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ORIGINAL RESEARCH
Synthesis and antimalarial activity of quinoline-substituted
furanone derivatives and their identification as selective
falcipain-2 inhibitors
Mymoona Akhter •Rikta Saha •Omprakash Tanwar •
Md. Mumtaz Alam •M. S. Zaman
Received: 10 September 2013 / Accepted: 30 June 2014 / Published online: 29 July 2014
ÓSpringer Science+Business Media New York 2014
Abstract 3-[(2-Chloroquinolin-3-yl) methylene]-5-phen-
ylfuran-2(3H)-one derivatives (6a–jand 7a–j) have been
synthesized and evaluated for their antimalarial activity.
Three compounds 7d,7f, and 7g showed excellent activity
(0.50–0.72 lg/mL). In addition, six compounds were
active in range below 5 lg/mL. A preliminary structure–
activity relationship analysis of the series suggested that
electropositive character is beneficial for antimalarial
activity. Falcipain-2 was identified as potential target for
the compounds by in silico studies. The docking studies of
synthesized compounds on falcipain-2 revealed vital
interactions and their binding conformation. Compound 7d
and 7f could be used as lead to develop selective falcipain-
2 inhibitors as they showed good inhibition of the enzyme
in enzyme assay studies.
Keywords Antimalarial Furanone Schizontocidal
Quinoline Falcipain
Abbreviations
FP-2 Falcipain-2
FP-3 Falcipain-3
DMF N,N-dimethylformamide
PDB Protein data bank
SAR Structure–activity relationship
Introduction
Malaria is one of the most severe and widespread parasitic
diseases because of its prevalence, virulence, and drug
resistance. It has a devastating impact on public health in
the developing regions of the world (Butcher et al., 2000).
Despite more than two decades of research effort, no
vaccine has been discovered for effective control of
malaria. In addition, increasing emergence of drug-resis-
tant strains of Plasmodium falciparum has created a need
for development of novel and effective antimalarial agents.
Heterocyclics, like the quinoline nucleus, are present in
several natural compounds. Compounds bearing a quino-
line moiety (Fig. 1) have been reported to exhibit a wide
spectrum of biological activities such as antifungal (Ko-
uznetsov et al., 2012; Musiol et al., 2006; Mele
´ndez
Go
´mez et al., 2008), antiparasitic (Lell et al., 2000), anti-
tubercular (Mao et al., 2009; Bermudez et al., 2004; Ja-
yaprakash et al., 2006), antibacterial (Martı
´nez et al.,
2005), antiviral (Luo et al., 2009; Chen et al., 2009),
cytotoxic and antineoplastic (Lamazzi et al., 2000), anti-
inflammatory (Pellerano et al., 1990), antioxidant (Dorey
et al., 2000), and immunosuppressive behaviors (Liu et al.,
2009). Quinoline moiety-bearing compounds have also
been reported to show excellent potential as lead moieties
in the search for novel antimalarial agents.
Furan derivatives are known to possess a broad spec-
trum of biological activities such as antimalarial, anti-
fungal, antibacterial, antiinflammatory, analgesic, and
antiviral properties. In addition, antioxidant and cardio-
tonic activities have been reported. Several natural com-
pounds containing furan ring have been reported to be
effective plasmodial inhibitors such as gersemolide alka-
loids (IC
50
=21.3 lg/mL) (Marrero et al., 2006), neuro-
lenin B (IC
50
=0.62 lM), hirsutinolides (IC
50
=1,800
M. Akhter R. Saha O. Tanwar Md. Mumtaz Alam
M. S. Zaman
Drug Design and Medicinal Chemistry Laboratory, Department
of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia
Hamdard, New Delhi 110062, India
M. Akhter (&)
Bioinformatics Facility, Jamia Hamdard, New Delhi 110062,
India
e-mail: mymoonaakhter@gmail.com
123
Med Chem Res (2015) 24:879–890
DOI 10.1007/s00044-014-1139-1
MEDICINAL
CHEMISTR
Y
RESEARCH
and 2,600 ng/mL, respectively), and bulaquine (BQ). The
2(5H)-furanone substructure present in mucochloric acid
and mucobromic acid (Pillay et al., 2007) is reported to be
active against the malaria parasite. The antiplasmodial
activities of mucochloric acid and mucobromic acid
(IC
50
=137 and 359 ng/mL, respectively) suggest that the
2(5H)-furanone unit is the key pharmacophore for anti-
plasmodial activity.
Many new strategies have been adopted to counteract
drug resistance and develop novel antimalarial compounds
possessing a broader spectrum of activities. An important
rationale for the development of newer effective chemical
entities is amalgamation of two biologically active moie-
ties, each possessing an independent mode of action.
Herein, we have designed novel inhibitors by combining
two biologically active units. In this paper, we present the
design, synthesis, in vitro evaluation, and molecular
docking study of quinoline-furanone derivatives as novel
antimalarial agents.
Results and discussion
Chemistry
The present study involved conjoining of furanone and
quinoline nucleus to yield newer effective compounds. The
synthetic methodology involves the synthesis of quinoline-
3-carbaldehyde derivatives by the reaction of N,N-
dimethylformamide (DMF) with an appropriate acetophe-
none oxime in the presence of phosphorus oxychloride
(POCl
3
) (Scheme 1). The reaction goes via Beckmann
rearrangement followed by Vilsmeier–Haack formylation.
The b-benzoyl propionic acids were prepared by Friedel–
Craft acylation of aromatic acids. Condensation of quino-
lone-3-carbaldehyde with b-benzoyl propionic acid in
presence of acetic anhydride and anhydrous sodium acetate
resulted in the title compounds. All the compounds were
characterized by IR,
1
HNMR,
13
CNMR, and Mass spectra.
The
1
H NMR signal at dvalue 6.5–6.7 indicates the for-
mation of furan ring. The absence of aldehydic proton and
presence of alkenic proton further indicate the conversion
of aldehydic group to the desired compound. The physi-
cochemical parameters of the synthesized compounds (6a–
6j and 7a–7j) are given in Table 1.
Antimalarial activity
All synthesized compounds were tested for antimalarial
activity, using the method reported by Dua et al. (2002).
All compounds (6a–j,7a–j) demonstrated good-to-excel-
lent activity against the parasite P. falciparum. Compounds
(7d,7f, and 7g) were found to be most active, with an IC
50
of 0.61, 0.50, and 0.72 lg/mL, respectively (Table 2). Six
other compounds (7a,7b,7c,7e,7i, and 7j) were found to
be moderately active (\5lg range). Structure–activity
relationship (SAR) studies have shown that replacement of
the methoxy group with a chloro group at the 6th position
of the quinolone ring enhances antimalarial activity. This
suggests that decreasing the electronegativity of the R
substituent increases the antimalarial activity (as demon-
strated by the 6 and 7 series). In addition, the replacement
of the chloro or dichloro group with a dimethyl group, in
N
HN
H
N
O
O
O
R
2
R
1
O
O
OO
O
O
Br
Br
HO
Mucochloric acid Mucobromic acid
O
O
Cl
Cl
HO
N
N
Antifungal
N
O
F
F
F
O
HN NH
2
Antiparasite
N
Cl
O
N
H
OOO
O O
H
Antiviral
Vitamin C or
Ascorbic acid
O
OH
HO
O
OH
HO H
Fig. 1 Known compounds
bearing quinoline and furan
moiety
880 Med Chem Res (2015) 24:879–890
123
case of R
1
substitution, augments antimalarial activity.
This indicates that substitutions with -rand ?pvalues or
with -rand -pvalues are important for antimalarial
activity. Similar results have been obtained in molecular
modeling studies of falcipain-2. Compounds 7d,7f, and 7g
allowed good cell viability when tested for cytotoxicity
(IC
50
) in VERO cells. The compounds tested showed
minimal toxicity and have been presented as excellent
leads for the present work.
Compounds 7d,7f, and 7g were also evaluated for their
inhibitory activity against falcipain-2 (FP-2) by using Cbz-
Phe-Arg-AMC as a fluorogenic substrate (Shenai et al.,
2000). Preliminary screening was performed at 10:l M
concentration, DMSO was used as a negative control, and
Table 1 Physicochemical parameters of the synthesized compounds (6a–6j and 7a–7j)
NCl
O
O
R1
R
6a-j
7a-j
Compounds R R
1
R
f
a
m.p. (°C) LogP ClogP % Yield
6a Cl H 0.8 240–242 5.41 5.53 37
6b Cl CH
3
0.8 272–274 5.89 6.03 36
6c Cl CH
3
CH
2
0.7 273–275 6.31 6.56 24
6d Cl Cl 0.8 280–282 5.96 6.24 20
6e Cl Br 0.7 288–289 6.23 6.39 15
6f Cl 2,4-di CH
3
0.6 215–217 6.38 6.53 30
6g Cl 3,4-di Cl 0.7 266–262 6.52 6.83 20
6h Cl 0.7 250–251 7.08 7.42 28
6i Cl 0.8 165–166 7.06 7.48 36
6j Cl OCH
3
0.7 160–162 5.28 5.45 33
7a OCH
3
H 0.8 245–246 4.72 5.04 38
7b OCH
3
CH
3
0.8 280–281 5.21 5.54 39
7c OCH
3
CH
3
CH
2
0.8 275–276 5.63 6.07 30
7d OCH
3
Cl 0.8 274–275 5.28 5.75 23
7e OCH
3
Br 0.8 264–266 5.55 5.90 25
7f OCH
3
2,4-di CH
3
0.8 250–252 5.69 6.04 22
7g OCH
3
3,4-di Cl 0.8 265–266 5.84 6.35 35
7h OCH
3
0.8 216–218 6.4 6.93 21
7i OCH
3
0.8 170–172 6.37 7.00 29
7j OCH
3
OCH
3
0.8 230–232 4.59 4.96 30
a
Mobile phase used for TLC 2:8 ethyl acetate: petroleum ether
Med Chem Res (2015) 24:879–890 881
123
the irreversible standard inhibitor E-64 was used as a
positive control. Assays were performed to determine the
percentage inhibition of the enzyme at a concentration of
10 lM. FP-2 activity was assessed by measuring the
release of fluorescent AMC group from the cleaved
fluorogenic substrate Cbz-Phe-Arg-AMC. Thus, a
decrease in fluorescence intensity in a sample represents
inhibition of enzyme activity. Of the three compounds
Table 2 IC
50
for the inhibition of gametocyte-producing P. falciparum culture lines (FDL-HD), cytotoxic results, and Inhibition of FP-2 in
percentage by the synthesized compounds (6a–6j and 7a–7j)
NCl
O
O
R
1
R
6a-j
7a-j
Compounds R R
1
Activity IC
50
(lg/mL)
a
Selectivity index (SI)
b
Falcipain-2 inhibition (%)
6a Cl H 38.02 ND ND
6b Cl CH
3
10.84 ND ND
6c Cl CH
3
CH
2
29.85 ND ND
6d Cl Cl 26.92 ND ND
6e Cl Br 26.45 ND ND
6f Cl 2,4-di CH
3
12.65 ND ND
6g Cl 3,4-di Cl 53.84 ND ND
6h Cl 49.65 ND ND
6i Cl 7.24 ND ND
6j Cl OCH
3
8.68 ND ND
7a OCH
3
H 2.31 ND ND
7b OCH
3
CH
3
1.03 ND ND
7c OCH
3
CH
3
CH
2
1.56 ND ND
7d OCH
3
Cl 0.61 12 58
7e OCH
3
Br 3.06 ND ND
7f OCH
3
2,4-di CH
3
0.50 18 62
7g OCH
3
3,4-di Cl 0.72 21 41
7h OCH
3
5.962 ND ND
7i OCH
3
2.05 ND ND
7j OCH
3
OCH
3
1.86 ND ND
Chloroquin 0.002 – –
a
IC50: Concentration corresponding to 50 % growth inhibition of the parasite
b
SI: IC
50
values of cytotoxic activity/IC
50
values of antimalarial activity
882 Med Chem Res (2015) 24:879–890
123
tested, compound 7d showed the highest level of inhibi-
tion (Table 2).
Molecular modeling
To provide additional insight into the inhibition modes of
the furanone derivatives, potential binding orientations and
interactions were explored via molecular docking studies.
To demonstrate these, BAITOC (freely available server)
(http://www.scfbio-iitd.res.in/software/drugdesign/baitocnew.
jsp) was used to conduct a systematic target prediction
search for the synthesized compounds. The falcipain-2
enzyme was found to be the best target for these molecules.
The probable binding mode of the title compound was then
determined using molecular modeling studies. The phar-
macophore features of the synthesized compounds were
generated (Fig. 2). Molecular docking studies were per-
formed on the recently published X-ray structure of falci-
pain-2 co-crystallized with E-64 (PDB ID 3BPF) (Kerr
et al., 2009). Binding of the furanone derivatives was
comparable to that of the reference compound, E64. All
molecules favorably occupied the active site of falcipain-2,
as shown by their negative binding energies. The com-
pounds form hydrogen bonds with the side chains of Cys 42
and Gln 36 of the falcipain-2 receptor. In silico binding
energies were also complementary to that experimental
biological activity. Compounds with a methoxy substitution
at the 6th position of quinoline showed a higher binding
score than the other compounds, as shown in Fig. 3. Fig-
ures 4and 5show the predicted binding orientations of
compounds 7d and 7f within the active site of falcipain-2.
Conclusion
A series of quinoline bearing furanones have been suc-
cessfully synthesized and screened for antimalarial activity.
The compounds showed good antimalarial activity with less
cytotoxicity. The results obtained suggest that compounds
O
R
N
R
HO
NCl
H
O
R
R
1
O
OH
O
R
1
NCl
O
O
R
1
R
ab
c
d
+
1-2 3-4
5a-j
6a-j
7a-j
NCl
H
O
R
3-4
O
OH
O
R
1
5a-j
Scheme 1 Reagents and conditions: (a)NH
2
OHHCl, NaOAc,
ethanol, water, reflux, 12 h. (b) DMF, POCl
3
, 0–5 °C then 12 h at
60 °C. (c) Succinic anhydride, AlCl
3
, rt. (d) NaOAc, Ac
2
O, 80 °C. In
this scheme R =Cl or OCH
3
,R
1
=H, CH
3
,CH
3
CH
2
, Cl, Br, 2,4-di
CH
3
, 3,4-di Cl, Diphenyl, Isobutyl, OCH
3
Fig. 2 Features of the synthesized compounds for interactions within
the active site of falcipain-2: AroC HAc HAc HAc AlaC (Avg.
RMSD: 0.142329)
Med Chem Res (2015) 24:879–890 883
123
7d,7f, and 7g with activity in low micromolar range could
be used as lead compound for development of more potent
antimalarial agents. Further these agents can be refined as
selective falcipain-2 inhibitors as supported by molecular
modeling studies.
Experimental section
Chemicals and instrumentation
1
H NMR and
13
CNMR spectra were obtained using a
Bruker AMX 400 and 100 MHz, respectively, with TMS as
internal standard. The IR spectrum was obtained using
spectrophotometer FT-IR (Shimadzu). The Mass spectra
(MS) were recorded on AMD-604 Mass spectrometer
operating at 70 eV. Melting points were uncorrected and
measured by open end capillary method on electrothermal
1A 9100 digital melting point apparatus. Reagents were
procured from Aldrich Chemical Company, MI, USA, SD-
fine chemical, and used as received. All anhydrous reac-
tions were carried out under nitrogen atmosphere. The
reactions were monitored by thin-layer chromatography
(TLC), using TLC sheets coated with silica gel 60 F
254
(Merck) and UV light visualization or iodine exposure. The
products were purified by flash chromatography on Com-
biflash on Merck silica gel 230–400 or recrystallization.
General procedure for the synthesis of 3-((2-
chloroquinolin-3-yl) methylene)-5-phenylfuran-2(3H)-
one derivatives (6a–jand 7a–j)
To a mixture of equimolar quantities of the corresponding
b-aroylacrylic acids (5a–j), 2-chloroquinoline-3-carbaldehyde
Fig. 3 Binding pose of ligands. aReference ligands binding pose
obtained from PBD, bbinding pose of compound 6i (H-Bond in
yellow color and ligand in green color), cBinding pose of E64
(reference compound). The figures have been generated with pdbsum
http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html
(Color figure online)
884 Med Chem Res (2015) 24:879–890
123
(3or 4), and anhydrous sodium acetate, acetic anhydride
(2 mL) was added dropwise. The mixture was heated to
80 °C and reaction progress was monitored by TLC. After
completion of the reaction, the reaction mixture was neu-
tralized with saturated sodium bicarbonate solution (10 %).
The organic layer was separated, dried over anhydrous
sodium sulfate, and concentrated under reduced pressure.
The crude product was purified by column chromatography
using silica gel as stationary phase and pet ether: chloroform
as mobile phase. The structure of all the synthesized com-
pounds was confirmed by spectral analysis. The intermedi-
ates, b-aroylacrylic acids (5a–j), required for the synthesis
were prepared through reported procedures (Oddy, 1923;
Papa et al., 1948). 2-Chloroquinolin-3-carbaldehyde deriv-
atives were obtained by reported procedure (Cohn et al.,
1981).
5-(4-Chlorophenyl)-3-((2-chloroquinolin-3-yl)
methylene)furan-2(3H)-one (6a)
Light yellow solid, yield 37 %, mp 240–242 °C. IRm
max
(KBr, cm
-1
): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-
C1 str.);
1
HNMR (CDCl
3
, 400 MHz) d8.33 (s, 1H), 7.99
(d, 1H, J=9.2 Hz), 7.93 (s, 1H), 7.80 (d, 2H,
J=3.2 Hz), 7.74 (s, 1H), 7.73 (m, 1H), 7.49(s, 1H),7.48
(d, 2H, J=3.2 Hz), 6.81(1H, s, furanone ring);
13
CNMR
(100 MHz, CDCl-
3
)d/ppm 167.2(1C, C-2), 149.6(1C, C00-
2), 146.8(1C, C00-9), 146.2(1C, C-5), 139.3(1C, =CH–),
136.2(1C, C-3), 136(1C, C00-4), 133.2(1C, C0-4), 131.6(1C,
C00-7), 130.6(1C, C00-3), 128.6(2C, C0-3,5), 128.5(1C, C0-1),
128.1(1C, C00-5), 127.5(2C, C0-2,6), 127.3(2C, C00-6,8),
126.6(1C, C00-10), 99(1C, C-4),; MS: m/e: 368(M?,
100 %); Anal. Calcd. for C
20
H
11
C
l2
NO
2
: C, 65.24; H, 3.01;
N, 3.80: Found: C, 65.14; H, 3.11; N, 3.58
3-((2,6-Dichloroquinolin-3-yl)methylene)-5-p-tolylfuran-
2(3H)-one (6b)
Cream colored solid, yield 36 %, mp 272–274 °C. IRm
max
(KBr, cm
-1
): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-
C1 str.);
1
HNMR (CDCl
3
, 400 MHz) d8.35 (s, 1H), 7.97
(d, 2H, J=8.4 Hz), 7.90 (s, 1H), 7.72–7.75 (m, 3H), 7.29
(d, 2H, J=8.4 Hz), 6.72(1H, s, furanone ring) 2.36 (s,
3H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 167.4(1C, C-2),
148.5(1C, C-5), 149.6(1C, C00-2), 145.3(1C, C00-9),
139.6(1C, =CH–), 137.2(1C, C0-4), 134.6(1C, C-3),
134(1C, C00-4), 132.6(1C, C00-7), 130.6(1C, C00-3), 129(2C,
C0-3,5), 128.9(1C, C00-5), 127.6(1C, C0-1), 127.5(1C, C00-
10), 127.2(1C, C00-8), 126.5(1C, C00-6), 126.1(2C, C0-2,6),
99.2(1C, C-4), 24.3(1C, CH
3
); MS: m/e: 382(M?, 100 %);
Anal. Calcd. for C
21
H
13
Cl
2
NO
2
: C, 65.99; H, 3.43; N, 3.66:
Found: C, 65.84; H, 3.39; N, 3.52
3-((2,6-Dichloroquinolin-3-yl)methylene)-5-(4-
ethylphenyl)furan-2(3H)-one (6c)
Light brown solid, yield 24 %, mp 273–275 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.33 (s, 1H), 7.99
(d, 2H, J=9.2 Hz), 7.92 (s, 1H), 7.70–7.74 (m, 3H), 7.31
(d, 2H, J=9.2 Hz), 6.76 (1H, s, Furanone ring), 2.72 (q,
2H, J=7.6 Hz), 1.28 (t, 3H, J=7.6 Hz);
13
CNMR
(100 MHz, CDCl-
3
)d/ppm 167.72(1C, C-2), 158.58(1C,
C00-2), 150.11(1C, C00-9), 147.5(1C, C-5), 147.59(1C, C00-
4), 144.96(1C, C00-6), 137.02(1C, =CH–), 132.6(1C, C-3),
Fig. 4 The predicted binding orientations of 7d (green) within the
active site of falcipain-2 (Color figure online)
Fig. 5 The predicted binding orientations of 7f (green) within the
active site of falcipain-2 (Color figure online)
Med Chem Res (2015) 24:879–890 885
123
131.83(1C, C00-3), 129.29(2C, C00-7,8), 128.73(2C, C00-
5,10), 128.03(1C, C0-6), 127.39(1C, C0-4), 126.57(2C, C0-
3,5), 125.46(1C, C0-2), 124.62(1C, C0-1), 98.62(1C, C-4),
28.36(1C, CH
3
–CH
2
–), 14.61(1C, CH
3
–CH
2
–); MS: m/e:
396 (M?, 100 %). Anal. Calcd. for C
22
H
15
Cl
2
NO
2
:C,
66.68; H, 3.82; N, 3.53: Found: C, 66.64; H, 3.76; N, 3.37
5-(4-Chlorophenyl)-3-((2,6-dichloroquinolin-3-
yl)methylene)furan-2(3H)-one (6d)
Yellowish solid, yield 20 %, mp 280–282 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.31 (s, 1H), 8.00
(d, 1H, J=8.8 Hz), 7.92 (s, 1H), 7.72–7.76 (m, 4H),
7.45–7.47 (m, 2H), 6.79 (1H, s, furanone ring);
13
CNMR
(100 MHz, CDCl-
3
)d/ppm 167.45(1C, C-2), 149.1(1C,
C-5), 148.96(1C, C00-2), 145.11(1C, C00-9), 138.02(1C,
=CH–), 135.6(1C, C-3), 133.5(1C, C0-4), 132.83(1C, C00-4),
132.3(2C, C00-6,7), 129.29(1C, C00-3), 128.87(2C, C0-3,5),
128.6(1C, C0-1), 128.58(1C, C00-8), 127.59(1C, C00-10),
126.57(2C, C0-2,6), 125.1(1C, C00-5), 99.2(1C, C-4);
MS:m/e: 402(M?, 100 %). Anal. Calcd. for
C
20
H
10
Cl
3
NO
2
: C, 59.66; H, 2.50; N, 3.48; Found: C,
59.60; H, 2.46; N, 3.35
5-(4-Bromophenyl)-3-((2,6-dichloroquinolin-3-
yl)methylene)furan-2(3H)-one (6e)
Yellowish solid, yield 15 %, mp 288–289 °C. IRm
max
(KBr): 1,690 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.34 (s, 1H), 7.89
(d, 1H, J=9.2 Hz), 7.80 (s, 1H), 7.77 (d, 2H,
J=3.2 Hz), 7.72 (s, 1H), 7.49 (s, 1H), 7.48 (d, 2H,
J=3.2 Hz), 6.60 (1H, s, furanone ring);
13
CNMR
(100 MHz, CDCl-
3
)d/ppm 167.2(1C, C-2), 149.96(1C, C00-
2), 146.5(1C, C-5), 145.11(1C, C00-9), 139.02(1C, =CH–),
136.6(1C, C-3), 133.83(1C, C00-4), 132.8(2C, C00-6,7),
129.87(2C, C0-3,5), 129.6(1C, C0-1), 129.58(1C, C00-8),
129.29(1C, C00-3), 128.57(2C, C0-2,6), 127.59(1C, C00-10),
125.8(1C, C00-5), 122.5(1C, C0-4), 98.9(1C, C-4),; MS: m/e:
447(M?, 100 %). Anal. Calcd. for C
20
H
10
BrCl
2
NO
2
:C,
53.73; H, 2.25; N, 3.13; Found: C, 53.65; H, 2.18; N, 2.96
3-((2,6-Dichloroquinolin-3-yl)methylene)-5-(2,4-
dimethylphenyl)furan-2(3H)-one (6f)
Brownish solid, yield 30 %, mp 215–218 °C. IRm
max
(KBr): 1,680 (CO str.), 1,476 (Ar–C=C-str.), 797 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.32 (s, 1H), 7.92
(d, 1H, J=9.2 Hz), 7.88 (s, 1H), 7.72 (m, 1H), 7.48 (s,
1H), 7.29 (d, 2H),7.11 (s, 1H), 6.82 (1H, s, furanone ring),
2.37 (s, 6H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm
167.4(1C, C-2), 149.86(1C, C00-2), 146.8(1C, C-5),
145.3(1C, C00-9), 139.6(1C, =CH–), 137.5(1C, C0-4),
136.3(1C, C-3), 135.57(1C, C0-2), 133.83(1C, C00-4),
133.8(2C, C00-6,7), 130.58(1C, C00-8), 129.39(1C, C0-3),
129.29(1C, C00-3), 127.69(1C, C00-10), 126.46(2C, C0-5,6),
125.6(1C, C0-1), 125.5(1C, C00-5), 99.1(1C, C-4), 24.3(1C,
4-CH
3
), 18.5(1C, 2-CH
3
); MS: m/e: 396(M?, 100 %).
Anal. Calcd. for C
22
H
15
Cl
2
NO
2
: C, 66.68; H, 3.82; N, 3.53;
Found: C, 66.60; H, 3.76; N, 3.40
5-(3,4-Dichlorophenyl)-3-((2,6-dichloroquinolin-3-
yl)methylene)furan-2(3H)-one (6g)
Pinkish solid, yield 20 %, mp 260–261 °C. IRm
max
(KBr):
1,688 (CO str.), 1,475 (Ar–C=C-str.), 795 (C-C1 str.)
cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.55 (s, 1H), 7.98(d,
1H), 7.92 (s, 1H), 7.76 (m, 1H), 7.59 (d, 1H), 7.52 (s, 1H),
7.45–7.47 (m, 2H), 6.85 (1H, s, furanone ring);
13
CNMR
(100 MHz, CDCl-
3
)d/ppm 167.6(1C, C-2), 149.96(1C, C00-
2), 146.3(1C, C-5), 145.6(1C, C00-9), 139.3(1C, =CH–),
136.5(1C, C-3), 133.83(1C, C00-4), 133.5(1C, C0-3),
132.87(1C, C0-4), 132.8(2C, C00-6,7), 129.6(1C, C0-1),
129.58(1C, C00-8), 129.39(1C, C0-5), 129.29(1C, C00-3),
127.69(1C, C00-10), 127.57(1C, C0-2), 125.6(1C, C00-5),
125.46(1C, C0-6) 99.6(1C, C-4); MS: m/e: 437(M?,
100 %). Anal. Calcd. for C
20
H
9
Cl
4
NO
2
: C, 54.96; H, 2.08;
N, 3.20; Found: C, 54.80; H, 2.01; N, 2.98
5-(Biphenyl-4-yl)-3-[(2,6-dichloroquinolin-3-
yl)methylidene]furan-2(3H)-one (6h)
Yellowish solid, yield 28 %, mp 250–251 °C. IRm
max
(KBr): 1,685 (CO str.), 1,466 (Ar–C=C-str.), 798 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.35 (s, 1H), 7.99
(d, 1H, J=8.8 Hz), 7.94 (s, 1H), 7.86 (d, 2H,
J=8.0 Hz), 7.71–7.74(m, 4H), 7.64 (d, 2H, J=7.2 Hz),
7.48(t, 2H, J=8.0 Hz), 7.41(d, 1H, J=6.8 Hz) 6.85 (1H,
s, furanone ring);
13
CNMR (100 MHz, CDCl-
3
)d/ppm
167.4(1C, C-2), 149.96(1C, C00-2), 146.2(1C, C-5),
145.6(1C, C00-9), 139.5(1C, =CH–), 136.4(1C, C-3),
135.69(1C, C00-4), 135.39(1C, C0-4), 132.4(1C, C00-7),
132.3(1C, C00-6), 131.83(1C, C00-3), 129.8(1C, C00-8),
129.3(1C, C0-1), 127.8(2C, C0-3,5), 127.58(1C, C00-10),
126.9(2C, C0-2,6), 125.6(1C, C00-5), 99.3(1C, C-4); MS:
m/e: 444 (M?, 100 %). Anal. Calcd. for C
26
H
15
Cl
2
NO
2
:C,
70.28; H, 3.40; N, 3.15; Found: C, 70.17; H, 3.34; N, 3.08.
3-((2,6-Dichloroquinolin-3-yl)methylene)-5-(4-
isobutylphenyl)furan-2(3H)-one (6i)
Pale yellow solid, yield 36 %, mp 165–166 °C. IRm
max
(KBr): 1,675 (CO str.), 1,475 (Ar–C=C-str.), 790 (C-C1
886 Med Chem Res (2015) 24:879–890
123
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.32 (s, 1H), 7.98
(m, 1H), 7.68 (d, 2H, J=3.2 Hz), 7.74 (s, 1H), 7.73 (m,
1H), 7.39 (s, 1H),7.28 (d, 2H, J=3.2 Hz), 6.78(1H, s,
furanone ring), 2.54(d, 2H), 2.30 (m, 1H), 1.12(d, 6H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 167.6(1C, C-2),
149.86(1C, C00-2), 146.7(1C, C-5), 145.3(1C, C00-9),
139.57(1C, C0-4), 139.4(1C, =CH–), 136.6(1C, C-3),
133.8(2C, C00-6,7), 130.58(1C, C00-8), 129.39(1C, C00-3),
128.5(2C, C00-3,5), 127.69(1C, C00-10), 127.6(1C, C0-1),
125.5(1C, C00-5), 125.46(2C, C00-2,6), 99.6(1C, C-4),
45.7(1C, CH
3
–CH
3
–CH–CH
2
), 29.3(1C, CH
3
–CH
3
–CH–
CH
2
), 22.8(2C, CH
3
–CH
3
–CH–CH
2
); MS: m/e: 424(M?,
100 %). Anal. Calcd. for C
24
H
19
Cl
2
NO
2
: C, 67.93; H, 4.51;
N, 3.30; Found: C, 67.84; H, 4.40; N, 3.13.
3-((2,6-Dichloroquinolin-3-yl)methylene)-5-(4-
methoxyphenyl)furan-2(3H)-one (6j)
Pale yellow color solid, yield 33 %, mp 160–162 °C.
IRm
max
(KBr): 1,680 (CO str.), 1,477 (Ar–C=C-str.), 795
(C-C1 str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.62(s,
1H), 7.99(d, 1H), 7.93 (s, 1H), 7.82(d, 2H, J=3.2 Hz),
7.72(s, 1H), 7.73(m, 1H), 7.49(s, 1H),7.51 (d, 2H,
J=3.2 Hz), 6.88(1H, s, furanone ring), 3.82 (s, 1H,
OCH
3
);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 166.7(1C,
C-2), 159.57(1C, C0-4), 149.86(1C, C00-2), 146.4(1C, C-5),
145.3(1C, C00-9), 139.2(1C, =CH–), 136.3(1C, C-3),
133.8(2C, C00-6,7), 130.58(1C, C00-8), 129.39(1C, C00-3),
127.69(1C, C00-10), 127.46(2C, C0-2,6), 125.5(1C, C00-5),
122.6(1C, C0-1), 114.5(2C, C0-3,5), 99.1(1C, C-4),
55.7(1C, O–CH
3
); MS: m/e: 398 (M?, 100 %). Anal.
Calcd. for C
21
H
13
Cl
2
NO
3
: C, 63.34; H, 3.29; N, 3.52;
Found: C, 63.21; H, 3.18; N, 3.32.
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-
phenylfuran-2(3H)-one (7a)
Yellowish solid, yield 38 %, mp 245–246 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.30 (s, 1H), 7.93
(d, 1H, J=8.8 Hz), 7.79–7.76 (m, 3H), 7.47–7.47 (m,
4H), 7.17 (s, 1H), 6.84 (1H, s, furanone ring), 3.98 (s, 3H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 166.9(1C, C-2),
157.8(1C, C00-6), 147.86(1C, C00-2), 146.2(1C, C-5),
142.3(1C, C00-9), 139.5(1C, =CH–), 136.6(1C, C-3),
134.39(1C, C00-4), 131.57(1C, C00-3), 130.6(1C, C0-1),
129.58(1C, C00-8), 128(3C, 3,4,5), 127.69(1C, C00-10),
126.46(2C, C0-2,6), 123.8(1C, C00-7), 105.5(1C, C00-5),
99.2(1C, C-4), 55.9(1C, OCH
3
); MS: m/e: 364(M?,
100 %); Anal. Calcd. for C
21
H
14
ClNO
3
: C, 69.33; H, 3.88;
N, 3.85; Found: C, 69.26; H, 3.81; N, 3.72;
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-p-
tolylfuran-2(3H)-one (7b)
Orange colored solid, yield 39 %, mp 280–281 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.30 (s, 1H), 7.91
(d, 1H, J=8 Hz), 7.75 (d, 2H, J=7.6 Hz), 7.69 (s, 1H),
7.44 (d, 1H, J=8.8 Hz), 7.32 (d, 2H, J=7.6 Hz), 7.16 (s,
1H), 6.81 (1H, s, furanone ring), 3.94 (s, 3H), 2.36 (s, 3H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 167.2(1C, C-2),
157.8(1C, C00-6), 147.2(1C, C00-2), 146.3(1C, C-5),
142.7(1C, C00-9), 139.6(1C, =CH–), 137(1C, C0-4),
136.4(1C, C-3), 134.39(1C, C00-4), 131.57(1C, C00-3),
129.58(1C, C00-8), 129.5(2C, C0-3,5), 127.69(1C, C00-10),
127.6(1C, C0-1), 126.46(2C, C0-2,6), 123.8(2C, C00-7,8),
105.5(1C, C00-5), 99.2(1C, C-4), 24.3(1C, CH
3
); MS: m/e:
378 (M?, 100 %). Anal. Calcd. for C
22
H
16
ClNO
3
:C,
69.94; H, 4.27; N, 3.71; Found: C, 69.86; H, 4.15; N, 3.51.
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-(4-
ethylphenyl)furan-2(3H)-one (7c)
Yellowish solid, yield 30 %, mp 275–276 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.30 (s, 1H), 7.94
(d, 1H, J=8.8 Hz), 7.73 (d, 2H, J=7.6 Hz), 7.7 (s, 1H),
7.44 (d, 1H, J=8.8 Hz), 7.30 (d, 2H, J=7.6 Hz), 7.17 (s,
1H), 6.79 (1H, s, furanone ring), 3.98(s, 3H), 2.72 (q, 2H,
J=7.6 Hz), 1.28(t, 3H, J=7.6 Hz);
13
CNMR
(100 MHz, CDCl-
3
)d/ppm 167.71(1C, C-2), 157.8(1C, C00-
6), 146.3(1C, C-5), 142.96(1C, =CH–), 142.7(1C, C00-9),
138.39(1C, C00-2), 136(1C, C-3), 129.58(2C, C00-5,7),
129.2(1C, C00-8), 128.57(1C, C00-3), 128.05(1C, C0-4),
127.5(1C, C0-5), 125.5(1C, C0-3), 124.46(1C, C0-6),
123.46(1C, C0-2), 104.89(1C, C0-1), 97.6(1C, C-4),
55.26(1C, OCH
3
), 28.4(1C, CH
3
–CH
2
–), 14.7(1C, CH
3
–
CH
2
–); MS: m/e: 392(M?, 100 %); Anal. Calcd. for
C
23
H
18
ClNO
3
: C, 70.50; H, 4.63; N, 3.57; Found: C, 70.38;
H, 4.54; N, 3.40.
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-(4-
chlorophenyl)furan-2(3H)-one (7d)
Brown solid, yield 23 %, mp 274–275 °C. IRm
max
(KBr):
1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1 str.)
cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.29 (s, 1H), 7.96 (d,
2H, J=8.8 Hz), 7.93 (s, 1H), 7.72 (d, 2H, J=8.0 Hz),
7.73 (s, 1H), 7.45 (d, 2H, J=8.0 Hz), 6.83 (1H, s, fura-
none ring), 3.97(s, 3H);
13
CNMR (100 MHz, CDCl-
3
)
d/ppm 167.9(1C, C-2), 149.86(1C, C00-2), 147.4(1C, C-5),
145.3(1C, C00-9), 138.2(1C, =CH–), 136.57(1C, C0-4),
135.3(1C, C-3), 133.8(1C, C00-6), 130.58(2C, C00-7,8),
Med Chem Res (2015) 24:879–890 887
123
129.39(1C, C00-3), 128.6(1C, C0-1), 128.5(2C, C0-3,5),
127.69(1C, C00-10), 127.46(2C, C0-2,6), 125.5(1C, C00-5)
98.1(1C, C-4), 56.9(1C, O–CH
3
); MS: m/e: 398(M?,
100 %). Anal. Calcd. for C
21
H
13
Cl
2
NO
3
: C, 63.34; H, 3.29;
N, 3.52; Found: C, 63.23; H, 3.15; N, 3.37.
5-(4-Bromophenyl)-3-((2-chloro-6-methoxyquinolin-3-
yl)methylene)furan-2(3H)-one (7e)
Light brown solid, yield 25 %, mp 264–266 °C. IRm
max
(KBr): 1,690 (CO str.), 1,477 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.25 (s, 1H), 7.93
(d, 1H, J=8.8 Hz), 7.87 (d, 2H, J=7.6 Hz), 7.75 (s,
1H), 7.61 (d, 1H, J=8.8 Hz), 7.38 (d, 2H, J=7.6 Hz),
7.15 (s, 1H), 6.67 (1H, s, furanone ring), 3.93 (s, 3H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 168.3(1C, C-2),
157.3(1C, C00-6),149.86(1C, C00-2), 148.4(1C, C-5),
141.8(1C, C00-9), 139.9(1C, =CH–), 137.3(1C, C-3),
133.8(1C, C00-7), 131.5(2C, C0-3,5), 130.58(1C, C00-8),
129.39(1C, C00-3), 128.6(1C, C0-1), 127.69(1C, C00-10),
127.46(2C, C0-2,6), 125.5(1C, C00-5), 122.57(1C, C0-4),
97.1(1C, C-4), 57.9(1C, O–CH
3
); MS: m/e: 443 (M?,
100 %). Anal. Calcd. for C
21
H
13
BrClNO
3
: C, 56.98; H,
2.96; N, 3.16; Found: C, 56.87; H, 2.74; N, 3.02.
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-(2,4-
dimethylphenyl)furan-2(3H)-one (7f)
Cream colored solid, yield 22 %, mp 250–252 °C. IRm
max
(KBr): 1,681 (CO str.), 1,476 (Ar–C=C-str.), 795 (C-C1 str.)
cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.33 (s, 1H), 7.94 (d,
1H, J=8.8 Hz), 7.90 (s, 1H), 7.78–7.76(m, 1H), 7.65(s,
1H), 7.54(d, 2H), 7.21(s, 1H) 6.69 (1H, s, furanone ring),
3.94 (s, 3H), 2.37 (s, 6H);
13
CNMR (100 MHz, CDCl-
3
)
d/ppm 168.6(1C, C-2), 149.86(1C, C00-2), 145.3(1C, C00-9),
144.4(1C, C-5), 141.2(1C, =CH–), 137.46(1C, C0-2),
137.3(1C, C-3), 136.57(1C, C0-4), 133.8(1C, C00-6),
130.58(2C, C00-7,8), 130.5(1C, C0-3), 129.39(1C, C00-3),
128.5(1C, C0-5), 127.69(1C, C0010), 126.46(1C, C0-6),
125.6(1C, C0-1), 125.5(1C, C00-5), 97.6(1C, C-4), 56.9(1C,
O–CH
3
), 24.6(1C, 4-CH
3
),18.5(1C, 2-CH
3
); MS: m/e:
392(M?, 100 %). Anal. Calcd. for C
23
H
18
ClNO
3
: C, 70.50;
H, 4.63; N, 3.57; Found: C, 70.41; H, 4.55; N, 3.41.
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-(3,4-
dichlorophenyl)furan-2(3H)-one (7g)
Light yellow solid, yield 35 %, mp 265–266 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.29 (s, 1H), 7.95
(d, 1H, J=9.2 Hz), 7.86 (d, 2H, J=13.6 Hz), 7.62 (d,
1H, J=8.2 Hz), 7.55 (d, 1H, J=8.2 Hz), 7.46 (d, 1H,
J=9.6 Hz), 7.18 (s, 1H), 6.85 (1H, s, furanone ring), 3.99
(s, 3H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 167.6(1C,
C-2), 149.86(1C, C00-2), 145.3(1C, C00-9), 143.4(1C, C-5),
143.2(1C, =CH–), 139.3(1C, C-3), 136.57(1C, C0-4),
133.8(1C, C00-6,7), 133.5(1C, C0-5), 130.58(1C, C00-8),
130.5(1C, C0-3), 129.6(1C, C0-1), 129.39(1C, C00-3),
127.69(1C, C00-10), 127.46(1C, C0-6), 126.46(1C, C0-2),
125.5(1C, C00-5), 98.6(1C, C-4), 57.9(1C, O–CH
3
); MS:
m/e: 432 (M?, 100 %). Anal. Calcd. for C
21
H
12
Cl
3
NO
3
:C,
58.29; H, 2.80; N, 3.24; Found: C, 58.18; H, 2.68; N, 3.10.
5-(Biphenyl-4-yl)-3-[(2-chloro-6-methoxyquinolin-3-
yl)methylidene]furan-2(3H)-one (7h)
Pale yellow solid, yield 21 %, mp 216–218 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.30(s, 1H),
7.97(d, 1H, J=8.8 Hz), 7.92(s, 1H), 7.89(d, 2H,
J=8.4 Hz), 7.77–7.79(m, 4H), 7.62(d, 2H, J=7.2 Hz),
7.52(t, 2H, J=8.0 Hz), 7.44(d, 1H, J=6.8 Hz) 6.83(1H,
s, furanone ring);
13
CNMR (100 MHz, CDCl-
3
)d/ppm
166.8(1C, C-2), 158.3(1C, C00-9), 151.86(1C, C00-2),
146.4(1C, C-5), 146.2(1C, =CH–), 139.7(1C, C-3),
136.6(1C, C0-1), 133.8(1C, C00-6), 130.58(2C, C00-7,8),
129.5(2C, C0-3,5), 129.39(1C, C00-3), 127.69(1C, C00-10),
127.57(1C, C0-4), 127.46(2C, C0-2,6), 125.5(1C, C00-5),
99.8(1C, C-4), 56.9(1C, O–CH
3
); MS: m/e: 440(M?,
100 %). Anal. Calcd. for C
27
H
18
ClNO
3
: C, 73.72; H, 4.12;
N, 3.18; Found: C, 73.62; H, 3.98; N, 3.05.
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-(4-
isobutylphenyl)furan-2(3H)-one (7i)
Light yellow solid, yield 29 %, mp 170–172 °C. IRm
max
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm
-1
;
1
HNMR (CDCl
3
, 400 MHz) d8.39 (s, 1H),
7.98–7.99 (m, 1H), 7.88 (d, 2H, J=3.2 Hz), 7.82(s, 1H),
7.75(s, 1H), 7.73 (m, 1H), 7.35 (d, 2H, J=3.2 Hz),
6.81(1H, s, furanone ring), 2.55(d, 2H), 2.29–2.30(m, 1H),
1.11(d, 6H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm
168.6(1C, C-2), 149.86(1C, C00-2), 145.3(1C, C00-9),
147.7(1C, C-5), 139.9(1C, =CH–), 139.57(1C, C0-4),
136.5(1C, C-3), 133.8(1C, C00-6), 130.58(2C, C00-7,8),
129.39(1C, C00-3), 128.5(2C, C0-3,5), 127.69(1C, C00-10),
127.6(1C, C0-1), 125.5(1C, C00-5), 125.46(2C, C0-2,6),
99.2(1C, C-4), 55.9(1C, O–CH
3
), 46.7(1C, CH
3
–CH
3
–CH–
CH
2
), 31.3(1C, CH
3
–CH
3
–CH–CH
2
), 21.8(2C, CH
3
–CH
3
–
CH–CH
2
); MS: m/e: 420(M?, 100 %). Anal. Calcd. for
C
25
H
22
ClNO
3
: C, 71.51; H, 5.28; N, 3.34; Found: C, 71.43;
H, 5.13; N, 3.16.
888 Med Chem Res (2015) 24:879–890
123
3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)-5-(4-
methoxyphenyl)furan-2(3H)-one (7j)
Brownish solid, yield 30 %, mp 230–232 °C. IRmmax
(KBr): 1,680 (CO str.), 1,470 (Ar–C=C-str.), 795 (C-C1
str.) cm-1; 1HNMR (CDCl3, 400 MHz) d8.32 (s, 1H),
7.93 (d, 1H, J=8 Hz), 7.74 (d, 2H, J=7.6 Hz), 7.69 (s,
1H), 7.45 (d, 1H, J=8.8 Hz), 7.31 (d, 2H, J=7.6 Hz),
7.17 (s, 1H), 6.83 (1H, s, furanone ring), 3.86 (s, 3H), 3.85
(s, 3H);
13
CNMR (100 MHz, CDCl-
3
)d/ppm 167.9(1C,
C-2), 159.57(1C, C0-4), 158.3(1C, C00-9), 149.86(1C, C00-2),
148.7(1C, C-5), 142.9(1C, =CH–), 139.5(1C, C-3),
133.8(1C, C00-6), 130.58(2C, C00-7,8), 129.39(1C, C00-3),
127.69(1C, C00-10), 127.46(2C, C0-2,6), 125.5(1C, C00-5),
122.6(1C, C0-1), 114.5(2C, C00-3,5), 99.5(1C, C-4),
55.9(1C, O–CH
3
); MS: m/e: 393(M?, 100 %). Anal.
Calcd. for C
22
H
16
ClNO
4
: C, 67.10; H, 4.10; N, 3.56;
Found: C, 67.03; H, 3.94; N, 3.38.
Antimalarial activity
The antimalarial activities of the synthesized compounds
were tested against gametocyte-producing P. falciparum
culture lines (FDL-HD), using a previously reported
method (Dua et al., 2002). Briefly, the P. falciparum cul-
ture lines (FDL-HD) were maintained in vitro in O
?ve
RBC
at 10 % hematocrit in AB
?ve
serum in RPMI 1640 medium
supplemented with D-glucose and L-glutamine. The cul-
tures were treated with selected concentrations of different
compounds incubated in CO
2
at 37 °C for 72 h. After
incubation, blood smears were prepared and stained with
Giemsa stain. Percentage inhibition for gametocytes and
asexual stages was calculated by comparing growth in
control sets. Chloroquine was used as a standard reference.
Falcipain-2 (FP2) enzyme assay
Compounds (7d,7f, and 7g) were tested for their binding to
falcipain-2, using the method reported by Mane et al.
(2012). Briefly, the enzyme was incubated for 10 min at
room temperature in 100 mM sodium acetate, pH 5.5,
10 mM DTT, with a fixed set of different concentrations of
the compounds to be tested or the standard (E64). The
substrate Z-Phe-Arg-AMC was added after incubation, and
the fluorescence intensity was monitored (excitation at
355 nm; emission at 460 nm) for 10 min at room temper-
ature with a Microplate Reader (BioTek). The inhibition
rate (%) was calculated using the following equation:
%Inhibition ¼1Ftest=FcontrolðÞ½100;
where Ftest is the fluorescence intensity of the test com-
pound, and Fcontrol is the fluorescence intensity of the
standard (E64). The results are shown in Table 2. All
values are the means of three independent determinations.
Cytotoxicity studies
Compounds (7d,7f and 7g) were tested for cytotoxicity
(IC
50
) in VERO cells at concentrations of 100 lg/mL.
After 72 h exposure, viability was assessed on the basis of
cellular conversion of MTT into a formazan product using
the Promega Cell Titer 96 non-radioactive cell proliferation
method.
Molecular docking
To identify the potential target of this novel inhibitor type,
a systemic search of antimalarial targets was carried out
using the BAITOC server provided by the Supercomputing
Facility of Bioinformatics & Computational Biology
(SCFBio), IIT Delhi, India. BAITOC is a useful online tool
for predicting probable binding receptors for new mole-
cules. The application screens thousands of protein struc-
tures against the input organic molecules in a time-efficient
manner and provides information on proteins (PDBID)
with high binding energy to the molecule under investi-
gation (http://www.scfbio-iitd.res.in/software/drugdesign/
baitocnew.jsp). After searching the possible targets (falci-
pain-2) using the BAITOC server, molecular docking
studies were performed on the PDB id 3BPF of falcipain-2
to determine potential binding modes of these molecules.
Molecular docking was performed using the Windows-
based Glide version of the Schrodinger-9 software (Glide,
2009). A model of the X-ray crystal structure of P. falci-
parum falcipain-2 co-crystallized with epoxysuccinate E64
(PDB code, 3BPF) was obtained from the Brookhaven
Protein Data Bank. Receptor and ligand preparations were
performed using the protein preparation wizard and the
ligprep module, respectively, by using the default options.
The co-crystallized ligands, water molecules, and back-
bone constraints were removed from the models during the
protein preparation step. The binding sites were identified
by analysis of the enzyme cavities, and a grid of 20 A
˚was
set for the docking calculations. The Glide XP module was
used for the final docking studies. The binding pose with
the receptor generated by Glide was analyzed using the
pdbsum site (http://www.ebi.ac.uk/thornton-srv/databases/
pdbsum/Generate.html).
Acknowledgments The authors wish to acknowledge the Univer-
sity Grants Commission, Govt. of India, New Delhi for providing
financial support (F. No. 36-110/2008 (SR)). We do not have any
competing financial interests with regard to this work. We also thank
DBT, Ministry of Science and Technology, Govt of India for pro-
viding Bioinformatics facility. The authors are thankful to NIMR,
Dwarka for helping us in carrying out the falcipain-2 activity.
Med Chem Res (2015) 24:879–890 889
123
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