Synthesis of novel spiropyrazoline oxindoles and evaluation of cytotoxicity in cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2014.04.023

A series of novel spiropyrazoline oxindole derivatives was synthesized by 1,3-dipolar cycloaddition reaction. The compounds were screened for their in vitro cytotoxic activity against MCF-7 breast cancer cell line (estrogen receptor positive (ER+) and hum

Full text of DOI:10.1016/j.ejmech.2014.04.023

European Journal of Medicinal Chemistry 79 (2014) 266e272
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of novel spiropyrazoline oxindoles and evaluation of
cytotoxicity in cancer cell lines
*
Ângelo Monteiro, Lídia M. Gonçalves, Maria M.M. Santos
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel spiropyrazoline oxindole derivatives was synthesized by 1,3-dipolar cycloaddition re-
action. The compounds were screened for their in vitro cytotoxic activity against MCF-7 breast cancer cell
line (estrogen receptor positive (ERþ) and human epidermal growth factor receptor 2 negative (HER2ꢀ)).
Received 2 December 2013
Received in revised form
10 March 2014
Accepted 5 April 2014
Available online 8 April 2014
Of the nineteen spiropyrazoline oxindoles tested, six compounds have a GI50 below 12 mM The most
potent compounds in this series were also evaluated against MDA-MB-231 breast cancer cell line (ERꢀ
and HER2ꢀ). Two spiropyrazoline oxindoles were highly selective between MCF-7 tumor cells and MDA-
MB-231 tumor cells. More importantly, they were noncytotoxic against HEK 293T non tumor derived cell
lines.
Keywords:
Spirooxindole
Pyrazoline
Ó 2014 Elsevier Masson SAS. All rights reserved.
Structureeactivity relationship
Cytotoxicity
Anticancer agents
1. Introduction
system is the core structure of a variety of medicinal agents and
natural products [4,5]. Spirooxindole derivatives were described
Cancer is one of the leading causes of mortality worldwide,
causing 7.6 million deaths in 2008. Moreover, World Health Orga-
nization projects a rise in deaths from cancer to 13.1 million in 2030
[1]. Despite a more sophisticated understanding of the etiology of
cancer and a greater emphasis placed on early detection of the
disease, overall mortality rates from cancer have not diminished
greatly and are not expected to decrease. As such, cancer continues
to pose a major threat to human health and further research
regarding new therapeutic strategies that more effectively combat
cancer are needed. Furthermore, the increase cases of multidrug
resistance (MDR) make a challenge for the development of new
drugs a milestone on the treatment of various types of cancers (e.g.
blood, breast, ovarian, lung, and lower gastrointestinal tract can-
cers). Various MDR mechanisms observed in cancer cells as well as
various strategies developed to overcome these mechanisms have
been extensively studied during the last few decades to enhance
the efficacy of chemotherapy by suppressing or evading the MDR
mechanisms [2].
with different biological activities, ranging from MDM2 antago-
nists, ion channel blockers and anti-inflammatory agents to an-
timalarials (Fig. 1) [6e9]. As a consequence, there is a huge
interest in industry and academia to develop novel spirooxindoles
with interesting biological activities. Pyrazoline is also a privi-
leged unit in medicinal chemistry. We now report the study of
spiropyrazoline oxindoles, containing
a five membered ring
(pyrazoline) with one more aromatic substituent (R4) (oxygen
atom in isoxazoline ring was replaced by a NeAr group). To
perform a structure-activity study, we synthesized and evaluated
the antiproliferative activity of a small library of novel spiropyr-
azoline oxindoles containing different substituents at the pyr-
azoline ring and in the aromatic ring of the oxindole moiety
(Fig. 2). Two breast cancer cell lines, one non-invasive estrogen
receptor (ER) positive (MCF-7) and, one invasive ER-negative
(MDA-MB-231), both derived from a metastatic adenocarcinoma
on the mammary gland, were chosen as models for testing the
new synthetized molecules. The ER-negative breast cancer [10] is
specifically studied due to a lack of cellular targets compared with
ER-positive breast cancer MCF-7 cell line, which can be effectively
inhibited by targeting the estrogen receptor with anti-estrogen
agents. The ER-positive breast cancer cells are described to often
develop MDR [2,11]. This makes very important the search for
potential new anticancer drugs independent from estrogens re-
ceptors to treat cancer.
We previously reported the potential use of spiroisoxazoline
oxindoles as anticancer agents [3]. In fact, the spirooxindole
* Corresponding author. Medicinal Chemistry Group, iMed.ULisboa, Faculdade de
Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
http://dx.doi.org/10.1016/j.ejmech.2014.04.023
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

Â. Monteiro et al. / European Journal of Medicinal Chemistry 79 (2014) 266e272
267
Fig. 1. Selected spirooxindoles with biological activity.
The hydrazonyl chlorides 3 required for the dipolar cycloaddi-
tion were obtained from reaction of N-chlorosuccinimide-dimethyl
sulphide complex with the appropriate N-arylhydrazones at ꢀ78 ꢁC
[15].
We started by synthesizing spiropyrazoline oxindoles 1aec,
with the nitrogen of the pyrazoline ring unprotected, by nucleo-
philic addition of hydrazine to the appropriate 3-methylene indo-
linones 2 (Scheme 2) [13].
Then, spiropyrazoline oxindoles 1des were synthesized from
the appropriate 3-methylene indolinones 2 with hydrazonyl chlo-
rides 3 in the presence of triethylamine (Scheme 3). The active
nitrile amine required for the 1,3-dipolar cycloaddition was
generated in situ by dehydrohalogenation of the corresponding
hydrazonyl chloride in the presence of base. The reaction was
Fig. 2. Chemical structure of spiroisoxazoline and spiropyrazoline oxindoles.
2. Results and discussion
regioselective, with the carbon end of the dipole adding to the b-
2.1. Chemistry
position of 3-methylene indolinones. The relative configuration of
the final products was established by comparison of their NMR
spectra with NMR spectra of other spiropyrazoline oxindoles
described in the literature, with published X-ray crystallography
structure [16]. Using compound 1n as an example, the proton
chemical shift observed for the hydrogen of the pyrazoline ring was
5.14 ppm and the carbon chemical shift observed for the spiro
carbon was 77.90 ppm.
Spiropyrazoline oxindoles 1 were synthesized by 1,3-dipolar
cycloaddition reaction, one of the most versatile methods to
obtain 5-membered heterocycles, between nitrile imines and 3-
methylene indolinones 2 (Scheme 1). The nitrile amines were
prepared in situ from hydrazonyl chlorides 3.
The 3-methylene indolinones 2 containing aromatic groups at
R2 were synthesized by aldolic condensation of substituted indolin-
2-ones with different aromatic aldehydes in the presence of
piperidine in yields between 92 and 98% [12].
2.2. In vitro cytotoxicity
Specifically, the 3-methylene indolinone required for the syn-
thesis of compounds 1aec and 1e was obtained by aldol conden-
sation between 5-chloro isatin with acetophenone in basic
medium, followed by dehydration using dilute alcoholic hydro-
chloric acid as described in the literature [13] and, the 3-methylene
indolinone required for the synthesis of compounds 1d was easily
prepared by Wittig reaction of 5-chloro isatin with (carbethoxy-
methylene)triphenylphosphorane in 92% yield [14].
The in vitro cytotoxicity of compounds 1aes was evaluated by
MTT assay in MCF-7 (human breast adenocarcinoma) tumor cell
line. The GI50 values obtained (Table 1) allows the following
observations:
1. Compounds 1aec, with an unsubstituted nitrogen and
hydrogen at R2, are inactive against MCF-7 cell line at tested
concentrations (GI50 > 100
2. Of the nineteen compounds tested, six compounds (1je1o and
1qe1r) have a GI50 below 12 M. These results show that the
mM).
m
substitution of an isoxazoline ring [3] by a pyrazoline ring leads
to an increase of activity as anticancer agents.
3. The order of inhibitory activity against MCF-7 cell line depends
in significant extent on the nature of the substituent at R2.
Compounds 1d and 1e with a CO2Et and a COPh substituent,
Scheme 1. Strategy to obtain spiropyrazoline oxindoles 1.
Scheme 2. Synthesis of spiropyrazoline oxindoles 1aec.

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Â. Monteiro et al. / European Journal of Medicinal Chemistry 79 (2014) 266e272
Scheme 3. Synthesis of spiropyrazoline oxindoles 1des.
respectively, are inactive, while compound 1g with a phenyl
substituent has an GI50 of 37.7 M. Also replacing the phenyl
group by a 4-OMe-phenyl (PMP) group leads to loss of activity
against MCF-7 tumor cells than against breast MDA-MB-231 tumor
cells. More remarkably, compounds 1j and 1n, exhibited more than
10-fold selectivity for MCF-7 cell line (Fig. 3).
m
(1h versus PMP-substituted counterpart 1s).
Finally, in order to evaluate the cytotoxicity of these compounds
on normal cells, we investigated the cytotoxicities of the most
active compounds (GI50 lower than 30 mM) on normal human HEK
293T cell line (Table 2). To our delight, we found that six spi-
ropyrazoline oxindoles (1he1j and 1ne1p) were noncytotoxic at
4. For compounds with a phenyl substituent at R2 the order of
inhibitory activity against MCF-7 cell line depends on the sub-
stituent on the aromatic ring of the oxindole and varies in the
order Br > Cl > H (compare 1j, 1g and 1f).
5. The presence of a Br on the aromatic ring of the oxindole seems
to improve the activity compared with the Cl-substituted
counterparts (e.g. 1j and 1k versus Cl-substituted counterparts
1g and 1h). The same trend was observed for compounds with a
PMP or a t-Bu substituent at R3 (spiropyrazoline oxindole 1o and
1r versus compounds 1m and 1p).
concentrations up to 100
spiropyrazoline oxindoles 1k, 1q, and 1r led to cytotoxicity at
concentrations <30 M, but even in this case, the selectivity index
mM in human HEK 293T cell line. Only,
m
was w3, which indicates low toxicity.
3. Conclusion
6. For 6-Br spiroxindoles containing phenyl groups at the nitrogen
and the R2, i.e. spiroxindoles 1k, 1o and 1r, the activity is almost
the same independently of the substituent at R3 (Ph, PMP or t-
Bu). However, for 7-Cl spiroxindoles containing phenyl groups
at the nitrogen and the R2, i.e. spiroxindoles 1i and 1q, the ac-
tivity of the t-Bu derivative (1q) is ca 2.6-fold higher than that of
its counterpart, with a phenyl group at R3 (1i).
Due to the lack of anticancer agents specific for tumours, the
development of novel scaffolds of highly selective anticancer
agents is still a very urgent topic of research. The studies herein
presented led to the discovery of novel spiropyrazoline oxindoles
active against tumor cells in vitro. Two compounds were highly
selective between two breast cancer cell lines (MCF-7 and MDA-
MB-231), and had minimal cytotoxicity in human cell line HEK
293T, thus representing useful lead compounds for the develop-
ment of more potent and selective anticancer agents. Our finding
thus adds, for the first time, the spiropyrazoline oxindole scaffold to
the list of chemotypes of anticancer agents small-molecules. The
mechanisms of cytotoxicity and lead optimization of the spiroox-
indole derivatives are currently under investigation.
The most active compounds (GI50 lower than 30 mM) were also
tested on human MDA-MB-231 tumor cell line (Table 2). Remark-
ably, spirooxindoles 1h, 1j, 1n, 1o, 1p and 1r, had higher potency
Table 1
Cytotoxicity of compounds 1aes against cancer cell line MCF-7.
4. Experimental section
4.1. Chemistry
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification.
Melting points were determined using a Kofler camera Bock
monoscope M and are uncorrected.
The infrared spectra were collected on a Shimadzu FTIR Affinity-
1 spectrophotometer.
Elemental analysis was performed in a Flash 2000 CHNSeO
analyzer (ThermoScientific, UK), and results were within ꢂ0.4% of
the theoretical values.
Compounds
R1
R2
R3
R4
MCF-7
GI50, mM
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
H
H
H
H
CO2Et
COPh
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
>100
>100
>100
>100
>100
>100
37.7 ꢂ 14.1
27.1 ꢂ 8.2
22.4 ꢂ 3.5
7.3 ꢂ 1.4
9.6 ꢂ 1.0
33.9 ꢂ 15.4
42.6 ꢂ 18.8
11.5 ꢂ 1.6
7.0 ꢂ 1.1
21.7 ꢂ 3.9
8.6 ꢂ 3.0
8.5 ꢂ 1.8
>100
5-Cl
5-Br
5-Cl
5-Cl
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Merck Silica Gel 60 F254 plates were used for analytical TLC;
flash column chromatography was performed on Merck Silica Gel
(200e400 mesh) and CombiFlash Rf from Teledyne ISCO (columns
RediSep Rf, silica).
5-Cl
6-Cl
7-Cl
5-Br
6-Br
5-Cl
6-Cl
7-Cl
6-Br
6-Cl
7-Cl
6-Br
6-Cl
Ph
Ph
Ph
1H and 13C NMR spectra were recorded on a Bruker 400 Ultra-
PMP
PMP
PMP
PMP
t-Bu
t-Bu
t-Bu
Ph
Shield instrument (400 MHz). 1H and 13C chemical shifts are
expressed in d (ppm) referenced to the solvent used and the proton
coupling constants (J) are given in hertz.
Ph
Ph
PMP
4.1.1. General preparation of spiropyrazoline oxindoles 1aec
A
mixture of the appropriate 3-methylene indolinone
(0.35 mmol, 1 eq.) and hydrazine hydrate (0.42 mmol, 1.2 eq.) in

Â. Monteiro et al. / European Journal of Medicinal Chemistry 79 (2014) 266e272
269
Table 2
4.1.2. General preparation of spiropyrazoline oxindoles 1des
Cytotoxicity of compounds 1hek and 1ner against cancer cell lines MCF-7 and
The appropriate 3-methylene indolinone (0.3 mmol, 1 eq.) was
dissolved in dry dichloromethane (2 ml) and the appropriate
hydrazonyl chloride (0.6 mmol, 2 eq.) was added. Then, triethyl-
amine (0.9 mmol, 3 eq.) was added and the reaction was stirred at
room temperature for 3 h. The reaction was then portioned with
7.5 ml of water, the phases were separated and the aqueous phase
was extracted twice with 10 ml of ethyl acetate. All organic extracts
were combined, dried and concentrated to afford the crude product
which was purified by flash column chromatography using ethyl
acetate and n-hexane as eluent.
MDA-MB-231 and non cancer cell line Hek 293T.
Compounds
MCF-7
GI50
MDA-MB-231
GI50
>50b
Hek 293T
GI50
>50b
>100
>100
SIa
,
mM
,
m
M
,
m
M
1h
1i
1j
1k
1n
1o
1p
1q
1r
27.1 ꢂ 8.2
22.4 ꢂ 3.5
7.3 ꢂ 1.4
9.6 ꢂ 1.0
11.5 ꢂ 1.6
7.0 ꢂ 1.1
21.7 ꢂ 3.9
8.6 ꢂ 3.0
8.5 ꢂ 1.8
>2
16.9 ꢂ 1.2
>4
>13
3
>8
>14
>2
2
>100
8.3 ꢂ 1.2
>100
28.9 ꢂ 1.2
>100
28.6 ꢂ 1.1
>100
>50b
>50b
6.4 ꢂ 1.2
15.0 ꢂ 1.2
17.8 ꢂ 1.2
20.9 ꢂ 1.2
2.5
4.1.2.1. Compound 1d: purified by flash column chromatography us-
ing ethyl acetate/n-hexane 1:1 and then, recrystallized from
dichloromethane. Obtained as a light yellow solid. Yield 85% (0.11 g).
IR (KBr): 3232,1736,1724,1618,1597,1495 cmꢀ1. 1H NMR (400 MHz,
a
Selectivity index toward MCF-7, which is expressed by the ratio GI50 Hek/GI50
MCF-7.
b
Solubility issues at concentrations of 100 mM.
CDCl3)
d
(ppm): 9.08 (s, 1H), 7.69 (d, J ¼ 6.8 Hz, 2H), 7.46e7.31 (m,
ethanol (2.5 ml) was refluxed for 1 h. The mixture was recrystal-
lized from i-PrOH [13].
3H), 7.21 (d, J ¼ 7.1 Hz, 2H), 7.13 (t, J ¼ 7.8 Hz, 2H), 6.92e6.75 (m, 4H),
4.93 (s, 1H), 3.90 (q, J ¼ 7.1 Hz, 2H), 0.94 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) d 177.24 (C]O), 166.63 (C]O), 144.17 (Cq), 142.89
(Cq), 138.94 (Cq), 131.27 (Cq), 130.60 (CH), 129.20 (CH), 129.16 (CH),
128.76 (Cq), 128.67 (CH), 126.72 (Cq), 126.45 (CH), 125.98 (CH),
121.40 (CH), 114.78 (CH), 112.07 (CH), 74.03 (Cq), 62.90 (CH), 62.04
(CH2), 13.65 (CH3). Anal. Calcd for C25H20ClN3O3: C 67.34, H 4.52, N
9.42, Found: C 67.03, H 4.53, N 9.31.
4.1.1.1. Compound 1a: obtained as a light yellow solid. Yield 40%
(42 mg). IR (KBr): 3296, 3186, 1705, 1618 cmꢀ1. 1H NMR (400 MHz,
CDCl3)
d
(ppm): 8.78 (s, 1H), 7.69 (d, J ¼ 7.3 Hz, 2H), 7.40 (m, 3H),
7.32 (d, J ¼ 7.3 Hz, 1H), 7.25 (m, 2H), 7.02 (t, J ¼ 7.5 Hz, 1H), 6.93 (d,
J ¼ 7.6 Hz, 1H), 3.72 (d, J ¼ 16.7 Hz, 1H), 3.44 (d, J ¼ 16.6 Hz, 1H).
Anal. Calcd for C16H13N3O.0.2H2O: C 72.00, H 5.07, N 15.75, Found: C
71.74, H 4.75, N 15.38.
4.1.2.2. Compound 1e: purified by flash column chromatography
using ethyl acetate/n-hexane 1:2 and then, recrystallized from
dichloromethane. Obtained as a light yellow solid. Yield 76% (0.10 g).
4.1.1.2. Compound 1b: obtained as a light orange solid. Yield 35%
(37 mg). IR (KBr): 3290, 3167, 1707, 1622 cmꢀ1. 1H NMR (400 MHz,
1H NMR (400 MHz, CDCl3)
d (ppm): 8.36 (s, 1H), 7.69e7.57 (m, 4H),
DMSO-d6)
d
(ppm): 10.52 (s, 1H), 7.96 (s, 1H), 7.64 (d, J ¼ 7.2 Hz, 2H),
7.48 (t, J ¼ 7.4 Hz, 1H), 7.40e7.28 (m, 5H), 7.12 (t, J ¼ 7.9 Hz, 2H), 7.03
(d, J ¼ 1.6 Hz, 1H), 6.97 (dd, J ¼ 8.3, 1.8 Hz, 1H), 6.93e6.79 (m, 3H),
6.51 (d, J ¼ 8.3 Hz, 1H), 5.94 (s, 1H). Anal. Calcd for C29H20ClN3O2: C
72.88, H 4.22, N 8.79, Found: C 73.01, H 4.34, N 8.78.
7.45e7.32 (m, 3H), 7.29 (d, J ¼ 6.1 Hz, 2H), 6.86 (d, J ¼ 8.9 Hz, 1H),
3.43 (s, 2H). Anal. Calcd for C16H12ClN3O.0.4H2O: C 63.15, H 4.25, N
13.81, Found: C 62.77, H 4.00, N 13.39.
4.1.1.3. Compound 1c: obtained as a light orange solid. Yield 37%
4.1.2.3. Compound 1f: purified by flash column chromatography us-
ing ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a light yellow solid. Yield 78% (0.11 g). IR (KBr):
(44 mg). IR (KBr): 3291, 3165, 1707, 1618 cmꢀ1. 1H NMR (400 MHz,
DMSO-d6)
7.49e7.31 (m, 5H), 6.81 (d, J ¼ 8.1 Hz,1H), 3.43 (s, 2H). Anal. Calcd for
16H12BrN3O: C 56.16, H 3.53, N 12.28, Found: C 56.54, H 3.68, N 12.05.
d
(ppm): 10.55 (s,1H), 7.95 (m, 1H), 7.64 (d, J ¼ 7.4 Hz, 2H),
3198, 1719, 1618, 1597, 1495 cmꢀ1 1H NMR (400 MHz, CDCl3)
.
C
d (ppm): 8.97 (s, 1H), 7.75e7.62 (m, 2H), 7.37e7.21 (m, 3H), 7.21e
Fig. 3. Effects of compounds 1j (A), and 1n (B) on MCF-7 (1) and MDA-MB-231 (2) cell viability using the MTT assay for 48 h of exposition. Results are given in % relative to control
and represent means ꢂ SD of three independent experiments performed in duplicate and carried out independently (* indicates P < 0.05 antiproliferative effect significantly higher
in MCF-7 than in MDA-MB-231).

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Â. Monteiro et al. / European Journal of Medicinal Chemistry 79 (2014) 266e272
6.88 (m, 10H), 6.87e6.69 (m, 2H), 6.55 (t, J ¼ 7.6 Hz, 1H), 6.35 (d,
4.1.2.8. Compound 1k: purified by flash column chromatography
using ethyl acetate/n-hexane 1:3 and then, recrystallized from
dichloromethane. Obtained as a white solid. Yield 85% (0.11 g). IR
(KBr): 3172, 1720, 1609, 1597, 1493 cmꢀ1. 1H NMR (400 MHz, DMSO-
J ¼ 7.5 Hz, 1H), 5.16 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 178.39 (C]
O), 148.93 (Cq), 144.33 (Cq), 140.24 (Cq), 134.78 (Cq), 131.81 (Cq),
129.48 (CH), 129.29 (CH), 129.03 (CH), 128.76 (CH), 128.66 (CH),
128.47 (CH), 128.02 (CH), 126.95 (CH), 126.54 (CH), 125.65 (Cq),
122.40 (CH), 121.08 (CH), 115.43 (CH), 110.62 (CH), 77.47 (Cq), 62.67
(CH). Anal. Calcd for C28H21N3O: C 80.94, H 5.09, N 10.11, Found: C
80,88, H 5.34, N 9.95.
d6)
d
(ppm): 11.01 (s, 1H), 7.66 (d, J ¼ 7.5 Hz, 2H), 7.36e7.31 (m, 3H),
7.20 (m, 3H), 7.14 (t, J ¼ 7.9 Hz, 2H), 7.01e7.02 (m, 3H), 6.82e6.78
(m, 3H), 6.73 (d, J ¼ 8.1 Hz, 1H), 6.16 (d, J ¼ 8.1 Hz, 1H), 5.43 (s, 1H).
13C NMR (100 MHz, DMSO-d6)
d 176.49 (C]O), 149.62 (Cq), 143.99
(Cq), 143.17 (Cq), 134.68 (Cq), 131.17 (Cq), 129.07 (CH), 129.02 (CH),
128.65 (CH), 128.02 (CH), 127.39 (CH), 126.59 (CH), 124.45 (Cq),
124.08 (CH), 122.37 (Cq), 120.72 (CH), 114.51 (CH), 113.24 (CH),
75.65 (Cq), 60.73 (CH). Anal. Calcd for C28H20BrN3O.0.05H2O: C
67.90, H 4.10, N 8.49, Found: C 67.61, H 4.13, N 8.25.
4.1.2.4. Compound 1g: purified by flash column chromatography
using ethyl acetate/n-hexane 1:4 and then, recrystallized from
dichloromethane. Obtained as a light yellow solid. Yield 82% (0.11 g).
IR (KBr): 3171, 1730, 1595, 1493 cmꢀ1. 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 7.68 (d, J ¼ 6.7 Hz, 2H), 7.42e7.27 (m, 3H), 7.28e7.08 (m,
4.1.2.9. Compound 1l: purified by flash column chromatography us-
ing ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 33% (0.05 g). IR (KBr): 3117,
6H), 7.02 (d, J ¼ 6.5 Hz, 2H), 6.89 (d, J ¼ 8.3 Hz, 1H), 6.83e6.79 (m,
3H), 6.14 (d, J ¼ 1.8 Hz, 1H), 5.45 (s, 1H). 13C NMR (100 MHz, DMSO-
d6)
d 176.32 (C]O), 149.54 (Cq), 143.94 (Cq), 140.42 (Cq), 134.62
1740, 1711, 1599, 1499 cmꢀ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
(Cq), 131.15 (Cq), 129.46 (CH), 129.09 (CH), 129.05 (CH), 128.65 (CH),
128.03 (CH), 127.10 (Cq), 126.61 (CH), 125.60 (CH), 125.55 (Cq),
120.70 (CH), 114.39 (CH), 111.74 (CH), 75.84 (Cq), 60.81 (CH). Anal.
Calcd for C28H20ClN3O.0.5CH2Cl2: C 69.63, H 4.31, N 8.55, Found: C
69.19, H 4.30, N 8.55.
10.97 (s, 1H), 7.61 (d, J ¼ 8.5 Hz, 2H), 7.27e7.08 (m, 6H), 7.00 (d,
J ¼ 6.7 Hz, 2H), 6.89 (dd, J ¼ 18.2, 8.4 Hz, 3H), 6.78 (dd, J ¼ 12.6,
7.8 Hz, 3H), 6.12 (s, 1H), 5.41 (s, 1H), 3.74 (s, 3H). 13C NMR (100 MHz,
DMSO-d6)
d 176.36 (C]O), 159.90 (Cq), 149.44 (Cq), 144.20 (Cq),
140.31 (Cq), 134.71 (Cq), 129.30 (Cq), 128.95 (CH), 128.50 (CH),
128.14 (CH), 127.88 (CH), 127.26 (CH), 125.52 (Cq), 125.46 (CH),
123.65 (Cq),120.31 (CH),114.23 (CH),114.08 (CH),111.59 (CH), 75.66
(Cq), 61.02 (CH), 55.20 (OCH3). Anal. Calcd for C29H22ClN3O2.1.1H2O:
C 69.80, H 4.90, N 8.42, Found: C 69.42, H 4.91, N 8.55.
4.1.2.5. Compound 1h: purified by flash column chromatography
using ethyl acetate/n-hexane 1:4 and then, recrystallized from
dichloromethane. Obtained as a light yellow solid. Yield 83% (0.11 g).
IR (KBr): 3171, 1722, 1611, 1597, 1495 cmꢀ1 1H NMR (400 MHz,
.
CDCl3)
d
(ppm): 8.95 (s, 1H), 7.68 (d, J ¼ 5.4 Hz, 2H), 7.28 (m, 3H),
4.1.2.10. Compound 1m: purified by flash column chromatography
using ethyl acetate/n-hexane 1:4 and then, recrystallized from
dichloromethane. Obtained as a light yellow solid. Yield 32% (0.04 g).
7.13 (m, 5H), 6.94 (m, 4H), 6.85 (t, J ¼ 7.0 Hz, 1H), 6.78 (s, 1H), 6.55
(d, J ¼ 8.1 Hz, 1H), 6.23 (d, J ¼ 8.1 Hz, 1H), 5.11 (s, 1H). 13C NMR
(100 MHz, CDCl3)
d 178.62 (C]O), 149.19 (Cq), 144.22 (Cq), 141.12
IR (KBr): 3210, 1714, 1701, 1597, 1497 cmꢀ1 1H NMR (400 MHz,
.
(Cq), 135.39 (Cq), 134.44 (Cq), 131.56 (Cq), 129.18 (CH), 128.99 (CH),
128.93 (CH), 128.57 (CH), 128.35 (CH), 127.39 (CH), 126.98 (CH),
124.09 (Cq),122.65 (CH), 121.48 (CH),115.52 (CH), 111.48 (CH), 76.84
(Cq), 62.60 (CH). Anal. Calcd for C28H20ClN3O.0.15H2O: C 74.29, H
4.53, N 9.29, Found: C 73.96, H 4.33, N 9.08.
DMSO-d6)
d
(ppm): 10.99 (s, 1H), 7.59 (d, J ¼ 8.6 Hz, 2H), 7.22e7.17
(m, 3H), 7.12 (t, J ¼ 7.9 Hz, 2H), 7.00 (d, J ¼ 6.8 Hz, 2H), 6.94e6.86 (m,
3H), 6.82e6.73 (m, 3H), 6.58 (d, J ¼ 8.1 Hz, 1H), 6.20 (d, J ¼ 8.1 Hz,
1H), 5.38 (s, 1H), 3.73 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d
176.75 (C]O), 159.94 (Cq), 149.58 (Cq), 144.31 (Cq), 143.00 (Cq),
134.87 (Cq), 133.82 (Cq), 129.03 (CH), 128.63 (CH), 128.21 (CH),
127.96 (CH), 127.05 (CH), 124.24 (Cq), 123.73 (Cq), 121.17 (CH),
120.39 (CH), 114.37 (CH), 114.15 (CH), 110.45 (CH), 75.44 (Cq), 61.01
(CH), 55.27 (OCH3). Anal. Calcd for C29H22ClN3O2.1.1H2O: C 69.80, H
4.90, N 8.42, Found: C 69.42, H 4.91, N 8.55.
4.1.2.6. Compound 1i: purified by flash column chromatography us-
ing ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 87% (0.12 g). 1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 11.31 (s, 1H), 7.67 (d, J ¼ 6.6 Hz, 2H),
7.36e7.30 (m, 3H), 7.21e7.12 (m, 6H), 7.02 (d, J ¼ 7.5 Hz, 2H), 6.83e
6.78 (m, 3H), 6.56 (t, J ¼ 7.9 Hz, 1H), 6.21 (d, J ¼ 7.5 Hz, 1H), 5.47 (s,
4.1.2.11. Compound 1n: purified by flash column chromatography
using ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 75% (0.10 g). IR (KBr): 3204,
1H). 13C NMR (100 MHz, DMSO-d6)
d 176.70 (C]O), 149.63 (Cq),
143.96 (Cq), 139.20 (Cq), 134.58 (Cq), 131.17 (Cq), 129.61 (CH),
129.08 (CH), 128.65 (CH), 128.60 (CH), 127.99 (CH), 127.01 (Cq),
126.61 (CH), 124.35 (CH), 122.63 (CH), 120.77 (CH), 114.54 (CH),
114.38 (Cq), 76.48 (Cq), 61.04 (CH). Anal. Calcd for
1732, 1608, 1595, 1497 cmꢀ1 1H NMR (400 MHz, CDCl3)
. d (ppm):
7.62 (s, 1H), 7.59 (d, J ¼ 8.8 Hz, 2H), 7.21e7.05 (m, 6H), 6.98 (s, 2H),
6.91 (d, J ¼ 7.9 Hz, 2H), 6.83 (m, 3H), 6.56 (t, J ¼ 7.9 Hz, 1H), 6.30 (d,
J ¼ 7.5 Hz, 1H), 5.14 (s, 1H), 3.78 (s, 3H). 13C NMR (100 MHz, CDCl3)
C
28H20ClN3O$0.15H2O: C 74.29, H 4.53, N 9.29, Found: C 74.07, H
4.69, N 8.89.
d
176.78 (C]O), 160.30 (Cq), 149.06 (Cq), 144.49 (Cq), 137.57 (Cq),
134.51 (Cq), 129.35 (CH), 129.29 (CH), 129.13 (CH), 128.80 (CH),
128.51 (CH), 128.24 (CH), 127.51 (Cq), 124.99 (CH), 124.23 (Cq),
123.30 (CH), 121.39 (CH), 115.70 (CH), 115.22 (Cq),114.00 (CH), 77.90
(Cq), 63.22 (CH), 55.39 (CH3). Anal. Calcd for C29H22ClN3O2: C 72.57,
H 4.63, N 8.76, Found: C 72.34, H 4.56, N 8.66.
4.1.2.7. Compound 1j: purified by flash column chromatography us-
ing ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 82% (0.10 g). IR (KBr): 3180,
1718, 1618, 1593, 1493 cmꢀ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
11.01 (s, 1H), 7.69 (d, J ¼ 7.6 Hz, 2H), 7.37e7.30 (m, 4H), 7.22 (m, 3H),
7.14 (t, J ¼ 7.9 Hz, 2H), 7.01 (d, J ¼ 7.4 Hz, 2H), 6.85e6.79 (m, 4H),
4.1.2.12. Compound 1o: purified by flash column chromatography
using ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 91% (0.12 g). IR (KBr): 3220,
6.24 (s,1H), 5.45 (s, 1H). 13C NMR (100 MHz, DMSO-d6)
d 176.15 (C]
O), 149.57 (Cq), 143.95 (Cq), 140.76 (Cq), 134.62 (Cq), 132.27 (CH),
131.14 (Cq), 129.10 (CH), 129.06 (CH), 128.66 (CH), 128.61 (CH),
128.36 (CH), 128.04 (CH), 127.38 (Cq), 126.61 (CH), 120.71 (CH),
114.38 (CH), 113.25 (CH), 112.20 (Cq), 75.84 (Cq), 60.78 (CH). Anal.
Calcd for C28H20BrN3O2.0.55H2O: C 66.68, H 4.23, N 8.33, Found: C
66.29, H 4.09, N 8.18.
1747, 1711, 1608, 1597, 1496 cmꢀ1 1H NMR (400 MHz, CDCl3)
.
d
(ppm): 8.76 (s, 1H), 7.61 (d, J ¼ 8.5 Hz, 2H), 7.22e7.06 (m, 5H), 6.92
(m, 5H), 6.83 (m, 8.2 Hz, 3H), 6.71 (d, J ¼ 8.1 Hz, 1H), 6.17 (d,
J ¼ 8.1 Hz, 1H), 5.07 (s, 1H), 3.78 (s, 3H). 13C NMR (100 MHz, CDCl3)
d
178.46 (C]O), 160.33 (Cq), 149.20 (Cq), 144.54 (Cq), 141.24 (Cq),

Â. Monteiro et al. / European Journal of Medicinal Chemistry 79 (2014) 266e272
271
134.54 (Cq), 129.23 (CH), 129.15 (CH), 128.92 (CH), 128.52 (CH),
128.32 (CH), 127.74 (CH), 125.54 (CH), 124.86 (Cq), 124.25 (Cq),
123.28 (Cq), 121.31 (CH), 115.51 (CH), 114.12 (CH), 114.04 (CH), 77.37
(Cq), 62.75 (CH), 55.41 (CH3). Anal. Calcd for C29H22BrN3O2.0.2H2O:
C 65.96, H 4.28, N 7.96, Found: C 65.60, H 4.39, N 7.70.
soluble tetrazolium dye that is converted by mitochondrial de-
hydrogenases in viable cells to a water-insoluble, purple formazan
[17]. The day before experiments cells obtained from the American
Type Culture Collection HEK 293T e human embryonic kidney
epithelial cell line (ATCC CRL-11268), two breast cancer cell lines
MCF-7 (ATCC HTB-22Ô) (estrogen receptor positive (ERþ) and
human epidermal growth factor receptor 2 negative (HER2ꢀ)) and
MDA-MB-231 (ATCC HTB-26Ô) (ERꢀ, and HER2ꢀ) were seeded at
4.1.2.13. Compound 1p: purified by flash column chromatography
using ethyl acetate/n-hexane 1:3 and then, recrystallized from
dichloromethane. Obtained as a white solid. Yield 80% (0.10 g). IR
(KBr): 3213, 1724, 1608, 1597, 1491 cmꢀ1. 1H NMR (400 MHz, CDCl3)
2 ꢃ 104 cells per well in 96 well tissue culture plates, in 100
ml of
RPMI 1640 culture medium supplemented with 10% fetal bovine
d
(ppm): 8.37 (s, 1H), 7.50e7.02 (m, 6H), 6.81 (m, 3H), 6.74 (m, 1H),
6.66 (s, 1H), 6.55 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.12 (d, J ¼ 8.2 Hz, 1H), 4.43
(s, 1H), 1.19 (s, 9H). 13C NMR (100 MHz, CDCl3)
177.91 (C]O),
serum, 100 units of penicillin G (sodium salt), 100
mg of strepto-
mycin sulfate and 2 mM -glutamine, at a concentration that allows
L
d
cells to grow exponentially during the assay. Moreover, the cell
lines have the doubling time of 24e30 h for Hek 293T, 29 h for MCF-
7, and 38 h for MDA-MB-231, during the 48 h of the assay all cell
lines duplicate.
161.88 (Cq), 145.72 (Cq), 141.38 (Cq), 135.18 (Cq), 134.99 (Cq), 128.99
(CH), 128.37 (CH), 127.39 (CH), 124.32 (Cq), 122.43 (CH), 121.41 (CH),
116.29 (CH), 111.05 (CH), 77.05 (Cq), 62.36 (CH), 34.91 (C(CH3)3),
29.49 (C(CH3)3). Anal. Calcd for C26H24ClN3O.0.5CH2Cl2: C 67.37, H
5.34, N 8.90, Found: C 67.14, H 5.39, N 8.84.
Tested compounds were dissolved in dimethyl sulfoxide
(DMSO) serially diluted in the culture medium and added to the
cells. The final concentration of DMSO in culture medium during
treatment did not exceed 0.5% (v/v), and the same concentration of
DMSO was added to the control. Each compound concentration was
tested in triplicate in a single experiment which was repeated at
least 3 times, controls contained equivalent concentrations of
DMSO. Cells were incubated at 37 ꢁC in humidified 5% CO2 atmo-
sphere. After 48 h, cell media was removed and replaced with fresh
medium, the MTT dye solution was added to each well (5 mg/mL in
10 mM phosphate buffer solution at pH 7.4), and after 3 h of in-
cubations the media was removed and intracellular formazan
crystals were solubilized and extracted with DMSO. After 15 min at
room temperature absorbance was measured at 570 nm in a
microplate reader (FLUOstar Omega, BMG Labtech, Germany), and
the percentage of viable cells was determined for each compound
concentration as described previously [18].
4.1.2.14. Compound 1q: purified by flash column chromatography
using ethyl acetate/n-hexane 1:3 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 87% (0.11 g). IR (KBr): 3177,
1752, 1712, 1618, 1598, 1498 cmꢀ1 1H NMR (400 MHz, CDCl3)
.
d
(ppm): 87.89 (s, 1H), 7.36 (s, 2H), 7.25 (m, 1H), 7.09 (m, 4H), 6.88e
6.78 (m, 3H), 6.68 (s, 1H), 6.53 (t, J ¼ 7.9 Hz, 1H), 6.15 (d, J ¼ 7.6 Hz,
1H), 4.49 (s, 1H), 1.19 (s, 9H). 13C NMR (100 MHz, CDCl3)
176.50
d
(C]O), 161.89 (Cq), 145.63 (Cq), 137.96 (Cq), 134.94 (Cq), 129.24
(CH), 129.01 (CH), 128.34 (CH), 127.46 (Cq), 124.85 (CH), 123.06
(CH), 121.55 (CH), 116.54 (CH), 115.12 (Cq), 78.17 (Cq), 62.59 (CH),
34.90
26H24ClN3O.0.4H2O: C 71.43, H 5.73, N 9.61, Found: C 71.11, H 5.70,
N 9.30.
(C(CH3)3),
29.48
(C(CH3)3).
Anal.
Calcd
for
C
4.1.2.15. Compound 1r: purified by flash column chromatography
using ethyl acetate/n-hexane 1:4 and then, recrystallized from diethyl
ether. Obtained as a white solid. Yield 92% (0.11 g). IR (KBr): 3218,
GI50s were determined by non-linear regression using GraphPad
PRISM software as previously published.
Statistical analysis of the experimental data was performed by
applying one-way ANOVA tests using the software package
GraphPad PRISM. Differences were considered to be significant
when P < 0.05.
1723, 1610, 1598, 1490 cmꢀ1 1H NMR (400 MHz, CDCl3)
. d (ppm):
8.48 (s, 1H), 7.34 (s, 2H), 7.26 (m, 1H), 7.15 (s, 1H), 7.07 (t, J ¼ 7.9 Hz,
2H), 6.90 (s, 1H), 6.81 (m, 3H), 6.73e6.58 (m, 2H), 6.05 (d, J ¼ 8.1 Hz,
1H), 4.43 (s, 1H), 1.18 (s, 9H). 13C NMR (100 MHz, CDCl3)
d 177.88
(C]O), 161.89 (Cq), 145.70 (Cq), 141.50 (Cq), 134.93 (Cq), 129.01
(CH), 128.38 (CH), 127.65 (CH), 125.35 (CH), 124.86 (Cq), 123.15 (Cq),
121.39 (CH), 116.21 (CH), 113.87 (CH), 77.09 (Cq), 62.29 (CH), 34.90
(C(CH3)3), 29.49 (C(CH3)3). Anal. Calcd for C26H24BrN3O.0.05H2O: C
65.70, H 5.12, N 8.84, Found: C 65.32, H 5.02, N 8.64.
Acknowledgments
This study was supported by FCT (Fundação para a Ciência e a
Tecnologia, Portugal) by research projects PTDC/QUI-QUI/111664/
2009, PTDC/SAU-FAR/110848/2009, and PEst-OE/SAU/UI4013/2014.
4.1.2.16. Compound 1s: purified by flash column chromatography
using ethyl acetate/n-hexane 1:3 and then, recrystallized from
dichloromethane. Obtained as a light yellow solid. Yield 78% (0.10 g).
Appendix A. Supplementary data
IR (KBr): 3171, 1720, 1611, 1597, 1491 cmꢀ1 1H NMR (400 MHz,
.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.023.
CDCl3)
d
(ppm): 8.90 (s, 1H), 7.68 (m, J ¼ 7.0, 1.9 Hz, 2H), 7.34e7.26
(m, 3H), 7.11 (t, J ¼ 7.9 Hz, 2H), 6.97e6.80 (m, 5H), 6.78 (d, J ¼ 1.3 Hz,
1H), 6.69 (d, J ¼ 8.6 Hz, 2H), 6.60 (dd, J ¼ 8.1, 1.5 Hz, 1H), 6.29 (d,
J ¼ 8.1 Hz, 1H), 5.06 (s, 1H), 3.73 (s, 3H). 13C NMR (100 MHz, CDCl3)
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178.59 (C]O), 159.42 (Cq), 149.49 (Cq), 144.32 (Cq), 141.15 (Cq),
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