Synthesis of novel quinoline–based 4,5–dihydro–1H–pyrazoles as potential anticancer, antifungal, antibacterial and antiprotozoal agents

Article abstract of DOI:10.1016/j.ejmech.2017.03.016

A new series of N–substituted 2–pyrazolines 9a–f, 10a–f, 11a–f, 12a–f and 13a–f were obtained from the cyclocondensation reaction of [(7–chloroquinolin–4–yl)amino]chalcones 8a–f with hydrazine hydrate and its derivatives. Fourteen of the synthesized compo

Full text of DOI:10.1016/j.ejmech.2017.03.016

Accepted Manuscript
Synthesis of novel quinoline–based 4,5–dihydro–1H–pyrazoles as potential
anticancer, antifungal, antibacterial and antiprotozoal agents
Jonathan Ramírez–Prada, B.Sc., Sara M. Robledo, Ph.D., Iván D. Vélez, Ph.D.,
María del Pilar Crespo, Ph.D., Jairo Quiroga, Ph.D., Rodrigo Abonia, Ph.D., Alba
Montoya, M.Sc., Laura Svetaz, Ph.D., Susana Zacchino, Ph.D., Braulio Insuasty,
Ph.D.
PII:
S0223-5234(17)30173-3
10.1016/j.ejmech.2017.03.016
EJMECH 9276
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 December 2016
Revised Date: 7 March 2017
Accepted Date: 10 March 2017
Please cite this article as: J. Ramírez–Prada, S.M. Robledo, I.D. Vélez, M.d.P. Crespo, J. Quiroga,
R. Abonia, A. Montoya, L. Svetaz, S. Zacchino, B. Insuasty, Synthesis of novel quinoline–based 4,5–
dihydro–1H–pyrazoles as potential anticancer, antifungal, antibacterial and antiprotozoal agents,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.016.
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GRAPHICAL ABSTRACT
Synthesis of novel quinoline–based 4,5–dihydro–1H–pyrazoles as potential anticancer,
antifungal, antibacterial and antiprotozoal agents
a
b
b
c,d
Jonathan Ramírez–Prada , Sara M. Robledo , Iván D. Vélez , María del Pilar Crespo , Jairo
a
a
a
e
e
Quiroga , Rodrigo Abonia , Alba Montoya , Laura Svetaz , Susana Zacchino , Braulio
a
Insuasty *
a
Grupo de Investigación de Compuestos Heterocíclicos, Departamento de Química, Universidad
del Valle, A. A. 25360 Cali, Colombia
b
PECET, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de
Antioquia, PO Box 1226, Medellín, Colombia
c
Grupo de Biotecnología e Infecciones Bacterianas, Departamento de Microbiología,
Universidad del Valle, Cali, Colombia
d
Departamento de Ciencias Biomédicas, Universidad Santiago de Cali, Cali, Colombia
e
Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario, Suipacha 531, (2000), Rosario, Argentina
*
braulio.insuasty@correounivalle.edu.co; Tel: +(57)–2–3392440
1

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2

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Synthesis of novel quinoline–based 4,5–dihydro–1H–pyrazoles as potential anticancer,
antifungal, antibacterial and antiprotozoal agents
Corresponding Author: Braulio Insuasty, Ph.D.
Corresponding Author's Institution: Universidad del Valle
First Author: Jonathan Ramírez–Prada, B.Sc.
Order of Authors: Jonathan Ramírez–Prada, B.Sc.; Sara M. Robledo, Ph.D.; Iván D. Vélez,
Ph.D.; María del Pilar Crespo, Ph.D.; Jairo Quiroga, Ph.D.; Rodrigo Abonia, Ph.D.; Alba
Montoya, M.Sc.; Laura Svetaz, Ph.D.; Susana Zacchino, Ph.D.; Braulio Insuasty, Ph.D.
Abstract
A new series of N–substituted 2–pyrazolines 9a–f, 10a–f, 11a–f, 12a–f and 13a–f were obtained
from the cyclocondensation reaction of [(7–chloroquinolin–4–yl)amino]chalcones 8a–f with
hydrazine hydrate and its derivatives. Fourteen of the synthesized compounds including the
starting chalcones were selected by US National Cancer Institute (NCI) for testing their
anticancer activity against 60 different human cancer cell lines, with the most important GI50
values ranging from 0.28 to 11.7 ꢀM (0.13 to 6.05 ꢀg/mL) and LC values ranging from 2.6 to >
5
0
1
00 ꢀM (1.2 to > 51.7 ꢀg/mL), for chalcones 8a,d and pyrazolines 10c,d. All compounds were
assessed for antibacterial activity against wild type and multidrug resistant gram negative and
gram positive bacteria, with MIC values ranging from 31.25 to 500 ꢀg/mL. Additionally, the
novel compounds were tested for antifungal and antiparasitic properties. Although these
compounds showed mild activity against Candida albicans, chalcones 8a and 8e showed high
3

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activity against Cryptococcus neoformans with MIC50 = 7.8 ꢀg/mL. For anti–Plasmodium
falciparum activity the 2–pyrazoline 11b was the most active with EC50 = 5.54 ꢀg/mL.
Regarding the activity against Trypanosoma cruzi, compound 10a was highly active with EC =
5
0
0
.70 ꢀg/mL. Chalcone 8a had good activity against Leishmania panamensis amastigotes with
EC = 0.79 ꢀg/mL.
50
*
Corresponding author: Dr. Braulio Insuasty, Facultad de Ciencias Naturales y Exactas,
Departamento de Química, Universidad del Valle, Santiago de Cali, Colombia.
E–mail: braulio.insuasty@correounivalle.edu.co
FAX: +57–2–3392440
Keywords: Quinoline, chalcones, 2–pyrazolines, anticancer activity, antifungal activity,
antibacterial activity, antimalarial activity, antitrypanosomal activity, antileishmanial activity
1
Introduction
The pharmacological properties of the quinoline ring are well illustrated by the large number of
commercially available drugs containing this heterocyclic system [1]. These compounds have
been studied for decades and structure–activity relationship analyses have been performed to
determine their biological effects [2]. Most studies on 4–aminoquinolines showed that the 7–
chloro–4–aminoquinoline ring present in pharmacologically active substances displays a broad
range of biological activities [3–6] and is determinant for antimalarial activity, because it
accumulates into the digestive vacuole of the Plasmodium parasite and inhibits β–hematin
4

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formation by accumulation of the FPIX–4–aminoquinoline complex, a highly toxic moiety which
induces parasite death [7–14]. Therefore, including this pharmacophore in the design of novel
heterocyclic compounds might improve their biological activity.
Chalcones have shown broad spectrum of biological activities as antifungal [15], antiparasitic
[16–19], anticancer [20] and antioxidant [21] compounds. They have also shown
immunomodulatory [22], anti–invasive [23,24] and anti–inflammatory effects [25–28]. However
their importance can also be associated with their recognized utility as intermediate compounds
in the synthesis of pyrazolines [29], which have also been largely studied for their
pharmacological activities. Pyrazolines (also called dihydropyrazoles) are known by their
activities including anticancer [30], anti–inflammatory [31], antiparasitic [32], antidepressive,
anticonvulsant [33], antimicrobial [34] and antinociceptive effects [35]. They have also been
recognized as nitric oxide synthase inhibitors used in Alzheimer’s and Huntington’s diseases and
inflammatory arthritis [36].
Regarding the synthesis and biological activities of 2–pyrazolines [37–39], in this study we are
reporting the efficient synthesis of five series of novel quinoline–based 4,5–dihydro–1H–
pyrazoles. The synthesized compounds including their synthetic intermediates quinoline–based
chalcones were tested against a panel of cancer cell lines in the Developmental Therapeutics
Program (DTP) at the National Cancer Institute (NCI). Given the emergence of antimicrobial and
antiparasitic resistance [40], all synthesized compounds were also tested for in vitro antifungal,
antibacterial and antiprotozoal activities.
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2
Results and discussion
2
.1 Chemistry
By using a previously reported methodology [41], 2–(3–nitrophenyl)–1,3–dioxolane 3 was
synthesized and subjected to reduction in methanol with hydrazine monohydrate. Then, the
precursor 3–(7–chloroquinolin–4–ylamino)benzaldehyde 6 was obtained in 98% yield through a
N
selective nucleophilic aromatic substitution (S Ar) of the 4–chlorine atom on the 4,7–
dichloroquinoline 5 with amine 4 previously synthetized. Then a Claisen–Schmidt condensation
reaction between aldehyde 6 and substituted acetophenones 7a–f led the novel quinoline–based
chalcones 8a–f (Scheme 1), in good yields (71–90%).
Scheme 1. General methodology for the synthesis of aldehyde 6 and chalcones 8a–f: i = PTSA,
2 2 2
toluene, reflux, 24 h. ii = NH NH ·H O, Ni–Raney, MeOH, reflux, 1 h. iii = 1) EtOH, reflux, 3
h. 2) HCl conc. iv = KOH, MeOH, rt, 12 h.
The structure elucidation of compounds 8a–f was performed from analysis of their spectroscopic
1
13
data (FTIR, H NMR, C NMR and mass spectrometry). The IR spectrum of compound 8a used
−
1
as representative compound of this series, showed an absorption band at 1659 cm associated to
1
the stretching vibration of the carbonyl group from the α,β–unsaturated fragment. In the H NMR
3
spectrum of compound 8a, two doublets at 7.78 and 7.95 ppm with coupling constant J = 15.6
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Hz assigned to H
β
and H
α
respectively are observed, indicating the E configuration for the
carbon–carbon double bond.
Treatment of chalcones 8a–f with hydrazine monohydrate in ethanol under reflux for 10 min,
and further treatment with acetic anhydride or formic acid, at room temperature for 10 min
(Scheme 2), afforded the acetylated and formylated products 9a–f and 10a–f respectively, in
good yields (75–90%).
Scheme 2. General methodology for the synthesis of compounds 9a–f and 10a–f: i =
NH NH ·H O, EtOH, reflux, 10 min. ii = Ac O (for 9a–f), rt, 10 min. iii = HCOOH (for 10a–f),
2
2
2
2
rt, 10 min.
The IR spectrum of compound 9a as a representative of the series 9a–f and 10a–f, showed an
−
1
absorption band at 3352 cm associated to the stretching vibration of the NH bond. Regarding
1
the H NMR spectrum of compound 9a, it showed a singlet at 2.32 ppm, assigned to the methyl
1
of the acetyl–inserted group. Instead, H NMR spectrum of compound 10a showed a singlet at
8
.93 ppm associated to the proton of the formyl–inserted group. In all cases, the two methylene
protons of C and the stereogenic proton in C generated an AMX spin system. For compound
4
’
5’
9
a, the signal associated to proton H appears as a double–doublet at 3.24 ppm with coupling
A
2
3
constant values J = 18.2 Hz and J = 4.9 Hz; for proton H the double–doublet appears at
AM
AX
M
2
3
3
.92 ppm, with coupling constant values JAM = 18.2 Hz and JMX = 12.0 Hz while the
corresponding signal of the H proton appeared as a double–doublet at 5.63 ppm with coupling
X
7

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3
3
13
constant values JMX = 12.0 Hz and JAX = 4.9 Hz. Furthermore, the C NMR spectrum of
compound 9a showed all the expected signals.
Reaction of the synthesized chalcones 8a–f with phenylhydrazine and its derivatives in methanol
under reflux led to the N–aryl pyrazolines 11a–f, 12a–f and 13a–f (Scheme 3) in acceptable to
excellent yields. The new pyrazolines were fully characterized by spectroscopic techniques such
as FTIR and 1D and 2D–NMR (see Experimental section).
Scheme 3. Synthesis of compounds 11a–f, 12a–f and 13a–f: i = C H NHNH (for 11a–f), 4–
6
5
2
ClC H NHNH ·HCl (for 12a–f), 3,5–diCl–C H NHNH ·HCl (for 13a–f), MeOH, reflux, 3 h.
6
4
2
6
3
2
The IR spectrum of compound 12a as a representative of the series 11a–f, 12a–f and 13a–f,
−
1
showed an absorption band at 3345 cm associated to the stretching vibration of the NH bond.
1
Regarding the H NMR spectrum, it showed two doublets at 7.03 and 7.24 ppm, each one
integrating for 2H assigned to the protons of the 4–Cl–aryl–inserted group. The signal associated
2
to proton H appears as a double–doublet at 3.25 ppm with coupling constant values J = 17.7
A
AM
3
Hz and J = 6.1 Hz; for proton H the double–doublet appears at 3.99 ppm, with coupling
AX
M
2
3
constant values J = 17.7 Hz and J = 12.3 Hz while the corresponding signal of the HX
AM
MX
3
proton appeared as a double–doublet at 5.64 ppm with coupling constant values J = 12.3 Hz
MX
3
13
and J = 6.1 Hz. Furthermore, the C NMR spectrum of compound 12a showed all the
AX
expected signals.
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2
.2 Anticancer activity
For a more comprehensive analysis of the results, we grouped all compounds into two series: (A)
that includes chalcones 8a–f and (B) that comprise pyrazolines 9a–f, 10a–f, 11a–f, 12a–f and
1
3a–f. Series (B) was in turn divided into two groups: (B ) that comprises compounds with an
1
acetyl (9a–f) or a formyl substituent (10a–f) on N–1 of the 2–pyrazoline ring; and (B ) that
2
includes compounds with an unsubstituted (11a–f) or substituted phenyl group [4–Cl (12a–f) or
3
,5–diCl (13a–f)], on N–1 of the 2–pyrazoline ring.
As a preliminary screening, structures of all new compounds (i.e. 6, 8a–f, 9a–f, 10a–f, 11a–f,
2a–f and 13a–f) were submitted to the Developmental Therapeutics Program (DTP) at National
1
Cancer Institute (NCI) for evaluation of their anticancer activity against different human cell
lines. Fourteen (i.e. 8a,c,d; 9a,c; 10a,c,d; 11a,c; 12a,c,d and 13a) compounds were selected and
subjected to the preliminary anticancer evaluation against the 60 cancer cell lines at a single dose
of 10 ꢀM after 48 h of incubation. The output from the single dose screening was analyzed by
the COMPARE program (data not shown). It was observed that only the chalcones and
pyrazolines with Cl and OCH on the ring B were selected for anticancer evaluation suggesting
3
that the substituents on this ring are modulators of the anticancer activity.
The results of this first screening showed that compounds 8c,d and 10a,c passed to a second
evaluation step in order to determine their cytostatic activity against 58 cancer cell lines from
lung, colon, brain, breast, ovary, kidney, prostate and leukemia and melanoma cell panels. None
of the compounds of series B
2
(11a,c; 12a,c,d and 13a) were selected for further testing
9

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suggesting that the substitution with a phenyl group on N–1 of the 2–pyrazoline ring decreases
the anticancer activity of the compounds. It is interesting to note that in a recent paper [37], we
found that analogues of compounds 11a–f and 12a–f, with scheleton type B but with N–phenyl
2
substituent attached to the vicinal N, exhibit anticancer activity suggesting that the position of
ring C is important for anticancer activity.
The results were expressed as follows according to previously published protocols: GI50, which
is the molar concentration of the compounds required to inhibit 50% of the growth of cell lines
(relative to untreated cells), and LC50, which is a parameter of cytotoxicity that reflects the molar
concentration needed to kill 50% of the cells [42]. The active compounds were evaluated at
several concentrations (100, 10, 1.0, 0.1, and 0.01 ꢀM) for a 48–h protocol in which
sulforhodamide B (SRB) protein assay was used to estimate cell growth according to protocols
described elsewhere [43,44]. As shown in Table 1, compounds 8c,d and 10a,c showed great
values of GI50 against several cell lines, some of them lower than 1.00 ꢀM. Compound 8c
showed GI50 values from 0.49 to 3.85 ꢀM (0.20 to 1.60 ꢀg/mL, 6 of them < 1.00 ꢀM) and LC50
values from 23.7 to > 100 ꢀM (9.8 to > 41.5 ꢀg/mL). The best cytostatic activity of 8c was
displayed against the SR leukemia cell line with GI50 = 0.49 ꢀM (0.20 ꢀg/mL) and the best
cytotoxic activity was shown against the U251 CNS cancer cell line with LC = 23.7 ꢀM (9.8
5
0
ꢀ
g/mL), better than adriamycin, the standard drug. Compound 8d was the most active with GI50
values from 0.31 to 2.49 ꢀM (0.15 to 1.18 ꢀg/mL) (20 of them < 1.00 ꢀM) and LC values from
5
0
2
.6 to > 100 ꢀM (1.2 to > 47.5 ꢀg/mL). The best cytostatic activity of 8d was displayed against
the RPMI–8226 leukemia cell line with GI50 = 0.31 ꢀM (0.15 ꢀg/mL) and the best cytotoxic
activity was shown against the SK–OV–3 ovarian cancer cell line with LC50 = 2.6 ꢀM (1.2
1
0

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ꢀ
g/mL), better than adriamycin. Compound 10a showed GI50 values from 0.28 to 9.42 ꢀM (0.13
to 4.35 ꢀg/mL) (12 of them < 1.00 ꢀM) and LC values from 5.1 to > 100 ꢀM (2.3 to > 46.1
5
0
ꢀ
g/mL). The best cytostatic activity of 10a was displayed against the MDA–MB–468 breast
cancer cell line with GI = 0.28 ꢀM (0.13 ꢀg/mL) and the best cytotoxic activity was shown
5
0
against the SK–MEL–5 melanoma cell line with LC = 5.1 ꢀM (2.3 ꢀg/mL). Compound 10c
5
0
showed GI values from 0.37 to 11.7 ꢀM (0.19 to 6.05 ꢀg/mL) (5 of them < 1.00 ꢀM) and LC
50
5
0
values from 17.4 to > 100 ꢀM (9.0 to > 51.7 ꢀg/mL). The best activity was shown against the
MDA–MB–435 melanoma cell line with GI50 = 0.37 ꢀM (0.19 ꢀg/mL) and against the SK–
MEL–5 melanoma cell line with LC50 = 17.4 ꢀM (9.0 ꢀg/mL). These findings make compounds
8
c,d and 10a,c, promising targets to perform structural modifications and improve their
anticancer activities.
Table 1. In vitro cytotoxic activities of compounds 8c,d and 10a,c expressed as growth
inhibition and lethal concentration of cancer cell lines and compared with the standard drug
a
adriamycin.
2
.3 Antifungal activity
Considering that some nitrogen–bearing heterocyclic compounds containing fragments like 2–
pyrazoline moiety have demonstrated antifungal activity in previous reports [37,45–48],
compounds 6, 8a–f, 9a–f, 10a–f, 11a–f, 12a–f and 13a–f were tested for antifungal activity
against two clinically important fungal species, C. albicans and C. neoformans. C. neoformans is
1
1

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an opportunistic fungus and causes significant mortality and morbidity in HIV–infected patients
[
(
[
49]. Moreover, C. albicans is the fourth leading cause of nosocomial bloodstream infection
BSI) in intensive care units, causing fatal invasive candidiasis in a high percentage of patients
50].
To assess antifungal activities, the broth microdilution method M27–A3 for yeasts of the Clinical
and Laboratory Standards Institute was used [51]. Growth inhibition was determined by testing
all compounds at the concentration range 3.9 – 250 µg/mL (Table 2 and Supplementary Table 1).
Table 2 shows the minimum concentration that inhibits 80% or 50% of fungal growth (MIC80
and MIC50 respectively) of C. albicans and C. neoformans. Most of the clinically used
antifungals are active at a MIC level ≤ 10 µg/mL [52]. Almost all compounds of series B (9a–f,
1
0a–f, 11a–f, 12a–f and 13a–f) lack of antifungal activity suggesting that cyclization of the
double bond of the chalcone derivatives 8a–f into an azole ring (9a–f, 10a–f, 11a–f, 12a–f and
3a–f) abrogates the antifungal effects of the compounds. In a recent paper [37] we found that
analogues of compounds 11a–f and 12a–f, with scheleton type B but with N–phenyl substituent
1
2
attached to the vicinal N, displayed relevant antifungal activity suggesting that the position of the
ring C is important for antifungal activity too. In addition, the results obtained with the aldehyde
6
clearly show that the 7–chloro–4–aminoquinoline nucleus does not confer antifungal activity.
Table 2. Antifungal activity expressed as MIC and MIC obtained for compounds 6, 8a–f, 9a–
8
0
50
f, 10a–f, 11a–f, 12a–f and 13a–f against C. albicans and C. neoformans.
1
2

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For the A series, the best activity was displayed by both, the compounds 8a (4–Cl benzene) and
8
e (4–CH benzene) against C. neoformans (MIC = 7.8 µg/mL). Instead, the unsubstituted 8f
3 50
was moderately active (MIC = 31.25 µg/mL); 8b (4–Br benzene) was marginally active (MIC
5
0
50
=
>
125 µg/mL) and 8c (4–OCH benzene) and 8d [3,4,5– (OCH ) benzene] were inactive (MIC
3 3 3
50
250 µg/mL) against C. neoformans and marginally active (MIC ≥ 250 µg/mL) against C.
albicans. Figure 1 shows the percentages of inhibition for all compounds of the series 8, at the
different concentrations tested, against C. albicans (Fig. 1A) and C. neoformans (Fig. 1B).
Figure 1. Comparative dose–response curves of compounds 8a–f against (A) C. albicans ATCC
1
0231 and (B) C. neoformans ATCC 32264. Amphotericin B, used as the control, displayed
00% of inhibition at all concentrations tested (curve not shown).
1
As shown in Figure 1, C. neoformans was more sensitive than C. albicans when tested with
compounds 8a–f, being 8a and 8e the most active compounds with low MIC80 (7.8 and 15.6
µg/mL respectively) and MIC (7.8 µg/mL for both compounds). Figure 2 compares the MIC
5
0
80
and MIC values for each compound of the series 8a–f against C. neoformans.
5
0
Figure 2. Comparative MIC80 and MIC50 values (µg/mL) for each compound of the series 8a–f
against C. neoformans ATCC 32264.
1
3

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In Figure 2, it is shown that 8a and 8e possess the lower MICs against C. neoformans, followed
by 8f. These findings clearly highlight that the quinoline–based chalcones 8a and 8e could be
used as leads for developing new series with promising anticryptococcal activity for further
development.
2
.4 Antibacterial activity
All the novel compounds were screened for their in vitro antibacterial activity against gram–
positive and gram–negative bacteria including wild type and emerging multidrug resistant
strains. The minimum inhibitory and bactericidal concentrations (MIC and MBC) were
determined and reported in Table 3. Compound 6 (the parent benzaldehyde chloroquinoline) was
the most active, showing inhibitory activity against Staphylococcus aureus ATCC 25923
(
methicillin sensitive, MIC 62.5 ꢀg/mL), vancomycin–intermediate Staphylococcus aureus
VISA) (MIC 125 ꢀg/mL) and Escherichia coli ATCC 25922 (MIC 62.5 ꢀg/mL). This
(
compound also showed discrete inhibition against a carbapenemase–positive Klebsiella
pneumoniae BAA 1705 (MIC 250 ꢀg/mL) which represents one of the therapeutically
challenging multidrug resistant strains and an emerging worldwide threat. Compound 9d, a
pyrazoline bearing a trimethoxyphenyl moeity, selectively inhibited Neisseria gonorrhoeae
growth with MIC of 31.25 ꢀg/mL. None of the compounds seemed to be active against P.
aeruginosa or K. pneumoniae ATCC 700603. The results obtained with the aldehyde 6 show that
the 7–chloro–4–aminoquinoline nucleus confer antibacterial activity, but this activity is
abolished when the nucleus is functionalized with benzene rings B and C. Compound 9d is an
exception to this observation probably by the presence of the trimethoxy substituent. Although
1
4

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the MICs for the active compounds are much higher than those for licensed antibiotics, they may
be a starting point to develop compounds with improved antibacterial activity particularly against
resistant strains.
Table 3. In vitro antibacterial activity (MIC/MBC values in ꢀg/mL)
2
.5 Cytotoxic activity in human U–937 macrophages
All compounds were tested first for toxicity on U–937 human cells at four serial dilution
concentrations (200, 50, 12.5 and 3.125 ꢀg/mL) to determine their 50% Lethal Concentration
(
LC ) (Table 4). All compounds of series A and B (6, 8a–f, 9a–f and 10a–f) and few
50 1
compounds of series B (11b, 11c, 11e and 12b) exhibiting LC < 100 ꢀg/mL were considered
2
50
as potentially cytotoxic whereas almost all compounds of the series B (11a, 11d, 11f, 12a, 12c–f
2
and 13a–f) showed a LC50 > 200 ꢀg/mL and therefore classified as potentially non cytotoxic.
Amphotericin B showed a LC50 = 36.0 ꢀg/mL (potentially cytotoxic) and benznidazole had a
LC50 = 180.0 ꢀg/mL (mildly cytotoxic). It is clear from Table 4 that the substitution with a
phenyl group on N–1 of the 2–pyrazoline ring decreases the cytotoxic activity of the compounds
and the decrease is greater when this ring is attached to one or two chlorine atoms. The results
obtained with the aldehyde 6 show that the 7–chloro–4–aminoquinoline nucleus confer cytotoxic
activity, but this activity is diminished when the nucleus is functionalized with rings A and B and
specially with ring C. However, these results cannot be misinterpreted, since amphotericin B has
1
5

ACCEPTED MANUSCRIPT
high cytotoxicity and is still a commercial drug. Therefore the results should be confirmed with
in vivo studies.
Table 4. In vitro cytotoxic activity of novel synthetic compounds 6, 8a–f, 9a–f, 10a–f, 11a–f,
1
2a–f and 13a–f.
2
.6 Antiplasmodial activity
All compounds were screened in vitro for antiplasmodial activity on asynchronic cultures of P.
falciparum (3D7 strain, sensitive to chloroquine) at four serial diluted concentration (100, 25,
6
.25 and 1.56 ꢀg/mL) to determine the Effective Concentration 50 (EC ) (See Table 5).
50
Table 5. In vitro antimalarial screening of novel synthetic compounds 6, 8a–f, 9a–f, 10a–f, 11a–
f, 12a–f and 13a–f.
Most compounds, except 8a, 9a, 9b, 12c, 12d and 13e, were highly active against P. falciparum
(EC50 ≤ 25 ꢀg/mL). Compound 11b was the most active (EC50 = 5.54 ꢀg/mL). The EC50
obtained with the aldehyde 6 and CQ show that the 7–chloro–4–aminoquinoline nucleus confer
antiplasmodial activity, and also that this activity can be improved when the nucleus is
functionalized with ring A in the 4–amino group. Although almost all active compounds showed
lower EC50 than CQ, these values do not vary significantly respect to compound 6 suggesting
1
6

ACCEPTED MANUSCRIPT
that the antiplasmodial activity is given by 7–chloro–4–aminoquinoline nucleus and the ring A
and it is not improved with the functionalization with rings B and C. Most compounds of series
B (11a, 11d, 11f, 12a, 12e, 12f, 13a–d and 13f) showed better EC than CQ, and also LC >
2
50
50
2
00 ꢀg/mL, these results suggest that those compounds have potential as antimalarial drug,
therefore could be further studied in vivo for drug development in malaria therapy.
2
.7 Antitrypanosomal activity
Antitrypanosomal activity was screened against intracellular amastigotes of Trypanosoma cruzi
Tulahuen strain) at four serial dilutions. The activity of these compounds (in terms of EC ) is
(
5
0
summarized in Table 6. The EC ranged from 0.70 to 22.58 ꢀg/mL for active compounds (EC
50
5
0
≤
25 ꢀg/mL). Compound 10a was highly active against T. cruzi (EC = 0.70 ꢀg/mL). It is clear
50
from Table 6, that almost all compounds of series A and B (8a–f, 9a–f and 10a–f) are highly
1
active against T. cruzi, but the activity is diminished when the N–1 of the 2–pyrazoline ring is
substituted with a phenyl group. The EC50 obtained with the aldehyde 6 show that the 7–chloro–
4
–aminoquinoline nucleus confer antitrypanosomal activity but this activity is diminished with
the functionalization with rings A, B and C. The results suggest that compounds 11a (EC =
5
0
1
6.21 ꢀg/mL), 11d (EC50 = 21.59 ꢀg/mL), 12d (EC50 = 22.58 ꢀg/mL), 12e (EC50 = 21.92
g/mL), 12f (EC = 15.11 ꢀg/mL), 13a (EC = 19.08 ꢀg/mL) and 13d (EC = 21.94 ꢀg/mL)
ꢀ
50
50
50
with LC > 200 ꢀg/mL have potential as antitrypanosomal drugs and therefore their potential as
5
0
leading compounds should be further studied.
1
7

ACCEPTED MANUSCRIPT
Table 6. In vitro testing of antitrypanosomal activity for novel compounds 6, 8a–f, 9a–f, 10a–f,
1
1a–f, 12a–f and 13a–f.
2
.8 Antileishmanial activity
Compounds were screened for antileishmanial activity in intracellular amastigotes of Leishmania
V) panamensis (MHOM/CO/87/UA140–EpiR–GFP strain). As done previously with
(
antiplasmodial and antitrypanosomal activities, the EC50 was determined after exposure to four
concentrations (Table 7). Most compounds evidenced good activity against Leishmania
amastigotes with EC50 varying from 0.79 to 22.67 ꢀg/mL for active compounds (EC50 ≤ 25
ꢀ
ꢀ
g/mL). The best antileishmanial activity was displayed by compound 8a which is active at 0.79
g/mL. Most compounds of series A and B (8a–f, 9a–f and 10b) are highly active against T.
1
cruzi, but the activity is diminished when the N–1 of the 2–pyrazoline ring is substituted with a
phenyl group. The results obtained with the aldehyde 6 show that the 7–chloro–4–
aminoquinoline nucleus confer antileishmanial activity but this activity is diminished with the
functionalization with rings A, B and C. None of the compounds had EC50 lower than
amphotericin B, however compounds 11a, 11d, 11f, 12d, 12f and 13a–f were active and showed
LC > 200 ꢀg/mL (better than amphotericin B) suggesting that those compounds have potential
5
0
as antileishmanial drug reducing the side effects of amphotericin B and therefore could be
considered as a hit for in vivo studies, especially compound 13b (EC = 2.77 ꢀg/mL).
5
0
1
8

ACCEPTED MANUSCRIPT
Table 7. In vitro antileishmanial activity of synthetic compounds 6, 8a–f, 9a–f, 10a–f, 11a–f,
1
2a–f and 13a–f.
3
Conclusions
A new series of novel 4,5–dihydro–1H–pyrazoles 9a–f, 10a–f, 11a–f, 12a–f and 13a–f were
efficiently synthetized in good yields by the reaction of hydrazine and its derivatives with
quinoline–based α,β–unsaturated carbonyl compounds 8a–f. Anticancer activity studies showed
that four compounds (8c,d and 10a,c) showed prominent values of GI against several cell lines,
5
0
some of them lower than 1.00 ꢀM. The studies on antifungal activity showed that compounds 8a
and 8e completely inhibited C. neoformans growth at 31.25 ꢀg/mL. We also found compound 9d
active against N. gonorrhoeae (MIC 31.25 ꢀg/mL) a bacteria becoming a resistant superbug and
a major public health problem. The parent compound 6 showed inhibition of MSSA, VISA and
E. coli. For antimalarial activity 2–pyrazoline 11b was the most active with EC50 = 5.54 ꢀg/mL.
Regarding to antitrypanosomal activity, the EC50 ranged from 0.70 to 22.58 ꢀg/mL for active
compounds, while compound 10a was highly active with EC = 0.70 ꢀg/mL. Antileishmanial
50
activity studies revealed that some compounds were active with EC varying from 0.79 to 22.67
5
0
ꢀ
g/mL for active compounds. The best antileishmanial activity was displayed by compound 8a
which is active at 0.79 ꢀg/mL. In short, in this paper are reported several novel compounds
exhibiting relevant in vitro anticancer and antiprotozoal activity that worth be further studied in
vivo.
1
9

ACCEPTED MANUSCRIPT
4
. Experimental
4
.1 General
Reagents and solvents used were obtained from commercial sources. Melting points were
measured using a Stuart SMP10 melting point device and are uncorrected. FTIR spectrum were
1
13
obtained with a Shimadzu IRAffinity–1. The H and C NMR spectrum were run on a BRUKER
DPX 400 spectrometer operating at 400 and 100 MHz respectively, using DMSO–d as solvent
6
and TMS as internal standard. The mass spectrum was obtained on a SHIMADZU–GCMS–
QP2010 spectrometer operating at 70 eV. The elemental analyses were obtained using a Thermo
Finnigan Flash EA1112 CHN (STIUJA) elemental analyzer. Thin layer chromatography (TLC)
was performed on a 0.2 mm pre–coated plates of silica gel 60 F254 (Merck).
4
4
.2 Chemistry
.2.1 Preparation of 2–(3–nitrophenyl)–1,3–dioxolane 3
A mixture of 3–nitrobenzaldehyde 1 (7.5 mmol), ethylene glycol 2 (11.25 mmol) and p–
toluenesulfonic acid (PTSA) (0.4 mmol) in toluene (25.0 mL) was heated under reflux for 24 h
using a Dean–Stark apparatus. After cooling, the crude was extracted with ethyl acetate and
NaOH 1 M. The organic layer was dried over anhydrous magnesium sulfate, filtered and
evaporated under reduced pressure. The solid was dried at room temperature and used without
further purification.
2
0

ACCEPTED MANUSCRIPT
4
.2.1.1. 2–(3–Nitrophenyl)–1,3–dioxolane 3. Beige solid; 91% yield; m.p. 53–55 °C. FTIR
–
1
(
ATR) v (cm ): 3093 (=C–H), 1620 (C=C), 1535 and 1360 (NO ), 1098 and 1070 (C–O–C). MS
2
+
(
70 eV) m/z (%): 195/196 [M ] (33/3), 194 (100), 148 (54), 77 (98), 73 (99).
4
.2.2 Preparation of 3–(1,3–dioxolan–2–yl)aniline 4
A mixture of the nitro compound 3 (6.5 mmol), hydrazine hydrate (18.5 mmol) and Raney nickel
356 mg) in methanol (25.0 mL) was heated under reflux for 1 h. The hot solution was filtered
(
and evaporated under reduced pressure. After cooling, the oil was filtered through a pad of silica
gel for purification.
–
1
4
3
d
.2.2.1. 3–(1,3–Dioxolan–2–yl)aniline 4. Yellow oil; 89% yield. FTIR (ATR) v (cm ): 3365 and
1
226 (NH ), 1625 (C=C), 1316 (=C–N), 1092 and 1070 (C–O–C). H NMR (400 MHz, DMSO–
2
6
) δ ppm 3.86–4.02 (m, 4H, H4’, H5’), 5.09 (s, 2H, NH
2
), 5.56 (s, 1H, H2’), 6.53–6.59 (m, 2H,
H
4
, H
6
), 6.66 (d, J = 1.7 Hz, 1H, H
2
), 7.01 (t, J = 7.7 Hz, 1H, H ). MS (70 eV) m/z (%): 165/166
5
+
[
M ] (100/11), 164 (49), 120 (26), 93 (40), 73 (23).
4
.2.3 Preparation of 3–((7–chloroquinolin–4–yl)amino)benzaldehyde 6
A mixture of the amine 4 (2.75 mmol), and 4,7–dichloroquinoline 5 (2.5 mmol) in ethanol (10.0
mL) was heated under reflux for 3 h. After cooling, HCl 37% (0.5 mL) was added and the
2
1

ACCEPTED MANUSCRIPT
solution was stirred at room temperature for 5 min. The solid formed was filtered and washed
with ethanol.
4
.2.3.1. 3–((7–Chloroquinolin–4–yl)amino)benzaldehyde 6. Beige solid; 98% yield; m.p. 195–
–
1
1
1
97 °C. FTIR (ATR) v (cm ): 3443 (NH), 3069 (=C–H), 2897 (–(C=O)–H), 1700 (C=O). H
NMR (400 MHz, DMSO–d ) δ ppm 6.90 (d, J = 7.0 Hz, 1H, H ), 7.80 (t, J = 7.7 Hz, 1H, H ),
6
3
m
7
1
1
1
2
.83–7.90 (m, 2H, H
H, H
H, NH). C NMR (100 MHz, DMSO–d
6
, H
o
), 7.96 (d, J = 7.4 Hz, 1H, H
p
), 8.01 (s, 1H, Ho’), 8.22 (d, J = 2.0 Hz,
), 10.08 (s, 1H, CHO), 11.44 (s,
) δ ppm 100.5, 116.2, 119.2, 125.5, 126.4, 127.5,
8
2 5
), 8.56 (d, J = 7.0 Hz, 1H, H ), 8.96 (d, J = 9.2 Hz, 1H, H
1
3
6
28.5, 130.9, 131.1, 137.6, 138.0, 138.5, 139.0, 143.6, 154.8, 192.6. MS (70 eV) m/z (%):
+
82/284 [M ] (100/32), 281 (52), 253 (60), 218 (58), 190 (17).
4
.2.4 General procedure for the synthesis of [(7–chloroquinolin–4–yl)amino]chalcones 8a–f
A mixture of the aldehyde 6 (1.00 mmol), the respective acetophenone 7a–f (1.2 mmol) and
potassium hydroxide (100 mg) in methanol (10.0 mL) was stirred at room temperature for 12 h.
The solid formed was filtered and washed with methanol.
4
.2.4.1. (E)–1–(4–Chlorophenyl)–3–(3–((7–chloroquinolin–4–yl)amino)phenyl)prop–2–en–1–
–
1
one 8a. Beige solid; 90% yield; m.p. 210–213 °C. FTIR (ATR) v (cm ): 3350 (NH), 3064 (=C–
1
H), 1659 (C=O), 1604 and 1569 (C=N and C=C). H NMR (400 MHz, DMSO–d ) δ ppm 6.94
6
3
(d, J = 5.2 Hz, 1H, H ), 7.46 (d, J = 7.8 Hz, 1H, HAo), 7.52 (t, J = 7.7 Hz, 1H, HAm), 7.58 (dd, J =
9
.0, 1.9 Hz, 1H, H ), 7.62 (d, J = 8.5 Hz, 2H, HBm), 7.69 (d, J = 7.4 Hz, 1H, HAp), 7.78 (d, J =
6
2
2

ACCEPTED MANUSCRIPT
1
5.6 Hz, 1H, H3’), 7.91 (s, 2H, H
8
, HAo’), 7.95 (d, J = 15.6 Hz, 1H, H2’), 8.18 (d, J = 8.5 Hz, 2H,
1
3
H ), 8.43–8.52 (m, 2H, H , H ), 9.31 (s, 1H, NH). C NMR (100 MHz, DMSO–d ) δ ppm
Bo
2
5
6
1
1
02.1, 118.4, 122.2, 122.9, 124.5, 124.8, 125.0, 125.1, 127.7, 128.9, 130.0, 130.5, 133.9, 135.9,
+
36.2, 138.2, 140.9, 144.2, 147.9, 149.6, 152.1, 188.1. MS (70 eV) m/z (%): 418/420 [M ]
(
100/64), 279 (40), 243 (77), 139 (56), 111 (50). Anal. Calcd. For C H Cl N O: C, 68.75; H,
24 16 2 2
3
.85; N, 6.68. Found: C, 68.76; H, 3.83; N, 6.65.
4
.2.4.2.
(E)–1–(4–Bromophenyl)–3–(3–((7–chloroquinolin–4–yl)amino)phenyl)prop–2–en–1–
–
1
one 8b. Beige solid; 87% yield; m.p. 206–208 °C. FTIR (ATR) v (cm ): 3354 (NH), 3069 (=C–
1
H), 1659 (C=O), 1603 and 1570 (C=N and C=C). H NMR (400 MHz, DMSO–d ) δ ppm 6.95
6
(
d, J = 5.3 Hz, 1H, H ), 7.46 (d, J = 8.0 Hz, 1H, H ), 7.53 (t, J = 7.8 Hz, 1H, H ), 7.59 (dd, J =
3 Ao Am
9
3
.0, 1.8 Hz, 1H, H ), 7.70 (d, J = 7.5 Hz, 1H, H ), 7.74–7.84 (m, 3H, H , H ), 7.87–7.98 (m,
6 Ap 3’ Bm
H, H , H , H ), 8.11 (d, J = 8.4 Hz, 2H, H ), 8.43–8.51 (m, 2H, H , H ), 9.15 (s, 1H, NH).
8
2’
Ao’
Bo
2
5
1
3
C NMR (100 MHz, DMSO–d ) δ ppm 102.1, 118.4, 122.2, 122.9, 124.4, 124.8, 125.0, 125.1,
6
1
27.4, 127.7, 130.0, 130.6, 131.9, 133.9, 135.9, 136.5, 141.0, 144.2, 147.9, 149.5, 152.0, 188.3.
+
MS (70 eV) m/z (%): 462/464 [M ] (77/100), 279 (49), 243 (75), 185 (38), 155 (33). Anal.
Calcd. For C H BrClN O: C, 62.16; H, 3.48; N, 6.04. Found: C, 62.18; H, 3.51; N, 6.03.
2
4
16
2
4
.2.4.3. (E)–3–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–1–(4–methoxyphenyl)prop–2–en–1–
–
1
one 8c. Yellow solid; 77% yield; m.p. 245–246 °C. FTIR (ATR) v (cm ): 3361 (NH), 3009
1
(
=C–H), 1650 (C=O), 1611 and 1573 (C=N and C=C). H NMR (400 MHz, DMSO–d ) δ ppm
6
3
.87 (s, 3H, OCH
3
), 6.95 (d, J = 4.8 Hz, 1H, H
3
), 7.08 (d, J = 8.5 Hz, 2H, HBm), 7.44 (d, J = 7.9
), 7.64–7.77 (m, 2H, H3’
Hz, 1H, HAo), 7.52 (t, J = 7.7 Hz, 1H, HAm), 7.60 (d, J = 9.1 Hz, 1H, H
6
,
2
3

ACCEPTED MANUSCRIPT
HAp), 7.87 (s, 1H, HAo’), 7.92 (s, 1H, H
8
), 7.95 (d, J = 15.8 Hz, 1H, H2’), 8.17 (d, J = 8.5 Hz, 2H,
1
3
H ), 8.46 (d, J = 9.1 Hz, 1H, H ), 8.49 (d, J = 4.8 Hz, 1H, H ), 9.19 (s, 1H, NH). C NMR (100
Bo
5
2
MHz, DMSO–d ) δ ppm 55.6, 102.0, 114.0, 118.4, 122.5, 122.8, 124.4, 124.6, 124.8, 125.0,
6
1
27.7, 130.0, 130.4, 131.0, 133.9, 136.1, 140.9, 142.8, 148.0, 149.6, 152.1, 163.3, 187.4. MS (70
+
eV) m/z (%): 414/416 [M ] (97/31), 385 (51), 243 (32), 135 (100), 107 (26). Anal. Calcd. For
C H ClN O : C, 72.37; H, 4.62; N, 6.75. Found: C, 72.33; H, 4.65; N, 6.74.
25
19
2
2
4
.2.4.4. (E)–3–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–1–(3,4,5–trimethoxyphenyl)prop–2–
–
1
en–1–one 8d. Yellow solid; 74% yield; m.p. 235–237 °C. FTIR (ATR) v (cm ): 3341 (NH),
1
3
005 (=C–H), 1647 (C=O), 1612 and 1566 (C=N and C=C). H NMR (400 MHz, DMSO–d ) δ
6
ppm 3.76 (s, 3H, OCH ), 3.89 (s, 6H, OCH ), 6.97 (d, J = 5.1 Hz, 1H, H ), 7.43 (s, 2H, H ),
3
3
3
Bo
7
7
.47 (d, J = 7.8 Hz, 1H, H ), 7.52 (t, J = 7.7 Hz, 1H, H ), 7.58 (dd, J = 9.0, 1.4 Hz, 1H, H ),
Ao Am 6
.69–7.81 (m, 2H, H , H ), 7.89 (s, 1H, H ), 7.90 (d, J = 1.4 Hz, 1H, H ), 7.95 (d, J = 15.6
3
’
Ap
Ao’
8
1
3
Hz, 1H, H ), 8.48 (d, J = 5.1 Hz, 1H, H ), 8.51 (d, J = 9.0 Hz, 1H, H ), 9.33 (s, 1H, NH). C
2
’
2
5
NMR (100 MHz, DMSO–d
6
) δ ppm 56.3, 60.2, 102.0, 106.3, 118.4, 122.4, 123.0, 124.5, 124.6,
1
1
24.8, 124.9, 127.6, 129.9, 132.9, 133.9, 136.0, 141.0, 142.1, 143.6, 147.9, 149.6, 152.0, 152.9,
+
87.9. MS (70 eV) m/z (%): 474/476 [M ] (81/26), 459 (100), 281 (39), 195 (47), 57 (76). Anal.
Calcd. For C H ClN O : C, 68.28; H, 4.88; N, 5.90. Found: C, 68.24; H, 4.83; N, 5.92.
2
7
23
2
4
4
.2.4.5. (E)–3–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–1–(p–tolyl)prop–2–en–1–one 8e.
–
1
Beige solid; 81% yield; m.p. 215–217 °C. FTIR (ATR) v (cm ): 3352 (NH), 3014 (=C–H), 1665
1
(
C=O), 1616 and 1565 (C=N and C=C). H NMR (400 MHz, DMSO–d
6
) δ ppm 2.39 (s, 3H,
3 3
CH ), 6.95 (d, J = 5.1 Hz, 1H, H ), 7.36 (d, J = 7.9 Hz, 2H, HBm), 7.46 (d, J = 7.5 Hz, 1H, HAo),
2
4

ACCEPTED MANUSCRIPT
7
7
8
1
1
.51 (t, J = 7.7 Hz, 1H, HAm), 7.58 (dd, J = 9.0, 2.1 Hz, 1H, H
6
), 7.68 (d, J = 7.3 Hz, 1H, HAp),
.74 (d, J = 15.6 Hz, 1H, H ), 7.85–7.98 (m, 3H, H , H , H ), 8.06 (d, J = 7.9 Hz, 2H, H ),
3
’
8
2’
Ao’
Bo
1
3
.44–8.55 (m, 2H, H , H ), 9.29 (s, 1H, NH). C NMR (100 MHz, DMSO–d ) δ ppm 21.2,
2
5
6
02.1, 118.4, 122.6, 122.8, 124.5, 124.6, 124.9, 125.0, 127.7, 128.7, 129.4, 130.0, 133.9, 135.0,
+
36.0, 140.9, 143.3, 143.7, 147.9, 149.6, 152.1, 188.6. MS (70 eV) m/z (%): 398/400 [M ]
(
100/32), 243 (33), 221 (36), 119 (58), 91 (74). Anal. Calcd. For C H ClN O: C, 75.28; H,
25 19 2
4
.80; N, 7.02. Found: C, 75.28; H, 4.82; N, 7.06.
4
.2.4.6.
(E)–3–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–1–phenylprop–2–en–1–one
8f.
–
1
Yellow solid; 71% yield; m.p. 220–222 °C. FTIR (ATR) v (cm ): 3380 (NH), 3054 (=C–H),
1
1
653 (C=O), 1613 and 1562 (C=N and C=C). H NMR (400 MHz, DMSO–d ) δ ppm 6.96 (d, J
6
=
5.3 Hz, 1H, H ), 7.47 (d, J = 7.9 Hz, 1H, H ), 7.50–7.60 (m, 4H, H , H , H ), 7.64–7.72
3 Ao 6 Bm Am
(
m, 2H, H , H ), 7.76 (d, J = 15.7 Hz, 1H, H ), 7.89 (s, 1H, H ), 7.91 (d, J = 2.0 Hz, 1H, H ),
Bp Ap 3’ Ao’ 8
7
1
1
1
.94 (d, J = 15.7 Hz, 1H, H ), 8.15 (d, J = 7.6 Hz, 2H, H ), 8.45–8.54 (m, 2H, H , H ), 9.29 (s,
2’ Bo 2 5
1
3
6
H, NH). C NMR (100 MHz, DMSO–d ) δ ppm 102.1, 118.4, 122.5, 122.8, 124.5, 124.6,
24.9, 125.0, 127.7, 128.5, 128.8, 130.0, 133.2, 133.9, 135.9, 137.5, 140.9, 143.7, 147.9, 149.6,
+
52.1, 189.2. MS (70 eV) m/z (%): 384/386 [M ] (100/37), 243 (50), 207 (53), 105 (55), 77 (75).
Anal. Calcd. For C H ClN O: C, 74.90; H, 4.45; N, 7.28. Found: C, 74.90; H, 4.49; N, 7.31.
2
4
17
2
4
.2.5 General procedure for the synthesis of 1–(3–aryl–5–(3–((7–chloroquinolin–4–
yl)amino)phenyl)–4,5–dihydro–1H–pyrazol–1–yl)ethan–1–ones 9a–f
2
5

ACCEPTED MANUSCRIPT
A mixture of the chalcone 8a–f (0.40 mmol) and hydrazine hydrate (2.00 mmol) in ethanol (1.0
mL) was heated under reflux for 10 min. After cooling, acetic anhydride (2.0 mL) was added and
the solution was stirred at room temperature for 10 min. The solid formed was filtered and
washed with water and ethanol.
4
1
.2.5.1.
1–(3–(4–Chlorophenyl)–5–(3–((7–chloroquinolin–4–yl)amino)phenyl)–4,5–dihydro–
H–pyrazol–1–yl)ethan–1–one 9a. White solid; 86% yield; m.p. 198–199 °C. FTIR (ATR) v
–
1
1
(
cm ): 3352 (NH), 3020 (=C–H), 1672 (C=O), 1603 and 1543 (C=N and C=C). H NMR (400
MHz, DMSO–d
6
) δ ppm 2.32 (s, 3H, CH
3
), 3.24 (dd, J = 18.2, 4.9 Hz, 1H, H
A
), 3.92 (dd, J =
1
8.2, 12.0 Hz, 1H, H ), 5.63 (dd, J = 12.0, 4.9 Hz, 1H, H ), 6.82 (d, J = 7.0 Hz, 1H, H ), 7.27
M
X
3
(
d, J = 7.8 Hz, 1H, H ), 7.30 (s, 1H, H ), 7.40 (d, J = 8.3 Hz, 1H, H ), 7.50–7.56 (m, 3H,
Ap Ao’ Ao
H , H ), 7.80 (d, J = 8.6 Hz, 2H, H ), 7.83 (dd, J = 9.1, 1.5 Hz, 1H, H ), 8.18 (s, 1H, H ),
Bm
Am
Bo
6
8
1
3
8
.52 (d, J = 7.0 Hz, 1H, H ), 8.82 (d, J = 9.1 Hz, 1H, H ), Not observed (1H, NH). C NMR
2 5
(
100 MHz, DMSO–d ) δ ppm 21.7, 41.9, 59.4, 100.3, 116.1, 119.5, 122.2, 123.8, 124.7, 126.1,
6
1
27.2, 128.4, 128.9, 129.9, 130.2, 134.9, 137.3, 138.2, 139.4, 143.6, 144.2, 153.2, 154.5, 167.7.
+
MS (70 eV) m/z (%): 474/476 [M ] (58/37), 403 (99), 254 (47), 179 (44), 43 (100). Anal. Calcd.
For C H Cl N O: C, 65.69; H, 4.24; N, 11.79. Found: C, 65.63; H, 4.25; N, 11.79.
2
6
20
2
4
4
1
.2.5.2.
1–(3–(4–Bromophenyl)–5–(3–((7–chloroquinolin–4–yl)amino)phenyl)–4,5–dihydro–
H–pyrazol–1–yl)ethan–1–one 9b. White solid; 90% yield; m.p. 211–213 °C. FTIR (ATR) v
–
1
1
(
cm ): 3348 (NH), 3018 (=C–H), 1672 (C=O), 1602 and 1543 (C=N and C=C). H NMR (400
MHz, DMSO–d ) δ ppm 2.32 (s, 3H, CH ), 3.24 (dd, J = 18.2, 4.9 Hz, 1H, H ), 3.92 (dd, J =
8.2, 12.0 Hz, 1H, H ), 5.63 (dd, J = 12.0, 4.9 Hz, 1H, H ), 6.83 (d, J = 7.0 Hz, 1H, H ), 7.26
6
3
A
1
M
X
3
2
6

ACCEPTED MANUSCRIPT
(d, J = 7.8 Hz, 1H, HAp), 7.30 (s, 1H, HAo’), 7.40 (d, J = 8.0 Hz, 1H, HAo), 7.53 (t, J = 7.8 Hz, 1H,
H ), 7.66 (d, J = 8.6 Hz, 2H, H ), 7.72 (d, J = 8.6 Hz, 2H, H ), 7.83 (d, J = 9.1 Hz, 1H, H ),
Am
Bm
Bo
6
8
.17 (d, J = 1.9 Hz, 1H, H ), 8.52 (d, J = 7.0 Hz, 1H, H ), 8.82 (d, J = 9.1 Hz, 1H, H ), Not
8 2 5
1
3
observed (1H, NH). C NMR (100 MHz, DMSO–d ) δ ppm 21.7, 41.9, 59.4, 100.3, 116.1,
6
1
1
19.5, 122.2, 123.7, 123.8, 124.6, 126.1, 127.2, 128.6, 130.1, 130.2, 131.8, 137.3, 138.2, 139.5,
+
43.6, 144.2, 153.3, 154.4, 167.7. MS (70 eV) m/z (%): 518/520 [M ] (52/66), 478 (25), 449
4
(100), 254 (32), 43 (72). Anal. Calcd. For C26H20BrClN O: C, 60.07; H, 3.88; N, 10.78. Found:
C, 60.10; H, 3.93; N, 10.85.
4
1
.2.5.3. 1–(5–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–3–(4–methoxyphenyl)–4,5–dihydro–
H–pyrazol–1–yl)ethan–1–one 9c. Yellow solid; 80% yield; m.p. 206–208 °C. FTIR (ATR) v
–
1
1
(
cm ): 3351 (NH), 3017 (=C–H), 1679 (C=O), 1604 and 1546 (C=N and C=C). H NMR (400
MHz, DMSO–d ) δ ppm 2.31 (s, 3H, CH ), 3.21 (dd, J = 18.0, 4.7 Hz, 1H, H ), 3.80 (s, 3H,
6
3
A
OCH ), 3.89 (dd, J = 18.0, 11.9 Hz, 1H, H ), 5.60 (dd, J = 11.9, 4.7 Hz, 1H, H ), 6.82 (d, J =
3
M
X
7
7
3
.0 Hz, 1H, H ), 7.01 (d, J = 8.8 Hz, 2H, HBm), 7.27 (d, J = 7.9 Hz, 1H, HAp), 7.29 (s, 1H, HAo’),
.40 (d, J = 8.1 Hz, 1H, HAo), 7.53 (t, J = 7.8 Hz, 1H, HAm), 7.72 (d, J = 8.8 Hz, 2H, HBo), 7.84
(
d, J = 9.1 Hz, 1H, H ), 8.19 (s, 1H, H ), 8.51 (d, J = 7.0 Hz, 1H, H ), 8.83 (d, J = 9.1 Hz, 1H,
6 8 2
1
3
H ), Not observed (1H, NH). C NMR (100 MHz, DMSO–d ) δ ppm 21.7, 42.1, 55.4, 59.0,
5
6
1
1
00.2, 114.2, 116.0, 119.2, 122.2, 123.5, 123.8, 124.7, 126.1, 127.3, 128.3, 130.2, 137.2, 138.3,
+
39.1, 143.3, 144.5, 154.0, 154.7, 161.0, 167.3. MS (70 eV) m/z (%): 470/472 [M ] (59/22), 428
(
31), 399 (100), 175 (32), 43 (60). Anal. Calcd. For C H ClN O : C, 68.86; H, 4.92; N, 11.90.
27 23 4 2
Found: C, 68.90; H, 4.89; N, 11.90.
2
7

ACCEPTED MANUSCRIPT
4
.2.5.4.
1–(5–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–3–(3,4,5–trimethoxyphenyl)–4,5–
dihydro–1H–pyrazol–1–yl)ethan–1–one 9d. Yellow solid; 77% yield; m.p. 210–212 °C. FTIR
–
1
1
(
ATR) v (cm ): 3383 (NH), 3028 (=C–H), 1674 (C=O), 1602 and 1568 (C=N and C=C). H
NMR (400 MHz, DMSO–d ) δ ppm 2.31 (s, 3H, CH ), 3.21 (dd, J = 18.0, 4.7 Hz, 1H, H ), 3.74
6
3
A
(
s, 3H, OCH ), 3.85–3.92 (m, 7H, OCH , H ), 5.60 (dd, J = 11.8, 4.7 Hz, 1H, H ), 6.81 (d, J =
3 3 M X
7
8
8
.0 Hz, 1H, H ), 7.03 (s, 2H, H ), 7.26 (d, J = 7.9 Hz, 1H, H ), 7.31 (s, 1H, H ), 7.40 (d, J =
3 Bo Ap Ao’
.0 Hz, 1H, HAo), 7.53 (t, J = 7.8 Hz, 1H, HAm), 7.84 (d, J = 9.0 Hz, 1H, H
.51 (d, J = 7.0 Hz, 1H, H ), 8.82 (d, J = 9.0 Hz, 1H, H
) δ ppm 21.7, 42.1, 56.4, 59.0, 60.5, 100.2, 114.2, 116.5, 119.2, 122.3,
6
8
), 8.17 (s, 1H, H ),
1
3
2
5
), Not observed (1H, NH). C NMR
(100 MHz, DMSO–d
6
1
1
23.5, 123.7, 124.9, 126.1, 127.5, 128.3, 130.2, 137.3, 138.3, 139.3, 143.4, 144.5, 154.0, 154.6,
+
61.0, 167.3. MS (70 eV) m/z (%): 530/532 [M ] (100/37), 499 (83), 254 (46), 239 (24), 43 (89).
Anal. Calcd. For C H ClN O : C, 65.60; H, 5.13; N, 10.55. Found: C, 65.59; H, 5.10; N, 10.57.
2
9
27
4
4
4
.2.5.5.
1–(5–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–3–(p–tolyl)–4,5–dihydro–1H–
–
1
pyrazol–1–yl)ethan–1–one 9e. White solid; 78% yield; m.p. 199–201 °C. FTIR (ATR) v (cm ):
1
3
338 (NH), 3016 (=C–H), 1679 (C=O), 1606 and 1545 (C=N and C=C). H NMR (400 MHz,
DMSO–d ) δ ppm 2.32 (s, 3H, CH ), 2.34 (s, 3H, CH ), 3.21 (dd, J = 18.1, 4.8 Hz, 1H, H ), 3.90
6
3
3
A
(
dd, J = 18.1, 11.9 Hz, 1H, H ), 5.61 (dd, J = 11.9, 4.8 Hz, 1H, H ), 6.82 (d, J = 7.0 Hz, 1H,
M X
H ), 7.23–7.28 (m, 3H, H , H ), 7.29 (s, 1H, H ), 7.40 (d, J = 8.1 Hz, 1H, H ), 7.53 (t, J =
3
Bm
Ap
Ao’
Ao
7
1
.8 Hz, 1H, H ), 7.67 (d, J = 8.1 Hz, 2H, H ), 7.84 (dd, J = 9.1, 1.9 Hz, 1H, H ), 8.19 (d, J =
Am Bo 6
.9 Hz, 1H, H ), 8.51 (d, J = 7.0 Hz, 1H, H ), 8.83 (d, J = 9.1 Hz, 1H, H ), Not observed (1H,
8
2
5
1
3
NH). C NMR (100 MHz, DMSO–d
6
) δ ppm 21.0, 21.7, 42.0, 59.1, 100.2, 116.0, 119.3, 122.2,
1
23.8, 124.7, 126.1, 126.6, 127.3, 128.2, 129.3, 130.2, 137.2, 138.3, 139.2, 140.2, 143.4, 144.4,
2
8

ACCEPTED MANUSCRIPT
+
1
54.2, 154.6, 167.5. MS (70 eV) m/z (%): 454/456 [M ] (48/17), 383 (63), 254 (13), 159 (21), 43
(
100). Anal. Calcd. For C H ClN O: C, 71.28; H, 5.10; N, 12.31. Found: C, 71.28; H, 5.15; N,
27 23 4
1
2.33.
4
1
.2.5.6. 1–(5–(3–((7–Chloroquinolin–4–yl)amino)phenyl)–3–phenyl–4,5–dihydro–1H–pyrazol–
–
1
–yl)ethan–1–one 9f. White solid; 75% yield; m.p. 193–195 °C. FTIR (ATR) v (cm ): 3340
1
(
NH), 3017 (=C–H), 1674 (C=O), 1601 and 1542 (C=N and C=C). H NMR (400 MHz, DMSO–
d
6
) δ ppm 2.33 (s, 3H, CH
), 5.63 (dd, J = 11.9, 4.8 Hz, 1H, H
H ), 7.31 (s, 1H, H ), 7.40 (d, J = 8.1 Hz, 1H, H ), 7.44–7.50 (m, 3H, H , H ), 7.54 (t, J =
3
), 3.24 (dd, J = 18.1, 4.8 Hz, 1H, H
A
), 3.93 (dd, J = 18.1, 12.0 Hz, 1H,
H
M
X
), 6.82 (d, J = 7.0 Hz, 1H, H
3
), 7.28 (d, J = 7.7 Hz, 1H,
Ap
Ao’
Ao
Bm
Bp
7
.8 Hz, 1H, H ), 7.76–7.81 (m, 2H, H ), 7.84 (d, J = 8.4 Hz, 1H, H ), 8.19 (s, 1H, H ), 8.51 (d,
Am Bo 6 8
1
3
J = 7.0 Hz, 1H, H ), 8.83 (d, J = 8.4 Hz, 1H, H ), Not observed (1H, NH). C NMR (100 MHz,
2
5
DMSO–d ) δ ppm 21.7, 42.0, 59.2, 100.3, 116.0, 119.2, 122.3, 123.9, 124.7, 126.1, 126.6, 127.3,
6
1
28.8, 130.2, 130.4, 131.0, 137.2, 138.3, 139.1, 143.4, 144.4, 154.2, 154.7, 167.6. MS (70 eV)
+
m/z (%): 440/442 [M ] (28/10), 369 (50), 254 (10), 145 (17), 43 (100). Anal. Calcd. For
26 4
C H21ClN O: C, 70.82; H, 4.80; N, 12.71. Found: C, 70.81; H, 4.79; N, 12.72.
4
.2.6 General procedure for the synthesis of 3–aryl–5–(3–((7–chloroquinolin–4–
yl)amino)phenyl)–4,5–dihydro–1H–pyrazole–1–carbaldehydes 10a–f
A mixture of the chalcone 8a–f (0.40 mmol) and hydrazine hydrate (2.00 mmol) in ethanol (1.0
mL) was heated under reflux for 10 min. After cooling, formic acid (2.0 mL) was added and the
2
9