Design, synthesis and biological evaluation of cinnamic acyl shikonin derivatives

Article abstract of DOI:10.1111/cbdd.12077

Inducing apoptosis is an important and promising therapeutic approach to overcome cancer. Here, we described a series of novel synthesized compounds, cinnamic acyl shikonin derivatives (1b-19b), which were synthesized starting from shikonin and cinnamic acids, which exhibit anticancer activity via inducing apoptosis in vitro. Our flow cytometry results showed that compound 8b((E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent -3-enyl-3-(3-(trifluoromethyl) phenyl)acrylate) (IC₅₀=0.69, 0.65, 1.62 μm for human SW872-s, A875 and A549 cell lines, respectively) exhibited conspicuous anticancer activities and has low cell toxicity in vitro. Therefore, we considered that compound 8b is potentially to be a candidate of anticancer agent. The proliferation inhibitory effect of compound 8b was associated with its apoptosis-inducing effect by activating caspase-3, caspase-7, caspase-9, and PARP. When the level of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved PARP are rise, apoptosis of cancer cells will be induced.

Full text of DOI:10.1111/cbdd.12077

Chem Biol Drug Des 2013; 81: 275–283
Research Article
Design, Synthesis and Biological Evaluation of
Cinnamic Acyl Shikonin Derivatives
caspase-9 to induce the activation of downstream
Hong-Yan Lin , Wei Chen , Jing Shi, Wen-Yao
Kong, Jin-Liang Qi, Xiao-Ming Wang* and
Yong-Hua Yang*
proteases (for example, caspase-3 and caspase-7) and
death response (6). Caspase-3 and PARP are important
performers of apoptosis that restrictively cleave all or part
of key proteins, and cleaved caspase-3 and cleaved PARP
are their activation form after being cut. Therefore, when
the level of cleaved caspase-3, cleaved caspase-7,
cleaved caspase-9, and cleaved PARP are rise, apoptosis
of cancer cells will be induced (7,8). Recently, we dedicate
ourselves to synthesizing some new compounds that can
cure cancer by inducing the apoptosis of cancer cells.
State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, China
*Corresponding authors: Xiao-Ming Wang,
wangxm07@nju.edu.cn; Yong-Hua Yang,
yangyh@nju.edu.cn
These two authors equally contribute to this paper.
Inducing apoptosis is an important and promising ther-
apeutic approach to overcome cancer. Here, we
described a series of novel synthesized compounds,
cinnamic acyl shikonin derivatives (1b–19b), which
were synthesized starting from shikonin and cinnamic
acids, which exhibit anticancer activity via inducing
apoptosis in vitro. Our flow cytometry results showed
that compound 8b((E)-1-(5,8-dihydroxy-1,4-dioxo-1,
Shikonin and its derivatives are active naphthoquinone
compounds that are isolated from the root of Chinese her-
bal medicine Lithospermum erythrorhizon (9). Historically,
Zicao root extracts were used in the treatment of burn,
inflammation, trauma, and ulcer (9–13). Recently, to find
more effective anticancer drugs, much attention was paid
to the research of the anticancer effects and molecular
mechanisms of shikonin and its derivatives (14–16).
4-dihydronaphthalen-2-yl)-4-methylpent
-3-enyl-3-(3-
(trifluoromethyl) phenyl)acrylate) (IC50 = 0.69, 0.65, 1.62
lM for human SW872-s, A875 and A549 cell lines,
respectively) exhibited conspicuous anticancer activi-
ties and has low cell toxicity in vitro. Therefore, we
considered that compound 8b is potentially to be a
candidate of anticancer agent. The proliferation inhibi-
tory effect of compound 8b was associated with its
apoptosis-inducing effect by activating caspase-3,
caspase-7, caspase-9, and PARP. When the level of
cleaved caspase-3, cleaved caspase-7, cleaved cas-
pase-9, and cleaved PARP are rise, apoptosis of can-
cer cells will be induced.
Meanwhile, the anticancer activities of various cinnamic
acids were explored by many research groups, especially
of the cinnamic acids derivatives in the past few years.
Now, cinnamoyl moiety has been applied to many drugs,
such as Mepramidil, Baclofen, Cinnarizine, and Ozagrel
Sodium (17–20). Luo Yin reported that the combination of
cinnamic acid and sulfonamide exhibits synergistic effect
in anticancer activities (21). At present, Zhou Wen com-
bined some cinnamic acids to 5, 8-O-dimethyl acylshiko-
nin so as to gain some potential anticancer agents (12).
Shen Chien-Chang synthesized several shikonin cinnamic
acyl derivatives that exhibit potent antimicrobial activities
(22). Therefore, in this study, we synthesized and evalu-
ated a series of cinnamic acyl shikonin derivatives so as to
find some active functional molecules that have novel
structure.
Key words: anticancer, apoptosis, caspase, cinnamic acids,
cinnamic acyl shikonin derivatives
Received 14 August 2012, revised 21 September 2012 and
accepted for publication 4 October 2012
Apoptosis is one of the evolutionary conserved pro-
grammed cell death mechanisms and predominantly
occurs in many cancer cells (1–3). Caspase-9 is one of
the most important caspase family proteins in mitochon-
drial apoptotic pathway (4). When the apoptosis is induced
in mitochondrial apoptotic pathway, cytochrome c will be
released from the intermembrane space of mitochondrial
and interactive with APAF1 and dATP to form composite
receptor (4,5). Then, the receptor will recruit and activate
Experiments Section
Materials and measurements
The All chemicals (reagent grade) used were purchased
from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China).
Separation of the compounds by column chromatography
was carried out with Silica Gel 60 (200–300 mesh ASTM;
E. Merck, Darmstadt, Germany). All the 1H NMR spectra
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12077
275

Lin et al.
were recorded on a Bruker DRX 500 spectrometer in
CDCl3. Chemical shifts (d) for 1H NMR spectra were
reported in ppm (d). Melting points (uncorrected) were
measured on a XT4 MP Apparatus (Taike Corp., Beijing,
China). ESI mass spectra were obtained on a Mariner Bio-
spectrometry Workstation (ESI-TOF) mass spectrometer.
TLC was carried out on the glass-backed silica gel sheets
1.63(s, 3H, C=C-CH3). ESI-TOF, calcd for C25H22O6
([M+Na]+), 441.1416, found 441.1357. Anal. Calcd for
C25H22O6: C, 71.76; H, 5.30; O, 22.94. Found: C, 70.51;
H, 6.21; O, 23.54.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)-4-methylpent-3-enyl-3-p-tolylacrylate (2b)
˚
(silica gel 60 A GF254) and visualized in UV light (254 nm).
Yield 73%. Red powder Mp: 120.2–121.7 ꢀC. 1H NMR
(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H, -OH);
7.61(d, J = 16 Hz, 1H, C=CH-Ar); 7.48(d, J = 8 Hz, 2H,
Ar-H); 7.24(d, J = 8 Hz, 2H, Ar-H); 7. 21(s, 2H); 7.07(s,
1H); 6.48(d, J = 15.5 Hz, 1H, CH=C-Ar); 6.16(t,
General procedure for preparation of compounds
1a–19a
A mixture of aromatic aldehydes (50 mmol), malonic acid
(60 mmol), piperidine (6.05 mmol) was dissolved in pyri-
dine and stirred on 80–90 ꢀC for 4–5 h. The pyridine was
removed at the vacuum. The reaction mixture was poured
into ice water and washed with HCl. And the precipitate
was filtered and washed with hexane for three times, then,
recrystallized by alcohol and dried under vacuum to afford
the cinnamic acids (Scheme 1).
J1 = 5.75 Hz, J2 = 6.5 Hz, 1H, -O-CH); 5.21(t, J1
=
7.25 Hz, J2 = 7.5 Hz, 1H, -C-C-CH=C); 2.74–2.69(m, 1H,
-C-CH2-C=C); 2.61–2.56(m, 1H, -C-CH2-C=C); 2.42(s,
3H, Ar-CH3); 1.72(s, 3H, C=C-CH3); 1.63(s, 3H, C=C-
CH3). ESI-TOF, calcd for C26H24O6 ([M+Na]+), 455.1573,
found 455.1447. Anal. Calcd for C26H24O6: C, 72.21; H,
5.59; O, 22.20. Found: C, 69.21; H, 6.02; O, 23.20.
General procedure for preparation of compounds
1b–19b
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)-4-methylpent-3-enyl-3 -(4-chlorophenyl) acrylate
(3b)
Yield 76%. Red powder Mp: 102.1–103.2 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H,
-OH); 7.70(d, J = 16 Hz, 1H, C=CH-Ar); 7.51(d, J =
8.5 Hz, 2H, Ar-H); 7. 41(d, J = 9 Hz, 2H, Ar-H); 7.22(s,
2H); 7.06(s, 1H); 6.50(d, J = 15.5 Hz, 1H, CH=C-Ar);
Shikonin 0.040 g (0.175 mmol) was dissolved in 16 mL of
dichloromethane, and 0.073 g (0.354 mmol) of N, N¢-
dicyclohexylcarbodiimide (DCC) was added into the reaction
system. The reaction mixture was stirred under nitrogen
atmosphere in the ice bath for 15 min. 0.004 g
(0.044 mmol) of 4-dimethylaminopyridine (DMAP) was
added, stirred in the ice bath, and continued for a further
15 min. Then, 0.175 mmol of carboxylic acid was added
to the reaction mixture and stirred in the ice bath for 12 h
to afford the target compounds (1b–19b) (Figure 1).
6.17(t, J1 = 5.5 Hz, J2 = 6.5 Hz, 1H, -O-CH); 5.19(t, J1
=
7 Hz, J2 = 7.25 Hz, 1H, -C-C-CH=C); 2.75–2.69(m, 1H, -
C-CH2-C=C); 2.61–2.55(m, 1H, -C-CH2-C=C); 1.72(s,
3H, Ar-CH3); 1.63(s, 3H, C=C-CH3); 1.63(s, 3H,
C=C-CH3). ESI-TOF, calcd for C25H21ClO6 ([M+Na]+),
475.1027, found 475.1011. Anal. Calcd for C25H21ClO6:
C, 66.30; H, 4.67; O, 21.20. Found: C, 64.91; H, 5.27; O,
22.34.
(E)-1-(5, 8-dihydroxy-1, 4-dioxo-1, 4-dihydronaph-
thalen-2-yl)-4-methylpent-3-enyl cinnamate (1b)
Yield 65%. Red powder Mp: 95.6–96.7 ꢀC. 1H NMR
(500 MHz, CDCl3) d: 12.65(s, 1H, -OH); 12.46(s, 1H, -OH);
7.76(d, J = 16.0 Hz, 1H, C=CH-Ar); 7.59–7.57(m, 2H, Ar-
H); 7.44–7.43(m, 3H, Ar-H); 7.22(s, 2H); 7. 07(s, 1H);
6.53(d, J = 16.5 Hz, 1H, CH=C-Ar); 6.17(t, J1 = 5.75 Hz,
J2 = 7.0 Hz, 1H, -O-CH); 5.21(t, J1 = 7.25 Hz, J2
7.0 Hz, 1H, -C-C-CH=C); 2.74–2.70(m, 1H, -C-CH2-C=C);
2.61–2.57(m, 1H, -C-CH2-C=C); 1.73(s, 3H, C=C-CH3);
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-4-methylpent-3-enyl -3-(4-
fluorophenyl) acrylate (4b)
Yield 32%. Red powder Mp: 97.2–98.7 ꢀC. 1H NMR
(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.45(s, 1H, -OH);
7.72(d, J = 16.5 Hz, 1H, C=CH-Ar); 7.59–7.56(m, 2H,
=
OH
OH
OH
O
a
O
CHO
OH
+
O
O
R
R
O
1a–19a
1–19
OH
O
O
Scheme 1: General synthesis of
OH
O
R
shikonin derivatives (1b–19b).
Reagents and conditions: (a)
piperidine, pyridine, 80–90 ꢀC,
4–5h. (b)CH2Cl2, DCC, DMAP,
)10 to 0 ꢀC, overnight.
b
+
HO
OH
O
R
OH
O
1a–19a
1b–19b
276
Chem Biol Drug Des 2013; 81: 275–283

Study of Cinnamic Acyl Shikonin Derivatives
Figure 1: Structure of compounds 1b–19b.
Ar-H); 7. 22(s, 2H); 7.13(t, J1 = 8.75 Hz, J2 = 9 Hz, 2H,
Ar-H); 7. 06(s, 1H); 6.46(d, J = 17 Hz, 1H, CH=C-Ar);
6.16(t, J1 = 5.5 Hz, J2 = 4.5 Hz, 1H, -O-CH); 5.20(t,
J1 = 6.75 Hz, J2 = 7.5 Hz, 1H, -C-C-CH=C); 2.75–2.70(m,
1H, -C-CH2-C=C); 2.61–2.56(m, 1H, -C-CH2-C=C);
1.72(s, 3H, Ar-CH3); 1.62(s, 3H, C=C-CH3); 1.63(s, 3H,
C=C-CH3). ESI-TOF, calcd for C25H21FO6 ([M+Na]+),
459.1322, found 459.1293. Anal. Calcd for C25H21FO6: C,
68.80; H, 4.85; O, 22.00. Found: C, 67.31; H, 5.37; O,
22.74.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)-4-methylpent-3-enyl-3 -o-tolylacrylate
(5b)
Yield 71%. Red powder Mp: 90.1–91.5 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H, -
OH); 7.70(d, J = 16 Hz, 1H, C=CH-Ar); 7.51(d, J = 8.5 Hz,
2H, Ar-H); 7. 41(d, J = 9 Hz, 2H, Ar-H); 7. 22(s, 2H);
7.06(s, 1H); 6.50(d, J = 15.5 Hz, 1H, CH=C-Ar); 6.17(t,
J1 = 5.5 Hz, J2 = 6.5 Hz, 1H, -O-CH); 5.19(t, J1 = 7 Hz,
J2 = 7.25 Hz, 1H, -C-C-CH=C); 2.75–2.69(m, 1H, -C-CH2-
Chem Biol Drug Des 2013; 81: 275–283
277

Lin et al.
C=C); 2.61–2.55(m, 1H, -C-CH2-C=C); 1.72(s, 3H, Ar-
CH3); 1.63(s, 3H, C=C-CH3); 1.63(s, 3H, C=C-CH3). ESI-
TOF, calcd for C26H24O6 ([M+Na]+), 455.1573, found
455.1498. Anal. Calcd for C26H24O6: C, 72.21; H, 5.59; O,
22.20. Found: C, 70.87; H, 6.27; O, 23.54.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-4-methylpent-3-enyl- 3-
(2,6-dichlorophenyl) acrylate (9b)
Yield 63%. Red powder Mp: 84.3–85.7 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H,
-OH); 7.90(d, J = 16.5 Hz, 1H, C=CH-Ar); 7.39(t,
J1 = 8.0 Hz, J2 = 8.0 Hz, 3H, Ar-H); 7. 22(s, 2H); 7. 09(s,
1H); 6.72(d, J = 16.5 Hz, 1H, CH=C-Ar); 6.16(t,
J1 = 5.5 Hz, J2 = 6.5 Hz, 1H, -O-CH); 5.20(t, J1 = 6.0 Hz,
J2 = 7.0 Hz, 1H, -C-C-CH=C); 2.76–2.71(m, 1H, -C-CH2-
C=C); 2.63–2.60(m, 1H, -C-CH2-C=C); 1.73(s, 3H, C=C-
CH3); 1.63(s, 3H, C=C-CH3). ESI-TOF, calcd for
C25H20Cl2O6 ([M+Na]+), 509.0637, found 509.0648. Anal.
Calcd for C25H20Cl2O6: C, 61.61; H, 4.14; O, 19.70.
Found: C, 58.35; H, 4.27; O, 21.74.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-4-methylpent-3-enyl-3-(4-
(trifluoromethyl) phenyl)acrylate (6b)
Yield 34%. Red powder Mp: 99.6–101.2 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.45(s, 1H,
-OH); 7.77(d, J = 16.0 Hz, 1H, C=CH-Ar); 7.69(s, 4H,
Ar-H); 7.22(s, 2H); 7.07(s, 1H); 6.61(d, J = 16.0 Hz, 1H,
CH=C-Ar); 6.17(t, J1 = 5.5 Hz, J2 = 6.5 Hz, 1H, -O-CH);
5.20(t, J1 = 7.0 Hz, J2 = 7.0 Hz, 1H, -C-C-CH=C); 2.76–
2.71(m, 1H, -C-CH2-C=C); 2.63–2.58(m, 1H, -C-CH2-
C=C); 1.73(s, 3H, C=C-CH3); 1.63(s, 3H, C=C-CH3). ESI-
TOF, calcd for C26H21F3O6 ([M+Na]+), 509.1290, found
509.1327. Anal. Calcd for C26H21F3O6: C, 64.20; H, 4.35;
O, 19.73. Found: C, 63.57; H, 5.27; O, 20.54.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)-4-methylpent-3-enyl -3-(4-
methoxyphenyl) acrylate (10b)
Yield 79%. Red powder Mp: 79.1–80.2 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H,
-OH); 7.71(d, J = 16.0 Hz, 1H, C=CH-Ar); 7.53(d,
J = 9.0 Hz, 2H, Ar-H); 7.21(s, 2H); 7. 07(s, 1H); 6.95(d,
J = 8.5 Hz, 2H, Ar-H); 6.39(d, J = 16.0 Hz, 1H, CH=C-Ar);
6.17–6.14(m, 1H, -O-CH); 5.25–5.19(m, 1H, -C-C-CH=C);
3.98(s, 3H,-OCH3); 2.75–2.66(m, 1H, -C-CH2-C=C); 2.60–
2.55(m, 1H, -C-CH2-C=C); 1.72(s, 3H, C=C-CH3); 1.63(s,
3H, C=C-CH3). ESI-TOF, calcd for C26H24O7 ([M+Na]+),
471. 1522, found 471. 1514. Anal. Calcd for C26H24O7: C,
69.63; H, 5.39; O, 24.97. Found: C, 68.45; H, 6.37; O,
25.21.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-4-methylpent-3-enyl-3- (2-
(trifluoromethyl) phenyl)acrylate (7b)
Yield 51%. Red powder Mp: 116.2–117.8 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H,
-OH); 8.16(d, J = 14 Hz, 1H, C=CH-Ar); 7.78(d,
J = 7.5 Hz, 1H, C=CH-Ar-H), 7.76(d, J = 8 Hz, 1H,
C=CH-Ar-H), 7.63(t, J1 = 7.5 Hz, J2 = 7.5 Hz, 1H, Ar-H);
7. 54(t, J1 = 7.75 Hz, J2 = 9.0 Hz, 1H, Ar-H); 7. 21(s, 2H);
7. 06(s, 1H); 6.50(d, J = 16.0 Hz, 1H, CH=C-Ar); 6.18(t,
J1 = 4.5 Hz,
J2 = 5.5 Hz,
1H,
-O-CH);
5.20(t,
J1 = 7.25 Hz, J2 = 7.0 Hz, 1H, -C-C-CH=C); 2.75–2.71(m,
1H, -C-CH2-C=C); 2.62–2.58(m, 1H, -C-CH2-C=C);
1.73(s, 3H, C=C-CH3); 1.64(s, 3H, C=C-CH3). ESI-TOF,
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen
-2-yl)-4-methylpent-3-enyl- 3-(2-chlorophenyl)
acrylate (11b)
calcd for
C
26H21F3O6 ([M+Na]+), 509.1290, found
Yield 54%. Red powder Mp: 69.1–70.3 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.45(s, 1H,
-OH); 8.19(d, J = 16.5 Hz, 1H, C=CH-Ar); 7.68(d,
J = 8.0 Hz, 1H, Ar-H); 7.46(d, J = 8.5 Hz, 1H, Ar-H);
7.38–7.31(m, 1H, Ar-H); 7.21(s, 2H); 7.07(s, 1H); 6.52(d,
J = 16.5 Hz, 1H, CH=C-Ar); 6.18–6.16(m, 1H, -O-CH);
5.21(t, J1 = 6.75 Hz, J2 = 6.5 Hz, 1H, -C-C-CH=C); 2.76–
2.71(m, 1H, -C-CH2-C=C); 2.62–2.56(m, 1H, -C-CH2-
C=C); 1.73(s, 3H, C=C-CH3); 1.64(s, 3H, C=C-CH3). ESI-
TOF, calcd for C25H21ClO6 ([M+Na]+), 475.1027, found
475.1029. Anal. Calcd for C25H21ClO6: C, 66.30; H, 4.67;
O, 21.20. Found: C, 65.45; H, 5.87; O, 23.21.
509.1326. Anal. Calcd for C26H21F3O6: C, 64.20; H, 4.35;
O, 19.73. Found: C, 63.47; H, 5.37; O, 20.59.
(E)-1-(5, 8-dihydroxy-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-4-methylpent-3-enyl-3-(3-
(trifluoromethyl) phenyl)acrylate (8b)
1
Yield 47%. Red oil. H NMR (500 MHz, CDCl3) d: 12.64(s,
1H, -OH); 12.44(s, 1H, -OH); 7.83(s, 1H, Ar-H); 7.77(d,
J = 16.5 Hz, 1H, C=CH-Ar); 7.75(d, J = 6.5 Hz, 1H, Ar-H);
7.69(d, J = 7.5Hz, 1H, Ar-H); 7.57(t, J1 = 7.5 Hz,
J2 = 7.5 Hz, 1H, Ar-H); 7.21(s, 2H); 7. 07(s, 1H); 6.61(d,
J = 16.0 Hz, 1H, CH=C-Ar); 6.18(t, J1 = 5.75 Hz, J2
=
6.5 Hz, 1H, -O-CH); 5.20 (t, J1 = 7.25 Hz, J2 = 7.5 Hz,
1H, -C-C-CH=C); 2.76- 2.71(m, 1H, -C-CH2-C=C); 2.62–
2.56(m, 1H, -C-CH2-C=C); 1.73(s, 3H, C=C-CH3); 1.62(s,
3H, C=C-CH3). ESI-TOF, calcd for C26H21F3O6 ([M+Na]+),
509.1290, found 509.1319. Anal. Calcd for C26H21F3O6:
C, 64.20; H, 4.35; O, 19.73. Found: C, 63.55; H, 5.27; O,
20.34.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)-4-methylpent-3-enyl -3-(3-chlorophenyl)
acrylate (12b)
Yield 27%. Red powder Mp: 107.6–108.7 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.45(s, 1H,
-OH); 7.68(d, J = 16 Hz, 1H, C=CH-Ar); 7.57(s, 1H, Ar-H);
7.45(d, J = 7.0 Hz, 1H, Ar-H); 7.42–7.35(m, 2H, Ar-H);
278
Chem Biol Drug Des 2013; 81: 275–283

Study of Cinnamic Acyl Shikonin Derivatives
7.21(s, 2H); 7.06(s, 1H); 6.54(d, J = 16.0 Hz, 1H, CH=C-
Ar); 6.17(t, J1 = 6.0 Hz, J2 = 7.0 Hz, 1H, -O-CH); 5.20(t,
J1 = 7.25 Hz, J2 = 7.5 Hz, 1H, -C-C-CH=C); 2.75–2.70(m,
1H, -C-CH2-C=C); 2.61–2.55(m, 1H, -C-CH2-C=C);
1.72(s, 3H, C=C-CH3); 1.63(s, 3H, C=C-CH3). ESI-TOF,
calcd for C25H21ClO6 ([M+Na]+), 475.1027, found
475.1023. Anal. Calcd for C25H21ClO6: C, 66.30; H, 4.67;
O, 21.20. Found: C, 65.42; H, 5.89; O, 23.24.
1.72(s, 3H, C=C-CH3); 1.63(s, 3H, C=C-CH3). ESI-TOF,
calcd for
459.1319. Anal. Calcd for C25H21FO6: C, 68.80; H, 4.85;
O, 22.00. Found: C, 65.32; H, 5.87; O, 21.29.
C
25H21FO6 ([M+Na]+), 459.1322, found
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)-4-methylpent-3-enyl- 3-(3,4-difluorop-
henyl) acrylate (16b)
Yield 48%. Red powder Mp: 98.2–99.6 ꢀC. 1H NMR
(500 MHz, CDCl3) d: 12.63(s, 1H, -OH); 12.44(s, 1H, -OH);
7.65(d, J = 16.5 Hz, 1H, C=CH-Ar); 7.43–7.39(m, 1H, Ar-
H); 7.32–7.30(m, 1H, Ar-H); 7. 25–7.22(m, 1H, Ar-H);
7.21(s, 2H); 7.05(s, 1H); 6.45(d, J = 16.0 Hz, 1H, CH=C-
Ar); 6.17–6.15(m, 1H, -O-CH); 5.19(t, J1 = 7.25 Hz,
J2 = 7.5 Hz, 1H, -C-C-CH=C); 2.75–2.70(m, 1H, -C-CH2-
C=C); 2.61–2.55(m, 1H, -C-CH2-C=C); 1.72(s, 3H, C=C-
CH3); 1.63(s, 3H, C=C-CH3). ESI-TOF, calcd for
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen
-2-yl)-4-methylpent-3-enyl -3-(4-isopropylphenyl)
acrylate (13b)
Yield 72%. Red powder Mp: 87.0–88.5 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H,
-OH); 7.74(d, J = 16 Hz, 1H, C=CH-Ar); 7.51(d, J =
8.0 Hz, 2H, Ar-H); 7.29(d, J = 8.5 Hz, 2H, Ar-H); 7. 22(s,
2H); 7.07(s, 1H); 6.49(d, J = 16.0 Hz, 1H, CH=C-Ar);
6.17(t, J1 = 5.75 Hz, J2 = 7.0 Hz, 1H, -O-CH); 5.21(t, J1 =
6.5 Hz, J2 = 5.5 Hz, 1H, -C-C-CH=C); 2.99–2.94(m, 1H, -
C-CH2-C=C); 2.74–2.70(m, 1H, -C-CH2-C=C); 2.61–
2.57(m, 1H, Ar-CH-C); 1.72(s, 3H, C=C-CH3); 1.63(s, 3H,
C=C-CH3); 1.30(s, 3H, Ar-C-CH3); 1.29(s, 3H, Ar-C-CH3).
ESI-TOF, calcd for C28H28O6 ([M+Na]+), 483.1886, found
483.1891. Anal. Calcd for C28H28O6: C, 73.03; H, 6.13;
O, 20.85. Found: C, 71.73; H, 6.89; O, 22.24.
C
25H20F2O6 ([M+Na]+), 477.1228, found 477. 1326. Anal.
Calcd for C25H20F2O6: C, 66.08; H, 4.44; O, 21.13.
Found: C, 65.06; H, 5.37; O, 22.27.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen
-2-yl)-4-methylpent-3-enyl -3-(2,3-dichlorophenyl)
acrylate (17b)
Yield 40%. Red oil. 1H NMR(500 MHz, CDCl3) d: 12.63(s,
1H, -OH); 12.44(s, 1H, -OH); 8.17(d, J = 15.5 Hz, 1H,
C=CH-Ar); 7.58(d, J = 8.0 Hz, 1H, Ar-H); 7.53(d, J =
7.0 Hz, 1H, Ar-H); 7.29–7.26(m, 1H, Ar-H); 7.21(s, 2H);
7.06(s, 1H); 6.50(d, J = 15.5 Hz, 1H, CH=C-Ar); 6.17–
6.16(m, 1H, -O-CH); 5.20(t, J1 = 7.0 Hz, J2 = 7.0 Hz, 1H,
-C-C-CH=C); 2.75–2.70(m, 1H, -C-CH2-C=C); 2.62–
2.56(m, 1H, -C-CH2-C=C); 1.72(s, 3H, C=C-CH3); 1.63(s,
3H, C=C-CH3). ESI-TOF, calcd for C25H20Cl2O6 ([M+Na]+),
509. 0637, found 509. 0649. Anal. Calcd for C25H20Cl2O6:
C, 61.61; H, 4.14; O, 19.70. Found: C, 60.06; H, 5.27; O,
20.66.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen
-2-yl)-4-methylpent-3-enyl -3-(3-bromophenyl)
acrylate (14b)
Yield 64%. Red powder Mp: 107.4–108.7 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.63(s, 1H, -OH); 12.44(s, 1H,
-OH); 7.73(s, 1H, Ar-H); 7.67(d, J = 16 Hz, 1H, C=CH-Ar);
7.55(d, J = 8.0 Hz, 1H, Ar-H); 7.49(d, J = 8.0 Hz, 1H,
Ar-H); 7.31(t, J1 = 7.75 Hz, J2 = 8.0 Hz, 1H, C=CH-Ar);
7.21(s, 2H); 7.06(s, 1H); 6.53(d, J = 16.0 Hz, 1H, CH=C-
Ar); 6.17(m, 1H, -O-CH); 5.20(t, J1 = 6.5 Hz, J2 = 6.0 Hz,
1H, -C-C-CH=C); 2.75–2.70(m, 1H, -C-CH2-C=C); 2.61–
2.55(m, 1H, -C-CH2-C=C); 1.72(s, 3H, C=C-CH3); 1.63(s,
3H, C=C-CH3). ESI-TOF, calcd for C25H21BrO6 ([M+Na]+),
519.0522, found 519.0517. Anal. Calcd for C25H21BrO6:
C, 60.38; H, 4.26; O, 19.30. Found: C, 59.32; H, 5.87; O,
20.27.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen
-2-yl)-4-methylpent-3-enyl- 3-(2-methoxyphenyl)
acrylate (18b)
Yield 44%. Red powder Mp: 97.1–98.5 ꢀC. 1H
NMR(500 MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.46(s, 1H,
-OH); 7.68(d, J = 16.0 Hz, 1H, C=CH-Ar); 7.56(d, J1
= 1.0 Hz, J2 = 7.5 Hz, 1H, Ar-H); 7.40(m, 1H, Ar-H);
7.21(s, 2H); 7.08(s, 1H); 7.01(t, J1 = 7.5 Hz, J2 = 7.0 Hz,
1H, Ar-H); 6.96(d, J = 8.0 Hz, 1H, Ar-H); 6.62(d,
J = 16.5 Hz, 1H, CH=C-Ar); 6.17–6.15(m, 1H, -O-CH);
5.21(t, J1 = 7.25 Hz, J2 = 7.5 Hz, 1H, -C-C-CH=C);
3.94(s, 3H, Ar-OCH3); 2.75–2.70(m, 1H, -C-CH2-C=C);
2.61–2.55(m, 1H, -C-CH2-C=C); 1.73(s, 3H, C=C-CH3);
1.64(s, 3H, C=C-CH3). ESI-TOF, calcd for C26H24O7
([M+Na]+), 471.1522, found 471.1523. Anal. Calcd for
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen
-2-yl)-4-methylpent-3-enyl-3-(3-fluorophenyl)
acrylate (15b)
Yield 30%. Red oil. 1H NMR(500 MHz, CDCl3) d: 12.63(s,
1H, -OH); 12.44(s, 1H, -OH); 7.70(d, J = 16.5 Hz, 1H,
C=CH-Ar); 7.43–7.39(m, 1H, Ar-H); 7.33(d, J = 7.5 Hz,
1H, Ar-H); 7. 27(d, J = 8.5 Hz, 1H, Ar-H); 7.21(s, 2H);
7.15–7.12(m, 1H, Ar-H); 7.06(s, 1H); 6.52(d, J = 16.0 Hz,
1H, CH=C-Ar); 6.18–6.16(m, 1H, -O-CH); 5.20(t,
J1 = 7.25 Hz, J2 = 7.0 Hz, 1H, -C-C-CH=C); 2.75–2.70(m,
1H, -C-CH2-C=C); 2.61–2.56(m, 1H, -C-CH2-C=C);
C
26H24O7: C, 69.63; H, 5.39; O, 24.97. Found: C, 67.77;
H, 5.48; O, 26.75.
Chem Biol Drug Des 2013; 81: 275–283
279

Lin et al.
(E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-4-methylpent-3-enyl -3-
(3,4-dimethoxyphenyl) acrylate (19b)
Both early apoptotic (AnnexinV positive and PI negative)
and late apoptotic (double positive of Annexin V and PI)
cells were detected.
Yield 44%. Red powder Mp: 65.3–66.7 ꢀC. 1H NMR(500
MHz, CDCl3) d: 12.64(s, 1H, -OH); 12.45(s, 1H, -OH);
7.70(d, J = 15.5 Hz, 1H, C=CH-Ar); 7.21(s, 2H); 7.17–
7.15(m, 1H, Ar-H); 7.11(d, J = 1.5 Hz, 1H, Ar-H); 7.08 (s,
1H); 6.91(d, J = 9.0 Hz, 1H, Ar-H); 6.40(d, J = 16.0 Hz,
1H, C-CH=C); 6.17(m, 1H, -O-CH); 5.21(t, J1 = 7.25 Hz,
J2 = 7.0 Hz, 1H, -C-C-CH=C); 3.97(s, 3H, -OCH3); 3.95(s,
3H, -OCH3); 2.74–2.70(m, 1H, -C-CH2-C=C); 2.61–2.56(m,
1H, -C-CH2-C=C); 1.72(s, 3H, C=C-CH3); 1.63(s, 3H, C=C-
CH3). ESI-TOF, calcd for C27H26O8 ([M+Na]+), 501.1628,
found 501.1637. Anal. Calcd for C27H26O8: C, 66.35; H,
6.49; O, 27.26. Found: C, 66.35; H, 6.49; O, 27.26.
Western blot analysis
Following drug treatment, cell lysates were prepared and
Western blot analysis was performed with primary antibod-
ies. The following antibodies were obtained from Becton-
Dickinson (San Jose, CA, USA): cleaved caspase-9,
cleaved caspase-3, cleaved caspase-7, and cleaved PARP
and GAPDH.
To ensure equivalent loading and transfer, all blots that
had been probed for proteins other than actin were
stripped and re-probed with an antibody against actin.
Cell lines and culture conditions
The cell lines used in this study were as follows: A549
(human), A875 (human), and SW872-s(human). They were
obtained from State Key Laboratory of Pharmaceutical Bio-
technology, Nanjing University. All cell lines were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) with L-glu-
tamine, supplemented with 10% fetal bovine serum (FBS) at
37 ꢀC in a humidified atmosphere containing 5% CO2.
Results and Discussion
Chemistry
The synthetic route for the novel shikonin esters (1b–19b)
is outlined in Scheme 1. Among them, compounds 2b–
19b are first reported, but compound 1b had been
reported by Shen Chien-Chang (22). These compounds
were synthesized from cinnamic acids (1a–19a) and
shikonin. Compounds (1a–19a) were prepared according
to the procedure reported by Davis et al. (23) with some
modifications. Aromatic aldehydes and malonic acid were
dissolved in a mixture of pyridine and piperidine and
heated and stirred for 4–5 h, and cinnamic acids were
obtained with yields of 70–80%. Then cinnamic acids
(1a–19a), shikonin, 4-dimethylaminopyridine (DMAP) and
N, N¢-dicyclohexylcarbodiimide (DCC) were dissolved in
dichloromethane and cooled and stirred for 12 h, after
that, adding proper amount of silica gel and condensing
solvent by vacuum concentration, and finally gaining
shikonin esters by ethyl acetate ⁄ petroleum ether
(V:V = 1:7) column chromatography.
Growth inhibition assays
Cells were plated in 96-well plates at appropriate densities
to ensure exponential growth throughout the experimental
period (2.0 · 103 cells ⁄ well) and then allowed to adhere
for 6 h. Cells were then treated for 72 h with four serial
dilutions (10, 100, 1000 nM, and 10 lM) of each com-
pound and shikonin as positive reference. Then, 20 lL of
MTT solution to a final concentration of 4 mg ⁄ mL was
added to each well. Plates were then incubated for a
further 4 h. Then, the entire medium was removed.
200 lL of DMSO was added to each well for coloration.
The plates were shaken vigorously to ensure complete sol-
ubilization for 10 min at room temperature, and the optic-
metric density (OD) was read on a microplate reader
ELx800 (BioTek, Highland Park, Winooski, VT, USA) at
wavelength of 570 nm. Data were analyzed using Origin
7.5, and IC50 values were obtained.
Biological activities
All of these compounds were evaluated as potential anti-
cancer agents against human SW872-s, A875, and A549
cell lines with shikonin as positive references by MTT
assay, respectively. The results are summarized in Table 1.
All the compounds exhibited significant antiproliferative
activity with IC50 values of <3 lM in A875 cell line. Com-
Annexin V ⁄ PI staining
For Annexin V ⁄ PI assays, cells were stained with Annexin V-
FITC and PI and then monitored for apoptosis by flow cytom-
etry. Briefly, 0.5 · 106 cells were washed with PBS and
stained with 5 lL of Annexin V-FITC and 2.5 lL of PI
(5 lg⁄ mL) in 1· binding buffer (10 mM HEPES, pH 7.4,
140 mM NaOH, 2.5 mM CaCl2) for 30 min at room tempera-
ture in the dark. Apoptotic cells were quantified using a FAC-
Scan cytofluorometer (PT. Madagasi Brosa Inc. Jl. Batang
Hari No. 73, Propinsi Sumatera Utara, Indonesia.). Statistical
analysis was performed using WINMDI software version 2.8
(The Scripps Research Institute(TSRI), San Diego, CA, USA).
pound with two fluorine atom substituted (16b) (IC50
=
0.36, 0.55, 2.1 lM for human SW872-s, A875 and A549
cell lines, respectively) exhibited significant antiproliferative
activities than other fluorine-substituted groups (4b, 15b).
The compounds with para halogen substituted (12b, 14b,
15b) exhibited slightly more potent activities in order of
Cl>Br>F against SW872-s (IC50 = 1.09, 1.11, 0.36 lM)
and A875 (IC50 = 0.79, 0.86, 1.29 lM) cell lines, while
showed more potent activities in order of Br>F>Cl against
A549 (IC50 = 8.94, 2.93, 3.92 lM) cell line. Compounds
280
Chem Biol Drug Des 2013; 81: 275–283

Study of Cinnamic Acyl Shikonin Derivatives
Table 1: Inhibition (IC50) of SW872-s, A875, and A549 cells’ pro-
liferation by compounds 1b–19b
0 μ
M
0.5 μ
M
1.0 μ
M
2.0 μ
M
4.0 μM
Cleaved Caspase-9
Cleaved Caspase-7
Cleaved Caspase-3
IC50 (lM)
Entry
SW872-s
A875
A549
1b
2b
3b
4b
5b
6b
7b
8b
4.74
1.15
1.06
3.99
1.49
3.47
3.57
0.69
7.63
0.91
2.41
1.09
1.87
1.11
1.64
0.36
3.51
1.59
1.07
0.76
0.93
1.03
0.83
2.07
0.81
2.52
2.60
0.65
7.05
0.78
1.91
0.79
1.02
0.86
1.29
0.55
2.12
1.16
0.95
0.58
3.61
3.17
3.14
2.66
5.11
2.20
2.67
1.62
5.59
3.75
3.83
8.94
5.50
2.93
3.92
2.10
27.8
4.72
4.77
1.8
PARP
Cleaved PARP
GAPDH
9b
Figure 3: Compound 8b increased the expression of cleaved
caspase-9, cleaved caspase-3, cleaved caspase-7, and cleaved
PARP in A875 cells. 5.0 · 105 A875 cells were seeded in 60 mm
dish and incubated with various concentrations of compound 8b
for 24 h. Then, cells were collected, and cell extracts were
acquired. Expression of cleaved caspase-9, cleaved caspase-3,
cleaved caspase-7, and cleaved PARP was analyzed through
Western blot analysis by anticleaved caspase-9 antibody, antic-
leaved caspase-3 antibody, anticleaved caspase-7 antibody, and
anticleaved PARP antibody. GAPDH was performed as a loading
control.
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
Shikonin
with chlorine atom substituted (3b) (IC50 = 1.06, 0.83,
3.14 lM) showed significant antiproliferative activities than
compounds 9b, 11b, 12b, and 17b. Compounds with
p-methoxyl substituted (10b) (IC50 = 0.91, 0.78, 3.75 lM)
showed better antiproliferative activities than those of
o-substituted (18b) and di-substituted group (19b). Most
importantly, the compound with m-trifluoromethyl-substi-
tuted group (8b)(IC50 = 0.69, 0.65, 1.62 lM) exhibited
more potent activities than those of o- and p-substituted
groups (6b, 7b).
Among all the compounds, 16b (IC50 = 0.36, 0.55,
2.10 lM) showed better anticancer activity against human
SW872-s, A875, and A549 cell lines compared with
5.77%
9.13%
21.63%
14.50%
8b
4.36%
5.77%
8.56%
19.59%
11.08%
71.32%
21.93%
0.3 μ
M
3.0 μM
con
1.0 μM
15.54%
16b
32.77%
67.30%
4.36%
8.81%
Annexin V
Figure 2: Cellular apoptosis study of compound 8b- and 16b-treated A875 cells. A875 cells were seeded in the 24-well cell culture clus-
ter and incubated with indicated concentrations of compound 8b and 16b for 24 h. Then, cells were collected and washed with the cold
PBS. Then, the cells were stained with Annexin V-FITC and PI protected from light for 15 min, and then cells were gently transferred into
tubes for flow cytometric analysis. The percentage of early apoptotic cells in the lower right quadrant (annexin V-FITC-positive ⁄ PI-negative
cells), as well as late apoptotic cells located in the upper right quadrant (annexin V-FITC-positive ⁄ PI-positive cells).
Chem Biol Drug Des 2013; 81: 275–283
281

Lin et al.
shikonin. Besides, 8b also exhibited better anticancer
activity against human A549 cell line with the IC50 of
1.62 lM than shikonin itself.
and the cross-talk between them. Cell Death Dif-
fer;16:966–975.
2. Hickman J.A. (1992) Apoptosis induced by anticancer
drugs. Cancer Metast Rev;11:121–139.
To determine the mechanism by which naphthazarin induced
cell death, we first treated A875 cells with varying concentra-
tions of compound 8b and 16b and analyzed cells for
changes in apoptotic markers by flow cytometer in vitro. The
results are shown in Figure 2, and after treated with increas-
ing concentrations of 8b and 16b, the percentage of killed
cells has a significant increase. However, we can reasonably
infer that compound 16b exhibits high cell toxicity when its
concentration rise to 3 lM compared with compound 8b
from Figure 2. Therefore, we considered compound 8b as
the best candidate of potential anticancer agent.
3. Acharya B.R., Bhattacharyya S., Choudhury D., Chak-
rabarti G. (2011) The microtubule depolymerizing agent
naphthazarin induces both apoptosis and autophagy in
A549 lung cancer cells. Apoptosis;16:924–939.
4. Johnstone R.W., Frew A.J., Smyth M.J. (2008) The
TRAIL apoptotic pathway in cancer onset, progression
and therapy. Nat Rev Cancer;8:782–798.
5. Fesik S.W. (2005) Promoting apoptosis as a strategy for
cancer drug discovery. Nat Rev Cancer;5:876–885.
6. Budihardjo I., Oliver H., Lutter M., Luo X., Wang X.
(1999) Biochemical pathway of caspase activation dug-
ing apoptosis. Annu Rev Cell Dev Biol;15:269–290.
7. Thompson C.B. (1995) Apoptosis in the pathogenesis
and treatment of disease. Science;267:1456–1462.
8. Hanahan D., Weinberg R.A. (2000) The hallmarks of
cancer. Cell;100:57–70.
To investigating the mechanism of apoptosis, we did the
Western blot about the expression of some related pro-
teins in the apoptosis-related pathway at the same time.
We found that the quantity of cleaved caspase-9, cleaved
caspase-3, cleaved caspase-7, and cleaved PARP in
A875 cells was increased after treated by compound 8b,
and this is a significant of apoptosis (Figure 3).
9. Chen X., Yang L., Oppenheim J.J., Zack Howard O.M.
(2002) Cellular pharmacology studies of shikonin deriv-
atives. Phytother Res;16:199–209.
10. Rao Z., Liu X., Zhou W., Yi J., Li S.S. (2011) Synthesis
and antitumour activity of beta-hydroxyisovalerylshiko-
nin analogues. Eur J Med Chem;46:3934–3941.
11. Kim S.H., Kang I.C., Yoon T.J., Park Y.M., Kang K.S.,
Song G.Y., Ahn B.Z. (2001) Antitumor activities of a
newly synthesized shikonin derivative, 2-hyim-DMNQ-
S-33. Cancer Lett;172:171–175.
12. Zhou W., Zhang X., Xiao L., Ding J., Liu Q.H., Li S.S.
(2011) Semi-synthesis and antitumor activity of 6-iso-
mers of 5, 8-O-dimethyl acylshikonin derivatives. Eur J
Med Chem;46:3420–3427.
13. Assimopoulou A.N., Papageorgiou V.P. (2005) Radical
scavenging activity of Alkanna tinctoria root extracts
and their main constituents, hydroxynaphthoquinones.
Phytother Res;19:141–147.
14. Su Y.H., Xie J.S., Wang Y.G., Hu X., Lin X.F. (2010)
Synthesis and antitumor activity of new shikonin glyco-
sides. Eur J Med Chem;45:2713–2718.
Conclusion
In this paper, a series of novel anticancer compounds (1b–
19b) containing naphthoquinone skeleton and cinnamoyl
moiety had been synthesized and evaluated their biological
activities. These compounds exhibited potent cell growth
inhibitory activities and antiproliferative activities against
human SW872-s, A875, and A549 cell lines. Compound 8b
showed the most potent inhibition activity with IC50 values
of 0.69, 0.65, and 1.62 lM against human SW872-s, A875,
and A549 cell lines, respectively. Besides, from the results
of flow cytometer, we found 8b can really inhibit A875 cell
proliferation and has low cell toxicity. Furthermore, the
results of Western blot also confirmed that 8b induces
apoptosis via up-regulation of caspase activation. To sum
up, we recommend 8b as the best candidate of potential
anticancer agent among these new compounds.
15. Nadine K., Beate R. (2012) Naphthoquinones from On-
osma paniculata induce cell-cycle arrest and apoptosis
in melanoma cells. J Nat Prod;75:865–869.
Acknowledgments
16. He H., Bai L.P., Jiang Z.H. (2012) Synthesis and
human telomeric G-quadruplex DNA-binding activity of
glucosaminosides of shikonin ⁄ alkannin. Bioorg Med
Chem Lett;22:1582–1586.
17. Hatano N., Sohji Y., Kawashim K. (1971) Pharmacolof-
ical studies of 3-(3,3-diphenylpropylamino)-propyl-
The authors are grateful to the National Natural Science
Foundation of China (30701041, 31171161), the Program
for Changjiang Scholars and Innovative Research Team in
University (IRT1020), the Fundamental Research Funds for
the Central Universities (1106020824, 1082020803) and the
Natural Science Foundations of the Jiangsu (BK2010053).
3¢,4¢,5¢-trimethoxybenzoate(PF-26),
a new coronary
vasodilator. Arzneimittel-Forschung;21:1628–1633.
18. Dias Lorraine S., Vivek G., Manthappa M. (2011) Role
of hemodialysis in baclofen overdose with normal renal
function. Indian J Pharmacol;43:722–723.
References
19. Cezarino P.Y., Bagnoli V.R., Fonseca A.M. (2011) The
effects of cinnarizine on menopausal symptoms in
women. Climacteric;14:492–496.
1. Eisenberg-Lerner A., Bialik S., Simon H.U., Kimchi A.
(2009) Life and death partners: apoptosis, autophagy
282
Chem Biol Drug Des 2013; 81: 275–283

Study of Cinnamic Acyl Shikonin Derivatives
20. Iizuka K., Akahane K., Momose D., Nakazawa M., Ta-
nouchi T., Kawamura M., Ohyama I., Kajiwara I., Iguchi
Y. (1981) Highly selective inhibitors of thrombitors of
thromboxane synthetase. 1. Imidazole derivatives. J
Med Chem;24:1139–1148.
21. Luo Y., Qiu K.M., Lu X., Liu K., Fu J., Zhu H.L. (2011)
Synthesis, biological evaluation, and molecular model-
ing of cinnamic acyl sulfonamide derivatives as novel
antitubulin agents. Bioorg Med Chem;19:4730–4738.
22. Shen C.C., Syu W.J., Li S.Y., Lin C.H., Lee G.H.,
Sun C.M. (2002) Antimicrobial Activities of Naphthaza-
rins from Arnebia euchroma. J Nat Prod;65:1857–
1862.
23. Davis R., Kumar N.S., Abraham S., Suresh C.H., Rath
N.P., Tamaoki N., Das S. (2008) Molecular packing
and solid-state fluorescence of alkoxy-cyano-substi-
tuted diphenylbutadienes: structure of the luminescent
aggregates. J Phys Chem C;112:2137–2146.
Chem Biol Drug Des 2013; 81: 275–283
283