Synthesis and in vitro antitumor activities of lupeol dicarboxylic acid monoester derivatives

Article abstract of DOI:10.1007/s12272-013-0155-x

Ten lupeol dicarboxylic acid monoester derivatives as new potent antitumor agents were synthesized and evaluated for in vitro antitumor activities against A549, LAC, HepG2 and HeLa cell lines. Among them, compounds 1-5 showed excellent antitumor activities against all tested tumor cell lines and compounds 6-10 exhibited high activities against A549, HepG2 and HeLa cells, exceeded lupeol, lupanol and doxorubicin. Compound 2 displayed the highest potent antitumor activities with IC₅₀ values of 5.78 μM against A549 cell, 2.38 μM against LAC cell, 6.14 μM against HepG2 cell and 0.00842 μM against HeLa cell.

Full text of DOI:10.1007/s12272-013-0155-x

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0155-x
RESEARCH ARTICLE
Synthesis and in vitro antitumor activities of lupeol dicarboxylic
acid monoester derivatives
•
Weijie Li Jing Hao Yeyu Xiao
•
Received: 14 January 2013 / Accepted: 28 April 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract Ten lupeol dicarboxylic acid monoester deriva-
tives as new potent antitumor agents were synthesized and
evaluated for in vitro antitumor activities against A549,
LAC, HepG2 and HeLa cell lines. Among them, compounds
1–5 showed excellent antitumor activities against all tested
tumor cell lines and compounds 6–10 exhibited high activ-
ities against A549, HepG2 and HeLa cells, exceeded lupeol,
lupanol and doxorubicin. Compound 2 displayed the highest
potent antitumor activities with IC50 values of 5.78 lM
against A549 cell, 2.38 lM against LAC cell, 6.14 lM
against HepG2 cell and 0.00842 lM against HeLa cell.
after revealing promising antiviral and antineoplastic
agents among these compounds (Tolstikova et al. 2006). In
particular, lupeol was found to possess various pharmaco-
logical properties like antioxaluric, anticalciuric (Anand
et al. 1995), antiplasmodial (De Almeida Alves et al.
1997), anticarcinogenic, antiinflammatory, antiulcerogenic,
antimicrobial, antiviral (Dzubak et al. 2006), antioxidant,
antiurolithic and hepatoprotective properties (Chaturvedi
et al. 2008). At the same time, semisynthetic analogues of
lupeol displayed various bioactivities as well. For example,
lupeol linoleate and lupeol palmitate exhibited antiarthritic
activity (Kweifio-Okai et al. 1995) and were selective
inhibitors of serine proteases, especially, trypsin, and they
did not affect the catalytic activity of, e.g., porcine pan-
creatic elastase (Flekhter et al. 2004). Furthermore, lupeol
linoleate was more effective in reverting the abnormalities
associated with hyperoxaluria than lupeol (Sudhahar et al.
2008). Recently, 3b-O-(a-D-mannopyranosyl)-lup-20(29)-
ene was demonstrated to reveal cytotoxic activity against
all of the tumor cell lines. However, this compound also
had significant toxicity toward normal cells (BJ fibroblasts)
(Cmoch et al. 2008). Except for structural determination
and modification of lupanol as a pentacyclic triterpene, the
biological properties of it and its derivatives were seldom
described in the literatures.
Keywords Lupeol derivative ꢀ Synthesis ꢀ Antitumor
activity
Introduction
In the last decade, the real burst of interest in the phar-
macological properties of lupane derivatives was observed
Weijie Li and Yeyu Xiao were contributed equally to this study.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-013-0155-x) contains supplementary
material, which is available to authorized users.
Lupeol derivatives attracted our attention due to their
availability and significant bioactivities. Lupeol, which is
produced by many medicinal herbs, vegetables and fruits
(Chaturvedi et al. 2008; Khan et al. 2008), was the subject of
comprehensive studies. In some cases, chemical modifica-
tion of lupeol leads to interesting results: new compounds
turn out to be more effective pharmacological properties
(Srinivasan et al. 2002; Tolstikova et al. 2006). Lupeol
acetate was found to exhibit inhibitory effect on melanoma
cell growth, exceeded lupeol (Hata et al. 2002). Lupeol long-
W. Li (&)
Department of Chemistry, Hanshan Normal University,
Chaozhou 521041, China
e-mail: weijieli1688@yahoo.com.cn
J. Hao
Guangxi Botanical Garden of Medicinal Plants,
Nanning 530023, China
Y. Xiao
The Second Affiliated Hospital, College of Medicine,
Shantou University, Shantou 515041, China
123

W. Li et al.
chain fatty acid ester derivatives, namely, 3-O-(30-hydrox-
yeicosanoyl)lupeol, 3-O-[20-(tetracosyloxy)acetyl]lupeol
and 3-O-[100-hydroxyoctadecyloxy]-20-hydroxypropanoy]
lupeol, were reported to display some in vitro inhibition
activity against the chloroquine-resistant strain FCR-3 and
the chloroquine-sensitive standard strain 3D7, and the
hydroxy group of the fatty acid side chain appeared to
decrease the observed activity (Fotie et al. 2006). 3{Beta}-
O-(30,30-dimethylsuccinyl)-lupeol (1) was recorded to show
anti-HIV activity because it was capable of inhibiting HIV-1
replication in lymphocyte (Yu et al. 2006), but its antitumor
activities has been not reported in the literatures. Herein, we
synthesized a series of lupeol dicarboxylic acid monoester
derivatives as new potent anticancer agents (Fig. 1) and
evaluated their in vitro antitumor activities against A549,
LAC, HepG2 and HeLa cell lines, and reported their struc-
ture–activity relationships.
out on a Perkin-Elmer 240C elemental analyzer. All
target compounds were analyzed for C and H and gave
values within 0.4 % of the theoretical values. Optical
rotation values were measured on a Polartronic HNQW 5
polarimeter. Succinic anhydride, 2,2-dimethylsuccinic
anhydride, glutaric anhydride, 3,3-dimethylglutaric anhy-
dride and 2,2-dimethylglutaric anhydride were purchased
from Alfa Aesar. Lupeol was extracted from Chinese
Holy Leaf and lupanol was prepared through catalytic
reduction of lupeol with hydrogen in the presence of
Pd/C catalyst in our laboratory. The other reagents were
of analytical grade. All solvents were dried and distilled
prior to use.
Procedures for synthesis of lupeol dicarboxylic acid
monoester derivatives (1–10)
A mixture of lupeol (50.0 mg, 0.12 mmol) or lupanol
(50.0 mg, 0.12 mmol), dicarboxylic anhydride (0.48 mmol)
and anhydrous cyclohexane (15 mL) was refluxed for 24 h.
After cooling to room temperature, cyclohexane was
removed under reduced pressure and the residue was poured
into ice water and was acidified to pH 1 by cautiously adding
dilute hydrochloric acid (2 mol/L), and the precipitate was
filtered, washed with ice water and dried. The solid was
purified by flash column chromatography on silica gel, using
dichloromethane–methanol (50:1) or chloroform–methanol
(55:1) as eluent to obtain the desired compound.
Materials and methods
Chemistry
Melting points were determined by the capillary method
and uncorrected. 1H NMR and 13C NMR spectra were
measured on a Bruker DRX-400 NMR spectrometer at
400 MHz, using CDCl3 as solvent and TMS as internal
standard. ESI–MS were taken on a MDS Sciex API 2000
LC/GC/MS instrument. Elemental analyses were carried
Fig. 1 Chemical structures of
lupeol derivatives
R1
R1
RO
RO
O
O
O
O
HO
HO
R1=
3. R=
1. R=
4. R=
2. R=
5. R=
HO
O
O
O
O
O
O
HO
HO
HO
O
O
O
O
HO
8. R=
6. R=
9. R=
7. R=
R1=
HO
O
O
O
O
O
O
10. R=
HO
HO
123

Synthesis and in vitro antitumor activities
3{Beta}-O-(30,30-dimethylsuccinyl)-lupeol (1) Yield 96.8 %;
white solid; [a]D20 ? 32.2 (c 0.20, CH2Cl2); m.p.
192–193 °C; H NMR (400 MHz, CDCl3): d 0.78, 0.80,
3{Beta}-O-(30,30-dimethylglutaryl)-lupeol (4) Yield 75.8 %;
white solid; [a]D20 =?23.7 (c 0.40, CH2Cl2); m.p.
202–203 °C; H NMR (400 MHz, CDCl3): d 0.79, 0.84,
1
1
0.83, 0.84, 0.94, 1.02 (s, 3H each, H-23, 24, 25, 26, 27 and
28), 1.05–1.26 (m, 6H), 1.28, 1.30 (s, 3H each (CH3-
30) 9 2), 1.33–1.65 (m, 17H), 1.68 (s, 3H, H-29),
1.84–1.97 (m, 1H, H-2), 2.34–2.41 (m, 1H, H-19), 2.58 (t,
J = 16.0 Hz, 1H), 2.67 (d, J = 16.0 Hz, 1H, H-20), 4.49
(dd, J = 6.0, 10.0 Hz, 1H, H-3), 4.56 (br s, 1H, H-30b),
4.68 (d, J = 1.6 Hz, 1H, H-30a). Among the protons
described above, an active hydrogen of CO2H was substi-
tuted by deuterium. 13C NMR (400 MHz, CDCl3): d 14.53,
15.98, 16.15, 16.51, 18.00, 18.19, 19.29, 20.95, 23.62,
25.03, 25.07, 25.61, 27.43, 27.92, 29.82, 34.19, 35.57,
37.06, 37.73, 38.03, 38.39, 40.00, 40.47, 40.84, 42.82,
43.00, 44.74, 48.01, 48.29, 50.32, 55.41, 81.62, 109.37,
150.97, 170.94, 182.89; ESI–MS m/z: 553 [M-H]-; Anal.
Calcd for C36H58O4: C, 77.93; H, 10.54. Found: C, 77.98;
H, 10.57.
0.85, 0.85, 0.94, 1.03 (s, 3H each, H-23, 24, 25, 26, 27 and
28), 0.97–1.00 (m, 2H), 1.13 (s, 3H each (CH3-30) 9 2),
1.18–1.39 (m, 14H), 1.47–1.50 (m, 2H), 1.59–1.65 (m,
6H), 1.68 (s, 3H, H-29), 1.89–1.92 (m, 1H, H-2), 2.32–2.47
(m, 5H, H-19, H-20 and 40), 4.47–4.51 (m, 1H, H-3), 4.57,
4.68 (br s, 1H each, H-30). Among the protons described
above, an active hydrogen of CO2H was substituted by
deuterium. 13C NMR (400 MHz, CDCl3): d 14.49, 15.93,
16.12, 16.56, 17.97, 18.16, 19.25, 20.90, 23.74, 25.02,
27.37, 28.00, 28.05, 28.11, 29.68, 29.78, 32.79, 34.14,
35.52, 37.03, 37.64, 37.98, 38.31, 39.96, 40.79, 42.79,
42.96, 45.53, 47.97, 48.23, 50.27, 55.33, 81.66, 109.34,
150.96, 172.79, 184.86; ESI–MS m/z: 567 [M-H]-; Ana-
l.Calcd for C37H60O4: C, 78.12; H, 10.63. Found: C, 78.25;
H, 10.69.
3{Beta}-O-(40,40-dimethylglutaryl)-lupeol (5) Yield 68.6 %;
[a]2D0 =?24.2 (c 0.30, CH2Cl2); m.p. 186–187 °C; 1H NMR
(400 MHz, CDCl3): d 0.76, 0.79, 0.84, 0.85, 0.94, 1.03 (s,
3H each, H-23, 24, 25, 26, 17 and 28), 0.97–0.98 (m, 2H),
1.05–1.13 (m, 1H), 1.22 (s, 3H each (CH3-40) 9 2),
1.15–1.28 (m, 3H), 1.31–1.54 (m, 11H), 1.57–1.66 (m,
6H), 1.68 (s, 3H, H-29), 1.84–1.97 (m, 3H, H-30 and H-2),
2.31–2.35 (m, 2H, H-20), 2.39 (dt, 1H, J = 6.0, 11.2 Hz,
H-19), 4.45–4.57 (m, 1H, H-3), 4.57 (br s, 1H, H-30b), 4.69
(d, 1H, J = 2.4 Hz, H-30a). Among the protons described
above, an active hydrogen of CO2H was substituted by
deuterium. 13C NMR (400 MHz, CDCl3): d 14.51, 15.96,
16.17, 16.58, 18.00, 18.18, 18.31, 19.28, 20.92, 23.68,
24.86, 25.06, 27.42, 27.99, 29.82, 30.54, 34.18, 34.93,
35.56, 37.06, 37.86, 38.02, 38.34, 39.99, 40.83, 41.53,
42.82, 42.99, 48.00, 48.27, 50.31, 55.35, 81.06, 109.36,
150.99, 173.17, 183.17; ESI–MS m/z: 567 [M-H]-; Anal.
Calcd for C37H60O4: C, 78.12; H, 10.63. Found: C, 78.29;
H, 10.70.
3{Beta}-O-succinyl-lupeol (2) Yield 81.6 %; white solid;
[a]2D0 =?33.9 (c 0.08, CH2Cl2); m.p. 222–223 °C; 1H NMR
(400 MHz, CDCl3): d 0.78, 0.83, 0.84, 0.85, 0.94, 1.03 (s,
3H each, H-23, 24, 25, 26, 27 and 28), 0.96–1.01 (m, 2H),
1.05–1.41 (m, 13H), 1.46–1.50 (m, 2H), 1.57–1.65 (m,
6H), 1.68 (s, 3H, H-29), 1.84–1.97 (m, 1H, H-2), 2.34–2.41
(m, 1H, H-19), 2.60–2.64 (m, 2H, H-20), 2.67–2.70 (m, 2H,
H-30), 4.48–4.52 (m, 1H, H-3), 4.57, 4.68 (br s, 1H each,
H-30), 5.30 (s, 1H, OH-40); 13C NMR (400 MHz, CDCl3):
d 14.49, 15.94, 16.13, 16.48, 17.97, 18.15, 19.25, 20.91,
23.59, 25.03, 27.39, 27.86, 28.93, 29.29, 29.79, 34.15,
35.53, 37.03, 37.81, 37.99, 38.31, 39.96, 40.80, 42.79,
42.96, 47.97, 48.24, 50.28, 55.35, 81.59, 109.34, 150.96,
171.82, 177.53; ESI–MS m/z: 525 [M-H]-; Anal.Calcd for
C34H54O4: C, 77.52; H, 10.33. Found: C, 77.74; H, 10.41.
3{Beta}-O-glutaryl-lupeol (3) Yield 68.9 %; [a]2D0 =?29.7
1
(c 0.30, CH2Cl2); m.p. 212–213 °C; H NMR (400 MHz,
CDCl3): d 0.78, 0.83, 0.84, 0.85, 0.94, 1.03 (s, 3H each,
H-23, 24, 25, 26, 27 and 28), 0.97–0.98 (m, 1H), 1.08–1.42
(m, 14H), 1.46–1.50 (m, 2H), 1.57–1.65 (m, 6H), 1.68 (s,
3H, H-29), 1.86–1.91 (m, 1H, H-2), 1.92–2.00 (m, 2H,
H-30), 2.34–2.36 (m, 1H, H-19), 2.37 (t, J = 8.0 Hz, 2H,
H-20), 2.43 (t, J = 8.0 Hz, 2H, H-40), 4.47–4.51 (m, 1H,
H-3), 4.57, 4.69 (br s, 1H each, H-30). Among the protons
described above, an active hydrogen of CO2H was substi-
tuted by deuterium. 13C NMR (400 MHz, CDCl3): d 14.51,
15.96, 16.16, 16.56, 17.99, 18.18, 19.28, 19.97, 20.93,
23.73, 25.06, 27.42, 28.00, 29.81, 33.01, 33.64, 34.17,
35.55, 37.06, 37.82, 38.01, 38.33, 39.99, 40.82, 42.81,
42.99, 48.00, 48.26, 50.30, 55.34, 81.14, 109.37, 150.98,
172.67, 178.76; ESI–MS m/z: 539 [M-H]-; Anal.Calcd for
C35H56O4: C, 77.73; H, 10.44. Found: C, 77.86; H, 10.49.
3{Beta}-O-(30,30-dimethylsuccinyl)-lupanol (6) Yield 93.5 %;
white solid; [a]2D0 =?22.0 (c 0.02, CH2Cl2); m.p. 244–
245 °C; H NMR (400 MHz, CDCl3): d 0.75, 0.77, 0.81,
1
0.82, 0.92, 1.03 (s, 3H each, H-23, 24, 25, 26, 27 and 28),
0.84 (d, J = 5.2 Hz, 6H, H-29 and 30), 0.94–1.00 (m, 3H),
1.07–1.15 (m, 1H), 1.18–1.24 (m, 1H), 1.25 (s, 4H), 1.29,
1.30 (s, 3H each (CH3-30) 9 2), 1.35–1.53 (m, 8H),
1.55–1.69 (m, 8H), 1.84–1.91 (m, 1H, H-19), 2.57, 2.68 (d,
J = 15.6 Hz, 1H each, H-20), 4.46–4.52 (m, 1H, H-3), 4.99
(s, 1H, OH-40); 13C NMR (400 MHz, CDCl3): d 14.37,
15.12, 15.95, 16.05, 16.49, 18.02, 18.16, 20.90, 21.88,
22.98, 23.57, 25.00, 25.60, 26.73, 27.26, 27.89, 29.35,
34.22, 35.46, 36.97, 37.69, 37.73, 38.34, 40.36, 40.42,
40.82, 42.98, 43.10, 44.64, 44.71, 47.55, 49.92, 55.32,
123

W. Li et al.
81.60, 170.94, 182.76; ESI–MS m/z: 555 [M-H]-; Anal.
Calcd for C36H60O4: C, 77.65; H, 10.86. Found: C, 77.76;
H, 10.91.
21.88, 22.98, 23.74, 26.73, 27.26, 28.01, 28.06, 28.14,
29.35, 32.81, 34.22, 35.46, 36.99, 37.64, 37.73, 38.29,
40.36, 40.83, 42.99, 43.11, 44.64, 45.22, 45.50, 47.55,
49.93, 55.29, 81.74, 172.81, 175.32; ESI–MS, m/z: 569 [M-
H]-; Anal.Calcd for C37H62O4: C, 77.84; H, 10.95. Found:
C, 77.95; H, 10.99.
3{Beta}-O-succinyl-lupanol (7) Yield 76.6 %; white solid;
[a]2D0 =?12.5 (c 0.03, CH2Cl2); m.p. 263–264 °C; 1H NMR
(400 MHz, CDCl3): d 0.75, 0.77, 0.84, 0.86, 0.92, 1.03 (s,
3H each, H-23, 24, 25, 26, 27 and 28), 0.83 (d, J = 6.0 Hz,
6H, H-29 and 30), 0.94–1.00 (m, 3H), 1.08–1.15 (m, 1H),
1.18–1.23 (m, 1H), 1.25–1.33 (m, 4H), 1.34–1.53 (m, 8H),
1.55–1.70 (m, 8H), 1.83–1.91 (m, 1H, H-19), 2.61–2.64
(m, 2H, H-20), 2.67–2.71 (m, 2H, H-30), 4.49–4.53 (m, 1H,
H-3). Among the protons described above, an active
hydrogen of CO2H was substituted by deuterium. 13C
NMR (400 MHz, CDCl3): d 14.46, 15.12, 15.94, 16.07,
16.50, 18.02, 18.16, 20.90, 21.88, 22.98, 23.58, 26.73,
27.26, 27.86, 28.96, 29.29, 29.34, 34.22, 35.46, 36.99,
37.74, 37.81, 38.29, 40.36, 40.83, 42.99, 43.11, 44.64,
47.55, 49.93, 55.30, 81.59, 171.81, 177.70; ESI–MS m/z:
527 [M-H]-; Anal.Calcd for C34H56O4: C, 77.22; H, 10.67.
Found: C, 77.35; H, 10.71.
3{Beta}-O-(40,40-dimethylglutaryl)-lupanol (10) Yield 72.8 %;
[a]2D0 =?2.3 (c 0.20, CH2Cl2); m.p. 229–230 °C; H NMR
1
(400 MHz, CDCl3): d 0.74, 0.77, 0.84, 0.86, 0.92, 1.03 (s,
3H each, H-23, 24, 25, 26, 27 and 28), 0.83 (d, J = 7.2 Hz,
6H, H-29 and 30), 0.94–1.00 (m, 3H), 1.10–1.15 (m, 1H),
1.17–1.20 (m, 1H), 1.22 (s, 6H, (CH3-40) 9 2), 1.25–1.33
(m, 4H), 1.35–1.53 (m, 8H), 1.55–1.70 (m, 8H), 1.82–1.88
(m, 1H, H-19), 1.89–1.93 (m, 2H, H-30), 2.32–2.36 (m, 2H,
H-20), 4.43–4.50 (m, 1H, H-3). Among the protons
described above, an active hydrogen of CO2H was substi-
tuted by deuterium. 13C NMR (400 MHz, CDCl3): d 14.35,
15.12, 15.94, 16.08, 16.57, 18.02, 18.16, 20.89, 21.88,
22.98, 23.64, 24.79, 24.83, 26.74, 27.26, 27.97, 29.34,
30.51, 34.23, 34.89, 35.46, 36.99, 37.74, 37.83, 38.29,
40.36, 40.82, 41.52, 42.99, 43.11, 44.64, 47.55, 49.93,
55.27, 81.04, 173.13, 183.51; ESI–MS m/z: 569 [M-H]-;
Anal.Calcd for C37H62O4: C, 77.84; H, 10.95. Found: C,
77.97; H, 10.98.
3{Beta}-O-glutaryl-lupanol (8) Yield 82.6 %; [a]2D0 =?6.1
1
(c 0.06, CH2Cl2); m.p. 238–239 °C; H NMR (400 MHz,
CDCl3): d 0.75, 0.77, 0.84, 0.86, 0.92, 1.03 (s, 3H each,
H-23, 24, 25, 26, 27 and 28), 0.83 (d, J = 5.6 Hz, 6H,
H-29 and 30), 0.94–1.00 (m, 3H), 1.08–1.15 (m, 1H),
1.18–1.24 (m, 1H), 1.25–1.33 (m, 4H), 1.34–1.53 (m, 8H),
1.55–1.70 (m, 8H), 1.85–1.89 (m, 1H, H-19), 1.93–2.00
(m, 2H, H-30), 2.40 (t, J = 8.0 Hz, 2H, H-20), 2.44 (t,
J = 8.0 Hz, 2H, H-40), 4.48–4.52 (m, 1H, H-3). Among the
protons described above, an active hydrogen of CO2H was
substituted by deuterium. 13C NMR (400 MHz, CDCl3): d
14.34, 15.11, 15.93, 16.06, 16.54, 18.01, 18.15, 19.93,
20.87, 21.87, 22.97, 23.68, 26.72, 27.25, 27.96, 29.33,
32.97, 33.60, 34.21, 35.45, 36.97, 37.72, 37.77, 38.27,
40.34, 40.81, 42.97, 43.09, 44.62, 47.53, 49.91, 55.25,
81.11, 172.63, 178.76; ESI–MS m/z: 541 [M-H]-; Anal.
Calcd for C35H58O4: C, 77.44; H, 10.77. Found: C, 77.58;
H, 10.80.
Biological assays
A549, HepG2 and HeLa were purchased from Biomedicine
Research and Development Centre of Jinan University
(Guangzhou, China), and LAC (human lung adenocarci-
noma) was obtained from Topical Medicine Institute, Gu-
angzhou University of Chinese Medicine (Guangzhou,
China). The cell lines were grown in RPMI-1640 medium
supplemented with 10 % fetal bovine serum (FBS).
MTT assay
The cytotoxicity assay of the test samples was determined
by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as described previously with modi-
fications (Mosmann 1983). Briefly, exponentially growing
cells were seeded in 96-well culture plates and allowed to
adhere overnight. Various concentrations (100, 50, 25,
12.5, 6.5 and 3.125 lM) of the test samples was added to
the cells and incubated for the indicated time periods.
Triplicate wells were used for each experimental condition.
In addition, cells were also treated with various concen-
trations (100, 50, 25, 12.5, 6.5 and 3.125 lM) of lupeol,
lupanol and doxorubicin as the positive control for 72 h,
respectively. At the end of the incubation time, 20 lL MTT
reagents (5 mg/mL) were added to each well for 4 h, and
the resulting crystals were dissolved in DMSO. Absorbance
3{Beta}-O-(30,30-dimethylglutaryl)-lupanol (9) Yield 81.4 %;
1
[a]2D0 =?3.4 (c 0.10, CH2Cl2); m.p. 246–247 °C; H NMR
(400 MHz, CDCl3): d 0.75, 0.77, 0.82, 0.86, 0.92, 1.04 (s,
3H each, H-23, 24, 25, 26, 27 and 28), 0.85 (d, J = 7.2 Hz,
6H, H-29 and 30), 0.94–1.00 (m, 3H), 1.08–1.12 (m, 1H),
1.14 (s, 6H (CH3-30) 9 2), 1.18–1.24 (m, 1H), 1.25–1.33
(m, 4H), 1.35–1.53 (m, 8H), 1.55–1.71 (m, 8H), 1.83–1.91
(m, 1H, H-19), 2.42 (t, J = 16.0 Hz, 2H), 2.49 (t,
J = 8.0 Hz, 2H), 4.49–4.53 (m, 1H, H-3). Among the
protons described above, an active hydrogen of CO2H was
substituted by deuterium. 13C NMR (400 MHz, CDCl3): d
14.36, 15.12, 15.94, 16.05, 16.57, 18.02, 18.17, 20.89,
123

Synthesis and in vitro antitumor activities
was measured in an ELISA reader (TECAN, Austria) at a
wavelength of 570 nm. Data were calculated as a per-
centage of inhibition using the following formula:
procedures were used for the synthesis of compounds 2–10
in the yields of 68.6–93.5 % (Scheme 1).
À
Á
In vitro antitumor activity
Inhibitory ratio ð%Þ ¼ Acontrol ꢁ Asample =ðAcontrol ꢁ Ablank
ꢂ 100:
Þ
In this present study, newly prepared pentacyclic triterpene
derivatives (1–10) were evaluated for their in vitro antitumor
activities against four different human tumor cell lines in
parallel with lupeol and lupanol, and doxorubicin was used
as positive control. The results are summarized in Table 1.
First, our new results confirm all the synthetic lupeol
derivatives (1–5) exhibited high inhibitory effects against all
tested human tumor cell lines, exceeded lupeol and doxo-
rubicin. Furthermore, the experimental results also reveal
that a 30,30-dimethylsuccinyl side chain surpassed 30,30-
dimethylglutaryl at the C-3 position of lupeol derivatives
while a succinyl side chain was superior to all other side
chain. Compound 1 with 3{beta}-(30,30-dimethylsuccinyl)
apparently exhibited excellent inhibitory effects against
A549, LAC, HepG2 and HeLa cells, their IC50 were 6.38,
14.64, 12.38 and 2.16 lM respectively, exceeded compound
4 with 3{beta}-(30,30-dimethylglutaryl). Compound 2 with
3{beta}-succinyl exhibited the highest potency with IC50
values of 5.78, 2.38, 6.14 and 0.00842 lM in A549, LAC,
HepG2 and HeLa cells, respectively. In the meantime, the
methyl side chainsof dicarboxyl groups at the C-3 position of
lupeol derivatives weaken their anticancer activities. Based
on the IC50 values, the antitumor activities of compound 2
with 3{beta}-succinyl surpassed those of compound 1 with
3{beta}-(30,30-dimethylsuccinyl) in the same assay. Similar
to those of compound 2, compound 3 with 3{beta}-glutaryl
Asample, Acontrol and Ablank refer to the absorbance of the
sample, control and blank, respectively.
The anticancer activity of each sample was evaluated
and expressed as the concentration of compound that
achieved 50 % inhibition (IC50) to cancer cells. Data were
expressed as mean S.D. (standard deviation) of three
different experiments. Statistical analysis was carried out
by SPSS 12.0 software.
Results and discussion
Chemistry
3{Beta}-O-(30,30-dimethylsuccinyl)–lupeol was reported to
hold an anti-HIV activity and synthesized in middle yield
by refluxing lupeol, 2,2-dimethylsuccinic anhydride and
4-dimethylamino pyridine (DMAP) in pyridine (Yu et al.
2006). However, pyridine has an unpleasant odor and
DMAP is too expensive. To address these issues, we had
successfully developed a facile efficient method for the
synthesis of compound 1. Lupeol was refluxed with excess
2,2-dimethylsuccinic anhydride in anhydrous cyclohexane
for 24 h to give compound 1 in 96.8 % yield. Similar
Scheme 1 Conditions:
a dicarboxylic anhydride,
cyclohexane, reflux 24 h,
b acidify with dilute
R1
R1
a
b
hydrochloride to pH 1
HO
RO
O
O
O
O
HO
HO
R1=
3. R=
1. R=
4. R=
2. R=
HO
O
O
O
O
O
O
5. R=
HO
HO
HO
O
O
O
O
HO
8. R=
6. R=
9. R=
7. R=
R1=
HO
O
O
O
O
O
O
10. R=
HO
HO
123

W. Li et al.
also displayed high potencies with IC50 values of 5.89, 10.49,
11.22 and 0.37 lM against A549, LAC, HepG2 and HeLa
cell lines, respectively, exceeded compound 4 with 3{beta}-
(30,30-dimethylglutaryl) and compound 5 with 3{beta}-
(40,40-dimethylglutaryl).
Our results also indicate that a terminal double bond
between C-20 and C-30 at lupeol scaffold had a significant
effect on its antitumor activities. Except for HepG2 cell,
lupeol was effective in reducing viability against A549,
LAC and HeLa cells. Lupanol scaffold has no double bond
between C-20 and C-30 in compare with lupeol, however it
had not any inhibitory activity against all the tested human
tumor cell lines. Interestingly, lupanol derivatives 6–10
exhibited strong antitumor activities in A549, HepG2 and
HeLa cells, exceeded lupanol and doxorubicin. Of lupanol
derivatives, only compounds 7 and 8 were effective to
inhibit LAC cell growth. The results also show that di-
carboxyl groups at the C-3 position of lupanol derivatives
could greatly increase their antitumor activities.
In conclusion, a facile method for the synthesis of lupeol
dicarboxylic acid monoester derivatives (1–10) as new
potent antitumor agents was described, and the in vitro
antitumor activities and the structure–activity relationship
for these derivatives were evaluated. Based on the above
structure–activity relationship results, the following con-
clusions can be drawn: (1) The terminal double bond
between C-20 and C-30 in lupeol derivatives plays an
important role in their anticancer activities. (2) The di-
carboxyl group at the C-3 position helps to strengthen the
antitumor activities of lupeol derivatives; However, the
methyl side chains of dicarboxyl group at the C-3 position
weaken their activities. (3) Of lupeol derivatives, com-
pound 2 with 3{beta}-succinyl was identified as the most
promising candidate for the antitumor activities.
Acknowledgments The study was supported by grants from the
Natural Science Foundation of Hanshan Normal University (No.
QD20110616) and the Basic and Clinical Research Foundation of
Medical College of Shantou University (No. LD030601).
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