Betulinic acid and its derivatives as anti-angiogenic agents

Article abstract of DOI:10.1016/j.bmcl.2004.02.044

Betulinic acid (1) significantly caused cytotoxicity to endothelial cell line ECV304 (IC₅₀ 1.26±0.44μg/mL) in a 5-day MTT assay. Novel and more potent derivatives of betulinic acid (2, 4, 6-8) have been synthesized with IC₅₀ less than 0.4μg/mL. The endothelial cell specificity against human tumor cell lines DU145, L132, A549, and PA-1 were determined. Further betulinic acid (1) inhibited TLS formation of ECV304 cells on Matrigel^TM by 5.5% while its derivatives caused an inhibition of 13.1-49.2%.

Full text of DOI:10.1016/j.bmcl.2004.02.044

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2181–2184
Betulinic acid and its derivatives as anti-angiogenic agents
Rama Mukherjee,a,b,c Manu Jaggi,a,* Praveen Rajendran,a Mohammad J. A. Siddiqui,b
Sanjay K. Srivastava,c,* Anand Vardhanb and Anand C. Burmana,b,c
aDivisions of Experimental Oncology, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201 010, UP, India
bChemical Research, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201 010, UP, India
cMedicinal Chemistry, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201 010, UP, India
Received 6 January 2004; accepted 5 February 2004
Abstract—Betulinic acid (1) significantly caused cytotoxicity to endothelial cell line ECV304 (IC50 1.26 0.44 lg/mL) in a 5-day
MTT assay. Novel and more potent derivatives of betulinic acid (2, 4, 6–8) have been synthesized with IC50 less than 0.4 lg/mL. The
endothelial cell specificity against human tumor cell lines DU145, L132, A549, and PA-1 were determined. Further betulinic acid (1)
inhibited TLS formation of ECV304 cells on MatrigelTM by 5.5% while its derivatives caused an inhibition of 13.1–49.2%.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
been shown to have some anti-angiogenic activity.4–6
These experiments suggest that only a few anti-cancer
agents have the ability to target both tumor cells and
cells of the vasculature (endothelial cells) and are
potentially effective angiogenesis inhibitors. We have
investigated the potential of betulinic acid (1) in angio-
genesis and have developed more potent betulinic acid
derivatives through bioactivity-guided structure–activity
studies. Here we report for the first time the anti-
angiogenic activity of betulinic acid (1) and its potent
derivatives (2, 4, 6–8).
Betulinic acid (1) (3-b-hydroxy-lup-20(29)-en-28-oic
acid) is a pentacyclic lupane-type triterpene that is
widely distributed throughout the plant kingdom. A
variety of biological activities have been ascribed to
betulinic acid (1) including anti-inflammatory and in
vitro anti-malarial effects.1 However, betulinic acid (1) is
most highly regarded for its anti-HIV-1 activity and
specific cytotoxicity against a variety of tumor cell lines.2
Betulinic acid (1) was previously reported to exhibit
selective cytotoxicity against several melanoma-derived
cell lines. However, more recent work has demonstrated
that betulinic acid is cytotoxic against other nonmel-
anoma human tumor varieties.3 Betulinic acid (1) ap-
pears to function by means of inducing apoptosis in cells
irrespective of their p53 status. Because of its selective
cytotoxicity against tumor cells and favorable thera-
peutic index, even at doses up to 500 mg/kg body weight,
betulinic acid (1) is a very promising new chemothera-
peutic agent for the treatment of HIV infection and
cancer.2
2. Chemistry
Synthesis of betulinic acid derivatives (2, 4, 6–8)7–9 has
been described in Scheme 1. Betulinic acid (1) was
converted to 3-oxo betulinic acid (2) by JonesÕ oxida-
tion. Compound 1 was hydrogenated with Pd/Cto
furnish 20,29-dihydro betulinic acid (3). Compound 3
was elaborated in two ways. In first, compound 3 was
brominated with liquid bromine in methylene chloride
to afford 2-bromo-20,29-dihydro betulinic acid (4). In
second, compound 3 was oxidized, as for 1, to afford
20,29-dihydro-3-oxo betulinic acid (5). Compound 5
was treated with different hydrazines to yield corre-
sponding 3-hydrazono-20,29-dihydro betulinic acid
derivatives (6–8). All the compounds (2, 4, 6–8) were
characterized by spectroscopic and analytical tools.
Anti-cancer agents have previously been evaluated for
their anti-angiogenic potential. Epirubicin, doxorubicin,
mitoxantrone, vinblastine, vincristine, and taxol have
* Corresponding authors. Tel.: +91-120-2777901-25; fax: +91-120-27-
77303; e-mail: jaggim@dabur.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.044

2182
R. Mukherjee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2181–2184
29
20
30
O
O
1
CrO3
OH
OH
3
H2SO4
O
HO
1
2
MeOH
Pd/C
O
CrO3
O
OH
OH
H2SO4
O
HO
3
5
RNHNH2
CH3COONa
EtOH
CH2Cl2
Br2
O
O
OH
H
Br
N
OH
R
N
HO
4
6-8
40 %
80%
70 %
25 %
6, R = C6H2(2,4,6-)Cl3
7, R = COC6H5
8, R = C6H4(4-)F
Scheme 1.
3. Results and discussion
6–8) showed IC50 < 0.4 lg/mL (<1 lM) on ECV304 cell
line. Betulinic acid and its derivatives were screened for
cytotoxicity on human tumor cell lines DU145 (pros-
tate), L132 (lung), A549 (lung), and PA-1 (ovary) and
the endothelial cell specificity (ECS) was calculated as
shown in Table 1. Betulinic acid (1) has shown low ECS
against all tumor lines (ECS < 10) while derivatives 2, 4,
6–8 have shown moderate to high ECS against A549;
compounds 6 and 8 have shown moderate ECS against
L132 and compound 2 has shown moderate ECS against
DU145.
Betulinic acid (1) is cytotoxic to endothelial cells
(ECV304) in a concentration-dependant manner (Fig.
1). Increasing concentrations of betulinic acid (1) were
tested against ECV304 cells in a 5-day MTT assay. The
half-maximal cytotoxic concentration (IC50) was
1.26 0.44 lg/mL (ꢀ2.77 lM). Data represented is mean
of three experiments. All betulinic acid derivatives (2, 4,
100
50
0
Figure 2 shows the effect of betulinic acid (1) and
derivatives (2, 4, 6–8) on tube-like structure (TLS) for-
mation of ECV304 cells in a MatrigelTM tube formation
assay. Betulinic acid (1) inhibited tube formation by
5.5% while the derivatives (2, 4, 6–8) screened inhibited
tube formation by 13.1–49.2% as measured by tube
length using image analysis. The inhibitory effect caused
by the derivatives (2, 4, 6–8) was equal to or greater than
betulinic acid (1) characterized by the inhibition of tube
length, endothelial sprouting, capillary network forma-
tion, and intussusception. Experiments were in dupli-
cates and each experiment was performed thrice.
0
2
4
6
8
10
Structure–activity relationship indicates that the cyto-
toxicity was increased about four to six times, after
converting betulinic acid (1) into 3-oxo betulinic acid
Betulinic acid (ug/mL)
Figure 1.

R. Mukherjee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2181–2184
2183
Table 1. IC50 and ECS ratios of betulinic acid (1) and its derivatives (2, 4, 6–8) on ECV304 cells
IC50 (lg/mL)
Endothelial cell specificity (ECS)a [IC50 tumor cell/IC50 endothelial cell]
S. no.
Compound
ECV304
DU145
L132
A549
PA-1
1
2
3
4
5
6
Betulinic acid (1)
1.26 0.44
0.28 0.005
0.27 0.02
0.21 0.03
0.39 0.07
0.35 0.07
2.2
14.2
7.0
1.0
3.2
1.1
9.8
3.2
9.2
7.4
5.2
4.3
1.4
2
4
6
7
8
3.1
11.5
19.0
27.7
20.0
6.6
7.7
11.4
1.0
3.7
11.1
a The IC50 for DU145, L132, A549, and PA-1 cell lines was determined using MTT assay (data not shown) and ECS ratios calculated as described in
methods.
4. Conclusion
5.5
1
6
8
4
2
7
It has been suggested, that anti-angiogenic compounds
given along with standard cytotoxic drugs in combina-
tion chemotherapy regimens are more effective than
cytotoxic drugs alone.10 Long duration combination
chemotherapy regimens involving cytotoxic and anti-
angiogenic drugs have the drawbacks of adverse toxic
effects. In such a scenario compounds such as betulinic
acid derivative 7, which possess both anti-tumor and
anti-angiogenic potential could be envisioned to act
effectively with acceptable toxicity profiles. Further
studies are under progress to determine the in vivo
efficacy of the compounds having in vitro anti-angio-
genic activity. However, recognition of the anti-angio-
genic potential of betulinic acid and its novel derivatives
may help in designing strategies to tackle angiogenesis-
dependant tumor growth.
13.1
Percent
reduction in
TLS
18.5
28.7
47.6
49.2
0
50
% reduction
100
Figure 2.
(2), and 20,29-dihydro betulinic acid derivatives (4, 6–8).
As far as ECS is concerned, all the betulinic acid
derivatives (2, 4, 6–8) have shown several fold higher
endothelial specificity than betulinic acid (1). The 3-hy-
drazono-20,29-dihydro betulinic acid derivatives (6–8)
exhibited high endothelial specificity against A549 cell
line while 3-oxo betulinic acid (2) and 2-bromo-20,29-
dihydro betulinic acid (4) have shown moderate speci-
ficity against DU145 and A549 cell lines, respectively.
Compounds 6 and 8 had moderate specificity against
L132 cell line. All betulinic acid derivatives except 8
have shown low endothelial specificity against PA-1 cell
line but were better than betulinic acid (1). It is predicted
that the high and moderate ECS compounds would
cause anti-cancer effect primarily due to anti-angiogenic
potential while low ECS compounds would supplement
their already known anti-tumor effects. Also all the
betulinic acid derivatives (2, 4, 6–8) have shown better
anti-TLS activity than betulinic acid (1). 3-Oxo betulinic
acid (2) and 3-(N-benzoyl)hydrazono-20,29-dihydro
betulinic acid (7) have shown about ninefold better anti-
TLS activity than betulinic acid (1).
5. Materials and methods
5.1. Chemicals
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide, Sigma, USA), MatrigelTM (Becton
Dickinson, USA), DMEM (DulbeccoÕs modified Eagles
medium), and Fetal bovine serum, FBS (Gibco BRL,
USA), DMSO (Merck, India). Chemicals used in syn-
thesis were purchased from Sigma, USA.
5.2. Cell culture
ECV304 cell line was generously gifted by Dr. Takah-
ashi (Tokyo University, Tokyo, Japan). Human tumor
cell lines DU145 (prostate), L132 (lung), A549 (lung),
and PA-1 (ovary) cell lines have been procured from
NCCS, Pune, India. Cell lines were grown in DMEM,
containing L-glutamine and 25 mM HEPES and sup-
plemented with 10% fetal bovine serum, penicillin
(100 units/mL), streptomycin (100 lg/mL), and ampho-
3-(N-Benzoyl)hydrazono-20,29-dihydro betulinic acid
(7), a potent cytotoxic agent on ECV304 cell lines, has
shown high endothelial specificity as well as high anti-
TLS activity. It seems that hydrazone group at position-
3 and 20,29-dihydro moiety in betulinic acid (1) play an
important role in eliciting high endothelial cytotoxicity,
specificity, and anti-TLS activity.
tericin B (0.25 lg/mL) at 37 ꢁC , 5% C O, 100% humidity.
2
5.3. Cytotoxicity assay
Cells (104) were incubated with betulinic acid (1) or its
derivatives (2, 4, 6–8), dissolved in DMSO (final DMSO

2184
R. Mukherjee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2181–2184
concn <0.1%), in triplicate wells to obtain drug con-
centration of 0.5–10 lg/mL. Cytotoxicity was measured
after 120 h using MTT assay as described by Mosmann.11
Each experiment was repeated thrice and mean IC50
values (half-maximal cytotoxicity) have been reported.
ECS ratios were calculated using the formula: IC50 (tu-
mor cell)/IC50 (endothelial cell). ECS < 10 was designated
low endothelial specificity, ECS between 10 and 20 as
moderate and ECS > 20 as high endothelial specificity.
References and notes
1. Anderson, B.M.; Musser, H.J. WO 95/04526.
2. Cichewicz, R. H.; Kouzi, S. A. Med. Res. Rev. 2004, 24,
90.
3. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.;
Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani,
M. C.; Wall, M. E.; Hieken, T. J.; Das Gupta, T. K.;
Pezzuto, J. M. Nat. Med. 1995, 1, 1046.
4. Steiner, R. EXS 1992, 61, 449.
5. Dordunoo, S. K.; Jackson, J. K.; Arsenault, A. L.;
Oktaba, A. M.; Hunter, W. L.; Burt, H. M. Cancer
Chemother. Pharmacol. 1995, 36, 279.
5.4. Tube-like structure (TLS) formation assay
6. Burt, H. M.; Jackson, J. K.; Bains, S. K.; Liggins, R. T.;
Oktaba, A. M.; Arsenault, A. L.; Hunter, W. L. Cancer
Lett. 1995, 88, 73.
7. Ramadoss, S.; Jaggi, M.; Siddiqui, M. J. A.; Khanna, A.
B. U.S. Patent 6,048,847, 2000.
8. Ramadoss, S.; Jaggi, M.; Siddiqui, M. J. A.; Khanna, A.
B. U.S. Patent 6,214,814, 2001.
9. Jaggi, M.; Ramadoss, S.; Rajendran, P.; Siddiqui, M. J. A.
U.S. Patent 6,228,850, 2001.
10. Folkman, J. Antiangiogenic therapy. In Cancer: Principles
& Practice of Oncology, 5th ed.; Vincent, T. D., Jr.,
Hellman, S., Steven, R., Eds.; Lippincott–Raven: Phila-
delphia, 1997; pp 3075–3085.
11. Mosmann, T. J. Immunol. Meth. 1983, 65, 55.
12. Shinji, S.; Yoshiaki, S.; Hiroshi, S. Biol. Pharm. Bull. 1997,
20, 1131.
The method as described by Shinji et al. was followed.12
Briefly, 1.5 · 104 ECV304 cells in growth medium
(DMEM containing 10% FBS) were seeded on Matri-
gelTM (70 lL). Betulinic acid (1) and derivatives (2, 4, 6–
8), solubilized in DMSO (final DMSO concn <0.1%)
were added in duplicate wells at 1 lg/mL (non-cytotoxic
concd at 18 h) and incubated. After 18 h the control cells
start to form an intense network of tube-like structures.
The total tube length was measured by image analysis
and percentage inhibition of tube formation was calcul-
ated compared to controls. A qualitative assessment was
performed by viewing the tube-like structures under the
microscope and scoring for inhibition of endothelial
sprouting, capillary network formation, and intussusception.