Anti-proliferative and anti-malarial activities of spiroisoxazoline analogues of artemisinin

Article abstract of DOI:10.1002/ardp.201800192

A series of spiroisoxazoline analogues of artemisinin was synthesized by employing 1,3-dipolar cycloaddition between various in situ generated nitrile oxides and artemisitene. All the synthesized compounds were tested for their anti-proliferative and anti

Full text of DOI:10.1002/ardp.201800192

Received: 28 June 2018
Revised: 22 October 2018
Accepted: 4 November 2018
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DOI: 10.1002/ardp.201800192
FULL PAPER
Anti-proliferative and anti-malarial activities of
spiroisoxazoline analogues of artemisinin
Surya Pratap1
Fatima Naaz2
Srinivas Reddy3,4
Kunal K. Jha5
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Kalicharan Sharma2
Devanna Nayakanti1
Dinakar Sahal6
Mymoona Akhter2
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Halmuthur M. S. Kumar3
Syed Shafi9
Vandana Kumari7
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Kailash Pandey7,8
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1 Research Scholar, Department of
Abstract
Biosciences, Jawaharlal Nehru Technological
University, Anantapur, India
A series of spiroisoxazoline analogues of artemisinin was synthesized by employing
1,3-dipolar cycloaddition between various in situ generated nitrile oxides and
artemisitene. All the synthesized compounds were tested for their anti-proliferative
and anti-malarial activities. Among thecompounds tested, compound 11a was foundto
be potent against the HCT-15 cancer cell line with IC50 = 4.04 μM when compared to
5-fluorouracil (IC50 = 35.53 μM). DNA cell cycle analysis shows that 11a was inhibiting
cell proliferation at the G2/M phase. Compound 11b was found to be most active
against Plasmodium falciparum with IC50 = 0.1 μM and also blocked host hemoglobin
hydrolysis by the falcipain-3 receptor. It was demonstrated to have better dynamics of
parasite killing efficiency than artemisinin. Molecular docking studies revealed that
these compounds interacted with falcipain-3 receptor sites.
2 Department of Pharmaceutical Chemistry,
School of Pharmaceutical Education and
Research, Jamia Hamdard, New Delhi, India
3 Vaccine Immunology Laboratory, Natural
Products Chemistry Division, CSIR-Indian
Institute of Chemical Technology, Hyderabad,
India
4 CNRS, Immunopathology and Therapeutic
Chemistry/Laboratory of Excellence Medalis,
Institut de Biologie Moléculaire et Cellulaire,
Strasbourg, France
5 Chemical and Biological Crystallography
Laboratory, Department of Chemistry, School
of Natural Science, Shiv Nadar University,
Tehsil Dadri, India
6 Malaria Research Laboratory, ICGEB, New
Delhi, India
K E Y W O R D S
1,3-dipolar cycloaddition, anti-malarial activity, anti-proliferative, artemisitene,
spiroisoxazoline
7 National Institute of Malaria Research, New
Delhi, India
8 National Institute for Research in
Environmental Health, Bhopal, India
9 Department of Chemistry, School of
Chemical and Life Science, Jamia Hamdard,
New Delhi, India
Correspondence
Dr. Syed Shafi, Department of Chemistry,
School of Chemical and Life Science, Jamia
Hamdard, Hamdard Nagar, New Delhi-
110062, India.
Email: syedshafi@jamiahamdard.ac.in
drugs for multi-drug resistance malaria Plasmodium falciparum.[1–3]
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1
INTRODUCTION
Although the exact mechanism of artemisinin and its analogues is still
Artemisinin 1 and its derivatives (dihydroartemisinin, artemether, and
unclear, it is believed to act by the generation of toxic carbon-centered
free radicals after activation by intra-parasitic heme-iron (which
artesunate) are currently recommended as front-line anti-malarial
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Arch Pharm Chem Life Sci. 2018;1–11.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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PRATAP ET AL.
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catalyzes the cleavage of this endoperoxide) and leading to the death
of the parasites.[4–7] Using artemisinin activity-based probes, a recent
study has identified multiple targets within the blood stage malaria
parasite. In these studies many deoxy analogs of the probes were
found to be inactive in targeting the proteins; but many of proteins
were also targeted in an iron-free environment created by deferox-
amine. This suggests that metals other than iron such as copper may
have the ability to cleave the endoperoxide bridge of artemisinin
(artemisinin activity-based probes).[8] World Health Organization
(WHO) has recommended artemisinin-based combination therapies
(ACT) as first-line treatment of uncomplicated falciparum malaria in the
malaria-affected areas.[9] Recently, there are certain alarming issues
about the signs of emerging resistance to such combination therapies
and artesunate monotherapy in the Thai–Cambodian border.[10–13] In
2014, WHO reported an artemisinin combination therapy (ACT)
resistance in North Kivu Province. In a case study, it was reported that
18 patients who were suffering from severe malaria did not responded
to ACT and intravenous artesunate monotherapy in this province even
after the treatment of 6 months. When these 18 patients were treated
with compressed leaf tablets of Artemisia annua (dried leaf Artemisia,
DLA), they were totally recovered.[14] Later on, mechanistic studies of
artemisinin resistance were explored and found that resistance of
artemisinins and ACTs was developed maybe due to the activation of
phosphatidylinositol 3-kinase (PI3P) which causes amplification of
PI3P vesicles and ultimately modifies both the parasite and host red
cells.[15,16] Artemisinin resistance would be disastrous for global
malaria control. Keeping in view of the emerging resistance, the
development of new artemisinin derivatives is an important area for
discovering new anti-malarial drugs.
artemanalogues were based on the scaffold modification of artemisinin
at the C10-carbonyl function (compounds 2–8, Figure 1). Generally
these C-10 modified analogues were found to be cysteine protease
falcipain-2 inhibitors. Cysteine protease falcipain-2 has been consid-
ered as one of the most attractive targets that cater the amino acids
required for the parasite protein biosynthesis through the hydrolysis of
host hemoglobin.[17,18]
To generate structurally diverse analogues, we aim to
generate some C-9 modified analogues in continuation of our
previous work on santonin.[19] Interestingly these C-9 modified
analogues were found to have excellent interactions with
falcipain-3 binding pocket of P. falciparum in contrast to the C-
10 artemanalogues that bind falcipain-2. A focused library of
spiroisoxazoline derivatives was synthesized and evaluated for
anti-proliferative and anti-malarial activities. Recently a simulta-
neous work has been carried out by Gang et al.[20] in which they
have reported the anti-proliferative activity of spiroisoxazoline
derivatives of artemisinin in human KB, KB/VCR, and A549 cell
lines. Though there are similarities in the present work, we have
independently carried out this work and synthesized a small
library of spiroisoxazoline derivatives and tested for anti-
proliferative activities in A-549, HCT-15, and Hep-G2 cancer
cell lines. Anti-malarial properties of these spiroisoxazoline
derivatives have been evaluated for the first time.
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RESULTS AND DISCUSSION
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2.1 Chemistry
The most common approach to overcome the resistance is to
continue modifying existing classes of artemisinins to provide new
analogues with improved attributes. Most of the recently reported
An exocyclic double bond was generated at the C-9 carbon of
artemisinin in two steps by employing reported method. Artemisitene
then was reacted with various in situ generated nitrile oxides (from
FIGURE 1 Artemisinin derivatives

PRATAP ET AL.
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SCHEME 1 Synthesis of spiroisoxazoline derivatives from artemisinin
chlorooximes and triethylamine) at 0–5°C to afford spiroisoxazoline
derivatives in 70–88% yields as illustrated in Scheme 1.
All the synthesized spiroisoxazoline derivatives were character-
ized by 1H NMR, 13C NMR, IR, ESI-MS. The regioselectivity of the 1,3-
dipolar cycloaddition was confirmed by the appearance two doublets
around δ 3.62 and 3.92 ppm in 1H NMR. The newly formed C9 R-
configuration was confirmed by the X-ray crystal of 11d shown in
Figure 2.
studies, it is evident that these spiroisoxazoline derivatives of
artemisinin are active against HCT-15 cancer cell lines.
Among all the compounds, 11a has shown significant cytotoxicity
against all the selected cells lines with IC50 of 32.43, 4.04, and
46.30 μM against A-549 cancer cell line, HCT-15, Hep-G2, respec-
tively. Whereas standard drug 5-FU showed IC50 of 52.82, 35.53,
33.68 μM against A-549 cancer cell line, HCT-15 cancer cell line, Hep-
G2 cancer cell line, respectively, and standard etoposide (VP-16)
exhibited IC50 of 8.13, 1.96, 16.23 μM against A-549 cancer cell line,
HCT-15 cancer cell line, and Hep-G2 cancer cell line, respectively.
Based on this cytotoxicity further cell cycle analysis was carried out
with this compound.
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2.2 Biological activity
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2.2.1 In vitro anti-proliferative activity
All the synthesized spirocyclic artemisinin analogues (artemalogues)
along with standards VP-16 and 5-FU were tested for cytotoxicity
against three human cancer cell lines such as A-549 (lung cancer cell
line), HCT-15 (colon cancer cell line), and Hep-G2 (liver cancer cell line)
by MTT assay method. IC50 (half maximal inhibitory concentration)
values of the test compounds were calculated and are presented in
Table 1. Among the three cell lines, colon cancer cell lines (HCT-15)
were found to be more susceptible to the compounds when compared
to other two cell lines. The anti-proliferative activity results are in
accordance with the previously reported results by Gang et al.[20]
against A-549 cancer cell lines. From the previous and the current
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2.2.2 Cell cycle analysis
Cell cycle analysis was studied to understand the cytotoxicity.
Compound 11a along with standard at IC50 concentration was chosen
for further evaluation. HCT-15 cancer cells were treated with 11a for
24 h and FACS (fluorescence-activated cell sorting) analysis was
carried out. Compound 11a showed 35.54% of cells at G2/M phase.
Moreover, decrease in cell population at G1 phase was observed
concomitantly.
Whereas the untreated cell (control), VP-16 (etoposide) and
compound 9 (artimesinine) exposed 21.20, 28.11, and 33.13% of cells
in G2/M phase, respectively, and 5-FU showed 74.02% cells in G1
phase. Based on these results from FACS analysis indicates 11a is
arresting at G2/M phase of cell cycle in HCT-15 cells as shown in
Figure 3.
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2.2.3 Anti-plasmodial activity
All the above-synthesized spiroisoxazoline derivatives were further
evaluated for their anti-plasmodial activity against Pf3D7 (P. falciparum
3D7) by using quantitative SYBR Green I-based estimation of DNA
replication after one developmental cycle (48 h) as a measure of
growth. IC50 values of the test compounds were calculated and are
presented in Table 1. All the newly synthesized spiroisoxazoline
derivatives were found to be less active when compared to the parent
artemisinin. Among the tested compounds, compound 11b was found
to be best with IC50 0.1 μM.
FIGURE 2 X-ray crystal of compound 11d (CCDC1819104)

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PRATAP ET AL.
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TABLE 1 Anti-proliferative and anti-plasmodial activity of spiroisoxazole analogs of artemisinin
Anti-proliferative activity (IC50 in μM)
Anti-plasmodial activity (IC50 in μM)
Code
11a
Compound
A-549 (lung)
HCT-15 (colon)
Hep-G2 (liver)
Pf3D7
32.43
4.04
46.30
0.3
11b
11c
>300
>300
34.79
39.45
121.33
86.64
0.1
0.45
11d
11e
>300
>300
74.84
55.11
53.64
0.6
204.36
0.25
11f
>300
>300
47.13
>300
82.63
>300
0.3
1.3
11g
11h
11i
9
208.57
>300
35.39
53.87
7.31
94.52
95.10
49.30
0.3
1.0
0.3
18.48
Artemisinin
VP-16 (etoposide)
5-FU
>300
8.13
>300
1.96
>300
16.23
33.68
<0.04
–
–
52.82
35.53
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2.2.4 Selectivity in the action of compound 11b
IC50 dose of compound 11b (700 nM), it was observed (Figure 4)
that after 48 h of culture, the rings had progressed only up to the
trophozoite stage, suggesting biochemical arrest and the resulting
interception of the progression to the schizont stage. It was
also observed that the rings did not mature even to the
trophozoite stage and the arrest of the parasite cycle was at the
ring stage.
In order to find whether there was any selectivity in the action of
compound 11b between ring and trophozoite, microscopic
evaluation of its action was studied against parasitized red cells
synchronized at ring and trophozoite (Figure 4) stages. When the
ring stage parasite culture was treated with seven times the

PRATAP ET AL.
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FIGURE 3 HCT-15 cells were treated with the compounds 9, 11a, VP-16, and 5-FU at IC50 concentration for 24 h; SS-1 = 11a, SS-11 = 9
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2.2.5 Hemoglobin hydrolysis assay of falcipain-3
and anti-malarial activities. Among all synthesized compounds,
compound 11a exerted significant anti-proliferative activity in HCT-
15 with low micromolar IC50 value (IC50 4.0 μM). Most of the
compounds demonstrated 8–10 fold of the activity compared with
parent artemisinin and also comparable with 5-FU. Cell cycle analysis
of compound 11a revealed that this compound induced apoptosis at
G2/M stage. With respect to anti-malarial activity, among all the tested
compounds, compound 11b was found to be potent against Pf3D7 and
was interacting with the falcipain-3 binding site. From the hemoglobin
hydrolysis assay, it was concluded that compound 11b effectively
blocked hemoglobin hydrolysis at 5 μM by falcipain-3 receptor and
in silico studies revealed that these compounds interacted with
falcipain-3 receptor. So, in silico data have fallen with experimental
data of compound 11b with falcipain-3 receptor. These analogues may
be further explored against this target to develop the new entities to
overcome the malarial resistance.
Hemoglobin hydrolysis is well-characterized function of falcipains (FP-
2 and FP-3), therefore we examined hemoglobin degradation by FP-3
(falcipain-3) in the presence of compound 11b. Interestingly, it was
found that compound 11b at 5 μM effectively blocked hemoglobin
hydrolysis by FP-3 as demonstrated in Figure 5, which ultimately
prevents parasite protein biosynthesis.
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2.3 Molecular docking study
Molecular docking studies of compounds 11a–i and 9 were performed
against falcipain-3 receptor to investigate the binding affinities and
interaction modes for the inhibitors using Maestro 9.6.[21] The docking
studies reveal a common binding orientation of compounds (11a–i and 9)
in the catalytic binding pocket of falcipain-3 receptor (PDBID: 3BWK).[22]
Herein, we report the binding pose of compound 11b and also compare
with the co-crystallized ligand C1P. As illustrated in Figures 6 and 7(a),
compound 11b is showing hydrogen binding interactions with Trp 215
and Gln 45 of the backbone of falcipain-3 receptor. In compound 11b,
both the spiroisoxazoline moiety and the phenyl ring attached to isoxazole
rings displayed hydrophobic and π–π stacking interactions with Trp 215
and His 183. Superimposition of compound 11b with co-crystallized
ligand (C1P) showed similar interactions at catalytic domain of falcipain-3
receptor as illustrated in Figure 7(b).
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EXPERIMENTAL
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4.1 Chemistry
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4.1.1 General
All commercial chemicals used as starting materials and reagents in
this study were purchased from Merck (India), Spectrochem, and
Sigma–Aldrich which were of reagent grade. All melting points were
uncorrected and measured using Electro-thermal IA 9100 apparatus
(Shimadzu, Japan); IR spectra were recorded on Bruker ALPHA FT-IR
spectrometer (Germany), 1H NMR spectra were determined on a
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CONCLUSION
In the current study, a focused library of spiroisoxazoline derivatives of
artemisinin were synthesized and evaluated for their anti-proliferative

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FIGURE 4 Continued.

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Bruker (200, 300, and 400 MHz) spectrometer and chemical shifts
were expressed as ppm against TMS as internal reference. Mass
spectra were recorded on 70 eV (EI Ms-QP 1000EX, Shimadzu, Japan),
column chromatography was performed on (Merck) silica gel 60
(particle size 0.06–0.20 mm).
(9R)-30-Phenyl-spiroisoxazoline artemalogue 11a
White solid, (67 mg, 84%); m.p. 145.6°C; IR (KBr) (cm−1): 2955, 2942,
2848, 1749, 1723, 1506, 1455, 1406, 1303, 1338, 1198, 1165, 1116,
1035, 992, 765, 696; 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.66–7.68
(m, 2H), 7.38–7.44 (m, 3H), 6.00 (s, 1H), 3.97 (d, 1H, J = 18.00 Hz), 3.66
(d, 1H, J = 18.40 Hz), 2.52–2.55 (m, 1H), 2.34–2.44 (m, 1H), 2.20–2.23
(m, 1H), 1.99–2.12 (m, 2H), 1.74–1.77 (m, 1H), 1.45–1.58 (m, 7H),
1.07–1.10 (m, 1H), 1.01 (d, 3H, J = 5.20 Hz); 13C NMR (CDCl3,
100 MHz): δ 169.11, 156.62, 130.46, 128.70, 128.59, 126.96, 105.70,
94.18, 83.64, 81.20, 50.36, 50.31, 49.72, 37.71, 35.86, 33.22, 25.12,
24.69, 24.16, 19.77; ESIMS: 400 (M++1).
The original spectra of the investigated compounds and their InChI
codes together with some biological activity data are provided as
Supporting Information.
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4.1.2 Procedure for the synthesis of spiroisoxazoline
artemalogues 11a–g
To
a
solution of hydroximoyl chloride 8a–g (0.21 mmol) and
(0.2 mmol) in THF (3 mL) was added Et3N
(9R)-30-(4-Chlorophenyl)-spiroisoxazoline artemalogue 11b
White solid, (74 mg, 86%); m.p. 202.5°C; IR (KBr) (cm−1): 2931, 2866,
1759, 1540, 1506, 1401, 1388, 1379, 1365, 1160, 1117, 1034, 1006,
984, 958, 898, 834, 788; 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.60 (d,
J = 8.00 Hz, 2H), 7.36 (d, J = 8.40 Hz, 2H), 6.00 (s, 1H), 3.92 (d, 1H,
J = 17.60 Hz), 3.66 (d, 1H, J = 18.00 Hz), 2.39–2.46 (m, 1H), 2.32–2.37
(m, 1H), 2.21 (dd, 1H, J = 12.80, 3.60 Hz), 2.09–2.13 (m, 1H), 2.01–2.03
artemisitene
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(0.25 mmol) at 0°C. The reaction mixture was stirred at 0–5°C for
6 h and then allowed to stir at room temperature for further 6 h. The
solvent was evaporated under vacuum and the residue was purified
by recrystallization in DCM/diethyl ether to provide compounds
11a–g.
FIGURE 4 Continued.
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FIGURE 4 Microscopy of anti-plasmodial action of compound 11b on Plasmodium falciparum 3D7: (A) untreated or 11b-treated or
artemisinin-treated schizont stage synchronized cultures were observed at 6–15 h. While schizonts with the characteristic rosette
arrangement of merozoites are intracellular at 6 h in untreated culture, they have (a) prematurely regressed and (b) lost the rosette
arrangement of merozoites in the 11b-treated or artemisinin-treated culture. At 15 h while the merozoites in control have invaded fresh red
cells to form rings, the merozoites of 11b-treated or artemisinin-treated cultures have failed to invade and form rings. Complete inhibition of
parasites was observed at this stage. (B) Untreated or 11b-treated or artemisinin-treated trophozoite-stage synchronized cultures were
observed after 24 h. Untreated culture shows intracellular rings while complete inhibition of parasites was observed in 11b-treated or
artemisinin-treated cultures. (C) Untreated or 11b-treated or artemisinin-treated, ring-stage synchronized cultures were observed after 48 h.
Untreated culture shows high ring-stage parasitemia, 11b-treated or artemisinin-treated cultures showed complete inhibition of parasites

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1.81–1.85 (m, 1H), 1.33–1.54 (m, 7H), 1.07–1.11 (m, 1H), 1.02 (d,
3H, J = 5.60 Hz); 13C NMR (CDCl3, 100 MHz): δ 168.89, 155.83,
131.96, 128.38, 127.58, 124.81, 116.00, 105.74, 94.18, 83.92, 81.17,
50.36, 50.34, 49.46, 37.72, 35.86, 33.21, 25.11, 24.69, 24.14, 19.78;
ESIMS: 477 (M+).
(9R)-3′-(4-Nitrophenyl)-spiroisoxazoline artemalogue 11e
White solid, (74 mg, 86%); m.p. 134.1°C; IR (KBr) (cm−1): 2950, 2939,
2877, 1744, 1666, 1562, 1543, 1500, 1457, 1379, 1366, 1238, 1164,
1158, 1114, 1030, 996, 980, 884, 813, 734, 667, 656; 1H NMR (CDCl3,
400 MHz) δ (ppm): 8.18 (d, J = 8.70 Hz, 2H), 7.77 (d, J = 8.40 Hz, 2H),
5.95 (s, 1H), 3.90 (d, J = 18.00 Hz, 1H), 3.60 (d, J = 17.70 Hz, 1H), 2.37–
2.41 (m, 1H), 2.25–2.33 (m, 1H), 2.15–2.21 (m, 1H), 1.94–2.08 (m, 2H),
1.77–1.81 (m, 1H), 1.37–1.42 (m, 7), 1.01–1.04 (m, 1H), 0.96 (d,
J = 5.10 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 168.53, 155.20,
148.72, 134.69, 127.69, 123.99, 105.83, 94.27, 84.68, 81.13, 50.37,
49.07, 37.70, 35.84, 33.17, 25.09, 24.67, 24.09, 19.76; ESIMS:
444.9 (M+).
(9R)-3′-(4-Methylphenyl)-spiroisoxazoline artemalogue 11f
White solid, (74 mg, 86%); m.p. – °C; IR (KBr) (cm−1): 2950, 2933, 2888,
1758, 1596, 1563, 1500, 1450, 1379, 1366, 1259, 1197, 1186, 1158,
1115, 1034, 1006, 984, 954, 897, 875, 822, 787, 688, 665; 1H NMR
FIGURE 5 Hemoglobin degradation assay by falcipain-3 in the
presence of compound 11b
(CDCl3, 400 MHz)
δ (ppm): 7.56 (d, J = 8.00 Hz, 2H), 7.18 (d,
J = 7.60 Hz, 2H), 6.00 (s, 1H), 3.95 (d, 1H, J = 17.60 Hz), 3.64 (d, 1H,
J = 18.00 Hz), 2.43–2.46 (m, 1H), 2.32–2.41 (m, 4H), 2.20 (dd,
J = 13.20, 4.00 Hz, 1H), 2.07–2.12 (m, 1H), 2.00–2.05 (m, 1H), 1.80–
1.86 (m, 1H), 1.32–1.58 (m, 7H), 1.06–1.13 (m, 1H), 1.01 (d, 3H,
J = 5.60 Hz); 13C NMR (CDCl3, 100 MHz): δ 169.13, 156.53, 140.72,
129.39, 126.91, 125.17, 105.68, 94.12, 83.44, 81.21, 50.39, 50.34,
49.87, 37.34, 35.88, 33.25, 25.14, 24.72, 24.20, 21.48, 19.80; ESIMS:
414 (M++1).
(m, 1H), 1.82–1.85 (m, 1H), 1.32–1.54 (m, 7H), 1.07–1.10 (m, 1H) 1.01
(d, 3H, J = 5.20 Hz); 13C NMR (CDCl3, 100 MHz): δ 168.87, 155.72,
136.45, 129.00, 128.18, 127.16, 105.73, 94.17, 83.90, 81.17, 50.38,
50.35, 49.53, 37.72, 35.87, 33.22, 25.11, 24.70, 24.15, 19.77; ESIMS:
433.9 (M+).
(9R)-30-(4-Fluorophenyl)-spiroisoxazoline artemalogue 11c
White solid, (74 mg, 86%); m.p. 116.9°C; IR (KBr) (cm−1): 2958, 2921,
2878, 1752, 1604, 1513, 1500, 1413, 1387, 1379, 1364, 1355,
1228, 1160, 1003, 976, 907, 880, 840, 803, 782, 658; 1H NMR
(9R)-3′-(4-Methoxylphenyl)-spiroisoxazoline artemalogue 11g
White solid, (74 mg, 86%); m.p. 150.1°C; IR (KBr) (cm−1): 2965, 2949,
2927, 2877, 1748, 1602, 1562, 1508, 1466, 1414, 1404, 1378,
1301, 1275, 1161, 1115, 1065, 1034, 1006, 906, 879, 833, 690,
668, 643; 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.56–7.68 (m, 2H),
6.89–6.98 (m, 2H), 6.00 (s, 1H), 3.94 (S, 3H), 3.86 (d, 1H,
J = 17.60 Hz), 3.61 (d, 1H, J = 17.60 Hz), 2.30–2.47 (m, 2H), 2.21
(dd, J = 13.60, 4.00 Hz, 1H), 2.10–2.13 (m, 1H), 2.01–2.04 (m, 1H),
1.81–1.85 (m, 1H), 1.36–1.59 (m, 7H), 1.07–1.13 (m, 1H), 1.02 (d,
3H, J = 5.20 Hz); 13C NMR (CDCl3, 100 MHz): δ 168.99, 161.32,
156.59, 156.14, 155.29, 128.79, 128.53, 126.69, 122.93, 122.02,
114.1, 111.88, 105.72, 94.16, 83.67, 83.35, 81.19, 56.26, 55.35,
50.38, 49.97, 49.66, 37.72, 35.87, 33.23, 25.12, 24.70, 24.16, 19.77;
ESIMS: 430 (M++1), 431 (M++2).
(CDCl3, 400 MHz)
δ (ppm): 7.65 (t, J = 8.00 Hz, 2H), 7.07 (t,
J = 8.00 Hz, 2H), 6.00 (s, 1H), 3.92 (d, 1H, J = 17.60 Hz), 3.62 (d,
J = 18.00 Hz, 1H), 2.39–2.45 (m, 1H), 2.32–2.35 (m, 1H), 2.19–2.23
(m, 1H), 2.08–2.12 (m, 1H), 1.98–2.07 (m, 1H), 1.81–1.84 (m, 1H),
1.36–1.47 (m, 7H), 1.06–1.12 (m, 1H) 1.01 (d, J = 4.00 Hz, 3H);
13C NMR (CDCl3, 100 MHz): δ 169.08, 155.69, 128.99, 128.91,
124.85, 116.00, 115.78, 105.73, 94.22, 83.78, 81.17, 50.34, 49.72,
45.90, 37.68, 35.84, 33.20, 25.09, 24.67, 24.13, 19.75; ESIMS: 418
(M++1), 419 (M++2).
(9R)-3′-(4-Bromophenyl)-spiroisoxazoline artemalogue 11d
White solid, (74 mg, 86%); m.p. 380°C; IR (KBr) (cm−1): 2965, 2949,
2877, 1746, 1500, 1413, 1379, 1371, 1200, 1160, 1116, 1063, 1034,
1001, 970, 909, 883, 824, 795, 775, 667; 1H NMR (CDCl3, 400 MHz) δ
(ppm): 7.50–7.57 (m, 4H), 6.00 (s, 1H), 3.92 (d, 1H, J = 18.00 Hz), 3.63
(d, 1H, J = 18.00 Hz), 2.39–2.46 (m, 1H), 2.32–2.37 (m, 1H), 2.22 (dd,
J = 13.60, 4.00 Hz, 1H), 2.08–2.13 (m, 1H), 2.00–2.03 (m, 1H),
(9R)-3′-(3,4-Dimethoxyphenyl)-spiroisoxazoline artemalogue
11h
White solid, (74 mg, 86%); m.p. 147.2°C; IR (KBr) (cm−1): 2961, 2943,
2876, 1748, 1601, 1513, 1405, 1368, 1378, 1344, 1231, 1214, 1162,
1115, 1034, 843, 789, 664; 1H NMR (CDCl3, 400 MHz) δ (ppm):

PRATAP ET AL.
9
|
FIGURE 6 (a) The binding mode of 11b (brown) is shown in the falcipain-3 receptor active site and important residues are highlighted with
cyan stick. (b) Ligaplot of 11b is shown in the falcipain-3 receptor active site. (c) The binding mode of co-crystallized ligand (CIP) (brown) is
shown in the falcipain-3 receptor active site and important residues are highlighted with cyan stick
7.26–7.27 (m, 2H), 6.83 (d, J = 9.20 Hz, 1H), 5.99 (s, 1H), 4.05 (d, 1H,
J = 18.40 Hz), 3.89–3.93 (m, 7H), 2.39–2.47 (m, 1H), 2.31–2.38 (m,
1H), 2.26 (dd, J = 13.20, 4.00 Hz, 1H), 2.00–2.11 (m, 2H), 1.82–1.85 (m,
1H), 1.36–1.49 (m, 7H), 1.10–1.13 (m, 1H), 1.02 (d, 3H, J = 5.20 Hz);
13C NMR (CDCl3, 100 MHz): δ 169.02, 156.63, 150.95, 147.84,
124.74, 119.92, 113.05, 112.40, 105.66, 94.19, 84.39, 81.08, 56.26,
56.22, 51.69, 50.37, 50.27, 37.74, 35.84, 33.26, 25.14, 24.72, 24.24,
19.80; ESIMS: 460 (M++1).
(s, 1H), 3.99 (d, J = 18.00 Hz, 1H), 3.68 (d, J = 18.00 Hz, 1H), 2.45–2.48
(m, 1H), 2.37–2.42 (dd, J = 18.80 and 4.0 Hz, 1H), 2.23–2.27 (dd,
J = 13.20 and 3.20 Hz, 1H), 2.10–2.14 (m, 1H), 2.02–2.10 (m, 1H),
1.87–1.84 (m, 1H), 1.33–1.55 (m, 6H), 1.26 (m, 1H), 1.09–1.15 (m, 1H),
1.03 (d, J = 5.20 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 168.68,
155.12, 148.42, 132.49, 130.48, 129.87, 124.91, 121.69, 105.84,
94.30, 84.49, 81.11, 50.33, 49.16, 37.68, 35.82, 33.17, 25.09, 24.67,
24.08, 19.75; ESIMS: 444 (M+).
(9R)-3′-(3-Nitrophenyl)-spiroisoxazoline artemalogue 11i
|
4.2 Biological assays
Pale yellow solid, (74 mg, 86%); m.p. 172.8°C; IR (KBr) (cm−1): 2964,
2943, 2928, 2871, 1753, 1650, 1521, 1509, 1433, 1388, 1379, 1365,
1340, 1199, 1161, 1114, 1034, 1011, 980, 933, 913, 882, 851, 753,
691, 666; 1H NMR (CDCl3, 400 MHz) δ (ppm): 8.43 (s, 1H), 8.27 (d,
J = 8.00 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 8.00 Hz, 1H), 6.02
|
4.2.1 Anti-proliferative activity
The cytotoxicity of the compounds was measured by using the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide

10
PRATAP ET AL.
|
stage, then cells were treated with SS-1 along with standards at IC50
concentration for 24 h. After 24 h incubation cells were harvested,
washed with PBS and fixed with 70% ice-cold ethanol for 1 h. Cells
were stained with propidium iodide and incubated 30 min in dark. Cell
cycle was performed by BD flow cytometry (FACSVerse).
|
4.2.3 In vitro cultivation of P. falciparum
3D7 strains of P. falciparum were cultivated by the method of Trager
and Jensen.[25] Cultures were maintained in fresh O+ human
erythrocytes at 4% hematocrit in complete medium (RPMI 1640
with 0.2% sodium bicarbonate, 0.5% Albumax, 45 mg/L hypoxanthine,
and 50 mg/L gentamicin) at 37°C under reduced O2 (gas mixture 5%
O2, 5% CO2, and 90% N2). Parasitemia was monitored by microscopic
examination of Giemsa-stained blood smears. Synchronized ring stage
parasite was obtained by 5% sorbitol treatment.[26] Trophozoites and
schizont-stage parasites were enriched by using Percoll gradient.[27]
|
4.2.4 Measurement of inhibition of P. falciparum
growth in culture
SYBR green I-based fluorescence assay was used to measure the
growth inhibition of P. falciparum as described by Smilkstein et al.[28] In
this study chloroquine sensitive Pf3D7 was used for in vitro culture.
Artemisinin was used as positive control. Artemisinin and test
compounds were dissolved in DMSO. All stocks were then diluted
with complete culture medium to achieve the required concentrations.
In all the cases, final concentration was 0.4% DMSO, which was found
to be nontoxic to the parasite. Artemisinin and test compounds were
then placed in triplicate wells of 96-well flat-bottom tissue culture
grade plates with drug concentrations ranging from 0 to 100 µM in a
final well volume of 100 µL for primary screening. Parasite culture was
synchronized at ring stage with 5% sorbitol. Synchronized culture was
aliquoted to drug containing 96-well plate at 2% hematocrit and 1%
parasitemia. After 48 h of incubation under standard culture con-
ditions, plates were harvested and read by the SYBR Green I
fluorescence-based method[25] using a 96-well fluorescence plate
reader (Victor, Perkin-Elmer), with excitation and emission wave-
lengths of 497 and 520 nm, respectively. The fluorescence readings
were plotted against drug concentration, and IC50 values were
obtained by visual matching of the drug concentration giving 50%
inhibition of growth.
FIGURE 7 (a) Ligplot of co-crystallized ligand (CIP) is shown in
the falcipain-3 active site. (b) Superimposition of 11b (green) with
co-crystallized ligand (CIP) (blue) at catalytic domain of falcipain-3
receptor
method.[23] Briefly, HCT-15, human colorectal adenocarcinoma cells
1 × 104 in 100 μL of media were seeded in 96-well plate and kept for
overnight incubation at 37°C in 5% CO2, cells were treated with
100 μL of serially diluted compounds at a final concentration of 200–
0.78 μM in triplicate each. The cell viability was studied after 48 h, by
adding 20 μL of MTT (5 mg/mL) in each well. The plates were further
incubated at 37°C for additional 4 h. The untransformed MTT in the
medium was discarded and the formazan crystals formed in the cells
were dissolved with 100 μL of DMSO and the optical density was
measured at 570 nm using TECAN multimode reader infinite
M200PRO.
|
4.2.5 Hemoglobin hydrolysis assay of falcipain-3
Expression, purification, and activation of FP-3 (falcipain-3) were done
as described earlier.[29] Briefly, Escherichia coli M15 cells containing FP-
3 recombinant plasmid were induced with 1 mM IPTG and purified
from inclusion bodies by Ni-NTA affinity chromatography (Ni-NTA
beads, Qiagen).[30] The recombinant proteins were refolded as
described earlier,[29,31] activated by addition of 100 mM sodium
acetate (NaAc, pH 5.5) and stored at −20°C. To analyze the activity of
|
4.2.2 Cell cycle analysis
Cell cycle analysis was performed according to method described by
Rosner et al.[24] Shortly, HCT-15 cells 1 × 105 cells were seeded in
6-well plate and synchronized for overnight to bring all cells in to G1

PRATAP ET AL.
11
|
recombinant FP-3 in the presence of compound 11b, hemoglobin
degradation was assessed. Briefly, 4 µg of human hemoglobin (Sigma)
was added to 10 µg of enzyme with 5 μM of 11b and incubated in
100 mM NaAc (pH 5.5), 5 mM DTT for 3 h at 37°C. Reactions were
analyzed on 15% SDS-PAGE followed by Coomassie Blue staining.
[8] H. M. Ismail, V. Barton, M. Phanchana, S. Charoensutthivarakul, M. H.
Wong, J. Hemingway, G. A. Biagini, P. M. O’Neill, S. A. Ward, Proc.
Natl. Acad. Sci. USA 2016, 113, 2080.
[9] Guidelines for the Treatment of Malaria. World Health Organization,
Geneva 2006.
[10] A. P. Alker, P. Lim, R. Sem, Am. J. Trop. Med. Hyg. 2007, 76, 641.
[11] M. B. Denis, R. Tsuyuoka, Y. Poravuth, Trop. Med. Int. Health 2006, 11,
1360.
|
4.3 Molecular docking
[12] M. B. Denis, R. Tsuyuoka, P. Lim, Trop. Med. Int. Health 2006, 11,
1800.
|
[13] H. Noedl, Y. Se, K. Schaecher, B. L. Smith, D. Socheat, M. M. Fukuda,
N. Engl. J. Med. 2008, 359, 2619.
4.3.1 Ligand and protein preparation
For better understanding of binding mode of compounds 11a–j at the
molecular level, we carried out molecular docking simulations for
compounds 11a–j at the falcipain-3 receptor catalytic ligand binding site
(PDBID: 3BWK).[22] The docking simulations of compounds 11a–j was
performed using Maestro, version 9.6 implemented from Schrodinger
software suite. The ligand was sketched in 3D format using build panel
and were prepared for docking using ligprep application. The protein for
docking study was taken from Protein Data Bank (PDB ID: 3BWK) and
prepared by removing solvent, adding hydrogen and further minimiza-
tion in the presence of bound ligand (C1P) using protein preparation
wizard. Grids for molecular docking were generated with bound
co-crystallized ligand. For the validation of docking parameters, the
co-crystal ligand (C1P) was re-docked at the catalytic site of protein and
the RMSD between co-crystal and re-docked pose was found to be
0.445 Å. Compounds 11a–j was docked using Glide extra-precision (XP)
mode, with up to three poses saved per molecule.
[14] B. D. Nsengiyumva, M. K. Luc, G. B. Pascal, L. W. Robert, J. T. Melissa,
J. W. Pamela, Phytomedicine 2017, 32, 37.
[15] S. Niraja, H. Kasturi, Curr. Opin. Pharmacol. 2018, 42, 46.
[16] H. Kasturi, B. Souvik, S. Innocent, Nat. Rev. Microbiol. 2018, 16,
156.
[17] P. J. Rosenthal, P. S. Sijwali, A. Singh, B. R. Shenai, Curr. Pharm. Des.
2002, 8, 1659.
[18] R. Ettari, F. Bova, M. Zappala, S. Grasso, N. Micale, Med. Res. Rev.
2010, 30, 136.
[19] K. Jabeena, P. S. Parvinder, D. M. Reddy, H. Irfan, S. Shafi, S. D.
Sawant, C. Gousia, M. Ajay, M. S. Alam, A. K. Saxena, S. Arvinda, B. D.
Gupta, H. M. S. Kumar, Eur. J. Med. Chem. 2013, 63, 279.
[20] L. Gang, S. Shanshan Song, S. Shiqi, M. Zehong, Z. Ao, D. Chunyong,
Eur. J. Med. Chem. 2015, 103, 17.
[21] Schrödinger Release 2017-4: Glide, Schrödinger, LLC, New York, NY,
2017.
[22] I. D. Kerr, J. H. Lee, C. J. Farady, R. Marion, M. Rickert, M. Sajid, K. C.
Pandey, C. R. Caffrey, J. Legac, E. Hansell, J. H. McKerrow, C. S. Craik,
P. J. Rosenthal, L. S. Brinen, J. Biol. Chem. 2009, 284, 25697.
[23] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
[24] M. Rosner, K. Schipany, M. Hengstschläger, Nat. Protocols 2013, 8,
602.
ACKNOWLEDGMENT
[25] W. Trager, J. B. Jensen, Science 1976, 193, 673.
[26] C. Lambros, J. P. Vanderberg, J. Parasitol. 1979, 65, 418.
[27] E. M. Rivadeneira, M. Wasserman, C. T. Espinal, J. Protozool. 1983, 30,
367.
The authors are thankful to Hon’ble Vice Chancellor, Jamia Hamdard
for providing the research facilities.
[28] M. Smilkstein, N. Sriwilaijaroen, J. X. Kelly, P. Wilairat, M. Riscoe,
Antimicrob. Agents Chemother. 2004, 48, 1803.
[29] P. S. Sijwali, L. S. Brinen, P. J. Rosenthal, Protein Expr. Purif. 2001, 22,
128.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
[30] C. P. Kailash, S. S. Puran, S. Ajay, N. Byoung-Kuk, J. R. Philip, J. Biol.
Chem. 2004, 279, 3484.
ORCID
[31] A. Herraez, Biochem. Mol. Biol. Educ. 2006, 34, 255.
Mymoona Akhter
http://orcid.org/0000-0003-4380-3965
Syed Shafi http://orcid.org/0000-0001-7657-0630
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
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