Synthesis of novel analogs of 2-pyrazoline obtained from [(7-chloroquinolin-4-yl)amino]chalcones and hydrazine as potential antitumor and antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2013.06.049

A new series of N-acetyl and N-formyl-pyrazoline derivatives 6 and 7-8 were synthesized by cyclocondensation reaction of [(7-chloroquinolin-4-yl)amino] chalcones with hydrazine hydrate in acetic acid and hydrazine hydrate in formic acid respectively. Thes

Full text of DOI:10.1016/j.ejmech.2013.06.049

European Journal of Medicinal Chemistry 67 (2013) 252e262
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel analogs of 2-pyrazoline obtained from
[(7-chloroquinolin-4-yl)amino]chalcones and hydrazine as
potential antitumor and antimalarial agents
,
a
a
a
a
Braulio Insuasty a , Alba Montoya , Diana Becerra , Jairo Quiroga , Rodrigo Abonia ,
*
Sara Robledo b, Ivan Darío Vélez b, Yulieth Upegui b, Manuel Nogueras c, Justo Cobo c
a Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, A. A. 25360 Cali, Colombia
b PECET-SIU, Universidad de Antioquia, PO Box 1226, Medellín, Colombia
c Department of Inorganic and Organic Chemistry, Universidad de Jaén, 23071 Jaén, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of N-acetyl and N-formyl-pyrazoline derivatives 6 and 7e8 were synthesized by cyclo-
condensation reaction of [(7-chloroquinolin-4-yl)amino]chalcones with hydrazine hydrate in acetic acid
and hydrazine hydrate in formic acid respectively. These compounds were evaluated in vitro as antitumor
and as antimalarial agents. Compounds 7b and 8bee showed remarkable antitumor activity against
Received 22 October 2012
Received in revised form
9 June 2013
Accepted 19 June 2013
Available online 2 July 2013
cancer cell lines, with the most important GI50 values ranging from 0.13 to 0.99
response was observed for compound 7a with an inhibition percentage of 50.8% for Plasmodium falcip-
arum, a hemolytic capacity of 3.2% and an IC50 of 14.1 g/mL.
Ó 2013 Elsevier Masson SAS. All rights reserved.
mM. The best antimalarial
m
Keywords:
Pyrazolines
Chalcones
Cyclocondensation reaction
Antitumor and antimalarial activities
1. Introduction
aminoquinolines as antimalarial agents suggested that the 7-
chloro-4-aminoquinoline nucleus is obligatory for antimalarial ac-
tivity, particularly, due to its accumulation into the vacuole of the
The quinoline nucleus is an important class of heterocyclic
structure found in many synthetic and natural occurring products
with a wide range of pharmacological activities [1], such as antiviral
[2], anticancer [3], antibacterial [4], antifungal [5], antiobesity [6]
and anti-inflammatory [7]. These properties are well illustrated
by the large number of commercially available drugs containing
this heterocyclic system.
The research work on aminoquinolines could be justified on the
basis of several considerations: (1) aminoquinolines have proved to
be the most highly successful class of compounds for the treatment
and prophylaxis of malaria; (2) 4-aminoquinolines are easily syn-
thesized and inexpensive to produce; (3) 4-aminoquinolines are
generally well tolerated, with acceptable toxicity profiles for
treatment of acute infection; (4) the 4-aminoquinolines has resul-
ted be effective against chloroquine-resistant strains of the parasite
[8]. The structureeactivity relationship studies on 4-
parasite, the inhibition of b-hematin formation and for instance,
permitting the accumulation of FPIX-4-aminoquinoline complex,
a highly toxic substance for the parasite which causes its dead
[9e16].
Chalcones have extensively been studied for their broad spec-
trum of biological activities as antifungal [17], antiparasitic [18],
antimitotic [19], antitumor and antioxidant [20], immunomodula-
tory [21], antileishmanial [22], antimalarial [23,24], anti-invasive
[25,26] and anti-inflammatory [27e30].
Reports on pyrazolines, reflects the biological activity that this
kind of compound have displayed as anti-inflammatory [31,32],
antitumor [33], antimycobacterial [34], antidepressants and anti-
convulsants [35], antimicrobial [36,37] and antifungal [38], among
others.
Continuing with our current studies directed toward the syn-
thesis of pyrazoline derivates with biological activity [39,40], in this
paper we describe the synthesis of new pyrazoline derivatives,
containing the 7-chloro-4-aminoquinoline moiety in their struc-
tures, and the evaluation of their cytotoxic and antimalarial
activities.
* Corresponding author. Fax: þ57 2 33392440.
E-mail
addresses:
braulio.insuasty@correounivalle.edu.co,
brainsu@
univalle.edu.co (B. Insuasty).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.049

B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
253
2. Results and discussion
2.1. Chemistry
against the 60 tumor cell lines at a single dose of 10
m
M after 48 h of
incubation. The output from the single dose screening was reported
as a mean graph available for analysis by the COMPARE program
(data are not shown). The results of this first assay showed that
compounds 6b, 7bef and 8bee were active. A structural feature
which worth be highlighted is the fact that none of the pyrazolines
bearing the Br-substituent, i.e. 6a, 7a and 8a were chosen to
examine their antitumor activity.
Then, active compounds passed to a second stage in order to
determine their cytostatic activity against the 60 tumor cell lines
represented in leukemia, melanoma, lung, colon, brain, breast, ovary,
kidney and prostate panels; where the testing results were
expressed according to the following three parameters: GI50 which
is the molar concentration of the compounds required to inhibit the
growing of the cell lines to 50% (relative to untreated cells). TGI as
the molar concentration that causes total growth inhibition, and
LC50 which is a parameter of cytotoxicity and reflects the molar
concentration needed to kill 50% of the cells [42]. The active com-
pounds were evaluated at five concentration levels (100, 10, 1.0, 0.1,
In order to obtain the key a,b-unsaturated carbonyl compounds
(chalcones 4 and 5), as starting materials for the synthesis of the
target products 6e8, precursors 4-(7-chloroquinolin-4-ylamino)
acetophenone 2 [41] and 3-(7-chloroquinolin-4-ylamino)aceto-
phenone 3 [41] were obtained through a selective nucleophilic
aromatic substitution (SNAr) of the 4-chlorine atom on the 4,7-
dichloroquinoline 1 with 3- and 4-aminoacetophenones respec-
tively (Scheme 1). Then the ClaiseneSchmidt condensation of ke-
tones 2 and 3 with different aromatic aldehydes led to the
formation of chalcones 4 and 5, respectively, in good yields
(Scheme 1).
The IR spectrum of compound 4a taken in KBr pellet showed an
absorption band at 1656 cmꢀ1 corresponding to the stretching vi-
bration of the carbonyl group. In the 1H NMR spectrum of com-
pound 4a is observed two doublets at 7.66 and 7.87 ppm
corresponding to protons H-200 and H-300 with J ¼ 16.3 Hz, con-
firming the E-configuration for the C]C bond.
Reaction of the synthesized chalcones 4 and 5 with hydrazine
hydrate in acetic acid or in formic acid by stirring at room tem-
perature or under reflux led to the formation of the N-acetyl and N-
formyl-pyrazolines 6 and 7,8 respectively (Scheme 2). Products 6e8
were obtained in acceptable to excellent yields. The new pyrazo-
lines 6e8, were fully characterized by means of spectroscopic
techniques such as FT-IR and 1D and 2D-NMR (see Experimental
section).
and 0.01
mM) and the test consisted of a 48 h continuous drug
exposure protocol using sulforhodamine B (SRB) protein assay to
estimate cell growth. Details of this evaluation method, and the
complementary information related with the activity pattern over
all cell lines, have been published [43e47].
As an outstanding result, compounds 6b, 7b,c and 8bee
exhibited significant activities, with GI50 values lower than
1.0 ꢁ 10ꢀ6 M against several cell lines. Particularly, compounds 8
were relatively more actives than compounds 6 and 7. Probably the
meta substitution of the ring C in compounds 8 could be related
with this finding. A raw comparison of the activities of our obtained
compounds 6e8 with respect to the activity reported for the
standard drug Adriamycin, used by NCI as control, reflects that the
activities displayed for our compounds against several cell lines
were comparable or even higher than those for the standard drug
control. In this sense, it is remarkable that compounds 7b, 8b, 8c, 8d
and 8e displayed activities with GI50 values of 0.58, 0.90, 0.52, 0.44
and 0.55 ꢁ 10ꢀ6 M, respectively, against the HCT-15 cell line (Colon
cancer panel), while this value was 6.46 ꢁ 10ꢀ6 M for the standard
drug Adriamycin; compounds 7b, 8c, 8d and 8e displayed activities
with GI50 values of 0.20, 0.13, 0.14 and 0.22 ꢁ 10ꢀ6 M, respectively,
against the MDA-MB-435 cell line (Melanoma panel), while this
value was 0.25 ꢁ 10ꢀ6 M for Adriamycin; as well as, compound 8b
had a GI50 value of 0.29 ꢁ 10ꢀ6 M against this cell line. Compounds
7b and 8c displayed activities with GI50 values of 0.35 and
0.28 ꢁ 10ꢀ6 M, respectively, against the OVCAR-3 cell line (Ovarian
cancer panel), while this value was 0.39 ꢁ 10ꢀ6 M for Adriamycin;
compounds 7b, 8b, 8c and 8d displayed activities with GI50 values
of 0.36, 0.76, 0.29 and 0.35 ꢁ 10ꢀ6 M, respectively, against the NCI/
ADR-RES cell line (Ovarian cancer panel), while this value was
As an example, in the IR spectrum for compound 6a, an ab-
sorption band is observed at 1646 cmꢀ1 which corresponds to the
stretching vibration of the C]O amide functionality. At 1640 and
1579 cmꢀ1 are observed the stretching bands of C]N and C]C
respectively. In the 1H NMR spectrum for compound 6a, the protons
on the diastereotopic center C-40, of the pyrazoline ring appears as
2
two double-doublets at
d
¼ 3.14 and 3.85 ppm with JAM ¼ 17.9,
3JAX ¼ 4.3 and 3JMX ¼ 11.9 Hz, while the H-50 proton is observed as a
double-doublet at 5.53 ppm with 3JMX ¼ 11.9 and 3JAX ¼ 4.3 Hz. All
types of carbon atoms were completely assigned by using DEPT-
135, HSQC and HMBC techniques.
2.2. Anticancer activity
As a preliminary screening, structures of all new compounds (i.e.
2e3, 4e5, and 6e8 series) were submitted to the Developmental
Therapeutics Program (DTP) at National Cancer Institute (NCI) for
evaluation of their anticancer activity against different human cell
lines. Twelve (i.e. 6b,d,e; 7bef and 8bee) of the submitted struc-
tures were selected and subjected to the preliminary evaluation
Scheme 1. Synthesis of [(7-chloroquinolin-4-yl)amino]chalcones 4 and 5.

254
B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
Scheme 2. Synthesis of new N-acetyl and N-formylpyrazolines 6 and 7e8.
7.16 ꢁ 10ꢀ6 M for Adriamycin. Compounds 7b, 8b, 8c, 8d and 8e
displayed activities with GI50 values of 0.41, 0.95, 0.33, 0.33 and
0.55 ꢁ 10ꢀ6 M, respectively, against the CAKI-1 cell line (Renal
cancer panel), while this value was 0.95 ꢁ 10ꢀ6 M for Adriamycin.
Compound 8c displayed GI50 values of 0.41, 0.29 and 0.21 ꢁ10ꢀ6 M
against UO-31 (Renal cancer panel), HS 578T and BT-549 (Breast
cancer panel) cell lines, respectively, while the values against the
same cell lines for Adriamycin were 0.49, 0.33 and 0.23 ꢁ 10ꢀ6 M,
respectively. Additionally comparable activities were found for
compounds 8c and 8d displaying GI50 values of 0.29 and
0.36 ꢁ 10ꢀ6 M against KM12 (Colon cancer panel) and HS 578T
(Breast cancer panel) cell lines, respectively, while the corre-
sponding values for Adriamycin were 0.27 and 0.33 ꢁ 10ꢀ6 M,
respectively. These findings make compounds 7b and 8cee,
promising targets to perform structural modifications pursuing to
improve their activities and hence to develop possible new leader
antitumor molecules based on their structures. Moreover, the
values of cytotoxicity of the evaluated compounds 6e8, measured
7d, 7f, 8c and 8f) of the 18 screened compounds (i.e. 6e8 series), at
concentrations of 20 g/mL, do not showed any growth inhibition
of P. falciparum. The remaining compounds showed changes in the
rate of growth and reproduction of the parasite.
m
The best doseeresponse observed was by compound 7a with a
percentage growth inhibition of 50.8%, followed by compounds
6bed with percentages growth inhibitions >30% and compounds
8e and 7c with percentage of inhibition of 24.6 ꢂ 3.2 and 20.2 ꢂ 4.4,
respectively (see Table 3). Although the percentages growth in-
hibitions for all assayed compounds were lower than that for the
standard (CQ), it is worth mention that compound 7a shown the
closer value (i.e. 50.8%) compared with reference drug (CQ) which
had a percentage growth inhibition of 68.4%, making compound 7a
as the more promising structure, of the obtained compounds, for
our future drug developing antimalarial studies.
2.3.2. Hemolytic activity
For the most active compounds (i.e. 6b, 6c, 6d and 7a), it was
determined their hemolytic activities to confirm that the action was
directed against the parasite and not host cells destruction
occurred. None of the compounds screened showed a marked he-
molytic capacity, i.e. ꢃ5%. Indeed, all of them displayed values
lower than that for the standard drug (CQ) (see Table 3).
as LC50, were higher than 100 mM, for the most cell lines, indicating
a low toxicity of such compounds for normal human cell lines, as
required for developing of potential antitumor agents (see Tables 1
and 2).
2.3. Antimalarial activity
2.3.3. Determination of the IC50
IC50 analysis was performed only for compounds 6b, 6c, 6d and
7a, since they showed percentages growth inhibitions higher than
30%. The results indicated that compound 7a displayed the lower
2.3.1. Antiplasmodial activity
The antiplasmodial activity was evaluated in vitro on asyn-
chronic cultures of Plasmodium falciparum (NF54 strain), see
Experimental section. It have been stated that an inhibition of
growing higher than 20% represents a significant antimalarial ac-
tivity for the compound evaluated. After our essays, only 5 (i.e. 6e,
IC50 value (i.e. 14.1
still too far in comparing with that for the standard drug (CQ) (i.e.
0.02 g/mL) (see Table 3).
mg/mL) of the assayed compounds, although it is
m

Table 1
In vitro testing expressed as growth inhibition of cancer cell lines for compounds 6b and 7bef.a
Panel/cell line
Compounds
6b
7b
7c
7d
7e
7f
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
(
m
M)
(
m
(
mM)
(
m
(m
M)
(
m
(
m
M)
(
m
(
mM)
(
m
(m
M)
(
m
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
e
e
e
e
2.51
2.66
2.28
2.55
1.77
>100
72.5
>100
>100
>100
2.71
2.23
2.54
2.71
2.83
>100
>100
>100
>100
>100
1.82
2.53
3.36
2.56
2.58
>100
75.8
>100
>100
>100
2.25
2.27
5.13
2.18
3.18
>100
>100
>100
>100
>100
3.56
2.42
e
>100
>100
e
0.38
0.53
e
>100
>100
e
0.61
>100
0.32
>100
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
8.81
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.99
>100
>100
>100
>100
>100
>100
>100
>100
>100
4.05
4.94
5.26
4.28
5.22
3.64
e
>100
>100
>100
>100
>100
>100
e
50.3
>100
43.7
>100
>100
>100
>100
>100
>100
e
6.90
11.7
4.13
1.83
34.5
20.4
e
>100
>100
96.1
>100
>100
>100
e
9.86
>100
>100
>100
>100
>100
>100
e
>100
>100
>100
>100
>100
>100
5.05
0.86
>100
4.66
2.77
>100
0.59
0.39
>100
2.14
>100
6.93
e
>100
5.06
e
3.96
e
>100
e
>100
e
>100
e
4.42
e
>100
e
>100
e
>100
e
5.93
>100
38.8
3.33
5.40
6.59
>100
>100
>100
>100
>100
>100
>100
>100
2.40
1.61
0.50
0.58
0.41
0.54
0.48
>100
>100
>100
>100
>100
>100
>100
2.21
3.50
3.58
2.54
3.21
3.41
2.73
>100
>100
>100
>100
>100
>100
68.7
3.56
18.1
9.97
2.97
3.81
16.5
35.8
>100
>100
>100
>100
>100
>100
>100
3.73
5.52
3.21
3.56
2.83
5.87
3.49
>100
>100
>100
>100
>100
>100
>100
4.59
7.44
25.2
2.78
6.09
6.64
>100
>100
>100
>100
>100
>100
>100
>100
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
9.10
3.58
>100
>100
>100
>100
>100
>100
>100
>100
0.85
0.27
0.58
1.92
0.42
0.51
>100
>100
>100
>100
>100
>100
3.96
2.41
4.57
5.13
5.03
3.12
>100
>100
99.2
>100
>100
>100
>100
24.8
>100
5.65
>100
1.50
>100
>100
>100
>100
>100
>100
28.0
5.63
2.75
18.2
5.19
2.06
>100
>100
37.6
>100
>100
19.4
8.61
4.00
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
SNB-75
U251
>100
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
3.15
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.60
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.61
4.21
4.12
1.59
e
6.88
>100
>100
>100
e
2.32
>100
7.67
1.72
e
>100
>100
>100
>100
e
1.89
3.20
3.75
2.10
e
7.83
>100
48.3
>100
e
4.11
>100
6.88
1.95
e
>100
>100
>100
>100
e
5.24
0.57
>100
4.88
2.20
>100
2.95
0.62
0.20
7.86
0.76
0.81
>100
0.57
8.35
1.38
10.1
1.55
>100
11.2
>100
17.4
20.9
0.62
4.39
5.99
>100
>100
>100
>100
2.22
1.98
2.84
2.20
14.0
35.5
55.4
59.3
>100
1.74
>100
4.13
>100
>100
>100
>100
>100
7.19
6.92
>100
>100
3.04
>100
>100
>100
>100
>100
>100
>100
5.01
0.35
6.48
6.46
3.22
0.36
0.57
>100
>100
>100
>100
>100
>100
>100
9.36
2.78
3.88
19.8
4.53
2.69
11.3
>100
43.2
43.1
14.5
13.4
>100
69.8
7.04
>100
>100
>100
>100
>100
>100
>100
>100
22.3
1.91
3.19
69.2
9.98
7.88
3.36
>100
6.03
26.6
6.82
5.15
>100
>100
5.22
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
55.8
>100
>100
>100
>100
3.96
>100
5.07
30.7
>100
3.87
>100
>100
>100
>100
>100
>100
>100
>100
0.91
>100
1.00
0.41
1.88
0.98
9.57
1.30
>100
>100
>100
>100
>100
>100
>100
>100
3.36
1.30
4.21
3.82
2.20
3.80
9.51
3.81
98.4
32.0
>100
7.02
>100
11.6
12.6
20.9
19.7
>100
>100
>100
>100
>100
>100
>100
>100
2.31
15.3
3.24
5.47
2.22
4.37
3.09
4.96
9.93
>100
47.0
>100
>100
>100
38.6
50.9
>100
>100
>100
>100
>100
>100
>100
>100
A498
ACHN
CAKI-1
RXF 393
SN12C
>100
>100
>100
>100
>100
>100
>100
3.27
>100
4.89
7.23
>100
5.49
TK-10
UO-31
67.5
Prostate cancer
PC-3
DU-145
3.02
>100
>100
>100
0.48
1.94
>100
>100
4.76
3.80
>100
>100
9.43
38.0
>100
>100
11.7
6.63
>100
>100
4.61
>100
>100
>100
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
4.04
>100
>100
5.40
1.57
2.41
>100
>100
>100
>100
>100
>100
0.48
1.43
3.93
4.77
1.56
0.32
>100
>100
>100
>100
>100
>100
3.42
2.43
3.77
3.81
2.18
0.98
>100
>100
>100
53.8
>100
10.0
8.78
20.1
>100
10.9
1.38
1.00
>100
>100
>100
>100
>100
>100
3.23
4.39
3.24
1.91
2.33
1.83
>100
>100
>100
8.23
>100
40.9
8.39
6.09
>100
>100
>100
>100
>100
>100
5.64
1.65
1.51
T-47D
MDA-MB-468
a
Data obtained from NCI's in vitro disease-oriented human tumor cell lines screen [42e47].
b
GI50 was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.
Determined at five concentration levels (100, 10, 1.0, 0.1, and 0.01 M).
LC50 is a parameter of cytotoxicity and reflects the molar concentration needed to kill 50% of the cells.
m
c

256
B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
3. Conclusions
determined in a Büchi melting point apparatus and are uncorrec-
ted. IR spectra were performed on a Shimadzu FTIR 8400 spectro-
photometer in KBr disks. The 1H and 13C NMR spectra were run on a
Bruker DPX 400 spectrophotometer operating at 400 MHz and
100 MHz, respectively, using dimethyl sulfoxide-d6 as solvent and
tetramethylsilane as internal reference. The mass spectra were
obtained on a Hewlett Packard HP Engine-5989 spectrometer
(equipped with a direct inlet probe) operating at 70 eV. The
elemental analyses were obtained using a Thermo Finnigan Flash
EA1112 CHN (STIUJA) elemental analyzer.
Starting from compounds 4 and 5 were synthesized three novel
series of acetyl 6aef and N-formyl 7aef and 8aef pyrazoline der-
ivates through a simple and easy synthetic methodology, con-
ducting to the obtained products in acceptable to excellent yields
and in short reaction times. The anticancer evaluation data against
60 cancer cell lines revealed that derivatives 7b and 8bee exhibited
the highest activities with GI50 values lower than 1.0 mM for the
most of the cell lines tested. Indeed, such compounds displayed
higher activity against several cell lines than the standard drug
Adriamycin. Regarding to the antimalarial activity, derivative 7a
was the most active compound inhibiting the growth of
P. falciparum parasites in 50.8%. Derivatives 6d, 6c and 6b were
moderately active inhibiting the growth of P. falciparum in 38.3,
33.0 and 32.9%, respectively. None of the evaluated compounds
showed marked hemolytic activity, moreover, their values were
lower than that for the standard drug chloroquine (CQ). Owing to
the significant obtained results, chemical studies are currently be-
ing conducted pursuing for an improvement of the antitumor and
antimalarial activities of such compounds.
4.1. Chemistry
4.1.1. General procedure for the synthesis of the precursors
Using a similar methodology like reported by Ferrer and co-
workers [41], it was carried out the synthesis of the precursors 2, 3,
4 and 5.
4.1.1.1. General procedure for the synthesis of [(7-chloroquinolin-4-yl)
amino]acetophenones 2 and 3. A mixture of 4,7-dichloroquinoline
(2.5 mmol) and 3- or 4-aminoacetophenone (2.75 mmol) in
ethanol (10 mL) was heated under reflux for 8 h. The solid formed
was filtered and recrystallized from ethanol.
4. Experimental
Commercially available starting materials, reagents and solvents
were used as supplied. TLC analyses were performed on Merck
silica gel 60 F254 aluminum plates. Melting points were
4.1.1.2. General procedure for the synthesis of [(7-chloroquinolin-4-
yl)amino]chalcones 4 and 5. A mixture of [(7-chloroquinolin-4-yl)
amino]acetophenone
2
or
3
(0.36 mmol), the respective
Table 2
In vitro testing expressed as growth inhibition of cancer cell lines for compounds 8bee and the standard drug Adriamycin.a
Panel/cell line
Compounds
Doxorubicin
(Adriamycin), NSC
123127
8b
8c
8d
8e
100
m
Md
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
(m
M)
(
m
(
m
M)
(
m
(
m
M)
(
m
(m
M)
(
m
(
mM)
(
m
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
2.34
>100
40.2
>100
>100
>100
>100
0.37
0.20
0.26
0.39
0.49
0.20
>100
4.56
>100
>100
>100
>100
0.68
>100
5.28
>100
>100
>100
>100
0.65
0.26
0.43
0.59
8.83
0.34
>100
3.97
>100
>100
>100
>100
0.08
0.12
0.19
0.03
0.08
0.03
100.00
89.33
100.00
100.00
100.00
100.00
1.10
0.41
1.92
2.45
0.37
0.25
0.29
0.49
16.7
0.24
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCIeH226
NCIeH23
NCIeH322M
NCIeH460
NCIeH522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
0.94
>100
>100
>100
>100
>100
>100
e
0.54
0.95
0.51
0.21
6.91
0.61
e
>100
>100
>100
>100
>100
>100
e
0.57
0.64
0.66
>100
>100
>100
>100
>100
>100
e
0.66
0.98
1.12
2.75
2.20
1.38
e
>100
>100
66.5
>100
>100
>100
e
0.06
0.41
0.07
0.10
0.05
0.15
0.54
0.02
0.03
100.00
47.97
67.61
42.27
6.40
13.15
67.76
51.29
2.80
>100
1.91
0.65
>100
>100
41.6
0.95
2.48
e
e
0.79
0.80
>100
>100
0.41
0.21
>100
>100
0.40
0.30
>100
>100
0.42
0.75
>100
>100
1.39
3.36
0.60
0.90
0.48
0.59
0.56
>100
>100
>100
>100
>100
>100
>100
0.36
1.22
0.39
0.52
0.32
0.29
0.35
7.92
>100
4.75
>100
4.32
0.45
1.32
0.38
0.44
0.34
0.44
0.36
13.9
>100
38.1
>100
>100
>100
>100
0.64
2.16
0.40
0.55
0.40
0.50
0.47
>100
>100
6.71
>100
4.99
0.18
0.26
0.08
6.46
0.12
0.27
0.09
4.33
21.68
54.58
100.00
67.45
92.68
58.61
>100
>100
>100
>100
4.37
1.06
3.78
3.48
1.58
1.29
>100
>100
>100
>100
>100
>100
1.04
0.37
0.31
0.60
0.22
0.37
>100
2.56
0.32
0.61
0.87
0.33
0.45
>100
>100
66.2
>100
>100
41.8
2.55
0.38
0.81
3.42
0.38
0.45
>100
>100
55.5
>100
51.0
>100
0.10
0.10
0.12
0.04
0.07
0.04
30.48
69.98
27.23
49.77
3.30
41.2
35.7
>100
>100
5.07
U251
30.62
Melanoma

B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
257
Table 2 (continued )
Panel/cell line
Compounds
Doxorubicin
(Adriamycin), NSC
123127
8b
8c
8d
8e
100
m
Md
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
GI50b
LC50c
M)
(
mM)
(
m
(
m
M)
(
m
(m
M)
(
m
(
mM)
(
m
(
m
M)
(
m
LOX IMVI
MALME-3M
M14
1.56
50.4
0.45
0.37
0.33
0.13
0.26
0.76
0.29
0.33
0.53
4.23
0.58
6.70
0.71
0.81
0.35
0.22
0.73
1.02
0.42
1.40
0.72
>100
>100
57.5
>100
50.1
97.1
23.5
>100
84.0
0.07
0.12
0.18
0.25
0.17
0.21
0.08
0.14
0.12
50.35
3.97
4.05
9.57
1.06
15.92
0.49
8.15
0.74
1.63
1.04
0.29
2.05
5.63
1.86
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
64.2
>100
>100
0.74
0.40
0.14
0.49
0.96
0.41
>100
>100
>100
>100
>100
>100
>100
>100
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
1.63
0.67
0.76
1.20
4.13
>100
>100
>100
>100
>100
>100
>100
0.82
0.28
0.56
1.38
0.51
0.29
0.33
>100
1.44
0.47
0.89
3.51
0.95
0.35
0.70
>100
77.4
3.29
0.52
1.13
4.32
2.06
1.36
1.40
>100
6.06
0.17
0.39
0.37
0.41
0.10
7.16
0.22
100.00
84.33
74.30
100.00
43.25
100.00
100.00
7.38
>100
>100
56.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.80
0.76
3.48
4.73
0.83
5.18
0.95
3.08
3.86
>100
>100
>100
>100
>100
>100
e
0.59
0.23
0.86
0.33
0.39
0.63
e
>100
>100
>100
0.53
0.26
0.87
0.33
0.85
0.78
>100
>100
>100
>100
>100
>100
e
0.84
0.29
1.27
0.55
1.54
0.78
e
76.3
>100
>100
12.1
73.4
>100
0.13
0.10
0.08
0.95
0.10
0.07
0.18
0.49
51.64
1.90
100.00
100.00
4.69
72.44
86.70
26.18
35.3
>100
>100
e
e
e
e
e
0.88
>100
0.41
>100
0.85
>100
1.30
>100
e
e
e
0.85
1.35
>100
>100
1.46
2.68
>100
>100
0.32
0.11
87.10
100.00
DU-145
4.17
>100
0.44
>100
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
0.81
2.12
1.97
1.36
2.63
0.42
>100
>100
>100
>100
>100
>100
0.34
1.15
0.29
0.21
0.34
0.22
>100
>100
>100
30.9
>100
>100
0.37
3.19
0.36
0.45
0.53
0.21
>100
>100
>100
55.6
>100
>100
0.37
1.60
0.68
0.39
0.65
0.30
>100
>100
>100
4.61
69.3
23.3
0.03
0.51
0.33
0.23
0.06
0.05
51.29
34.75
85.70
21.33
85.70
2.52
MDA-MB-468
a
Data obtained from NCI’s in vitro disease-oriented human tumor cell lines screen [42e47].
b
GI50 was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.
Determined at five concentration levels (100, 10, 1.0, 0.1, and 0.01 M).
m
c
LC50 is a parameter of cytotoxicity and reflects the molar concentration needed to kill 50% of the cells.
d
The values of activity against human tumor cell lines displayed by Adriamycin correspond to the reported by NCI at highest concentration of 10ꢀ4 M. Please visit: http://
dtp.nci.nih.gov/dtpstandard/cancerscreeningdata/index.jsp.
benzaldehyde (0.40 mmol), and potassium hydroxide (one pellet)
in methanol (8 mL) was stirred at room temperature for 12 h. Then,
water (2 mL) was added and the resulting precipitate was collected
by filtration, washed with water and recrystallized from a mixture
ethanol/water (1:0.5).
4.1.1.2.2. (E)-3-(4-Bromophenyl)-1-[3-(7-chloroquinolin-4-ylami
no)phenyl]prop-2-en-1-one 5a. Yellow solid; 94% yield; mp: 201e
203 ꢄC. FTIR (KBr) (cmꢀ1): 3347 (NH), 3059 (]CeH), 1660 (C]O)
y
and 1576 (C]N and C]C). 1H NMR (400 MHz, DMSO-d6)
d ppm
7.01 (d, J ¼ 5.3 Hz, 1H, H-3), 7.57e7.98 (m, 11H, H-6, H-200, H-300, Ho,
Ho0, Hm, Hp, Ho00, Hm00), 8.08 (s, 1H, H-8), 8.47 (d, J ¼ 9.0 Hz, 1H, H-
5), 8.51 (d, J ¼ 5.3 Hz, 1H, H-2), Not observed (br s, 1H, NH). 13C NMR
Analytical data for compounds 2, 3, 4bef and 5bed were
consistent with those reported for the same compounds in Ref. [41].
4.1.1.2.1. (E)-3-(4-Bromophenyl)-1-[4-(7-chloro-quinolin-4-ylami
no)phenyl]prop-2-en-1-one 4a. Yellow solid; 81% yield; mp: 225e
(100 MHz, DMSO-d6)
d ppm 78.9, 101.9, 118.3, 121.6, 122.6, 123.7,
124.2, 124.8, 126.4, 127.4, 129.6, 130.5, 131.6, 133.6, 133.7, 138.5,
140.8, 142.5, 147.3, 149.4, 151.7, 188.6. Anal. Calcd. For
226 ꢄC. FTIR (KBr) (cmꢀ1): 3388 (NH), 3059 (]CeH), 1656 (C]O),
y
1608 and 1573 (C]N and C]C). 1H NMR (400 MHz, DMSO-d6)
C
24H16BrClN2O: C, 62.16; H, 3.48; N, 6.04. Found: C, 62.10; H, 3.56;
N, 6.10.
4.1.1.2.3. (E)-1-[3-(7-Chloroquinolin-4-ylamino)phenyl]-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one 5e. Yellow solid; 71% yield; mp:
256e258 ꢄC. FTIR (KBr) (cmꢀ1): 3307 (NH), 3062 (]CeH), 1647
(C]O) and 1575 (C]N and C]C). 1H NMR (400 MHz, DMSO-d6)
d
ppm 7.27 (d, J ¼ 3.7 Hz, 1H, H-3), 7.46 (d, J ¼ 8.5 Hz, 2H, Ho0), 7.57
(d, J ¼ 9.1 Hz, 1H, H-6), 7.63 (d, J ¼ 8.5 Hz, 2H, Ho), 7.66 (d,
J ¼ 16.3 Hz, 1H, H-200), 7.78 (d, J ¼ 8.5 Hz, 2H, Hm0), 7.87 (d,
J ¼ 16.3 Hz, 1H, H-300), 7.96 (s, 1H, H-8), 8.15 (d, J ¼ 8.5 Hz, 2H, Hm),
8.37 (d, J ¼ 9.1 Hz, 1H, H-5), 8.64 (d, J ¼ 3.7 Hz, 1H, H-2), 9.26 (s, 1H,
y
NH). 13C NMR (100 MHz, DMSO-d6)
d
ppm 119.0, 121.9, 122.9, 123.7,
d
ppm 3.73 (s, 3H, OCH3), 3.87 (s, 6H, OCH3), 7.04 (d, J ¼ 5.3 Hz,1H, H-
124.7, 125.6,127.8, 128.3,130.6,131.1, 131.4, 131.8, 134.2, 134.6,136.6,
141.8, 146.0, 150.0, 152.1, 187.1. Anal. Calcd. For C24H16BrClN2O: C,
62.16; H, 3.48; N, 6.04. Found: C, 62.22; H, 3.51; N, 5.97.
3), 7.24 (s, 2H, Ho00), 7.56e8.00 (m, 7H, H-6, H-200, H-300, Ho, Hm, Hp,
Ho0), 8.06 (s,1H, H-8), 8.48 (d, J ¼ 9.0 Hz,1H, H-5), 8.52 (d, J ¼ 5.3 Hz,
1H, H-2), Not observed (br s,1H, NH). 13C NMR (100 MHz, DMSO-d6)

258
B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
2H, Hm0), 7.60 (d, J ¼ 8.4 Hz 1H, H-6), 7.79 (d, J ¼ 8.3 Hz, 2H, Hm),
7.93 (s, 1H, H-8), 8.41 (d, J ¼ 8.4 Hz, 1H, H-5), 8.54 (d, J ¼ 4.9 Hz, 1H,
Table 3
In vitro testing antimalarial and hemolytic activity for compounds 6aef, 7aef and
8aef and the standard drug chloroquine (CQ).a
H-2), 9.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d ppm 22.2,
b
Compound Inhibition
Coefficient of Hemolysis (%) IC50
variation
(
mg/mL) ꢂ SD
42.4, 59.4, 103.9, 119.4, 120.7, 121.4, 125.1, 125.8, 126.1, 128.2, 128.3,
128.4, 132.0, 134.6, 142.3, 143.1, 147.3, 150.1, 152.5, 154.3, 167.8. MS
(70 eV) m/z (%): 518/520 [Mþ] (100/75), 478 (63), 480 (45), 43 (64).
Anal. Calcd. For C26H20BrClN4O: C, 60.07; H, 3.88; N,10.78. Found: C,
60.20; H, 3.95; N, 10.82.
(%) ꢂ SD
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
6.8 ꢂ 0
0
n.e.
n.e.
32.9 ꢂ 1.8
5.5
3.4 ꢂ 0.05
2.8 ꢂ 0.6
3.1 ꢂ 0.2
n.e.
54.6 ꢂ 1.5
38.0 ꢂ 1.9
>100
n.e.
33.0 ꢂ 8.0 24.0
38.3 ꢂ 3.0
0 ꢂ 0
7.8
0
4.1.2.2. 1-(5-(4-Chlorophenyl)-3-[4-(7-chloroquinolin-4-ylamino)
phenyl]-4,5-dihydro-1H-pyrazole-1-yl)ethanone 6b. White solid;
10.6 ꢂ 2.8 26.5
n.e.
n.e.
50.8 ꢂ 4.6
9.0
0
3.2 ꢂ 0.1
14.1 ꢂ 1.7
y
(cmꢀ1): 3316 (NH), 3068
1.1
n.e.
n.e.
96% yield; mp: 139e142 ꢄC. FTIR (KBr)
20.2 ꢂ 4.4 22.2
3.8 ꢂ 0.6
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
(]CeH), 1645 (C]O), 1614 and 1577 (C]N and C]C). 1H NMR
(400 MHz, DMSO-d6)
0 ꢂ 0
0
0
0
d
ppm 2.30 (s, 3H, CH3), 3.11 (dd, J ¼ 17.8,
5.8 ꢂ 0
0 ꢂ 0
3.9 Hz, 1H, H-40a), 3.83 (dd, J ¼ 17.8, 12.0 Hz, 1H, H-40b), 5.53 (dd,
J ¼ 12.0, 3.9 Hz, 1H, H-50), 7.13 (d, J ¼ 5.3 Hz, 1H, H-3), 7.22 (d,
J ¼ 8.3 Hz, 2H, Ho0), 7.39 (d, J ¼ 8.3 Hz, 2H, Hm0), 7.44 (d, J ¼ 8.0 Hz,
2H, Ho), 7.59 (d, J ¼ 9.7 Hz,1H, H-6), 7.80 (d, J ¼ 8.0 Hz, 2H, Hm), 7.93
(s, 1H, H-8), 8.40 (d, J ¼ 9.7 Hz,1H, H-5), 8.54 (d, J ¼ 5.3 Hz, 1H, H-2),
n.e.
8a
8b
8c
8d
8e
8f
12.5 ꢂ 2.0 16.0
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
7.0 ꢂ 1.2
0 ꢂ 0
16.6
0
4.1 ꢂ 0
0
n.e.
n.e.
24.6 ꢂ 3.2 13.1
3.4 ꢂ 0.2
n.e.
n.e.
9.29 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d ppm 22.2, 42.4,
0 ꢂ 0
0
n.e.
CQc
68.4 ꢂ 3.6
5.5
17.2 ꢂ 0.6
0.02 ꢂ 0.01
59.4, 103.9, 119.4, 120.7, 121.4, 125.1, 125.8, 126.1, 128.2, 128.3, 128.4,
132.0, 134.6, 142.3, 143.1, 147.2, 150.1, 152.5, 154.3, 167.8. MS (70 eV)
m/z (%): 474/476 [Mþ] (100/71), 432 (73), 434 (49), 321 (14), 43 (25).
Anal. Calcd. For C26H20Cl2N4O: C, 65.69; H, 4.24; N, 11.79. Found: C,
65.59; H, 4.29; N, 11.88.
SD: Standard deviation.
n.e.: not evaluated.
a
Data obtained from PECET in vitro for the antimalarial activity [48e52].
b
IC50
parasite.
(mg/mL): concentration corresponding to 50% growth inhibition of the
c
CQ: chloroquine diphosphate salt.
4.1.2.3. 1-(3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-phenyl-4,5-
dihydro-1H-pyrazole-1-yl)ethanone 6c. White solid; 89% yield; mp:
141e143 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3276 (NH), 3172 (]CeH), 1642
d
ppm 55.7, 59.6, 78.7, 101.7, 106.2, 118.7, 120.9, 121.2, 121.9, 123.5,
(C]O), 1614 and 1573 (C]N and C]C). 1H NMR (400 MHz, DMSO-
124.0,124.6,125.9,126.6,127.2,129.3,130.3,134.2,139.2,140.1,141.1,
144.3, 151.5, 153.3, 189.1. Anal. Calcd. For C27H23ClN2O4: C, 68.28; H,
4.88; N, 5.90. Found: C, 68.17; H, 4.96; N, 5.98.
d6)
d
ppm 2.30 (s, 3H, CH3), 3.10 (dd, J ¼ 18.0, 4.4 Hz,1H, H-40a), 3.84
(dd, J ¼ 18.0, 11.7 Hz, 1H, H-40b), 5.53 (dd, J ¼ 11.7, 4.4 Hz, 1H, H-50),
7.13 (d, J ¼ 5.3 Hz, 1H, H-3), 7.19 (d, J ¼ 7.3 Hz, 2H, Ho0), 7.25 (m, 1H,
Hp), 7.32 (d, J ¼ 7.3 Hz, 2H, Hm0), 7.43 (d, J ¼ 8.8 Hz, 2H, Ho), 7.58
(dd, J ¼ 9.1, 2.2 Hz 1H, H-6), 7.80 (d, J ¼ 8.8 Hz, 2H, Hm), 7.93 (d,
J ¼ 2.2, 1H, H-8), 8.40 (d, J ¼ 9.1 Hz, 1H, H-5), 8.54 (d, J ¼ 5.3 Hz, 1H,
4.1.1.2.4. (E)-1-[3-(7-Chloroquinolin-4-ylamino)phenyl]3-(p-
tolyl)-prop-2-en-1-one 5f. Yellow solid; 86% yield; mp: 216e217 ꢄC.
FTIR (KBr)
and 1570 (C]N and C]C). 1H NMR (400 MHz, DMSO-d6)
y
(cmꢀ1): 3392 (NH), 3061 (]CeH), 1661 (C]O), 1595
d
ppm
H-2), 9.29 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d ppm 21.7,
2.35 (s, 3H, CH3), 7.01 (d, J ¼ 5.4 Hz, 1H, H-3), 7.27 (d, J ¼ 8.0 Hz, 2H,
Ho00), 7.53e7.97 (m, 9H, H-6, H-200, H-300, Ho, Hm, Hp, Ho0, Hm00), 8.06
(s, 1H, H-8), 8.46 (d, J ¼ 8.8 Hz, 1H, H-5), 8.51 (d, J ¼ 5.4 Hz, 1H, H-2),
42.2, 58.8, 103.4, 118.5, 120.9, 124.6, 125.4, 125.7, 127.9, 128.7, 131.3,
132.0, 134.1, 134.6, 139.5, 142.5, 146.3, 149.6, 152.0, 153.8, 167.2. MS
(70 eV) m/z (%): 440/442 [Mþ] (100/38), 398 (99), 400 (34), 321
(18), 43 (23). Anal. Calcd. For C26H21ClN4O: C, 70.82; H, 4.80; N,
12.71. Found: C, 70.73; H, 4.91; N, 12.73.
Not observed (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d ppm
21.1, 79.1, 102.2, 118.5, 121.0, 121.8, 123.9, 124.5, 125.1, 126.5, 127.7,
128.9, 129.5, 129.8, 131.9, 134.0, 138.9, 140.8, 144.3, 147.6, 149.6,
152.0, 188.9. Anal. Calcd. For C25H19ClN2O: C, 75.28; H, 4.80; N, 7.02.
Found: C, 75.19; H, 4.86; N, 6.97.
4.1.2.4. 1-(3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-(4-methoxy
phenyl)-4,5-dihydro-1H-pyrazole-1-yl)ethanone 6d. Yellow solid;
62% yield; mp: 163e165 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3261 (NH), 3075
4.1.2. General procedure for the synthesis of 1-(3-[4-((7-chloroqui
nolin-4-yl)amino)phenyl]-5-substitutedphenyl-4,5-dihydro-1H-pyr
azole-1-yl)ethanones 6aef
(]CeH), 1640 (C]O), 1612 and 1573 (C]N and C]C). 1H NMR
(400 MHz, DMSO-d6)
d
ppm 2.28 (s, 3H, CH3), 3.48 (dd, J ¼ 20.4,
5.6 Hz, 1H, H-40a), 3.74 (s, 3H, OCH3), 3.81 (dd, J ¼ 20.4, 9.3 Hz, 1H,
H-40b), 5.49 (dd, J ¼ 9.3, 5.6 Hz, 1H, H-50), 6.87 (d, J ¼ 8.4 Hz, 2H,
Ho0), 7.07 (d, J ¼ 5.2 Hz, 1H, H-3), 7.13 (d, J ¼ 8.3 Hz, 2H, Ho), 7.40 (d,
J ¼ 8.4 Hz, 2H, Hm0), 7.52 (d, J ¼ 8.0 Hz, 1H, H-6), 7.78 (d, J ¼ 8.3 Hz,
2H, Hm), 7.91 (s, 1H, H-8), 8.38 (d, J ¼ 8.0 Hz, 1H, H-5), 8.53 (d,
J ¼ 5.2 Hz, 1H, H-2), Not observed (br s, 1H, NH). 13C NMR (100 MHz,
A
mixture of 4-(7-chloroquinolin-4-yl)aminochalcone
4
(0.67 mmol), hydrazine hydrate (0.87 mmol) and acetic acid
(2.0 mL) was heated under reflux for 6 h until complete con-
sumption of the chalcone (TLC control). After cooling, the resulting
solution was neutralized with concentrate ammonium hydroxide.
Then, the adding of crushed ice to the solution precipitated a solid
which was filtered and washed with water. Pure compounds 6 were
obtained by crystallization from ethanol.
DMSO-d6)
d ppm 21.1, 41.7, 54.8, 58.7, 103.4, 118.6, 120.7, 124.0,
124.7, 125.8, 126.0, 126.3, 127.1, 132.0, 133.7, 134.6, 141.4, 142.5,
146.9, 150.0, 153.2, 158.2, 166.8. MS (70 eV) m/z (%): 470/472 [Mþ]
(100/30), 428 (66), 426 (33), 321 (13), 295 (19), 280 (22), 43 (75).
Anal. Calcd. For C27H23ClN4O2: C, 68.86; H, 4.92; N, 11.90. Found: C,
68.79; H, 4.96; N, 11.99.
4.1.2.1. 1-(5-(4-Bromophenyl)-3-[4-(7-chloroquinolin-4-ylamino)
phenyl]-4,5-dihydro-1H-pyrazole-1-yl)ethanone 6a. White solid;
95% yield; mp: 140e142 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3320 (NH), 3060
(]CeH), 1646 (C]O), 1640 and 1579 (C]N and C]C). 1H NMR
(400 MHz, DMSO-d6)
d
ppm 2.30 (s, 3H, CH3), 3.14 (dd, J ¼ 17.9,
4.1.2.5. 1-(3-[4-(7-Chloroquinolin-4-ylamino) phenyl]-5-(3,4,5-trime
4.3 Hz, 1H, H-40a), 3.85 (dd, J ¼ 17.9, 11.9 Hz, 1H, H-40b), 5.53 (dd,
J ¼ 11.9, 4.3 Hz, 1H, H-50), 7.13 (d, J ¼ 4.9 Hz, 1H, H-3), 7.17 (d,
J ¼ 8.3 Hz, 2H, Ho0), 7.43 (d, J ¼ 8.3 Hz, 2H, Ho), 7.52 (d, J ¼ 8.3 Hz,
thoxyphenyl)-4,5-dihydro-1H-pyrazole-1-yl)ethanone
solid; 59% yield; mp: 120e123 ꢄC. FTIR (KBr) (cmꢀ1): 3274 (NH), 3002
(]CeH), 1639 (C]O) and 1589 (C]N and C]C). 1H NMR (400 MHz,
6e. Yellow
y

B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
259
DMSO-d6)
d
ppm 2.03 (s, 3H, CH3), 2.87 (dd, J ¼ 16.1, 11.2 Hz, 1H, H-40a),
H-50), 7.12 (d, J ¼ 5.5 Hz, 1H, H-3), 7.27 (d, J ¼ 8.5 Hz, 2H, Ho0), 7.41
(d, J ¼ 8.5 Hz, 2H, Ho), 7.44 (d, J ¼ 8.5 Hz, 2H, Hm0), 7.60 (dd,
J ¼ 9.0, 2.1 Hz, 1H, H-6), 7.80 (d, J ¼ 8.5 Hz, 2H, Hm), 7.93 (d,
J ¼ 2.1 Hz, 1H, H-8), 8.13 (s, 1H, NH), 8.41 (d, J ¼ 9.0 Hz, 1H, H-5),
8.55 (d, J ¼ 5.5 Hz, 1H, H-2), 8.88 (s, 1H, CHO). 13C NMR (100 MHz,
3.43 (dd, J ¼ 16.1, 10.5 Hz, 1H, H-40b), 3.64 (s, 3H, OCH3), 3.77 (s, 6H,
OCH3), 4.80 (t, J ¼ 10.92, 1H, H-50), 6.72 (s, 2H, Ho0), 7.00 (d, J ¼ 5.3 Hz,
1H, H-3), 7.37 (d, J ¼ 8.4 Hz, 2H, Ho), 7.57 (dd, J ¼ 9.0, 2.0 Hz 1H, H-6),
7.66 (d, J ¼ 8.4 Hz, 2H, Hm), 7.90 (d, J ¼ 2.0,1H, H-8), 8.42 (d, J ¼ 9.0 Hz,
1H, H-5), 8.49 (d, J ¼ 5.3 Hz, 1H, H-2), Not observed (br s, 1H, NH). 13
C
DMSO-d6) d ppm 42.1, 57.9, 103.5, 114.1, 118.8, 121.0, 124.7, 125.4,
NMR (100 MHz, DMSO-d6)
d
ppm 21.3, 41.9, 55.3, 58.7, 59.6, 103.5,
125.5, 127.8, 128.1, 128.7, 132.0, 134.3, 140.3, 142.7, 155.6, 155.7,
159.0, 159.4, 159.5. MS (70 eV) m/z (%): 460/462 [Mþ] (100/68), 431
(25), 433 (18), 321 (47), 323 (16), 280 (44), 282 (14). Anal. Calcd.
For C25H18Cl2N4O: C, 65.09; H, 3.93; N, 12.14. Found: C, 65.00; H,
3.99; N, 12.15.
119.6, 121.7, 123.0, 124.9, 126.1, 126.6, 127.7, 132.3, 133.9, 134.1, 134.5,
141.3,142.2,146.1,151.8,153.7,158.5,167.0. MS (70 eV) m/z (%): 530/532
[Mþ] (66/14), 488 (34), 43 (100). Anal. Calcd. For C29H27ClN4O4: C,
65.59; H, 5.13; N, 10.55. Found: C, 65.49; H, 5.14; N, 10.63.
4.1.2.6. 1-(3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-(p-tolyl)-
4.1.3.3. 3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-phenyl-4,5-
4,5-dihydro-1H-pyrazole-1-yl)ethanone 6f. Yellow solid; 85% yield;
dihydro-1H-pyrazole-1-carbaldehyde 7c. Yellow solid; 73% yield;
mp: 162e164 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3314 (NH), 3086 (]CeH),
mp: 120e123 ꢄC. FTIR (KBr) (cmꢀ1): 3473 (NH), 3048 (]CeH),
y
1642 (C]O) and 1585 (C]N and C]C). 1H NMR (400 MHz, DMSO-
d6)
1675 (C]O), 1615 and 1593 (C]N and C]C). 1H NMR (400 MHz,
d
ppm 1.87 (s, 3H, CH3), 2.26 (s, 3H, CH3), 3.16 (dd, J ¼ 18.0,
DMSO-d6)
d
ppm 3.24 (dd, J ¼ 18.1, 4.8 Hz, 1H, H-40a), 3.96 (dd,
4.7 Hz, 1H, H-40a), 3.89 (dd, J ¼ 18.0, 11.7 Hz, 1H, H-40b), 5.48 (dd,
J ¼ 11.7, 4.7 Hz, 1H, H-50), 7.04e7.16 (m, 5H, H-3, Ho0, Hm0), 7.44 (d,
J ¼ 8.5 Hz, 2H, Ho), 7.62 (dd, J ¼ 9.0, 2.1 Hz, 1H, H-6), 7.81 (d,
J ¼ 8.5 Hz, 2H, Hm), 7.93 (d, J ¼ 2.1, 1H, H-8), 8.42 (d, J ¼ 9.0 Hz 1H,
H-5), 8.54 (d, J ¼ 5.5 Hz, 1H, H-2), 8.87 (s, 1H, NH). 13C NMR
J ¼ 18.1, 11.7 Hz, 1H, H-40b), 5.57 (dd, J ¼ 11.7, 4.8 Hz, 1H, H-50), 7.02
(d, J ¼ 6.5 Hz,1H, H-3), 7.25 (d, J ¼ 7.3 Hz, 2H, Ho0), 7.35 (d, J ¼ 7.3 Hz,
2H, Hm0), 7.54 (d, J ¼ 8.5 Hz, 2H, Ho), 7.82 (dd, J ¼ 9.0, 2.0 Hz, 1H, H-
6), 7.92 (d, J ¼ 8.5 Hz, 2H, Hm), 7.89 (d, J ¼ 2.0 Hz, 1H, H-8), 8.13 (d,
J ¼ 5.8 Hz,1H, Hp), 8.38 (s, 1H, NH), 8.57 (d, J ¼ 6.5 Hz, 1H, H-2), 8.63
(d, J ¼ 9.0 Hz,1H, H-5), 8.92 (s,1H, CHO). 13C NMR (100 MHz, DMSO-
(100 MHz, DMSO-d6)
d ppm 21.1, 23.3, 41.9, 55.1, 56.7, 103.2, 118.1,
120.7, 122.0, 124.7, 125.8, 126.3, 127.1, 132.0, 133.9, 134.6, 141.4,
142.5, 147.9, 150.0, 152.2, 158.2, 166.8. MS (70 eV) m/z (%): 454/456
[Mþ] (15/5), 440 (100), 442 (39), 411 (32), 321 (37), 295 (45), 280
(45), 43 (16). Anal. Calcd. For C27H23ClN4O: C, 71.28; H, 5.10; N,
12.31. Found: C, 71.19; H, 5.16; N, 12.41.
d6) d ppm 42.3, 58.6, 101.5, 116.8, 119.7, 121.4, 124.1, 125.5, 125.6,
127.1, 127.5, 128.3, 128.5, 128.8, 137.6, 139.8, 141.3, 149.0, 155.4,
159.0, 159.7. MS (70 eV) m/z (%): 426/428 [Mþ] (100/36), 397 (28),
399 (10), 321 (46), 323 (15), 280 (29), 282 (10). Anal. Calcd. For
C25H19ClN4O: C, 70.34; H, 4.49; N, 13.12. Found: C, 70.28; H, 4.59; N,
13.21.
4.1.3. General procedure for the synthesis of 1-(3-[4-(7-chloroquin
olin-4-yl)amino)phenyl]-5-substitutedphenyl-4,5-dihydro-1H-pyra
zole-1-carbaldehyde 7aef
4.1.3.4. 3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-(4-methoxyph-
enyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde 7d. Yellow solid;
A
mixture of 4-(7-chloroquinolin-4-yl)aminochalcone
4
67% yield; mp: 208e211 ꢄC. FTIR (KBr) (cmꢀ1): 3478 (NH), 3025
y
(0.38 mmol), DMF (1.0 mL), boron trifluoride diethyl etherate (3
drops) and hydrazine hydrate (0.49 mmol) was heated to reflux for
6 h until complete consumption of the chalcone 4 (TLC control).
Then, formic acid (2.0 mL) was added to the reaction mixture and
the refluxing was continued by 2 h further. After cooling, the adding
of crushed ice precipitated a solid which was filtered and washed
thoroughly with water and dried at ambient temperature to afford
compounds 7.
(]CeH), 1676 (C]O), 1618 and 1593 (C]N and C]C). 1H NMR
(400 MHz, DMSO-d6)
d
ppm 3.18 (dd, J ¼ 18.0, 4.7 Hz, 1H, H-40a),
3.72 (s, 3H, OCH3), 3.88 (dd, J ¼ 18.0, 11.7 Hz, 1H, H-40b), 5.48 (dd,
J ¼ 11.7, 4.7 Hz, 1H, H-50), 6.89 (d, J ¼ 8.5 Hz, 2H, Ho0), 7.12 (d,
J ¼ 5.5 Hz, 1H, H-3), 7.16 (d, J ¼ 8.5 Hz, 2H, Hm0), 7.44 (d, J ¼ 8.5 Hz,
2H, Ho), 7.61 (dd, J ¼ 9.0, 2.3 Hz, 1H, H-6), 7.81 (d, J ¼ 8.5 Hz, 2H,
Hm), 7.93 (d, J ¼ 2.3 Hz, 1H, H-8), 8.13 (s, 1H, NH), 8.42 (d, J ¼ 9.0 Hz,
1H, H-5), 8.54 (d, J ¼ 5.5 Hz, 1H, H-2), 8.86 (s, 1H, CHO). 13C NMR
(100 MHz, DMSO-d6) d ppm 42.3, 55.1, 58.0, 103.4, 114.1, 118.7, 121.1,
4.1.3.1. 5-(4-Bromophenyl)-3-[4-(7-chloroquinolin-4-ylamino)
phenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde 7a. Yellow solid;
124.7, 125.4, 125.6, 127.0, 127.2, 128.0, 133.4, 134.4, 142.6, 147.2,
148.9, 151.4, 155.7, 158.6, 159.4. MS (70 eV) m/z (%): 456/458 [Mþ]
(100/36), 427 (27), 429 (10), 321 (20), 295 (33), 297 (10), 280 (38),
282 (12). Anal. Calcd. For C26H21ClN4O2: C, 68.34; H, 4.63; N, 12.26.
Found: C, 68.28; H, 4.53; N, 12.19.
80% yield; mp: 201e203 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3472 (NH), 3062
(]CeH), 1674 (C]O), 1618 and 1593 (C]N and C]C). 1H NMR
(400 MHz, DMSO-d6)
d
ppm 3.21 (dd, J ¼ 18.1, 4.8 Hz, 1H, H-40a),
3.91 (dd, J ¼ 18.1, 11.7 Hz, 1H, H-40b), 5.53 (dd, J ¼ 11.7, 4.8 Hz, 1H, H-
50), 7.12 (d, J ¼ 5.4 Hz, 1H, H-3), 7.21 (d, J ¼ 8.4 Hz, 2H, Ho0), 7.44 (d,
J ¼ 8.8 Hz, 2H, Ho), 7.54 (d, J ¼ 8.4 Hz, 2H, Hm0), 7.62 (dd, J ¼ 9.0,
2.1 Hz, 1H, H-6), 7.81 (d, J ¼ 8.8 Hz, 2H, Hm), 7.94 (d, J ¼ 2.1 Hz, 1H,
H-8), 8.12 (s,1H, NH), 8.42 (d, J ¼ 9.0 Hz,1H, H-5), 8.55 (d, J ¼ 5.4 Hz,
4.1.3.5. 3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde
Yellow solid; 63% yield; mp: 203e205 ꢄC. FTIR (KBr) (cmꢀ1): 3484
(NH), 3061 (]CeH), 1670 (C]O), 1622 and 1594 (C]N and C]C).
1H NMR (400 MHz, DMSO-d6)
7e.
y
1H, H-2), 8.88 (s, 1H, CHO). 13C NMR (100 MHz, DMSO-d6)
d
ppm
d
ppm 3.28 (dd, J ¼ 18.1, 5.3 Hz,1H, H-
42.1, 57.9, 103.4, 118.8, 120.5, 121.1, 124.7, 125.4, 125.5, 128.1, 128.2,
131.6, 133.5, 140.7, 151.2, 155.7, 159.0, 159.5, 161.5, 163.0, 166.8. MS
(70 eV) m/z (%): 504/506 [Mþ] (76/100), 475 (15), 477 (21), 321 (47),
323 (15), 280 (40), 282 (13). Anal. Calcd. For C25H18BrClN4O: C,
59.37; H, 3.59; N, 11.08. Found: C, 59.44; H, 3.66; N, 10.17.
40a), 3.64 (s, 3H, OCH3), 3.75 (s, 6H, OCH3), 3.94 (dd, J ¼ 18.1, 11.8 Hz,
1H, H-40b), 5.52 (dd, J ¼ 11.8, 5.3 Hz, 1H, H-50), 6.54 (s, 2H, Ho0), 6.99
(d, J ¼ 7.0 Hz, 1H, H-3), 7.58 (d, J ¼ 8.5 Hz, 2H, Ho), 7.91 (dd, J ¼ 9.2,
2.0 Hz, 1H, H-6), 7.96 (d, J ¼ 8.5 Hz, 2H, Hm), 8.02 (d, J ¼ 2.0 Hz, 1H,
H-8), 8.59 (d, J ¼ 7.0 Hz, 1H, H-2), 8.70 (d, J ¼ 9.2 Hz, 1H, H-5), 8.95
(s, 1H, CHO), Not observed (br s, 1H, NH). 13C NMR (100 MHz,
4.1.3.2. 5-(4-Chlorophenyl)-3-[4-(7-chloroquinolin-4-ylamino)
DMSO-d6) d ppm 42.5, 55.9, 58.9, 59.9, 100.9, 102.8, 116.3, 119.9,
phenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde 7b. Yellow solid;
124.9, 125.6, 125.7, 127.6, 128.3, 129.5, 136.8, 136.9, 138.5, 139.0,
139.5, 144.4, 153.1, 154.1, 159.9. MS (70 eV) m/z (%): 516/518 [Mþ]
(100/35), 487 (13), 321 (14), 280 (19). Anal. Calcd. For
87% yield; mp: 216e218 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3472 (NH), 3065
(]CeH), 1676 (C]O), 1618 and 1594 (C]N and C]C). 1H NMR
(400 MHz, DMSO-d6)
d
ppm 3.21 (dd, J ¼ 18.1, 4.8 Hz, 1H, H-40a),
C28H25ClN4O4: C, 65.05; H, 4.87; N, 12.84. Found: C, 65.16; H, 4.80;
3.91 (dd, J ¼ 18.1, 11.7 Hz, 1H, H-40b), 5.54 (dd, J ¼ 11.7, 4.8 Hz, 1H,
N, 12.77.

260
B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
4.1.3.6. 3-[4-(7-Chloroquinolin-4-ylamino)phenyl]-5-(p-tolyl)-4,5-
C25H18Cl2N4O: C, 65.09; H, 3.93; N, 12.14. Found: C, 65.02; H, 3.90;
dihydro-1H-pyrazole-1-carbaldehyde 7f. Yellow solid; 70% yield;
N, 12.19.
mp: 208e211 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3481 (NH), 3059 (]CeH),
1679 (C]O), 1618 and 1593 (C]N and C]C). 1H NMR (400 MHz,
DMSO-d6)
4.1.4.3. 3-[3-(7-Chloroquinolin-4-ylamino)phenyl]-5-phenyl-4,5-
d
ppm 2.27 (s, 3H, CH3), 3.17 (dd, J ¼ 18.0, 4.6 Hz, 1H, H-
dihydro-1H-pyrazole-1-carbaldehyde 8c. Yellow solid; 61% yield;
40a), 3.89 (dd, J ¼ 18.0, 11.7 Hz, 1H, H-40b), 5.48 (dd, J ¼ 11.7, 4.6 Hz,
1H, H-50), 7.09e7.17 (m, 5H, Ho0, Hm0, H-3), 7.43 (d, J ¼ 8.5 Hz, 2H,
Ho), 7.61 (dd, J ¼ 9.0, 2.1 Hz, 1H, H-6), 7.80 (d, J ¼ 8.5 Hz, 2H, Hm),
7.93 (d, J ¼ 2.1 Hz, 1H, H-8), 8.13 (s, 1H, NH), 8.41 (d, J ¼ 9.0 Hz, 1H,
H-5), 8.54 (d, J ¼ 5.3 Hz, 1H, H-2), 8.87 (s, 1H, CHO). 13C NMR
mp: 134e135 ꢄC. FTIR (KBr) (cmꢀ1): 3356 (NH), 3048 (]CeH),
y
1670 (C]O) and 1570 (C]N and C]C). 1H NMR (400 MHz, CDCl3)
d
ppm 3.10 (dd, J ¼ 17.8, 4.9 Hz, 1H, H-40a), 3.68 (dd, J ¼ 17.8, 11.7 Hz,
1H, H-40b), 5.43 (dd, J ¼ 11.7, 4.9 Hz,1H, H-50), 6.90 (d, J ¼ 5.5 Hz,1H,
H-3), 7.11e7.24 (m, 5H, H-6, Ho, Hm, Hp, Hp00), 7.28e7.39 (m, 4H,
Ho00, Hm00), 7.63 (s, 1H, Ho0), 7.88 (d, J ¼ 9.0 Hz, 1H, H-5), 7.92 (d,
J ¼ 2.0 Hz, 1H, H-8), 8.46 (d, J ¼ 5.5 Hz, 1H, H-2), 8.90 (s, 1H, CHO),
(100 MHz, DMSO-d6)
d ppm 20.6, 42.3, 58.2, 103.4, 118.7, 121.0,
124.7, 125.4, 125.5, 125.6, 127.2, 128.0, 129.2, 134.3, 136.7, 138.5,
142.6, 147.1, 151.5, 155.7, 159.0, 159.4. MS (70 eV) m/z (%): 440/442
[Mþ] (100/35), 411 (31), 413 (12), 321 (32), 323 (11), 280 (35), 282
(12). Anal. Calcd. For C26H21ClN4O: C, 70.82; H, 4.80; N, 12.71.
Found: C, 70.76; H, 4.89; N, 12.73.
Not observed (br s, 1H, NH). 13C NMR (100 MHz, CDCl3)
d ppm 42.6,
59.3, 102.8, 118.5, 120.1, 122.0, 122.8, 124.3, 125.6, 126.2, 128.1, 128.7,
129.1, 130.1, 132.5,135.5, 140.3, 140.6, 147.5, 149.7, 151.8, 155.4, 160.3.
MS (70 eV) m/z (%): 426/428 [Mþ] (100/35), 369 (44), 371 (15), 321
(34), 323 (11), 280 (17). Anal. Calcd. For C25H19ClN4O: C, 70.34; H,
4.49; N, 13.12. Found: C, 70.25; H, 4.54; N, 13.19.
4.1.4. General procedure for the synthesis of 1-(3-[3-(7-
chloroquinolin-4yl)amino) phenyl]-5-substitutedphenyl-4,5-
dihydro-1H-pyrazole-1-carbaldehyde 8aef
4.1.4.4. 3-[3-(7-Chloroquinolin-4-ylamino) phenyl]-5-(4-methox
A
mixture of 3-(7-chloroquinolin-4-yl)aminochalcone
5
yphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde 8d. Yellow solid;
(0.32 mmol), boron trifluoride diethyl etherate (2 drops) and hy-
drazine hydrate (0.42 mmol) was stirred at room temperature for
4 h until complete consumption of the chalcone 5 (TLC control).
Then, formic acid (2.0 mL) was added to the reaction mixture and
the stirring, at room temperature, was continued for 1 h further.
Once the reaction finished, ethanol (3.0 mL) was added and the
solid formed was removed by filtration and discarded. Then the
resulting filtrate was concentrate to dryness under reduced pres-
sure and the crude obtained was purified by column chromatog-
raphy on silica gel by using a mixture CHCl3:MeOH (30:1) as
eluent.
47% yield; mp: 222e224 ꢄC. FTIR (KBr) (cmꢀ1): 3340 (NH), 3058 (]
y
CeH), 1666 (C]O), 1609 and 1567 (C]N and C]C). 1H NMR
(400 MHz, CDCl3)
d
ppm 3.22 (dd, J ¼ 17.8, 4.7 Hz, 1H, H-40a), 3.78
(m, 4H, OCH3, H-40b), 5.51 (dd, J ¼ 11.5, 4.7 Hz, 1H, H-50), 6.86 (d,
J ¼ 8.3 Hz, 2H, Ho00), 6.90e6.97 (m, 1H, H-3), 7.19 (d, J ¼ 8.3 Hz, 2H,
Hm00), 7.28 (s, 1H, H-6), 7.37e7.54 (m, 4H, Ho, Hm, Hp, NH), 7.77 (s,
1H, Ho0), 7.97e8.05 (m, 2H, H-5, H-8), 8.43e8.50 (m, 1H, H-2), 8.96
(s, 1H, CHO). 13C NMR (100 MHz, CDCl3)
d ppm 42.8, 59.5, 60.8, 101.2,
117.2, 121.2, 123.8, 124.1, 124.4, 125.2, 127.0, 130.4, 132.7, 136.0, 137.6,
137.7, 139.5, 144.7, 147.1, 150.9, 153.8, 155.1, 160.3, 168.6. MS (70 eV)
m/z (%): 456/458 [Mþ] (100/35), 427 (43), 429 (15), 321 (16), 280
(64), 282 (20). Anal. Calcd. For C26H21ClN4O2: C, 68.34; H, 4.63; N,
12.26. Found: C, 68.27; H, 4.57; N, 12.29.
4.1.4.1. 5-(4-Bromophenyl)-3-[3-(7-chloroquinolin-4-ylamino)
phenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde 8a. Yellow solid;
75% yield; mp: 154e155 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3481 (NH), 3061
4.1.4.5. 3-[3-(7-Chloroquinolin-4-ylamino)phenyl]-5-(3,4,5-trimetho-
(]CeH), 1670 (C]O) and 1569 (C]N and C]C). 1H NMR
xyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde 8e. Yellow solid;
(400 MHz, CDCl3)
d
ppm 3.19 (dd, J ¼ 17.7, 4.6 Hz, 1H, H-40a), 3.83
53% yield; mp: 127e129 ꢄC. FTIR (KBr) (cmꢀ1): 3262 (NH), 3065 (]
y
(dd, J ¼ 17.7, 11.7 Hz, 1H, H-40b), 5.52 (dd, J ¼ 11.7, 4.6 Hz, 1H, H-
50), 7.00 (d, J ¼ 4.9 Hz, 1H, H-3), 7.15 (d, J ¼ 7.8 Hz, 2H, Ho00), 7.38e
7.58 (m, 7H, H-6, Hm00, Ho, Hm, Hp, NH), 7.75 (s, 1H, Ho0), 8.06 (s,
1H, H-8), 8.59 (d, J ¼ 4.9 Hz, 1H, H-2), 7.92 (d, J ¼ 9.0 Hz, 1H, H-5),
CeH), 1674 (C]O) and 1593 (C]N and C]C). 1H NMR (400 MHz
CDCl3)
d
ppm 3.21 (dd, J ¼ 17.8, 4.7 Hz, 1H, H-40a), 3.67e3.94 (m, 10H,
(OCH3 ꢁ 3), H-40b), 5.49 (dd, J ¼ 11.4, 4.7 Hz, 1H, H-50), 6.44 (s, 2H,
Ho00), 6.83 (d, J ¼ 4.8 Hz,1H, H-3), 7.32e7.61 (m, 4H, H-6, Ho, Hm, Hp),
7.81 (s, 1H, Ho0), 7.93 (s, 1H, H-8), 8.18 (d, J ¼ 8.5 Hz,1H, H-5), 8.29 (d,
8.95 (s, 1H, CHO). 13C NMR (100 MHz, CDCl3)
d ppm 42.4, 58.7,
100.1, 120.1, 121.3, 122.1, 123.0, 124.5, 126.5, 127.5, 128.7, 129.5,
130.3, 132.3, 132.5, 136.7, 137.2, 139.4, 140.3, 146.2, 151.5, 154.8,
160.1. MS (70 eV) m/z (%): 504/506 [Mþ] (76/100), 475 (18), 477
(19), 447 (27), 449 (35), 321 (33), 280 (25). Anal. Calcd. For
J ¼ 4.8 Hz,1H, H-2), 8.99 (s, 1H, CHO), Not observed (br s,1H, NH). 13
C
NMR (100 MHz, CDCl3) d ppm 42.8, 56.2, 59.5, 60.8,101.4,102.5,117.2,
121.2, 123.6, 124.3, 125.2, 126.9, 130.4, 132.7, 136.0, 137.6, 137.7, 139.3,
144.6, 147.0, 150.9, 153.8, 155.1, 160.3, 168.6. MS (70 eV) m/z (%): 516/
518 [Mþ] (64/23), 501 (100), 487 (18), 321 (11), 280 (65), 282 (20).
Anal. Calcd. For C28H25ClN4O4: C, 65.05; H, 4.87; N, 12.84. Found: C,
65.13; H, 4.92; N, 12.94.
C
25H18BrClN4O: C, 59.37; H, 3.59; N, 11.08. Found: C, 59.43; H,
3.64; N, 10.15.
4.1.4.2. 5-(4-Chlorophenyl)-3-[3-(7-chloroquinolin-4-ylamino)
phenyl]-4,5-dihydro-1H-pyrazole-1-carbaldehyde 8b. Yellow solid;
4.1.4.6. 3-[3-(7-Chloroquinolin-4-ylamino)phenyl]-5-(p-tolyl)-4,5-
71% yield; mp: 139e141 ꢄC. FTIR (KBr)
y
(cmꢀ1): 3484 (NH), 3061
dihydro-1H-pyrazole-1-carbaldehyde 8f. Yellow solid; 60% yield;
(]CeH), 1669 (C]O) and 1566 (C]N and C]C). 1H NMR
mp: 261e263 ꢄC. FTIR (KBr) (cmꢀ1): 3288 (NH), 3060 (]CeH),1662
y
(400 MHz, CDCl3)
d
ppm 3.26 (dd, J ¼ 18.2, 5.1 Hz, 1H, H-40a), 3.96
(C]O), 1608 and 1567 (C]N and C]C). 1H NMR (400 MHz, CDCl3)
(dd, J ¼ 18.2, 11.9 Hz, 1H, H-40b), 5.59 (dd, J ¼ 11.9, 5.1 Hz, 1H, H-50),
6.93 (d, J ¼ 6.8 Hz, 1H, H-3), 7.29 (d, J ¼ 8.4 Hz, 2H, Ho00), 7.42 (d,
J ¼ 8.4 Hz, 2H, Hm00), 7.57e7.63 (m, 1H, Ho), 7.68 (t, J ¼ 7.8 Hz, 1H,
Hm), 7.79 (d, J ¼ 7.8 Hz, 1H, Hp), 7.86e7.92 (m, 2H, H-6, Ho0), 8.10e
8.18 (br s, 1H, NH), 8.13e8.17 (m, 1H, H-8), 8.56 (d, J ¼ 6.8 Hz, 1H, H-
2), 8.67 (d, J ¼ 9.0 Hz,1H, H-5), 8.90 (s,1H, CHO). 13C NMR (100 MHz,
d
ppm 2.31 (s, 3H, CH3), 3.21 (dd, J ¼ 17.8, 4.9 Hz,1H, H-40a), 3.79 (dd,
J ¼ 17.8,11.8 Hz,1H, H-40b), 5.52 (dd, J ¼ 11.8, 4.9 Hz,1H, H-50), 6.85 (d,
J ¼ 5.0 Hz, 1H, H-3), 7.09e7.55 (m, 9H, H-6, Ho, Hm, Hp, Ho00, Hm00,
NH), 7.80 (s,1H, Ho0), 7.95 (s,1H, H-8), 8.14 (d, J ¼ 8.8 Hz,1H, H-5), 8.32
(d, J ¼ 5.0 Hz, 1H, H-2), 8.96 (s, 1H, CHO). 13C NMR (100 MHz, CDCl3)
d
ppm 21.1, 42.6, 59.2, 101.2, 117.1, 121.2, 123.7, 124.0, 124.2, 125.2,
CDCl3)
d
ppm 42.1, 58.1,100.8,116.3,120.4,122.8,125.5,125.6,126.8,
125.6, 127.0, 129.7, 130.4, 132.9, 137.4, 137.8, 137.9, 139.2, 144.1, 146.6,
151.2, 154.9, 160.2. MS (70 eV) m/z (%): 440/442 [Mþ] (100/35), 411
(16), 321 (16), 280 (17). Anal. Calcd. For C26H21ClN4O: C, 70.82; H,
4.80; N, 12.71. Found: C, 70.72; H, 4.82; N, 12.81.
127.4, 127.8, 128.7, 130.6, 132.1, 132.5, 137.9, 138.1, 140.1, 144.9, 154.0,
155.5, 159.0, 159.8. MS (70 eV) m/z (%): 460/462 [Mþ] (100/66), 431
(19), 403 (42), 405 (28), 321 (33), 280 (24). Anal. Calcd. For

B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
261
4.2. Biological evaluation
expressed as greater or less than the maximum or minimum con-
centration tested [44e47].
4.2.1. Anticancer activity
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and
2 mM L-glutamine. For a typical screening experiment, cells are
4.2.2. Antiplasmodial activity
The antiplasmodial activity was evaluated in vitro on asyn-
chronic cultures of P. falciparum (NF54 strain), maintained in
standard culture conditions [48]. The effect of the evaluated
compounds over the growing of the parasites was evaluated ac-
cording to the fluorometric method by quantifying the parasite
DNA by staining with ethidium bromide (EtBr). The fluorescence
intensity is directly proportional to the amount of DNA and this, in
turn, is proportional to the amount of viable parasites. The amount
of ADN detected is directly related with the concentration of the
evaluated compound and in turn with the inhibition of the repli-
cation process of the parasite [49e51]. Briefly, the P. falciparum
cultures with total forms were adjusted to a parasitemia (1.5%e2%)
and hematocrit (5%) in 10% RPMI medium of human serum (total
medium). Then, in each well of a 24-wells plate it was simulta-
inoculated into 96 well microtiter plates. After cell inoculation, the
microtiter plates were incubated at 37 ꢄC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of tested compounds.
After 24 h, two plates of each cell line were fixed in situ with TCA, to
represent a measurement of the cell population for each cell line at
the time of sample addition (Tz). The samples were solubilized in
dimethyl sulfoxide (DMSO) at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
compounds addition, an aliquot of frozen concentrate was thawed
and diluted to twice, the desired final maximum test concentration
with complete medium containing 50
tional four, 10-fold or ½ log serial dilutions were made to provide a
total of five drug concentrations plus control. Aliquots of 100 L of
these different sample dilutions were added to the appropriate
microtiter wells already containing 100 L of medium, resulting in
mg/mL gentamicin. Addi-
m
neously placed 500
of dilutions of each compound to be evaluated (Dilutions were
prepared from a concentrate mother solution of 100 g/mL).
Concentration of the positive chloroquine (CQ) control was
22.2 g/mL. A well with the suspension of the parasites free of any
mL of a suspension of the parasites and 500 mL
m
m
the required final sample concentrations [44]. After the tested
compounds were added, the plates were incubated for an addi-
tional 48 h at 37 ꢄC, 5% CO2, 95% air, and 100% relative humidity. For
adherent cells, the assay was terminated by the addition of cold
m
compound was used for both growing and viability control. The
plates were incubated during 48 h at 37 ꢄC in N2 (90%), CO2 (5%)
and O2 (5%) atmosphere. After the incubation finished, extraction
and purification of DNA was carried out by using a lysis solution
containing proteinase K.
In this sense, the anti-Plasmodium activity of each evaluated
compound was evidenced by the reduction of the fluorescence
(Flu). Indeed, the viability percentage was calculated through the
following formula:
TCA. Cells were fixed in situ by the gentle addition of 50 mL of cold
50% (w/v) TCA (final concentration, 10% TCA) and incubated for
60 min at 4 ꢄC. The supernatant was discarded, and plates were
washed five times with tap water and air dried. Sulforhodamine B
(SRB) solution (100 mL) at 0.4% (w/v) in 1% acetic acid was added to
each well, and plates were incubated for 10 min at room temper-
ature. After staining, unbound dye was removed by washing five
ðFluÞ of parasites with compound ꢀ ðFluÞ of the medium
ðFluÞ of parasites without compound ꢀ ðFluÞ of the medium
Viability ð%Þ ¼
*100
times with 1% acetic acid and the plates were air dried. Bound stain
was subsequently solubilized with 10 mM trizma base, and the
absorbance was read on an automated plate reader at a wavelength
of 515 nm. Using the seven absorbance measurements [time zero
(Tz), control growth in the absence of drug (C), and test growth in
the presence of drug at the five concentration levels (Ti)], the per-
centage growth was calculated at each of the drug concentrations
levels. Percentage growth inhibition was calculated as: [(Ti ꢀ TZ)/
(C ꢀ TZ)] ꢁ 100 for concentrations for which Ti > Tz, and [(Ti ꢀ TZ)/
TZ] ꢁ 100 for concentrations for which Ti < Tz. Three dose response
parameters were calculated for each compound. Growth inhibition
of 50% (GI50) was calculated from [(Ti ꢀ TZ)/(C ꢀ TZ)] ꢁ 100 ¼ 50,
which is the drug concentration resulting in a 50% lower net protein
increase in the treated cells (measured by SRB staining) as
compared to the net protein increase seen in the control cells. The
drug concentration resulting in total growth inhibition (TGI) was
calculated from Ti ¼ Tz. The LC50 (concentration of drug resulting in
a 50% reduction in the measured protein at the end of the drug
treatment as compared to that at the beginning) indicating a net
loss of cells following treatment was calculated from [(Ti ꢀ TZ)/
TZ] ꢁ 100 ¼ ꢀ50. Values were calculated for each of these three
parameters if the level of activity is reached; however, if the effect
was not reached or was exceeded, the value for that parameter was
Then, the inhibition growing percentage was calculated ac-
cording to the following formula:
Inhibition ð%Þ ¼ 100 ꢀ Viability ð%Þ
All determinations were carried out by triplicate. CQ was used as
control of activity for each case.
4.2.3. Hemolytic activity
The ability to induce hemolysis was evaluated specifically to
compounds which showed antiplasmodial activity following the
method of cytotoxicity by spectrophotometry on 96-wells plates.
Red blood cells (hematocrit 5%) were placed into the wells along
with the RPMI medium 1640 and subsequently were exposed
against three different concentrations of compounds (20, 40 and
80 mg/mL). Detection of free hemoglobin, after 48 h of incubation at
37 ꢄC, should be an evidence of the capability of the compound to
induce hemolysis. The measurement of the concentration of free
hemoglobin was performed spectrophotometrically at 542 nm.
4.2.4. Determination of the IC50
The inhibitory concentration (IC50) corresponding to the con-
centration of the compound which produces a 50% reduction of

262
B. Insuasty et al. / European Journal of Medicinal Chemistry 67 (2013) 252e262
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growing of the parasite was determined by mean of the statistical
program Probit. It is a parametric method of linear regression
which permits the analysis of the doseeresponse relationship
[52].
Acknowledgments
The authors wish to credit The Developmental Therapeutics
Program (DTP) of the National Cancer Institute of the United States
(U.S.) for performing the screening of compounds. This work was
supported financially by Colciencias, Universidad del Valle and
Universidad de Antioquia. Authors thank ‘‘Servicios Técnicos de
Investigación de la Universidad de Jaén’’ and the staff for data
collection.
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