Synthesis of Artemisinin-Derived Dimers, Trimers and Dendrimers: Investigation of Their Antimalarial and Antiviral Activities Including Putative Mechanisms of Action

Article abstract of DOI:10.1002/chem.201800729

Generation of dimers, trimers and dendrimers of bioactive compounds is an approach that has recently been developed for the discovery of new potent drug candidates. Herein, we present the synthesis of new artemisinin-derived dimers and dendrimers and inve

Full text of DOI:10.1002/chem.201800729

A Journal of
Accepted Article
Title: Synthesis of artemisinin-derived di/tri-mers and dendrimers,
investigation of their activity against P. falciparum and
cytomegalovirus, and insights into their mechanisms of action
Authors: Tony Fröhlich, Friedrich Hahn, Lucid BELMUDES, Maria
Leidenberger, Oliver Friedrich, Barbara Kappes, Yohann
Couté, Manfred Marschall, and Svetlana B. Tsogoeva
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201800729
Link to VoR: http://dx.doi.org/10.1002/chem.201800729
Supported by

10.1002/chem.201800729
Chemistry - A European Journal
FULL PAPER
Synthesis of artemisinin-derived di/tri-mers and dendrimers,
investigation of their activity against P. falciparum and
cytomegalovirus, and insights into their mechanisms of action
Tony Fröhlich,‡[a] Friedrich Hahn,‡[b] Lucid Belmudes, [c] Maria Leidenberger,[d] Oliver Friedrich,[d]
Barbara Kappes,[d] Yohann Couté,[c] Manfred Marschall*[b] and Svetlana B. Tsogoeva*[a]
Dedicated to Professor Youyou Tu
Abstract: Generation of dimers, trimers and dendrimers of bioactive Introduction
compounds is one of the most recent approaches for the discovery of
new potent drug candidates. Herein we present the synthesis of new
artemisinin-derived dimers and dendrimers, which were investigated
against malaria parasite Plasmodium falciparum 3D7 strain and
human cytomegalovirus (HCMV). Dimer 7 was, with an EC50 value of
1.4 nM, the most active compound in terms of antimalarial efficacy
and was even more effective than the standard drugs
dihydroartemisinin (EC50 = 2.4 nM), artesunic acid (EC50 = 8.9 nM)
and chloroquine (EC50 = 9.8 nM). Trimer 4 stood out as the most
active agent against HCMV in vitro replication and exerted an EC50
value of 0.026 µM stating an even higher activity than two reference
drugs ganciclovir (EC50 = 2.60 µM) and artesunic acid (EC50 = 5.41
µM). In addition, artemisinin-derived dimer 13 and trimer 15 were for
the first time both immobilized on TOYOPEARL® AF-Amino-650M
beads and used for mass spectrometry-based target identification
experiments using total lysates of HCMV-infected primary human
fibroblasts. Two major groups of novel target candidates, namely
cytoskeletal and mitochondrial proteins were obtained. Two putatively
compound-binding viral proteins, namely major capsid protein (MCP)
and envelope glycoprotein pUL132, which are both essential for
HCMV replication, were successfully identified.
Over the last 20 years, the dimerization of the natural product
artemisinin (1) (Figure 1) has emerged as a powerful tool for the
design of new lead compounds for the field of medicinal
chemistry.[1] Just like the hybridization of bioactive natural
products, which was applied by our group in past projects with
promising results,[2] this strategy reduces synthesis efforts to a
minimum and therefore, is relatively time and cost saving. In
addition, the dimerization concept has the potential to improve
pharmacological properties of the monomeric compounds,
possibly including their biological activity, reduced toxicity, higher
bioavailability, increased metabolic stability and might even
counteract the problem of drug resistance.[1g,1h,3] Artesmisinin (1)
is an enantiomerically pure sesquiterpene containing
a
1,2,4-trioxane ring and was extracted for the first time in 1972
from the Chinese medicinal plant Artemisia annua L. by Youyou
Tu, for which she received the 2015 Nobel Prize in Physiology or
Medicine.[4] Since then, many studies have been conducted on
the pharmacological activity of artemisinin, which revealed that
artemisinin not only exhibits great antimalarial properties, for
which it has been initially used in Traditional Chinese Medicine
(TCM), but it also possesses great pharmacological potential as
an antiviral and anticancer agent just like its semi-synthetic
derivatives,
including
artesunic
acid (2a)
and
dihydroartemisinin (2b) (Figure 1).[5] These two derivatives are
valuable starting materials for artemisinin-based compounds, as
they already contain functional groups (alcohol and carboxylic
acid function) that are useful for chemical transformations.
[a]
[b]
T. Fröhlich, Prof. Dr. S. B. Tsogoeva
Organic Chemistry Chair I and Interdisciplinary Center for Molecular
Materials (ICMM), Friedrich-Alexander University of Erlangen-
Nürnberg, Nikolaus-Fiebiger-Straße, 10, 91058, Germany,
e-mail: svetlana.tsogoeva@fau.de
Recently,
we
applied
artesunic
acid (2a)
and
dihydroartemisinin (2b) for the synthesis of four novel
Dr. F. Hahn, Prof. Dr. M. Marschall
Institute for Clinical and Molecular Virology, Friedrich-Alexander
University of Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen,
Germany, e-mail: manfred.marschall@fau.de
[c]
[d]
L. Belmudes, Dr. Y. Couté
Université Grenoble Alpes, CEA, INSERM, BIG-BGE, 38000
Grenoble, France
M. Leidenberger, Prof. Dr. O. Friedrich, Prof. Dr. B. Kappes
Institute of Medical Biotechnology, Friedrich-Alexander University of
Erlangen-Nürnberg, Paul-Gordon-Straße 3, 91052 Erlangen,
Germany
‡
These authors contributed equally to this work.
Supporting information (SI) for this article can be found under
https://doi.org/...
Figure 1.
Structures
of
artemisinin (1),
artesunic
acid (2a),
dihydroartemisinin (2b) and artemisinin-derived trimer 3.
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10.1002/chem.201800729
Chemistry - A European Journal
FULL PAPER
artemisinin-derived compounds: two dimers and two trimers,
which were screened for their potency against malaria, leukemia
and HCMV.[6] Trimer 3 (Figure 1) turned out to be the most
bioactive compound out of the four and surpassed its monomer
of 12 artemisinin-derived compounds 4-15 (monomers, dimers
and dendrimers) (Figure 2), which were investigated for their
biological activity against malaria parasites (Plasmodium
falciparum 3D7) and human cytomegalovirus (HCMV).
Furthermore, artemisinin-derived acids 13 (dimer) and 15 (trimer)
as well as artesunic acid (monomer) were immobilized on
TOYOPEARL® AF-Amino-650M beads applying two different
techniques, and were subsequently used for the first time in mass
spectrometry-based target identification experiments (target ID)
using total lysates of HCMV-infected primary human fibroblasts.
This should provide a valuable basis for further molecular
investigations of putative target proteins of these
artemisinin-based compounds.
artesunic acid by a factor of 35 in terms of antileukemia (EC50
=
0.002 µM) and by a factor of 95 in terms of anticytomegaloviral
activity (EC50 = .04 µM). This result piqued our interest about the
differences of the underlying mechanisms of action of artesunic
acid and trimer 3. Up until now, the main focus of mechanistic
studies and target identification in connection with
artemisinin-based compounds was set almost entirely on
monomeric compounds.[7] In order to fill this gap and get more
insights into the activities and mechanisms of artemisinin
monomers, dimers and trimers, we herein present the synthesis
H
H
H
H
H
O
O
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
O
O
OH
O
H
O
O
O
O
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
6a: C10, C10'-
O
4
O
5
O
O
6b:
C10, C10'-
O
O
O
H
H
H
H
H
H
H
O
H
O
H
H
O
H
O
O
O
O
O
O
O
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
3
O
O
N
O
O
O
X
X
O
O
O
O
O
NH
O
O
3
O
O
O
O
O
O
3
9
10
7:
O
X = H
8:
X, X = C60
=
H
O
H
H
H
O
O
H
H
O
O
O
O
H
O
H
O
O
O
O
H
H
O
O
O
O
O
O
O
3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
3
O
O
O
O
O
O
O
OH
O
3
11
12
13
O
OH
H
O
H
O
O
O
H
H
H
O
H
O
H
H
O
O
H
O
O
O
O
O
O
O
O
O
O
O
O
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
14
15
H
H
O
O
O
OH
O
Figure 2. Compounds 4-15 applied for biological tests against CCRF-CEM, CEM/ADR5000 cells, HCMV and P. falciparum 3D7.
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1:2) and because it was not possible to separate these two isomers
from each other, the mixture was applied for biological tests.
To be able to perform the intended target ID experiments for
dimeric and trimeric artemisinin structures, dimer 13 and trimer 15
were synthesized. Both compounds contain a free carboxylic acid
function, which is a suitable anchoring group and can be utilized for
Results and Discussion
Chemistry. Artemisinin-derived trimer 4 was prepared in 80% yield
by performing a Steglich-type esterification reaction between
artesunic acid and diol 17 utilizing EDCI and DMAP as coupling
agents (Scheme 1). Precursor 17 was synthesized from olefin 16
according to a published protocol.[1c] Dimer 16 can also be
converted into primary alcohol 18 by borane reduction and in situ
oxidation,[1c] which was used for the preparation of dendrimer 5 or
acid 19. Applying 2.0 equivalents of alcohol 18 and 1.0 equivalent
of succinic anhydride in combination with DCC and DMAP as
coupling agents yielded the desired dendrimer 5 in 47%, whereas
3.0 equivalents of succinic anhydride and 1.0 equivalent of
alcohol 18 together with DMAP as the only catalyst afforded
artemisinin-derived acid 19 in 82% yield. Dimer 6 was obtained in
58% yield by the reaction of two molecules of
dihydroartemisinin (2b) with malonyl chloride. The malonylation
reaction was conducted in CH2Cl2 as solvent and pyridine served
as a base. The desired product was isolated in form of two
diastereomers (6a: C10, 10’-α, α; 6b: C10, 10’-α, β), which were
separated from each other via column chromatography (yield for
6a: 37%; yield for 6b: 21%). The configuration at C10, C10’ was
determined via 1H-NMR spectroscopy in analogy to our previously
published work.[2a,6] In order to investigate the radical scavenging
effect of fullerene on artemisinin dimers, a Bingel-Hirsch reaction
was performed between artesunic acid dimer 7, which contains a
malonyl moiety and fullerene (C60), resulting in the desired
product 8 with a yield of 43% after 3 days. DBU was applied as
base, CBr4 as reagent and toluene as solvent. The starting
compound 7 was obtained in 63% yield by using the same method
as described for dimer 6. Alcohol 20, in turn, was prepared by
Steglich esterification between artesunic acid and ethylene glycol
in 91% yield. The idea behind the two artemisinin monomers 10
and 11 is to improve the water-solubility of artesunic acid and
dihydroartemisinin and to see how it influences its pharmacological
properties. This concept was introduced for the first time in 2013 by
Axelsson and Ek within a patent, where they described the
synthesis of ether 11 and similar dihydroartemisinin derivatives,[8]
but up until now only antimalarial and no antiviral data were
reported. For both compounds 10 and 11, the main building block
was the branched polyethylene glycol ether 21, which contains a
free primary alcohol group that was used to attach the two
artemisinin derivatives. Ether 21 was synthesized according to an
already published procedure (Scheme S2; SI).[8-9] Artesunic acid
was directly coupled to this precursor via a Steglich-type
esterification reaction, and it was possible to obtain the desired
product 10 in 62% yield. Compound 11 was prepared in 51% yield
by an etherification reaction between alcohol 21 and
dihydroartemisinin using phosphotungstic acid as catalyst instead
of BF3 etherate. This procedure was already reported in literature
in the context of synthesis of other dihydroartemisinin ether
derivatives.[10] It has to be noted that ether 11 was isolated as a
mixture of C10-α- and C10-β-diastereomers (ratio C10-α/C10-β =
immobilization onto
a solid support. Dimer 12, which was
necessary for the synthesis of acid 13, can be obtained via an
esterification reaction between artesunic acid and glycerin. The
outcome of this reaction was controlled over the applied
equivalents of the two reactants: an excess of glycerin (5.0
equivalents) afforded monomer 22 in 71% yield, a large excess of
artesunic acid (6.0 equivalents) yielded dendrimer 14 in 73% and
using 2.1 equivalents of artesunic acid and 1.0 equivalent of
glycerin resulted in reasonable amounts of the desired dimer 12
(54% yield). Protection chemistry was not necessary, as the two
primary alcohols of glycerin are more reactive and less sterically
hindered compared to the secondary alcohol function and therefore,
always reacted first. Dimer 12 was then brought to reaction with
succinic anhydride to gain acid 13 in 68% yield. Trimer 15 was
synthesized in a similar fashion: Dimeric artemisinin-derived
acid 19 was coupled with alcohol 22 yielding trimer 23 in 53%,
which in turn was reacted with succinic anhydride in the presence
of DMAP as catalyst and resulted in the desired trimeric
artemisinin-derived acid 15 in 64% yield.
With compounds 13 and 15 generated, it was now possible to
prepare the specific tools for target ID experiments. For this
purpose, these compounds and artesunic acid (2a) were
immobilized on TOYOPEARL® AF-Amino-650M beads using two
different methods: direct amide coupling of the artemisinin-derived
acids to the beads (Scheme 2) and indirect coupling via an UV-
cleavable linker (Scheme 3). These two methods were chosen and
compared with each other to circumvent the problem of unselective
binding of proteins to the solid matrix, which is a common issue in
target ID. The direct amide coupling is very straight-forward and
involves the overnight reaction of each artemisinin-derived acid
(artesunic acid (2a), dimer 13 and trimer 15) with the
amino-functionalized resin in the presence of DIC as coupling agent,
followed by several washing steps and finally blocking the
remaining amine groups with acetic acid.
In order to confirm that the artemisinin derivatives withstand the
slightly acidic reaction conditions (pH = 4-5), which are necessary
for an efficient ligand coupling to TOYOPEARL® AF-Amino-650M
beads, a test reaction was performed. Artesunic acid (2) was
dissolved in DMF and stirred overnight in the presence of DCC at
pH 4-5, which was realized with small amounts of hydrochloric acid
(c = 0.1 M). After following this procedure, it was possible to isolate
active ester 9 in 89% yield, which contained a completely intact
artemisinin moiety and consequently, this method can be regarded
as suitable for artemisinin-based compounds. To our surprise, the
obtained compound 9 turned out to be relatively stable and it was
even possible to store it for 3 days at room temperature without any
visible degradation in the NMR spectra. Therefore, we decided to
test the compound for its biological activity. Only in the presence of
nucleophiles, such as alcohols or amines, it reacted to the
corresponding esters or amides.
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Immobilization via the UV-cleavable linker proved to be more final step was the coupling of artemesinin-derived acids 13 and 15
complicated and included a total of four reaction steps. The first as well as artesunic acid (2a) to the modified beads using the free
step was the attachment of the Fmoc-protected UV-sensitive primary amine function of the UV-cleavable linker applying
linker 25, which was prepared in analogy to an already published standard amide coupling conditions: EDCI, HOBt as coupling
procedure by Holmes and Jones (Scheme S3; SI),[11] to the agents, DIPEA as base and DMF as solvent. In addition to these
amino-activated beads using DIC as coupling agent and DMF as various different artemisinin containing bead constructs, we also
solvent. After blocking the residual free amine groups of the resin performed a direct coupling reaction between only acetic acid and
with acetic acid, the Fmoc-protecting group of the UV-sensitive TOYOPEARL® AF-Amino-650M beads, which served as a control
linker was removed by treatment with 30% piperidine in DMF. The system for the biological experiments (Scheme S4; SI).
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
artesunic acid (2a)
1. BH3 SMe2
THF, 0 °C rt, 3 h
OsO4, NMO
acetone, rt, 24 h
EDCI, DMAP
4
OH
CH2Cl2
2. NaBO3 4H2O
THF/H2O, rt, 18 h
OH
OH
0 °C
rt, o/n
18
16
17
H
H
succinic
anhydride
(DCC,) DMAP
H
H
O
O
H
O
O
O
O
CH2Cl2
O
O
malonyl chloride
pyridine, CH2Cl2
H
O
0 °C
rt, o/n
O
O
5
or
6a/b
°
O
4 A molecular sieves
0 °C, 2 h
O
O
HO
OH
2b
O
H
19
H
O
O
O
H
O
H
O
O
OH
O
O
HO
DCC, DMAP, CH2Cl2
0 °C rt, o/n
malonyl chloride
pyridine, CH2Cl2
O
O
C60, CBr4, DBU
toluene, rt, 3 d
O
O
7
8
°
O
OH
4 A molecular sieves
0 °C, 2 h
OH
O
O
2a
20
HO
artesunic acid (2a)
EDCI, DMAP
dihydroartemisinin(2b)
phosphotungstic acid
O
O
O
O
11
10
3
3
DMF/CH3CN, 0 °C
rt, o/n
O
CH2Cl2/CH3CN/DMF, rt, 2 h
O
3
21
H
O
H
O
O
H
O
H
O
O
glycerin
EDCI, DMAP
succinic anhydride
DMAP, CH2Cl2
O
O
O
O
or
14
or
12
13
O
O
DMF/CH3CN
0 °C
rt, o/n
O
0 °C
rt, o/n
OH
O
OH
O
OH
2a
22
H
H
H
H
H
O
H
O
O
O
O
O
O
O
O
O
H
O
H
O
O
artemisinin-derived
19
EDCI, DMAP
O
O
O
O
acid
succinic anhydride
DMAP, CH2Cl2
O
O
O
O
O
15
O
CH2Cl2/CH3CN
0 °C
rt, 2 d
O
OH
O
O
0 °C
rt, o/n
OH
O
OH
23
O
22
Scheme 1. Synthesis route for artemisinin-based compounds 4-15.
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Art
Art
DIC, pH = 4-5
DMF, rt, o/n
O
O
R
HW-65
R
+
NH2
H2N
O
O
O
O
R
HW-65
R
O
O
H2N
O
O
O
O
N
H
HO
OH
OH
OH
OH
O
O
2a 13 15
, or
TOYOPEARL AF-Amino-650M
artemisinin-derived acids
O
CH3
CH3
OH
CH2
C
C
O
R
O
C
C
CH2
C
C
O
R
O
C
C
DIC, pH = 4-5
DMF, rt, o/n
O
O
Art
HW-65
=
(OH)
(OH)
O
O
O
R
HW-65
R
O
N
H
O
O
O
O
N
H
OH
24a:
OH
O
O
2a
Art = subunit of artesunic acid (
24b: Art = subunit of artemisinin-derived acid 13
24c: 15
)
CH2
CH2
R = Hydroxylated
aliphatic group
Art = subunit of artemisinin-derived acid
CH3
CH3
Scheme 2. Synthesis route for immobilized artemisinin-derived acids (artesunic acid (2a), dimer 13 and trimer 15) on TOYOPEARL® AF-Amino-650M beads without
UV-cleavable-linker (constructs 24a, 24b and 24c).
H
N
O
O
Fmoc
O
R
O
R
O
O2N
O
DIC, pH = 4-5
DMF, rt, o/n
HW-65
+
R
HW-65
R
O
O
N
O
O
O
NH2
H
HO
O
OH
O
OH
O
TOYOPEARL AF-Amino-650M
25
O2N
Fmoc
N
O
H
O
O
OH
NH
NH
OH
DIC, pH = 4-5
DMF, rt, o/n
OH
O
R
O
R
O
O
O
O
Piperidine (30%) in DMF
rt, 1 h
HW-65
HW-65
R
R
O
O
N
O
O
O
N
O
H
H
OH
O
OH
O
O2N
O2N
O
Fmoc
NH2
N
NH
H
Art
OH
O
O
R
O
EDCI, HOBt, DIPEA
DMF, rt, o/n
HO
O
O
O
HW-65
R
artemisinin-derived
2a 13 15
O
O
N
O
acids
,
or
H
OH
O
Art
O2N
O
26a: Art = subunit of artesunic acid (2a)
26b:
26c: Art = subunit of artemisinin-derived acid 15
O
13
Art = subunit of artemisinin-derived acid
N
H
O
Scheme 3. Synthesis route for immobilized artemisinin-derived acids (artesunic acid (2a), dimer 13 and trimer 15) on TOYOPEARL® AF-Amino-650M beads via an
UV-cleavable-linker (constructs 26a, 26b and 26c).
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Having prepared all prerequisites for target ID experiments, we
furthermore intended to verify the compound immobilization to the
solid matrix, and we also addressed the possibility to cleave the
bond of our formed construct upon irradiation with UV-light. To
was the most effective compound and was even more active than
the reference compound dihydroartemisinin (EC50 = 2.4 nM).
However, as soon as a fullerene moiety is connected to dimer 7,
this strong antimalarial effect is strikingly decreased (dimer 8:
EC50 = 226 nM), which might be an evidence that the biological
activity of artemisinin dimers is also mediated via radicals, as
fullerene is well-known for its radical scavenging and antioxidant
properties.[12] Comparing the EC50 values of the two
diastereomers of dimer 6, it can be seen that the stereochemistry
at the C10 position does not affect antimalarial activity. The same
observation can be made by looking at the data published by
Posner et al.[1h,13] This is quite interesting as for other artemisinin
dimers, for example where the two artemisinin subunits are
covalently joined at C9, the opposite finding was reported and
consequently, it seems to be dependent on how the artemisinin
moieties are connected.[1h,14] However, in order to make a definite
statement, whether stereochemistry at the C10 position in
artemisinin dimers is important for antimalarial activity or not,
more data are necessary.
this end,
a control experiment was performed, in which
immobilized artesunic acid 26a was suspended in DMSO and
exposed for 3 h to UV-light under gentle rotation (Scheme 4).
After this time period, several washing steps were carried out,
from which the supernatant liquids were collected and examined
by TLC. Indeed, after developing with phosphomolybdic acid
staining solution, TLC revealed one specific spot (Figure S2; SI),
which was isolated by column chromatography and analyzed by
NMR spectroscopy and HRMS. The isolated spot could be
identified as artesunic amide 27, which is exactly the product we
expected from this reaction and consequently, this can be seen
as a proof for a working immobilization of our compounds to the
solid support, as all compounds of this study had similar structural
motifs and were connected in the same way to the beads.
O
NH
Table 1. EC50 values for chloroquine, dihydroartemisinin (2b), artesunic
acid (2a), previously published trimer 3[6] and target compounds 4-15 tested
against P. falciparum 3D7 parasite-infected RBCs.
OH
O
R
compound
MW (g/mol)
319.87
284.35
384.42
973.21
989.21
1295.65
636.74
636.74
924.99
1643.63
590.76
941.12
841.04
824.91
924.99
1191.32
1247.43
P. f. 3D7 EC50 (nM)
9.8 ± 2.8
2.4 ± 0.4
8.9 ± 1.9
12.8 ± 1.2
13.5 ± 0.3
343 ± 80
2.4 ± 0.4
2.7 ± 0.2
1.4 ± 0.2
226 ± 17.5
4.5 ± 1.0
4.5 ± 1.6
46 ± 2.6
O
O
HW-65
R
chloroquine[a]
H
H
O
O
O
N
H
O
O
O
OH
O
O
dihydroartemisinin (2b)[a]
O
26a
O2N
artesunic acid (2a)
O
N
H
3[b]
4
O
O
UV light
DMSO, rt, 1.5 h
NH
OH
H
O
5
O
H
O
O
O
6a
6b
7
R
O
+
O
O
O
HW-65
R
H2N
O
O
N
H
O
O
OH
O
27
8
O2N
9
O
10
11
12
13
14
15
Scheme 4. Reaction for the chemical verification for immobilized artesunic
acid (2a) on TOYOPEARL® AF-Amino-650M beads via an UV-cleavable-linker
(construct 26a) resulting in artesunic amide 27.
2.9 ± 0.1
4.8 ± 0.7
5.4 ± 0.5
5.7 ± 0.8
Antiplasmodial activity. All synthesized target compounds 4-15
were examined in vitro for their antimalarial activity towards P.
falciparum 3D7 malaria parasite-infected red blood cells (RBCs)
and compared to their parent compounds artesunic acid (2a) and
dihydroartemisinin (2b), as well as the standard drug chloroquine
(Table 1). Compounds 6, 7, 9, 10 and 12-15 showed excellent
antimalarial efficacies (inhibiting parasite growth) with EC50
values (1.4-5.7 nM) below those of artesunic acid (EC50 = 8.9 nM)
and chloroquine (EC50 = 9.8 nM). Dimer 7 with an EC50 of 1.4 nM
[a] EC50 values have been previously reported.[15] [b] EC50 value has been
previously reported.[6]
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10.1002/chem.201800729
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To our surprise, dendrimer 5 was the least effective substance out
of the synthesized with an EC50 value of 343 nM. This result was
not expected, especially because the precursor 18 used for the
synthesis of dendrimer 5 is one of the most active compounds
among artemisinin-derived dimers and was applied for a series of
other biologically effective derivatives.[1c,1h,16] Furthermore, 4
(EC50 = 13.5 nM) showed about the same activity as our
previously published compound 3 (EC50 = 12.8 nM)[6] and was
considerably more active than dendrimer 5, even though these
compounds contain similar structural motifs. An explanation might
be that the uptake of dendrimer 5 into the malaria parasites is
limited due to the extremely high lipophilicity of the compound
(clogP = 13.32). The size of the molecule on the other hand
should not be the cause, as other compounds, such as
dendrimers 14 and 15, have a similar molecular weight and still
biological activity (HCMV-specific EC50 = 3.13 µM). In addition, it
should be mentioned that our first artemisinin-derived dendrimer
analyzed in this group, compound 5, exhibited an excellent
antiviral potency (EC50 = 0.27 µM) and was seen as the second
most active compound in the current series.
Surprisingly, dimer 13 revealed to be partially cytotoxic towards
HFFs within the analyzed range of concentrations, so that in this
case it was not possible to determine an EC50 value. All other
compounds 4-12 and 15 showed no cytotoxic side effects and
were selectively inhibitory towards HCMV. In summary, we have
the following compounds for the aspired target ID experiments:
artesunic acid (2a) representing the monomeric compound with
an EC50 value of 5.41 µM, artemisinin-derived dimer acid 13,
which exhibited cytotoxic side effects, and artemisinin-derived
trimer acid 15 with an antiviral activity that is nine times higher
(EC50 = 0.59 µM) compared to that of artesunic acid.
possess
a
high antimalarial potency. Substituting the
dihydroartemisinin subunit in compound 11 (EC50 = 46 nM) with
artesunic acid resulted in a 10 times more active compound 10
(EC50 = 4.5 nM). This result highlights that artesunic acid should
Table 2. EC50 values of anti-HCMV activity for ganciclovir, artemisinin (1),
dihydroartemisinin (2b), artesunic acid (2a), trimer 3[6] and target
compounds 4-15.
be considered more often as
a
viable alternative to
dihydroartemisinin, despite the fact that artesunic acid ester
derivatives are less hydrolytically stable. However, this should not
pose a problem, if the drug is administered via injection or
intravenous infusion,[17] as the half-life of artesunic acid at
moderate pH values like those in the blood plasma (pH ≈ 7.4) is
much longer (t1/2 = 7.3 h).[18]
compound
MW (g/mol)
579.98
282.14
284.35
384.42
973.21
989.21
1295.65
636.74
636.74
924.99
1643.63
590.76
941.12
841.04
824.91
924.99
1191.32
1247.43
HCMV EC50 (µM)
2.60 ± 0.5
> 10
ganciclovir[a]
artemisinin (1)[b]
dihydroartemisinin (2b)[b]
> 10
Anticytomegaloviral activity. The antiviral activity of target
compounds 4-15 was evaluated for the HCMV strain AD169-GFP,
which expresses the green fluorescent protein (GFP) in
quantifiable amounts, and can be reliably used for the
determination of the efficiency or the compound-mediated
inhibition of viral replication (Table 2). Infection experiments were
carried out with cultures of primary human foreskin fibroblasts
(HFFs) and the measurement of antiviral activity was performed
according to a previously published protocol.[19] Artesunic acid
(2a) and ganciclovir were applied as reference compounds and
both exerted strong anti-HCMV activity at EC50 levels of 5.41 µM
and 2.60 µM, respectively. Compound 4 turned out to be the most
active compound with an EC50 value of 0.026 µM, thus even more
artesunic acid (2a)[c]
5.41 ± 0.61
0.04 ± 0.01
0.026 ± 0.002
0.27 ± 0.001
0.74 ± 0.10
1.20 ± 0.28
0.57 ± 0.04
11.2 ± 2.4
1.34 ± 0.19
3.13 ± 0.89
27.5 ± 2.9
1.36 ± 0.40
> 10[e]
3[d]
4
5
6a
6b
7
8
active than our previously published hit compound 3 (EC50
=
9
0.04 µM). Of note, the measured anti-HCMV activity of 4 was
higher than the activity of two reference drugs ganciclovir and
artesunic acid, so that it is considered as a potential experimental
candidate for further development. The antiviral efficacies for
compounds 5, 6, 7, 9, 12, 14 and 15 were less pronounced than
related compounds analyzed earlier (in particular 3 and 4), i.e. the
current compounds exerted EC50 values between 0.27 and
1.36 µM, and still all of these remained in a range of activity
comparable to the reference compounds. Comparing the
antimalarial and antiviral data, a trend may be deduced, in that
stereochemistry at C10 of dimers 6 did not have a major impact
on their biological activity. Moreover, the fullerene-containing
dimer 8 (HCMV-specific EC50 = 11.2 µM) was considerably less
potent than dimer 7 (EC50 = 0.57 µM). Finally, the substitution of
the dihydroartemisinin subunit in compound 11 (EC50 = 27.5 µM)
with artesunic acid to generate compound 10 increased its
10
11
12
13
14
15
0.53 ± 0.16
0.59 ± 0.43
[a] EC50 value has been previously reported.[20] [b] EC50 values have been
previously reported.[21] [c] EC50 value has been previously reported.[22] [d]
EC50 value has been previously reported.[6] [e] microscopically detectable
cytotoxicity approx. 50% at 1 µM, no antiviral activity detectable.
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10.1002/chem.201800729
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Table 3. Proteins identified by a mass spectrometry-based approach using linker-coupled artemisinin-related compounds.
mean
WSC
subcellular
localization
protein name
UniProt I.D.
fold enrichment over control
known function
cellular proteins
adapter protein implicated in the
regulation of a large spectrum
signaling pathways
14-3-3 protein epsilon
P62258
P10809
8
6.5; absent from control
7; absent from control
C N
Mt
60 kDa heat shock protein,
mitochondrial
mitochondrial protein import,
assembly of proteins, protein folding
13
ATP synthase subunit alpha
ATP synthase subunit beta
P25705
P06576
8
8.7; absent from control
7.25; absent from control
Mt
Mt
ATP synthesis
ATP synthesis
8.8
ER-membrane PM
Cs
cytoskeleton associated protein 4
Q07065
O43175
P52907
P47756
7.3
6
7.5; absent from control
absent from control
anchoring of ER to microtubules
serine biosynthesis
D-3-phosphoglycerate
dehydrogenase
C
F-actin-capping protein subunit
alpha-1
3.6
7.5
6.8; absent from control
11; absent from control
Cs
Cs
regulation of actin cytoskeleton
Regulation of actin cytoskeleton
F-actin-capping protein subunit
beta
mitochondrial carrier homolog 2
myosin, heavy chain 9
N-acetyl-D-glucosamine kinase
prohibitin
Q9Y6C9
P35579
Q9UJ70
P35232
Q99623
P68371
P68371
Q9BUF5
3
absent from control
5.5; absent from control
absent from control
8; absent from control
absent from control
absent from control
6.8
Mt
Cs
unknown function
7.6
4.5
7.6
5.5
10.9
11.4
5.7
cytoskeleton organization
C
metabolism of N-acetylglucosamine
inhibits DNA synthesis
Mt
prohibitin 2
N Mt C
Cs
transcriptional repression
tubulin beta
major constituent of microtubules
major constituent of microtubules
major constituent of microtubules
tubulin beta-4B chain
tubulin beta-6 chain
Cs
11.4; absent from control
Cs
constituent of class-III intermediate
filaments
vimentin
P08670
P21796
6
6
C
diffusion of small hydrophilic
molecules through outer Mt
membrane, apoptosis
voltage-dependent anion-selective
channel protein 1
23.5
43; absent from control
Mt PM
diffusion of small hydrophilic
molecules through outer Mt
membrane
voltage-dependent anion-selective
channel protein 2
P45880
8.7
11.5; absent from control
Mt
HCMV proteins
absent from control
7
unknown function, essential for viral
replication
envelope glycoprotein pUL132
HCMV major capsid protein
P69338
P16729
3.6
7
Vi
Vi
main constituent of viral capsids
C: cytoplasm; Cs: cytoskeleton; N: nucleus, Mt: mitochondrion; ER: endoplasmatic reticulum; Vi: virion; PM: plasma membrane
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Target identification. Recently, we and others studied putative
inhibitory mechanisms of artemisinin-related compounds, in order
to understand the basis of their outstanding broad-spectrum
anti-infective,[5f,22-23] antimalarial[24] and anticancer[25] activities. It
has been known since the beginning of the molecular studies on
this class of compounds that sesquiterpenes/trioxanes possess a
pronounced potency in alkylating proteins, whereby the specificity
of alkylation reaction and the resulting covalent compound linkage
to individual target proteins remained in question. To date, one
mode of antiviral action has repeatedly been suggested and could
experimentally been confirmed, namely the inhibition of the
nuclear factor kappa B (NF-B) signaling pathway. Our study by
Hutterer and colleagues provided evidence based on a number of
different methodologies that artesunic acid has the capacity to
interfere with the canonical NF-B pathway by directly targeting
RelA/p65.[22] This statement is currently further substantiated by
novel data from our group to be published elsewhere. In addition
to NF-B, it appears also generally accepted that more target
proteins may very probably be involved in the inhibitory
mechanism of these compounds and might contribute to their
broad activity. Of note, Ravindra and colleagues recently
At this stage, it appears too early to conclude about the
importance of individual target candidates from this list. A series
of further target validation experiments will be required to address
this question and to clarify the role of binding proteins in the
supposed complex mode of antiviral action. The fact that the
formation of HCMV resistance against artemisinin-related
compounds could not be observed yet,[20,22,27] makes it probable
that primarily cellular proteins, possibly combined with one or
more viral proteins, may represent a functionally relevant
targeting network. In addition, cell type-specific differences may
also contribute to the compounds’ mechanistic complexity, which
has to be unraveled in future studies.
identified potential artesunate target proteins by using
a
biotinylated dihydroartemisinin in a human bronchial epithelial cell
line-based model for allergic asthma.[7b] Best hits included
mitochondrial proteins and enzymes involved in glycolysis, but to
date it is unclear whether these proteins have relevance for our
investigated system of HCMV-infected cells.
Thus, to either confirm or identify novel cellular (or viral) proteins
with the capacity to bind to artemisinin-related compounds, we
performed a target binding analysis using the newly generated
linker-coupled versions of these active compounds (Scheme 5).
The compound binding capacity of putative target proteins should
thus be investigated by the use of total protein lysates prepared
from primary human fibroblasts. To this end, the lysates derived
from HCMV AD169 GFP-infected HFFs were incubated with
immobilized artemisinin-derived acids (constructs 24 and 26).
Binding proteins were eluted, separated by SDS PAGE and
visualized by Instant blue and silver staining. Bands being
considered as enriched in the samples of interest, but absent from
controls, were cut from Instant blue-stained gels and utilized for
target ID by mass spectrometry-based proteomic analysis
(Supplemental files X1 and X2). In Table 3, candidate proteins are
summarized, which were (i) at least 5-fold enriched over negative
controls according to the weighted spectral counts (WSC) and (ii)
could be detected in at least two independent samples.
Importantly, we obtained two major groups of novel target
candidates, namely cytoskeletal and mitochondrial proteins
(Table 3. Cs and Mt). Interestingly, for a number of these proteins
their correlation with HCMV replication has been published by
various groups, particular for prohibitin, prohibitin 2, vimentin,
14-3-3 protein epsilon and myosin heavy chain 9. For these
proteins, either upregulation during HCMV replication, their
presence in virions or functional importance, respectively, has
been demonstrated.[26] In addition, our analysis identified two
putatively compound-binding viral proteins, namely major capsid
protein (MCP) and envelope glycoprotein pUL132, which are both
essential for HCMV replication.
Scheme 5. Principle of the mass spectrometry-based target protein
identification using linker-coupled artemisinin-related compounds.
Conclusions
In conclusion, we state that the artemisinin skeleton provides an
excellent basis for the further development of new improved
derivatives with highly interesting antimicrobial properties. During
this study, a series of artemisinin-derived monomers, di/tri-mers
and dendrimers was synthesized and investigated for their in vitro
pharmacological activity against the malaria parasite Plasmodium
falciparum 3D7 strain in infected RBCs and HCMV-infected HFFs.
Among tested compounds 4-15, trimer 4 (EC50 = 0.026 µM) stood
out as the most effective compound in terms of anti-HCMV
efficacy and was even more active than our previously published
trimer 3 (EC50 = 0.04 µM), as well as two reference drugs
ganciclovir (EC50 = 2.60 µM) and artesunic acid (EC50 = 5.41 µM).
Regarding antimalarial activity, dimer 7 exhibited the highest
efficacy and was with an EC50 value of 1.4 nM even more active
than the standard drugs dihydroartemisinin (EC50 = 2.4 nM),
artesunic acid (EC50 = 8.9 nM) and chloroquine (EC50 = 9.8 nM).
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10.1002/chem.201800729
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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, J. Med. Chem.
1999, 42, 4275-4280; c) G. H. Posner, I. H. Paik, S. Sur, A. J. McRiner,
K. Borstnik, S. Xie, T. A. Shapiro, J. Med. Chem. 2003, 46, 1060-1065;
d) J. P. Jeyadevan, P. G. Bray, J. Chadwick, A. E. Mercer, A. Byrne, S.
A. Ward, B. K. Park, D. P. Williams, R. Cosstick, J. Davies, A. P. Higson,
E. Irving, G. H. Posner, P. M. O'Neill, J. Med. Chem. 2004, 47, 1290-
1298; e) J. Chadwick, A. E. Mercer, B. K. Park, R. Cosstick, P. M. O'Neill,
Bioorg. Med. Chem. 2009, 17, 1325-1338; f) A. M. Galal, W. Gul, D.
Slade, S. A. Ross, S. Feng, M. G. Hollingshead, M. C. Alley, G. Kaur, M.
A. ElSohly, Bioorg. Med. Chem. 2009, 17, 741-751; g) D. Chaturvedi, A.
Goswami, P. Pratim Saikia, N. C. Barua, P. G. Rao, Chem. Soc. Rev.
2010, 39, 435-454; h) T. Fröhlich, A. Çapcı Karagöz, C. Reiter, S. B.
Tsogoeva, J. Med. Chem. 2016, 59, 7360-7388.
Both compounds 4 and 7 showed no major signs of cytotoxic
effects in healthy HFFs and thus, can be considered as selective
antimicrobial drug candidates. Furthermore, we easily
immobilized artesunic acid (2a), artemisinin-derived dimer 13 and
trimer 15 onto a solid support (TOYOPEARL® AF-Amino-650M)
via two different methods, which were utilized for the first time in
mass spectrometry-based target ID experiments using total
lysates of HCMV-infected primary human fibroblasts. We
successfully identified a series of proteins (Table 3), some of
which are known from literature to be associated with HCMV
replication. Further experiments are underway to validate the
involvement of these target candidates in the mechanism of action
of artesunic acid and artemisinin-derived di/tri-mers and
dendrimers.
[2]
a) T. Fröhlich, B. Ndreshkjana, J. K. Muenzner, C. Reiter, E. Hofmeister,
S. Mederer, M. Fatfat, C. El-Baba, H. Gali-Muhtasib, R. Schneider-Stock,
S. B. Tsogoeva, Chem. Med. Chem. 2017, 12, 226-234; b) T. Fröhlich,
C. Reiter, M. M. Ibrahim, J. Beutel, C. Hutterer, I. Zeitträger, H. Bahsi, M.
Leidenberger, O. Friedrich, B. Kappes, T. Efferth, M. Marschall, S. B.
Tsogoeva, ACS Omega 2017, 2, 2422-2431; c) F. E. Held, A. A. Guryev,
T. Fröhlich, F. Hampel, A. Kahnt, C. Hutterer, M. Steingruber, H. Bahsi,
C. von Bojničić-Kninski, D. S. Mattes, T. C. Foertsch, A. Nesterov-
Mueller, M. Marschall, S. B. Tsogoeva, Nature communications 2017, 8,
15071.
Combined, the findings strongly suggest a continuation of these
investigations in terms of the compounds’ inhibitory potency
against plasmodium, cytomegalovirus and possibly even more
human pathogens.
Experimental Section
[3]
a) I. H. Paik, S. Xie, T. A. Shapiro, T. Labonte, A. A. Narducci Sarjeant,
A. C. Baege, G. H. Posner, J. Med. Chem. 2006, 49, 2731-2734; b) A. A.
Alagbala, A. J. McRiner, K. Borstnik, T. Labonte, W. Chang, J. G.
D’Angelo, G. H. Posner, B. A. Foster, J. Med. Chem. 2006, 49, 7836-
7842; c) C. Horwedel, S. B. Tsogoeva, S. Wei, T. Efferth, J. Med. Chem.
2010, 53, 4842-4848; d) C. Reiter, A. Herrmann, A. Capci, T. Efferth, S.
B. Tsogoeva, Bioorg. Med. Chem. 2012, 20, 5637-5641; e) S. Wang, T.
Sasaki, Bioorg. Med. Chem. Lett. 2013, 23, 4424-4427.
The following experimental details can be found in the supporting
information (SI), which is available under https://doi.org/...: Experimental
conditions and procedures for precursors and target compounds 4-15;
experimental
conditions
and
procedures
for
immobilized
artemisinin-derived acids 13 and 15 as well as artesunic acid (2a) on
TOYOPEARL® AF-Amino-650M beads (constructs 24 and 26); spectral
data of precursors and target compounds 4-15; recorded spectra of target
compounds 4-15; details of cell lines and reagents as well as cell viability
assay for biological evaluation; conditions and procedures for target ID
experiments.
[4]
[5]
Y. Tu, Nat. Med. 2011, 17, 1217-1220.
a) Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J.
(Engl. Ed.) 1979, 12, 811-816; b) D. Klayman, Science 1985, 228, 1049-
1055; c) Y. Li, Y. L. Wu, Curr. Med. Chem. 2003, 10, 2197-2230; d) N. P.
Singh, H. C. Lai, Anticancer Res. 2004, 24, 2277-2280; e) T. Efferth, Curr.
Drug Targets 2006, 7, 407-421; f) T. Efferth, M. R. Romero, D. G. Wolf,
T. Stamminger, J. J. Marin, M. Marschall, Clin. Infect. Dis. 2008, 47, 804-
811; g) L. H. Miller, X. Su, Cell 2011, 146, 855-858; h) B. N. Sharma, M.
Marschall, C. H. Rinaldo, Antimicrob. Agents Chemother. 2014, 58,
6724-6734; i) X. G. Zhang, G. X. Li, S. S. Zhao, F. L. Xu, Y. H. Wang, W.
Wang, Parasitol. Res. 2014, 113, 1769-1773; j) A. Flobinus, N. Taudon,
M. Desbordes, B. Labrosse, F. Simon, M. C. Mazeron, N. Schnepf, J.
Antimicrob. Chemother. 2014, 69, 34-40; k) C.-j. Hu, L. Zhou, Y. Cai,
Cancer Biol. Ther. 2014, 15, 279-288; l) M. Lucibello, S. Adanti, E.
Antelmi, D. Dezi, S. Ciafre, M. L. Carcangiu, M. Zonfrillo, G. Nicotera, L.
Sica, F. De Braud, P. Pierimarchi, Oncotarget 2015, 6, 5275-5291.
C. Reiter, T. Fröhlich, L. Gruber, C. Hutterer, M. Marschall, C.
Voigtländer, O. Friedrich, B. Kappes, T. Efferth, S. B. Tsogoeva, Bioorg.
Med. Chem. 2015, 23, 5452-5458.
Acknowledgements
S.B.T. is grateful to the Deutsche Forschungsgemeinschaft
(DFG) for generous funding by grant TS 87/16-3 and to the
Interdisciplinary Center for Molecular Materials (ICMM), the
Graduate School Molecular Science (GSMS), as well as
Emerging Fields Initiative (EFI) “Chemistry in Live Cells”
supported by Friedrich-Alexander-Universität Erlangen-Nürnberg
for research funding. M.M. greatly acknowledges excellent
technical assistance by Hanife Strojan and funding support
through the Deutsche Forschungsgemeinschaft (grant MA
1289/7-3). L.B. and Y.C. thank the support of the discovery
platform and informatics group at EDyP. Proteomic experiments
were partly supported by the Proteomics French Infrastructure
(ANR-10-INBS-08-01 grant) and Labex GRAL (ANR-10-LABX-
49-01).
[6]
[7]
a) N. Edikpo, S. Ghasi, A. Elias, N. Oguanobi, Mol. Cell. Pharmacol. 2013,
5, 75-89; b) K. C. Ravindra, W. E. Ho, C. Cheng, L. C. Godoy, J. S.
Wishnok, C. N. Ong, W. S. F. Wong, G. N. Wogan, S. R. Tannenbaum,
Chem. Res. Toxicol. 2015, 28, 1903-1913; c) H. M. Ismail, V. E. Barton,
M. Panchana, S. Charoensutthivarakul, G. A. Biagini, S. A. Ward, P. M.
O'Neill, Angew. Chem. Int. Ed. 2016, 55, 6401-6405; d) H. M. Ismail, V.
Barton, M. Phanchana, S. Charoensutthivarakul, M. H. L. Wong, J.
Hemingway, G. A. Biagini, P. M. O’Neill, S. A. Ward, Proc. Nat. Acad.
Sci. U.S.A. 2016, 113, 2080-2085; e) Y. Zhou, W. Li, Y. Xiao, ACS Chem.
Biol. 2016, 11, 882-888; f) C.-J. Zhang, J. Wang, J. Zhang, Y. M. Lee, G.
Feng, T. K. Lim, H.-M. Shen, Q. Lin, B. Liu, Angew. Chem. Int. Ed. 2016,
55, 13770-13774; g) C. Sun, B. Zhou, Microbial Cell 2016, 3, 196-205;
h) Y. K. Wong, C. Xu, K. A. Kalesh, Y. He, Q. Lin, W. S. F. Wong, H.-M.
Shen, J. Wang, Med. Res. Rev. 2017, 37, 1492-1517.
Keywords: Artemisinin • artemisinin-derived dendrimers •
antimalarial activity • antiviral activity • target identification
[1]
a) 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, Bioorg. Med. Chem. 1997, 5, 1257-1265; b) G. H.
This article is protected by copyright. All rights reserved.

10.1002/chem.201800729
Chemistry - A European Journal
FULL PAPER
[8]
[9]
O. Axelsson, F. Ek, 2013, EP 2573089 A1.
[20] S. J. F. Kaptein, T. Efferth, M. Leis, S. Rechter, S. Auerochs, M. Kalmer,
C. A. Bruggeman, C. Vink, T. Stamminger, M. Marschall, Antiviral Res.
2006, 69, 60-69.
a) C. G. Overberger, Y. Okamoto, V. Bulacovschi, Macromolecules 1975,
8, 31-36; b) O. Axelsson, F. Ek, 2011, US 2013022669 A1.
[10] P. P. Bora, N. Baruah, G. Bez, N. C. Barua, Synth. Commun. 2012, 42,
1218-1225.
[21] S. Chou, G. Marousek, S. Auerochs, T. Stamminger, J. Milbradt, M.
Marschall, Antiviral Res. 2011, 92, 364-368.
[11] C. P. Holmes, D. G. Jones, J. Org. Chem. 1995, 60, 2318-2319.
[12] a) P. J. Krusic, E. Wasserman, P. N. Keizer, J. R. Morton, K. F. Preston,
Science 1991, 254, 1183-1185; b) R. Bakry, R. M. Vallant, M. Najam-ul-
Haq, M. Rainer, Z. Szabo, C. W. Huck, G. K. Bonn, Int. J. Nanomedicine
2007, 2, 639-649.
[22] C. Hutterer, I. Niemann, J. Milbradt, T. Fröhlich, C. Reiter, O. Kadioglu,
H. Bahsi, I. Zeitträger, S. Wagner, J. Einsiedel, P. Gmeiner, N. Vogel, S.
Wandinger, K. Godl, T. Stamminger, T. Efferth, S. B. Tsogoeva, M.
Marschall, Antiviral Res. 2015, 124, 101-109.
[23] a) W. E. Ho, H. Y. Peh, T. K. Chan, W. S. Wong, Pharmacol. Ther. 2014,
142, 126-139; b) J. Li, T. Casteels, T. Frogne, C. Ingvorsen, C. Honoré,
M. Courtney, K. V. M. Huber, N. Schmitner, R. A. Kimmel, R. A. Romanov,
C. Sturtzel, C.-H. Lardeau, J. Klughammer, M. Farlik, S. Sdelci, A. Vieira,
F. Avolio, F. Briand, I. Baburin, P. Májek, F. M. Pauler, T. Penz, A.
Stukalov, M. Gridling, K. Parapatics, C. Barbieux, E. Berishvili, A. Spittler,
J. Colinge, K. L. Bennett, S. Hering, T. Sulpice, C. Bock, M. Distel, T.
Harkany, D. Meyer, G. Superti-Furga, P. Collombat, J. Hecksher-
Sørensen, S. Kubicek, Cell 2017, 168, 86-100.
[13] G. H. Posner, J. Northrop, I.-H. Paik, K. Borstnik, P. Dolan, T. W. Kensler,
S. Xie, T. A. Shapiro, Bioorg. Med. Chem. 2002, 10, 227-232.
[14] S. Ekthawatchai, S. Kamchonwongpaisan, P. Kongsaeree, B.
Tarnchompoo, Y. Thebtaranonth, Y. Yuthavong, J. Med. Chem. 2001, 44,
4688-4695.
[15] C. Reiter, T. Fröhlich, M. Zeino, M. Marschall, H. Bahsi, M. Leidenberger,
O. Friedrich, B. Kappes, F. Hampel, T. Efferth, S. B. Tsogoeva, Eur. J.
Med. Chem. 2015, 97, 164-172.
[16] a) G. H. Posner, W. Chang, L. Hess, L. Woodard, S. Sinishtaj, A. R.
Usera, W. Maio, A. S. Rosenthal, A. S. Kalinda, J. G. D'Angelo, K. S.
Petersen, R. Stohler, J. Chollet, J. Santo-Tomas, C. Snyder, M.
Rottmann, S. Wittlin, R. Brun, T. A. Shapiro, J. Med. Chem. 2008, 51,
1035-1042; b) A. S. Rosenthal, X. Chen, J. O. Liu, D. C. West, P. J.
Hergenrother, T. A. Shapiro, G. H. Posner, J. Med. Chem. 2009, 52,
1198-1203.
[24] J. Wang, C.-J. Zhang, W. N. Chia, C. C. Y. Loh, Z. Li, Y. M. Lee, Y. He,
L.-X. Yuan, T. K. Lim, M. Liu, C. X. Liew, Y. Q. Lee, J. Zhang, N. Lu, C.
T. Lim, Z.-C. Hua, B. Liu, H.-M. Shen, K. S. W. Tan, Q. Lin, Nature
communications 2015, 6, 10111.
[25] J. Wang, J. Zhang, Y. Shi, C. Xu, C. Zhang, Y. K. Wong, Y. M. Lee, S.
Krishna, Y. He, T. K. Lim, W. Sim, Z.-C. Hua, H.-M. Shen, Q. Lin, ACS
Central Science 2017, 3, 743-750.
[17] a) K. T. Batty, K. F. Ilett, M. E. Davis, Timothy, J. Pharm. Pharmacol.
1996, 48, 22-26; b) Q. Li, P. Weina, Pharmaceuticals 2010, 3, 2322-
2332; c) E. Esu, E. E. Effa, O. N. Opie, A. Uwaoma, M. M. Meremikwu,
Cochrane Database Syst. Rev. 2014, 1-83.
[26] a) S. M. Varnum, D. N. Streblow, M. E. Monroe, P. Smith, K. J. Auberry,
L. Pasa-Tolic, D. Wang, D. G. Camp, 2nd, K. Rodland, S. Wiley, W. Britt,
T. Shenk, R. D. Smith, J. A. Nelson, J. Virol. 2004, 78, 10960-10966; b)
S. Spaderna, B. Kropff, Y. Ködel, S. Shen, S. Coley, S. Lu, W. Britt, M.
Mach, J. Virol. 2005, 79, 11837-11847; c) M. S. Miller, L. Hertel, J. Virol.
2009, 83, 7015-7028; d) A. Zhang, C. D. Williamson, D. S. Wong, M. D.
Bullough, K. J. Brown, Y. Hathout, A. M. Colberg-Poley, Mol. Cell.
Proteomics 2011, 10, M111.009936; e) S. Reyda, N. Buscher, S. Tenzer,
B. Plachter, Viruses 2014, 6, 172-188; f) F. Chai, H. Y. Li, W. Wang, X.
J. Zhu, Y. Li, S. Wang, L. Guo, L. K. Zhang, G. Xiao, Biochim. Biophys.
Acta 2015, 1854, 967-978.
[18] E. H. R. Rajab, A. E. M. Saeed, Asian J. Chem. Sci. 2017, 2, 1-10.
[19] a) M. Marschall, M. Freitag, S. Weiler, G. Sorg, T. Stamminger,
Antimicrob. Agents Chemother. 2000, 44, 1588-1597; b) M. Marschall, I.
Niemann, K. Kosulin, A. Bootz, S. Wagner, T. Dobner, T. Herz, B. Kramer,
J. Leban, D. Vitt, T. Stamminger, C. Hutterer, S. Strobl, Antiviral Res.
2013, 100, 640-648; c) C. Hutterer, J. Eickhoff, J. Milbradt, K. Korn, I.
Zeitträger, H. Bahsi, S. Wagner, G. Zischinsky, A. Wolf, C. Degenhart, A.
Unger, M. Baumann, B. Klebl, M. Marschall, Antimicrob. Agents
Chemother. 2015, 59, 2062-2071.
[27] D. G. Wolf, A. Shimoni, I. B. Resnick, T. Stamminger, A. U. Neumann, S.
Chou, T. Efferth, O. Caplan, J. Rose, A. Nagler, M. Marschall, Antiviral
Res. 2011, 90, 183-186.
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10.1002/chem.201800729
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Convenient synthesis of new
artemisinin-derived di/tri-mers
and dendrimers, highly active
against malaria parasites and
viruses, are presented. Both
the immobilization of artesunic
acid and selected di/tri-mers
on TOYOPEARL® beads and
their application for mass
spectrometry-based target
identification has been
T. Fröhlich, F. Hahn, L. Belmudes,
M. Leidenberger, O. Friedrich, B.
Kappes, Y. Couté, M. Marschall*
and S. B. Tsogoeva*
Page No. – Page No.
Synthesis of artemisinin-
derived di/tri-mers and
dendrimers, investigation of
their activity against
P. falciparum and
demonstrated for the first time.
Two major groups of novel
target candidates, namely
cytoskeletal and mitochondrial
proteins were successfully
obtained.
cytomegalovirus, and insights
into their mechanisms of action
This article is protected by copyright. All rights reserved.