Synthesis of a series of novel dihydroartemisinin monomers and dimers containing chalcone as a linker and their anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2016.06.035

A new series of monomer and dimer derivatives of dihydroartemisinin (DHA) containing substituted chalcones as a linker were synthesized and investigated for their cytotoxicity in human cancer cell lines HL-60 (leukemia), Mia PaCa-2 (pancreatic cancer), PC

Full text of DOI:10.1016/j.ejmech.2016.06.035

Accepted Manuscript
Synthesis of a series of novel dihydroartemisinin monomers and dimers containing
chalcone as a linker and their anticancer activity
Rashmi Gaur, Anup Singh Pathania, Fayaz Ahmad Malik, Rajendra Singh Bhakuni,
Ram Kishor Verma
PII:
S0223-5234(16)30514-1
10.1016/j.ejmech.2016.06.035
EJMECH 8696
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2015
Revised Date: 17 June 2016
Accepted Date: 19 June 2016
Please cite this article as: R. Gaur, A.S. Pathania, F.A. Malik, R.S. Bhakuni, R.K. Verma, Synthesis of
a series of novel dihydroartemisinin monomers and dimers containing chalcone as a linker and their
anticancer activity, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.035.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
R
R
2
1
O
O
O
O
O
O
O
O
O
O
O
O
O
O
BrCH2CH2OH
BF3.OEt2 ,
Chalcone,
O
O
O
O
DMF, KI, K2CO3
Reflux, 60 oC
O
R
CH2Cl2, 0oC
O
OH
Br
O
O
O
O
O
R
Compounds DHA, 8 and 15 trigger mitochondrial potential loss in HL-60 cells and
induced apoptotic bodies formation in human leukemia HL-60 cells.

ACCEPTED MANUSCRIPT
Synthesis of a series of novel dihydroartemisinin monomers and dimers
containing chalcone as a linker and their anticancer activity
Rashmi Gaur†, ‡, Anup Singh Pathania§, ‡, Fayaz Ahmad Malik§, ‡*, Rajendra Singh Bhakuni†,
‡*, Ram Kishor Verma†
*†Medicinal Chemistry Division; Central Institute of Medicinal and Aromatic Plants,
Lucknow-226015, India.
*§Department of Cancer Pharmacology; Indian Institute of Integrative Medicine; Canal Road
Jammu, Jammu and Kashmir, 180001, India.
‡Academy of Scientific and Innovative Research (AcSIR), New Delhi, 110001, India.

ACCEPTED MANUSCRIPT
Abstract
A new series of monomer and dimer derivatives of dihydroartemisinin (DHA) containing
substituted chalcones as a linker were synthesized and investigated for their cytotoxicity in
human cancer cell lines HL-60 (leukemia), Mia PaCa-2 (pancreatic cancer), PC-3 (prostate
cancer), LS180 (colon cancer) and HEPG2 (hepatocellular carcinoma). Some of these
derivatives have greater antiproliferative and cytotoxic effects in tested cell lines than parent
1
compound DHA. The structures of the all compounds were confirmed by IR, H-NMR and
mass spectral data. Among the new derivatives, compounds 8, 14, 15, 20 and 24 were found
to be more active than parent DHA against tested human cancer cell lines. DHA derivatives
were found to be most active in human leukemia cell lines with compounds 8, 14, 15, 20 and
24 showed IC50 values less than 1 µM for 48 h whereas DHA has IC50 value of 2 µM at same
time period. The most potent compounds 8 with IC50= 0.3 µM (at par with doxorubicin
(IC50= 0.3 µM)) and 15 with IC50= 0.4 µM, of the series, six and three times active than DHA
(with IC50= 2 µM) respectively were selected for further mechanistic work in human
leukemia HL-60 cells.
Keywords: Artemisinin, Monomer and dimer, Apoptosis, Leukemia, Synthesis

ACCEPTED MANUSCRIPT
1. Introduction
Artemisinin (1), isolated from Artemisia annua L.[1] contains a 1, 2, 4-trioxane
moiety (Figure 1). Artemisinin and its derivatives dihydroartemisinin (DHA, 2), artemether
(3), arteether (4), and artesunate (5) have been developed as a new type of antimalarial drugs
[2a]. Recently we have synthesized twenty five ether and ester derivatives having significant
antimalarial activity [2b]. In addition to their antiparasitic properties, it is of considerable
interest to note that artemisinin derivatives are cytotoxic towards cancer cell lines in vitro [3].
Interestingly, the recent discovery of artemisinins as anticancer agents against various cancer
cell lines have evoked many interests on this class of compounds [4-21a]. Artemisinin (1) and
its derivatives are significantly cytotoxic towards murine lymphocytic leukaemia (P-388),
human lung carcinoma (A-549), human colon adenocarcinoma (HT-29) and other tumour cell
lines[21b]. However, one of its derivatives appeared to be more cytotoxic than artemisinin
(1) towards Ehrlich ascites tumour (EAT) cells [21c, 21d].
Artemisinin dimers are obtained by joining two artemisinin molecules without
destroying their endoperoxide bridge. Endoperoxide bridge in artemisinin is the key factor for
its outstanding medicinal value. Due to sensitive nature of endoperoxide ring of artemsisinin
majority of artemisinin analogues were synthesized via chemical modification of artemisinin
at its C-10/C-13 position [22,23]. Beekman et al. synthesized C-10 acetal dimers where two
artemisinin units are connected through an ether-linkage possessing good anticancer activity
[24]. Posner et al. synthesized a series of C-10 acetal artemisinin dimers linked through a
polyethylene glycol or carbon chain link or disulfide linker, with varying length and
flexibility [25,26]. Lee et al. reported that introduction of a sulfur atom to an artemisinin
moiety affords new derivatives which selectively control tumor related angiogenesis [27,28].
Although different classes of cytotoxic artemisinin derivatives have been prepared,
identification of the key factors contributing to their cytotoxicities and rational design of new
classes of cytotoxic artemisinin analogues remain difficult. In this connection, it is of
importance to conduct systematic structure-activity relationship (SAR) studies to assist the
design and synthesis of new cytotoxic artemisinin derivatives in a rational manner.
Chalcone is an open chain flavonoid α, β-unsaturated carbonyl group and is one of the
important compound groups derived from nature identified in Angelica Keiskei and exhibit
interesting pharmacological activities [29,30]. Chemically, they are open-chained molecules
bearing two aromatic rings linked by a three-carbon enone [31]. Both natural and synthetic
chalcones exhibit various activities like antimicrobial, antimalarial, anti-inflammatory, anti-
cancer, antioxidant and many more [29]. These activities are largely being attributed due to

ACCEPTED MANUSCRIPT
the unsaturated ketone moiety. Chalcone and its derivatives have been reported to be
cytotoxic for cancer cells including leukemia cells [32].
Some of the new dimeric trioxane also have selective and potent anticancer activity
[33]. Increasingly widespread evidence indicates that human cancer cells, richer than normal
cells in iron-transport transferrin receptors, [34,35] selectively activate trioxanes to produce
various cytotoxic intermediates; this process is similar to that in the triggering of trioxanes by
heme iron in malaria-infected human erythrocytes [8]. The anticancer properties of trioxanes
have been reviewed [36-40]. Here we disclose that some of the new dimeric trioxane and
monomers powerfully inhibit the growth (submicromolar IC50 values) of various cancer cells
in vitro.
As more analogs were evaluated for antitumor activity, the unsymmetrical DHA
acetal dimer was reported as being highly cytotoxic and more potent than cis-platin [1], while
the symmetrical DHA acetal dimer also exhibited pronounced cytotoxic effects [41a]. In
continuation of our work on artemsinin and chalcone in the area of antimalarial and
anticancer drug discovery [41b-41e], these findings stimulated an interest in preparing
additional DHA acetal dimers with various linkers. Synthesis and evaluations of many
diversified dimers have been reported. However chalcone dimers are new and reported for the
first time in this paper. We designed a new group of DHA monomers and dimers containing
chalcone with a different substituent, linked by ether and chalcone as a linker in dimers and
examined their vitro cytotoxic activities on human leukemia HL-60 cells, pancreatic cancer
Mia PaCa-2 cells, prostate cancer PC-3 cells, colon cancer LS180 cells, hepatocellular
carcinoma HEP G2. Since chalcones are known to have a wide variety of biological activities
including anti-proliferative activity against leukemia cells, this study seems is an approach to
develop drug like candidates.
In general, the cytotoxicities of artemisinin derivatives depends on type of linker
[42,43] which decides efficient diffusion of compounds through membranes [44]. Yet, the
effects of solubility, the stereochemistry (configuration and conformation) [42,45-51] and
nature of functional groups of artemisinin derivatives could not be distinguished [47-51].
Therefore, structure modification might improve their anti-cancer activities. Several groups
have performed modifications at the C-12 position of artemisinin and reported that the
addition of an alkyl carbon chain (C8H17 to C16H33) or a cyanoarylmethyl group significantly
improved its antitumor activities [51]. In addition, it has been found that the endoperoxide in
artemisinin is required for the cytotoxic activity. These data provide a rationale for the
modification of artemisinin’s structure in order to improve its antitumor activity.

ACCEPTED MANUSCRIPT
2. Results and discussion
We have designed and synthesized a new group of DHA monomers and dimers
containing chalcone moiety with a different substituent, linked by C-12 ether linkage/s. The
synthetic pathways are shown in Scheme 1. Firstly, various chalcone based (basic structure:
1, 3-diphenyl-2-propene-1-one, Figure 2.) analogues were synthesized by Claisen-Schmidt
condensation based on a method reported previously [52,53]. In this process, an
acetophenone was reacted with the corresponding aldehyde under acid / base catalyst. In the
second step, the DHA prototype, 2-(12β-dihydroartemisinoxy)-ethyl bromide (2P) was
synthesized according to a reported procedure [40], by the reaction of bromoethanol and
DHA in presence of BF3.OEt2 in dichloromethane. The intermediate compound 2P was
crystallised with methanol and filtered as white crystals. Finally, in the third step the targeted
artemisinin monomers 6-20 (using 1 eq. of 2P and 1 eq. of respective chalcone) (Scheme 1.)
and dimers, 21-25 (using 2 eq. of 2P and 1 eq. of respective chalcone) (Scheme 2.) were
successfully obtained by the reaction of the DHA prototype, 2P and appropriate chalcones in
the presence of K2CO3 and KI in N,N-dimethylformamide (DMF) at 60°C, respectively. The
products obtained were purified by silica gel column chromatography.
The stereochemistry (αH-12 or βH-12) of these compounds was confirmed by the
application of 1H NMR technique which analyzes the chemical shift of H-12 and the coupling
constant between H-11 and H-12. The DHA aliphatic ethers are 12β-isomers as indicated by
a chemical shift (4.60-4.90 ppm) and a small coupling constant (J = ~3.3 Hz) [28]. The
geometrical configuration of the chalcone (α, β-unsaturated ketone) side chain was
determined to be trans by observing the coupling constant (J = ~15.6 Hz) between H-α (H-2)
and H-β (H-3). Among the synthesized compounds, 8, 14, 15, 20 and 24 were found to be
more active than parent DHA against tested human cancer cell lines. DHA derivatives were
found to be most active in human leukemia cell lines with compounds 8, 14, 15, 20 and 24
showed IC50 values less than 1 µM for 48 h whereas DHA has IC50 value of 2 µM at same
time period. It is evident from Table 2 that a progressive increase in electron density on ring
A/B of chalcone resulted in progressive enhancement in anticancer potency, with the para
hydroxyl substituted hybrids 8 and 15 displaying the most promising activity (IC50= 0.3 µM
and IC50= 0.4 µM respectively) followed by para hydroxyl, dimethoxy (20, IC50= 0.5 µM).
On the other hand, the replacement of ring A with heteroaromatic moieties like heterocyclic
benzofuran ring led to only moderate activities (14, IC50= 0.7 µM).

ACCEPTED MANUSCRIPT
The presence of an electron withdrawing (Cl) group on ringA/B of chalcone resulted
in loss of activity. In view of the immense pharmacological importance of hydroxy
substituted chalcones, we were inspired to evaluate the anticancer profiles of various
artemsinin-chalcone hybrids possessing a hydroxylated chalcone ring [29-32]. Gratifyingly,
our above premise was confirmed, as the hydroxy artemsinin-chalcone hybrids were in
general found to display comparatively enhanced potency with anticancer activity,
progressively increasing upon an increase in electron density at chalcone ringA/B, (8, 15, 20
and 14 with IC50 of 0.3, 0.4, 0.5, and 0.7 ꢀM, respectively). In particular, 8 possessing free
hydroxyl group in ring A, showed the best anticancer activity (IC50 = 0.3 ꢀM) against the HL-
60 cell line. Further the declining potency in dimethoxy positional hybrids 20 (IC50 = 0.5 ꢀM)
and 14 (IC50 = 0.7 ꢀM) indicated the importance of dimethoxy substitution on chalcone ring
for the most potent anticancer effect. The above observation was further reinforced when
dimers 21-25 were synthesized by a similar procedure as given in Scheme 2, with 24
possessing electron donating group on chalcone ring, displayed higher potency (IC50 of 0.3
ꢀM).
In order to discern the role of relative positions of rings A and B in the above
scaffolds (A−C=CH−CO−B), the artemsinin-chalcone hybrids possessing chalcone moiety
with reversal in rings A and B (A−CO−HC=C−B) were also evaluated for anticancer activity.
Interestingly, such a study revealed the beneficial role of the presence of a strong electron
withdrawing (carbonyl) group adjacent to the artemisinin moiety, as the artemsinin-chalcone
hybrids possessing an exocyclic olefinic bond adjacent to the artemisinin moiety generally
displayed lower activity, with 15 possessing an IC50 of 0.4 ꢀM compared to 8 (IC50 = 0.3
ꢀM). Having identified the core framework of an artemsinin-chalcone hybrid responsible for
potent anticancer activity, we were encouraged to evaluate the effect of further diversification
on the above motif by installing various functional groups on the chalcone rings. However,
methyl group in the ring led to decreased activity, causing no change in potency (Table 1).
Since progressive increments in the number of electron donating methoxy groups on chalcone
rings led to molecules with progressively increasing anticancer potency, it is apparent that
electron density in chalcone ring is a determinant of anticancer potency of artemsinin-
chalcone. Hybrids with chalcones bearing free hydroxyl groups were most potent. The most
potent compounds 8 with IC50= 0.3 µM and 15 with IC50= 0.4 µM, of the series, six and three
times active than DHA (with IC50= 2 µM) respectively were selected for further mechanistic
work in human leukemia HL-60 cells.

ACCEPTED MANUSCRIPT
Artesunic acid, the hemisuccinate derivative of dihydroartemisinin, is the only clinically
useful water-soluble derivative of artemisinin. However, being a lactol ester, it is rapidly
hydrolyzed back to dihydroartemisinin in aqueous alkaline solution.
The log P values for all of these dimers and monomers range between 5.34-7.45 and aqueous
solubility with Log S values in the range of -4.76 to -6.58. The log P value of the active
compounds 8 and 15 is 6.33. The log P of artemether (2) is 3.5. The derivatives are relatively
polar. The compounds have aqueous solubility <10 mgL-1 at pH 7. Notably, compounds 8
and 15, have solubility at pH 7 of >100 mgL-1, with Log P value -5.96, a value which renders
these compounds suitable for examination as an anticancer molecule, a purpose for which it
would be more suited than DHA. Compound 8 and 15 possess anticancer activity similar to
that of doxorubicin.
2.1. Dihydroarteminsin analogues inhibit cell proliferation in different cancer cell lines
Antiproliferative effect of dihydroarteminsin analogues 6-20 and dimers 21-25 were analysed
in different human cancer cell lines by using MTT assay. Most of the compounds inhibited
cellular proliferation in different cancer cell lines and showed much potent activity than
parent compound DHA (Table. 1). Some highly potent DHA derivatives (8, 14, 15, 20 and
24) were selected from series to further evaluate their IC50 values in human cancer cell lines
HL-60, MiaPaCa-2, PC-3, HEP-G2 and LS180 cells for 48 h time period (Table. 2. - Table.
6.). These compounds showed most potent cytotoxicity in human leukemia HL-60 cells with
IC50 values were less than 1 µM while the parent compound DHA showed IC50 value of
around 2 µM.
2.2. Compounds 8 and 15 induced subG0 apoptotic population in human leukemia HL-
60 cells.
We have selected two most potent compounds 8 and 15 along with their parent compound
DHA for further mechanistic work in human leukemia HL-60 cells. Compound 8 and 15
induced concentration dependent apoptotic DNA damage in cells treated for 24 h time period.
Compound 8 treatment induced 28%, 95% and 96% apoptotic subG0 population at 0.5, 1 and
5 µM concentrations while 15 showed 9%, 65% and 70% DNA damage at same
concentrations. DHA is less effective as compared to these analogues in inducing DNA
damage as only 27% cells showed damaged DNA at 5 µM concentrations (Figure. 3).
2.3. Compounds 8 and 15 triggered mitochondrial potential (MMP) loss in human
leukemia HL-60 cells.
Perturbation in mitochondrial membrane causes the release of many factors that induced
apoptotic signalling in cancer cells. We explored the effect of 8 and 15 along with

ACCEPTED MANUSCRIPT
comparative DHA treatment in inducing mitochondrial damage in HL-60 cells by using
mitochondrial sensor dye rhodamine-123. Control cells that didn’t get any compound
treatment are almost healthy without any significant MMP loss whereas compound 8
treatment induced 27% MMP loss at 0.5 µM concentration, represented by M1 population in
the figure 4. At 1 and 5 µM the loss was 53% and 61% while 15 showed 50% and 58% MMP
loss at 1 and 5µM concentrations when given for 24 h time period. DHA is less effective then
compounds 8 and 15 in triggering MMP loss as 1 and 5 µM concentrations of BA145 showed
only 32 and 41% cells showed M1 population (Figure. 4).
2.4. Compounds 8 and 15 induced apoptotic bodies’ formation in human leukemia HL-
60 cells.
To check the effect of compounds 8 and 15 on HL-60 cells nuclei, we performed hoechst
staining in cells. Hoechst dye stains the condensed chromatin of apoptotic cells more brightly
then chromatin of normal cells Compound 8 and 15 cause condensation of nuclei and induced
morphological alterations in HL-60 cells in concentration dependent manner. Both
compounds induced apoptotic bodies’ induction even at 0.5 µM concentration when given for
24 h time period. DHA showed no any sign of apoptotic bodies’ formation in HL-60 cells at
these concentrations. Number of apoptotic bodies formed in HL-60 cells by compound 8 and
15 is much higher than DHA treatment (Figure 5). Phase contrast microscopy of treated cells
showed cell shrinkage and blebs formation in 8 and 15 treated HL-60 cells in dose dependent
manner (Figure 6).
3. Conclusion
We have prepared a new series of chalcone linker based monomers and dimers of
artemisinin, several of which have shown better anticancer activity than dihydroatemisinin
against HL-60 cell line. Monomers 8 with IC50= 0.3 µM and 15 with IC50= 0.4 µM, the most
active compounds of the series, are six and three times active than DHA (with IC50= 2 µM)
respectively. The resulting artemisinin derivatives showed potent activity against cancer cell
lines with high selectivity Index.
4. Materials and methods
4.1. General Methods
Melting point was determined on a Toshniwal melting point apparatus and is uncorrected.
IR spectra were recorded on a Perkin Elmer 1719 FT-IR spectrophotometer. NMR
spectra were obtained in acetone-d6 on a Bruker Avance, 300 and 400 MHz instrument
using TMS as internal standard. The chemical shift values are reported in ppm and

ACCEPTED MANUSCRIPT
coupling constants in Hz. ESI-MS spectra were recorded on a Perkin Elmer Turbo
Mass/ Shimadzu LC-MS. TLC analyses were carried out on precoated silica gel 60 F254
plates (Merck) using solvent system, hexane: ethyl acetate (6:4). The compounds were
visualized by either exposure of TLC plates to I2 vapours or by spraying with vanillin-
sulfuric acid reagent, followed by heating at 110°C for 15 minutes. Si-gel, 60-120 mesh
(spectrochem) was used in the column chromatography for the purification of metabolites.
13
The compounds were identified by their spectral IR, ID (1H, C, DEPT) and 2D (COSY,
HSQC, HMBC) NMR and ESIMS analysis. The purity of all synthesized compounds was
determined by HPLC method. HPLC analysis was carried out on a reversed-phase waters 30
mm × 4.6 mm, 5 ꢀm C18 column maintained at ambient temperature with a flow rate of 0.4
mL/min and 10 ꢀL samples were injected in all experiments. The retention time was
expressed in min at the UV detection of 240 nm. The mobile phase was composed of
MeCN/H2O (7:3, v/v). According to HPLC analysis, the purity of all compounds is >95%.
4.2. General procedure for the synthesis of artemisinin monomers /dimers (6-25)
4.2.1. Synthesis of 2-(12β-dihydroartemisinoxy) ethyl bromide (DHA-prototype, 2P):
BF3.Et2O (16 mL) was added to a solution of DHA (3, 20.0 g, 70.4 mmol) and 2-
bromoethanol (13.1 g, 105.6 mmol) in 100 ml of CH2Cl2 at 0°C. The mixture was stirred at
room temperature until the reaction completed. The reaction mixture was washed with
saturated NaHCO3 solution (100 ml), water (100 ml) and brine (100ml). The organic layer
was dried and concentrated. The residue was recrystallized to afford white crystals of
compound 2P (26.17 g, 90.0%) as, mp 154-157°C; 1H-NMR (300 MHz, Acetone-d6): δ 0.87
(3H, d, J=7.5 Hz, H3-13), 0.90 (3H, d, J=6.5 Hz, H3-14), 1.31 (3H, s, H3-15 ), 2.41 (1H, m,
H-11a), 4.77 (1H, d, J=3.4 Hz, Ha-1a’), 3.94 (1H, td, J=9.3, 4.1 Hz, 1b’), 3.68 (2H, m, H2-
2a’), 4.83 (1H, d, J=3.5 Hz); 5.43 (1H, s, H-5); ESI-MS: m/z 413 [M+Na]+, molecular
formula C17H27O5Br.
4.2.2. Synthesis of linker moieties (chalcones): Chalcone linkers for synthesized artemisinin
monomers and dimers were synthesized as per method reported in literarture. A mixture of
the acetophenone (10 mmol) and the appropriate aldehyde (11 mmol) in anhydrous MeOH
(30 mL) was stirred at room temperature for 5 min. Then, SOCl2 (2 ml) was added. The
reaction mixture was stirred at room temperature for 2 h. Added water (30 mL). The
precipitate obtained was filtered and recrystallized from 95% EtOH to yield crystals of
chalcones.

ACCEPTED MANUSCRIPT
4.2.3. Synthesis of DHA monomers and dimers: K2CO3 (0.03 mol) was added to a stirred
solution of 2-(12β-dihydroartemisinoxy)-ethyl bromide (2P, 10 mmol for monomers and 20
mmol for dimers) in DMF (50 mL) at 60 °C for 30 min followed by adding appropriate
chalcones: 4’-hydroxy-2, 4-dimethoxychalcone (for 6); 3-chloro-4’-hydroxy- 4-
methoxychalcone (for 7); 4, 4’- dihydroxychalcone (for 8, 23); 3, 4’-dihydroxychalcone (for
9, 15); 2-chloro-4’-hydroxychalcone (for 10); 4-chloro-4’-hydroxychalcone (for 11); 1-(1”-
benzofuranyl)-3-(4’-hydroxyphenyl)-2-propen-1-one (for 12); 1-(3”-chloro-1”-thienyl)-3-(4’-
hydroxyphenyl)-2-propen-1-one (for 13); 1-(1”-furanyl)-3-(4’-hydroxyphenyl)-2-propen-1-
one (for 14); 1-(4”-hydroxyphenyl)-3-(1’-indolyl)- 2-propen-1-one (for 16); 3’-ethoxy-4, 4’-
dihydroxychalcone (for 17); 4-hydroxy-2’, 3’, 4’-trimethoxychalcone (for 18); 4-hydroxy-
2’, 4’ -dimethoxychalcone (for 19); 4, 4’-dihydroxy, 3’, 5’- dimethoxychalcone (for 20, 24);
1-(3”-hydroxyphenyl)-3-(1’-indolyl)-2-propen-1-one
(for
21);
4,
4’-dihydroxy-3-
methylchalcone (for 22); 7, 4’- dihydroxyflavone (LTG, for 25) (9 mmol) and KI as catalysts
and stirring further the reaction at 120 °C for 5 to 6 h. After completion of the reaction, the
reaction mixture was washed with 5% NaOH (30ml) and water (30ml) and extracted with
ethyl acetate. The organic layer (ethyl acetate) was dried (Na2SO4) and concentrated in vacuo
to furnish the crude products. Pure DHA monomers and dimers (6-25) were obtained by
column chromatography (silica gel) using hexane-ethyl acetate (8-5:2-5 v/v) as eluent.
3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-1-(2”, 4”-dimethoxyphenyl)-2-
propen-1-one (6): Elution of the column with n-hexane-ethyl acetate (70:20) yielded a
viscous compound, 90% (w/w) yield; IR ν max(neat): 1654 (chalcone), 1253, 1169, 1109
1
(ether), 1601, 1459, 1362, 1026, 984 (aromatics) cm-1; H, COSY- NMR (300 MHz,
Acetone-d6): δ 0.84 (3H, d, J =9.3 Hz, H3-13a), 0.88 (3H, d, J =7.5 Hz, H3-14a), 1.21 (3H, s,
H3-15a), 2.46 (1H, m, H-11a), 3.88, 3.95 (3H each, s, 2 x OCH3), 3.79 (1H, m, Ha-1a’), 4.05
(1H, m, Hb-1a’), 4.27 (2H, d, J=6.3 Hz, H2-2a’), 4.78 (1H, d, J=3.0 Hz, αH-12a), 5.42 (1H, s,
H-5a), 6.60 (1H, d, J = 2.4 Hz, H-3”), 6.64 (1H, dd, J = 8.7, 2.1 Hz, H-5”), 7.02 (2H, dd, J=
8.7, 1.8 Hz, H-3’, H-5’), 7.48 (1H, d, J= 15.9 Hz, H-2), 7.58 (1H, d, J= 15.9 Hz, H-3), 7.64
(1H, m, H-6”), 7.66 (2H, d, J= 8.7 Hz, H-2’, H-6’); 13C, DEPT- NMR (75 MHz, Acetone-d6):
δ 12.82 (C-13a), 20.18 (C-14a), 24.64 (C-8a), 25.07 (C-2a), 25.75 (C-15a), 31.29 (C-11a),
34.96 (C-9a), 36.68 (C-3a), 37.57 (C-10a), 44.90 (C-7a), 53.00 (C-1a), 55.51, 55.79 (2 x
OCH3), 66.54 (C-1a’), 67.92 (C-2a’), 81.01 (C-6a, q), 87.91 (C-5a), 98.79 (C-3”), 101.99 (C-
12a), 103.88 (C-4a, q), 106.13 (C-5”), 115.54 (C-5’, C-5’), 122.73 (C-1”, q), 125.58 (C-2),
128.60 (C-1’, q), 130.34 (C-2’, C-6’), 132.68 (C-6”), 141.33 (C-3), 160.89 (C-2”, q), 161.17

ACCEPTED MANUSCRIPT
(C-4’, q), 164.64 (C-4”, q), 189.52 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for
C34H43O9, 595.2901; found, 595.2903. ESI-MS(positive): m/z 595 [M+H]+, 617 [M+Na]+ and
(negative): 593 [M-H]+, molecular formula C34H42O9.
1-(3”-Chloro-4”-methoxyphenyl)-3-[4’-{1a’-(12aβ-dihydroxyartemisinoxy)}-
ethoxyphenyl]-2-propen-1-one (7): Elution of the column with n-hexane-ethyl acetate
(70:20) yielded a clorless solid, 90% (w/w) yield; mp. 62 °C; IR ν max(neat): 1655 (chalcone),
1254, 1173, 1107 (ether), 1595, 1509, 1469, 1203, 1029, 984, (aromatics), 788 (Cl group)
1
cm-1; H, COSY- NMR (300 MHz, Acetone-d6): δ 0.87 (6H, d, J =6.9 Hz, H3-13a, H3-14a),
1.30 (3H, s, H3-15a), 2.48 (1H, m, H-11a), 3.99 (3H, s, OCH3), 4.03 (2H, m, H2-1a’), 4.26
(2H, t, J=4.2 Hz, H2-2a’), 4.78 (1H, d, J=3.3 Hz, αH-12a), 5.42 (1H, s, H-5a), 7.01 (2H, dd,
J= 8.7, 1.8 Hz, H-3’, H-5’), 7.22 (1H, d, J = 8.7 Hz, H-5”), 7.71 (1H, d, J= 16.5 Hz, H-2),
7.74 (1H, s, H-2”), 7.77 (2H, d, J= 8.7 Hz, H-2’, H-6’), 8.11 (1H, d, J=8.7 Hz, H-6”), 8.13
13
(1H, d, J= 16.5 Hz, H-3); C, DEPT- NMR (75 MHz, Acetone-d6): δ12.81 (C-13a), 20.18
(C-14a), 24.64 (C-8a), 25.07 (C-2a), 25.74 (C-15a), 31.29 (C-11a), 34.95 (C-9a), 36.66 (C-
3a), 37.58 (C-10a), 44.89 (C-7a), 52.99 (C-1a), 56.46 (OCH3), 66.50 (C-1a’), 67.96 (C-2a’),
81.01 (C-6a, q), 87.91 (C-5a), 101.98 (C-12a), 103.88 (C-4a, q), 112.38 (C-5”), 115.43,
116.29 (C-3’/ C-5’), 119.36 (C-2), 122.71 (C-3”, q), 128.27 (C-1’, q), 129.55 (C-6”), 130.63
(C-2”), 130.94, 131.14 (C-2’/ C-6’), 131.14 (C-2”), 132.30 (C-1”, q), 144.24 (C-3), 158.97
(C-4”, q), 161.53 (C-4’, q), 186.76 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for
C33H40O8Cl, 599.2406; found, 599.2407. ESI-MS(positive): m/z 599 [M+H]+, 621 [M+Na]+,
637 [M+K]+, molecular formula C33H39O8Cl.
3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-1-(4”-hydroxyphenyl)-2-
propen-1-one (8): Elution of the column with n-hexane-ethyl acetate (70:30) yielded a
viscous compound, 55% (w/w) yield; IR ν max(neat): 3567 (OH), 1654 (chalcone), 1256,
1
1173, 1107 (ether), 1602, 1514, 1464, 1201, 1031, 981, (aromatics) cm-1; H, COSY- NMR
(300 MHz, Acetone-d6): δ 0.81 (6H, d, J =7.20 Hz, H3-13a, H3-14a), 1.17 (3H, s, H3-15a),
2.42 (1H, m, H-11a), 3.70 (1H, m, Ha-1a’), 4.09 (1H, m, Hb-1a’), 4.19 (2H, d, J=4.2 Hz, H2-
2a’), 4.71 (1H, d, J=3.0 Hz, αH-12a), 5.36 (1H, s, H-5a), 6.84 (2H, d, J=8.4 Hz, H-3’, H-5’),
7.00 (2H, d, J=8.7 Hz, H-3”, H-5”), 7.59 (1H, d, J=15.3 Hz, H-2), 7.62 (1H, d, J=15.3 Hz, H-
3), 7.68 (2H, d, J=8.7 Hz, H-2’, H-6’), 8.05 (2H, d, J=8.7 Hz, H-2”, H-6”); 13C, DEPT- NMR
(75 MHz, Acetone-d6): δ 12.60 (C-13a), 20.13 (C-14a), 24.64 (C-8a), 25.60 (C-2a), 25.72 (C-
15a), 31.30 (C-11a), 34.96 (C-9a), 36.76 (C-3a), 37.58 (C-10a), 44.91 (C-7a), 53.02 (C-1a),
66.50 (C-1a’), 67.94 (C-2a’), 81.02 (C-6a, q), 87.93 (C-5a), 102.00 (C-12a), 103.89 (C-4a, q),

ACCEPTED MANUSCRIPT
114.82 (C-3’, C-5’), 116.29 (C-3”, C-5”), 119.07 (C-2), 127.19 (C-1’, q), 130.93, 131.02 (C-
2’/C-6’, C-2”/C-6”), 131.89 (C-1”, q), 144.14 (C-3), 160.36 (C-4”, q), 163.45 (C-4’, q),
187.78 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for C32H39O8, 551.2639; found,
551.2640. ESI-MS(positive): m/z 551 [M+H]+, 573 [M+Na]+, 589 [M+K]+, molecular
formula C32H38O8.
3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-1-(3”-hydroxyphenyl)-2-
propen-1-one (9): Elution of the column with n-hexane-ethyl acetate (70:40) yielded a
viscous compound,, 38% (w/w) yield, more polar than monomer 60; IR ν max(neat): 3425
(OH), 1661 (chalcone), 1252, 1175, 1108 (ether), 1590, 1510, 1443, 1030, 985, (aromatics)
1
cm-1; H, COSY- NMR (300 MHz, Acetone d6): δ 0.79 (6H, d, J =6.0 Hz, H3-13a, H3-14a),
1.12 (3H, s, H3-15a), 2.41 (1H, m, H-11a), 3.69 (1H, m, Ha-1a’), 4.04 (1H, m, Hb-1a’), 4.22
(2H, d, J=4.2 Hz, H2-2a’), 4.72 (1H, d, J=3.0 Hz, αH-12a), 5.35 (1H, s, H-5a), 6.97 (2H, d,
J=8.4 Hz, H-3’, H-5’), 7.12 (1H, m, H-4”), 7.39 (2H, m, H-2”, H-5”), 7.60 (1H, d, J=15.9 Hz,
13
H-2), 7.66 (4H, m, H-3, H-2’, H-6’, H-6”), 7.83 (1H, brs, 3’-OH); C, DEPT- NMR (75
MHz, Acetone-d6): δ 12.78 (C-13a), 20.14 (C-14a), 24.64 (C-8a), 25.05 (C-2a), 25.72 (C-
15a), 31.30 (C-11a), 34.97 (C-9a), 36.68 (C-3a), 37.58 (C-10a), 44.93 (C-7a), 53.04 (C-1a),
66.52 (C-1a’), 67.98 (C-2a’), 81.00 (C-6a, q), 87.91 (C-5a), 102.00 (C-12a), 103.85 (C-4a, q),
114.52 (C-2” ), 115.47 (C-3’, C-5’), 119.48, 120.09, 121.24 (C-2, / C-4”, / C-6”), 128.29 (C-
1’, q), 130.10 (C-5”), 130.89 (C-2’, C-6’), 140.37 (C-1”, q), 144.32 (C-3), 159.82 (C-3”, q),
161.63 (C-4’, q), 189.04 (C-1, q); HR-MS (m/z) [M + H]+ (ESI+) calcd for C32H39O8,
551.2639; found, 551.2639. ESI-MS(positive): m/z 551 [M+H]+, (Negative): 549 [M-H]-,
molecular formula C32H38O8.
1-(2”-Chlorophenyl)-3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-2-
propen-1-one (10): Elution of the column with n-hexane-ethyl acetate (70:30) yielded a
viscous compound,, 75% (w/w) yield,; IR ν max(neat): 1656 (chalcone), 1250, 1173, 1102
1
(ether), 1591, 1519, 1440, 1029, 982, (aromatics), 783 (Cl group) cm-1; H, COSY- NMR
(300 MHz, Acetone d6): δ 0.81 (6H, d, J =7.5 Hz, H3-13a, H3-14a), 1.28 (3H, s, H3-15a), 2.49
(1H, m, H-11a), 3.96 (1H, m, Ha-1a’), 4.01 (1H, m, Hb-1a’), 4.21 (2H, m, H2-2a’), 4.71 (1H,
d, J=3.0 Hz, αH-12a), 5.32 (1H, s, H-5a), 6.89 (2H, d, J= 7.2 Hz, H-3’, H-5’), 7.01 (2H, m,
13
H-4’’, H-5’’), 7.33 (1H, d, J= 15.6 Hz, H-2), 7.46 (4H, m Hz, H-2’, H-6’, H-3, H-6”); C,
DEPT- NMR (75 MHz, Acetone-d6): δ 17.77 (C-13a 20.14 (C-14a), 24.63 (C-8a), 25.05 (C-
2a), 25.72 (C-15a), 31.29 (C-11a), 34.96 (C-9a), 36.67 (C-3a), 37.58 (C-10a), 44.90 (C-7a),
53.03 (C-1a), 66.50 (C-1a’), 67.99 (C-2a’), 80.99 (C-6a, q), 87.91 (C-5a), 102.00 (C-12a),

ACCEPTED MANUSCRIPT
103.86 (C-4a, q), 115.58 (C-3’, C-5’), 124.55 (C-2), 127.50 (C-5”), 129.57 (C-3”), 127.67
(C-1’), 131.13 (C-2’, C-6’), 131.56 (C-6”), 131.66 (C-4”), 139.00 (C-2”, q), 140.09 (C-1”, q),
146.30 (C-3), 161.93 (C-4’, q), 193.15 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for
C32H38O7Cl, 569.2300; found, 569.2300. ESI-MS(positive): m/z 569 [M+H]+, 591 [M+Na]+,
molecular formula C32H37O7Cl.
1-(4”-chlorophenyl)-3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-2-
propen-1-one (11): Elution of the column with n-hexane– ethyl acetate (70:30) yielded a
max
viscous compound, 75% (w/w); IR ν
(neat): 1647 (chalcone), 1250, 1170, 1107 (ether),
1595, 1511, 1435, 1027, 983, (aromatics), 766 (Cl group) cm-1; 1H, COSY- NMR (300 MHz,
Acetone d6): δ 0.75 (6H, d, J =6.9 Hz, H3-13a, H3-14a), 1.21 (3H, s, H3-15a), 2.39 (1H, m, H-
11a), 3.62 (1H, m, Ha-1a’), 3.96 (1H, m, Hb-1a’), 4.14 (2H, t, 4.2 Hz, H2-2a’), 4.66 (1H, d,
J=2.7 Hz, αH-12a), 5.30 (1H, s, H-5a), 6.89 (2H, d, J= 8.4 Hz, H-3’, H-5’), 7.42 (2H, d,
J=8.4, H-2”, H-6”), 7.53 (3H, m, H-2’, H-6’, H-2), 7.66 (4H, d, J=15.00 Hz, 3), 8.00 (2H, d,
J=8.7 Hz, H-3”, H-5”); 13C, DEPT- NMR (75 MHz, Acetone-d6): δ 12.82 (C-13a), 20.19 (C-
14a), 24.65 (C-8a), 25.07 (C-2a), 25.77 (C-15a), 31.29 (C-11a), 34.97 (C-9a), 36.68 (C-3a),
37.60 (C-10a), 44.90 (C-7a), 52.99 (C-1a), 66.53 (C-1a’), 67.99 (C-2a’), 81.03 (C-6a, q),
87.94 (C-5a), 102.02 (C-12a), 103.92 (C-4a. q), 115.49 (C-3’, C-5’), 119.59 (C-2), 128.14
(C-1’,q), 129.16 (C-3”, C-5”), 130.50 (C-2’, C-6’), 131.10 (C-2”, C-6”), 137.59 (C-1”, q),
138.60 (C-4”, q), 144.91 (C-3), 161.74 (C-4’, q), 188.32 (C-1, q); HR-MS (m/z) [M+H]+
(ESI+) calcd for C32H38O7Cl, 569.2300; found, 569.2300. ESI-MS(positive): m/z 569 [M +
H]+, molecular formula C32H37O7Cl.
1-(1”-Benzofuranyl)-3-[4’-{1a’-(12aβ-dihydroxyartemisinoxy)}-ethoxyphenyl]-2-
propen-1-one (12): Elution of the column with n-hexane– ethyl acetate (70:20) yielded a
max
clourless solid, 78% (w/w); mp. 65 °C; IR ν
(neat): 1659 (chalcone), 1254, 1165 1108
(ether), 1592, 1510, 1452, 1031, 984, (aromatics), 750 (furan moiety) cm-1; 1H, COSY- NMR
(300 MHz, Acetone d6): δ 0.81 (3H, d, J =7.5 Hz, H3-13a), 0.82 (3H, d, J =6.0 Hz, H3-14a),
1.28 (3H, s, H3-15a), 2.43 (1H, m, H-11a), 3.73 (1H, m, Ha-1a’), 3.96 (1H, m, Hb-1a’), 4.22
(2H, t, J=3.9 Hz, H2-2a’), 4.72 (1H, d, J=3.6 Hz, αH-12a), 5.36 (1H, s, H-5a), 6.98 (2H, dd,
J=8.7, 1.2 Hz, H-3’, H-5’), 7.29 (1H, dd, J= 7.8, 0.9 Hz, H-5”), 7.46 (1H, dd, J= 8.1, 1.2 Hz,
H-6”), 7.49 (1H, d, J = 8.1 Hz, H-7”), 7.61 (1H, d, J=15.8 Hz, H-2), 7.73 (3H, dd, J=8.7, 2.1
Hz, H-2’, H-6’, H-4 ”), 7.70 (1H, d, J=15.6 Hz, H-3), 7.87 (1H, brs, H-2”); 13C, DEPT- NMR
(75 MHz, Acetone-d6): δ 12.98 (C-13a), 20.15 (C-14a), 24.64 (C-8a), 25.06 (C-2a), 25.73 (C-
15a), 31.29 (C-11a), 34.96 (C-9a), 36.67 (C-3a), 37.59 (C-10a), 44.91 (C-7a), 53.02 (C-1a),

ACCEPTED MANUSCRIPT
66.50 (C-1a’), 67.99 (C-2a’), 81.00 (C-6a, q), 87.92 (C-5a), 101.99 (C-12a), 103.87 (C-4a, q),
112.50 (C-2”), 113.46 (C-7”), 115.54 (C-3’, C-5’), 119.68 (C-4”), 123.79 (C-5”), 124.31 (C-
6”), 127.92 (C-1’, q), 127.96 (C-3”, q), 128.50 (C-2), 131.04 (C-2’, C-6’), 143.92 (C-3),
154.50 (C-8”, q), 156.09 (C-1”, q), 161.86 (C-4’, q), 179.00 (C-1, q); HR-MS (m/z) [M+H]+
(ESI+) calcd for C34H39O8, 575.2639; found, 575.2642. ESI-MS(positive): m/z 597 [M+Na]+,
(Negative): 573 [M-H]-, molecular formula C34H38O8.
1-(3”-Chloro-2”-thienyl)-3-[4’-{1a’-(12aβ-dihydroxyartemisinoxy)}-ethoxyphenyl]-2-
propen-1-one (13): Elution of the column with n-hexane-ethyl acetate (70:30) yielded a
max
viscous compound, 85% (w/w); IR ν
(neat): 1647 (chalcone), 1258, 1174, 1107 (ether),
1
1588, 1511, 1028, 984, (aromatics), 1421, 826 (thiophene group), 789 (Cl group) cm-1; H,
COSY- NMR (300 MHz, Acetone d6): δ 0.78 (3H, d, J =9.3 Hz, H3-13a), 0.82 (3H, d, J=6.0,
H3-14a), 1.28 (3H, s, H3-15a), 2.44 (1H, m, H-11a), 3.65 (1H, m, Ha-1a’), 3.98 (1H, m, Hb-
1a’), 4.20 (2H, d, J=4.5 Hz, H2-2a’), 4.72 (1H, d, J=3.0 Hz, αH-12a), 5.35 (1H, s, H-5a), 6.96
(2H, d, J= 8.7 Hz, H-3’, H-5’), 7.12 (1H, d, J=3.6, H-2”), 7.76 (3H, m, H-2, H-2’, H-6’), 7.53
(1H, d, J=15.9 Hz, H-3), 7.94 (1H, d, J=4.2 Hz, H-3”); 13C, DEPT- NMR (75 MHz, Acetone-
d6): δ 12.76 (C-13a), 20.14 (C-14a), 24.63 (C-8a 25.06 (C-2a), 25.71 (C-15a), 31.29 (C-11a),
34.97 (C-9a), 36.66 (C-3a), 37.59 (C-10a), 44.91 (C-7a), 53.02 (C-1a), 66.49 (C-1a’), 67.98
(C-2a’), 80.98 (C-6a, q), 87.91 (C-5a), 101.98 (C-12a), 103.85 (C-4a, q), 115.48 (C-3’, C-5’),
118.50 (C-2), 127.93 (C-1’, q), 128.77 (C-5”), 131.04 (C-2’, C-6’), 132.29 (C-2”), 138.44 (C-
3”, q), 145.79 (C-1”, q), 144.08 (C-3), 161.82 (C-4’, q), 180.94 (C-1, q); HR-MS (m/z) [M +
H]+ (ESI+) calcd for C30H36O7SCl, 575.1864; found, 575.1864. ESI-MS(positive): m/z 575
[M+H]+ , 597 [M+Na]+ , 613 [M+K]+, molecular formula C30H35O7SCl.
3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-1-(1”-furanyl)-2-propen-1-one
(14): Elution of the column with n-hexane-ethyl acetate (70:25) yielded a light yellow
viscous compound, 75% (w/w); IR ν max (neat): 1657 (chalcone), 1257, 1174, 1106, (ether),
1
1594, 1511, 1463, 1029, 985 (aromatics), 793 (furan moiety) cm-1; H, COSY- NMR (300
MHz, Acetone-d6): δ 0.88 (6H, d, J =6.3 Hz, H3-13a, H3-14a), 1.31 (3H, s, H3-15a), 2.51 (1H,
m, H-11a), 3.79 (1H, m, Ha-1a’), 4.11 (1H, m, Hb-1a’), 4.27 (2H, t, J=4.2 Hz, H2-2a’), 4.79
(1H, d, J=3.3 Hz, αH-12a), 5.42 (1H, s, H-5a), 6.69 (1H, d, J=3.6, H-4”), 7.02 (2H, d, J= 8.7
Hz, H-3’, H-5’), 7.52 (1H, d, J=3.0, H-3”),7.51 (1H , d, J=15.6, H-2), 7.72 (2H, d, J=3.6 Hz,
H-2”), 7.74 (2H, d, J= 8.7, H-2’, H-6’), 7.76 (1H, d, J=15.6 Hz, H-3); 13C, DEPT- NMR (75
MHz, Acetone-d6): δ 12.83 (C-13a), 20.19 (C-14a), 24.66 (C-8a), 25.08 (C-2a), 25.78 (C-
15a), 31.30 (C-11a), 34.98 (C-9a), 36.70 (C-3a), 37.60 (C-10a), 44.92 (C-7a), 53.02 (C-1a),

ACCEPTED MANUSCRIPT
66.53 (C-1a’), 67.99 (C-2a’), 81.01 (C-6a, q), 87.93 (C-5a), 102.01 (C-12a), 103.89 (C-4a, q),
112.77 (C-3”), 115.51 (C-3’, C-5’), 117.74 (C-2”), 119.74 (C-2), 128.07 (C-1’, q), 130.82 (C-
2’, C-6’), 143.21 (C-3), 147.39 (C-4”, q), 154.34 (C-1”, q), 161.64 (C-4’, q), 177.32 (C-1, q);
HR-MS (m/z) [M+H]+ (ESI+) calcd for C30H37O8, 525.2482; found, 525.2482. ESI-
MS(positive): m/z 525 [M+H]+, 547 [M+Na]+, 563 [M+K]+, molecular formula C30H36O8.
1-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-3-(3”-hydroxyphenyl)-2-
propen-1-one (15): Elution of the column with n-hexane-ethyl acetate (70:40) yielded a
viscous compound, 52% (w/w) yield, less polar than monomer 9; IR ν max(neat): 3367 (OH),
1654 (chalcone), 1256, 1173, 1107 (ether), 1578, 1509, 1448, 1028, 984, (aromatics) cm-1;
1H, COSY- NMR (300 MHz, Acetone d6): δ 0.81 (6H, d, J =6.9 Hz, H3-13a, H3-14a), 1.17
(3H, s, H3-15a), 2.42 (1H, m, H-11a), 3.70 (1H, m, Ha-1a’), 4.09 (1H, m, Hb-1a’), 4.19 (2H,
d, J= 4.2 Hz, H2-2a’), 4.71 (1H, d, J=3.0 Hz, αH-12a), 5.36 (1H, s, H-5a), 6.96 (2H, d, J=8.4
Hz, H-3’, H-5’), 7.03 (1H, dd, J=8.10, 2.10 Hz, H-4”), 7.29 (1H, t, J=7.80 Hz, H-5”), 7.47
(1H, brs, H-2”), 7.56 (1H, d, J=15.3 Hz, H-2), 7.65(1H, d, J=15.3 Hz, H-3), 7.72(3H, d,
13
J=8.4 Hz, H-2’, H-6’, H-6”), 7.90 (1H, brs, 4’-OH); C, DEPT- NMR (75 MHz, Acetone-
d6): δ 12.78 (C-13a), 20.16 (C-14a), 24.64 (C-8a), 25.06 (C-2a), 25.73 (C-15a), 31.30 (C-
11a), 34.96 (C-9a), 36.67 (C-3a), 37.58 (C-10a), 44.91 (C-7a), 53.02 (C-1a), 66.53 (C-1a’),
67.97 (C-2a’), 81.02 (C-6a, q), 87.93 (C-5a), 102.00 (C-12a), 103.89 (C-4a, q), 115.13 (C-2”
), 115.48 (C-3’, C-5’), 120.06, 120.11, 120.23 (C-2, / C-4”, / C-6”), 128.30 (C-1’, q), 130.05
(C-5”), 130.82 (C-2’, C-6’), 140.44 (C-1”, q), 144.14 (C-3), 158.17 (C-4’, q), 161.58 (C-3”,
q), 189.33 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for C32H39O8, 551.2639; found,
551.2639. ESI-MS(positive): m/z 551 [M+H]+, (Negative): 549 [M-H]-, molecular formula
C32H38O8.
1-[4”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-3-(1’-indolyl)-2-propen-1-one
(16): Elution of the column with n-hexane– ethyl acetate (70:20) yielded a viscous
compound, 30% (w/w); IR ν max (neat): 3340, 2370 (NH), 1654 (chalcone), 1254, 1169, 1108,
1
(ether), 1598, 1526, 1451, 1029, 983 (aromatics), 744 (pyrrole moiety) cm-1; H, COSY-
NMR (300 MHz, Acetone-d6): δ 0.76 (6H, m, H3-13a, H3-14a,), 1.28 (3H, s, H3-15a), 2.41
(1H, m, H-11a), 3.70 (1H, m, Ha-1a’), 3.92 (1H, m, Hb-1a’), 4.21 (2H, m, H2-2a’), 4.67 (1H,
brs, αH-12a), 5.31 (1H, s, H-5a), 6.97 (2H, d, J=9.0 Hz, H-3”, H-5”), 7.12 (2H, dd, J =6.6,
2.1 Hz, H-5’, H-6’), 7.40 (1H, d, J=6.0, H-4’), 7.59 (1H, d, J=15.6 Hz, H-2), 7.83 (1H, brs,
H-2’, COSY correlation with NH), 7.96 (1H, d, J=15.6 Hz, H-3), 7.99 (1H, m, H-7’), 8.02
(2H, d, J=9.0 Hz, H-2”, H-6”), 10.80 (1H brs, NH); 13C, DEPT- NMR (75 MHz, Acetone-d6):

ACCEPTED MANUSCRIPT
δ 12.74 (C-13a), 20.10 (C-14a), 24.63 (C-8a), 25.05 (C-2a), 26.69 (C-15a), 31.30 (C-11a),
34.96 (C-9a), 36.66 (C-3a), 37.58 (C-10a), 44.93 (C-7a), 53.06 (C-1a), 66.53 (C-1a’), 67.99
(C-2a’), 81.00 (C-6a, q), 87.92 (C-5a), 102.01 (C-12a), 103.81 (C-4a, q), 112.64 (C-4’),
113.51 (C-1’, q), 114.76 (C-3”, C-5”), 120.10, 120.76, 121.48 (C-2, / C-6’, / C-7’), 123.16
(C-5’), 126.21 (C-3’, q), 130.71 (C-2”, C-6”), 131.20 (C-2”), 132.01 (C-1”, q), 138.08 (C-3),
138.70 (C-8’, q), 162.00 (C-4”, q), 185.50 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for
C34H40O7N, 574.2799; found, 574.2803. ESI-MS(positive): m/z 574 [M+H]+. molecular
formula C34H39O7N.
1-[4”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-3-(3’-ethoxy-4’-
hydroxyphenyl)-2-propen-1-one (17): Elution of the column with n-hexane-ethyl acetate
(70:30) yielded a colorless viscous compound, 75% (w/w); IR ν max (neat): 3422 (OH),, 1654
1
(chalcone), 1600, 1509, 1458, 1029, 984 (aromatics), 1560, 1261, 1168 (ether) cm-1; H,
COSY- NMR (300 MHz, Acetone-d6): δ 0.88 (6H, d, J =6.6 Hz, H3-13a, H3-14a), 1.27 (3H,
brs, H3-15a), 1.37 (3H, t, J=7.2 Hz, CH3), 2.48 (1H, m, H-11a), 3.76 (1H, m, Ha-1a’), 4.10
(1H, m, Hb-1a’), 4.16 (2H, q, J=7.5 Hz, OCH2), 4.31 (2H, d, J=3.9 Hz, H2-2a’), 4.79 (1H, d,
J=3.3 Hz, αH-12a), 5.42 (1H, s, H-5a), 6.89 (1H, d, J=8.1, H-5’), 7.07 (2H, d, J=8.2 Hz, H-
3”, H-5”), 7.28 (1H, dd, J=8.1, 1.5 Hz, H-6’), 7.44 (1H, s, H-2’), 7.70 (2H, m, H-2, H-3),
13
8.11 (2H, d, J=8.7 Hz, H-2”, H-6”); C, DEPT- NMR (75 MHz, Acetone-d6): δ 12.78 (C-
13a), 14.56 (CH3), 20.14 (C-14a), 24.64 (C-8a), 25.06 (C-2a), 25.73 (C-15a), 31.29 (C-11a),
34.95 (C-9a), 36.67 (C-3a), 37.58 (C-10a), 44.90 (C-7a), 53.02 (C-1a), 64.79 (OCH2-), 66.49
(C-1a’), 68.03 (C-2a’), 81.00 (C-6a, q), 87.92 (C-5a), 102.01 (C-12a), 103.87 (C-4a, q),
112.50 (C-2’), 114.79 (C-3”, C-5”), 115.72 (C-5’), 119.43 (C-2), 123.75 (C-6’), 127.72 (C-
1’, q), 130.98 (C-2”, C-6”), 131.95 (C-1”, q), 144.29 (C-3), 147.47 (C-4’, q), 149.91 (C-3’,
q), 163.12 (C-4”, q), 187.64 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for C34H43O9,
595.2901; found, 595.2900. ESI-MS(positive): m/z 595 [M+H]+, 517 [M+Na]+, 633 [M+K]+,
molecular formula C34H42O9.
1-[4”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-3-(2’,
3’,
4’-trimethoxy
phenyl)-2-propen-1-one (18): Elution of the column with n-hexane– ethyl acetate (70:30)
max
yielded a yellow viscous compound, 75% (w/w); IR ν
(neat): 1656 (chalcone), 1601,
1
1494, 1463, 1030, 986 (aromatics), 1257, 1171 (ether) cm-1; H, COSY- NMR (300 MHz,
Acetone d6): δ 0.88 (6H, d, J =6.3 Hz, H3-13a, H3-14a), 1.27 (3H, s, H3-15a), 2.48 (1H, m, H-
11a), 3.78 (1H, m, Ha-1a’), 3.82, 3.89, 3.92,, (3H each, s, 3xOCH3), 4.12 (1H, m, Hb-1a’),
4.31 (2H, d, J=4.2 Hz, H2-2a’), 4.79 (1H, d, J=3.0 Hz, αH-12a), 5.42 (1H, s, H-5a), 6.86 (1H,

ACCEPTED MANUSCRIPT
d, J=8.7 Hz, H-5’), 7.08 (2H, d, J=8.7 Hz, H-3”, H-5”), 7.62 (1H, d, J=8.7, H-6’), 7.75 (1H,
13
d, J=15.6 Hz, H-2), 7.98 (1H, d, J=15.6 Hz, H-3), 8.11 (3H, d, J=8.7 Hz, H-2”, H-6”); C,
DEPT- NMR (75 MHz, Acetone-d6): δ 12.72 (C-13a), 20.21 (C-14a), 24.65 (C-8a), 25.07 (C-
2a), 25.74 (C-15a), 31.29 (C-11a), 34.96 (C-9a), 36.67 (C-3a), 37.59 (C-10a), 44.89 (C-7a),
53.01 (C-1a), 56.01, 60.48, 61.37 (3xOCH3), 66.49, 68.04 (C-1a’, C-2a’), 81.00 (C-6a, q),
87.91 (C-5a), 102.01 (C-12a), 103.88 (C-4a, q), 108.60 (C-5’), 114.85 (C-3”, C-5”), 120.94
(C-2), 122.22 (C-1’, q), 123.65 (C-6’), 131.90 (C-1”, q), 131.00 (C-2”, C-6”), 138.52 (C-3),
142.91 (C-3’, q), 153.98 (C-2’, q), 156.42 (C-4’, q), 163.17 (C-4”, q), 187.83 (C-1, q); HR-
MS (m/z) [M+H]+ (ESI+) calcd for C35H45O10, 625.3007; found, 625.3007. ESI-MS(positive):
m/z 625 [M+H]+, 647 [M+Na]+, 663 [M + K]+, molecular formula C35H44O10.
1-[4”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-1-(2’,4’-dimethoxyphenyl)-2-
propen-1-one (19): Elution of the column with n-hexane-ethyl acetate (70:25) yielded a
max
viscous compound, 75% (w/w); IR ν
(neat): 1653 (chalcone), 1600, 1506, 1459, 1028,
1
985 (aromatics), 1260, 1168 (ether) cm-1; H, COSY- NMR (300 MHz, Acetone-d6): δ 0.95
(3H, d, J =9.3 Hz, H3-13a), 0.91 (3H, d, J=7.2Hz, H3-14a), 1.43 (3H, brs, H3-15a), 2.61 (1H,
m, H-11a), 3.83 (1H, m, Ha-1a’), 3.86, 3.91 (3H each, s, 2xOCH3), 3.97 (1H, m, Hb-1a’), 4.19
(2H, m, H2-2a’), 4.91 (1H, d, J=3.3 Hz, αH-12a), 5.48 (1H, s, H-5a), 6.49 (1H, s, H-3’), 6.54
(1H, dd, J=8.4, 2.1 Hz, H-5’), 6.97 (2H, d, J=8.4 Hz, H-3”, H-5”), 7.54 (1H, d, J=15.6 Hz, H-
13
2), 7.57 (1H, d, J=8.1, H-6’), 8.01 (2H, d, J= 8.4 Hz, H-2”, H-6”); C, DEPT- NMR (75
MHz, Acetone-d6): δ 13.33 (C-13a), 20.72 (C-14a), 24.81 (C-8a), 25.11 (C-2a), 26.56 (C-
15a), 31.27 (C-11a), 35.00 (C-9a), 36.82 (C-3a), 37.86 (C-10a), 44.81 (C-7a), 52.95 (C-1a),
55.89, 55.96 (2x OCH3), 66.79 (C-1a’), 67.90 (C-2a’), 81.51 (C-6a, q), 88.34 (C-5a), 98.88
(C-3’), 102.62 (C-12a), 104.53 (C-4a, q), 105.80 (C-5’), 114.67 (C-3”, C-5”), 117.70 (C-1’,
q), 120.60 (C-2), 131.10 (C-2”, C-6”), 131.25 (C-6’), 132.17 (C-1”, q), 140.23 (C-3), 160.74
(C-4’, q), 162.83 (C-2’, q), 163.30 (C-4”, q), 189.90 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+)
calcd for C34H43O9, 595.2901; found, 595.2902. ESI-MS(positive): m/z 595 [M+H]+,
617[M+Na]+, 633[M+K]+, molecular formula C34H42O9.
1-[4”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-3-(3’-hydroxy-2’,4’-
dimethoxyphenyl)-2-propen-1-one (20): Elution of the column with n-hexane– ethyl acetate
max
(70:30) yielded a viscous compound, 75% (w/w); IR ν
(neat): 3398 (OH), 1654
1
(chalcone), 1598, 1508, 1459 1027 (aromatics), 1279, 1125 (ether) cm-1; H, COSY- NMR
(300 MHz, Acetone-d6): δ 0.74 (3H, d, J =7.2 Hz, H3-13a), 0.76 (3H, d, J =6.0 Hz, H3-14a),
1.17 (3H, brs, H3-15a), 2.31 (1H, m, H-11a), 3.57 (6H, s, 2xOCH3), 3.84 (1H, m, Ha-1a’),

ACCEPTED MANUSCRIPT
3.90 (1H, m, Hb-1a’), 4.08 (2H, m, H2-2a’), 4.61 (1H, d, J=3.3 Hz, αH-12a), 5.20 (1H, s, H-
5a), 6.82 (2H, m, H-3”, H-5”), 7.03 (2H, brs, H-2’, H-6’), 7.55 (1H, d, J=15.6 Hz, H-2), 7.67
13
(1H, d, J=15.6 Hz, H-3), 7.93 (2H, d, J=8.7 Hz, H-2”, H-6”), 9.18 (1H, m, 4’-OH); C,
DEPT- NMR (75 MHz, Acetone-d6): δ 12.75 (C-13a), 20.25 (C-14a), 24.42 (C-8a), 25.05 (C-
2a), 25.72 (C-15a), 31.31 (C-11a), 35.07 (C-9a), 36.69 (C-3a), 37.41 (C-10a), 45.00 (C-7a),
53.16 (C-1a), 56.16 (2xOCH3), 68.58, 72.55 (C-1a’, C-2a’), 81.06 (C-6a, q), 87.90 (C-5a),
102.23 (C-12a), 103.79 (C-4a, q), 106.55 (C-2’, C-6’), 115.66 (C-3”, C-5”), 121.42 (C-2),
130.75, 131.04 (C-1’/ C-1”, q), 131.31 (C-2”, C-6”), 139.60 (C-4’, q), 143.75 (C-3), 154.08
(C-3’, C-5’, q), 162.23 (C-4”, q), 187.56 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for
C34H43O10, 611.2850; found, 611.2851. ESI-MS(positive): m/z 611 [M+H]+, 633 [M+Na]+,
649 [M+K]+, molecular formula C34H42O10.
1-[3”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxyphenyl]-3-[N-{1ba’-(12bβ-
dihydroxyartemisinoxy)}-ethoxy-1H-1’-indolyl]-2-propen-1-one (21): Elution of the
column with n-hexane-ethyl acetate (70:40) yielded a viscous compound, 30% (w/w); IR ν
max
(neat): 2923, 2854, 1653 (chalcone), 1561, 1257, 1107 (ether), 1577, 1459, 1376, 1029,
1
984 (aromatics) cm-1; H, COSY- NMR (400 MHz, Acetone-d6): δ 0.63 (6H, m, H3-13a, H3-
13b), 0.76 (6H, m, H3-14a, H3-14b), 1.08, 1.17 (3H each, brs, H3-15a, H3-15b), 2.20, 2.41
(1H each, m, H-11a, H-11b), 3.62, 4.00, 4.19 (6H, m, H2-1a’, H2-1b’, H2-2a’), 4.48 (2H
each, m, H2-2b’), 4.52, 4.67 (1H each, d, J=2.7 and 2.4 Hz, αH-12a, αH-12b), 5.33 (2H, brs,
H-5a, H-5b ), 7.08 (2H, d, J=8.12 Hz, H-4”), 7.16 (2H, m, H-5’, H-6’), 7.34 (1H, t, J=7.40
Hz, H-5”), 7.48 (1H, d, J=7.56 Hz, H-4’), 7.50 (1H, d, J=2.56 Hz, H-2”), 7.54 (1H, d,
J=15.52 Hz, H-2), 7.59 (1H, d, J=7.64 Hz, H-6”), 7.89 (1H, brs H-2’), 7.94 (1H, d, J=6.64
Hz, H-7’), 7.97 (1H, d, J=15.60 Hz, H-3); 13C, DEPT and 2D-NMR (100 MHz, Acetone-d6):
δ 13.06, 13.22 (C-13a, C-13b), 20.51, 20.56 (C-14a, C-14b), 24.85, 25.06 (C-8a, C-8b),
25.19, 25.44 (C-2a, C-2b), 26.02, 26.12 (C-15a, C-15b), 31.47, 31.72 (C-11a, C-11b), 35.11,
35.36 (C-9a, C-9b), 36.91, 37.05 (C-3a, C-3b), 37.42, 37.98 (C-10a, C-10b), 44.92, 45.34 (C-
7a, C-7b), 46.87 (C-2b’), 53.12, 53.44 (C-1a, C-1b), 66.29, 66.93, 68.40 (C-1a’, C-1b’, C-
2a’), 81.11, 81.43 (C-6a, C-6b, q), 87.99, 88.32 (C-5a, C-5b), 101.84, 102.33 (C-12a, C-12b),
104.09, 104.26 (C-4a, C-4b, q), 111.66 (C-4’), 113.63 (C-1’, q), 114.67 (C-2”), 117.07 (C-2),
119.38 (C-4”), 121.28 (C-5’), 121.41 (C-6”), 122.15 (C-6’), 123.59 (C-7’), 127.30 (C-3’, q),
130.44 (C-5”), 135.82 (C-2’), 138.10 (C-8’, q), 138.99 (C-3), 141.60 (C-1”, q), 160.19 (C-3”,
q), 189.21 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for C51H66NO2, 884.4579; found,

ACCEPTED MANUSCRIPT
884.4588. ESI-MS(positive): m/z 884 [M+H]+, 906 [M+Na]+, 922 [M+K]+, molecular
formula C51H65NO2.
1-[4”-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxy-3”-methylphenyl]-3-[4’-{1ba’-(12bβ-
dihydroxyartemisinoxy)}-ethoxyphenyl]-2-propen-1-one (22): Elution of the column with
n-hexane-ethyl acetate (70:30) yielded a viscous compound, 75% (w/w); IR ν max: 2927,
2876, 1655 (chalcone), 1255, 1131 (ether), 1598, 1508, 1454, 1030, 985 (aromatics) cm-1; 1H,
COSY- NMR (300 MHz, Acetone-d6): δ 0.76 (12H, d, J =7.2 Hz, H3-13a, H3-13b , H3-14a,
H3-14b), 1.18 (6H, s, H3-15a, H-15b), 2.14 (3H, s, CH3), 2.35 (2H, m, H-11a, H-11b), 3.65
(2H, m, Ha-1a’, Ha-1b’), 4.01 (2H, m, Hb-1a’, Hb-1b’), 4.15 (4H, m, H2-2a’, H2-2b’), 4.66
(2H, d, J=3.3 Hz, αH-12a, αH-12b), 5.30 (1H, s, H-5a, H-5b), 6.90 (1H, d, J=7.8 Hz, H-5”),
6.95 (2H, d, J=8.7 Hz, H-3’, H-5’), 7.61 (3H, m, H-2, H-3, H-2”), 7.63 (1H, d, J=8.40 Hz, H-
13
6”), 7.88 (2H, m, H-2’, H-6’); C, DEPT- NMR (75 MHz, Acetone-d6): δ 12.77 (C-13a, C-
13b), 15.97 (CH3), 20.11 (C-14a, C-14b), 24.62 (C-8a, C-8b), 25.05 (C-2a, C-2b), 25.71 (C-
15a, C-15b), 31.29 (C-11a, C-11b), 34.96 (C-9a, C-9b), 36.65 (C-3a, C-3b), 37.58 (C-10a, C-
10b), 44. 89 (C-7a, C-7b), 52.98 (C-1a, C-1b), 66.53, 66.88 (C-1a’, C-1b’), 67.95, 68.07 (C-
2a’C-2b’), 81.00 (C-6a, C-6b, q), 87.92 (C-5a, C-5b), 101.99, 102.19 (C-12a, C-12b), 103.88
(C-4a, C-4b, q), 111.04 (C-5”), 115.43 ( C-3’, C-5’), 120.06 (C-2), 126.93 (C-3”, q), 128.50
(C-1’, C-1”, q), 128.87 (C-6”), 130.66 (C-2’, C-6’), 131.37 (C-2”), 143.18 (C-3), 161.33 (C-
4’, C-4”, q), 187.23 (C-1, q); HR-MS (m/z) [M+H]+ (ESI+) calcd for C50H67O13, 875.4576;
found, 875.4578. ESI-MS(positive): m/z 875 [M+H]+, 897 [M+Na]+, 913 [M+K]+, molecular
formula C50H66O13.
1-[4”-{1a’-(12aβ-dihydroxyartemisinoxy)-ethoxyphenyl]-3-[4’-{1b’-(12bβ-
dihydroxyartemisinoxy)}-ethoxyphenyl]-2-propen-1-one (23): Elution of the column with
n-hexane– ethyl acetate (70:30) yielded a viscous compound, 75% (w/w); IR ν max: 1657
1
(chalcone), 1258, 1169, 1107 (ether), 1599, 1509, 1029, 985 (aromatics) cm-1; H COSY-,
NMR (300 MHz, Acetone-d6): δ 0.80 (12H, d, J =7.5 Hz, H3-13a, H3-13b, H3-14a, H3-14b),
1.27 (6H, s, H3-15a, H3-15b), 2.48 (2H, m, H-11a, H-11b), 3.72 (2H, m, Ha-1a’, Ha-1b’), 4.20
(2H, m, Hb-1a’, Hb-1b’), 4.23 (4H, m, H2-2a’, H2-2b’), 4.71 (2H, d, J=3.0 Hz, αH-12a, αH-
12b), 5.35 (2H, s, H-5a, H-5b), 6.95 (2H, d, J=8.7 Hz, H-3’, H-5’), 7.00 (2H, d, J= 9.0 Hz, H-
3”, H-5”), 7.69 (3H, m, H-2, H-2’, H-6’), 7.88 (2H, m, H-2, H-3), 8.05 (2H, d, J=9.0 Hz, H-
13
2”, H-6”); C, DEPT- NMR (75 MHz, Acetone-d6): δ 12.81 (C-13a, C-13b), 20.20 (C-14a,
C-14b), 24.65 (C-8a, C-8b), 25.06 (C-2a, C-2b), 25.75 (C-15a, C-15b), 31.29 (C-11a, C-11b),
34.96 (C-9a, C-9b), 35.87 (C-10a, C-10b), 36.67 (C-3a, C3b), 44.90 (C-7a, C-7b), 53.00 (C-

ACCEPTED MANUSCRIPT
1a, C-1b), 66.54 (C-1a’, C-1b’), 67.97 (C-2a’), 68.07 (C-2a’), 81.03 (C-6a, C-6b, q), 87.93
(C-5a, C-5b), 102.00 (C-12a, 12b), 103.93 (C-4a, C-4b, q), 114.86 (C-3’, C-5’), 115.46 (C-
3”, C-5”), 119.92 (C-2), 128.43 (C-1’,q), 130.76 (C-2’, C-6’), 131.08 (C-2”, C-6”), 131.79
(C-1”), 143.54 (C-3), 161.44 (C-4’, q), 163.23 (C-4”, q), 187.72 (C-1, q); HR-MS (m/z) [M +
H]+ (ESI+) calcd for C49H65O13, 861.4419; found, 861.4423. ESI-MS(positive): m/z 861
[M+H]+, 899 [M+K]+, molecular formula C49H64O13.
3-[4’-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxy-3’, 5’-dimethoxyphenyl]-1-[4”-{1ba’-
(12bβ-dihydroxyartemisinoxy)}-ethoxyphenyl]-2-propen-1-one (24): Elution of the
column with n-hexane-ethyl acetate (70:30) yielded a colorless solid, 75% (w/w); mp. 68 °C;
IR ν max (neat): 1655 (chalcone), 1253, 1160, 1109 (ether), 1591, 1023, 982 (aromatics) cm-1;
1H, COSY- NMR (300 MHz, Acetone-d6): δ 0.88 (12H, m, H3-13a, H3-13b, H3-14a, H3-14b),
1.16 (6H, s, H3-15a, H3-15b), 2.48 (2H, m, H-11a, H-11b), 3.75 (2H, m, Ha-1a’, Ha-1b’), 3.91
(6H, brs, 2xOCH3), 4.12 (2H, m, Hb-1a’, Hb-1b’), 4.32 (2H, m, H2-2a’), 4.33 (2H, m, H2-2b’),
4.72, 4.81 (1H each, d, J=3.0 Hz, αH-12a, αH-12b), 5.33 (2H, s, H-5a, H-5b), 7.08 (2H, d,
J=8.7 Hz, H-3”, H-5”), 7.16 (2H, brs, H-2’, H-6’), 7.70 (1H, d, J=15.3 Hz, H-2), 7.79 (1H, d,
J=15.3 Hz, H-3), 8.12 (2H, d, J=8.7 Hz, H-2”, H-6”); 13C, DEPT- NMR (75 MHz, Acetone-
d6): δ 12.77 (C-13a, C-13b), 20.13 (C-14a), 20.28 (C-14b), 24.43 (C-8a), 24.64 (C-8b), 25.07
(C-2a, C-2b), 25.74 (C-15a, C-15b), 31.31 (C-11a, C-11b), 34.96 (C-9a), 35.09 (C-9b), 36.66
(C-3a), 36.71(C-3b), 37.42 (C-10a), 37.59 (C-10b), 44.90 (C-7a), 45.02 (C-7b), 53.09 (C-1a,
C-1b), 56.18 (2xOCH3), 66.48, 68.05 (C-1a’, C-1b’), 68.58, 72.56 (C-2a’, C-2b’), 80.98,
81.05 (C-6a, C-6b), 87.90 (C-5a, C-5b), 102.01, 102.23 (C-12a , C-12b), 103.78 , 103.86 (C-
4a, C-4b, q), 106.64 (C-2’, C-6’), 114.84 (C-3”, C-5”), 121.34 (C-2), 130.97, 131.73 (C-1’,
C-1”, q), 131.08 (C-2”, C-6”), 144.05 (C-3), 154.10 (C-3’, C-4’ C-5’, q), 163.27 (C-4”, q),
187.60 (C-1, q); HR-MS (m/z) [M + H]+ (ESI+) calcd for C51H69O15, 921.4630; found,
921.4630. ESI-MS(positive): m/z 921 [M+H]+, 943 [M+Na]+, 959 [M+K]+, molecular
formula C51H68O15.
7-{1a’-(12aβ-Dihydroxyartemisinoxy)}-ethoxy-4’-{2a’-(12bβ-dihydroxyartemisinoxy)]-
ethoxyflavone (25):
Elution of the column with n-hexane-ethyl acetate (70:30) yielded a
max
colorless solid, 55% (w/w), mp. 64 °C; IR ν
: 1457, 1368, 1029, 985 (aromatics), 1684
1
(αβ unsaturated CO) cm-1; H, COSY- NMR (400 MHz, Acetone-d6): δ 0.90 (12H, m, H3-
13a, H3-13b, H3-14a, H3-14b), 1.31 (6H, s, H3-15a, H3-15b), 2.52 (2H, m, H-11a, H-11b),
3.79 (2H, m, Ha-1a’, Ha-1b’), 4.12 (2H, m, Hb-1a’, Hb-1b’), 4.32 (4H, m, H2-2a’, H2-2b’),
4.79 (2H, d, J=2.92 Hz, αH-12a, αH-12b), 5.42, 5.44 (1H each, s, H-5a, H-5b), 6.51 (1H, d,

ACCEPTED MANUSCRIPT
J= 2.4 Hz, H-8), 6.58 (1H, m, H-6), 7.06 (2H, d, J=8.68, H-3’, H-5’), 7.84 (2H, d, 8.72 Hz,
13
H-2’, H-6’), 7.86 (1H, s, H-3), 8.20 (1H, d, J=8.96, H-5); C, DEPT- NMR (100 MHz,
Acetone-d6): δ 12.36, 12.37 (C-13a, C-13b), 19.47, 19.74 (C-14a, C-14b), 24.23 (C-8a, C-
8b), 24.66 (C-2a, C-2b), 25.30 (C-15a, C-15b), 31.72 (C-11a, C-11b), 34.56 (C-9a, C-9b),
37.19 (C-10a, C-10b), 36.25 (C-3a, C3b), 44.33 (C-7a, C-7b), 52.62 (C-1a, C-1b), 66.82,
66.93 (C-1a’, C-1b’), 68.42, 68.57 (C-2a’, C-2b’), 81.44 (C-6a, C-6b, q), 88.34 (C-5a, C-5b),
102.40, 102.49 (C-12a, 12b), 104.31 (C-4a, C-4b, q), 115.47, 115.94 (C-3’, C-5’), 118.97 (C-
6), 128.57 (C-5), 128.80 (C-4a*, q), 131.64, 132.87 (C-2’, C-6’), 132.36 (C-1’, q), 145.09 (C-
3), 162.29 (C-4’), 164.37 (C-8a, q), 166.50 (C-7, q), 167.53 (C-2, q), 192.95 (C-4, q); HR-
MS (m/z) [M+H]+ (ESI+) calcd for C49H63O14, 875.4212; found, 875.4213. ESI-MS(positive):
m/z 875 [M+H]+ , molecular formula C49H62O14.
3.3. In vitro cytotoxic studies of artemisinin derivatives
3.3.1. Cell culture, growth conditions and treatment
Human leukemia cell line HL-60, pancreatic cancer cell line Mia PaCa-2, prostate cancer cell
line PC-3, hepatic cancer cell line HEP-G2 and colon cancer cell line LS180 and VERO cells
were obtained from ECACC, England. Cells were grown in RPMI-
1640/DMEM/MEM/McCoy’s medium containing 10% FCS and 1X antibiotic antimycotic
solution from invitrogen. Cells were grown in CO2 incubator (Thermocon Electron
Corporation, USA) at 370C with 95% humidity and 5% CO2 gas environment. Test
compounds used in cells treatment were dissolved in DMSO while the untreated cultures
received only the vehicle (DMSO, <0.2%, v/v).
3.3.2. Chemicals
Propidium iodide, Dihydrorhodamine 123 (DHR123), DNase-free RNase, 3-(4,5,-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, ethanol
were purchased from M/s Sigma chemicals Co., India. Fetal bovine serum was obtained from
M/s GIBCO Invitrogen Corporation, USA.
3.3.3. MTT Cell proliferation Assay
In vitro cytotoxic effects of test compounds were determined by MTT dye, which is
converted into formazan crystals by mitochondria of healthy cells. Cells (1.5×104/200µl
media for suspension cell line and 70-75% confluent for adherent cell lines) were cultured in
96-well culture plates and treated with various concentrations of compounds for 48 h. MTT
dye was added 3 h prior to experiment termination. The MTT formazan crystals formed were
dissolved in 150 µL of DMSO and OD measured at 570 nm (reference wave length 620 nm).

ACCEPTED MANUSCRIPT
% growth inhibition was calculated by comparing the absorbance of treated verses untreated
cells. Selectivity index (SI) of each compound with different cell lines was calculated as
follow, SI= IC50 of VERO cell/ IC50 of compound on different tested cell line. Doxorubicin
was used as a reference drug.
3.3.4. Cell Cycle Analysis
HL-60 cells (0.5×106cells/ml/12well plate) were treated with compounds dihydroarteminsin
(DHA), 8 and 15 at 0.2, 0.5, 1 and 5 µM concentrations for 24 h time period. Cells were
collected at 400×g for 5 min in 5 ml polystyrene tubes. Cell pellets were washed twice with
PBS and fixed in 70% ethanol at 4◦C overnight. Cells were collected next day, washed with
PBS and resuspended in 250 µl of PBS. Cells were incubated with RNaseA (200 µg/ml) at 37
◦C for 1.30 h and then after stained with PI (10 µg/ml) for 20 min. The cells were acquired
for PI-DNA fluorescence by flow cytometer and data were collected in list mode on 10,000
events for FL2-A vs. FL2-W. Apoptotic population were analysed by ModFit software from
Verity software house.
3.3.5. Mitochondrial Potential Loss
Mitochondrial membrane potential (Ψm) was measured by using dihydrorhodamine 123
(Rh123) dye which is used to monitor membrane potential of mitochondria. Cells
(0.5×106cells/ml/12well plate) were treated with dihydroarteminsin (DHA), 8 and 15 at 0.2,
0.5, 1 and 5 µM for 24 h. Cells were stained with the dye (200 nM concentration) 30 min
prior to experiment termination. Cells were collected at 400×g, washed with PBS and
analyzed on FL-1 channel of flow cytometer (BD FACS Calibur). The decrease in FL-1
fluorescence indicates the loss of mitochondrial membrane potential in cells.
3.3.6. Hoechst Staining and Phase contrast Microscopy
HL-60 cells (0.5×106cells/ml/12well plate) were seeded in culture plates and treated with
compounds dihydroarteminsin (DHA), 8 and 15 at 0.2, 0.5, 1 and 5 µM concentrations for 24
h. Cells were collected at 400×g, washed with PBS twice and fixed in 1 ml of fixing solution
(cold acetic acid: methanol (1:3), v/v) for 1 h on ice. Cells were centrifuged again at 400 ×g
and resuspended in 100 µl of fixing solution. Cells were spreaded on a clean cold slide and
dried overnight at room temperature. Next day, hoechst 33258 (5 µg/ml in 0.01 M citric acid
and 0.45 M disodium phosphate containing 0.05% Tween 20) were poured on the slides for
30 min. After incubation, slides were washed with PBS twice and while wet, 30 µl of
mounting fluid containing glycerol: PBS at equal ratio were poured over the slides. The slides
was covered with glass cover slip and sealed with nail polish. Photographs were taken under

ACCEPTED MANUSCRIPT
microscope at 20x by using Olympus IX70 microscope. For phase contrast microscopy,
treated and control cells were photographed under microscope after treatment.
Author information
Corresponding Author
Tel.: +91 522 2718622 (O), +91 522 2359628; fax: +91522 2342666.
E-mail address: bhakunirs2000@gmail.com, rs.bhakuni@cimap.res.in, fmalik@iiim.ac.in
Acknowledgements
We wish to thank Director CIMAP, Lucknow for providing the necessary facilities and
constant encouragement to carry out this study. The work was carried out under a major
laboratory project (MLP-02) on “Exploration of bioactive molecules from Natural sources
and value addition through semi-synthetic approach”.
References
[1] D. L. Klayman. Qinghaosu (artemisinin): an antimalarial drug from China. Science.
228 (1985) 1049-1055.
[2] (a) T. Efferth. Mechanistic perspectives for 1,2,4-trioxanes in anti-cancer therapy.
Drug Res. Update. 8 (2005) 85-97. (b) R. Gaur, H. S. Cheema, Y. Kumar, S. P. Singh,
D. K. Yadav, M. P. Darokar, F. Khan, R. S. Bhakuni. In vitro antimalarial activity and
molecular modeling studies of novel artemisinin derivatives. RSC Adv., 5 (2015)
47959.
[3] (a). G. H. Posner, I. H. Paik, S. Sur, A. J. McRiner, K. Borstnik, S. Xie, T. A. Shapiro.
Orally active, antimalarial, anticancer, artemisinin derived trioxane dimers with high
stability and efficacy. J. Med. Chem. 46 (2003) 1060-1065. (b). Y. Li, F. Shan, J. M.
Wu, G. S. Wu, J. Ding, D. Xiao, W. Y. Yang, G. Atassi, S. Leonce, D. H. Caignard,
P. Renard. Synthesis and cytotoxicity of dihydroartemisinin ethers containing
cyanoarylmethyl group. Bioorg. Med. Chem. 11 (2003) 977-984.
[4] T. Efferth, M. Davey, A. Olrich, G. Rucker, E. Gedhart, R. Davey. Activity of drugs
from traditional Chinese medicine toward sensitive and MDR1- or MRP1-

ACCEPTED MANUSCRIPT
overexpressing multidrug-resistant human CCRF-CEM leukemia cells. Blood Cells
Mol. Dis. 28 (2002) 160.
[5] J. C. Moore, H. Lai, J. R. Li, R. L. Ren, J. A. McDougall, N. P. Singh, C. K. Chou.
Oral administration of dihydroartemisinin and ferrous sulfate retarded implanted
fibrosarcoma growth in the rat. Cancer Lett. 98 (1995) 83-87.
[6] P. Y. Lin, J. Q. Pan, Z. M. Feng, D. Zhang, L. Y. Xiao. in Progress in Immuno-
pharmacology, ed. J. H. Zhou, X. Y. Li and K. T. Rong, Chinese Science and
Technique. 3 (1993) 25.
[7] Jung, Lee S, Ham J, Lee K, Kim H, Kim SK. Antitumor activity of novel
deoxoartemisinin monomers, dimers, and trimer. J. Med. Chem. 2003;46:987–994.
[8] M. T. Efferth, Dunstan H, Sauerbrey A, Miyachi H, Chitambar CR. The anti-malarial
artesunate is also active against cancer. Int. J. Oncol. 2001;18:767.
[9] Y. Liu, V. K. Wong, B. C. Ko, M. K. Wong, C. M. Che. Synthesis and Cytotoxicity
Studies of Artemisinin Derivatives Containing Lipophilic Alkyl Carbon Chains. Org.
Lett. 2005; 7:1561
[10] R. Dell’Eva, U. Pfeffer, R. Vene, L. Anfosso, A. Forlani, A. Albini, T. Efferth.
Inhibition of angiogenesis in vivo and growth of Kaposi's sarcoma xenograft tumors
by the anti-malarial artesunate. Biochem Pharmacology. 68 (2004) 2359-2366.
[11] C. Muller, V. Diehl, F. W. Lichtenthaler. Building blocks from sugars. Part 23.
Hydrophilic 3-pyridinols from fructose and isomaltulose. Tetrahedron.
1998;54:10703-10712.
[12] L. Sun, F. Shah, M. A. Helal, Y. Wu, Y. Pedduri, A. G. Chittiboyina, J. Gut, P. J.
Rosenthal, M. A. Avery. Design, synthesis, and development of novel guaianolide-
endoperoxides as potential antimalarial agents. J. Med. Chem. 53 (2010) 7864-7868.
[13] C. P. Hencken, L. Jones-Brando, C. Bordon, R. Stohler, B. T. Mott, R. Yolken, G.
H. Posner, L. E. Woodard. Thiazole, Oxadiazole, and Carboxamide Derivatives of
Artemisinin are Highly Selective and Potent Inhibitors of Toxoplasma gondii. J. Med.
Chem. 53 (2010) 3594-3601.
[14] B. Pacorel, S. C. Leung, A. V. Stachulski, J. Davies, L. Vivas, H. Lander, S. A.
Ward, M. Kaiser, R. Brun, P. M. O’Neill. Modular Synthesis and in Vitro and in Vivo
Antimalarial Assessment of C-10 Pyrrole Mannich Base Derivatives of Artemisinin.
J. Med. Chem. 53 (2010) 633-640.
[15] S. Ekthawatchai, S. Kamchonwongpaisan, P. Kongsaeree, B. Tarnchompoo, Y.
Thebtaranonth, Y. Yuthavong. C-16 Artemisinin Derivatives and Their Antimalarial

ACCEPTED MANUSCRIPT
and Cytotoxic Activities:ꢁ Syntheses of Artemisinin Monomers, Dimers, Trimers, and
Tetramers by Nucleophilic Additions to Artemisitene. J. Med. Chem. 44 (2001) 4688-
4695.
[16]
[17] C. Horwedel, S. B. Tsogoeva, S. Wei, T. Efferth. Cytotoxicity of artesunic acid
homo- and heterodimer molecules toward sensitive and multidrug-resistant CCRF-
CEM leukemia cells. J. Med. Chem. 53 (2010) 4842-4848.
[18] I. H. Paik, S. Xie, T. A. Shapiro, T. Labonte, A. A. N. Sarjeant, A. C. Baege, G. H.
Posner. Second generation, orally active, antimalarial, artemisinin-derived trioxane
dimers with high stability, efficacy, and anticancer activity. J. Med. Chem. 49 (2006)
2731.
[19] S. Cho, S. Oh, Y. Um, J. H. Jung, J. Ham, W. S. Shin, S. Lee. Synthesis of 10-
substituted triazolyl artemisinins possessing anticancer activity via Huisgen 1,3-
dipolar cylcoaddition. Bioorg. Med. Chem. Lett. 19 (2009) 382.
[20] M. Jung. Synthesis and cytotoxicity of novel artemisinin analogs. Bioorg. Med.
Chem. Lett. 7 (1997) 1091.
[21] (a) Lombard MC, N’Da DD, Breytenbach JC, Smith PJ, Lategan CA. Artemisinin-
quinoline hybrid-dimers: synthesis and in vitro antiplasmodial activity. Bioorg. Med.
Chem. Lett. 20 (2010) 6975-6977. (b) C. H. Lee, H. D. Hong, J. Shin, M. K. Jung, I.
Shin, J. Yoon, W. Lee. NMR studies on novel antitumor drug candidates,
deoxoartemisinin and carboxypropyldeoxoartemisinin. Biochem. Biophys. Res.
Comm. 274 (2000) 359-369. (c) H. J. Woerdenbag, T. A. Moskal, N. Pras, T. M.
Maringle, F. S. ElFeraly, H. H. Kampinga, A. W. T. Konings. Cytotoxicity of
Artemisinin-Related Endoperoxides to Ehrlich Ascites Tumor Cells. J. Nat. Prod. 56
(1993) 849-856. (d) A. J. L. Q. Lin, S. L. Anderson, O. L. Klayman. Antimalarial
activity of new dihydroartemisinin derivatives. 5. Sugar analogs. J. Med. Chem. 35
(1992) 1639-1642.
[22]
H. H. Chen, H. J. Zhou, X. Fang. Inhibition of human cancer cell line growth and
human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro.
Pharmacol. Res. 48 (2003) 231-236.
[23] G. H. Posner, P. Ploypradith, W. Hapangama, D. Wang, J. N. Cumming, P. Dolan,
T. W. Kensler, D. Klinedinst, T. A. Shapiro, Q. Y. Zheng, C. K. Murray, L. G.
Pilkington, L. R. Jayasinghe, J. F. Bray, R. Daughenbaugh. Trioxane dimers have

ACCEPTED MANUSCRIPT
potent antimalarial, antiproliferative and antitumor activities in vitro. Bioorg. Med.
Chem. 5 (1997) 1257-1265.
[24] A. C. Beekman, A. R. W. Barentsen, H. J. Wperdenbag, W. V. Uden, N. Pras, A.
W. Konings, F. S. El-Feraly, A. M. Galal, H. V. Wikstrom. Stereochemistry-
dependent cytotoxicity of some artemisinin derivatives. J. Nat. Prod. 60 (1997) 325-
330.
[25] G. H. Posner, P. Ploypradith, M. H. Parker, H. O’Dowd, S. H. Woo, J. Northrop,
M. Krasavin, P. Dolan, T. W. Kensler, S. Xie, T. A. Shapiro. Antimalarial,
antiproliferative, and antitumor activities of artemisinin-derived, chemically robust,
trioxane dimmers. J. Med. Chem. 42 (1999) 4275-4280.
[26] S. Oh, I. H. Jeong, W. S. Shin, S. Lee. Growth inhibition activity of thioacetal
artemisinin derivatives against human umbilical vein endothelial cells. Bioorg. Med.
Chem. Lett. 13 (2003) 3665.
[27] S. Oh, I. H. Jeong, C. M. Ahn, W. S. Shin, S. Lee. Synthesis and antiangiogenic
activity of exo-olefinated deoxoartemisinin derivatives. Bioorg. Med. Chem. 12
(2004) 3783-3786.
[28] Y. C. Liu, C. W. Hsieh, C. C. Wu, B. S. Wung. Chalcone inhibits the activation of
NF-kappaB and STAT3 in endothelial cells via endogenous electrophile. Life Sci. 80
(2007) 1420-1430.
[29] (a) S. A. Aherne, N. M. O’Brien. Dietary flavonols: chemistry, food content, and
metabolism. Nutrition. 18 (2002) 75-81. (b) R. Gaur, D. K. Yadav, S. M. P. Kumar,
Darokar, F. Khan, R. S. Bhakuni. Molecular modelling based synthesis and evaluation
of in vitro anticancer activity of indolyl chalcones. Curr. Top. in Med. Chem. 15
(2015) 1003-1012. (c) A. Modzelewska, C. Pettit, G. Achanta, N. E..Davidson, P.
Huang, S. R. Khan. Anticancer activities of novel chalcone and bischalcone
derivatives. Bioorg. Med. Chem., 14 (2006) 3491-5. (d) R.J. Anto, K. Sukumaran, G.
Kuttana, M.N.A. Rao, V. Subbaraju, R. Kuttana. Anticancer and antioxidant activity
of synthetic chalcones and related compounds. Cancer Lett. 97 (1995) 33-7. (e) Y.L.
Hsu, P.L. Kuo, W.S. Tzeng, C.C. Lin. Chalcone inhibits the proliferation of human
breast cancer cell by blocking cell cycle progression and inducing apoptosis. Food
Chem. Toxicol. 44 (2006) 704-13. (f) N.J. Lawrence, R.P. Patterson, L.L. Ooi, D.
Cook, S.Ducki. Effects of alpha-substitutions on structure and biological activity of
anticancer chalcones. Bioorg. Med. Chem. Lett. 16 (2006) 5844-8.

ACCEPTED MANUSCRIPT
[30] Z. Nowakowska. A review of anti-infective and anti-inflammatory chalcones. Eur.
J. Med. Chem. 42 (2007) 125-137.
[31] R. Borges-Argáez, L. Balnbury, A. Flowers, A. Giménez-Turba, G. Ruiz, P. G.
Waterman, L. M. Peña-Rodríguez. Cytotoxic and antiprotozoal activity of flavonoids
from Lonchocarpusspp. Phytomedicine. 14 (2007) 530
[32] A. A. Alagbala, A. J. McRiner, K. Borstnik, T. Labonte, W. Chang, J. G. D-
Angelo, G. H. Posner, B. A. Foster. Biological mechanism of action of Novel C-10
non-acetal trioxane dimers in prostate cancer cell lines. J. Med. Chem. 49 (2006)
7836-7842.
[33] K. C. Gatter, G. Brown, I. S. Townbridge, R. E. Woolston, D. Y. Mason.
Transferrin receptors in human tissues; their distribution and possible clinical
relevance. J. Clin. Pathol. 36 (1983) 539–545.
[34] H. J. Zhou, Z. Wang, A. Li. Dihydroartemisinin induces apoptosis in human
leukemia cells HL60 via downregulation of transferring receptor expression. Anti-
Cancer Drugs. 19 (2008) 247-255.
[35] H. Lai, T. Sasaki, N. P. Singh. Targeted treatment of cancer with artemisinin and
artemisinin-tagged iron-carrying compounds. Expert Opin. Ther. Targets. 9 (2005)
995-1007.
[36]
G. H. Posner, J. D’Angelo, P. M. O’Neill, A. Mercer. Anticancer activity of
artemisinin-derived trioxanes. Expert Opin. Ther. Patents. 16 (2006) 1665-1672.
[37] M. Jung, K. Lee, H. Kim, M. Park. Recent advances in artemisinin and its
derivatives as antimalarial and antitumor agents. Curr. Med. Chem. 11 (2004) 1265–
1284.
[38] L. L. Song, J. W. Kosmeder, S. K. Lee, C. Gerhäuser, D. Lantvit, R. C. Moon, R.
B. Moriarty, J. M. Pezzuto. Cancer chemopreventive activity mediated by 4'-
bromoflavone, a potent inducer of phase II detoxification enzymes. Cancer Res. 59
(1999) 578.
[39] L. Xie, Y. Zhao, X. Zhai, P. Li, C. Liu, Y. Li, P. Gong. The Application of Tandem
Aza-Wittig Reaction to Synthesize Artemisinin–Guanidine Hybrids and Their Anti-
Tumor Activity Arch. Pharm. Chem. Life Sci. 344 (2011) 631-638
[40] A. C. Beekman, H. J. Woerdenbag, H. H. Kampigna, A. W. T. Konings.
Cytotoxicity of artemisinin, a dimer of dihydroartemisinin, artemisitene and
eupatoriopicrin as evaluated by the MTT and clonogenic assay. Phytother. Res. 10
(1996) 140-144.

ACCEPTED MANUSCRIPT
[41] (a) G. H. Posner, A. J. McRiner, I. H. Paik, S. Sur, K. Borstnik, S. J. Xie, T. A.
Shapiro, A. Alagbala, B. Foster. Anticancer and Antimalarial Efficacy and Safety of
Artemisinin-Derived Trioxane Dimers in Rodents. J. Med. Chem. 47 (2004) 1299-
1301. (b) S. Patel, R. Gaur, P. Verma, A. Mathur, R. S. Bhakuni. Biotech. Lett. 32
(2010) 1167-1171. (c) S. Patel, R. Gaur, M. Upadhyaya, A. Mathur, A. K. Mathur, R.
S. Bhakuni. J. Nat.Med. 65 (2011) 646-650. (d) R. Gaur, S. Patel, R. K. Verma, A.
Mathur, R. S. Bhakuni. Med. Chem. Res. 23 (2014) 1202-2120. (e) R. Gaur, M. P.
Darokar, P. V. Ajayakumar, R. S. Shukla, R. S. Bhakuni. In vitro antimalarial studies
of novel artemisinin biotransformed products and its derivatives. Phytochem. 107
(2014) 135-140
[42] H. Lai, N. P. Singh. Selective cancer cell cytotoxicity from exposure to
dihydroartemisinin and holotransferrin. Cancer Lett. 91(1) (1999) 41-6.
[43] Z. S. Yang, W. L. Zhou, Y. Sui, J. X. Wang, J. M. Wu, Y. Zhou, Y. Zhang, P. L.
He, J. Y. Han, W. Tang, Y. Li, J. P. Zuo. Synthesis and immunosuppressive activity
of
new
artemisinin
derivatives.
1.
[12(beta
or
alpha)-
Dihydroartemisininoxy]phen(ox)yl aliphatic acids and esters. J. Med. Chem. 48
(2005) 4608-4617.
[44] S. M. Kupehan, M. A. Eakin, A. M. Thomas. Tumor inhibitors. 69. Structure-
cytotoxicity relationships among the sesquiterpene lactones. J. Med. Chem. 14 (1971)
1147.
[45] A. C. Beekman, A. R. W. Barentsen, H. J. Woerdenbag. Artemisinin-derived
sesquiterpene lactones as potential antitumour compounds: cytotoxic action against
bone marrow and tumour cells. Planta Medica. 64(7) (1998) 615-619.
[46] G. H. Posner, D. Wang, J. N. Cumming, C. H. Oh, A. N. French, A. L. Bodley, T.
A. Shapiro. Further Evidence Supporting the Importance of and the Restrictions on a
Carbon-Centered Radical for High Antimalarial Activity of 1,2,4-Trioxanes Like
Artemisinin. J. Med. Chem. 38 (1995) 2273-2275.
[47] J. N. Cumming, D. Wang, S. B. Park, T. A. Shapiro, G. H. Posner. Design,
synthesis, derivatization, and structure-activity relationships of simplified, tricyclic,
1,2,4-trioxane alcohol analogues of the antimalarial artemisinin. J. Med.Chem. 41
(1998) 952-964.
[48] G. H. Posner, J. P. Maxwell, H. O'Dowd, M. Krasavin, S. Xie, T. A. Shapiro.
Antimalarial sulfide, sulfone, and sulfonamide trioxanes. Bioorg. Med. Chem. 8
(2000) 1361-1370.