Hz, 2H), 7.99-8.03 (m, 2H), 9.13 (s, 1H). C NMR
(DMSO-d6, 125 MHz APT) δ ppm: 32.2, 78.4, 121.8, 122.6, 123.9, 125.4, 125.7, 129.8,
131.2, 132.4, 133.1, 133.3, 134.3, 135.2, 146.5, 158.7, 177.0, 181.4. HRMS-ESI m/z [M
+ H]+ calcd for C20H13ClN3O3: 378.06400; found: 378.06399. ∆ = 0 ppm.
2.1.1.12. 2-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-b]furan-
4,5-dione (7f). Orange solid. m.p. 236-237 ºC. IR (KBr, cm-1): ν 1694, 1655, 1620,
1574, 1502, 1453, 1408, 1357, 1274, 1249, 1218 1142, 1080, 990, 868, 826, 777, 756,
698, 657. 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.40 (dd, J 15.4 and 7.4 Hz, 1H), 3.54
(dd, J 15.4 and 10.3 Hz, 1H), 6.44 (dd, J 10.3 and 7.4 Hz, 1H), 7.63-7.71 (m, 3H), 7.75-
13
7.78 (m, 1H), 7.93-7.99 (m, 3H), 9.14 (s, 1H). C NMR (DMSO-d6, 125 MHz APT) δ
ppm: 31.5, 79.8, 114.9, 121.8, 122.6, 124.1, 126.8, 128.6, 129.7, 130.5, 132.0, 133.1,
134.7, 135.2, 146.5, 167.4, 174.8, 180.3. HRMS-ESI m/z [M + H]+ calcd for
C20H13ClN3O3: 378.06400; found: 378.06399. ∆ = 0 ppm.

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2.1.1.13.
2-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-
b]furan-4,9-dione (6g). Yellow solid, m.p. 225-226 ºC. IR (KBr, cm-1): ν 1680, 1624,
1581, 1487, 1437, 1370, 1282, 1240, 1190, 1132, 1053, 1026, 956, 881, 856, 812, 750,
721, 679. 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.49 (dd, J 16.9 and 7.9 Hz, 1H), 3.67
(dd, J 16.9 and 10.8 Hz, 1H), 6.33 (dd, J 10.8 and 7.9 Hz, 1H), 7.81-7.89 (m, 3H), 7.95-
8.03 (m, 3H), 8.25 (d, J 3.0 Hz, 1H), 9.18 (s, 1H). 13C NMR (DMSO-d6, 125 MHz
APT) δ ppm: 32.2, 78.2, 120.1, 121.9, 122.8, 123.8, 125.4, 125.7, 131.2, 131.7, 132.2,
132.4, 133.3, 134.4, 133.3, 135.9, 146.7, 158.6, 177.0, 181.4. HRMS-ESI m/z [M + H]+
calcd for C20H12Cl2N3O3: 412.02502; found: 412.02502. ∆ = 0 ppm.
2.1.1.14.
2-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-
b]furan-4,5-dione (7g). Orange solid, m.p. 242-243 ºC. IR (KBr, cm-1): ν 1697, 1651
(C-H Ar), 1614, 1572, 1487, 1438, 1410, 1362, 1279, 1251, 1219, 1166, 1133, 1083,
1
1030, 1005, 868, 813, 770, 719, 677, 625. H NMR (DMSO-d6, 500 MHz) δ ppm: 3.38
(dd, J 15.4 and 7.2 Hz, 1H), 3.55 (dd, J 15.4 and 10.4 Hz, 1H), 6.45 (dd, J 10.4 and 7.2
Hz, 1H), 7.64 (d, J 8.0 Hz, 1H), 7.67-7.79 (m, 3H), 7.89 (d, J 8.5 Hz, 3H), 7.96-7.99
13
(m, 2H), 8.26 (d, J 2.5 Hz, 1H), 9.21 (s, 1H). C NMR (DMSO-d6, 125 MHz APT) δ
ppm: 31.6, 79.7, 114.9, 120.2, 121.9, 122.8, 124.2, 126.8, 128.6, 130.5, 131.2, 131.7,
132.0, 132.3, 134.7, 135.9, 146.7, 167.4, 174.8, 180.3. HRMS-ESI m/z [M + H]+ calcd
for C20H12Cl2N3O3: 412.02502; found: 412.02502. ∆ = 0 ppm.
2.1.1.15.
2-(1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-
b]furan-4,9-dione (6h). Yellow solid, m.p. 214-215 ºC. IR (KBr, cm-1): ν 1672, 1624,
1591, 1491, 1487, 1491, 1487, 1451, 1371, 1332, 1292, 1236, 1191, 1097, 1050, 946,
825, 718, 688. 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.53 (dd, J 16.8 and 8.3 Hz, 1H),
3.66 (dd, J 16.8 and 10.5 Hz, 1H), 6.35 (dd, J 10.5 and 8.3 Hz, 1H), 7.74 (dd, J 8.3 and

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2.5 Hz, 1H), 7.81-7.84 (m, 2H), 7.85-7.88 (m, 1H), 7.94 (d, J 2.5 Hz, 1H), 7.99-8.02
13
(m, 2H), 8.89 (s, 1H). C NMR (DMSO-d6, 125 MHz APT) δ ppm: 31.1, 78.2, 123.9,
125.4, 125.7, 126.5, 127.4, 128.1, 131.2, 131.5, 131.9, 132.4, 132.4, 133.3, 134.3,
135.1, 145.3, 158.7, 177.0, 181.4. HRMS-ESI m/z [M + H]+ calcd for C20H12Cl2N3O3:
412.02502; found: 412.02502. ∆ = 0 ppm.
2.1.1.16.
2-(1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2-
b]furan-4,5-dione (7h). Orange solid, m.p. 207-208 ºC. IR (KBr, cm-1): ν 1697, 1657,
1624, 1588, 1486, 1448, 1407, 1354, 1277, 1235, 1211, 1140, 1101, 1081, 1030, 1010,
1
860, 826, 772, 720, 698, 657, 631. H NMR (DMSO-d6, 500 MHz) δ ppm: 3.40 (dd, J
15.2 and 7.5 Hz, 1H), 3.55 (dd, J 15.2 and 10.4 Hz, 1H), 6.47 (dd, J 10.4 and 7.5 Hz,
1H), 7.64 (dd, J 7.5 and 1.0 Hz, 1H), 7.68-7.79 (m, 4H), 7.82 (d, J 9.0 Hz, 1H), 7.95 (d,
13
J 2.5 Hz, 1H), 7.98 (dd, J 7.8 and 0.8 Hz,1H), 8.91 (s, 1H). C NMR (DMSO-d6, 125
MHz APT) δ ppm: 31.6, 79.6, 114.9, 124.2, 126.4, 126.8, 127.4, 128.1, 128.6, 130.5,
131.5, 131.8, 132.0, 132.4, 134.6, 135.1, 145.4, 167.4, 174.8, 180.2. HRMS-ESI m/z [M
+ H]+ calcd for C20H12Cl2N3O3: 412.02502; found: 412.02502. ∆ = 0 ppm.
2.2. Biological Assays
2.2.1. Materials
The Caco-2, Calu-3, MDA-MB231 and Vero cell lines were purchased from the
Rio de Janeiro Cell Bank, Brazil. Dulbecco’s Modified Eagles Medium (DMEM), Ross
Park Memorial Institute Medium (RPMI-1640), Hank’s Balanced Salt Solution (HBSS),
foetal bovine serum (FBS), antibiotic solution (10,000 U/mL penicillin and 10mg/mL
streptomycin), antifungal solution (25-30 µg/mL amphotericin B), trypsin–EDTA

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solution (2.5 mg/mL trypsin, 0.2mg/mL EDTA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were all supplied by Sigma–Aldrich (São Paulo,
Brazil). Dimethyl sulfoxide (DMSO) and other reagents were from analytical grade.
2.2.2. Cell line culture conditions
The Caco-2 (colon adenocarcinoma), Calu-3 (lung adenocarcinoma), MDA-
MB231 (mammary gland adenocarcinoma) and Vero cell lines were maintained in
DMEM supplemented with 4.5 mg/ml glucose, 0.1 mg/ml penicillin, 0.14 mg/ml
streptomycin and 10 % inactivated FBS. Cultured cells were maintained at 37 °C in an
atmosphere containing 95% air and 5 % CO2. Cells were sub-cultivated every 48h by
trypsin–EDTA solution.
2.2.3. Cytotoxicity by MTT assay
Metabolically active cells were assessed using the MTT reduction colorimetric
assay, as reported by Mosmann [16] and Alley [17]. Cells were seeded in 96-well plates
(Corning) at density of 32,000 cells/well, distributed in a total volume of 200 µL/well.
Plates were taken to cell incubator at 37 °C and 5 % CO2 for 24 hours. After incubation,
cells were placed in contact with the samples (5 to 400 µM) for 48 hours. Samples were
solubilized in 10 % FBS DMEM or RPMI containing 0.5 % of DMSO. The control
group was represented by the mixture between the culture media and DMSO (0.5 %).
The samples were aspirated and treated with MTT reagent by adding100 µL of HBSS
and 25µL of MTT solution (2.5 mg/mL) per well. The plates containing the cells were
incubated with MTT for 3 hours at 37 °C and 5 % CO2. At the end of incubation time,
MTT was aspirated and the cells were washed with phosphate buffer solution (PBS)

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(pH 7.4). The phosphate buffer was then aspirated and 100 µL/well of DMSO were
added to break cell membrane and solubilize formazan crystals. The absorbance
readings were performed in Microplate Absorbance Reader iMARK™ (Bio-Rad
Laboratories Srl, Segrate, Italy), with reference to 570 nm and 690 nm after vigorous
shaking during 60 seconds [16, 17].
Cytotoxicity was expressed as the percentage of cells surviving after treatment
with samples in comparison to untreated cells. Drug concentration required to inhibit
cell growth by 50 % (IC50 for tumor cells and CC50 for Vero cells) and selectivity index
(SI) were calculated with GraphPad Prism 5 (version 5.00; GraphPad Software, Inc.,
San Diego, CA, USA). In some circumstances, the samples that presented statistically
significant differences from control group were not selected for IC50 assays due to the
lack of biological relevance of the data, i.e., statistical significance without adequate
level of cell viability. Thus, IC50 studies were carried out only with substances with high
toxicity (cell viability decreased by 50 %) on at least one tumor cell line and
simultaneously cell viability percentage above 70 % in Vero cells, which meets the
criteria defined by ISO 10993:5 [18].
2.2.4. In vitro hemocompatibility studies
All human blood samples were obtained from adult volunteers, healthy, 18-35
years, not using drugs or other substances that could interfere with the experiment, for
at least 15 days (Ethics Committee – ID number: 2.364.834).
The hemocompatibility evaluation of the best dihydronaphthoquinone derivative
with antitumor activity was performed through the hemolysis, platelet aggregation,
prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays. All
samples were used at 100 µM and the vehicle (DMSO) did not exceeded 1 % in the

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tests. In hemolysis test, healthy erythrocytes were washed 3 times with PBS (pH 7.4)
by centrifugation. Then, the derivative was incubated with the erythrocytes suspension
for 3 h at 37 °C. The release of hemoglobin was determined by the optical density of the
supernatant at 540 nm. The experiment was performed in triplicate and the complete
hemolysis (positive control) was determined by using 1 % Triton X-100 (Sigma
Aldrich, USA) [19-21]. The platelet aggregation assay was performed by turbidimetric
method using Chronolog® Model 560 lumi-aggregometer (Chrono-Log Corporation,
Havertown, PA). In this evaluation, the derivative (100 µM) was pre-incubated for 2
minutes at 37 °C in fresh PRP (platelet-rich plasma) before the addition of the platelet
agonist. Maximal aggregation was obtained by stimulating platelets with arachidonic
acid (500 mM) (Cayman Chemical Co., Inc., Ann Arbor, MI). The inhibition of platelet
aggregation was obtained using acetylsalicylic acid (100 µM) (Sigma Aldrich, USA) as
positive control [22,23]. The PT and aPTT assays were performed by using citrated
platelet poor plasma (PPP). Assays were performed in coagulation analyzer CoagLab®
IV (Beijing Shining Sun Technology Co., Ltd., China) and anticoagulant profile
(positive control) was obtained using rivaroxaban (100 µM) (BioChemPartner, Wuhan,
China) [23,24]. Negative control, for all hemocompatibility tests, was DMSO 1%.
2.2.5. Molecular modeling studies
The molecular structure of 7a, (the most promising compound according to the
biological assays) was constructed in neutral state using Spartan’10 program
(Wavefunction Inc. Irvine, CA, USA). The neutral state of this compound was selected
for this study accordingly to the protonation state at pH 7.4 predicted by MarvinSketch
v. 16.2.29.0, 2016, ChemAxon (http://www.chemaxon.com). This structure was
submitted to a conformational analysis using the Merck Molecular Force Field

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(MMFF). Then, the lowest energy conformer was selected for further geometry
optimization in vacuum using the RM1 semi-empirical method. Finally, the structure
was submitted to a single point ab initio calculation using the Hartree-Fock method
with 6-31G* basis set. The final optimized structure with electrostatic charges was used
for docking studies.
Meanwhile, the structures of the potential biological targets were obtained from
Protein Data Bank with the following codes: human topoisomerase I (1K4T),
topoisomerase IIα including ATPase domain (1ZXM) and DNA-binding domain
(5GWK) and topoisomerase IIβ (3QX3) [25-27]. Solvent molecules and ligands were
removed from protein structures.
Molecular docking studies were performed using Autodock Vina 1.1.2 [28].
Initially, the protein structures were prepared using Autodock Tools 1.5.7 by adding
polar hydrogen and assigning Gasteiger charges and all rotatable bonds of the ligand
were allowed to rotate freely during docking studies. The cubic grid box was centered
on the ligand and presented the dimensions of 20 Å3 for topoisomerase I, 18 Å3 for
topoisomerase IIα ATPase domain, 26 Å3 for topoisomerase IIα DNA-binding domain
and 20 Å3 for topoisomerase IIβ. The default search parameters values were used. The
lowest binding energy pose obtained in the docking of 7a with each enzyme was
selected and interactions analysis were carried out using Pymol v. 1.2r2 (The PyMOL
Molecular Graphics System, Version 1.2r2 Schrödinger, LLC).
2.2.6. Statistical Analysis
All experimental results (average and standard deviation) were statistically
evaluated in Sigma Plot 12.5 software. The t-test was used for comparative evaluations
of these results where a p-value ≤ 0.05 was considered statistically significant.

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3. Results and Discussion
3.1. Chemistry
Initially, the 4-vinyl-1H-1,2,3-triazoles 4a-h were prepared according to the
reported methodology [29, 30] from the protocol based on a Huisgen 1,3-dipolar
cycloaddition. The reaction between several aryl azides (from anilines 3a-h) and a
propargylic alcohol was catalyzed by Cu(I), providing only the 1,4-disubstituted
regioisomer, followed by partial oxidation of the alcohol to generate the 4-
carboxaldehyde-1H-1,2,3-triazoles. The Wittig reaction allowed to prepare the 4-vinyl-
1H-1,2,3-triazoles (4a-h) with overall yields ranging from 80-99 % (Fig. 2). All
obtained derivatives presented spectroscopic data in agreement with described report.
The reaction proposed to synthetize the hybrids naphthoquinone-1,2,3-triazoles
was a radical electrocyclic reaction between 2-hydroxy-1,4-naphthoquinone 5 and the 4-
vinyl-1H-1,2,3-triazoles (4a-h) that have been reported in the literature by several
authors (Fig. 2) [30]. Thus, the reactions were carried out in presence of an excess
cerium (IV) ammonium nitrate as one-electron oxidizing agent, in dried acetonitrile, at
low temperature and monitored by thin layer chromatography (TLC). After 3 hours of
reaction time it was observed total consumption of 5 a remaining small amounts of
unreacted 4 and the formation of two more polar products (a yellow spot and an orange
more polar spot). After the evaporation of the solvent and purification of the reaction
mixture by column chromatography using chloroform/ethyl acetate gradient as eluent,
followed by spectroscopic analysis of each synthetized compound, it was possible to
identify the compounds 6a-h (yellow solid compounds) and 7a-h (orange solid
compounds). Considering the conversions of 5 in these isolated two isomers the yields

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ranged from 40 to 59 %. In the Table 1 are described the ratio between 6:7 and the
overall.
Fig. 2. Synthesis of isomeric hybrids 6a-h and 7a-h
Table 1. Chemical conversion of compounds 6 and 7
Entry Compounds
R
H
Ratio (6 : 7)
Conversion (%)
55
1
2
6a
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
1 : 1.3
3
4-CH3
4-OCH3
4-NO2
4-F
1 : 1.7
1 : 1.7
1 : 1.3
1 : 1.4
1 : 1.4
1 : 1.4
1 : 1.5
59
47
55
40
59
56
58
4
5
6
7
8
9
10
11
12
13
14
15
16
4-Cl
7f
6g
7g
6h
7h
3,4-diCl
2,5-diCl

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The structures of the compounds were elucidated by spectroscopic techniques
1
(see Experimental Section and Supplementary Information). The H NMR spectrum
analysis of compound 6b, it could be observed two double doublets at 3.52 ppm (dd, J
17.0 and 7.9 Hz, 1H) and 3.65 ppm (dd, J 17.0 and 10.9 Hz, 1H) corresponding to H-3
protons; it was also observed a double doublet at 6.31 ppm (dd, J 10.9 and 7.9 Hz, 1H)
corresponding to H-2 proton (see Fig. 2 for numbering) and the typical benzoaromatic
pattern of 1,4-naphthoquinone isomers as multiplets at 7.80-7.88 ppm (m, 2H) and 7.99-
1
8.03 ppm (m, 2H). The H NMR spectrum of 7b, it can be observed two double
doublets at 3.40 ppm (dd, J 15.3 and 7.5 Hz, 1H) and 3.53 ppm (dd, J = 10.5 and
15.3Hz, 1H) corresponding to H-3 protons; it was also observed a double doublet at
6.43 ppm (dd, J 10.5 and 7.5 Hz, 1H) corresponding to H-2 proton and the typical
benzoaromatic pattern of 1,2-naphthoquinone isomers as one multiplet at 7.62-7.74 ppm
corresponding to three protons (H-7, H-8 and H-9) and at a lower field one double
doublet at 7.97 ppm (dd, J 8.0 and 1.0 Hz, 1H) referring to H-6 proton. Moreover, in the
aromatic region, the singlet at 9.03 ppm assigned to the resonance of the H-5’ proton for
isomer 6b and at 9.07 ppm for 7b, a very characteristic signal of the proton resonance of
triazolic ring CH.
3.2. Biological Assays
3.2.1. In vitro evaluation of the anticancer and toxicological profile
The hybrids naphthoquinone-1,2,3-triazoles 6a-h and 7a-h were screened in
vitro activity against MDA-MB231, Caco-2 and Calu-3 human cancer cell lines and by

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the absence of toxic effects (cell viability ≥ 70 %) on Vero cells, using MTT reduction
assay. The results are expressed in Table 2.
Table 2. Cell viability of cancer cell lines (Caco-2, Calu-3 and MDA-MB231) and Vero
cells after 48 hours of exposition to hybrids naphthoquinone-1,2,3-triazoles 6a-h and
7a-h at 5 µM. Data are presented as mean ± standard deviation
Cell lines
Compounds (5 µM)
Vero
Caco-2
Calu-3
MDA-MB231
108.73 ± 2.01
87.72 ± 3.44
95.33 ± 2.41
90.71 ± 3.37
99.56 ± 5.69
41.10 ± 5.59
97.44 ± 2.46
99.24 ± 4.35
97.90 ± 4.45
41.49 ± 2.97
103.37 ± 3.89
46.55 ± 5.82
96.62 ± 4.49
76.21 ± 14.33
67.97 ± 10.20
74.03 ± 6.37
41.80 ± 5.48
64.79 ± 7.54
34.14 ± 1.10
63.64 ± 3.78
88.65 ± 25.20
72.98 ± 8.08
55.84 ± 1.85
78.11 ± 16.08
59.29 ± 9.67
78.67 ± 9.35
88.29 ± 15.44
23.92 ± 0.58
88.03 ± 3.63
33.27 ± 2.84
81.81 ± 2.11
23.04 ± 3.43
95.49 ± 10.18
86.89 ± 4.26
77.32 ± 5.79
37.89 ± 8.28
82.12 ±7.68
46.42 ± 17.74
83.94 ± 5.66
113.49 ± 9.48
110.34 ± 17.78
111.60 ± 19.72
110.88 ± 14.63
125.48 ± 2.82
112.65 ± 5.16
119.97 ± 2.33
132.93 ± 6.43
104.43 ± 4.92
117.46 ± 8.99
134.31 ± 7.88
122.25 ± 8.43
110.98 ± 11.39
6a
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
7f
6g
80.99 ± 8.41
90.37 ± 14.71
74.70 ± 4.41
94.38 ± 4.52
85.75 ± 4.17
78.12 ± 4.95
110.70 ± 8.94
139.05 ± 4.92
117.82 ± 7.99
90.22 ± 1.13
103.83 ± 5.69
82.09 ± 6.29
7g
6h
7h
Significance levels for statistical tests (t-test) are marked with * when p-value ≤ 0.001
and ** when 0.001 < p-value ≤ 0.05. The groups with no statistically significant
differences were not marked (p-value > 0.05).
In vitro screening clearly demonstrated that only lung adenocarcinoma cells
(Calu-3) were not affected by derivatives in MTT assay, since cell viability values

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exceeded 100 %, i.e., cells were stimulated by the hybrids naphthoquinone-1,2,3-
triazoles 6a-h and 7a-h. The compounds 7a-h were selectively cytotoxic activity
against colon and / or mammary gland adenocarcinoma cells.
MDA-MB231 cells were sensitized by 7c, 7e and 7f (cell viability lower than 50
% - p-value ≤ 0.05), however these compounds were also toxic to Vero cells, and for
this reason, determination of selectivity index between Vero and MDA-MB231 cells
would not be relevant in this study.
Caco-2 cells have their viabilities decreased when exposed to derivatives 7a, 7b,
7c, 7e, 7f (p-value ≤ 0.05). However, these derivatives also decreased Vero cells
viability (< 70 %), with exception of 7a (67.97 ± 10.20), which was evaluated by IC50 in
Caco-2 cells (4.48 ± 0.71 µM) and CC50 in Vero cells (28.00 ± 7.88 µM). Finally, we
found that 7a was six times more selective for Caco-2 than Vero cells (SI = 6.25),
indicating the potential of this compound as future drug candidate.
Through the hemocompatibility studies it was possible to observe that 7a does
not affect the platelet aggregation induced by arachidonic acid, in addition, this
derivative did not interfere in prothrombin time (PT) and activated thromboplastin time
(APTT), preserving integrates the activation of blood coagulation by extrinsic and
intrinsic pathway. Interesting, 7a showed no significant hemolytic profile after 3 hours
incubation reinforcing their good safety degree (Fig. 3).

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Fig. 3. Hemocompatibility Evaluation. (A) Platelet Aggregation Assay: (C-) DMSO 1
% and (C+) ASA 100µM. (B) Hemolysis Assay: (C-) DMSO 1 % and (C+) Triton x-
100 1%. (C) Prothrombin Time (PT) Test: (C-) DMSO 1 % and (C+) Rivaroxaban
100µM. (D) Activated partial thromboplastin time (APTT) Test: (C-) DMSO 1 % and
(C+) Rivaroxaban 100 µM 7a (100 µM).
3.2.2 Molecular docking studies
The naphthoquinones are widely explored as potential anticancer agents and one
of their mechanisms of action described is the apoptosis induction through
topoisomerase enzymes inhibition 31-33]. Additionally, the dihydronaphthoquinone
moiety can mimic the polycyclic nucleus of known topoisomerase inhibitors (Fig. 4).
Thereby, in order to investigate the possible mechanism of action of 7a, we carried out

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docking studies with topoisomerase enzymes and compared with the crystal structure of
topoisomerases with known inhibitors.
Fig. 4. 2D structures of the co-crystallized inhibitor of topoisomerase I (topotecan) and
of topoisomerase II (etoposide and AMP-PNP).
First, we validated our docking protocol by redocking the co-crystalized
inhibitors topotecan (8) in topoisomerase I, etoposide (10) in topoisomerase IIα and IIβ
and adenylyl-imidodiphosphate (AMP-PNP) (9) in ATPase domain of topoisomerase
IIα. All the ligands in the docking complexes conserved the same binding mode and
interactions in the docking complexes in comparison to the crystal structures, which
were evidenced by the root mean square deviation (RMSD) values obtained: topotecan
(0.77 Å), AMP-PNP (0.49 Å), etoposide in topoisomerase IIα (0.63 Å) and etoposide in
topoisomerase IIβ (0.34 Å). According to the literature, successful docking prediction is
achieved when RMSD is lower than 2.0 [34-36].

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Thus, we applied these protocols for docking studies of 7a. Since this compound
has one chiral center, we docked both enantiomers, (R)-7a and (S)-7a, into the enzymes
active site. Both compounds bound between the base pairs +1G/+1C and -1T/-1A, but
they exhibited different binding modes. The results suggested that the S-enantiomer
may be the most efficient enantiomer and main responsible for the activity as it
presented the most similar binding mode in comparison to the known ligands, then, we
focused our discussion on this enantiomer binding mode with topoisomerase enzymes
(Fig. 5).
Docking of 7a in topoisomerase I showed that this compound bound between the
base pairs +1G/+1C and -1T/-1A (Fig. 5). The dihydronaphthoquinone moiety of this
compound was π-stacked with +1G, -1T and -1A as observed for the drug topotecan (8).
In addition, this nucleus was involved in hydrogen bonds with +1G and R364 which are
important interactions described in the literature for other topoisomerase I inhibitors
[37]. Meanwhile, the triazole and phenyl rings were positioned towards DNA major
groove, allowing van der Waals contacts with M428 which may also contribute to the
complex stabilization. The similar binding mode of 7a with topotecan resulted in a great
theoretical affinity to topoisomerase I, with a binding energy value of -11.5 Kcal/mol.

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Fig. 5. Binding mode of 7a in topoisomerases I and II in comparison to known
inhibitors of these enzymes. In the first line are the complexes of topoisomerase I with
(A) (S)-7a and (B) topotecan (8); in the second line are the complexes of topoisomerase
IIβ with (C) (S)-7a and (D) etoposide (10); in the third line are the complexes of the
DNA-binding domain of topoisomerase IIα with (E) (S)-7a, and (F); in the fourth line

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are the complexes of the ATPase domain of topoisomerase IIα with (G) (S)-7a, and (H)
AMP-PNP (9). Carbon atoms of the ligands were depicted in green, nucleotides in
yellow and amino acid residues in white. Ionic interactions and hydrogen bonds are
represented as dashed lines.
Moreover, we performed the docking studies of 7a in topoisomerase IIβ which
showed that this compound intercalated between base pairs +1T/+4A and -1C/+5G
similar to etoposide (Fig. 5). We observed that the triazole ring of 7a was π-stacked
with -1C and +1T while phenyl group and tricyclic nucleus protruded towards DNA
major and minor grooves, respectively, allowing van der Waals contacts with Q778 and
M782 and also a hydrogen bond interaction with R503. 7a conserved contacts observed
for etoposide and other topoisomerase IIβ inhibitors (e.g. R503, M782 and Q778) [38].
However, the dihydronaphthoquinone moiety of this compound was not stacked with
the nitrogenous-containing bases as expected for naphthoquinones with topoisomerase
IIβ inhibitory activity.
Further, we investigated whether this compound may act on topoisomerase IIα
(Fig. 5). As observed for etoposide, the compound bound between the base pairs +5G/-
1C and +4A/+1T with low binding energy (-10.1 Kcal/mol). The
dihydronaphthoquinone moiety was positioned similar to the polycyclic nucleus of the
etoposide and π-stacked with +1T and -1C nucleobases while hydrogen bond interaction
was observed with +5G. R487 and G488 were also involved in van der Waals contacts
with this nucleus. Interestingly, the triazole and phenyl rings of this compound was
positioned towards DNA major groove as observed for the glycoside moiety of
etoposide, conserving van der Waals contact with M766.
As the inhibition of the ATPase function of topoisomerase IIα is also a possible
mechanism of action for the cytotoxic activity of naphthoquinone derivatives [39], we

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docked 7a in the ATP binding site of this enzyme (Fig. 5). It exhibited a similar binding
mode than the known inhibitor with low binding energy (-11.2 Kcal/mol). The
dihydronaphthoquinone nucleus bound in the same site of adenine and ribose groups of
AMP-PNP, exploring van der Waals contacts with N91, I141 and F142 and,
additionally, the hydrogen bond interaction with S149 was conserved. The triazole and
phenyl groups were positioned in the phosphate binding site region, making contacts
with G161, R162, N163, G164, Y165, G166, A167 and K168 like AMP-PNP [26].
4. Conclusions
A series of hybrids naphthoquinone-1,2,3-triazoles have been evaluated for in
vitro cytotoxic activity against human tumor cell lines (MDA-MB231, Calu-3 and
Caco-2) and healthy cells (Vero). Compound 7a exhibited the most promising profile
due to its selective cytotoxic action against colon adenocarcinoma cells. Based on
docking studies, we suggest that 7a may inhibit topoisomerase I and IIα due to the
similarities in the binding mode and interactions in comparison to known inhibitors but
not topoisomerase IIβ and the blockage of topoisomerase I and IIα may be one of the
mechanisms of action responsible for the cytotoxic effect of 7a in Caco-2 cells. These
results encourage new studies of molecular mechanism and the development of more
potent and selective derivatives.
Supporting information
Supporting information includes physical and spectroscopic information for
compounds

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Acknowledgments
The authors would like to thank to National Council of Research of Brazil
(CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES)
and Carlos Chagas Filho Research Support Foundation of the state of Rio de Janeiro
(FAPERJ) for funding this work and for Research and Post-graduation fellowships.
Conflict of Interest
The authors declare that there are no conflicts of interest.
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