Synthesis and pharmacological in vitro investigations of novel shikonin derivatives with a special focus on cyclopropane bearing derivatives

Article abstract of DOI:10.3390/ijms22052774

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constitu

Full text of DOI:10.3390/ijms22052774

International Journal of
Molecular Sciences
Article
Synthesis and Pharmacological In Vitro Investigations of Novel
Shikonin Derivatives with a Special Focus on Cyclopropane
Bearing Derivatives
Nadine Kretschmer 1, Antje Hufner 2 , Christin Durchschein 1, Katrin Popodi 1, Beate Rinner 3, Birgit Lohberger 4
and Rudolf Bauer 1,
*
1
Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Beethovenstr. 8,
8010 Graz, Austria; nadine.kretschmer@uni-graz.at (N.K.); christin.durchschein@hotmail.com (C.D.);
katrin.popodi@gmx.at (K.P.)
2
3
4
Department of Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, University of Graz,
Universitaetsplatz 1, 8010 Graz, Austria; antje.huefner@uni-graz.at
Division of Biomedical Research, Medical University of Graz, Roseggerweg 48, 8036 Graz, Austria;
beate.rinner@medunigraz.at
Department of Orthopedics and Trauma, Medical University Graz, Auenbruggerplatz 5, 8036 Graz, Austria;
birgit.lohberger@medunigraz.at
*
Correspondence: rudolf.bauer@uni-graz.at
Abstract: Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all
skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates
are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata
as well as a synthetic derivative of the most active constituent showed promising results in metastatic
melanoma cell lines. In the current study, we address the question whether we can generate further
derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin
derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells)
using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-
yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity.
Furthermore, pharmacological investigations including the ApoToxGloTM Triplex assay, LDH assay,
and cell cycle measurements revealed that this compound induced apoptosis and reduced cells
in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed
hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against
non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be
promising drug leads in the treatment of melanoma.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kretschmer, N.; Hufner, A.;
Durchschein, C.; Popodi, K.; Rinner,
B.; Lohberger, B.; Bauer, R. Synthesis
and Pharmacological In Vitro
Investigations of Novel Shikonin
Derivatives with a Special Focus on
Cyclopropane Bearing Derivatives.
Int. J. Mol. Sci. 2021, 22, 2774.
https://doi.org/10.3390/ijms22052774
Academic Editor: Serena Lembo
Received: 25 January 2021
Accepted: 5 March 2021
Published: 9 March 2021
Keywords: shikonin derivatives; synthesis; apoptosis; cell cycle; melanoma
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Malignant melanoma belongs to the most dangerous type of skin cancer and arises
from melanocytes [1]. Only 2–3% of all diagnosed skin cancers are melanomas, however,
they account for approx. 75% of all skin cancer deaths [
2
]. Melanomas are divided into four
different stages: stages I and II display local primary tumors, stage III represents tumors
with locoregional metastases, and stage IV tumors with distant metastases. In addition,
Copyright:
© 2021 by the authors.
tumors are typically categorized by thickness and ulceration [
are detected at an early clinical stage and treated appropriately, the 10-year survival
rate is around 98% [ ]. However, when the tumor metastasizes, the survival rate drops
dramatically and therapeutic success is limited due to tumor recurrence, severe side effects,
and/or the development of resistances [ ]. According to the Melanoma Research Alliance,
1,3]. When melanomas
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
4
2
the five-year survival rate for metastatic melanoma is currently only 22.5%. Therefore, the
search for novel drug leads remains an important task.
Int. J. Mol. Sci. 2021, 22, 2774. https://doi.org/10.3390/ijms22052774
https://www.mdpi.com/journal/ijms

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In the discovery of novel therapeutics, natural products have always played a cen-
tral role. In the case of anticancer drugs, the FDA approved in total 247 new chemical
entities between 1981 and 2019, including 185 (75%) small molecules. Only 15.7% of the
small molecules were totally synthetic compounds. All others were natural products or
inspired by them [5]. Some of them, such as vinblastine, campthothecin, or taxol are most
important in cancer chemotherapy today [
6
]. Shikonin ( ) and its derivatives are naturally
1
occurring, biologically active naphthoquinones and can be found in several members of
the Boraginaceae family such as Arnebia euchroma (Royle ex Benth.) I.M. Johnst., Lithos-
permum erythrorhizon Siebold et Zucc., and Onosma paniculata Bureau & Franchet Their
pharmacological spectrum comprises anti-oxidative, anti-inflammatory, anti-virus, and
anti-cancer activities [7–10]. Plants containing these constituents are traditionally used for
the treatment of eruptive exanthema, eczema, skin infections, burns, constipation, scalds,
and cancer [11]. Concerning the anti-cancer activity, it has been reported that shikonin and
derivatives led to induction of apoptosis, cell cycle arrest, and autophagy and inhibited cell
growth and metastasis [7–10]. Shikonin was also shown to kill cancer cells synergistically
in combination with established cancer therapeutics such as erlotinib [12].
In previous studies, we could show that
cytotoxic compound isolated from the roots of O. paniculata and exhibited the strongest
cytotoxicity toward several melanoma cell lines [13 14]. Ongoing studies revealed that
favored catabolic processes and caused generation of reactive oxygen species, loss of mito-
chondrial membrane potential, and the upregulation of NOXA expression [15 16]. Finally,
this led to apoptosis, autophagy, and cell cycle arrest. Using a mouse xenograft model,
we could show that exhibited also promising in vivo effects [16]. However, we were
wondering whether the structure of can be modified to further improve and optimize the
pharmacological effects. In a first attempt, we ended up with cyclopropylacetylshikonin ( ),
which exhibited lower IC50 values than in metastatic melanoma cell lines and induced
apoptosis as well [17]. In the current study, we followed a two-fold strategy: (a) optimiz-
ing the cyclopropylacetyl group of our last hit and (b) mounting a broad spectrum of
β,β-dimethylacrylshikonin (2) was the most
,
2
,
2
2
3
2
3
structural features within the acyl residue of shikonin. To cover a broader spectrum of
compounds and for comparison reasons, we also included some already known synthetic
shikonin derivatives. However, most of the compounds are novel or not yet investigated in
this context.
2. Results and Discussion
2.1. Syntheses of Shikonin Derivatives
Shikonin derivatives have been shown to exhibit potent anti-cancer activities [
It was reported that the naphthoquinone scaffold with its hydroxyl groups is necessary for
the pharmacological activity [18 21] and that the side chain modifies the activity. Similar
7–10].
–
features in the well-known anthracycline antitumor antibiotics and mitoxantrone seem to
be important for DNA binding and bioavailability [22]. Therefore, we decided to focus on
modifying the side chain. Shikonin (
Acylation of was accomplished via Steglich esterification in dichloromethane with the
corresponding carboxylic acid, as well as dicyclohexylcarbodiimide (DCC) as a coupling
reagent and 4-dimethylaminopyridine (DMAP) as a catalyst (Figure 1) [17 20 23 27]. NMR
1, 100% R-isomer [17]) was chosen as starting material.
1
,
,
–
spectra of all synthesized derivatives can be found in supplementary material (chapter 2).
Figure 1. Acylation of shikonin (1).

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The first strat0egy was the optimization of the cyclopropylacetate in
3
[
17]. In the
0
bicyclus
4
, 1 and 2 positions of cyclopropylacetate are connected as part of a cyclohexane.
Replacement of the methylene spacer of
3 with a CO group, a C,C double bond, and an
ethylene group resulted in 5, 6, and 7, respectively.
The cyclopropane precursor acids were synthesized in our laboratory. As outlined in
Figure 2, the bicyclic acetic acid p1 was prepared from the corresponding (1-cyclohexenyl)-
2-acetic via Furukawa modification of the Simmons–Smith reaction analogously to a
procedure of Renaud and Fox [28]. 3-Cyclopropylpropanoic acid (p2) was prepared by
α
-alkylation of acetic acid in analogy to a procedure described by Barczak and Jarvo [29
(Figure 3). The Knoevenagel condensation of cyclopropanecarbaldehyde with malonic
acid [30] resulted in -cyclopropylacrylic acid p3. 2-Cyclopropyl-2-oxoacetic acid (p4
]
β
)
was obtained by oxidation of acetylcyclopropane with KMnO4 in a combination of the
procedures given by Prokopenko et al. [31] and Xu et al. [32] (Figure 4). Details of the
syntheses of the precursor acids are described in material and methods. Their NMR spectra
can be found in the supplementary material (chapter 3). In all cases (precursor acids and
shikonin derivatives), the structure and purity of each compound were analyzed using 1D
and 2D NMR, LC-MS, and IR experiments.
Figure 2. Synthesis of the bicyclic acetic acid p1.
Figure 3. Synthesis of 3-cyclopropylpropanoic acid (p2).
Figure 4. Synthesis of precursor acids p3 and p4.
The second goal was to produce a broad spectrum of structural features within the acyl
residue of shikonin to find potential novel drug leads. For better comparison and a more
complete picture, we also included some already known derivatives. Phenylacetate
cinnamate showed, for example, cytotoxicity in previous studies and were synthesized for
comparison reasons [26 33]. As there have been no reports about any shikonin alkynylacyl
8 and
9
,
esters yet, we synthesized tetrolate 10, 2-butynoate 11, and 3-phenylpropiolate 12 to
investigate the influence of a triple bond.

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Except for three recently published studies about benzoylacrylates [34], succinamides,
and maleinamides [35
nin esters were part of acetoxy groups mainly derived from
,
36], all carbonyl groups in the acyl chain of shikonin or alkan-
-hydroxy acylates. How-
β
ever, the substances of Sun et al. [34] are of unknown chirality, among them the p-
methylbenzoylacrylate 13. We also had a look on derivatives with the carbonyl carbon
as a part of the carbon chain—either as a ketone or as part of a diester. Pyruvate 14 and
2-oxo-2-phenylacetate 15 represent α-ketoacylates. γ-Carbonyls are found in the keto esters
13 and 16 as well as in the diesters derived from monomethyl succinate 17 and monoethyl
fumarate 18.
The known short chain alkyl and alkenyl esters 19 (isobutyrate) [37], 20 (isovaler-
ate) [38], 21 (crotonate) [39], and 22 (sorbate) [38] were used to examine the influence
of the saturation and branching on the activity. Compounds 19, 20, and 22 are already
known to inhibit the growth of other types of cancer cells [23,37]. However, there have
been no reports about their effects in melanoma cells. Derivative 21 was one of the very
first non-natural 10-O-acyl shikonines and shialkines [39], but no data about the biological
activity have been reported so far.
Other features, which we investigated, were carbocyclic and heterocyclic aryl groups.
The most basic structures, i0.e., phenylacetyl, benzoyl, and diphenylacetyl (
8, 23, 24), were
among the early synthetic 1 -O-acyl shikonines and shialkines. They were reported to bind
to tubuline [38] and were cytotoxic against several cancer cell lines [23
no data were published about their effects in melanoma cells. Shikonin cinnamate
intensively explored for its anticancer properties, too [26 41 42]. We used these derivatives
double bond with the help of 3-phenylpropionate
,28
,40], however,
9
was
,
,
to investigate the influence of the α,β
25 [36].
Additionally, as a p-dimethylamino group improved the activity of phenylacetate 8 [40]
we prepared the p-dimethylaminocinnamate 26. Nicotinate 27 was reported as quite inac-
tive [27 43], but more active than benzoate 23 44]. Therefore, we tested nicotinate 27 and,
,
,
[
additionally, isonicotinate 28. According to literature data, 3-(3-indolyl)propionate 29 showed
up as the most active compound in another study [21]. As shikonin 2-furylcarboxylate was
reported to be active [45] and we found that cinnamate was more active than benzoate,
we prepared 3-(2-furyl)acrylate 30. The cytotoxicity of bromoacetate 31 was reported
among other halogenated acetates [46]. Therefore, we prepared 31 together with novel
ω-bromoalkylacylates 32, 33, and 34 to study the influence of chain length.
2.2. Results of the XTT Screening
All prepared shikonin derivatives (see Table 1, compounds
4 to 34) were subjected to
a cytotoxicity screening using the XTT viability assay. This assay is based on the activity
of mitochondrial dehydrogenases. These enzymes cleave the yellow tetrazolium salt XTT
leading to an orange formazan. This conversion only occurs in viable cells and can be
directly quantified by measuring the absorbance [47]. Melanoma cells were treated with
1.0
the Supplementary Material). In brief, the most active derivatives were
29, with being the most cytotoxic. Its IC50 values are listed in Table 2. Moreover, the
derivatives 11 and 28 showed no cytotoxicity at 10 M, the derivatives 16 20, and 27
exhibited only a very weak cytotoxicity at 10 µM (Supplementary Figures S1 and S2).
µM, 5.0
µM, and 10.0
µM of each derivative for 72 h (Figure 5; Figures S1 and S2 in
5
,
6
,
9,
14, 24, and
5
µ
4
,
,

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Table 1. Structures of shikonin (1) and derivatives thereof.
OH
O
OR
5'
8
1
4
4'
1'
6'
5
OH
O
Compound
1
R
Compound
2
R
O
H
∗
O
∗
3
5
4
6
O
O
O
O
7
8
O
O
9
10
12
14
O
11
13
15
16
O
C2H5O
17
19
18
20
O
O
O

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Table 1. Cont.
Compound
21
R
Compound
22
R
O
O
23
25
24
26
O
O
O
27
29
28
30
N
N
O
O
N
O
H
31
33
32
34
O
Br
Table 2. IC50 values (
µM) of 2, 3, and 5 after 72 h of treatment and as determined using SigmaPlot
14.0 and the four-parameter logistic curve (n = 6, mean ± sem). n.d. = not determined.
IC50 Values of 5
IC50 Values of 3 [17]
IC50 Values of 2 [14]
Cell Line
(µM)
(µM)
(µM)
WM9
WM164
MUG-Mel2
HEK293
1.5 ± 0.1
4.5 ± 0.5
2.4 ± 0.2
3.4 ± 0.2
3.2 ± 0.8
4.9 ± 1.7
3.2 ± 0.3
5.4 ± 0.7
2.7 ± 0.3
8.3 ± 0.3
7.2 ± 0.5
n.d.

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Figure 5. Results of the XTT assay. For clarity reasons, only the results of the treatment with 5.0
for 72 h are shown. The complete results can be found in the Supplementary Material (Figures S1
and S2). was tested as a reference at 5.0 M. The strongest cytotoxicity was found for (n = 6,
mean sem). Vinblastine was used as positive control. At a concentration of 0.01 nM, it reduced the
cell viability compared to control cells to: WM9 cells: 23.8 1.5%, WM164 cells: 59.4 4.4 %, and
MUG-Mel2 cells: 65.0 ± 6.9 % (n = 6, mean ± sem, 72 h of treatment).
µM
2
µ
5
±
±
±
Discussed in more detail, the results showed that the sensitivity of the three cell lines
used was different. In general, WM164 cells reacted least to the treatment. Only a few
compounds showed moderate activity, for example, the known derivatives 9, 24, and 29,

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as well as the novel derivative 14. WM9 cells were most affected by the derivatives. Next
to
derivatives in this cell line. MUG-Mel2 cells reacted in the case of some compounds (18
21 22 30 33, and 34) more sensitive to the treatment than the WM9 and WM164 cells. This
5
(novel), compounds
6
(novel),
9
(known), and 14 (novel) were the most cytotoxic
,
19
,
,
,
,
is of special interest, because MUG-Mel2 cells are NRAS mutated, while WM9 cells and
WM164 cells are BRAF mutated. The mutational status is another category in melanoma
diagnosis because some mutations lead to a poorer prognosis than others do. Around 50%
of all melanomas exhibit a mutation in the BRAF gene and another 25% a mutation in the
NRAS gene [48]. Tumors carrying a BRAF mutation are currently typically treated with a
combination of MEK and BRAF inhibitors, however, tumor resistances often develop [49].
In the case of NRAS mutated melanoma, the therapeutic success is even lower because they
are more difficult to treat [50]. This means that the most active derivatives of our study, can
also display lead compounds for the development of further novel shikonin derivatives
with a special focus on NRAS mutated melanoma cells.
Returning to the present results, we firstly modified the structure of our previous
hit
significantly. The modification of the spacer showed unequal effects: replacement of the
methylene group with carbonyl ( ) or ethylene ( ) showed a similar activity on the cell
lines with being more cytotoxic towards WM9 cells. An unsaturation resulted in less
3. Further substituents at the cyclopropane reduced the activity against all cell lines
5
7
5
α,β
activity towards MUG-Mel2 cells but in an increased effect in WM9 cells.
When analyzing the other investigated structural features, it became obvious that
hydrogenation of the acyl side chain of
cell lines. Shortening the side chain and removal of the branching
2
reduced the cytotoxicity to a very low level in all
-methyl are among the
β
few modifications, which had a bigger influence on the activity in WM9 cells than in MUG-
Mel2 cells. The additional conjugated double bond in the sorbate 22 had no significant effect.
However, the attachment of a phenyl group restored the properties to the activity level
of
of the exocyclic double bond of the cinnamic residue lowered activity significantly. The
removal or shortening of the spacer slightly improved the activity (compare and 23).
The replacement of the phenyl ring of with a furan moiety (30) abolished the activity
in WM9 cells but kept some effects in MUG-Mel2 cells. The diphenylacetate 24 and
indolylpropionate 29 appeared as active as the cinnamate and as . The replacement of
the phenyl ring in benzoate 23 by a 3-pyridine or 4-pyridine abolished the cytotoxicity.
Moreover, the activity of the oxo esters 13 16 17, and 18 was generally low. While
2. The p-dimethylamino group had no effect on the activity and, again, hydrogenation
8
9
9
2
γ
,
,
WM9 cells were less affected by saturated and unsaturated compounds, MUG-Mel2 cells
showed a slight sensitivity towards unsaturated compounds. The introduction of an
α
carbonyl in 10-O-acetylshikonin resulted in pyruvate 14 which showed an improved
activity in WM9 cells, but a reduced one in MUG-Mel2 cells. Also,
benzoate 23, resulting in 2-oxo-2-phenylacetate 15, affected the cell lines differently. All
the alkynylacyl esters (10 11, and 12) showed no or only marginal effects on all cell lines.
Even though -bromo compounds 31 32, 33, and 34 appear more active, their potency is
inferior to 2 and display no regularity concerning chain length and activity.
In summary, appeared as the most cytotoxic compound in this series and, there-
fore, was investigated pharmacologically in more detail. First, was also tested in non-
tumorigenic HEK293 cells. As shown in Table 2, the IC50 value after 72 h was 3.4 M,
α-carbonylation of the
,
ω
,
5
5
µ
which is 2.3-fold higher than in WM9 cells, but also 1.3-fold lower than in WM164 cells.
The cytotoxicity of chemotherapeutics against non-tumorigenic cells is a known problem
in cancer therapy and one reason of undesirable side effects. For example, vinblastine
and doxorubicin are two well-known and often clinically administered chemotherapeutics.
In in vitro experiments, both also show cytotoxicity towards non-tumor cells to a similar
or even greater extent [14
in vivo effects. Nevertheless, there is a certain risk that
they are already known for clinically used chemotherapeutics. However, it has already
been shown for shikonin that there are ways to overcome this problem. Fayez et al. [53
,
51
,52]. However, it is difficult to extrapolate from in vitro to
5
will also cause side effects as
]

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reported recently that a combination of shikonin and silver nanoparticles synergistically
inhibited the growth of lung cancer cells. Wang et al. [54] have shown that shikonin and
JQ1, a bromodomain and extra-terminal motif (BET) inhibitor, encapsulated in lactoferrin
nanoparticles changed the tumor immune microenvironment, activated immunogenic cell
death, repolarized protumor phenotype, tumor-associated macrophages, and repressed
glucose metabolism. They concluded that their system can be developed as a novel cancer
immunotherapy due to several synergistic advantages. Another strategy could be a further
development of our compounds to oxime derivatives. Huang et al. [55] demonstrated
that such derivatives exhibited strong cytotoxicity towards cancer cells, but only a low
cytotoxicity towards human skin fibroblasts.
2.3. Pharmacological Effects of Cyclopropyloxoacetate 5
To investigate the effects of
performed. Since was most active in the metastatic cell line WM9 (BRAF mutated), we
decided to use this cell line and, in addition, another BRAF mutated cell line (WM164) for
comparison. Using the ApoToxGlo Triplex Assay, we investigated the effects regarding
5 in more detail, several pharmacological assays were
5
™
cell viability, cytotoxicity, and apoptosis induction in more detail (Figure 6). The advantage
of this assay is the combination of three assays in one single assay well. It has already
been shown by our [14
apoptosis in a variety of cancer types. We treated the cells with up to 20
to 48 h. Regarding cell viability, we found no statistically significant changes up 10
after 4 h and 24 h and up to 5 M after 48 h. In the case of cytotoxicity, the fluorescence
,
16] and other groups [
8
,9
] that several shikonin derivatives induced
µ
M of for up
5
µM
µ
intensity decreased time- and dose-dependently, which can be an indicator for primary
necrosis in combination with a reduced viability. Concerning apoptosis, we found a clear
increase in caspase 3/7 activity after 24 h and 48 h in WM9 cells and a slight increase after
24 h in WM164. This agrees with the effects of our previous hit
3 [17] even if the activation
of caspase 3/7 was weaker in case of
activated during the treatment with
cell death.
5
5
. In summary, our results indicate that caspases are
further indicating that the cells undergo apoptotic
To investigate necrosis induction, we performed the CytoTox 96® Non-Radioactive
Cytotoxicity Assay (LDH assay) (Figure 7). Lactate dehydrogenase (LDH) is released into
the cell culture medium when the cell membrane is damaged. Therefore, this enzyme can
be used as a marker for measuring necrotic cell death [56]. Cells were treated with
up to 20 M for up to 72 h. We found no LDH release up to 10 M, which is 6.7-fold higher
than the IC50 in WM9 cells and 2.2-fold higher than the IC50 in WM164 cells after 72 h.
When the cells were treated with 20 , a slight increase of LDH release was found.
5 with
µ
µ
µM 5
However, compared to the maximal possible LDH release during complete cell lysis, the
measured amount of LDH released by treated cells was generally quite low. In addition,
these changes at 20
48 h: p = 0.115; 72 h: p = 0.269. In WM9 cells, the changes were statistically significant
only at the highest tested concentration of 20 M (p = 0.0197). In summary, necrosis seems
µM were not statistically significant in WM164 cells: 24 h: p = 0.182;
µ
to play only a minor role during the observed cells death at the concentrations tested.
Nevertheless, it has been reported for shikonin that it induced necrosis in cancer cells such
as lung and gastric cancer cells [57
,58]. Cellular lysis appears also during the process of
necroptosis [59] which has also been reported for shikonin [60]. Therefore, the increase
of LDH release could also point to the induction of necroptosis by
investigated in future studies.
5, which should be

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Figure 6. Results of the ApoToxGlo
M, 10 M, and 20 M of for 4 h, 24 h, or 48 h (n = 6, mean
measured as relative fluorescence of control cells. (
as relative fluorescence of control cells. (
induction. Staurosporine (25 M) served as positive control (apoptosis increase in WM9 cells after
24 h: 1193.9 % and after 48 h: 297.4%; in WM164 cells after 24 h: 989.1% and after 48 h: 362.6%).
™
Triplex Assay. WM9 and WM164 cells were treated with 1
µ
M,
) Viability of the cells
towards the cells measured
5
µ
µ
µ
5
±
sem). (A
5
B
) Cytotoxicity of
C) Activity of caspases 3 and 7 indicative for apoptosis
µ

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Figure 7. Results of the LDH assay. WM9 and WM164 cells were treated with 1
µM, 5 µM, 10 µM,
and 20 M of for 24 h, 48 h, or 72 h (n = 9, mean sem). Results are displayed as percentage of
µ
5
±
cell lysis. Control = vehicle treated cells (0.5% EtOH). Only at 20
were found.
µM, slight increases in LDH release
Finally, we investigated the effect of
shikonin derivatives are able to bind to tubulin and, therefore, lead to cell cycle arrest [43
Also, in the case of , we found a cell cycle arrest in different types of melanoma cell
lines [14]. Therefore, we treated the cells with up to 20 M of for up to 48 h (Figure 8).
Only at higher concentrations, changed the cell cycle distribution statistically significant
(Table 3). We also tested the effect of 5 on the cell cycle. However, had no effect on
the cell cycle at this concentration (data not shown). Our results agree with other studies
5
on the cell cycle. It has been reported that
,
61].
2
µ
5
5
µM
5
5
since Baloch et al. [43] reported a quite high IC50 of 25.28
inhibitory effect on tubulin polymerization.
µM for shikonin concerning its
Figure 8. Effects of 5 on the cell cycle. (A) WM9 and (B) WM164 cells were treated with 10 µM and 20 µM of 5 for 24 h or
48 h (n = 6, mean). Results are displayed as percentage of cell cycle distribution. Control = vehicle treated cells (0.5% EtOH).
5 influence the distribution of the cell cycle only at high concentrations.
Table 3. Calculated p-values when comparing the amount of control and treated cells in the G2/M
phase (student’s t-test, n = 6). The number of cells in the S-phase was not statistically signifi-
cantly changed.
Cell Line
10 µM (24 h)
20 µM (24 h)
10 µM (48 h)
20 µM (48 h)
WM9
WM164
0.0311
0.3313
0.0014
0.0153
0.1467
0.0553
0.0515
0.0065

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3. Materials and Methods
3.1. Chemicals
Shikonin was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu,
China). -Dimethylacrylshikonin ( ) was isolated from dried roots of Onosma paniculata
β,β
2
Bureau & Franchet (Boraginaceae) and identified as reported previously [14].
3.2. Synthesis of Shikonin Derivatives
The synthesis of shikonin derivatives is described below. Their NMR spectra can
be found in the supplementary material, chapter 2. The purity of all compounds was
analyzed using NMR experiments and always exceeded 95%. LC-ESI-MS measurements
were performed on a Dionex Ultimate 3000 UHPLC (Thermo, San José, CA, USA). It was
coupled with a Thermo LTQ XL linear ion trap mass spectrometer equipped with an
H-ESI II probe (n◦egative mode). The acquisition wavelength was 500 nm, source heater
◦
temperature: 250 C, capillary temperature: 200 C, source voltage: 3.5 kV, sheath gas flow:
50 arbitrary units, capillary voltage:
−
14 V, and auxiliary gas flow: 10 arbitrary units. A
Kinetex C18 column (2.6 m, 100 2.10 mm, Phenomenex, Torrance, CA, USA) was used
µ
×
as stationary phase. Water (A) and acetonitrile (B) were used as mobile phases (gradient
program: 0–45 min: 55–100% B, flow rate: 0.2 mL/min, column temperature: 30 ◦C).
3.3. General Procedure for the Acylation of Shikonin
A solution of shikonin in abs. CH2Cl2 (0.1 mmol/5 mL) was cooled to 0 ◦C under
argon atmosphere and DCC was added. After 15 min of stirring, DMAP was added.
After an additional 15 min stirring, the corresponding acid was added and stirred for
another 5.5 h to 5 days with slowly warming up to room temperature. Afterwards, 1 mL
cyclohexane/0.1 mmol shikonin was added and the mixture was concentrated at room
temperature and under reduced pressure to ca. 0.5 mL/0.1 mmol shikonin. The mixture
was then filtered over 3 mm silica and 2 mm celite® (eluent: petroleum ether/CH2Cl2 = 1:0
to 1:2). The resulting fractions were evaporated and subjected to flash CC and/or repeated
PTLC (cyclohexane/CH2Cl2 mixtures).
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-(bicyclo[4
.1.0]heptan-1-yl)acetate (4), 50 µmol Shikonin, 0.18 mmol DCC, 15 µmol DMAP and 57 µmol
2-(1-bicyclo[4.1.0]heptyl)acetic acid (p1); reaction time 15 h; CC on silica (4 g; cyclohexane
to cyclohexane/CH2Cl2 = 2:1; PTLC on silica with cyclohexane/CH2Cl2 = 2:1 (three times
developed);
4
, yield: 25%.
4
: Rf = 0.49 (silica, CH2Cl2); IR (ATR): 2990 (w),
≈
2950 (br) (OH),
2927 (w), 2856 (w), 1736 (m) (C=O), 1607 (s), 1568 (m), 1451 (m), 1263(m), 1230 (m), 1202
(m) (COC), 1141 (s), 782 (m) cm−1
;
H-NMR (CDCl3): 0.33, 0.35 (2t, 2H J
≈
5 Hz H-70’),
1
0.50–0.58 (m, 2H, H-70’), 0.84–0.92 (m, 2H, H-20’), 1.09–1.37 (m, 8H, H-40’, H-50’), 1.55–1.64
(m, 2H, H-30’), 1.58 (s, 6H, H-60), 1.69 (s, 6H, H-50), 1.68–1.80 (m, 4H, H-60’), 1.90–2.00 (m,
2H, H-30’), 2.20–2.34 (m, 4H, H- ), 2.42–2.52 (m, 2H, H-20), 2.58–2.67 (m, 2H, H-20), 5.14
α
(tquint, J = 7.3, 0.9 Hz, 2H, H-30), 6.04 (J = 7.3, 4.4, 0.8 Hz, 2H, H-10), 7.00 (d, J = 0.9 Hz,
H-3), 7.01 (d, J = 1.0 Hz, 1H, H-3), 7.19 (s, 4H, H-6, H-7), 12.43 (s, 2H, C5-OH), 12.59 (s,
2H, C8-OH); 13 C-NMR (CDCl3):
δ
16.7 (C-10’), 2x 17.0 (C-70’), 17.7, 17.8 (C-20’), 18.0 (C-60),
2x 21.0 (C-40’), 2
×
21.4 (C-50’), 2
), 2
69.2 (C-10), 111.6 (C-4a), 111.8 (C-8a), 118.0 (C-30), 2
132.8 (C-6), 135.9 (C-40), 148.5 (C-2), 2
×
23.7 (C-30’), 25.8 (C-50), 28.6, 28.7 (C-60’), 32.9, 33.0
131.5 (C-3),
167.4 (C-8), 171.5
(C-20), 2x 46.3 (C-
α
×
×
132.8 (C-7), 2
×
×
166.9 (C-5), 2
×
(COO), 2 × 176.8 (C-1), 2 × 177.3, (C-4); MS (ESI-) m/z (%): 423.24 (100) [M-H]−, [M-H]−
calculated for C25H28O6: 423.1808.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-
2-oxoacetate (5), 0.1 mmol Shikonin, 0.22 mmol DCC, 50 µmol DMAP and 0.15 mmol 2-
cyclopropyl-2-oxoacetic acid (p4) reaction time 15 h; CC on silica (4 g; hexanes/CH2Cl2 = 1:0
to hexanes/CH2Cl2 = 0:1); PTLC on silica (developed four times with cyclohexane/
CH2Cl2 = 2:1 and twice with cyclohexane/CH2Cl2 = 1:1);
(silica, CH2Cl2); IR (ATR): 2971 (w), 2950 (br) (OH), 2915 (w), 2857 (w), 1735 (m) (C=O),
1715 (m) (C=O), 1608 (s), 1568 (m), 1451 (m), 1261 (s), 1230 (s), 1203 (s) (COC), 1057 (s),
5, yield: 45%. 5: Rf = 0.34
≈

Int. J. Mol. Sci. 2021, 22, 2774
13 of 25
778 (m) cm−1; 1H-NMR (CDCl3): 1.16–1.22 (m, 2H, H-20’, H-30’), 1.25–1.31 (m, 2H, H-20’,
H-30’), 1.59 (s, 3H, H-60), 1.68 (s, 3H, H-50), 2.61 (dtm, J = 15.0, 7.5 Hz, 1H, H-20), 2.67–2.76
(m, 2H, H-20, H-10’), 5.14 (tm, J = 7.3 Hz, 1H, H-30), 6.17 (ddd, J = 7.1, 4.8, 0.5 Hz, 1H, H-10),
7.11 (d, J = 0.7 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.40 (s, 1H, C5-OH), 12.59 (s, 1H,
C8-OH); 13C-NMR (CDCl3):
δ
2
×
14.3 (C-20’, C-30’), 18.0 (C-60), 18.2 (C-10’), 25.8 (C-50),
32.8 (C-20), 71.4 (C-10), 111.6 (C-4a), 111.8 (C-8a), 117.0 (C-30), 131.4 (C-3), 133.3 (C-7), 133.6
0
(C-6), 136.9 (C-4 ), 146.2 (C-2), 159.9 (COO), 168.8 (C-5), 169.3 (C-8), 174.6 (C-1), 176.2 (C-4),
193.3 (
383.1131.
(R,E)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-cyclopropyl-
2-propenoate ( ), 0.1 mmol Shikonin, 0.20 mmol DCC, 30 mol DMAP and 0.10 mmol
cyclopropylacrylic acid (p3); reaction time 17 h; two consecutive PTLC on silica (developed
twice with cyclohexane/CH2Cl2 = 2:1 each). , yield: 8 %. : Rf = 0.29 (silica, CH2Cl2). IR
α
-CO); MS (ESI-) m/z (%): 383.06 (100) [M-H]−, [M-H]− calculated for C21H20O7:
6
µ
β-
6
6
(ATR): 2917 (m),
≈
2950 (br) (OH), 2851 (w), 1717 (s) (C=O), 1643 (m), 1609 (vs), 1570 (m),
1453 (m), 1263 (s), 1203 (s) (COC), 1140 (s), 779 (w) cm−1; 1H-NMR (CDCl3): 0.63–0.73 (m,
2H, H-20’, H-30’), 0.95–1.04 (m, 2H, H-20’, H-30’), 1.58 (s, 3H, H-60), 1.58–1.68 (m, 1H, H-10’),
0
0
0
1.69 (s, 3H, H-5 ), 2.50 (dtm, J = 15.0, 7.3 Hz, 1H, H-2 ), 2.64 (dtm, J = 14.9, 5.9 Hz, 1H, H-2 ),
5.14 (tm, J = 7.3 Hz, 1H, H-30), 5.97 (d, J = 15.4 Hz, 1H, H-
), 6.04 (ddd, J = 7.1, 4.4, 0.8 Hz,
α
1H, H-10), 6.48 (dd, J = 15.4, 10.1 Hz, 1H, H-
β
), 6.98 (d, J = 0.9 Hz, 1H, H-3), 7.18 (s, 20H,
0
δ 8.9 (C-2 ’, C-3 ’),
H-6, H-7), 12.43 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH); 13C-NMR (CDCl3):
14.6 (C-10’), 18.0 (C-60), 25.8 (C-50), 32.9 (C-20), 69.2 (C-10), 111.6 (C-4a), 111.7 (C-8a), 117.3
(C-α β),
), 117.9 (C-30), 131.6 (C-3), 132.5 (C-7), 132.6 (C-6), 135.9 (C-40), 148.8 (C-2), 155.8 (C-
165.3 (COO), 166.3 (C-5), 166.8 (C-8), 177.4 (C-1), 178.8 (C-4); MS (ESI-) m/z (%): 785.31
(9) [2(M-H)+Na]−, 382.14 (54) [M]−, 381.25 (100) [M-H]−; [M]− calculated for C22H22O6:
382.1416.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-cyclopr
opylpropanoate (
cyclopropylpropanoic acid (p2) reaction time 15 h; CC on silica (8 g; hexanes/CH2Cl2 = 1:0
to hexanes/CH2Cl2 = 1:2); , yield: 40%. : Rf = 0.49 (silica, CH2Cl2); IR (ATR): 3080 (w),
2972 (w),
7), 0.1 mmol Shikonin, 0.26 mmol DCC, 50 µmol DMAP and 0.15 mmol 3-
7
7
≈
2950 (br) (OH), 2916 (w), 2857 (w), 1742 (m) (C=O), 1610 (s), 1569 (m), 1454 (m),
1228 (s), 1204 (s) (CO0C), 784 (m) cm−1; 1H-NMR (CDCl3): 0.04–0.09 (m, 2H, H-20’, H-30’),
0
0
0.40–0.49 (m, 2H, H-2 ’, H-3 ’), 0.66–0.77 (m, 1H, H-1 ’), 1.54 (q, J = 7.3 Hz, 2H, H-β), 1.57 (s,
3H, H-60), 1.68 (s, 3H, H-50), 2.42–2.51 (m, 1H, 1H, H-20), 2.48 (td, J = 7.5, 2.2 Hz, 2H, H-α),
2.61 (dtm, J = 15.2, 5.6 Hz, 1H, H-20), 5.12 (tm, J = 7.4 Hz, 1H, H-30), 6.02 (ddd, J = 7.2, 4.4,
0.8 Hz, 1H, H-10), 6.98 (d, J = 1.0 Hz, 1H, H-3), 7.18 (s, 02H, H-6, H-7), 12.042 (s, 1H, C5-OH),
12.58 (s, 1H, C8-OH); 13C-NMR (CDCl3):
0 0
δ 4.4, 4.5 (C-2 ’, C-3 ’), 10.4 (C-1 ’), 17.9 (C-6 ), 25.7
(C-50), 30.0 (C- ), 32.9 (C-20), 34.4 (C- ), 69.3 (C-10), 111.6 (C-4a), 111.8 (C-8a), 117.8 (C-30),
β α
131.5 (C-3), 132.7 (C-7), 132.8 (C-6), 136.0 (C-40), 148.4 (C-2), 166.8 (C-5), 167.4−(C-8), 172.4
(COO), 176.8 (C-1), 178.3 (C-4); MS (ESI-) m/z (%): 383.30 (100) [M-H] , [M-H] calculated
−
for C22H24O6: 383.4144.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl phenylac-
etate (8), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and two portions of 0.1 mmol
phenylacetic acid each; reaction time 17 h; CC on silica (8 g; CH2Cl2);
8, yield: 25%.
8:
1
Rf = 0.25 (silica, cyclohexane/CH2Cl2 = 1:4); H- and 13C-NMR data fit with literature
values [23,38].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cinnamate
(9), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and 0.1 mmol cinnamic acid;
reaction time 17 h; CC on silica (8 g; CH2Cl2); 9, yield: 17%. 9: Rf = 0.31 (silica, CH2Cl2);
1H-NMR data fit with literature values [26] and the 13C-NMR data fit with those of the
corresponding alkannin derivative [25].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-butynoate
(
10), 0.1 mmol Shikonin, 0.20 mmol DCC, 25
acid; reaction time 14 h; CC on silica (4 g; CH2Cl2); 10, yield: 42%. 10: Rf = 0.29 (sil-
ica, cyclohexane/CH2Cl2 = 1:4); IR (ATR): 3000 (vbr) (OH), 3052 (w), 2973 (w), 2928 (w),
µmol DMAP and 0.1 mmol 2-butynoic
≈

Int. J. Mol. Sci. 2021, 22, 2774
14 of 25
2858 (w), 2238 (m) (C
≡
C), 1708 (s) (C=O), 1605 (s), 1568 (m), 1451 (m), 1238 (s) 1198 (s)
(COC), 1066 (s), 768 (m), 738 (m) cm−1; 1H-NMR (CDCl3): 1.58 (s, 3H, H-60), 1.70 (s, 3H,
H-50), 2.03 (s, 3H, H- ), 2.51 (dtm, J = 15.0, 7.4 Hz, 1H, H-20), 2.65 (dtm, J = 15.0, 6.0 Hz,
γ
1H, H-20), 5.12 (tm, J = 7.3 Hz, 1H, H-30), 6.08 (ddd, J = 7.2, 4.6, 1.0 Hz, 1H, H-10), 7.07 (d,
J = 1.0 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.42 (s, 1H, C5-OH), 12.57 (s, 1H, C8-OH);
13C-NMR (CDCl3):
87.0 (C-
δ 3.9 (C-γ α),
), 18.0 (C-60), 25.7 (C-50), 32.7 (C-20), 70.3 (C-10), 72.4 (C-
β
), 111.6 (C-4a), 111.8 (C-8a), 117.8 (C-30), 131.5 (C-3), 133.0 (C-7), 133.2 (C-6), 136.5
(C-40), 147.1 (C-2), 152.4 (COO), 167.7 (C-5), 168.2 (C-8), 175.8 (C-1), 177.4 (C-4); MS (ESI-)
m/z (%): 354.07 (36) [M]−, 353.15 (100) [M-H]−; [M]− calculated for C20H18O6: 354.1103.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-pentynoate
(
11), 0.1 mmol Shikonin, 0.15 mmol DCC, 25
reaction time 16 h; CC on silica (4 g; cyclohexane / CH2Cl2 = 1:4); 11, yield: 35%. 11: Rf = 0.38
(silica, cyclohexane/CH2Cl2 = 1:4); IR (ATR): 3052 (w), 2980 (w), 2970 (vbr) (OH), 2937 (w),
2858 (w), 2236 (m) (C C), 1710 (s) (C=O), 1609 (s), 1569 (m), 1453 (m), 1232 (s) 1200 (s) (COC),
µmol DMAP and 0.1 mmol 2-pentynoic acid;
≈
≡
1080 (s), 1052 (s), 777 (m), 750 (m) cm−1; 1H-NMR (CDCl3): 1.24 (t, J = 7.5 Hz, 3H, H-
δ), 1.58 (s,
3H, H-60), 1.70 (s, 3H, H-50), 2.39 (0q, J = 7.5 Hz, 2H, H-
γ
), 2.51 (dtm, J = 14.6, 7.5 Hz, 1H, H-20),
0
2.65 (dtm, J = 15.0, 5.9 Hz, 1H, H-2 ), 5.14 (tm, J = 7.3 Hz, 1H, H-3 ), 6.08 (ddd, J = 7.1, 4.6, 0.9 Hz,
1H, H-10), 7.07 (d, J = 0.9 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.43 (s, 1H, C5-OH), 12.57 (s, 1H,
C8-OH); 13C-NMR (CDCl3):
0 0 0 0
δ 12.4 (C-δ), 12.5 (C-γ), 18.0 (C-6 ), 25.8 (C-5 ), 32.7 (C-2 ), 70.8 (C-1 ),
72.0 (C- ), 92.1 (C-β
α
), 111.6 (C-4a), 111.8 (C-8a), 117.3 (C-30), 131.6 (C-3), 132.9 (C-7), 133.1 (C-6),
136.5 (C-40), 147.2 (C-2), 152.6 (COO), 167.6 (C-5), 168.1 (C-8), 175.9 (C-1), 177.5 (C-4); MS (ESI-)
m/z (%): 368.18 (24) [M]−, 367.21 (100), [M]− calculated for C21H20O6: 368.1260.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-phenylpro
pynoate (12), 50 µmol Shikonin, 0.12 mmol DCC, 15 µmol DMAP and 68 µmol phenyl-
propynoic acid; reaction time 19 h; PTLC on silica with cyclohexane/CH2Cl2 = 1:1 (twice
developed); 12, yield: 67%. 12: Rf = 0.42 (silica, cyclohexane/CH2Cl2 = 1:4); IR (ATR): 3058
(w), 2971 (w), 2915 (w), 2857 (w), 2212 (m) (CΞC), 1711 (s) (C=O), 1608 (s), 1568 (m), 1452
(m), 1275 (s), 1238 (s) 1164 (s), 1164 (vs) (COC), 1111 (m), 755 (s), 687 (m) cm-1; 1H-NMR
(CDCl3): 1.61 (s, 3H, H-60), 1.72 (s, 3H, H-50), 2.57 (dtm, J 0= 14.9, 7.4 Hz, 1H, H-20), 2.70
0
(dtm, J = 14.8, 5.7 Hz, 1H, H-2 ), 5.19 (tm, J = 7.3 Hz, 1H, H-3 ), 6.17 (ddd, J = 7.3, 4.7, 1.0 Hz,
0
0
1H, H-1 ), 7.14 (d, J = 0.9 Hz, 1H, H-3), 7.19 (s, 2H, H-6, H-7), 7.40 (tm, J = 7.4 Hz, 2H, H-3 ’,
H-50’), 7.48 (tt, J = 7.5, 1.1 Hz, 1H, H-40’), 7.63 (m, 2H, H-20’, H-60’), 12.43 (s, 1H, C5-OH),
12.59 (s, 1H, C8-OH); 13C-NMR (CDCl3):
δ
18.0 (C-60), 25.8 (C-50), 32.8 (C-20), 71.1 (C-10),
80.2 (C-α), 87.6 (C-β
), 111.6, (C-4a), 111.9 (C-8a), 117.2 (C-30), 119.3 (C-10’), 128.6 (C-30’,
C-50’), 130.9 (C-40’), 131.6 (C-3), 133.0 (C-7), 133.1 (C-20’, C-30’), 133.3 (C-6), 136.6 (C-40),
147.0 (C-2), 152.8 (COO), 167.9 (C-5), 168.4 (C-8), 175.6 (C-1), 177.2 (C-4); MS (ESI-) m/z
(%):416.18 (27) [M]-, 415.12 (100) [M-H]−, [M]− calculated for C25H20O6: 416.1260.
(R,E)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 4-oxo-
4-(4-methylphenyl)-2-butenoate (13), 50
and 95 mol (E) 4-(4-methylphenyl)-3-oxo-2-butenoic acid; reaction time 16 h; PTLC on
silica with cyclohexane/CH2Cl2 = 2:1 (four times developed); 13, yield: 4%. 13: Rf = 0.51
(silica, CH2Cl2); IR (ATR): 2967 (w), 2918 (m), 2950 (br) (OH), 2851 (w), 1728 (m) (C=O),
µmol Shikonin, 0.15 mmol DCC, 15 µmol DMAP
µ
≈
1669 (m), 1607 (vs), 1570 (m), 1453 (m), 1297 (s) (C-O-C), 1205 (m), 1161 (m), 756 (w) cm−1
;
1H-NMR (CDCl3): 1.61 (s, 3H, H-60), 1.70 (s, 3H, H-50), 2.45 (s, 3H, Ph-CH3), 2.58 (dtm,
J = 15.2, 7.6 Hz, 1H, H-20), 2.70 (dtm, J = 15.2, 5.4 Hz, 1H, H-20), 5.16 (tm, J = 7.2 Hz, 1H,
H-30), 6.17 (dd, J = 7.3, 4.7 Hz, 1H, H-10), 6.95 (d, J = 15.5 Hz, 1H, H-
α
), 7.04 (d, J = 0.8 Hz,
1H, H-3), 7.19 (s, 2H, H-6, H-7), 7.32 (d, J = 8.2 Hz, 2H, H-30’, H-50’), 7.91 (d, J = 8.2 Hz,
2H, H-20’, H-60’), 7.96 (d, J = 15.5 Hz, 1H, H-
), 12.41 (s, 1H, C5-OH), 12.61 (s, 1H, C8-OH);
13C-NMR (CDCl3): 18.0 (C-60), 21.8 (Ph-CH3), 25.8 (C-50), 32.9 (C-20), 70.5 (C-10), 111.7
(C-4a), 111.8 (C-8a), 117.4 (C-30), 129.0 (C-20’, C-60’), 129.7 (C-30’, C-50’), 131.2 (C-
), 131.3
(C-3), 133.1 (C-7), 133.3 (C-6), 134.0 (C-40’), 136.5 (C-40), 137.8 (C- ), 145.2 (C-10’), 147.2
); MS (ESI-)
β
δ
α
β
(C-2), 164.5 (COO), 168.0 (C-5), 168.6 (C-8), 175.5 (C-1), 177.0 (C-4), 188.6 (C-
γ
m/z (%): 1417.45 (100) [3M-2H+K]−, 1401.56 (91) [3M-2H+Na]-, 1001.40 (55), 269.13 (42)
[M-H-RCOOH]-; [3M-2H+K]− calculated for C27H24O7: 1417.4047.

Int. J. Mol. Sci. 2021, 22, 2774
15 of 25
Synthesis and spectroscopic data of this compound were published mislabeled as
Z-isomer. 13C-NMR shift values fit with our data, but 1H-NMR data and assignment of
13C-NMR signals are deficient [34].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-oxopropanoate
(
14), 0.25 mmol Shikonin, 0.75 mmol and 0.20 mmol DCC, 62
and 0.68 mmol pyruvic acid; reaction time 240 min plus 90 min; flash filtration on silica (10 g;
cyclohexane/CH2Cl2 = 2:1 to 0:1); 14, from the acrylester derived from HBr elimination. 14
yield: 44%. 14: Rf = 0.31 (silica, CH2Cl2); IR (ATR): 2969 (w), 2950 (br) (OH), 2928 (w), 2856
µmol DMAP and 0.25 mmol
,
≈
(w), 1733 (s) (C=O), 1607 (s), 1569 (m), 1435 (m), 1411 (m), 1262 (m), 1231 (s), 1202 (s) (COC),
1113 (m), 779 (m) cm−1; 1H-NMR (CDCl3): 1.59 (s, 3H, H-60), 1.69 (s, 3H, H-50), 2.50 (s, 3H,
0 0
H-β), 2.61 (dtm, J = 14.9, 7.4 Hz, 1H, H-2 ), 2.70 (dtm, J = 15.0, 5.9 Hz, 1H, H-2 ), 5.13 (tm, J = 7.1
Hz, 1H, H-30), 6.15 (dd, J = 7.1, 5.1 Hz, 1H, H-10), 7.09 (s, 1H, H-3), 7.17 (s, 2H, H-6 and H-7),
12.38 (s, 1H, C5-OH), 12.58 (s, 1H, C8-OH); 13C-NMR (CDCl3):
δ
17.9 (C-60), 25.7 (C-50), 26.7
0
0
0
0
(C-2 ), 32.8 (C-
β
), 70.3 (C-1 ), 71.5 (C-2 ’), 111.6, (C-4a), 111.8 (C-8a), 116.9 (C-3 ), 131.5 (C-3), 133.5
0
(C-7), 133.7 (C-6), 136.9 (C-4 ), 145.9 (C-2), 159.6 (COO), 169.2 (C-5), 169.7 (C-8), 174.1 (C-1), 175.7
(C-4), 190.9 (C-
α
); MS (ESI−) m/z (%): 1111.26 (100) [3M-2H+K]−, 269.25 (40) [M-H-RCOOH]-,
[3M-2H+K]− calculated for C19H18O7: 1111.2638.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-oxo-phen
ylacetate (15), 50 µmol Shikonin, 0.12 mmol DCC, 15 µmol DMAP and 67 µmol 2-oxo-
phenylacetic acid; reaction time 19 h; PTLC on silica with cyclohexane/CH2Cl2 = 1:1 (twice
developed); 15, yield: 57%. 15: Rf = 0.33 (silica, cyclohexane/CH2Cl2 = 1:4); IR (ATR): 3064
(w), 2972(w), 2930 (w) 2857 (w), 1742 (m) (C=O), 1688 (m) (C=O), 1609 (s), 1570 (m), 1451
(m), 1193 (s) 1173 (s) (COC), 982 (m), 682 (m) cm−1; 1H-NMR (CDCl3): 1.59 (s, 3H, H-600),
0
0
1.72 (s, 3H, H-5 ), 2.61 (dtm, J = 15.1, 7.7 Hz, 1H, H-2 ), 2.76 (dtm, J = 15.1, 5.5 Hz, 1H, H-2 ),
5.20 (tm, J = 7.3 Hz, 1H, H-30), 6.37 (ddd, J = 7.1, 4.3, 0.8 Hz, 1H,0H-10), 7.18 (d, J = 0.8 Hz
,
0
1H, H-3), 7.19 (s, 2H, H-6, H-7), 7.53 (tm, J = 7.8 Hz, 2H, H-3 ’, H-5 ’), 7.69 (tt, J = 7.7, 1.2 Hz,
1H, H-40’), 8.00 (m, 2H, H-20’, H-60’), 12.41 (s, 1H, C5-OH), 12.62 (s, 1H, C8-OH); 13C-NMR
(CDCl3):
δ
18.0 (C-60), 25.8 (C-50), 33.0 (C-20), 71.2 (C-10), 111.6, (C-4a), 111.8 (C-8a), 117.2
(C-30), 129.0 (C-30’, C-50’), 130.0 (C-20’, C-30’), 131.4 (C-3), 132.2 (C-10’), 133.4 (C-7), 133.8
(C-6), 135.1 (C-40’), 137.0 (C-40), 146.0 (C-2), 162.6 (COO), 169.1 (C-5), 169.6 (C-8), 174.3
(C-1), 175.9 (C-4), 185.6 (C-α
); MS (ESI-) m/z (%): 1297.25 (100) [3M-2H+K]−, 1281.41
(85) [3M-2H+Na]−, 269.18 (22) [M-H-RCOOH]-, [3M-2H+K]− calculated for C24H20O7:
1297.3108.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-(2-oxocyc
lopentyl)acetate (16), 0.1 mmol Shikonin, 0.25 mmol DCC, 25 µmol DMAP and 0.18 mmol 2-
(2-oxocyclopentyl)acetic acid; reaction time 15 h; CC on silica (4 g; cyclohexane/CH2Cl2 = 1:0
to cyclohexane/CH2Cl2 = 0:1); PTLC on silica (developed seven times with cyclohexane
/CH2Cl2 = 2:1); 16, yield: 33%. 16: Rf = 0.11 (silica, CH2Cl2); IR (ATR): 2966 (w),
≈
2950 (br)
(OH), 2917 (w), 2878 (w), 1736 (m) (C=O), 1609 (s), 1569 (m), 1452 (m), 1261(m), 1231 (m),
0
1202 (m) (COC), 1160 (m), 782 (m) cm−1; 1H-NMR (CDCl3): 1.58 (s, 6H, H-6 ), 1.58-1.68 (m,
2H, H-50’), 1.69 (s, 6H, H-50), 1.74-1.88 (m, 2H, H-40’), 2.01–2.10 (m, 2H, H-400’), 2.11–2.23
0
0
0
0
(m, 2H, H-3 ’), 2.25–2.41 (m, 4H, H-5 ’, H-4 ’), 2.41–2.56 (m, 6H, H-2 , H-
α
, H-1 ’), 2.57–2.67
(m, 2H, H-20), 7.74–2.88 (m, 2H, H-
(d, J = 1.6 Hz, 1H, H-3), 7.03 (d, J = 0.8 Hz, 1H, H-3), 7.18 (s, 4H, H-6, H-7), 12.42 (s, 1H,
C5-OH), 12.43 (s, 1H, C5-OH), 12.58 (s, 2H, C8-OH); 13C-NMR (CDCl3): 18.0 (C-60), 20.6
(C-20’), 25.7 (C-50), 29.2, 29.3 (C-50’), 32.8, 32.9 (C-20), 33.9, 34.0 (C- 37.3 (C-30’), 45.6
), 2
(C-10’), 2 69.8 (C-10), 111.6 (C-4a), 111.9 (C-8a), 117.7 (C-30), 2x 131.5 (C-3), 2x 132.8 (C-7),
α
), 5.07–5.15 (m, 2H, H-30), 6.00–6.07 (m, 2H, H-10), 7.00
δ
α
×
×
2
×
133.0 (C-6), 136.1, 136.2 (C-40), 147.9 (C-2), 167.3, 167.5 (C-5), 167.7, 167.8 (C-8), 171.0
(COO), 176.3, 176.3 (C-1), 177.8, 177.9 (C-4), 218.8 (C-20’); MS (ESI-) m/z (5): 411.27 (100)
[M-H]−, [M-H]− calculated for C23H24O7: 411.1444.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl methyl bu-
tanedioate (17), 50 µmol Shikonin, 0.12 mmol DCC, 12.5 µmol DMAP and 76 µmol monomethyl
succinate (4-methoxy-4-oxobutanoic acid); reaction time 18 h; CC on silica (8 g; cyclohex
ane/CH2Cl2 = 2:1 to 0:1); 17, yield: 15 %. 17: Rf = 0.36 (silica, CH2Cl2); IR (ATR): 2961 (m),

Int. J. Mol. Sci. 2021, 22, 2774
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2918 (w), 2855 (w), 1735 (m) (C=O), 1607 (s), 1568 (m), 1452 (m), 1436 (m), 1410 (m), 1345
(m), 1261 (m), 1229 (m), 1200 (s) (COC), 1150 (s), 1111 (m), 1020 (m), 781 (m) cm−1; 1H-NMR
0
0
0
(CDCl3): 1.58 (s, 3H, H-6 ), 1.69 (s, 3H, H-5 ), 2.49 (dtm, J = 14.9, 7.3 Hz, 1H, H-2 ), 2.58-2.68
(m, 03H, H-20, H-
α
*), 2.70–2.75 (m, 2H, H-
β
*), 3.70 (s, 3H, OCH3), 5.12 (tm, J = 7.3 Hz, 1H,
0
H-3 ), 6.04 (dd, J = 7.1, 4.6 Hz, 1H, H-1 ), 7.01 (d, J = 0.8 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7),
12.42 (s, 1H, C5-OH), 12.57 (s, 1H, C8-OH); 13C-NMR (CDCl3):
δ
17.9 (C-60), 25.7 (C-50),
28.8, 29.2 (C-α, C-β
), 32.8 (C-20), 51.9 (OCH3), 69.9 (C-10), 111.6, (C-4a), 111.8 (C-8a), 117.6
(C-30), 131.5 (C-3), 132.8 (C-7), 132.9 (C-6), 136.2 (C-40), 147.9 (C-2), 167.2 (C-5), 167.7 (C-8),
171.1 (C-1 ’), 172.4 (COOCH3), 176.4 (C-1), 177.9 (C-4); MS (ESI-) m/z (%): 402.10 (28) [M] ,
0
−
401.15 (100) [M-H]−, [M]- calculated for C21H22O8: 402.1315.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl ethyl (2E)-
but-2-enedioate (18), 0.1 mmol Shikonin, 0.15 mmol DCC, 20 µmol DMAP and 0.11 mmol
monoethyl fumarate ((E)-4-methoxy-4-oxobut-2-enoic acid); reaction time 17 h; CC on
silica (4 g; CH2Cl2); 18, yield: 7%. 18: Rf = 0.24 (silica, CH2Cl2); IR (ATR): 3074 (w), 2980
(m),
≈
2950 (br) (OH), 2930 (w), 2857 (w), 1719 (s) (C=O), 1608 (s), 1569 (m), 1452 (m),
1294 (s), 1256 (s), 1230 (s), 1202 (s) (COC), 1149 (s), 1112 (m), 1026 (m), 771 (m) cm−1
;
1H-NMR (CDCl3): 1.35 (t, J = 7.1 Hz, O-CH2-CH3), 1.57 (s, 3H, H-60), 1.69 (s, 3H, H-50),
2.54 (dtm, J = 15.0, 7.3 Hz, 1H, H-20), 2.67 (dtm, J = 15.0, 5.0 Hz, 1H, H-20), 4.29 (q, J = 7.1
Hz, O-CH2-CH3), 5.13 (tm, J = 7.3 Hz, 1H, H-10), 6.17 (ddd, J = 7.3, 4.6, 0.8 Hz, 1H, H-10),
6.90, 6.92 (2d, J = 15.9 Hz, 2H, H-
H-7), 12.41 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH); 13C-NMR (CDCl3):
18.0 (C-60), 25.8 (C-50), 32.8 (C-20), 61.5 (O-CH2-CH3), 70.5 (C-10), 111.6 (C-4a), 111.8 (C-8a),
117.3 (C-30), 131.2 (C-3), 132.7 (C-
or C- ), 133.2 (C-7), 133.4 (C-6), 134.8 (C- or C- ),
136.6 (C-40), 147.1 (C-2), 163.7 (C-
α
, H-
β), 7.00 (d, J = 0.8 Hz, 1H, H-3), 7.19 (s, 2H, H-6,
δ
14.1 (O-CH2-CH3),
α
β
α
β
α
-COO), 164.7 (COOEt), 168.2 (C-5), 168.7 (C-8), 175.3
(C-1), 176.8 (C-4); MS (ESI-) m/z (%):1279 (5) [3M-2H+K]−, 1265 (8) [3M-2H+Na]−, 1264
(14) [3M-2H+Na]−, 1263 (19) [3M-2H+Na]−, 414.14 (28) [M]−, 413.21 (100) [M-H]−; [M]−
calculated for C22H22O8: 414.1315.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-methyl
propanoate (19), 0.1 mmol Shikonin, 0.20 mmol DCC, 25 µmol DMAP and 0.11 mmol
isobutyric acid; reaction time 22 h; PTLC on silica (1 mm layer; developed once with
cyclohexane/CH2Cl2 = 1:4); 19, yield: 14%. 19: Rf = 0.24 (silica, cyclohexane/CH2Cl2 = 1:4);
1H- and 13C-NMR data fit with literature values [62–64].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-methyl-butan
oate (20), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and 0.1 mmol isovaleric acid;
reaction time 17 h; PTLC on silica (developed three times with cyclohexane/CH2Cl2 = 2:5); 20
,
yield: 11 %. 20: Rf = 0.29 (silica, cyclohexane/CH2Cl2 = 1:4); 1H- and 13C-NMR data fit with
literature values [62,64].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl (E)-2-butenoate
(21), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and two portions of 0.1 mmol
crotonoic acid each; reaction time 16 plus 24 h; CC on silica (4 g; cyclohexane/CH2Cl2 = 1:1 to
0:1); 21, yield: 17%. 21: Rf = 0.27 (silica, cyclohexane/ CH2Cl2 = 1:4); 1H-NMR (CDCl3): 1.58
(s, 3H, H-60), 1.69 (s, 3H, H-50), 1.93 (dd, J = 6.8 Hz, 1.9, 3H, H-
γ
), 2.50 (dtm, J = 14.9, 7.2 Hz,
1H, H-20), 2.64 (dtm, J = 15.1, 5.5 Hz, 1H, H-20), 5.14 (tquint, J = 7.3, 1.3 Hz, 1H, H-30), 5.91 (dq,
J = 15.5, 1.5 Hz 2H, H-
), 6.07 (ddd, J = 6.2, 3.7, 0.8 Hz, 1H, H-10), 6.98 (d, J = 1.0 Hz, 1H, H-3),
7.05 (dq, J = 15.5, 6.9 Hz, 1H, H-
α
β
), 7.18 (s, 2H, 0H-6, H-7), 012.43 (s, 1H, C5-OH),012.59 (s, 1H,
C8-OH); 13C-NMR (CDCl3):
δ
17.9, 18.1 (C-
γ
, C-6 ), 25.8 (C-5 ), 29.7 (C-
β
), 32.9 (C-2 ), 69.3 (C-1 ),
0
0
0
111.6 (C-4a), 111.8 (C-8a), 117.8 (C-3 ), 122.1 (C-
α), 131.5 (C-3), 132.6, 132.7 (C-7, C-6), 136.0 (C-4 ),
146.1 (C-ß), 148.5 (C-2), 165.1 (COO), 166.6 (C-5), 167.1 (C-8), 177.1 (C-1), 178.6 (C-4); 1H-NMR
shift values fit with literature values [39].
(R,E,E)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2,4-
hexadienoate (22), 0.1 mmol Shikonin, 0.35 mmol DCC, 37.5 µmol DMAP and 0.1 mmol sorbic
acid each; reaction time 17 h; PTLC on silica four times developed with cyclohexane/CH2Cl2
1
= 1:1 and once with CH2Cl2; 22, yield: 8%. 22: Rf = 0.40 (silica, CH2Cl2). H- and 13C-NMR
data fit with literature values [23,38].

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(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl benzoate
(
23), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and 0.1 mmol benzoic acid
each; reaction time 17 h; PTLC on silica (twice developed with cyclohexane/CH2Cl2 = 1:1
and twice developed with cyclohexane/CH2Cl2 = 1:2); 23, yield: 5%. 23: Rf = 0.27 (silica,
cyclohexane/CH2Cl2 = 1:4); 1H- and 13C-NMR data fit with literature values [23,38].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl dipheny-
lacetate (24), 50 µmol Shikonin, 0.12 mmol DCC, 15 µmol DMAP and 61 µmol diphenylacetic
acid each; reaction time 13 h; CC on silica (8 g; cyclohexane/CH2Cl2 = 1:1 to 4:1); PTLC
on silica with cyclohexane/CH2Cl2 = 1:1 (three times developed); PTLC on silica with
cyclohexane/CH2Cl2 = 2:1 (three times developed); 24, yield: 29%. 24: Rf = 0.69 (silica,
CH2Cl2);
1H- and 13C-NMR data fit with literature values [23,38].
(R,E)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-phenyl-
propanoate (25), 50
µmol Shikonin, 0.12 mmol DCC, 20 µmol DMAP and 73 µmol 3-
phenylpropanoic acid; reaction time 15 h; PTLC on silica with cyclohexane/CH2Cl2 =
1:1 (three times developed); 25, yield: 14%. 25: Rf = 0.45 (silica, cyclohexane/CH2Cl2 = 1:4);
IR (ATR): 3028 (vw), 2965 (w), 2918 (w),
≈
2950 (br) (OH), 2851 (w), 1738 (s) (C=O), 1608 (s),
1568 (m), 1453 (m), 1342 (m), 1264 (m), 1229 (m), 1201 (s) (C-O-C), 1146 (m), 1112 (m), 735
(m), 698 (m) cm-1; 1H- and 13C-NMR data fit with literature values [36].
(R,E)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-(4-dim
ethylaminophenyl)2-propenoate (26), 0.5 mmol Shikonin, 1.5 mmol DCC, 0.125 mmol DMAP
and 0.675 mmol p-dimethylaminocinnamic acid; reaction time 24 h; flash CC on silica with
cyclohexane/CH2Cl2 = 2:1 to 0:1; 26, yield: 6%. 26: Rf = 0.13 (silica, CH2Cl2). IR (ATR):
2920 (m, br), 2852 (w), 1707 (m), (C=O), 1597 (vs), 1526 (m), 1444 (m), 1232 (m), 1206 (m),
1182 (m), 1146 (s) (COC), 1113 (m), 815 (w) cm−1; 1H-NMR (CDCl3): 1.60 (d, J = 0.8 Hz,
3H, H-60), 1.69 (d, J = 0.8 Hz, 3H, H-50), 2.56 (dtm, J = 15.0, 7.1 Hz, 1H, H-20), 2.68 (dtm,
J = 15.0, 5.7 Hz, 1H, H-20), 3.06 (s, 6H, NCH3), 5.19 (tm, J = 7.2 Hz, 1H, H-30), 6.12 (ddd,
J = 7.0, 4.4, 0.7 Hz, 1H, H-10), 6.31 (d, J = 15.8 Hz, 1H, H-
7.04 (d, J = 1.0 Hz, 1H, H-3), 7.19 (s, 2H, H-6 and H-7), 7.47 (d, J = 8.6 Hz, 1H, H-30’), 7.67
(d, J = 15.8 Hz, 1H, H-
), 12.43 (s, 1H, C5-OH), 12.61 (s, 1H, C8-OH); 13C-NMR (CDCl3):
α
), 6.83 (d, J = 8.0 Hz, 1H, H-30’),
β
δ
18.0 (C-60), 25.8 (C-50), 32.9 (C-20), 40.9 (NCH3), 69.2 (C-10), 111.6 (C-4a), 111.9 (C-8a), 112.4
(C-α
), 112.9 (C-30’, C-50’), 117.9 (C-30), 123.6 (C-30’), 129.9 (C-20’, C-60’), 131.7 (C-3), 132.4
0
(C-7), 132.5 (C-6), 135.8 (C-4 ), 146.2 (C-
β
), 148.8 (C-2), 166.2 (C-8), 166.3 (COO), 166.8 (C-5),
177.5 (C-4), 178.9 (C-1); MS no spectra available via (ESI-) or (ESI+), [M]− calculated for
C27H27NO6: 461.1838.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl pyridine-
3-carboxylate (27), 50 µmol Shikonin, 0.12 mmol DCC, 55 µmol DMAP and 85 µmol nico-
tinic acid; reaction time 15 h; PTLC on silica with CH2Cl2 (three times developed) and
PTLC on silica with CH2Cl2 (three times developed); 27, yield: 10%. 27: Rf = 0.50 (silica,
CH2Cl2/MeOH = 40:1); 1H-NMR data fit with literature values [27].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylpyridine-4-carbo
xylate (28), 50 µmol Shikonin, 100 µmol DCC, 7.5 µmol DMAP and 75 µmol isonicotinic acid;
reaction time 22 h; flash CC on silica (8 g; CH2Cl2 to CH2Cl2/MeOH = 40:1), PTLC on silica
with CH2Cl2 (three times developed) and PTLC on silica with cyclohexane/CH2Cl2 = 1:4 (four
times developed); 28, yield: 1%. 28: Rf = 0.41 (silica, CH2Cl2 / MeOH = 40:1); IR (ATR): 2961
(w), 2918 (m),
≈
2950 (br) (OH), 2851 (w), 1734 (s) (C=O), 1610 (s), 1570 (m), 1559 (m), 1456 (m),
1406 (m), 1269 (s) (C-O-C), 1205 (m), 1117 (m), 757 (w) cm−1; 1H-NMR (CDCl3): 1.62 (s, 3H,
H-60), 1.69 (s, 3H, H-50), 2.68 (dtm, J = 15.2, 7.2 Hz, 1H, H-20), 2.78 (dtm, J = 15.0, 5.6 Hz, 1H,
H-20), 5.18 (tm, J = 7.3 Hz, 1H, H-30), 6.30 (ddd, J = 7.2, 4.8, 0.7 Hz, 1H, H-10), 7.06 (d, J = 0.9
Hz, 1H, H-3), 7.19 (s, 2H, H-6 and H-7), 8.02 (s, 2H, H-30’ and H-50’), 8.87 (d, J = 7.3 Hz, 2H,
H-20’ and H-60’), 12.38 (s, 1H, C5-OH), 12.63 (s, 1H, C8-OH); 13C-NMR (CDCl3 / MeOD = 2:1):
δ
170.6 (C-60), 25.8 (C-50), 32.6 (C-20), 70.9 (C-10), 111.4, (C-4a), 111.8 0(C-8a), 117.1 (C-30), 123.0
0
0
0
(C-3 ’, C-5 ’), 130.6 (C-3), 133.2, 133.4 (C-6, C-7), 136.5 (C-4 ), 137.2 (C-4 ’), 146.4 (C-2), 150.0 (C-2 ’,

Int. J. Mol. Sci. 2021, 22, 2774
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C-60’), 163.6 (COO), 168.7 (C-8), 169.1 (C-5), 173.8 (C-1), 175.3 (C-4); MS (ESI-) m/z (%): 807 (14)
−
−
−
[2(M-H)+Na], 393.09 (28) [M] , 392.07 (100) [M-H] ; [M] calculated for C22H19NO6: 393.1212.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-(1H-indol-
3-yl)-propanoate (29), 50 µmol Shikonin, 0.12 mmol DCC, 15 µmol DMAP and 63 µmol 3-(1H-
indol-3-yl)-propanoic acid; reaction time 19 h; PTLC on silica CH2Cl2 (twice developed); 29
,
yield: 22%. 29: Rf = 0.22 (silica, CH2Cl2); 1H-NMR (CDCl3): 1.53 (s, 3H, H-60), 1.64 (s, 3H,
H-50), 2.41 (dtm, J = 14.9, 7.5 Hz, 1H, H-20), 2.54 (dtm, J = 14.7, 5.7 Hz, 1H, H-20), 2.77-2.83
0
(m, 2H, H-
α
), 3.13 (tm, J = 7.4 Hz, 2H, H-
β
), 5.03 (tquint, J = 7.2, 1.1 Hz, 1H, H-3 ), 5.99 (ddd,
0
0
J = 6.9, 4.8, 0.7 Hz, 1H, H-1 ), 6.70 (d, J = 0.7 Hz, 1H, H-3), 6.99 (d, J = 2.1 Hz, 1H, H-2 ’), 7.11
(td, J = 7.4 Hz, 0.8 Hz, 1H, H-60’), 7.16 (td, J = 7.8 Hz, 1.3 Hz, 1H, H-70’), 7.18 (s, 2H, H-6,
H-7), 7.34 (d, J = 7.3 Hz, 1H, H-80’), 7.59 (d, J = 7.7 Hz, 1H, H-50’), 8.00 (s br, 1H, NH), 12.43
(s, 1H, C5-OH), 12.56 (s, 1H, C8-OH); 1H-NMR data fit with literature values (Wang et al.,
2014). 13C-NMR (CDCl3): 17.9 (C-60), 20.7 (C- ), 25.7 (C-50), 32.8 (C-20), 35.0 (C-
δ β α), 69.4
(C-10), 111.2 (C-80’), 111.6 (C-4a), 111.8 (C-8a), 114.6 (C-3‘’), 117.6 (C-30), 118.6 (C-50’), 119.4
(C-600’), 121.5 (C-20’), 122.1 (C-70’), 127.1 (C-40’), 131.5 (C-3), 132.5 (C-7), 132.7 (C-6), 136.0
(C-4 ), 136.3 (C-9“), 148.3 (C-2), 166.5 (C-5), 167.0 (C-8), 172.3 (COO), 177.1 (C-1), 178.6 (C-4).
(R,E)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-(furan-
2-yl)-2-propenoate (30), 50 µmol Shikonin, 0.12 mmol DCC, 15 µmol DMAP and 72 µmol 3-
(furan-2-yl)-2-propenoic acid; reaction time 13 h; PTLC on silica with cyclohexane/CH2Cl2
= 1:1 (three times developed); 30, yield: 5%. 30: Rf = 0.43 (silica, cyclohexane/CH2Cl2 =
1:4); IR (ATR): 2969 (w), 2918 (m, br), 2853 (w), 1713 (m), (C=O), 1637 (m), 1609 (s), 1569 (m),
1454 (m), 1263 (m), 1232 (m), 1204 (s), 1155 (s), (COC), 1017 (m), 752 (m) cm−1; 1H-NMR
(CDCl3): 1.60 (d, J = 0.8 Hz, 3H, H-60), 1.69 (s, 3H, H-50), 2.55 (dtm, J0= 14.9, 7.4 Hz, 1H,
0
0
H-2 ), 2.68 (dtm, J = 14.8, 5.7 Hz, 1H, H-2 ), 5.17 (tm, J = 7.2 Hz, 1H, H-3 ), 6.12 (ddd, J = 7.2
,
4.4, 0.5 Hz, 1H, H-10), 6.38 (d, J = 15.7 Hz, 1H, H-
6.66 (d, J = 3.4 Hz, 1H, H-30’), 7.03 (d, J = 0.6 Hz, 1H, H-3), 7.19 (s, 2H, H-6 and H-7), 7.46
(d, J = 15.7 Hz, 1H, H- ), 7.52 (d, J = 1.5 Hz, 1H, H-5‘’), 12.42 (s, 1H, C5-OH), 12.60 (s, 1H,
C8-OH); 13C-NMR (CDCl3): 18.0 (C-60), 25.8 (C-50), 32.9 (C-20), 69.6 (C-10), 111.6 (C-4a),
111.9 (C-8a), 112.4 (C-40’), 114.9 (C- ), 115.5 (C-30’), 117.8 (C-30), 131.6 (C-3), 132.0 (C-
),
α
), 6.50 (dd, J = 3.4, 1.8 Hz, 1H, H-40’),
β
δ
α
β
132.6 (C-7), 132.8 (C-6), 136.1 (C-40), 145.1 (C-50’), 148.4 (C-2), 150.7 (C-20’), 165.7 (COO),
166.8 (C-8), 167.3 (C-5), 177.0 (C-4), 178.4 (C-1); MS (ESI-) m/z (%): 837 (51) [2(M-H)+Na] ,
−
408.07 (41) [M]−, 407.20 (67) [M-H]−, 137 (100), [M]- calculated for C23H20O7: 408.1209.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl bromoac-
etate (31), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and 0.1 mmol bromoacetic
acid each; reaction time 17 h; CC on silica (8 g; CH2Cl2) and PTLC on silica with cyclohex-
ane/ CH2Cl2 = 2:1 (four times developed); 31, yield: 12%. 31: Rf = 0.33 (silica, CH2Cl2).
0
0
0
1H-NMR (CDCl3): 1.51 (s, 3H, H-6 ), 1.70 (s, 3H, H-5 ), 2.52 (dtm, J = 15.1, 7.5 Hz, 1H, H-2 ),
2.66 (dtm, J = 15.1, 5.6 Hz, 1H, H-20)), 3.87 (d, J = 12.1 Hz, 1H, CH2Br), 3.90 (d, J = 12.1
Hz,01H, CH2Br), 5.13 (tquint, J = 7.3, 1.3 Hz, 1H, H-30), 6.09 (ddd, J = 7.3, 4.5, 0.9 Hz, 1H,
H-1 ), 7.08 (d, J = 0.9 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.41 (s, 1H, C5-OH), 12.57 (s, 1H,
C8-OH); 13C-NMR (CDCl3):
δ
18.0 (C-60), 25.3 (CH2Br), 25.8 (C-50), 32.8 (C-20), 70.2 (C-10),
111.6 (C-4a), 111.9 (C-8a), 117.2 (C-30), 131.2 (C-3), 133.1 (C-7), 133.4 (C-6), 136.6 (C-40),
146.8 (C-2), 166.1 (COO), 168.3 (C-5), 168.8 (C-8), 175.2 (C-1), 176.8 (C-4); disregarding the
assignment of the signals the 1H-NMR spectrum fits with literature values [46].
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 3-bromopr
opanoate (32), 0.3 mmol Shikonin, 16 mL CH2Cl2, two portions 0.45 mmol DCC, 75 µmol
DMAP and two portions of 0.3 mmol 3-bromopropanoic acid; reaction time 17 h plus 4
days; CC on silica (10 g; CH2Cl2) and PTLC on silica with cyclohexane/CH2Cl2 = 2:1 (six
times developed). It was impossible to separate 32, from the acryl ester derived from HBr
elimination. 32, yield: 2%. 32: Rf = 0.38 (silica, CH2Cl2); IR (ATR): 2972 (w),
≈
2950 (br)
(OH), 2926 (w), 2857 (w), 1742 (m) (C=O), 1608 (s), 1568 (m), 1435 (m), 1408 (m), 1228 (s),
1201 (s) (COC), 1112 (m), 774 (m) cm−1; 1H-NMR (CDCl3): 1.59 (s, 3H, H-60), 1.69 (s, 3H,
H-50), 2.51 (dtm, J = 14.8, 7.2 Hz, 1H, H-20), 2.64 (dtm, J = 14.9, 5.5 Hz, 1H, H-20), 3.01 (t,
0
J = 6.7 Hz, 2H, H-
α
), 3.60 (2t, J = 6.8 Hz 2H, H-
β
), 5.13 (tm, J = 7.3 Hz, 1H, H-3 ), 6.09 (ddd,

Int. J. Mol. Sci. 2021, 22, 2774
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J = 7.3, 4.6, 0.9 Hz, 1H, H-10), 7.04 (d, J = 1.0 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.41 (s,
1H, C5-OH), 12.58 (s, 1H, C8-OH); 13C-NMR (CDCl3):
δ β),
18.0 (C-60), 25.5 (C-50), 29.7 (C-
32.9 (C-20), 37.7 (C-
α
), 70.2 (C-10), 111.6 (C-4a), 111.8 (C-8a), 117.5 (C-30), 131.5 (C-3), 133.0
0
(C-7), 133.2 (C-6), 136.3 (C-4 ), 147.5 (C-2), 167.7 (C-5), 168.2 (C-8), 169.4 (COO), 175.8 (C-1),
177.4 (C-4); MS (ESI−) m/z (%): 269.12 (100) [M-RCOOH-H]−, 241.08 (61) [269-CO]; [M]−
calculated for C19H19BrO6: 422.0365 and 424.0345.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 4-bromo-
butanoate (33), 0.1 mmol Shikonin, 0.25 mmol DCC, 50 µmol DMAP and 0.5 mmol 4-
bromobutanoic acid; reaction time 5 days; CC on silica (10 g; cyclohexane/CH2Cl2 = 2:5);
33, yield: 23%. 33: Rf = 0.34 (silica, cyclohexane/CH2Cl2 = 1:4); IR (ATR): 3055 (w), 2967(w),
2920 (m) (OH), 2855 (w), 1738 (s) (C=O), 1607 (s), 1568 (m), 1453 (m), 1198 (s) (COC), 1112 (s),
0
0
775 (m) cm−1; 1H-NMR (CDCl3): 1.59 (s, 3H, H-6 ), 1.70 (s, 3H, H-5 ), 2.20 (quint, J = 6.8 Hz
,
2H, H-
β
), 2.49 (dtm, J = 14.9, 7.0 Hz, 1H, H-20), 2.57-2.67 (m, 3H, H-20, H-
α), 3.47 (t, J = 6.4
Hz, 2H, H-
γ
), 5.12 (tm, J = 7.3 Hz, 1H, H-30), 6.05 (ddd, J = 7.4, 4.6, 0.9 Hz, 1H, H-10), 7.00
(d, J = 1.0 Hz, 1H, H-3), 7.19 (s, 2H, H-6, H-7), 12.42 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH);
13C-NMR (CDCl3):
δ
18.0 (C-60), 25.8 (C-50), 27.5 (C-
β
), 32.38, 32.44 (C-
α
, C-γ
), 32.9 (C-20),
0
0
69.6 (C-1 ), 111.6 (C-4a), 111.8 (C-8a), 117.6 (C-3 ), 131.3 (C-3), 132.9 (C-7), 133.1 (C-6), 136.3
(C-40), 147.9 (C-2), 167.5 (C-5), 168.0 (C-8), 171.4 (COO), 176.1 (C-1), 177.6 (C-4); MS (ESI-)
m/z (%): 438.11 (27) [M]-, 437.17 (83) [M-H]−, 436.36 (35) [M]−, 435.30 (100) [M-H]−; [M]-
calculated for C20H21BrO6: 436.0522 and 438.0501.
(R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 5-bromo
pentanoate (34), 0.1 mmol Shikonin, 0.15 mmol DCC, 25 µmol DMAP and 0.1 mmol 5-
brompentanoic acid each; reaction time 17 h; CC on silica (8 g; CH2Cl2) and PTLC on silica
with cyclohexane/CH2Cl2 = 2:1 (four times developed); 34, yield: 20%. 34: Rf = 0.32 (silica,
CH2Cl2); IR (ATR): 3055 (w), 2967(w), 2920 (m) (OH), 2855 (w), 1738 (s) (C=O), 1607 (s),
1568 (m), 1453 (m), 1198 (s) (COC), 1112 (s), 775 (m) cm−1; 1H-NMR (CDCl3): 1.59 (s, 3H,
H-60), 1.70 (s, 3H, H-50), 1.77-1.86 (m, 2H, H-
1.7 Hz, 2H, H-
H-20), 3.42 (t, J = 6.5 Hz, 2H, H-
β
), 1.87-1.96 (m, 2H, H-
), 2.49 (dtm, J = 14.9, 7.4 Hz, 1H, H-20), 2.61 (dtm, J = 14.7, 5.4 Hz, 1H,
), 5.12 (tm, J = 7.3 Hz, 1H, H-30), 6.05 (dd, J = 7.1, 6.1, Hz,
γ), 2.44 (td, J = 7.1,
α
δ
1H, H-10), 6.99 (s, 1H, H-3), 7.18 (s, 2H, H-6 and H-7), 12.41 (s, 1H, C5-OH), 12.58 (s, 1H,
C8-OH); 13C-NMR (CDCl3):
δ
18.0 (C-60), 23.4 (C-
β
), 25.7 (C-50), 31.8 (C-
γ
), 32.8 (C-
δ
), 32.9
0
0
0
(C-2 ), 33.3 (C-
α
), 69.5 (C-1 ), 111.6 (C-4a), 111.8 (C-8a), 117.7 (C-3 ), 131.3 (C-3), 132.9 (C-7),
133.0 (C-6), 136.1 (C-40), 148.0 (C-2), 167.4 (C-5), 167.9 (C-8), 171.8 (COO), 176.2 (C-1), 177.7
(C-4); MS (ESI-) m/z (%): 452.04 (30) [M]-, 451.08 (100) [M-H]−, 450.16 (30) [M]−, 449.16
(98) [M-H]−; [M]− calculated for C21H23BrO6: 450.0678 and 452.0658.
3.4. Synthesis of Precursor Acids
3.4.1. 2-(1-Bicyclo[4.1.0]heptyl)acetic Acid (p1)
Under argon and at 0 ◦C, a solution of (1-cyclohexenyl)-2-acetic (226 mg, 1.61 mmol)
in 1.3 mL toluene and and CH2I2 (1.06 g, 4.97 mmol) were added to a solution of ZnEt2
in toluene (3.2 mL, 1 M). After stirring overnight, the mixture was poured into HCl (2 M).
The organic layer was separated, and the aqueous phase was extracted with Et2O. The
combined organic layers were extracted with NaOH (1 M). The aqueous phase was acidified
with 2 M HCl and extracted with CH2CI2. Evaporation of the solvent gave p1
[
65], which
1
was used without further purification. Yield: 60 %. H- and 13C NMR data fit with literature
values [66].
3.4.2. 3-Cyclopropylpropanoic Acid (p2)
A solution of LDA (2. 5 mL, 2 M in THF/toluene/heptane) was added to THF (25 mL)
under argon. The mixture was cooled to 0 ◦C and acetic acid (150 mg, 2.5 mmo◦l) in THF
(4 mL) were added dropwise. The mixture was then stirred for 30 min at 45 C. After
cooling to room temperature, bromomethylcy◦clopropane (405 mg, 3 mmol) was added
within 15 min. The mixture was stirred at 45 C for 5.5 h, cooled to room temperature,
and poured into a mixture of Et2O and water (25 mL and 37 mL resp.). The organic layer

Int. J. Mol. Sci. 2021, 22, 2774
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was acidified with 2 N HCl and extracted AcOEt (3
×
25 mL). The extract was dried
over Na2SO4 and most of the solvent was evaporated under reduced pressure resulting
in a 74% solution of p2 in THF (67 mg, yield: 23%). This solution was used without
further purification.
1H-NMR (CDCl3): 0.06 (q, J = 4.9 Hz, 2H, cyclpropyl-CH2), 0.39–49 (m, 2H, cyclpropyl-
CH2), 0.67–0.79 (m, 1H), 1.53 (q, J = 7.3 Hz, 2H), 2.45 (t, J = 7.4 Hz, 2H, CH2-COOH), 10.72
(br.s, COOH); data fit with literature [67]; 13C NMR (CDCl3),
δ: 4.45 (cyclpropyl-CH2),
10.4 (CH); 29.8 (CH2-CH2-COOH), 34.2 (CH2-CH2-COOH), 180.4 (COOH); data fit with
literature, except δ(CO) [68,69].
3.4.3. (E) 3-Cyclopropylpropenoic Acid (p3)
A mixture of 500 mg (7,13 mmol) cyclopropanecarbaldehyde, 1.04 g (10 mmol) malonic
acid, and 0.94 mL pyridine was stirred under argon for 6 h at 105 ◦C bath temperature,
cooled to room temperature, acidified with ca. 7 mL 2 M HCl, and cooled in an ice bath.
The precipitate was filtered with suction, washed with water, and dried over P2O5 in
1
vacuo resulted in p3 (270 mg, yield 34%; drying caused loss due to sublimation) [70]. H-
NMR (CDCl3) data fit with literature values [31]; 13C-NMR (CDCl3):
δ 9.0 (CH2), 14.6
(cyclopropane CH), 117.4 (C-α), 157.1 (C-β), 172.2 (COOH).
3.4.4. 2-Cyclopropyl-2-Oxoacetic Acid (p4)
KMnO4 (4.42 g, 28 mmol) was solved in water (38 mL). Acetylcyclopropane (1.26 g,
15 mmol) was added under stirring, the mixture was heated to 30 ◦C, and 10% aq. KOH
(6 mL) was added. The mixture was then gently heated with an oil bath and stirred for a
◦
further hour at 60 C, cooled to room temperature, and filtered. The filter cake was washed
with water (10 mL). The filtrate and washing solutions were combined, concentrated
to about 15 mL, acidified with concentrated HCl to pH 2 and extracted with CH2CI2
(
5 × 30 mL). Drying over Na2SO4 and evaporation of the solvent gave p4 (1.33 g; 78%) [71].
1H and 13C; NMR data fit with literature [32].
3.5. Cell Culture
WM9, WM164, and MUG-Mel2 melanoma cell lines were cultivated in RPMI1640
medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The medium was sup-
plemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS, Gibco), and 1% peni-
cillin/streptomycin solution (Pen/Strep, Gibco). HEK-293 (human epithelial cells) were
grown in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco®),
supplemented with L-glutamine (2mM), 10 % FBS, and 1 % Pen/Strep. All cells were grown
in a humidified 5% CO2 atmosphere and at 37 ◦C. They were passaged by trypsinization
when reaching 90% confluence using a 0.25% trypsin-EDTA solution (Gibco).
3.6. XTT Viability Assay
To study cell viability, the cell proliferation kit II (XTT) (Roche Diagnostics, Mannheim,
Germany; cat. no. 11 465 015 001) was used and performed according to the manufacturer’s
instructions. Cell suspensions with 50,000 c/mL were prepared and 100 µL seeded in each
well of a 96 well plate (clear plate, flat bottom). To allow the cells to adhere, cells were
incubated for 24 h before the treatment was started. Control cells were then treated with
0.5% ethanol; all other cells were treated with different concentrations (1.0
10.0 M) of one of the shikonin derivatives. After 72 h of incubation, 50
prepared XTT solution consisting of 5 mL XTT solution plus 100
reagent were added to each well and incubated for another 2 h at 37 C. Finally, the
absorbance was measured with a Hidex Sense Microplate Reader (Hidex, Turku, Finland)
µ
M, 5.0
µM, and
µ
µL of a freshly
µ
L electron coupling
◦
at 490 nm and a reference wavelength of 650 nm.
5.0 M. The assay was performed with three replicates and two different cell passages
at least.
2 was always tested as a reference at
µ

Int. J. Mol. Sci. 2021, 22, 2774
21 of 25
3.7. ApoToxGloTM Triplex Assay
The ApoToxGloTM Triplex Assay was purchased from Promega (Fitchburg, WI, USA,
cat. no. G6320) and performed according to the manufacturer’s instructions. In brief,
10,000 cells/well (WM9 or MUG-Mel2 cells) in 100
well plates (white plates, flat bottom), incubated for 24 h and then treated with different
concentrations of for 4 h, 24 h and 48 h. Staurosporine (Abcam, Cambridge, UK) at a
concentration of 25.0 M is known to induce apoptosis and served as positive control.
Subsequently, the viability/cytotoxicity reagent was prepared and 20 L added to each
µL of medium were pipetted in 96-
5
µ
µ
well. After mixing for 30 s (orbital shaking, 300–500 rpm), the plates were incubated
for another 30 min at 37 ◦C. Fluorescence was then measured at 400Ex/505Em (viability)
and 485Ex/520Em (cytotoxicity) using a Hidex Sense Microplate Reader. Afterwards, the
Caspase-Glo® 3/7 reagent was prepared and 100
µ were added to each well, followed
by mixing (orbital shaking, 300–500 rpm, 30 s), and incubation at room temperature for
another 30 min. Finally, luminescence was measured using a Hidex Sense Microplate
Reader. The assay was performed at least two times, with three replicates each.
3.8. LDH Assay
To measure LDH release, the CytoTox 96® Non-Radioactive Cytotoxicity Assay
(Promega) were used in accordance with the manufacturers protocol. Cell suspension of
100,000 c/mL were prepared and seeded into 96 well plates (clear, flat bottom, 100
To allow the cells to adhere, cells were incubated for 24 h before the treatment was started.
Afterwards, cells were treated with 1 M, 5 M, 10 M, or 20 M of for 24 h, 48 h, or 72 h.
To measure maximum LDH release, cells were lysed using the lyse reagent included in
the assay kit. To quantify LDH release, the plates were centrifuged and 50 L of each well
were transferred into another 96 well plate. After addition of 50 L CytoTox96 Reagent for
30 min, 50 L of the stop solution were added and absorption were measured at 490 nm
µL/well).
µ
µ
µ
µ
5
µ
µ
µ
using a Hidex Sense Microplate Reader. Finally, using the following formula, the amount
of LDH release was calculated: 100 × OD490 test well/OD490 maximum LDH release.
3.9. Cell Cycle Analysis
For cell cycle analysis, 2 mL of a 150,000 c/mL cell suspension were seeded into
6 well plates and incubated for 24 h. Afterwards, the cells were treated with 10
20 M of for 24 h and 48 h. The cells were collected including supernatants and by using
trypsinization, centrifugation, and washing with RPMI cell culture medium. Finally, cells
were resuspended in 500 L PBS and fixed with 5 mL ice cold ethanol for 10 min. For
FACS analyses, cells were centrifuged and resuspended with 200 L PI lysis buffer. After
flow cytometer (BD
µM or
µ
5
µ
µ
incubation for 20 min in the dark, cells were analyzed using a LSRII
™
Biosciences, Franklin Lakes, NJ, USA) and ModFit software.
4. Conclusions
In previous studies, shikonin and derivatives have been demonstrated to be potent
cytotoxic substances. In this study, we prepared 31 shikonin derivatives by synthesis.
Most of them are novel derivatives, which have not yet been reported. One goal was to
further optimize our previous hit 3. Another goal was to synthesize a broad spectrum
of structural features to gain more insight into the structure-activity relationship. All
derivatives were screened for their cytotoxicity in several melanoma cell lines. The results
indicate that there is no strict structure-activity relationship and the different cell lines
exhibited distinct sensitivities towards the derivatives. The most potent derivative was
5
, which is a cyclopropyloxoacetate derivative of shikonin and, thus, structurally related
to our previous hit . Compared to was more cytotoxic. Subsequent pharmacological
investigations revealed that leads to caspase 3/7 activation, no significant LDH release,
3
3, 5
5
and to a G2/M phase cell cycle arrest at higher concentrations. Nevertheless, it was also
cytotoxic towards non-tumorigenic cells, which needs to be evaluated in more detail in

Int. J. Mol. Sci. 2021, 22, 2774
22 of 25
future studies. In summary, our results indicate that shikonin derivatives might be potential
drug leads for the development of novel melanoma treatment options.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006
7/22/5/2774/s1.
Author Contributions: Design and supervision of the study: N.K., A.H., B.R., R.B.; synthesis
of derivatives, NMR recording and analyzing: A.H.; screening of derivatives: N.K., C.D., K.P.,
pharmacological investigations: N.K., C.D., K.P., B.L.; data processing and writing of the manuscript:
N.K., A.H. and R.B. All authors have read and agreed to the published version of the manuscript.
Funding: This project was funded in whole by the Austrian Science Fund (FWF) [P27505].
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Eva-Maria Pferschy-Wenzig for her help in LC-MS measurements and
Ing. Andrea Fleck for recording the IR spectra. Open Access Funding by the Austrian Science Fund
(FWF) is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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