Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.08.003

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC₅₀ of 2.44, 2.5, and 2.7 μg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC₅₀ of 0.67 μg/ml on MIAPaCa cell line.

Full text of DOI:10.1016/j.bmcl.2008.08.003

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5058–5062
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic
acid derivatives
a
a
b
b
b
Vivek Kumar a, , Nidhi Rani , Pawan Aggarwal , Vinod K. Sanna , Anu T. Singh , Manu Jaggi ,
*
Narendra Joshi a, Pramod K. Sharma c, Raghuveer Irchhaiya c, Anand C. Burman a,b
a Chemical Research, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201010, UP, India
b Preclinical Research, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad 201010, UP, India
c Institute of Pharmacy, Bundelkhand University, Jhansi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 June 2008
Revised 8 July 2008
Accepted 1 August 2008
Available online 6 August 2008
A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between
C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-
20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance
the selectivity. Compound 11 resulted in IC50 of 2.44, 2.5, and 2.7
lg/ml on MIAPaCa, PA-1, and SW620
cancer cell lines, respectively. Compound 38 resulted in IC50 of 0.67
l
g/ml on MIAPaCa cell line.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Betulinic acid
Anticancer
Cytotoxicity
Natural products played a major role in the anticancer drug dis-
covery. Over 60% of the anticancer drugs are of natural origin. Bet-
ulinic acid (1), 3b-hydroxy-lup-20(29)-en-28-oic acid, a naturally
occurring pentacyclic lupane-type triterpene, is widely distributed
throughout the tropics. A variety of biological properties are as-
cribed to betulinic acid, but betulinic acid is recognized for its anti-
cancer and anti-HIV activities.1–4 Previous reports revealed that
betulinic acid is a melanoma-specific cytotoxic agent,5 but recent
evidence has indicated that betulinic acid also possesses a broader
spectrum of cytotoxic activity against other cancer cell lines.
Various modifications of substituents at positions 2, 3, 20, and
28 of betulinic acid have been the subject matter of all research ef-
forts to obtain potent lead compounds.6–11 All the above men-
tioned reports collectively disclose a large number of betulinic
acid derivatives, with a vast majority of them found to possess
antitumor activity. However, due to various reasons they are not
particularly good candidates, clinically as well as do not have the
best of pharmacokinetic properties. A need therefore exists for no-
vel betulinic acid derivatives, which are not only potent, but also
clinically safe and moreover, have better pharmacokinetic proper-
ties. In our efforts, we have found that in betulinic acid (1) hetero-
cyclic ring-like indole, benzofuran and pyrrole at C-2 and C-3
positions, imparts the desired characteristics. Further changes
were also carried out by reduction of C-20(29) unsaturated bond
and substitution of C-28 carboxyl group by ester and amide linkage
to enhance the selectivity (Table 1).12
Synthesis of betulinic acid derivatives has been described in
Schemes 1–3.13 Betulinic acid (1) was acetylated with acetic anhy-
dride in presence of pyridine to afford 3-acetyloxy betulinic acid
(2). Compound 2 upon hydrogenation with Pd/C in presence of
hydrogen gas afforded 3-acetyloxy-20,29-dihydrobetulinic acid
(3).14 Dihydrobetulinic acid (4) was obtained by deacetylation of
3-O-acetyl 20,29-dihydrobetulinic acid (3) under basic conditions
as shown in Scheme 1.
Both betulinic acid (1) and dihydrobetulinic acid (4) were oxi-
dized in presence of Jones reagent to their respective betulonic acid
(5) and 3-O-dihydrobetulinic acid (6), respectively.15 Compounds 5
and 6 undergo Fischer indole synthesis with various arylhydra-
zines by loss of ammonia to afford compounds 7-19.16 N-Benzyl-
pyrrolo substituted betulinic acid derivative (20) was afforded by
the reaction of intermediate formed from the reaction of benzyl-
amine with dihydrobetulinic acid (6), then with Michael acceptor
nitroalkene.17 Benzofuran derivatives (21 and 22) were synthe-
sized from betulonic acid (5) and 3-O-dihydrobetulinic acid (6)
with O-phenylhydroxylamine in presence of methanesulfonic acid
as shown in Scheme 2.18
Ester derivatives 23–27 were synthesized by the reaction of
compounds 7 and 11 with corresponding halides under basic con-
dition. To synthesize amide derivatives (28–37), C-28 carboxylic
group was converted to acyl chloride intermediate, which was fur-
ther reacted with the corresponding amines. Compounds 28 and
29 upon hydrolysis under basic condition afforded compounds
* Corresponding author. Tel.: +91 120 4378501; fax: +91 120 4376501.
E-mail addresses: kumarv@dabur.com (V. Kumar), jaggim@dabur.com (M. Jaggi).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.003

V. Kumar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5058–5062
5059
Table 1
Betulinic acid derivatives
R
ZR2
X
R4
Y
3'
2'
O
R3
1'
R1
Compound
X
Y
R
R1
Z
R2
R3
R4
7
8
9
H
H
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
N
N
N
N
N
N
N
N
C(@CH2)CH3
CH(CH3)2
C(@CH2)CH3
CH(CH3)2
C(@CH2)CH3
CH(CH3)2
C(@CH2)CH3
CH(CH3)2
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
CH(CH3)2
H
H
CH3
CH3
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
50-Cl
50-Cl
50-F
50-F
70-Cl
70-F
40-Cl, 60-Cl
50-Cl, 70-Cl
50-OCH3
H
—
H
H
H
H
H
50-Cl
H
C6H5CH2
H
Ph
C(@CH2)CH3
CH(CH3)2
—
—
H
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
O
O
O
O
CH2C(O)OC(CH3)3
CH2CH@CH2
CH2Ph
CH2Ph
O
CH2PhNO2(4)
CH2CO2CH3
CH2CO2CH3
CH2C„CH
H
50-Cl
50-Cl
NH
NH
NH
31
50-Cl
N
C(@CH2)CH3
H
NH
Ph
HN
32
50-Cl
N
C(@CH2)CH3
H
NH
Ph
HN
33
34
35
50-Cl
50-Cl
H
N
N
N
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
H
H
H
NH
NH
NH
C6H4CF3 (400)
C6H4OCF3 (400)
CH2Ph
Ph
Ph
Ph
50-Cl
50-Cl
N
N
C(@CH2)CH3
C(@CH2)CH3
H
H
NH
NH
Ph
Ph
HN
N
36
37
N
HN
S
38
39
40
41
H
N
N
N
N
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
C(@CH2)CH3
H
H
NH
NH
NH
NH
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
Ph
Ph
Ph
Ph
50-Cl
H
C6H5CH2
C6H5CH2
50-Cl
38 and 39. In a similar way, compounds 40 and 41 were synthe-
sized by benzylation at N-1 position using sodium hydride, fol-
lowed by hydrolysis as shown in Scheme 3.
compound 8. Introduction of electron-donating methyl group at
N-1 position in indolo betulinic acid derivative (9) showed selec-
tivity toward MIAPaCa cancer cell line with IC50 of 5.34 lg/ml.
Results and discussion. All the synthesized betulinic acid deriva-
tives (7–41) were tested for in vitro cytotoxicity on seven tumor
cell lines as well as on one non-tumorous cell line, and IC50 values
No major affect on activity was observed (10) upon reduction of
C-20(29) double bond.
Introduction of the electron-withdrawing halogen group in
indolo ring imparted potent cytotoxicity. Compound 11 having
chloro group at C-50 position is the most potent compound of this
were calculated in micromole (l
g/ml).19,20 The human tumor cell
lines used in the study are ovary (PA-1), prostate (DU145), colon
(SW620), breast (HBL100), pancreas (MIAPaCa2), lung (A-549),
and leukemia (K562) cancers. Compounds (7–41) were also
screened against normal mouse fibroblast (NIH3T3) to evaluate
their cancer cell specificity (safety index).19 The cytotoxicity data
are summarized in Table 2.
series. Compound 11 showed IC50 of 2.44, 2.5, and 2.7 lg/ml on
MIAPaCa, PA-1, and SW620 cancer cell lines, respectively, with
safety index of ꢀ2 on MIAPaCa. Reduction of C-20(29) double bond
(12) caused 2- to 3-fold decrease in the activity. However, when we
replaced the chloro group with fluoro at C-50 position (13), major
fall in activity was observed. Reduction of double bond (14) caused
some improvement in activity on MIAPaCa and A549 cancer cell
lines. On changing the position of halo group from C-50 to C-70 in
indolo ring, chloro-substituted derivative 15 resulted in maximum
Unsubstituted indolo betulinic acid derivative (7) resulted in
broad spectrum of cytotoxicity with IC50 of 5.15, 6.01, and
6.7
lg/ml on SW620, PA-1, and MIAPaCa cancer cell lines, respec-
tively. Reduction of C-20(29) double bond led to inactive

5060
V. Kumar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5058–5062
29
20
Acetic anhydride/
pyridine
OH
28
O
OH
O
O
H3C
O
HO
2
3
1
H2 /Pd/C
,
OH
OH
NaOH/ MeOH
O
O
O
H3C
O
HO
4
3
Scheme 1.
R
OH
R
O
OH
HO
X
R
O
1 C(=CH2)CH3
4 CH(CH3)2
C6H5(X)NR1NH2/
EtOH/ HCl
N
R1
7-19
Jones reagent
R
(i) Benzyl amine/
PTSA/ EtOH/
Mol. sieves
OH
O
OH
O
NO2
(ii)
O
N
R
20
5 C(=CH2)CH3
6 CH(CH3)2
H
PhONH2/ MeSO3H
R
OH
X
O
O
21-22
Scheme 2.
activity on SW620 cancer line with IC50 of 2.00
index of ꢀ6. Exchange of chloro with fluoro led to compound 16,
which showed potent activity with IC50 of <3.5 g/ml on three can-
cer cell lines. Substitution of the indolo ring with dihalo electron-
withdrawing group provided the selectivity toward A549 cancer
cell line in compound 17, but compound 18 was inactive. Replacing
the electron-withdrawing halo group with electron donating
methoxy group (19) did not result in substantial change in the
activity.
l
g/ml, with safety
N-Benzyl pyrrolo-substituted betulinic acid derivative (20) led
to complete loss in the activity. Replacement of the indolo part
with isosteric benzofuran led to inactive compound 21. While
reduction of C-20(29) double bond in benzofuran derivative (22)
resulted in moderate activity on A549 cancer cell line.
As indolo-substituted betulinic acid derivatives 7 and 11 might
have broad spectrum of cytotoxicity, their C-28 carboxyl group was
further replaced with different ester and amide linkage. Replace-
ment of the C-28 carboxyl group with ester groups like Boc (23),
l

V. Kumar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5058–5062
5061
R
R
R2-Cl/ R2-Br
OH
OR2
X
K2CO3
X
O
O
N
N
R1
7, 11
R1
23-27
(i) Oxalyl chloride
(ii) Amine
R
NHR2
MeOH/THF
4N NaOH
NHCH2COOH
X
X
O
O
(For 28-29)
N
N
H
R1
28-37
(i) NaH
38-39
(For 28-29)
(ii) BnBr
NHCH2COOH
X
O
N
40-41
Scheme 3.
Table 2
IC50 values of in vitro cytotoxicity of betulinic acid (BA) derivatives
Compound
IC50
HBL100
(l
g/ml) for cell lines
PA-1
DU145
SW620
MIAPaCa
A549
K562
NIH3T3
1
7
9
11.53 0.8
6.01 0.9
>20
>20
13.26 0.64
5.15 0.7
>20
5.02 0.7
10.3 0.9
19.5 2.1
14.14 0.8
11.75 1.65
>20
>20
3.00 0.7
7.6 0.57
6.9 0.21
6.5 3.4
7.14 0.5
8.8 0.74
17.8 1.79
8.89 0.84
8.7 0.19
3.0 0.6
5.5 1.1
8.2 0.14
7.1 1.6
3.53 0.82
12.4 1.78
3.25 1.2
10.05 0.61
7.73 0.17
10.85 1.5
9.61 0.78
>20
10.95 0.93
14.01 0.75
9.26 1.6
8.7 0.9
>20
4.37 0.7
6.9 0.76
15.12 3.04
>20
4.6 0.14
19.03 0.21
>20
14.59 0.33
11.8 0.19
2.1 0.55
4.10 0.9
7.5 0.48
20 5.6
8.86 0.2
20 0.6
>20
4.9 0.9
6.5 1.2
>20
>20
11.66 0.71
>20
>20
5.75 0.75
>20
7.0 0.7
10.32 0.9
6.7 0.59
5.34 0.96
5.4 0.43
2.44 0.26
15.19 1.06
17.4 2.6
8.4 0.76
11.6 0.49
3.3 0.2
10
11
12
13
14
15
16
17
19
22
38
39
>20
>20
2.5 0.6
6.6 0.9
>20
>20
6.39 0.1
ND
>20
5.8 0.9
>20
3.0 0.9
6.66 0.43
2.7 0.2
5.9 0.8
9.16 0.8
7.28 0.3
2.00 0.3
>20
>20
8.4 0.9
>20
>20
>20
11.8 0.35
3.5 0.9
>20
12.8 0.39
>20
>20
6.4 0.44
8.8 0.36
0.67 0.03
10.7 0.63
>20
15.28 0.18
11.92 1.37
>20
8.7 0.1
10.42 0.5
>20
16.5 0.6
0.68 0.003
11.2 0.63
Cytotoxicity was determined by MTT assay, as described.19 Data shown are IC50 SD of three independent experiments. If IC50 was not achieved, it was represented as greater
than highest concentration tested, that is, 20
ND, not done.
lg/ml.
allyl (24), and benzyl groups (25–27) led to the complete loss of the
activity. Similarly, replacement of the C-28 carboxyl group with
amide groups like amino acid ester (28 and 29), alkyl (30) cyclo-
alkyl (31 and 32), aryl (33–35), and heteroaryl (36 and 37) amide
led to the inactive compounds.
However, when we carried out the hydrolysis of ester com-
pound 28, it led to potent molecule 38, which resulted in IC50 of
0.67, 3.0, and 3.53 lg/ml on MIAPaCa, PA-1 and A549 cancer cell
lines, respectively. While compound 39 showed moderate activity
with IC50 of 6.66 g/ml on PA-1 cancer cell line. Substitution of N-1
position with benzyl group along with hydrolysis in compounds 28
l
and 29 resulted in inactive compounds 40 and 41.
In the present study, several derivatives have shown better
cytotoxicity than betulinic acid. The halo-substituted hetrocyclic
ring (indolo) at C-2 and C-3 positions in betulinic acid afforded
highly potent cytotoxic compounds 11. Substitution of N-1 posi-

5062
V. Kumar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5058–5062
15. Mukherjee, R.; Jaggi, M.; Rajendran, P.; Siddiqui, M. J. A.; Srivastava,
S. K.; Vardhan, A.; Burman, A. C. Bioorg. Med. Chem. Lett. 2004, 14,
2181.
16. Robinson, B. The Fischer Indole Synthesis; John Wiley & Sons: New York, 1982.
pp 487.
17. Shefali; Srivastava, S. K.; Hall, L. D.; Lewis, J. W.; Husbands, S. M. Helv. Chim.
Acta 2002, 85, 1790.
18. Portoghese, P. S.; Nagase, H.; MaloneyHuss, K. E.; Lin, C. E.; Takemori, A. E. J.
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tion of indolo ring led to the loss of activity. So, N-1 position should
be kept unoccupied. In most of the cases, hydrogenation of C-
20(29) double bond decreased the cytotoxicity. At C-28 position,
carboxylic group is essential for activity, replacement of carboxyl
group with ester and amide ester leads to inactive compounds.
However, hydrolysis of amide ester provided highly potent com-
pound 38. Compounds 11 and 38 have been selected for further
studies.
19. Derivatives of betulinic acid (7–41) were screened for cytotoxic activity at the
highest soluble concentration of 20 lg/ml and at four lower concentrations on
seven human tumor cell lines and one non-tumorous cell line. Briefly, cells
were collected from 70–80% confluent adherent cultures by trypsinization
(0.25% trypsin and 0.02% EDTA) and seeded in 96-well plates at a density of
5000 cells/well, except K562 which was seeded at15,000 cells/well in cell
culture medium (DMEM) for 24 h in a CO2 incubator. The test substance was
dissolved in DMSO (Merck, India), and further dilutions were made in cell
culture medium such that the final DMSO concentration in the well even at the
highest concentration is less than 1%. After 24 h, the cells were incubated with
the above-mentioned test substance to obtain drug concentrations in the range
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l
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