Synthesis of a new class of naphthoquinone glycoconjugates and evaluation of their potential as antitumoral agents

Article abstract of DOI:10.1039/c5ra19192k

A novel series of carbohydrate-based naphthoquinones was synthesized and evaluated for cytotoxicity against different cancer cell lines. The compounds derived from 5-hydroxy-1,4-naphthoquinone (juglone) showed better cytotoxicity profiles against HCT-116, A-549 and MDA-MB 435 human cancer cells than the parent compound. The results suggest that the hydroxyl group on the aromatic ring increased the pro-oxidant activity of these new naphthoquinone derivatives. Furthermore, two derivatives were found to be more active against melanoma cells (MDA-MB435) than the clinically useful anticancer agent doxorubicin, and none of the compounds caused mouse erythrocyte lysis.

Full text of DOI:10.1039/c5ra19192k

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Synthesis of a new class of naphthoquinone
glycoconjugates and evaluation of their potential
Cite this: RSC Adv., 2015, 5, 96222
as antitumoral agents†
a
a
Vinicius R. Campos, Anna C. Cunha, Wanderson A. Silva,a Vitor F. Ferreira,a
*
Carla Santos de Sousa,b Patrıcia D. Fernandes,b Vinıcius N. Moreira,a David R. da
´
´
Rocha,a Flaviana R. F. Dias,a Raquel C. Montenegro,c Maria C. B. V. de Souza,a
Fernanda da C. S. Boechat,a Caroline F. J. Francoa and Jackson A. L. C. Resended
A novel series of carbohydrate-based naphthoquinones was synthesized and evaluated for cytotoxicity
against different cancer cell lines. The compounds derived from 5-hydroxy-1,4-naphthoquinone
(juglone) showed better cytotoxicity profiles against HCT-116, A-549 and MDA-MB 435 human cancer
cells than the parent compound. The results suggest that the hydroxyl group on the aromatic ring
increased the pro-oxidant activity of these new naphthoquinone derivatives. Furthermore, two
derivatives were found to be more active against melanoma cells (MDA-MB435) than the clinically useful
anticancer agent doxorubicin, and none of the compounds caused mouse erythrocyte lysis.
Received 17th September 2015
Accepted 2nd November 2015
DOI: 10.1039/c5ra19192k
www.rsc.org/advances
events lead to the depletion of glutathione in cells as well as
damage to other intracellular components.3
1 Introduction
Natural glycoconjugated compounds,1–3 1–7 (Fig. 1), are useful
drugs that are commonly used clinically in the treatment of
cancer. The importance of the carbohydrate residues in these
structures has been well established: these moieties are
important for solubilization and also decrease the toxicity and
improve the pharmacokinetics of the compounds.4 Indeed,
several cases in the literature5 show the benet to biological
activity of a carbohydrate moiety in the structure of a molecule.
For example, the sugar moieties in antibiotic anthracycline
derivatives 1–3 (Fig. 1) participate in the molecular recognition
of the main cellular targets, DNA and topoisomerase II, forming
a DNA–drug–topoisomerase II ternary complex in which the
enzyme is covalently linked to a broken DNA strand, which is
critical for the induction of apoptosis and cell death.6,7
The importance of exploring the synthesis of sugar-conjugated
compounds to increase the selectivity of drug uptake is based on
the Warburg effect,8 which is related to the large consumption of
glucose by cancerous cells compared to normal cells due to the
high rate of aerobic glycolysis in the former.8
Recently, our group described the synthesis and pharmaco-
logical evaluation of a series of carbohydrate-based quinones.
In this study,5 compounds 8a and 8b (Fig. 2) (IC50 below 3.0 mM)
were found to exhibit signicant activity against a melanoma
cell line (MDA-MB 435).
Quinones containing heterocyclic aromatic rings in their
structures have been extensively explored by many research
groups.9,10 Structure–activity relationship studies have revealed
that the number and position of nitrogen (N) atoms in the ve-
or six-membered heterocyclic ring are critical factors for
enhancing antitumor activity.9,10
Among nitrogen-containing heterocycles, 1,2,3-triazole
derivatives exhibit a broad spectrum of biological properties11–15
and may serve as scaffold structures for the development of new
pharmaceutical compounds. This heterocyclic system is resis-
tant to metabolic degradation and can interact with biological
targets through hydrogen bonds and dipole interactions,
properties that may be responsible for the enhanced biological
activities of many synthetically versatile molecules.16
Other mechanisms can be involved in quinone toxicity of
such as redox cycles, which result in the production of semi-
quinone radicals and reactive oxygen species (ROS). These
a
´
ˆ
Universidade Federal Fluminense, Departamento de Quımica Organica, Programa de
´
˜
´
˜
˜
´
Pos-Graduaçao em Quımica, Outeiro de Sao Joao Batista, 24020-141 Niteroi, RJ,
Brazil. E-mail: annac@vm.uff.br; Fax: +55 21 26292145; Tel: +55 21 26292148
b
ˆ
´
´
Universidade Federal do Rio de Janeiro, Instituto de Ciencias Biomedicas, Laboratorio
˜
´
de Farmacologia da Dor e da Inamaçao, CCS, Bloco J, sala 10. Cidade Universitaria,
RJ, Brazil
c
´
ˆ
´
´
Universidade Federal do Para, Instituto de Ciencias Biologicas, Belem, PA, Brazil
To extend our investigation into the synthesis and biological
evaluation of quinone glycoconjugate compounds5,17 and 1,2,3-
triazole derivatives,11–15 we prepared a new series of naphtho-
triazoles, 9a–c (Scheme 1), and assessed their activity against
three types of cancer cells: human colorectal carcinoma
d
´
ˆ
´
Universidade Federal Fluminense, Departamento de Quımica Inorganica, Laboratorio
˜
´
Regional de Difraçao de Raios X (LDRX), 24020-141, Niteroi, RJ, Brazil
† Electronic supplementary information (ESI) available. CCDC 1055524 and
1055525. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c5ra19192k
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Fig. 1 Examples of natural glycoconjugated compounds (1–7) with antitumoral activity.
Naturally occurring hydroxy naphthoquinone (juglone, 12)
exhibits a toxicological effect through its ability to undergo
redox cycling to damage macromolecules, such as DNA, lipids
and proteins.18 Several studies have shown that the introduction
of a hydroxyl group at the carbon of the benzene moiety
improves the pro-oxidant properties of naphthoquinone deriv-
atives, stimulating lipid peroxidation and adduct formation
with macromolecules.18
Based on these reports, we also decided to prepare a new
series of hydroxylated naphthoquinones, 11a–c, exploring
cycloaddition between juglone (12) and azide-functionalized
carbohydrates 13a–c (Scheme 1), and to investigate the inu-
ence on biological activity of a hydroxyl group at position 5 of
the benzene ring.
Fig. 2 Examples of synthetic molecules (8a and 8b) with antitumoral
activities.
(HCT-116), human lung adenocarcinoma (A-549) and human
melanoma (MDA-MB435).
The synthesis of derivatives17 10a–c, which possess amino-
carbohydrate chains at the C-2 position of the quinone moiety
(Scheme 1), was also performed to verify the possible pharma-
cophoric effects of the 1,2,3-triazole moiety fused to
naphthoquinone.
The compounds 10a–c and 11a–c were also evaluated for
their cytotoxic activities against cancer cell lines HCT-116, A-549
and MDA-MB435.
Scheme 1 Strategy for the synthesis of naphthoquinone derivatives 9a–c, 10a–c and 11a–c.
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Scheme 2 Synthesis of the novel naphthotriazole derivatives 9a–c and 2-aminonaphthoquinones 10a–c.
a catalytic amount of 10% Pd/C (Scheme 3) led to the corre-
sponding amines 16a–c.
2 Results and discussion
2.1 Chemistry
Juglone 12, prepared in 85% yield via the methodology
described by Ferreira and coworkers,21 was reacted with azide
compounds 13a and 13b. Although it is possible to form
a mixture of the 5- and 8-hydroxy-naphthotriazole regioisomers
(Scheme 4) via this thermal 1,3-dipolar cycloaddition reaction,
only 5-hydroxy-1-substituted-1H-naphtho[2,3-d][1,2,3-]triazole-
4,9-dione derivatives 11a and 11b were isolated, in moderate
yields. Furthermore, unexpected products 22a and 22b, pos-
sessing a carbohydrate chain at the C-2 position of the quinone
ring, were also isolated.
Naphthotriazole derivatives 9a–c (Scheme 2) were obtained in
moderate yields via a [3 + 2] cycloaddition reaction between
glycosyl azides 13a–c and 1,4-naphthoquinone (14) (Scheme 2).
The probable mechanism for the formation of these
compounds involves an initial 1,3-dipolar cycloaddition reac-
tion between the azide carbohydrates 13a–c and 1,4-benzoqui-
none (14), producing the corresponding triazoline
intermediates 15a–c, which are tautomerized and oxidized to
form the desired glycotriazole derivatives 9a–c.
The reaction of compound 13c with juglone (12) gave a crude
product which upon TLC analysis showed to be a mixture of two
major compounds with retention factors (Rf) very similar. The
1H NMR spectrum of this mixture led to the conclusion that one
of the substances corresponded to 11c, the desired compound,
and that the other substance was the amine 22c. Attempts to
separate the products by chromatography on silica gel column
were unsuccessful (Scheme 5).
Glycosyl azides 13a–c were prepared from their correspond-
ing commercially available reagents D-ribose, D-xylose and D-
galactose using previously described methods for carbohydrate
derivatization19,20 (Scheme 3).
The aminonaphthoquinone analogs 10a–c (Scheme 2) were
synthesized in moderate yields via ultrasound-accelerated 1,4-
cycloaddition between 1,4-naphthoquinone (14) and different
aminocarbohydrates (16a–c), according to our previous report.17
Reduction of the azide derivatives 13a–c in the presence of
Scheme 3 Preparation of derivatives of carbohydrates 13a–c and 16a–c.
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2.2 Biological assay
Compounds 9a–c, 10a–c, 11a, 11b, 12, 14, 22a and 22b were
tested in vitro against three cancer cell lines and normal cells,
human blood peripheral leukocytes, using the MTT assay.
Doxorubicin was used as the positive control. Erythrocyte lysis
was evaluated at a concentration of 200 mM. The concentrations
inducing 50% inhibition of cell growth (IC50) are reported in
Table 2. The compounds were classied according to their
activity as highly active (IC50 < 2 mM), moderately active (2 mM <
IC50 < 10 mM), or inactive (IC50 > 10 mM).22
The naphthoquinone derivatives 9a–c, 10a–c, 11a, 11b, 12,
14, 22a and 22b (Table 2) did not exhibit a lytic effect against
either erythrocytes or normal human leukocytes.
Scheme 4 Preparation of isomeric glycotriazole derivatives 11a and
b and aminonaphthoquinone derivatives 22a and b.
Among naphthotriazoles 9a–c, only derivative 9b showed
signicant activity against HCT-116 cells, with an IC50 value of
4.85 mM. This result indicates that the presence of the glycol
1,2,3-triazole subunit determines the biological activity
assessed. Aminoquinones 10a–c showed no activity against the
three cell lines, allowing us to speculate that the presence of the
amino group attached to carbon 2 has no effect on the biolog-
ical activity of the compounds.
Aminoquinone analogs 22a and 22b showed higher cytotoxic
activities than compounds 10a and 10b. This can be attributed
to the presence of the hydroxyl group connected to the benzene
ring in these derivatives, possibly contributing to their pro-
oxidant activities.18
Scheme 5 Synthesis of naphthoquinone compounds 11c and 22c.
In addition, the new glycoconjugate compounds 22a and 22b
were also more active than juglone (12). We speculate that
cellular permeability of these 5-hydroxy-1,4-naphthoquinonic
derivatives could be improved by the presence of the carbohy-
drate chain at the C-2 position of the quinone moiety. As 22a
and 22b were active against different cell lines, it appears that
they can bind to different targets in a specic background.
The series of quinones 11a–c displayed greater activity in all
tested cancer cell lines than naphthoquinone derivatives 9a and
9b, possibly indicating that these quinones have increased pro-
oxidant capacity. One can speculate that the hydroxyl attached
to the C5 carbon in these derivatives is responsible for their
increased anti-tumor activity.
The structures of the new compounds 9a–c, 11a, 11b, 22a
and 22b were established by spectral data: one- and two-
dimensional 1H and 13C NMR spectra [1H, 13C-APT, COSY-1H
1
ꢀ H, HSQC and HMBC], IR and elemental analysis.
1
The H NMR spectra of 11a, 11b, 22a and 22b indicate that
only one of the two possible isomers was formed. The regio-
chemistry of the reaction was investigated based on X-ray crys-
tallographic analysis of substances 11a and 22a (Fig. 3). Fig. 3
shows the ORTEP diagrams of these compounds, and crystal
data and renements are provided in Table 2. The diagram
shows that the carbohydrate chains are in the anti-position to
the hydroxyl group attached to the quinone ring of 11a and 22a.
The carbohydrate moieties of 11a and 22a adopt envelope and
twist conformations, respectively.
Fig. 3 ORTEP diagrams of compounds 11a (a) and 22a (b), showing 50% probability ellipsoids and the atom-numbering scheme.
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Table 1 X-ray crystallographic data for compounds 11a and 22a
3 Conclusion
11a
22a
The thermal cycloaddition reaction of glycosyl azides 13a–c with
1,4-naphthoquinone (14) produced the new glyconaphtho-
triazoles 9a–c in moderate yields. Under the same conditions,
parent compound 21 afforded the corresponding glyconaph-
thotriazoles, 11a and 11b, in moderate yields as well as the
unexpected aminonaphthoquinones 22a and 22b. These
carbohydrate-based naphthoquinones, 11a, 11b, 22a and 22b,
derived from juglone (12) showed the best cytotoxicity prole
against human cell lines HCT-116, A-549 and MDA-MB 435
when compared with the parent compounds, 9a, 9b, 10a and
10b, respectively. These data show the importance of the
hydroxyl group to the pro-oxidant action of these naph-
thoquinone compounds. Indeed, derivatives 11a and 11b were
more active than the clinically useful anticancer agent doxoru-
bicin against melanoma cancer cells (MDA-MB435 cells).
However, none of the compounds exhibited lytic effects against
mouse erythrocytes.
Chemical formula
Mr
Crystal size/mm
C
19H19N3O7
C19H21NO7
375.37
401.37
0.44 ꢀ 0.19 ꢀ
0.43 ꢀ 0.33 ꢀ
0.07 mm
0.21 mm
Crystal color, habit
Crystal system,
space group
Yellow, plate
Monoclinic, P21
Yellow, prism
Monoclinic, P21
Temperature (K)
150
150
˚
a, b, c (A)
5.9539 (2),
5.7856 (2),
25.9610 (11)
91.139 (4)
894.10 (6)
2
Mo Ka
0.12
0.810, 1
7869, 3770, 3232
5.8376 (2),
11.9174 (4),
12.2331 (5)
94.745 (4)
848.13 (5)
2
Mo Ka
0.11
0.918, 1
9555, 3667, 3075
b (ꢁ)
3
˚
V (A )
Z
Radiation type
m (mmꢂ1
)
Tmin, Tmax
No. of measured,
independent and
observed [I > 2s(I)]
reections
Molecules 11a, 11b, 22a and 22b can be considered prom-
ising prototypes for cancer therapy.
Rint
0.036
0.042, 0.098, 1.10
263
0.19, ꢂ0.20
0.030
0.035, 0.078, 0.97
248
0.19, ꢂ0.21
R[F2 > 2s(F2)], wR(F2), S
No. of parameters
ꢂ3
4 Experimental
4.1 Materials and measurements
˚
Drmax, Drmin (e A
)
Melting points were determined with a Fisher-Johns – Melting
Point Apparatus instrument, and the values are uncorrected.
Infrared (IR) spectra were recorded on a Perkin-Elmer FT-IR
1600 spectrophotometer using KBr pellets. NMR spectra were
recorded on a Varian Unity Plus 300 MHz or 500 MHz spec-
trometer using the specied solvents. Chemical shis (d) are
reported in ppm, and the coupling constants (J) are expressed in
Hz. Column chromatography was performed with silica gel
ash from Acros. The reactions were routinely monitored by
thin layer chromatography (TLC) on silica gel pre-coated F254
Derivatives 11a and 11b were also more active against
melanoma cancer cells (MDA-MB435) than the clinically useful
anticancer agent doxorubicin. Although doxorubicin is consid-
ered an important drug for cancer chemotherapy, it has several
clinical limitations, such as cardiotoxic effects and a high
incidence of multi-drug resistance. Therefore, glycoconjugate
compounds 11a, 11b, 22a and 22b can be considered promising
prototypes in drug development for cancer therapy.
Table 2 Cytotoxic activity expressed as the IC50 (mM) of the compounds against different cell linesd
Cancer cell linea
Compound
HCT-116
A-549
MDA-MB 435
Human blood leukocytes
Erythrocytesc
9a
9b
9c
10a
10b
10c
11a
11b
>10
4.85
>10
>10
>10
>10
0.67
0.52
1.60
>10
1.26
0.43
0.10
>10
>10
>10
>10
>10
>10
1.22
0.99
3.7
>10
>10
>10
8.9
>10
>10
0.47
0.29
NT
NT
5.0
NT
0.88
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.41
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>25
12
14
22a
NT
0.77
1.76
0.47
22b
Doxorubicinb
a
Data are presented as IC50 values (mM), and 95% condence intervals were obtained by nonlinear regression for all cell lines from three
independent experiments. Doxorubicin was used as a positive control. Only compounds with an IC50 value lower than 5 mg mLꢂ1 for at least
b
one cell line were considered active. c Concentration of compound that induced erythrocyte lysis. NT – not tested.
d
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1-(50-Deoxy-10,20-O-isopropylidene-D-xylofuranos-50-yl)-1H-
naphtho[2,3-d][1,2,3]triazole-4,9-dione (9c). The 1,3-dipolar
cycloaddition reaction between naphthoquinone 12 and
glycosyl azide 13c yielded glyconaphthotriazole 9c (90.0 mg,
Merck plates. Microanalyses were performed using a Perkin-
Elmer Model 2400 instrument, and all values were within
ꢃ0.4% of the calculated compositions.
48%) as a light yellow solid: mp 240–243 C; IR (KBr) n (cmꢂ1):
ꢁ
1687 and 1592 (C]O). 1H NMR (500.00 MHz, DMSO) d: 1.21 (s,
3H, CH3), 1.31 (s, 3H, CH3), 4.18 (d, 1H, J ¼ 2.7 Hz, H-30), 4.50 (d,
1H, J ¼ 3.6 Hz, H-20), 4.61–4.64 (m, 1H, H-40), 5.00–5.01 (m, 2H,
H-50a and H-50b), 5.88 (d, 1H, J ¼ 3.6 Hz, H-10), 7.92 (td, 1H, J ¼
7.5 and 1.5 Hz, H-7), 7.95 (td, 1H, J ¼ 7.5 and 1.5 Hz, H-6), 8.17
(dd, 1H, J ¼ 7.5 and 1.5 Hz, H-8), 8.19 (dd, 1H, J ¼ 6.5 and 1.5
Hz, H-5) ppm. 13C NMR (125.0 MHz, DMSO) d: 26.1 (CH3), 26.7
(CH3), 49.4 (C-50), 73.8 (C-30), 78.5 (C-40), 85.1 (C-20), 104.5 (C-10),
110.9 (–OCO–), 126.8 (C-5), 126.9 (C-8), 132.9 (C-8a), 133.1 (C-
4a), 134.3 (C-9a), 134.5 (C-7), 135.1 (C-6), 144.8 (C-3a), 175.1
(C-9), 176.8 (C-4) ppm. Anal. calc. for C18H17N3O6: C, 58.22; H,
4.61; N, 11.32. Found: C, 58.61; H, 4.35; N, 11.52%.
4.2 General procedure for the preparation of new
naphthotriazoles 9a–c
The glycosyl azides 13a–c (1.0 mmol) and 1,4-naphthoquinone
(14) (0.158 mg, 1.0 mmol) were dissolved in toluene and heated
at 100 ꢁC for 24 hours. Aer completion of the reaction, which
was monitored using TLC (eluted with hexane/EtOAc ¼ 1/1), the
mixture was cooled, and the solvent was removed. The residual
solid product was puried by column chromatography on silica
gel and eluted with an increasing polarity gradient mixture of
hexane and ethyl acetate (100% to 50%) to afford compounds
9a–c.
(Methyl-50-deoxy-20,30-O-isopropylidene-b-D-ribofuranosid-50-
yl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (9a). The 1,3-
dipolar cycloaddition reaction between naphthoquinone 12 and
glycosyl azide 13a yielded glyconaphthotriazole 9a (121.0 mg,
4.3 General procedure for the preparation of new
naphthoquinone derivatives 11a, 11b, 22a and 22b
63%) as an orange solid: mp 225–227 ꢁC; IR (KBr) n (cmꢂ1): 1588 The glycosyl azides 13a and 13b (1.0 mmol) and juglone 12
and 1686 (C]O).
(0.174 mg, 1.0 mmol) were dissolved in toluene and heated at
1H NMR (500.00 MHz, CDCl3) d: 1.30 (s, 3H, CH3), 1.44 (s, 3H, 100 ꢁC for 24 hours. The reaction mixture was concentrated
CH3), 3.41 (s, 3H, OCH3), 4.72–4.73 (m, 1H, H-40), 4.74 (d, 1H, J under reduced pressure and the resulting residue was puried
¼ 6.0 Hz, H-20), 4.86 (d, 1H, J ¼ 6.0 Hz, H-30), 5.01 (dd, 2H, J ¼ by column chromatography using silica gel and eluted with an
7.5 and 1.0 Hz, H-50a and H-50b), 5.02 (s, 1H, H-10), 7.82 (td, 1H, J increasing polarity gradient mixture of hexane and ethyl acetate
¼ 7.5 and 1.5 Hz, H-7), 7.86 (td, 1H, J ¼ 7.5 and 1.5 Hz, H-6), 8.24 (100% to 70%) to give the desired compounds 11a and 11b and
(dd, 1H, J ¼ 7.5 and 1.5 Hz, H-8), 8.34 (dd, 1H, J ¼ 7.5 and 1.5 the unexpected aminoquinones 22a and 22b.
Hz, H-5) ppm.
13C NMR (125.0 MHz, CDCl3) d: 25.1 (CH3), 26.5 (CH3), 53.2 furanosid-50-yl)-1H-nao[2,3-d][1,2,3]triazole-4,9-dione
(C-50), 55.9 (OCH3), 81.8 (C-30), 84.6 (C-40), 85.3 (C-20), 110.4 (C- and 5-hydroxy-2-(methyl-50-deoxy-20,30-O-isopropylidene-b-D-
5-Hydroxy-(methyl-50-deoxy-20,30-O-isopropylidene-b-D-ribo-
(11a)
10), 113.1 (–OCO–), 127.5 (C-8), 128.5 (C-5), 132.8 (C-8a), 133.5 ribofuranosid-50-yl)-amino-1,4-naphthoquinone (22a). The 1,3-
(C-4a), 133.6 (C-9a), 134.5 (C-7), 135.4 (C-6), 145.7 (C-3a), 175.6 dipolar cycloaddition reaction between juglone 12 and glycosyl
(C-9), 176.7 (C-4) ppm. Anal. calc. for C19H19N3O6: C, 59.22; H, azide 13a gave glyconaphthotriazole 11a (136.0 mg, 68%) and
4.97; N, 10.90. Found: C, 59.05; H, 5.29; N, 10.79%.
amino compound 22a (5.6 mg, 3%).
1-(60-Deoxy-10,20:30,40-di-O-isopropylidene-D-galactopyranos-
The compound 11a was obtained as a yellow solid: mp 144–
60-yl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (9b). The 1,3- 147 ꢁC; IR (KBr) n (cmꢂ1): 3434 (OH), 1649 (C]O), 1449 (C]C).
dipolar cycloaddition reaction between naphthoquinone 12 and 1H NMR (500.00 MHz, CDCl3) d: 1.31 (s, 3H, CH3), 1.45 (s, 3H,
glycosyl azide 13b yielded glyconaphthotriazole 9b (126.0 mg, CH3), 3.41 (s, 3H, OCH3), 4.70–4.72 (m, 1H, H-40), 4.72 (d, 1H, J
57%) as a light yellow solid: mp 160–162 C; IR (KBr) n (cmꢂ1): ¼ 6.0 Hz, H-20), 4.85 (d, 1H, J ¼ 6.0 Hz, H-30), 4.98 (dd, 2H, J ¼
ꢁ
1689 and 1593 (C]O). 1H NMR (300.00 MHz, CDCl3) d: 1.22 (s, 7.5 and 2.5 Hz, H-50a and H-50b), 5.02 (s, 1H, H-10), 7.38 (dd, 1H,
3H, CH3), 1.35 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.57 (s, 3H, CH3), J ¼ 8.5 and 1.0 Hz, H-6), 7.68 (dd, 1H, J ¼ 8.5 and 7.5 Hz, H-7),
4.31 (dd, 1H, J ¼ 5.0 and 2.7 Hz, H-20), 4.36 (dd, 1H, J ¼ 7.8 and 7.80 (dd, 1H, J ¼ 7.5 and 1.0 Hz, H-8), 12.31 (s, 1H, OH) ppm.
2.0 Hz, H-30), 4.46–4.49 (m, 1H, H-50), 4.68 (dd, 1H, J ¼ 7.8 and
13C/APT NMR (125.0 MHz, CDCl3) d: 25.1 (CH3), 26.5 (CH3),
2.6 Hz, H-40), 4.82 (dd, 1H, J ¼ 13.9 and 3.4 Hz, H-60a), 5.32 (dd, 53.3 (C-50), 56.0 (OCH3), 81.8 (C-40), 84.6 (C-20), 85.3 (C-30), 110.4
1H, J ¼ 13.9 and 9.6 Hz, H-60b), 5.40 (d, 1H, J ¼ 5.0 Hz, H-10), (C-10), 113.1 (–OCO–), 115.5 (C-4a), 120.8 (C-8), 126.9 (C-6), 133.2
7.80 (td, 1H, J ¼ 7.5 and 1.5 Hz, H-7), 7.85 (td, 1H, J ¼ 7.5 and 1.5 (C-8a), 133.9 (C-9a), 136.9 (C-7), 145.5 (C-3a), 163.6 (C-5), 174.8
Hz, H-6), 8.24 (dd, 1H, J ¼ 7.6 and 1.2 Hz, H-8), 8.34 (dd, 1H, J ¼ (C-9), 182.7 (C-4) ppm. Anal. calc. for C19H19N3O7: C, 56.86; H,
7.6 and 1.2 Hz, H-5) ppm.
4.77; N, 10.47. Found: C, 58.04; H, 5.25; N, 9.05%.
13C NMR (75.0 MHz, CDCl3) d: 24.8 (CH3), 24.9 (CH3), 25.8
The compound 22a was obtained as a red solid: mp 128–130
(CH3), 26.2 (CH3), 50.7 (C-60), 67.2 (C-50), 70.4 (C-20), 71.2 (C-40), ꢁC; IR (KBr) n (cmꢂ1): 3401 (OH), 3263 (N–H); 1623, 1592 (C]O),
71.4 (C-30), 96.3 (C-10), 109.1 (–OCO–), 110.4 (–OCO–), 127.4 (C- 1468 (C]C). 1H NMR (500.00 MHz, CDCl3) d: 1.32 (s, 3H, CH3),
5), 127.9 (C-8), 133.0 (C-8a), 133.6 (C-4a), 134.3 (C-7), 134.4 (C- 1.50 (s, 3H, CH3), 3.29–3.34 (m, 2H, H-50a and H-50b), 3.41 (s,
9a), 135.2 (C-6), 145.6 (C-3a), 175.5 (C-9), 176.9 (C-4) ppm. 3H, OCH3), 4.49–4.51 (m, 1H, H-40), 4.63 (dd, 1H, J ¼ 6.0 and 1.0
Anal. calc. for C22H23N3O7: C, 59.86; H, 5.25; N, 9.52. Found: C, Hz, H-30), 4.66 (d, 1H, J ¼ 6.0 Hz, H-20), 5.05 (s, 1H, H-10), 5.64 (s,
59.32; H, 5.63; N, 9.25%.
1H, H-3), 6.66–6.68 (m, 1H, N–H), 7.25 (dd, 1H, J ¼ 8.5 and 1.0
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Hz, H-6), 7.47 (dd, 1H, J ¼ 8.5 and 7.5 Hz, H-7), 7.59 (dd, 1H, J ¼ least squares on F2 with the SHELX-97 soware package.23 The
7.5 and 1.0 Hz, H-8), 12.98 (s, 1H, OH) ppm. 13C/APT NMR CCDC reference numbers for 11a and 22a are 1055525 and
(125.0 MHz, CDCl3) d ppm: 25.1 (CH3), 26.6 (CH3), 45.6 (C-50), 1055524, respectively.†
55.7 (OCH3), 82.3 (C-30), 84.5 (C-40), 85.4 (C-20), 100.5 (C-3), 109.8
(C-10), 113.1 (–OCO–), 115.0 (C-4a), 119.2 (C-8), 126.1 (C-6), 130.7
(C-8a), 134.2 (C-7), 149.0 (C-2), 161.3 (C-5), 181.1 (C-1), 189.2 (C-
6 Biology
6.1 Cytotoxicity against cancer cell lines
4) ppm. Anal. calc. for C19H21NO7: C, 60.79; H, 5.64; N, 3.73.
Found: C, 61.61; H, 6.00; N, 3.21%.
The compounds (0.15–20 mM) were tested for cytotoxic activity
against cell lines HCT-116 (human colorectal carcinoma, ATCC
# CCL-247), A-549 (human lung adenocarcinoma, ATCC # CCL-
185) and MDA-MB435 (human melanoma, ATCC # HTB-129) as
well as against freshly prepared human blood leukocytes and
erythrocytes. All cell lines were maintained in DMEM medium
supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 U mLꢂ1 penicillin, and 100 mg mLꢂ1 streptomycin at 37 ꢁC
in an atmosphere containing 5% CO2. Each compound was
dissolved with DMSO and diluted with the cell culture medium
to obtain a concentration of 100 mM, which was then incubated
with the cells for 72 hours. The negative control received the
same amount of DMSO (0.005% at the highest concentration).
Doxorubicin was used as a positive control. Cell viability was
determined by the reduction of the yellow dye 3-(4,5-dimethyl-2-
thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue
formazan product aer 3 hours of incubation, as described by
Denizot (1986).24
5-Hydroxy-1-(60-deoxy-10,20:30,40-di-O-isopropylidene-D-gal-
actopyranos-60-yl)-1H-nao[2,3-d][1,2,3]triazole-4,9-dione (11b)
and 5-hydroxy-2-(60-deoxy-10,20:30,40-di-O-isopropylidene-D-gal-
actopyranos-60-yl)-amino-1,4-naphthoquinone (22b). The 1,3-
dipolar cycloaddition reaction between juglone 12 and glycosyl
azide 13b gave glyconaphthotriazole 11b (64.0 mg, 28%) and
amino compound 22b (4.3 mg, 2%).
The compound 11b was obtained a yellow solid: mp 160–
162 ꢁC; IR (KBr) n (cmꢂ1): 3416 (OH), 1654 (C]O), 1453 (C]C).
1H NMR (300.00 MHz, CDCl3) d: 1.23 (s, 3H, CH3), 1.36 (s, 3H,
CH3), 1.38 (s, 3H, CH3), 1.57 (s, 3H, CH3), 4.32 (dd, 1H, J ¼ 5.0
and 2.5 Hz, H-20), 4.36 (dd, 1H, J ¼ 7.5 and 2.0 Hz, H-40), 4.44–
4.47 (m, 1H, H-50), 4.69 (dd, 1H, J ¼ 7.5 and 2.5 Hz, H-30), 4.81
(dd, 1H, J ¼ 14.0 and 3.5 Hz, H-60a), 5.31 (dd, 1H, J ¼ 14.0 and
10.0 Hz, H-60b), 5.39 (d, 1H, J ¼ 5.0 Hz, H-10), 7.36 (dd, 1H, J ¼
8.5 and 1.5 Hz, H-6), 7.66 (dd, 1H, J ¼ 8.5 and 7.5, H-7), 7.77 (dd,
1H, J ¼ 7.5 and 1.0 Hz, H-8), 12.34 (s, 1H, OH) ppm. 13C/APT
NMR (75.0 MHz, CDCl3) d: 24.9 (CH3), 25.0 (CH3), 25.8 (CH3),
26.2 (CH3), 50.8 (C-60), 67.1 (C-50), 70.4 (C-20), 71.2 (C-30), 71.4 (C-
40), 96.3 (C-10), 109.1 (–OCO–), 110.5 (–OCO–), 115.6 (C-4a), 120.5
(C-8), 126.6 (C-6), 133.4 (C-8a), 134.4 (C-9a), 136.7 (C-7), 145.2 (C-
3a), 163.5 (C-5), 174.7 (C-9), 183.0 (C-4) ppm. Anal. calc. for
6.2 Cell membrane disruption
Cytotoxicity testing was performed as described previously25 in
96-well plates using a 2% mouse erythrocyte suspension in
0.85% NaCl containing 10 mM CaCl2. The compounds, which
were diluted as mentioned above, were tested at a concentration
of 200 mM. Aer incubation at room temperature for 30 min and
centrifugation, the supernatant was removed, and the liberated
hemoglobin was measured spectrophotometrically at 540 nm.
DMSO was used as a negative control and Triton X-100 (1%) was
used as a positive control.
C
22H23N3O8: C, 57.76; H, 5.07; N, 9.19. Found: C, 57.99; H, 5.34;
N, 8.78%.
The compound 22b was obtained as a red solid: mp 203–205
ꢁC; IR (KBr) n (cmꢂ1): 3400 (OH), 3341 (N–H), 1615 (C]O), 1469
(C]C). 1H NMR (300.00 MHz, CDCl3) d: 1.33 (s, 3H, CH3), 1.37
(s, 3H, CH3), 1.49 (s, 3H, CH3), 1.52 (s, 3H, CH3), 3.44–3.48 (m,
2H, H-60a and H-60b), 4.00–4.05 (m, 1H, H-50), 4.25 (dd, 1H, J ¼
7.8 and 1.8 Hz, H-40), 4.35 (dd, 1H, J ¼ 5.1 and 2.4 Hz, H-20), 4.65
(dd, 1H, J ¼ 7.8 and 2.4 Hz, H-30), 5.55 (d, 1H, J ¼ 5.1 Hz, H-10),
5.67 (s, 1H, H-3), 6.52–6.56 (m, 1H, N–H), 7.23 (dd, 1H, J ¼ 8.4
and 1.2 Hz, H-6), 7.46 (dd, 1H, J ¼ 8.4 and 7.5 Hz, H-7), 7.59 (dd,
1H, J ¼ 7.5 and 1.2 Hz, H-8), 13.05 (s, 1H, OH) ppm. 13C/APT
NMR (75.0 MHz, CDCl3) d: 24.5 (CH3), 25.0 (CH3), 26.1 (CH3),
26.2 (CH3), 43.3 (C-60), 64.9 (C-50), 70.5 (C-30), 70.6 (C-40), 71.9 (C-
20), 96.5 (C-10), 99.9 (C-3), 109.0 (–OCO–), 110.0 (–OCO–), 115.1
(C-4a), 119.2 (C-8), 125.9 (C-6), 130.7 (C-8a), 134.0 (C-7), 149.1 (C-
2), 161, 2 (C-5), 181.0 (C-1), 189.1 (C-4) ppm. Anal. calc. for
Acknowledgements
This work was supported by the Brazilian agency FAPERJ-
PRONEX. Fellowships granted to UFF, by FAPERJ, CAPES, and
CNPq-PIBIC are gratefully acknowledged. We would like to
thank the LabCri/UFMG for the use of the X-ray facilities.
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