Quinoline carboxamide core moiety-based compounds inhibit P. falciparum falcipain-2: Design, synthesis and antimalarial efficacy studies

Article abstract of DOI:10.1016/j.bioorg.2020.104514

Targeting Falcipain-2 (FP2) for the development of antimalarials is a promising and established concept in antimalarial drug discovery and development. FP2, a member of papain-family cysteine protease of the malaria parasite Plasmodium falciparum holds an important role in hemoglobin degradation pathway. A new series of quinoline carboxamide-based compounds was designed, synthesized and evaluated for antimalarial activity. We integrated molecular hybridization strategy with in-silico drug design to develop FP2 inhibitors. In-vitro results of FP2 inhibition by Qs17, Qs18, Qs20 and Qs21 were found to be in low micromolar range with IC₅₀ 4.78, 7.37, 2.14 and 2.64 μM, respectively. Among the 25 synthesized compounds, four compounds showed significant antimalarial activities. These compounds also depicted morphological and food-vacuole abnormalities much better than that of E-64, an established FP2 inhibitor. Overall these aromatic substituted quinoline carboxamides can serve as promising leads for the development of novel antimalarial agents.

Full text of DOI:10.1016/j.bioorg.2020.104514

Journal Pre-proofs
Quinoline carboxamide core moiety-based compounds inhibit P. falcipa-
rumfalcipain-2: Design, synthesis and antimalarial efficacy studies
Anju Singh, Md Kalamuddin, Mudasir Maqbool, Asif Mohmmed, Pawan
Malhotra, Nasimul Hoda
PII:
S0045-2068(20)31812-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104514
YBIOO 104514
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Bioorganic Chemistry
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21 July 2020
7 September 2020
19 November 2020
Please cite this article as: A. Singh, M. Kalamuddin, M. Maqbool, A. Mohmmed, P. Malhotra, N. Hoda,
Quinoline carboxamide core moiety-based compounds inhibit P. falciparumfalcipain-2: Design, synthesis and
antimalarial efficacy studies, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104514
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Quinoline carboxamide core moiety-based compounds inhibit P. falciparum falcipain-2:
Design, synthesis and antimalarial efficacy studies
Anju Singh1, Md Kalamuddin2, Mudasir Maqbool1, Asif Mohmmed2, Pawan Malhotra2*,
Nasimul Hoda1*
1Drug Design and Synthesis Lab., Department of Chemistry, Jamia Millia Islamia, Jamia Nagar,
New Delhi-110025, India
2International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg,
New Delhi-110067, India
*Corresponding authors
Nasimul Hoda
Pawan Malhotra
Professor
Department of Chemistry,
Jamia Millia Islamia,
Jamia Nagar, New Delhi-110025, India
Tel.: +0091-9910200655;
fax: +0091-11-26985507;
e-mail: nhoda@jmi.ac.in
Group Leader, Malaria Biology
International Centre for Genetic
Engineering and Biotechnology
Aruna Asaf Ali Marg-110067
New Delhi, India
Tel: +91-11-267423
E-mail: pawanm@icgeb.res.in
1

Graphical abstract
Abstract
Targeting Falcipain-2 (FP2) for the development of antimalarials is a promising and
established concept in antimalarial drug discovery and development. FP2, a member of papain-
family cysteine protease of the malaria parasite plasmodium falciparum holds an important role in
hemoglobin degradation pathway. A new series of quinoline carboxamide based compounds was
designed, synthesized and evaluated for antimalarial activity. We integrated molecular
hybridization strategy with in-silico drug design to develop FP2 inhibitors. In- vitro results of FP2
inhibition by Qs17, Qs18, Qs20 and Qs21 were found to be in low micromolar range with IC50
4.78, 7.37, 2.14 and 2.64 µM, respectively. Among the 25 synthesized compounds four compounds
showed significant antimalarial activities. These compounds also depicted morphological and
food-vacuole abnormalities much better than that of E-64 an established FP2 inhibitor. Overall
these aromatic substituted quinoline carboxamides can serve as promising leads for the
development of novel antimalarial agents.
Keywords: Quinoline carboxamide; Falcipain-2 (FP2); P. falciparum; Malaria; drug
development.
2

1.
Introduction
Malaria is one of the most life menacing neglected disease caused by protozoan parasites
of genus plasmodium. Approximately two hundred million infections and 438,000 deaths in a year,
predominantly children have been reported by WHO due to malaria [1]. Majority of the
antimalarial drugs reported till date target the asexual blood-stages of the parasite where it
replicates within human erythrocytes [2]. However, liver- and transmission-stage of the parasite
do not cause malaria symptoms. Due to the complex life cycle of the parasite and increasing
emergence of resistance against conventional antimalarials and artemisinin combination therapy
malaria remains a challenging issue for the humanity. The identification of new apt drug targets
and the development of effective anti-malarial molecules that target important parasite functions
with a potential to act against multi-drug-resistant strains of the parasite is an indispensable need
to prevent malaria pathogenesis and its transmission [3].
Proteases are among the attractive drug targets for the development of antimalarial therapy.
Falcipain-2 (FP2) and falcipain-3 (FP3) are papain-family cysteine proteases of erythrocytic stages
of P. falciparum that localize to the food vacuole and readily hydrolyze hemoglobin [4,5].
Disruption of the FP2 gene led to the accumulation of undegraded hemoglobin in trophozoites,
confirming a critical role of this enzyme in hemoglobin hydrolysis [6]. Inhibition of FP2 and
related proteases led to the inhibition of parasite development [7,8] and cured mice with murine
malaria [9,10].
E64 (Figure 1), an irreversible inhibitor of cysteine proteases, displayed a significant
outcome on growth, adherence and viability of parasite trophozoites [11]. Chiyanzu et al. [12]
designed a new class of thiosemicarbazones with isatin scaffold as FP2 inhibitors. The most
promising FP2 inhibitor of this series (Figure 1a) showed an inhibition with IC50 4.4 µM. From
such results, chalcones proved to be the robust inhibitors of cysteine proteases. Firstly, Li and co-
workers in 1995 started working over the development of antimalarial chalcones as one of the
derivatives: 1-(2,5-dichlorophenyl)-3(4-quinolinyl)- 2-propen-1-one (Figure 1b) showed an IC50
value of 200 nM against both CQ-resistant strain (W2) and CQ-sensitive strain (D6) of P.
falciparum [13,14]. Several peptide-based antitypanosomal agents such as compound c (Figure
1c) have been found to inhibit FP2 of cysteine protease rhodesain and P. falciparum of brucei
rhodesiense.
3

S
NH2
O
HN
O
O
NH
N NH2
N
H
O2N
N
HO
N
H
H
O
O
N
H
a
E64
H
Ph
N
O
H
N
COMe
O
N
O
H
O
Cl
H
Cl
c
b
Figure 1. Structure of some known potential FP2 inhibitors.
1.1. Design strategy for the new cysteine proteases inhibitors of P. falciparum
Recently, (DDD107498), a quinoline-4-carboxamide has been reported with excellent
pharmacokinetic and antimalarial properties, including activity against multiple life-cycle stages
of the malaria parasite [15]. Compound SC81458 and the clinical development candidate,
SC83288 (Figure 2), both containing sulfonamide and piperazine groups, showed noteworthy
results to cure a P. falciparum infection with tolerable toxicity. Properties like quick parasite
killing, good safety margin, a potentially different mode of action and a distinct chemotype support
SC83288 for the clinical trials against malaria [16]. The quinoline-4-carboxlic acid and its analogs
show diverse range of therapeutic activity such as antimalarial, antifungal and anti-leishmanial
effects [17,18].
Dual inhibitors of FP2 and FP3 have been identified through virtual screening of 241000
compounds against homology models of FP2 and FP3 in three consecutive stages of docking [19].
Benzothiazole containing sulfonyl-2-nitrobenzene based compounds, for example compound 1
(Figure 2) have been found to inhibit FP2 with IC50 11.14 μM. These compounds have been
prophesied to bury into the S2 pockets of FP2 and FP3, thereby inhibit both the enzymes
significantly [20]. Same way, potential antimalarial agents containing 4-aminoquinoline with
natural product isatin scaffold as in compound 2 (Figure 2) have been discovered with IC50 1.3–
0.079 and 2.0–0.050 µM against a chloroquine-sensitive (D10) and two resistants (K1 and W2)
strains of P. falciparum. Two such isatin based compounds 3 and 4 (Figure 2) displayed in-vitro
4

activity against K1 and W2 strains with IC50 values of 51 and 54 nM, respectively. In addition,
these compounds were found to inhibit parasitic cysteine protease FP2 [21]. Keeping these things
into consideration, we attempted to incorporate different functional units of reported FP2 inhibitors
and different clinical candidate antimalarials in one molecule as represented in (Figure 2).
Integration of structure based drug design technique and molecular hybridization was used to
incorporate potential scaffolds in the designed molecules.
Figure 2. Rational for the design and synthesis of quinoline-carboxamide as FP2 inhibitors.
Here, we report the design, synthesis and biological evaluation of quinoline-4-carboxamide
based analogues as FP2 inhibitors. The core moiety quinoline was prepared from isatin and
acetophenone (Scheme 1) using the Pfitzinger reaction. We utilized diversity-oriented route to
design and synthesize the target molecules. The 25 synthesized molecules were identified as
potential inhibitors of FP2, among which the best compound QS20 displayed FP2 inhibition with
IC50 value = 2.14 μM. In addition, the anti-plasmodial activity of these molecules against P.
falciparum was tested in which QS20 exhibited better parasite inhibition with IC50 = 0.81 μM.
2. Results and discussion
2.1.
In silico design of potential FP2 inhibitors
In an effort to investigate the plausible modes of action of compounds to act as
antimalarials as well as to predict molecular orientation at the active site, the docking simulations
5

were performed via AutoDock Vina program [22]. The docking study was performed on reported
crystal structure of FP2 (PDB ID: 3BPF) which was retrieved from RCSB. The computational
binding free energy (∆G) of the designed compounds was calculated and is mentioned in Table 1.
Docking results suggested that compounds Qs17, Qs18, Qs20 and Qs21 could effectively bind to
the active site of FP2 (Figure 3) with binding energy of -10.9, -11.3, -11.9 and -10.9 kcal/mol,
respectively. E64 a well-known FP2 inhibitor was taken as the reference. The binding energy for
all these compounds was found to be optimistically favorable than E64 having free energy of
complexation -7.9 kcal/mol. As observed from the docking studies, compounds Qs17 and Qs18
were found to show π-π interaction with Trp206 and H-bond with Asp170 (Figure 3a,c). In
addition, Qs20 displayed π-π stacking interactions with Trp206, Trp210, Phe156 (Figure 3e). In
addition, a strong H-bond was observed between oxygen of p-methoxy benzene of Qs20 and H of
Gln171. Same way, Qs21 showed π-π stacking interactions with Trp206, Trp210, His174 (Figure
3g). Qs21 also participated strongly in H-bonding with Asp154 and Gln171. These cumulative
effects could be the favorable factor behind high potency of these molecules. Along with that, the
docking pose analysis suggested that quinoline-carboxamide scaffold wraps around the key
residues of the enzyme and provide perfect snug fit at the active site. In addition to that,
geometrical complementary is further strengthened by favorable residue interaction with the
molecule. However, the standard FP2 inhibitor, displayed only H-bonds with Trp206, Val152,
Gln209 and Asn16, Glu14 Lys37 as shown in (Figure 3i,j). Since E64 lacks an aromatic system
and is unable to take part in π-π interactions, may be the reason for its less binding affinity with
the enzyme.
6

7

Figure 3. (a) Binding interaction of compound Qs17 with active site residues of FP2. (b) One of
the docking poses of Qs17 with FP2. (c)Binding interaction of compound Qs18 with FP2 (d) one
the docking poses of Qs18 with FP2. (e)(f) Docking interaction of Qs20 with FP2 active site. (g)(h)
Interaction of Qs21 with the active site residues of FP2. (i)(j) Interaction of E64 with the active
site residues of FP2. Figures were generated with Pymol. H-bonds are indicated with yellow
dashed lines and π-π stacking are shown by red dashed lines.
Table 1: The calculated free energies of selected derivatives in molecular docking using ParDock
[23] and AutoDock Vina program. [22].
Compounds Complexation energy (kcal/mol) of
compounds with FP2 using
ParDOCK
Complexation energy (kcal/mol) of
compounds with FP2 using
AutoDock
-9.1
QS1
QS2
-8.81
-10.11
-9.42
-10.07
-10.11
-8.91
-9.01
-8.81
-9.51
-9.98
-8.20
-10.01
-10.89
-7.98
-8.41
-10.01
-9.98
-10.45
-9.34
-10.98
-10.45
-10.01
-8.01
-7.30
-8.01
-6.14
-10.4
-10.1
-10.1
-10.1
-9.8
-8.9
-9.9
-9.3
-10.1
-8.0
-9.0
-11.0
-8.2
-8.6
-9.0
-10.9
-11.3
-9.1
-11.9
-10.9
-9.2
QS3
QS4
QS5
QS6
QS7
QS8
QS9
QS10
QS11
QS12
QS13
QS14
QS15
QS16
QS17
QS18
QS19
QS20
QS21
QS22
QS23
QS24
QS25
E64
-8.1
-9.0
-8.1
-7.9
8

2.2. Chemistry
The target compounds were synthesized via different organic synthesis routes as depicted in the
following reaction schemes. In addition, the synthesis of some substituents is described in the
supporting information file.
Scheme 1
O
OH
O
O
O
(ii)
HON
(i)
O
(iii)
CH3
N
N
H
H
H
H2N
N
1
4
2
3
5
Reagents and conditions: (i) Cl3CC(OH)2 NH2OH, HCl, H2O, ∆ (ii) H2SO4, 60-80
̊
KOH, EtOH/ water, 125 ̊C.
Scheme 2
H
N
Cl
Cl
N
NH2
O
O
(ii)
(iii)
(i)
HO
OH
N
H
O
N
H
7
Cl
N
H
9
O
1
6
8
Reagents and conditions: (i) POCl3 (ii) HCl or 1:4 dioxane (iii) piperazine, K2CO3, ethylene
glycol
Scheme 3
H
N
CH3
H
N
O
CH3
O
H
S
N
CH3
NH2
(iii)
O
N
O
(i)
(ii)
O
O
S
HN
O
Cl
O
Cl
13
12
11
1
10
Reagents and conditions: (i) DCM,0
̊
̊C,
2 h.
9

O
R
N
5
Compounds name
Qs1
R
Reagents and conditions
CN
N
TEA, HBTU, DMSO, 12 h, rt [24]
N
NH
H
TEA, HBTU, DMSO, 10 h, rt [25]
N
N
Qs2
Qs3
Qs4
Cl
N
H
N
TEA, HBTU, DMSO, 18 h, rt.
TEA, HBTU, DMSO, 18 h, rt.
N
N
N
N
H3C
N
H
N
N
N
N
N
F3C
N
H
N
TEA, HBTU, DMSO, 12 h, rt.
N
Qs5
Qs6
N
H
O
H
TEA, HBTU, EDC, DMSO, 7 h, rt.
N
CH3
O
O
S
N
O
HN
10

H
N
TEA, HBTU, EDC, DMSO, 7 h, rt.
CH3
O
O
Qs7
S
F
N
O
HN
TEA, HBTU, EDC, DMSO, 6 h, rt.
TEA, HBTU, EDC, DMSO, 7 h, rt.
TEA, HBTU, EDC, DMSO, 7 h, rt.
NH
O
S
N
Qs8
Qs9
O
Cl
Cl
CH3
O
S
O
N
HN
Br
O
Qs10
S
N
O
HN
Scheme 4
H
N
N
N
HN
H2N
Cl
O
OH
N
O
O
N
N
N
N
(iii)
(i)
N
(ii)
N
14
N
N
N
15
N
R
N
R
17
R
N
18
16
Reagents and conditions: (i) acetic acid, 110
piperazine 110
̊
̊C (iii) 1,4-dioxane, K2CO3
̊C
11

Scheme 5
N
CH3
N
H
N
N
CF3
N
N
Cl
Cl
N
N
EtO2C
CH3
N
N
N
N
N
N
Cl
20
N
N
N
N
19
N
(ii)
(i)
R
N
EtO2C
CH3
18
R=CH3,CF3
Cl
N
Qs11
N
O
S Cl
O
Qs12
HN
O
(iii)
CH3
H
21
O
N
CH3
O
S
N
O
N
H3C
N
N
Qs13
N
N
Reagents and conditions: (i)1,4-dioxane, 100
(iii) 1,4-dioxane, 100 C, K2CO3 48 h.
̊
̊C, K2CO3 8h
̊
12

Scheme 6
H
N
CH3
O
O
S
N
O
O
OH
O
N
O
OH
O
(v)
(iii)
(i)
(vi)
O
N
H
N
Cl
N
Cl
N
OH
23
24
Qs18
22
N
H
N
(ii)
N
CH3
(iv)
HN
Cl
O
O
S
O
R3
N
O
N
O
N
N
O
N
N
OH
Cl
N
Qs19-Qs25
Qs14-Qs16
R4
N
Cl
Qs17
Qs19; R4=p-tolylboronic acid
Qs20; R4=(4-methoxyphenyl)boronic acid
Qs14; R3= 1-((2,4-dichlorophenyl)sulfonyl)piperazine
Qs15; R3= 1-tosylpiperazine
Qs21; R4=(4-(trifluoromethoxy)phenyl)boronic acid
Qs22; R4=(3,4-difluorophenyl)boronic acid
Qs23; R4=(4-fluorophenyl)boronic acid
Qs16; R3=7-chloro-4-(piperazin-1-yl)quinoline
Qs24; R4=(3,4-dichlorophenyl)boronic acid
Qs25; R4=(4-trifluromethylphenyl)boronic acid
Reagents and conditions: (i) malonic acid, acetic acid, 12 h reflux (ii) R3, TEA, HBTU, DMSO,
7 h at rt. (iii) POCl3 reflux 2 h (iv) TEA, HBTU, DMSO, 7 h at rt. (v) TEA, HBTU, DMSO, 7 h
at rt (vi) Pd (PPh3)4, K2CO3, Aryl boronic acid, Toluene, reflux, 8 h.
2.3. Anti-malarial screening of the synthesized compounds
2.3.1. Inhibition of FP2
The inhibitory activities of the synthesized compounds were analyzed by assessing their ability
to block the in vitro protease activity of recombinant FP2, as well as their antiplasmodial activities
(Table 2). A total of 25 compounds were evaluated in the protease inhibitory assay, and the most
potent inhibitors from this enzymatic assay were further evaluated in the parasite cultures as
antimalarial agents. In the assays targeting FP2 (Table 2), four compounds, Qs17, Qs18, Qs20
and Qs21 exhibited micromolar inhibition values ranging from 2.14 to 7.37 µM. Although, all the
13

compounds inhibited FP2 moderately however few compounds exhibit an average inhibition of
FP2.
2.3.2. Effect of Quinoline-4-carboxamide derivatives target compounds on FP2 activity and
parasite growth
The inhibition effect of quinoline carboxamide compounds were analyzed on the activity
of recombinant FP2 protein as well as on P. falciparum growth and development. Recombinant
FP2 was produced by following a protocol described by Shenai et al.[4] and Kumar et al [26]
shows the expression, purification and refolding of FP2 protein. The refolded protein was
catalytically active as it cleaved the enzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin
hydrochloride (ZFR-AMC), a substrate of FP2, in a dose dependent manner. Twenty-five
quinoline carboxamide derivatives were evaluated protease assay, as shown in (Table 2), all the
tested compounds showed the inhibitory activity in micromolar range towards the inhibition of
FP2. Compounds Qs8, Qs9, Qs10, Qs16, Qs17, Qs18, Qs20, Qs21 showed the best activity with
IC50 values less than (10 µM).
Furthermore, the inhibitors were then analyzed for their antimalarial activities in a P.
falciparum 3D7 culture. Ten compounds (Qs3,Qs4,Qs11,Qs12,Qs16,Qs17,Qs18,Qs20,Qs21,
Qs24) inhibited P. falciparum 3D7 cultures with IC50 ranging from 0.81 ± 0.31 µM to 5.47± 0.74
µM. Compound Qs20 was found to be the most potent parasite growth inhibitor, with an IC50 of
0.81 ± 0.31µM, which correlated well with the inhibition of FP2 (IC50= 2.14 ± 0.64 µM).
Interestingly the best results for FP2 inhibition was obtained for the two compounds Qs20 and
Qs21 obtained via modification of quinoline carboxamides core moiety containing quinolin-
2(1H)-one by the sulfonyl-phenyl-acetamide along with 4-methoxy benzene attached to the
quinolin-piperazin-methanone, which specifies the importance of aromatic substitution and
sulphonyl actamide incorporation for enhanced anti-malarial activity. The in-silico results
obtained were in accordance with the in vitro studies and indicates the potential of compounds
Qs20 and Qs21 as effective FP2 inhibitors.
14

Table 2: In vitro inhibitory effect of Quinoline-4-carboxamide compounds against recombinant
FP2 and P. falciparum 3D7 (IC50) growth in vitro.
Compounds Inhibition of FP2 IC50
(µM)
Std.
error
Inhibition of P. falciparum
Std.
error
IC50 (µM)
Qs1
Qs2
18.20
13.79
25.20
17.44
13.01
15.82
20.22
4.2
0.70
1.12
1.95
0.60
1.31
1.34
1.53
0.57
0.50
1.02
NA
19.70
NA
1.05
NA
Qs3
2.82
1.47
20.19
20.45
NA
0.52
0.41
1.07
0.09
NA
Qs4
Qs5
Qs6
Qs7
Qs8
19.99
NA
1.05
NA
Qs9
3.47
6.14
NA
Qs10
Qs11
Qs12
Qs13
Qs14
Qs15
Qs16
Qs17
Qs18
Qs19
Qs20
Qs21
Qs22
Qs23
Qs24
Qs25
NA = Not Applicable
23.5
1.87
1.07
NA
0.24
0.53
0.35
NA
16.55
NA
1.43
NA
NA
NA
65.53
NA
0.65
NA
NA
NA
3.19
4.78
7.37
NA
1.21
1.12
1.13
NA
5.47
1.05
1.95
20.32
0.81
1.43
25.31
NA
0.74
0.45
1.95
0.74
0.31
0.45
0.90
NA
2.14
2.64
32.72
35.3
29.72
13.28
0.64
0.28
4.3
1.76
2.4
3.82
NA
0.76
NA
2
15

2.3.3. Effect of Quinoline-4-carboxamide derivatives on parasite development
Cysteine protease inhibitors are well known to cause morphological and developmental
abnormalities, mainly in the food vacuole of the parasites [6]. Based on the overall potency against
FP2, P. falciparum growth inhibition and in silico activity of compounds Qs17, Qs18, Qs20 and
Qs21 were selected to study the effect over morphology and development of P. falciparum through
its asexual stage. Briefly, the ring stage (8-10 h) parasites were treated with these compounds or
solvent alone as a control. In control, parasites developed normally from ring to trophozoite stage,
and then turned to the schizont stage and merozoites. These merozoites reinvaded fresh RBC to
form rings. Cultures treated with a known cysteine protease inhibitor E64 (10 µM) showed severe
food vacuole abnormalities at trophozoite stage along with clumps of malarial pigment, indicating
abrogation of hemozoin production in these parasites. P. falciparum cultures when treated with
Qs17, Qs18, Qs20 and Qs21 morphological and food-vacuole abnormalities were seen followed
by developmental arrest at the trophozoite stage. These phenotypes observed were like those
depicted in parasites treated with E64. However, the effect for all four compounds was better than
the E64 (Figure 4).
16

Figure 4. Effect of E-64, compounds Qs17, Qs18, Qs20 and Qs21 on the growth and development
of P. falciparum. Light microscopy images of parasitized red blood cells at different time points
after the treatment with E64, Qs17, Qs18, Qs20 and Qs21.
17

3. Structure activity relationship studies (SAR)
Overall, the effect of various chemical entities attached to the core structure quinolin-
piperazin-methanone displayed a considerable role in the FP2 and parasite inhibiting ability of the
test compounds (as depicted in Figure 5). It was observed that replacement of quinolin-2(1H)-one
of Qs5 with 2,4-dichloro-sulfonylbenzene increased the FP2 inhibiting ability of compound Qs8
by 3-fold. The FP2 and parasite growth inhibition was found to further improve by the replacement
of 2,4-dichloro-sulfonylbenzene of Qs8 with sulfonyl-phenyl-acetamide in Qs20. The parasite
growth inhibition by Qs20 was recorded to be 810 nM. Further the replacement of 4-methoxy
benzene of Qs20 with simple chloro group in Qs18 resulted in the lowering of FP2 inhibiting
capability of the compound by 3.4-fold. The replacement of sulfonyl-phenyl-acetamide of Qs18
with chloroquinoline in Qs17 resulted in the enhancement of FP2 inhibition by almost 2-fold,
however, the parasite growth inhibiting capacity was found to be unaltered. From the above, we
concluded that the presence of both sulfonyl-phenyl-acetamide group and 4-methoxy benzene
attached to the quinolin-piperazin-methanone core moiety as in Qs20 resulted in the most potential
antimalarial molecule in the series which can serve as a lead for the further development of novel
effective antimalarials.
18

Figure 5. Diagrammatic illustration of Structure activity relationship studies.
4. Conclusions
A series of quinoline carboxamide-containing compounds was designed, synthesized and
evaluated for its efficacy to inhibit FP2. These molecules arrest the parasite growth at trophozoite
stage. Docking simulation and molecular hybridization techniques were used for the design of
inhibitors. The designed compounds were synthesized in the wet lab. Computational binding
affinity of the compounds was calculated in terms of kcal/mol and it was prophesied that the
quinoline carboxamide-based compounds bind to FP2 (PDB ID 3BPF) with high affinity, inhibit
its catalytic activity at sub-micromolar concentrations, thereby can arrest the parasite growth. In
the series, Compounds Qs17, Qs18, Qs20 and Qs21 were predicted to show most effective and
favorable interaction with the active site residues of FP2. Later, in-vitro results followed almost
same trend, as Qs17, Qs18, Qs20 and Qs21 inhibited FP2 with IC50 values 4.78, 7.37, 2.14 and
2.64 µM, respectively. These compounds also inhibited the growth of P. falciparum 3D7 with IC50
1.05, 1.95, 0.81 and 1.43 µM, respectively. From these results, we concluded that compounds
containing sulfonyl phenyl acetamide group along with an aromatic substitution to the quinoline
19

system as in Qs20 have greater FP2 inhibiting and antimalarial effect than the compounds with
mere quinoline structures. Overall, these compounds can be used as promising leads for the
development of novel and robust antimalarial agents.
5.
Experimental section
5.1.
Docking protocol
Crystal structure of FP2 (PDB ID 3BPF) in pdb format was downloaded from Protein Data
Bank (www.rcsb.org) [27]. AutoDock Vina version 1.5.6 was used to perform docking with the
ligand molecules. PyMOL visualization tool was used to visualize the molecular interactions.
Weak non-covalent interactions like π-π stacking interaction and H-bonding were observed
between the active site residues of FP2 and the ligand molecules lying within a range of 1.9 Ǻ,2Ǻ
and 2.1 Å. In addition, binding free energy was calculated based on which the designed molecules
were screened.
5.2.
Chemistry
All the chemicals and reagents were purchased from Sigma-Aldrich, Alfa-Aesar, Spectrochem
and SD Fine chemicals Pvt. Ltd. India, and used as received. The reactions were monitored and Rf
values were determined using analytical thin layer chromatography (TLC) with Merck silica gel
60-120 and F254 pre-coated plates (0.25 mm) thickness. Spot on the TLC plates were visualized
using ultraviolet light both at short (254 nm) and long wave (365 nm) UV light. Products were
purified by flash chromatography on silica gel (mesh size 200-400). The 1H NMR spectra and 13C
NMR were recorded on Bruker 300 spectrometers and 400 spectrometers. Chemical shifts are
reported in ppm (TMS,δ 0.00) or with the solvent reference relative to TMS employed as the
internal standard (CDCl3 ,δ 7.26; DMSO-d6 δ 2.54) and multiplicities of NMR signals are
designated as s (singlet), d (doublet), dd (double doublet), t (triplet),q (quartet), br (broad
coupling), m (multiplet, for unresolved lines). Elemental analyses of the compounds were found
to be within ±0.4% of the theoretical values.
5.2.1. General method of preparation of the compound 5
To a solution of chloral hydrate (0.54 mole) in 10 mL water, a solution of sodium sulfate was
added (Solution A). On the other hand, to a solution of aniline (0.5 mol) in 5 mL water concentrated
hydrochloric acid was added (Solution B). Now the solution B was added to solution B and the
reaction mixture could stir at 60–80 ̊C for half an hour. Formation of isonitrosoacetanilide needle
shaped crystals were observed and the reaction was cooled to room temperature. The product
20

obtained (2) was filtered, dried and produced for further reactions without purification [28]. Dry
isonitrosoacetanilide (0.46 moles) in a round bottom flask was added with 20 mL concentrated
sulfuric acid and the reaction mixture was stirred at 80 ̊C for half an hour. The mixture was cooled
to room temperature and quenched with crushed ice to obtain a yellow colored precipitate. The
precipitate was filtered and washed with cold water to remove the excessive sulfuric acid to obtain
pure 1H-indole-2,3-dione, 3 (yield 70-75%). To a solution of 1H-indole-2,3-dione (4 mmol) in
ethanol (20 mL), acetophenone (5 mmol), water (10 mL), and an aqueous solution of potassium
hydroxide (2.80 g, 50 mmol) were added. The reaction mixture was heated to reflux at 80 ̊C for 5
h. The reaction was monitored by TLC (80 % EtoAc/hexane). After that completion of the reaction,
crushed ice was added, and the organic solution was extracted by EtOAc. The combined organic
layers were dried over sodium sulphate, filtered and concentrated to give the crude yellow colored
product, (5) [15], which was purified by column chromatography using 80% EtOAC/Hexane. The
yield of the compound was recorded to be 75-80% and LC−MS m/z (M + H) + = 250.2.
5.2.2. General methods of the preparation of compound 18
A mixture of 3-amino-1,2,4-triazole (14) (20 mmol), ethyl acetoacetate (15) (20 mmol) and
acetic acid (10 mL) was refluxed for 4-5 h. After that, the reaction mixture was cooled to room
temperature to obtain a white precipitated which was filtered, washed with acetic acid followed by
ethanol, dried under vacuum to obtain the desired product with 65-70% yield.
5- Methyl- [1,2,4] triazolo [1,5-a] Pyrimidin-7-ol (16) (10 mmol) was added to 2.75 mL (30 mmol)
of phosphorous oxychloride and heated under reflux for 1 h in a round bottom flask. Excessive
POCl3 was removed under reduced pressure and the residue was triturated with ice water. The
product was extracted from the aqueous mixture with CH2Cl2, evaporated, and purified by column
chromatographed using 30% MeOH/chloroform. Yield of the product was found to be 55%. A
mixture of appropriate compound 17 (10 mmol), piperazine, (10 mmol) and K2CO3 (12 mmol) in
1,4-dioxane was refluxed at 100 ̊C for 3 h. The reaction mixture was cooled down to room
temperature, filtered and washed with 1,4-dioxane to obtained compound 18 [29][30].
5.2.3. General methods for the preparation of compound 9
6.5 g amine (1) and 6.73 g malonic acid (6) were dissolved in 39 mL of POCl3 and the reaction
mixture was refluxed at 100 ̊C for 9 h. On the completion of the reaction, the reaction mixture was
cooled to room temperature, poured dropwise into ice cold water and then neutralizes by sodium
carbonate. White precipitate obtained was filtered, washed with water and dried. The crude product
21

was purified by column chromatography to obtained pure compound 7. Then compound 7 was
treated with HCl in presence of 1-4 dioxane to obtain 4-chloroquinolin-2(1H)-one (8). A mixture
of compound 8 (10 mmol) in ethylene glycol (20 mL) and piperazine (30 mmol) was refluxed for
1.5 h in a round bottom flask. After the completion of the reaction (monitored by TLC) was
concentrated under vacuum, washed with water (3 x 20 mL) and the brined organic layer was
dried over Na2SO4 to obtain 4-(piperazin-1-yl)quinolin-2(1H)-one (9) [31].
5.2.4. Procedure for the preparation of compound 13
76.0 mmol of aniline (1) was dissolved in 120 mL of dichloromethane and cool to 0 ̊C using
an ice bath. Triethylamine (83.6 mmol, 1.10 eq) and the substituted acid chloride 10 (76.0 mmol)
were added to the amine solution drop wise for 30 minutes. After the completion of the reaction,
the solvents were removed under pressure, cold water was added, and the organic layer was
extracted with CH2Cl2. The combined organic layers were washed with brine and dried over
Na2SO4. The resulting off white solid compound 11 was used for the forward reaction step without
purification. The yield obtained was 80–85 %. Further, 195 mmol of chloro sulfonic acid was
added drop wise to compound 11 (5.0g, 37mmol) in a round bottom flask at 0
̊
of chloro sulfonic acid, the reaction mixture could stir under reflux conditions at 60
̊
reaction mixture was cooled room temperature and poured into crushed ice to obtain white solid
precipitate of (4-acetamidobenzene-1-sulfonyl chloride) 12 which was purified by column
chromatography [32]. Piperazine (0.01 mmol) was dissolved in 30 mL CH2Cl2 and Et3N (4.4 mL,
0.033 mml) was added to the solution at 0 ̊C for half an hour. 4-acetamidobenzene-1-sulfonyl
chloride 12 (2.29 g, 0.01 mmol) was added to the reaction mixture. The reaction was stirred for 2
h, quenched with water and then poured into a separating funnel and extracted with CH2Cl2 (3x20
mL) and then the brined organic layer was dried with Na2SO4 to obtain N-(4-(piperazin-1-
ylsulfonyl) phenyl) acetamide 13 as white colored powder with 60–70% yield.
5.2.5. General procedure for the preparation of compound Qs1-Qs10
To a stirring solution of differently substituted piperazine complexes (20 mmol) in 30 mL
DMSO, HBTU (50 mmol), TEA (50 mmol) and EDC were added at room temperature. Then the
reaction mixture was added with 2-phenylquinoline-4-carboxylic acid (5) (20 mmol). The resulting
mixture was stirred at room temperature for 8 h. The reaction monitored via TLC. On completion
of the reaction, the reaction was quenching with ice and extracted with ethyl acetate (3x50 mL).
The combined organic layers were washed with brine solution (50 mL), dried over Na2SO4 and
22

concentrated under vacuum. The crud products (Qs1–Qs10) were purified by column
chromatography using 70% EtOAc/Hexane.
5.2.5.1.
2-[4-(2-phenylquinoline-4-carbonyl) piperazin-1-yl] pyridine-3-carbonitrile
(Qs1)
◦
1
Light yellow solid, m.p. 180-182 C, 50% yield obtained after column chromatography. H
NMR (300 MHz, CDCl3) δ 8.36 (dd, J = 4.8, 1.8 Hz, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.17 (d, J =
6.7 Hz, 2H), 7.86 (m, 4H), 7.54 (m,= 4H), 6.85 (dd, J = 7.5, 4.8 Hz, 1H), 4.15 (d, J = 26.6 Hz,
2H), 3.88 (s, 2H), 3.55 (d, J = 11.5 Hz, 2H), 3.42 (d, J = 4.2 Hz, 2H). 13C NMR (75 MHz, CDCl3)
δ167.461, 160.875, 157.119, 151.863, 148.498, 143.637, 142.707, 138.919, 130.483, 129.783,
128.966, 127.546, 124.243, 123.072, 117.413, 115.963, 115.401, 96.510, 48.863, 48.407, 46.932,
+
41.685 HRMS: (ESI, m/z): [M+H]+ calcd for 419.17 found 420.1719. and [M+Na] : m/z =
442.1703. Anal. Calcd for C26H21N5O C, 74.44; H, 5.05; N, 16.70; found C, 74.11; H, 5.35; N,
16.40.
5.2.5.2.
(4-(7-chloroquinolin-3-yl) piperazin-1-yl) (2-phenylquinolin-4-yl) methanone
(Qs2)
Yellow solid, m.p. 200-202 ◦C, 63 % yield obtained after column chromatography. 1H NMR
(300 MHz, CDCl3) δ 8.89 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.16 (d, J =
8.2 Hz, 1H), 8.09 (s, 1H), 7.87 (t, J = 6.7 Hz, 1H), 7.81 – 7.74 (m, 1H), 7.64 – 7.28 (m, 1H), 6.84
(d, J = 5.1 Hz, 1H), 3.45 (d, J = 33.7 Hz, 1H), 2.80 (s, 1H), 2.08 (s, 1H). 13C NMR (75 MHz,
CDCl3) δ 175.13, 167.32, 156.97, 156.68, 150.96, 148.73, 148.23, 142.38, 138.56, 135.62, 130.45,
130.25, 129.76, 128.85, 127.75, 127.47, 127.40, 126.81, 124.58, 124.03, 122.84, 121.37, 115.91,
109.20, 52.33, 46.93, 41.58, 38.4 HRMS: (ESI, m/z): [M+H]+ calcd found 478.16 found 479.1547
Anal. Calcd for C29H23ClN4O C, 72.72; H, 4.84; N, 11.70; found C, 72.5.0; H, 4.64; N, 11.60.
5.2.5.3.
4-(4- {5-methyl- [1,2,4] triazolo[1,5-a] pyrimidin-6-yl} piperazine-1-carbonyl)-
2phenylquinoline (Qs3)
Dull yellow solid, m.p. 238 -240◦C, 53.5 % yield obtained after column chromatography. 1H
NMR (300 MHz, CDCl3) δ 8.27 (s, 1H), 8.22 (s, 1H), 8.17 (d, J = 6.9 Hz, 1H), 7.85 (s, 1H), 7.79
(t, J =8.7 Hz,1H), 7.59 (t, J =7.5 Hz,1H, 1H), 7.53 (d, J = 7.5 Hz, 1H), 6.16 (s, 1H), 4.23 (d, J =
26.4 Hz, 2H), 3.95 (d, J = 5.4 Hz, 2H), 3.70 (d, J = 15.9 Hz, 2H), 3.49 (d, J = 12.0 Hz, 2H), 2.58
13
(s, 3H). C NMR (75 MHz, CDCl3) δ 167.43, 165.20, 157.09, 154.44, 149.79, 148.50, 142.21,
138.77, 130.60, 130.56, 129.90, 129.01, 127.60, 127.50, 124.05, 122.97, 115.98, 94.97, 48.30,
23

47.89, 46.51, 41.13, 25.25 [M+H]+ m/z calcd 449.20 for found 450.1. Anal. Calcd for C26H23N7O
C, 69.47; H, 5.16; N, 21.81; found C, 69.27; H, 5.46; N, 21.41.
5.2.5.4.
(2-phenylquinolin-4-yl) (4-(5-(trifluoromethyl)- [1,2,4] triazolo[1,5-a] pyrimidin-
6-yl) piperazin-1-yl) methanonein (Qs4)
White solid, m.p. 250-252 ̊
C, 55 % yield obtained after column chromatography.1H NMR (300
MHz, CDCl3) δ 8.43 (s, 1H), 8.25 (d, J = 8.7 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 7.83 ( J = m, 3H),
7.57 (J = m, 4H), 7.27 (s, 1H), 6.59 (s, 1H), 4.37 – 3.87 (m, 4H), 3.56 (s, 1H), 2.81 (s, 1H), 2.07
(d, J = 13.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 167.53, 157.11, 156.54, 155.58, 150.74,
148.44, 141.96, 138.64, 130.66, 130.57, 129.97, 129.02, 127.72, 127.51, 123.93, 122.88, 116.05,
90.25, 48.61, 48.21, 46.42, 41.15 HRMS: (ESI, m/z): [M+H] + calcd 503.17 found 504.1682. and
[M+Na] +: m/z = 526.1543 Anal. Calcd for C26H20F3N7O C, 62.02; H, 4.00; N, 19.47; found C,
61.02; H, 4.51; N, 19.37.
5.2.5.5.
4-(4-(2-phenylquinoline-4-carbonyl) piperazin-1-yl) quinolin-2(1H)-one (Qs5)
1
Pale yellow solid, m.p. 199-202
̊
NMR (300 MHz, CDCl3) δ 12.29 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 7.8 Hz, 2H), 7.86
(d, J = 5.4 Hz, 2H), 7.82 (t, J=7.8Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.64 – 7.46 (m, 5H), 7.37 (d,
J = 8.1Hz, 1H), 7.22 (dd, J = 17.3, 9.7 Hz, 1H), 6.18 (s, 1H), 4.24 (d, J = 32.4 Hz, 2H), 3.44 (d, J
= 36.9 Hz, 4H), 3.07 (s, 2H).13C NMR (75 MHz, CDCl3) δ 176.59, 167.49, 165.54, 160.02, 157.15,
148.44, 142.54, 138.80, 134.96, 130.94, 130.57, 130.46, 129.87, 129.02, 127.56, 124.20, 124.08,
123.02, 122.55, 117.29, 116.44, 116.06, 105.97, 52.04, 51.77, 47.03, 41.68. HRMS: (ESI, m/z):
+
+
[M+H] calcd 460.19 for found 461.1902. and [M+Na] : m/z = 483.1706 Anal. Calcd for
C29H24N4O2 C, 75.63; H, 5.25; N, 12.17; found C, 75.43; H, 5.35; N, 12.25.
5.2.5.6.
N-(4-(4-(2-phenylquinoline-4-carbonyl)
acetamide (Qs6)
piperazin-1-ylsulfonyl)
phenyl)
Deep yellow solid, m.p. 200◦C, 72 % yield obtained after column chromatography. 1H NMR
(300 MHz, CDCl3) δ 8.50 (s, 1H), 8.21 (d, J = 9.00 Hz, 1H), 8.05 (dd, J = 9.0, 3.0 Hz, 1H), 7.76
(dd, J = 9.0, 3.0 Hz, 1H), 7.72 (s, 1H), 7.70 (s, 1H), 7.61 (m, J = 7.7 Hz, 3H), 7.49 (m, J = 7.4 Hz,
4H), 7.36 (dd, J = 6.0, 3.0 Hz, 1H), 4.00 (m, 2H), 3.30 (t, J = 4.9 Hz, 2H), 2.86 (d, J = 6.0 Hz, 1H),
2.80 (s, 1H), 2.18 (s, 3H), 2.06 (d, J = 6.0 Hz, 1H), 1.26 (t, J = 6.00 Hz, 1H). 13C NMR (75 MHz,
24

CDCl3) δ 169.65, 167.38, 157.21, 148.10, 143.00, 142.16, 138.42, 130.83, 130.12, 129.99, 129.38,
129.00, 128.82, 127.79, 127.58, 126.67, 126.59, 123.93, 122.73, 119.67, 116.13, 46.48, 46.26,
45.80, 41.15, 38.68, 24.48 HRMS: (ESI, m/z): [M+H]+ calcd 514.60 for found 515.1666. and
[M+Na]+: m/z = 537.1516 Anal. Calcd for C28H26N4O4S C, 65.35; H, 5.09; N, 10.89; found C,
65.55; H, 5.05; N, 10.35.
5.2.5.7.
N-(2-fluoro-4-((4-(2-phenylquinoline-4-carbonyl) piperazin-1-yl) sulfonyl)
phenyl) acetamide (Qs7)
Crystalline white solid, m.p. 260-262 ̊C, 61 % yield obtained after column chromatography.
1H NMR (300 MHz, CDCl3) δ 8.76 (d, J = 8.7 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.13 (d, J =
6.6Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.72 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.51 (m = Hz, 5H),
7.26 (t, J = 9.4 Hz, 2H), 4.22 (m, 2H), 3.33 (t, J = 4.8 Hz, 3H), 3.19 (s, 1H), 2.98 (d, J = 26.3 Hz,
2H), 2.24 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 165.13, 160.87, 158.92, 157.19, 156.16, 154.43,
152.06, 150.19, 135.25, 129.18, 126.73, 124.64, 121.78, 116.77, 111.36, 109.43, 94.94, 51.60,
47.97, 25.32. HRMS: (ESI, m/z): [M+H] + calcd for found 532.16 found [M+Na] +:m/z = 555.1405
Anal. Calcd for C28H25FN4O4S C, 63.15; H, 4.73; N, 10.52; found C, 62.15; H, 4.53; N, 10.72.
5.2.5.8.
(4-((2,4-dichlorophenyl) sulfonyl) piperazin-1-yl) (2-phenylquinolin-4-yl)
methanone (QS8)
White powder, m.p. 252-254 ̊
C, 66 % yield obtained after column chromatography.1H NMR
(500 MHz, CDCl3) δ 8.25 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 7.0 Hz, 2H), 8.07 (d, J = 1.0 Hz, 1H),
7.79 (t, J = 5.9 Hz, 2H), 7.60 (d, J = 7.1 Hz, 1H), 7.56 (s, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.51 (s,
2H), 7.29 (s, 2H), 4.07 (d, J = 13.0 Hz, 2H), 3.57 (s, 2H), 3.33 (d, J = 2.5 Hz, 2H), 3.26 (d, J = 5.5
Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 167.42, 157.08, 138.76, 137.47, 133.99, 133.92, 133.48,
133.35, 131.85, 130.68, 130.58, 130.42, 129.98, 129.90, 129.01, 127.61, 127.52, 124.01, 122.89,
115.93, 47.05, 46.12. HRMS: (ESI, m/z): [M+H] + calcd 525.07 for found 526.0. and [M+Na]+:
m/z = 548.0531 Anal. Calcd for C26H21Cl2N3O3S C, 59.32; H, 4.02; N, 7.98; found C, 58.32; H,
4.46; N, 7.40.
5.2.5.9.
(2-phenylquinolin-4-yl) (4-tosylpiperazin-1-yl) methanone (QS9)
C, 59 % yield obtained after column chromatography. H NMR
1
Off powder, m.p. 270-272
̊
(500 MHz, CDCl3) δ 8.26 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 7.5 Hz, 2H), 7.79 (t, J = 7.6 Hz, 1H),
7.73 (s, 1H), 7.66 (t, J = 8.1 Hz, 3H), 7.53-7.30 (m, 4H), 7.38 (d, J = 8.0 Hz, 2H), 4.06 (d, J = 24.0
Hz, 2H), 3.39 – 3.15 (m, 4H), 2.91 (d, J = 32.0 Hz, 2H), 2.50 (s, 3H). 13C NMR (126 MHz, CDCl3)
25

δ 167.42, 157.08, 138.76, 137.47, 133.99, 133.92, 133.48, 133.35, 131.85, 130.68, 130.58, 130.42,
129.98, 129.90, 129.01, 127.61, 127.52, 124.01, 122.89, 115.93, 47.05, 46.12. HRMS: (ESI, m/z):
[M+H] + calcd 471.16 for found 472.1624. Anal. Calcd for C27H25N3O3S C, 68.77; H, 5.34; N,
8.91; found C, 68.04; H, 5.74; N, 7.91.
5.2.5.10. (4-((4-bromophenyl)
methanone (QS10)
sulfonyl)
piperazin-1-yl)
(2-phenylquinolin-4-yl)
1
Yellowish powder, m.p. 265-267
̊
NMR (500 MHz, CDCl3) δ 8.24 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.0 Hz, 2H), 7.79 (t, J = 7.7 Hz,
1H), 7.75 – 7.73 (m, 3H), 7.66 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.58 – 7.48 (m, 4H),
4.07 (d, J = 18.0 Hz, 2H), 3.39 – 3.17 (m, 4H), 2.91 (d, J = 17.5 Hz, 2H). 13C NMR (126 MHz,
CDCl3) δ 167.31, 157.03, 148.43, 142.03, 138.73, 134.59, 132.71, 130.56, 129.92, 129.17, 129.01,
128.54, 127.55, 127.52, 123.96, 122.84, 115.91, 46.46, 46.31. HRMS: (ESI, m/z): calcd 536.44
found [M + 2] + = 538.1. Anal. Calcd for C26H22BrN3O3S C, 58.21; H, 4.13; N, 7.83; found C,
57.61; H, 4.23; N, 7.09.
5.2.6. General procedure for the preparation of compound Qs11, Qs12, Qs13
1 mmol solution of compound 18 was reacted separately with the compound 19, 20 and 21 to
obtain Qs11,Qs12 and Qs13, respectively [33]. Compounds 19, 20 and 21 (1.5 mmol each) were
added to compound 18 (1.5 mmol) in presence of K2CO3 (3 mmol) and anhydrous 1,4 dioxane (20
mL) and the reaction mixture was refluxed at 100 ̊C for 12 h. After the completion of the reaction,
the mixture was cooled to room temperature. The reaction mixture was concentrated under vacuum
and the crude obtained was diluted with chloroform and washed with water (100 mL x 3). The
organic layer was separated and dry with Na2SO4. The crude mixture was purified by column
chromatography using 80% EtOH/ Hexane.
5.2.6.1.
7-chloro-4-(4-(7-methyl- [1,2,4] triazolo[1,5-b] pyridazin-8-yl) piperazin-1-yl)
quinoline (Qs11)
Pale yellow , m.p. 200-205 ̊
C, 69.2 % yield obtained after column chromatography.1H NMR
(300 MHz, CDCl3) δ 8.79 (s, 1H), 8.35 (s, 1H), 8.09 (s, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.48 (d, J =
8.7 Hz, 1H), 6.95 (s, 1H), 6.28 (s, 1H), 4.10 (s, 4H), 3.48 (s, 4H), 2.64 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 165.13, 157.19, 156.16, 154.43, 152.06, 150.19, 135.25, 132.72, 129.18, 126.73, 124.64,
121.78, 109.43, 94.94, 51.60, 47.97, 25.32. HRMS: (ESI, m/z): [M+H] + calcd 379.13 for found
26

380.1368. Anal. Calcd for C19H18ClN7 C, 60.08; H, 4.78; N, 25.81 found C, 60.00; H, 4.45; N,
25.51.
5.2.6.2.
Ethyl 7-methyl-4-(4-(5-(trifluoromethyl)- [1,2,4] triazolo[1,5-a] pyrimidin-7-yl)
piperazin-1 yl) quinoline-3-carboxylate (Qs12)
Light Yellow solid, m.p. 250-252 ̊C, 65 % yield obtained after column chromatography.
1HNMR(300 MHz, CDCl3) δ 8.95 (s, 1H), 8.50 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.5
Hz, 1H), 7.27 (s, 1H), 6.70 (s, 1H), 4.47 (q, J = 14.2, 7.2 Hz, 2H), 4.33 (s, 2H), 3.65 (t, J = 7.3 Hz
4H), 2.61 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H), 1.25 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 175.13,
167.23, 156.97, 156.68, 150.96, 148.73, 148.23, 142.38, 138.56, 135.62, 130.45, 130.25, 129.76,
128.85, 127.75, 127.40, 126.81, 124.58, 124.03, 122.84, 121.37, 115.91, 109.20, 46.93, 41.58,
+
38.48, 29.55. HRMS: (ESI, m/z): [M+H] calcd 485.18 for found 486.0. Anal. Calcd for
C23H22F3N7O2 C, 56.90; H, 4.57; N, 20.20; found C, 56.40; H, 4.23; N, 20.50.
5.2.6.3.
sulfonyl) phenyl) acetamide (Qs13)
Cotton white, m.p. 270-272
C, 74 % yield obtained after column chromatography. 1H NMR
N-(4-((4-(5-methyl- [1,2,4] triazolo[1,5-a] pyrimidin-7-yl) piperazin-1 yl)
̊
(300 MHz, CDCl3) δ 9.52 (s, 1H), 8.54 (s, 1H), 8.28 (s, 1H), 7.70 (dd, J = 23.2, 7.6 Hz, 2H), 7.28
(s, 1H), 6.17 (s, 1H), 3.89 (s, 4H), 3.24 (s, 2H), 2.58 (d, J = 1.8 Hz, 2H), 2.21 (s, 3H), 2.10 (s,
3H).13C NMR (75 MHz, CDCl3) δ 177.38, 165.65, 153.69, 151.68, 149.64, 142.89, 129.19, 128.94,
+
119.49, 95.25, 47.30, 45.47, 29.70, 20.91 HRMS: (ESI, m/z): [M+H] calcd 415.14 for found
416.1300. Anal. Calcd for C18H21N7O3S C, 52.04; H, 5.09; N, 23.60; found C, 51.04; H, 5.49; N,
23.30.
5.2.7. General procedure for the preparation of compounds Qs14–Qs16
To a solution of 2-hydroxyquinoline-4-carboxylic acid 23 (2mmol) in (20 mL) DMSO was
added with R3, HBTU (5 mmol), TEA (5mmol) and EDC a round bottom flask, at stirred room
temperature overnight. After the completion reaction, the reaction mixture was dissolved in 50 mL
ethyl acetate. The organic phase washed with water and dried with Na2SO4. The crude product was
purified by column chromatography 75 % ethyl acetate/ Hexane to obtain target products Qs14–
Qs16.
5.2.7.1.
(4-((2,4-dichlorophenyl) sulfonyl) piperazin-1-yl) (2-hydroxyquinolin-4-yl)
methanone (QS14)
27

White, m.p. 240-242 ̊C, 70% yield obtained after column chromatography HRMS: (ESI, m/z):
[M+H] + calcd 466.33 for found [M+Na] + m/z: 488.0247 Anal. Calcd for C20H17Cl2N3O4S C,
51.51; H, 3.67; N, 9.01; found C, 50.81; H, 3.37; N, 8.81; 1H NMR (500 MHz, DMSO) δ 11.82
(s, 1H), 7.92 (d, J = 14.7 Hz, 1H), 7.48 (d, J = 14.7 Hz, 2H), 7.41 (d, J = 7.6 Hz, 1H), 7.35 – 7.26
(m, 2H), 7.10 (dd, J = 14.9, 7.4 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 3.84 (d, J = 35.0 Hz, 2H), 3.31
(dd, J = 73.9, 40.0 Hz, 4H), 2.51 (d, J = 14.0 Hz, 2H).13C NMR (126 MHz, DMSO) δ 165.81,
161.91, 145.65, 139.44, 137.20, 134.15, 133.61, 133.25, 131.57, 131.35, 124.91, 122.70, 119.09,
119.03, 116.54, 116.00, 46.74, 45.99.
5.2.7.2.
(2-hydroxyquinolin-4-yl) (4-tosylpiperazin-1-yl) methanone (QS15)
1
Yellow, m.p. 230-235
̊
MHz, CDCl3) δ 8.75 (s, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.09 (s, 1H), 7.87
(t, J = 6.7 Hz, 1H), 7.81 – 7.70 (m, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.42
(d, J = 9.0 Hz, 1H), 6.84 (d, J = 5.1 Hz, 1H), 4.26 (d, J = 34.8 Hz, 2H), 3.45 (d, J = 33.7 Hz, 2H),
3.09 (s, 2H), 2.80 (s, 2H), 2.08 (s, 3H). 13C NMR (126 MHz, DMSO) δ 160.87, 157.29, 140.94,
139.53, 134.45, 127.23, 126.63, 125.21, 122.85, 120.14, 118.01, 113.97, 111.81, 111.29, 41.46,
+
41.04, 36.07 HRMS: (ESI, m/z): [M+H] + calcd 411.13 for found [M+Na] m/z := 434.1065.
Anal. Calcd for C21H21N3O4S C, 61.30; H, 5.14; N, 10.21; found C, 60.79; H, 5.54; N, 10.51.
5.2.7.3.
(4-(7-chloroquinolin-4-yl) piperazin-1-yl) (2-hydroxyquinolin-4-yl) methanone
(QS16)
1
White, m.p. 280-282 ̊C, 52 % yield obtained after column chromatography. H NMR (300
MHz, CDCl3) δ 8.78 (d, J = 4.9 Hz, 1H), 8.10 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 7.7
Hz, 2H), 7.46 (dd, J = 10.8, 3.9 Hz, 2H), 7.36 – 7.25 (m, 2H), 6.87 (d, J = 5.1 Hz, 1H), 6.72 (s,
1H), 4.19 (d, J = 3.6 Hz, 2H), 3.64 (d, J = 10.5 Hz, 2H), 3.37 (s, 1H), 3.14 (d, J = 16.8 Hz, 2H).13C
NMR (126 MHz, DMSO) δ 161.18, 157.32, 141.12, 134.60, 126.59, 122.25, 120.24, 120.17,
118.00, 114.30, 111.79, 111.37, 104.10, 47.54, 47.23. HRMS: (ESI, m/z): [M+H] + calcd 418.12
for found 419.1245. Anal. Calcd for C23H19ClN4O2 C, 65.95; H, 4.57; N, 13.38; found C, 65.45;
H, 3.87; N, 13.58.
5.2.8. Procedure for the synthesis of Qs17
To stirred solution of isatin 22 (10.00 g, 61 mmol) in acetic acid (400 mL), malonic acid (18.91
g, 182 mmol) was added and the reaction mixture was refluxed for 16 h. The excessive solvent
was removed under vacuum, and the suspended solid was dissolved in water (400 mL), filtered,
28

and washed with water (300 mL) to obtain brown solid. The brown solid was dissolved in saturated
aqueous solution of NaHCO3 (800 mL), and the insoluble material was filtered off. The filtrate
was acidified to pH 1−2 with concentrated HCl, the resulting precipitate was filtered, washed with
water (300 mL), and dried. The resulting pale-yellow solid (23) was used directly for further
synthesis without purification. The yield obtained was 60%. Then a solution of 2-
hydroxyquinoline-4-carboxylic acid 23 (10.6 g) in POCl3 (70 mL) was refluxed for 2 h. the
reaction mixture was cooled to room temperature and the excessive POCl3 was evaporated under
reduced pressure. The crude viscous reaction mixture was poured into ice water (400 mL) and
stirred for 30 minutes. The resulting precipitate was filtered and washed with water. The solid
residue obtained was then stirred in 100 mL of 0.5 M KOH until fully dissolved and pH was
adjusted to 2 with HCl (3N). Precipitate obtained was filtered and dried. The desired product 24
was solid and off white in color [15]. The product obtained was used for the next step without
further purification. Qs17 was prepared by reacting 2-chloroquinoline-4-carboxylic acid 24 and 7-
chloro-4-(piperazin-1-yl) quinoline in presence of DMSO, HBTU and TEA at the room
temperature overnight. After the completion of the reaction, the crud product was purified by
column chromatography using 65% ethyl acetate/hexane.
5.2.8.1.
(2-chloroquinolin-4-yl) (4-(7-chloroquinolin-4-yl) piperazin-1-yl) methanone
(QS17)
1
Yellow, m.p. 220-222 ̊C, 58 % yield obtained after column chromatography. H NMR (500
MHz, CDCl3) δ 8.77 (d, J = 5.0 Hz, 1H), 8.17 (dd, J = 5.2, 3.1 Hz, 2H), 7.96 (d, J = 9.0 Hz, 1H),
7.85 (d, J = 10.0Hz, 1H), 7.67 (d, J = 5.0 Hz, 1H), 7.53 – 7.49 (m, 2H), 7.35 (s, 1H), 6.93 (d, J =
5.0 Hz, 1H), 4.28 (d, J = 20.0Hz, 2H), 3.65 – 3.40 (m, 4H), 3.24 (d, J = 25.0Hz, 2H). 13C NMR
(126 MHz, CDCl3) δ 165.97, 159.95, 156.91, 147.07, 145.91, 143.58, 131.50, 129.04, 128.68,
127.90, 127.38, 127.17, 124.84, 124.76, 124.41, 120.45, 109.18, 109.02, 106.05, 52.42, 52.15.
HRMS: (ESI, m/z): [M+H] + calcd 436.09 for found 437.0851. Anal. Calcd for C23H18Cl2N4O C,
63.17; H, 4.15; N, 12.81; found C, 62.87; H, 4.25; N, 11.85.
5.2.9. Procedure for the synthesis of Qs18
Later, to a solution of 2-chloroquinoline-4-carboxylic acid 24 (10 mmol) in DMSO (10 mL),
EDC (528 mg, 2.7 mmol, 2 eq) and HBTU (371 mg, 2.7 mmol, 2 eq) were added. Then, N-(4-
(piperazin-1-ylsulfonyl) phenyl) acetamide (10mmol) and TEA (30 mmol) was added to the
reaction mixture. The reaction was stirred for 8 h at room temperature. The reacting mixture was
29