Naphthoquinone-based chalcone hybrids and derivatives: Synthesis and potent activity against cancer cell lines

Article abstract of DOI:10.1039/c4md00371c

Novel naphthoquinone-based chalcones were prepared from the reaction between 3-bromo-nor-β-lapachone and amino-chalcones. Lapachone derivatives are also described here. All the substances were evaluated against cancer and normal cell lines and several compounds demonstrated potent antitumor activity. This journal is

Full text of DOI:10.1039/c4md00371c

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CONCISE ARTICLE
Naphthoquinone-based chalcone hybrids and
derivatives: synthesis and potent activity against
cancer cell lines†
Cite this: Med. Chem. Commun., 2015,
6, 120
Guilherme A. M. Jardim,a Tiago T. Guimaraes,b Maria do Carmo F. R. Pinto,c
˜
Bruno C. Cavalcanti,d Kaio M. de Farias,d Claudia Pessoa,de Claudia C. Gatto,f
g
g
a
*
*
Divya K. Nair, Irishi N. N. Namboothiri and Eufranio N. da Silva Junior
ˆ
´
Received 28th August 2014
Accepted 19th September 2014
Novel naphthoquinone-based chalcones were prepared from the reaction between 3-bromo-nor-b-
lapachone and amino-chalcones. Lapachone derivatives are also described here. All the substances were
evaluated against cancer and normal cell lines and several compounds demonstrated potent antitumor
activity.
DOI: 10.1039/c4md00371c
www.rsc.org/medchemcomm
Generally, quinones are able to provoke apoptosis and act as
topoisomerase inhibitors via DNA intercalation. Their toxicity
1. Introduction
can also be explained by inducing oxidative stress through
reactive oxygen species (ROS) generation.11 Recently, described
by Bolognesi and coworkers, quinones have attracted attention
due to their activity being intrinsically related by a multitarget
mechanism.12
In recent years, lapachones were employed as key substrates
for the synthesis of complex diazaazulenones,13 spirolactones,14
oxazoles15 and other potentially bioactive heterocyclic
compounds.15 Our research group has explored the potential of
quinones against cancer, mainly, via structural modication of
the nor-b-lapachone, particularly, the C-ring and redox centre
modication16 since these moieties are deeply related with the
generation of ROS (Scheme 1).
Earlier, we reported the synthesis of nor-b-lapachone-based
1,2,3-triazole and 3-arylamino derivatives with potent antitumor
activity via C-ring modication of nor-b-lapachone and molec-
ular hybridization19 with the junction of 1,2,3-triazole groups
(Scheme 1).17,18 Recently, we described a derivative of nor-b-
lapachone coupled benzothiadiazole (Scheme 1) with potent
activity against twenty cancer cell lines and low cytotoxicity
against three normal cells.20 These results were very promising
and the mechanism of action of this substance in tumor cells is
being currently investigated in our laboratories and it will be
reported in due course.
The population growth and aging associated with some risk
factors have increased the incidence of new cases of cancer and
related deaths in developed countries and in developing coun-
tries.1 In a four-year period, the estimated number increased
from 12.7 million new cancer cases with 7.6 million cancer-
related deaths in 2008, to 14.1 million new cancer cases and 8.2
million cancer-related deaths in 2012. The projection for the
year 2030, estimates 27 million new cancer cases with 17 million
cancer-related deaths.2,3
Naphthoquinone containing natural products belong to an
important class of naturally occurring secondary metabolites
found in the bignoniaceae family.4 Among these, naph-
thoquinoidal compounds obtained from natural products, such
as lapachol and lawsone (2-hydroxy-1,4-naphthoquinone), have
received considerable attention because of their antitumor
potential.5–8 1,4- and 1,2-naphthoquinones, and for instance,
dehydro-a-lapachone, an important anti-vascular agent,9 and b-
lapachone, a potent antitumoral compound, have also been
used as prototypes for the development of new drugs.10
aInstitute of Exact Sciences, Department of Chemistry, Federal University of Minas
Gerais, CEP 31270-901, Belo Horizonte-MG, Brazil. E-mail: eufranio@ufmg.br; Fax:
+55 31 34095700; Tel: +55 31 34095720
b
ˆ
ˆ
˜
Instituto Nacional de Cancer, Hospital do Cancer – Unidade I – Seçao de Medicina
Using redox centre modication, imidazoles and an oxirane
derivative were obtained from nor-b-lapachone and b-lapa-
chone (Scheme 1), with antimycobacterial21 and trypanocidal22
activities, indicating the importance of this approach. The C-
ring modication in the a-lapachone was also accomplished
and thio-derivatives with antitumor activities and 1,2,3-triazoles
with leishmanicidal activity were recently reported.23,24
Finally, following our program to develop new bioactive
molecules, particularly, new nor-b-lapachones with activity
Nuclear, 20230-130, Rio de Janeiro, RJ, Brazil
c
´
Nucleo de Pesquisas de Produtos Naturais, UFRJ, 21944-971, Rio de Janeiro, RJ, Brazil
dDepartamento de Fisiologia e Farmacologia, UFC, 60430-270, Fortaleza, CE, Brazil
e
´
Fiocruz – Ceara, 60180-900, Fortaleza, CE, Brazil
fInstitute of Chemistry, University of Brasilia, CEP 70904970, Brasilia-DF, Brazil
gDepartment of Chemistry, Indian Institute of Technology Bombay, Mumbai 400 076,
India. E-mail: irishi@iitb.ac.in; Fax: +91-22-2576-7152; Tel: +91-22-2576-7196
† Electronic supplementary information (ESI) available. CCDC 1018940. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4md00371c
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Scheme 1 Structural modification of the prototype lapachones.
against cancer cell lines and low cytotoxicity against normal ethanol. Compounds 1–6 were prepared with electron-donating
cells, the synthesis of quinone-based chalcones, is described (methyl, phenyl, methoxy and isopropyl groups) and an elec-
herein for the rst time. The strategies of molecular hybrid- tron-withdrawing group (nitro) in addition to the unsub-
ization and C-ring modication were used because of chal- stituted, 1. All the substances are known in the literature.26–28
cone's known antitumor activity.25 Redox centre modication in
Using the Hooker oxidation methodology29 nor-lapachol 8
lapachones to obtain hydrazone derivatives and C-ring modi- was prepared from lapachol 7 and used to obtain the classic
cation in a-lapachone in addition to nor-b-lapachone coupled intermediate 3-bromo-nor-b-lapachone 9 in quantitative yield.
with para-quinones were also accomplished. All the substances Compound 9 was reacted with the previously prepared amine-
were evaluated against cancer and normal cell lines.
chalcones 1–6 to provide nor-b-lapachone-based chalcones 10–
15 (Scheme 3).
New compounds 10–15 were well characterized by 1H and
13C NMR and high resolution mass spectrometry and all the
data are in accordance with the proposed structures. It was
observed in the 1H NMR spectrum of chalcone derivatives that
the respective doublet signals with coupling constant, J ¼ 15.6
Hz indicated the presence of the trans-olen of the quinone
coupled chalcone. Another important observation is a singlet at
around d 4.90–5.00 corresponding to the hydrogen attached to
the C-3 in the chalcone derivatives. It is more shielded,
compared to its counterpart in the bromo derivative 9, which is
observed at d 5.40. In addition, the 13C NMR spectra showed all
the expected signals. The structure of quinone-based chalcone
14 was reconrmed by X-ray crystallography (Fig. 1).
2. Results and discussion
Chemistry
To obtain the rst class of compounds, nor-b-lapachone-based
chalcones, initially amine-chalcones were prepared in good
yields (Scheme 2). The syntheses of chalcones were easily
accomplished by condensing 40-aminoacetophenone with
different aldehydes in the presence of sodium hydroxide in
Recently, the second class of substances was reported by us
(Scheme 4).30 Lawsone was reacted with Morita–Baylis–Hillman
acetates of nitroalkenes in the presence of
a quinine–
Scheme 2 Synthesis of the chalcone intermediates 1–6.
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Scheme 3 Nor-b-lapachone-based chalcones obtained from lapachol 7.
squaramide organocatalyst to provide enantioenriched disub-
stituted a-lapachones. This methodology allowed us to access
asymmetric lapachones that are C-ring modied in a single
step. In general, the products were obtained in high yields with
excellent diastereo- and enantioselectivities.30 The catalyst
shown in Scheme 4 was the most effective.
The compounds 16–25 assayed against cancer cell lines here
were previously described.30
In view of the ease of access to lapachone derivatives by
Fig. 1 An ORTEP-3 representation (50% probability displacement selective chemical transformation of lapachol 7, we invested to
ellipsoids) of the asymmetric unit of compound 14.
study the corresponding hydrazone derivatives. In addition, the
hydrazones of the b-lapachone 26, 3-iodo-b-lapachone 27, 3-
Scheme 4 a-Lapachone derivatives 16–25.
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bromo-b-lapachone 28, a-lapachone 32, nor-b-lapachone 34, evident from 1H NMR data that the peri-hydrogen of the
nor-a-lapachone 36 and 3-hydroxy-nor-b-lapachone 38, were naphthalene ring appeared at d 7.55 to 7.14 ppm, due to the
obtained in high yields. In fact, the hydrazones of naturally anisotropic hydrazo group at carbon C-6. In addition, the 13C
occurring quinones have been considered for a long time as NMR spectra of 30, 35 and 39, for instance, showed similar
simple derivatives to conrm the identity of quinones. To the chemical shis for the carbonyls, at d 179.1, 178.2 and 177.0
best of our knowledge, the use of hydrazones from quinones of ppm, respectively, which is consistent with carbonyl at carbon
the lapachol's group was not considered for major antitumor C-6 (for 30) and C-5 (for 35 and 39). A common feature observed
studies until now.
for compounds 30, 35 and 39 in the 1H NMR spectra using
Initially, b-lapachone 26, its derivatives 27 and 28 and a- CDCl3 as solvent is the presence of sharp singlet signals in the
lapachone 32, were prepared from lapachol 7, by reaction with downeld region (d 15.5–16.5 ppm) attributed to one N–H,
sulfuric acid, iodine, bromine and CH3COOH/HCl, respectively, which could form an internal hydrogen bond with the carbonyl
as previously reported.31,32 Nor-lapachol 8 was used to obtain 3- of the ortho-hydrazone moiety, a strong H-bond acceptor.
hydroxy-nor-b-lapachone 38, nor-b-lapachone 34 and nor-a-
a-Lapachone 32 gave the regioselective para-hydrazone 33,
lapachone 36.33 The hydrazone derivatives were prepared which is conrmed by the presence in its 1H NMR spectrum of
according to the methodology previously published with minor an isolated downeld doublet in the aromatic region, attributed
modications.34 The reaction of the appropriate quinones with to one naphthalene peri-hydrogen. Thus, as in the case of the
phenylhydrazine hydrochloride in acetic acid yielded the above ortho-hydrazones, the anisotropic effect of the hydrazine
respective derivatives 29–31, 33, 35, 37 and 39 in excellent yields moiety on the peri-hydrogen of naphthalene is also observed in
(Scheme 5).
the case of para-hydrazones. On the other hand, in comparison
The spectroscopic data of hydrazone derivatives are in to the original methylene hydrogens of the pyran ring in 32, the
accordance with the proposed structures. The formation of 1H NMR of the corresponding ones of 33, shows only negligible
these compounds followed the regioselective pattern previously effects, indicating that the hydrazo group has little inuence
observed and it is possible to conclude that the benzylic over the pyran ring.
carbonyl group formed the hydrazone in all the cases.34 This is
Scheme 5 Synthesis of the hydrazone derivatives 29–31, 33, 35, 37 and 39. (i) To obtain compound 26: H2SO4, 27: I2, Py, CH2Cl2, 0 ꢀC, 28: Br2,
CH2Cl2.
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Scheme 6 Lapachones 41–43 obtained from lapachol (7).
Recently, the trypanocidal activity of nor-b-lapachone-
The selected compounds have different patterns of substitu-
derived
1,2,3-triazole-conjugated
aminoquinones
was tion with an aryl ring substituted by electron withdrawing (Cl and
described.35 These compounds present ortho- and para-carbonyl NO2) and donating (OMe and CH3) groups in different positions,
moieties that are able to generate ROS and these can be in addition to furan and thiophene rings. Unfortunately, in this
considered as important structures for evaluation against study our strategy has failed and none of the compounds pre-
cancer cell lines because the activity of quinoidal molecules is sented antitumor activity with IC50 values > 12.1 mM (Table 1). At
deeply related with ROS generation as previously discussed.11,12 present, several modications of the asymmetric lapachones, for
Bearing in mind the importance of these substances, we instance the insertion of 1,2,3-triazoles, are being currently
considered the compounds 41–43, obtained from lapachol 7 investigated and will be reported in due course.
(Scheme 6) for antitumor studies. Nor-lapachol 8 was cyclized to
Recently, redox centre modication in quinones was
3-bromo-nor-b-lapachone that was used to prepare compound successfully employed to obtain antitumor compounds.16 Imine
40. The azido derivative 40 was used to synthesize 41–43 and all derivatives obtained from b-lapachone 26 presented potent
spectroscopy data were in accordance with the literature.35
antitumor activity.37 In this context, we conducted the synthesis
of hydrazone derivatives from b-lapachone 26, their iodine and
bromine derivatives 27 and 28, a-lapachone 32, nor-b-lapa-
chone 34, nor-a-lapachone 36 and 3-hydroxy-nor-b-lapachone
38, all the substances from the lapachol group. The derivatives
29–31, 33, 35, 37 and 39 were evaluated against four cancer cell
lines. In the same manner as the asymmetric quinones, the
hydrazones were not active against all the lineages evaluated
(Table 1). In comparison of these structures with the imines,
previously obtained from b-lapachone 26,37 the simple insertion
of the NH-bond was enough to provoke the suppression of the
activity. As suggested by Burton and coworkers,37 the change in
the redox centre does not affect the overall activity and selec-
tivity of the lapachone imine derivatives and it was described as
an alternative in the search for new anticancer agents consid-
ering the high cytotoxicity of the b-lapachone 26 in normal cell
lines. On the basis of this principle, we considered the redox
centre modication of lapachone derivatives as an important
strategy to obtain novel antitumor drugs despite the lack of
activity of the compounds described herein.
3. Biology
All the substances described (Schemes 2–5), were evaluated in
vitro using the MTT assay against four cancer cell lines: HL-60
(human promyelocytic leukemia), OVCAR-8 (human ovarian
adenocarcinoma), SF295 (human glioblastoma) and HCT-116
(human colon carcinoma). Doxorubicin was used as the positive
control (Table 1).36 The selectivity of the compounds toward a
normal proliferating cell line was investigated using the MTT
assay with human peripheral blood mononuclear cells (PBMC)
aer 72 h of drug exposure.
Our strategy was based on the modication of the prototypes
b-lapachone 26, a-lapachone 32, nor-b-lapachone 34 and nor-a-
lapachone 36. Recently, 1,2,3-triazole-, arylamino- and thio-
substituted a-lapachones were synthesized and evaluated
against eleven cancer cell lines.24,35,36 Most of them were
considered moderately active and several compounds were
considered highly active (IC50 < 2 mM). Following this previous
work, herein, the antitumor activities of asymmetric a-lapa-
chone C-ring modied 16–25 were evaluated.
The third group of compounds was strategically planned by
C-ring modication16 of nor-b-lapachone 34 and molecular
hybridization19 with chalcone moieties (Scheme 7).
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Table 1 Cytotoxic activities expressed by IC50 mM (95% CI) for compounds 1–6, 10–25, 29–31, 33, 35, 37, 39 and 41–43 and for the positive
control doxorubicin and the precursors lapachonea
Compounds
HL-60
HCT-116
OVCAR-8
SF295
PBMC
1
2
3
4
6.40 (5.24–7.84)
1.75 (0.89–3.46)
0.46 (0.08–1.94)
>16.71
14.1 (12.86–15.59)
10.96 (9.99–12.01)
15.35 (12.73–18.47)
>16.71
>22.4
>18.65
>21.08
>16.71
>22.4
>18.65
>21.08
>16.71
20.2 (19.54–22.00)
13.83 (13.54–14.54)
>21.08
>16.71
5
6
10
11
12
13
14
15
10.50 (8.53–12.99)
10.0 (12.52–17.53)
0.04 (0.02–0.04)
0.20 (0.16–0.26)
0.10 (0.04–0.21)
2.20 (1.84–2.60)
0.10 (0.08–0.12)
0.14 (0.10–0.18)
>14.92
>19.75
>18.85
>19.75
>18.85
>19.75
>18.85
>19.75
>18.85
0.71 (0.62–0.82)
2.22 (1.94–2.57)
1.94 (1.77–2.15)
>9.5
1.54 (0.91–1.89)
1.56 (1.18–2.23)
>14.92
1.53 (1.33–1.75)
4.35 (3.78–4.81)
2.13 (1.81–2.52)
>9.5
0.64 (0.33–1.08)
2.54 (1.99–2.91)
>14.92
0.37 (0.26–0.53)
2.89 (2.50–3.39)
4.57 (4.14–5.05)
>9.5
1.08 (0.56–1.46)
1.62 (1.30–2.13)
>14.92
2.20 (1.82–2.64)
6.51 (5.80–6.88)
7.90 (7.66–8.26)
>9.5
1.33 (0.85–1.73)
2.28 (1.77–2.64)
>14.92
16
17
>12.98
>12.98
>12.98
>12.98
>12.98
18
>12.65
>12.65
>12.65
>12.65
>12.65
19
>13.54
>13.54
>13.54
>13.54
>13.54
20
>14.32
>14.32
>14.32
>14.32
>14.32
21
>12.10
>12.10
>12.10
>12.10
>12.10
22
>13.54
>13.54
>13.54
>13.54
>13.54
23
>13.15
>13.15
>13.15
>13.15
>13.15
24
>14.66
>14.66
>14.66
>14.66
>14.66
25
>15.38
>15.38
>15.38
>15.38
>15.38
29
>15.05
>15.05
>15.05
>15.05
>15.05
30
>10.91
>10.91
>10.91
>10.91
>10.91
31
>12.19
>12.19
>12.19
>12.19
>12.19
33
>15.05
>15.05
>15.05
>15.05
>15.05
35
>15.71
>15.71
>15.71
>15.71
>15.71
37
>15.71
>15.71
>15.71
>15.71
>15.71
39
>14.96
>14.96
>14.96
>14.96
>14.96
41
42
43
0.45 (0.39–0.54)
1.76 (1.43–2.17)
1.75 (1.37–2.25)
1.75, 1.59–1.83
1.65, 1.49–1.78
8.18, 7.55–8.92
0.03, 0.01–0.05
0.58 (0.52–0.66)
1.90 (1.65–2.21)
2.32 (2.25–2.72)
1.41, 1.28–1.54
0.97, 0.81–1.04
19.11, 18.75–19.88
0.02, 0.01–0.03
1.33 (1.12–1.58)
3.32 (2.82–4.06)
4.36 (3.83–4.78)
1.25, 1.07–1.43
1.16, 0.97–1.25
14.79, 14.42–18.15
0.09, 0.04–0.12
1.29 (1.14–1.43)
1.80 (1.55–2.10)
2.21 (1.52–2.79)
1.58, 1.31–1.88
0.91, 0.74–1.11
18.77, 18.15–19.03
0.48, 0.37–0.52
0.27 (0.14–0.37)
1.10 (0.83–1.18)
1.00 (0.87–1.07)
>21.90
>20.60
>20.66
Nor-b-lapachone
b-Lapachone
a-Lapachone
Doxorubicin
0.37, 0.30–0.43
a
Results obtained by nonlinear regression for all assayed cell lines from three independent experiments (deviations in parenthesis).
Chalcones are recognized by their biological activities, for
For the insertion of chalcone in the naphthoquinoidal
instance, antitumor,38 leishmanicidal39 and antimalarial.40 moiety, compounds 1–6 were prepared. The structures 1–3 were
Considering the potential of the chalcones, naphthoquinoidal also evaluated and showed activity against HL-60, HCT-116 and
compounds 10–15 were prepared and evaluated against cancer OVCAR-8 with IC50 values in the range of 0.46 to 15.35 mM.
and normal cell lines (Table 1). The selectivity index was Compound 4 was not active and the structures 5 and 6 pre-
considered to establish the potentialof thesestructures (Table 2). sented activity only for HL-60 with IC50 values ¼ 10.5 and 10.0
mM, respectively.
Scheme 7 Strategy used to obtain new antitumor naphthoquinonoid compounds.
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Table Selectivity index for the most active compounds [ratio
between the cytoxicities, expressed as IC50 (mM), against PBMC and
four cancer cell lines]. Nd ¼ not determined
Concise Article
2
against all the cancer cell lines evaluated, but these compounds
were more cytotoxic than the other derivatives of this class.
In general terms, lapachone-based chalcone hybrids pre-
sented important activity against cancer cell lines with low
cytotoxicity in normal cancer cell lines. This manuscript
represents the rst report on this class of compounds with
promising activity, and based on our experience with lapachone
derivatives, the compounds herein described deserve further
subsequent studies.
The last class of compounds herein evaluated has been
chosen because of the intrinsic ability of these structures to
generate ROS. In fact, the compounds 41–43 were considered
highly active (IC50 < 2 mM) against all the cancer lineages eval-
uated with some exceptions. The main problem observed for
these compounds was the high cytotoxicity observed in normal
cell lines (PBMC). For instance, compound 41 was highly active
against HL-60 cell lines with IC50 ¼ 0.45 mM, but it presented
IC50 value ¼ 0.27 mM for normal cells. In general, 41–43 are
more cytotoxic for normal cell than cancer cell lines.
PBMC vs.
PBMC vs.
PBMC vs.
PBMC vs.
Compounds
HL 60
HCT-116
OVCAR-8
SF295
1
2
3
10
11
12
13
14
15
41
42
43
3.1
7.9
1.4
1.2
1.3
3.0
2.9
4.0
1.0
0.8
1.4
0.4
0.5
0.4
18.5
0.9
0.7
1.0
1.4
1.4
3.7
1.0
2.0
0.8
0.2
0.3
0.2
4.1
0.9
0.7
1.0
5.9
2.2
1.7
1.0
1.2
1.4
0.2
0.6
0.4
0.7
45.8
55.0
32.5
79.0
4.3
13.3
16.2
0.6
0.6
0.5
12.0
Doxorubicin
Lately, microencapsulation aimed controlled release is an
important strategy to increase the efficiency of antitumor
drugs.41 Recent studies using b-lapachone were described to try
to diminish the cytotoxicity of this important antitumor
quinone.42 Considering the promising results observed for 41–
43, in four types of cancer cell lineage, studies aiming controlled
delivery systems, could be an interesting possibility to solve the
problem of cytotoxicity.
Aer evaluating nor-b-lapachone based chalcone 10–15, the
data conrmed the success of our approach. As previously
described,8 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). The structures 10–15
were considered highly active against HL-60 and HCT-116 with
IC50 values between 0.04 to 2.2 mM. Compounds 3, 10–12, 14
and 15 were considered very promising against HL-60 aer
assaying these structures against peripheral blood mono-
nuclear cells (PBMC). The selectivity index (SI), which is an
excellent parameter to determine the efficiency of the
compounds in cancer cells with low cytotoxicity in normal cell
lines is expressed by the ratio of cytotoxicities between normal
cell (PBMC) and different cancer cell lines. For compounds 3,
10, 11, 12, 14 and 15 the values of SI (considering HL-60) were
45.8, 55.0, 32.5, 79, 13.3 and 16.2 (Table 2), respectively. For
doxorubicin, the drug used clinically against several types of
cancer, the SI value is 12.0. In comparison with the positive
control, doxorubicin, nor-b-lapachone-based chalcone 12 is
more active and this structure could be considered as an
important prototype for further studies. To the best of our
knowledge, 12 can be considered as one of the most active
substances for HL-60 cancer cell line with a high selectivity
index obtained from lapachol 7 already reported.
4. Conclusions
We evaluated thirty-two compounds against four cancer cell
lines and peripheral blood mononuclear cells (PBMC). These
compounds were obtained by C-ring and redox centre modi-
cations based on the prototypes nor-b-lapachone, a-lapachone,
nor-a-lapachone and b-lapachone analogues. Fourteen
compounds were discovered as important substances with
potent antitumor activity. In general, all the compounds were
highly active (IC50 < 2 mM) and the substances were more active
than doxorubicin, a clinically used drug against several types of
cancer. Compound 12 presented good selectivity for HL-60 cell
line with high selectivity index even better than doxorubicin the
positive control. This compound presents a promising prole
for further experimental investigations in this study.
Another important characteristic that points to the success
of the strategy herein employed was the increase of the activity
of nor-b-lapachone 34 aer the insertion of the chalcone
moieties (IC50 values for 34 in the range of 1.25–1.75 mM), Table
1. The hybrid compounds were also more active than the non-
hybridized chalcones 1–4.
5. Experimental section
Melting points were obtained on a Thomas Hoover apparatus
and are uncorrected. Analytical grade solvents were used.
Column chromatography was performed on silica gel (Acros
Organics 0.035–0.070 mm, pore diameter ca. 6 nm). 1H and 13
C
Finally, compound 13 was not active against HCT-116,
OVCAR-8 and SF295, but this molecule presented selective
activity for HL-60 with IC50 value ¼ 2.20 mM. In terms of selec-
tivity index this structure was not promising, but further
modications in this prototype are under investigation in our
research group to develop drugs with selectivity against a
certain type of cancer. Compounds 14 and 15 were highly active
NMR were recorded at room temperature using a Bruker
AVANCE DRX 200 and Varian Mercury 400 and 500 MHz spec-
trometers in the solvents indicated with TMS as an internal
reference. Chemical shis (d) are given in ppm. Electron-impact
mass spectra (70 eV) were obtained using a VG Autospec appa-
ratus (Micromass, Manchester, UK). The main fragments were
described as a relation between atomic mass units and the
126 | Med. Chem. Commun., 2015, 6, 120–130
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charge (m/z) and the relative abundance as a percentage of the
base peak intensity. Lapachol (5) (2-hydroxy-3-(30-methyl-20-
butenyl)-1,4-naphthoquinone) was extracted from the heart-
wood of Tabebuia sp. (Tecoma) and puried by a series of
recrystallizations in an appropriate solvent. From this quinone,
(E)-3-(4-(3-(Biphenyl-4-yl)acryloyl)phenylamino)-2,2-
dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (13). The
compound 13 was obtained as a orange solid (157 mg, 0.3
mmol, 30% yield); mp 248–252 ꢀC. 1H NMR (400 MHz, CDCl3) d
8.12 (dd, 1H, J ¼ 7.4, 1.3 Hz), 7.98 (d, 2H, J ¼ 8.7 Hz), 7.82 (d, 1H,
J ¼ 15.6 Hz), 7.75–7.73 (m, 1H), 7.73–7.69 (m, 3H), 7.69–7.67 (m,
1H), 7.67–7.61 (m, 5H), 7.58 (d, 1H, J ¼ 15.6 Hz), 7.50–7.44 (m,
2H), 7.41–7.36 (m, 1H), 6.64 (d, 2H, J ¼ 8.8 Hz), 1.72 (s, 3H), 1.59
(s, 3H). 13C NMR (100 MHz, DMSO-d6) d 186.4, 181.0, 175.3,
168.5, 152.8, 141.9, 141.7, 139.7, 135.5, 135.4, 134.7, 133.1,
132.9, 131.6, 131.4, 130.6, 129.7, 129.5, 127.6, 127.5, 127.2,
126.6, 125.0, 122.7, 115.5, 96.2, 59.7, 27.1, 21.8. EI/MS (m/z) [M +
Na]+: 548.1836. Calcd for [C35H27NNaO4]+: 548.1832.
(E)-3-((4-(3-(4-Methoxyphenyl)acryloyl)phenyl)amino)-2,2-
dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (14). The
compound 14 was obtained as a red solid (191 mg, 0.4 mmol,
40% yield); mp 230–232 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.06 (d,
1H, J ¼ 7.4 Hz), 7.90 (d, 2H, J ¼ 8.7 Hz), 7.74–7.59 (m, 4H), 7.54
(d, 2H, J ¼ 8.7 Hz), 7.38 (d, 1H, J ¼ 15.6 Hz), 6.89 (d, 2H, J ¼ 8.7
Hz), 6.59 (d, 2H, J ¼ 8.8 Hz), 4.91 (d, 1H, J ¼ 7.2 Hz), 4.60 (d, 1H,
J ¼ 7.2 Hz), 3.82 (s, 3H), 1.68 (s, 3H), 1.55 (s, 3H). 13C NMR (100
MHz, DMSO-d6) d 187.9, 180.7, 175.2, 170.0, 151.3, 151.2, 143.1,
134.6, 132.9, 132.7, 131.1, 131.0, 129.5, 128.4, 128.1, 127.1,
126.9, 125.1, 120.9, 114.4, 113.9, 112.1, 96.7, 60.7, 27.4, 21.7. EI/
MS (m/z) [M + Na]+: 502.1624. Calcd for [C30H25NO5Na]+:
502.1630.
(E)-3-((4-(3-(4-Isopropylphenyl)acryloyl)phenyl)amino)-2,2-
dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (15). The
compound 15 was obtained as a orange solid (171 mg, 0.3
mmol, 35% yield); mp 212–214 ꢀC. 1H NMR (400 MHz, CDCl3) d:
8.10 (d, 1H, J ¼ 7.5 Hz), 7.94 (d, 2H, J ¼ 8.6 Hz), 7.77–7.71 (m,
2H), 7.69–7.64 (m, 1H), 7.56 (d, 2H, J ¼ 8.1 Hz), 2.97–2.92 (m,
1H), 7.27 (t, 3H, J ¼ 4.1 Hz), 6.62 (d, 2H, J ¼ 8.7 Hz), 4.94 (d, 1H, J
¼ 7.2 Hz), 4.59 (d, 1H, J ¼ 7.2 Hz), 2.97–2.92 (m, 1H), 1.71 (s,
3H), 1.58 (s, 3H), 1.28 (s, 3H), 1.26 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d: 187.9, 180.7, 175.2, 170.0, 151.3, 151.8, 143.1,
134.6, 132.9, 132.7, 131.1, 130.9, 129.5, 128.4, 128.1, 127.1,
127.0, 126.9, 125.1, 120.9, 114.4, 113.8, 112.1, 96.7, 60.7, 34.1,
27.4, 23.4, 21.7. EI/MS (m/z) [M + Na]+: 514.1979. Calcd for
[C32H29NO4Na]+: 514.1994.
nor-lapachol
(2-hydroxy-3-(20-methyl-propenyl)-1,4-naph-
thoquinone) was obtained by the Hooker oxidation method.28
General procedure to prepare the quinone-based chalcones
An excess of bromine (2 mL) was added to a cooled solution of
nor-lapachol (228 mg, 1 mmol) in 25 mL of dichloromethane.
The brominated intermediate 9 was obtained as an orange
solid. Aer the removal of excess bromine, a solution of the
respective chalcone (1 mmol) in 25 mL of dichloromethane was
added and stirred overnight. The reaction mixture was
concentrated under reduced pressure and the residue was
puried by column chromatography in silica gel by eluting with
an increasing polarity gradient mixture of hexane and ethyl
acetate.
(E)-3-(4-Cinnamoylphenylamino)-2,2-dimethyl-2,3-dihy-
dronaphtho[1,2-b]furan-4,5-dione (10). The compound 10 was
obtained as a red solid (157 mg, 0.35 mmol, 35% yield); mp 196–
199 ꢀC. 1H NMR (500 MHz, CDCl3) d 7.98 (d, 1H, J ¼ 7.4 Hz), 7.88
(d, 2H, J ¼ 8.4 Hz), 7.69 (dt, 3H, J ¼ 14.8, 11.6 Hz), 7.59 (t, 3H, J ¼
7.4 Hz), 7.49 (d, 1H, J ¼ 15.6 Hz), 7.39–7.35 (m, 3H), 6.61 (d, 2H,
J ¼ 8.7 Hz), 4.91 (d, 1H, J ¼ 7.3 Hz), 1.69 (s, 3H), 1.57 (s, 3H). 13
C
NMR (125 MHz, CDCl3) d 187.8, 180.7, 175.2, 170.0, 151.3, 142.9,
135.3, 134.6, 132.7, 131.1, 131.0, 129.9, 129.5, 128.8, 128.2,
128.0, 127.2, 125.2, 121.9, 114.5, 112.2, 96.7, 60.8, 27.4, 21.8. EI/
MS (m/z) [M + Na]+: 472.1511. Calcd for [C29H23NO4Na]+:
472.1519.
(E)-2,2-Dimethyl-3-(4-(3-(4-nitrophenyl)acryloyl)phenyl-
amino)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (11). The
compound 11 was obtained as a brown solid (144 mg, 0.3 mmol,
30% yield); mp 224–227 ꢀC. 1H NMR (400 MHz, acetone) d 8.39–
8.28 (m, 3H), 8.17 (d, 1H, J ¼ 9.1 Hz), 8.15–8.01 (m, 3H), 7.92 (d,
1H, J ¼ 15.7 Hz), 7.82–7.75 (m, 2H), 7.01 (d, 2H, J ¼ 8.5 Hz), 6.84
(d, 2H, J ¼ 8.8 Hz), 5.06 (s, 1H), 1.76 (s, 3H), 1.60 (s, 3H). 13C
NMR (125 MHz, CDCl3) d 189.2, 183.9, 180.6, 171.1, 151.6, 148.2,
141.6, 139.8, 134.7, 132.7, 131.2, 131.2, 129.6, 128.7, 127.6,
127.1, 125.8, 125.1, 124.1, 114.3, 112.2, 96.4, 60.8, 27.4, 21.8. EI/
General procedure to prepare the hydrazone-derivatives
MS (m/z) [M + Na]+: 517.1378. Calcd for [C29H22N2NaO6]+: Phenylhydrazine hydrochloride (288 mg, 2 mmol) was added all
517.1370. at once to a solution of respective quinone (1 mmol) in acetic
(E)-2,2-Dimethyl-3-(4-(3-p-tolylacryloyl)phenylamino)-2,3- acid (15 mL). The mixture was stirred at reux and monitored by
dihydronaphtho[1,2-b]furan-4,5-dione (12). The compound 12 TLC until complete consumption of the quinone. The residue
was obtained as a red solid (153 mg, 0.3 mmol, 33% yield); mp was poured into water and the precipitate was ltered and
217–220 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.14 (d, 1H, J ¼ 7.3 Hz), washed with plenty of water. Aer dried, the crude hydrazones
7.96 (d, 2H, J ¼ 8.6 Hz), 7.76 (d, 1H, J ¼ 16.0 Hz), 7.74–7.62 (m, were puried by column chromatography in silica gel by eluting
3H), 7.53 (d, 2H, J ¼ 8.1 Hz), 7.50 (d, 1H, J ¼ 15.9 Hz), 7.21 (d, with an increasing polarity gradient mixture of hexane and ethyl
2H, J ¼ 7.8 Hz), 6.62 (d, 2H, J ¼ 8.7 Hz), 4.95 (s, 1H), 2.39 (s, 3H), acetate.
1.71 (s, 3H), 1.58 (s, 3H). 13C NMR (100 MHz, CDCl3) d 187.9,
3-Iodo-2,2-dimethyl-6-(2-phenylhydrazono)-3,4-dihydro-2H-
180.7, 175.3, 170.1, 151.2, 143.2, 140.4, 134.6, 132.7, 132.5, benzo[h]chromen-5(6H)-one (30). The compound 30 was
131.1, 131.0, 129.6, 128.3, 128.1, 127.2, 125.2, 120.9, 114.4, obtained as a red solid (435 mg, 0.9 mmol, 95% yield); mp 157–
112.1, 96.7, 60.7, 27.4, 21.8, 21.5. EI/MS (m/z) [M + Na]+: 158 ꢀC. 1H NMR (200 MHz, CDCl3) d 16.11 (s, 1H), 8.25 (d, 1H, J
486.1669. Calcd for [C30H25NNaO4]+: 486.1676.
¼ 8.1 Hz), 7.80 (d, 1H, J ¼ 7.8 Hz), 7.43–7.22 (m, 6H), 7.08–7.01
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(m, 1H), 4.29 (dd, 1H, J ¼ 8.9, 5.7 Hz), 3.44 (dd, 1H, J ¼ 5.5, 7.3 using SHELXL-97.45 Molecular graphics, ORTEP-3,46 soware
Hz), 3.22 (dd, 1H, J ¼ 8.6, 8.0 Hz) 1.59 (s, 3H), 1.50 (s, 3H). 13
C
was used to prepare material for publication, WinGX-
NMR (50 MHz, CDCl3) d 178.0, 158.7, 142.6, 133.0, 129.4, 127.6, Routine.47†
125.9, 124.9, 123.7, 122.5, 121.6, 116.2, 110.6, 79.4, 30.9, 29.9,
27.3, 24.1. EI/MS (m/z) [M + Na]+: 481.0371. Calcd for [C21H19
-
Crystal data for compound 14
IN2O2Na]+: 481.0389.
C30H25NO5; M ¼ 479.51; monoclinic, space group C2/c; a ¼
3-Bromo-2,2-dimethyl-6-(2-phenylhydrazono)-3,4-dihydro-
2H-benzo[h]chromen-5(6H)-one (31). The compound 31 was
obtained as an orange solid (369 mg, 0.9 mmol, 90% yield); mp
187–189 ꢀC. 1H NMR (200 MHz, CDCl3) d 16.17 (s, 1H), 8.31 (d,
1H, J ¼ 7.9 Hz), 7.87 (d, 1H, J ¼ 7.8 Hz), 7.58–7.33 (m, 6H), 7.12
(m, 1H), 4.22 (dd, 1H, J ¼ 8.1, 5.7 Hz), 3.29 (dd, 1H, J ¼ 5.5, 12.2
ꢀ
˚
˚
˚
37.446(2) A, b ¼ 10.070(7) A, c ¼ 13.198(10) A; a ¼ g ¼ 90 , b ¼
101.015(5) ; V ¼ 4885.3(6) A ; Z ¼ 8; Dc ¼ 1.304 g cmꢁ1; F(000) ¼
3
ꢀ
˚
2016; red prism, size 0.39 ꢂ 0.22 ꢂ 0.11 mm; 4944 independent
measured reections, renement based on F2 to give R1[F2
>
4s(F2)] ¼ 0.055; w2 ¼ 0.106 for 17 276 observed reections, and
393 parameters.
Hz), 3.03 (dd, 1H, J ¼ 8.4, 9.4 Hz), 1.61 (s, 3H), 1.52 (s, 3H). 13
C
NMR (50 MHz, CDCl3) d 178.3, 158.5, 142.6, 133.0, 129.4, 127.7,
Cytotoxicity against cancer and normal cell lines
125.9, 124.9, 123.6, 122.6, 121.6, 116.2, 109.8, 79.4, 51.3, 28.3,
Compounds (0.001–5 mg mLꢁ1) were tested for cytotoxic activity
against several human cancer cell lines obtained from the
National Cancer Institute, NCI (Bethesda, MD). Peripheral
blood mononuclear cells (PBMC) were isolated from heparin-
ized blood from healthy, non-smoker donors who had not taken
any medication at least 15 days prior to sampling by a standard
method of density-gradient centrifugation on Histopaque-1077
(Sigma Aldrich Co. – St. Louis, MO/USA). All cancer cell lines
and PBMC were maintained in RPMI 1640 medium. All culture
media were supplemented with 20% (PBMC) or 10% (HL-60,
HCT-116, OVCAR-8 and SF295) fetal bovine serum, 2 mM L-
glutamine, 100 IU mLꢁ1 penicillin, 100 mg mLꢁ1 streptomycin at
37 ꢀC with 5% CO2. PBMC cultures were also supplemented
with 2% phytohaemagglutinin. In cytotoxicity experiments,
cells were plated in 96-well plates (1 ꢂ 106 cells per well for
leukemia HL-60 cells and 1 ꢂ 106 cells per well for solid tumors
cell lines and PBMC). All the tested compounds were dissolved
with DMSO. The nal concentration of DMSO in the culture
medium was kept constant (0.1%, v/v). Doxorubicin (0.001–1.10
mM) was used as the positive control, and negative control
groups received the same amount of vehicle (DMSO). The cell
viability was determined by reduction of the yellow dye 3-(4,5-
26.6, 22.4. EI/MS (m/z) [M + Na]+: 433.0522. Calcd for [C21H19
-
BrN2O2Na]+: 433.0527.
2,2-Dimethyl-5-(2-phenylhydrazono)-2,3-dihydronaphtho-
[1,2-b]furan-4(5H)-one (35). The compound 35 was obtained as
an orange solid (334 mg, 0.9 mmol, 95% yield); mp 186–188 ꢀC.
1H NMR (200 MHz, CDCl3) d 16.30 (s, 1H), 8.42 (d, 1H, J ¼ 7.8
Hz), 7.74 (d, 1H, J ¼ 7.8 Hz), 7.64–7.32 (m, 6H), 7.15 (t, 1H, J ¼
7.2 Hz), 3.02 (s, 2H), 1.60 (s, 6H). 13C NMR (50 MHz, CDCl3) d
177.3, 165.9, 142.7, 135.0, 129.7, 129.4, 129.3, 125.9, 124.6,
123.0, 122.4, 120.4, 116.0, 113.6, 91.6, 39.5, 28.5. Mass spectra
(relative intensity): 318 (M+c, 100), 303 (10), 226 (85), 213 (27),
130 (20). Elemental analysis calcd for C20H18N2O2 (318.36): C ¼
75.45, H ¼ 7.55, N ¼ 8.79. Found: C ¼ 75.52, H ¼ 7.60, N ¼ 8.33.
2,2-Dimethyl-9-(2-phenylhydrazono)-2,3-dihydronaphtho-
[2,3-b]furan-4(9H)-one (37). The compound 37 was obtained as
an orange solid (270 mg, 0.8 mmol, 85% yield); mp 165–167 ꢀC.
1H NMR (200 MHz, CDCl3) d 10.86 (s, 1H), 8.26 (d, 1H, J ¼ 7.7
Hz), 8.16 (d, 1H, J ¼ 7.4 Hz), 7.51–7.28 (m, 6H), 7.04–7.01 (m,
1H), 2.92 (s, 2H), 1.61 (s, 6H). 13C NMR (50 MHz, CDCl3) d 181.1,
157.7, 142.7, 133.8, 130.8, 130.5, 129.5, 127.2, 125.3, 123.2,
122.7, 116.2, 114.5, 93.5, 38.8, 28.7. EI/MS (m/z) [M + Na]+:
341.1258. Calcd for [C20H18N2O2Na]+: 341.1266.
dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium
bromide
3-Hydroxy-2,2-dimethyl-5-(2-phenylhydrazono)-2,3-dihy-
dronaphtho[1,2-b]furan-4(5H)-one (39). The compound 39 was
obtained as a red solid (300 mg, 0.9 mmol, 90% yield); mp 197–
198 ꢀC. 1H NMR (200 MHz, CDCl3) d 16.11 (s, 1H), 8.34 (d, 1H, J
¼ 8.2 Hz), 7.74 (d, 1H, J ¼ 7.8 Hz), 7.58–7.32 (m, 6H), 7.15 (t, 1H,
J ¼ 14.1 Hz), 5.12 (s, 1H), 1.67 (s, 3H), 1.48 (s, 3H). 13C NMR (50
MHz, CDCl3) d 178.0, 167.8, 142.7, 135.9, 130.9, 129.7, 129.5,
126.3, 125.2, 123.9, 122.8, 120.3, 116.4, 116.2, 94.7, 27.0, 21.0.
EI/MS (m/z) [M + Na]+: 357.1208. Calcd for [C20H18N2O3Na]+:
357.1215.
(MTT) to a blue formazan product as described by Mosmann.48
At the end of the incubation time (72 h), the plates were
centrifuged and the medium was replaced by fresh medium
(200 mL) containing 0.5 mg mLꢁ1 MTT. Three hours later, the
MTT formazan product was dissolved in DMSO (150 mL) and the
absorbance was measured using a multiplate reader (Spectra
Count, Packard, Ontario, Canada). Drug effect was quantied as
the percentage of control absorbance of the reduced dye at 550
nm. All cell treatments were carried out with three replicates.
Acknowledgements
Crystallographic data for compound 14
X-ray data collection was accomplished on a Bruker CCD This work was partially supported by grants from the Conselho
´
´
SMART APEX II single crystal diffractometer with Mo Ka radi- Nacional de Desenvolvimento Cientıco e Tecnologico (CNPq)
ation (0.71073 A) at 296 K. The data were processed with SAINT
43
˚
Edital Jovens pesquisadores 550579/2012-5, CAPES, FAPEMIG,
and were corrected for absorption using SADABS.44 The struc- and RENORBIO (Rede Nordeste de Biotecnologia) and
˜
´
tures were solved by direct methods using SHELXS-97 and Fundaçao Cearense de Apoio ao Desenvolvimento Cientıco –
subsequent Fourier-difference map analyses yielded the posi- FUNCAP. A research grant to INNN by DST India and a research
tions of the nonhydrogen atoms, the renement was performed felowship to DKN by CSIR India are gratefully acknowledged.
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