Anticancer activity of 3-O-acylated betulinic acid derivatives obtained by enzymatic synthesis

Article abstract of DOI:10.1271/bbb.90917

An easy and efficient strategy to prepare betulinic acid esters with various anhydrides was used by the enzymatic synthesis method. It involves lipase-catalyzed acylation of betulinic acid with anhydrides as acylating agents in organic solvent. Lipase from Candida antarctica immobilized on an acrylic resin (Novozym 435) was employed as a biocatalyst. Several 3-O-acyl-betulinic acid derivatives were successfully obtained by this procedure. The anticancer activity of betulinic acid and its 3-O-acylated derivatives were then evaluated in vitro against human lung carcinoma (A549) and human ovarian (CAOV3) cancer cell lines. 3-O-glutarylbetulinic acid, 3-O-acetyl-betulinic acid, and 3-O-succinyl-betulinic acid showed IC₅₀ < 10 μg/ml against A549 cancer cell line tested and showed better cytotoxicity than betulinic acid. In an ovarian cancer cell line, all betulinic acid derivatives prepared showed weaker cytotoxicity than betulinic acid.

Full text of DOI:10.1271/bbb.90917

Biosci. Biotechnol. Biochem., 74 (5), 1025–1029, 2010
Anticancer Activity of 3-O-Acylated Betulinic Acid Derivatives Obtained
by Enzymatic Synthesis
y
Faujan Bin H. AHMAD,1; Mansour GHAFFARI MOGHADDAM,1
1;2
Mahiran BASRI,1 and Mohd Basyaruddin ABDUL RAHMAN
1Department of Chemistry, Faculty of Science, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
2Structural Biology Research Center, Malaysia Genomic Institute, MTDC-UKM Smart Technology Center,
UKM Bangi, 43600 Bangi, Selangor, Malaysia
Received December 10, 2009; Accepted January 31, 2010; Online Publication, May 7, 2010
[doi:10.1271/bbb.90917]
An easy and efficient strategy to prepare betulinic
acid esters with various anhydrides was used by the
enzymatic synthesis method. It involves lipase-catalyzed
acylation of betulinic acid with anhydrides as acylating
agents in organic solvent. Lipase from Candida antarc-
tica immobilized on an acrylic resin (Novozym 435) was
employed as a biocatalyst. Several 3-O-acyl-betulinic
acid derivatives were successfully obtained by this
procedure. The anticancer activity of betulinic acid
and its 3-O-acylated derivatives were then evaluated
in vitro against human lung carcinoma (A549) and
human ovarian (CAOV3) cancer cell lines. 3-O-glutaryl-
betulinic acid, 3-O-acetyl-betulinic acid, and 3-O-
succinyl-betulinic acid showed IC50 < 10 ꢀg/ml against
A549 cancer cell line tested and showed better cytotox-
icity than betulinic acid. In an ovarian cancer cell line,
all betulinic acid derivatives prepared showed weaker
cytotoxicity than betulinic acid.
it can be carried out under mild reaction conditions, high
selectivity, and product purity.14,15) Study of the enzy-
matic acylation of betulinic acid was initiated in our
laboratory for the synthesis of 3-O-acetyl-betulinic acid
using Novozym 435 (Candida antarctica lipase), giving
the expected product in 85% yield.16) Recently, the
synthesis of 3-O-benzoyl-betulinic acid using Candida
antarctica lipase as biocatalyst in an organic solvent was
reported by Yasin et al.17) giving betulinic acid ester at
48.5% yield using benzoyl chloride as acylating agent.
We herein report the enzymatic synthesis of several
3-O-acyl-betulinic acid derivatives (2–11, Scheme 1)
using various anhydrides and Candida antarctica lipase
(Novozym 435) as the catalyst in organic solvent. The
cytotoxicity of the synthesized compounds was evalu-
ated on human lung carcinoma (A549) and human
ovarian (CAOV3) cancer cell lines.
Materials and Methods
Key words: enzymatic synthesis; Novozym 435;
3-O-acyl-betulinic acid; betulinic acid;
anticancer agents
Enzyme. Immobilized lipase (triacylglycerol hydrolase, EC 3.1.1.3;
Novozym 435, 10000 PLU/g) from Candida antarctica, supported on
a macroporous acrylic resin with a water content of 3% (w/w) was
purchased from Novo Nordisk A/S (Bagsvaerd, Denmark).
Betulinic acid (1) (3ꢀ-hydroxy-lup-20(29)-en-28-oic
acid), pentacyclic lupane triterpene, is a known natural
product which possess several pharmacological activ-
ities, including inhibition of human immunodeficiency
virus (HIV), anti-bacterial, anti-malarial, anti-inflamma-
tory, anthelmintic, antioxidant, and anticancer proper-
ties.1) It has been identified as a highly selective growth
inhibitor against human melanoma,2,3) neuroectoder-
mal,4) and malignant5) tumor cells and was reported to
induce apoptosis in these cells.1) Nevertheless, further
clinical development of betulinic acid in the pharma-
ceutical industry is strongly hampered because of its
poor hydrosolubility and pharmacokinetic properties
(absorption, distribution, metabolism, and elimination).6)
Thus, much work has been focused on modification
of betulinic acid at the C-3 and/or C-28 positions in
order to increase its hydrosolubility and biological
activity.7–10) Methods for the synthesis of 3-O-acyl-
betulinic acid based on chemical catalytic esterification
have been described.10–13) However, the application of
enzymes in organic synthesis provides advantages, since
Chemicals. Chloroform and n-hexane (Fisher Chemical,
Loughborough, UK) were used as the organic solvents. Betulinic acid
was isolated from Malaysian Callistemon speciosus by a previous
method.18) Phthalic anhydride, 3-methyl phthalic anhydride, succinic
anhydride, maleic anhydride, glutaric anhydride, 3,3-dimethyl glutaric
anhydride, acetic anhydride, butyric anhydride, isobutyric anhydride,
and valeric anhydride were purchased from Acros Organics (Geel,
Belgium). Ethyl acetate, dimethyl sulfoxide (DMSO), celiteꢀ545,
Na2SO4, K2CO3, and HCl were from Merck (Darmstadt, Germany).
MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) and RPMI 1640 medium were from Sigma (St. Louis, MO).
Fetal bovine serum was from BioWhittaker Inc. (Walkersville, USA).
All chemicals were of analytical reagent grade.
Cell lines. Cell lines A549 and CAOV3 were purchased from
American Type Culture Collection (ATCC, Manassas, VA, USA).
The cells were grown and maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum at 37 ꢀC, 5% CO2, and
90% humidity.
Analytical methods. The progress of the reactions for the synthesis
of 3-O-acyl-betulinic acid derivatives was monitored by thin layer
y
To whom correspondence should be addressed. Tel: +603-89466784; Fax: +603-89435380; E-mail: faujan@fsas.upm.edu.my

1026
F. B. H. AHMAD et al.
Scheme 1. Esterification of Betulinic Acid with Various Anhydrides by Lipase as Biocatalyst in Organic Solvent.
chromatography (TLC) on silica gel plates eluted with a mixture of
n-hexane/ethyl acetate (9:1, v/v). The plates were visualized under a
UV lamp and/or iodine vapor. NMR spectra were recorded with a
Varian Unity Inova 500 NMR spectrometer operating at a resonance
frequency of 499.89 MHz for the 1H-NMR spectra and 125.71 MHz for
the 13C-NMR spectra. The products were also analyzed by Fourier
Transform Infrared (FT-IR) spectroscopy (Perkin Elmer, Spectrum 100
FTIR, MA, USA). The mass spectra of the products were recorded
using a gas chromatograph-mass spectrometer (Thermo Finnigan,
Trace GC Polaris Q, MA, USA). The products were also analyzed
using CHN analyzer (Leco, CHNS-932, MI, USA). The melting points
of purified products were determined using the melting point equip-
ment (Barnstead Electrothermal, ESSEX, UK).
395, 248, 220, 203, 189. Anal. Calcd. for C38H52O6: C, 75.39; H, 8.60.
Found: C, 75.69; H, 8.61.
3-O-(30-Methyl phthalyl)-betulinic acid (3). Starting with 3-methyl
phthalic anhydride (59.0 mg); after crystallization from CH3CN–H2O
gave colorless needles (101 mg, 49.7%); mp 238–241 ꢀC. IR ꢁmax
(CHCl3) cmꢁ1: 3468 (OH of COOH), 1722 (C=O ester), 1688 (C=O
acid), 1644 (C=C), 1600 and 1580 (C=C aromatic), 1290 (C–O).
1H NMR (CDCl3, 500 MHz): ꢂ 0.76, 0.83, 0.94, 0.97, 0.98, 1.69, 2.46
(each 3H, s, 7 ꢂ CH3), 3.00 (1H, m), 4.50 (1H, dd, J ¼ 4:5, 12.0 Hz),
4.61 and 4.75 (each 1H, br s), 7.47 (1H, d, J ¼ 7:5 Hz), 7.41 (1H, t,
J ¼ 8:0 Hz), 7.91 (1H, d, J ¼ 8:0 Hz). 13C NMR (CDCl3, 125 MHz):
ꢂ 14.46 (C-27), 15.56 (C-24), 16.36 (C-25), 16.41 (C-26), 17.96 (C-6),
18.53 (C-30), 19.50 (30-CH3), 21.10 (C-11), 25.75 (C-12), 27.63 (C-2),
28.22 (C-23), 29.66 (C-21), 30.78 (C-15), 32.38 (C-16), 34.57 (C-7),
37.27 (C-10), 37.44 (C-22), 38.61 (C-13), 38.96 (C-4), 39.06 (C-1),
40.94 (C-8), 42.66 (C-14), 47.10 (C-18), 49.50 (C-19), 50.77 (C-9),
55.58 (C-5), 56.70 (C-17), 79.25 (C-3), 109.96 (C-29), 123.51 (C-40),
123.76 (C-60), 131.87 (C-20), 135.68 (C-50), 138.11 (C-10), 140.69
(C-30), 150.62 (C-20), 165.35 (C=O ester), 169.30 (20-COOH), 181.55
(C-28, COOH). MS m=z: 618 (Mþ), 456, 438, 423, 395, 248, 220, 203,
189. Anal. Calcd. for C39H54O6: C, 75.62; H, 8.73. Found: C, 75.89;
H, 8.90.
General procedure for enzymatic synthesis of 3-O-acyl-betulinic
acid derivatives. Ester synthesis was performed in a 150 ml glass-
stoppered round bottom flask. To a magnetically stirred solution of
betulinic acid (150 mg, 0.328 mmol), acid anhydride (0.364 mmol),
celiteꢀ545 (1.0 g), K2CO3 (36.0 mg, 0.260 mmol), chloroform (50 ml)
and n-hexane (50 ml) was added Novozym 435 (0.873 g). The reaction
mixture was magnetically stirred at 54 ꢀC for 20 h at 150 rpm. After
20 h of reaction, the enzyme was removed by filtration and washed
twice with chloroform. The filtrate was evaporated and ethyl acetate
was then added and the mixture was washed twice with aqueous
solution of HCl and twice with water. The organic layer was dried over
Na2SO4 and concentrated under reduced pressure. The residue was
chromatographed on silica gel 60 (n-hexane/ethyl acetate, 9:1–5:1,
v/v) as eluent. The structures of the products were analyzed using
1H-NMR, 13C-NMR, FT-IR, CHN analyzer and MS spectroscopy.
3-O-Glutaryl-betulinic acid (4). Starting with glutaric anhydride
(41.5 mg); after crystallization from MeOH–H2O gave colorless
needles (87 mg, 46.4%); mp 273–275 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:
2500–300 (OH of COOH), 1726 (C=O ester), 1687 (C=O acid), 1644
(C=C), 1237, 1190 and 1142 (C–O). 1H NMR (CDCl3, 500 MHz):
ꢂ 0.76, 0.83, 0.94, 0.98, 0.97, 1.65 (each 3H, s, 6 ꢂ CH3), 2.99 (1H, m),
4.50 (1H, dd, J ¼ 4:0, 11.5 Hz), 4.61 and 4.74 (each 1H, br s), 2.37
(2H, d, J ¼ 7:0 Hz), 2.53 (2H, t, J ¼ 7:5 Hz), 2.57 (2H, t, J ¼ 7:5 Hz).
13C NMR (CDCl3, 125 MHz): ꢂ 14.88 (C-27), 16.26 (C-26), 16.56
(C-24), 16.69 (C-25), 18.52 (C-6), 19.92 (C-30), 19.92 (C-30), 21.04
(C-11), 25.68 (C-12), 27.59 (C-2), 28.17 (C23), 29.90 (C-21), 30.67
(C-15), 32.26 (C-16), 32.26 (C-40), 33.18 (C-20), 34.57 (C-7), 37.43
(C-10), 38.54 (C-22), 38.95 (C-13), 38.95 (C-4), 39.08 (C-1), 40.94
(C-8), 42.66 (C-14), 47.12 (C-18), 49.39 (C-19), 50.89 (C-9), 55.58
(C-5), 56.59 (C-17), 81.26 (C-3), 109.97 (C-29), 150.97 (C-20), 172.51
(C-10, C=O ester), 178.68 (C-50, COOH), 181.53 (C-28, COOH). MS
m=z: 570 (Mþ), 456, 438, 423, 395, 248, 220, 203, 189. Anal. Calcd.
for C35H54O6: C, 73.58; H, 9.46. Found: C, 73.31; H, 9.55
3-O-Phthalyl-betulinic acid (2). Starting with phthalic anhydride
(53.9 mg); after crystallization from CH3CN–H2O gave colorless
needles (128 mg, 64.7%); mp 256–258 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:
3474 (OH), 1761 (C=O ester), 1696 (C=O acid), 1653 (C=C), 1600
and 1582 (C=C aromatic), 1292 (C–O). 1H NMR (CDCl3, 500 MHz):
ꢂ 0.76, 0.83, 0.94, 0.97, 0.98, 1.69 (each 3H, s, 6 ꢂ CH3), 3.00 (1H, m),
4.40 (1H, dd, J ¼ 4:5, 12.0 Hz), 4.61 and 4.74 (each 1H, br s), 7.55–
7.61 (2H, m), 7.72 (1H, d, J ¼ 7:0 Hz), 7.92 (1H, d, J ¼ 7:5 Hz).
13C NMR (CDCl3, 125 MHz): ꢂ 14.83 (C-27), 16.16 (C-24), 16.38
(C-26), 16.72 (C-25), 18.39 (C-6), 19.52 (C-30), 23.83 (C-11), 25.68
(C-12), 28.15 (C-2), 28.31 (C-23), 29.85 (C-21), 30.75 (C-15), 32.31
(C-16), 34.47 (C-7), 37.34 (C-10), 38.10 (C-22), 38.19 (C-13), 38.56
(C-4), 39.50 (C-1), 40.99 (C-8), 42.65 (C-14), 47.12 (C-18), 48.42
(C-19), 50.66 (C-9), 55.65 (C-5), 57.97 (C-17), 80.83 (C-3), 109.95
(C-29), 128.76 (C-60), 129.18 (C-30), 130.57 (C-20), 131.06 (C-10),
133.10 (C-40 and C-50), 150.59 (C-20), 168.32 (C=O ester), 169.24
(20-COOH), 181.11 (C-28, COOH). MS m=z: 604 (Mþ), 456, 438, 423,
3-O-(30,30-Dimethyl glutaryl)-betulinic acid (5). Starting with
3,3-dimethylglutaric anhydride (51.7 mg); after crystallization from
MeOH–H2O gave colorless needles (79 mg, 40.2%); mp 217–220 ꢀC.
IR ꢁmax (CHCl3) cmꢁ1: 2500–300 (OH of COOH), 1722 (C=O ester),

Enzymatic Synthesis of Betulinic Acid Esters as Anticancer Agents
1027
1642 (C=C), 1237 and 1150 (C–O). 1H NMR (CDCl3, 500 MHz):
ꢂ 0.76, 0.82, 0.93, 0.96, 0.97, 1.15, 1.17, 1.69 (each 3H, s, 8 ꢂ CH3),
2.60 (2H, s), 2.51 (2H, s), 2.98 (1H, m), 4.41 (1H, dd, J ¼ 5:0,
11.0 Hz), 4.60 and 4.73 (each 1H, br s). 13C NMR (CDCl3, 125 MHz):
ꢂ 14.57 (C-27), 15.56 (C-24), 16.21 (C-26), 16.76 (C-25), 18.52 (C-6),
19.54 (C-30), 21.11 (C-11), 25.69 (C-12), 27.43 (C-2), 27.79 (30-CH3),
28.07 (30-CH3), 28.16 (C-23), 29.89 (C-21), 30.77 (C-15), 32.34
(C-16), 32.54 (C-30), 34.55 (C-7), 37.42 (C-10), 38.18 (C-22), 38.58
(C-13), 38.93 (C-4), 38.98 (C-1), 40.98 (C-8), 42.67 (C-14), 44.04
(C-20), 44.83 (C-40), 47.14 (C-18), 49.45 (C-19), 50.77 (C-9), 55.55
(C-5), 56.65 (C-17), 80.63 (C-3), 109.96 (C-29), 150.62 (C-20), 183.21
(C-28, COOH), 174.86 (C-10, C=O ester), 177.52 (C-50, COOH). MS
m=z: 598 (Mþ), 456, 438, 423, 395, 248, 220, 203, 189. Anal. Calcd for
C37H58O6: C, 74.14; H, 9.69. Found: C, 74.68; H, 9.58.
3462 (OH of COOH), 1728 (C=O ester), 1685 (C=O acid), 1642
(C=C), 1272, 1236, 1189 and 1145 (C–O). 1H NMR (CDCl3,
500 MHz): ꢂ 0.76, 0.84, 0.85, 0.94, 0.95, 1.69 (each 3H, s, 6 ꢂ CH3),
0.98 (3H, t, J ¼ 7:0 Hz), 2.99 (1H, m), 4.49 (1H, dd, J ¼ 5:0, 11.0 Hz),
4.61 and 4.74 (each 1H, br s), 2.34 (2H, t, J ¼ 7:5 Hz), 1.62–1.69 (2H,
m). 13C NMR (CDCl3, 125 MHz): ꢂ 13.85 (C-40), 14.85 (C-27), 16.18
(C-24), 16.35 (C-26), 16.73 (C-25), 18.39 (C-2), 18.86 (C-30), 19.55
(C-30), 21.08 (C-6), 23.97 (C-11), 25.58 (C-12), 28.15 (C-23), 29.87
(C-21), 30.77 (C-15), 32.35 (C-16), 34.48 (C-7), 36.18 (C-20), 37.00
(C-22), 37.34 (C-10), 38.06 (C-4), 38.59 (C-13), 39.07 (C-1), 40.94
(C-8), 42.66 (C-14), 47.15 (C-19), 49.45 (C-18), 50.65 (C-9), 55.64
(C-5), 56.64 (C-17), 80.88 (C-3), 109.94 (C-29), 150.62 (C-20), 173.85
(C-10, C=O ester), 180.08 (C-28, COOH). MS m=z: 526 (Mþ), 456,
438, 423, 395, 248, 220, 203, 189. Anal. Calcd. for C34H54O4: C,
77.45; H, 10.25. Found: C, 77.70; H, 10.21.
3-O-Succinyl-betulinic acid (6). Starting with succinic anhydride
(36.4 mg); after crystallization from MeOH–H2O gave colorless
3-O-Isobutyryl-betulinic acid (10). Starting with isobutyric anhy-
dride (57.5 mg); after crystallization from MeOH–H2O gave colorless
needles (80 mg, 46.2%); mp 260–262 ꢀC. ꢁmax (CHCl3) cmꢁ1: 3463
(OH of COOH), 1726 (C=O ester), 1685 (C=O acid), 1642 (C=C),
1237 and 1148 (C–O). 1H NMR (CDCl3, 500 MHz): ꢂ 0.76, 0.85, 0.94,
0.98, 1.17, 1.69 (each 3H, s, 6 ꢂ CH3), 1.20 and 1.21 (each 3H, d,
J ¼ 7:0 Hz), 2.51–2.69 (1H, m), 3.00 (1H, m), 4.46 (1H, dd, J ¼ 4:5,
11.0 Hz), 4.61 and 4.73 (each 1H, br s). 13C NMR (CDCl3, 125 MHz):
ꢂ 14.90 (C-27), 16.15 (C-30b), 16.22 (C-26), 16.38 (C-24), 16.77
(C-25), 16.98 (C-30a), 18.39 (C-6), 19.42 (C-30), 21.10 (C-11), 25.69
(C-12), 27.52 (C-2), 28.16 (C-23), 29.88 (C-21), 30.78 (C-15), 32.35
(C-16), 34.08 (C-20), 34.71 (C-7), 37.34 (C-10), 38.18 (C-22), 38.59
(C-13), 38.94 (C-4), 39.06 (C-1), 40.95 (C-8), 42.66 (C-14), 47.14
(C-18), 49.45 (C-19), 50.66 (C-9), 55.58 (C-5), 56.65 (C-17), 80.64
(C-3), 109.96 (C-29), 150.62 (C-20), 177.17 (C-10, C=O ester), 183.76
(C-28, COOH). MS m=z: 526 (Mþ), 456, 438, 423, 395, 248, 220, 203,
189. Anal. Calcd. for C34H54O4: C, 77.45; H, 10.25. Found: C, 77.50;
H, 10.11.
needles (132 mg, 72.4%); mp 276–279 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:
3463 (OH of COOH), 1727 (C=O ester), 1686 (C=O acid), 1640
(C=C), 1237 and 1157 (C–O). 1H NMR (CDCl3, 500 MHz): ꢂ 0.76,
0.83, 0.94, 0.97, 0.98, 1.69 (each 3H, s, 6 ꢂ CH3), 3.00 (1H, m), 2.75
(4H, s), 4.50 (1H, dd, J ¼ 4:5, 12.0 Hz), 4.61 and 4.74 (each 1H, br s).
13C NMR (CDCl3, 125 MHz): ꢂ 14.62 (C-27), 15.58 (C-24), 18.52
(C-26), 18.70 (C-25), 18.89 (C-6), 19.60 (C-30), 21.08 (C-11), 25.73
(C-12), 27.66 (C-2), 28.22 (C-23), 28.59 (C-20 and C-30), 29.94 (C-21),
30.78 (C-15), 32.38 (C-16), 34.57 (C-7), 37.44 (C-10), 38.10 (C-13),
38.27 (C-4), 38.92 (C-22), 39.45 (C-1), 40.93 (C-8), 42.67 (C-14),
47.11 (C-18), 49.06 (C-19), 50.75 (C-9), 55.52 (C-5), 56.50 (C-17),
81.23 (C-3), 109.73 (C-29), 150.43 (C-20), 172.76 (C-10, C=O ester),
174.33 (C-40, COOH), 180.73 (C-28, COOH). MS m=z: 557 (Mþ), 511,
483, 456, 438, 423, 395, 248, 220, 203, 189. Anal. Calcd. for
C
34H52O6: C, 73.19; H, 9.33. Found: C, 73.11; H, 9.36.
3-O-Maleyl-betulinic acid (7). Starting with maleic anhydride
(35.6 mg); after crystallization from MeOH–H2O gave colorless
needles (45 mg, 24.7%); mp 258–260 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:
3-O-Valeryl-betulinic acid (11). Starting with valeric anhydride
(67.7 mg); after crystallization from MeOH–H2O gave colorless
3467 (OH of COOH), 1729 (C=O ester), 1685 (C=O acid), 1642
(C=C), 1272, 1237, 1191 and 1147 (C–O). 1H NMR (CDCl3,
500 MHz): ꢂ 0.76, 0.82, 0.88, 0.94, 0.97, 1.69 (each 3H, s, 6 ꢂ CH3),
3.00 (1H, m), 6.86 (1H, d, J ¼ 5:5 Hz), 7.23 (1H, d, J ¼ 5:0 Hz), 4.50
(1H, dd, J ¼ 4:5, 11.5 Hz), 4.61 and 4.74 (each 1H, br s). 13C NMR
(CDCl3, 125 MHz): ꢂ 14.32 (C-27), 15.57 (C-24), 18.51 (C-26), 18.76
(C-25), 18.85 (C-6), 19.52 (C-30), 21.16 (C-11), 25.75 (C-12), 27.55
(C-2), 28.21 (C-23), 29.98 (C-21), 30.76 (C-15), 32.34 (C-16), 34.57
(C-7), 37.41 (C-10), 38.14 (C-4), 38.84 (C-22), 39.10 (C-13), 39.45
(C-1), 40.94 (C-8), 42.63 (C-14), 47.21 (C-18), 49.13 (C-19), 50.77
(C-9), 55.53 (C-5), 56.52 (C-17), 80.25 (C-3), 110.21 (C-29), 136.36
(C-20), 137.24 (C-30), 150.42 (C-20), 173.29 (C-10, C=O ester), 174.23
(C-40, COOH), 181.52 (C-28, COOH). MS m=z: 555 (Mþ), 456, 438,
423, 395, 248, 220, 203, 189. Anal. Calcd. for C34H50O6: C, 73.54; H,
9.01. Found: C, 73.24; H, 9.36.
needles (77 mg, 43.8%); mp 259–261 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:
3466 (OH of COOH), 1728 (C=O ester), 1685 (C=O acid), 1642
(C=C), 1272, 1236, 1189 and 1144 (C–O). 1H NMR (CDCl3,
500 MHz): ꢂ 0.76, 0.84, 0.85, 0.90, 0.97, 1.69 (each 3H, s, 6 ꢂ CH3),
0.93 (3H, t, J ¼ 7:5 Hz), 2.99 (1H, m), 4.48 (1H, dd, J ¼ 5:0, 11.0 Hz),
4.61 and 4.73 (each 1H, br s), 2.36 (2H, t, J ¼ 7:5 Hz), 1.34–1.42 (2H,
m), 1.60–1.66 (2H, m). 13C NMR (CDCl3, 125 MHz): ꢂ 13.89 (C-50),
14.80 (C-27), 16.20 (C-26), 16.38 (C-24), 16.76 (C-25), 18.40 (C-6),
19.54 (C-30), 22.40 (C-40), 23.96 (C-11), 25.69 (C-12), 26.98 (C-30),
27.46 (C-2), 28.17 (C-23), 29.87 (C-21), 30.77 (C-15), 32.34 (C-16),
34.03 (C-20), 34.81 (C-7), 37.33 (C-10), 38.06 (C-22), 38.58 (C-13),
38.94 (C-4), 39.06 (C-1), 40.95 (C-8), 42.66 (C-14), 47.13 (C-18),
49.44 (C-19), 50.65(C-9), 55.65 (C-5), 56.64 (C-17), 80.88 (C-3),
109.96 (C-29), 150.61 (C-20), 174.04 (C-10, C=O ester), 180.39 (C-28,
COOH). MS m=z: 540 (Mþ), 456, 438, 423, 395, 248, 220, 203, 189.
Anal. Calcd. for C35H56O4: C, 77.66; H, 10.35. Found: C, 77.70;
H, 10.41.
3-O-Acetyl-betulinic acid (8). Starting with acetic anhydride
(37.1 mg); after crystallization from MeOH–H2O gave colorless
needles (130 mg, 79.3%); mp 288–290 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:
2500–3500 (OH of COOH), 1729 (C=O ester), 1698 (C=O acid),
1644 (C=C), 1247 (C–O). 1H NMR (CDCl3, 500 MHz): ꢂ 0.84, 0.85,
0.86, 0.94, 0.98, 1.70, 2.05 (each 3H, s, 7 ꢂ CH3), 3.00 (1H, m), 4.48
(1H, dd, J ¼ 5:5, 10.5 Hz), 4.62 and 4.75 (each 1H, br s). 13C NMR
(CDCl3, 125 MHz): ꢂ 14.89 (C-27), 16.28 (C-24), 16.40 (C-25), 16.70
(C-26), 18.39 (C-2), 19.58 (C-30), 21.08 (C-6), 21.55 (C-20), 23.93
(C-11), 25.67 (C-12), 28.18 (C-23), 29.93 (C-21), 30.80 (C-15), 32.39
(C-16), 34.47 (C-7), 37.29 (C-22), 37.35 (C-10), 38.03 (C-4), 38.61
(C-13), 38.66 (C-1), 40.93 (C-8), 42.65 (C-14), 47.17 (C-19), 49.50
(C-18), 50.62 (C-9), 55.65 (C-5), 56.65 (C-17), 81.20 (C-3), 109.98
(C-29), 150.60 (C-20), 171.31 (C-10, C=O ester), 182.56 (C-28,
COOH). MS m=z: 498 (Mþ), 483, 456, 438, 423, 395, 248, 220, 203,
189. Anal. Calcd. for C32H50O4: C, 76.99; H, 10.02. Found: C, 77.03;
H, 10.12.
MTT cytotoxic assay. In vitro cytotoxic activity of betulinic acid (1)
and its 3-O-acylated derivatives (2–11) was done against human lung
carcinoma (A549) and human ovarian (CAOV3) cancer cell lines by
Microculture Tetrazolium Salt (MTT) assay. The assay was carried out
in 96-well microtiter plates. Various concentrations of the compound
were added into the 96-well microtiter plates before the cells were
seeded. The control contained only untreated cells, included for each
sample. The assay was performed in duplicate and the culture plates
were incubated for 72 h at 37 ꢀC in a 5% CO2 humidified incubator.
After 72 h of incubation, the fractions of surviving cells were measured
relative to the untreated cell population by colorimetric MTT assay. A
volume of 20 ml of MTT (5 mg/ml) in phosphate buffer solution was
added to each microtiter well and incubated for 3 to 4 h. After this
incubation period, 100 ml of Dimethyl sulfoxide (DMSO) was added to
each well to dissolve the resulting MTT formazan crystals by pipetting
up and down 10–20 times. The plate was left at room temperature for
15–30 min. Then the optical density (OD) was measured on an ELIZA
microplate reader at 570 nm. The percentage of cell viability was
3-O-Butyryl-betulinic acid (9). Starting with butyric anhydride
(57.5 mg); after crystallization from MeOH–H2O gave colorless
needles (81.8 mg, 47.6%); mp 265–269 ꢀC. IR ꢁmax (CHCl3) cmꢁ1
:

1028
F. B. H. AHMAD et al.
Table 1. Cytotoxicity Assay of Betulinic Acid (1) and 3-O-Acyl-
betulinic Acid Derivatives (2–11) against Human Lung Carcinoma
(A549) and Human Ovarian (CAVO3) Cancer Cell Lines
exhibited moderate cytotoxicity towards CAVO3 cell
line (IC50 ¼ 15:0 mg/ml).
By comparing the cytotoxic activities of compounds
8–11, it was found that the cytotoxic potency may be
dependent on the length of the alkyl chain on the acyl
group at the C-3 position. The compounds having a
shorter alkyl chain on the acyl group at the C-3 position
were found to be more toxic on the cancer cell line. A
similar trend was observed for compounds 4 and 5.
These results suggest that increasing the bulkiness or the
chain length of the alkyl on acyl group at C-3 position
may decrease cytotoxicity against the cancer cell line.
3-O-Phthalyl-betulinic acid (2) had weak cytotoxicity
on the A549 cell line (IC50 > 30 mg/ml). Incorporation
of a methyl group on the aromatic ring (compound 3) led
to a further increase in cytotoxic potency, suggesting
that the presence of an electron-donating group might
change the electrostatic properties. The presence of a
double bond on the acyl group at the C-3 position may
not be critical for the cytotoxic activity; the saturated
form in compound 6 was observed to be more cytotoxic
than its unsaturated form in compound 7.
IC50 (mg/ml)
Compounds
A549
CAOV3
1
2
8.4
>30
18.4
6.4
9.4
>30
>30
>30
>30
15
3
4
5
>30
7.4
6
7
26.6
6.8
>30
>30
>30
>30
>30
8
9
11.4
12.1
>30
10
11
calculated using the following equation: % Viability ¼ (OD sample=
OD controlÞ ꢂ 100. A plot of percentage of cell viability against the
concentration of the drug gives a measure of cytotoxicity. The
cytotoxic index used was IC50, the drug concentration lethal to 50% of
the tumor cells as calculated from the plot.
Results and Discussion
Conclusions
In initial work in our laboratory, the enzymatic
synthesis of 3-O-phthalyl-betulinic acid (2) was chosen
as a reaction model for optimization the reaction
parameters using response surface mythology (RSM).
Several betulinic acid esters (2–11) were synthesized
under the optimal operation conditions, obtained by the
RSM technique. Betulinic acid (1) and its derivatives
(compounds 2–11) were then screened for cytotoxicity
in vitro against human lung carcinoma (A549) and
human ovarian (CAOV3) cancer cell lines by MTT
assay. The results presented in Table 1 are expressed as
the concentration inhibiting 50% of the cell growth
(IC50).
Based on the IC50values, compounds with IC50 < 10
mg/ml were considered strongly active, those with IC50
ranging from 10 to 30 mg/ml were considered moder-
ately active, and those with IC50 > 30 mg/ml were
weakly active. Betulinic acid (1), 3-O-glutaryl-betulinic
acid (4), 3-O-succinyl-betulinic acid (6), and 3-O-acetyl-
betulinic acid (8) showed high activity against the lung
A549 cell line (IC50 < 10 mg/ml), while 3-O-(30-methyl
phthalyl)-betulinic acid (3), 3-O-maleyl-betulinic acid
(7), 3-O-butyryl-betulinic acid (9), and 3-O-isobutyryl-
betulinic acid (10) showed moderate activity against
the A549 (10 mg/ml < IC50 < 30 mg/ml). In contrast,
3-O-phthalyl-betulinic acid (2), 3-O-(30,30-dimethyl
glutaryl)-betulinic acid (5), and 3-O-valeryl-betulinic
acid (11) showed weakly cytotoxicity against the A549
cell line (IC50 > 30 mg/ml). Betulinic acid (1) and
compounds 2–11 are arranged in order of decreasing
activity for the lung A549 cell line: 4 > 8 > 6 > 1 >
9 > 10 > 3 > 7 > 2 (about 5 and 11).
This is the first report on the enzymatic synthesis of
3-O-acyl-betulinic acid derivatives using acid anhy-
drides. It was found that Candida antarctica lipase
performed esterification of the betulinic acid with
anhydrides. On the basis of our in vitro cytotoxic results
and the structure-activity relationship (SAR), we con-
cluded that (i) 3-O-glutaryl-betulinic acid (4), 3-O-
succinyl-betulinic acid (6), and 3-O-acetyl-betulinic acid
(8) were the most active compounds as compared to
betulinic acid (1) against human lung carcinoma (A549),
(ii) 3-O-glutaryl-betulinic acid (4) exhibited the best
cytotoxicity against A549 cell line, (iii) all the betulinic
acid derivatives (compounds 2–11) showed weaker
cytotoxicity than betulinic acid (1) against a human
ovarian cell line (CAVO3).
Acknowledgments
We wish to thank all the staff in the Department of
Chemistry of Universiti Putra Malaysia for their help in
this research. We would also like to thank the Institute of
Biosciences (IBS) of Universiti Putra Malaysia for
providing the facilities to carry out this study. We are
grateful to Mr. Salahuddin for measurement of NMR
spectra. This research was supported by Universiti Putra
Malaysia under a University Research Grant (RUGS
9135500).
References
1) Yogeeswari P and Sriram D, Curr. Med. Chem., 12, 657–666
(2005).
2) Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E,
Gambacorti-Passerini C, and Formelli F, Cancer Lett., 175,
17–25 (2002).
Compared to betulinic acid (1), 3-O-glutaryl-betulinic
acid (4), 3-O-succinyl-betulinic acid (6), and 3-O-acetyl-
betulinic acid (8) exhibited stronger cytotoxicity against
the lung A549 cell line. All the betulinic acid derivatives
(compounds 2–11) reported here showed weaker cyto-
toxicity than betulinic acid (1) against the ovarian
CAVO3 cancer cell line. 3-O-succinyl-betulinic acid (6)
3) Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR,
Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM,
Wani MC, Wall ME, Hieken TJ, Das Gupta TK, and Pezzuto
JM, Nat. Med., 1, 1046–1051 (1995).
4) Fulda S and Debatin KM, Med. Pediatr. Oncol., 35, 616–618
(2000).

Enzymatic Synthesis of Betulinic Acid Esters as Anticancer Agents
1029
5) Fulda S, Jeremias I, Steiner HH, Pietsch T, and Debatin KM,
Int. J. Cancer, 82, 435–441 (1999).
12) Mukherjee R, Jaggi M, Rajendran P, Srivastava SK, Siddiqui
MJA, Vardhan A, and Burman AC, Bioorg. Med. Chem. Lett.,
14, 3169–3172 (2004).
6) Udeani GO, Zhao GM, Geun Shin Y, Cooke BP, Graham J,
Beecher CW, Kinghorn AD, and Pezzuto JM, Biopharm. Drug
Dispos., 20, 379–383 (1999).
13) Kashiwada Y, Hashimoto F, Cosentino LM, Chen CH, Garrett
PE, and Lee KH, J. Med. Chem., 39, 1016–1017 (1996).
14) Zarevuka M and Wimmer Z, Int. J. Mol. Sci., 9, 2447–2473
(2008).
7) Gauthier C, Legault J, Lavoie S, Rondeau S, Tremblay S, and
Pichette A, Tetrahedron, 64, 7386–7399 (2008).
8) Thibeault D, Gauthier C, Legault J, Bouchard J, Dufour P, and
Pichette A, Bioorg. Med. Chem., 15, 6144–6157 (2007).
9) Jeong HJ, Chai HB, Park SY, and Kim DSHL, Bioorg. Med.
Chem. Lett., 9, 1201–1204 (1999).
15) Sonnet PE, J. Am. Oil Chem. Soc., 65, 900–904 (1998).
16) Ahmad FBH, Issak A, Basri M, Hana N, Yasin Y, and Ali AM,
Chem. Environ. Res., 14, 207–213 (2005).
17) Yasin Y, Basri M, Ahmad F, and Salleh AB, J. Chem. Technol.
Biotechnol., 83, 694–698 (2008).
10) Kvasnica M, Sarek J, Klinotova E, Dzubak P, and Hajduch M,
Bioorg. Med. Chem., 13, 3447–3454 (2005).
18) Ahmad F, Omar J, and Ali AM, Ultra Sci., 11, 357–360
(1999).
11) Mukherjee R, Kumar V, Srivastava SK, Agarwal SK, and
Burman AC, Anticancer Agents Med. Chem., 6, 271–279 (2006).