A new and potent class of quinoline derivatives against cancer

Article abstract of DOI:10.1007/s00706-015-1570-0

A new class of 4-quinolinylhydrazone derivatives has been synthesized and evaluated for their cytotoxic potential against three cancer cell lines using the MTT assay. Compounds displaying more than 90 % of growth inhibition were evaluated for in vitro anticancer activities against four human cancer cell lines. The results were expressed as the concentrations that induce 50 % inhibition of cell growth (IC₅₀) in μg/cm³. These compounds exhibited good cytotoxic activity against at least three cancer cell lines, with IC₅₀ values between 0.314 and 4.65 μg/cm³. These derivatives are useful starting points for further study for new anticancer drugs and confirm the potential of quinoline derivatives as lead compounds in anticancer drug discovery. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-015-1570-0

Monatsh Chem
DOI 10.1007/s00706-015-1570-0
ORIGINAL PAPER
A new and potent class of quinoline derivatives against cancer
Marcelle de L. F. Bispo1,2 Camila C. de Alcantara3 Manoel O. de Moraes4
•
•
•
Claudia do O Pessoa Felipe A. R. Rodrigues4 Carlos R. Kaiser2
Solange M. S. V. Wardell5 James L. Wardell1,6 Marcus V. N. de Souza1,3
4
´
•
•
•
´
•
•
Received: 12 July 2015 / Accepted: 3 September 2015
Ó Springer-Verlag Wien 2015
Abstract A new class of 4-quinolinylhydrazone deriva-
tives has been synthesized and evaluated for their cytotoxic
potential against three cancer cell lines using the MTT
assay. Compounds displaying more than 90 % of growth
inhibition were evaluated for in vitro anticancer activities
against four human cancer cell lines. The results were
expressed as the concentrations that induce 50 % inhibition
of cell growth (IC50) in lg/cm3. These compounds exhib-
ited good cytotoxic activity against at least three cancer
cell lines, with IC50 values between 0.314 and 4.65 lg/
cm3. These derivatives are useful starting points for further
study for new anticancer drugs and confirm the potential of
quinoline derivatives as lead compounds in anticancer drug
discovery.
Graphical Abstract
Keywords Drug research Á Antitumor agents Á
Heterocycles Á Hydrazones Á Quinoline compounds
Introduction
Cancer is a growing public health problem that in 2012 was
responsible for 8.2 million of deaths worldwide [1]. This
disease accounted for nearly one-quarter of all deaths in the
United States in 2012, exceeded only by heart diseases [2].
After 70 years of rapid advances in the identification
and development of curative treatment for many malignant
processes, the introduction of potential drugs, such as
methotrexate and azathioprine, helped in the treatment of
several tumors that were previously untreatable or only
accessible through surgery and/or radiation [3]. Despite the
introduction of new anticancer drugs in the market and new
therapeutically approaches, nearly 10 million new cases
occur each year worldwide [4]. The main disadvantage of
conventional drugs is their nonspecific cytotoxicity to
tumor cells, which lead to a narrow therapeutic window
and low therapeutic index [5]. Furthermore, resistance to
drugs such as imatinib and trastuzumab, used to target
specific tyrosine kinases, is now evident [6, 7].
& Marcus V. N. de Souza
Marcos_souza@far.fiocruz.br
1
´
´
Centro de Desenvolvimento Tecnologico em Saude (CDTS),
Fundaca˜o Oswaldo Cruz (FIOCRUZ), Casa Amarela,
Campus de Manguinhos, Av. Brasil 4365, Rio de Janeiro, RJ
21040-900, Brazil
2
3
´
Departamento de Quımica Organica, Universidade Federal
do Rio de Janeiro, Instituto de Quımica, CP 68563,
21945-970 Rio de Janeiro, RJ, Brazil
ˆ
´
Fundac¸a˜o Oswaldo Cruz, Instituto de Tecnologia em
´
Farmacos-Far Manguinhos, 21041-250 Rio de Janeiro, RJ,
Brazil
4
5
6
´
Laboratorio de Oncologia Experimental, Universidade
Federal do Ceara, Fortaleza, CE, Brazil
´
CHEMSOL, 1 Harcourt Road, Aberdeen AB15 5NY,
Scotland, UK
Department of Chemistry, University of Aberdeen,
Old Aberdeen AB24 3UE, Scotland, UK
123

M. L. F. Bispo et al.
Due to the high impact of this disease and the elevated
cytotoxicity profiles of anticancer drugs, we urgently need
new drugs and strategies to treat efficiently this disease.
Moreover, new drugs must be less cytotoxic, safer, and
more effective for the patient.
Results and discussion
Synthesis and characterization
The preparation of series B (4-quinolinylhydrazones
derivatives 6a–6i) is shown in Scheme 1. First, 7-chloro-4-
methoxyquinoline (2) was prepared from 4,7-dichloro-
quinoline on reaction with sodium methoxide (1 M) at
50 °C, and was subsequently dehalogenated to
4-methoxyquinoline (3) using hydrogen and Pd/C. 4-Hy-
drazinylquinoline (4) was prepared from 3 using hydrazine
hydrate (80 %) in ethanol under reflux. Finally, the
hydrazones 5a–5i (Table 2) were obtained through reaction
between 4-hydrazinoquinoline and appropriate benzalde-
hydes. Identification of the isolated series B compounds as
hydrochloride salts, with protonated quinolinyl nitrogen
atoms and with (E) geometries at the C=N center, was
achieved by spectroscopic data in all cases, and specifically
by X-ray crystallography for two hydrated salts, namely
[(5: R1=Cl; R2=R3=R4=R5=H)Á2H2O] [10] and [(5:
R1=R3=Cl; R2=R4=R5=H)ÁH2O] [11], see later. Generally,
In our studies on the anticancer activities of heteroaro-
matic compounds, we identified a series of 7-chloro-4-
quinolinylhydrazone derivatives (series A, 1a-1i) with
promising antitumoral activities (Table 1) [8, 9]. Following
on from this earlier work, we have studied the anticancer
activities of two new series of 4-quinolinylhydrazones,
namely series B (5a–5i) and series C (6a–6i), see Fig. 1.
These series B and C were synthesized to evaluate the
importance of a 7-chloro substituent in the quinoline
moiety and the effect of methylation on the activities of
quinolinylhydrazone derivatives. Furthermore, the selec-
tion of the phenyl substituents in the series B and
C compounds was based on the best biological results
obtained with series A compounds (Fig. 1). Our results
have enabled us to construct important structure–activity
relationships.
Table 1 Selected IC50 results for derivatives 1a–1i of the series A against cancer cell lines
R4
R3
R5
N
R2
HN
N
R1
-
1a 1i
Cl
A
Series
Compound series A
Substituents
Best IC50/lg cm-3
Cancer cell line
1a
1b
1c
1d
1e
1f
R1=R2=R3=R4=R5=H
2.085
3.206
1.238
1.588
0.8018
1.086
0.6888
2.837
0.7483
SF-295a
HTC-8b
SF-295
HL-60c
HTC-8
HTC-8
SF-295
HL-60
R2=F; R1=R3=R4=R5=H
R2=Cl; R1=R3=R4=R5=H
R2=Br; R1=R3=R4=R5=H
R2=OMe; R1=R3=R4=R5=H
R1=R5=Cl; R2=R3=R4=H
R2=R3=OMe; R1=R4=R5=H
R1=R4=OMe; R2=R3=R5=H
R2=R3=R4=OMe; R1=R5=H
1g
1h
1i
HTC-8
a
SF-295 (nervous system)
b
HTC-8 (colon)
c
HL-60 (leukemia)
123

A new and potent class of quinoline derivatives against cancer
Fig. 1 Design concept for new series B (5a–5i) and series C (6a–6i) of 4-quinolinylhydrazones
in the 1H NMR spectra of 5a–5i, the chemical shifts of the
N = CH protons are found in the range 8.37–8.81 ppm,
X-ray structural studies
The structure of 6e, an active compound, was confirmed in
while in the IR spectra, the N–H and N=C stretching
vibrations occur in the ranges 3197–3247 and
1570–1585 cm-1, respectively.
this study by X-ray crystallography: the triclinic space
group, P-1, was assigned. Figure 1 shows the atom
arrangements: there is a weak C5-H5–N2 intramolecular
hydrogen bond. Selected bond lengths and angles are also
listed in Fig. 2. The structure determination confirmed that
methylation of the precursor, 1e, had occurred on the
quinoline nitrogen, with loss of the aromatic structure of
the pyridine ring and the formation of a (E,E)-CH=N–
N=CH-C6H4OMe-m fragment. The molecule overall is
very near planar with only a small angle of 4° between the
planes of the quinolinyl and phenyl groups. The only
intermolecular interactions are weak C–H–O and C–H–N
hydrogen bonds, and p–p stacking interactions.
Series C derivatives were prepared by direct methyla-
tion of compounds 1a-1i (series A) [8, 9], using MeI and
K2CO3 in acetone (Scheme 2). The products were obtained
as yellow or orange solids in moderate to excellent yields
(Table 2). Identification of the isolated series C compounds
6 as substituted benzaldehyde 7-chloro-1-methyl-4H-
quinolinyl-4-ylidene hydrazones, with methylated quino-
line nitrogen atoms, was generally achieved by
spectroscopic data, and specifically by X-ray crystallogra-
1
phy for 6e, see later. The NMe signal in the H NMR
spectra of 6a-6i occurs as a singlet between 3.62 and
3.74 ppm. In addition, the IR spectra showed N–CH3 and
N=C stretching vibrations at 2855–2939 and
1626–1635 cm-1, respectively.
X-ray crystal structures have been previously reported
for a number of anhydrous [12–14] and hydrated [15–18]
series A compounds 1. These include both active and non-
123

M. L. F. Bispo et al.
Scheme 1
(a) MeONa (1M), 50 °C, 3 h, 95%; (b) H2, Pd/C, MeOH, r.t., 6 h, 85%; (c) N2H4 H2O
(80%), EtOH, 80 °C, 4 h, 80%; (d) corresponding benzaldehyde, EtOH, r.t., 4-24 h, 60-
95%.
Table 2 Yields and melting points of series B and C compounds
R4
R4
R3
R2
R3
R2
R5
R5
N
N
N
HN
R1
R1
a-
6
6i
Series C
a-
5 5i
Series B
l
C
N
C
N
H
Cl
H
3
Series B/C
Substituents
Yield/%
M.p./°C
Series B
R1
R2
R3
R4
R5
Series B
Series C
Series C
5a/6a
5b/6b
5c/6c
5d/6d
5e/6e
5f/6f
H
H
H
H
H
H
H
H
H
Cl
H
H
H
76
82
80
65
77
89
82
85
68
65
58
61
62
68
62
82
90
77
202–203
214–215
223–225
304
156–157
173–175
185–187
189–190
144–145
142–143
190–191
151–152
214–215
H
F
H
H
H
Cl
Br
H
H
H
H
H
H
OMe
H
H
H
261–262
227–229
240–241
284–286
288–289
Cl
H
H
H
5g/6g
5h/6h
5i/6i
OMe
H
OMe
H
H
OMe
H
OMe
OMe
OMe
OMe
123

A new and potent class of quinoline derivatives against cancer
Scheme 2
N1-C2
1.362(3)
1.355(3)
1.439(3)
1.469(3)
1.413(3)
1.315(2)
1.403(2)
1.285(3)
1.472(3)
1.466(3)
1.389(2)
2.4
C2-C3
C3-C4
C4-C5A
C5A-C8A
C4-N2
N2-N3
N3-C9
C9-C10
N1-C16
C8A-N1
C3-C4-N2-N3
C4-N2-N3-C9
N2-N3-C9-C10
179.39(17)
180.00(17)
(a)
(b)
˚
Fig. 2 a Atom arrangements and numbering scheme for compound 6e, b selected bond lengths and angles (A, °)
Anticancer activity
active compounds. All these compounds, including the
hydrated derivatives, have (E)-stereochemistries at the
C=N group and all show only slight distortions from
overall planarity as indicated by the small angles, up to
13°, between the phenyl and quinoline groups.
Initially, all compounds were tested in vitro against three
cancer cells at 25 lg/cm3 using the MTT assay (Table 3)
[20]. The compounds were classified by their growth
inhibition (GI) percentage at least in one cell line as active
if 95 \ GI \100 %, moderately active if 70 \ GI \90 %,
or inactive if GI \50 %. For series B, compounds 5a and
5c-5 h were active against at least one cancer cell line with
GI values greater than 90 %, while 5b and 5i only exhib-
ited moderately active. For series C, compounds 6b-6e and
6g-6i were active, 6a was moderately so and 6f was inac-
tive against all cancer cell lines at 25 lg/cm3.
The active compounds were selected for in vitro anti-
cancer activity evaluations against four human cancer cell
lines (HCT-116, OVCAR-8, HL-60, and SF-295) using the
MTT assay (Table 4) [19]. The concentrations that induced
50 % inhibition of cell growth (IC50) in lg/cm3 are pre-
sented in Table 4. None of the compounds was able to
disrupt the cell membrane integrity of erythrocytes in the
mouse model (data not shown) [21].
Generally, series B salts were found to be difficult to
obtain in suitable form for X-ray studies. However, crystal
structures have been reported for two hydrates of two series
B salts, namely [(5: R1=Cl; R2=R3=R4=R5=H)Á2H2O] [10]
and [(5: R1=R3=Cl; R2=R4=R5=H)ÁH2O] [11] as well as the
non-hydrated 3-chlorobenzoate salt of (5: R2=Cl; R1-
=R3=R4=R5=H) [19]. In all three cases, it was confirmed
that the site of protonation in the cation was the quinolinyl
nitrogen and that the C=N moiety had an (E)-stereochem-
istry. The cations exhibited a slight twist in the link
between the phenyl group and the rest of the cation: the
twist between phenyl and quinolinium rings are 7.51(12)°
and 4.33(3)°, respectively, for [(5: R1=Cl; R2=R3=
R4=R5=H)Á2H2O] [10] and [(5: R1=R3=Cl; R2=R4=R5=H)Á
H2O] [11]. In the 3-chlorobenzoate salt, the cation exhibits
a greater twist of 18.98(10)°.
123

M. L. F. Bispo et al.
Table 3 Growth inhibition percentage (GI %) for three tumors cells line by the MTT assay
Entry
Nr
SF-295
GI %
SD %
HCT-116
GI %
SD %
OVCAR-8
GI %
SD %
Substituents
Series B
5a
H
73.02
24.14
98.93
98.63
100.00
54.54
100.00
98.04
83.05
5.19
3.56
0.00
0.93
1.35
4.18
0.76
1.43
1.49
100.00
57.75
0.17
1.35
1.90
5.35
4.46
0.43
1.08
1.24
1.71
100.00
92.06
100.00
92.36
100.00
101.52
90.33
92.36
36.05
0.25
0.08
0.43
2.59
2.88
0.17
1.97
3.60
3.53
5b
3-F
5c
3-Cl
100.00
100.00
100.00
101.81
52.29
5d
3-Br
5e
3-OMe
2,6-Cl2
3,4-(OMe)2
2,5-(OMe)2
2,3,4-(OMe)3
5f
5g
5h
97.56
5i
11.20
Series C
6a
H
64.08
86.71
87.68
55.88
100.38
45.88
99.88
101.69
100.70
0.76
2.79
0.00
2.95
0.06
0.34
0.53
0.06
0.76
89.55
99.49
1.41
0.18
0.54
1.03
0.36
1.82
0.13
0.00
0.13
52.95
98.56
0.50
0.31
0.16
0.00
0.16
0.91
0.17
0.17
0.08
6b
3-F
6c
3-Cl
100.38
94.27
100.67
100.00
101.33
26.90
6d
3-Br
6e
3-OMe
2,6-Cl2
3,4-(OMe)2
2,5-(OMe)2
2,3,4-(OMe)3
100.51
56.13
6f
6g
102.45
101.82
101.36
101.05
100.93
100.64
6h
6i
The results in Tables 3 and 4 show that compounds of both
series exhibit good cytotoxic activity against the four cancer
cell lines. The results obtained for series B clearly indicate the
importance of the degree and nature of the substitution in the
phenyl group. Mono-meta-substituted compounds (5c–5e),
irrespective of the substituent being electron donating or
electron withdrawing, are more active than the non-substi-
tuted compound 5a. Interestingly, the most active mono-
substituted compound is the bromo derivative 5d and, fur-
thermore, the sequence of activity for the mono-halo
derivatives is 5d [3-Br] [5c [3-Cl] [5b [3-F]. The 3-OMe
derivative 5e is less reactive than the 3-Cl derivative 5c.
Considering just the mono-halogeno derivatives, the activity
sequence correlates with their electronic donating effects, as
well as the bulk of the substituent, but a correlation fails if the
methoxy substituent is also included. The addition of another
chloro group does increase the activity (compare results for 5c
and 5f). In contrast, the addition of further methoxy groups
does not improve the cytotoxic activity.
halogeno substituents follows their mesomer donor abili-
ties. Further differences between series B and C are (1) the
2,6-dichloro substituted compound 5f is more active than
the mono-chloro compound 5c and (2) the di- and tri-
methoxy derivatives 6g–6i are more active than the mono-
methoxy 6e compound in the C series. Indeed, the tri-
methoxy derivative 6i is the most active compound.
Series C derivatives showed the best activity against the
HL-60 cancer cell line, the one exception being 6e, which
was most active against the HCT-116 cell line. This cell
line specificity was not shown by compounds in series B.
Conclusion
Comparisons of the activities of selected members of the
three series, series A, B, and C are available in Table 5.
General conclusions can be made as follows: (1) the
activity of the best compounds in series B and C is greater
than those of the corresponding compounds in series A and
(2) derivatives of series C showed greater cytotoxic
activities than did the derivatives of series B.
There are some interesting contrasts and similarities
between the results of the two series B and C. First, the
similarity: mono-substituted derivatives, no matter whether
electron donating or withdrawing (6b–6e), are more active
than the unsubstituted derivative, 6a. However, in contrast,
the sequence of activity is 6e [3-OMe] [6b [3-F] [6c [3-
Cl] [6d [3-Br]. This time the activity sequence of the
Three compounds, 5d, 5f, and 6i, in particular, show
encouraging results and thus can be considered as useful
starting points for further study. The structural modifica-
tions of series A made to form series B and C have led to
123

A new and potent class of quinoline derivatives against cancer
Table 4 Cytotoxic activity of quinoline derivatives of series B and C on tumor cells linesa
Entry
Series B
Substituents
IC50/lg cm-3
HCT-116
OVCAR-8
HL-60
SF-295
5a
5c
5d
5e
5f
H
2.807
1.931
0.7914
1.789
2.245–3.510
1.874
1.702–2.189
2.655
0.6837–0.9161
0.9133
1.363–2.349
2.643
3-Cl
1.672–2.101
1.023
2.414–2.919
0.5728
0.8180–1.020
0.6808
2.229–3.133
0.5178
3-Br
0.8716–1.200
2.165
0.369–0.889
3.573
0.542–0.854
0.8338
0.1643–1.632
4.654
3-OMe
2,6-Cl2
3,4-(OMe)2
2,5-(OMe)2
1.901–2.466
0.7148
3.136–4.071
0.6792
0.4698–1.480
0.8270
2.741–7.902
1.084
0.4724–1.082
2.169
0.4786–0.9638
1.346
0.6797–1.006
2.751
0.7747–1.518
1.351
5g
5h
1.437–3.274
2.828
0.7149–2.536
3.628
1.437–3.274
2.091
0.7030–2.596
3.894
1.606–4.982
HCT-116
0.8188
3.254–4.045
OVCAR-8
1.120
0.9105–4.803
HL-60
1.660–9.134
SF-295
Series C
Substituents
3-F
6b
0.7325
2.564
0.5596–1.198
1.924
0.8357–1.501
1.953
0.6273–0.8553
1.315
0.7498–8.769
1.966
6c
3-Cl
1.655–2.236
3.620
1.666–2.290
2.202
0.8834–1.958
1.979
1.672–2.312
2.373
6d
3-Br
2.883–4.545
0.6963
1.728–2.806
0.7034
1.655–2.366
0.9317
2.084–2.701
2.167
6e
3-OMe
0.4607–1.052
1.507
0.4875–1.015
0.9223
0.7603–1.142
0.3318
0.4367–10.75
1.661
6g
3,4-(OMe)2
2,5-(OMe)2
2,3,4-(OMe)3
1.132–2.005
0.8303
0.7493–1.135
0.8408
0.2673 –0.4118
0.6436
1.223–2.255
1.569
6h
0.7691 0.8963
0.5080
0.6976–1.013
0.4333
0.5717 –0.7247
0.3139
1.331–1.850
0.8190
6i
0.3498–0.7375
0.01
0.3469–0.5411
0.265
0.2866–0.3438
0.02
0.7071–0.9486
0.23
Doxorubicin
0.01–0.02
0.11–0.305
0.01–0.02
0.19–0.25
a
Data are presented as IC50 values, with 95 % confidence intervals obtained by nonlinear regression for all cell lines leukemia (HL-60), ovarium
(OVCAR-8), nervous system (SF-295), colon (HCT-116) from three independent experiments. Doxorubicin (Dox) was used as positive control.
The trials were performed in triplicate; IC50, concentrations that induce 50 % inhibition of cell growth in lg cm-3
useful and interesting findings and suggest that further
modifications are well worth making. Further studies on the
activity and structure–activity relationships of modified
quinoline derivatives are underway.
spectra were recorded in a Bruker Avance 400 operating at
400.00 MHz (1H) and 100.0 MHz (13C), and Bruker Avance
500 spectrometer operating at 500.00 MHz (1H) and
125.0 MHz (13C), in deuterated dimethylsulfoxide. Chemical
shifts are reported in ppm (d) relative to tetramethylsilane and
J-coupling in Hertz (Hz). Proton and carbon spectra were
typically obtained at room temperature. TLC plates, coated
with silica gel, were run in a chloroform/methanol (9:1)
mixture and spots were developed in ultraviolet.
Experimental
Melting points were determined on a Bu¨chi apparatus.
Infrared spectra were recorded in a Thermo Nicolet Nexus
670 spectrometer, as potassium bromide pellets and fre-
quencies are expressed in cm-1. High-resolution mass
spectrum was performed on a Bruker compact QTOF. NMR
7-Chloro-4-methoxyquinoline (2)
To a solution of sodium methoxide (1 M) in methanol were
portionwise added 2.5 g of 4,7-dichloroquinoline
123

M. L. F. Bispo et al.
Table 5 Comparison of the best anticancer activities of series A, B, and C compounds
Series A
Best IC50/lg cm-3
[cancer cell line]
Series B
Best IC50/lg cm-3
[cancer cell line]
Series C
Best IC50/lg cm-3
[cancer cell line]
1a
1b
1c
1d
1e
1f
2.085
5a
5b
5c
5d
5e
5f
0.7914
[HL-60]
–
6a
6b
6c
6d
6e
6f
–
[SF-295]
3.206
0.7325
[HL-60]
1.315
[HTC-8]
1.238
0.9133
[SF-295]
1.588
[HL-60]
0.5178
[HL-60]
1.979
[HL-60]
0.8018
[HTC-8]
1.086
[SF-295]
0.8338
[HL-60]
0.6963
[HCT-116]
–
[HL-60]
0.6792
[HTC-8]
0.6888
[SF-295]
2.837
[OVCAR-8]
1.346
1g
1h
1i
5g
5h
5i
6g
6h
6i
0.3318
[OVCAR-8]
2.091
[HL-60]
0.6436
[HL-60]
0.7483
[HTC-8]
[HL-60]
–
[HL-60]
0.3139
[HL-60]
(12.6 mmol). The mixture reaction was stirred at 40 °C for
4 h. After that, the solvent was removed under reduced
pressure. Then, the crude product was extracted with
30 cm3 water and ethyl acetate (3 9 30 cm3) and the
combined organic phases were dried over anhydrous
sodium sulfate and concentrated under reduced pressure
to yield the intermediate 2 as a white solid. Yield: 2.32 g
(95 %); m.p.: 139–141 °C (Ref. [22] 141–143 °C).
General procedure for synthesis of 4-quinolinylhydrazone
hydrochlorides 5a-5i
The 4-quinolinylhydrazone derivatives 5a–5i were
obtained by the reaction between 4-hydrazinylquinoline
(4, 1.03 mmol) and the appropriate benzaldehyde
(1.24 mmol) in 5 cm3 ethanol. After stirring for 4–24 h
at room temperature, the resulting mixture was concen-
trated under reduced pressure and the residue purified by
washing with cold Et2O (3 9 10 cm3), leading to the pure
derivatives 5a–5i as solids in 60–95 % yields.
4-Methoxyquinoline hydrochloride (3)
To a solution of 2.0 g intermediate 2 (10.3 mmol) in
30 cm3 methanol catalytic amounts of 10 % Pd–C
(5 mol %) were added, and the mixture was treated with
H2 for 6 h. The catalyst was filtered off and washed with
methanol (2 9 10 cm3), and the filtrate was concentrated
to lead the intermediate 3 as a white solid. Yield: 1.17 g
(89 %); m.p.: 165–167 °C (dec.) [Ref. [23] 165 °C (dec.)].
4-(Benzylidenehydrazinyl)quinoline hydrochloride
(5a, C16H14ClN3)
1
Yield: 76 %; yellow solid; m.p.: 202–203 °C; H NMR
(400 MHz, DMSO-d6): d = 11.79 (1H, s), 8.49 (1H, s, H3
8.45 (1H, d, J = 5.8 Hz, H2), 8.41 (1H, d, J = 8.0 Hz, H5),
7.85–7.80 (3H, m, H5, H9, H8), 7.73 (1H, t, J = 7.5 Hz,
H7), 7.54 (1H, t, J = 7.5 Hz, H6), 7.49–7.42 (3H, m, H6
7, H8
), 7.37 (1H, d, J = 5.8 Hz, H3) ppm; 13C NMR
´
),
´
´
´
,
H
´
´
4-Hydrazinylquinoline hydrochloride (4)
(100 MHz, DMSO-d6): d = 149.2, 147.0, 145.5, 145.2,
134.7, 130.1, 129.5, 128.8, 126.8, 125.8, 124.7, 122.3,
117.1, 100.4 ppm; IR (KBr):
Hydrazine hydrate (80 %, 7.6 cm3, 126 mmol) in 25 cm3
absolute ethanol was added to 1.0 g 4-methoxyquinoline
hydrochloride (3, 6.3 mmol) dissolved in 20 cm3 absolute
ethanol. The mixture was refluxed for 6 h. After that, the
mixture was allowed to cool at 5 °C for 12 h. The pallid
yellow precipitate was filtered and washed with 5 cm3
absolute ethanol and 20 cm3 diethyl ether to give the
compound 4 as a white solid. Yield: 0.851 g (85 %); m.p.:
301–303 °C (dec.) [Ref. [12] 300–301 °C (dec.)].
vꢀ = 3178 (N–H), 2915 (N?–
H), 1616 (C=N) cm-1; HRMS (ESI): m/z = 246.1113
([M?H]?).
4-(3-Fluorobenzylidenehydrazinyl)quinoline hydrochloride
(5b, C16H13ClFN3)
1
Yield: 82 %; yellow solid; m.p.: 214–215 °C; H NMR
(400 MHz, DMSO-d6): d = 8.46 (1H, s, H3), 8.43–8.41
´
123

A new and potent class of quinoline derivatives against cancer
(1H, m, H5, H2), 7.84 (1H, d, J = 8.2 Hz, H5), 7.74 (1H, t,
4-(2,6-Dichlorobenzylidenehydrazinyl)quinoline
J = 7.5 Hz, H7), 7.67–7.63 (2H, m, H6, H5
(2H, m, H7, H9), 7.41 (1H, d, J = 6.0 Hz, H3), 7.27–7.23
(1H, m, H
8) ppm; 13C NMR (100 MHz, DMSO-d6):
´
), 7.57–7.48
hydrochloride (5f, C16H12Cl3N3)
1
´
´
Yield: 89 %; yellow solid; m.p.: 227–229 °C; H NMR
(400 MHz, DMSO-d6): d = 11.46 (1H, s, NH), 8.70 (1H,
´
d = 155.5, 146.2, 145.5, 143.2, 135.9, 130.6, 129.5,
128.9, 127.8, 125.8, 124.9, 123.2, 117.2, 100.3 ppm; IR
s, H
(1H, d, J = 8.0 Hz, H8), 7.70 (1H, t, J = 8.0 Hz, H7),
7.60–7.58 (2H, m, H6, H8), 7.52 (1H, t, J = 8.0 Hz, H6),
7.40 (1H, t, J = 8.0 Hz, H9), 7.27 (1H, d, J = 5.2 Hz, H3)
´
3), 8.43 (1H, s, H2), 8.38 (1H, d, J = 8.0 Hz, H5), 7.83
(KBr): vꢀ = 3189 (N–H), 2908 (N?–H), 1615 (C=N) cm-1
HRMS (ESI): m/z = 264.1015 ([M?H]?).
;
´
´
´
ppm; 13C NMR (100 MHz, DMSO-d6): d = 153.2, 152.3,
150.4, 144.5, 137.3, 132.8, 126.8, 123.5, 117.7, 115.1,
113.7, 110.2, 99.8 ppm; IR (KBr): vꢀ = 3485 (N–H), 1579
(C=N) cm-1; HRMS (ESI): m/z = 314.0332 ([M?H]?).
4-(3-Chlorobenzylidenehydrazinyl)quinoline hydrochloride
(5c, C16H13Cl2N3)
1
Yield: 80 %; yellow solid; m.p.: 223–225 °C; H NMR
(400 MHz, DMSO-d6): d = 8.63 (1H, s, H3), 8.60 (1H, d,
´
4-(3,4-Dimethoxybenzylidenehydrazinyl)quinoline
J = 8.4 Hz, H5), 8.52 (1H, d, J = 6.0 Hz, H2), 7.94–7.92
hydrochloride (5g, C18H18ClN3O2)
´
(2H, m, H5, H8), 7.86–7.78 (2H, m, H6, H7), 7.64 (1H, t,
1
Yield: 82 %; yellow solid; m.p.: 240–241 °C; H NMR
J = 7.5 Hz, H
8´), 7.56–7.46 (3H, m, H3, H7
´
, H9
) ppm; 13C
´
(400 MHz, DMSO-d6): d = 14.70 (1H, s, NH), 12.88 (1H,
s, NH), 9.16 (1H, s, H3´), 8.84 (1H, d, J = 7.2 Hz, H5), 8.68
NMR (100 MHz, DMSO-d6): d = 150.4, 145.7, 145.3,
142.7, 136.7, 133.8, 131.5, 130.6, 129.7, 126.3, 125.9,
125.6, 123.8, 123.1, 116.7, 100.6 ppm; IR (KBr): vꢀ = 3163
(N–H), 2908 (N?–H), 1608 (C=N) cm-1; HRMS (ESI): m/
z = 282.0717 ([M?H]?).
(1H, d, J = 6.8 Hz, H2), 8.10 (1H, d, J = 8.4 Hz, H8), 8.03
(1H, dd, J = 8.4, 7.6 Hz, H7), 7.81 (1H, dd, J = 7.6,
´
7.2 Hz, H6), 7.55 (2H, m, H3, H5), 7.19 (1H, d, J = 8.3 Hz,
H
´
8, H9
), 3.87 (6H, s, OCH3) ppm; 13C NMR (100 MHz,
´
DMSO-d6): d = 152.7, 152.4, 148.5, 146.4, 142.2, 138.2,
133.6, 127.0, 126.7, 124.5, 123.9, 120.0, 117.1, 115.0,
99.8, 61.4, 55.8 ppm; IR (KBr): vꢀ = 2766 (N?–H), 1612
(C=N) cm-1; HRMS (ESI): m/z = 308.1321 ([M?H]?).
4-(3-Bromobenzylidenehydrazinyl)quinoline hydrochloride
(5d, C16H13BrClN3)
Yield: 65 %; yellow solid; m.p.: 304 °C; 1H NMR
(400 MHz, DMSO-d6): d = 14.77 (1H, s, NH), 13.10
(1H, s, NH), 8.91–8.89 (2H, m, H
J = 6.9 Hz, H2), 8.11–8.09 (1H, m, H8, H5), 8.03 (1H, t,
J = 7.7 Hz, H7), 7.87 (1H, d, J = 7.8 Hz, H7), 7.81 (1H, t,
J = 7.7 Hz, H6), 7.73 (2H, d, J = 8.4 Hz, H5, H9), 7.70–
7.73 (2H, m, H3, H8), 7.49 (1H, d, J = 7.8 Hz, H9) ppm;
´
3, H5), 8.69 (1H, d,
4-(2,5-Dimethoxybenzylidenehydrazinyl)quinoline
´
hydrochloride (5h, C18H18ClN3O2)
1
´
Yield: 85 %; yellow solid; m.p.: 284–286 °C; H NMR
´
´
(400 MHz, DMSO-d6): d = 14.61 (1H, s, NH), 12.75 (1H,
s, NH), 9.13 (1H, s, H3´), 8.78 (1H, d, J = 8.3 Hz, H5), 8.65
´
´
13C NMR (100 MHz, DMSO-d6): d = 152.6, 148.6, 172.5,
138.2, 135.9, 135.6, 133.8, 133.4, 131.1, 129.4, 126.9,
123.9, 122.4, 120.1, 115.1, 100.2 ppm; IR (KBr): vꢀ = 3152
(N–H), 2684 (N?–H), 1612 (C=N) cm-1; HRMS (ESI): m/
z = 326.0217 ([M?H]?).
(1H, d, J = 6.8 Hz, H2), 8.08 (1H, d, J = 8.3 Hz, H8), 8.02
(1H, dd, J = 8.3, 7.2 Hz, H7), 7.80 (1H, dd, J = 8.3,
7.2 Hz, H6), 7.68 (1H, d, J = 6.8 Hz, H3), 7,56 (1H, s, H9´
),
7.14-7.09 (2H, m, H6, H7), 3.87 (3H, s, OCH3), 3.81 (3H, s,
´
´
OCH3) ppm; 13C NMR (100 MHz, DMSO-d6): d = 153.4,
152.9, 152.3, 145.9, 142.6, 138.3, 133.9, 127.1, 123.5,
122.3, 120.3, 118.7, 115.1, 113.7, 109.8, 100.1, 56.4,
4-(3-Methoxybenzylidenehydrazinyl)quinoline hydrochlo-
55.7 ppm; IR (KBr): vꢀ = 2771 (N?–H), 1589 (C=N) cm-1
HRMS (ESI): m/z = 308.1319 ([M?H]?).
;
ride (5e, C17H16ClN3O)
1
Yield: 77 %; yellow solid; m.p.: 261–262 °C; H NMR
(400 MHz, DMSO-d6): d = 14.76 (1H, s, NH), 13.12 (1H,
s, NH), 9.01-8.99 (2H, m, H3´, H5), 8.63 (1H, d,
4-(3,4,5-Trimethoxybenzylidenehydrazinyl)quinoline
hydrochloride (5i, C19H20ClN3O3)
Yield: 68 %; E/Z ratio = 67:33; yellow solid; m.p.: 288–
J = 7.0 Hz, H2), 8.10 (1H, d, J = 8.0 Hz, H8), 7.98 (1H,
t, J = 8.0 Hz, H7), 7.75 (1H, t, J = 8.0 Hz, H6), 7.61 (1H,
1
289 °C; H NMR (400 MHz, DMSO-d6): d = 14.52 (1H,
s, NH), 12.97 (1H, s, NH), 8.83 (1H, s, H3´), 8.89 (1H, d,
d, J = 7.0 Hz, H3), 7.42-7.37 (3H, m, H5
´
H8´
, H9´
), 7.06
7), 3.85 (3H, s, OCH3) ppm; 13C
(1H, d, J = 7.5 Hz, H
´
J = 8.4 Hz, H5), 8.67–8.65 [1.6H, d, J = 6.8 Hz, H2, H3´
NMR (100 MHz, DMSO-d6): d = 159.6, 152.5, 150.3,
142.3, 138.2, 134.9, 133.7, 130.1, 126.8, 124.4, 120.4,
120.0, 116.9, 115.1, 111.8, 99.9, 55.3 ppm; IR (KBr):
vꢀ = 3414 (N–H), 2688 (N?–H), 1589 (C=N) cm-1; HRMS
(ESI): m/z = 278.1213 ([M?H]?).
(Z)], 8.05 (1H, d, J = 8.4 Hz, H8), 8.03 (1H, t, J = 8.4 Hz,
H7), 7.80 (1H, t, J = 8.4 Hz, H6), 7.69 (1H, d, J = 6.8 Hz,
H3), 7.22 [1H, s, H5
´
, H
´
9(Z)], 7.19 (2H, s, H5
´
, H9´), 3.90 (6H,
s, OCH3), 3.84 (6H, s, OCH3), 3.75–3.73 [4.7H, m, OCH3
123

M. L. F. Bispo et al.
(Z)] ppm; 13C NMR (100 MHz, DMSO-d6): d = 161.1,
153.3, 153.1, 152.4, 150.4, 142.4, 140.2, 140.1, 138.1,
133.7, 129.2, 128.9, 126.8, 123.9, 120.1, 115.1, 105.6,
105.0, 99.9, 60.2, 60.1, 56.1, 55.9 ppm; IR (KBr):
vꢀ = 2770 (N?–H), 1589 (C=N) cm-1; HRMS (ESI):
m/z = 338.1422 ([M ? H]?).
H9
´
), 7.34 (dd, J = 8.7, 1.6 Hz, 1H, H6), 6.91 (1H, d,
J = 7.8 Hz, H3), 3.66 (3H, s, NCH3) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 156.6, 151.4, 140.7, 140.5,
138.2, 135.9, 133.5, 130.5, 128.9, 126.4, 126.2, 125.9,
123.4, 120.7, 115.3, 99.7 ppm; IR (KBr): vꢀ = 2864 (N–
CH3), 1631 (C=N) cm-1; HRMS (ESI): m/z = 329.0491
([M?H]?).
Synthesis of quinoline derivatives of series C (6a–6i)
To a solution of 4-chloro-4-quinolinylhydrazone deriva-
tives [8, 9] (1 eq., 0.20 g) in 10 cm3 acetone was added
potassium carbonate (4.0 eq.). The reaction mixture was
stirred at room temperature for 30 min, methyl iodide
(4.0 eq.) was added, and the reaction mixture was heated to
40 °C for 3–36 h. The mixture was evaporated and 20 cm3
water was added to the residue. The mixture was extracted
with ethyl acetate (3 9 10.0 cm3), the combined organic
phases were dried over anhydrous MgSO4, and rotary
evaporated to yield impure solids. These were purified by
column chromatography using a mixture of hexane and
ethyl acetate (50 %) as eluent to leave the desired products
6a–6i as yellow solids in 61–90 % yield.
3-Bromobenzaldehyde
(E)-(7-chloro-1-methyl-4(1H)-
quinolinylidene)hydrazone (6d, C17H13BrClN3)
1
Yield: 62 %; yellow solid; m.p.: 189–190 °C; H NMR
(400 MHz, DMSO-d6): d = 8.45 (1H, s, H3), 8.42 (1H, d,
J = 8.7 Hz, H5), 8.01 (1H, s, H5), 7.81 (1H, d, J = 7.8 Hz,
7), 7.58–7.55 (2H, m, H6, H8), 7.47 (1H, d, J = 7.9 Hz,
H2), 7.37 (1H, t, J = 7.8 Hz, H8), 7.35 (1H, dd, J = 8.7,
´
´
H
´
´
´
1.8 Hz, H6), 6.91 (1H, d, J = 7.9 Hz, H3), 3.67 (3H, s,
NCH3) ppm; 13C NMR (100 MHz, DMSO-d6): d = 156.5,
151.3, 140.7, 140.4, 138.9, 135.9, 130.8, 128.6, 129.3,
126.3, 126.2, 123.4, 122.1, 120.7, 115.2, 99.7 ppm; IR
(KBr): vꢀ = 2918 (N–CH3), 1629 (C=N) cm-1; HRMS
(ESI): m/z = 373.9989 ([M?H]?).
Benzaldehyde (E)-(7-chloro-1-methyl-4(1H)-quinolinyli-
3-Methoxybenzaldehyde
quinolinylidene)hydrazone (6e, C18H16ClN3O)
Yield: 68 %; yellow solid; m.p.: 144–145 °C; H NMR
(400 MHz, DMSO-d6): d = 8.45 (1H, s, H3), 8.41 (1H, d,
(E)-(7-chloro-1-methyl-4(1H)-
dene)hydrazone (6a, C17H14ClN3)
1
1
Yield: 65 %; yellow solid; m.p.: 156–157 °C; H NMR
(400 MHz, DMSO-d6): d = 8.48 (1H, s, H3
J = 8.7 Hz, H5), 7.82 (2H, d, J = 7.0 Hz, H5
(1H, d, J = 1.6 Hz, H8), 7.45–7.39 (4H, m, H2, H6
´
), 8.41 (1H, d,
, H9), 7.51
H7, H8),
´
´
´
J = 8.7 Hz, H5), 7.51 (1H, d, J = 1.8 Hz, H8), 7.43–7.31
´
(5H, m, H2, H6, H5, H7, H
´
´
´
8´), 6.96 (1H, d, J = 8.0 Hz, H9´),
´
7.33 (dd, J = 8.7, 1.6 Hz, 1H, H6), 6.87 (1H, d,
J = 7.8 Hz, H3), 3.64 (3H, s, NCH3) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 155.8, 152.9, 140.4, 140.3,
135.8, 135.7, 129.5, 128.6, 127.3, 126.0, 123.2, 120.7,
115.3, 99.6, 39.5 ppm; IR (KBr): vꢀ = 2855 (N–CH3), 1635
(C=N) cm-1; HRMS (ESI): m/z = 296.0872 ([M?H]?).
6.87 (1H, d, J = 7.9 Hz, H3), 3.82 (3H, s, OCH3), 3.64
(3H, s, NCH3) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 159.4, 155.9, 152.9, 140.4, 137.3, 135.7, 129.7, 126.1,
123.2, 120.7, 120.1, 115.2, 115.1, 111.8, 99.6, 55.1 ppm;
IR (KBr): vꢀ = 2924 (N–CH3), 1630 (C=N) cm-1; HRMS
(ESI): m/z = 326.0980 ([M?H]?).
3-Fluorobenzaldehyde
(E)-(7-chloro-1-methyl-4(1H)-
2,6-Dichlorobenzaldehyde (E)-(7-chloro-1-methyl-4(1H)-
quinolinylidene)hydrazone (6b, C17H13ClFN3)
quinolinylidene)hydrazone (6f, C17H12Cl3N3)
1
1
Yield: 58 %; yellow solid; m.p.: 173–175 °C; H NMR
(400 MHz, DMSO-d6): d = 8.54 (1H, d, J = 8.6 Hz, H5),
Yield: 62 %; yellow solid; m.p.: 142–143 °C; H NMR
(400 MHz, DMSO-d6): d = 8.66 (1H, s, H3
J = 8.7 Hz, H5), 8.02 (1H, d, J = 1.7 Hz, H8), 7.56 (2H, d,
J = 8.0 Hz, H6, H8), 7.51 (1H, d, J = 7.9 Hz, H3), 7.39–
7.35 (2H, m, H6, H7), 6.81 (1H, d, J = 7.9 Hz, H3), 3.67
´
), 8.47 (1H, d,
´
3), 7.67–7.63 (2H, m, H5, H9
8.49 (1H, s, H
´
´
), 7.49–7.45 (2H,
m, H8, H8´
), 7.35 (1H, d, J = 7.8 Hz, H2), 7.29 (dd,
´
´
J = 8.6, 1.6 Hz, 1H, H6), 7.15 (1H, dd, J = 8.4, 1.6 Hz,
H7‘), 7.02 (1H, d, J = 7.8 Hz, H3), 3.75 (3H, s, NCH3)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 173.5, 167.5,
162.4, 151.4, 150.5, 149.7, 146.9, 141.0, 137.1, 134.4,
133.7, 131.9, 126.3, 125.3, 123.3, 110.7, 48.9 ppm; IR
(KBr): vꢀ = 2835 (N–CH3), 1626 (C=N) cm-1; HRMS
(ESI): m/z = 314.0783 ([M?H]?).
´
(3H, s, NCH3) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 151.7, 150.0, 147.5, 140.4, 133.6, 132.7, 129.3, 127.8,
125.8, 123.7, 120.2, 115.4, 110.9, 99.9 ppm; IR (KBr):
vꢀ = 2922 (N–CH3), 1627 (C=N) cm-1; HRMS (ESI):
m/z = 364.0093 ([M?H]?).
3,4-Dimethoxybenzaldehyde
(E)-(7-chloro-1-methyl-
3-Chlorobenzaldehyde
quinolinylidene)hydrazone (6c, C17H13Cl2N3)
Yield: 61 %; yellow solid; m.p.: 185–187 °C; H NMR
(400 MHz, DMSO-d6): d = 8.46 (1H, s, H3), 8.42 (1H, d,
J = 8.7 Hz, H5), 7.87 (1H, s, H5), 7.78–7.77 (1H, m, H7),
7.55 (1H, d, J = 1.6 Hz, H8), 7.48-7.44 (3H, m, H2, H8
(E)-(7-chloro-1-methyl-4(1H)-
4(1H)-quinolinylidene)hydrazone (6g, C19H18ClN3O2)
1
Yield: 82 %; yellow solid; m.p.: 190–191 °C; H NMR
1
(400 MHz, DMSO-d6): d = 8.40–8.37 (2H, m, H3
7.48 (2H, m, H8, H5), 7.39 (1H, d, J = 7.9 Hz, H2), 7.32–
7.27 (2H, m, H6, H8), 7.01 (1H, d, J = 8.3 Hz, H7), 6.86
(1H, d, J = 7.9 Hz, H3), 3.84 (3H, s, OCH3), 3.81 (3H, s,
´
, H5),
´
´
´
´
´
´
´
,
123

A new and potent class of quinoline derivatives against cancer
OCH3), 3.62 (3H, s, NCH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 155.0, 153.2, 150.4, 148.9, 140.5, 140.2,
135.6, 128.6, 126.0, 123.2, 121.8, 120.8, 115.1, 111.5,
109.0, 99.7, 55.6, 55.4 ppm; IR (KBr): vꢀ = 2929 (N–CH3),
attached to methine C9. The coordinates, along with iso-
tropic displacement parameters, were fully refined. All
other hydrogen atoms were placed in calculated positions.
Crystal data and structure refinement details are listed in
Table 1. Crystal data of 6e (colorless crystal) were col-
lected at 120(2) K: 0.40 9 0.34 9 0.16 mm. Formula:
1630 (C=N) cm-1
; HRMS (ESI): m/z = 356.1090
([M?H]?).
C18H16ClN3O, M = 325.79; triclinic, P-1; a = 8.8352(3)
o
2,5-Dimethoxybenzaldehyde
(E)-(7-chloro-1-methyl-
˚
˚
˚
A, b = 8.9684(5) A, c = 10.3083(7) A, a = 83.525(6) ,
4(1H)-quinolinylidene)hydrazone (6h, C19H18ClN3O2)
1
Yield: 90 %; yellow solid; m.p.: 151–152 °C; H NMR
(400 MHz, DMSO-d6): d = 8.70 (1H, s, H3
J = 8.7 Hz, H5), 7.54 (1H, d, J = 2.9 Hz, H9
J = 1.6 Hz, H8), 7.41 (1H, d, J = 7.9 Hz, H2), 7.31 (1H,
dd, J = 8.7, 1.6 Hz, H6), 7.03 (1H, d, J = 9.0 Hz, H6),
b = 76.717(5)o, c = 79.485(6)o, Z = 2, V = 779.46(7)
3
A , independent reflections 3545 [R(int) = 0.0188], 2636
˚
´
), 8.41 (1H, d,
observed reflections [I [ 2r(I)]: parameters refined 213;
number of restraints 0; R(F) 0.037 (obs data), largest diff
peak 0.366 eA-3. Atomic coordinates, bond lengths,
˚
´
), 7.50 (1H, d,
´
angles, and thermal parameters have been deposited at the
Cambridge Crystallographic Data Centre, deposition
number 1028262.
6.97 (1H, dd, J = 9.0, 2.9 Hz, H6), 6.87 (1H, d,
J = 7.9 Hz, H3), 3.81 (3H, s, OCH3), 3.77 (3H, s,
OCH3), 3.64 (3H, s, NCH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 155.7, 153.2, 152.3, 148.2, 140.5, 140.3,
135.7, 126.1, 124.5, 123.2, 120.8, 116.6, 115.1, 113.2,
110.0, 99.7, 56.2, 55.4 ppm; IR (KBr): vꢀ = 2930 (N–CH3),
Cytotoxicity assays
Cytotoxicity against cancer cell lines
1632 (C=N) cm-1
([M?H]?).
; HRMS (ESI): m/z = 356.1089
Compounds 5a–5i and 6a–6i (0.009–5 lg/cm3) were tested
for cytotoxic activity against three four cancer cell lines:
SF-295 (glioblastoma), HCT-116 (colon), OVCAR-8
(ovarium), HL-60 (leukemia) (National Cancer Institute,
Bethesda, MD). All cell lines were maintained in RPMI
1640 medium supplemented with 10 % fetal bovine serum,
2 mM glutamine, 100 U/cm3 penicillin, 100 lg/cm3
streptomycin at 37 °C with 5 % CO2. Each compound was
dissolved with DMSO to obtain a concentration of 1 mg/
cm3. The final concentration of DMSO in the culture
medium was kept constant, below 0.1 % (v/v). Compounds
5a–5i and 6a–6i were incubated with the cells for 72 h.
The negative control received the same amount of DMSO
(0.001 % in the highest concentration). The cell viability
was determined by reduction of the yellow dye 3-(4,5-
dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) to a blue formazan product as described by Mos-
mann [11].
3,4,5-Trimethoxybenzaldehyde
(E)-(7-chloro-1-methyl-
4(1H)-quinolinylidene)hydrazone (6i, C20H20ClN3O3)
1
Yield: 77 %; yellow solid; m.p.: 214–215 °C; H NMR
(400 MHz, DMSO-d6): d = 8.41 (2H, m, H5, H3
(1H, d, J = 1.6 Hz, H8), 7.41 (1H, d, J = 7.9 Hz, H2), 7.31
(1H, dd, J = 8.7, 1.6 Hz, H6), 7.15 (2H, s, H5, H9), 6.87
´
), 7.50
´
´
(1H, d, J = 7.9 Hz, H3), 3.85 (6H, s, OCH3), 3.70 (3H, s,
OCH3), 3.64 (3H, s, NCH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 156.6, 153.2, 148.2, 140.2, 136.3, 125.1,
123.6, 121.6, 115.1, 113.3, 110.2, 99.9, 56.5, 55.6 ppm; IR
(KBr): vꢀ = 2938 (N–CH3), 1630 (C=N) cm-1; HRMS
(ESI): m/z = 385.1197 ([M?H]?).
X-ray crystallographic analysis
Data were collected at 120(2) K with Mo–Ka radiation
using a Rigaku Saturn724 ? (2 9 2 bin mode) instrument
of the UK EPSRC crystallographic service, based at the
University of Southampton. Data collection, data reduc-
tion, and unit cell refinement were carried out under the
control of the program CrystalClear-SM Expert 2.0 r7 [24].
Correction for absorption was achieved in all cases by a
semi-empirical method based upon the variation of equiv-
alent reflections with the program SADABS [25]. The
programs ORTEP-3 for Windows [26] and MERCURY
[27] were used in the preparation of the figures.
SHELXL97 [28] and PLATON [29] were used in the cal-
culation of molecular geometry. The structure was solved
by direct methods using SHELXS-97 [28] and fully refined
by means of the program SHELXL-97 [28]. Difference
map peaks provided positions for the hydrogen atom
Cell membrane disruption
The test was performed in 96-well plates using a 2 %
mouse erythrocyte suspension in 0.85 % NaCl containing
10 mM CaCl2 [12]. The compounds 3a–3u were diluted as
mentioned above and were tested at 250 lg/cm3. After
incubation at room temperature for 30 min and centrifu-
gation, the supernatant was removed and the liberated
hemoglobin was measured spectrophotometrically at
540 nm. DMSO was used as a negative control and Triton
X-100 (1 %) was used as positive control. After incubation
at room temperature for 1 h and centrifugation, the
supernatant was removed and the liberated hemoglobin
was measured spectrophotometrically at 540 nm. EC50 is
123

M. L. F. Bispo et al.
12. Howie RA, De Souza MVN, Ferreira ML, Kaiser CR, Wardell
JL, Wardell SMSV (2010) Z Krist 225:440
13. Ferreira ML, De Souza MVN, Wardell SMSV, Tiekink ERT,
Wardell JL (2012) Acta Cryst E 68:1214
the calculated effective concentration that induced lysis on
50 % that of the Triton X-100.
14. De Souza MVN, Ferreira ML, Wardell SMSV, Tiekink ERT,
Wardell JL (2012) Acta Cryst E 68:1244
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123