Semi-synthesis and antitumor activity of 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.006

We recently discovered that 5, 8-O-dimethyl acylshikonin derivatives displayed the selectivity towards MCF-7 and no toxicity to normal cells. Herein, a series of the corresponding 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives were synthesized star

Full text of DOI:10.1016/j.ejmech.2011.05.006

European Journal of Medicinal Chemistry 46 (2011) 3420e3427
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Semi-synthesis and antitumor activity of 6-isomers of 5, 8-O-dimethyl
acylshikonin derivatives
,
,
,
*
Wen Zhou a 1, Xu Zhang a 1, Ling Xiao b, Jing Ding a, Quan-Hua Liu b, Shao-Shun Li a
a School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China
b Shanghai Institute of Pharmaceutical Industry, 1111 North Zhongshan Road, Shanghai 200437, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently discovered that 5, 8-O-dimethyl acylshikonin derivatives displayed the selectivity towards
MCF-7 and no toxicity to normal cells. Herein, a series of the corresponding 6-isomers of 5, 8-O-dimethyl
acylshikonin derivatives were synthesized starting from shikonin. In vitro evidence of the cytotoxicities
indicated that most of thecompounds were more active than or comparative to shikonin and retained the
selectivity against MCF-7, MDA-MB-231 besides no toxicity in the normal cells. Also, in vivo anticancer
activity of the positional isomers 5p, 6c further showed that 6-isomers of 5, 8-O-dimethyl acylshikonin
derivatives were more active than their corresponding 2-isomers. Thus, we may conclude that the
position of the side chain of shikonin attached to 5,8-dimethoxy -1,4-naphthoquinone is associated with
the antitumor activity.
Received 6 February 2011
Received in revised form
2 May 2011
Accepted 4 May 2011
Available online 12 May 2011
Keywords:
Shikonin derivatives
Semi-synthesis
Antitumor activity
Positional isomer
Structure-activity relationship
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
shikonin is certainly no exception. As evidenced by Ahn and his
team, compared with 2-isomers, 6-isomers demonstrated a higher
The pharmacophore of shikonin was found to be naphthazarin
ring [1], which was probably complicated in forming covalent bond
with cellular nucleophiles such as DNA, proteins etc and generating
superoxide anions via the redox cycling to kill cells [2e4]. The
permethylated products of naphthazarin and its derivatives,
5,8-dimethoxy-1,4- naphthoquinone (DMNQ) and its analogues,
decreased slightly the cytotoxicity but appeared to increase the T/C
values, implying that the disappearance of tautomerism of naph-
thazarin and the electron density increased in the quinonoid
moiety of DMNQ will benefit antitumor activities in vivo [5]. In our
continuing drug discovery program most of 5, 8-O- dimethyl
acylshikonin derivatives, expressing higher selectivity to MCF-7
and no toxicity to the normal cell, exhibited a greater potency in
the growth inhibition of subcutaneous S-180 carcinoma, as well as
less toxicity in vivo than lead compound shikonin [6]. However, it is
well-known that the permethylation of naphthazarin derivatives
could form two positional isomers as 6-substituted DMNQ
(6-isomer) and 2-substituted DMNQ (2-isomer) (Fig. 1), and
potency in the inhibitory effects on DNA topoisomerase-I and the
cytotoxicity against L1210 cell [7e9], to a large extent suggesting
the importance of the position of side chain on quinonoid moiety in
effecting the antitumor bioactivities. Therefore, studies on the
synthesis and antitumor activities of 6-isomers of 5, 8-O-dimethyl
shikonin derivatives could be crucial for deriving a novel class for
promising anti-cancer scaffolds.
Additionally, previous studies demonstrated that acylation of
10-hydroxyl contained in the side chain of DMNQ derivatives
potentiated the cytotoxic activity due to the enhancing effect of
acylating on the electrophilicity of the quinonoid unit [6,10,11], thus
raising renewed interest in the induction of the various organic
acids to the hydroxyl group at the 10 -position of 6-isomer of 5, 8-O-
dimethyl shikonin as an available approach to producing prospec-
tive anti-tumor candidates. To testify the feasibility of this line of
reasoning as well as to provide access to diverse analogues of the
positional isomers, it would be useful to develop a synthetic
method that allowed 6-isomers of 5,8-O-dimethyl acylshikonin
derivatives, 1-(1,4-dihydro-5,8- dimethoxy-1,4- dioxonaphthalen
-6-yl) -4-methylpent -3-enyl carboxylates, to be synthesized
expediently. So in the study, we designed and prepared a series of
6-isomers of 5, 8-O-dimethyl acylshikonin derivatives starting from
shikonin to give insight into their cytotoxic activities in vitro so as
* Corresponding author. Tel./fax: þ86 21 34204775.
E-mail addresses: wzhou60@sjtu.edu.cn (W. Zhou), ssli@sjtu.edu.cn (S.-S. Li).
Wen Zhou, Xu Zhang contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.006

W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
3421
speaking, two hydrogens of benzene ring of 2-isomers of
5,8-O-dimethyl shikonin and its derivatives appeared at lower field
than one hydrogen of their benzoquinone, while two hydrogens of
benzoquinone of all 6-isomers indicated at the relatively higher
field than one hydrogen of their benzene ring.
Finally, the target compounds 5ae5v derived from the corre-
sponding organic acids by treatment of dicyclohexylcarbodiimide
(DCC) and dimethylaminopyridine (DMAP) with compound 4 at
room temperature overnight [16].
O
OCH3
8
OCH3O
1
4
4
1
5
6
5
2
8
R
R
O
OCH3
R: substituent group
OCH3O
2-isomer
6-isomer
Fig. 1. Structures of 2-isomer and 6-isomer.
to acquire more potent and selective agents, and ultimately further
evaluated for the antitumor activities on S-180 xenograft models
with a pair of the positional isomers 5p, 6c as a representative
example.
3. Results and discussion
The in vitro cytotoxic activity of all the compounds prepared
against the cancerous cells and normal cells was evaluated by the
cell-based MTT Assay [6,17] using shikonin as a positive control.
Antitumor potency of the tested derivatives was showed by IC50
values that were calculated by linear regression analysis of the
concentration-response curves afforded for each compound. The
data are described in Table 1 and Table 2. Afterwards, two typical
positional isomers (5p, 6c) were further evaluated with an S-180
xenograft model in male KM mice.
To identify whether the position of the side chain linked to
DMNQ was related to the antitumor activity, we compared the
cytotoxicities of several 2-isomers of 5,8-O-dimethyl acylshikonin
derivatives, which possessed the remarkably effective antitumor
activities among the tested compounds [6], with their corre-
sponding 6-isomers against K562 (leukemia), MCF-7 (breast
cancer), HCT-15(colon cancer), CNE (nasopharyngeal cancer), MDA-
MB-231(breast cancer), L-929 (mouse fibroblast growth) and
HSF(human skin fibroblasts). As indicated in Table 1, all the
compound tested were effective against five tumorigentic cell lines
2. Chemistry
The published synthesis of 2-isomer of 5, 8-O-dimethyl shikonin
(the same structure of 5, 8-O-dimethyl shikonin mentioned in
reference 6.) was not suitable for the preparation of the key inter-
mediate 4 [6]. Consequently, a novel synthetic pathway was needed
to be established in order to prepare the derivatives of compound 4.
The preparation of 6-isomers of 5, 8-O-dimethyl acylshikonin
derivatives (5a-5v) is outlined in Scheme 1.
Shikonin 1, prepared in bulk according to the same procedure
reported previously [6,12], was converted to 5,8-O-dimethox-
ymethyl shikonin 2 by treatment of newly distilled chloromethyl
ether with anhydrous K2CO3 in DMF at 40 ꢀC. Subsequently,
synthesis of compound 3 was performed in accordance with the
modified standard procedure of reductive methylation proposed by
George [13], which was involved in treating compound 2 with
sodium hydrosulfite in the mixture of THF and water as the ratio of
4: 1, and followed by adding sodium hydroxide and dimethyl
sulfate and heating under the atmosphere of N2. Noteworthy, the
catalytic amount of tetrabutylammonium bromide was crucial for
the yield of compound 3 [14].
Next, the removal of methoxymethyl groups was conducted
smoothly by means of 10% HCl [15] and further exposed to air to
afford compound 4, 6-isomer of 5,8-O-dimethyl shikonin, in 65.5%
yield. The structure of the resulting product was explained
distinctly that the chemical shifts of hydrogens on the naph-
thoquinone ring were used to determine the detailed difference
with 2-isomers of 5,8-O-dimethyl shikonin. In fact, the significant
difference was attributed to the presence of the substituent posi-
tion of the side chain, which resulted in the differentiation of three
hydrogens of naphthoquinone displayed in 1H-NMR. Generally
with the IC50 values ranging from 0.2 mM to 58.0 mM. Compound 5a,
5f, 5p, 5r exhibited 2e9-fold more active and selective toward
MDA-MB-231 and MCF-7 cell lines than the corresponding isomers
6ae6d, and the tendency was in good accordance with the rela-
tionship of 6-isomers and 2-isomers of DMNQ analogues against
L1210 cell [7,8]. The greater activities of 6-isomers than 2-isomers
were due to the introduction of the same side chain with an ester
group into different positions of 6- and 2-isomers and the exposure
of quinone moiety as a Michael acceptor to cellular nucleophiles
[9,18]. Although compound 6ae6d were shown to have slightly
more effective on K562, CNE and HCT-15 cell lines than those of the
corresponding 6-isomers except compound 6a against K562, on the
whole the inhibitory activities to these cell lines decreased signif-
icantly than the lead compound shikonin. Therefore, a great
difference between IC50 values of the two positional isomers
OCH3
OCH3
OH
O
O
O
O
OCH3
i
5
8
4
1
ii
iii
2
1'
OH
O
OH
O
OCH3OH
O
O
OH
OCH3
OCH3
2
3
1
O
OCH3
O
OCH3
iv
O
OCH3O
R
O
OCH3OH
O
5a~5v
4
Scheme 1. Reagents and conditions: (i) CH3OCH2Cl/K2CO3/DMF, 1 h, 40 ꢀC, N2; (ii) Na2S2O4/(CH3)2SO4/NaOH/N(But)4Br/THF/H2O, 2h, reflux; (iii) i-PrOH/THF/10%HCl, r.t. overnight;
(iv) DCC/DMAP/CH2Cl2/RCOOH, r.t, overnight.

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W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
Table 1
Cytotoxicity comparision of 6- and 2- isomers of 5,8-O-dimethyl acylshikonin derivatives against K562, MCF-7, HCT-15, CNE, MDA-MB-231, L-929, HSF.
O
OCH3
OCH3O
O
OCH3O
R
OCH3O
O
R
O
O
5a, 5f, 5q, 5r
6a~ 6d
Compound
R
Tumorigenic cell lines IC50
(
mM)
Normal cell lines IC50
L-929
(mM)
K562
15.3
MCF-7
4.9
HCT-15
19.3
CNE
58.0
MDA-MB-231
3.4
HSF
5a
>100
>100
6a
5f
18.2
16.8
13.4
0.9
29.6
21.9
23.1
49.2
7.2
1.7
>100
>100
87.5
>100
6b
5p
11.7
17.6
3.3
19.5
16.4
27.6
46.2
6.8
0.2
>100
>100
>100
>100
0.35
OH
O
6c
5r
15.3
11.2
2.2
2.5
13.1
18.6
32.1
20.6
1.7
0.6
>100
>100
>100
>100
6d
Shikonin
11.5
0.6
5.7
1.9
12.7
7.3
16.5
0.7
2.1
6.6
>100
3.2
>100
1.3
The data of antitumor activities of compound 6awd came from reference 6; Mean IC50 values were calculated from at least three independent experiments.
Table 2
Cytotoxicity of 6- isomers of 5,8-O-dimethyl acylshikonin derivatives against MDA-MB-231, MCF-7, HSF.
O
OCH3
O
OCH3O
R
O
5a~5v
Compound
R
IC50 (mM)
MDA-MB-231
3.4
MCT-7
4.9
HSF
5a
5b
5c
5d
>100
2.5
4.3
2.8
6.2
5.1
3.7
78.6
86.3
69.3
5e
1.5
3.3
74.9
5f
1.7
5.9
0.9
4.5
>100
5g
93.6

W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
3423
Table 2 (continued )
Compound
R
IC50 (mM)
MDA-MB-231
MCT-7
6.7
HSF
5h
5i
2.2
>100
>100
3.7
2.4
5.9
3.9
5j
>100
>100
88.3
5k
5l
16.8
1.4
15.2
8.3
NO2
OCH3
5m
5n
4.4
2.3
21.3
5.5
>100
>100
H3CO
OCH3
OCH3
5o
5p
5.0
0.2
7.7
>100
>100
OCH3
0.35
OH
O
5q
5r
5s
1.3
0.6
0.4
2.7
2.5
1.3
>100
>100
>100
O
O
O
N
5t
0.2
1.1
69.4
5u
5v
6.0
1.2
17.4
6.3
91.7
>100
N
4
1
4.1
6.6
9.2
1.9
79.6
1.3
shikonin
Mean IC50 values were calculated from at least three independent experiments.

3424
W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
Table 3
Tumor growth inhibition in KM mice implanted with S-180 sarcoma.
Group
Dose (mg/kg)
Routes
Numbers
BWC (%)
TW(g) mean ꢁ SD
TWI (%)
D0
D10
Control
5p
5p
5p
6c
i.p.
i.p
i.p
i.p.
i.p.
i.p.
10
10
10
10
10
10
10
10
10
10
10
10
þ39.1%
þ32.6%
þ25.3%
þ20.5%
þ22.9%
þ34.7%
2.07 ꢁ 0.42
3
6
12
12
25
1.24 ꢁ 0.29**
0.91 ꢁ 0.07**
0.58 ꢁ 0.10**
0.89 ꢁ 0.08**
1.11 ꢁ 0.05**
40.0
55.9
72.4
57.1
46.5
5-FU
Data were presented as X ꢁ SD, and significance was assessed with Student’s t-test. Differences were considered significant at **p < 0.01, compared with control group; D0
means the day before beginning to dose.; D10 means the 10th day; BWC means average body weight change; TW average means tumor weight; TWI means the average
inhibition rate of tumor weight.
implied that diverse mechanisms need to be considered to explain
the cytotoxicity against measured cancer cell lines, and that the
importance of the position of the side chain connected to DMNQ
had a profound influence on the antitumor activities. It was
noticeable that the tested 6-isomers of 5,8-O-dimethyl acyl-
shikonin derivatives (5a, 5f, 5p, 5r) also displayed no cytotoxicity to
form Table 2 indicated that the switch of heteroatom’ positions and
the introduction of double bonds to the ring could lead to signifi-
cant change of the cytotoxic activity.
Unlike our previous report [6,16], sharp drop in cytotoxic activity
did not appear when replacing the alkylgroup with the aryl moiety
(5he5o) in cancer cell lines, suggesting that an increase in steric
hindrance beneficial to the enhancement of the antitumor effects
was associated probably with other mechanisms. Although the
position of methoxy substituent on aryl moiety had not significant
influenceonthecytotoxicity, thepara-position(5l)hadanadvantage
over the ortho-position (5m) against the cancer lines. Replacement
of the methoxy group with an electron-withdrawing nitro-group
(5k) led to a dramatic decrease of antitumor potency against MDA-
DB-231 compared with compound 5l. Additionally, the phenethyl
and cinnamenyl in place of the phenyl resulted in the corresponding
compounds 5i, 5j, which had similar effects on the cytotoxic activity.
In order to compare the ability of 2-isomer of 5,8-O-dimethyl
acylshikonin derivative 6c to inhibit the tumor growth with that
of its corresponding 6-isomer 5p and their toxic effects in vivo
test, KM mice bearing subcutaneous sarcoma S-180 were per-
formed as an evaluation model [6], which shikonin derivatives
had possessed good antitumor activities towards [20]. As
demonstrated in Table 3, compound 5p, 6c significantly inhibited
the growth of sarcoma S-180 with 72.4%, 57.1% respectively when
administered intraperitoneally (i.p.) at 12 mg/kg once a day for 9
days in comparison with the control group. However, 5-FU used
as a positive control was showed only 46.5% suppression via i.p.at
the dose of 25 mg/kg once day. Consistent with the previous
report [6], 2-isomer of 5,8-O-dimethyl acylshikonin derivative 6c
at 12 mg/kg showed the similar inhibition rate of 57.1%, which
was comparative to the antitumor activity of the corresponding
isomer 5p at 6 mg/kg. From Table 3 the dose-dependent rela-
tionship was observed in the tumor growth suppressed by
compound 5p. Treatment of the model mice with compound 5p
at 3 mg/kg, 6 mg/kg and 12 mg/kg resulted in the inhibitory
effects of 40.0%, 55.9%, 72.4% respectively, which positively
related with concentrations. It was noteworthy that no signifi-
cant toxicity was found in two dimethylated shikonin
derivatives-treated mice. Evidences from the antitumor effects
in vivo indicated the two positional derivatives were more potent
antitumor agents than the positive control, and 2-isomer of
5,8-O-dimethyl acylshikonin derivative 6c less active than its
6-isomer 5p, further reflecting the fact that the position of the
side chain attached to DMNQ was vital for enhancing the anti-
tumor potency of shikonin derivatives.
the non-tumerigentic cell lines (IC50 ＞ 100 mM) and retained the
same selectivity to breast cancer cells as most of 2-isomers reported
previously [6]. Taken together, besides the significant increase of
the selectivity, the 6-isomers of 5,8-O-dimethyl acylshikonin
derivatives were comparative to, or more active against MDA-MB-
231 and MCF-7 cell lines than the reference compound shikonin,
and thus it was suggested that 6-isomer of 5,8-O-dimethyl shikonin
4 was a useful lead compound for the synthesis of naphthoquinone
derivatives against certain specific cancer cell types.
As it was proved that the introduction of various substituents to
10-position in the side chain of shikonin was closely related with an
increase in cytotoxicity [6], it was appealing that more 6-isomers of
5,8-O-dimethyl acylshikonin derivatives were synthesized to
undertake more indepth investigations into the cytotoxicities
against selected MDA-MB-231, MCF-7, which turned out to be the
most sensitive cell lines suppressed by 5a, 5f, 5p, 5r and 6ae6d. It
had been observed from Table 2 that all the compounds but
compound 5k, 5m, 5u had higher cytotoxicities than compound 4
with IC50 values of less than 8.3 mM in breast cancer lines, implying
that the positive effect of the attachment of various organic acids to
the side chain of shikonin. The introduction of saturated alkylgroup
to 10-hydroxyl group in the side chain could benefit the increase of
antitumor potency to some extent (5a-5d), and the unsaturated
alkylgroups could further promote the enhancement of the cyto-
toxic activity (5e, 5f). As demonstrated in Table 2, compound 5f was
approximately 10-fold more active against MCF-7 with the IC50
values of 0.9
m
M than compound 4 (IC50 ¼ 9.2
mM). In addition,
whether the ester containing heteroatom had a significant influ-
ence on the cytotoxicity to the target compounds against cancer
lines. For instance, compound 5p bearing hydroxyl group was
found to be 5-fold and 33-fold more potent than the lead
compound shikonin against corresponding MCF-7 (IC50 ¼ 0.35
vs. IC50 1.9 M) and MDA-DB-231 (IC50 0.2
IC50 ¼ 6.6 M), and be more markedly potent than other tested
mM
¼
m
¼
mM vs.
m
compounds as well. It remained to be further studied whether
there was a unique mechanism of action responsible for its high
antitumor activities as the analogue of
b-hydroxyisovaleryl-
shikonin, an ATP-noncompetitive inhibitor of tyrosine kinases such
as v-Src and EGFR [19]. There was similarity in compounds 5qe5t
bearing respective 20-furyl, 20-tetrahydrofuryl, 30-furyl and 30-tet-
rahydrofuryl to show stronger cytotoxic activity with the IC50 value
4. Conclusion
of less than 1.3
mM against MDA-DB-231 than compound 4
(IC50 ¼ 4.1
m
M). Apparently, the relative positions and types of the
In summary, we have designed and synthesized twenty-two
6-isomers of 5, 8-O-dimethyl acylshikonin derivatives, and evalu-
ated for their antitumor effects using two susceptible cancer cells
heteroatom and whether it was saturated seemed to play an
important part in the cytotoxic activity (5qe5v). In vitro evidence

W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
3425
and the normal cell and KM mice implanted S-180 carcinoma
subcutaneously. Most of the prepared compounds displayed the
selective cytotoxic activities toward breast cancer cells MCF-7,
MDA-MB-231 together with no cytotoxicity to the normal cell,
and they were also found to exhibit more active than or compar-
ative to the positive control shikonin. More importantly, the in vitro
and in vivo evidences unfolded that they achieved greater activities
than the corresponding 2-isomers of 5, 8-O-dimethyl acylshikonin
derivatives and displayed more potent than 5-Fu, a typical drug
clinically. This study may provide a wealth of knowledge that the
position of the side chain of shikonin attached to DMNQ, together
with the introduction of an appropriate oxygen-containing group
to the 10-hydroxyl in the side chain of shikonin, hold great promise
for future development as antitumor agents with higher selectivity
and lower toxicity.
stirred for 2 h under refluxing, the reaction mixture was poured
into iced water (100 ml), and then extracted with dichloromethane
in twice (200 ml). The combined organic phase was dried over
anhydrous MgSO4, afterwards filtrated, and concentrated in vacuo.
The residues were chromatographed on a silica gel column with
ethyl acetate/petroleum ether (v/v: 1/4) to give compound 3
(2.86 g, 70.4%) as pale-orange oil. 1H NMR (300 MHz, CDCl3):
(m, 3H, benzene ring H), 5.28 (m, 6H), 3.94 (s, 6H), 3.57 (s, 6H), 2.61
(m, 2H), 1.74 (s, 3H), 1.68 (s, 3H). 13C NMR (75 MHz, CDCl3):
153.3,
d 7.47
d
149.5, 148.9, 146.4, 135.6, 134.0, 120.4, 116.2, 115.2, 105.7, 98.2, 97.7,
68.7, 63.0, 57.1, 56.7, 56.6, 37.6, 26.1,18.2. ESI-MS: 429.46 (M þ Na) þ
.
5.4. 6-(1-hydroxy-4-methylpent-3-enyl)-5,8-dimethoxynaphthalene-
1,4-dione (4)
10% hydrochloric acid (10 ml) was added to a solution of 1-[1,
4-dimethoxy-5,8-bis -(methoxymethoxy)-naphthalen-2-yl]-4-
5. Experimental protocols
methylpent-3-en-1-ol 3 (2.03 g, 0.005 mol) in the mixture of
THF(20 ml) and isopropanol (5 ml), and the reaction was stirred for
3 h under the monitor of TLC. Then the suitable amount of saturated
NaHCO3 and ethyl acetate (80 ml) were added respectively. The
organic layer was washed with water (20 ml) and brine (40 ml),
then dried by anhydrous Na2SO4, and filtrated, and then evaporated
under the reduced pressure. The crude product was purified by
silica gel column with ethyl acetate/petroleum ether (v/v: 1/2) to
obtain the compound 4 (1.03 g, 65.5%) as orange oil. 1H NMR
5.1. Chemistry
Reagents and solvents were commercially available. Solvents
were dried and purified using standard techniques. All reactions
involving air or moisture sensitive or intermediates were carried
out under the atmosphere of nitrogen. Melting points were
measured on an SGW X-4 micro-melting point apparatus and are
uncorrected. NMR spectra were determined on Varian Mercury-
300 spectrometer (300 MHz for 1H and 75 MHz for 13C), chemical
shifts of 1H and 13C spectra were recorded with tetramethylsilane as
internal standard. Mass spectra were recorded on a Shimadzu
LCMS-2010EV mass spectrometer. Column chromatography was
run on silica gel (200e300 mesh) from Qingdao Ocean Chemical
Factory.
(300 MHz, CDCl3):
d 7.55 (s, 1H, benzene ring H), 6.79 (s, 2H,
quinone ring H), 5.24 (m, 1H), 5.10 (t, 1H, J ¼ 5.7 Hz), 3.97 (s, 3H),
3.89 (s, 3H), 2.35 (m, 2H),1.76 (s, 3H),1.65 (s, 3H). 13C NMR (75 MHz,
CDCl3):
d 185.1, 184.5, 156.5, 150.9, 147.9, 139.2, 137.9, 136.9, 125.1,
68.8, 62.4, 56.9, 37.2, 26.1, 18.2. ESI-MS: 339.13 (M þ Na) þ
.
5.5. General procedure for preparation of 1-(1,4-dihydro-5,
5.2. 2-(1-hydroxy-4-methylpent-3-enyl)-5,8-bis-(methoxymethoxy)-
8-dimethoxy-1,4-dioxonaphthalene -6-yl) -4-methlypent-3-enyl
naphthalene-1,4-dione (2)
caroxylates (5ae5v)
Shikonin 1 (2.88 g, 0.01 mol) and potassium carbonate(13.8 g,
0.1 mol) in dimethyl formamide (20 ml) were stirred for 30 min at
40 ꢀC, and then newly distilled chloromethyl ether was added in
batches. The reaction proceeded for 1 h under the monitoring of
TLC. Ethyl acetate (30 ml) and distilled water (20 ml) were poured
into the reaction mixture. The organic layer was washed by water
and brine respectively, dried over anhydrous MgSO4 for 1 h and
concentrated in vacuo. The residual oil was purified by flash chro-
matography on silica gel with ethyl acetate and petroleum ether
(V:V ¼ 1:2) to afford 2-(1-hydroxy-4-methylpent-3-enyl)-5,8-bis-
(methoxymethoxy)- naphthalene-1,4-dione 2 (2.24 g, 77.1％) as
To
6-(1-hydroxy-4-methylpent-3-enyl)-5,8-dimethoxynaph-
thalene-1,4-dione 4 (0.1 mol) and carboxylic acid (0.15 mol) in
anhydrous CH2Cl2 were added DCC (0.2 mol) and DMAP (0.05 mol).
After stirring overnight at room temperature under nitrogen
atmosphere, petroleum ether was added to the reaction mixture to
facilitate precipitates, and then the solution was filtered, and
concentrated in vacuo. The residual oil was purified by flash chro-
matography to give 5ae5v as yellow oil.
5.5.1. 1-(1,4-dihydro-5,8-dimethoxy -1,4-dioxonaphthalen-6-yl)-
4-methylpent-3-enyl acetate (5a)
yellow oil. 1H NMR (300 MHz, CDCl3):
d
7.47 (s, 2H, benzene ring H),
Yield 73.7%, yellow oil. 1HNMR (300 MHz, CDCl3):
d 7.26 (s, 1H,
6.80 (s, 1H, quinone ring H), 5.30 (m, 5H), 4.76 (t, 1H, J ¼ 6.3 Hz),
benzene ring H), 6.79 (s, 2H, quinone ring H), 6.16 (t, 1H, J ¼ 7.8 Hz),
3.56 (s, 6H), 2.55 (m, 2H),1.72 (s, 3H),1.62 (s, 3H). 13C NMR (75 MHz,
5.11 (t, 1H, J ¼ 7.5 Hz), 3.97 (s, 3H), 3.82 (s, 3H), 2.63 (m, 2H), 2.10 (s,
CDCl3):
d
185.1, 184.8, 152.6, 152.1, 136.7, 134.4, 125.6, 125.0, 120.4,
3H),1.68 (s, 3H),1.52 (s, 3H).13C NMR (75 MHz, CDCl3):
d 184.6,184.1,
119.1, 102.6, 96.3, 96.2, 68.9, 57.9, 56.9, 56.8, 35.7, 26.1, 18.3. ESI-MS:
169.8, 155.9, 150.5, 144.3,138.8, 137.7,135.6,125.1, 120.0,118.0,116.7,
399.39 (M þ Na) þ
.
70.4, 61.9, 56.6, 33.9, 25.5, 21.0, 17.7. ESI-MS: 381.14 (M þ Na) þ
.
5.3. 1-[1,4-dimethoxy-5,8-bis-(methoxymethoxy)-naphthalen-2-yl]-
5.5.2. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
4-methylpent-3-en-1-ol (3)
4-methylpent-3-enyl propionate (5b)
Yield 54.3%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.25 (s, 1H,
To a solution of 2-(1-hydroxy-4-methylpent-3-enyl)-5,8-bis-
(methoxymethoxy)-naphthalene- 1,4-dione 2 (3.76 g, 0.01 mol) in
THF (40 ml) and water (10 ml) was added sodium hydrosulfite
(17.4 g, 0.1 mol) at room temperature under the atmosphere of N2.
When the color of the reaction solution turned into pale-yellow,
sodium hydroxide (4.0 g, 0.1 mol) and tetrabutyl ammonium
bromide (200 mg) was added successively, and then dimethyl
sulfate (13.41 ml, 0.08 mol) was syringed at 40 ꢀC, After being
benzene ring H), 6.77 (s, 2H, quinone ring H), 6.11 (t, 1H, J ¼ 7.2 Hz),
5.14 (t, 1H, J ¼ 7.2 Hz), 3.98 (s, 3H), 3.91(s, 3H), 2.55 (m, 4H), 1.68 (s,
3H), 1.53 (s, 3H), 1.15 (t, 3H, J ¼ 6.9 Hz). ESI-MS: 395.15 (M þ Na) þ
.
5.5.3. 1-(1,4-dihydro-5,8-dimethoxy -1,4-dioxonaphthalen-6-yl)-4-
methylpent-3-enyl butyrate (5c)
Yield 50.1%, yellow oil. 1H NMR (300 MHz, CDCl3):
d
7.24 (s, 1H,
benzene ring H), 6.77 (s, 2H, quinone ring H), 6.16 (t, 1H,

3426
W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
J ¼ 7.8 Hz), 5.12 (t, 1H, J ¼ 5.4 Hz), 3.95 (s, 3H), 3.90 (s, 3H), 2.47 (m,
5.5.12. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
4H), 1.71 (m, 5H), 1.62 (s, 3H), 0.98 (t, 3H, J ¼ 7.5 HZ). ESI-MS:
4-methylpent-3-enyl 4-methoxybenzoate (5l)
þ
409.15 (M þ Na)
.
Yield 36.8%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 8.10 (d, 1H,
J ¼ 9.0 Hz, benzene ring H), 8.09 (d, 1H, J ¼ 9.0 Hz, benzene ring H),
7.31 (s, 1H, benzene ring H), 6.99 (d, 1H, J ¼ 9.0 Hz, benzene ring
H), 6.98 (d, 1H, J ¼ 9.0 Hz, benzene ring H), 6.78 (s, 2H, quinone
ring H), 6.31 (m, 1H), 5.23 (t, 1H, J ¼ 6.9 Hz), 3.97 (s, 3H), 3.88 (s,
3H), 3.83 (s, 3H),þ2.68 (m, 2H), 1.67 (s, 3H), 1.56 (s, 3H). ESI-MS:
5.5.4. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-4-
methylpent-3-enyl isobutyrate (5d)
Yield 57.8%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.24 (s, 1H,
benzene ring H), 6.77 (s, 2H, quinone ring H), 6.10 (t, 1H, J ¼ 5.1 Hz),
5.13 (m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 2.66 (m, 3H), 1.49 (s, 3H), 1.47
(s, 3H), 1.21 (d, 3H, J ¼ 2.1 Hz), 1.16 (d, 3H, J ¼ 2.1 Hz). ESI-MS: 409.15
473.16 (M þ Na)
.
(M þ Na) þ
.
5.5.13. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
4-methylpent-3-enyl 2-methoxybenzoate (5m)
5.5.5. (E)-1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-
Yield 35.9%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.86 (m, 1H,
yl)-4-methylpent-3-enyl but-2-enoate (5e)
benzene ring H), 7.53 (m, 1H, benzene ring H), 7.41 (s, 1H, benzene
ring H), 7.04 (m, 2H, benzene ring H), 6.77 (d, 2H, J ¼ 1.2 Hz,
quinone ring H), 6.32 (m, 1H), 5.24 (t, 1H, J ¼ 6.9 Hz), 3.98 (s, 3H),
3.93 (s, 3H), 3.90 (s, 3H), 2.67 (m, 2H), 1.68 (s, 3H), 1.52 (s, 3H). ESI-
Yield 47.7%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.24 (s, 1H,
benzene ring H), 7.04 (m, 1H), 6.78 (s, 2H, quinone ring H), 6.17 (m,
1H), 5.94 (m, 1H), 5.12 (t, 1H, J ¼ 7.8 Hz), 3.96 (s, 3H), 3.93 (s, 3H),
2.56 (m, 2H), 1.93 (m, 3H), 1.67 (s, 3H), 1.53 (s, 3H). ESI-MS: 407.17
MS: 473.16 (M þ Na) þ
.
(M þ Na) þ
.
5.5.14. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
5.5.6. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-4-
4-methylpent-3-enyl 2-(4-methoxyphenyl)acetate (5n)
methylpent-3-enyl 3-methylbut-2-enoate (5f)
Yield 33.4%, yellow oil, 1H NMR (300 MHz, CDCl3):
d 7.25 (d, 2H,
Yield 56.9%, yellow oil. 1H NMR (300 MHz, CDCl3):
d
7.24 (s, 1H,
J ¼ 8.1 Hz, benzene ring H), 6.93 (s, 1H, benzene ring H), 6.87 (d, 2H,
J ¼ 8.1 Hz, benzene ring H), 6.53 (s, 2H, quinone ring H), 6.11 (m,1H),
5.13 (t, 1H, J ¼ 8.4 Hz), 3.91 (s, 3H), 3.88 (s, 3H), 3.73 (s, 3H), 3.63 (s,
benzene ring H), 6.78 (s, 2H, quinone ring H), 5.90 (m, 1H), 5.43 (s,
1H), 5.13(m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 2.53 (m, 2H), 2.15 (s, 3H),
þ
1.93 (s, 3H), 1.65 (s, 3H), 1.54 (s, 3H). ESI-MS: 421.16 (M þ Na)
.
2H), 2.55 (m, 2H),1.67 (s, 3H),1.52 (s, 3H). ESI-MS: 487.17 (M þ Na) þ
.
5.5.7. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-4-
5.5.15. (E)-1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-
methylpent-3-enyl 2-methylbutanoate (5g)
yl)-4-methylpent-3-enyl 3-(3,4-dimethoxyphenyl)acrylate (5o)
Yield 42.7%, yellow oil. 1H NMR (300 MHz, CDCl3):
d
7.24 (d, 1H,
Yield 33.7%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.67 (d, 1H,
J ¼ 1.5 Hz, benzene ring H), 6.77 (s, 2H, quinone ring H), 6.10 (m,
1H), 5.13 (t, 1H, J ¼ 5.4 Hz), 3.93 (s, 3H), 3.92 (s, 3H), 2.64 (m, 3H),
1.63 (s, 3H), 1.52 (s, 3H), 1.19 (m, 2H), 1.16 (m, 6H). ESI-MS: 423.18
J ¼ 15.9 Hz,), 7.29 (s, 1H, benzene ring H), 7.67 (d, 1H, J ¼ 8.7 Hz,
benzene ring H), 7.06 (s, 1H, benzene ring H), 7.67 (d, 1H, J ¼ 8.7 Hz,
benzene ring H), 6.37 (s, 2H, quinone ring H), 6.34 (d, 1H,
J ¼ 15.6 Hz), 6.22 (m, 1H), 5.20 (t,1H, J ¼ 6.0 Hz), 3.95 (s, 3H), 3.94 (s,
3H), 3.93 (s, 3H), 3.81 (s, 3H), 2.61 (m, 1H), 2.37 (m, 1H), 1.68 (s, 3H),
(M þ Na) þ
.
5.5.8. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-4-
1.58 (s, 3H). ESI-MS: 529.18 (M þ Na) þ
.
methylpent-3-enyl benzoate (5h)
Yield 27.2%, yellow oil. 1H NMR (300 MHz, CDCl3):
d
8.08 (m, 2H,
5.5.16. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
benzene ring H), 7.61 (m, 3H, benzene ring H), 7.32 (s, 1H, benzene
ring H), 6.74 (s, 2H, quinone ring H), 6.17 (m, 1H), 5.21 (t, 1H,
4-methylpent-3-enyl 2-hydroxy-2-methylpropanoate (5p)
Yield 45.5%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.27 (s, 1H,
J ¼ 6.9 Hz), 4.01 (s, 3H), 3.97 (s, 3H), 2.72 (m, 2H),1.66 (s, 3H),1.59 (s,
benzene ring H), 6.67 (s, 2H, quinone ring H), 6.18 (m, 1H), 5.04 (t,
þ
3H). ESI-MS: 443.15 (M þ Na)
.
1H, J ¼ 8.4 Hz), 3.95 (s, 3H), 3.94 (s, 3H), 2.58 (m, 4H), 1.68 (s, 3H),
1.55 (s, 3H), 1.29 (s, 3H), 1.26 (s, 3H). ESI-MS: 439.17 (M þ Na) þ
.
5.5.9. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-4-
methylpent-3-enyl 3-phenylpropanoate (5i)
5.5.17. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
Yield 31.9%, yellow oil. 1H NMR (300 MHz, CDCl3):
d7.29 (m, 6H,
4-methylpent-3-enyl furan-2-carboxylate (5q)
benzene ring H), 6.78 (s, 2H, quinone ring H), 6.17 (m, 1H), 5.10 (t,
1H, J ¼ 7.5 Hz), 3.90 (s, 3H), 3.87 (s, 3H), 2.99 (t, 2H, J ¼ 7.8 Hz), 2.75
(t, 2H, J ¼ 7.8 Hz), 2.53 (m, 2H), 1.67 (s, 3H), 1.52 (s, 3H). ESI-MS:
Yield 49.2%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.69 (m, 1H,
furan ring), 7.34 (s, 1H, benzene ring H), 7.25 (m, 1H, furan ring H),
6.79 (s, 2H, quinone ring H), 6.56 (m, 1H, furan ring H), 6.34 (t, 1H,
471.18 (M þ Na) þ
.
J ¼ 5.1 Hz), 5.10 (t, 1H, J ¼ 2.7 Hz), 3.96 (s, 3H), 3.92 (s, 3H), 2.67 (m,
þ
2H), 1.68 (s, 3H), 1.57 (s, 3H). ESI-MS: 433.13 (M þ Na)
.
5.5.10. 1-(1,4 -dihydro-5,8-dimethoxy -1,4-dioxoaphthalent-6-yl)-
4-methypent-3-enyl cinnamate (5j)
5.5.18. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
Yield 35.6%, yellow oil. 1H NMR (300 MHz, CDCl3):
d
7.75 (d, 1H,
4-methylpent-3-enyl tetrahydrofuran-2-carboxylate (5r)
J ¼ 16.2 Hz), 7.58 (m, 6H, benzene ring H), 6.79 (s, 2H, quinone ring
Yield 42.8%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.28 (d, 1H,
H), 6.55 (d, 1H, J ¼ 16.2 Hz), 6.25 (dd, 1H, J ¼ 7.5, 4.8 Hz), 5.19 (t, 1H,
J ¼ 3.3 Hz, benzene ring H), 6.79 (s, 2H, quinone ring H), 6.12 (m,
1H), 5.14 (t, 1H, J ¼ 6.3 Hz), 4.55 (t, 1H, J ¼ 4.8 Hz), 4.02 (m, 8H), 2.58
(m, 2H), 2.25 (m, 1H), 2.08 (m, 3H), 1.68 (s, 3H), 1.55 (s, 3H). ESI-MS:
J ¼ 6.0 Hz), 3.96 (s, 3H), 3.94 (s, 3H), 2.57 (m, 2H),1.69 (s, 3H),1.56 (s,
þ
3H). ESI-MS: 469.16 (M þ Na)
.
437.16 (M þ Na) þ
.
5.5.11. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-4-
methylpent-3-enyl 4-nitrobenzoate (5k)
5.5.19. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
Yield 68.3%, yellow oil. 1H NMR (300 MHz, CDCl3):
d
8.35 (m, 4H,
4-methylpent-3-enyl furan-3- carboxylate (5s)
benzene ring H), 7.29 (s, 1H, benzene ring H), 6.80 (s, 2H, quinone
Yield 49.2%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 8.10 (d, 1H,
ring H), 6.40 (m, 1H), 5.20 (t,1H, J ¼ 7.2 Hz), 3.98 (s, 3H), 3.90 (s, 3H),
J ¼ 1.2 Hz, furan ring H), 7.49 (d, 1H, J ¼ 1.2 Hz, furan ring H), 7.29 (s,
2.70 (m, 2H), 1.69 (s, 3H), 1.59 (s, 3H). ESI-MS: 488.13 (M þ Na) þ
.
1H, benzene ring H), 6.82 (s, 2H, quinone ring H), 6.80 (s, 1H, furan

W. Zhou et al. / European Journal of Medicinal Chemistry 46 (2011) 3420e3427
3427
ring H), 6.52 (dd, 1H, J ¼ 4.8, 4.8 Hz), 5.19 (t, 1H, J ¼ 7.5 Hz), 3.97 (s,
5.7. Antitumor activity in vivo
3H), 3.94 (s, 3H), 2,63 (m, 2H), 1.69 (s, 3H) 1.58 (s, 3H). ESI-MS:
433.13 (M þ Na) þ
.
Sarcoma S-180 cells (2 ꢂ 106) in 0.2 ml of physiological saline
were subcutaneously injected between the femur of the male KM
mice (Seven-week-old specific pathogen free (SPF) male KM mice
with the weight of 18e22 g each were obtained from Shanghai
Laboratory Animal Center, Chinese Academy of Sciences).Tumor-
bearing mice were randomly subdivided into 5 groups of 10. After
24 h, mice were treated with compound 5p via an intraperitoneal
(i.p.) injection at a dose of 3, 6, 12 mg/kg once a day for 9 days (9
times) respectively while compound 6c was administrated with
12 mg/kg. Similarly, 5-fluorouracil (5-FU, 25 mg/kg, i.p.) was
administered as a positive control. It was noticeable that all
compound tested were dissolved in 0.5% DMSO and 1% Tween 80,
and diluted to the given concentration by physiological saline. The
body weight of mice was observed. Subsequently mice were killed,
and the tumors were excised and weighed in day 10. Control mice
were injected with 0.5% DMSO and 1% Tween 80 in physiological
5.5.20. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
4-methylpent-3-enyl tetrahydrofuran-3-carboxylate (5t)
Yield 57.4%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.24 (d, 1H,
J ¼ 3.3 Hz, benzene ring H), 6.78 (s, 2H, quinone ring H), 6.16 (m,
1H), 5.11 (t, 1H, J ¼ 6.3 Hz), 4.02 (m, 10H), 3.19 (m, 1H), 2.53 (m, 2H),
2.24 (m, 2H), 1.68 (s, 3H), 1.54 (s, 3H). ESI-MS: 437.16 (M þ Na) þ
.
5.5.21. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
4-methylpent-3-enyl nicotinate (5u)
Yield 47.6%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 9.28 (s,
1H,pyridine ring H), 8.81 (d,1H, J ¼ 3.6 Hz, pyridine ring H), 8.33 (m,
1H,pyridine ring H), 7.46 (d, 1H, J ¼ 3.6 Hz, pyridine ring H), 7.31 (s,
1H, benzene ring H), 6.79 (s, 2H, quinone ring H), 6.41 (m, 1H), 5.20
(t, 1H, J ¼ 7.2 Hz), 3.97 (s, 3H), 3.94 (s, 3H), 2.76 (m, 2H), 1.66 (s, 3H)
1.59 (s, 3H) ESI-MS: 444.15(M þ Na) þ
.
saline (vehicle). Tumor growth inhibition was calculated as T/C
P
(Treatment/control) by the following formula: T/C ¼ [( C10/10-
P
P
5.5.22. 1-(1,4-dihydro-5,8-dimethoxy-1,4-dioxonaphthalen-6-yl)-
T10/10)/( C10/10)] ꢂ 100%, where C10 is the tumor weight at day
4-methylpent-3-enyl isonicotinate (5v)
10 in control group and T10 is the tumor weight at day 10 in treated
group.
Yield 59.7%, yellow oil. 1H NMR (300 MHz, CDCl3):
d 8.84 (d, 2H,
J ¼ 4.5 Hz,pyridine ring H), 7.88 (d, 2H, J ¼ 4.5 Hz,pyridine ring H),
7.28 (s, 1H, benzene ring H), 6.80 (s, 2H, quinone ring H), 6.39 (m,
1H), 5.19 (m, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 2.70 (m, 2H), 1.69 (s, 3H),
Acknowledgments
1.58 (s, 3H). ESI-MS: 444.15 (M þ Na) þ
5.6. Antitumor activity in vitro
5.6.1. Cell culture
.
We thank National Natural Science Foundation of China
(No.30973604, No.91013012) and Shanghai Natural Science Fund
(No.11ZR1416300) for financial supports.
HCT-15, NCE, MDA-DB-231, MCF-7, K562, L-929 and HSF were
grown in RPMI-1640 or Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM
nonessential amino acids, 0.1 mM sodium pyruvate, 100 U/mL
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supernatant was removed, and then 20
mL) was added to each well. After re-incubated for another 4 h,
100 L of DMSO was added to each well for dissolving the formazan
crystals. The percentage of cell viability was determined by
measuring the absorbance (Abs) at
mL of MTT solution (5 mg/
m
l
¼ 570 nm using a Multiskan
MK3 microplate reader (Thermo, USA). Survival percentage
was calculated using the following equation: inhibitory
rate ¼ (Abs570control cells ꢃ Abs570 treated cells)/Abs570control cells ꢂ 100%.
IC50 values were obtained from linear regression analysis of the
concentration-response curves plotted for each compounds tested.