Design, synthesis and biological evaluation of 3-[4-(7-chloro-quinolin-4- yl)-piperazin-1-yl]-propionic acid hydrazones as antiprotozoal agents

Article abstract of DOI:10.1016/j.ejmech.2014.01.023

N-Acylhydrazones derived from 7-chloro-4-piperazin-1-yl-quinoline were synthesized and biologically evaluated for blood-stage of Plasmodium falciparum and Entamoeba histolytica trophozoites. N-Acylhydrazone F12 was found to inhibit the P. falciparum growt

Full text of DOI:10.1016/j.ejmech.2014.01.023

European Journal of Medicinal Chemistry 75 (2014) 67e76
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and biological evaluation of 3-[4-(7-chloro-quinolin-
4-yl)-piperazin-1-yl]-propionic acid hydrazones as antiprotozoal
agents
Afreen Inam a, Shadab Miyan Siddiqui a, Taís Soares Macedo b,
Diogo Rodrigo Magalhaes Moreira b, Ana Cristina Lima Leite c,
,
,
*
Milena Botelho Pereira Soares b d, Amir Azam a
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025 New Delhi, India
b Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, CEP 40296-710 Salvador, BA, Brazil
c Universidade Federal de Pernambuco, Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, CEP 50740-520 Recife, PE, Brazil
d Hospital São Rafael, Centro de Biotecnologia e Terapia Celular, CEP 41253-190 Salvador, BA, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Acylhydrazones derived from 7-chloro-4-piperazin-1-yl-quinoline were synthesized and biologically
evaluated for blood-stage of Plasmodium falciparum and Entamoeba histolytica trophozoites. N-Acylhy-
drazone F12 was found to inhibit the P. falciparum growth as well as its life cycle with good selectivity,
which was achieved by inhibiting hematin formation. Compound F24 showed better IC50 value than the
amoebicidal drug metronidazole.
Received 4 June 2013
Received in revised form
10 January 2014
Accepted 11 January 2014
Available online 24 January 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Malaria
Amoebiasis
Aminoquinoline
Piperazinyl derivatives
Acylhydrazone
1. Introduction
In our previous works, chalcones and hydrazones derived from
chloroquinoline exhibited antiamoebic activity of higher potency
Entamoeba histolytica and Plasmodium falciparum represent
major threats to the human health worldwide, with over one billion
clinical cases and between four million deaths annually [1]. The
protozoan parasites have now become resistant to some of the
antiprotozoal medicaments, thereby pressurizing the control
measures in place to treat patients infected with malaria and
amoebiasis. This scenario has necessitated the search for novel
drugs to contribute to the global chemotherapeutic regimens [2,3].
Quinolines are useful source for drug development of anti-
protozoal agents [4e6]. The most notable is chloroquine, which is
not only an antimalarial, but also a broad-spectrum antiparasitic
agent, including antiamoebic activity [7]. Indeed, a large number of
naturally and synthetically-derived quinolines are antiprotozoal
agents, such as iodoquinol, mefloquine, and amodiaquine [8e10].
than the parent quinoline [11,12]. However, no substantial anti-
malarial activities were observed. The N-acylhydrazones have
reactivity as Michael-acceptors and are classically employed as
warheads during drug design [13]. The attachment of N-acylhy-
drazones has been employed both in the design of ligands for
further complexation with transition metals [14] and during the
processes of hit-to-lead conversion [15]. N-Acylhydrazone is a well-
known group of antiparasitic agents, which includes antimalarial
and antiamoebic properties [16]. Besides, the synthetic redesign of
antiparasitics in N-acylhydrazones is a cost-efficient and timely
manner for developing chemical libraries [17].
In view of these functional properties, it was envisaged to
conjugate aminoquinoline with N-acylhydrazone. However, recent
literature survey revealed that the most likely way of optimizing
antimalarial quinolines is by using a chemical linker between the
quinoline ring and a second pharmacophoric group [18,19]. In the
present study piperazine was used as a linker between the
aminoquinoline and N-acylhydrazone because of the potent
* Corresponding author. Tel.: þ91 11 2698 1717, þ91 11 2698 3254; fax: þ91 11
2698 0229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.023

68
A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
Table 1
antimalarial activities observed for trioxaquines [20]. Considering
this perspective, we designed, synthesized and pharmacologically
evaluated a chemical library of N-acylhydrazones (F1eF33) derived
from 7-chloro-4-piperazin-1-yl-quinoline.
Antiparasitic activity of N-acylhydrazones F1eF20.
Cpd Ar
P. falciparum IC50 ꢀ SD (
mM)
inhibitiona,c
P. falciparumb,c E. histolyticad Splenocytese
4
e
74.1
93.3
81.4
97.8
97.5
41.7 ꢀ 2.5
e
>100
2. Results and discussion
F1 Ph
0.2 ꢀ 0.05
6.6 ꢀ 0.2
8.1 ꢀ 0.15
11.1 ꢀ 0.11
0.3 ꢀ 0.2
F2 4-CH3Ph
F3 4-C2H5Ph
F4 4-iPrPh
e
e
e
e
2.1. Chemistry
1.7 ꢀ 0.31
17.1 ꢀ 0.15 7.9 ꢀ 1.5
F5 4-(CH3)2NPh 98.0
1.3 ꢀ 0.45
15.2 ꢀ 0.2
11.3 ꢀ 0.12
12.6 ꢀ 0.22
>100
e
The synthesis of N-acylhydrazones (F1eF33) was accomplished
in four steps as outlined in Scheme 1. The nucleophilic substitution
of 4,7-dichloroquinoline (1) with piperazine afforded 7-chloro-4-
piperazin-1-yl-quinoline (2). Synthesized quinoline (2) was then
reacted under neat conditions with methyl acrylate, forming 3-[4-
(7-chloro-quinolin-4-yl)piperazin-1-yl]propionic acid methyl ester
(3). The reaction of compound (3) with hydrazine hydrate furnished
hydrazide (4) which was further reacted with commercially avail-
able aldehydes to give N-acylhydrazones (F1eF33).
F6 2-NO2Ph
F7 3-NO2Ph
F8 4-NO2Ph
F9 2-ClPh
F10 3-ClPh
F11 4-ClPh
F12 2-OHPh
F13 4-OHPh
F14 2-OC2H5Ph
F15 4-OCH3Ph
F16 4-OC2H5Ph
F17 4-OiPrPh
F18 2-Py
73.1
19.9
38.4
97.9
97.3
98.4
98.9
80.6
28.1
43.0
14.9
98.0
0
e
e
e
e
0.95 ꢀ 0.11 12.1 ꢀ 2.4
12.3 ꢀ 0.13 10.8 ꢀ 0.46
0.78 ꢀ 0.05
e
7.7 ꢀ 0.23
87.2 ꢀ 0.2
>100
0.2 ꢀ 0.04
1.4 ꢀ 0.3
0.33 ꢀ 0.095
25.6 ꢀ 0.15 11.5 ꢀ 0.4
e
>100
>100
e
e
e
e
19.7 ꢀ 0.32
12.0 ꢀ 0.24
5.3 ꢀ 0.22
>100
e
e
e
0.8 ꢀ 0.5
3.6 ꢀ 2.2
2.2. Biology
e
e
F19 2-CF3Ph
F20 4-(OEt)2CHPh 71.7
59.6
e
8.1 ꢀ 0.31
e
e
>100
e
2.2.1. Antiamoebic activity
Mtz
Mfq
e
e
e
1.5 ꢀ 0.1
e
5.4 ꢀ 1.6
9.5 ꢀ 0.46
All the desired compounds were screened for antiamoebic ac-
tivity against HM1:IMSS strain of E. histolytica and metronidazole
(Mtz) was used as reference amoebicidal drug. Table 1 displays the
antiamoebic activity of N-acylhydrazone (F1eF20). The IC50 of
e
0.04 ꢀ 0.01
a
%
Of parasite inhibition using the concentration of 1.0
m
g/mL for each
compound.
b
IC50s were calculated using at least five compound concentrations.
Determined 24 h after incubation of W2 strain P. falciparum (erythrocytic stage)
c
compound F1 was 6.6 ꢀ 0.2
m
M, which was fourfold higher than the
with the respective compounds.
IC50 observed for Mtz (1.5 ꢀ 0.1
mM). Incorporation of an alkyl
d
Determined 72 h after incubation of HM1:IMSS strain trophozoites with the
substituent at para-position decreased the antiamoebic activity in
following order: methyl (F2) > ethyl (F3) > isopropyl groups (F4).
Replacing a phenyl by 2-pyridinyl (F18) was deleterious for activity.
Electron-donating substituents attached at para-position, such as
N,N-dimethylamino (F5), hydroxy (F12, F13) and alkoxy (F14, F15,
F16, F20) produced weaker amoebicidal than the compound having
phenyl group (F1). Attaching a chloro atom at ortho (F9) and meta
(F10) produced active amoebicidal compounds, albeit less potent
than the observed for the compound incorporating phenyl group
(F1). However, attaching a chloro at para-position (F11) was dele-
terious for the amoebicidal activity. Furthermore, it was observed
that a trifluoromethyl group when attached to the ortho-position
(F19) led to a compound as potent as the compound bearing phenyl
group (F1) in inhibiting the E. histolytica growth. Interestingly,
compounds. IC50 was calculated from at least five concentrations using concentra-
tions in triplicate.
e
IC50 values for mouse splenocytes after 24 h of incubation in the presence of the
compounds. SD means standard deviation. Mtz is metronidazole; Mfq is mefloquine.
(IC50 ¼ 0.95 ꢀ 0.11
(IC50 ¼ 12.1 ꢀ 2.4
m
M) without affecting mouse splenocyte viability
mM). The compounds containing a nitro group at
ortho (F6) and meta (F7) were less active than compound (F8).
The screening of N-acylhydrazones F21eF33 (Table 2) revealed
two other promising antiamoebic compounds F24 and F33. Com-
pound (F24) (IC50 ¼ 0.13 ꢀ 0.02
mM) was more potent against
E. histolytica than metronidazole. Indeed, compound (F24) was the
most potent antiamoebic agent among the studied compounds. It
has low cytotoxicity to mouse splenocytes (IC50 ¼ 15.8 ꢀ 2.0
mM).
the 4-nitro compound (F8) is
a potent amoebicidal agent
Scheme 1. Synthesis of N-acylhydrazones (F1eF33).

A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
69
Table 2
Antiparasitic activity of N-acylhydrazones (F21eF33).
Cpd
Ar
P. falciparum
IC50 ꢀ SD (
mM)
% Inhibitiona,c
P. falciparumb,c
E. histolyticad
Splenocytese
F21
F22
99.1
98.4
2.1 ꢀ 0.53
21.3 ꢀ 0.31
9.4 ꢀ 1.5
e
11.6 ꢀ 0.32
15.5 ꢀ 0.15
e
F23
98.6
4.5 ꢀ 1.2
8.8 ꢀ 1.9
F24
F25
F26
0
e
e
e
0.13 ꢀ 0.02
>100
15.8 ꢀ 2.0
27.8
71.7
e
e
>100
F27
F28
F29
37.4
47.4
42.9
e
e
e
8.2 ꢀ 0.2
12.4 ꢀ 0.35
3.7 ꢀ 0.31
e
9.6 ꢀ 0.21
F30
F31
41.5
28.0
e
e
20.5 ꢀ 0.32
6.6 ꢀ 0.35
e
e
F32
F33
81.5
10.9
3.3 ꢀ 0.63
9.6 ꢀ 0.25
1.1 ꢀ 0.10
5.7 ꢀ 1.2
8.2 ꢀ 1.0
e
Mtz
Mfq
e
e
e
e
1.5 ꢀ 0.1
e
0.04 ꢀ 0.01
e
9.5 ꢀ 0.46
a
% Of parasite inhibition using the concentration of 1.0
IC50s were calculated using at least five compound concentrations.
Determined 24 h after incubation of W2 strain P. falciparum (erythrocytic stage) with the respective compounds.
m
g/mL for each compound.
b
c
d
Determined 72 h after incubation of HM1:IMSS strain trophozoites with the compounds. IC50 was calculated from at least five concentrations using concentrations in
triplicate.
e
IC50 values for mouse splenocytes after 24 h of incubation in the presence of the compounds. SD means standard deviation. Mtz is metronidazole; Mfq is mefloquine.
Therefore, compound (F24) has a selectivity index (splenocyte/
M)
for E. histolytica, had comparable ameobicidal activity to that of
metronidazole. However, compound (F33) displayed selectivity
index of 7.5, which is relatively low. All these N-acylhydrazone (F1e
F33) showed IC50 value in the range (0.95 0.11 M-
87.2 ꢀ 0.02 M). In terms of IC50 value, it was concluded that among
the N-acylhydrazone (F1eF20) compound (F8) exhibited the most
E. histolytica) of 121. Compound (F33), with IC50 value (1.1 ꢀ 0.1
m
ꢀ
m
m

70
A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
promising antiamoebic activity (IC50 ¼ 0.95 ꢀ 0.11
m
M), whereas
growth. N-Acylhydrazones (F4, F5, F9, F12, F21, F23, F32) exhibited
antimalarial activities for the blood-stage parasite. It was found that
none of the compounds caused lysis in uninfected erythrocytes
the remaining compounds showed higher IC50 value than Mtz and
therefore, were considered of little interest (Table 1). Further,
screening of N-acylhydrazones (F21eF33) for amoebicidal activity
when tested for hemolytic properties at concentration of 25 mM
revealed that two compounds, (F24, IC50 ¼ 0.13 ꢀ 0.02
m
M) and
(Fig. 1). Ideally, an antimalarial compound should inhibit the intra-
erythrocytic life cycle of parasite. Thus we examined the ability of
the potent antimalarial compound (F12) to inhibit intra-
erythrocytic life cycle of W2 strain of P. falciparum, which was
synchronized to ring stage and then incubated in erythrocytes. The
distribution of rings, schizonts and trophozoites in erythrocytes
was determined by microscopy. At the beginning, the parasitemia
in untreated-infected erythrocytes is composed of immature tro-
phozoites (ring stage). After 24 h of incubation, most ring-stage
cells maturate to trophozoites and at 48 h of incubation, a signifi-
cant number of schizonts were observed. Compound (F12) at
(F33, IC50 ¼ 1.1 ꢀ 0.1
m
M) exhibited promising antiamoebic activity.
The compound (F24) was approximately tenfold more potent than
Mtz in this assay. In comparison to our previous investigated sul-
fonamides and thiosemicarbazones [21,22], N-acylhydrazones were
found more potent against E. histolytica.
2.2.2. Antimalarial activity
All the target compounds were screened for antimalarial ac-
tivity against W2 strain of P. falciparum which is chloroquine
resistant, therefore, mefloquine (Mfq) was used as reference
inhibitory drug. The IC50 values of chloroquine and mefloquine
0.5
1.0
m
M substantially reduced the number of schizonts, while at
against P. falciparum cells are (0.15 ꢀ 0.3
m
M) and (0.04 ꢀ 0.01
mM)
mM, no schizonts were observed in the cell culture (Fig. 2). The
respectively. Toxicity to mammalian cells (IC50) was determined by
using mouse splenocytes (Tables 1 and 2). Compound (F9) having
a chloro group at ortho-position was found to exhibit sub-
mode of action of N-acylhydrazones is structurally similar to
quinoline-based antimalarials, which are well-known to inhibit the
b
-hematin formation [24]. Hence, the ability of selected com-
pounds to inhibit the formation of -hematin was evaluated in vitro
(Table 3). Interestingly, a trend showing an increase in in vitro
antimalarial activity with the increase in -hematin formation
could be observed (Fig. 3). The -hematin formation assay showed
micromolar IC50 value (0.78 ꢀ 0.05
group by a hydroxyl group subsequently yielded a two-fold more
potent compound (F12, IC50 0.33 ꢀ 0.09 M). Since the incorpo-
mM). Replacing this chloro
b
m
b
ration of a chloro group (F9) or hydroxyl group (F12) in ortho of
the phenyl ring exerted significant inhibitory activity, it was
concluded that polar substituents at ortho position are favorable
for antimalarial activity. Although (F9) and (F12) exhibited
promising antimalarial activity yet these were less potent than the
reference drug mefloquine (Mfq). These compounds do not show
toxicity against mouse splenocytes at concentrations of up to
b
that the potent antimalarial compounds F4, F5, F9 and F12 were the
best inhibitors, while compound F21 & F32 were moderate in-
hibitors, and compounds 4 and F23 were least inhibitor as
compared to chloroquine, which showed 87.1% inhibition at the
concentration 2.5 mM.
10
at 25
m
M. Moreover, uninfected erythrocytes exposed to (F9) or (F12)
3. Conclusion
m
M showed no hemolytic action. At concentration of 1.0 M,
m
the N-acylhydrazone (F12) was able to efficiently inhibit the
parasite differentiation into schizont forms showing that this
compound affects the parasite development in the blood-stage.
Thus the data suggest that the compounds (F9) and (F12) impair
erythrocytic life cycle of P. falciparum. However, the notable
advantage is that they did not overtly affect the viability of host
cells. The introduction of a nitro (compounds F6eF8) or alkoxy
groups (compounds F14eF17) on the phenyl ring proved to be
detrimental to antimalarial activity (Table 1). However, attaching a
nitro group in ortho position (F6) maintained some level of anti-
malarial activity (73.1% inhibition). Compounds with tri-
fluoromethyl (F19) or ethoxy (F14) groups at ortho-position and
methoxy (F15) or ethoxy (F16) groups attached to the para-posi-
tion were less potent than (F12). Likewise, a 2-pyridinyl ring (F18)
was not beneficial, being inactive.
An analysis of the antiplasmodial and antiamoebic properties
for the compounds (F1eF33) revealed a potency enhancement of
antiparasitic activity when compared to the hydrazide (4). We were
able to identify compounds (F12) and (F24) as the most potent and
The effect of substitution (F21eF33) on the phenyl ring was
analyzed. The 3-methoxyl-4-hydroxyl derivative (F21, 99.1% inhi-
bition) is more active against P. falciparum than 4-hydroxyl
derivative (F13, 80.6% inhibition). The same was observed for the
3,4-dimethoxy (F22) and 3-methoxy-4-isopropyl (F23) derivatives.
When 3-methoxy-4-O-acetyl (F24) or 1,3-benzodioxole (F25) is
attached, no antimalarial activity was observed. Regarding the
methoxyl group, optimal antimalarial activity was observed for the
3,4-dimethoxy compound (F22), whereas the 4-methoxy (F15),
2,5-dimethoxy (F26), and 3,4,5-trimethoxy (F28) derivatives were
weaker antimalarial agents. Accordingly, attaching bromo group
did not improve the activity as compounds (F29), (F30) and (F31)
were inactive. In comparison to (F1), which inhibited 93.3% of
parasite growth, indolyl derivative (F32) also exhibited antimalarial
activity (IC50 ¼ 3.3 ꢀ 0.63
(IC50 ¼ 5.7 ꢀ 1.2 M to splenocytes). Though ferrocenyl derivatives
are well-known antimalarial agents [23], yet the ferrocenyl deriv-
ative (F33) (1.0 g/mL) was unable to inhibit the P. falciparum
mM) but without enough selectivity
m
Fig. 1. Hemolytic assay for the N-acylhydrazones. Activity was assayed in uninfected
human erythrocytes (type Oþ) after 1 h of incubation with compounds (25
mM) in
m
triplicate. ***p < 0.0001 (one-way ANOVA followed by Tukey’s test).

A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
71
Fig. 2. Effects of N-acylhydrazone (F12) in the cell-cycle development of blood-stage
P. falciparum. Erythrocytes were infected with W2 strain P. falciparum and incubated
with compound F12 (at 0.5 or 1.0 M) or solvent (control). Each parasite forms were
m
analyzed by confocal microscopy in different times. Assay represents two replicates per
plate. Significance in reference to % of parasitemia: *p < 0.05; ***p < 0.001 (one-way
ANOVA).
Fig. 3. Comparison between the %
hydrazones. %
concentration. pIC50 was calculated as (ꢂlog IC50) by using IC50 values determined
against P. falciparum W2 strain. Dot diagonal line is for visualization only and does not
represent statistical analysis.
b
-hematin inhibition and pIC50 values for N-acyl-
b
-Hematin inhibition was determined by using 2.5 mM compound
selective antimalarial and antiamoebal agents of this series,
respectively. Specifically, (F12) inhibited the parasite growth as
well as its life cycle with good potency and selectivity, in addition to
4.1. Synthesis of 7-chloro-4-(piperazin-1-yl)quinoline (2)
inhibiting the
b-hematin formation. Against E. histolytica, com-
Piperazine (30.4 g, 353.4 mmol) was added to a mixture of 4,7-
dichloro-quinoline 1 (10 g, 50.4 mmol) in ethanol (150 mL). The
reaction mixture was refluxed and stirred for 12 h. The solvent was
removed in vacuo and the resulting solid mixture was dissolved in
dichloromethane (200 mL) and washed with saturated sodium
bicarbonate solution until the complete removal of piperazine in
the organic layer (TLC). The combined organic phases were dried
over anhydrous Na2SO4, concentrated and then purified by
recrystallization in 30% dichloromethane/hexane, (yield: 9.7 g 77%)
of compound 2. Mp: 118e120 ꢁC; 1H NMR (300 MHz, CDCl3):
pound (F24) exhibited higher potency than metronidazole, while
displayed lower cytotoxicity to mouse splenocytes. In overall, this
work has confirmed that N-acylhydrazones are antiparasitic agents,
and also suggested that converting antiparasitic agents in N-acyl-
hydrazone derivatives is an appealing synthetic tool for the anti-
parasitic drug discovery.
4. Experimental protocol
The melting points were recorded on Buchi melting point
apparatus Model No. M-650 and are uncorrected. Elemental ana-
lyses were performed on Elementar Vario analyzer and the results
are within ꢀ0.3% of the theoretical values. IR spectra (KBr) were
acquired at Bruker FT-IR spectrophotometer. 1H and 13C NMR were
recorded on a Bruker Spectrospin DPX 300 MHz and Bruker Spec-
trospin DPX 75 MHz spectrometer respectively, using CDCl3 as a
solvent and trimethylsilane (TMS) as the internal standard. Split-
ting patterns are designated as follows; s, singlet; d, doublet; t,
triplet; m, multiplet. Chemical shift values are given in ppm. The
FAB mass spectra of the compounds were recorded on JEOL SX 102/
DA-6000 mass spectrometer using Argon/Xenon 6 KV, 10 mA as the
FAB gas and m-nitrobenzyl alcohol (NBA) was used as the matrix.
d
¼ 8.72 (d, 1H, J ¼ 5.1 Hz), 8.06 (dd, 1H, J ¼ 2.1 and 9 Hz), 7.97 (t, 1H,
J ¼ 9 Hz), 7.42 (dd, 1H, J ¼ 2.1 and 9.0 Hz), 6.83 (d, 1H, J ¼ 5.1 Hz),
3.33 (s, 1H), 3.14e3.21 (m, 8H); IR (KBr) nmax ¼ 3342, 3024, 1579,
823 cmꢂ1; FAB-MS (m/z): [M þ 1]þ, 248; Anal. calcd for C13H14N3Cl:
C 63.03, H 5.70, N 16.96%; found: C 63.23, H 5.61, N 17.02%.
4.2. Synthesis of methyl 3-[4-(7-chloro-4-quinolyl)piperazin-1-yl]
propanoate (3)
7-Chloro-4-(piperazin-1-yl)quinoline 2 (1 g, 4.0 mmol) and
methyl acrylate (8 mL) were stirred under neat conditions for 12 h
at room temperature. The reaction mixture was concentrated in
vacuo gave a crude solid, which was purified by recrystallization in
20% (DCM/hexane), (yield: 1.1 g, 85%) of compound 3. Mp: 101 ꢁC;
1H NMR (300 MHz, CDCl3):
d
¼ 8.71 (d, 1H, J ¼ 5.1 Hz), 8.03 (d, 1H,
Table 3
Inhibition of
b
-hematin formation.
Substituent (Ar)
J ¼ 2.1 Hz), 7.93 (d, 1H, J ¼ 9 Hz), 7.40 (dd, 1H, J ¼ 2.1 and 9.0 Hz),
6.82 (d, 1H, J ¼ 5.1 Hz), 3.71 (s, 3H), 3.23 (t, 4H, J ¼ 4.8 Hz), 2.83 (t,
2H, J ¼ 7.2 Hz), 2.75 (t, 4H, J ¼ 4.8 Hz), 2.58 (t, 2H, J ¼ 7.2 Hz); IR
(KBr) nmax ¼ 3435, 2881, 2827, 1668, 1626, 1545 cmꢂ1. FAB-MS (m/
z): 333.4 [M þ 1]þ, 100%; Anal. calcd for C17H20N3O2Cl: C 61.17, H
6.04; N 12.59%; found: C 61.38, H 6.29, N 12.51%.
Cpd
% Of hematin inhibitiona
4
F4
F5
F9
F12
F21
F23
F32
Cqn
e
21.7
94.0
94.2
87.3
83.2
50.7
15.7
43.3
87.1
4-iPrPh
4-(CH3)2NPh
2-ClPh
2-OHPh
4-C(OEt)2Ph
3-OCH3e4-OiPrPh
3-Indole
e
4.3. Synthesis of 3-[4-(7-chloro-4-quinolyl)piperazin-1-yl]
propanehydrazide (4)
a
Hydrazine hydrate(7.3 mL,149.7 mmol) was added drop wise toa
solution of 3-(4-(7-chloroquinolin-4-yl) piperazin-1-yl) propionate
Percentage of inhibition hematin formation using the concentration of 2.5 mM
for each compound. Values determined 24 h after incubation.

72
A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
3 (5 g,14.9 mmol) in ethanol (50 mL). The reaction mixture was kept
under vigorous stirring and refluxed for 12 h. The mixture was
cooled to room temperature, concentrated in vacuo, and the
resulting product was recrystallized in ethanol (yield: 4.1 g, 82%)
4.4.4. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
isopropylphenyl)methyleneamino]propanamide (F4)
Yellow solid (yield: 77%); mp: 149 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.4 (s, 1H), 8.94 (s, 1H), 8.03 (d, 1H, J ¼ 2.1 Hz), 7.95 (d,
yellow crystals. Mp: 155e156 ꢁC; 1H NMR (300 MHz, CDCl3):
d
¼ 8.9
1H, J ¼ 9 Hz), 7.64 (s, 1H), 7.58 (d, 2H, J ¼ 8.1 Hz), 7.42e7.39 (dd, 1H,
J ¼ 2.1 and 9 Hz), 7.28 (d, 2H, J ¼ 8.1 Hz), 6.81 (d, 1H, J ¼ 5.1 Hz), 3.25
(t, 4H, J ¼ 3.9 Hz), 3.08e2.84 (m, 9H), 1.26 (d, 6H, J ¼ 6.9 Hz); 13C
(s, 1H), 8.74 (d, 1H, J ¼ 4.8 Hz), 8.05 (d, 1H, J ¼ 2.1 Hz), 7.93 (d, 1H,
J ¼ 9 Hz), 7.45e7.41 (dd,1H, J ¼ 2.1 and 9.0 Hz), 6.85 (d,1H, J ¼ 4.8 Hz),
3.9 (bs, 2H), 3.28 (t, 4H, J ¼ 4.8 Hz), 2.79e2.75 (m, 6H), 2.50 (t, 2H,
J ¼ 6 Hz); IR (KBr) nmax ¼ 3320, 3249, 3021, 2902, 2811, 1669, 1626,
1545 cmꢂ1; FAB-MS (m/z): 333 (Mþ, 100%); Anal. calcd for
NMR (75 MHz, CDCl3):
d
¼ 174.7, 168.4, 157.0, 151.8, 150.1, 144.0,
134.9, 131.4, 128.7, 127.2, 126.9, 126.1, 125.2, 121.9, 108.9, 53.6, 52.8,
52.1, 34.1, 30.5, 23.8; IR (KBr) nmax ¼ 3465, 3053, 2958, 2816, 1677,
1604, 1564, 1378, 1359, 1197 cmꢂ1; FAB-MS (m/z): 464.21 [M þ 1]þ,
100%; 449.21 [M ꢂ CH3]þ, 21%; Anal. calcd for C26H30N5OCl: C 67.30,
H 6.52, N 15.09%, found: C 67.42, H 6.67, N 15.21%.
C
16H20N5OCl: C 57.57, H 6.04, N 20.98 found: C 57.58, H 6.29, N
20.81%.
4.4. General procedure for the preparation of compounds (F1eF33)
4.4.5. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-N,N-
dimethylaminophenyl)methyleneamino]propanamide (F5)
Yellow solid (yield: 69%); mp: 176 ꢁC; 1H NMR (300 MHz,
A mixture of 3-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)pro-
panehydrazide 4 (1 mmol) and the respective aldehyde (1 mmol) in
ethanol was stirred at room temperature for 12 h. The resulting
precipitate, filtered and recrystallized in ethanol yielded 60e86% of
N-acylhydrazones.
CDCl3):
d
¼ 11.2 (s, 1H), 8.7 (d, 1H, J ¼ 4.8 Hz), 8.66 (s, 1H), 8.02 (d,
1H, J ¼ 1.8 Hz), 7.95 (d, 1H, J ¼ 9 Hz), 7.53 (d, 2H, J ¼ 7.5 Hz), 7.42e
7.39 (dd, 1H, J ¼ 2.1 and 9 Hz), 6.81 (d, 1H, J ¼ 4.8 Hz), 6.7 (d, 2H,
J ¼ 8 Hz), 3.25 (t, 4H, J ¼ 4.8 Hz), 3.02 (s, 6H), 2.98e2.84 (m, 8H); 13
C
NMR (75 MHz, CDCl3):
d
¼ 174.0, 168.0, 157.0, 151.9, 150.0, 144.4,
134.8, 129.2, 128.4, 126.0, 125.2, 121.9, 121.3, 111.8, 108.9, 53.6, 52.8,
52.1, 40.1, 30.5; IR (KBr) nmax ¼ 2231, 3068, 2892, 2824, 1651, 1596,
1574,1545,1364,1130 cmꢂ1; FAB-MS (m/z): 464.2 (Mþ,100%); 465.1
[M þ 1]þ, 77%; Anal. calcd for C25H29N6OCl: C 64.57, H 6.29, N 18.07,
found: C 64.52, H 6.37, N 18.19%.
4.4.1. N-[(E)-Benzylideneamino]-3-[4-(7-chloro-4-quinolyl)
piperazin-1-yl]propanamide (F1)
Colorless solid (yield: 78%); mp: 110 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.4 (s, 1H), 8.79 (s, 1H), 8.6 (d, 1H, J ¼ 4.8 Hz), 8.02 (d,
1H, J ¼ 1.8 Hz), 7.95 (d, 1H, J ¼ 9 Hz), 7.75 (m, 2H), 7.65 (d, 1H,
J ¼ 3.6 Hz), 7.47e7.40 (m, 3H), 6.81 (d, 1H, J ¼ 4.8 Hz), 3.25 (t, 4H,
4.4.6. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(2-
nitrophenyl)methyleneamino]propanamide (F6)
J ¼ 4.8 Hz), 3.05e2.84 (m, 8H); 13C NMR (75 MHz, CDCl3):
¼ 182.4,
d
174.3, 156.8, 151.7, 147.8, 143.6,134.7, 133.5, 130.1, 128.6, 126.9, 125.9,
125.8, 121.7, 108.8, 53.3, 52.7, 51.9, 30.3; IR (KBr) nmax ¼ 3363, 3067,
2921, 2852, 1655, 1604,yield 1572, 1545, 1336 cmꢂ1; FAB-MS (m/z):
422.14 [M þ 1]þ, 100%; Anal. calcd for C23H24N5OCl: C 65.47, H, 5.73,
N 16.60%; found: C 65.51, H 5.79, N 16.41%.
Colorless solid (yield: 58%); mp: 217e219 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 12.2 (s,1H), 9.4 (s,1H), 8.77 (d,1H, J ¼ 4.8 Hz), 8.3 (s,1H),
8.28 (d, 1H, J ¼ 7.8 Hz), 8.08e8.01 (m, 2H), 7.95 (d, 1H, J ¼ 8.7 Hz),
7.43 (t, 2H, J ¼ 9.6 Hz), 6.94 (d,1H, J ¼ 5.1 Hz), 3.26 (t, 4H, J ¼ 4.8 Hz),
3.04e2.88 (m, 8H); 13C NMR (75 MHz, CDCl3):
d
¼ 174.3, 164.8,
155.2,151.7,145.7,143.9,137.8,132.6,127.3,127.0,126.4,125.0,122.9,
120.7, 108.0, 56.2, 51.8, 50.9, 17.5; IR (KBr) nmax ¼ 3376, 3071, 2926,
2810, 1682, 1574, 1514, 1350, 1426, 1350, 1132 cmꢂ1; FAB-MS (m/z):
467.05 [M þ 1]þ,100%; Anal. calcd for C23H23N6O3Cl: C 59.16, H 4.97,
N 18.00, found: C 59.14, H 4.76, N 18.27%.
4.4.2. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(p-tolyl)
methyleneamino]propanamide (F2)
Colorless solid (yield: 69%); mp: 153e154 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
¼ 11.4 (s, 1H), 8.79 (s, 1H), 8.03 (d, 1H, J ¼ 2.1 Hz), 7.94 (d,
1H, J ¼ 9 Hz), 7.7 (s, 1H), 7.55 (d, 2H, J ¼ 8.1 Hz), 7.42e7.39 (dd, 1H,
J ¼ 1.8 and 8.8 Hz), 7.22 (d, 2H, J ¼ 8.1 Hz), 6.81 (d, 1H, J ¼ 5.1 Hz),
3.24 (t, 4H, J ¼ 6.6 Hz), 2.98e2.84 (m, 8H), 2.17 (s, 3H); 13C NMR
4.4.7. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(3-
nitrophenyl)methyleneamino]propanamide (F7)
Yellow solid (yield: 76%); mp: 232e234 ꢁC; 1H NMR (300 MHz,
(75 MHz, CDCl3):
d
¼ 183.4, 175.3, 155.8, 157.7, 148.8, 147.6, 136.7,
CDCl3):
d
¼ 11.03 (s, 1H), 8.71 (s, 1H), 8.51 (d, 1H, J ¼ 4.8 Hz), 8.18 (s,
131.5, 130.1, 127.6, 126.9, 125.9, 125.8, 121.7, 108.8, 54.9, 53.3, 52.7,
31.3; IR (KBr) nmax ¼ 3435, 3152, 2980, 2881, 1665, 1604, 1566, 1332,
1134 cmꢂ1; FAB-MS (m/z): 436.08 [M þ 1]þ, 82%; Anal. calcd for
1H), 8.02e7.94 (m, 4H), 7.61e7.56 (m, 1H), 7.44e7.42 (m, 1H), 6.86
(d, 1H, J ¼ 5.1 Hz), 3.26 (m, 4H), 3.02e2.95 (m, 8H); 13C NMR
(75 MHz, CDC):
d
¼ 174.3, 164.8, 155.2, 151.7, 145.7, 143.9, 137.8,
C
24H26N5OCl: C 66.12, H 6.01, N 16.06%; found: C 66.23, H 6.07, N
132.6, 127.3, 127.0, 126.4, 125.0, 122.9, 120.7, 108.0, 56.2, 51.8, 50.9;
IR (KBr) nmax ¼ 3254, 3063, 2951, 2824, 1667, 1606, 1568, 1527, 1376,
1140 cmꢂ1; FAB-MS (m/z): 466.15 [M þ 1]þ 100%; Anal. calcd for
16.31%.
C23H23N6O3Cl: C 59.16, H 4.97, N 18.00, found: C 59.21, H 4.79, N
4.4.3. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
ethylphenyl)methyleneamino]propanamide (F3)
18.18%.
Colorless solid (yield: 74%); mp: 115e117 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.4 (s, 1H), 8.79 (s, 1H), 8.6 (d, 1H, J ¼ 4.8 Hz), 8.02 (d,
4.4.8. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
nitrophenyl)methyleneamino]propanamide (F8)
1H, J ¼ 1.8 Hz), 7.95 (d, 1H, J ¼ 9 Hz), 7.75 (m, 2H), 7.65 (d, 1H,
J ¼ 3.6 Hz), 7.47e7.40 (m, 2H), 6.81 (d, 1H, J ¼ 4.8 Hz), 3.25 (t, 4H,
J ¼ 4.8 Hz), 3.05e2.84 (m, 8H), 1.34 (m, 5H); 13C NMR (75 MHz,
Yellow solid (yield: 55%); mp: 210e211 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.4 (s, 1H), 8.79 (s, 1H), 8.6 (d, 1H, J ¼ 4.8 Hz), 8.02 (d,
CDCl3):
d
¼ 174.6, 168.4, 157.0, 151.9, 150.1, 146.9, 143.9, 134.8, 131.2,
1H, J ¼ 1.8 Hz), 7.95 (d, 1H, J ¼ 9 Hz), 7.75 (m, 2H), 7.65 (d, 1H,
128.7, 128.3, 127.1, 126.1, 125.2, 121.9, 108.9, 53.5, 52.9, 52.1, 30.5,
28.8, 15.3; IR (KBr) nmax ¼ 3247, 3056, 2971, 2823, 1655, 1606, 1571,
1543, 1374, 1133 cmꢂ1; FAB-MS (m/z): 450.11 [M þ 1]þ, 100%; Anal.
calcd for C25H28N5OCl: C 66.73, H 6.2, N 15.56, found: C 66.54, H
6.21, N 15.45%.
J ¼ 3.6 Hz), 7.47e7.40 (m, 3H), 6.81 (d, 1H, J ¼ 4.8 Hz), 3.25 (t, 4H,
J ¼ 4.8 Hz), 3.05e2.84 (m, 8H); 13C NMR (75 MHz, CDCl3):
¼ 173.3,
d
167.8, 155.9, 150.7, 148.7, 146.9, 139.8, 133.6, 127.3, 127.0, 126.4,
125.0, 122.9, 120.7, 108.0, 56.2, 51.8, 50.9, 17.5; IR (KBr) nmax ¼ 3173,
3075, 2922, 2853, 1667, 1579, 1518, 1495, 1337, 1141 cmꢂ1; FAB-MS

A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
73
(m/z): 466.15 [M þ 1]þ, 100%; Anal. calcd for C23H23N6O3Cl: C 59.16,
(75 MHz, CDCl3):
d
¼ 174.7, 168.4, 157.0, 151.8, 150.1, 144.0, 134.9,
H 4.97, N,18.00, found: C 59.21, H 4.82, N 18.07%.
131.4, 128.7, 127.2, 126.9, 126.1, 125.2, 121.9, 108.9, 53.6, 52.8, 52.1,
34.1, 30.5, 23.8; IR (KBr) nmax ¼ 3358, 3227, 3067, 2886, 2822, 1649,
1604, 1574, 1512, 1368, 1133 cmꢂ1; FAB-MS (m/z): 438.26 [M þ 1]þ,
100%; Anal. calcd For C23H24N5O2Cl: C 63.08, H 5.52, N 15.99, found:
C 63.15, H 5.34, N 15.82%.
4.4.9. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(2-
chlorophenyl)methyleneamino]propanamide (F9)
Colorless solid, (yield: 59%); mp: 178e179 ꢁC; 1H NMR
(300 MHz, CDCl3):
d
¼ 11.57 (s, 1H), 9.23 (s, 1H), 8.7 (d, 1H,
J ¼ 5.1 Hz), 8.13e8.03 (m, 2H), 7.94 (d, 1H, J ¼ 9 Hz), 7.76e7.7 (m,
4.4.14. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(2-
ethoxyphenyl)methyleneamino]propanamide (F14)
2H), 7.51e7.31 (m, 3H), 6.81 (d, 1H, J ¼ 4.8 Hz), 3.26 (t, 4H,
J ¼ 6.9 Hz), 3.05e2.86 (m, 8H); 13C NMR (75 MHz, CDCl3):
¼ 179.9,
d
Colorless solid (yield: 62%); mp: 183e185 ꢁC; 1H NMR (300 MHz,
167.7, 156.8, 154.4, 150.4, 145.4, 143.2, 136.3, 135.9, 132.1, 128.8,
126.5, 126.1, 125.5, 125.2, 121.7, 111.9, 54.5, 53.9, 51.1, 30.5; IR (KBr)
nmax ¼ 3363, 3067, 2921, 2852, 1655, 1604, 1572, 1545, 1336,
1133 cmꢂ1; FAB-MS (m/z): 455.13 (Mþ, 100%); Anal. calcd for
CDCl3):
d
¼ 11.4 (s, 1H), 8.94 (s, 1H), 8.03 (d, 1H, J ¼ 2.1 Hz), 7.95 (d,
1H, J ¼ 9 Hz), 7.64 (s, 1H), 7.58 (d, 2H, J ¼ 8.1 Hz), 7.42e7.39 (dd, 1H,
J ¼ 2.1 and 9 Hz), 7.28 (d, 2H, J ¼ 8.1 Hz), 6.81 (d, 1H, J ¼ 5.1 Hz), 3.25
(t, 4H, J ¼ 3.9 Hz), 3.08e2.84 (m, 8H), 1.46 (t, 3H, J ¼ 6.9 Hz), 1.32 (m,
C
23H23N5OCl2: C 60.53, H 5.08, N 15.35, found: C 60.28, H 5.17, N
2H). 13C NMR (75 MHz, CDCl3):
d
¼ 174.7, 168.4, 157.0, 151.8, 150.1,
15.29%.
144.0, 134.9, 131.4, 128.7, 127.2, 126.9, 126.1, 125.2, 121.9, 108.9, 53.6,
52.8, 52.1, 34.1, 30.5, 23.8; IR (KBr) nmax ¼ 3203, 3059, 2977, 2879,
1649,1602, 1574, 1551, 1359, 1250,1127 cmꢂ1; FAB-MS (m/z): 465.19
[M þ 1]þ, 100%; Anal. calcd for C25H28N5O2Cl: C 64.44, H 6.06, N
15.03, found: C 64.38, H 6.19, N 15.33%.
4.4.10. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(3-
chlorophenyl)methyleneamino]propanamide (F10)
Colorless solid, (yield: 63%); mp: 197e198 ꢁC; 1H NMR
(300 MHz, CDCl3):
d
¼ 11.57 (s, 1H), 9.23 (s, 1H), 8.7 (d, 1H,
J ¼ 5.1 Hz), 8.13e8.03 (m, 2H), 7.94 (d, 1H, J ¼ 9 Hz), 7.76e7.7 (m,
2H), 7.51e7.31 (m, 3H), 6.81 (d, 1H, J ¼ 4.8 Hz), 3.26 (t, 4H,
4.4.15. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-anisyl)
methyleneamino]propanamide (F15)
J ¼ 6.9 Hz), 3.05e2.86 (m, 8H); 13C NMR (75 MHz, CDCl3):
¼ 174.9,
d
168.7, 156.9, 151.9, 150.1, 146.3, 142.2, 135.5, 134.9, 130.1, 128.8,
126.5, 126.1, 125.5, 125.2, 121.7, 108.9, 53.5, 52.9, 52.1, 30.5; IR (KBr)
nmax ¼ 3181, 3075, 2959, 2826, 1665, 1601, 1575, 1495, 1382,
1140 cmꢂ1; FAB-MS (m/z): 455.13 (Mþ, 100%), 456.33 [M þ 1]þ, 72%;
Anal. calcd for C23H23N5OCl2: C 60.53, H 5.08, N 15.35, found: C
60.74, H 5.29, N 15.52%.
Colorless solid (yield: 81%); mp: 126e128 ꢁC; 1H NMR
(300 MHz, CDCl3):
d
¼ 11.34 (s,1H), 8.87 (s,1H), 8.72 (m,1H), 8.02 (s,
1H), 7.94 (d, 1H, J ¼ 9 Hz), 7.6 (d, 2H, J ¼ 8.7 Hz), 7.4 (dd, 1H, J ¼ 1.8
and 9 Hz), 6.93 (d, 2H, J ¼ 8.4 Hz), 6.89 (d, 1H, J ¼ 4.5 Hz), 3.84 (s,
3H), 3.24 (t, 4H, J ¼ 4.8 Hz), 3.04e2.89 (m, 8H); 13C NMR (75 MHz,
CDCl3):
d
¼ 174.6, 168.4, 161.2, 157.0, 151.8, 150.0, 143.7, 134.8, 129.2,
128.6, 126.1, 125.2, 121.9, 111.2, 108.9, 55.3, 53.6, 52.9, 52.0, 30.4; IR
(KBr) nmax ¼ 3137, 3001, 2886, 2833, 1665, 1602, 1566, 1509, 1303,
1234, 1135 cmꢂ1; FAB-MS (m/z): 452.09 [M þ 1]þ, 100%; Anal. calcd
for C24H26N5O2Cl: C 63.78, H 5.80, N 15.50, found: C 63.73, H 5.61, N
15.45%.
4.4.11. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
chlorophenyl)methyleneamino]propanamide (F11)
Colorless solid (yield: 87%); mp: 155e157 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.46 (s, 1H). 8.97 (s, 1H), 8.7 (d, 1H, J ¼ 5.1 Hz), 8.13 (s,
1H), 7.93 (d, 1H, J ¼ 9 Hz), 7.6 (d, 2H, J ¼ 8.4 Hz), 7.47e7.43 (dd, 1H,
J ¼ 2.1 Hz and 9 Hz), 7.38 (d, 2H, J ¼ 8.4 Hz), 6.81 (d, 1H, J ¼ 5.1 Hz),
3.24 (t, 4H, J ¼ 4.2 Hz), 3.06e2.85 (m, 8H); 13C NMR (75 MHz,
4.4.16. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
ethoxyphenyl)methyleneamino]propanamide (F16)
CDCl3):
d
¼ 174.1, 167.7, 156.8, 151.8, 149.8, 142.0, 135.2, 134.5, 132.8,
Colorless solid (yield: 67%); mp: 186e187 ꢁC; 1H NMR (300 MHz,
128.7, 127.9, 125.8, 125.3, 121.7, 108.9, 53.1, 52.7, 51.9, 30.2; IR (KBr)
nmax ¼ 3419, 3038, 2955, 2836, 1670, 1595, 1565, 1422, 1136 cmꢂ1
;
CDCl3):
d
¼ 11.35 (s, 1H), 8.79 (s, 1H), 8.7 (d, 1H, J ¼ 5.1 Hz), 8.03 (s,
FAB-MS (m/z): 455.03 (Mþ, 100%), 456.53 [M þ 1]þ, 85%; Anal. calcd
for C23H23N5OCl2: C 60.53, H 5.08, N 15.35, found: C 60.88, H 5.12, N
15.29%.
1H), 7.94 (d, 1H, J ¼ 9 Hz), 7.58 (d, 2H, J ¼ 8.7 Hz), 7.43e7.39 (dd, 1H,
J ¼ 2.1 and 9 Hz), 6.92 (d, 2H, J ¼ 8.4 Hz), 6.81 (d,1H, J ¼ 4.8 Hz), 4.05
(q, 2H, J ¼ 6.9 Hz), 3.33 (t, 4H, J ¼ 5.1 Hz), 3.06e2.89 (m, 8H), 1.43 (t,
3H, J ¼ 6.9 Hz); 13C NMR (75 MHz, CDCl3):
d
¼ 174.5, 168.3, 160.7,
4.4.12. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-2-
(hydroxyphenyl)methyleneamino]propanamide (F12)
157.0, 151.8, 150.1, 143.8, 134.8, 129.7, 128.6, 126.1, 125.2, 121.9, 114.7,
108.9, 63.6, 53.6, 52.9, 52.1, 30.4, 14.7; IR (KBr) nmax ¼ 3201, 3060,
2944, 2887, 1653, 1605, 1568, 1509, 1369, 1252, 1131 cmꢂ1; FAB-MS
(m/z): 466.08 [M þ 1]þ,100%; Anal. calcd for C25H28N5O2Cl: C 64.44,
H 6.06, N 15.03, found: C 64.48, H 6.16, N 15.07%.
Colorless solid (yield: 66%); mp: 203e205 ꢁC; 1H NMR
(300 MHz, CDCl3):
d
¼ 11.56 (s, 1H), 11.07 (s, 1H), 8.77 (d, 1H,
J ¼ 4.8 Hz), 8.34 (s, 1H), 8.08e8.01 (m, 2H), 7.95 (d, 1H, J ¼ 8.7 Hz),
7.57e7.48 (m, 2H), 7.43 (m, 2H), 6.94 (d, 1H, J ¼ 5.1 Hz), 3.26 (t, 4H,
J ¼ 4.8 Hz), 3.04e2.88 (m, 8H); 13C NMR (75 MHz, CDCl3):
¼ 174.3,
d
164.8,155.2,151.7,145.7,143.9,137.8,132.6,127.3,127.0,126.4,125.0,
122.9, 120.7, 108.0, 56.2, 51.8, 50.9, 17.5; IR (KBr) nmax ¼ 3363, 3067,
2921, 2852, 1655, 1604, 1572, 1545, 1336, 1133 cmꢂ1; FAB-MS (m/z):
438.1 [M þ 1]þ, 100%; Anal. calcd for C23H24N5O2Cl: C 63.08, H 5.52,
N 15.99, found: C 63.24, H 5.66, N 15.71%.
4.4.17. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
isopropoxyphenyl)methyleneamino]propanamide (F17)
Colorless solid (yield: 73%); mp: 167e168 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.32 (s, 1H), 8.7 (d, 1H, J ¼ 4.8 Hz), 8.68 (s, 1H), 8.02 (d,
1H, J ¼ 1.8 Hz), 7.94 (d,1H, J ¼ 9 Hz), 7.67 (d, 2H, J ¼ 8.4 Hz), 7.41 (dd,
1H, J ¼ 2.1 and 9 Hz), 6.90 (d, 2H, J ¼ 8.4 Hz), 6.81 (d, 1H, J ¼ 5.1 Hz),
4.63e4.55 (m, 1H), 3.33 (t, 4H, J ¼ 4.8 Hz), 3.03e2.85 (m, 8H), 1.35
4.4.13. 3-[4-(7-Chloro-4-quinolyl) piperazin-1-yl]-N-[(E)-(4-
hydroxyphenyl)methyleneamino]propanamide (F13)
(d, 6H, J ¼ 6 Hz). 13C NMR (75 MHz, CDCl3):
d
¼ 174.3, 168.2, 159.7,
Colorless solid (yield: 84%); mp: 162e164 ꢁC; 1H NMR
157.0, 151.9, 150.1, 143.5, 134.8, 129.3, 128.6, 126.1, 125.2, 121.9, 115.8,
108.9, 70.0, 53.5, 52.9, 52.1, 30.5, 21.9; IR (KBr) nmax ¼ 3244, 3081,
2976, 2887, 1655, 1605, 1563, 1507, 1373, 1250, 1127 cmꢂ1; FAB-MS
(m/z): 480.1 [M þ 1]þ, 100%; Anal. calcd for C26H30N5O2Cl: C 65.06,
H 6.30, N 14.59, found: C 65.18, H 6.26, N 14.57%.
(300 MHz, CDCl3):
d
¼ 11.67 (s, 1H), 11.34 (s, 1H), 8.75 (d, 1H,
J ¼ 4.8 Hz), 8.69 (d, 1H, J ¼ 4.5 Hz), 8.58 (s, 1H), 7.96 (m, 1H), 7.64 (s,
1H), 7.53 (d, 2H, J ¼ 8.1 Hz), 7.28 (d, 2H, J ¼ 8.1 Hz), 6.84 (d, 1H,
J ¼ 5.1 Hz), 3.33e3.26 (m, 4H), 3.03e2.86 (m, 8H), 13C NMR

74
A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
4.4.18. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-2-
pyridylmethyleneamino]propanamide (F18)
110.0, 107.9, 107.2, 54.7, 52.5, 51.6, 50.9, 29.2; IR (KBr) nmax ¼ 3228,
3062, 2958, 2936, 1655, 1599, 1575, 1551, 1333, 1270, 1238,
1133 cmꢂ1; FAB-MS (m/z): 482.21 [M þ 1]þ, 100%; Anal. calcd for
Colorless solid (yield: 76%); mp: 115e116 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.91 (s, 1H), 8.9 (s, 1H), 8.75 (d, 1H, J ¼ 4.8 Hz), 8.59 (d,
C25H28N5O3Cl: C 62.30, H 5.86, N 14.53, found: C 62.48, H 5.72, N
1H, J ¼ 4.8 Hz), 8.05 (d, 1H, J ¼ 10.8 Hz), 7.95 (d, 1H, J ¼ 8.7 Hz), 7.87
(s, 1H), 7.74 (t, 1H, J ¼ 7.8 Hz), 7.43e7.45 (m, 1H), 7.32e7.30 (m, 1H),
6.9 (d, 1H, J ¼ 5.1 Hz), 3.34 (t, 4H, J ¼ 5.1 Hz), 3.07 (t, 2H, J ¼ 6.6 Hz),
14.47%.
4.4.23. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
isopropoxy-3-methoxyphenyl)methyleneamino]propanamide (F23)
Colorless solid (yield: 78%); mp: 134e135 ꢁC; 1H NMR (300 MHz,
2.99e2.84 (m, 6H). 13C NMR (75 MHz, CDCl3):
d
¼ 174.5, 168.8,
156.9, 152.9, 151.9, 149.5, 147.6, 144.0, 136.5, 134.9, 128.7, 126.1,
125.2, 124.2, 121.2, 120.2, 108.9, 52.9, 52.5, 52.0, 30.4; IR (KBr)
CDCl3):
d
¼ 11.34 (s, 1H), 8.7 (d, 1H, J ¼ 4.8 Hz), 8.53 (s, 1H), 8.02 (d,
nmax ¼ 3467, 3040, 2970, 2829, 1687, 1604, 1579, 1378, 1215 cmꢂ1
;
1H, J ¼ 1.8 Hz), 7.93 (d, 1H, J ¼ 9 Hz), 7.65 (s, 1H), 7.41 (dd, 1H, J ¼ 1.8
and 9 Hz), 7.11 (d, 1H, J ¼ 8.1 Hz), 6.9 (d, 1H, J ¼ 8.3 Hz), 6.81 (d, 1H,
J ¼ 4.8 Hz), 4.62e4.57 (m, 1H), 3.91 (s, 3H), 3.24 (t, 4H, J ¼ 5.1 Hz),
3.04e2.85 (m, 8H), 1.4 (d, 6H, J ¼ 6 Hz). 13C NMR (75 MHz, CDCl3):
FAB-MS (m/z): 422.16 [M þ 1]þ, 100%; Anal. calcd for C22H23N6OCl:
C 62.48, H 5.48, N 19.87, found: C 62.58, H 5.46, N 19.77%.
4.4.19. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-[2-
(trifluoromethyl)phenyl]methyleneamino]propanamide (F19)
Yellow solid (yield: 85%); mp: 187e189 ꢁC; 1H NMR (300 MHz,
d
¼ 174.4, 168.4, 156.9, 151.8, 150.4, 150.0, 149.5, 148.1, 144.0, 134.9,
128.7, 126.1, 124.9, 122.5, 121.5, 114.5, 109.0, 71.3, 56.0, 53.5, 52.9,
52.1, 30.5, 21.9; IR (KBr) nmax ¼ 3199, 3060, 2994, 2822, 1651, 1600,
1571, 1549, 1331, 1271, 1237, 1132 cmꢂ1; FAB-MS (m/z): 510.12
[M þ 1]þ, 100%; Anal. calcd for C27H32N5O3Cl: C 63.58, H 6.32, N
13.73; found: C 63.43, H 6.36, N 13.72%.
CDCl3):
d
¼ 12.2 (s, 1H), 9.0 (s, 1H), 8.79 (d, 1H, J ¼ 5.1 Hz), 8.43 (s,
1H), 8.37 (d, 1H, J ¼ 7.8), 8.07 (d, 1H, J ¼ 1.8 Hz), 7.93 (d, 1H,
J ¼ 4.8 Hz), 7.68 (t, 2H, J ¼ 8.7 Hz), 7.48e7.40 (m, 2H), 3.25 (t, 4H,
J ¼ 4.8 Hz), 3.04 (t, 2H, J ¼ 6.9 Hz), 2.83 (t, 4H, J ¼ 4.8 Hz), 2.65 (t, 2H,
J ¼ 6 Hz); 13C NMR (75 MHz, CDCl3):
d
¼ 182.4, 174.3, 156.8, 151.7,
147.8, 143.6, 134.7, 133.5, 130.1, 128.6, 126.9, 125.9, 125.8, 121.7,
108.8, 53.3, 52.7, 51.9, 30.3; IR (KBr) nmax ¼ 3395, 3068, 2950, 2821,
1682, 1606, 1570, 1376, 1193, 1131 cmꢂ1; FAB-MS (m/z): 489.15
[M þ 1]þ, 100%; Anal. calcd for C24H23F3N5OCl: C 58.84, H 4.73, N
14.29, found: C 58.65, H 4.76, N 14.35%.
4.4.24. 4-[(E)-[3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]
propanoylhydrazono]methyl]-2-methoxyphenyl]acetate (F24)
Colorless solid (yield: 83%); mp: 184e185 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.47 (s, 1H), 8.87 (s, 1H), 8.70 (d, 1H, J ¼ 4.8 Hz), 8.09 (s,
1H), 7.93 (d, 1H, J ¼ 9 Hz), 7.71 (s, 1H), 7.43e7.39 (dd, 1H, J ¼ 2.1 and
6.6 Hz), 7.13e7.06 (m, 2H), 6.81 (d, 1H, J ¼ 5.1 Hz), 3.88 (s, 3H), 3.24
(t, 4H, J ¼ 4.8 Hz), 3.06e2.83 (m, 8H), 2.33 (s, 3H); 13C NMR
4.4.20. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-4-
(diethoxymethyl)phenyl]methyleneamino]propanamide (F20)
Colorless solid (yield: 65%); mp: 164e166 ꢁC; 1H NMR
(75 MHz, CDCl3):
d
¼ 174.1, 168.1, 157.4, 156.9, 156.4, 151.9, 150.1,
143.6, 139.7, 134.8, 131.4, 128.8, 126.0, 125.2, 122.2, 120.8, 112.1,
(300 MHz, CDCl3):
d
¼ 11.5 (s, 1H), 9.11 (s, 1H), 8.7 (d, 1H, J ¼ 1.8 Hz),
108.9, 63.9, 53.5, 52.9, 52.1, 30.5, 14.8; IR (KBr) nmax ¼ 3244, 3072,
8.13 (d, 1H, J ¼ 1.8 Hz); 7.94 (d, 1H, J ¼ 9 Hz), 7.7 (s, 1H), 7.66 (d, 2H,
J ¼ 8.1 Hz), 7.52 (d, 2H, J ¼ 8.1 Hz), 6.8 (d, 1H, J ¼ 4.8 Hz), 3.59 (q, 4H,
J ¼ 6.9 Hz), 3.24 (t, 4H, J ¼ 5.1 Hz), 3.05e2.84 (m, 8H), 1.24 (t, 6H,
2945, 2815, 1752, 1660, 1604, 1576, 1552, 1371, 1278, 1128 cmꢂ1
;
FAB-MS (m/z): 525.11 [M þ 1]þ, 100%; Anal. calcd for C27H29N4O5Cl:
C 61.77, H 5.57, N 10.67%; found: C 61.78, H 5.76, N 10.72%
J ¼ 6.9 Hz). 13C NMR (75 MHz, CDCl3):
d
¼ 174.1, 168.1, 157.4, 156.9,
156.4, 151.9, 150.1, 143.6, 139.7, 134.8, 131.4, 128.8, 126.0, 125.2,
122.2, 120.8, 112.1, 108.9, 63.9, 53.5, 52.9, 52.1, 30.5, 14.8; IR (KBr)
4.4.25. N-[(E)-1,3-Benzodioxol-5-ylmethyleneamino]-3-[4-(7-
chloro-4-quinolyl) piperazin-1-yl]propanamide (F25)
nmax ¼ 3360, 3055, 2970, 2879, 1664, 1606, 1576, 1336, 1135 cmꢂ1
;
Colorless solid (yield: 86%); mp: 210e211 ꢁC; 1H NMR (300 MHz,
FAB-MS (m/z): 524.4 [M þ 1]þ, 100%; Anal. calcd for C28H34N5O3Cl:
CDCl3):
d
¼ 11.34 (s, 1H), 8.94 (s, 1H), 8.7 (d, 1H, J ¼ 4.8 Hz), 8.03 (s,
C 64.17, H 6.54, N 13.36, found: C 64.18, H 6.76, N 13.27%.
1H), 7.94 (d, 1H, J ¼ 9 Hz), 7.67 (s, 1H), 7.42e7.39 (dd, 1H, J ¼ 1.8 and
8.7 Hz), 7.01 (d, 1H, J ¼ 8.4 Hz), 6.89e6.78 (m, 2H), 6.02 (s, 2H), 3.25
(t, 4H, J ¼ 4.8 Hz), 3.02e2.84 (m, 8H); 13C NMR (75 MHz, CDCl3):
4.4.21. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(4-
hydroxy-3-methoxyphenyl)methyleneamino]propanamide (F21)
Colorless solid (yield: 61%); mp: 125e127 ꢁC; 1H NMR (300 MHz,
d
¼ 174.6, 168.8, 156.9, 151.9, 150.1, 143.0, 141.4, 134.8, 132.6, 128.8,
126.1, 125.2, 123.2, 121.9, 120.4, 109.9, 109.0, 55.9, 53.5, 52.9, 52.1,
30.6, 20.6; IR (KBr) nmax ¼ 3186, 3007, 2941, 2823, 1661, 1607, 1574,
1334, 1258, 1142 cmꢂ1; FAB-MS (m/z): 466.16 [M þ 1]þ, 100%; Anal.
calcd for C24H24N5O3Cl: C 61.87, H 5.19, N 15.03, found: C 61.81, H
5.26, N 15.17%.
CDCl3):
d
¼ 11.4 (s, 1H), 8.94 (s, 1H), 8.03 (d, 1H, J ¼ 2.1 Hz), 7.95 (d,
1H, J ¼ 9 Hz), 7.64 (s, 1H), 7.58 (d, 2H, J ¼ 8.1 Hz), 7.42e7.39 (dd, 1H,
J ¼ 2.1 and 9 Hz), 7.28 (d, 2H, J ¼ 8.1 Hz), 6.81 (d, 1H, J ¼ 5.1 Hz), 3.85
(s, 3H), 3.25 (t, 4H, J ¼ 3.9 Hz), 3.08e2.84 (m, 8H). 13C NMR (75 MHz,
CDCl3):
d
¼ 174.5, 168.8, 156.9, 152.9, 151.9, 149.5, 147.6, 144.0, 136.5,
134.9, 128.7, 126.1, 125.2, 124.2, 121.2, 120.2, 108.9, 52.9, 52.5, 52.0,
30.4; IR (KBr) nmax ¼ 3396, 3378, 3045, 2947, 2832, 1664, 1597, 1571,
1545, 1376, 1276, 1123 cmꢂ1; FAB-MS (m/z): 468.27 [M þ 1]þ, 100%;
Anal. calcd for C24H26N5O3Cl: C, 61.60; H, 5.60; N, 14.97%; found: C,
61.48; H, 5.76; N, 14.74%.
4.4.26. Ethyl-2-[4-[(E)-[3-[4-(7-chloro-4-quinolyl)piperazin-1-yl]
propanoylhydrazono]methyl]-2-methoxyphenoxy]acetate (F26)
Yellow solid (yield: 74%); mp: 137e138 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.41 (s, 1H), 8.81 (s, 1H), 8.70 (d, 1H, J ¼ 4.8 Hz), 8.04 (s,
1H), 7.96e7.92 (dd, 1H, J ¼ 1.5 and 9 Hz), 7.68 (s, 1H), 7.43e7.39 (dd,
1H, J ¼ 2.1 and 9 Hz), 7.13e7.09 (dd, 1H, J ¼ 1.8 and 8.1 Hz), 6.82e
6.75 (m, 2H), 4.78 (s, 2H), 4.25 (q, 2H, J ¼ 7.2 Hz), 3.94 (s, 3H), 3.24
(q, 4H, J ¼ 5.1 Hz), 3.04e2.87 (m, 8H), 1.28 (t, 3H, J ¼ 7.2 Hz); 13C
4.4.22. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(3,4-
dimethoxyphenyl)methyleneamino]propanamide (F22)
Colorless solid (yield: 65%); mp: 180e182 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.35 (s, 1H), 8.90 (s, 1H), 8.7 (d, 1H, J ¼ 5.1 Hz), 8.03 (s,
NMR (75 MHz, CDCl3):
d
¼ 174.5, 168.8, 156.9, 152.9, 151.9, 149.5,
1H), 7.93 (d, 1H, J ¼ 9 Hz), 7.7 (s, 1H), 7.42e7.39 (dd, 1H, J ¼ 1.8 Hz
and 8 Hz), 7.1 (d, 1H, J ¼ 8.4 Hz), 6.88 (d, 1H, J ¼ 8.4 Hz), 6.82 (d, 1H,
J ¼ 5.4 Hz), 3.94 (s, 3H), 3.92 (s, 3H), 3.34 (t, 4H, J ¼ 5.1 Hz), 3.08e
147.6, 144.0, 136.5, 134.9, 128.7, 126.1, 125.2, 124.2, 121.2, 120.2,
108.9, 52.9, 52.5, 52.0, 30.4; IR (KBr) nmax ¼ 3244, 3072, 2945, 2815,
1752, 1660, 1604, 1576, 1552, 1371, 1278, 1128 cmꢂ1; FAB-MS (m/z):
553.1 [M þ 1]þ, 100%; Anal. calcd for C28H32N5O5Cl: C 60.70, H 5.82,
N 12.64, found: C 60.58, H 5.76, N 12.77%.
2.95 (m, 8H); 13C NMR (75 MHz, CDCl3):
d
¼ 172.7,167.1,155.7,150.8,
149.4, 148.0, 142.2, 133.3, 127.3, 126.3, 124.7, 124.4, 120.7, 120.2,

A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
75
4.4.27. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-(2,5-
dimethoxyphenyl)methyleneamino]propanamide (F27)
51.9, 30.3; IR (KBr) nmax ¼ 3363, 3067, 2921, 2852, 1655, 1604, 1572,
1545, 1336, 1133 cmꢂ1; FAB-MS (m/z): 594.02 [M þ 1]þ, 100%; Anal.
calcd for C23H22Br2N5O2Cl: C 46.37, H 3.72, N 11.76, found: C 46.48,
H 3.76, N 11.67%.
Colorless solid (yield: 76%); mp: 158e160 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.54 (s, 1H), 8.93 (s, 1H), 8.7 (d, 1H, J ¼ 5.1 Hz), 8.04 (dd,
1H, J ¼ 1.8 and 12 Hz), 7.94 (d, 1H, J ¼ 9 Hz), 7.43e7.38 (m, 2H),
6.95e6.81 (m, 3H), 3.82 (s, 3H), 3.8 (s, 3H), 3.24 (t, 4H, J ¼ 5.1 Hz),
4.4.32. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-[(E)-1H-
indole-3-ylmethylene-amino]propanamide (F32)
3.06e2.83 (m, 8H); 13C NMR (75 MHz, CDCl3):
d
¼ 174.5, 168.8,
156.9, 152.9, 151.9, 149.5, 147.6, 144.0, 136.5, 134.9, 128.7, 126.1,
125.2, 124.2, 121.2, 120.2, 108.9, 52.9, 52.5, 52.0, 30.4; IR (KBr)
nmax ¼ 3126, 3049, 2943, 2899, 1677, 1600, 1572, 1336, 1263, 1244,
1137 cmꢂ1; FAB-MS (m/z): 482.1 [M þ 1]þ, 100%; Anal. calcd for
Yellow solid (yield: 78%); mp: 221e223 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.53 (s, 1H), 10.97 (s, 1H), 8.69 (s, 1H), 8.25e8.17 (m,
2H), 8.04e7.98 (m, 2H), 7.77 (s, 1H), 7.57e7.44 (m, 2H), 7.20e7.13
(m, 2H), 6.98 (s, 1H), 3.2 (s, 4H), 2.93e2.77 (m, 8H); 13C NMR
C
25H28N5O3Cl: C 62.30, H 5.86, N 14.53, found: C 62.48, H 5.71, N
(75 MHz, CDCl3):
d
¼ 184.4, 175.5, 157.8, 153.7, 148.8, 144.6, 138.7,
14.47%.
134.5, 132.1, 124.6, 123.9, 122.9, 122.8, 121.7, 108.8, 53.3, 52.7, 51.9,
31.1; IR (KBr) nmax ¼ 3482, 3027, 2934, 1640, 1608, 1572, 1376,
1133 cmꢂ1. FAB-MS (m/z): 461.28 [M þ 1]þ, 100%; Anal. calcd for
4.4.28. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-(E)-3,4,5-
(trimethoxyphenyl)methyleneamino]propanamide (F28)
Yellow solid (yield: 62%); mp: 86 ꢁC; 1H NMR (300 MHz, CDCl3)
C25H25N6OCl: C 65.14, H 5.47, N 18.23 found: C 65.18, H 5.47, N
18.27%.
d
¼ 11.33 (s, 1H), 9.35 (s, 1H), 8.68 (d, 1H, J ¼ 4.8 Hz), 8.06 (s, 1H),
7.94e7.9 (dd, 1H, J ¼ 2.4 and 8.7 Hz), 7.7 (s, 1H), 7.41e7.38 (dd, 1H,
J ¼ 1.8 and 9 Hz), 6.86 (s, 1H), 6.79 (d, 1H, J ¼ 4.8 Hz), 3.87 (s, 9H),
3.24 (m, 4H), 3.05e2.84 (m, 8H); 13C NMR (75 MHz, CDCl3):
4.4.33. (E)-N-(Ferrocenyl)-3-(4-(7-chloroquinolin-4-yl)piperazin-
1-yl)propanehydrazide (F33)
Red solid (yield: 71%); mp: 121e123 ꢁC; 1H NMR (300 MHz,
d
¼ 174.5, 168.6, 156.9, 153.5, 151.8, 150.0, 143.8, 140.2, 134.9, 128.7,
CDCl3):
d
¼ 11.004 (s, 1H), 9.03 (s, 1H), 8.7 (d, 1H, J ¼ 5.1 Hz), 8.06 (s,
126.1, 125.1, 121.8, 108.9, 104.3, 60.9, 56.2, 52.9, 52.0, 30.5. IR (KBr)
nmax ¼ 3393, 3054, 2934, 2832, 1648, 1608, 1572, 1358, 1230,
1120 cmꢂ1; FAB-MS (m/z): 512.2 [M þ 1]þ, 29%; Anal. calcd for
1H), 7.5 (d,1H, J ¼ 8.1 Hz), 7.64 (s, 1H), 7.43 (t,1H, J ¼ 9.9 Hz), 7.82 (d,
1H, J ¼ 5.1 Hz), 4.59 (s, 1H), 4.39 (s, 3H), 4.19 (s, 5H), 3.26 (s, 4H),
2.86e2.85 (m, 8H); 13C NMR (75 MHz, CDCl3):
d
¼ 174.1, 168.1, 157.4,
C
26H30N5O4Cl: C 60.99, H 5.91, N 13.68, found: C 60.86, H 5.76, N
156.9,156.4,151.9,150.1,143.6,139.7,134.8,131.4,128.8,126.0,125.2,
122.2, 120.8, 112.1, 108.9, 63.9, 53.5, 52.9, 52.1, 30.5, 14.8; IR (KBr)
13.72%.
nmax ¼ 3456, 3183, 2956, 2890, 1672, 1602, 1579, 1327, 1217 cmꢂ1
;
4.4.29. N-[(E)-(2-Bromo-3,4,5-trimethoxyphenyl)
methyleneamino]-3-[4-(7-chloro-4-quinolyl)piperazin-1-yl]
propanamide (F29)
FAB-MS (m/z): 515 (Mþ, 100%); Anal. calcd for C27H29N5O2ClFe: C
64.44, H 6.06, N 15.03, found: C 64.71, H 6.29, N 13.43%.
Yellow solid (yield: 65%); mp: 170 ꢁC; 1H NMR (300 MHz,
5. Pharmacological evaluation
CDCl3):
d
¼ 12.3 (s, 1H), 8.76 (d, 1H, J ¼ 5.1 Hz), 8.4 (s, 1H), 8.18 (s,
1H), 8.07e8.03 (dd, 1H, J ¼ 2.1 and 11 Hz), 7.93 (m 1H), 7.47e7.43
(dd, 1H, J ¼ 2.1 and 11 Hz), 6.87 (d, 1H, J ¼ 5.1 Hz), 3.87 (s, 3H), 3.91
(s, 6H), 3.34 (m, 4H), 3.04e2.84 (m, 8H); 13C NMR (75 MHz, CDCl3):
5.1. Toxicity to mouse splenocytes
BALB/c mouse splenocytes were placed into a 96-well plate at a
d
¼ 174.5, 168.8, 156.9, 152.9, 151.9, 149.5, 147.6, 144.0, 136.5, 134.9,
cell density of 5 ꢃ 106 cells/well in RPMI-1640 medium supple-
128.7, 126.1, 125.2, 124.2, 121.2, 120.2, 108.9, 52.9, 52.5, 52.0, 30.4; IR
(KBr) nmax ¼ 3363, 3067, 2921, 2852, 1655, 1604, 1572, 1545, 1336,
1133 cmꢂ1; FAB-MS (m/z): 590.12 [M þ 1]þ, 100%; Anal. calcd for
mented with 10% of FCS and 50 m
g mLꢂ1 of gentamycin. Each
compound was tested in five concentrations in triplicate. To each
well, an aliquot of test inhibitor suspended in DMSO was added, in
addition to wells only containing solvent (untreated cells). Plate
was incubated for 24 h at 37 ꢁC and 5% CO2. After incubation, [3H]-
thymidine was added to each well, and the plate was returned to
the incubator. The plate was then transferred to a beta-radiation
counter, and the percent of [3H]-thymidine was determined. Cell
viability was measured as the percent of [3H]-thymidine incorpo-
ration for treated cells in comparison to untreated cells [25].
C
26H29BrN5O4Cl: C 52.85, H 4.95, N 11.85, found: C 52.68, H 4.86, N
11.79%.
4.4.30. N-[(E)-(3-Bromo-4-hydroxy-5-methoxyphenyl)
methyleneamino]-3-[4-(7-chloro-4-quinolyl)piperazin-1-yl]
propanamide (F30)
Colorless solid (yield: 70%); mp: 184 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.4 (s, 1H), 11.2 (s, 1H), 8.7e8.68 (m, 1H), 8.04e7.88 (m,
3H), 7.57e7.51 (m, 1H), 7.4 (s, 1H), 7.27 (d, 1H, J ¼ 9 Hz), 6.99 (t, 1H,
5.2. Antiamoebic activity
J ¼ 6.6 Hz), 3.88 (s, 3H), 3.18 (s, 4H), 2.86e2.73 (m, 8H); 13C NMR
(75 MHz, CDCl3):
d
¼ 182.4, 174.3, 156.8, 151.7, 147.8, 143.6, 134.7,
The compounds were tested for antiamoebic activity against the
HM1:IMSS strain of E. histolytica by microdilution method [26].
Trophozoites were cultured in Diamond TYIS-33 growth medium
[27]. The test compounds (1 mg) were dissolved in DMSO (40 mL,
level at which no inhibition of amoeba occurs). The stock solutions
of the compounds were prepared freshly before use at a concen-
tration of 1 mg/mL. Two-fold serial dilutions were made in the
wells of 96-well microtiter plate (costar). Each plate included
metronidazole as standard amoebicidal drug, negative control
(culture medium plus amoeba) and a blank (culture medium only).
All the compounds were used in triplicate concentrations. Com-
pounds were added to the respective wells and the plate was
sealed, gassed for 10 min with nitrogen and then incubated at 37 ꢁC
for 72 h. After incubation, the amoeba growth was checked under
microscope. The culture medium was removed by inverting the
133.5, 130.1, 128.6, 126.9, 125.9, 125.8, 121.7, 108.8, 53.3, 52.7, 51.9,
30.3; IR (KBr) nmax ¼ 3363, 3067, 2921, 2852, 1655, 1604, 1572, 1545,
1336, 1133 cmꢂ1; FAB-MS (m/z): 545.3 (Mþ, 100%); Anal. calcd for
C
24H25BrN5O3Cl: C 52.71, H 4.61, N 12.81%, found: C 52.68, H 4.46, N
12.75%.
4.4.31. 3-[4-(7-Chloro-4-quinolyl)piperazin-1-yl]-N-(E)-(3,5-
dibromo-4-hydroxyphenyl)methyleneamino]propanamide (F31)
Yellow solid (yield: 81%); mp: 220 ꢁC; 1H NMR (300 MHz,
CDCl3):
d
¼ 11.5 (s, 1H), 11.33 (s, 1H), 8.69 (d, 1H, J ¼ 4.8 Hz), 8.04e
7.99 (m, 2H), 7.86 (s, 2H), 7.82 (s, 1H), 7.55 (d, 1H, J ¼ 8.7 Hz), 7.01 (d,
1H, J ¼ 4.5 Hz), 3.23 (s, 4H), 2.91 (s, 4H), 2.81 (s, 2H), 2.51 (s, 2H); 13
C
NMR (75 MHz, CDCl3):
d
¼ 182.4, 174.3, 156.8, 151.7, 147.8, 143.6,
134.7, 133.5, 130.1, 128.6, 126.9, 125.9, 125.8, 121.7, 108.8, 53.3, 52.7,

76
A. Inam et al. / European Journal of Medicinal Chemistry 75 (2014) 67e76
plate and shaking gently. Plate was then immediately washed with
NaCl (0.9%) at 37 ꢁC, dried at room temperature and the cells were
fixed with methanol and once dried, stained with aqueous eosin
(0.5%) for 15 min. The optical density of the resulting solution in
each well was determined at 490 nm in a microplate reader. The %
of inhibition of parasite growth was calculated from the optical
densities of the control and test compounds. A non-linear regres-
sion analysis was used to determine the best fitting line from which
the IC50 values were estimated. Each compound was tested at least
in two independent experiments.
positive control. The absorption was determined at 405 nm using a
spectrophotometer [30]. Results are expressed as percentage of
inhibition of hematin formation in comparison to negative control
(without compound). Experiments were carried out at least twice
using compound concentrations in triplicate.
Acknowledgments
This work was supported by Council of Scientific and Industrial
Research (grant # 01(2278)/08/EMR-II, New Delhi, India), CNPq and
FAPESB (Brazil). D.R.M.M. is recipient of a FAPESB post-doctoral
scholarship.
5.3. Antimalarial activity
It was performed using the [3H]-hypoxanthine incorporation
assay, as previously described [28]. W2 strain P. falciparum was
maintained in continuous culture of human erythrocytes (blood
group Oþ) using the RPMI-1640 medium supplemented with 10% of
human plasma. Parasites grown at 1e2% parasitemia and 2.5% he-
matocrit were distributed into 96-wells culture plate and incubated
with the compounds (previously diluted with 4% DMSO and culture
medium). After 24 h of incubation, [3H]-hypoxanthine was added,
the plate incubated again and parasites were harvested using a cell
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50
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m