Newer series of trioxane derivatives as potent antimalarial agents

Article abstract of DOI:10.1007/s00044-017-2090-8

Among synthesized 1,2,4-trioxane derivatives, six compounds were found to be considerably potent, with better activity against resistant strain of P. falciparum than the sensitive strain. The IC₅₀ values of the best compound with 4-hydroxypheny

Full text of DOI:10.1007/s00044-017-2090-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2090-8
ORIGINAL RESEARCH
Newer series of trioxane derivatives as potent antimalarial agents
Mithun Rudrapal1 Zartaj Washmin Banu1 Dipak Chetia1
●
●
Received: 13 June 2017 / Accepted: 25 September 2017
© Springer Science+Business Media, LLC 2017
Abstract Among synthesized 1,2,4-trioxane derivatives,
six compounds were found to be considerably potent, with
better activity against resistant strain of P. falciparum than
the sensitive strain. The IC50 values of the best compound
with 4-hydroxyphenyl substitution were found to be 0.391
and 0.837 µg/mL against sensitive and resistant strain of P.
falciparum, respectively. Results of the tested compounds
were comparable with that of the standard drug, chloroquine
(IC50 = 0.044 and 0.205 µg/mL against sensitive and resis-
tant strain of P. falciparum, respectively). Docking simu-
lation, in silico drug-likeness and ADMET studies further
validated the results of in vitro antimalarial activity.
Trioxane derivatives exhibited good binding affinity for the
P. falciparum cysteine protease falcipain 2 receptor (PDB
id: 3BPF) with well defined drug-like and pharmacokinetic
properties based on Lipinski’s rule of five with additional
physicochemical and ADMET parameters. In view of
having antimalarial potential, newly reported 1,2,4-trioxane
derivative(s) may be useful as novel antimalarial lead(s) in
the discovery and development of future antimalarial drug
candidates as P. falciparum falcipain 2 inhibitors against
resistant malaria.
Introduction
Malaria continues to be a major lethal infectious disease of
human beings, affecting around 200–300 million people
and causing approximately 430,000 deaths every year
globally (WHO 2016). Plasmodium falciparum is the most
prevalent and deadly species that causes severe malaria such
as cerebral malaria, and it is also responsible for most of the
malaria-related deaths in humans (Rudrapal and Chetia
2016; Pandey et al. 2013; Gogoi et al. 2016). Every year
over 50% of all malarial infections are caused by P. falci-
parum with about 20–50% of total mortality cases, parti-
cularly in children younger than 5 years old and pregnant
women (Geleta and Ketema 2016). During the past two
decades, the emergence of drug resistant strains of P. fal-
ciparum has become an increasingly serious concern in
malaria control and prevention worldwide (Rudrapal and
Chetia 2016). Artemisinin (ART)-based combination
therapies (ACTs) such as artemether + lumefantrine +
amodiaquine and artesunate + mefloquine + piperaquine
recommended by WHO for the treatment of multi-drug
resistant P. falciparum malaria have also been reported to
develop resistance in some parts of the world (Rudrapal and
Chetia 2016; Kyle et al. 1998). This increasing burden of
resistant malaria has stimulated drug discovery scientists to
search for new antimalarial drugs or alternative therapeutic
options to combat the problem of drug resistance. In view of
this, the development of new antimalarial drugs with novel
mode(s) of action could be an attractive strategy to address
the above challenging issue. In recent years, research into
several synthetic endoperoxide scaffolds such as 1,2,4-
trioxane, 1,2,4-trioxolane and 1,2,4,5-teraoxane derivatives
and their hybrid analogs have gained significant interest
in discovery and development of potent antimalarial
drugs. Some endoperoxide-based antimalarial compounds
●
●
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Keywords P. falciparum Resistance 1,2,4-Trioxane
●
Falcipain 2 inhibitors Antimalarial
* Mithun Rudrapal
rudrapal.m03@gmail.com
1
Department of Pharmaceutical Sciences, Dibrugarh University,
Dibrugarh 786 004 Assam, India

Med Chem Res
H
O
O
O
O
O
O
O
O
O
O
H
NH
NH2
O
O
N
N
S
O
O
O
BAY 44 9585 (Artemisone)
OZ 277 (Arterolane)
Fig. 1 Structures of some endoperoxide antimalarials under clinical development
OZ 439 (Artefenomel)
(Rudrapal and Chetia 2016) that are currently under clinical
phase of development are represented in Fig. 1. Endoper-
oxide molecules bearing 1,2,4-trioxane core have been
identified to exhibit a broad spectrum of biological effects
such as anticancer, antimicrobial, and anti-parasitic effects
(Rubush et al. 2012). Several design strategies have been
initiated by medicines for malaria venture (MMV) program
for the development of effective and affordable synthetic
peroxides as alternative therapy to existing ART-based
drugs (Rudrapal and Chetia 2016; Tang et al. 2004).
However, considering all above facts, development of
peroxide-based synthetic compounds is expected provide
effective antimalarials agents with limited toxicity issues
and ready to be available at affordable cost. It would offer a
better therapeutic option for the treatment of malaria with
good clinical outcomes (desired potency with optimal
pharmacokinetics) and resistant preventing action over
ART, its semi-synthetic analogs and related endoperoxides.
It may therefore represent a new class of synthetic, new-
generation, and orally active peroxides, with optimal anti-
malarial potency against resistant P. falciparum malaria.
As part of our research program towards developing
potent antimalarial drugs that would be active against
resistant P. falciparum malaria, novel molecular manipula-
tion approach was adopted to design and develop newer
series of 1,2,4-trioxane derivatives as single drug conjugates
(single molecular entity rather than hybrid drugs) based on
the basic structural scaffold of 1,2,4-trioxane ring system.
After the synthesis of compounds, in vitro antimalarial
screening, molecular docking and drug-likeness studies
including ADMET prediction using in silico tools were also
carried out. Since, ART and its semi-synthetic analogs or
ART derived peroxides possess unfavorable pharmacoki-
netic issues and certain tissue toxicities because of their
high structural complexities and some physicochemical
insufficiencies (Garner and Graves 2005; Park et al. 1998),
it is expected that designed compounds would possess
modified pharmacokinetic properties and toxicities due to
less complexities in the molecular structure. Moreover,
single drug conjugate-based trioxane molecule can be given
as single drug therapy that would also be advantageous over
hybrid antimalarial drugs and combination therapies.
Molecular docking study was performed to validate the
antimalarial efficacy of synthesized 1,2,4-trioxane deriva-
tives by investigating binding modes as well as orientation
of ligands in the receptor pocket of target falcipain 2 protein
(P. falciparum cysteine protease enzyme). In our study,
three dimensional structure of falcipain 2 protein molecule
was used as possible antimalarial drug target for 1,2,4-
trioxane derivatives. Docking rationalizes finding new
antimalarial lead molecules and also gives an insight into
structure-activity relationships and mode(s) of antimalarial
action based on scoring function and further binding modes
analysis from ligand-protein binding/interaction studies
(Singh and Konwar 2012). Drug-likeness study includes
calculation of fundamental molecular (physicochemical)
properties and Lipinski’s parameters, which assesses the
acceptability of compounds as drug molecules. They
represent the combined physicochemical, pharmacokinetic,
and pharmacodynamic properties (collectively termed as
drug-like properties) of molecules to exhibit good drug-
likeness behavior in human body (Shukla et al. 2013). In
fact, molecular properties are the fundamental structural
parameters which determine the physicochemical (solubility
and permeability) and biochemical (metabolic stability,
transport property, and protein/tissue affinity) properties,
which ultimately determine molecule’s in vivo pharmaco-
kinetics (bioavailability and half life), toxicity and phar-
macodynamics (receptor affinity and efficacy) (Sharma
et al. 2016) ADMET (absorption, distribution, metabolism,
excretion, and toxicity) properties have a predictable influ-
ence on pharmacokinetic and pharmacodynamic effects of
drug molecules. The calculation of ADMET properties is
therefore intended as the first step towards optimizing the
new drug molecules, whether to check the failure of lead
molecules which may cause toxicity and one unable to cross
the intestinal membranes or metabolized by the body in to
an inactive form (Singh and Srivastava 2015).

Med Chem Res
Experimental
from ethyl acetate/ethanol mixture (3:1) to obtain a pure
product of white crystalline solid.
Synthesis and analysis
The intermediate compound, p-peroxiquinol (4-methyl-
4-hydroperoxycyclohexa-2,5-dienone)
2 (1 mmol) was
All of the chemicals were procured commercially from
Sigma-Aldrich Corporation (USA), Merck Specialists Pvt.
Ltd. (Germany), HiMedia Lab. Pvt. Ltd. (Germany) or
Spectrochem Pvt. Ltd. (India) and were used without further
purification. All the reactions were performed in oven dried
glass ware using synthetic grade chemicals. Melting points
(MP) were measured in open capillaries on an electrically
heated melting point apparatus. Ultraviolet (UV)–Visible
spectra were recorded on Shimadzu UV-1700 UV–Visible
spectrophotometer and the wave lengths of maximum
absorption (λmax, nm) are reported. Infrared (IR) spectra
were obtained on a Bruker Alpha Fourier transform (FT-IR)
spectrometer using KBr disc and are reported in terms of
frequency of absorption (υ, cm−1). 1H & 13C nuclear
magnetic resonance (NMR) spectra were recorded on Bru-
ker Avance II 400 FT-NMR spectrometer at 400 and 100
MHz, respectively using tetramethylsilane (TMS) as an
internal standard (δ 0.00 ppm) and CDCl3 as a solvent.
Chemical shift (δ) values were expressed in parts per mil-
added slowly into a mixture of aldehyde (1.5 mmol) and
dichloromethane (4.0 mL), followed by the addition of an
acid catalyst, p-toluenesulfonic acid (PTSA, 0.05 mmol).
The reaction mixture was stirred at 45–50 °C till the com-
pletion of reaction (8–12 h) and then concentrated in
vacuum. Solvent recrystallization of the resulting residue
with ethanol the desired pure product of target compounds,
3a–j as crystalline solids or semi-solids (Rubush et al. 2012;
Carreno et al. 2006).
8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-6(8aH)-one
(3a)
UV spectrum (MeOH), λmax, nm: 202.5; IR (KBr, cm−1) υ:
2932.02, 2894.01 (C–H, CH3), 1656.48 (C=O, conj.),
1145.20 (C–O), 892.28 (O–O); 1H NMR (400 MHz,
CDCl3) δ: 1.23 (s, 3H, CH3), 2.46 (d, J = 3.0 Hz, 2H,
CO–CH2–CH), 4.12 (d, J = 3.0 Hz, 1H, CO–CH2–CH),
6.04 (d, J = 9.0, 1H, CO–CH=CH), 6.32 (d, J = 9.0, 1H,
CO–CH=CH); 13C NMR (100 MHz, CDCl3) δ: 18.24
(CH3), 24.23, 28.78 (CH2), 36.81, 42.37 (CH), 84.77, 92.40
(CH=CH), 188.27 (C=O); MS (ES+), m/z (%): 170.57
(100), [M]+.
1
lion (ppm) relative to TMS (δ 0.00 ppm). HNMR data are
assigned in order: peak multiplicity (s, singlet; d, doublet; t,
triplet; m, multiplet), number of protons (numerical integral
value), coupling constants (J value) in hertz. 13C NMR data
represents different types of structural carbons with respect
to their corresponding chemical shift values. Mass spectra
were obtained on a LC–MS Water 4000 ZQ instrument
using electrospray ionization. The m/z values were recorded
in the range of m/z between 100–500 and the m/z values of
the most intense molecular ion [M]+ peak, with relative
intensities in parentheses, are given followed by peaks
corresponding to major fragment ions.
3-Methyl-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-6
(8aH)-one (3b)
UV spectrum (MeOH), λmax, nm: 212.7; IR (KBr, cm−1) υ:
2940.31, 2883.27 (C–H, CH3), 1643.20 (C=O, conj.),
1145.32 (C–O), 898.20 (O–O); 1H NMR (400 MHz,
CDCl3) δ: 0.84 (s, 3H, CH3), 1.22 (s, 3H, CH3), 2.48 (d, J
= 3.0 Hz, 2H, CO–CH2–CH), 4.20 (d, J = 3.0 Hz, 1H,
CO–CH2–CH), 6.12 (d, J = 9.0, 1H, CO–CH=CH), 6.42
(d, J = 9.0, 1H, CO–CH=CH); 13C NMR (100 MHz,
CDCl3) δ: 14.20, 18.12 (CH3), 26.23 (CH2), 36.45, 42.12
(CH), 84.42, 90.20 (CH=CH), 188.19 (C=O); MS (ES+),
m/z (%): 184.67 (100), [M]+.
General procedure of synthesis 3a–j
A mixture of p-cresol, 1 (1 mmol, 0.1045 mL), distilled
water (16.0 mL) and acetonitrile (CH3CN, 4.0 mL) was
vigorously stirred until complete solution was achieved. A
mixture of oxone (5 mmol, 1.537 g) and NaHCO3 (15
mmol, 1.261 g), previously ground into powder, was slowly
added in portion, and a septum with an empty balloon was
immediately placed into the flask in order to avoid over-
pressure and loss of generated singlet oxygen. The mixture
was vigorously stirred at room temperature until total dis-
appearance of the phenol. After about 1 h of stirring, the
reaction was quenched with water and extracted several
times (3–4 times) with ethyl acetate (EtOAc). Combined
ethyl acetate extracts were dried in Na2SO4 and the solvent
was removed under reduced pressure to yield the product of
p-peroxyquinol, 2. The crude compound was recrystallized
3-Isopropyl-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-6
(8aH)-one (3c)
UV spectrum (MeOH), λmax, nm: 219.4; IR (KBr, cm−1) υ:
2964.42, 2884.03 (C–H, CH3), 1666.23 (C=O, conj.),
1178.23 (C–O), 887.21 (O–O); 1H NMR (400 MHz,
CDCl3) δ: 0.82 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.46 (m,
2H, CH2–CH2–CH3), 2.42 (d, J = 3.0 Hz, 2H,
CO–CH2–CH), 4.26 (d, J = 3.0 Hz, 1H, CO–CH2–CH),
4.89 (m, CH(CH)3), 6.08 (t, J = 8.0 Hz, 1H,
CH–CH2–CH3), 6.14 (d, J = 9.0, 1H, CO–CH=CH), 6.42

Med Chem Res
(d, J = 9.0, 1H, CO–CH=CH); 13C NMR (100 MHz,
CDCl3) δ: 15.84, 18.12 (CH3), 32.20 (CH2), 34.02, 46.29
(CH), 84.50, 94.02 (CH=CH), 188.12 (C=O); MS (ES+),
m/z (%): 212.42 (100), [M]+.
86.10, 94.16 (CH=CH), 116.34, 133.00, 138.14, 142.20,
147.56, 152.02 (Ar–C), 186.37 (C=O); MS (ES+), m/z (%):
278.53 (100), [M]+.
3-(4-Hydroxyphenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-
3-Phenyl-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-6
trioxin-6(8aH)-one (3g)
(8aH)-one (3d)
UV spectrum (MeOH), λmax, nm: 229.5; IR (KBr, cm−1) υ:
3186.13 (O–H, bonded), 2968.12, 2882.40 (C–H, CH3),
1682.45 (C=O, conj.), 1584.00, 1549.12, 1468.45 (C=C,
UV spectrum (MeOH), λmax, nm: 223.0; IR (KBr, cm−1) υ:
3162.24 (O–H, bonded), 2968.00, 2874.10 (C–H, CH3),
1667.12 (C=O, conj.), 1582.19, 1560.32, 1532.21, 1426.14
(C=C, aryl), 1163.21 (C–O), 836.27 (O–O); 1H NMR (400
MHz, CDCl3) δ: 1.12 (s, 3H, CH3), 2.42 (d, J = 8.0 Hz, 2H,
CO–CH2–CH), 4.20 (d, J = 8.0 Hz, 1H, CO–CH2–CH),
6.22 (d, J = 9.0, 1H, CO–CH=CH), 6.34 (d, J = 9.0, 1H,
CO–CH=CH), 7.24 (d, J = 8.0 Hz, 1H, Ar–H), 7.37 (d, J
= 7.0 Hz, 1H, Ar–H), 7.81 (d, J = 8.0 Hz, 1H, Ar–H); 13C
NMR (100 MHz, CDCl3) δ: 15.20 (CH3), 38.42 (CH),
86.30, 92.77 (CH=CH), 116.11, 130.12, 134.04, 148.55,
156.12, 161.40 (Ar–C), 191.34 (C=O); MS (ES+), m/z (%):
246.42 (100), [M]+.
1
aryl), 1162.30 (C–O), 845.20 (O–O); H NMR (400 MHz,
CDCl3) δ: 1.22 (s, 3H, CH3), 2.48 (d, J = 8.0 Hz, 2H, CO–
CH2–CH), 4.24 (d, J = 8.0 Hz, 1H, CO–CH2–CH), 6.02 (d,
J = 9.0, 1H, CO–CH=CH), 6.40 (d, J = 9.0, 1H,
CO–CH=CH), 7.13 (d, J = 8.0 Hz, 1H, Ar–H), 7.22 (d, J
= 8.0 Hz, 1H, Ar–H), 7.49 (d, J = 8.0 Hz, 1H, Ar–H), 7.89
(d, J = 8.0 Hz, 1H, Ar–H), 9.84 (Ar–OH); 13C NMR (100
MHz, CDCl3) δ: 15.36 (CH3), 36.28 (CH), 84.10, 92.52
(CH=CH), 118.20, 130.56, 134.22, 144.21, 156.03, 168.42
(Ar–C), 190.12 (C=O); MS (ES+), m/z (%): 262.82 (100),
[M]+.
3-(3-Hydroxyphenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-
3-(4-Methoxyphenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-
trioxin-6(8aH)-one (3e)
trioxin-6(8aH)-one (3h)
UV spectrum (MeOH), λmax, nm: 217.5; IR (KBr, cm−1) υ:
3174.10 (O–H, bonded), 2970.21, 2879.23 (C–H, CH3),
1667.13 (C=O, conj.), 1595.12, 1514.23, 1423.14 (C=C,
UV spectrum (MeOH), λmax, nm: 279.5; IR (KBr, cm−1) υ:
2956.33, 2880.16 (C–H, CH3), 1674.20 (C=O, conjugated),
1642.13, 1582.30, 1424.10 (C=C, aryl), 1152.82 (C–O),
1
1
aryl), 1172.70 (C–O), 832.19 (O–O); H NMR (400 MHz,
878.41 (O–O); H NMR (400 MHz, CDCl3) δ: 1.19 (s, 3H,
CDCl3) δ: 1.19 (s, 3H, CH3), 2.42 (d, J = 3.0 Hz, 2H, CO–
CH2–CH), 4.22 (d, J = 3.0 Hz, 1H, CO–CH2–CH), 6.08 (d,
J = 10.0, 1H, CO–CH=CH), 6.32 (d, J = 10.0, 1H,
CO–CH=CH), 7.14 (d, J = 7.0 Hz, 1H, Ar–H), 7.22 (d, J
= 7.0 Hz, 1H, Ar–H), 7.48 (d, J = 8.0 Hz, 1H, Ar–H), 7.80
(d, J = 8.0 Hz, 1H, Ar–H), 9.52 (Ar–OH); 13C NMR (100
MHz, CDCl3) δ: 15.47 (CH3), 36.42 (CH), 86.20, 96.00
(CH=CH), 118.12, 132.04, 136.24, 140.10, 148.02, 158.12
(Ar–C), 188.24 (C=O); MS (ES+), m/z (%): 262.28 (100),
[M]+.
CH3), 2.43 (d, J = 4.0 Hz, 2H, CO–CH2–CH), 4.39 (s, 3H,
OCH3), 4.23 (d, J = 4.0 Hz, 1H, CO–CH2–CH), 6.04 (d, J
= 10.0, 1H, CO–CH=CH), 6.37 (d, J = 10.0, 1H,
CO–CH=CH), 7.16 (d, J = 7.0 Hz, 1H, Ar–H), 7.28 (d, J
= 7.0 Hz, 1H, Ar–H), 7.44 (d, J = 8.0 Hz, 1H, Ar–H), 7.82
(d, J = 8.0 Hz, 1H, Ar–H); 13C NMR (100 MHz, CDCl3) δ:
15.68 (CH3), 28.67 (OCH3), 38.40 (CH), 84.65, 92.28
(CH=CH), 118.18, 126.12, 132.17, 140.72, 152.32, 162.25
(Ar–C), 190.20 (C=O); MS (ES+), m/z (%): 276.42 (100),
[M]+.
3-(3,4-Dihydroxyphenyl)-8a-methyl-4a,5-dihydrobenzo-
3-(Furan-2-yl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-
1,2,4-trioxin-6(8aH)-one (3f)
6(8aH)-one (3i)
UV spectrum (MeOH), λmax, nm: 254.5; IR (KBr, cm−1) υ:
3182.67 (O–H, bonded), 2970.21, 2879.23 (C–H, CH3),
1667.13 (C=O, conj.), 1595.12, 1514.23, 1423.14 (C=C,
UV spectrum (MeOH), λmax, nm: 219.5, 259.2; IR (KBr,
cm−1) υ: 2932.10, 2872.63 (C–H, CH3), 1662.12 (C=O,
conj.), 1128.00 (C–O), 882.12 (O–O); 1H NMR (400 MHz,
CDCl3) δ: 1.18 (s, 3H, CH3), 2.20 (d, J = 3.0 Hz, 2H,
CO–CH2–CH), 4.27 (d, J = 3.0 Hz, 1H, CO–CH2–CH),
6.20 (d, J = 9.0, 1H, CO–CH=CH), 6.44 (d, J = 9.0, 1H,
CO–CH=CH), 7.06 (d, J = 8.0 Hz, 1H, furan-2-yl-H3),
7.10 (t, 1H, J = 8.0 Hz, furan-2-yl-H4), 7.48 (d, 1H, J = 9.0
Hz, furan-2-yl); 13C NMR (100 MHz, CDCl3) δ: 15.10
(CH3), 36.12 (CH), 86.52, 88.46 (CH=CH), 109.00,
1
aryl), 1161.15 (C–O), 834.70 (O–O); H NMR (400 MHz,
CDCl3) δ: 1.18 (s, 3H, CH3), 2.44 (d, J = 8.0 Hz, 2H, CO–
CH2–CH), 4.26 (d, J = 7.0 Hz, 1H, CO–CH2–CH), 6.07 (d,
J = 10.0, 1H, CO–CH=CH), 6.22 (d, J = 10.0, 1H,
CO–CH=CH), 7.42 (d, J = 8.0 Hz, 1H, Ar–H), 7.47 (d, J
= 8.0 Hz, 1H, Ar–H), 7.86 (s, 1H, Ar–H), 9.64 (Ar–OH);
13C NMR (100 MHz, CDCl3) δ: 15.42 (CH3), 36.70 (CH),

Med Chem Res
114.26, 118.40, 122.35 (Ar–C, 1H-pyrrole-2-yl), 188.20
(C=O); MS (ES+), m/z (%): 236.77 (100), [M]+.
parasitaemia in 5% HCT was used for the antimalarial assay
using 96-well microtitre plate. A stock solution of 1.0 mg/
mL of the test compound was prepared in dimethyl sulph-
oxide and subsequent dilutions were made with incomplete
RPMI to obtain different concentrations (50, 25, 12.5, 6.25,
3.13, 1.56, 0.78, 0.39, and 0.19 µg/mL) of samples (test/
standard) in duplicate. In addition, drug free negative con-
trol to assess the parasite growth and chloroquine dipho-
sphate as positive control to assess the integrity of the assay
were also maintained in duplicate in the microtitre plate.
After 40 h of incubation at 37 °C the smears were prepared
from each well (depending on the maturation of schizonts in
negative control, ≥10%), stained with 3% Giemsa and
examined under light microscope to ascertain drug sensi-
tivity by assessing the level of parasitaemia i.e., the inhi-
bition of parasite growth in terms of percent inhibition of
schizonts’ maturation (Rudrapal et al. 2013; Roy et al.
2013).
3-(Pyridin-2-yl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-
trioxin-6(8aH)-one (3j)
UV spectrum (MeOH), λmax, nm: 262.5; IR (KBr, cm−1) υ:
3023.22, 3010.23 (C–H, aryl), 2932, 2822.11 (C–H, CH3),
1667.34 (C=O, conj.), 1481.36, 1450.25 (C=C, conj.,
1
aryl), 1154.20 (C–O), 872.13 (O–O); H NMR (400 MHz,
CDCl3) δ: 1.26 (s, 3H, CH3), 2.63 (d, J = 4.0 Hz, 2H,
CO–CH2–CH), 4.32 (d, J = 4.0 Hz, 1H, CO–CH2–CH),
6.21 (d, J = 8.0, 1H, CO–CH=CH), 6.40 (d, J = 8.0, 1H,
CO–CH=CH), 7.48 (d, J = 8.0 Hz, 1H, 4-pyridyl-H2/6),
7.68 (d, J = 8.0 Hz, 1H, 4-pyridyl-H3/5); 13C NMR (100
MHz, CDCl3) δ: 15.65 (CH3), 34.12 (CH), 84.72, 92.68
(CH=CH), 116.20, 122.58, 128.34, 140.67 (Ar. C, 4-pyr-
idyl), 188.42 (C=O); MS (ES+), m/z (%): 247.03 (100),
[M]+.
Observation and assessment of activity
Evaluation of antimalarial activity
Each test compound was assayed in duplicate and number
of schizonts was counted against 200 asexual parasites per
replica under light microscope. The number of schizonts
count was also assessed in negative control maintained in
duplicate in the microtitre plate. Mean number of schizonts
for duplicate observations was calculated at each con-
centration of test/standard samples compounds. Test values
were compared with the control values. The IC50 (con-
centration at which the inhibition of parasite growth repre-
sents 50%) values in µg/mL were also calculated using the
NonLin v1.1 software (Kashyap et al. 2016). Test results
were compared with the standard results of CQ.
Chemicals and parasite strains
All the synthesized compounds, 3a–j were evaluated for
in vitro antimalarial activity at multiple doses against CQ-
sensitive (3D7) and CQ-resistant (RKL9) strains of P. fal-
ciparum using blood (erythrocytic) stage of parasite. The
solvents and reagents used in the antimalarial study were of
analytical grade and were procured from Sigma-Aldrich
Corporation (USA) and HiMedia Lab. Pvt. Ltd. (Germany).
Assay procedure
The in vitro antimalarial activity screening was carried out
by microculture Giemsa Stained slide method based on light
microscopy. Schizont maturation inhibition assay technique
was used for the quantitative assessment of parasitaemia
and evaluation of drug sensitivity. The assay was conducted
using the blood parasite of in vitro P. falciparum culture
according to the previously reported methods (Trager and
Jensen 1976; Lambros and Vanderberg 1976). Briefly, a
continuous culture of P. falciparum strain was maintained
in vitro in A+ human red blood cells (RBCs) diluted to 5%
hematocrit (HCT) in RPMI (Roswell Park Memorial Insti-
tute)-1640 medium supplemented with 25 mM HEPES [(N-
2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)] buffer,
1% D-glucose, 5.0% sodium bicarbonate, gentamycin (40
μg/mL), and 10% human AB+ serum. Incubations were
done at 37 °C and 5% CO2 level in a modular incubator.
The culture was passaged with fresh mixture of erythrocytes
and complete RPMI medium for every day to maintain cell
growth. D-sorbitol (5%) synchronized 1% ring stage
In silico studies
Molecular docking study
Molecular modeling studies were carried out using Dell
Precision work station T3400 running Intel Core2 Duo
Processor, 4 GB RAM, 250 GB hard disk and NVidia
Quodro FX 4500 graphics card. Two-dimensional (2D)
structures of all compounds were built on Chemdraw Ultra
10.0 (Cambridge Soft Co., USA, 2010) and Marvin Sketch
(ChemAxon LLC, Cambridge, USA, 2015) software. The
2D structures were later transformed into three-dimensional
(3D) structures using the converter module of Biovia Dis-
covery Studio (DS) v 4.5 (2015) software. All the con-
formations generated were then energetically minimized by
a single step Steepest Descent method using CHARMM
(Chemistry at HARvard Macromolecular Mechanics,
Cambridge, USA) Force Field with 5000 iterations and a

Med Chem Res
minimum root mean square (RMS) gradient of 0.01 kcal/
mol/Å (Singh et al. 2016).
receptor–ligand complexes. Receptor–ligand interaction
study out for better understanding the molecular interactions
between the binding site residues of receptor molecule and
complimentary groups/atoms of ligands involved in
interactions.
The x-ray crystal structure of falcipain 2-E-64 (PDB id:
3BPF) was retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/) and Chain A of the protein
determined at a resolution of 2.9 Å was used in the study
(Sashidhara et al. 2012a, b). Prior to docking, protein was
prepared using protein preparation wizard tool of DS. Polar
hydrogen atoms were added to the proteins and charges
were assigned. All bound water molecules, other heteroa-
toms and ligands were excluded from the crystal structure as
they were not significant for the proteins’ function. Subse-
quently, the 3D structure of protein was optimized by
energy minimization using CHARMM Force Field. It was
done in two steps to remove the bad steric clashes using
Steepest Descent and Conjugate Gradient methods for
5000 steps at RMS gradients of 0.01 and 0.05 kcal/mol/Å,
respectively (Singh et al. 2016).
Molecular docking studies were carried out using Lib-
dock program available in Biovia Discovery Studio (DS) v
4.5 (2015) software packages. After energy minimization,
the Chain A of falcipain 2 protein was defined as a receptor
and the binding site sphere was selected based on the ligand
binding location of E-64. A receptor grid was thereby
generated around the binding cavity (active sites) of protein
by specifying the key amino acid residues (Cys 42, Gly 83,
and His 174) (Sashidhara et al. 2012a, b). In DS, binding
site sphere was set with a radius of 20 Å and x, y, z
dimensions of −52.25, −4.46, −19.25, respectively. Flex-
ible molecular docking was performed where the protein
was held rigid while the ligands were allowed to be flexible
during refinement. During docking, the falcipain 2-E-64
complex was imported and E-64 molecule (co-crystal
ligand) was removed, and ligands were placed in the pre-
dicted binding site (grid box) and docking was performed
using the Dock Ligands module of LibDock genetic algo-
rithm program (Usha et al. 2014) of DS. All other docking
and consequent scoring parameters used were kept at their
default settings. The LibDock scores of the docked ligands
were calculated. A number of dock poses were studied to
know the best binding mode of receptor–ligand complex in
terms of scoring function. The number of dock poses gen-
erated was 20 for each compound. All docked poses were
scored, ranked and the best pose of each compound having
the highest score was selected and it was later used for the
receptor–ligand interaction analysis. Interaction of ligands
with receptor was studied to know the best binding orien-
tation of receptor–ligand complex having maximum Lib-
Dock score. Binding modes of the best pose for each
compound was also analyzed with the help of 3D
receptor–ligand complex. Different non-bonding interac-
tions (hydrogen bonding and hydrophobic) were also ana-
lyzed with the help of 2D diagram of docked
Molecular properties calculation and drug-likeness studies
In silico calculations of the molecular properties and drug-
likeness parameters for all compounds, 3a–j were per-
formed based on theoretical approaches to identify the
compounds which violate the optimum requirements for
drug-likeness. Molecular properties (molecular weight,
LogP value, number of hydrogen bond acceptor(s), number
of hydrogen bond donor(s), total polar surface area (TPSA))
incorporated in Lipinski’s rule of five (Lipinski et al. 1997)
and other physicochemical parameters like aqueous solu-
bility (LogS), molar refractivity and molar volume were
calculated using Calculation of Molecular Properties mod-
ule of Biovia DS v 4.5 software. The number of rotatable
bonds was predicted using Molsoft Online software (http://
www.molsoft.com), and non-violation of drug-likeness and
drug-likeness score were calculated using Molinspiration
online software (http://www.molindpiration.com).
ADMET prediction
ADME-toxicity (ADMET) for all the target compounds was
calculated in silico using ADMET descriptor module of
Biovia DS v 4.5 software. Six mathematical models (aqu-
eous solubility, blood-brain barrier (BBB) penetration,
cytochrome P450 2D6 inhibition, hepatotoxicity, human
intestinal absorption, and plasma protein binding) were used
to quantitatively predict properties related to ADMET
characteristics or pharmacokinetics of molecules (Alam and
Khan 2014). These properties influence oral bioavailability,
cell permeation, and metabolism of drug molecules.
Results and discussion
Design of compounds
Rationale behind the design strategy involved selection of
1,2,4-trioxane ring system as the parent peroxide scaffold
considering the pharmacodynamic importance of 1,2,4-
trioxane component for antimalarial activity of ART and
related endoperoxides. Our study was aimed at developing
1,2,4-trioxane derivatives as newer synthetic peroxide-
based antimalarial agents having comparatively simpler
molecular framework than conventional antimalarial drug
molecules with activity against resistant malaria parasites.
Target compounds (Fig. 2) were designed with diverse

Med Chem Res
CH3
H
was a [4 + 2] cycloaddition between electron rich p-alkyl
phenol and O2 (singlet oxygen) generated in situ in the
reaction system from oxone, which gave p-peroxyquinol
Peroxide bond
CH3
1
H
O
H
4
5
8
O
O
O
R
O
intermediate through
a 1,4-endoperoxide i.e., perox-
3
2
6
O
O
yhemiacetal. Subsequent desymmetrization of p-perox-
yquinol after reaction with aldehydes with the help of PTSA
catalyst yielded different trioxane derivatives (Fig. 4)
(Carreno et al. 2006). The progress of reactions was mon-
itored by the silica gel-G thin-layer chromatography (TLC)
and the spots were visualized by iodine vapors. The purity
of the synthesized compounds was ascertained by melting
point determinations and silica gel G TLC. All the com-
pounds were obtained in good yields with high purity. The
physicochemical details of synthesized compounds are
summarized in Table 1.
7
O
1
H3C
CH3
Trioxane
Lactone
O
Trioxane
(Tetracyclic endoperoxide)
Artemisinin
(Bicyclic endoperoxide)
Trioxane derivatives
R= alkyl/aryl/heteroaryl
Fig. 2 Design of 1,2,4-trioxane derivatives
1
The spectral (UV, IR, HNMR, 13CNMR, mass) data
OH
O
depicted in the experimental section are in close agreement
with the structures of synthesized compounds. In UV
spectra (in methanol), λmax values were observed at the
range of 202.5–279.5 which was due to the presence of
chromophoric trioxane ring system along with alkyl/aryl/
heteroaryl substituents at the C-3 position of the parent
scaffold. IR spectral data showed absorption frequencies
characteristic to specific functional groups and/or bonds
present in the structure of synthesized compounds. The
appearance of a broad peak at 3174.10, 3182.67, and
3186.13 cm−1 confirmed the presence of phenolic –OH
group in the structure of 3e, 3f, and 3g, respectively. The
carbonyl (C=O) group was assigned with a strong and
sharp absorption band in the region of 1682.45–1643.20
cm−1 for all the synthesized compounds. Distinguished
C–O stretching band was appeared in the range of
1078.23–1128.00 cm−1. A weak absorption band observed
at 898.20–832.119 cm−1 was due to O–O stretching. The
presence of aliphatic C–H (CH3/CH), aromatic C–H
(CH=C) and aromatic C=C groups in the structure of
synthesized compounds were also confirmed by character-
istic peaks as depicted in the experimental section.
H
O
O
O
R
a
b
O
CH3
H3C
OOH
CH3
1
3
2
Fig. 3 Scheme of synthesis of target compounds, 3a–j. Reagents and
conditions: a Oxone, NaHCO3/CH3CN/H2O, rt; b RCHO, 40–50 °C,
8–12 h, CH2Cl2, PTSA
substitution patterns (alkyl/aryl/heteroaryl groups) at C-3
position of the 1,2,4-trioxane structural scaffold, based on
the molecular manipulation approach of drug design with
due consideration to all structure and property parameters
that are relevant to biological activity. The 1,2,4-trioxane
ring system was considered as the basic requirement for the
antimalarial activity and substitutions with groups/moieties
having electronic property of (s) was the main motif behind
the design task.
Chemistry
1HNMR spectra exhibited resonance signals with che-
mical shift (δ, ppm) values characteristic to various struc-
tural protons which concorded the anticipated structure of
synthesized compounds. A singlet for three protons of CH3
group of the trioxane ring system was observed in the range
of 1.12–1.26 ppm. Depending on the nature of the sub-
stituent at C-3 position of the trioxane ring, distinct δ values
were also observed with expected splitting patterns (doub-
lets, triplets or multiplets) and coupling constant (J) values.
A broad singlet at 9.52, 9.64, and 9.84 ppm was observed
due to aromatic OH proton for compounds, 3e, 3f, and 3g,
respectively. Distinct singlet due to three protons of the
OCH3 group was appeared at 4.39 ppm for compound 3h.
Aromatic protons including heteroaryl substituents were
appeared in the range of 7.14–7.89 ppm. Aliphatic methyl
In our study, new series of 1,2,4-trioxane derivatives were
synthesized and evaluated in vitro for their antimalarial
activity. Substitutions with different aliphatic, aromatic, and
heteroaromatic groups at the C-3 position of 1,2,4-trioxane
ring system afforded target trioxane derivatives obtained as
single drug conjugates. The standard reaction procedure
(Rubush et al. 2012) involving a facile synthetic route
depicted in Fig. 3 was employed for the preparation of
1,2,4-trioxane derivatives. The oxidative dearomatization of
p-cresol, 1 with oxone first yielded the intermediate com-
pound, p-peroxyquinol, 2 which was later allowed to react
with different aldehydes (aliphatic/aromatic/heteroaromatic)
in presence of an acid catalyst called PTSA, to obtain
desired trioxane deivatives, 3a–j. The first step of reaction

Med Chem Res
Fig. 4 Reaction mechanism of
synthesis of 1,2,4-trioxane
derivative(s)
O
OH
O
OH
H
O
O
O
R
H
1O2
R
H2O
O
O
O
CH3CN/H2O
rt
CH2Cl2/H2O
40-50oC
CH3
CH3
H3C OOH
CH3
Trioxane derivative(s)
p-Cresol
p-Peroxyquinol
1,4-Endoperoxide
Table 1 Physicochemical data
Comp.
R
MF
Color (state)
% Yield
MP (oC)
Rfa
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
C8H10O4
C9H12O4
Colorless (solid)
Colorless (solid)
Colorless (solid)
Colorless (solid)
84
78
68
74
102–104
108–110
118–121
122–124
–
0.67
0.72
0.76
0.72
0.68
0.66
0.76
0.72
0.65
0.69
Methyl
Isopropyl
C
C
C
C
C
C
C
11H16O4
14H14O4
14H14O5
14H14O6
14H14O5
15H16O5
12H12O5
Phenyl
3-Hydroxyphenyl
3,4-Dihydroxyphenyl
4-Hydroxyphenyl
4-Methoxyphenyl
Furan-2-yl
Brown (semi-solid)
Brown (semi-solid)
Colorless (solid)
68
70
72
74
65
68
–
120–124
128–130
–
Pale yellow (solid)
Brown (semi-solid)
Brown (semi-solid)
3j
a
Pyridine-2-yl
C13H13O4
–
Diethyl ether:EtOAc:acetic acid (1:3:0.5)
(CH3) and methine (CH) protons were observed in multiplet
pattern (doublet or triplet) at δ values in the downfield
region for compounds 3b and 3c, as depicted in experi-
mental section (Silverstein and Webster 2005).
Table 2 In vitro antimalarial activity data
Comp.
IC50 in µg/mL SDa
Pf 3D7
Pf RKL9
In 13C NMR spectra, distinguished resonance signal due
to the carbonyl (>C=O) carbon was appeared in the range
of 188.12–191.34 ppm. Aromatic/heteroaromatic carbons
were appeared at 109.00–168.42 ppm. Characteristic
downfield signals for aliphatic carbons were observed for
CH3 (14.20–18.24 ppm), OCH3 (28.67 ppm, 3h) and CH
(34.12–46.39 ppm) groups. Olefinic (CH=CH) carbons
were also assigned and depicted in the experimental section.
The mass spectra of 3a–j exhibited prominent molecular ion
peaks, [M]+ which finally confirmed the structure of com-
pounds with the anticipated mass corresponding to their
respective molecular formula.
3a
3b
3c
3d
3e
3f
0.652 0.002
0.648 0.003
0.642 0.005
0.638 0.006
0.476 0.003
0.403 0.002
0.391 0.002
0.478 0.004
0.492 0.003
0.478 0.005
0.044 0.006
2.167 0.004
2.198 0.002
2.240 0.002
2.018 0.004
1.925 0.003
1.222 0.002
0.827 0.002
1.973 0.003
1.989 0.005
1.958 0.004
0.205 0.006
3g
3h
3i
3j
CQb
a
Calculated using NonLin v1.1 software, mean SD (standard
deviation) of two replicate observations (counted against 200 asexual
cells per replicate)
Antimalarial activity
b
CQ—Chloroquine used as standard reference drug
Results of in vitro antimalarial activity (Table 2) revealed
that all the synthesized compounds (3a–j) showed activity
against both CQ-sensitive (3D7) and CQ-resistant (RKL9)
strains of P. falciparum in a dose dependant manner with
IC50 values ranging from 0.391 to 0.652 and 0.827 to 2.240
µg/mL against CQ-sensitive and CQ-resistant strains of P.
falciparum, respectively. Out of ten tested compounds, five
compounds (3e–j) exhibited good activity as compared to
rest of the analogs, with better activity profile against
resistant strain than the sensitive one. The IC50 value of the
best compound (3g) was found to be 0.391 and 0.837 µg/
mL against sensitive strain and resistant strain of P. falci-
parum, respectively. Results of tested compounds were
comparable with that of the standard drug, CQ (IC50
=
0.044 and 0.205 µg/mL against sensitive and resistant strain

Med Chem Res
of P. falciparum, respectively). Figure 5 shows photographs
of the effect of the most active compound, 3g against both
CQ-sensitive (3D7) and CQ-resistant (RKL9) strains of P.
falciparum.
Results indicate different structural substitutions such as
non-bulky alkyl and bulky aryl/heteroaryl groups at C-3
position of the ring system modulates the antimalarial effi-
cacy of prepared 1,2,4-trioxane derivatives. Some degree of
variations in activity among synthesized analogs might be
(4-hydroxyphenyl, compound 3g). The findings of our
present study are also consistent with earlier report
(Sashidhara et al. 2012a, b) on trioxane-based compounds
shows excellent antiparasitic activity upon incorporation of
an aryl group. It is seen that bulkiness of substituents is
important for the activity. However, aryl substitution seems
to be more important than alkyl or heteroaryl substitution. It
is important to note here that besides bulkiness of sub-
stituents, lipophilicity (hydrophobicity) and ionization
potential (basicity) of the molecule are also considered to
play a crucial role for the activity. The lipophilicity (logP) is
inevitable for permeation of parasitic membrane and basi-
city (pKa) is required for accumulation of trioxane molecule
in the acidic food vacuole of parasites.
Literature report that 1,2,4-trioxane structure lacking the
lactone ring play an important role for the antimalarial
activity, but 1,2,4-trioxane ring alone is insufficient for
optimal antimalarial potency as compared to ART and its
semi-synthetic analogs (Liu et al. 2011). The reductive
cleavage of the peroxide bond in vivo by haem [Fe(II)]
(released during hemoglobin degradation process in para-
sitized RBC) and subsequent production of cytotoxic
carbon-centered radicals target cell proteins/enzymes and
destroys parasites (Meshnick 2002; Cumming et al. 1996;
Paul et al. 2004).
due to diverse structural substitutions.
A
brief
structure–activity relationship (SAR) study can be depicted
as follows: no substitution or non-bulky small alkyl sub-
stituents like methyl, or isopropyl moieties are of con-
siderable importance for the activity. Substitution with
bulky aryl moieties also contributes good antimalarial
potency to a similar or greater extent as compared to alkyl
substituted analogs. Compounds with phenyl substitutions
sufficiently retains the activity, whereas, 3-hydroxyphenyl,
3,4-dihydroxyphenyl, 4-methoxyphenyl substitution con-
siderably increases the activity. Substitution with 4-
hydroxyphenyl significantly increases the activity. With
bulky heteroaryl moieties like furan-2-yl and pyridine-2-yl
substitutions also potentiate the activity. It is also clear that
compounds with five membered heterocyclic rings like
furan-2-yl are less important for the activity than six
membered heteroaryl systems like pyridyl ring. Upon cri-
tical analysis of SAR, the most notable point found is that
substitutions electron releasing groups (alkyl, aryl rings
substituted with electron releasing groups) considerably
increases the activity. More illustratively, phenyl substituent
with electron releasing (OH, OCH3 etc.) groups sig-
nificantly increases the activity (Fig. 6). Substitutions of
electron releasing groups at para or both at meta and para
have greater contributing effects than meta substitutions.
Di-substitution (3,4-dihydroxy, compound 3f) is less
important for the activity than mono meta-substitution
(3-hydroxyphenyl, compound 3e), and para-substitution
H
4
5
Substitution
O
O
O
R
(modulates activity)
6
7
3
O
- R: e releasing gr. increases activity
2
8
1
CH3
Peroxide bond
Key Pharmacophore
(for antimalarial activity)
Fig. 6 Effect of substitutions on antimalarial activity of 1,2,4-trioxane
derivatives
Fig. 5 Photographs showing
in vitro antimalarial activity of
the most active compound, 3g
against a CQ-sensitive 3D7 and
b CQ-resistant RKL9 strains of
P. falciparum
Ring
Ring
Ring
Trophozoite
Schizont
(a)
(b)

Med Chem Res
Fig. 7 a Optimized co-crystal
structure of falcipain 2 (Chain
A)-E-64, b receptor grid for
docking
Docking simulations
Docking results revealed that LibDock program suc-
cessfully docked all 1,2,4-trioxane derivatives into the
binding pocket of falcipain 2 enzyme. All compounds
which were active in in vitro test could bind with the active
site of falcipain-2 with high docking score (LibDock) and
binding affinities in the range of 75.682–125.232. The
LibDock scores are summarized in Table 3. All trioxane
derivatives except 3a, 3b, and 3c exhibited more binding
affinity (higher dock scores) than the co-crystal ligand, E-64
(Libdock score = 100.364). Five compounds which showed
more potency in in vitro study also ranked top in docking
study with LibDock scores of 120.143, 122.807, 125.232,
114.923, 101.911, and 118.049 for 3a, 3b, 3c, 3d, 3e, 3f,
and 3g, respectively. It is important to note here that
docking results are generally analyzed by a statistical
scoring function which converts interacting energy (binding
energy) into numerical values called as the docking score,
and therefore docking score is nothing but an expression of
binding energy (Lionta et al. 2004). Binding energy (ΔG)
includes the sum of all non-bonded interactions including
hydrogen bonding, hydrophobic, and Vander Walls
between protein residues and bound ligand (Kitchen et al.
2004). LibDock score is useful to assess the antimalarial
activity of ligands as P. falciparum falcipain 2 inhibitors.
Docking scores of compounds supported the results of
in vitro antmalarial activity of synthesized 1,2,4-trioxane
derivatives.
Plasmodium cysteine protease falcipain 2 enzyme plays an
essential role in the degradation of host hemoglobin into
smaller peptides which takes place in the acidic food
vacuole of P. falciparum. Recent literature (Oliveria et al.
2013) claims that endoperoxide antimalarials have potential
to inhibit falcipain 2 enzyme and therefore, drug targeting
of this particular enzyme would be an attractive avenue in
the design and development of new antimalarial drugs.
Accordingly, a molecular docking study was performed for
all newly designed compounds 3a–j, using the falcipain 2
enzyme as receptor molecule.
The protein model used for docking study was validated
as follows: the three dimensional crystal structure of falci-
pain 2 co-crystallized with the inhibitor trans-epox-
ysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) with
active site (receptor grid model) as defined by Cys 42, Gln
36, and His 174 residues was optimized and used for the
study. The co-crystal structure of falcipain 2-E-64 (PDB:
3BPF) and the receptor grid model used for docking study
are represented in Fig. 7. Co-crystallized ligand, E-64 was
re-docked using flexible docking simulations (LibDock
program of DS) into the original structure of the target
protein, falcipain-2 by non-covalent docking procedure. For
this study, docking parameters were set to the software’s
default values. E-64 was successfully re-docked to the
predicted active sites of falcipain 2 with an acceptable
RMSD value of 1.124 Å. Further, in order to reproduce an
experimentally observed ligand-binding mode, the co-
crystallized ligand, E-64 (a selective falcipain 2 inhibitor)
was used as reference ligand. Results confirmed experi-
mental binding conformations of E-64 in the binding
pocket of receptor molecule. The binding modes and
ligand–protein interaction diagrams obtained in re-docking
of E-64 are shown in Fig. 8a.
Protein–ligand docking was performed to generate the
bioactive binding poses of designed inhibitors in the active
site of falcipain 2 enzyme. LibDock is a high throughput
docking algorithm that finds various conformations of the
ligands in the protein active site based on polar interaction
sites (hotspots) (Usha et al. 2014). The 3D poses of bound
ligands were visualized which revealed that the best
orientation of the ligand relative to the receptor as well as
the conformation of the ligand and receptor (best fit of

Med Chem Res
Fig. 8 a Redocked conformer
(pose) of co-crystal ligand, E-64
in the active site of the protein
falcipain 2 (left) and 2D
representation of the binding
interaction (right); b Binding
mode (left) and 2D receptor-
ligand interaction diagram
(right) of compound 3g at
binding pocket of falcipain 2
Table 3 LibDock scores and no. of H-bonds
Table 4 revealed the hydrogen bonding interactions of
five most potent compounds (3e–j) with active site residues
such as Asn 38, Asp 154, Asp 170, Arg 12, Gln 36, Glu 15,
Glu 138, Gly 13, Gly 15, Gly 169, Cys 39, and His 19.
Compound 3g which showed the highest antimalarial
activity among the series, could bind the active sites of
falcipain-2 mainly by hydrogen bonding interactions. The
3D binding modes and 2D interaction diagrams of the most
potent compounds, 3g are shown in Fig. 8b. Compound 3g
formed four strong hydrogen bonds with residues like Arg
12 (O··H··O), Cys 39 (O··H··O), Gln 36 (O··H··O) and Asp
154 (O··H··O) with bonding distances of 1.686, 2.038,
2.745, and 2.934 Ao, respectively. Analysis of best docking
pose of 3g revealed that the trioxane scaffold was oriented
in the binding cavity (active site residues) of falcipain 2
receptor molecule. In 2D diagram, the trioxane moiety
could occupy the binding sites of falcipain-2 through strong
H-bonding interactions along with hydrophobic interac-
tions. Such interactions afforded good stability between
receptor molecule and ligand. Trioxane moiety interacted
with multiple amino acid residues such as Arg 12, Gln 36,
and Asp 154 residues interacted with the 4-hydroxyphenyl
in compound 3g, respectively. Strong H-bonding interac-
tions between C=O group of the trioxane and amino acid
residues were also observed. The OH groups were involved
for H-bonding interactions from the substituent moieties for
compound 3g. Further analysis of docking interactions, it
Comp.
LibDock score
No. of H-bond(s)
3a
3b
3c
75.682
2
2
2
4
4
4
4
6
3
5
7
77.062
97.656
3d
3e
107.807
120.143
122.807
125.232
114.923
101.911
118.049
100.364
3f
3g
3h
3i
3j
E-64a
a
Co-crystal ligand
ligand in the receptor molecule). Analysis of 2D diagram
indicated that various non-bonded interactions mainly polar
hydrogen bonding interactions were involved between
binding site residues (active site amino acids) and ligand
moieties/atoms. Table 3 also depicts the number of hydro-
gen bonds (H-bonds) for all docked compounds. Higher the
number of hydrogen bonds, higher is the binding affinity.
Apart from hydrogen bonding interactions other non-
bonded interactions like hydrophobic bonding were also
observed.

Med Chem Res
Table 4 Details of hydrogen
bonding ligands and receptor
molecule for five most active
compounds
Comp.
H-bond(s)
4
H-binding ligand
H-binding receptor
H-bond distance (Ao)
Element
Type
Residue
Element
Type
3e
O
H
H
O
H
H
O
H
O
H
O
O
O
O
O
O
H
H
O
N
O
H
H
O
A
D
A
A
A
D
A
A
A
D
A
A
A
A
A
A
D
D
A
A
A
D
D
A
ARG 12
CYS 39
ASN 38
GLU 15
GLY 13
CYS 39
HIS 19
H
O
O
H
O
O
H
O
H
O
H
H
H
H
H
H
O
O
H
H
H
O
O
H
D
A
D
D
D
A
D
D
D
A
D
D
D
D
D
D
A
A
D
D
D
A
A
D
1.467
2.123
2.056
2.078
1.458
2.032
2.490
2.123
1.686
2.038
2.745
2.934
2.005
3.074
2.826
2.438
3.056
2.995
2.999
2.866
2.474
2.827
2.885
2.999
3f
4
4
6
ASP 154
ARG 12
CYS 39
GLN 36
ASP 154
ASP 170
ARG 12
GLY 169
HIS 19
3g
3h
GLY 13
GLU 15
ARG 12
ARG 12
HIS 19
3j
6
GLY 15
GLU 15
ARG 12
was found that trioxane ring played a crucial role in
protein–ligand binding. Substituents increased binding
strength by forming additional H-bonds that facilitated
much stronger interaction of ligands with the receptor
(falcipain-2 protein) molecule with optimum binding affi-
nity to achieve desired antimalarial activity.
and the affinity between the ligand and the receptor with the
correct poses of ligands in the binding pocket of a protein
(Lionta et al. 2004; Usha et al. 2014; Nikam et al. 2015;
Sapre et al. 2008).
Molecular properties and drug-likeness
Docking is generally used to find the best binding
orientation of small molecules bound to their target protein
molecules in order to predict the binding affinity and
thereby biological activity of the small molecules. Hence,
docking plays an important role in the rational drug design.
To evaluate the ligand–receptor interactions the best pose of
receptor–ligand complex should possess the lowest free
energy (binding energy) which differ from experimentally
observed other structure and estimate the binding affinity.
Search algorithm is used to generate different poses of the
ligand within the active site of molecules, orientations of
particular conformations of the molecule in the binding site.
Scoring function is used to identify the most likely pose for
an individual ligand to assign a priority order to a set of
diverse ligands docked to the same protein and hence esti-
mate binding affinity. Using scoring function, one can
predict the binding conformation of a ligand in its receptor
The results of predicted Lipinski’s parameters and other
drug-likeness properties of the synthesized compounds, 3a–
j are depicted in Table 5. Results revealed that all the
compounds possessed good drug-like properties based on
Lipinski’s rule of five with additional parameters such as
molar solubility (MS), molar volume (MV), molar refrac-
tivity (MR), and number of rotable bonds (nRotB). All
compounds obeyed Lipinski’s rule of five and Veber rule.
Lipinski rule of five is a rule to evaluate drug likeness to
determine if a chemical compound has a certain pharma-
cological or biological activity to make it an orally active
drug (Khoshneviszadeh et al. 2016). According to Lipins-
ki’s rule, compounds are more likely to be drug-like and
orally bioavailable if they obey the following criteria:
LogPo/w (octanol/water partition coefficient) ≤ 5, MW
(molecular weight) ≤ 500, HBA (hydrogen bond

Med Chem Res
Table 5 Calculated molecular properties and drug-likeness parameters
Comp.
Lipinski’s parameters
MS
MR
MV (A3)
nRotB
DL score
MW
LogP
nHBA
nHBD
TPSA (A2)
nViolations
3a
3b
3c
3d
3e
3f
170.16
184.18
212.24
246.26
262.26
278.26
262.26
276.29
236.23
247.25
0.11
0.45
1.28
2.17
1.79
2.12
1.79
2.26
1.32
2.39
4
4
4
4
5
4
5
5
4
4
0
0
0
0
1
0
1
0
0
1
44.76
44.76
44.76
44.76
64.99
44.76
64.99
53.99
57.90
60.55
0
0
0
0
0
0
0
0
0
0
−0.57
−1.00
−1.64
−2.68
−2.21
−2.42
−2.21
−2.81
−2.08
−2.21
40.78
45.58
54.46
65.27
66.97
68.79
66.97
71.74
57.68
71.87
187.56
202.89
236.53
255.79
268.63
262.02
268.63
287.64
241.85
296.39
0
0
1
1
1
1
1
2
1
1
−1.09
−0.83
−0.85
−0.82
−0.17
−0.72
−0.17
−0.54
−0.70
−1.24
3g
3h
3i
3j
MW molecular weight, LogP log of octanol/water partition coefficient, nHBA no. of hydrogen bond accepror(s), nHBD no. of hydrogen bond donor
(s), TPSA total polar surface area, nViolations no. of rule of five violations, MS molar aqueous solubility, MR molar refractivity, MV molar volume,
nRotB no. of rotable bonds, DL drug-likeness
acceptors) ≤ 10 and HBD (hydrogen bond donors) ≤ 5
(Sharma et al. 2016). To further substantiate Veber et al.
stated that compounds with ≤10 rotatable bonds and TPSA
of ≤140 A2 are more likely to show membrane permeability
and good bioavailability (Khoshneviszadeh et al. 2016;
Faidallah et al. 2016). Lipinski rule of five is considered
predictive for oral bioavailability; however, 16% of oral
drugs violate at least one of the criteria and 6% fail in two or
more (Khoshneviszadeh et al. 2016). In our study, com-
pounds did not violate Lipinski rule of five parameters. Poor
absorption or permeation of a ligand is more likely if a
drug-like molecule have more than one of five rule viola-
tions. Values of LogP, MW, and TPSA indicated that
compounds possessed good membrane permeability and
oral bioavailability, whereas, nRotb bonds suggested that
compounds had good intestinal availability. MS data indi-
cated good bioavailability of compounds if given by oral
route. MV and MR values were also found in permissible
range which indicated good oral bioavailaility for all the
compounds. Hydrophobicity, membrane permeability and
bioavailability are dependent on molecule’s MW, LogP,
MS, HBA, and HBD. Molecules violating more than one of
these rules fail to exhibit optimum bioavailability. Sufficient
water solubility is also important for optimal bioavailability
of drugs. Number of rotable bonds is important for mole-
cular conformational studies (i.e., stereoselectivity of drug
molecules) for optimal binding with the receptor molecule.
Reduced molecular flexibility, as measured by the number
of rotatable bonds, and polar surface area or total hydrogen
bond count (sum of donors and acceptors) are some
important predictors of good oral bioavailability, indepen-
dent of molecular weight (Sharma et al. 2016). Further,
TPSA, MV, and MR are also useful parameters for drug’s
transport and biodistribution (Sharma et al. 2016). The drug
score combines drug-likeness, lipophilicity, solubility,
molecular weight, and the risk of toxicity into a single
numerical value that can be used to predict a global value
for each compound as a potential new drug candidate
(Kashyap et al. 2016). Drug-likeness score were obtained in
the range of −1.24 and −0.17. Even though compounds
exhibited negative drug-likeness values, scores were in
acceptable range and indicated that compounds possessed
potential as new drug candidates. The overall analysis of
drug-likeness studies strongly suggested that newly
designed 1,2,4-trioxane derivatives possessed good drug-
likeness behavior favorable for optimal membrane perme-
ability, transport and bioavailability, and eventual interac-
tion with the receptor molecule.
Since all newly designed compounds showed favorable
drug-like properties a reasonable correlation can be drawn
between their calculated drug-like properties and in vitro
antimalarial activity profile. LogPo/w is a direct indicator of
lipophilicity of drug substances. Higher the value of logP,
better the biological membrane permeability. It is inevitable
for a molecule to have sufficient lipophilicity which is
required for its optimal bioavailability and biological action.
Hydrogen bond acceptor and donor groups are of para-
mount importance required to achieve optimal drug action.
PSA is also closely related to the hydrogen bonding
potential of a drug molecule. Practically, a compound with
all drug-like properties in the desired range appears to
exhibit high levels of therapeutic potency. Good drug-like
properties and activity are complementary, and hence
balanced attention to both properties and bioactivity could
suitably transform a ligand to a good drug lead (Grover
et al. 2014; Debnath and Ganguly 2016; Kerns and Di 2008;
Kashid et al. 2013). Better antimalarial activity of the top
six compounds, 3a–j might probably due to their sufficient

Med Chem Res
PP binding
Table 6 Theoretical ADMET
parameters
Comp. Aqueous
solubility
BBB
penetration
CYP P450 2D6 Hepatotoxicity Intestinal
inhibition
absorption
3a
3b
3c
3d
3e
3f
3
3
3
2
3
3
3
2
3
3
2
2
1
1
2
1
2
2
2
2
False
False
False
False
False
False
False
False
False
False
True
True
True
False
False
False
False
False
True
False
0
0
0
0
0
0
0
0
0
0
False
False
False
True
False
False
False
True
True
False
3g
3h
3i
3j
Aqueous solubility: 3-good, 2-low; Blood brain barrier (BBB) penetration: 3-low, 2-medium, 1-moderate;
Cytochrome (CYP) P450 2D6 inhibition: false-non-inhibitor; Hepatotoxicity: true-toxic, false-non-toxic;
Intestinal absorption: 0-good; Plasma protein (PP) binding: true-highly bounded, false-poorly bounded
lipophilicity along with balanced polar properties likes
HBD, HBA groups, rotable bonds, and PSA. They collec-
tively determine membrane permeability as well as transport
property of drug molecule. Because of having optimal
lipophilicity compound readily penetrated parasite’s cell
membrane that led to attain an intracellular concentration
for desired antimalarial activity.
along with various substitution patterns on the overall
antimalarial activity of 1,2,4-trioxane analogs. Pharmaco-
dynamic significance and unique structural simplicity of
trioxane molecules highlighted their potential to be used as
future antimalarial agents. In silico studies also confirms
that newer 1,2,4-trioxane derivatives developed as single
drug conjugates possess target specific action against P.
falciparum falcipain 2 receptor with well defined physico-
chemical, pharmacokinetic and toxicity properties. All these
results yielded valuable information for optimization of
1,2,4-trioxane-based compounds to be developed as novel
as falcipain 2 inhibitors with the help of structure-based
ADMET prediction
The ADMET values of newly designed 1,2,4-trioxane
derivatives presented in Table 6 were found in acceptable
range with favorable ADMET properties. All the com-
pounds were predicted to have good intestinal absorption
and non-inhibitors of cytochrome P450 2D6 (CYP2D6)
with medium to moderate BBB penetration. BBB penetra-
tion is mandatory for the drug to be used in the treatment of
cerebral malaria. The CYP2D6 enzyme is one of the
important enzymes involved in drug metabolism. The
aqueous solubility prediction (defined in water at 25 °C)
indicated that most of the compounds were soluble in water.
The predictive hepatotoxicity was observed for a few
compounds among three series. Some of the compounds
were found to be highly bound with plasma protein, while
some were poorly bound with plasma protein.
drug
design
approach.
Further,
QSAR
and
pharmacokinetic-based optimization studies are also
required to develop new antimalarial drug candidates from
present series of 1,2,4-trioxane lead molecules.
Acknowledgements Authors are thankful to Bioinformatics Infra-
structure Facility, Center for Biotechnology and Bioinformatics,
Dibrugarh University, Dibrugarh, Assam (India) for carrying out in
silico studies. Authors are also thankful to SAIF, Panjab University,
Chandigarh for providing spectral data of synthesized compounds.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Conclusion
Since newly reported 1,2,4-trioxane derivatives have sig-
nificant in vitro antimalarial effectiveness, further studies
are required for the evaluation of their toxicity, antimalarial
efficacy and pharmacokinetics in animal models.
Structure–activity relationship study helped us to under-
stand the importance of the 1,2,4-trioxane core system
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