Synthesis of Novel Hybrids of Thymoquinone and Artemisinin with High Activity and Selectivity Against Colon Cancer

Article abstract of DOI:10.1002/cmdc.201600594

Colorectal cancer causes 0.5 million deaths each year. To combat this type of cancer the development of new specific drug candidates is urgently needed. In the present work seven novel thymoquinone–artemisinin hybrids with different linkers were synthesized and tested for their in vitro anticancer activity against a panel of various tumor cell lines. The thymoquinone–artesunic acid hybrid 7 a, in which both subunits are connected via an ester bond, was found to be the most active compound and selectively decreased the viability of colorectal cancer cells with an IC₅₀value of 2.4 μm (HCT116) and 2.8 μm (HT29). Remarkably, hybrid 7 a was up to 20-fold more active than its parent compounds (thymoquinone and artesunic acid), while not affecting nonmalignant colon epithelial HCEC cells (IC₅₀>100 μm). Moreover, the activity of hybrid 7 a was superior to that of various 1:1 mixtures of thymoquinone and artesunic acid. Furthermore, hybrid 7 a was even more potent against both colon cancer cell lines than the clinically used drug 5-fluorouracil. These results are another excellent proof of the hybridization concept and confirm that the type and length of the linker play a crucial role for the biological activity of a hybrid drug. Besides an increase in reactive oxygen species (ROS), elevated levels of the DNA-damage marker γ-H2AX were observed. Both effects seem to be involved in the molecular mechanism of action for hybrid 7 a in colorectal cancer cells.

Full text of DOI:10.1002/cmdc.201600594

Accepted Article
Title: Synthesis of Novel Hybrids of Thymoquinone and Artemisinin
with High Activity and Selectivity Against Colon Cancer
Authors: Tony Fröhlich, Benardina Ndreshkjana, Julienne K.
Muenzner, Christoph Reiter, Elisabeth Hofmeister, Sandra
Mederer, Maamoun Fatfat, Chirine El-Baba, Hala Gali-
Muhtasib, Regine Schneider-Stock, and Svetlana B.
Tsogoeva
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ChemMedChem
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Synthesis of Novel Hybrids of Thymoquinone and Artemisinin
with High Activity and Selectivity Against Colon Cancer
Tony Fröhlich,[a]‡ Benardina Ndreshkjana,[b]‡ Julienne K. Muenzner, Christoph Reiter,
[b]
[a]
[
a]
[b]
[c]
[b]
[c]
Elisabeth Hofmeister, Sandra Mederer, Maamoun Fatfat, Chirine El-Baba, Hala Gali-Muhtasib,
[
b]
[a]
Regine Schneider-Stock, * and Svetlana B. Tsogoeva *
Abstract: Colorectal cancer causes 0.5 million deaths each year. To
combat this type of cancer the development of new specific drug
candidates is urgently needed. In the present work seven novel
thymoquinone-artemisinin hybrids with different linkers were
synthesized for the first time and tested for their in vitro anticancer
activity in a panel of different tumor cell lines. The thymoquinone-
artesunic acid hybrid 7a was found to be the most active compound
and selectively reduced the viability of colorectal cancer cells with an
IC50 of 2.4 µM (HCT116) and 2.8 µM (HT29). Remarkably, hybrid 7a
was up to 20-fold more active than its parental compounds
of the currently available drugs are no longer effective due to
enhanced drug resistance in tumor cells or undesirable side
effects because of their unselectivity for tumor versus normal
cells.[2] One of the most promising and fundamentally novel
approaches in order to obtain new specific anticancer active
compounds with improved pharmacological properties is the
hybridization of bioactive natural products: Two or more natural
product fragments are combined and linked with each other via
covalent bonds forming new hybrid molecules.[ These synthetic
hybrids containing partial structures of natural compounds are in
3]
[
3b,4]
(
thymoquinone and artesunic acid), while not affecting non-malignant
many cases more active than their parental compounds.
colon epithelial HCEC cells (IC50 >100 µM). Moreover, the activity of
hybrid 7a was superior to that of different 1:1 mixtures of
thymoquinone and artesunic acid. Furthermore, hybrid 7a was even
more potent against both colon cancer cell lines than the clinically
used drug 5-fluorouracil. These results are another excellent proof of
the hybridization concept and confirm that the type and the length of
the linker play a crucial role for the biological activity of a hybrid drug.
Besides an increase in ROS, elevated levels of DNA-damage marker
γ-H2AX, were observed. Both effects seem to be involved in the
molecular mechanism of action for hybrid 7a in colorectal cancer cells.
In addition, they are able to overcome drug resistance[5] and open
up the possibility of combining positive properties of different
natural products in one single structure, thus leading to lower
toxicity. Many examples presented in literature have already
documented the high potential of the hybridization concept.[4,6] In
the search for new drug candidates that specifically target colon
cancer cells, we focused on the concept of hybridization,
encouraged also by our previous results and experiences with
artemisinin based hybrids.[5a,7]
We hypothesized that linking the natural products artemisinin (1)
and/or artesunic acid (Art, 2) with thymoquinone (TQ, 3) (Figure 1)
might yield highly effective anticancer agents having low toxicity
towards normal cells. To our knowledge, no thymoquinone-
artemisinin hybrids have been reported yet.
Introduction
Colorectal cancer is the third most common cancer with more than
1
.3 million new cases reported annually and nearly 0.5 million
[
1]
deaths each year. To control this type of cancer the
development of new drug candidates is urgently needed, as most
[
a]
b]
T. Fröhlich, Dr. C. Reiter, E. Hofmeister, Prof. Dr. S. B. Tsogoeva
Organic Chemistry Chair I and Interdisciplinary Center for Molecular
Materials (ICMM), Friedrich-Alexander University of Erlangen-
Nürnberg, Henkestr. 42, 91054 Erlangen, Germany.
E-mail: svetlana.tsogoeva@fau.de
Figure 1. Structures of artemisinin (1), artesunic acid (Art, 2) and thymoquinone
(TQ, 3).
[
B. Ndreshkjana, Dr. J. K. Muenzner, S. Mederer, Dr. Chirine El-
Baba, Prof. Dr. R. Schneider-Stock
Experimental Tumorpathology, Institute of Pathology, Friedrich-
Alexander-University of Erlangen-Nürnberg, Universitätsstr. 22,
Artemisinin (1) is an enantiomerically pure sesquiterpene
containing a 1,2,4-trioxane ring, which was extracted from the
Chinese medicinal plant Artemisia annua L. in 1972 by Youyou
Tu (Nobel Prize 2015).[ It is widely known as a traditional
9
1054 Erlangen, Germany.
E-mail: regine.schneider-stock@uk-erlangen.de
Dr. M. Fatfat, Prof. Dr. H. Gali-Muhtasib
Department of Biology, and Department of Anatomy, Cell Biology
and Physiological Science, American University of Beirut, Beirut,
Lebanon.
[
c]
8]
antimalarial drug,[9] but also possesses great anticancer
potential.[
10]
Its semisynthetic derivative, Art (2), also
‡
These authors contributed equally to this work.
demonstrated to be a very effective anticancer agent and
exhibited remarkable cytotoxic activities against a wide range of
cancer cell lines including prostate, ovarian, leukemia, melanoma,
Supporting information for this article is given via a link at the end of
the document.
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ChemMedChem
10.1002/cmdc.201600594
FULL PAPER
breast, colorectal and renal cancer cells.[11] Furthermore, in vivo
experiments revealed its anticancer activity in pancreatic cancer,
liver cancer, lung cancer and Kaposi sarcoma.[
potent and specific cytotoxic activity against colon cancer cell
lines without toxic effects on normal colon cells. Further
evaluation of the molecular mechanism of action of hybrid 7a
showed no direct interaction with DNA, but ROS-induced DNA
damage and apoptosis.
12]
Although the mechanism of action of artemisinin is still not
completely understood, it is generally accepted that the
endoperoxide moiety within the 1,2,4-trioxane system is essential
for its anticancer activity.[ It is assumed that the peroxide bridge
is cleaved by intracellular Fe(II) leading to the formation of
reactive oxygen species (ROS) and carbon-centered free radicals.
These species induce oxidative stress, alkylation of target
proteins, DNA damage, and apoptosis.[ TQ (3) (Figure 1), a
naturally occurring phytochemical compound, is the main
constituent of the volatile oil of Nigella sativa (black seed) and was
first extracted in 1963 by El-Dakhakhany.[15] This monoterpene
was found to have strong anticancer effects both in vitro and in
vivo.[16] Recent reports have shown that TQ induces apoptosis in
vitro by p53-dependent and independent pathways, while
exhibiting no toxicity in normal cells.[17] Being a short-chain
ubiquinone-derivative, TQ is able to act as a pro-oxidant and
consequently induce oxidative stress by triggering ROS
13]
Results and Discussion
Chemistry: To be able to synthesize the novel thymoquinone-
artemisinin hybrids 7a-c, 9a/b and 11a/b (Scheme 2), first TQ (3)
has to be converted into the corresponding alcohol
derivatives 6a-c (Scheme 1).
This was achieved by treating TQ and different sodium hydroxyl
carboxylate salts 5a-c (which were obtained in quantitative yield
by simple ester hydrolysis of commercially available
lactones 4a-c) with (NH ) S O and catalytic amounts of AgNO
4 2 2 8 3
in a mixture of water and acetonitrile. This procedure, which is
already reported in literature in the context of synthesis of other
derivatives of TQ,[24] afforded alcohols 6a-c. Subsequently, these
14]
[
18]
production, which is revealed to be directly linked to its pro-
apoptotic effect in colon cancer and leukemia cells.[ Recently, it
has been shown that TQ suppresses metastasis through NF-kB
inhibition and activation of JNK and p38 in CPT-11-R LoVo colon
cancer cells, and to abrogate epithelial–mesenchymal transition
in cancer cells mainly through the inhibition of PI3K/AKT signaling
alcohols were reacted with either Art (2) or the artemisinin-derived
carboxylic acid 8[25] under Steglich esterification conditions to
furnish ester hybrids 7a-c in 80-86% yield and 9a/b in
19]
3
2/39% yield. Hybrids 9a/b belong to the group of C-10
non-acetals, which are known to be more hydrolytically stable
than common artemisinin derivatives such as Art.[26]
[
20]
axis.
In addition, in vivo studies showed that the general toxicity of TQ
In order to investigate the effect of the type of linkage between the
artemisinin and the TQ subunit on biological activity, two ether
hybrids 11a/b were prepared, as ethers are known to be more
[
21]
is relatively low, making the compound worth considering for
clinical applications.[22] Interestingly, it was shown that TQ
enhances in vitro and in vivo the efficacy of many
chemotherapeutic agents - even in resistant types of cancer -
documented procedure:[27]
stable than esters, using
Dihydroartemisinin acetate 10 and alcohols 6b/c were stirred at
°C for 1 h in the presence of catalytic amounts of TMSOTf and
a
0
[
16b,23]
such as cisplatin in lung cancer and many solid tumors.
thereby ethers 11a/b could be isolated in 43/52% yield. It has to
Thus, both natural products, Art and TQ, can be regarded as very
promising anticancer active compounds, and, therefore are
perfectly suitable for applying the hybridization concept in order to
obtain new potent anticancer agents.
be mentioned that both products occurred as a mixture of
1
0α‐ and 10β-diastereomers. In both cases the 10-isomer
3.3/3.4 Hz) was predominantly formed (ratio
0/10-isomers = 8:1). The ratio between both isomers was
H-9;H-10
(J =
1
In this article, we report on seven novel TQ-Art hybrids
determined by comparing the integrals of corresponding protons
(compounds 7a-c, 9a/b and 11a/b, Scheme 2) whose synthesis
1
at C-10 and C-9 in the recorded H-NMR spectra. This was
consists of only a few steps. Their biological activity against
various colon cancer cell lines (HCT116, HT29, Caco-2, DLD-1)
and other cancer cell lines, such as breast cancer (MCF-7),
prostate cancer (PC-3), liver cancer (HEPG2) and leukemia (JKT,
HUT102) have been evaluated. The hybrid compounds exhibited
possible, because a large coupling constant (7-10 Hz) is generally
found for the 10-isomer, indicating a relative trans-configuration,
whereas a small coupling constant (3-5 Hz) appears for the
0-isomer, indicating a cis-configuration.[28]
1
Scheme 1. Synthesis of TQ derivatives 6a-c.
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ChemMedChem
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Scheme 2. Synthesis of thymoquinone-artemisinin hybrids 7, 9 and 11.
Biological evaluation: To study the cytotoxic effect of the novel
seven hybrids we first investigated cell viability by crystal violet
assay in colon (HCT116) and prostate (PC-3) cancer cell lines as
well as in a normal colon epithelial cell line (HCEC), Table 1. We
compared different TQ-Art hybrids 7a-c, 9a/b and 11a/b
harbouring different linkers.
The most effective hybrid on the colorectal HCT116 cancer cells
was 7a with IC50 of 2.4 µM. Hybrids 9b and 11a revealed
IC50 values of 9.1 and 12.7 µM, respectively. The least active
values in the different tumor cell lines (approx. 40-60 µM),
reflecting its strong universal anticancer effect.
Table 1. IC50 values (in µM) of thymoquinone-artemisinin hybrids 7, 9
and 11 in two cancer and a normal cell line after 24 h of incubation
Compound
HCT116
PC-3
HCEC
>100
7a
2.4 ± 0.19[a]
3.7 ± 0.24
26.2 ± 7.3
15.5 ± 6.3
9.1 ± 2.7
93.0 ± 18.5
84.6 ± 8.3
>100
compounds were hybrids 11b (IC50 = 18 µM) and 7c (IC50
=
7b
>100
2
6.2 µM). Intriguingly, one additional CH -group or less between
2
7
9
9
1
c
>100
the natural product subunits is enough to considerably decrease
anticancer activity. These results demonstrate that a linker in a
hybrid drug is important and in fact its length and nature is
relevant for the observed activities. Notably, the lowest IC50 value
in PC-3 cells was found for hybrid 11b (51.7 µM) and the highest
a
>100
>100
b
96.8 ± 10.7
71.3 ± 3.4
51.7 ± 2.5
>100
1a
12.7 ± 0.8
18.0 ± 2.7
72.4 ± 1.8
36.7 ± 1
one for hybrids 7c and 9a (IC50 values >100 µM). Interestingly, the
activity of all hybrids was significantly lower on normal HCEC cells
when compared to the other two tumor cell lines, suggesting a
tumor specific effect (Table 1).
To study the effect of the most potent hybrid 7a in comparison to
the parental compounds Art (2) and TQ (3), we investigated cell
viability of a panel of cancer cell lines in response to these three
drugs (Table 2, Suppl. Figure 1). TQ showed comparable IC50
11b
[a] the corrisponding viability graphs for hybrid 7a are given in Suppl. Figure 1
as an example.
Interestingly, Art was significantly more effective in HCT116 cells
(5.3 µM) whereas the other cancer types required higher
concentrations of Art (20 µM to higher than 500 µM) to induce
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ChemMedChem
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5
0% of cell death. Similarly, the IC50 values of hybrid 7a in
single drugs TQ and Art (1:1, 2.5:2.5, 5:5 mixtures, Figure 3 A-D,
Suppl. Figure 2). Interestingly, hybrid 7a was always more
effective than the three different combination treatments in
HCT116 cells after 24 h and 48 h of and in HT29 cells after 48 h
of incubation.
HCT116 and HT29 cells were remarkably lower (2.4 µM and 2.8
µM respectively) than in all the other cancer cell lines (60 µM to
higher than 1 mM), which suggests a specific drug action in
colorectal cancer cells (Tables 1-3, Figure 2 and Suppl. Fig. 1 and
2
).
In a next step, we determined the protein level of poly (ADP-
ribose) polymerase (PARP) in HCT116 and HT29 cells which
plays a critical role in the maintenance of DNA integrity. Its main
role is to detect and report single-strand DNA breaks (SSB) to the
enzymatic machinery involved in the SSB repair. Cleaved PARP
is indicating a loss of repair signaling and the induction of
apoptotic cell death.[29] For this, the cells were treated with 5 µM
of each single drug (Art and TQ), the combination of both (5:5 µM),
and the hybrid 7a (5 µM). Treatment with Art, the combination,
and 7a led to PARP cleavage in HCT116 cells after 24 and 48 h,
whereas PARP cleavage was detectable for TQ only after 48 h
(Figure 3 E). In contrast, HT29 cells were more resistant to Art,
the combination, and hybrid 7a treatment and showed PARP
cleavage only after 48 h (Figure 3 F). TQ did not induce cell death
at either one of the analyzed time points in HT29 cells.
Furthermore, TQ/ART combination treatment, and hybrid 7a
induced caspase 9 cleavage but only after 48h in both cell lines
In addition, we evaluated the cytotoxicity of the synthetic TQ
derivative 6a (a subunit of hybrid 7a) in HCT116 cells. Notably, 6a
showed an IC50 of >100 µM (Suppl. Figure 3) indicating that that
the observed superior performance and reduced toxicity of 7a
(Table 1) is the result of covalent linking of TQ derivative 6a with
Art (2) to a hybrid molecule and that the hybridization concept is
a powerful strategy to develop new effective anticancer drug
candidates.
Table 2. IC50 values (in µM) of Art, TQ and hybrid 7a in different cancer cell
lines after 24 h of incubation*
Comp.
Art
HCT
16
Hep G2
PC-3
MCF-7
JKT
HUT
102
1
5.3
21.9
± 5.4
39.5
± 1.5
25.0
± 2.5
>500
>250
±
0.1
(
Suppl. Figure 4 A, B) suggesting the involvement of the intrinsic
TQ
50.1
37.6
± 9.7
59.0
± 7.7
42.0
± 8.5
43.1
± 3.6
40.3
± 4.2
apoptotic pathway.
±
6.1
To examine the drug-induced DNA damage we treated HCT116
and HT29 cells with the single drugs, the combination of both, and
hybrid 7a. Art, the combination treatment and 7a, but not TQ
induced strong DNA damage as determined by an increased level
of γ-H2AX representing a marker of DNA double-strand breaks
7
a
2.4
0.19
63.0
± 8.4
93.0
± 18.5
78.4
± 14.1
>1000
>300
±
*
1
IC50 value of clinically used anticancer drug 5-fluorouracil: IC50 (HCT116) =
0 ± 1.6 µM.
(
DSBs), an ultimate result of missing SSB repair.[30] In HT29 cells
this effect was less pronounced, which possibly explains the
higher general drug resistance observed in the cytotoxicity assays
We next aimed to investigate the effect of hybrid 7a in two other
colorectal cancer cells (DLD-1 and Caco-2) to confirm our
assumption that 7a could act as a colon-specific drug (Table 3).
Indeed, the IC50 values of hybrid 7a in Caco-2 and DLD-1 cells
were 15.3 and 8.2 µM, respectively, thus significantly lower than
for hepatic, prostate, breast and blood cancer cells (Table 2, 3).
Notably, hybrid 7a again selectively affected cancer cells and not
normal cells (Table 3). Moreover, all compounds showed solid
tumor selectivity, as no effect was observed in leukemia cells (JKT
and HUT102) even at very high doses (> 250 µM).
(Figure 4 A, B).
To assess if this DNA damage might be caused by a direct
interaction with DNA, pBR322 plasmid DNA was incubated with
increasing concentrations of hybrid 7a or the single drugs (Art and
TQ). Afterwards, the electrophoretic mobility of the plasmid DNA
was analyzed by agarose gel electrophoresis (Figure 4 C).
Doxorubicin (Dox), which is known to intercalate into the DNA
double helical structure,[31] was used as a positive control. Due to
its interaction with double-stranded pBR322 DNA and the hereby
caused extensive topological changes, treatment with Dox led to
a strong shift of the plasmid DNA bands in a concentration-
dependent manner. Retardation of the front most DNA band,
which corresponds to the covalently closed circular (ccc) form of
the plasmid, results from an unwinding of this supercoiled DNA to
an open circular form (oc). In contrast to the positive Dox control,
no band shift and hence no interaction with DNA could be
observed for hybrid 7a or the single drugs, Art and TQ. These
results suggest that a direct interaction with DNA cannot be the
cause of drug-induced DNA damage.
Table 3. IC50 values (in µM) of Art, TQ and hybrid 7a in different colorectal
cancer cells and a normal colon epithelial cell line after 24 h of incubation*
Comp.
Art
HT29
Caco-2
DLD-1
HCEC
19.0 ± 5.4
60.3 ± 4.1
2.8 ± 0.2
25.5 ± 7.2
7.8 ± 0.8
15.3 ± 5.5
13.0 ± 2.9
35.0 ± 1.8
8.2 ± 2.4
84.8 ± 18.3
141 ± 30.7
>100
TQ
7a
*
IC50 value of clinically used anticancer drug 5-fluorouracil: IC50 (HT29) =
In a second ethidium bromide fluorescence-based (EtBr) assay
we analyzed the potential interaction of the single drugs (Art &
TQ) and hybrid 7a with linear double-stranded DNA (linearized
pBR322 plasmid), again applying Dox as a positive control. None
of the three tested drugs showed any interaction with the
linearized plasmid.
3
1.4 ± 5.2 µM.
For the next experiments we selected HCT116 and HT29
colorectal cancer cells that differ in their p53 status (HCT116:
wildtype, HT29: mutant p53-R273H). We studied the effects of
hybrid 7a versus combination treatment with 1:1 mixture of both
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Figure 2. Viability of HCT116 and HT29 cells after treatment with hybrid 7a in different concentrations (0.05 – 500 µM). Cell viability was assessed after 24 h and
8 h by crystal violet assay and is expressed as percentage of DMSO control. Significance between hybrid treatment and the DMSO control is indicated by * (p<0.05)
4
and ** (p<0.001) as determined by one-way Anova analysis (SPSS, version 24). Error bars denote standard deviation of means calculated from six technical
replicates. Note: viability measurements for hybrid 7a have been done several times during a period of 12 months and a range of IC50 between 2.3 µM and 11.2 µM
have been detected.
Figure 3. Viabiltiy of HCT116 (A, B) and HT29 (C, D) cells after treatment with TQ (1, 2.5, 5 µM), Art (1, 2.5, 5 µM), the combination of TQ and Art (1:1; 2.5:2.5,
5:5 µM), or hybrid 7a (1, 2.5, 5 µM) for 24 h (A, C), and 48 h (B, D). The viability was assessed by crystal violet assay and is expressed as percentage of DMSO
control. Significances between hybrid and other treatments are indicated by * (p<0.05) and ** (p<0.001), while all treatments that significantly differed from the
DMSO group are marked with # (p<0.001). Error bars denote standard deviations of eight technical replicates. Levels of active and cleaved PARP as assessed by
western blot analysis. The effects are shown for TQ, Art, the combination of the single drugs, and hybrid 7a in HCT116 (E) and HT29 (F) cells after 24 and 48 h.
GAPDH was used as a loading control. In Figure 3F two bands have been spliced out (*).
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Figure 4. (A, B) Detection of -H2AX by western blot analysis in HCT116 (A) and HT29 (B) cells after treatment with 5 M of Art, TQ, the combination of the single
drugs or the hybrid 7a. The cells were harvested after different time points (24, 48 h). The specific primary antibodies used are -H2AX and GAPDH was used as a
loading control. (C) Interaction of doxorubicin (Dox), Art, TQ or hybrid 7a with circular pBR322 plasmid DNA as determined by electrophoretic mobility shift assays
(
EMSAs) after 24 h of incubation. Pictures are representative of at least two independent experiments ( oc: open circular; ccc: covalently closed circular). (D)
Relative ROS levels in HCT116 cells after treatment with increasing concentrations of Dox, Art, TQ or hybrid 7a (1 – 50 µM) as assessed in NBT assays. Values
represent means ± SD of two independent experiments in duplicate. (E) ROS dependent cytotoxicity of Dox, Art, TQ, hybrid 7a and the combination of the single
drugs (Art and TQ) in HCT116 and HT29 cells as determined in MTT assays after 24 h of incubation with different concentrations of the test compounds (25 µM,
5
0 µM and 100 µM) in the absence or presence of 10 mM NAC. Values represent means ± standard deviation (SD) of two independent experiments in quadruplicate.
F) Detection of -H2AX, PARP, H2AX by western blot analysis in HCT116 and HT29 cells after 48 h treatment with 25 µM of hybrid 7a in the absence or presence
of 10 mM NAC. GAPDH was used as a loading control. In figure 4 A, B two bands have been spliced out (*).
(
Only Dox strongly reduced EtBr fluorescence in the applied lower
concentrations (5 and 10 µM) due to an inhibition of intercalation
production. For Art and TQ ROS-dependent mechanisms of
action have already been reported.[19,33] Thus we evaluated the
intracellular ROS levels in HCT116 cells after treatment with
different doses of Dox, Art, TQ, hybrid 7a or the combination of
the single drugs (Art and TQ) after 24 h of incubation with the test
compounds (Figure 4 D). We saw that ROS induction was clearly
dose-dependent in all treatment settings (Figure 4 D). In a next
sites in the DNA (Suppl. Figure 5). However, Dox is
a
fluorochrome itself emitting light in the same wavelength range as
EtBr and its fluorescence intensity is increased strongly upon
intercalation into DNA just as it is the case for EtBr.[32] Thus, in the
samples of Dox treated DNA a stronger fluorescence intensity
was detected at the applied higher concentrations (25 and 50 µM), step, HCT116 cells were incubated with 25, 50, and 100 µM Dox,
which is most likely only the result of a great amount of Dox
molecules interacting with the DNA (= higher fluorescence
intensity).
Another possibility for DNA damage induction might be drug-
associated intracellular reactive oxygen species (ROS)
Art, TQ, hybrid 7a or the combination of the single drugs, in the
absence or presence of the ROS scavenger N-acetyl-cysteine
(NAC) in order to evaluate whether the cytotoxicity of the test
compounds is dependent on ROS (Figure 4 E). Although the
toxicity of all tested compounds measured by MTT-assay was
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considerably decreased by the addition of NAC in HCT116 cells,
the generation of H2AX protein and PARP cleavage after hybrid
treatment were not affected in both, HCT116 and HT29 cells
starting material 6a: 12.0 mg (0.05 mmol, 0.8 eq). R
(hexane/EtOAc 1:1, UV and molybdato phosphate). Anal. calcd.
for C32 10: C, 65.29; H, 7.53; Found: C, 65.01; H, 7.32.
Thymoquinone-artesunic acid hybrid 7b. Art (119 mg,
0.31 mmol, 1.5 eq), DMAP (11.4 mg, 0.09 mmol, 30 mol%), TQ
alcohol 6b (43.0 mg, 0.21 mmol, 1.0 eq), dry CH Cl (3.0 mL),
DCC (63.9 mg, 0.31 mmol, 1.5 eq). Column conditions:
hexane/EtOAc 4:1. Yield: 97.7 mg, 0.17 mmol, 80%. R = 0.81
(hexane/EtOAc 1:1, UV and molybdato phosphate). Anal. calcd.
for C31 10: C, 64.79; H, 7.37; Found: C, 64.62; H, 7.53.
Thymoquinone-artesunic acid hybrid 7c. Art (146 mg,
.38 mmol, 1.5 eq), DMAP (13.9 mg, 0.11 mmol, 30 mol%), TQ
alcohol 6c (60.0 mg, 0.25 mmol, 1.0 eq), dry CH Cl (3.5 mL),
DCC (78.6 mg, 0.38 mmol, 1.5 eq). Column conditions:
f
= 0.82
44
H O
(Figure 4 F). Thus, we suggest other ROS species that are not
scavenged by NAC to be responsible for H2AX formation and
apoptosis induction by the hybrid 7a. Otherwise, Lambert et al.[
reported that ROS might interact with a quinone-derived drug
34]
2
2
(epigallocatechin-3-gallate) to produce an intracellular active
f
compound, which finally could form toxic mediators with NAC.
42
H O
0
Conclusions
2
2
In conclusion, in our study seven thymoquinone-artemisinin
hybrids 7a-c, 9a/b and 11a/b were successfully synthesized and
investigated for the first time for their anticancer activity in different
cancer cell lines (colon, breast, prostate, liver and leukemia).
Remarkably, all novel thymoquinone-artemisinin hybrids showed
a high specificity for colon cancer cells. In particular, hybrid 7a
was the most active compound against the tested colorectal
cancer cell lines (with a IC50 of 2.4 µM in HCT116 cells and 2.8
µM in HT29 cells) and thus was more potent, with up to 20-fold
higher activity, than the parental compounds Art and TQ, while
being nontoxic to non-malignant colon epithelial cells (IC50
hexane/EtOAc 4:1. Yield: 131 mg, 0.22 mmol, 86%. R
f
= 0.22
(
hexane/EtOAc 4:1, UV and molybdato phosphate). Anal. calcd.
for C33 10: C, 65.76; H, 7.69; Found: C, 65.65; H, 7.81.
Thymoquinone-artemisinin-derived acid hybrid 9a.
46
H O
Acid 8
(
3
(
41.0 mg, 0.13 mmol, 1.0 eq), DMAP (46.2 mg, 0.38 mmmol,
.0 eq), TQ alcohol 6a (28.0 mg, 0.13 mmol, 1.0 eq), dry CH Cl
4.2 mL), DCC (77.9 mg, 0.38 mmol, 3.0 eq). Column conditions:
2
2
hexane/Et
hexane/Et
: C, 67.90; H, 7.98; Found: C, 68.18; H, 8.11.
Thymoquinone-artemisinin-derived hybrid 9b.
2
O 1:1. Yield: 26.0 mg, 0.05 mmol, 39%. R
2
O 1:1, UV and molybdato phosphate). Anal. calcd. for
f
= 0.49
(
30 42 8
C H O
Acid 8
>100 µM). Moreover, selected hybrid 7a was more effective than
(
1
(
59.4 mg, 0.18 mmol, 1.0 eq), DMAP (27.8 mg, 0.23 mmmol,
.3 eq), TQ alcohol 6c (43.0 mg, 0.18 mmol, 1.0 eq), dry CH Cl
3.0 mL), DCC (46.9 mg, 0.23 mmol, 1.3 eq). Column conditions:
hexane/EtOAc 1:1 and hexane/EtOAc 2:1. Yield: 31.5 mg,
.06 mmol, 32% yield. R = 0.67 (hexane/EtOAc 1:1, UV and
molybdato phosphate). Anal. calcd. for C31 : C, 68.36; H,
.14; Found: C, 68.37; H, 8.04.
the combination treatment with different 1:1 mixtures of both
single drugs. This is an encouraging result since the activity of
hybrid 7a against both colon cancer cell lines (HCT116 and less
sensitive cells HT29) was even superior to that of a clinically used
drug 5-FU. The molecular mechanism for the specific activity of
hybrid 7a in colorectal cancer cells seems to be accumulation of
double strand breaks via specific ROS-subspecies that are not
captured by NAC. Further in vitro and in vivo investigations will
provide a deeper understanding of the involved signaling
pathways and molecular targets of this promising novel drug
candidate. Finally, these results are another excellent proof of the
hybridization concept and also confirm that the type of linkage
2
2
0
f
44 8
H O
8
General procedure for hybrids 11a/b: Dihydroartemisinin
acetate 10 (1.0 eq) and the corresponding TQ alcohol 6a/c
(
1.1 eq) were dissolved under N
cooled to 0 °C. Then TMSOTf (0.1 eq) was added under N
°C. The resulting reaction mixture was stirred at 0 °C for 1 h
and afterwards quenched with sat. Na CO (1.5 mL). Additional
O was added and the aqueous phase extracted with either
CHCl (3 x 2 mL) in the case of ether hybrid 11a or with EtOAc (3
x 20 mL) in the case of ether hybrid 11b. The combined organic
layers were washed with H O, dried over NaSO , filtered and
2
in dry CHCl
3
(1.4 mL) and
2
at
0
(
ester or ether) and the linker length play a crucial role for the
2
3
H
2
biological activity of a hybrid drug.
3
2
4
Experimental Section
concentrated under reduced pressure. The crude product was
purified by column chromatography in order to obtain hybrid 11a/b
as an orange to yellow gum.
Experimental Details. The purity of all hybrids was approved with
Elemental Analysis and was >95%.
General procedure for hybrids 7a-c and 9a/b: A solution of
Art (2) or artemisinin-derived acid 8, the corresponding TQ
Thymoquinone-artemisinin hybrid 11a
(configuration:
0β/10α 8:1). Dihydroartemisinin acetate 10 (62.5 mg, 0.19 mmol,
.0 eq), TQ alcohol 6a (46.8 mg, 0.21 mmol, 1.1 eq), TMSOTf
1
1
2 2
alcohol 6a/b or c and DMAP in dry CH Cl was cooled to 0 °C.
(
3.47 l, 4.26 mg, 0.02 mmol, 0.1 eq). Column conditions:
hexane/EtOAc 3:1, 2:1. Yield: 39.8 mg, 0.08 mmol, 43%.
= 0.34 (hexane/Et O 1:1, UV and molybdato phosphate). Anal.
calcd. for C28 : C, 68.83; H, 8.25; Found: C, 69.29; H, 8.27.
Thymoquinone-artemisinin hybrid 11b (configuration:
0β/10α 8:1). Dihydroartemisinin acetate 10 (45.1 mg, 0.14 mmol,
.0 eq), TQ alcohol 6c (36.0 mg, 0.15 mmol, 1.1 eq), TMSOTf
After addition of DCC the reaction mixture was slowly warmed to
room temperature and stirred overnight. The precipitated
dicyclohexylurea was removed by filtration and the solvent was
removed under reduced pressure. The residue was purified by
column chromatography and thereby hybrid 7a-c or 9a/b was
obtained either as a yellow solid or a yellow gum.
R
f
2
40 7
H O
1
1
Thymoquinone-artesunic acid hybrid 7a.
.07 mmol, 1.0 eq), DMAP (2.60 mg, 0.02 mmol, 30 mol%), TQ
alcohol 6a (31.0 mg, 0.14 mmol, 2.0 eq), dry CH Cl (2.0 mL),
Art
(27.0 mg,
(
2.50 µL, 3.07 mg, 0.01 mmol, 0.1 eq). Column conditions:
hexane/EtOAc 9:1. Yield: 36.0 mg, 0.07 mmol, 52%. R = 0.19
hexane/EtOAc 9:1, UV and molybdato phosphate). Anal. calcd.
for C29 : C, 69.30; H, 8.42; Found: C, 69.19; H, 8.79.
0
f
2
2
(
DCC (15.9 mg, 0.08 mmol, 1.1 eq). Column conditions:
hexane/EtOAc 4:1. Yield: 34.0 mg, 0.06 mmol, 83%; regained
42 7
H O
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Supporting Information
F. Nogueira, R. Amewu, J. Gut, P. J. Rosenthal, R. Oliveira, M. M. Mota,
R. Moreira, F. Marti, M. Prudêncio, P. M. O’Neill, F. Lopes, ACS Med.
Chem. Lett. 2013, 5, 108-112.
Experimental conditions and procedures for intermediates 4a-c
and 6a-c; spectral data of intermediates 4a-c, 6a-c and target
compounds 7a-c, 9a/b and 11a/b; recorded spectra of target
compounds; details of cell lines and reagents as well as cell
viability assay for biological evaluation. This material is available
free of charge via the Internet at http://.
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Entry for the Table of Contents
FULL PAPER
T. Fröhlich, B. Ndreshkjana, J. K. Muenzner,
C. Reiter, E.Hofmeister, S. Mederer, M.
Fatfat, C. El-Baba, H. Gali-Muhtasib,
R. Schneider-Stock* and S.B. Tsogoeva*
Page No. – Page No.
Synthesis of Novel Hybrids of
Thymoquinone and Artemisinin with
High Activity and Selectivity
Against Colon Cancer
[
a]
Organic Chemistry Chair I and Interdisciplinary Center for Mole
Materials (ICMM), Friedrich-Alexander University of Erlangen-
Nürnberg, Henkestr. 42, 91054 Erlangen, Germany.
E-mail: svetlana.tsogoeva@fau.de
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