Design, Synthesis, and Biological Evaluation of Novel 1,2,4-Trioxanes as Potential Antimalarial Agents

Article abstract of DOI:10.1002/ardp.201600335

A series of substituted 1,2,4-trioxanes were synthesized and evaluated for their antimalarial potential, in silico ADME properties and cytotoxicity on neuronal cell lines. Among the 15 synthesized substituted 1,2,4-trioxanes, two compounds (compound 15, IC₅₀ = 25.71 nM; compound 21, IC₅₀ = 19.6 nM) exhibited promising in vitro antimalarial potential comparable to those of the existing drugs chloroquine and artemisinin. Both of these compounds were found to be nontoxic up to 20 μM concentration in neuronal PC-12 cells. Compound 21 may serve as an optimized lead compound because of its less in vitro toxicity and lower probability to cross the blood brain barrier.

Full text of DOI:10.1002/ardp.201600335

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Full Paper
Design, Synthesis, and Biological Evaluation of Novel
1,2,4-Trioxanes as Potential Antimalarial Agents
Amit K. Gupta1, Kanika Varshney1, Vivek Kumar2, Kumkum Srivastava3, Aditya B. Pant2, Sunil K. Puri3,
and Anil K. Saxena1
1
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, India
In Vitro Toxicology Laboratory, CSIR-Indian Institute of Toxicology Research, Lucknow, India
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India
2
3
A series of substituted 1,2,4-trioxanes were synthesized and evaluated for their antimalarial potential,
in silico ADME properties and cytotoxicity on neuronal cell lines. Among the 15 synthesized
substituted 1,2,4-trioxanes, two compounds (compound 15, IC50 ¼ 25.71 nM; compound 21, IC50 ¼ 19.6
nM) exhibited promising in vitro antimalarial potential comparable to those of the existing drugs
chloroquine and artemisinin. Both of these compounds were found to be nontoxic up to 20 mM
concentration in neuronal PC-12 cells. Compound 21 may serve as an optimized lead compound
because of its less in vitro toxicity and lower probability to cross the blood brain barrier.
Keywords: 1,2,4-Trioxanes / Antimalarial / Drug design / Synthesis
Received: November 14, 2016; Revised: January 22, 2017; Accepted: January 25, 2017
DOI 10.1002/ardp.201600335
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
from this class of compounds, a number of groups have
attempted to produce totally synthetic endo-peroxide ana-
Introduction
Malaria, one of the most devastating infectious diseases,
affects about half of the global population [1]. About 212
million of new cases and 429000 attributed deaths were
covered in year 2015 by this disease worldwide [2]. The
scenario is aggravated by the persistent tendency of the most
common malaria parasite Plasmodium falciparum, to rapidly
develop resistance against any newly introduced drug [3, 4].
Artemisinin and its analogs are found suitable for the
treatment of cerebral malaria caused by multi-drug-resistant
P. falciparum [5, 6]. The major drawback of all semi-synthetic
trioxanes is that they require artemisinin as starting material
for their synthesis, but the current production of artemisinin
from natural sources is relatively low. In view of it and in
search of small molecules incorporating the pharmacophore
logs, some of which have demonstrated remarkable antima-
larial activity [7–11].
We have earlier reported a validated quantitative pharma-
cophore model utilizing this class of molecules [12]. Based on
it, a focused virtual library was designed and screened. This
led to the identification of simple substituted 1,2,4-trioxane
(compound 5a) having antimalarial activity up to 35 nM
(Fig. 1B) with drug likeness property. Encouraged by the
results of our previous work, and in order to further improve
the antimalarial potential of our above identified hit we
herein report the novel substituted 1,2,4-trioxanes as poten-
tial antimalarial agent.
Results and discussion
Lead optimization
The judicious addition/replacement in our earlier identified
lead trioxane molecule (5a) was performed on the basis of
different hydrophobic aliphatic and aromatic substituents
requirements at site-1 and site-3 (Fig. 1A and B). The n-alkyl
substituents at site-2 were also evaluated for their potential in
Correspondence: Dr. Anil K. Saxena, Medicinal and Process
Chemistry Division, CSIR-Central Drug Research Institute,
Lucknow 226031, India.
E-mail: anilsak@gmail.com
Fax: þ91-522-26123405
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Figure 1. (A) Pharmacophore mapping of the
lead compound 5a previously reported by our
research group; H-bond acceptor (HBA) and
H-bond acceptor lipid (HBAL) as green, aliphatic
hydrophobic (HYAl-1, HYAl-2) as dark blue and
aromatic hydrophobic (HYAr) as light blue. (B)
Site-1, site-2, and site-3 are the three sites of the
lead compound 5a targeted for the optimiza-
tion studies presented here.
modulating the antimalarial activity. Table 1 outlines the site-
specific modifications carried out at the two sites of the lead
compound 5a as envisaged.
compounds. Interestingly, the pharmacophore well discrim-
inates themolecules withhigh andlow activity. Theerrorfactor
between estimated and experimental activity of all the
molecules was within ꢀ3 (the uncertainty ratio in the Discovery
Studio software) except compound 15 which carries the error
factor of ꢀ4.5 (Table 2). It was observed from the mapping of
the synthesized molecules to the pharmacophore, that among
all these molecules, the 1,2,4-trioxane ring itself provided the
HBA and HBAL functionalities to the molecules. Four of the
highly active molecules (7, 11, 15, and 21) among the 15
synthesized compounds were simple trioxanes where the
required pharmacophoric features were better placed than
others. The pharmacophore mapping of the two highly active
compounds (15 and 21) among the 15 synthesized compounds
revealed that the di-substituted phenyl ring of the compounds
15 and 21 occupied the HYAr feature of the pharmacophore
(Fig. 2A andB). Whereas the admantyl ring attached at position
30 of the trioxane ring in compound 15 provided the bulky
aliphatic hydrophobic functionality and occupied the HYAl-1
and HYAl-2 feature. Addition of amino phenyl ring to the
cyclohexyl group of compound 21 does not appear to provide
any required pharmacophoric features. However its cyclohexyl
group provided the HYAl-1 functionality to the molecule. The
high antimalarial activity of this compound is because of total
high fit value for the HBA, HBAl, and HYAr feature (Fig. 2A).
Similartocompounds15and21admantylandcylcohexylgroup
of compounds 7 and 11 occupied the aliphatic hydrophobic
feature (HYAl-1 and 2) while their aromatic rings provided the
HYAr feature to the molecule resulting their high activity. The
similar study with the two less active compounds (compounds
10 and 13) revealed that their poor activity may be because of
thepooraccessibility ofthestructuralfeaturestomap theHYAr
in case of compound 13 and HYA1-1 and 2 feature in case of
compound 10 (Fig. 2C and D).
In silico ADME calculation
Various in silico ADME properties of these compounds were
calculated in the ADME module of acccelrys DS 2.0 software
packages [13] and used to predict the pharmacokinetic
behavior. Descriptor functions viz. Fast Polar Surface Area
(FPSA) and AlogP of the ellipse model were used to predict the
blood-brain penetration and intestinal absorption at 95% and
99% confidence. The molecule lying under the region
common to both the confidence ellipses for a given property
is considered robust with 99.9% confidence.
Chemistry
Allylic alcohols (3a–d) were prepared from the correspond-
ing ketones (1a–d) according to the reported procedure [14]
and were photo-oxygenated to give b-hydroxy hydroper-
oxides (4a–d). The photo-oxygenated products of allylic
alcohols (b-hydroxy hydroperoxides) were not isolated and
were condensed in situ where 4a–d on condensation with
different substituted ketones gave trioxanes 5–16 (Scheme 1)
in the presence of catalytic amount of HCl to furnish these
trioxanes with 49–58% overall yields (Table 1). However, the
b-hydroxy hydroperoxides (4a–c) on in situ condensation
with 1,4-cyclohexadiones gave compounds 17–19 which in
48–68% yields. Reductive amination of 17–19 with substi-
tuted anilines using NaBH(OAc)3 furnished the amino
functionalized trioxanes 20, 21, and 22 as mixture of
diastereomers [15] in 46–87% yields (Scheme 2).
Structure–activity relationship interpretation
The test results of all the 15 synthesized molecules are
summarized in Table 2 where four molecules (7, 11, 15, and
21) have shown antimalarial activity less than 40 nM, while two
molecules (15 and 21) have shown antimalarial activity
comparable to the existing drug chloroquine and artemisinin,
however, these two molecules were less active than the drug
artemether and arteether (Table 2). The earlier developed
pharmacophore model was explored to understand the
structure–activity relationships (SAR) among these synthesized
The inherent problems associated with the most of the
artemisinin derivatives as well as current synthetic trioxanes
are their poor intestinal absorption and neurotoxicity [16].
Therefore, only those substituted trioxanes with good
absorption rate and least neurotoxicity should be forwarded
to drug development. The graphical plot analysis revealed
that all the clinically used drugs (artemisinin, artemether,
arteether) fell under the common region of 95% and 99%
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Table 1. Site-specific modifications of the synthesized compounds (5–22) based on the lead molecule 5a.
Site-3
Ar
Site-2
R1
Site-1
S. N.
Compd. no.
5
R2/R3
R4
1
2,4-dimethylphenyl
CH3
–
2
3
6
7
2,4-dimethylphenyl
2,4-dimethylphenyl
CH3
CH3
–
–
4
5
6
7
8
8
2,4-dichlorophenyl
2,4-dichlorophenyl
phenyl
CH3
–
–
–
–
–
9
CH3
10
11
12
propyl
propyl
propyl
phenyl
phenyl
9
13
14
2,4-difluorophenyl
2,4-difluorophenyl
CH3
CH3
–
–
10
11
15
2,4-difluorophenyl
CH3
–
12
13
16
20
phenyl
phenyl
propyl
propyl
–
–
–
14
15
21
22
2,4-dimethylphenyl
2,4-dichlorophenyl
CH3
CH3
confidence ellipses for probability of intestinal absorption
and blood-brain penetration, thus showing that these drugs
may have a higher risk of neurotoxicity (Fig. 3). Although the
value of BBB penetration of two highly active synthetic
trioxanes (compounds 15 and 21) was in close proximity
(Table 2) yet they fall under different confidence ellipses. As
the compound 15 fell under the common region of 95% and
99% confidence ellipses for both blood-brain penetration and
Scheme 1. Synthesis of the trioxanes 5–16. Reagents and conditions: (i) Zn, BrCH2COOC2H5, C6H6 (CaCl2-dried), I2 (catalyst), reflux
(Refortmasky reaction); (ii) pTSA, C6H6, reflux, 10% HCl; (iii) LAH, dry ether, N2 atmosphere, <0°C; (iv) O2, hv, methylene blue, MeCN-
CH2Cl2, rt; (v) ketones/aldehydes, HCl, rt.
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Scheme 2. Synthesis of the fuctionalized trioxanes 20–22. Reagents and conditions: (i) 1,4-Cyclohexadione, HCl, rt; (ii) R4NH2,
NaBH(OAc)3, C6H6, 0°C, 1 h.
intestinal absorption properties, it was expected it may have
good intestinal absorption but may cause neuronal effect
because of its permeability to BB barrier.
On the other hand, though the compound 21 did not fall
under common region of 95% and 99% confidence ellipses
for blood-brain penetration and intestinal absorption prop-
erties, thus the compound 21 may have some poorer intestinal
absorption than compound 15 yet it may not cross the BB
barrier to cause neuronal effects.
brainstem neurons, and paradoxial increase in the reactive
oxygen species at therapeutic concentrations [17, 18]. In vitro
study on neuronal cells using arteether, artemether, and
dihydroartemisinin (DHA) at 300 nM drug concentrations was
found to significantly inhibit neurite outgrowth and MTT
metabolism [19]. Another in vitro study where neurotoxicity
was monitored by uptake of radiolabeled leucine into mouse
neuroblastoma–rat glioma hybrid NG108-15 cells growing in
cultures suggested artemisinin as least neurotoxic with an IC50
value of 100 mM whereas DHA and artesunic acid with IC50
value of 0.46 mM as most neurotoxic compound among
clinically active trioxanes [20]. In vivo neurohistopathological
studies on clinically active trioxanes including arteether,
artemether, DHA, artelinic acid, artesunate indicated to
produce manifest neurotoxicity [21].
In vitro cytotoxic evaluation on neuronal
cell lines
The trioxane molecules are known to carry inherent
neurotoxic properties causing severe energy depletion in
Table 2. Experimental and predictive in vitro antimalarial activity of compounds against 3D7 strain of P. falciparum and
their predictive ADME properties.
Antimalarial activity
ADMET properties
Hepatotoxicity
Comp.
no./name
Experimental
Predicted
IC50 (nM)
IC50 (nM)
Errora)
BBBb)
probability
A logP
PSAc)
5
6
7
8
9
10
11
12
13
14
15
16
20
21
22
110
201
36
68.1
229
667
39.4
142
3271.4
286.85
25.71
168.6
364
19.6
311.9
2.86
3.27
11.2
16.3
61.61
168.50
41.70
44.28
243.24
577.01
29.80
218.04
1137.58
214.07
118.24
209.12
362.98
40.00
239.51
8.2
ꢁ1.79
ꢁ1.19
1.16
0.92
1.06
1.27
1.17
1.38
1.03
1.17
1.37
0.46
0.89
1.09
1.57
–
0.50
0.44
0.74
0.35
0.69
0.37
0.36
0.70
0.34
0.37
0.71
0.37
0.40
0.20
0.48
0.158
0.132
0.90
0.25
4.86
5.31
5.97
5.67
6.33
5.20
5.65
6.31
3.36
4.75
5.41
6.94
7.08
6.07
7.09
3.17
3.52
3.14
4.35
26.79
26.79
26.79
26.79
26.79
26.79
26.79
26.79
26.79
26.79
26.79
26.79
39.6
39.6
39.6
44.65
44.65
53.02
27.42
ꢁ1.54
1.06
ꢁ1.16
ꢁ1.32
1.54
ꢁ2.88
ꢁ1.34
4.60
1.24
ꢁ1.00
2.04
1.10
–
ꢁ1.30
2.86
2.26
Artemether
Arteether
Artemisinin
Chloroquine
0.12
0.23
ꢁ0.023
0.76
7.4
6.8
ꢁ1.63
–
–
a) Values in the error column represent the ratio of estimated activity to experimental activity, or its negative inverse if the ratio is
less than 1; b)BBB, blood brain barrier; c)PSA, polar surface area.
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Figure 2. Pharmacophore mapping of the two highly active compounds (A) 21 and (B) 15 and of the two less active compounds (C) 13
and (D) 10.
Therefore, the four highly active molecules (7, 11, 15, and 21)
among the 15 synthesized trioxanes were evaluated for their
neurotoxicity on neuronal PC-12 cell lines. Results of percent
cell viability of neuronal PC-12 cells exposed to various
concentrations (1–80 mM) of compounds (7, 11, 15, and 21)
for 24 h are summarized in Fig. 4 where compounds 15 and
21 showed better results in terms of cell viability at higher
dose than other molecules. At 40 mM concentration com-
pounds 15 and 21 were found to be cytotostatic with 21
having less cytotostatic effect, as this concentration seems to
be comparatively less lethal and causes a physiological stress
to the cells. A total of 80 mM concentration was found to be
cytotoxic for both compounds 15 and 21; however,
compound 21 was less toxic than 15 at this concentration.
For compounds 7, 11, and 15 the concentration of 40 mM was
found to be cytotoxic while 20 mM concentration was found
cytostatic, thus confirming the in silico predictions for the
compounds 15 and 21. The rest of the lower doses (1 and
10 mM) were found non-cytotoxic for all the molecules used
in this study.
trioxanes which led to the identification of the two
promising novel antimalarial compounds 15 (IC50 ¼ 25.71
nM) and 21 (IC50 ¼ 19.6 nM) having in vitro antimalarial
potential comparable to the existing drugs chloroquine and
artemisinin. Compound 21 has been identified as optimized
lead for further studies due to its less neurotoxicity and less
probability to cross the blood brain barrier among the
compounds tested.
Experimental
Chemistry
General
Melting points were determined in open capillaries on a
COMPLAB melting point apparatus and are uncorrected. IR
spectra were recorded on a PerkinElmer RXI FT-IR spectro-
photometer. 1H NMR and 75 MHz for 13C NMR was recorded
on a Bruker Supercon Magnet DPX-200 (operating at
200 MHz for 1H) spectrometer using deuterated as solvent.
Tetramethylsilane (0.00 ppm) served as an internal standard
in 1H NMR. Multiplicities are represented by s (singlet), d
(doublet), dd (doublet of doublet), t (triplet), q (quartet), bs
(broad singlet), and m (multiplet). Reactions were moni-
tored on silica gel TLC plates. All the compounds tested
were 95% pure. Column chromatography was performed
over silica gel (60–120 mesh) procured from Qualigens
(India) using freshly distilled solvents. All the chemicals and
reagents were obtained from Aldrich (USA), Lancaster
Conclusion
In conclusion, in this study, we embarked on the design,
synthesis and biological evaluation of novel 1,2,4-trioxanes
as potential antimalarial agents where the optimization of
our earlier discovered lead compound was performed. For
this, we have synthesized and evaluated a series of novel
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Figure 3. Plot of polar surface area (PSA) versus ALogP for the synthesized compounds along with marketed drugs showing 95% and
99% confidence limit ellipses corresponding to the blood brain barrier and intestinal absorption models.
(England), or Spectrochem (India) and were used without
purification.
The InChI codes of the investigated compounds are
provided as Supporting Information.
Trioxane 5
ESMS (m/z): 274 [M}þ, IR (KBr, cmꢁ1) 3015.8, 2960.8, 2873.3,
2368.5, 1449.2, 1332.3, 1216.9, 1110, 971.5, 922.6, 1H NMR
(300 MHz.CDCl3) 1.60–1.89 (m,8H); 2.28 (s,3H); 2.33 (s,3H);
3.71–3.79 (m,2H); 4.99–5.03 (m,1H); 5.18 (s,1H); 5.47 (s,1H);
6.9–7.35 (m,3H); 13C NMR (CDCl3, 75 MHz) d 19.85, 21.05,
23.01, 25.01, 32.7, 37.5, 63.1, 81.2, 102.4, 118.1, 126.31, 128.69,
131.11, 135.26, 136.13, 137.47, 144.01.
Trioxane 6
ESMS (m/z): 288 [M}þ, IR (KBr, cmꢁ1) 3018, 2963, 2365, 1449.6,
1340.5, 1218.4, 1103.5, 984.3, 925.6, 1H NMR (300 MHz.CDCl3)
1.28–1.60 (m,10H); 2.28 (s,3H); 2.32 (s,3H); 3.66–3.71 (m,1H);
3.87–3.95 (m,1H); 4.76–4.79 (m,1H); 5.17 (s,1H); 5.48 (s,1H);
6.94–7.02 (m,3H); 13C NMR (CDCl3, 75 MHz) d 19.90, 21.62,
22.25, 26.4, 29.6, 32.5, 63.3, 81.5, 102.5, 116.9, 126.3, 128.72,
131.16, 135.29, 136.15, 137.50, 144.02.
Trioxane 7
ESMS (m/z): 340 [M]þ; IR (KBr, cmꢁ1) 3429.2, 3015.7, 2922.8,
2369.1, 1451.9, 1219.3, 1111.2, 924.4; 1H NMR (300 MHz.
CDCl3) 1.43–2.06 (m,14H); 2.20 (s,3H); 2.30 (s,3H); 3.63–3.70
(m,1H); 3.81–3.93 (m,1H); 4.90–4.97 (m,1H); 5.15 (s,1H); 5.44
(s,1H); 6.9–7.0 (m,3H); 13C NMR (CDCl3, 75 MHz) d 19.87, 21.2,
27.9, 32.9, 36.4, 37.1, 63.4, 81.6, 112.6, 118.1, 126.4, 128.6,
131.2, 135.4, 136.2, 137.6, 144.02.
Figure 4. MTT assay of compounds 7, 11, 15, and 21 on the
neuronal PC-12 cell line. Error bars in the graph represent the
SEM for the values obtained from at least three independent
experiments. The values of ꢂp < 0.05 were considered as
significant and of ꢂꢂp < 0.01 as more significant.
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Trioxane 8
(m,10H); 3.66–3.99 (m,2H); 5.08–5.12 (m,1H); 5.34 (s,1H); 5.44
(s,1H); 6.81–6.90 (m,2H); 7.27–30 (m,1H); 13C NMR (CDCl3,
75 MHz) 22.24, 26.4, 29.8, 32.7, 63.10, 81.23, 102.4, 118.1,
120.3, 121.2, 122.9, 127.2, 144.01, 153.5, 155.9.
ESMS (m/z): 329 [M]þ; IR (KBr, cmꢁ1) 3021.1, 2940.3, 2359.0,
1448.3, 1216.7, 1041.7, 927.8; 1H NMR (300MHz.CDCl3)
0.94–2.31 (m,10H); 3.79–3.83 (m,1H); 3.92–3.99 (m,1H);
5.03–5.07 (m,1H); 5.34 (s,1H); 5.61 (s,1H); 7.1–7.43 (m,3H);
13C NMR (CDCl3, 75MHz) d 22.23, 26.6, 29.5, 32.5, 63.5, 81.7,
102.7, 116.5, 125.31, 127.9, 129.2, 130.16, 133.29, 133.9, 144.03.
Trioxane 15
ESMS (m/z): 348 [M]þ; IR (KBr, cmꢁ1) 3453, 3021, 2360, 1451,
1217, 1043, 920.7; 1H NMR (300 MHz.CDCl3) 1.60–2.0 (m,14H);
3.78–3.97 (m,2H); 5.09–5.13 (m,1H); 5.44 (s,1H); 5.55 (s,1H);
6.79–6.89 (m,2H); 7.21–29 (m,1H); 13C NMR (CDCl3, 75 MHz) d
27.8, 32.7, 36.8, 37.9, 63.2, 81.3, 112.4, 118.3, 120.4, 121.3,
123.4, 125.6, 144.01, 153.6, 156.5.
Trioxane 9
ESMS (m/z): 380 [M]þ; IR (KBr, cmꢁ1) 3452.5, 3020.9, 2359.5,
1449.4, 1216.6, 1042.6, 925.7; 1H NMR (300 MHz.CDCl3)
1.63–2.44 (m,14H); 3.54–3.59 (m,1H); 3.93–3.96 (m,1H);
5.49–5.53 (m,1H); 5.86–5.91 (m,2H); 7.34–7.39 (m,3H);
13C NMR (CDCl3, 75 MHz) d 27.9, 32.7, 36.4, 37.5, 63.4,
81.6, 112.4, 116.1, 125.41, 127.6, 129.6, 130.6, 133.6, 133.6,
144.03.
Trioxane 16
ESMS (m/z): 378 [M]þ; IR (KBr, cmꢁ1) 3448.5, 3019, 2360.5,
1444, 1215.6, 1040.6, 920.7; 1H NMR (300 MHz.CDCl3) 0.98
(m,3H), 1.45–2.13 (m,10H); 2.95 (m,1H); 3.54–3.65 (m,2H);
5.54–5.59 (m,1H); 6.14 (m,1H); 6.50–7.56 (m,10H); 13C NMR
(CDCl3, 75 MHz) d 14.3, 20.5, 63.5, 81.32, 111.4, 115.1, 127.2,
127.3, 127.6, 127.6, 128.3, 128.9, 137.7, 139.4, 144.01.
Trioxane 10
ESMS (m/z): 289 [Mþ1}þ, IR (KBr, cmꢁ1) 3019.7, 2961.8,
2872.6, 2361.3, 1448.3, 1331.2, 1216.2, 1111.5, 970.1;
1H NMR (300 MHz.CDCl3) 1.42 (t,3H, J ¼ 6 Hz); 1.39–1.81
(m,8H); 2.35–2.41 (m,2H); 2.98 (t,2H, J ¼ 9 Hz); 3.58–3.62
(m,1H); 3.83–3.90 (m,1H); 5.48–5.50 (m,1H); 5.80–5.85 (m,1H);
7.3–7.99 (m,3H); 13C NMR (CDCl3, 75 MHz) d 14.8, 20.5, 23.01,
25.01, 32.7, 37.5, 63.2, 81.2, 102.4, 117.7, 126.9, 128.5, 128.9,
139.4, 139.1, 144.01.
Trioxane 20
ESMS (m/z): 428 [Mþ1}þ; IR (KBr, cmꢁ1) 3019, 2960, 2930, 2401,
1497.8, 1216, 1089, 929.9; 1H NMR (300 MHz.CDCl3) 0.99 (t,3H,
J ¼6 Hz); 1.3–1.99 (m,10H); 2.31–3.37 (m,2H); 3.51–3.54 (m,1H);
3.90–3.97 (m, 1H); 4.22–4.25 (m,1H); 5.42–5.46 (m,1H);
5.80–5.86 (m,1H); 6.49–6.53 (m,2H); 7.1–7.12 (m,2H);
7.5–7.99 (m,5H); 13C NMR (CDCl3, 75 MHz) d 14.7, 20.5, 22.3,
26.3, 26.6, 62.01, 63.5, 81.32, 102.4, 111.6, 115.1, 126.2, 126.5,
128.2, 128.8, 129.9, 140.1, 144.01, 145.9.
Trioxane 11
ESMS (m/z): 303 [Mþ1}þ, IR (KBr, cmꢁ1) 3018.5, 2935.7, 2871.6,
2361.2, 2338.9, 1448.8, 1329.2, 1216.5, 1086.4, 924.2; 1H NMR
(300 MHz.CDCl3) 0.95 (t,3H, J¼ 6Hz); 0.95–1.60 (m,10H); 2.32–2.36
(m,2H); 2.97 (t,2H, J¼ 12 Hz); 3.50–3.51 (m,1H); 3.85–3.96 (m,1H);
5.39–5.43 (m,1H); 5.80–5.84 (m,1H); 7.26–7.99 (m,5H); 13C NMR
(CDCl3, 75MHz) d 14.8, 20.5, 22.30, 26.1, 30.7, 33.2, 63.2, 81.2,
102.4, 118.1, 126.9, 128.5, 128.9, 135.4, 144.01.
Trioxane 21
ESMS (m/z): 367 [M]þ; IR (KBr, cmꢁ1) 3020.6, 2923.1, 2358.9,
1430.8, 1216.5, 1105.2, 1042.9, 928.9; 1H NMR (300 MHz.
CDCl3) 1.50–1.80 (m,8H); 2.27 (s,3H); 2.33 (s,3H); 2.78 (m, 1H);
3.60–3.70 (m,1H); 3.72–3.96 (m,1H); 4.94–4.97 (m,1H); 5.19
(s,1H); 5.47 (s,1H); 6.7–7.30 (m,8H); 13C NMR (CDCl3, 75 MHz) d
19.8, 20.09, 22.4, 27.6, 61.8, 63.9, 81.23, 102.27, 111.2, 118.07,
120.6, 126.31, 128.4, 129.6, 131.1, 135.26, 136.13, 137.4,
144.01, 146.3.
Trioxane 12
ESMS (m/z): 354 [M}þ; IR (KBr, cmꢁ1) 3020, 2928.6, 2872.6,
2360.4, 1455.9, 1217.4, 1111.5, 1038.4, 920.9;1H NMR(300 MHz.
CDCl3) 0.99 (t,3H, J ¼ 6 Hz); 1.25–1.80 (m,14H); 2.0–2.17 (m,2H);
2.80–2.88 (m,2H); 3.44–3.52 (m,1H); 3.83–3.94 (m,1H); 5.40–5.47
(m,1H); 5.77–5.80 (m,1H); 7.61–7.98 (m,5H); 13C NMR (CDCl3,
75 MHz) d 14.6, 20.4, 27.8, 32.7, 36.7, 37.8, 63.4, 81.4, 112.4,
118.1, 127.01, 128.6, 129.0, 135.4, 144.01.
Trioxane 22
ESMS (m/z): 453 [M]þ; IR (KBr, cmꢁ1) 3423, 2941, 2363.0, 1500.4,
1217.0, 1043.7, 931.0; 1H NMR (300 MHz.CDCl3) 0.88–1.66
(m,8H); 2.69–2.73 (m,1H): 3.33–3.38 (m,1H); 3.85–3.90 (m,2H);
5.08–5.10 (m,1H); 5.32 (s,1H); 5.36 (s,1H); 5.61 (s,1H); 6.55–6.60
(m,2H); 7.12–7.17 (m,4H); 7.43 (s,1H); 13C NMR(CDCl3, 75 MHz) d
22.9, 27.5, 62.01, 63.10, 81.23, 102.4, 118.07, 120.6, 126.31,
126.9, 129.6, 131.2, 132.3, 135.26, 136.13, 144.01, 145.4.
Trioxane 13
ESMS (m/z): 256 [M]þ; IR (KBr, cmꢁ1) 3450.5, 3019.5, 2361.5,
1450.4, 1211.6, 1042.6, 923.7; 1H NMR (300 MHz.CDCl3) 1.60
(s,3H); 1.63 (s,3H); 3.81–3.97 (m,2H); 5.07–5.11 (m,1H); 5.44
(s,1H); 5.55 (s,1H); 6.80–6.90 (m,2H); 7.27–30 (m,1H); 13C NMR
(CDCl3, 75 MHz) d 25.38, 63.10, 81.23, 102.4, 118.1, 120.1,
120.9, 122.9, 126.8, 144.01, 153.6, 156.5.
Pharmacology
Antimalarial screening
All the 15 synthesized compounds along with the standard
drugs artemether, arteether, and chloroquine were evalu-
ated for their in vitro antimalarial activity against chloro-
quine (CQ) sensitive 3D7 strain of P. falciparum using a
Trioxane 14
ESMS (m/z): 296 [M]þ; IR (KBr, cmꢁ1) 3455.5, 3022.9, 2361,
1451, 1215, 1040.4, 924.5; 1H NMR (300 MHz.CDCl3) 1.10–2.19
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A. K. Gupta et al.
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standardized inexpensive assay based on SYBER Green I
(Table 2) [22]. The IC50 values were calculated from experi-
ments carried out in triplicate. The compounds were
dissolved in DMSO at 5 ng/mL. For the assays, fresh dilutions
of all compounds in screening medium were prepared and
50 mL of highest starting concentration (500 ng/mL) was
dispensed in duplicate wells in row B of 96-well tissue culture
plates. The highest concentration of chloroquine was 25 ng/
mL. Subsequently, twofold serial dilutions were prepared up
to row H (seven concentrations). Finally, 50 mL of 2.5%
parasitized cell suspension containing 0.5% parasitemia was
added to each well except four wells in row “A” which
The authors (A.K.G. and V.K.) are thankful to the I.C.M.R.,
New Delhi for the financial assistance in the form of a
fellowship, and SAIF Department of CSIR-CDRI, Lucknow for
the characterization of compounds. The technical assistance
of Mr. Akhilesh K. Srivastava, and Ms. Shashi Rastogi is also
acknowledged. CSIR-CDRI communication number allotted to
this manuscript is 9426.
The authors have declared no conflict of interest.
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a non infected cell suspension. These wells
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