Synthesis and cytotoxic activity of 17-carboxylic acid modified 23-hydroxy betulinic acid ester derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.09.096

New 17-carboxylic acid modified 23-hydroxy betulinic acid ester derivatives were prepared and tested for cytotoxic activity on five cancer cell lines in vitro: all tested compounds showed stronger cytotoxic activity than 23-hydroxy betulinic acid and betulinic acid. In addition, compound 5a was tested for anti-tumor activity in vivo: it had much better anti-tumor activity than 23-OH betulinic acid and had similar anti-tumor activity with cyclophosphamide and 5-fluorouracil.

Full text of DOI:10.1016/j.bmcl.2006.09.096

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1475–1478
Synthesis and cytotoxic activity of 17-carboxylic acid modified
23-hydroxy betulinic acid ester derivatives
Yi Bi,a Jinyi Xu,a Xiaoming Wu,a,* Wencai Ye,b Shengtao Yuanc and Luyong Zhangc
aDepartment of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
bDepartment of Phytochemistry, China Pharmaceutical University, Nanjing 210009, China
cNational Drug Screening Center of China Pharmaceutical University, Nanjing 210038, China
Received 4 July 2006; revised 18 September 2006; accepted 30 September 2006
Available online 4 October 2006
Abstract—New 17-carboxylic acid modified 23-hydroxy betulinic acid ester derivatives were prepared and tested for cytotoxic activ-
ity on five cancer cell lines in vitro: all tested compounds showed stronger cytotoxic activity than 23-hydroxy betulinic acid and bet-
ulinic acid. In addition, compound 5a was tested for anti-tumor activity in vivo: it had much better anti-tumor activity than 23-OH
betulinic acid and had similar anti-tumor activity with cyclophosphamide and 5-fluorouracil.
Ó 2006 Elsevier Ltd. All rights reserved.
Lupane derivatives are prevalent in natural sources and
have various biological activities. 23-Hydroxy betulinic
acid was isolated from the root of Pulsatilla chinensis
which is a Chinese medicinal herb for ‘blood-cooling’
and detoxification in traditional Chinese medicine, and
as such has been used for the treatment of amoebic dys-
entery and malaria. 23-Hydroxy betulinic acid was
recently turned out to have good anti-tumor and anti-
HIV selectivity, such as the cytotoxicity and apoptotic
response induced by 23-hydroxy betulinic acid in human
leukemia HL-60 cells. The telomerase activity during
apoptosis was examined by telomeric repeat amplifica-
tion protocol (TRAP) assay, and B cell leukemia/lym-
phoma 2 gene (bcl-2) was analyzed to determine
whether their expressions correlated with telomerase
activity. 23-Hydroxy betulinic acid inhibited telomerase
activity and suppressed the expression of bcl-2 in HL-60
cells. The inhibition of telomerase activity of HL-60 cells
was closely associated with the apoptotic events induced
by 23-hydroxy betulinic acid.1–4 Betulinic acid, one of
well-known lupines, showed the selective cytotoxic
activity toward many cell lines.5–10
now. In this manuscript, we report a series of derivatives
of 23-hydroxy betulinic acid with modification of
17-carboxylic acid. The study results revealed that all
derivatives have better cytotoxicity than 23-hydroxy
betulinic acid in vitro and many of them have better
cytotoxicity than betulinic acid. Compound 5a shows
more potent anti-tumor activity than 23-hydroxy betuli-
nic acid and has slightly less anti-tumor activity than
cyclophosphamide and 5-fluorouracil.
Synthesis of 17-carboxylic acid ester derivatives of 23-
hydroxy betulinic acid has been described in Scheme
1. 23-Hydroxy betulinic acid (1) was provided by the
laboratory of Wencai Ye. It was treated with acetic
anhydride in pyridine to afford 3,23-O-diacetyl deriva-
tive 2. Compound 2 upon reaction with (COCl)2 in
CH2Cl2 yielded 3,23-O-diacetyl-17-acyl chloride deriva-
tive 3. Upon reaction with ethylene glycol, 1,4-butane-
diol, 1,6-hexanediol, butyne-1,4-diol, allyl alcohol,
propargyl alcohol, respectively, yielded corresponding
3,23-O-diacetyl-17-carboxylate derivatives 4a–f, and
4a–f were then hydrolyzed to give derivatives 5a–f. In
the process of hydrolysis reaction, the esters of 17-po-
sition remained the esters but not acids. We think the
steric hindrance of this position is the reason. We
found the esters were not hydrolyzed in the condition
of NaOH/THF/CH3OH, but they could be transmitted
to the acids in the condition of LiI/DMF.12 All the pre-
pared compounds were characterized by spectroscopic
tools.5–8
The research of derivatives of 23-hydroxy betulinic acid
about their cytotoxicity has not been reported up to
Keywords: 23-Hydroxy betulinic acid; 17-Carboxylic acid modified
ester derivatives; Anti-tumor activity.
*
Corresponding author. Tel.: +86 25 83271543; fax: +86 25
83302827; e-mail: xmwu@cpu.edu.cn
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.096

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Y. Bi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1475–1478
i
ii
COOH
COCl
COOH
HO
AcO
AcO
OH
OAc
OAc
1
3
2
iii
R
CH2CH=CH2
CH2C CH
a
iv
b
c
d
e
f
COOR
CH2CH2OH
(CH2)4OH
COOR
HO
OH
(CH2)6OH
AcO
OAc
5a-f
CH2C CCH3
4a-f
Scheme 1. Reagents and conditions: (i) Ac2O/Pyr (yield 85%); (ii) (COCl)2/CH2Cl2 (yield 90%); (iii) ROH/CH2Cl2 (yields 45–70%); (iv) NaOH/THF/
CH3OH (yields 80–90%).
The cytotoxic activity of betulinic acid, 23-hydroxy bet-
ulinic acid and its derivatives (5a–f) in vitro was deter-
mined by performing the MTT cytotoxicity assay, and
the result is summarized in Table 1. Many different cell
lines were used: A549 (human lung carcinoma), B16
(mice melanoma), SF-763 (human cerebroma), BEL-
7402 (human hepatoma), and C6 (mice neuroglioma).
The activity of derivatives was compared with that of
23-hydroxy betulinic acid and betulinic acid in each
panel.9,10 It is the first time to report the cytotoxicity
of the derivatives of the 23-hydroxy betulinic acid. In
human lung carcinoma cell line (A549), compounds
5a–f have better cytotoxicity than betulinic acid and
have similar cytotoxicity as 23-OH betulinic acid. Com-
pound 5a has the best cytotoxicity in A549. In human
hepatoma cell line (BEL-7402), compound 5f is the best
compound which has similar cytotoxicity as betulinic
acid. Compounds 5a–f have better cytotoxicity than
23-OH betulinic acid but none of them have better than
betulinic acid. In human cerebroma cell line (SF-763),
compound 5a is the most potent which is almost twofold
more potent than betulinic acid and 23-OH betulinic
acid. Compounds 5b, 5e, and 5f are almost 1.5-fold
more potent than betulinic acid and 23-OH betulinic
acid. Compound 5d is slightly better than betulinic acid
and 23-OH betulinic acid. Compound 5c is a little better
than 23-OH betulinic acid but worse than betulinic acid.
In mice melanoma cell line (B16), compound 5e is the
best compound which has twofold more potency than
23-OH betulinic acid and is little better than betulinic
acid. Compounds 5a–d and 5f have better cytotoxicity
than 23-betulinic acid but are worse than betulinic acid.
In mice neuroglioma cell line (C6), compound 5e has
twofold better cytotoxicity than betulinic acid and 23-
OH betulinic acid. Compounds 5c and 5d are 1.5-fold
more potent than betulinic acid and 23-OH betulinic
acid. Compounds 5a, 5b, and 5f are slightly better than
betulinic acid and 23-OH betulinic acid.
In conclusion, compounds 5a–f are more potent than
23-betulinic acid in all cell lines and more potent than
betulinic acid in A549 and C6. In SF-763 all compounds
have better cytotoxicity than betulinic acid but com-
pound 5c. In B16 none of compounds are more potent
than betulinic acid but compound 5e. In BEL-7402,
none of compounds have better cytotoxicity than betu-
linic acid. Compounds 5a and 5f were the best com-
pounds. So compound 5a was chosen to test in vivo.
In vivo cytotoxic activity of 23-hydroxy betulinic acid
and its derivative 5a was determined by performing
the cytotoxicity assay in murine having H22 liver tumor
Table 1. The cytotoxicity data of 23-hydroxy betulinic acid and its derivatives (5a–f) [IC50 (lmol/L) SD]
Compound
Cell line
A549
BEL-7402
SF-763
B16
C6
5a
5b
74.22 17.61*
87.82 7.24
63.86 2.51
67.01 14.65
78.66 21.30
74.47 9.21
69.17 15.67
52.96 13.36*
97.32 10.94
43.39 9.22
54.72 16.38*
63.41 25.19
101.15 1.756
78.60 9.06
64.31 5.80
64.31 7.16
104.54 1.04
92.07 5.06
80.54 2.99
72.93 7.52
80.87 1.56
66.03 7.03
41.06 3.62*
63.15 7.52
83.04 4.06
53.46 9.47
82.05 5.44
82.73 8.22
65.00 24.12
69.30 9.11
49.68 30.01*
75.88 14.89
99.48 21.44
90.69 21.09
5c
5d
83.78 13.70
84.145 11.03
78.28 18.50
81.06 77.74
87.60 8.67
5e
5f
HBA
BA
97.51 11.78
Concentration (lmol/L): 26.20, 32.80, 41.00, 51.20, 64.00, 80.00, and 100.00. Data are means of three experiments. BA, betulinic acid; HBA, 23-
hydroxy betulic acid.
* The significant differences among IC50 values of these compounds in a cell line.

Y. Bi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1475–1478
1477
Table 2. The cytotoxicity data of 23-hydroxy betulinic acid and its derivatives 5a in mice H22 in vivo
Drugs
Dose
Injection Number of Weight of mice (g)
mice
Weight of tumor Ratio of inhibition (%) P value
SD (g)
X
Start End Start
End
Normal saline
Cyclophosphamide
5a
0.4 ml/mouse iv
10
10
10
10
10
10
10
10
18.1 0.6 27.3 2.8 1.58 0.49
18.1 0.5 26.2 2.3 0.69 0.30
18.5 0.9 25.0 2.7 0.90 0.53
18.3 0.9 26.9 1.3 1.42 0.36
30 mg/kg
20 mg/kg
iv
ip
ip
56.3
43.0
10.1
<0.01
<0.05
>0.05
23-OH betulinic acid 20 mg/kg
Table 3. The cytotoxicity data of 23-hydroxy betulinic acid and its derivatives 5a in mice B16 in vivo
Drugs
Dose
Injection Number of Weight of mice (g)
mice
Weight of tumor Ratio of inhibition (%) P value
SD (g)
X
Start End Start
End
18.9 2.3 21.3
19.0 1.4 18.8 1.7 0.56 0.25
19.0 1.4 17.9 1.18 0.99
18.8 2.4 20.5 2.9 1.76 0.8
Normal saline
5-Fluorouracil
5a
0.4 ml/mouse iv
9
9
9
9
9
9
9
9
3
2.03 0.67
25 mg/kg
20 mg/kg
iv
ip
ip
72.4
41.9
13.3
<0.01
<0.05
>0.05
2
23-OH betulinic acid 20 mg/kg
and B16 melanoma and the cytotoxicity data are sum-
marized in Tables 2 and 3. ICR female murine with
body weight of 18–22 g were transplanted with H22
and B16 subcutaneously into the right oxter according
to protocols of transplant tumor research. After 24 h
of tumor transplantation, murine were weighed, and
each model group was at random divided into 4 groups,
each of which had 10 murine in H22 group and 9 murine
in B16 group. The groups with 23-OH betulinic acid and
5a were administered intraperitoneously 20 mg/kg in a
vehicle of 20% DMSO/80% saline. respectively. The
positive control group was treated with cyclophospha-
mide (30 mg/kg) in H22 group and 5-fluorouracil
(25 mg/kg) in B16 group through intravenous injection
in a vehicle of 20% DMSO/ 80% saline. The negative
control group received 0.9% normal saline through
intravenous injection. All test compounds were given
through injections 24 h after tumor transplantation (or
inoculation). Treatments were done at a frequency of
intravenous or intraperitoneal injection one dose per
day for a total of four consecutive days in H22 group
and for a total of 11 consecutive days in B16 group.
After the treatments, all murine were killed and weighed
simultaneously, and then tumor segregated and
weighed. Tumor inhibitory ratio was calculated by the
following formula and perform T test:11
Acknowledgments
The authors thank National Natural Science Fund
Committee for the investigation support
(No.30472083), and thank to National Drug Screening
Center of China pharmaceutical University for the
MTT cytotoxicity assay and the anti-tumor activity
assay in mice.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.09.096.
References and notes
1. Pisha, E.; Chai, H.; Lee, I.-S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.;
Fong, H. H. S.; Dogglas Kinghorn, A.; Brown, D. M.;
Wani, M. C.; Wall, M. E.; Hieken, T. J.; Das Gupta, T.
K.; Pezzut, J. M. Nat. Med. 1995, 1, 1046.
2. Ye, W.-C.; Ji, N.-N.; Zhao, S.-X.; Liu, J.-H.; Ye, T.;
Mckervey, M. A.; Stevensin, P. Phytochemistry 1996, 42,
799.
3. Ji, Z.-N.; Ye, W.-C.; Liu, G.-G.; Wendy Hsiao, W. L. Life
Sci. 2002, 72, 1.
4. Shi-Jun, Y.; Hong, G. J. Liaoning Normal Univ. (Nat. Sci.
Ed.) 2002, 26, 56.
5. Sun, I.-C.; Wang, H.-K.; Shen, Y. K. J.-K.; Cosentino, L.
M.; Chen, C.-H.; Yang, L.-M.; Lee, K.-H. J. Med. Chem.
1998, 41, 4648.
Tumor inhibitory ratio ð%Þ
¼ ð1 ꢀ average tumor weight of treated group=
average tumor weight of control groupÞ ꢁ 100%
Compound 5a has better anti-tumor activity than 23-
OH betulinic acid in mice with H22 liver tumor and
with B16 melanoma which has slight potent anti-tumor
activity than cyclophosphamide in H22 group and
5-fluorouracil in B16 group. Cyclophosphamide and
5-fluorouracil are the normal anti-tumor drugs in
clinic.
6. Sun, I.-C.; Chen, C.-H.; Wu, Y. K. J.-H.; Wang, H.-K.;
Lee, K.-H. J. Med. Chem. 2002, 45, 4271.
7. Evers, M.; Poujade, C.; Ribeill, F. S. Y.; James, C.;
Lelievre, Y.; Gueguen, J.-C.; Reisdorf, D.; Morize, I.;
Pauwels, R.; De Clercq, E.; Henin, Y.; Bousseau, A.;
Mayaux, J.-F.; Le Pecq, J.-B.; Dereu, N. J. Med. Chem.
1996, 39, 1056.

1478
Y. Bi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1475–1478
8. Soler, F.; Poujade, C.; Evers, M.; Carry, J.-C.; Henin, Y.;
10. Mukherjee, R.; Jaggi, M.; Siddiqui, M. J. A.; Srivastava,
S. K.; Rajendran, P.; Vardhan, A.; Burman, A. C. Bioorg.
Med. Chem. Lett. 2004, 14, 4087.
11. Xu, S. Y.; Bian, R. L.; Chen, X. Pharmacological
Experimental Method; People’s Medical Publishing House:
Beijing, 1991, 1424.
Bousseau, A.; Huet, T.; Pauweis, R.; De Clercq, E.;
Mayaux, J.-F.; Le Pecq, J.-B.; Dereu, N.. J. Med. Chem.
1996, 39, 1069.
9. Mukherjee, R.; Jaggi, M.; Rajendran, P.; Srivast-
ava, S. K.; Siddiqui, M. J. A.; Vardhan, A.;
Burman, A. C. Bioorg. Med. Chem. Lett. 2004,
14, 3169.
12. Finlay, H. J.; Honda, T.; Gribble, G. W. ARKIVOC 2002,
xii, 38.