Synthesis of 3-O-acyl/3-benzylidene/3-hydrazone/3-hydrazine/17- carboxyacryloyl ester derivatives of betulinic acid as anti-angiogenic agents

Article abstract of DOI:10.1016/j.bmcl.2004.04.010

New 3-O-acyl, 3-benzylidene, 3-hydrazone, 3-hydrazine, 17-carboxyacryloyl ester derivatives of betulinic acid (2-6, 8-11, 13, 17, 18, 21, and 22) were synthesized and evaluated in vitro for anti-angiogenic activity on endothelial cell cytotoxicity, specificity, and tube-formation ability. All derivatives reported here showed IC₅₀<4μg/mL. Compounds 3, 9, 10, 17, 21, and 22 have shown better cytotoxicity (IC₅₀<1.2μg/mL) than betulinic acid (1) and improved endothelial cell specificity (ECS>10) in some cases. Compounds 10, 17, and 18 have shown 20%, 32%, and 48% reduction in TLS, respectively, and were found better than betulinic acid (1). We have shown that 20,29-dihydrobetulinic acid derivatives have better anti-angiogenic activity as compared to betulinic acid or its other derivatives.

Full text of DOI:10.1016/j.bmcl.2004.04.010

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3169–3172
Synthesis of 3-O-acyl/3-benzylidene/3-hydrazone/3-hydrazine/
17-carboxyacryloyl ester derivatives of
betulinic acid as anti-angiogenic agents
Rama Mukherjee,a,b,c Manu Jaggi,a,* Praveen Rajendran,a Sanjay K. Srivastava,b,*
Mohammad J. A. Siddiqui,c Anand Vardhanc and Anand C. Burmana,b,c
aDivisions of Experimental Oncology, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201 010, U.P., India
bMedicinal Chemistry, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201 010, U.P., India
cChemical Research, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201 010, U.P., India
Received 11 March 2004; accepted 2 April 2004
Abstract—New 3-O-acyl, 3-benzylidene, 3-hydrazone, 3-hydrazine, 17-carboxyacryloyl ester derivatives of betulinic acid (2–6, 8–11,
13, 17, 18, 21, and 22) were synthesized and evaluated in vitro for anti-angiogenic activity on endothelial cell cytotoxicity, specificity,
and tube-formation ability. All derivatives reported here showed IC50 < 4 lg/mL. Compounds 3, 9, 10, 17, 21, and 22 have shown
better cytotoxicity (IC50 < 1.2 lg/mL) than betulinic acid (1) and improved endothelial cell specificity (ECS > 10) in some cases.
Compounds 10, 17, and 18 have shown 20%, 32%, and 48% reduction in TLS, respectively, and were found better than betulinic acid
(1). We have shown that 20,29-dihydrobetulinic acid derivatives have better anti-angiogenic activity as compared to betulinic acid or
its other derivatives.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
report the synthesis of 3-O-acyl, 3-benzylidene, 3-
hydrazone, 3-hydrazine, 17-carboxyacryloyl ester deriv-
atives of betulinic acid (2–6, 8–11, 13, 17, 18, 21, and 22)
with IC50 < 4 lg/mL on ECV304 cell line, their endothe-
lial cell specificity (ECS) and percentage inhibition of
tube-like structure (TLS) formation of ECV304 cells.
Betulinic acid (1), a pentacyclic triterpene has been
reported to be a selective inducer of apoptosis in various
human cancers.1–3 Recent studies have revealed that
betulinic acid (1) is cytotoxic to endothelial cells4 and
inhibits invasion and tube formation of endothelial cells
at noncytotoxic concentrations through a modulation of
mitochondrial function.4;5 It also inhibits in vitro enzy-
matic activity of aminopeptidase N, which is known to
play an important role in angiogenesis.6
2. Chemistry
Synthesis of 3-O-acyl, 3-phenylhydrazone, and 17-
carboxyacryloyl ester derivatives of betulinic acid (2–6,
8–11, and 13)4 has been described in Scheme 1. Betulinic
acid (1) on treatment with acryloyl chloride in presence
of sodium hydride and solvent DMF afforded to 17-
carboxyacryloyl ester derivative 2. Betulinic acid (1),
upon reaction separately with 3-(trifluoromethyl)benz-
oyl chloride, 4-pentylbenzoyl chloride and 4-hept-
ylbenzoyl chloride, yielded corresponding 3-O-acyl
derivatives 3, 4, and 6, respectively. Upon treatment of 1
with (2,5-dimethoxyphenyl)acetyl chloride afforded
compound 5. Hydrogenation of betulinic acid (1) with
Pd/Cgave 20,29-dihydro betulinic acid ( 7) and which
was later converted, using the similar method as
Previously, we reported the in vitro anti-cancer activity
of derivatives of betulinic acid on leukemia, lymphoma,
prostate, lung, and ovarian cancer cell lines.7 Recently,
the anti-angiogenic activity of some betulinic acid
derivatives has been reported by us.4;8 In order to better
understand the structure–activity relationships, we have
synthesized several derivatives of betulinic acid and
studied their effect on endothelial cells in vitro. Here we
* Corresponding authors. Tel.: +91-120-277-7901 to 25; fax: +91-120-
277-7303; e-mail addresses: jaggim@dabur.com; srivastavasa@
dabur.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.010

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R. Mukherjee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3169–3172
29
20
30
19
17
28
O
R1 = H
iii
O
O
R1 = H
1
OR1
2
OR1
ii
OH
3
HO
HO
RO
7, R1 = H
8, R1 = COCH=CH2
1, R1 = H
2, R1 = COCH=CH2
i
3, R = COC6H4(CF3)[3]
4, R = COC6H4(C5H11)[4]
i
5, R = COCH2C6H3(OCH3)2[2,5]
6, R = COC6H4(C7H15)[4]
R1 = H
ii
iv
R1 = H
O
O
O
v
OH
OH
RO
OH
H
N
Ph
N
O
9, R = COC6H3F2[3,5]
10, R = COC6H3F2[2,4]
11, R = COC6H4CF3[2]
12
13
Scheme 1. Reagents and conditions: (i) R1-Cl/NaH/DMF; (ii) R-Cl/DCM/Py; (iii) H2/Pd–C/MeOH; (iv) Jones’ reagent; (v) PhNHNH2/NaOAc/
MeOH.
discussed for the synthesis of 2, to its 17-carboxyacryloyl
ester derivative 8. Similarly as discussed for the synthesis
of 3–6, 20,29-dihydro betulinic acid (7) was converted to
3-O-acyl-20,29-dihydro betulinic acid derivatives (9–11).
On the other hand, upon Jones’ oxidation of betulinic
acid (1), betulonic acid (12) was formed and which, was
further reacted with phenyl hydrazine and sodium ace-
tate to provide 3-phenylhydrazone betulonic acid (13).
Jones’ reagent and which, was then elaborated in two
ways. In the first, compound 14 was treated with
hydroxylamine hydrochloride in pyridine to afford 3-
hydroxyloxime 20,29-dihydrobetulonic acid (15) and
which, upon treatment with platinum oxide in acetic
acid, yielded 3-amino 20,29-dihydrobetulinic acid (16).
The 3-benzylidene 20,29-dihydrobetulinic acid deriva-
tives 17 and 18 were prepared from the reaction of 16
with 3,4-difluorobenzaldehyde and 2,4-difluorobenz-
aldehyde, respectively. In the second, compound 14 was
treated separately with phenylhydrazine and 4-meth-
oxyphenyl hydrazine to afford 3-hydrazone derivatives
19 and 20, respectively. Upon hydrogenation of 19 and
Scheme 2 describes the synthesis of 3-benzylidene and 3-
hydrazine 20,29-dihydrobetulinic acid derivatives (17,
18, 21, and 22).4 The 20,29-dihydrobetulinic acid (7) was
oxidized to 20,29-dihydrobetulonic acid (14) using
O
O
O
ii
iii
i
7
OH
HO
OH
OH
O
N
H2N
14
16
15
iv
v
O
O
O
vi
OH
OH
OH
H
C
R
R
R
N
N
N
H
21, R = NH-C6H5
22, R = NH-C6H4(OCH3)[4]
19, R = NH-C6H5
20, R = NH-C6H4(OCH3)[4]
17, R = C6H3F2[3,4]
18, R = C6H3F2[2,4]
Scheme 2. Reagents and conditions: (i) Jones’ reagent; (ii) H2NOHÆHCl/Py; (iii) H2/PtO2/AcOH; (iv) RCHO/MeOH; (v) RNH2/NaOAc/EtOH; (vi)
H2/Pt/AcOH.

R. Mukherjee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3169–3172
3171
20 in the presence of platinum sponge catalyst in glacial
acetic acid afforded 3-hydrazine 20,29-dihydrobetulinic
acid derivatives 21 and 22, respectively. All the com-
pounds were characterized by spectroscopic and ana-
lytical tools.
18
17
10
Reduction in TLS
3. Results and discussion
Betulinic acid
Betulinic acid derivatives (2–6, 8–11, 13, 17, 18, 21, and
22) were screened for cytotoxicity on human endothelial
cells (ECV304) and tumor cell lines DU145 (Prostate),
L132 (Lung), PA-1 (Ovary) and HT-29 (Colon) and the
endothelial cell specificity (ECS) was calculated as
shown in Table 1. All betulinic acid derivatives reported
here showed IC50 < 4 lg/mL on ECV304 cell line and
compounds (3, 9, 10, 17, 21, and 22) have shown better
cytotoxicity (IC50 < 1.2 lg/mL) than betulinic acid (1)
and improved endothelial cell specificity (ECS > 10) in
some cases. Compound 17 exhibited the best cytotox-
icity (IC50 ¼ 0.35 lg/mL) and compounds 21 and 22 have
shown the best ECS, particularly, against DU145 cell
line.
0
20
40
60
80
Percent reduction in TLS
Figure 1. Inhibition of TLS by betulinic acid (1) and derivatives 10, 17,
and 18 at noncytotoxic concentration.
All the betulinic acid derivatives were screened for
inhibition of tube-like structure (TLS) formation of
ECV304 cells in a MatrigelTM tube formation assay.
Compounds 10, 17, and 18 have shown 20%, 32%, and
48% reduction in TLS, respectively, and were found
better than betulinic acid (1) as shown in Figure 1.
Figure 2 shows the formation of TLS by ECV304 cells
on matrigel (left panel) and its inhibition by incubation
for 48 h with compound 18.
Figure 2. Representative image analysis photograph of formation of
TLS by ECV304 cells on Matrigel (left panel) and inhibition of TLS by
incubation with 18 after 48 h (right panel).
by a bulky group like pentyl or heptyl (compounds 4
and 6) or electron donating group in the aromatic ring in
5, lowered the cytotoxicity as well as ECS. Unlike
compounds 3–6, in the 3-O-acyl 20,29-dihydrobetulinic
acid series (9–11), compounds 9 and 10, possessing flu-
oro group, exhibited better cytotoxicity and ECS while
compound 11, bearing an electron withdrawing group,
lowered the cytotoxicity and ECS. Introduction of a
phenylhydrazone function at position-3 (13) in betulinic
acid also lowered the cytotoxicity. It indicated that
Structure–activity relationship indicated that upon
protection of C-28 carboxylic acid in betulinic acid or its
20,29-dihydro derivative (compounds 2 and 8), cyto-
toxicity was lowered. In the 3-O-acyl betulinic acid
derivatives (3–6), compound 3, having an electron
withdrawing group in aromatic ring, was the most
cytotoxic with low to moderate ECS while substitution
Table 1. IC50 and ECS ratios of betulinic acid (1) and its derivatives (2–6, 8–11, 13, 17, 18, 21, and 22) on ECV304 cells
Compound
IC50 (lg/mL)
ECS ratio
ECV304 (endothelial)
DU145 (prostate)
L132 (lung)
PA-1 (ovary)
HT-29 (colon)
2
4.0 0.74
0.9 0.10
1.9 0.33
1.8 0.40
1.7 0.49
2.0 0.61
0.7 0.34
0.7 0.55
2.6 0.80
2.4 0.75
0.35 0.14
2.4 0.68
0.5 0.31
0.4 0.27
1.2 0.44
>2.5
>11.1
1.05
0.55
>5.8
>2.0
4.5
0.43
3.0
3.1
2.5
4.1
2.0
1.7
1.5
1.5
1.9
7.1
1.4
1.6
––
>1
2.5
>11.1
1.84
>5.6
>5.89
5.0
3
>4.4
>2.1
––
4
5
6
>2.3
0.75
––
8
9
10
>14.3
2.4
3.5
2.3
11
13
>1.5
2.7
>1.53
2.08
3.7
3.84
4.2
17
18
6.2
11.4
2.04
3.5
1.04
19.8
21.1
2.2
0.54
7.0
21
22
––
0.87
––
Betulinic acid (1)
1.0
3.2
‘––’ Not done; ‘>’ IC50 in tumor cells greater than highest concentration tested.

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R. Mukherjee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3169–3172
electron withdrawing group in betulinic acid derivatives
(3–6) is playing a crucial role for eliciting better activity
while it did not improve the activity in 20,29-dihydro-
betulinic acid derivatives (9–11).
plemented with 10% fetal bovine serum, penicillin
(100 units/mL), streptomycin (100 lg/mL), and ampho-
tericin B (0.25 lg/mL) at 37 ꢁC , 5% C O, 100% humidity.
2
Between the 3-benzylideno 20,29-dihydrobetulinic acid
derivatives 17 and 18, compound 17 showed high cyto-
toxicity and low to moderate ECS along with good anti-
TLS while compound 18 showed lowered cytotoxicity
and ECS with high anti-TLS activity. It seemed that the
position of fluoro group in the aromatic ring has a vital
role in eliciting both cytotoxicity and anti-TLS activity.
Compounds 21 and 22 of 20,29-dihydro-3-hydrazine
series, exhibited high cytotoxicity and high ECS indi-
cating that substitution of an electron donating group in
the aromatic ring has slightly improved the activity. It
clearly pointed that the electron donating group in
20,29-dihydrobetulinic acid was required for eliciting
better activity than betulinic acid.
5.3. Cytotoxicity assay
Cells (1.5 · 104) were incubated with the compounds
dissolved in DMSO (final DMSO concn <0.1%), in
triplicate wells to obtain drug concentration of 0.5–4 lg/
mL. Cytotoxicity was measured after 72 h using MTT
assay as described by Mosmann.9 Each experiment was
repeated thrice and mean IC50 values (half-maximal
cytotoxicity) have been reported. ECS ratios were cal-
culated using the formula: IC50 (tumor cell)/IC50
(endothelial cell). ECS < 10 was designated low, between
10 and 20 as moderate and >20 as high endothelial
specificity.
5.4. Tube-like structure (TLS) formation assay
The method as described by Shinji et al. was followed.10
Briefly, 104 ECV304 cells in growth medium (DMEM
containing 10% FBS) were seeded on MatrigelTM
(70 lL). Compounds were solubilized in DMSO and
were added in duplicate wells at noncytotoxic concen-
tration and incubated for 18 h following which the con-
trol cells start to form an intense network of tube-like
structures. The absence of cytotoxicity of betulinic acid
and its derivatives on ECV304 cells at the above time
point was confirmed by suitable controls. The total tube
length was measured by image analysis and percentage
inhibition of tube formation was calculated 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.
4. Conclusion
In the present study we have shown that 20,29-dihy-
drobetulinic acid derivatives have better anti-angiogenic
acivity as compared to the other derivatives of betulinic
acid. In our earlier work, we had speculated that the
double bond between the position-20 and 29 in betulinic
acid was playing a crucial role in eliciting high endo-
thelial cell cytotoxicity, the endothelial cell specificity,
and inhibition of tube-like structures.8 The present
studies confirmed our earlier results. We predict that the
‘high’ and ‘moderate’ ECS compounds specifically tar-
get endothelial cells and can be grouped under potent
anti-angiogenic compounds while ‘low’ ECS compounds
would supplement their already reported cytotoxic
activity against tumor cells. Further studies would be
done to test the best derivatives in vivo to ascertain the
effect on in vivo experimental angiogenesis as well as
determine the mechanism of action of these compounds.
References and notes
1. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W.; Fong,
H. H.; Kinghorn, A. D.; Brown, D. M.; Wani, M. C.;
Wall, M. E.; Hieken, T. J.; Gupta, T. D.; Pezzuto, J. M.
Nat. Med. 1995, 1, 1046.
5. Materials and methods
5.1. Chemicals
2. Cichewicz, R. H.; Kouzi, A. A. Med. Res. Rev. 2004, 24,
90.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide, Sigma, USA], MatrigelTM (Becton Dick-
inson, USA), DMEM (Dulbeccos modified Eagles
medium), and fetal bovine serum, FBS (Gibco BRL,
USA), DMSO (Merck, India). Chemicals used in syn-
thesis were purchased from Sigma, USA.
3. Wick, W.; Grimmel, C.; Wagenknecht, B.; Dichgans, J.;
Weller, M. J. Pharmacol. Exp. Ther. 1999, 289, 1306.
4. Jaggi, M.; Ramadoss, S.; Rajendran, P.; Siddiqui, M. J. A.
U.S. Patents 6,228,850, 2001; 6,403,816, 2002.
5. Ho, J. K.; Joong, S. S.; Jin, H. K.; Hyun, Y. C.; Young,
N. Y.; Seung, H. K.; Jaehoon, Y. Jpn. J. Cancer Res. 2002,
93, 417.
6. Melzig, M. F.; Bormann, H. Planta Med. 1998, 64, 655.
7. Ramadoss, S.; Jaggi, M.; Siddiqui, M. J. A. U.S. Patents
6,048,847, 2000; 6,214,814, 2001; 6,670,345, 2003.
8. Mukherjee, R.; Jaggi, M.; Rajendran, P.; Siddiqui, M. J.
A.; Vardhan, A.; Burman, A. C. Bioorg. Med. Chem. Lett.
2004, 14, 2181.
9. Mosmann, T. J. Immunol. Meth. 1983, 65, 55.
10. Shinji, S.; Yoshiaki, S.; Hiroshi, S. Biol. Pharm. Bull. 1997,
20, 1131.
5.2. Cell culture
ECV304 cell line was generously gifted by Dr. Taka-
hashi (Tokyo University, Tokyo, Japan). Human tumor
cell lines DU145 (prostate), L132 (lung), HT-29 (colon),
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-