Synthesis and in vitro anti-proliferative activity of β-elemene monosubstituted derivatives in HeLa cells mediated through arrest of cell cycle at the G1 phase

Article abstract of DOI:10.1016/j.bmc.2008.12.040

β-Elemene monosubstituted amine, ether and rhenium coordinated complex were synthesized. Their structures were characterized by IR, ¹H NMR, ¹³C NMR, HRMS or EA. Their IC₅₀ on HeLa cell lines, cell cycle and protein express

Full text of DOI:10.1016/j.bmc.2008.12.040

Bioorganic & Medicinal Chemistry 17 (2009) 1118–1124
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro anti-proliferative activity of b-elemene
monosubstituted derivatives in HeLa cells mediated through
arrest of cell cycle at the G1 phase
*
Yanhong Sun , Guifeng Liu , Yuangqing Zhang, Hua Zhu, Yunfeng Ren, Yu-Mei Shen
Research Center of Radiopharmaceuticals, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 2019 BaoJia Road, Shanghai 201800, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Elemene monosubstituted amine, ether and rhenium coordinated complex were synthesized. Their
structures were characterized by IR, 1H NMR, 13C NMR, HRMS or EA. Their IC50 on HeLa cell lines, cell
cycle and protein expression of G1 phase (Cyclin D1, Rb, P-Rb) were detected respectively by the method
of WST-1, Flow Cytometry and Western Blot. The Results showed that the in vitro anti-proliferative activ-
ity of b-elemene monosubstituted amine and Re(CO)3-b-elemene derivatives in human cervix epitheloid
carcinoma HeLa cells were improved significantly compared with both of ether derivatives and parent b-
elemene. These derivatives could reduce Rb phosphorylation and cyclin D1 protein expression to arrest
the cell cycle at G1 phase.
Received 21 October 2008
Revised 16 December 2008
Accepted 17 December 2008
Available online 25 December 2008
Keywords:
b-Elemene derivatives
Synthesis
Anticancer activity
Cell cycle arrest
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ported the b-elemene 13,14-disubstituted amine derivatives’
activity in cancer cells improved significantly.7 Peng et al.6 summa-
b-Elemene, (5S,7R,10S)-(À)-(1-methyl-1-vinyl-2,4-diisoprope-
nyl-cyclohexane), a natural sesquiterpene extracted from the Tra-
ditional Chinese Herb Medicinal Curcuma wenyujin,1 is the main
effective monomer of elemene emulsion. b-Elemene exhibits anti-
cancer effects in human and murine tumour cells in vitro and
in vivo and has substantial clinical activity against various tumours
without severe side effects.2–4 No bone marrow suppression and
drug resistance have been observed in the clinical studies; on the
contrary, patient immunity was improved during the therapy with
b-elemene.5,6 In China b-elemene has been effective for the treat-
ment of cervical cancer as well as carcinomas of the liver, brain
and other vital tissues, and now application for clinical studies in
the US. However b-elemene suffers from limited bioavailability
due to poor water solubility, short half lives and rapid clearance
from the body. The mechanism of action of b-elemene in cancer re-
mains unknown (Fig. 1).
rized the methodological quality of these trials for b-elemene
derivatives’ synthesis is relatively poor. We for the first time to
yield the pure 13-monosubstituted ether, amine and rhenium
complex derivatives by carefully controlling the reaction condi-
tions, to our pleasure some of the derivatives has much higher
anti-proliferative activities compared with that of parent b-elem-
ene, furthermore we detected the cell growth and cell cycle of
some derivatives and explored their possible mechanism of their
anti-proliferative activities. Herein we report our preliminary
result.
2. Results and discussion
2.1. Chemical syntheses
The monosubstituted b-elemene ether and amine derivatives
were synthesized according to the procedure outlined in Scheme
1. The synthesis began with chlorination of b-elemene with NaClO,
the resulting product chlorinated b-elemene 2 reacted further with
alcohols or amine to yield the desired b-elemene monosubstituted
ether or amine derivatives 3, 4 (a–h). Generally speaking the first
step product is mixture of 13-monosubstituted, 14-monosubsti-
tuted, disubstituted chlorinated b-elemene. In order to get the
13-monosubstituted chlorinated b-elemene as the absolutely
major product the reaction temperature must be kept below 0 °C,
and the reactant NaClO should be fresh and its mole ratio to
Some of the b-elemene derivatives with better properties have
already been developed.7,8 As we know the mixture of b-elemene
13-monosubstituted, 14-monosubstituted and 13,14-disubstituted
derivatives is difficult to separate each other, so an easy way to
solve the problem is to let b-elemene react with much excess an-
other reactant to give 13,14-disubstituted derivatives. Dong re-
* Corresponding author. Tel.: +86 21 59554692; fax: +86 21 59554696.
E-mail address: shenyumei@sinap.ac.cn (Y.-M. Shen).
These two authors contributed equally to this paper.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.040

Y. Sun et al. / Bioorg. Med. Chem. 17 (2009) 1118–1124
1119
Figure 1. Chemical structure of b-elemene.
NaClO
ROH
CH3COOH
R NH
or R1
2
3(a-h)
4(a-h)
CH2Cl
2
1
OR (NR1R2)
3a) R = Me
3b) R = Et
3c) R = Bu
3d) R = t-Bu
3e) R = (CH2)10CH3,
3f) R = cyclohexyl
4b) R1R2 =t-BuH
3g) R =PhCH2
3h) R = CH2CH2OCH3
4d)R1R2 = H Cyclopentyl
4a) R1R2 =BuH
4c) R R2 = H Cyclohexyl
1
4e)R1R2 = H Cyclooctyl
4f) R1 R2 = Imidazole
4g) R =R2=Et
4h) R1R2=i-PrH
1
Scheme 1. Synthetic procedure of b-elemene monosubstituted derivatives.
b-elemene keeps between 1.1 and 1.2, the chlorinated b-elemene
mixture was used directly without further purification. In the sec-
ond step, the 13-monosubstituted b-elemene chloride is more ac-
tive than the other two chloride products, so when alcohol was
added to the chlorinated b-elemene mixture, the reaction mixture
was heated and refluxed for about 2 h. After purified with chroma-
tography to give pure b-elemene 13-monosubstituted ether deriv-
13 (Scheme 2). All of them were characterized by IR, 1H NMR,
HRMS, HPLC or elemental analysis.
2.2. Anti-proliferative activity
With pure b-elemene derivatives in hands we tested their
in vitro antiproliferative activities on HeLa cell lines. Their antipro-
liferative activities were evaluated by WST-1 method. Results were
shown in Table 1, generally speaking the proliferative activity for
amine derivatives is much higher than that of the ether derivatives,
especially for isopropyl amine, cyclopentyl amine or cyclohexyl
amine, their IC50 value were decreased dramatically. Compared
with the similar work reported by Dong,7 the IC50 for 13,14-disub-
stituted amine derivatives is similar to our 13-monosubstituted
derivative, it seems that b-elemene 14-monosubstituted deriva-
tives contributes extremely weak antiproliferative activities. At
the same time, for most of b-elemene ether derivatives, their anti-
proliferative activity did not increase at all. The methyl, butyl and
2-methoxyl ethyl ether derivatives, their antiproliferative activity
is not higher than that of parent b-elemene, the ethyl ether deriv-
ative holds a little higher antiproliferative activity. For benzyl, tert-
butyl and undecyl and cyclohexyl ether derivatives, their IC50 value
is even higher than that of parent b-elemene (These data was not
shown in Table 1). The three rhenium coordinated derivatives
shows similar anti-proliferative activities, all of their IC50 values
decreased significantly compared with parent b-elemene which
provides us opportunity for further research other metal coordi-
nated b-elemene derivatives’ activity.
atives, Scheme
1 shows the procedure. Under the similar
conditions the expected pure 13-monosubstituted amine deriva-
tives cannot be given due to amine is more active than alcohol,
the added amine in the second step not only reacts with 13-mon-
osubstituted chloride b-elemene, but also reacts with the other
two byproducts. So we had to run the second step at room temper-
ature and luckily got the pure b-elemene 13-monosubstitued
amine derivatives after silica gel purification.
For the syntheses of the rhenium coordinated derivatives, we
first prepared various chelating systems with the spacer entities
according to the literature.9,10 Their structures were shown in Fig-
ure 2.
These intermediates including commercial available compound
di-(2-picoyl) amine were coupled to the b-elemene chlorinated
compound 2 under basic conditions to form the corresponding
intermediates 8, 9, 10 in very good yields.
The complex of [N(CH2CH3)4]2[ReBr3(CO)3] (7) was prepared
according to a previously published procedure.9,11 This complex
is an important starting material for compounds containing the
fac-Re(CO)3 moiety since the three bromide ligands are very weak
bound. When [N(CH2CH3)4]2[ReBr3(CO)3] complex was dissolved in
water, the three bromide ligands were quantitatively exchanged by
three H2O molecules to form the complex of fac-[Re(CO)3(H2O)3]+,
which is stable in aqueous solution even when exposed to air for
several weeks.
FACS data on cell cycle analysis supported the effect of b-elem-
ene and its derivatives on cancer cells proliferation. The results of
cell cycle analysis (Fig. 3) indicated the percentage of cells in G1
phase increased treated by the derivatives, so the percentage of
cells in S and G2-M phase decreased. Therefore the G1 phase arrest
was one of possible mechanisms of anti-proliferative activities for
both of b-elemene and its derivatives.
These substrates (8, 9, 10) are easy to coordinate with
[N(CH2CH3)4]2[ReBr3(CO)3] to afford the compounds 11, 12 and
It was found that the cell cycle protein Cyclin D1 and the cell
survival checkpoint protein p-Rb decreased while the protein level
of Rb did not decrease (Fig. 4). Cyclin D1 is an important regulation
protein at the G1/S checkpoint,12 Rb is an important regulator of
the G1 to S phase transition in the cell cycle and represses gene
transcription required for this transition by interacting with E2F
transcription factors.13 Rb is regulated by CDK phosphorylation,
and the Rb phosphorylation state continues from the late G1 phase
of the cell cycle until mitosis.14 The present data showed that onset
N
N
N
N
N
O
N
H2N
H2N
O
5
6
Figure 2. Structure of compound 5 and 6.

1120
Y. Sun et al. / Bioorg. Med. Chem. 17 (2009) 1118–1124
CO
CO
CO
N
N
+
N
Re
N
H
d
Br-
a
11
N
N
N
N
H
8
b
e
N
N
Cl
N
2
Br-
Re
CO
N
N
9
+
CO
CO
N
12
c
N
N
O
N
f
N
H
O
10
CO
CO
CO
N
+
N
Re
O
Br-
N
H
O
N
13
Scheme 2. Synthesis of compounds 8–13. Reagents and conditions: (a) compound 5, dry CH3CN, reflux, 8 h; (b) di-(2-picolyl)amine, dry CH3CN, reflux, 8 h; (c) compound 6,
dry CH3CN, reflux, 10 h; (d)–(f) compound 7, CH3OH, rt, 40 min.
Table 1
method. For monosubstituted amine and rhenium coordinated
derivatives their activities were improved significantly, but for
monosubstituted ether derivatives their antiproliferative activities
are higher than that of parent b-elemene only in some cases. All of
them block the cell cycle at the G1 phase in HeLa cells. These deriv-
atives could reduce Rb phosphorylation and cyclin D1 protein
expression to arrest the cell cycle at G1 phase which is probably
one of the anti-proliferative activities’ mechanisms for b-elemene
derivatives.
Anti-proliferative activity of b-elemene monosubstituted derivatives in HeLa cell lines
Compounds
IC50 (lM)
1
236.2 3.2
211.9 2.8
3a
3b
3c
3h
4a
4b
4c
4d
4e
4f
4g
4h
11
12
13
105.9 24.4**
265.9 17.9
237.6 40.3
10.81 0.21**
25.37 0.45**
3.39 1.40**
0.52 0.20**
5.90 2.42**
15.67 1.80**
14.35 1.05**
0.04 0.01**
10.9 1.2**
11.2 1.5**
10.5 2.9**
3. Experimental
b-Elemene was obtained from WenTe Research Institute of
Oleum Curcumae Wenchowensis in Yue Qing city, Zhejiang prov-
ince (purity 98%). Other materials were purchased from Fluka Co.
and Sinopharm Chemical Reagent Co. Ltds. The NMR data were
obtained using a Bruker DRX 500 MHz FT spectrometer. The chem-
ical shifts as d are reported in ppm relative to TMS. Infrared (IR)
spectra were recorded on a Perkin–Elmer FT-IR spectrometer. Mass
spectral data were collected using positive mode on a Finnigan LCQ
classic mass spectrometer. Elemental analysis was performed
using a Perkin–Elmer Series III analyzer.
HeLa cells were treated with b-elemene mono-substituted ether, amine and
Re(CO)3-b-elemene derivatives in five different concentrations for 24 h, anti-pro-
liferative activity was evaluated by WST-1 assay, absorbance values were read by a
96-well Opsys Microplate Reader at 450 nm. The IC50 were calculated according to
the absorbance. Data are presented as mean SD, n = 3. Differences were consid-
ered significant when p < 0.05 (*) and p < 0.01 (**).
3.1. Chemistry
of differentiation initiated Rb phosphorylation. However b-elem-
ene and its derivatives, especially compound 13 reduced the Rb
phosphorylation suppress the cell cycle progression at the G1
checkpoint by interacting with E2F and repressing transcription.
These results indicate that b-elemene and its derivatives blocks
the cell cycle at the G1 phase transition and causes HeLa cells to re-
main at the G1 phase.
In conclusion, the b-elemene monosubstituted amine, ether and
rhenium coordinated derivatives were successfully synthesized for
the first time by carefully controlling the reaction conditions and
their in vitro anti-proliferative activity were screened by WST-1
3.1.1. A General procedure for the formation of 13-chloro-b-
elemene (2)7
The formation of 2: To a solution of b-elemene in glacial acetic
acid, sodium hypochlorite was added over 4 h period under stirring
at 0 °C, then the mixture was stirred for 2 h at room temperature
and extracted with EtOAc. The combined organic extracts were
washed with water, dried over anhydrous MgSO4, and filtered.
The filtrate was concentrated in vacuo to yield the mixture as yel-
low oil which was used for the next step directly without further
purification.

Y. Sun et al. / Bioorg. Med. Chem. 17 (2009) 1118–1124
1121
Figure 3. Effects of 4a and 13 on cell cycle distribution. HeLa cells were treated with compound 4a and 13 (IC20) for 24 h. DNA content was analyzed by flow cytometry using
PI staining, as described in Section 3. The percentage of cells in each phase of the cell cycle was calculated using Cell Quest Pro and is indicated in the top right of each cell
cycle profile.
Figure 4. Effects of 4a and 13 on protein expression of Cyclin D1 and p-Rb. (a) HeLa cells were treated with compound 4a and 13 (concentration is their IC20 values) for 24 h. A
total of 50 lg cell extract protein isolated from the drug-treated and untreated HeLa cells was subjected to SDS–PAGE and immunoblotted with antibodies against cyclin D1,
Rb. b-Actin was used as a loading control. (b) The comparative level of Rb, p-Rb and cyclin D1 protein expression were normalized by density ratio of themselves to b-actin on
the same lane. Differences were considered significant when p < 0.05 (*).
3.1.2. A general procedure for the formation of b-elemene
monosubstituted ether derivatives 3
53.5, 42.0, 40.5, 40.4, 33.9, 27.8, 25.5, 17.3; FTIR (KBr, cmÀ1):
3081, 2927, 2856, 1641, 1452, 1374, 1107, 1007, 905; EI-HRMS:
calcd for C16H27O ([M+H])+ 235.2062, found 235.2063.
To alcohol solution of b-elemene chloride mixture which con-
tains 13-monosubstituted chloride, 14-monosubstituted chloride
and 13,14-disubstituted chloride, 1.5 equiv of base NaOH was
added. The mixture was stirred under reflux for 2–6 h, after that
the reaction mixture was cooled to rt and washed with water
and extracted with EtOAc, dried over anhydrous MgSO4, filtered,
concentrated and purified with chromatography to yield the final
product 13-monosubstituted ether derivatives 3.
The formation of compound 3b: A colorless oil, yield 64%; 1H
NMR (CDCl3, TMS, 500 MHz), d (ppm): 0.94 (s, 3H), 1.15 (t,
J = 7.01, 3H), 1.36–1.44 (m, 3H), 1.49–1.62 (m, 3H), 1.94–2.08 (m,
2H), 3.40 (q, J = 6.97, 2H), 3.89 (s, 2H), 4.52 (s, 1H), 4.75 (s, 1H),
4.82 (s, 1H), 4.85 (d, J = 6.37, 1H), 4.89 (s, 1H), 4.96 (s, 1H), 5.75
(dd, J = 17.36, 11.04, 1H); 13C NMR (CDCl3, 125 MHz) d: 147.9,
150.7, 149.5, 111.3, 112.6, 110.5, 73.3, 58.7, 54.0, 41.8, 40.2, 39.3,
33.7, 28.9, 25.3, 16.0, 14.7; FTIR (KBr, cmÀ1): 3081, 2929, 2856,
1638, 1442, 1375, 1103, 1008, 909; EI-HRMS: calcd for C17H28O, re-
quires ([M+H])+ 249.2218; found: M++H, 249.2219.
The formation of compound 3a: A colorless oil, yield 58%; 1H
NMR (CDCl3, TMS, 500 MHz), d: 0.94 (s, 3H), 1.36–1.71 (m, 6H),
1.64 (s, 3H), 1.94–2.00 (m, 2H), 3.24 (s, 3H), 3.84 (s, 2H), 4.52 (s,
1H), 4.75 (s, 1H), 4.81 (s, 1H), 4.84 (d, J = 3.46, 1H), 4.90 (s, 1H),
4.956 (s, 1H), 5.74 (dd, J = 11.20, 6.47, 1H); 13C NMR (CDCl3,
125 MHz) d: 151.2, 150.9, 148.3, 112.8, 110.9, 110.6, 75.7, 58.5,
The formation of compound 3c: A colorless oil, yield 42%; 1H
NMR (CDCl3, TMS, 500 MHz), d (ppm): 0.85 (t, J = 7.38, 3H), 0.94
(s, 3H), 1.27–1.35 (m, 3H), 1.35–1.42 (m, 3H), 1.44–1.54 (m, 4H),

1122
Y. Sun et al. / Bioorg. Med. Chem. 17 (2009) 1118–1124
1.63 (s, 3H), 1.92–2.00 (m, 2H), 3.32 (t, J = 6.58, 2H), 3.87 (s, 2H),
4.51 (s, 1H), 4.74 (s, 1H), 4.81 (s, 1H), 4.84 (d, J = 7.65, 1H), 4.87
(s, 1H), 4.95 (s, 1H), 5.74 (dd, J = 17.37, 10.89, 1H); 13C NMR (CDCl3,
125 MHz) d: 149.9, 149.2, 146.6, 111.1, 108.7, 107.3, 72.1, 69.0,
51.8, 40.7, 40.4, 38.7, 32.2, 30.9, 26.1, 25.7, 15.6, 15.0, 12.9; FTIR
(KBr, cmÀ1): 3081, 2930, 2862, 1640, 1457, 1375, 1102, 1006,
904; EI-HRMS: calcd for C19H33O requires ([M+H])+, 277.2531;
found: M++H, 277.2539.
and 13,14-disubstituted chloride, 1.5 equiv of NaOH was added fol-
lowed by 1.2 equiv of BuNH2. The mixture was stirred at room
temperature for 3 h, then washed with water and extracted with
EtOAc, dried over anhydrous MgSO4, filtered, concentrated and
purified with chromatography to yield the final product 13-mono-
substituted amine derivatives.
The formation of compound 4a: A yellow oil, yield 48%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 0.92 (t, J = 7.32 Hz, 3H), 1.01 (s,
3H), 1.36–1.74 (m, 10H), 1.71 (s, 3H), 2.00–2.10 (m, 2H), 2.68 (t,
J = 7.49 Hz, 2H), 3.33 (s, 2H), 4.06 (s, 1H, NH), 4.59 (s, 1H), 4.82
(s, 1H), 4.89 (s, 1H), 4.91 (d, J = 7.78 Hz, 1H), 4.99 (s, 1H), 5.04 (s,
1H), 5.82 (dd, J = 17.37, 10.94 Hz, 1H); 13C NMR (CDCl3, 125 MHz)
d: 150.6, 148.4, 148.0, 112.8, 110.7, 109.1, 53.0, 52.3, 48.3, 42.7,
40.4, 40.3, 33.7, 30.2, 27.8, 25.6, 20.9, 17.2, 14.3; FTIR (KBr, cm
À1): 3081, 2927, 2856, 1641, 1452, 1374, 1107, 1007, 905; MS
(EI+) calcd for C19H33N [M+] 275, found 275.
The formation of compound 3d: A colorless oil, yield 48%; 1H
NMR (CDCl3, TMS, 500 MHz), d (ppm): 0.94 (s, 3H), 1.19 (s, 9H),
1.34–1.41 (m, 3H), 1.46–1.51 (m, 3H), 1.64 (s, 3H), 1.92–2.00 (m,
2H), 3.88 (d, J = 3.74, 2H), 4.52 (s, 1H), 4.75 (s, 1H), 4.82 (s, 1H),
4.85 (d, J = 6.64, 1H), 4.90 (s, 1H), 4.97 (s, 1H), 5.75 (dd, J = 17.45,
11.17, 1H); 13C NMR (CDCl3, 125 MHz) d: 151.4, 150.9, 148.3,
112.8, 110.8, 110.6, 77.5, 73.0, 53.5, 42.1, 40.7, 40.5, 32.6, 30.4,
30.0, 25.5, 17.3; FTIR (KBr, cmÀ1): 3080, 2924, 2853, 1640, 1463,
1375, 1080, 905; EI-HRMS: calcd for C19H33O ([M+H])+, 277.2531;
found: M++H, 277.2533.
The formation of compound 4b: A yellow oil, yield 43%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 0.99 (s, 3H), 1.41–1.64 (m, 6H),
1.49 (s, 9H), 1.71 (s, 3H), 2.12 (dd, J = 12.76, 3.08 Hz, 1H), 2.28–
2.40 (m, 1H), 3.52 (q, J = 13.67 Hz, 2H), 4.57 (s, 1H), 4.82 (s, 1H),
4.89 (s, 1H), 4.92 (d, J = 7.05 Hz, 1H), 5.83 (dd, J = 17.23, 11.17 Hz,
1H); 13C NMR (CDCl3, 125 MHz) d: 150.5, 148.0, 145.3, 114.8,
112.8, 110.7, 58.5, 52.9, 45.7, 42.7, 40.3, 40.2, 27.8, 27.5, 26.5,
21.8, 17.1; FTIR (KBr, cmÀ1): 3434, 3080, 2929, 2757, 1637, 1442,
1377, 1008, 907; MS (EI+) calcd for C19H33N [M+] 275, found 275.
The formation of compound 4c: A yellow oil, yield 36%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 0.99 (s, 3H), 1.17–1.30 (m, 4H),
1.35–1.59 (m, 10H), 1.71 (s, 3H), 1.78–1.90 (m, 2H), 2.05–2.26
(m, 2H), 2.91–3.02 (m, 1H), 3.50–3.66 (m, 2H), 4.57 (s, 1H), 4.82
(s, 1H), 4.88 (s, 1H), 4.90–4.92 (m, 1H), 5.23 (s, 1H), 5.35 (s, 1H),
5.82 (dd, J = 17.76, 11.1 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d:
149.9, 147.4, 144.5, 112.2, 110.1, 108.5, 56.5, 53.8, 52.2, 41.7,
39.7, 39.6, 32.9, 29.0, 27.1, 25.1, 25.0, 24.7, 16.6; FTIR (KBr, cm
À1): 3395, 3079, 2934, 2858, 1637, 1454, 1375, 1007, 907; MS
(EI+) calcd for C21H35N [M+] 301, found 301.
The formation of compound 3e: A colorless oil, yield 47%; 1H
NMR (CDCl3, TMS, 500 MHz), d (ppm): 0.88 (t, J = 6.57, 3H), 1.01
(s, 1H), 1.20–1.1.38 (m, 20H), 1.41–1.65 (m, 6H), 1.71 (s, 3H),
1.99–2.09 (m, 2H), 3.39 (t, J = 6.60, 2H), 3.95 (s 2H), 4.59 (s, 1H),
4.88 (s, 1H), 4.92 (d, J = 7.76, 2H), 4.94 (s, 1H), 5.82 (dd, J = 17.37,
10.95, 1H); 13C NMR (CDCl3, 125 MHz) d: 146.5, 149.8, 149.2,
111.1, 108.9, 107.3, 72.1, 69.3, 51.8, 40.4, 39.0, 38.8, 32.2, 30.9,
28.70, 28.64, 28.63, 28.58, 28.55, 28.4, 26.1, 25.1, 23.8, 21.7, 15.6,
13.1; FTIR (KBr, cmÀ1): 3081, 2926, 2854, 1640, 1461, 1375,
1106, 1005, 905; EI-HRMS: calcd for C26H47O ([M+H])+, 375.3627;
found: M++H, 375.3618.
The formation of compound 3f: A colorless oil, yield 50%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 0.94 (s, 1H), 1.10–1.26 (m, 6H),
1.34–1.48 (m, 4H), 1.50–1.54 (m, 2H), 1.58–1.72 (m, 2H), 1.80–
1.86 (m, 2H), 1.93–2.02 (m, 2H), 3.16–3.20 (m, 1H), 3.92 (s, 2H),
4.52 (s, 1H), 4.75 (s, 1H), 4.82 (s, 1H), 4.83–4.86 (m, 2H), 4.98 (s,
1H), 5.75 (dd, J = 17.35, 10.98, 1H); 13C NMR (CDCl3, 125 MHz) d:
152.2, 151.0, 148.3, 112.8, 110.5, 110.0, 77.4, 70.7, 53.5, 42.2,
40.5, 40.4, 33.9, 32.9, 27.8, 26.5, 25.4, 24.8, 17.3; FTIR (KBr, cm
À1): 3080, 2930, 2856, 1640, 1450, 1373, 1088, 1006, 904; EI-
HRMS: calcd for C21H34O, requires ([M+H])+ 303.2688; found:
M++H, 303.2684.
The formation of compound 4d: A yellow oil, yield 42%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 1.01 (s, 3H), 1.42–1.60 (m, 10H),
1.67–1.75 (m, 4H), 1.71 (s, 3H), 1.83–1.92 (m, 2H), 1.98–2.15 (m,
2H), 3.15–3.20 (m, 1H), 3.30 (s, 2H), 4.59 (s, 1H), 4.82 (s, 1H),
4.89 (s, 1H), 4.91 (d, J = 7.04 Hz, 1H), 4.95 (s, 1H), 5.00 (s, 1H),
5.84 (dd, J = 17.43, 11.05 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d:
153.6, 150.9, 148.3, 112.8, 110.5, 108.5, 60.0, 53.5, 52.9, 43.4,
40.7, 40.4, 33.8, 28.0, 25.4, 24.8, 21.7, 17.3; FTIR (KBr, cmÀ1):
3419, 3080, 2930, 2862, 1638, 1440, 1375, 1006, 907; MS (EI+)
calcd for C20H33N [M+] 287, found 287.
The formation of compound 3g: A colorless oil, yield 53%; 1H
NMR (CDCl3, TMS, 500 MHz), d (ppm): 0.93 (s, 3H), 1.36–1.43 (m,
3H), 1.49–1.56 (m, 3H), 1.63 (s, 3H), 1.94 (dd, J = 11.82, 4.07, 1H),
1.99–2.06 (m, 1H), 3.95 (s, 2H), 4.42 (s 2H), 4.51 (s, 1H), 4.75 (s,
1H), 4.81 (s 1H), 4.84 (d, J = 6.95, 1H), 4.92 (s, 1H), 5.01 (s, 1H),
5.75 (dd, J = 17.17, 11.16), 7.25–7.29 (m, 5H); 13C NMR (CDCl3,
125 MHz) d: 151.2, 150.8, 148.2, 139.1, 129.0, 128.3, 128.1, 112.8,
111.1, 110.5, 73.1, 72.6, 53.4, 42.1, 40.6, 40.4, 33.8, 30.4, 27.7,
17.3; FTIR (KBr, cmÀ1): 3081, 2926, 2854, 1640, 1453, 1374,
1093, 1007, 905, 734, 697; EI-HRMS: calcd for C22H31O ([M+H])+,
311.2375; found: M++H, 311.2372.
The formation of compound 4e: A yellow oil, yield 58%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 1.01 (s, 3H), 1.40–1.65 (m, 16H),
1.67–1.81 (m, 4H), 1.71 (s, 3H), 1.98–2.06 (m, 2H), 2.69–2.77 (m,
1H), 3.27 (s, 2H), 4.59 (s, 1H), 4.82 (s, 1H), 4.88 (s, 1H), 4.89–4.94
(m, 2H), 4.97 (s, 1H), 5.82 (dd, J = 17.41, 10.95 Hz, 1H); 13C NMR
(CDCl3, 125 MHz) d: 149.6, 148.9, 147.4, 111.9, 111.7, 110.0, 57.4,
53.7, 52.3, 42.1, 39.7, 39.6, 33.2, 31.1, 26.7, 26.5, 25.9, 24.9, 24.0,
16.5; FTIR (KBr, cmÀ1): 3447, 3080, 2923, 2855, 1638, 1445,
1375, 1006, 906; MS (EI+) calcd for C23H39N [M+] 329, found 329.
The formation of compound 4f: A yellow oil, yield 48%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 0.99 (s, 3H), 1.41–1.48 (m, 3H),
1.53–1.64 (m, 3H), 1.70 (s, 3H), 1.82–1.88 (m, 1H), 1.93–1.98 (m,
1H), 4.53 (s, 2H), 4.57 (s, 1H), 4.59 (s, 1H), 4.83 (s, 1H), 4.88 (d,
J = 2.42 Hz, 1H), 4.91 (d, J = 2.63 Hz, 1H), 5.04 (s, 1H), 5.77 (dd,
J = 17.71, 10.52 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d: 150.4,
150.1, 147.9, 138.2, 123.0, 120.0, 113.0, 112.4, 110.9, 54.5, 53.2,
42.2, 40.4, 40.3, 33.9, 27.8, 25.5, 16.3; FTIR (KBr, cmÀ1): 3398,
3080, 2929, 2859, 1637, 1504, 1439, 1375, 1008, 905; MS (EI+)
calcd for C18H26N2 [M+] 270, found 270.
The formation of compound 3h: A colorless oil, yield 46%; 1H
NMR (CDCl3, TMS, 400 MHz), d (ppm): 1.01 (s, 3H), 1.41–1.53 (m,
3H), 1.55–1.74 (m, 3H), 1.71 (s, 3H), 1.99–2.09 (m, 2H), 3.39 (s,
3H), 3.55 (s, 4H), 4.02 (s, 2H), 4.59 (s, 1H), 4.82 (s, 1H), 4.88 (s,
1H), 4.92 (d, J = 6.35, 1H), 4.97 (s, 1H), 5.03 (s, 1H), 5.82 (dd,
J = 17.34, 10.98, 1H); 13C NMR (CDCl3, 125 MHz) d: 149.4, 148.7,
146.6, 111.1, 109.3, 107.3, 72.6, 71.0, 68.0, 58.0, 51.7, 40.2, 38.9,
38.7, 32.1, 26.1, 23.8, 15.6; FTIR (KBr, cmÀ1): 3080, 2927, 2868,
1638, 1456, 1374, 1115, 1008, 905, 734, 697; EI-HRMS: calcd for
C18H31O2 ([M+H])+, 279.2324; found: M++H, 279.2281.
3.1.3. A general procedure for the formation of b-elemene
monosubstituted amine derivatives 4
To CH3CN solution of b-elemene chloride mixture which con-
tains 13-monosubstituted chloride, 14-monosubstituted chloride
The formation of compound 4g: A yellow oil, yield 40%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 0.99 (t, J = 7.05 Hz, 6H), 1.01 (s,

Y. Sun et al. / Bioorg. Med. Chem. 17 (2009) 1118–1124
1123
3H), 1.41–1.65 (m, 6H), 1.71 (s, 3H), 2.02–2.10 (m, 2H), 2.45 (t,
J = 7.05 Hz, 4H), 2.96 (s, 2H), 4.59 (s, 1H), 4.81 (s, 1H), 4.88 (s,
2H), 4.91 (d, J = 8.94 Hz, 1H), 4.95 (s, 1H), 5.83 (dd, J = 17.44,
10.86 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d: 153.4, 151.1, 148.6,
112.6, 110.4, 108.9, 59.1, 53.4, 47.4, 42.4, 40.8, 40.5, 34.0, 28.0,
25.5, 17.3, 12.3; FTIR (KBr, cmÀ1): 3432, 3081, 2931, 2869, 1639,
1454, 1375, 1004, 906; MS (EI+) calcd for C19H33N [M+] 275, found
275.
(dt, 1J = 7.71 Hz, 2J = 1.66 Hz, 2H), 8.50 (d, J = 4.13 Hz, 2H); FTIR
(KBr, cmÀ1): 2924, 2028, 1917; EI-HRMS: calcd for C18H26N4
([M+H])+ 298.2157, found 298.2157.
Compound 6: A colorless oil, 87% yield; 1H NMR (CDCl3, TMS,
500 MHz), d: 2.64 (s, 2H), 2.76–2.85 (m, 4H), 3.48–3.68 (m, 8H),
3.87 (s, 4H), 7.12 (t, J = 6.40 Hz, 2H), 7.51 (d, J = 7.60 Hz, 2H), 7.62
(dt, 1J = 7.60 Hz, 2J = 1.60 Hz, 2H), 8.49 (d, J = 4.80 Hz, 2H); FTIR
(KBr, cmÀ1): 3458, 2987, 1999, 1858; ESI-HRMS: calcd for
C18H27N4O2 ([M+H])+ 331.2134, found 331.2123.
The formation of compound 4h: A yellow oil, yield 51%; 1H NMR
(CDCl3, TMS, 500 MHz), d (ppm): 1.00 (s, 3H), 1.17 (d, J = 6.12 Hz,
6H), 1.42–1.76 (m, 6H), 1.71 (s, 3H), 1.99–2.17 (m, 2H), 2.96–
3.05 (m, 1H), 3.36 (s, 2H), 4.36 (s, 1H, NH), 4.58 (s, 1H), 4.71 (s,
1H), 4.88 (s, 1H), 4.90–4.95 (m, 1H), 4.97 (s, 1H), 5.03 (s, 1H),
5.83 (dd, J = 18.67, 11.13 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d:
150.0, 147.5, 145.1, 114.9, 112.3, 110.2, 52.2, 50.2, 48.0, 41.9,
39.8, 39.7, 33.1, 27.1, 25.1, 19.5, 16.7; FTIR (KBr, cmÀ1): 3423,
3081, 2929, 2862, 1638, 1439, 1378, 1006, 906; MS (EI+) calcd
for C19H31N [M+] 261, found 261.
3.1.5. General procedure for the preparation of compounds 11–
13
Complexes 11–13 were prepared according to the following
general procedure: 2 mmol [N(CH2CH3)4]2[ReBr3(CO)3], and
2 mmol corresponding derivative (8–10) was dissolved in CH3OH
and stirred for 30 min. The mixture was evaporated, and dried in
a vacuum and the desired product was recrystallized with n-hex-
ane/dichloromethane = 1:2.
The formation of compound 11. A white solid, 78% yield; mp
161.7–162.1 °C; 1H NMR (CDCl3, TMS, 500 MHz), d: 0.99 (s, 3H),
1.23–1.33 (m, 2H), 1.38–1.46 (m, 2H), 1.47–1.59 (m, 3H), 1.62–
1.83 (m, 4H), 1.70 (s, 3H), 2.10–2.21 (m, 4H), 2.27–2.33 (m, 1H),
3.05 (t, J = 7.16 Hz, 2H), 3.70 (q, J = 14.00 Hz, 2H), 3.81 (t,
J = 8.08 Hz, 2H), 4.57 (s, 1H), 4.73 (d, J = 16.75 Hz, 2H), 4.81 (s,
1H), 4.87–4.89 (m, 1H), 4.91 (s, 1H), 5.26 (s, 1H), 5.45 (s, 1H),
5.59 (d, J = 17.02 Hz, 2H), 5.82 (dd, J = 17.49 Hz, 10.85, 1H), 7.23
(t, J = 6.71 Hz, 2H), 7.84 (dt, J = 7.74 Hz, 0.38, 2H), 7.93 (d,
3.1.4. General procedure for the preparation of compounds 8–
10
A solution of Cl-b-elemene, (2 mmol), 5 or Di-(2-picolyl) amine
or 6 (4 mmol), and sodium hydroxide (8 mmol) in 10 mL dry ace-
tonitrile was refluxed for 8–10 h. Then water (10 mL) was added
and the mixture was extracted with ethyl ether (4 Â 30 mL). The
combined organic extracts were dried over anhydrous sodium sul-
fate and filtered. The filtrate was concentrated in vacuo. The resi-
due was purified on a silica gel column with dichloromethane–
methanol as eluent to give a target product.
13
C
NMR
J = 7.81 Hz, 2H), 8.67 (d, J = 5.4 Hz, 2H);
(CD3OD,
125 MHz), d: 197.6, 196.7, 162.5, 153.4, 151.5, 148.9, 147.4,
141.9, 129.7, 127.1, 114.6, 113.2, 110.9, 73.2, 72.0, 71.6, 69.1,
59.4, 54.1, 52.3, 48.3, 43.9, 41.2, 41.0, 34.6, 31.0, 27.5, 27.3, 26.2,
The formation of compound 8: A brown oil, 80% yield; 1H NMR
(CDCl3, TMS, 500 MHz), d: 0.97 (s, 3H), 1.19–1.32 (m, 4H), 1.35–
1.60 (m, 8H), 1.62–1.75 (m, 2H), 1.69 (s, 3H), 1.96–2.12 (m, 2H),
2.55 (t, J = 6.88 Hz, 2H), 2.89 (t, J = 7.78 Hz, 2H), 3.61 (s, 2H), 3.84
(s, 4H), 4.55 (s, 1H), 4.80 (s, 1H), 4.87–4.94 (m, 2H), 5.15 (s, 1H),
5.17 (s, 1H), 5.79 (dd, 1J = 17.71 Hz, 2J = 10.54 Hz, 1H), 7.17 (t,
25.6, 17.4; FTIR (KBr, cmÀ1): : 3472, 2926, 2027, 1912 cmÀ1; Anal.
m
Calcd for C36H48BrN4O3ReÁCH2Cl2: C, 47.49; H, 5.39; N, 5.99. Found:
C, 47.29; H, 5.64; N, 6.26.
The formation of compound 12. A white solid, 82% yield; 1H NMR
(CDCl3, TMS, 500 MHz), d: 1.03 (s, 3 H, CH3), 1.51–1.58 (m, 3H),
1.68 (s, 3 H, CH3), 1.72–1.82 (m, 2H), 1.97–2.02 (m, 1H), 2.17–
2.19 (m, 1H), 2.30–2.39 (m, 1H), 4.36–4.49 (m, 4H), 4.61 (s, 1H),
4.85 (s, 1H), 4.91–4.99 (m, 2H), 5.53 (s, 1H), 5.62 (s, 1H), 5.77–
5.89 (q, 1H), 6.05 (q, J = 18.53 Hz, 2H), 7.18 (t, J = 6.39 Hz, 2H),
7.80 (t, J = 7.63 Hz, 2H), 8.00 (d, J = 7.64 Hz, 2H), 8.63 (d,
1
J = 5.6 Hz, 2H), 7.50 (d, J = 7.79 Hz, 2H), 7.67 (dt, J = 7.67 Hz,
2J = 1.64 Hz, 2H), 8.53 (d, J = 4.19 Hz, 2H); FTIR (KBr, cmÀ1): 2928,
2362, 1388; ESI-HRMS calcd for C33H49N4 ([M+H])+ 501.3957,
found 501.3922.
The formation of compound 9: A brown oil, 87% yield; 1H NMR
(CDCl3, TMS, 500 MHz), d: 0.90 (s, 3H), 1.47–1.27 (m, 6H), 1.61 (s,
3H), 1.92–1.89 (m, 1H), 2.07–2.04 (m, 1H), 3.06 (q,
2
H,
J = 5.34 Hz, 2H); FTIR (KBr, cmÀ1): 3080, 2027, 1910 cmÀ1 13C
;
J = 14.0 Hz), 3.73 (q, J = 14.1 Hz, 4H), 4.47 (s, 1H), 4.73 (s, 1H),
4.84 (dd, J = 5.82 Hz, 1.08 Hz, 1H), 4.85 (d, J = 1.3 Hz, 1H), 4.89 (s,
1H), 5.06 (s, 1H), 5.77 (dd, J = 10.48 Hz, 7.38 Hz, 1H), 7.09–7.07
(m,H), 7.50–7.47 (m,H), 7.60 (dt, J = 7.65 Hz, 1.57 Hz, 2H), 8.45 (d,
J = 4.3 Hz, 2H); 13C NMR (CDCl3, TMS, 125 MHz), d: 17.39, 25.31,
27.68, 33.73, 40.57, 40.74, 42.56, 53.74, 59.71, 60.80, 110.53,
111.63, 112.71, 122.62, 123.41, 137.10, 148.14, 149.61, 150.94,
152.06, 160.46; FTIR (KBr, cmÀ1): 3080, 1640; ESI-HRMS calcd for
C27H35N3 ([M+H])+ 401.2831, found 401.2842.
NMR (CDCl3, TMS, 125 MHz), d: 17.23, 25.64, 28.00, 34.65, 40.44,
40.82, 44.52, 52.92, 67.78, 67.92, 74.48, 110.86, 113.00, 121.66,
125.85, 126.36, 140.96, 147.12, 148.02, 150.49, 151.07, 161.84,
161.95, 196.22, 196.86; Anal. Calcd for C30H36BrN4O3ReÁCH2Cl2:
C, 46.99; H, 4.73; N, 7.31. Found: C, 46.63; H, 4.69; N, 7.08.
The formation of compound 13. A white solid, 75% yield; 1H NMR
(CDCl3, TMS, 500 MHz), d: 1.00 (s, 3H), 1.38–1.47 (m, 2H), 1.48–
1.61 (m, 2H), 1.74 (s, 3H), 1.65–1.93 (m, 10H), 2.08–2.31 (m, 6H),
3.01–3.09 (m, 2H), 3.62–3.74 (m, 2H), 3.76–3.85 (m, 2H), 4.58 (s,
1H), 4.73 (d, J = 16.80 Hz, 2H), 4.81 (s, 1H), 4.87 (s, 1H), 4.91 (d,
J = 8.8 Hz, 1H), 5.27 (s, 1H), 5.48 (s, 1H), 5.64 (d, J = 16.80 Hz, 2H),
5.82 (dd, J = 17.49 Hz, 10.85, 1H), 7.23 (t, J = 6.80 Hz, 2H), 7.84 (t,
J = 7.60 Hz, 2H), 7.95 (d, J = 7.60 Hz, 2H), 8.68 (d, J = 6.80 Hz, 2H);
13C NMR (CD3OD, 125 MHz), d: 197.6, 196.8, 162.5, 155.6, 153.5,
148.9, 143.2, 141.9, 127.2, 125.0, 124.9, 113.2, 113.0, 99.4, 72.1,
72.0, 69.1, 62.0, 44.0, 41.2, 41.0, 36.1, 31.1, 28.8, 27.7, 27.3, 26.6,
The formation of compound 10: A brown oil, 81% yield; 1H NMR
(CDCl3, TMS, 500 MHz), d: 0.97 (s, 3H), 1.35–1.63 (m, 6H), 1.67 (s,
3H), 1.97–2.16 (m, 2H), 2.89 (s, 2H), 3.22 (t, J = 4.32 Hz, 2H),
3.46–3.51 (m, 2H), 3.57–3.65 (m, 4H), 3.69 (s, 2H), 3.81–3.95 (m,
6H), 4.54 (s, 1H), 4.80 (s, 1H), 4.86–4.94 (m, 2H), 5.18 (s, 1H),
5.32 (s, 1H), 5.78 (dd, 1J = 17.85 Hz, 2J = 6.85 Hz, 1H), 7.17 (t,
J = 6.65 Hz, 2H), 7.38 (d, J = 7.45 Hz, 2H), 7.66 (t, J = 7.64 Hz, 2H),
8.55 (d, J = 3.97 Hz, 2H); FTIR (KBr, cmÀ1): 3371, 2928, 1639,
1591, 765; ESI-HRMS calcd for C33H49N4O2 ([M+H])+ 533.3856,
found 533.3845.
26.4, 25.6, 17.5; FTIR (KBr, cmÀ1): : 1914, 2028, 2928, 3437 cmÀ1
m .
Anal. Calcd for [C36H48N4O5Re]BrÁ1/3CH2Cl2: C, 47.89; H, 5.38; N,
6.15. Found: C, 47.80; H, 5.45; N, 6.11.
The synthesis of compound 5 and 6 according to literature.9,10
Compound 5: A colorless oil, 92% yield; 1H NMR (CDCl3, TMS,
500 MHz), d: 1.20–1.29 (m, 4H), 1.35–1.41 (m, 2H), 1.50–1.55 (m,
2H), 1.75 (s, 2H), 2.52 (t, J = 7.29, 2H), 2.62 (t, J = 7.06 Hz, 2H),
3.79 (s, 4H), 7.12 (t, J = 5.20 Hz, 2H), 7.52 (d, J = 7.80 Hz, 2H), 7.63
3.2. Cell culture and cytotoxicity essay
HeLa cell line (human uterine cervix carcinoma cells) were
obtained from Shanghai Cell Repository of Chinese Academy of

1124
Y. Sun et al. / Bioorg. Med. Chem. 17 (2009) 1118–1124
Sciences. cells were maintained in RPMI 1640(Gibico) with 10%
inactivated fetal bovine serum(Gibico), penicillin (100 IU/mL) and
15 min, and protein concentrations were determined using the
Bradford assay. Cell lysate (50 g) was separated by 12% SDS–
l
streptomycin (100
grown in logarithmic growth at 37 °C in a humidified atmosphere
consisting of 5% CO2 and 95% air.
The HeLa cells were harvested using 0.25% trypsin-EDTA and
seeded 5 Â 103 cells per well of a 96-well plate and incubated for
12 h. Then the b-elemene monosubstituted amines, ether and
PEGylation derivatives were added to wells of the plate at five dif-
ferent concentrations and continued to culture in CO2 incubator for
l
g/mL), and
L
-glutamine. The cell lines were
PAGE and transferred to PVDF membranes. Blots were incubated
with blocking buffer (0.1 M Tris–HCl, PH 7.4, 0.9% NaCl, 0.1%
Tween 20 and 5% milk) for 2 h at room temperature. Then, the
rabbit polyclonal anti-Cycin D1and anti-Rb antibody (Santa Cruz
Biotechnology, Inc.) at 1:100 dilution in 5% milk solution in TTBS
was added and incubated with PVDF for 1 h at 37 °C with con-
stant shaking. After three 15 min wash washes in TTBS, the PVDF
membranes were incubated for 1 h at 37 °C with secondary anti-
body (goat anti-rabbit biotynylated IgG) at 1:400 dilution. After
being washed three times in TTBS, PVDF membranes were incu-
bated in ABC (Boster Biological Technology. Ltd) reagent for
30 min. When the color developed, the membranes were washed
extensively with distilled water.
24 h, respectively. 10 lL WST-1[2- (4-lodophenyl)-3(4-nitro-
phenyl)-5-(2,4-disulfonphenyl)-2H-tetrazoliumÁNa] solution was
added to wells, absorbance values were taken using a 96-well
Opsys Microplate Reader at 450 nm.15 IC50 were calculated accord-
ing to absorbance.
IC50 were calculated with SPSS 11.5 Statistical software. Data
are presented as mean SD.
Acknowledgments
3.3. Cell cycle analysis
This project was supported by Hundred Talent Program of Chi-
nese Academy of Sciences (No. 26200601) and the foundation of
Innovation Project of the Chinese Academy of Sciences (No.
55200701).
HeLa cells were incubated with b-elemene monosubstituted
amines, ether, Rhenium coordinated derivatives and b-elemene
(IC20) for 24 h at 37 °C. Cells suspensions from either control cul-
tures or drug-treated cultures were prepared by trypsinization.
Approximately 1 Â 106 Cells were washed twice with cold PBS,
fixed in chilled methanol (75%) at À20 °C overnight. Then ethanol
was removed by centrifuging the cells and washing them with
PBS. The cells were then incubated in 0.1% Triton X-100-containing
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This method was used according to the method of Janecka
et al. with slightly modification.16,17 HeLa cells treated with b-
elemene, the monosubstituted amines, derivatives and the
Rhenium coordinated derivatives which were harvested by tryp-
sinization followed by 24 h incubation. After wash with ice-cold
PBS, the cells were lysed on ice in RIPA buffer, containing
10 ml/ml 200 mM phenylmethylsulfonyl fluoride (PMSF). Cellular
extracts were clarified by centrifugation at 12,000 rpm at 4 °C for
17. Peng, Yi-Chen; Kuo, Hsien-Shou; Tsai, Hsin-Da; Yang, Yu-Ping; Lin, Yuh-Ling
Bioorg. Med. Chem. 2006, 14, 263.