Synthesis and evaluation of novel alkannin and shikonin oxime derivatives as potent antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2014.07.012

A set of forty alkannin and shikonin oxime derivatives were firstly designed and synthesized. Their cytotoxicities against three kinds of tumor cells and a normal cell line were tested and compared with alkannin and shikonin. The cell-based investigation demonstrated that some oxime derivatives were more or comparatively effective to the lead compounds, especially their selective and excellent antitumor activities towards K562 cells with no toxicity in normal cells. We may conclude that oximate modification to the mother nucleus of alkannin and shikonin is an available approach to acquire potent antitumor agents.

Full text of DOI:10.1016/j.bmcl.2014.07.012

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4304–4307
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel alkannin and shikonin oxime
derivatives as potent antitumor agents
⇑
Rubing Wang , Xu Zhang , Hualong Song, Shanshan Zhou, Shaoshun Li
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of forty alkannin and shikonin oxime derivatives were firstly designed and synthesized. Their
cytotoxicities against three kinds of tumor cells and a normal cell line were tested and compared with
alkannin and shikonin. The cell-based investigation demonstrated that some oxime derivatives were
more or comparatively effective to the lead compounds, especially their selective and excellent antitumor
activities towards K562 cells with no toxicity in normal cells. We may conclude that oximate modifica-
tion to the mother nucleus of alkannin and shikonin is an available approach to acquire potent antitumor
agents.
Received 10 February 2014
Revised 15 June 2014
Accepted 5 July 2014
Available online 30 July 2014
Keywords:
Alkannin
Shikonin
Ó 2014 Elsevier Ltd. All rights reserved.
Oxime derivatives
Antitumor activity
Nearly half of the clinical anticancer drugs are either natural
products or directly derived from naturally occurring lead com-
pounds, such as paclitaxel, topotecan, irinotecan, vinblastine and
colchicines.1,2 Alkannin (S-isomer) and shikonin (R-isomer),
extracted and identified, respectively, from the roots of Alkanna
tinctoria in Europe and Lithospermum erythrorhizon in the Orient
as a pair of enantiomers, have attracted great interest as hallmark
molecules because of their fascinating biological activities.2–6 Hun-
dreds of alkannin and shikonin derivatives have been isolated, syn-
thesized and evaluated as to their tumor inhibitory potency.2–18 So
far, most modifications have focused on the hydroxyl group in the
side chain and some of these modified compounds have shown
comparable or stronger cytotoxicities than their parent natural
products in vitro,4–13,16 such as b-hydroxyisovalerylshikonin,
isovalylshikonin, acetyl-shikonin and so on. However, none of
them enter into clinical trials because of their serious toxic effects,
even though great tumor inhibitory effects were observed for
many shikonin derivatives in cell culture studies.
tumor cells but also in normal cells. In order to develop less toxic
alkannin and shikonin derivatives, modifications of structures on
naphthazarin ring besides the hydroxyl group of the side chain
seem reasonable and practical. Part of the existing data displays
that dimethylation of the naphthazarin ring for some compounds
containing naphthazarin moiety will improve the antitumor activ-
ity.14–18 However, adverse effects such as weight reduction, hypo-
trichosis and much bloody ascites were further observed in in vivo
experiments. It is important to point out that O-dimethyl alkannin
and shikonin derivatives have comparable level of ROS and alkyl-
ation to naphthoquinone compounds, and this should also be
responsible for the unselective profile of cell damage. Therefore,
only the modifications on the alcoholic hydroxyl group of the side
chain and phenolic hydroxyl groups are insufficient to overcome
drawbacks in the structure of alkannin and shikonin itself.
In this study, the modification of alkannin and shikonin was
focused on the naphthoquinone moiety basing on the structures
of O-dimethyl alkannin and shikonin derivatives. Many oxime
compounds have recently exhibited satisfactory anticancer effects
recently.24–28 This research project presents a series of novel oxime
alkannin and shikonin derivatives, both their synthesis and antitu-
mor activities against different cancer cell lines in vitro.
The key pharmacophores of both alkannin and shikonin were
found to be the naphthazarin ring and hydroxyl side chain. It has
been demonstrated that the naphthazarin ring has a strong ability
to generate reactive oxygen species (ROS) via the redox cycling and
bio-reductive and Michael addition alkylation processes,19–23
which induce the apoptotic death of many cancer cell lines indis-
A general synthetic route for O-dimethyl acylalkannin and acyls-
hikonin oxime derivatives was illustrated in Scheme 1. (R)- or (S)-4-
methyl-1-(1,4,5,8-tetramethoxynaphthalen-2-yl)pent-3-en-1-ol (2)
was prepared in high optical purity (>99% ee) using 1,4,5,8-tetra-
methoxy-2-naphthaldehyde (1) as the starting material according
to the procedures previously reported by our group.29–31 Condensa-
tion of 2 with different kinds of carboxylic acid in the presence of
dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP)
criminately cause damages to
a wide variety of biological
macromolecules, such as nucleic acids and proteins, not only in
⇑
Corresponding author.
E-mail address: ssli@sjtu.edu.cn (S. Li).
R.B. Wang and X. Zhang contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.07.012
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

R. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4304–4307
4305
OMe OMe
OMe OMe
OMe OMe
Ref 22
i
CHO
OMe OMe
OMe OMe OH
OMe OMe OCOR
2
1
3a 3p
~
ii
HO
N
OMe
O
O
OMe
R, shikonin derivatives
iii
5
8
4
1
1'
S, alkannin derivatives
N
OMe OCOR
5a 5p
OMe OCOR
4a 4p
HO
~
~
Scheme 1. Synthesis of the oxime derivatives 5a–5p. Reagents and conditions: (i) RCO2H/DCC/DMAP/CH2Cl2, rt, 1–12 h; (ii) CAN/H2O/CH2Cl2, 0 °C, 15 min; (iii) HONH3Cl/Py/
EtOH, 50 °C, 8 h.
resulted in acyl derivatives (3a–3p) and subsequent oxidation
with cerium (IV) ammonium nitrate (CAN) gave corresponding
O-dimethyl alkannin and shikonin compounds (4a–4p).17 Stirring
at room temperature of compounds 4a–4p with hydroxylamine
hydrochloride respectively in the presence of pyridine
subsequently produced alkannin and shikonin oxime derivatives
(5a–5p).32,33
compounds were comparable to the lead compounds alkannin
and shikonin, and some even better. Among these oxime deriva-
tives, IC50 values of the most effective alkannin derivatives 5b
and 5e against K562 were as low as 0.7
lM, which showed higher
cytotoxicities than alkannin (IC50 = 1.3
lM). Meanwhile, the IC50
values of alkannin derivative 5e against MCF-7 and DU145 were
7.5 and 19.3 M, respectively. Almost all the IC50 values of other
l
An appropriately designed route for the synthesis of ether
derivatives of O-dimethyl alkannin and shikonin oxime derivatives
was shown in Scheme 2. The nucleophilic substitution reaction of
compound 2 with organic halides produced ether derivatives
(6a–6d), and subsequent oxidation of 6a–6d with CAN yielded
key intermediates O-dimethyl alkannin and shikonin.17 Targeted
compounds 8a–8d were easily obtained by the condensation reac-
tion (Scheme 2) between 7a–7d and hydroxylamine hydrochloride
in the presence of pyridine.32,33
The in vitro cytotoxicities of the prepared alkannin and shikonin
derivatives against MCF-7 (breast cancer), K562 (leukemia), DU145
(prostate cancer) and HSF (human skin fibroblasts) cells were eval-
uated by the standard MTT assay using alkannin and shikonin as
reference compounds. Antitumor potencies of the forty compounds
are displayed as IC50 values that were calculated by linear regres-
sion analysis of the concentration–response curves afforded for
each compound. The results are summarized in Table 1.
compounds in this category showed the same trend among tested
cancer cell lines with the best inhibitory effect in K562, moderate
in MCF-7, but little in DU145, reflecting excellent selectivity for a
particular leukemia cell line. Moreover, it is exciting to note that
none of the prepared compounds displayed cytotoxicity towards
normal cell line HSF (IC50 >50 lM). Though great cytotoxicities
on cancer cell lines (K562, MCF-7 and DU145) were observed for
the lead compounds alkannin and shikonin, comparable inhibitory
activities towards normal cell HSF and no selectivity was also
observed between cancer and normal cells.
It can be observed from Table 1 that the side chain of oxime
derivatives had a great effect on their cytotoxicities. Compounds
with ester moieties in the side chain showed higher cytotoxicities
than those with ether groups, especially for K562 cells. For exam-
ple, alkannin derivative 5e containing ester groups (IC50 = 0.7
was approximately 20-fold more cytotoxic than relative ether
compound 8d (IC50 = 13.5 M). For compounds 8a with hydroxyl
lM)
l
From the cytotoxic activity results of alkannin and shikonin
derivatives shown in Table 1, we can see that most target oxime
derivatives except shikonin compound 8a displayed potent activity
against three kinds of cancer cell lines. The potencies of some
in the side chain, its cytotoxicity sharply decreased. It can be con-
cluded that the cytotoxicities of resulting oxime derivatives are
significantly correlated with the nature of the substituent group
at 10-position in the side chain. Within the series 5a–5p which bear
O
OMe
OMe OMe
OMe OMe
ii
i
O
7a~7d
OMe OR
OMe OMe OR
6a~6d
OMe OMe OH
2
iii
HO
HO
N
OMe
R, shikonin derivatives
S, alkannin derivatives
5
8
4
1
1'
N
OMe OR
8a 8d
~
Scheme 2. Synthesis of the oxime derivatives 8a–8d. Reagents and conditions: (i) NaH/RBr/THF, 0–20 °C, 24 h; (ii) CAN/H2O/CH2Cl2, 0 °C, 15 min; (iii) HONH3Cl/Py/EtOH, rt,
12 h.

4306
R. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4304–4307
Table 1
In vitro inhibitory activity of alkannin and shikonin derivatives against DU145, MCF-7, K562 and HSF cell lines
HO
HO
N
OMe
N
OMe
R, shikonin derivatives
S, alkannin derivatives
N
OMe OCOR
N
OMe OR
8a~8d
HO
HO
5a~5p
Compd
Substituents
IC50 (lM)
DU145
MCF-7
K562
HSF
R
S
R
S
R
S
R & S
5a
5b
5c
5d
5e
5f
5g
5h
5i
Methyl
25.1
11.3
28.9
10.2
27.7
12.3
18.0
19.4
19.9
18.4
18.0
16.8
16.3
19.7
12.1
16.9
>50
17.1
18.1
32.8
18.0
19.3
20.6
19.5
16.9
14.3
17.0
16.8
21.4
16.9
12.5
20.7
16.1
30.2
21.1
10.6
30.8
20.9
6.0
26.0
4.9
16.7
7.1
4.9
4.5
7.7
3.6
5.8
8.1
1.6
8.2
3.7
7.5
3.1
2.4
2.8
1.8
1.5
1.7
2.4
1.9
1.2
6.3
1.4
9.3
5.3
0.7
4.7
3.0
0.7
3.7
2.6
1.6
1.5
1.3
1.3
1.3
2.1
1.7
3.6
1.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.2
3-Methylbut-2-enyl
3-Hydroxyisobutyl
Isopropyl
Isobutyl
Ethyl
1.4
9.5
3.3
2.1
5.4
3.1
1.9
1.7
1.5
2.9
2.1
2.5
2.6
3.8
2.0
Methylvinyl
Phenyl
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
4-Chlorophenyl
4-Methoxyphenyl
4-Nitrophenyl
Pyridin-2-yl
Thiophen-2-yl
Hydrogen
Methyl
Ethyl
Isopentyl
Shikonin
5j
5k
5l
6.6
6.3
3.9
7.6
5m
5n
5o
5p
8a
8b
8c
8d
SK
AK
5.7
>50
30.2
15.7
16.2
1.8
21.9
17.3
17.1
17.6
42.7
21.1
17.1
12.4
0.7
13.8
14.6
15.4
13.5
32.4
17.1
26.3
16.0
19.9
Alkannin
2.4
1.3
1.5
Data represent the mean values of three independent determinations.
ester groups in the side chain, the great majority of compounds
displayed comparable activities to those of alkannin and shikonin.
When the ester groups were linked with aliphatic hydrocarbon,
their selectivities on three cancer cell lines and cytotoxicities were
higher than those linked with aromatic hydrocarbon. Most alkan-
nin oxime derivatives (S-isomer) showed generally better cytotoxic
activities than corresponding shikonin derivatives (R-isomer),
especially for MCF-7 and K562 cell lines, though there were some
opposite outcomes in DU145 cell line. For synthesized ether deriv-
atives (8a–8d), not only were their cytotoxicities on selective can-
cer cell lines not as good as ester compounds, but also no similar
selectivities were observed as shown by the ester derivatives.
ROS, Michael addition and bioreductive alkylation were poten-
tial mechanisms leading to the cytotoxicity of shikonin com-
pounds.19,20,23 In this study, two representative compounds (R-5b
and S-5b) and acetylshikonin (SK-02) (Fig. 1) were chosen to be
tested and compared for their corresponding ROS and alkylation
levels. A quantitative assay using single cells (MCF-7) analyzed
by flow cytometry was adopted for testing ROS levels induced by
these derivatives. DCFH-DA (20,70-dichloro-dihydrofluorescein
diacetate; Sigma) was used as a ROS-capturing reagent in the
method reported.34 The data was summarized in Table 2. Accord-
ing to the data shown in Table 2, it is obvious that ROS generated
by oxime derivatives significantly decreased. Compared to SK-02,
which has the same side chain and was assumed as 100%, shikonin
oxime derivative (R-5b) and alkannin oxime derivative (S-5b)
decreased to 24.3% and 33.0%, respectively. Additionally, the alkyl-
ation level of oxime derivatives was tested and the results were
summarized in Table 3. In this study, p-thiocresol, a kind of nucle-
ophilic and reducing agent, was applied to react with the three
compounds under the same conditions to determine their ability
of alkylation. As shown in Table 3, for the two chosen oxime deriv-
atives (R-5b and S-5b), both Michael addition and bioreductive
alkylation pathways were blocked, and thus no alkylation product
was obtained. Based on the ROS and alkylation data above, we con-
clude that the excellent cytotoxicities of these target oxime deriv-
atives are not resulted from ROS and alkylation, but probably
associated with other unknown mechanisms.
In summary, new series of oxime derivatives of alkannin and
shikonin were synthesized and evaluated for their antitumor effects
HO
HO
OH
O
N
N
OMe
OMe
OH
O
O
N
OMe O
N
OMe O
HO
HO
SK-02
R-5b
O
S-5b
O
O
Figure 1. Structures of the selected three compounds for ROS and alkylation examination.

R. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4304–4307
4307
Table 2
Test of ROS level for the selected three compounds in MCF-7 cells by flow cytometer
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