Synthesis and evaluation of the cytotoxic activity of 1,2- furanonaphthoquinones tethered to 1,2,3-1H-triazoles in myeloid and lymphoid leukemia cell lines

Article abstract of DOI:10.1016/j.ejmech.2014.07.079

Leukemia is the most common blood cancer, and its development starts at diverse points, leading to distinct subtypes that respond differently to therapy. This heterogeneity is rarely taken into account in therapies, so it is still essential to look for ne

Full text of DOI:10.1016/j.ejmech.2014.07.079

European Journal of Medicinal Chemistry 84 (2014) 708e717
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of the cytotoxic activity of
1,2-furanonaphthoquinones tethered to 1,2,3-1H-triazoles
in myeloid and lymphoid leukemia cell lines
,
,
Mariana F.C. Cardoso b 1, Patrícia C. Rodrigues a 1, Maria Eduarda I.M. Oliveira a,
Ivson L. Gama b, Illana M.C.B. da Silva b, Isabela O. Santos b, David R. Rocha b,
Rosa T. Pinho c, Vitor F. Ferreira b, Maria Cecília B.V. de Souza b, Fernando de C. da Silva b
,
*
,
,
*
Floriano Paes Silva-Jr a
a
~
ꢀ
Fundaçao Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratorio de Bioquímica de Proteínas e Peptídeos, 21040-360, Rio de Janeiro, RJ, Brazil
Universidade Federal Fluminense, Instituto de Química, Departamento de Química Organica, Campus do Valonguinho, 24210-141, Niteroi, RJ, Brazil
Fundaçao Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratorio de Imunologia Clínica, 21040-360, Rio de Janeiro, RJ, Brazil
b
c
^
ꢀ
~
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Leukemia is the most common blood cancer, and its development starts at diverse points, leading to
distinct subtypes that respond differently to therapy. This heterogeneity is rarely taken into account in
therapies, so it is still essential to look for new specific drugs for leukemia subtypes or even for therapy-
resistant cases. Naphthoquinones (NQ) are considered privileged structures in medicinal chemistry due
to their plethora of biological activities, including antimicrobial and anticancer effects. Nitrogen-
containing heterocycles such as 1,2,3-1H-triazoles have been identified as general scaffolds for gener-
ating glycosidase inhibitors. In the present study, the NQ and 1,2,3-1H-triazole cores have been combined
to chemically synthesize 18 new 1,2-furanonaphthoquinones tethered to 1,2,3-1H-triazoles (1,2-FNQT).
Their cytotoxicities were evaluated against four different leukemia cell lines, including MOLT-4 and CEM
(lymphoid cell lines) and K562 and KG1 (myeloid cell lines), as well as normal human peripheral blood
mononucleated cells (PBMCs). The new 1,2-FNQT series showed high cytotoxic potential against all
leukemia cell lines tested, and some compounds (12o and 12p) showed even better results than the
classical therapeutic compounds such as doxorubicin or cisplatin. Others compounds, such as 12b, are
promising because of their high selectivity against lymphoblastic leukemia and their low activity against
normal hematopoietic cells. The cells of lymphoid origin (MOLT and CEM) were generally more sensitive
than the myeloid cell lines to this series of compounds, and most of the compounds that showed the
highest cytotoxicity were similarly active against both cell lines.
Received 18 May 2014
Received in revised form
20 July 2014
Accepted 22 July 2014
Available online 23 July 2014
Keywords:
Naphthoquinone
1,2,3-Triazole
Organic synthesis
Cytotoxic activity
Leukemia
Cancer
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
hematopoietic stem cell malignancies thought to follow a “Two Hit”
model of disease origin [1], in which at least two genetic mutations
Acute leukemia is one of the most aggressive hematopoietic
malignancies and is characterized by the abnormal proliferation of
the immature cells of the hematopoietic system. As a set of very
heterogeneous diseases, acute leukemia is commonly divided into
Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia
(AML) depending on the precursor cell. Both forms are clonal
are necessary to trigger the expansion of a clonal population of cells
that is blocked from differentiating into mature cells. The leukemic
clone may arise at different stages of differentiation of the
lymphoid and myeloid precursors, which results in the develop-
ment of multiple subtypes of leukemia that can be characterized by
distinct biochemical and morphological aspects [2,3].
ALL is the most common type of cancer in children and also has
diverse subtypes. Whereas ALL prognosis and survival have shown
a large improvement over the last forty years, reaching 70%e80%
[4,5], children with AML experience a worse prognosis, with overall
survival rates of only 40%e60% under intensive chemotherapy
[6e8]. Furthermore, the complete remission rate differs, with 5%e
* Corresponding authors.
E-mail addresses: gqofernando@vm.uff.br (F.C. da Silva), floriano@ioc.fiocruz.br
(F.P. Silva-Jr).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.07.079
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
709
10% induction failures in AML resulting from refractory disease and
toxicity as opposed to only 1%e2% induction failures in ALL.
Despite these advances in survival rates during the several de-
cades, the expected 5-year survival rate for all patients diagnosed
with AML in the United States is a dismal 21% while ALL has a 5-year
survival rate of 65% [9]. Survival becomes even worse in patients
over 50 years of age, in which case the 5-year survival is 11% (AML)
and 16% (ALL) [10]. The drug resistance mechanisms causing
treatment failure remains a challenge in the fight against leukemia,
especially for several AML subtypes or for patients who either do
not achieve remission or relapse. Moreover, resistance is observed
not only with conventional chemotherapy but also with targeted
therapies, i.e., the so-called “smart drugs”, such as kinase inhibitors
and tamoxifen, which binds to the estrogen receptor [11].
Each patient harbors a different cancer with respect to which
genes are mutated, the nature of each mutation (i.e., different
mutations for the same gene have been detected in several pa-
tients) [12], and the order of appearance of these mutations.
Furthermore, tumors are very heterogeneous due to the clonal
evolution of tumor cell populations driven by genomic instability
[13]. These observations partly explain why different patients
harboring the “same” cancer may respond differently to the same
treatment regimen.
synthetic derivative that can be obtained either by a degradation
reaction with H2O2, called Hooker degradation [26,27], or by
condensation of 2-hydroxy-1,4-naphthoquinone (Lawsone) with
isobutyraldehyde [28].
Nor-lapachol can be cyclized under acid catalysis to yield nor-b-
lapachone (8), which has selective cytotoxicity to human lympho-
cytes and HL-60 leukemia cells and murine fibroblasts V79 [29].
This 1,4-naphthoquinone can also be reacted with bromine in the
presence of nucleophiles to substitute nor-b-lapachone at the
dihydrofuran ring. Several 3-arylamino-nor-
b
-lapachone de-
rivatives were synthesized by this route and were found to be
highly potent against some cancer cells including HL60, with IC50
below
2 mM (9aec) [30,31]. Other synthetic furanonaph-
thoquinones (FNQ) obtained by effective one-pot cascade reactions
of 3-phenyliodonio-1,2,4-trioxo-1,2,3,4-tetrahydronaphthalenides
with 3-butyn-2-ol showed moderate cytotoxicity against human
leukemia U937 and HL-60 cells with IC50 below 4.9 mM [32].
The 1,2,3-1H-triazole nucleus is a remarkably important phar-
macophore group in medicinal chemistry [33,34] because it has
been incorporated into several compounds that present important
biological activities. Novel and improved anticancer agents have
recently been designed by molecular hybridization of known
cytotoxic compounds with 1,2,3-1H-triazol [35]. Five 1,2,3-1H-tri-
Quinones have diverse biological activities [14] and some are in
use or under clinical trials for cancer treatment. These include the
natural products daunorubicin (1) and doxorubicin (2); streptoni-
grin (3) and mytomicin C (4) (N-heterocyclic quinones) (Fig. 1); and
lapachol (5) [15] (Fig. 2). Several studies have shown that quinones
owe their activity against cancer cells either to inhibition of the
nuclear enzymes topoisomerases I and II [16,17] or to the generation
of reactive oxygen species (ROS), ultimately inducing DNA damage
that is followed bycell apoptosis [18e20]. The anti-tumor specificity
of quinones is generally due to overexpression of topoisomerases
andthe associateddeficiencyofprotectionmechanismsagainstDNA
damage in cancer cells, or to the increase in topoisomerase bio-
activation bythe NAD(P)H:quinoneoxidoreductase 1(NQO1), which
is an abundant enzyme in many tumors [21].
azoles coupled to b-lapachone have been synthesized and assayed
against some cancer cell lines [36], and compounds 10a and 10b
were found to be the most active against HL-60 leukemia.
The diversity of responses to therapy and the need to improve
therapies against certain subtypes of AML, acute leukemia in
older people, and even disease relapse, prompted us to look for
more specific and safer drugs that could efficiently personalize
therapy or improve patient survival in standard protocols against
leukemia. In view of the promising results achieved with 1,2-
furanonaphthoquinones tethered to 1,2,3-1H-triazole (1,2-FNQT)
on the HL-60 cell line, we synthesized an extended series of such
hybrid compounds. In investigating the cytotoxicity and speci-
ficity of these compounds, we considered cell origin and used
four different leukemia cell lines: CCRF-CEM and MOLT-4
(lymphoid origin); and K562 and KG1 (myeloid origin). Further-
more, we evaluated the selectivity for leukemia over normal
hematopoietic cells by assaying the compounds on peripheral
blood mononuclear cells obtained from normal human donors.
Fig. 2 shows a group of natural (5, 6) and synthetic (7e10)
naphthoquinones, of which lapachol (5) and b-lapachone (6) [22]
are the best known members [23,24] due to several biological ac-
tivities that include anti-tumor effects [25]. Nor-lapachol (7) is a
Fig. 1. Examples of quinones that are in clinical use or trials for cancer treatment.

710
M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
Fig. 2. Some bioactive synthetic and natural naphthoquinones. Shown IC50 values are for HL60 cells (see Ref. [30] for (9aec) and Ref. [36] for 10a,b).
Fig. 3. General scheme used for preparation of 1,2-furanaphthoquinone triazoles 10a,b and 12aep.
Finally, we used simple computational models to predict the
drug-like characteristics and infer the predicted physicochemical
and toxicological properties of 1,2-FNQTs to determine their
potential as new anti-leukemia drugs.
by Cu(I) and provided only the 1,4-regioisomers (Fig. 3). Most
compounds were obtained in high yields (>75%).
2.2. Hemolytic activity
Hemolysis was assayed to evaluate possible adverse effects
induced by surfactant compounds, which could lead to cellular
membrane damage and therefore unspecific cytotoxicity. All com-
2. Results and discussion
2.1. Chemistry
pounds were evaluated at a concentration of 1000
cases where hemolysis was higher than 5% at 1000
m
m
M (Fig. 4). In
M, the com-
All 2,2-dimethyl-3-(4-(alkyl or aryl)-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione were prepared by known
synthetic routes [36]. The methodology was based on the Huisgen
1,3-dipolar cycloaddition reaction between the key intermediate 11
(3-azido-2,2-dimethyl-2,3-dihydro-naphtho[1,2-b]furan-4,5-
dione) and the appropriate terminal alkynes, which were catalyzed
pound was re-tested at 200
Of the compounds tested, only 12d and 12o presented any sig-
nificant hemolysis (>5%) at 1000 M. After re-testing 12d and 12o
at 200 M, neither showed significant hemolysis (data not shown).
Thus, the concentration of 200 M was utilized as the maximum
concentration in cytotoxicity assays with Alamar Blue®.
mM (e.g., for compounds 12d and 12o).
m
m
m

M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
711
2.3. Cytotoxicity in leukemia cell lines
All compounds were characterized for their cytotoxic effects on
the four leukemia cell lines by their cell viability inhibition (IC50
values and the results are summarized in Table 2. Potencies for IC50
were directly determined without performing preliminary
screening step at a fixed compound concentration due to the
)
a
known cytotoxic effects of the nor-b-lapachone moiety present on
these hybrid molecules.
Our results showed that all compounds were cytotoxic to the four
cell lines with different potencies (IC50 values ranging from 0.05 to
83.44
showed an improved cytotoxic potential with the four cell lines
tested when compared with the precursor nor- -lapachone. Overall,
the hybrid compounds were more active than nor- -lapachone
mM). Many compounds within this new series of 1,2-FNQTs
Fig. 4. Hemolytic activity of the 1,2,3-1H-triazol derivatives of nor-
b-lapachone at
1000 M. Osmotic shock with milli-Q H2O (100% hemolysis) was used as a positive
m
b
control and PBS was added as a negative control.
b
against the lymphoid cell lines MOLT and CEM but showed less of a
pronounced improvement in cytotoxicity for the K562 cell line.
The most potent compounds (12p, 12m, 12o, 12b and 12i) were
the same for the MOLT and CEM lymphoid cell lines, while com-
pound 12c had low cytotoxicity in both cell lines.
Table 1
Standardized conditions for the cytotoxic assay with AlamarBlue®.
Cell/cell line
Cells/well
Incubation time with AlamarBlue®
CCRF-CEM
MOLT-4
K562
KG1
Normal PBMC
2.5 ꢀ 104
2.5 ꢀ 104
2.5 ꢀ 104
5.0 ꢀ 104
5.0 ꢀ 104
2 h
6 h
4 h
6 h
24 h
Among the compounds showing submicromolar IC50 values, we
identified the following: 10b on MOLT (0.28 mM); 12b on MOLT
Table 2
Cytotoxic activity on 48 h incubation of the nor-
b-lapachone tethered triazoles with the four leukemia cell lines MOLT, CEM, KG1 and K562.
Compounds MOLT
CEM
KG1
IC50
K562
IC50
(
m
M)
SD
IC50
(
mM)
SD
(
m
M)
SD
IC50
(
mM)
SD
Nor-
11 (azide deriv.)
Cisplatin
b
-Lapachone
13.6
3.64
nt
0.4
0.24
e
15.2
3.18
nt
2.1
0.36
e
17.2
6.06
113
nt
1.6
0.67
37
2.28
1.58
304
nt
0.11
0.36
88
Doxorubicin
0.07
0.02
0.15
0.02
e
e
R
10a
10b
2.58
0.28
0.32
0.22
2.79
12.6
0.18
0.9
4.92
8.41
0.15
0.86
1.53
1.11
0.29
3.75
12a
12b
2.13
0.75
0.22
0.15
1.97
1.96
0.82
0.19
7.62
12.8
0.76
1.5
3.80
14.4
0.41
3.9
12c
12d
34.5
2.62
1.8
11.7
13.8
1.9
1.5
76.2
3.92
7.1
9.06
8.03
0.82
0.81
0.31
0.44
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
Pr
Bu
Pentyl
Hexyl
1.47
1.92
2.28
2.17
1.35
2.16
1.98
1.47
0.16
2.04
0.48
0.05
0.22
0.39
0.25
0.69
2.78
0.42
0.58
0.59
0.01
0.28
0.02
0.03
3.14
3.73
27.6
7.30
1.53
1.73
3.57
1.77
0.31
3.16
0.67
0.34
0.81
0.06
2.0
1.55
3.79
30.0
5.58
5.33
2.00
5.63
1.32
9.58
5.63
0.42
0.23
3.6
60.9
3.78
2.68
e
10.4
0.33
0.42
e
7.53
0.58
0.96
0.13
0.05
0.06
0.14
0.03
0.01
0.39
0.19
1.57
0.48
0.06
0.98
2.53
4.89
0.93
CH2OH
1.58
2.43
1.39
1.91
8.35
1.84
1.38
2.07
0.47
0.15
0.25
0.05
0.20
0.33
0.13
0.23
C(Me)2OH
COH(Et)(Me)
C(iBu)(Me)OH
CH2OAc
CH2OTHP
CH2Cl
12.27
11.07
(CH2)3Cl
Cisplatin and Doxorubicin were used as standard anticancer compounds. The precursor compounds and intermediates, nor-lapachol and nor-b-lapachone and its azide
derivative, were assayed for comparative purposes. Cytotoxicity, expressed by IC50, was measured by the Alamar Blue method. nt ¼ not tested.

712
M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
(0.75
(0.98
MOLT (0.05
m
M); 12m on MOLT (0.16
m
M), CEM (0.31
M) and CEM (0.67
M) and KG1 (0.93 m
m
M) and KG1
M); and 12p on
M). With the
mM); 12o on MOLT (0.48
m
m
m
M), CEM (0.34
m
exception of 10b, the anticancer activities of all compounds are
reported here for the first time.
Although the activity data are complex due to intrinsic differ-
ences for each cell line and multiple factors affecting the observed
cytotoxicity (e.g., cell permeability Vs macromolecular target bind-
ing), some trends in SAR (structureeactivity relationship) could still
be derived. By comparing the IC50 values of the derivatives bearing a
cyclic R group substituting C-4 of the 1,2,3-1H-triazol ring on the
MOLTcell line, we observed that all these bulk substituents except 4-
OMe phenyl contributed to the improved potency of the derivatives
over the parent nor-b-lapachone. Among the hybrid triazoles car-
rying aliphatic hydroxylated R groups in C-4 (12iel), the effects of
the alkyl chain length or branching were negligible, with IC50 values
ranging from 1.35 to 2.16 mM. On the other hand, acetylation of the
Fig. 5. Toxicity in normal PBMCs from three donors. Over 48 h, 5 ꢀ 104 PBMCs of
donors were treated with compounds at doses 10ꢀ the IC50 determined in leukemia
cell lines. Cytotoxicity was measured by AlamarBlue method.
hydroxyl of 12i resulted in compound 12m, which was 5 to 10-times
more active on lymphoid cell lines MOLT and CEM but not on the
myeloid cell lines KG1 and K562. Finally, the presence of a chlorine
atom in the alkyl-chloride derivatives 12o and 12p seemed tofurther
improve the potency of these 1,2-FNQTs. Compound 12p performed
comparably to doxorubicin and even better than cisplatin, which are
classical agents used in anticancer therapy.
PBMC viability. Compound 12b presented the lowest toxicity,
reducing cell viability by only 30% on average (Fig. 5). The addition
of a phenyl group to the triazol ring in 12b led to a good compro-
mise between cytotoxic potential and specificity.
Beta-lapachone, a natural 1,2-furanonaphthoquinone related to
the parental compound of the series studied in this work, presented
Although all compounds of this series were cytotoxic to myeloid
cell lines, the potency in myeloid cells was lower than in lymphoid
cells and the compounds with stronger cytotoxicity were different
for KG1 and K562. This is evidence for the higher resistance of AML
to drugs in comparison with ALL [37,38] as well as the heteroge-
neity of myeloid leukemias [1,39].
a similar level of toxicity with normal hematopoietic cells (IC50
¼
14e16 M, calculated from unstimulated PBMCs from 8 normal
m
donors) [51]. Nor-beta-lapachone itself seems to be slightly less
toxic considering its weak cytotoxicity against human lymphocytes
(IC50 > 22
mM) and rodent V79 fibroblasts (IC50 ¼ 13
mM) [27].
1,2,3-1H-triazol has recently been identified as a general scaffold
for generating glycosidase inhibitors because it was postulated to
mimic the partial positive charge of the anomeric carbon of the re-
action catalyzed by glycosidases [40e42] Glycosidases are involved
in a variety of metabolic disorders and diseases such as diabetes,
viral or bacterial infections, and cancer. Thus, the inhibition of these
enzymes has many potential clinical applications [43e48].
It has been realized that the glycans expressed by cancer cells
are either displayed at different levels or with different structures
than those expressed by normal cells. Glycans regulate tumor
proliferation, differentiation, invasion, metastasis, immune sur-
veillance and angiogenesis. Altered glycosylation results in altered
expression of cell surface oligosaccharides, protein folding, or sta-
bilization of tertiary structures, resulting in defective recognition
and elimination of glycoproteins by cell quality control mechanisms
[49,50]. Hence, the incorporation of the 1,2,3-1H-triazol nucleus
into the 1,2-FNQTs has improved the anticancer properties of such
molecules. The anticancer effects may be a result of the observed
glycosidase inhibition associated with the triazol scaffold.
2.5. Predicted toxicity and other drug relevant properties
To anticipate any toxicity risks and evaluate the drug-like
characteristics of the synthesized compounds, we computed a set
of drug relevant properties from their 2D chemical structures
(Table 3). The calculated properties indicated that most 1,2,3-
triazole derivatives of nor-b-lapachone present a better toxicolog-
ical profile than both their parent compound and the classical
cytotoxic anticancer agent doxorubicin. To predict intestinal
Table 3
Predicted drug-relevant properties of the 1,2-furanonaphthoquinones tethered to
1,2,3-1H-triazoles.
Compounds
cLogP Solubility
TPSA nON nOH/
N
[LogS(mol/L)] (Å2)
NH
violationsa
Nor-b-Lapachone 2.36
ꢁ3.06
ꢁ4.66
ꢁ3.39
ꢁ3.39
ꢁ3.89
ꢁ3.88
ꢁ3.9
43.4
206.1 12
3
0
7
1
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
0
0
0
3
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
Doxorubicin
10a
10b
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
0.42
2.51
2.76
3.26
2.97
2.86
4.58
2.47
2.93
3.40
3.86
0.71
1.36
1.83
2.63
1.20
2.02
1.90
2.65
94.3
74.1
74.1
74.1
83.3
74.1
74.1
74.1
74.1
74.1
94.3
94.3
94.3
94.3
100.4
92.6
74.1
74.1
7
6
6
6
7
6
6
6
6
6
7
7
7
7
8
8
6
6
2.4. Cytotoxic activity in normal peripheral blood mononuclear
cells
ꢁ5.38
ꢁ2.90
ꢁ3.17
ꢁ3.44
ꢁ3.71
ꢁ2.01
ꢁ2.49
ꢁ2.76
ꢁ3.19
ꢁ2.42
ꢁ3.04
ꢁ3.24
ꢁ3.30
To find compounds that presented potent cytotoxicity to leu-
kemia cells and low toxicity to normal cells, we screened all com-
pounds for cytotoxic activity in normal PBMCs from human donors
at concentrations 10ꢀ higher than the respective IC50 found in
leukemia cells. Such high concentrations were chosen to allow for
rapid identification of compounds showing any potential cytotox-
icity against normal cells. The majority of the compounds showed
high toxicity (>80%) in this screening (data not shown) except for
12a, 12b, 12k and 12o (Fig. 5).
At 10 mM, a concentration that would kill nearly all leukemia
cells (according to their IC50 values in Table 2), compounds 12a,12b,
12k and 12o showed only a partial disruption of human donors'
a
Number of violations to the Lipinski's “rule of five”: logP ꢂ 5, molecular weight
ꢂ500, number of hydrogen bond acceptors ꢂ10, and number of hydrogen bond
donors ꢂ5.

M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
713
absorption, we estimated the molecular polar surface areas (PSA) of
the triazol compounds from the calculated TPSA. PSA has been
extensively used in medicinal chemistry for modeling absorption
phenomena and to optimize a drug's ability to permeate cells. The
PSA of a molecule is defined as the surface sum over all polar atoms,
which primarily consist of oxygen and nitrogen as well as their
attached hydrogens. The PSA of the triazol compounds suggested a
better intestinal absorption (PSA < 140 Å2) than doxorubicin
(PSA ¼ 206.1 Å2). On the other hand, most triazol derivatives differ
compound 12p is also promising due to its high cytotoxic activity
against some leukemia cells (IC50 ranging from 0.48 to 1.38 M) and
its lower toxicity against normal hematopoietic cells (estimated
IC50 > 10 M). Compounds such as these could be the key to
developing more effective therapies against leukemia with fewer
side effects, especially for cases of resistance or relapse that prevent
the improvement of survival rates in lymphoid leukemia.
m
m
4. Experimental section
from nor-
b
-lapachone (PSA ¼ 43.4 Å2) in that they are unlikely to
cross the bloodebrain barrier (PSA > 60 Å2). Molecules with a polar
surface area greater than 140 Å2 tend to be poor at permeating cell
membranes, while a PSA of less than 60 Å2 is usually needed for
molecules to penetrate the bloodebrain barrier and thus act on the
brain and other central nervous system tissues [52]. Thus, the
calculated PSA values for these hybrid nor-b-lapachone-triazol
derivatives indicate a possible better oral bioavailability with a
smaller chance of CNS toxicity.
Doxorubicin hydrochloride (Dox.HCl) is approved to be used
alone or with other drugs to treat both ALL and AML (U.S. National
Cancer Institute). However, Dox.HCl shows very low oral bioavail-
ability (~5%) mainly due to its limited intestinal absorption [53] but
also to other contributing factors that include substrate specificity
to the P-glycoprotein (P-gp) efflux pump, acid catalyzed hydrolysis
in the stomach, and susceptibility to cytochrome P450 (CYP450),
resulting in high first-pass metabolism [54]. Therefore, only the
injectable form of Dox.HCl is used in the clinic under the trade
names Adriamycin® and Rubex® (Dox.HCl in solution form). For-
mulations designed to improve doxorubicin clinical efficacy exist,
such as non-PEGylated (Myocet®) and PEGlyated liposomal for-
mulations (Doxil®, Caelyx®, and LipoDox®), but their safety is still
hampered by the well-known doxorubicin induced cardiotoxicity
[55]. The latter is a result of a complex interplay of different
mechanisms, whereas interference with iron metabolism and
oxidative stress induced by doxorubicin-generated ROS are
considered the primary mechanisms [56].
4.1. Chemistry
Reagents were purchased from Sigma Aldrich Brazil LTDA and
were used without further purification. Column chromatography
was performed with silica gel 60 (Merck 70e230 mesh). Analytical
thin-layer chromatography was performed with silica gel plates
(Merck, TLC silica gel 60 F254) and the plots were visualized using
UV light or aqueous solutions of ammonium sulfate. Yields refer to
chromatographically and spectroscopically homogeneous mate-
rials. Melting points were obtained on a Fischer-Johns apparatus
and were uncorrected. Infrared spectra were measured using KBr
pellets on a PerkineElmer model 1420 FT-IR Spectrophotometer
calibrated relative to the 1601.8 cmꢁ1 absorbance of polystyrene.
NMR spectra were recorded on a Varian Unity Plus VXR (500 MHz)
instrument in DMSO-d6 and CDCl3 solutions. The chemical shift
data were reported in units of
d (ppm) downfield from tetrame-
thylsilane or the solvent, which were used as an internal standard.
Coupling constants (J) are reported in Hertz and refer to apparent
peak multiplicities. The high resolution mass spectra (electrospray
ionization) were obtained using a QTOF Micro (Waters, Manchester,
UK) mass spectrometer (HRESIMS).
4.1.1. General procedure for preparation of 10a,b, 11 and 12aep
The general protocol for the synthesis of 2,2-dimethyl-3-(4-
(alkyl or aryl)-1H-1,2,3-triazol-1-yl)-2,3-dihydronaphtho[1,2-b]
furan-4,5-dione has been reported elsewhere [36]. Briefly, nor-b-
Computational prediction of the drug-relevant properties for
nor-b-lapachone and its 1,2,3-1H-triazol hybrid derivatives indi-
lapachol (7) was reacted with excess of bromine in chloroform,
followed by nucleophilic substitution with sodium azide in
dichloromethane to yield 11 (3-azido-2,2-dimethyl-2,3-dihydro-
naphtho[1,2-b]furan-4,5-dione). This key intermediate was
employed in Huisgen [59] 1,3-dipolar cyclization with an appro-
priate terminal alkyne catalyzed by copper (I) ion, also known as a
click reaction [60], to obtain the 2,2-dimethyl-3-(4-(alkyl or aryl)-
1H-1,2,3-triazol-1-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione
10a,b and 12aep [61e63].
cated that many of the problems associated with doxorubicin may
be absent or weaker in these compounds. For instance, admetSAR
server results indicated that none of the most promising com-
pounds revealed in this work (e.g., 12a, 12b, 12i, 12k, 12o and 12p)
had been predicted with high probabilities to be substrates of P-gp
or potent inhibitors of the Human Ether-a-go-go-Related Gene
(hERG) (data not shown). P-gp is implicated in diminished intes-
tinal absorption and drug-induced resistance in many cancers [57],
while hERG is a potassium channel with critical importance to
ventricular repolarization whose inhibition has been use as a major
parameter for preclinical assessment of drug-induced ventricular
arrhythmia [58].
4.1.1.1. 3-(4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (10a). Compound 10a was
obtained in 58% yield as yellow solid; m.p. 100e101 ꢃC. IR (KBr, cmꢁ1):
n
1651, 1590, 1574, 1494, 1410, 1257, 1222, 1160, 1084, 1051; 1H NMR
(DMSO-d6, 500 MHz): d 1.12 (3H, s), 1.40e1.36 (2H, m), 1.48e1.45 (2H,
3. Conclusion
m), 1.78e1.57 (4H, m), 1.80 (3H, s), 2.03e1.95 (2H, m), 6.10 (1H, s), 7.88
(1H, d, J ¼ 7.8 Hz), 7.97e7.90 (2H, m), 8.18 (1H, d, J ¼ 6.9 Hz), 8.24 (1H,
s); 13C NMR (DMSO-d6, 125 MHz): 20.3, 21.5, 25.0, 26.7, 37.6, 65.7, 67.6,
95.1, 111.0, 121.1, 124.7, 126.5, 128.4, 131.6, 132.7, 134.3, 155.1, 169.4,
174.3, 179.6.
In the present work, we synthesized a series of eighteen new
1,2-furanonaphthoquinones tethered to 1,2,3-1H-triazoles and
investigated their antileukemic activity. The improved cytotoxic
potential when compared with the parent nor-b-lapachone in-
dicates that the attachment of a 1,2,3-1H-triazol to a variety of C-4
substituents mostly had a positive effect on the anticancer activity
of the newly synthesized compounds.
Compound 12b has great potential for further development as
an anti-leukemia drug not only because of its potent and selective
cytotoxicity (normal x cancer cells) but also because of its selec-
tivity against leukemia lymphoid cell lines (approximately 7e19
times more effective than in leukemia myeloid cells). Additionally,
4.1.1.2. 3-(4-(Cyclohex-1-en-1-yl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (10b). Compound 10b was
obtained in 48% yield as yellow solid; m.p. 187e188 ꢃC. IR (KBr,
cmꢁ1): 1650, 1612, 1567, 1410, 1216; 1H NMR (CDCl3, 500 MHz):
n
d
1.20 (3H, s), 1.61e1.66 (2H, m), 1.70e1.74 (2H, m), 1.74 (3H, s),
2.15e2.19 (2H, m), 2.31e2.33 (2H, m), 5.94 (1H, s), 6.50e6.52 (1H, m),
7.31 (1H, s), 7.72 (1H, dt, J ¼ 1.5, 7.3 Hz), 7.75 (1H, dt, J ¼ 1.5, 7.8 Hz),
7.80 (1H, dd, J ¼ 1.0, 7.3 Hz), 8.18 (1H, dd, J ¼ 1.5, 7.3 Hz); 13C NMR

714
M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
ꢄ
(CDCl3, 125 MHz): 21.0, 22.0, 22.2, 25.1, 26.1, 27.5, 66.5, 95.9, 111.2,
117.6, 125.3, 125.4, 126.7, 126.8, 129.7, 131.3, 133.1, 134.7, 149.1, 170.9,
174.4,179.9; HRESIMS m/z 376.1658 [MþH]þꢄ (Calcd. for C22H22N3O3þ:
376.1656).
134.9, 148.1, 171.1, 174.6, 180.1; HRESIMS m/z 338.1502 [MþH]þ
(Calcd. for C19H20N3Oþ3 : 338.1499).
4.1.1.8. 3-(4-Butyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (12f). Compound 12f was
obtained in 90% yield as yellow solid; m.p. 219e220 ꢃC. IR (KBr,
4.1.1.3. 3-(4-Cyclohexyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (12a). Compound 12a was
obtained in 52% yield as orange solid; m.p. 92e93 ꢃC. IR (KBr,
cmꢁ1):
n
3.128, 3.077, 2.957, 2.930, 2.850, 1.657, 1.621, 1.589, 1.572,
1.408, 1.221, 776; 1H NMR (DMSO-d6, 500 MHz):
d
0.86 (3H, t,
cmꢁ1):
n
3129, 2925, 1654, 1493, 1451, 1221; 1H NMR (CDCl3,
J ¼ 7.2 Hz), 1.01 (3H, s), 1.29 (2H, dt, J ¼ 7.2, 14.9 Hz), 1.58e1.52 (2H,
m), 1.68 (3H, m), 1.68 (3H, s), 2.59 (2H, dt, 2.2, 7.7 Hz), 5,96 (1H, s),
7,88e7,75 (3H, m), 8.05 (1H, s), 8.07 (1H, s); 13C NMR (DMSO,
125 MHz): 13.5, 21.5, 21.8, 22.4, 23.6, 30.8, 92.8, 110.8, 118.5, 124.8,
126.0, 128.9, 132.5, 134.7, 135.2, 172.5, 178.9, 185.6, 197.2; HRESIMS
m/z 352.1651 [MþH]þꢄ (Calcd. for C20H21N3Oþ3 : 351.1583).
500 MHz):
d
1.09 (3H, s), 1.12e1.34 (6H, m), 1.57e1.70 (2H, m), 1.67
(3H, s), 1.92e1.94 (2H, m), 2.64e2.70 (1H, m), 5.86 (1H, s), 7.10 (1H,
s), 7.65 (1H, dt, J ¼ 1.5, 7.3 Hz), 7.69 (1H, dt, J ¼ 1.5, 7.3 Hz), 7.73 (1H,
dd, J ¼ 1.5, 7.3 Hz), 8.12 (1H, dd, J ¼ 1.5, 7.3 Hz); 13C NMR (CDCl3,
125 MHz): 20.8, 25.8, 25.9, 27.5, 29.5, 32.7, 32.8, 35.1, 66.7, 96.0,
111.3, 118.6, 125.4, 126.6, 129.8, 131.3, 133.1, 134.7, 153.4, 170.9, 174.4,
180.0; HRESIMS m/z 378.1809 [MþH]þ (Calcd. for C22H24N3O3þ:
4.1.1.9. 2,2-Dimethyl-3-(4-pentyl-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (12g). Compound 12g was
obtained in 70% yield as yellow solid; m.p. 65e66 ꢃC. IR (KBr, cmꢁ1):
ꢄ
378.1812).
4.1.1.4. 2,2-Dimethyl-3-(4-phenyl-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (12b). Compound 12b was
obtained in 92% yield as yellow solid. m.p. 178e179 ꢃC. IR (KBr,
n
3.128, 3.079, 2.955, 2.930, 2.859, 1.657, 1.621, 1589, 1.572, 1.409,
1.222, 776; 1H NMR (DMSO-d6, 500 MHz):
d
0.83 (3H, t, 7.0 Hz),
1.30e1.23 (4H, m), 1.61e1.53 (2H, m), 2.60e2.55 (2H, m), 3.31 (6H,
s), 5.97 (1H, s), 7.77e7.74 (1H, m), 7.81e7.82 (1H, m), 7.87e7.82 (1H,
m), 8.05 (1H, dd, 7.3 and 1.5 Hz); 8.06 (1H, s); 13C NMR (DMSO,
125 MHz): 13.7, 21.7, 22.0, 22.4, 28.3, 30.6, 92.8, 124.8, 128.9, 132.5,
cmꢁ1):
1H NMR (CDCl3, 500 MHz):
n
3414, 2362, 2342, 1655, 1618, 1588, 1572, 1409, 1222, 768;
1.25 (3H, s), 1.78 (3H, s), 6.02 (1H, s),
d
7.30 (1H, s), 7.38 (2H, t, J ¼ 7.3 Hz), 7.71e7.74 (2H, m), 7.75e7.79 (1H,
m), 7.75e7.79 (1H, m), 7.75e7.79 (1H, m), 7.82 (1H, d, J ¼ 7.3 Hz),
8.20 (1H, d, J ¼ 7.3 Hz); 13C NMR (CDCl3, 125 MHz): 21.0, 27.6, 66.8,
95.9, 111.1, 118.8, 125.6, 126.5, 128.1, 128.6, 129.9, 130.0, 131.4, 125.5,
133.3, 134.8, 147.5, 171.1, 174.4, 179.9; HRESIMS m/z 372.1359
[MþH]þꢄ (Calcd. for C22H18N3O3þ: 372.1343).
134.7, 135.2, 172,5, 185.6, 197.2; HRESIMS m/z 366.1813 [MþH]þ
ꢄ
(Calcd. for C21H23N3Oþ4 : 365.4256).
4.1.1.10. 3-(4-Hexyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (12h). Compound 12h was
obtained in 88% yield as yellow solid; m.p. 178e179 ꢃC. IR (KBr,
4.1.1.5. 3-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (12c). Compound 12c
was obtained in 56% yield as yellow solid; m.p. 128e129 ꢃC. IR (KBr,
cmꢁ1):
(CDCl3, 500 MHz):
n
2927, 1654, 1620, 1588, 1568, 1409, 1218, 1112; 1H NMR
d
0.85 (3H, t, J ¼ 7.2 Hz), 1.17 (3H, s), 1.23e1.32
(2H, m),1.23e1.32 (2H, m),1.23e1.32 (2H, m),1.62 (2H, t, J ¼ 7.2 Hz),
1.74 (3H, s), 2.66 (2H, t, J ¼ 7.2 Hz), 5.93 (1H, s), 7.21 (1H, s),
7.69e7.80 (1H, m), 7.69e7.80 (1H, m), 7.69e7.80 (1H, m), 8.18 (1H,
d, J ¼ 7.7 Hz); 13C NMR (CDCl3, 125 MHz): 13.8, 20.8, 22.3, 25.4, 27.5,
28.6, 29.1, 31.3, 66.4, 95.9, 111.2, 120.0, 126.6, 129.7, 131.3, 125.4,
133.1, 134.7, 148.2, 170.9, 174.4, 179.9; HRESIMS m/z 380.1973
cmꢁ1):
1H NMR (CDCl3, 500 MHz):
n
1657, 1618, 1572, 1497, 1411, 1252, 1225, 1178, 1085, 1030;
1.24 (3H, s), 1.77 (3H, s), 3.81 (3H, s),
d
6.00 (1H, s), 6.90 (2H, d, J ¼ 8.8 Hz), 7.62 (1H, s), 7.70 (2H, d,
J ¼ 8.8 Hz), 7.71e7.74 (1H, m), 7.76 (1H, dt, J ¼ 1.5, 7.8 Hz), 7.82 (1H,
dd, J ¼ 1.5, 7.8 Hz), 8.19 (1H, dd, J ¼ 1.5, 7.8 Hz); 13C NMR (CDCl3,
125 MHz): 21.0, 27.5, 55.1, 66.7, 95.9, 111.2, 114.0, 118.2, 122.8, 125.4,
126.5, 126.9, 129.7, 131.3, 133.2,134.7, 147.3, 159.5, 171.1, 174.4, 180.0;
HRESIMS m/z 402.1452 [MþH]þꢄ (Calcd. for C23H20N3Oþ4 : 402.1448).
[MþH]þ (Calcd. for C22H26N3O3þ: 380.1969).
ꢄ
4.1.1.11. 3-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (12i). Compound 12i was
obtained in 88% yield as yellow solid; m.p. 212e213 ꢃC. IR (KBr,
4.1.1.6. 2,2-Dimethyl-3-(4-(4-pentylphenyl)-1H-1,2,3-triazol-1-yl)-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (12d). Compound 12d
was obtained in 65% yield as yellow solid; m.p. 161e162 ꢃC. IR (KBr,
cmꢁ1):
(CDCl3, 500 MHz):
n
3626, 1652, 1614, 1584, 1567, 1416, 1218, 795; 1H NMR
d
1.21 (3H, s), 1.76 (3H, s), 4.77 (2H, s), 5.97 (1H,
cmꢁ1):
n
2929, 1656, 1616, 1571, 1588, 1414, 1222, 777; 1H NMR
s), 7.50 (1H, s), 7.73e7.81 (1H, m), 7.73e7.81 (1H, m), 7.73e7.81 (1H,
m), 8.10 (1H, d, J ¼ 7.3 Hz); 13C NMR (CDCl3, 125 MHz): 22.5, 27.0,
55.1, 66.0, 95.2, 111.3, 122.9, 126.6, 128.7, 131.9, 124.9, 132.9, 134.6,
(CDCl3, 500 MHz):
d
0.87 (3H, t, J ¼ 6.8 Hz), 1.24 (3H, s), 1.28e1.36
(4H, m), 1.57e163 (2H, m), 1.77 (3H, s), 2.59 (2H, t, J ¼ 7.34 Hz), 6.01
(1H, s), 7.19 (2H, d, J ¼ 7.8 Hz), 7.69e7.74 (2H, m), 7.69e7.74 (1H, m),
7.69e7.74 (1H, m), 7.76 (1H, dt, J ¼ 1.0, 7.3 Hz), 7.82 (1H, dd, J ¼ 1.0,
7.3 Hz), 8.19 (1H, dd, J ¼ 1.5, 7.8 Hz); 13C NMR (CDCl3, 125 MHz):
14.0, 21.2, 22.5, 27.7, 31.0, 31.4, 35.7, 66.9, 96.0, 111.3, 118.8, 125.6,
125.7, 126.7, 127.6, 128.8,129.9,131.5, 133.3, 134.8,143.2,147.7, 171.2,
148.0, 169.7, 174.6, 179.8; HRESIMS m/z 326.1136 [MþH]þ (Calcd.
ꢄ
for C17H16N3Oþ4 : 326.1135).
4.1.1.12. 3-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)-
2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione
Compound 12j was obtained in 83% yield as yellow solid; m.p.
220e221 ꢃC. IR (KBr, cmꢁ1):
3422, 2981, 2968, 2925, 1657, 1615,
1588, 1570, 1412, 1224, 780; 1H NMR (CDCl3, 500 MHz):
1.17
(12j).
174.6, 180.1; HRESIMS m/z 442.2128 [MþH]þ (Calcd. for
ꢄ
C
27H28N3Oþ3 : 442.2125).
n
d
4.1.1.7. 2,2-Dimethyl-3-(4-propyl-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (12e). Compound 12e was
obtained in 80% yield as yellow solid. m.p. 219e220 ꢃC. IR (KBr,
(3H, s), 1.60 (6H, s), 1.75 (3H, s), 5.95 (1H, s), 7.38 (1H, s),
7.70e7.80 (1H, m), 7.70e7.80 (1H, m), 7.70e7.80 (1H, m), 8.17
(1H, d, J ¼ 7.9 Hz); 13C NMR (CDCl3, 125 MHz): 21.5, 27.7, 30.5,
30.6, 66.8, 68.8, 96.2, 111.4, 118.7, 126.7, 130.6, 131.5, 125.6, 133.4,
cmꢁ1):
n
3412, 3123, 1651, 1611, 1586, 1567, 1411, 1219, 779; 1H NMR
134.9, 155.7, 171.3, 174.6, 180.1; HRESIMS m/z 354.1447 [MþH]þ
ꢄ
(CDCl3, 500 MHz):
d
0.85 (3H, t, J ¼ 7.3 Hz), 1.11 (3H, s), 1.58 (2H, q,
J ¼ 7.3 Hz), 1.67 (3H, s), 2.58 (2H, td, J ¼ 1.9, 7.3 Hz), 5.86 (1H, s), 7.15
(1H, s), 7.63e7.74 (1H, m), 7.63e7.74 (1H, m), 7.63e7.74 (1H, m), 8.11
(1H, dd, J ¼ 1.5, 7.3 Hz); 13C NMR (CDCl3, 125 MHz): 13.6, 20.9, 22.5,
27.5, 27.6, 66.6, 96.0, 111.4, 120.3, 126.7, 129.8, 131.4, 125.5, 133.3,
(Calcd. for C19H20N3Oþ4 : 354.1448).
4.1.1.13. 3-(4-(2-Hydroxybutan-2-yl)-1H-1,2,3-triazol-1-yl)-
2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione
(12k).

M.F.C. Cardoso et al. / European Journal of Medicinal Chemistry 84 (2014) 708e717
715
Compound 12k was obtained in 90% yield as yellow solid; m.p.
193e195 ꢃC. IR (KBr, cmꢁ1):
3416, 2970, 1656, 1615, 1570, 1570,
1411, 1223, 778; 1H NMR (CDCl3, 500 MHz):
0.80 (3H, dd, J ¼ 5.4,
was obtained in 86% yield as yellow solid; m.p. 63e64 ꢃC. IR (KBr,
cmꢁ1): 1643, 1570, 1492, 1408, 1219, 1173, 1083; 1H NMR (DMSO-
d6, 500 MHz): 1.03 (3H, s), 1.69 (3H, s), 2.01e2.06 (2H, m), 2.74
(2H, t, J ¼ 7.3 Hz), 3.63 (2H, dt, J ¼ 2.4, 6.3 Hz), 5.98 (1H, s), 7.76e7.85
(3H, m), 8.06 (1H, d, J ¼ 6.8 Hz), 8.10 (1H, s); 13C NMR (DMSO-d6,
125 MHz): 22.4, 22.8, 31.5, 44.5, 88.0, 92.7, 110.8, 121.8, 124.7, 126.0,
128.8, 129.8, 132.5, 134.6, 135.2, 172.5, 178.5, 185.5, 197.1; HRESIMS
m/z 372.1097 [MþH]þꢄ (Calcd. for C19H18ClN3Oþ3 : 371.1037).
n
n
d
d
7.9 Hz), 1.17 (3H, s), 1.54 (3H, d, J ¼ 5.4 Hz), 1.75 (3H, s), 1.80e1.92
(2H, m), 5.95 (1H, d, J ¼ 11.5 Hz), 7.37 (1H, d, J ¼ 3.0 Hz),
7.70e7.81 (1H, m), 7.70e7.81 (1H, m), 7.70e7.81 (1H, m), 8.17
(1H, d, J ¼ 7.3 Hz); 13C NMR (CDCl3, 125 MHz): 8.1, 20.9, 22.9, 27.5,
35.7, 66.6, 71.3, 96.0, 111.2, 119.5, 126.5, 129.8, 131.4, 125.5, 133.2,
134.7, 154.3, 171.1, 174.4, 180.0; HRESIMS m/z 368.1608 [MþH]þ
ꢄ
(Calcd. for C20H22N3Oþ4 : 368.1605).
4.2. Biology
4.1.1.14. 3-(4-(2-Hydroxy-4-methylpentan-2-yl)-1H-1,2,3-triazol-1-
4.2.1. Leukemia cell lines
yl)-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione
Compound 12l was obtained in 77% yield as yellow solid; m.p.
163e164 ꢃC. IR (KBr, cmꢁ1):
3128, 3077, 2957, 2930, 2850, 1657,
1621, 1589, 1572, 1408, 1221, 776; 1H NMR (DMSO, 500 MHz):
0.70
(12l).
A panel of human leukemia cell lines that included myeloid
(K562, KG1) and lymphoid cells (MOLT-4, CCRF-CEM) was used. Cell
lines were obtained from ATCC (Philadelphia, PA, USA) and cultured
in RPMI 1640 medium (SigmaeAldrich®) supplemented with 10%
n
d
(3H, dd, J ¼ 2.9, 6.4 Hz), 0.78 (3H, dd, J ¼ 2.9, 6.4 Hz), 1.02 (3H, s),
1.43 (3H, d, J ¼ 5.3 Hz), 1.54e1.67 (3H, m), 1.69 (3H, d, J ¼ 5.3 Hz),
6.00 (1H, d, J ¼ 6.4 Hz), 7.75e7.85 (3H, m), 8.04 (1H, s), 8.06 (1H, s);
13C NMR (DMSO-d6,125 MHz): 20.4, 20.5, 23.6, 23.6, 24.0, 24.1, 24.2,
24.3, 26.8, 26.9, 29.4, 29.5, 51.3, 51.4, 65.8, 65.9, 69.7, 69.9, 95.0, 95.1,
111.0, 111.3, 121.2, 121.3, 124.7, 124.8, 126.5, 126.6, 128.4, 128.5, 131.7,
131.8,132.7, 132.8,134.4,134.5,155.0,155.1,169.4,169.5,174.4,174.5,
inactivated fetal bovine serum (SigmaeAldrich®) and 50
gentamicin at 37 ꢃC in a humidified incubator with 5% CO2.
mg/ml
4.2.2. Normal peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated by
density gradient centrifugation (Hystopaque; Sigma) from buffy
coat preparations from healthy donors (C.E. Fiocruz protocol
number 535/09). PBMCs were counted and seeded in 96-well plates
with RPMI 1640 (SigmaeAldrich®) supplemented with 10% inacti-
vated fetal bovine serum.
179.6, 179.7; HRESIMS m/z 396.1923 [MþH]þ (Calcd. for
ꢄ
C
19H20N3Oþ4 : 395.1845).
4.1.1.15. (1-(2,2-Dimethyl-4,5-dioxo-2,3,4,5-tetrahydronaphtho
[1,2-b]furan-3-yl)-1H-1,2,3-triazol-4-yl)methyl acetate (12m).
Compound 12m was obtained in 75% yield as yellow solid; m.p.
183e185 ꢃC. IR (KBr, cmꢁ1):
3147, 1747, 1623, 1561, 1554, 1405,
1240, 1221, 1035, 782; 1H NMR (CDCl3, 500 MHz):
1.17 (3H, s), 1.75
The compounds were tested at a concentration 10ꢀ higher than
their IC50 determined in leukemia cell lines.
n
4.2.3. Hemolysis assay
d
This assay was performed using fresh lamb blood from CECAL/
Fiocruz (Breeding center of laboratory animals). Erythrocytes were
collected by centrifugation at 1500 rpm for 15 min, washed three
times with Phosphate buffered saline (PBS) with 10 mM of glucose
at pH 7.4 and counted in a Countess®Automated Cell Counter (Life
Technologies). Erythrocytes were seeded in 96-well plates at a
(3H, s), 2.04 (3H, s), 5.16 (2H, s), 5.96 (1H, s), 7.55 (1H, s), 7.70e7.80
(1H, m), 7.70e7.80 (1H, m), 7.70e7.80 (1H, m), 8.18 (1H, dd, J ¼ 1.5,
7.3 Hz); 13C NMR (CDCl3, 125 MHz): 20.6, 21.0, 27.5, 57.3, 66.7, 95.7,
110.9,123.2,126.4,129.7, 131.3, 125.4,133.2, 134.7,142.5,170.7,171.2,
174.3, 179.8; HRESIMS m/z 368.1245 [MþH]þ (Calcd. for
ꢄ
C
19H18N3Oþ5 : 368.1241).
concentration of 4 ꢀ 108/well in triplicate, and 10
m
m
L of the com-
M in glucose-
pounds were added at a final concentration of 1000
4.1.1.16. 2,2-Dimethyl-3-(4-(((tetrahydro-2H-pyran-2-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-
dione (12n). Compound 12n was obtained in 98% yield as yellow
PBS (100
m
L final volume). Ten microliters of PBS was used as a
negative control (0% hemolysis) and 10
mL of Milli-Q H2O was used
as a positive control (100% hemolysis). The solutions were mixed
and incubated in a shaker for 10 min at room temperature. After
centrifugation at 13,000 rpm for 15 min, hemolysis was measured
by reading the supernatant absorbance at 540 nm on a FlexStation
3 Benchtop Multi-Mode Microplate Reader (Molecular Devices,
Sunnyvale, CA). All hemolysis data points were presented as the
percentage of the complete hemolysis control obtained by osmotic
shock with deionized water.
solid; m.p. 139e140 ꢃC. IR (KBr, cmꢁ1):
n
2943, 1664, 1625, 1589,
1573, 1410, 1221, 1118, 1028, 779; 1H NMR (CDCl3, 500 MHz):
d
1.13
(3H, s),1.42e1.65 (6H, m),1.69 (3H, s), 3.41e3.46 (1H, m), 3.75e3.80
(1H, m), 4.55e4.79 (3H, m), 5.81 (1H, s), 5.89 (1H, s), 5.90 (1H, s),
7.41 (1H, s), 7.42 (1H, s), 7.69 (3H, m), 8.11 (1H, dt, J ¼ 1.5, 7.3 Hz),
8.12 (1H, dt, J ¼ 1.5, 7.3 Hz); 13C NMR (CDCl3, 125 MHz): 19.2, 19.3,
23.0, 24.8, 25.3, 27.7, 30.3, 30.4, 60.5, 60.6, 62.3, 62.4, 66.7, 66.8,
95.9, 96.0, 111.2, 111.3, 125.5, 126.6, 129.9, 130.3, 131.5, 133.3, 134.8,
135.1, 171.2, 171.3, 174.5, 174.6, 180.0; HRESIMS m/z 410.1706
[MþH]þꢄ (Calcd. for C22H22N3O3þ: 410.1710).
4.2.4. Cytotoxic activity
Leukemia cell lines and normal PBMCs were seeded in 96-well
microculture plates and incubated for 48 h with the studied com-
pounds before in vitro cytotoxicity was assessed using the Ala-
marBlue® kit (Life Technologies). This kit is based on the principle
that only viable cells are able to reduce Resazurin, a blue reagent
present in AlamarBlue®, to a pink colored and highly red fluores-
cent product, Resorufin, which can be spectrophotometrically
quantitated using lex ¼ 560 nm and lem ¼ 590 nm. For each cell or
cell line used, an optimal time and number of cells were stan-
dardized for the incubation step with AlamarBlue® (Table 1). Ala-
marBlue® was added to cells at different times according to the
optimal incubation times listed in Table 1 before the 48 h total
incubation time was complete. Each of the 19 compounds was
tested in triplicate wells at six or seven different concentrations
4.1.1.17. 3-(4-(Chloromethyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (12o). Compound 12o
was obtained in 79% yield as yellow solid; m.p. 212e213 ꢃC. IR (KBr,
cmꢁ1):
n
1655, 1614, 1586, 1568, 1412, 1218; 1H NMR (CDCl3,
500 MHz):
d
1.14 (3H, s), 1.70 (3H, s), 4.60 (2H, m), 5.91 (1H, s), 7.49
(1H, d, J ¼ 4.4 Hz), 7.66e7.75 (1H, m), 7.66e7.75 (1H, m), 7.66e7.75
(1H, m), 8.12 (1H, dd, J ¼ 0.9, 7.3 Hz); 13C NMR (CDCl3, 125 MHz):
21.0, 27.5, 35.8, 66.8, 95.8, 110.8, 122.3, 126.4, 129.8, 131.3, 125.5,
133.3, 134.7, 144.5, 171.2, 174.4, 179.9; HRESIMS m/z 344.0792
[MþH]þꢄ (Calcd. for C17H15ClN3O3þ: 344.0796).
4.1.1.18. 3-(4-(3-Chloropropyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-
2,3-dihydronaphtho[1,2-b]furan-4,5-dione (12p). Compound 12p
varying from 0.01 mM to 200 mM (5 times lower than the highest

716
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Acknowledgments
Fellowships granted by CNPq, CAPES and FAPERJ are gratefully
acknowledged. This work was partially supported by Capes grant
number 058862/2010 and CNPq-FAPERJ-PRONEX grant number E-
26/110.574/2010. We thank the Hemotherapy Service of the Hos-
pital Clementino Fraga Filho (Federal University of Rio de Janeiro,
Brazil) for providing buffy coats.
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