In vitro antimalarial activity and molecular modeling studies of novel artemisinin derivatives

Article abstract of DOI:10.1039/c5ra07697h

Cerebral malaria is a serious and sometimes fatal disease caused by a Plasmodium falciparum parasite that infects a female anopheles mosquito which feeds on humans. The parasites responsible for mosquito-borne infectious diseases are increasingly resistant to current drug approaches, and almost half of the world is at risk of contracting an illness. A series of twenty five new ether and ester derivatives of dihydroartemisinin (DHA) have been prepared based on in silico studies and in vitro antimalarial activity and later assessed against the chloroquine sensitive NF-54 strain of Plasmodium falciparum. In general the incorporation of nitro functionality in ester derivatives enhances the activity relative to artemisinin. Most of the ether derivatives were found to be as active as DHA, while 11-OH ether derivatives were not as active as DHA. The most potent analogue in the series was compound 21 which was several fold more active than artemisinin against P. falciparum used in the study. Molecular docking and ADMET studies were performed to explore the possible mode of interaction of active compounds in to the binding site pocket of malaria parasite target enzyme plasmepsin-II and evaluated compliance with oral bioavailability and pharmacokinetics parameters. The ester derivatives 19 and 20 were found to be twice active than DHA, having nitro functionality showing IC₅₀ 10.58 nM and 8.54 nM respectively.

Full text of DOI:10.1039/c5ra07697h

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1
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Page 1 of 43
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DOI: 10.1039/C5RA07697H
In vitro antimalarial activity and molecular modeling studies of novel
artemisinin derivartives
a,d
b,d
c
a
Rashmi Gaur , Harveer S Cheema , Yogesh Kumar , Suriya P Singh , Dharmendra K
c
b,d
c,d
a,d
Yadav , Mahendra P Darokar , Feroz Khan , Rajendra S Bhakuni *
a
b
c
Medicinal Chemistry Division
,
Molecular Bioꢀprospection Department, Metabolic and Structural Biology Department
,
CSIRꢀCentral
Institute of Medicinal and Aromatic Plants, Lucknowꢀ226015, India
d
Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi 110 001, India
*Corresponding authors. Tel: +915222718622; Fax: 915222342666
Eꢀmail addresses: bhakunirs2000@yahoo.com and bhakunirs2000@gmail.com

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DOI: 10.1039/C5RA07697H
ABSTRACT
Cerebral malaria is a serious and sometimes fatal disease caused by a Plasmodium falciparum
that infects a certain female anopheles mosquito which feeds on humans. The parasites
responsible for the mosquitoꢀborne infectious disease are increasingly resistant to current
drug approaches, and almost half of the world is at risk of contracting an illness. A series of
twenty five new ether and ester derivatives of dihydroartemisinin (DHA) have been prepared
based on in silico studies and in vitro antimalarial activity and later assessed against the
chloroquine sensitive NFꢀ54 strain of Plasmodium falciparum. In general the incorporation of
nitro functionality in ester derivatives enhances the activity relative to artemisinin. Most of
the ether derivatives were found to be as active as DHA, while 11ꢀOH ether derivatives were
not as active as DHA. The most potent analogue in the series was compound 21 which was
several fold more active than artemisinin against P. falciparum used in the study. Molecular
docking and ADMET studies were performed to explore the possible mode of interaction of
active compounds in to the binding site pocket of malaria parasite target enzyme plasmepsinꢀ
II and evaluated compliance with oral bioavailability and pharmacokinetics parameters. The
ester derivatives 19 and 20 were found to be twice active than DHA, having nitro
functionality showing IC50 10.58nM and 8.54nM respectively.
Keywords: Dihydroartemisinin, P. falciparum, in vitro, antimalarial, docking, plasmepsinꢀII
___________________________________________________
Abbreviations: MIC, minimum inhibitory concentrations; DEPT, distortionless enhancement by polarization
transfer; ESI-MS, electrospray ionization mass spectrometry, COSY, correlation spectroscopy,
HSQC,heteronuclear single quantum correlation, HMBC, heteronuclear multiple-bond correlation
spectroscopy; MIC, Minimum Inhibitory Concentration
*Corresponding author Tel: Fax: +91 522 2342666, +91 522 2342666; Eꢀmail address:
bhakunirs2000@gmail.com, rs.bhakuni@cimap.res.in

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Introduction
Malaria is one of the world's most deadly diseases and is becoming an increasingly serious
problem as malaria parasites have developed resistance to drugs such as chloroquine and
mefloquine. Malaria is endemic in many parts of the world. Globally, an estimated 1.2 billion
people are at high risk (>1 in 1000 chance of getting malaria in a year). According to the
latest estimates, 198 million cases of malaria occurred globally in 2013 and the disease led to
1
5
84 000 deaths. .Malaria is a major parasitic disease of the tropical and subtropical countries
1
including India . Hence the malaria situation is getting worse with rapid spread of multidrug
resistant Plasmodium falciparum. Isolation of artemisinin 1 from Artemisia annua, as the
active principle of the Chinese traditional drug against malaria, is a major breakthrough in
2
ꢀ12
malaria chemotherapy . Artemisinin owes its antimalarial activity due to the presence of
1,2,4ꢀtrioxane system. It is active against both chloroquineꢀsensitive and chloroquine resistant
malaria. The semisynthetic derivatives of artemisinin such as dihydroartemisinin 2,
artemether 3, arteether 4, and artesunic acid 5 are more active than artemisinin and are
currently used as drugs of choice for the treatment of malaria caused by multidrugꢀ resistant
13ꢀ14
P. falciparum
. Therefore, there is considerable urgency to develop new classes of
antimalarials. Artemisinin (1) (qinghaosu) is an unusual 1,2,4ꢀtrioxane which has been used
clinically in China for the treatment of multidrug resistant P. falciparum malaria. However
the clinical application of artemisinin has been limited by the drug's pharmacokinetic
properties. This has provided the impetus for the investigation of derivatives of this
compound, some of which include esters and ethers of the corresponding lactol,
dihydroartemisinin (DHA) (2). Analogues of this type are currently being developed as
2
potent and rapidly acting antimalarials . Chloroquineꢀresistant P. falciparum is present in
most of the countries of Asia, Africa, and South America. Resistance to the sulphonamideꢀ

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pyrimethamine combination is widespread in southeast Asia and South America. Field trials
with mefloquine have met with rapid emergence of malarial parasites resistant to the drug.
Resistance to quinine is not common, but the duration of the treatment with the drug
is long and requires hospitalization. Currently, artemisinin 1 and its derivatives
dihydroartemisinin 2, artemether 3, arteether 4, and artesunic acid 5 (Figure 1) are the only
2ꢀ11
class of drugs that are effective against multidrug resistant malaria . Artesunic acid, the
hemisuccinate ester of dihydroartemisinin 2, is one of the clinically useful derivative of
1
5ꢀ19
artemisinin
. Through strong worldwide programs malaria has been eradicated from many
countries, resistance to current malarial drugs continues and about 600,000 people a year die
from the disease, mostly children. As the disease and carrier mosquitoes build up resistances
to the current drugs, researchers are looking for the individual molecules that will kill the
1
2
malaria parasite to combat resistance. In continuation of our work to meet these objectives,
we have prepared a series of novel ether and ester derivatives of dihydroartemisinin 6ꢀ18, 19ꢀ
25 and 28ꢀ32.
MATERIALS AND METHODS:
General Experimental Procedures
Melting point was determined on a Toshniwal melting point apparatus. IR spectra were
recorded on a Perkin Elmer 1719 FTꢀIR spectrophotometer. NMR spectra were obtained in
acetoneꢀd , CDCl on a Bruker Avance, 300 MHz and 400 MHz instrument using TMS as
6
3
internal standard. The chemical shift values are reported in ppm and coupling constants in
Hz. ESIꢀMS spectra were recorded on a Perkin Elmer Turbo Mass/Shimadzu LCꢀMS. TLC
analyses were carried out on precoated silica gel 60 F plates (Merck Mumbai, India) were
2
54
used using solvent system, hexane:ethylacetate (7:3). All the required solvents and reagents
were purchased from Merck (Mumbai, India) and Sigma Aldrich., India. The compounds
2
were visualized by either exposure of TLC plates to I vapors or by spraying with vanillinꢀ

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sulfuricacid reagent, followed by heating at 110°C for 15 minutes. Siꢀgel, 60ꢀ120 mesh
spectrochem) was used in the column chromatography for the purification of metabolites.
(
1
13
The compounds were identified by their spectral IR, ID ( H, C, DEPT) NMR and ESIMS
analysis.
Test compound preparation. Isolation of secondary metabolite artemisinin from the plant
Artemisia annua, grown at the Central Institute of Medicinal and Aromatic Plants (CIMAP)
experimental farms, Lucknow, India, was carried out essentially according to the patented
19b
procedure . A schematic outline for the extraction protocol is provided in Fig. 2 summarizes
the outline for the preparation of a series of new and known derivatives of artemisinin.
Briefly, artemisinin (1) was reduced with sodium borohydride at 0 to ꢀ5°C to prepare
dihydroartemisinin (2) in 85% (wt/wt) yield in 2 h.
Synthesis of ether derivatives of artemisinin: After crystallization and drying,
Dihydroartemisinin (1.0 g, 3mmol) was dissolved in dichloromethane (20 ml), stirred for 12
min to 4 h at 0ꢀ10°C in the presence of appropriate alkylating reagents (3mmol), viz., oꢀ
nitrobenzylalcohol, mꢀnitrobenzylalcohol (for 6 and 7), 2,3ꢀdimethoxybenzoylalcohol, 2,5ꢀ
dimethoxybenzoylalcohol, 3,5ꢀdimethoxybenzoylalcohol (for 8, 9 and 10), oꢀbromo, pꢀ
cyanoꢀbenzoylalcohol,
oꢀchloro,pꢀflourobenzoylalcohol
(for
11
and
12),
3ꢀ
phenoxybenzylalcohol (for 13), 3ꢀmethylꢀ2ꢀbuteneꢀ1ꢀol (for 14), and octadecaneꢀ1ꢀol (for 15)
and the catalyst pꢀTSA (Fig. 3). After completion of the reaction (as monitored by thinꢀlayer
chromatography [TLC], the reaction mixture was quenched with cooled 1% aqueous
NaHCO
3
(50 ml) and extracted with ethyl acetate (three times with 50 ml each time). The
SO ), and concentrated to get
combined extract was washed with water till neutral, dried (Na
2
4
the crude mixture of β/α products (1.2 to 1.8 g). The mixture was subjected to silica gel
column chromatography to obtain the pure βꢀisomer.

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12βꢀArteꢀ3’ꢀnitrobenzylether (6): Elution of the column with nꢀhexaneꢀethyl acetate (95:5)
yielded an oily compound in 93% (w/w) yield). IR λmax (neat): 1254, 1171, 1103 (ether),
ꢀ1
1
1
,527, 1343 (Ar NO ), 2875, 2925, 1029, 875 cm ; H, COSYꢀ NMR (300MHz, CDCl )
2
3
δ0.87 (6H, d, J =6.0 Hz, H
3
ꢀ13, H
3
ꢀ14), 1.37 (3H, s, H
3
ꢀ15), 2.61 (1H, m, Cꢀ11), 4.77 (1H, d,
J=14.4 Hz, Haꢀ1’), 4.87 (1H, brs, αHꢀ12), 5.18 (1H, d, J=14.7 Hz, Hbꢀ1’), 5.37 (1H, s, Hꢀ5),
.37 (1H, d, J = 7.8 Hz, Hꢀ7’), 7.56 (2H, m, Hꢀ5’, Hꢀ6’), and 7.95 (1H, d, J=8.1 Hz, Hꢀ4’);
7
1
3
C, DEPTꢀ NMR(75 MHz, CDCl
3
) : δ13.3 (Cꢀ13), 20.7 (Cꢀ14), 24.8 (Cꢀ8), 25.0 (Cꢀ2), 26.5
(Cꢀ15), 31.3 (Cꢀ11), 34.9 (Cꢀ9), 36.8 (Cꢀ3), 37.7 (Cꢀ10), 44.6 (Cꢀ7), 52.9 (Cꢀ1), 67.80 (Cꢀ1’),
81.4 (Cꢀ6, q), 88.4 (Cꢀ5), 102.5 (Cꢀ12), 104.5 (Cꢀ4,q), 125.1 (Cꢀ4’), 128.5 (Cꢀ7’), 129.5 (Cꢀ
+
5’), 133.7 (Cꢀ6’), 134.7 (Cꢀ2’, q), and 148.1 (Cꢀ3’, q); ESIꢀMS(positive): m/z 420 [M+H]
ꢀ
calcd. for C22
H
30
O
7
N found 420 and (negative): 418 [MꢀH] calcd. for C22
H
28
O
7
N and found
418.
12βꢀArteꢀ4’ꢀnitrobenzylether (7): Elution of the column with nꢀhexaneꢀethyl acetate (95:5)
yielded a colorless oil in 90% (w/w) yield. IR λmax (neat): 1195, 1102 (ether), 1531, 1349
ꢀ1 1
(
Ar NO ), 2925, 2874, 1034, 875 cm ; H, COSYꢀ NMR (300 MHz, CDCl ) δ0.94 (3H, d, J
2
3
=
5.7 Hz, H
3
ꢀ13), 0.98 (3H, d, J =4.2 Hz, H
3
ꢀ14), 1.44 (3H, s, H
3
ꢀ15), 2.69 (1H, m, Hꢀ11),
4.62 (1H, d, J=17.6 Hz, Haꢀ1’), 4.91 (1H, brs, αHꢀ12), 4.97 (1H, d, J=17.6 Hz, Hbꢀ1’), 5.44
(1H, s, Hꢀ5), 7.48 (1H, dd, J = 7.5, 3.3 Hz, Hꢀ6’)7.61 (1H, d, J = 6.9 Hz, Hꢀ7’), 8.11 (1H, d, J
13
=
3
7.2 Hz, Hꢀ5’), 8.23 (1H, s, Hꢀ3’); C, DEPTꢀ NMR (75 MHz, CDCl ): δ 13.4 (Cꢀ13), 20.7
(Cꢀ14), 24.9 (Cꢀ8), 25.0 (Cꢀ2), 26.5 (Cꢀ15), 31.2 (Cꢀ11), 34.9 (Cꢀ9), 36.7 (Cꢀ3), 37.8 (Cꢀ10),
4
4.6 (Cꢀ7), 52.91 (Cꢀ1), 68.9 (Cꢀ1’), 81.4 (Cꢀ6, q), 88.4 (Cꢀ5), 102.1 (Cꢀ12), 104.6 (Cꢀ4, q),
22.3 (Cꢀ3’), 122.8 (Cꢀ5’), 129.6 (Cꢀ6’), 133.3 (Cꢀ7’), 141.0 (Cꢀ2’, q), 148.6 (Cꢀ4’, q); ESIꢀ
1
+
ꢀ
MS(positive): m/z 420 [M+H] calcd. for C22
30
H O
7
N found 420 and (negative): 418 [MꢀH]
+
calcd. for C22
H
28
O
7
N and found 418 and 442 [M+Na] .

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12βꢀArteꢀ3’, 4’ꢀdimethoxybenzylether (8): Elution of the column with nꢀhexaneꢀethyl
acetate (95:5) yielded a colorless oil in 90% (w/w) yield. IR λmax (neat): 1278, 1098 (ether),
ꢀ1
1
2
939, 2875, 1481, 1378, 1012, 787 cm ; H, COSYꢀ NMR (300 MHz, Acetoneꢀd ): δ0.92
6
(
6H, d, J =6.9 Hz, H
3
3 3
ꢀ13, H ꢀ14), 1.32 (3H, s, H ꢀ15), 2.56 (1H, m, Hꢀ11), 3.75, 3.79 (3H
each, s, 2 x OCH
3
), 4.47 (1H, d, J=12.3 Hz, Haꢀ1’), 4.86 (1H, d, J=12.3 Hz, Hbꢀ1’), 4.83
1
3
(
1H, d, J=4.5 Hz, αHꢀ12), 5.46 (1H, s, Hꢀ5), 6.97 (1H, m, Hꢀ5’, Hꢀ6’, Hꢀ7’ ); C, DEPTꢀ
NMR (75 MHz, Acetoneꢀd ): δ12.9 (Cꢀ13), 20.1 (Cꢀ14), 24.7 (Cꢀ8), 25.0 (Cꢀ2), 25.7 (Cꢀ15),
1.3 (Cꢀ11), 34.9 (Cꢀ9), 36.6 (Cꢀ3), 37.6 (Cꢀ10), 44.9 (Cꢀ7), 53.0 (Cꢀ1), 55.6, 60.3 (2 x
6
3
3
OCH ), 64.8 (Cꢀ1’), 81.0 (Cꢀ6, q), 88.0 (Cꢀ5), 101.5 (Cꢀ12), 103.9 (Cꢀ4, q), 112.5 (Cꢀ5’),
121.1 (Cꢀ7’), 124.1 (Cꢀ6’), 132.4 (Cꢀ2’, q), 147.4 (Cꢀ4’, q), and 153.0 (Cꢀ3’, q); ESIꢀ
+
34 7.
MS(positive): m/z 457 [M + Na] , molecular formula C24H O
12βꢀArteꢀ3’, 6’ꢀdimethoxybenzylether (9): Elution of the column with nꢀhexane– ethyl
acetate (95:5) yielded a colorless oil in 90% (w/w) yield. IR λmax (neat): 1276, 1082 (ether),
ꢀ1
1
2
934, 2872, 1476, 1381, 1019 cm ; H, COSYꢀ NMR (300 MHz, CDCl
3
): δ0.92 (3H, d, J
=
6.6 Hz, H ꢀ13), 0.96 (3H, d, J =7.5 Hz, H ꢀ14), 1.31 (3H, s, H ꢀ15), 2.58 (1H, m, Hꢀ11),
3
3
3
3
1
6
.73, 3.78 (3H each, s, 2xOCH
3
), 4.41 (1H, d, J=13.2 Hz, Haꢀ1’), 4.84 (1H, d, J=2.7 Hz, αHꢀ
2), 4.86 (1H, d, J=13.2 Hz, Hbꢀ1’), 5.46 (1H, s, Hꢀ5), 6.78 (1H, dd, J = 8.7, 3.0 Hz, Hꢀ5’),
1
3
.88 (1H, d, J = 8.7 Hz, Hꢀ4’), 6.97 (1H, d, J=3.0 Hz, Hꢀ7’); C, DEPTꢀ NMR (75 MHz,
): δ12.9 (Cꢀ13), 20.1 (Cꢀ14), 24.7 (Cꢀ8), 25.0 (Cꢀ2), 25.7 (Cꢀ15), 31.4 (Cꢀ11), 35.0
Cꢀ9), 36.6 (Cꢀ3), 37.6 (Cꢀ10), 44.9(Cꢀ7), 53.0 (Cꢀ1), 55.3, 55.6 (2 x OCH ), 65.0 (Cꢀ1’),
Acetoneꢀd
6
(
3
81.0 (Cꢀ6, q), 87.9 (Cꢀ5), 101.7 (Cꢀ12), 103.8 (Cꢀ4, q), 111.5 (Cꢀ5’), 112.6 (Cꢀ4’), 114.4 (Cꢀ
+
7’), 128.4 (Cꢀ2’, q), 151.3 (Cꢀ3’, q), 154.0 (Cꢀ6’, q); ESIꢀMS(positive): m/z 435 [M+H]
35 7 .
calcd. for C24H O found 435

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12βꢀArteꢀ4’, 6’ꢀdimethoxybenzylether (10): Elution of the column with nꢀhexaneꢀethyl
acetate (95:5) yielded a colorless oil in 90% (w/w) yield. IR λmax (neat): 1203, 1155 (ether),
ꢀ
1
1
2
927, 2873, 1600, 1476, 1381, 1029,875 cm ; H, COSYꢀ NMR (300 MHz, Acetoneꢀd6):
δ0.92 (3H, d, J =6.6 Hz, H ꢀ13), 0.96 (3H, d, J =7.5 Hz, H ꢀ14), 1.31 (3H, s, H ꢀ15), 2.55
1H, m, Hꢀ11), 3.74, 3.76 (3H each, s, 2 x OCH ), 4.44 (1H, d, J=12.6 Hz, Haꢀ1’), 4.76 (1H,
d, J=12.6 Hz, Hbꢀ1’), 4.79 (1H, d, J=3.6 Hz, αHꢀ12), 5.42 (1H, s, Hꢀ5), 6.38 (1H, s, Hꢀ5’),
3
3
3
(
3
1
3
6
1
4
1
.53 (2H, brs, Hꢀ3’, Hꢀ7’); C, DEPTꢀ NMR (75 MHz, Acetoneꢀd
4), 22.1 (Cꢀ8), 24.8 (Cꢀ2), 25.0 (Cꢀ15), 31.3 (Cꢀ11), 34.9 (Cꢀ9), 36.6 (Cꢀ3), 37.6 (Cꢀ10),
4.8 (Cꢀ7), 53.0 (Cꢀ1), 55.0 (2 x OCH ), 69.5 (Cꢀ1’), 81.0 (Cꢀ6, q), 88.0(Cꢀ5), 99.5 (Cꢀ5’),
01.3 (Cꢀ12), 103.9 (Cꢀ4, q), 105.2 (Cꢀ3’, Cꢀ7’), 141.5 (Cꢀ2’, q), 161.4 (Cꢀ4’, Cꢀ6’, q); ESIꢀ
6
): δ 13.0 (Cꢀ13), 20.1 (Cꢀ
3
+
+
MS(positive): m/z 435 [M+H] calcd. for C24
2αꢀArteꢀ2’ꢀbromoꢀ4’ꢀcyanophenylether (11): Elution of the column with nꢀhexane– ethyl
acetate (95:5) yielded white crystals in 70% w/w yield. IR λmax (KBr): 1378, 1138 (ether),
35 7
H O found 435 and 457 [M+Na] .
1
ꢀ1 1
2
926, 2873, 1450, 875 cm ; H NMR (300 MHz, CDCl
3 3 3
) δ0.90 (6H, d, J =6.3 Hz, H ꢀ13, H ꢀ
1
4), 1.43 (3H, brs, H ꢀ15), 2.83 (1H, m, Hꢀ11), 5.44 (1H, d, J=7.2 Hz, βHꢀ12), 5.54 (1H, s,
3
Hꢀ5), 6.90 (1H, dd, J=8.1, 8.1 Hz, Hꢀ6’), 7.08 (1H, d, J=6.9, Hꢀ5’), 7.26 (1H, d, J=1.8 Hz, Hꢀ
+
3
’); ESIꢀMS(positive): m/z 464 [M+H] calcd. for C22
H
27
O
5
NBr found 464
.
12βꢀArteꢀ2’ꢀchloro, 4’ꢀflourophenylether (12): Elution of the column with nꢀhexaneꢀethyl
acetate (95:5) yielded a colorless oil, in 80% (w/t) yield. IR λmax (neat): 1371, 1132 (ether),
ꢀ
1 1
2
870, 1456, 1592, 1102 and 753 (Cl groups) cm ; H, COSYꢀ NMR (300 MHz, CDCl
3
): δ
0
.86 (3H, d, J =6.0 Hz, H ꢀ13), 0.96 (3H, d, J =5.1 Hz, H ꢀ14), 1.42 (3H, s, H ꢀ15), 2.81 (1H,
3
3
3
m, Hꢀ11), 5.45 (1H, brs, αHꢀ12), 5.54 (1H, s, Hꢀ5), 6.92 (1H, dd, Hꢀ5’), 7.08 (1H, d, J = 8.4
1
3
Hz, Hꢀ5’), 7.30 (1H, brs, Hꢀ3’); C, DEPTꢀ NMR (75 MHz, CDCl ): δ 13.4 (Cꢀ13), 20.7 (Cꢀ
3
14), 24.6 (Cꢀ8), 25.0 (Cꢀ2), 26.4 (Cꢀ15), 31.5 (Cꢀ11), 35.0 (Cꢀ9), 36.7 (Cꢀ3), 37.8 (Cꢀ10),

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4
4.6 (Cꢀ7), 52.9 (Cꢀ1), 81.3 (Cꢀ6, q), 88.9 (Cꢀ5), 102.4 (Cꢀ12), 104.7 (Cꢀ4, q), 114.6 (Cꢀ5’),
17.4 (Cꢀ6’), 124.7 (Cꢀ2’, q), 150.3 (Cꢀ3’), 155.9 (Cꢀ1’, q), 159.2 (Cꢀ4’, q); ESIꢀ
1
+
+
MS(positive): m/z 413 [M+H] calcd. for C21
2βꢀArteꢀ4’ꢀphenoxybenzylether (13): Elution of the column with nꢀhexaneꢀethyl acetate
95:5) yielded a colorless oil, 95% (w/w) in yield. IR λmax (neat): 3067, 3040, 1585, 1487,
27 5
H O ClF found 413 and 452 [M +H+ K] .
1
(
ꢀ
1 1
1
253, 1214, 1030, 875, 789, 693 (aromatics), 2873, 1448, 1377, 1140 (ether) cm ; H NMR
): δ 0.83 (3H, d, J =7.2 Hz, H ꢀ13), 0.88 (3H, d, J =5.4 Hz, H ꢀ14), 1.38
3H, s, H ꢀ15), 2.60 (1H, m, Hꢀ11), 4.44 (1H, d, J=12.6 Hz, Haꢀ1’), 4.81 (1H, d, J=12.3 Hz,
(300 MHz, CDCl
3
3
3
(
3
Hbꢀ1’), 4.83 (1H, d, J=3.0, αHꢀ12), 5.36 (1H, s, Hꢀ5), 6.91 (5H, m, Hꢀ3’, Hꢀ5’, Hꢀ7’, Hꢀ9’,
1
3
Hꢀ13’), 7.04 (1H, t, J=7.5 Hz, Hꢀ11’), 7.27 (3H, m, Hꢀ6’, Hꢀ10’, Hꢀ12’); C, DEPTꢀ NMR
75 MHz, CDCl ): δ 13.4 (Cꢀ13), 20.7 (Cꢀ14), 24.8 (Cꢀ8), 25.0 (Cꢀ2), 26.5 (Cꢀ15), 31.3 (Cꢀ
1), 35.0 (Cꢀ9), 36.8 (Cꢀ3), 37.7 (Cꢀ10), 44.8 (Cꢀ7), 52.9 (Cꢀ1), 69.6 (Cꢀ1’), 81.4 (Cꢀ6, q),
(
3
1
8
1
8.4 (Cꢀ5), 101.8 (Cꢀ12), 104.5 (Cꢀ4, q), 117.4 (Cꢀ3’), 118.0 (Cꢀ5’), 119.5 (Cꢀ9’, Cꢀ13’),
22.0 (Cꢀ10’, Cꢀ12’), 123.7 (Cꢀ11’), 129.9 (Cꢀ7’), 130.1 (Cꢀ6’) 140.9 (Cꢀ2’, q) 157.4 (Cꢀ4’,
+
q), 157.9 (Cꢀ8’, q); ESIꢀMS(positive): m/z 467 [M+H] calcd. for C28
H O
35 6
found 467 and
ꢀ
(
negative): 465 [MꢀH] calcd. for C28
H
33
O
6
found 465.
2βꢀArteꢀ3’ꢀmethylꢀ2’ꢀbutenylether (14): Elution of the column with nꢀhexaneꢀethyl acetate
(95:5) yielded a colorless oil, 90% (w/w) in yield. IR λmax (neat): 1655 (double bond), 1377,
ꢀ
1 1
1
007 (ether), 2922, 2874,1450, cm ; H, COSYꢀ NMR (400 MHz, Acetoneꢀd
ꢀ13), 0.94 (3H, d, J =8.0 Hz, H ꢀ14), 1.32 (3H, s, H ꢀ15), 1.69, 1.74 (3H
ꢀ5’), 3.63 (1H, m, Haꢀ1’), 4.20 (1H, m, Hbꢀ1’), 4.71 (1H, d, J=3.4 Hz, αHꢀ
6
): δ 0.89 (3H,
d, J =7.4 Hz, H
each, s, H ꢀ4’, H
2), 5.31 (1H, t, J =4.5 Hz, Hꢀ2’), 5.37 (1H, s, Hꢀ5); C, DEPTꢀ NMR (100 MHz, Acetoneꢀ
): δ 12.5 (Cꢀ13), 17.3, 24.3 (Cꢀ4’, Cꢀ5’), 19.8 (Cꢀ14), 24.6 (Cꢀ2), 24.9 (Cꢀ8), 25.3 (Cꢀ15),
0.8 (Cꢀ11), 34.6 (Cꢀ9), 36.3 (Cꢀ3), 37.2 (Cꢀ10), 44.5 (Cꢀ7), 52.6 (Cꢀ1), 63.9 (Cꢀ1’), 80.6
3
3
3
3
3
1
3
1
d
3
6

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(Cꢀ6, q), 87.5 (Cꢀ5), 100.5 (Cꢀ12), 103.4 (Cꢀ4, q), 121.3 (Cꢀ2’), 135.8 (Cꢀ3’, q); ESIꢀ
+
+
30 5
MS(positive): m/z 353 [M+H] calcd. for C20H O found 353; 375 [M+Na] .
12βꢀArteoctadecanylether (15): Elution of the column with nꢀhexaneꢀethyl acetate (95:5)
max
yielded a colorless oil, 90% (w/w) yield. IR ν
(neat): IR λmax (neat): 1377, 1007 (ether)
): δ 0.91 (3H,
ꢀ17’), 0.97 (3H, d, J =5.7 Hz, H ꢀ14), 1.27
3
ꢀ15), 3.37 (1H, m, Haꢀ1’), 4.79 (1H, m, Hbꢀ1’), 4.80 (1H,
ꢀ
1 1
7
14, 725 (long chain), 2922 , 2874,1450 cm ; H NMR (300 MHz, Acetoneꢀd
d, J =7.2 Hz, H ꢀ13), 0.93 (3H, t, J =6.9 Hz, H
28H, brs, H ꢀ2’ꢀH ꢀ16’), (3H, s, H
d, J=2.4 Hz, αHꢀ12), 5.35 (1H, s, Hꢀ5); C, DEPTꢀ NMR (300 MHz, Acetoneꢀd ): δ 13.4,
6
3
3
3
(
2
2
13
6
14.5 (Cꢀ13, Cꢀ18’), 20.7 (Cꢀ14), 23.0 (Cꢀ8), 25.1 (Cꢀ2), 26.6 (Cꢀ15), 24.4, 29.7, 30.11, 32.3
(
Cꢀ2’ to Cꢀ17’), 31.3 (Cꢀ11), 35.1 (Cꢀ9), 36.8 (Cꢀ3), 37.8 (Cꢀ10), 44.9 (Cꢀ7), 53.0 (Cꢀ1), 68.8
Cꢀ1’), 81.5 (Cꢀ6, q), 88.3 (Cꢀ5), 102.3 (Cꢀ12), 104.4 (Cꢀ4, q); ESIꢀMS(positive): m/z 537
(
+
[
M+H] calcd. for C H O found 537.
3
3
62
5
2.4.3. General Procedure II: Preparation of 12αꢀartethioethers 16ꢀ18
DHA (1.0 g, 3mmol) was dissolved in dichloromethane (20 ml), stirred for 12 min to 4 h at 0ꢀ
0°C in the presence of appropriate alkylating reagents, viz., admantanethiol (3mmol for 16)
1
and butanedithiol (5mmol for 16 and 18) and the catalyst boron trifluoride etherate. After
completion of the reaction, monitored by TLC, the reaction mixture was quenched with
cooled 1% aqueous NaHCO
3
(50 ml) and extracted with ethyl acetate (3 x 50 ml). The
SO ) and concentrated to
combined extract was washed with water till neutral pH, dried (Na
2
4
get the crude mixture of β/α products (1.3 to 1.7g). The mixture was subjected to silica gel
column chromatography to obtain the pure βꢀisomer.
12αꢀArteadmantanethioether (16): Elution of the column with nꢀhexane– ethyl acetate
ꢀ
1 1
(
95:5) yielded a white solid, 71% (w/w) yield. IR λmax (KBr): 3178, 2923, 1410 cm ; H,
COSYꢀNMR (300 MHz, CDCl ): δ 0.90 (3H, m, H ꢀ14), 1.13 (3H, d, J=5.1 Hz, H ꢀ13), 1.38
3H, s, H ꢀ15), 1.66 (4H, brs, H ꢀ9’, H ꢀ10’), 1.92 (1H, m, Hꢀ8’), 1.99 (8H, brs, H ꢀ2’, H ꢀ4’,
3
3
3
(
3
2
2
2
2

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2 2
H ꢀ6’, H ꢀ7’), 2.27 (1H, t, J=13.65 Hz, Hꢀ3’, Hꢀ5’), 5.42 (1H, s, Hꢀ5), 5.44 (1H d, J=11.1 Hz,
1
3
βHꢀ12); C, DEPTꢀNMR (75 MHz, CDCl
3
): δ 20.2 (Cꢀ14), 21.7 (Cꢀ13), 25.2 (Cꢀ2, Cꢀ8),
26.0 (Cꢀ15), 30.2 (Cꢀ11, Cꢀ3’, Cꢀ5’), 32.0 (Cꢀ9’), 34.5 (Cꢀ9), 36.7 (Cꢀ4’, Cꢀ10’), 36.9 (Cꢀ3),
37.7 (Cꢀ10), 41.4 (Cꢀ8’), 44.4 (Cꢀ2’, Cꢀ6’), 44.5 (Cꢀ7’), 46.5 (Cꢀ1’, q 47.9 (Cꢀ7), 51.7 (Cꢀ1),
+
78.8 (Cꢀ5), 82.5 (Cꢀ6, q), 91.4 (Cꢀ12), 102.6 (Cꢀ4, q); ESIꢀMS(positive): m/z 435 [M+H]
+
+
39 4
calcd. for C25H O S found 435 m/z; 457 [M + Na] , 501 [M+K] .
12αꢀArtebutaneꢀ4’ꢀthiol, 1’ꢀthioether (17): Elution of the column with nꢀhexaneꢀethyl
acetate (90:10) yielded a viscous compound, 51% (w/w) yield. IR λmax (neat): 689 (CꢀSꢀ
ꢀ1 1
linkage), 2927, 2872, 1453, 1377, 1129,1038, 788 cm ; H NMR (400 MHz, CDCl ): δ 0.93
3
(
3H, d, J=5.4 Hz, H
ꢀ2’, H ꢀ3’), 2.70 (2H, m, H
.26 (1H, d, J=3.87 Hz, SH), 5.56 (1H, s, Hꢀ5); C, DEPTꢀNMR (100 MHz, CDCl
Cꢀ13), 20.6 (Cꢀ14), 25.1 (Cꢀ8), 25.4 (Cꢀ2), 26.2 (Cꢀ15), 32.9 (Cꢀ11), 35.1 (Cꢀ9), 37.1 (Cꢀ3),
7.8 (Cꢀ10), 46.0 (Cꢀ7), 53.6 (Cꢀ1), 81.1 (Cꢀ6, q), 88.5 (Cꢀ5), 92.8 (Cꢀ12), 104.5 (Cꢀ4, q);
3
ꢀ13), 0.97 (3H, d, J= 4.5 Hz, H
3
ꢀ14), 1.33 (3H, s, H
ꢀ1’), 4.67 (1H, d, J=7.86 Hz, βHꢀ12a),
): δ 15.1
3
ꢀ15), 1.73 (4H, m,
H
2
2
2
ꢀ4’), 2.93 (1H, m, H
2
13
5
3
(
3
+
+
ESIꢀMS(positive): m/z 389 [M+H] calcd. for C H O S found 389; m/z 411[M + Na] .
1
9
33
4 2
12αꢀDiartebutaneꢀ1’, 4’ꢀdithioether (18, dimer): Elution of the column with nꢀhexane–
ethyl acetate (92:8) yielded a viscous compound, 36% (w/w) yield. IR λmax (KBr): 690 (Cꢀ
ꢀ
1 1
Sꢀlinkage), 2926, 2871, 1451, 1377, 1129,1037 cm ; H NMR (400 MHz, CDCl
6H, d, J=7.2 Hz,, H ꢀ13a, H ꢀ13b), 0.97 (6H, m, H ꢀ14a, H ꢀ14b,), 1.32 (6H, s, H
5b, ), 1.73 (4H, m, H ꢀ2’, H ꢀ3’), 2.62 (4H, m, H ꢀ1’, H ꢀ4’), 4.60 (1H, d, J=8.02 Hz, βHꢀ
2a), 4.75 (1H, d, J=8.22 Hz, βHꢀ12b), 5.37 (1H, s, Hꢀ5a), 5.98 (1H, s, Hꢀ5b); C NMR
3
): δ 0.92
(
3
3
3
3
3
ꢀ15a, H
3
ꢀ
1
1
2
2
2
2
1
3
(
(
(
100 MHz, CDCl ): δ 14.8, 15.3 (Cꢀ13a, 13b), 20.5, 20.7 (Cꢀ14a, 14b), 22.3 (Cꢀ8a, 8b), 24.4
3
Cꢀ2a, 2b), 25.5, 26.1 (Cꢀ15a, 15b), 28.1 (Cꢀ2’), 31.2 (Cꢀ11a, 11b), 32.8 (Cꢀ3’), 33.7, 33.8
Cꢀ1’, Cꢀ4’), 34.9, 35.3 (Cꢀ9a, 9b), 36.1 (Cꢀ3a, Cꢀ3b), 36.9, 37.8 (Cꢀ10a, 10b), 47.0, 49.1 (Cꢀ

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7
1
6
a, 7b), 52.7, 56.0 (Cꢀ1a, 1b), 80.6, 81.1 (Cꢀ6a, 6b, q), 83.5 (Cꢀ5a, 5b), 92.7, 93.3 (Cꢀ12a,
+
2b), 104.4 (Cꢀ4a, 4b, q); ESIꢀMS(positive): m/z 655 [M+H] calcd. for C34
H
55
O
8
S
2
found
+
+
55; 677 [M + Na] , 693 [M+K] .
2.4.5. General Procedure IV: Preparation of 12αꢀarteesters 19ꢀ25
Dihydroartemisinin (1.0 g, 3mmol) was dissolved in dichloromethane (20 ml), stirred for 12
min to 4 h at 0ꢀ10°C in the presence of appropriate acylating reagents (3mmol), viz., mꢀ
nitrobenzoylchloride, pꢀnitrobenzoylchloride, (for 19 and 20), piperonyloylchloride, 4ꢀ
phenylazobenzoylchloride, lauroyl chloride and palmitoyl chloride (for 21, 22, 23 and 24)
and furanꢀ3ꢀcarboxylic acid (for 25), catalyst triethyamine for 19ꢀ24 and DCC/DMAP for
25(Fig. 4). After completion of the reaction, monitored by TLC, the reaction mixture was
worked up as usual to get crude mixture of β/α products (1.1 to 1.8 g). The mixture was
subjected to silica gel column chromatography to obtain the pure αꢀisomer.
12αꢀDihydroartemisinylꢀ4’ꢀnitrobenzoate (19): Elution of the column with nꢀhexaneꢀethyl
acetate (95:5) yielded a colorless oil, 90% (w/w) yield. IR λmax (neat): 3087, 1439, 1261,
ꢀ
1 1
1
040, 858, 719 (aromatics), 1732 (ester CO), 1538, 1556 (Ar NO
CDCl ): δ 0.93 (3H, d, J =7.2 Hz, H ꢀ13), 0.97 (3H, d, J =5.7 Hz, H
.80 (1H, m, Hꢀ11), 5.52 (1H, s, Hꢀ5), 6.01 (1H, d, J=9.9 Hz βHꢀ12), 7.66 (1H, m, Hꢀ6’),
2
) cm ; H NMR (300 MHz,
3
3
3
ꢀ14), 1.41 (3H, s, H ꢀ15),
3
2
8
1
3
.42 (2H, d, J = 8.4 Hz, Hꢀ5’, Hꢀ7’), 8.91 (1H, s, Hꢀ3’); C, DEPTꢀ NMR (70 MHz, CDCl ):
3
δ 12.2 (Cꢀ13), 20.1 (Cꢀ14), 22.0 (Cꢀ8), 24.5 (Cꢀ2), 25.8 (Cꢀ15), 31.8 (Cꢀ11), 34.0 (Cꢀ9), 36.1
Cꢀ3), 37.2 (Cꢀ10), 45.2 (Cꢀ7), 51.5 (Cꢀ1), 80.0 (Cꢀ6, q), 91.6 (Cꢀ5), 93.3 (Cꢀ12 ), 104.5 (Cꢀ4,
(
q), 124.9 (Cꢀ3’), 127.7 (Cꢀ5’), 129.6 (Cꢀ6’), 131.3 (Cꢀ2’, q), 135.6 (Cꢀ7’), 148.2 (Cꢀ2’, q),
+
1
63.2 (Cꢀ1’, q); EIꢀMS(positive): m/z 434 [M+H] calcd. for C H O N found 434; 456
2
2
28
8
+
+
[
M+Na] , 472 [M+K] .
12αꢀDihydroartemisinylꢀ5’ꢀnitrobenzoate (20): Elution of the column with nꢀhexane– ethyl
acetate (95:5) yielded an oily compound in 95% (w/w) yield. IR λmax (neat): 3081, 1437,

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ꢀ1
1
1
2
256, 1034, 851, 711 (aromatics), 1734 (ester CO), 1,533, 1558 (Ar NO ) cm ; H NMR
(300 MHz, CDCl
3
3 3 3
): δ 0.92 (6H, m, H ꢀ13, H ꢀ14), 1.41 (3H, s, H ꢀ15), 2.75 (1H, m, Hꢀ11),
1
3
5
.52 (1H, s, Hꢀ5), 6.01 (1H, d, J=9.0 Hz, βHꢀ12), 8.27 (4H, brs, Hꢀ3’, Hꢀ4’, Hꢀ6’, Hꢀ7’); C,
DEPTꢀ NMR(70 MHz, CDCl ): δ 12.62 (Cꢀ13), 20.60 (Cꢀ14), 22.42, (Cꢀ8), 24.96 (Cꢀ2), 26.2
3
(Cꢀ15), 32.3 (Cꢀ11), 34.4 (Cꢀ9), 36.6 (Cꢀ3), 37.6 (Cꢀ10), 45.6 (Cꢀ7), 51.9 (Cꢀ1), 81.5 (Cꢀ6, q),
92.0 (Cꢀ5), 93.7 (Cꢀ12), 104.9 (Cꢀ4, q), 123.9 (Cꢀ4’, Cꢀ6’), 131.6 (Cꢀ3’, Cꢀ7’), 135.4 (Cꢀ2’,
+
q), 151.1 (Cꢀ5’, q), 163.9 (Cꢀ1’, q); EIꢀMS(positive): m/z 434 [M+H] calcd. for C22
H
28
O
8
N
+
found 434; 456 [M+Na] .
12αꢀArtepiperonyloylester (21) Elution of the column with nꢀhexaneꢀethyl acetate (95:5)
yielded an oily compound, 90% (w/w) yield. IR λmax (neat): 1626, 1508, 1444, 1260, 1017,
ꢀ1 1
878, 861 (aromatics), 1734 (ester CO), 921 (ꢀOꢀCH
2
ꢀOꢀ group) cm ; H NMR (300 MHz,
CDCl ): δ 0.89 (3H, d, J =7.2 Hz, H ꢀ13), 0.96 (3H, d, J =5.7 Hz, H ꢀ14), 1.41 (3H, s, H ꢀ15),
3
3
3
3
2.70 (1H, m, Hꢀ11), 5.50 (1H, s, Hꢀ5), 5.94 (1H, d, J=9.9 Hz, βHꢀ12), 6.03 (2H, s, Hꢀ8’), 6.83
1
3
(
(
1H, d, J = 7.8 Hz, Hꢀ6’), 7.53 (1H, s, Hꢀ3’), 7.73 (1H, d, J = 7.8 Hz, Hꢀ7’); C, DEPTꢀNMR
70 MHz, CDCl ) = 12.1 (Cꢀ13), 20.1 (Cꢀ14), 21.9 (Cꢀ8), 24.5 (Cꢀ2), 25.8 (Cꢀ15), 31.9 (Cꢀ
1), 34.0 (Cꢀ9), 36.2 (Cꢀ3), 37.2 (Cꢀ10), 45.2 (Cꢀ7), 51.5(Cꢀ1), 80.1 (Cꢀ6, q), 91.5 (Cꢀ5), 92.4
3
1
(Cꢀ12), 101.8 (Cꢀ8’), 104.3 (Cꢀ4, q), 107.8 (Cꢀ6’), 109.8 (Cꢀ7’), 123.4 (Cꢀ2’, q), 126.0 (Cꢀ3’),
+
1
47.6 (Cꢀ4’, q), 151.9 (Cꢀ5’, q), 164.5 (Cꢀ1’, q); ESIꢀMS(positive): m/z 455 [M+Na] calcd.
for C23
H
28
O
8
Na found 455m/z.
2αꢀDihydroartemisinylꢀ5’ꢀphenylazobenzoate (22): Elution of the column with nꢀhexane–
ethyl acetate (95:5) yielded orange crystals of compound 24, 85% (w/w) yield, mp. 138ꢀ
40°C. IR λmax (KBr): 2344 (ꢀN=Nꢀ group), 1654, 1508, 1458, 1271, 1028, 882, 864, 783
1
1
ꢀ
1 1
(
aromatics), 1732 (ester CO) cm ; H NMR (300 MHz, CDCl
ꢀ13), 0.98 (3H, d, J =6.0 Hz, H ꢀ14), 1.49 (3H, s, H ꢀ15), 2.79 (1H, m, Hꢀ11), 5.54 (1H,
brs, Hꢀ5), 6.02 (1H, d, J=9.9 Hz, βHꢀ12), 7.53 (3H, m, Hꢀ10’, Hꢀ11’, Hꢀ12’), 7.95 (4H, d, J =
3
): δ 0.92 (3H, d, J =8.1 Hz,
H
3
3
3

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1
3
8
.7 Hz, Hꢀ4’, Hꢀ6’, Hꢀ9’, Hꢀ13’), 8.27 (2H, d, J=8.7 Hz, Hꢀ3’, Hꢀ7’); C, DEPTꢀ NMR (75
MHz, CDCl ): δ 12.6 (Cꢀ13), 20.6 (Cꢀ14), 22.4 (Cꢀ8), 25.0 (Cꢀ2), 26.3 (Cꢀ15), 32.4 (Cꢀ11),
4.5 (Cꢀ9), 36.6 (Cꢀ3), 37.6 (Cꢀ10), 45.7 (Cꢀ7), 52.0 (Cꢀ1), 80.6 (Cꢀ6), 92.0 (Cꢀ5), 93.2 (Cꢀ
3
3
1
3
2), 104.8 (Cꢀ4, q), 122.9 (Cꢀ4’, Cꢀ6’), 123.5 (Cꢀ9’, Cꢀ13’), 129.5 (Cꢀ10’, Cꢀ12’), 131.5 (Cꢀ
’, Cꢀ7’), 132.1 (Cꢀ11’), 152.9 (Cꢀ8’, q), 155.7 (Cꢀ5’, q), 165.1 (Cꢀ1’, q) ; ESIꢀMS(positive):
+
m/z 493 [M+H] calcd. for C28
H
33
O
6
N
2
found 493.
1
2αꢀDihydroartemisinyllauroyloate (23): Elution of the column with nꢀhexaneꢀethyl
acetate (95:5) yielded a colorless oil, 90% (w/w) in yield. IR λmax (neat): 1736 (ester CO),
ꢀ
1
1
7
14, 725(long chain), 2922, 2874, 1450, 1377, 1007 cm ; H, COSYꢀ NMR (300 MHz,
CDCl ): δ 0.78 (3H, t, J= 7.2, H ꢀ12’), 0.79 (3H, d, J =7.2 Hz, H ꢀ13), 0.89 (3H, d, J =5.4 Hz,
ꢀ14), 1.18 (18H, m, H ꢀ3’ ꢀ H ꢀ11’), 1.36 (3H, s, H ꢀ15), 2.31 (2H, t, J=7.2, H ꢀ2’), 2.48
1H, m, Hꢀ11), 5.37 (1H, s, Hꢀ5), 5.72 (1H, d, J=9.6 Hz, βHꢀ12); C, DEPTꢀ NMR (75 MHz,
CDCl ): δ 12.4 (Cꢀ12’), 14.4 (Cꢀ13), 20.6 (Cꢀ14), 22.4 (Cꢀ8), 23.0 (Cꢀ2), 24.9, 25.0, 29.4,
9.6, 29.7, 29.8, 29.9, 30.0, 32.2, 34.7 (Cꢀ2’ to Cꢀ11’,Cꢀ8), 26.3 (Cꢀ15), 32.2 (Cꢀ11), 34.5
3
3
3
H
3
2
2
3
2
1
3
(
3
2
(Cꢀ9), 36.6 (Cꢀ3), 37.6 (Cꢀ10), 45.6 (Cꢀ7), 52.0 (Cꢀ1), 80.5 (Cꢀ6, q), 91.8 (Cꢀ5), 92.0 (Cꢀ12),
+
1
04.8 (Cꢀ4, q), 172.9 (Cꢀ1’, q); ESIꢀMS(positive): m/z 467 [M+H] calcd. for C27
H
47
O
6
+
ꢀ
found 467; 489 [M+Na] (negative): 465 [MꢀH] .
12αꢀDihydroartemisinylpalmitoyloate (24): Elution of the column with nꢀhexaneꢀethyl
acetate (95:5) yielded a colorless oil, 90% (w/w) in yield. IR λmax (neat): 1751 (ester CO),
ꢀ
1
1
7
22 (long chain), 2924, 2853, 1459, 1376, 1037 cm ; H, COSYꢀ NMR (300 MHz, CDCl
δ 0.93 (6H, m, H ꢀ13, H ꢀ16’ ), 0.97 (3H, d, J =4.2 Hz, H3ꢀ14), 1.23 (26H, m, H ꢀ3’ ꢀ H
5’), 1.41 (3H, s, H3ꢀ15), 2.34 (2H, t, J=8.4, H ꢀ2’), 2.54 (1H, m, Hꢀ11), 5.42 (1H, s, Hꢀ5),
3
):
3
3
2
2
ꢀ
1
5
1
2
2
1
3
.77 (1H, d, J=9.9 Hz, βHꢀ12); C, DEPTꢀ NMR (75 MHz, CDCl ); δ 12.5 (Cꢀ16’), 14.5 (Cꢀ
3
3), 20.6 (Cꢀ14), 22.4 (Cꢀ8), 23.0 (Cꢀ2), 24.9, 25.0, 29.4, 29.6, 29.7, 29.8, 29.9 (12 x CH
2
),
6.3 (Cꢀ15), 32.2 (Cꢀ11), 30.0, 34.7 (2 x CH ), 34.7 (Cꢀ9), (Cꢀ2’), 36.6 (Cꢀ3), 37.6 (Cꢀ10),
2

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44.6 (Cꢀ7), 52.0 (Cꢀ1), 80.5 (Cꢀ6, q), 91.8 (Cꢀ5), 92.0 (Cꢀ12), 104.8 (Cꢀ4, q), 172.9 (Cꢀ1’, q);
+
ESIꢀMS(positive): m/z 561 [M + K] calcd. for C31
H
54
O
6
K found 561.
12αꢀDihydroartemisinylfuranoate (25): Elution of the column with nꢀhexane– ethyl acetate
(
(
95:5) yielded creamish crystals of compound 27, 85% (w/w) yield, mp. 140°C IR λmax
ꢀ1
1
KBr): 3145, 1509, 876 (furan moiety), 1739 (ester CO), 2926, 1306, 1164, 1036 cm ; H
): δ 0.86 (3H, d, J =6.9 Hz, H ꢀ13), 0.95 (3H, d, J =5.4 Hz, H ꢀ14),
ꢀ15), 2.65 (1H, s, Hꢀ11), 5.47 (1H, s, Hꢀ5), 5.91 (1H, d, J=9.6 Hz, βHꢀ12),
NMR (300 MHz, CDCl
3
3
3
1
.31 (3H, s, H
3
1
3
6
.77 (1H, s, Hꢀ5’), 7.41 (1H, s, Hꢀ4’), 8.08 (1H, s, Hꢀ3’); C, DEPTꢀ NMR (75 MHz,
CDCl ): δ 12.5 (Cꢀ13), 20.6 (Cꢀ14), 22.4 (Cꢀ8), 24.9 (Cꢀ2), 26.3 (Cꢀ15), 32.3 (Cꢀ11), 34.4 (Cꢀ
3
9), 36.6 (Cꢀ3), 37.6 (Cꢀ10), 45.6 (Cꢀ7), 52.0 (Cꢀ1), 80.5 (Cꢀ6, q), 91.9 (Cꢀ5), 92.4 (Cꢀ12),
104.8 (Cꢀ4, q), 110.3 (Cꢀ5’), 119.2 (Cꢀ2’, q), 144.1 (Cꢀ4’), 148.9 (Cꢀ3’), 162.1 (Cꢀ1’, q); ESIꢀ
+
MS(positive): m/z 417 [M + K] calcd. for C20
H
26
O
7
K found 417; (negative): m/z 377 [MꢀH].
2.4.4.1. Preparation ofanhydrodihydroartemisinin (26): To a solution of DHA (10g) in
diethylether 500 ml added dropwise BF .OEt solution and stirred the reaction mixture
3
2
overnight at RT. After completion of reaction it was washed with 5% NaHCO
3
solution
1
9c
(
3x100 ml), dried (Na SO ) and concentrated . The residue, obtained was then purified by
2 4
column chromatography (10% EtOAc in hexane) followed by crystallisation (EtOAcꢀhexane)
to afford shiny white crystals of 9, 10ꢀanhydrodihydroartemisinin (26), (7.1 gm), mp 98ꢀ100
ꢀ
1 1
°
C ;IR λmax(KBr): 1684 (double bond), 2928, 2850, 1452, 1109 cm ; H NMR (300 MHz,
CDCl ) δ 0.92 (3H, d, J=6.0 Hz, H ꢀ14), 1.42 (3H, s, H ꢀ15), 1.56 (3H, d, J =1.2, H ꢀ13),
.66 (2H, m), 1.87 (3H, m), 2.04 (2H, m), 1.92 (1H, m, Hꢀ1), 2.39 (1H, dt, J =13.9, 4.8 Hz,
3
3
3
3
1
1
3
Hꢀ7), 5.52 (1H, s, Hꢀ5), 6.16 (1H, s, Hꢀ12); C, DEPTꢀNMR (75 MHz, CDCl ) δ 16.5 (Cꢀ
3
13), 20.6 (Cꢀ14), 24.8 (Cꢀ2), 26.2 (Cꢀ15), 30.3 (Cꢀ8), 34.5 (Cꢀ9), 36.6 (Cꢀ3), 37.8 (Cꢀ10),

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44.8 (Cꢀ7), 51.8 (Cꢀ1), 79.3 (Cꢀ6, q), 90.0 (Cꢀ5), 104.9 (Cꢀ4, q), 108.4 (Cꢀ11, q), 135.3 (Cꢀ
2
7a
+
1
23 4
2) ; ESIꢀMS(positive): m/z 267 [M +H] calcd. for C15H O found 417.
2.4.4.2. Preparation of11β, 12βꢀarteepoxide (27): Anhydrodihydroartemisinin (26) (6g)
was dissolved in CH Cl (150 ml) and mꢀchloroperbenzoic acid (5.7g in 0.5 N NaHCO ) was
2
2
3
19c
19d
added . The reaction mixture was stirred at room temperature for 2h . After completion of
the reaction, monitored by TLC the reaction mixture was diluted to 300 ml with CH Cl and
was washed with 5% Na SO (3 x 200 ml), 5% NaHCO (200 ml x 3) and finally with water
200 ml x 3). After drying over anhydrous Na SO and evaporation of solvent followed by
column chromatography of the crude residue afforded 11β, 12βꢀarteepoxide, 4.2g (27), mp
2
2
2
3
3
(
2
4
ꢀ
1
1
1
12 °C ; IR λmax(KBr): 2928, 2857, 1680, 1449, 1109, 876 cm ; H, COSYꢀ NMR (300
MHz, CDCl ) δ 0.91 (3H, d, J= 4.8 Hz, H ꢀ14), 1.09 (1H, m, Hꢀ9α), 1.24 (1H, m, Hꢀ10), 1.28
3H, s, H ꢀ13), 1.38ꢀ1.56 (2H, m, Hꢀ8β, Hꢀ2β), 1.71 (2H, m, Hꢀ1, Hꢀ9β), 1.76ꢀ1.96 (3H, m,
Hꢀ8α, Hꢀ7, Hꢀ2α), 2.05 (1H, m, Hꢀ3β), 2.32 (1H, ddd, J=4.2, 12.8, 14.2 Hz, Hꢀ3α), 4.91 (1H,
3
3
(
3
1
3
s, Hꢀ12), 5.24 (1H,s, Hꢀ5); C, DEPTꢀ NMR (75 MHz, CDCl ) :δ 20.0 (Cꢀ14), 22.0 (Cꢀ13),
3
24.1 (Cꢀ8), 24.2 (Cꢀ2), 25.9 (Cꢀ15), 33.3 (Cꢀ9), 36.1 (Cꢀ3), 36.9(Cꢀ10), 45.0 (Cꢀ7), 51.6 (Cꢀ
1), 56.2 (Cꢀ11, q), 78.1 (Cꢀ6, q), 82.5 (Cꢀ12), 90.4 (Cꢀ5), 104.5 (Cꢀ4, q) ESIꢀMS(positive):
+
m/z 283 [M +H] calcd. for C15
H
23
O
5
found 283.
2
.4.4.3. General Procedure III: Preparation of 11βꢀhydoxy12βꢀarteethers 28ꢀ33:
Further, the epoxide 27 was alkylated, using appropriate alcohols, 1ꢀadmantaneethanol,
cinnamyl alcohol, cyclopropylmethanol, 2,5ꢀdimethoxybenzyl alcohol, allyl
alcoholpiperonylyl alcohol, catalyst BF .OEt in CH Cl to yield 11ꢀhydoxy12βꢀarteethers
3
2
2
2
28ꢀ33 (Fig. 5).
11βꢀHydroxyꢀ12βꢀarteallylether (28): Elution of the column with nꢀhexane– ethyl acetate
(90:10) yielded a white solid in 85% (w/w) yield. IR λmax (KBr): 3423 (OH), 1103, 1029

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ꢀ
1 1
(
ether linkage), 1655 (double bond), 2923, 2853, 1463, 1376, 877 cm . H, COSYꢀ NMR
400 MHz, Acetoneꢀd ): δ 0.96 (3H, d, J =4.76 Hz, H ꢀ14), 1.34 (3H, s, H ꢀ15), 1.47 (3H, s,
ꢀ13), 4.08 (1H, dd, J =12.62, 4.94 Hz, Haꢀ1’), 4.32, d, J= 10.12 Hz, Hbꢀ1’), 4.60 (1H, brs,
αHꢀ12), 5.18 (1H, d, J=10.44 Hz, Haꢀ3’), 5.33 (1H, d, J=17.16 Hz, Hbꢀ3’), 5.42 (1H, s, Hꢀ5),
(
6
3
3
H
3
13
5
.99 (1H, m, Hꢀ2’); C, DEPTꢀ NMR (100 MHz, Acetoneꢀd ): δ 19.7 (Cꢀ14), 24.3 (Cꢀ2, Cꢀ
6
8), 25.0 (Cꢀ15), 28.3 (Cꢀ13 ), 34.2 (Cꢀ9), 36.2 (Cꢀ3), 37.2 (Cꢀ10), 50.4 (Cꢀ7), 52.7 (Cꢀ1), 68.9
(Cꢀ1’), 70.0 (Cꢀ11, q), 82.6 (Cꢀ6, q), 87.4 (Cꢀ5), 103.0 (Cꢀ12), 104.3 (Cꢀ4, q), 116.9 (Cꢀ3’),
+
1
35.5 (Cꢀ2’); ESIꢀMS(positive): m/z 341 [M +H] calcd. for C18
H
29
O
6
found 341; (negative):
ꢀ
3
39 [MꢀH] .
11βꢀHydroxyꢀ12βꢀartecyclopropylmethylether (29): Elution with nꢀhexane– ethyl acetate
(90:10) yielded a white solid, 78% (w/w) yield. IR λmax (KBr):3363 (OH), 1272, 1103, (ether
ꢀ
1
1
linkage), 2921, 2863, 1469, 1378 cm ; H NMR (300 MHz, CDCl
m H ꢀ3’/H ꢀ4’), 0.97 (3H, brs, H ꢀ14), 1.43 (3H, s, H ꢀ15), 1.56 (3H, s, H
Hꢀ2’), 3.43 (1H, m, Haꢀ1’), 3.60 (1H, m, Hbꢀ1’), 4.67 (1H, d, J=2.1 Hz, αHꢀ12), 5.42 (1H, s,
3
): δ 0.25/0.53 (2H each,
2
2
3
3
3
ꢀ13), 2.36 (1H, m,
1
3
Hꢀ5); C, DEPTꢀNMR (75 MHz, CDCl
3
): δ 3.1/3.6 (Cꢀ3’ , Cꢀ4’), 10.9 (Cꢀ2’), 20.6 (Cꢀ14),
2
5
4.7 (Cꢀ2, Cꢀ8), 26.2 (Cꢀ15), 28.3 (Cꢀ13), 34.5 (Cꢀ9), 36.8 (Cꢀ3), 37.9 (Cꢀ10), 50.6 (Cꢀ7),
3.0 (Cꢀ1), 69.9 (Cꢀ1’), 74.0 (Cꢀ11, q), 82.3 (Cꢀ6, Q), 88.2 (Cꢀ5), 103.6 (Cꢀ12), 104.3(Cꢀ4,
+
q); ESIꢀMS(positive): m/z 355 [M +H] calcd. for C19
H
31
O
6
found 355; (negative): 353 [Mꢀ
ꢀ
H] .
11β ꢀHydroxyꢀ12βꢀarteꢀ2’ꢀadmantanethylether (30): Elution of the column with nꢀhexane–
ethyl acetate (90:10) yielded a white solid, 80% (w/w) yield. IR λmax (KBr): 3448 (OH),
ꢀ1 1
1
301, 1141, 1091, 1010 (ether linkage), 2857, 3178, 2923, 1410 cm ; H, COSYꢀNMR (300
MHz, CDCl ): δ 0.95 (3H, d, J=5.1 Hz, H ꢀ14), 1.45 (3H, s, H ꢀ15 ), 1.51 (7H, brs, H ꢀ13,
3
3
3
3
H ꢀ11’, H ꢀ12’), 1.63 (10H, m, H ꢀ4’, H ꢀ6’, Hꢀ7’, H ꢀ8’, H ꢀ9’, Hꢀ10’ ) 1.87 (2H, t, J=18.9
2
2
2
2
2
2
2
Hz, H ꢀ2’), 2.35 (1H, t, J=13.95 Hz, Hꢀ5’), 3.51 (1H, m, Haꢀ1’), 3.96 (1H, m, Hbꢀ1’), 4.65

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1
3
(
(
(
3
1H, brs, αHꢀ12), 5.40 (1H, s, Hꢀ5); C, DEPTꢀNMR (75 MHz, CDCl ): δ 20.6 (Cꢀ14), 24.6
Cꢀ8), 24.8 (Cꢀ2), 26.2 (Cꢀ15), 28.3 (Cꢀ13), 29.0(Cꢀ5’, Cꢀ7’), 32.3(Cꢀ3’, q), 34.5 (Cꢀ9), 36.8
Cꢀ3), 37.4 (Cꢀ6’, Cꢀ11’, Cꢀ12’), 37.8 (Cꢀ10), 43.1 (Cꢀ2’, Cꢀ4’, Cꢀ8’, Cꢀ9’), 44.0 (Cꢀ10’),
50.5 (Cꢀ7), 53.0 (Cꢀ1), 65.6 (Cꢀ1’), 70.0 (Cꢀ11, q), 82.3 (Cꢀ6, q), 88.2 (Cꢀ5), 104.1 (Cꢀ12),
+
1
04.4 (Cꢀ4, q); ESIꢀMS(positive): m/z 485 [M+Na] calcd. for C H O Na found 485.
27
42
6
11βꢀHydroxyꢀ12βꢀartecinnamylether (31): Elution of the column with nꢀhexaneꢀethyl
acetate (90:10) yielded a white solid, 88% (w/w) yield. IR λmax (KBr): 3405 (OH), 1276,
119, (ether linkage), 1652 (double bond), 2855, 1603, 1508, 1444, 1260, 1017, 878, 861
1
ꢀ1 1
(
aromatics) cm ; H NMR (300 MHz, CDCl ): δ 0.96 (3H, d, J =6.0 Hz, H ꢀ14), 1.40 (3H, s,
3
3
H
3 3
ꢀ15), 1.58 (3H, s, H ꢀ13), 4.28 (1H, t, J= 6.0 Hz Haꢀ1’), 4.53 (1H, m, Hbꢀ1’), 4.82 (1H, brs,
αHꢀ12), 5.47 (1H, brs, Hꢀ5), 6.30 (1H, m, Hꢀ2’), 6.64 (1H, m, Hꢀ3’), 7.40 (5 H, m, Hꢀ5’, Hꢀ
1
3
6
2
1
1
3
’, Hꢀ7’, Hꢀ8’, Hꢀ9’); C, DEPTꢀ NMR (75 MHz, CDCl ); δ 20.6 (Cꢀ14), 24.7 (Cꢀ2, Cꢀ8),
6.2 (Cꢀ15), 28.4 (Cꢀ13), 34.5 (Cꢀ9), 36.8 (Cꢀ3), 37.8 (Cꢀ10), 50.5 (Cꢀ7), 53.0 (Cꢀ1), 69.6 (Cꢀ
’), 70.0 (Cꢀ11, q), 82.3 (Cꢀ6, q), 88.3 (Cꢀ5), 103.2 (Cꢀ12), 104.4 (Cꢀ4, q), 125.2 (Cꢀ2’),
27.0 (Cꢀ6’, Cꢀ8’), 127.8 (Cꢀ7’), 128.3 (Cꢀ5’, Cꢀ9’), 133.7 (Cꢀ3’), 137.08(Cꢀ4’, q); ESIꢀ
+
MS(positive): m/z 417 [M+H] calcd. for C24
H
33
O
6
found 417.
1
1βꢀHydroxyꢀ12βꢀartepiperonylylether (32): Elution of the column with nꢀhexane– ethyl
acetate (90:10) yielded an oily compound, 90% (w/w) yield. IR λmax (neat): 3398 (OH), 1121,
031 (ether linkage), 1606, 1514 1432, 1256, 1011, 871 (aromatics), 914 (ꢀOꢀCH ꢀOꢀ group)
1
2
ꢀ
1 1
cm ; H NMR (300 MHz, CDCl
.55 (3H, s, H ꢀ13), 4.45 (1H, d, J=11.4 Hz, Haꢀ1’), 4.75 (1H, d, J=11.4 Hz, Hbꢀ1’), 4.78
1H, brs, αHꢀ12), 5.45 (1H, brs, Hꢀ5), 5.95 (2H, s, Hꢀ8’), 6.78 (3H, m, Hꢀ3’, Hꢀ4’, Hꢀ5’); C,
DEPTꢀ NMR (70 MHz, CDCl ): δ 20.6 (Cꢀ14), 24.7 (Cꢀ2, Cꢀ8), 26.2 (Cꢀ15), 28.4 (Cꢀ13),
4.4 (Cꢀ9), 36.8 (Cꢀ3), 37.8 (Cꢀ10), 50.5 (Cꢀ7), 52.9 (Cꢀ1), 70.0 (Cꢀ11, q), 70.8 (Cꢀ1’), 82.3
Cꢀ6, q), 88.3 (Cꢀ5), 101.5 (Cꢀ8’), 103.0 (Cꢀ12), 104.4 (Cꢀ4, q), 108.6 (Cꢀ5’), 109.0 (Cꢀ3’),
3 3 3
): δ 0.93 (3H, d, J =5.4 Hz, H ꢀ14), 1.44 (3H, s, H ꢀ15),
1
3
13
(
3
3
(

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122.1 (Cꢀ4’), 131.2 (Cꢀ2’, q), 148.2 (Cꢀ7’), 148.2 (Cꢀ6’); ESIꢀMS(positive): m/z 435
+
[
M+H] calcd. for C23
31
H O
8
found 435.
11βꢀHydroxyꢀ12βꢀarteꢀ3’, 6’ꢀdimethoxybenzylether (33): Elution of the column with nꢀ
hexaneꢀethyl acetate (90:10) yielded a colourless oil in 90% (w/w) yield. IR λmax (neat): 3408
ꢀ1
(
OH), 1102, 1038 (ether linkage), 1606, 1501, 1432, 1262, 1011, 874, 864 (aromatics) cm ;
1
H NMR (300 MHz, CDCl
.77, 3.79 (3H each, s, 2xOCH
1H, d, J=11.4 Hz, Hbꢀ1’), 5.55 (1H, s, Hꢀ5), 6.69 (2H, dd, J = 6.9, 3.0 Hz, Hꢀ4’, Hꢀ5’), 6.86
3
): δ 0.94 (3H, m, H
3 3 3
ꢀ14), 1.47 (3H, s, H ꢀ15), 1.56 (3H, s, H ꢀ13),
3
3
), 4.53 (1H, d, J=11.4 Hz, Haꢀ1’), 4.80 (1H, brs, αHꢀ12), 4.92
(
(
1
3
1H, s, Hꢀ7’); C, DEPTꢀ NMR (75 MHz, Acetoneꢀd ): δ 20.1 (Cꢀ14), 24.6 (Cꢀ8), 24.8 (Cꢀ
6
2
), 26.2 (Cꢀ15), 28.3 (Cꢀ13), 34.5 (Cꢀ9), 36.8 (Cꢀ3), 37.8 (Cꢀ10), 50.5 (Cꢀ7), 53.0 (Cꢀ1), 56.1
2 x OCH ), 67.6 (Cꢀ1’), 69.9 (Cꢀ11, q), 82.4 (Cꢀ6, q), 88.3 (Cꢀ5), 104.1 (Cꢀ12), 104.4 (Cꢀ4,
q), 111.5 (Cꢀ5’), 112.6 (Cꢀ4’), 114.4 (Cꢀ7’), 128.12 (Cꢀ2’, q), 152.2 (Cꢀ3, q’), 153.7 (Cꢀ6’, q);
(
3
+
+
ESIꢀMS(positive): m/z 451 [M+H] calcd. for C24
H
35
O
8
found 451; 473 [M+Na]
.
In vitro antiꢀmalarial activity
Antiꢀmalarial assay: The in vitro inhibitory assays were performed using P. falciparam
NF54 strain) in human blood. P. falciparum cultures were carried out according to the
(
20
method described . Compounds were dissolved in DMSO and then diluted. Synchronous
cultures with parasitaemia of 1ꢀ1.5% and 2% final haematocrit were incubated in 96 well
microtitre plates with multiple concentrations of compounds for 48 h at 37°C. Parasite
growth was determined spectrophotometrically by measuring the activity of the pLDH, in
control and drugꢀtreated cultures. At the end of the incubation, the cultures were carefully
resuspended, and aliquots of 20 ꢁL were removed and added to 0.1mL of Malstat reagent in a
96 well microtitre plates. The spectrophotometric assessment of pLDH activity is facilitated
by adding 25 ꢁL of a solution of 1.9 ꢁM NBT and 0.24 ꢁM PES. As APADH is formed, the
NBT is reduced and forms a blue formazan product that can be measured at 650nm. The antiꢀ

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malarial activity of the test compound was expressed as the 50% inhibitory concentration
IC50) determined from the doseꢀresponse curve (Table 1, Table 2, Table 3).
(
In silico molecular docking study
Molecular docking and visualization studies of artemisinin and its derivatives was
performed through AutoDock Vina (www.vina.scripps.edu/), an openꢀsource program for
doing molecular docking with 78% binding mode prediction accuracy on test set (Molecular
21
Graphics Lab at The Scripps Research Institute, USA).
PyRx v0.8
(http://pyrx.sourceforge.net), an openꢀsource virtual screening software of computational
drug discovery used to screen libraries of compounds against potential drug targets. The 3D
crystallographic structure of antiꢀmalarial protein target was retrieved through RCSB Protein
Data Bank (PDB) (www.rcsb.org/). To find the possible bioactive conformations of
artemisinin derivatives, these programs were used to dock the artemisinin derivatives into the
22
binding site of P. falciparum target hydrolase enzyme PlasmepsinꢀII (PDB: 2IGY). During
docking procedure all parameters were assigned to their default values.
Screening through in silico pharmacokinetic parameters
Most of drugs fail during discovery process to cross human clinical trials because of
23
poor pharmacokinetic (PK). The important parameters of PK are absorption, distribution,
metabolism, excretion, and toxicity (ADMET). These PK parameters are important
descriptors for human therapeutic use of any compounds. These PK parameters were
calculated and later evaluated through ADMET modules in Discovery Studio v3.5 software
(Accelrys, USA).From this module, mathematical predictive ADMET QSAR models for
different pharmacokinetics parameters namely, aqueous solubility, blood–brain barrier
penetration, cytochrome P450 2D6 inhibition, hepatotoxicity, human intestinal absorption
and plasma protein binding were used to quantitatively predict properties of a set of rules that

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specify ADMET characteristics of the chemical structure of the artemisinin derivatives.
These ADMET descriptors allow us to eliminate compounds with unfavourable ADMET
characteristics early, to avoid expensive reformulation, preferably before synthesis and also
help to evaluate proposed structural refinements that are designed to improve ADMET
properties. The studied compounds were also evaluated against Lipinski’s rule of five for oral
24
bioavailability, since 90% orally active existing drugs/compounds follows Lipinski’s rule.
To predict a variety of toxicities that are often used in drug development, the recommended
toxicity screening models for carcinogenicity, developmental toxicity, mutagenicity and skin
irritancy or sensitization were used for calculations of artemisinin derivatives through
25
DS_TOPKAT module of Discovery Studio 3.5 software (Accelrys, USA). These
predictions will help in optimizing therapeutic ratios of lead compounds for further
development and assessing their potential safety concerns. These predictions will also help in
evaluating intermediates, metabolites and pollutants, along with setting dose range for animal
assays.
RESULT AND DISCUSSION
Dihydroartemisinin (2) was prepared by NaBH
4
reduction of artemisinin using the
ꢀcatalyzed reaction of 2 with appropriate alcohols (Figure 2) in
at subzero temperature (ꢀ10°C to ꢀ5°C) furnished the corresponding ether derivatives
ꢀ18 and 28ꢀ32 in 65ꢀ99% yields as diastereomeric mixtures of β and α isomers, with βꢀ
19
known procedure . BF
3 2
.OEt
CH ꢀCl
2
2
6
isomers isolated as the major products. In the case of ester derivatives, α isomers appeared as
major spot on TLC and was separated by column chromatography, and the pure isomers were
evaluated for antimalarial activity. βꢀisomer of ether derivatives and αꢀisomers of ester
derivatives were used for bioevaluation. The acid chlorides were reacted with
dihydroartemisinin in the presence of triethylamine in dry dichloromethane at 0°C for 2h to
furnish corresponding ester derivatives 19ꢀ24 in 40ꢀ90% yield. Ester derivative 25 was

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synthesised by reacting furanꢀ2ꢀcarboxylic acid and dihydroartemisinin (2) in the presence of
DCC and DMAP in dry dichloromethane at RT for 2h.
Derivatives 28ꢀ32 were synthesized by dissolving anhydrodihydroartemisinin
(
200mg) in CH Cl (15 ml) and mꢀchloroperbenzoic acid (190 mg in 0.5 N NaHCO ) was
2
2
3
added. The reaction mixture was vigorously stirred at room temp. for 75 min. Further epoxide
7 was hydrolysed by using BF .OEt in CH Cl in the presence of appropriate alcohols
2
3
2
2
2
26
which resulted in the products 28ꢀ32 . The configuration at Cꢀ12 stereocenter is assigned
based on the vicinal coupling constant JHꢀ9:Hꢀ10. A large coupling constant (7ꢀ10 Hz) is
generally found for the 10αꢀisomer, indicating the relative transꢀconfiguration. The 10βꢀ
isomer, indicating cisꢀconfiguration at Cꢀ10, on the other hand, has a smaller coupling
26
constant (2ꢀ5 Hz).
Artemisinin derivatives such as artemether 3, arteether 4, and artesunic acid 5 have
excellent antimalarial activity when given by systemic routes. They are fast acting drugs and
are increasingly being used for the treatment of complicated cases of malaria caused by
multidrugꢀresistant P. falciparum. These drugs, however, have serious limitation such as
2
7
short halfꢀlife and poor bioavailability when given by oral route . Both the short halfꢀlife and
poor oral bioavailability are believed to be due to the poor stability of C10ꢀO linkage which is
2
8
prone to acid hydrolysis and P450ꢀcatalyzed oxidation . Since Cꢀ10 acetal derivatives are
unstable, several workers have in recent past prepared C10ꢀC linked derivatives which are
2
9ꢀ33
more stable and have shown improved antimalarial activity by oral route
. In a parallel
program on synthetic antimalarial 1,2,4ꢀtrioxanes, we had observed that incorporation of
34
particular functionality enhances the activity relative to artemisinin . Also there are several
35
reports in the literature, ether and ester derivatives show promising biological activities On
the basis of these considerations, we have prepared ether derivatives 6ꢀ18 and ester

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derivatives 19ꢀ25 and 11ꢀhydroxy ether derivatives of artemisinin 28ꢀ32 and compared their
biological activity against P. falciparum.
Among ether derivatives 6, 8, 13, 14 and 16 displayed better activity than arteether,
and 16 was most active with IC 9.21nM. Except 7 and 15 having nitro group functionality
5
0
at meta position and long chain ether group respectively all the derivatives were better than
artemisinin. Thus, against NF54 strain, the esters were found still more active than the ethers.
All esters were distinctively the most active of all artemsinin derivatives synthesized during
this study. They were significantly more potent than arteether and artemisinin. As the
3
6,37
lipophilicity of the compound increased, there was an increase in activity
. This increase in
1
6
activity is in accordance with earlier observations that lipophilic derivatives are more active
than their more polar counterparts; i.e. esters and ethers are more active than the
corresponding alcohols. Decrease in lipophilicity decreases activity. In the 11ꢀOHꢀ12ꢀether
series, none of the derivatives were as active as artemisinin.
Molecular docking revealed binding affinity against Plasmepsin II
The aim of docking studies was to explore the binding conformation of active
artemisinin derivatives against antimalarial target plasmepsin II. The results of docking
studies suggest that active compounds inhibit the activity of plasmepsin II due to high
binding affinity as indicated by docking score. Predicted results were also complimentary to
3
8
our prior studies. In the studied work, we explored the orientations and binding affinity of
dihydroartemisinin derivatives towards antimalarial target plasmepsin II (PDB: 2IGY).
Plasmepsins are a class of at least 10 enzymes produced by the plasmodium parasite.
Through their haemoglobinꢀdegrading activity, they are an important cause of symptoms in
malaria sufferers. Consequently this family of enzymes is a potential target for antimalarial
drugs. The aspartic protease of plasmodium species are known as plasmepsins. Plasmepsins
are aspartic acid protease, which means their active site contains two aspartic acid residues.

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The binding affinity obtained in the docking study allowed the activity of the artemisinin
derivatives to be compared to that of the standard antimalarial compounds artemisinin and
DHA. The docking study suggest that studied artemisinin and their derivatives inhibit the
plasmepsin II enzymatic activity by indicating acceptable binding affinity score. All the
derivatives showed significant binding affinity against plasmepsin II.
When we compared how the binding site pocket residues of plasmepsins interacted
with the artemisinin derivatives, we found that compounds 19, 20 and 21 showed interaction
with specific amino acid residues thus lead to more stability and potency in these cases and
also showed the hydrogen (Hꢀ) bond formation between the ligand and receptor (Table 4).
During docking, compounds 19, 20 and 21 showed quiet lower binding energy (i.e., higher
binding affinity) in compare to reference drugs DHA (binding energy ꢀ6.9 Kcal/mol) (Fig.
6b) and artemisinin (binding energy ꢀ6.8 Kcal/mol) (Fig. 6c). The docking results for the
active artemisinin derivatives namely, compounds 19, 20 and 21, docked on plasmepsin
enzyme of P. falciparum with high docking score (i.e., low docking binding energy) ofꢀ8.0
Kcal/mol (Fig. 6d), ꢀ7.6 Kcal/mol (Fig. 6e) and ꢀ8.2 Kcal/mol (Fig. 6f), respectively. For
compound 19, docking result showed formation of Hꢀbond with binding site polar basic
residue HISꢀ164 having bond length of 2.19 Å. Likewise compound 20, also showed Hꢀbond
formation with same polar basic residue histidine (HISꢀ164) having bond length of 1.99 Å.
Similarly, compound 21, showed Hꢀbond formation with chemically similar family i.e., polar
basic amino acid residue arginine (ARGꢀ307) having bond length of 2.37Å. Compound 21
seems best docked artemisinin derivative even potent then DHA and artemisinin. During
docking against plasmepsin II, results showed similar binding site residues for most
favourable conformations of predicted active artemisinin derivatives as well as for DHA and
artemisinin (Tableꢀ4).
Compliance with in silico pharmacokinetics properties

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Since the docking studies were found to be promising, the chemical descriptors for the
pharmacokinetic properties were also calculated, so as to check the compliance of studied
compounds with standard range. Calculating these ADMET properties was intended as the
first step toward analysing the novel chemical entities in order to check the failure of lead
candidates, which may cause toxicity or be metabolized by the body into an inactive
form or one unable to cross the intestinal membranes. Results revealed that artemisinin and
its derivatives viz., compound 19, 20 and 21 follows Lipinski’s rule of five for oral
bioavailability (Tableꢀ5). The biꢀplot showed two confidence ellipses of 95% and 99% for the
blood–brain barrier penetration and human intestinal absorption models (Fig.7). The polar
surface area (PSA) was shown to have an inverse relationship with percent human intestinal
absorption and membrane permeability. Compounds 19, 20 showed comparatively higher
PSA and logP, therefore reflect moderate intestinal absorption and very low or no bloodꢀbrain
barrier penetration. Compound 21 showed comparatively low PSA and logP, therefore reflect
good intestinal absorption and medium bloodꢀbrain barrier penetration, similar to DHA &
artemisinin. However, compounds 19, 20 and 21 showed no or very low aqueous solubility,
while DHA and artemisinin comparatively showed higher but low aqueous solubility
(calculated for water at 25°C). All derivatives showed poorly bound property while
evaluating plasmaꢀprotein binding prediction, similar to reference drugs DHA and
artemisinin. Besides, artemisinin derivatives were found to be nonꢀinhibitors of cytochrome
P450 2D6, similar to reference drugs DHA and artemisinin, thus may not metabolized
frequently. The CYP2D6 enzyme is one of the important enzyme involved in drug
metabolism (Tableꢀ6).
Toxicity risk assessment screening
Toxicity risk assessment screening was performed for artemisinin derivatives. Predicted
results showed that compounds 19, 20 have rodent (mouse & rat both male & female)

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carcinogenicity similar to DHA and artemisinin, while compound 21 is nonꢀcarcinogenic.
Similarly, compounds 19, 20, 21 showed nonꢀmutagenicity (Ames) similar to DHA &
artemisinin. Compounds 19, 20, 21 showed mild skin irritancy similar to DHA and
artemisinin. Except compound 21, other two compounds 19, 20 showed
developmental/reproductive toxicity, similar to DHA and artemisinin. Likewise, compounds
19, 20 showed nonꢀdegradable properties (aerobic), while compound 21 showed degradable
property similar to DHA & artemisinin (Tableꢀ7).
Acknowledgements
We thank the Director, CIMAP, Lucknow and AcSIRꢀCIMAP for providing the necessary
research facilities. The work was carried out under a supraꢀinstitutional project, ChemBio
(BSCꢀ0203)ꢀChemical biology of Ocimum and other aromatic plants. Author DKY and YK
acknowledge the Indian Council of Medical Research (ICMR), New Delhi and University
Grant Commission (UGC), New Delhi for RA and RGNF (JRF) fellowship support. Research
work in the Bioinformatics lab is supported by grants from CSIR, New Delhi networking
project BSCꢀ0121 (GENESIS), SERBꢀDepartment of Science & Technology, New Delhi and
CST, UP, Lucknow, India.

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