3-Epihydroxy lup-20(29)-en-19(28)-olide: partial synthesis, antitopoisomerase activity, and 3D molecular docking

Article abstract of DOI:10.1007/s00044-016-1551-9

A novel method for the partial synthesis of the rare triterpenoid, 3-epihydroxy lup-20(29)-en-19(28)-olide, and 1 from betulinic acid is reported. The binding efficiency, mode of binding for different compounds to the central catalytic domain of topoisomerase IIα, was calculated from a complete 3D molecular docking study on the crystal structure of the enzyme (1bgw, pdb). The compounds 1, 2b, and 2d showed a dose-dependent inhibition of catalytic activity of topoisomerase IIα.

Full text of DOI:10.1007/s00044-016-1551-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2016) 25:1087–1095
DOI 10.1007/s00044-016-1551-9
ORIGINAL RESEARCH
3-Epihydroxy lup-20(29)-en-19(28)-olide: partial synthesis,
antitopoisomerase activity, and 3D molecular docking
1
,3
1
2
1
•
Amitava Mandal
Asim Kumar Bothra
•
Ashim Ghosh
•
•
Shilpi Ghosh
1
•
Suranjan Shil
3
Pranab Ghosh
Received: 26 March 2015 / Accepted: 27 February 2016 / Published online: 14 March 2016
Ó Springer Science+Business Media New York 2016
Abstract A novel method for the partial synthesis of the
rare triterpenoid, 3-epihydroxy lup-20(29)-en-19(28)-olide,
and 1 from betulinic acid is reported. The binding effi-
ciency, mode of binding for different compounds to the
central catalytic domain of topoisomerase IIa, was calcu-
lated from a complete 3D molecular docking study on the
crystal structure of the enzyme (1bgw, pdb). The com-
pounds 1, 2b, and 2d showed a dose-dependent inhibition
of catalytic activity of topoisomerase IIa.
field of natural product drug discovery is the inaccessibility
to supply of important natural products from their respective
natural sources (Harvey, 2008). Hence, the synthesis of
complex NPs from the frequently abundant core structures
rather than its isolation from the natural sources is more
efficient and an economical approach for developing bio-
logically relevant agents (Camp et al., 2012). The recent
trends of research in bioactive NPs are to develop anticancer,
antiviral, antiprotozoal, antidiabetic, antifertility, and car-
diovascular drugs. Therefore, NPs with reliably proven
biological activity are target for synthetic transformation.
DNA topoisomerase is an ever-present indispensable
enzyme. It is involved in chromosomal segregation during
the progress of some vital cellular processes such as repli-
cation, recombination, and transcription. The enzyme func-
tions as a dimer that passes a DNA segment through a
transient double-stranded break (Vosberg, 1985). Under
normal condition, topoisomerase-DNA covalent complexes
are transient and are found in very low levels because the
relegation step is faster than the cleavage step. Stabilization
of this complex (cleavable complex) generates DNA lesions,
thereby initiating cellular responses that induce cell cycle
arrest and apoptosis (Ganguly et al., 2007). Frequent studies
have shown that Topo IIa levels increase during cell prolif-
eration and this enzyme appears to be the isoform involved in
mitosis (Syrovets et al., 2000). The pharmacological inhi-
bition of this enzyme has been identified as target for anti-
cancer drug development. The plant-derived topoisomerase
inhibitors, such as camptothecin and podophyllotoxins/
etoposides, have notable therapeutic efficacy as antitumor
drugs. However, despite their clinical success, a significant
need for less toxic and morechemically stable topoisomerase
inhibitor still persists (Dexheimer and Pommier, 2008).
Triterpenes represent a varied and important class of
natural compounds. Among these, pentacyclic lupane-type
Keywords Triterpenoid Á Antitopoisomerase activity Á
3
D molecular docking
Introduction
A tailback in the lead invention for pharmaceutical research
and for chemical biology lies in the discovery of new bio-
logically applicable substances (Wess et al., 2001). Natural
products (NPs) represent one of the most enduring approa-
ches to drug discovery for therapeutics (Camp et al., 2012).
They have high chemical diversity and biochemical speci-
ficity, which differentiate them from synthetic and combi-
natorial compounds. However, the most difficult task in the
&
Pranab Ghosh
pizy12@yahoo.com
1
Natural Products and Polymer Chemistry Laboratory,
Department of Chemistry, University of North Bengal,
Darjeeling, West Bengal 734 013, India
2
3
Department of Biotechnology, University of North Bengal,
Darjeeling, West Bengal 734 013, India
Department of Chemistry, Raiganj University, Raiganj,
North Dinajpur, West Bengal, India
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Med Chem Res (2016) 25:1087–1095
(
6–6–6–6–5 type) triterpenes are one of the most signifi-
purified by column chromatography to brownish crystalline
compound 2d having melting point (mp) 192 °C. In mass
spectrum, the compound showed a molecular ion peak at
cant subclass that has been shown to possess several
medicinal properties. A number of reviews (Williams,
?
2
009) highlight the past achievements of lupane triter-
m/z 410 (M ). Its elemental analysis revealed the presence
penoids in affording new lead molecules. Recently, a
triterpene 3-epi hydroxy lup-20(29)-en-19(28)-olide (1)
isolated from the bark of Microtropis fokienensis and
Perrottetia arisanensis has shown significant cytotoxicity
against seven different cancer cell lines (Wu et al., 2011).
But the compound could not be further biologically eval-
uated because of its low availability (Wu et al., 2011). In
our previous studies, we have described the synthesis of
some biologically active triterpenoids (Ghosh et al., 2012;
Mandal et al., 2012), and in continuation of our effort in
the present communication, we report the very first partial
synthesis of compound 1 from betulinic acid, 2 (Fig. 1) and
its antitopoisomerase activity. The 3D molecular docking
of these molecules on crystal structure of topoisomerase IIa
of 79.53 % carbon and 10.18 % oxygen. The above data
infer the molecular formula of the compound as C H O .
31 48 3
The molecular formula of 2d was further corroborated by
1
13 13
H and C NMR spectra. The C and DEPT spectral data
revealed the presence of 31 carbon atoms including an ester
carbonyl (d 171.0), nine quaternary, three tertiary, twelve
C
secondary, and seven primary carbon atoms (including the
Me-ester group). The eight degrees of unsaturation inherent
in the molecule coupled with the NMR data showed the
presence of one carbonyl group, five rings, and two olefinic
double bonds in the molecule which indicated that dehy-
drogenation has occurred during the mercuric acetate oxi-
dation of compound 2c.
In IR spectrum, 2d showed peaks at 3440, 1733, 1461,
-
1
1
(
1bgw for topoisomerases IIa, PDB (Berger et al., 1996)
1373, 1020, and 967 cm . Its H NMR spectrum showed
the presence of five tertiary methyl groups at dH 0.76 (s,
3H, 24-Me), 0.86 (s, 3H, 25-Me), 0.90 (s, 3H, 26-Me), 0.93
(s, 3H, 23-Me), and 0.97 (s, 3H, 27-Me). The methyl group
of the isopropylene moiety appeared at dH 1.61 (s, 3H).
was performed for the first time to evaluate the binding
energies as well as their mode of interaction with the
enzyme. We have also developed the electrostatic potential
(ESP) surface of compound 1 that will determine its best
fitting to the active site of the enzyme.
C-28 carbomethoxy group appeared at d 3.69 (s, 3H). The
H
d-H at C-3 appeared at d 3.61 (m, 1H). The absence of
H
any multiplet around d 3.00 (due to d-H at C-19) and a
H
Results and discussion
Chemistry
singlet at d 1.64 (due to d-H at C-18) (Wu et al., 2011)
H
signified the incorporation of an additional unsaturation at
C-18 during the mercuric acetate dehydrogenation. 2d on
acetylation and subsequent purification gave compound 2e
as slightly yellowish crystals of m.p 176 °C. In IR spec-
trum, it gave peaks at 1734, 1461, 1373, 1248, 1020, and
Betulinic acid (2) used in this study was isolated from
Biscofia javanica Linn. The synthetic approach involved
the epimerization of betulinic acid by the method described
by Symon et al. (2003) with suitable modifications, fol-
lowed by its esterification, mercuric acetate dehydrogena-
tion, and lead tetra acetate (LTA) oxidation, to get the
target molecule, 1 (Scheme 1).
-
1
1
967 cm . In the H NMR spectrum, the acetoxy methyl
appeared at d 2.00 (s, 3H). All other peaks were compa-
H
rable to that of 2d. All these observations could be
explained clearly if compound 2d is 3a-hydroxy-28-car-
bomethoxy lup-18(19), 20(29)-diene.
Methyl ester of 3-epi betulinic acid (Symon et al., 2003)
Compound 2d on controlled LTA oxidation (Mandal
et al., 2012) and subsequent chromatographic purifica-
tion yielded white pure compound, 1 (62 %) of m.p
(
2c) was dehydrogenated by mercuric acetate in glacial
acetic acid, followed by a reaction with hydrogen sulfide
for 3 h in pyridine. The product obtained was (vide infra)
?
300–302 °C. From mass spectral data (m/z, 454.46 M )
and elemental analysis (carbon 79.12 %, oxygen 10.43 %),
the molecular formula of the compound was inferred as
13
3
0
C H O . The C NMR spectral data revealed the pres-
3
0 46 3
2
0
1
9
21
ence of 30 carbon atoms including a lactone carbonyl (dC
2
9
1
O
2
18
2
2
178.9), and DEPT experiment confirmed the presence of
1
3
1
9
1
28
2
5
26
17
C
1
4
COOH
eight quaternary, four tertiary, twelve secondary, and six
primary carbon atoms. Here also, the eight degrees of
unsaturation inherent in the molecule coupled with the
NMR data showed the presence of one lactone carbonyl
group, five rings, one olefinic double bond, and an addi-
tional ring in the molecule. In its IR spectrum, it showed
peaks at 3442 (–OH), 1761 (lactone carbonyl), 1461, 1373
14
1
6
2
8
1
0
15
O
3
7 27
HO
5
HO
6
2
4
23
2
1
Fig. 1 Structure of the 3-epihydroxy lup-20(29)-en-19(28)-olide (1)
and betulinic acid (2)
1
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Med Chem Res (2016) 25:1087–1095
1089
Scheme 1 Partial synthesis of
3
1
a-hydroxy lup-20(29)-en-
9(28)-olide; a, Jones reagent,
acetone, ice cold water, ether,
anhydrous Na SO ; b,
L-selectride, THF, 2 N NaOH,
, CH , ice cold ether,
glacial acatic acid, cold water;
c, mercuric acetate, H S, ether,
anhydrous Na SO ; d, LTA,
glacial acetic acid, ice cold
water, ether, anhydrous Na SO
4
COOH
6%
COOH
72%
b
COOR
2
4
7
2
H O
2
N
2 2
HO
a
O
HO
2b, R = H
2c, R = -CH
2a
2
3
2
c
2
2
4
c
56%
2
O
COOMe
C
6
2%
O
d
RO
RO
2
d, R = H
1
, R = H
2
e, R = Ac
1
a, R = Ac
-
gem dimethyl), 1149, 1114, and 967 cm . In H NMR
1
1
(
2D NMR (COSY) spectroscopy. a-H18 appeared at d 1.64.
H
spectrum, five tertiary methyl groups appeared at d 0.82
This proton was considered as reference to elucidate the
H
(
s, 3H, 24-Me), 0.84 (s, 6H, 25 and 26-Me), 0.87 (s, 3H,
3-Me), and 0.93 (s, 3H, 27-Me) and the methyl group of
the isopropylene side chain of lupane skeleton appeared at
.62 (s, 3H, 30-Me). Two olefinic protons at C-29 each
exact structure of the present compound. In the COSYqf45
2
pulse program (solvent CDCl ) spectrum, two-bond and
3
three-bond correlations were observed for the proton pairs
at C21 and C22 and each one is quadrupledly coupled due to
rigid molecular framework (Fig. 2b). Proton at C18 was not
the part of this coupling/correlation sequence; rather, it was
observed that it correlated with a-H13 and proton pair at
C . As a-H is not correlated with protons on C21 and
1
appeared as a singlet at d 4.95 (s, 1H) and 5.32 (s, 1H).
The d-H atom at C-3 gave a multiplet centered at d 3.29
H
H
1
m, 1H). In the C NMR spectrum, a carbonyl carbon
3
(
appeared at d 178.9, which was due to the lactone car-
C
12
18
bonyl at C-28. The olefinic carbon atoms of the side chain
C , it can be inferred that structure I (Fig. 2) is the exact
22
appeared at d 92.3 (C-20) and 112.7 (C-29). C-3 appeared
structure of the compound. Hence, from the results, it can
be concluded that the compound synthesized was the target
molecule, 1.
C
at d 78.5. All the above data gave evidences in favor of
C
the plausible formation of the lactone ring during the LTA
oxidation of 2d. The carbon atoms either at C-18 or at C-19
might be involved in the formation of lactone ring (Fig. 2).
Therefore, a five or four-membered lactone involving C-19
or C-18 carbon, respectively, was anticipated. However,
On acetylation followed by purification 1 gave the
acetylated product 1a. Its IR spectrum showed peaks
at 1761 (five-membered lactone carbonyl), 1733
(acetate [ C=O), 1461, 1373 (gem dimethyl), 1248
-
1
-1
1
13
the IR data (1761 cm ) and the absence of any multiplet
1
(–OCOMe), 1149, 1114, and 967 cm . The H and
C
in the H NMR spectrum of 1 between d 2.50 and 3.00
NMR spectra were in full agreement with the proposed
H
(
for the b-H ) clearly signified the formation of a c-lactone
structure. Acetyl methyl group appeared at d 2.13 (s, 3H)
1
9
H
involving C-19. Its quaternary nature has also been verified
by DEPT spectra. Further confirmation was obtained from
and C-3 appeared at d 80.8. The acetoxy carbonyl group
C
appeared at d 171.1.
C
Fig. 2 a Possible formation of
two lactone rings; b COSY
correlation of compound 1
H
H
H
H
O
H
H
O
18
H
O
H
H
C
O
C
O
H
C
O
I
II
(a)
(b)
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Med Chem Res (2016) 25:1087–1095
Compound 1 was also prepared from a direct oxidation
of 3-epi betulinic acid by mercuric acetate; however, the
percent yield was low (17 %).
Fig. 3. In the compound 3-epibetulinic acid (2b), the C-3
hydroxyl group and the C-28 carboxylic group represent
the exclusive pharmacophore, generating H-bond interac-
tions. The C-3 hydroxyl group forms two H-bonds with the
neighboring Lys 776 and Tyr 511, whereas the acidic
carboxylic group at C-28 forms two hydrogen bonds with
the amino group of the Arg 782. The bulk of intermolecular
interaction between 2b and topoisomerase IIa is hydropho-
bic involving Ala 778, Ala 779, Ala 780, Tyr 511, and Ile
553 in the binding pocket (Fig. 3a).
3
D molecular docking
We evaluated compounds 1, 2b, and 2d into the central
catalytic domain (Deweese and Osheroff, 2009) of the
enzyme (1bgw, PDB for topoisomerase II alpha) using
AutoDock 4. Small molecules geometry optimization has
been carried out by steepest descent followed by BFGS.
It was reported in the literature that for lupane-type
triterpenoids, the amino acids Arg 1016, His 1012, Tyr
From the Fig. 3b, it is evident that compound 2d also
interact with the hydrophobic domain containing Ala 778,
Ala 779, Ala 780, Lys 776, and Tyr 511. Unlike 2b, it does
not form H-bond interactions with neighboring amino acids
and thus could not fully optimize the intermolecular
interaction within the binding pocket. The result is further
supported by a lower value binding energy minimization in
comparison with 1 and 2b.
8
04, Lys 812 and Lys 713 were present in the binding
domain of Topo IIa (Berger et al., 1996). In the present
docking studies, the catalytic Tyr 804 residue was con-
sidered as flexible for binding of the present triterpenoid
derivatives. Interestingly, all the derivatives (1, 2b, and 2d)
showed significant binding energies (-8.57 kcal/mol for 1,
The docking result of compound 1 (Fig. 3d) is quite
different from that of 2d. The presence of the lactone ring
in the compound changes the binding characteristics to the
enzyme. The C-3 hydroxyl group formed three H-bond
interaction with the[C=O group of Ala 779, Lys 776, and
Tyr 511 residues. The bulk of intermolecular interaction
-
8.81 kcal/mol for 2b, and -4.32 kcal/mol for 2d). Since
all the molecules had lupane skeleton, they showed inter-
action with quite similar drug-binding motif with small
variation in the nature of the amino acid residues. The
results of docking experiments are overlaid as shown in
Fig. 3 Detailed docked view of different compounds: a for compound 2b, a for compound 2d, c electrostatic view of compound 2d and d for
compound 1
1
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Med Chem Res (2016) 25:1087–1095
1091
between 1 and topoisomerase IIa is hydrophobic as it binds
to a pocket of the enzyme rich in hydrophobic amino acids,
involving Ala 778, Ala 779, Ala 743, Ala 781, Gly 633,
Lys 776, and Tyr 511n the binding pocket (Fig. 3d).
The analysis of enzyme–drug interaction indicated that
the compound 1 solely binds in the interior of the topoi-
somerase, formed by two winged-helix domain (WHD)
that contains the catalytic tyrosines responsible for DNA
cleavage and separation (Noble and Maxwell, 2002). Other
two compounds 2b and 2d bind to an intermediate/inter-
stitial space between the WHD domain, and the metal
binding TOPRIM (TOposisomerase/PRIMase) domain is
believed to assist formation of the protein-DNA interme-
is achieved when the shape and the charge distribution of
the receptor cavity are optimally matched by the shape and
the charge distribution of the binding molecule. This ESP
surface is valuable in computer-aided drug design because
it helps in optimization of electrostatic interactions
between the enzyme and the binding molecule.
Each protein has a unique shape that determines its
function, location in the body, relationship to cellular
membranes and binding interactions with drug molecules.
Most often, the binding site of a drug is the active site
where the enzymatic transformation is catalyzed; therefore,
the catalytic activity of the enzyme is inhibited. In cases
where the binding site of an enzyme is not at the active site
of the enzyme (allosteric site), the drug molecule causes an
enzymatic conformational change that prevents the enzyme
molecule to perform/play the accurate physiologic or
pathophysiologic functioning. To determine whether the
binding of compounds 1, 2b, and 2d cause any confor-
mational change in the enzyme or not, we further analyzed
the Ramachandran plots (Fig. 5) of the free protein mole-
cule and the different docked complexes with different
compounds. At the same time, it serves as a check about
the site of binding of compounds 1, 2b, and 2d in the
enzyme.
2
?
diate by coordinating Mg ions indispensable for DNA
cleavage (Dong and Berger, 2007). Therefore, from the
above results, it can be inferred that the synthesized lupane
triterpenoids can bind to topoisomerase IIa at the central
binding domain either by inhibiting DNA to bind to the
o
enzyme or by hindering the enzyme to enforce a 150 DNA
bend necessary to position the DNA backbone near a
reactive tyrosine and a coordinated magnesium ion (Dong
and Berger, 2007).
The study is helpful for the development of target-ori-
ented anticancer drugs. It is also evident from the docking
study that a small structural modification of the compound
changes the binding site on the target molecule. The phe-
nomenon is especially important for future drug designing,
because prolonged application of single drug causes an
obvious conformational change at the binding site of the
host molecule, and thereby developing drug resistance. A
finer modification in the structural arrangement of the
molecule leads its probable binding to other nearby resi-
dues within the radius of binding domain, regaining the
effectiveness of the drug.
As all the analyzed Ramachandran plots (Fig. 5) are
indistinguishable, it can be concluded that in our case, the
binding of the drug molecules did not induce any confor-
mational change in the enzyme, and hence, compounds 1,
2b, and 2d bind to the active site of the enzyme.
Biological part
Topoisomerase inhibitory activity of lupane derivatives
Furthermore, we have analyzed the ESP surface and
ESP charge of compound 1 (Fig. 4), because tight binding
Inhibition of catalytic activity of topoisomerase consti-
tutes a valuable approach for the identification of
Fig. 4 a ESP surface of compound 1 and b ESP charge on individual atoms
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Med Chem Res (2016) 25:1087–1095
Fig. 5 Representative Ramachandran plot of the free protein (a) and the docked complex with compound 1
Fig. 6 Effect of compounds 2b (A), 2d (B), and 1 (C) on DNA
relaxation activity by DNA topoisomerase IIa. A Lane a supercoiled
DNA alone; lane b control (no drug, 3 units of topo II); lanes c, d, e,
and f 100, 50, 25, and 12.5 lM of 2b, respectively; B lane a
supercolied DNA alone; lane b control (no drug, 2 unit of topo II);
lanes c, d, e, and f 100, 50, 25, and 12.5 lM of 2d, respectively;
C lane a supercoiled DNA alone; lane b control (no drug, 3 units of
topo II); lanes c, d, e, and f 100, 50, 25, and 12.5 lM of 1,
respectively
potential antitumor agents. Topo IIa creates transient
breaks in supercoiled DNA resulting in DNA relaxation.
The relaxed DNA can be distinguished from supercoiled
DNA by gel electrophoresis analysis. The results of
molecular docking studies on interaction of lupane
derivatives with DNA binding domain of human Topo IIa
were further confirmed by examining DNA relaxation
activity of Topo IIa. The ATP-dependent relaxation of
supercoiled DNA by the enzyme was monitored in the
absence or presence of the triterpenoid derivatives 1, 2b,
and 2d. From the results shown in Fig. 6, it is evident that
the inhibitory effects of these compounds were dose-de-
pendent. They showed complete inhibition of the catalytic
activity of Topo IIa at 100 and 50 lM concentrations,
whereas partial inhibition of the activity was observed at
a concentration of 25 and 12.5 lM. Compounds 2c and 2e
could not be studied due to their very poor solubility in
DMSO.
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Med Chem Res (2016) 25:1087–1095
1093
Conclusion
dry THF (10 mL) at cold under nitrogen atmosphere. The
reaction mixture was stirred for 5 h, and then, aqueous
NaOH (2 N, 18 mL) was added followed by 38 % aqueous
In summary, we have developed an efficient method for the
partial synthesis of 3-epihydroxy lup-20(29)-en-19(28)-
olide and studied its antitopoisomerase activity. It appeared
that the study is helpful for the development of target-
oriented anticancer drugs. It is also evident from the 3D
molecular docking study that a minute structural modifi-
cation of the drug molecule changes the binding site on the
target molecule. The phenomenon is especially important
for future drug designing, because prolonged application of
single drug causes an obvious conformational change at the
binding site of the host molecule which may lead
to developing drug resistance. As all the analyzed
Ramachandran plots (Fig. 4) are indistinguishable, it can
be infer that the synthesized molecules binded to the cen-
tral catalytic domain of the enzyme.
H O (4 mL). The reaction mixture continued to stir at
2 2
room temperature for 1 h; then, the solvent was concen-
trated under reduced pressure. To the residue, 10 %
aqueous HCl (15 mL) and ether (20 mL) were added and
the mixture was stirred until the all the precipitate was
dissolved. The organic layer was separated, the solvent was
evaporated, and the residue was purified by silica gel col-
umn chloroform to afford compound 3-epihydroxy lup-
20(29)-en-28-oic acid (2b, Symon et al., 2003) (127 mg,
38 % yield) as a colorless solid.
Esterification of 3-epihydroxy lup-20(29)-en-28-oic
acid (2b)
The acid 2b (0.8 g, 1.75 mmol) was dissolved in diethyl
ether (100 mL) at cold (0–5 °C) in a 250-mL conical flask.
To this, an excess of diazomethane dissolved in ether at
cold (0–5 °C) was added slowly with constant shaking. The
resulting yellowish solution was kept overnight under dark
and then galcial acetic acid (2 mL) was added to neutralize
excess of diazomethane. The reaction mixture was diluted
with water (200 mL) and extracted with ether (3 9
100 mL). The combined ether layers were evaporated
under reduced pressure. The gummy residue was purified
by silica gel column chromatography eluting with 1 %
ethyl acetate in petroleum ether to afford the methyl ester
2c (Berger et al., 1996).
Experimental
General
Melting points were recorded in open capillary method. IR
spectra were recorded in Shimadzu 800 FT-IR spec-
trophotometer using both KBr disk as well as Nujol. NMR
spectra were recorded in Bruker-Avance 300 MHz FT-
NMR instrument at ambient temperature with a 5-mm
BBO probe. The NMR chemical shift was reported in ppm
relative to 7.20 and 77.0 ppm of CDCl solvent as the
3
standards. Coupling constant ‘J’ was calculated in Hz. The
mass spectra were taken in 4800 (ABSciex) MALDI-TOF/
TOF tandem mass spectrometer. Elemental analyses were
done in Vario EL-III. Friedelin and cerin were isolated
from cork through soxhlet apparatus using petroleum ether
Mercuric acetate oxidation of the methyl ester of
3-epihydroxy lup-20(29)-en-28-oic acid (2c)
To compound 2c (600 mg 1.28 mmol) dissolved in chlo-
roform (50 mL) was added a solution of mercuric acetate
(1.30 mmol) in hot acetic acid (50 mL) and the reaction
mixture was maintained at 100 °C in oil bath for 4 h. Upon
cooling, the solid was filtered and was further diluted with
water (100 mL) and the organic layer was separated. The
organic layer was dried over anhydrous sodium sulfate, and
the solvent was concentrated under reduced pressure. The
residue was dissolved in dry pyridine, and hydrogen sulfide
gas was passed for 3 h. The black reaction mixture was
filtered using Kiselguhr, and pyridine was removed under
reduced pressure. The brownish black gummy residue was
purified by silica gel column chromatography eluting with
chloroform to afford compound 2d.
(60–80 °C) as the solvent. All the chemicals used were
commercial grade and were purified prior to their use.
Oxidation of 3-hydroxy lup-20(29)-en-28-oic acid
(
betulinic acid, 2)
Oxidation of 3-hydroxy lup-20(29)-en-28-oic acid (be-
tulinic acid, Symon et al., 2003) was done following the
same method as described by Symon et al. (2003). All
physical data were in good agreement to that reported
earlier for betulonic acid, 2a.
Synthesis of 3-epihydroxy lup-20(29)-en-28-oic acid
(
2b)
Compound 2d, white needles (MeOH) mp 192 °C, m/z
10 (M ), elemental analysis carbon 79.53 % and oxygen
?
4
1
10.18 %; H NMR (CDCl
A 1 M solution of L-Selectride in THF (5.6 mL) was added
3
, 300 MHz): d = 0.76 (s, 3H,
24-Me), 0.86 (s, 3H, 25-Me), 0.90 (s, 3H, 26-Me), 0.93 (s,
to a solution of betulonic acid (2a) (1.0 g, 2.20 mmol) in
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Med Chem Res (2016) 25:1087–1095
3
3
H, 23-Me) and 0.97 (s, 3H, 27-Me), 1.61 (s, 3H), 3.69 (s,
H), 3.29 (m, 1H).
adopted for acetylation of methyl ester of 3-epihydroxy
lup-20(29), 18(19)-diene-28-oic acid, 2d.
IR (KBr) mmax 1761 (five-membered lactone carbonyl),
1733, 1461, 1373 (gem dimethyl), 1248 (–OCOMe), 1149,
Acetylation of methyl ester of 3-epihydroxy lup-
2
-
1 1
0(29), 18(19)-diene (2d)
1114, 967 cm ; H NMR (CDCl , 300 MHz): d = 0.82
3
(s, 3H, 24-Me), 0.84 (s, 6H, 25 and 26-Me), 0.87 (s, 3H,
5
00 mg (1.06 mmol) compound 2d was dissolved in
0 mL dry pyridine in a 100-mL round bottom flask, and to
23-Me), 0.93 (s, 3H, 27-Me), 1.62 (s, 3H, 30-Me), 2.13 (3H
(–OCOMe), 4.95 (s, 1H) and 5.32 (s, 1H), 4.5–4.7 (m, 1H,
b-H C-3).
5
this, 10 mL acetic anhydride was added. The reaction
mixture was warmed over water bath for 6 h. After cool-
ing, it was poured into 150 mL ice cold water and worked
up with ether. The ether layer was washed with 6 N HCl
and again with water till neutral. After drying and evapo-
ration of solvent at reduced pressure, a brown residue was
obtained and then purified over a column of silica gel
3D molecular docking
Three-dimensional molecular docking studies were carried
out with AutoDock 4. Initially, we selected the drug
molecule, and by neighbor selection through Arguslab, we
located amino acid residues within 3.5A surrounding the
binding molecule. Molecular viewing was performed by
Molegro molecular viewer as well as in AutoDock 4.
ESP calculations have been done using GO9 W soft-
ware. The b3lyp functional with 6-31G (d) basis set is used
to optimize the geometry of the molecule (Frisch et al.,
2009).
(
60–120 mesh).
Compound 2e, mp 176 °C, m/z 468.25 (M ? 1), ele-
mental analysis carbon 79.53 % and oxygen 10.18 %; H
1
NMR (CDCl , 300 MHz): d = 0.76 (s, 3H, 24-Me), 0.86
3
(
s, 3H, 25-Me), 0.90 (s, 3H, 26-Me), 0.93 (s, 3H, 23-Me)
and 0.97 (s, 3H, 27-Me), 1.61 (s, 3H), 2.00 (s, 3H), 3.69 (s,
H).
3
LTA oxidation of methyl ester of 3-epihydroxy lup-
0(29), 18(19)-diene-28-oic acid (2d)
Topoisomerase II assay
2
Human TOPO IIa activity was measured by relaxation of
supercoiled pBR322 plasmid DNA. Reaction mixture
contained 10 mM Tris (pH-6.9), 50 mM KCl, 50 mM
A mixture of compound 2d (450 mg, 0.96 mmol), LTA
744.8 mg, 1.6 mmol), and glacial acetic acid (10 mL) was
heated at 30 °C with stirring under nitrogen atmosphere for
h. After cooling, ethane diol (15 mL) was added to
(
NaCl, 5 mM MgCl , 1 mM ATP, pBR 322 plasmid DNA
2
4
(100 ng), and three units of Topo IIa, in a final volume of
20 lL. For inhibition studies, the compounds were pre-
incubated with human TOPO IIa and DNA for 15 min.
Compounds were used at the appropriate concentrations by
dissolving in 2 % (v/v) DMSO. DMSO did not show
detrimental effect on the enzyme activity at concentration
up to 2 % v/v. Reaction mixture was incubated at 37 °C for
30 min and stopped by addition of 2 ml of 7 mM EDTA.
Reaction product was mixed with DNA loading dye and
electrophoresed on 1 % TAE-Agarose. The gel was stained
with ethidium bromide (0.5 lg/mL) for 20 min, destained
twice in TAE buffer and then visualized using a Gel Doc-
Imaging system (Spectronics, USA).
neutralize excess LTA and then the mixture was poured
into ice cold water. This aqueous portion was extracted
with ether, washed with 10 % sodium bicarbonate solution,
and water until the ether layer was neutral. The organic
layer was over anhydrous sodium sulfate, filter solid and
concentrated under reduced pressure. The residue was
purified by column chromatography eluting with chloro-
form to afford compound 1 (62 % yield) obtained as a
?
colorless solid: mp 300–302 °C, m/z 454.46 (M ), ele-
mental analysis (carbon 79.12 %, oxygen 10.43 %), IR
(
KBr) mmax 3442 (–OH), 1761 (five-membered lactone
carbonyl), 1461, 1373 (gem dimethyl), 1149, 1114 and
-
1 1
9
2
0
5
67 cm ; H NMR (CDCl , 300 MHz): d = 0.82 (s, 3H,
3
Acknowledgments Financial support from UGC, New Delhi, India,
was cordially acknowledged to carry out the work.
4-Me), 0.84 (s, 6H, 25 and 26-Me), 0.87 (s, 3H, 23-Me),
.93 (s, 3H, 27-Me), 1.62 (s, 3H, 30-Me), 4.95 (s, 1H) and
.32 (s, 1H), 4.46 (m, 1H, b-H C-3).
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