Synthesis, antimalarial activity evaluation and docking studies of some novel tetraoxaquines

Article abstract of DOI:10.1007/s00044-016-1644-5

Tetraoxaquines, molecular hybrids of 1,2,4,5-tetraoxane and 4-aminoquinoline, were designed via molecular docking analysis against Falcipain-2. Among the studied compounds, 11 top scoring analogues showing low binding energy were further selected for synthesis and evaluated for their in vitro antimalarial activity. In inhibitory assay, five compounds showed significant activity against chloroquine-resistant strain of P. falciparum-RKL-9 with IC₅₀ values of 3.906, 3.942, 4.272, 3.906, 4.814 μg/ml.

Full text of DOI:10.1007/s00044-016-1644-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1644-5
ORIGINAL RESEARCH
Synthesis, antimalarial activity evaluation and docking studies of some
novel tetraoxaquines
Mukesh Kumar Kumawat1,2 Pratap Parida3 Dipak Chetia1
●
●
Received: 19 June 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract Tetraoxaquines, molecular hybrids of 1,2,4,5-
tetraoxane and 4-aminoquinoline, were designed via mole-
cular docking analysis against Falcipain-2. Among the
studied compounds, 11 top scoring analogues showing low
binding energy were further selected for synthesis and
evaluated for their in vitro antimalarial activity. In inhibi-
tory assay, five compounds showed significant activity
against chloroquine-resistant strain of P. falciparum-RKL-9
with IC50 values of 3.906, 3.942, 4.272, 3.906, 4.814 µg/ml.
falciparum, Plasmodium vivax, Plasmodium malariae,
Plasmodium ovale and Plasmodium knowlesi are the cau-
sative organisms of malaria. These parasites are transmitted
from an infected person to the healthy human by female
mosquitoes. Among them, infection due to P. falciparum
(Pf) accounts for majority of morbidity (i.e., 495 %) and
mortality cases.
Malaria is a curable disease if treated in the earlier stage
of infection. However, the resistant strains of malaria has
seriously jeopardized the clinical efficacy of currently used
drugs. This has made the urgent necessity for the discovery
and development of novel drugs that would be effective
against the resistant strains of malarial parasites (Kumawat
et al., 2011; Casteel, 1997).
●
●
Keywords Antimalarial activity Tetraoxaquine
●
●
P. falciparum Molecular docking studies Falcipain-2
Quinoline-based drugs such as chloroquine and its deri-
vatives are known to affect the parasite metabolism and
interfering with its survival by suppressing the polymeriza-
tion of toxic haem into an insoluble and non-toxic pigment,
hemozoin. It results in lysis of cell. Compounds belonging to
the family of 1,2,4,5-tetraoxane show excellent antimalarial
activity via reaction with haem (or free Fe (II)) to generate
cytotoxic radicals for its antimalarial activity (O’Neill et al.,
2010). Therefore, it has been postulated that the development
of hybrid conjugates comprising of quinoline and tetraoxane
(Tetraoxaquines) into single scaffold could result into new
class of antimalarials with considerable efficacy than the
molecules used alone. More recently, Opsenica et al. (2008)
synthesized such hybrid molecules which showed excellent
in vitro and in vivo activity against both chloroquine-
sensitive and chloroquine-resistant strains of Pf. This obser-
vation supports the rational for the design and synthesis of
tetraoxaquines as a new class of antimalarial drugs.
Introduction
Malaria is a global infectious disease responsible for major
public health problem in tropical and subtropical regions of
the world. It accounted for the death of nearly half million
people against the 207 million cases of infection in 2013
(WHO, 2013). The African subcontinent has the highest
burden of the disease, for instance, more than 75 % malaria-
related deaths are reported in pregnant women and children
under the age of five (www.rbm.who.int). Plasmodium
* Mukesh Kumar Kumawat
phmukesh@gmail.com
1
Department of Pharmaceutical Sciences, Dibrugarh University,
Dibrugarh 786004 Assam, India
2
Anand College of Pharmacy, Keetham, Agra 282006 Uttar
The present study was undertaken to design and syn-
thesize hybrid conjugates of 4-aminoquinoline and 1,2,4,5-
tetraoxane (Tetraoxaquines) via covalent linker (Scheme 1).
Pradesh, India
3
Regional Medical Research Centre, ICMR (NE Region), Post Bag
No. 105, Dibrugarh, Assam, India

Med Chem Res
These compounds were subsequently tested against
CQ-resistant strain (RKL-9) of Pf. Docking study was also
performed with these inhibitors against falcipain-2.
and were used without further purification. The TLC plates
coated with silica gel-G was utilized for monitoring the
completion of the reaction using various solvent combina-
tions. The spots were detected with iodine vapours and
observed under UV-light. The melting point of the inter-
mediates, as well as the target compounds were determined
by open capillary method using Veego-MPI melting point
apparatus and are uncorrected. The UV-Visible spectra of
the synthesized compounds were recorded on Shimadzu
UV-1800 UV-visible spectrophotometer. The Infrared
spectra were recorded on FTIR Perkin-Elmer spectrometer.
Materials & Methods
Chemistry
Structural investigation
1
The H and 13C NMR spectra were recorded at 400 MHz
All the Chemicals were procured either from Sigma-Aldrich
Corporation, USA or Merck Specialties Pvt. Ltd., Mumbai
and 100 MHz, respectively, on a Bruker Avance-II 400
Scheme 1 Reagents and condi-
tions: a Reflux for 8–10 h;
b Reflux for 8–10 h; c CH2Cl2/
CH3CN mixture (1:3 v/v),
Concn HCl, Stirring for 2 h at rt;
d CH3CN, CH2Cl2, Concn
H2SO4, stirring at 0–10 °C

Med Chem Res
NMR spectrometer using either DMSO-d6 or CDCl3 as
solvent with tetramethylsilane as an internal standard. Mass
spectra were obtained on a Waters Q-TOF MICROMASS
LC mass spectrometer. Elemental analysis (CHN & O) were
carried out on Eager Xperience Elemental analyzer (Coates,
2000; Pasto et al., 1992; Mathieson, 1965; Silverstein and
Webster, 1963).
separated out and the precipitate in the water layer was
collected by filtration to furnish the target compounds.
N-(1-aminopropan-2-yl)-7-chloroquinolin-4-amine (3a) Odour-
less yellow solid; soluble in ethanol, chloroform; melting
range 105–107 °C; %yield 87; Rf value 0.75 (acetone:
ethanol: 2:1); Spectroscopic analysis: λmax (in CHCl3)
255.06 nm; FTIR spectrum (νmax, in cm−1, Film) 3501.81,
3435.96 (sym. and asymm. N–H stretching, –NH2),
3222.98 (N–H stretching, 4NH), 3100.50–3061.27 (aro-
matic C–H stretching), 3016.10–2823.23 (C–H stretching,
4CH2 and –CH3), 2024.98–1534.00 (N–H bending),
1517.60–1425.72 (C=C–C, aromatic ring stretching),
1396.47–1132.19 (C–N stretching), 1238.63–960.92 (aro-
matic C–H in-plane bend), 1084.98 (C–Cl stretching,
Ar–Cl), 907.53–676.06 (aromatic C–H out-of-plane bend).
General Procedure
Synthesis of 4-substituted-7-chloroquinolines (3a–3b) A
mixture of 4,7-dichloroquinoline (1) (12.6 mmol) and dia-
mine, different in each case, (2a–2c) (56.8 mmol) was
heated slowly with stirring at elevated temperature (80 °C)
for 1 h. The mixture was then heated at 120–130 °C for 6–8
h with stirring to drive the reaction to completion. The
reaction mixture was cooled to room temperature and taken
up in dichloromethane with subsequent washing with 1 M
sodium hydroxide and brine solution to afford an aqueous
layer, an organic layer and a particulate precipitate. The
resulting precipitate was filtered-out and dried to obtain the
corresponding product.
N1-(7-chloroquinolin-4-yl)benzene-1,2-diamine (3b) Odour-
less blackish brown solid; soluble in dichloromethane;
melting range 150–152 °C; %yield 89; Rf value 0.51
(Acetone: Pet. ether: 1:2); Spectroscopic analysis: λmax (in
CHCl3) 240.51 nm; FTIR Spectrum (νmax, in cm−1, Film)
3565.24, 3461.94 (sym. & asymm. N–H stretching, –NH2),
3374.62 (N–H stretching, 4NH), 3034.39 (aromatic C–H
stretching), 2926.08 (aliphatic C–H stretching), 1915.54-
1567.15 (N–H bending), 1531.00-1421.82 (C=C-C, aro-
matic ring stretching), 1368.59-1277.77 (C-N stretching),
1237.15–913.02 (aromatic C–H in-plane bend), 1080.16
(C–Cl stretching, Ar–Cl), 875.15–592.35 (aromatic C-H
out-of-plane bend).
Synthesis of Intermediate 2-butanone derivatives (5a–5c)
A mixture of 4-substituted-7-chloroquinolines (3a–3c)
(12.6 mmol) and 3-chlorobutan-2-one (4) (56.8 mmol) in
dry acetone was heated slowly with stirring and temperature
was raised to 80 °C for 1 h, subsequently at 120–130 °C for
6–8 h. Then the reaction mixture was cooled to room tem-
perature and taken up in dichloromethane followed by
washing with 1 M sodium hydroxide and brine solution to
afford an aqueous layer, an organic layer, and a particulate
precipitate. The resulting precipitate was filtered-out and
dried to obtain the corresponding product.
7-chloro-4-piperazine-1-ylquinoline (3c) A mixture of
4,7-dichloroquinoline (1) (0.505 mol), piperazine (2c) (1.5
mol) and potassium iodide (0.12 mol) in isopropyl alcohol
(20 ml) was refluxed for 10 h, and cooled to room tem-
perature. The precipitated solid was filtered, washed with
isopropyl alcohol and evaporated in vacuo. The crude
product, thus obtained was diluted further with dichlor-
omethane (20 ml) and washed twice with water. The pH of
dichloromethane layer was adjusted using conc. HCl. The
separated water layer was made alkaline with 10 % NaOH
solution and extracted again with dichloromethane. The
organic layer was evaporated to dryness to get 7-chloro-4-
piperazine-1-ylquinoline. Odourless off-white solid; solu-
ble in acetone, dichloromethane; melting range 161–162 °C;
%Yield 50; Rf value 0.76 (dichloromethane: ethanol: 1:1);
Spectroscopic analysis: λmax (in DMSO) 321.52 nm; FTIR
Spectrum (νmax, in cm−1, film) 3250.01 (N–H stretching,
4NH piprazinyl), 3035.30 (aromatic C–H stretching, qui-
nolyl), 2925.41–2830.10 (C–H stretching, 4CH2 piprazi-
nyl), 1918.46–1496.85 (N–H bending, 4NH piprazinyl),
1452.82–1419.76 (C=C–C, aromatic ring stretching, qui-
nolyl), 1373.06–1281.11 (C–N stretching), 1250.50–922.34
(aromatic C–H in-plane bend), 1067.64 (C–Cl stretching,
Synthesis of dihydroproxides (8a–8e) Cyclic or alicyclic
ketone (6a–6e) (1 ml, 10 mmol) was taken in a mixture of
CH2Cl2/CH3CN (20 ml, 1:3 v/v) at room temperature fol-
lowed by addition of 30 % H2O2 (10.4 ml, 0.1 mol) and
concentrated HCl (3 ml). The resulting mixture was further
stirred for 2 h at room temperature and then quenched with
saturated NaHCO3 and CH2Cl2. The organic layer thus
obtained was separated out, and the water layer was filtered
to collect the precipitate and dried to obtain the desired
product
Synthesis of targeted tetraoxaquines (9a–9k) The
2-butanone derivative (5a–5c) (2.3 mmol) prepared was
added to a cooled solution (ice bath) of dihydroperoxide
(8a–8e) (2.3 mmol) in CH2Cl2. The resultant mixture was
stirred for 30 min followed by the dropwise addition of 20
ml of cooled H2SO4/CH3CN mixture (1:10 v/v). After an
additional 50 min of stirring, the mixture was quenched
with saturated NaHCO3 and CH2Cl2. The organic layer was

Med Chem Res
quinolyl–Cl), 867.05–618.50 (aromatic C–H out-of-plane
bend, quinolyl).
Spectrum (νmax, in cm−1, film) 3247.73-3061.40 (aromatic
C–H stretching, quinolyl), 2960.36-2858.89 (C–H stretching,
4CH2, alkyl and piprazinyl), 1730.72 (4C=O stretching),
1608.58–1424.47 (C=C–C, aromatic ring stretching,
quinolyl), 1367.37–908.15 (aromatic C–H in-plane bend,
quinolyl), 1015.32 (C–Cl stretching, quinolyl–Cl),
873.44–619.00 (aromatic C–H out-of-plane bend, quinolyl).
3-(2-(7-chloroquinolin-4-ylamino)propylamino)butan-2-
one (5a) Reddish brown solid with characteristic odour;
soluble in dichloromethane, chloroform; melting range
251–252 °C; %Yield 95.78; Rf value 0.72 (acetone: carbon
tetrachloride: 1:2); Spectroscopic analysis: λmax (in CHCl3)
252.53 nm; FTIR Spectrum (νmax, in cm−1, film) 3565.11,
3219.34 (N–H stretching, 4NH), 3063.37 (aromatic
C–H stretching), 2923.94–2856.30 (C–H stretching,
4CH2 and –CH3), 1742.19–1645.48 (C=O stretching),
1918.13–1532.77 (N–H bending), 1518.54–1425.51
(C=C–C, aromatic ring stretching), 1368.29–1140.06 (C–N
stretching), 1238.63–960.92 (aromatic C–H in-plane bend),
1085.31 (C–Cl stretching, Ar–Cl), 845.69–602.69 (aromatic
1,1-dihydroperoxycyclohexane (8a) White solid char-
acteristic odour; soluble in Pet. Ether; melting range 63 °C;
%Yield 30.8; Rf value 0.61 (Ethanol: acetone: 1:1);
Spectroscopic analysis: λmax (in DMSO) 275 nm; FTIR
Spectrum (νmax, in cm−1, film) 3419.09–3387.32 (–OH
stretching, dihydroperoxide), 2938.52–2855.97 (C–H
stretching, cyclohexyl), 1551.89 (–OH bending,
dihydroperoxide), 1447.21–1342.74 (C–H bending, cyclo-
hexyl), 1272.68–1158.23 (C–C–O symmetrical stretching),
1
C–H out-of-plane bend); H NMR (400 MHz, CHCl3) δ
1.01–1.03 (d, 3H, J = 8Hz, –CH3), 1.05–1.07 (d, 3H, J =
8Hz, –CH3), 2.50 (s, 1H, 4NH), 3.58 (s, 3H, –CH3),
2.02–2.31 (m, 1H, –CH2), 3.87–3.89 (d, 1H, J = 8Hz,
4C–H), 3.92–3.94 (d, 1H, J = 8Hz, 4C–H), 4.44 (s, 1H,
4NH), 6.09–6.10 (d, 1H, J = 4Hz, quinolinyl-H),
7.27–7.29 (d, 1H, J = 8Hz, quinolinyl-H), 7.56–7.58 (d, 1H,
J = 8Hz, quinolinyl-H), 7.71–7.74 (d, 1H, J = 12Hz, qui-
nolinyl-H), 7.89 (s, 1H, quinolinyl-H).
946.13–910.15
(C–C–O
asymmetrical
stretching),
868.21–823.88 (peroxide, C–O–O stretching); 1H NMR
(400 MHz, DMSO-d6) δ 1.40–1.42 (t, 2H, J = 8Hz,
cyclohexyl–H), 1.46–1.48 (t, 2H, J = 8Hz, cyclohexyl–H),
1.49–1.75 (m, 2H, cyclohexyl–H), 1.79–1.85 (m, 2H,
cyclohexyl–H), 2.28 (s, 2H, 2× –OH); 13C NMR (100 MHz,
DMSO-d6)
δ
22.07 (cyclohexane –CH3–), 24.36
(cyclohexane–CH2–), 25.04 (cyclohexane–CH2–), 26.47
(cyclohexane–CH2–), 29.38 (cyclohexane–CH2–), 108.02
(cyclohexane–C–O–); Mass Spectrum (TOF MS, m/z)
Calculated 148.07, Observed 142.2 (100 %), 137.1
(38.06 %), 173(52.92 %).
3-(2-(7-chloroquinolin-4-ylamino)phenylamino)butan-2-
one (5b) Odourless blackish solid; soluble in dichlor-
omethane; melting range112–113 °C; %Yield 72.69; Rf
value 0.71 (Pet. ether: acetone: 1: 2); Spectroscopic
analysis: λmax (in CHCl3) 240.51 nm; FTIR Spectrum
(νmax, in cm−1, film) 3565.76, 3451.26 (sym. and asymm.
N–H stretching, –NH2), 3373.43 (N–H stretching,
4NH), 3105.25 (aromatic C–H stretching), 2927.29 (ali-
phatic C–H stretching), 1916.62–1566.15 (N–H bending),
1743.95–1706.32(4C=O stretching) 1531.70–1425.58
(C=C–C, aromatic ring stretching), 1368.53–1318.67 (C–N
stretching), 1211.26–904.97 (aromatic C–H in-plane bend),
1088.47 (C–Cl stretching, Ar–Cl), 849.91–581.16 (aromatic
C–H out-of-plane bend); 1H NMR (400 MHz, CDCl3)
δ 2.11–2.33 (m, 1H, 4C–H), 2.49–2.50 (d, 3H, J = 4Hz,
–CH3), 2.99 (s, 3H, –CH3), 4.49 (bs, 2H, 2×4NH),
6.19–6.21(d, 1H, J = 8Hz, Ar–H), 6.61–6.65 (t, 1H, J =
16Hz, Ar–H), 6.83–6.85 (d, 1H, J = 8Hz, Ar–H), 7.02–7.07
(m, 1H, Ar–H), 7.40–7.46 (m, 1H, quinolinyl-H), 7.09–7.10
(d, 1H, J = 4Hz, quinolinyl-H), 8.02 (s, 1H, quinolinyl-H),
8.28–8.29 (d, 1H, J = 4Hz, quinolinyl-H), 8.53–8.55 (d, 1H,
J = 8Hz, quinolinyl-H).
1,1-dihydroperoxycyclopentane (8b) Odourless off-white
solid; soluble in dichloromethane; melting range 80–82 °C;
%Yield 23; Rf value 0.71(chloroform: dichloromethane:
3:1); Spectroscopic analysis: λmax (in CHCl3) 243.04 nm;
FTIR Spectrum (νmax, in cm−1, film) 3417.72 (–OH
stretching, dihydroperoxide), 2949.97–2873.68 (C–H
stretching, cyclopentyl), 1454.31–1395.70 (C–H bending,
cyclopentyl), 1161.99–1073.47 (C–C–O symmetrical
stretching), 977.25–952.31 (C–C–O asymmetrical stretch-
ing), 827.85 (peroxide, C–O–O– stretching); 1H NMR
(400 MHz, DMSO-d6) δ 1.79–1.81 (t, 2H, J = 8Hz,
cyclopentane–CH2–), 1.83–1.86 (t, 2H, J = 12Hz, cyclo-
pentane–CH2–), 1.87–1.89 (t, 2H, J = 8Hz, cyclopen-
tane–CH2–), 1.90–1.98 (m, 2H, cyclopentane–CH2–), 2.06
(s, 2H, 2× –OH); 13C NMR (100 MHz, DMSO-d6) δ 22.65
(cyclopentane–CH2–), 24.11 (cyclopentane–CH2–), 32.67
(cyclopentane–CH2–), 37.68 (cyclopentane–CH2–), 119.90
(cyclopentane–C–O–); Mass Spectrum (TOF MS, m/z)
Calculated 134.06; Observed 341.1 (100 %), 101.1(63.35
%), 441.2 (56.04 %), 241.1(32.91 %).
3-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)butan-2-one
(5c) Odourless off-white solid; soluble in acetone,
dichloromethane; melting range 140–142 °C; %Yield
51.18; Rf value 0.77 (Pet. ether: acetone: 1:1); Spectro-
scopic analysis: λmax (in DMSO) 325.48 nm; FTIR
1,1-dihydroperoxycycloheptane (8c) White solid with
characteristic in odour; soluble in Chloroform; melting
range 90–92 °C; %Yield 47.5; Rf value 0.65 (n-Butanol:

Med Chem Res
Benzene: 1:1); Spectroscopic analysis: λmax (in CHCl3)
249.05 nm; FTIR Spectrum (νmax, in cm−1, film) 3797.13
(–OH stretching, dihydroperoxide), 2924.03–2851.74 (C–H
stretching, cycloheptyl), 1454.56–1348.56 (C–H bending,
cycloheptyl), 1168.70–1017.26 (C–C–O symmetrical
stretching), 991.80–895.47 (C–C–O asymmetrical stretch-
ing), 791.90 (peroxide, C–O–O- stretching).
2.05–2.38 (m, 1H, 4C–H), 2.50 (s, 1H, 4N–H), 3.01–3.18
(m, 1H, 4C–H), 3.62 (bs, 1H, 4N–H), 7.56–7.67 (d, 1H,
J = 8Hz, quinolinyl-H), 7.90 (s, 1H, quinolinyl-H),
8.19–8.20 (d, 1H, J = 4Hz, quinolinyl-H), 8.36–8.38 (d, 1H,
J = 8Hz, quinolinyl-H), 8.46–8.50 (d, 1H, J = 16Hz, qui-
nolinyl-H); 13C NMR (100 MHz, DMSO-d6) δ 13.48 (1×C,
–CH3), 22.07 (1×C, –CH3), 24.85 (1×C, –CH3), 28.68
(2×C, –CH3) 28.98 (1×C, 4C–H), 31.27 (1×C, 4CH2),
38.93 (1×C, 4C–H), 39.14, 39.34, 39.55, 39.76 (2×C,
tetraoxane), 39.97, 40.18, 78.45, 78.78, 79.11 (9×C, qui-
nolinyl); Mass Spectrum (TOF MS, m/z) Calculated 395.88,
Observed 342.2 (100 %), 378.3 (34.83 %), 330.2 (29.70 %),
396.89 (11.34 %) [M+H]+; Elemental Analysis for
C22H30ClN3O4; Calculated C, 57.64; H, 6.62; N 10.61; O,
16.17 Observed C, 57.373; H, 6.329; N, 10.933; O, 16.490.
1,1-dihydroperoxy-2-methylcyclohexane (8d) Pale yellow
liquid with characteristic odour; soluble in Dichlor-
omethane, Chloroform; Boiling range 95–96 °C; %Yield
28.12; Rf value 0.72 (Pet ether: chloroform: 1:1); Spectro-
scopic analysis: λmax (in CHCl3) 242.00 nm; FTIR Spec-
trum (νmax, in cm−1, film) 3565.12 (–OH stretching,
dihydroperoxide), 2935.66–2865.04 (C–H stretching,
methylcyclohexyl), 1540.24–1513.22 (–OH bending, dihy-
droperoxide), 1454.81–1376.00 (C–H bending, cyclo-
hexyl), 1234.67–1089.90 (C–C–O symmetrical stretching),
990.33–935.39 (C–C–O asymmetrical stretching), 841.58
(peroxide, C–O–O– stretching).
N2-(7-chloroquinolin-4-yl)-N1-(1-(3-methyl-1,2,4,5-tetra-
oxaspiro[5.5] undecan-3-yl)ethyl)propane-1,2-diamine (9b)
Blackish brown solid with characteristic odour; soluble in
dichloromethane, DMSO; melting range 245–247 °C; %
Yield 11.12; Rf value 0.73 (Pet. ether: acetone: 3:1);
Spectroscopic analysis: λmax (in DMSO) 341.77 nm;
FTIR Spectrum (νmax, in cm−1, film) 3254.20 (N–H
stretching, 4NH), 3109.22 (aromatic C–H stretching),
2,2-dihydroperoxypropane (8e) White solid with a char-
acteristic odour; soluble in ethanol, dichloromethane,
DMSO; melting range 95 °C; %Yield 70; Rf value 0.64
(Pet. ether: ether: 4:1); Spectroscopic analysis: λmax (in
ethanol) 268.40 nm; FTIR Spectrum (film, in cm−1) 3374.77
(–OH stretching, dihydroperoxide), 3006.71–2946.00 (C–H
stretching, gem dimethyl group), 1633.47–1549.83 (–OH
bending, dihydroperoxide), 1456.45–1360.31 (C–H bend-
ing, gem dimethyl group), 1273.60–1174.54 (C–C–O
symmetrical stretching), 996.75–841.08 (C–C–O asymme-
2924.53–2853.14
(aliphatic
C–H
stretching),
1710.60–1577.31 (N–H bending), 1451.97–1335.61
(C=C–C, aromatic ring stretching), 1238.85–1212.91 (C–N
stretching), 1082.01 (aromatic C–H in-plane bend), 1048.66
(C–Cl stretching, Ar–Cl), 897.70 (C–C–O stretching),
871.10 (aromatic C–H out-of-plane bend), 809.33–764.82
(peroxide, C–O–O stretching); 1HNMR (400 MHz, DMSO-
d6) δ 1.23–1.29 (d, 6H, J = 24Hz, 2× CH3), 1.38–1.42 (t,
6H, J = 16Hz, 3×4CH2, cyclohexyl), 1.51 (bs, 3H, –CH3),
1.70–1.76 (t, 4H, J = 24Hz, 2×4CH2, cyclohexyl),
2.20–2.28 (d, 2H, J = 32Hz, 4C–H), 2.50 (s, 1H, 4N–H),
2.67–2.89 (m, 1H, 4C–H), 3.08–3.82 (m, 1H, 4C–H), 3.99
(s, 1H, 4N–H), 7.00–7.02 (d, 1H, J = 8Hz, quinolinyl-H),
7.57–7.62 (m, 1H, quinolinyl-H), 7.68–7.90 (m, 1H, qui-
nolinyl-H), 8.34 (s, 1H, quinolinyl–H), 8.46–8.51 (d, 1H, J
= 20Hz, quinolinyl–H); 13C NMR (100 MHz, DMSO-d6) δ
24.06 (1×C, –CH3), 24.39 (1×C, –CH3), 24.85, 24.93 (2×C,
2×4CH2, cyclohexyl), 25.09 (1×C, –CH3), 27.76, 27.83,
28.69 (3×C, 3×4CH2, cyclohexyl), 33.33 (1×C, 4C–H),
33.48 (1×C, 4C–H), 33.61 (1×C, 4CH2), 38.83, 39.04
(2×C, tetraoxane), 39.25, 39.46, 39.67, 39.88, 40.8,
63.47, 162, 174, 176 (9×C, quinolinyl); Mass Spectrum
(TOF MS, m/z) Calculated 435.19, Observed 342.1 (100
%), 344.1 (27.13 %), 412.1 (18.88 %); Elemental Analysis
for C19H26ClN3O4; Calculated C, 60.61; H, 6.94; N 9.64;
O, 14.68; Observed C, 60.577; H, 6.942; N, 9.269; O,
14.841.
1
trical stretching); H NMR (400 MHz, DMSO-d6) δ 1.38(s,
6H, 2× –CH3), 1.54(s, 1H, –OH), 1.69(s, 1H, –OH); 13C
NMR (100 MHz, DMSO-d6) δ 20.96 (–CH3), 21.06(–CH3),
106.68(aliphatic C–C); Mass Spectrum (TOF MS, m/z)
Calculated 108.04; Observed 173 (100 %), 247.1(87.43 %),
99(48.37 %), 141(33.53 %).
N2-(7-chloroquinolin-4-yl)-N1-(1-(3,6,6-trimethyl-1,2,4,5-
tetraoxan-3-yl)ethyl)propane-1,2-diamine (9a) Odourless
pink solid; soluble in dichloromethane, DMSO; melting
range 258–260 °C; %Yield 22; Rf value 0.90 (Pet. ether:
methanol: 1: 1); Spectroscopic analysis: λmax (in DMSO)
339.56 nm; FTIR Spectrum (νmax, in cm−1, film) 3247.25
(N–H stretching, 4NH), 3111.60 (aromatic C–H stretch-
ing), 2922.43–2853.03 (aliphatic C–H stretching),
1730.33–1554.45 (N–H bending), 1453.33–1371.06
(C=C–C, aromatic ring stretching), 1213.62 (C–N stretch-
ing), 1087.09 (aromatic C–H in-plane bend), 1049.08 (C–Cl
stretching, Ar–Cl), 896.76 (C–C-O stretching), 868.95
(aromatic C–H out-of-plane bend), 813.19–766.68 (per-
oxide, C–O–O stretching); 1H NMR (400 MHz, DMSO-d6)
δ 0.83–0.84 (d, 6H, J = 4Hz, 2× CH3), 1.22 (d, 6H, J = 4Hz,
2× CH3), 1.49 (bs, 3H, –CH3), 1.54–2.01 (m, 2H, 4C–H),
N2-(7-chloroquinolin-4-yl)-N1-(1-(8-methyl-6,7,9,10-tetra-
oxaspiro[4.5]decan-8-yl)ethyl)propane-1,2-diamine (9c)

Med Chem Res
Odourless brownish solid; soluble in dichloromethane,
DMSO; melting range 270–271 °C; %Yield 13; Rf value
0.90 (Pet. ether: methanol: 1:1); Spectroscopic analysis:
λmax (in DMSO) 341.46 nm; FTIR Spectrum (νmax, in cm−1,
film): 3255.64 (N–H stretching, 4NH), 3114.47 (aromatic
C–H stretching), 2923.16–2853.42 (aliphatic C–H stretch-
ing), 1729.34–1611.90 (N–H bending), 1453.28–1373.58
(C=C–C, aromatic ring stretching), 1210.50–1164.69 (C–N
stretching), 1092.67 (aromatic C–H in-plane bend), 1022.68
(C–Cl stretching, Ar–Cl), 900.12 (C–C–O stretching),
868.86 (aromatic C–H out-of-plane bend), 813.23-764.15
(peroxide, C–O–O- stretching).
methylcyclohexyl), 39.33 (1×C, –CH3), 39.54, 39.75 (3×C,
3×4CH2, methylcyclohexyl), 39.95 (1×C, 4C–H), 40.16
(1×C, 4C–H), (1×C, 4CH2), 78.44, 78.76 (2×C, tetra-
oxane), 79.09, 143.01 (quinolinyl-C).
(7-chloroquinolin-4-yl)-N2-(1-(3,6,6-trimethyl-1,2,4,5-
tetraoxan-3-yl)ethyl)benzene-1,2-diamine (9f) Odourless
brownish red solid; soluble in methanol; melting range
179–180 °C; %yield 10.56; Rf value 0.87 (acetone: metha-
nol: 1:4); spectroscopic analysis: λmax (in methanol) 323.15
nm; FTIR spectrum (νmax, in cm−1, film) 3222.09
(N–H stretching, 4NH), 3060.81 (aromatic C–H stretch-
ing), 2920.32–2851.80 (aliphatic –C–H stretching),
1917.89–1581.53 (N–H bending), 1533.04–1451.67
(C=C–C, aromatic ring stretching), 1370.82 (C–N stretch-
ing), 1207.90–1027.49 (aromatic C–H in-plane bend),
1093.02 (C–Cl stretching, Ar–Cl), 873.94–571.28 (aromatic
C–H out–of-plane bend), 815.97 (C–C–O stretching),
757.39–720.21 (peroxide, C–O–O– stretching).
N2-(7-chloroquinolin-4-yl)-N1-(1-(3-methyl-1,2,4,5-tetra-
oxaspiro[5.6]dodecan-3-yl)ethyl)propane-1,2-diamine
(9d) Odourless blackish brown solid; soluble in DMSO;
melting range 265–266 °C; %Yield 13.6; Rf value 0.83 (Pet.
ether: methanol: 1:1); Spectroscopic analysis: λmax (in
DMSO) 335.76 nm; FTIR Spectrum (νmax, in cm−1, film)
3246.65 (N–H stretching, 4NH), 3106.87 (aromatic C–H
stretching), 2923.15–2853.33 (aliphatic C–H stretching),
1731.61–1545.44 (N–H bending), 1452.40–1369.82
(C=C–C, aromatic ring stretching), 1240.41–1160.56 (C–N
stretching), 1084.70 (aromatic C–H in-plane bend), 1018.65
(C–Cl stretching, Ar–Cl), 896.90 (C–C–O stretching),
871.17 (aromatic C–H out–of-plane bend), 806.82–765.03
(peroxide, C–O–O– stretching).
N1-(7-chloroquinolin-4-yl)-N2-(1-(8-methyl-6,7,9,10-tetra-
oxaspiro[4.5]decan-8-yl)ethyl)benzene-1,2-diamine (9g) Odo-
urless black solid; soluble in Dichloromethane; melting
range 275–277 °C; %Yield 15.23; Rf value 0.88 (Pet. ether:
methanol: 1:4); Spectroscopic analysis: λmax (in dichlor-
omethane) 288.81 nm; FTIR Spectrum (νmax, in cm−1, film)
3223.23 (N–H stretching, 4NH), 3061.54 (aromatic C–H
stretching), 2922.23–2852.55 (aliphatic –C–H stretching),
1917.56–1517.18 (N–H bending), 1447.00 (C=C–C, aro-
matic ring stretching), 1368.72–1332.57 (C–N stretching),
1207.44–1091.81 (aromatic C–H in-plane bend), 1028.05
(C–Cl stretching, Ar–Cl), 906.14–500.00 (aromatic C–H
out-of-plane bend), 817.47 (C–C–O stretching), 759.43
(peroxide, C–O–O– stretching); 1H NMR (400 MHz,
DMSO-d6) δ 1.20 (s, 3H, –CH3), 1.53–1.58 (d, 3H, J =
20Hz, –CH3), 1.82–196 (m, 4H, 2×4CH2, cyclopentyl),
2.24–2.51 (m, 4H, 2×4CH2, cyclopentyl), 2.69–3.04 (m,
1H, 4C–H), 3.55 (s, 2H, 2×4N–H), 6.34–6.35 (d, 2H,
J = 4Hz, Ar–H), 6.43–7.14 (m, 2H, Ar–H), 7.54–7.60
(d, 1H, J = 24Hz, quinolinyl-H), 7.86–8.58 (m, 2H, qui-
nolinyl-H), 8.70 (s, 1H, quinolinyl-H), 8.89–8.93 (d, 1H,
J = 16Hz, quinolinyl-H); 13C NMR (100 MHz, DMSO-d6)
δ 29.03 (2×C, –CH3), 38.98 (2×C, 2×4CH2, cyclopentyl),
39.19 (2×C, 2×4CH2, cyclopentyl), 39.40 (1×C, 4C–H),
39.60, 39.81(2×C, tetraoxane), 40.02, 40.23 (4×C, Ar),
77.79, 78.12, 78.45 (9×C, quinolinyl); Mass Spectrum
(TOF MS, m/z) Calculated 455.93 Observed 270.2 (100 %),
272.2 (24.02 %), 410.3 (7.08 %); Elemental Analysis for
C24H26ClN3O4; Calculated C, 63.22; H, 5.75; N 9.22; O,
14.04 Observed C, 62.928; H, 5.994; N, 08.925; O, 14.143.
N2-(7-chloroquinolin-4-yl)-N1-(1-(3,7-dimethyl-1,2,4,5-
tetraoxaspiro[5.5]undecan-3-yl)ethyl)propane-1,2-diamine
(9e) Odourless dark brown solid; soluble in dichlor-
omethane, DMSO; melting range 260–262 °C; %Yield
08.98; Rf value 0.89 (Pet. ether: Methanol: 1: 1); Spectro-
scopic analysis: λmax (in DMSO) 340.19 nm; FTIR Spec-
trum (νmax, in cm−1, film) 3230.87 (N–H stretching, 4NH),
3060.40 (aromatic C–H stretching), 2920.67–2851.15 (ali-
phatic C–H stretching), 1991.73–1517.05 (N–H bending),
1477.87–1340.47 (C=C–C, aromatic ring stretching),
1214.62 (C–N stretching), 1087.18 (aromatic C–H in-plane
bend), 1042.15 (C–Cl stretching, Ar–Cl), 896.27 (C–C–O
stretching), 868.05 (aromatic C–H out-of-plane bend),
809.08–765.95 (peroxide, C–O–O- stretching); 1H NMR
(400 MHz, DMSO-d6): δ 0.25-0.32 (t, 3H, J = 28Hz, –CH3,
methylcyclohexyl), 0.98–1.23 (d, 3H, J = 100Hz, 2×-CH3),
1.49 (s, 3H, –CH3), 1.65–1.93 (m, 4H, 3×4CH2, methyl-
cyclohexyl), 2.03–2.09 (t, 4H, J = 24Hz, 2×4CH2,
methylcyclohexyl), 2.27 (s, 1H, 4N–H), 2.49–2.61 (t, 2H,
J = 48Hz, 4CH2), 2.75–2.99 (m, 1H, 4C–H), 3.60 (s, 1H,
4N–H), 4.23–4.42 (m, 1H, 4C–H), 5.95–5.96 (d, 1H,
J = 4Hz, quinolinyl-H), 6.30–6.54 (m, 1H, quinolinyl-H),
7.23–7.82 (m, 1H, quinolinyl–H), 7.96–8.17 (m, 1H,
quinolinyl–H), 8.44 (s, 1H, quinolinyl-H); 13C NMR
N1-(7-chloroquinolin-4-yl)-N2-(1-(3-methyl-1,2,4,5-tetra-
oxaspiro[5.5]undecan-3-yl)ethyl)benzene-1,2-diamine (9h)
Black solid with characteristic in odour; soluble in ethanol,
(100 MHz, DMSO-d6)
δ 29.1 (2×C, –CH3), 38.91
(1×C, –CH3, methylcyclohexyl), 39.12 (2×C, 2×4CH2,

Med Chem Res
dichloromethane; %Yield 08.08; Rf value 0.86 (Pet. ether:
of-plane bend, quinolyl), 876.70 (C–C–O stretching),
722.73–696.00 (peroxide, C–O–O– stretching); 1HNMR
(400 MHz, CHCl3) δ 0.85-0.91 (m, 3H, –CH3), 1.27 (s, 6H,
2×–CH3), 2.96 (s, 3H, –CH3), 2.00–2.10 (m, 1H, 4C–H),
2.33–2.37 (t, 4H, J = 16Hz, 2×4CH2, piperazinyl–H),
2.91–2.94 (t, 2H, J = 12Hz, 2×4CH2, piperazinyl–H),
7.00–7.18 (m, 1H, quinolinyl-H), 7.28 (s, 1H, quinolinyl-
H), 7.48–7.52 (t, 1H, J = 16Hz, quinolinyl-H), 7.59–7.61
(d, 1H, J = 8Hz, quinolinyl-H), 7.81–7.83 (t, 1H, J = 8Hz,
quinolinyl-H); 13C NMR (100 MHz, DMSO-d6) δ 14.12
(1×C, –CH3), 22.69 (1×C, –CH3), 24.76 27.09 (2×C,
–CH3), 29.27, 29.36, 29.70 (1×C, 4CH), 31.93, 33.84
(4×C, piperazine), 76.70 (1×C, tetraoxane), 77.02 (1×C,
tetraoxane), 77.34 (1×C, quinolinyl), 128.11 (2×C, quino-
linyl), 130.67 (4×C, quinolinyl).
methanol:
1:4);
Spectroscopic
analysis:
λmax
(in dichloromethane) 228.57 nm; FTIR Spectrum (νmax, in
cm−1, film) 3235.48 (N–H stretching, 4NH), 3062.80
(aromatic C–H stretching), 2925.57–2857.58 (aliphatic
–C–H stretching), 1707.19–1541.02 (N–H bending),
1455.56 (C=C–C, aromatic ring stretching), 1394.14 (C–N
stretching), 1167.25–1097.28 (aromatic C–H in-plane
bend), 1042.33 (C–Cl stretching, Ar–Cl), 960.80–580.83
(aromatic C–H out-of-plane bend), 819.77 (C–C–O
stretching), 736.52–695.67 (peroxide, C–O–O– stretching).
N1-(7-chloroquinolin-4-yl)-N2-(1-(3-methyl-1,2,4,5-tetra-
oxaspiro[5.6]dodecan-3-yl)ethyl)benzene-1,2-diamine
(9i) Odourless black solid; soluble in methanol; melting
range 280-282 °C; %Yield 07.78; Rf value 0.88 (Pet. ether:
methanol: 1:1); Spectroscopic analysis: λmax (in DMSO)
341.46 nm; FTIR Spectrum (νmax, in cm−1, film) 3221.07
(N–H stretching, 4NH), 3061.02 (aromatic C–H stretch-
ing), 2919.95–2851.39 (aliphatic –C–H stretching),
1917.59–1516.78 (N–H bending), 1451.38 (C=C–C, aro-
matic ring stretching), 1368.82 (C–N stretching), 1200.85
(aromatic C–H in-plane bend), 1024.68 (C–Cl stretching,
Ar–Cl), 908.69–572.72 (aromatic C–H out-of-plane bend),
813.79 (C–C–O stretching), 754.81 (peroxide, C–O–O–
stretching).
Antimalarial Activity Evaluation All the synthesized
compounds were evaluated for in vitro antimalarial activity
against chloroquine-resistant strain (RKL-9) of Pf using 96
well-microtitre plates. The laboratory adapted strain of Pf
was routinely cultured at 37 °C temperature and 5 % CO2
environment in RPMI-1640 medium supplemented with
25 mM HEPES, 1 % D-glucose, 0.23 % sodium bicarbonate
and 10 % heat inactivated human serum. For antimalarial
activity testing, the asynchronous parasites of Pf were
synchronized to obtain only the ring stage parasitized cells
by 5 % D-sorbitol treatment. For carrying out the assay, the
initial ring stage parasitaemia of 0.8–1.5 % at 3 % haema-
tocrit in a total volume of 100 ml of medium RPMI-1640
was uniformly maintained. A stock solution of 1 mg/ml was
prepared by dissolving the test compounds in DMSO and
subsequent dilutions were made with the culture medium.
Hundred microlitres of the test compounds at 100 µg/ml
concentrations in triplicate wells was incubated with para-
sitized cell preparation at 37 °C and 5 % CO2 in a CO2
incubator. After an incubation period of 36–40 h, the blood
smears were prepared from each well and stained with 3 %
Giemsa stain. The slides were microscopically observed and
the per cent dead rings and schizonts were scored against
200 asexual parasites with respect to the control group.
Chloroquine was used as the standard reference drug
(Trager and Jensen, 1976).
N1-(7-chloroquinolin-4-yl)-N2-(1-(3,7-dimethyl-1,2,4,5-
tetraoxaspiro[5.5]undecan-3-yl)ethyl)benzene-1,2-diamine
(9j) Blackish brown semisolid with characteristic odour;
soluble in ethanol, dichloromethane; %Yield 06.96;
Rf value 0.77 (acetonitrile: ethanol: 1:1); Spectroscopic
analysis: λmax (in CHCl3) 239.56 nm; FTIR Spectrum
(νmax, in cm−1, film) 3224.09 (N–H stretching, 4NH),
3063.32 (aromatic C–H stretching), 2927.32–2860.35
(aliphatic –C–H stretching), 1706.98–1540.20 (N–H bend-
ing), 1454.88–1414.69 (C=C–C, aromatic ring stretching),
1375.30 (C–N stretching), 1169.57 (aromatic C–H
in-plane bend), 1089.94–1043.16 (C–Cl stretching,
Ar–Cl), 928.68–582.08 (aromatic C–H out-of-plane
bend), 822.13 (C–C–O stretching), 756.76 (peroxide,
C–O–O– stretching).
7-chloro-4-(4-(1-(3,6,6-trimethyl-1,2,4,5-tetraoxan-3-yl)
ethyl)piperazin-1-yl)quinoline (9k) Black semisolid with
characteristic odour; soluble in dichloromethane, chloro-
form; %Yield 05.54; Rf value 0.91 (Pet. ether: methanol:
1:4); Spectroscopic analysis: λmax(in CHCl3) 251.27 nm;
FTIR Spectrum (νmax, in cm−1, film) 3060.00 (aromatic
C–H stretching), 2921.16–2852.83 (C–H stretching,
4CH2, alkyl and piprazinyl), 1867.16–1458.33 (C=C–C,
aromatic ring stretching, quinolyl), 1373.86–876.70 (aro-
matic C–H in-plane bend, quinolyl), 1038.55 (C–Cl
stretching, quinolyl–Cl), 917.60-578.47 (aromatic C–H out-
Molecular Docking Studies The three dimensional (3D)
crystal structure of Falcipain-2 (PDB code 3BPF) was
retrieved from the protein data bank (PDB) (www.rcsb.org/
pdb). The native auto inducer and all water molecules were
removed. The CHARMm force field (FF) was used to add
atom types and hydrogens in the proteins. 3D structures of
all synthesized compounds were constructed and energy
minimized using the Discovery Studio 2.5/Builder module.
Docking studies were performed using the CDOCKER
module of Discovery Studio 2.5. CDOCKER is a grid-
based molecular docking method, where the receptor is held

Med Chem Res
rigid while the ligands are allowed to flex during the
refinement. The CHARMm FF was used as an energy grid
FF for docking and scoring function calculations. Random
ligand conformations were generated from the initial
structure through high temperature molecular dynamics,
followed by random rotations which were further refined by
grid-based (GRID 1) simulated annealing and a final grid-
based minimization. Of the 10 best poses, one (conforma-
tion) having highest docking score (−CDOCKER energy)
was used for the binding energy calculations and further
analysis. The higher negative value of CDOCKER energy
represents more favourable binding of the complex. This
means that ligands with high docking scores are able to fit
snugly in the active site pocket with the minimal steric
clashes. CDOCKER score (−CDOCKER Energy) includes
internal ligand strain energy and receptor–ligand interaction
energy, and is used to sort the different conformations of
each input ligand (Oliveira et al., 2013; Liu et al., 2012).
6.72–8.26 are due to Ar–H or quinolinyl-H. The analytical
and spectral data of the compounds were found in com-
pliance with the structure of the synthesized compounds.
Biological activity
In vitro antimalarial activity
All the eleven synthesized compounds were tested for
in vitro antimalarial activity against the chloroquine-
resistant strain RKL-9 (Rourkela, Orissa (India)) of Pf.
Among the tested compounds, compounds 9a, 9b, 9g, 9e
and 9k showed activity against chloroquine-resistant Pf
Table 1 In vitro antimalarial activity profile of the synthesized
compounds against chloroquine-resistant Pf strain RKL-9
S. No. Compound
code
MIC (µM) MIC
IC50
IC90
(µg/ml) (µg/ml) (µg/ml)
1
9a
9b
9c
9d
9e
9f
78.94
71.81
296.26
ND
31.25
31.25
125
3.906
3.942
4.686
4.573
4.272
5.629
3.906
4.886
5.024
4.271
4.814
0.393
4.498
2
24.982
101.230
167.736
52.761
96.637
4.166
Results and Discussion
Chemistry
3
4
ND
5
138.90
1163.06
68.69
1063.92
258.80
ND
62.5
500
6
The synthesis of target tetraoxaquine hybrids was achieved
in three step reactions as shown in scheme 1.
7
9g
9h
9i
31.25
500
8
145.485
94.339
153.491
46.129
1.218
The step 1 corresponds to the synthesis of 2-butanone
derivatives (5a–5c), which was afforded by the reaction
between 4-substituted-7-chloroquinolines (3a–3c) and
3-chlorobutan-2-one (4). The template, i.e., compound 3a–c
was synthesized by reacting 4,7-dichloroquinoline (1) with
corresponding diamines 2a–c.
9
125
10
11
12
9j
ND
9k
CQ
153.23
78.32
62.5
25
ND = MIC was not determined
The corresponding cyclic or alicyclic ketone (6a–6e) was
allowed to react with hydrogen peroxide (7) to afford
resultant dihydroperoxides (8a–8e), which was then con-
jugated with derivatives of step 1 (5) to furnish target
compounds (9a–9k).
MIC, IC50 and IC90 values were means of three independent
experiments
The structures of the synthesized compounds were con-
firmed with the aid of various spectroscopic techniques.
FTIR spectra showed the stretching in the frequency range
between 3200–3400 cm−1 attributable to N–H. The presence
of stretching band amid 3100–3000 cm−1 corresponds to
aromatic C–H. The other stretching bands in range of
2800–2950 cm−1 were attributable to aliphatic –C–H, while
1250–900 cm−1 confirmed the presence of C-C-O stretch-
ing. The peroxide, C–O–O– of compounds showed
1
stretching at 900–750 cm−1. The H NMR spectra of the
compounds showed the characteristic singlet, doublet, tri-
plet or multiplet at δ (ppm). More specifically the 0.50–2.50
was attributable to aliphatic –C–H, a singlet at 2.50–3.50
caused by N–H. A doublet, triplet or multiple at
3.00–3.70 showed the presence of tetraoxane –C–H.
Moreover, a singlet, doublet, triplet or multiple at δ
Fig. 1 Comparison in MIC against CQ-resistant Pf strain RKL-9,
where ND is not determined

Med Chem Res
strain RKL-9 with MIC 31.25, 31.25, 31.25, 62.50, 62.50
µg/ml and IC50 values 3.906, 3.942, 3.906, 4.272, 4.814 µg/
ml, respectively. In comparison test, CQ showed prominent
inhibition with MIC 25 µg/ml and IC50 0.393 µg/ml
(Table 1 and Fig. 1).
The designed hybrid compounds exhibited significant
activity against CQ-resistant strain. Compounds containing
spiro-cycloheptane substitution on tetraoxane moiety of
tetraoxaquine showed less antimalarial activity as compared
to other congeners having dimethyl, spiro-cyclohexane,
spiro-cyclopentane. It has been observed that nature of
linkers (1,2-diaminopropane, o-phenylenediamine and
piperazine) have its unique role on the pharmacological
activity. Particularly, tetraoxaquines with 1,2-diaminopro-
pane and o-phenylenediamine as linkers exhibit prominent
activity than compounds having piperazine linker.
were performed into the binding pocket of Falcipain-2
(PDB code 3BPF). The results and docked conformations of
the ligands in the active site are illustrated in Table 2 and
Fig. 2 and 3. The results showed that the targeted molecules
were snugly fitted into the active site with considerable and
diverse CDOCKER energy (−1.9658 to −17.3049) along
with the formation of hydrogen bonds and hydrophobic
interactions.
The results indicate that most promising inhibitor, i.e.,
compound 9b showed very low binding energy (−15.3656)
against falcipain-2; other compounds, e.g., 9f also showed
very low binding energy (−17.3049) with less antimalarial
activity. The formation of two hydrogen bonds with Glu14
and Asn173 through the involvement of 1,2,4,5-tetraoxane
nucleus and 7-chloroquinoline nucleus, respectively, was
noted. The stability of these complexes (post-docked
ligand–receptor) and their low binding energies are attrib-
uted to the formation of greater intermolecular hydrogen
bonding and hydrophobic interactions with the amino acid
residues of falcipain-2 (Fig. 2).
Molecular Docking Studies
In order to gain insight into the key structural requirements
and the basis of the distinct activity profile of the test
compounds in Pf parasite, molecular docking study was
carried out. The docking studies of the target compounds
The 2,3-diaminopropyl bridge used to join 4-
aminoquinoline with 1,2,4,5-tetraoxane was predicted to
be predominantly engaged in hydrogen bonding with
Fig. 2 Docked complex of
compound 9b in the binding
pocket of FP-2

Med Chem Res

Med Chem Res
Fig. 3 Docked complex of
compound 9h in the binding
pocket of FP-2
key amino acids, i.e.,Glu14 and Asn173. The hydrophobic
interactions through the participation of 1,2,4,5-tetraoxane,
the cyclohexyl and its amine linkage with Glu15, Asn16,
Gln36, Gly40, Cys42, Gly82, Gly83, Leu84, Ile85, Trp43,
Ser149, Gln171, Leu172, Asn173, His174, Ala175,
Trp206 suggest the role of hydrophobic interactions in
biological activity. Compound 9b showed formation of two
hydrogen bonds with Glu14 and Asn173 through oxygen
atom of 1,2,4,5-tetraoxane and nitrogen of amino group of
side chain modified 7-chloroquinoline. Compounds 9a, 9c
and 9d showed similar fashion of interactions like 9b. In the
case of compounds 9e, 9f, 9g, 9h (Fig. 3), 9i, 9j and 9k,
where a diaminophenyl and piperazinyl group are present at
the positions of the diaminopropyl revealed a diverse pat-
tern of interaction with the binding site. The Hydrogen
bonding with Glu14, Gly83, ASN173, as well as hydro-
phobic interaction with Glu15, Asn16, Val152, Asn173,
Val150, Leu172, Gln171, Ile85, Leu84, Gly83, Gly82,
Ser41, Asn81, Cys80, Cys39, Lys37, Asn38, Trp206,
Asp234, Ser149, Asn173, His174, Ala175 were observed
for rest of the compounds.
The in vitro evaluation against chloroquine-resistant strain
of Pf RKL-9 showed activity in the designed compounds.
The promising antimalarial activity exhibited by the novel
tetraoxaquine hybrids reported in the present study
emphasizes their potential for further development as
antimalarial drugs.
Acknowledgments The authors are thankful to Ipca Laboratories
Ltd., Indore, India for providing gift sample of 4,7-dichloroquinoline.
The authors are also thankful to S.A.I.F., Punjab University, Chandi-
garh, India for carrying out spectroscopic analysis of the synthesized
compounds.
Compliance with ethical standards
Conflict of interest The authors declare that they have no com-
peting interests.
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