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The structure–activity relationship was studied keeping
in mind the activity on different cancer sub-types that is
leukemia/lymphoma, prostate, ovary, and lung cancer.
The activity of derivatives was compared with betulinic
acid in each panel.
dihydrobetulinic acid (19) was the most potent on L132
cell line. It clearly showed that oxime group in 20,29-
dihydrobetulinic acid plays a crucial role in eliciting
anti-lung cancer activity.
Amongst leukemia/lymphoma cancer cell lines, 2-bromo
20,29-dihydrobetulinic acid (23) was threefold more
potent than betulinic acid on MOLT-4 and CEM.CM3
cell lines and had similar activity as betulinic acid on
JurkatE6.1 cell line. The 20,29-dihydrobetulinic acid
derivative (9) was twofold more potent than betulinic
acid on MOLT-4 and had similar activity as betulinic
acid on CEM.CM3 and U937 cell lines. Betulonic acid
(13) was 1.5-fold more potent than betulinic acid on
JurkatE6.1 and had similar activity as betulinic acid on
MOLT-4 cell line. Compounds 2 and 10, having 2-
acetoxypropionyl and acetyl substituent at position-3,
respectively, have shown similar activity as betulinic
acid on MOLT-4 cell line. Compound 10 also had
similar activity as betulinic acid on CEM.CM3 cell line.
The 20,29-dihydrobetulinic acid derivative 20 and 17-
carboxyalkyl carboxylate derivatives (22 and 25) of 2-
bromo betulinic acid had similar activity as betulinic
acid on MOLT-4 cell line. Compound 22 also had
similar activity as betulinic acid on CEM.CM3 cell line.
This indicated that although O-acyl, oxime and hydra-
zone substituents at position-3 had some effect on the
cytotoxicity but it did not significantly improve the
activity of betulinic acid. In this cancer subtype, it
seemed that the bromo group at position-2 in betulinic
acid derivative, was the most suitable substituent for
eliciting better anti-leukemia/lymphoma activity.
4. Conclusion
In the present study, several derivatives have shown
better cytotoxicity than betulinic acid. The 2-bromo
20,29-dihydrobetulonic acid derivative (23) had shown
broad spectrum cytotoxicity. It had better cytotoxic
activity than betulinic acid in two out of five cancer cell
lines of leukemia/lymphoma and similar activity as
betulinic acid on JurkatE6.1 cell line. The betulonic acid
(13) and 3-O-acyl 20,29-dibromobetulinic acid (27) were
more active than betulinic acid in one out of five cell
lines of leukemia/lymphoma. The 3-hydroxyloxime
20,29-dihydrobetulonic acid (19) had shown better
activity than betulinic acid in one of the two lung cancer
cell lines. The 3-phenylhydrazone 20,29-dihydrobetu-
lonic acid (20) had several fold better anti-prostate as
well as anti-ovarian cancer activity than betulinic acid.
The present study pointed that bromo group in betulinic
acid is vital for anti-leukemia/lymphoma activity. The
oxime group at position-3 is essential for anti-lung
activity while the hydrazone substituent is responsible
for eliciting anti-prostate and anti-ovarian activities.
And in general, C-28 carboxylic acid group in betulinic
acid and its derivatives was found essential for providing
cytotoxic activity. Earlier studies have shown that C-20
side chain is not good for structural modifications.14 On
the contrary, it was interesting to note that in the present
study, C-20 double bond in betulinic acid was not crit-
ical for the cytotoxic activity in the cell lines tested since
upon its hydrogenation, the cytotoxicity of the deriva-
tives was affected significantly. Based on these studies,
compounds 13, 19, 20, 23, and 27 have been selected as
‘LEAD’ molecules and further studies are under pro-
gress to determine the ADME characteristics and in vivo
activity in animal models.
In prostate cancer cell line (DU145), the best compound
was the 3-phenylhydrazone derivative (20) of 20,29-di-
hydrobetulinic acid, which has shown around two-fold
more activity than betulinic acid, while its 3-hydroxyl-
oxime derivative 19 had similar activity as betulinic acid.
The 20,29-dihydrobetulinic acid (9) and its 3-acetyl and
3-(2-acetoxy) propionyl derivatives (10 and 11) were
slightly better than betulinic acid. Compound 5, 3-O-
trimethylacetyl derivative of betulinic acid, had similar
activity as betulinic acid. It indicated that the hydrazone
functionality at position-3 in 20,29-dihydrobetulinic
acid was the best substituent for anti-prostate activity.
5. Materials and methods
5.1. Chemicals
In ovarian cancer cell line, all the betulinic acid deriva-
tives (1–27) have shown several fold better cytotoxicity
than betulinic acid. In particular, the 20,29-dihydro-
betulinic acid derivatives 9–12, 19, 20, and 23 were rel-
atively highly potent. As in the case of anti-prostate
agents, here also, compound 20 was the most potent. It
pointed that hydrazone substituent at position-3 in
20,29-dihydrobetulinic acid plays a key role in eliciting
anti-ovarian as well as anti-prostate activity.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide) was obtained from Sigma, USA,
DMEM (Dulbecco’s modified Eagles medium) from
Gibco BRL, USA, fetal bovine serum (FBS) from Gibco
BRL, USA, dimethyl sulfoxide (DMSO) from Merck,
India, antibiotic solution (containing penicillin and
streptomycin) from Hyclone, USA. Chemicals used in
synthesis were purchased from Sigma, USA.
In lung cancer cell lines (A549 and L132), the cytotox-
icity against A549 cell line, was several fold increased
after converting betulinic acid into 20,29-dihydrobetu-
linic acid derivatives 9–11 and 19. In L132 cell line,
though, none of the derivatives (1–27) were found better
than betulinic acid. However, 3-hydroxyloxime 20,29-
5.2. Cell culture
Human tumor cell lines MOLT-4 (human lymphoblastic
leukemia), JurkatE6.1 (human lymphoblastic leukemia),
CEM.CM3 (human lymphoblastic leukemia), BRIS-
TOL8 (human B-cell lymphoma), U937 (human histio-