Synthesis, characterization, and in vitro antiproliferative activity of novel β-elemene monosubstituted derivatives

Article abstract of DOI:10.1007/s00044-013-0615-3

A series of β-elemene monosubstituted ester, carbamate, acylamide, and carbamidine derivatives were synthesized via intermediates, β-elemene alcohol and β-elemene amine, which were synthesized from the traditional Chinese medicine, β-elemene. The structur

Full text of DOI:10.1007/s00044-013-0615-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3536–3540
DOI 10.1007/s00044-013-0615-3
ORIGINAL RESEARCH
Synthesis, characterization, and in vitro antiproliferative activity
of novel b-elemene monosubstituted derivatives
•
Guifeng Liu Zhenwu Kong Yumei Shen
•
Received: 22 December 2012 / Accepted: 3 May 2013 / Published online: 16 May 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of b-elemene monosubstituted ester,
carbamate, acylamide, and carbamidine derivatives were
synthesized via intermediates, b-elemene alcohol and
b-elemene amine, which were synthesized from the tradi-
tional Chinese medicine, b-elemene. The structures of all
the new compounds were characterized by NMR, IR, and
HRMS. Their in vitro antiproliferative activities on HeLa
cell line were tested through the WST-1 assay. The results
show that the in vitro antiproliferative activities of the
novel compounds are improved compared to that of the
parent b-elemene.
et al., 1983), is the main effective monomer of elemene
emulsion, and the chemical structure are shown in Fig. 1.
b-Elemene has many advantages when it is used as
anticancer drug: it has a broad-spectrum antitumor effect in
many types of cancer (lung, breast, prostate, cervical,
gastric, ovarian, bladder cancer, and osteosarcoma) without
severe side effects (Li et al., 2005, 2010, 2013; Wang et al.,
2005; Liu et al., 2011; Liang et al., 2012; Zhang et al.,
2012). No bone marrow suppression and drug resistance
have been observed in the clinical studies, as well as
patient immunity was improved during the therapy with
b-elemene (Peng et al., 2006). Furthermore, it has low
toxicity and is therefore well-tolerated and accepted by
patients.
Keywords b-Elemene Á Carbamate Á Acylamide Á
Carbamidine Á Antiproliferative activity
However, b-elemene suffers from limited bioavailability
due to poor water solubility, short half life, and rapid
clearance from the body (Cheng et al., 2008). As we know,
the water solubility of compounds could be improved by
adding polar groups such as an ether, ester, amide, and so
on into the molecular structure. Although many b-elemene
derivatives with better properties have already been syn-
thesized (Jia et al., 1991; Peng et al., 2006; Xu et al., 2006;
Ren et al., 2009; Sun et al., 2009), b-elemene derivatives
with carbamate, acylamide, and carbamidine groups have
not been reported. In this study, a series of novel b-elem-
ene-monosubstituted ester, carbamate, acylamide, and
carbamidine derivatives were synthesized from intermedi-
ates b-elemene amine and b-elemene alcohol at ambient
temperature. And by controlling the reaction conditions,
we prepared b-elemene 13-monosubstituted derivatives as
the major product. The in vitro antiproliferative activities
of pure b-elemene and its derivatives over HeLa cell lines
were evaluated through the WST-1 assay. To our pleasure
that some of the derivatives has much higher antiprolifer-
ative activities compared to that of parent b-elemene.
Introduction
b-Elemene, (5S,7R,10S)-(-)-(1-methyl-1-vinyl-2,4-diiso-
propenyl-cyclohexane), a natural sesquiterpene extracted
from the traditional Chinese drugs curcuma wenyujin (Guo
G. Liu Á Z. Kong
Institute of Chemical Industry of Forest Products, CAF,
Nanjing 210042, China
G. Liu Á Z. Kong
Institute of New Technology of Forestry, CAF,
Beijing 100091, China
Y. Shen (&)
Shanghai Center for Systems Biomedicine, Key Laboratory
of Systems Biomedicine (Ministry of Education), Shanghai Jiao
Tong University, Shanghai 200240, China
e-mail: yumeishen@sjtu.edu.cn
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Med Chem Res (2013) 22:3536–3540
3537
Fig. 1 Chemical structure of b-
elemene
A general procedure for the formation of b-elemene
amine 4
A b-elemene chlorination mixture (0.78 g) containing
chlorinated b-elemene (1.6 mmol) was dissolved in 5-mL
CH3CN, and then KI (78 mg) was added to the solution.
Liquid ammonia was introduced into the autoclave until the
pressure is up to 4 atm. The reaction mixture was stirred at
60 °C for 24 h. Purification was accomplished by chro-
matograph to give b-elemene amine 4 (70 mg, 19.4 %).
1H NMR (CDCl3, TMS, 500 MHz): d 1.00 (s, 3H, CH3),
1.32–1.77(m, 6H), 1.71 (s, 3H, CH3), 2.04–2.18 (m, 2H),
3.62 (s, 2H), 4.58 (s, 1H), 4.83 (s, 1H), 4.89 (s, 1H), 4.92
(d, J = 4.66, 1H), 5.15 (s, 1H), 5.23 (s, 1H), 5.81 (dd,
J = 17.70, 10.65, 1H). IR (cm-1): 3390 (N–H), 3081
(=CH), 2976, 2867 (CH), 1642 (C=C), 1442, 1375 (CH),
909 (CH). HRMS (m/z): 219.17.
14
13
Experimental
b-Elemene was obtained from WenTe Research Institute of
Oleum Curcumae Wenchowensis in Yue Qing city, Zhe-
jiang province (purity 98 %). Other materials were pur-
chased from Fluka Co. and Sinopharm Chemical Reagent
Co. Ltds. The NMR data were obtained using a Bruker
DRX 500 MHz FT spectrometer. The chemical 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 anal-
ysis was performed using a Perkin-Elmer Series III
analyzer.
A general procedure for the formation of b-elemene
carbamate and ester 5
A mixture of b-elemene alcohol 3 (50 mg, 0.23 mmol) and
isocyanate (0.5 mmol) was dissolved in 10-mL toluene and
stirred at 67 °C for 10 h. Concentrated in vacuo to give the
mixture as yellow oil, purification was accomplished by
chromatograph to give compounds 5.
A general procedure for the formation of 13-chloro-b-
elemene 2
The formation of compound 5a. A colorless oil, yield
58 %;
b-Elemene in acetic acid was cooled to 0 °C, and sodium
hypochlorite was added over a 4 h period, 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 give the mixture as yellow oil
used in the next step directly without further purification.
1H NMR (CD3OD, TMS, 400 MHz): d 1.02 (s, 3H),
1.51–1.77 (m, 6H), 1.71 (s, 3H), 2.01–2.16 (m, 2H), 4.60
(s, 1H), 4.84 (s, 2H), 4.84 (s, 1H), 4.89 (s, 1H), 4.92 (d,
J = 6.63, 1H), 5.07 (s, 1H), 5.16 (s, 1H), 5.82 (dd,
J = 17.69, 11.07, 1H), 7.45 (t, J = 7.64, 2H), 7.57 (t,
J = 6.55, 1H). IR (cm-1): 3321 (N–H), 3081 (=CH), 2929,
2865 (CH), 1701 (C=O), 1643 (C=C), 1463, 1374 (CH),
907(CH). HRMS (m/z): 324.21.
A general procedure for the formation of b-elemene
alcohol 3
The formation of compound 5b. A colorless oil, yield
65 %;
A chlorinated b-elemene mixture (1.2 g) containing chlo-
rinated b-elemene (2.5 mmol) was dissolved in dry
CH2Cl2, then anhydrous CH3COONa (0.82 g, 10 mmol),
HMPT (1.0 mL), and KI (0.76 g, 4.5 mmol) were added to
the solution. The reaction mixture was stirred at 95 °C for
12 h, then extracted with hexane, dried over anhydrous
Na2SO4, filtered, and evaporated. Purification was accom-
plished by chromatograph to give b-elemene acetate, which
was hydrolyzed to give b-elemene alcohol (0.33 g, 60 %).
1H NMR (CDCl3, TMS, 500 MHz): d 1.06 (s, 3H, CH3),
1.34–1.66 (m, 6H), 1.73 (s, 3H, CH3), 2.01–2.11 (m, 2H),
4.14 (s, 2H), 4.59 (s, 1H), 4.82 (s, 1H), 4.91 (s, 1H), 4.92(d,
J = 5.55, 1H), 4.94 (s, 1H), 5.05 (s, 1H), 5.82 (dd,
J = 17.12, 11.18, 1H). IR (cm-1): 3334 (O–H), 3081
(=CH), 2968, 2858 (CH), 1638 (C=C), 1440, 1375 (CH),
910 (CH). HRMS (m/z): 220.20.
1H NMR (CDCl3, TMS, 500 MHz): d 0.93 (t, J = 7.34,
3H), 1.01 (s, 3H), 1.31–1.38 (m, 2H), 1.42–1.53 (m, 5H),
1.55–1.62 (m, 2H), 1.65–1.75 (m, 2H), 1.71 (s, 3H),
1.99–2.05 (m, 1H), 3.19 (t, J = 6.32, 2H), 4.57 (s, 2H),
4.58 (s, 1H), 4.82 (s, 1H), 4.89 (s, 1H), 4.92 (d, J = 5.29,
1H), 4.98 (s, 1H), 5.04 (s, 1H), 5.82 (dd, J = 17.96, 10.48,
1H); IR (cm-1): 3338 (N–H), 3081 (=CH), 2931, 2863
(CH), 1702 (C=O), 1643 (C=C), 1463, 1375 (CH), 907
(CH). HRMS (m/z): 319.25.
The formation of compound 5c. A colorless oil, yield
70 %;
1H NMR (CDCl3, TMS, 500 MHz): d 1.01 (s, 3H), 1.16
(d, J = 6.51, 6H), 1.44–1.74 (m, 6H), 1.68 (s, 3H),
1.99–2.02 (m, 2H), 3.80–4.02 (m, 1H), 4.56 (s, 1H), 4.58
(s, 2H), 4.82–5.04 (m, 5H), 5.81 (dd, J = 18.00, 11.25,
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Med Chem Res (2013) 22:3536–3540
1H). IR (cm-1): 3329 (N–H), 3081 (=CH), 2931, 2863
(CH), 1701 (C=O), 1643 (C=C), 1457, 1368 (CH), 907
(CH). HRMS (m/z): 305.23.
The antiproliferative evaluation by WST-1 method
The antiproliferative effect of the monosubstituted ether
derivatives of b-elemene in human HeLa cervix carcinoma
cell (ATCC CCL-2) was evaluated by WST-1 method
(Beyotime). HeLa cells were maintained in RPMI 1640
(GIBCO) with 10 % inactivated fetal bovine serum (GIB-
CO). The cell lines were 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 %
The formation of compound 5d. A colorless oil, yield
67 %;
1H NMR (CDCl3, TMS, 500 MHz): d 1.01 (s, 3H),
1.44–1.52 (m, 3H), 1.58–1.66 (m, 3H), 1.71 (s, 3H),
1.69–1.75 (m, 1H), 2.00–2.09 (m, 1H), 4.59 (s, 1H), 4.68
(s, 2H), 4.83 (s, 1H), 4.89 (s, 1H), 4.92 (d, J = 6.14, 1H),
5.03 (s, 1H), 5.10 (s, 1H), 5.82 (dd, J = 17.86, 10.41, 1H),
6.70 (s, 1H, NH), 7.05–7.08 (m, 1H), 7.29–7.32 (m, 2H),
7.37–7.39 (m, 2H). IR (cm-1): 3319 (N–H), 3081 (=CH),
2929, 2859 (CH), 1708 (C=O), 1640 (C=C), 1444, 1375
(CH), 907 (CH). HRMS (m/z): 339.21.
NaClO
CH3COOH
The formation of compound 5e. A colorless oil, yield
74 %;
1H NMR (CDCl3, TMS, 500 MHz): d 1.01 (s, 3H),
1.09–1.20 (m, 3H), 1.30–1.40 (m, 2H), 1.42–1.51 (m, 3H),
1.56–1.62 (m, 3H), 1.66–1.76 (m, 3H), 1.71 (s, 3H),
1.90–1.98 (m, 2H), 1.99–2.07 (m, 2H), 3.45–3.55 (m, 1H),
4.55 (s, 1H), 4.58 (s, 2H), 4.82 (s, 1H), 4.89 (s, 1H), 4.92
(d, J = 5.35, 1H), 4.98 (s, 1H), 5.04 (s, 1H), 5.82 (dd,
J = 17.94, 10.44, 1H). IR (cm-1): 3332 (N–H), 3081
(=CH), 2931, 2855 (CH), 1705 (C=O), 1643 (C=C), 1451,
1374 (CH), 907 (CH). HRMS (m/z): 345.26.
CH2Cl
2
1
Scheme 1 Synthesis of b-elemene chloride 2
NaOH
A general procedure for the formation of b-elemene
acylamide and carbamide 6
CH2OH
3
CH2OOCCH3
NaAc
HMPT
A mixture of b-elemene amine 4 (1.3 g) and excess iso-
cyanate was dissolved in 10-mL toluene and stirred at room
temperature for 3 h. Concentrated in vacuo to give the
mixture as yellow oil, purification was accomplished by
chromatograph to give 6.
NH3
KI
CH2NH2
4
CH2Cl
2
The formation of compound 6a. A colorless oil, yield
58 %;
Scheme 2 Synthesis of the intermediate b-elemene alcohol 3 and
b-elemene amine 4
1H NMR (CDCl3, TMS, 500 MHz): d 1.00 (s, 3H),
1.40–1.66 (m, 6H), 1.71 (s, 3H), 2.04–2.27 (m, 2H), 3.62
(s, 2H), 4.58 (s, 1H), 4.83 (s, 1H), 4.89 (s, 1H), 4.92 (d,
J = 4.66, 1H), 5.16 (s, 1H), 5.23 (s, 1H), 5.81 (dd,
J = 17.7, 10.65, 1H), 7.31–7.45 (m, 5H). IR (cm-1): 3339
(N–H), 3071 (=CH), 2924, 2852 (CH), 1685 (C=O), 1641
(C=C), 1452, 1383 (CH), 907 (CH). HRMS (m/z): 323.20.
The formation of compound 6b. A colorless oil, yield
58 %;
Table 1 The antiproliferative activity of b-elemene monosubstituted
derivatives over HeLa cell lines
Compounds
IC50 (lmol L-1
)
b-elemene
336.2 7.2
105.9 14.4
5.90 3.4
3
4
1H NMR (CDCl3, TMS, 500 MHz): d 1.01 (s, 3H), 1.16
(d, J = 6.39, 6H), 1.45–1.52 (m, 4H), 1.57–1.75 (m, 3H),
1.70 (s, 3H), 1.93–2.03 (m, 1H), 3.89–4.00 (m, 1H), 4.40
(d, J = 3.34, 2H), 4.58 (s, 1H), 4.83 (s, 1H), 4.89 (s, 1H),
4.91–4.95 (m, 2H), 5.05 (s, 1H), 5.81 (dd, J = 17.74,
10.56, 1H). IR (cm-1): 3418 (N–H), 3081 (=CH), 2930,
2868 (CH), 1646 (C=C), 1456, 1367 (CH), 906 (CH).
HRMS (m/z): 305.24.
5a
5b
5c
5d
5e
6a
6b
529.9 7.0
529.7 7.7
280.8 6.5
264.1 6.7
318.0 15.0
219.0 12.4
364.6 16.5
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Med Chem Res (2013) 22:3536–3540
3539
5 (a-e)
6 (a-b)
R' = PhC(O); i-PrNHC(O)
CH2OH
3
CH2OR
CH2NHR'
CH2NH2
4
R = PhC(O); BuNHC(O); i-PrNHC(O); PhNHC(O); CyNHC(O)
Scheme 3 Synthesis of b-elemene ester, carbamate, acylamide, and carbamidine derivatives
trypsin–EDTA (Beyotime) and seeded 5 9 103 cells in
each well of a 96-well plate and incubated for 12 h. The
b-elemene derivatives were added to wells of the plate at
different concentrations (500, 250, 125, 62.5, 31.2 lg/mL)
and continued to culture in CO2 incubator for 48 h. WST-1
solution (10 lL) was added to wells absorbance values
were got using a 96-well Opsys Microplate Reader (BIO-
RAD) at 450 nm. IC50 were calculated with SPSS 11.5
statistical software according to the absorbance values. The
differences between mean values were analyzed with stu-
dents’ tests. Differences were considered significant when
p \ 0.05. Data are presented as mean SD.
The reactions of intermediate 3 with benzoyl chloride
and several isocyanates at room temperature forming the
ester and carbamate derivatives. The products are purified
by chromatograph. Intermediate 4 is more reactive than
intermediate 3, to produce less byproduct, the reactions of
4 with benzoylchloride and i-PrNCO at 0 °C. We tested the
pure b-elemene and its derivatives antiproliferative activity
in vitro by the WST-1 method. Results show that the
b-elemene derivatives were synthesized from 13-mono-
substituted chlorinated b-elemene with higher proliferative
activity over HeLa cell lines than that of the mother
b-elemene (Table 1).
Results and discussion
Conclusion
The novel derivatives were synthesized from chlorination
of b-elemene according to the procedure outlined in
Scheme 1. In order to make the 13-monosubstituted chlo-
rinated b-elemene as the absolutely major product, we
optimized the reaction conditions and found that the
reaction temperature should be kept below 0 °C, NaClO
should be fresh and its mole ratio to b-elemene should be
kept between 1.1 and 1.2, the mixture of products includes
little 14-monosubstituted and disubstituted chlorinated
b-elemene derivatives. Finally, the chlorinated b-elemene
mixture was used directly without further purification.
The b-elemene monosubstituted alcohol 3 was synthe-
sized according to literature (Jia et al., 1991) with a little
modification. The chlorinated b-elemene reacts with Ac2O
promoted by HMPT to give the ester at room temperature,
followed by hydroxylation under basic conditions to yield
the important intermediate 3. For the intermediate 4, it is
the first time to be synthesized successfully. At first, we
attempted to prepare it by the reaction of compound 2 with
saturated ammonia hydroxide under reflux conditions,
unfortunately, no product was identified. To improve the
reaction’s activity, liquid ammonia was employed instead
of ammonia hydroxide in autoclave under basic conditions.
The expected reaction took place efficiently. Purification
was accomplished by chromatograph to give compound 4
in good yield (Scheme 2).
In summary, a series of novel b-elemene monosubstituted
ester, carbamate, acylamide, and carbamidine derivatives
were synthesized via intermediates b-elemene alcohol and
b-elemene amine at ambient temperature, especially,
intermediate b-elemene amine is the first time to be syn-
thesised successfully (Scheme 3). The monosubstituted
derivatives in vitro antiproliferative activities over HeLa
cell lines were tested by WST-1 method and some of their
activities were improved compared to pure b-elemene.
Acknowledgments The authors gratefully acknowledge the finan-
cial supports provided by the National Natural Science Foundation of
China (81071250), Major Projects in National Science and Technol-
ogy, ‘‘Creation of major new drugs’’ (2011ZX09501-001-01), Insti-
tute of New Technology of Chinese Academy of Forestry (Special
Fund for Fundamental Research) (CAFINT2012C04).
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