Design, Synthesis, Structure-Activity Relationship and Docking Studies of Novel Functionalized Arylvinyl-1,2,4-Trioxanes as Potent Antiplasmodial as well as Anticancer Agents

Article abstract of DOI:10.1002/cmdc.202000045

A novel series of synthetic functionalized arylvinyl-1,2,4-trioxanes (8 a–p) has been prepared and assessed for their in vitro antiplasmodial activity against the chloroquine-resistant Pf INDO strain of Plasmodium falciparum by using a SYBR green-I fluorescence assay. Compounds 8 g (IC₅₀=0.051 μM; SI=589.41) and 8 m (IC₅₀=0.059 μM; SI=55.93) showed 11-fold and >9-fold more potent antiplasmodial activity, respectively, as compared to chloroquine (IC₅₀=0.546 μM; SI=36.63). Different in silico docking studies performed on many target proteins revealed that the most active arylvinyl-1,2,4-trioxanes (8 g and 8 m) showed dihydrofolate reductase (DHFR) binding affinities on a par with those of chloroquine and artesunate. The in vitro cytotoxic potentials of 8 a–p were also evaluated against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines. Following screening, five derivatives viz. 8 a, 8 h, 8 l, 8 m and 8 o (IC₅₀=1.65–31.7 μM; SI=1.08–10.96) were found to show potent cytotoxic activity against (A549) lung cancer cell lines, with selectivity superior to that of the reference compounds artemisinin (IC₅₀=100 μM), chloroquine (IC₅₀=100 μM) and artesunic acid (IC₅₀=9.85 μM; SI=0.76). In fact, the most active 4-naphthyl-substituted analogue 8 l (IC₅₀=1.65 μM; SI >10) exhibited >60 times more cytotoxicity than the standard reference, artemisinin, against A549 lung cancer cell lines. In silico docking studies of the most active anticancer compounds, 8 l and 8 m, against EGFR were found to validate the wet lab results. In summary, a new series of functionalized aryl-vinyl-1,2,4-trioxanes (8 a–p) has been shown to display dual potency as promising antiplasmodial and anticancer agents.

Full text of DOI:10.1002/cmdc.202000045

Accepted Article
Title: Design, Synthesis, Structure-Activity Relationship and Docking
Studies of Novel Functionalized Arylvinyl-1,2,4-Trioxanes as
Potent Antiplasmodial as well as Anticancer Agents
Authors: Mohit K. Tiwari, Paolo Coghi, Prakhar Agrawal, Bharti Rajesh
K. Shyamlal, Li Jun Yang, Lalit Yadav, Yuzhong Peng, Richa
Sharma, Dharmendra K. Yadav, Dinkar Sahal, Vincent Kam
Wai Wong, and Sandeep Chaudhary
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000045
Link to VoR: https://doi.org/10.1002/cmdc.202000045
01/2020

10.1002/cmdc.202000045
ChemMedChem
FULL PAPER
Design, Synthesis, Structure-Activity Relationship and Docking
Studies of Novel Functionalized Arylvinyl-1,2,4-Trioxanes as
Potent Antiplasmodial as well as Anticancer Agents
Mohit K. Tiwari,a,† Paolo Coghi,b,† Prakhar Agrawal,c,† Bharti Rajesh K. Shyamlal,a Li Jun Yang,d Lalit
Yadav,a Yuzhong Peng,b Richa Sharma,a Dharmendra K. Yadav,e Dinkar Sahal,c,* Vincent Kam Wai
Wong,d,* Sandeep Chaudharya,*
Supporting information for this article is given via a link at the end of the document.
Abstract: A novel series of synthetic functionalized arylvinyl-1,2,4-
trioxanes (8a-p) has been prepared and assessed for their in vitro
antiplasmodial activity against chloroquine-resistant Pf INDO strain
of Plasmodium falciparum using SYBR green-I fluorescence assay.
Compounds 8g (IC50 = 0.051 µM; SI = 589.41) and 8m (IC50 = 0.059
the wet lab results. In summary, a new series of functionalized aryl-
vinyl-1,2,4-trioxanes (8a-p) has been recognized to display dual
potency as promising antiplasmodial as well as anticancer agents.
µM; SI
= 55.93) showed 11-fold and >9-fold more potent
Introduction
antiplasmodial activity, respectively, as compared to chloroquine
(IC50 = 0.546 µM; SI = 36.63). Different in silico docking studies
performed on many target proteins revealed that the most active
arylvinyl-1,2,4-trioxanes (8g and 8m) showed dihydrofolate
reductase (DHFR) binding affinities at par with chloroquine and
artesunate. The in vitro cytotoxic potentials of 8a-p were also
evaluated against human lung (A549) and liver (HepG2) cancer cell
lines along with immortalized normal lung (BEAS-2B) and liver (LO2)
cell lines. Following screening five derivatives viz. 8a, 8h, 8l, 8m and
8o (IC50 = 1.65-31.7 µM; SI = 1.08-10.96) were found to show potent
cytotoxic activity with superior selectivity than the reference
compounds, artemisinin (IC50 = 100 µM), chloroquine (IC50 = 100 µM)
as well as artesunic acid (IC50 = 9.85 µM; SI = 0.76) against (A549)
lung cancer cell lines. Infact, the most active 4-naphthyl substituted
analogue 8l (IC50 = 1.65 µM; SI > 10), exhibited >60-fold more
cytotoxicity than the standard reference artemisinin against A549
lung cancer cell lines. In silico docking studies of most active
anticancer compounds 8l and 8m against EGFR were found validate
Malaria is a mosquito-borne parasitic infectious disease affecting
mainly the tropical and subtropical countries. According to “The
World Malaria Report (2018)”, there have been around 3 million
more cases of malaria in 2017 compared to 2016; a total of 219
million clinical cases were reported out of which 4,35,000
causalities occurred in 2017.1 This life-threatening infection is
caused by malaria parasites like P. falciparum and P.vivax with
symptoms of fever and anemia appearing, when the “parasite”
are passed through the “liver stage” and enters into the “blood
stage” by invading human red blood cells.2 The emergence of
drug resistant parasite strains has further complicated its
treatment.
[a]
Mohit K. Tiwari, Bharti Rajesh K. Shyamlal, Lalit Yadav, Richa
Sharma, Dr. S. Chaudhary
Laboratory of Organic and Medicinal Chemistry
Department of Chemistry
Malaviya National Institute of Technology
Jawaharlal Nehru Marg, Jaipur-302017, India.
E-mail: schaudhary.chy@mnit.ac.in
[b]
[c]
Dr. Paolo Coghi, Yuzhong Peng
School of Pharmacy, Macau University of science and technology,
Avenida wai long, Taipa, Macau, China.
Prakhar Agrawal, Dr. Dinkar Sahal
Malaria Drug Discovery Laboratory,
Figure 1. Artemisinin and its derivatives (1a-f).
International Centre for Genetic Engineering and Biotechnology,
Aruna Asaf Ali Marg, 110 067, New Delhi, India.
E-mail: dinkar@icgeb.res.in
The isolation and identification of artemisinin (1a),
a
[d]
[e]
[†]
Li J. Yang, Dr. Vincent K. W. Wong
State Key Laboratory of Quality Research in Chinese Medicine,
Macau University of Science and Technology,
Avenida Wai Long, Taipa, Macau, China.
E-mail: bowaiwong@gmail.com
Dr. Dharmendra K. Yadav
College of Pharmacy,
Gachon University of Medicine and Science,
Hambakmoeiro 191, Yeonsu-gu, Incheon city, 406-799, South
Korea.
sesqueterpene lactone endoperoxide from Artemisia annua has
been a milestone achievement in the treatment of malaria. After
the debacle of widespread drug resistance against chloroquine,
artemisinin (1a) and its semisynthetic derivatives such as
dihydroartemisinin (DHA, 1b), artemether (1c), arteether (1d),
artesunic acid (AS,1e), and artelinic acid (1f) emerged as a new
hope and changed the current scenario of malaria
These authors contributed equally.
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
1
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10.1002/cmdc.202000045
ChemMedChem
FULL PAPER
chemotherapy for which Prof. Tu Youyou got the Nobel prize in
physiology and medicine in 2015.3 It has been reported that till
2015, more than 200 million people have been treated
successfully using artemisinin and its derivatives (1a-e) (Figure
1).4 However, during the last two decades, the advent of
artemisinin-resistant parasite strains has further made treatment
more problematic and more challenging.5 Unlike monotherapy
which is prone to the development of quick resistance, WHO has
recognized artemisinin-based combination therapy (ACT) as a
way for delaying the advent of resistance in the treatment of
clinical cases of malaria.6
activity against multidrug-resistant P. yoelii nigeriensis in Swiss
mice via intramuscular and oral routes (Figure 2).16 However, the
anticancer activity of synthetic arylvinyl-1,2,4-trioxanes has been
less explored and is still an area of investigation.17,18
Cancer is also a fatal disease responsible for 9.6 million deaths
in 2018 (the second-largest after heart-related deaths); out of
which 1.76 million casualties are due to lung cancer in a
calendar year.7 Apart from antiplasmodial activity, artemisinin
derived endoperoxides have also shown promising in vitro as
well as in vivo anticancer activity. It has been well-established
that the peroxide bridge present in trioxane-based drugs, is the
main pharmacophore responsible for both antiplasmodial as well
as anticancer activities.8 Molecular targets of artemisinin and
synthetic peroxides in tumor cells and also in P. falciparum
parasites are still under debate.9-12
In spite of being an antimalarial drug and showing promising
anticancer activity; the tetracyclic framework of artemisinin is
associated with several drawbacks such as; “less
abundance“ from natural resources, “less stability“ due to
breakage of peroxide linkage, lower solubility in both oil and
water (Log P value), poor oral bioavailability, high rate of
recrudescence etc.13 In order to circumvent these problems, the
synthetic versions of artemisinin i.e., 1,2,4-trioxanes-based
derivatives have emerged as prominent alternatives to the
naturally occurring drug. In this context, different research
groups14 including ours15 have prepared several in vitro / in vivo
active synthetic 1,2,4-trioxanes having promising antiplasmodial
activity. Singh et al. have reported several synthetic
functionalized arylvinyl-1,2,4-trioxanes (1g-k) of general
structure A which showed high order of in vivo antiplasmodial
Figure 2. Structures of potent antiplasmodial synthetic arylvinyl-1,2,4-
trioxanes (1g-k) congeners of general structure A.
In our endeavour to develop structurally simple novel synthetic
1,2,4-trioxanes embraced with dual activities (antiplasmodial as
well as anticancer), herein, we report the synthesis of a new
series of synthetic C-6 arylvinyl-1,2,4-trioxanes (8a-p). We have
screened these synthetic 1,2,4-trioxanes for (a) in vitro
antiplasmodial activity against chloroquine-resistant Pf INDO
strain of P. falciparum using SYBR green-I fluorescence assay
and (b) cytotoxic potential against human lung (A549) and liver
(HepG2) cancer cell lines along with immortalized normal lung
(BEAS-2B) and liver (LO2) cell lines. The in-silico virtual
interactions between ligand and different protein targets (DHFR;
PDB: 3QG2 and EGFR; PDB: 1M17) have also been carried out
for the assessment of the probable mechanism of actions of the
C-6 arylvinyl-1,2,4-trioxanes against Plasmodium and cancer
cells, respectively.
Scheme 1. General procedure for the synthesis of functionalized aryl-vinyl-1,2,4-trioxanes (8a-p). Reagents and conditions: (i) (a) Zn dust, I2, Dry C6H6, 1 h; (b) 3,
Dry C6H6, reflux, 5-6 h. (ii) p-TSA, Dry C6H6, reflux, 5-6 h. (iii) LAH, anhyd. ether, 0 °C, 1h; (iv) O2, methylene blue, MeCN, hν, -10-0 °C, 4-6h; (v)
Aldehydes/ketones, p-TSA, 1-10h, rt.
2
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10.1002/cmdc.202000045
ChemMedChem
FULL PAPER
Structure-activity relationship studies (antiplasmodial
activity):
Inspired by previous pioneering findings in the field of
antiplasmodial chemotherapy,16 we prepared the set of sixteen
functionalized aryl-vinyl-1,2,4-trioxanes (8a-p) with variations at
three different sites i.e., site A, B and C as mentioned in trioxane
of general structure A (Figure 2). As it can be seen from Table 2,
8a (IC50 = 1.720 µM) having a cyclohexylphenyl group at site A
and p-OMe phenyl group at site C has shown more promising
antiplasmodial activity than chloroquine (IC50 = 0.546 µM);
Results and Discussion
Chemistry
The syntheses of several functionalized arylvinyl-1,2,4-
trioxanes (8a-p) were performed using the literature procedure
as shown in Scheme 1.19 Substituted acetophenones (2a-f)
along with Zn dust and ethyl bromoacetate (3) were subjected to
Reformatsky reaction in the presence of iodine as activator,
which furnished the corresponding β-hydroxy esters (4a-f),
which were (without further purification) subjected to dehydration
with p-TSA furnished the more stable α,β-unsaturated allylic
esters (5a–f) in 35–74% yield range. LAH reduction of 5a-f
furnished corresponding allylic alcohols (6a–f) in 67–85% yield
range. Singlet oxygen-mediated photo-oxygenation reaction of
allylic alcohols (6a-f) in the presence of methylene blue dye as a
photosensitizer at −5 °C to −10 °C in acetonitrile as a solvent
furnished β-hydroxyhydroperoxides (7a-f), which on (without
further purification) subjecting to in-situ p-TSA-catalyzed
condensation with various ketones/aldehydes in one-pot
furnished the functionalized aryl-vinyl-1,2,4-trioxanes (8a-p) in
29–78% yield range.
however, it showed lower activity than sodium artesunate (IC50
=
0.006µM) against chloroquine-resistant PfINDO strain of P.
falciparum (Table 2, entry 1). Similarly, 8b (IC50 = 2.19 µM)
having a biphenyl group at site A and 8c (IC50 = 1.41 µM) having
a biphenyl group at site C also showed promising antiplasmodial
activities (Table 2, entries 2-3). In addition, compound 8d (IC50
=
2.67 µM), with a p-OMe group at site A and the substituted
phenyl group at site C also showed promising activity (Table 2,
entry 4). In further attempt to improve the activity profile of 1,2,4-
trioxanes, we targeted the C-6 vinylic position of general
structure A and prepared the compounds 8e and 8f having a
phenyl ring at C-6 position. While, 1,2,4-trioxane 8e (IC50
=
0.328 µM) was found more potent than chloroquine (IC50 = 0.546
µM); 8f (IC50 = 0.643 µM) turned out to be nearly equipotent to
chloroquine (Table 2, entries 5-6). Next, derivatives with alicyclic
fused aromatic ring at site A were analyzed. Thus, 8g-k having a
1,2,3,4-tetrahydronaphthalene moiety at site A with various
aliphatic and aromatic substitution at site C were prepared.
1,2,4-trioxane 8g (IC50 = 0.051 µM), the most active compound
of the series, showed 11 fold more activity than chloroquine
(Table 2, entry 7). Similarly, 1,2,4-trioxane 8h (IC50 = 0.126 µM)
and 8i (IC50 = 0.386 µM) were also found 4-fold and 1.4-fold
more potent than chloroquine, respectively (Table 2, entries 8-9).
Contrary to the spiro compounds 8g-i; aromatic substituted
compounds 8j (IC50 = 8.910 µM) and 8k (IC50 = 3.170 µM)
showed poorer activity than chloroquine (IC50 = 0.546 µM; SI =
36.63) (Table 2, entries 10-11).
The melting point and isolated yields of synthesized 1,2,4-
trioxanes 8a-p are presented in Table 1. It was observed that
biphenyl-based 1,2,4-trioxanes 8b and 8c displayed higher
melting point than the other analogues.
Table 1. Melting points and percentage yields of aryl-vinyl 1,2,4-trioxanes (8a-
p).
Entry
Trioxane
Mol. Formula
m.p. (ºC)
70-72
150-152
148-150
Yield (%)
1
2
8a
8b
C24H28O4
C24H22O4
52
63
38
3
8c
C24H22O4
4
5
6
7
8
8d
8e
8f
8g
8h
8i
8j
8k
8l
C20H22O6
C22H24O3
C19H20O3
C20H26O3
C19H24O3
C17H22O3
C22H24O3
C22H24O4
C26H26O3
----
35
45
51
62
42
71
29
40
71
66-68
62-64
----
46-48
----
56-58
50-52
79-81
9
10
11
12
Table 2. In vitro antiplasmodial activity of synthetic aryl-vinyl-1,2,4-trioxanes
(8a-p) against chloroquine-resistant PfINDO strain of P. falciparum.a
Selectivity
index
(CC50
HEK293/IC50
INDO)
13
8m
C25H24O3
111-113
70
Cell
cytotoxicity
HEK293
Antiplasmodial
activity
14
15
16
8n
8o
8p
C23H22O3
C30H30O3
C28H24O4
88-90
119-121
76-78
78
50
52
Entry
Compound
PfINDO IC50 (µM)
CC50 (µM)
1
2
8a
8b
1.720
2.190
> 100
> 100
>58.14
>45.66
3
8c
1.410
> 100
>70.92
Antiplasmodial Activity
4
5
6
7
8
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
CQ
2.670
0.328
0.643
0.051
> 100
36.1
> 100
30.06
16.98
> 100
93.8
64.5
18.26
3.3
66.57
> 100
79.53
20
>37.45
110.06
>155.52
589.41
134.76
> 259.06
10.53
20.35
134.26
55.93
In vitro antiplasmodial activity of functionalized aryl-vinyl-
1,2,4-trioxanes against Pf INDO strain:
0.126
0.386
8.910
3.170
0.136
0.059
0.300
0.209
3.860
0.546
9
10
11
12
13
14
15
16
17
All the synthesized arylvinyl-1,2,4-trioxanes (8a-p) were
assessed for their in vitro antiplasmodial activity against
chloroquine-resistant Pf INDO strain of P. falciparum using
SYBR green-I fluorescence assay, where chloroquine (CQ) or
sodium artesunate (AS) were utilized as positive controls for the
study.20 Further, the cell cytotoxicity of all the prepared
compounds was evaluated against mammalian cell line (HEK
293) using the MTT assay. The selectivity indices of all the
compounds were also determined. The results are summarized
in Table 2.
221.9
> 478.47
20.60
36.63
18
AS
0.006
> 26
4333.33
aIC50: Concentration corresponding to 50% inhibition of growth of P. falciparum
in culture. 8g and 8m with IC50 <100 nM are shown in bold.
3
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10.1002/cmdc.202000045
ChemMedChem
FULL PAPER
Earlier, we have observed that peroxide molecules assembled
with bulkier groups at site A are associated with increased
lipophilicity which are accountable for displaying potent
antiplasmodial activity.21 Consequently, a naphthyl group at site
A was introduced along with spiro as well as aromatic
substitutions at site C for the preparation of 1,2,4-trioxanes 8l-p
(Table 2, entries 12-16). While the second best compound of the
series i.e., 8m (IC50 = 0.059 µM) having a cyclopentyl group at
site C showed > 9-fold more antiplasmodial potency than
chloroquine (IC50 = 0.546 µM; SI = 36.63); 8l (IC50 = 0.136 µM),
8n (IC50 = 0.300 µM) and 8o (IC50 = 0.209 µM) having a
cyclohexyl, methyl and adamantyl group at site C, respectively,
also showed promising antiplasmodial activity profiles (Table 2,
entries 12-15). 8p (IC50 = 3.860 µM) having an aromatic ring
substitution at site C showed detrimental effect on the activity
(Table 2, entry 16). It is to be noted that all the 1,2,4-trioxanes
(8a-p) were found to be less active than sodium artesunate (IC50
= 0.006µM) against chloroquine-resistant PfINDO strain of P.
falciparum.
artesunate (IC50 = 9.85 µM, SI = 0.76) against lung cancer cell
lines (Table 3, entries 5-6).
In addition, 8g and 8i-k alicyclic-aromatic fused derivatives
having a 1,2,3,4-tetrahydronaphthalene moiety at site A and
aliphatic and aromatic substitution at site C also displayed low
potency (IC50 = ~50-100 µM) in comparison to reference drug;
the only exception was 8h (IC50 = 12.2 µM, SI = 2.57), a 1,2,3,4-
tetrahydronaphthalene derivative with
a
cyclopentyl ring
substitution at site C, which showed nearly equipotent
anticancer activity against lung cancer cell line (A549) in
comparison to standard drug artesunate (IC50 = 9.85 µM, SI =
0.76) (Table 3, entries 7-11).
Finally, we concentrated our attention towards derivatives built
around trioxane of general structure A having bulkier groups with
increased lipophilicity. These derivatives showed better in vitro
activity profile in anticancer studies. Thus, 8l (IC50 = 1.65 µM, SI
= 10.96) having a naphthyl group at site A and spiro cyclohexyl
ring at site C has shown ~ 6-fold more cytotoxic potency against
lung cancer (A549) cell lines than artesunate (lung, IC50 = 9.85
µM, SI = 0.76) and was found to be the most active compound of
the series (Table 3, entry 12). Similarly, 8m (IC50 = 3.36 µM, SI =
5.23) having a cyclopentyl ring at site C was found to be the
second most potent derivative of the series and displayed ~ 3-
fold more anticancer potency than artesunate (IC50 = 9.85 µM, SI
= 0.76) against lung cancer (A549) cell lines (Table 3, entry 13).
Naphthyl derivatives 8n (IC50 = 45.4µM) and 8o (IC50 = 27.3 µM,
SI = 3.66) substituted with a methyl and adamantyl ring at site C
were also found to have promising anticancer activity profile in
comparison to reference drug artesunate against lung cancer
(A549) cell lines (Table 3, entries 14-15). Compound 8p having
an aryl ring substitution at site C showed diminished cytotoxic
activity in comparison to reference drug artesunate (IC50 = 9.85
µM, SI = 0.76) (Table 3, entry 16).
It is noteworthy to mention that the 11-fold and 9-fold higher
potency of 8g and 8m, respectively, against the chloroquine
resistant Pf INDO strain makes these two lead compounds to be
nearly equipotent against the CQ sensitive Pf 3D7 strain (IC50
=
CQ ~ 40 nM).22 Overall, it can be interpreted that aryl
substitutions (naphthyl and 1,2,3,4-tetrahydronaphthalene) in
aryl-vinyl-1,2,4-trioxanes at sites
A
and
B
increases
antiplasmodial activity whereas the activity decreases with the
introduction of aryl substitution at the site C.
Cytotoxicity Studies
In vitro anticancer activity against lung/liver cancer and
normal cell lines:
For the first time, all the synthesized 1,2,4-trioxanes (8a-p) of
the general structure A having sites A, B and C as points of
variation have been assessed for in vitro activities against
different lung and liver cancerous as well as normal cell lines for
their cytotoxic potential.
Table 3. Cytotoxicity of functionalized aryl-vinyl-1,2,4-trioxanes (8a-p) against
human lung A549 cancer cell lines and the immortalized lung BEAS-2B cell
lines.
BEAS-2B
A549 cells
Entry
Compound
cells
S.I.b
IC50 [µM]a
IC50 [µM]a
Anticancer activity against lung cancer was evaluated on human
lung (A549) cancer cell line versus immortalized (BEAS-2B)
normal cell lines; while the liver cancer was probed using
cancerous (HepG2) liver cell line versus (LO2) normal cell lines.
Artemisinin, artesunic acid, chloroquine and paclitaxel were
taken as standard reference compounds for the study (Table 3
and Table 4, supporting information Fig 6-9).
1
2
3
4
5
6
7
8
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
31.7
100
100
95.5
34.32
> 100
> 100
52.15
> 100
70.5
> 100
31.4
73.6
64.42
35.6
18.1
17.6
30.7
1.08
---
---
---
1.29
---
---
2.57
---
---
---
10.96
5.23
---
3.66
---
77.50
> 100
>100
12.2
>100
>100
48.4
1.65
3.36
45.4
9
Structure-activity relationship studies (anticancer activity):
As it can be observed from Table 3, 8a (IC50 = 31.7 µM, SI =
1.08) with a cyclohexylphenyl ring at site A and p-OMe phenyl
group at site C, the analogous of CDRI-99/411 (1g), has shown
promising anticancer activity against lung cancer (A549) cell
lines as compared to reference drug artesunate (IC50 = 9.85 µM,
SI = 0.76) (Table 3, entry 1).
Similarly, 8b-d having different aryl substitutions at site A
(biphenyl, p-OMe) and site C (biphenyl, substituted-OMe),
analogues to another potent compound CDRI-99/357 (1h),
demonstrated less activity (IC50 = ~100 µM) than artesunate
against lung cancer (IC50 = 9.85 µM, SI = 0.76) cell lines (Table
3, entries 2-4). The attempt to switchover the aryl substitution at
10
11
12
13
14
15
16
17
8o
8p
ART
27.3
>100
100
> 100
> 100
100
---
18
19
20
AS
CQ
9.85
100
7.53
3.07
0.1
0.76
0.03
33.3
Paclitaxelc
0.003
aIC50: Concentration at which the inhibition of 50% cells was observed. Values
expressed are means from at least three independent experiments conducted
in duplicates; bS.I.: Selectivity index lung (IC50 for cytotoxicity against normal
cells/ IC50 for cytotoxicity against cancer cells); cSee ref. 23.
vinylic site B did not enhanced the activity since both 8e (IC50
=
77.50 µM) and 8f (IC50 = ~100 µM) were found less active than
4
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In general, it can be interpreted from the in vitro anticancer
studies that 37% of the cyclic peroxides showed better
selectivity index for lung cancer (A549) cell lines compared to
artemisinin (IC50 = 100 µM, SI = 100), artesunic acid (IC50 = 9.85
µM, SI = 7.53) and chloroquine (IC50 = 100 µM, SI = 3.07). On
the other hand, although all the synthesized aryl-vinyl-1,2,4-
trioxanes (8a-p) displayed moderate activity against liver cancer
ID: NAS91-11, LC55, pyrimethamine (CP6), flavin adenine
dinucleotide (FAD), E64, TIT) utilizing the Glide module of the
Schrodinger maestro software and XP (extra precision) method
(for details, see Table 2 in SI).
Earliar, we had demonstrated the effect of artemisinin on yeast
glutathione reductase (GR; used as a surrogate for Plasmodium
falciparum) particularly in the oxidation of cofactors (NADPH and
FAD).26 Based on our previous studies28 on both parasite and
the mammalian thioredoxin reductase (TrxR) which had shown
susceptiblity towards artemisinin and TrxR (4B1B) was selected
in the present study. It has been observed that the binding
affinity values were found comparatively lower than the control
ligand (for details, see Table 2 in SI). Based on these results, we
are currently continuing research work to evaluate oxidizing of
dihydroflavin in TrxR by 1,2,4- trioxanes (8a-p) in
(HepG2) cells in comparison to standard drug artesunate (IC50
=
4.09 µM, SI = 2.01) and paclitaxel (IC50 = 0.19 + 0.4 µM, SI =
0.52); ten-derivatives demonstrated higher cytotoxicity and
selectivity index than parental endoperoxide drug artemisinin
(IC50 = >100 µM, SI = >100) (Table 4, entries 1-16).
As can be seen from the in vitro anticancer assessment, the
introduction of bulkier and lipophilic group substitutions (naphthyl
and 1,2,3,4-tetrahydronaphthalene) in aryl-vinyl-1,2,4-trioxanes
skeleton at site A increases the anticancer activity of compounds, acetonitrile/aqueous buffer condition as previously reported for
while the activity diminishes drastically on substituting the
general structure A with aryl ring at sites B and C.
GR.26
Table 5. Docking scores of the ary-vnyl-1,2,4-trioxanes (8a-p) and control
drugs against PDB ID: 3QG2.a,b
Table 4. Cytotoxicity of functionalizedaryl-vinyl-1,2,4-trioxanes (8a-p) against
human liver HepG2 cancer cell lines, and the immortalized liver LO2 cell lines.
Docking Score
Entry
Compound
(3QG2)
HepG2 cells
IC50 [µM]a
LO2 cells
IC50 [µM]a
Entry
Compound
S.I.b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
-5.46
-6.44
-5.95
-6.21
-6.91
-6.48
-6.56
-6.22
-5.64
-7.63
-6.43
-7.27
-7.34
-6.92
-7.34
-6.28
-6.06
-4.01
-5.70
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
71.6
>100
>100
79.4
> 100
89.9
>100
32.9
>100
75.4
57.5
10.2
10.9
84.5
46.4
>100
73.3
> 100
> 100
67.9
> 100
90.6
68.7
10.2
71.9
48
74.5
5.58
5.33
23.3
36
1.02
---
---
---
---
1.007
---
---
---
---
1.29
---
---
---
---
---
Pyrimethamine
> 100
CQ
AS
17
18
ART
AS
>100
>100
8.25
15
---
aDetailed account of protein targets are mentioned in materials and methods
section. Inhibitors are the ligands that are co-crystallized with the targeted
protein. bdocking scores in bold indicate that these molecules have stronger
affinity than the respective known inhibitors.
4.09+0.4
49.02+0.4
0.19+0.4
2.01
0.30
0.52
19
20
CQ
Paclitaxelc
<0.1
aIC50: Concentration at which the inhibition of 50% cells was observed. Values
expressed means at least three independent experiments conducted in
duplicates; bS.I.: Selectivity index liver. (IC50 for cytotoxicity against normal
cells/ IC50 for cytotoxicity against cancer cells); cSee ref. 24.
The aryl-vinyl-1,2,4-trioxanes (8a-p) showed good docking score
against dihydrofolate reductase protein (DHFR; PDB ID: 3QG2),
another system that using cofactors NADPH, as an electron
donor, reduces dihydrofolic acid to tetrahydrofolic acid (Table 5).
In-silico Studies
Molecular Docking Simulation Studies with Dihydrofolate
Reductase (DHFR) Protein (PDB: 3QG2):
Wang et al.25 evidenced 124 possible artemisinin covalent
binding protein targets, many of which were involved in
fundamental biological processes in parasite. Based on these
facts and to further gain insights on antiplasmodial activity, some
of these binding protein targets were selected to study
interactions in detail. The in-silico molecular docking studies of
the aryl-vinyl-1,2,4-trioxanes (8a-p) were performed against P.
falciparum target proteins which were chosen based on our
previous results26 (PDB ID: 3QG2, 4B1B, 3AZB, 5JC1, 3QGT,
and 3BPF27) along with their respective known inhibitors (PDB
Figure 3: Ligand interaction diagram of compound 8g with DHFR protein
(PDB: 3QG2).
5
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The compound 8g showed good docking score i.e. -6.56 with
DHFR protein and forms an H-bond with the hydrophilic amino
acid Serine-111 and pi-pi stacking with the hydrophobic aromatic
residue Phe-58. In the docking complex, binding site residues
within a radius of 3Å are mostly hydrophobic residues (Val 45,
Leu 40, 46, 164, 119, 164, Ile 112, Phe 116, Met 55, Phe 58),
hydrophilic residues (Asn 108, Ser 111) and neutral residue (Gly
41, Pro 113) as shown in figure 3 and table 6.
Similarly, the docking results for 8m against the DHFR also
showed good binding affinity as indicated by a score of -7.34,
8m was seen to form a pi-pi stacking with Phe-58, 116 and pi-
cation interaction with Arg-122. In the docking complex, binding
site residues within a radius of 3Å are hydrophilic residues (Asn-
the Leu-164 and 40, and has steric clashes with Asp-54, Asn-
108 and Met 55. The binding site around chloroquine (3Å radius)
is constituted by mainly hydrophobic residues (Met-55, Phe-58,
Ala-16, Cys-15, Ile-14, Leu-164, Trp-48, Leu-46, Val-45, Leu-40,
Tyr-170, Ile-112, Val-195) with only four hydrophilic residues
(Asn-42, Thr-107, Asn-108, Ser-111, 167) as shown in table 6.
Similarly, AS showed a docking score of –5.70 and very low -dG
binding energy (-34.87 kcal/mol). Artesunate showed an
association with Ser-167 through a hydrogen bond together with
steric clashes with Ile-112, Leu-46, Cys-15, Ala-16, Leu-164,
Asn-108, Gly-166, Leu-40 and Leu-46. In the docking complex,
majority of the residues within a radius of 3Å are hydrophobic
such as Ile-112, Leu-119, Phe-58, Met-55, Leu-164, Ala-16,
108, Asp-54, Arg-59) and hydrophobic residues (Leu-40, 46, 164, Cys-15, Val-195, Tyr-170, Leu-40, Leu-46 along with Asp-46 as
Tyr-170, Met-55, Trp-48, Ile-112, Phe-116) and neutral residues
(Cys-15, Ala-16, Pro-113) as shown in figure 4 and table 6.
charged amino acid and Asn-108 and Ser-167 as polar residues
as shown in table 6.
Molecular Docking Simulation Studies of Epidermal Growth
Factor Receptor (EGFR) Protein:
Artemisinin and artesunate have shown profound activity not
only against malaria, but also against cancer in vivo and in
clinical trials. It is well reported that reactive oxygen species
(ROS) are involved in the mutation of EGFR-mediated signaling
pathways, which further regulates the tumorigenesis in lung
cancer.29c Many lung cancer patients diagnosed in advanced
stages have shown mutations of epidermal growth factor
receptor (EGFR). Recently, 1a and 1e have been used in
combination therapy with EGFR-tyrosine kinase inhibitor.29
Earlier, mechanistic studies of synthetic arylvinyl-1,2,4-trioxanes
which were performed in the presence of different Fe(II) salts
such as FeCl2.4H2O, FeBr2 and a combination of hemin (bovine)
and GSH (glutathione) under both aerobic and anaerobic
conditions revealed that the generation of intermediate reactive
oxygen species (ROS) with hemin/GSH in aerobic conditions
could be toxic against Plasmodia and responsible for the various
therapeutic responses of these aryl-vinyl-1,2,4-trioxanes.30
The naphthyl substituted aryl-vinyl-1,2,4-trioxanes, 8l and 8m,
were found more active in lung cancer cell lines. Here, we
studied the virtual interaction of these two arylvinyl-1,2,4-
trioxanes along with control compounds chloroquine, artesunic
acid and artemisinin toward the anticancer target EGFR. The
protein target EGFR complex with 4-anilinoquinazoline inhibitor
erlotinib (PDB ID: 1M17) was obtained from the RCSB protein
data bank having a resolution of 2.5Å.31 Whole moiety docking
was done using Schrodinger maestro for the evaluation of
binding energy and interaction pattern for the total set of the
molecules. The results have been summarized in Table 7.
The docking results for 8l against the EGFR showed good
binding affinity as indicated by docking score of -4.943. Detailed
analysis revealed a H-bond of length 2.1 Å. The results disclose
that the key conserved hydrogen bonds are between the ligands
and the binding-cavity residues, notably with the flap residue
Cys-773. In docking pose, the chemical nature of binding site
residues within a radius of 3Å from bound compound included
acidic (polar, negative charged) Glu-738, Asp-831, Asp-776;
hydrophobic Ala-719, Gly-772, Ile-720, Leu-694, Leu-764, Leu-
768, Leu-820, Leu-753 and Val-702; and nucleophilic (polar,
hydrophobic) Thr-830 and Thr-766; nucleophilic (polar,
hydrophobic) CYS-773. The coming together of these diverse
interactions between 8l and EGFR seem to be contributing to
strong affinity and activity in this compound (Figure 5C).
Figure 4: Ligand interaction diagram of compound 8m with DHFR protein
(PDB: 3QG2).
The docking results for the control molecule pyrimethamine with
the DHFR showed a docking score -6.06. Pyrimethamine forms
hydrogen bonds with Leu-164, Ile-14, Asp-54; pi-pi stacking with
Phe-58 and a halogen bond with Asn-108. In the docking
complex, binding site residues within a radius of 3Å are
hydrophobic residues (Leu-164, Tyr-170, Ile-14,112, Cys-15,
Ala-16, Met-55, Phe-58) and hydrophilic residues (Thr-185, Asp-
54, Asn-108) as shown in table 6.
Table 6: Docking scores, glide XP and Emodel energy of top two hit trioxanes
(8g and 8m) and Artemisinin together with Pyrimethamine (positive control)
and Chloroquine ( negative control) against PfDHFR protein (PDB ID: 3QG2).a
Glide XP
Energy
(kcal/mol)
-44.60
-47.15
-37.65
Glide XP
Emodel
(kcal/mol)
-53.51
-59.95
-53.36
Docking
score
Entry
Compound
1.
2.
3.
4.
5.
8g
8m
-6.56
-7.34
-6.06
-4.01
-5.70
Pyrimethamine
Chloroquine
Artesunate
-43.98
-34.87
-47.01
-50.53
aFor details interaction studies of the standard references, see SI.
Consequently, since chloroquine (CQ) and artesunate (AS) were
taken as standard reference in the antiplasmodial studies; we
carried out the docking studies of CQ and AS against target
protein (PDB ID: 3QG2). Interestingly, compounds 8g and 8m
showed higher docking scores than that of CQ and AS. Further,
insights in docking score revealed that lipophilic and Van der
Waals interaction are major forces for more negative docking
scores than control drugs. Interaction analysis of CQ suggested
that it has a pi-pi stacking formed with the Phe-58, a H-bond with
6
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Similarly, the docking results for compound 8m with EGFR also
showed good docking score of -5.248. 8m forms a H-bond of
length 2.61 Å to the hydrophobic aliphatic residue i.e., Lysine-
721. In the docking complex, binding site residues within a
radius of 3Å included basic (polar, hydrophobic, positive
charged) Lys-721; acidic (polar, negative charged) Glu-738 and
Asp-831 and Asp-776; hydrophobic Ala-719, Gly-772, Leu-694
and Val-702; nucleophilic (polar, hydrophobic) Thr-830, Thr-766;
nucleophilic (polar, hydrophobic) CYS-773. These interactions
are well suited to contribute to the observed high docking scores
and potent biological activity of 8m (Figure 5E).
Figure 5: Molecular docking poses of 8l and 8m in 1M17 protein binding pocket. (A) Superposition of docked pose; Binding complex of 8l (B) and 8m (D) with
1M17 protein; The binding site amino acid residues displaying interactions within 3Å is shown only (C) 8l and (E) 8m.
Table 7: Docking scores of synthesized aryl-vinyl-1,2,4-trioxanes (8l and 8m)
and standard compound against protein (PDB ID: 1M17).
= 0.051 µM, SI = > 588) and 8m (IC50 = 0.059 µM, SI = > 56)
were found to be the most active compounds of the series and
Docking
score
(kcal/mol)
-4.943
-5.248
-4.514
-3.759
-3.155
Glide XP
Energy
(kcal/mol)
-44.184
-43.901
-30.943
-32.464
-24.726
Glide XP
Emodel
(kcal/mol)
-60.620
-58.684
-32.510
-44.549
-28.918
showed ~11-fold and >9-fold more in vitro antiplasmodial
activity in comparison to chloroquine (IC50 = 0.546µM, SI = 36.6),
respectively against chloroquine-resistant Pf INDO strain of
Plasmodium falciparum. Different in silico docking studies
performed on many target proteins revealed that the most active
arylvinyl-1,2,4-trioxanes (8g and 8m) showed higher binding
affinity against dihydrofolate reductase (DHFR) in comparison to
chloroquine. Moreover, in vitro anticancer studies against lung
(A549) and liver (HepG2) cancer cell lines revealed five
derivatives (8a, 8h, 8l, 8m and 8o) (IC50 = 1.65-31.7µM; SI =
1.08-10.96) with high order of cytotoxicity and selectivity against
lung (A549) cancer cell lines in comparison to reference
molecules such as artemisinin (IC50 = 100 µM), artesunic acid
(IC50 = 9.85 µM) and chloroquine (IC50 = 100 µM). The two most
potent analogues from the series, 8l (IC50 = 1.65 µM, SI = 10.96)
and 8m (IC50 = 3.36 µM, SI = 0.23) showed >60-fold and ~30-
fold in vitro more potency in comparison to reference drug
artemisinin (IC50 = 100 µM), respectively against lung (A549)
cancer cell lines. In silico docking studies of most active
Entry
Compound
1.
2.
3.
4.
5.
8l
8m
Artesunic acid
Chloroquine
Artemisinin
The docking results for the control molecules chloroquine,
artesunic acid and artemisinin with the anticancer target protein
EGFR were found to show low binding affinity which are
indicated as -3.759, -4.514 and -3.155, respectively, without any
H-bond formation. Thus, the docking procedure of Glide
software in reproducing the experimental binding affinity seems
reliable (Table 7).
Conclusion
In conclusion, we have synthesized a novel series of synthetic
aryl-vinyl-1,2,4-trioxanes (8a-p); out of which compound 8g (IC50
7
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workup followed by purification of the crude mixture via column
chromatography furnished (5a) as a pure white solid in 74% yield (4.75g,
based on 2a).
anticancer compounds 8l and 8m against EGFR validates the
wet results. Succinctly, our findings uncover a structurally
modified and versatile novel series of aryl-vinyl-1,2,4-trioxanes
(8a-p) having dual potency as a promising antiplasmodial as
well as anticancer agents.
Compound 5a: Ethyl(E)-3-(4-(naphthalen-1-yl)phenyl)but-2-enoate)
The crude product was purified by column chromatography over silica gel
(230-400 mesh) using 0.2% ethyl acetate: n-Hexane as eluent. Yield:
74%; White Solid, m.p. 91-93 ºC; 1H NMR (400 MHz, CDCl3) δ 7.91 –
7.85 (m, 3H), 7.62 – 7.60 (m, 2H), 7.54 – 7.41 (m, 6H), 6.25 (d, J = 1.2
Hz, 1H), 4.24 (q, J = 7.2 Hz, 2H), 2.66 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 167.03, 155.15, 141.67, 141.09, 139.52,
133.89, 131.51, 130.29, 128.44, 128.02, 127.00, 126.35, 126.27, 125.97,
125.90, 125.47, 117.25, 59.99, 17.97, 14.46; HRMS (ESI/QTOF) m/z: [M
+ H]+ Calcd for [C22H21O2]+: 317.1536; found: 317.1538.
Experimental Section
General experimental conditions:
All the oven dried laboratory glassware’s were used for all synthetic
procedures. Melting points were taken in open capillaries on complab
melting point apparatus and are presented uncorrected. Infrared spectra
were recorded on
a Perkin-Elmer FT-IR RXI spectrophotometer
(Spectrum Version 10.4.00) using neat, thin film or KBr pellets. 1H NMR
and 13C NMR spectra were recorded on a JEOL ECS-400 spectrometer
(2-channel console with a flexible broadband RF performance), which
was operating at 400 MHz for 1H and 100 MHz for 13C NMR and utilized
CDCl3 as a solvent for all the sample preparation. Tetramethylsilane (δ
0.00 ppm) and CDCl3 both were served as an internal standard in 1H
NMR (δ 7.246 ppm) and 13C (δ 77.0 ppm) NMR. Pattern of chemical
shifts were reported in parts per million. Peak splitting patterns were
described as singlet (s), doublet (d), double doublet (dd), triplet (t),
multiplet (m) and broad (br). Coupling constant (J) were reported in Hertz
(Hz). High-Resolution Electron Impact Mass Spectra (HR-EIMS) were
obtained on Xevo G2-S Q-Tof (Waters, USA) compatible with ACQUITY
UPLC® and nano ACQUITY UPLC® systems. Column chromatography
was performed over normal (particle size: 60-120 Mesh, 100-200 Mesh)
and flash (particle size: 230-400 Mesh) silica gel, which were procured
from QualigensTM (India), Rankem (India), and Spectrochem (India). TLC
plates coated with silica gel (Kiesel 60-F254, Merck (India) were used for
the monitoring of the progress of the reactions. Visualizing agents used
for TLC were UV light, Iodine vapours or spraying with an aq. solution of
vanillin in 10% sulphuric acid followed by heating at 100 °C. BUCHI’s
Rotavapor R-210 was used for all drying and concentration procedures.
All the commercially grade supplied anhydrous solvents such as
benzene, diethyl ether and THF were kept over benzophenone-Na wires
and refluxed prior to use. Where, benzophenone used as indicator for
drying solution, which turns the colour of solvent from colourless to deep
blue/purple due to the formation of ketyl.32 The activation of the zinc dust
was carried out using the literature procedure, which involves treatment
of zinc dust with acid followed by the removal of moisture content under
reduced pressure, and stored under N2 atmosphere. All the remaining
chemicals and reagents obtained from Sigma Aldrich (USA), or Merck
(India) and TCI (India) etc. were used without further purification.
Compound 5c: Ethyl (E)-3,4-diphenylbut-2-enoate
The crude product was purified by column chromatography over silica gel
(230–400 mesh) using n-Hexane as an eluent. Yield: 35%; Viscous; 1H
NMR (400 MHz, CDCl3) δ 7.43 – 7.38 (m, 2H), 7.31 – 7.28 (m, 3H), 7.21
– 7.09 (m, 5H), 6.24 (s, 1H), 4.50 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 1.30
(t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.56, 157.27, 141.08,
138.79, 129.01, 128.64, 128.55, 128.42, 127.07, 126.09, 118.84, 60.16,
36.49, 14.39; HRMS (ESI/QTOF) m/z: [M+H]+ Calcd for [C18H19O2]+
267.1380; found: 267.1381.
General procedure for the preparation of substituted allylic alcohols
(6a-f): Preparation of ((E)-3-(4-(naphthalen-1-yl)phenyl)but-2-en-1-ol)
(6a) as a representative:
To a magnetically stirred mixture of LiAlH4 (LAH) (359 mg, 9.49 mmol) in
anhyd. diethyl ether (60 mL) at 0 ºC under N2 atmosphere; a solution of
α,β-unsaturated allylic ester (5a, 2.5 g, 7.90 mmol) in anhyd. diethyl ether
(60 mL) was added dropwise over 30 min. The reaction mixture was
stirred at 0 ºC for additional 1h. The reaction mixture was neutralized
using water (10 mL) followed by 5% NaOH solution (3 mL) under N2
atmosphere. The organic layer was separated and extracted with diethyl
ether (4 × 50 mL), dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude mass was purified by column
chromatography which furnished (6a) as pure white solid in 85% yield
(1.84 g).
Compound 6a: ((E)-3-(4-(naphthalen-1-yl)phenyl)but-2-en-1-ol)
The crude product was purified through column chromatography over
silica gel (100-200 mesh) using 10% ethyl acetate: n-Hexane as eluent.
Yield: 85%; Solid, m.p. 111-113 ºC; 1H NMR (400 MHz, CDCl3) δ 7.94 –
7.83 (m, 3H), 7.55 – 7.48 (m, 4H), 7.47 – 7.40 (m, 4H),6.10 (td, J = 6.8,
1.2 Hz, 1H), 4.42 (d, J = 6.4Hz, 2H), 2.16 (s, 3H), 1.49 (Br, 1H); 13C NMR
(100 MHz, CDCl3) δ 141.75, 139.92, 139.86, 137.63, 133.89, 131.65,
130.09, 128.37, 127.74, 126.97, 126.67, 126.12, 126.06, 125.87, 125.73,
General procedure for the preparation of substituted allylic esters
(5a-f): Preparation of ethyl (E)-3-(4-(naphthalen-1-yl)phenyl)but-2-
enoate) (5a) as a representative:
In the refluxing mixture of substituted acetophenone (1-(4-(naphthalen-1-
yl)phenyl)ethan-1-one) (2a, 5g, 20.29 mmol), Zn dust (2.65g, 40.59
125.48, 60.14, 16.12;HRMS (ESI/QTOF) m/z: [M
[C20H19O]+: 275.1431; found:275.1433.
+
H]+ Calcd for
mmol), I2 (15 mg) in dry benzene (50 mL);
a solution of ethyl
bromoacetate (3) (5.09g, 30.44 mmol) in dry benzene (50 mL) was
added dropwise over 1 h under N2 atmosphere. The reaction mixture was
allowed to stir for additional 5-6 h. The progression of the reaction was
observed using TLC. On completion of the reaction, the reaction mixture
was allowed to cool down at 0 °C and quenched using 10% aqueous HCl
solution (120 mL). Further, the organic layer was extracted with benzene
(2 × 50 mL), washed with 5% aqueous NaHCO3 solution (2 × 50 mL),
then with brine (2 × 40 mL), and finally dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Subsequently, without further
purification, the crude unstable substituted β-hydroxy ester (4a) was
treated with p-TSA (200 mg) in refluxing benzene (50 mL) for 5-6 h,
which afforded crude α,β-unsaturated allylic esters (5a). The reaction
mixture was neutralized with a satd. NaHCO3 solution (100 mL). Usual
Compound 6c: (E)-3,4-diphenylbut-2-en-1-ol
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 10% ethyl acetate: n-Hexane as an eluent. Yield:
67%; Viscous oil, 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.35 (m, 2H), 7.28
– 7.21 (m, 5H), 7.16 – 7.13 (m, 3H), 6.15 (t, J = 6.8 Hz, 1H), 4.39 (d, J =
6.8 Hz, 2H), 3.90 (s, 2H), 1.45 (br, 1H); 13C NMR (100 MHz, CDCl3) δ
142.03, 140.46, 139.35, 128.84, 128.63, 128.41, 128.16, 127.44, 126.50,
126.20, 60.17, 36.11; HRMS (ESI/QTOF) m/z: [M+H]+ Calcd for
[C16H17O]+ 225.1274; found 225.1273.
8
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General procedure for the preparation of aryl-vinyl-1,2,4-trioxanes
(8a-p): Preparation of (3-(1-(4-(naphthalen-1-yl)phenyl)vinyl)-1,2,5-
trioxaspiro[5.5]undecane) (8l) as a representative (1O2 -mediated
photooxygenation reaction):
Viscous; 1H NMR (400 MHz, CDCl3) δ 7.37-7.35 (m, 2H), 7.08-7.04 (m,
2H), 6.89-6.85 (m, 2H), 6.78-6.73 (m, 1H), 6.14 (s, 1H), 5.45-5.42 (m,
2H), 5.26 (s, 1H), 4.20 (dd, J = 12.0, 2.4 Hz, 1H), 3.96-3.92 (m, 1H), 3.90
(s, 3H), 3.88 (s, 3H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 159.74,
150.36, 148.99, 142.21, 130.97, 127.70, 126.66, 120.14, 116.09, 115.34,
114.86, 114.05, 110.81, 109.68, 104.14, 80.62, 69.94, 56.00, 55.97,
55.41; HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for [C20H23O6]+: 359.1489;
found 359.1489.
A solution of 6a (0.300 g, 1.09 mmol) and methylene blue (5 mg) in
acetonitrile (35 mL) solvent were taken in a double-jacketed round-
bottom flask (100 mL) maintained between 0 °C to -10 °C. The reaction
mixture was irradiating with 500 W tungsten-halogen-lamp along with
continuous bubbling of a slow stream of O2 for 4 h. The progression of
the reaction was monitored using TLC plates. After completion of the
reaction, without further purification, the crude solution of β-
hydroxyhydroperoxide (7a) was reacted with cyclohexanone (1.03 mL,
9.84 mmol) in the presence of p-TSA (0.018 g, 0.109 mmol) as catalyst in
the same pot for 2 h at room temperature. The acetonitrile solvent was
evaporated and the crude product was separated and extracted with
ethyl acetate (3 × 10 mL), washed with satd. aq. NaHCO3 solution (15
mL), and then with brine (2 × 15 mL) and dried over anhyd. Na2SO4 and
concentrated under reduced pressure. The crude product was purified by
column chromatography which furnished pure (8l) in 71% yield (0.300 g).
Compound
8e:
(E)-3-(1,2-diphenylvinyl)-1,2,5-
trioxaspiro[5.5]undecane
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using n-Hexane as eluent. Yield: 45%; Solid; m.p. 66-68
ºC; 1H NMR (400 MHz, CDCl3) δ 7.54 – 7.27 (m, 10H), 6.91 (s, 1H), 5.49
- 5.46 (m, 1H), 4.01 – 3.80 (m, 1H), 3.44 (dd, J = 12.0, 2.8 Hz, 1H), 2.27
– 2.02 (m, 1H), 2.00 – 1.77 (m, 1H), 1.62 – 1.41 (m, 8H); 13C NMR (100
MHz, CDCl3) δ 140.25, 138.92, 136.42, 135.98, 129.11, 128.66, 128.43,
128.22, 128.02, 127.65, 102.39, 78.99, 61.00, 34.92, 29.59, 25.62,
22.45, 22.32; HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for [C22H25O3]+:
337.1798; found 337.1797.
Compound 8a: 6-(1-(4-cyclohexylphenyl)vinyl)-3-(4-methoxyphenyl)-
1,2,4-trioxane
Compound 8f: (E)-6-(1,2-diphenylvinyl)-3,3-dimethyl-1,2,4-trioxane
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 3% ethyl acetate: n-Hexane as eluent. Yield: 52%;
Solid; m.p. 70-72 ºC; 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.41 (m, 2H),
7.38 – 7.32 (m, 2H), 7.21-7.19 (m, 2H), 6.92-6.89 (m, 2H), 6.15 (s, 1H),
5.51 (s, 1H),5.47 – 5.44 (m, 1H), 5.31 (s, 1H), 4.21 (dd, J = 11.6, 2.4 Hz,
1H), 3.94-3.89 (m, 1H), 3.81 (s, 3H), 2.55 – 2.45 (m, 1H), 1.88-183 (m,
5H), 1.43 – 1.35 (m, 5H); 13C NMR (100 MHz, CDCl3) δ 160.94, 148.42,
142.76, 136.02, 128.61, 127.15, 126.51, 126.39, 116.01, 113.92, 104.10,
80.54, 70.01, 55.40, 44.33, 34.45, 26.94, 26.21; HRMS (ESI/QTOF) m/z:
[M + H]+ Calcd for [C24H29O4]+: 381.2061; found 381.2062.
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using n-Hexane as eluent. Yield: 51%; Solid; m.p. 62-64
ºC; 1H NMR (400 MHz, CDCl3) δ 7.46 – 7.30 (m, 10H), 6.91 (s, 1H), 5.46-
5.43 (m, 1H), 3.91-3.85 (m, 1H), 3.46 (dd, J = 12.0, 2.8 Hz, 1H), 1.59 (s,
3H), 1.33 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 140.17, 138.92, 136.25,
135.93, 129.08, 128.65, 128.41, 128.24, 128.03, 127.67, 102.23, 78.78,
61.65, 25.71, 20.70; HRMS (ESI/QTOF) m/z: [M
[C19H21O3]+: 297.1485; found 297.1486.
+
H]+ Calcd for
Compound 8g: ((R)-3-(1-(5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)-
1,2,5-trioxaspiro[5.5]undecane)
Compound 8b: 6-(1-([1,1'-biphenyl]-4-yl)vinyl)-3-(4-methoxyphenyl)-
1,2,4-trioxane
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using n-Hexane as eluent. Yield: 62%; Viscous oil; FT-IR
(KBr, cm-1) 1624.63, 1446.42, 1093.59, 919.94, 828.61; 1H NMR (400
MHz, CDCl3) δ 7.16-7.08 (m, 2H), 7.02 (d, J = 8.0 Hz, 1H), 5.45 (s, 1H),
5.24 (s, 1H), 5.21-5.19 (m, 1H), 3.99 – 3.86 (m, 1H), 3.75 (dd, J = 12.0,
2.8 Hz, 1H), 2.75 (s, 4H), 2.24-2.19 (m, 1H), 2.03-1.98 (m, 1H), 1.86 –
1.57 (m, 9H), 1.53 – 1.39 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 143.51,
137.46, 137.37, 135.85, 129.41, 126.99, 123.53, 115.33, 102.63, 80.35,
63.01, 34.79, 29.80, 29.54, 29.21, 29.04, 25.64, 23.21, 22.42, 22.38;
HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for [C20H27O3]+: 315.1955; found
315.1954.
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 5% ethyl acetate: n-Hexane as eluent. Yield: 63%;
Solid; m.p. 150-152 ºC;1H NMR (400 MHz, CDCl3) δ 7.62 – 7.56 (m, 4H),
7.54 – 7.41 (m, 6H), 7.39 – 7.32 (m, 1H), 6.96 – 6.84 (m, 2H), 6.17 (s,
1H), 5.60 (s, 1H), 5.51-5.48 (m, 1H), 5.39 (s, 1H), 4.26-4.23 (m, 1H),
3.98-3.93 (m, 1H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.96,
142.47, 141.20, 140.49, 137.44, 128.93, 128.61, 127.61, 127.40, 127.12,
126.93, 126.43, 116.82, 113.94, 104.15, 80.46, 69.90, 55.41; HRMS
(ESI/QTOF) m/z: [M + H]+ Calcd for [C24H23O4]+: 375.1591; found
375.1592.
Compound 8h: ((R)-8-(1-(5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)-
6,7,10-trioxaspiro[4.5]decane)
Compound 8c: 3-([1,1'-biphenyl]-4-yl)-6-(1-(4-methoxyphenyl)vinyl)-
1,2,4-trioxane
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using n-Hexane as eluent. Yield: 42%; Solid; mp 46-
48ºC;1H NMR (400 MHz, CDCl3) δ 7.20 – 7.06 (m, 2H), 7.03 (d, J = 7.6
Hz, 1H), 5.45 (s, 1H), 5.28 (dd, J = 10.0, 4.0 Hz, 1H), 5.23 (s, 1H), 3.91 –
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 5% ethyl acetate: n-Hexane as eluent. Yield: 38%;
Solid; m.p. 148-150 ºC;1H NMR (400 MHz, CDCl3) δ 7.68 – 7.52 (m, 6H),
7.50 – 7.29 (m, 5H), 6.90-6.88 (m, 2H), 6.24 (s, 1H), 5.48 (m, 2H), 5.29
(s, 1H), 4.23 (dd, J = 11.6, 2.4 Hz, 1H), 3.98-3.93 (m, 1H), 3.82 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 159.76, 143.02, 142.22, 140.60, 133.02,
130.96, 128.90, 127.71, 127.54, 127.33, 127.31, 115.40, 114.07, 104.04,
3.71 (m, 2H), 2.75 (s, 4H), 2.60 – 2.43 (m, 1H), 1.94 – 1.62 (m, 11H); 13
C
NMR (100 MHz, CDCl3) δ 143.29, 137.49, 137.38, 135.75, 129.42,
127.03, 123.55, 115.39, 114.63, 80.37, 65.41, 37.16, 32.84, 29.81,
29.54, 29.21, 24.89, 23.47, 23.21; HRMS (ESI/QTOF) m/z: [M + H]+
Calcd for [C19H25O3]+: 301.1798; found 301.1797.
80.73, 69.90, 55.42; HRMS (ESI/QTOF) m/z: [M
[C24H23O4]+: 375.1591; found 375.1592.
+
H]+ Calcd for
Compound 8i: ((R)-3,3-dimethyl-6-(1-(5,6,7,8-tetrahydronaphthalen-
2-yl)vinyl)-1,2,4-trioxane)
Compound
methoxyphenyl)vinyl)-1,2,4-trioxane
8d:
3-(3,4-dimethoxyphenyl)-6-(1-(4-
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using n-Hexane as eluent. Yield: 71%; Viscous oil; 1H
NMR (400 MHz, CDCl3) δ 7.14 – 7.05 (m, 2H), 7.03 (d, J = 8.0 Hz, 1H),
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 5% ethyl acetate: n-Hexane as eluent. Yield: 35%;
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000045
ChemMedChem
FULL PAPER
5.45 (s, 1H), 5.25 (s, 1H), 5.23 – 5.20 (m, 1H), 3.94-3.88 (m, 1H), 3.77
(dd, J = 12.0, 3.2 Hz, 1H), 2.75 (s, 4H), 1.80-1.78 (m, 4H), 1.67 (s, 3H),
1.40 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 143.43, 137.49, 137.39,
135.78, 129.41, 127.04, 123.57, 115.34, 102.48, 80.29, 63.76, 30.13,
29.80, 29.54, 29.21, 25.58, 23.20; HRMS (ESI/QTOF) m/z: [M + H]+
Calcd for [C17H23O3]+: 275.1642; found 275.1643.
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 0.5% ethyl acetate: n-Hexane as eluent. Yield:
78%; Solid; m.p. 88-90ºC; 1H NMR (400 MHz, CDCl3) δ 7.98 – 7.80 (m,
3H), 7.58 – 7.37 (m, 8H), 5.62 (s, 1H), 5.39 (s, 1H),5.34 – 5.30 (dd, J =
10.0, 2.8 Hz, 1H), 4.05-3.99 (m, 1H), 3.88 (dd, J = 12.0, 2.8 Hz, 1H), 1.71
(s, 3H), 1.43 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 143.10, 140.77,
139.63, 137.51, 133.87, 131.54, 130.36, 128.41, 127.91, 126.99, 126.36,
126.22, 125.96, 125.93, 125.47, 116.45, 102.61, 80.21, 63.62, 25.55,
20.26; HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for [C23H23O3]+: 347.1642;
found 347.1643.
Compound 8j: 6-(1-(5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)-3-(p-
tolyl)-1,2,4-trioxane
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 3% ethyl acetate: n-Hexane as eluent. Yield: 29%;
Solid; m.p. 56-58ºC; 1H NMR (400 MHz, CDCl3) δ 7.41-7.39 (m, 2H),
7.21 – 7.12 (m, 4H), 7.06-7.04 (m, 1H), 6.17 (s, 1H), 5.49 (s, 1H), 5.46-
5.43 (m, 1H), 5.30-5.29 (m, 1H), 4.21 (dd, J = 12.0, 2.4 Hz, 1H), 3.94-
3.88 (m, 1H), 2.78-2.75 (m, 4H), 2.36 (s, 3H), 1.82 – 1.78 (m, 4H); 13C
NMR (100 MHz, CDCl3) δ 142.90, 140.11, 137.59, 137.45, 135.76,
131.31, 129.46, 129.23, 127.08, 127.03, 123.61, 115.79, 104.25, 80.58,
70.01, 29.56, 29.22, 23.21, 21.48; HRMS (ESI/QTOF) m/z: [M + H]+
Calcd for [C22H25O3]+: 337.1798; found 337.1797.
Compound
8o:
((6'R)-6'-(1-(4-(naphthalen-1-
yl)phenyl)vinyl)spiro[adamantane-2,3'-[1,2,4]trioxane])
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using n-Hexane as eluent. Yield: 50%; Solid; m.p. 119-
121 ºC; FT-IR (KBr, cm-1) 1737.21, 1448.15, 1222.23, 1094.52, 913.42,
783.00; 1H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 16.0, 8.4 Hz, 3H),
7.58 – 7.37 (m, 8H), 5.62 (s, 1H), 5.37 (s, 1H), 5.34 (d, J = 2.8 Hz, 1H)),
4.09 – 3.93 (m, 1H), 3.88-3.84 (m, 1H), 2.99 (s, 1H), 2.15 – 1.58 (m,
13H); 13C NMR (100 MHz, CDCl3) δ 143.18, 140.72, 139.66, 137.64,
133.87, 131.55, 130.36, 128.40, 127.90, 126.99, 126.29, 126.21, 125.97,
125.93, 125.47, 116.35, 104.82, 80.08, 62.33, 37.28, 36.33, 33.68,
33.57, 33.34, 33.09, 29.46, 27.23; HRMS (ESI/QTOF) m/z: [M + H]+
Calcd for [C30H31O3]+: 439.2268; found 439.2267.
Compound
8k:
3-(4-methoxyphenyl)-6-(1-(5,6,7,8-
tetrahydronaphthalen-2-yl)vinyl)-1,2,4-trioxane
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 3% ethyl acetate: n-Hexane as eluent. Yield: 40%;
Solid; m.p. 50-52 ºC; 1H NMR (400 MHz, CDCl3) δ 7.46-7.42 (m, 2H),
7.15 – 7.11 (m, 2H), 7.05 – 7.03 (m, 1H), 6.92-6.88 (m, 2H), 6.14 (s, 1H),
5.48 (s, 1H), 5.44 (dd, J = 10.4, 1.6 Hz, 1H), 5.29 (s, 1H), 4.21 (dd, J =
12.0, 2.4 Hz, 1H), 3.93-3.87 (m, 1H), 3.81 (s, 3H), 2.77-2.76 (m, 4H),
1.81-1.77 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 160.93, 142.91, 137.59,
137.45, 135.77, 129.45, 128.59, 127.08, 126.51, 123.61, 115.78, 113.92,
104.09, 80.53, 70.06, 55.40, 29.56, 29.23, 23.21; HRMS (ESI/QTOF)
m/z: [M + H]+ Calcd for [C22H25O4]+: 353.1748; found 353.1749.
Compound
yl)phenyl)vinyl)-1,2,4-trioxane
8p:
3-(4-methoxyphenyl)-6-(1-(4-(naphthalen-1-
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 5% ethyl acetate: n-Hexane as eluent. Yield: 52%;
Solid; m.p. 76-78 ºC; 1H NMR (400 MHz, CDCl3) δ 7.96 – 7.82 (m, 3H),
7.58 – 7.40 (m, 10H), 6.92-6.90 (m, 2H), 6.20 (s, 1H), 5.66 (s, 1H), 5.56-
5.53 (m, 1H), 5.43 (s, 1H), 4.32-4.29 (m, 1H), 4.02 (dd, J = 11.6, 10.0 Hz,
1H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.98, 142.60, 140.87,
139.61, 137.46, 133.88, 131.54, 130.41, 128.63, 128.42, 127.94, 126.99,
126.41, 126.23, 125.95, 125.48, 116.97, 113.95, 104.18, 80.51, 69.94,
55.41; HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for [C28H25O4]+: 425.1748;
found 425.1749.
Compound
8l:
(3-(1-(4-(naphthalen-1-yl)phenyl)vinyl)-1,2,5-
trioxaspiro[5.5]undecane)
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 1.0% ethyl acetate: n-Hexane as eluent. Yield:
71%; Solid; m.p. 79-81ºC; 1H NMR (400 MHz, CDCl3) δ 7.92 – 7.84 (m,
3H), 7.54 – 7.40 (m, 8H), 5.62 (s, 1H), 5.38 (s, 1H), 5.34 (d, J = 10.4 Hz,
1H), 4.08-4.02 (m, 1H), 3.87-3.84 (m, 1H), 2.31 – 2.19 (m, 1H), 2.10-2.05
(m, 1H), 1.94 – 1.56 (m, 8H); 13C NMR (100 MHz, CDCl3) δ 143.17,
140.74, 139.65, 137.58, 133.87, 131.55, 130.36, 128.40, 127.90, 126.99,
126.32, 126.21, 125.97, 125.93, 125.47, 116.42, 102.77, 80.27, 62.85,
34.76, 29.10, 25.64, 22.44, 22.40; HRMS (ESI/QTOF) m/z: [M + H]+
Calcd for [C26H27O3]+: 387.1955; found 387.1954.
Materials and Methods for antiplasmodial activity:
In vitro cultivation and maintenance of Plasmodium falciparum
culture:
CQ resistant (Pf INDO) strains of P. falciparum were cultivated in vitro by
the method of Trager & Jensen, 1976 with minor modifications.33
Cultures were maintained in fresh O+ve human erythrocytes at 4%
haematocrit in complete medium at 37 °C under reduced O2 (gas mixture
5% O2, 5% CO2, and 90% N2). Serum supplemented cRPMI was used
for cultivation of Pf INDO. Thin cell smears were prepared from culture
plate and fixed by methanol. Percentage of parasitemia was calculated
by microscopic examination of Giemsa-stained blood smears. Medium
was changed daily (every 24 h) and the parasitemia was maintained at 5-
10 %. Cultures at high percentage parasitemia (>10 %) were diluted to
low percentage of parasitemia using fresh RBC.
Compound 8m: ((R)-8-(1-(4-(naphthalen-1-yl)phenyl)vinyl)-6,7,10-
trioxaspiro[4.5]decane)
The crude product was purified by column chromatography over silica gel
(100-200 mesh) using 0.5% ethyl acetate: n-Hexane as eluent. Yield:
70%; Solid; m.p. 111-113 ºC; FT-IR (KBr, cm-1) 1737.60, 1445.82,
1219.95, 1097.59, 912.08, 782.67; 1H NMR (400 MHz, CDCl3) δ 7.91-
7.84 (m, 3H), 7.56 – 7.38 (m, 8H), 5.62 (s, 1H), 5.41 – 5.37(m, 2H), 4.03
– 3.85 (m, 2H), 2.63 – 2.47 (m, 1H), 2.07 – 1.59 (m, 7H); 13C NMR (100
MHz, CDCl3) δ 142.97, 140.77, 139.63, 137.48, 133.87, 131.54, 130.36,
128.41, 127.91, 126.99, 126.34, 126.22, 125.96, 125.93, 125.47, 116.54,
114.74, 80.29, 65.25, 37.15, 32.91, 24.90, 23.48; HRMS (ESI/QTOF)
m/z: [M + H]+ Calcd for [C25H25O3]+: 373.1798; found 373.1797.
Preparation of drug stock and dilutions:
All test compounds (25 mM) and artesunate (1 mM) were dissolved in
100 % DMSO. Chloroquine (1 mM) stock solutions were prepared in
water (milliQ grade). All stocks were then diluted with 10 % DMSO (in
RPMI) to achieve the required concentrations. In all cases the final
concentration of DMSO maintained at 0.4%, which was found to be non-
toxic to the parasite. Further, antimalarial drugs and test compounds
were placed in 96-well flat-bottom tissue culture grade plates to yield
triplicate wells with drug concentrations ranging from 0-100 μM in a final
Compound
8n:
((R)-3,3-dimethyl-6-(1-(4-(naphthalen-1-
yl)phenyl)vinyl)-1,2,4-trioxane)
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000045
ChemMedChem
FULL PAPER
well volume of 100 μL for antiplasmodial drug screening. The values of
IC50 given are means where standard deviations in all cases were <2%.
structure (PDB id: 5JC1) was reported with fosmidomycin analogue,
LC55 paved the pathway for in silico screening for small molecules
against this protein.
3.
Dihydrofolate reductase: Thymidylate synthase (DHFR-TS): this
enzyme recycles folates in parasites for use in different metabolic
pathways. Early drug discovery against this protein led to mutation
in protein at N51I+C59R+S108N+I164L which is attributed to
pyrimethamine resistance. Two crystal structure of this protein have
been reported with pyrimethamine one wild type (PDB id: 3QGT)
and mutant (PDB id: 3QG2).
Thioredoxin reductase (TrxR): This protein is involved in
managing oxidative stress response in parasite. Crystal structure of
this protein in association with FAD as ligand was deposited by
Boumis et.al with PDB id 4B1B.
Falcipain II: This protein is involved in haemoblobin degradation
pathway. These cysteine proteases breakdown haemoglobin into
smaller peptides. Crystal srtucture of falcipin II is reported with its
inhibitor epoxysuccinate 64 (E64) with PDB id 3BPF.
Plasmepsin II: Plasmepsins are aspartic proteases that are
involved in hemoglobin degradation in the food vacuole where they
initiate hemoglobin degradation by hydrolyzing the peptide bond
between residues Phe33 and Leu34 in the α chain of native
hemoglobin. Crystal srtucture of plasmepsin II is reported with its
inhibitor n-((3s,4s)-5-[(4-bromobenzyl)oxy]-3-hydroxy-4-{[n-(pyridin-
Cytotoxicity Assay
Cytotoxicity was assessed using the 3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) (5 mg/mL) assay. Briefly, 5 × 103
cells per well were seeded in 96-well plates before drug treatments. After
overnight cell culture, the cells were exposed to different concentrations
of selected compounds (0–100 µM) for 24 h. Cells without drug treatment
and with 10% DMSO treatment were used as controls. Subsequently, 10
µL of 5 mg/mL MTT solution was added to each well and incubated at
37 °C for 4 h, followed by the centrifugation at 4000 rpm for 5 min and
removing all spent media. Formazan crystals formed were dissolved in
200 µL DMSO. The absorbance, A570 nm, was then determined in each
well. The percentage cell viability was calculated using the expression: %
Viability = Atreated / Acontrol × 100.
4.
5.
6.
Material and Methods for anticancer activity:
Cell Culture, Reagent and cells
Adenocarcinomic human alveolar basal epithelial (A549) and human
hepatoma (HepG2) cancer cell lines, immortalized normal human liver
(LO2), human bronchial epithelial (BEAS-2B cells were purchased from
ATCC (ATCC, Manassas, VA, USA). The cells were cultured in RPMI-
1640 (A549 cell lines) and DMEM (HepG2, LO2 and BEAS-2B) medium
supplemented with 10% fetal bovine serum and antibiotics: Penicillin (50
U/Ml) and streptomycin (50 µg/mL; Invitrogen, Paisley, Scotland, UK). All
cells were incubated at 37 °C in a 5% humidified CO2 incubator). All test
compounds were dissolved in DMSO at a final concentration of 50 mM
and stored at −20 °C before use.
2-ylcarbonyl)-lvalyl]amino}pentanoyl)-l-alanyl-l-leucinamide
PDB id 1W6H.
with
7.
EGFR protein: The X-ray crystal structure of Epidermal Growth
Factor Receptor tyrosine kinase (EGFR) a target for cancer, in
complex with inhibitor is obtained from the Protein Data Bank.34
Protein and ligand preparation
Protein preparation wizard of Schrodinger Maestro suite used to prepare
and water molecules were removed, missing side chains, hydrogen
atoms were added to the proteins and all atom force field (OPSL3)
charges and atom types were assigned.35-43 Optimization and
minimization of the protein was performed by applying restrains on heavy
atoms within RMSD of 0.30 Å.44 Dihydrofolate reductase (DHFR) enzyme
recycles folates in parasites for use in different metabolic pathways. Early
drug discovery against this protein led to mutation in this protein, which is
attributed to pyrimethamine resistance, mutant protein structure in
complex with inhibitor were obtained from the Protein Data Bank.
Cytotoxicity Assay
Cytotoxicity was assessed by the 3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) (5 mg/mL) assay, as described
previously.12 Briefly, 5 × 103 cells per well were seeded in 96-well plates
before drug treatments. After overnight cell culture, the cells were
exposed to different concentrations of selected compounds (0.19–100
µM) for 72 h. Cells without drug treatment were used as controls.
Subsequently, 10 µL of 5 mg/mL MTT solution was added to each well
and incubated at 37 °C for 4 h, followed by the addition of 100 µL
solubilization buffer (12 mMHCl in a solution of 10% SDS) and overnight
incubation. The absorbance, A570 nm, was then determined in each well
on the next day. The percentage cell viability was calculated using the
expression: % Viability = Atreated / Acontrol × 100, and was given as
cytotoxicity in Table (1-4) (See supporting infornation Fig 6-9).
Associated Content
Supporting Information
Table 1. Percentage yields of intermediate compounds 5a-6f; 1H NMR
and 13C NMR spectra of all compounds 5a-c, 6a-c and 8a-p and figures
1-9 are presented in the supporting information and available free of
charge on the journal website.
Docking studies of trioxanes with P. falciparum and EGFR proteins
Sequencing of P. falciparum genome has led to novel molecular targets
for drug design. However, for our study we chose targets from different
functional process which are as follows.
Author Contribution
SC, VKWW and DS conceived and designed the study. MKT synthesized
all the compounds with assistance from BRKS, LY, and RS. PC carried
out anticancer studies along with VKWW. PA carried out antiplasmodial
studies along with DS. SC and MKT carried out the SAR analysis and
analytical interpretation of all the synthesized compounds. PA and DS
carried out in silico antiplasmodial docking studies. DKY carried out in
silico anticancer docking studies. SC, VKWW and DS wrote the
manuscript. All authors have read and approved the final version of the
manuscript.
1.
Fatty acid biosynthesis pathway: Parasites and humans have
different pathways for fatty acid synthesis that makes it a good drug
target. In this pathway, FabZ protein i.e. β-hydroxyacyl-acyl carrier
protein dehydratase catalyzes dehydration of β-hydroxyacyl-acyl
carrier protein to trans-2-enoyl-ACPs. FabZ has been reported in its
crystal structure (PDB id: 3AZB) with its inhibitor NAS91-11 which
binds to active site of this enzyme and stabilizes the substrate
binding loop, which exist in disordered state in absence of its
substrate.
Competing interests
2.
Mevalonate pathway of isoprenoid synthesis: This pathway is
absent in humans and is attractive target for antimalarial discovery.
1-deoxy-D-xyulose-5-phosphate reductoisomerase (DXR) crystal
The authors(s) confirm that this article content have no conflicts of
interests.
11
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as anti-malarial agents and a process of producing the substituted 1,2,4-
trioxane,” US Patent no. 6316493 B1, 2001.
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S. C. acknowledges DST, New Delhi for DST-ARRS Indo-
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M.K.T acknowledges CSIR, New Delhi (Award No.:
[09/964(0013)2K18] EMR-1) for providing financial assistance in
the form of senior research fellowship (SRF). This work was
supported by a FDCT grant from the Macao Science and
Technology Development Fund to VKWW (Project code:
0022/2018/A1). D.K.Y. acknowledges the National Research
Foundation of Korea (NRF), which is funded by the Ministry of
Education, Science, and Technology (No: 2017R1C1B2003380)
at the Gachon University, Incheon city, Korea. B.R.K.S, L.Y and
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Entry for the Table of Contents
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A new series of functionalized arylvinyl-1,2,4-trioxanes 8a-p has been prepared and assessed for their in vitro antiplasmodial and
cytotoxic activities. Compounds, 8g and 8m, showed 11-fold and >9-fold more potent antiplasmodial activity, respectively, as
compared to chloroquine (IC50 = 0.546 µM). Compounds 8l exhibited >60-fold more cytotoxicity than artemisinin (IC50 = 100 µM)
against lung cancer cell lines (A549). In silico docking studies validated the wet results. Thus, 8a-p exhibited dual potency as
antiplasmodial and anticancer agents.
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